U.S. patent application number 16/165993 was filed with the patent office on 2019-02-21 for treatment of central nervous system disorders by intranasal administration of immunoglobulin g.
The applicant listed for this patent is Baxalta Incorporated, Baxalta Incorporated. Invention is credited to William H. Frey, II, Leah Ranae Bresin Hanson, Sharon Pokropinski, Francisco M. Rausa, III.
Application Number | 20190055307 16/165993 |
Document ID | / |
Family ID | 50424697 |
Filed Date | 2019-02-21 |
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United States Patent
Application |
20190055307 |
Kind Code |
A1 |
Frey, II; William H. ; et
al. |
February 21, 2019 |
TREATMENT OF CENTRAL NERVOUS SYSTEM DISORDERS BY INTRANASAL
ADMINISTRATION OF IMMUNOGLOBULIN G
Abstract
The present invention provides, among other aspects, methods and
compositions for treating a central nervous system (CNS) disorder
by delivering a therapeutically effective amount of a composition
of pooled human immunoglobulin G (IgG) to the brain via intranasal
administration of the composition directly to the olfactory
epithelium of the nasal cavity. In particular, methods and
compositions for treating Alzheimer's disease are provided.
Inventors: |
Frey, II; William H.; (White
Bear Lake, MN) ; Hanson; Leah Ranae Bresin; (Vadnais
Heights, MN) ; Pokropinski; Sharon; (Schaumburg,
IL) ; Rausa, III; Francisco M.; (Vernon Hills,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baxalta Incorporated
Baxalta Incorporated |
Bannockburn
Zug |
IL |
US
CH |
|
|
Family ID: |
50424697 |
Appl. No.: |
16/165993 |
Filed: |
October 19, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15335027 |
Oct 26, 2016 |
10144776 |
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16165993 |
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14189981 |
Feb 25, 2014 |
9556260 |
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15335027 |
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61769673 |
Feb 26, 2013 |
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61862814 |
Aug 6, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/16 20180101;
C07K 2317/92 20130101; A61K 9/08 20130101; A61P 25/08 20180101;
C07K 16/06 20130101; A61K 31/4172 20130101; A61P 25/28 20180101;
C07K 2317/55 20130101; A61P 25/14 20180101; A61P 25/00 20180101;
A61K 31/401 20130101; A61K 2039/543 20130101; A61P 43/00 20180101;
A61P 25/22 20180101; A61K 9/0043 20130101; A61K 31/198 20130101;
C07K 16/18 20130101; A61K 2039/505 20130101; C07K 2317/21 20130101;
A61P 25/18 20180101 |
International
Class: |
C07K 16/18 20060101
C07K016/18; C07K 16/06 20060101 C07K016/06; A61K 31/198 20060101
A61K031/198; A61K 31/401 20060101 A61K031/401; A61K 31/4172
20060101 A61K031/4172; A61K 47/18 20170101 A61K047/18; A61K 9/00
20060101 A61K009/00 |
Claims
1-91. (canceled)
92. A method for treating a central nervous system (CNS) disorder
in a subject in need thereof, the method comprising: delivering a
therapeutically effective amount of a composition comprising pooled
human immunoglobulin G (IgG) to the brain of the subject, wherein
delivering the composition to the brain comprises intranasally
administering the composition to a nasal epithelium of the subject
associated with trigeminal nerve endings, wherein at least 40% of
the pooled human IgG administered to the subject contacts the nasal
epithelium of the subject associated with trigeminal nerve
endings.
93. The method of claim 92, wherein the CNS disorder is selected
from the group consisting of a neurodegenerative disorder of the
central nervous system, a systemic atrophy primarily affecting the
central nervous system, an extrapyramidal and movement disorder, a
demyelinating disorder of the central nervous system, an episodic
or paroxysmal disorder of the central nervous system, a paralytic
syndrome of the central nervous system, a nerve, nerve root, or
plexus disorder of the central nervous system, an organic mental
disorder, a mental or behavioral disorder caused by psychoactive
substance use, a schizophrenia, schizotypal, or delusional
disorder, a mood (affective) disorder, neurotic, stress-related, or
somatoform disorder, a behavioral syndrome, an adult personality or
behavior disorder, a psychological development disorder, and a
child onset behavioral or emotional disorder.
94. The method of claim 92, wherein the CNS disorder is selected
from the group consisting of Alzheimer's disease, Parkinson's
disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS),
Huntington's disease, cerebral palsy, bipolar disorder,
schizophrenia, and Pediatric acute-onset neuropyschiatric syndrome
(PANS).
95. The method of claim 92, wherein the CNS disorder is selected
from the group consisting of Alzheimer's disease, multiple
sclerosis, and Parkinson's disease.
96. The method of claim 92, wherein the CNS disorder is Alzheimer's
disease.
97. (canceled)
98. The method of claim 92, wherein intranasal administration of
the composition comprises administration of a liquid drop of the
composition directly onto the nasal epithelium associated with
trigeminal nerve endings.
99. (canceled)
100. The method of claim 92, wherein intranasal administration of
the composition comprises directed administration of a liquid
aerosol of the composition to the nasal epithelium of the subject
associated with trigeminal nerve endings.
101. The method of claim 92, wherein intranasal administration of
the composition comprises directed administration of a powder
aerosol of the composition to the nasal epithelium of the subject
associated with trigeminal nerve endings.
102. (canceled)
103. The method of claim 92, wherein at least 50% of the pooled
human IgG administered to the subject contacts the nasal epithelium
associated with trigeminal nerve endings of the subject.
104. The method of claim 92, wherein at least 60% of the pooled
human IgG administered to the subject contacts the nasal epithelium
associated with trigeminal nerve endings of the subject.
105. (canceled)
106. The method of claim 92, wherein the composition comprising
pooled human IgG consists essentially of pooled human IgG and an
amino acid selected from the group consisting of glycine,
histidine, and proline.
107-108. (canceled)
109. The method of claim 92, wherein the composition comprising
pooled human IgG is an aqueous composition comprising: (a) from 10
mg/mL to 250 mg/mL pooled human IgG, and (b) from 50 mM to 500 mM
glycine.
110. (canceled)
111. The method of claim 109, wherein the pH of the composition is
from 4.0 to 6.0.
112. (canceled)
113. The method of claim 109, wherein the pH of the composition is
from 6.0 to 7.5.
114. The method of claim 92, wherein the composition comprising
pooled human IgG is a dry powder composition prepared from an
aqueous solution comprising: (a) from 10 mg/mL to 250 mg/mL pooled
human IgG, and (b) from 50 mM to 500 mM glycine.
115. (canceled)
116. The method of claim 114 or 115, wherein the dry powder
composition is prepared from an aqueous solution having a pH of
from 4.0 to 6.0.
117. (canceled)
118. The method of claim 114, wherein the dry powder composition is
prepared from an aqueous solution having a pH of from 6.0 to
7.5.
119. The method of claim 92, wherein the method comprises
intranasally administering to the subject a dose of from 0.08 mg to
100 mg pooled human IgG per kg body weight of the subject (mg
IgG/kg).
120-123. (canceled)
124. The method of claim 92, wherein the method comprises
intranasally administering to the subject a fixed dose of from 50
mg to 10 g pooled human IgG.
125-126. (canceled)
127. The method of claim 92, wherein the method comprises
intranasally administering to the subject a dose of pooled human
IgG at least twice monthly.
128-229. (canceled)
230. The method of claim 92, wherein at least 70% of the pooled
human IgG administered to the subject contacts the nasal epithelium
of the subject associated with trigeminal nerve endings.
231. The method of claim 100, wherein at least 50% of the pooled
human IgG administered to the subject contacts the nasal epithelium
of the subject associated with trigeminal nerve endings.
232. The method of claim 100, wherein at least 60% of the pooled
human IgG administered to the subject contacts the nasal epithelium
of the subject associated with trigeminal nerve endings.
233. The method of claim 100, wherein at least 70% of the pooled
human IgG administered to the subject contacts the nasal epithelium
of the subject associated with trigeminal nerve endings.
234. The method of claim 101, wherein at least 50% of the pooled
human IgG administered to the subject contacts the nasal epithelium
of the subject associated with trigeminal nerve endings.
235. The method of claim 101, wherein at least 60% of the pooled
human IgG administered to the subject contacts the nasal epithelium
of the subject associated with trigeminal nerve endings.
236. The method of claim 101, wherein at least 70% of the pooled
human IgG administered to the subject contacts the nasal epithelium
of the subject associated with trigeminal nerve endings.
Description
CROSS REFERENCES TO APPLICATIONS
[0001] This application is a Continuation of U.S. patent
application Ser. No. 15/335,027, filed Oct. 26, 2016, which is a
Continuation of U.S. patent application Ser. No. 14/189,981, filed
Feb. 25, 2014 (now issued as U.S. Pat. No. 9,556,260), which claims
priority to U.S. Provisional Patent Application Ser. Nos.
61/769,673 filed Feb. 26, 2013, and 61/862,814 filed Aug. 6, 2013,
the disclosures of which are hereby incorporated herein by
reference in their entireties for all purposes.
BACKGROUND OF THE INVENTION
[0002] The central nervous system (CNS) is the processing center
for the nervous system. CNS disorders can affect the brain, the
spinal cord, and nerve endings, resulting in neurological and/or
psychiatric disorders. CNS disorders can be caused by genetic
inheritance, trauma, infection, autoimmune disorders, structural
defects, tumors, and stroke. Certain CNS disorders are
characterized as neurodegenerative disease, many of which are
inherited genetic diseases. Examples of neurodegenerative diseases
include Huntington's disease, ALS, hereditary spastic hemiplegia,
primary lateral sclerosis, spinal muscular atrophy, Kennedy's
disease, Alzheimer's disease, a polyglutamine repeat disease, or
Parkinson's disease. Treatment of CNS disorders, e.g., genetic
diseases of the brain such as Parkinson's disease, Huntington's
disease, and Alzheimer's disease, remain an ongoing problem.
[0003] Alzheimer's disease is a common form of age-related dementia
that causes gradual loss of cognitive function, including memory
and critical thinking abilities. Alzheimer's disease is diagnosed
clinically by through a finding of progressive memory loss and
decrease in cognitive abilities. However, confirmation of
Alzheimer's disease does not occur until after death.
[0004] Alzheimer's disease is becoming more prevalent in developed
nations, where an increase in the population of elder persons has
occurred due in part to improved healthcare. While less than 1% of
the population under the age of 60 is affected by Alzheimer's, it
is estimated that 25% to 33% of persons develop some form of
Alzheimer's by the age of 85. As 0 of 2012, 5.4 million Americans
were diagnosed with Alzheimer's. As life expectancy continues to
increase worldwide, the prevalence of Alzheimer's and other
age-related dementia should continue to grow as well.
[0005] Alzheimer's disease is typically classified as either "early
onset," referring to cases that begin to manifest at between 30 and
60 years of age in affected individuals, and the more common "late
onset" Alzheimer's, in which symptoms first become apparent after
the age of 60. Although only about 10% of all Alzheimer's cases are
familial, early onset Alzheimer's disease has been linked to
mutations in the amyloid precursor protein (app), presenilin 1
(psen1), and presenilin 2 (psen2) genes, while late onset
Alzheimer's disease has been linked to mutations in the
apolipoprotein E (apoE) gene (Ertekin-Taner N., Neurol Clin.,
25:611-667 (2007)).
[0006] Histopathologically, this neurodegenerative disease is
characterized by the formation of amyloid plaques, neurofibrillary
tangles, amyloid angiopathy, and granolovacuolar degeneration in
the cerebral cortex (Mirra et al., Arch Pathol Lab Med.,
117:132-144 (1993); Perl D P, Neurol Clin., 18:847-864 (2000)). The
characteristic amyloid plaques, used to confirm Alzheimer's disease
post-mortem, are formed largely by deposition of a small
amyloid-beta (A.beta.) peptide derived from the amyloid precursor
protein (APP).
[0007] To date, the U.S. Food and Drug Administration (FDA) has
approved two types of medications for the management of Alzheimer's
disease: cholinesterase inhibitors, including donepezil (e.g.,
ARICEPT.RTM.), rivastigmine (e.g., EXELON.RTM.), galantamine (e.g.,
RAZADYNE.RTM.), and tacrine (e.g., COGNEX.RTM.); and the NMDA-type
glutamate receptor inhibitor memantine (marketed under a number of
different brands). Although a cure for Alzheimer's disease has not
been identified, these therapies serve to alleviate cognitive
symptoms such as memory loss, confusion, and loss of critical
thinking abilities in subjects diagnosed with age-related dementia
(e.g., Alzheimer's disease). In all, it is estimated that
healthcare spending on Alzheimer's disease and related age-related
dementias in 2012 will be $200 billion in the United States alone
(Factsheet, Alzheimer's Association, March 2012).
[0008] In addition to these approved therapies, several studies
have suggested that pooled intravenous immunoglobulin (IVIG) is
effective in slowing the progression of symptoms in Alzheimer's
patients (Dodel R C et al., J Neurol Neurosurg Psychiatry, October;
75(10):1472-4 (2004); Magga J. et al., J Neuroinflammation,
December 7; 7:90 (1997); Relkin N R et al., Neurobiol Aging,
30(11):1728-36 (2008); Puli L. et al., J Neuroinflammation May 29;
9:105 (2012)).
[0009] Immune globulin products from human plasma were first used
in 1952 to treat immune deficiency. Initially, intramuscular or
subcutaneous administration of immunoglobulin isotype G (IgG)
isolated from plasma were the methods of choice. However, IgG
products that could be administered intravenously, referred to as
intravenous immunoglobulin (IVIG), were later developed to allow
for the administration of larger amounts of IgG necessary for
effective treatment of various diseases. Usually, IVIG contains the
pooled immunoglobulin G (IgG) immunoglobulins from the plasma of
multiple donors, e.g., more than a hundred or more than a thousand
blood donors. These purified IgG products are primarily used in
treating three main categories of medical conditions: (1) immune
deficiencies: X-linked agammaglobulinemia, hypogammaglobulinemia
(primary immune deficiencies), and acquired compromised immunity
conditions (secondary immune deficiencies), featuring low antibody
levels; (2) inflammatory and autoimmune diseases; and (3) acute
infections.
[0010] Specifically, many people with primary immunodeficiency
disorders lack antibodies needed to resist infection. In certain
cases these deficiencies can be supplemented by the infusion of
purified IgG, commonly through intravenous administration (i.e.,
IVIG therapy). Several primary immunodeficiency disorders are
commonly treated in the fashion, including X-linked
agammaglobulinemia (XLA), Common Variable Immunodeficiency (CVID),
Hyper-IgM Syndrome (HIM), Severe Combined Immunodeficiency (SCID),
and some IgG subclass deficiencies (Blaese and Winkelstein, J
Patient & Family Handbook for Primary Immunodeficiency
Diseases. Towson, Md.: Immune Deficiency Foundation; 2007).
[0011] While IgG treatment can be very effective for managing
primary immunodeficiency disorders, this therapy is only a
temporary replacement for antibodies that are not being produced in
the body, rather than a cure for the disease. Accordingly, patients
depend upon repeated doses of IgG therapy, typically about once a
month for life. This therapy places a great demand on the continued
production of IgG compositions. However, unlike other biologics
that are produced via in vitro expression of recombinant DNA
vectors, IgG is fractionated from human blood and plasma donations.
Thus, the level of commercially available IgG is limited by the
available supply of blood and plasma donations.
[0012] Several factors drive the demand for IgG, including the
acceptance of IgG treatments, the identification of additional
indications for which IgG therapy is effective, and increasing
patient diagnosis and IgG prescription. Notably, the global demand
for IgG more than quadrupled between 1990 and 2009, and continues
to increase at an annual rate between about 7% and 10% (Robert P.,
Pharmaceutical Policy and Law, 11 (2009) 359-367). For example, the
Australian National Blood Authority reported that the demand for
IgG in Australia grew by 11.1% for the 2010-2011 fiscal year
(National Blood Authority Australia Annual Report 2010-2011).
[0013] It has been reported that in 2007, 26.5 million liters of
plasma were fractionated, generating 75.2 metric tons of IgG, with
an average production yield of 2.8 grams per liter (Robert P.,
supra). This same report estimated that global IgG yields are
expected to increase to about 3.43 grams per liter by 2012.
However, due to the continued growth in global demand for IgG,
projected at between about 7% and 14% annually between now and
2015, further improvement of the overall IgG yield will be needed
to meet global demand. One of the factors that may drive increased
demand for pooled human immunoglobulins (e.g., IVIG) over the next
decade is whether or not IgG is approved for the treatment of
Alzheimer's disease. It is estimated that if these treatments are
approved by major regulatory agencies, an additional 5% increase in
demand for IVIG will be seen (Robert P., supra).
[0014] Due in part to the increasing global demand and fluctuations
in the available supply of immunoglobulin products, several
countries, including Australia and England, have implemented demand
management programs to protect supplies of these products for the
highest demand patients during times of product shortages. Thus,
the development of methodologies that reduce the amount of pooled
immunoglobulin G needed to treat various indications will be
critical as the increase in demand for pooled immunoglobulin begins
to outpace the increase in global manufacturing output.
[0015] Pooled human immunoglobulin G (IgG) is manufactured
according to different processes depending upon the specific
manufacturer. However, the origin of most manufacturing processes
is found in the fourth installment of a series of seminal papers
published on the preparation and properties of serum and plasma
proteins, Cohn et al. (J. Am. Chem. Soc., 1946, 68(3): 459-475).
This paper first described a method for the alcohol fractionation
of plasma proteins (method 6), which allows for the isolation of a
fraction enriched in IgG from human plasma.
[0016] The Cohn procedures were initially developed to obtain
albumin at relatively high (95%) purity by fractional precipitation
with alcohol. However, it was realized by Oncley et al. (J. Am.
Chem. Soc., 1949, 71(2): 541-550), Deutsch et al. (J. Biol. Chem.,
1946, 164, 109-118), and Kistler and Nitschmann (Vox Sang., 1962,
7, 414-424), that particular protein precipitates (Fraction II and
Fraction II+III) from the Cohn method could be used as a starting
material for the purification of highly enriched immunoglobulin
compositions. In order to achieve the higher purity and safety
required for the intravenous administration of IgG compositions,
several purification and polishing steps (e.g. adsorption in
general or all different chromatographic techniques,
Cross-flow-filtration, etc.) have been added to IgG manufacturing
processes after the alcohol fractionation steps.
[0017] Current IgG manufactures typically rely on either a Cohn
method 6 Fraction II+III precipitate or a Kistler-Nitschmann
precipitate A as the starting material for downstream processing.
Both fractions are formed by a two step process in which proteins
such as fibrinogen and Factor XIII are removed by an initial
precipitation step (Fraction I precipitation) performed at high pH
(7.2) with low ethanol concentration (8-10% v/v), followed by a
second precipitation step in which IgG is precipitated from the
Fraction I supernatant at pH 6.8 with 20-25% (v/v) ethanol
(Fraction II+III) or at pH 5.85 with 19% ethanol (v/v) ethanol
(precipitate A), while albumin and a significant portion of A1PI
remain in the supernatant.
[0018] These methods, while laying the foundation for an entire
industry of plasma derived blood proteins, were unable to provide
IgG preparations having sufficiently high purity for the chronic
treatment of several immune-related diseases, including Kawasaki
syndrome, immune thrombocytopenic purpura, and primary immune
deficiencies, without an undue occurrence of serious side effects.
As such, additional methodologies employing various techniques,
such as ion exchange chromatography, were developed to provide
higher purity IgG formulations. Hoppe et al. (Munch Med Wochenschr
1967 (34): 1749-1752), Falksveden (Swedish Patent No. 348942), and
Falksveden and Lundblad (Methods of Plasma Protein Fractionation
1980) were among the first to employ ion exchange chromatography
for this purpose.
[0019] It is common practice to administer IgG by intravenous (IV)
injection (Imbach et al., Lancet 1(8232): 1228-31 (1981)).
Intravenous IgG (IVIG) may be administered alone or in combination
with other compositions. IVIG is often administered over a 2 to 5
hour period, once a day for 2 to 7 days, with follow-up doses every
10 to 21 days or every 3 to 4 weeks. Such an administration regime
is time consuming and inconvenient for many patients. This
inconvenience may be aggravated in the case of Alzheimer's
patients, who may have difficulty sitting quietly during the
infusion period, and may have to rely on their caregiver to bring
them to an infusion center or supervise their infusion.
[0020] Systemic IVIG administration may cause adverse side effects.
For example, IVIG may cause backache, headache, migraine, joint or
muscle pain, general feeling of discomfort, leg cramps, rash, pain
at the injection site, hives, dizziness, unusual fatigue or
tiredness or weakness, chills, fever, sweating, increased heart
rate, increased blood pressure, cough, redness of the face, upset
stomach, upper abdominal pain, and vomiting. Immediate adverse
effects post-IVIG administration which have been observed include
headache, flushing, malaise, chest tightness, fever, chills,
myalgia, fatigue, dyspnea, back pain, nausea, vomiting, diarrhea,
blood pressure changes, tachycardia, and anaphylactic reactions.
Orbach et al., Clin. Rev. Allergy Immunol., 29(3): 173-84
(2005).
[0021] Furthermore, the adverse side effects may vary based on the
IVIG manufacturer. Most manufactures preparations contain between
90% and 99% purified IgG in combination with stabilizers and
liquid(s) for reconstitution. Orange et al. 2006 (J. Allergy Clin.
Immunol. 117(4 Suppl.): S525); Vo et al. 2006 (Clin. J. Am. Soc.
Nephrol. 1(4): 844; Stiehm et al. 2006 (J. Pediatr. 148(1): 6). For
example, some manufacturers use maltose as a stabilizer while
others use sucrose or amino acids.
[0022] The sodium and sugar content in IVIG, along with varying
amounts of IgA and additional chemicals used in the IVIG production
can affect the tolerability and efficacy of the brand of IVIG in
patients. Specifically, older patients often suffer from co-morbid
conditions that increase the risk of IVIG adverse side effects. For
example, subjects with renal disorders, vascular disorders, or
diabetes also have a heightened risk of renal insufficiency and
thrombotic events following IVIG administration because IVIG
compositions are commonly hyper-viscous and contain high
concentrations of sugar and salt.
[0023] IVIG also carries the risk of catheter-related infection,
i.e., an infection where the catheter or needle enters a subject's
vein or skin. Examples of catheter-related infection are
tenderness, warmth, irritation, drainage, redness, swelling, and
pain at the catheter site. Accordingly, alternate modes of
administration would be beneficial from the standpoint of time,
convenience, and adverse side effects.
[0024] In addition to adverse side effects of systemic
administration of IVIG, penetration of IVIG across the blood-brain
barrier has been shown to be unpredictable and intraventricular or
intrathecal IgG may be necessary. For example, Haley et al.
administered IVIG in the treatment of meningeal inflammation caused
by West Nile virus encephalitis. Haley et al. found that
penetration of IVIG was unpredictable and posited that intrathecal
or intraventricular administration may be required. Haley et al.
2003 (Clin. Inf. Diseases 37: e88-90).
[0025] It is difficult to target the CNS with IV administration
therapeutic compositions because of the blood-brain barrier (BBB).
The BBB provides an efficient barrier, preventing and/or limiting
access to the CNS of therapeutic compositions administered
intravenously into the peripheral circulation. Specifically, the
BBB prevents diffusion of most therapeutic compositions, especially
polar compositions, into the brain from the circulating blood.
[0026] At least three methods for increasing the passage of
molecules through the BBB have been developed. First, lipophilic
compounds such as lipid-soluble drugs and polar drugs encased in a
lipid membrane have been developed. Lipophilic compounds with a
molecular weight of less than 600 Da can diffuse through the BBB.
Second, therapeutic compounds can be bound to transporter molecules
which can cross the BBB through a saturable transporter system.
Examples of saturable transporter molecules are transferrin,
insulin, IGF-1, and leptin. Third, therapeutic compounds can cross
the BBB by binding the therapeutic compounds to polycationic
molecules such as positively-charged proteins that preferentially
bind to the negatively-charged endothelial surface of the BBB.
Patridge et al. 2003 (Mol. Interv. 3(2): 90-105); Patridge et al.
2002 (Nature Reviews-Drug Discovery 1:131-139). However, each of
the above-described approaches for increasing the delivery of
therapeutics through BBB to gain access to the CNS are limited. One
such limitation is that the above-described approaches rely on
systemic delivery systems, e.g., administration directly or
indirectly to the blood stream, which results in non-specific
delivery of the therapeutic agent to other parts in the body,
increasing the chance of adverse side effects.
[0027] Intranasal administration of therapeutics has become an
increasingly explored method for delivering therapeutic agents to
the brain because it circumvents the BBB and is a localized,
non-invasive method for delivery. Furthermore, intranasal
administration offers the advantages, over more traditional methods
of delivery (e.g., intravenous, subcutaneous, oral transmucosal,
oral or rectal administration), of being simple to administer,
providing rapid onset of action, and avoiding first-pass
metabolism. Unfortunately, intranasal administration has only been
shown effective for the transport of small molecules, and to a
certain extent smaller Fc fusion proteins, to the brain. The
delivery of larger molecules, such as intact antibodies, has not
yet been demonstrated. The difficulty in transporting larger
proteins is believed to be due to the limited permeability of tight
junctions present in the olfactory epithelia, which likely excludes
globular molecules having a hydrodynamic radius of more than 3.6
.ANG. (Stevenson B R, et al., Mol Cell Biochem., 1988 October;
83(2):129-45).
[0028] U.S. Pat. No. 5,624,898 to Frey describes compositions and
methods for transporting neurologic agents, which promote nerve
cell growth and survival or augment the activity of functioning
cells, to the brain by means of the olfactory neural pathway. The
neurological agents of the '898 patent are transported to the brain
by means of the nervous system, rather than the circulatory system,
so that potentially therapeutic agents that are unable to cross the
blood-brain barrier may be delivered to damaged neurons in the
brain. The compositions described in the '898 patent include a
neurologic agent in combination with a pharmaceutical carrier
and/or additive which promote the transfer of the agent within the
olfactory system. The '898 patent does not teach intranasal
administration of pooled human immunoglobulins.
[0029] PCT publications WO 2006/091332 and WO 2009/058957, both by
Bentz et al., describe methods for the delivery of therapeutic
polypeptides to the brain by fusing the polypeptide to an antibody
or antibody fragment and administering the resulting fusion protein
intranasally. Although the examples of the '332 and '957 PCT
publications suggest that Fc-fusion "mimetibodies" may be
administered intranasally, neither publication demonstrates
delivery of larger, intact antibodies. In fact, the '957 PCT
publication, published three years after the '332 PCT publication,
states that "[i]n published delivery studies to date, intranasal
delivery efficiency to the CNS has been very low and the delivery
of large globular macromolecules, such as antibodies and their
fragments, has not been demonstrated." The '957 PCT publication
purports to solve this problem through the use of a permeability
enhancer (e.g., membrane fluidizers, tight junction modulators, and
medium chain length fatty acids and salts and esters thereof, as
described below), which enhances intranasal administration to the
central nervous system. Neither PCT publication teaches intranasal
administration of pooled human immunoglobulins.
[0030] PCT publication WO 2003/028668 by Barstow et al., describes
the treatment of immune-mediated diseases by alimentary
administration (i.e., administration to the digestive tract) of
pooled immunoglobulins. Although the '668 PCT publication discloses
nasal administration of a composition of pooled immunoglobulins, it
is in the context of delivering the composition to the digestive
tract. The '668 PCT publication does not teach the delivery of
pooled human immunoglobulins to the brain via intranasal
administration.
[0031] PCT publication WO 2001/60420 by Adjei et al., describes
aerosol formulations of therapeutic polypeptides, including
immunoglobulins, for pulmonary delivery. These aerosolizable
compositions are formulated such that after oral or nasal
inhalation, the therapeutic agent is effectively delivered to the
patient's lungs. The '420 PCT publication does not teach the
delivery of therapeutic agents to the brain via intranasal
administration.
[0032] Accordingly, there is a need in the art for methods of
treating central nervous system disorders, such as Alzheimer's
disease, using pooled human immunoglobulin G that provide specific
targeting to the CNS (e.g., administration primarily to the brain),
reduce systemic distribution of the pooled immunoglobulins, and
lower the therapeutically effected dose needed for
administration.
BRIEF SUMMARY OF INVENTION
[0033] The present disclosure provides solutions to these and other
problems by providing methods and compositions for the treatment of
central nervous system disorders via intranasal administration of
pooled human immunoglobulin G. Advantageously, intranasal
administration provides directed delivery of pooled IgG to the
brain, bypassing the requirement that it pass through the blood
brain barrier (BBB). As shown herein, intranasal administration
allows the delivery of intact IgG to the brain. This results in
greater efficiency for the treatment and reduces the necessary IgG
dose that must be administered to achieve the desired effect. As
pooled human IgG is isolated from donated human plasma, pooled IgG
is a limited resource. The reduction in the effective dose of IgG
provided by the present disclosure effectively increases the
therapeutic potential provided by the world's supply of pooled
human IgG. Furthermore, as demonstrated herein, intranasal
administration of IgG nearly eliminates the systemic exposure
caused by intravenous administration, improving the overall safety
profile of the treatment. Finally, it was surprisingly found that
IgG is efficiently transported to the brain when intranasally
administered in the absence of permeability enhancers, some of
which have neurostimulatory effects themselves.
[0034] In one aspect, the disclosure provides a method for treating
a central nervous system (CNS) disorder in a subject in need
thereof, the method including delivering a therapeutically
effective amount of a composition comprising pooled human
immunoglobulin G (IgG) to the brain of the subject, where
delivering the composition to the brain includes intranasally
administering the composition directly to the olfactory epithelium
of the nasal cavity of the subject.
[0035] In another aspect, the disclosure provides a method for
treating a central nervous system (CNS) disorder in a subject in
need thereof, the method including delivering a therapeutically
effective amount of a composition comprising pooled human
immunoglobulin G (IgG) to the brain of the subject, where
delivering the composition to the brain includes intranasally
administering the composition to a nasal epithelium of the subject
associated with trigeminal nerve endings.
[0036] In another aspect, the disclosure provides a method for
treating a central nervous system (CNS) disorder in a subject in
need thereof, the method including delivering a therapeutically
effective amount of a composition comprising pooled human
immunoglobulin G (IgG) to the brain of the subject, where
delivering the composition to the brain includes intranasally
administering the composition to the upper third of the nasal
cavity of the subject.
[0037] In another aspect, the disclosure provides a method for
treating a central nervous system (CNS) disorder in a subject in
need thereof, the method including delivering a therapeutically
effective amount of a composition comprising pooled human
immunoglobulin G (IgG) to the brain of the subject, where
delivering the composition to the brain includes intranasally
administering the composition to one or both maxillary sinus of the
subject.
[0038] In one embodiment of the methods described above, the CNS
disorder is selected from the group consisting of a
neurodegenerative disorder of the central nervous system, a
systemic atrophy primarily affecting the central nervous system, an
extrapyramidal and movement disorder, a demyelinating disorder of
the central nervous system, an episodic or paroxysmal disorder of
the central nervous system, a paralytic syndrome of the central
nervous system, a nerve, nerve root, or plexus disorder of the
central nervous system, an organic mental disorder, a mental or
behavioral disorder caused by psychoactive substance use, a
schizophrenia, schizotypal, or delusional disorder, a mood
(affective) disorder, neurotic, stress-related, or somatoform
disorder, a behavioral syndrome, an adult personality or behavior
disorder, a psychological development disorder, and a child onset
behavioral or emotional disorder.
[0039] In one embodiment of the methods described above, the CNS
disorder is selected from the group consisting of Alzheimer's
disease, Parkinson's disease, multiple sclerosis, amyotrophic
lateral sclerosis (ALS), Huntington's disease, cerebral palsy,
bipolar disorder, schizophrenia, and Pediatric acute-onset
neuropyschiatric syndrome (PANS).
[0040] In one embodiment of the methods described above, the CNS
disorder is selected from the group consisting of Alzheimer's
disease, Parkinson's disease, multiple sclerosis, Pediatric
Autoimmune Neuropsychiatric Disorders Associated with Streptococcal
infections (PANDAS), and Pediatric acute-onset neuropyschiatric
syndrome (PANS).
[0041] In one embodiment of the methods described above, the CNS
disorder is selected from the group consisting of Alzheimer' s
disease, multiple sclerosis, and Parkinson's disease.
[0042] In one embodiment of the methods described above, the CNS
disorder is Alzheimer's disease.
[0043] In one embodiment of the methods described above, intranasal
administration of the composition includes the use of a
non-invasive intranasal delivery device.
[0044] In one embodiment of the methods described above, intranasal
administration of the composition includes administration of a
liquid drop of the composition directly onto the nasal epithelium,
the nasal epithelium of the subject associated with trigeminal
nerve endings, or the upper third of the nasal cavity of the
subject.
[0045] In one embodiment of the methods described above, intranasal
administration of the composition includes directed administration
of an aerosol of the composition to the nasal epithelium, the nasal
epithelium of the subject associated with trigeminal nerve endings,
or the upper third of the nasal cavity of the subject.
[0046] In one embodiment of the methods described above, the
aerosol of the composition is a liquid aerosol.
[0047] In one embodiment of the methods described above, the
aerosol of the composition is a powder aerosol.
[0048] In one embodiment of the methods described above, at least
40% of the pooled human IgG administered to the subject contacts
the nasal epithelium of the subject, the olfactory epithelium of
the nasal cavity of the subject, a nasal epithelium of the subject
associated with trigeminal nerve endings, the upper third of the
nasal cavity of the subject, or one or both maxillary sinus of the
subject.
[0049] In one embodiment of the methods described above, at least
50% of the pooled human IgG administered to the subject contacts
the olfactory epithelium of the nasal cavity of the subject, the
nasal epithelium of the subject associated with trigeminal nerve
endings, the upper third of the nasal cavity of the subject, or one
or both maxillary sinus of the subject.
[0050] In one embodiment of the methods described above, at least
60% of the pooled human IgG administered to the subject contacts
the olfactory epithelium of the nasal cavity of the subject, the
nasal epithelium of the subject associated with trigeminal nerve
endings, the upper third of the nasal cavity of the subject, or one
or both maxillary sinus of the subject.
[0051] In one embodiment of the methods described above, the pooled
human IgG composition does not contain a permeability enhancer.
[0052] In one embodiment of the methods described above, the pooled
human IgG composition consists essentially of pooled human IgG and
an amino acid.
[0053] In one embodiment of the methods described above, the amino
acid is selected from the group consisting of glycine, histidine,
and proline. In a specific embodiment of the methods provided
above, the amino acid is glycine.
[0054] In one embodiment of the methods described above, the pooled
human IgG composition is an aqueous composition.
[0055] In one embodiment of the methods described above, the pooled
human IgG composition includes from 10 mg/mL to 250 mg/mL pooled
human IgG and from 50 mM to 500 mM glycine.
[0056] In one embodiment of the methods described above, the pH of
the composition is from 4.0 to 6.0. In another embodiment of the
methods provided above, the pH of the composition is from 4.0 to
7.5. In another embodiment of the methods provided above, the pH of
the composition is from 6.0 to 7.5.
[0057] In one embodiment of the methods described above, the pooled
human IgG composition is a dry powder composition.
[0058] In one embodiment of the methods described above, the dry
powder composition is prepared from an aqueous solution including
from 10 mg/mL to 250 mg/mL pooled human IgG and from 50 mM to 500
mM glycine.
[0059] In one embodiment of the methods described above, the dry
powder composition is prepared from an aqueous solution having a pH
of from 4.0 to 6.0. In another embodiment of the methods provided
above, the pH of the composition is from 4.0 to 7.5 In another
embodiment of the methods provided above, the pH of the composition
is from 6.0 to 7.5
[0060] In one embodiment of the methods described above, the method
includes intranasally administering to the subject a dose of from
0.08 mg to 100 mg pooled human IgG per kg body weight of the
subject (mg IgG/kg). In a specific embodiment of the methods
provided above, the method includes intranasally administering to
the subject a dose of from 0.2 mg to 40 mg pooled human IgG per kg
body weight of the subject (mg IgG/kg). In a specific embodiment of
the methods provided above, the method includes intranasally
administering to the subject a dose of from 5 mg to 20 mg pooled
human IgG per kg body weight of the subject (mg IgG/kg). In a
specific embodiment of the methods provided above, the method
includes intranasally administering to the subject a dose of from 5
mg to 10 mg pooled human IgG per kg body weight of the subject (mg
IgG/kg). In a specific embodiment of the methods provided above,
the method includes intranasally administering to the subject a
dose of from 1 mg to 5 mg pooled human IgG per kg body weight of
the subject (mg IgG/kg).
[0061] In one embodiment of the methods described above, the method
includes intranasally administering to the subject a fixed dose of
from 50 mg to 10 g pooled human IgG. In a specific embodiment of
the methods provided above, the method includes intranasally
administering to the subject a fixed dose of from 100 mg to 5.0 g
pooled human IgG. In a specific embodiment of the methods provided
above, the method includes intranasally administering to the
subject a fixed dose of from 500 mg to 2.5 g pooled human IgG.
[0062] In one embodiment of the methods described above, the method
includes intranasally administering to the subject a dose of pooled
human IgG at least twice monthly. In a specific embodiment of the
methods described above, the method includes intranasally
administering to the subject a dose of pooled human IgG at least
once weekly. In a specific embodiment of the methods described
above, the method includes intranasally administering to the
subject a dose of pooled human IgG at least twice weekly. In a
specific embodiment of the methods described above, the method
includes intranasally administering to the subject a dose of pooled
human IgG at least once daily. In a specific embodiment of the
methods described above, the method includes intranasally
administering to the subject a dose of pooled human IgG at least
twice daily.
[0063] In one embodiment of the methods described above, the pooled
human IgG composition includes at least 0.1% anti-amyloid .beta.
IgG.
[0064] In one embodiment of the methods described above, the method
further includes administering a second therapy for the CNS
disorder to the subject in need thereof.
[0065] In one embodiment of the methods described above, the second
therapy for the CNS disorder is a cholinesterase inhibitor. In a
specific embodiment of the methods described above, the
cholinesterase inhibitor is selected from the group consisting of
donepezil (e.g., ARICEPT.RTM.), rivastigmine (e.g., EXELON.RTM.),
galantamine (e.g., RAZADYNE.RTM.), and tacrine (e.g.,
COGNEX.RTM.).
[0066] In one embodiment of the methods described above, the second
therapy for the CNS disorder is an inhibitor of NMDA-type glutamate
receptor. In a specific embodiment of the methods described above,
the inhibitor of NMDA-type glutamate receptor is memantine.
[0067] In another aspect, the disclosure provides the use of a
composition comprising pooled human immunoglobulin G (IgG) for the
treatment of a central nervous system (CNS) disorder in a subject
in need thereof by intranasal administration.
[0068] In some embodiments of the uses described above, intranasal
administration includes administration to the nasal epithelium of
the subject. In other embodiments of the uses described above,
intranasal administration comprises administration to the olfactory
epithelium of the nasal cavity of the subject. In other embodiments
of the uses described above, intranasal administration includes
administration to a nasal epithelium of the subject associated with
trigeminal nerve endings. In other embodiments of the uses
described above, intranasal administration includes administration
to the upper third of the nasal epithelium of the nasal cavity of
the subject. In yet other embodiments, of the uses described above,
intranasal administration includes administration to one or both
maxillary sinus of the subject.
[0069] In one embodiment of the uses described above, the CNS
disorder is selected from the group consisting of a
neurodegenerative disorder of the central nervous system, a
systemic atrophy primarily affecting the central nervous system, an
extrapyramidal and movement disorder, a demyelinating disorder of
the central nervous system, an episodic or paroxysmal disorder of
the central nervous system, a paralytic syndrome of the central
nervous system, a nerve, nerve root, or plexus disorder of the
central nervous system, an organic mental disorder, a mental or
behavioral disorder caused by psychoactive substance use, a
schizophrenia, schizotypal, or delusional disorder, a mood
(affective) disorder, neurotic, stress-related, or somatoform
disorder, a behavioral syndrome, an adult personality or behavior
disorder, a psychological development disorder, and a child onset
behavioral or emotional disorder.
[0070] In one embodiment of the uses described above, the CNS
disorder is selected from the group consisting of Alzheimer's
disease, Parkinson's disease, multiple sclerosis, amyotrophic
lateral sclerosis (ALS), Huntington's disease, cerebral palsy,
bipolar disorder, schizophrenia, and Pediatric acute-onset
neuropyschiatric syndrome (PANS).
[0071] In one embodiment of the uses described above, the CNS
disorder is selected from the group consisting of Alzheimer's
disease, Parkinson's disease, multiple sclerosis, Pediatric
Autoimmune Neuropsychiatric Disorders Associated with Streptococcal
infections (PANDAS), and Pediatric acute-onset neuropyschiatric
syndrome (PANS).
[0072] In one embodiment of the uses described above, the CNS
disorder is selected from the group consisting of Alzheimer's
disease, multiple sclerosis, and Parkinson's disease.
[0073] In one embodiment of the uses described above, the CNS
disorder is Alzheimer's disease.
[0074] In one embodiment of the uses described above, intranasal
administration of the composition includes the use of a
non-invasive intranasal delivery device.
[0075] In one embodiment of the uses described above, intranasal
administration of the composition includes administration of a
liquid drop of the composition directly onto the nasal epithelium,
the nasal epithelium of the subject associated with trigeminal
nerve endings, or the upper third of the nasal cavity of the
subject.
[0076] In one embodiment of the uses described above, intranasal
administration of the composition includes directed administration
of an aerosol of the composition to the nasal epithelium, the nasal
epithelium of the subject associated with trigeminal nerve endings,
or the upper third of the nasal cavity of the subject.
[0077] In one embodiment of the uses described above, the aerosol
of the composition is a liquid aerosol.
[0078] In one embodiment of the uses described above, the aerosol
of the composition is a powder aerosol.
[0079] In one embodiment of the uses described above, at least 40%
of the pooled human IgG administered to the subject contacts the
nasal epithelium of the subject, the olfactory epithelium of the
nasal cavity of the subject, a nasal epithelium of the subject
associated with trigeminal nerve endings, the upper third of the
nasal cavity of the subject, or one or both maxillary sinus of the
subject.
[0080] In one embodiment of the uses described above, at least 50%
of the pooled human IgG administered to the subject contacts the
nasal epithelium of the subject, the olfactory epithelium of the
nasal cavity of the subject, a nasal epithelium of the subject
associated with trigeminal nerve endings, the upper third of the
nasal cavity of the subject, or one or both maxillary sinus of the
subject.
[0081] In one embodiment of the uses described above, at least 60%
of the pooled human IgG administered to the subject contacts the
nasal epithelium of the subject, the olfactory epithelium of the
nasal cavity of the subject, a nasal epithelium of the subject
associated with trigeminal nerve endings, the upper third of the
nasal cavity of the subject, or one or both maxillary sinus of the
subject.
[0082] In one embodiment of the uses described above, the pooled
human IgG composition does not contain a permeability enhancer.
[0083] In one embodiment of the uses described above, the pooled
human IgG composition consists essentially of pooled human IgG and
an amino acid.
[0084] In one embodiment of the uses described above, the amino
acid is selected from the group consisting of glycine, histidine,
and proline. In a specific embodiment of the methods provided
above, the amino acid is glycine.
[0085] In one embodiment of the uses described above, the pooled
human IgG composition is an aqueous composition.
[0086] In one embodiment of the uses described above, the pooled
human IgG composition includes from 10 mg/mL to 250 mg/mL pooled
human IgG and from 50 mM to 500 mM glycine.
[0087] In one embodiment of the uses described above, the pH of the
composition is from 4.0 to 6.0. In another embodiment of the uses
described above, the pH of the composition is from 4.0 to 7.5. In
another embodiment of the methods provided above, the pH of the
composition is from 6.0 to 7.5.
[0088] In one embodiment of the uses described above, the pooled
human IgG composition is a dry powder composition.
[0089] In one embodiment of the uses described above, the dry
powder composition is prepared from an aqueous solution including
from 10 mg/mL to 250 mg/mL pooled human IgG and from 50 mM to 500
mM glycine.
[0090] In one embodiment of the uses described above, the dry
powder composition is prepared from an aqueous solution having a pH
of from 4.0 to 6.0. In another embodiment of the uses described
above, the pH of the composition is from 4.0 to 7.5 In another
embodiment of the uses described above, the pH of the composition
is from 6.0 to 7.5
[0091] In one embodiment of the uses described above, the use
includes intranasally administering to the subject a dose of from
0.08 mg to 100 mg pooled human IgG per kg body weight of the
subject (mg IgG/kg). In a specific embodiment of the uses described
above, the use includes intranasally administering to the subject a
dose of from 0.2 mg to 40 mg pooled human IgG per kg body weight of
the subject (mg IgG/kg). In a specific embodiment of the uses
described above, the use includes intranasally administering to the
subject a dose of from 5 mg to 20 mg pooled human IgG per kg body
weight of the subject (mg IgG/kg). In a specific embodiment of the
uses described above, the use includes intranasally administering
to the subject a dose of from 5 mg to 10 mg pooled human IgG per kg
body weight of the subject (mg IgG/kg). In a specific embodiment of
the uses described above, the use includes intranasally
administering to the subject a dose of from 1 mg to 5 mg pooled
human IgG per kg body weight of the subject (mg IgG/kg).
[0092] In one embodiment of the uses described above, the use
includes intranasally administering to the subject a fixed dose of
from 50 mg to 10 g pooled human IgG. In a specific embodiment of
the uses provided above, the use includes intranasally
administering to the subject a fixed dose of from 100 mg to 5.0 g
pooled human IgG. In a specific embodiment of the uses provided
above, the use includes intranasally administering to the subject a
fixed dose of from 500 mg to 2.5 g pooled human IgG.
[0093] In one embodiment of the uses described above, the method
includes intranasally administering to the subject a dose of pooled
human IgG at least twice monthly. In a specific embodiment of the
uses described above, the method includes intranasally
administering to the subject a dose of pooled human IgG at least
once weekly. In a specific embodiment of the uses described above,
the method includes intranasally administering to the subject a
dose of pooled human IgG at least twice weekly. In a specific
embodiment of the uses described above, the method includes
intranasally administering to the subject a dose of pooled human
IgG at least once daily. In a specific embodiment of the uses
described above, the method includes intranasally administering to
the subject a dose of pooled human IgG at least twice daily.
[0094] In one embodiment of the uses described above, the pooled
human IgG composition includes at least 0.1% anti-amyloid .beta.
IgG.
[0095] In one embodiment of the uses described above, the method
further includes administering a second therapy for the CNS
disorder to the subject in need thereof.
[0096] In one embodiment of the uses described above, the second
therapy for the CNS disorder is a cholinesterase inhibitor. In a
specific embodiment of the uses described above, the cholinesterase
inhibitor is selected from the group consisting of donepezil (e.g.,
ARICEPT.RTM.), rivastigmine (e.g., EXELON.RTM.), galantamine (e.g.,
RAZADYNE.RTM.), or tacrine (e.g., COGNEX.RTM.).
[0097] In one embodiment of the uses described above, the second
therapy for the CNS disorder is an inhibitor of NMDA-type glutamate
receptor. In a specific embodiment of the uses described above, the
inhibitor of NMDA-type glutamate receptor is memantine.
BRIEF DESCRIPTION OF DRAWINGS
[0098] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0099] FIGS. 1A-1F show brain slices from rats used to assess the
biodistribution of intranasally administered IgG in Example 2. Six
2 mm slices (3 rostral to the optic chiasm and 3 caudal) were
acquired.
[0100] FIG. 1G illustrates a brain bisected along the midline. The
bisected brain is further dissected in to midbrain, pons, medulla,
and cerebellum for biodistribution analysis.
[0101] FIGS. 2A-2B illustrate results of average brain tissue
.sup.125I IgG concentrations (nM) 30 and 90 minutes after
intranasal administration of IgG. FIG. 2A illustrates results of
brain tissue .sup.125I IgG concentrations (nM) 30 (n=8) and 90
(n=6) minutes after administration of a liquid protein IgG
preparation, normilized to a 6.0 mg dose. FIG. 2B illustrates
results of brain tissue .sup.125I IgG concentrations (nM) 30 (n=12)
and 90 (n=6) minutes after administration of a solid microsphere
IgG preparation, normalized to a 6.0 mg dose.
[0102] FIGS. 3A-3E illustrate IHC data on cortical and hippocampal
brain slices. Plaque content was determined for 12 mice from each
cohort (WT-Saline, WT-High, TG-Saline, TG-Low, and TG-High; shown
left to right in the charts, respectively). FIG. 3A shows the
percent area covered by beta-amyloid plaques. Four slides from the
cortex of each mouse were analyzed using ImageJ software. The data
is distributed in the given range by plaque radius size (in
micrometers). Significant differences between transgenic treatment
groups are marked in the graph with the p-value. FIG. 3B shows the
average number of beta-amyloid plaques. Four slides from the cortex
of each mouse were analyzed using ImageJ software. The data is
distributed in the given range by plaque radius size (in
micrometers). Significant differences between transgenic treatment
groups are marked in the graph with the p-value. FIG. 3C shows the
average number of beta-amyloid plaques. Four slides from the
hippocampus of each mouse were analyzed using ImageJ software. The
data is distributed in the given range by plaque radius size (in
micrometers). FIG. 3D shows the percent area covered by
beta-amyloid plaques. Four slides from the hippocampus of each
mouse were analyzed using ImageJ software. The data is distributed
in the given range by plaque radius size (in micrometers). FIG. 3E
shows immunofluorescent staining of amyloid plaques in the
hippocampus and cortex of aged TG2576 transgenic mice (field of
view=5.3 mm). For the immunofluorescent staining, mice were
intranasally administered saline, low dose IgG, or high dose IgG
three times weekly over a period of 8 months. Each value is
reported as the mean value for the cohorts .+-.standard error.
[0103] FIG. 4 illustrates a Kaplan-Meier curve for survival rates
of transgenic and wild-type mice administered IgG intranasally.
These mice belong to a different cohort than the mice used for
plaque analysis in FIG. 3.
[0104] FIG. 5 Illustrates the seven coronal brain slices which were
hemisected from intranasal .sup.125I IgG treated rats used to
assess CNS delivery in Example 8.
[0105] FIGS. 6A-6B show comparative results of the intactness of
IgG sprayed through a device designed for intranasal delivery with
that of non-sprayed control. FIG. 6A shows a Coomassie stained,
non-reducing gel of sprayed and non-sprayed (control) IgG. FIG. 6B
shows a Western blot of a reducing gel probed with an anti-IgG
antibody.
[0106] FIG. 7 shows results demonstrating the highly efficient
olfactory epithelium targeting of IN device administration in rats.
The upper panel shows the deposition of IN IgG after device
administration of 15 .mu.L of 25% IVIG solution spiked with 0.01%
fluorescein tracer in a rat. The lower panel shows the deposition
pattern after deposition of 15 .mu.L of 25% IVIG solution spiked
with 0.01% fluorescein tracer administered via nose drops.
OB=olfactory bulb, OE=olfactory epithelium, RE=respiratory
epithelium, NS=naris.
[0107] FIGS. 8A-8C illustrate data showing a decrease of amyloid
load in the low IgG and high IgG intranasal treatment groups. FIG.
8A shows the total amyloid area (plaque and vasculature). FIG. 8B
shows the number (#) of amyloid deposits (plaque and vasculature).
FIG. 8C shows the total intensity of all amyloid deposits (i.e.,
the Sum Intensity).
[0108] FIGS. 9A-9C illustrate data showing a decrease in amyloid is
a result of a decrease in plaque load. FIG. 9A shows the total
amyloid area (plaque and vasculature). FIG. 9B shows the number (#)
of amyloid deposits (plaque and vasculature). FIG. 9C shows the
total intensity of all amyloid deposits (i.e., the Sum
Intensity).
[0109] FIGS. 10A-10C illustrate data showing that the vascular
component of the amyloid was found to increase slightly when a
decrease in amyloid as a result of a decrease in plaque load was
observed (FIG. 9). FIG. 10A shows the vascular amyloid area. FIG.
10B shows the number (#) of vascular deposits. FIG. 10C shows the
total intensity of vascular deposits (i.e., the Sum Intensity).
[0110] FIGS. 11A-11B illustrate data showing the relative
proportions of vascular and plaque amyloid as it contributes to
total amyloid. FIG. 11A shows the relative plaque contribution to
total amyloid. FIG. 11B shows the relative vascular contribution to
total amyloid.
[0111] FIGS. 12A-12F show Congo Red stained sagittal sections
captured with confocal fluorescent microscopy. FIG. 12A shows a
z-stack max intensity projection image created from five individual
images at 10.times. with a 512.times.512 resolution. FIGS. 12B-12F
show single images created from thirty of z-stacks projections as
shown in FIG. 12A, encompassing the whole tissue section that were
tiled (6.times.5, 5% overlap). Representative images from the
groups: Tg-Saline with Thresholding, Full-Resolution, Portion of
the cortex and hippocampus (FIG. 12A); Tg-Low without Thresholding
(FIG. 12B); WT-Saline with thresholding (FIG. 12C); Tg-Saline with
thresholding (FIG. 12D); Tg-Low with thresholding (FIG. 12E); and
Tg-High with thresholding (FIG. 12F).
[0112] FIGS. 13A-13B illustrate data for the average staining
intensity for the Astrocyte marker GFAP (FIG. 13A) and the
microglial marker CD11b (FIG. 13B).
[0113] FIG. 14 is an example image of amyloid (blue), GFAP (green)
and CD11b (red) staining from a Tg2576 mouse brain that had been
treated with a high dose of IN IgG. CD11b staining was often
observed surrounding the amyloid plaques.
DETAILED DESCRIPTION OF INVENTION
Introduction
[0114] The present disclosure provides methods and compositions for
treating a central nervous system (CNS) disorder in a subject by
intranasal delivery of a therapeutically effective amount of pooled
human immunoglobulin G (IgG) directly to the epithelium of the
nasal cavity of the subject. In a specific embodiment, pooled human
IgG is administered directly to the olfactory epithelium of the
nasal cavity. In some embodiments, pooled IgG is delivered to the
upper third of the nasal cavity, e.g., above the lower turbinates.
In some embodiments, pooled IgG is delivered to the brain via the
trigeminal nerve after intranasal administration to the nasal
respiratory epithelium. In a specific embodiment, pooled IgG is
delivered to the brain via the maxillary nerve after intranasal
administration to the nasal respiratory epithelium. In other
embodiments, pooled IgG is delivered to the brain after
administration to the maxillary sinus.
[0115] In some embodiments, methods and compositions for the
treatment of Alzheimer's disease, multiple sclerosis, and
Parkinson's disease via intranasal administration of pooled human
IgG are provided herein. In other embodiments, the methods and
compositions provided herein are useful for the treatment of CNS
disorder known to one of skill in the art including, without
limitation, a neurodegenerative disorder of the central nervous
system, a systemic atrophy primarily affecting the central nervous
system, an extrapyramidal and movement disorder, a demyelinating
disorder of the central nervous system, an episodic or paroxysmal
disorder of the central nervous system, a paralytic syndrome of the
central nervous system, a nerve, nerve root, or plexus disorder of
the central nervous system, an organic mental disorder, a mental or
behavioral disorder caused by psychoactive substance use, a
schizophrenia, schizotypal, or delusional disorder, a mood
(affective) disorder, neurotic, stress-related, or somatoform
disorder, a behavioral syndrome, an adult personality or behavior
disorder, a psychological development disorder, or a child onset
behavioral or emotional disorder. In some embodiments, the CNS
disorder is selected from the group consisting of Alzheimer's
disease, Parkinson's disease, multiple sclerosis, amyotrophic
lateral sclerosis (ALS), Huntington's disease, cerebral palsy,
bipolar disorder, schizophrenia, or Pediatric acute-onset
neuropyschiatric syndrome (PANS). In some embodiments, the CNS
disorder is selected from the group consisting of Alzheimer's
disease, Parkinson's disease, multiple sclerosis, Pediatric
Autoimmune Neuropsychiatric Disorders Associated with Streptococcal
infections (PANDAS), or Pediatric acute-onset neuropyschiatric
syndrome (PANS).
[0116] Advantageously, it is shown herein that intranasal
administration of IgG increased weight and survival time of
Alzheimer's disease mice models. For example, it is shown in
Example 6 that intranasal administration of IgG, at either high
(0.8 g/kg once every two weeks) or low (0.4 g/k once every two
weeks) doses, prolonged the lifespan of TG2576 mice. This result
shows that intranasal administration of IgG is capable of
increasing lifespan in the Alzheimer's mouse model, indicating
efficacy in Alzheimer's treatment.
[0117] Moreover, intranasal administration of IgG significantly
reduced plaques in the cerebral cortex of in the Alzheimer's mouse
model. It is shown in Example 4 that treatment with pooled human
IgG reduced the percent area covered by plaques in the Alzheimer's
mouse model by about 25%, when administered intranasally at either
low (0.4 mg/kg/2 wk; p=0.014) or high (0.8 mg/kg/2 wk; p=0.037)
dosage. This is further indication of the efficacy of intranasal
administration of IgG in the treatment of Alzheimer's disease.
[0118] As further demonstrated herein, intranasal administration
results in a much more discriminate delivery of pooled human IgG to
the brain, as compared to intravenous administration. For example,
it is shown in Example 9 that intranasal administration of pooled
human immunoglobulin G resulted in a 6-fold lower blood exposure as
compared to intravenous administration. The lower system exposure
of IgG provided by intranasal administration advantageously reduces
the risk of side effects associated with the systemic exposure of
IgG.
[0119] Advantageously, it was also found that pooled human
immunoglobulin G was efficiently delivered to the brain following
intranasal administration in the absence of a permeability enhancer
(e.g., membrane fluidizers, tight junction modulators, and medium
chain length fatty acids and salts and esters thereof, as described
below). Previous reports have suggested that in order to achieve
efficient transport of biotherapeutics (e.g., mimetibodies and Fc
fusions) through the olfactory epithelium, a permeability enhancer
is required (WO 2009/058957). However, as shown in the examples
provided herein, pooled human IgG is efficiently delivered to the
brain when intranasally administered as a liquid or dry powder
formulated with only an amino acid (e.g., glycine).
[0120] Advantageously, it is also shown herein that the dose of
pooled human IgG can be significantly reduced when administered
intranasally, as compared to intravenous administration. For
example, it is shown in Example 9 that administration of a low dose
of pooled human IgG (0.002 g/kg IgG) intranasally delivered
directly to the olfactory epithelium results in substantially the
same amount of IgG being delivered to the right and left
hemispheres of the brain as for intravenous administration of a
ten-fold higher dose of pooled human IgG (0.02 g/kg IgG; compare
corrected AUC values for right and left hemisphere IgG delivery in
Table 71 and Table 72). A ten-fold reduction in the amount of
pooled human IgG required for administration is significant because
of the limited supply of pooled human IgG and the high cost
associated therewith.
[0121] The results described above, which taken together suggest
that low doses of intranasally administered pooled human IgG is
effective for the treatment of Alzheimer's disease, are surprising
given the difficulty of delivering full-length immunoglobulins to
the brain via intranasal administration. First, although antibody
fragments (e.g., Fabs) have previously been administered
intranasally, the inventors are unaware of any reports
demonstrating delivery of full-length antibodies to the brain via
intranasal administration. In fact, it has been reported that the
delivery of full-length antibodies poses a great difficulty in the
field of medicine (Harmsen M M et al., Appl Microbiol Biotechnol.,
2007, 77(1): 13-22; Athwal G S, Innovations in Pharmaceutical
Technology, July 2009; WO 2006/091332; and WO 2009/058957).
Consistent with these reports, Applicants found that antibody
fragments are delivered much more readily to the brain, as compared
to full-length immunoglobulins, after intranasal administration.
For example, it is shown in Example 2 that, on average, the
concentration of Fabs in brain tissue post-intranasal
administration is 19-times higher than the concentration of
full-length immunoglobulins post-intranasal administration. Given
the significantly lower delivery of full-length immunoglobulins to
the brain, it is surprising that intranasal administration of
pooled immunoglobulins provides the effective results shown
herein.
[0122] Advantageously, intranasal delivery of the pooled human IgG
composition disclosed herein can be accomplished by a non-invasive
means, as compared to intravenous, subcutaneous, and intramuscular
administration, all of which require puncture of the skin of the
subject. For example, it is shown in Example 3 that pooled human
IgG can be efficiently delivered to the brain using nasal drops or
a nasal spray.
[0123] Another benefit provided by the methods and compositions
provided herein for intranasal administration of pooled human IgG
is improved patient compliance. Treatment with intravenous IgG
(IVIG) requires a lengthy administration period under medical
supervision, generally taking place at hospitals and medical
facilities. For example, initial administration of IVIG occurs over
a 2 to 5 hour period, once a day for 2 to 7 consecutive days.
Follow-up doses, also typically administered at a hospital over a
period of 2 to 5 hours, are required every 1 to 4 weeks depending
on the indication being treated and dosing regimen. Such an
administration regime is time consuming and inconvenient for many
patients. In comparison, intranasal administration can be
administered at home without medical supervision. Also, intranasal
administration can be performed quickly, over several minutes
depending on the number of drops/sprays required, rather than
several hours as required for intravenous administration. Thus,
treatment can be prescribed more frequently at lower doses to
maintain an effective level of IgG in the CNS with minimal
inconvenience because administration occurs at home in a shorter
period of time.
[0124] Furthermore, IVIG therapy requires catheterization which can
cause discomfort and infection at the site of the catheter. IVIG
solutions are often high in sodium and glucose to create
isotonicity, causing increased risk to the elderly population,
which already have increased rates of diabetes and high blood
pressure. On the other hand, intranasal administration is
non-invasive, i.e. there is no catheterization and does not carry
invasive-procedure related risks such as infection and discomfort
at the site of the catheter. Intranasal administration of pooled
human IgG compositions is an improved procedure for elderly persons
because it does not require IV perfusion and thus does not create a
systemic increase in concentrations of salt or glucose in the
blood.
[0125] Thus, as compared to currently utilized modes of
administering pooled human IgG (e.g., intravenous, subcutaneous,
and intramuscular) intranasal administration increases the ease of
administration, decreases overall administration time, decreases
the number of hospital visits required, and eliminates the risks
associated with catheter-based administration (e.g., IV
administration). Thus, implementation of intranasal administration
of pooled human IgG will result in improved patient compliance.
Definitions
[0126] As used herein, the terms "disorder of the central nervous
system," "central nervous system disorder," "CNS disorder," and the
like refer to a disorder affecting either the spinal cord (e.g., a
myelopathy) or brain (e.g., an encephalopathy) of a subject, which
commonly presents with neurological and/or psychiatric symptoms.
CNS disorders include many neurodegenerative diseases (e.g.,
Huntington's disease, Amyotrophic lateral sclerosis (ALS),
hereditary spastic hemiplegia, primary lateral sclerosis, spinal
muscular atrophy, Kennedy's disease, Alzheimer's disease, ataxias,
Huntington's disease, Lewy body disease, a polyglutamine repeat
disease, and Parkinson's disease) and psychiatric disorders (e.g.,
mood disorders, schizophrenias, and autism). Non-limiting examples
of ataxia include Friedreich's ataxia and the spinocerebellar
ataxias. Specifically for this application, CNS disorders do not
include disorders resulting from acute viral and bacterial
infections.
[0127] Non-limiting examples of CNS disorders include
neurodegenerative disorders of the central nervous system, systemic
atrophies primarily affecting the central nervous system,
extrapyramidal and movement disorders, demyelinating disorders of
the central nervous system, episodic or paroxysmal disorders of the
central nervous system, paralytic syndromes of the central nervous
system, nerve, nerve root, or plexus disorders of the central
nervous system, organic mental disorders, mental or behavioral
disorders caused by psychoactive substance use, schizophrenic,
schizotypal, or delusional disorders, mood (affective) disorders,
neurotic, stress-related, or somatoform disorders, behavioral
syndromes, adult personality or behavior disorders, psychological
development disorders, and child onset behavioral or emotional
disorders. (Diagnostic and Statistical Manual of Mental Disorders,
4th Edition (DSM-IV); The World Health Organization, The
International Classification of Diseases, 10th revision (ICD-10),
Chapter V. Further exemplary CNS disorders are provided herein
below.
[0128] Neurodegenerative CNS disorders are typically characterized
by progressive dysfunction and/or cell death in the central nervous
system. The hallmark of many neurodegenerative CNS disorders is the
accumulation of misfolded proteins, such as beta-amyloid, tau,
alpha-synuclein, and TDP-43, both intracellularly and
extracellularly. Many neurodegenerative diseases are also
associated with gross mitochondrial dysfunction. Common examples of
neurodegenerative CNS disorders include Alzheimer's disease (AD),
Parkinson's disease (PD), Huntington's disease, and Amyotrophic
lateral sclerosis (ALS).
[0129] Psychiatric disorders (also referred to as mental illnesses)
commonly present with cognitive deficits and mood dysregulation.
Psychiatric disorders are generally defined by a combination of how
a person feels, acts, thinks or perceives. Well established systems
for the classification of psychiatric disorders include the
International Statistical Classification of Diseases and Related
Health Problems, 10th Revision (World Health Organization, tenth
revision (2010), the content of which is hereby expressly
incorporated by reference in its entirety for all purposes) and the
Diagnostic and Statistical Manual of Mental Disorders (DSM-IV;
American Psychiatric Association, DS-IV-TR (2000), the content of
which is hereby expressly incorporated by reference in its entirety
for all purposes). Common examples of psychiatric disorders include
mood disorders, schizophrenia, and autism.
[0130] As used herein, the terms "pooled human immunoglobulin G"
and "pooled human IgG" refer to a composition containing polyvalent
immunoglobulin G (IgG) purified from the blood/plasma of multiple
donors, e.g., more than a hundred or more than a thousand blood
donors. Typically, the composition will be at least 80% IgG (w/w,
e.g., weight IgG per weight total protein), preferably at least
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% IgG (w/w). In certain embodiments, the pooled human IgG
composition contains intact IgG immunoglobulins. In other
embodiments, the pooled human IgG composition contains IgG
fragments, for example those prepared by treatment of intact
antibodies with trypsin. In certain embodiments, the pooled human
IgG compositions used in the treatments disclosed herein contain
natural or synthetic modifications, e.g., post-translational
modifications and/or chemical modifications.
[0131] As used herein, the terms "high titer anti-amyloid .beta.
pooled immunoglobulin G" and "high titer anti-amyloid .beta. pooled
IgG" refer to a composition containing polyvalent immunoglobulin G
(IgG) purified from the blood/plasma of multiple donors, e.g., more
than a hundred or more than a thousand blood donors, having a
relative titer of anti-amyloid immunoglobulin G that is greater
than the expected titer of anti-amyloid .beta. immunoglobulins in a
pooled IgG composition prepared from the blood/plasma of more than
a thousand random individuals. Commercially available intravenous
immunoglobulin G (IVIG) preparations contain IgGs that specifically
recognize epitopes of various conformers of amyloid .beta., e.g.,
amyloid .beta. monomers, amyloid .beta. fibrils, and cross-linked
amyloid .beta. protein species (CAPS). It has been reported that a
commercial preparation of GAMMAGARD LIQUID.RTM. (10% Immune
Globulin Infusion (Human); Baxter International Inc., Deerfield,
Ill.) contains 0.1% anti-amyloid .beta. fibril IgG, 0.04% anti-CAPS
IgG, and 0.02% anti-amyloid .beta. monomer IgG, having EC.sub.50
affinities of 40 nM, 40 nM, and 350 nM for their target amyloid
.beta. conformer, respectively (O'Nuallain B. et al., Biochemistry,
2008 Nov. 25; 47(47):12254-6, the content of which is hereby
incorporated by reference in its entirety for all purposes). In
some embodiments, a high titer anti-amyloid .beta. pooled
immunoglobulin G composition contains a high titer of IgG specific
for one or more conformer of amyloid .beta.. In other embodiments,
a high titer anti-amyloid .beta. pooled immunoglobulin G
composition contains a high titer of IgG specific for amyloid
.beta. monomers, amyloid .beta. fibrils, and cross-linked amyloid
.beta. protein species (CAPS).
[0132] Accordingly, in one embodiment, a high titer anti-amyloid
.beta. pooled immunoglobulin G composition contains at least 0.1%
anti-amyloid .beta. IgG (e.g., 0.1% IgG with specific affinity for
any amyloid .beta. conformer). In another embodiment, a high titer
anti-amyloid .beta. pooled immunoglobulin G composition contains at
least 0.2% anti-amyloid .beta. IgG. In yet other embodiments, a
high titer anti-amyloid .beta. pooled immunoglobulin G composition
contains at least 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%,
0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%,
1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% or more
anti-amyloid .beta. IgG.
[0133] In one embodiment, a high titer anti-amyloid .beta. pooled
immunoglobulin G composition contains at least 0.1% anti-amyloid
.beta. fibril IgG. In another embodiment, a high titer anti-amyloid
.beta. pooled immunoglobulin G composition contains at least 0.2%
anti-amyloid .beta. fibril IgG. In yet other embodiments, a high
titer anti-amyloid .beta. pooled immunoglobulin G composition
contains at least 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%,
0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%,
1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% or more
anti-amyloid .beta. fibril IgG.
[0134] In one embodiment, a high titer anti-amyloid .beta. pooled
immunoglobulin G composition contains at least 0.04% anti-CAPS IgG.
In another embodiment, a high titer anti-amyloid .beta. pooled
immunoglobulin G composition contains at least 0.08% anti-CAPS IgG.
In yet other embodiments, a high titer anti-amyloid .beta. pooled
immunoglobulin G composition contains at least 0.04%, 0.05%, 0.06%,
0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%,
0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%,
0.95%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% or more
anti-CAPS IgG.
[0135] In one embodiment, a high titer anti-amyloid .beta. pooled
immunoglobulin G composition contains at least 0.02% anti-amyloid
.beta. monomer IgG. In another embodiment, a high titer
anti-amyloid .beta. pooled immunoglobulin G composition contains at
least 0.04% anti-amyloid .beta. monomer IgG. In yet other
embodiments, a high titer anti-amyloid .beta. pooled immunoglobulin
G composition contains at least 0.02%, 0.03%, 0.04%, 0.05%, 0.06%,
0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%,
0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%,
0.95%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0% or more
anti-amyloid .beta. monomer IgG.
[0136] High titer anti-amyloid .beta. pooled IgG can be prepared
according to standard methods for the manufacture of pooled IgG
starting with a standard pool of blood/plasma of multiple donors,
e.g., more than a hundred or more than a thousand blood donors, and
subsequently enriched for anti-amyloid .beta. immunoglobulin G.
Methods for the enrichment of target-specific immunoglobulin G
molecules are well known in the art (for example, see U.S. Patent
Application Publication No. 2004/0101909, the content of which is
hereby expressly incorporated by reference herein in its entirety
for all purposes). Alternatively, high titer anti-amyloid .beta.
pooled IgG can be prepared according to standard methods for the
manufacture of pooled IgG starting with an enriched pool of
blood/plasma from at least fifty, one hundred, two hundred, five
hundred, or one thousand donors having a high relative titer of
anti-amyloid .beta.. immunoglobulin G. As compared to the
manufacture of standard IgG for intravenous administration,
hyperimmune IgG preparations are commonly prepared from smaller
donor pools. These enriched pools of blood/plasma can be formed,
for example, by selectively pooling blood/plasma donations or
donors with a high relative titer of anti-amyloid .beta.
immunoglobulin G, e.g., by selection of high titer blood/plasma
donations or donors. Alternatively, an enriched pool of
blood/plasma can be formed by screening for blood/plasma donations
or donors with a low relative titer of anti-amyloid .beta.
immunoglobulin G and excluding these donations or donors from the
starting blood/plasma pool, e.g., screening for low titer
blood/plasma donations or donors.
[0137] As used herein, the term "intactness" refers to a percentage
of therapeutic agent that has not been at least partially degraded
at a particular point in time following administration. In one
embodiment, intactness is a measure of the total administered dose
of the therapeutic agent that has not been at least partially
degraded at the particular point in time (i.e., systemic
intactness). In another embodiment, intactness is a measure of the
therapeutic agent present at a particular site of the subject,
e.g., brain or bloodstream, which has not been at least partially
degraded (i.e., local intactness). In one embodiment, the
intactness of administered immunoglobulin (e.g., pooled human IgG)
is measured by mass spectroscopy. For example, the intactness of
the administered immunoglobulins is determined by analyzing a
biological sample from the subject, or proteins extracted from the
biological sample, by mass spectroscopy. In some embodiments, the
intactness of the administered immunoglobulins is determined by
separating proteins present in a biological sample from the subject
by molecular weight, size, or shape (e.g., by electrophoresis or
size exclusion chromatography) and determining the size
distribution of administered immunoglobulins in the sample.
[0138] In one embodiment, the intactness of immunoglobulin (e.g.,
pooled human IgG) in the brain of a subject following intranasal
administration is at least 40%. In preferred embodiments, the
intactness of immunoglobulin (e.g., pooled human IgG) in the brain
of a subject following intranasal administration is at least 50%,
preferably at least 60%. In certain embodiments, the intactness of
immunoglobulin (e.g., pooled human IgG) in the brain of a subject
following intranasal administration is at least 35%, 36%, 37%, 38%,
39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, or higher.
[0139] As used herein, the terms "intranasal administration" and
"nasal administration" refer to administration of a therapeutic
composition to the nasal cavity of a subject such that a
therapeutic agent in the composition is delivered directly to one
or more epithelium located in the nose. In certain embodiments,
intranasal administration is achieved using a liquid preparation
(e.g., an aqueous preparation), an aerosolized preparation, or a
dry powder preparation, each of which can be administered via an
externally propelled or self-propelled (e.g., via inhalation)
non-invasive nasal delivery device, or via a gel, cream, ointment,
lotion, or paste directly applied to one or more nasal epithelium
(e.g., olfactory epithelium or nasal respiratory epithelium).
[0140] As used herein, the term "nasal epithelium" refers to the
tissues lining the internal structure of the nasal cavity. The term
nasal epithelium includes both the nasal respiratory epithelium,
located in the lower two-thirds of the nasal cavity in humans, and
the olfactory epithelium, located in the upper third of the nasal
cavity of humans.
[0141] As used herein, the term "olfactory epithelium" refers to a
specialized epithelial tissue inside the nasal cavity involved in
smell. In humans, the olfactory epithelium is located in the upper
third of the nasal cavity.
[0142] As used herein, the term "directed administration" refers to
a process of preferentially delivering a therapeutic agent to a
first location in a subject as compared a second location or
systemic distribution of the agent. For example, in one embodiment,
directed administration of a therapeutic agent results in at least
a two-fold increase in the ratio of therapeutic agent delivered to
a targeted site to therapeutic agent delivered to a non-targeted
site, as compared to the ratio following systemic or non-directed
administration. In other embodiments, directed administration of a
therapeutic agent results in at least a 3-fold, 4-fold, 5-fold,
6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold,
30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 60-fold, 70-fold,
80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold,
750-fold, 1000-fold, or greater increase in the ratio of
therapeutic agent delivered to a targeted site to therapeutic agent
delivered to a non-targeted site, as compared to the ratio
following systemic or non-directed administration. In a particular
embodiment, directed administration of an agent is contrasted to
intravenous administration of the agent. For example, in one
embodiment, the ratio of therapeutic agent present at a targeted
site to therapeutic agent present in the blood stream is increased
at least two-fold when the agent is subject to directed
administration (e.g., by delivery to the brain via intranasal
administration), as compared to when the therapeutic agent is
administered intravenously.
[0143] As used herein, the term "non-invasive nasal delivery
device" refers an instrument that is capable of delivering a
therapeutic composition (e.g., pooled human IgG) to the nasal
cavity without piercing the epithelium of the subject. Non-limiting
examples of non-invasive nasal delivery devices include propellant
(e.g., a pressurized inhaler) and non-propellant (e.g., a pump-type
inhaler) types of aerosol or atomizer devices, particle dispersion
devices, nebulizers, and pressurized olfactory delivery devices for
delivery of liquid or powder formulations.
[0144] The term "treatment" or "therapy" generally means obtaining
a desired physiologic effect. The effect may be prophylactic in
terms of completely or partially preventing a disease or condition
or symptom thereof and/or may be therapeutic in terms of a partial
or complete cure for an injury, disease or condition and/or
amelioration of an adverse effect attributable to the injury,
disease or condition and includes arresting the development or
causing regression of a disease or condition. Treatment can also
refer to any delay in onset, amelioration of symptoms, improvement
in patient survival, increase in survival time or rate, improvement
in cognitive function, etc. The effect of treatment can be compared
to an individual or pool of individuals not receiving the
treatment.
[0145] As used herein, a "therapeutically effective amount or dose"
or "sufficient/effective amount or dose," refers to a dose that
produces effects for which it is administered. The exact dose will
depend on the purpose of the treatment, and will be ascertainable
by one skilled in the art using known techniques (see, e.g.,
Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd,
The Art, Science and Technology of Pharmaceutical Compounding
(1999); Pickar, Dosage Calculations (1999); and Remington: The
Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed.,
Lippincott, Williams & Wilkins).
[0146] As used here, the terms "dose" and "dosage" are used
interchangeably and refer to the amount of active ingredient given
to an individual at each administration. The dose will vary
depending on a number of factors, including frequency of
administration; size and tolerance of the individual; severity of
the condition; risk of side effects; and the route of
administration. One of skill in the art will recognize that the
dose can be modified depending on the above factors or based on
therapeutic progress. The term "dosage form" refers to the
particular format of the pharmaceutical, and depends on the route
of administration. For example, a dosage form can be a liquid or
dry powder, formulated for intranasal administration.
[0147] As used herein, a therapeutic composition "consisting
essentially of a buffering agent and pooled human IgG" may also
contain residual levels of chemical agents used during the
manufacturing process, e.g., surfactants, buffers, salts, and
stabilizing agents, as well as chemical agents used to pH the final
composition, for example, counter ions contributed by an acid
(e.g., hydrochloric acid or acetic acid) or base (e.g., sodium or
potassium hydroxide), and/or trace amounts of contaminating
proteins.
[0148] As used herein, the term "permeability enhancer" refers to a
component of a therapeutic composition formulated for intranasal
administration which promotes the passage of biotherapeutics (e.g.,
mimetibodies and Fc-fusion polypeptides) through the nasal
epithelium. Non-limiting examples of permeability enhancers include
membrane fluidizers, tight junction modulators, and medium chain
length fatty acids and salts and esters thereof. Non-limiting
examples of medium chain length fatty acids and salts and esters
thereof included mono-, di-, and triglycerides (such as sodium
caprylate, sodium caprate, glycerides (CAPMUL, GELUCIRE 44/14 PEG32
glyceryl laurate EP); lipids; pegylated peptides; and liposomes.
Surfactants and similarly acting compounds can also be used as
permeability enhancers. Non-limiting examples of surfactants and
similarly acting compounds include polysorbate-80,
phosphatidylcholine, N-methylpiperazine, sodium salicylate,
melittin, and palmitoyl carnitine chloride (PCC). Generally, the
pooled human immunoglobulin G compositions described herein are
formulated for intranasal administration in the absence of
permeability enhancers.
[0149] As used herein, the term "dry powder composition" refers to
a lyophilized or spray dried form of a therapeutic pooled human IgG
formulation. In one embodiment, a dry powder composition contains
less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less residual
water content.
[0150] A "control" is used herein, refers to a reference, usually a
known reference, for comparison to an experimental group. One of
skill in the art will understand which controls are valuable in a
given situation and be able to analyze data based on comparisons to
control values. Controls are also valuable for determining the
significance of data. For example, if values for a given parameter
vary widely in controls, variation in test samples will not be
considered as significant.
[0151] Before the present disclosure is described in greater
detail, it is to be understood that this invention is not limited
to particular embodiments described, as such may, of course, vary.
It is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting, since the scope of the present invention
will be limited only by the appended claims.
[0152] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0153] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, representative illustrative methods and materials are
now described.
[0154] It is noted that, as used herein and in the appended claims,
the singular forms "a," "an," and "the" include plural referents
unless the context clearly dictates otherwise. It is further noted
that the claims may be drafted to exclude any optional element. As
such, this statement is intended to serve as antecedent basis for
use of such exclusive terminology as "solely," "only," and the like
in connection with the recitation of claim elements, or use of a
"negative" limitation.
[0155] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
Administration
[0156] Intranasal (IN) administration is an advantageous mode of
delivering a drug to the brain because it is non-invasive and there
is a direct connection between the olfactory system and the brain.
Intranasal administration of IgG (INIG) to treat neurological
diseases is particularly advantageous because the direct connection
between the olfactory system and the brain obviates delivery
concerns associated with the blood-brain barrier (BBB) and
minimizes systemic exposure to the drug, thereby minimizing side
effects of the drug. Furthermore, IN delivery allows compositions
such as powders, granules, solutions, ointments, and creams,
thereby obviating the need for intravenous and intramuscular
administration. For example, when a drug is administered
intranasally, it is transported through the nasal mucosa and along
the olfactory neural pathway. The drug can be administered alone or
can be combined with a carrier molecule(s) to promote transport
through the nasal mucosa and along the olfactory neural pathway.
The drug can also be administered in combination with an absorption
enhancer. Absorption enhancers promote the absorption of the drug
through the nasal mucosa and along the olfactory neural pathway.
Furthermore, additional molecules can be added to facilitate drug
transport across the olfactory neural pathway.
[0157] IN administration can also be used to deliver therapeutic
drugs to the brain via the trigeminal pathway. Specifically, IN
administration can be used to deliver IgG via the trigeminal
pathway. The olfactory and trigeminal nerves receive high
concentrations of a drug with IN administration because the
absorbent respiratory and olfactory pseudoepithelium are innervated
by the trigeminal nerve. These nerves can then transport the drug
into the brain and other connected structures. For example, the
trigeminal nerve branches directly or indirectly reach the
maxillary sinus, brainstem, hindbrain, cribriform plate, forebrain
(e.g., cortex and diencephalon), orofacial structures (e.g., teeth,
masseter muscle, and the temporomandibular joint), midbrain,
cerebellum, cervical spinal cord, thoracic spinal cord, and lumbar
spinal cord. Accordingly, INIG can be carried across the trigeminal
pathway to reach and treat neurological diseases.
[0158] In certain embodiments, methods are provided for the
treatment of CNS disorders by administration of pooled human
immunoglobulins to tissue innervated by the olfactory and/or
trigeminal nerves. Surprisingly, it was found that therapeutically
effective amounts of pooled human immunoglobulin are delivered to
the CNS when administered intranasally. For example, it is shown
herein that intranasal administration of pooled human
immunoglobulins is effective to reduce total amyloid plaque load in
a rodent model of Alzheimer's disease. Moreover, by specifically
targeting the nasal epithelium, as opposed to the respiratory
system (lung, pharynx, etc.), systemic exposure of the pooled human
immunoglobulins is reduced.
[0159] Many types of intranasal delivery devices can be used to
practice the methods provided herein. In some embodiments, the
delivery device is an intranasal device for the administration of
liquids. Non-limiting examples of devices useful for the
administration of liquid compositions (e.g., liquid pooled IgG
compositions) include vapor devices (e.g., vapor inhalers), drop
devices (e.g., catheters, single-dose droppers, multi-dose
droppers, and unit-dose pipettes), mechanical spray pump devices
(e.g., squeeze bottles, multi-dose metered-dose spray pumps, and
single/duo-dose spray pumps), bi-directional spray pumps (e.g.,
breath-actuated nasal delivery devices), gas-driven spray
systems/atomizers (e.g., single- or multi-dose HFA or nitrogen
propellant-driven metered-dose inhalers, including traditional and
circumferential velocity inhalers), and electrically powered
nebulizers/atomizers (e.g., pulsation membrane nebulizers,
vibrating mechanical nebulizers, and hand-held mechanical
nebulizers). In some embodiments, the delivery device is an
intranasal device for the administration of powders or gels.
Non-limiting examples of devices useful for the administration of
powder compositions (e.g., lyophilized or otherwise dried pooled
IgG compositions) include mechanical powder sprayers (e.g.,
hand-actuated capsule-based powder spray devices and hand-actuated
powder spray devices, hand actuated gel delivery devices),
breath-actuated inhalers (e.g., single- or multi-dose nasal
inhalers and capsule-based single- or multi-dose nasal inhalers),
and insufflators (e.g., breath-actuated nasal delivery devices). In
some embodiments, the pooled human immunoglobulins are
preferentially administered to the olfactory area, located in the
upper third of the nasal cavity, and particularly to the olfactory
epithelium. Fibers of the olfactory nerve are unmyelinated axons of
olfactory receptor cells, which are located in the superior
one-third of the nasal cavity. The olfactory receptor cells are
bipolar neurons with swellings covered by hair-like cilia that
project into the nasal cavity. At the other end, axons from these
cells collect into aggregates and enter the cranial cavity at the
roof of the nose. Surrounded by a thin tube of pia, the olfactory
nerves cross the subarachnoid space containing CSF and enter the
inferior aspects of the olfactory bulbs. Once the pooled human
immunoglobulin is dispensed into the nasal cavity, the
immunoglobulin can undergo transport through the nasal mucosa and
into the olfactory bulb and interconnected areas of the brain, such
as the hippocampal formation, amygdaloid nuclei, nucleus basalis of
Meynert, locus ceruleus, the brain stem, and the like (e.g.,
Johnson et al., Molecular Pharmaceutics (2010) 7(3):884-893).
[0160] In certain embodiments, pooled human immunoglobulin is
administered to tissue innervated by the trigeminal nerve. The
trigeminal nerve innervates tissues of a mammal's (e.g., human)
head including skin of the face and scalp, oral tissues, and
tissues surrounding the eye. The trigeminal nerve has three major
branches, the ophthalmic nerve, the maxillary nerve, and the
mandibular nerve. In some embodiments, the methods provided herein
include targeted administration of pooled human immunoglobulin to
one or more of these trigeminal branches, i.e. the trigeminal
pathway. In some embodiments, the methods provided herein include
targeted administration of pooled human immunoglobulin to the
maxillary sinus, thereby reaching the brainstem, hindbrain,
cribriform plate, forebrain (e.g., cortex and diencephalon),
midbrain, cerebellum, cervical spinal cord, thoracic spinal cord,
and lumbar spinal cord through the trigeminal pathway. In certain
embodiments, methods provided herein include targeted
administration of pooled human immunoglobulin for treatment of a
disorder of the CNS (e.g., Alzheimer's disease).
[0161] In some embodiments, the pooled human immunoglobulin is
administered to nasal tissues innervated by the trigeminal nerve,
for example, to nasal tissues including the sinuses, the inferior
two-thirds of the nasal cavity and the nasal septum. In certain
embodiments, the pooled human immunoglobulin is targeted to the
inferior two-thirds of the nasal cavity and/or the nasal
septum.
[0162] In some embodiments, the pooled human immunoglobulin is
administered to one or both maxillary sinus of the individual.
Methods and devices for administration to the maxillary sinus are
known in the art, for example, see United States Patent Application
Publication Number 2011/0151393, the contents of which are hereby
incorporated by reference in their entirety for all purposes.
[0163] The maxillary sinus is in fluid communication with the
patient's nasal cavity and comprises right and left maxillary
sinuses. Each maxillary sinus communicates with the corresponding
nasal passage via the orifice of the maxillary sinus. The maximum
volume of the maxillary sinus in adults is approximately 4 to 15
ml, though individual sinuses may comprise volumes outside of this
range.
[0164] The pathway from the nasal passages to the corresponding
orifice of maxillary sinus, and ultimately to the corresponding
maxillary sinus, allows for a device to be inserted into the nasal
passage to the orifice of the maxillary sinus, whereupon at least
one effective amount or dose of pooled human immunoglobulins may be
administered and delivered into the maxillary sinus. The pathway to
the maxillary sinus is tortuous and requires: traversing the
nostril, moving through the region between the lower and middle
concha, navigating over and into the semilunar hiatus, traveling
superiorly into the maxillary sinus opening, resisting the ciliated
action of the ostium/tube passing into the maxillary sinus and
ultimately moving into the sinus itself.
[0165] Since the trigeminal nerve passes through the maxillary
sinus, the pooled human immunoglobulins in the maxillary sinus
after delivery therein will be moved along the trigeminal nerve to
structures innervated by the trigeminal nerve. In this fashion,
pooled human IgG administered to one or both of the maxillary sinus
is delivered to the brain via the trigeminal nerve.
[0166] In one embodiment, the pooled human IgG compositions
provided herein for the treatment of a CNS disorder (e.g.,
Alzheimer's disease) are intranasally administered as a liquid
preparation, e.g., an aqueous based preparation. For example, in
one embodiment, nasal drops are instilled in the nasal cavity by
tilting the head back sufficiently and apply the drops into the
nares. In another embodiment, the drops are snorted up the nose. In
another embodiment, nasal drops are applied with an applicator or
tube onto the upper third of the nasal mucosa. In another
embodiment, nasal drops are applied with an applicator or tube into
one or both of maxillary sinus of the subject. In another
embodiment, the liquid preparation may be placed into an
appropriate device so that it may be aerosolized for inhalation
through the nasal cavity. For example, in one embodiment, the
therapeutic agent is placed into a plastic bottle atomizer. In a
specific embodiment, the atomizer is advantageously configured to
allow a substantial amount of the spray to be directed to the upper
one-third region or portion of the nasal cavity (e.g., the
olfactory epithelium). In another embodiment, the liquid
preparation is aerosolized and applied via an inhaler, such as a
metered-dose inhaler (for example, see, U.S. Pat. No. 6,715,485).
In a specific embodiment, the inhaler is advantageously configured
to allow a substantial amount of the aerosol to be directed to the
upper one-third region or portion of the nasal cavity (e.g., the
olfactory epithelium). In certain embodiments, a substantial amount
of the pooled human immunoglobulin refers to at least 25%, 30%,
35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or
100% of the composition, which is administered to the upper
one-third region of the nasal cavity (e.g., administered to the
upper one-third of the nasal epithelium).
[0167] In one embodiment, the pooled human IgG compositions
provided herein for the treatment of a CNS disorder (e.g.,
Alzheimer's disease) are intranasally administered as a dry powder.
Dry powder nasal delivery devices are well known in the art, for
example, see PCT publication No. WO 1996/222802. In one embodiment,
following intranasal administration, pooled human IgG is absorbed
across the olfactory epithelium, which is found in the upper third
of the nasal cavity. In another embodiment, following intranasal
administration, pooled human IgG is absorbed across the nasal
respiratory epithelium, which is innervated with trigeminal nerves,
in the lower two-thirds of the nasal cavity. The trigeminal nerves
also innervate the conjunctive, oral mucosa, and certain areas of
the dermis of the face and head, and absorption after intranasal
administration of the IgG from these regions may also occur. In
other embodiments, following intranasal administration, pooled
human IgG is absorbed across the maxillary sinus epithelium. In yet
other embodiments, pooled human IgG may be absorbed across more
than one of these nasal epitheliums and subsequently delivered to
the brain of the subject.
[0168] Although administration is referred to herein as a single
event that may occur according to some regular or irregular
frequency of the course of a treatment, a single administration
even may include multiple administrations. In this regard, a single
dosage of pooled human IgG may be partitioned into two or more
physical compositions for administration. For example, a 200 mg
dose of pooled human IgG in a liquid composition formulated at 200
g/L IgG may be administered to a 50 kg subject (4 mg/kg IgG) in
four drops having a volume of 250 .mu.L each. Likewise, a dry
powder composition containing a single dosage of pooled human IgG
may be administered, for example, in two or more distinct puffs. In
some embodiments, pooled human IgG is administered in one or more
puffs or sprays into each nare of the individual (e.g., one or more
puff into the right nare and one or more puffs into the left
nare).
[0169] In certain embodiments, the methods described herein for
treating a CNS disorder include intranasal administration of pooled
human IgG via a non-invasive intranasal delivery device. In one
embodiment, the non-invasive intranasal delivery device is a
non-propellant type aerosol or atomizer device, a propellant type
aerosol or atomizer device, a non-propellant pump-type device, a
particle dispersion device, a nebulizer device, or a pressurized
olfactory delivery device.
[0170] In one embodiment the non-invasive intranasal delivery
device delivers a liquid drop of a pooled human IgG composition to
the nasal cavity of a subject. In a particular embodiment, the
non-invasive intranasal delivery device delivers a liquid drop of
pooled human IgG directly to a nasal epithelium of the subject. In
a more specific embodiment, the non-invasive intranasal delivery
device delivers a liquid drop of pooled human IgG directly to the
olfactory epithelium of the subject. In one embodiment, the liquid
drop is administered by tilting the head of the subject back and
administering the drop into a nare of the subject. In another
embodiment, the liquid drop is administered by inserting the tip of
a non-invasive intranasal delivery device into a nare of the
subject and squirting or spraying the drop into the nasal cavity of
the subject.
[0171] In another embodiment, the non-invasive intranasal delivery
device delivers a liquid or a powder aerosol of a pooled human IgG
composition to the nasal cavity of a subject. In a particular
embodiment, the non-invasive intranasal delivery device delivers a
liquid or a powder aerosol of pooled human IgG directly to a nasal
epithelium of the subject. In a more specific embodiment, the
non-invasive intranasal delivery device delivers a liquid or a
powder aerosol of pooled human IgG directly to the olfactory
epithelium of the subject.
[0172] In another embodiment, the non-invasive intranasal delivery
device delivers a dry powder composition of pooled human IgG
composition to the nasal cavity of a subject. In a particular
embodiment, the non-invasive intranasal delivery device delivers a
dry powder composition of pooled human IgG directly to a nasal
epithelium of the subject. In a more specific embodiment, the
non-invasive intranasal delivery device delivers a dry powder
composition of pooled human IgG directly to the olfactory
epithelium of the subject.
[0173] In another embodiment, the non-invasive intranasal delivery
device delivers a sustained release or controlled release
composition of pooled human IgG composition to the nasal cavity of
a subject. In a specific embodiment the sustained release
composition comprises a dry powder composition of pooled human IgG.
In some embodiments, the sustained release composition is a gel,
paste, hydrogel, cream, lotion, film, or similar form that coats at
least a portion of the nasal epithelium (e.g., all or a portion of
the olfactory epithelium, all or a portion of a nasal epithelium
associated with trigeminal nerve endings, all or a portion of the
upper third of the nasal epithelium, all or a portion of the lower
third of the nasal epithelium, or all or a portion of the nasal
maxillary epithelium.
[0174] In one embodiment, the intranasal device is a single-use,
disposable device. In another embodiment, the intranasal device is
a multi- or repeat-use device. In certain embodiments, the
single-use or multi-use device is pre-metered. In a specific
embodiment, the single-use or multi-use device is pre-filled. In
certain embodiments, the multi- or repeat-use device is refillable.
In certain embodiments, the device is refilled by inserting a
pooled human IgG composition into a chamber of the device. In other
embodiments, a chamber of the multi- or repeat-use device designed
to hold the pooled human IgG composition is replaced with a new,
pre-filled chamber.
[0175] In certain embodiments, the pooled human immunoglobulin
compositions are administered by a pressurized nasal delivery (PND)
device. In one embodiment, the PND device can be used to deliver a
liquid IgG composition to the nasal cavity. In one embodiment, the
PND device can be used to deliver a powder IgG composition to the
nasal cavity. In one embodiment, the PND device administers an IgG
composition into one nostril. In one embodiment, the Impel device
administers an IgG composition into both nostrils.
[0176] In some embodiments, the PND device is configured to deliver
the liquid or powder IgG compositions to a particular epithelium,
location, and/or structure of the nasal cavity. For example, in one
embodiment, the PND device is configured to deliver the IgG
composition to the upper nasal cavity. In one embodiment, the PND
device is configured to deliver the IgG composition to the
olfactory epithelium of the nasal cavity. In one embodiment, the
PND device is configured to deliver the IgG composition to the
lower two thirds of the nasal epithelium. In one embodiment, the
PND device is configured to deliver the IgG composition to a nasal
epithelium associated with trigeminal nerve endings. In one
embodiment, the PND device is configured to deliver the IgG
composition to the nasal maxillary sinus epithelium.
[0177] Methods for configuring pressurized delivery devices to
achieve a particular delivery profile are known in the field. For
example, in one embodiment, a pressurized nasal delivery device is
configured to produce a stream, spray, puff, etc., have a
particular characteristic. For example, in one embodiment, to
achieve administration to the upper third of the nasal epithelium,
the device is configured to produce a strong, focused stream,
spray, puff, etc. In one embodiment, the strong focused spray is
created by imparting circumferential and/or axial velocity onto the
stream of the therapeutic composition (e.g., pooled human IgG)
being administered into the nose. In another embodiment, to achieve
administration to a greater portion of the nasal epithelium (e.g.,
the entire or the lower two thirds of the nasal epithelium), the
device is configured to produce a diffuse and/or weaker stream,
spray, puff, etc. In some embodiments, the tip of the delivery
device is configured to physically direct the stream, spray, puff,
etc., to the desired intranasal location when inserted into the
subject's nare. For example, a kink or bend may be introduced into
the tip of the delivery device to "point" the stream, spray, puff,
etc., at a targeted epithelium. In some embodiments, the delivery
pattern of the device is adjustable, such that the device can be
differentially configured to target the therapeutic agent (e.g.,
pooled human IgG) to a particular epithelium, structure, or
location within the nose. In certain embodiments, the pooled human
immunoglobulin compositions are administered by a breath-powered
technology device. In certain embodiments, the breath-powered
technology provides positive pressure during administration. In
certain embodiments, the positive pressure expands narrow nasal
passages. In certain embodiments, the expansion of the nasal
passages allows reliable delivery of liquid or powder pooled human
immunoglobulin compositions described herein to the CNS. In some
embodiments, exhalation into the device propels the therapeutic
(e.g., pooled human IgG) into the nose, while at the same time
closing the soft-palette, thereby reducing deposition of the
therapeutic into the throat and/or lungs. In one embodiment, the
breath-powered technology device administers an IgG composition
described herein into one nostril. In one embodiment, the
breath-powered technology device administers an IgG composition
described herein into two nostrils.
[0178] Non-limiting examples of commercial intranasal delivery
devices include the EQUADEL.RTM. nasal spray pump (Aptar Pharma),
the Solovent dry powder device (BD Technologies), the Unidose nasal
drug delivery device (Consort Medical PLC), the NasoNeb.RTM. Nasal
Nebulizer (MedInvent, LLC), the VeriDoser.RTM. nasal delivery
device (Mystic Pharmaceuticals), the VRx2.TM. nasal delivery device
(Mystic Pharmaceuticals), the DirectHaler.TM. Nasal device
(Direct-Haler A/S), the TriViar.TM. single-use unit-dose dry powder
inhaler (Trimel Pharmaceuticals), the SinuStar.TM. Aerosol Delivery
System (Pari USA), the Aero Pump (Aero Pump GmbH), the
Fit-Lizer.TM. nasal delivery device (Shin Nippon Biomedical
Laboratories), the LMA MAD Nasal.TM. device (LMA North America,
Inc.), the Compleo intranasal bioadhesive gel delivery system
(Trimel Pharmaceuticals), Impel's Pressurized Olfactory Delivery
(POD) device (Impel Neuropharma), the ViaNase.TM. electronic
atomizer (Kurve Technology, Inc.), the OptiNose powder delivery
device (OptiNose US Inc.), and the Optinose liquid delivery device
(OptiNose US Inc.)
[0179] In one embodiment, an intranasal device described herein can
deliver 10%-20% of the metered IgG dose to the olfactory region. In
one embodiment, an intranasal device described herein can deliver
20%-30% of the metered IgG dose to the olfactory region. In one
embodiment, an intranasal device described herein can deliver
5%-20% of the metered IgG dose to the olfactory region. In one
embodiment, an intranasal device described herein can deliver
30%-40% of the metered IgG dose to the olfactory region. In one
embodiment, an intranasal device described herein can deliver
40%-50% of the metered IgG dose to the olfactory region. In one
embodiment, an intranasal device described herein can deliver
60%-70% of the metered IgG dose to the olfactory region. In one
embodiment, an intranasal device described herein can deliver
60%-80% of the metered IgG dose to the olfactory region. In one
embodiment, an intranasal device described herein can deliver
70%-80% of the metered IgG dose to the olfactory region. In one
embodiment, an intranasal device described herein can deliver
80%-90% of the metered IgG dose to the olfactory region. In one
embodiment, an intranasal device described herein can deliver
60%-80% of the metered IgG dose to the olfactory region.
[0180] In certain embodiments, the pooled human immunoglobulin
compositions are administered by an intranasal device described
above in one or more doses. In one embodiment the more than one
dose is administer by the intranasal device in alternating
nostrils. In one embodiment, the more than one does is administered
by the intranasal device at different time points throughout the
day. In certain embodiments the more than one dose is two, three,
four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,
fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or
twenty or more doses. In certain embodiments the more than one dose
is administered by the intranasal device one, two, three, four,
five, six, seven, eight, nine, or ten or more time points
throughout the day.
[0181] In certain embodiments, the pooled human immunoglobulin
compositions are administered by an intranasal device described
above in an initial dose or set of doses followed by repeat
maintenance doses. In certain embodiments the initial dose is one,
two, three, four, five, six, seven, eight, nine, ten, eleven,
twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,
nineteen, or twenty or more doses.
[0182] In another embodiment, a gel, cream, ointment, lotion, or
paste containing pooled human IgG is applied onto the nasal
epithelium, for example, by use of an application stick or swab. In
a particular embodiment, a gel, cream, ointment, lotion, or paste
containing pooled human IgG is applied directly onto a nasal
epithelium of the subject. In a more specific embodiment, a gel,
cream, ointment, lotion, or paste containing pooled human IgG is
applied directly onto the olfactory epithelium of the subject.
[0183] In certain embodiments, a substantial fraction of the
therapeutic agent present in the composition is delivered directly
to one or more nasal epithelium. In certain embodiments, at least
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, or 100% of the therapeutic agent present in the
composition is delivered directly to a nasal epithelium. In a
specific embodiment, a substantial fraction of the therapeutic
agent present in the composition is delivered directly to the
olfactory epithelium. In a more specific embodiment, at least 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, or 100% of the therapeutic agent present in the composition is
delivered directly to the olfactory epithelium. In another specific
embodiment, a substantial fraction of the therapeutic agent present
in the composition is delivered directly to nasal epithelium
innervated with trigeminal nerves. In a more specific embodiment,
at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, or 100% of the therapeutic agent present in the
composition is delivered directly to nasal epithelium innervated
with trigeminal nerves.
[0184] In some embodiments, pooled human IgG can be administered to
a subject as a combination therapy with another treatment, e.g.,
another treatment for a disorder of the central nervous system
(e.g., Alzheimer's disease, age-related dementia, Parkinson's
disease, or multiple sclerosis). For example, the combination
therapy can include administering to the subject (e.g., a human
patient) one or more additional agents that provide a therapeutic
benefit to the subject who has, or is at risk of developing, a
disorder of the central nervous system, e.g., Alzheimer's disease.
In some embodiments, the pooled human IgG and the one or more
additional agents are administered at the same time. In other
embodiments, the pooled human IgG is administered first in time and
the one or more additional agents are administered second in time.
In some embodiments, the one or more additional agents are
administered first in time and the pooled human IgG is administered
second in time.
[0185] The pooled human IgG can replace or augment a previously or
currently administered therapy. For example, upon treating with
pooled human IgG, administration of the one or more additional
agents can cease or diminish, e.g., be administered at lower
levels. In other embodiments, administration of the previous
therapy is maintained. In some embodiments, a previous therapy will
be maintained until the level of polyclonal IgG reaches a level
sufficient to provide a therapeutic effect. The two therapies can
be administered in combination.
[0186] In one embodiment, a human receiving a first therapy for a
disorder of the central nervous system, e.g., Alzheimer's disease,
who is then treated with pooled human IgG, continues to receive the
first therapy at the same or a reduced amount. In another
embodiment, treatment with the first therapy overlaps for a time
with treatment with pooled human IgG, but treatment with the first
therapy is subsequently halted.
[0187] In a particular embodiment, pooled human IgG may be
administered in combination with a treatment for an age-related
dementia, e.g., Alzheimer's disease. In certain embodiments, the
treatment for an age-related dementia co-administered with pooled
human IgG is administration of a cholinesterase inhibitor (e.g.,
ARICEPT (donepezil), EXELON (rivastigmine), RAZADYNE (galantamine),
or COGNEX (tacrine), or an inhibitor of the NMDA-type glutamate
receptor (e.g., memantine).
[0188] In further embodiments the second therapy is levodopa
(L-DOPA). The second therapy can also be a dopamine agonist.
Non-limiting examples of dopamine agonists include bromocriptine,
pergolide, pramipexole, ropinirole, piribedil, cabergoline,
apomorphine and lisuride. The second therapy can be a MAO-B
inhibitor. Non-limiting examples of MAO-B inhibitors are selegiline
and rasgiline. Addition second therapies can include amantaine,
anticholinergic compositions, clozapine, modafinil, and
non-steroidal anti-inflammatory drugs.
[0189] In further embodiments the second therapy is CAMPATH
(alemtuzumab), ZENAPX (daclizumab), rituximab, dirucotide,
BHT-3009, cladribine, dimethyl fumarate, estriol, laquinimod,
pegylated interferon-.beta.-1a, minocycline, statins, temsirolimus,
teriflunomide, and low dose naltexone.
[0190] In certain embodiments the second therapy is psychotherapy.
Non-limiting examples of psychotherapy are psychosocial
intervention, behavioral intervention, reminiscence therapy,
validation therapy, supportive psychotherapy, sensory integration,
simulated presence therapy, cognitive retraining, and
stimulation-oriented therapies such as art, music, pet, exercise,
and recreational activities.
[0191] Furthermore, two or more second therapies can be combined
with therapeutic intranasal IgG. For example, therapeutic
intranasal IgG can be combined with memantine and donepezil.
Dosing
[0192] The use of intravenous immunoglobulin G (IVIG) for the
treatment of disorders of the central nervous system (CNS) is
currently under investigation (Awad et al. 2011 (Current
Neuropharmacology, 9:417428); Pohl et al. 2012 (Current Treatment
Options in Neurology, 14:264-275); Krause et al. 2012 (European J.
of Paediatric Neurology, 16:206-208); Elovaara et al. 2011
(Clinical Neuropharmacology, 34(2):84-89); Perlmutter, et al. 1999
(The Lancet, 354:1153-1158); Snider et al. 2003 (J. of Child and
Adolescent Psychopharmacology, 13(supp 1): S81-S88). In these
trials, subjects are administered between 0.4 g/kg body weight and
2.0 g/kg body weight IVIG per dosage. Specifically, the treatment
regimes of CNS disorders with IVIG range from 0.4 g/kg body weight
IVIG administered once daily for 5 consecutive days to 2.0 g/kg
body weight IVIG administered once daily for 2 consecutive days.
There are several variations of these IVIG treatment regimes. For
example, IVIG treatment regimes may be 1.0 g/kg body weight IVIG
administered twice a day (total 2.0 g/kg body weight IVIG per day).
The initial 2 to 5 day IVIG dosages can also be followed with
maintenance doses ranging from 0.4 g/kg to 0.5 g/kg body weight
IVIG. Due to the limited supply of pooled human IgG, and high cost
associated therewith, large-scale implementation of these
treatments may prove problematic if they are approved by major
regulatory bodies.
[0193] Typical intravenous dosing of IgG in human Alzheimer's
trials ranges from 200 mg/kg to 400 mg/kg every two weeks.
Advantageously, the inventors have found that levels of pooled
human IgG seen in the brain after intravenous administration can
also be achieved by intranasal administration. For example, it is
shown in Example 3 that administration of pooled human IgG (0.02
g/kg IgG) intranasally as drops (IN1) or a liquid spray delivered
directly to the olfactory epithelium (IN3) results in substantially
the same amount of IgG being delivered to the right and left
hemispheres of the brain as for intravenous administration of
pooled human IgG (0.02 g/kg IgG; compare corrected AUC values for
right and left hemisphere IgG delivery in Table 69, Table 71, and
Table 72). Significantly, intranasal administration of IgG liquid
drops at concentrations ten-fold lower (0.002 g/kg IgG) also
resulted in the delivery of intact IgG to the cerebral cortex (see,
Table 70). Any reduction in the amount of pooled human IgG required
for administration is significant because of the limited supply of
pooled human IgG and the high cost associated therewith.
[0194] Accordingly, in certain embodiments, the methods for
treating a CNS disorder provided herein include intranasally
administering from about 0.05 mg of pooled human IgG per kg body
weight (mg/kg IgG) to about 500 mg/kg IgG in a single dosage.
[0195] In certain embodiments, the methods for treating a CNS
disorder provided herein include intranasally administering a low
dose of pooled human IgG. In one embodiment, a low dose of pooled
human IgG is from about 0.05 mg/kg IgG to about 10 mg/kg IgG. In
specific embodiments, a low dose of pooled human IgG is about 0.05
mg/kg, 0.06 mg/kg, 0.07 mg/kg, 0.08 mg/kg, 0.09 mg/kg, 0.10 mg/kg,
0.15 mg/kg, 0.20 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.35 mg/kg, 0.40
mg/kg, 0.45 mg/kg, 0.50 mg/kg, 0.55 mg/kg, 0.60 mg/kg, 0.65 mg/kg,
0.70 mg/kg, 0.75 mg/kg, 0.80 mg/kg, 0.85 mg/kg, 0.90 mg/kg, 0.95
mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 3.0 mg/kg, 3.5
mg/kg, 4.0 mg/kg, 4.5 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0
mg/kg, 9.0 mg/kg, or 10.0 mg/kg IgG. In yet other embodiments, a
low dose of pooled human IgG is from 0.1 mg/kg to 5 mg/kg, 0.5
mg/kg to 5 mg/kg, 1 mg/kg to 5 mg/kg, 2 mg/kg to 5 mg/kg, 0.5 mg/kg
to 10 mg/kg, 1 mg/kg to 10 mg/kg, 2 mg/kg to 10 mg/kg, 1 mg/kg to 8
mg/kg, 2 mg/kg to 8 mg/kg, 3 mg/kg to 8 mg/kg, 4 mg/kg to 8 mg/kg,
5 mg/kg to 8 mg/kg, 1 mg/kg to 6 mg/kg, 2 mg/kg to 6 mg/kg, 3 mg/kg
to 6 mg/kg, 4 mg/kg to 6 mg/kg, 5 mg/kg to 6 mg/kg, 1 mg/kg to 4
mg/kg, 2 mg/kg to 4 mg/kg, or 3 mg/kg to 4 mg/kg IgG.
[0196] In certain embodiments, the methods for treating a CNS
disorder provided herein include intranasally administering a
medium dose of pooled human IgG. In one embodiment, a medium dose
of pooled human IgG is from about 10 mg/kg IgG to about 100 mg/kg
IgG. In specific embodiments, a medium dose of pooled human IgG is
about 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg,
16 mg/kg, 17 mg/kg, 18 mg/kg, 19 mg/kg, 20 mg/kg, 21 mg/kg, 22
mg/kg, 23 mg/kg, 24 mg/kg, 25 mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg,
29 mg/kg, 30 mg/kg, 31 mg/kg, 32 mg/kg, 33 mg/kg, 34 mg/kg, 35
mg/kg, 36 mg/kg, 37 mg/kg, 38 mg/kg, 39 mg/kg, 40 mg/kg, 41 mg/kg,
42 mg/kg, 43 mg/kg, 44 mg/kg, 45 mg/kg, 46 mg/kg, 47 mg/kg, 48
mg/kg, 49 mg/kg, 50 mg/kg, 55, 60, 65, 70, 75, 80, 85, 90, 95, or
100 mg/kg IgG. In yet other embodiments, a medium dose of pooled
human IgG is from 10 mg/kg to 100 mg/kg, 25 mg/kg to 100 mg/kg, 50
mg/kg to 100 mg/kg, 75 mg/kg to 100 mg/kg, 10 mg/kg to 75 mg/kg, 25
mg/kg to 75 mg/kg, 50 mg/kg to 75 mg/kg, 10 mg/kg to 50 mg/kg, 25
mg/kg to 50 mg/kg, or 10 mg/kg to 25 mg/kg IgG.
[0197] In some embodiments, the methods for treating a CNS disorder
provided herein include intranasally administering a high dose of
pooled human IgG. In one embodiment, a high dose of pooled human
IgG is from about 100 mg/kg IgG to about 400 mg/kg IgG. In specific
embodiments, a high dose of pooled human IgG is about 100 mg/kg,
110, 120 mg/kg, 130 mg/kg, 140 mg/kg, 150 mg/kg, 175 mg/kg, 200
mg/kg, 225 mg/kg, 250 mg/kg, 275 mg/kg, 300 mg/kg, 325 mg/kg, 350
mg/kg, 375 mg/kg, 400 mg/kg, or higher. In yet other embodiments, a
high dose of pooled human IgG is from 100 mg/kg to 400 mg/kg, 150
mg/kg to 400 mg/kg, 200 mg/kg to 400 mg/kg, 250 mg/kg to 400 mg/kg,
300 mg/kg to 400 mg/kg, 350 mg/kg to 400 mg/kg, 100 mg/kg to 300
mg/kg, 150 mg/kg to 300 mg/kg, 200 mg/kg to 300 mg/kg, 250 mg/kg to
300 mg/kg, 100 mg/kg to 200 mg/kg, 150 mg/kg to 200 mg/kg, or 100
mg/kg to 150 mg/kg IgG.
[0198] In some embodiments, pooled human IgG is administered at a
set dosage, regardless of the weight of the subject. Without being
bound by theory, unlike intravenous administration, the final
concentration of IgG in the brain should be independent of total
body weight when administered intranasally since the therapeutic
will travel directly from the nose to the brain. Accordingly, a
standard dose of intranasal pooled human IgG, which is independent
of body weight, may simplify the process of dosing individual
subjects.
[0199] Accordingly, in one embodiment, the methods described herein
include intranasal administration of a fixed dose of pooled human
IgG of from about 50 mg to about 10 g. In some embodiments, the
fixed dose of IgG is about 50 mg, 75 mg, 100 mg, 125 mg, 150 mg,
175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 350 mg, 400 mg, 450
mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg,
900 mg, 950 mg, 1.0 g, 1.25 g, 1.5 g, 1.75 g, 2.0 g, 2.5 g, 3.0 g,
3.5 g, 4.0 g, 4.5 g, 5.0 g, 5.5 g, 6.0 g, 6.5 g, 7.0 g, 7.5 g, 8.0
g, 8.5 g, 9.0 g, 9.5 g, 10.0 g, or more IgG. In other embodiments,
the methods described herein include intranasal administration of
from 50 mg to 5 g, 100 mg to 5 g, 250 mg to 5 g, 500 mg to 5 g, 750
mg to 5 g, 1 g to 5 g, 2.5 g to 5 g, 50 mg to 2.5 g, 100 mg to 2.5
g, 250 mg to 2.5 g, 500 mg to 2.5 g, 750 mg to 2.5 g, 1 g to 2.5 g,
50 mg to 1 g, 100 mg to 1 g, 250 mg to 1 g, 500 mg to 1 g, 750 mg
to 1 g, 50 mg to 500 mg, 100 mg to 500 mg, 250 mg to 500 mg, 50 mg
to 250 mg, 100 mg to 250 mg, or 50 mg to 100 mg pooled human
IgG.
[0200] Depending upon the CNS disorder being treated and the
progression of the disorder in the subject, the pooled human IgG
compositions described herein are intranasally administered to a
subject anywhere from several times daily to monthly. For example,
a subject diagnosed with a CNS disorder in an early stage of
progression may require only a low dosage and/or low dosage
frequency, while a subject diagnosed with a CNS disorder in a late
stage of progression may require a high dose and/or high dosage
frequency. In yet another embodiment, a subject having a high
likelihood of developing a CNS disorder may also be prescribed a
low dose and/or low dosing frequency as a prophylactic treatment or
to delay onset of symptoms associated with a CNS disorder. For
example, a subject with a familial history of an age-related
dementia (e.g., Alzheimer's disease) may be intranasally
administered pooled human IgG at a low dosage and/or low frequency
to delay the onset of symptoms associated with the age-related
dementia. A skilled physician will readily be able to determine an
appropriate dosage and dosing frequency for a subject diagnosed
with or having a high likelihood of developing a CNS disorder.
[0201] In one embodiment, where the progression of a particular CNS
disorder in a subject requires frequent dosing, the methods
provided herein for treating a disorder of the central nervous
system include administering a composition comprising pooled human
immunoglobulin G (IgG) to the subject at least once a week. In
other embodiments, the method includes administering a composition
comprising pooled human immunoglobulin G (IgG) to the subject at
least two, three, four, five, or six times a week. In yet another
embodiment, the method includes administering a composition
comprising pooled human immunoglobulin G (IgG) to the subject at
least once daily. In other embodiments, the method includes
administering a composition comprising pooled human immunoglobulin
G (IgG) to the subject at least two, three, four, five, or more
times daily. In a specific embodiment, the CNS disorder is an
age-related dementia, Parkinson's disease, or multiple sclerosis.
In a more specific embodiment, the CNS disorder is Alzheimer's
disease.
[0202] In another embodiment, where the progression of a particular
CNS disorder in a subject requires less frequent dosing, the
methods provided herein for treating a disorder of the central
nervous system include administering a composition comprising
pooled human immunoglobulin G (IgG) to the subject at least once a
month. In other embodiments, the method includes administering a
composition comprising pooled human immunoglobulin G (IgG) to the
subject at least two, three, four, five, six, or more times a
month. In yet another embodiment, the method includes administering
a composition comprising pooled human immunoglobulin G (IgG) to the
subject at least once daily. In other embodiments, the method
includes administering a composition comprising pooled human
immunoglobulin G (IgG) to the subject at least two, three, four,
five, or more times daily. In a specific embodiment, the CNS
disorder is an age-related dementia, Parkinson's disease, or
multiple sclerosis. In a more specific embodiment, the CNS disorder
is Alzheimer's disease.
[0203] In certain embodiments, the composition can be administered
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 times a month.
The composition can be administered between equally spaced days of
the month, for example, on the 1.sup.st and the 15.sup.th of each
month. Alternatively, the composition can be administered in block
dosing at the beginning, end, or middle of the month. For example,
the composition can be administered only on the 1.sup.st,
1.sup.st-2.sup.nd, 1 st-3.sup.rd 1.sup.st-4.sup.th,
1.sup.st-5.sup.th, 1.sup.st-6.sup.th, or 1.sup.st-7.sup.th days of
the month. Similar dosing schemes can be administered toward the
middle or end of the month.
[0204] In certain embodiments the dosing can change between dosing
days. For example, on the first day of dosing a subject can receive
10 mg/kg IgG and on the second day of dosing the subject can
receive 20 mg/kg IgG. Similarly, a subject who is administered two
or more doses per day of intranasal IgG can receive two different
doses. For example, the first dose of the day can be 10 mg/kg IgG
and the second dose of the day can be 5 mg/kg IgG.
[0205] In certain embodiments, the methods provided herein for the
treatment of a CNS disorder include intranasally administering from
0.05 mg/kg to 50 mg/kg pooled human immunoglobulin to a subject in
need thereof daily. In other embodiments, the methods provided
herein for the treatment of a CNS disorder include intranasally
administering pooled human IgG in a dosage/frequency combination
selected from variations 1 to 816 found in Table 1 and Table 2.
TABLE-US-00001 TABLE 1 Exemplary combinations of dosage and
frequency for methods of treating a CNS disorder by intranasal
administration of pooled human IgG. One Two Three Four One Two
Three Every Time Times Times Times Time Times Times Other Monthly
Monthly Monthly Monthly Weekly Weekly Weekly Day 0.05 Var. 1 Var.
52 Var. 103 Var. 154 Var. 205 Var. 256 Var. 307 Var. 358 mg/kg 0.1
Var. 2 Var. 53 Var. 104 Var. 155 Var. 206 Var. 257 Var. 308 Var.
359 mg/kg 0.25 Var. 3 Var. 54 Var. 105 Var. 156 Var. 207 Var. 258
Var. 309 Var. 360 mg/kg 0.5 Var. 4 Var. 55 Var. 106 Var. 157 Var.
208 Var. 259 Var. 310 Var. 361 mg/kg 0.75 Var. 5 Var. 56 Var. 107
Var. 158 Var. 209 Var. 260 Var. 311 Var. 362 mg/kg 1.0 Var. 6 Var.
57 Var. 108 Var. 159 Var. 210 Var. 261 Var. 312 Var. 363 mg/kg 1.5
Var. 7 Var. 58 Var. 109 Var. 160 Var. 211 Var. 262 Var. 313 Var.
364 mg/kg 2.0 Var. 8 Var. 59 Var. 110 Var. 161 Var. 212 Var. 263
Var. 314 Var. 365 mg/kg 2.5 Var. 9 Var. 60 Var. 111 Var. 162 Var.
213 Var. 264 Var. 315 Var. 366 mg/kg 3.0 Var. 10 Var. 61 Var. 112
Var. 163 Var. 214 Var. 265 Var. 316 Var. 367 mg/kg 3.5 Var. 11 Var.
62 Var. 113 Var. 164 Var. 215 Var. 266 Var. 317 Var. 368 mg/kg 4.0
Var. 12 Var. 63 Var. 114 Var. 165 Var. 216 Var. 267 Var. 318 Var.
369 mg/kg 4.5 Var. 13 Var. 64 Var. 115 Var. 166 Var. 217 Var. 268
Var. 319 Var. 370 mg/kg 5.0 Var. 14 Var. 65 Var. 116 Var. 167 Var.
218 Var. 269 Var. 320 Var. 371 mg/kg 6.0 Var. 15 Var. 66 Var. 117
Var. 168 Var. 219 Var. 270 Var. 321 Var.372 mg/kg 7.0 Var. 16 Var.
67 Var. 118 Var. 169 Var. 220 Var. 271 Var. 322 Var. 373 mg/kg 8.0
Var. 17 Var. 68 Var. 119 Var. 170 Var. 221 Var. 272 Var. 323 Var.
374 mg/kg 9.0 Var. 18 Var. 69 Var. 120 Var. 171 Var. 222 Var. 273
Var. 324 Var.375 mg/kg 10 Var. 19 Var. 70 Var. 121 Var. 172 Var.
223 Var. 274 Var. 325 Var. 376 mg/kg 11 Var. 20 Var. 71 Var. 122
Var. 173 Var. 224 Var. 275 Var. 326 Var.377 mg/kg 12 Var. 21 Var.
72 Var. 123 Var. 174 Var. 225 Var. 276 Var. 327 Var. 378 mg/kg 13
Var. 22 Var. 73 Var. 124 Var. 175 Var. 226 Var. 277 Var. 328 Var.
379 mg/kg 14 Var. 23 Var. 74 Var. 125 Var. 176 Var. 227 Var. 278
Var. 329 Var. 380 mg/kg 15 Var. 24 Var. 75 Var. 126 Var. 177 Var.
228 Var. 279 Var. 330 Var. 381 mg/kg 16 Var. 25 Var. 76 Var. 127
Var. 178 Var. 229 Var. 280 Var. 331 Var. 382 mg/kg 17 Var. 26 Var.
77 Var. 128 Var. 179 Var. 230 Var. 281 Var. 332 Var. 383 mg/kg 18
Var. 27 Var. 78 Var. 129 Var. 180 Var. 231 Var. 282 Var. 333 Var.
384 mg/kg 19 Var. 28 Var. 79 Var. 130 Var. 181 Var. 232 Var. 283
Var. 334 Var. 385 mg/kg 20 Var. 29 Var. 80 Var. 131 Var. 182 Var.
233 Var. 284 Var. 335 Var. 386 mg/kg 22.5 Var. 30 Var. 81 Var. 132
Var. 183 Var. 234 Var. 285 Var. 336 Var. 387 mg/kg 25 Var. 31 Var.
82 Var. 133 Var. 184 Var. 235 Var. 286 Var. 337 Var. 388 mg/kg 27.5
Var. 32 Var. 83 Var. 134 Var. 185 Var. 236 Var. 287 Var. 338 Var.
389 mg/kg 30 Var. 33 Var. 84 Var. 135 Var. 186 Var. 237 Var. 288
Var. 339 Var. 390 mg/kg 32.5 Var. 34 Var. 85 Var. 136 Var. 187 Var.
238 Var. 289 Var. 340 Var. 391 mg/kg 35 Var. 35 Var. 86 Var. 137
Var. 188 Var. 239 Var. 290 Var. 341 Var. 392 mg/kg 37.5 Var. 36
Var. 87 Var. 138 Var. 189 Var. 240 Var. 291 Var. 342 Var. 393 mg/kg
40 Var. 37 Var. 88 Var. 139 Var. 190 Var. 241 Var. 292 Var. 343
Var. 394 mg/kg 45 Var. 38 Var. 89 Var. 140 Var. 191 Var. 242 Var.
293 Var. 344 Var. 395 mg/kg 50 Var. 39 Var. 90 Var. 141 Var. 192
Var. 243 Var. 294 Var. 345 Var. 396 mg/kg 0.5-40 Var. 40 Var. 91
Var. 142 Var. 193 Var. 244 Var. 295 Var. 346 Var. 397 mg/kg 0.5-30
Var. 41 Var. 92 Var. 143 Var. 194 Var. 245 Var. 296 Var. 347 Var.
398 mg/kg 0.5-20 Var. 42 Var. 93 Var. 144 Var. 195 Var. 246 Var.
297 Var. 348 Var. 399 mg/kg 0.5-20 Var. 43 Var. 94 Var. 145 Var.
196 Var. 247 Var. 298 Var. 349 Var. 400 mg/kg 0.5-10 Var. 44 Var.
95 Var. 146 Var. 197 Var. 248 Var. 299 Var. 350 Var. 401 mg/kg
0.5-5 Var. 45 Var. 96 Var. 147 Var. 198 Var. 249 Var. 300 Var. 351
Var. 402 mg/kg 1-20 Var. 46 Var. 97 Var. 148 Var. 199 Var. 250 Var.
301 Var. 352 Var. 403 mg/kg 1-15 Var. 47 Var. 98 Var. 149 Var. 200
Var. 251 Var. 302 Var. 353 Var. 404 mg/kg 1-10 Var. 48 Var. 99 Var.
150 Var. 201 Var. 252 Var. 303 Var. 354 Var. 405 mg/kg 1-5 Var. 49
Var. 100 Var. 151 Var. 202 Var. 253 Var. 304 Var. 355 Var. 406
mg/kg 2-10 Var. 50 Var. 101 Var. 152 Var. 203 Var. 254 Var. 305
Var. 356 Var. 407 mg/kg 2-5 Var. 51 Var. 102 Var. 153 Var. 204 Var.
255 Var. 306 Var. 357 Var. 408 mg/kg * Var. = variation
TABLE-US-00002 TABLE 2 Exemplary combinations of dosage and
frequency for methods of treating a CNS disorder by intranasal
administration of pooled human IgG. Four Five Six One Two Three
Four Five Times Times Times Time Times Times Times Times Weekly
Weekly Weekly Daily Daily Daily Daily Daily 0.05 Var. 409 Var. 460
Var. 511 Var. 562 Var. 613 Var. 664 Var. 715 Var. 766 mg/kg 0.1
Var. 410 Var. 461 Var. 512 Var. 563 Var. 614 Var. 665 Var. 716 Var.
767 mg/kg 0.25 Var. 411 Var. 462 Var. 513 Var. 564 Var. 615 Var.
666 Var. 717 Var. 768 mg/kg 0.5 Var. 412 Var. 463 Var. 514 Var. 565
Var. 616 Var. 667 Var. 718 Var. 769 mg/kg 0.75 Var. 413 Var. 464
Var. 515 Var. 566 Var. 617 Var. 668 Var. 719 Var. 770 mg/kg 1.0
Var. 414 Var. 465 Var. 516 Var. 567 Var. 618 Var. 669 Var. 720 Var.
771 mg/kg 1.5 Var. 415 Var. 466 Var. 517 Var. 568 Var. 619 Var. 670
Var. 721 Var. 772 mg/kg 2.0 Var. 416 Var. 467 Var. 518 Var. 569
Var. 620 Var. 671 Var. 722 Var. 773 mg/kg 2.5 Var. 417 Var. 468
Var. 519 Var. 570 Var. 621 Var. 672 Var. 723 Var. 774 mg/kg 3.0
Var. 418 Var. 469 Var. 520 Var. 571 Var. 622 Var. 673 Var. 724 Var.
775 mg/kg 3.5 Var. 419 Var. 470 Var. 521 Var. 572 Var. 623 Var. 674
Var. 725 Var. 776 mg/kg 4.0 Var. 420 Var. 471 Var. 522 Var. 573
Var. 624 Var. 675 Var. 726 Var. 777 mg/kg 4.5 Var. 421 Var. 472
Var. 523 Var. 574 Var. 625 Var. 676 Var. 727 Var. 778 mg/kg 5.0
Var. 422 Var. 473 Var. 524 Var. 575 Var. 626 Var. 677 Var. 728 Var.
779 mg/kg 6.0 Var. 423 Var. 474 Var. 525 Var. 576 Var. 627 Var. 678
Var. 729 Var. 780 mg/kg 7.0 Var. 424 Var. 475 Var. 526 Var. 577
Var. 628 Var. 679 Var. 730 Var. 781 mg/kg 8.0 Var. 425 Var. 476
Var. 527 Var. 578 Var. 629 Var. 680 Var. 731 Var. 782 mg/kg 9.0
Var. 426 Var. 477 Var. 528 Var. 579 Var. 630 Var. 681 Var. 732 Var.
783 mg/kg 10 Var. 427 Var. 478 Var. 529 Var. 580 Var. 631 Var. 682
Var. 733 Var. 784 mg/kg 11 Var. 428 Var. 479 Var. 530 Var. 581 Var.
632 Var. 683 Var. 734 Var. 785 mg/kg 12 Var. 429 Var. 480 Var. 531
Var. 582 Var. 633 Var. 684 Var. 735 Var. 786 mg/kg 13 Var. 430 Var.
481 Var. 532 Var. 583 Var. 634 Var. 685 Var. 736 Var. 787 mg/kg 14
Var. 431 Var. 482 Var. 533 Var. 584 Var. 635 Var. 686 Var. 737 Var.
788 mg/kg 15 Var. 432 Var. 483 Var. 534 Var. 585 Var. 636 Var. 687
Var. 738 Var. 789 mg/kg 16 Var. 433 Var. 484 Var. 535 Var. 586 Var.
637 Var. 688 Var. 739 Var. 790 mg/kg 17 Var. 434 Var. 485 Var. 536
Var. 587 Var. 638 Var. 689 Var. 740 Var. 791 mg/kg 18 Var. 435 Var.
486 Var. 537 Var. 588 Var. 639 Var. 690 Var. 741 Var. 792 mg/kg 19
Var. 436 Var. 487 Var. 538 Var. 589 Var. 640 Var. 691 Var. 742 Var.
793 mg/kg 20 Var. 437 Var. 488 Var. 539 Var. 590 Var. 641 Var. 692
Var. 743 Var. 794 mg/kg 22.5 Var. 438 Var. 489 Var. 540 Var. 591
Var. 642 Var. 693 Var. 744 Var. 795 mg/kg 25 Var. 439 Var. 490 Var.
541 Var. 592 Var. 643 Var. 694 Var. 745 Var. 796 mg/kg 27.5 Var.
440 Var. 491 Var. 542 Var. 593 Var. 644 Var. 695 Var. 746 Var. 797
mg/kg 30 Var. 441 Var. 492 Var. 543 Var. 594 Var. 645 Var. 696 Var.
747 Var. 798 mg/kg 32.5 Var. 442 Var. 493 Var. 544 Var. 595 Var.
646 Var. 697 Var. 748 Var. 799 mg/kg 35 Var. 443 Var. 494 Var. 545
Var. 596 Var. 647 Var. 698 Var. 749 Var. 800 mg/kg 37.5 Var. 444
Var. 495 Var. 546 Var. 597 Var. 648 Var. 699 Var. 750 Var. 801
mg/kg 40 Var. 445 Var. 496 Var. 547 Var. 598 Var. 649 Var. 700 Var.
751 Var. 802 mg/kg 45 Var. 446 Var. 497 Var. 548 Var. 599 Var. 650
Var. 701 Var. 752 Var. 803 mg/kg 50 Var. 447 Var. 498 Var. 549 Var.
600 Var. 651 Var. 702 Var. 753 Var. 804 mg/kg 0.5-40 Var. 448 Var.
499 Var. 550 Var. 601 Var. 652 Var. 703 Var. 754 Var. 805 mg/kg
0.5-30 Var. 449 Var. 500 Var. 551 Var. 602 Var. 653 Var. 704 Var.
755 Var. 806 mg/kg 0.5-20 Var. 450 Var. 501 Var. 552 Var. 603 Var.
654 Var. 705 Var. 756 Var. 807 mg/kg 0.5-20 Var. 451 Var. 502 Var.
553 Var. 604 Var. 655 Var. 706 Var. 757 Var. 808 mg/kg 0.5-10 Var.
452 Var. 503 Var. 554 Var. 605 Var. 656 Var. 707 Var. 758 Var. 809
mg/kg 0.5-5 Var. 453 Var. 504 Var. 555 Var. 606 Var. 657 Var. 708
Var. 759 Var. 810 mg/kg 1-20 Var. 454 Var. 505 Var. 556 Var. 607
Var. 658 Var. 709 Var. 760 Var. 811 mg/kg 1-15 Var. 455 Var. 506
Var. 557 Var. 608 Var. 659 Var. 710 Var. 761 Var. 812 mg/kg 1-10
Var. 456 Var. 507 Var. 558 Var. 609 Var. 660 Var. 711 Var. 762 Var.
813 mg/kg 1-5 Var. 457 Var. 508 Var. 559 Var. 610 Var. 661 Var. 712
Var. 763 Var. 814 mg/kg 2-10 Var. 458 Var. 509 Var. 560 Var. 611
Var. 662 Var. 713 Var. 764 Var. 815 mg/kg 2-5 Var. 459 Var. 510
Var. 561 Var. 612 Var. 663 Var. 714 Var. 765 Var. 816 mg/kg * Var.
= variation
[0206] Formulation
[0207] Pharmaceutical compositions of pooled human immunoglobulin G
described herein can be prepared in accordance with methods well
known and routinely practiced in the art. See, e.g., Remington: The
Science and Practice of Pharmacy, Mack Publishing Co., 20.sup.th
ed., 2000; and Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.
Pharmaceutical compositions are preferably manufactured under GMP
conditions. Typically, a therapeutically effective dose or
efficacious dose of the pooled human IgG preparation is employed in
the pharmaceutical compositions described herein. The
pharmaceutical composition can be formulated into dosage forms by
conventional methods known to those of skill in the art. Dosage
regimens are adjusted to provide the optimum desired response
(e.g., a therapeutic response). For example, a single bolus may be
administered, several divided doses may be administered over time
or the dose may be proportionally reduced or increased as indicated
by the exigencies of the therapeutic situation. It can be
advantageous to formulate parenteral compositions in dosage unit
form for ease of administration and uniformity of dosage. Dosage
unit form as used herein refers to physically discrete units suited
as unitary dosages for the subjects to be treated; each unit
contains a predetermined quantity of active compound calculated to
produce the desired therapeutic effect in association with the
required pharmaceutical carrier.
[0208] Actual dosage levels can be varied so as to obtain an amount
of the active ingredient which is effective to achieve the desired
therapeutic response for a particular patient without being toxic
to the patient. A physician can start doses of the pharmaceutical
composition at levels lower than that required to achieve the
desired therapeutic effect and gradually increase the dosage until
the desired effect is achieved. In general, effective doses vary
depending upon many different factors, including the specific
disease or condition to be treated, its severity, physiological
state of the patient, other medications administered, and whether
treatment is prophylactic or therapeutic.
[0209] In one embodiment, a therapeutic composition of pooled human
IgG formulated for intranasal administration does not contain a
permeability enhancer. Permeability enhancers facilitate the
transport of molecules through the mucosa, including the mucous,
and the nasal epithelium. Non-limiting examples of absorption
enhancers include mucoadhesives, ciliary beat inhibitors, mucous
fluidizers, membrane fluidizers, and tight junction modulators.
Specific non-limiting examples include bile salts, phospholipids,
sodium glycyrrhetinate, sodium caprate, ammonium tartrate, gamma.
aminolevulinic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and oxaloacetic acid.
[0210] In addition to pooled human IgG, the pharmaceutical
compositions provided herein include one or more stabilizing
agents. In a specific embodiment, the stabilizing agent is a
buffering agent suitable for intranasal administration.
Non-limiting examples of buffering agents suitable for formulating
the pooled human IgG compositions provided herein include an amino
acid (e.g., glycine, histidine, or proline) a salt (e.g., citrate,
phosphate, acetate, glutamate, tartrate, benzoate, lactate,
gluconate, malate, succinate, formate, propionate, or carbonate),
or any combination thereof adjusted to an appropriate pH.
Generally, the buffering agent will be sufficient to maintain a
suitable pH in the formulation for an extended period of time. In a
particular embodiment, the buffering agent is sufficient to
maintain a pH of 4 to 7.5. In a specific embodiment, the buffering
agent is sufficient to maintain a pH of approximately 4.0, or
approximately 4.5, or approximately 5.0, or approximately 5.5, or
approximately 6.0, or approximately 6.5, or approximately 7.0, or
approximately 7.5.
[0211] In a particular embodiment, a pooled human IgG composition
described herein for the treatment of a CNS disorder via intranasal
administration contains a stabilizing amount of an amino acid. In
certain embodiments, a stabilizing amount of an amino acid is from
about 25 mM to about 500 mM
[0212] In a particular embodiment, the stabilizing agent employed
in the pooled human IgG compositions provided herein is an amino
acid. Non-limiting examples of amino acids include isoleucine,
alanine, leucine, asparagine, lysine, aspartic acid, methionine,
cysteine, phenylalanine, glutamic acid, threonine, glutamine,
tryptophan, glycine, valine, proline, selenocysteine, serine,
tyrosine, arginine, histidine, ornithine, taurine, combinations
thereof, and the like. In one embodiment, the stabilizing amino
acids include arginine, histidine, lysine, serine, proline,
glycine, alanine, threonine, and a combination thereof. In a
preferred embodiment, the amino acid is glycine. In another
preferred embodiment, the amino acid is proline. In yet another
preferred embodiment, the amino acid is histidine.
[0213] For purposes of stabilizing the compositions provided
herein, the buffering agent (e.g., glycine, histidine, or proline)
will typically be added to the formulation (or to a solution from
which a dry powder composition is to be prepared) at a
concentration from 5 mM to 0.75 M. In one embodiment, at least 100
mM of the buffering agent is added to the formulation. In another
embodiment, at least 200 mM of the buffering agent is added to the
formulation. In yet another embodiment, at least 250 mM of the
buffering agent is added to the formulation. In yet other
embodiments, the formulations provided herein contains at least 25
mM, 50 mM, 75 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM,
400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, or
more of the buffering agent. In a specific embodiment, the
buffering agent is glycine.
[0214] In one embodiment, the concentration of buffering agent
(e.g., glycine, histidine, or proline) in the formulation (or in
the solution from which a dry powder composition is to be prepared)
is at or about from 5 mM to 500 mM. In certain embodiments, the
concentration of the buffering agent in the formulation will be at
or about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 50 mM, 75 mM, 100 mM,
125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325
mM, 350 mM, 375 mM, 400 mM, 425 mM, 450 mM, 475 mM, 500 mM or
higher. In a specific embodiment, the buffering agent is
glycine.
[0215] In yet other embodiments, the concentration of the buffering
agent (e.g., glycine, histidine, or proline) in formulation (or in
the solution from which a dry powder composition is to be prepared)
is from 50 mM to 500 mM, 100 mM to 500 mM, 200 mM to 500 mM, 250 mM
to 500 mM, 300 mM to 500 mM, 50 mM to 300 mM, 100 mM to 300 mM, 200
mM to 300 mM, or 225 mM to 275 mM. In yet other specific
embodiments, the concentration of the buffering agent (e.g.,
glycine, histidine, or proline) in formulations provided herein is
250.+-.50 mM, 250.+-.40 mM, 250.+-.30 mM, 250.+-.25 mM, 250.+-.20
mM, 250.+-.15 mM, 250.+-.10 mM, 250.+-.5 mM, or 250 mM.
[0216] In some embodiments, the pooled human immunoglobulins are
formulated with between 100 mM and 400 mM histidine; no more than
10 mM of an alkali metal cation; and a pH between 5.0 and 7.0.
[0217] In some embodiments of the pooled human immunoglobulin
histidine formulation, the concentration of histidine is between 5
mM and 500 mM. In another embodiment, the concentration of
histidine in the formulation will be between 100 mM and 400 mM. In
another embodiment, the concentration of histidine in the
formulation will be between 200 mM and 300 mM. In another
embodiment, the concentration of histidine in the formulation will
be between 225 mM and 275 mM. In another embodiment, the
concentration of histidine in the formulation will be between 240
mM and 260 mM. In a particular embodiment, the concentration of
histidine will be 250 mM. In certain other embodiments, the
concentration of histidine in the formulation will be 5.+-.0.5 mM,
10.+-.1 mM, 15.+-.1.5 mM, 20.+-.2 mM, 25.+-.2.5 mM, 50.+-.5 mM,
75.+-.7.5 mM, 100.+-.10 mM, 125.+-.12.5 mM, 150.+-.15 mM,
175.+-.17.5 mM, 200.+-.20 mM, 225.+-.22.5 mM, 250.+-.25 mM,
275.+-.27.5 mM, 300.+-.30 mM, 325.+-.32.5 mM, 350.+-.35 mM,
375.+-.37.5 mM, 400.+-.40 mM, 425.+-.42.5 mM, 450.+-.45 mM,
475.+-.47.5 mM, 500.+-.50 mM or higher. In yet other embodiments,
the concentration of histidine in the formulation will be 5 mM, 10
mM, 15 mM, 20 mM, 25 mM, 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 175
mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 325 mM, 350 mM, 375 mM,
400 mM, 425 mM, 450 mM, 475 mM, 500 mM or higher.
[0218] In some embodiments of the pooled human immunoglobulin
histidine formulation, the pH of the histidine formulation is from
4.0 to 7.5. In some embodiments, the pH of the histidine
formulation is from 4.0 to 6.0. In some embodiments, the pH of the
histidine formulation is from 4.0 to 4.5. In some embodiments, the
pH of the histidine formulation is from 4.5 to 5.0. In some
embodiments, the pH of the histidine formulation is from 4.0 to
5.5. In some embodiments, the pH of the histidine formulation is
from 4.0 to 6.5. In some embodiments, the pH of the histidine
formulation is from 4.0 to 7.0. In some embodiments, the pH of the
histidine formulation is from 4.5 to 6.0. In some embodiments, the
pH of the histidine formulation is from 4.5 to 6.5. In some
embodiments, the pH of the histidine formulation is from 4.5 to
7.0. In some embodiments, the pH of the histidine formulation is
from 4.5 to 7.5. In some embodiments, the pH of the histidine
formulation is from 5.5 to 7.0. In some embodiments, the pH of the
histidine formulation is from 6.0 to 7.0. In some embodiments, the
pH of the histidine formulation is from 6.5 to 7.0. In some
embodiments, the pH of the histidine formulation is from 5.0 to
6.5. In some embodiments, the pH of the histidine formulation is
from 5.0 to 7.0. In some embodiments, the pH of the histidine
formulation is from 5.5 to 6.5. In some embodiments, the pH of the
histidine formulation is from 6.0 to 6.5. In some embodiments, the
pH of the histidine formulation is from 5.0 to 6.0. In some
embodiments, the pH of the histidine formulation is from 5.5 to
6.0. In some embodiments, the pH of the histidine formulation is
from 5.0 to 5.5. In some embodiments, the pH of the histidine
formulation is from 7.0 to 7.5. In some embodiments, the pH of the
histidine formulation is from 6.0 to 7.5. In some embodiments, the
pH of the histidine formulation is from 5.5 to 7.5. In some
embodiments, the pH of the histidine formulation is from 5.0 to
7.5. In some embodiments, the pH of the histidine formulation is
5.0.+-.0.2, 5.1.+-.0.2, 5.2.+-.0.2, 5.3.+-.0.2, 5.4.+-.0.2,
5.5.+-.0.2, 5.6.+-.0.2, 5.7.+-.0.2, 5.8.+-.0.2, 5.9.+-.0.2,
6.0.+-.0.2, 6.1.+-.0.2, 6.2.+-.0.2, 6.3.+-.0.2, 6.4.+-.0.2,
6.5.+-.0.2, 6.6.+-.0.2, 6.7.+-.0.2, 6.8.+-.0.2, 6.9.+-.0.2, or
7.0.+-.0.2. In some embodiments, the pH of the histidine
formulation is 5.0.+-.0.1, 5.1.+-.0.1, 5.2.+-.0.1, 5.3.+-.0.1,
5.4.+-.0.1, 5.5.+-.0.1, 5.6.+-.0.1, 5.7.+-.0.1, 5.8.+-.0.1,
5.9.+-.0.1, 6.0.+-.0.1, 6.1.+-.0.1, 6.2.+-.0.1, 6.3.+-.0.1,
6.4.+-.0.1, 6.5.+-.0.1, 6.6.+-.0.1, 6.7.+-.0.1, 6.8.+-.0.1,
6.9.+-.0.1, or 7.0.+-.0.1.
[0219] In one embodiment, the pooled human IgG compositions
described herein for the treatment of a CNS disorder via intranasal
administration is formulated at a pH from about 4.0 to about 7.0.
In particular embodiments, a pooled human IgG compositions is
formulated at a pH of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,
4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0,
6.1, 6.2, 6.3, 6.4, 6.5, 6.7, 6.8, 6.9, or 7.0. In other
embodiments, a pooled human IgG composition is formulated at a pH
from 4.0 to 6.5, 4.0 to 6.0, 4.0 to 5.5, 4.0 to 5.0, 4.0 to 4.5,
4.5 to 6.5, 4.5 to 6.0, 4.5 to 5.5, 4.5 to 5.0. In yet other
embodiments, a pooled human IgG composition is formulated at a pH
of 4.8.+-.0.5, 4.8.+-.0.4, 4.8.+-.0.3, 4.8.+-.0.2, 4.8.+-.0.1, or
about 4.8.
[0220] In one embodiment, liquid compositions of pooled human IgG
formulated for intranasal administration are provided for the
treatment of CNS disorders (e.g., Alzheimer's disease, Parkinson's
disease, and multiple sclerosis). In a specific embodiment, the
liquid composition is an aqueous composition. In a particular
embodiment, an aqueous therapeutic composition formulated for
intranasal administration provided herein consists essentially of a
buffering agent and pooled human IgG.
[0221] In one embodiment, a liquid composition formulated for
intranasal administration contains from about 1.0 g pooled human
IgG per liter (g/L IgG) to about 250 g/L IgG. In other embodiments,
the liquid composition formulated for intranasal administration
contains about 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8
g/L, 9 g/L, 10 g/L, 12.5 g/L, 15 g/L, 17.5 g/L, 20 g/L, 25 g/L, 30
g/L, 35 g/L, 40 g/L, 45 g/L, 50 g/L, 55 g/L, 60 g/L, 65 g/L, 70
g/L, 75 g/L, 80 g/L, 85 g/L, 90 g/L, 95 g/L, 100 g/L, 110 g/L, 120
g/L, 130 g/L, 140 g/L, 150 g/L, 160 g/L, 170 g/L, 180 g/L, 190 g/L,
200 g/L, 210 g/L, 220 g/L, 230 g/L, 240 g/L, 250 g/L, or higher
concentration of pooled human IgG. In certain embodiments, the
liquid composition formulated for intranasal administration
contains from 5.0 g/L to 250 g/L, 10 g/L to 250 g/L, 20 g/L to 250
g/L, 30 g/L to 250 g/L, 40 g/L to 250 g/L, 50 g/L to 250 g/L, 60
g/L to 250 g/L, 70 g/L to 250 g/L, 80 g/L to 250 g/L, 90 g/L to 250
g/L, 100 g/L to 250 g/L, 125 g/L to 250 g/L, 150 g to 250 g/L, 175
g/L to 250 g/L, 200 g/L to 250 g/L IgG.
[0222] In certain embodiments, the methods for treating a CNS
disorder provided herein include intranasally administering a
liquid composition containing a low concentration of pooled human
IgG. In one embodiment, a low concentration of pooled human IgG
contains from 1.0 g/L to 100 g/L, 5.0 g/L to 100 g/L, 10 g/L to 100
g/L, 20 g/L to 100 g/L, 30 g/L to 100 g/L, 40 g/L to 100 g/L, 50
g/L to 100 g/L, 60 g/L to 100 g/L, 70 g/L to 100 g/L, 75 g/L to 100
g/L, 80 g/L to 100 g/L, 1.0 g/L to 50 g/L, 5.0 g/L to 50 g/L, 10
g/L to 50 g/L, 20 g/L to 50 g/L, 30 g/L to 50 g/L, or 40 g/L to 50
g/L IgG.
[0223] In certain embodiments, the methods for treating a CNS
disorder provided herein include intranasally administering a
liquid composition containing an intermediate concentration of
pooled human IgG. In one embodiment, an intermediate concentration
of pooled human IgG contains from 75 g/L to 200 g/L, 100 g/L to 200
g/L, 110 g/L to 200 g/L, 120 g/L to 200 g/L, 130 g/L to 200 g/L,
140 g/L to 200 g/L, 150 g/L to 200 g/L, 160 g/L to 200 g/L, 170 g/L
to 200 g/L, 175 g/L to 200 g/L, 180 g/L to 200 g/L, 75 g/L to 150
g/L, 100 g/L to 150 g/L, 110 g/L to 150 g/L, 120 g/L to 150 g/L,
130 g/L to 150 g/L, or 140 g/L to 150 g/L IgG.
[0224] In certain embodiments, the methods for treating a CNS
disorder provided herein include intranasally administering a
liquid composition containing a high concentration of pooled human
IgG. In one embodiment, a high concentration of pooled human IgG
contains from 175 g/L to 250 g/L, 200 g/L to 250 g/L, 210 g/L to
250 g/L, 220 g/L to 250 g/L, 230 g/L to 250 g/L, or 240 g/L to 250
g/L IgG.
[0225] In a particular embodiment, a liquid compositions of pooled
human IgG formulated for intranasal administration consists
essentially of from 100 g/L to 250 g/L pooled human IgG and from
150 mM to 350 mM glycine.
[0226] In another particular embodiment, a liquid compositions of
pooled human IgG formulated for intranasal administration consists
essentially of from 150 g/L to 250 g/L pooled human IgG and from
200 mM to 300 mM glycine.
[0227] In yet another particular embodiment, a liquid compositions
of pooled human IgG formulated for intranasal administration
consists essentially of from 200 g/L to 250 g/L pooled human IgG
and 250.+-.25 mM glycine.
[0228] In certain embodiments, the liquid compositions of pooled
human IgG formulated for intranasal administration provided herein
further include a humectant. Non-limiting examples of humectants
include glycerin, polysaccharides, and polyethylene glycols.
[0229] In certain embodiments, the liquid compositions of pooled
human IgG formulated for intranasal administration provided herein
further include an agent that increases the flow properties of the
composition. Non-limiting examples of agents that increase to flow
properties of an aqueous composition include sodium carboxymethyl
cellulose, hyaluronic acid, gelatin, algin, carageenans, carbomers,
galactomannans, polyethylene glycols, polyvinyl alcohol,
polyvinylpyrrolidone, sodium carboxymethyl dextran, and
xantham.
[0230] In one embodiment, dry powder compositions of pooled human
IgG formulated for intranasal administration are provided for the
treatment of CNS disorders (e.g., Alzheimer's disease, Parkinson's
disease, and multiple sclerosis). In a specific embodiment, a dry
powder therapeutic composition formulated for intranasal
administration provided herein consists essentially of a buffering
agent and pooled human IgG.
[0231] In one embodiment, a dry powder composition of pooled human
IgG formulated for intranasal administration further comprises a
bulking agent. Non-limiting examples of bulking agents include
oxyethylene maleic anhydride copolymer, polyvinylether,
polyvinylpyrrolidone polyvinyl alcohol, polyacrylates, including
sodium, potassium or ammonium polyacrylate, polylactic acid,
polyglycolic acid, polyvinyl alcohol, polyvinyl acetate,
carboxyvinyl polymer, polyvinylpyrrolidone, polyethylene glycol,
celluloses (including cellulose, microcrystalline cellulose, and
alpha-cellulose), cellulose derivatives (including methyl
cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl
cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl
cellulose and ethylhydroxy ethyl cellulose), dextrins (including
alpha-, beta-, or gamma-cyclodexthn, and
dimethyl-beta-cyclodexthn), starches (including hydroxyethyl
starch, hydroxypropyl starch, carboxymethyl starch),
polysaccharides (including dextran, dextrin and alginic acid,
hyaluronic acid, and pectic acid), carbohydrates (such as mannitol,
glucose, lactose, fructose, sucrose, and amylose), proteins
(including casein, gelatin, chitin, and chitosan), gums (such as
gum arabic, xanthan gum, tragacanth gum, and glucomannan),
phospholipids, and combinations thereof.
[0232] In certain embodiments, a dry powder composition of pooled
human IgG formulated for intranasal administration further
comprises a mucosal penetration enhancer. Non-limiting examples of
mucosal penetration enhancers are bile salts, fatty acids,
surfactants and alcohols. Specific non-limiting examples of mucosal
penetration enhancers are sodium cholate, sodium dodecyl sulphate,
sodium deoxycholate, taurodeoxycholate, sodium glycocholate,
dimethylsulfoxide or ethanol.
[0233] In certain embodiments, a dry powder composition of pooled
human IgG formulated for intranasal administration further
comprises a dispersant. A dispersant is an agent that assists
aerosolization of the IgG or the absorption of the IgG in
intranasal mucosal tissue, or both. Non-limiting examples of
dispersants are a mucosal penetration enhancers and
surfactants.
[0234] In certain embodiments, a dry powder composition of pooled
human IgG formulated for intranasal administration further
comprises a bioadhesive agent. Non-limiting examples of bioadhesive
agents include chitosan or cyclodextrin. In certain embodiments, a
dry powder composition of pooled human IgG formulated for
intranasal administration further comprises a filler. Non-limiting
examples of fillers include sugars, including lactose, sucrose,
mannitol, or sorbitol; cellulose preparations such as: for example,
maize starch, wheat starch, rice starch, potato starch, gelatin,
gum tragacanth, methylcellulose, microcrystalline cellulose,
hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or
others such as: polyvinylpyrrolidone (PVP or povidone) or calcium
phosphate.
[0235] The particle size a dry powder composition of pooled human
IgG can be determined by standard methods in the art. For example,
the particles can be screened or filtered through a mesh sieve. In
certain embodiments, the dry particles have an average diameter
from about 0.1 .mu.m to about 250 .mu.m. In some embodiments, the
dry particles have an average diameter between from 1 .mu.m to
about 25 .mu.m. In some embodiments, the dry particles have an
average diameter between from 10 .mu.m to about 100 .mu.m. In yet
other embodiments, the dray particles have an average diameter of
about 0.1 .mu.m.+-.10%, 0.2 .mu.m.+-.10%, 0.3 .mu.m.+-.10%, 0.4
.mu.m.+-.10%, 0.5 .mu.m.+-.10%, 0.6 .mu.m.+-.10%, 0.7 .mu.m.+-.10%,
0.8 .mu.m.+-.10%, 0.9 .mu.m.+-.10%, 1.0 .mu.m.+-.10%, 2 .mu.m 10%,
3 .mu.m.+-.10%, 4 .mu.m.+-.10%, 5 .mu.m.+-.10%, 6 .mu.m.+-.10%, 7
.mu.m.+-.10%, 8 .mu.m.+-.10%, 9 .mu.m 10%, 10 .mu.m.+-.10%, 11
.mu.m.+-.10%, 12 .mu.m.+-.10%, 13 .mu.m.+-.10%, 14 .mu.m.+-.10%, 15
.mu.m.+-.10%, 16 .mu.m.+-.10%, 17 .mu.m.+-.10%, 18 .mu.m.+-.10%, 19
.mu.m.+-.10%, 20 .mu.m.+-.10%, 25 .mu.m.+-.10%, 30 .mu.m.+-.10%, 35
.mu.m.+-.10%, 40 .mu.m.+-.10%, 45 .mu.m.+-.10%, 50 .mu.m.+-.10%, 60
.mu.m.+-.10%, 65 .mu.m.+-.10%, 70 .mu.m.+-.10%, 75 .mu.m.+-.10%, 80
.mu.m.+-.10%, 85 .mu.m.+-.10%, 90 .mu.m.+-.10%, 95 .mu.m.+-.10%,
100 .mu.m.+-.10%, 110 .mu.m.+-.10%, 120 .mu.m.+-.10%, 130
.mu.m.+-.10%, 140 .mu.m.+-.10%, 150 .mu.m.+-.10%, 160 .mu.m.+-.10%,
170 .mu.m.+-.10%, 180 .mu.m.+-.10%, 190 .mu.m.+-.10%, 200
.mu.m.+-.10%, 225 .mu.m.+-.10%, 250 .mu.m.+-.10%, 275 .mu.m.+-.10%,
300 .mu.m.+-.10%, 350 .mu.m.+-.10%, 400 .mu.m.+-.10%, 450
.mu.m.+-.10%, 500 .mu.m.+-.10%, or greater.
[0236] In one embodiment, gel, cream, or ointment compositions of
pooled human IgG formulated for intranasal administration are
provided for the treatment of CNS disorders (e.g., Alzheimer's
disease, Parkinson's disease, and multiple sclerosis). In a
specific embodiment, a gel, cream, or ointment therapeutic
composition formulated for intranasal administration provided
herein consists essentially of a buffering agent and pooled human
IgG.
[0237] In one embodiment, a gel, cream, or ointment composition of
pooled human IgG formulated for intranasal administration further
comprises a carrier agent. Non-limiting examples of carrier agents
for gel and ointment compositions include natural or synthetic
polymers such as hyaluronic acid, sodium alginate, gelatin, corn
starch, gum tragacanth, methylcellulose, hydroxyethylcellulose,
carboxymethylcellulose, xanthan gum, dextrin, carboxymethylstarch,
polyvinyl alcohol, sodium polyacrylate, methoxyethylene maleic
anhydride copolymer, polyvinylether, polyvinylpyrrolidone, fats and
oils such as beeswax, olive oil, cacao butter, sesame oil, soybean
oil, camellia oil, peanut oil, beef fat, lard, and lanolin, white
petrolatum, paraffins, hydrocabon gel ointments, fatty acids such
as stearic acid, alcohols such as cetyl alcohol and stearyl
alcohol, polyethylene glycol, water, and combinations thereof.
[0238] In certain embodiments, the pooled human immunoglobulins are
co-formulated with one or more vasoconstrictor agents. When
present, the vasoconstrictor agent reduces non-target exposure
(e.g., systemic exposure) of the pooled human immunoglobulin, by
reducing absorption of the immunoglobulins into the blood,
effectively increasing the targeting of the immunoglobulin to the
CNS (e.g., to the brain). Methods for the co-formulation of other
pharmaceuticals and vasoconstrictors can be found in U.S. Patent
Application Publication No. 2008/0305077, the content of which is
expressly incorporated herein by reference in its entirety for all
purposes. Non-limiting examples of vasoconstrictors that may be
co-formulated with pooled human immunoglobulins in this fashion
include tetrahydrozoline, methoxamine, phenylephrine, ephedrine,
norepinephrine, oxymetazoline, tetrahydrozoline, xylometazoline,
clonidine, guanabenz, guanfacine, .alpha.-methyldopa, arginine
vasopressin, and pseudoephedrine.
[0239] Disorders of the Central Nervous System--
[0240] IVIG treatment has been used in the treatment of CNS
disorders. Specifically, IVIG has been studied or used in the
treatment of Multiple Sclerosis (MS), stiff-person syndrome,
Alzheimer's disease (AD), postpolio syndrome, narcolepsy, stroke,
and fibromyalgia and other pain syndromes. Stangle 2008
(Therapeutic Advances in Neurological Disorders, 1(2):115-124).
[0241] IVIG has also been used to treat neuromyelitis optica (NMO).
NMO, also known as Devic's disease or Devic's syndrome, is an
autoimmune, inflammatory disorder of the optic nerves and spinal
cord. For example, a 2 g/kg induction dose of IVIG followed by
0.4-0.5 g/kg monthly maintenance doses of IVIG has been used to
treat NMO. Awad et al. 2011 (Current Neuropharmacology,
9:417428).
[0242] IVIG has also been used and studied for the treatment of
acute disseminated encephalomyelitis (ADEM). ADEM is an immune
mediated disease of the brain. Specifically, ADEM involves
autoimmune demyelination and is classified as a MS borderline
disease. For example, a standard dose of 2 g/kg IVIG given over 2-5
days can be used to treat ADEM. Pohl et al. 2012 (Current Treatment
Options in Neurology, 14:264-275).
[0243] IVIG has also studied and used in the treatment of
Parkinson's disease (PD). For example, studies have shown that IVIG
may reduce .alpha.-synuclein neurotoxicity, a possible contributing
factor to the pathogenesis of PD, through an unknown mechanism.
Smith et al. 2012 (International Immunopharmacology, 14:550-557)
and Patrias et al. (Clinical and Experimental Immunology,
161:527-535).
[0244] IVIG has also been used and studied for the treatment of MS.
For example, IVIG has been used successfully in the treatment of
Schilder's disease (SD), a rare variant of MS. Krause et al. 2012
(European J. of Paediatric Neurology, 16:206-208). IVIG has also
been suggested to be beneficial in the treatment of acute relapses
in MS patients. Elovaara et al. 2011 (Clinical Neuropharmacology,
34(2):84-89).
[0245] IVIG has also been used and studied for the treatment of
obsessive-compulsive disorders (OCD) and tic disorders. For
example, IVIG was shown to lessen the severity of symptoms of OCD
and tic disorders in children with infection-triggered OCD and tic
disorders. Perlmutter, et al. 1999 (The Lancet, 354:1153-1158).
Similarly, it has been shown that IVIG is effective in reducing
neuropsychiatric symptom severity in a subgroup OCD and tic
disorder patients with childhood-onset OCD and tic disorders.
Snider et al. 2003 (J. of Child and Adolescent Psychopharmacology,
13(supp 1): S81-S88).
[0246] In one aspect, the present invention provides a method for
treating a central nervous system (CNS) disorder in a subject in
need thereof by delivering a therapeutically effective amount of a
composition comprising pooled human immunoglobulin G (IgG) to the
brain of the subject, wherein delivering the composition to the
brain comprises intranasally administering the composition directly
to an epithelium of the nasal cavity of the subject. In a specific
embodiment, the composition is administered directly to the
olfactory epithelium of the nasal cavity. In certain embodiments,
the CNS disorder is selected from the group consisting of a
systemic atrophy primarily affecting the central nervous system, an
extrapyramidal and movement disorder, a neurodegenerative disorder
of the central nervous system, a demyelinating disorder of the
central nervous system, an episodic or paroxysmal disorder of the
central nervous system, a paralytic syndrome of the central nervous
system, a nerve, nerve root, or plexus disorder of the central
nervous system, an organic mental disorder, a mental or behavioral
disorder caused by psychoactive substance use, a schizophrenia,
schizotypal, or delusional disorder, a mood (affective) disorder,
neurotic, stress-related, or somatoform disorder, a behavioral
syndrome, an adult personality or behavior disorder, a
psychological development disorder, or a child onset behavioral or
emotional disorder. In some embodiments, the CNS disorder is
selected from the group consisting of Alzheimer's disease,
Parkinson's disease, multiple sclerosis, amyotrophic lateral
sclerosis (ALS), Huntington's disease, cerebral palsy, bipolar
disorder, schizophrenia, or Pediatric acute-onset neuropyschiatric
syndrome (PANS). In some embodiments, the CNS disorder is selected
from the group consisting of Alzheimer's disease, Parkinson's
disease, multiple sclerosis, Pediatric Autoimmune Neuropsychiatric
Disorders Associated with Streptococcal infections (PANDAS), or
Pediatric acute-onset neuropyschiatric syndrome (PANS).
[0247] In one embodiment, the CNS disorder is a systemic atrophy
primarily affecting the central nervous system. Non-limiting
examples of systemic atrophies that primarily affect the central
nervous system include: Huntington's disease; hereditary ataxias
(e.g., congenital non-progressive ataxia, early-onset cerebellar
ataxias--such as early-onset cerebellar ataxia with essential
tremor, Hunt's ataxia, early-onset cerebellar ataxia with retained
tendon reflexes, Friedreich's ataxia, and X-linked recessive
spinocerebellar ataxia--late-onset cerebellar ataxia, ataxia
telangiectasia (Louis-Bar syndrome), or hereditary spastic
paraplegia); a spinal muscular atrophy or related disorder thereof
(e.g., Werdnig-Hoffman disease (Type 1), progressive bulbar palsy
of childhood (Fazio-Londe syndrome), Kugelberg-Welander disease
(Type 3), or a motor neuron disease--such as familial motor neuron
disease, amyotrophic lateral sclerosis (ALS), primary lateral
sclerosis, progressive bulbar palsy, and progressive spinal
muscular atrophy); paraneoplastic neuromyopathy and neuropathy;
systemic atrophy primarily affecting the central nervous system in
neoplastic disease; paraneoplastic limbic encephalopathy; and
systemic atrophy primarily affecting the central nervous system in
myxoedema.
[0248] In one embodiment, the CNS disorder is an extrapyramidal and
movement disorder. Non-limiting examples of extrapyramidal and
movement disorders that affect the central nervous system include:
Parkinson's disease; a secondary parkinsonism (e.g., malignant
neuroleptic syndrome or postencephalitic parkinsonism); a
degenerative disease of the basal ganglia (e.g., Hallervorden-Spatz
disease, progressive supranuclear ophthalmoplegia
(Steele-Richardson-Olszewski disease), or striatonigral
degeneration), a dystonia (e.g., drug-induced dystonia, idiopathic
familial dystonia, idiopathic non-familial dystonia, spasmodic
torticollis, idiopathic orofacial dystonia--such as orofacial
dyskinesia--or blepharospasm); an essential tremor; a drug-induced
tremor, myoclonus, drug-induced chorea, drug-induced tics; restless
legs syndrome; and stiff-man syndrome.
[0249] In one embodiment, the CNS disorder is a neurodegenerative
disorder of the central nervous system. Non-limiting examples of
neurodegenerative disorders that affect the central nervous system
include: Alzheimer's disease; a circumscribed brain atrophy (e.g.,
Pick's disease); senile degeneration of brain; a degeneration of
nervous system due to alcohol; grey-matter degeneration (e.g.,
Alpers' disease); Lewy body dementia, subacute necrotizing
encephalopathy (e.g., Leigh's disease); and subacute combined
degeneration of spinal cord. In certain embodiments, the CNS
disorder is disorder characterized by dementia. In certain
embodiments, the dementia is a cortical dementia (associated, for
example, with Alzheimer's) arising from a disorder affecting the
cerebral cortex. In certain embodiments, the dementia is a
subcortical dementia (associated, for example, with Parkinson's
disease and Huntington's disease) resulting from dysfunction in the
parts of the brain that are beneath the cortex. In certain
embodiments, the dementia is a side effect of drug administration.
In specific embodiments, the dementia is a side effect of the
administration of a chemotherapeutic agent. In specific
embodiments, the dementia is a result of undergoing cardiac bypass.
In specific embodiments, the dementia is a result of a vascular
disorder (e.g., myocardial infarction, stroke, high blood
pressure). In specific embodiments, the dementia is a result of
depression.
[0250] In one embodiment, the CNS disorder is a demyelinating
disorder of the central nervous system. Non-limiting examples of
demyelinating disorders that affect the central nervous system
include: multiple sclerosis; an acute disseminated demyelination
disorder (e.g., neuromyelitis optica (Devic's syndrome) or acute
and subacute hemorrhagic leukoencephalitis (Hurst's disease));
diffuse sclerosis; central demyelination of corpus callosum;
central pontine myelinolysis; acute transverse myelitis in
demyelinating disease of central nervous system; subacute
necrotizing myelitis; and concentric sclerosis (Balo disease).
[0251] In one embodiment, the CNS disorder is an episodic or
paroxysmal disorder of the central nervous system. Non-limiting
examples of episodic and paroxysmal disorders that affect the
central nervous system include: epilepsy (e.g.,
localization-related (focal)(partial) idiopathic epilepsy and
epileptic syndromes with seizures of localized onset,
localization-related (focal)(partial) symptomatic epilepsy and
epileptic syndromes with simple partial seizures;
localization-related (focal)(partial) symptomatic epilepsy and
epileptic syndromes with complex partial seizures; a benign
epileptic syndrome--such as myoclonic epilepsy in infancy and
neonatal convulsions (familial)--childhood absence epilepsy (e.g.,
pyknolepsy), epilepsy with grand mal seizures on awakening, a
juvenile epilepsy--such as absence epilepsy or myoclonic epilepsy
(impulsive petit mal)--a nonspecific epileptic seizure--such as an
atonic, clonic, myoclonic, tonic, or tonic-clonic epileptic
seizure, epilepsy with myoclonic absences or myoclonic-astatic
seizures, infantile spasms, Lennox-Gastaut syndrome, Salaam
attacks, symptomatic early myoclonic encephalopathy, West's
syndrome, epilepsia partialis continua (Kozhevnikov epilepsy),
grand mal seizures, or petit mal); headaches (e.g., a
migraine--such as a migraine without aura (common migraine), a
migraine with aura (classical migraine), status migrainosus, and
complicated migraine--cluster headache syndrome, a vascular
headache, a tension-type headache, a chronic post-traumatic
headache, or a drug-induced headache); a cerebrovascular episodic
or paroxysmal disorder (e.g., a transient cerebral ischaemic
attacks or related syndrome--such as vertebrobasilar artery
syndrome, carotid artery syndrome (hemispheric), a multiple and
bilateral precerebral artery syndrome, amaurosis fugax, and
transient global amnesia--a vascular syndrome of the brain--such as
middle cerebral artery syndrome, anterior cerebral artery syndrome,
posterior cerebral artery syndrome, a brain stem stroke syndrome
(e.g., Benedikt syndrome, Claude syndrome, Foville syndrome,
Millard-Gubler syndrome, Wallenberg syndrome, or Weber syndrome),
cerebellar stroke syndrome, pure motor lacunar syndrome, pure
sensory lacunar syndrome, or a lacunar syndromes); and a sleep
disorder (e.g., insomnia, hyperinsomnia, a disruption in circadian
rhythm, sleep apnoea, narcolepsy, or cataplexy).
[0252] In one embodiment, the CNS disorder is a paralytic syndrome
of the central nervous system. Non-limiting examples of paralytic
syndromes that affect the central nervous system include: a
cerebral palsy (e.g., spastic quadriplegic cerebral palsy, spastic
diplegic cerebral palsy, spastic hemiplegic cerebral palsy,
dyskinetic cerebral palsy, or ataxic cerebral palsy); a hemiplegia
(e.g., flaccid hemiplegia or spastic hemiplegia); a paraplegia or
tetraplegia (e.g., flaccid paraplegia, spastic paraplegia,
paralysis of both lower limbs, lower paraplegia, flaccid
tetraplegia, spastic tetraplegia, or quadriplegia); diplegia of
upper limbs; monoplegia of a lower limb, monoplegia of an upper
limb; cauda equina syndrome; and Todd's paralysis
(postepileptic).
[0253] In one embodiment, the CNS disorder is a nerve, nerve root,
or plexus disorder of the central nervous system. Non-limiting
examples of nerve, nerve root, or plexus disorders that affect the
central nervous system include: a disorder of the trigeminal nerve
(V; e.g., trigeminal neuralgia); a facial nerve disorders (VII;
e.g., bell's palsy, facial palsy, geniculate ganglionitis,
melkersson's syndrome, melkersson-Rosenthal syndrome, a clonic
hemifacial spasm, facial myokymia); a disorder of the olfactory
nerve (I); a disorder of the glossopharyngeal nerve (IX); a
disorder of the vagus nerve (X); a disorder of the hypoglossal
nerve (XII); a disorder of multiple cranial nerves; and a nerve
root or plexus disorder affecting the CNS (e.g., a brachial plexus
disorder--such as thoracic outlet syndrome--a lumbosacral plexus
disorder, a cervical root, a thoracic root disorder, a lumbosacral
root disorder, a neuralgic amyotrophy--such as
Parsonage-Aldren-Turner syndrome--or phantom limb syndrome with or
without pain).
[0254] In one embodiment, the CNS disorder is an otherwise
classified disorder of the central nervous system. Non-limiting
examples of these disorders include: hydrocephalus; a toxic
encephalopathy, a cerebral cyst; anoxic brain damage; benign
intracranial hypertension; postviral fatigue syndrome; an
encephalopathy; compression of brain; cerebral oedema; reye's
syndrome; postradiation encephalopathy; traumatic brain injury;
syringomyelia; syringobulbia; a vascular myelopathy; spinal cord
compression; myelopathy; a cerebrospinal fluid leak; a disorder of
the meninges (e.g., cerebral or spinal meningeal adhesion); and a
post-procedural disorder of nervous system (e.g., cerebrospinal
fluid leak from spinal puncture, an adverse reaction to a spinal or
lumbar puncture, or intracranial hypotension following ventricular
shunting).
[0255] In one embodiment, the CNS disorder is an organic mental
disorder. Non-limiting examples of organic mental disorders that
affect the central nervous system include: dementia (e.g., dementia
associated with Alzheimer's disease, Pick's disease,
Creutzfeldt-Jakob disease, Huntington's disease, Parkinson's
disease, or human immunodeficiency virus (HIV) disease, or vascular
dementia--such as multi-infarct dementia); organic amnesic syndrome
not induced by alcohol and other psychoactive substances); delirium
not induced by alcohol and other psychoactive substances; a mental
disorder due to brain damage and dysfunction and to physical
disease (e.g., organic hallucinosis, organic catatonic disorder,
organic delusional (schizophrenia-like) disorder, organic mood
(affective) disorder, organic anxiety disorder, organic
dissociative disorder; organic emotionally labile (asthenic)
disorder; a mild cognitive disorder, or organic brain syndrome);
and a personality and behavioral disorders due to brain disease,
damage and dysfunction (e.g., organic personality disorder,
postencephalitic syndrome, or postconcussional syndrome).
[0256] In one embodiment, the CNS disorder is a mental or
behavioral disorder caused by psychoactive substance use.
Non-limiting examples of mental or behavioral disorders caused by
psychoactive substance use that affect the central nervous system
include: acute intoxication (e.g., from alcohol, opioid, cannabis,
benzodiazepine, or cocaine use); a dependence syndrome (e.g., from
alcohol, opioid, cannabis, benzodiazepine, cocaine, or nicotine
addiction); a withdrawal syndrome (e.g., an alcohol or
benzodiazepine withdrawal syndrome); delirium tremens; and a
psychotic disorder (e.g., alcoholic hallucinosis or stimulant
psychosis); an amnesic syndrome (e.g., Korsakoff's syndrome); a
residual and late-onset psychotic disorder (e.g., posthallucinogen
perception disorder).
[0257] In one embodiment, the CNS disorder is an autism spectrum
disorder. In certain embodiments, the CNS disorder is autism,
Asperger syndrome, pervasive developmental disorder not otherwise
specified (PDD-NOS), childhood disintegrative disorder, or Rett
syndrome.
[0258] In one embodiment, the CNS disorder is a schizophrenia,
schizotypal, or delusional disorder. Non-limiting examples of
schizophrenia, schizotypal, and delusional disorders that affect
the central nervous system include: schizophrenia (e.g., paranoid
schizophrenia, hebephrenic schizophrenia (disorganized
schizophrenia), catatonic schizophrenia, undifferentiated
schizophrenia, post-schizophrenic depression, residual
schizophrenia, simple schizophrenia, cenesthopathic schizophrenia,
schizophreniform disorder, or schizophreniform psychosis);
schizotypal disorder; a persistent delusional disorder (e.g.,
delusional disorder, delusional dysmorphophobia, involutional
paranoid state, or paranoia querulans); an acute or transient
psychotic disorder (e.g., acute polymorphic psychotic disorder
without symptoms of schizophrenia, acute polymorphic psychotic
disorder with symptoms of schizophrenia, or acute
schizophrenia-like psychotic disorder); an induced delusional
disorder (e.g., folie a deux, induced paranoid disorder, or induced
psychotic disorder); a schizoaffective disorder (e.g., manic type,
depressive type, or mixed type schizoaffective disorder); and
chronic hallucinatory psychosis.
[0259] In one embodiment, the CNS disorder is a mood (affective)
disorder. Non-limiting examples of mood (affective) disorders that
affect the central nervous system include: a manic episode (e.g.,
hypomania, mania without psychotic symptoms, or mania with
psychotic symptoms); a bipolar affective disorder (e.g., bipolar
affective disorder--current episode hypomanic, bipolar affective
disorder--current episode manic without psychotic symptoms, bipolar
affective disorder--current episode manic with psychotic symptoms,
bipolar affective disorder--current episode mild or moderate
depression, bipolar affective disorder--current episode severe
depression without psychotic symptoms, bipolar affective
disorder--current episode severe depression with psychotic
symptoms, bipolar affective disorder--current episode mixed,
bipolar affective disorder--currently in remission, bipolar II
disorder, or recurrent manic episodes); a depressive episode (e.g.,
mild depressive episode, moderate depressive episode, severe
depressive episode without psychotic symptoms, severe depressive
episode with psychotic symptoms, atypical depression, or single
episodes of "masked" depression); a recurrent depressive disorder
(e.g., recurrent depressive disorder--current episode mild,
recurrent depressive disorder--current episode moderate, recurrent
depressive disorder--current episode severe without psychotic
symptoms, recurrent depressive disorder--current episode severe
with psychotic symptoms, or recurrent depressive
disorder--currently in remission); a persistent mood (affective)
disorder (e.g., cyclothymia or dysthymia); mixed affective episode;
and recurrent brief depressive episodes.
[0260] In one embodiment, the CNS disorder is a neurotic,
stress-related, or somatoform disorder. Non-limiting examples of
neurotic, stress-related, or somatoform disorders that affect the
central nervous system include: a phobic anxiety disorder (e.g.,
agoraphobia, anthropophobia, social neurosis, acrophobia, animal
phobias, claustrophobia, or simple phobia); an otherwise
categorized anxiety disorder (e.g., panic disorder (episodic
paroxysmal anxiety) or generalized anxiety disorder);
obsessive-compulsive disorder; an adjustment disorder (e.g., acute
stress reaction; post-traumatic stress disorder, or adjustment
disorder); a dissociative (conversion) disorder (e.g., dissociative
amnesia, dissociative fugue, dissociative stupor; trance disorder,
possession disorder, dissociative motor disorder, dissociative
convulsions, dissociative anaesthesia and sensory loss, mixed
dissociative (conversion) disorder, Ganser's syndrome, or multiple
personality disorder); a somatoform disorder (e.g., Briquet's
disorder, multiple psychosomatic disorder, a hypochondriacal
disorder--such as body dysmorphic disorder, dysmorphophobia
(nondelusional), hypochondriacal neurosis, hypochondriasis, and
nosophobia--a somatoform autonomic dysfunction--such as cardiac
neurosis, Da Costa's syndrome, gastric neurosis, and
neurocirculatory asthenia--or psychalgia); neurasthenia;
depersonalization-derealization syndrome; Dhat syndrome,
occupational neurosis (e.g., writer's cramp); psychasthenia;
psychasthenic neurosis; and psychogenic syncope.
[0261] In one embodiment, the CNS disorder is a behavioral syndrome
associated with physiological disturbances or physical factors.
Non-limiting examples of behavioral syndromes associated with
physiological disturbances or physical factors that affect the
central nervous system include: an eating disorder (e.g., anorexia
nervos, atypical anorexia nervosa, bulimia nervosa, atypical
bulimia nervosa, overeating associated with other psychological
disturbances, vomiting associated with other psychological
disturbances, or pica in adults); a nonorganic sleep disorder
(e.g., nonorganic insomnia, nonorganic hypersomnia, nonorganic
disorder of the sleep-wake schedule, sleepwalking (somnambulism),
sleep terrors (night terrors), or nightmares); a sexual dysfunction
not caused by organic disorder or disease; a mental or behavioral
disorder associated with the puerperium (e.g., postnatal
depression, postpartum depression, or puerperal psychosis); and
abuse of non-dependence-producing substances.
[0262] In one embodiment, the CNS disorder is an adult personality
or behavior disorder. Non-limiting examples of adult personality
and behavior disorders that affect the central nervous system
include: a specific personality disorder (e.g., paranoid
personality disorder, schizoid personality disorder, a dissocial
personality disorder--such as antisocial personality disorder--an
emotionally unstable personality disorder--such as borderline
personality disorder--histrionic personality disorder, an
anankastic personality disorder--such as obsessive-compulsive
personality disorder, anxious (avoidant) personality disorder,
dependent personality disorder, eccentric personality disorder,
haltlose personality disorder, immature personality disorder,
narcissistic personality disorder, passive-aggressive personality
disorder, or psychoneurotic personality disorder); mixed
personality disorder; a habit or impulse disorder (e.g.,
pathological gambling, pathological fire-setting (pyromania),
pathological stealing (kleptomania), or trichotillomania); and
Munchausen syndrome.
[0263] In one embodiment, the CNS disorder is a psychological
development disorder. Non-limiting examples of psychological
development disorders that affect the central nervous system
include: a developmental disorder of speech or language (e.g.,
specific speech articulation disorder, expressive language
disorder, receptive language disorder (receptive aphasia), acquired
aphasia with epilepsy (Landau-Kleffner disorder), or lisping); a
developmental disorder of scholastic skills (e.g., a specific
reading disorder--such as developmental dyslexia--specific spelling
disorder, a specific disorder of arithmetical skills--such as
developmental acalculia and Gerstmann syndrome--or a mixed disorder
of scholastic skills); a developmental disorder of motor function
(e.g., developmental dyspraxia); a mixed specific developmental
disorder; and a pervasive developmental disorder (e.g., childhood
autism, atypical autism, Rett's syndrome, overactive disorder
associated with mental retardation and stereotyped movements, or
Asperger's syndrome).
[0264] In one embodiment, the CNS disorder is a behavioral or
emotional disorder with onset usually occurring in childhood and
adolescence. Non-limiting examples of behavioral or emotional
disorders with onset usually occurring in childhood and adolescence
that affect the central nervous system include: a hyperkinetic
disorder (e.g., a disturbance of activity and attention--such as
attention-deficit hyperactivity disorder and attention deficit
syndrome with hyperactivity--or hyperkinetic conduct disorder); a
conduct disorder (e.g., conduct disorder confined to the family
context, unsocialized conduct disorder, socialized conduct
disorder, or oppositional defiant disorder); a mixed disorder of
conduct or emotions (e.g., depressive conduct disorder); an
emotional disorder with onset specific to childhood (e.g.,
separation anxiety disorder of childhood, phobic anxiety disorder
of childhood, social anxiety disorder of childhood, sibling rivalry
disorder, identity disorder, or overanxious disorder); a disorder
of social functioning with onset specific to childhood and
adolescence (e.g., elective mutism, reactive attachment disorder of
childhood, or disinhibited attachment disorder of childhood); a tic
disorder (e.g., transient tic disorder, chronic motor or vocal tic
disorder, or combined vocal and multiple motor tic disorder (de la
Tourette); and an otherwise classified behavioral or emotional
disorder with onset usually occurring in childhood and adolescence
(e.g., nonorganic enuresis, nonorganic encopresis, feeding disorder
of infancy and childhood, pica of infancy and childhood,
stereotyped movement disorders, stuttering (stammering),
cluttering, attention deficit disorder without hyperactivity,
Pediatric Autoimmune Neuropsychiatric Disorders Associated with
Streptococcal infections (PANDAS), or Pediatric acute-onset
neuropyschiatric syndrome (PANS)).
[0265] In one embodiment of the method for treating a CNS disorder,
the method includes intranasally administering a dry powder
composition containing from 0.05 mg/kg to 50 mg/kg pooled human
immunoglobulin to a subject in need thereof daily. In other
embodiments, the methods provided herein for the treatment of a CNS
disorder include intranasally administering a dry powder
composition of pooled human IgG in a dosage/frequency combination
selected from variations 1 to 816 found in Table 1 and Table 2. In
a particular embodiment, the method comprises administering the dry
powder composition directly to a nasal epithelium of the subject.
In a particular embodiment, the method comprises administering the
dry powder composition directly to the olfactory epithelium of the
subject.
[0266] In one embodiment of the method for treating a CNS disorder,
the method includes intranasally administering a liquid (e.g., an
aqueous) composition containing from 0.05 mg/kg to 50 mg/kg pooled
human immunoglobulin to a subject in need thereof daily. In other
embodiments, the methods provided herein for the treatment of a CNS
disorder include intranasally administering a liquid (e.g., an
aqueous) composition of pooled human IgG in a dosage/frequency
combination selected from variations 1 to 816 found in Table 1 and
Table 2. In a particular embodiment, the method comprises
administering the composition drop-wise directly to a nasal
epithelium of the subject. In a particular embodiment, the method
comprises administering the composition drop-wise directly to the
olfactory epithelium of the subject. In another particular
embodiment, the method comprises administering the composition via
a spray directly to a nasal epithelium of the subject. In a
particular embodiment, the method comprises administering the
composition via a spray directly to the olfactory epithelium of the
subject.
[0267] In one embodiment of the method for treating a CNS disorder,
the method includes intranasally administering a gel, cream, or
ointment composition containing from 0.05 mg/kg to 50 mg/kg pooled
human immunoglobulin to a subject in need thereof daily. In other
embodiments, the methods provided herein for the treatment of a CNS
disorder include intranasally administering a gel, cream, or
ointment composition of pooled human IgG in a dosage/frequency
combination selected from variations 1 to 816 found in Table 1 and
Table 2. In a particular embodiment, the method comprises
administering the gel, cream, or ointment composition directly to a
nasal epithelium of the subject. In a particular embodiment, the
method comprises administering the gel, cream, or ointment
composition directly to the olfactory epithelium of the
subject.
[0268] Alzheimer's Disease
[0269] IVIG has been used in the treatment of Alzheimer's disease.
It has been proposed that IVIG contains antibodies against
.beta.-amyloid. Relkin et al. 2009 (Neurobiol. Aging 30(11):
1728-36). In this study, pooled human IgG was administered
intravenously (IVIG therapy) to eight subjects diagnosed with mild
Alzheimer's disease (AD). The patients received IVIG therapy for 6
months, discontinued treatment, and then resumed treatment for 9
more months. It was found that .beta.-amyloid antibodies in the
serum from AD patients increased in proportion to IVIG dose and
plasma levels of .beta.-amyloid increased transiently after each
infusion. After 6 months of treatment, mini-mental state tests were
performed on the patients. The mini-mental state scores increased
an average of 2.5 points after 6 months, returned to baseline
during washout and remained stable during subsequent IVIG
treatment.
[0270] In one aspect, the present invention provides a method for
treating Alzheimer's disease in a subject in need thereof by
delivering a therapeutically effective amount of a composition
comprising pooled human immunoglobulin G (IgG) to the brain of the
subject, wherein delivering the composition to the brain comprises
intranasally administering the composition directly to an
epithelium of the nasal cavity of the subject. In a specific
embodiment, the composition is administered directly to the
olfactory epithelium of the nasal cavity. In one embodiment, the
Alzheimer's disease is early-onset Alzheimer's disease. In another
embodiment, the Alzheimer's disease is late-onset Alzheimer's
disease.
[0271] In one embodiment of the method for treating Alzheimer's
disease, the method includes intranasally administering a dry
powder composition containing from 0.05 mg/kg to 50 mg/kg pooled
human immunoglobulin to a subject in need thereof daily. In other
embodiments, the methods provided herein for the treatment of
Alzheimer's disease include intranasally administering a dry powder
composition of pooled human IgG in a dosage/frequency combination
selected from variations 1 to 816 found in Table 1 and Table 2. In
a particular embodiment, the method comprises administering the dry
powder composition directly to a nasal epithelium of the subject.
In a particular embodiment, the method comprises administering the
dry powder composition directly to the olfactory epithelium of the
subject. In one embodiment, the Alzheimer's disease is early-onset
Alzheimer's disease. In another embodiment, the Alzheimer's disease
is late-onset Alzheimer's disease.
[0272] In one embodiment of the method for treating Alzheimer's
disease, the method includes intranasally administering a liquid
(e.g., an aqueous) composition containing from 0.05 mg/kg to 50
mg/kg pooled human immunoglobulin to a subject in need thereof
daily. In other embodiments, the methods provided herein for the
treatment of Alzheimer's disease include intranasally administering
a liquid (e.g., an aqueous) composition of pooled human IgG in a
dosage/frequency combination selected from variations 1 to 816
found in Table 1 and Table 2. In a particular embodiment, the
method comprises administering the composition drop-wise directly
to a nasal epithelium of the subject. In a particular embodiment,
the method comprises administering the composition drop-wise
directly to the olfactory epithelium of the subject. In another
particular embodiment, the method comprises administering the
composition via a spray directly to a nasal epithelium of the
subject. In a particular embodiment, the method comprises
administering the composition via a spray directly to the olfactory
epithelium of the subject. In one embodiment, the Alzheimer's
disease is early-onset Alzheimer's disease. In another embodiment,
the Alzheimer's disease is late-onset Alzheimer's disease.
[0273] In one embodiment of the method for treating Alzheimer's
disease, the method includes intranasally administering a gel,
cream, or ointment composition containing from 0.05 mg/kg to 50
mg/kg pooled human immunoglobulin to a subject in need thereof
daily. In other embodiments, the methods provided herein for the
treatment of Alzheimer's disease include intranasally administering
a gel, cream, or ointment composition of pooled human IgG in a
dosage/frequency combination selected from variations 1 to 816
found in Table 1 and Table 2. In a particular embodiment, the
method comprises administering the gel, cream, or ointment
composition directly to a nasal epithelium of the subject. In a
particular embodiment, the method comprises administering the gel,
cream, or ointment composition directly to the olfactory epithelium
of the subject. In one embodiment, the Alzheimer's disease is
early-onset Alzheimer's disease. In another embodiment, the
Alzheimer's disease is late-onset Alzheimer's disease.
[0274] Multiple Sclerosis
[0275] Multiple sclerosis (MS) is a chronic neurodegenerative and
inflammatory disease of the central nervous system (CNS) that
represents one of the most prevalent human autoimmune diseases.
Multiple sclerosis (MS) is an autoimmune disease that specifically
affects the brain and spinal cord. MS is caused by damage to the
myelin sheath, the protective covering that surrounds nerve cells.
When the myelin sheath is damaged, nerve signals slow down or stop.
Damage to the myelin sheath is caused by inflammation which occurs
when the body's own immune cells attack the nervous system. This
can occur along any area of the brain, optic nerve, and spinal
cord.
[0276] MS is classified into four subtypes based on the disease's
progression: Relapsing-Remitting MS (RMSS), Secondary Progressive
MS (SPMS), Primary-Progressive MS (PPMS), and Progressive-Relapsing
MS (PRMS). More than 80 percent of patients who are diagnosed with
MS exhibit initial signs of RMSS. RMSS is characterized by relapse
(characterized by symptom flare-ups) followed by remission. The
relapses can be mild to severe flare-ups and the remissions can
last for days to months. RMSS patients often develop SPMS. SPMS is
characterized by relapses followed by only partial recoveries.
During the partial recovery phase, the symptoms may lessen but do
not go into full remission. SPMS is a progressive subtype of MS
wherein the symptoms steadily worsen until a chronic disability
replaces the cycles of recovery and partial recovery. PPMS accounts
for approximately 15 percent of MS occurrences. It is characterized
by a slow and steady progression without periods of remission or
partial recovery. PRMS is the least common subtype of MS. PRMS is
characterized by steadily worsening symptoms and attacks followed
by periods of remission.
[0277] There are peptide-induced and transgenic mouse model for MS.
Experimental autoimmune encephalomyelitis (EAE) is an animal model
of brain inflammation. EAE is an inflammatory demyelinating disease
of the CNS. Acute and relapsing EAE is characterized by the
formation of focal inflammatory demyelinating lesions in the white
matter of the brain. This phenotype can be induced in normal SJL
mice through the administration of PLP139-151 peptide. Chronic
progressive EAE is pathologically associated with a widespread
axonal damage in the normal appearing white matter and massive
demyelination in the grey matter, particularly in the cortex. This
phenotype can be induced in normal C57BL/6 mice through the
administration of MOG35-55 peptide.
[0278] There is also evidence that tumor necrosis factor (TNF)
ligand/receptor superfamily, particularly TNF and Fas/Fas ligand
(FasL) are involved in the pathogenesis of MS. Akassoglou et al.
1998 (Am J Pathol. 153(3): 801-813). Accordingly, mouse models
deficient in TNF can be used to study the pathologies of MS. The
genotype of transgenic TNF knockout mouse models include p55TNFR
(p55-/-), p75TNFR (p75-/-), and TNF (TNF-/-).
[0279] IVIG has proven useful in the treatment of a number of
autoimmune diseases; however its role in the treatment of MS
remains uncertain. IVIG trials in different types of MS patients
have produced variable results ranging from reports of monthly IVIG
being beneficial to IVIG administration not slowing disease
progression or reversing disease-induced deficits.
[0280] In one aspect, the present invention provides a method for
treating multiple sclerosis in a subject in need thereof by
delivering a therapeutically effective amount of a composition
comprising pooled human immunoglobulin G (IgG) to the brain of the
subject, wherein delivering the composition to the brain comprises
intranasally administering the composition directly to an
epithelium of the nasal cavity of the subject. In a specific
embodiment, the composition is administered directly to the
olfactory epithelium of the nasal cavity.
[0281] In one embodiment of the method for treating multiple
sclerosis, the method includes intranasally administering a dry
powder composition containing from 0.05 mg/kg to 50 mg/kg pooled
human immunoglobulin to a subject in need thereof daily. In other
embodiments, the methods provided herein for the treatment of
multiple sclerosis include intranasally administering a dry powder
composition of pooled human IgG in a dosage/frequency combination
selected from variations 1 to 816 found in Table 1 and Table 2. In
a particular embodiment, the method comprises administering the dry
powder composition directly to a nasal epithelium of the subject.
In a particular embodiment, the method comprises administering the
dry powder composition directly to the olfactory epithelium of the
subject.
[0282] In one embodiment of the method for treating multiple
sclerosis, the method includes intranasally administering a liquid
(e.g., an aqueous) composition containing from 0.05 mg/kg to 50
mg/kg pooled human immunoglobulin to a subject in need thereof
daily. In other embodiments, the methods provided herein for the
treatment of multiple sclerosis include intranasally administering
a liquid (e.g., an aqueous) composition of pooled human IgG in a
dosage/frequency combination selected from variations 1 to 816
found in Table 1 and Table 2. In a particular embodiment, the
method comprises administering the composition drop-wise directly
to a nasal epithelium of the subject. In a particular embodiment,
the method comprises administering the composition drop-wise
directly to the olfactory epithelium of the subject. In another
particular embodiment, the method comprises administering the
composition via a spray directly to a nasal epithelium of the
subject. In a particular embodiment, the method comprises
administering the composition via a spray directly to the olfactory
epithelium of the subject.
[0283] In one embodiment of the method for treating multiple
sclerosis, the method includes intranasally administering a gel,
cream, or ointment composition containing from 0.05 mg/kg to 50
mg/kg pooled human immunoglobulin to a subject in need thereof
daily. In other embodiments, the methods provided herein for the
treatment of multiple sclerosis include intranasally administering
a gel, cream, or ointment composition of pooled human IgG in a
dosage/frequency combination selected from variations 1 to 816
found in Table 1 and Table 2. In a particular embodiment, the
method comprises administering the gel, cream, or ointment
composition directly to a nasal epithelium of the subject. In a
particular embodiment, the method comprises administering the gel,
cream, or ointment composition directly to the olfactory epithelium
of the subject.
[0284] Parkinson's Disease
[0285] Parkinson's disease (PD) is a degenerative disorder of the
CNS. PD is notably linked to a decrease in motor control. The loss
of motor control caused by PD results from the death of
dopamine-generating cells in the substantia nigra, a region of the
midbrain. Early in the progression of the disease, the most common
symptoms include shaking, rigidity, slowness of movement and
difficulty with walking and gait. As the disease progresses,
cognitive and behavioral problems arise, with dementia occurring in
the advanced stages of the disease. Additional symptoms include
sensory, sleep and emotional problems. PD is more common in the
elderly, with symptoms most commonly occurring after the age of
50.
[0286] There are numerous transgenic mouse models for PD. These
models include, for example, Park2 (parkin) transgenic strains,
LRRK2 transgenic strains, and synuclein transgenic strains (Jackson
Laboratories, Bar Harbor, Me.). In addition to transgenic models,
parkinsonian symptoms can also be induced in mice by administering
the compounds MPTP, rotenone, paraquat, or maneb.
[0287] In one aspect, the present invention provides a method for
treating Parkinson's disease in a subject in need thereof by
delivering a therapeutically effective amount of a composition
comprising pooled human immunoglobulin G (IgG) to the brain of the
subject, wherein delivering the composition to the brain comprises
intranasally administering the composition directly to an
epithelium of the nasal cavity of the subject. In a specific
embodiment, the composition is administered directly to the
olfactory epithelium of the nasal cavity.
[0288] In one embodiment of the method for treating Parkinson's
disease, the method includes intranasally administering a dry
powder composition containing from 0.05 mg/kg to 50 mg/kg pooled
human immunoglobulin to a subject in need thereof daily. In other
embodiments, the methods provided herein for the treatment of
Parkinson's disease include intranasally administering a dry powder
composition of pooled human IgG in a dosage/frequency combination
selected from variations 1 to 816 found in Table 1 and Table 2. In
a particular embodiment, the method comprises administering the dry
powder composition directly to a nasal epithelium of the subject.
In a particular embodiment, the method comprises administering the
dry powder composition directly to the olfactory epithelium of the
subject.
[0289] In one embodiment of the method for treating Parkinson's
disease, the method includes intranasally administering a liquid
(e.g., an aqueous) composition containing from 0.05 mg/kg to 50
mg/kg pooled human immunoglobulin to a subject in need thereof
daily. In other embodiments, the methods provided herein for the
treatment of Parkinson's disease include intranasally administering
a liquid (e.g., an aqueous) composition of pooled human IgG in a
dosage/frequency combination selected from variations 1 to 816
found in Table 1 and Table 2. In a particular embodiment, the
method comprises administering the composition drop-wise directly
to a nasal epithelium of the subject. In a particular embodiment,
the method comprises administering the composition drop-wise
directly to the olfactory epithelium of the subject. In another
particular embodiment, the method comprises administering the
composition via a spray directly to a nasal epithelium of the
subject. In a particular embodiment, the method comprises
administering the composition via a spray directly to the olfactory
epithelium of the subject.
[0290] In one embodiment of the method for treating Parkinson's
disease, the method includes intranasally administering a gel,
cream, or ointment composition containing from 0.05 mg/kg to 50
mg/kg pooled human immunoglobulin to a subject in need thereof
daily. In other embodiments, the methods provided herein for the
treatment of Parkinson's disease include intranasally administering
a gel, cream, or ointment composition of pooled human IgG in a
dosage/frequency combination selected from variations 1 to 816
found in Table 1 and Table 2. In a particular embodiment, the
method comprises administering the gel, cream, or ointment
composition directly to a nasal epithelium of the subject. In a
particular embodiment, the method comprises administering the gel,
cream, or ointment composition directly to the olfactory epithelium
of the subject.
Specific Embodiments
[0291] In a first aspect, the disclosure provides a method for
treating a central nervous system (CNS) disorder in a subject in
need thereof, the method comprising: delivering a therapeutically
effective amount of a composition comprising pooled human
immunoglobulin G (IgG) to the brain of the subject, wherein
delivering the composition to the brain comprises intranasally
administering the composition directly to a nasal epithelium of the
subject.
[0292] In one embodiment of the first aspect, at least 40% of the
pooled human IgG administered to the subject contacts the nasal
epithelium of the subject.
[0293] In one embodiment of the first aspect, at least 50% of the
pooled human IgG administered to the subject contacts the nasal
epithelium of the subject.
[0294] In one embodiment of the first aspect, at least 60% of the
pooled human IgG administered to the subject contacts the nasal
epithelium of the subject.
[0295] In one embodiment of the first aspect, the nasal epithelium
is the olfactory epithelium of the subject.
[0296] In one embodiment of the first aspect, at least 40% of the
pooled human IgG administered to the subject contacts the olfactory
epithelium of the subject.
[0297] In one embodiment of the first aspect, at least 50% of the
pooled human IgG administered to the subject contacts the olfactory
epithelium of the subject.
[0298] In one embodiment of the first aspect, at least 60% of the
pooled human IgG administered to the subject contacts the olfactory
epithelium of the subject.
[0299] In one embodiment of the first aspect, the nasal epithelium
is a nasal epithelium of the subject associated with trigeminal
nerve endings.
[0300] In one embodiment of the first aspect, at least 40% of the
pooled human IgG administered to the subject contacts the nasal
epithelium of the subject associated with trigeminal nerve
endings.
[0301] In one embodiment of the first aspect, at least 50% of the
pooled human IgG administered to the subject contacts the nasal
epithelium of the subject associated with trigeminal nerve
endings.
[0302] In one embodiment of the first aspect, at least 60% of the
pooled human IgG administered to the subject contacts the nasal
epithelium of the subject associated with trigeminal nerve
endings.
[0303] In one embodiment of the first aspect, delivering the
composition to the brain comprises intranasally administering the
composition to the upper third of the nasal cavity of the
subject.
[0304] In one embodiment of the first aspect, at least 40% of the
pooled human IgG administered to the subject contacts the upper
third of the nasal cavity of the subject.
[0305] In one embodiment of the first aspect, at least 50% of the
pooled human IgG administered to the subject contacts the upper
third of the nasal cavity of the subject.
[0306] In one embodiment of the first aspect, at least 60% of the
pooled human IgG administered to the subject contacts the upper
third of the nasal cavity of the subject.
[0307] In one embodiment of any of the methods provided above, the
CNS disorder is a neurodegenerative disorder of the central nervous
system. In a specific embodiment, the neurodegenerative disorder of
the central nervous system is Alzheimer's disease. In a specific
embodiment, the neurodegenerative disorder of the central nervous
system is Parkinson's disease.
[0308] In one embodiment of any of the methods provided above, the
CNS disorder is a systemic atrophy primarily affecting the central
nervous system. In a specific embodiment, the systemic atrophy
primarily affecting the central nervous system is amyotrophic
lateral sclerosis (ALS). In a specific embodiment, the systemic
atrophy primarily affecting the central nervous system is
Huntington's disease.
[0309] In one embodiment of any of the methods provided above, the
CNS disorder is an extrapyramidal and movement disorder.
[0310] In one embodiment of any of the methods provided above, the
CNS disorder is a demyelinating disorder of the central nervous
system. In a specific embodiment, the demylelinating disorder of
the central nervous system is multiple sclerosis.
[0311] In one embodiment of any of the methods provided above, the
CNS disorder is an episodic or paroxysmal disorder of the central
nervous system.
[0312] In one embodiment of any of the methods provided above, the
CNS disorder is a paralytic syndrome of the central nervous system.
In a specific embodiment, the CNS disorder is a paralytic syndrome
of the central nervous system is cerebral palsy
[0313] In one embodiment of any of the methods provided above, the
CNS disorder is a nerve, nerve root, or plexus disorder of the
central nervous system.
[0314] In one embodiment of any of the methods provided above, the
CNS disorder is an organic mental disorder.
[0315] In one embodiment of any of the methods provided above, the
CNS disorder is a mental or behavioral disorder caused by
psychoactive substance use.
[0316] In one embodiment of any of the methods provided above, the
CNS disorder is a schizophrenia, schizotypal, or delusional
disorder. In a specific embodiment, the schizophrenia, schizotypal,
or delusional disorder is schizophrenia.
[0317] In one embodiment of any of the methods provided above, the
CNS disorder is a mood (affective) disorder. In a specific
embodiment, the mood (affective) disorder is bipolar disorder.
[0318] In one embodiment of any of the methods provided above, the
CNS disorder is a neurotic, stress-related, or somatoform
disorder.
[0319] In one embodiment of any of the methods provided above, the
CNS disorder is a behavioral syndrome.
[0320] In one embodiment of any of the methods provided above, the
CNS disorder is an adult personality or behavior disorder.
[0321] In one embodiment of any of the methods provided above, the
CNS disorder is a psychological development disorder.
[0322] In one embodiment of any of the methods provided above, the
CNS disorder is a child onset behavioral or emotional disorder. In
a specific embodiment, the child onset behavioral or emotional
disorder is Pediatric acute-onset neuropyschiatric syndrome (PANS).
In another specific embodiment, the the child onset behavioral or
emotional disorder is Pediatric Autoimmune Neuropsychiatric
Disorders Associated with Streptococcal infections (PANDAS).
[0323] In one embodiment of any of the methods provided above,
intranasal administration of the composition comprises the use of a
non-invasive intranasal delivery device.
[0324] In one embodiment of any of the methods provided above,
intranasal administration of the composition comprises
administration of a liquid drop of the composition directly onto
the nasal epithelium.
[0325] In one embodiment of any of the methods provided above,
intranasal administration of the composition comprises directed
administration of an aerosol of the composition to the nasal
epithelium. In a specific embodiment, the aerosol of the
composition is a liquid aerosol. In a specific embodiment, the
aerosol of the composition is a powder aerosol.
[0326] In one embodiment of any of the methods provided above, the
composition comprising pooled human IgG does not contain a
permeability enhancer.
[0327] In one embodiment of any of the methods provided above, the
composition comprising pooled human IgG consists essentially of
pooled human IgG and an amino acid. In a specific embodiment, the
amino acid is glycine. In another specific embodiment, the amino
acid is histidine. In another specific embodiment, the amino acid
is proline.
[0328] In one embodiment of any of the methods provided above, the
composition comprising pooled human IgG is an aqueous composition.
In one embodiment, the composition comprises: from 10 mg/mL to 250
mg/mL pooled human IgG; and from 50 mM to 500 mM glycine. In a
specific embodiment, the pH of the composition is from 4.0 to 7.5.
In another specific embodiment, the pH of the composition is from
4.0 to 6.0. In another specific embodiment, the pH of the
composition is from 6.0 to 7.5.
[0329] In one embodiment of any of the methods provided above, the
composition comprising pooled human IgG is a dry powder
composition. In one embodiment, the dry powder composition is
prepared from an aqueous solution comprising: from 10 mg/mL to 250
mg/mL pooled human IgG; and from 50 mM to 500 mM glycine. In a
specific embodiment, the dry powder composition is prepared from an
aqueous solution having a pH of from 4.0 to 7.5. In another
specific embodiment, the pH of the composition is from 4.0 to 6.0.
In another specific embodiment, the pH of the composition is from
6.0 to 7.5.
[0330] In one embodiment of any of the methods provided above, the
method includes intranasally administering to the subject a dose of
from 0.08 mg to 100 mg pooled human IgG per kg body weight of the
subject (mg IgG/kg).
[0331] In one embodiment of any of the methods provided above, the
method includes intranasally administering to the subject a dose of
from 0.2 mg to 40 mg pooled human IgG per kg body weight of the
subject (mg IgG/kg).
[0332] In one embodiment of any of the methods provided above, the
method includes intranasally administering to the subject a dose of
from 0.5 mg to 20 mg pooled human IgG per kg body weight of the
subject (mg IgG/kg).
[0333] In one embodiment of any of the methods provided above, the
method includes intranasally administering to the subject a dose of
from 0.5 mg to 10 mg pooled human IgG per kg body weight of the
subject (mg IgG/kg).
[0334] In one embodiment of any of the methods provided above, the
method includes intranasally administering to the subject a dose of
from 1 mg to 5 mg pooled human IgG per kg body weight of the
subject (mg IgG/kg).
[0335] In one embodiment of any of the methods provided above, the
method includes intranasally administering to the subject a fixed
dose of from 50 mg to 10 g pooled human IgG.
[0336] In one embodiment of any of the methods provided above, the
method includes intranasally administering to the subject a fixed
dose of from 100 mg to 5 g pooled human IgG.
[0337] In one embodiment of any of the methods provided above, the
method includes intranasally administering to the subject a fixed
dose of from 500 mg to 2.5 g pooled human IgG.
[0338] In one embodiment of any of the methods provided above, the
method includes intranasally administering to the subject a dose of
pooled human IgG at least twice monthly.
[0339] In one embodiment of any of the methods provided above, the
method includes intranasally administering to the subject a dose of
pooled human IgG at least once weekly.
[0340] In one embodiment of any of the methods provided above, the
method includes intranasally administering to the subject a dose of
pooled human IgG at least twice weekly.
[0341] In one embodiment of any of the methods provided above, the
method includes intranasally administering to the subject a dose of
pooled human IgG at least once daily.
[0342] In one embodiment of any of the methods provided above, the
method includes intranasally administering to the subject a dose of
pooled human IgG at least twice daily.
[0343] In one embodiment of any of the methods provided above, the
composition comprising pooled human IgG comprises at least 0.1%
anti-amyloid .beta. IgG.
[0344] In one embodiment of any of the methods provided above, the
method includes administering a second therapy for the CNS disorder
to the subject in need thereof. In one embodiment, the second
therapy for the CNS disorder is a cholinesterase inhibitor. In a
specific embodiment, the cholinesterase inhibitor is donepezil. In
another specific embodiment, the cholinesterase inhibitor is
rivastigmine. In another specific embodiment, the cholinesterase
inhibitor is galantamine. In another specific embodiment, the
cholinesterase inhibitor is tacrine. In another embodiment, the
second therapy for the CNS disorder is an inhibitor of NMDA-type
glutamate receptor. In a specific embodiment, the inhibitor of
NMDA-type glutamate receptor is memantine.
EXAMPLES
Example 1--Tolerability of Intranasal Administration of IgG in
Rats
[0345] A study was conducted to examine the tolerability of
intranasal administration of IgG in rats. The purpose of this study
was to determine the tolerability of rats to intranasal IgG
administration at various concentrations and preparations.
[0346] Experimental Design:
[0347] IgG was prepared as a liquid protein solution or as a
microsphere preparation. The liquid IgG protein solution was
prepared in glycine at 200 mg/mL and 100 mg/mL and had a pH of
5.1-5.3. The IgG microsphere preparation was prepared at 200 mg/mL
and 150 mg/mL in PEG. The IgG preparations were administered to 8
anesthetized, adult male Sprague Dawley rats.
[0348] Prior to anesthesia, each rat was weighed. An anesthesia
cocktail was prepared and full, half, and quarter anesthesia doses
were calculated according to the animal's weight with a full dose
containing 30 mg/kg ketamine, 6 mg/kg xylazine, and 1 mg/kg
acepromazine. The anesthesia was administered subcutaneously into
the left hind leg, above the thigh. Anesthesia was monitored
throughout the procedures by assessing reflexes using pinching of
the hind paw or tail. If a reflex was present, a half or quarter
dose booster was administered as necessary. During drug
administration, animals received a half dose booster roughly 20-25
min after initial dose if needed.
[0349] Anesthetized rats were placed on their backs on a heating
pad in a metal surgical tray. The heating pad was connected to a
thermostat and was automatically regulated to maintain a 37.degree.
C. temperature based on continuous measurement from a rectal probe.
A 2''.times.2'' gauze pad was rolled tightly into a pillow, taped
together, and under the neck to maintain a correct neck position
horizontal with the counter.
[0350] A 6 .mu.L drop was loaded into a pipette and wiped dry with
a tissue. A cotton swab covered in parafilm was used to occlude one
naris completely (the flat part of the swab was pushed gently
against the naris to prevent airflow), while the 6 .mu.L drop was
expelled slowly from the pipette (held at a 45.degree. angle from
the rat's midline), forming a drop on the pipette tip. The drop was
lowered onto the open naris to be inhaled. The IgG preparations
were administered intranasally as described in Table 3.
TABLE-US-00003 TABLE 3 Intranasal administration of IgG to 8 rats
to test for intranasal tolerability. # Drops Time to Rat Weight (g)
Drug/Dose delivered perfusion 1 259.87 Liquid protein 10 @ 6
.mu.L/drop 23 min solution - 200 mg/mL (60 .mu.L total) 2 272.61
Microsphere - 50 mg/mL 10 @ 6 .mu.L/drop 60 min (60 .mu.L total) 3
309.14 Liquid protein 8 @ 6 .mu.L/drop 60 min solution - 200 mg/mL
(60 .mu.L total) 4 309.00 Liquid protein 10 @ 6 .mu.L/drop 60 min
solution - 100 mg/mL (60 .mu.L total) 5 342.62 Microsphere - 10 @ 6
.mu.L/drop 60 min 200 mg/mL (60 .mu.L total) 6 355.1 Microsphere -
10 @ 6 .mu.L/drop 60 min 150 mg/mL (60 .mu.L total) 7 364.28
Microsphere - 10 @ 6 .mu.L/drop 60 min 200 mg/mL (60 .mu.L total) 8
348.93 Microsphere - 28 @ 6 .mu.L/drop 60 min 150 mg/mL (162 .mu.L
total)
[0351] Results.
[0352] Three rats received the liquid preparation of intranasal
IgG. One rat received 60 .mu.L at 100 mg/ml and it was well
tolerated. Two rats received 60 .mu.L at 200 mg/mL. The first rat
had some difficulty breathing, most likely due to a problem with
light anesthesia. The second rat had some difficulties breathing,
but survived. Tracheotomies were not necessary.
[0353] Four rats received the microsphere preparation. Two rats
received 60 .mu.L at 150 mg/ml. One rat received 60 .mu.L at 200
mg/ml. One rat received 162 .mu.L at 150 mg/ml. These rats
tolerated the highest concentration available at 200 mg/ml very
well.
[0354] The rats tolerated the liquid and microsphere preparations;
however, the rats did tolerate the microsphere preparation better
than the protein preparation.
Example 2--Comparison of Liquid, Microsphere, and Fragment
Biodistribution at 30 and 90 Minutes
[0355] The purpose of this study was to quantify the amount of
intranasally administered IgG that reaches the central nervous
system and peripheral tissues in anesthetized rats. Specifically,
the biodistribution of different formulations and modes of
administration were compared. The different formulations and modes
of administration are described in Table 4.
TABLE-US-00004 TABLE 4 Formulations and modes of administration
used in biodistribution study. .sup.125I radiolabeled IgG
Formulation Mode of Administration Liquid protein Intranasal
(biodistribution measured at 30 min formulation post
administration) Liquid protein Intravenous biodistribution measured
at 30 min formulation post administration) Liquid protein
Intranasal (biodistribution measured at 90 min formulation post
administration) Microsphere formulation Intranasal (biodistribution
measured at 30 min post administration) Microsphere formulation
Intranasal (biodistribution measured at 90 min post administration)
Microsphere formulation Intranasal (biodistribution measured at 30
min (low .mu.Ci) post administration) Antibody fragment (FAb)
Intranasal (biodistribution measured at 30 min post
administration)
[0356] Experimental Design:
[0357] 40 male Sprague-Dawley rats were given one of three
preparations of .sup.125I radiolabeled IgG. These included liquid
IgG protein solution in glycine at pH 5.1-5.3, IgG in a microsphere
preparation including PEG, or as Fab antibody fragments in
phosphate buffered saline (PBS). Drug administration was either
intranasal or intravenous. Rats were sacrificed either 30 or 90 min
after the onset of delivery of the IgG preparations for
biodistribution studies.
[0358] For intranasal delivery, the rats were anesthetized and
placed on their backs on a heating pad in a metal surgical tray.
The heating pad was connected to a thermostat and was automatically
regulated to maintain a 37.degree. C. temperature based on
continuous measurement from a rectal probe. A 2''.times.2'' gauze
pad was rolled tightly into a pillow, taped together, and under the
neck to maintain a correct neck position horizontal with the
counter. A lead impregnated shield was placed between the surgical
tray and the experimenter for protection against radiation. The
dose solution, pipette, pipette tips, and waste receptacle were
arranged behind the shield for easy access.
[0359] A 6 .mu.L drop was loaded into the pipette behind the shield
and wiped dry with a tissue. A cotton swab covered in parafilm was
used to occlude one naris completely (the flat part of the swab was
pushed gently against the naris to prevent airflow), while the 6
.mu.L drop was expelled slowly from the pipette (held at a
45.degree. angle from the rat's midline), forming a drop on the
pipette tip. The drop was lowered onto the open naris to be
inhaled. After two minutes, the alternate naris was occluded and a
6 .mu.L drop was administered in the same fashion. A drop was
administered as described above every two minutes to alternating
nares until a total of 8 drops was delivered (4 to each naris) over
14 min. Delivered time of each drop was noted as well as any
details regarding the animal's respiration or success of the
delivery. Three 3 .mu.L aliquots of each dosing solution were gamma
counted to determine the measured specific activity.
[0360] For intravenous IgG delivery, the rats required cannulation
of the femoral artery. Anesthetized animals were positioned on
their backs in surgical tray on a heating pad maintained at
37.degree. C. Both hind legs were secured by loosely tying a suture
around the limbs and weighting them with a hemostat. Small,
superficial cuts with blunt scissors were made at the mid inguinal
point, making sure not to cut the superficial blood vessels.
Gentle, blunt dissection using cotton swabs exposed the femoral
vein from the great saphenous vein to the inguinal ligament. Blunt
scissors were used to cut away the skin to get a better view the
area. Overlying muscle was retracted by threading a 4-0 suture with
a curved needle through the muscle, attaching a curved hemostat to
the end of the suture and weighting it in place. Connective tissue
surrounding the femoral vein and artery was carefully removed with
blunt dissection (cotton swabs). Connective tissue between the vein
and artery was teased apart using two pairs of forceps carefully
using a motion running parallel to the blood vessels and being
careful not to rupture the vessels. Saline was applied if the area
was dry.
[0361] In an area free of branches, the angled forceps was inserted
underneath the vein, the tip poked through the connective tissue,
and the forceps slowly opened to pull a 12 inch 4-0 suture through
very carefully. If the vein collapsed, a cotton swab was used to
gently pump the vein full of blood. A second suture was pulled
through in a similar manner. The medial and lateral sutures were
tied into loose knots. A cotton swab was used to pump the vein full
of blood. The lateral suture (closest to the knee) was tied into a
tight knot. A hemostat was attached to the suture strings of the
medial suture and some tension was added to occlude blood flow.
[0362] A 1 mm transverse incision was made in the femoral vein and
a blunted 25 G butterfly needle connected to tubing previously
filled with 0.9% NaCl and attached to a 3-way stopcock was
immediately inserted. The medial suture was tied down around the
needle to secure it in place. To confirm placement within the vein,
a small amount of blood was withdrawn then saline was pushed. Free
suture strings were tied to the butterfly needle securing the
cannula in place. Muscles were protracted, sutures securing the
limbs removed, and the surgical area was covered with gauze wet
with saline.
[0363] For the intravenous infusion of .sup.125I IgG, a syringe
pump was placed in the hood behind the lead shield. Parts of the
pump were covered with parafilm (or saran wrap) to prevent
contamination with radiation. The pump was set for 4.75 mm diameter
and rate of 50 .mu.L/min. The dose solution (48 .mu.L) was mixed
with 452 .mu.L of saline (0.9% NaCl, total volume 500 .mu.L) in a
1.5 mL microcentrifuge tube. A 1 cc syringe filled with saline was
attached to the 3-way stopcock attached to the butterfly needle and
placed in the pump. A piece of parafilm was used to secure the
saline syringe to the stopcock. With the stopcock closed to the
rat, the pump was started to fill the stopcock with saline.
[0364] A 1 cc syringe attached to a 27 G or 30 G needle was used to
collect the drug from the microcentrifuge tube and then the syringe
was connected to the 3-way stopcock. The stopcock was turned so
that the flow was open between the dose solution and the rat. The
tubing was filled with dose solution making sure that no air
bubbles are pushed into the rat and that fluid does not pool near
the femoral vein (this would indicate the needle was not in the
vein). The stopcock was turned so that flow was open to the saline
syringe and the rats.
[0365] The time and start volume of the saline syringe was noted
and the pump was started. The stop volume of the saline syringe was
also noted at the end of the 14 min infusion. At least 700 .mu.L of
saline was infused (50 .mu.L/min over 14 min). The volume of saline
administered was slightly more than the volume of the tubing which
ensured that all of the dose solution was administered.
[0366] Two minutes prior to the desired end point time,
anesthetized animals were laid flat on their backs in a metal
surgical tray. The heating pad, rectal probe, and neck pillow were
removed. Tape was used to secure the front limbs to the pan. The
back of the pan was elevated slightly to allow blood to run away
from the animal. The sternum was exposed by cutting through the
skin. The sternum was clamped with a hemostat and the rib cage was
cut open laterally, exposing the diaphragm. The diaphragm was cut
laterally to expose the pleural cavity.
[0367] Surgical scissors were used to cut up the sides of the
ribcage toward the armpits of the animal, creating a `V` shaped
incision exposing the heart. The hemostat holding the sternum was
taped above the head to hold the cavity open. The heart was
stabilized using the blunt forceps while a small cut was made into
the left ventricle. A 1 cc-syringe with 18 G, 1'' blunt needle was
inserted into the left ventricle and approximately 0.1 mL of blood
was removed and placed into a pre-weighed tube for gamma counting.
A second 18 G blunt needle attached to an extension set filled with
60 cc of saline was inserted through the left ventricle and into
the aorta. A large bulldog clamp was placed just above the heart on
the aorta, securing the blunt needle in place.
[0368] The animal was perfused with 60 mL of saline followed by 360
mL of paraformaldehyde using a syringe pump at a rate of 15
mL/min.
[0369] Throughout experimental procedures, strict precautions were
followed to prevent radioactive contamination of animal tissues,
surgical tools, and equipment. Geiger counters were placed at each
work station to continuously screen tools, workspace, and staff.
Personal protective equipment including double layered gloves, lab
coats, eye protection, masks, and bouffant caps were worn at all
times. Lead impregnated shields were used to minimize exposure to
radiation. Radioactive monitoring badges were also worn by staff
throughout experimental procedures to quantify exposure.
[0370] Immediately after collection, each tissue sample was placed
into a pre-labeled and pre-weighed gamma tube for later
measurement.
[0371] For brain dissection, skin and muscle around the neck were
cut with a scalpel just above the shoulder blades and a large pair
of scissors used to decapitate the animal, cutting dorsal to
ventral to avoid contamination from the trachea and esophagus. To
expose the brain, a midline incision was made on the dorsal side of
the skull, then skin was peeled away, and a straight hemostat was
used to break the bone, taking care to leave the dorsal dura
attached. Dorsal dura was collected.
[0372] To remove the brain from the skull, the head was inverted
and a small spatula was used to free it from the cavity. The
posterior optic nerve and trigeminal nerves were cut close to the
brain. The brain was then placed into a clean Petri dish for
dissection.
[0373] From the base of the skull, the ventral dura was collected
by scraping a forceps on the ventral skull walls. The pituitary,
optic chiasm, and trigeminal nerves were collected. The anterior
portion of the trigeminal nerve consisted of the portion before the
visible branch in the skull, while the remainder containing the
trigeminal ganglion was considered as the posterior section. The
head was then set aside and covered with a kim-wipe for later
dissection.
[0374] A microscope was used to help remove vessels from the brain.
Using surgical forceps, microscissors, and a 30 G needle, the
basilar artery and circle of Willis were removed and placed onto
pre-weighed paper (paper was used because of the small weight of
this tissue). The needle was used to lift the vessels away from the
brain, the forceps to grab hold, and the microscissors to make the
cuts. This tissue was weighed immediately upon collection and then
the entire paper was crumpled and placed into the bottom of
tube).
[0375] Prior to placing the brain into the coronal matrix, the
olfactory bulbs were cut off at the natural angle using a razor
blade. In the coronal brain matrix, a razor blade was inserted at
the center of where optic chiasm was before removal to normalize
each animal to the same location (bregma). Additional blades were
placed every 2 mm from the first blade, resulting in 6.times.2 mm
slices, 3 rostral to the optic chiasm and 3 caudal.
[0376] Blades were removed and tissues were dissected from each
slice (FIG. 1A-1F). The remaining section of cortex and hippocampus
was dissected from the remaining brain tissue in the matrix and
placed in respective tubes. The upper cervical spinal cord was
collected. The remaining brain was then bisected along the midline
and dissected into midbrain, pons, medulla, and cerebellum
according to FIG. 1G.
[0377] Returning to the head, the ventral side of the neck was cut
anteriorly and skin peeled back exposing lymph nodes, salivary
glands, and neck muscles. The superficial nodes, deep cervical
nodes, carotid arteries, and thyroid gland were dissected and
cleared of connective tissue. A razor blade was used to bisect the
skull along the midline. The olfactory epithelium and respiratory
epithelium were collected.
[0378] For body dissection, bodies were placed on their backs and a
longitudinal cut using a scalpel was used to open the peritoneal
cavity down to the bladder. 3 mm square samples of liver
(superficial right lobe), kidney (left, tip), renal artery, spleen
(tip), lung (right, top lobe), and heart were collected.
Approximately 0.1-0.2 mL of urine was collected.
[0379] Bodies were flipped over onto the stomach and a superficial
incision was made down the length of the animal from shoulders to
hips, following the spine. The skin was peeled away from the
underlying tissue on both sides to expose the shoulder blades.
Axillary nodes in the armpits were dissected and cleared of
connective tissue. A piece of right deltoid muscle was collected
(.about.3 mm.sup.2).
[0380] The muscles overlying the spine were scored with a scalpel.
To expose the spinal cord, a small hemostat was inserted into the
spinal column and used to chip away overlying vertebrae and
tissues. A small spatula was used to loosen the cord from the
spinal cavity and forceps used to remove it and place into a petri
dish. The dura was peeled off of the cord using forceps. The cord
was dissected into lower cervical, thoracic, and lumbar portions.
The top .about.2 mm of lower cervical segment was discarded.
[0381] A 2 cm segment of trachea and esophagus was dissected from
the body and connective tissues were removed. The top 0.5 cm
(closest to the decapitation point) of each was discarded.
[0382] Pre-weighed gamma tubes containing samples were reweighed to
determine tissue weight. Tissue samples from the rats were counted
using a COBRA II Auto-Gamma Counter using a standard .sup.125I
protocol and a 5 min count time. Counters were normalized weekly to
ensure a counting efficiency at or above 80%. Background counts
were subtracted.
[0383] Mean and standard error of the nM concentration of each
tissue sample were calculated. Any value outside two standard
deviations of the mean for each tissue was considered an outlier
and removed from the data set. nM IgG concentrations were
calculated for each tissue using the measured specific activity of
dosing solutions, the CPM of each tissue, and the volume of each
tissue (assuming 1 g=1 mL).
[0384] Results, Intranasal IgG Liquid Preparation Distribution at
30 min End Point.
[0385] Eight rats received IN IgG liquid preparation at an average
dose of 6.0 mg in 47.4 .mu.L containing 69.6 with a 30 min end
point. Animals tolerated the IN administration well and all
survived until the 30 min desired end point.
[0386] At the site of IN drug administration, the average IgG
concentrations in the respiratory and olfactory epithelia were
136,213 nM and 442 nM respectively. A rostral to caudal gradient of
13.1 nM to 6.0 nM IgG was observed in the trigeminal nerve. A
similar gradient from the olfactory bulb to the anterior olfactory
nucleus of 4.1 nM to 1.5 nM IgG was observed. The average cortex
concentration of IgG after IN administration was 1.3 nM.
Concentrations of IgG in other brain regions ranged from a low of
0.7 nM in the striatum to a high of 1.7 nM in the hypothalamus. The
hippocampus was found to contain 0.6 nM IgG. A rostral to caudal
concentration gradient (1.6 nM to 0.7 nM) was observed within the
extra brain material sampled. Similarly, a rostral to caudal
concentration gradient (1.2 nM to 0.3 nM) was observed in the
spinal cord. The average concentration of IgG in the dura of the
brain was 15.2 nM compared to a spinal cord dura concentration of
2.8 nM. The dura likely also contains some or most of the arachnoid
membrane and together comprise two of the three components of the
meninges. Other tissues sampled from the cavity of the ventral
skull (pituitary and optic chiasm) contained 8.2 nM and 7.4 nM IgG
respectively.
[0387] The blood concentration of IgG at the 30 min end point was
13.9 nM. Concentrations of IgG in peripheral organs ranged from a
low of 1.3 nM in the heart to a high of 6.1 nM in the spleen and
kidney, with urine containing 8.1 nM. Concentrations of IgG in the
basilar and carotid arteries were considerably higher than the
renal artery (11.7 and 14.1 nM versus 4.4 nM). Average
concentration of IgG in the sampled lymph nodes was 4.7 nM. Levels
of IgG in tissues measured to assess variability of IN
administration and breathing difficulty (lung, esophagus, and
trachea) were consistent across animals.
[0388] Results, Intranasal IgG Microsphere Preparation (low .mu.Ci)
Distribution at 30 Min End Point.
[0389] Four rats received IN IgG microsphere preparation (low at an
average dose of 7.2 mg in 48.0 .mu.L containing 24.7 .mu.Ci with a
30 min end point. The raw data from the four rats is provided in
Table 5. The measured specific activity from this dosing solution
was much lower than expected based upon the provided specific
activity. Animals tolerated the IN administration well and all
survived until the 30 min desired end point. Zero statistically
significant outliers and fourteen non-statistically significant
outliers were identified out of a total of 211 concentration
values.
TABLE-US-00005 TABLE 5 Biodistribution (nM concentrations) of
intranasally administered IgG microsphere preparations (with low
uCi) at the 30 min end point with outliers included. BAX-17 BAX-18
BAX-19 BAX-20 Avg SE Volume Delivered (.mu.L) 48.0 48.0 48.0 48.0
48.0 .+-.0.00 uCi Delivered 20.9 20.9 28.6 28.6 24.7 .+-.2.2 mg
Delivered 7.2 7.2 7.2 7.2 7.2 .+-.0.00 Olfactory Epithelium 6,806.0
3,931.1 15,573.6 203.9 6,628.6 .+-.3,273.6 Respiratory Epithelium
559,241.5 268,256.5 219,595.4 25,412.0 268,126.3 .+-.110,307.7
Anterior Trigeminal 9.7 28.6 11.4 4.6 13.6 .+-.5.2 Nerve Posterior
Trigeminal 5.4 14.5 6.3 4.1 7.6 .+-.2.4 Nerve Olfactory Bulbs 5.9
3.4 3.7 4.1 4.3 .+-.0.6 Anterior Olfactory 1.9 2.4 2.1 1.4 1.9
.+-.0.2 Nucleus Frontal Cortex 1.2 1.6 2.0 1.4 1.6 .+-.0.2 Parietal
Cortex 0.8 1.2 1.1 0.5 0.9 .+-.0.2 Temporal Cortex 0.9 * 1.2 0.6
0.9 .+-.0.2 Occipital Cortex 0.0 1.3 0.4 1.6 0.8 .+-.0.4 Extra
Cortex 1.6 1.4 1.1 0.9 1.3 .+-.0.2 Amygdala 1.6 5.1 1.9 0.7 2.3
.+-.0.9 Striatum 0.8 16.5 0.6 0.6 4.6 .+-.4.0 Septal Nucleus 1.8
4.2 0.6 0.3 1.7 .+-.0.9 Hypothalamus 1.8 3.9 2.5 1.1 2.3 .+-.0.6
Thalamus 0.3 0.9 0.6 0.4 0.5 .+-.0.1 Midbrain 0.8 1.6 0.7 0.6 0.9
.+-.0.2 Hippocampus 0.6 1.3 0.7 0.4 0.7 .+-.0.2 Pons 0.6 1.9 1.2
0.8 1.1 .+-.0.3 Medulla 0.7 1.2 1.1 0.8 1.0 .+-.0.1 Cerebellum 0.6
1.2 0.8 0.6 0.8 .+-.0.2 Extra Slice #1 1.3 2.6 2.4 1.5 2.0 .+-.0.3
Extra Slice #2 1.0 1.1 1.2 1.1 1.1 .+-.0.05 Extra Slice #3 0.7 1.1
1.0 0.7 0.9 .+-.0.1 Extra Slice #4 0.7 1.2 0.8 0.6 0.8 .+-.0.1
Extra Slice #5 0.6 1.0 0.8 0.5 0.7 .+-.0.1 Extra Slice #6 0.7 1.3
1.0 0.6 0.9 .+-.0.2 Pituitary 7.0 18.1 6.2 3.6 8.7 .+-.3.2 Optic
Chiasm 14.9 19.0 8.2 8.1 12.5 .+-.2.7 Dorsal Dura 12.0 20.7 15.1
20.6 17.1 .+-.2.1 Ventral Dura 18.5 56.5 16.2 15.2 26.6 .+-.10.0
Spinal Dura 2.9 1.0 1.7 3.6 2.3 .+-.0.6 Upper Cervical Spinal 1.0
1.2 1.0 1.5 1.2 .+-.0.1 Cord Lower Cervical Spinal 0.4 0.3 0.4 0.9
0.5 .+-.0.1 Cord Thoracic Spinal Cord 0.5 0.4 0.6 0.7 0.5 .+-.0.1
Lumbar Spinal Cord 0.2 0.2 0.2 0.3 0.2 .+-.0.03 Circle of Willis
& Basilar 24.9 29.7 17.7 9.3 20.4 .+-.4.4 Artery Carotid Artery
207.3 17.9 14.1 13.1 63.1 .+-.48.1 Renal artery (L) 6.1 2.4 4.5 2.4
3.8 .+-.0.9 Superficial Nodes (2) 30.8 17.1 6.9 0.8 13.9 .+-.6.6
Cervical Nodes (2) 7.7 3.4 9.2 62.7 20.8 .+-.14.0 Axillary Nodes
(2) 4.2 2.1 2.2 2.8 2.8 .+-.0.5 Blood Sample 2,889.3 8.0 1,730.1
7.1 1,158.6 .+-.705.4 Muscle (R, deltoid) 3.7 2.1 1.2 1.4 2.1
.+-.0.6 Liver (R, superficial lobe) 1.2 1.0 0.9 0.9 1.0 .+-.0.1
Kidney (L, tip) 13.1 2.5 6.9 3.0 6.4 .+-.2.5 Urine 5.4 4.0 9.3 3.0
5.4 .+-.1.4 Spleen (tip) 3.1 1.2 3.0 1.8 2.3 .+-.0.5 Heart 4.4 2.9
0.4 0.7 2.1 .+-.0.9 Lung (R, top lobe) 3.4 5.2 2.3 2.6 3.4 .+-.0.6
Thyroid 28,623.9 102.6 30,320.6 15.7 14,765.7 .+-.8,497.9 Esophagus
3.7 2.5 2.7 4.7 3.4 .+-.0.5 Trachea 2.7 1.5 2.0 4.9 2.8 .+-.0.8
Drug Standard CPM 2,316,335 2,316,335 3,256,120 3,256,120 2,786,228
.+-.271,292.6 Drug Standard CPM 2,380,434 2,380,434 3,216,298
3,216,298 2,798,366 .+-.241,293.2 Drug Standard CPM 2,259,775
2,259,775 3,051,466 3,051,466 2,655,621 .+-.228,541.5 * = negative
tube weight, so nM could not be calculated
[0390] Results, Intranasal IgG Microsphere Preparation Distribution
at 30 Min End Point.
[0391] Eight rats received IN IgG microsphere preparation at an
average dose of 7.2 mg in 48.0 .mu.L containing 60.0 .mu.Ci with a
30 min end point. The raw data from the eight rats is provided in
Table 6. Animals tolerated the IN administration well and all
survived until the 30 min desired end point.
TABLE-US-00006 TABLE 6 Biodistribution (nM concentrations) of
intranasally administered IgG microsphere preparations at the 30
min end point with outliers excluded. BAX-21 BAX-22 BAX-23 BAX-25
BAX-26 BAX-28 Avg SE Volume Delivered 48.0 48.0 48.0 48.0 48.0 48.0
48.0 .+-.0.00 (.mu.L) uCi Delivered 59.9 56.3 73.4 60.8 53.1 56.5
60.0 .+-.2.9 mg Delivered 7.2 7.2 7.2 7.2 7.2 7.2 7.2 .+-.0.00
Olfactory Epithelium X 377.5 629.1 X 97.9 201.0 326.4 .+-.116.3
Respiratory 23,108.2 20,219.7 33,657.6 87,547.5 183,182.6 101,353.0
74,844.8 .+-.25,792.8 Epithelium Anterior Trigeminal 2.0 1.7 2.1
1.3 0.8 1.2 1.5 .+-.0.2 Nerve Posterior Trigeminal 2.0 1.2 1.3 0.8
0.7 0.9 1.1 .+-.0.2 Nerve Olfactory Bulbs 2.7 1.0 1.1 0.6 1.0 0.7
1.2 .+-.0.3 Anterior Olfactory 0.7 1.2 0.4 0.4 0.3 0.3 0.6 .+-.0.1
Nucleus Frontal Cortex 0.8 0.3 0.8 0.4 0.4 0.4 0.5 .+-.0.1 Parietal
Cortex 0.3 0.6 0.6 0.2 0.4 0.4 0.4 .+-.0.1 Temporal Cortex 0.2 0.4
0.2 1.3 0.2 0.3 0.5 .+-.0.2 Occipital Cortex 0.3 0.1 0.4 0.4 2.3
0.6 0.7 .+-.0.3 Extra Cortex 0.3 0.3 0.9 0.3 0.2 0.3 0.4 .+-.0.1
Amygdala 0.2 X 0.2 0.4 0.3 0.3 0.3 .+-.0.04 Striatum 0.6 1.5 0.3
1.1 X 2.1 1.1 .+-.0.3 Septal Nucleus 0.4 0.7 0.1 1.1 0.6 0.6 0.6
.+-.0.1 Hypothalamus 0.7 0.5 0.3 0.4 0.3 0.6 0.5 .+-.0.1 Thalamus
0.1 0.1 0.2 0.1 0.1 0.1 0.1 .+-.0.01 Midbrain 0.2 0.2 0.5 0.2 0.2
0.2 0.3 .+-.0.05 Hippocampus 0.2 0.3 0.2 0.2 0.1 0.1 0.2 .+-.0.03
Pons 0.4 0.3 0.5 0.3 0.5 0.3 0.4 .+-.0.04 Medulla 0.3 0.2 0.4 0.3
0.2 0.2 0.3 .+-.0.04 Cerebellum 0.3 1.1 X 1.7 0.2 0.2 0.7 .+-.0.3
Extra Slice #1 1.1 0.3 0.4 0.4 0.3 0.4 0.5 .+-.0.1 Extra Slice #2
0.6 0.2 0.2 0.2 0.2 2.4 0.6 .+-.0.4 Extra Slice #3 0.4 0.3 0.3 0.2
0.2 0.5 0.3 .+-.0.04 Extra Slice #4 0.3 0.2 0.2 0.2 0.1 2.8 0.6
.+-.0.4 Extra Slice #5 0.2 0.2 0.3 0.2 0.2 0.2 0.2 .+-.0.02 Extra
Slice #6 0.2 0.2 0.2 0.2 0.2 0.2 0.2 .+-.0.01 Pituitary 1.8 1.3 3.2
2.9 1.7 2.6 2.2 .+-.0.3 Optic Chiasm 2.2 1.4 2.5 2.1 1.4 0.7 1.7
.+-.0.3 Dorsal Dura 5.4 7.8 5.0 2.4 5.1 1.6 4.6 .+-.0.9 Ventral
Dura 9.4 3.1 2.9 3.7 3.1 1.7 4.0 .+-.1.1 Spinal Dura 0.6 0.4 0.8
0.2 X 0.6 0.5 .+-.0.1 Upper Cervical 0.50 0.36 0.52 0.28 0.34 0.53
0.42 .+-.0.04 Spinal Cord Lower Cervical 0.06 0.14 0.13 0.10 0.12
0.12 0.11 .+-.0.01 Spinal Cord Thoracic Spinal 0.06 0.03 0.08 0.09
X 0.04 0.06 .+-.0.0 Cord Lumbar Spinal Cord 0.08 0.07 0.10 0.05
0.07 0.03 0.06 .+-.0.01 Circle of Willis & 11.5 X 15.7 12.4 2.0
5.2 9.3 .+-.2.5 Basilar Artery Carotid Artery 4.6 5.4 1.6 1.9 X 1.9
3.1 .+-.0.8 Renal artery (L) 0.9 0.4 0.5 0.7 0.6 0.5 0.6 .+-.0.1
Superficial Nodes 0.8 0.7 0.9 X 4.3 0.8 1.5 .+-.0.7 (2) Cervical
Nodes (2) 1.2 1.9 1.1 0.8 X 0.5 1.1 .+-.0.2 Axillary Nodes (2) 0.4
X 0.3 0.5 1.0 0.5 0.5 .+-.0.1 Blood Sample 156.7 261.5 1.1 1.9
362.3 268.7 175.4 .+-.61.1 Muscle (R, deltoid) 0.1 0.9 0.3 0.3 0.7
0.2 0.4 .+-.0.1 Liver (R, superficial 0.0 X 0.1 0.2 0.3 0.3 0.2
.+-.0.05 lobe) Kidney (L, tip) 0.6 0.3 0.4 1.0 1.0 1.2 0.8 .+-.0.1
Urine 0.6 1.1 0.9 0.9 3.5 2.7 1.6 .+-.0.5 Spleen (tip) 0.3 0.4 0.6
0.6 0.4 0.9 0.5 .+-.0.1 Heart 0.3 0.4 0.3 0.1 0.1 0.3 0.3 .+-.0.04
Lung (R, top lobe) 0.5 0.4 0.3 2.2 0.2 1.3 0.8 .+-.0.3 Thyroid
1,697.8 3,275.2 16.1 36.2 X 35.6 1012.2 .+-.651.5 Esophagus 0.6 0.4
0.1 0.7 1.3 0.4 0.6 .+-.0.2 Trachea 0.5 1.0 0.3 0.6 0.8 0.6 0.6
.+-.0.1 Drug Standard 6,936,801 6,170,223 8,071,624 7,024,714
6,006,357 6,587,524 6,799,540.2 .+-.303,198.0 CPM Drug Standard
6,854,563 6,687,656 8,239,126 6,958,531 6,134,932 6,360,075
6,872,480.3 .+-.300,895.5 CPM Drug Standard 6,894,326 6,596,846
9,035,030 7,046,819 6,205,338 6,576,363 7,059,120.2 .+-.412,602.5
CPM X = outlier removed from analysis
[0392] At the site of IN drug administration, the average IgG
concentrations in the respiratory and olfactory epithelia were
74,844.8 nM and 326 nM respectively. A rostral to caudal gradient
of 1.5 nM to 1.1 nM IgG was observed in the trigeminal nerve. A
similar gradient from the olfactory bulb to the anterior olfactory
nucleus of 1.2 nM to 0.6 nM IgG was observed. The average cortex
concentration of IgG after IN administration was 0.5 nM.
Concentrations of IgG in other brain regions ranged from a low of
0.1 nM in the thalamus to a high of 1.1 nM in the striatum. The
hippocampus was found to contain 0.2 nM IgG. The average
concentration of IgG in the extra brain material sampled was 0.4
nM, similar to the average cortex concentration, and a
concentration gradient was not observed. A rostral to caudal
concentration gradient (0.42 nM to 0.06 nM) was observed in the
spinal cord. The average concentration of IgG in the dura of the
brain was 4.3 nM compared to a spinal cord dura concentration of
0.6 nM. Other tissues sampled from the ventral skull, the pituitary
and optic chiasm, contained 2.2 nM and 1.7 nM IgG respectively.
[0393] The blood concentration of IgG at the 30 min end point was
175 nM. Concentrations of IgG in peripheral organs ranged from a
low of 0.2 nM in the liver to a high of 0.8 nM in the kidney, with
urine containing 1.6 nM. Concentrations of IgG in the basilar and
carotid arteries were considerable greater than the concentration
in the renal artery (9.3 and 3.1 nM versus 0.6 nM). Average
concentration of IgG in the sampled lymph nodes was 1.0 nM. Levels
of IgG in tissues measured to assess variability of IN
administration and breathing difficulty (lung, esophagus, and
trachea) were consistent across animals. IgG levels in the thyroid
varied greatly ranging from 16.1 nM to 3.275 nM, even after the
removal of outliers.
[0394] Results, Intranasal IgG Fragment Preparation Distribution at
30 Min End Point.
[0395] Four rats received an IN IgG Fab antibody fragment
preparation at an average dose of approximately 3.3 mg in 48.2
.mu.L containing 76.4 .mu.Ci. The raw data from the four rats is
provided in Table 7. All four experiments were completed with an
end point of 30 min, and as expected the animals tolerated the IN
administration well and all survived until the desired end
point.
TABLE-US-00007 TABLE 7 Biodistribution (nM concentrations) of
intranasally administered IgG Fab preparations at the 30 min end
point with outliers excluded. BAX-41 BAX-42 BAX-43 BAX-44 Avg SE
Volume Delivered (.mu.L) 48.1 48.1 48.2 48.2 48.2 .+-.0.0 uCi
Delivered 76.7 76.7 76.0 76.0 76.4 .+-.0.2 mg Delivered 3.3 3.3 3.3
3.3 3.3 .+-.0.0 Olfactory Epithelium 232.4 435.2 271.2 X 312.9
.+-.62.1 Respiratory Epithelium 93,166.9 138,501.7 59,830.3
140,806.9 108076.4 .+-.19,465.7 Anterior Trigeminal 72.8 101.4
141.5 73.3 97.2 .+-.16.2 Nerve Posterior Trigeminal 32.4 34.2 33.9
19.8 30.1 .+-.3.4 Nerve Olfactory Bulbs 54.0 26.3 45.2 23.7 37.3
.+-.7.3 Anterior Olfactory 20.4 14.1 25.0 15.9 18.8 .+-.2.4 Nucleus
Frontal Cortex 20.0 11.9 21.5 X 17.8 .+-.3.0 Parietal Cortex 7.0
5.8 11.6 6.7 7.8 .+-.1.3 Temporal Cortex 5.6 4.3 9.5 4.3 5.9
.+-.1.2 Occipital Cortex 9.3 5.6 7.1 10.0 8.0 .+-.1.0 Extra Cortex
8.9 7.0 8.5 4.2 7.2 .+-.1.1 Amygdala 10.3 14.3 15.2 6.9 11.7
.+-.1.9 Striatum 5.0 5.0 8.6 3.8 5.6 .+-.1.0 Septal Nucleus 8.4 6.8
10.8 5.1 7.8 .+-.1.2 Hypothalamus 18.0 18.1 22.7 6.3 16.3 .+-.3.5
Thalamus 5.1 8.2 9.8 2.9 6.5 .+-.1.5 Midbrain 8.9 10.3 11.0 4.0 8.6
.+-.1.6 Hippocampus 6.1 7.4 7.2 2.6 5.8 .+-.1.1 Pons 11.0 12.4 12.4
4.9 10.2 .+-.1.8 Medulla 11.0 10.5 11.3 5.0 9.4 .+-.1.5 Cerebellum
9.2 5.5 6.1 8.3 7.3 .+-.0.9 Extra Slice #1 27.6 16.8 31.2 32.5 27.0
.+-.3.6 Extra Slice #2 12.5 9.8 16.0 X 12.8 .+-.1.8 Extra Slice #3
8.5 8.1 11.5 13.9 10.5 .+-.1.4 Extra Slice #4 7.4 6.5 9.2 6.2 7.3
.+-.0.7 Extra Slice #5 6.8 X 8.1 14.3 9.7 .+-.2.3 Extra Slice #6
6.0 5.5 7.4 4.1 5.7 .+-.0.7 Pituitary 41.6 44.2 50.6 34.0 42.6
.+-.3.4 Optic Chiasm 31.8 21.8 36.4 12.4 25.6 .+-.5.4 Dorsal Dura
138.2 115.3 129.9 101.5 121.2 .+-.8.1 Ventral Dura 123.1 109.7
106.0 81.1 105.0 .+-.8.8 Spinal Dura 3.4 8.1 2.7 4.5 4.7 .+-.1.2
Upper Cervical Spinal 20.5 13.7 16.7 7.9 14.7 .+-.2.7 Cord Lower
Cervical Spinal 1.0 0.7 0.9 1.3 1.0 .+-.0.1 Cord Thoracic Spinal
Cord 0.9 0.7 0.8 1.3 0.9 .+-.0.1 Lumbar Spinal Cord 0.7 0.6 0.6 0.8
0.7 .+-.0.1 Circle of Willis & 64.0 84.6 69.8 44.4 65.7 .+-.8.3
Basilar Artery Carotid Artery X 36.0 35.5 42.9 38.1 .+-.2.4 Renal
artery (L) 9.9 14.8 4.0 5.9 8.6 .+-.2.4 Superficial Nodes (2) 9.0
9.4 5.5 6.7 7.6 .+-.0.9 Cervical Nodes (2) 19.5 X 23.8 32.0 25.1
.+-.3.7 Axillary Nodes (2) 3.2 6.2 3.6 4.1 4.3 .+-.0.7 Blood Sample
31.2 38.4 28.9 33.2 32.9 .+-.2.0 Muscle (R, deltoid) 2.87 5.05 2.26
2.18 3.1 .+-.0.7 Liver (R, superficial 3.8 3.3 4.0 2.4 3.4 .+-.0.3
lobe) Kidney (L, tip) 11.1 21.5 4.0 13.1 12.4 .+-.3.6 Urine 10.6
10.3 19.9 9.0 12.4 .+-.2.5 Spleen (tip) 9.7 12.9 3.4 9.0 8.7
.+-.2.0 Heart 0.8 3.0 4.5 1.5 2.5 .+-.0.8 Lung (R, top lobe) 3.5
9.1 6.7 4.4 5.9 .+-.1.2 Thyroid 228.2 411.7 230.1 273.2 285.8
.+-.43.2 Esophagus 4.1 6.4 X 5.8 5.4 .+-.0.7 Trachea 5.6 8.7 11.3
4.8 7.6 .+-.1.5 Drug Standard CPM 7,158,905 7,158,905 6,994,454
6,994,454 7076679.3 .+-.47,472.8 Drug Standard CPM 6,974,631
6,974,631 7,215,418 7,215,418 7095024.0 .+-.69,509.2 Drug Standard
CPM 7,280,104 7,280,104 7,020,805 7,020,805 7150454.3 .+-.74,853.3
X = outlier removed from analysis
[0396] At the site of IN drug administration, the average IgG Fab
concentrations in the respiratory and olfactory epithelia were
108,076 nM and 313 nM respectively. A rostral to caudal gradient of
97.2 nM to 30.1 nM IgG Fab was observed in the trigeminal nerve. A
similar gradient from the olfactory bulb to the anterior olfactory
nucleus of 37.3 nM to 18.8 nM IgG Fab was observed. The average
cortex concentration of IgG Fab after IN administration was 9.3 nM.
Concentrations of IgG in other brain regions ranged from a low of
5.6 nM in the striatum to a high of 16.3 nM in the hypothalamus.
The hippocampus was found to contain 5.8 nM IgG Fab. A rostral to
caudal concentration gradient (27.0 nM to 5.7 nM) was observed
within the extra brain material sampled. Similarly, a rostral to
caudal concentration gradient (14.7 nM to 0.7 nM) was observed in
the spinal cord. The average concentration of IgG Fab in the dura
of the brain was 113.1 nM compared to a spinal cord dura
concentration of 4.7 nM. Other tissues sampled from the cavity of
the ventral skull (pituitary and optic chiasm) contained 42.6 nM
and 25.6 nM IgG Fab respectively.
[0397] The blood concentration of IgG Fab at the 30 min end point
was 32.9 nM. Concentrations of IgG Fab in peripheral organs ranged
from a low of 2.5 nM in the heart to a high of 12.4 nM in the
kidney and urine, with the spleen containing 8.7 nM. Concentrations
of IgG Fab in the basilar and carotid arteries were considerably
higher than the renal artery (65.7 and 38.1 nM versus 8.6 nM).
[0398] Results, Comparison of 30 Min and 90 Min End Points.
[0399] Concentrations of IgG in brain tissues were generally
similar or slightly higher with the extended 90 min end point as
compared to the 30 min end point for the IgG liquid preparation.
There was more variability in the IgG microsphere preparation, with
some tissues containing much more (thalamus, midbrain) and some
tissues containing much less (striatum, occipital cortex) at the 90
min vs. the 30 min end points. Summaries of the IgG concentrations
in tissues are provided in Table 8 and Table 9.
TABLE-US-00008 TABLE 8 Summary of tissue concentrations (nM .+-.
SE) of IN, IV, and Fab IgG at 30 min and 90 min endpoints with
outliers removed. Treatment IgG Protein (mean nM .+-. SE) IgG
Microspheres (mean nM .+-. SE) Route Intravenous Intranasal
Intranasal Time Point 30 min 90 min 30 min Sample Size n = 7 n = 8
n = 6 n = 6 Volume Delivered (.mu.L) 47.7 .+-. 0.2 47.4 .+-. 0.2
47.6 .+-. 0.1 48.0 .+-. 0.00 uCi Delivered 69.5 .+-. 0.3 69.6 .+-.
0.3 70.0 .+-. 0.01 60.0 .+-. 2.9 mg Delivered 6.0 .+-. 0.03 6.0
.+-. 0.02 7.4 .+-. 0.00 7.2 .+-. 0.00 Olfactory Epithelium 43.0
.+-. 3.7 441 .+-. 185 355 .+-. 71 326 .+-. 116 Respiratory
Epithelium 41.1 .+-. 4.3 136,213 .+-. 27,325 163,627 .+-. 16,376
74,845 .+-. 25793 Anterior Trigeminal 10.5 .+-. 1.0 13.1 .+-. 2.6
19.3 .+-. 2.8 1.5 .+-. 0.2 Nerve Posterior Trigeminal 6.3 .+-. 1.0
6.0 .+-. 1.1 8.4 .+-. 1.7 1.1 .+-. 0.2 Nerve Olfactory Bulbs 3.4
.+-. 0.5 4.1 .+-. 0.9 9.9 .+-. 1.6 1.2 .+-. 0.3 Anterior Olfactory
1.9 .+-. 0.3 1.5 .+-. 0.2 2.5 .+-. 0.3 0.6 .+-. 0.1 Nucleus Frontal
Cortex 2.9 .+-. 0.5 1.4 .+-. 0.1 3.8 .+-. 0.6 0.5 .+-. 0.1 Parietal
Cortex 3.3 .+-. 0.7 0.9 .+-. 0.1 1.5 .+-. 0.1 0.4 .+-. 0.1 Temporal
Cortex 2.9 .+-. 0.7 1.1 .+-. 0.1 1.4 .+-. 0.2 0.5 .+-. 0.2
Occipital Cortex 2.3 .+-. 0.2 1.8 .+-. 0.3 2.5 .+-. 0.2 0.7 .+-.
0.3 Extra Cortex 1.8 .+-. 0.3 1.0 .+-. 0.1 1.9 .+-. 0.2 0.4 .+-.
0.1 Amygdala 1.9 .+-. 0.1 1.4 .+-. 0.2 1.6 .+-. 0.2 0.3 .+-. 0.04
Striatum 1.8 .+-. 0.2 0.7 .+-. 0.1 0.9 .+-. 0.1 1.1 .+-. 0.3 Septal
Nucleus 1.8 .+-. 0.1 0.9 .+-. 0.1 1.1 .+-. 0.1 0.6 .+-. 0.1
Hypothalamus 2.0 .+-. 0.2 1.7 .+-. 0.3 1.9 .+-. 0.2 0.5 .+-. 0.1
Thalamus 1.7 .+-. 0.3 0.4 .+-. 0.03 0.6 .+-. 0.04 0.1 .+-. 0.01
Midbrain 1.8 .+-. 0.3 0.7 .+-. 0.1 1.3 .+-. 0.1 0.3 .+-. 0.05
Hippocampus 1.1 .+-. 0.1 0.6 .+-. 0.1 1.0 .+-. 0.1 0.2 .+-. 0.03
Pons 1.7 .+-. 0.2 0.9 .+-. 0.1 1.6 .+-. 0.2 0.4 .+-. 0.04 Medulla
1.8 .+-. 0.3 0.9 .+-. 0.1 1.6 .+-. 0.2 0.3 .+-. 0.04 Cerebellum 1.9
.+-. 0.3 0.8 .+-. 0.1 1.7 .+-. 0.2 0.7 .+-. 0.3 Extra Slice #1 2.0
.+-. 0.2 1.6 .+-. 0.3 3.3 .+-. 0.4 0.5 .+-. 0.1 Extra Slice #2 2.1
.+-. 0.3 1.0 .+-. 0.1 1.9 .+-. 0.2 0.6 .+-. 0.4 Extra Slice #3 2.2
.+-. 0.3 0.8 .+-. 0.1 1.6 .+-. 0.2 0.3 .+-. 0.04 Extra Slice #4 2.4
.+-. 0.4 0.7 .+-. 0.1 1.2 .+-. 0.1 0.6 .+-. 0.4 Extra Slice #5 2.6
.+-. 0.6 0.7 .+-. 0.1 1.2 .+-. 0.1 0.2 .+-. 0.02 Extra Slice #6 2.6
.+-. 0.5 0.9 .+-. 0.1 1.3 .+-. 0.1 0.2 .+-. 0.01 Pituitary 10.1
.+-. 0.8 8.2 .+-. 1.8 8.4 .+-. 1.1 2.2 .+-. 0.3 Optic Chiasm 5.1
.+-. 0.7 7.4 .+-. 1.7 8.0 .+-. 0.7 1.7 .+-. 0.3 Dorsal Dura 27.6
.+-. 3.0 15.3 .+-. 2.6 31.1 .+-. 4.0 4.6 .+-. 0.9 Ventral Dura 23.5
.+-. 3.1 15.0 .+-. 2.5 32.3 .+-. 4.4 4.0 .+-. 1.1 Spinal Dura 47.2
.+-. 3.0 2.8 .+-.0.3 3.3 .+-. 0.7 0.5 .+-. 0.1 Upper Cervical
Spinal 2.0 .+-. 0.2 1.2 .+-. 0.1 2.0 .+-. 0.3 0.4 .+-. 0.04 Cord
Lower Cervical Spinal 2.6 .+-. 0.3 0.6 .+-. 0.1 0.6 .+-. 0.1 0.1
.+-. 0.01 Cord Thoracic Spinal Cord 1.6 .+-. 0.2 0.4 .+-. 0.1 0.5
.+-. 0.1 0.1 .+-. 0.01 Lumbar Spinal Cord 2.1 .+-. 0.3 0.3 .+-.
0.04 0.4 .+-. 0.1 0.1 .+-. 0.01 Circle of Willis & 18.1 .+-.
2.8 11.7 .+-. 2.5 14.8 .+-. 1.1 9.3 .+-. 2.5 Basilar Artery Carotid
Artery 33.2 .+-. 3.3 14.1 .+-. 2.0 16.1 .+-. 2.3 3.1 .+-. 0.8 Renal
artery (L) 111.2 .+-. 10.1 4.4 .+-. 1.0 11.4 .+-. 3.3 0.6 .+-. 0.1
Superficial Nodes (2) 25.3 .+-. 2.9 4.8 .+-. 0.4 10.4 .+-. 2.2 1.5
.+-. 0.7 Cervical Nodes (2) 62.6 .+-. 9.2 5.6 .+-. 0.7 6.9 .+-. 0.8
1.1 .+-. 0.2 Axillary Nodes (2) 42.8 .+-. 12.8 3.7 .+-. 0.5 6.0
.+-. 0.6 0.5 .+-. 0.1 Blood Sample 1,361 .+-. 42.5 13.9 .+-. 0.9
19.7 .+-. 1.4 175 .+-. 61 Muscle (R, deltoid) 19.1 .+-. 3.8 2.7
.+-. 0.5 2.9 .+-. 0.7 0.4 .+-. 0.1 Liver (R, superficial 135 .+-.
23.7 1.7 .+-. 0.2 2.6 .+-. 0.4 0.2 .+-. 0.05 lobe) Kidney (L, tip)
355 .+-. 30.8 6.1 .+-. 0.8 8.5 .+-. 1.6 0.8 .+-. 0.1 Urine 92.6
.+-. 26.0 8.1 .+-. 1.4 17.5 .+-. 2.2 1.6 .+-. 0.5 Spleen (tip) 228
.+-. 17.5 6.1 .+-. 1.0 6.8 .+-. 0.4 0.5 .+-. 0.1 Heart 63.2 .+-.
11.7 1.3 .+-. 0.2 2.7 .+-. 0.6 0.3 .+-. 0.04 Lung (R, top lobe) 261
.+-. 51.3 2.9 .+-. 0.4 4.5 .+-. 0.7 0.8 .+-. 0.3 Thyroid 534 .+-.
65.0 148 .+-. 12.8 620 .+-. 30.8 1,012 .+-. 652.sup. Esophagus 28.1
.+-. 3.9 4.3 .+-. 0.6 7.7 .+-. 1.3 0.6 .+-. 0.2 Trachea 28.2 .+-.
6.2 3.9 .+-. 0.6 6.6 .+-. 1.4 0.6 .+-. 0.1 Drug Standard CPM
7,448,243 .+-. 128,562.sup. 7,630,853 .+-. 169,309.sup. 7,166,204
.+-. 76,377 6,799,540 .+-. 303,198.sup. Drug Standard CPM 7,089,796
.+-. 272,234.sup. 7,470,182 .+-. 171,868.sup. 7,200,437 .+-.
154,753.sup. 6,872,480 .+-. 300,896.sup. Drug Standard CPM
7,390,784 .+-. 351,624.sup. 7,689,073 .+-. 214,590.sup. 7,022,761
.+-. 10,481 7,059,120 .+-. 412,602.sup. Treatment IgG Microspheres
(mean nM .+-. SE) IgG FAB (mean nM .+-. SE) Route Intranasal
Intranasal Time Point 90 min 30 min Sample Size n = 5 n = 4 Volume
Delivered (.mu.L) 48.0 .+-. 0.00 48.2 .+-. 0.0 uCi Delivered 59.7
.+-. 2.0 76.4 .+-. 0.2 mg Delivered 7.2 .+-. 0.00 3.3 .+-. 0.0
Olfactory Epithelium 3,192 .+-. 1,625 312.9 .+-. 62.1 Respiratory
Epithelium 124,509 .+-. 20,723 108076.4 .+-. 19,465.7 Anterior
Trigeminal 8.0 .+-. 1.3 97.2 .+-. 16.2 Nerve Posterior Trigeminal
3.1 .+-. 0.2 30.1 .+-. 3.4 Nerve Olfactory Bulbs 1.5 .+-. 0.2 37.3
.+-. 7.3 Anterior Olfactory 0.6 .+-. 0.1 18.8 .+-. 2.4 Nucleus
Frontal Cortex 0.7 .+-. 0.1 17.8 .+-. 3.0 Parietal Cortex 0.3 .+-.
0.1 7.8 .+-. 1.3 Temporal Cortex 0.5 .+-. 0.1 5.9 .+-. 1.2
Occipital Cortex 0.3 .+-. 0.1 8.0 .+-. 1.0 Extra Cortex 0.5 .+-.
0.1 7.2 .+-. 1.1 Amygdala 0.4 .+-. 0.1 11.7 .+-. 1.9 Striatum 0.6
.+-. 0.2 5.6 .+-. 1.0 Septal Nucleus 0.6 .+-. 0.4 7.8 .+-. 1.2
Hypothalamus 0.6 .+-. 0.1 16.3 .+-. 3.5 Thalamus 0.3 .+-. 0.1 6.5
.+-. 1.5 Midbrain 0.5 .+-. 0.1 8.6 .+-. 1.6 Hippocampus 0.5 .+-.
0.1 5.8 .+-. 1.1 Pons 0.5 .+-. 0.1 10.2 .+-. 1.8 Medulla 0.4 .+-.
0.04 9.4 .+-. 1.5 Cerebellum 0.5 .+-. 0.1 7.3 .+-. 0.9 Extra Slice
#1 0.8 .+-. 0.1 27.0 .+-. 3.6 Extra Slice #2 0.5 .+-. 0.1 12.8 .+-.
1.8 Extra Slice #3 0.4 .+-. 0.04 10.5 .+-. 1.4 Extra Slice #4 0.3
.+-. 0.03 7.3 .+-. 0.7 Extra Slice #5 0.3 .+-. 0.04 9.7 .+-. 2.3
Extra Slice #6 0.3 .+-. 0.05 5.7 .+-. 0.7 Pituitary 2.8 .+-. 0.5
42.6 .+-. 3.4 Optic Chiasm 1.9 .+-. 0.9 25.6 .+-. 5.4 Dorsal Dura
5.8 .+-. 1.8 121.2 .+-. 8.1 Ventral Dura 11.4 .+-. 3.8 105.0 .+-.
8.8 Spinal Dura 0.7 .+-. 0.1 4.7 .+-. 1.2 Upper Cervical Spinal 0.6
.+-. 0.2 14.7 .+-. 2.7 Cord Lower Cervical Spinal 0.3 .+-. 0.1 1.0
.+-. 0.1 Cord Thoracic Spinal Cord 0.2 .+-. 0.05 0.9 .+-. 0.1
Lumbar Spinal Cord 0.2 .+-. 0.02 0.7 .+-. 0.1 Circle of Willis
& 5.8 .+-. 1.1 65.7 .+-. 8.3 Basilar Artery Carotid Artery 6.3
.+-. 0.4 38.1 .+-. 2.4 Renal artery (L) 3.7 .+-. 1.5 8.6 .+-. 2.4
Superficial Nodes (2) 2.4 .+-. 0.1 7.6 .+-. 0.9 Cervical Nodes (2)
2.6 .+-. 0.03 25.1 .+-. 3.7 Axillary Nodes (2) 2.6 .+-. 0.6 4.3
.+-. 0.7 Blood Sample 223 .+-. 84.2 32.9 .+-. 2.0 Muscle (R,
deltoid) 0.9 .+-. 0.3 3.1 .+-. 0.7 Liver (R, superficial 0.8 .+-.
0.2 3.4 .+-. 0.3 lobe) Kidney (L, tip) 2.6 .+-. 0.6 12.4 .+-. 3.6
Urine 6.3 .+-. 1.7 12.4 .+-. 2.5 Spleen (tip) 2.0 .+-. 0.5 8.7 .+-.
2.0 Heart 0.6 .+-. 0.1 2.5 .+-. 0.8 Lung (R, top lobe) 1.2 .+-. 0.4
5.9 .+-. 1.2 Thyroid 216 .+-. 50 285.8 .+-. 43.2 Esophagus 4.7 .+-.
1.3 5.4 .+-. 0.7 Trachea 2.0 .+-. 0.6 7.6 .+-. 1.5 Drug Standard
CPM 6,861,351 .+-. 210,321.sup. 7076679.3 .+-. 47,472.8.sup. Drug
Standard CPM 6,758,588 .+-. 176,717.sup. 7095024.0 .+-.
69,509.2.sup. Drug Standard CPM 7,027,097 .+-. 316,344.sup.
7150454.3 .+-. 74,853.3.sup.
TABLE-US-00009 TABLE 9 Summary of tissue concentrations of IgG
normalized to a 6 mg dose. Treatment IgG Protein (mean nM) IgG
Microspheres (mean nM) IgG FAB (mean nM) Route Intravenous
Intranasal Intranasal Intranasal Time Point 30 min 90 min 30 min 90
min 30 min Sample Size n = 7 n = 8 n = 6 n = 6 n = 5 n = 4 Volume
Delivered (.mu.L) 47.7 47.4 47.6 48.0 48.0 48.2 uCi Delivered 69.5
69.6 70.0 60.0 59.7 76.4 mg Delivered 6.0 6.0 7.4 7.2 7.2 3.3
Olfactory Epithelium 43.0 441 288 272 2,660 569.0 Respiratory
Epithelium 41.1 136,213 132,671 62,371 103,758 196502.6 Anterior
Trigeminal 10.5 13.1 15.6 1.3 6.7 176.8 Nerve Posterior Trigeminal
6.3 6.0 6.8 0.9 2.6 54.7 Nerve Olfactory Bulbs 3.4 4.1 8.0 1.0 1.2
67.8 Anterior Olfactory 1.9 1.5 2.1 0.5 0.5 34.3 Nucleus Frontal
Cortex 2.9 1.4 3.1 0.4 0.5 32.3 Parietal Cortex 3.3 0.9 1.3 0.3 0.3
14.1 Temporal Cortex 2.9 1.1 1.1 0.4 0.4 10.8 Occipital Cortex 2.3
1.8 2.0 0.6 0.3 14.5 Extra Cortex 1.8 1.0 1.6 0.3 0.4 13.0 Amygdala
1.9 1.4 1.3 0.2 0.3 21.2 Striatum 1.8 0.7 0.7 0.9 0.5 10.2 Septal
Nucleus 1.8 0.9 0.9 0.5 0.5 14.2 Hypothalamus 2.0 1.7 1.6 0.4 0.5
29.6 Thalamus 1.7 0.4 0.5 0.1 0.3 11.8 Midbrain 1.8 0.7 1.1 0.2 0.4
15.6 Hippocampus 1.1 0.6 0.8 0.2 0.4 10.6 Pons 1.7 0.9 1.3 0.3 0.4
18.5 Medulla 1.8 0.9 1.3 0.2 0.3 17.1 Cerebellum 1.9 0.8 1.3 0.6
0.4 13.2 Extra Slice #1 2.0 1.6 2.7 0.4 0.6 49.1 Extra Slice #2 2.1
1.0 1.6 0.5 0.4 23.2 Extra Slice #3 2.2 0.8 1.3 0.2 0.3 19.1 Extra
Slice #4 2.4 0.7 0.9 0.5 0.2 13.3 Extra Slice #5 2.6 0.7 1.0 0.2
0.3 17.7 Extra Slice #6 2.6 0.9 1.0 0.2 0.3 10.4 Pituitary 10.1 8.2
6.8 1.9 2.3 77.5 Optic Chiasm 5.1 7.4 6.5 1.4 1.6 46.5 Dorsal Dura
27.6 15.3 25.2 3.8 4.9 220.4 Ventral Dura 23.5 15.0 26.2 3.3 9.5
190.8 Spinal Dura 47.2 2.8 2.7 0.4 0.6 8.5 Upper Cervical Spinal
2.0 1.2 1.6 0.4 0.5 26.7 Cord Lower Cervical Spinal 2.6 0.6 0.5 0.1
0.3 1.8 Cord Thoracic Spinal Cord 1.6 0.4 0.4 0.1 0.2 1.7 Lumbar
Spinal Cord 2.1 0.3 0.4 0.1 0.1 1.2 Circle of Willis & 18.1
11.7 12.0 7.8 4.8 119.4 Basilar Artery Carotid Artery 33.2 14.1
13.1 2.6 5.3 69.3 Renal artery (L) 111.2 4.4 9.2 0.5 3.1 15.7
Superficial Nodes (2) 25.3 4.8 8.5 1.2 2.0 13.9 Cervical Nodes (2)
62.6 5.6 5.6 0.9 2.2 45.6 Axillary Nodes (2) 42.8 3.7 4.9 0.4 2.2
7.8 Blood Sample 1,361 13.9 16.0 146 186 59.8 Muscle (R, deltoid)
19.1 2.7 2.3 0.4 0.8 5.6 Liver (R, superficial 135 1.7 2.1 0.2 0.7
6.1 lobe) Kidney (L, tip) 355 6.1 6.9 0.6 2.1 22.6 Urine 92.6 8.1
14.2 1.3 5.2 22.6 Spleen (tip) 228 6.1 5.5 0.4 1.7 15.9 Heart 63.2
1.3 2.2 0.2 0.5 4.5 Lung (R, top lobe) 261 2.9 3.6 0.7 1.0 10.8
Thyroid 534 148 502 843 180 519.6 Esophagus 28.1 4.3 6.2 0.5 3.9
9.9 Trachea 28.2 3.9 5.4 0.5 1.6 13.8
[0400] Results, Intranasal IgG Liquid Preparation Distribution at
90 Min End Point.
[0401] Six rats received IN IgG liquid preparation at an average
dose of 7.4 mg in 47.6 .mu.L containing 70.0 .mu.Ci with a 90 min
end point. Animals tolerated the IN administration well and all
survived until the 90 min desired end point. The nanomolar IgG
concentrations in tissues for IN IgG liquid preparation
administrations taken at the 90 min end point are presented in
Table 10.
TABLE-US-00010 TABLE 10 Tissue concentrations (nM) of IgG after
intranasal IgG liquid preparation administration at the 90 min end
point with outliers excluded. BAX-24 BAX-33 BAX-34 BAX-35 BAX-36
BAX-40 Avg SE Volume Delivered 47.8 47.4 47.4 47.5 47.5 47.8 47.6
.+-.0.1 (.mu.L) uCi Delivered 70.0 70.0 70.0 70.0 70.0 70.0 70.0
.+-.0.01 mg Delivered 7.4 7.4 7.4 7.4 7.4 7.4 7.4 .+-.0.00
Olfactory 669.3 389.3 196.5 203.8 307.7 365.8 355.4 .+-.70.8
Epithelium Respiratory 205,721.0 194,945.7 189,621.1 139,524.3
150,482.2 101,469.9 163,627.4 .+-.16,376.5 Epithelium Anterior
Trigeminal 16.8 30.0 15.0 10.6 20.1 23.1 19.3 .+-.2.8 Nerve
Posterior Trigeminal X 13.4 5.2 6.7 5.6 11.3 8.4 .+-.1.7 Nerve
Olfactory Bulbs 15.5 9.1 10.1 10.5 3.6 10.8 9.9 .+-.1.6 Anterior
Olfactory 3.0 3.4 2.3 2.7 1.5 2.4 2.5 .+-.0.3 Nucleus Frontal
Cortex 3.3 5.9 2.4 5.0 2.7 3.3 3.8 .+-.0.6 Parietal Cortex 1.4 1.6
1.3 2.1 1.4 X 1.5 .+-.0.1 Temporal Cortex 1.1 0.9 1.7 1.4 1.2 2.0
1.4 .+-.0.2 Occipital Cortex 2.1 3.43 1.8 2.6 2.4 2.8 2.5 .+-.0.2
Extra Cortex 1.6 1.6 1.6 2.6 1.9 2.5 1.9 .+-.0.2 Amygdala 1.7 1.6
1.3 1.2 1.1 2.6 1.6 .+-.0.2 Striatum 0.9 0.4 0.6 1.0 1.2 1.0 0.9
.+-.0.1 Septal Nucleus 1.4 1.1 1.1 0.9 1.4 0.9 1.1 .+-.0.1
Hypothalamus 2.5 1.8 1.6 2.3 1.3 2.2 1.9 .+-.0.2 Thalamus 0.6 0.6
0.6 0.5 0.6 0.78 0.6 .+-.0.04 Midbrain 1.4 1.1 1.4 1.0 1.2 1.8 1.3
.+-.0.1 Hippocampus 1.0 0.9 1.1 0.7 0.8 1.2 1.0 .+-.0.1 Pons 1.9
1.2 1.1 1.9 1.2 2.2 1.6 .+-.0.2 Medulla 1.7 1.0 1.1 1.9 1.5 2.4 1.6
.+-.0.2 Cerebellum 1.4 1.0 1.6 2.1 1.4 2.5 1.7 .+-.0.2 Extra Slice
#1 3.6 4.2 2.5 4.1 1.6 3.7 3.3 .+-.0.4 Extra Slice #2 X 2.4 1.4 2.1
1.4 2.3 1.9 .+-.0.2 Extra Slice #3 1.4 1.9 1.1 1.6 1.1 2.3 1.6
.+-.0.2 Extra Slice #4 1.2 1.56 0.8 1.2 1.1 1.2 1.2 .+-.0.1 Extra
Slice #5 1.2 1.3 0.9 1.1 1.1 1.43 1.2 .+-.0.1 Extra Slice #6 1.5
1.2 1.0 1.1 1.0 1.9 1.3 .+-.0.1 Pituitary 12.7 5.5 5.6 8.1 8.4 10.4
8.4 .+-.1.1 Optic Chiasm 7.2 8.4 7.1 9.9 5.7 9.6 8.0 .+-.0.7 Dorsal
Dura 12.3 33.0 37.7 37.7 29.6 36.5 31.1 .+-.4.0 Ventral Dura 21.6
47.6 41.2 26.5 21.4 35.4 32.3 .+-.4.4 Spinal Dura 2.0 3.2 1.4 2.6
4.3 6.4 3.3 .+-.0.7 Upper Cervical 2.8 1.2 1.9 1.8 1.5 2.8 2.0
.+-.0.3 Spinal Cord Lower Cervical 0.7 0.6 0.4 0.7 0.7 X 0.6
.+-.0.1 Spinal Cord Thoracic Spinal 0.5 0.4 0.4 0.3 0.5 0.9 0.5
.+-.0.08 Cord Lumbar Spinal Cord 0.4 0.3 0.2 0.4 0.5 0.8 0.4
.+-.0.08 Circle of Willis & 16.8 11.7 15.1 X 17.7 12.9 14.8
.+-.1.1 Basilar Artery Carotid Artery 17.4 13.5 12.6 13.0 13.5 26.9
16.1 .+-.2.3 Renal artery (L) 22.4 8.4 7.7 14.6 3.7 X 11.4 .+-.3.3
Superficial Nodes 7.4 20.5 5.0 8.7 9.6 11.5 10.4 .+-.2.2 (2)
Cervical Nodes (2) 6.7 8.9 4.4 5.2 6.9 9.1 6.9 .+-.0.8 Axillary
Nodes (2) 7.2 5.8 4.3 4.5 7.9 6.3 6.0 .+-.0.6 Blood Sample 17.7
23.3 16.1 X 22.5 19.0 19.7 .+-.1.4 Muscle (R, deltoid) 2.6 3.2 1.0
1.3 5.4 3.6 2.9 .+-.0.7 Liver (R, superficial 2.8 1.4 3.9 1.1 2.7
3.4 2.6 .+-.0.4 lobe) Kidney (L, tip) 16.0 7.6 8.8 4.7 6.6 7.3 8.5
.+-.1.6 Urine 25.9 10.8 18.7 12.3 18.0 19.3 17.5 .+-.2.2 Spleen
(tip) 5.8 7.8 6.8 7.1 5.3 7.7 6.8 .+-.0.4 Heart 2.1 4.8 1.5 X 1.8
3.4 2.7 .+-.0.6 Lung (R, top lobe) 5.4 2.0 4.6 7.3 4.5 3.1 4.5
.+-.0.7 Thyroid 543.6 566.7 700.5 X 680.7 606.1 619.5 .+-.30.8
Esophagus 13.5 8.7 6.3 7.4 5.5 4.8 7.7 .+-.1.3 Trachea 13.5 6.6 5.6
3.4 5.6 5.0 6.6 .+-.1.4 Drug Standard 7,390,846 7,130,719 7,130,719
6,977,049 6,977,049 7,390,846 7166204.3 .+-.76,377.4 CPM Drug
Standard 7,285,169 7,575,479 7,575,479 6,740,664 6,740,664
7,285,169 7200437.0 .+-.154,753.0 CPM Drug Standard 6,990,473
7,032,426 7,032,426 7,045,383 7,045,383 6,990,473 7022760.5
.+-.10,480.7 CPM
[0402] X=outlier removed from analysis At the site of IN drug
administration, the average IgG concentrations in the respiratory
and olfactory epithelia were 163/,627 nM and 355 nM respectively. A
rostral to caudal gradient of 19.3 nM to 8.4 nM IgG was observed in
the trigeminal nerve. A similar gradient from the olfactory bulb to
the anterior olfactory nucleus of 9.9 nM to 2.5 nM IgG was
observed. The average cortex concentration of IgG after IN
administration was 2.2 nM. Concentrations of IgG in other brain
regions ranged from a low of 0.6 nM in the thalamus to a high of
1.9 nM in the hypothalamus. The hippocampus was found to contain
1.0 nM IgG. A rostral to caudal concentration gradient (3.3 nM to
1.2 nM) was observed within the extra brain material sampled.
Similarly, a rostral to caudal concentration gradient (2.0 nM to
0.4 nM) was observed in the spinal cord. The average concentration
of IgG in the dura of the brain was 31.7 nM compared to a spinal
cord dura concentration of 3.3 nM. Other tissues sampled from the
ventral skull, the pituitary and optic chiasm, contained 8.4 nM and
8.0 nM IgG respectively.
[0403] The blood concentration of IgG at the 30 min end point was
19.7 nM. Concentrations of IgG in peripheral organs ranged from a
low of 2.6 nM in the liver to a high of 7.7 nM in the spleen, with
urine containing 17.5 nM. Concentrations of IgG in the basilar and
carotid arteries were similar to the concentration in the renal
artery (14.8 and 16.1 nM versus 11.4 nM). Average concentration of
IgG in the sampled lymph nodes was 7.8 nM. Levels of IgG in tissues
measured to assess variability of IN administration and breathing
difficulty (lung, esophagus, and trachea) were consistent across
animals.
[0404] Results, Intranasal IgG Microsphere Preparation Distribution
at 90 Min End Point.
[0405] Six rats received IN IgG microsphere preparation at an
average dose of 7.2 mg in 48.0 .mu.L containing 59.7 .mu.Ci with a
90 min end point. Animals tolerated the IN administration well and
all survived until the 90 min desired end point. The nanomolar IgG
concentrations in tissues for IN IgG microsphere preparation
administrations taken at the 90 min end point in five of the six
rats are presented in Table 11.
TABLE-US-00011 TABLE 11 Tissue concentrations (nM) of IgG after
intranasal IgG microsphere preparation administration at the 90 min
end point with outliers excluded. BAX-31 BAX-32 BAX-37 BAX-38
BAX-39 Avg SE Volume Delivered (.mu.L) 48.0 48.0 48.0 48.0 48.0
48.0 .+-.0.00 uCi Delivered 57.5 52.7 62.8 62.8 62.8 59.7 .+-.2.0
mg Delivered 7.2 7.2 7.2 7.2 7.2 7.2 .+-.0.00 Olfactory Epithelium
293.5 3,632.7 853.2 1,898.5 9,281.1 3,191.8 .+-.1,625 Respiratory
Epithelium 169,083.6 169,807.3 128,471.3 69,460.0 85,723.8
124,509.2 .+-.20,723 Anterior Trigeminal 11.1 5.9 10.7 7.6 4.5 8.0
.+-.1.3 Nerve Posterior Trigeminal 2.6 3.3 3.6 X 3.1 3.1 .+-.0.2
Nerve Olfactory Bulbs 2.0 1.9 1.2 1.2 1.2 1.5 .+-.0.2 Anterior
Olfactory 0.7 0.5 0.5 0.4 0.8 0.6 .+-.0.1 Nucleus Frontal Cortex
0.7 0.8 0.4 0.8 0.6 0.7 .+-.0.1 Parietal Cortex 0.3 X 0.1 0.4 0.5
0.3 .+-.0.1 Temporal Cortex 0.7 0.7 0.3 0.5 0.5 0.5 .+-.0.1
Occipital Cortex 0.6 0.4 0.1 0.4 0.2 0.3 .+-.0.1 Extra Cortex 0.6
0.81 0.4 0.4 X 0.5 .+-.0.1 Amygdala 0.2 X 0.3 0.49 0.53 0.4 .+-.0.1
Striatum 0.2 1.3 0.2 0.4 0.9 0.6 .+-.0.2 Septal Nucleus 0.4 1.9 0.1
0.2 X 0.6 .+-.0.4 Hypothalamus 0.6 0.8 0.4 0.4 0.9 0.6 .+-.0.1
Thalamus 0.2 0.6 0.1 0.2 0.4 0.3 .+-.0.1 Midbrain 0.3 0.5 0.2 X 0.8
0.5 .+-.0.1 Hippocampus 0.3 0.5 0.2 0.2 1.0 0.5 .+-.0.1 Pons 0.5
0.7 0.4 0.5 0.5 0.5 .+-.0.1 Medulla 0.5 0.4 0.3 0.4 0.5 0.4
.+-.0.04 Cerebellum 0.5 0.8 0.2 0.4 0.7 0.5 .+-.0.1 Extra Slice #1
1.0 1.0 0.4 0.8 0.7 0.8 .+-.0.1 Extra Slice #2 0.4 0.50 0.2 0.4
0.96 0.5 .+-.0.1 Extra Slice #3 0.3 0.4 0.2 0.4 0.5 0.4 .+-.0.04
Extra Slice #4 0.3 X 0.2 0.3 0.3 0.3 .+-.0.03 Extra Slice #5 0.3
0.4 X 0.5 0.3 0.3 .+-.0.04 Extra Slice #6 0.4 X 0.2 0.4 0.4 0.3
.+-.0.05 Pituitary 4.4 2.1 2.9 2.7 1.6 2.8 .+-.0.5 Optic Chiasm X
3.4 X 1.9 0.4 1.9 .+-.0.9 Dorsal Dura X 11.3 3.8 3.7 4.5 5.8
.+-.1.8 Ventral Dura 11.8 26.0 7.4 3.9 8.1 11.4 .+-.3.8 Spinal Dura
0.6 0.8 0.7 X X 0.7 .+-.0.1 Upper Cervical Spinal 0.5 0.3 1.1 0.9
0.3 0.6 .+-.0.2 Cord Lower Cervical Spinal 0.2 0.2 0.1 0.4 0.6 0.3
.+-.0.1 Cord Thoracic Spinal Cord 0.1 0.1 0.1 0.3 0.3 0.2 .+-.0.05
Lumbar Spinal Cord X 0.1 0.1 0.2 0.2 0.2 .+-.0.02 Circle of Willis
& 8.9 5.0 3.4 X 5.9 5.8 .+-.1.1 Basilar Artery Carotid Artery
5.3 7.1 5.9 7.5 5.9 6.3 .+-.0.4 Renal artery (L) 1.8 3.3 1.9 9.6
1.8 3.7 .+-.1.5 Superficial Nodes (2) 2.3 2.1 2.7 2.6 2.3 2.4
.+-.0.1 Cervical Nodes (2) 2.5 2.6 2.7 2.5 2.7 2.6 .+-.0.0 Axillary
Nodes (2) 2.2 1.4 1.9 4.6 3.1 2.6 .+-.0.6 Blood Sample 249.6 388.4
53.0 6.6 417.6 223.0 .+-.84.2 Muscle (R, deltoid) 0.0 0.9 1.2 X 1.5
0.9 .+-.0.3 Liver (R, superficial 1.1 0.5 0.6 0.5 1.5 0.8 .+-.0.2
lobe) Kidney (L, tip) 1.6 1.9 1.3 3.7 4.3 2.6 .+-.0.6 Urine 4.7 4.6
6.7 2.7 12.8 6.3 .+-.1.7 Spleen (tip) 1.4 1.5 0.8 2.9 3.4 2.0
.+-.0.5 Heart 0.5 0.7 0.2 0.5 0.9 0.6 .+-.0.1 Lung (R, top lobe)
0.9 2.3 0.8 0.9 X 1.2 .+-.0.4 Thyroid 181.3 153.4 X 314.3 X 216.4
.+-.49.6 Esophagus 2.4 5.6 1.2 5.3 8.8 4.7 .+-.1.3 Trachea 1.7 1.6
0.9 3.7 X 2.0 .+-.0.6 Drug Standard CPM 6,696,942 6,103,589
7,168,742 7,168,742 7,168,742 6,861,351 .+-.210,321 Drug Standard
CPM 6,548,447 6,157,644 7,028,950 7,028,950 7,028,950 6,758,588
.+-.176,717 Drug Standard CPM 6,631,733 5,962,084 7,513,889
7,513,889 7,513,889 7,027,097 .+-.316,344
[0406] X=outlier removed from analysis at the site of IN drug
administration, the average IgG concentrations in the respiratory
and olfactory epithelia were 124,509 nM and 3,191 nM respectively.
A rostral to caudal gradient of 8.0 nM to 3.1 nM IgG was observed
in the trigeminal nerve. A similar gradient from the olfactory bulb
to the anterior olfactory nucleus of 1.5 nM to 0.6 nM IgG was
observed. The average cortex concentration of IgG after IN
administration was 0.5 nM. Concentrations of IgG in other brain
regions ranged from a low of 0.3 nM in the thalamus to a high of
0.65 nM in the septal nucleus. The hippocampus was found to contain
0.5 nM IgG. The average concentration of IgG in the extra brain
material sampled was 0.4 nM, similar to the average cortex
concentration, and a rostral to caudal concentration gradient was
observed. Similarly, a rostral to caudal concentration gradient
(0.6 nM to 0.2 nM) was observed in the spinal cord. The average
concentration of IgG in the dura of the brain was 8.6 nM compared
to a spinal cord dura concentration of 0.7 nM. Other tissues
sampled from the ventral skull, the pituitary and optic chiasm,
contained 2.8 nM and 1.9 nM IgG respectively.
[0407] The blood concentration of IgG at the 30 min end point was
223.0 nM. Concentrations of IgG in peripheral organs ranged from a
low of 0.6 nM in the heart to a high of 2.6 nM in the kidney, with
urine containing 6.3 nM. Concentrations of IgG in the basilar and
carotid arteries were similar to the concentration in the renal
artery (5.8 and 6.3 nM versus 3.7 nM). Average concentration of IgG
in the sampled lymph nodes was 2.5 nM. Levels of IgG in tissues
measured to assess variability of IN administration and breathing
difficulty (lung, esophagus, and trachea) were fairly consistent
across animals. IgG levels in the thyroid varied greatly prior to
the removal of outliers.
[0408] Overall, IN administration of the IgG liquid preparation
resulted in higher brain concentrations than the microsphere
preparation when normalizing to a 6.0 mg dose with brain
concentrations ranging from 0.4 to 1.7 nM. A summary of the IN, IV
and Fab data is presented in Table 8. This could be explained by
lower concentrations of the microsphere IgG reaching the olfactory
and respiratory epithelium. Intranasal microsphere preparation also
resulted in about ten times higher concentrations of IgG in the
blood than the liquid preparation.
[0409] Normalized to a 6 mg IN dose, Fab tissue concentrations were
on average 19-fold higher in the brain than the liquid IgG
preparations. A summary of the tissue concentrations of IgG
normalized to a 6 mg dose is presented in Table 9. The three times
smaller molecular weight of Fab versus intact IgG is likely
responsible for the increased efficiency of direct delivery from
the nasal cavity to the CNS. If the Fab has similar biological
effects as IgG for the treatment of Alzheimer's disease, it would
be a promising candidate for IN delivery.
[0410] Comparisons of brain tissue concentrations (nM) after
intranasal IgG liquid and microsphere preparations at 30 and 90 min
end points are depicted in FIG. 2A and FIG. 2B.
[0411] Results of IN and IV Delivery of the Liquid Protein
Preparation after 30 Min.
[0412] On average, IN administration of the IgG liquid preparation
resulted in lower brain concentrations than an equivalent IV dose
administered at the 30 min end point (for example the average
cortex concentration of 1.3 nM vs. 2.6 nM). However, to achieve
these brain concentrations of IgG, IV administration resulted in
blood concentrations that were a hundred times higher than IN
administration (1,361 nM vs. 13.9 nM). Higher IgG concentrations in
peripheral organs and systems were also observed with IV vs. IN
administration. For example, IgG concentrations in the lymphatic
system were ten times greater with IV vs. IN administration (43.6
nM vs. 4.7 nM).
[0413] When normalizing tissue concentrations to blood, liver, or
lymphatic concentrations, it was apparent that IN administration
targets the central nervous system. The ratio of tissue
concentrations to blood concentrations of intranasal and
intravenous IgG is presented in Table 12. For example for frontal
cortex, IN administration results in a 48 fold higher concentration
than IV when normalizing for blood concentration, 40 fold higher
when normalizing to liver concentration, and 5 fold higher when
normalizing to average lymph concentration. Intranasal
administration increased IgG targeting about 50-fold more than IV
administration (relative to the blood) to areas of the brain known
to accumulate .beta.-amyloid and heme (both known to bind IgG)
including the frontal cortex, hippocampus, and the blood vessel
walls of the cerebrovasculature. Importantly, .beta.-amyloid
tightly binds heme and heme is both a strong pro-oxidant and
pro-inflammatory agent known to inactivate brain receptors involved
in memory.
TABLE-US-00012 TABLE 12 Comparison of intranasal and intravenous
targeting of IgG. Tissue to Blood Ratios Tissue to Liver Ratios
Tissue to Avg Lymph Ratios IV IN IN/IV IV IN IN/IV IV IN IN/IV
Olfactory 0.032 31.649 1002.1 0.319 266.650 836.2 0.986 94.481 95.8
Epithelium Respiratory 0.030 9764.511 323269.6 0.305 82267.965
269754.1 0.943 29149.726 30898.8 Epithelium Anterior 0.008 0.937
122.0 0.078 7.896 101.8 0.240 2.798 11.7 Trigeminal Nerve Posterior
0.005 0.427 92.8 0.046 3.600 77.5 0.144 1.276 8.9 Trigeminal Nerve
Olfactory Bulbs 0.002 0.294 119.4 0.025 2.478 99.6 0.077 0.878 11.4
Anterior 0.001 0.105 73.3 0.014 0.883 61.2 0.045 0.313 7.0
Olfactory Nucleus Frontal Cortex 0.002 0.102 48.1 0.022 0.863 40.1
0.067 0.306 4.6 Parietal Cortex 0.002 0.066 26.9 0.025 0.556 22.5
0.077 0.197 2.6 Temporal Cortex 0.002 0.081 38.2 0.022 0.686 31.9
0.067 0.243 3.7 Occipital Cortex 0.002 0.130 78.1 0.017 1.093 65.2
0.052 0.387 7.5 Extra Cortex 0.001 0.073 54.9 0.013 0.611 45.8
0.041 0.217 5.3 Amygdala 0.001 0.103 73.5 0.014 0.867 61.3 0.044
0.307 7.0 Striatum 0.001 0.052 39.2 0.014 0.442 32.7 0.042 0.156
3.7 Septal Nucleus 0.001 0.065 48.9 0.013 0.549 40.8 0.042 0.194
4.7 Hypothalamus 0.001 0.120 80.8 0.015 1.008 67.4 0.046 0.357 7.7
Thalamus 0.001 0.030 24.7 0.012 0.254 20.6 0.038 0.090 2.4 Midbrain
0.001 0.049 37.7 0.013 0.411 31.4 0.040 0.146 3.6 Hippocampus 0.001
0.041 51.8 0.008 0.346 43.2 0.025 0.123 5.0 Pons 0.001 0.062 48.3
0.013 0.522 40.3 0.040 0.185 4.6 Medulla 0.001 0.062 47.4 0.013
0.526 39.5 0.041 0.186 4.5 Cerebellum 0.001 0.056 40.0 0.014 0.470
33.4 0.044 0.166 3.8 Extra Slice #1 0.001 0.116 77.8 0.015 0.978
64.9 0.047 0.346 7.4 Extra Slice #2 0.002 0.071 47.2 0.015 0.602
39.4 0.047 0.213 4.5 Extra Slice #3 0.002 0.059 36.5 0.016 0.497
30.5 0.050 0.176 3.5 Extra Slice #4 0.002 0.050 28.2 0.018 0.422
23.5 0.056 0.149 2.7 Extra Slice #5 0.002 0.053 28.1 0.019 0.451
23.5 0.059 0.160 2.7 Extra Slice #6 0.002 0.066 33.9 0.020 0.553
28.3 0.060 0.196 3.2 Pituitary 0.007 0.585 79.0 0.075 4.928 65.9
0.231 1.746 7.5 Optic Chiasm 0.004 0.528 141.8 0.038 4.450 118.3
0.116 1.577 13.6 Dorsal Dura 0.020 1.100 54.2 0.205 9.268 45.2
0.634 3.284 5.2 Ventral Dura 0.017 1.075 62.2 0.174 9.053 51.9
0.539 3.208 5.9 Spinal Dura 0.035 0.200 5.8 0.351 1.688 4.8 1.084
0.598 0.6 Upper Cervical 0.001 0.089 60.3 0.015 0.749 50.3 0.046
0.265 5.8 Spinal Cord Lower Cervical 0.002 0.044 23.2 0.019 0.372
19.4 0.059 0.132 2.2 Spinal Cord Thoracic Spinal 0.001 0.032 27.7
0.012 0.269 23.1 0.036 0.095 2.6 Cord Lumbar Spinal 0.002 0.022
14.4 0.016 0.188 12.0 0.049 0.067 1.4 Cord Circle of Willis &
0.013 0.837 62.8 0.135 7.048 52.4 0.416 2.497 6.0 Basilar Artery
Carotid Artery 0.024 1.013 41.6 0.246 8.537 34.7 0.761 3.025 4.0
Renal artery (L) 0.082 0.315 3.9 0.825 2.651 3.2 2.552 0.939 0.4
Superficial 0.019 0.341 18.4 0.187 2.876 15.3 0.580 1.019 1.8 Nodes
(2) Cervical Nodes (2) 0.046 0.398 8.7 0.465 3.356 7.2 1.438 1.189
0.8 Axillary Nodes (2) 0.031 0.265 8.4 0.318 2.235 7.0 0.983 0.792
0.8 Blood Sample 1.000 1.000 1.0 10.097 8.425 0.8 31.233 2.985 0.1
Muscle (R, 0.014 0.190 13.6 0.142 1.603 11.3 0.438 0.568 1.3
deltoid) Liver (R, 0.099 0.119 1.2 1.000 1.000 1.0 3.093 0.354 0.1
superficial lobe) Kidney (L, tip) 0.261 0.440 1.7 2.635 3.711 1.4
8.150 1.315 0.2 Urine 0.068 0.584 8.6 0.687 4.917 7.2 2.124 1.742
0.8 Spleen (tip) 0.168 0.434 2.6 1.693 3.658 2.2 5.236 1.296 0.2
Heart 0.046 0.095 2.0 0.469 0.803 1.7 1.452 0.284 0.2 Lung (R, top
0.192 0.210 1.1 1.936 1.769 0.9 5.990 0.627 0.1 lobe) Thyroid 0.393
10.607 27.0 3.963 89.366 22.5 12.259 31.665 2.6 Esophagus 0.021
0.312 15.1 0.209 2.627 12.6 0.645 0.931 1.4 Trachea 0.021 0.281
13.5 0.209 2.365 11.3 0.647 0.838 1.3
[0414] Eight rats received IV IgG liquid preparation at an average
dose of 6.0 mg in 47.4 containing 69.5 .mu.Ci (diluted in saline to
a total volume of 500 .mu.L for injection) with a 30 min end point.
Animals tolerated the IV administration well and all survived until
the 30 min desired end point. One animal (BAX-3) was removed from
analysis of mean, standard error, and outliers because the blood
concentration was less than 20% of the value observed in all other
animals, suggesting the IV infusion was not successful. Nanomolar
concentrations of intravenously administered IgG liquid preparation
were measured in seven rats at the 30 min end point and presented
in Table 13.
TABLE-US-00013 TABLE 13 Tissue concentrations of intravenously
administered IgG liquid preparation was measured in rats at the 30
min end point and outliers were removed. BAX-5 BAX-7 BAX-9 BAX-10
BAX-11 BAX-13 BAX-15 Avg SE Volume Delivered 47.0 47.0 48.0 48.0
48.0 48.0 48.0 47.7 .+-.0.2 (.mu.L) uCi Delivered 69.7 69.5 70.5
70.3 70.1 68.3 68.3 69.5 .+-.0.3 mg Delivered 6.0 6.0 6.0 6.0 6.0
5.9 5.9 6.0 .+-.0.03 Olfactory 33.0 40.5 40.4 43.0 56.5 32.0 55.5
43.0 .+-.3.7 Epithelium Respiratory 30.4 33.5 46.7 39.1 59.5 29.0
49.4 41.1 .+-.4.3 Epithelium Anterior 7.4 14.8 13.5 10.3 10.1 7.9
9.2 10.5 .+-.1.0 Trigeminal Nerve Posterior 4.2 11.0 8.3 6.7 5.4
3.5 4.8 6.3 .+-.1.0 Trigeminal Nerve Olfactory Bulbs 2.2 2.8 5.5
3.7 3.2 1.9 4.2 3.4 .+-.0.5 Anterior Olfactory 1.1 2.1 3.3 1.8 1.9
1.2 2.2 1.9 .+-.0.3 Nucleus Frontal Cortex 2.5 4.0 3.2 1.8 2.7 1.2
4.9 2.9 .+-.0.5 Parietal Cortex 3.3 5.2 3.0 1.6 2.6 1.3 6.4 3.3
.+-.0.7 Temporal Cortex 1.7 3.7 2.5 2.2 5.8 1.5 X 2.9 .+-.0.7
Occipital Cortex 1.9 2.8 2.5 2.3 X 1.8 X 2.3 .+-.0.2 Extra Cortex
1.4 2.1 2.6 1.8 X 1.1 X 1.8 .+-.0.3 Amygdala 1.5 1.9 X 2.1 2.0 1.8
2.2 1.9 .+-.0.1 Striatum 2.4 1.6 1.6 1.3 1.5 1.8 2.6 1.8 .+-.0.2
Septal Nucleus 1.6 1.4 2.0 1.6 X 2.0 2.2 1.8 .+-.0.1 Hypothalamus
1.2 2.4 2.7 1.7 1.9 1.5 2.7 2.0 .+-.0.2 Thalamus 1.2 1.3 1.8 1.3
2.1 0.9 3.1 1.7 .+-.0.3 Midbrain 1.1 1.4 2.3 1.3 2.2 1.1 2.9 1.8
.+-.0.3 Hippocampus 1.1 1.3 0.6 1.3 X 1.1 X 1.1 .+-.0.1 Pons 1.1
1.6 2.4 1.4 1.6 1.3 2.8 1.7 .+-.0.2 Medulla 1.2 1.4 2.7 1.5 X 1.2
2.7 1.8 .+-.0.3 Cerebellum 1.3 1.7 2.5 1.8 2.9 1.2 X 1.9 .+-.0.3
Extra Slice #1 1.5 2.8 2.7 1.7 2.2 1.3 2.1 2.0 .+-.0.2 Extra Slice
#2 1.6 3.6 2.2 1.4 1.8 1.2 2.7 2.1 .+-.0.3 Extra Slice #3 1.9 3.3
2.1 1.4 2.0 1.1 3.6 2.2 .+-.0.3 Extra Slice #4 1.9 3.1 2.5 1.4 2.6
1.1 4.3 2.4 .+-.0.4 Extra Slice #5 1.7 3.0 2.1 1.5 3.4 1.1 5.3 2.6
.+-.0.6 Extra Slice #6 1.9 2.4 2.2 1.6 3.9 1.3 5.3 2.6 .+-.0.5
Pituitary 10.9 X 12.7 9.4 8.7 7.1 11.8 10.1 .+-.0.8 Optic Chiasm
5.9 5.2 8.4 3.9 4.7 2.9 4.6 5.1 .+-.0.7 Dorsal Dura 14.8 31.7 30.7
31.0 29.5 18.2 37.4 27.6 .+-.3.0 Ventral Dura 16.4 31.0 34.4 19.6
18.9 13.8 30.4 23.5 .+-.3.1 Spinal Dura 52.3 45.9 54.8 37.6 X 53.4
39.5 47.2 .+-.3.0 Upper Cervical 1.4 2.3 2.7 1.8 2.1 1.7 2.0 2.0
.+-.0.2 Spinal Cord Lower Cervical 2.5 2.5 3.7 3.4 1.4 2.4 2.2 2.6
.+-.0.3 Spinal Cord Thoracic Spinal 1.6 1.9 2.8 1.1 1.0 1.2 1.3 1.6
.+-.0.2 Cord Lumbar Spinal 2.8 1.8 2.5 2.0 1.3 1.3 3.1 2.1 .+-.0.3
Cord Circle of Willis & 16.8 23.8 X 14.2 15.7 9.9 28.4 18.1
.+-.2.8 Basilar Artery Carotid Artery 21.7 33.8 37.2 37.5 37.4 43.9
20.6 33.2 .+-.3.3 Renal artery (L) 98.8 129.2 76.5 94.4 129.3 139.0
X 111.2 .+-.10.1 Superficial Nodes 20.3 29.6 22.9 31.9 35.3 12.6
24.1 25.3 .+-.2.9 (2) Cervical Nodes (2) 32.5 39.6 78.7 43.4 83.9
65.5 94.9 62.6 .+-.9.2 Axillary Nodes (2) 103.2 31.9 75.4 18.6 37.9
14.1 18.6 42.8 .+-.12.8 Blood Sample 1,224.9 1,234.2 1,543.3
1,322.6 1,364.7 1,413.4 1,422.9 1,360.9 .+-.42.5 Muscle (R,
deltoid) 39.06 19.5 24.6 13.3 13.0 10.7 13.4 19.1 .+-.3.8 Liver (R,
74.2 72.2 115.0 126.1 122.2 186.3 247.5 134.8 .+-.23.7 superficial
lobe) Kidney (L, tip) 347.7 313.9 287.3 459.1 397.7 441.0 238.9
355.1 .+-.30.8 Urine 32.9 174.5 187.3 41.1 68.3 122.8 21.1 92.6
.+-.26.0 Spleen (tip) 234.3 241.9 196.8 317.5 232.6 175.8 198.1
228.1 .+-.17.5 Heart 57.7 42.1 87.5 53.5 44.2 35.6 122.1 63.2
.+-.11.7 Lung (R, top lobe) 392.8 289.6 219.1 104.5 482.5 177.0
161.4 261.0 .+-.51.3 Thyroid 317.8 651.8 832.2 522.9 545.5 372.0
496.9 534.2 .+-.65.0 Esophagus 24.8 41.3 28.0 42.8 24.4 20.5 15.1
28.1 .+-.3.9 Trachea 14.6 29.2 17.9 39.2 13.4 59.0 24.2 28.2
.+-.6.2 Drug Standard 7,378,277 7,493,218 7,635,815 7,367,611
7,809,027 6,770,035 7,683,717 7,448,243 .+-.128,561.8 CPM Drug
Standard 7,962,330 7,709,707 6,369,627 -- 6,846,596 6,401,005
7,249,509 7,089,796 .+-.272,233.7 CPM Drug Standard 7,947,735
8,077,594 -- -- 6,447,049 6,626,261 7,855,283 7,390,784
.+-.351,624.3 CPM X = outlier removed from analysis
[0415] The blood concentration of IgG at the 30 min end point was
1,361 nM. Concentrations of IgG in the respiratory and olfactory
epithelia were low as expected (43 nM and 41 nM respectively). A
rostral to caudal gradient of 10.5 nM to 6.3 nM IgG was observed in
the trigeminal nerve. A similar gradient from the olfactory bulb to
the anterior olfactory nucleus of 3.4 nM to 1.9 nM IgG was
observed. The average cortex concentration of IgG after IV
administration was 2.6 nM. Concentrations of IgG in other brain
regions ranged from a low of 1.1 nM in the hippocampus to a high of
2.0 nM in the hypothalamus. The average concentration of IgG in the
extra brain material sampled was 2.3 nM, similar to the average
cortex concentration, and a concentration gradient was not
observed. Similarly, a concentration gradient was not observed in
the spinal cord and the average IgG concentration was 2.1 nM. The
average concentration of IgG in the dura of the brain was 25.6 nM
compared to a spinal cord dura concentration of 47.2 nM. Other
tissues sampled from the ventral skull, the pituitary and optic
chiasm, contained 10.1 nM and 5.1 nM IgG respectively.
[0416] Concentrations of IgG in peripheral organs ranged from a low
of 19.1 nM in the muscle to a high of 355.1 in the kidney, with
urine containing 92.6 nM. IgG concentrations in basilar and carotid
arteries were considerably lower than the renal artery (18.1 and
33.2 nM versus 111.2 nM). Average concentration of IgG in the
sampled lymph nodes was 43.6 nM.
Example 3--The Effect of IN and IV Delivery on the Intactness of
IgG
[0417] A study was conducted to examine whether IgG remains intact
after IN and IV administration. Specifically, rats were
administered .sup.125I radiolabeled IgG either intranasally or
intravenously and the total intact and degraded IgG was determined
30 min after administration.
[0418] Experimental Design: The rats were anesthetized and IgG was
administered as described above in Example 2. Blood and brain was
sampled and intact IgG was detected.
[0419] Blood was sampled approximately 30 minutes after intranasal
administration prior to perfusing with at least 100 mL of saline
containing protease inhibitors and serum was processed.
[0420] Each blood sample (1.0 mL) was added to glass/tissue
homogenizer containing 2.0 mL of homogenization buffer (H.B., 10 mM
tris buffer, pH 8.0 containing protease inhibitors) and aprotinin
(100 .mu.L per mL blood). The sample was manually homogenized (30
passes) and then transferred into a pre-weighed conical tube (15
mL) and stored on ice. Triplicate 25 samples were removed for gamma
counting.
[0421] The sample was centrifuged at 1,000.times.g (3,160 rpm) for
10 minutes at 4.degree. C. Blood supernatant was removed into a
pre-weighed ultracentrifuge tube and stored on ice. The extraction
procedure was repeated on the blood pellet a second time (i.e. same
volume of homogenization buffer added to conical test tube
containing pellet, inverted several times to dislodge the pellet,
transferred into glass homogenizer, homogenized with 15 passes,
transferred to same pre-weighed conical test tube, centrifuged, and
blood supernatant removed). All blood supernatant was pooled and
stored in the same pre-weighed conical tube. The extraction
procedure was repeated on the blood pellet a third time. Triplicate
25 .mu.L samples from pooled blood supernatant were remove for
gamma counting.
[0422] 2 mL of the pooled blood supernatant was ultracentrifuged at
5,000.times.g (7,071 rpm) for 90 minutes at 4.degree. C. to in a
100 kDa filter tube. After the first two rats, it was found that 2
mL took a lot of time to filter so for and animals that followed,
we centrifuged only 1 mL of the pooled blood supernatant. At the
same time, 2 mL of the pooled blood supernatant in the
ultracentrifuged at 5,000.times.g (7,071 rpm) for 90 minutes at
4.degree. C. to in a 30 kDa filter tube. After the first two rats,
it was found that 2 mL took a lot of time to filter so for and
animals that followed, only 1 mL of the pooled blood supernatant
was centrifuged.
[0423] And 2 mL of the pooled blood supernatant was
ultracentrifuged at 5,000.times.g (7,071 rpm) for 90 minutes at
4.degree. C. to in a 10 kDa filter tube. After the first two rats,
it was found that 2 mL took a lot of time to filter, for subsequent
animals only 1 mL of the pooled blood supernatant was centrifuged.
Triplicate 25 .mu.L samples were removed for gamma counting from
the filtrate (100 kDa filter tube), the retentate (100 kDa filter
tube), the filtrate (30 kDa filter tube), the retentate (30 kDa
filter tube), the filtrate (10 kDa filter tube) for gamma counting,
the retentate (10 kDa filter tube) for gamma counting.
[0424] Each brain was removed (on ice), weighed, and placed into a
glass tissue homogenizer. the brain was manually homogenized (40-50
passes) with homogenization buffer at a 1:3 dilution (i.e., 2 mL
buffer per g wet brain) and the homogenate was transferred into a
pre-weighed conical tube (15 mL) and stored on ice. Triplicate 25
.mu.L samples from brain homogenate were removed for gamma
counting. The sample was centrifuged at 1,000.times.g (3,160 rpm)
for 10 minutes at 4.degree. C. Brain supernatant was removed into
pre-weighed ultracentrifuge tube and stored on ice.
[0425] The extraction procedure was repeated a second time on the
pellet (i.e., added same volume of homogenization buffer to conical
test tube containing pellet, inverted several times to dislodge the
pellet, transferred into glass homogenizer, homogenized with 20-30
passes, transferred to same pre-weighed conical test tube,
centrifuged, and removed supernatant). All brain supernatant was
pooled and stored in the same pre-weighed conical tube. The
extraction procedure was repeated a third time on the pellets.
Triplicate 25 .mu.L samples from pooled brain supernatant were
removed for gamma counting.
[0426] 2 mL of the pooled brain supernatant was ultracentrifuged at
5,000.times.g (7,071 rpm) for 90 minutes at 4.degree. C. to in a
100 kDa filter tube. After the first two rats, it was found that 2
mL took a lot of time to filter, for subsequent animals only 1 mL
of the pooled blood supernatant was centrifuged. At the same time,
2 mL of the pooled brain supernatant in the ultracentrifuged at
5,000.times.g (7,071 rpm) for 90 minutes at 4.degree. C. to in a 30
kDa filter tube. After the first two rats, it was found that 2 mL
took a lot of time to filter, for subsequent animals only 1 mL of
the pooled blood supernatant was centrifuged. And 2 mL of the
pooled brain supernatant was ultracentrifuged at 5,000.times.g
(7,071 rpm) for 90 minutes at 4.degree. C. to in a 10 kDa filter
tube. After the first two rats, it was found that 2 mL took a lot
of time to filter, for subsequent animals only 1 mL of the pooled
blood supernatant was centrifuged.
[0427] Triplicate 25 .mu.L samples were removed for gamma counting
from the filtrate (100 kDa filter tube), the retentate (100 kDa
filter tube), the filtrate (30 kDa filter tube), the retentate (30
kDa filter tube), the filtrate (10 kDa filter tube) for gamma
counting, the retentate (10 kDa filter tube) for gamma
counting.
[0428] Results: Two rats received IV IgG liquid preparation and two
rats received IN IgG liquid preparation at an average dose of 52
.mu.L containing 56 .mu.Ci (diluted in saline to a total volume of
500 .mu.L for IV injection) with a 30 min end point. Animals
tolerated the administration well and all survived until the 30 min
desired end point.
[0429] In the brain, approximately 80% of gamma counts from
.sup.125I-labeled IgG after both IN and IV delivery were greater
than 100 kD, suggesting intact protein. In the blood, 100% gamma
counts from .sup.125I-labeled IgG after IV delivery were greater
than 100 kD, suggesting all was intact. With IN delivery, only 33%
of gamma counts from .sup.125I-labeled IgG found in blood was
greater than 100 kD, suggesting that .sup.125I-labeled IgG may be
broken down and enter the blood as part of the clearance process
from the nasal cavity, the brain or both. This also provides
additional evidence that IgG entering the CNS after IN
administration does not travel from the nasal cavity to the blood
to the brain, but rather along direct pathways involving the
olfactory and trigeminal nerves. A summary of the intactness of IgG
in the brain and blood after intranasal or intravenous
administration is presented in Table 14.
TABLE-US-00014 TABLE 14 Summary of Intactness of IgG in the Brain
and Blood. IN IV R1 R3 Avg R2 R4 Avg BLOOD % greater than 100 kD 30
36 33 123 113 118 % greater than 30 kD 34 34 34 123 110 116 %
greater than 10 kD 67 57 62 99 108 104 BRAIN % greater than 100 kD
93 70 81 78 77 77 % greater than 30 kD 87 78 82 83 84 83 % greater
than 10 kD 88 78 83 88 93 91
Example 4--Effect of Intranasal Administration of IgG on Amyloid
Plaque Loads
[0430] A study was conducted to examine whether intranasal
administration of IgG decreases amyloid plaque loads in the brain
in vivo. The purpose of the study was to determine whether chronic
treatment with intranasally delivered IgG at two doses (0.4 g/kg/2
wk and 0.8 g/kg/2 wk) would have any effect on the amyloid plaque
load in a transgenic amyloid mouse model of Alzheimer's
disease.
[0431] Experimental Design: The TG2576 ("TG") amyloid mouse model
was used in this study as a mouse model for Alzheimer's disease and
C57 mice were used as controls. TG2576 mice (cat. #1349-RD1-M) were
acquired from Taconic, Inc. in two batches of 50 spaced one month
apart (Batch 1 and Batch 2). Animals were individually housed with
free access to food and water, and were kept on a 12 hour light
cycle. For dosing with drug in a mg/kg dosing scheme, mice were
divided into three size classes within each treatment group, small,
medium, and large. Size groups were made to include 1/3 of animals
in each size group. Mice were re-evaluated to make new size groups
every two weeks. The mice were divided into five treatment groups
of 20 mice as described in Table 15.
TABLE-US-00015 TABLE 15 Treatment groups assigned for intranasal
administration of IgG. Mouse Strain Drug Administration Description
Tg2576 IN IgG 0.4 g/kg/2 wk "TG-High" Tg2576 IN IgG 0.8 g/kg/2 wk
"TG-Low" Tg2576 IN Saline (control) "TG-Saline" C57 IN IgG 0.8
g/kg/2 wk "WT-High" C57 IN Saline (control) "WT-Saline"
[0432] The mice were ordered and received in the animal facility at
2 months of age and were singly housed and aged for 6 months. At 8
months of age, the mice were acclimated to handling for awake
intranasal delivery over the course of 1 month. Mice were then
intranasally treated with IgG or saline three times/week for 7
months. At 16 months of age, behavioral testing occurred for 5
weeks while intranasal treatment continued. At .about.17 months of
age, 12 mice/group were euthanized and brain tissue was collected
for analysis.
[0433] IgG and saline for IN delivery was prepared Friday
afternoons from stocks sent by Baxter, and stored at
.about.4.degree. C. for use the following week. Solutions were made
to deliver a dose of either 0.4 mg/kg/2 wk IgG or 0.8 mg/kg/2 wk
IgG, and were made to deliver a total of 24 .mu.L. Drug was also
made to cater to each of the three size classes within a treatment
group.
[0434] Mice were acclimated to handling for a period of two-four
weeks before the onset of intranasal dosing. Acclimation to
handling was important, as it helped ensure a correct body position
for maximum effectiveness of awake intranasal drug delivery. In
addition, mice that have not been properly accustomed to this
process can have a severe anxiety reaction after dosing. Mice spent
about 1-3 days on each of nine steps before progressing to the next
step, depending upon the animal's comfort to handling. The mouse's
stress level was used as a measure of progress. This means
monitoring the mouse's movements, the amount/frequency of
urination, defecation, trembling, and biting. If a mouse had a high
stress response, it remained on that step before progressing to the
next until the response is reduced. A sample acclimation schedule
can be seen in Table 16. Acclimation of the mice progressed through
the following once-a-day steps. The steps were not performed more
than once per day in order to minimize the anxiety in the mice.
[0435] First, the mouse was placed in the palm of the hand for a
period of two to three minutes, no more than one foot above the
cage top, as animals frequently jumped during this introductory
step. If the mouse attempted to crawl out of the hand and up one's
arm, the mouse was lifted by the base of the tail and placed back
in one's hand. Second, the mouse was placed in the palm of the hand
for three minutes and petted gently. The mouse was petted
directionally from head to tail, while allowing the animal to move
about freely. Third, the mouse was placed in the palm of the hand
for three minutes while massaging behind the ears (lightly pinching
together the skin on the back of the neck using the thumb and
pointer finger). Fourth, the mouse was held/lifted by the scruff of
its neck for 30 seconds, letting the mouse rest on the cage top for
30 seconds before repeating the hold again. Fifth, the mouse was
held using the intranasal grip, without inverting the animal, for a
period of 30 seconds and then released back to the cage top. This
was repeated a second time after a one-minute rest period. Sixth,
the mouse was held with the intranasal grip while inverting the
animal so its ventral side was facing up towards the ceiling with
the animal's neck is parallel to the floor. This position was held
for 30 seconds and was then repeated a second time after a
one-minute rest period. If the mouse freed itself from the grip,
the mouse was put back on the cage top and re-gripped. If the
mouse's stress level increased too much, the mouse was returned it
to the cage. Seventh, the mouse was held with the intranasal grip,
inverted and a pipettor with an empty tip was briefly placed over
each nostril for 30 seconds. This step was repeated after a
one-minute rest period. Eighth, the mouse was held with the
intranasal grip, inverted, and intranasally administered 6 .mu.l of
saline into the left and right nare. Ninth, the mouse was held with
the intranasal grip, inverted, and intranasally administered 6
.mu.l of saline into the left and right nare twice placing the
animal back on the cage top in between.
TABLE-US-00016 TABLE 16 Sample schedule for acclimation to awake IN
drug delivery. Day # Day Action 1 M Hold for ~2-3 min 2 Tu Hold for
~2-3 min 3 W Hold and pet ~2-3 min 4 Th Hold and pet ~2-3 min 5 F
Lightly pinch/scruff 6 M Lightly pinch/scruff 7 Tu Scruff and lift
8 W Scruff and lift 9 Th Intranasal Grip 10 F Intranasal Grip 11 M
Intranasal (IN) Grip and Invert 12 Tu Intranasal (IN) Grip and
Invert 13 W IN Grip, Invert, empty pipette tip 14 Th IN Grip,
Invert, empty pipette tip 15 F IN Grip, Invert, deliver 1 round
saline to each nare 16 M IN Grip, Invert, deliver 1 round saline to
each nare 17 Tu IN Grip, Invert, deliver 2 rounds saline to each
nare 18 W IN Grip, Invert, deliver 2 rounds saline to each nare
[0436] For awake intranasal delivery of drug, the intranasal grip,
each mouse was restrained twice and held with their necks parallel
to the floor while a volume of 24 .mu.l of liquid was administered.
Specifically, un-anesthetized mice were grabbed by the scruff of
their necks and held gently, but firmly, in an inverted position so
that the mouse cannot move around. Each mouse was given four 6
.mu.l nose drops (alternating nares) using a 20-.mu.l pipettor.
Intranasal drug delivery began when mice were 9 months of age.
[0437] At 16 months of age, mice were subjected to a five week
battery of behavioral tests to assess for memory, sensorimotor, and
anxiolytic changes. These included Morris water maze hidden and
visual platform tests (reference memory, visual ability), radial
arm water maze (working memory), passive avoidance task (memory),
Barnes maze (memory), open field test (exploratory behavior),
elevated plus maze (anxiety), and rotarod (motor skills).
[0438] After behavior, 12 mice from each treatment group were
euthanized and their brains collected for biochemical analyses.
These analyses include immunohistochemistry (IHC) for amyloid
plaques, inflammatory markers, and soluble and insoluble
amyloid.
[0439] Prior to euthanasia via transcardial perfusion, mice were
anesthetized with sodium pentobarbital (60 mg/kg diluted 1:4 with
sterile saline). A first booster of half the full dose was given
followed by additional quarter-dose boosters, if necessary. The
level of anesthesia and sensitivity to pain was monitored every
five minutes throughout the procedure by testing reflexes including
pinching the hind paw and tail. Mice were then euthanized with
transcardial perfusion with 15 ml ice cold saline (no protease
inhibitor needed) and blood was collected from the heart. Briefly,
the arms of the mouse were taped down. The skin was cut to expose
the sternum. A hemostat was used to hold the sternum while blunt
dissection scissors were used to cut vertically on both sides of
the sternum making an incision with a V-shape to expose the heart.
Blood was collected from the heart prior to perfusion and processed
into serum. A small hole in the left ventricle was made using a
24-gauge cannula. The cannula was inserted into the aorta and held
in place. Extension tubing (filled with 5 mL of 0.9% NaCl) was
attached to the cannula and the animal was manually perfused with
15 ml saline.
[0440] Blood was spun down and serum divided into two aliquots. One
aliquot was 50 and will be eventually pooled and sent for analyses
of overall health of the treatment group. The remaining serum was
placed into its own tube and snap frozen for other analyses.
[0441] The brain was collected and hemisected sagitally in a mouse
brain matrix. The left half of the brain was dissected into
olfactory bulbs, cortex/hippocampus mix, septum,
midbrain/diencephalon, brainstem (down to the V of the upper
cervical spinal cord), and cerebellum. These tissues were placed
into microcentrifuge tube and snap frozen in liquid nitrogen. The
right half was left in the matrix and sliced 3 mm from the
centerline. The inner portion towards the center of the brain was
post-fixed in formalin (in a 15 ml conical tube filled with 10 ml
formalin) and sliced for IHC analyses. The outer portion was snap
frozen in liquid nitrogen for eventual analysis for
inflammation.
[0442] The medial 3 mm sagittal section of the right half of the
mouse brain was fixed by placing them each into 20 mL of 10%
formalin. These samples were fixed for several hours at room
temperature and then overnight at 4.degree. C. on slow moving
rocker. The fixed sagittal brain sections were placed medial side
down into labeled pathology cassettes. The pathology department at
Region's Hospital conducted the paraffin processing and embedding
(dehydrate, infiltrate with paraffin, mount into paraffin blocks).
The paraffin blocks were blinded by coding/relabeling.
[0443] The paraffin blocks were sectioned at a thickness of 5 .mu.m
using the Leica RM2235 microtome and collected on Superfrost Plus
microscope slides (Cardinal Health, cat# M6146-PLUS). Seven
sections were collected per mouse, with at least/approximately 100
.mu.m of tissue removed between tissue section collections (labeled
1-6 from, medial to lateral). To increase the quality of the
sections to be stained, a dissection microscope was used to
identify and remove one of the seven sections.
[0444] Slides were deparaffinized and hydrated. Specifically, the
slides were placed in a glass staining jar rack for easy transfer
between staining dishes. The paraffin wax was removed with xylene
washes (clearing) and then hydrated with ethanol/water.
Specifically, the slides were washed in xylene three times for five
minute intervals, washed in 100% ethanol two times for five minute
intervals, washed in 95% ethanol one time for five minutes, rinsed
in running water for five minutes, and rinsed in PBS for five
minutes.
[0445] Heat induced epitope retrieval (HIER) was used to pretreat
the slides prior to antibody staining. A Tris/EDTA Buffer (pH 9)
was used. The slides were immersed in a steamer proof dish
containing the Target Retrieval Solution (Tris/EDTA pH 9)
pre-warmed to 70.degree. C. The dish with slides was then placed in
the steamer and incubate for 30 minutes at 97.degree. C. The
steamer was turned off and allowed to cool to at least 65.degree.
C. The container of slides was removed from the steamer and allowed
to cool for another 10-15 minutes. The slides were then removed
from the container and rinsed in PBS for 10 min in a coplan
jar.
[0446] Non-specific binding sites were then blocked with normal
serum blocking solution (300 .mu.L/slide) for 1 hour in a humidity
chamber. Sections were incubated in a humid box with primary
antibody against amyloid (purified Anti-Beta-Amyloid, 17-24 (4G8)
Monoclonal Antibody, from Covance (SIG-39220)) at a 1:200 dilution
in primary antibody dilution buffer (0.01 M PBS pH 7.2) for 1 hour
at room temperature. Sections were incubated in secondary antibody
(Goat anti-mouse IgG, Alexa Fluor 647 (2 mg/ml) from Invitrogen
(A21235)) dilution buffer (0.01 M PBS, pH 7.2) with a 1:200
secondary concentration for 1 hr at room temperature.
[0447] Slides were then counterstained with DAPI. Diluted 300 nM
DAPI in PBS was used. 1 .mu.l of 14.3 mM DAPI stock was diluted
into 48 ml PBS, vortexed, and mix thoroughly. The DAPI solution was
poured into coplan jar containing the slides. The slides were
incubate for 20 min at RT. The slides were rinsed quickly in PBS,
then 2.times.10 min in washing buffer, followed by a 10 min
incubation in PBS.
[0448] Immediately after staining, the slides were then dehydrated,
cleared, and mounted. Specifically, the slides were incubated in
95% ethanol for 5 minutes, 100% ethanol for two five minute
increments, xylene for three five minute increments, and mounted
with a coverslip in DPX without letting the specimen dry. The
mounted slides were stored at room temperature.
[0449] Images of the fluorescently stained plaques were captured
with the AZ100 Multizoom Macroscope with the C1si Spectral Confocal
attachment and an AZ Plan Apo 4.times. objective. Initial
localization and focusing of the hippocampus and cortex was
conducted through epifluorescence imaging using filters for the
DAPI stain. The scope was then switched to confocal imaging using
the 637 nm laser for acquisition of the IHF-labeled amyloid. Fine
tuning within the z-axis for optimal signal detection was confirmed
with a 512.times.512 pixel resolution. Images were then captured at
1024.times.1024 with the Nikon EZ-C1 software and the raw image
files were saved in Nikon's ".ids" file format. Corresponding tiff
files of the 637 nm channel were generated using Fiji (ImageJ). The
tiff files were then converted to 8-bit images (from 16-bit) and
the contrast was enhanced by 0.5% through batch processing (Macro
programming) in Fiji (ImageJ).
[0450] Plaques were quantitated in selected regions of interest in
the hippocampus and cortex by determining the average number of
plaques detected in each region and by determining the percent area
covered by plaques within each region. Image processing and
analysis was conducted in Fiji. Plaques were defined within Fiji by
using the particle analysis and the threshold function to select a
minimum pixel value that defined each identified particle as
qualifying as a plaque. These values were determined by analyzing
multiple positive and negative controls and verifying which values
correctly identified the plaques in these control slides. The
region of interest within each image was chosen by a blinded
researcher who was instructed to place the region of interest in
the position that would maximize the inclusion of plaques. The size
(pixels) and number of plaques identified were copied into excel
for data analysis. The plaques were then characterized by their
relative size. The plaque sizes reported in this study refer to the
calculated radius of a plaque assuming the particle conformed to
the shape of a perfect circle. The number of plaques and percent
area covered by plaques calculated from each region of interest was
used as a single data point in comparing the treatment groups. Two
tailed t-tests were used to assess the significance between
groups.
[0451] Prior to staining the complete set of collected tissue
sections, an initial verification of the staining and microscopy
analysis was conducted with relevant staining controls. These
controls included, a positive control using sections from one of
the transgenic mice receiving saline, negative controls in which
either the primary or secondary antibody incubation was omitted
from the staining procedure and a negative control using sections
from one of the wild-type mice receiving saline. Additional
controls, including the titration of primary and secondary
antibodies and the comparison of different epitope retrieval
methods have been conducted previously in our lab using these
antibodies and the same experimental procedure.
[0452] Tissue supernatants were analyzed using kits from Life
Technologies (formerly Invitrogen; Carlsbad, Calif.; part #s
KHB3482 (A.beta.40) and KHB3442 (A.beta.42)). Generally, the proper
dilutions were first determined with three samples from either TG
or WT mice, and then all samples were run at that dilution. Samples
were quantified using a polynomial equation fit to a standard
curve. Quantities of AB measured in the wells were then corrected
for dilutions and total protein (as determined by a BCA assay).
[0453] Results:
[0454] Immunohistochemical measurement of amyloid plaques in brain
tissue slices demonstrated that there was a significant drug
effect. Both groups of TG mice administered IgG intranasally had
significantly decreased plaque loads in the cortex (FIGS. 3A, 3B,
and 3E).
[0455] Nasal administration of both the low dose and high dose of
IgG significantly reduced the total percent area covered by plaques
in the cortex of TG2576 mice (FIG. 3A). The percent area covered by
plaques decreased by 25.7% (low dose; p=0.014) and 24.3%, (high
dose; p=0.037), respectively. The change in the percent area
covered by plaques was slightly more pronounced at 27.1% for the
low dose and 26.0% for the high dose when the minimum threshold for
defining a plaque was increased from a radius of 25 .mu.m to 50
.mu.m (p values of 0.01 and 0.026, respectively). The decrease in
plaque load was also found to be significant when the minimum
threshold was set at 100 .mu.m (p values of 0.035 and 0.021,
respectively). A change in the percent area covered by plaques was
not apparent when the smaller plaques (less than 50 .mu.m radius)
were used exclusively in the analysis. Thus, plaque reduction in
the cortex appears to be more pronounced plaques larger than 50
.mu.m.
[0456] The number of plaques in the cortex of both low dose and
high dose IgG treatment groups showed a trend toward a decrease in
the numbers of plaques detected (FIG. 3B). This decrease reached
significance in the low dose IgG treatment group when small plaques
(less than 50 .mu.m radius) were not included in the analysis.
Specifically, treatment with intranasally administered IgG provided
a significant reduction in plaque load when the data were analyzed
by inclusion of plaques having a radius of from 50 .mu.m to 100
.mu.m, greater than 100 .mu.m and greater than 50 .mu.m. The
decrease in plaque load reached significance for the high dose IgG
treatment group when the radius of analyzed plaques was set at
greater than 100 .mu.m.
[0457] In contrast to the results seen in the brain cortex, IgG
treatments did not result in a significant change in either the
percent area covered by plaques or the numbers of plaques detected
in the hippocampus (FIGS. 3C and 3D). Although intranasal
administration of both low and high dose IgG appeared to result in
a slightly reduced plaque load in the hippocampus, the reduction
was minimal and did not reach significance in this region of the
brain.
[0458] Immunofluorescent staining of amyloid plaques in the
hippocampus and cortex of aged TG mice is depicted in FIG. 3E. As
show, there is a decrease staining for amyloid plaques in the
hippocampus and cortex in mice that were treated with low and high
IgG doses compared to TG mice treated with saline.
Example 5--Effect of Intranasally Administered IgG on Soluble and
Insoluble AB40 and AB42
[0459] A study was conducted to assess the efficacy of chronic
intranasal (IN) administration of IgG at two doses in a transgenic
amyloid mouse model. Specifically, measurements of the soluble and
insoluble amyloid beta peptides A.beta.40 and A.beta.42 were taken
in wild type and Tg2576 (amyloid mouse model) pre- and
post-intranasal IgG administration. The purpose of the study was to
determine whether chronic treatment with intranasally delivered IgG
at two doses (0.4 g/kg/2 wk and 0.8 g/kg/2 wk) would have any
effect on the amyloid plaque load in a transgenic amyloid mouse
model of Alzheimer's disease.
[0460] Experimental Design:
[0461] As described in Example 4, the TG2576 ("TG") amyloid mouse
model was used in this study as a mouse model for Alzheimer's
disease and C57 mice were used as controls. The handling of the
mice, preparation of drug, and administration of drug was conducted
as described above in Example 4.
[0462] The mice were divided into five treatment groups of 20 mice
as described in Table 15. At approximately 17 months of age and 12
months of treatment, 12 mice from each treatment group were
euthanized and the concentration of the A.beta.40 and A.beta.42
amyloid peptides in the brains of the TG and control mice were
measured by ELISA to determine whether amyloid plaque
concentrations changes could be detected.
[0463] A.beta.40 and A.beta.42 were measured by ELISA using
Invitrogen ELISA kits. The ELISA kits were stored in refrigerator
until they were ready to use. The kits were removed from
refrigerator and allowed to warm to room temperature before
use.
[0464] Standards and samples were run in duplicate. The samples and
standards were run in a protease inhibitor cocktail with 1 mM AEBSF
(a serine protease inhibitor). AEBSF was important because serine
proteases can rapidly degrade A.beta. peptides. The samples were
kept on ice until they were ready to be applied to the ELISA
Plate.
[0465] Sample matrix has a dramatic impact on A.beta. recovery. To
ensure accurate quantitation, the standard curves were generated in
the same diluent as the samples. A standard reconstitution buffer
was prepared by dissolving 2.31 grams of sodium bicarbonate in 500
mL of deionized water and the pH was adjusted using 2 N sodium
hydroxide until the pH was 9.0.
[0466] The standards for a quantitative standard curve were
prepared. The Hu A.beta.42 Standard was used. The Hu A.beta.42
Standard was allowed to equilibrate to room temperature (RT) and
then reconstituted to 100 ng/mL with Standard Reconstitution Buffer
(55 mM sodium bicarbonate, pH 9.0). The standard mixture was
swirled and mixed gently and allowed to sit for 10 minutes to
ensure complete reconstitution. The standard was then briefly
vortexed prior to preparing standard curve. Generation of the
standard curve using the AP peptide standard was performed using
the same composition of buffers used for the diluted experimental
samples. 0.1 mL of the reconstituted standard was added to a tube
containing 0.9 mL of the Standard Diluent Buffer and labeled as
10,000 pg/mL Hu A.beta.40. The standard was mixed and then 0.1 mL
of the 10,000 pg/mL standard was added to a tube containing 1.9 mL
Standard Diluent Buffer and labeled as 500 pg/mL Hu A.beta.40. Mix.
The standard was mixed and then 0.15 mL of Standard Diluent Buffer
was added to each of 6 tubes labeled 250, 125, 62.5, 31.25, 15.63,
7.81, and 0 pg/mL Hu A.beta.40 to make serial dilutions of the
standard.
[0467] The samples were then prepared for the plate. Specifically,
the samples were remove from the freezer, allowed to thaw, and
diluted to the desired dilution using dilution buffer provided with
the kit mixed with a protease inhibitor tablet. The samples were
kept on ice until loaded into the wells on the plate.
[0468] The plates were labeled as being either AB40 or AB42 with a
sharpie. 50 ul of standards and sample were added to the
pre-labeled wells. 50 .mu.L of Hu A.beta.40 or A.beta.42 Detection
Antibody solution provided with the kit was added to each well. The
plate was covered and incubated for 3 hours at room temperature
with shaking. Shortly before the 3 hours expired, the Anti-Rabbit
IgG HRP Working Solution was prepared. To make this, 10 .mu.L of
Anti-Rabbit IgG HRP (100.times.) concentrated solution was diluted
in 1 mL of HRP Diluent for each 8-well strip used in the assay and
labeled as Anti-Rabbit IgG HRP Working Solution.
[0469] The solution was thoroughly decanted from wells and the
wells were washed 5 times with 300 .mu.L of wash solution. The
plates were banged hard on lab bench to be sure it was dry. 100
.mu.L of the Anti-Rabbit IgG HRP working solution was added to each
well. The plate was covered and allowed to sit at room temp for 30
min. The solution was thoroughly decanted from wells and the wells
were washed 5 times with 300 .mu.L of wash solution. The plates
were banged hard on lab bench to be sure it was dry. 100 .mu.L of
Stabilized Chromogen was added to each well and the plate was
immediately placed in the dark and allowed to sit for 20 min. 100
.mu.L of Stop Solution was added to each well and the sides of the
plate were gently tapped to mix.
[0470] The absorbance of each well was read at 450 nm having
blanked the plate reader within 30 minutes after adding the Stop
Solution. The concentrations were determined using the standard
curve.
[0471] Results:
[0472] The ELISA plates for both A.beta.40 and A.beta.42 purchased
from Invitrogen yielded consistent standard curves. The best
dilutions of brain supernatant for samples for soluble A.beta.40
and A.beta.42, and insoluble A.beta.40 and A.beta.42 were
10.times., undiluted, 10000.times., and 2500.times., respectively.
Brain concentrations of each protein were analyzed by first
determining the concentration of the sample in the well in the
ELISA plate based on the standard curve. These values were then
corrected for dilution of supernatant, dilution from the extraction
process, and then given a correction factor from a BCA analysis of
total protein extracted. For each protein, between 1 and 4 samples
were excluded for either being statistical outliers or being too
high/low to fit within the standard curve. A summary of the soluble
and insoluble A.beta.40 concentrations are presented in Table 17
and Table 18. A summary of the soluble and insoluble A.beta.42
concentrations are presented in Table 19 and Table 20. The ratios
of soluble A.beta.40/A.beta.42 are provided in Table 21 and the
ratios of insoluble A.beta.40/A.beta.42 are provided in Table
22.
TABLE-US-00017 TABLE 17 Soluble A.beta.40 detected in brain. Mouse
sac Date Concentration Mouse sac Date Concentration order # Group
measured (pg/ml) order # Group measured (pg/ml) 1 TG-Low 1-Oct 9078
33 TG-Saline 1-Oct 3940 6 TG-Low 1-Oct 3964 38 TG-Saline 1-Oct 1328
11 TG-Low 1-Oct 3110 43 TG-Saline 1-Oct 1983 16 TG-Low 1-Oct 2788
48 TG-Saline 1-Oct 3656 21 TG-Low 1-Oct 3934 53 TG-Saline 1-Oct
6650 26 TG-Low 1-Oct 3747 58 TG-Saline 1-Oct 6159 31 TG-Low 1-Oct
3796 4 WT-High 9-Oct 0 36 TG-Low 1-Oct 5450 9 WT-High 9-Oct 0 41
TG-Low 27-Sep 5261 14 WT-High 9-Oct 0 46 TG-Low 1-Oct 2082 19
WT-High 9-Oct 0 51 TG-Low 1-Oct 2520 24 WT-High 9-Oct 0 56 TG-Low
1-Oct 9448 29 WT-High 9-Oct 0 2 TG-High 1-Oct 3061 34 WT-High 9-Oct
0 7 TG-High 1-Oct 1814 39 WT-High 9-Oct 0 12 TG-High 1-Oct 4681 44
WT-High 9-Oct 0 17 TG-High 1-Oct 2509 49 WT-High 9-Oct 0 22 TG-High
1-Oct 7869 54 WT-High 9-Oct 0 27 TG-High 1-Oct 6363 59 WT-High
9-Oct 0 32 TG-High 1-Oct 5541 5 WT-Saline 9-Oct 0 37 TG-High 27-Sep
5190 10 WT-Saline 9-Oct 0 42 TG-High 1-Oct 3609 15 WT-Saline 9-Oct
0 47 TG-High 1-Oct 1122 20 WT-Saline 9-Oct 0 52 TG-High 1-Oct 12163
25 WT-Saline 9-Oct 0 57 TG-High 1-Oct 1502 30 WT-Saline 9-Oct 0 3
TG-Saline 27-Sep 3708 35 WT-Saline 9-Oct 0 8 TG-Saline 1-Oct 4833
40 WT-Saline 9-Oct 0 13 TG-Saline 1-Oct 1673 45 WT-Saline 9-Oct 0
18 TG-Saline 1-Oct 4039 50 WT-Saline 9-Oct 0 23 TG-Saline 1-Oct
2373 55 WT-Saline 9-Oct 0 28 TG-Saline 1-Oct 4133 60 WT-Saline
9-Oct 0 Average Std deviation Std error TG-Low 4598.418 2395.218
691.4399 TG-High 3932.644 1782.644 630.2598 TG-Saline 3706.334
1570.737 473.595 WT-High 0 0 0 WT-Saline 0 0 0
TABLE-US-00018 TABLE 18 Insoluble A.beta.40 detected in brain.
Mouse Date Concentration Concentration Mouse Date Concentration
Concentration sac order # Group measured (pg/ml) (ug/ml) sac order
# Group measured (pg/ml) (ug/ml) 1 TG-Low 8-Oct 10257199 10.26 33
TG-Saline 8-Oct 25512730 25.51 6 TG-Low 8-Oct 11697779 11.70 38
TG-Saline 8-Oct 19414980 19.41 11 TG-Low 8-Oct 7575663 7.58 43
TG-Saline 8-Oct 26032547 26.03 16 TG-Low 8-Oct 8322854 8.32 48
TG-Saline 8-Oct 39277004 39.28 21 TG-Low 8-Oct 28084221 28.08 53
TG-Saline 8-Oct 19280789 19.28 26 TG-Low 8-Oct 22248049 22.25 58
TG-Saline 8-Oct 39064072 39.06 31 TG-Low 8-Oct 14817934 14.82 4
WT-High 18-Oct 79052 0.07905 36 TG-Low 8-Oct 25661660 25.66 9
WT-High 18-Oct 48296 0.04830 41 TG-Low 3-Oct 25537069 25.54 14
WT-High 18-Oct 48256 0.04826 46 TG-Low 8-Oct 9547715 9.55 19
WT-High 18-Oct 9511 0.00951 51 TG-Low 8-Oct 5688511 5.69 24 WT-High
18-Oct 249003 0.24900 56 TG-Low 8-Oct 9606698 9.61 29 WT-High
18-Oct 31520 0.03152 2 TG-High 8-Oct 4410637 4.41 34 WT-High 18-Oct
39666 0.03967 7 TG-High 8-Oct 15713013 15.71 39 WT-High 18-Oct
25225 0.02522 12 TG-High 8-Oct 18125865 18.13 44 WT-High 18-Oct
134629 0.13463 17 TG-High 8-Oct 4945207 4.95 54 WT-High 18-Oct
15163 0.01516 22 TG-High 8-Oct 33296598 33.30 59 WT-High 18-Oct
23228 0.02323 27 TG-High 8-Oct 68491264 68.49 5 WT-Saline 18-Oct
3764 0.00376 32 TG-High 8-Oct 50062749 50.06 10 WT-Saline 18-Oct 37
TG-High 3-Oct 36070736 36.07 15 WT-Saline 18-Oct 10815 0.01081 42
TG-High 8-Oct 26864520 26.86 20 WT-Saline 18-Oct 38643 0.03864 47
TG-High 8-Oct 3774286 3.77 25 WT-Saline 18-Oct 52 TG-High 8-Oct
42493407 42.49 30 WT-Saline 18-Oct 4356 0.00436 57 TG-High 8-Oct
8907543 8.91 35 WT-Saline 18-Oct 3 TG-Saline 3-Oct 13934212 13.93
40 WT-Saline 18-Oct 5701 0.00570 8 TG-Saline 8-Oct 28128069 28.13
45 WT-Saline 18-Oct 14256 0.01426 13 TG-Saline 8-Oct 18908021 18.91
50 WT-Saline 18-Oct 2986 0.00299 18 TG-Saline 8-Oct 18777770 18.78
55 WT-Saline 18-Oct 3072 0.00307 28 TG-Saline 8-Oct 20470065 20.47
60 WT-Saline 18-Oct 3008 0.00301 Average Std deviation Std error
TG-Low 14.92045 8.129698 2.346842 TG-High 24.60567 20.93301 6.31154
TG-Saline 24.43639 8.31837 2.401306 WT-High 0.063959 0.070783
0.021342 WT-Saline 0.009622 0.011582 0.003861
TABLE-US-00019 TABLE 19 Soluble A.beta.42 detected in brain. Mouse
sac Date Concentration Mouse sac Date Concentration order # Group
measured (pg/ml) order # Group measured (pg/ml) 1 TG-Low 1-Oct 1455
33 TG-Saline 2-Oct 626 6 TG-Low 2-Oct 551 38 TG-Saline 2-Oct 393 11
TG-Low 2-Oct 511 43 TG-Saline 2-Oct 562 16 TG-Low 2-Oct 744 48
TG-Saline 2-Oct 432 21 TG-Low 2-Oct 705 53 TG-Saline 1-Oct 1295 26
TG-Low 2-Oct 623 58 TG-Saline 1-Oct 1361 31 TG-Low 2-Oct 463 4
WT-High 9-Oct 0 36 TG-Low 2-Oct 609 9 WT-High 9-Oct 0 41 TG-Low
27-Sep 1564 14 WT-High 9-Oct 0 46 TG-Low 2-Oct 606 19 WT-High 9-Oct
0 51 TG-Low 1-Oct 825 24 WT-High 9-Oct 0 56 TG-Low 1-Oct 1526 29
WT-High 9-Oct 0 2 TG-High 2-Oct 579 34 WT-High 9-Oct 0 7 TG-High
2-Oct 446 39 WT-High 9-Oct 0 12 TG-High 2-Oct 880 44 WT-High 9-Oct
0 17 TG-High 2-Oct 410 49 WT-High 9-Oct 0 22 TG-High 1-Oct 1198 54
WT-High 9-Oct 0 27 TG-High 2-Oct 851 59 WT-High 9-Oct 0 32 TG-High
2-Oct 877 5 WT-Saline 9-Oct 0 37 TG-High 27-Sep 1470 10 WT-Saline
9-Oct 0 42 TG-High 2-Oct 880 15 WT-Saline 9-Oct 0 47 TG-High 2-Oct
290 20 WT-Saline 9-Oct 0 52 TG-High 1-Oct 3050 25 WT-Saline 9-Oct 0
57 TG-High 2-Oct 385 30 WT-Saline 9-Oct 0 3 TG-Saline 27-Sep 791 35
WT-Saline 9-Oct 0 8 TG-Saline 2-Oct 990 40 WT-Saline 9-Oct 0 13
TG-Saline 2-Oct 562 45 WT-Saline 9-Oct 0 18 TG-Saline 2-Oct 733 50
WT-Saline 9-Oct 0 23 TG-Saline 2-Oct 521 55 WT-Saline 9-Oct 0 28
TG-Saline 2-Oct 737 60 WT-Saline 9-Oct 0 Average Std deviation Std
error TG-Low 848.4637 414.464 119.6455 TG-High 751.4925 368.8014
111.1978 TG-Saline 750.1043 315.7884 91.16026 WT-High 0 0 0
WT-Saline 0 0 0
TABLE-US-00020 TABLE 20 Insoluble A.beta.42 detected in brain.
Mouse sac Date Concentration Concentration Mouse sac Date
Concentration Concentration order # Group measured (pg/ml) (ug/ml)
order # Group measured (pg/ml) (ug/ml) 1 TG-Low 3-Oct 4493237 4.49
33 TG-Saline 3-Oct 3705386 3.71 6 TG-Low 3-Oct 7320913 7.32 38
TG-Saline 3-Oct 6032562 6.03 11 TG-Low 3-Oct 2641985 2.64 43
TG-Saline 3-Oct 6871640 6.87 16 TG-Low 3-Oct 1644845 1.64 48
TG-Saline 3-Oct 12292890 12.29 21 TG-Low 3-Oct 10670528 10.67 53
TG-Saline 3-Oct 6847851 6.85 26 TG-Low 3-Oct 2902253 2.90 58
TG-Saline 3-Oct 11978111 11.98 31 TG-Low 3-Oct 5435473 5.44 4
WT-High 18-Oct 32757 0.0328 36 TG-Low 3-Oct 5458033 5.46 9 WT-High
18-Oct 53457 0.0535 41 TG-Low 2-Oct 5807761 5.81 14 WT-High 18-Oct
38889 0.0389 46 TG-Low 3-Oct 2751646 2.75 19 WT-High 18-Oct 5910
0.0059 51 TG-Low 3-Oct 1474153 1.47 24 WT-High 18-Oct 76083 0.0761
56 TG-Low 3-Oct 6022267 6.02 29 WT-High 18-Oct 19765 0.0198 2
TG-High 3-Oct 1984182 1.98 34 WT-High 18-Oct 26479 0.0265 7 TG-High
3-Oct 2733892 2.73 39 WT-High 18-Oct 20134 0.0201 12 TG-High 3-Oct
10072470 10.07 44 WT-High 18-Oct 79499 0.0795 17 TG-High 3-Oct
1786008 1.79 54 WT-High 18-Oct 19179 0.0192 22 TG-High 3-Oct
7201686 7.20 59 WT-High 18-Oct 23360 0.0234 27 TG-High 3-Oct
11756896 11.76 5 WT-Saline 18-Oct 3891 0.0039 32 TG-High 3-Oct
7338861 7.34 10 WT-Saline 18-Oct 3916 0.0039 37 TG-High 2-Oct
10547976 10.55 15 WT-Saline 18-Oct 9542 0.0095 42 TG-High 3-Oct
6116270 6.12 20 WT-Saline 18-Oct 13878 0.0139 47 TG-High 3-Oct
954559 0.95 25 WT-Saline 18-Oct 52 TG-High 3-Oct 14015195 14.02 30
WT-Saline 18-Oct 4040 0.0040 57 TG-High 3-Oct 1353498 1.35 35
WT-Saline 18-Oct 3 TG-Saline 2-Oct 3362018 3.36 40 WT-Saline 18-Oct
4698 0.0047 8 TG-Saline 3-Oct 6261392 6.26 45 WT-Saline 18-Oct
15173 0.0152 13 TG-Saline 3-Oct 6056766 6.06 50 WT-Saline 18-Oct
2862 0.0029 18 TG-Saline 3-Oct 5685368 5.69 55 WT-Saline 18-Oct
3151 0.0032 28 TG-Saline 3-Oct 3122150 3.12 60 WT-Saline 18-Oct
3320 0.0033 Average Std deviation Std error TG-Low 4.718591
2.656887 0.766977 TG-High 5.622391 4.045875 1.219877 TG-Saline
6.565103 3.065648 0.884976 WT-High 0.035956 0.024034 0.007247
WT-Saline 0.006447 0.004669 0.001477
TABLE-US-00021 TABLE 21 Ratios of soluble A.beta.40/A.beta.42.
Ratio of Mouse sac Ratio of Mouse sac AB42/ order # Group AB42/AB40
order # Group AB40 1 TG-Low 0.160295 33 TG-Saline 0.1588125 6
TG-Low 0.13894 38 TG-Saline 0.2960768 11 TG-Low 0.164329 43
TG-Saline 0.2834986 16 TG-Low 0.266987 48 TG-Saline 0.1180902 21
TG-Low 0.179196 53 TG-Saline 0.19472 26 TG-Low 0.16629 58 TG-Saline
0.2209363 31 TG-Low 0.1219 4 WT-High 0 36 TG-Low 0.111683 9 WT-High
0 41 TG-Low 0.297232 14 WT-High 0 46 TG-Low 0.290904 19 WT-High 0
51 TG-Low 0.327174 24 WT-High 0 56 TG-Low 0.161546 29 WT-High 0 2
TG-High 0.189314 34 WT-High 0 7 TG-High 0.245964 39 WT-High 0 12
TG-High 0.188097 44 WT-High 0 17 TG-High 0.163459 49 WT-High 0 22
TG-High 0.152272 54 WT-High 0 27 TG-High 0.13377 59 WT-High 0 32
TG-High 0.158201 5 WT-Saline 0 37 TG-High 0.283163 10 WT-Saline 0
42 TG-High 0.243714 15 WT-Saline 0 47 TG-High 0.258574 20 WT-Saline
0 52 TG-High 0.250769 25 WT-Saline 0 57 TG-High 0.25651 30
WT-Saline 0 3 TG-Saline 0.2132 35 WT-Saline 0 8 TG-Saline 0.204815
40 WT-Saline 0 13 TG-Saline 0.335658 45 WT-Saline 0 18 TG-Saline
0.181432 50 WT-Saline 0 23 TG-Saline 0.219518 55 WT-Saline 0 28
TG-Saline 0.1783 60 WT-Saline 0 Average Std deviation Std error
TG-Low 0.198873 0.075008 0.0217 TG-High 0.20664 0.05204 0.0157
TG-Saline 0.217088 0.061325 0.0177 WT-High 0 0 0 WT-Saline 0 0
0
TABLE-US-00022 TABLE 22 Ratios of insoluble A.beta.40/A.beta.42.
Mouse Mouse sac Ratio of sac Ratio of order # Group AB42/AB40 order
# Group AB42/AB40 1 TG-Low 0.43806 33 TG-Saline 0.14524 6 TG-Low
0.62584 38 TG-Saline 0.31072 11 TG-Low 0.34875 43 TG-Saline 0.26396
16 TG-Low 0.19763 48 TG-Saline 0.31298 21 TG-Low 0.37995 53
TG-Saline 0.35516 26 TG-Low 0.13045 58 TG-Saline 0.30663 31 TG-Low
0.36682 4 WT-High 0.41437 36 TG-Low 0.21269 9 WT-High 1.10685 41
TG-Low 0.22742 14 WT-High 0.80589 46 TG-Low 0.2882 19 WT-High
0.62142 51 TG-Low 0.25915 24 WT-High 0.30555 56 TG-Low 0.62688 29
WT-High 0.62708 2 TG-High 0.44986 34 WT-High 0.66754 7 TG-High
0.17399 39 WT-High 0.79817 12 TG-High 0.5557 44 WT-High 0.5905 17
TG-High 0.36116 54 WT-High 1.26484 22 TG-High 0.21629 59 WT-High
1.00566 27 TG-High 0.17166 5 WT-Saline 1.0335 32 TG-High 0.14659 10
WT-Saline 37 TG-High 0.29242 15 WT-Saline 0.88232 42 TG-High
0.22767 20 WT-Saline 0.35914 47 TG-High 0.25291 25 WT-Saline 52
TG-High 0.32982 30 WT-Saline 0.92744 57 TG-High 0.15195 35
WT-Saline 3 TG-Saline 0.24128 40 WT-Saline 0.82407 8 TG-Saline
0.2226 45 WT-Saline 1.06434 13 TG-Saline 0.32033 50 WT-Saline
0.95864 18 TG-Saline 0.30277 55 WT-Saline 1.02571 28 TG-Saline
0.15252 60 WT-Saline 1.10371 Average Std deviation Std error TG-Low
0.3418191 0.15905 0.04591 TG-High 0.2727456 0.13256 0.04192
TG-Saline 0.2667446 0.06933 0.02001 WT-High 0.7461702 0.28933
0.08724 WT-Saline 0.9087633 0.22479 0.07493
[0473] The most obvious and expected result was that both soluble
and insoluble A.beta.40 and A.beta.42 were drastically higher in
all TG mice than WT mice. Soluble A.beta.40 and A.beta.42 were not
detectable in WT mice, while insoluble A.beta.40 and A.beta.42 were
present, though at about 1000 times lower than in TG mice. The next
most obvious result was that in all TG mice, the concentration of
insoluble A.beta.40 and A.beta.42 was much higher than soluble
A.beta.40 and A.beta.42, roughly about 5000 and 7500 times higher,
respectively.
[0474] Regarding group comparisons among the three TG groups, there
were no significant differences among any of the groups for either
soluble or insoluble A.beta.40 or A.beta.42 using an ANOVA. This
was somewhat surprising for insoluble amyloid as there were clear
differences in plaques in the cortex between drug-treated and
saline-treated TG mice. The most likely explanation is that the
ELISA was not as sensitive to these differences as the IHC slides
of plaques.
Example 6--Effect of Intranasal Administration of IgG on Weight and
Survival
[0475] A study was conducted to assess the efficacy of chronic
intranasal (IN) administration of IgG at two doses in a transgenic
amyloid mouse model. The purpose of the study was to determine
whether chronic treatment with intranasally delivered IgG at two
doses (0.4 g/kg/2 wk and 0.8 g/kg/2 wk) would have any effect on
the mouse weight and survival.
[0476] Experimental Design:
[0477] As described in Example 4, the TG2576 ("TG") amyloid mouse
model was used in this study as a mouse model for Alzheimer's
disease and C57 mice were used as controls. The handling of the
mice, preparation of drug, and administration of drug was conducted
as described above in Example 4.
[0478] The mice were divided into five treatment groups of 20 mice
as described in Table 15. The weight and survival of the mice were
monitored for 103 weeks. The weight of each mouse was recorded
weekly (data not shown).
[0479] Results: These experiments showed that intranasal IgG
increases the lifespan of TG mice. FIG. 4A shows that TG mice have
an increased lifespan when they are administered a high (0.8 g/kg/2
wk) or a low (0.4 g/kg/2 wk) dose of intranasal IgG compared to TG
mice administered saline intranasally (control). FIG. 4B shows that
TG mice administered intranasal IgG had longer lifespans than WT
mice. Although this study begun with 20 mice in each cohort, due to
the mass euthanasia performed to evaluate amyloid plaque content
(as described in Example 5), Kaplan-Meier survival analysis was
performed using the sub-group of 8 mice in each cohort that were
not euthanized. Dosing to the mice in the sub-groups was continued
as described above through the entirety of the experiment.
Example 7--Effect of Intranasal Administration of IgG on Memory
[0480] A study was conducted to examine whether intranasal
administration of IgG affects the memory in the brain in vivo. The
purpose of this study was to examine whether chronic treatment with
intranasally delivered IgG at two doses (0.4 g/kg/2 wk and 0.8
g/kg/2 wk) would have any effect on memory in a transgenic amyloid
mouse model of Alzheimer's disease.
[0481] Experimental Design:
[0482] At 15 months of age, the mice described in Example 4 were
subjected to a six week battery of behavioral tests to assess for
memory, sensorimotor, and anxiolytic changes. These included Morris
water maze hidden and visual platform tests (reference memory,
visual ability), radial arm water maze (working memory), passive
avoidance task (memory), Barnes maze (memory), open field test
(exploratory behavior), elevated plus maze (anxiety), and rotarod
(motor skills).
[0483] Results:
[0484] For each behavioral test, comparison data was analyzed using
T-tests as described above in Table 23. Statistical tests were
performed on data after removal of both statistical outliers and
non-compliant mice, which were specified for each behavioral test.
Data was first analyzed by comparing WT-saline (WT-Sal) mice to
TG-saline (TG-Sal) mice to determine whether there is a transgenic
(model) effect for that test. Comparisons between all TG and all WT
mice were also performed. Although the latter analysis is
confounded by drug treatment, it gains power by increasing sample
size and serves to give an overall picture of a potential
transgenic (model) effect. Comparisons were made among individual
drug treatment groups. Specifically, the drug treated TG groups
were compared directly to the TG-saline group to determine whether
the drug had any effect.
TABLE-US-00023 TABLE 23 T-tests used to evaluate results of
behavioral studies in wild type and Alzheimer's disease mouse
models administered IgG intranasally. Comparison Reason for
Comparison WT-saline vs. TG-saline To determine whether there is a
transgenic effect of the model. WT-all vs. TG-all (all = saline and
To provide a larger scale view of the transgenic effect of the
model. IN IgG) TG-saline vs TG-low dose IN IgG To determine whether
TG mice treated with the low dose of IgG performed differently than
TG mice treated with saline. TG-saline vs TG-high dose IN IgG To
determine whether TG mice treated with the high dose of IgG
performed differently than TG mice treated with saline.
[0485] Overall, in the three visio-spatial memory tests, mice
learned over time, and there was generally improved performance in
the WT mice as compared to the TG mice, which was expected. There
was also a difference between WT and TG mice in the Elevated Plus
Maze. There were minimal observed differences in the Rotarod and
Open Field Tests, but differences were not expected. Compliance was
only a problem in the Barnes Maze, however, when non-compliant mice
were removed the learning trends were present, and the model effect
mirrored those seen in the MWM and RAWM.
[0486] The Morris Water Maze (MWM) Hidden Platform.
[0487] MWM is a standard test of spatial memory. MWM performance
was assessed using hidden-platform testing (4 days, 4 trials/day).
Before trials began, the mice were acclimated to swimming in the
water. For each of these blocks of trials, mice were randomly
dropped into four quadrants within the MWM (round tub with water)
and allowed to swim for 60 seconds or until they reached the
platform. The mouse's ability to reach the platform depended on his
ability to remember visual cues from previous trials and their
location in relation to the platform. Mice that did not reach the
platform after 60 seconds were placed on the platform. Mice were
allowed to remain/rest on the platform for 20 seconds between
trials. All data was recorded using MouseApp software, which
records escape latency.
[0488] The Morris Water Maze Visual Platform is designed to assess
visual ability. It was run just like the MWM hidden platform,
except the platform was raised just above the surface of the water,
has a flag on top to identify it, and stripes along the side to
make it more visual. It was only run for one day. Analysis was
performed the same as with the MWM hidden platform tests.
[0489] Overall, the Morris Water Maze Hidden Platform tests showed
that there was a clear trend of learning both throughout the week
and during individual days, demonstrating that the test was
effective for measuring memory. Escape latencies were lowest during
days 3 and 4, and were especially lowest during trials 3 and 4 on
these days.
[0490] There was evidence of a transgenic model effect. Table 25
and Table 26 show that both WT groups had lower escape latencies
than all three TG groups on days 3 and 4. WT-Sal mice had lower
escape latencies than TG-Sal mice (Table 24, Table 25, Table 26,
Table 27, and Table 28). However, when the WT and TG groups were
put together, there were several significant differences, including
B1-T2, B3-T4, B4-T1, B4-T3, and B4-T4 (p<0.05 or 0.01; Table 24,
Table 27, and Table 28). Much of the power for this difference came
from the TG-high mice, which performed particularly well in this
task.
TABLE-US-00024 TABLE 24 Summary of T-tests for specific comparisons
in behavior tests. Tests are 2-sided and unpaired. Reported numbers
are p-values. WT-Sal vsTG- WT-All vs TG-Sal vsTG- TG-Sal vsTG- Test
Measure Block Trial Sal TG-All Low High RAWM Escape 1 1 0.023 0
0.883 0.539 RAWM Escape 1 2 0.689 0.558 0.141 0.298 RAWM Escape 1 3
0.088 0.215 0.592 0.15 RAWM Escape 1 4 0.615 0.358 0.803 0.335 RAWM
Escape 2 1 0.159 0.215 0.653 0.607 RAWM Escape 2 2 0.194 0.926
0.675 0.13 RAWM Escape 2 3 0.161 0.497 0.06 0.046 RAWM Escape 2 4
0.446 0.219 0.271 0.918 RAWM Escape 3 1 0.959 0.767 0.619 0.65 RAWM
Escape 3 2 0.069 0.001 0.5 0.202 RAWM Escape 3 3 0.995 0.806 0.185
0.597 RAWM Escape 3 4 0.281 0.002 0.198 0.257 RAWM Escape 4 1 0.785
0.487 0.217 0.701 RAWM Escape 4 2 0.35 0.274 0.433 0.627 RAWM
Escape 4 3 0.357 0.149 0.348 0.292 RAWM Escape 4 4 0.232 0.008
0.583 0.513 RAWM Errors 1 1 0.538 0.001 0.154 0.881 RAWM Errors 1 2
0.284 0.105 0.06 0.233 RAWM Errors 1 3 0.062 0.196 0.236 0.089 RAWM
Errors 1 4 0.443 0.255 0.577 0.293 RAWM Errors 2 1 0.656 0.753
0.223 0.136 RAWM Errors 2 2 0.227 0.642 0.606 0.022 RAWM Errors 2 3
0.17 0.706 0.247 0.139 RAWM Errors 2 4 0.719 0.385 0.601 0.954 RAWM
Errors 3 1 0.86 0.678 0.783 0.551 RAWM Errors 3 2 0.043 0.002 0.207
0.336 RAWM Errors 3 3 0.946 0.75 0.55 0.526 RAWM Errors 3 4 0.393
0.02 0.998 0.391 RAWM Errors 4 1 0.437 0.229 0.397 0.814 RAWM
Errors 4 2 0.064 0.048 0.154 0.263 RAWM Errors 4 3 0.357 0.214
0.296 0.432 RAWM Errors 4 4 0.135 0.007 0.935 0.566 MWM hid Escape
1 1 0.262 0.186 0.007 0.095 MWM hid Escape 1 2 0.069 0.086 0.663
0.532 MWM hid Escape 1 3 0.62 0.26 0.5 0.419 MWM hid Escape 1 4
0.663 0.171 0.111 0.189 MWM hid Escape 2 1 0.882 0.555 0.357 0.702
MWM hid Escape 2 2 0.24 0.568 0.091 0.963 MWM hid Escape 2 3 0.393
0.71 0.802 0.276 MWM hid Escape 2 4 0.986 0.256 0.638 0.963 MWM hid
Escape 3 1 0.475 0.419 0.906 0.163 MWM hid Escape 3 2 0.681 0.173
0.109 0.549 MWM hid Escape 3 3 0.905 0.106 0.908 0.864 MWM hid
Escape 3 4 0.355 0.072 0.874 0.6 MWM hid Escape 4 1 0.672 0.045
0.044 0.102 MWM hid Escape 4 2 0.147 0.127 0.264 0.991 MWM hid
Escape 4 3 0.592 0.03 0.585 0.802 MWM hid Escape 4 4 0.507 0.02
0.436 0.192 Barnes Escape 1 1 0.681 0.35 0.946 0.696 Barnes Escape
1 2 0.925 0.643 0.587 0.337 Barnes Escape 1 3 0.098 0.277 0.876
0.408 Barnes Escape 2 1 0.478 0.576 0.542 0.63 Barnes Escape 2 2
0.673 0.64 0.132 0.534 Barnes Escape 2 3 0.501 0.529 0.284 0.496
Barnes Escape 3 1 0.943 0.313 0.189 0.764 Barnes Escape 3 2 0.764
0.88 0.678 0.626 Barnes Escape 3 3 0.581 0.274 0.826 0.657 Barnes
Escape 4 1 0.623 0.052 0.072 0.606 Barnes Escape 4 2 0.138 0.21
0.29 0.482 Barnes Escape 4 3 0.916 0.986 0.925 0.845 Barnes Errors
1 1 0.485 0.851 0.807 0.75 Barnes Errors 1 2 0.057 0.033 0.436
0.416 Barnes Errors 1 3 0.231 0.414 0.541 0.603 Barnes Errors 2 1
0.48 0.519 0.731 0.434 Barnes Errors 2 2 0.085 0.15 0.41 0.383
Barnes Errors 2 3 0.423 0.079 0.17 0.341 Barnes Errors 3 1 0.979
0.894 0.875 0.759 Barnes Errors 3 2 0.54 0.741 0.802 0.535 Barnes
Errors 3 3 0.864 0.952 0.806 0.764 Barnes Errors 4 1 0.928 0.245
0.185 0.355 Barnes Errors 4 2 0.885 0.965 0.736 0.758 Barnes Errors
4 3 0.013 0.116 0.19 0.707 MWM vis Escape 1 1 0.074 0.282 0.134
0.589 MWM vis Escape 1 2 0.507 0.222 0.665 0.597 MWM vis Escape 1 3
0.863 0.237 0.516 0.959 MWM vis Escape 1 4 0.898 0.448 0.46 0.593
Open field Line n/a n/a 0.534 0.138 0.112 0.688 Crossings Open
field Velocity n/a n/a 0.38 0.057 0.25 0.618 Elev. plus Time in
open n/a n/a 0.036 0.001 0.726 0.225 arms Elev. plus Frequency in
n/a n/a 0.034 0 0.372 0.13 open arms Rotarod Best run n/a n/a 0.98
0.153 0.64 0.875 Rotarod Average run n/a n/a 0.856 0.131 0.557
0.973 Pass. Avoid Escape Learn n/a 0.032 0.001 0.952 0.825 Pass.
Avoid Escape Test n/a 0.072 0 0.34 0.207 Underlined cells p <
0.05; Boxed
TABLE-US-00025 TABLE 25 Average escape latencies (sec) from the
Morris Water Maze tests. Group Day 1 Day 2 Day 3 Day 4 TG-Low (N =
18) 34.54 30.47 25.03 24.68 TG-High (N = 18) 33.38 27.06 24.50
30.51 TG-Saline (N = 16) 24.80 22.20 24.25 24.02 WT-High (N = 16)
23.38 23.58 17.73 16.11 WT-Saline (N = 18) 27.26 26.82 24.85
26.82
TABLE-US-00026 TABLE 26 Average escape latencies (sec) from the
Morris Water Maze tests with non-compliance removed. Group Day 1
Day 2 Day 3 Day 4 TG-Low (N = 15-18) 31.36 25.53 25.03 23.46
TG-High (N = 15-16) 28.73 20.55 20.06 25.57 TG-Saline (N = 14-15)
23.25 19.68 21.87 19.68 WT-High (N = 14-15) 20.93 19.30 14.92 13.18
WT-Saline (N = 13-16) 21.65 22.67 18.07 15.87
TABLE-US-00027 TABLE 27 Average daily escape latencies (sec) from
the Morris Water Maze tests. Group Day 1 Day 2 Day 3 Day 4 TG ALL
(N = 52) 31.14 26.75 24.61 26.50 WT ALL (N = 34) 25.43 25.29 21.50
21.78
TABLE-US-00028 TABLE 28 Average daily escape latencies (sec) from
the Morris Water Maze tests with noncompliance removed. Group Day 1
Day 2 Day 3 Day 4 TG ALL (N = 45-49) 27.86 21.92 22.44 22.99 WT ALL
(N = 28-30) 21.29 21.10 16.49 14.43
[0491] Like with RAWM, the three transgenic groups are grouped
closely in Table 25 and Table 26. The only significant differences
between TG-Sal and TG-low came on B1-T1, B2-T2, and B4-T1 (Table
24), and in each case, TG-Sal mice had shorter escape latencies
than TG-low mice, who performed particularly poor in this task.
There was only one example in which there was a statistical
difference between TG-high and TG-Sal (B1-T1). In this instance,
TG-Sal did very well and outperformed the TG-high mice. However, it
should be noted that the WT-high mice consistently outperformed all
other groups in this task. Although T-tests performed at each trial
showed no statistical differences between WT-high and WT-Saline,
repeated measures ANOVA would demonstrate a difference between
these two groups.
[0492] For the MWM hidden platform test, the escape latency (time
to find the platform) was collected. T-tests were conducted for
each day of each trial (1-4). Data was analyzed with non-compliant
mice removed in order to more accurately represent memory.
Non-compliant mice were defined as any mice that had escape
latencies of 60 seconds (the full time allotted) for trials 3 and
4, when they should have been learning to some extent. The percent
of non-compliant mice for each group was recorded. For hidden
platform tests non-compliance was as follows: TG-low=8.3%;
TG-high=15.3%; TG-saline=7.8%; WT-high=7.8%; and
WT-saline=18.1%.
[0493] The Radial Arm Water Maze (RAWM).
[0494] RAWM is used to evaluate short-term, working memory. Similar
to a MWM, this test has a round tub with water, visual cues
throughout the room and a hidden platform. It is unique in that
inserts are placed into the tank to create six radially distributed
arms of equal size that emanate from the center. Before trials
began, the mice were acclimated to swimming in the water. Mice were
dropped into 4 radial arms, in an order selected randomly for each
trial, and given 1 minute to find the platform, with 20 seconds of
rest between each trial. Trials occurred daily for twelve days and
each day the platform was moved to a new location. Halfway through
the testing, an extra intra-maze visual cue was added to the tank
in an effort to make the test a little easier. The visual cue was a
large `X` made of tape and placed on the inner wall of the maze
above the arm with the escape platform. Both errors and escape
latency were recorded.
TABLE-US-00029 TABLE 29 RAWM escape latency (seconds) of mice
grouped in blocks 1-4. Block 1 Block 2 Block 3 Block 4 T1(1) T2(1)
T3(1) T4(1) T1(2) T2(2) T3(2) T4(2) T1(3) T2(3) T3(3) T4(3) T1(4)
T2(4) T3(4) T4(4) TG-Low (N = 18) 49.20 50.48 49.44 45.02 48.06
42.98 40.42 46.66 44.17 35.45 36.25 40.67 37.46 26.44 22.72 29.89
TG-High (N = 18) 49.72 47.13 44.94 47.33 47.78 34.09 42.11 42.96
40.00 45.02 35.56 41.59 32.70 27.20 29.76 29.57 TG-Saline (N = 16)
50.58 44.13 48.96 42.94 49.92 41.58 34.98 43.50 39.94 36.21 28.45
32.98 32.75 29.06 28.04 29.42 WT-High (N = 18) 39.96 47.11 43.04
42.02 44.81 41.61 34.72 41.70 41.91 33.52 39.54 34.93 40.15 28.28
24.78 24.43 WT-Saline (N = 18) 40.62 38.50 41.76 40.26 43.28 35.98
40.15 40.81 42.41 31.70 33.83 33.70 33.09 27.87 24.96 25.20
[0495] Overall, RAWM was too difficult for mice in blocks 1 and 2,
as evidenced by a general trend for the escape latency not to go
below about 35 seconds (Table 30). After the addition of the extra
visual cue in blocks 3 and 4, a clear trend of decreased time to
find the platform and errors became apparent in all treatment
groups from trial 1 to trial 4 (Table 30, Table 31, and Table 32).
This demonstrated that the test was effective for measuring
memory.
TABLE-US-00030 TABLE 30 RAWM escape latency (seconds) of blocks 1
and 2. ESCAPE LATENCY (BLOCK) T1(1) T2(1) T1(2) T2(2) T1(3) T2(3)
T1(4) T2(4) TG-Low 49.20 50.48 48.06 42.98 44.17 35.45 37.46 26.44
(N = 18) TG-High 49.72 47.13 47.78 34.09 40.00 45.02 32.70 27.20 (N
= 18) TG-Saline 50.58 44.13 49.92 41.58 39.94 36.21 32.75 29.06 (N
= 16) WT-High 39.96 47.11 44.81 41.61 41.91 33.52 40.15 28.28 (N =
18) WT-Saline 40.62 38.50 43.28 35.98 42.41 31.70 33.09 27.87 (N =
18)
TABLE-US-00031 TABLE 31 RAWM escape latency (seconds) of blocks 1
and 3. ESCAPE LATENCY (BLOCK) T1(1) T3(1) T1(2) T3(2) T1(3) T3(3)
T1(4) T3(4) TG-Low 49.20 49.44 48.06 40.42 44.17 36.25 37.46 22.72
(N = 18) TG-High 49.72 44.94 47.78 42.11 40.00 35.56 32.70 29.76 (N
= 18) TG-Saline 50.58 48.96 49.92 34.98 39.94 28.45 32.75 28.04 (N
= 16) WT-High 39.96 43.04 44.81 34.72 41.91 39.54 40.15 24.78 (N =
18) WT-Saline 40.62 41.76 43.28 40.15 42.41 33.83 33.09 24.96 (N =
18)
TABLE-US-00032 TABLE 32 RAWM escape latency (seconds) of blocks 1
and 4. ESCAPE LATENCY (BLOCK) T1(1) T4(1) T1(2) T4(2) T1(3) T4(3)
T1(4) T4(4) TG-Low 49.20 45.02 48.06 46.66 44.17 40.67 37.46 29.89
(N = 18) TG-High 49.72 47.33 47.78 42.96 40.00 41.59 32.70 29.57 (N
= 18) TG-Saline 50.58 42.94 49.92 43.50 39.94 32.98 32.75 29.42 (N
= 16) WT-High 39.96 42.02 44.81 41.70 41.91 34.93 40.15 24.43 (N =
18) WT-Saline 40.62 40.26 43.28 40.81 42.41 33.70 33.09 25.20 (N =
18)
[0496] There was clear evidence of a transgenic model effect in
RAWM (Table 33 and Table 34). In Table 35 an overall summary of all
groups averaged out over all days shows that in all four trials,
both WT groups had lower times to find the platform than all three
TG groups. This was also true of errors for trials 2-4 (Table 36).
In Table 33, Table 34, Table 35, and Table 36, individual blocks
and trials can be seen. For escape latency, WT-Sal mice had
significantly shorter escape latencies than TG-Sal mice in B1-T1
(Batch 1-Trial 1), B1-T3, and B3-T2 (p<0.05 or 0.1) (Table 24).
For errors (Table 36), WT-Sal mice had significantly fewer errors
than TG-Sal mice in B1-T3, B3-T2, and B4-T2 (p<0.05 or 0.1)
(Table 24). When all WT mice were combined and compared to all TG
mice (irrespective of treatment), it was clear that WT mice
outperformed TG mice. When all days were combined, WT mice had
shorter escape latency and fewer errors than TG mice in all trials
(Table 35 and Table 36). Similarly, in individual blocks and
trials, all WT mice had shorter escape latency and fewer errors in
all trials in blocks 2-4 (Table 35 and Table 36). Statistically, WT
mice had shorter escape latencies than TG mice in B1-T1, B3-T2,
B3-T4, and B4-T4 (p<0.05) (Table 24). Statistically, WT mice had
fewer errors than TG mice in B1-T1, B3-T2, B3-T4, B4-T2, and B4-T4
(p<0.05) (Table 24).
TABLE-US-00033 TABLE 33 RAWM escape latencies (seconds) recorded
for 12 days of RAWM testing. TG ALL (N = 52) WT ALL (N = 36) Arm
Arm Arm Arm Arm Arm Arm Arm 1 2 3 4 1 2 3 4 Day 1 51.67 53.33 49.08
45.24 45.94 45.56 46.81 45.22 Day 2 51.15 45.58 48.54 46.87 36.11
40.22 40.06 37.47 Day 3 46.60 43.19 45.60 43.41 38.86 42.64 40.33
40.72 Day 4 50.29 37.31 39.87 43.27 43.17 37.67 37.03 34.83 Day 5
49.85 40.62 38.27 44.76 41.94 38.14 36.61 44.08 Day 6 45.41 40.45
39.84 45.20 47.00 40.58 38.67 44.86 Day 7 45.76 38.76 37.14 42.38
41.53 32.08 34.53 40.67 Day 8 38.79 41.61 39.20 38.92 43.36 28.36
38.36 28.97 Day 9 39.75 36.79 24.71 34.63 41.57 37.39 37.17 33.31
Day 10 34.42 29.90 29.69 29.94 39.81 27.19 27.50 25.47 Day 11 34.13
23.69 24.10 31.15 35.50 27.50 26.08 28.94 Day 12 34.54 28.94 26.60
27.81 34.56 29.53 21.03 20.03
TABLE-US-00034 TABLE 34 RAWM escape latencies (seconds) of blocks
1-4. Block 1 Block 2 Block 3 Block 4 T1(1) T2(1) T3(1) T4(1) T1(2)
T2(2) T3(2) T4(2) T1(3) T2(3) T3(3) T4(3) T1(4) T2(4) T3(4) T4(4)
TG ALL (N = 52) 49.81 47.37 47.73 45.18 48.54 39.45 39.32 44.40
41.42 39.04 33.62 38.62 34.37 27.51 26.79 29.63 WT ALL (N = 36)
40.29 42.81 42.40 41.14 44.06 38.80 37.44 41.26 42.16 32.61 36.69
34.31 36.62 28.07 24.87 24.81
TABLE-US-00035 TABLE 35 12 day average of RAWM escape latencies
(seconds). Trial 1 Trial 2 Trial 3 Trial 4 TG ALL (N = 52) 43.53
38.35 36.89 39.46 WT ALL (N = 36) 40.78 35.57 35.35 35.38
TABLE-US-00036 TABLE 36 12 day average of RAWM errors (trial
averages). Trial 1 Trial 2 Trial 3 Trial 4 TG ALL (N = 52) 4.77
4.64 4.28 4.42 WT ALL (N = 36) 4.52 3.84 3.71 3.72
[0497] There was evidence of a TG model effect in RAWM. A summary
of all groups averaged out over all days (Table 35) shows that in
all four trials, both WT groups had lower times to find the
platform than all three TG groups. This was also true of errors for
trials 2-4 (Table 35 and Table 36). In Table 35 and Table 36,
individual blocks and trials can be seen. For escape latency,
WT-Sal mice had significantly shorter escape latencies than TG-Sal
mice in B1-T1 (Batch 1-Trial 1), B1-T3, and B3-T2 (p<0.05 or
0.1) (Table 24). As shown in Table 36, WT-Sal mice had
significantly fewer errors than TG-Sal mice in B1-T3, B3-T2, and
B4-T2 (p<0.05 or 0.1) (Table 24). When all WT mice were combined
and compared to all TG mice (irrespective of treatment), it was
clear that WT mice outperformed TG mice. When all days were
combined, WT mice had shorter escape latency and fewer errors than
TG mice in all trials (Table 35). Similarly, in individual blocks
and trials, all WT mice had shorter escape latency and fewer errors
in all trials in blocks 2-4 (Table 35 and Table 36). Statistically,
WT mice had shorter escape latencies than TG mice in B1-T1, B3-T2,
B3-T4, and B4-T4 (p<0.05) (Table 24). Statistically, WT mice had
fewer errors than TG mice in B1-T1, B3-T2, B3-T4, B4-T2, and B4-T4
(p<0.05) (Table 24).
[0498] The Barnes Maze.
[0499] The Barnes maze is a visual memory task based on finding an
escape hole on a table, aided by visual cues throughout the room.
The table was round, elevated 1 m from the floor, and had 40 escape
holes spaced equally around the periphery of the table. One of
these holes had an escape box directly underneath, while the others
were open. The motivation to find the escape box was aversive
stimuli in the form of bright lights and fans blowing above the
surface of the table. The escape box was located in one location
for the duration of the study. The mouse was given 4 days, with 3
trials/day to learn the location of the escape box. Mice were given
up to two minutes on the table to find the escape hole. If after 2
minutes they did not find the escape box, they were placed into the
box. Both escape latency to find the hole and errors were recorded
and analyzed. Errors were defined as head-pokes through holes that
do not have the escape box.
[0500] Overall, the Barnes maze test did not work well for the mice
in this study. This was the only behavior test in which
non-compliance was an issue (roughly 50% of all mice did not
perform the task). While running the tests, the mice were generally
not scared of the aversive stimuli. However, among the mice that
were compliant and included in the analyses, there was a learning
trend across the days and trials, which can be seen in the escape
latencies.
[0501] There was evidence of a model effect with this test. Table
37 and Table 38 shows that both WT groups have lower escape
latencies on days 3 and 4 than all three TG groups. This mirrors
data collected with the RAWM and MWM tests, the other two long-term
memory tasks. This difference is also seen when all WT mice and TG
mice were combined as in Table 39 and Table 40.
TABLE-US-00037 TABLE 37 Average escape latencies (sec) from the
Barnes Water Maze by treatment. Time (s) Day 1 Day 2 Day 3 Day 4
TG-Low (N = 18) 105.15 99.76 95.44 85.67 TG-High (N = 18) 107.74
94.57 100.30 97.33 TG-Saline (N = 16) 95.48 89.10 90.10 82.31
WT-High (N = 17) 99.06 95.98 93.65 82.04 WT-Saline (N = 18) 94.15
97.41 93.43 87.63
TABLE-US-00038 TABLE 38 Average escape latencies (sec) from the
Barnes Water Maze by treatment with noncompliance removed. Time (s)
Day 1 Day 2 Day 3 Day 4 TG-Low (N = 7-12) 82.81 78.25 83.60 72.75
TG-High (N = 6-8) 84.28 72.14 75.71 68.86 TG-Saline (N = 7-9) 79.71
65.38 74.30 71.48 WT-High (N = 8-10) 83.74 79.17 66.29 60.13
WT-Saline (N = 7-10) 69.17 68.43 73.00 60.74
TABLE-US-00039 TABLE 39 Average escape latencies (sec) from the
Barnes Water Maze by genotype. Time (s) Day 1 Day 2 Day 3 Day 4 TG
ALL (N = 52) 103.07 94.69 95.48 88.67 WT ALL (N = 35) 96.53 96.71
93.53 84.91
TABLE-US-00040 TABLE 40 Average escape latencies (sec) from the
Barnes Water Maze by genotype with noncompliance removed. Time (s)
Day 1 Day 2 Day 3 Day 4 TG All (N = 21-28) 82.05 72.21 78.16 71.37
WT All (N = 17-19) 76.88 74.75 70.02 60.42
[0502] There was no evidence of a drug effect in the Barnes Maze
tests (Table 37, Table 38, Table 39, Table 40, Table 41, Table 42).
The only statistical significance was in B4-T1, in which TG-low
mice performed very poorly and had longer escape latency than
TG-Sal mice (p<0.1; Table 24).
TABLE-US-00041 TABLE 41 Average number of errors from the Barnes
Water Maze by treatment. Day 1 Day 2 Day 3 Day 4 TG-Low (N = 18)
8.48 5.57 6.24 5.39 TG-High (N = 18) 6.85 5.54 4.15 4.04 TG-Saline
(N = 16) 11.90 8.02 6.29 5.69 WT-High (N = 17) 6.96 6.45 4.69 4.00
WT-Saline (N = 18) 9.46 8.44 5.35 4.89
TABLE-US-00042 TABLE 42 Average errors from the Barnes Water Maze
by genotype. Day 1 Day 2 Day 3 Day 4 TG ALL (N = 52) 8.97 6.31 5.53
5.01 WT ALL (N = 35) 8.25 7.48 5.03 4.46
[0503] For Barnes Maze, both the escape latency (time to find the
escape hole) and errors (number of times a mouse pokes his head
into a hole that does not have the escape box) were collected.
T-tests were conducted for each day of each trial (1-3). Data was
analyzed with non-compliant mice removed in order to more
accurately represent memory. Non-compliant mice were defined as any
mice that had escape latencies of 120 seconds (the full time
allotted) for trials 3, when they should have been learning to some
extent. The percent of non-compliant mice for each group was
recorded and was as follows: TG-low=48.6%; TG-high=61.1%;
TG-saline=48.4%; WT-high=45.6%; and WT-saline=52.8%.
[0504] Elevated Plus Maze.
[0505] The Elevated Plus Maze is a standard test of baseline
anxiety in which the animal is placed in the center of an elevated
4-arm maze that consists of two arms that are open and two arms
that are enclosed. The number of times the animal entered each of
the arms and the time spent in each arm over 4 minutes was
recorded. The test was used to determine the unconditioned response
to a potentially dangerous environment (the open, unprotected arms)
and anxiety-related behavior was measured by the degree to which
the rodent avoids the open arms of the maze.
[0506] There was a transgenic effect in the Elevated Plus Maze. In
this model, all TG mice spent more time and made more frequent arm
entries into the open arms of the maze than all WT mice,
demonstrating inhibition of exploratory behavior and anxiety that
WT mice have regarding open spaces. When WT-Sal mice were compared
to TG-Sal mice, TG mice spent significantly more time and have
significantly more arm entries into the open arms (Table 24, Table
43, and Table 44). When all WT-mice and all TG-mice were combined,
the same results were seen (Table 44 and Table 45), p<0.05;
Table 24).
TABLE-US-00043 TABLE 43 Average time spent in open arms during the
Elevated Plus Maze. TIME (SEC) SUM PERCENTAGE Avg. Time Avg Time
Std Error Std Error Avg. Time Avg Time Std Error Std Error Enclosed
Open Enclosed Open Enclosed Open Enclosed Open TG-Low (N = 18)
115.2 31.4 10.8 5.3 48.0 13.1 4.5 2.2 TG-High (N = 16) 128.7 48.9
11.2 8.7 53.7 20.4 4.7 3.6 TG-Saline (N = 15) 117.5 34.6 11.6 7.4
49.0 14.4 4.9 3.1 WT-High (N = 16) 151.9 20.6 8.6 3.7 63.4 8.6 3.6
1.6 WT-Saline (N = 16) 169.8 15.9 11.6 4.4 70.8 6.6 4.8 1.8 TG ALL
(N = 49) 120.3 38.1 6.4 4.2 50.2 15.9 2.7 1.8 WT ALL (N = 32) 160.8
18.3 7.3 2.9 67.1 7.6 3.0 1.2
TABLE-US-00044 TABLE 44 Average frequency of entries into open arms
during the Elevated Plus Maze. FREQUENCY SUM PERCENTAGE Avg. Freq
Avg. Freq Std Error Std Error Avg. Freq Avg. Freq Std Error Std
Error Enclosed Open Enclosed Open Enclosed Open Enclosed Open
TG-Low (N = 18) 16.6 10.8 2.0 2.0 60.9 39.1 5.1 5.1 TG-High (N =
16) 16.0 14.8 2.2 3.6 57.8 42.2 5.2 5.2 TG-Saline (N = 15) 15.9 8.1
2.4 2.2 69.6 30.4 5.8 5.8 WT-High (N = 16) 13.8 3.4 1.5 0.5 82.1
17.9 2.4 2.4 WT-Saline (N = 16) 9.8 3.0 1.1 0.8 81.9 18.1 3.8 3.8
TG ALL (N = 49) 16.2 11.3 1.2 1.6 62.5 37.5 3.1 3.1 WT ALL (N = 32)
11.8 3.2 1.0 0.5 82.0 18.0 2.2 2.2
[0507] There was no evidence of a drug effect in the Elevated Plus
Maze tests. Although the TG-high group had the most arm-entries and
spent the most time in the open arms, it was not significantly
different from any other groups (Table 24, Table 43, and Table
44).
[0508] For the Elevated Plus Maze, both the time spent in open and
enclosed arms and the number of arm entries (also called frequency
of arm entries) were recorded. Mice were not included in the
analyses if they fell off the maze in less than 120 seconds. There
were 3 mice that fell off, all from different groups. For outliers,
mice were removed if both their time spent in open arms and
frequency of entries into open arms were more than two standard
deviations from the mean of their treatment group. Outliers
included 3 mice, all from different groups.
[0509] The Open Field Maze Test.
[0510] The Open Field Maze Test is used to detect any change in
spontaneous locomotor activity due to drug treatment or anxiety.
Each mouse was given 4 minutes to individually explore a
rectangular box, while being tracked by the EthoVision video
tracking system. For analysis, the box was subdivided into 16
equally sized squares that are separated by manually drawn lines
using the "line draw" feature in EthoVision. The number of line
crossings and patterns of exploration were measured.
[0511] There was no evidence of a transgenic or drug effect in the
Open Field Maze tests. All groups of mice had very similar line
crossings and velocity (Table 24, Table 45, Table 46, Table 47, and
Table 48).
TABLE-US-00045 TABLE 45 Average velocity of mice. Avg Velocity Std
Dev Std Error TG Low (N = 18) 7.66 2.48 0.58 TG High (N = 18) 9.32
3.73 0.88 TG Saline (N = 15) 8.73 2.78 0.72 WT High (N = 16) 10.03
2.50 0.63 WT Saline (N = 17) 9.71 3.38 0.82
TABLE-US-00046 TABLE 46 Average velocity of mice, averaged by
genotype. Avg Velocity Std Error TG ALL (N = 51) 8.66 0.44 WT ALL
(N = 33) 9.46 0.56
TABLE-US-00047 TABLE 47 Average number of line crossings by mice.
Avg Line Crossings Std Dev Std Error TG Low (N = 18) 87.56 32.93
7.76 TG High (N = 18) 110.94 44.01 10.37 TG Saline (N = 15) 105.53
29.47 7.61 WT High (N = 16) 113.06 30.10 7.53 WT Saline (N = 17)
112.59 33.44 8.11
TABLE-US-00048 TABLE 48 Average number of line crossings by mice,
averaged by genotype. Avg Line Crossings Std Error TG ALL (N = 51)
102.65 5.33 WT ALL (N = 33) 107.83 6.21
[0512] For the Open Field Maze, both the number of line crossings
and the overall velocity were measured. Outliers were removed if an
individual mouse's line crossings were more than 2 standard
deviations from the mean of the treatment group. This included 3
mice, each from different treatment groups. Analysis was performed
for both line crossings and velocity.
[0513] The Rotarod Performance Test.
[0514] The Rotarod Performance Testis used to detect any changes in
endurance, balance, and coordination. Mice were placed on an
automated rotating bar and allowed to walk on the bar for up to 60
seconds. The speed of rotation was gradually increased and the
rodent's ability to remain on the rotating bar was recorded as the
total time spent on the bar. Mice were given three trials, and the
best time is used for analysis.
[0515] There was no transgenic model effect on the Rotarod tests.
All groups performed essentially the same and there were no
statistical differences among groups (Table 24 and Table 49, Table
50, and Table 51). There was a non-significant trend for all WT
mice to outperform all TG mice (Table 49, Table 50, and Table
51).
TABLE-US-00049 TABLE 49 Longest average runs on the rotarod by
treatment group. Best Trial (Average) (sec) TG-Low (N = 18) 30.59
TG-High (N = 18) 37.33 TG-Saline (N = 16) 35.38 WT-High (N = 16)
54.13 WT-Saline (N = 18) 35.67 TG ALL (N = 52) 37.33 WT ALL (N =
34) 35.38
TABLE-US-00050 TABLE 50 Average run time on the rotarod by
treatment group. Avg. Time (sec) TG-Low (N = 18) 19.43 TG-High (N =
18) 23.30 TG-Saline (N = 16) 22.35 WT-High (N = 16) 35.24 WT-Saline
(N = 18) 22.25 TG ALL (N = 52) 23.30 WT ALL (N = 34) 22.35
TABLE-US-00051 TABLE 51 Trial averages of run time (sec) on the
rotarod by treatment group. Trial 1 Trial 2 Trial 3 TG-Low (N = 18)
10.53 21.25 27.06 TG-High (N = 18) 15.72 20.44 33.72 TG-Saline (N =
16) 16.60 25.63 24.60 WT-High (N = 16) 19.56 43.00 44.20 WT-Saline
(N = 18) 17.00 17.06 32.39 TG ALL (N = 52) 15.72 20.44 33.72 WT ALL
(N = 34) 16.60 25.63 24.60
[0516] There was no evidence of a drug effect among transgenic
groups (Table 49, Table 50, and Table 51). However, it was observed
that the WT-high mice had longer times on the rotarod than the
WT-Sal mice. A t-test between WT-Sal and WT-high yielded a p-value
of 0.089 for the longest run, and a p-value of 0.041 for the
average run (T-tests not shown, Table 49, Table 50, and Table
51).
[0517] For the Rotarod test, the time on the rotating bar before
the mouse fell off was recorded. Three trials were conducted. If a
mouse reached 120 seconds (the maximum time) before trial 3,
subsequent runs were not conducted. For each treatment group, both
the average time on the bar and the maximum time on the bar for
each mouse were analyzed. Data could not be recorded if the mouse
did not stay on the rod long enough before starting (.about.3
seconds), and there was only 1 mouse that did not stay on long
enough to start for all three trials.
[0518] The Passive Avoidance Task.
[0519] The Passive Avoidance Task is a classical conditioning test
used to assess short-term or long-term memory for mice and rats.
The passive avoidance apparatus consists of equal-sized light and
dark compartments with a light bulb fixed in the center of the roof
of the light compartment. The floor consists of a metal grid
connected to a shocker. The two compartments are separated by a
trap door. On the learning day (day 1), a mouse was placed in the
light compartment and the time taken to enter the dark compartment
was recorded and termed as initial latency. Immediately after the
mouse entered the dark chamber a door was automatically closed and
an electric footshock (0.7 mA) was delivered for 3 seconds.
Twenty-four hours after the acquisition trial, a second retention
trial was conducted and the time the mouse takes to enter the dark
compartment as designated retention latency (RL; recorded to a
maximum of 500 seconds, no shock is administered during this
entry). T-tests were performed to compare the effects of IN IgG WT
vs. TG.
[0520] Whereas RAWM, MWM hidden platform, and Barnes maze tests all
showed evidence of learning and improved learning in WT mice over
TG mice, this test consistently showed the opposite effect,
regardless of drug treatment. There was no evidence of a drug
effect among transgenic groups (Table 24, Table 52, Table 53, and
Table 54).
TABLE-US-00052 TABLE 52 Passive avoidance learn day escape latency
(sec). Learn Esc. St. Err TG-Low (N =17) 44.5 9.4 TG-High (N = 19)
46.3 7.6 TG-Saline (N = 15) 43.7 9.2 WT-High (N = 17) 21.6 6.4
WT-Saline (N = 18) 22.4 3.9 TG ALL (N = 51) 44.9 4.9 WT ALL (N =
35) 22 3.6
TABLE-US-00053 TABLE 53 Passive avoidance test day escape latency
(sec). Test Esc. St. Err TG-Low (N = 15) 224.6 8.5 TG-High (N = 17)
229.5 8.3 TG-Saline (N = 13) 207.0 16.8 WT-High (N = 16) 114.3 22.0
WT-Saline (N = 18) 153.8 20.9 TG ALL (N = 45) 221.4 4.9 WT ALL (N =
34) 135.2 3.6
TABLE-US-00054 TABLE 54 Passive avoidance average of escape latency
differences (sec). Average of Differences St. Err TG-Low (N = 15)
190.2 8.5 TG-High (N = 17) 191.9 8.2 TG-Saline (N = 13) 175.1 16.8
WT-High (N = 16) 98.8 22.4 WT-Saline (N = 18) 131.4 21.4 TG ALL (N
= 45) 186.5 6.3 WT ALL (N = 34) 116.1 15.5
[0521] This test demonstrated an unexpected TG effect. Whereas TG
mice with impaired memory should normally have trouble remembering
not to enter the dark chamber and receive a shock after training,
this was not the case. TG mice generally did not enter the chamber
on the test day, whereas WT mice seemed not to care whether they
received a shock on the test day. These results can be seen in
Table 52, Table 53, and Table 54. The poor performance of the WT
mice compared to the TG mice is statistically significant
(p<0.05; Table 24). The same willingness for WT mice to enter
the dark chamber can be seen in the learning phase and may play a
role in the willingness of normal, WT mice to go receive a painful
shock.
[0522] For the Passive Avoidance Task, the escape latency on both
the learning day (day 1) and the test day (day 2) were recorded and
the difference between the escape latency between the test and
learn day were calculated. Mice were not run on the test day (day
2) if they did not receive a shock on day 1, which included 7 mice
spread across 4 groups. Mice did not receive a shock simply because
they did not enter the dark chamber. There were no outliers
calculated. Analyses were performed for the learn trial and the
test trial.
[0523] Morris Water Maze--Visual Platform.
[0524] Differences in performance in this test were not expected as
all mice were genetically tested for the RD1 gene and the mice did
not have problems with vision. There was no transgenic model
effect. All groups performed essentially the same and there were no
statistical differences among groups (Table 24). The one
statistical difference came in trial 1, due to a strong performance
by WT-Sal that did not carry over into subsequent trials. There was
also no evidence of a drug effect among transgenic groups (Table
24, Table 55, Table 56, Table 57, and Table 58). However, much like
with the MWM hidden platform tests, there was a trend for WT-high
mice to outperform all other groups (Table 55, Table 56, Table 57,
and Table 58). T-test comparisons between WT-Sal and WT-high for
each individual trial were not significant, but a T-test for all
trials between these two groups had a p-value of 0.06.
TABLE-US-00055 TABLE 55 Visual escape (sec) by treatment group.
Group Trial 1 Trial 2 Trial 3 Trial 4 Average TG-Low (N = 18) 34.83
33.44 39.17 30.22 33.44 TG-High (N = 18) 31.44 33.67 35.33 37.89
33.67 TG-Saline (N = 16) 23.19 36.56 29.75 28.94 36.56 WT-High (N =
16) 28.25 23.44 23.13 22.00 23.44 WT-Saline (N = 18) 29.78 29.06
26.11 25.50 29.06
TABLE-US-00056 TABLE 56 Visual escape (sec) by treatment group,
with non-compliance removed. Group Trial 1 Trial 2 Trial 2 Trail 3
Average TG-Low (N = 13) 30.46 29.15 31.15 18.77 29.15 TG-High (N =
13) 21.69 27.92 25.85 29.38 27.92 TG-Saline (N = 14) 17.93 33.21
25.43 24.50 33.21 WT-High (N = 14) 25.07 18.57 17.86 16.57 18.57
WT-Saline (N = 17) 31.41 27.24 24.12 23.47 27.24
TABLE-US-00057 TABLE 57 Visual escape (sec) by genotype group.
Group Trial 1 Trial 2 Trial 3 Trial 4 Average TG ALL (N = 52) 30.08
34.48 34.94 32.48 34.48 WT ALL (N = 34) 29.06 26.41 24.71 23.85
26.41
TABLE-US-00058 TABLE 58 Visual escape (sec) by genotype, with
non-compliance removed. Group Trial 1 Trial 2 Trial 3 Trial 4
Average TG ALL (N = 40) 23.23 30.18 27.43 24.23 30.18 WT ALL (N =
31) 28.55 23.32 21.29 20.35 23.32
[0525] For the visual platform MWM, the escape latency (time to
find the platform) was collected. T-tests were conducted for each
day of each trial (1-4). Data was analyzed with non-compliant mice
removed in order to more accurately represent memory. Non-compliant
mice were defined as any mice that had escape latencies of 60
seconds (the full time allotted) for trials 3 and 4, when they
should have been learning to some extent. The percent of
non-compliant mice for each group was recorded. For visual platform
tests non-compliance was as follows: TG-low=6.9%; TG-high=6.9%;
TG-saline=3.1%; WT-high=3.1%; and WT-saline=1.4%.
Example 8--Radiolabeled .sup.125I IgG Reaches the CNS with
Intranasal Delivery
[0526] A study was conducted to determine the feasibility and to
optimize the methods used to determine the amount of intravenously
and intranasally delivered radiolabed .sup.125I IgG reaching the
CNS in rats and mice at a two hour time point.
[0527] Experimental Design: There were two phases of this
experiment. In phase 1, six mice and rats were used to test a
variety of different methods including anesthesia with 2 hour
survival, drug administration methods (intravenous infusion through
cannulations of the jugular vein in rats and mice, intranasal tube
method in rats), transcardial perfusion (with and without a
non-ionic detergent), and tissue processing for capillary depletion
and gamma counting. Animals and the methods tested with each are
shown in Table 51.
TABLE-US-00059 TABLE 59 Experimental design of phase 1 of Example
8. Ani- IV IN Brain mal Surgery delivery delivery Perfusion
Dissection 1a- Jugular Vein No infusion IN tube Saline Whole Brain
R-1 Cannulation method removal 1a- Jugular Vein 2 mg/mL BSA IN tube
0.05% Whole Brain R-2 Cannulation until death method Triton X
removal 1a- Jugular Vein 2 mg/mL BSA IN tube Saline Capillary R-3
Cannulation over 1 hour method Depletion 1a- Jugular Vein No
infusion No IN 0.1% Whole Brain R-4 Cannulation delivery Triton X
removal 1a- Jugular Vein No infusion IN tube 0.1% Whole Brain R-5
Cannulation method Triton X removal 1a- Jugular Vein No infusion No
IN Saline Capillary R-6 Cannulation delivery Depletion 1a- Jugular
Vein No infusion No IN Saline Capillary M-1 Cannulation delivery
Depletion 1a- Jugular Vein 2 mg/mL BSA No IN 0.05% Whole Brain M-2
Cannulation over 1 hour delivery Triton X removal 1a- Jugular Vein
2 mg/mL BSA No IN 0.1% Whole Brain M-3 Cannulation over 1 hour
delivery Triton X removal 1a- Jugular Vein 2 mg/mL BSA No IN Saline
Whole Brain M-4 Cannulation over 1 hour delivery removal 1a-
Jugular Vein 8 g/kg IgG No IN 0.05% Whole Brain M-5 Cannulation
over 1 hour delivery Triton X removal 1a- Jugular Vein 8 g/kg IgG
No IN 0.05% Whole Brain M-6 Cannulation over 1 hour delivery Triton
X removal R = rat and M = mouse.
[0528] In Phase 2, three tissue processing techniques after
administration of high IVIG does in 18 rats were tested in order to
determine the optimal technique of subsequent Phase 1 experiments.
The 18 rats were divided into 3 experimental groups (Table 60).
TABLE-US-00060 TABLE 60 Experimental groups for Phase 2. Group 1
Group 2 Group 3 .sup.125I-IVIG dose 200 mg 200 mg 200 mg Perfusion
140 mL saline 140 mL saline 90 mL saline, with capillary 25 mL
0.025% depletion Triton X-100, 25 mL saline n= 6 rats 6 rats 6
rats
[0529] Adult male Sprague Dawley rats (N=6, average weight 250 g)
and adult male C57blk mice (n=6, 7-8 weeks) were used for Phase 1.
Adult male Sprague Dawley rats (N=18, average weight 264 g) were
used for Phase 2. The animals were housed in pairs with free access
to food and water and were kept on a 12 h light cycle.
[0530] Prior to commencing the Phase 1 and 2 experiments, the
animals were allowed to normalize in the facility for a period of
three days before handling occurred. Animals were slowly acclimated
to human handling over a period of about two weeks. Enrichment food
treats are given after handling to encourage a human-animal bond
while the acclimation process proceeds. Restraint techniques were
kept brief and facilitated by using a towel, restraint device, or
scruffing, when working with mice.
[0531] An anesthesia cocktail containing Ketamine HCl (30 mg/kg),
Xylazine HCl (6 mg/kg), and Acepromazine (1 mg/kg) was used. All
anesthesia was administered as subcutaneous injections. Boosters
alternated between the Cocktail above and 50 mg/kg Ketamine.
Reflexes were tested to assess level of anesthesia every 10-15
minutes throughout the study.
[0532] Intranasal deliver in rats was performed using a specialized
pipette tip. The specialized pipette tip was inserted into the rat
naris. The pipette tip was created by cutting 23 mm off the end of
a gel loading pipette tip and attaching a 16 mm length of tubing
(ID=0.04 mm, OD=0.07 mm). The tubing was placed over the wide end
of the pipette tip with an overlap of 5.5 mm, and a black mark with
a sharpie was made at 14.5 mm from the narrowest end of the pipette
tip. The narrow end was ultimately inserted into the rat's nose up
to the black mark.
[0533] For intranasal delivery, the fully anesthetized rat was
placed on its back on a heating pad in a metal surgical tray. The
heating pad and rectal probe was used to maintain the rat's core
temperature at 37.degree. C. A 2''.times.2'' gauze pad was rolled
into a pillow and was securely taped. The pillow was then placed
under the rat's neck to ensure that the underside from nostril to
torso was planar and horizontal.
[0534] A lead impregnated shield was placed between the surgical
tray and the experimenter for protection against radiation. The
dose solution, pipette, pipette tips, and waste receptacle were
arranged behind the shield for easy access. The modified pipette
tip was inserted into the rat naris up to the black mark. The
sample to be delivered (40-50 .mu.l) was drawn into a pipettor, the
tip of the pipettor placed into the open tube at the end of the
modified pipette tip (while carefully holding the modified pipette
tip in place in the rat's nose), and then the entire dose was
slowly expelled into the rat's nostril.
[0535] After the animals were euthanized, their brains were removed
for analysis. With a large surgical scissors, the head of the
animal was removed by cutting dorsal to ventral to avoid
contamination. Using a scalpel, the fur and skin on the top of the
skull was cut from nose to point of decapitation. The skin was
folded back and held with a small gauze pad to expose the top of
the skull. Using a small hemostat, the remainder of the spinal
column was chipped away exposing the upper cervical spinal cord and
posterior brain (cerebellum). Next, the top of the skull was
removed to the olfactory bulbs exposing the entire dorsal side of
the brain. The hemostat was inserted with one blade scraping the
ventral surface of the skull. This ensured the integrity of the
dorsal surface of the brain was maintained. A small spatula was
used to loosen the lateral surfaces of the brain from the skull and
dura. The brain was inverted over a clean Petri dish. The optic
nerve was severed, which released the brain from the skull. The
brain was assessed for quality of perfusion.
[0536] The brain was placed dorsal side up. A single edged razor
blade was used to sever the olfactory bulbs from the brain at the
natural angle. Olfactory bulbs were collected. Razor blades were
used to cut the brain into seven coronal sections (see FIG. 5).
Each section was hemisected and placed into tubes for counting.
[0537] For capillary deletion, each brain section was weighed and
transferred to an ice cold ground glass homogonizer. A volume of
2.857 times the tissue sample weight of buffer, pH 7.4 (10 mM
HEPES, 141 mM NaCl, 4 mM KCl, 2.8 mM CaCl.sub.2), 1 mM
MgSO.sub.4--H.sub.2O, 1 mM NaH.sub.2PO.sub.4, and 10 mM D-Glucose),
was added to the homogonizer. The brain sample was homogenized
using vertical strokes. A small volume of 26% dextran solution was
added to the homogenized brain sample in order to provide a final
concentration of 15.5% Dextran in the homogenate. The homogenate
was then vortexted, homogenized for a second time with vertical
strokes, and then decanted into a small glass centrifuge tube. The
homogenate was then centrifuged in a swinging bucket rotor for 15
minutes at 4.degree. C. at a speed of 5400.times.g. The homogenate
was separated into the following layers: a bottom pellet containing
the capillary segments, a clear liquid layer, and a top "cream"
layer containing the nervous tissue. Using a transfer pipette, the
cream and clear liquid layers were transferred into new tubes. The
radioactivity of the supernatant and the pellet was determined
using a gamma counter.
[0538] Results:
[0539] The data from Phase 2 shows that intravenous .sup.125I-IVIG
reached the central nervous system. The animals with capillary
depletion tissue processing had the most IVIG in the brain tissue
(49,791 ng). The animals perfused with 0.025% Triton X as a second
perfusate had the least IVIG in the brain tissue (33,855 ng) (Table
61 and Table 62). The capillary depletion pellet which should hold
all of the IVIG stuck to the capillary walls only accounted for
.about.3% of the whole brain IVIG in those animals (Table 63). The
low amount of IVIG in the capillary pellet could be a result of
homogenization friction during processing, releasing the IVIG stuck
to the capillary walls and allowing it to be mixed in with the
supernatant instead of staying with the capillaries in the
pellet.
TABLE-US-00061 TABLE 61 .sup.125I-IVIG present in the central
nervous system measured in CPM. Total CPM Total CPM Total CPM Total
CPM Total CPM Perfusate(CPM/ul) Rat Method Whole Brain Liquid
Pellet R. Hemisphere L. Hemisphere (2nd) (3rd) 1b-1 Cap Dep 68,554
65,326 3,228 30,687 37,867 1b-4 Cap Dep 40,791 39,372 1,419 28,352
12,439 1b-7 Cap Dep 29,048 28,229 819 13,374 15,674 1b-10 Cap Dep
15,498 14,851 647 8,104 7,393 1b-13 Cap Dep 47,908 46,533 1,376
28,757 19,151 1b-16 Cap Dep 69,964 68,128 1,836 29,458 40,505 1b-3
Control 98,341 52,972 45,368 278 356 1b-6 Control 21,141 10,557
10,584 112 144 1b-11 Control 36,457 19,077 17,380 141 121 1b-15
Control 28,303 14,228 14,075 126 66 1b-17 Control 20,524 9,508
11,016 231 127 1b-18 Control 38,683 19,350 19,333 125 73 1b-2
Triton X 36,984 16,622 20,362 540 216 1b-5 Triton X 49,882 25,617
24,264 98 219 1b-8 Triton X 19,194 11,031 8,163 243 no sample 1b-9
Triton X 33,716 15,026 18,690 422 82 1b-12 Triton X 21,255 7,639
13,616 527 151 1b-14 Triton X 14,013 6,712 7,301 441 117 Average
Cap Dep 45,294 43,740 1,554 23,122 22,172 Average Control 40,575
20,949 19,626 169 148 Average Triton X 29,174 13,775 15,399 379
157
TABLE-US-00062 TABLE 62 ng by Group Total ng Total ng Total ng
Total ng Total ng Perfusate(ng/ul) Rat Method Whole Brain Liquid
Pellet R. Hemisphere L. Hemisphere (2nd) (3rd) 1b-1 Cap Dep 68,537
65,310 3,227 30,679 37,858 1b-4 Cap Dep 45,383 43,804 1,579 31,544
13,840 1b-7 Cap Dep 32,060 31,156 904 14,761 17,300 1b-10 Cap Dep
18,231 17,470 761 9,534 8,697 1b-13 Cap Dep 57,258 55,614 1,644
34,369 22,889 1b-16 Cap Dep 77,276 75,248 2,028 32,537 44,739 1b-3
Control 108,404 58,393 50,011 306 392 1b-6 Control 24,824 12,397
12,428 132 169 1b-11 Control 35,411 18,530 16,881 137 118 1b-15
Control 36,686 18,442 18,244 163 86 1b-17 Control 25,940 12,017
13,923 292 160 1b-18 Control 50,757 25,390 25,367 165 95 1b-2
Triton X 46,547 20,921 25,626 680 272 1b-5 Triton X 56,294 28,910
27,383 111 247 1b-8 Triton X 22,577 12,975 9,601 285 no sample 1b-9
Triton X 39,032 17,396 21,637 488 95 1b-12 Triton X 22,099 7,943
14,157 548 157 1b-14 Triton X 16,581 7,942 8,639 522 138 Average
Cap Dep 49,791 48,101 1,690 25,571 24,220 Average Control 47,004
24,195 22,809 199 170 Average Triton X 33,855 16,014 17,841 439
182
TABLE-US-00063 TABLE 63 ng by Group Percent Percent Percent est. of
ng Percent Percent of of ng of ng est. ng est. ng Percent delivered
of whole whole delivered delivered est. ng in 2nd in 3rd ng in
(Whole brain brain (2nd (3rd in blood* perfusate perfusate blood
Brain) (Liquid) (Pellet) perfusate) perfusate) Average 124,564,379
62% 0.02% 97% 3% Cap Dep Average 151,853,766 4,978,470 4,249,634
76% 0.02% 2.5% 2.1% Control Average 134,662,521 10,980,039
4,543,372 67% 0.02% 5.5% 2.3% Triton X *The total estimated blood
volume was determined as the body weight times 0.06 plus 0.77 (Lee
and Blaufox, 1985).
[0540] The Triton X perfusion methods resulted in a 28% reduction
of IVIG whole brain concentration versus the saline perfusion
control. The perfusate should show the amount of IVIG cleared from
the blood vessels over the course of the 25 ml (perfused at a rate
of 15 ml/min). Three 250 .mu.l samples of each perfusate were
counted in the gamma counter. Averages of the three were than
calculated. To determine the total amount of IVIG in each
perfusate, the ng/.mu.l IVIG concentration was determined and
multiplied by 25000 (the 25 ml of perfusate used). The first
perfusates (.about.90 ml at 15 ml/min) were not collected since
this step was the same in all of the animals in the study. In the
group perfused with 0.025% Triton X, more .sup.125I-IVIG was
removed (439 ng/.mu.1) than the groups perfused with saline (199
ng/.mu.1). This difference was not seen in the 3.sup.rd perfusate,
meant to clear any remaining Triton X from the blood vessels, (170
ng/.mu.1 and 182 ng/.mu.1, respectively) (Tables 1 and 2)
suggesting that the maximum clearance of IVIG from the vessels at
this concentration of Triton X was achieved. A higher Triton X
concentration in the perfusate may yield a further reduction.
[0541] In these results, approximately 0.02% of the total delivered
IVIG that was infused reached the brain (Table 55) in all methods.
During the Phase 2 experiments it was noted that the brain tissues
were slightly pinkish, suggesting the total volume perfused was not
adequate to completely remove blood from the brain. This slight
coloration appeared consistent throughout all animals in each
experimental group. An increase in the total volume of perfusate in
the next Phase should solve this issue.
Example 9--Biodistribution of IgG Administered Intranasally and
Intravenously in Mice
[0542] A study was conducted to compare the biodistribution of
pooled human immunoglobulin G (IgG) administered to mice
intranasally and intravenously. Delivery of IgG to the brain and
residual IgG in the bloodstream were determined.
[0543] Experimental Design:
[0544] IgG radiolabeled with iodine-125 (.sup.125I-IgG) was either
infused into the left femoral vein (intravenous administration; IV)
or intranasally administered (IN) as drops to anesthetized rats
over 14 minutes. Animals were sacrificed and concentrations of
.sup.125I-IgG were determined in the brain, blood, and body of the
mice at nine different time points (15 min, 30 min, 1 h, 2 h, 4 h,
8 h, 12 h, 24 h, and 72 h post-IgG administration). Blood samples
were taken from the heart, animals were perfused, and brains
removed. Radiolabeled IgG was detected with a gamma counter for
quantitative analysis. Half of each brain was processed into
supernatant and run through a size exclusion column to explore
intactness of the .sup.125I-label. The three experimental cohorts
were administered IgG as described in Table 64.
TABLE-US-00064 TABLE 64 Treatment groups assigned for intranasal
administration of IgG. IgG Cohort Dosage Administration Intranasal
Drops - 0.02 g/kg one drop every 2 minutes to alternating High Dose
naris; infusion of saline (IN Drop - high) Intranasal Drops - 0.002
g/kg one drop every 2 minutes to alternating Low Dose naris;
infusion of saline (IN Drop - low) Intranasal 0.02 g/kg two puffs
to alternating naris at 0 and Device - 10 minutes, with
accompanying control (IN Device) intravenous infusion of saline
Intravenous (IV) 0.02 g/kg infusion to left femoral vein over four-
teen minutes *3 rats/time point for a total of 27 rats per
experimental group
[0545] On the day of delivery, each .sup.125I-IgG aliquot was
removed from the freezer and allowed to come to room temperature
(about 20 minutes). The aliquots were then gently vortexed. A
sample of 1 .mu.l was placed into 999 .mu.l of water and vortexed
(1:1,000 dilution). Three 20 .mu.l samples were removed from the
dilution and placed into labeled gamma tubes. An additional 10
.mu.l was placed into 90 .mu.l of water and vortexed (1:10,000
dilution). Three 20 .mu.l samples were removed from the 1:10,000
dilution and placed into labeled gamma tubes. Standards were later
quantified through gamma counting. All doses within groups were
equalized for volume, weight (mg), and radioactivity (.mu.Ci) by
varying the dilution with saline to account for the decay of
.sup.125I.
[0546] Adult male Sprague-Dawley rats (Animal Care Facility at
Regions Hospital from Harlan) with the left femoral vein canulated
were used in this experiment. All rats weighed approximately 250 g
to ensure accurate dosing. The animals were housed individually
with free access to food and water. Animals were kept on a 12-hour
light cycle.
[0547] For the IN Drop, IN Device, and IV administrations, an
anesthesia cocktail containing ketamine HCl (30 mg/kg), xylazine
HCl (6 mg/kg), and acepromazine (1 mg/kg) was used. All anesthesia
was administered as subcutaneous injections. Boosters alternated
between the cocktail described above and 50 mg/kg ketamine.
Reflexes were tested to assess level of anesthesia every 10-15
minutes throughout the study. Animals in groups sacrificed at 4 hr
and beyond were allowed to recover from anesthesia and were
re-anesthetized prior to euthanasia.
[0548] For IN Drop delivery, anesthetized rats were placed on their
backs on a heating pad. .sup.125I-IgG was administered intranasally
as 8.times.6 .mu.L nose drops with an Eppendorf pipettor to
alternating nares every 2 minutes for a total volume of 48 .mu.L.
Animals were then monitored for adverse effects and anesthesia
levels until the euthanasia time point was reached. During
intranasal delivery, a 500 .mu.L sample of saline was infused over
14 minutes through the left femoral vein. All animals were rolled
off of their backs at 15 minutes after the completion of
delivery.
[0549] For IN Device delivery, anesthetized animals were placed on
their backs and a tube was inserted about 14 mm deep into the
nostril. The tube was connected to an actuator that delivered 15
.mu.L of dosing solution toward the olfactory epithelium. One bolus
was sprayed at the start of delivery, one was sprayed at 10 minutes
after the onset of delivery. Animals were then monitored for
adverse effects and anesthesia levels until the euthanasia time
point was reached. During intranasal delivery, a 500 .mu.l sample
of saline was infused over 14 minutes through the left femoral
vein. All animals were rolled off of their backs at 15 min after
the completion of delivery.
[0550] For IV delivery, anesthetized animals were placed on their
backs. A blunt 22 gauge needle attached to a 1 cc syringe was
inserted into the femoral vein canula. .sup.125I-IgG was prepared
in 500 .mu.l aliquots and infused over 14 minutes. Animals were
then monitored for adverse effects and anesthesia levels until the
euthanasia time point was reached.
[0551] At the experimental end time, blood was drawn directly from
the heart and animals were perfused with 120 ml ice cold saline
directly through the heart. One small drop of blood was placed into
a pre-weighed, labeled gamma tube and approximately 0.6 mL was
placed into a labeled serum separator tube. The serum separator
tube was spun and serum was collected. The serum was diluted in
homogenization buffer. The diluted serum was further spun down in a
100 kDa size exclusion filtration device. Samples were collected
from both the top of the filter and the bottom and placed into
labeled gamma tubes. The filter was also collected and placed into
a labeled gamma tube.
[0552] The brain was extracted from the skull and hemisected. The
left hemisphere was further processed as described below. The right
hemisphere was weighed, cut into 7 pieces and placed into labeled
gamma tubes.
[0553] Additionally, the olfactory and respiratory epithelia were
collected separately. The epithelia were expected to contain higher
amounts of .sup.125I than the quantitation limit of the gamma
counter, so both were split into multiple pieces. Each piece of
epithelia was placed into a pre-weighed, labeled gamma tube.
[0554] The left hemisphere was weighed after removal from the
skull. It was homogenized and spun down to retrieve supernatant.
The supernatant was further spun down in a 100 kDa size exclusion
filtration device. Samples were collected from both the top of the
filter and the bottom and placed into labeled gamma tubes. The
pellet was collected and placed into a pre-weighed labeled gamma
tube. The filter was also collected and placed into a labeled gamma
tube.
[0555] 3-5 mm samples of body tissues were collected and placed
into pre-weighed, labeled gamma tubes. Body tissues include: liver,
spleen, kidney, small intestine, lung, esophagus, trachea, and
blood (drawn directly from the heart as described above). The gamma
tubes containing samples were counted using a COBRA II Auto-Gamma
Counter.
[0556] Results:
[0557] Intactness of IgG in the brain was slightly less with
intranasal administrations (example: IN high--49%, IN low--49%, IN
device--40% at 15 minutes) as compared to intravenous
administration (69% at 15 minutes) in the earlier time points
(Table 65, Table 66, Table 67, and Table 68). However, because of
the non-validated method of calculating the intactness and the
limitations of the gamma counting machine, non-intact or "free"
.sup.125I may be magnified. The CPM counts from gamma tubes for
aliquots representing the "bottom" of the filtration device tubes
(where the non-intact IgG would be expected) were rather low in
many of IN treated animals. It is usually desired that the counts
reach at least two times background (in this study would be
.about.50 CPM).
TABLE-US-00065 TABLE 65 Biodistribution and intactness of IgG
administered to rats via high dose nasal drops (0.02 g IgG/kg).
ug/g IN-Drops High IN IN IN IN IN High High High High High Time 15
min 30 min 1 hr 2 hr 4 hr Raw ug/g 92,625,403 99,889,203 97,886,218
111,043,619 101,049,672 Dosed ug/g (60 uCi) Total ug/g Olfactory
585 127 120 938 167 Epithelium Respiratory 8,614 11,790 13,222
16,686 5,189 Epithelium R. Hemisphere 0.24 0.22 0.18 0.10 0.11 L.
Hemisphere 0.11 0.272 0.200 0.126 0.095 (total recovered) Dosing
Solution 38,594 41,621 40,786 46,268 42,104 (1:1,000) ug/g Blood
3.1 3.3 4.4 4.0 3.7 Liver 0.23 0.46 0.51 0.43 0.44 Spleen 0.55 1.1
1.4 1.2 1.4 Kidney 0.9 1.9 2.7 1.5 1.6 Small Intestines 0.32 0.4
0.91 0.75 2.2 Lung 0.9 1.8 1.5 1.0 1.3 Esophagus 0.51 0.61 1.1 0.9
33 Trachea 0.75 0.77 4.0 1.7 3.0 Intactness IN1 Brain 49% 46% 40%
48% 51% IN1 Blood 39% 32% 35% 33% 16% ug/g IN-Drops High IN IN IN
IN High High High High Time 8 hr 12 hr 24 hr 72 hr Raw ug/g
99,398,932 78,063,258 108,114,877 76,689,700 Dosed ug/g (60 uCi)
Total ug/g Olfactory 118 20 10 0.56 Epithelium Respiratory 1,312 41
10 2.4 Epithelium R. Hemisphere 0.15 0.22 0.16 0.039 L. Hemisphere
0.128 0.231 0.145 0.029 (total recovered) Dosing Solution 41,416
32,526 45,048 31,954 (1:1,000) ug/g Blood 5.3 7.3 5.4 0.8 Liver 1.0
0.9 1.1 0.24 Spleen 1.2 2.4 1.5 0.16 Kidney 2.8 3.8 2.2 0.39 Small
Intestines 2.5 6.3 1.3 0.09 Lung 2.1 2.4 1.7 0.26 Esophagus 126 4.9
6 0.22 Trachea 2.4 17 5 0.25 Intactness IN1 Brain 53% 49% 49% 66%
IN1 Blood 27% 30% 27% 54%
TABLE-US-00066 TABLE 66 Biodistribution and intactness of IgG
administered to rats via low dose nasal drops (0.002 g IgG/kg).
ug/g IN Drops-Low IN IN IN IN IN Low Low Low Low Low Time 15 min 30
min 1 hr 2 hr 4 hr Dosed ug/g 91,152,030 71,045,179 83,024,122
109,042,942 102,934,060 (60 uCi) Total ug/g Olfactory 118 57.6 58.7
58.0 56 Epithelium Respiratory 9,930 12,284 10,402 6,716 3,055
Epithelium R. Hemisphere 0.060 0.048 0.031 0.020 0.015 L.
Hemisphere 0.057 0.042 0.023 0.018 0.016 (total recovered) Dosing
Solution 37,980 29,602 34,593 45,435 42,889 (1:1,000) ug/g Blood
0.41 0.56 0.51 0.44 0.37 Liver 0.091 0.09 0.06 0.086 0.061 Spleen
0.15 0.21 0.31 0.19 0.12 Kidney 0.22 0.26 0.27 0.1 0.20 Small
Intestines 0.075 0.16 0.10 0.13 0.18 Lung 0.14 0.25 0.09 0.15 0.17
Esophagus 0.076 0.13 0.13 0.17 14 Trachea 0.14 0.36 0.26 0.19 0.58
Intactness IN2 Brain 49% 46% 45% 48% 50% IN2 Blood 28% 22% 29% 19%
26% ug/g IN Drops-Low IN IN IN IN Low Low Low Low Time 8 hr 12 hr
24 hr 72 hr Dosed ug/g 64,471,560 78,549,717 72,139,899 64,455,268
(60 uCi) Total ug/g Olfactory 1.9 25.9 6.69 0.571 Epithelium
Respiratory 101 111 7.8 1.20 Epithelium R. Hemisphere 0.026 0.032
0.015 0.0044 L. Hemisphere 0.027 0.030 0.014 0.0040 (total
recovered) Dosing Solution 26,863 32,729 30,058 26,856 (1:1,000)
ug/g Blood 0.78 0.99 0.57 0.067 Liver 0.15 0.19 0.12 0.036 Spleen
0.38 0.30 0.20 0.023 Kidney 0.53 0.63 0.28 0.042 Small Intestines
0.33 0.29 0.058 0.012 Lung 0.26 0.43 0.29 0.032 Esophagus 3.4 0.69
0.47 0.028 Trachea 4 0.50 0.30 0.034 Intactness IN2 Brain 65% 48%
49% 72% IN2 Blood 37% 25% 31% 52%
TABLE-US-00067 TABLE 67 Biodistribution and intactness of IgG
administered to rats via high dose intranasal device (0.02 g
IgG/kg). ug/g IN Device IN IN IN IN IN Device Device Device Device
Device Time 15 min 30 min 1 hr 2 hr 4 hr Dosed ug/g 99,099,000
125,565,000 60,108,000 77,362,000 73,446,000 (60 uCi) Total ug/g
Olfactory 5,076 5,276 2,016 3,917 2,134 Epithelium Respiratory
5,658 5,970 3,285 6,850 3,099 Epithelium R. Hemisphere 0.6 1.07 1.5
0.22 0.2 L. Hemisphere 0.831 1.32 0.365 0.229 0.139 (total
recovered) Dosing Solution 66,067 83,710 40,072 51,575 48,964
(1:1,000) ug/g Blood 11 18 11 8.3 4.7 Liver 0.45 4.3 1.6 0.57 0.4
Spleen 1.4 3.2 4.8 1.7 1.3 Kidney 1.5 4.9 5.6 2.3 1.3 Small
Intestines 0.52 1.2 3.2 1.3 1.2 Lung 1.4 4.5 5.9 2.0 1.8 Esophagus
1.5 3.1 4.9 3.4 488 Trachea 1.8 2.5 4.2 22 5.2 Intactness IN3 Brain
40% 44% 46% 43% 45% IN3 Blood 34% 29% 34% 30% 26% ug/g IN Device IN
IN IN IN Device Device Device Device Time 8 hr 12 hr 24 hr 72 hr
Dosed ug/g 67,726,000 87,418,000 83,486,000 74,898,000 (60 uCi)
Total ug/g Olfactory 381 103 14 1.7 Epithelium Respiratory 262 46
10 1.4 Epithelium R. Hemisphere 0.36 0.23 0.14 0.042 L. Hemisphere
0.15 0.202 0.122 0.0527 (total recovered) Dosing Solution 45,151
58,279 55,657 49,932 (1:1,000) ug/g Blood 6.7 5.9 4.7 0.61 Liver
0.8 1.1 0.76 0.23 Spleen 2.1 2.3 1.2 0.13 Kidney 3.3 3.2 1.0 0.26
Small Intestines 6.6 2.5 0.61 0.079 Lung 3.1 5.1 3.2 0.17 Esophagus
19 2.5 1.3 0.12 Trachea 3.6 3.3 1.7 0.22 Intactness IN3 Brain 46%
51% 47% 66% IN3 Blood 32% 25% 30% 67%
TABLE-US-00068 TABLE 68 Biodistribution and intactness of IgG
administered to rats via high dose intravenous infusion (0.02 g
IgG/kg). ug/g IV High IV IV IV IV IV Time 15 min 30 min 1 hr 2 hr 4
hr Dosed ug/g 125,946,138 76,865,000 88,715,556 150,181,389
86,270,833 (60 uCi) Total ug/g Olfactory 14.4 19 21 24 7 Epithelium
Respiratory 9.8 14 17 13.1 5.3 Epithelium R. Hemisphere 0.425 0.5
0.6 0.63 0.36 L. Hemisphere 0.533 0.52 0.56 0.548 0.411 (total
recovered) Dosing Solution 5,038 3,075 3,549 6,007 3,451 (1:1,000)
ug/g Blood 141 96 79 59 35 Liver 80 34 28 34 25 Spleen 57 30 28 32
20 Kidney 112 100 77 59 38 Small Intestines 13.4 8 9 9.2 7.1 Lung
24 89 21 33 13.1 Esophagus 7.0 4 4 5.0 5.7 Trachea 9.7 9 11 9.4 8.8
Intactness IV Brain 69% 68% 63% 59% 56% IV Blood 94% 92% 94% 90%
83% ug/g IV High IV IV IV IV Time 8 hr 12 hr 24 hr 72 hr Dosed ug/g
105,588,889 81,584,098 74,669,134 64,916,672 (60 uCi) Total ug/g
Olfactory 18 18 12 0.41 Epithelium Respiratory 20 10 7 0.7
Epithelium R. Hemisphere 0.34 0.39 0.14 0.038 L. Hemisphere 0.306
0.315 0.134 0.036 (total recovered) Dosing Solution 4,224 3,263
2,987 2,597 (1:1,000) ug/g Blood 26 13 8 0.8 Liver 15 15 10 3.6
Spleen 14 14 10 2.6 Kidney 29 29 19 10 Small Intestines 5.2 3.1 2.4
0.29 Lung 7.2 6.8 4.2 0.7 Esophagus 5.5 2.8 4.3 0.28 Trachea 6.3
2.1 5.4 0.34 Intactness IV Brain 56% 48% 51% 68% IV Blood 74% 57%
53% 79%
TABLE-US-00069 TABLE 69 Biodistribution and intactness of IgG
administered to rats via high dose nasal drops (0.02 g IgG/kg), as
corrected for intactness of immunoglobulin G. ug/g IN Drops High -
corrected for intactness IN IN IN IN IN IN IN IN IN Drops High
Drops High Drops High Drops High Drops High Drops High Drops High
Drops High Drops High Minutes 15 30 60 120 240 480 720 1,440 4,320
Corrected R. Hemisphere 0.12 0.10 0.07 0.05 0.05 0.08 0.11 0.08
0.03 L. Hemisphere 0.05 0.12 0.08 0.06 0.05 0.07 0.11 0.07 0.02
(total recovered) Blood 1.20 1.07 1.53 1.331 0.604 1.427 2.20 1.47
0.454 Uncorrected R. Hemisphere 0.24 0.22 0.18 0.10 0.11 0.15 0.22
0.16 0.04 L. Hemisphere 0.11 0.27 0.20 0.13 0.10 0.13 0.23 0.15
0.03 (total recovered) Blood 3.05 3.32 4.38 4.01 3.73 5.27 7.31
5.44 0.84 Trap. Calcs AUC R. Hemisphere 1.6 2.6 3.5 6.0 16.0 22.2
66.6 151.9 270.4 L. Hemisphere 1.3 3.1 4.2 6.5 13.9 21.6 65.7 129.0
245.4 (total recovered) Blood 17.0 39.0 86.0 116.1 243.7 435.3
1,323.3 2,777.1 5,038 Uncorrected Trap. Calcs R. Hemisphere 3.4 5.9
8.2 12.1 30.7 44.0 137.1 293.1 534.5 L. Hemisphere 2.9 7.1 9.8 13.3
26.8 43.0 135.2 250.8 488.8 (total recovered) Blood 47.8 115.5
251.6 464.2 1,079.2 1,508.9 4,587.5 9,041.8 17,096 Intactness IN1
Brain 49% 46% 40% 48% 51% 53% 49% 49% 66% IN1 Blood 39% 32% 35% 33%
16% 27% 30% 27% 54%
TABLE-US-00070 TABLE 70 Biodistribution and intactness of IgG
administered to rats via low dose nasal drops (0.002 g IgG/kg), as
corrected for intactness of immunoglobulin G. ug/g IN Drops Low -
corrected for intactness IN IN IN IN IN IN IN IN IN Low Low Low Low
Low Low Low Low Low Minutes 15 30 60 120 240 480 720 1,440 4,320
Corrected R. Hemisphere 0.029 0.022 0.014 0.0096 0.0073 0.017 0.016
0.0072 0.0032 L. Hemisphere 0.028 0.019 0.010 0.009 0.0078 0.0175
0.014 0.0068 0.0029 (total recovered) Blood 0.12 0.12 0.14 0.08
0.10 0.28 0.25 0.17 0.035 Uncorrected R. Hemisphere 0.060 0.048
0.031 0.020 0.015 0.026 0.032 0.015 0.004 L. Hemisphere 0.057 0.042
0.023 0.018 0.016 0.027 0.030 0.014 0.004 (total recovered) Blood
0.41 0.56 0.51 0.44 0.37 0.78 1.0 0.57 0.067 Corrected AUC Trap.
Calcs R. Hemisphere 0.4 0.5 0.7 1.0 2.9 3.9 8.2 15.0 33 L.
Hemisphere 0.4 0.4 0.6 1.0 3.0 3.8 7.7 14.0 31 (total recovered)
Blood 1.8 4.0 6.9 10.8 45.7 63.7 151.3 302.0 586 Uncorrected Trap.
Calcs R. Hemisphere 0.8 1.2 1.5 2.1 4.9 7.1 17.0 27.6 62 L.
Hemisphere 0.7 1.0 1.2 2.0 5.1 6.9 15.9 25.9 59 (total recovered)
Blood 7.3 15.9 28.4 48.8 137.7 212.1 562.4 919.3 1,932 Intactness
IN2 Brain 49% 46% 45% 48% 50% 65% 48% 49% 72% IN2 Blood 28% 22% 29%
19% 26% 37% 25% 31% 52%
TABLE-US-00071 TABLE 71 Biodistribution and intactness of IgG
administered to rats via high dose intranasal device (0.02 g
IgG/kg), as corrected for intactness of immunoglobulin G. ug/g IN
Device - corrected for intactness IN3 IN3 IN3 IN3 IN3 IN3 IN3 IN3
IN3 Minutes 15 30 60 120 240 480 720 1,440 4,320 Corrected R.
Hemisphere 0.23 0.47 0.71 0.09 0.08 0.16 0.12 0.07 0.03 L.
Hemisphere 0.33 0.58 0.17 0.10 0.06 0.07 0.10 0.06 0.03 (total
recovered) Blood 3.9 5.3 3.9 2.5 1.2 2.2 1.5 1.4 0.41 Uncorrected
R. Hemisphere 0.6 1.1 1.5 0.2 0.2 0.4 0.2 0.1 0.0 L. Hemisphere 0.8
1.3 0.4 0.2 0.1 0.2 0.2 0.1 0.1 (total recovered) Blood 11.5 18.2
11.4 8.3 4.7 6.7 5.9 4.7 0.6 Trap. Calcs AUC R. Hemisphere 5.3 17.7
24.0 10.2 28.9 33.6 65.4 133.2 318 L. Hemisphere 6.9 11.2 8.0 9.7
15.8 20.5 57.4 132.1 262 (total recovered) Blood 68.8 137.6 189.9
221.6 409.6 436.8 1,047.2 2,669.4 5,181 Uncorrected Trap. Calcs R.
Hemisphere 12.3 39.3 52.8 23.1 63.3 70.5 132.6 259.6 654 L.
Hemisphere 16.2 25.3 17.8 22.0 34.6 42.2 116.4 250.8 525 (total
recovered) Blood 222.4 444.2 591.4 778.3 1,369.6 1,516.1 3,826.3
7,703.8 16,452 Intactness IN3 Brain 40% 44% 46% 43% 45% 46% 51% 47%
66% IN3 Blood 34% 29% 34% 30% 26% 32% 25% 30% 67%
TABLE-US-00072 TABLE 72 Biodistribution and intactness of IgG
administered to rats via high dose intravenous infusion (0.02 g
IgG/kg), as corrected for intactness of immunoglobulin G. ug/g IV
High - corrected for intactness IV IV IV IV IV IV IV IV IV Minutes
15 30 60 120 240 480 720 1,440 4,320 Corrected R. Hemisphere 0.29
0.35 0.40 0.37 0.20 0.19 0.19 0.07 0.03 L. Hemisphere 0.37 0.36
0.35 0.32 0.23 0.17 0.15 0.07 0.02 (total recovered) Blood 132.3
88.4 74.0 53.2 28.6 19.2 7.2 4.2 0.6 Uncorrected R. Hemisphere 0.43
0.52 0.628 0.632 0.36 0.34 0.39 0.14 0.038 L. Hemisphere 0.53 0.52
0.56 0.55 0.41 0.31 0.32 0.13 0.036 (total recovered) Blood 141.1
96.2 79.1 58.8 34.6 26.1 12.7 7.8 0.80 Trap. Calcs AUC R.
Hemisphere 4.8 11.2 23.1 34.6 47.4 45.5 92.7 139.0 398 L.
Hemisphere 5.4 10.6 20.3 33.3 48.1 38.6 78.6 133.5 369 (total
recovered) Blood 1,655.5 2,437.1 3,817.6 4,908.0 5,738.1 3,165.3
4,074.7 6,898.1 32,694 Uncorrected Trap. Calcs R. Hemisphere 7.1
17.2 37.8 59.6 84.8 88.3 191.1 254.9 741 L. Hemisphere 7.69 16.56
34.25 67.06 93.15 76.73 230.14 360.25 886 (total recovered) Blood
1,779.9 2,630.8 4,138.5 5,602.1 7,279.5 4,651.1 7,366.9 12,398.1
45,847 Intactness IV Brain 69% 68% 63% 59% 56% 56% 48% 51% 68% IV
Blood 94% 92% 94% 90% 83% 74% 57% 53% 79%
[0558] The maximum brain delivery of intact IgG achieved with the
intranasal IN Device (0.71 .mu.g IgG/g tissue) was almost twice the
maximum brain delivery achieved with intravenous (IV)
administration (0.40 .mu.g IgG/g tissue) of the same dose (0.02
g/kg), while resulting in a maximum blood concentration of 25 times
less (IN Device--5.3 .mu.g IgG/g, IV--132 .mu.g IgG/g) (Table 71
and Table 72).
[0559] The AUC of brain exposure over 72 hr was fairly equivalent
with the IN Device versus intravenous administration (318/262 IN
Device vs 398/369 IV in right/left hemisphere as corrected for
intactness) while the AUC of blood exposure was almost six times
greater with intravenous (5,181 Device vs. 32,694 as corrected for
intactness; Table 73).
TABLE-US-00073 TABLE 73 Area under the curve and maximum
concentrations of IN and IV .sup.125I-IgG over time in rats
administered pooled human IgG via INI, IN2, or IV. AUC - Corrected
IN1 IN2 IN3 IV R. Hemisphere 270 33 318 398 L. Hemisphere (total
recovered) 245 31 262 369 Blood 5,038 586, 5,181 32,694 Max
Concentrations - Corrected ug/g time (min) ug/g time ug/g time ug/g
time R. Hemisphere 0.12 15 0.029 15 0.71 60 0.40 60 L. Hemisphere
(total recovered) 0.12 30 0.028 15 0.58 30 0.37 15 Blood 2.2 720
0.28 480 5.3 30 132 15 AUC - Uncorrected IN1 IN2 IN3 IV R.
Hemisphere 534 62 654 741 L. Hemisphere (total recovered) 489 59
525 682 Blood 17,096 1,932 16,452 45,847 Max Concentrations -
Uncorrected ug/g time (min) ug/g time ug/g time ug/g time R.
Hemisphere 0.24 15 0.060 15 1.54 60 0.63 120 L. Hemisphere (total
recovered) 0.27 30 0.057 15 1.32 30 0.56 60 Blood 7.3 720 0.99 720
18.2 30 141 15
[0560] All four delivery methods showed decreasing concentrations
of IgG in the brain over time (Table 65, Table 66, Table 67, Table
68, Table 69, Table 70, Table 71, and Table 72). Intranasal drop
delivery to the brain was dose dependent (Table 65, Table 66, Table
69, and Table 70). Animals treated with the high dose of IgG had a
maximum brain concentration when corrected for intactness (0.12
.mu.g IgG/g) that was approximately four times higher than the
brain concentration (0.029 .mu.g IgG/g) of animals treated with the
lower dose of IgG (Table 69, Table 70, and Table 73).
[0561] A second .sup.125I-IgG peak was observed most of the tissues
in the IN administration groups (Table 65, Table 66, and Table 67).
This second peak may be due to an artifact of the animal model. The
animals began waking up from anesthesia around 2 hours and were
able to eat, drink, and groom normally. As a result, they may have
ingested some of the residue IgG that was on their noses and passed
through the nasopharynx into the mouth and esophagus once they were
upright. Therefore this second peak that occurred after 4 hours is
likely a result of blood-to-brain delivery of degraded IgG instead
of direct nose to brain delivery of intact IgG.
[0562] A small group of three rats was also treated intranasally
with a non-enhancer based dosing solution (Table 74). This group
had only one time point (30 minutes) and was dosed with the same
concentration and method as the IN high group described in Example
9 and Table 65. At the 30 minute time point, the concentration was
much lower in both the respiratory (2,097 IgG/g) and olfactory (37
.mu.g IgG/g) compared to the IN high group at 15 minutes (8,614
.mu.g IgG/g and 585 .mu.g IgG/g) and 30 minutes (11,790 .mu.g IgG/g
and 127 .mu.g IgG/g) respectively. The right hemisphere is equal
(0.22 .mu.g IgG/g for both the non-enhancer and IN high groups),
however the left hemisphere is much lower (0.04 .mu.g IgG/g)
compared to the IN high group
TABLE-US-00074 TABLE 74 Biodistribution and intactness of IgG
administered to rats via high dose nasal drops (0.02 g IgG/kg) (IN
High) compared to IgG administered with a non-enhancer based dosing
solution (IN4). ug/g IN High IN High IN High IN4 Raw ug/g 15 min 30
min 30 min Dosed ug/g (60 uCi) 92,625,403 99,889,203 685,291,111
Total ug/g Olfactory Epithelium 585 127 37 Respiratory Epithelium
8,614 11,790 2,097 R. Hemisphere 0.24 0.22 0.22 L. Hemisphere 0.11
0.27 0.04 (total recovered) Dosing Solution 38,594 41,621 27,412
(1:1,000) ug/g Blood 3.1 3.3 6.9 Liver 0.23 0.46 0.3 Spleen 0.55
1.1 0.8 Kidney 0.9 1.9 2.6 Small Intestines 0.32 0.4 0.6 Lung 0.9
1.8 1.1 Esophagus 0.51 0.61 1.1 Trachea 0.75 0.77 1.3 Intactness
IN1 Brain 49% 46% 39% IN1 Blood 39% 32% 37%
[0563] The bioavailability was calculated as the percent of CPMs
measured in the brain (or estimated blood volume) of the total
amount of CPMs delivered. Delivery via the intranasal device
resulted in the highest bioavailability of all methods (0.037% at
30 min) and was twice as high as the maximum bioavailability with
intravenous delivery (0.018% at 2 hr) (Table 75 and Table 76).
TABLE-US-00075 TABLE 75 Bioavailability - Brain (% of CPMs
delivered that reached the brain - not corrected for intactness).
Time IN high IN low IN device IV 15 min 0.0037% 0.016% 0.020%
0.015% 30 min 0.0072% 0.014% 0.037% 0.016% 1 h 0.0056% 0.008%
0.030% 0.018% 2 h 0.0034% 0.0053% 0.007% 0.0180% 4 h 0.0030%
0.0047% 0.004% 0.012% 8 h 0.0041% 0.008% 0.006% 0.010% 12 h 0.0068%
0.010% 0.006% 0.011% 24 h 0.0047% 0.0045% 0.0039% 0.0041% 72 h
0.0010% 0.0012% 0.0014% 0.0011%
TABLE-US-00076 TABLE 76 Bioavailability - Blood (% of CPMs
delivered that reached the brain - not corrected for intactness).
Time IN high IN low IN device IV 15 min 1.09% 1.47% .sup. 4% 50.3%
30 min 1.18% 2.0% .sup. 6% 34.3% 1 h 1.56% 1.8% .sup. 4% 28.2% 2 h
1.43% 1.58% .sup. 3% 21.0% 4 h 1.33% 1.32% 1.7% 12.3% 8 h 1.9% 2.8%
2.4% 9.3% 12 h 2.6% 3.5% 2.1% 4.5% 24 h 1.9% 2.0% 1.7% 2.79% 72 h
0.30% 0.24% 0.22% 0.29%
[0564] As well as being a less invasive option than intravenous
infusion, the increased targeting to the brain (i.e. less blood and
systemic exposure) achieved using IN Drops or an IN Device would be
expected to reduce the risk of systemic side effects of IgG
therapy.
Example 10--Stability of IgG Administered by IN Drops and IN Device
& Olfactory Epithelium Targeting
[0565] The stability of IgG administered by IN Drops or IV and IN
Device was compared to determine optimal modes of administration.
Degradation and aggregation of the IgG administered via the IN
Device was measured and olfactory targeting was assessed.
[0566] Stability of IgG:
[0567] Samples of sprayed IgG from the IN Device were compared to
unsprayed IgG solution (representative of IVIG and IN Drops). Five
IgG formulations were prepared and divided into spray and solution
trial groups (A=25% pooled IgG, B=5% pooled IgG, C=10% pooled IgG,
D=20% pooled IgG, E=25% pooled IgG). The sprayed and solution IgG
samples were run on non-reducing and reducing gels. The gels were
either stained or blotted as follows: 1) a Coomassie stained,
non-reduced gel, and 2) a reducing SDS-Page gel which was Western
blotted and Coomassie stained.
[0568] In the non-reduced gel (FIG. 6A), there were no apparent
higher order aggregates or IgG degradation forms for both the
solution and sprayed IgG. In the reduced Western blot (FIG. 6B),
intact IgG was seen as well as the heavy chain (HC) and light chain
(LC) fragments of the IgG. Combinations of heavy chain and light
chain (HC/LC) were also seen on the reducing gel. Based on these
results, spraying the IgG through an IN Device does not increase
IgG degradation or increase IgG aggregation.
[0569] Olfactory Epithelium Targeting:
[0570] IgG was administered to rats with the intranasal Device and
with intranasal drops. A 25% solution of IgG formulated in
histidine buffer was spiked with 0.01% fluorescein tracer. It was
then intranasally delivered to a rat using an intranasal Device or
drops and the brain of the rat was imaged to detect neural
IgG-fluorescein deposition.
[0571] Representative images of the deposition pattern of
intranasal IgG (less than 2 minutes after drug administration) are
depicted in FIGS. 7A and 7B. FIG. 7A shows the deposition after
Device administration of 15 .mu.L of 25% IgG solution spiked with
0.01% fluorescein tracer in a rat. FIG. 7B shows the deposition
patter after deposition of the same compound that was administered
with nose drops. As can be seen by FIG. 7, there is greater
olfactory epithelium (OE) staining from Device administration.
[0572] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
Example 11--Intranasal IgG Administration Decreases the Area of the
Brain Covered by Plaques
[0573] An experiment was designed to assess the effect of
intranasal IgG on brain plaques and vascular amyloid. Congo Red
staining of Tg2576 mouse brains revealed a decrease in the area
covered by plaques, the number of plaques and the total intensity
of the plaques in both the low-dose and high-dose IgG intranasal
treatment groups. The decrease approximates the reduction of
.beta.-amyloid identified with immuno-staining in the cortex. For
example, in the low dose group amyloid decreased by 25.7% when
assessed with IHC and 22% when analyzed with Congo Red. However,
unlike the immuno-staining, the reduction in Congo Red plaque
staining did not reach a level of statistical significance.
[0574] Plaque amyloid reduction was not observed with the vascular
amyloid in either the low or high dose IgG groups. Although not
statistically significant, a slight increase in vascular amyloid
was observed with the high-dose IgG group.
[0575] Experimental Design:
[0576] The aim of this study was to determine if intranasal IgG
treatment alters plaque and/or vascular amyloid in the brains of
Tg2576 mice. At 9 months of age, the mice were intranasally treated
with IgG or saline three times per week for 7 months (a description
of the experimental groups is provided in Table 15). At 16 months
of age, behavioral testing occurred and at .about.17 months of age,
12 mice per group were euthanized (transcardial perfusion with
saline) and brain tissue was collected for analysis. The brain was
sagittally hemisected, the right half was fixed in formalin,
embedded in paraffin and sectioned at 5 .mu.m. Sections
approximately 2.5 mm from sagittal midline were used for Congo Red
staining.
[0577] Quantification of both the plaque and vascular amyloid in
the brains of the control groups, WT-PBS and WT-High Dose, and the
experimental Tg2576 groups, Tg-PBS, Tg-Low Dose (400 .mu.g/kg/2 wk)
and Tg-High Does (800 .mu.g/kg/2 wk) were analyzed using Congo Red
staining and fluorescent microscopy. In this procedure sagittal
sections were stained with a standard Congo Red procedure and
imaged fluorescently using a Nikon A1 Spectral Confocal Microscope.
Specifically, NIS Elements imaging software was used to control
acquisition and analysis. An objective with 10.times. magnification
was used for capturing images. With this magnification the smaller
blood vessels could be easily distinguish from plaques. At this
magnification a 6.times.5 tiling of 30 images (5% overlap) was
needed to capture the whole brain section. Each of these 30 images
was created from a max intensity projection of a z-stack of 5
individual images. This corrected any change in the captured focal
plane across the whole tissue section. Laser excitation at 561 nm
(voltage at 10%) was used to excite the Congo Red and the spectrum
between 570-620 nm was captured for quantifying the
fluorescence.
[0578] A single image of the complete sagittal section was obtained
by stitching together 150 individual images (thirty in the x-y
plane and five in z dimension). Representative examples of these
images are included in FIG. 12. The plaque and vasculature amyloid
deposits were distinguished manually by a blinded researcher
through the de-selection of the vascular components from the total
amyloid using Nikon's Elements AR software. The area covered by the
amyloid (Area), the number of individual amyloid deposits (#) and
the sum of the intensities of these objects (Sum Intensity) were
determined for the total of the amyloid deposits (both plaque and
vascular), and for the plaque and vascular deposits
individually.
[0579] Specifically, image analysis and quantification consisted of
first adjusting look-up tables (LUTs) for optimal and consistent
viewing to distinguish vascular and plaque amyloid. Second, the
whole brain area of the brain section was quantified to determine
the fraction of total brain covered by amyloid wherein the
threshold was set so all tissue was highlighted and the cerebellum
was deselected. Third, the total amyloid occupied areas were
quantified by setting the threshold to accurately select the
amyloid stained objects, deselect any staining due to background or
tissue/staining artifacts prior to quantification, and deselect
object in the cerebellum. Fourth, the vasculature amyloid was
deselected by manually zooming into the image to a 1:1 resolution
and individually deselecting the highlighted selections that were
associated with blood vessels. Fifth, the plaque amyloid occupied
areas were quantified.
[0580] For each sagittal section, the total amyloid stained objects
(Total), the amyloid stained plaques (Plaques), and the amyloid
stained vascular deposits (Vasculature) were measured and data
collected. Separate measurements were directly captured for Total
and Plaques. Values for Vascular were obtained by subtracting
Plaques from Total. For each of the data sets, parapeters were
determined, including 1) number of objects, 2) area (% area
occupied by the collection of objects), and 3) SumIntensity (equals
the sum of [Mean Intensity*Area] for each individual object). Two
separate values were calculated for the area parameter: 1) AreaImg
(areas summed from image divided by total area of Image*100), and
2) AreaTis (Areas summed from image divided by the area occupied by
brain tissue*100).
[0581] Average values, standard deviations, and standard errors
were calculated for each of the three parameters for each of the
five experimental groups described in Table 15. The five
experimental groups were analyzed for significance using a
two-tailed t-test and the following comparisons were performed:
WT-Saline vs. Tg-Saline, Tg-Saline vs. Tg-Low, Tg-Saline vs.
Tg-High, and Tg-Low vs. Tg-High.
[0582] Results:
[0583] Changes in the accumulation of total, plaque or vascular
amyloid did not reach significance with t-tests in the comparison
of the Tg-PBS group to either the Tg-low or Tg-high group (Table
77). However, for each of the three parameters assessed, area
covered by the amyloid (FIG. 8A and FIG. 8B), the number of
individual amyloid deposits (FIG. 8B and FIG. 9B) and the sum of
the intensities of these objects (Sum Intensity, which represents
the total quantity of amyloid, FIG. 8C and FIG. 9C), for both total
amyloid and plaque amyloid were found to decrease with both the low
and high IgG intranasal treatments. The area covered by plaques was
reduced by 22% for the low dose and 20% for the high dose. The
number of plaques was reduced by 17% for low dose and 19% for the
high dose. The sum intensity of these plaques decreased 16% in the
low dose group and 24% in the high dose group.
TABLE-US-00077 TABLE 77 Percent change between groups and
associated t-test p-values. % change T-test (p=) Total Amyloid Area
(Plaque and Vascular) WT-saline vs TG-saline 2517% 0.000109
TG-saline vs TG-low -14% 0.520 TG-saline vs TG-high -7% 0.809
TG-low vs TG-high -7% 0.824 # Amyloid Depoits (Plaque and Vascular)
WT-saline vs TG-saline 1633% 0.000010 TG-saline vs TG-low -8% 0.381
TG-saline vs TG-high -1% 0.667 TG-low vs TG-high -1% 0.745 Total
Intestity All Amyloid Deposits (Sum Intensity) WT-saline vs
TG-saline 3854% 0.000132 TG-saline vs TG-low -9% 0.713 TG-saline vs
TG-high -11% 0.715 TG-low vs TG-high -11% 0.958 Plaque Amyloid Area
WT-saline vs TG-saline 3919% 0.000220 TG-saline vs TG-low -22%
0.345 TG-saline vs TG-high -20% 0.478 TG-low vs TG-high -20% 0.931
# Amyloid Plaques WT-saline vs TG-saline 6355% 0.000109 TG-saline
vs TG-low -20% 0.381 TG-saline vs TG-high -11% 0.667 TG-low vs
TG-high -11% 0.745 Total Intestity Amyloid Plaques (Sum Intensity)
WT-saline vs TG-saline 4520% 0.000171 TG-saline vs TG-low -16%
0.531 TG-saline vs TG-high -24% 0.391 TG-low vs TG-high -24% 0.801
Vascular Amyloid Area WT-saline vs TG-saline 1708% 0.000357
TG-saline vs TG-low -1% 0.887 TG-saline vs TG-high 9% 0.822 TG-low
vs TG-high 9% 0.750 # Vascular Deposits WT-saline vs TG-saline 787%
0.000001 TG-saline vs TG-low 6% 0.710 TG-saline vs TG-high 12%
0.598 TG-low vs TG-high 12% 0.828 Total Intestity Vascular Deposits
(Sum Intensity) WT-saline vs TG-saline 3037% 0.000648 TG-saline vs
TG-low 3% 0.927 TG-saline vs TG-high 11% 0.796 TG-low vs TG-high
11% 0.855
[0584] The reduction of amyloid was absent in the vasculature
(FIGS. 10A, 10B, and 10C). For the vascular amyloid, each of the
three parameters increased in the high IgG intranasal treatment
group, whereas this increase was either absent or was present to a
lesser degree in the low dose group (FIGS. 10A, 10B, and 10C). The
sum intensity of these vascular amyloid deposits increased 3% in
the low dose group and 11% in the high dose group (Table 77).
[0585] The relative proportions of vascular and plaque amyloid as
it contributes to total amyloid is depicted in FIG. 11A and FIG.
11B. The average values from each group, along with standard
deviations and percent error are provided in
TABLE-US-00078 TABLE 78 Average values of amyloid plaques by
treatment group. Group Avg St. Dev St. Err Total Amyloid Area
(Plaque and Vascular) WT-saline 2329 4235 1223 WT-high 5039 4875
1407 TG-saline 60930 31891 9206 TG-low 52249 31572 9114 TG-high
56385 54901 15849 # Amyloid Deposits (Plaque and Vascular)
WT-saline 7 2.09 0.60 WT-high 18 1.51 0.43 TG-saline 114 34.36 9.92
TG-low 104 34.60 9.99 TG-high 113 46.17 13.33 Total Intestity of
Amyloid Deposits (Sum Intensity) WT-saline 891830 2038864 588569
WT-high 1426429 1390829 401498 TG-saline 35260904 19089383 5510630
TG-low 31921724 23768445 6861359 TG-high 31318149 30948336 8934015
Plaque Amyloid Area WT-saline 852 2442 705 WT-high 647 687 198
TG-saline 34241 19804 5717 TG-low 26539 18268 5273 TG-high 27338
25815 7452 # Amyloid Plaques WT-saline 1 2.09 0.60 WT-high 2 1.51
0.43 TG-saline 65 34.36 9.92 TG-low 52 34.60 9.99 TG-high 57 46.17
13.33 Total Intestity of Amyloid Plaques (Sum Intensity) WT-saline
491018 1538511 444130 WT-high 207397 217296 62728 TG-saline
22685550 12752488 3681326 TG-low 18985208 15068946 4350030 TG-high
17345869 16402272 4734928 Vascular Amyloid Area WT-saline 1477 687
198 WT-high 4391 19804 5717 TG-saline 26688 18268 5273 TG-low 26539
25815 7452 TG-high 29047 31592 9120 # Vascular Deposits WT-saline 6
5.33 1.54 WT-high 16 15.02 4.34 TG-saline 50 15.49 4.47 TG-low 53
24.61 7.10 TG-high 55 34.12 9.85 Total Intestity of Vascular
Deposits (Sum Intensity) WT-saline 400812 550529 158924 WT-high
1219032 1387106 400423 TG-saline 12575353 8294807 2394505 TG-low
12936517 10455665 3018290 TG-high 13972280 16240149 4688127
TABLE-US-00079 TABLE 79 Raw data of the areas and sum intensities
for each mouse (identified by ID number). ID Area Area Area
SumIntensity SumIntensity SumIntensity Number Group Total Plaques
Vascular Total Plaques Vascular 6 TG-saline 115,428 49,388 66,039
65,768,632 32,797,559 32,971,072 12 TG-saline 9,615 3,207 6,408
4,122,348 1,775,361 2,346,986 13 TG-saline 44,759 32,566 12,193
23,331,489 18,754,125 4,577,364 17 TG-saline 45,902 20,360 25,542
24,817,330 14,365,335 10,451,995 19 TG-saline 100,853 61,581 39,272
55,289,299 38,307,916 16,981,383 24 TG-saline 40,275 16,232 24,043
28,219,039 14,592,692 13,626,348 26 TG-saline 72,873 40,008 32,864
50,896,735 32,669,266 18,227,469 40 TG-saline 72,892 51,395 21,497
42,116,237 31,329,942 10,786,294 46 TG-saline 83,078 55,167 27,911
49,487,815 37,301,209 12,186,606 58 TG-saline 59,409 38,548 20,862
31,689,695 22,410,388 9,279,308 59 TG-saline 25,142 8,199 16,943
12,131,324 5,237,262 6,894,062 2 TG-Low 37,468 20,017 17,451
20,321,550 13,258,997 7,062,553 7 TG-Low 61,365 34,477 26,888
49,667,940 34,128,193 15,539,747 10 TG-Low 52,240 33,836 18,404
28,447,707 21,346,439 7,101,268 25 TG-Low 68,827 43,997 24,831
37,245,908 25,626,879 11,619,029 27 TG-Low 132,727 61,346 71,380
95,657,774 52,780,178 42,877,596 32 TG-Low 32,299 9,761 22,538
19,011,353 6,252,635 12,758,717 35 TG-Low 10,110 1,270 8,840
4,705,869 625,181 4,080,687 39 TG-Low 14,232 3,239 10,993 6,755,635
2,258,758 4,496,877 42 TG-Low 50,544 38,522 12,022 29,879,434
24,362,743 5,516,691 49 TG-Low 68,719 39,481 29,238 38,885,021
27,024,755 11,860,266 51 TG-Low 49,382 18,550 30,832 27,695,601
12,602,421 15,093,180 52 TG-Low 49,071 13,978 35,093 24,786,898
7,555,312 17,231,586 1 TG-high 2,813 641 2,172 1,048,809 237,175
811,634 15 TG-high 103,101 57,180 45,921 61,230,873 37,704,161
23,526,712 16 TG-high 53,649 34,611 19,039 31,405,116 23,587,366
7,817,749 20 TG-high 195,070 86,114 108,956 110,420,271 54,319,412
56,100,859 23 TG-high 69,989 41,806 28,184 37,222,228 26,023,396
11,198,831 34 TG-high 7,887 1,975 5,912 3,920,088 1,385,403
2,534,685 37 TG-high 57,257 37,868 19,388 31,515,587 23,230,486
8,285,101 41 TG-high 94,661 27,898 66,763 46,735,215 14,998,264
31,736,951 44 TG-high 23,751 4,985 18,766 13,317,820 3,773,642
9,544,178 47 TG-high 38,637 11,895 26,742 21,140,702 7,803,449
13,337,253 53 TG-high 10,580 8,478 2,102 6,247,449 5,468,454
778,995 55 TG-high 19,223 14,606 4,617 11,613,632 9,619,224
1,994,408 4 WT-saline 775 -- 775 175,280 -- 175,280 8 WT-saline 203
-- 203 63,844 -- 63,844 9 WT-saline 2,197 -- 2,197 549,057 --
549,057 14 WT-saline 2,274 -- 2,274 537,918 -- 537,918 18 WT-saline
279 191 89 99,865 75,732 24,133 21 WT-saline -- -- -- -- -- -- 29
WT-saline 15,165 8,503 6,662 7,272,935 5,363,736 1,909,198 30
WT-saline -- -- -- -- -- -- 33 WT-saline 2,547 133 2,413 806,288
64,381 741,907 43 WT-saline 3,836 1,397 2,439 1,030,568 388,371
642,196 57 WT-saline 667 -- 667 166,208 -- 166,208 60 WT-saline --
-- -- 0 -- -- 5 WT-high -- -- -- -- -- -- 11 WT-high 946 546 400
262,652 162,969 99,683 22 WT-high 5,233 1,035 4,198 1,418,585
303,890 1,114,696 28 WT-high 2,826 1,988 838 951,551 710,345
241,205 31 WT-high 9,405 362 9,043 2,931,770 228,181 2,703,589 36
WT-high 4,591 171 4,420 1,260,094 90,200 1,169,894 38 WT-high
15,883 -- 15,883 4,399,983 -- 4,399,983 45 WT-high -- -- -- -- --
-- 48 WT-high 11,393 1,638 9,754 3,248,816 451,611 2,797,205 50
WT-high 1,892 1,067 826 465,341 245,731 219,610 54 WT-high 4,477
959 3,518 1,191,920 295,835 896,086 56 WT-high 3,817 -- 3,817
986,431 -- 986,431
[0586] Congo Red stained sagittal sections captured with confocal
fluorescent microscopy are shown in FIGS. 12A-12F. Five individual
images at 10.times. with a 512.times.512 resolution were used to
create a z-stack max intensity projection image (FIG. 12A). Thirty
of these z-stacks projections, encompassing the whole tissue
section were tiled (6.times.5, 5% overlap) to create a single image
for analysis (FIGS. 12B-12F). Laser excitation at 561 nm excited
the Congo Red and the spectrum between 570-620 nm was captured for
quantification. Selection of the amyloid deposits was conducted
with the thresholding function in Nikons Elements AR software.
Examples of thresholding are depicted in FIGS. 12A and 12C-12F by
highlighting in red. FIG. 12A depicts a portion of the cortex and
hippocampus at full resolution, highlighting both amyloid plaques
and vascular amyloid. FIG. 12A is an example of a section from a
Tg-Low mouse without highlighting. Representative images from the
groups, WT-Saline (FIG. 12C), Tg-Saline (FIG. 12D), Tg-Low (FIG.
12E) and Tg-High (FIG. 12F) are shown with thresholding.
[0587] Analysis:
[0588] Congo Red staining revealed a decrease of amyloid load in
both the low IgG and high IgG intranasal treatment groups (FIGS.
8A-8C). This decrease in amyloid was a result of a decrease in
plaque load (FIGS. 9A-9C) as the vascular component of the amyloid
was found to increase slightly (FIGS. 10A-10C). Although the Congo
Red results did not reach statistical significance, the reduction
in plaque load is supported by the statistically significant
reduction of plaques in the cortex of these mice as determined by
the immuno-detection of .beta.-amyloid (IHC study). In that study,
using the 4G8 antibody to target .beta.-amyloid, the percent area
covered by plaques decreased by 25.7% for the low dose IgG group
and 24.3%, for the high dose IgG group, respectively, with p values
of 0.014 and 0.037. In the current study using Congo Red
fluorescent staining, the area covered by plaques was reduced by
22% for the low dose and 20% for the high dose, respectively, with
p values of 0.35 and 0.48. Thus, a similar degree of plaque
reduction observed in the IHC analysis was also detected with the
Congo Red analysis.
[0589] Several experimental parameters may be responsible for the
difference of statistical power observed between the IHC and Congo
Red analysis. The IHC analysis was limited to either the cortex or
hippocampus of the mouse brain and a significant decrease was only
observed in the cortex. In the Congo Red analysis, the complete
brain section that is anterior to the pons and cerebellum, with the
exception of the olfactory bulb was included. Variability and
dilution of the plaque load throughout these additional areas of
the brain may have contributed to a less significant p value.
Additionally, three tissue sections from each mouse were included
in the IHC analysis, whereas a single section was analyzed with the
Congo Red staining. The difference could also be related to the
staining properties of each method. The Congo Red detects the
insoluble fibrous protein aggregates of .beta.-sheets of amyloid,
whereas the IHC detects all Tg human .beta.-amyloid protein.
[0590] Amyloid staining in the vasculature is, for the most part,
observed only in the larger vessels of the brain. It has been
suggested that this is at least partially due to the absence of the
efflux amyloid transporter, LRP1 in these vessels. Consistent with
this point, vascular amyloid was observed almost exclusively in the
larger vessels in this study and IgG either did not affect the
aggregation of vascular amyloid or may have slightly increased it.
Although the brain does not have a separate lymphatic system, the
perivascular space surrounding the larger cerebral vessels provides
a path by which interstitial fluid and extracellular solutes,
including .beta.-amyloid can exit the brain.
Example 12--Immunofluorescent Staining of Intranasal IgG Treated
Tg2576 Mice Astrocyte (GFAP) and Microglial (CD11b)
Quantification
[0591] Human immunoglobulins are reactive to a wide array of
inflammatory proteins and intravenously administered IgG has been
shown to induce anti-inflammatory properties under a variety of
different conditions (Nimmerjahn F. et al., Annu Rev Immunol, 2008;
26: 513-533). The present study assessed the expression of two
inflammatory markers in the brains of Tg2576 mice in response to
low and high doses of chronically administered IN IgG.
[0592] Brain tissues were analyzed for GFAP and CD11b from the 3
transgenic groups of mice [TG-saline n=2, TG-low (0.4 g/kg/2 wk)
n=4, and TG-high (0.8 g/kg/2 wk) n=6] for which frozen samples were
available. Quantification of both GFAP and CD11b staining in the
brains of the experimental Tg2576 mice were analyzed using
fluorescent microscopy. In this procedure fixed frozen brain
sections (1 millimeter from the mid-sagittal plane) were triple
stained using antibodies for amyloid, a marker for activated
astrocytes (GFAP) and a marker for activated microglial (CD11b).
Fluorescence was captured with a Nikon A1 Spectral Confocal
Microscope. A sagittal section encompassing a portion of the
frontal cortex and a portion of the hippocampus was used for
quantifying average intensity of GFAP and CD11b staining. An
example of the images is shown in FIG. 14.
[0593] Briefly, brains were sectioned into 2 mm sagittal sections
and placed into 20% sucrose. Sections were then stored at 4.degree.
C. until all animals from the study were collected. Once all
samples were collected the tissue was mounted in OCT (frozen
quickly with dry ice) and sectioned on the Leica CM3050 cryostat at
20 .mu.m. Slide were allowed to dry at room temp overnight and
stored in the -20.degree. C. freezer. Prepared sections were then
stained according to standard IHC staining protocols.
[0594] The fluorescence of stained sections was imaged using three
channels corresponding to AlxaFluor405, AlexaFluor488, and
AlexaFluor 568 with a Nikon A1 Spectral Confocal Microscope. The
system was mounted on a Nikon Ti2000E widefield, inverted,
fluorescence microscope. NIS Elements imaging software was used to
control acquisition and analysis. An objective with 20.times.
magnification was used for capturing images. Individual
512.times.512 images that included a portion of the frontal cortex
and hippocampus were captured for quantitative analysis. Image
quantification was conducted with Nikon's Elements AR software and
total and average intensity values (corresponding to GFAP and
CD11b) were determined for each image.
[0595] The results show that the Tg-PBS group was not statistically
significant from the Tg-low or Tg-high groups for either the GFAP
or CD11b comparisons (Table 80). The average intensity of GFAP
staining was nearly identical in all three groups, differing by
only a few percentage points (Table 80, FIG. 13A). The average
intensity of CD11b staining decreased by 17% with the low dose of
IN IgG (p=0.741) and the magnitude of this decrease was larger, at
47%, with high dose of IN IgG (p=0.379) (Table 80, FIG. 13B).
TABLE-US-00080 TABLE 80 Results of GFAP and CD11b staining. GFAP
CD11b Ratio Ttest Ratio Ttest Tg-Low vs Tg-Saline 0.993 0.963 0.828
0.741 Tg-High vs Tg-Saline 0.987 0.946 0.565 0.379 Tg-Low vs
Tg-High 1.006 0.965 1.467 0.416
[0596] The immunofluorescent staining of the Tg2576 mouse brains
for the markers of inflammation, GFAP and CD11b, did not reveal a
significant difference between the saline and the low or high IN
IgG treated groups. The statistical power of this analysis was
limited by the low number of saline treated Tg mice available.
Values obtained for the astrocyte staining using the GFAP antibody
revealed practically no change in the quantification with either
the low or high IN IgG treatment (Table 80). Although significance
was not reached, there was an apparent reduction in microglial
staining using an antibody against CD11b. Average intensity of
CD11b marker decreased by 17% with the low IN IgG dose and
decreased by 47% with the high IN IgG dose (Table 80).
[0597] In this study, GFAP, a marker of astrocyte activation, was
not altered with either of the two treatment concentrations of IgG.
This result is consistent with previous work showing that IVIG
treatment of APP/PS1dE9 mice did not significantly affect the
expression of GFAP (Puli L. et al., Journal of neuroinflammation,
2012; 9: 105.). In the current study, CD11b, a marker of microglial
activation, did show a dose dependent decrease in protein
expression. However, neither the low dose nor the high dose values
reached statistical significance. In the APP/PS1dE9 study mentioned
above, Puli et. al. (Id.) found a significant reduction in the
microglia marker CD45 and an elevation of the microglial marker
Ibal with IV IgG treatment. Magga, et. al. (Journal of
neuroinflammation, 2010; 7: 90) also found that IVIG functioned in
a APP/PS1 mouse model through a mechanism involving microglial, but
not through a mechanism involving astrocytes. Although the affects
identified in the present study did not reach statistical
significance, the results do support a mechanism by which IgG
influences the inflammatory state of the CNS through microglial
modulation.
* * * * *