U.S. patent application number 12/726308 was filed with the patent office on 2010-07-22 for compositions and methods for enhanced mucosal delivery of parathyroid hormone.
This patent application is currently assigned to MDRNA, INC.. Invention is credited to Henry R. Costantino, Mary S. Kleppe, Ching-Yuan Li, Steven C. Quay.
Application Number | 20100184688 12/726308 |
Document ID | / |
Family ID | 35207584 |
Filed Date | 2010-07-22 |
United States Patent
Application |
20100184688 |
Kind Code |
A1 |
Quay; Steven C. ; et
al. |
July 22, 2010 |
COMPOSITIONS AND METHODS FOR ENHANCED MUCOSAL DELIVERY OF
PARATHYROID HORMONE
Abstract
Pharmaceutical compositions and methods are described comprising
at least a parathyroid hormone peptide (PTH) preferably
PTH.sub.1-34 and one or more mucosal delivery-enhancing agents for
enhanced nasal mucosal delivery of PTH, for treating or preventing
osteoporosis or osteopenia in a mammalian subject, preferably a
human.
Inventors: |
Quay; Steven C.;
(Woodinville, WA) ; Costantino; Henry R.;
(Woodinville, WA) ; Kleppe; Mary S.; (Snohomish,
WA) ; Li; Ching-Yuan; (Bellevue, WA) |
Correspondence
Address: |
Eckman Basu LLP
2225 E. Bayshore Road, Suite 200
Palo Alto
CA
94303-3220
US
|
Assignee: |
MDRNA, INC.
Bothell
WA
|
Family ID: |
35207584 |
Appl. No.: |
12/726308 |
Filed: |
March 17, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11744745 |
May 4, 2007 |
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12726308 |
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11126996 |
May 10, 2005 |
7244709 |
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11744745 |
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60570113 |
May 10, 2004 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61K 47/183 20130101;
A61K 31/724 20130101; A61P 43/00 20180101; A61P 19/10 20180101;
A61P 19/08 20180101; A61K 38/29 20130101; A61K 9/0043 20130101;
A61P 5/18 20180101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61P 19/08 20060101 A61P019/08; A61P 19/10 20060101
A61P019/10 |
Claims
1. A pharmaceutical composition for intranasal delivery comprising
an aqueous mixture of a PTH, a cyclodextrin, a phospholipid,
ethylene diamine tetraacetic acid, a polyol, and a preservative,
wherein the composition has at least 6% bioavailability of the PTH
upon intranasal administration of the composition to a human.
2. The pharmaceutical composition of claim 1, wherein the
composition has at least 8% bioavailability of the PTH upon
intranasal administration of the composition to a human.
3. The pharmaceutical composition of claim 1, wherein the PTH is
selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
4. The pharmaceutical composition of claim 1, wherein the
cyclodextrin is hydroxypropyl-.beta.-cyclodextrin,
sulfobutylether-.beta.-cyclodextrin, or
methyl-.beta.-cyclodextrin.
5. The pharmaceutical composition of claim 1, wherein the
cyclodextrin is methyl-.beta.-cyclodextrin at a concentration of
4.5% (w/w).
6. The pharmaceutical composition of claim 1, wherein the
phospholipid is didecanoylphosphatidylcholine (DDPC).
7. The pharmaceutical composition of claim 1, wherein the polyol is
selected from sucrose, mannitol, sorbitol, lactose, L-arabinose,
D-erythrose, D-ribose, D-xylose, D-mannose, trehalose, D-galactose,
lactulose, cellobiose, and gentibiose.
8. The pharmaceutical composition of claim 1, wherein the
preservative is chlorobutanol or sodium benzoate.
9. The pharmaceutical composition of claim 1, wherein the
composition has a pH of from about 3 to about 6.
10. The pharmaceutical composition of claim 1, wherein the
composition is in the form of liquid droplets.
11. The pharmaceutical composition of claim 10, wherein the liquid
droplets have an average volume-mean particle size (Dv,50) of from
about 1 micron to about 1000 microns.
12. The pharmaceutical composition of claim 10, where in the liquid
droplets have an average volume-mean particle size (Dv,50) of from
about 5 microns to about 500 microns.
13. The pharmaceutical composition of claim 10, where in the liquid
droplets have an average volume-mean particle size (Dv,50) of from
about 10 microns to about 100 microns.
14. A method for treating osteoporosis or osteopenia in a human
subject comprising administering intranasally to the human a
pharmaceutical composition of claim 1.
15. The method of claim 14, wherein upon intranasal administration
to the human subject the PTH has a maximum serum concentration,
Cmax, of about 100 pg/mL or greater.
16. The method of claim 14, wherein the dose of PTH administered to
the human subject is from about 1 microgram to about 2000
microgram.
17. A method for preventing the onset of osteoporosis or osteopenia
in a human subject comprising administering intranasally to the
human a pharmaceutical composition of claim 1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of prior U.S. application
Ser. No. 11/744,745, filed May 4, 2007, which is a continuation of
prior U.S. application Ser. No. 11/126,996, filed May 10, 2005,
which claimed the benefit under 35 U.S.C. .sctn.119(e) of U.S.
Provisional Application No. 60/570,113, filed May 10, 2004, each of
which is hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0002] This application includes a Sequence Listing submitted
herewith via EFS-Web as an ASCII file created on Mar. 16, 2010,
named MDR-04-04CON2_SeqList.txt, which is 1,882 bytes in size, and
is hereby incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] The teachings of all the references cited in the present
specification are incorporated in their entirety by reference.
[0004] Osteoporosis can be defined as a systemic skeletal disease
characterized by low bone mass, microarchitectural deterioration of
bone tissue, and increased bone fragility and susceptibility to
fracture. It most commonly affects older populations, primarily
postmenopausal women.
[0005] The prevalence of osteoporosis poses a serious health
problem. The National Osteoporosis Foundation has estimated that 44
million people are experiencing the effects of osteoporosis or
osteopenia. By the year 2010, osteoporosis will affect more than 52
million people and, by 2020, more than 61 million people. The
prevalence of osteoporosis is greater in Caucasians and Asians than
in African-Americans, perhaps because African-Americans have a
higher peak bone mass. Women are affected in greater numbers than
men are because men have a higher peak bone density. Furthermore,
as women age the rate of bone turnover increases, resulting in
accelerated bone loss because of the lack of estrogen after
menopause.
[0006] The goal of pharmacological treatment of osteoporosis is to
maintain or increase bone strength, to prevent fractures throughout
the patient's life, and to minimize osteoporosis-related morbidity
and mortality by safely reducing the risk of fracture. The
medications that have been used most commonly to treat osteoporosis
include calcium, and vitamin D, estrogen (with or without
progestin), bisphonates, selective estrogen receptor modulators
(SERMs), and calcitonin.
[0007] Parathyroid hormone (PTH) has recently emerged as a popular
osteoporosis treatment. Unlike other therapies that reduce bone
resorption, PTH increases bone mass, which results in greater bone
mineral density (BMD). PTH has multiple actions on bone, some
direct and some indirect. PTH increases the rate of calcium release
from bone into blood. The chronic effects of PTH are to increase
the number of bone cells both osteoblasts and osteoclasts, and to
increase the remodeling bone. These effects are apparent within
hours after PTH is administered and persist for hours after PTH is
withdrawn. PTH administered to osteoporotic patients leads to a net
stimulation of bone formation especially in trabecular bone in the
spine and hip resulting in a highly significant reduction in
fractures. The bone formation is believed to occur by the
stimulation of osteoblasts by PTH as osteoblasts have PTH
receptors.
[0008] Parathyroid hormone (PTH) is a secreted, 84 amino acid
residue polypeptide having the amino acid sequence
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-G-
lu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe Val
Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser Gln Arg Pro Arg
Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu Lys Ser Leu Gly Glu
Ala Asp Lys Ala Asn Val Asp Val Leu Thr Lys Ala Lys Ser Gln (SEQ ID
NO: 1). Studies in humans with certain forms of PTH have
demonstrated an anabolic effect on bone, and have prompted
significant interest in its use for the treatment of osteoporosis
and related bone disorders.
[0009] Using the N-terminal 34 amino acids of the bovine and human
hormone
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-G-
lu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe (SEQ
ID NO: 2) for example, which by all published accounts are deemed
biologically equivalent to the full length hormone, it has been
demonstrated in humans that parathyroid hormone enhances bone
growth particularly when administered in pulsatile fashion by the
subcutaneous route. A slightly different form of PTH, human
PTH(1-38) has shown similar results.
[0010] PTH preparations have been reconstituted from fresh or
lyophilized hormone, and incorporate various forms of carrier,
excipient and vehicle. Most are prepared in water-based vehicles
such as saline, or water acidified typically with acetic acid to
solubilize the hormone. The majority of reported formulations also
incorporate albumin as a stabilizer. See for example, Reeve et al.,
Br. Med. J. 280:6228, 1980; Reeve et al., Lancet 1:1035, 1976;
Reeve et al., Calcif. Tissue Res. 21:469, 1976; Hodsman et al.,
Bone Miner 9(2):137, 1990; Tsai et al., J. Clin. Endocrinol Metab.
69(5):1024, 1989; Isaac et al., Horm. Metab. Res. 12(9):487, 1980;
Law et al., J. Clin Invest. 72(3):1106, 1983; and Hulter, J. Clin
Hypertens 2(4):360, 1986. Other reported formulations have
incorporated an excipient such as mannitol, which is present either
with the lyophilized hormone or in the reconstitution vehicle.
[0011] PTH1-34 also called teriparatide is currently on the market
under the brand name FORTEO.RTM., Eli Lilly, Indianapolis, Ind. for
the treatment of postmenopausal women with osteoporosis who are at
high risk of fracture. This drug is administered by a once daily
subcutaneous injection of 20 .mu.g in a solution containing acetate
buffer, mannitol, and m-cresol in water, pH 4. However, many people
are adverse to injections, and thus become non-compliant with the
prescribed dosing of the PTH. Thus, there is a need to develop an
intranasal formulation of a parathyroid hormone peptide that has
suitable bioavailability such that therapeutic levels can be
achieved in the blood to be effective to treat osteoporosis or
osteopenia. FORTEO.RTM. is manufactured by recombinant DNA
technology using an Escherichia coli strain. PTH.sub.1-34 has a
molecular weight of 4117.87 daltons. Reviews on PTH.sub.1-34 and
its clinical that have been published, including, e.g., Brixen et
al., 2004; Dobnig, 2004; Eriksen and Robins, 2004; Quattrocchi and
Kourlas 2004, are hereby incorporated by reference. Forsteo is
currently licensed in the United States (as FORTEO.RTM.) and
Europe. The safety of teriparatide has been evaluated in over 2800
patients in doses ranging from 5 to 100 .mu.g per day in short term
trials. Doses of up to 40 .mu.g per day have been given for up to
two years in long term trials. Adverse events associated with
Forsteo were usually mild and generally did not require
discontinuation of therapy. The most commonly reported adverse
effects were dizziness, leg cramps, nausea, vomiting and headache.
Mild transient hypercalcemia has been reported with Forsteo which
is usually self limiting within 6 hours.
[0012] Teriparatide has been previously been administered
intranasally to humans at doses of up to 500 .mu.g per day for 7
days in one study (Suntory News Release, Suntory Establishes Large
Scale Production of recombinant human PTH.sub.1-34 and obtains
promising results from Phase 1 Clinical Trials using a Nasal
Formulation. February 1999,
http://www.suntory.com/news/1999-02.html accessed 15 Apr. 2004) and
in another study subjects received up to 1,000 .mu.g per day for 3
months (Matsumoto et al., "Daily Nasal Spray of hPTH.sub.1-34 for 3
Months Increases Bone Mass In Osteoporotic Subjects," (ASBMR 2004
presentation 1171 Oct. 4, 2004, Seattle Wash.). No safety concerns
were noted with this route.
[0013] Currently Forsteo is administered as a daily subcutaneous
injection. It would be preferable for patient acceptability if a
non-injected route of administration were available, including
nasal, buccal, gastrointestinal and dermal.
DISCLOSURE OF THE INVENTION
[0014] Preferably the parathyroid hormone and the mammal is a
human. In a most preferred embodiment the parathyroid hormone
peptide, is PTH.sub.1-34, also known as teriparatide. Tregear, U.S.
Pat. No. 4,086,196, described human PTH analogues and claimed that
the first 27 to 34 amino acids are the most effective in terms of
the stimulation of adenylyl cyclase in an in vitro cell assay. Pang
et al., WO 93/06845, published Apr. 15, 1993, described analogues
of hPTH which involve substitutions of Arg.sup.25, Lys.sup.26,
Lys.sup.27 with numerous amino acids, including alanine,
asparagine, aspartic acid, cysteine, glutamine, glutamic acid,
glycine, histidine, isoleucine, leucine, methionine, phenylalanine,
proline, serine, threonine, tryptophan, tyrosine, or valine. Other
PTH analogues are disclosed in the following patents, hereby
incorporated by reference: U.S. Pat. No. 5,317,010; U.S. Pat. No.
4,822,609; U.S. Pat. No. 5,693.616; U.S. Pat. No. 5,589,452; U.S.
Pat. No. 4,833,125; U.S. Pat. No. 5,607,915; U.S. Pat. No.
5,556,940; U.S. Pat. No. 5,382,658; U.S. Pat. No. 5,407,911; U.S.
Pat. No. 6,583,114; U.S. Pat. No. 6,541,450; U.S. Pat. No.
6,376,502; U.S. Pat. No. 5,955,425; U.S. Pat. No. 6,316,410; U.S.
Pat. No. 6,110,892; U.S. Pat. No. 6,051,686; U.S. Pat. No.
5,695,955; U.S. Pat. No. 4,771,124; and U.S. Pat. No.
6,376,502.
[0015] PTH operates through activation of two second messenger
systems, G.sub.s-protein activated adenylyl cyclase (AC) and
G.sub.q-protein activated phospholipase C.sub..beta.. The latter
results in a stimulation of membrane-bound protein kinase Cs (PKC)
activity. The PKC activity has been shown to require PTH residues
29 to 32 (Jouishomme et al., J. Bone Mineral Res. 9:1179-1189,
1994. It has been established that the increase in bone growth,
i.e. that effect which is useful in the treatment of osteoporosis,
is coupled to the ability of the peptide sequence to increase AC
activity. The native PTH sequence has been shown to have all of
these activities. The hPTH-(1-34) sequence is typically shown
as:
TABLE-US-00001 (SEQ ID NO: 2) Ser Val Ser Glu Ile Gln Leu Met His
Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys
Lys Leu Gln Asp Val His Asn Phe.
[0016] The following linear analogue, hPTH.sub.1-31NH.sub.2, has
only AC-stimulating activity and has been shown to be fully active
in the restoration of bone loss in the ovariectomized rat model
[Rixon, R. H. et al., J. Bone Miner. Res. 9:1179-1189, 1994;
Whitfield et al., Calcified Tissue Int. 58:81-87, 1996]; Willick et
al., U.S. Pat. No. 5,556,940, hereby incorporated by reference:
TABLE-US-00002 (SEQ ID NO: 3) Ser Val Ser Glu Ile Gln Leu Met His
Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys
Lys Leu Gln Asp Val.
[0017] The above molecule, SEQ ID NO: 3, and its counterpart with a
Leu.sub.27 substitution SEQ ID NO:2 may have a free carboxyl ending
instead of the amide ending. Another PTH analog is
[Leu.sub.27]cyclo(Glu.sub.22-Lys.sub.26)PTH.sub.1-31.
[0018] In other embodiments of the present invention, the PTH
composition is administered in droplets exiting from an actuator
form a spray plume with a measured ellipsoid (ratio of length of
longest to shortest axes) of 1-2,the droplets exiting from the
actuator form a spray plume with a measured ellipsoid (ratio of
length of longest to shortest axes) of 1-1.3, the volume median
droplet size is between 10 and 1000 microns (10<Dv,50<1000),
where the Dv,50 is between 30 and 300 microns, the percentage of
droplets having a diameter 10 microns or less is 10% or less and
the percentage of droplets having a diameter 10 microns or less is
1% or less.
[0019] The present invention is also directed to an intranasal
formulation of a PTH-agonist that is substantially free of proteins
or polypeptides that stabilize the formulation. In particular, the
preferred formulation is free of such proteins as albumin, and
collagen-derived proteins such as gelatin.
[0020] In other aspects of the present invention a transmucosal PTH
peptide formulation is comprised of a PTH peptide, water and a
solubilizing agent having a pH of 3-6.5. In a preferred embodiment,
the solubilization agent is a cyclodextrin.
[0021] In another embodiment of the present invention a
transmucosal PTH peptide formulation is comprised of a PTH peptide,
water, a solubilizing agent, preferably a cyclodextrin, and at
least one polyol, preferably 2 polyols. In alternate embodiments
the formulation may contain one or all of the following: a
chelating agent, a surface-acting agent and a buffering agent.
[0022] In another embodiment of the present invention the
formulation is comprised of a PTH peptide, water, chelating agent
and a solubilization agent.
[0023] In another embodiment of the present invention the
formulation is comprised of a PTH peptide, water and a chelating
agent having a pH of 3-6.5.
[0024] In another embodiment of the present invention the
formulation is comprised of a PTH peptide, water, chelating agent
and at least one polyol, preferably two polyols. Additional
embodiments may include one or more of the following: a
surface-active agent, a solubilizing agent and a buffering
agent.
[0025] In another embodiment of the present invention the
formulation is comprised of a PTH peptide, water, and at least two
polyols, such as lactose and sorbitol. Additional agents, which can
be added to the formulation, include, but are not limited to, a
solubilization agent, a chelating agent, one or more buffering
agents and a surface-acting agent.
[0026] The enhancement of intranasal delivery of a PTH peptide
agonist according to the methods and compositions of the invention
allows for the effective pharmaceutical use of these agents to
treat a variety of diseases and conditions in mammalian
subjects.
[0027] The present invention fills this need by providing for a
liquid or dehydrated PTH peptide formulation wherein the
formulation is substantially free of a stabilizer that is a
polypeptide or a protein. The liquid parathyroid hormone
formulation is comprised of water, parathyroid hormone and at least
one of the following additives selected from the group consisting
of polyols, surface-active agents, solubilizing agents and
chelating agents. The pH of the formulation is preferably 3 to
about 7.0, preferably 4.5 to about 6.0, most preferably about
5.0.+-.0.3.
[0028] Another embodiment of the present invention is an aqueous
PTH formulation of the present invention is comprised of water, a
PTH peptide, a polyol and a surface-active agent wherein the
formulation has a pH of about 3 to about 6.5, and the formulation
is substantially free of a stabilizer that is a protein or
polypeptide.
[0029] Another embodiment of the present invention is an aqueous
PTH peptide formulation comprised of water, PTH peptide, a polyol
and a solubilizing agent wherein the formulation has a pH of about
3.0 to about 6.5, and the formulation is substantially free of a
stabilizer that is a protein or polypeptide.
[0030] Another embodiment of the present invention is an aqueous
PTH peptide formulation comprised of water, PTH peptide, a
solubilizing agent and a surface-active agent wherein the
formulation has a pH of about 3.0 to about 6.5, and the formulation
is substantially free of a stabilizer that is a protein or
polypeptide.
[0031] Another embodiment of the invention is an aqueous PTH
peptide formulation comprised of water, a PTH peptide, a
solubilizing agent, a polyol and a surface-active agent wherein the
formulation has a pH of about 3.0 to about 6.5, and the formulation
is substantially free of a stabilizer that is a protein or
polypeptide.
[0032] In another aspect of the present invention, the stable
aqueous formulation is dehydrated to produce a dehydrated PTH
peptide formulation comprised of PTH peptide and at least one of
the following additives selected from the group consisting of
polyols, surface-active agents, solubilizing agents and chelating
agents, wherein said dehydrated PTH peptide formulation is
substantially free of a stabilizer that is a protein or polypeptide
such as albumin, collagen or collagen-derived protein such as
gelatin. The dehydration can be achieved by various means such as
lyophilization, spray-drying, salt-induced precipitation and
drying, vacuum drying, rotary evaporation, or supercritical
CO.sub.2 precipitation.
[0033] In one embodiment, the dehydrated PTH peptide is comprised
of PTH peptide, a polyol and a solubilizing agent, wherein the
formulation is substantially free of a stabilizer that is a
protein.
[0034] In another embodiment, the dehydrated PTH peptide
formulation is comprised of a PTH peptide, a polyol, and a
surface-active agent wherein the PTH peptide formulation is
substantially free of a stabilizer that is a protein or
polypeptide.
[0035] In another embodiment, the dehydrated PTH peptide
formulation is comprised of a PTH peptide, a surface-active agent,
and a solubilizing agent wherein the PTH peptide formulation is
substantially free of a stabilizer that is a protein or
polypeptide.
[0036] In another embodiment of the present invention, the
dehydrated PTH peptide formulation is comprised of a PTH peptide, a
polyol, a surface-active agent and a solubilizing agent wherein the
PTH peptide formulation is substantially free of a stabilizer that
is a protein or polypeptide.
[0037] Another aspect of the present invention is an intranasal PTH
peptide formulation contain within an actuator able to produce an
aerosol of said solution, wherein the spray pattern ellipticity
ratio of said aerosol is between 1.00 and 1.40 when measured at a
height of between 0.5 cm and 10 cm distance from the actuator tip,
which has preferably an ellipticity of between 1.00 and 1.30 and
produces an aerosol of between 20 and 200 microliters per
actuation.
[0038] In another embodiment, the intranasal PTH peptide solution
is in an actuator, which produces an aerosol of said solution,
wherein the spray pattern major and minor axes of said aerosol are
between 10 and 50 mm when measured at a height of between 0.5 cm
and 10 cm distance from the actuator tip. In another embodiment, an
aqueous solution of a PTH peptide is in a container attached an
actuator so that an aerosol of the solution is produced wherein
less than 10% of the droplets produced are smaller than 10 microns
in size and aerosol containing the PTH peptide contains 20 and 200
microliters solution per actuation. In another embodiment, a
solution of the PTH peptide is in a container attached to an
actuator so that the aerosol of the solution produced upon
actuation has droplets between 25 and 700 microns.
[0039] Any solubilizing agent can be used but a preferred one is
selected from the group consisting of
hydroxypropyl-.beta.-cyclodextran,
sulfobutylether-.beta.-cyclodextran, methyl-.beta.-cyclodextrin and
chitosan.
[0040] Generally a polyol is selected from the group consisting of
lactose, sorbitol, trehalose, sucrose, mannose and maltose and
derivatives and homologs thereof.
[0041] A satisfactory surface-active agent is selected from the
group consisting of L-.alpha.-phosphatidylcholine didecanoyl
(DDPC), polysorbate 20 (Tween 20), polysorbate 80 (Tween 80),
polyethylene glycol (PEG), cetyl alcohol, polyvinylpyrolidone
(PVP), polyvinyl alcohol (PVA), lanolin alcohol, and sorbitan
monooleate.
[0042] In a preferred formulation, the PTH peptide formulation is
also comprised of a chelating agent such as ethylene diamine
tetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA).
Also a preservative such as chlorobutanol, methyl paraben, propyl
paraben, butyl paraben, benzalkonium chloride, benzethonium
chloride, sodium benzoate, sorbic acid, phenol, or ortho-, meta- or
paracresol.
[0043] The pH is generally regulated using a buffer such as sodium
citrate and citric acid, and sodium acetate and acetic acid. An
alternative buffer would be acetic acid and sodium acetate or
succinic acid and sodium hydroxide.
[0044] The present invention also comprehends a formulation wherein
the concentration of the PTH peptide is 0.1-15.0 mg/mL, preferably
1.0-2 mg/mL and the pH of the aqueous solution is 3.0-6.5
preferably about 5.0.+-.0.3.
[0045] The present invention further includes PTH peptide
formulation wherein the concentration of the polyol is between
about 0.1% and 10% (w/v) and additionally wherein the concentration
of the polyol is in the range from about 0.1% to about 3%
(w/v).
[0046] The instant invention also includes a formulation, wherein
the concentration of the surface-active agent is between about
0.00001% and about 5% (w/v), and wherein the concentration of the
surface-active agent is between about 0.0002% and about 0.1%
(w/v).
[0047] The instant invention also includes a formulation, wherein
the concentration of the solubilzation agent is 1% -10% (w/v), and
wherein the concentration of the solubilizing agent is 2% to 5%
(w/v).
[0048] The finished solution can be filtered and freeze-dried,
lyophilized, using methods well known to one of ordinary skill in
the art, and by following the instructions of the manufacturer of
the lyophilizing equipment. This produces a dehydrated PTH peptide
formulation substantially free of a stabilizer that is a
protein.
[0049] In another embodiment of the present invention, a PTH
peptide formulation is comprised of an PTH peptide and a
pharmaceutically acceptable carrier wherein the PTH-bind peptide
formulation has at least 1%, preferably 3% and most preferably at
least 6% higher permeation in an in vitro tissue permeation assay
than a control formulation consisting of water, sodium chloride, a
buffer and the PTH peptide, as determined by the transepithelial
electrical resistance assay shown in Examples 2 and 7. In a
preferred embodiment, the PTH formulation is further comprised of
at least one excipient selected from the group consisting of a
surface-active agent, a solubilization agent, a polyol, and a
chelating agent.
[0050] In exemplary embodiments, the enhanced delivery methods and
compositions of the present invention provide for therapeutically
effective mucosal delivery of the PTH peptide agonist for
prevention or treatment of osteoporosis or osteopenia in mammalian
subjects. In one aspect of the invention, pharmaceutical
formulations suitable for intranasal administration are provided
that comprise a therapeutically effective amount of a PTH peptide
and one or more intranasal delivery-enhancing agents as described
herein, which formulations are effective in a nasal mucosal
delivery method of the invention to prevent the onset or
progression of osteoporosis or osteopenia in a mammalian subject.
Nasal mucosal delivery of a therapeutically effective amount of a
PTH peptide agonist and one or more intranasal delivery-enhancing
agents yields elevated therapeutic levels of the PTH peptide
agonist in the subject and promotes the increase in bone mass in an
individual.
[0051] The enhanced delivery methods and compositions of the
present invention provide for therapeutically effective mucosal
delivery of a PTH peptide for prevention or treatment of
osteoporosis or osteopenia in mammalian subjects. PTH peptide can
be administered via a variety of mucosal routes, for example by
contacting the PTH peptide to a nasal mucosal epithelium, a
bronchial or pulmonary mucosal epithelium, the oral buccal surface
or the oral and small intestinal mucosal surface. In exemplary
embodiments, the methods and compositions are directed to or
formulated for intranasal delivery (e.g., nasal mucosal delivery or
intranasal mucosal delivery).
[0052] In one aspect of the invention, pharmaceutical formulations
suitable for intranasal administration are provided that comprise a
therapeutically effective amount of a PTH peptide agonist and one
or more intranasal delivery-enhancing agents as described herein,
which formulations are effective in a nasal mucosal delivery method
of the invention to prevent or treat osteoporosis.
[0053] In another aspect of the invention, pharmaceutical
formulations and methods are directed to administration of a PTH
peptide agonist in combination with calcium, vitamin D,
bisphosphonates, calcitonin or a bone morphogenic protein. See U.S.
Pat. No. 5,616,560 and U.S. Pat. No. 5,700,774, hereby incorporated
by reference.
[0054] The foregoing mucosal PTH peptide formulations and
preparative and delivery methods of the invention provide improved
mucosal delivery of a PTH peptide to mammalian subjects. These
compositions and methods can involve combinatorial formulation or
coordinate administration of one or more PTH peptides with one or
more mucosal delivery-enhancing agents. Among the mucosal
delivery-enhancing agents to be selected from to achieve these
formulations and methods are (A) solubilization agents; (B) charge
modifying agents; (C) pH control agents; (D) degradative enzyme
inhibitors; (E) mucolytic or mucus clearing agents; (F) ciliostatic
agents; (G) membrane penetration-enhancing agents (e.g., (i) a
surfactant, (ii) a bile salt, (iii) a phospholipid or fatty acid
additive, mixed micelle, liposome, or carrier, (iv) an alcohol, (v)
an enamine, (iv) an NO donor compound, (vii) a long-chain
amphipathic molecule (viii) a small hydrophobic penetration
enhancer; (ix) sodium or a salicylic acid derivative; (x) a
glycerol ester of acetoacetic acid (xi) a cyclodextrin or
beta-cyclodextrin derivative, (xii) a medium-chain fatty acid,
(xiii) a chelating agent, (xiv) an amino acid or salt thereof, (xv)
an N-acetylamino acid or salt thereof, (xvi) an enzyme degradative
to a selected membrane component, (xvii) an inhibitor of fatty acid
synthesis, (xviii) an inhibitor of cholesterol synthesis; or (xiv)
any combination of the membrane penetration enhancing agents of
(i)-(xviii)); (H) modulatory agents of epithelial junction
physiology, such as nitric oxide (NO) stimulators, chitosan, and
chitosan derivatives; (I) vasodilator agents; (J) selective
transport-enhancing agents; (K) stabilizing delivery vehicles,
carriers, supports or complex-forming species with which the PTH
peptide (s) is/are effectively combined, associated, contained,
encapsulated or bound to stabilize the active agent for enhanced
mucosal delivery; and (L) alcohols such as ethanol.
[0055] In various embodiments of the invention, a PTH peptide is
combined with one, two, three, four or more of the mucosal
delivery-enhancing agents recited in (A)-(K), above.
[0056] These mucosal delivery-enhancing agents may be admixed,
alone or together, with the PTH peptide, or otherwise combined
therewith in a pharmaceutically acceptable formulation or delivery
vehicle. Formulation of a PTH peptide with one or more of the
mucosal delivery-enhancing agents according to the teachings herein
(optionally including any combination of two or more mucosal
delivery-enhancing agents selected from (A)-(K) above) provides for
increased bioavailability of the PTH peptide following delivery
thereof to a mucosal surface of a mammalian subject.
[0057] Thus, the present invention is a method for treating
osteoporosis or osteopenia in a mammal comprising transmucosally
administering a formulation comprised of a PTH peptide, such that
when at 50 .mu.g of the PTH is administered transmucosally to the
mammal the concentration of the PTH peptide in the plasma of the
mammal increases by at least 5 pmol, preferably at least 10 pmol
per liter of plasma.
[0058] Intranasal delivery-enhancing agents are employed which
enhance delivery of PTH into or across a nasal mucosal surface. For
passively absorbed drugs, the relative contribution of paracellular
and transcellular pathways to drug transport depends upon the pKa,
partition coefficient, molecular radius and charge of the drug, the
pH of the luminal environment in which the drug is delivered, and
the area of the absorbing surface. The intranasal
delivery-enhancing agent of the present invention may be a pH
control agent. The pH of the pharmaceutical formulation of the
present invention is a factor affecting absorption of PTH via
paracellular and transcellular pathways to drug transport. In one
embodiment, the pharmaceutical formulation of the present invention
is pH adjusted to between about pH 3.0 to 6.5. In a further
embodiment, the pharmaceutical formulation of the present invention
is pH adjusted to between about pH 3.0 to 5.0. In a further
embodiment, the pharmaceutical formulation of the present invention
is pH adjusted to between about pH 4.0 to 5.0. Generally, the pH is
5.0.+-.0.3.
[0059] As noted above, the present invention provides improved
methods and compositions for mucosal delivery of PTH peptide to
mammalian subjects for treatment or prevention of osteoporosis or
osteopenia. Examples of appropriate mammalian subjects for
treatment and prophylaxis according to the methods of the invention
include, but are not restricted to, humans and non-human primates,
livestock species, such as horses, cattle, sheep, and goats, and
research and domestic species, including dogs, cats, mice, rats,
guinea pigs, and rabbits.
[0060] In order to provide better understanding of the present
invention, the following definitions are provided:
[0061] According to the present invention a parathyroid hormone
peptide also includes the free bases, acid addition salts or metal
salts, such as potassium or sodium salts of the peptides, and
parathyroid hormone peptides that have been modified by such
processes as amidation, glycosylation, acylation, sulfation,
phosphorylation, acetylation, cyclization and other well known
covalent modification methods.
[0062] Osteopenia is a decreased calcification or density of bone,
a descriptive term applicable to all skeletal systems in which the
condition is noted.
[0063] "Mucosal delivery enhancing agents" are defined as chemicals
and other excipients that, when added to a formulation comprising
water, salts and/or common buffers and PTH peptide (the control
formulation) produce a formulation that produces a significant
increase in transport of PTH peptide across a mucosa as measured by
the maximum blood, serum, or cerebral spinal fluid concentration
(C.sub.max) or by the area under the curve, AUC, in a plot of
concentration versus time. A mucosa includes the nasal, oral,
intestional, buccal, bronchopulmonary, vaginal, and rectal mucosal
surfaces and in fact includes all mucus-secreting membranes lining
all body cavities or passages that communicate with the exterior.
Mucosal delivery enhancing agents are sometimes called
carriers.
[0064] "Non-infused administration" means any method of delivery
that does not involve an injection directly into an artery or vein,
a method which forces or drives (typically a fluid) into something
and especially to introduce into a body part by means of a needle,
syringe or other invasive method. Non-infused administration
includes subcutaneous injection, intramuscular injection,
intraparitoneal injection and the non-injection methods of delivery
to a mucosa.
[0065] As noted above, the instant invention provides improved and
useful methods and compositions for nasal mucosal delivery of a PTH
peptide to prevent and treat osteoporosis or osteopenia in
mammalian subjects. As used herein, prevention and treatment of
osteoporosis or osteopenia means prevention of the onset or
lowering the incidence or severity of clinical osteoporosis by
reducing increasing bone mass, decreasing bone resporption or
reducing the incidence of fractured bones in a patient.
[0066] The PTH peptide can also be administered in conjunction with
other therapeutic agents such as bisphonates, calcium, vitamin D,
estrogen or estrogen-receptor binding compounds, selective estrogen
receptor modulators (SERMs), bone morphogenic proteins or
calcitonin.
[0067] Improved methods and compositions for mucosal administration
of PTH peptide to mammalian subjects optimize PTH peptide dosing
schedules. The present invention provides mucosal delivery of PTH
peptide formulated with one or more mucosal delivery-enhancing
agents wherein PTH peptide dosage release is substantially
normalized and/or sustained for an effective delivery period of PTH
peptide release ranges from approximately 0.1 to 2.0 hours; 0.4 to
1.5 hours; 0.7 to 1.5 hours; or 0.8 to 1.0 hours; following mucosal
administration. The sustained release of PTH peptide achieved may
be facilitated by repeated administration of exogenous PTH peptide
utilizing methods and compositions of the present invention.
[0068] Improved compositions and methods for mucosal administration
of PTH peptide to mammalian subjects optimize PTH peptide dosing
schedules. The present invention provides improved mucosal (e.g.,
nasal) delivery of a formulation comprising PTH peptide in
combination with one or more mucosal delivery-enhancing agents and
an optional sustained release-enhancing agent or agents. Mucosal
delivery-enhancing agents of the present invention yield an
effective increase in delivery, e.g., an increase in the maximal
plasma concentration (C.sub.max) to enhance the therapeutic
activity of mucosally-administered PTH peptide. A second factor
affecting therapeutic activity of PTH peptide in the blood plasma
and CNS is residence time (RT). Sustained release-enhancing agents,
in combination with intranasal delivery-enhancing agents, increase
C.sub.max and increase residence time (RT) of PTH peptide.
Polymeric delivery vehicles and other agents and methods of the
present invention that yield sustained release-enhancing
formulations, for example, polyethylene glycol (PEG), are disclosed
herein. The present invention provides an improved PTH peptide
delivery method and dosage form for treatment or prevention of
osteoporosis or osteopenia in mammalian subjects.
[0069] Within the mucosal delivery formulations and methods of the
invention, the PTH peptide is frequently combined or coordinately
administered with a suitable carrier or vehicle for mucosal
delivery. As used herein, the term "carrier" means a
pharmaceutically acceptable solid or liquid filler, diluent or
encapsulating material. A water-containing liquid carrier can
contain pharmaceutically acceptable additives such as acidifying
agents, alkalizing agents, antimicrobial preservatives,
antioxidants, buffering agents, chelating agents, complexing
agents, solubilizing agents, humectants, solvents, suspending
and/or viscosity-increasing agents, tonicity agents, wetting agents
or other biocompatible materials. A tabulation of ingredients
listed by the above categories, can be found in the U.S.
Pharmacopeia National Formulary, 1990, pp 1857-1859. Some examples
of the materials which can serve as pharmaceutically acceptable
carriers are sugars, such as lactose, glucose and sucrose; starches
such as corn starch and potato starch; cellulose and its
derivatives such as sodium carboxymethyl cellulose, ethyl cellulose
and cellulose acetate; powdered tragacanth; malt; gelatin; talc;
excipients such as cocoa butter and suppository waxes; oils such as
peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,
corn oil and soybean oil; glycols, such as propylene glycol;
polyols such as glycerin, sorbitol, mannitol and polyethylene
glycol; esters such as ethyl oleate and ethyl laurate; agar;
buffering agents such as magnesium hydroxide and aluminum
hydroxide; alginic acid; pyrogen free water; isotonic saline;
Ringer's solution, ethyl alcohol and phosphate buffer solutions, as
well as other non toxic compatible substances used in
pharmaceutical formulations. Wetting agents, emulsifiers and
lubricants such as sodium lauryl sulfate and magnesium stearate, as
well as coloring agents, release agents, coating agents,
sweetening, flavoring and perfuming agents, preservatives and
antioxidants can also be present in the compositions, according to
the desires of the formulator. Examples of pharmaceutically
acceptable antioxidants include water soluble antioxidants such as
ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium
metabisulfite, sodium sulfite and the like; oil-soluble
antioxidants such as ascorbyl palmitate, butylated hydroxyanisole
(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,
alpha-tocopherol and the like; and metal-chelating agents such as
citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,
tartaric acid, phosphoric acid and the like. The amount of active
ingredient that can be combined with the carrier materials to
produce a single dosage form will vary depending upon the
particular mode of administration.
[0070] Within the mucosal delivery compositions and methods of the
invention, various delivery-enhancing agents are employed which
enhance delivery of PTH peptide into or across a mucosal surface.
In this regard, delivery of PTH peptide across the mucosal
epithelium can occur "transcellularly" or "paracellularly." The
extent to which these pathways contribute to the overall flux and
bioavailability of the PTH peptide depends upon the environment of
the mucosa, the physico-chemical properties the active agent, and
on the properties of the mucosal epithelium. Paracellular transport
involves only passive diffusion, whereas transcellular transport
can occur by passive, facilitated or active processes. Generally,
hydrophilic, passively transported, polar solutes diffuse through
the paracellular route, while more lipophilic solutes use the
transcellular route. Absorption and bioavailability (e.g., as
reflected by a permeability coefficient or physiological assay),
for diverse, passively and actively absorbed solutes, can be
readily evaluated, in terms of both paracellular and transcellular
delivery components, for any selected PTH peptide within the
invention. For passively absorbed drugs, the relative contribution
of paracellular and transcellular pathways to drug transport
depends upon the pKa, partition coefficient, molecular radius and
charge of the drug, the pH of the luminal environment in which the
drug is delivered, and the area of the absorbing surface. The
paracellular route represents a relatively small fraction of
accessible surface area of the nasal mucosal epithelium. In general
terms, it has been reported that cell membranes occupy a mucosal
surface area that is a thousand times greater than the area
occupied by the paracellular spaces. Thus, the smaller accessible
area, and the size- and charge-based discrimination against
macromolecular permeation would suggest that the paracellular route
would be a generally less favorable route than transcellular
delivery for drug transport. Surprisingly, the methods and
compositions of the invention provide for significantly enhanced
transport of biotherapeutics into and across mucosal epithelia via
the paracellular route. Therefore, the methods and compositions of
the invention successfully target both paracellular and
transcellular routes, alternatively or within a single method or
composition.
[0071] As used herein, "mucosal delivery-enhancing agents" include
agents which enhance the release or solubility (e.g., from a
formulation delivery vehicle), diffusion rate, penetration capacity
and timing, uptake, residence time, stability, effective half-life,
peak or sustained concentration levels, clearance and other desired
mucosal delivery characteristics (e.g., as measured at the site of
delivery, or at a selected target site of activity such as the
bloodstream or central nervous system) of PTH peptide or other
biologically active compound(s). Enhancement of mucosal delivery
can thus occur by any of a variety of mechanisms, for example by
increasing the diffusion, transport, persistence or stability of
PTH peptide, increasing membrane fluidity, modulating the
availability or action of calcium and other ions that regulate
intracellular or paracellular permeation, solubilizing mucosal
membrane components (e.g., lipids), changing non-protein and
protein sulfhydryl levels in mucosal tissues, increasing water flux
across the mucosal surface, modulating epithelial junctional
physiology, reducing the viscosity of mucus overlying the mucosal
epithelium, reducing mucociliary clearance rates, and other
mechanisms.
[0072] As used herein, a "mucosally effective amount of PTH
peptide" contemplates effective mucosal delivery of PTH peptide to
a target site for drug activity in the subject that may involve a
variety of delivery or transfer routes. For example, a given active
agent may find its way through clearances between cells of the
mucosa and reach an adjacent vascular wall, while by another route
the agent may, either passively or actively, be taken up into
mucosal cells to act within the cells or be discharged or
transported out of the cells to reach a secondary target site, such
as the systemic circulation. The methods and compositions of the
invention may promote the translocation of active agents along one
or more such alternate routes, or may act directly on the mucosal
tissue or proximal vascular tissue to promote absorption or
penetration of the active agent(s). The promotion of absorption or
penetration in this context is not limited to these mechanisms.
[0073] As used herein "peak concentration (C.sub.max) of PTH
peptide in a blood plasma", "area under concentration vs. time
curve (AUC) of PTH peptide in a blood plasma", "time to maximal
plasma concentration (t.sub.max) of PTH peptide in a blood plasma"
are pharmacokinetic parameters known to one skilled in the art.
Laursen et al., Eur. J. Endocrinology, 135:309-315, 1996. The
"concentration vs. time curve" measures the concentration of PTH
peptide in a blood serum of a subject vs. time after administration
of a dosage of PTH peptide to the subject either by intranasal,
intramuscular, subcutaneous, or other parenteral route of
administration. "C.sub.max" is the maximum concentration of PTH
peptide in the blood serum of a subject following a single dosage
of PTH peptide to the subject. "t.sub.max" is the time to reach
maximum concentration of PTH peptide in a blood serum of a subject
following administration of a single dosage of PTH peptide to the
subject.
[0074] While the mechanism of absorption promotion may vary with
different mucosal delivery-enhancing agents of the invention,
useful reagents in this context will not substantially adversely
affect the mucosal tissue and will be selected according to the
physicochemical characteristics of the particular PTH peptide or
other active or delivery-enhancing agent. In this context,
delivery-enhancing agents that increase penetration or permeability
of mucosal tissues will often result in some alteration of the
protective permeability barrier of the mucosa. For such
delivery-enhancing agents to be of value within the invention, it
is generally desired that any significant changes in permeability
of the mucosa be reversible within a time frame appropriate to the
desired duration of drug delivery. Furthermore, there should be no
substantial, cumulative toxicity, nor any permanent deleterious
changes induced in the barrier properties of the mucosa with
long-term use.
[0075] Within certain aspects of the invention,
absorption-promoting agents for coordinate administration or
combinatorial formulation with PTH peptide of the invention are
selected from small hydrophilic molecules, including but not
limited to, dimethyl sulfoxide (DMSO), dimethylformamide, ethanol,
propylene glycol, and the 2-pyrrolidones. Alternatively, long-chain
amphipathic molecules, for example, deacylmethyl sulfoxide, azone,
sodium laurylsulfate, oleic acid, and the bile salts, may be
employed to enhance mucosal penetration of the PTH peptide. In
additional aspects, surfactants (e.g., polysorbates) are employed
as adjunct compounds, processing agents, or formulation additives
to enhance intranasal delivery of the PTH peptide. Agents such as
DMSO, polyethylene glycol, and ethanol can, if present in
sufficiently high concentrations in delivery environment (e.g., by
pre-administration or incorporation in a therapeutic formulation),
enter the aqueous phase of the mucosa and alter its solubilizing
properties, thereby enhancing the partitioning of the PTH peptide
from the vehicle into the mucosa.
[0076] Additional mucosal delivery-enhancing agents that are useful
within the coordinate administration and processing methods and
combinatorial formulations of the invention include, but are not
limited to, mixed micelles; enamines; nitric oxide donors (e.g.,
S-nitroso-N-acetyl-DL-penicillamine, NOR1, NOR4--which are
preferably co-administered with an NO scavenger such as
carboxy-PITO or doclofenac sodium); sodium salicylate; glycerol
esters of acetoacetic acid (e.g., glyceryl-1,3-diacetoacetate or
1,2-isopropylideneglycerine-3-acetoacetate); and other
release-diffusion or intra- or trans-epithelial
penetration-promoting agents that are physiologically compatible
for mucosal delivery. Other absorption-promoting agents are
selected from a variety of carriers, bases and excipients that
enhance mucosal delivery, stability, activity or trans-epithelial
penetration of the PTH peptide. These include, inter alia,
cyclodextrins and .beta.-cyclodextrin derivatives (e.g.,
2-hydroxypropyl-.beta.-cyclodextrin and
heptakis(2,6-di-O-methyl-.beta.-cyclodextrin). These compounds,
optionally conjugated with one or more of the active ingredients
and further optionally formulated in an oleaginous base, enhance
bioavailability in the mucosal formulations of the invention. Yet
additional absorption-enhancing agents adapted for mucosal delivery
include medium-chain fatty acids, including mono- and diglycerides
(e.g., sodium caprate--extracts of coconut oil, Capmul), and
triglycerides (e.g., amylodextrin, Estaram 299, Miglyol 810).
[0077] The mucosal therapeutic and prophylactic compositions of the
present invention may be supplemented with any suitable
penetration-promoting agent that facilitates absorption, diffusion,
or penetration of PTH peptide across mucosal barriers. The
penetration promoter may be any promoter that is pharmaceutically
acceptable. Thus, in more detailed aspects of the invention
compositions are provided that incorporate one or more
penetration-promoting agents selected from sodium salicylate and
salicylic acid derivatives (acetyl salicylate, choline salicylate,
salicylamide, etc.); amino acids and salts thereof (e.g.,
monoaminocarboxlic acids such as glycine, alanine, phenylalanine,
proline, hydroxyproline, etc.; hydroxyamino acids such as serine;
acidic amino acids such as aspartic acid, glutamic acid, etc.; and
basic amino acids such as lysine, etc.--inclusive of their alkali
metal or alkaline earth metal salts); and N-acetylamino acids
(N-acetylalanine, N-acetylphenylalanine,
[0078] N-acetylserine, N-acetylglycine, N-acetyllysine,
N-acetylglutamic acid, N-acetylproline, N-acetylhydroxyproline,
etc.) and their salts (alkali metal salts and alkaline earth metal
salts). Also provided as penetration-promoting agents within the
methods and compositions of the invention are substances which are
generally used as emulsifiers (e.g., sodium oleyl phosphate, sodium
lauryl phosphate, sodium lauryl sulfate, sodium myristyl sulfate,
polyoxyethylene alkyl ethers, polyoxyethylene alkyl esters, etc.),
caproic acid, lactic acid, malic acid and citric acid and alkali
metal salts thereof, pyrrolidonecarboxylic acids,
alkylpyrrolidonecarboxylic acid esters, N-alkylpyrrolidones,
proline acyl esters, and the like.
[0079] Within various aspects of the invention, improved nasal
mucosal delivery formulations and methods are provided that allow
delivery of PTH peptide and other therapeutic agents within the
invention across mucosal barriers between administration and
selected target sites. Certain formulations are specifically
adapted for a selected target cell, tissue or organ, or even a
particular disease state. In other aspects, formulations and
methods provide for efficient, selective endo- or transcytosis of
PTH peptide specifically routed along a defined intracellular or
intercellular pathway. Typically, the PTH peptide is efficiently
loaded at effective concentration levels in a carrier or other
delivery vehicle, and is delivered and maintained in a stabilized
form, e.g., at the nasal mucosa and/or during passage through
intracellular compartments and membranes to a remote target site
for drug action (e.g., the blood stream or a defined tissue, organ,
or extracellular compartment). The PTH peptide may be provided in a
delivery vehicle or otherwise modified (e.g., in the form of a
prodrug), wherein release or activation of the PTH peptide is
triggered by a physiological stimulus (e.g. pH change, lysosomal
enzymes, etc.). Often, the PTH peptide is pharmacologically
inactive until it reaches its target site for activity. In most
cases, the PTH peptide and other formulation components are
non-toxic and non-immunogenic. In this context, carriers and other
formulation components are generally selected for their ability to
be rapidly degraded and excreted under physiological conditions. At
the same time, formulations are chemically and physically stable in
dosage form for effective storage.
[0080] Included within the definition of biologically active
peptides and proteins for use within the invention are natural or
synthetic, therapeutically or prophylactically active, peptides
(comprised of two or more covalently linked amino acids), proteins,
peptide or protein fragments, peptide or protein analogs, and
chemically modified derivatives or salts of active peptides or
proteins. A wide variety of useful analogs and mimetics of PTH
peptide are contemplated for use within the invention and can be
produced and tested for biological activity according to known
methods. Often, the peptides or proteins of PTH peptide or other
biologically active peptides or proteins for use within the
invention are muteins that are readily obtainable by partial
substitution, addition, or deletion of amino acids within a
naturally occurring or native (e.g., wild-type, naturally occurring
mutant, or allelic variant) peptide or protein sequence.
Additionally, biologically active fragments of native peptides or
proteins are included. Such mutant derivatives and fragments
substantially retain the desired biological activity of the native
peptide or proteins. In the case of peptides or proteins having
carbohydrate chains, biologically active variants marked by
alterations in these carbohydrate species are also included within
the invention.
[0081] As used herein, the term "conservative amino acid
substitution" refers to the general interchangeability of amino
acid residues having similar side chains. For example, a commonly
interchangeable group of amino acids having aliphatic side chains
is alanine, valine, leucine, and isoleucine; a group of amino acids
having aliphatic-hydroxyl side chains is serine and threonine; a
group of amino acids having amide-containing side chains is
asparagine and glutamine; a group of amino acids having aromatic
side chains is phenylalanine, tyrosine, and tryptophan; a group of
amino acids having basic side chains is lysine, arginine, and
histidine; and a group of amino acids having sulfur-containing side
chains is cysteine and methionine. Examples of conservative
substitutions include the substitution of a non-polar (hydrophobic)
residue such as isoleucine, valine, leucine or methionine for
another. Likewise, the present invention contemplates the
substitution of a polar (hydrophilic) residue such as between
arginine and lysine, between glutamine and asparagine, and between
threonine and serine. Additionally, the substitution of a basic
residue such as lysine, arginine or histidine for another or the
substitution of an acidic residue such as aspartic acid or glutamic
acid for another is also contemplated. Exemplary conservative amino
acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine. By aligning a peptide or protein analog
optimally with a corresponding native peptide or protein, and by
using appropriate assays, e.g., adhesion protein or receptor
binding assays, to determine a selected biological activity, one
can readily identify operable peptide and protein analogs for use
within the methods and compositions of the invention. Operable
peptide and protein analogs are typically specifically
immunoreactive with antibodies raised to the corresponding native
peptide or protein.
[0082] An approach for stabilizing solid protein formulations of
the invention is to increase the physical stability of purified,
e.g., lyophilized, protein. This will inhibit aggregation via
hydrophobic interactions as well as via covalent pathways that may
increase as proteins unfold. Stabilizing formulations in this
context often include polymer-based formulations, for example a
biodegradable hydrogel formulation/delivery system. As noted above,
the critical role of water in protein structure, function, and
stability is well known. Typically, proteins are relatively stable
in the solid state with bulk water removed. However, solid
therapeutic protein formulations may become hydrated upon storage
at elevated humidities or during delivery from a sustained release
composition or device. The stability of proteins generally drops
with increasing hydration. Water can also play a significant role
in solid protein aggregation, for example, by increasing protein
flexibility resulting in enhanced accessibility of reactive groups,
by providing a mobile phase for reactants, and by serving as a
reactant in several deleterious processes such as beta-elimination
and hydrolysis.
[0083] Protein preparations containing between about 6% to 28%
water are the most unstable. Below this level, the mobility of
bound water and protein internal motions are low. Above this level,
water mobility and protein motions approach those of full
hydration. Up to a point, increased susceptibility toward
solid-phase aggregation with increasing hydration has been observed
in several systems. However, at higher water content, less
aggregation is observed because of the dilution effect.
[0084] In accordance with these principles, an effective method for
stabilizing peptides and proteins against solid-state aggregation
for mucosal delivery is to control the water content in a solid
formulation and maintain the water activity in the formulation at
optimal levels. This level depends on the nature of the protein,
but in general, proteins maintained below their "monolayer" water
coverage will exhibit superior solid-state stability.
[0085] A variety of additives, diluents, bases and delivery
vehicles are provided within the invention that effectively control
water content to enhance protein stability. These reagents and
carrier materials effective as anti-aggregation agents in this
sense include, for example, polymers of various functionalities,
such as polyethylene glycol, dextran, diethylaminoethyl dextran,
and carboxymethyl cellulose, which significantly increase the
stability and reduce the solid-phase aggregation of peptides and
proteins admixed therewith or linked thereto. In some instances,
the activity or physical stability of proteins can also be enhanced
by various additives to aqueous solutions of the peptide or protein
drugs. For example, additives, such as polyols (including sugars),
amino acids, proteins such as collagen and gelatin, and various
salts may be used.
[0086] Certain additives, in particular sugars and other polyols,
also impart significant physical stability to dry, e.g.,
lyophilized proteins. These additives can also be used within the
invention to protect the proteins against aggregation not only
during lyophilization but also during storage in the dry state. For
example sucrose and Ficoll 70 (a polymer with sucrose units)
exhibit significant protection against peptide or protein
aggregation during solid-phase incubation under various conditions.
These additives may also enhance the stability of solid proteins
embedded within polymer matrices.
[0087] Yet additional additives, for example sucrose, stabilize
proteins against solid-state aggregation in humid atmospheres at
elevated temperatures, as may occur in certain sustained-release
formulations of the invention. Proteins such as gelatin and
collagen also serve as stabilizing or bulking agents to reduce
denaturation and aggregation of unstable proteins in this context.
These additives can be incorporated into polymeric melt processes
and compositions within the invention. For example, polypeptide
microparticles can be prepared by simply lyophilizing or spray
drying a solution containing various stabilizing additives
described above. Sustained release of unaggregated peptides and
proteins can thereby be obtained over an extended period of
time.
[0088] Various additional preparative components and methods, as
well as specific formulation additives, are provided herein which
yield formulations for mucosal delivery of aggregation-prone
peptides and proteins, wherein the peptide or protein is stabilized
in a substantially pure, unaggregated form using a solubilization
agent. A range of components and additives are contemplated for use
within these methods and formulations. Exemplary of these
solubilization agents are cyclodextrins (CDs), which selectively
bind hydrophobic side chains of polypeptides. These CDs have been
found to bind to hydrophobic patches of proteins in a manner that
significantly inhibits aggregation. This inhibition is selective
with respect to both the CD and the protein involved. Such
selective inhibition of protein aggregation provides additional
advantages within the intranasal delivery methods and compositions
of the invention. Additional agents for use in this context include
CD dimers, trimers and tetramers with varying geometries controlled
by the linkers that specifically block aggregation of peptides and
protein. Yet solubilization agents and methods for incorporation
within the invention involve the use of peptides and peptide
mimetics to selectively block protein-protein interactions. In one
aspect, the specific binding of hydrophobic side chains reported
for CD multimers is extended to proteins via the use of peptides
and peptide mimetics that similarly block protein aggregation. A
wide range of suitable methods and anti-aggregation agents are
available for incorporation within the compositions and procedures
of the invention.
[0089] To improve the transport characteristics of biologically
active agents (including PTH peptide, other active peptides and
proteins, and macromolecular and small molecule drugs) for enhanced
delivery across hydrophobic mucosal membrane barriers, the
invention also provides techniques and reagents for charge
modification of selected biologically active agents or
delivery-enhancing agents described herein. In this regard, the
relative permeabilities of macromolecules is generally be related
to their partition coefficients. The degree of ionization of
molecules, which is dependent on the pK.sub.a. of the molecule and
the pH at the mucosal membrane surface, also affects permeability
of the molecules. Permeation and partitioning of biologically
active agents, including PTH peptide and analogs of the invention,
for mucosal delivery may be facilitated by charge alteration or
charge spreading of the active agent or permeabilizing agent, which
is achieved, for example, by alteration of charged functional
groups, by modifying the pH of the delivery vehicle or solution in
which the active agent is delivered, or by coordinate
administration of a charge- or pH-altering reagent with the active
agent.
[0090] Consistent with these general teachings, mucosal delivery of
charged macromolecular species, including PTH peptide and other
biologically active peptides and proteins, within the methods and
compositions of the invention is substantially improved when the
active agent is delivered to the mucosal surface in a substantially
un-ionized, or neutral, electrical charge state.
[0091] Certain PTH peptide and other biologically active peptide
and protein components of mucosal formulations for use within the
invention will be charge modified to yield an increase in the
positive charge density of the peptide or protein. These
modifications extend also to cationization of peptide and protein
conjugates, carriers and other delivery forms disclosed herein.
Cationization offers a convenient means of altering the
biodistribution and transport properties of proteins and
macromolecules within the invention. Cationization is undertaken in
a manner that substantially preserves the biological activity of
the active agent and limits potentially adverse side effects,
including tissue damage and toxicity.
[0092] Effective delivery of biotherapeutic agents via intranasal
administration must take into account the decreased drug transport
rate across the protective mucus lining of the nasal mucosa, in
addition to drug loss due to binding to glycoproteins of the mucus
layer. Normal mucus is a viscoelastic, gel-like substance
consisting of water, electrolytes, mucins, macromolecules, and
sloughed epithelial cells. It serves primarily as a cytoprotective
and lubricative covering for the underlying mucosal tissues. Mucus
is secreted by randomly distributed secretory cells located in the
nasal epithelium and in other mucosal epithelia. The structural
unit of mucus is mucin. This glycoprotein is mainly responsible for
the viscoelastic nature of mucus, although other macromolecules may
also contribute to this property. In airway mucus, such
macromolecules include locally produced secretory IgA, IgM, IgE,
lysozyme, and bronchotransferrin, which also play an important role
in host defense mechanisms.
[0093] The coordinate administration methods of the instant
invention optionally incorporate effective mucolytic or
mucus-clearing agents, which serve to degrade, thin or clear mucus
from intranasal mucosal surfaces to facilitate absorption of
intranasally administered biotherapeutic agents. Within these
methods, a mucolytic or mucus-clearing agent is coordinately
administered as an adjunct compound to enhance intranasal delivery
of the biologically active agent. Alternatively, an effective
amount of a mucolytic or mucus-clearing agent is incorporated as a
processing agent within a multi-processing method of the invention,
or as an additive within a combinatorial formulation of the
invention, to provide an improved formulation that enhances
intranasal delivery of biotherapeutic compounds by reducing the
barrier effects of intranasal mucus.
[0094] A variety of mucolytic or mucus-clearing agents are
available for incorporation within the methods and compositions of
the invention. Based on their mechanisms of action, mucolytic and
mucus clearing agents can often be classified into the following
groups: proteases (e.g., pronase, papain) that cleave the protein
core of mucin glycoproteins; sulfhydryl compounds that split
mucoprotein disulfide linkages; and detergents (e.g., Triton X-100,
Tween 20) that break non-covalent bonds within the mucus.
Additional compounds in this context include, but are not limited
to, bile salts and surfactants, for example, sodium deoxycholate,
sodium taurodeoxycholate, sodium glycocholate, and
lysophosphatidylcholine.
[0095] The effectiveness of bile salts in causing structural
breakdown of mucus is in the order
deoxycholate>taurocholate>glycocholate. Other effective
agents that reduce mucus viscosity or adhesion to enhance
intranasal delivery according to the methods of the invention
include, e.g., short-chain fatty acids, and mucolytic agents that
work by chelation, such as N-acylcollagen peptides, bile acids, and
saponins (the latter function in part by chelating Ca.sup.2+ and/or
Mg.sup.2+ which play an important role in maintaining mucus layer
structure).
[0096] Additional mucolytic agents for use within the methods and
compositions of the invention include N-acetyl-L-cysteine (ACS), a
potent mucolytic agent that reduces both the viscosity and
adherence of bronchopulmonary mucus and is reported to modestly
increase nasal bioavailability of human growth hormone in
anesthetized rats (from 7.5 to 12.2%). These and other mucolytic or
mucus-clearing agents are contacted with the nasal mucosa,
typically in a concentration range of about 0.2 to 20 mM,
coordinately with administration of the biologically active agent,
to reduce the polar viscosity and/or elasticity of intranasal
mucus.
[0097] Still other mucolytic or mucus-clearing agents may be
selected from a range of glycosidase enzymes, which are able to
cleave glycosidic bonds within the mucus glycoprotein.
.alpha.-amylase and .beta.-amylase are representative of this class
of enzymes, although their mucolytic effect may be limited. In
contrast, bacterial glycosidases which allow these microorganisms
to permeate mucus layers of their hosts.
[0098] For combinatorial use with most biologically active agents
within the invention, including peptide and protein therapeutics,
non-ionogenic detergents are generally also useful as mucolytic or
mucus-clearing agents. These agents typically will not modify or
substantially impair the activity of therapeutic polypeptides.
[0099] Because the self-cleaning capacity of certain mucosal
tissues (e.g., nasal mucosal tissues) by mucociliary clearance is
necessary as a protective function (e.g., to remove dust,
allergens, and bacteria), it has been generally considered that
this function should not be substantially impaired by mucosal
medications. Mucociliary transport in the respiratory tract is a
particularly important defense mechanism against infections. To
achieve this function, ciliary beating in the nasal and airway
passages moves a layer of mucus along the mucosa to removing
inhaled particles and microorganisms.
[0100] Ciliostatic agents find use within the methods and
compositions of the invention to increase the residence time of
mucosally (e.g., intranasally) administered PTH peptide, analogs
and mimetics, and other biologically active agents disclosed
herein. In particular, the delivery these agents within the methods
and compositions of the invention is significantly enhanced in
certain aspects by the coordinate administration or combinatorial
formulation of one or more ciliostatic agents that function to
reversibly inhibit ciliary activity of mucosal cells, to provide
for a temporary, reversible increase in the residence time of the
mucosally administered active agent(s). For use within these
aspects of the invention, the foregoing ciliostatic factors, either
specific or indirect in their activity, are all candidates for
successful employment as ciliostatic agents in appropriate amounts
(depending on concentration, duration and mode of delivery) such
that they yield a transient (i.e., reversible) reduction or
cessation of mucociliary clearance at a mucosal site of
administration to enhance delivery of PTH peptide, analogs and
mimetics, and other biologically active agents disclosed herein,
without unacceptable adverse side effects.
[0101] Within more detailed aspects, a specific ciliostatic factor
is employed in a combined formulation or coordinate administration
protocol with one or more PTH peptide proteins, analogs and
mimetics, and/or other biologically active agents disclosed herein.
Various bacterial ciliostatic factors isolated and characterized in
the literature may be employed within these embodiments of the
invention. Ciliostatic factors from the bacterium Pseudomonas
aeruginosa include a phenazine derivative, a pyo compound
(2-alkyl-4-hydroxyquinolines), and a rhamnolipid (also known as a
hemolysin). The pyo compound produced ciliostasis at concentrations
of 50 .mu.g/ml and without obvious ultrastructural lesions. The
phenazine derivative also inhibited ciliary motility but caused
some membrane disruption, although at substantially greater
concentrations of 400 .mu.g/ml. Limited exposure of tracheal
explants to the rhamnolipid resulted in ciliostasis, which was
associated with altered ciliary membranes. More extensive exposure
to rhamnolipid was associated with removal of dynein arms from
axonemes.
[0102] Within more detailed aspects of the invention, one or more
membrane penetration-enhancing agents may be employed within a
mucosal delivery method or formulation of the invention to enhance
mucosal delivery of PTH peptide analogs and mimetics, and other
biologically active agents disclosed herein. Membrane penetration
enhancing agents in this context can be selected from: (i) a
surfactant, (ii) a bile salt, (iii) a phospholipid additive, mixed
micelle, liposome, or carrier, (iv) an alcohol, (v) an enamine,
(vi) an NO donor compound, (vii) a long-chain amphipathic molecule
(viii) a small hydrophobic penetration enhancer; (ix) sodium or a
salicylic acid derivative; (x) a glycerol ester of acetoacetic acid
(xi) a cyclodextrin or beta-cyclodextrin derivative, (xii) a
medium-chain fatty acid, (xiii) a chelating agent, (xiv) an amino
acid or salt thereof, (xv) an N-acetylamino acid or salt thereof,
(xvi) an enzyme degradative to a selected membrane component,
(xvii) an inhibitor of fatty acid synthesis, or (xviii) an
inhibitor of cholesterol synthesis; or (xix) any combination of the
membrane penetration enhancing agents recited in (i)-(xviii).
[0103] Certain surface-active agents are readily incorporated
within the mucosal delivery formulations and methods of the
invention as mucosal absorption enhancing agents. These agents,
which may be coordinately administered or combinatorially
formulated with PTH peptide proteins, analogs and mimetics, and
other biologically active agents disclosed herein, may be selected
from a broad assemblage of known surfactants. Surfactants, which
generally fall into three classes: (1) nonionic polyoxyethylene
ethers; (2) bile salts such as sodium glycocholate (SGC) and
deoxycholate (DOC); and (3) derivatives of fusidic acid such as
sodium taurodihydrofusidate (STDHF). The mechanisms of action of
these various classes of surface-active agents typically include
solubilization of the biologically active agent. For proteins and
peptides which often form aggregates, the surface active properties
of these absorption promoters can allow interactions with proteins
such that smaller units such as surfactant coated monomers may be
more readily maintained in solution. Examples of other
surface-active agents are L-.alpha.-Phosphatidylcholine Didecanoyl
(DDPC) polysorbate 80 and polysorbate 20. These monomers are
presumably more transportable units than aggregates. A second
potential mechanism is the protection of the peptide or protein
from proteolytic degradation by proteases in the mucosal
environment. Both bile salts and some fusidic acid derivatives
reportedly inhibit proteolytic degradation of proteins by nasal
homogenates at concentrations less than or equivalent to those
required to enhance protein absorption. This protease inhibition
may be especially important for peptides with short biological
half-lives.
[0104] The present invention provides pharmaceutical composition
that contains one or more PTH peptides, analogs or mimetics, and/or
other biologically active agents in combination with mucosal
delivery enhancing agents disclosed herein formulated in a
pharmaceutical preparation for mucosal delivery.
[0105] The permeabilizing agent reversibly enhances mucosal
epithelial paracellular transport, typically by modulating
epithelial junctional structure and/or physiology at a mucosal
epithelial surface in the subject. This effect typically involves
inhibition by the permeabilizing agent of homotypic or heterotypic
binding between epithelial membrane adhesive proteins of
neighboring epithelial cells. Target proteins for this blockade of
homotypic or heterotypic binding can be selected from various
related junctional adhesion molecules (JAMs), occludins, or
claudins. Examples of this are antibodies, antibody fragments or
single-chain antibodies that bind to the extracellular domains of
these proteins.
[0106] In yet additional detailed embodiments, the invention
provides permeabilizing peptides and peptide analogs and mimetics
for enhancing mucosal epithelial paracellular transport. The
subject peptides and peptide analogs and mimetics typically work
within the compositions and methods of the invention by modulating
epithelial junctional structure and/or physiology in a mammalian
subject. In certain embodiments, the peptides and peptide analogs
and mimetics effectively inhibit homotypic and/or heterotypic
binding of an epithelial membrane adhesive protein selected from a
junctional adhesion molecule (JAM), occludin, or claudin.
[0107] One such agent that has been extensively studied is the
bacterial toxin from Vibrio cholerae known as the "zonula occludens
toxin" (ZOT). This toxin mediates increased intestinal mucosal
permeability and causes disease symptoms including diarrhea in
infected subjects. Fasano et al., Proc. Nat. Acad. Sci., U.S.A.
8:5242-5246, 1991. When tested on rabbit ileal mucosa, ZOT
increased the intestinal permeability by modulating the structure
of intercellular tight junctions. More recently, it has been found
that ZOT is capable of reversibly opening tight junctions in the
intestinal mucosa. It has also been reported that ZOT is capable of
reversibly opening tight junctions in the nasal mucosa. U.S. Pat.
No. 5,908,825.
[0108] Within the methods and compositions of the invention, ZOT,
as well as various analogs and mimetics of ZOT that function as
agonists or antagonists of ZOT activity, are useful for enhancing
intranasal delivery of biologically active agents--by increasing
paracellular absorption into and across the nasal mucosa. In this
context, ZOT typically acts by causing a structural reorganization
of tight junctions marked by altered localization of the junctional
protein ZO1. Within these aspects of the invention, ZOT is
coordinately administered or combinatorially formulated with the
biologically active agent in an effective amount to yield
significantly enhanced absorption of the active agent, by
reversibly increasing nasal mucosal permeability without
substantial adverse side effects.
[0109] The compositions and delivery methods of the invention
optionally incorporate a selective transport-enhancing agent that
facilitates transport of one or more biologically active agents.
These transport-enhancing agents may be employed in a combinatorial
formulation or coordinate administration protocol with one or more
of the PTH peptides, analogs and mimetics disclosed herein, to
coordinately enhance delivery of one or more additional
biologically active agent(s) across mucosal transport barriers, to
enhance mucosal delivery of the active agent(s) to reach a target
tissue or compartment in the subject (e.g., the mucosal epithelium,
liver, CNS tissue or fluid, or blood plasma). Alternatively, the
transport-enhancing agents may be employed in a combinatorial
formulation or coordinate administration protocol to directly
enhance mucosal delivery of one or more of the PTH peptides,
analogs and mimetics, with or without enhanced delivery of an
additional biologically active agent.
[0110] Exemplary selective transport-enhancing agents for use
within this aspect of the invention include, but are not limited
to, glycosides, sugar-containing molecules, and binding agents such
as lectin binding agents, which are known to interact specifically
with epithelial transport barrier components. For example, specific
"bioadhesive" ligands, including various plant and bacterial
lectins, which bind to cell surface sugar moieties by
receptor-mediated interactions can be employed as carriers or
conjugated transport mediators for enhancing mucosal, e.g., nasal
delivery of biologically active agents within the invention.
Certain bioadhesive ligands for use within the invention will
mediate transmission of biological signals to epithelial target
cells that trigger selective uptake of the adhesive ligand by
specialized cellular transport processes (endocytosis or
transcytosis). These transport mediators can therefore be employed
as a "carrier system" to stimulate or direct selective uptake of
one or more PTH peptides, analogs and mimetics, and other
biologically active agent(s) into and/or through mucosal epithelia.
These and other selective transport-enhancing agents significantly
enhance mucosal delivery of macromolecular biopharmaceuticals
(particularly peptides, proteins, oligonucleotides and
polynucleotide vectors) within the invention. Lectins are plant
proteins that bind to specific sugars found on the surface of
glycoproteins and glycolipids of eukaryotic cells. Concentrated
solutions of lectins have a `mucotractive` effect, and various
studies have demonstrated rapid receptor mediated endocytocis (RME)
of lectins and lectin conjugates (e.g., concanavalin A conjugated
with colloidal gold particles) across mucosal surfaces. Additional
studies have reported that the uptake mechanisms for lectins can be
utilized for intestinal drug targeting in vivo. In certain of these
studies, polystyrene nanoparticles (500 nm) were covalently coupled
to tomato lectin and reported yielded improved systemic uptake
after oral administration to rats.
[0111] In addition to plant lectins, microbial adhesion and
invasion factors provide a rich source of candidates for use as
adhesive/selective transport carriers within the mucosal delivery
methods and compositions of the invention. Two components are
necessary for bacterial adherence processes, a bacterial `adhesin`
(adherence or colonization factor) and a receptor on the host cell
surface. Bacteria causing mucosal infections need to penetrate the
mucus layer before attaching themselves to the epithelial surface.
This attachment is usually mediated by bacterial fimbriae or pilus
structures, although other cell surface components may also take
part in the process. Adherent bacteria colonize mucosal epithelia
by multiplication and initiation of a series of biochemical
reactions inside the target cell through signal transduction
mechanisms (with or without the help of toxins). Associated with
these invasive mechanisms, a wide diversity of bioadhesive proteins
(e.g., invasin, internalin) originally produced by various bacteria
and viruses are known. These allow for extracellular attachment of
such microorganisms with an impressive selectivity for host species
and even particular target tissues. Signals transmitted by such
receptor-ligand interactions trigger the transport of intact,
living microorganisms into, and eventually through, epithelial
cells by endo- and transcytotic processes. Such naturally occurring
phenomena may be harnessed (e.g., by complexing biologically active
agents such as PTH peptides with an adhesin) according to the
teachings herein for enhanced delivery of biologically active
compounds into or across mucosal epithelia and/or to other
designated target sites of drug action.
[0112] Various bacterial and plant toxins that bind epithelial
surfaces in a specific, lectin-like manner are also useful within
the methods and compositions of the invention. For example,
diptheria toxin (DT) enters host cells rapidly by RME. Likewise,
the B subunit of the E. coli heat labile toxin binds to the brush
border of intestinal epithelial cells in a highly specific,
lectin-like manner. Uptake of this toxin and transcytosis to the
basolateral side of the enterocytes has been reported in vivo and
in vitro. Other researches have expressed the transmembrane domain
of diphtheria toxin in E. coli as a maltose-binding fusion protein
and coupled it chemically to high-Mw poly-L-lysine. The resulting
complex was successfully used to mediate internalization of a
reporter gene in vitro. In addition to these examples,
Staphylococcus aureus produces a set of proteins (e.g.,
staphylococcal enterotoxin A (SEA), SEB, toxic shock syndrome toxin
1 (TSST-1) which act both as superantigens and toxins. Studies
relating to these proteins have reported dose-dependent,
facilitated transcytosis of SEB and TSST-I in Caco-2 cells.
[0113] Viral haemagglutinins comprise another type of transport
agent to facilitate mucosal delivery of biologically active agents
within the methods and compositions of the invention. The initial
step in many viral infections is the binding of surface proteins
(haemagglutinins) to mucosal cells. These binding proteins have
been identified for most viruses, including rotaviruses, varicella
zoster virus, semliki forest virus, adenoviruses, potato leafroll
virus, and reovirus. These and other exemplary viral hemagglutinins
can be employed in a combinatorial formulation (e.g., a mixture or
conjugate formulation) or coordinate administration protocol with
one or more of the PTH peptide, analogs and mimetics disclosed
herein, to coordinately enhance mucosal delivery of one or more
additional biologically active agent(s). Alternatively, viral
hemagglutinins can be employed in a combinatorial formulation or
coordinate administration protocol to directly enhance mucosal
delivery of one or more of the PTH peptide proteins, analogs and
mimetics, with or without enhanced delivery of an additional
biologically active agent.
[0114] A variety of endogenous, selective transport-mediating
factors are also available for use within the invention. Mammalian
cells have developed an assortment of mechanisms to facilitate the
internalization of specific substrates and target these to defined
compartments. Collectively, these processes of membrane
deformations are termed `endocytosis` and comprise phagocytosis,
pinocytosis, receptor-mediated endocytosis (clathrin-mediated RME),
and potocytosis (non-clathrin-mediated RME). RME is a highly
specific cellular biologic process by which, as its name implies,
various ligands bind to cell surface receptors and are subsequently
internalized and trafficked within the cell. In many cells the
process of endocytosis is so active that the entire membrane
surface is internalized and replaced in less than a half hour. Two
classes of receptors are proposed based on their orientation in the
cell membrane; the amino terminus of Type I receptors is located on
the extracellular side of the membrane, whereas Type II receptors
have this same protein tail in the intracellular milieu.
[0115] Still other embodiments of the invention utilize transferrin
as a carrier or stimulant of RME of mucosally delivered
biologically active agents. Transferrin, an 80 kDa
iron-transporting glycoprotein, is efficiently taken up into cells
by RME. Transferrin receptors are found on the surface of most
proliferating cells, in elevated numbers on erythroblasts and on
many kinds of tumors. The transcytosis of transferrin (TO and
transferrin conjugates is reportedly enhanced in the presence of
Brefeldin A (BFA), a fungal metabolite. In other studies, BFA
treatment has been reported to rapidly increase apical endocytosis
of both ricin and HRP in MDCK cells. Thus, BFA and other agents
that stimulate receptor-mediated transport can be employed within
the methods of the invention as combinatorially formulated (e.g.,
conjugated) and/or coordinately administered agents to enhance
receptor-mediated transport of biologically active agents,
including PTH peptide proteins, analogs and mimetics.
[0116] In certain aspects of the invention, the combinatorial
formulations and/or coordinate administration methods herein
incorporate an effective amount of peptides and proteins which may
adhere to charged glass thereby reducing the effective
concentration in the container. Silanized containers, for example,
silanized glass containers, are used to store the finished product
to reduce adsorption of the polypeptide or protein to a glass
container.
[0117] In yet additional aspects of the invention, a kit for
treatment of a mammalian subject comprises a stable pharmaceutical
composition of one or more PTH peptide compound(s) formulated for
mucosal delivery to the mammalian subject wherein the composition
is effective for treating or preventing osteoporosis or osteopenia.
The kit further comprises a pharmaceutical reagent vial to contain
the one or more PTH peptide compounds. The pharmaceutical reagent
vial is composed of pharmaceutical grade polymer, glass or other
suitable material. The pharmaceutical reagent vial is, for example,
a silanized glass vial. The kit further comprises an aperture for
delivery of the composition to a nasal mucosal surface of the
subject. The delivery aperture is composed of a pharmaceutical
grade polymer, glass or other suitable material. The delivery
aperture is, for example, a silanized glass.
[0118] A silanization technique combines a special cleaning
technique for the surfaces to be silanized with a silanization
process at low pressure. The silane is in the gas phase and at an
enhanced temperature of the surfaces to be silanized. The method
provides reproducible surfaces with stable, homogeneous and
functional silane layers having characteristics of a monolayer. The
silanized surfaces prevent binding to the glass of polypeptides or
mucosal delivery enhancing agents of the present invention.
[0119] The procedure is useful to prepare silanized pharmaceutical
reagent vials to hold PTH peptide compositions of the present
invention. Glass trays are cleaned by rinsing with double distilled
water (ddH.sub.2O) before using. The silane tray is then be rinsed
with 95% EtOH, and the acetone tray is rinsed with acetone.
Pharmaceutical reagent vials are sonicated in acetone for 10
minutes. After the acetone sonication, reagent vials are washed in
ddH.sub.2O tray at least twice. Reagent vials are sonicated in 0.1M
NaOH for 10 minutes. While the reagent vials are sonicating in
NaOH, the silane solution is made under a hood. (Silane solution:
800 mL of 95% ethanol; 96 L of glacial acetic acid; 25 mL of
glycidoxypropyltrimethoxy silane).
[0120] After the NaOH sonication, reagent vials are washed in
ddH.sub.2O tray at least twice. The reagent vials are sonicated in
silane solution for 3 to 5 minutes. The reagent vials are washed in
100% EtOH tray. The reagent vials are dried with prepurified
N.sub.2 gas and stored in a 100.degree. C. oven for at least 2
hours before using.
[0121] In certain aspects of the invention, the combinatorial
formulations and/or coordinate administration methods herein
incorporate an effective amount of a nontoxic bioadhesive as an
adjunct compound or carrier to enhance mucosal delivery of one or
more biologically active agent(s). Bioadhesive agents in this
context exhibit general or specific adhesion to one or more
components or surfaces of the targeted mucosa. The bioadhesive
maintains a desired concentration gradient of the biologically
active agent into or across the mucosa to ensure penetration of
even large molecules (e.g., peptides and proteins) into or through
the mucosal epithelium. Typically, employment of a bioadhesive
within the methods and compositions of the invention yields a two-
to five-fold, often a five- to ten-fold increase in permeability
for peptides and proteins into or through the mucosal epithelium.
This enhancement of epithelial permeation often permits effective
transmucosal delivery of large macromolecules, for example to the
basal portion of the nasal epithelium or into the adjacent
extracellular compartments or a blood plasma or CNS tissue or
fluid.
[0122] This enhanced delivery provides for greatly improved
effectiveness of delivery of bioactive peptides, proteins and other
macromolecular therapeutic species. These results will depend in
part on the hydrophilicity of the compound, whereby greater
penetration will be achieved with hydrophilic species compared to
water insoluble compounds. In addition to these effects, employment
of bioadhesives to enhance drug persistence at the mucosal surface
can elicit a reservoir mechanism for protracted drug delivery,
whereby compounds not only penetrate across the mucosal tissue but
also back-diffuse toward the mucosal surface once the material at
the surface is depleted.
[0123] A variety of suitable bioadhesives are disclosed in the art
for oral administration, U.S. Pat. Nos. 3,972,995; 4,259,314;
4,680,323; 4,740,365; 4,573,996; 4,292,299; 4,715,369; 4,876,092;
4,855,142; 4,250,163; 4,226,848; 4,948,580; U.S. Pat. Reissue No.
33,093, hereby incorporated by reference, which find use within the
novel methods and compositions of the invention. The potential of
various bioadhesive polymers as a mucosal, e.g., nasal, delivery
platform within the methods and compositions of the invention can
be readily assessed by determining their ability to retain and
release PTH peptide, as well as by their capacity to interact with
the mucosal surfaces following incorporation of the active agent
therein. In addition, well known methods will be applied to
determine the biocompatibility of selected polymers with the tissue
at the site of mucosal administration. When the target mucosa is
covered by mucus (i.e., in the absence of mucolytic or
mucus-clearing treatment), it can serve as a connecting link to the
underlying mucosal epithelium. Therefore, the term "bioadhesive" as
used herein also covers mucoadhesive compounds useful for enhancing
mucosal delivery of biologically active agents within the
invention. However, adhesive contact to mucosal tissue mediated
through adhesion to a mucus gel layer may be limited by incomplete
or transient attachment between the mucus layer and the underlying
tissue, particularly at nasal surfaces where rapid mucus clearance
occurs. In this regard, mucin glycoproteins are continuously
secreted and, immediately after their release from cells or glands,
form a viscoelastic gel. The luminal surface of the adherent gel
layer, however, is continuously eroded by mechanical, enzymatic
and/or ciliary action. Where such activities are more prominent or
where longer adhesion times are desired, the coordinate
administration methods and combinatorial formulation methods of the
invention may further incorporate mucolytic and/or ciliostatic
methods or agents as disclosed herein above.
[0124] Typically, mucoadhesive polymers for use within the
invention are natural or synthetic macromolecules which adhere to
wet mucosal tissue surfaces by complex, but non-specific,
mechanisms. In addition to these mucoadhesive polymers, the
invention also provides methods and compositions incorporating
bioadhesives that adhere directly to a cell surface, rather than to
mucus, by means of specific, including receptor-mediated,
interactions. One example of bioadhesives that function in this
specific manner is the group of compounds known as lectins. These
are glycoproteins with an ability to specifically recognize and
bind to sugar molecules, e.g., glycoproteins or glycolipids, which
form part of intranasal epithelial cell membranes and can be
considered as "lectin receptors."
[0125] In certain aspects of the invention, bioadhesive materials
for enhancing intranasal delivery of biologically active agents
comprise a matrix of a hydrophilic, e.g., water soluble or
swellable, polymer or a mixture of polymers that can adhere to a
wet mucous surface. These adhesives may be formulated as ointments,
hydrogels (see above) thin films, and other application forms.
Often, these adhesives have the biologically active agent mixed
therewith to effectuate slow release or local delivery of the
active agent. Some are formulated with additional ingredients to
facilitate penetration of the active agent through the nasal
mucosa, e.g., into the circulatory system of the individual.
[0126] Various polymers, both natural and synthetic ones, show
significant binding to mucus and/or mucosal epithelial surfaces
under physiological conditions. The strength of this interaction
can readily be measured by mechanical peel or shear tests. When
applied to a humid mucosal surface, many dry materials will
spontaneously adhere, at least slightly. After such an initial
contact, some hydrophilic materials start to attract water by
adsorption, swelling or capillary forces, and if this water is
absorbed from the underlying substrate or from the polymer-tissue
interface, the adhesion may be sufficient to achieve the goal of
enhancing mucosal absorption of biologically active agents. Such
`adhesion by hydration` can be quite strong, but formulations
adapted to employ this mechanism must account for swelling which
continues as the dosage transforms into a hydrated mucilage. This
is projected for many hydrocolloids useful within the invention,
especially some cellulose-derivatives, which are generally
non-adhesive when applied in pre-hydrated state. Nevertheless,
bioadhesive drug delivery systems for mucosal administration are
effective within the invention when such materials are applied in
the form of a dry polymeric powder, microsphere, or film-type
delivery form.
[0127] Other polymers adhere to mucosal surfaces not only when
applied in dry, but also in fully hydrated state, and in the
presence of excess amounts of water. The selection of a
mucoadhesive thus requires due consideration of the conditions,
physiological as well as physico-chemical, under which the contact
to the tissue will be formed and maintained. In particular, the
amount of water or humidity usually present at the intended site of
adhesion, and the prevailing pH, are known to largely affect the
mucoadhesive binding strength of different polymers.
[0128] Several polymeric bioadhesive drug delivery systems have
been fabricated and studied in the past 20 years, not always with
success. A variety of such carriers are, however, currently used in
clinical applications involving dental, orthopedic,
ophthalmological, and surgical uses. For example, acrylic-based
hydrogels have been used extensively for bioadhesive devices.
Acrylic-based hydrogels are well suited for bioadhesion due to
their flexibility and nonabrasive characteristics in the partially
swollen state, which reduce damage-causing attrition to the tissues
in contact. Furthermore, their high permeability in the swollen
state allows unreacted monomer, un-crosslinked polymer chains, and
the initiator to be washed out of the matrix after polymerization,
which is an important feature for selection of bioadhesive
materials for use within the invention. Acrylic-based polymer
devices exhibit very high adhesive bond strength. For controlled
mucosal delivery of peptide and protein drugs, the methods and
compositions of the invention optionally include the use of
carriers, e.g., polymeric delivery vehicles that function in part
to shield the biologically active agent from proteolytic breakdown,
while at the same time providing for enhanced penetration of the
peptide or protein into or through the nasal mucosa. In this
context, bioadhesive polymers have demonstrated considerable
potential for enhancing oral drug delivery. As an example, the
bioavailability of 9-desglycinamide, 8-arginine vasopressin (DGAVP)
intraduodenally administered to rats together with a 1% (w/v)
saline dispersion of the mucoadhesive poly(acrylic acid) derivative
polycarbophil, was 3-5-fold increased compared to an aqueous
solution of the peptide drug without this polymer.
[0129] Mucoadhesive polymers of the poly(acrylic acid)-type are
potent inhibitors of some intestinal proteases. The mechanism of
enzyme inhibition is explained by the strong affinity of this class
of polymers for divalent cations, such as calcium or zinc, which
are essential cofactors of metallo-proteinases, such as trypsin and
chymotrypsin. Depriving the proteases of their cofactors by
poly(acrylic acid) was reported to induce irreversible structural
changes of the enzyme proteins which were accompanied by a loss of
enzyme activity. At the same time, other mucoadhesive polymers
(e.g., some cellulose derivatives and chitosan) may not inhibit
proteolytic enzymes under certain conditions. In contrast to other
enzyme inhibitors contemplated for use within the invention (e.g.
aprotinin, bestatin), which are relatively small molecules, the
trans-nasal absorption of inhibitory polymers is likely to be
minimal in light of the size of these molecules, and thereby
eliminate possible adverse side effects. Thus, mucoadhesive
polymers, particularly of the poly(acrylic acid)-type, may serve
both as an absorption-promoting adhesive and enzyme-protective
agent to enhance controlled delivery of peptide and protein drugs,
especially when safety concerns are considered.
[0130] In addition to protecting against enzymatic degradation,
bioadhesives and other polymeric or non-polymeric
absorption-promoting agents for use within the invention may
directly increase mucosal permeability to biologically active
agents. To facilitate the transport of large and hydrophilic
molecules, such as peptides and proteins, across the nasal
epithelial barrier, mucoadhesive polymers and other agents have
been postulated to yield enhanced permeation effects beyond what is
accounted for by prolonged premucosal residence time of the
delivery system. The time course of drug plasma concentrations
reportedly suggested that the bioadhesive microspheres caused an
acute, but transient increase of insulin permeability across the
nasal mucosa. Other mucoadhesive polymers for use within the
invention, for example chitosan, reportedly enhance the
permeability of certain mucosal epithelia even when they are
applied as an aqueous solution or gel. Another mucoadhesive polymer
reported to directly affect epithelial permeability is hyaluronic
acid and ester derivatives thereof. A particularly useful
bioadhesive agent within the coordinate administration, and/or
combinatorial formulation methods and compositions of the invention
is chitosan, as well as its analogs and derivatives. Chitosan is a
non-toxic, biocompatible and biodegradable polymer that is widely
used for pharmaceutical and medical applications because of its
favorable properties of low toxicity and good biocompatibility. It
is a natural polyaminosaccharide prepared from chitin by
N-deacetylation with alkali. As used within the methods and
compositions of the invention, chitosan increases the retention of
PTH peptides, analogs and mimetics, and other biologically active
agents disclosed herein at a mucosal site of application. This mode
of administration can also improve patient compliance and
acceptance. As further provided herein, the methods and
compositions of the invention will optionally include a novel
chitosan derivative or chemically modified form of chitosan. One
such novel derivative for use within the invention is denoted as a
.beta.-[1.fwdarw.4]-2-guanidino-2-deoxy-D-glucose polymer
(poly-GuD). Chitosan is the N-deacetylated product of chitin, a
naturally occurring polymer that has been used extensively to
prepare microspheres for oral and intra-nasal formulations. The
chitosan polymer has also been proposed as a soluble carrier for
parenteral drug delivery. Within one aspect of the invention,
o-methylisourea is used to convert a chitosan amine to its
guanidinium moiety. The guanidinium compound is prepared, for
example, by the reaction between equi-normal solutions of chitosan
and o-methylisourea at pH above 8.0.
[0131] The guanidinium product is -[14]-guanidino-2-deoxy-D-glucose
polymer. It is abbreviated as Poly-GuD in this context (Monomer
F.W. of Amine in Chitosan=161; Monomer F.W. of Guanidinium in
Poly-GuD=203).
[0132] Additional compounds classified as bioadhesive agents for
use within the present invention act by mediating specific
interactions, typically classified as "receptor-ligand
interactions" between complementary structures of the bioadhesive
compound and a component of the mucosal epithelial surface. Many
natural examples illustrate this form of specific binding
bioadhesion, as exemplified by lectin-sugar interactions. Lectins
are (glyco) proteins of non-immune origin which bind to
polysaccharides or glycoconjugates.
[0133] Several plant lectins have been investigated as possible
pharmaceutical absorption-promoting agents. One plant lectin,
Phaseolus vulgaris hemagglutinin (PHA), exhibits high oral
bioavailability of more than 10% after feeding to rats. Tomato
(Lycopersicon esculeutum) lectin (TL) appears safe for various
modes of administration.
[0134] In summary, the foregoing bioadhesive agents are useful in
the combinatorial formulations and coordinate administration
methods of the instant invention, which optionally incorporate an
effective amount and form of a bioadhesive agent to prolong
persistence or otherwise increase mucosal absorption of one or more
PTH peptides, analogs and mimetics, and other biologically active
agents. The bioadhesive agents may be coordinately administered as
adjunct compounds or as additives within the combinatorial
formulations of the invention. In certain embodiments, the
bioadhesive agent acts as a `pharmaceutical glue,` whereas in other
embodiments adjunct delivery or combinatorial formulation of the
bioadhesive agent serves to intensify contact of the biologically
active agent with the nasal mucosa, in some cases by promoting
specific receptor-ligand interactions with epithelial cell
"receptors", and in others by increasing epithelial permeability to
significantly increase the drug concentration gradient measured at
a target site of delivery (e.g., liver, blood plasma, or CNS tissue
or fluid). Yet additional bioadhesive agents for use within the
invention act as enzyme (e.g., protease) inhibitors to enhance the
stability of mucosally administered biotherapeutic agents delivered
coordinately or in a combinatorial formulation with the bioadhesive
agent.
[0135] The coordinate administration methods and combinatorial
formulations of the instant invention optionally incorporate
effective lipid or fatty acid based carriers, processing agents, or
delivery vehicles, to provide improved formulations for mucosal
delivery of PTH peptides, analogs and mimetics, and other
biologically active agents. For example, a variety of formulations
and methods are provided for mucosal delivery which comprise one or
more of these active agents, such as a peptide or protein, admixed
or encapsulated by, or coordinately administered with, a liposome,
mixed micellar carrier, or emulsion, to enhance chemical and
physical stability and increase the half life of the biologically
active agents (e.g., by reducing susceptibility to proteolysis,
chemical modification and/or denaturation) upon mucosal
delivery.
[0136] Within certain aspects of the invention, specialized
delivery systems for biologically active agents comprise small
lipid vesicles known as liposomes. These are typically made from
natural, biodegradable, non-toxic, and non-immunogenic lipid
molecules, and can efficiently entrap or bind drug molecules,
including peptides and proteins, into, or onto, their membranes.
The attractiveness of liposomes as a peptide and protein delivery
system within the invention is increased by the fact that the
encapsulated proteins can remain in their preferred aqueous
environment within the vesicles, while the liposomal membrane
protects them against proteolysis and other destabilizing factors.
Even though not all liposome preparation methods known are feasible
in the encapsulation of peptides and proteins due to their unique
physical and chemical properties, several methods allow the
encapsulation of these macromolecules without substantial
deactivation.
[0137] A variety of methods are available for preparing liposomes
for use within the invention, U.S. Pat. Nos. 4,235,871, 4,501,728,
and 4,837,028, hereby incorporated by reference. For use with
liposome delivery, the biologically active agent is typically
entrapped within the liposome, or lipid vesicle, or is bound to the
outside of the vesicle.
[0138] Like liposomes, unsaturated long chain fatty acids, which
also have enhancing activity for mucosal absorption, can form
closed vesicles with bilayer-like structures (so called
"ufasomes"). These can be formed, for example, using oleic acid to
entrap biologically active peptides and proteins for mucosal, e.g.,
intranasal, delivery within the invention.
[0139] Other delivery systems for use within the invention combine
the use of polymers and liposomes to ally the advantageous
properties of both vehicles such as encapsulation inside the
natural polymer fibrin. In addition, release of biotherapeutic
compounds from this delivery system is controllable through the use
of covalent crosslinking and the addition of antifibrinolytic
agents to the fibrin polymer.
[0140] More simplified delivery systems for use within the
invention include the use of cationic lipids as delivery vehicles
or carriers, which can be effectively employed to provide an
electrostatic interaction between the lipid carrier and such
charged biologically active agents as proteins and polyanionic
nucleic acids. This allows efficient packaging of the drugs into a
form suitable for mucosal administration and/or subsequent delivery
to systemic compartments.
[0141] Additional delivery vehicles for use within the invention
include long and medium chain fatty acids, as well as surfactant
mixed micelles with fatty acids. Most naturally occurring lipids in
the form of esters have important implications with regard to their
own transport across mucosal surfaces. Free fatty acids and their
monoglycerides which have polar groups attached, have been
demonstrated in the form of mixed micelles to act on the intestinal
barrier as penetration enhancers. This discovery of barrier
modifying function of free fatty acids (carboxylic acids with a
chain length varying from 12 to 20 carbon atoms) and their polar
derivatives has stimulated extensive research on the application of
these agents as mucosal absorption enhancers.
[0142] For use within the methods of the invention, long chain
fatty acids, especially fusogenic lipids (unsaturated fatty acids
and monoglycerides such as oleic acid, linoleic acid, linoleic
acid, monoolein, etc.) provide useful carriers to enhance mucosal
delivery of PTH peptide, analogs and mimetics, and other
biologically active agents disclosed herein. Medium chain fatty
acids (C6 to C12) and monoglycerides have also been shown to have
enhancing activity in intestinal drug absorption and can be adapted
for use within the mocosal delivery formulations and methods of the
invention. In addition, sodium salts of medium and long chain fatty
acids are effective delivery vehicles and absorption-enhancing
agents for mucosal delivery of biologically active agents within
the invention. Thus, fatty acids can be employed in soluble forms
of sodium salts or by the addition of non-toxic surfactants, e.g.,
polyoxyethylated hydrogenated castor oil, sodium taurocholate, etc.
Other fatty acid and mixed micellar preparations that are useful
within the invention include, but are not limited to, Na caprylate
(C8), Na caprate (C 10), Na laurate (C 12) or Na oleate (C 18),
optionally combined with bile salts, such as glycocholate and
taurocholate.
[0143] Additional methods and compositions provided within the
invention involve chemical modification of biologically active
peptides and proteins by covalent attachment of polymeric
materials, for example dextrans, polyvinyl pyrrolidones,
glycopeptides, polyethylene glycol and polyamino acids. The
resulting conjugated peptides and proteins retain their biological
activities and solubility for mucosal administration. In alternate
embodiments, PTH peptide proteins, analogs and mimetics, and other
biologically active peptides and proteins, are conjugated to
polyalkylene oxide polymers, particularly polyethylene glycols
(PEG). U.S. Pat. No. 4,179,337, hereby incorporated by
reference.
[0144] Peptides could be linked to PEG directly as described in the
art. PEG can be a molecule having a molecular mass ranging between
300 and 60,000. Also included are various PEG molecules, including
linear, branched, attached to a peptide at a single moiety or
multiple attachment sites. Amine-reactive PEG polymers for use
within the invention include SC-PEG with molecular masses of 2000,
5000, 10000, 12000, and 20 000; U-PEG-10000; NHS-PEG-3400-biotin;
T-PEG-5000; T-PEG-12000; and TPC-PEG-5000. PEGylation of
biologically active peptides and proteins may be achieved by
modification of carboxyl sites (e.g., aspartic acid or glutamic
acid groups in addition to the carboxyl terminus). The utility of
PEG-hydrazide in selective modification of carbodiimide-activated
protein carboxyl groups under acidic conditions has been described.
Alternatively, bifunctional PEG modification of biologically active
peptides and proteins can be employed. In some procedures, charged
amino acid residues, including lysine, aspartic acid, and glutamic
acid, have a marked tendency to be solvent accessible on protein
surfaces.
[0145] In addition to PEGylation, biologically active agents such
as peptides and proteins for use within the invention can be
modified to enhance circulating half-life by shielding the active
agent via conjugation to other known protecting or stabilizing
compounds, for example by the creation of fusion proteins with an
active peptide, protein, analog or mimetic linked to one or more
carrier proteins, such as one or more immunoglobulin chains.
[0146] Mucosal delivery formulations of the present invention
comprise PTH peptides, analogs and mimetics, typically combined
together with one or more pharmaceutically acceptable carriers and,
optionally, other therapeutic ingredients. The carrier(s) must be
"pharmaceutically acceptable" in the sense of being compatible with
the other ingredients of the formulation and not eliciting an
unacceptable deleterious effect in the subject. Such carriers are
described herein above or are otherwise well known to those skilled
in the art of pharmacology. Desirably, the formulation should not
include substances such as enzymes or oxidizing agents with which
the biologically active agent to be administered is known to be
incompatible. The formulations may be prepared by any of the
methods well known in the art of pharmacy.
[0147] Within the compositions and methods of the invention, the
PTH peptides, analogs and mimetics, and other biologically active
agents disclosed herein may be administered to subjects by a
variety of mucosal administration modes, including by oral, rectal,
vaginal, intranasal, intrapulmonary, or transdermal delivery, or by
topical delivery to the eyes, ears, skin or other mucosal surfaces.
Optionally, PTH peptides, analogs and mimetics, and other
biologically active agents disclosed herein can be coordinately or
adjunctively administered by non-mucosal routes, including by
intramuscular, subcutaneous, intravenous, intra-atrial,
intra-articular, intraperitoneal, or parenteral routes. In other
alternative embodiments, the biologically active agent(s) can be
administered ex vivo by direct exposure to cells, tissues or organs
originating from a mammalian subject, for example as a component of
an ex vivo tissue or organ treatment formulation that contains the
biologically active agent in a suitable, liquid or solid
carrier.
[0148] Compositions according to the present invention are often
administered in an aqueous solution as a nasal or pulmonary spray
and may be dispensed in spray form by a variety of methods known to
those skilled in the art. Preferred systems for dispensing liquids
as a nasal spray are disclosed in U.S. Pat. No. 4,511,069, hereby
incorporated by reference. The formulations may be presented in
multi-dose containers, for example in the sealed dispensing system
disclosed in U.S. Pat. No. 4,511,069. Additional aerosol delivery
forms may include, e.g., compressed air-, jet-, ultrasonic-, and
piezoelectric nebulizers, which deliver the biologically active
agent dissolved or suspended in a pharmaceutical solvent, e.g.,
water, ethanol, or a mixture thereof.
[0149] Nasal and pulmonary spray solutions of the present invention
typically comprise the drug or drug to be delivered, optionally
formulated with a surface-active agent, such as a nonionic
surfactant (e.g., polysorbate-80), and one or more buffers. In some
embodiments of the present invention, the nasal spray solution
further comprises a propellant. The pH of the nasal spray solution
is optionally between about pH 3.0 and 6.0, preferably 5.0.+-.0.3.
Suitable buffers for use within these compositions are as described
above or as otherwise known in the art. Other components may be
added to enhance or maintain chemical stability, including
preservatives, surfactants, dispersants, or gases. Suitable
preservatives include, but are not limited to, phenol, methyl
paraben, paraben, m-cresol, thiomersal, chlorobutanol,
benzylalkonimum chloride, and the like. Suitable surfactants
include, but are not limited to, oleic acid, sorbitan trioleate,
polysorbates, lecithin, phosphotidyl cholines, and various long
chain diglycerides and phospholipids. Suitable dispersants include,
but are not limited to, ethylenediaminetetraacetic acid, and the
like. Suitable gases include, but are not limited to, nitrogen,
helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs),
carbon dioxide, air, and the like.
[0150] Within alternate embodiments, mucosal formulations are
administered as dry powder formulations comprising the biologically
active agent in a dry, usually lyophilized, form of an appropriate
particle size, or within an appropriate particle size range, for
intranasal delivery. Minimum particle size appropriate for
deposition within the nasal or pulmonary passages is often about
0.5.mu. mass median equivalent aerodynamic diameter (MMEAD),
commonly about 1.mu. MMEAD, and more typically about 2.mu. MMEAD.
Maximum particle size appropriate for deposition within the nasal
passages is often about 10.mu. MMEAD, commonly about 8.mu. MMEAD,
and more typically about 4.mu. MMEAD. Intranasally respirable
powders within these size ranges can be produced by a variety of
conventional techniques, such as jet milling, spray drying, solvent
precipitation, supercritical fluid condensation, and the like.
These dry powders of appropriate MMEAD can be administered to a
patient via a conventional dry powder inhaler (DPI), which rely on
the patient's breath, upon pulmonary or nasal inhalation, to
disperse the power into an aerosolized amount. Alternatively, the
dry powder may be administered via air-assisted devices that use an
external power source to disperse the powder into an aerosolized
amount, e.g., a piston pump.
[0151] Dry powder devices typically require a powder mass in the
range from about 1 mg to 20 mg to produce a single aerosolized dose
("puff"). If the required or desired dose of the biologically
active agent is lower than this amount, the powdered active agent
will typically be combined with a pharmaceutical dry bulking powder
to provide the required total powder mass. Preferred dry bulking
powders include sucrose, lactose, dextrose, mannitol, glycine,
trehalose, human serum albumin (HSA), and starch. Other suitable
dry bulking powders include cellobiose, dextrans, maltotriose,
pectin, sodium citrate, sodium ascorbate, and the like.
[0152] To formulate compositions for mucosal delivery within the
present invention, the biologically active agent can be combined
with various pharmaceutically acceptable additives, as well as a
base or carrier for dispersion of the active agent(s). Desired
additives include, but are not limited to, pH control agents, such
as arginine, sodium hydroxide, glycine, hydrochloric acid, citric
acid, etc. In addition, local anesthetics (e.g., benzyl alcohol),
isotonizing agents (e.g., sodium chloride, mannitol, sorbitol),
adsorption inhibitors (e.g., Tween 80), solubility enhancing agents
(e.g., cyclodextrins and derivatives thereof), stabilizers (e.g.,
serum albumin), and reducing agents (e.g., glutathione) can be
included. When the composition for mucosal delivery is a liquid,
the tonicity of the formulation, as measured with reference to the
tonicity of 0.9% (w/v) physiological saline solution taken as
unity, is typically adjusted to a value at which no substantial,
irreversible tissue damage will be induced in the nasal mucosa at
the site of administration. Generally, the tonicity of the solution
is adjusted to a value of about 1/3 to 3, more typically 1/2 to 2,
and most often 3/4 to 1.7.
[0153] The biologically active agent may be dispersed in a base or
vehicle, which may comprise a hydrophilic compound having a
capacity to disperse the active agent and any desired additives.
The base may be selected from a wide range of suitable carriers,
including but not limited to, copolymers of polycarboxylic acids or
salts thereof, carboxylic anhydrides (e.g., maleic anhydride) with
other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.),
hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl
alcohol, polyvinylpyrrolidone, cellulose derivatives such as
hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural
polymers such as chitosan, collagen, sodium alginate, gelatin,
hyaluronic acid, and nontoxic metal salts thereof Often, a
biodegradable polymer is selected as a base or carrier, for
example, polylactic acid, poly(lactic acid-glycolic acid)
copolymer, polyhydroxybutyric acid, poly(hydroxybutyric
acid-glycolic acid) copolymer and mixtures thereof. Alternatively
or additionally, synthetic fatty acid esters such as polyglycerin
fatty acid esters, sucrose fatty acid esters, etc., can be employed
as carriers. Hydrophilic polymers and other carriers can be used
alone or in combination, and enhanced structural integrity can be
imparted to the carrier by partial crystallization, ionic bonding,
crosslinking and the like. The carrier can be provided in a variety
of forms, including, fluid or viscous solutions, gels, pastes,
powders, microspheres and films for direct application to the nasal
mucosa. The use of a selected carrier in this context may result in
promotion of absorption of the biologically active agent.
[0154] The biologically active agent can be combined with the base
or carrier according to a variety of methods, and release of the
active agent may be by diffusion, disintegration of the carrier, or
associated formulation of water channels. In some circumstances,
the active agent is dispersed in microcapsules (microspheres) or
nanocapsules (nanospheres) prepared from a suitable polymer, e.g.,
isobutyl 2-cyanoacrylate and dispersed in a biocompatible
dispersing medium applied to the nasal mucosa, which yields
sustained delivery and biological activity over a protracted
time.
[0155] To further enhance mucosal delivery of pharmaceutical agents
within the invention, formulations comprising the active agent may
also contain a hydrophilic low molecular weight compound as a base
or excipient. Such hydrophilic low molecular weight compounds
provide a passage medium through which a water-soluble active
agent, such as a physiologically active peptide or protein, may
diffuse through the base to the body surface where the active agent
is absorbed. The hydrophilic low molecular weight compound
optionally absorbs moisture from the mucosa or the administration
atmosphere and dissolves the water-soluble active peptide. The
molecular weight of the hydrophilic low molecular weight compound
is generally not more than 10000 and preferably not more than 3000.
Exemplary hydrophilic low molecular weight compound include polyol
compounds, such as oligo-, di- and monosaccarides such as sucrose,
mannitol, sorbitol, lactose, L-arabinose, D-erythrose, D-ribose,
D-xylose, D-mannose, trehalose, D-galactose, lactulose, cellobiose,
gentibiose, glycerin and polyethylene glycol. Other examples of
hydrophilic low molecular weight compounds useful as carriers
within the invention include N-methylpyrrolidone, and alcohols
(e.g., oligovinyl alcohol, ethanol, ethylene glycol, propylene
glycol, etc.). These hydrophilic low molecular weight compounds can
be used alone or in combination with one another or with other
active or inactive components of the intranasal formulation.
[0156] The compositions of the invention may alternatively contain
as pharmaceutically acceptable carriers substances as required to
approximate physiological conditions, such as pH adjusting and
buffering agents, tonicity adjusting agents, wetting agents and the
like, for example, sodium acetate, sodium lactate, sodium chloride,
potassium chloride, calcium chloride, sorbitan monolaurate,
triethanolamine oleate, etc. For solid compositions, conventional
nontoxic pharmaceutically acceptable carriers can be used which
include, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharin, talcum, cellulose,
glucose, sucrose, magnesium carbonate, and the like.
[0157] Therapeutic compositions for administering the biologically
active agent can also be formulated as a solution, microemulsion,
or other ordered structure suitable for high concentration of
active ingredients. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), and suitable mixtures thereof. Proper
fluidity for solutions can be maintained, for example, by the use
of a coating such as lecithin, by the maintenance of a desired
particle size in the case of dispersible formulations, and by the
use of surfactants. In many cases, it will be desirable to include
isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the biologically active agent can be
brought about by including in the composition an agent which delays
absorption, for example, monostearate salts and gelatin.
[0158] In certain embodiments of the invention, the biologically
active agent is administered in a time-release formulation, for
example in a composition which includes a slow release polymer. The
active agent can be prepared with carriers that will protect
against rapid release, for example a controlled release vehicle
such as a polymer, microencapsulated delivery system or bioadhesive
gel. Prolonged delivery of the active agent, in various
compositions of the invention can be brought about by including in
the composition agents that delay absorption, for example, aluminum
monosterate hydrogels and gelatin. When controlled release
formulations of the biologically active agent is desired,
controlled release binders suitable for use in accordance with the
invention include any biocompatible controlled-release material
which is inert to the active agent and which is capable of
incorporating the biologically active agent. Numerous such
materials are known in the art. Useful controlled-release binders
are materials that are metabolized slowly under physiological
conditions following their intranasal delivery (e.g., at the nasal
mucosal surface, or in the presence of bodily fluids following
transmucosal delivery). Appropriate binders include but are not
limited to biocompatible polymers and copolymers previously used in
the art in sustained release formulations. Such biocompatible
compounds are non-toxic and inert to surrounding tissues, and do
not trigger significant adverse side effects such as nasal
irritation, immune response, inflammation, or the like. They are
metabolized into metabolic products that are also biocompatible and
easily eliminated from the body.
[0159] Exemplary polymeric materials for use in this context
include, but are not limited to, polymeric matrices derived from
copolymeric and homopolymeric polyesters having hydrolysable ester
linkages. A number of these are known in the art to be
biodegradable and to lead to degradation products having no or low
toxicity. Exemplary polymers include polyglycolic acids (PGA) and
polylactic acids (PLA), poly(DL-lactic acid-co-glycolic acid)(DL
PLGA), poly(D-lactic acid-coglycolic acid)(D PLGA) and
poly(L-lactic acid-co-glycolic acid)(L PLGA). Other useful
biodegradable or bioerodable polymers include but are not limited
to such polymers as poly(epsilon-caprolactone),
poly(epsilon-aprolactone-CO-lactic acid),
poly(-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyric
acid), poly(alkyl-2-cyanoacrilate), hydrogels such as
poly(hydroxyethyl methacrylate), polyamides, poly(amino acids)
(i.e., L-leucine, glutamic acid, L-aspartic acid and the like),
poly (ester urea), poly (2-hydroxyethyl DL-aspartamide), polyacetal
polymers, polyorthoesters, polycarbonate, polymaleamides,
polysaccharides and copolymers thereof. Many methods for preparing
such formulations are generally known to those skilled in the art.
Other useful formulations include controlled-release compositions
e.g., microcapsules, U.S. Pat. Nos. 4,652,441 and 4,917,893, lactic
acid-glycolic acid copolymers useful in making microcapsules and
other formulations, U.S. Pat. Nos. 4,677,191 and 4,728,721, and
sustained-release compositions for water-soluble peptides, U.S.
Pat. No. 4,675,189, all patents hereby incorporated by
reference.
[0160] Sterile solutions can be prepared by incorporating the
active compound in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle that contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders, methods of preparation include vacuum drying
and freeze-drying which yields a powder of the active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The prevention of the action of
microorganisms can be accomplished by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like.
[0161] Mucosal administration according to the invention allows
effective self-administration of treatment by patients, provided
that sufficient safeguards are in place to control and monitor
dosing and side effects. Mucosal administration also overcomes
certain drawbacks of other administration forms, such as
injections, that are painful and expose the patient to possible
infections and may present drug bioavailability problems. For nasal
and pulmonary delivery, systems for controlled aerosol dispensing
of therapeutic liquids as a spray are well known. In one
embodiment, metered doses of active agent are delivered by means of
a specially constructed mechanical pump valve, U.S. Pat. No.
4,511,069.
[0162] For prophylactic and treatment purposes, the biologically
active agent(s) disclosed herein may be administered to the subject
intranasally once daily. In this context, a therapeutically
effective dosage of the PTH peptide may include repeated doses
within a prolonged prophylaxis or treatment regimen that will yield
clinically significant results to alleviate or prevent osteoporosis
or osteopenia. Determination of effective dosages in this context
is typically based on animal model studies followed up by human
clinical trials and is guided by determining effective dosages and
administration protocols that significantly reduce the occurrence
or severity of targeted disease symptoms or conditions in the
subject. Suitable models in this regard include, for example,
murine, rat, porcine, feline, non-human primate, and other accepted
animal model subjects known in the art. Alternatively, effective
dosages can be determined using in vitro models (e.g., immunologic
and histopathologic assays). Using such models, only ordinary
calculations and adjustments are typically required to determine an
appropriate concentration and dose to administer a therapeutically
effective amount of the biologically active agent(s) (e.g., amounts
that are intranasally effective, transdermally effective,
intravenously effective, or intramuscularly effective to elicit a
desired response). The actual dosage of biologically active agents
will of course vary according to factors such as the disease
indication and particular status of the subject (e.g., the
subject's age, size, fitness, extent of symptoms, susceptibility
factors, etc.), time and route of administration, other drugs or
treatments being administered concurrently, as well as the specific
pharmacology of the biologically active agent(s) for eliciting the
desired activity or biological response in the subject. Dosage
regimens may be adjusted to provide an optimum prophylactic or
therapeutic response. A therapeutically effective amount is also
one in which any toxic or detrimental side effects of the
biologically active agent are outweighed in clinical terms by
therapeutically beneficial effects. A non-limiting range for a
therapeutically effective amount of a PTH peptide within the
methods and formulations of the invention is 0.7 .mu.g/kg to about
25 .mu.g/kg. To treat osteoporosis or osteopenia, an intranasal
dose of PTH peptide is administered at dose high enough to promote
the increase in bone mass but low enough so as not to induce any
unwanted side-effects such as nausea. A preferred intranasal dose
of parathyroid hormone 1-34 is about 1 .mu.g-10 .mu.g/kg weight of
the patient, most preferably from about 1.5 .mu.g/kg to about 3
.mu.g/kg weight of the patient. In a standard dose a patient will
receive 50 .mu.g to 1600 .mu.g, more preferably about between 75
.mu.g to 800 .mu.g, most preferably 100 .mu.g, 150 .mu.g, 200 .mu.g
to about 400 .mu.g. Alternatively, a non-limiting range for a
therapeutically effective amount of a biologically active agent
within the methods and formulations of the invention is between
about 0.001 pmol to about 100 pmol per kg body weight, between
about 0.01 pmol to about 10 pmol per kg body weight, between about
0.1 pmol to about 5 pmol per kg body weight, or between about 0.5
pmol to about 1.0 pmol per kg body weight. Per administration, it
is desirable to administer at least one microgram of the
biologically active agent (e.g., one or more PTH peptide proteins,
analogs and mimetics, and other biologically active agents), more
typically between about 10 .mu.g and 5.0 mg, and in certain
embodiments between about 100 .mu.g and 1.0 or 2.0 mg to an average
human subject. For certain oral applications, doses as high as 0.5
mg per kg body weight may be necessary to achieve adequate plasma
levels. It is to be further noted that for each particular subject,
specific dosage regimens should be evaluated and adjusted over time
according to the individual need and professional judgment of the
person administering or supervising the administration of the
permeabilizing peptide(s) and other biologically active agent(s).
An intranasal dose of a parathyroid hormone will range from 50
.mu.g to 1600 .mu.g of parathyroid hormone, preferably 75 .mu.g to
800 .mu.g, more preferably 100 .mu.g to 400 .mu.g with a most
preferred dose being between 100 .mu.g to 200 .mu.g with 150 .mu.g
being a dose that is considered to be highly effective. Repeated
intranasal dosing with the formulations of the invention, on a
schedule ranging from about 0.1 to24 hours between doses,
preferably between 0.5 and 24.0 hours between doses, will maintain
normalized, sustained therapeutic levels of PTH peptide to maximize
clinical benefits while minimizing the risks of excessive exposure
and side effects. The goal is to mucosally deliver an amount of the
PTH peptide sufficient to raise the concentration of the PTH
peptide in the plasma of an individual to promote increase in bone
mass.
[0163] Dosage of PTH agonists such as parathyroid hormone may be
varied by the attending clinician or patient, if self administering
an over the counter dosage form, to maintain a desired
concentration at the target site.
[0164] In an alternative embodiment, the invention provides
compositions and methods for intranasal delivery of PTH peptide,
wherein the PTH peptide compound(s) is/are repeatedly administered
through an intranasal effective dosage regimen that involves
multiple administrations of the PTH peptide to the subject during a
daily or weekly schedule to maintain a therapeutically effective
elevated and lowered pulsatile level of PTH peptide during an
extended dosing period. The compositions and method provide PTH
peptide compound(s) that are self-administered by the subject in a
nasal formulation between one and six times daily to maintain a
therapeutically effective elevated and lowered pulsatile level of
PTH peptide during an 8 hour to 24 hour extended dosing period.
[0165] The instant invention also includes kits, packages and
multicontainer units containing the above described pharmaceutical
compositions, active ingredients, and/or means for administering
the same for use in the prevention and treatment of diseases and
other conditions in mammalian subjects. Briefly, these kits include
a container or formulation that contains one or more PTH peptide
proteins, analogs or mimetics, and/or other biologically active
agents in combination with mucosal delivery enhancing agents
disclosed herein formulated in a pharmaceutical preparation for
mucosal delivery.
[0166] The intranasal formulations of the present invention can be
administered using any spray bottle or syringe. An example of a
nasal spray bottle is the, "Nasal Spray Pump w/Safety Clip,"
Pfeiffer SAP #60548, which delivers a dose of 0.1 mL per squirt and
has a diptube length of 36.05 mm. It can be purchased from Pfeiffer
of America of Princeton, N.J. Intranasal doses of a PTH peptide
such as parathyroid hormone can range from 0.1 .mu.g/kg to about
1500 .mu.g/kg. When administered in as an intranasal spray, it is
preferable that the particle size of the spray is between 10-100
.mu.m (microns) in size, preferably 20-100 .mu.m in size.
[0167] We have discovered that the parathyroid hormone peptides can
be administered intranasally using a nasal spray or aerosol. This
is surprising because many proteins and peptides have been shown to
be sheared or denatured due to the mechanical forces generated by
the actuator in producing the spray or aerosol. In this area the
following definitions are useful.
[0168] 1. Aerosol--A product that is packaged under pressure and
contains therapeutically active ingredients that are released upon
activation of an appropriate valve system.
[0169] 2. Metered aerosol--A pressurized dosage form comprised of
metered dose valves, which allow for the delivery of a uniform
quantity of spray upon each activation.
[0170] 3. Powder aerosol--A product that is packaged under pressure
and contains therapeutically active ingredients in the form of a
powder, which are released upon activation of an appropriate valve
system.
[0171] 4. Spray aerosol--An aerosol product that utilizes a
compressed gas as the propellant to provide the force necessary to
expel the product as a wet spray; it generally applicable to
solutions of medicinal agents in aqueous solvents.
[0172] 5. Spray--A liquid minutely divided as by a jet of air or
steam. Nasal spray drug products contain therapeutically active
ingredients dissolved or suspended in solutions or mixtures of
excipients in nonpressurized dispensers.
[0173] 6. Metered spray--A non-pressurized dosage form consisting
of valves that allow the dispensing of a specified quantity of
spray upon each activation.
[0174] 7. Suspension spray--A liquid preparation containing solid
particles dispersed in a liquid vehicle and in the form of course
droplets or as finely divided solids.
[0175] The fluid dynamic characterization of the aerosol spray
emitted by metered nasal spray pumps as a drug delivery device
("DDD"). Spray characterization is an integral part of the
regulatory submissions necessary for Food and Drug Administration
("FDA") approval of research and development, quality assurance and
stability testing procedures for new and existing nasal spray
pumps.
[0176] Thorough characterization of the spray's geometry has been
found to be the best indicator of the overall performance of nasal
spray pumps. In particular, measurements of the spray's divergence
angle (plume geometry) as it exits the device; the spray's
cross-sectional ellipticity, uniformity and particle/droplet
distribution (spray pattern); and the time evolution of the
developing spray have been found to be the most representative
performance quantities in the characterization of a nasal spray
pump. During quality assurance and stability testing, plume
geometry and spray pattern measurements are key identifiers for
verifying consistency and conformity with the approved data
criteria for the nasal spray pumps.
DEFINITIONS
[0177] Plume Height--the measurement from the actuator tip to the
point at which the plume angle becomes non-linear because of the
breakdown of linear flow. Based on a visual examination of digital
images, and to establish a measurement point for width that is
consistent with the farthest measurement point of spray pattern, a
height of 30 mm is defined for this study
[0178] Major Axis--the largest chord that can be drawn within the
fitted spray pattern that crosses the COMw in base units (mm)
[0179] Minor Axis--the smallest chord that can be drawn within the
fitted spray pattern that crosses the COMw in base units (mm)
[0180] Ellipticity Ratio--the ratio of the major axis to the minor
axis
[0181] D.sub.10--the diameter of droplet for which 10% of the total
liquid volume of sample consists of droplets of a smaller diameter
(.mu.m)
[0182] D.sub.50--the diameter of droplet for which 50% of the total
liquid volume of sample consists of droplets of a smaller diameter
(.mu.m), also known as the mass median diameter
[0183] D.sub.90--the diameter of droplet for which 90% of the total
liquid volume of sample consists of droplets of a smaller diameter
(.mu.m)
[0184] Span--measurement of the width of the distribution, The
smaller the value, the narrower the distribution. Span is
calculated as
( D 90 - D 10 ) D 50 . ##EQU00001##
[0185] % RSD--percent relative standard deviation, the standard
deviation divided by the mean of the series and multiplied by 100,
also known as % CV.
[0186] A nasal spray device can be selected according to what is
customary in the industry or acceptable by the regulatory health
authorities. One example of a suitable device is described in
described in U.S. application Ser. No. 10/869,649 (S. Quay and G.
Brandt: Compositions and Methods for Enhanced Mucosal Delivery of
Y2 Receptor-binding Peptides and Methods for Treating and
Preventing Obesity, filed Jun. 16, 2004).
[0187] To treat osteoporosis or osteopenia, an intranasal dose of a
PTH peptide parathyroid hormone is administered at dose high enough
to promote an increase in bone mass but low enough so as not to
induce any unwanted side-effects such as nausea. A preferred
intranasal dose of a PTH peptide such as parathyroid hormone(1-34)
is about 3 .mu.g-10 .mu.g/kg weight of the patient, most preferably
about 6 .mu.g/kg weight of the patient. In a standard dose a
patient will receive 50 .mu.g to 800 .mu.g, more preferably about
between 100 .mu.g to 400 .mu.g, most preferably 150 .mu.g to about
200 .mu.g. The a PTH peptide such as parathyroid hormone (1-34) is
preferably administered once a day.
[0188] The following examples are provided by way of illustration,
not limitation.
EXAMPLE 1
[0189] An exemplary formulation for enhanced nasal mucosal delivery
of PTH following the teachings of the instant specification can be
prepared and evaluated as shown in Table 1.
EXAMPLE 2
Nasal Mucosal Delivery--Permeation Kinetics and Cytotoxicity
[0190] The following methods are generally useful for evaluating
nasal mucosal delivery parameters, kinetics and side effects for
PTH within the formulations and method of the invention, as well as
for determining the efficacy and characteristics of the various
intranasal delivery-enhancing agents disclosed herein for
combinatorial formulation or coordinate administration with
PTH.
[0191] Permeation kinetics and cytotoxicity are also useful for
determining the efficacy and characteristics of the various mucosal
delivery-enhancing agents disclosed herein for combinatorial
formulation or coordinate administration with mucosal
delivery-enhancing agents. In one exemplary protocol, permeation
kinetics and lack of unacceptable cytotoxicity are demonstrated for
an intranasal delivery-enhancing agent as disclosed above in
combination with a biologically active therapeutic agent,
exemplified by PTH.
[0192] The EPIAIRWAY.RTM. system was developed by MatTek Corp
(Ashland, Mass.) as a model of the pseudostratified epithelium
lining the respiratory tract. The epithelial cells are grown on
porous membrane-bottomed cell culture inserts at an air-liquid
interface, which results in differentiation of the cells to a
highly polarized morphology. The apical surface is ciliated with a
microvillous ultrastructure and the epithelium produces mucus (the
presence of mucin has been confirmed by immunoblotting). The
inserts have a diameter of 0.875 cm, providing a surface area of
0.6 cm.sup.2. The cells are plated onto the inserts at the factory
approximately three weeks before shipping.
[0193] On arrival, the units are placed onto sterile supports in
6-well microplates. Each well receives 5 mL of proprietary culture
medium. This DMEM-based medium is serum free but is supplemented
with epidermal growth factor and other factors. The medium is
always tested for endogenous levels of any cytokine or growth
factor, which is being considered for intranasal delivery, but has
been free of all cytokines and factors studied to date except
insulin. The 5 mL volume is just sufficient to provide contact to
the bottoms of the units on their stands, but the apical surface of
the epithelium is allowed to remain in direct contact with air.
Sterile tweezers are used in this step and in all subsequent steps
involving transfer of units to liquid-containing wells to ensure
that no air is trapped between the bottoms of the units and the
medium.
[0194] The units in their plates are maintained at 37.degree. C. in
an incubator in an atmosphere of 5% CO.sub.2 in air for 24 hours.
At the end of this time the medium is replaced with fresh medium
and the units are returned to the incubator for another 24
hours.
[0195] A "kit" of 24 EPIAIRWAY.RTM. units can routinely be employed
for evaluating five different formulations, each of which is
applied to quadruplicate wells. Each well is employed for
determination of permeation kinetics (4 time points),
transepithelial resistance, mitochondrial reductase activity as
measured by MTT reduction, and cytolysis as measured by release of
LDH. An additional set of wells is employed as controls, which are
sham treated during determination of permeation kinetics, but are
otherwise handled identically to the test sample-containing units
for determinations of transepithelial resistance and viability. The
determinations on the controls are routinely also made on
quadruplicate units, but occasionally we have employed triplicate
units for the controls and have dedicated the remaining four units
in the kit to measurements of transepithelial resistance and
viability on untreated units or we have frozen and thawed the units
for determinations of total LDH levels to serve as a reference for
100% cytolysis.
[0196] In all experiments, the nasal mucosal delivery formulation
to be studied is applied to the apical surface of each unit in a
volume of 100 .mu.L, which is sufficient to cover the entire apical
surface. An appropriate volume of the test formulation at the
concentration applied to the apical surface (no more than 100 .mu.L
is generally needed) is set aside for subsequent determination of
concentration of the active material by ELISA or other designated
assay.
[0197] The units are placed in 6 well plates without stands for the
experiment: each well contains 0.9 mL of medium which is sufficient
to contact the porous membrane bottom of the unit but does not
generate any significant upward hydrostatic pressure on the
unit.
[0198] To minimize potential sources of error and avoid any
formation of concentration gradients, the units are transferred
from one 0.9 mL-containing well to another at each time point in
the study. These transfers are made at the following time points,
based on a zero time at which the 100 .mu.L volume of test material
was applied to the apical surface: 15 minutes, 30 minutes, 60
minutes, and 120 minutes.
[0199] In between time points the units in their plates are kept in
the 37.degree. C. incubator. Plates containing 0.9 mL medium per
well are also maintained in the incubator so that minimal change in
temperature occurs during the brief periods when the plates are
removed and the units are transferred from one well to another
using sterile forceps.
[0200] At the completion of each time point, the medium is removed
from the well from which each unit was transferred, and aliquotted
into two tubes (one tube receives 700 .mu.L and the other 200
.mu.L) for determination of the concentration of permeated test
material and, in the event that the test material is cytotoxic, for
release of the cytosolic enzyme, lactic dehydrogenase, from the
epithelium. These samples are kept in the refrigerator if the
assays are to be conducted within 24 hours, or the samples are
subaliquotted and kept frozen at -80.degree. C. until thawed once
for assays. Repeated freeze-thaw cycles are to be avoided.
[0201] In order to minimize errors, all tubes, plates, and wells
are prelabeled before initiating an experiment.
[0202] At the end of the 120 minute time point, the units are
transferred from the last of the 0.9 mL containing wells to 24-well
microplates, containing 0.3 mL medium per well. This volume is
again sufficient to contact the bottoms of the units, but not to
exert upward hydrostatic pressure on the units. The units are
returned to the incubator prior to measurement of transepithelial
resistance.
[0203] Respiratory airway epithelial cells form tight junctions in
vivo as well as in vitro, restricting the flow of solutes across
the tissue. These junctions confer a transepithelial resistance of
several hundred ohms.times.cm.sup.2 in excised airway tissues; in
the MatTek EpiAirway units, the transepithelial resistance (TER) is
claimed by the manufacturer to be routinely around 1000
ohms.times.cm.sup.2. We have found that the TER of control
EPIAIRWAY.RTM. units which have been sham-exposed during the
sequence of steps in the permeation study is somewhat lower
(700-800 ohms.times.cm.sup.2), but, since permeation of small
molecules is proportional to the inverse of the TER, this value is
still sufficiently high to provide a major barrier to permeation.
The porous membrane-bottomed units without cells, conversely,
provide only minimal transmembrane resistance (5-20
ohms.times.cm.sup.2).
[0204] Accurate determinations of TER require that the electrodes
of the ohmmeter be positioned over a significant surface area above
and below the membrane, and that the distance of the electrodes
from the membrane be reproducibly controlled. The method for TER
determination recommended by MatTek and employed for all
experiments here employs an "EVOM.TM." epithelial voltohmmeter and
an "ENDOHM.TM." tissue resistance measurement chamber from World
Precision Instruments, Inc., Sarasota, Fla.
[0205] The chamber is initially filled with Dulbecco's phosphate
buffered saline (PBS) for at least 20 minutes prior to TER
determinations in order to equilibrate the electrodes.
[0206] Determinations of TER are made with 1.5 mL of PBS in the
chamber and 350 .mu.L of PBS in the membrane-bottomed unit being
measured. The top electrode is adjusted to a position just above
the membrane of a unit containing no cells (but containing 350
.mu.L of PBS) and then fixed to ensure reproducible positioning.
The resistance of a cell-free unit is typically 5-20
ohms.times.cm.sup.2 ("background resistance").
[0207] Once the chamber is prepared and the background resistance
is recorded, units in a 24-well plate which had just been employed
in permeation determinations are removed from the incubator and
individually placed in the chamber for TER determinations.
[0208] Each unit is first transferred to a petri dish containing
PBS to ensure that the membrane bottom is moistened. An aliquot of
350 .mu.L PBS is added to the unit and then carefully aspirated
into a labeled tube to rinse the apical surface. A second wash of
350 .mu.L PBS is then applied to the unit and aspirated into the
same collection tube.
[0209] The unit is blotted free of excess PBS on its exterior
surface only before being placed into the chamber (containing a
fresh 1.5 mL aliquot of PBS). An aliquot of 350 .mu.L PBS is added
to the unit before the top electrode is placed on the chamber and
the TER is read on the EVOM meter.
[0210] After the TER of the unit is read in the ENDOHM chamber, the
unit is removed, the PBS is aspirated and saved, and the unit is
returned with an air interface on the apical surface to a 24-well
plate containing 0.3 mL medium per well.
[0211] The units are read in the following sequence: all
sham-treated controls, followed by all formulation-treated samples,
followed by a second TER reading of each of the sham-treated
controls. After all the TER determinations are complete, the units
in the 24-well microplate are returned to the incubator for
determination of viability by MTT reduction.
[0212] MTT is a cell-permeable tetrazolium salt which is reduced by
mitochondrial dehydrogenase activity to an insoluble colored
formazan by viable cells with intact mitochondrial function or by
nonmitochondrial NAD(P)H dehydrogenase activity from cells capable
of generating a respiratory burst. Formation of formazan is a good
indicator of viability of epithelial cells since these cells do not
generate a significant respiratory burst. We have employed a MTT
reagent kit prepared by MatTek Corp for their units in order to
assess viability.
[0213] The MTT reagent is supplied as a concentrate and is diluted
into a proprietary DMEM-based diluent on the day viability is to be
assayed (typically the afternoon of the day in which permeation
kinetics and TER were determined in the morning). Insoluble reagent
is removed by a brief centrifugation before use. The final MTT
concentration is 1 mg/mL.
[0214] The final MTT solution is added to wells of a 24-well
microplate at a volume of 300 .mu.L per well. As has been noted
above, this volume is sufficient to contact the membranes of the
EPIAIRWAY.RTM. units but imposes no significant positive
hydrostatic pressure on the cells.
[0215] The units are removed from the 24-well plate in which they
were placed after TER measurements, and after removing any excess
liquid from the exterior surface of the units, they are transferred
to the plate containing MTT reagent. The units in the plate are
then placed in an incubator at 37.degree. C. in an atmosphere of 5%
CO.sub.2 in air for 3 hours.
[0216] At the end of the 3-hour incubation, the units containing
viable cells will have turned visibly purple. The insoluble
formazan must be extracted from the cells in their units to
quantitate the extent of MTT reduction. Extraction of the formazan
is accomplished by transferring the units to a 24-well microplate
containing 2 mL extractant solution per well, after removing excess
liquid from the exterior surface of the units as before. This
volume is sufficient to completely cover both the membrane and the
apical surface of the units. Extraction is allowed to proceed
overnight at room temperature in a light-tight chamber. MTT
extractants traditionally contain high concentrations of detergent,
and destroy the cells.
[0217] At the end of the extraction, the fluid from within each
unit and the fluid in its surrounding well are combined and
transferred to a tube for subsequent aliquotting into a 96-well
microplate (200 .mu.L aliquots are optimal) and determination of
absorbance at 570 nm on a VMax multiwell microplate
spectrophotometer. To ensure that turbidity from debris coming from
the extracted units does not contribute to the absorbance, the
absorbance at 650 nm is also determined for each well in the VMax
and is automatically subtracted from the absorbance at 570 nm. The
"blank" for the determination of formazan absorbance is a 200 .mu.L
aliquot of extractant to which no unit had been exposed. This
absorbance value is assumed to constitute zero viability.
[0218] Two units from each kit of 24 EPIAIRWAY.RTM. units are left
untreated during determination of permeation kinetics and TER.
These units are employed as the positive control for 100% cell
viability. In all the studies we have conducted, there has been no
statistically significant difference in the viability of the cells
in these untreated units vs cells in control units which had been
sham treated for permeation kinetics and on which TER
determinations had been performed. The absorbance of all units
treated with test formulations is assumed to be linearly
proportional to the percent viability of the cells in the units at
the time of the incubation with MTT. It should be noted that this
assay is carried out typically no sooner than four hours after
introduction of the test material to the apical surface, and
subsequent to rinsing of the apical surface of the units during TER
determination.
[0219] While measurement of mitochondrial reductase activity by MTT
reduction is a sensitive probe of cell viability, the assay
necessarily destroys the cells and therefore can be carried out
only at the end of each study. When cells undergo necrotic lysis,
their cytotosolic contents are spilled into the surrounding medium,
and cytosolic enzymes such as lactic dehydrogenase (LDH) can be
detected in this medium. An assay for LDH in the medium can be
performed on samples of medium removed at each time point of the
two-hour determination of permeation kinetics. Thus, cytotoxic
effects of formulations which do not develop until significant time
has passed can be detected as well as effects of formulations which
induce cytolysis with the first few minutes of exposure to airway
epithelium.
[0220] The recommended LDH assay for evaluating cytolysis of the
EPIAIRWAY.RTM. units is based on conversion of lactate to pyruvate
with generation of NADH from NAD. The NADH is then reoxidized along
with simultaneous reduction of the tetrazolium salt INT, catalyzed
by a crude "diaphorase" preparation. The formazan formed from
reduction of INT is soluble, so that the entire assay for LDH
activity can be carried out in a homogenous aqueous medium
containing lactate, NAD, diaphorase, and INT.
[0221] The assay for LDH activity is carried out on 50 .mu.L
aliquots from samples of "supernatant" medium surrounding an
EPIAIRWAY.RTM. unit and collected at each time point. These samples
were either stored for no longer than 24 h in the refrigerator or
were thawed after being frozen within a few hours after collection.
Each EPIAIRWAY.RTM. unit generates samples of supernatant medium
collected at 15 min, 30 min, 1 h, and 2 h after application of the
test material. The aliquots are all transferred to a 96 well
microplate.
[0222] A 50 .mu.L aliquot of medium which had not been exposed to a
unit serves as a "blank" or negative control of 0% cytotoxicity. We
have found that the apparent level of "endogenous" LDH present
after reaction of the assay reagent mixture with the unexposed
medium is the same within experimental error as the apparent level
of LDH released by all the sham-treated control units over the
entire time course of 2 hours required to conduct a permeation
kinetics study. Thus, within experimental error, these sham-treated
units show no cytolysis of the epithelial cells over the time
course of the permeation kinetics measurements.
[0223] To prepare a sample of supernatant medium reflecting the
level of LDH released after 100% of the cells in a unit have lysed,
a unit which had not been subjected to any prior manipulations is
added to a well of a 6-well microplate containing 0.9 mL of medium
as in the protocol for determination of permeation kinetics, the
plate containing the unit is frozen at -80.degree. C., and the
contents of the well are then allowed to thaw. This freeze-thaw
cycle effectively lyses the cells and releases their cytosolic
contents, including LDH, into the supernatant medium. A 50 .mu.L
aliquot of the medium from the frozen and thawed cells is added to
the 96-well plate as a positive control reflecting 100%
cytotoxicity.
[0224] To each well containing an aliquot of supernatant medium, a
50 .mu.L aliquot of the LDH assay reagent is added. The plate is
then incubated for 30 minutes in the dark.
[0225] The reactions are terminated by addition of a "stop"
solution of 1 M acetic acid, and within one hour of addition of the
stop solution, the absorbance of the plate is determined at 490
nm.
[0226] Computation of percent cytolysis is based on the assumption
of a linear relationship between absorbance and cytolysis, with the
absorbance obtained from the medium alone serving as a reference
for 0% cytolysis and the absorbance obtained from the medium
surrounding a frozen and thawed unit serving as a reference for
100% cytolysis.
[0227] The procedures for determining the concentrations of
biologically active agents as test materials for evaluating
enhanced permeation of active agents in conjunction with coordinate
administration of mucosal delivery-enhancing agents or
combinatorial formulation of the invention are generally as
described above and in accordance with known methods and specific
manufacturer instructions of ELISA kits employed for each
particular assay. Permeation kinetics of the biologically active
agent is generally determined by taking measurements at multiple
time points (for example 15 min., 30 min., 60 min. and 120 min)
after the biologically active agent is contacted with the apical
epithelial cell surface (which may be simultaneous with, or
subsequent to, exposure of the apical cell surface to the mucosal
delivery-enhancing agent(s)).
[0228] The procedures for determining the concentrations of PTH
peptide in blood serum, central nervous system (CNS) tissues or
fluids, cerebral spinal fluid (CSF), or other tissues or fluids of
a mammalian subject may be determined by immunologic assay for PTH.
The procedures for determining the concentrations of PTH as test
materials for evaluating enhanced permeation of active agents in
conjunction with coordinate administration of mucosal
delivery-enhancing agents or combinatorial formulation of the
invention are generally as described above and in accordance with
known methods and specific manufacturer instructions for
radioimmunoassay (RIA), enzyme immunoassay (EIA), and antibody
reagents for immunohistochemistry or immunofluorescence for PTH
peptide. Bachem AG (King of Prussia, Pa.).
[0229] EPIAIRWAY.RTM. tissue membranes are cultured in phenol red
and hydrocortisone free medium (MatTek Corp., Ashland, Mass.). The
tissue membranes are cultured at 37.degree. C. for 48 hours to
allow the tissues to equilibrate. Each tissue membrane is placed in
an individual well of a 6-well plate containing 0.9 mL of serum
free medium. 100 .mu.L of the formulation (test sample or control)
is applied to the apical surface of the membrane. Triplicate or
quadruplicate samples of each test sample (mucosal
delivery-enhancing agent in combination with a biologically active
agent, PTH) and control (biologically active agent, PTH, alone) are
evaluated in each assay. At each time point (15, 30, 60 and 120
minutes) the tissue membranes are moved to new wells containing
fresh medium. The underlying 0.9 mL medium samples is harvested at
each time point and stored at 4.degree. C. for use in ELISA and
lactate dehydrogenase (LDH) assays.
[0230] The ELISA kits are typically two-step sandwich ELISAs: the
immunoreactive form of the agent being studied is first "captured"
by an antibody immobilized on a 96-well microplate and after
washing unbound material out of the wells, a "detection" antibody
is allowed to react with the bound immunoreactive agent. This
detection antibody is typically conjugated to an enzyme (most often
horseradish peroxidase) and the amount of enzyme bound to the plate
in immune complexes is then measured by assaying its activity with
a chromogenic reagent. In addition to samples of supernatant medium
collected at each of the time points in the permeation kinetics
studies, appropriately diluted samples of the formulation (i.e.,
containing the subject biologically active test agent) that was
applied to the apical surface of the units at the start of the
kinetics study are also assayed in the ELISA plate, along with a
set of manufacturer-provided standards. Each supernatant medium
sample is generally assayed in duplicate wells by ELISA (it will be
recalled that quadruplicate units are employed for each formulation
in a permeation kinetics determination, generating a total of
sixteen samples of supernatant medium collected over all four time
points).
[0231] It is not uncommon for the apparent concentrations of active
test agent in samples of supernatant medium or in diluted samples
of material applied to the apical surface of the units to lie
outside the range of concentrations of the standards after
completion of an ELISA. No concentrations of material present in
experimental samples are determined by extrapolation beyond the
concentrations of the standards; rather, samples are rediluted
appropriately to generate concentrations of the test material which
can be more accurately determined by interpolation between the
standards in a repeat ELISA.
[0232] The ELISA for a biologically active test agent, for example,
PTH, is unique in its design and recommended protocol. Unlike most
kits, the ELISA employs two monoclonal antibodies, one for capture
and another, directed towards a nonoverlapping determinant for the
biologically active test agent, e.g., PTH, as the detection
antibody (this antibody is conjugated to horseradish peroxidase).
As long as concentrations of PTH that lie below the upper limit of
the assay are present in experimental samples, the assay protocol
can be employed as per the manufacturer's instructions, which allow
for incubation of the samples on the ELISA plate with both
antibodies present simultaneously. When the PTH levels in a sample
are significantly higher than this upper limit, the levels of
immunoreactive PTH may exceed the amounts of the antibodies in the
incubation mixture, and some PTH which has no detection antibody
bound will be captured on the plate, while some PTH which has
detection antibody bound may not be captured. This leads to serious
underestimation of the PTH levels in the sample (it will appear
that the PTH levels in such a sample lie significantly below the
upper limit of the assay). To eliminate this possibility, the assay
protocol has been modified:
[0233] The diluted samples are first incubated on the ELISA plate
containing the immobilized capture antibody for one hour in the
absence of any detection antibody. After the one hour incubation,
the wells are washed free of unbound material.
[0234] The detection antibody is incubated with the plate for one
hour to permit formation of immune complexes with all captured
antigen. The concentration of detection antibody is sufficient to
react with the maximum level of PTH which has been bound by the
capture antibody. The plate is then washed again to remove any
unbound detection antibody.
[0235] The peroxidase substrate is added to the plate and incubated
for fifteen minutes to allow color development to take place.
[0236] The "stop" solution is added to the plate, and the
absorbance is read at 450 nm as well as 490 nm in the VMax
microplate spectrophotometer. The absorbance of the colored product
at 490 nm is much lower than that at 450 nm, but the absorbance at
each wavelength is still proportional to concentration of product.
The two readings ensure that the absorbance is linearly related to
the amount of bound PTH over the working range of the VMax
instrument (we routinely restrict the range from 0 to 2.5 OD,
although the instrument is reported to be accurate over a range
from 0 to 3.0 OD). The amount of PTH in the samples is determined
by interpolation between the OD values obtained for the different
standards included in the ELISA. Samples with OD readings outside
the range obtained for the standards are rediluted and run in a
repeat ELISA.
Measurement of Transepithelial Resistance by TER Assay
[0237] After the final assay time points, membranes are placed in
individual wells of a 24-well culture plate in 0.3 mL of clean
medium and the trans epithelial electrical resistance (TER) is
measured using the EVOM Epithelial Voltohmmeter and an Endohm
chamber (World Precision Instruments, Sarasota, Fla.). The top
electrode is adjusted to be close to, but not in contact with, the
top surface of the membrane. Tissues are removed, one at a time,
from their respective wells and basal surfaces are rinsed by
dipping in clean PBS. Apical surfaces were gently rinsed twice with
PBS. The tissue unit is placed in the Endohm chamber, 250 .mu.L of
PBS added to the insert, the top electrode replaced and the
resistance measured and recorded. Following measurement, the PBS is
decanted and the tissue insert is returned to the culture plate.
All TER values are reported as a function of the surface area of
the tissue.
[0238] The final numbers are calculated as:
TER of cell membrane=(Resistance (R) of Insert with membrane-R of
blank Insert).times.Area of membrane (0.6 cm.sup.2).
EXAMPLE 3
Preparation of a Parathyroid Hormone Formulation Free of a
Stabilizer that is a Protein
[0239] A parathyroid hormone formulation suitable for intranasal
administration of parathyroid hormone, which was substantially free
of a stabilizer that is a protein was prepared having the
formulation listed below.
[0240] About 3/4 of the water was added to a beaker and stirred
with a stir bar on a stir plate and the sodium citrate was added
until it was completely dissolved. The EDTA was then added and
stirred until it was completely dissolved. The citric acid was then
added and stirred until it was completely dissolved. The
methyl-.beta.-cyclodextrin was added and stirred until it was
completely dissolved. The DDPC was then added and stirred until it
was completely dissolved. The lactose was then added and stirred
until it was completely dissolved. The sorbitol was then added and
stirred until it was completely dissolved. The chlorobutanol was
then added and stirred until it was completely dissolved. The
parathyroid hormone 1-34 was added and stirred gently until it
dissolved. The pH was adjuste to 5.0.+-.0.25 by addition of HCl or
NaOH. Water was added to final volume.
EXAMPLE 4
PTH and Enhancer Effects on Human Chondrocytes
[0241] The effect of intranasal PTH.sub.1-34 formulation on human
chondrocytes was measured in vitro, specifically measuring the
effect of permeation enhancers on chondrocyte proliferation and
production of collagen in comparison to calcitonin and IGF-1.
[0242] The cell lines used were derived from human articular
cartilage. Articular chondrocytes are phenotypically very similar
to nasal chondrocytes (Shikani et al., 2004), and thus provided a
good model for the current study. The cells were provided in two
different forms, the first as a monolayer (proliferation model) and
the second as cells encapsulated in alginate beads (redifferention
model).
[0243] The cell monolayer model was employed to examine cell
proliferation. The objective was to examine cell proliferation in
the presence of PTH.sub.1-34 in a simple formulation (citrate
buffer) or a formulation containing formulation enhancers, and then
compare these data to cell proliferation for a positive control
(media containing antibiotics, insulin, TGF-beta and IGF-1) and
negative control (media devoid of any cell growth components). A
placebo solution used to make peptide-containing formulations is
described in Table 3. Peptide was added to this solution to achieve
the desired concentration. PTH.sub.1-34 and salmon calcitonin used
were from Nastech. IGF-1 was purchased from Sigma.
[0244] Chondrocyte monolayers (Cell Applications, Inc., San Diego,
Calif.) derived from normal human cartilage were adhered on to a
24-well plate and shipped following the first doubling.
Approximately 16000 cells per well were expected at the time the
cells were received. Two plates were treated identically with
PTH.sub.1-34 and appropriate control samples. Controls included
cells treated with chondrocyte growth media for positive control
(Cell Applications) and cells treated with basal media for negative
control (Cell Applications).
[0245] Once treated, one of the two plates was analyzed for cell
viability (t=0 sample). The second plate was placed at 37.degree.
C., 5% CO.sub.2 incubator for 4 days, after which the plates were
analyzed using the MTT assay.
[0246] For MTT analysis (Cell Applications), a volume of 100 .mu.L
MTT concentrate solution was added to each well containing 1000
.mu.L sample. Plates were sealed and placed at 37.degree. C. for 4
hours. Each plate was removed from 37.degree. C. after 4 h
incubation and placed on a bench top. Supernatant from each well
was carefully removed and discarded. Visible purple crystals were
seen attached to the bottom of the plated in each well. A volume of
500 .mu.L of extraction solution was then added into each well.
Plates were sealed with parafilm immediately after adding
extraction solution, and then gently rocked and/or swirled to
solubilize the purple crystals. Absorbance was read at 570 nm.
[0247] Approximately 3 million human chondrocyte cells (Cell
Applications) encapsulated in alginate beads were received in 25 mL
total volume of re-differentiated medium. Human chondrocytes in
alginate beads produce their phenotypic markers such as aggrecan
and Type II collagen (Benya et al., 1982; Guo et al., 1982; Kato et
al., 1984) unlike in monolayer culture where chondrocytes lose
their phenotypic characteristics and de-differentiate to
fibroblast-like cells (Kuettmer et al., 1982; Jennings et al.,
1983; Kato et al., 1984). Alginate based cell system was pursued to
assess Type II collagen expression as result of PTH.sub.1-34 dosing
and therefore indication of any cartilage growth.
[0248] The cell-containing alginate bead suspension was transferred
from cell culture flask to 50 mL conical tubes. A volume of 500
.mu.L (.about.62,500 cells per well) suspension was dispensed per
well in 24-well plates. Samples were added to each well and final
volume was brought to one milliliter with appropriate growth media.
Controls included cells treated with re-differentiated media as
positive control (Cell Applications), cells treated with
chondrocyte growth media (Cell Applications) and basal media (Cell
Applications) as negative controls.
[0249] Plates were swirled gently to allow homogenous mixture of
each sample. Plates were placed in 37.degree. C., 5% CO.sub.2
incubator. Human chondrocytes were grown for up to 12 days on
longer. Appropriate growth media were replaced every second or
third day. Alginate beads were prepared for Type II collagen
extraction on the last day of the experiment and extracted collagen
was quantitated using Native Type II Collagen Detection Kit
(Chondrex Inc., Redmond Wash.). A capture ELISA kit (Chondrex) was
used to measure native type II collagen. Glycosaminoglycans were
measured by a kit (Accurate Chemical & Scientific Corp.).
[0250] The MTT assay was employed to test for cell proliferation.
The MTT assay measures cell viability, and an increase or decrease
in the MTT assay values reflect an increase or decrease in the
population of viable cells. To test for stimulation of the growth
of chondrocytes, various test solutions containing PTH.sub.1-34
were applied to the apical side of the chondrocyte monolayers, and
the MTT assay was conducted at the beginning and then after 4 days
incubation at 37 .degree. C./5% CO.sub.2. The results showed that
PTH.sub.1-34 did not stimulate the growth of chondrocytes whether
formulated as a simple solution or in the presence of permeation
enhancers.
[0251] A cartilage growth model was examined, in which cells were
provided in a form where they were encapsulated in alginate beads.
In this form, human chondrocytes exhibit their phenotypic markers
such as aggrecan and Type II collagen (Benya et al., 1982; Guo et
al., 1982; Kato et al., 1984) unlike in monolayer culture where
chondrocytes lose their phenotypic characteristics and
de-differentiate to fibroblast-like cells (Kuettmer et al., 1982;
Jennings et al., 1983; Kato et al., 1984). This alginate-based cell
system was used to assess effect of PTH.sub.1-34 dosing on Type II
collagen expression as an indication of cartilage growth.
[0252] PTH.sub.1-34 was tested for its ability to stimulate
chondrocytes to produce cartilage. Cell-containing alginate beads
were incubated in the presence of various test solutions for 12
days at 37.degree. C., 5% CO.sub.2. After the incubation, the
alginate beads were processed using an extraction method in order
to quantitate the production of Type II collagen (a major component
of extracellular matrix of nasal cartilage (Shikani et al.,
2004)).
[0253] Production of type II collagen was measured in positive
(re-differentiation media) and negative (growth media) controls as
well as the effect of exposing the cells to either 20 or 200 .mu.g
PTH.sub.1-34. Low levels of type II collagen were produced in the
presence of the re-differentiation media but not in the growth
media. Application of 20 mg of PTH.sub.1-34 did not cause
production of type II collagen from the chondrocytes, either in
citrate buffer or with permeation enhancers. When 200 mg of
PTH.sub.1-34 was applied to the cell culture in a simple
formulation, the production of type II collagen was increased.
Surprisingly, when the same amount of peptide was given in the
formulation containing permeation enhancers, type II collagen was
not produced by the cells. As a control, the cell system was
validated in tests showing that production of type II collagen was
increased by culturing with 5 mg IGF-I.
[0254] These results are consistent with a role of permeation
enhancers such as methyl-b-cyclodextrin and/or DDPC in blocking the
local activity of the PTH.sub.1-34.
EXAMPLE 5
PK Study of PTH.sub.1-34 in Rabbits
[0255] The objective of this study was to determine the plasma
pharmacokinetics and relative intranasal and subcutaneous
bioavailability of PTH.sub.1-34 following intranasal or
subcutaneous dose administration to rabbits. In addition, the
absorption profile of subcutaneous recombinant and synthetic
PTH.sub.1-34was evaluated.
[0256] This was a randomized, single treatment parallel study in
four groups of four animals per group. Following dose
administration, serial blood samples were obtained from each animal
by direct venipuncture of a marginal ear vein. Serial blood samples
(about 2 mL each) were collected by direct venipuncture from a
marginal ear vein into blood collection tubes containing EDTA as
the anticoagulant. After collection of the blood, the tubes were
gently rocked several times immediately for ani-coagulation.
Aprotinin at 100 .mu.L was added to the collection tubes. Blood
samples were collected at 0, 5, 10, 20, 30, 45, 60, 120 and 240
minutes post-dosing. Clinical observations were observed at least
once daily and at all times of blood sampling.
[0257] Doses were based on the most recently recorded body weight.
For intranasal and subcutaneous administration animals were dosed
at the volume of 50 .mu.l/kg and 5 .mu.l/kg, respectively. The dose
multiples were based on a nasal surface measurements of the rabbit
and human. The nasal surface dose multiples of the rabbit compared
to the human is approximately 2 fold in this study based on a
unilateral nasal surface area of 30 cm.sup.2 and 80 cm.sup.2 for
rabbit and humans, respectively. The dosing groups are presented in
Table 4.
[0258] The nasal and subcutaneous formulations for Group 1, 2, 3
and 4 were according to the methods described above. An enzyme
immunoassay was developed to measure the concentration of Human
PTH.sub.1-34 in rabbit serum. Samples were collected with protease
inhibitor (aprotinin) and frozen. Samples, Standards, and Quality
Control samples are assayed using a modified Human Bioactive
PTH.sub.1-34 ELISA kit. Standards, Samples, and Quality Control
samples are added to streptavidin coated strip wells in duplicate.
These samples are then incubated with a mixture of biotinylated
human PTH.sub.1-34 antibody and HRP-conjugated human PTH.sub.1-34
antibody. The plate is washed with the kit Wash solution and TMB
substrate solution is added to each well. Color is allowed to
develop for 10 minutes before solution is added to each well. OD is
measured on an absorbance plate reader. Concentration is calculated
by interpolation of a standard curve and assay performance is
controlled with Quality Control samples. Pharmacokinetic
calculations were performed using WinNonLin software (Pharsight
Corporation, Version 4.0, Mountain View, Calif.) using a
non-compartmental model.
[0259] Group 1 and 2 were dosed at 50 .mu.g/kg by the intranasal
route using synthetic PTH from Bachem as the active ingredient.
Group 1 was the marketed FORTEO.RTM. formulation and Group 2 was
Nastech's formulation. Group 3 and 4 were dosed at 5 .mu.g/kg by
the subcutaneous route using recombinant and synthetic PTH,
respectively. The excipients used for the subcutaneous route were
the same as FORTEO.RTM. marketed product for both groups.
[0260] The mean C.sub.max was 1,921.13, 2,559.28, 1,538.10 and
2,526.43 pg/mL for Group 1, 2, 3 and 4, respectively. The mean
T.sub.max was 35, 20, 20 and 10 minutes for group 1, 2, 3 and 4,
respectively. The C.sub.max for group 2 was 1.3, 1.7 fold greater
and approximately the same compared to groups 1, 3, and 4,
respectively. However, the dose for group 2 was 10 fold higher than
group 3 and 4. The relative bioavailability for Group 2 corrected
for dose was 16.6 and 10.0% compared to groups 3 and 4 for
C.sub.max, respectively. The C.sub.max for Group 2 was 1.3 fold
higher than Group 1. The relative bioavailability comparing the
subcutaneous routes C.sub.max was 61% for group 3 versus group
4.
[0261] The mean AUC.sub.max was 111,850.81, 123,498.63, 173,992.88
and 194,895.25 min*pg/mL for group 1, 2, 3, and 4, respectively.
The mean AUC.sub.inf was 118,022.48, 130,377.44, 177,755.35 and
206,317.05 for group 1, 2, 3 and 4 respectively. The relative
bioavailability for Group 2 corrected for dose was 7.1 and 6.3%
compared to groups 3 and 4 for AUC.sub.last, respectively. The
relative bioavailability for Group 2 corrected for dose was 7.3 and
6.3% compared to groups 3 and 4 for AUC.sub.inf, respectively. The
AUC.sub.last and infinity for Group 2 was 1.1 fold higher than
Group 1. The relative bioavailability comparing the subcutaneous
routes AUC.sub.last was 89.3% for group 3 versus group 4. The
relative bioavailability comparing the subcutaneous routes
AUC.sub.inf was 86.2% for group 3 versus group 4.
[0262] Based on the human pharmacokinetic data that tested the
approved subcutaneous FORTEO.RTM. at a dose of 20 .mu.g, the human
dose systemic exposure multiple for Group 2 are approximately 20,
8.4 and 7.4 fold for C.sub.max, AUC.sub.last, and AUC.sub.inf,
respectively. The nasal exposure in the study is approximately 2
fold based on the mean rabbit weight of 2.5 kg and nasal surface
area of 30 cm.sup.2 and 80 cm.sup.2 for rabbits and humans,
respectively.
[0263] The t.sub.1/2 of PTH.sub.1-34 ranges from approximately
43-55 minutes for all groups. The mean t.sub.max was 35, 20, 20 and
10 minutes for groups 1, 2, 3, and 4, respectively. Kel was 0.018,
0.017, 0.016 and 0.014 for groups 1, 2, 3, and 4, respectively. No
adverse clinical signs were observed following dosing of any
formulation.
[0264] On a C.sub.max and AUC basis the relative bioavailability of
formulation of the invention was approximately 10%. Other
intranasal studies conducted in rabbits with PYY.sub.3-36 as the
active with a similar formulation matrix also had an approximate
19% bioavailability and when tested in humans resulted in a similar
bioavailability. Therefore, human intranasal dose is 200 .mu.g in
contrast to the FORTEO.RTM. subcutaneous dose of 20 .mu.g.
[0265] Even though formulation of the invention had a higher
C.sub.max, AUC and a faster T.sub.max, Group 1 had an unexpected
high absorption rate as compared to Group 2, 3 and 4. consistent
with in-vitro studies.
[0266] Synthetic PTH.sub.1-34 has a higher absorption rate than the
recombinant PTH.sub.1-34 given by the subcutaneous route. The
levels reached in this study with formulation of the invention are
approximately 20 and 8.4 fold for C.sub.max and AUC.sub.last based
on a human dose of 20 .mu.g. Based on a nasal surface area basis,
the doses tested in this study are approximately 2 fold. Therefore,
the doses chosen in the 14 day toxicity studies of 50 and 250 .mu.g
give an appropriate comparison to the human dose for nasal and
systemic exposure.
EXAMPLE 6
PK Study of PTH.sub.1-34
[0267] This study was conducted to determine the pharmacokinetic
profile of teriparatide (human parathyroid hormone 1-34
(hPTH.sub.1-34)) in selected formulations following intranasal
administration in the rabbit.
[0268] The overall study design is provided in Table 5. The dose
level was selected based on previous studies with teriparatide
dosed via intranasal instillation.
[0269] Each animal was dosed by intranasal instillation into the
left nare. Blood samples were taken from the marginal ear vein,
pre-dose and 5, 10, 20, 30, 45 minutes and 1 (60 minutes), 2 (120
minutes) and 4 hours (240 minutes) post-dose. Blood sampling and
handling were conducted per protocol, with no deviations that were
considered to impact sample quality. Blood samples were collected
with a protease inhibitor (Aprotinin), processed for harvest of
serum and plasma, and frozen and stored at -70.degree. C. until
analyzed.
[0270] Five nasal formulations of teriparatide were evaluated in
the study. The vehicle composition for each formulation is provided
in Table 6. The nasal formulations of the invention were
manufactured at Nastech Pharmaceutical Company Inc. (Bothell,
Wash.).
[0271] An enzyme immunoassay was developed (serum and plasma) and
validated (serum) to measure the concentration of human
PTH.sub.1-34 in blood samples from rabbit. Study samples,
Standards, and Quality Control samples were assayed using a
modified Human Bioactive PTH.sub.1-34 ELISA kit. Standards,
Samples, and Quality Control samples were added to streptavidin
coated strip wells, with each sample analyzed in duplicate. Samples
were incubated with a mixture of biotinylated anti-human
PTH.sub.1-34 antibody and HRP-conjugated anti-human PTH.sub.1-34
antibody. The plate was washed with the kit wash solution and TMB
substrate solution was added to each well. Color was allowed to
develop for 10 minutes before the reaction was stopped by the
addition of 1 M sulfuric acid to each well. The OD.sub.450 for each
well was measured on an absorbance plate reader, and the
concentration was calculated by interpolation from the standard
curve. Assay performance was monitored with Quality Control
samples. The assay Lower Limit of Quantification (LLOQ) was
determined to be 7.8 pg/mL using human PTH.sub.1-34 as the standard
analyte and normal rabbit serum or plasma.
[0272] Due to species similarity between rabbit and human
parathyroid hormones, it was anticipated the assay would detect
endogenous (i.e., rabbit) parathyroid hormone and
rabbit-PTH.sub.1-34. Pharmacokinetic calculations as described
above.
[0273] Pre-dose concentrations of PTH (assumed to represent rabbit
parathyroid hormone or its fragments) in serum or plasma were
generally below 100 pg/mL, with several samples determined to be
<62.4 pg/mL. Limitations on sample volume precluded repeated
analysis to obtain definitive results or the assay LLOQ. The value
provided is the lowest value (or estimated value) obtained at which
no further analysis was possible.
[0274] Individual animal serum and plasma values for teriparatide
exceeded 100 pg/mL at the 5 through 60 minute time points. At the
final time points, particularly 240 minutes, the majority of the
animals had teriparatide concentrations of <31.2 pg/mL (the last
point at which sample volume was insufficient for further
evaluation). Thus the absorption and elimination phases for
teriparatide were captured by the sampling time frame employed for
the study.
Formulation 1/Group 1
[0275] The C.sub.max, t.sub.1/2, and AUC.sub.last results for serum
and plasma of the Group 1 animals greatly exceed the expected
values based on dose level. Pre-dose values for each of the four
animals in Group 1 were within the expected range, indicating the
animals endogenous levels were not a factor in this observation.
Animals #931 had teriparatide values in serum and plasma at the 5-
and 10-minute that that were within the expected range. The
5-minute time point for Animal #932 was within the expected range,
while at 10-minutes post-dose the assay value for plasma was
>250,000 pg/mL. Subsequent time points for both animals were
found to have concentrations of >30,000 pg/mL in serum or
plasma. For Animals #933 and #934, all post-dose time points for
serum and plasma were found to have teriparatide concentrations
that were >30,000 pg/mL. As a result, C.sub.max was >45,000
pg/mL and the t.sub.1/2 was >300 minutes for the majority of
animals. Mean AUC.sub.last was >7,000,000 min*pg/mL.
[0276] The mean T.sub.max of 29 and 20 minutes for serum and
plasma, respectively, were consistent with the expected range for
intranasal dosing. However, the measured C.sub.max of 30,000 to
almost 50,000 pg/mL were an order of magnitude high than the
expected maximal concentrations of 3000 to 5000 pg/mL.
Formulation 2/Group 2
[0277] In serum, the group mean C.sub.max and T.sub.max were 4396
pg/mL and 36 minutes, respectively. The group mean t.sub.1/2 was
70.7 minutes, however, this was influenced by Animal #935 in which
the calculated t.sub.1/2 was 198 minutes; the t.sub.1/2 for the
other three animals was 37.7, 22.7, and 24.6 minutes. The mean
AUC.sub.last was 307213 min*pg/mL. The mean C.sub.max and
AUC.sub.last in plasma were 3819 pg/mL and 105144 min*pg/mL,
respectively. T.sub.max was estimated to be 9 minutes and the
t.sub.1/2 was 66.9 minutes.
Formulation 3/Group 3
[0278] C.sub.max, T.sub.max, t.sub.1/2, and AUC.sub.last in serum
were 1224 pg/mL, 9 minutes, 53 minutes, an 35111 min*pg/mL,
respectively. In plasma, C.sub.max, T.sub.max, .sup.and
AUC.sub.last were 1102 pg/mL, 8 minutes, and 53283 min*pg/mL,
respectively. The mean t.sub.1/2 was estimated to be 99.7 mutes,
however, this was attributable to a long t.sub.1/2 (216.6 minutes)
for Animal #939.
Formulation 4/Group 4
[0279] In serum, C.sub.max, T.sub.max, t.sub.1/2, and AUC.sub.last
were 653 pg/mL, 35 minutes, 22.3 minutes, and 30807 min*pg/mL,
respectively. In plasma, these same parameters were 915 pg/mL, 35
minutes, 73.9 minutes, and 56383 min*pg/mL, respectively.
Formulation 5/Group 5
[0280] In serum, C.sub.max, T.sub.max, t.sub.1/2, and AUClast were
549 pg/mL, 45 minutes, 57.6 minutes, and 41489 min*pg/mL,
respectively. In plasma, these same parameters in serum, and 734
pg/mL, 41 minutes, 48.8 minutes, and 23710.9 min*pg/mL,
respectively.
[0281] The post-dose data for Group 1 demonstrate the unexpected
high concentrations and absence of a clear elimination phase. This
would be consistent with a contamination of a common solution in
the collection procedure, and concentrations that are independent
of dose administration.
[0282] For Groups 3 and 5 the mean concentration of teriparatide at
each time point post-dose was similar between serum and plasma. The
shape of the concentration vs. time curves was also similar between
serum and plasma. In Group 2, peak concentrations of teriparatide
were similar is serum and plasma, but serum was noted with higher
concentrations at the latter time points. For Group 4, plasma
concentrations of teriparatide were higher at the latter time
points, as compared to serum; similar concentrations of were found
in serum and plasma at the early time points after dosing. Overall,
these results suggest that either plasma or serum may be an
appropriate matrix for the determination of teriparatide
concentrations after dose administration.
[0283] The data for Formulation 1/Group 1 was intended to be the
set used for calculation of the comparative bioavailability for the
other formulation, however, with consideration of the data obtained
for this group any comparison was considered not to be appropriate.
As an alternative, data from previous studies, above, were compiled
to obtain a value for C.sub.max and AUC.sub.last. Although the
formulation components for these studies were slightly different,
the primary components--methyl-.beta. cyclodextrin,
phosphatidylcholine didecanoyl, edetate disodium, and sodium
benzoate--were present at similar concentrations to Formulation
1.
[0284] Using the compiled value for C.sub.max and AUC.sub.last, the
comparative bioavailability of teriparatide for each intranasal
formulation/group was calculated. The C.sub.max and AUC.sub.last
for Formulation 2/Group 2 were approximately equal to (serum) or
slightly greater (plasma) than the compiled values, indicating
comparable bioavailability. The primary difference for Formulation
2 was the concentration of phosphatidylcholine didecanoyl was
decreased to 0.1 mg/mL (from 1.0 mg/mL).
[0285] Formulation 3 was dosed at half the dose volume, but
contained 6.6 mg/mL teriparatide to achieve the same total dose.
The concentration of methyl-.beta. cyclodextrin,
phosphatidylcholine didecanoyl, and edetate disodium were
increased; sodium benzoate remained at the same concentration. The
comparative bioavailability for Formulation 3 was approximately 40%
for both serum and plasma.
[0286] Formulation 4 contains the components at the concentrations
listed for FORTEO.RTM., a subcutaneously delivered form of
teriparatide.
[0287] Formulation 5 was this formulation with m-cresol removed.
Relative bioavailability for Group 4 (Formulation 4) and Group 5
(Formulation 5) was approximately 30% -35% for serum or plasma. The
data suggest that the presence of m-cresol did not have a
significant impact of teriparatide bioavailability.
[0288] These results show that the concentration of
phosphatidylcholine didecanoyl can be decreased from 1.0 mg/mL to
0.1 mg/mL without significantly decreasing bioavailability.
Increasing the concentration of teriparatide and the absorption
enhancers had an apparent negative impact upon the bioavailability.
The cause for this drop may be related to reduction in surface area
of contact with the nasal mucosa due to reduced volume. In the
absence of absorption enhancers, the relative bioavailability was
further decreased. This decreased bioavailability was expected due
to the absence of any specific permeation enhancers, and is also
consistent with in vitro permeation data. Subcutaneous injection is
the route of administration most often used for nonclinical studies
with teriparatide, and is the only route approved for clinical use.
For Group 2, a relative bioavailability in the range of 5 to 17% is
consistent previous estimate of 6-10%. Increasing the concentration
of teriparatide and the absorption enhancers (Group 3) decreased
the relative bioavailability to 2-5%. In the absence of absorption
enhancers (groups 4 and 5) the relative bioavailability for
teriparatide was approximately 2-3%.
EXAMPLE 7
PK Study of PTH.sub.1-34 in Humans
[0289] The primary objectives of this study are to: evaluate the
absorption of three formulations of the invention in a nasal spray
at two dose levels; evaluate the safety of these formulations at
two dose levels and to compare the bioavailability of Forsteo given
subcutaneously with these formulations at two dose levels.
[0290] This is a phase I, crossover, dose ranging study involving 6
healthy male and 6 healthy female volunteers. There are five study
periods as follows:
[0291] Period 1: All 12 subjects receive Forsteo 20 .mu.g
subcutaneously.
[0292] Period 2: 6 (3 male and 3 female) subjects receive 500 .mu.g
of Formulation #2 and 6 (3 male and 3 female) subjects receive 500
.mu.g of Formulation #3. The results of a total calcium level drawn
four hours after dosing are used to determine the doses of these
Formulations to be administered during Periods 4 and 5 as
follows:
[0293] If none of the six subjects who receive a given Formulation
have a total calcium level.gtoreq.3 mmol/L (12.0 mg/dL) at four
hours post dose, then all doses of that Formulation in periods 4 or
5 are at 1000 .mu.g.
[0294] If a single subject of the six who receive a given
Formulation has a total calcium level.gtoreq.3 mmol/L (12.0 mg/dL)
at four hours post dose, that subject receive only a 500 .mu.g dose
of Formulations 2 and 3 during Periods 4 and 5.
[0295] If 2 or more subjects of the six who receive a given
Formulation have a total calcium level.gtoreq.3 mmol/L (12.0 mg/dL)
at four hours post dose, all subjects receive a 500 .mu.g dose of
that Formulation during Periods 4 or 5.
[0296] Period 3: All subjects receive 1000 .mu.g of Formulation
#1.
[0297] Period 4: All subjects receive a dose of Formulation #2 at
either 500 .mu.g or 1000 .mu.g as described above.
[0298] Period 5: All subjects receive a dose of Formulation #3 at
either 500 .mu.g or 1000 .mu.g as described above.
[0299] Subjects are confined to the study center from Day-1 until
all study activities have been completed on Dosing Day #5. Safety
assessments include vital signs, clinical laboratory evaluations,
nasal tolerance and adverse events.
[0300] Blood samples for PK analysis of teriparatide levels are
collected via an indwelling catheter and/or via direct
venipuncture. These samples will be collected at 0 (i.e.,
pre-first-dose), 5, 10, 15, 30, 45, 60, 90 minutes and 2, 3, and 4
hours post-dose.
[0301] At each time point, 7 mL of blood will be collected. Plasma
concentrations of teriparatide are determined using a validated
analytical procedure.
[0302] Although the foregoing invention has been described in
detail by way of example for purposes of clarity of understanding,
it will be apparent to the artisan that certain changes and
modifications are comprehended by the disclosure and may be
practiced without undue experimentation within the scope of the
appended claims, which are presented by way of illustration not
limitation.
TABLE-US-00003 TABLE 1 PTH Formulation Composition Formula-
PTH.sub.1-34 tions Per 100 ml Sample Mucosal Delivery Enhancing
Agent A 60 .mu.g Phosphate-buffered saline (0.8%) pH 7.4 (Control
1) B 60 .mu.g Phosphate-buffered saline (0.8%) pH 5.0 (Control 2) C
60 .mu.g L-Arginine (10% w/v) D 60 .mu.g Poly-L-Arginine (0.5% w/v)
E 60 .mu.g Gamma-Cyclodextrin (1% w/v) F 60 .mu.g
.alpha.-Cyclodextrin (5% w/v) G 60 .mu.g Methyl-.beta.-Cyclodextrin
(3% w/v) H 60 .mu.g n-Capric Acid Sodium (0.075% w/v) I 60 .mu.g
Chitosan (0.5% w/v) J 60 .mu.g L-.alpha.-phosphatidilcholine
didecanyl (3.5% w/v) K 60 .mu.g S-Nitroso-N-Acetyl-Penicillamine
(0.5% w/v) L 60 .mu.g Palmotoyl-DL-Carnitine (0.02% w/v) M 60 .mu.g
Pluronic-127 (0.3% w/v) N 60 .mu.g Sodium Nitroprusside (0.3% w/v)
O 60 .mu.g Sodium Glycocholate (1% w/v) P 60 .mu.g F1: Gelatin,
DDPC, MBCD, EDTA F 1 L-.alpha.-phosphatidilcholine didecanyl (0.5%
w/v) Methyl .beta. Cyclodextrin (3% w/v) EDTA (0.1% w/v, Inf. Conc.
0.5 M) Gelatin (0.5% w/v)
TABLE-US-00004 TABLE 2 Theoretical Weight Ingredient Name g/100 mL
(Grams) PTH.sub.1-34, GMP grade 0.022 0.0066 Chlorobutanol, NF
(anhydrous) 0.50 1.25 Methyl-.beta.-Cyclodextrin 4.50 11.25
L-.alpha.-Phosphatidylcholine Didecanoyl 0.10 0.25 Edetate
Disodium, USP 0.10 0.25 Sodium Citrate dihydrate, USP 0.1800 0.45
Citric acid anhydrous, USP 0.0745 0.1863 Lactose monohydrate, NF
0.90 2.25 Sorbitol, NF 1.82 4.55 Hydrochloric acid, NF TAP* TAP
Sodium Hydroxide, USP TAP TAP Sterile Water for Irrigation, USP
94.03** 235.075
TABLE-US-00005 TABLE 3 Formula for PTH1-34 Nasal Spray solution
Theoretical Weight Ingredient Name g/100 mL (Grams) Chlorobutanol,
NF (anhydrous) 0.50 1.25 Methyl-.beta.-Cyclodextrin 4.50 11.25
L-.alpha.-Phosphatidylcholine Didecanoyl 0.10 0.25 Edetate
Disodium, USP 0.10 0.25 Sodium Citrate dehydrate, USP 0.18 0.45
Citric acid anhydrous, USP 0.0745 0.1863 Lactose monohydrate, NF
0.90 2.25 Sorbitol, NF 1.82 4.55 Hydrochloric acid, NF TAP TAP
Sodium hydroxide, USP TAP TAP Sterile Water for Irrigation, USP
94.03* 235.075 TAP = To adjust pH. *Using experimentally determined
diluent density of 1.022 g/mL
TABLE-US-00006 TABLE 4 Dosing Groups for Toxicokinetic Animals
Route of Dose Dose Dose Administration Conc Vol Level Group Animals
Formulation (mg/mL) (mL/kg) (.mu.g/kg) 1 4M Intranasal 3.33 0.015
50 (Bachem PTH Marketed Formulation) 2 4M Intranasal 3.33 0.015 50
(Bachem PTH Nastech Formulation) 3 4M Subcutaneous (Sub-Q) 0.025
0.2 5 (Marketed Product) 4 4M Subcutaneous (Sub-Q) 0.025 0.2 5
(Bachem PTH Marketed Formulation)
TABLE-US-00007 TABLE 5 Dosing Groups for Pharmacokinetic Evaluation
Route of Teriparatide Dose Teriparatide Study Ani- Administration
Concentration Volume Dose Level Groups mals (Formulation) (mg/mL)
(mL/kg) (.mu.g/kg) 1 4M Intranasal 3.33 0.015 50 (Formulation 1) 2
4M Intranasal 3.33 0.015 50 (Formulation 2) 3 4M Intranasal 6.6
0.0075 50 (Formulation 3) 4 4M Intranasal 3.33 0.015 50
(Formulation 4) 5 4M Intranasal 3.33 0.015 50 (Formulation 5)
TABLE-US-00008 TABLE 6 Vehicle Composition for Formulations 1-5
Component mg/mL (% w/w) Formulation 1 Methyl-.beta. Cyclodextrin 45
(4.5) Phosphatidylcholine didecanoyl (DDPC) 1.0 (0.1) Edetate
Disodium, USP 1.0 (0.1) Sodium Benzoate, NF 4.0 (0.4) Sodium
Hydroxide, USP TAP Hydrochloric Acid, NF TAP Sterile Water for
Irrigation, USP q.s pH = 4.5 Formulation 2 Methyl-.beta.
Cyclodextrin 45 (4.5) Phosphatidylcholine didecanoyl (DDPC) 0.1
(0.01) Edetate Disodium, USP 1.0 (0.1) Sodium Benzoate, NF 4.0
(0.4) Sodium Hydroxide, USP TAP Hydrochloric Acid, NF TAP Sterile
Water for Irrigation, USP q.s pH = 4.5 Formulation 3 Methyl-.beta.
Cyclodextrin 90 (9.0) Phosphatidylcholine didecanoyl (DDPC) 2.0
(0.2) Edetate Disodium, USP 2.0 (0.2 Sodium Benzoate, NF 4.0 (0.4)
Sodium Hydroxide, USP TAP Hydrochloric Acid, NF TAP Sterile Water
for Irrigation, USP q.s pH = 4.5 Formulation 4 Mannitol, USP 30.8
(3.08) M-Cresol 3.0 (0.3) Glacial Acetic Acid, USP 0.42 (0.042)
Sodium Acetate, Anhydrous, USP 0.1 (0.01) Sodium Hydroxide, USP TAP
Hydrochloric Acid, NF TAP Sterile Water for Irrigation, USP q.s pH
= 4.0 Formulation 5 Mannitol, USP 30.8 (3.08) Glacial Acetic Acid,
USP 0.42 (0.042) Sodium Acetate, Anhydrous, USP 0.1 (0.01) Sodium
Hydroxide, USP TAP Hydrochloric Acid, NF TAP Sterile Water for
Irrigation, USP q.s pH = 4.0 TAP = added to adjust final pH
Sequence CWU 1
1
3184PRTHomo sapiens 1Ser Val Ser Glu Ile Gln Leu Met His Asn Leu
Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg Lys
Lys Leu Gln Asp Val His 20 25 30Asn Phe Val Ala Leu Gly Ala Pro Leu
Ala Pro Arg Asp Ala Gly Ser 35 40 45Gln Arg Pro Arg Lys Lys Glu Asp
Asn Val Leu Val Glu Ser His Glu 50 55 60Lys Ser Leu Gly Glu Ala Asp
Lys Ala Asn Val Asp Val Leu Thr Lys65 70 75 80Ala Lys Ser
Gln234PRTHomo sapiens 2Ser Val Ser Glu Ile Gln Leu Met His Asn Leu
Gly Lys His Leu Asn1 5 10 15Ser Met Glu Arg Val Glu Trp Leu Arg Lys
Lys Leu Gln Asp Val His 20 25 30Asn Phe331PRTHomo sapiens 3Ser Val
Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn1 5 10 15Ser
Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val 20 25
30
* * * * *
References