U.S. patent application number 09/733168 was filed with the patent office on 2001-11-22 for method for administering a cytokine to the central nervous system and the lymphatic system.
This patent application is currently assigned to Chiron Corporation. Invention is credited to Frey, William H. II.
Application Number | 20010043915 09/733168 |
Document ID | / |
Family ID | 22742853 |
Filed Date | 2001-11-22 |
United States Patent
Application |
20010043915 |
Kind Code |
A1 |
Frey, William H. II |
November 22, 2001 |
Method for administering a cytokine to the central nervous system
and the lymphatic system
Abstract
The present invention is directed to a method for delivering
cytokines to the central nervous system and the lymphatic system by
way of a tissue innervated by the trigeminal nerve and/or olfactory
nerve. Cytokines include tumor necrosis factors, interleukins,
interferons, particularly interferon-.beta. and its muteins such as
IFN-.beta..sub.ser17. Such a method of delivery can be useful in
the treatment of central nervous system disorders, brain disorders,
proliferative, viral, and/or autoimmune disorders such as Sjogren's
disorder.
Inventors: |
Frey, William H. II; (North
Oaks, MN) |
Correspondence
Address: |
Joseph H. Guth, Esq.
Corporate Patent Counsel
CHIRON CORPORATION
P.O. Box 8097
Emeryville
CA
94662-8097
US
|
Assignee: |
Chiron Corporation
|
Family ID: |
22742853 |
Appl. No.: |
09/733168 |
Filed: |
December 8, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60200708 |
Dec 9, 1999 |
|
|
|
Current U.S.
Class: |
424/85.5 ;
424/43; 424/85.1 |
Current CPC
Class: |
A61P 31/18 20180101;
A61P 25/28 20180101; A61P 35/00 20180101; A61K 38/217 20130101;
A61P 37/02 20180101; A61K 38/212 20130101; A61K 38/215 20130101;
A61P 31/12 20180101; A61P 25/00 20180101; A61P 29/00 20180101 |
Class at
Publication: |
424/85.5 ;
424/85.1; 424/43 |
International
Class: |
A61K 038/21; A61K
038/19 |
Claims
That which is claimed:
1. A method for transporting a cytokine to a central nervous system
of a mammal, comprising: administering a composition comprising the
cytokine to a tissue of the mammal innervated by the trigeminal
nerve, the olfactory nerve, or a combination thereof, wherein the
cytokine is absorbed through the tissue and transported to the
central nervous system of the mammal.
2. The method of claim 1, wherein the tissue comprises a nasal
cavity tissue, a conjunctiva, an oral tissue, or a skin.
3. The method of claim 2, wherein administering the cytokine to the
conjunctiva comprises administering the cytokine between a lower
eyelid and an eye.
4. The method of claim 2, wherein administering the cytokine to the
skin comprises administering the cytokine to a face, a forehead, an
upper eyelid, a lower eyelid, a dorsum of the nose, a side of the
nose, an upper lip, a cheek, a chin, a scalp, or a combination
thereof.
5. The method of claim 2, wherein administering the cytokine to the
oral tissue comprises sublingual administration.
6. The method of claim 1, wherein said cytokine is selected from
the group consisting of interferon-alpha (IFN-.alpha.),
interferon-beta (IFN-.beta.), interferon-gamma (IFN-.gamma.), and
biologically active variants thereof.
7. The method of claim 6, wherein the IFN-.beta. is human
IFN-.beta. or a biologically active variant thereof.
8. The method of claim 7, wherein the IFN-.beta. is biologically
active and comprises an amino acid sequence having at least 70%
sequence identity to human IFN-.beta..
9. The method of claim 1, wherein the cytokine is administered to
an upper one third of a nasal cavity.
10. The method of claim 1, wherein the cytokine is transported to a
cerebellum, a superior colliculus, a periventricular white matter,
an optic nerve, a midbrain, a pons, an olfactory bulb, an anterior
olfactory nucleus, or any combination thereof.
11. The method of claim 1, wherein the cytokine is transported to a
spinal cord, a brain stem, a cortical structure, a subcortical
structure, or any combination thereof.
12. The method of claim 1, wherein the cytokine is administered in
a dosage range of about 0.14 nmol/kg of brain weight to about 138
nmol/kg of brain weight.
13. The method of claim 12, wherein the cytokine is human
IFN-.beta. or a biologically active variant thereof.
14. A method for administering a cytokine to a central nervous
system of a mammal, comprising: administering a composition
comprising an effective amount of the cytokine to a tissue of the
mammal innervated by the trigeminal nerve, the olfactory nerve, or
a combination thereof, wherein the cytokine is absorbed through the
tissue and transported into the central nervous system of the
mammal in an amount effective to provide a diagnostic, protective,
or therapeutic effect on a cell of the central nervous system.
15. The method of claim 14, wherein the tissue comprises a tissue
of a nasal cavity, a conjunctiva, an oral tissue, or a skin.
16. The method of claim 15, wherein administering the cytokine to
the conjunctiva comprises administering the cytokine between a
lower eyelid and an eye.
17. The method of claim 15, wherein administering the cytokine to
the skin comprises administering the cytokine to a face, a
forehead, an upper eyelid, a lower eyelid, a dorsum of the nose, a
side of the nose, an upper lip, a cheek, a chin, a scalp, or a
combination thereof.
18. The method of claim 15, wherein administering the cytokine to
the oral tissue comprises sublingual administration.
19. The method of claim 14, wherein the cytokine is transported to
lymphatics associated with the central nervous system.
20. The method of claim 14, wherein said cytokine is selected from
the group consisting of IFN-.alpha., IFN-.beta., IFN-.gamma., and
biologically active variants thereof.
21. The method of claim 20, wherein said IFN-.beta. is human
IFN-.beta. or a biologically active variant thereof.
22. The method of claim 21, wherein said IFN-.beta. or variant
thereof retains biological activity and comprises an amino acid
sequence having at least 70% sequence identity to the sequence of
human IFN-.beta..
23. The method of claim 14, wherein the cytokine is delivered to an
upper one third of a nasal cavity.
24. The method of claim 14, wherein the cytokine is transported to
the central nervous system of the mammal in an amount effect for
preventing or reducing a viral infection.
25. The method of claim 24, wherein said viral infection is
selected from the group consisting of viral meningitis, herpes
simplex, hepatitis C, and human immunodeficiency (HIV).
26. The method of claim 14, wherein the cytokine is transported to
the central nervous system of the mammal in an amount effective to
treat or prevent a disorder characterized by an immune or an
inflammatory response.
27. The method of claim 26, wherein said disorder is selected from
the group consisting of Alzheimer's disease, meningitis, Primary
Sjogren's Syndrome, multiple sclerosis, and HIV.
28. The method of claim 14, wherein the cytokine is transported to
the central nervous system of the mammal in an amount effective to
treat or prevent a proliferative disorder.
29. The method of claim 28, wherein said proliferative disorder is
a glioma.
30. The method of claim 14, wherein the cytokine is administered in
a dosage range from about 0.14 nmol/kg of brain weight to about 138
nmol/kg of brain weight.
31. The method of claim 30, wherein the cytokine is human
IFN-.beta. or a biologically active variant thereof.
32. A method for transporting a cytokine to a lymphatic system of a
mammal, comprising: administering a composition comprising the
cytokine to a tissue of the mammal innervated by the trigeminal
nerve, the olfactory nerve, or a combination thereof, wherein the
cytokine is absorbed through the tissue and transported to the
lymphatic system of the mammal.
33. The method of claim 32, wherein the tissue comprises a nasal
cavity tissue, a conjunctiva, an oral tissue, or a skin.
34. The method of claim 33, wherein administering the cytokine to
the conjunctiva comprises administering the cytokine between a
lower eyelid and an eye.
35. The method of claim 33, wherein administering the cytokine to
the skin comprises administering the cytokine to a face, a
forehead, an upper eyelid, a lower eyelid, a dorsum of the nose, a
side of the nose, an upper lip, a cheek, a chin, a scalp, or a
combination thereof.
36. The method of claim 33, wherein administering the cytokine to
the oral tissue comprises sublingual administration.
37. The method of claim 32, wherein said cytokine is selected from
the group consisting of interferon-alpha (IFN-.alpha.),
interferon-beta (IFN-.beta.), interferon-gamma (IFN-.gamma.), and
biologically active variants thereof.
38. The method of claim 37, wherein the IFN-.beta. is human
IFN-.beta. or a biologically active variant thereof.
39. The method of claim 38, wherein the IFN-.beta. is biologically
active and comprises an amino acid sequence having at least 70%
sequence identity to human IFN-.beta..
40. The method of claim 32, wherein the cytokine is administered to
an upper one third of a nasal cavity.
41. The method of claim 32, wherein the cytokine is transported to
a deep cervical node, a superficial cervical node, or a combination
thereof.
42. The method of claim 32, wherein the cytokine is administered in
a dosage range of about 0.14 nmol/kg of brain weight to about 138
nmol/kg of brain weight.
43. The method of claim 42, wherein the cytokine is human
IFN-.beta. or a biologically active variant thereof.
44. A method for administering a cytokine to a lymphatic system of
a mammal, comprising: administering a composition comprising an
effective amount of the cytokine to a tissue of the mammal
innervated by the trigeminal nerve, the olfactory nerve, or a
combination thereof, wherein the cytokine is absorbed through the
tissue and transported into the lymphatic system of the mammal in
an amount effective to modulate an immune or inflammatory
response.
45. The method of claim 44, wherein the tissue comprises a tissue
of a nasal cavity, a conjunctiva, an oral tissue, or a skin.
46. The method of claim 45, wherein administering the cytokine to
the conjunctiva comprises administering the cytokine between a
lower eyelid and an eye.
47. The method of claim 45, wherein administering the cytokine to
the skin comprises administering the cytokine to a face, a
forehead, an upper eyelid, a lower eyelid, a dorsum of the nose, a
side of the nose, an upper lip, a cheek, a chin, a scalp, or a
combination thereof.
48. The method of claim 45, wherein administering the cytokine to
the oral tissue comprises sublingual administration.
49. The method of claim 44, wherein said cytokine is selected from
the group consisting of IFN-.alpha., IFN-.beta., IFN-.gamma., and
biologically active variants thereof.
50. The method of claim 49, wherein said IFN-.beta. is human
IFN-.beta. or a biologically active variant thereof.
51. The method of claim 50, wherein said IFN-.beta. or variant
thereof retains biological activity and comprises an amino acid
sequence having at least 70% sequence identity to the sequence of
human IFN-.beta..
52. The method of claim 44, wherein the cytokine is delivered to an
upper one third of a nasal cavity.
53. The method of claim 44, wherein the cytokine is administered in
a dosage range of about 0.14 nmol/kg of brain weight to about 138
nmol/kg of brain weight.
54. The method of claim 53, wherein the cytokine is human
IFN-.beta. or a biologically active variant thereof.
55. The method of claim 44, wherein the cytokine is transported to
the lymphatic system of the mammal in an amount effect for
preventing or reducing a viral infection.
56. The method of claim 55, wherein said viral infection is
selected from the group consisting of viral meningitis, herpes
simplex, hepatitis C, and human immunodeficiency (HIV).
57. The method of claim 44, wherein the cytokine is transported to
the lymphatic system of the mammal in an amount effective to treat
or prevent a disorder characterized by an immune or an inflammatory
response.
58. The method of claim 57, wherein said disorder is selected from
the group consisting of Alzheimer's disease, meningitis, Primary
Sjogren's Syndrome, multiple sclerosis, and HIV.
59. The method of claim 44, wherein the cytokine is transported to
the lymphatic system of the mammal in an amount effective to treat
or prevent a proliferative disorder.
60. The method of claim 59, wherein said proliferative disorder is
a glioma.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Application No. 60/200,708, filed Dec. 9, 1999, herein incorporated
by reference.
FIELD OF THE INVENTION
[0002] The present invention is directed to a method for delivering
cytokines to the central nervous system and by the lymphatic system
by way of a tissue innervated by the trigeminal nerve and/or
olfactory nerve. Cytokines include tumor necrosis factors,
interleukins, interferons, particularly .beta.-interferon and its
muteins such as IFN-.beta..sub.ser17. Such a method of delivery can
be useful in the treatment of central nervous system and/or brain
disorders.
BACKGROUND OF THE INVENTION
[0003] The central nervous system (CNS) includes several tissues
and organs, such as the brain, the brain stem, and the spinal cord.
Each of these organs and tissues is made up of a variety of
different types of cells and subcellular structures, e.g., neurons,
glial cells, dendrites, axons, myelin, and various membranes. The
CNS is isolated from the external world by several membranes that
both cushion and protect these organs, tissues, cells, and
structures. For example, the membranes that form the blood-brain
barrier protect the brain from certain contents of the blood. The
blood-cerebrospinal fluid barrier protects other portions of the
CNS from many chemicals and microbes.
[0004] Access to the CNS for some substances is provided by
specialized active transport systems or through passive diffusion
through the protective membrane into the CNS. Present methods for
delivering desired therapeutic agents to the CNS are typically
invasive. For example, a pump implanted into the chest cavity (an
intracerebroventricular pump) can effectively deliver a variety of
useful compounds to the brain. However, implanting such a pump
requires surgery, which can entail a variety of serious
complications. Certain compounds (e.g., epidural painkillers) can
be injected directly through the protective membrane into the CNS.
Such injection is, however, impractical for most medications.
Better methods for administering desired agents to the CNS, brain,
spinal cord, and lymphatic channels are needed.
SUMMARY OF THE INVENTION
[0005] The present invention relates to a method for transporting
or delivering a cytokine, such as an interferon, an interleukin, or
a tumor necrosis factor, preferably interferon-.beta., to the
central nervous system of a subject. The method employs
administration of the cytokine to a tissue innervated by the
trigeminal nerve and/or olfactory nerve.
[0006] In one embodiment, the method administers the cytokine
through the mucosa or epithelium of the nasal cavity, tongue,
mouth, skin, or conjunctiva. In another embodiment, the method
includes administering a composition of the cytokine to the nasal
cavity, under the tongue, to the skin, or to the conjunctiva of the
subject. The cytokine can then be absorbed through a mucosa or
epithelium and transported to the central nervous system of the
mammal.
[0007] In another embodiment, the method includes administering the
cytokine in a manner such that the cytokine is absorbed through the
tissue and transported into the central nervous system of the
mammal by a neural pathway and in an amount effective to provide a
protective or therapeutic effect on a cell of the central nervous
system.
[0008] The present invention further relates to a method for
transporting or delivering a cytokine, such as an interferon, an
interleukin, or a tumor necrosis factor, preferably
interferon-.beta., to the lymphatic system of a subject. The method
employs administration of the cytokine to a tissue innervated by
the trigeminal nerve and/or olfactory nerve.
[0009] In another embodiment, the method includes administering the
cytokine in a manner such that the cytokine is absorbed through the
tissue and transported into the central nervous system of the
mammal by a neural pathway and in an amount effective to modulate
an immune or inflammatory response.
[0010] In other embodiments, the method of administering a cytokine
is used for the treatment and/or prevention of central nervous
system disorders, brain disorders, proliferative, viral, and/or
autoimmune disorders.
[0011] The composition can be of any form suitable for
administration by these routes and can include a carrier that
facilitates absorption of the cytokine, transport of the cytokine
by a neural pathway, and/or transport of the cytokine to the
lymphatic system, CNS, brain, and/or spinal cord. Preferred
compositions include one or more of a solubility enhancing
additive, a hydrophilic additive, an absorption promoting additive,
a cationic surfactant, a viscosity enhancing additive, or a
sustained release matrix or composition, a lipid-based carrier,
preferably a micellar or liposomal composition, a bilayer
destabilizing additive, or a fusogenic additive. The composition
can be formulated as a cosmetic for dermal delivery.
BRIEF DESCRIPTION OF FIGURES
[0012] FIG. 1 shows the level of Betaseron in the blood stream over
time following both intravenous administration (I.V.) and
intranasal administration (I.N.) in a rat.
DETAILED DESCRIPTION OF THE INVENTION
[0013] Routes of Administration
[0014] The method of the invention administers the cytokine to
tissue innervated by the trigeminal and olfactory nerves. Such
nerve systems can provide a direct connection between the outside
environment and the brain, thus providing advantageous delivery of
a cytokine to the CNS, including brain, brain stem, and/or spinal
cord. Cytokines are unable to cross or inefficiently cross the
blood-brain barrier from the bloodstream into the brain. The
methods of the present invention allow for the delivery of the
cytokine by way of the olfactory and/or trigeminal nerve rather
than through the circulatory system. This method of administration
allows for the efficient delivery of a cytokine to the CNS, brain,
or spinal cord.
[0015] The Olfactory Nerve
[0016] The method of the invention includes administration of a
cytokine to tissue innervated by the olfactory nerve. Preferably,
the cytokine is delivered to the olfactory area in the upper third
of the nasal cavity and particularly to the olfactory
epithelium.
[0017] Fibers of the olfactory nerve are unmyelinated axons of
olfactory receptor cells that are located in the superior one-third
of the nasal mucosa. The olfactory receptor cells are bipolar
neurons with swellings covered by hair-like cilia that project into
the nasal cavity. At the other end, axons from these cells collect
into aggregates and enter the cranial cavity at the roof of the
nose. Surrounded by a thin tube of pia, the olfactory nerves cross
the subarachnoid space containing CSF and enter the inferior
aspects of the olfactory bulbs. Once the cytokine is dispensed into
the nasal cavity, the cytokine can undergo transport through the
nasal mucosa and into the olfactory bulb and interconnected areas
of the brain, such as the hippocampal formation, amygdaloid nuclei,
nucleus basalis of Meynert, locus ceruleus, the brain stem, and the
like.
[0018] The Trigeminal Nerve
[0019] The method of the invention administers the cytokine to
tissue innervated by the trigeminal nerve. The trigeminal nerve
innervates tissues of a mammal's (e.g., human) head including skin
of the face and scalp, oral tissues, and tissues of and surrounding
the eye. The trigeminal nerve has three major branches, the
ophthalmic nerve, the maxillary nerve, and the mandibular nerve.
The method of the invention can administer the cytokine to tissue
innervated by one or more of these branches.
[0020] The Ophthalmic Nerve and its Branches
[0021] The method of the invention can administer the cytokine to
tissue innervated by the ophthalmic nerve branch of the trigeminal
nerve. The ophthalmic nerve innervates tissues including
superficial and deep parts of the superior region of the face, such
as the eye, the lacrimal gland, the conjunctiva, and skin of the
scalp, forehead, upper eyelid, and nose.
[0022] The ophthalmic nerve has three branches known as the
nasociliary nerve, the frontal nerve, and the lacrimal nerve. The
method of the invention can administer the cytokine to tissue
innervated by the one or more of the branches of the ophthalmic
nerve. The frontal nerve and its branches innervate tissues
including the upper eyelid, the scalp, particularly the front of
the scalp, and the forehead, particularly the middle part of the
forehead. The nasociliary nerve forms several branches including
the long ciliary nerves, the ganglionic branches, the ethmoidal
nerves, and the infratrochlear nerve. The long ciliary nerves
innervate tissues including the eye. The posterior and anterior
ethmoidal nerves innervate tissues including the ethmoidal sinus
and the inferior two-thirds of the nasal cavity. The infratrochlear
nerve innervates tissues including the upper eyelid and the
lacrimal sack. The lacrimal nerve innervates tissues including the
lacrimal gland, the conjunctiva, and the upper eyelid. Preferably,
the present method administers the cytokine to the ethmoidal
nerve.
[0023] The Maxillary Nerve and its Branches
[0024] The method of the invention can administer the cytokine to
tissue innervated by the maxillary nerve branch of the trigeminal
nerve. The maxillary nerve innervates tissues including the roots
of several teeth and facial skin, such as skin on the nose, the
upper lip, the lower eyelid, over the cheekbone, over the temporal
region. The maxillary nerve has branches including the infraorbital
nerve, the zygomaticofacial nerve, the zygomaticotemporal nerve,
the nasopalatine nerve, the greater palatine nerve, the posterior
superior alveolar nerves, the middle superior alveolar nerve, and
the interior superior alveolar nerve. The method of the invention
can administer the cytokine to tissue innervated by the one or more
of the branches of the maxillary nerve.
[0025] The infraorbital nerve innervates tissue including skin on
the lateral aspect of the nose, upper lip, and lower eyelid. The
zygomaticofacial nerve innervates tissues including skin of the
face over the zygomatic bone (cheekbone). The zygomaticotemporal
nerve innervates tissue including the skin over the temporal
region. The posterior superior alveolar nerves innervate tissues
including the maxillary sinus and the roots of the maxillary molar
teeth. The middle superior alveolar nerve innervates tissues
including the mucosa of the maxillary sinus, the roots of the
maxillary premolar teeth, and the mesiobuccal root of the first
molar tooth. The anterior superior alveolar nerve innervates
tissues including the maxillary sinus, the nasal septum, and the
roots of the maxillary central and lateral incisors and canine
teeth. The nasopalantine nerve innervates tissues including the
nasal septum. The greater palatine nerve innervates tissues
including the lateral wall of the nasal cavity. Preferably, the
present method administers the cytokine to the nasopalatine nerve
and/or greater palatine nerve.
[0026] The Mandibular Nerve and its Branches
[0027] The method of the invention can administer the cytokine to
tissue innervated by the mandibular nerve branch of the trigeminal
nerve. The mandibular nerve innervates tissues including the teeth,
the gums, the floor of the oral cavity, the tongue, the cheek, the
chin, the lower lip, tissues in and around the ear, the muscles of
mastication, and skin including the temporal region, the lateral
part of the scalp, and most of the lower part of the face.
[0028] The mandibular nerve has branches including the buccal
nerve, the auriculotemporal nerve, the inferior alveolar nerve, and
the lingual nerve. The method of the invention can administer the
cytokine to one or more of the branches of the mandibular nerve.
The buccal nerve innervates tissues including the cheek,
particularly the skin of the cheek over the buccinator muscle and
the mucous membrane lining the cheek, and the mandibular buccal
gingiva (gum), in particular the posterior part of the buccal
surface of the gingiva. The auriculotemporal nerve innervates
tissues including the auricle, the external acoustic meatus, the
tympanic membrane (eardrum), and skin in the temporal region,
particularly the skin of the temple and the lateral part of the
scalp. The inferior alveolar nerve innervates tissues including the
mandibular teeth, in particular the incisor teeth, the gingiva
adjacent the incisor teeth, the mucosa of the lower lip, the skin
of the chin, the skin of the lower lip, and the labial mandibular
gingivae. The lingual nerve innervates tissues including the
tongue, particularly the anterior two-thirds of the tongue, the
floor of the mouth, and the gingivae of the mandibular teeth.
Preferably, the method of the invention administers the cytokine to
one or more of the inferior alveolar nerve, the buccal nerve,
and/or the lingual nerve.
[0029] Tissues Innervated by the Trigeminal Nerve
[0030] The method of the invention can administer the cytokine to
any of a variety of tissues innervated by the trigeminal nerve. For
example, the method can administer the cytokine to skin,
epithelium, or mucosa of or around the face, the eye, the oral
cavity, the nasal cavity, the sinus cavities, or the ear.
[0031] Preferably, the method of the invention administers the
cytokine to skin innervated by the trigeminal nerve. For example,
the present method can administer the cytokine to skin of the face,
scalp, or temporal region. Suitable skin of the face includes skin
of the chin; the upper lip, the lower lip; the forehead,
particularly the middle part of the forehead; the nose, including
the tip of the nose, the dorsum of the nose, and the lateral aspect
of the nose; the cheek, particularly the skin of the cheek over the
buccinator muscle or skin over the cheek bone; skin around the eye,
particularly the upper eyelid and the lower eyelid; or a
combination thereof. Suitable skin of the scalp includes the front
of the scalp, scalp over the temporal region, the lateral part of
the scalp, or a combination thereof. Suitable skin of the temporal
region includes the temple and scalp over the temporal region.
[0032] Preferably, the method of the invention administers the
cytokine to mucosa or epithelium innervated by the trigeminal
nerve. For example, the present method can administer the cytokine
to mucosa or epithelium of or surrounding the eye, such as mucosa
or epithelium of the upper eyelid, the lower eyelid, the
conjunctiva, the lacrimal system, or a combination thereof. The
method of the invention can also administer the cytokine to mucosa
or epithelium of the sinus cavities and/or nasal cavity, such as
the inferior two-thirds of the nasal cavity and the nasal septum.
The method of the invention can also administer the cytokine to
mucosa or epithelium of the oral cavity, such as mucosa or
epithelium of the tongue; particularly the anterior two-thirds of
the tongue and under the tongue; the cheek; the lower lip; the
upper lip; the floor of the oral cavity; the gingivae (gums), in
particular the gingiva adjacent the incisor teeth, the labial
mandibular gingivae, and the gingivae of the mandibular teeth; or a
combination thereof. Preferably, the method of the invention
administers the cytokine to mucosa or epithelium of the nasal
cavity. Other preferred regions of mucosa or epithelium for
administering the cytokine include the tongue, particularly
sublingual mucosa or epithelium, the conjunctiva, the lacrimal
system, particularly the palpebral portion of the lacrimal gland
and the nasolacrimal ducts, the mucosa of the lower eyelid, the
mucosa of the cheek, or a combination thereof.
[0033] Preferably, the method of the invention administers the
cytokine to nasal tissues innervated by the trigeminal nerve. For
example, the present method can administer the cytokine to nasal
tissues including the sinuses, the inferior two-thirds of the nasal
cavity and the nasal septum. Preferably, the nasal tissue for
administering the cytokine includes the inferior two-thirds of the
nasal cavity and the nasal septum.
[0034] Preferably, the method of the invention administers the
cytokine to oral tissues innervated by the trigeminal nerve. For
example, the present method can also administer the cytokine to
oral tissues such as the teeth, the gums, the floor of the oral
cavity, the cheeks, the lips, the tongue, particularly the anterior
two-thirds of the tongue, or a combination thereof. Suitable teeth
include mandibular teeth, such as the incisor teeth. Suitable
portions of the teeth include the roots of several teeth, such as
the roots of the maxillary molar teeth, the maxillary premolar
teeth, the maxillary central and lateral incisors, the canine
teeth, and the mesiobuccal root of the first molar tooth, or a
combination thereof. Suitable portions of the lips include the skin
and mucosa of the upper and lower lips. Suitable gums include the
gingiva adjacent the incisor teeth and the gingivae of the
mandibular teeth, such as the labial mandibular gingivae, or a
combination thereof. Suitable portions of the cheek include the
skin of the cheek over the buccinator muscle, the mucous membrane
lining the cheek, and the mandibular buccal gingiva (gum), in
particular the posterior part of the buccal surface of the gingiva,
or a combination thereof. Preferred oral tissue for administering
the cytokine includes the tongue, particularly sublingual mucosa or
epithelium, the mucosa inside the lower lip, the mucosa of the
cheek, or a combination thereof.
[0035] Preferably, the method of the invention administers the
cytokine to a tissue of or around the eye that is innervated by the
trigeminal nerve. For example, the present method can administer
the cytokine to tissue including the eye, the conjunctiva, and the
lacrimal gland including the lacrimal sack, the skin or mucosa of
the upper or lower eyelid, or a combination thereof. Preferred
tissue of or around the eye for administering the cytokine includes
the conjunctiva, the lachrimal system, the skin or mucosa of the
eyelid, or a combination thereof. Cytokine that is administered
conjunctivally but not absorbed through the conjunctival mucosa can
drain through nasolachrimal ducts into the nose, where it can be
transported to the CNS, brain, and/or spinal cord as though it had
been intranasally administered.
[0036] Preferably, the method of the invention administers the
cytokine to a tissue of or around the ear that is innervated by the
trigeminal nerve. For example, the present method can administer
the cytokine to tissue including the auricle, the external acoustic
meatus, the tympanic membrane (eardrum), and the skin in the
temporal region, particularly the skin of the temple and the
lateral part of the scalp, or a combination thereof. Preferred
tissue of or around the ear for administering the cytokine includes
the skin of the temple.
[0037] Cytokines
[0038] Cytokines can be administered to the CNS, brain, and/or
spinal cord according to the present invention. Cytokines that can
be administered by the method of the invention are cytokines that
are immunomodulators, such as interleukins (i.e., IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 and IL-10), interferons, and
tumor necrosis factor (i.e., TNF-.alpha. and TNF-.beta.), and that
have activities directed at cells of the immune system. These
cytokines are of interest as therapeutic cytokines, for example,
for treatment of viral diseases and control of cancer. It is
believed that such cytokines have not been observed to have
neurotrophic activity, or to have other direct, beneficial effects
on neurons characteristic of nerve growth factor and like
compounds. Thus, it was not expected that such cytokines should be
transported into the CNS, brain, and or spinal cord, particularly
not by a neural pathway, or from tissues innervated by the
olfactory and/or trigeminal nerves.
[0039] A preferred cytokine for use in the practice of the
invention are members of the interferon family. Interferons (IFNs)
are a family of molecules encompassing over 20 different proteins
and are members of the cytokine family that induce antiviral,
antiproliferative, antitumor, and/or cytokine effects. IFNs are
relatively small, species-specific, single chain polypeptides,
which are produced in response to a variety of inducers, such as
mitogens, polypeptides, viruses, and the like. In humans, IFNs are
produced in forms .alpha., .beta., .gamma., .omega., and .tau..
Synthetic interferons are also known in the art. See, for example,
6,114,145, herein incorporated by reference. Upon secretion from
mammalian cells, interferon molecules bind to a receptor on the
surface of a target cell and elicit a chain of events, which can
alter the amount and activity of protein in the target cell. Such
alterations can include, for example, changes in gene transcription
or enzymatic activity. A preferred interferon for use in the
practice of the invention is interferon-.beta. (IFN-.beta.),
interferon-.alpha. (IFN-.alpha.), and interferon-.gamma.
(IFN-.gamma.).
[0040] Biologically active variants of cytokines are also
encompassed by the method of the present invention. Such variants
should retain the biological activity of the cytokine. For example,
when the cytokine is an interferon, such as IFN-.alpha.,
IFN-.beta., IFN-.gamma., the ability to bind their respective
receptor sites will be retained. Such activity may be measured
using standard bioassays. Representative assays detecting the
ability of the variant to interact with an interferon receptor type
I can be found in, for example, U.S. Pat. No. 5,766,864, herein
incororpated by reference. Preferably, the variant has at least the
same activity as the native molecule. Alternatively, the biological
activity of a variant of the cytokine of the invention can be
assayed by measuring the ability of the variant to increase viral
resistance in a cell line using a standard viral reduction assay.
See for example, U.S. Pat. No. 5,770,191, herein incorporated by
reference. Other assays for biological activity include,
anti-proliferative assays as described in U.S. Pat. No.
5,690,925.
[0041] Suitable biologically active variants can be fragments,
analogues, and derivatives of the cytokine polypeptides. By
"fragment" is intended a protein consisting of only a part of the
intact cytokine polypeptide sequence. The fragment can be a
C-terminal deletion or N-terminal deletion of the cytokine
polypeptide. By "analogue" is intended an analogue of either the
full length polypeptide having biological activity or a fragment
thereof, that includes a native sequence and structure having one
or more amino acid substitutions, insertions, or deletions.
Peptides having one or more peptoids (peptide mimics) are also
encompassed by the term analogue (see i.e., International
Publication No. WO 91/04282). By "derivative" is intended any
suitable modification of the native polypeptide or fragments
thereof, or their respective analogues, such as glycosylation,
phosphorylation, or other addition of foreign moieties, so long as
the activity is retained.
[0042] Preferably, naturally or non-naturally occurring variants of
a cytokine have amino acid sequences that are at least 70%,
preferably 80%, more preferably, 85%, 90%, 91%, 92%, 93%, 94% or
95% identical to the amino acid sequence to the reference molecule,
for example, the native human interferon, or to a shorter portion
of the reference interferon molecule. More preferably, the
molecules are 96%, 97%, 98% or 99% identical. Percent sequence
identity is determined using the Smith-Waterman homology search
algorithm using an affine gap search with a gap open penalty of 12
and a gap extension penalty of 2, BLOSUM matrix of 62. The
Smith-Waterman homology search algorithm is taught in Smith and
Waterman, Adv. Appl. Math. (1981) 2:482-489. A variant may, for
example, differ by as few as 1 to 10 amino acid residues, such as
6-10, as few as 5, as few as 4, 3, 2, or even 1 amino aid
residue.
[0043] With respect to optimal alignment of two amino acid
sequences, the contiguous segment of the variant amino acid
sequence may have additional amino acid residues or deleted amino
acid residues with respect to the reference amino acid sequence.
The contiguous segment used for comparison to the reference amino
acid sequence will include at least 20 contiguous amino acid
residues, and may be 30, 40, 50, or more amino acid residues.
Corrections for sequence identity associated with conservative
residue substitutions or gaps can be made (see Smith-Waterman
homology search algorithm).
[0044] The art provides substantial guidance regarding the
preparation and use of such variants, as discussed further below. A
fragment of a cytokine polypeptide will generally include at least
about 10 contiguous amino acid residues of the full-length
molecule, preferably about 15-25 contiguous amino acid residues of
the full-length molecule, and most preferably about 20-50 or more
contiguous amino acid residues of full-length cytokine
polypeptide.
[0045] For example, conservative amino acid substitutions may be
made at one or more predicted, preferably nonessential amino acid
residues. A "nonessential" amino acid residue is a residue that can
be altered from the wild-type sequence of a cytokine, such as an
interferon (i.e., IFN-.alpha., IFN-.beta., or IFN-.gamma.) without
altering its biological activity, whereas an "essential" amino acid
residue is required for biological activity. A "conservative amino
acid substitution" is one in which the amino acid residue is
replaced with an amino acid residue having a similar side chain.
Families of amino acid residues having similar side chains have
been defined in the art. These families include amino acids with
basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g., aspartic acid, glutamic acid), uncharged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine,
tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine), and aromatic side chains (e.g., tyrosine,
phenylalanine, tryptophan, histidine). Such substitutions would not
be made for conserved amino acid residues, or for amino acid
residues residing within a conserved motif.
[0046] Alternatively, variant cytokine nucleotide sequences can be
made by introducing mutations randomly along all or part of a
cytokine coding sequence, such as by saturation mutagenesis, and
the resultant mutants can be screened for cytokine biological
activity to identify mutants that retain activity. Following
mutagenesis, the encoded protein can be expressed recombinantly,
and the activity of the protein can be determined using standard
assay techniques described herein.
[0047] Alternatively, the cytokine can be synthesized chemically,
by any of several techniques that are known to those skilled in the
peptide art. See, for example, Li et al. (1983) Proc. Natl. Acad.
Sci. USA 80:2216-2220, Steward and Young (1984) Solid Phase Peptide
Synthesis (Pierce Chemical Company, Rockford, Ill.), and Baraney
and Merrifield (1980) The Peptides: Analysis, Synthesis, Biology,
ed. Gross and Meinhofer, Vol. 2 (Academic Press, New York, 1980),
pp. 3-254, discussing solid-phase peptide synthesis techniques; and
Bodansky (1984) Principles of Peptide Synthesis (Springer-Verlag,
Berlin) and Gross and Meinhofer, eds. (1980) The Peptides:
Analysis, Synthesis, Biology, Vol. 1 (Academic Press, New York),
discussing classical solution synthesis. The cytokine can also be
chemically prepared by the method of simultaneous multiple peptide
synthesis. See, for example, Houghten (1984) Proc. Natl. Acad. Sci.
USA 82:5131-5135; and U.S. Pat. No. 4,631,211.
[0048] The cytokine used in the methods of the invention can be
from any animal species including, but not limited to, avian,
canine, bovine, porcine, equine, and human. Preferably, the
cytokine is from a mammalian species when the cytokine is to be
used in treatment of a mammalian viral, immunomodulatory, or
neurologic disorder of the CNS, brain or spinal cord, and more
preferably is from a mammal of the same species as the mammal
undergoing treatment for such a disorder.
[0049] Interferon-.beta.
[0050] The term "IFN-.beta." as used herein refers to IFN-.beta. or
variants thereof, sometimes referred to as IFN-.beta.-like
polypeptides. Human IFN-.beta. variants, which may be naturally
occurring (e.g., allelic variants that occur at the IFN-.beta.
locus) or recombinantly produced, have amino acid sequences that
are the same as, similar to, or substantially similar to the mature
native IFN-.beta. sequence. DNA sequences encoding human IFN-.beta.
are also available in the art. See, for example, Goeddel et al.
(1980) Nucleic Acid Res. 8:4057 and Tanigachi et al. (1979) Proc.
Japan Acad. Sci. 855:464. Fragments of IFN-.beta. or truncated
forms of IFN-.beta. that retain their activity are also
encompassed. These biologically active fragments or truncated forms
of IFN-.beta. are generated by removing amino acid residues from
the full-length IFN-.beta. amino acid sequence using recombinant
DNA techniques well known in the art. IFN-.beta. polypeptides may
be glycosylated or unglycosylated, as it has been reported in the
literature that both the glycosylated and unglycosylated forms of
IFN-.beta. show qualitatively similar specific activities and that,
therefore, the glycosyl moieties are not involved in and do not
contribute to the biological activity of IFN-.beta..
[0051] The IFN-.beta. variants encompassed herein include muteins
of the native mature IFN-.beta. sequence, wherein one or more
cysteine residues that are not essential to biological activity
have been deliberately deleted or replaced with other amino acids
to eliminate sites for either intermolecular crosslinking or
incorrect intramolecular disulfide bond formation. IFN-.beta.
variants of this type include those containing a glycine, valine,
alanine, leucine, isoleucine, tyrosine, phenylalanine, histidine,
tryptophan, serine, threonine, or methionine substituted for the
cysteine found at amino acid 17 of the mature native amino acid
sequence. Serine and threonine are the more preferred replacements
because of their chemical analogy to cysteine. Serine substitutions
are most preferred. For example, an IFN-.beta. variant can comprise
a serine residue replacing the cysteine found at amino acid 17 of
the mature native sequence. Cysteine 17 may also be deleted using
methods known in the art (see, for example, U.S. Pat. No.
4,588,585, herein incorporated by reference), resulting in a mature
IFN-.beta. mutein that is one amino acid shorter than the native
mature IFN-.beta.. Thus, IFN-.beta. variants with one or more
mutations that improve, for example, their pharmaceutical utility
are also encompassed by the present invention.
[0052] The skilled artisan will appreciate that additional changes
can be introduced by mutation into the nucleotide sequences
encoding IFN-.beta., thereby leading to changes in the IFN-.beta.
amino acid sequence, without altering the biological activity of
the interferon. Thus, an isolated nucleic acid molecule encoding an
IFN-.beta. variant having a sequence that differs from human
IFN-.beta. can be created by introducing one or more nucleotide
substitutions, additions, or deletions into the corresponding
nucleotide sequence disclosed herein, such that one or more amino
acid substitutions, additions or deletions are introduced into the
encoded IFN-.beta.. Mutations can be introduced by standard
techniques, such as site-directed mutagenesis and PCR-mediated
mutagenesis. Such IFN-.beta. variants are also encompassed by the
present invention. Variants of IFN-.beta. are described in European
Patent Application No. 18545981, and U.S. Pat. Nos. 4,518,584,
4,588,585, and 4,737,462, all of which are incorporated herein by
reference.
[0053] Biologically active IFN-.beta. variants encompassed by the
invention also include IFN-.beta. polypeptides that have covalently
linked with, for example, polyethylene glycol (PEG) or albumin.
[0054] Biologically active variants of IFN-.beta. encompassed by
the invention should retain IFN-.beta. activities, particularly the
ability to bind to IFN-p receptors or retain immunomodulatory or
anti-viral activities. In some embodiments, the IFN-.beta. variant
retains at least about 25%, about 50%, about 75%, about 85%, about
90%, about 95%, about 98%, about 99% or more of the biological
activity of the native IFN-.beta. polypeptide. IFN-.beta. variants
whose activity is increased in comparison with the activity of the
native IFN-.beta. polypeptide are also encompassed. The biological
activity of IFN-p variants can be measured by any method known in
the art. Examples of such assays can be found in Fellous et aL
(1982) Proc. Natl. Acad. Sci USA 79:3082-3086; Czemiecki et al.
(1984) J. Virol. 49(2):490-496; Mark et al (1984) Proc. Natl Acad.
Sci. USA 81:5662-5666; Branca et al. (1981) Nature 277:221-223;
Williams et al. (1979) Nature 282:582-586; Herberman et aL (1979)
Nature 277:221-223; and Anderson etal. (1982) J. Biol. Chem.
257(19):11301-11304.
[0055] Non-limiting examples of IFN-.beta. polypeptides and
IFN-.beta. variant polypeptides encompassed by the invention are
set forth in Nagata et al. (1980) Nature 284:316-320; Goeddel et
al. (1980) Nature 287:411-416 ; Yelverton etaL (1981) Nucleic Acids
Res. 9:731-741; Streuli et al. (1981) Proc. Natl. Acad. Sci. U.S.A.
78:2848-2852; EP028033B1, and EP109748B1. See also U.S. Pat. Nos.
4,518,584; 4,569,908; 4,588,585; 4,738,844; 4,753,795; 4,769,233;
4,793,995; 4,914,033; 4,959,314; 5,545,723; and 5,814,485. These
disclosures are herein incorporated by reference. These citations
also provide guidance regarding residues and regions of the
IFN-.beta. polypeptide that can be altered without the loss of
biological activity.
[0056] In one embodiment of the present invention, the IFN-.beta.
used in the methods of the invention is the mature native human
IFN-.beta. polypeptide. In another embodiment, the IFN-.beta. is
the mature IFN-.beta. C17S polypeptide. However, the present
invention encompasses other embodiments where the IFN-.beta. is any
biologically active IFN-.beta. polypeptide or variant as described
elsewhere herein.
[0057] In some embodiments of the present invention, the IFN-.beta.
is recombinantly produced. By "recombinantly produced IFN-.beta."
is intended IFN-.beta. that has comparable biological activity to
native IFN-.beta. and that has been prepared by recombinant DNA
techniques. IFN-.beta. can be produced by culturing a host cell
transformed with an expression vector comprising a nucleotide
sequence that encodes an IFN-.beta. polypeptide. The host cell is
one that can transcribe the nucleotide sequence and produce the
desired protein, and can be prokaryotic (for example, E. coli) or
eukaryotic (for example a yeast, insect, or mammalian cell).
Examples of recombinant production of IFN-.beta. are given in
Mantei et al. (1982) Nature 297:128; Ohno et al. (1982) Nucleic
Acids Res. 10:967; Smith et al. (1983) Mol. Cell. Biol. 3:2156, and
U.S. Pat. No. 4,462,940, 5,702,699, and 5,814,485; herein
incorporated by reference.
[0058] Interferon-.alpha.
[0059] The term "IFN-.alpha." as used herein refers to IFN-.alpha.
or variants thereof, sometimes referred to as IFN-.alpha.-like
polypeptides. Human alpha interferons comprise a family of about 30
protein species, encoded by at least 14 different genes and about
16 alleles. Such IFN-.alpha. polypeptides include IFN-.alpha.a,
IFN-.alpha.B, IFN-.alpha.C, IFN-.alpha.D, IFN-.alpha.H,
IFN-.alpha.J, IFN-.alpha.J1, IFN-.alpha.J2 and IFN-.alpha.K. Human
IFN-.alpha. variants, which may be naturally occurring (e.g.,
allelic variants that occur at the IFN-.alpha. locus) or
recombinantly produced, have amino acid sequences that are the same
as, similar to, or substantially similar to the mature native
IFN-.alpha. sequence. DNA sequences encoding human IFN-.alpha. are
also available in the art. See, for example, Goeddel et al. (1981)
Nature 290:20-26 (Genbank Accession No. V00551 J00209); Nagata et
al. (1980) Nature 284:3126-320; Bowden et al. (1984) Gene 27:87-99
(Genbank Accession No. NM.sub.--000605); and Ohara et al. (1987)
FEBS Letters 211:78-82; all of which are herein incorporated by
reference. Fragments of IFN-.alpha. or truncated forms of
IFN-.alpha. that retain their activity are also encompassed. These
biologically active fragments or truncated forms of IFN-.alpha. are
generated by removing amino acid residues from the full-length
IFN-.alpha. amino acid sequence using recombinant DNA techniques
well known in the art. IFN-.alpha. polypeptides may further be
glycosylated or unglycosylated.
[0060] The skilled artisan will appreciate that additional changes
can be introduced by mutation into the nucleotide sequences
encoding IFN-.alpha., thereby leading to changes in the IFN-.alpha.
amino acid sequence, without altering the biological activity of
the interferon. Thus, an isolated nucleic acid molecule encoding an
IFN-.alpha. variant having a sequence that differs from human
IFN-.alpha. can be created by introducing one or more nucleotide
substitutions, additions, or deletions into the corresponding
nucleotide sequence disclosed herein, such that one or more amino
acid substitutions, additions or deletions are introduced into the
encoded IFN-.alpha.. Mutations can be introduced by standard
techniques. Such variants of IFN-.alpha., include, for example,
IFN-.alpha.-2a (Roferon-A.TM.), IFN-.alpha.-2b (Intron A.TM.), and
IFN-.alpha.con-1 (Infergen.TM.). Another variant useful in the
methods of the present invention is IFN-.alpha.2a, which is
disclosed in, for example, EP 43980; Meada et al. (1980) PNAS
77:7010; and Levy et al. (1981) PNAS 78:6186; all of which are
herein incorporated by reference. Further, variants of IFN-.alpha.
can be found, for example, in U.S. Pat. No. 5,676,942, herein
incorporated by reference. These citations also provide guidance
regarding residues and regions of the IFN-.alpha. polypeptide that
can be altered without the loss of biological activity.
[0061] Biologically active IFN-.alpha. variants encompassed by the
invention also include IFN-.alpha. polypeptides that have
covalently linked with, for example, polyethylene glycol (PEG) or
albumin. See, for example, U.S. Pat. No. 5,762,923, herein
incorporated by reference.
[0062] Biologically active variants of IFN-.alpha. encompassed by
the invention should retain IFN-.alpha. activities, particularly
the ability to bind to IFN-.alpha. receptors or retain
immunomodulatory, antiviral, or anit-proliferative activities. In
some embodiments, the IFN-.alpha. variant retains at least about
25%, about 50%, about 75%, about 85%, about 90%, about 95%, about
98%, about 99% or more of the biological activity of the native
IFN-.alpha. polypeptide. IFN-.alpha. variants whose activity is
increased in comparison with the activity of the native IFN-.alpha.
polypeptide are also encompassed. The biological activity of
IFN-.alpha. variants can be measured by any method known in the
art. Examples of such assays are describe above.
[0063] In one embodiment of the present invention, the IFN-.alpha.
used in the methods of the invention is the mature native human
IFN-.alpha. polypeptide. However, the present invention encompasses
other embodiments where the IFN-.alpha. is any biologically active
IFN-.alpha. polypeptide or variant as described elsewhere
herein.
[0064] In some embodiments of the present invention, the
IFN-.alpha. is recombinantly produced. By "recombinantly produced
IFN-.alpha." is intended IFN-.alpha. that has comparable biological
activity to native IFN-.alpha. and that has been prepared by
recombinant DNA techniques. IFN-.alpha. can be produced by
culturing a host cell transformed with an expression vector
comprising a nucleotide sequence that encodes an IFN-.alpha.
polypeptide. The host cell is one that can transcribe the
nucleotide sequence and produce the desired protein, and can be
prokaryotic (for example, E. coli) or eukaryotic (for example a
yeast, insect, or mammalian cell). Details of the cloning of
interferon-cDNA and the direct expression thereof, especially in E.
coli, have in the meantime been the subject of many publications.
Thus, for example, the preparation of recombinant interferons is
known. See, for example, (1982) Nature 295: 503-508; (1980) Nature
284: 316-320; (1981) Nature 290: 20-26; (1980) Nucleic Acids Res.
8: 4057-4074, as well as from European Patents Nos. 32134, 43980
and 211 148. Further examples of recombinant production of
IFN-.alpha.-2 are provided in Nagata et al. (1980) Nature 284:316
and European Patent 32,134. All of these references are herein
incorporated by reference.
[0065] Interferon-.gamma.
[0066] The term "IFN-.gamma." as used herein refers to IFN-.gamma.
or variants thereof, sometimes referred to as IFN-.gamma.-like
polypeptides. IFN-.gamma. is a glycoprotein whose mature form has
143 amino acids and a molecular weight of about 63-73 kilodaltons.
The amino acid sequence of IFN-.gamma. can be found in, for
example, U.S. Pat. No. 6,046,034, herein incorporated by reference.
Human IFN-.gamma. variants, which may be naturally occurring (e.g.,
allelic variants that occur at the IFN-.gamma. locus) or
recombinantly produced, have amino acid sequences that are the same
as, similar to, or substantially similar to the mature native
IFN-.gamma. sequence. DNA sequences encoding human IFN-.gamma. are
also available in the art. See, for example, Grey et al. (1983)
Proc. Natl. Acad. Sci. USA 80:5842-5846, herein incorporated by
reference. Fragments of IFN-.gamma. or truncated forms of
IFN-.gamma. that retain their activity are also encompassed. These
biologically active fragments or truncated forms of IFN-.gamma. are
generated by removing amino acid residues from the full-length
IFN-.gamma. amino acid sequence using recombinant DNA techniques
well known in the art. IFN-.gamma. polypeptides may be glycosylated
or unglycosylated.
[0067] The IFN-.gamma. variants encompassed herein include muteins
of the native mature IFN-.gamma. sequence. Thus, IFN-.gamma.
variants with one or more mutations that improve, for example,
their pharmaceutical utility are also encompassed by the present
invention.
[0068] Such IFN-.gamma. variants are also encompassed by the
present invention. Variants of IFN-.gamma. are well known in the
art. For example, U.S. Pat. No. 5,770,191, herein incorporated by
reference, discloses peptides comprising the C-terminus of
IFN-.gamma. that retain the biological activity of the mature
IFN-.gamma.. Additionally, in EP 0 306870 A2, variants of human
IFN-.gamma. were identified whose activity was significantly
increased by deleting the C-terminal 7-11 amino acids. In addition,
WO 92-08737 discloses a variant of recombinant human
IFN-.gamma.(IFN-.gamma. C-10L) that has increased biological
activity. Further variants of IFN-.gamma. can be found in, for
example, U.S. Pat. No. 5,690,925 and U.S. Pat. No. 6,046,034 both
of which provide guidance as to the amino acid substitutions and
deletions that can be made in IFN-.gamma. without losing biological
activity. Each of these references is herein incorporated by
reference. The above examples represent non-limiting examples of
IFN-.gamma. polypeptides and IFN-.gamma. variant polypeptides
encompassed by the invention. These citations also provide guidance
regarding residues and regions of the IFN-.gamma. polypeptide that
can be altered without the loss of biological activity.
[0069] Biologically active IFN-.gamma. variants encompassed by the
invention also include IFN-.gamma. polypeptides that have
covalently linked with, for example, polyethylene glycol (PEG) or
albumin.
[0070] Biologically active variants of IFN-.gamma. encompassed by
the invention should retain IFN-.gamma. activities, particularly
the ability to bind to IFN-.gamma. receptors or retain
immunomodulatory, antiviral, or antiproliferative activities. In
some embodiments, the IFN-.gamma. variant retains at least about
25%, about 50%, about 75%, about 85%, about 90%, about 95%, about
98%, about 99% or more of the biological activity of the native
IFN-.gamma. polypeptide. IFN-.gamma. variants whose activity is
increased in comparison with the activity of the native IFN-.gamma.
polypeptide are also encompassed. The biological activity of
IFN-.gamma. variants can be measured by any method known in the
art. Examples of such assays are described above.
[0071] In one embodiment of the present invention, the IFN-.gamma.
used in the methods of the invention is the mature native human
IFN-.gamma. polypeptide. However, the present invention encompasses
other embodiments where the IFN-.gamma. is any biologically active
IFN-.gamma. polypeptide or variant as described elsewhere
herein.
[0072] In some embodiments of the present invention, the
IFN-.gamma. is recombinantly produced. By "recombinantly produced
IFN-.gamma." is intended IFN-.gamma. that has comparable biological
activity to native IFN-.gamma. and that has been prepared by
recombinant DNA techniques. IFN-.gamma. can be produced by
culturing a host cell transformed with an expression vector
comprising a nucleotide sequence that encodes an IFN-.gamma.
polypeptide. The host cell is one that can transcribe the
nucleotide sequence and produce the desired protein, and can be
prokaryotic (for example, E. coli) or eukaryotic (for example a
yeast, insect, or mammalian cell). Examples of recombinant
production of IFN-.gamma. are given in U.S Pat. Nos. 6,046,034 and
5,690,925; both of which are herein incorporated by reference.
[0073] Pharmaceutical Composition
[0074] Increases in the amount of cytokine in the CNS, brain,
and/or spinal cord to a therapeutically effective level may be
obtained via administration of a pharmaceutical composition
including a therapeutically effective dose of this cytokine. By
"therapeutically effective dose" is intended a dose of cytokine
that achieves the desired goal of increasing the amount of this
cytokine in a relevant portion of the CNS, brain, and/or spinal
cord to a therapeutically effective level enabling a desired
biological activity of the cytokine.
[0075] The invention is, in particular, directed to a composition
that can be employed for delivery of a cytokine to the CNS, brain,
and/or spinal cord upon administration to tissue innervated by the
olfactory and/or trigeminal nerves. The composition can include,
for example, any pharmaceutically acceptable additive, carrier, or
adjuvant that is suitable for administering a cytokine to tissue
innervated by the olfactory and/or trigeminal nerves. Preferably,
the pharmaceutical composition can be employed in diagnosis,
prevention, or treatment of a disease, disorder, or injury of the
CNS, brain, and/or spinal cord. Preferably, the composition
includes a cytokine in combination with a pharmaceutical carrier,
additive, and/or adjuvant that can promote the transfer of the
cytokine within or through tissue innervated by the olfactory
and/or trigeminal nerves. Alternatively, the cytokine may be
combined with substances that may assist in transporting the
cytokine to sites of nerve cell damage. The composition can include
one or several cytokines.
[0076] The composition typically contains a pharmaceutically
acceptable carrier mixed with the cytokine and other components in
the pharmaceutical composition. By "pharmaceutically acceptable
carrier" is intended a carrier that is conventionally used in the
art to facilitate the storage, administration, and/or the healing
effect of the cytokine. A carrier may also reduce any undesirable
side effects of the cytokine. A suitable carrier should be stable,
i.e., incapable of reacting with other ingredients in the
formulation. It should not produce significant local or systemic
adverse effect in recipients at the dosages and concentrations
employed for treatment. Such carriers are generally known in the
art.
[0077] Suitable carriers for this invention include those
conventionally used for large stable macromolecules such as
albumin, gelatin, collagen, polysaccharide, monosaccharides,
polyvinylpyrrolidone, polylactic acid, polyglycolic acid, polymeric
amino acids, fixed oils, ethyl oleate, liposomes, glucose, sucrose,
lactose, mannose, dextrose, dextran, cellulose, mannitol, sorbitol,
polyethylene glycol (PEG), and the like.
[0078] Water, saline, aqueous dextrose, and glycols are preferred
liquid carriers, particularly (when isotonic) for solutions. The
carrier can be selected from various oils, including those of
petroleum, animal, vegetable or synthetic origin, for example,
peanut oil, soybean oil, mineral oil, sesame oil, and the like.
Suitable pharmaceutical excipients include starch, cellulose, talc,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, magnesium stearate, sodium stearate, glycerol
monostearate, sodium chloride, dried skim milk, glycerol, propylene
glycol, water, ethanol, and the like. The compositions can be
subjected to conventional pharmaceutical expedients, such as
sterilization, and can contain conventional pharmaceutical
additives, such as preservatives, stabilizing cytokines, wetting,
or emulsifying agents, salts for adjusting osmotic pressure,
buffers, and the like.
[0079] A composition formulated for intranasal delivery may
optionally comprise an odorant. An odorant agent is combined with
the cytokine to provide an odorliferous sensation, and/or to
encourage inhalation of the intranasal preparation to enhance
delivery of the active cytokine to the olfactory neuroepithelium.
The odorliferous sensation provided by the odorant agent may be
pleasant, obnoxious, or otherwise malodorous. The odorant receptor
neurons are localized to the olfactory epithelium that, in humans,
occupies only a few square centimeters in the upper part of the
nasal cavity. The cilia of the olfactory neuronal dendrites which
contain the receptors are fairly long (about 30-200 um). A 10-30
.mu.m layer of mucus envelops the cilia that the odorant agent must
penetrate to reach the receptors. See Snyder et al. (1998) J Biol.
Chem. 263:13972-13974. Use of a lipophillic odorant agent having
moderate to high affinity for odorant binding protein (OBP) is
preferred. OBP has an affinity for small lipophillic molecules
found in nasal secretions and may act as a carrier to enhance the
transport of a lipophillic odorant substance and cytokines to the
olfactory receptor neurons. It is also preferred that an odorant
agent is capable of associating with lipophillic additives such as
liposomes and micelles within the preparation to farther enhance
delivery of the cytokines by means of OBP to the olfactory
neuroepithelium. OBP may also bind directly to lipophillic agents
to enhance transport of the cytokines to olfactory neural
receptors.
[0080] Suitable odorants having a high affinity for OBP include
terpanoids such as cetralva and citronellol, aldehydes such as amyl
clnnamaldehyde and hexyl cinnamaldehyde, esters such as octyl
isovalerate, jasmines such as C1S-jasmine and jasmal, and musk 89.
Other suitable odorant agents include those which may be capable of
stimulating odorant-sensitive enzymes such as aderrylate cyclase
and guanylate cyclase, or which may be capable of modifying ion
channels within the olfactory system to enhance absorption of the
cytokine.
[0081] Other acceptable components in the composition include, but
are not limited to, pharmaceutically acceptable agents that modify
isotonicity, including water, salts, sugars, polyols, amino acids
and buffers, such as, phosphate, citrate, succinate, acetate, and
other organic acids or their salts. Typically, the pharmaceutically
acceptable carrier also includes one or more stabilizers, reducing
agents, anti-oxidants and/or anti-oxidant chelating agents. The use
of buffers, stabilizers, reducing agents, anti-oxidants and
chelating agents in the preparation of protein based compositions,
particularly pharmaceutical compositions, is well known in the art.
See Wang et al. (1980) J. Parent. Drug Assn., 34(6):452-462; Wang
et al. (1988) J Parent. Sci. and Tech. 42:S4-S26 (Supplement);
Lachman, et al. (1968) Drug and Cosmetic Industry, 102(1): 36-38,
40 and 146-148; Akers, M. J. (1988) J Parent. Sci. and Tech.,
36(5):222-228; and Colowick et al. Methods in Enzymology, Vol. XXV,
p. 185-188.
[0082] Suitable buffers include acetate, adipate, benzoate,
citrate, lactate, maleate, phosphate, tartarate, borate,
tri(hydroxymethyl aminomethane), succinate, glycine, histidine, the
salts of various amino acids, or the like, or combinations thereof.
See Wang (1980) supra at page 455. Suitable salts and
isotonicifiers include sodium chloride, dextrose, mannitol,
sucrose, trehalose, or the like. Where the carrier is a liquid, it
is preferred that the carrier is hypotonic or isotonic with oral,
conjunctival or dermal fluids and have a pH within the range of
4.5-8.5. Where the carrier is in powdered form, it is preferred
that the carrier is also within an acceptable non-toxic pH
range.
[0083] Suitable reducing agents, which maintain the reduction of
reduced cysteines, include dithiothreitol (DTT also known as
Cleland's reagent) or dithioerythritol at 0.01% to 0.1% wt/wt;
acetylcysteine or cysteine at 0.1% to 0.5% (pH 2-3); and
thioglycerol at 0.1% to 0.5% (pH 3.5 to 7.0) and glutathione. See
Akers (1988) supra at pages 225 to 226. Suitable antioxidants
include sodium bisulfite, sodium sulfite, sodium metabisulfite,
sodium thiosulfate, sodium formaldehyde sulfoxylate, and ascorbic
acid. See Akers (1988) supra at pages 225. Suitable chelating
agents, which chelate trace metals to prevent the trace metal
catalyzed oxidation of reduced cysteines, include citrate,
tartarate, ethylenediaminetetraacetic acid (EDTA) in its disodium,
tetrasodium, and calcium disodium salts, and diethylenetriamine
pentaacetic acid (DTPA). See, e.g., Wang (1980) supra at pages
457-458 and 460-461, and Akers (1988) supra at pages 224-227.
[0084] The composition can include one or more preservatives such
as phenol, cresol, p-aminobenzoic acid, BDSA, sorbitrate,
chlorhexidine, benzalkonium chloride, or the like. Suitable
stabilizers include carbohydrates such as trehalose or glycerol.
The composition can include a stabilizer such as one or more of
microcrystalline cellulose, magnesium stearate, mannitol, sucrose
to stabilize, for example, the physical form of the composition;
and one or more of glycine, arginine, hydrolyzed collagen, or
protease inhibitors to stabilize, for example, the chemical
structure of the composition. Suitable suspending additives include
carboxymethyl cellulose, hydroxypropyl methylcellulose, hyaluronic
acid, alginate, chondroitin sulfate, dextran, maltodextrin, dextran
sulfate, or the like. The composition can include an emulsifier
such as polysorbate 20, polysorbate 80, pluronic, triolein, soybean
oil, lecithins, squalene and squalanes, sorbitan treioleate, or the
like. The composition can include an antimicrobial such as
phenylethyl alcohol, phenol, cresol, benzalkonium chloride,
phenoxyethanol, chlorhexidine, thimerosol, or the like. Suitable
thickeners include natural polysaccharides such as mannans,
arabinans, alginate, hyaluronic acid, dextrose, or the like; and
synthetic ones like the PEG hydrogels of low molecular weight and
aforementioned suspending cytokines.
[0085] The composition can include an adjuvant such as cetyl
trimethyl ammonium bromide, BDSA, cholate, deoxycholate,
polysorbate 20 and 80, fusidic acid, or the like, and in the case
of DNA delivery, preferably, a cationic lipid. Suitable sugars
include glycerol, threose, glucose, galactose, mannitol, and
sorbitol. A suitable protein is human serum albumin.
[0086] Preferred compositions include one or more of a solubility
enhancing additive, preferably a cyclodextrin; a hydrophilic
additive, preferably a monosaccharride or oligosaccharide; an
absorption promoting additive, preferably a cholate, a
deoxycholate, a fusidic acid, or a chitosan; a cationic surfactant,
preferably a cetyl trimethyl ammonium bromide; a viscosity
enhancing additive, preferably to promote residence time of the
composition at the site of administration, preferably a
carboxymethyl cellulose, a maltodextrin, an alginic acid, a
hyaluronic acid, or a chondroitin sulfate; or a sustained release
matrix, preferably a polyanhydride, a polyorthoester, a hydrogel, a
particulate slow release depo system, preferably a polylactide
co-glycolides (PLG), a depo foam, a starch microsphere, or a
cellulose derived buccal system; a lipid-based carrier, preferably
an emulsion, a liposome, a niosomes, or a micelles. The composition
can include a bilayer destabilizing additive, preferably a
phosphatidyl ethanolamine; a fusogenic additive, preferably a
cholesterol hemisuccinate.
[0087] Other preferred compositions for sublingual administration
including, for example, a bioadhesive to retain the cytokine
sublingually; a spray, paint, or swab applied to the tongue;
retaining a slow dissolving pill or lozenge under the tongue; or
the like. Other preferred compositions for transdermal
administration include a bioadhesive to retain the cytokine on or
in the skin; a spray, paint, cosmetic, or swab applied to the skin;
or the like.
[0088] These lists of carriers and additives is by no means
complete and a worker skilled in the art can choose excipients from
the GRAS (generally regarded as safe) list of chemicals allowed in
the pharmaceutical preparations and those that are currently
allowed in topical and parenteral formulations.
[0089] For the purposes of this invention, the pharmaceutical
composition comprising the cytokine can be formulated in a unit
dosage and in a form such as a solution, suspension, or emulsion.
The cytokine may be administered to tissue innervated by the
trigeminal and/or olfactory neurons as a powder, a granule, a
solution, a cream, a spray (e.g., an aerosol), a gel, an ointment,
an infusion, an injection, a drop, or sustained-release
composition, such as a polymer disk. For buccal administration, the
compositions can take the form of tablets or lozenges formulated in
a conventional manner. For administration to the eye or other
external tissues, e.g., mouth and skin, the compositions can be
applied to the infected part of the body of the patient as a
topical ointment or cream. The compounds can be presented in an
ointment, for instance with a water-soluble ointment base, or in a
cream, for instance with an-oil-in water cream base. For
conjunctival applications, the cytokine can be administered in
biodegradable or non-degradable ocular inserts. The drug may be
released by matrix erosion or passively through a pore as in
ethylene-vinylacetate polymer inserts. For other mucosal
administrations, such as sublingual, powder discs may be placed
under the tongue and active delivery systems may for in situ by
slow hydration as in the formulation of liposomes from dried lipid
mixtures or pro-liposomes.
[0090] Other preferred forms of compositions for administration
include a suspension of a particulate, such as an emulsion, a
liposome, an insert that releases the cytokine slowly, and the
like. The powder or granular forms of the pharmaceutical
composition may be combined with a solution and with a diluting,
dispersing, or surface-active cytokine. Additional preferred
compositions for administration include a bioadhesive to retain the
cytokine at the site of administration; a spray, paint, or swab
applied to the mucosa or epithelium; a slow dissolving pill or
lozenge; or the like. The composition can also be in the form of
lyophilized powder, which can be converted into a solution,
suspension, or emulsion before administration. The pharmaceutical
composition including cytokine is preferably sterilized by membrane
filtration and is stored in unit-dose or multi-dose containers such
as sealed vials or ampoules.
[0091] The method for formulating a pharmaceutical composition is
generally known in the art. A thorough discussion of formulation
and selection of pharmaceutically acceptable carriers, stabilizers,
and isomolytes can be found in Remington's Pharmaceutical Sciences
(18.sup.th ed.; Mack Publishing Company, Eaton, Pa., 1990), herein
incorporated by reference.
[0092] The cytokine of the present invention can also be formulated
in a sustained-release form to prolong the presence of the
pharmaceutically active cytokine in the treated mammal, generally
for longer than one day. Many methods of preparation of a
sustained-release formulation are known in the art and are
disclosed in Remington's Pharmaceutical Sciences (18.sup.th ed.;
Mack Publishing Company, Eaton, Pennsylvania, 1990), herein
incorporated by reference.
[0093] Generally, the cytokine can be entrapped in semipermeable
matrices of solid hydrophobic polymers. The matrices can be shaped
into films or microcapsules. Examples of such matrices include, but
are not limited to, polyesters, copolymers of L-glutamic acid and
gamma ethyl-L-glutamate (Sidman et al. (1983) Biopolymers
22:547-556), polylactides (U.S. Pat. No. ,773,919 and EP 58,481),
polylactate polyglycolate (PLGA) such as polylactide-co-glycolide
(see, for example, U.S. Pat. Nos. 4,767,628 and 5,654,008),
hydrogels (see, for example, Langer et al. (1981) J Biomed. Mater.
Res. 15:167-277; Langer (1982) Chem. Tech. 12:98-105),
non-degradable ethylene-vinyl acetate (e.g. ethylene vinyl acetate
disks and poly(ethylene-co-vinyl acetate)), degradable lactic
acid-glycolic acid copolyers such as the Lupron Depot.TM.,
poly-D-(-)-3-hydroxybutyric acid (EP 133,988), hyaluronic acid gels
(see, for example, U.S. Pat. No. 4,636,524), alginic acid
suspensions, and the like.
[0094] Suitable microcapsules can also include
hydroxymethylcellulose or gelatin-microcapsules and polymethyl
methacrylate microcapsules prepared by coacervation techniques or
by interfacial polymerization. See the PCT publication WO 99/24061
entitled "Method for Producing Sustained-release Formulations,"
wherein a protein is encapsulated in PLGA microspheres, herein
incorporated by reference. In addition, microemulsions or colloidal
drug delivery systems such as liposomes and albumin microspheres,
may also be used. See Remington's Pharmaceutical Sciences
(18.sup.th ed.; Mack Publishing Company Co., Eaton, Pa., 1990).
Other preferred sustained-release compositions employ a bioadhesive
to retain the cytokine at the site of administration.
[0095] Among the optional substances that may be combined with the
cytokine in the pharmaceutical composition are lipophilic
substances that can enhance absorption of the cytokine through the
mucosa or epithelium of the nasal cavity, or along a neural,
lymphatic, or perivascular pathway to damaged nerve cells in the
CNS. The cytokine may be mixed with a lipophilic adjuvant alone or
in combination with a carrier, or may be combined with one or
several types of micelle or liposome substances. Among the
preferred lipophilic substances are cationic liposomes included of
one or more of the following: phosphatidyl choline, lipofectin,
DOTAP, a lipid-peptoid conjugate, a synthetic phospholipid such as
phosphatidyl lysine, or the like. These liposomes may include other
lipophilic substances such as gangliosides and phosphatidylserine
(PS). Also preferred are micellar additives such as GM-1
gangliosides and phosphatidylserine (PS), which may be combined
with the cytokine either alone or in combination. GM-1 ganglioside
can be included at 1-10 mole percent in any liposomal compositions
or in higher amounts in micellar structures. Protein cytokines can
be either encapsulated in particulate structures or incorporated as
part of the hydrophobic portion of the structure depending on the
hydrophobicity of the active cytokine.
[0096] One preferred liposomal formulation employs Depofoam. A
cytokine can be encapsulated in multivesicular liposomes, as
disclosed in the WO publication 99/12522 entitled "High and Low
Load Formulations of IGF-I in Multivesicular Liposomes," herein
incorporated by reference. The mean residence time of cytokine at
the site of administration can be prolonged with a Depofoam
composition.
[0097] Administering the Cytokine
[0098] According to this embodiment of the invention, the total
amount of cytokine administered per dose should be in a range
sufficient to delivery a biologically relevant amount of the
cytokine (i.e., an amount sufficient to produce a therapeutical
effect). The pharmaceutical composition having a unit dose of
cytokine can be in the form of solution, suspension, emulsion, or a
sustained-release formulation. The total volume of one dose of the
pharmaceutical composition can range from about 10 .mu.l to about
100 .mu.l, for example, for nasal administration. It is apparent
that the suitable volume can vary with factors such as the size of
the tissue to which the cytokine is administered and the solubility
of the components in the composition.
[0099] It is recognized that the total amount of cytokine
administered as a unit dose to a particular tissue will depend upon
the type of pharmaceutical composition being administered, that is
whether the composition is in the form of, for example, a solution,
a suspension, an emulsion, or a sustained-release formulation. For
example, where the pharmaceutical composition comprising a
therapeutically effective amount of cytokine is a sustained-release
formulation, cytokine is administered at a higher concentration.
Needle-free subcutaneous administration to an extranasal tissue
innervated by the trigeminal nerve may be accomplished by use of a
device which employs a supersonic gas jet as a power source to
accelerate an agent that is formulated as a powder or a
microparticle into the skin. The characteristics of such a delivery
method will be determined by the properties of the particle, the
formulation of the agent and the gas dynamics of the delivery
device. Similarly, the subcutaneous delivery of an aqueous
composition can be accomplished in a needle-free manner by
employing a gas-spring powered hand held device to produce a high
force jet of fluid capable of penetrating the skin. Alternatively,
a skin-patch formulated to mediate a sustained release of a
composition can be employed for the transdermal delivery of a
neuroregulatory agent to a tissue innervated by the trigeminal
nerve. Where the pharmaceutical composition comprises a
therapeutically effective amount of an agent, or a combination of
agents, in a sustained-release formulation, the agent(s) is/are
administered at a higher concentration.
[0100] It should be apparent to a person skilled in the art that
variations may be acceptable with respect to the therapeutically
effective dose and frequency of the administration a cytokine in
this embodiment of the invention. The amount of the cytokine
administered will be inversely correlated with the frequency of
administration. Hence, an increase in the concentration of cytokine
in a single administered dose, or an increase in the mean residence
time in the case of a sustained-release form of cytokine, generally
will be coupled with a decrease in the frequency of
administration.
[0101] In the practice of the present invention, additional factors
should be taken into consideration when determining the
therapeutically effective dose of cytokine and frequency of its
administration. Such factors include, for example, the size of the
tissue, the area of the surface of the tissue, the severity of the
disease or disorder, and the age, height, weight, health, and
physical condition of the individual to be treated. Generally, a
higher dosage is preferred if the tissue is larger or the disease
or disorder is more severe.
[0102] Some minor degree of experimentation may be required to
determine the most effective dose and frequency of dose
administration, this being well within the capability of one
skilled in the art once apprised of the present disclosure.
[0103] For the treatment of a disorder of the CNS in a human,
including neurologic, viral, proliferative or immunomodulatory
disorders, a therapeutically effective amount or dose of a cytokine
is about 0.14 nmol/kg of brain weight to about 138 nmol/kg brain
weight and about 0.14 nmol/kg of brain weight to about 69 nmol/kg
of brain weight. In some regimens, therapeutically effective doses
for administration of a cytokine include about 0. 13, 0.2, 0.4,
0.6, 0.8, 1.0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,
130, or 140 nmoles per kg of brain weight. For the treatment of a
disorder of the CNS in a human, including neurologic, viral,
proliferative or immunomodulatory disorders, the therapeutically
effective amount or dose of IFN-.beta. or biologically active
variant thereof is about 0.14 nmol/kg of brain weight to about 138
nmol/kg of brain weight and about 0.14 nmol/kg of brain weight to
about 69 nmol/kg of brain weight. In some regimens, therapeutically
effective doses for administration of IFN-.beta. include about
0.13, 0.2, 0.4, 0.6, 0.8, 1.0, 10, 20, 30, 40, 50, 60, 70, 80, 90,
100, 110, 120, 130, or 140 nmoles per kg of brain weight.
[0104] It is further recognized that the therapeutically effective
amount or dose of a cytokine to a human may be lower when the
cytokine is administered via the nasal lymphatics to various
tissues of the head and neck for the treatment or prevention of
disorders or diseases characterized by immune and inflammatory
responses (i.e., diseases resulting in acute or chronic
inflammation and/or infiltration by lymphocytes). In these
embodiments, while the cytokine can be administered in the dosage
range provided above, the cytokine may also be administered from
about 0.02 to about 138 pmol/kg of brain weight. Alternatively, the
cytokine may be administered from about 0.02, 0.03, 0.08, 0.1, 0.2,
0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 10, 20, 30, 40, 50, 60, 70,
80, 90, 100, 110, 120, 130, or 140 pmol per kg of brain weight.
Similarly, when the cytokine is IFN-.beta., the dosage range may
also be from about 0.02 to about 138 pmol/kg of brain weight.
Alternatively, the cytokine may be administered from about 0.02,
0.03, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 10, 20,
30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, or 140 pmol per kg
of brain weight.
[0105] These doses depend on factors including the efficiency with
which cytokine IFN-.beta. is transported to the CNS or lymphatic
system. A larger total dose can be delivered by multiple
administrations of the agent.
[0106] Intermittent Dosing
[0107] In another embodiment of the invention, the pharmaceutical
composition comprising the therapeutically effective dose of
cytokine is administered intermittently. By "intermittent
administration" is intended administration of a therapeutically
effective dose of cytokine, followed by a time period of
discontinuance, which is then followed by another administration of
a therapeutically effective dose, and so forth. Administration of
the therapeutically effective dose may be achieved in a continuous
manner, as for example with a sustained-release formulation, or it
may be achieved according to a desired daily dosage regimen, as for
example with one, two, three or more administrations per day. By
"time period of discontinuance" is intended a discontinuing of the
continuous sustained-released or daily administration of cytokine.
The time period of discontinuance may be longer or shorter than the
period of continuous sustained-release or daily administration.
During the time period of discontinuance, the cytokine level in the
relevant tissue is substantially below the maximum level obtained
during the treatment. The preferred length of the discontinuance
period depends on the concentration of the effective dose and the
form of cytokine used. The discontinuance period can be at least 2
days, preferably is at least 4 days, more preferably is at least 1
week and generally does not exceed a period of 4 weeks. When a
sustained-release formulation is used, the discontinuance period
must be extended to account for the greater residence time of
cytokine at the site of injury. Alternatively, the frequency of
administration of the effective dose of the sustained-release
formulation can be decreased accordingly. An intermittent schedule
of administration of cytokine can continue until the desired
therapeutic effect, and ultimately treatment of the disease or
disorder, is achieved.
[0108] In yet another embodiment, intermittent administration of
the therapeutically effective dose of cytokine is cyclic. By
"cyclic" is intended intermittent administration accompanied by
breaks in the administration, with cycles ranging from about 1
month to about 2, 3, 4, 5, or 6 months. For example, the
administration schedule might be intermittent administration of the
effective dose of cytokine, wherein a single short-term dose is
given once per week for 4 weeks, followed by a break in
intermittent administration for a period of 3 months, followed by
intermittent administration by administration of a single
short-term dose given once per week for 4 weeks, followed by a
break in intermittent administration for a period of 3 months, and
so forth. As another example, a single short-term dose may be given
once per week for 2 weeks, followed by a break in intermittent
administration for a period of 1 month, followed by a single
short-term dose given once per week for 2 weeks, followed by a
break in intermittent administration for a period of 1 month, and
so forth. A cyclic intermittent schedule of administration of
cytokine to subject may continue until the desired therapeutic
effect, and ultimately treatment of the disorder or disease, is
achieved.
[0109] Neuronal Transport
[0110] One embodiment of the present method includes administration
of the cytokine to the subject in a manner such that the cytokine
is transported to the lymphatic system, the lacrimal gland, CNS,
brain, and/or spinal cord along a neural pathway. A neural pathway
includes transport within or along a neuron, through or by way of
lymphatics running with a neuron, through or by way of a
perivascular space of a blood vessel running with a neuron or
neural pathway, through or by way of an adventitia of a blood
vessel running with a neuron or neural pathway, or through an
hemangiolymphatic system. The invention prefers transport of a
cytokine by way of a neural pathway, rather than through the
circulatory system, so that cytokines that are unable to, or only
poorly, cross the blood-brain barrier from the bloodstream into the
brain can be delivered to the lymphatic system, CNS, brain, and/or
spinal cord. The cytokine, once past the blood-brain barrier and in
the CNS, can then be delivered to various areas of the brain or
spinal cord through lymphatic channels, through a perivascular
space, or transport through or along neurons. In one embodiment,
the cytokine preferably accumulates in areas having the greatest
density of receptor or binding sites for that cytokine.
[0111] Use of a neural pathway to transport a cytokine to the
lymphatic system, lacrimal gland, brain, spinal cord, or other
components of the central nervous system obviates the obstacle
presented by the blood-brain barrier so that medications that
cannot normally cross that barrier, can be delivered directly to
the brain, cerebellum, brain stem, or spinal cord. Although the
cytokine that is administered may be absorbed into the bloodstream
as well as the neural pathway, the cytokine preferably provides
minimal effects systemically. In addition, the invention can
provide for delivery of a more concentrated level of the cytokine
to neural cells since the cytokine does not become diluted in
fluids present in the bloodstream. As such, the invention provides
an improved method for delivering a cytokine to the lymphatic
system, CNS, brain, and/or spinal cord.
[0112] The Olfactory Neural Pathway
[0113] One embodiment of the present method includes delivery of
the cytokine to the subject in a manner such that the cytokine is
transported into the CNS, brain, and/or spinal cord along an
olfactory neural pathway. Typically, such an embodiment includes
administering the cytokine to tissue innervated by the olfactory
nerve and inside the nasal cavity. The olfactory neural pathway
innervates primarily the olfactory epithelium in the upper third of
the nasal cavity, as described above. Application of the cytokine
to a tissue innervated by the olfactory nerve can deliver the
cytokine to damaged neurons or cells of the CNS, brain, and/or
spinal cord. Olfactory neurons innervate this tissue and can
provide a direct connection to the CNS, brain, and/or spinal cord
due, it is believed, to their role in olfaction.
[0114] Delivery through the olfactory neural pathway can employ
lymphatics that travel with the olfactory nerve to the various
brain areas and from there into dural lymphatics associated with
portions of the CNS, such as the spinal cord. Transport along the
olfactory nerve can also deliver cytokines to an olfactory bulb. A
perivascular pathway and/or a hemangiolymphatic pathway, such as
lymphatic channels running within the adventitia of cerebral blood
vessels, can provide an additional mechanism for transport of
therapeutic cytokines to the brain and spinal cord from tissue
innervated by the olfactory nerve.
[0115] A cytokine can be administered to the olfactory nerve, for
example, through the olfactory epithelium. Such administration can
employ extracellular or intracellular (e.g., transneuronal)
anterograde and retrograde transport of the cytokine entering
through the olfactory nerves to the brain and its meninges, to the
brain stem, or to the spinal cord. Once the cytokine is dispensed
into or onto tissue innervated by the olfactory nerve, the cytokine
may transport through the tissue and travel along olfactory neurons
into areas of the CNS including the brain stem, cerebellum, spinal
cord, olfactory bulb, and cortical and subcortical structures.
[0116] Delivery through the olfactory neural pathway can employ
movement of a cytokine into or across mucosa or epithelium into the
olfactory nerve or into a lymphatic, a blood vessel perivascular
space, a blood vessel adventitia, or a blood vessel lymphatic that
travels with the olfactory nerve to the brain and from there into
meningial lymphatics associated with portions of the CNS such as
the spinal cord. Blood vessel lymphatics include lymphatic channels
that are around the blood vessels on the outside of the blood
vessels. This also is referred to as the hemangiolymphatic system.
Introduction of a cytokine into the blood vessel lymphatics does
not necessarily introduce the cytokine into the blood.
[0117] The Trigeminal Neural Pathway
[0118] One embodiment of the present method includes delivery of
the cytokine to the subject in a manner such that the cytokine is
transported into the CNS, brain, and/or spinal cord along a
trigeminal neural pathway. Typically, such an embodiment includes
administering the cytokine to tissue innervated by the trigeminal
nerve including inside and outside the nasal cavity. The trigeminal
neural pathway innervates various tissues of the head and face, as
described above. In particular, the trigeminal nerve innervates the
nasal, sinusoidal, oral and conjunctival mucosa or epithelium, and
the skin of the face. Application of the cytokine to a tissue
innervated by the trigeminal nerve can deliver the cytokine to
damaged neurons or cells of the CNS, brain, and/or spinal cord to
cells of the lymphatic system. Trigeminal neurons innervate these
tissues and can provide a direct connection to the CNS, brain,
and/or spinal cord due, it is believed, to their role in the common
chemical sense including mechanical sensation, thermal sensation
and nociception (for example detection of hot spices and of noxious
chemicals).
[0119] Delivery through the trigeminal neural pathway can employ
lymphatics that travel with the trigeminal nerve to the pons and
other brain areas and from there into dural lymphatics associated
with portions of the CNS, such as the spinal cord. Transport along
the trigeminal nerve can also deliver cytokines to an olfactory
bulb. A perivascular pathway and/or a hemangiolymphatic pathway,
such as lymphatic channels running within the adventitia of
cerebral blood vessels, can provide an additional mechanism for
transport of therapeutic cytokines to the spinal cord from tissue
innervated by the trigeminal nerve.
[0120] The trigeminal nerve includes large diameter axons, which
mediate mechanical sensation, e.g., touch, and small diameter
axons, which mediate pain and thermal sensation, both of whose cell
bodies are located in the semilunar (or trigeminal) ganglion or the
mesencephalic trigeminal nucleus in the midbrain. Certain portions
of the trigeminal nerve extend into the nasal cavity, oral and
conjunctival mucosa and/or epithelium. Other portions of the
trigeminal nerve extend into the skin of the face, forehead, upper
eyelid, lower eyelid, dorsum of the nose, side of the nose, upper
lip, cheek, chin, scalp and teeth. Individual fibers of the
trigeminal nerve collect into a large bundle, travel underneath the
brain and enter the ventral aspect of the pons. A cytokine can be
administered to the trigeminal nerve, for example, through the
nasal cavity's, oral, lingual, and/or conjunctival mucosa and/or
epithelium; or through the skin of the face, forehead, upper
eyelid, lower eyelid, dorsum of the nose, side of the nose, upper
lip, cheek, chin, scalp and teeth. Such administration can employ
extracellular or intracellular (e.g., transneuronal) anterograde
and retrograde transport of the cytokine entering through the
trigeminal nerves to the brain and its meninges, to the brain stem,
or to the spinal cord. Once the cytokine is dispensed into or onto
tissue innervated by the trigeminal nerve, the cytokine may
transport through the tissue and travel along trigeminal neurons
into areas of the CNS including the brain stem, cerebellum, spinal
cord, olfactory bulb, and cortical and subcortical structures.
[0121] Delivery through the trigeminal neural pathway can employ
movement of a cytokine across skin, mucosa, or epithelium into the
trigeminal nerve or into a lymphatic, a blood vessel perivascular
space, a blood vessel adventitia, or a blood vessel lymphatic that
travels with the trigeminal nerve to the pons and from there into
meningial lymphatics associated with portions of the CNS such as
the spinal cord. Blood vessel lymphatics include lymphatic channels
that are around the blood vessels on the outside of the blood
vessels. This also is referred to as the hemangiolymphatic system.
Introduction of a cytokine into the blood vessel lymphatics does
not necessarily introduce the cytokine into the blood.
[0122] Neural Pathways and Nasal Administration
[0123] In one embodiment, the method of the invention can employ
delivery by a neural pathway, e.g., a trigeminal or olfactory
neural pathway, after administration to the nasal cavity. Upon
administration to the nasal cavity, delivery via the trigeminal
neural pathway may employ movement of a cytokine through the nasal
mucosa and/or epithelium to reach a trigeminal nerve or a
perivascular and/or lymphatic channel that travels with the nerve.
Upon administration to the nasal cavity, delivery via the olfactory
neural pathway may employ movement of a cytokine through the nasal
mucosa and/or epithelium to reach the olfactory nerve or a
perivascular and/or lymphatic channel that travels with the
nerve.
[0124] For example, the cytokine can be administered to the nasal
cavity in a manner that employs extracellular or intracellular
(e.g., transneuronal) anterograde and retrograde transport into and
along the trigeminal and/or olfactory nerves to reach the brain,
the brain stem, or the spinal cord. Once the cytokine is dispensed
into or onto nasal mucosa and/or epithelium innervated by the
trigeminal and/or olfactory nerve, the cytokine may transport
through the nasal mucosa and/or epithelium and travel along
trigeminal and/or olfactory neurons into areas of the CNS including
the brain stem, cerebellum, spinal cord, olfactory bulb, and
cortical and subcortical structures. Alternatively, administration
to the nasal cavity can result in delivery of a cytokine into a
blood vessel perivascular space or a lymphatic that travels with
the trigeminal and/or olfactory nerve to the pons, olfactory bulb,
and other brain areas, and from there into meningeal lymphatics
associated with portions of the CNS such as the spinal cord.
Transport along the trigeminal and/or olfactory nerve may also
deliver cytokines administered to the nasal cavity to the olfactory
bulb, midbrain, diencephalon, medulla, and cerebellum. A cytokine
administered to the nasal cavity can enter the ventral dura of the
brain and travel in lymphatic channels within the dura.
[0125] In addition, the method of the invention can be carried out
in a way that employs a perivascular pathway and/or an
hemangiolymphatic pathway, such as a lymphatic channel running
within the adventitia of a cerebral blood vessel, to provide an
additional mechanism for transport of cytokine to the spinal cord
from the nasal mucosa and/or epithelium. A cytokine transported by
the hemangiolymphatic pathway does not necessarily enter the
circulation. Blood vessel lymphatics associated with the circle of
Willis as well as blood vessels following the trigeminal and/or
olfactory nerve can also be involved in the transport of the
cytokine.
[0126] Administration to the nasal cavity employing a neural
pathway can deliver a cytokine to the lymphatic system, brain stem,
cerebellum, spinal cord, and cortical and subcortical structures.
The cytokine alone may facilitate this movement into the CNS,
brain, and/or spinal cord. Alternatively, the carrier or other
transfer-promoting factors may assist in the transport of the
cytokine into and along the trigeminal and/or olfactory neural
pathway. Administration to the nasal cavity of a therapeutic
cytokine can bypass the blood-brain barrier through a transport
system from the nasal mucosa and/or epithelium to the brain and
spinal cord.
[0127] Neural Pathways and Transdermal Administration
[0128] In one embodiment, the method of the invention can employ
delivery by a neural pathway, e.g., a trigeminal neural pathway,
after transdermal administration. Upon transdermal administration,
delivery via the trigeminal neural pathway may employ movement of a
cytokine through the skin to reach a trigeminal nerve or a
perivascular and/or lymphatic channel that travels with the
nerve.
[0129] For example, the cytokine can be administered transdermally
in a manner that employs extracellular or intracellular (e.g.,
transneuronal) anterograde and retrograde transport into and along
the trigeminal nerves to reach the brain, the brain stem, or the
spinal cord. Once the cytokine is dispensed into or onto skin
innervated by the trigeminal nerve, the cytokine may transport
through the skin and travel along trigeminal neurons into areas of
the CNS including the brain stem, cerebellum, spinal cord,
olfactory bulb, and cortical and subcortical structures.
Alternatively, transdermal administration can result in delivery of
a cytokine into a blood vessel perivascular space or a lymphatic
that travels with the trigeminal nerve to the pons, olfactory bulb,
and other brain areas, and from there into meningeal lymphatics
associated with portions of the CNS such as the spinal cord.
Transport along the trigeminal nerve may also deliver transdermally
administered cytokines to the olfactory bulb, midbrain,
diencephalon, medulla and cerebellum. The ethmoidal branch of the
trigeminal nerve enters the cribriform region. An transdermally
administered cytokine can enter the ventral dura of the brain and
travel in lymphatic channels within the dura.
[0130] In addition, the method of the invention can be carried out
in a way that employs a perivascular pathway and/or an
hemangiolymphatic pathway, such as a lymphatic channel running
within the adventitia of a cerebral blood vessel, to provide an
additional mechanism for transport of cytokine to the spinal cord
from the skin. A cytokine transported by the hemangiolymphatic
pathway does not necessarily enter the circulation. Blood vessel
lymphatics associated with the circle of Willis as well as blood
vessels following the trigeminal nerve can also be involved in the
transport of the cytokine.
[0131] Transdermal administration employing a neural pathway can
deliver a cytokine to the brain stem, cerebellum, spinal cord and
cortical and subcortical structures. The cytokine alone may
facilitate this movement into the CNS, brain, and/or spinal cord.
Alternatively, the carrier or other transfer-promoting factors may
assist in the transport of the cytokine into and along the
trigeminal neural pathway. Transdermal administration of a
therapeutic cytokine can bypass the blood-brain barrier through a
transport system from the skin to the brain and spinal cord.
[0132] Neural Pathways and Sublingual Administration
[0133] In another embodiment, the method of the invention can
employ delivery by a neural pathway, e.g., a trigeminal neural
pathway, after sublingual administration. Upon sublingual
administration, delivery via the trigeminal neural pathway may
employ movement of a cytokine from under the tongue and across the
lingual epithelium to reach a trigeminal nerve or a perivascular or
lymphatic channel that travels with the nerve.
[0134] For example, the cytokine can be administered sublingually
in a manner that employs extracellular or intracellular (e.g.,
transneuronal) anterograde and retrograde transport through the
oral mucosa and then into and along the trigeminal nerves to reach
the brain, the brain stem, or the spinal cord. Once the cytokine is
administered sublingually, the cytokine may transport through the
oral mucosa by means of the peripheral processes of trigeminal
neurons into areas of the CNS including the brain stem, spinal cord
and cortical and subcortical structures. Alternatively, sublingual
administration can result in delivery of a cytokine into lymphatics
that travel with the trigeminal nerve to the pons and other brain
areas and from there into meningeal lymphatics associated with
portions of the CNS such as the spinal cord. Transport along the
trigeminal nerve may also deliver sublingually administered
cytokines to the olfactory bulbs, midbrain, diencephalon, medulla
and cerebellum. The ethmoidal branch of the trigeminal nerve enters
the cribriform region. A sublingually administered cytokine can
enter the ventral dura of the brain and travel in lymphatic
channels within the dura.
[0135] In addition, the method of the invention can be carried out
in a way that employs an hemangiolymphatic pathway, such as a
lymphatic channel running within the adventitia of a cerebral blood
vessel, to provide an additional mechanism for transport of a
cytokine to the spinal cord from the oral submucosa. A cytokine
transported by the hemangiolymphatic pathway does not necessarily
enter the circulation. Blood vessel lymphatics associated with the
circle of Willis as well as blood vessels following the trigeminal
nerve can also be involved in the transport of the cytokine.
[0136] Sublingual administration employing a neural pathway can
deliver a cytokine to the brain stem, cerebellum, spinal cord and
cortical and subcortical structures. The cytokine alone may
facilitate this movement into the CNS, brain, and/or spinal cord.
Alternatively, the carrier or other transfer-promoting factors may
assist in the transport of the cytokine into and along the
trigeminal neural pathway. Sublingual administration of a
therapeutic cytokine can bypass the blood-brain barrier through a
transport system from the oral mucosa to the brain and spinal
cord.
[0137] Neural Pathways and Conjunctival Administration
[0138] In another embodiment, the method of the invention can
employ delivery by a neural pathway, e.g. a trigeminal neural
pathway, after conjunctival administration. Upon conjunctival
administration, delivery via the trigeminal neural pathway may
employ movement of a cytokine from the conjunctiva through the
conjunctival epithelium to reach the trigeminal nerves or lymphatic
channels that travel with the nerve.
[0139] For example, the cytokine can be administered conjunctivally
in a manner that employs extracellular or intracellular (e.g.,
transneuronal) anterograde and retrograde transport through the
conjunctival mucosa and then into and along the trigeminal nerves
to reach the brain, the brain stem, or the spinal cord. Once the
cytokine is administered conjunctivally, the cytokine may transport
through the conjunctival mucosa by means of the peripheral
processes of trigeminal neurons into areas of the CNS including the
brain stem, spinal cord and cortical and subcortical structures.
Alternatively, conjunctival administration can result in delivery
of a cytokine into lymphatics that travel with the trigeminal nerve
to the pons and other brain areas and from there into meningeal
lymphatics associated with portions of the CNS such as the spinal
cord. Transport along the trigeminal nerve may also deliver
conjunctivally administered cytokines to the olfactory bulbs,
midbrain, diencephalon, medulla and cerebellum. The ethmoidal
branch of the trigeminal nerve enters the cribriform region. An
conjunctivally administered cytokine can enter the ventral dura of
the brain and travel in lymphatic channels within the dura.
[0140] In addition, the method of the invention can be carried out
in a way that employs an hemangiolymphatic pathway, such as a
lymphatic channel running within the adventitia of cerebral blood
vessel, to provide an additional mechanism for transport of a
cytokine to the spinal cord from the conjunctival submucosa. A
cytokine transported by the hemangiolymphatic pathway does not
necessarily enter the circulation. Blood vessel lymphatics
associated with the circle of Willis as well as blood vessels
following the trigeminal nerve can also be involved in the
transport of the cytokine.
[0141] Conjunctival administration employing a neural pathway can
deliver a cytokine to the brain stem, cerebellum, spinal cord and
cortical and subcortical structures. The cytokine alone may
facilitate this movement into the CNS, brain, and/or spinal cord.
Alternatively, the carrier or other transfer-promoting factors may
assist in the transport of the cytokine into and along the
trigeminal neural pathway. Conjunctival administration of a
therapeutic cytokine can bypass the blood-brain barrier through a
transport system from the conjunctival mucosa to the brain and
spinal cord.
[0142] Articles and Methods of Manufacture
[0143] The present invention also includes an article of
manufacture providing a cytokine for administration to the CNS,
brain, and/or spinal cord. The article of manufacture can include a
vial or other container that contains a composition suitable for
the present method together with any carrier, either dried or in
liquid form. The article of manufacture further includes
instructions in the form of a label on the container and/or in the
form of an insert included in a box in which the container is
packaged, for the carrying out the method of the invention. The
instructions can also be printed on the box in which the vial is
packaged. The instructions contain information such as sufficient
dosage and administration information so as to allow the subject or
a worker in the field to administer the cytokine. It is anticipated
that a worker in the field encompasses any doctor, nurse,
technician, spouse, or other care-giver that might administer the
cytokine. The cytokine can also be self-administered by the
subject.
[0144] According to the invention, a cytokine can be used for
manufacturing a cytokine composition or medicament suitable for
intranasal, conjunctival, transdermal, and/or sublingual
administration. For example, a liquid or solid composition can be
manufactured in several ways, using conventional techniques. A
liquid composition can be manufactured by dissolving a cytokine in
a suitable solvent, such as water, at an appropriate pH, including
buffers or other excipients, for example to form a solution
described herein above.
[0145] Disorders of the Central Nervous System
[0146] In one embodiment, the present method can be employed to
deliver cytokines, particularly IFN-.beta., to the brain for
diagnosis, treatment or prevention of disorders or diseases of the
CNS, brain, and/or spinal cord. IFN-.beta. increases the astrocytic
production of nerve growth factor (NGF) (Boutros et al (1997)
Journal of Neurochemistry 69:939-946) and IFN-.beta. sustains
neuronal growth in cell culture (Plioplys et al. (1995)
Neuroimmunodulation 2:131-135). IFN-.beta. has therefore been
associated with neurotrophic activity; hence, the methods of the
present invention can be used for the delivery of a cytokine to the
CNS to treat or prevent disorders or diseases of the CNS, brain,
and/or spinal cord.
[0147] Disorders of the CNS, brain and/or spinal cord can be
neurologic or psychiatric disorders, and include, for example,
brain diseases such as Alzheimer's disease, Parkinson's disease,
Lewy body dementia, multiple sclerosis, epilepsy, cerebellar
ataxia, progressive supranuclear palsy, amyotrophic lateral
sclerosis, affective disorders, anxiety disorders, obsessive
compulsive disorders, personality disorders, attention deficit
disorder, attention deficit hyperactivity disorder, Tourette
Syndrome, Tay Sachs, Nieman Pick, and other lipid storage and
genetic brain diseases and/or schizophrenia. The method can also be
employed in subjects suffering from or at risk for nerve damage
from cerebrovascular disorders such as stroke in the brain or
spinal cord, from CNS infections including meningitis and HIV, from
tumors of the brain and spinal cord, or from a prior disease. The
method can also be employed to deliver cytokines to counter CNS
disorders resulting from ordinary aging (e.g., anosmia or loss of
the general chemical sense), brain injury, or spinal cord
injury.
[0148] Multiple sclerosis is a preferred disease or disorder of the
CNS, brain, and/or spinal cord. Despite its possible presence in
the periphery, multiple sclerosis is a disease of the CNS.
Accordingly, multiple sclerosis may be targeted more efficiently by
a method delivering interferons to the CNS, brain and/or spinal
cord.
[0149] Another preferred disease of the CNS, brain, and/or spinal
cord is meningitis.
[0150] An "effective amount" of a cytokine is an amount sufficient
to prevent, treat, reduce and/or ameliorate the symptoms and/or
underlying causes of any of the above disorders or diseases
discussed herein. In some instances, an "effective amount" is
sufficient to eliminate the symptoms of those diseases and,
perhaps, overcome the disease itself. In the context of the present
invention, the terms "treat" and "therapy" and the like refer to
alleviate, slow the progression, prophylaxis, attenuation or cure
of existing disease. Prevent, as used herein, refers to delaying,
slowing, inhibiting, reducing or ameliorating the onset of the CNS
or brain diseases or disorders. It is preferred that a sufficient
quantity of the cytokine be applied in non-toxic levels in order to
provide an effective level of activity within the CNS to prevent or
treat the disease. The method of the present invention may be used
with any mammal. Exemplary mammals include, but are not limited to
rats, cats, dogs, horses, cows, sheep, pigs, and more preferably
humans.
[0151] Further Embodiments
[0152] Modulation of Immune and Inflammatory Responses
[0153] The method of cytokine administration provided by the
present invention allows for the directed administration of the
cytokine to the nasal lymphatic system. Following entry of the
cytokine into the nasal lymphatics, the cytokine can be distributed
throughout the lymphatics of the head and neck region. Hence, the
method of the present invention can be employed to deliver
cytokines to the lymphatic system including, for example, the deep
and superficial cervical nodes, and to various tissues of the head
and neck for the treatment or prevention of disorders or diseases
characterized by immune and inflammatory responses (i.e., diseases
resulting in acute or chronic inflammation and/or infiltration by
lymphocytes). As such the present invention provides a method to
modulate the immune response. By modulate is intended any up or
down regulation of the immune or inflammatory response (i.e.,
influencing systemic immune function, antigen presentation,
cytokine production, and entry of leukocytes into the CNS).
[0154] Of particular interest in the methods of the invention is
the administration of IFN-.beta.. IFN-.beta., like many of the
interferons, reportedly serves as an immunomodulator on a number of
target cells (Hall et al. (1997) J Neuroimmunol. 72:11-19). For
instance, IFN-.beta. appears to exert antiproliferative action on
macrophages, counteract "the mitogenic stimulus of certain
cytokines", augment natural killer cell activity to induce an
increase in the production of cytotoxic T lymphocytes, and act on
large, granular lymphocytes to increase killer cell activity.
Additionally, IFN-.beta. augments the proliferation of B cells and
the secretion of IgM, IgG, and IgA. It has been shown to upregulate
class I MHC expression to produce an increase in the presentation
of class I restricted antigen CD8 cells (Hall et al. (1997) J
NeuroimmunoL 72:11-19). Conversely, IFN-.beta. exerts an inhibitory
effect on the upregulation of class II surface expression. Hence,
the immunomodulatory activities of IFN-.beta. include, for example,
influencing systemic immune function, antigen presentation,
cytokine production, and entry of leukocytes into the CNS (Yong et
al. (1998) Neurology 51:582-689). Direct delivery of the cytokine
to the lymphatics of the head and neck using the administration
methods of the present invention allows the cytokine to modulate
the immune response, i.e., influence chronic and acute
inflammation, wound healing, and the autoimmune response; modulate
the function by lymphocytes (reduce lymphocyte infiltration of the
injured tissue); etc.
[0155] Given the immunomodulatory role of cytokines, the present
invention can be employed to deliver cytokines, preferably
IFN-.beta., to various tissues of the head and neck for the
treatment and/or prevention of diseases or disorders characterized
by immune and inflammatory responses. Disorders or diseases of
particular interest include Multiple Sclerosis (MS), meningitis,
and Primary Sjogren's Syndrome.
[0156] MS presents in the white matter of the CNS and spinal cord
as a number of sclerotic lesions or plaques (Prineas (1985)
Demyelinating Diseases, Elsvevier: Amsterdam; Raine (1983) Multiple
Sclerosis, Williams and Wilkins: Baltimore; Raine et al. (1988) J.
Neuroimmunol. 20:189-201; and Martin (1997) J. Neural Transmission
(Suppl) 49:53-67). The characteristic MS lesion is inflamed,
exhibits axonal demyelination, axonal degeneration, and is found
around small venules. These characteristics typically evolve early
in plaque development and are hypothesized to occur as a result of
a breakdown in the blood-brain barrier (BBB). As a consequence of
BBB breakdown, infiltrates consisting of various lymphocytes and
macrophages enter the brain. The infiltrates cause a decrease in
inflammation while increasing the presence of glial scar tissue,
and elicit incomplete remyelination (Martin (1997) J. Neural
Transmission (Suppl) 49:53-67). Further, it is hypothesized that
this apparent immunologic attack targets not only the myelin
sheath, but also the oligodendrocytes imperative to CNS myelin
production. Cytokines are known to effectively reduce the symptoms
of MS. For example, interferon-.beta. (IFN-.beta.) has received
interest as a treatment for relapsing-remitting MS. In addition,
interest has also developed in the use of interferon-i as an
effective treatment in autoimmune diseases, such as MS. See, for
example, U.S. Pat. No. 6,060,450, herein incorporated by
reference.
[0157] The immunomodulating activity of IFN-.beta. influences the
clinical symptoms of MS. Hence, IFN-.beta. can be administered
according to the methods of the present invention to treat MS.
While the present invention is not bound by the mechanism of
IFN-.beta. action, the central nervous system damage that ensues in
MS patients is believed to be due to the delayed-type
hypersensitivity response. This is a cell-mediated response. First,
T cells are activated by antigens and conveyed to the lymphoid
organ (activation). The lymphoid organ then activates these T cells
while continuing to recruit more T cells to its site (recruitment).
The activated lymphocytes proliferate and return to circulation
(expansion). Once returned to circulation, the activated
lymphocytes migrate through the blood stream, crossing endothelial
cells lining the capillaries (migration). These migrating
lymphocytes and macrophages target, and are attracted to the area
of inflammation (attraction). Resulting from this attraction, other
lymphocytes continue to the area of inflammation and tissue is
destroyed (tissue destruction). Subsequently, the acute response is
suppressed (via tissue destruction), and repair of the area of
inflammation, which is quite limited in MS, may commence (repair)
(Kelley (1996) J. of Neuroscience Nursing 28:114-120). Therefore,
the migration of activated lymphocytes from the blood initiates the
immune response, thereby allowing BBB penetration of activated
lymphocytes.
[0158] Evidence suggests that the immunomodulatory activity of
IFN-.beta. inhibits IFN-.gamma. upregulation by inhibiting the
expansion stage of the delayed-type hypersensitivity response and
thereby influences the clinical symptoms of MS. Particularly, the
reduction of myelin damage appears to occur as a result of two
hypothesized mechanisms of IFN-.beta. action: (1) inhibition of
IFN-.gamma.-induced macrophage activation, and (2) inhibition of
monocytotic TNF release (Kelly (1996) J. Neuroscience Nursing
28:114-120). Potential sites of IFN-.beta. action construed by
these hypotheses involve systemic immune function, antigen
presentation, cytokine production, and entry of leukocytes into the
CNS (Yong et al. (1998) Neurology 51:682-689). Each of these sites
has been elaborated in human and animal experiments of MS.
[0159] An "effective amount" of a cytokine to treat MS using the
administration methods of the present invention will be sufficient
to reduce or lessen the clinical symptoms of MS. For instance,
experimental allergic encephalomyelitis (EAE) is commonly used as
an animal model of MS. A therapeutically effect amount of a
cytokine delivered by the methods of the present invention will be
such as to improve the clinical symptoms of EAE in the experimental
animal (i.e., rats or mice). EAE in rats is scored on a scale of
0-4:0, clinically normal; 1, flaccid tail paralysis; 2, hind limb
weakness; 3, hind limb paralysis; 4, front and hind limb affected.
An effective amount of cytokine delivered by the methods of the
present invention will be effective if there is at least a 30%,
40%, 50% or greater reduction in the mean cumulative score over
several days following the onset of disease symptoms in comparison
to the control group.
[0160] Furthermore, effective treatment of MS may be examined in
several alternative ways including, EDSS (extended disability
status scale), appearance of exacerbations, or MRI. Satisfying any
of the following criteria evidences effective treatment.
[0161] The EDSS is a means to grade clinical impairment due to MS
(Kurtzke (1983) Neurology 33:1444). Eight functional systems are
evaluated for the type and severity of neurologic impairment.
Briefly, prior to treatment, impairment in the following systems is
evaluated: pyramidal, cerebellar, brainstem, sensory, bowel and
bladder, visual, cerebral, and other. Follow-ups are conducted at
defined intervals. The scale ranges from 0 (normal) to 10 (death
due to MS). A decrease of one full step defines an effective
treatment in the context of the present invention (Kurtzke (1994)
Ann. NeuroL 36:573-79).
[0162] Exacerbations are defined as the appearance of a new symptom
that is attributable to MS and accompanied by an appropriate new
neurologic abnormality (IFN-.beta. MS Study Group, supra). In
addition, the exacerbation must last at least 24 hours and be
preceded by stability or improvement for at least 30 days. Standard
neurological examinations result in the exacerbations being
classified as either mild, moderate, or severe according to changes
in aNeurological Rating Scale (Sipe et al. (1984) Neurology
34:1368). An annual exacerbation rate and proportion of
exacerbation-free patients are determined. Therapy is deemed to be
effective if there is a statistically significant difference in the
rate or proportion of exacerbation-free patients between the
treated group and the placebo group for either of these
measurements. In addition, time to first exacerbation and
exacerbation duration and severity may also be measured. A measure
of effectiveness as therapy in this regard is a statistically
significant difference in the time to first exacerbation or
duration and severity in the treated group compared to control
group.
[0163] MRI can be used to measure active lesions using
gadolinium-DTPA-enhanced imaging (McDonald et al (1994) Ann.
Neurol. 36:14) or the location and extent of lesions using
T.sub.2-weighted techniques. Briefly, baseline MRIs are obtained.
The same imaging plane and patient position are used for each
subsequent study. Areas of lesions are outlined and summed slice by
slice for total lesion area. Three analyses may be done: evidence
of new lesions, rate of appearance of active lesions, and
percentage change in lesion area (Paty et al. (1993) Neurology
43:665). Improvement due to therapy is established when there is a
statistically significant improvement in an individual patient
compared to baseline or in a treated group versus a placebo
group.
[0164] It is further recognized that additional compounds can be
administered with the cytokine to produce a therapeutic effect. For
instance, IGF-1 has been implicated in preventing the depletion of
mature oligodendrocytes and promoting recovery from demyelination
in MS and other demyelinating disorders. See, for example, Mason et
al. (2000) J. Neuroscience 20:5703-5708, herein incorporated by
reference. Hence, IFN-.beta. can be administered in conjunction
with IGF-1 for the treatment of MS. The compounds can be
administered by the methods of the invention. Alternatively, one of
the compounds can be administered by any method known in the art
including, for example, subcutaneous and intramuscular routes.
[0165] The IGF-1 used according to the methods of the present
invention can be in its substantially purified, native,
recombinantly produced, or chemically synthesized forms. For
example, IGF-1 can be isolated directly from blood, such as from
serum or plasma, by known methods. See, for example, Phillips
(1980) New Eng. J Med. 302:371-380; Svoboda et al. (1980)
Biochemistry 19:790-797; Cornell and Boughdady (1982) Prep.
Biochem. 12:57; Cornell and Boughdady (1984) Prep. Biochem. 14:123;
European Patent No. EP 123,228; and U.S. Pat. No. 4,769,361. IGF-1
may also be recombinantly produced in the yeast strain Pichia
pastoris and purified essentially as described in U.S. Pat. Nos.
5,324,639, 5,324,660, and 5,650,496 and International Publication
No. WO 96/40776; all of which are herein incorporated by
reference.
[0166] Alternatively, IGF-1 can be synthesized chemically, by any
of several techniques that are known to those skilled in the
peptide art. See, for example, Li et al. (1983) Proc. Natl. Acad.
Sci. USA 80:2216-2220, Stewart and Young (1984) Solid Phase Peptide
Synthesis (Pierce Chemical Company, Rockford, Ill.), and Barany and
Merrifield (1980) The Peptides: Analysis, Synthesis, Biology, ed.
Gross and Meienhofer, Vol. 2 (Academic Press, New York, 1980), pp.
3-254, for solid phase peptide synthesis techniques; and Bodansky
(1984) Principles of Peptide Synthesis (Springer-Verlag, Berlin);
and Gross and Meienhofer, eds. (1980) The Peptides: Analysis,
Synthesis, Biology, Vol. 1 (Academic Press, New York), for
classical solution synthesis. IGF-1 can also be chemically prepared
by the method of simultaneous multiple peptide synthesis. See, for
example, Houghten (1985) Proc. Natl. Acad. Sci. USA 82:5131-5135;
and U.S. Pat. No. 4,631,211. These references are herein
incorporated by reference. Furthermore, methods to prepare a highly
concentrated, low salt-containing, biologically active form of
IGF-1 or variant thereof are provided in WO 99/24062, entitled
Novel IGF-1 Compositions and Its Use.
[0167] Methods for making IGF-1 fragments, analogues, and
derivatives are available in the art. See generally U.S. Pat. Nos.
4,738,921, 5,158,875, and 5,077,276; International Publication Nos.
WO 85/00831, WO 92/04363, WO 87/01038, and WO 89/05822; and
European Patent Nos. EP 135094, EP 123228, and EP 128733; herein
incorporated by reference.
[0168] In addition, several IGF-1 variants are known in the art and
include those described in, for example, Proc. Natl. Acad. Sci. USA
83 (1986):4904-4907; Biochem. Biophys. Res. Commun. 149
(1987):398-404; J Biol. Chem. 263 (1988):6233-6239; Biochem.
Biophys. Res. Commun. 165 (1989):766-771; Forsbert et al. (1990)
Biochem. J. 271:357-363; U.S. Pat. Nos. 4,876,242 and 5,077,276;
and International Publication Nos. WO 87/01038 and WO 89/05822.
Representative variants include one with a deletion of Glu-3 of the
mature molecule, a variant with up to 5 amino acids truncated from
the N-terminus, a variant with a truncation of the first 3
N-terminal amino acids (referred to as des(1-3)-IGF-1, des-IGF-1,
tIGF-1, or brain IGF), and a variant including the first 17 amino
acids of the B chain of human insulin in place of the first 16
amino acids of human IGF-1.
[0169] Meningitis refers to an inflammatory process of the
leptomeninges and CSF within the subarachnoid space.
Meningoencephalitis applies to inflammation of the meninges and
brain parenchyma. Meningitis is usually caused by an infection, but
chemical meningitis may also occur in response to a non-bacterial
irritant introduced into the subarachonoid space. Infiltration of
the subarachnoid space by carcinoma is referred to as meningeal
carcinomatosis and by lymphoma as lymphomapyogenic (usually
bacterial), aseptic (usually viral), and chronic (most any
infectious agent).
[0170] It has been suggested that the central nervous system damage
that occurs in viral and bacterial meningitis may be more related
to invasion of the surface of the brain by the host's own
lymphocytes in response to the meningitis pathogen, rather than to
the pathogen itself or any toxin produced by the pathogen (Lewis
(1979) The Medusa and The Snail, Penguin Books). In fact, many
patients fall victim to the disease despite the prompt
sterilization of the cerebrospinal fluid using the current
aggressive treatments, such as the third generation cephalosporins.
This unexpected outcome may result from harmful interactions
between host cells/tissues and bacterial components released by
treatment with lytic antibiotics (Scand et al. (1991) J Infect.,
Dis. Supp. 74:173-179). The burst of peptidoglycan, capsular
polysaccharide, and lipopolysaccharide liberated from the bacteria
induce the production of a number of mediators including TNF in the
central nervous system leading to meningeal and perivascular
inflammation in the subarachnoid space. Disruption of the
blood-brain barrier ensues, leading to cerebral edema, ischemia,
and a dramatic increase in intracranial pressure. Those that
survive the acute phase of the disease are often left with multiple
neurological sequelae. Previous results from trials utilizing
steroid-based anti-inflammatories either prior to or concomitant
with antibiotic administration suggest that such an approach may
have value. See, for example, Mustafa et al. (1990) Amer. J.
Diseases of Children 144:883-887. Hence, administration of a
cytokine, particularly interferon-.beta., using the methods of the
present invention could be effective in preventing damage by
activated lymphocytes. The methods of the invention could be used
in conjunction with the existing treatments for meningitis to help
prevent brain damage. Such treatments are described in Harrison's
Principles of Internal Medicine (McGraw Hill, 1994), pp. 2296-2309,
herein incorporated by reference.
[0171] An "effective amount" of a cytokine to treat meningitis
using the administration method of the present invention will be
sufficient to reduce or lessen the clinical symptoms of meningitis.
In preferred embodiments, the cytokine is administered in
conjunction with an antibiotic regiment. As such, an effective
amount of the cytokine augments the activity of the antibiotics and
leads to enhanced survival and/or improved clinical status of the
animals in comparison to animals treated with antibiotics alone.
Such clinical manifestations may include, for example, 1) a more
rapid normalization of the CNS inflammatory indices compared to a
control; 2) a more rapid disappearance in fever as compared to a
control; 3) a reduction in the overall neurologic sequelae; and/or,
4) an improved mortality as compared to a control. More extensive
details regarding the clinical manifestations of meningitis that
can be improved upon the administration of an effective
concentration of a cytokine can be found in Harrison's Principles
of Internal Medicine (McGraw Hill, 1994), pp. 2296-2309, herein
incorporated by reference.
[0172] Primary Sjogren's Syndrome, also known as Dry Eye Syndrome,
is characterized by decreased secretion of the lacrimal glands that
make the aqueous layer of the tear film that lubricates the eyes.
Many patients afflicted with Sjogren's Syndrome also experience dry
mouth due to decreased secretion of the salivary glands. This is an
autoimmune disease characterized by chronic inflammation and
infiltration of the lacrimal and salivary glands by lymphocytes.
Activated T cells of the CD4.sup.+ type that infiltrate the
lacrimal gland mediate tissue destruction (Tabbara et al. (1999)
Eur. J. Ophthalomol. 9:1-7). Recently, nHu-IFN-alpha administered
by the oral mucosa route has been shown to stimulate output (Ship
et al. (1999) J. Interferon Cytokine Res. 19:480-488).
[0173] Hence, the present invention provides a method of
administering cytokines, particularly, IFN-.alpha. and IFN-.beta.,
such that the compounds directly enter the nasal lymphatic system.
The interferon will then be distributed to the lymphatics of the
head and neck region altering the function of the lymphocytes that
affect the lacrimal and salivary glands. It is further recognized
that delivery of the cytokine via the trigeminal or the olfactory
nerve can result in the direct delivery of the cytokine to the
lacrimal gland. This direct delivery of the interferon to the
lymphatics of the head and neck region or directly to the lacrimal
gland will reduce lymphocyte infiltration of the lacrimal and
salivary glands and treat Sjogren's Syndrome.
[0174] An "effective amount" of a cytokine to treat Sjogren's
Syndrome using the administration method of the present invention
will be sufficient to reduce or lessen the clinical symptoms of
Sjogren's Syndrome. As such, an effective amount of the cytokine
leads to an improved clinical status of a patient suffering from
Sjogren's Syndrome in comparison to an untreated patient. For
instance, an improved clinical status of the oral symptoms of
Sjogren's Syndrome includes, for example, an overall increase in
mouth wetness, an improvement in the ability to swallow dry food,
an improvement in the ability to speak continuously, etc. Further,
an effective concentration encompasses any improvement in the
ocular manifestations of Sjogren's Syndrome including, for example,
increase in the wetness of eyes (i.e., a lessening of the sandy or
gritty feeling under the eyelids), an increase in tearing, and a
decrease in burning sensations, redness, itching, and eye fatigue.
Improvements also encompass an improvement in lacrimal function
(i.e., a reduction in lymphocyte infiltration into the lacrimal
gland). A more extensive description of the clinical manifestation
of Sjogren's Syndrome can be found in Harrison's Principles of
Internal Medicine (McGraw Hill, 1994), pp. 1662-1664, herein
incorporated by reference.
[0175] Treatment of Viral Infections
[0176] In another embodiment, the present method can be employed to
deliver cytokines and/or antiviral agents to the lymphatic system,
CNS, brain, and/or spinal cord for the treatment, diagnosis or
prevention of disorders or disease resulting from viral
infection.
[0177] As used herein "treating or preventing viral infection"
means to inhibit virus transmission, or to prevent the virus from
establishing itself in its host CNS, brain or spinal cord, or to
ameliorate or alleviate the symptoms of the disease caused by viral
infection. The treatment is considered therapeutic if there is a
reduction in viral load in the CNS, brain, or spinal cord, decrease
in mortality, and/or morbidity. Of particular interest is the
administration of a cytokine (particularly IFN-.alpha. or
IFN-.beta.) by the methods of the invention for the treatment or
prevention of viral hepatitis.
[0178] Viral hepatitis refers to an infection of the liver caused
by a group of viruses having a particular affinity for the liver
and include hepatitis A virus, hepatitis B virus, hepatitis C
virus, hepatitis D virus, and hepatitis E virus. Of particular
interest is the use of the present invention for the treatment of
hepatitis C.
[0179] Acute infection with hepatitis C virus results in persistent
viral replication and progression to chronic hepatitis in
approximately 90% of cases. While chronic hepatitis C infection is
commonly treated with IFN-.beta. and IFN-.alpha., less than 50% of
the patients have sustained remission following treatment (i.e.,
the eradication of hepatitis C virus). See, for example, Barbaro et
al. (1999) Scand. J. Gastroenterol. 9:928-933; Oketani et al.
(1999) J. Clin. Gastroenterol. 28:49-51; and, Kakizaki et al.
(1999) J. Viral Hepatitis 6:315-319; all of which are herein
incorporated by reference. Similarly, IFN therapy has also been
demonstrated to be an effective treatment for chronic hepatitis B,
however only 25-40% of the patients profit from a long-term
beneficial response to the current interferon therapies.
Combination therapies for viral hepatitis have also been developed,
which combine IFN-therapy with antiviral agents such as ribavirin.
These IFN/antiviral therapies are usually given systemically (i.e.,
intravenously), and hence, the therapeutic agents are not able to
cross the blood-brain barrier. Thus, the hepatitis virus can harbor
in the central nervous system where the therapeutic agents cannot
penetrate. Re-infection and relapse to viral hepatitis symptoms
subsequent to treatment frequently occurs. In addition, viral
hepatitis infection of the CNS can have serious neurologic
consequences. See, for example, Bolay et al. (1996) Clin. Neurol.
Neurosurg. 98:305-308, herein incorporated by reference. Therefore,
new methods of treatment are necessary in the treatment of viral
hepatitis. The methods of the present invention can be used to
administer a cytokine and/or an antiviral agent or any combination
thereof, to the lymphatic system, CNS, brain and/or spinal cord for
the treatment or prevention of viral hepatitis. The methods of the
invention can be used in conjunction with the existing treatments
for viral hepatitis to aid in reducing the clinical symptoms of
hepatitis.
[0180] As used herein, an "effective amount" of a cytokine or an
antiviral agent for the treatment of viral hepatitis using the
administration method of the present invention will be sufficient
to reduce or lessen the clinical symptoms of hepatitis. As such, an
effective amount of the cytokine or antiviral agent administered by
the methods of the present invention will augment the activity of
the systemically administered antiviral/immunomodulatory compounds
used in the art for the treatment of viral hepatitis. As such, the
methods of the invention enhance survival and/or improve clinical
status of the treated animals in comparison to animals treated with
systemic administration methods alone. Improvement in clinical
status includes, for example, the prevention of the progression of
acute viral hepatitis to chronicity, the reduction of the viral
load in chronic hepatitis, and/or the prevention or reduction in
the frequency of re-infection and relapse of viral hepatitis
symptoms, and/or prevent or reduce the neurologic damage resulting
from the viral infection.
[0181] Antiviral agents and cytokines of particular interest
include, for example, ribavirin, thymosins, and cytokines, such as,
IFN-.beta., IFN-.alpha., and IFN-.gamma.. See, for example, Musch
et al. (1998) Hepato-Gastroenterology 45:2282-2294; Barbaro et al.
(1999) Scand. J. Gastroenterol. 9(34):928-933; Oketani et al.
(1999) J. Clin. Gastroenterol. 28:49-51; Kakizaki et al. (1999) J.
Viral Hepatitis 6:315-319; U.S. Pat. No. 6,030,785; U.S. Pat. No.
5,676,942; and U.S. Pat. No. 6,001,799; all of which are herein
incorporated by reference.
[0182] The course of the viral hepatitis and its response to the
treatments administered by the methods of the present invention may
be followed by clinical examination and laboratory findings that
are commonly performed in the art. For instance, elevated serum
alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
are known to occur in uncontrolled hepatitis C. A complete response
to treatment is generally defined as the normalization of these
serum enzymes, particularly ALT (Davis et al. (1989) New England J.
Med. 321:1501-6). Alternatively, hepatitis C virus replication in
subjects in response to the antiviral/immunomodulatory treatment of
the present invention can be followed by measuring hepatitis C
virus RNA in serum samples by, for example, a nested polymerase
chain reaction assay that uses two sets of primers derived from the
NS3 and NS4 non-structural gene regions of the HCV genome (Farci et
al. (1991) New England J. Med. 325:98-104; Ulrich et al. (1990) J.
Clin. Invest. 86:1609-14).
[0183] In another embodiment, the methods of the present invention
can be used to treat or prevent herpes simplex viral infection.
Herpes simplex viruses (HSV-1 and HSV-2) produce a variety of
infections involving mucocutaneous surfaces, the central nervous
system, and occasionally visceral organs. For instance, acute viral
replication at a peripheral site such as the cornea is followed by
viral entry into neuronal termini. Corneal infection is followed by
intra-axonal transport, which moves the virus to the trigeminal
ganglia, where further replication may occur before clearance of
infectious virus and the establishment of latency. Failure to clear
the virus may result in central nervous system infection,
encephalitis, and death. Latency may periodically break down in
response to certain stimuli, leading to viral reactivation and
shedding. The present invention provides a method of administering
a cytokine (via, for example, the trigeminal or olfactory nerve) to
the trigeminal ganglia and/or the CNS, thereby allowing for the
treatment and/or prevention of herpes simplex viral infection.
[0184] The immune response to acute herpes simplex virus infection
involves both innate and acquired immunity. Key mediators of innate
resistance to viral infection include cytokines, particularly
interferons such as IFN-.alpha., IFN-.beta., and IFN-.gamma.. For
instance, IFN-.alpha. has been shown to inhibit the onset of
immediate-early herpes simplex virus gene expression (Oberman et
al. (1988) J. Gen. Virol. 69:1167-1177). Furthermore, in mice
IFN-.alpha. and IFN-.beta. are potent inhibitors of replication in
the cornea. Specifically, studies have shown that following corneal
inoculation in mice, herpes simplex viral titer in both the eyes
and trigeminal ganglia was enhanced by up to 1000 fold in mice
mutant for IFN-.alpha. or IFN-.beta. compared to wild-type control
mice (Leib et al. (1999) J. Exp. Med. 189:663-672, herein
incorporated by reference). The same study further demonstrated
that IFNs significantly reduce productive viral infection and
reduce the spread of virus from intact corneas. Related studies
have also been preformed by Minagawa et al (1997) Antiviral Res.
36:99-105.
[0185] In addition, IFN-.alpha. and IFN-.beta. activate host
defenses such as natural killer cells, which have themselves been
shown to be important in controlling herpes simplex virus infection
and pathology (Bouley et al. (1996) Clin. Immunol. Immunopathol.
80:23-30). IFN-.alpha. and IFN-.beta. have also been suggested to
be important for limiting progress of infection from peripheral
tissues to the nervous system (Halford et al. (1997) Virology
236:328-337). Furthermore, IFN-.gamma. appears to play an important
role in the clearance of herpes simplex virus from the cornea and
in resistance to encephalitis, possibly by inhibiting apoptosis of
neurons (Bouley et al. (1995) J. Immunol. 155:3964-3971, Geiger et
al. (1997) Virology 238:189-197, and Imanishi et al. (2000) J.
Biochem. 127:525-530). Hence, interferons, particularly
IFN-.alpha., IFN-.beta., and IFN-.gamma., play a major role in
limiting herpes simplex viral replication in the cornea, trigiminal
ganglia, and in the nervous system.
[0186] An "effective amount" of a cytokine for the treatment of
herpes simplex virus using the administration method of the present
invention will be sufficient to reduce or lessen the clinical
symptoms of herpes simplex virus. As such, an effective amount of
the cytokine administered by the methods of the present invention
will attenuate the activity of the virus and thereby enhance
survival and/or improve clinical status of the treated animal in
comparison to the untreated control. Improvement in clinical status
includes, for example, the prevention or reduction of encephalitis
and/or apoptosis in the central nervous system (i.e, increase in
neuroprotection), a decrease in the severity of infection (i.e.,
enhancing viral clearance from the cornea, the trigeminal ganglia,
and the CNS), a decrease in viral spread, an increase in the
maintenance of latency, and/or a decrease in the frequency of
herpes simplex recurrences. More extensive details regarding the
clinical manifestations of herpes simplex that can be improved upon
the administration of an effective concentration of a cytokine can
be found in Harrison's Principles of Internal Medicine (McGraw
Hill, 1994), pp. 782-787, herein incorporated by reference.
[0187] In another embodiment, the methods of the invention can be
used for the treatment of human immunodeficiency virus (HIV). HIV
is an infectious disease of the immune system characterized by a
progressive deterioration of the immune system in most infected
subjects. During disease progression, key cells associated with the
immune system become infected with HIV, including, e.g., CD4.sup.+
T cells, macrophages/monocytes, and glial cells. Prolonged HIV
infection frequently culminates in the development of AIDS. In the
late stages of this disease, the immune system is severely
compromised due to loss or dysfunction of CD4.sup.+ T cells
(Shearer et al. (1991) AIDS 5:245-253). The nervous system is also
a major target of HIV infection. The virus is carried to the brain
by infected monocytes and the neurologic manifestations of HIV
infection are thought to arise from viral products and soluble
factors produced by the infected macrophages/microglia. Thus, the
HIV virus can harbor in the central nervous system where the
therapeutic agents cannot penetrate. Re-infection and relapse to
HIV symptoms subsequent to treatment frequently occurs.
Accordingly, the present invention provides a method of
administering a cytokine, particularly an interferon such as
IFN-.alpha., IFN-.beta., and IFN-.gamma., to the CNS or the
lymphatic system for the treatment or prevention of HIV
infection.
[0188] Interferons are known to exert pleiotropic antiretroviral
activities and affect many different stages of the HIV infectious
cycle. For instance, IFN-.beta. influences uptake of HIV particles
(Vieillard et aL (1994) Proc. Natl. Acad. Sci. USA 91:2689-2693);
reverse transcription of viral genomic RNA into proviral DNA
(Baca-Regen et al. (1994) J. Virol. 68:7559-7565: Kombluth et al.
(1990) Clin. Immunol. Immunopathol. 54:200-219 and Shirazi et al.
(1993) Virology 193:303-312); viral protein synthesis (Coccia et aL
(1994) J. Biol Chem. 269:23087-23094); and packaging and release of
viral particles (Poli et al. (1989) Science 244:575-577). In
addition, virions released from IFN-.beta. treated cells are up to
1,000-fold less infectious than equal numbers of virions released
from untreated cells (Hansen et al. (1992) J. Virol. 66:7543-7548).
Furthermore, recent studies have shown that genetically engineered
human CD4.sup.+ T cells producing constitutively low amounts of
IFN-.beta. can eradicate HIV in vivo using a mouse animal model
that supports persistent, replicative HIV infection. These results
indicated that a therapeutic strategy based upon IFN-.beta.
transduction of CD4.sup.+ T cells may be successful in controlling
a preexisting HIV infection and allowing immune restoration. See,
for example, Vieillard et aL (1999) J. Virol. 73:10281-10288,
herein incorporated by reference. IFN-.gamma. has also been shown
to modulate the susceptibility of macrophages to HIV (Zaitseva et
al. (2000) Blood 96:3109-3117).
[0189] It is recognized that administration of the cytokine via the
methods of the present invention for the treatment of HIV can be
used in combination with any other HIV treatment or therapy known
in the art. Therapies used in the treatment of HIV infection
include, for example, anti-retroviral drugs, such as reverse
transcriptase inhibitors, viral protease inhibitors, and viral
entry inhibitors (Caliendo et aL (1994) Clin. Infect. Dis.
18:516-524). More recently, treatment with combinations of these
agents, known as highly active antiretroviral therapy (HAART), has
been used to effectively suppress replication of HIV (Gulick et al.
(1997) N. Engl. J. Med. 337:734-9 and Hammer et al. (1997) N. Engl.
J. Med. 337:725-733).
[0190] An "effective amount" of a cytokine for the treatment of HIV
using the administration method of the present invention will be
sufficient to reduce or lessen the clinical symptoms of HIV. As
such, an effective amount of the cytokine administered by the
methods of the present invention will attenuate the activity of the
virus (i.e., have a direct antiviral effect) and/or improve the
HIV-induced immunological dysfuntions (i.e., enhance the ability of
an HIV-infected patient to effectively mount a cellular immune
defense against actively replicating HIV). Regardless of the
mechanism of action, an effective amount of a cytokine will enhance
survival and/or improve clinical status of the treated animals in
comparison to the untreated control. Improvement in clinical status
includes, for example, a reduction in preexisting HIV infection
and/or the rate of disease progression; enhanced CD4.sup.+ T-cell
survival; suppression of cytokine dysregulation caused by HIV
(i.e., enhanced Th1-like cytokine expression); inhibition of viral
replication; and improvement in the proliferative expansion of
antigen-selected lymphocytes, more particularly the HIV
antigen-specific CD8.sup.+ subset of T cells, in response to an
increase in viral load. Assays to measure these various
improvements are known in the art. See, for example, Vieillard et
al. (1999) J. Virol. 73:10281-10288, Vieillard et al. (1997) Proc.
Natl. Acad. Sci. USA 94:11595-11600; U.S. Pat. No. 5,911,990 and
U.S. Pat. No. 5,681,831; all of which are herein incorporated by
reference. More extensive details regarding the clinical
manifestations of HIV that can be improved upon the administration
of an effective concentration of a cytokine can be found in
Harrison's Principles of Internal Medicine (McGraw Hill, 1994), pp.
1559-1617, herein incorporated by reference.
[0191] Treatment of Proliferative Disorders of the CNS
[0192] In another embodiment, the present method can be employed to
deliver cytokines to the lymphatic system, CNS, brain, and/or
spinal cord for the treatment, diagnosis or prevention of a
proliferation disorder or disease.
[0193] Cytokines have anti-proliferative activity. For instance,
interferons have been shown to have both a direct cytotoxic effect
on tumor cells and an indirect cytotoxic effect through the
activation of natural killer cells, macrophages, or other immune
cells. Specifically, studies have suggested IFN-.gamma. mediated
anti-tumor activity results from modulating the interplay of B and
T cell components of the immune system, as well as the inhibition
of tumor angiogenesis (Saleh et al. (2000) Gene Ther 7:1715-24).
IFN-.alpha. has also been shown to significantly decrease average
tumor size and increase the average survival time of the treated
mammal (Wang et al. (1999) J Neuropathol Exp. Neurol 58:847-58).
Intratumoral injection of liposomes containing the human IFN-.beta.
gene in nude mice inhibits tumor growth, with complete tumor
regression occurring following multiple Intratumoral injections of
the gene. Furthermore, IFN-.beta. has been demonstrated to be an
effective treatment of high grade astrocytomas (Natsume et al.
(1999) Gene Ther. 9:1626-33 and Fine et al. (1997) Clin. Cancer Res
3: 381-7). The antiproliferative effect of IFN-.beta. appears to
occurs through an arrest in the ordered progression through S phase
or decreasing the entry into G2/M phase of the cell cycle (Garrison
et al. (1996) J Neurooncol 30:213-23). Hence, interferons,
particularly IFN-.alpha., IFN-.beta., and IFN-.gamma., are
effective agents for the treatment or prevention of a proliferation
disorder of the CNS, spinal cord, brain and lymphatic system.
[0194] By "proliferation disorder" is intended any disorder
characterized by cellular division occurring in defiance of the
normal tissue homeostasis mechanism. The proliferation disorder can
be either malignant or benign and result from either an increase in
the rate of cell proliferation or a decrease in the rate of cell
death. The proliferative disorder treated by the methods of the
invention may be at any stage of development (i.e., an early stage
with minimal or microscopic tumor burdens or at advanced stages of
tumor development).
[0195] Proliferative disorders of the central nervous system,
brain, or spinal cord include, for example, gliomas, neuronal
tumors, poorly differentiated neoplasms, and meningiomas. Gliomas
derived from glial cells include astrocytomas (i.e., fibrillary
astrocytomas, glioblastoma multiforme, pilocytic astrocytoma,
pleomorphic xanthastrocytoma, and brain stem glioma),
oligodendrogliomas, and ependymomas and paraventricular mass
lesions (i.e., myxopapillary ependymomas, subependymomas, choroid
plexus papillomas). Neural tumors comprise CNS tumors that contain
mature-appearing neurons (ganglion cells) that may constitute the
entire cell population of the lesion or, alternatively, the lesion
is an admixture with a glial neoplasm. Poorly differentiated
neoplasms include, for example, medulloblastomas. Other
proliferative disorders of the CNS, brain or spinal cord include,
-primary brain lymphoma, meningiomas, and metastatic tumors.
[0196] It is recognized that administration of the cytokine via the
methods of the present invention for the treatment of a
proliferative disorder can be used in combination with any other
treatment or therapy known in the art for the treatment of
proliferation disorders. Therapies used in the treatment of
proliferative disorders include, for example, any form of radiation
and chemotherapy treatments. See, for example, Hatano et al. (2000)
Acta Neurochir 142:633-8, Burton et al. (1999) Curr Opin Oncol.
11:157-61, and Brandes et al. (2000) Anticancer Res 20:1913-20; all
of which are herein incorporated by reference.
[0197] An "effective amount" of a cytokine for the treatment of a
proliferative disease or disorder using the administration method
of the present invention will be sufficient to reduce or lessen the
morphological and/or clinical symptoms of the proliferative
disorder. As such, an effective amount of the cytokine administered
by the methods of the present invention will exert any
physiological response that decreases proliferation of tumor cells
and thereby enhances survival and/or improves clinical status of
the treated animal in comparison to the untreated control. Such
physiological responses include, for example, activation of immune
cells, inhibition of cell proliferation, induction of cell
differentiation, up-regulation of class I major histocompatiblity
complex antigens, inhibition of angiogenesis, and establishment of
the T helper 1 (Th1)-type response. Improvement in clinical status
includes, for example, an increase in the survival rate of the
treated mammal (i.e., an increase in either the one or two year
survival rate) and a decrease in tumor size. Assays to measure
these various improvements are known in the art. See, for example,
Hong et al. (2000) Clin. Cancer Res. 6:3354-60); Knupfer et al.
(2000) Cytokine 12:409-12; Natsume et al. (1999) Gene Ther
6:1626-33; and U.S. Pat. No. 4,846,782, all of which are herein
incorporated by reference. More extensive details regarding the
clinical manifestations of proliferative disorders of the CNS,
brain, spinal cord, or lymphatic system that can be improved upon
the administration of an effective concentration of a cytokine can
be found in Harrison's Principles of Internal Medicine (McGraw
Hill, 1994), herein incorporated by reference.
[0198] The present invention may be better understood with
reference to the following examples. These examples are intended to
be representative of specific embodiments of the invention, and are
not intended as limiting the scope of the invention.
EXPERIMENTAL
Example 1
[0199] Intranasal Administration of IFN-.beta. to the CNS
[0200] Introduction
[0201] Administering interferon-P (IFN-.beta.) intranasally is an
effective means for delivering this cytokine to the CNS of an
animal.
[0202] Materials and Methods
[0203] Intranasal Delivery to the CNS:
[0204] Male Sprague-Dawley rats, 199 and 275 grams, were
anesthetized with intraperitoneal pentobarbital (40 mg/kg). Drug
delivery to the CNS was assessed after intranasal administration of
51 picomoles and 57 picomoles of .sup.125I-IFN-.beta. in 20 mM
Hepes, pH 7.5, to the light and heavy rat, respectively. Rats were
placed on their backs and administered.about.100 microliters
.sup.125I-IFN-.beta. to each naris over 10-22 minutes, alternating
drops every 2-3 minutes between the left and right nares. During
the intranasal administration of IFN-.beta., one side of the nose
and the mouth were held closed. This method of administering the
cytokine allows for both pressure and gravity to deliver the agent
into the upper one third of the nasal cavity. Rats subsequently
underwent perfusion-fixation within minutes following the
completion of .sup.125I-IFN-.beta. administration.
Perfusion-fixation was performed with 50-100 ml physiologic saline
followed by 500 ml of fixative containing 4% paraformaldehyde in
0.1 M Sorenson's phosphate buffer, pH 7.4, prior to brain and
spinal cord dissection and 1251 measurement by gamma counting.
Areas dissected included the spinal cord, olfactory bulbs, frontal
cortex, anterior olfactory nucleus, hippocampal formation, choroid
plexus, diencephalon, medulla, pons, and cerebellum.
[0205] Results
[0206] Rapid appearance of radiolabel was observed throughout the
spinal cord, brain stem, and brain, with the concentrations ranging
from about 3 pM to about 93 pM. Detailed results are shown below in
Table 1. The observation of substantial concentrations of
interferon-.beta. in the olfactory and trigeminal nerves suggests
that this cytokine is transported through or along these nerves.
Tissues with biologically significant levels of interferon-P
include the olfactory bulbs, frontal cortex, caudate putamen,
anterior olfactory nerve, hippocampal formation, choroid plexus,
diencephalon, pons, medulla, ventral dura, trigeminal nerve,
olfactory epithelium, circle of Willis, and upper cervical spinal
cord.
1TABLE 1 Data for the intranasal (I.N.) delivery of Betaseron to
the CNS Concentration (pM) Concentration (pM) Tissue type (51
picomole dose) (57 picomole dose) Left olfactory bulb 89.5 51.4
Right olfactory bulb 92.9 67.7 Frontal cortex 9.19 29.1
Caudate/putamen 7.09 34.0 Anterior olfactory nerve 46.9 97.4
Hippocampal formation (left) 5.81 11.7 Hippocampal form (right)
11.1 21.0 Choroid plexus 79.0 33.2 Diencephalon 15.5 24.0 Midbrain
10.9 19.8 Pons 16.9 49.4 Medulla 24.7 90.2 Cerebellum 10.2 30.4
Dura (ventral) 263.0 896 Trigeminal nerve 36.7 362 Left olfactory
epithelium 3697 Circle of Willis 189 Upper Cervical Spinal Cord
24.3 455 Cervical spinal cord 6.88 Thoracic spinal cord 4.0 2.55
Lumbar spinal cord 2.08 3.5 Right olfactory epithelium 22,540
[0207] Further quantitation studies for the intranasal delivery of
[.sup.125I]Betaseron were performed in Sprague-Dawley rats
essentially described above. The results are summarized in Table 2.
Scans of coronal brain tissue sections showed prominent labeling of
the olfactory bulb, caudate/putamen, septal nucleus,
periventricular white matter, optic nerve, and superior colliculus
(data not shown). These results are in agreement with the results
provided in Table 1. The quantitative studies performed in six
animals, following internasal administration of about 6 nmol of
Betaseron, demonstrated consistent delivery to a wide variety of
CNS structures. Highest concentrations of IFN-.beta. were found in
the olfactory bulbs (9 nM), anterior olfactory nucleus (3.3 nM),
midbrain (1.9 nM), medulla (1.8 nM), pons (1.6 nM), and cerebellum
(1.4 nM). Moderate concentrations were observed in the hippocampal
formation (1.3 nM), diencephalon (1.3 nM), frontal cortex (1.1 nM),
cervical spinal cord (1.1 nM), and caudate/putamen (0.83 nM).
[0208] The very high concentrations of [.sup.1251]Betaseron
observed in the trigeminal nerve (14 nM) and ventral dura mater (19
nM) strongly suggest that delivery to the CNS involves movement not
only along the olfactory neural pathway but also along the
trigeminal nerve pathway. Trigeminal delivery should result in high
levels in both the olfactory areas and midbrain and brain stem
regions. Delivery to the spinal cord probably occurs via the
trigeminal pathway. Consistent with trigeminal delivery,
[.sup.125I]Betaseron reaches the spinal cord within 25 minutes, and
exhibits decreasing concentration as you move down the spinal
cord.
[0209] These results indicate the direct transport of IFN-.beta.
along one or more neural pathways into the CNS, brain, and spinal
cord.
2TABLE 2 Concentration (nM) IFN-.beta. (Betaseron) in Different Rat
Tissues Following I.N. Administration of .sup.125I-IFN-.beta. +
IFN-.beta.. Tissue IF11 IF12 IF13 IF14 IF15 IF16 Mean SE Blood
Sample 1 1.12 1.53 0.92 0.74 1.70 0.85 1.1 0.2 Blood Sample 2 2.77
3.26 1.44 2.11 3.13 2.74 2.6 0.3 Blood Sample 3 3.72 6.62 2.81 3.87
5.02 4.22 4.4 0.5 Blood Sample 4 5.37 6.35 7.38 5.4 7.32 5.50 6.2
0.4 Blood Sample 5 7.29 6.69 8.15 7.95 7.5 0.3 Left Olfactory Bulb
9.02 6.11 3.01 5.93 18.29 18.46 10 3 Right Olfactory Bulb 5.6 6.99
3.39 4.84 15.26 12.48 8.1 1.9 Frontal Cortex 1.12 1.24 1.09 0.44
1.72 1.19 1.1 0.2 Caudate/Putamen 0.68 0.91 0.83 0.36 1.08 1.11
0.83 0.11 Ant. Olf. Nucleus 2.11 2.55 1.96 1.09 6.82 5.50 3.3 0.9
L. Hippocampal Form. 0.84 1.63 1.24 0.37 2.23 1.71 1.3 0.3 R.
Hippocampal Form. 0.85 1.77 1.24 0.40 1.84 1.91 1.3 0.3
Diencephalon 0.86 1.52 1.39 0.44 2.05 1.72 1.3 0.2 Midbrain 0.80
1.69 1.53 0.44 5.07 1.91 1.9 0.7 Pons 0.76 1.91 1.76 0.38 2.71 2.04
1.6 0.4 Medulla 0.63 2.41 2.90 0.42 2.29 2.08 1.8 0.4 Cerebellum
0.89 1.72 1.56 0.36 2.19 1.84 1.4 0.3 Ventral Dura 2.47 46.16 10.89
7.13 21.35 23.52 19 6 Trigeminal Nerve 7.94 12.14 19.89 4.57 24.44
17.63 14 3 Spinal Dura 0.59 0.13 0.29 0.34 0.13 Cervical Spinal
Cord 0.33 0.88 3.12 0.38 0.98 1.00 1.1 0.4 Thoracic Spinal Cord
0.14 0.11 0.39 0.29 0.33 0.15 0.24 0.05 Lumbar Spinal Cord 0.13
0.12 0.27 0.22 0.32 0.10 0.19 0.04 Deltoid Muscle 0.62 0.58 0.50
1.10 0.67 0.22 0.62 0.12 Liver 0.58 0.78 1.01 1.38 0.54 0.31 0.77
0.16 Kidney 0.67 0.73 2.08 5.26 0.56 1.81 1.9 0.7 Lung 1.87 0.56
2.18 0.72 0.85 0.99 1.2 0.3 Esophagus 1.10 1.50 68.2 5234.83 1.44
22.40 888 869 Trachea 1.48 3.11 83.46 4.67 1.45 5.91 17 13 L.
Olfact. Epithelium 1175.9 75.64 14.08 1431.14 454.41 227.29 563 244
R. Olfact. Epithelium 2083.1 411.32 45.66 1113.87 191.13 2765.47
1102 453 IF 11-16 represent individual rats Average weight (g.)
across rats: 243 g. (range = 203 g-268 g) Average concentration
administered: 6.0 n moles (range = 4.8 nmol-6.9 nmol) Average
radioactivity (uCi): 39 uCi (range = 32 uCi-52 uCi)
Example 2
[0210] Intranasal Administration of IFN-.beta. Retains
Pharmacological Activity in the CNS
[0211] Assays were performed to determine if IFN-.beta., delivered
intranasally, retained pharmacological activity in the CNS.
IFN-.beta. activates signal transduction pathways via a cell
surface IFN receptor. The IFN receptor is part of a prototypical
JAK-STAT signaling complex. It has two transmembrane chains that
associate with intracellular signaling proteins including TYK2,
JAK1, and two latent transcription factors termed "signal
transducers and activators of transcription" (STATs). Binding of
IFN-.beta. to the receptor brings the two Janus kinases (TYK2 and
JAK1) near each other, and they become activated by
phosphorylation. The kinases then activate the cytoplasmic tails of
the IFN receptors by phosphorylating tyrosine residues. These
phosphotyrosines provide docking sites for the STATs, bringing them
into appropriate positions for phosphorylation by the nearby Janus
kinases. Upon phosphorylation STATs translocate to the nucleus,
bind specific DNA elements and direct transcription. Hence, the
pharmacological activity of IFN-.beta. following intranasal
delivery can be effectively assayed by monitoring the
phosphorylation states of TYK2 and STAT1 throughout the brain
cortex.
[0212] Methods:
[0213] Control/Drug Treatment:
[0214] Harlan Sprague-Dawley rats were anesthetized with
pentabarbitol (50 mg/kg). 80 .mu.l of either water or IFN-.beta.
was intranasally administered in 5 doses over a 20 minute time
period. Specifically, 8 ,.mu.l was administered in 5 doses at 2
minute intervals for each nostril. Recombinant rat interferon-:
(rrIFN-.beta.) (35 picomoles) was intranasally administered to rat
IF35 (drug-treated) and H.sub.2O (vehicle used to dilute rrIFN-0)
was administered to rat IF33 (control-treated). After
administration the animal was perfused with 100 ml of saline and
fixed with 200 ml of 10% formalin. The brain was then removed and
sliced in a brain matrix into 2 mm sections. The slices were
collected in cassettes and paraffin embedded. Tissue was sliced to
4 .mu.m and placed on microscope slides.
[0215] Immunohistological Staining:
[0216] The antibodies to the phosphorylated forms of proteins TYK2
and STAT1 were purchased from Cell Signaling Technology (product
numbers 9321L and 9171S, respectively).
[0217] The method of immunohistological staining was as follows.
Tissue sections were deparaffinized and hydrated by placing the
slides in the following solutions for the indicated times: Xylene
for 10 min; 100% EtOH for 5 min; 95% EtOH for 5 min; 70% EtOH for 5
min; and, 50% EtOH for 5 min. The slides were removed from Coplin
jars and washed in H.sub.2O for 2 min on a rocking platform. The
antigen (TYK2 and/or STAT 1) was unmasked by incubating slides in
citrate buffer (pH 6.0) and heating in a vegetable steamer for 45
min. The slides were removed and washed in cold running tap
H.sub.2O for 10 min. Slices were incubated in 3% H.sub.2O for 10
min at room temperature (RT) in a humid chamber and subsequently
washed in H.sub.2O for 5 min. Next, slides were washed in a tris
buffered saline solution (50 mM tris, 150 mm NaCl) with 0.2% Triton
X-100 (TBST) for three 5 min washes. Following the wash, the slides
were blocked with 2% goat serum in TBST (GSTBST) for 1 hr at RT.
Following three 5 min washes in TBST, the slides were incubated
with primary antibody (rabbit anti-TYK2 polycolonal antibody;
diluted in GSTBST 1:250) in a humid chamber at RT for 30 min and
incubated overnight at 4.degree. C. The next day, the slides were
wash in TBST for three 5 min washes and incubated with goat
anti-rabbit secondary antibody. The secondary antibody was diluted
1:400 in 10 mM phosphate buffered solution (PBS; 137 mM sodium
chloride, 2.7 mM potassium chloride) at RT for 1 hr. For the last
15 min of this incubation, the ABC reagent was made (5 ml PBS, 2
drops of reagent A, mix, 2 drops of reagent B, mix; Vector
Technology product # PK-6101) and allowed to stand at RT. Slides
underwent an additional three 5 minute washes in TBST, followed by
incubation with ABC reagent at RT for 1 hr in a humid chamber. An
additional three 5 minute washes in TBST followed. Approximately
100-150 .mu.l, enough to cover the tissue, of diaminobenzidine
tetrahydrochloride (DAB) was added and allowed to incubate at RT
for 10 min. The reaction was stopped by a 2 minute wash with
H.sub.2O. Slides were subsequently washed in H.sub.2O until the
solution was clear. Slides were dehydrated in the following
solutions for the indicated times: 50% EtOH for 2 min; 70% EtOH for
2 min; 95% EtOH for 2 min; 100% EtOH for 2 min; 50/50 Xylene/ROH
for 2 min; and Xylene for 5 min. Excess xylene was removed and
slides were mounted by adding 2-3 drops of Vectamount and covered
with coverslip. The Vectamount was allowed to dry before
viewing.
[0218] Results:
[0219] Induction of the IFN-.alpha./.beta. pathway is characterized
by the phosphorylation of TYK2 and STAT1. Therefore, antibodies
specific to the phosphorylated forms of TYK2 and STAT 1 were used
to measure the level of the activated from of these proteins prior
to and following intranasal delivery of IFN-.beta.. Quantitation
revealed that the levels of phosphorylated TYK2 increased
throughout the brain cortex following intranasal delivery of 35
pmol of recombinant rat IFN-.beta. (data not shown). These results
demonstrate that IFN-.beta. retains pharmacological activity in the
CNS following the intranasal delivery methods of the present
invention.
Example 3
[0220] Intranasal Administration of IFN-.beta. to the Lymphatic
System
[0221] Intranasal delivery of [125I] Betaseron was performed in
Sprague-Dawley Rats as essentially described in Example 1. 3.9-7.9
nmol Betaseron was administered in a 44-96 .mu.l volume over the
course of 20-29 minutes. Animals were perfused at 30 minutes. Data
obtained from eight individual animals is provided in Table 3.
Experimental means from this set of experiments are provided in
Table 4. These quantitative studies demonstrated delivery of [1251]
Betaseron to the superficial cervical nodes and to the deep
cervical nodes of the lymphatic system. On average, 6.1 nM
Betaseron was found in the superficial cervical nodes, and 31.5 nM
was found in the deep cervical nodes following the administration
methods of the invention. These results are summarized in Table
5.
3TABLE 3 Betaseron Concentration (nM) Following I.N. Administration
of .sup.125I-IFN.beta. + rhIFN.beta. Experiment IF34 IF36 IF37 IF38
MicroCi 31 47 61 48 Nmol 3.9 6.9 7.9 6.6 Blood Sample #1 0.53 0.58
1.1 1.6 Blood Sample #2 1.6 4.3 2.8 3.9 Blood Sample #3 2.4 3.1 4.6
6.2 Blood Sample #4 3.6 4.7 5.1 8.3 Blood Sample #5 4.1 7.0 7.0 10
Blood Sample #6 8.2 8.5 10 Left Olfactory Epithelium 65 862 388 643
Right Olfactory Epithelium 62 1103 1447 1876 Left Olfactory Bulb
1.6 3.5 5.6 Right Olfactory Bulb 1.3 8.1 7.1 Antenor Olfactory
Nucleus 0.96 1.3 2.5 Frontal Cortex 0.28 0.84 0.97 Caudate/Putamen
0.09 0.57 1.7 L + R Hippocampus 0.38 0.62 0.82 Left Hippocampus
Right Hippocampus Diencephalon 0.65 0.74 0.95 Midbrain 0.48 0.61
0.88 Pons 0.45 0.75 0.91 Medulla 0.36 0.76 0.95 Cerebellum 0.34
0.54 0.69 Ventral Brain Dura 6.1 9.7 12 14 Optic Nerve + Chiasm 1.2
6.3 4.4 25 Trigeminal Nerve 5.8 12 8.5 20 Spinal Dura 0.09 0.16
0.67 1.1 Upper Cervical Cord 0.43 2.3 0.92 1.1 Cervical Spinal Cord
0.17 0.21 0.47 1.3 Thoracic Spinal Cord 0.13 0.20 0.35 0.58 Lumbar
Spinal Cord 0.17 0.29 0.39 0.49 Superficial Cervical Nodes 8.1 6.3
4.0 6.1 L. Superficial Cervical Node 3.9 R. Superficial Cervical
Node 4.2 Deep Cervical Nodes 9.7 16 68 Left Deep Cervical Node
Right Deep Cervical Node Common Carotids 14 27 38 22 Thyroid 250
462 830 725 Esophagus 145 196 394 715 Trachea 177 41863 692 553
Muscle 0.52 0.64 0.74 1.1 Liver 0.47 1.9 0.83 1.2 Kidney 1.0 0.79
2.92 1.8 Lung 0.66 1.7 2.4 27
[0222]
4TABLE 4 Experimental Means of Betaseron Concentrations (nM)
following I.N. Administration of .sup.125I-IFN.beta. + rhIFN.beta.
Avg for IF34,37,38 microCi/nmol 46.67 6.13 Mean Std Dev Blood
Sample #1 1.1 0.53 Blood Sample #2 2.7 1.2 Blood Sample #3 4.4 1.9
Blood Sample #4 5.7 2.4 Blood Sample #5 7.1 3.1 Blood Sample #6 9.5
1.4 Left Olfactory Epithelium 365 290 Right Olfactory Epithelium
1128 948 Left Olfactory Bulb 3.6 2.0 Right Olfactory Bulb 5.5 3.7
Anterior Olfactory Nucleus 1.6 0.81 Frontal Cortex 0.70 0.37
Caudate/Putamen 0.80 0.85 L + R Hippocampus 0.61 0.22 Left
Hippocampus Right Hippocampus Diencephalon 0.78 0.16 Midbrain 0.66
0.20 Pons 0.71 0.24 Medulla 0.69 0.30 Cerebellum 0.52 0.17 Ventral
Brain Dura 11 4.3 Optic Nerve + Chiasm 10 13 Trigeminal Nerve 11
7.5 Spinal Dura 0.61 0.49 Upper Cervical Cord 0.83 0.36 Cervical
Spinal Cord 0.65 0.59 Thoracic Spinal Cord 0.35 0.23 Lumbar Spinal
Cord 0.35 0.17 Superficial Nodes 6.0 2.1 Left Superficial Cervical
Node Right Superficial Cervical Node Deep Cervical Nodes 39 41 Left
Deep Cervical Node Right Deep Cervical Node Common Carotids 25 12
Thyroid 602 309 Esophagus 418 286 Trachea 474 266 Muscle 0.78 0.27
Liver 0.82 0.35 Kidney 1.9 0.95 Lung 10 15
[0223]
5TABLE 5 Summary of Betaseron Concentration (nM) in the Cervical
Lymph Nodes Following I.N. Administration of .sup.125I-IFN.beta. +
rhIFN.beta. Experiment IF34 IF36 IF37 IF38 MicroCi 31 47 61 48 Nmol
3.9 6.9 7.9 6.6 Average Std Dev Superficial Cervical 8.1 6.3 4.0
6.1 6.1 1.7 Nodes Deep Cervical Nodes 9.7 16 68 31 32 Average Dose
Administered = 46.75 uCi and 6.32 nmol
Example 4
[0224] Intravenous Administration of Betaseron
[0225] Intravenous administration of Betaseron was studied in order
to determine the extent to which delivery to the CNS and/or
lymphatic system following intranasal administration may be due to
absorption from the nasal cavity into the circulation, followed by
subsequent delivery to the CNS and lymphatics.
[0226] Male Harlan Sprague-Dawley rats weighing 263-318 g were used
for these experiments. Rats were anaesthetized with sodium
pentobarbital (Nembutal, 50 mg/kg). For each rat, a 500 .mu.l
solution containing .sup.125I-IFN-.beta. and rhIFN-.beta. in 0.9%
NaCl was delivered intravenously over 60-90 seconds through a
cannula into the femoral vein. On average, 560 pmol and 49 .mu.Ci
of IFN-.beta. were administered to each rat. Then 0.2 ml of blood
was collected from the descending aorta cannula every 5 minutes for
a total of five blood samples. Lastly, the rat was perfused through
the descending aorta cannula with 60-90 ml of 0.9% NaCl followed by
400 ml of fixative (4% paraformaldehyde in Sorenson's phosphate
buffer). Individual tissue sections were dissected out, placed in 5
ml Startedt tubes, and then counted for gamma rays in the Packard
Cobra II autogamma counter.
[0227] The methods described above created the same general blood
level of Betaseron with intravenous delivery as that achieved in
the intranasal administration studies. Tables 6 and 7 provide the
level of Betaseron in the blood following either intravenous
injection and intranasal administration. The level of Betaseron in
the blood following intravenous administration and intranasal
administration over time is shown graphically in FIG. 1.
[0228] This study demonstrated that very little of the
intravenously administered Betaseron reaches either the CNS or
lymphatics. Consequently, it is clear that the intranasal method of
delivery described in this application is very beneficial in
targeting the CNS and lymphatics of the head and neck region. This
method of delivery does not utilize the circulation to reach the
CNS or lymphatics, but rather bypasses the circulation and
blood-brain barrier to accomplish delivery. Because it is not
necessary to use the circulatory system to deliver the medication
to the CNS and/or lymphatics, systemic side effects can be
significantly reduced.
6TABLE 6 Level of Betaseron in the blood following intravenous
administration. Experiment # IF47 IF49 IF50 Mean Std Err Delivered
nmol 0.521 0.579 0.579 0.560 0.019 Delivered uCi 56 46 45 49 4 5
min Blood Sample 6.30 4.47 6.78 5.85 0.70 10 min Blood Sample 5.35
4.41 5.29 5.01 0.30 15 min Blood Sample 5.90 4.14 5.95 5.33 0.60 20
min Blood Sample 5.95 4.48 5.92 5.45 0.49 25 min Blood Sample 6.40
4.16 6.30 5.62 0.73
[0229]
7TABLE 7 Level of Betaseron in the blood stream following
intranasal administration. Experiment # IF36 IF37 IF38 IF40 Mean
Std Err Delivered nmol 6.890 7.947 6.583 7.360 7.195 0.30 Delivered
uCi 47 61 48 51 52 3 5 min Blood Sample 0.58 1.08 1.60 2.44 1.43
0.40 10 min Blood Sample 1.43 2.76 3.89 5.91 3.50 0.95 15 min Blood
Sample 3.06 4.62 6.22 8.30 5.55 1.12 20 min Blood Sample 4.70 5.14
8.27 10.17 7.07 1.30 25 min Blood Sample 6.93 7.04 10.16 12.85 9.25
1.42
[0230]
8TABLE 8 Concentration (nM) following intravenous administration of
IFN-.beta.. IF47 IF49 IF50 Mean Std Err Delivered nmol 0.521 0.579
0.579 0.560 0.019 Delivered uCi 56 46 45 49 4 Blood Sample #1 (5
min) 6.30 4.47 6.78 5.85 0.70 Blood Sample #2 (10 min) 5.35 4.41
5.29 5.02 0.30 Blood Sample #3 (15 min) 5.90 4.14 5.95 5.33 0.60
Blood Sample #4 (20 min) 5.95 4.47 5.92 5.45 0.49 Blood Sample #5
(25 min) 6.40 4.16 6.30 5.62 0.73 Left Olfactory Epithelium 0.27
0.72 0.86 0.62 0.18 Right Olfactory Epithelium 0.19 0.78 1.04 0.67
0.25 Left Olfactory Bulb 0.63 0.23 0.31 0.39 0.12 Right Olfactory
Bulb 1.01 0.22 0.23 0.49 0.26 Anterior Olfactory Nucleus 0.15 0.13
0.17 0.15 0.01 Frontal Cortex 0.15 0.16 0.18 0.16 0.01
Caudate/Putamen 0.21 0.11 0.15 0.16 0.03 Hippocampus 0.13 0.11 0.14
0.13 0.01 Cerebellum 0.15 0.12 0.16 0.14 0.01 Diencephalon 0.14
0.12 0.14 0.13 0.01 Midbrain 0.16 0.12 0.14 0.14 0.01 Pons 0.13
0.11 0.03 0.09 0.03 Medulla 0.14 0.11 0.14 0.13 0.01 Dorsal Brain
Dura 0.41 0.43 0.42 0.01 Ventral Brain Dura 1.32 0.28 0.17 0.59
0.37 Optic Nerve + Chiasm 0.18 0.29 0.24 0.04 Trigeminal Nerve 0.28
0.21 0.26 0.25 0.02 Spinal Dura 0.07 0.13 0.12 0.11 0.02 Upper
Cervical Cord 0.15 0.12 0.09 0.12 0.02 Cervical Cord 0.10 0.10 0.09
0.10 0.00 Thoracic Spinal Cord 0.08 0.09 0.11 0.09 0.01 Lumbar
Spinal Cord 0.11 0.12 0.14 0.12 0.01 Superficial Nodes 0.42 0.28
0.64 0.45 0.10 Deep Cervical Nodes 0.10 0.34 0.40 0.28 0.09 Axial
Nodes 0.33 0.64 0.49 0.13 Common Carotids 0.13 0.11 0.09 0.11 0.01
Thyroid 52.65 56.49 11.03 40.06 14.56 Esophagus 0.92 0.91 0.29 0.71
0.21 Trachea 0.81 0.49 0.46 0.59 0.11 Deltoid Muscle 0.29 0.19 0.30
0.26 0.04 Liver 15.17 11.82 16.90 14.63 1.49 Kidney 1.30 1.51 1.49
1.43 0.07 Lung 16.02 30.02 33.14 26.39 5.26
[0231] It should be noted that, as used in this specification and
the appended claims, the singular forms "a," "an," and "the"
include plural referents unless the content clearly dictates
otherwise. Thus, for example, reference to a composition containing
"a compound" includes a mixture of two or more compounds.
[0232] All publications and patent applications in this
specification are indicative of the level of ordinary skill in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated by reference.
[0233] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications may be
made while remaining within the spirit and scope of the
invention.
* * * * *