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Title:  Method for administering a cytokine to the central nervous system and the lymphatic system
United States Patent: 
6,991,785
Issued: 
January 31, 2006

Inventors:  Frey, II; William H. (North Oaks, MN)
Assignee: 
Chiron Corporation (Emeryville, CA)
Appl. No.: 
102163
Filed: 
March 20, 2002


 

Woodbury College's Master of Science in Law


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-β and its muteins such as IFN-βser17. 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.

DETAILED DESCRIPTION OF THE INVENTION

Routes of Administration

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.

The Olfactory Nerve

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.

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.

The Trigeminal Nerve

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.

The Ophthalmic Nerve and its Branches

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.

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.

The Maxillary Nerve and its Branches

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.

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.

The Mandibular Nerve and its Branches

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.

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.

Tissues Innervated by the Trigeminal Nerve

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.

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.

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.

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.

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.

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.

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.

Cytokines

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-α and TNF-β), 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.

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 α, β, γ, ω, and τ. Synthetic interferons are also known in the art. See, for example, U.S. Pat. No. 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-β (IFN-β), interferon-α (IFN-α), and interferon-γ (IFN-γ).

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-α, IFN-β, IFN-γ, 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.

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.

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.

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).

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.

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-α, IFN-β, or IFN-γ) 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.

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.

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.

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.

Interferon-β

The term "IFN-β" as used herein refers to IFN-β or variants thereof, sometimes referred to as IFN-β-like polypeptides. Human IFN-β variants, which may be naturally occurring (e.g., allelic variants that occur at the IFN-β locus) or recombinantly produced, have amino acid sequences that are the same as, similar to, or substantially similar to the mature native IFN-β sequence. DNA sequences encoding human IFN-β 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-β or truncated forms of IFN-β that retain their activity are also encompassed. These biologically active fragments or truncated forms of IFN-β are generated by removing amino acid residues from the full-length IFN-β amino acid sequence using recombinant DNA techniques well known in the art. IFN-β polypeptides may be glycosylated or unglycosylated, as it has been reported in the literature that both the glycosylated and unglycosylated forms of IFN-β 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-β.

The IFN-β variants encompassed herein include muteins of the native mature IFN-β 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-β 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-β 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-β mutein that is one amino acid shorter than the native mature IFN-β. Thus, IFN-β variants with one or more mutations that improve, for example, their pharmaceutical utility are also encompassed by the present invention.

The skilled artisan will appreciate that additional changes can be introduced by mutation into the nucleotide sequences encoding IFN-β, thereby leading to changes in the IFN-β amino acid sequence, without altering the biological activity of the interferon. Thus, an isolated nucleic acid molecule encoding an IFN-β variant having a sequence that differs from human IFN-β 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-β. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such IFN-β variants are also encompassed by the present invention. Variants of IFN-β 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.

Biologically active IFN-β variants encompassed by the invention also include IFN-β polypeptides that have covalently linked with, for example, polyethylene glycol (PEG) or albumin.

Biologically active variants of IFN-β encompassed by the invention should retain IFN-β activities, particularly the ability to bind to IFN-β receptors or retain immunomodulatory or anti-viral activities. In some embodiments, the IFN-β 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-β polypeptide. IFN-β variants whose activity is increased in comparison with the activity of the native IFN-β polypeptide are also encompassed. The biological activity of IFN-β 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 et al. (1982) J. Biol. Chem. 257(19):11301-11304.

Non-limiting examples of IFN-β polypeptides and IFN-β 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 et al. (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-β polypeptide that can be altered without the loss of biological activity.

In one embodiment of the present invention, the IFN-β used in the methods of the invention is the mature native human IFN-β polypeptide. In another embodiment, the IFN-β is the mature IFN-β C17S polypeptide. However, the present invention encompasses other embodiments where the IFN-β is any biologically active IFN-β polypeptide or variant as described elsewhere herein.

In some embodiments of the present invention, the IFN-β is recombinantly produced. By "recombinantly produced IFN-β" is intended IFN-β that has comparable biological activity to native IFN-β and that has been prepared by recombinant DNA techniques. IFN-β can be produced by culturing a host cell transformed with an expression vector comprising a nucleotide sequence that encodes an IFN-β 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-β 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. Nos. 4,462,940, 5,702,699, and 5,814,485; herein incorporated by reference.

Interferon-α

The term "IFN-α" as used herein refers to IFN-α or variants thereof, sometimes referred to as IFN-α-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-α polypeptides include IFN-αa, IFN-αB, IFN-αC, IFN-αD, IFN-αH, IFN-αJ, IFN-αJ1, IFN-αJ2 and IFN-αK. Human IFN-α variants, which may be naturally occurring (e.g., allelic variants that occur at the IFN-α locus) or recombinantly produced, have amino acid sequences that are the same as, similar to, or substantially similar to the mature native IFN-α sequence. DNA sequences encoding human IFN-α 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. NM000605); and Ohara et al. (1987) FEBS Letters 211:78-82; all of which are herein incorporated by reference. Fragments of IFN-α or truncated forms of IFN-α that retain their activity are also encompassed. These biologically active fragments or truncated forms of IFN-α are generated by removing amino acid residues from the full-length IFN-α amino acid sequence using recombinant DNA techniques well known in the art. IFN-α polypeptides may further be glycosylated or unglycosylated.

The skilled artisan will appreciate that additional changes can be introduced by mutation into the nucleotide sequences encoding IFN-α, thereby leading to changes in the IFN-α amino acid sequence, without altering the biological activity of the interferon. Thus, an isolated nucleic acid molecule encoding an IFN-α variant having a sequence that differs from human IFN-α 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-α. Mutations can be introduced by standard techniques. Such variants of IFN-α, include, for example, IFN-α-2a (ROFERON-A™), IFN-α-2b (INTRON A™), and IFN-αcon-1 (INFERGEN™). Another variant useful in the methods of the present invention is IFN-α2a, 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-α 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-α polypeptide that can be altered without the loss of biological activity.

Biologically active IFN-α variants encompassed by the invention also include IFN-α 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.

Biologically active variants of IFN-α encompassed by the invention should retain IFN-α activities, particularly the ability to bind to IFN-α receptors or retain immunomodulatory, antiviral, or anit-proliferative activities. In some embodiments, the IFN-α 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-α polypeptide. IFN-α variants whose activity is increased in comparison with the activity of the native IFN-α polypeptide are also encompassed. The biological activity of IFN-α variants can be measured by any method known in the art. Examples of such assays are described above.

In one embodiment of the present invention, the IFN-α used in the methods of the invention is the mature native human IFN-α polypeptide. However, the present invention encompasses other embodiments where the IFN-α is any biologically active IFN-α polypeptide or variant as described elsewhere herein.

In some embodiments of the present invention, the IFN-α is recombinantly produced. By "recombinantly produced IFN-α" is intended IFN-α that has comparable biological activity to native IFN-α and that has been prepared by recombinant DNA techniques. IFN-α can be produced by culturing a host cell transformed with an expression vector comprising a nucleotide sequence that encodes an IFN-α 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-α-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.

Interferon-γ

The term "IFN-γ" as used herein refers to IFN-γ or variants thereof, sometimes referred to as IFN-γ-like polypeptides. IFN-γ 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-γ can be found in, for example, U.S. Pat. No. 6,046,034, herein incorporated by reference. Human IFN-γ variants, which may be naturally occurring (e.g., allelic variants that occur at the IFN-γ locus) or recombinantly produced, have amino acid sequences that are the same as, similar to, or substantially similar to the mature native IFN-γ sequence. DNA sequences encoding human IFN-γ 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-γ or truncated forms of IFN-γ that retain their activity are also encompassed. These biologically active fragments or truncated forms of IFN-γ are generated by removing amino acid residues from the full-length IFN-γ amino acid sequence using recombinant DNA techniques well known in the art. IFN-γ polypeptides may be glycosylated or unglycosylated.

The IFN-γ variants encompassed herein include muteins of the native mature IFN-γ sequence. Thus, IFN-γ variants with one or more mutations that improve, for example, their pharmaceutical utility are also encompassed by the present invention.

Such IFN-γ variants are also encompassed by the present invention. Variants of IFN-γ 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-γ that retain the biological activity of the mature IFN-γ. Additionally, in EP 0 306870 A2, variants of human IFN-γ 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-γ (IFN-γ C-10 L) that has increased biological activity. Further variants of IFN-γ 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-γ without losing biological activity. Each of these references is herein incorporated by reference. The above examples represent non-limiting examples of IFN-γ polypeptides and IFN-γ variant polypeptides encompassed by the invention. These citations also provide guidance regarding residues and regions of the IFN-γ polypeptide that can be altered without the loss of biological activity.

Biologically active IFN-γ variants encompassed by the invention also include IFN-γ polypeptides that have covalently linked with, for example, polyethylene glycol (PEG) or albumin.

Biologically active variants of IFN-γ encompassed by the invention should retain IFN-γ activities, particularly the ability to bind to IFN-γ receptors or retain immunomodulatory, antiviral, or antiproliferative activities. In some embodiments, the IFN-γ 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-γ polypeptide. IFN-γ variants whose activity is increased in comparison with the activity of the native IFN-γ polypeptide are also encompassed. The biological activity of IFN-γ variants can be measured by any method known in the art. Examples of such assays are described above.

In one embodiment of the present invention, the IFN-γ used in the methods of the invention is the mature native human IFN-γ polypeptide. However, the present invention encompasses other embodiments where the IFN-γ is any biologically active IFN-γ polypeptide or variant as described elsewhere herein.

In some embodiments of the present invention, the IFN-γ is recombinantly produced. By "recombinantly produced IFN-γ" is intended IFN-γ that has comparable biological activity to native IFN-γ and that has been prepared by recombinant DNA techniques. IFN-γ can be produced by culturing a host cell transformed with an expression vector comprising a nucleotide sequence that encodes an IFN-γ 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-γ are given in 6,046,034 and 5,690,925; both of which are herein incorporated by reference.

Pharmaceutical Composition

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.

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.

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.

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.

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.

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 μm 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 further 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 (18th ed.; Mack Publishing Company, Eaton, Pa., 1990), herein incorporated by reference.

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 (18th ed.; Mack Publishing Company, Eaton, Pa., 1990), herein incorporated by reference.

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. 3,773,919 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™, 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.

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 (18th 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.

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.

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.

Administering the Cytokine

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 μl to about 100 μl, 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.

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.

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.

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.

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.

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-β 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-β 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.

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-β, the dosage range may also be from about 0.02 to about 138 pmol/kg of brain weight. Alternatively, the cytokine maybe 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.

These doses depend on factors including the efficiency with which cytokine IFN-β is transported to the CNS or lymphatic system. A larger total dose can be delivered by multiple administrations of the agent.

Intermittent Dosing

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.

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.

Neuronal Transport

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.

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.

The Olfactory Neural Pathway

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.

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.

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.

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.

The Trigeminal Neural Pathway

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).

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.

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.

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.

Neural Pathways and Nasal Administration

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.

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.

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.

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.

Neural Pathways and Transdermal Administration

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.

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.

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.

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.

Neural Pathways and Sublingual Administration

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.

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.

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.

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.

Neural Pathways and Conjunctival Administration

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.

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.

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.

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.

 

Claim 1 of 48 Claims

1. A method for administering a human interferon-β (IFN-β) or a biologically active variant thereof to the central nervous system or the lymphatic system of a mammal, said mammal in need of said IFN-β to reduce or treat an infection or disorder by producing a therapeutic effect on the central nervous system or the lymphatic system, comprising:

administering a pharmaceutical composition comprising IFN-β or a variant thereof to a tissue of a nasal cavity of the mammal wherein a therapeutically effective amount of the IFN-β or the variant thereof is transported via an olfactory neural pathway to the central nervous system or the lymphatic system of the mammal, wherein the IFN-β or variant thereof provides the therapeutic effect on the central nervous system or the lymphatic system, wherein said biologically active variant has at least 70% sequence identity to the human interferon-β and retains antiviral activity or anti-proliferative activity, and wherein said infection or disorder is selected from viral meningitis, herpes simplex, hepatitis-C, HIV, multiple sclerosis, and a glioma.
 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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