This invention relates to the use of ADNF polypeptides in the treatment of neurotoxicity induced by chemical agents or by disease processes. The ADNF polypeptides include ADNF I and ADNF III (also referred to as ADNP) polypeptides, analogs, subsequences such as NAP and SAL, and D-amino acid versions (either wholly D-amino acid peptides or mixed D- and L-amino acid peptides), and combinations thereof which contain their respective active core sites.

Description of the Invention

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for treating peripheral neurotoxicity in a subject, the method comprising administering a therapeutically effective amount of an ADNF polypeptide to a subject in need thereof.

In one embodiment, the ADNF polypeptide is a member selected from the group consisting of: (a) an ADNF I polypeptide comprising an active core site having the following amino acid sequence: Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:1), or an analogue thereof; (b) an ADNF III polypeptide comprising an active core site having the following amino acid sequence: Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2), or an analogue thereof, and (c) a mixture of the ADNF I polypeptide of part (a) and the ADNF III polypeptide of part (b), or their respective analogues.

In another embodiment, the ADNF polypeptide is a member selected from the group consisting of a full length ADNF I polypeptide, a full length ADNF III polypeptide (ADNP), and a mixture of a full length ADNF I polypeptide and a full length ADNF III polypeptide.

In one embodiment, the ADNF polypeptide is prepared by recombinant DNA methodology. In another embodiment, the active core site of the ADNF polypeptide comprises at least one D-amino acid. In another embodiment, the active core site of the ADNF polypeptide comprises all D-amino acids.

In one embodiment, the ADNF I polypeptide has the formula (R1)x-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-(R2)y (SEQ ID NO:20), or an analogue thereof, in which R1 is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs; R2 is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs; and x and y are independently selected and are equal to zero or one.

In one embodiment, the ADNF I polypeptide is selected from the group consisting of -- see Original Patent.

In one embodiment, the ADNF I polypeptide comprises up to about 20 or 40 amino acids at either or both of the N-terminus and the C-terminus of the active core site.

In another embodiment, the ADNF III polypeptide has the formula (R1)x-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-(R2)y (SEQ ID NO:13), or an analogue thereof, in which R1 is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs; R2 is an amino acid sequence comprising from 1 to about 40 amino acids wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs; and x and y are independently selected and are equal to zero or one.

In another embodiment, the ADNF III polypeptide is a member selected from the group consisting of -- see Original Patent.

In another embodiment, the ADNF III polypeptide comprises up to about 20 amino acids at at least one of the N-terminus and the C-terminus of the active core site.

In one embodiment, an ADNF I polypeptide of part (a) and an ADNF III polypeptide of part (b) are administered to the subject.

In one embodiment, the ADNF polypeptide is administered intranasally. In another embodiment, the ADNF polypeptide is administered orally. In another embodiment, the ADNF polypeptide is administered intravenously or subcutaneously.

In one aspect the invention provides the use of an ADNF polypeptide in the manufacture of a medicament for the treatment of peripheral neurotoxicity.

In one embodiment, the symptoms of said peripheral neurotoxicity are measured by motor dysfunction, muscle wasting, or a change selected from among a change in sense of smell, vision or hearing, deep tendon reflexes, vibratory sense, cutaneous sensation, gait and balance, muscle strength, orthostatic blood pressure, and chronic or intermittent pain.

In another embodiment, the peripheral neurotoxicity is a consequence of treatment with one or more chemical agents. In another embodiment, the peripheral neurotoxicity is a consequence of treatment with a chemical agent selected from among chemical agents for cancer, multiple sclerosis, gout, arthritis, Bechet's disease, psychiatric disorder, immunosuppression and infectious disease.

In another embodiment, one or more chemical agents is selected from among the vinca alkaloids (e.g., vincristine, vindesine, vinorelbine and vinblastine), platinum drugs (e.g., cisplatinum, carboplatinum), L-asparaginase and the taxanes (e.g., taxol, taxotere). In addition to anti-cancer agents, neurotoxicity may be caused by thalidomide, methotrexate, colchicine and anti-infective agents (including but not limited to nucleoside analogs such as lamivudine, zalcitabine, didanosine and stavudine).

In another embodiment, peripheral neurotoxicity is a consequence of a disease process. In another embodiment, the disease process selected from among diabetes, leprosy, Charcot-Marie-Tooth Disease, hereditary sensory and autonomic neuropathies (HSAN), Guillain-Barre syndrome, viral illnesses, (e.g., cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, and human immunodeficiency virus (HIV)), bacterial infection (including Campylobacter jejuni and Lyme disease), chronic alcoholism, botulism, poliomyelitis, uremia, chronic kidney failure, and atherosclerosis.

In another aspect, the present invention provides, the treatment of cancer or neoplasia comprising a) administering an anti-cancer agent; and b) administering, contemporaneously or sequentially with the anti-cancer agent of step a), an ADNF polypeptide in a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a method of testing for response to a therapeutic agent for a neurodegenerative disease or peripheral neurotoxicity comprising the following steps, a) measuring olfaction capacity in a subject having a neurodegenerative disease or potential peripheral neurotoxicity; b) administering a therapeutic agent to the subject; c) measuring olfaction capacity in the subject subsequent to step b); d) comparing olfaction capacity from step a) and step c).

In another embodiment, the therapeutic agent is an ADNF polypeptide. In another embodiment, the neurodegenerative disease is Alzheimer's disease. In another embodiment, the subject has potential peripheral neurotoxicity associated with treatment by a chemotherapeutic agent.

In another aspect, the present invention provides a method of treatment of tauopathy in a subject comprising administering to a subject having or suspected of having a tauopathy, a therapeutically effective amount of an ADNF polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

This invention discloses the surprising finding that an ADNF polypeptide that was shown previously to be neuroprotective of the CNS and to provide cognitive enhancement can alternatively be used in the treatment of peripheral neurotoxicity induced by chemical agents or disease processes. The invention is supported by the findings set out in the Examples that in vivo administration of NAP peptide significantly reduces peripheral neurotoxicity induced by chemical agents.

ADNF Polypeptides: Composition and Synthesis

In one embodiment, the ADNF polypeptides of the present invention comprise the following amino acid sequence:(R.sup.1).sub.x-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-(R.sup.2).sub.y (SEQ ID NO:13) and conservatively modified variations thereof. In this designation, R.sup.1 denotes the orientation of the amino terminal (NH.sub.2 or N-terminal) end and R.sup.2 represents the orientation of the carboxyl terminal (COOH or C-terminal) end.

In the above formula, R.sup.1 is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs. The term "independently selected" is used herein to indicate that the amino acids making up the amino acid sequence R.sup.1 may be identical or different (e.g., all of the amino acids in the amino acid sequence may be threonine, etc.). Moreover, as previously explained, the amino acids making up the amino acid sequence R.sup.1 may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs). Suitable amino acids that can be used to form the amino acid sequence R.sup.1 include, but are not limited to, those listed in Table I (see Original Patent), infra. The indexes "x" and "y" are independently selected and can be equal to one or zero.

As with R.sup.1, R.sup.2, in the above formula, is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs. Moreover, as with R.sup.1, the amino acids making up the amino acid sequence R.sup.2 may be identical or different, and may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs). Suitable amino acids that can be used to form R.sup.2 include, but are not limited to, those listed in Table I, infra.

As used herein, "NAP" or "NAP peptide" refers to the formula above where x and y both equal 0. "NAP related peptide" refers to any of the other variants of NAP which are described the formula.

R.sup.1 and R.sup.2 are independently selected. If R.sup.1 R.sup.2 are the same, they are identical in terms of both chain length and amino acid composition. For example, both R.sup.1 and R.sup.2 may be Val-Leu-Gly-Gly-Gly (SEQ ID NO:14). If R.sup.1 and R.sup.2 are different, they can differ from one another in terms of chain length and/or amino acid composition and/or order of amino acids in the amino acids sequences. For example, R.sup.1 may be Val-Leu-Gly-Gly-Gly (SEQ ID NO:14), whereas R.sup.2 may be Val-Leu-Gly-Gly (SEQ ID NO:15). Alternatively, R.sup.1 may be Val-Leu-Gly-Gly-Gly (SEQ ID NO:14), whereas R.sup.2 may be Val-Leu-GIy-Gly-Val (SEQ ID NO:16). Alternatives, R.sup.1 may be Val-Leu-Gly-Gly-Gly (SEQ ID NO:14), whereas R.sup.2 may be Gly-Val-Leu-Gly-Gly (SEQ ID NO:17).

Within the scope of the above formula, certain NAP and NAP related polypeptides are preferred, namely those in which x and y are both zero (i.e. NAP). Equally preferred are NAP and NAP related polypeptides in which x is one; R.sup.1 Gly-Gly; and y is zero. Also equally preferred are NAP and NAP related polypeptides in which is one; R.sup.1 is Leu-Gly-Gly; y is one; and R.sup.2 is -Gin-Ser. Also equally preferred are NAP and NAP related polypeptides in which x is one; R.sup.1 is Leu-Gly-Leu-Gly-Gly-(SEQ ID NO:18; y is one; and R.sup.2 is -Gln-Ser. Also equally preferred are NAP and NAP related polypeptides in which x is one; R.sup.1 is Ser-Val-Arg-Leu-Gly-Leu-Gly-Gly-(SEQ ID NO:19); y is one; and R.sup.2 is -Gln-Ser. Additional amino acids can be added to both the N-terminus and the C-terminus of the active peptide without loss of biological activity.

In another aspect, the present invention provides pharmaceutical compositions comprising one of the previously described NAP and NAP related polypeptides in an amount sufficient to exhibit desired therapeutic activity, in a pharmaceutically acceptable diluent, carrier or excipient. In one embodiment, the NAP or NAP related peptide has an amino acid sequence selected from the group consisting of SEQ ID NO:2, and 9-12, and conservatively modified variations thereof.

In another embodiment, the ADNF polypeptide comprises the following amino acid sequence: (R.sup.1).sub.x-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-(R.sup.2).sub.y (SEQ ID NO:27) and conservatively modified variations thereof. In this designation, R.sup.1 denotes the orientation of the amino terminal (NH.sub.2 or N-terminal) end and R.sup.2 represents the orientation of the carboxyl terminal (COOH or C-terminal) end.

In the above formula, R.sup.1 is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs. The term "independently selected" is used herein to indicate that the amino acids making up the amino acid sequence R.sup.1 may be identical or different (e.g., all of the amino acids in the amino acid sequence may be threonine, etc.). Moreover, as previously explained, the amino acids making up the amino acid sequence R.sup.1 may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs). Suitable amino acids that can be used to form the amino acid sequence R.sup.1 include, but are not limited to, those listed in Table I, infra. The indexes "x" and "y" are independently selected and can be equal to one or zero.

As with R.sup.1, R.sup.2, in the above formula, is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs. Moreover, as with R.sup.1, the amino acids making up the amino acid sequence R.sup.2 may be identical or different, and may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs). Suitable amino acids that can be used to form R.sup.2 include, but are not limited to, those listed in Table I, infra.

As used herein, "SAL" or "SAL peptide" refers to the formula above where x and y both equal 0. "SAL related peptide" refers to any of the other variants of SAL which are described the formula.

R.sup.1 and R.sup.2 are independently selected. If R.sup.1 R.sup.2 are the same, they are identical in terms of both chain length and amino acid composition. Additional amino acids can be added to both the N-terminus and the C-terminus of the active peptide without loss of biological activity.

In another aspect, the present invention provides pharmaceutical compositions comprising one of the previously described SAL and SAL-related polypeptides in an amount sufficient to desired therapeutic activity, in a pharmaceutically acceptable diluent, carrier or excipient. In one embodiment, the SAL or SAL related peptide has an amino acid sequence selected from the group consisting of SEQ ID NO:1 and 3-8, and conservatively modified variations thereof. In a further embodiment, the SAL related peptide comprises SALLRSIPAPAGASRLLLLTGEIDLP (SEQ ID NO:21). The sequence SALLRSIPAPAGASRLLLLTGEIDLP (SEQ ID NO:21) is also known as Colivelin and is a combination of the SAL active site and a derivative of the Humanin protein named AGA-(C8R)HNG17. Colivelin is described in Chiba et al., J. Neurosci. 25:10252-10261 (2005), which is herein incorporated by reference for all purposes.

It will be readily apparent to those of ordinary skill in the art that preferred ADNF polypeptides can readily be selected for peripheral neuroprotective activity by employing suitable assays and animal models known to those skilled in the art, some of which are disclosed herein.

In addition, one of skill in the art will recognize that a variety of chemical modifications can be made to the peptides without diminishing their biological activity. In addition to replacement of specific amino acids with other amino acids, there may also be a wide range of modifications to specific amino acids, and conjugates with a wide variety of polymers, proteins, carbohydrates or other organic moieties.

The peptides of the invention may be prepared via a wide variety of well-known techniques. Peptides of relatively short size are typically synthesized on a solid support or in solution in accordance with conventional techniques (see, e.g., Merrifield, Am. Chem. Soc. 85:2149-2154 (1963)). Various automatic synthesizers and sequencers are commercially available and can be used in accordance with known protocols (see, e.g., Stewart & Young, Solid Phase Peptide Synthesis (2nd ed. 1984)). Solid phase synthesis in which the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the preferred method for the chemical synthesis of the peptides of this invention. Techniques for solid phase synthesis are described by Barany & Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield et al 1963; Stewart et al. 1984). NAP and related peptides are synthesized using standard Fmoc protocols (Wellings & Atherton, Methods Enzymol. 289:44-67 (1997)).

Other synthetic methods for peptides include liquid phase synthesis (e.g. Fischer and Zheleva J Pept Sci. 8(9):529-42 (2002).

In addition to the foregoing techniques, the ADNF peptides, in particular the full length proteins ADNF I and ADNF III for use in the invention may be prepared by recombinant DNA methodology. Generally, this involves creating a nucleic acid sequence that encodes the protein, placing the nucleic acid in an expression cassette under the control of a particular promoter, and expressing the protein in a host cell. Recombinantly engineered cells known to those of skill in the art include, but are not limited to, bacteria, yeast, plant, filamentous fungi, insect (especially employing baculoviral vectors) and mammalian cells.

Use of ADNF Polypeptides for Treating Peripheral Neurotoxicity

Peripheral neurotoxicity may be identified and diagnosed in a subject by a variety of techniques. Typically it may be measured by motor dysfunction, muscle wasting, or a change in sense of smell, vision or hearing, or changes in deep tendon reflexes, vibratory sense, cutaneous sensation, gait and balance, muscle strength, orthostatic blood pressure, and chronic or intermittent pain. In humans these symptoms are also sometimes demonstrative of toxic effects in both the PNS and the CNS. Ultimately, there are hundreds of possible peripheral neuropathies that may result from neurotoxicity. Reflecting the scope of PNS activity, symptoms may involve sensory, motor, or autonomic functions. They can be classified according to the type of affected nerves and how long symptoms have been developing.

Peripheral neurotoxicity can be induced by chemotherapeutic agents (anti-cancer, anti-microbial and the like) and by disease processes. These two different areas are discussed separately below.

Regarding chemotherapeutic agents, it is well known that patients exposed to agents such as Vinca alkaloids, suramin, taxanes, and cisplatin can develop peripheral neurotoxicity. Neurological observations published in recent years indicate that administration of taxanes and cisplatin in patients affected by neoplasm induces nerve deficits in a dose- and time-dependent manner. (Bedikian A. Y., et al. 1995. J. Clin. Oncol., 13: 2895-2899). Moreover, when platinum compounds and taxanes are used in combination, the patients develop more severe peripheral neuropathies. The pathophysiology of chemotherapeutic agent-induced neuropathy is still not clear, although a variety of studies have shown that taxanes interfere with axonal transport, causing axonal distal sensory-motor lesions, whereas platinum compounds induce sensory neuropathy acting mainly on the neuronal cell bodies of the spinal ganglion. Pathological and electrophysiological studies have also indicated that neurons of the dorsal root ganglion are selectively damaged after cisplatin treatment. It has been reported that the development of this peripheral neurotoxicity can induce clinicians to interrupt therapy to prevent more severe neurological deficits (Amato A. A., Collins M. P. Semin. Neurol., 18: 125-142, 1998.). Because of the neurotoxic effects, much effort has been devoted to the identification of potential neuroprotective agents. It is reasonable, therefore, to hypothesize that ADNF polypeptides, which can prevent neurotoxicity and/or promote peripheral innervation after chemotherapy, will be clinically useful. Those skilled in the art are familiar with chemotherapeutic agents that may cause peripheral neurotoxicities. In general such chemotherapeutic agents are used in the treatment of cancer, multiple sclerosis, gout, arthritis, Bechet's disease, psychiatric disorders, familial Mediterranean fever, amyloidosis, immunosuppression and infectious disease. A representative list includes vinca alkaloids (vincristine, vindesine, vinorelbine and vinblastine), platinum drugs (cisplatinum, carboplatinum), L-asparaginase and the taxanes (taxol, taxotere). In addition to anti-cancer agents, neurotoxicity may be cause by thalidomide, methotrexate, colchicine and anti-infective agents (including but not limited to nucleoside analogs such as lamivudine, zalcitabine, didanosine and stavudine).

The method of the invention recognizes that administration of a therapeutically effective amount of an ADNF polypeptide is useful to treat or prevent peripheral neurotoxicity in a subject receiving a chemotherapeutic agent, such as those described above. Relative to the administration of the chemotherapeutic agent, the administration of the ADNF polypeptide can occur before, at the same time, subsequent to or on an irregular basis. Those skilled in the art are able to identify a suitable temporal relationship between the agents which is designed to establish peripheral neuroprotection before the consequences of the neurotoxicity develop. Treatment may continue until chemotherapeutic agent is discontinued, or until the neurotoxicity resulting from the agent is resolved and not expected to worsen.

When administered non-contemporaneously (e.g. sequentially) with the chemotherapeutic agent, the ADNF polypeptide will typically be formulated separately from the agent. When administered contemporaneously, it may be advantageous to provide the ADNF polypeptide in a dosage form in combination with the agent. Thus the invention recognizes a formulation of a chemotherapeutic agent and an ADNF polypeptide, wherein the dose of the ADNF polypeptide is effective to reduce or eliminate the peripheral neurotoxicity associated with the chemotherapeutic agent. Those skilled in the art are able to select a proper dose of ADNF polypeptide based this disclosure and on the anticipated neurotoxic effects of the selected chemotherapeutic agent.

As mentioned previously, certain disease processes can also result in peripheral neurotoxicity. For example, the diabetes/peripheral neuropathy link has been well established. A typical pattern of diabetes-associated neuropathic symptoms includes sensory effects that first begin in the feet. The associated pain or pins-and-needles, burning, crawling, or prickling sensations form a typical "stocking" distribution in the feet and lower legs.

Other diseases that may result in peripheral neurotoxicity include inherited or acquired disorders, including infectious diseases. Such diseases include leprosy, Charcot-Marie-Tooth Disease, Inherited neurological disorders such as the hereditary sensory and autonomic neuropathies (HSAN), Guillain-Barre syndrome which may arise from complications associated with viral illnesses, such as cytomegalovirus, Epstein-Barr virus, and human immunodeficiency virus (HIV), or bacterial infection, including Campylobacter jejuni and Lyme disease. Other well-known causes of peripheral neuropathies include chronic alcoholism, infection varicella-zoster virus, botulism, and poliomyelitis. Peripheral neuropathy may develop as a primary symptom, or it may be less significant. Uremia, or chronic kidney failure, carries a 10-90% risk of eventually developing neuropathy, and there may be an association between liver failure and peripheral neuropathy. Accumulation of lipids inside blood vessels (atherosclerosis) can choke-off blood supply to certain peripheral nerves.

As recognized in the case of chemotherapeutic agents, use of ADNF polypeptides to treat or prevent neurotoxicity from disease processes requires administration of a therapeutically effective amount of an ADNF polypeptide sufficient to treat or prevent peripheral neurotoxicity in a subject suffering from such disease. In this case, relative to the onset of the peripheral neurotoxicity, the administration of the ADNF polypeptide can occur before, at the same time, subsequent to or on an irregular basis. Those skilled in the art are able to identify a suitable temporal relationship between the agents which is designed to establish peripheral neuroprotection before the consequences of the disease induced neurotoxicity develop. Treatment may continue until the underlying disease resolves, or until the neurotoxicity resulting from the disease is resolved and not expected to worsen. In some cases, administration of ADNF polypeptides may be chronic.

Because the disease processes of concern to this invention are often treated with other therapeutic agents, the invention recognizes that it may be advantageous to provide the ADNF polypeptide in a dosage form in combination with such an agent. Thus the invention recognizes a formulation of a therapeutic agent and an ADNF polypeptide, wherein the dose of the ADNF polypeptide is effective to reduce or eliminate the peripheral neurotoxicity associated with the chemotherapeutic agent. Those skilled in the art are able to select a proper dose of ADNF polypeptide based this disclosure and on the anticipated neurotoxic effects of the selected therapeutic agent.

Use of ADNF Polypeptides to Treat Tauopathy and Related Diseases

Tauopathy means the accumulation of microtubule-associated protein tau in the neuronal and glial cytoplasm. This terminology is relatively new, but it relates to neurodegenerative diseases evidencing widespread accumulation of tau epitopes both in neurons and glia, sometimes without deposition of amyloid beta protein. Tauopathy is now considered to be one of the primary causes of neuronal degeneration, with about one third of the very elderly presenting with deposition of abnormally phosphorylated tau proteins with relative paucity of amyloid beta protein (Abeta). In the course of neurofibrillary tangle formation (including tau aggregates), the major proteinaceous components of these lesions undergo post-translational modifications. In the case of tau, these include phosphorylation of mainly serine and threonine, but also tyrosine residues. In addition, tau is subject to ubiquitination, nitration, truncation, prolyl isomerization, association with heparan sulfate proteoglycan, glycosylation, glycation and modification by advanced glycation end-products (AGEs). Human tauopathies include Alzheimer's disease and frontotemporal dementia with parkinsonism linked to chromosome 17 (Chen et al. Curr Drug Targets. 5(6):503-15 (2004)). Furthermore, recent studies have shown that as a consequence of chemotherapy there was an increase in cerebrospinal fluid tau, which is a marker of neurodegeneration (Van Gool et al. Leukemia. 14:2076-84 (2000); Lee et al., Biochem. Biophys Acta. 1739: 251-9 (2005))

The instant invention relates to a method of treatment of tauopathy in a subject comprising administering to the subject a therapeutically effective amount of an ADNF polypeptide. Treatment of tauopathy with the NAP peptide is a specific embodiment of this invention.

The inventors have recognized, based on the instant disclosure, that ADNF polypeptides such as NAP effectively prevent neurotoxic damage by the vinca alkaloid vincristine (see Examples), and without wishing to be bound to any particular theory or mechanism of action, that this effect of NAP can be combined with the teachings of PCT publication WO 2004/080957 (Gozes et al.) and Divinski et al. J Biol Chem. 279(27):28531-8. (2004) that demonstrate that NAP interacts with tubulin to enhance microtubule formation and stabilize microtubular structure in glial and neural cells, to establish for the first time that NAP is useful for the treatment of tauopathy. Other peptides of the ADNF family including ADNF-9 (or SAL) and all D-amino acids SAL (termed D-SAL, Brenneman et al. (2004), infra) as well as full length ADNP (ADNFIII) interact with tubulin. (Furman et al., Neuron Glia Biology 1:193-9 (2004).

It is well recognized that tau performs an important function of stabilizing and maintaining the microtubular network, that in turn is important for axonal transport in neurons. The formation of the pathological neurofibrillary tangles which results from the hyperphosphorylation of tau, leads to microtubule breakdown and impaired axonal transport (Ishihara et al. Neuron 24:751-62 (1999); Lee et al., Annu Rev Neurosci. 24:1121-59 (2001); Morfini et al. Neuromolecular Med. 2:89-99 (2002); Gozes. J Mol Neurosci. 19(3):337-8 (2002)). Divinski et al. J Biol Chem. 279(27):28531-8. (2004) have demonstrated that exposure to zinc toxicity resulted in microtubule breakdown in astrocytes and neurons and that NAP protects these cells from this toxicity by promoting the reorganization of the microtubular network. In the same experiments, tubulin was identified as a NAP binding molecule. Furthermore, in the presence of NAP, there is an increase in the ratio of non-phosphorylated tau to phosphorylated tau (Gozes & Divinski, Journal of Alzheimer's Disease 6(6 Suppl.):S37-41 (2004)) and increased neurite outgrowth, a process that is dependent on slow axoplasmic transport (Lagreze et al., Invest Opthalmol Vis Sci. 46:933-8 (2005); Gozes. Neurochem Int. 4:101-20 (1982); Smith-Swintosky et al. J Mol Neurosci. 25:225-38 (2005). Therefore, it is possible that NAP functions to promote the assembly and stability of the microtubular network either directly by binding to tubulin or indirectly through changes in the levels of the different forms of tau. The promotion of proper microtubule assembly is also important in the case of vicristine treatment, as vincristine and related compounds facilitate the tubulin spiral filaments and aggregated spiral formation (Verdier-Pinard et al. Biochem Pharmacol. 58(6):959-71 (1999)) Any other tubulin binding and modifying agents including, but not limited to vinca alkaloids (vincristine, vindesine, vinorelbine and vinblastine), the taxanes (taxol, taxotere), nocodazole and colchicines will affect axoplasmic transport which can in turn be protected by the specific neuroprotective effect of NAP treatment (Gozes et al. J Mol Neurosci. 20(3):315-22, (2003)).

Use of Olfaction Testing to Measure Effectiveness of Neurological Therapeutics, such as ADNF Polypeptides

Olfaction disabilities, including hyposmia (reduction in ability to taste and smell) or anosmia (total loss of ability to taste and smell), are associated with neurodegenerative disease (such as Alzheimer's disease, multiple sclerosis, Huntington disease, amyotrophic lateral sclerosis, Parkinson's disease and others) and peripheral neurotoxicity induced by chemotherapeutic agents and by disease processes. In all these cases, olfaction disabilities are generally progressive.

The present invention provides a method to identify whether a subject having a neurodegenerative disease or peripheral neurotoxicity is responding to therapeutic agents administered to treat the disease by measuring olfaction in the subject. A response to therapy is indicated either by an improvement in olfaction capacity or quality of the subject after treatment with a therapeutic agent, or at least a reduction, after such treatment, in the degree of hyposmia or the progress of hyposmia to anosmia that would be expected in subjects with untreated disease.

While the method can be used to test response to any therapeutic agent for the treatment of the neurodegenerative disease or the peripheral neurotoxicity, in particular, this invention provides a method to identify a response to therapy with ADNF polypeptide.

The method involves testing for response to a therapeutic agent for a neurodegenerative disease comprising the following steps: a) measuring olfaction capacity in a subject having a neurodegenerative disease or potential peripheral neurotoxicity; b) administering a therapeutic agent to the subject; c) measuring olfaction capacity in the subject subsequent to step b); and d) comparing olfaction capacity from step a) and step c).

Based on the results of the comparison of step d), the subject and care-giver can determine whether there is either an improvement in olfaction capacity or quality of the subject after treatment with a therapeutic agent, or at least a reduction, after such treatment, in the degree of hyposmia or the progress of hyposmia to anosmia that would be expected in subjects with untreated disease, thus indicating a response to the therapeutic agent, or not. Patients and care-givers can then go on to decide whether treatment with the therapeutic agent should continue or be halted.

This method provides many advantages for assessing a response to a therapeutic agent, in particular because olfaction is one of the first senses to be lost or diminished as a result of a neurodegenerative disease or the onset of peripheral neurotoxicity.

Pharmaceutical Administration

ADNF polypeptides of the invention are generally administered in a pharmaceutical formulation. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences (17th ed. 1985). In addition, for a brief review of methods for drug delivery, see Langer, Science 249:1527-1533 (1990).

As such, the present invention provides for therapeutic compositions or medicaments comprising one or more of the ADNF polypeptides described herein in combination with a pharmaceutically acceptable excipient, wherein the amount of the ADNF polypeptide is sufficient to provide a therapeutic effect.

The ADNF polypeptides of the present invention are embodied in pharmaceutical compositions intended for administration by any effective means, including parenteral, topical, nasal, oral, pulmonary (e.g. by inhalation) or local administration. Preferably, the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, intramuscularly, or intranasally.

Thus, the invention provides compositions for parenteral administration that comprise a solution of ADNF polypeptide, as described above, dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used including, for example, water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques or, they may be sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions including pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, such as, for example, sodium acetate, sodium lactate, sodium chloride potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

For solid compositions, conventional nontoxic solid carriers may be used that include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient and more preferably at a concentration of 25%-75%.

For aerosol administration, the ADNF polypeptides are preferably supplied in finely divided form along with a surfactant and propellant. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery. An example includes a solution in which each milliliter included 7.5 mg NaCl, 1.7 mg citric acid monohydrate, 3 mg disodium phosphate dihydrate and 0.2 mg benzalkonium chloride solution (50%) (Gozes et al., J Mol Neurosci. 19(1-2):167-70 (2002)). The ADNF polypeptides of the invention can therefore be used in the manufacture of a medicament for the treatment or prevention of peripheral neurotoxicity. The medicament can comprise any of the pharmaceutical formulations contemplated herein, with any amount of active ingredient (e.g. ADNF polypeptide) contemplated herein.

In therapeutic applications, the ADNF polypeptides of the invention are administered to a patient in an amount sufficient to reduce or eliminate symptoms of peripheral neurotoxicity. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on, for example, the particular NAP or ADNF polypeptide employed, the type of disease or disorder to be prevented, the manner of administration, the weight and general state of health of the patient, and the judgment of the prescribing physician.

For example, an amount of polypeptide falling within the range of a 100 ng to 10 mg dose given intranasally once a day (e.g., in the evening) would be a therapeutically effective amount. Alternatively, dosages may be outside of this range, or on a different schedule. For example, dosages may range from 0.0001 mg/kg to 1000 mg/kg, and will preferably be about 0.001 mg/kg, 0.1 mg/kg, 1 mg/kg, 5 mg/kg, 50 mg/kg or 500 mg/kg per dose. Doses may be administered hourly, every 4, 6 or 12 hours, with meals, daily, every 2, 3, 4, 5, 6, or 7 days, weekly, every 2, 3, 4 weeks, monthly or every 2, 3 or 4 months, or any combination thereof. The duration of dosing may be single (acute) dosing, or over the course of days, weeks, months, or years, depending on the condition to be treated. Those skilled in the art can determine the suitable dosage, and may rely on preliminary data reported in Gozes et al., 2000, Gozes et al., 2002), Bassan et al. 1999; Zemlyak et al., Regul. Pept. 96:39-43 (2000); Brenneman et al., Biochem. Soc. Trans. 28: 452-455 (2000); Erratum Biochem Soc. Trans. 28:983; Wilkemeyer et al. Proc. Natl. Acad. Sci. USA 100:8543-8548 (2003)). Suitable dose ranges are described in the examples provided herein, as well as in WO 9611948.
 

Claim 1 of 24 Claims

1. A method for treating peripheral neurotoxicity in a subject, the method comprising administering a therapeutically effective amount of an ADNF polypeptide to a subject in need thereof, wherein the ADNF polypeptide is a member selected from the group consisting of: (a) an ADNF I polypeptide comprising an active core site having the following amino acid sequence: Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:1); (b) an ADNF III polypeptide comprising an active core site having the following amino acid sequence: Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2), and (c) a mixture of the ADNF I polypeptide of part (a) and the ADNF III polypeptide of part (b); wherein said peripheral neurotoxicity is a consequence of treatment with one or more chemical agents.

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