Peptides for the Treatment of Multiple Sclerosis and Related Diseases

A method of treatment of neurodegenerative illness in a patient comprising the steps of preparing a composition comprising a D peptide and a pharmaceutically acceptable carrier. The D peptide has the general structure: A-B-C-D-E-F-G-H in which A is Ala, or absent, B is Ser, Thr or absent, C is Ser, Thr or absent, D is Ser, Thr, Asn, Glu, Arg, Ile, Leu, E is Ser, Thr, Asp, Asn, F is Thr, Ser, Asn, Arg, Gln, Lys, Trp, G is Tyr, and H is Thr, Ser, Arg, Gly. All amino acids in the D peptide are the D stereoisomeric configuration. The peptide composition is administered in a therapeutically effective dose.

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Description

This application claims the benefit of U.S. Provisional Application Ser. No. 61/816,565, filed Apr. 26, 2013.

The present invention relates, broadly to the treatment or prevention of excessive inflammation, whether caused by injury, bacteria, viruses and/or other infective agents, opportunistic infections (which may be consequential to an immunodepressed state, for example resulting from cancer or therapy, particularly cytotoxic drug therapy or radiotherapy), autoimmunity, cessation of immunosuppressive treatments, or initiation of antiviral therapies, or otherwise.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the chemotaxis of human monocytes for several all-D analogues of Peptide T.

FIG. 2 illustrates RAP-103 potently blocking both MCP-1- and MIP-1β-elicited chemotaxis of human monocytes.

FIG. 3 illustrates the effects of several peptides in blocking CCL2 (MCP-1) chemotaxis at low concentration.

FIG. 4 illustrates five all-D-pentapeptides, with unrelated sequences, all inhibiting CCR2 (MCP-1) elicited chemotaxis of human monocytes.

FIG. 5 illustrates all-D-(IDNYT) potently blocking MCP-1 elicited adhesion of human monocytes.

FIG. 6 illustrates lipopolysaccharide (LPS) induction TNFα secretion in monocyte-derived iDC.

In particular embodiments, the invention relates to the prevention or treatment of neurodegenerative illnesses which may include demyelinating diseases such as HTLV-1-associated myelopathy (TSP/HAM), multiple sclerosis (MS), amyotrophic lateral sclerosis and symptoms or diseases in humans which are associated with chronic immune activation that occurs via cytokine, chemokine, and toll-receptor inflammatory pathways. The invention also relates to pharmaceutical compositions useful in such treatment and/or prevention and to certain active peptides per se.

It is now appreciated that the CNS exhibits features of inflammation mediated for example by the resident CNS cells astrocytes and microglia, which in response to injury, infection or disease, generate inflammatory mediators, including proinflammatory cytokines, prostaglandins, free radicals and complement, which in turn induce chemokines and adhesion molecules, recruit immune cells, and activate glial cells. When excessive, this defense response causes pathologies. Much of the key evidence demonstrating that inflammation and inflammatory mediators contribute to CNS disorders points to this delicate balance of inflammation and modulation as the tightrope under which so called normalcy functions.

IL-1b, IL-6, and tumor necrosis factor-α (TNF-α) are known to be among the major cytokines up-regulated during the acute-phase response to CNS stress and injury. The release of TNF-α and other inflammatory cytokines exacerbates the activation of glial cells and the physiological response switching the Th1 to Th2 cycle and promoting gliosis, inhibiting astrocytic glutamate uptake and inducing apoptosis, particularly in oligodendrocytes thereby contributing to damaging demyelination. The ability to inhibit the action of TNF-α and other inflammatory activators during ongoing CNS disease processes may yield a salutary clinical outcome.

The invention finds particular use in the prevention or treatment of MS, TSP/HAM and other inflammatory myelopathies (particularly those previously specifically mentioned) and/or symptoms or diseases in humans which are associated with immune activation, either acute or chronic. More particularly, the invention is useful in treating chronic progressive, relaxing or remitting neurogegenerative diseases, chronic fatigue syndromes, including fatigue in MS, which causes substantial patient morbidities, toxic shock syndrome associated with microbial infection, arthritis, inflammatory bowel disease and host-versus-graft response in transplant patients. Such efficacious results in the use of the above compounds is thought to be due, without being limited to any particular theory, to the immunonormaltive activities of these compounds to treat chronic inflammatory states.

Multiple sclerosis (MS) is generally considered by many authorities to be a chronic inflammatory disease. Both MS and HTLV-1 associated myelopathy (TSP/HAM) affect the central and the peripheral nervous systems and both may present clinically as a myelopathy affecting both the spinal nerves and the spinal myelinated nerve fibers. Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system and is the commonest chronic neurological disease of young adults. The incidence of MS and its pattern of distribution have been unchanged for decades.

Myelopathy, as already mentioned in being a disorder of the spinal cord, can have many different etiologies most of which are mediated by inflammation include the following:

neurosyphillis;

B12 or folate deficiency;

sarcoidosis;

transverse myelitis;

arachonoiditis;

cervical spondylitis;

motor neuron disease;

neurofibromatosis;

spinal cord compression from tumour, disc or arthritis;

lupus erythematosus of the spinal cord; and

viral encephalomyelitis.

In particular the compounds here described would also have significant patient benefit in the immune reconstitution inflammatory syndrome (IRIS)—a paradoxical deterioration of a preexisting condition, often an infection, related to the recovery of the immune system. MS patients who stop their immunosuppressive treatments often experience an IRIS reaction, which can be severe. If immune function improves rapidly following the commencement of a medical treatment intervention or therapy, systemic or local inflammatory reactions may occur. This restoration of immunity may result in additional immunopathological reactions. In brain, neurological symptoms can worsen and do not always spontaneously resolve, and may be life-threatening. In MS an IRIS event typically follows discontinuation of immunosuppressive therapies in the context of JC virus infection.

Chronic fatigue syndrome (CFS) or chronic fatigue immune dysfunction syndrome (DeFritas et al, Proc. Natl. Acad. Sci. 88, 2922-2926 (1991)) is a condition of unknown etiology characterized by a diverse set of signs and systems including severe fatigue, post-exertional malaise, headaches, night sweats, myalgia, ataxia, low grade fever and lymphadenophathy. The serum and cerebrospinal fluid of patients with CFS has been shown to contain increased levels of IL-2, IFN and IL-1 (Wallace et al, Arth. Rheum. 32 1334-1335 (1989) and IL-6 (Chao et al, J. Infect. Dis. 162 1412 (1990)). MS patients may experience fatigue reactions which are immunological in origin, disabling, and not treated with current therapies.

Toxic shock syndrome (TSS) is produced by a Staphylococcus aureus enterotoxin, toxic shock syndrome toxin-1 (TSST-1) acting to stimulate innate immunity via Toll receptors, (TLRs). The symptoms of TSS (such as fever, rash, hypotension, nausea, vomiting and diarrhea) are consistent with over activation of the immune system and production of cytokines. These symptoms have been reproduced in animal models by the administration of tumor necrosis factor (TNF) (Miethke et al. J. Exp. Med. 175 91-98 (1992)).

Rheumatoid arthritis is a disease characterized by chronic inflammation and erosion of joints that may affect up to 3% of the population, including children. Symptoms of rheumatoid arthritis include morning stiffness, swelling and pain upon motion in at least one joint and joint swelling.

Extra-articular manifestations of rheumatoid arthritis include vasculitis, cataracts, uveitis, interstitial fibrosis, pericarditis and myocarditis, peripheral neuropathy, myeloid deposits, chronic anemia and subcutaneous and pulmonary nodules.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition that fulfills some of the criteria of an autoimmune disease (Snook, Gur 31 961-963 (1990)). Inflammation and tissue damage involves the recruitment and activation of neutrophils, macrophages and lymphocytes (MacDermott et al, Adv. Immunol. 42 285-328 (1988)) which generate cytokines and proinflammatory molecules

such as prostaglandins and leukotrienes. Drugs used to treat IBD include anti-inflammatory agents such as sulphasalazine (5-ASA), corticosteroids,
cyclosporin A and azathioprine.

The invention may be useful in the prevention or treatment of illness or medical conditions, particularly those involving inflammation, such as: Viral, bacterial or drug-induced hepatitis or meningitis; rheumatoid, psoriatic, reactive, or osteo-arthritisor other arthritides; sepsis/septic shock; dermal inflammation; ARDS (adult respiratory distress syndrome); graft rejection; inflammation secondary to the chemotherapy or radiotherapy of neoplastic disease. Excessive inflammation that occurs due to initiation or cessation of therapy may also be treated.

Approximately 85% of newly diagnosed MS patients have an episodic course termed relapsing-remitting MS (RRMS) where attacks are followed by partial or complete recovery, such that symptoms may be inactive for months. Another type is secondary-progressive MS (SPMS) which manifests as occasional relapses but generally constant symptoms with no remissions. A primary-progressive MS form (PPMS) affects 10% of the MS population and has slow onset but continuous worsening conditions. A progressive-relapsing MS form (PRMS) affects 5% and shows steady worsening of condition from the onset.

There is no cure for multiple sclerosis. Available therapies reduce inflammation, delay the progression of the disease, reduce the frequency and severity of acute attacks, and improve walking Physical, occupational, speech, and cognitive therapy also are used for improving function. Steroids are used for treating the acute episodes of inflammation in the brain because of the ability of steroids to suppress inflammation. Since their use is associated with important long-term side effects they are used only for short periods of time. Interferon beta-1a and Interferon beta-1b are used for the treatment of patients with relapsing forms of MS to slow the progression of physical disability and decrease the frequency of flare ups. The mechanism of action of interferons in MS is unknown.

Tysabri, a recombinant antibody that binds to integrins expressed on the surface of white blood cells, blocks inflammation in MS by inhibiting adhesion of the white blood cells to their receptors. It is used to delay the progression of physical disability and reduce the frequency of clinically important flare-ups in patients with relapsing MS. Tysabri increases the risk of progressive multifocal leukoencephalopathy, a viral infection of the brain that usually leads to death or severe disability. Therefore, Tysabri generally is reserved for patients who have had an inadequate response or are unable to tolerate other therapies for MS.

Thus the major current therapies for MS have anti-inflammatory mechanisms, but all can have serious, even life-threatening, side effects that limit their use. An unmet medical need exists for treatments to treat underlying disease processes of MS with low or no toxicities.

New therapies for MS and the other mentioned illnesses should seek additional inflammatory targets, not covered by the mentioned current treatments. Chemokines have been shown to play an important role in the activation and directional migration of cells to sites of CNS inflammation. The chemokine monocyte chemoattractant protein (MCP)-1/CCL2 and its receptor CCR2 have been strongly implicated in disease pathogenesis in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS). Simpson [Simpson, J., P. Rezaie, J. Newcombe, M. L. Cuzner, D. Male, and M. N. Woodroofe. 2000. Expression of the beta-chemokine receptors CCR2, CCR3 and CCR5 in multiple sclerosis central nervous system tissue. J Neuroimmunol. 108:192-200] examined the expression of the beta-chemokine receptors CCR2, CCR3 and CCR5 in post-mortem MS CNS tissue. Low levels of CCR2, CCR3 and CCR5 were expressed by microglial cells throughout control CNS tissue. In contrast, chronic active MS lesions expressed high CCR2, CCR3 and CCR5 which were associated with foamy macrophages and activated microglia. CCR2 and CCR5 were also present on large numbers of infiltrating lymphocytes. A smaller number of CCR3-positive lymphocytes were present. Ligands for CCR2 and CCR3 include MCP-1 and MCP-3 which were co-localized around vessels with the infiltrating leukocytes, but were also present in unaffected areas of cortex. The elevated expression of CCR2, CCR3 and CCR5 in the CNS in MS suggests these beta-chemokine receptors and their ligands play a role in the pathogenesis of MS.

Baloshov [Balashov, K. E., J. B. Rottman, H. L. Weiner, and W. W. Hancock. 1999. CCR5(+) and CXCR3(+) T cells are increased in multiple sclerosis and their ligands MIP-1alpha and IP-10 are expressed in demyelinating brain lesions. Proc Natl Acad Sci USA. 96:6873-6878] found both CCR5(+) and CXCR3(+) T cells increased in progressive MS compared with controls. Furthermore, peripheral blood CCR5(+) T cells secreted high levels of IFN-gamma. In the brain, the CCR5 ligand, MIP-1alpha, was strongly associated with microglia/macrophages, and the CXCR3 ligand, IP-10, was expressed by astrocytes in MS lesions but not unaffected white matter of control or MS subjects. Areas of plaque formation were infiltrated by CCR5-expressing and, to a lesser extent, CXCR3-expressing cells. The authors suggest their results “provide a rationale for the use of agents that block CCR5 and/or CXCR3 as a therapeutic approach in the treatment of MS”.

All compounds disclosed in these specifications are useful for the present invention. The compounds block multiple chemokine receptors implicated in the discussed neuroinflammatory conditions, with particular relevance to MS and TSP/HAM, and the lead compound DAPTA, has shown patient benefits with no toxicities.

The lead compound DAPTA was derived from the octapeptide Ala-Ser-Thr-Thr-Thr-AsnTyr-Tyr. This all-L amino acid octapeptide was called Peptide T because 50% of the amino acid residues are threonines. This peptide has been identified from the V2 subregion of the human immune deficiency virus (HIV) external glycoprotein molecule gp120, specifically near the bridging sheet to the V3 loop, a region which is responsible for virus binding via the CCR5 and related chemokine receptors, such as CCR2, CCR8, CX3CR1 all of which may function as HIV entry receptors. The peptides we here describe are antagonists of multiple HIV entry chemokine receptors. Peptide T is not stable in the body, however its close derivative Dala1-peptide T-amide, or “DAPTA” has a single D-amino acid in position 1 and a terminal amide (—NH2) group which confers stability in the blood to some proteases. The DAPTA peptide however easily aggregates upon storage in liquid solutions due to defects in manufacturing and an improved process was developed to overcome this limitation. Further modifications have been created that confer oral bioavailability.

The peptides can be used in pharmaceutical compositions and compositions of matter for treating and preventing any disease or condition caused by an organism, compound or immune dysfunction that results in an inflammatory reaction of the immune system.

The peptides or peptide formulations may be used alone or in combination with any other pharmaceutically active compound, such as an anti-infective agent, for example an antibiotic and/or antiviral agent and/or antifungal agent, or another pharmaceutically active compound, such as an antineoplastic agent or an excipient that enhances delivery through nasal or oral routes of administration.

The peptides may be administered orally, bucally, parenterally, topically, rectally, vaginally, by intranasal inhalation spray, by intrapulmonary inhalation or in other ways. In particular, the peptides according to the invention may be formulated for topical use, for inhalation with spray or powder, for injection (for example subcutaneous, intramuscular, intravenous, intra-articular or intra-cisternal injection), for infusion or for oral administration and may be presented in unit dose form in ampoules or tablets or in multidose vials or other containers with an added perservative. The compositions may take such forms as suspensions, solutions, or emulsions or gels in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder and/or lyophilised form for direct administration or for constitution with a suitable vehicle (e.g. sterile, pyrogen-free water, normal saline or 5% dextrose) before use. The pharmaceutical compositions containing peptides(s) may also contain other active ingredients such as antimicrobial agents, or preservatives.

The compositions may contain from 0.001-99% (w/v or, preferably, w/w) of the active material.

The compositions are administered in therapeutically or prophylactic effective does, i.e. 0.05-1000 mg of peptide per day, in particular 5-500 mg per day. Very large doses may be used as the peptide according to the invention is non-toxic. However, normally this is not required. The dose administered daily of course depends on the degree of inflammation and inflammatory response.

For administration by injection or infusion of the compositions, the daily dosage, as employed for treatment of adults of approximately 70 kg of body weight, will often range from 5-500 mg of active material which may be administered in the form of 1 to 4 doses over each day, The invention may be useful in the prevention or treatment of illness or medical conditions, particularly those involving inflammation, such as: neurodegeneration, viral, bacterial or drug-induced hepatitis or meningitis; rheumatoid, psoriatic, reactive, or osteo-arthritisor other arthritides; sepsis/septic shock; dermal inflammation; ARDS (adult respiratory distress syndrome); graft rejection; inflammation secondary to the chemotherapy or radiotherapy of neoplastic disease,

The invention finds particular use in the prevention or treatment of MS, TSP/HAM and other inflammatory myelopathies (particularly those previously specifically mentioned) and/or symptoms or diseases in humans which are associated with acute or chronic immune activation. More particularly, the invention is useful in treating neurodegeneration, IRIS reactions, chronic fatigue syndromes, toxic shock syndrome associated with Staphylococcus aureus infection, arthritis, inflammatory bowel disease and host-versus-graft response in transplant patients. Such efficacious results in the use of the above compounds is thought to be due, without being limited to any particular theory, to the immunosuppressive activities of these compounds in chronic inflammatory states.

An unexpected and non-obvious aspect of the present invention is the use of all-D amino-acids in the creation of the bioactive peptides.

The result is surprising in view of previous work, Pert, PNAS, 83:9254, 1986, FIGS. 3 and 4, which showed that that D for L substitutions in linear peptide of General Formula 1 (see below), of which the specific example is ASTTTNYT (SEQ ID NO:1) can cause great loss of potency.

Having one D substitution, in the specific position No 1, (the D-ala) retains potency.

Making an additional D substitution, in the specific position No 8 (the D-Thr) results in loss of 99 to 99.9% of the activity. Thus it is shown that introduction of L to D substitutions can not be made in a general fashion, and that these modifications can, and typically do, destroy biopotency by disrupting the peptide structure required for receptor potency.

This point is further made in Brenneman, Drug Dev Res 15:361, 1988, with specific reference to the peptide TTNYT (SEQ ID NO:2). See FIG. 2 and Table 1. Upon making the L to D substitution in position 4 (Tyr), the peptide completely loses activity. This directly contradicts Andersen, U.S. Pat. No. 6,265,374 because each of the amino-acids cannot be in the D-form and retain biopotency. The objection is not overcome as Andersen provides no evidence that each amino-acid can be in a D-form.

A detailed study of the peptide TTNYT (SEQ ID NO:2) and L to D substitutions was published in Smith, Drug Dev Res, 1988. Refer to FIG. 3. Introduction of single L to D substitutions in each position 1, 2, 3, 4, results in loss of potency, and all of the D form substitutions are substantially less active (50×) to completely inactive.

As such the use of D-substitutions by Andersen in “each” position has not been reduced to practice. The data shows that in no instance does a D for L amino-acid substitution achieve comparable potency to the all-L form, rather D substitutions result in loss of activity, sometimes complete loss of biopotency in a position dependent fashion.

The notion that an all-D peptide would retain significant potency is furthermore novel in consideration of long the established understanding that such modifications are not possible as shown in Stewart and Woolley, Nature, 206:619, 1965 who prepared all-D peptides. For example, from their article, “In contrast to the change of a single residue, the inversion of all the amino-acid residues in a pentapeptide which has hormonal activity of MSH was found to cause loss of hormonal activity . . . .”

Further in this paper the authors stated that because there is as yet no general method for predicting the structural requirements required to make antimetabolites of peptides, we synthesized all-D bradykinin (note 9 amino acids, similar size to the 8 amino acid Formula 1 peptide of Andersen) in an effort to find out whether inversion of all the amino-acids of a peptide may be a generally applicable method for synthesis of peptide antagonists.”

The authors then concluded that “amounts of all-D-bradykinin up to 50,000 times the standard challenge of bradykinin showed neither any inhibition of the response to bradykinin, or any bradykinin-like effect. It would thus seem that inversion of all the amino-acid residues may not be a generally applicable method for formation of antimetabolites of biologically active peptides”.

In contrast to the repeated findings of numerous authors (op. cit. above) the author of the current study discovered, while seeking to construct a negative control, inactive version of linear octapeptide of General Formula 1, that a linear octapeptide of General Formula 1 comprised of all-D-amino-acid substitutions, as well as all-D-amino-acid substitutions of linear pentapeptide analogs of the linear octapeptide of General Formula 1, do retain comparable potency as the all-L or single-D-substituted peptides first described in Pert, 1985. Thus there was little loss of potency, a result unexpected in view of Smith, Drug Dev Res, 1988 and Brenneman, Drug Dev Res 15:361, 1988, with specific reference to the peptide TTNYT (SEQ ID NO:2).

An example is provided in FIG. 1. We synthesized three all-D-amino acid peptide analogs of DAPTA, such as “all-D-DAPTA” (RAP-107), all-D-Peptide T (RAP-106), and the shorter pentapeptide that contains the core bioactive moiety of Peptide T (Ruff, M. R., P. L. Hallberg, J. M. Hill, and C. B. Pert. 1987. Peptide T[4-8] is core HIV envelope sequence required for CD4 receptor attachment [letter]. Lancet. 2:751).

The results show that three “all-D” peptides, comprised of D, not L, amino acids, retained nearly full potency. As expected DAPTA was most potent and the others were some 2 to 35-fold reduced in potency. The potency reductions will not affect clinical usefulness as all of the compounds are active at sub-pM concentrations to antagonize chemokine receptors and have in vivo benefits in animal models of inflammation (Padi, 2012).

All-D Pentapeptide Analogs of Peptide T (ASTTTNYT, Pert, 1985) Inhibit CL2-Mediated Human Monocyte Chemotaxis.

FIG. 1 shows the effect of all-D amino acid derivatives (“RAPs”, generic names) of Peptide T (all-L-ASTTTNYT (SEQ ID NO:1))(Pert, 1986), the V2 derived antagonist of CCR5/CCR2 mediated HIV infection and inflammation, to block CCL2 (MCP-1) chemotaxis. Triplicate determinations were made and results are expressed as the mean plus or minus SEM. The experiment shown is a direct comparison among all RAPs. Statistical analysis was by unpaired t-test, with significance set at the p<0.01 (*) level for difference from CCL2 only chemotaxis.

Converting peptides of general formula Ito all-D-amino acids retain similar receptor targets, and similar receptor potencies to block innate immune responses. FIG. 1 illustrates the activity of all-D compared to mostly L-form peptides of formula I. It is shown that three related peptides of general formula I, that have identical primary sequence or that share partial sequence, differing only in enantiomeric form, block CCL2 chemotaxis. The results are unexpected in view of {Brenneman; Ruff; Smith, ruff; Stewart and Woolley, Nature, 206:619, 1965} which show that most L to D substitutions show reduced activity, and some cause complete loss of biopotency. The tyrosine moiety is particularly sensitive and the peptides are not active in L-form with a D-tyrosine. RAP-101 is the monomeric form of DAPTA and shows greater potency.

In order to show receptor targets we evaluated the ability of one of the new class of all-D peptides, (all-D-TTNYT (SEQ ID NO:2), aka RAP-103) to block chemokine chemotaxis caused by the CCR2 and CCR5 receptors. The results are presented in FIG. 2.

FIG. 2 shows all-D (TTNYT (SEQ ID NO:2)), generic name RAP-103, is an antagonist of CCR5 and CCR2 human monocyte chemotaxis. MCP-1 is CCL1, and MIP-113 is CCL4, Data are from [Padi, 2012, FIG. 1]. The result shows a further un-anticipated action of this family of peptides related to the HIV V2-region derived Peptide T related to its ability to block CCR2 chemotaxis. Previously an ability of Peptide T and DAPTA to block CCR5 was shown (Redwine, L. S., C. B. Pert, J. D. Rone, R. Nixon, M. Vance, B. Sandler, M. D. Lumpkin, D. J. Dieter, and M. R. Ruff. 1999. Peptide T blocks GP120/CCR5 chemokine receptor-mediated chemotaxis. Clin Immunol. 93:124-131). The peptides described herein therefore are at least dual-chemokine receptor antagonists as they block both CCR2 and CCR5. Dual-chemokine receptor antagonists may have added therapeutic value by blocking multiple inflammatory pathways.

FIG. 2 shows RAP-103 potently blocking both MCP-1- and MIP-1β-elicited chemotaxis of human monocytes. Monocytes were treated with the indicated doses of RAP-103 for 30 min before chemotaxis against human MCP-1 or MIP-1β (both 50 ng/mL) for 90 min. Data are the average of 2 separate experiments, conducted with triplicate determinations. Data (chemotactic index) are presented as mean±SEM. The IC50 for inhibition of MCP-1 or MIP-1β was generated by a nonlinear inhibition curve fit in GraphPad Prism software, version 5.0. The chemotactic index for MCP-1 without RAP-103 was 2.5-3.5 times over control, whereas for MIP-1β without RAP-103, it was approximately 2 times over control. Data are presented as mean±SEM. *P<0.05, **P<0.01 vs RAP-103 untreated.

In order to show the generalizability of the all-D-amino acid modifications we synthesized additional examples and tested them for antagonism of chemokines receptors implicated in the subject diseases, FIG. 3
Additional all-D-(Pepntapeptides) from V2 Region Block CCR5/CCR2 Human Monocyte Chemotaxis

In FIG. 3, the effects of RAPS in blocking CCL2 (MCP-1) chemotaxis was illustrated. Compounds were tested at a concentration of 10−14 M. All of the compounds were highly active to antagonize CCL2. Triplicate determinations were performed and results are expressed as the mean plus or minus SEM. The experiment shown is a direct comparison among all RAPs. Statistical analysis was by unpaired t-test, with significance set at the p<0.01 (*) level for difference from CCL2 only chemotaxis.

The results show that all-D-versions of additional HIV gp120 V2-region pentapeptides (SSTYR (SEQ ID NO:3), TTSYT (SEQ ID NO:4), NTSYR (SEQ ID NO:7)) retain potency and are antogonists of chemokine receptors.

We broadened the list of efficacious all-D-peptides to include five more unique examples (NYRYR (SEQ ID NO:5), IDNYT (SEQ ID NO:6), IDNYT (SEQ ID NO:8), NTSYG (SEQ ID NO:9), ETWYS (SEQ ID NO:10)) of HIV envelope protein derived peptides related to Peptide T that potently block CCR2/CCR5 chemotaxis.

Data on these five examples is shown in FIG. 4. These all-D-pentapeptides inhibit CCR2 (MCP-1) elicited chemotaxis of human monocytes. Purified human monocytes were treated with 20 pM of All-D-pentapeptides for 30 minutes prior to chemotaxis against human MCP-1 (0.6 nM) for 2 hours. The chemotactic index (ratio of migration for CCR2/buffer) for MCP-1 was 3-4. A representative experiment is shown comprising triplicate determinations and is presented as relative fluorescence units, Mean±SEM. The activity of All-D-pentapeptide TTNYT (SEQ ID NO:2) (RAP-103) to block MCP-1 human monocyte chemotaxis has been published, {Padi, 2012}.

The usefulness of the subject compounds in MS is further suggested by additional actions of the all-D peptides to inhibit B-Integrin-mediated adhesion to human fibronectin. The results are of interest as approved treatments for MS include the immunosupressive monoclonal antibody therapy “Tysabri”, which blocks T cell binding to cellular integrins, and thereby infiltration of inflammatory cells into brains of MS patients. The results suggest further anti-inflammatory mechanisms to block infiltration of T cells or monocytes into brain, useful in treating MS patients.

All-D-(IDNYT) Inhibition of Human Monocyte and THP-1 Cell β-Integrin-Mediated Adhesion to Hu-Fibronectin

FIG. 5 illustrates all-D-(IDNYT) potently blocking MCP-1 elicited adhesion of human monocytes. Human monocytes were treated with the indicated doses of all-D-(IDNYT) for 10 minutes prior to adherence to the β-integrin human fibronectin. Data is the average of two separate experiments, conducted with triplicate determinations. Data are presented as Mean±SEM of the normalized adherence response from two experiments. The IC50 for inhibition of MCP-1 stimulated adherence was generated using a nonlinear inhibition curve fit in GraphPad Prism Version 5.0.

D-Ala1-Peptide T-Amide (DAPTA/RAP-101) and all-D-TTNYT (RAP-103) have Anti-Inflammatory Effects by Lowering Neurotoxic Cytokines in People and Animals.

A further action of the subject peptides relevant to degenerative diseases of inflammation is the ability to decrease the inflammatory cytokines, chemokines, and receptors which underly disease processes in MS, TSP/HAM and the other inflammatory conditions. Here we show that Dala1-peptide T-amide (DAPTA, RAP-101), which has only 1 of 8 amino acids in the D-configuration, lowers inflammatory cytokine levels in humans. The effect is shared by the pentapeptide all-D-TTNYT (SEQ ID NO:2) (RAP-103), which was administered by oral gavage, (0.05-1 mg/kg) for 7 days to sciatic nerve injured rats. The specific experimental details are provided in Padi, 2012. Both Dala1-peptide T-amide and all-D-TTNYT (SEQ ID NO:2) share receptor targets, and biological effects indicating they are analogs that target the same pathological processes. All of the members of the class of HIV gp120, V2 region derived peptides that we describe are therefore expected to share the same actions, benefits, and therapeutic mechanisms, as is expected from structurally related analogs.

TABLE 1 Summary of Inflammatory Biomarker Changes for DAPTA/RAP-101 and all-D-TTNYT (RAP-103). Biomarker Species Change DRUG Reference IL-1 Hu decrease RAP-101 Ruff, 2003 IL-6 Hu decrease RAP-101 Ruff, 2003 IL-8 Hu decrease RAP-101 Ruff, 2003 TNFα Hu decrease RAP-101 Ruff, 2003 MCP-1 Rat decrease RAP-103 unpublished MIP-1α Rat decrease RAP-103 unpublished TNFα Rat decrease RAP-103 unpublished CCL2 Rat decrease RAP-103 unpublished CCL3 Rat decrease RAP-103 unpublished CCR2 Rat decrease RAP-103 unpublished CCR5 Rat decrease RAP-103 unpublished IL-1β Rat decrease RAP-103 Padi, 2012 IL-6 Rat decrease RAP-103 Padi, 2012

Septic shock is an illustration of a disease involving inflammation. Many of the clinical features of Gram-negative septic shock may be reproduced in animals by the administration of lipopolysaccharide (LPS). The administration of LPS to animals can prompt severe metabolic and physiological changes that can lead to death. Associated with the injection of LPS is the extensive production of tumour necrosis factor alpha (TNFα). TNFα and IL-1β, with their common functional activities such as pyrogenicity, somnogenicity and being mediators of inflammation, have been implicated in the pathology of other diseases associated with chronic inflammation, apart from toxic shock and cancer-related cachexia. TNFα has been detected in synovial fluid in patients with both rheumatoid and reactive arthritis and in the serum of patients with rheumatoid arthritis (Saxne et al, 1988, Arthrit. Rheumat. 31, 1041). Raised levels of TNF have been detected in renal transplant patients during acute rejection episodes (Maury and Teppo 1987, J. Exp. Med. 166, 1132). In animals, TNFα has been shown to be involved in the pathogenesis of graft-versus-host disease in skin and gut following allogenic marrow transplantation.

In view of the effects of the subject peptides, including the all-D-peptide analogs of Peptide T such as all-D-TTNYT (SEQ ID NO:2) (RAP-101) to lower TNF levels in humans and animals, we explored the effect of all-D-TTNYT (SEQ ID NO:2) (RAP-101) to block TNFα production from cultured human dendritic cells (iDCs). Such an effect would have benefits in septic shock or other conditions with elevated TNFα levels.

RAP-103 (All-D-TTNYT) Blocks TLR4

FIG. 6 shows that LPS induces TNFα secretion in monocyte-derived iDC. Cells were incubated for 5 h in medium alone or LPS only at a concentration of 100 ng/mL (black columns), or medium containing LPS plus RAP103 (All-D-TTNYT (SEQ ID NO:2)) (light grey columns). Supernatants were analyzed for secreted TNF-α after 5 hours. The bars represent absorbance in an ELISA assay and correspond to TNFα levels.

We tested a high (100 ng/ml) concentration of LPS, nevertheless, all-D-TTNYT (SEQ ID NO:2)/RAP103 was able to effectively blunt this response at low (pM) concentrations, results consistent with other data showing that the parent compound Dala1-peptide T-amide/RAP-101 also blocks LPS activation of microglia and NfKb activation {Rosi et al., 2005, #40595} and TNF-α secretion {Phipps and MacFadden, 1996, #14397}.

The IC50 for all-D-TTNYT (SEQ ID NO:2)/RAP103 inhibition of TNFα production was approximately 10−13 M. The effect of RAP103 to block TLR4 may be allosteric or act via an accessory protein, and may be upstream of chemokine receptor activation since TLR4 signaling typically releases chemokines and cytokines Treatment uses in lowering TNFα levels are suggested by these in vitro and in vivo (Table 1) data.

Oral Bioavailability

The ability to substitute a D for an L amino acid and retain biopotency creates the possibility to make peptides orally deliverable drug compounds. Stability of peptides in biological fluids, such as plasma, or digestive enzymes has limited their utility as drugs. The ability to create all-D peptides that retain potency is an unexpected general method of creating peptides of General Formula 1, and likely many others, which may be stabilized to proteolysis, while retaining biopotency, so a therapeutic may be administered to people via oral dosing or otherwise enjoy enhanced bioavailability in the body.

A pharmacokinetic study of all-D-TTNYT (SEQ ID NO:2) (RAP-103) following intravenous and oral administration was conducted at a target dose level of 1 mg/kg in the male Sprague Dawley rat. The concentration of RAP-103 in each plasma and brain sample was measured using a suitable LC-MS/MS assay. The assay used was a research grade assay (RGA-1) which was established by assessing the accuracy, precision and the linearity of the method. Plasma concentrations generated were used to evaluate the pharmacokinetic parameters of all-D-TTNYT (SEQ ID NO:2) (RAP-103).

The dose formulation was administered intravenously to some animals as a slow bolus over ca 30 s via the tail vein and to a different group of animals orally via gastric gavage at a target dose volume of 1 mL/kg, to achieve a target dose level of 1 mg/kg. The dose volume administered was calculated according to the bodyweight of the animal on the day of dosing. The weight of administered dose was recorded. All dose administrations were well tolerated and no adverse effects from the treatments were observed. The results of the study are in Table 1. Results expressed as ng/mL

TABLE 1 CONCENTRATION OF ALL-D-TTNYT (RAP-103) IN MALE RAT PLASMA FOLLOWING INTRAVENOUS ADMINISTRATION AT A TARGET DOSE LEVEL OF 1 MG/KG. Animal Number Nominal 007 008 009 010 011 012 013 014 015 016 017 Time (h) M M M M M M M M M M M 0.083 3050 2490 — — — — — — — — — 0.167 — — 2400 2500 — — — — — — — 0.25 — — — — 2050 2270 1860 — — — — 0.5 — — — — — — — 558 561 — — 1 — — — — — — — — — 405 433

The results show that as quickly as can be determined, 5 minutes (0.083 hrs) after dosing, all-D-TTNYT (SEQ ID NO:2) (RAP-103) is detected in plasma, and continues to be detected at 1 hr. Peak levels occur at 10-15 minutes.

The biological relevance is shown in the publication by Padi et al., [Padi, S. S., X. Q. Shi, Y. Q. Zhao, M. R. Ruff, N. Baichoo, C. B. Pert, and J. Zhang. 2012. Attenuation of rodent neuropathic pain by an orally active peptide, RAP-103, which potently blocks CCR2- and CCR5-mediated monocyte chemotaxis and inflammation. Pain. 153:95-106. doi:10.1016/j.pain.2011.09.022] that shows oral administration of RAP-103 (0.05-1 mg/kg) for 7 days fully prevents mechanical allodynia and inhibits the development of thermal hyperalgesia after partial ligation of the sciatic nerve in rats. Administered from days 8 to 12, RAP-103 (0.2-1 mg/kg) reverses already established hypersensitivity. RAP-103 relieves behavioral hypersensitivity through either or both CCR2 and CCR5 blockade. Moreover, RAP-103 is able to reduce spinal microglial activation and monocyte infiltration, and to inhibit inflammatory responses evoked by peripheral nerve injury that cause chronic pain. The findings suggest that targeting CCR2/CCR5 should provide greater efficacy than targeting CCR2 or CCR5 alone, and that the dual CCR2/CCR5 antagonist RAP-103 has the potential for broad clinical use in neuropathic pain treatment.

In this way the peptides can be used in pharmaceutical compositions and compositions of matter for treating and preventing any disease or condition caused by an organism, compound or immune dysfunction that results in an inflammatory reaction of the immune system. The peptides or peptide formulations may be used alone or in combination with any other pharmaceutically active compound, such as an anti-infective agent, for example an antibiotic and/or antiviral agent and/or antifungal agent, or another pharmaceutically active compound, such as an antineoplastic agent.

The peptides may be administered orally, bucally, parenterally, topically, rectally, vaginally, by intranasal inhalation spray, by intrapulmonary inhalation or in other ways. In particular, the peptides according to the invention may be formulated for topical use, for inhalation with spray or powder, for injection (for example subcutaneous, intramuscular, intravenous, intra-articular or intra-cisternal injection), for infusion or for oral administration and may be presented in unit dose form in ampoules or tablets or in multidose vials or other containers with an added perservative. The compositions may take such forms as suspensions, solutions, or emulsions or gels in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder and/or lyophilised form for direct administration or for constitution with a suitable vehicle (e.g. sterile, pyrogen-free water, normal saline or dextrose or mannose) before use. The pharmaceutical compositions co taining peptides(s) may also contain other active ingredients such as antimicrobial agents, or preservatives.

The compositions may contain from 0.001-99% (w/v or, preferably, w/w) of the active material. The invention will now be illustrated by the following non-limiting clinical examples.

EXAMPLE 1 HTLV-1 Myelopathy

A 59 year old female with a nine year history of spastic paraparesis was diagnosed as HTLV-1 seropositive. Her neurological examination confirmed increased deep tendon reflexes, spasticity of the lower limbs and pyramidal weakness of the legs. Therapy, consisting of 6.0 mg of Peptide T daily, was self-administered via 4 metered intranasal sprays, delivered three times daily. The patient reported definite improvements after 2 weeks which continued to the end of week 10; she took only 15 secs to walk 10 meters at this time, compared to 65 seconds initially. Four weeks after treatment was discontinued stiffness in her legs returned.

EXAMPLE 2 Multiple Sclerosis

To evaluate the effect of Dala1-peptide T-amide (DAPTA) on overall health and symptom burden, five MS patients were treated and followed using three self-report quality of life instruments. Three patients who stopped taking DAPTA and then restarted drug some weeks later after establishing a new baseline, appear twice in the analysis. In this manner 8 baseline and repeated measures (wks 0, 4, 8, 12) were defined and analyzed. The MOS-HIV was originally developed by Wu, [Wu, Qual Life Res. 1997 August; 6(6):481-93. Evidence for reliability, validity and usefulness of the Medical Outcomes Study HIV Health Survey (MOS-HIV)], and the Psychological general Well-Being (PGWB) Index developed by Dupuy. [Dupuy H J. The Psychological general Well-Being (PGWB) Index. In: Assessment of Quality of Life in clinical trials of cardiovascular therapies. Edited by Wenger N K, Mattson M E, Furberg C D, Elinson J. Le Jacq Publishing 1984; Chap 9:170-183].

The MS modified MOS-HIV showed a trend or significant improvement in physical discomfort, mental health and health distress dimensions. Improvement was statistically significant at all measurement points for energy, general quality of life, well being and un-weighted MOS summary score (p=0.015) at three months. In PGWB vitality and Summary were significantly improved at all measurement points (PGWB Summary Score, p=0.009 at week 12). The MS symptom checklist was particularly significant for fatigue associated with MS (p=0.001), and significant for Slow Thinking (p=0.042). Results appear in Table 2.

TABLE 2 DAPTA IN MULTIPLE SCLEROSIS (MS) PATIENTS MS trial; Two Tailed Paired T tests; MS patients Baseline Week 4 Week 8 Week 12 Mean SD Mean SD p value Mean SD p value Mean SD p value MOS-HIV (0-100) n = 8 n = 8 n = 6 n = 5 Discomfort 30 15.1 55 25.6 0.019 80 29.2 0.092 52 30.3 0.109 Mental Heath 58.5 21.4 72.5 9.7 0.102 81.3 10 0.125 76.3 11.8 0.07 Energy 35.6 19.4 63.8 12.2 0.009 69.2 13.6 0.006 67 14.4 0.031 Health Distress 69.4 28.5 89.4 9 0.078 95.8 4.9 0.1 89 14.7 0.096 Quality of life 53.12 16 81.75 11.6 0.007 83.3 129 0.012 75 17.7 0.015 Well Being 49.3 17.25 72.4 9.3 0.000 77.9 9.3 0.023 72 14.3 0.013 MOS summary 47.7 19.8 61.9 145 0.003 69.7 14.4 0.003 71.5 15.8 0.015 PGWB n = 7 n = 7 n = 5 n = 5 Vitality (0-20) 9.1 3.3 15 2.6 0.008 14.8 1.5 0.03 15 2.1 0.011 PGWB summary (0-100) 63.5 13.2 78.8 7.5 0.007 85.8 10.7 0.003 81 13.3 0.009 MS symptom Checklist n = 7 n = 7 n = 6 n = 6 Fatigue (0-4) 1.375 0.916 2.86 0.8 0.025 3.17 0.75 0.001 3.33 0.52 0.007 Malaise(0-4) 2.29 1.1 3.33 0.052 0.042 3.83 0.41 0.025 3.4 0.53 0.14 Forgetfulness(0-4) 3 1.1 3.6 0.053 0.048 3.7 0.52 0.101 3.83 0.41 0.101 Slow Thinking(0-4) 2.68 0.99 3.43 0.79 0.103 3.67 0.52 0.042 3.83 0.41 0.042 Higher numbers indicate improvement.

As stated previously, immune reconstitution inflammatory syndrome (IRIS), also called immune restoration disease, occurs in MS patients upon cessation of immunosuppressive therapy. Return of immune competence may cause worsening of symptoms and neurological disease, which can be severe, even fatal. Even in those who survive disabilities typically persist and recovery is partial. An unmet medical need is to prevent or treat IRIS reactions. The set of peptides that are effective in treating IRIS will be essentially the same peptides that are effective in treating MS.

According to a first aspect of the present invention, there is provided the use of a linear peptide of General Formula 1 wherein all amino acids are in the D-stereoisomeric configuration:

A-B-C-D-E-F-G-H. General Formula 1) in which:
A is Ala, or absent,
B is Ser, Thr or absent,
C is Ser, Thr or absent,

D is Ser, Thr, Asn, Glu, Arg, Ile, Leu, E is Ser, Thr, Asp, Asn, F is Thr, Ser, Asn, Arg, Gln, Lys, Trp, G is Tyr, H is Thr, Ser, Arg, Gly.

All of the amino acids referred to in General Formula 1 will be in the D-stereoisomeric configuration and candidates for H may be esterified or amidated. The peptide comprises at least 5 amino acids. Alternatively, the total length of the peptide can be at least about twenty (20) amino acids, or alternatively at least about twelve (12) amino acids. Still another alternative is at least about eight (8) amino acids. The all-L amino acid version of a linear peptide of General Formula 1 has been called Peptide T. Peptides useful in the invention may be administered as a composition in conjunction with a pharmaceutically acceptable carrier.

Converting peptides of general formula 1 (ASTTTNYT (SEQ ID NO:1)) to all-D-amino acids retain similar receptor targets, and similar receptor potencies to block innate immune responses. FIG. 1 illustrates the activity of all-D compared to mostly L-form peptides of formula I. It is shown that three related peptides of general formula I, that have identical primary sequence or that share partial sequence, differing only in enantiomeric form, block CCL2 chemotaxis. The results are unexpected in view of {Pert, 1986; Brenneman et al., 1988, #9180; Ruff; Smith et al., 1988, #34265; Stewart and Woolley, 1965, #22293} which show that most L to D substitutions show reduced activity, and some cause complete loss of biopotency. The tyrosine moiety is particularly sensitive and the peptides are not active in L-form with a D-tyrosine.

In a preferred embodiment, the list of efficacious all-D-peptides includes eight more unique examples (Ser-Ser-Thr-Tyr-Arg (SEQ ID NO:3), Thr-Thr-Ser-Tyr-Thr (SEQ ID NO:4), Asn-Thr-Arg-Tyr-Arg (SEQ ID NO:5), Ile-Asp-Asn-Tyr Thr (SEQ ID NO:6), Asn-Thr-Ser-Tyr-Arg (SEQ ID NO:7), Ile-Asn-Asn-Tyr-Thr (SEQ ID NO:8), Asn-Thr-Ser-Tyr-Gly (SEQ ID NO:9), Glu-Thr-Trp-Tyr-Ser (SEQ ID NO:10)) of HIV envelope gp160 and more commonly gp120-V2 region, near the bridging sheet, at approximately amino-acid 185 in the V2-loop of the envelope protein, depending on HIV env isolate. The synthetic derived all-D-pentapeptides with a tyrosine in the fourth position, and Ser, Thr, or Asp in the second position, that potently block CCR2/CCR5 chemotaxis are broad spectrum receptor antagonists useful as potential orally active peptide therapeutics. The difficulty of development of orally active peptides has greatly impeded their development as therapies. Here we suggest a general method to stabilize peptides to proteolytic degradation that preserves biopotency, either as an agonist or as an antagonist of cell surface receptors.

Claims

1. A method of treatment of neurodegenerative illness in a patient comprising the steps of:

preparing a composition comprising a D peptide and a pharmaceutically acceptable carrier, said D peptide further comprises the general structure: A-B-C-D-E-F-G-H in which: A is Ala, or absent, B is Ser, Thr or absent, C is Ser, Thr or absent, D is Ser, Thr, Asn, Glu, Arg, Ile, Leu, E is Ser, Thr, Asp, Asn, F is Thr, Ser, Asn, Arg, Gln, Lys, Trp, G is Tyr, and H is Thr, Ser, Arg, Gly, and wherein all amino acids are the D stereoisomeric configuration, and
administering said composition to the patient in a therapeutically effective dose,
wherein said composition acts to treat the loss of brain function in the patient.

2. The method as defined in claim 1 wherein the neurodegenerative illness results in a condition characterized by loss of brain function.

3. The method as defined in claim 2 wherein said condition is selected from the group consisting of: HTLV-1-associated myelopathy (HAM), multiple sclerosis (MS) and amyotrophic lateral sclerosis.

4. The method as defined in claim 1 wherein said administering said composition to the patient is selected from the group consisting of administrating: orally, bucally, parenterally, topically, rectally, vaginally, by intranasal inhalation spray, by intrapulmonary inhalation.

5. The method as defined in claim 1 further comprising, SEQ ID NO: 3 Ser-Ser-Thr-Tyr-Arg, SEQ-ID NO: 4 Thr-Thr-Ser-Tyr-Thr, SEQ ID NO: 5 Asn-Thr-Arg-Tyr-Arg, SEQ ID NO: 6 Ile-Asp-Asn-Tyr-Thr, SEQ ID NO: 7 Asn-Thr-Ser-Tyr-Arg, SEQ ID NO: 8 Ile-Asn-Asn-Tyr-Thr, SEQ ID NO: 9 Asn-Thr-Ser-Tyr-Gly, and SEQ ID NO: 10 Glu-Thr-Trp-Tyr-Ser.

said D peptide is at most twenty (20) D amino acid residues in length and contains five contiguous D amino acid residues that have a sequence selected from the group consisting of:

6. The method as defined in claim 5 further comprising,

said D peptide derivative is at most twelve (12) D amino acid residues in length.

7. The method as defined in claim 5 further comprising,

said D peptide derivative is at most eight (8) D amino acid residues in length.

8. The method as defined in claim 5 further comprising,

said D peptide is five (5) D amino acid residues in length.

9. A method of treatment of neurodegenerative illness in a patient comprising the steps of:

treating loss of brain function in a patient comprising the steps of:
preparing a composition comprising a peptide analog and a pharmaceutically acceptable carrier, said peptide analog is [D-Ala1]-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-NH2 SEQ ID NO:1 in which the first amino acid is a D stereoisomer and the remaining amino acids are L stereoisomers and the last amino acid has an amide cap, and
administering said peptide analog to the patient in a therapeutically effective dose,
wherein said composition acts to treat the brain neurodegenerative condition in the patient.

10. The method as defined in claim 9 wherein the brain neurodegenerative results in a condition characterized by loss of brain function.

11. The method as defined in claim 10 wherein said condition is selected from the group consisting of: HTLV-1-associated myelopathy (HAM), multiple sclerosis (MS) and amyotrophic lateral sclerosis.

Patent History
Publication number: 20140322250
Type: Application
Filed: Apr 28, 2014
Publication Date: Oct 30, 2014
Inventor: Michael Ruff (Potomac, MD)
Application Number: 14/263,493
Classifications
Current U.S. Class: Amino Acid Sequence Disclosed In Whole Or In Part; Or Conjugate, Complex, Or Fusion Protein Or Fusion Polypeptide Including The Same (424/185.1)
International Classification: C07K 7/08 (20060101); C07K 7/06 (20060101);