WO1993006128A1

WO1993006128A1 - Tnf antagonist peptides

Info

Publication number: WO1993006128A1
Application number: PCT/AU1992/000487
Authority: WO
Inventors: Deborah Ann Rathjen; Fred Widmer
Original assignee: Peptide Technology Limited
Priority date: 1991-09-16
Filing date: 1992-09-16
Publication date: 1993-04-01

Abstract

The present invention provides TNF antagonist peptides which have the ability to reduce TNF toxicity. The present invention further relates to compositions including these peptides as the active ingredient and to methods of treatment involving the administration of this composition.

Description

TNF ANTAGONIST PEPTIDES

Field of the Invention

The present invention relates to TNF antagonist peptides which have the ability to reduce TNF toxicity as manifest by decreased blood glucose levels and weight loss (cachexia). The present invention further relates to compositions including these peptides as the active ingredient and methods of treatment involving the

administration of this composition.

Background of the invention

Many of the clinical features of Gram-negative septicemic shock may be reproduced in animals by the administration of bacterial lipopolysaccharide (LPS). The administration of LPS to animals can prompt severe

metabolic and physiological changes which can lead to death. Associated with the injection of LPS is the extensive production of tumour necrosis factor alpha

(TNF). Mice injected with recombinant human TNF develop piloerection of the hair (ruffling), diarrhea, a

withdrawn, unkempt appearance and die if sufficient amounts are given. Rats treated with TNF become

hypotensive, tachypneic and die of sudden respiratory arrest (Tracey et al., 1986 Science 234, 470). Severe acidosis, marked hemoconcentration and biphasic changes in blood glucose concentration were also observed.

Histopathology revealed severe leukostatsis in the lungs, haemorraghic necrosis in the adrenals, pancreas and other organs and tubular necrosis of the kidneys. All these changes were prevented if the animals were pretreated with a neutralizing monoclonal antibody against TNF.

The massive accumulation of neutrophils in the lungs of TNF-treated animals reflects the activation of

neutrophils by TNF. TNF causes neutrophil degranulation, respiratory burst, enhanced antimicrobiocidal and

anti-tumour activity (Klebanoff et al., 1986 J. Immunol. 136, 4220; Tsujimoto et al., 1986 Biochem Biophys Res Commun 137, 1094). Endothelial cells are also an

important target for the expression of TNF toxicity. TNF diminishes the anticoagulant potential of the endothelium, inducing procoagulant activity and down regulating the expression of thrombomodulin (Stern and Nawroth, 1986 J Exp Med 163, 740).

TNF, a product of activated macrophages produced in response to infection and malignancy, was first identified as a serum factor in LPS treated mice which caused the haemorraghic necrosis of transplantable tumours in murine models and was cytoxoic for tumour cells in culture

(Carswell et al., 1975 PNAS 72, 3666; Helson et al., 1975 Nature 258, 731). Cachexia is a common symptom of

advanced malignancy and severe infection. It is

characterised by abnormal lipid metabolism with

hypertriglyceridemia, abnormal protein and glucose

metabolism and body wasting. Chronic administration of TNF (also known as cachectin in the early literature) to mice causes anorexia, weight loss and depletion of body lipid and protein within 7 to 10 days (Cerami et al., 1985 Immunol Lett 11, 173, Fong et al., 1989 J Exp Med 170, 1627). These effects were reduced by concurrent

administration of antibodies against TNF, TNF has been measured in the serum of patients with cancer and chronic disease associated with cachexia. The results are

inconclusive since large differences in TNF levels have been reported. These may be due to the short half-life of TNF (6 minutes), differences in TNF serum binding protein or true differences in TNF levels in chronic disease states.

TNFα, as a mediator of inflammation, has been

implicated in the pathology of other diseases apart from toxic shock and cancer related cachexia. TNF has been measured 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 TNF has been shown to be involved in the pathogenesis of graft versus host disease in skin and gut following allogeneic marrow transplantation.

Administration of a rabbit anti-murine TNF was

demonstrated to prevent the histological changes

associated with graft versus host disease and reduced mortality (Piquet et al., 1987 J Exp Med 166, 1280).

TNF has also been shown to contribute significantly to the pathology of malaria (Clark et al., 1987; Am. J. Pathol. 129: 192-199). Further, elevated serum levels of TNF have been reported in malaria patients (Scuderi et al., 1986; Lancet 2: 1364-1365). TNF may also

contribute to the brain pathology and consequent dementia observed in late stage HIV infections (Grimaldi et al Ann Nevrol 29 : 21)

The biological response to TNF is mediated by

specific cell surface receptors. At least two cell surface molecules of molecular weight 55 and 75 kd

specifically bind TNFα and TNF/3 with high affinity

(Hohmann et al, 1989 J. biol. Chem. 264 14927). These receptors have now been cloned (Loetscher et al, 1990 Cell 61, 351; Smith et al, 1990 Science 248 1019). Both TNF type I and II receptors are shed into serum following proteolytic cleavage to form serum binding proteins which reversibly inactivate TNF and which may regulate the activity of TNF in vivo (Kohno et al., 1990 PNAS 87 8331; Seckinger et al, 1988 J Biol Chem 264 11966). Infusion of TNF has been shown to cause an increase in circulating TNF-binding protein in humans (Lantz et al, 1990 Cytokine 2 1). The present inventors have produced peptides which are able to reduce TNF toxicity as manifest by reduced mortality decreased blood glucose levels and weight loss (cachexia) in tumour-bearing mice treated with human recombinant TNF.

Summary of the Invention

In a first aspect the present invention consists in a linear or cyclic peptide of the general formula:- x₁-x₂-x₃-x₄-x₅-x₆-x₇-x₈-x₉-x₁₀- in which x₁ is null, Cys or R₁;

x₂ is null, Cys, R₁ or

A₁-A₂-A₃-A₄-A₅-A₆-A₇

in which A₁ is Ser or Thr or Ala,

A₂ is Lys or Arg or His,

A₃ is Cys or Arg or His,

A₄ is His or Lys or Arg or Phe or Tyr or Trp,

A₅ is Lys or Arg or His,

A₆ is Gly or Ala,

A₇ is Thr or Ser or Ala,

X₃ is Null, Cys, R₁ or A₈-A₉

in which A₈ is absent or Gly or Ala or Tyr or Phe or

Trp or His

A₉ is Leu or lle or Val or Met

X₄ is Cys, R₁ or

Tyr or Phe or Trp or His or Gly or Ala,

A₁₁ is Asn or Gln,

A₁₂ is Asp or Glu,

A₁₃ is Cys or Arg or His,

A₁₄ is Pro or Not - alkyl amino acid

X₅ is Gly or Ala,

X₆ is Cys , R₂ or A₁₅-A₁₆

in which A₁₅ is Pro or Nα - alkyl amino acid, A _{1 6} is

Gly or Ala,

X₇ is null, Cys , R₂ or A₁₇-A₁₈-A_{1 9} in which A_{1 7} is Gln or Asn,

A₁₈ is Asp or Glu,

A₁₉ is Thr or Ser or Ala,

X₈ is null , Cys , R₂ , Asp, Glu, Gly or Ala,

X₉ is null , Cys , R₂ or

A₂₀-A₂₁-A₂₂-A₂₃-A₂₄-A₂₅-A₂₆-A₂₇

-A ₂₈-A₂₉

in which A₂₀ is Cys or Arg or His,

A₂₁ is Arg or Lys or His,

A₂₂ is Glu or Asp,

A₂₃ is Cys or Arg or His,

A₂₄ is Glu or Asp,

A₂₅ is Ser or Thr or Ala,

A₂₆ is Gly or Ala,

A₂₇ is Ser or Thr or Ala,

A₂₈ is Phe or Tyr or Trp or His,

A₂₉ is Thr or Ser or Ala,

X ₁₀ is null, Cys or R₂

R₁ is R-CO, where R is H, straight, branched or cyclic alkyl up to C20, optionally containing double bonds and/or substituted with halogen, nitro, amino, hydroxy, sulfo, phospho or carboxyl groups (which may be substituted themselves), or aralkyl or aryl optionally substituted as listed for the alkyl and further including alkyl, or R₁ is glycosyl, nucleosyl, lipoyl or R₁ is an L- or D-α amino acid or an oligomer thereof consisting of up to 5 residues R₁ is absent when the amino acid adjacent is a desamino-derivative.

R₂ is -NR₁₂R₁₃, wherein R₁₂ and R₁₃ are

independently H, straight, branched or cyclic alkyl, aralkyl or aryl optionally substituted as defined for R₁ or N-glycosyl or N-lipoyl -OR₁₄, where R₁₄ is H,

straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R₁-O-glycosyl, -O-lipoyl or - an L- or D-α-amino acid or an oligomer thereof consisting of up to 5 residues or R₂ is absent, when the adjacent amino acid is a decarboxy derivative of cysteine or a homologue thereof or the peptide is in a N-C cyclic form.

with the proviso that:

X₁ is always and only null when X₂ is R₁, Cys or null

X₂ is always and only null when X₃ is R₁, Cys or null

X₃ is always and only null when X₄ is R₁ or Cys

X₇ is always and only null when X₆ is R₂ or Cys

X₈ is always and only null when X₇ is null, R₂ or Cys

X₉ is always and only null when X₈ is null, R₂ or Cys

X₁₀ is always and only null when X₉ is null, R₂ or

Cys

when X₄ is R₁ or Cys then X₆ is A₁₅-A₁₆, X₇ is

A₁₇-A₁₈-A₁₉ and X₈ is Asp, Glu, Gly or Ala,

when X₆ is R₂ or Cys then X₄ is

A₁₀-A₁₁-A₁₂-A₁₃-A₁₄

when X₇ is null, R₂ or Cys then X. is

A₁₀-A₁₁-A₁₂-A₁₃-A₁₄.

In a preferred embodiment of the first aspect of the present invention the peptide is selected from the group consisting of:-

H-Leu-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gly-Gln-Asp-Thr-

Asp-OH,

Leu-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gln-Asp-Thr-Asp-

Cys(Acm)-Arg-Glu-Cys(Acm)-Glu-Ser-Gly-Ser-Phe-Thr,

Thr-Lys-Cys(Acm)-His-Lys-Gly-Thr-Tyr-Leu-Tyr-Asn-Asp-

Cys(Acm)-Pro-Gly-Pro-Gly-Gln-Asp-Thr-Asp,

Ac-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly,

Gly-Leu-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly,

Gly-Leu-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gly,

Gly-Pro-Gly-Gln-Asp-Thr-Asp,

Ac-Gly-Pro-Gly-Gln-Asp-Thr-Asp-NH₂,

Gly-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gly-Gln-Asp-Thr-Gly, and Ac-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gly-Gln-Asp-Thr-NH₂ In a particularly preferred embodiment of the present invention the peptide is H-Leu-Tyr-Asn-Asp-Cys-Pro-Gly- Pro-Gly-Gln-Asp-Thr-Asp-OH.

In a second aspect the present invention consists in a linear or cyclic peptide of the general formula:-

Y₁-Y₂-Y₃-Y₄-Y₅-Y₆-Y₇-Y₈-Y₉-Y₁₀- Y₁₁-Y₁₂-Y₁₃-Y₁₄-Y₁₅-Y₁₆-Y₁₇

in which Y₁ is null, Cys or R₁

Y₂ is Gly or Ala

Y₃ is Ala or Gla

Y₄ is Gln or Asn

Y₅ is Met or Gal or lle or Leu

Y₆ is Cys or Arg or His

Y₇ is Cys or Arg or His

Y₈ is Ser or Thr or Ala

Y₉ is Lys or Arg or His

Y₁₀ is Cys or Arg or His

Y₁₁ is Ser or Thr or Ala

Y₁₂ is Pro or nα - alkyl amino acid

Y₁₃ is Gly or Ala

Y₁₄ is Gln or Asn

Y₁₅ is His or Lys or Arg

Y₁₆ is Gly or Ala

Y₁₇ is null, Cys or A₂

R₂ is -NR₁₂R₁₃, wherein R₁₂ and R₁₃ are independently H, straight, branched or cyclic alkyl, aralkyl or aryl optionally substituted as defined for R₁ or N-glycosyl or N-lipoyl -OR₁₄, where R₁₄ is H,

straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R.-O-glycosyl, -O-lipoyl or - an L- or D-α-amino acid or an oligomer thereof consisting of up to 5 residues or R₂ is absent, when the adjacent amino acid is a decarboxy derivative of cysteine or a homologue thereof or the peptide is in a N-C cyclic form.

In a preferred embodiment of the second apect of the present invention the peptide is Gly-Ala-Gln-Met-Cys(Acm)- Cys(Acm)-Ser-Lys-Cys(Acm)-Ser-Pro-mly-Gln-His-Gly.

The amino acids may be D or L isomers, however, generally the peptide will primarily consist of L-amino acids. In addition, the cysteine residues may also include an Acn group protecting the cysteine residues.

In a third aspect the present invention consists in a pharmaceutical composition for use in treating subjects suffering from TNF toxicity, the composition comprising a peptide of the first or second aspect of the present invention in combination with a pharmaceutically

acceptable sterile carrier.

In a fourth aspect the present invention consists in a method of treating a subject suffering from the toxic effects of TNF, the method comprising administering to the subject the composition of the third aspect of the present invention.

In a preferred embodiment of the present invention the peptide is Peptide 371 as hereinafter defined.

The peptide of the present invention may be used in therapy to prevent TNF pathology associated with decreased blood glucose levels and weight loss and may be a useful therapy in the treatment of septic shock.

Further the composition and method of the present invention would be expected to be useful as an

anti-inflammatory agent in a wide range of disease states including toxic shock, adult respiratory distress

syndrome, hypersensitivity pneumonitis, systemic lupus erythromatosis, cystic fibrosis, asthma, bronchitis, drug withdrawal, schistosomiasis, sepsis, rheumatoid arthritis, acquired immuno-deficiency syndrome, multiple sclerosis, leperosy, malaria, systemic vasculitis, bacterial

meningitis, cachexia, dermatitis, psoriasis, diabetes, neuropathy associated with infection or autoimmune

disease, ischemia/reperfusion injury, encephalitis,

Guillame Barre Syndrome, atherosclerosis, chronic fatigue syndrome, TB, other viral and parasitic diseases, OKT3 therapy, and would be expected to be useful in conjunction with radiation therapy, chemotherapy and transplantation, to ameliorate the toxic effects of such treatments or procedures.

As the peptide of the present invention suppresses activation of neutrophils the composition and method of the present invention may also be useful in the treatment of diseases with an underlying element of local, systemic, acute or chronic inflammation. In general, it is believed the composition and method of the present invention will be useful in treatment of any systemic or local infection leading to inflammation.

The peptides of the present invention may also be administered in cancer therapy in conjunction with

cytotoxic drugs which may potentiate the toxic effects of TNFα (Watanabe et al., 1988; Immunopharmacol.

Immunotoxicol. 10: 117-127) such as vinblastin, acyclovir, interferon alpha, cyclosporin A, IL-2, actinomycin D, adriamycin, mitomycin C, AZT, cytosine arabinoside, daunororubin, cis-platin, vincristine, 5-flurouracil and bleomycin; in cancer patients undergoing radiation

therapy; and in AIDS patients (or others suffering from viral infection such as viral meningitis, hepatitis, herpes, green monkey virus etc.) and in patients receiving immunostimulants such as thymopentin and muramyl peptides or cytokines such as IL-2 and GM-CSF. In this use

peptides of the present invention will serve to abrogate the deleterious effects of TNFα .

It will be appreciated by those skilled in the art that a number of modifications may be made to the peptide of the present invention without deleteriously effecting the biological activity of the peptide. This may be achieved by various changes, such as insertions, deletions and substitutions (e.g., sulfation, phosphorylation, nitration, halogenation), either conservative or

non-conservative (e.g., W-amino acids, desamino acids) in the peptide sequence where such changes do not

substantially altering the overall biological activity of the peptide. By conservative substitutions the intended combinations are:-

G, A; V, I, L, M; D, E; N, Q; S, T; K, R, H;

F, Y, W, H; and P, Nα-alkylamino acids.

It may also be possible to add various groups to the peptide of the present invention to confer advantages such as increased potency or extended half-life in vivo, without substantially altering the overall biological activity of the peptide.

The term peptide is to be understood to embrace peptide bond replacements and/or peptide mimetics, i.e. pseudopeptides, as recognised in the art (see for example: Proceedings of the 20th European Peptide Symposium, edt. G. Jung. E. Bayer, pp. 289-336, and references therein), as well as salts and pharmaceutical preparations and/or formulations which render the bioactive peptide(s)

particularly suitable for oral, topical, nasal spray, ocular pulmonary, I.V., subcutaneous, as the case may be, delivery. Such salts, formulations, amino acid replacements and pseudopeptide structures may be necessary and desirable to enhance the stability, formulation, deliverability (e.g., slow release, prodrugs), or to improve the economy of production, and they are

acceptable, provided they do not negatively affect the required biological activity of the peptide.

Apart from substitutions, three particular forms of peptide mimetic and/or analogue structures of particular relevance when designating bioactive peptides, which have to bind to a receptor while risking the degradation by proteinases and peptidases in the blood, tissues and elsewhere, may be mentioned specifically, illustrated by the following examples: Firstly, the inversion of backbone chiral centres leading to D-amino acid residue structures may, particularly at the N-terminus, lead to enhanced stability for proteolytical degradation while not

impairing activity. An example is given in the paper "Tritriated D-ala¹-Peptide T Binding", Smith, C.S. et al. Drug Development Res. 15, pp. 371-379 (1988).

Secondly, cyclic structure for stability, such as N to C interchain imides and lactames (Ede et al in Smith and Rivier (Eds) "Peptides: Chemistry and Biology", Escom, Leiden (1991), p268-270), and sometimes also receptor binding may be enhanced by forming cyclic analogues. An example of this is given in "Confirmationally restricted thymopentin-like compounds", U.S. pat. 4,457,489 (1985), Goldstein, G. et al. Finally, the introduction of

ketomethylene, methylsulfide or retroinverse bonds to replace peptide bonds, i.e. the interchange of the CO and NH moieties may both greatly enhance stability and

potency. An example of the latter type is given in the paper "Biologically active retroinverso analogues of thymopentin", Sisto A. et al in Rivier, J.E. and Marshall, G.R. (eds.) "Peptides, Chemistry, Structure and Biology", Escom, Leiden (1990), p.722-773. The peptides of the invention can be synthesized by various methods which are known in principle, namely by chemical coupling methods (cf. Wunsch, E.: "Methoden der organischen Chemie", Volume 15, Band 1 + 2, Synthese von Peptiden, Thieme Verlag, Stuttgart (1974), and Barrany, G.; Merrifield, R.B: "The Peptides", eds. E. Gross,

J. Meienhofer., Volume 2, Chapter 1, pp. 1-284, Academic Press (1980)), or by enzymatic coupling methods

(cf. Widmer, F., Johansen, J.T., Carlsberg Res. Commun., Volume 44, pp. 37-46 (1979), and Kullmann, W.: "Enzymatic Peptide Synthesis", CRC Press Inc., Boca Raton, Florida (1987), and Widmer, F., Johansen, J.T. in "Synthetic

Peptides in Biology and Medicine:, eds., Alitalo, K.,

Partanen, P., Vatieri, A., pp. 79-86, Elsevier, Amsterdam (1985)), or by a combination of chemical and enzymatic methods if this is advantageous for the process design and economy.

It will be seen that one of the alternatives embraced in the general formula set out above is for a cysteine residue to be positioned at both the amino and carboxy terminals of the peptide. This will enable the cylisation of the peptide by the formation of di-sulphide bond.

It is intended that such modifications to the peptide of the present invention which do not result in a decrease in biological activity are within the scope of the present invention.

As would be recognized by those skilled in the art there are numerous examples to illustrate the ability of anti-idiotypic (anti-Ids) antibodies to an antigen to function like that antigen in its interaction with animal cells and components of cells. Thus, anti-Ids to a

peptide hormone antigen can have hormone-like activity and interact specifically with the receptors to the hormone. Conversely, anti-Ids to a receptor can interact

specifically with a mediator in the same way as the receptor does. (For a review of these properties see: Gaulton, G.N. and Greane, M.I. 1986. Idiotypic mimicry of biological receptors, Ann. Rev. Immunol. 4, 253-280;

Sege, K and Peterson, P.A., 1978. Use of anti-idiotypic antibodies as cell surface receptor probes. Proc. Natl. Acad. Sci. U.S.A. 75, 2443-2447).

As might be expected from this functional similarity of anti-Id and antigen, anti-Ids bearing the internal image of an antigen can induce immunity to such an

antigen. (This nexus is reviewed in Hiernaux, J.R. 1988. Idiotypic vaccines and infectious diseases. Infect.

Immun. 56, 1407-1413.)

As will be appreciated by persons skilled in the art from the disclosure of this application it will be

possible to produce anti-idiotypic antibodies to the peptide of the present invention which will have similar biological activity. It is intended that such

anti-idiotypic antibodies are included within the scope of the present invention.

Accordingly, in a fifth aspect the present invention consists in an anti-idiotypic antibody to the peptide of the first aspect of the present invention, the

anti-idiotypic antibody being capable of reducing TNF toxicity.

The individual specificity of antibodies resides in the structures of the peptide loops making up the

Complementary Determining Regions (CDRs) of the variable domains of the antibodies. Since in general, the amino acid sequences of the CDR peptide loops of an anti-Id are not identical to or even similar to the amino acid

sequence of the peptide antigen from which it was

originally derived, it follows that peptides whose amino acid sequence is quite dissimilar, in certain contexts can take up a very similar three-dimensional structure. The concept of this type of peptide, termed a "functionally equivalent sequence" or mimotope by Geyson is familiar to those expert in the field. (Geyson, H.M. et al 1987.

Strategies for epitope analysis using peptide synthesis. J. Immun. Methods. 102, 259-274).

Moreover, the three-dimensional structure and function of the biologically active peptides can be simulated by other compounds, some not even peptidic in nature, but which mimic the activity of such peptides. This field of science is summarised in a review by

Goodman, M. (1990). (Synthesis, spectroscopy and computer simulations in peptide research. Proc. 11th American Peptide Symposium published in Peptides-Chemistry,

Structure and Biology pp 3-29. Ed Rivier, J.E. and

Marshall, G.R. Publisher ESCOM.)

As will be recognized by those skilled in the art, armed with the disclosure of this application, it will be possible to produce peptide and non-peptide compounds having the same three-dimensional structure as the peptide of the present invention. These "functionally equivalent structures" or "peptide mimics" will react with antibodies raised against the peptide of the present invention and may also be capable of reducing TNF toxicity. It is intended that such "peptide mimics" are included within the scope of the present invention.

Accordingly, in a sixth aspect the present invention consists in a compound the three-dimensional structure of which is similar as a pharmacophore to the three- dimensional structure of the peptide of the first or second aspect of the present invention, the compound being characterized in that it reacts with antibodies raised against the peptide of the first aspect of the present invention and that the compound is capable of reducing TNF toxicity.

More detail regarding pharmacophores can be found in Bolin et al. p 150, Polinsky et al. p 287, and Smith et al. p 485 in Smith and Rivier (Eds) "Peptides: Chemistry and Biology", Escom, Leiden (1991).

Detailed Description of the Invention

In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following examples and Figures in which:-

Figure 1 shows changes in blood glucose levels in ascites tumour-bearing mice in response to TNF treatment □ 0 hrs, ■ 24 hrs; and

Figure 2 shows weight loss by tumour-bearing mice as a result of TNF treatment; and

Figure 3 shows the effect of peptides on TNF-induced lethality, ■ 24 hrs post TNF; ♡ 30 hrs post TNF.

Peptide Synthesis

Peptides numbered 363, 369-374 listed below were synthesized either by the Boc or Fmoc strategies.

(Abbreviations used in the following description: Acm

Acetamidomethyl, OCxl Cyclohexyl ester, Pmc

2,2,5,7,8-Pentamethylchroman-6-sulfonyl, NMM

N-Methylmorpholine).

363 H-Pro-Gln-Gly-Lys-Tyr-OH

369 H-Arg-Asp-Thr-Val-Cys(Acm)-Gly-Cys(Acm)-Arg-Lys-Asn-Gln

-Tyr-Arg-His-OH

370 Ac-Gln-Asp-Thr-Asp-Cys(Acm)-Arg-Glu-Cys(Acm)-Glu-Ser-

Gly-Ser-Phe-Thr-Ala-Ser-Glu-Asn-His-Leu-Arg-His-OH

371 H-Leu-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gly-Gln-Asp-Thr -Asp-OH

372 H-Asp-Ser-Val-Cys(Acm)-Pro-Gln-Gly-Lys-Tyr-Ile-His-Pro- Gln-Asn-Asn-Ser-OH

373 H-Thr-Lys-Cys(Acm)-His-Lys-Gly-Thr-OH

374 H-Glu-Asn-Val-Lys-Gly-Thr-Glu-Asp-Ser-Gly-Thr-Thr-OH 466 Leu-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gln

Asp-Thr-Asp-Cys(Acm)-Arg-Glu-Cys(Acm)-Glu-Ser-Gly-Ser- Phe-Thr 467 Thr-Lys-Cys(Acm)-His-Lys-Gly-Thr-Tyr-Leu-Tyr-Asn-Asp- Cys (Acm) -Pro-Gly-Pro-Gly-Gln-Asp-Thr-Asp

536 Arg-Glu-Asn-Glu-Cys(Acm)-Val-Ser-Cys(Acm)-Ser-Asn- Cys(Acm)-Thr-Lys-Leu

630 Ac-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly

631 Gly-Leu-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly

632 Gly-Leu-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gly

633 Gly-Pro-Gly-Gln-Asp-Thr-Asp

634 Ac-Gly-Pro-Gly-Gln-Asp-Thr-Asp-NH₂

635 Gly-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gly-Gln-Asp-Thr-Gly

636 Ac-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gly-Gln-Asp-Thr- NH₂

637 Gly-Ala-Gln-Met-Cys(Acm)-Cys(Acm)-Ser-Lys-Cys(Acm)- Ser-Pro-Gly-Gln-His-Gly

SYNTHESIS OF PEPTIDES USING THE FMOC-STRATEGY

Peptides were synthesized either on the continuous flow system using the standard Fmoc-polyamide method of solid phase peptide synthesis (Atherton et al, 1978, J. Chem. Soc. Chem. Commun., 13 , 537-539) or alternatively on the stirred cell system using polystyrene based resins.

For peptides with free carboxyl at the C-terminus, the solid resin used was PepSyn KA which is a

polydimethylacrylamide gel on Kieselguhr support with 4-hydroxymethylphenoxyacetic acid as the functionalised linker (Atherton et al., 1975, J.Am.Chem.Soc. 97,

6584-6585). The carboxy terminal amino acid was attached to the solid support by a DCC/DMAP-mediated

symmetrical-anhydride esterification. The alternative is the Wang resin with Fmoc-amino acid previously attached.

For peptides with carboxamides at the C-terminus, the solid resin used was Fmoc-PepSyn K Am which is analogous polyamide resin with a Rink Linker, p-[(R,S)-α[1-(9H- fluoren-9-yl)-methoxyformamido]-2,4-dimethoxybenzyl]- phenoxyacetic acid (Bernatowicz et al, 1989, Tet. Lett. 30, 4645). The synthesis starts by removing the Fmoc-group with an initial piperidine wash and

incorporation of the first amino acid is carried out by the usual peptide coupling procedure. In the stirred cell system, this is replaced by the Rink resin which is a polystyrene-based support.

All Fmoc-groups during synthesis were removed by 20% piperidine/DMF and peptide bonds were formed either of the following methods except as indicated in Table 1:

1. Pentafluorophenyl active esters - the starting Fmoc amino acids are already in the active ester form.

2. Hydroxybenzotriazol esters - these are formed in situ either using Castro's reagent, BOP/NMM/HOBt (Fournier et al, 1989, Int. J. Peptide Protein Res., 33., 133-139) or using Knorr's reagent, HBTU/NMM/HOB-t (knorr et al, 1989, Tet. Lett., 10, 1927)

Side chain protection chosen for the amino acids was removed concomitantly during cleavage with the exception of Acm on cysteine which was left on after synthesis.

Cleavage Conditions

Peptides were cleaved from the PepSyn KA and PepSyn K Am using 5% water and 95% TFA where Arg(Pmc) is not present. Where Arg(Pmc) is present a mixture of 5% thioanisole in TFA is used. The cleavage typically took 3 h at room temperature with stirring. Thionanisole was removed by washing with ether or ethyl acetate and the peptide was extracted into an aqueous fraction. Up to 30% acetonitrile was used in some cases to aid dissolution. Lyophilization of the aqueous/acetonitrile extract gave the crude peptide.

Peptides from the Wang resin were cleaved in TFA containing 5% phenol, for up to 2 h at ambient temperature with stirring.

N-TERMINAL ACETYLATION

The peptide resin obtained after the synthesis (with Fmoc removed in the usual manner) was placed in a 0.3 M DMF solution of 10 equivalents of Ac-ONSu or acetic anhydride for 60 minutes. The resin was filtered, washed with DMF, Ch₂Cl₂, ether and dried.

SYNTHESIS OF PETIODES USING THE BOC-STRATEGY

Syntheses of these peptides were carried out using polystyrene based resins. For peptide with C-terminal acids, the appropriate Merrifield resin Boc-amino

acid-O-resin or the 100-200 mesh PAM resin is used.

Peptides with C-terminal amides are synthesized on MBHA resins.

Couplings of Boc-amino acids (Table 2) were carried out either using Symmetrical anhydride method or a HOBt ester method mediated by DCC or HTBU.

CLEAVAGE

Peptides were cleaved in HF with p-cresol or anisole as scavenger for up to 90 min. For His with Dnp

protection, the resin required pre-treatment with

mercaptoethanol:DIPEA:DMF (2:1:7), for 30 min. After removal of scavengers by ether wash, the crude peptide is extracted into 30% aqueous acetonitrile.

N-TERMINAL ACETYLATION

Acetylation was achieved by treating the deblocked resin with acetic anhydride in DMF solution.

PURIFICATION OF PEPTIDES

Crude peptide is purified by reverse phase

chromatography using either a C4 or C18 column and the

Buffer system: Buffer A - 0.1% aqueous TFA, Buffer B - 80% Acetonitrile and 20% A. Fractions are monitored by an analytical h.p.l.c. system with a diode array detection. Structures were confirmed by amino acid analysis, proton n.m.r. and FAB mass spectrometry.

Example 1

371 H-Leu-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gly-Gln-Asp-Thr-A sp-OH

The resin Boc-Asp (Cxl)-O-Resin (100-200 mesh 0.75 mM/g) was used in the synthesis. This is followed by the coupling of the subsequent residues in the following order:

Boc-Thr(Bzl)-OH DCC S.A. (Symmetrical anhydride)

Boc-Asp-(OCxl)-OH DCC S.A.

Boc-Gln-OH DCC/HOBt

Boc-Gly-OH DCC S.A.

Boc-Pro-OH DCC S.A.

Boc-Gly-OH DCC S.A.

Boc-Pro-OH DCC S.A.

Boc-Cys(Acm)-OH DCC/HOBt

Boc-Asp(OCxl)-OH DCC S.A.

Boc-Asn-OH DCC/HOBt

Boc-Tyr(2Br-Z) DCC S.A.

Boc-Leu-OH DCC S.A.

The peptide resin (1.5 g) was treated with 15 ml HF at 0°C for 60 min. in the presence of p-cresol (1.5 g).

The peptide resin was washed with diethyl ether and then extracted with 30% aqueous acetonitrile. The peptide collected in the aqueous fraction was lyophilized to give

570 mg.

Purification was carried out in a Waters Delta Prep instrument using a Delta Pak, C18 Prep-Pak cartridge, 300

A, 15 μm. Buffer A: 0.1% TFA/water and buffer B is 90% acetonitrile/water (0.1% TFA). The gradient is 0-40% B over 60 min. (linear) monitored at 230 nm at a flow rate of 20m./min. A yield of 90 mg of 94% pure peptide was obtained.

Amino acid analysis gave Asp 4.0 (4.0) Glu 1.1(1.0) Gly 2.0(2.0) Leu 1.0(1.0) Pro 1.9(2.0) Thr 1.0(1.0) Tyr

0.7(1.0) and Cys was present.

Fast atom bombardment mass spectroscopy using argon gas gave a M+H peak at 1465.

IV Example 2

374 H-Glu-Asn-Val-Lys-Gly-Thr-Glu-Asp-Ser-Gly-Thr-Thr-OH Pepsyn KA (2.0 g, 0.25 mMol/g) was esterified with

Fmoc-Thr-OH using 8 equiv of Fmoc-Thr(But) -OH, 4 equiv of

DCC and 0.4 equiv of DMAP.

The following amino acid derivatives and coupling conditions were used in subsequent steps:

Fmoc-Thr(But)-OH BOP/HOBt/NMM

Fmoc-Gly-OH BOP/HOBt/NMM

Fmoc-Ser(But)-OH BOP/HOBt/NMM

Fmoc-Asp(OBut)-OH BOP/HOBt/NMM

Fmoc-Glu(OBut)-OH BOP/HOBt/NMM

Fmoc-Thr(But)-OH BOP/HOBt/NMM

Fmoc-Gly-OH BOP/HOBt/NMM

Fmoc-Lys(Boc)-OH BOP/HOBt/NMM

Fmoc-Val-OH BOP/HOBt/NMM

Fmoc-Asn-OPfp

Fmoc-Glu(OBut)-OH BOP/HOBt/NMM

The peptide resin, after removal of the final Fmoc group weighed 2.76 g. Cleavage was carried in 95% aqueous

TFA and the crude peptide, after lyophilization gave

813 mg.

The crude peptide was purified on a Waters' Delta

Prep using a C18 Prep Pak cartridge 300 A 15 μm particle size. Buffer A is 0.1% TFA and Buffer B is 80%

Acetonitrile and 20% buffer A and a linear gradient of 0-80%B over 40 min monitored at 23 nm with a flow rate of

30 ml/min, gave 262 mg of the 90% pure peptide.

Amino acid analysis gave Asp 1.9(2.0) Glu 2.1(2.0)

Ser 0.9(1.0) Gly 2.3(2.0) Thr 2.9(3.0) Val 1.0(1.0) Lys

1.0(1.0). FAB mass spectrum of the peptide gives a M+H peak at 1237.

In Vivo Experiments

Balb/c mice (female, aged 10-12 weeks) were

inoculated with Meth A ascites tumour cells (2 x 10 ) two weeks before treatment with TNF (10μg) alone or in combination with TNF inhibiting peptide (10 mg, Fig. 1). At the time of treatment and 24 hrs after treatment

measurements were taken of weight and blood glucose levels. Figure 1 shows changes in blood glucose levels in ascites tumour-bearing mice inn response to TNF treatment. It can be seen that peptide 371 inhibited the TNF-induced

decrease in blood glucose levels which occurred within the 24 hours following TNF treatment. Peptide 371 also

inhibited weight loss by the tumour-bearing animals as a result of TNF treatment (Figure 2).

In lethality experiments Balb/C mice (female, aged 10 - 12 weeks) were primed intraperitoneally with pristane (0.5 ml) 10 days prior to peritoneal implantation of MethA tumour cells. Approximately 10 days later the mice were challenged with 25μg recombinant human TNF administered subcutaneously and the number of mice surviving at 24 and 30 hours recorded. Mice were also treated with peptide 1 mg or anti-TNF monoclonal antibody (M47) by innoculation at a separate subcutaneous site. The results of these experiments are shown in Figure 3 and Table 3. TABLE 3

EFFECT OF PEPTIDE 371 ON SURVIVAL OF MICE ADMINISTERED A LETHAL DOSE OF TNF

* 24 hours post TNF treatment As will be seen from the above results the peptides of the present invention, and in particular peptide 371, are capable of preventing a decrease in blood glucose levels and weight loss in tumour-bearing mice treated with human recombinant tumour necrosis factor. In the light of these results it is believed these peptides have utility in the treatment of numerous disease states which are due to the deleterious effects of TNF.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodimentswithout departing from the spirit or scope of the

invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS :

1. A linear or cyclic peptide of the general formula:- x₁-x₂-x₃-x₄-x₅-x₆-x₇-x₈-x₉-x₁₀- in which x. is null, Cys or R₁;

x₂ is null, Cys, R₁ or

A₁-A₂-A₃-A₄-A₅-A₆-A₇

in which A₁ is Ser or Thr or Ala,

A₂ is Lys or Arg or His,

A₃ is Cys or Arg or His,

A₄ is His or Lys or Arg or Phe or Tyr or Trp,

A₅ is Lys or Arg or His,

A₆ is Gly or Ala,

A₇ is Thr or Ser or Ala,

X₃ is Null, Cys, R₁ or A₈-A₉

in which A₈ is absent or Gly or Ala or Tyr or Phe or Trp or His

A₉ is Leu or lle or Val or Met

X₄ is Cys, R₁ or

A₁₀-A₁₁-A₁₂-A₁₃-A₁₄ in Which A₁₀ is

Tyr or Phe or Trp or His or Gly or Ala,

A₁₁ is Asn or Gln,

A₁₂ is Asp or Glu,

A₁₃ is Cys or Arg or His,

A₁₄ is Pro or Nα - alkyl amino acid

X₅ is Gly or Ala,

X₆ is Cys, R₂ or A₁₅-A₁₆

in which A₁₅ is Pro or Nα - alkyl amino acid, A _{1 6} is

Gly or Ala,

X₇ is null, Cys, R₂ or A₁₇-A₁₈-A_{1 9}

in which A₁₇ is Gln or Asn,

A₁₈ is Asp or Glu,

A₁₉ is Thr or Ser or Ala,

X₈ is null, Cys, R₂, Asp, Glu, Gly or Ala, X₉ is null, Cys, R₂ or

A₂₀-A₂₁-A₂₂-A₂₃-A₂₄-A₂₅-A₂₆-A₂₇ -A ₂₈-A₂₉

in which A₂₀ is Cys or Arg or His,

A₂₁ is Arg or Lys or His,

A₂₂ is Glu or Asp,

A₂₃ is Cys or Arg or His,

A₂₄ is Glu or Asp,

A₂₅ is Ser or Thr or Ala,

A₂₆ is Gly or Ala,

A₂₇ is Ser or Thr or Ala,

A₂₈ is Phe or Tyr or Trp or His,

A₂₉ is Thr or Ser or Ala,

X ₁₀ is null, Cys or R₂

R₁ is R-CO, where R is H, straight, branched or cyclic alkyl up to C20, optionally containing double bonds and/or substituted with halogen, nitro, amino, hydroxy, sulfo, phospho or carboxyl groups (which may be substituted themselves), or aralkyl or aryl optionally substituted as listed for the alkyl and further including alkyl, or R₁ is glycosyl, nucleosyl, lipoyl or R₁ is an L- or D-α amino acid or an oligomer thereof consisting of up to 5 residues R₁ is absent when the amino acid adjacent is a- desamino-derivative.

R₂ is -NR₁₂R₁₃, wherein R₁₂ and R₁₃ are

straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R₁-O-glycosyl, -O-lipoyl or - an L- or D-α-amino acid or an oligomer thereof consisting of up to 5 residues or R₂ is absent, when the adjacent amino acid is a decarboxy derivative of cysteine or a homologue thereof or the peptide is in a N-C cyclic form,

with the proviso that:

X₁ is always and only null when X₂ is R₁, Cys or null X₂ is always and only null when X₃ is R₁, Cys or null X₃ is always and only null when X₄ is R₁ or Cys

X₇ is always and only null when X₆ is R₂ or Cys

X₈ is always and only null when X₇ is null, R₂ or Cys X₉ is always and only null when X₈ is null, R₂ or Cys X₁₀ is always and only null when X₉ is null, R₂ or

Cys

when X₄ is R₁ or Cys then X₆ is A₁₅-A₁₆, X₇ is

A₁₇-A₁₈-A₁₉ and X₈ is Asp, Glu, Gly or Ala,

when X₆ is R₂ or Cys then X₄ is

A₁₀-A₁₁-A₁₂-A₁₃-A₁₄

when X₇ is null, R₂ or Cys then X₄ is

A₁₀-A₁₁-A₁₂-A₁₃-A₁₄.

2. A linear or cyclic peptide as claimed in claim 1 in which the peptide is selected from the group consisting of:-

H-Leu-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gly-Gln-Asp-Thr-

Asp-OH,

Leu-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gln-Asp-Thr-Asp- Cys(Acm)-Arg-Glu-Cys(Acm)-Glu-Ser-Gly-Ser-Phe-Thr,

Thr-Lys-Cys(Acm)-His-Lys-Gly-Thr-Tyr-Leu-Tyr-Asn-Asp-

Cys(Acm)-Pro-Gly-Pro-Gly-Gln-Asp-Thr-Asp,

Ac-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly,

Gly-Leu-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly,

Gly-Leu-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gly,

Gly-Pro-Gly-Gln-Asp-Thr-Asp,

Ac-Gly-Pro-Gly-Gln-Asp-Thr-Asp-NH₂,

Gly-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gly-Gln-Asp-Thr-Gly, and

Ac-Tyr-Asn-Asp-Cys(Acm)-Pro-Gly-Pro-Gly-Gln-Asp-Thr-NH₂

3. A linear or cyclic peptide as claimed in claim 1 in which the peptide is;

H-Leu-Tyr-Asn-Asp-Cys-Pro-Gly-Pro-Gly-Gln-Asp-Thr-Asp-OH.

4. A linear or cyclic peptide of the general formula:-

Y₁-Y₂-Y₃-Y₄-Y₅-Y₆-Y₇-Y₈-Y₉-Y₁₀- Y₁₁-Y₁₂-Y₁₃-Y₁₄-Y₁₅-Y₁₆-Y₁₇ in which Y₁ is null, Cys or R₁

Y₂ is Gly or Ala

Y₃ is Ala or Gla

Y₄ is Gln or Asn

Y₅ is Met or Gal or lle or Leu

Y₆ is Cys or Arg or His

Y₇ is Cys or Arg or His

Y₈ is Ser or Thr or Ala

Y₉ is Lys or Arg or His

Y₁₀ is Cys or Arg or His

Y₁₁ is Ser or Thr or Ala

Y₁₂ is Pro or nα - alkyl amino acid

Y₁₃ is Gly or Ala

Y₁₄ is Gln or Asn

Y₁₅ is His or Lys or Arg

Y₁₆ is Gly or Ala

Y₁₇ is null, Cys or A₂

R₂ is -NR₁₂R₁₃, wherein R₁₂ and R₁₃ are

straight, branched or cyclic alkyl, aralkyl or aryl, optionally substituted as defined for R₁-O-glycosyl, -O-lipoyl or - an L- or D-α-amino acid or an oligomer thereof consisting of up to 5 residues or R₂ is absent. when the adjacent amino acid is a decarboxy derivative of cysteine or a homologue thereof or the peptide is in a N-C cyclic form.

5. A linear or cyclic peptide as claimed in claim 4 in which the peptide is;

Gly-Ala-Gln-Met-Cys(Acm)-Cys(Acm)-Ser-Lys-Cys(Acm)-Ser-Pro- Gly-Gln-His-Gly.

6. A pharmaceutical composition for use in treating subjects suffering from TNF toxicity, the composition comprising a peptide as claimed in any one of claims 1 to 5 in combination with a pharmaceutically acceptable

sterile carrier.

7. A method of treating a subject suffering from the toxic effects of TNF, the method comprising administering to the subject the composition as claimed in claim 6.

8. A method as claimed in claim 7 in which the subject is suffering from TNF pathology associated with decreased blood glucose levels and/or weight loss or from septic shock.

9. An anti-idiotypic antibody to the peptide as claimed in any one of claims 1 to 5, the anti-idiotypic antibody being characterised in that it is capable of reducing TNF toxicity.

10. A compound the three-dimensional structure of which is similar as a pharmacophore to the three- dimensional structure of the peptide as claimed in any one of claims 1 to 5, the compound being characterized in that it reacts with antibodies raised against the peptide as claimed in any one of claims 1 to 5 and that the compound is capable of reducing TNF toxicity.