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WO1995000538A1 - Peptides derives de l'interleukine-2 humaine destines a etre utilises en medecine - Google Patents

Peptides derives de l'interleukine-2 humaine destines a etre utilises en medecine Download PDF

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Publication number
WO1995000538A1
WO1995000538A1 PCT/GB1994/001349 GB9401349W WO9500538A1 WO 1995000538 A1 WO1995000538 A1 WO 1995000538A1 GB 9401349 W GB9401349 W GB 9401349W WO 9500538 A1 WO9500538 A1 WO 9500538A1
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Prior art keywords
peptide
medicine
leu
peptides
ome
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Inventor
Vadim Tikhonovich Ivanov
Inessa Ivanovna MIKHALEVA
Ludmilla Viktorovna ONOPRIENKO
Boris Olegovich Voitenkov
Valery Borisovich OKULOV
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Peptech UK Ltd
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Peptech UK Ltd
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Priority to AU69779/94A priority Critical patent/AU6977994A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to interleu in-2 (IL-2) derived peptides that are useful in the treatment of inflammation, in tissue reparation and in the treatment of cancer in a broad sense.
  • IL-2 interleu in-2
  • IL-2 is a 15.5 kiloDalton glycoprotein synthesised by lymphocytes following their activation by mitogens or antigens. In addition to being an essential growth factor for T lymphocytes, IL-2 has a range of other biological functions. These include the induction of antigen specific cytotoxic T-lymphocytes, and the activation of "natural killer cells" or lymphokine activated killer cells. These and other activities have demonstrated the importance of IL-2 in a number of physiological processes involving the immune system, particularly involving defence against tumours and activation of cells at sites of infection and defence. Moreover, associations have also been reported between II.-2 and diseases such as lupus erythematosus and primary immunodeficiencies.
  • IL-2 is a toxic therapeutic agent (Vial, T & Descotes, J; Drug Safety, 7, 417-433 (1992)) causing the manifestation of serious side effects, including cancer of the kidney and therefore it is desirable to develop agents which mimic aspects of IL-2 activity but which do not have the harmful side effects of IL-2.
  • agents which regulate IL-2 activity or which can modulate the production of other cytokines may have useful therapeutic application in immunoregulation, inflammation , tissue repair or cancer.
  • a conventional strategy to develop such agents would be to isolate a part of the IL-2 molecule which is able to reproduce some of its beneficial activities, whilst avoiding its unwanted side effects. The obvious choice for such agents would be the part of the molecule suspected of interacting with the receptor.
  • IL-2 derived peptides that appear to interact with the receptor have been described (Zav'yalov et al , Immunology Letters 31, 285-288, 1992), and some of these compete with binding of full-sized IL-2 to its receptor.
  • the regions claimed to antagonise IL-2 receptor binding were in the region of amino acid residues 27-35. Furthermore, this region was previously suggested to be involved in receptor binding from X-ray analysis of IL-2 structure (Zurawski and Zurawski, EMBO J. 8, 2583-2590, 1989) .
  • the present inventors have surprisingly found a completely different region of the IL-2 molecule which appears to be implicated in its biological activity and, in particular its tissue reparation, anti-inflammatory and anti-cancer properties.
  • peptides have the desirable properties of IL-2 mentioned above whilst lacking the undesirable side effects of full length IL-2.
  • the activity of the peptides is particularly surprising since the region from amino acids 58 to 72 of IL-2 is structurally distinct from the receptor binding region at residues 27 to 35.
  • the term "derivative" relates to a peptide having the amino and/or carboxy terminus blocked, for example by conversion of the carboxy terminus into an amide or ester derivative or to a peptide in which one or more of the amino acids is substituted or has the D configuration.
  • substantially homologous would be well understood by one skilled in the art who would easily be able to determine whether or not two sequences were substantially homologous.
  • amino acid sequences can be described as substantially homologous when they have at least 40% homology although, for the purpose of the present invention, it is preferable for a sequence to have at least 50%, 60%, 70%, 80%, 90% or 95% homology (in increasing order of preference) to the amino acid sequence 58 to 72 of IL-2.
  • the residues which are compared need not be in exactly the same positions in two sequences which are substantially homologous but rather, one of the sequences may have various inserted or deleted amino acid residues or regions with respect to the sequence with which it is compared.
  • C ⁇ C ⁇ alkyl refers to a straight or branched chain alkyl group containing from one to 20 carbon atoms. Examples of such groups include methyl, ethyl, n-butyl, tertiary butyl, decyl, dodecyl and stearyl.
  • C 2 -C 20 alkenyl refers to a straight or branched chain hydrocarbon having at least one double bond. Examples include 1-propenyl and 1-hexenyl.
  • the region of the human IL-2 molecule, with the amino acid positions numbered from 58 to 72 is as follows:
  • the present invention relates to peptides comprising this region or a fragment of this region of at least five amino acids in length, peptides containing a region substantially homologous to the above region or a fragment of at least five amino acids of the above region or derivatives of either of the above.
  • the peptides may also be either linear or cyclic although linear forms are preferred.
  • the peptide may be up to 50 amino acids in length, it is preferred that it is somewhat shorter than this, for example no more than 40, 30 or even 20 amino acids long.
  • the C-terminal carboxylic acid may be converted to a substituted or unsubstituted C ⁇ C- JO alkyl amide, C 2 - C 20 alkenyl amide, aryl amide, C 1 -C 20 alkyl or C 2 -C 20 alkenyl ester, aryl ester, C ⁇ C ⁇ alkyl or C 2 -C 20 alkenyl ether or aryl ether.
  • Examples of such groups include phenyl ester, benzyl ether or thiophenylethyl ester but C 1 -C 3 alkyl ethers and esters, and especially the methyl ester, are particularly suitable.
  • Suitable substituents for alkyl, alkenyl and aryl groups include hydroxy, amino and halogen substituents.
  • Cysteine residues of the peptides of the invention may be substituted by C 1 -C 20 alkyl or C ⁇ ⁇ alkenyl amide or substituted amide, triphenylmethyl, a C 1 -C 20 alkyl or C 2 -C 20 alkenyl substituted carbamoyl group or a benzyl group.
  • the N-terminal amino acid of the peptide may be N- substituted with a group such a saturated or unsaturated aliphatic C 1 -C 2Q acyl group or by an aromatic acyl group.
  • Suitable substituents include formyl, acetyl and propionyl groups.
  • the derivatisation of the N and C terminals of the peptide and the cysteine groups helps to confer resistance to enzymatic degradation.
  • Suitable substituents include those in which one or more nitrogen atoms are replaced by oxygen atoms such that the amide bonds are replaced by ester bonds.
  • hydroxy side chains may be derivatised to form c ⁇ _C 2o ester or ether groups and/or at least one amino acid may be substituted by a monomeric or polymeric carbohydrate or a derivative thereof via a hydroxy, amino or amido group of the amino acid.
  • an S-S bridge can then be formed between two cysteine residues.
  • the peptides can be substituted in other ways but it is generally preferred that the substitutions are conservative in nature. That is to say, that the substituents will resemble the groups they replace in size, polarity and acidity or basicity.
  • Particularly preferred peptides of the present invention include the following:
  • Leu-amide or ester may be advantageous at position 15 of peptide C-I-7 (equivalent to position 72 of IL-2) .
  • a D-Leu at position 2 of C-I-7 or indeed any D-amino acid at position 2 of any of the peptides may provide the advantage of increased half-life in vivo by preventing the actions of aminopeptidases.
  • introduction of a D-amino acid at any position in the peptide may help confer resistance to peptidases.
  • one of the sensitive sites has already been localised at the bond between Leu (66) -Glu(67) .
  • peptides of the invention do have some IL-2 like properties, they do not mimic all of the actions of IL-2.
  • CTLL assay Robot, R.J. in Methods in Enzymology, 165, Eds G. diSabato, J.J. Langone, H. van Vunakis, Academic Press Inc, Orlando pp 493-525, 1985
  • peptides of the invention are tested in the CTLL assay (Robb, R.J. in Methods in Enzymology, 165, Eds G. diSabato, J.J. Langone, H. van Vunakis, Academic Press Inc, Orlando pp 493-525, 1985) , which is classically used to detect IL-2 activity, it does not induce the proliferation characteristic of IL-2 in these cells.
  • the peptides of the invention are able further to enhance proliferation when added to cultures 24 h later.
  • the apparent potency of the IL-2 is increased, i.e. half maximal stimulation of the cells is achieved with a lower dose of IL-2.
  • the peptides of the present invention are potentially useful in a wide variety of areas .
  • One property of the peptides is their effect on the activation of macrophages.
  • a number of investigators have described the so-called growth stimulating phase of macrophages which is distinguished from the (tumour cell) cytotoxicity phase (Okulov et al , J. Cancer Res . Clin . Oncol . , 118, 537-541, 1992) . It has now been found that the peptides of the present invention can act on activated macrophages which are expressing heightened cytotoxic properties to switch them into a growth stimulating mode.
  • the peptides of the present invention also have the ability to enhance the processes of tissue regeneration and reparation.
  • the peptides significantly enhanced wound healing of permanently disturbed skin wounds when applied either topically or intraperitoneally.
  • the peptides have been found to be effective in promoting restoration of liver function. It has been shown that the peptides are capable of restoring the protein synthesising function of the liver and significantly inhibiting carbon tetrachloride induced elevation of aspartate and alanine-aminotransferases, ⁇ - glutamyl transpeptidase and alkaline phosphatase.
  • the peptides of the invention have, moreover, been found to have immunosuppressant properties and, in particular, it has been demonstrated that they are capable of significantly reducing delayed type hypersensitivity reactions. Clearly, therefore, the peptides of the present invention have useful biological properties. Moreover, they do not simply mimic the action of IL-2 but, rather, enhance certain useful activities whilst being free of the majority of the toxic side effects associated with IL-2. The inflammatory properties associated with IL-2 seem to be absent in many cases and under some circumstances it seems that the peptides can act as anti-inflammatory agents.
  • the peptides of the present invention are likely to be of therapeutic use in a method for the treatment of a number of diseases and conditions, the method comprising administering to a patient an effective amount of a peptide according to the invention.
  • the general type of disease or illness in which the invention will be useful includes any condition in which IL-2 may be beneficial although, because the peptides of the present invention fail to exhibit some of the toxic effects of IL-2 (for example the inflammatory effects) , the peptides may occasionally be useful for conditions where IL-2 would not be expected to be beneficial.
  • the peptides are expected to be useful in the treatment of cancers.
  • the ability in some circumstances to transform macrophages into a "stimulatory" mode may manifest as an antiinflammatory activity.
  • all circumstances where tissue regeneration and repair is of importance should be improved by the peptides.
  • a peptide of not more than 50 amino acids in length comprising a fragment of at least 5 amino acids taken from the region extending from amino acid residues 58 to 72 of IL-2 and having the sequence:
  • the type of diseases and conditions in which the peptides of the present invention may be beneficial include the following:
  • ARDS adult respiratory distress syndrome
  • in organ or bone marrow transplant patients cancer (optionally in combination with other treatments such as chemotherapy and/or radiation) ; wound, tissue and organ healing; recovery from toxicity induced by consumption of alcohol, medication and drugs of abuse; recovery from surgical trauma; Crohn's disease; ulcerative colitis; inflammatory bowel disease; auto-immune disease; multiple sclerosis; AIDS; inflammation of the muscles and joints; regeneration of peripheral nerves; and restoration of functional activity of liver enzymes.
  • the peptides can be used to increase the immune response to an antigen, thereby acting as an adjuvant and increasing the production of antibodies either as a vaccine, or for experimental purposes.
  • the peptides may either be administered alone or in combination with other agents for example known antibacterial, antifungal or antiviral agents or steroidal or non steroidal anti inflammatory agents.
  • the peptides may be used in combination with other anti- cancer therapy including chemotoxic agents and immunomodulators such as - or ⁇ -interferon, IL-2 or other IL-2 derivatives or TNF-c- and, when used in the treatment of transplant patients, other agents will preferably be used in combination with the peptides.
  • linear peptides described in this invention may be prepared by any process, such as conventional solid phase peptide synthetic techniques, (see “Solid Phase Peptide Synthetic Techniques", 2nd ed., J.ll. Stewart, J.D. Young, Pierce Chemical Company, 1984, ISBN: 0-9350,40-03-0) . Another possibility is solution phase techniques. Cyclic forms of these peptides may be prepared by known techniques, such as, for example, described in Y. Hamada, Tetrahedron Letters, 26, 5155-5158 (1985) .
  • the cyclic peptides may be established in the form of an S-S bridge between two Cys-residues and/or reacting the carboxy terminal amino acid residue with the amino terminal residue and/or reacting the amino terminal residue with for example the gamma carboxyl group of Glu.
  • peptides will generally be included in a pharmaceutical or veterinary formulation and, therefore, in a third aspect of the invention, there is provided a pharmaceutical or veterinary composition comprising a peptide of not more than 50 amino acids in length and comprising a fragment of at least 5 amino acids taken from the region extending from amino acid residues 58 to 72 of IL-2 and having the sequence:
  • compositions may also include other components, for example any known antibacterial and/or antiviral agent and/or antifungal agent or steroidal or non-steroidal anti-inflammatory agents.
  • compositions of the present invention may be prepared simply by admixing the ingredients.
  • the compositions may be adapted for administration by the oral, parenteral, topical, nasal, buccal, rectal, or vaginal routes, or by inhalation spray or in other ways.
  • the peptides of the invention may be formulated for topical use, for inhalation by spray, for injection, for infusion or for oral administration and may be presented in unit-dose form in ampoules, tablets or in multidose containers with an added preservative.
  • the compositions may take such forms as suspensions, solutions, emulsions in oily or aqueous vehicles or liposomal formulations, and may contain formulating agents such as suspending, stabilising and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
  • compositions may contain from lxl0 "6 % to 99% of the active material.
  • Other ingredients which may be included in the compositions include antimicrobial agents, preservatives and flavouring and/or colouring agents.
  • compositions are administered in therapeutically or prophylactic effective doses.
  • the dose administered daily will, however, be determined by the physician and will, of course, depend on the type and severity of the condition to be treated, for example, in the case of inflammation, on the degree of inflammation and inflammatory response.
  • the size of dose will also, of course, depend on the size of the patient to be treated. However, in general, the dose will generally be in the region of 0.001-10000 mg of peptide per day, in particular 1-1000 mg per day.
  • FIGURE 1 is a histogram showing the effect of an IL-
  • FIGURE 2 shows the potentiation of IL-2 activity in the CTLL assay by a peptide of the invention when the cells have been pre-incubated with the peptide;
  • FIGURE 3 shows the potentiation of IL-2 activity in the CTLL assay by a peptide of the invention when the cells have been co-incubated with the peptide;
  • FIGURE 4 shows the effect of peptides of the invention on induction of growth stimulating potential in macrophage supernates
  • FIGURE 5 compares the effects of incubating resting macrophages with TPA or with a peptide of the present invention on tumour cell growth stimulation
  • FIGURE 6 is a plot of the healing of full-thickness cutaneous wounds either untreated or treated with a peptide of the invention
  • FIGURE 7 is a plot showing the action of various peptides of the invention on the liver function of mice after treatment with carbon tetrachloride.
  • FIGURE 8 shows the action of a peptide of the invention on liver function of mice suffering galactosamine induced toxicity.
  • PBMC peripheral blood mononuclear cells
  • the assay was performed in 96-well U-bottomed tissue culture plates (Falcon, Oxnard, California, USA) .
  • 100 ⁇ l of PBMC (1 x 106 cells/ ml) were incubated with lOO ⁇ l of peptide solution (at concentrations of 200 ⁇ g/ml, 20 ⁇ g/ml, 2 ⁇ g/ml and 0.2 ⁇ g/ml) or lOO ⁇ l of recombinant interleukin 2 (IOS, Riga, Lithuania, 10000 U/ ⁇ g) at a concentration of 1000 U/ ml in complete RPMI 1640 medium.
  • Control cultures had 100 ⁇ l of complete medium or 100 ⁇ l of PHA (lO ⁇ l/ml) .
  • IL-2 increased proliferation of PBMC over control values, and to a greater degree than was obtained simply by addition of further PHA (Note that all cultures, even “controls”, had been exposed to PHA the day before performing the "IL-2 related activity assay") .
  • the peptide C-I-6 which comprises only a part of ' the sequence of IL-2, was also able to increase proliferation.
  • CTLL cells were incubated with C-I-6 at a range of concentrations for 90 min at 37°C and then washed three times to remove excess peptide. These pretreated cells were then plated at 104 cells/well in a range of dilutions of IL-2. After 24 h, 0.04 MBq of [3H] -thymidine was added to each well, and incorporation of radioactivity over the subsequent 24 h was determined as a measure of proliferation.
  • CTLL cells are dependent upon IL-2 for their growth, and the potency of a batch of IL-2 can be described as the amount (or dilution) necessary to cause 50% maximal growth of these cells.
  • preincubation of cells in peptide C-I-6 resulted in half-maximal stimulation of cells at a lower concentration of IL-2, i.e. the apparent potency was increased.
  • CTLL cells were incubated in a range of dilutions of IL-2, in the presence or absence of C-I-6 at a number of different concentrations. After 24 h incubation, treatment was as described in Example 2.
  • Examples 1 to 3 demonstrate that some of the activity of the peptides of the invention is related to IL-2 activity.
  • IL-2 is able further to enhance proliferation when added to cultures 24 hours later and Peptide C-I-6 has a similar effect.
  • the apparent potency of the IL-2 is increased, i.e. half maximal stimulation of the cells is achieved with a lower dose of IL-2.
  • mice weighing 18-22g were obtained from the Rappoplovo animal breeding facility of the Academy of Medical Sciences, USSR.
  • Peritoneal macrophages isolation Mice were killed by cervical dislocation. Peritoneal cells were collected by lavage with 5 ml of basal Eagle's medium (BEM, Institute of Poliomyelitis and Viral Encephalitis, Moscow) , centrifuged at 200g for 10 min, and resuspended in the same medium at 1 x 106 cells/ml. lOO ⁇ l of cell suspension was plated in each well of 96-well flat-bottomed plates (Nunc. Denmark) and incubated in 5% C02, 95% air, 100% humidity at 37°C for 90 min. Non-adherent cells were removed by washing. 4-5 x 104 adherent cells remaining per well had a morphology typical of macrophages.
  • Basal Eagle's medium BEM, Institute of Poliomyelitis and Viral Encephalitis, Moscow
  • Macrophages obtained as described above were cultivated in 200 ⁇ l of complete medium (BEM supplemented with 10% heat-inactivated neonatal bovine serum) containing 2 mM L-Gln, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin and, to achieve activation, well known activating agents such as lipopolysaccharide.
  • complete medium BEM supplemented with 10% heat-inactivated neonatal bovine serum
  • C-I-2, C-I-3, C-I-4, C-I-5 or C-I-7 (10 ⁇ g/ ml).
  • cultures were washed and incubated for 18h in serum free medium. Supernatants were then aspirated and again tested in the tumour growth stimulation/inhibition assay.
  • P3X-63-Ag/8 mouse myeloma cells were maintained in complete medium and cultured under conventional conditions.
  • cells were seeded in 96-well plates (5 x 103 cells per well) in 100 ⁇ l of serum free medium.
  • 100 ⁇ l of serum free macrophage culture supernatants were added to myeloma cells, which were cultured for 24h under conventional conditions.
  • Cells were then pulsed with 1.0 ⁇ Ci of [3H] -thymidine for 2h.
  • Cells were harvested on filters and radioactivity was determined with a liquid scintillation counter Delta-300 (Nuclear Chicago) .
  • cpm C counts per min in control wells (target cells incubated with supernatants obtained from unstimulated macrophages) .
  • cpm E counts per min in experimental wells (target cells incubated with supernatants obtained from macrophages having been in contact with IL-2 derived peptides) .
  • Peritoneal macrophages obtained as described in Example 4 from SPF mice (breeding facility of the Central Institute of Microbiology and Epidemiology, Jena, DDR) were treated with TPA (1 nmol/ml) or C-I-6 (1 ⁇ g/ml) in complete culture medium under conventional conditions for 24 h. On days 1, 2, 3, 4, 5, 6 and 7, cultures were washed and incubated for a further 18 h in serum free medium. These supernatants were aspirated and used for tumour growth stimulation/inhibition assay as described in Example 4.
  • Examples 4 and 5 relate to the activation of macrophages by peptides of the present invention.
  • a number of investigators have described the so-called growth stimulating phase of macrophages, which is distinguished from the (tumour cell) cytotoxicity phase (Okulov et al, J. Cancer Res . Clin . Oncol . , 118, 537-541, 1992) .
  • activated macrophages which are expressing heightened cytotoxic properties can be switched into a growth-stimulating mode by incubation with a number of the peptides of the present invention.
  • this activity was detectable at 10 pg/ml and maximal at 1 ⁇ g/ml.
  • stimulation of growth promoting activity was detectable at 250 ⁇ g/kg and maximal at 2.5 mg/kg.
  • Supernatants derived from stimulated macrophage cultures treated with peptides of the present invention contained no TNF-alpha/cachectin and low amounts of IL-1 indicating a reduction of inflammatory state and immune cell activation.
  • both TNF-o. and IL-l/S are potent inflammatory mediators.
  • the peptides differ from TGF-/3, in that the latter inhibits both macrophage-mediated cytotoxicity and lymphocyte activity.
  • mice were narcotised (80 mg of hexenal, i.p.) and full-thickness skin wounds were made with a round steel biopsy punch (9mm in diameter) on the depilated dorsal surface. On the 4th day the blood clot was removed. Blood clot removal was performed every day thereafter, until termination of the experiment .
  • C-I-6 was given every day i.p. or topically (in distilled water) at a dose of 2.5 mg/kg from day 1 to day 10 of the experiment.
  • Results indicate that C-I-6 peptide enhances the rate of wound healing after either topical or intraperitineal administration.
  • mice Male HP mice weighing 18-22g were obtained from the Rappoplovo animal breeding facility of the Academy of Medical Sciences USSR. Peptides were administered daily i.p. in 0.2 ml of saline at a dose of 2.5 mg/kg/day for
  • Peptides C-I-3, C-I-4, C-I-5, C-I-6, C-I-7 caused a reduction in duration of narcosis. This indicates that they were able to facilitate recovery from liver damage previously induced by CC1 4 .
  • mice For three days before hepatectomy, rats (10 per each group) received C-I-6 treatment (at a dose of 2.5 mg/kg, i.p.) , or control treatment.
  • C-I-6 treatment at a dose of 2.5 mg/kg, i.p.
  • control treatment for three days before hepatectomy, rats (10 per each group) received C-I-6 treatment (at a dose of 2.5 mg/kg, i.p.) , or control treatment.
  • C-I-6 treatment at a dose of 2.5 mg/kg, i.p.
  • control treatment for three days before hepatectomy, rats (10 per each group) received C-I-6 treatment (at a dose of 2.5 mg/kg, i.p.) , or control treatment.
  • General histology, mitotic index and the index of labelled nuclei was assessed 24 and 48 h after the hepatectomy after pulse labelling in vivo with [3H] -thymidine. The results are shown in Table 3.
  • the experiment was carried out according to method of Higgins and Anderson 1931.
  • C-I-6 exerted some inhibition of proliferative processes in the regenerating liver. However, it later stimulated proliferation of hepatocytes significantly. Moreover, histological examination revealed more intensive cell destruction in the first 24 h of the experiment in C-I-6 treated livers. In addition, in treated animals substantially more glycogen granules were observed in hepatocytes, as comparison to controls.
  • C-I-6 was found to be effective in promoting restoration of liver function.
  • a model of tetrachloromethane (CC1 4 ) -induced hepatitis the duration of thiopental narcosis was determined after treatment with various IL-2 derived peptides.
  • the duration of narcosis of mice was increased from 7.5 minutes to 113.5 minutes following tetrachloromethane treatment (7.5 ml/kg) indicating that the tetrachloromethane had caused damage to the liver.
  • Peptide C-I-6 intraperitoneally administered at a dose of 2.5 mg/kg/day for 3 days after
  • CC1 4 administration caused a significant reduction in animal recovery time, indicating improved recovery of liver damage induced by CC1 4 .
  • Example 8 where hepatotoxicity was induced by galactosamine, C-I-6 was effective in inducing hepatic recovery and consequently facilitating recovery from thiopental narcosis.
  • mice given a sub-lethal dose of hepatotoxin C-I-6 was able to reduce the incidence of mortality induced by CC1 4 .
  • mice were sensitised with live BCG vaccine (500 ⁇ g/mouse, intradermal injection) .
  • the tuberculin test was performed: Purified protein derivative (PPD) (900 units) was injected into one hind foot pad and saline into the contralateral foot pad.
  • PPD Purified protein derivative
  • C-I-6 0.4, 2.0 or 10 mg/kg was introduced i.p. 24 hours later, the DTH reaction was evaluated by measuring and comparing the thickness of foot pads of right and left legs. Groups of mice which received no C-I-6, or a mixture of amino acids were used as controls.
  • mice received C-I-6 24 hours after PPD injection.
  • DTH reaction was evaluated 48 hours after PPD injection.
  • mice Outbred NMRI male mice, 2 months old, with a body weight of 25-29 g were used. The animals were maintained under SPF conditions, at 20+2°C, 45-55% RH, and food and water were provided ad libi tum.
  • Test materials C-I-6 was dissolved in Dulbecco's balanced salt solution (DBSS) . Cyclophosphamide was used as a positive control material.
  • DBSS Dulbecco's balanced salt solution
  • Antigen SRBC obtained by jugular puncture were stored as a 50% suspension at 4°C under sterile conditions before use. Immediately before use, 1 ml (for sensitisation) or 3 ml (for challenge) of 50% suspension were washed 3 times in saline and resuspended in saline.
  • Treatment schedule 2 Effect on challenge stage: Animals were treated twice (20 hour interval) with intraperitoneal injections of C-I-6 (0.4, 2.0 or 10.0 mg/kg) , dexamethasone (10 mg/kg) or drug free vehicle
  • mice (10 per group) were sensitised to SRBC by a single injection of 2 x 10 7 SRBC in ml saline (day 0) . Five days later, mice were given 108 cells in 50 ⁇ l by injection into the left hind footpad. 50 ⁇ l injected into the left hind footpad served as control. 24 hours after the last injection mice were euthanised in C02 and control and challenge footpads excised above the heel joint and weighed.
  • DTH index ( % ) Challenge paw weight - Control paw weight x 100
  • C-I-6 caused a significant reduction in the DTH reaction. This reduction was less marked than that caused by the known immunosuppressant cyclophosphamide, but the latter agent was used at near toxic doses, whereas the C-I-6 showed no toxic effect.
  • the peptides claimed in this invention have surprising biological properties. However, they do not simply mimic activities of the molecule from which they are derived (IL-2) , although they do enhance certain activities of this molecule. They have been shown to have activity in animals, and thus can be expected to have therapeutic effect in man. In addition, acute toxicity evaluation of C-I-6 in mice revealed no adverse reactions at doses up to 25 mg/mouse, suggesting that toxicity would not be a limiting factor in the use of this peptide, unlike full-sized IL-2.

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Abstract

Des peptides, dérivés de l'interleukine-2 et contenant des acides aminés provenant de la région s'étendant des restes 58 à 72 de l'IL-2, se révèlent utiles dans le traitement de maladies induites par l'IL-2, alors même qu'ils ne contiennent pas la région de liaison reconnue de l'IL-2.
PCT/GB1994/001349 1993-06-22 1994-06-22 Peptides derives de l'interleukine-2 humaine destines a etre utilises en medecine Ceased WO1995000538A1 (fr)

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AU69779/94A AU6977994A (en) 1993-06-22 1994-06-22 Peptides derived from human interleukin-2 for use in medicine

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GB939312874A GB9312874D0 (en) 1993-06-22 1993-06-22 Peptides for use in medicine

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WO2000011028A3 (fr) * 1998-08-21 2000-07-06 Yeda Res & Dev Peptides anti-inflammatoires derives d'il-2 et analogues correspondants

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EP0157643A2 (fr) * 1984-04-05 1985-10-09 Scripps Clinic And Research Foundation Anticorps à l'interleukine-2-humaine induite à l'aide de polypeptides synthetiques
WO1989003221A1 (fr) * 1987-10-13 1989-04-20 Genelabs Incorporated Agent antisyncytial
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EP0157643A2 (fr) * 1984-04-05 1985-10-09 Scripps Clinic And Research Foundation Anticorps à l'interleukine-2-humaine induite à l'aide de polypeptides synthetiques
WO1989003221A1 (fr) * 1987-10-13 1989-04-20 Genelabs Incorporated Agent antisyncytial
WO1991006562A1 (fr) * 1989-10-24 1991-05-16 University Of North Carolina At Chapel Hill Domaines immunologiques de l'antigene du virus delta de l'hepatite

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L.V. ONOPRIENKO ET AL: "Synthesis and immunogenic properties of peptides corresponding to 59-72 and 25-36 sequences of human iL-2", BIOORGANITCHESKAJA CHIMIA, vol. 15, no. 7, 1989, pages 908 - 921 *
T. BALASHOVA ET AL: "Conformation of the complex of human interleukin-2 fragment (66-72) with the anti-interleukin-2 monoclonal antibody", BIOORGANITCHESKAJA CHIMIA, vol. 19, no. 1, January 1993 (1993-01-01), pages 21 - 32 *
T.A. BALASHOVA ET AL: "Proton NMR study of the complexes formed by monoclonal antibody against human interleukin-2 and its Fab-fragment with synthetic peptides,the fragments of interleukin-2", BIOORGANITCHESKAJA CHIMIA, vol. 17, no. 11, 1991, pages 1470 - 1486 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000011028A3 (fr) * 1998-08-21 2000-07-06 Yeda Res & Dev Peptides anti-inflammatoires derives d'il-2 et analogues correspondants
US6906170B1 (en) 1998-08-21 2005-06-14 Yeda Research And Development Co. Ltd. Anti-inflammatory peptides derived from IL-2 and analogues thereof

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AU6977994A (en) 1995-01-17

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