Peptide T and related peptides in the treatment of inflammation, including multiple sclerosis

The present invention relates, broadly, to the treatment or prevention of inflammation, whether caused by bacteria, viruses and/or other infective agents, opportunities infections (which may be consequent on an immunodepressed state, for example resulting from cancer or therapy, particularly cytotoxic drug therapy or radiotherapy) autoimmunity or otherwise. In particular embodiments, the invention relates to the prevention or treatment of neurodegenerative or demyelinating diseases such as HTLV-1-associated myelopathy (HAM), multiple sclerosis (MS) and symptoms or diseases in humans which are associated with chronic immune activation. The invention also relates to pharmaceutical compositions useful in such treatment and/or prevention and to certain active peptides per se.
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 which can lead to death. Associated with the injection of LPS is the extensive production of tumour necrosis factor alpha (TNF-.alpha.). Mice injected with recombinant human TNF develop piloerection of the hair (ruffling), diarrhoea and a withdrawn and unkempt appearance, followed by death if sufficient amounts are given.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 haemoconcentration and biphasic changes in blood glucose concentration were also observed.
Histopathology of such rats revealed severe leukostatsis in the lungs, haemorraphic necrosis in the adrenals, pancreas and other organs and tubular necrosis of the kidneys. All of these changes were prevented if the animals were pretreated with a neutralizing monoclonal antibody against TNF.
The massive accumulation of neutrophilis in the lungs of TNF-treated animals reflects the activation of neutrophils by TNF. TNF causes neutrophil degranulation, respiratory burst as well as enhanced neutrophil 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 is a product of activated macrophages and is produced in response to infection and malignancy. It was first discovered in LPS-treated mice as a serum factor which caused the haemorraghic necrosis of transplanted tumours in murine models and was cytotoxic for tumour cells in culture (Carswell et al, 1975 PNAS 72, 3666; Helson et al, 1975, Nature 258, 731). Cachexia, which is characteristic of chronic exposure to TNF, is a common symptom of advanced malignancy and severe infection. It is characterised by abnormal lipid metabolism with hypertriglyceridaemia, abnormal protein and glucose metabolism and body wasting. Chronic administration of TNF and IL-1 in mice, rats and/or humans 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, Moldawer et al, Am. J. Physiol., 254 G450-G456, 1988; Fong et al, Am. J. Physiol. 256 R659-R665 (1989); McCarthy et al, Am. J. Clin. Nutr. 42 1179-1182, 1982). TNF levels have been measured in patients with cancer and chronic disease associated with cachexia. The results are inconclusive since large differences in TNF levels have been reported. These may have been explicable by the short half-life of TNF (6 minutes), differences in TNF serum binding protein or true differences in TNF levels in chronic disease states.
TNF-.alpha. and IL-1, 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. TNF 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, TNF has been shown to be involved in the pathogenesis of graft-versus-host disease in skin and gut following allogenic marrow transplantation.
Administration of a rabbit anti-murine TNF antibody was shown to prevent the histological changes associated with graft-versus-host disease and to reduce mortality (Piquet et al, 1987, J. Exp. Med. 166, 1220). 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).
Multiple sclerosis (MS) is generally considered by many authorities to be a chronic inflammatory disease.
Both MS and HTLV-1 associated myelopathy (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 fibres.
Multiple sclerosis (MS) is a chronics 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. The disease remains essentially untreatable.
MS has always been regarded as a disease of the temperate zones and has a prevalence in the northern United States, Canada and Europe of 1:1000. The disease has a gender predilection of 1.5:1 (female:male).
MS usually affects multiple areas of white matter in the central nervous system (CNS), most frequently, the periventricular white matter, brainstem, spinal cord and the optic nerves. The primary process destroys myelin sheaths and eventually kills oligodendrocytes creating the characteristic plaque of MS.
The early development of the plaque is characterised by the development of perivascular inflammation followed by the migration of lymphocytes, plasma cells and macrophages into the lesion. This is followed by astrocyte gliosis and the attempts of remyelination by oligodendrocytes. The plaque is surrounded by lymphocytes.
Although the aetiology of MS is still unknown, the focus of research efforts that have led to plausible hypotheses have been those of immune dysregulation including autoimmunity and genetic predisposition, both of which may play a role in the actual development of disease.
Multiple immunological abnormalities are reproducibly found in patients in the acute stage of the disease. The synthesis of immunoglobulins, although normal in the periphery, is increased in the central nervous system and the antibodies produced have a characteristic banding pattern. The antigenic specificity of these antibodies is not known and it is unclear whether they have a role to play in the progression of disease.
Various stressors known to activate the immune system such as viral infection or surgery can also produce an exacerbation of MS. Other activators such as .delta.-interferon produce similar effects when administered. In addition, immunosuppressive anti-inflammatory therapy with corticosteroids for example, can produce modest remission or at least palliation for short periods of time, although this therapy is controversial.
Lymphocyte reactivity against two neuronal antigens, myelin basic protein and proteolipid has been demonstrated. Although not proven, this activity would form the basis for an autoimmune response against neuronal tissue.
The discovery of the neurotropic capacity of HTLV-1 in patients from Martinique with tropical spastic paraparesis (TSP) and in Japan with chronic myelopathy, has demonstrated HTLV-1 as the common aetiologic agent of these diseases. It has subsequently been shown that the neurologic manifestations of HTLV-1 infection are the same despite the varied geographic regions in which they are described.
The neurological signs of this chronic retroviral infection including slowly progressive spastic paraparesis with spastic bladder and minimal sensory deficits result from involvement of the pyramidal tracts in a bilateral and symmetrical fashion predominately at the thoracic level of the spinal cord.
The peripheral nervous system has been shown to be involved, resulting in slowing of nerve conduction velocities in the lower limbs. Systemic manifestations of HTLV-1 in patients with HTLV-1 myelopathy have been described and include inflammatory involvement of the lungs, skin, eyes and striated muscle producing a myositis. In addition, patients experience profound fatigue similar to MS. The clinical manifestations of the disease are very similar to MS and are frequently confused with the latter.
There are at least four possible pathogenetic mechanisms whereby HTLV-1 can involve the CNS to produce HAM. These may include a slow virus infection, a cell-mediated immune response and a predominantly humoral immune mediated mechanism and the development of an autoimmune phenonmenon. The slowly progressive course supports the hypothesis of a slow virus infection. The finding of peri-vascular cuffing in post mortem specimens as well as transiently favourable response to steroids supports the hypothesis that an inflammatory immune reaction, probably a result of viral infection, is responsible for the development of HAM.
These two diseases have many similarities and dissimilarities, both clinical and neurological. Both diseases are a form of demyelinating disease whereby the myelin sheath of the nervous system is destroyed by one of many mechanisms common to both diseases and also peculiar to either of the diseases. MS is a multi-faceted disease in that it can be both a central nervous system disease which can include a myelopathy. Conversely, HTLV-1-associated myelopathy is predominantly a myelopathy which can occasionally demonstrate central nervous system effects. Furthermore, MS can affect the peripheral nervous system in wants that arecommon in HTLV-1.
Myelopathy, as already mentioned in being a disorder of the spinal cord, can have many different aetiologies from MS and HAM. Various forms of myelopathy, most of which are mediated by inflammation, include the following:
neurosyphillis;
B.sub.12 or folate deficiency;
sarcoidosis;
transverse myelitis;
arachnoiditis;
cervical spondylitis;
motor neuron disease;
neurofibromatosis;
spinal cord compression from tumour, disc or
arthritis;
lupus erythematosus of the spinal cord; and
viral encephalomyelitis.
Chronic inflammation or, as more commonly known, chronic immune system activation occurs in response to persistent antigen whose origin may be exogenous or endogenous or may resule from an autoimmune state. Such chronic inflammation results in local tissue destruction and depending upon the type of inflammation can result in systemic effects due to the sustained production of inflammatory mediators. Such inflammatory mediators include the cytokines which are soluble mediators produced by activated lymphocytes and macrophages and effect cellular communication and physiological response. Chronic immune activation can occur as a result of infectious diseases, such as chronic fatigue syndrome or toxic shock syndrome or through autoimmune mechanisms resulting in such conditions as rheumatoid arthritis, inflammatory bowel disease and variants such as graft versus host interactions.
The immune response to antigen may be divided into four overlapping phases: initiation (antigen presentation), amplification (cell activation), effector and regulation (Roitt et al, "Immunology", Gower Medical Publ. London, EK, 1989; "Basic and Clinical Immunology", Stites et al. Eds, Appleton and Lange, Norwalk, Conn., 1991). Briefly antigen is phagocytosed by antigen presenting cells (APC) which must express major histocompatibility (MHC) Class II molecules on their surface. In this respect, cells of the macrophage/monocyte lineage (CD4 positive) and B cells (CD4 negative) may act as APC. Following phagocytosis, antigen is processed intracytoplasmically and expressed on the surface as antigenic fragments in association with MHC-II molecules. The combination of antigen/MHC-II induces the activation of T helper cells (CD4 positive) in an antigen-specific manner and primes them to receive a second antigen non-specific activating signal. Activated T helper cells then induce the activation of effector T cells (cytotoxic lymphocytes, CD4 negative) and B cells which produce antibody. Effector cells and molecules facilitate the elimination of antigen by a variety of antigen specific and non-specific mechanisms that may result in host tissue damage if effector mechanisms are expressed inappropriately by deposition of antigen or immune complexes on host tissues, responding to self-antigens or as a result of a prolonged (chronic) immune response. Regulation of the immune response via removal of antigen, active suppression or idiotypic regulation limits the normal immune response to a duration of one to three weeks.
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 aetiology characterised by a diverse set of signs and symptoms including severe fatigue, post-exertional malaise, headaches, night sweats, myalgia, ataxia, low grade fever and lymphadenopathy (CDWR 1-3: Joncas J H, Welcome and Introduction in: Proceedings of a Workshop: Chronic Fatigue Syndrome, Can. Dis. Weekly Report vol. 17S1E, January 1991 pages 1-3).
Although the origin of CFS is unknown its symptoms are consistent with over-production of cytokines (Landay et al, Lancet 338 707-712 (1991)). CFS-like symptoms have been observed following the therapeutic administration of interferons (IFNs) (Lloyd et al. Med. J. Aust. 151 122-124 (1989); Lever et al, Lancet 2 101 (1988); Moworay et al, Br. Med. Bull. 47 886-894 (1991)) and interleukin-2 (IL-2) (Cheney et al, Annal. Intern. Med. 110 321 (1989)). In a trial of IFN-.alpha. in patients with CFS, the drug exacerbated the condition further supporting a cytokine-mediated pathogenesis (McBride et al, Br. Med. Bull. 47 895-907 (1991)). 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); Shepperd The Practitioner 233 41-46 (1989)) as well as IL-6 (Chao et al, J. Infect. Dis. 162 1412 (1990)). In addition neopterin, a marker of macrophage activation (Chao et al, J. Infect. Dis. 162 1412 (1990)) and the IFN-associated enzyme 2'-5'-oligoadenylate synthetase (Klimas et al, J. Clin. Microbiol. 28 1403-1410 (1990)) are both increased in CFS as are other markers of macrophage activation such as ICAM-1 and LFA-1 (Gupta et al, Scand. J. Immunol. 33 319-327 (1991)). CFS-associated anergy to skin test antigens (Johnson et al. FASEB J. 5 2706-2712 (1991)), reduction in lymphosyte response to mitogens (Klimas et al, J. Clin. Microbiol. 28 1403-1410 (1990)) and soluble antigens (Gupta et al, Scand. J. Immunol. 33 319-327 (1991)) are consistent with macrophage dysfunction (Prieto et al, Scand. J. Immunol. 30 13-20 (1989)) and may be explained by an autocrine exhaustion of immunocompetent cells by chronic activation resulting in immunodysregulation (CDWR 1-3: Joncas J H, Welcome and Introduction in: Proceedings of a Workshop: Chronic Fatigue Syndrome, Can. Dis. Weekly Report vol. 17S1E, January 1991, pages 49-50).
CFS and the acquired immune deficiency syndrome (AIDS, see below) share many symptoms (Miller et al, Neurology 41 1603-1607 (1991)) and laboratory findings (Gupta et al, Scand. J. Immunol. 33 319-327 (1991)); and one study has demonstrated an association between infection with the HIV related retrovirus HTLV-II and CFS (Gupta et al, Scand. J. Immunol. 33 319-327 (1991)).
There is no accepted drug therapy for CFS. There have been anecdotal reports of beneficial effects following administration of amantidine, monoamine oxidase inhibitors (ie phenelzine), fatty acid supplements (McBride et al, Br. Med. Bull. 47 895-907 (1991)) and 5-hydroxytryptophan (caruso et al, J. Int. Med. Res. 18 201-209 (1990)) among others, but controlled studies have not demonstrated efficacy.
Toxic shock syndrome (TSS) is produced by a Staphylococcus aureus enterotoxin, toxic shock syndrome toxin-1 (TSST-1). TSST-1 belongs to a family of staphylococcal enterotoxins which are mitogenic for T cells expressing particular V.beta. genes (Kappler st al, Science 244 811-813 (1989)). As a result of their non-specific mitogenicity, staphylococcal enterotoxins can induce the proliferation of up to 20% of T-cells and have been called "superantigens" (Johnson et al, Sci. Am. 266 92-101 (1992)). Macrophages are required to present TSST-1 to T-cells (Poindexter et al. J. Infect. Dis. 151 65-72 (1985)); however, like other staphylococcal enterotoxins, TSST-1 does not require antigen processing for T-cell activation (Pontzer et al. Proc. Natl. Acad. 88 125-128 (1991)). The native molecule binds outside the antigen binding cleft of MHC Class II molecules to a non-polymorphic region of the .beta. chain (Fraser Nature 339 221-223 (1989)); Johnson et al, FASEB J. 5 2706-2712 (1991)).
The symptoms of TSS (such as fever, rash, hypotension, nausea, vomiting and diarrhoea) are consistent with over-activation of the immune system (Johnson et al, Sci. Em. 266 92-101 (1992)) and over-production of cytokines (Ikejima et al, J. Clin. Invest. 73 1312-1320 (1984); Micussan et al, Immunology 58 203-208 (1986)). These symptoms have been reproduced in animal models by the administration of tumour necrosis factor (TNF) (Miethke et al, J. Exp. Med. 175 91-98 (1992)). Massive immune activitation of this nature could lead to exhaustion of immunocompetent cells and may explain the immunosuppression associated with enterotoxin shock (Langford et al, Infect. Immun. 22 62-68 (1978)) and in vitro enterotoxin-induced T-cell anergy (O'hehir et al. Immunol. Lett. 30 165-170 (1991)).
Therapy of TSS involves immediate replacement of lost fluid to counter hypovolaemia. If the patient fails to respond to anti-staphylococcal antibiotics then steroid therapy (ie methylprednisone) may be required for those in severe shock (Todd, Drugs 39 856-861 (1990)).
Rheumatoid arthritic (Marrow et al, "Autoimmune Rheumatic Disease", Blackwell Scientific Publ., Oxford, UK, Chapter 4, pp148-207 (1987)) is a disease characterised by chronic inflammation and erosion of joints that may affect up to 3% of the population, including children. Symptons of rheumatoid arthritis include morning stiffness, swelling and pain upon motion in at least one joint and joint swelling. Non-specific symptoms including lethargy, anorexia and weakness as well as fever and lymphadenopathy (characteristic of immune activation) may antedate joint involvement. Extra-articular manifestations of rheumatcid arthritis include vasculitis, cataracts, uveitis, interstitial fibrosis, pericarditis and myocarditis, peripheral neuropathy, myeloid deposits, chronic anaemia and subcutaneous and pulmonary nodules.
Genetic factors and infectious agents including bacteria, fungi, mycoplasmas and viruses have been associated with the development of rheumatoid arthritis. Mild rheumatoid arthritis may be treated with non-steroidal anti-inflammatory drugs while severe cases require systemic corticosteroids, anti-metabolites or cytotoxic agents. Experimentally, anti-CD4 monoclonal antibodies have been used to treat rheumatoid arthritis (Horneff et al, Cytokine 3 266-267 (1991); Horneff et al, Arth. Rheum. 34 129-140 (1991) and Shoenfeld et al, Clin. Exp. Rheum. 9 663-673 (1991)).
Inflammatory bowel disease (IBD) is a chronic inflammatory condition that fulfils some of the criteria of an autoimmune disease (Snook, Gut 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 (MacDermott, Mt. Sinai J. Med. 57 273-278 (1990)). As a result of chronic activiation of immunocompetent cells, IL-1, IL-6 (Starter, Immunol. Res. 10 465-471 (1991). Fiocchi, Immunol. Res. 10 239-246 (1991)) and TNF-.alpha.(MacDermott, Mt. Sinai J. Med. 57 273-278 (1990)) are all elevated in IBD patients.
Drugs used to treat IBD include anti-inflammatory agents such as sulphasalazine (5-ASA), corticosteroids, cyclosporin A and azathioprine (Hanauer, Scand. J. Gastroenterol. 25 (Suppl. 175) 97-106 (1990); Peppercorn, Annal. Intern. Med. 112 50-60 (1990)). Experimentally, anti-CD4 monoclonal antibodies have been used to successfully treat ulcerative colitis (Emmrich et al. Lancet 338 570-571 (1991)).
While a host may react against a genetically incompatible graft producing a host-versus-graft response, an immunocompetent graft (such as bone marrow or intestinal tissue) may react against the host resulting in graft-versus-host disease. These reactions are mediated by allogenic responses directed against a foreign MHC molecule and are mimicked in vitro by the mixed lymphocyte reaction (MLR). Graft/host interactions result in chronic inflammation surrounding the grafted tissue with an increase in markers of immune activation such as are seen in AIDS (Grant, Immunol. Today 12 171-172 (1991)). Treatment of the graft/host interactions currently include either azathioprine, cyclosporin A or methylprednisone and, more recently, rapamycin (Stepkowski et al. Transplantation 53 258-264 (1992); Huber et al, Bibliotheca Cardiologica, 43 103-110 (1988)). Monoclonal antibodies specific for CD3 (Wissing et al, Clin. Exp. Immunol. 83 333-337 (1991)) and CD4 (Reinke et al, Lancet 338 702-703 (1991)) have been used experimentally to inhibit graft/host reactions.
The present invention deals with the identification of a group of peptides that alleviates the inflammatory response in number of diseases. These include: autoimmune disease; organ transplantation; neoplasia; viral, bacterial, fungal or other infections; and, in particular, any disease wherein infection can manifest in an opportunistic fashion, eg during cytotoxic or radiation therapy or in any situation where an immunodepressed state exists. Peptides useful in the present invention do not necessarily interfere directly with the pathogenic mechanisms of the disease-causing component. As will be described below in the experimental data, the mechanism whereby these peptides can alleviate the symptoms of these diseases is dependent on their capability of modulating the production and effect of cytokines produced by activated cells of the immune system. The modulation of cytokines may not be limited to TNF but may also be valid for a whole range of interleukins, for example from interleukin-1 to interleukin-10. The data presented are at present not direct evidence, but rather a powerful indirecit model. Thus, the model uses one of the most powerful inflammatory compounds known, LPS, which binds to receptors on neutrophils, monocytes and macrophages; these cells consequently become activated and start production of IL-1 and TNF, among other cytokines, thus staring the inflammatory cascade. One parameter used to measure this effect of LPS is the concentration of blood glucose, whcih normally decreases on exposure to TNF or LPS. From what is known in the literature about the mechanism of Peptide T at a cellular level, it is therefore highly surprising that Peptide T and its analogues are able significantly to reduce the negative effects of LPS. LPS normally combines with LPS-Binding
Protein (LBP) and exerts its dramatic effect through the CD14 receptor. In the literature up to date, the peptides useful in this invention have only been connected to the CD4 receptor, which is not believed to be involved in the primary inflammatory response associated with cytokines, such as TNF, or LPS.
More specifically, it has been discovered that a particular group of peptides, particularly those within the group having at least 5 amino acid residues, are very effective agents useful in the treatment, inter alia, MS and HAM, and are likely to be useful in treating other myelopathies, most of which have similar disease mechanisms.
From the above discussion, it is apparent that many symptoms and diseases are associated with chronic inflammation; however, several of these diseases appear to involve different mechanisms. It is therefore important that particular compounds have been found which are useful in treating symptoms and diseases associated with chronic inflammation where it appears that these compounds interact in some manner with CD4 receptors of immune system cells. The compounds relate, as indicated above, to Peptide T and its various derivatives. It was originally thought that such compounds had no effect on the immune system other than being very useful in blocking attachment of HIV virus to CD4 receptor cells (Ruff et al, IV international Conference on AIDS, Stockholm June 1988).
Originally, many of the peptides useful in the invention were described as being effective in the prevention of infection and replication of HIV in vitro; see EP-A-024390, EP-A-0249394 and WO-A-8809338, all of which are incorporated by reference to the maximum extent allowed by law, as are all other documents referred to in this specification. All compounds disclosed in these specifications are useful for the present invention. The original peptide has its basic point of origin in the octapeptide Ala--Ser--Thr--Thr--Asn--Tyr--Thr. It is called Peptide T because 50% of the amino acid residues are threonines. This peptide has been identified from a subregion of the human immune deficiency virus (HIV) external glycoprotein molecule gp120, which is responsible for binding to any cell carrying the CD4 molecule and, in particular, helper lymphocytes, microglial cells in the CNS, monocytes and dendritic cells. Binding occurs via specific attachment of gp120to the CD4 molecule. Treating individuals infected with HIV with this peptide and its derivatives, which are described below, consequently has the effect of potentially inhibiting binding of the whole virus or the neurotoxic gp120 molecule to the cell receptor CD4. In this way, the cell is protected from infection, and so the virus, being unable to replicate, is destroyed by the immune defence.
According to a first aspect of the present invention, there is provided the use of a linear or cyclic peptide of General Formula 1:
I-A-B-C-D-E-F-G-H-II (General Formula1)(SEQ ID NO:1)
wherein
A is Ala, Gly, Val, Ser, Thr or absent,
B is Ala, Gly, Val, Ser, Thr or absent,
C is Ser, Thr or absent,
D is Ser, Thr, Asn, Glu, Arg, Ile, Leu or absent,
E is Ser, Thr, Asp or absent,
F is Thr, Ser, Asn, Arg, Gln, Lys, Trp or absent,
G is Tyr or absent,
H is Thr, Arg, Gly, Met, Met(O), Cys, Thr, Gly or absent,
I is Cys or absent,
II is Cys or absent,
at least one of the amino acids optionally being substituted by a monomeric or polymeric carbohydrate or derivative thereof, such substitution being accomplished through hydroxyl and/or amino and/or amido groups of the amino acids,
and wherein the peptide comprises at least four amino acid residues,
or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating or preventing inflammation.
Each of the amino acids referred to in General Formula 1 may be in the L-- or D-- stereoisomeric configuration and candidates for H may be esterified or amidated. The peptide comprises at least 4 amino acids.
Tetra-, penta-, hexa-, hepta-, octa- and nona-peptides useful in the invention are all of the peptides chosen from the sequence:
I-A-B-C-D-E-F-G-H-II
by deleting residues, for example, one at a time, from either the carboxyl or amino terminal, or from within the sequence.
It is appreciated that peptides having the core sequence of Thr--Thr--Asn--Tyr--Thr-- may have at both ends additional amino acid residues, some of which are represented by General Formula 2:
X--Ser--Thr--Thr--Thr--Asn--Tyr--Y General Formula 2)(SEQ ID NO:2)
wherein X is an amino acid terminal residue selected from Ala and D--Ala and Y is a carboxy terminal residue selected from Thr and Thr-amide.
A particular preferred peptide of the group of peptides has the aforementioned core sequence of --Thr--Thr--Asn--Tyr--Thr--. These peptides of the above General Formula 2, and in particular a variant Peptide T of the formula --Ser--Thr--Thr--Asn--Tyr--, were found to be very useful in inhibiting binding of the human immunodeficiency virus (HIV) to human cells by blocking receptor sites on the cell surfaces. The term Peptide T is used throughout the specification to reference, unless the context otherwise requires, peptides of General Formula 2 which all include the core peptide sequence. It is therefore intended that Peptide T encompass all of the compounds of General Formula 2 where it is understood that all such compounds are variants of the normally understood octapeptide T, also referred to as prototype Peptide T, of the particular formula D--Ala--Ser--Thr--Thr--Thr--Asn--Tyr--Thr-amide. (SEQ ID NO:3).
The invention may be useful in both clinical (human) and veterinary medicine. The invention therefore has application in a method for treating or preventing inflammation, the method comprising administering to a human or other animal subject, for example on a repeated basis, a peptide of General Formula 1. The peptide will generally be administered in an effective, non-toxic amount or in such an amount that strikes an acceptable balance between efficacy and toxicity, having regard to the circumstances of the case.
Preferred peptides useful in the invention have, as their active portion, an amino acid sequence of the formula:
--Thr--Thr--Asn--Tyr--Thr-- (SEQ ID NO:4)
These peptides, while being useful for all prophylactic and therapeutic utilities within the invention, are particularly preferred for the prevention or treatment of MS and HAM and for the prevention or treatment of symptoms or diseases, in humans or other animals, associated with chronic immune activation, chronic inflammation and chronic autoimmune disease.
Most preferred peptides useful in the invention, then, are the following:
1. D--Ala--Ser--Thr--Thr--Thr--Asn--Tyr--Thr--NH.sub.2 (prototype Peptide T) (SEQ ID NO:3)
2. Ala--Ser--Thr--Thr--Thr--Asn--Tyr--Thr (SEQ ID NO:5)
3. D--Ala--Ser--Thr--Thr--Thr--Asn--Tyr--Thr (SEQ ID NO:3)
4. D--Ala--Ala--Ser--Ser--Ser--Asn--Tyr--Met (SEQ ID NO:6)
5. Thr--Asp--Asn--Tyr--Thr (SEQ ID NO:7)
6. Thr--Thr--Ser--Tyr--Thr (SEQ ID NO:8)
7. Thr--Thr--Asn--Tyr--Thr (SEQ ID NO:4)
8. D--Thr--Thr--Tyr--D--Thr (SEQ ID NO:9)
9. D--Ala--Ser--D--Thr--Thr--D--Thr--Asn--Tyr--D--Thr--NH.sub.2 (SEQ ID NO:1)
10. D--Ser--Ser--D--Thr--Thr--D--Thr--Thr--Tyr--D--Thr--NH.sub.2 (SEQ ID NO:11)
Quite often it may be an advantage to have the amino terminal amino acid as a D-stereoisomer, to protect the molecule from degradation from aminopeptidases; alternatively or additionally, the carboxy terminal amino acid may be an amino acid amide to protect the molecule from degradation from carboxypeptidases. In this connection, compound 5, 6 and 7, listed above, include analogues with D--Thr as the amino terminal residue and/or an amide derivative at the carboxy terminal.
Furthermore, it should be understood that one more of the amino acids in the peptides may be substituted N-alkyl (eg (C.sub.1 -C.sub.4) alkyl) amino acids instead of primary amino acids; examples include methyl and ethyl. The hydroxyl group side chains of one or more of the amino acids (Ser, Thr, Tyr) may be derivatised into an ether or ester group. Any (optionally substituted) alkyl ester or ether may be formed, such as (C.sub.1 -C.sub.4) alkyl, aryl or aryl (C.sub.1 -C.sub.4) alkyl esters, ethers, thioesters and thioethers, for example phenylester, benzylether or thiophenol ethylester. The presently preferred ethers are methyl, ethyl and propyl ethers and presently preferred esters are methyl, ethyl and propyl esters.
The hydroxyl side chains of the amino acids Ser, Thr and/or Tyr and the amido groups of the amino acids Asn and/or Gln may be substituted with different carbohydrates or derivatives of carbohydrates. Carbohydrate derivatives may be as discussed above.
Linear peptides useful in this invention may be prepared by any suitable process, such as conventional solid phase peptide synthetic techniques; see "Solid Phase Peptide Synthetic Techniques", 2nd ed, J. M. Stewart, J. D. Young, Pierce Chemical Company, 1984, ISBN: 0-935940-03-0. A frequently used solid phase method is the Merrifield technique. Another possibility is solution phase techniques. The preferred peptide, prototype Peptide T, is readily obtainable from Carlbiotech A/S, Copenhagen, Denmark.
Cyclic peptides useful in the invention may be prepared by know techniques, such as, for example, described in Y. Hamada in Tetrahedron Letters, 26 5155 (1985). Cyclic peptides may be established in the form of a disulphide bridge between two Cys residues and/or by reacting the carboxy terminal amino acid residue with the amino terminal residue and/or by reacting the amino terminal residue with for example the .gamma.-carboxyl group of Glu, when Glu is at position D.
Carbohydrate derivatives may be prepared by methods know in the art.
Certain peptide derivatives useful in the invention are new and themselves form another aspect of the invention according to which there is provided a linear or cyclic peptide of General Formula 1:
I-A-B-C-D-E-F-G-H-II (General Formula1)
wherein
A is Ala, Gly, Val, Ser, Thr or absent,
B is Ala, Gly, Val, Ser, Thr or absent,
C is Ser, Thr or absent,
D is Ser, Thr, Asn, Glu, Arg, Ile, Leu or absent,
E is Ser, Thr, Asp or absent,
F is Thr, Ser, Asn, Arg, Gln, Lys, Trp or absent,
G is Tyr or absent,
H is Thr, Arg, Gly, Met, Met(O), Cys, Thr, Gly or absent,
I is Cys or absent,
II is Cys or absent,
at least one of the amino acids being substituted by a monomeric or polymeric carbohydrate or derivative thereof, such substitution being accomplished through hydroxyl and/or amino and/or amido groups of the amino acids,
and wherein the peptide comprises at least four amino acid residues,
except for glycosylated prototype Peptide T,
or a pharmaceutically acceptable salt thereof.
Glycosylated Peptide T is disclosed in Urge et al., Biochem. Biophys. Res. Comms. 184(2) 1125-1132 (1992), published Apr. 30, 1992, but the utility of the present invention is neither disclosed nor suggested.
Preferred features of this aspect of the invention are as for the first aspect.
Peptides useful in the invention may be administered as a composition in conjunction with a pharmaceutically acceptable carrier.
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 anti-neoplstic 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 intr-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 preservative. The compositions may take such forms as suspensions, solutions, or emulsions or gels in oily or aqueous vehicles, an 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 (eg sterile, pyrogen-free water, normal saline or 5% dextrose) before use. The pharmaceutical compositions containing peptide(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. Peptide T obtainable from Carlbiotech A/S is usually formulated and packaged in a sterile manner in 5% dextrose solution in multi-dose vials. It will be appreciated that the peptide may be packaged in other carriers, such as saline. Preferably, the concentration of peptide in each dose is in the order of 8.5 mg/ml for subcutaneous injection in one ml doses.
The compositions are administered in therapeutically or prophylactic effective doses, ie 0.05-10000 mg of peptide per day, in particular 5-1000 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 composition, the daily dosage, as employed for treatment of adults of approximately 70 kg of body weight, will often range from 0.2 mg to 20 mg of active material which may be administered in the form of 1 to 4 doses over each day, such dosage ranges depending upon the route of administration and the condition of the patient.
Compositions as described above may be prepared by mixing or otherwise bringing into association the ingredients.
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.
The invention finds particular use in the prevention or treatment of MS, HAM and other inflammatory myelopathies (particularly those previously specifically mentioned) and/or symptoms or diseases in humans which are associated with chronic immune activation. More particularly, the invention is useful in treating chronic fatigue syndrome, 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.
In order to provide a guideline for the administration of and insight into the use of the peptides according to the invention, particularly the treatment of MS, myelopathies such as HAM and chronic inflammation, and the formulation of the compositions, the following is offered as a guide based on the extensive work already conducted in the use of peptide T for treating HIV infection.
Peptide T is an octapeptide homologous to a region of gp120, an HIV envelope glycoprotein, and to human vasoactive intestinal peptide (VIP). It was originally developed by Pert et al (EP-A-0249394), to block the binding of gp120 (an HIV envelope glycoprotein) and thus also block binding of HIV to CD4, the specific membrane-bound vial receptor, thereby blocking internalisation of the virus into the cell--a process necessary for viral replication. The CD4 molecule necessary for the entry of HIV into cells has been localised on the surface of lymphocytes, macrophages, microglial cells, neurons and numerous other cells. Binding of HIV to the CD4 receptor has been demonstrated to effect viral entry; and binding of free (non-viral related) gp120 has resulted in neuronal toxicity in both in vitro and in vivo studies.
The efficacy of Peptide T in reversing signs of HIV-induced dementia has been demonstrated in both the Peptide T Phase I clinical trial at the University of Southern California in Los Angeles and in the Phase II clinical trial at the Fenway Clinic in Boston. Both studies have demonstrated improvement in the HIV-induced neurocognitive impairment in patients with AIDS.
To date, the Peptide T/gp120/VIP homology has been used to explain at least two possible mechanisms of action of Peptide T. Firstly, that it competitively binds to CD4 (the known receptor for HIV) on human cell surfaces and competes with both HIV and gp120 for binding sites.
The binding of Peptide T and its analogues of General Formula 1, or more particularly General Formula 2, to CD4 could produce a blocking effect to prevent the binding of any other molecule capable of binding to that receptor; alternatively, or in addition, the binding of Peptide T to CD4 could induce a reaction similar to that caused by the endogenous ligand.
CD4 is the differentiation antigen that defines that T lymphocyte subgroup of helper/inducer cells, but it is also present on a wide variety of cells including neurons, activated macrophages and B cells. CD4 is the predominant receptor for HIV and was originally thought to be necessary for cellular infection. Using the monoclonal antibody OKT4, Pert et al (Proc. Natl. Sci. USA 83 9254-9258 (1986); Pert et al (Clin. Neuropharmacol. 9(4) S198 (1986)) demonstrated the presence of this antigen throughout the human CNS and showed that it is present in highest concentration in the dentate gyrus, hippocampus, amygdala and deep cortex. This distribution was found to be similar to that found in other higher mammals. Peptide T and similar analogues were found to inhibit the binding of radiolabelled gp120 to rat hippocampal membranes and to do so in 0.1 nM concentrations.
Using Peptide T and the same analogues, Pert et al (Proc. Natl. Sci. USA 83 9254-9258 (1986); Pert et al (Clin. Neuropharmacol. 9(4) S198 (1986)) were able to demonstrate a reduction in the detectable levels of HIV reverse transcriptase when these peptides were present in an assay of HIV infectivity. A ninefold reduction of reverse transcriptase took place at 100 nM concentrations of Peptide T.
Since gp120 is not identical in all isolated strains of HIV, a comparison was made with nine different HIV isolates Pert et al (Clin. Neuropharmacol. 9(4) S198 (1986)). Significant homology was found between the isolates examined and Peptide T when comparison was made with the core pentapeptide, (Ruff et al, FEBS Lett. 211 17-22 (1987); Brenneman et al, Nature, 335 639-642 (1988); Brenneman et al, Drug Dev. Res. 15 361-369 (1988); Komisaruk et al, Annals of the NY Acad. Sci. 527 650-654 (1988); Buzy et al, The Lancet 22 July, 226-227 (1989)). This comparison has now been extended to over twenty isolates.
The inhibition of gp120 and HIV binding to CD4, as well as the demonstration of reduced infectivity of HIV in the presence of Peptide T and its analogues, provides one possible mechanism of action to explain the clinical effects of Peptide T. In this regard, Peptide T in sufficient concentration may prevent new cellular infection with HIV. Initial research in this area was focused on the CNS for two reasons: a high concentration of CD4 molecules was found on neurons and one of the major effect of HIV infection is the development of neurocognitive dysfunction. These fats are particularly important given that Peptide T is transported from the blood to the brain by an active, saturable transport system, while its exit is by diffusion only, (Barrera et al, Brain Res. Bull. 19 629-633 (1987)).
Although it is well accepted that HIV can infect not only lymphocytes but also neurons, it is difficult to ascribe the neurologic dysfunction see in HIV patients to active CNS HIV infection, since only a small number of neurons are actively infected. It has been suggested that the neurological deficits seen in HIV infection may occur not only as a result of infection but also as a result of a viral "toxin", such as gp120. Brenneman et al, Nature 335 639-642 (1988)) found that purified gp120 from two isolates as well as recombinant gp120 produced significant neuronal cell death in cultures of mouse foetal hippocampal neurons. Neurotoxicity could be reduced by pretreatment with antibody to CD4 and was completely eliminated by VIP. Since mouse neurons cannon be infected with HIV, it is evident that neurotoxicity is gp120-induced and is not a result of viral entry or replication.
VIP (66,67,68,69: TDNYT) and the core peptide (61-65: TTNYT) share the homologous sequence that binds CD4, and that is also found in isolates of the much larger gp120. Peptide T, when used in the same mouse hippocampal neuronal culture system, completely, antagonised the gp120-induced neurotoxicity, Brenneman et al, Drug Dev. Res, 15 361-369 (1988)). In addition, CSF from a patient with AIDS dementia produced substantial neurotoxicity in this system (44-49% killing at 1:100,000 dilution). This effect was inhibited by Peptide T, Buzy et al, The Lancet, July 22, pp 226-227, (1989)). Normally gp120 is produced in vast excess of amounts required for viral replication; this excess gp120 may exert a neurotoxic effect far out of proportion to the number of microglial cells or neurons actively infected with HIV.
Peptide T may act as an agonist in addition to or even in the absence of its neuroprotective effects against viral infection and neurotoxicity. Direct agonist activity has been demonstrated in two ways. Ruff et al, (FEBS Lett. 211 17-22 (1987)) showed that Peptide T and two analogues were potent agonists of human monocyte chemotaxis. Their rank order potency as chemotactic agents corresponding to their relative ability to inhibit both gp120 binding and HIV T cell infectivity.
As a further demonstration of the agonist activity of Peptide T, both Peptide T and VIP exert their cellular effects via the regulation of protein kinase C: Zorn et al. The Endoc. Society. Abstract (1989)). Agonist activity of Peptide T is thus implied by the production of a transmembrane signal that can influence the regulation of protein kinase C. It has also been demonstrated in six individual experiments, as part of the present invention, that Peptide T can down-regulate the enzyme p56.sup.lck, a tyrosine kinase linked to the cytoplasmic portion of membrane-bound CD4, thus implying that the binding of Peptide T to the CD4 molecule can produce a transmembrane signal.
Further evidence of Peptide T's potential VIP-like agonist activity is provided by results from experimental testing of the hypothesis of Komisaruk et al, Annals of the NY Acad. Sci. 527 650-654 (1988)) that VIP released from pelvic nerve terminals into the spinal cord can produce analgesia. Knowing that naloxone-independent analgesia produced by administration of VIP to the periaqueductal grey matter in rats had been shown, the investigators administered VIP directly to rat spinal cord and measured the pain threshold to distal noxious sitmuli to test the hypothesis. Spinal administration of VIP produced analgesia as measured by the tail-flick latency response and the tail-shock induced vocalisation test by action on both opiate and non-opiate modulated pain pathways, Komisaruk et al, Annals of the NY Acad. Sci. 527 650-654 (1988)).
The existence of clinical benefits from the administration of Peptide T to humans has been suggested in all studies to date: in HIV disease, by the Pilot Swedish Study, the USC Phase I and Fenway/CRI studies, and the Toronto Western Hospital Compassionate Use Program involving 51 patients; in psoriasis and the other medical conditions, in case reports from Sweden (Marcusson, Lazega et al, 1989-9, and Marcusson and Wetterberg, 189-10) and in 8 patients with psoriasis or other medical conditions in Toronto.
Neurocognitive improvement found in HIV positive patients and improvement in constitutional symptoms in both HIV positive and HIV negative patients may well depend primarily on Peptide T's VIP-like neurotropic and agonist effects, as well as the anti-inflammatory and anti-TNF effects discussed below.
Not wishing to be bound by any particular theory, with respect to the use of these peptide with treatment of MS and HAM, and in view of the above guidelines and discussions in relation to the use of various peptides of General Formula 2 and their analogues in the treatment of HIV, it is hypothesised that there are numerous similarities of disease expression and potential similarities of disease etiology, Peptide T appears to act as an agonist and as a blocker of CD4-mediated immune function rather than as an antiviral drug. In investigations associated with the present invention, patients with non-HIV disease such as multiple sclerosis, HTLV-1 associated myelopathy, and psoriasis have all been treated with Peptide T.
Now that the effectiveness of the peptides of General Formula 1, and particularly those of General Formula 1, is shown, the following is suggested as a hypothesis as to why the compounds do work:
1) Both HAM and MS are chronic CNS inflammatory and demyelinating diseases as is HIV disease.
2) Both diseases have possible viral aetiologies; it is now generally accepted that HAM is caused by the retrovirus, HTLV-1, a virus in the same family as HIV; MS has also been suggested as a manifestation of HTLV-1 infection and the chronic fatigue syndrome has recently been linked to a number of possible viral infections both of DNA (eg HHV-6) and retroviral aetiologies.
3) The two diseases share a number of common symptoms, for example, fatigue, lack of balance and signs of autoimmune phenomena; it is worth while noting that HTLV-1 disease exhibits numerous signs of autoimmunity such that it may be expected that some retroviral diseases have a concomitant expression in autoimmune phenomena. One common theme among these diseases may be peripheral neuropathy which is bases on the process of demyelination.
4) The basis appears to be the common denominator of both demyelination whether it be in the central or peripheral nervous system and the common autoimmune manifestations in HAM, MS and HIV disease.
5) Demyelination is associated with inflammation of any aetiology and appears to be mediated at least in part by TNF.
Not wishing to be bound by any particular theory with respect to the use of peptides with the treatment of symptoms and diseases associated with chronic immune activation, and in view of the above guidelines and discussions in relation to the use of various peptides of General Formula 2 and their analogues in the treatment of HIV, it is hypothesised that there is an immunomodulatory effect of the Peptide T binding to CD4. Such effect is demonstrated in the following examples where Peptide T has been found to inhibit mitogen induced proliferation of peripheral blood mononuclear cells (PBMC) at picomolar and lower concentrations. We have found that Peptide T allowed PBMC to proliferate in response to mitogen, but at a reduced level compared to the growth of PBMC in its absence. Pre-incubation of PBMC with Peptide T for less than 30 minutes had no effect on mitogen stimulation. However, exposure of PBMC to Peptide T for 2 hours followed by washing of the cells before exposure to the mitogen resulted in inhibition of proliferation similar to that seen when cells are incubated in the presence of both Peptide T and mitogen. It was also found that Peptide T did not significantly affect the growth of PBMC cultured in the absence of mitogen. It is therefore thought that Peptide T is able to suppress the normal proliferative response of PBMC to non-CD4 associated proliferation signals.
Now that the effectiveness of the peptides of General Formula 1, and particularly those General Formula 2,in treating symptoms and diseases associated with chronic immune activations or chronic inflammation has been discovered, the following is suggested as hypothesis of the mechanism of action of Peptide T and, therefore, why the compounds useful in the invention are effective.
Peptide T binds to CD4. It has been established in the following tests that Peptide T inhibits mitogen and MLR induced lymphocyte proliferation. It is therefore thought that Peptide T may serve as an immunomodulatory drug which would down-regulate the enhanced immune response occuring in the chronic presence of antigen or for other reasons mentioned and hence reduce chronic inflammation.
Underlying all these utilities runs the common theme of inflammation.
In accordance with various embodiments of this invention and in view of the above guidelines gained from the use of peptides of General Formula 1, and particularly those of General Formula 2, in the treatment of HIV, similar doses of Peptide T and its analogues can be administered to humans or other animals for purposes of treating inflammation.
The invention will now be illustrated by the following non-limiting examples. The examples refer to the accompanying drawings, in which:
FIG. 1 is referred to in Example 6 and shows the effect of Peptide T on TNF-induced tissue factor expression on human umbilical vein endothelial cells.
FIG. 2a, 2b and 2c relate to Example 17 and show that Peptide T was able to inhibit lymphoproliferation in response to mitogens. 10.sup.5 PBMC were cultured in the presence of PWM (2a), Con A (2b) or PHA (2c) with dilutions of Peptide T as follows: A: no Peptide T; B: 10.sup.-5 M; C: 10.sup.-7 M, D: 10.sup.-9 M; E: 10.sup.-11 M; F: 10.sup.-13 M; G: 10.sup.-15 M; H: 10.sup.-17 M. Cultures were incubated for 5 days, pulsed with .sup.3 H-thymidine and the amount of incorporated radioactivity converted into proliferation index as described in Example 17. Asterisk (*) indicates the difference in proliferation; = is statistically insignificant (p>0.05).
FIG. 3 relates to Example 17 and shows that Con A-induced lymphocyte proliferation in the presence of Peptide T parallels, but at a reduced rate, growth in the absence of Con A. 10.sup.5 PBMC were cultured in the presence of Con A for 8 days. Cultures were pulsed and harvested daily and the degree of lymphoproliferation expressed as counts per minute (CPM) of incorporated .sup.3 H-thymidine. Peptide T (PT, 10.sup.-9 M), was added concurrently with Con A.
FIG. 4 relates to Example 18 . In the presence of non-mitogen stimuli such as recall antigens (measles, mumps, rubella vaccine: MMR), superantigens such as Staphylococcal enterotoxins SEB and TSST-1, and antibodies to CD3 (anti-CD3) PBMC will proliferate as indicated by a proliferation index greater than 1 (-Peptide T). The presence of Peptide T (+Peptide T) added on the first day of this six day culture had no effect on the growth of PBMC in response to these stimuli.
FIG. 5 also relates to Example 18. When PBMC from one person (A) are cultured in the presence of PBMC from another person (B) they will proliferate [(A+B(P-PT)] as a result of recognising foreign antigens (alloantigens) on the cell surface. In the presence of Peptide T [A+B(+PT)]alloresponsive cells are still able to proliferate.
FIG. 6 also relates to Example 18. Molt-4 is a spontaneously growing human malignant T cell line. In the presence of PBMC growth is inhibited due to killing of Molt-4 by PBMC (PBMC/Molt-4-PT). In the presence of Peptide T added on the first day of a four day culture, PBMC mediated killing of Molt-4 remained unchanged (PBMC/Molt-4+PT).
FIG. 7 relates to Example 19, in which is given the sequence of steps involved in this cytotoxicity assay. Activated macrophages are able to kill K562 target cells in the absence of Peptide T (noPT). If Peptide T is added to macrophage cultures concurrently with the activators (PTstep1) it is unable to affect cytotoxicity. If Peptide T is added to activated macrophage cultures concurrently with the target cells (PTstep2) cytotoxicity is inhibited. Altering the timing of addition of Peptide T does not alter the Peptide T- mediated inhibition of macrophage-mediated cytotoxicity.
FIG. 8, which also relates to Example 19, shows that the supernatant of macrophage cultures stimulated with LPS contain cytolytic substances capable of killing target K562 cells. Target cells are labelled with intracellular radioactivity that is released upon cell death. More radiolabel release (measured as counters per minute, cpm) indicates more killing. In the absence of Peptide T (A) more killing occurred than in its presence (B-F). As the concentration of Peptide T was dropped below 1.times.10.sup.-15 M cytolytic activity the degree of cytotoxicity returned to control (A) levels. The Peptide T concentrations were as follows:
A: no Peptide T
B: Peptide T, 10.sup.-5 M
C: Peptide T, 10.sup.-7 M
D: Peptide T, 10.sup.-9 M
E: Peptide T, 10.sup.-11 M
F: Peptide T, 10.sup.-13 M
G: Peptide T, 10.sup.-15 M
FIG. 9 is referred to in Example 20 and shows that Peptide T has a similar effect to anti-TNF in inhibiting the effects of TNF.
FIG. 10 is referred to in Example 21 and shows that Peptide T decreases serum TNF levels in HIV patients.
FIG. 11 is referred to in Example 23 and shows that Peptide T analogue 623 prolongs survival of sensitised mice administered a lethal dose of LPS.
FIG. 12 is referred to in Example 24 and shows that Peptide T inhibits activation of human neutrophils by TNF.
FIG. 13 is also referred to in Example 24 and shows that Peptide T analogue 505 inhibits TNF priming and activation of human neutrophils.
FIG. 14 is again referred to in Example 24 and shows that Peptide T analogue 505 inhibits LPS activation of human neutrophils.
FIG. 15 is referred to in Example 26 and shows that Peptide T reduces TNF-induced expression of tissue factor in HUVECs.

Examples 1 to 4 demonstrate the significant effects that the peptides useful in the invention have clinically on MS and HAM. The peptide used in each of Examples 1 to 4 is a variant Peptide T of General Formula 2 having he sequence D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-Amide.
Example 1
HTLV-1 MYELOPATHY
A 45 year old female was diagnosed as HTLV-1 seropositive after experiencing dysaesthesia of the feet, pain in the feet and legs, weakness of the legs and urinary frequency. Her neurological examination confirmed increased deep tendon reflexes, spasticity of the lower limbs and pyramidal weakness of the legs. The diagnosis was confirmed by somatosensory and auditory evoked potentials and HTLV-1 seropositivity. Therapy, consisting of 8.5 mg of Peptide T, was self-administered subcutaneously each morning. After two months of taking Peptide T, she stated that she was substantially improved and now able to lift both feet instead of dragging hem along the floor. In addition, she was able to work without a cane, could stand without the support of a walker or a cane, could climb stairs for the first time in two years and reported decreased burning pain in the feet to the knees and reported decreased urinary frequency and nocturia. Her most functionally debilitating symptom had been the painful peripheral neuropathy which had severely limited her mobility and had limited her social contacts and disturbed her sleep; she described the pain as severe to intolerable. The pain, previously refractory to known therapy, became manageable, tolerable and of minor significance to daily function.
After a subsequent two months of Peptide T therapy, which was her only new medication, she reported continual decrease in symptoms and continuing improved mobility. Her neurological examination remained unchanged other than her improved functional capacity, eg walking.
Although previously confined to her apartment, her improved post-treatment mobility enabled her to travel to her Caribbean home. During this trip she used her entire supply of drug and within five days noted progressive fatigue and became bed-bound with fatigue, weakness and pain. Within two weeks of stopping Peptide T. her symptoms essentially resolved within one month. On two subsequent occasions she terminated treatment, with similar recurrance of symptoms and similar resolution upon reinstitution of Peptide T therapy.
EXAMPLE 2
MULTIPLE SCLEROSIS
A 40 year old female was diagnosed as having MS after presenting with weakness, loss of balance, double vision, and paraesthesia in her right arm at age 23. She was treated with prednisone and later with physiotherapy, with little effect. Upon presentation, she remained unable to walk without a cane or a walker, showed depression, horizontal nystagmus, and weakness of hip flexors bilaterally, weakness of knee flexors and of dorsilflexors of the feet. Tone was increased in both lower limbs and she had bilateral ankle clonus. Deep tendon reflexes were 2+ in the arms, 3+in both knees and ankles and the plantar responses were extensor. She was treated with LIORESAL.TM. (baclofen) with some improvement; however, she experienced progressive worsening until she became wheelchair dependent and complained of weakness. spasticity, clonus, urinary incontinence and recurrent urinary tract infections. When she was evaluated her MRI brain scan showed findings consistent with multiple sclerosis.
She was started on Peptide T, 8.5 mg subcutaneously daily. She reported that many of her symptoms including spasticity, fine motor co-ordination, nocturia and urinary frequency, concentration, memory and emotional lability had greatly improved. She was assessed after five weeks of treatment and both reported and demonstrated functional improvement. After six months, she demonstrated sustained improvement in these symptoms. Her frequency of nocturia was reduced, fine motor control improved, stuttering decreased, her legs were less spastic and she could stand with minimal support. She showed marked symptomatic improvement in intellectual and motor functioning within a few weeks after starting treatment with Peptide T, such improvement being sustained over a 6 month period. Worsening of symptoms occurred when the patient stopped Peptide T for 3 weeks, but improved when she restarted the drug. The patient has remained stable rather than following her normal progressive downhill course for the months she has remained on Peptide T.
EXAMPLE 3
MULTIPLE SCLEROSIS
A 28 year old female was diagnosed with optic neuritis secondary to multiple sclerosis after presenting with numbness, impaired motor function, and blurred vision (20/300 bilaterally). Her somatic neurological examination was within normal limits. Minor episodes of numbness and impaired speech, balance and co-ordination had occurred over 6 months, as well as headache and loss of vision.
Peptide T therapy was started. Six days later a reassessment showed remarkably improved visual acuity (21/30) bilaterally). The patient noted functional improvement over the next week. A prior episode of optic neuritis took six months to recover (compatible with the natural history of MS) compared to this episode which recovered much more quickly than would be expected for an episode of MS-induced optic neuritis.
EXAMPLE 4
MULTIPLE SCLEROSIS
A 34 year old woman with her first episode of multiple sclerosis as manifest by optic neuritis and complete blindness in the right eye for 2 weeks, numbness in her right leg and then both legs. Her symptoms worsened and she was given prednisone and experienced some improvement. She had worsening of her ataxias with blurred vision and oscillopsia and was given prednisone 80 mg per day. An MRI scan showed multiple high intensity signals periventricularly as well as in the brain stem consistent with the diagnosis of MS. The ataxia and some visual problems seemed to improve on steroids. Another MRI scan about 16 months later showed extensive periventricular white matter disease with involvement of the corpus collosum. Findings were typical of demyelination consistent with MS. At that time the patient showed resting oscillatory lateral beating nystagmus and lateral nystagmus with bilateral gaze, an ataxia gait, a slight impairment on her heel shin test but good finger nose testing. The tone was increased in her lower limbs and she had non-sustained ankle clonus bilaterally. Her knee jerk was increased on the right compared to the left. Plantar responses were both upgoing. She had decreased position, vibration, light touch and cold sensation in the feet compared to the hands.
About 2 months later the patient was started on Peptide T 8.5 mg subcutaneously daily. Within 1 to 2 weeks she reported subjective improvement. She experienced improved feelings in her fingers, fine movements of her hands and improved cognitive function. When she stopped Peptide T therapy she noticed an increased fatigue and ataxia. She obtained minimal symptomatic improvement from prednisone 150 mg daily. After restarting Peptide T, she noted a remarkable improvement of her fatigue, ataxia and fine motor function.
EXAMPLE 5
MULTIPLE SCLEROSIS
A 56 year old female with a history of dizziness, ataxia, episodes of vertigo, impairment of motor function and generalised weakness of the legs was treated with dilantin and admitted to hospital. Subsequent examination showed vertical nystagmus, some saccadic smooth pursuit and an impaired tandem gait. She was reassessed about 2 years later for episodes of dizziness, and complaints of leg spasms. Her physical examination showed decreased visual fields with a partial left lateral hemianopsia in the lateral field and a right constricted field. She showed impaired tandem gait, decreased right hip flexion and strength and decreased deep tendon reflexes in the right brachioradialis compared to the left and in the right knee compared to the left. Plantar response was downgoing and she had upwards gaze vertical nystagmus as previously noted.
The patient began Peptide T 8.5 mg subcutaneously daily and within 10 days reported increased energy and intellectual function, and improved vision. When Peptide T therapy was stopped, she regressed symptomatically to her pre-drug status. Her physical examination at that time showed no change from her examination before Peptide T Therapy was begun.
Examples 5 to 7 show experimentally certain significant effects exhibited by peptides useful in the invention.
EXAMPLE 6
LPS-INDUCED INFLAMMATION
Normal balb/c mice (female, 12 weeks) were administered LPS (250 .mu.g, E. coli K-235, Sigma cat. no. L-2018) at time zero. One group of mice (50 animals) were then treated at 30 minutes intervals by i.p. injections of bovine serum albumin (BSA) (Sigma cat. no. 6793) dissolved in pyrogen-free, sterile, isotonic water (2.5 mg BSA per animal per injection, each injection containing 100 .mu.l.)
The second group of mice (50 animals) were treated at 30 minutes intervals by i.p. injections of Peptide T (Carlbiotech A/S batch 109401) dissolved pyrogen-free, sterile, isotonic water (2.5 mg Peptide T per animal per injection, each injection 100 .mu.l).
Glucose levels were determined on blood samples after 3 hours. Subjective parameters such as secretion from the eye, diarrhoea and general well being are indicated on a scale of (-) to (+++).
As can be seen from Table I, the difference in glucose levels between Peptide T-treated animals and BSA-(placebo) treated animals is highly significant. The subjective parameters also strongly indicate a general higher index of well being for animals treated with Peptide T.
TABLE I______________________________________ General Secretion wellDiarrhoea from eves being.sup.t 0 Blood Glucose .sup.t 0 .sup.t 0.sup.t 3 hrs .sup.t 0 .sup.3 3 hrs .sup.t 3 hrs .sup.t 3 hrs______________________________________Control - +++ 4.4 .+-. 0.7 2.0 .+-. 0.2 - +++ - +++(BSA)Peptide T - + 4.3 .+-. 0.6 2.4 .+-. 0.5 - + - +treated______________________________________ t-test = 98 P < 0.001 for peptide T treated group vs. control (BSAtreated group).
EXAMPLE 7
PEPTIDE T ANTAGONISES TNF
Stimulation of endothelial cells with TNF results in the induction of procoagulant activity through the expression of tissue factor activity and a concomitant reduction of the anticoagulant potential of the cells through reduced expression of thrombomodulin (Bevilacqua et al, Nawroth and Stern, J. Exp. Med. 163 740 (1986)). Septic shock is frequently accompanied by disseminated intravascular coagulation (DIC, Semeraro Acta Clinica Belgica 31 377 (1976)) and there is considerable evidence that tissue factor is an essential requirement for DIC to be induced by endotoxin (LPS). A monoclonal antibody against tissue factor has been shown to inhibit DIC induced by the administration of endotoxin (Warr et al, Blood, 75 1481 (1990)).
Human umbilical vein endothelial cells were cultured in M199 medium supplemented with 10% foetal calf serum, endothelial cell growth factor, penicillin, streptomycin and L-glutamine in 96-well dishes using a modification of the procedure of Bevilaqua et al, Proc. Natl. Acad. Sci. U.S.A. 83 4533 (1986)). TNF treatment of cultures (100 units.ml) was for 4 h at 37.degree. C. in the presence of growth medium, after which the cells were washed and fixed. Cultures were also treated with Peptide T+TNF or Peptide T alone. In these cultures Peptide T was at a concentration which ranged from 0.5 to 100 .mu.g/ml. Tissue factor expression by the endothelial cells treated with TNF with or without peptide T was determined in an ELISA using a monoclonal antibody which neutralises the activity of human tissue factor (Carlbiochem A/S). Bound anti-tissue factor antibody was detected using peroxidase-conjugated rabbit anti-mouse Ig and the substrate ABST. Colour intensity was determined by the optical density of each well at 405 nm.
The data obtained from triplicate cultures show that peptide T inhibited the expression of tissue factor on endothelial cells stimulated by 100 units/ml human recombinant TNF and are illustrated in FIG. 1.
EXAMPLE 8
PEPTIDE T INSTIGATES LPS-INDUCED MORTALITY
Mice bearing Meth A ascites tumours were given 100 .mu.g LPS subcutaneously with either bovine serum albumin (BSA) or peptide T analogue (504 or 505, 1 mg). The number of animals showing signs of LPS toxicity (diarrhoea, exudate from the eyes and general level of activity) in each group was recorded at 19, 24 and 41 hours after injection. At the completion of the experiment the number of surviving animals was recorded (see Table II below) and the survivors bled for later determination of TNF, IL-1 , IL-6 and RNI levels.
The sequence of the most effective analogue (505 is :
H-D-Ser-Ser-D-Thr-Thr-D-Thr-Thr-Tyr-D-Thr-NH.sub.2 (SEQ ID NO: 11)
and the sequence of the 504 is :
H-D-Ala-Ser-D-Thr-Thr-D-Thr-Asn-Tyr-D-Thr-NH.sub.2 (SEQ ID NO: 10).
TABLE II______________________________________EFFECTS OF PEPTIDE T ANALOGUES ON SURVIVAL OFLPS-TREATED TUMOUR BEARING MICE % mice with % mice with % mice surviving diarrhoea exudate atTreatment 41 hours at 19 hrs 19 hrs______________________________________LPS + BSA 60 100 100LPS + 505 100 40 20LPS + 504 100 40 60______________________________________
Examples 9 to 15 illustrate the effectiveness of peptides useful in the invention in relation to various diseases and/or symptoms associated with chronic inflammation.
EXAMPLE 9
INHIBITION OF MITOGEN INDUCED PROLIFERATION OF PBMC
PBMC (10.sup.5) were cultured in 200 .mu.l RPMI containing 10% heat inactivated foetal bovine serum, 2 mM L-glutamine and gentamycin 100 .mu.g/ml with mitogen and Peptide T added on the first day of culture. Growth was assessed in triplicate by .sup.3 H thymidine uptake following a 6 day incubation and expressed as a proliferation index (cpm in presence of mitogen .+-. Peptide T + cpm in absence of stimulation). A proliferation index>1.00 implies growth beyond that in media alone. At physiologically obtainable concentrations, Peptide T inhibits proliferation of normal (HIV seronegative) peripheral blood mononuclear cells (PBMC) without causing significant cytotoxicity as shown in Table III. this effect is long term in that Peptide T given on the first day of a six day culture will inhibit mitogen induced proliferation.
TABLE III______________________________________Proliferation Index .+-. SD.sup.1Peptide T.sup.2 Media.sup.3 PWM.sup.4 ConA.sup.4 PHA.sup.4______________________________________0 1.00 7.47 .+-. 0.50 4.55 .+-. 0.66 317.0 .+-. 41.010.sup.-5 1.14 .+-. 0.15 ND.sup.5 2.72 .+-. 1.20 149.0 .+-. 38.410.sup.-7 0.73 .+-. 0.16 4.47 .+-. 0.36 3.19 .+-. 0.06 123.6 .+-. 18.110.sup.-9 2.17 .+-. 1.10 4.69 .+-. 0.42 1.98 .+-. 0.04 78.6 .+-. 23.110.sup.-12 1.52 .+-. 0.15 4.15 .+-. 0.23 1.69 .+-. 0.48 103.8 .+-. 22.8______________________________________ .sup.1 SD standard deviation .sup.2 Concentration of peptide T in mol/l .sup.3 Growth in absence of mitogens .sup.4 Pokeweed mitogen (PWM), Concanavalin A (Con A) and phytohemagglutinin (PHA) were used at 25 .mu.g/ml .sup.5 ND not done
EXAMPLE 10
INHIBITION OF MLR
A two way MLR was performed using 10.sup.5 allogeneic PBMC (cpm of A=142 .+-.86, B=148 .+-.84) cocultured in 200 .mu.l of media as described in Table III.
Peptide T given on the first day of an MLR will inhibit allogeneic induced proliferation of PBMC (Table IV).
TABLE IV______________________________________Inhibition of MLR Peptide T.sup.1 cpm .+-. SD.sup.2______________________________________ 0 1365 .+-. 335 10.sup.-5 649 .+-. 263 10.sup.-7 534 .+-. 108 10.sup.-9 764 .+-. 77 10.sup.-11 681 .+-. 222 10.sup.-13 235 .+-. 4 10.sup.-15 371 .+-. 117 10.sup.-17 672 .+-. 261______________________________________ .sup.1 Concentration of peptide T in mol/l .sup.2 Counts per minute .+-. standard deviation
EXAMPLE 11
EFFECT OF PEPTIDE T ON ACTIVITY OF p56.sup.lck IN PBL BLASTS
PBL (peripheral blood leukocyte) blasts were prepared by incubating PBMC at 37.degree. C. with PHA (10 .mu.g/ml) in RPMI+10% FBS for 48 hours followed by addition of interleukin-2 and incubation for a subsequent 96 hours. Cells were washed free of IL-2 and incubated for 24 hours then washed free of FBS and incubated for 24 hours. PBL blasts remained either stimulated or were treated with OKT4A (anti-CD4) at 10 .mu.g/ml for 1 hour followed by rat anti-mouse Ig at 5 .mu.g/ml for 5 minutes to enhance lck activity. PBL blasts were pretreated with Peptide T (10.sup.-9 M) for 2 hours, then washed extensively before anti-CD4 stimulation. Following immunoprecipitation with anti-lck-Protein A conjugates and incubation (37.degree. C. 10 minutes) with .sup.32 P-ATP, p56.sup.lck autophosphorylation was determined by autoradiography and used as a measure of p56.sup.lck activity.
Peptide T will inhibit the PHA-induced activation and anti-CD4 dependent enhancement of p56.sup.lck activity as we have determined (Table V) by an lck specific tyrosine kinase assay. p56.sup.lck is a CD4 associated tyrosine kinase involved in T cell activation and transmembrane signalling (Janeway C.A. Ann. Re. Immunol. 10 645-674 (1992)). In lieu of a T cell receptor associated signal, cross-linking of CD4 by monoclonal anti-CD4 inhibits T cell activation (Burgess et al, Eur. J. Immunol. 21 1663-1668 (1991)), possibly by activating p56.sup.lck including autophosphoryllation of the inhibitory Tyr 505 (Abraham et al, Nature 350 62-66 (1991); Abraham et al, Cancer Invest. 9 455-463 (1991)). Peptide T is able to inhibit p56.sup.lck activation (Table V) thus achieving the same result as anti-CD4 (inhibition of T cell activation) although by a different mechanism.
TABLE V______________________________________EFFECT OF PEPTIDE T ON ACTIVITY OFp56.sup.LCK IN PB < BLASTSUnstimulated Anti-CD4-P.T..sup.1 +P.T. -P.T. +P.T.______________________________________+++.sup.2 + ++++ ++______________________________________ .sup.1 P.T. means Peptide T .sup.2 ++++ highly active + slightly active
It is known that anti-CD4 antibodies will inhibit mitogen-induced as well as and antigen-induced in vitro proliferation of PBMC, lymphokine release, T helper function and MLR (Bank et al, J. Exp. Med. 162 1294-1303 (1985)). There are two possible mechanisms of anti-CD4 inhibition of T cell function.
1) Anti-CD4 antibodies cause steric hinderance of the CD4/MHC II interaction that occurs during T cell activation. Peptide T would probably not share this effect since it is derived from a sequence of gp120 known to bind to CD4 and the binding site of gp120 on the CD4 molecule is distant from the MHC II binding site (Fleury et al (1991)).
2)) Cross-linking of CD4 by multivalent anti-CD4 antibodies, in lieu of TcR/CD3 stimulation sends a negative signal to the T cell (Bank et al, J. Exp. Med. 162 1294-1303 (1985)). Peptide T is monovalent, hence cannot cross-link CD4 receptors; therefore, its mechanism of inhibition of T cell activation must be distinct from that of anti-CD4. Treatment of inflammatory conditions with mouse anti-CD4 has resulted in a number of undesirable side effects including urticaria, chills, fever and tremors that occasionally require medical intervention. In addition, following administration of anti-CD4 there has been reported a reduction in CD4 cell numbers, a reduction in proliferation to mitogens and antigens and a reduction in delayed hypersensitivity reactions, a measure of cell mediated immune function (Horneff et al, Cytokine 3 266-267 (1991); Horneff et al, Arth. Rheum. 34 129-140(1991); Wofsey et al, Semin. Immuno. 2 419-425 (1990)). Administration of anti-CD4 concurrently with antigen can inhibit both primary and secondary immune responses (Waldor N.K. Immunol. Ser. 45 573-586 (1990)). In addition, the use of xenogeneic (non-human) proteins as therapeutic agents is limited due to the potential formation of antibodies directed against the foreign protein which can mediate shock and type III hypersensitivity reactions upon repeated administration.
EXAMPLE 12
EFFECT OF PEPTIDE T ON ANTIGEN INDUCED PBMC PROLIFERATION
PBMC were incubated as described in relation to Table III in Example 9 in the presence of live, attenuated measles, mumps and rubella vaccine (Merck Sharp and Dohme, Canada) at a final dilution of 1:20. Peptide T was added either on day 1 of incubation or on a daily basis. Results are expressed as a proliferation index .+-. standard deviation.
TABLE VI______________________________________EFFECT OF PEPTIDE T ON ANTIGEN INDUCED PMBCPROLIFERATIONConc. Peptide T Proliferation Index .+-. S.D.(Mol/L) Day 1 Daily______________________________________0 3.26 .+-. 0.5510.sup.-7 4.02 .+-. 0.78 1.83 .+-. 0.3610.sup.-9 1.91 .+-. 0.46 0.98 .+-. 0.1610.sup.-11 5.35 .+-. 1.46 1.56 .+-. 0.1410.sup.-13 5.46 .+-. 1.37 1.38 .+-. 0.5110.sup.-15 5.56 .+-. 0.60 1.56 .+-. 0.6110.sup.-17 3.99 .+-. 0.18 1.60 .+-. 0.6010.sup.-19 6.15 .+-. 2.59 2.27 .+-. 1.09______________________________________
Although Peptide T can inhibit antigen-induced proliferation of PBMC when administered in vitro on a daily basis, we do not believe this will have any significant effect on the normal immune response when administered in vivo (as demonstrated by Goodwin et al V. International Conf. Aids, Montreal, July 1989 Abstract #WBP286). There are two possible explanations for this that are not mutually exclusive.
1. Macrophages may be more sensitive to Peptide T than are T cells, since T cells receive many more potential activating signals than the CD4/MHC II interaction. Thus macrophages may be down regulated by Peptide T administration on the first day of in vitro culture inhibiting their ability to present mitogens and antigens to T cells. B cells can act as APC in place of macrophages when presenting antigen, but not when presenting mitogens which are antigen non-specific (explaining the results of Table III). Resting B cells are resistant to Peptide T as they are CD4 negative. In the presence of a single dose of Peptide T, they could present antigen to T cells allowing proliferation of PBMC in response to antigen. When Peptide T is given on a daily basis in cultured T cells, which may be less sensitive to the effects of Peptide T, then macrophages may be inhibited. This would explain the results shown in Table 4. Daily administration of Peptide T in vivo, may not function as it does on a daily basis in vitro, since the half life of Peptide T in vivo is shorter than in vitro. In vitro serum supplements are heat inactivated to destroy proteolytic enzymes which actively degrade Peptide T in vivo. Thus daily administration of Peptide T in vivo would result in the inhibition of macrophage activity, but not T cell activity and would allow the continued expression of antigen driven immune responses. This would explain the efficacy of Peptide T in chronic inflammatory conditions such as rheumatoid arthritis (see below) where the characteristic inflammatory cell is the macrophage. Acute inflammation is characterised by neutrophil (CD4 negative) infiltrates and would not be affected by Peptide T administration. The requirement for antigen presentation (phagocytosis, degradation and presentation) in an antigen driven immune response results in a delay in T cell signalling. Hence Peptide T present on the first day of in vitro culture will not affect T cell signalling on subsequent days. Mitogen stimulation of PBMC proliferation (Table III) is an immediate event not requiring antigen processing and may be inhibited on a long term basis by concurrent Peptide T administration. Peptide T needs to be present concurrently with an activating signal to manifest its immunodepressive effects. During chronic inflammation or immune activation, all responding cells will be receiving proliferation signals all the time. Daily Peptide T administration in vivo will inhibit chronic immune activation. Since the half life of Peptide T is <1 hour (Ruff et al, Prog. Neuro-Psychopharmacol & Biol. Psychiat Vol. 15, pp 791-801 (1991)), those proliferation signals active during the first hour after Peptide T administration will be inhibited. since all cells responding to chronic activation signals respond all the time, they will be down regulated during this 1 hour period and remain down regulated on a long term basis. Due to the discrete timing of antigen presentation and the fact that not all epitopes of the antigen are presented at the same time (therefore not all T cells capable of responding are activated at the same time), there are 23 hours before the next injection of Peptide T in which to mount antigen specific immune responses. Although some antigen specific immune responses may be inhibited in the 60 minutes post Peptide T administration, the majority which are happening at discrete times, will be allowed to proceed.
In this fashion, Peptide T is able to inhibit chronic immune activation and inflammation while allowing the expression of antigen specific immune responses.
EXAMPLE 13
ARTHRITIS
A 67 year old man was treated with daily Peptide T (8.5 mg s.c.) for severe psoriasis refractory to all known treatment. In addition, he complained of painful arthritis of the knees which although present for over 10 years had progressed to the point of preventing him form participating in his normal daily activities for six months prior to his first visit. Although his psoriasis was only transiently improved by Peptide T, knee pain began to resolve three weeks after starting on the drug. The patient remained pain free for the three months he continued on Peptide T. He was still pain-free at his last follow up two months after discontinuing Peptide T and he had been able to resume daily physical activities. He remained pain free eight months after discontinuing Peptide T. The severity of his arthritis was such that, prior to Peptide T, he had been advised to have knee joint replacement. This dramatic relief from pain is extraordinarily rare in patients with erosive rheumatoid arthritis even when treated aggressively with conventional therapies. His only medication during this time was a non-steroidal anti-inflammatory drug which he had been taking for the previous two years.
EXAMPLE 14
ARTHRITIS
A 47 year old woman was diagnosed as having facet joint arthritis in 1977 with recurrences on a number of occasions the following year. A back education program and exercises resulted in remission until 1992. Following a compressive spinal injury in May 1992, the facet joint arthritis recurred manifest as intense hyperaesthesia of the left hip and thigh progressing within 8 hours to involve the entire upper leg, characteristic of previous episodes which usually took 3-4 weeks to resolve on bed rest and non-steroidal anti-inflammatory medication. The following day the pain intensified and was characterised as an intense burning sensation accompanied by severe pain in the lumbar region unaffected by Toradol.RTM. or Naprosyn.RTM. at full dose. The patient was bedridden the following day and sleep disturbance was severe through the following 48 hours. Five days after the episode the patient was given the MOS questionnaire for chronic disease and chronic back pain with an unweighted average score of 40.77 out of 100. Later that day, Peptide T was given (10 mg s.c.) with diminution of the hyperaesthesia and facilitated sleep within 12 hours. Oral medicatins were discontinued. By the following day, the hyperaesthesia had resolved, sleep was normal and only very mild pain was present in the paraspinal muscles. Peptide T was given for the subsequent 2 days with total relief of pain and improving MOS scores of T1 and 87.4 (this score is comparable to a population with no acute or chronic illness). Peptide T was continued for a total of 10 days. On discontinuation, the hyperaesthesia returned and Naprosyn and Toradol were reinstituted with no noticeable effect for 2 days. Peptide T was resumed as the only medication for the next four days with complete relief of pain and hyperaesthesia and reinstitution of normal sleep habits. On discontinuation of Peptide T 5 days later, no recurrence of the symptoms occurred and no oral medication was required.
EXAMPLE 15
CHRONIC FATIGUE SYNDROME
A 45 year old woman with CFS presented with severe fatigue requiring frequence naps, headache, tinnitus, episodic diarrhoea, depression, lightheadedness and a reduction in auditory working memory. She was started on daily Peptide T (8.5 mg s.c.) and noticed improvement within one week. After two months she reported that 50% of her day was normal. Her fatigue had reduced and remaining fatigue was associated only with increased activity possible only after starting Peptide T. her memory improved and headaches became less frequent. Three months after starting on the drug she was still improving. She could walk for extended periods for the first time in two years and the frequency of her headaches was reduced almost to zero. After five months on the drug, her fatigue had resolved, she experienced no lightheadedness and was gardening, caring for her children and driving distances again.
EXAMPLE 16
CHRONIC FATIGUE SYNDROME
A 32 year old, EBV+ woman presented with a four month history of severe fatigue that prevented her from working more than 11/2hours per day. A putative diagnosis of post-infectious neuromyasthenia was made based on fatigue, decreased concentration, slowness of thought, myalgias and sore throat. Within five days of Peptide T administration (8.5 mg daily s.c.) her fatigue had remarkably decreased and had fully resolved within one month to the point that she was able to resume work fulltime and attend the gym three times per week. She remained on Peptide T for 35 days and she has remained asymptomatic for six months. The Patient Symptom Inventory (a self rating instrument which measures symptom severity on a scale of 0 to 4) was administered before Peptide T was begun and after 30 days of treatment revealed marked improvement in all symptoms:
______________________________________ Day 0 Day 30______________________________________Fatigue 0 3Stamina 0 2Concentration 2 3Slowness of thought 1 3Difficulty word finding 2 4______________________________________
Peptide T appears to act as an anti-inflammatory agent. Peptide T would therefore be a novel non-steroidal anti-inflammatory drug (NSAID). Traditional NSAIDs, such as aspirin and ibuprofen, have two potential mechanisms of action: inhibition of cyclooxygenase reducing prostaglandin production and inhibition of neutrophil function (Altman R.D. Arth. Rheum. 19 (Suppl. 2) 1-5 (1990)); Vane et al, Postgrad. Med J. 66(Suppl. 4) S2-S17 (1990)). It had been reported that inhibition of cyclooxygenase will enhance (rather than suppress mitogen driven PBMC proliferation (Vane et al, Int. J. Immunopharmacol. 5 107-114 (1983)) and neutrophils (CD4negative) would be resistant to Peptide T; therefore, Peptide T may have a unique mechanism of anti-inflammatory action.
The anti-inflammatory azathioprine inhibits lymphocyte proliferation by inhibiting DNA and RNA synthesis. The proliferation of both B and T lymphocytes are inhibited by the action of azathiporine (Briggs J.D. Immunol. Lett. 29 89-94 (1991)). Since B cells are CD4 negative and can function as APC in the presence of Peptide T (see above), Peptide T must have a mechanism of action distinct from that of azathiporine.
Cyclosporine is an anti-inflammatory drug that inhibits lymphocyte proliferation by inhibiting IL-2 and IFN .gamma. gene transcription by binding to and inactivating cyclophilin, an isomerase required for signal transduction (Halloran et al, Clin. Biochem. 24 3-7 (1991)). While Peptide T may also affect signal transduction (Table 3), it does not have the severe side effects associated with cyclosporine therapy. Cyclosporine induced side effects include renal tubular atrophy, nephrotoxicity, hypertension, hirsutism, gingival hypertrophy, tremors, convulsions and paresthesia (Briggs J.D. Immunol. Lett. 29 89-94 (1991)). In the experience of the present invention, no significant side effects have been attributable to Peptide T therapy.
EXAMPLE 17
PEPTIDE T INHIBITS MITOGEN-INDUCED LYMPHOPROLIFERATION
Concanavalin A (Con A), pokeweed mitogen, (PWM) and phytohaemagglutin-P (PHA-P) were obtained from Sigma Chemical Co. (St. Louis, Mo., U.S.A.). Peptide T was synthesised by Carlbiotech Ltd (Copenhagen, Denmark) and supplied as the chloride salt at 8.5 mg/ml in 0.9% benzyl alcohol as a preservative and was stored at 4.degree. C. Peptide T was diluted to working solutions in culture medium (RPMI). Dilute solutions of Peptide T were prepared fresh from stock Peptide T for each experiment. .sup.3 H-thymidine (5 Ci/mmol) was obtained from Amersham.
Isolation of PBMC: Whole blood from healthy, adult volunteers was drawn into heparin and peripheral blood mononuclear cells (PBMC) isolated by density gradient centrifugation on HISTOPAQUE.TM. (Sigma Chemical Co). PBMC were removed from the interface, washed three times in RPMI containing gentamycin (100 .mu.g/ml) and resuspended to 1.times.10.sup.6 PBMC/ml in RPMI containing 10% foetal bovine serum and gentamycin. 10.sup.5 PBMC were cultured in the presence of 100 .mu.l of mitogen suspension in 96 well plates. The final concentration of mitogen was: PWM 25 .mu.g/ml; PHA 12.5 .mu.g/ml and Con A 6.25 .mu.g/ml. 10 .mu.l of Peptide T working solution (or RPMI as negative control) was added and cell cultures incubated for a total of 5 days in a humid atmosphere, 5% CO.sub.2, 37.degree. C. Cultures were then pulsed with .sup.3 H-thymidine (in RPMI) 18-24 hours prior to harvesting. The amount of incorporated .sup.3 H-thymidine was determined using a BECKMAN.TM. LS8000 liquid scintillation counter and proliferation expressed as either counts per minute (cpm) or as a proliferation index (PI). ##EQU1##
a PI of 2 indicates that cells have grown to twice their resting (unstimulated) number.
All experiments were performed in triplicate. Results reported are characteristic of replicated experiments.
Production of Tumor Necrosis Factor .alpha. (TNF.alpha.): 5.times.10.sup.6 PBMC/ml were incubated in the presence of Con A (6.25 .mu.g/ml) for 48 hours in a humid atmosphere, 5% CO.sub.2, 37.degree. C. Peptide T (10.sup.-9 M) was added at t=0 and 24 hours. At the end of incubation, SEPHADEX.TM. G25 (Pharmacia) was added (10 mg/ml) and incubated for 10 minutes to remove mitogen. PBMC and SEPHADEX were pelleted by centrifugation and the supernatant tested for levels of TNF.alpha. using a TNF.alpha. radioimmune assay kit from Amersham. Samples were analysed in duplicate.
Statistical Analyses: Data were analysed by a two sample ANOVA test using 95% confidence limits such that a value of p.ltoreq.0.05 was considered a significant difference.
TABLE VII______________________________________1 .times. 10.sup.5 PBMC were cultured in the presenceof mitogens with Peptide T (1 .times. 10.sup.-9 M) addedconcurrently with mitogens (Day 1) or on subsequentdays as indicated. Cells were cultured for a totalof 6 days. Results are expressed as proliferationindex .+-. standard deviation PWM Con A PHA______________________________________no Peptide T 4.29 .+-. 0.25 21.0 .+-. 5.7 197 .+-. 3Peptide T:Day 1 3.13 .+-. 0.21 6.2 .+-. 0.5 148 .+-. 13Day 2 3.20 .+-. 0.30 6.0 .+-. 1.1 101 .+-. 7Day 3 2.65 .+-. 0.17 9.9 .+-. 0.1 148 .+-. 7Day 4 3.22 .+-. 0.28 9.5 .+-. 1.6 131 .+-. 10______________________________________
Results
When added on the first day of culture, Peptide T was able to inhibit lymphoproliferation in response to PWM (FIG. 2a), Con A (FIG. 2b) and PHA (FIG. 2c). Peptide T was able to inhibit PHA stimulated PBMC cultures at a concentration of 1.times.10.sup.-13 M. Cultures stimulated with Con A or PWM were inhibited by as little as 1.times.10.sup.-15 M Peptide T. Peptide T reaches peak plasma concentrations of 5.5.times.10.sup.-8 M following administration in vivo; therefore, Peptide T mediated immunomodulation would be active at concentrations of the drug readily attainable in plasma and CSF. The culture medium, in which Peptide T was diluted, had no effect on lymphoproliferation. At the most effective concentration of Peptide T used, lymphoproliferation in response to PHA was inhibited 26%, PWM 38 and Con A 36%.
Inhibition of lymphoproliferation could be a result of cytotoxicity, a delay in initiation of proliferation, a reduction in growth rate or any combination of the above
When the Con A-induced proliferation of PBMC was measured daily, during the growth period (day 2-4) lymphocyte proliferation in the presence of Peptide T paralleled, but at a reduced rate, growth in the absence of the drug (FIG. 3). In the absence of mitogen, Peptide T had negligible effects on PBMC. Therefore, Peptide T was not cytotoxic for responding lymphocytes nor did it delay the onset of proliferation. Peptide T allowed cells to grow, but at a reduced rate.
Peptide T was able to inhibit mitogen induced lymphoproliferation when added to PBMC after they had been exposed to mitogens (Table VII). This effect was present even when Peptide T was added three days after exposure of PBMC to mitogen. Peptide T mediated inhibition of lymphoproliferation was most pronounced when added to cultures that had been stimulated with Con A.
In addition to inhibition of lymphoproliferation, the presence of Peptide T reduced the amount of TNF.alpha. secreted into the supernatant (SN) of a 48 hour Con A stimulated culture of PBMC (Table VIII). Neither Peptide T nor RPMI had any reactivity in the RIA for TNF.alpha.. In the absence of Con A, no TNF.alpha. was detected in the culture supernatant.
TABLE VIII______________________________________PBMC (5 .times. 10.sup.6 PBMC/ml) were cultured for48 hours in the presence or absence of Con A(6.25 .mu.g/ml) = Peptide T (10.sup.-9 M, added concurrentlywith mitogen). Levels of TNF.alpha. in culturesupernatants were determined as described. TNF.alpha. (fmol/100 ul)______________________________________RPMI + Peptide T <1PBMC + RPMI + Peptide T <1PBMC + Con A 69.0 .+-. 6.9PBMC + Con A + Peptide T 47.0 .+-. 0.1______________________________________
EXAMPLE 18
PEPTIDE T INHIBITS NON-MITOGEN-INDUCED LYMPHOPROLIFERATION
In contrast to mitogens which induce lymphocyte proliferation signals by ligating MO/MAC and T cells via carbohydrate moieties on a variety of cell surface receptors, non-mitogen stimulated lymphoproliferation mediated by recall antigens, superantigens, antibodies to CD3 (anti-CD3) and allogeneic MHC molecules interact solely with the T cell antigen receptor/CD3 complex (TcR/CD3). In this respect, non-mitogen stimuli are more specific than mitogens.
Only those lymphocytes with TcR complementary to processed antigen epitopes will respond to recall antigens. Typically this represents a small fraction of the total lymphocyte pool. Reactions to allogeneic MHC, characterised by the mixed lymphocyte reaction (MLR) is mediated by recognition of foreign MHC Class II molecules by the TCR of responding cells (Adv. Immunol. 31 271 (1981)). Approximately 10% of T cells are alloreactive (Lancet 339 824 (1992)). Superantigens such as Staphylococcal enterotoxins A and B (SEA and SEB) and toxic shock syndrome toxin-1 (TEST-1) stimulate growth of approximately 20% of the T cell pool (Sci. Am. 266 92 (1992)) by interaction with defined V.sub..beta. segments of the TcR (Science 244 811 (1989)). Anti-CD3 is able to stimulate the growth of 100% of T cells independent of accessory cell function by ligating TcR/CD3 complexes directly.
Since mitogens represent an artificial, non-specific form of lymphocyte activation, the effect of Peptide T on lymphoproliferation in response to recall antigens, anti-CD3, superantigens and MLR was assessed. In addition, T cell effector functions and the growth of spontaneously proliferating malignant cell lines were examined in the presence of Peptide T.
Unless otherwise stated, experimental conditions were generally as described in Example 17.
Peptide T does not inhibit non-mitogen induced lymphoproliferation
When added on the first day of a six day culture, Peptide T was able to inhibit Con A-induced lymphoproliferation, but had negligible effects on lymphocytes responding to recall antigen (measles, mumps, rubella vaccine, MMR), antibodies to CD3 or the superantigens SEB and TSST-1 (FIG. 4). Similarly, Peptide T did not affect the growth of lymphocytes in a mixed lymphocyte reaction (FIG. 5).
Molt-4 is a human T lymphoma cell line, p56.sup.lck negative, and Jurkat is a human cell line, p56.sup.lck positive, that will grow spontaneously in the absence of exogenous stimuli. As the results of Table IX demonstrate, Peptide T did not significantly affect the growth of either Molt-4 or Jurkat cell lines.
TABLE IX______________________________________Effect of Peptide T on Growth of Cell LinesTwo spontaneously growing human malignant T cell lines(Jurkat and Molt-4) were grown in the absence (Media-P.T.) or presence (Media + P.T.) of Peptide T added on thefirst day of culture. Growth was determined bymeasurement of incorporated .sup.3 H-thymidine; counters perminute (cpm) were not converted into a proliferationindex. A higher cpm represents greater cell growth.Media - P.T. (CPM .+-. S.D.) Media + P.T. (CPM .+-. S.D.)______________________________________JurkatDay 1 7445 .+-. 261 8345 .+-. 131Day 2 14728 .+-. 1349 17441 .+-. 1360Day 3 41203 .+-. 6127 40002 .+-. 1572Day 4 50930 .+-. 1030 49852 .+-. 1011Day 5 80046 .+-. 1072 79748 .+-. 8474Day 6 109201 .+-. 8805 114967 .+-. 7342Day 8 113667 .+-. 7266 109144 .+-. 14052Molt - 4Day 1 6182 .+-. 1296 6639 .+-. 726Day 2 13111 .+-. 370 15001 .+-. 1509Day 3 29175 .+-. 5594 32401 .+-. 850Day 4 39920 .+-. 3407 39839 .+-. 10740Day 5 54686 .+-. 1472 50269 .+-. 5436Day 6 56824 .+-. 3580 57570 .+-. 4968Day 8 10835 .+-. 264 19510 .+-. 4217______________________________________
Peptide T does not inhibit PBMC mediated killing of Molt-4 cells
When PBMC are mixed with allogeneic Molt-4 cells the growth of Molt-4 is inhibited (FIG. 6). Alloreactive PBMC recognise Molt-4 as foreign and generate cytotoxic effector cells that kill Molt-4. Molt-4 is not a cytotoxic cell line and can not generate alloreactive effector cells. When Peptide T is added to this reaction, PBMC-mediated inhibition of Molt-4 proliferation remains unaffected (FIG. 6) suggesting that Peptide T does not affect the generation of cytotoxic effector functions. It is not possible to distinguish growth of alloreactive T cells from the growth of Molt-4 survivors since neither the growth of alloreactive T cells (FIG. 5) nor the growth of Molt-4 (Table IX) would be affected by Peptide T.
EXAMPLE 19
PEPTIDE T's EFFECT ON MACROPHAGE AND THE FUNCTION
Cells of the monocyte/macrophage lineage (MO/MAC) are long lived, non-proliferating cells that characterise chronic inflammatory reactions. MO/MAC function as non-specific phagocytes, antigen presenting cells (initiating antigen specific immune responses), as immunoregulatory cells and cytotoxic effector cells. MO/MAC generate a variety of intracellular and extracelluar cytotoxic effector molecules including reactive oxygen intermediates, lysosomal enzymes and tumour necrosis factor alpha (TNF).
MO/MAC have been implicated in the pathogenesis of HIV disease. All MO/MAC express CD4 (J. Immunol. Meth. 135 59 (1990)) and are therefore capable of being infected with HIV. It has even been suggested that MO/MAC maybe the first cell to be infected with HIV during the infection process (Annal. N.Y. Acad. Sci. 616 1 (1990)). MO/MAC are not as susceptible to the cytopathic effects of HIV as are CD4 positive T cells (TIPS 12 28 (1991)) and may be responsible for the establishment of a persistent or latent HIV infection. MO/MAC may be responsible for transmission of HIV into the CNS where it comprises the primary cell type infected with HIV (Compr. Ther. 17 57 (1991)). In the CNS, MO/MAC may mediate neurological damage by releasing neurotoxic gp120 (Science 248 364 (1990)), tat (J. Virol. 65 961 (1991)) or MO/MAC derived neurotoxins (Science 250 1593 (1990)). It is interesting to note that activated MO/MAC have also been implicated in the pathogenesis of other neurological disorders such as multiple sclerosis (Acta. Neuropathol. 80 208 (1990)); Pathol. Immunopathol. Res. 6 241 (1987); Ann. Rev. Immunol. 10 153 (1992 )) and Alzheimer's disease (Eur. Neurol. 28 30 (1988)).
HIV-infected MO/MAC exhibit accessory cell dysfunction (Clin. Immunol. Immunopathol. 59 436 (1991); J. Immunol. 146 2297 (1991)). In addition, HIV induces MO/MAC activation as indicated by enhanced levels of neopterin, a marker of T cell dependent MO/MAC activation, and enhanced levels of monokines (TNF, IL-1 and IL-6).
In vitro, MO/MAC may be activated by IFN-.gamma. and lipopolysaccharide (LPS) which initiate at leat four distinct intracellular signals including a calcium flux (Immunol. Today 10 33 (1989)). The production of TNF may be initiated by IFN activation of MO/MAC but requires LPS to trigger its release (J. Exp. Med. 175 405 (1992)).
A role for TNF in HIV disease has been suggested based on the observations that macrophages from AIDS patients release greater amounts of TNF than those from normal controls (Am. Rev. Respir. Dis. 144 195 (1991)), and increased levels of TNF have been associated with progression from latent HIV infection to AIDS (Am. J. Med. 85 289 (1988)). Potential effects of TNF contributing to HIV pathogenesis include TNF mediated cachexia (New Eng. J. Med 327 329 (1992)); J. Nutr. 122 749 (1992)), autocrine/paracrine enhancement of HIV replication (Immunol. Today 11 176 (1990)), hyperactivation of the immune system (Biotherapy 3 127 (1991)), inhibition of zidovudine (AZT), ddI and ddC (VIII Int'l Conf. AIDS Abst.#PoA 2326 (1992)); J. Intern. Med. 228 549 (1990)), expansion of autoreactive CD4 specific CTL (VIII Int'l Conf. AIDS Abst.#PoA 2365 (1992)), damage to myelin and oligodendrocytes (AIDS 5 1405 (1991)), cytopenia (Am. Rev. Respir. Dis. 144 195 (1991)) and apoptosis (Immunol. Ser. 56 315 (1992)).
TNF has also been implicated in the pathogenesis of multiple sclerosis. TNF is cytotoxic for myelin producing oligodendrocytes (Ann. Rev. Immunol. 10 153 (1992)) and can cause myelin damage directly by inducing blebbing and swelling of the myelin sheath (Annal. Neurol. 23 339 (1988)).
Methods
Macrophage Cytotoxicity Assay
The MO/MAC cytotoxicity assay is a two step assay. After isolation of adherent cells (mainly MO/MAC) from PBMC, MO/MAC are activated by lymphokines (including IFN gamma and LPS. Subsequent to washing which removes activators, .sup.3 H-thymidine labelled K562 target cells were added to activated MO/MAC and incubated for 3 days. The degree of cytotoxicity was proportional to the amount of radioactive label released into the culture supernatant, is shown in the following scheme:
______________________________________ PBMC.vertline.Adherent Cells (1 hours, 37.degree. C. on plastic).vertline.Step 1: Activation (8 hour): cytokines LPS (1 ug/ml).vertline.Step 2: Effector (3 days): .sup.3 H-T K562.vertline.Release of .sup.3 H-T______________________________________
MO/MAC Supernatant Cytotoxicity
Supernatant (SN) of activated MO/MAC were collected from monolayers of PBMC derived adherent cells that had been exposed to LPS for 23 hours. This SN was tested for cytolytic activity against .sup.3 H-thymidine labelled K562 cells in a 48 hour, 37.degree. C. cytotoxicity assay. SN were stored at 4.degree. C. overnight or stored frozen at -20.degree. C. for longer periods before using.
Results
Peptide T inhibits MO/MAC mediated cytotoxicity
The presence of Peptide T at different stages of the cytotoxicity assay demonstrated that while Peptide T did not affect the ability of resting MO/MAC to be activated, it was able to inhibit the cytotoxicity of activated MO/MAC (FIG. 7). Whether Peptide T was added concurrently with target cells or every day of the culture did not affect the drug's activity.
Peptide T treatment reduces neopterin levels but Peptide T-dependent decreases in serum neopterin were not associated with decreases in serum IL-1.
When sera from patients with Peptide T dependent decreases in elevated neopterin levels were tested for IL-1, no correlation between the two could be demonstrated (Table X). However, neopterin levels were elevated prior to Peptide T administration whereas serum IL-1 levels were normal.
TABLE X______________________________________Serum IL-1 and Neopterin ConcentrationSerum was collected from patients with HIV disease and storedat - 20.degree. C. Neopterin and IL-1 concentrations weredetermined by RIA. Pre- and post-Peptide T values of IL-1 andneopterin were determined for the same serumsamples [Neopterin](nM)PA- [IL-1](fmol/100 .mu.l) Pre-TIENT pre-Peptide T post-Peptide T Peptide T post-Peptide T______________________________________norm. 4 .+-. 0.03 N/A ND NDBL 4.1 .+-. 0.03 4.7 .+-. 0.05 ND NDCJ 3.7 .+-. 0.03 4.2 .+-. 0.02 ND NDTS 4.2 .+-. 0.14 4.2 .+-. 0.26 ND NDLB 3.9 .+-. 0.13 4.35 .+-. 0.003 ND NDBG 4.35 .+-. 0.06 4.6 .+-. 0.02 52.0 .+-. 4.0 16.85 .+-. 0.05BC 3.5 .+-. 0.14 3.8 .+-. 0.12 47.3 .+-. 0.8 32.5 .+-. 3.5JM 5.23 .+-. 0.17 4.2 .+-. 0.07 27.8 .+-. 0.8 19.75 .+-. 0.25RH 4.1 .+-. 0.01 4.6 .+-. 0.11 13.15 .+-. 2.85 8.9 .+-. 0.6______________________________________
Peptide T inhibits cytotoxicity of MO/MAC culture supernatants.
The ability of MO/MAC culture SN to lyse target K562 cells was determined in the presence of Peptide T. MO/MAC culture SN was used at a final dilution of 1:6 in the culture well. As shown in FIG. 8, Peptide T was able to block MO/MAC SN mediated lysis of K562 even when added at 1.times.10.sup.-13 M.
EXAMPLE 20
PEPTIDE T HAS A SIMILAR EFFECT TO ANTI-TINE IN INHIBITING THE EFFECTS OF TNF
10.sup.5 PBMC were cultured in the presence of PWM, Con A or PHA (see Example 17). Peptide T (10.sup.-9 M), anti-TNF MoAb #47 at a 1:200 dilution (antiTNF) or a combination of them both were added to cultures concurrently with mitogens. Following a five day incubation, cultures were pulsed with .sup.3 H-thymidine 18-24 hours prior to harvesting. Incorporated .sup.3 H-thymidine was converted to a proliferation index (see Example 17). Data were analysed by two sample ANOVA using 95% confidence limits.
EXAMPLE 21
PEPTIDE T DECREASES SERUM TNF LEVELS IN HIV PATIENTS
Serum levels of TNF were determined in patients with stage IV HIV disease pre- and post-Peptide T treatment. Patients had been receiving drug from 2 weeks to 11 months. Results are shown in FIG. 10. The increase in serum TNF levels in patient BG was associated with the development of an opportunistic infection.
EXAMPLE 22
PEPTIDE T PROLONGS SURVIVAL OF D-GALACTOSAMINE-SENSITIZED MICE IN A SEPTIC SHOCK MODEL
Fifty BALB/c/Swiss (1st cross) female mice approximately 8 weeks old were split into five groups and dosed as follows:
1. 16 mg D-galactosamine+20 .mu.g TNF+H.sub.2 O
2. 16 mg D-galactosamine+20 .mu.g TNF+MAb 47
3. 16 mg D-galactosamine+20 .mu.g TNF+100 .mu.g Peptide T
4. 16 mg D-galactosamine+20 .mu.g TNF+10 .mu.g Peptide T
5. 16 mg D-galactosamine+20 .mu.g TNF+1 .mu.g Peptide T
D-galactosamine sensitizes the mice to the effects of TNF in this experimental septic shock model. MAb 47 is a broad spectrum anti-TNF monoclonal antibody available from Peptide Technology Limited.
the protective effect of Peptide T was assessed after 12 hours. The results in the following Table XI were obtained.
TABLE XI______________________________________A B C D E F G H Alive Dose______________________________________1 0 0 0 0 0 0 0 0 0 TNF2 0 4 10 4 2 3 6 10 14 TNF + <An3 0 1 8 1 1 1 2 8 9 TNF + 100 .mu.g PT4 1 1 5 3 1 1 0 5 7 TNF + 10 .mu.g PT5 1 1 2 2 1 0 0 1 4 TNF + 1 .mu.g______________________________________ PT Parameters A Cold B Cool C Warm D Ruffled E Diarrhoea F Discharge from Eyes G Touch Sensitive H Activity
EXAMPLE 23
D-Thr-Thr-Tyr-D-Thr-NH.sub.2 PROLONGS SURVIVAL OF SENSITISED MICE ADMINISTERED A LETHAL DOSE OF LPS
Meth A ascites tumour cells were injected (10.sup.6 cells i.p.) into mice and allowed to grow. The Meth A tumour-bearing mice were thereby sensitised to the effects of TNF/LPS. The mice were administered with 50 .mu.g LPS, formulated in phosphate-buffered saline (PBS), by injecton i.p. and divided into three groups of ten. Treatment of each group was then as follows:
1. Positive control. 1 mg polymyxin B (PMB) in PBS injected i.p. (PMB is an inhibitor of LPS activity.)
2. Negative control. PBS injected i.p.
3. Compound under test. 1 mg Peptide T analogue 623 (D-Thr-Thr-Tyr-D-Thr-NH.sub.2) in PBS injected i.p.
The results are shown in FIG. 11. Survival of the mice treated with analogue 623 was at least as good as that of PMB-treated mice.
EXAMPLE 24
PEPTIDE T INHIBITS ACTIVATION OF HUMAN NEUTROPHILS BY TNF AND LPS
Neutrophils.
Neutrophils were isolated from whole blood from healthy blood donors. Separation was achieved by the rapid-single-step method of centrifugation of whole blood on FICOLL-HYPAQUE.TM.. These were >99% viable and usually >98% pure.
Peptide and TNF.
TNF was a recombinant product produced in E. coli. Peptide T and analogue 505 (D-Ser-Ser-D-Thr-Thr-D-Thr-Thr-Tyr-D-Thr) were resuspended in Hank's Balanced Salt Solution (HBSS).
Examination of Peptide T's and analogue 505's effect on stimuli-induced neutrophil (100 .mu.g) by chemiluminescence.
To 100 .mu.l of 500-1000 U/ml of TNF was added 10 .mu.l of peptide and after 5 min 100 .mu.l of 1.times.10.sup.7 neutrophils/ml was added. The cells further incubated at 37.degree. C. /20 min and then tested for chemiluminescence activity. In some cases an agonist such as fMLP (10.sup.-7 M) was added. Chemiluminescence was measured in a luminometer (LKB). The results for Peptide T are presented in FIG. 12 as peak rate of CL release. Lucigenin-dependent chemiluminescence was measured: this represents superoxide measurement. In another set of experiments the effects of analogue 505 on the LPS-induced neutrophil chemiluminescence were examined. In this case the LPS was essentially substituted for the TNF. The concentration of LPS was 0.005 .mu.g/ml. The results are presented in FIGS. 13 and 14, which show respectively that analogue 505 (a) inhibits TNF priming and activation of human neutrophils and (b) inhibits LPS activation of human neutrophils.
EXAMPLE 25
PEPTIDE T REDUCES TNF-MEDIATED PROTEOGLYCAN DEGRADATION AND REDUCES TNF'S ABILITY TO STIMULATE NEUTROPHIL-MEDIATED DEGRADATION OF CARTILAGE
(a) In one group of experiments various concentrations of peptide T were mixed with TNF and then added to .sup.35 S-labelled cartilage. The cartilage was cultured for 3 days at 37.degree. C. and on each day the supernatant was removed to determine the amount of degradation. The cartilage culture was replenished daily with TNF or peptide T-TNF. (b) In another group of experiments the human neutrophils were treated with TNF or peptide T-TNF and then added to cartilage. The neutrophil mediated cartilage degradation determined.
Results 1. Effects of peptide T on TNF-induced cartilage degradation
TABLE XII______________________________________Effect of peptide T on TNF-inducedcartilage proteoglycan degradation % Cumulative release (day 1,2,3)Cartilage treatment Exp 1 Exp 2______________________________________TNF* 45.5 23.1TNF + 1 .mu.g peptide T 11.1 -0.5TNF + 10 .mu.g peptide T 3.2 1.7TNF + 100 .mu.g peptide T 15.9 13.2______________________________________ *100 U of TNF
2. Effects of peptide T on TNF-primed neutrophil for proteoglycan degradation
Peptide T reduced the ability of TNF to stimulate neutrophil-mediated degradation of cartilage (Table XIII).
TABLE XIII______________________________________The effect of peptide T onTNF priming of neutrophils for cartilagedamage % Cumulative releaseTreatment of neutrophils Exp 1 Exp 2 Exp 3______________________________________TNF* 17.4 6.8 5.3TNF + 10 .mu.g peptide T -5.2 9.9 1.9TNF + 100 .mu.g peptide T -0.8 0.9 0.6______________________________________ *100 U or TNF
This example effectively gives good in vitro support to the anecdotal clinical evidence presented in Examples 13 and 14.
EXAMPLE 26
PEPTIDE T REDUCES TISSUE FACTOR EXPRESSION INDUCED BY TNF
In acute systemic inflammation, for example in a septic shock patient, disseminated intravascular coagulation can be observed. During the course of the coagulation reaction, endothelial cells are involved as mediators of cell adhesion. One factor which is produced and which is involved in the coagulation cascade is tissue factor, also known as endothelial cell procoagulant activity; it is induced by TNF. In this example, it is demonstrated that Peptide T inhibits tissue factor expression by endothelial cells, thereby showing that Peptide T inhibits some of the pathology associated with acute inflammatory reactions.
Methodology
Human umbilical vein endothelial cells were cultured essentially by the method of Bevilacqua et al. Proc. Natl. Acad. Sci. USA 83 4533-4537 (1986). Cell cultures were treated with TNF.alpha. at 3 .mu.g/ml for 4 hours at 37.degree. in the presence or absence of Peptide T. Tissue factor expression was measured by an ELISA, following absorbence at 450 nm. The results are shown in FIG. 15, from which it can be seen that increasing concentrations of Peptide T progressively reduce tissue factor expression.
EXAMPLE 27
HEALTH-RELATED QUALITY OF LIFE AND SYMPTOM ASSESSMENT IN MS PATIENTS
To evaluate the impact of drug-related change on overall health and symptom burden, MS patients were followed routinely with both recognized and newly developed health-related quality of life instruments: an MS-modified version of the 30-item Medical Outcomes Study-HIV Survey (MOS-HIV) originally developed by Dr. Albert Wu, the 22-item Psychological General Well-Being Schedule (PGWB) developed by Dr. Harold Dupuy, and the MS Symptom Checklist developed for this study. These instruments, of which the first two have previously been validated and shown to be responsive to change in other disease states, allow quantitation of self-reported symptom variation (see Brook et al., Medical Care 17 (7 Suppl.) 1-54 (1979), Fazio "A Concurrent Validation survey of the NCHS General Well-Being schedule", U.S. Dept of Health, Education and Welfare, National Center for Health Statistics, Hyattsville, Md., U.S.A. 1977, McDowell and Newell "Measuring Health: A Guide to Rating Scales and Questionnaires", Oxford University Press 1987, Stewart et al., Medical Care 26 724-35 (1988), JAMA 262 907-13 (1989), and "Measuring Functioning and Well-Being of Patients with Chronic Conditions: the Medical Outcomes Study Approach", Duke University Press 1992, Wachtel et al., Ann. Int. Med. 116 129-137 (1992), Ware et al., "Conceptualization and Measurement of Health for Adults in the Health Insurance Survey, Vol III: Mental Health", Rand Corporation 1979, and Wu et al., Medical Care 29 (8) 786-98 (1991)).
Given the small number of patients involved in this report, some of whom received Peptide T in an on-off-on design, it would not be expected that statistical significance might be obtained. To facilitate establishment of any trends, three patients who stopped taking Peptide T and then restarted the drug some weeks later after a new baseline period appear twice in the following analysis. In this manner, 8 baseline and repeated measures (at weeks 0, 4, 8, and 12) were defined and analysed using two-tailed paired t-tests: see Table XIV.
TABLE XIV__________________________________________________________________________Two-tailed Paired T-Tests, MS Patients BASELINE Week 4 Week 8 Week 12ITEM MEAN SD MEAN SD P-value MEAN SD P-value MEAN SD P-value__________________________________________________________________________MOS-HIV n = 8 n = 8 n = 6 n = 5(all scored 0-100)Discomfort 30 15.1 55 25.6 .019 60 25.3 .082 52 30.3 .109Mental Health 58.5 21.4 72.5 9.7 .102 81.3 10 .125 76.8 11.8 .07Energy 35.6 19.4 63.8 12.2 .005 69.2 13.6 .006 67 14.4 .031Health Distress 69.4 28.5 89.4 9 .078 95.8 4.9 .1 89 14.7 .086Quality of life 53.12 16 81.25 11.6 .007 83.3 12.9 .012 75 17.7 .016Well-Being 49.3 17.25 72.4 9.3 .006 77.9 9.3 .023 72 14.3 .013MOS Summary 47.7 19.8 61.9 14.5 .003 69.7 14.4 .003 71.5 15.6 .015PGWB n = 7 n = 7 n = 5 n = 5Vitality (0-20) 9.1 3.3 15 2.6 .003 14.8 1.6 .03 15 2.1 .011PGWB Summary (0-110) 63.5 13.2 79.8 7.5 .007 85.8 10.7 .003 81 13.3 .009SYMPTOM CHECKLIST n = 7 n = 7 n = 6 n = 6Fatigue (0-4) 1.375 0.916 2.86 0.9 .025 3.17 0.75 .001 3.33 0.52 .007Malaise (0-4) 2.29 1.1 3.33 0.52 .042 3.83 0.41 .025 3.4 0.55 .14Forgetfulness (0-4) 3 1.1 3.6 0.53 .046 3.7 0.52 .101 3.83 0.41 .101Slow thinking (0-4) 2.88 0.99 3.43 0.79 .103 3.67 0.52 .042 3.83 0.41 .042__________________________________________________________________________ Note: Starts and restarts after washout are considered as separate examples. In all scales, a higher score indicates improvement.
In the MS-modified MOS-HIV, either a trend or statistically significant improvement was observed at weeks 4, 8, and 12 even in this small sample in such dimensions of health as physical discomfort, mental health, and health distress. Improvement was statistically significant at all three measurement points in energy, general quality of life, well-being and the unweighted summary score. General overall improvement was corroborated by the summary score of the PGWB.
Remarkable improvement in energy was corroborated by the vitality subscale of the PGWB and by ratings of the impact of fatigue on daily activities.
Improvement in malaise was statistacally significant at 4 and 8 weeks. In regard to neurocognitive manifestations of MS, there was a trend towards improvement in forgetfulness and slowness in thinking.
In health related quality of life assessment, a statistically significant change of greater than 0.5 standard deviations is usually taken as indication of moderate clinically significant change (see Testa, j. Hypertension 5 S9-S13 (1987)). All improvements noted above easily exceeded this test, with many improvements being greater than 1 standard deviation. Mean improvements in measures of energy at week 12 on all three instruments exceeded 2 standard deviations, an indication of very great clinical change in a common and debilitating symptom of MS.
These quantifiable assessments over time support the clinical observations of significant improvement.
__________________________________________________________________________# SEQUENCE LISTING- (1) GENERAL INFORMATION:- (iii) NUMBER OF SEQUENCES: 11- (2) INFORMATION FOR SEQ ID NO:1:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 8 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: both- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..8#/note= "Where Xaa 1 is Ala, Gly, Ser,#absent" Thr or- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: l..8#/note= "where Xaa 2 is Ala, Gly, Val,#or absent" Ser, Thr- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: l..8#/note= "where Xaa 3 is Ser, Thr or absent"- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: l..8#/note= "where Xaa 4 is Ser, Thr, Asn, Glu, Arg, - # Ile, Leu or absent"- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: l..8#/note= "where Xaa 5 is Ser, Thr, Asp or absent"- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: l..8#/note= "where Xaa 6 is Thr, Ser, Asn, Arg, Gln, - # Lys, Trp or absent"- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: l..8#/note= "where Xaa 7 is Tyr or absent"- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: l..8#/note= "where Xaa 8 is Thr, Arg, Gly, Met, Met( - #o), Cys, Tyr, Gly or absent"- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: l..8#/note= "where the ends may have CYS or are absen - #t"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:- Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5- (2) INFORMATION FOR SEQ ID NO:2:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 8 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: both- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..8#/note= "where Xaa 1 is Ala, D-Ala; and#is Thr or Thr-amide"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:- Xaa Ser Thr Thr Thr Asn Tyr Xaa 1 5- (2) INFORMATION FOR SEQ ID NO:3:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 8 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: both- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..8#/note= "Ala 1 is D-Ala"RMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:- Ala Ser Thr Thr Thr Asn Tyr Thr 1 5- (2) INFORMATION FOR SEQ ID NO:4:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 5 amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: both- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..5#/note= "core"OTHER INFORMATION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:- Thr Thr Asn Tyr Thr 1 5- (2) INFORMATION FOR SEQ ID NO:5:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 8 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: circular- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:- Ala Ser Thr Thr Thr Asn Tyr Thr 1 5- (2) INFORMATION FOR SEQ ID NO:6:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 8 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: both- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..8#/note= "aa 1 is D-Alanine"TION:- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:- Ala Ala Ser Ser Ser Asn Tyr Met 1 5- (2) INFORMATION FOR SEQ ID NO:7:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 5 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: both- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:- Thr Asp Asn Tyr Thr 1 5- (2) INFORMATION FOR SEQ ID NO:8:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 5 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: both- (ii) MOLECULE TYPE: peptide- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:- Thr Thr Ser Tyr Thr 1 5- (2) INFORMATION FOR SEQ ID NO:9:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 4 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: both- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..4#/note= "aa1 is D-Thr, aa4 is D-Thr"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:- Thr Thr Tyr Thr 1- (2) INFORMATION FOR SEQ ID NO:10:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 8 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: both- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: l..8#/note= "aa1 is D-Ala, aa3 is D-Thr,#D-thr, aa8 is D-Thr"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:- Ala Ser Thr Thr Thr Asn Tyr Thr 1 5- (2) INFORMATION FOR SEQ ID NO:11:- (i) SEQUENCE CHARACTERISTICS:#acids (A) LENGTH: 8 amino (B) TYPE: amino acid (C) STRANDEDNESS: Not R - #elevant (D) TOPOLOGY: both- (ii) MOLECULE TYPE: peptide- (ix) FEATURE: (A) NAME/KEY: Peptide (B) LOCATION: 1..8#/note= "aa1 is D-Ser, aa3 is D-Thr, aa5 is D-Thr, - # aa8 is D-Thr"- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:- Ser Ser Thr Thr Thr Thr Tyr Thr 1 5__________________________________________________________________________

Number	Name	Date	Kind
5686417	McFadden et al.	Nov 1997
5756449	Andersen et al.	May 1998

Number	Date	Country
87304913	Dec 1987	EPX
120657	Dec 1989	WOX
09338	Dec 1989	WOX
19257	Nov 1992	WOX

Peptide T and related peptides in the treatment of inflammation, including multiple sclerosis

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Cotelle, et al Biochem&BiophysResComm. vol. 171, No. 2. pp. 596-602, Sep. 14, 1990.
Urge, et al; Biochem&BiophysResComm. vol. 184, No. 2, pp. 1125-1132. Apr. 30, 1992.