Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/482—Serine endopeptidases (3.4.21)
  • A61K38/4826—Trypsin (3.4.21.4) Chymotrypsin (3.4.21.1)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/4873—Cysteine endopeptidases (3.4.22), e.g. stem bromelain, papain, ficin, cathepsin H
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21004—Trypsin (3.4.21.4)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/22—Cysteine endopeptidases (3.4.22)
  • C12Y304/22002—Papain (3.4.22.2)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55516—Proteins; Peptides

Definitions

• the present invention relates to the use of proteases, other than bromelain, in the treatment of a variety of diseases and conditions which are mediated by intracellular signals.
• the invention relates to the use of such proteases in the treatment of diseases and conditions such as cancer and autoimmune diseases and as an immunosuppressive agent .
• proteases may be used as vaccine adjuvants.
• Bromelain has previously been used in the treatment of a variety of conditions including inflammation and, in particular, it has been used in the treatment of diarrhoea.
• the use of bromelain in the treatment of infectious diarrhoea is described in WO-A-9301800, where it is suggested that bromelain works by destroying intestinal receptors for pathogens by proteolysis, and in WO-A-8801506 which teaches that bromelain detaches pathogens from intestinal receptors.
• bromelain is also disclosed in the treatment of a variety of diseases and conditions which are mediated by intracellular signals.
• bromelain can reduce toxin binding activity and can inhibit the secretory effect of toxins such as heat labile toxin (LT) and cholera toxin (CT) and also toxins such as heat stable toxin (ST) .
• LT heat labile toxin
• CT cholera toxin
• ST heat stable toxin
• LT and ST are both produced by enterotoxigenic strains of E . coli (ETEC) .
• ETEC E . coli
• Some ETEC strains also produce pilus adhesins called colonisation factor antigens. These adhesins promote attachment of ETEC strains to the small intestinal mucosa, thereby facilitating colonisation and delivery of enterotoxin. Diarrhoeal disease is ultimately dependent on production and efficient delivery of enterotoxin.
• the enterotoxins stimulate secretion by cells by activation of signal pathways. Internal signals within cells are carried by "second messengers".
• Every cell of the human body is constantly bombarded with various signals from its environment. Normal cells receive and process these signals which may promote growth, differentiation or death or control other functions of the cell, such as secretion of fluids in the cells of the intestinal epithelium. Therefore, signals are the keys to understanding the processes in the cell which ultimately determine its fate. Signals are received through receptors with distinct biochemical activities on cell surfaces and transmit the messages further down to responder proteins. These proteins, in turn, process the signals and transduce them to other molecules within the cell. The series of biochemical events that take place after the interaction of a cell with a growth factor and before the cellular response occurs is referred to as signal transduction.
• cellular signals are transmitted via GTP-binding proteins, various protein kinases, protein phosphatases, enzymes that modify lipids, and second messengers such as Ca 2* and cyclic adenosine monophosphate (cyclic AMP or cAMP) .
• the instructions are finally interpreted in the nucleus by transcription factors that initiate gene expression and subsequent translation of cellular proteins.
• At least three signal pathways are known to be important for secretion.
• One pathway employs the second messenger, cyclic AMP.
• Another employs the second messenger cyclic guanosine monophosphate (cyclic GMP or cGMP) .
• cyclic GMP or cGMP second messenger cyclic guanosine monophosphate
• the third signal pathway (Ca 2 *-dependent pathway) requires Ca 2* as the second messenger.
• WO-A-9400147 teaches that stem bromelain protease is capable of preventing diarrhoea by interfering with cyclic nucleotide and Ca 2 *-dependent pathways and thus affecting secretion.
• PCT/GB95/01501 teaches that bromelain can be used to treat a variety of conditions that are mediated by intracellular signal pathways mediated by inositol phosphates, protein kinases and/or protein phosphates .
• proteases in general appear to affect pathways, in particular pathways which are modulated by inositol phosphates, protein kinases and/or protein phosphatases.
• a protease other than bromelain, in the preparation of an agent for modulating intracellular signalling pathways which depend on the action of inositol phosphates, protein kinases and/or protein phosphatases.
• inositol phosphate refers to any phosphorylated inositol molecule, regardless of the degree of phosphorylation or the positions of the phosphate groups.
• inositol phosphates include phosphatidyl-4, 5-biphosphate (PIP 2 ) and inositol-1, 4, 5-triphosphate (IP 3 ) .
• PIP 2 5-biphosphate
• IP 3 5-triphosphate
• Protein kinases and protein phosphatases refer to any molecule capable of converting an inactive form of a protein to an active form by either the addition or removal of phosphate molecules .
• proteases are particularly useful for controlling inositol phosphate, protein kinase or protein phosphatase dependent signalling pathways which lead to the production of non synaptic extracellular signalling molecules such as vasopressin and thrombin, and particularly signalling molecules which affect growth and proliferation of cells, for example interleukins and other growth factors.
• Cancer occurs when tumour suppressor genes are inactivated. Similarly, cells receiving hyper-stimulation arising from defects in the stimulatory signalling cascade, exhibit excess proliferation. Oncogenes are genes which produce a protein with altered function and their activation provides the cell with a strong, unrelenting impetus to grow. An oncogene disrupts the carefully balanced molecular controls on cell proliferation to such an extent that malignant growth ensues.
• Protein tyrosine kinases such as v-src and the related v-abl protein have proved to be among the most frequently implicated proteins in experimental and human cancer, c-src is a kinase which is found in normal cells and is regulated by other kinases. This regulation is lost in v-src, found in cancer cells.
• the v-src kinase is persistently hyperactive as a result of a few amino acid differences between c-src and v-src proteins. Unbridled catalytic activity of the mutant protein-tyrosine kinase can have a detrimental effect on the control of cell growth.
• Protein tyrosine phosphorylation cascades (or kinase cascades) play a significant role in regulating events throughout signal transduction. Many receptors for growth factors possess tyrosine kinase activity and, when activated, trigger the phosphorylation of multiple cellular proteins on tyrosine residues. The result of this phosphorylation process causes the target protein to gain or lose function.
• p21c-ras plays a critical role in mediating mitogenic and differentiating signals received from receptor tyrosine kinases (Wood et al . , Cell , 68, 1041-1050, 1992; Thomas et al .
• kinases that include members of the protein kinase C (PKC) , Raf, Mitogen-activated protein (MAP) , and S6 kinase families (Cantley et al . , Cell , 64, 281-302, 1991) .
• PLC protein kinase C
• MAP Mitogen-activated protein
• S6 kinase families Cantley et al . , Cell , 64, 281-302, 1991.
• These kinases can integrate signals from multiple membrane receptors.
• MAPk mitogen-activated protein kinases
• MAPk are serine/threonine kinases that are activated by various growth factors and tumour promoters in cells.
• the best studied of these kinases are p42MAPK and p44MAPK (also referred to as ERK2 and ERK1 respectively, pp42mapk/erk2 and pp44mapk/erkl /mpk, also known as microtubule associated protein kinase; myelin basic protein (MBP) kinase; and RSK I and
• Substrates of MAP kinase include pp90 and 70S rsk kinases and several transcription factors, notably Jun (Pulverer et al . , Nature, 353, 670-674, 1991) , Myc and p62TCF. Proteins that affect transcriptional activity are the most widely implicated in the cancer process.
• MAPk exists as a dephosphorylated form in quiescent cells and become activated when both tyrosine and threonine residues are phosphorylated (Boulton et al . , Cell , 65, 663-675, 1991) . In vi tro, this activation is almost completely reversed if either residue is dephosphorylated (Anderson et al . , Nature, 343, 651-653, 1990) .
• PAC1 has recently been reported to be a MAP kinase phosphatase which inhibits MAP-kinase-regulated reporter gene expression (Ward et al .
• MAP kinase kinase kinases that include the proto-oncogene product Raf (Anderson et al . , Biochem. J. , 277, 573-576, 1991) and MEKK, which in turn are regulated by protein kinase C (PKC) .
• PKC protein kinase C
• proteases are capable of interfering with signalling pathways which are important for growth, in particular, signalling pathways which lead to the production of growth factors such as IL-2, platelet derived growth factor (PDGF) and insulin like growth factor (IGF) .
• T-lymphocytes were used as a cell model to demonstrate the mode of action of the growth-promoting mechanism of cells.
• the growth of T-lymphocytes is regulated through growth factor production, receptor function, cytoplasmic signal processing and gene responses in the nucleus.
• T- lymphocytes are a commonly used model for measurement of proliferation because of the ease of access to the cells and the well documented role of interleukin 2 (IL-2) , the T-cell growth factor which is required for growth and proliferation.
• IL-2 interleukin 2
• T-cells and, indeed, other types of cell require stimulation in order to " initiate the series of events required for proliferation.
• the immune system contains billions of white blood cells or lymphocytes which are divided into two classes, B lymphocytes and T lymphocytes.
• B-cells function to protect the host from extracellular pathogens and T-cells protect the host from intracellular pathogens.
• B-cells and T-cells recognise distinct forms of different antigens using B-cell receptors (BCR) and T-cell receptors (TCR) respectively.
• T cells The activation of T cells is a complex process requiring protein tyrosine kinase activity that results in cell growth and differentiation. Activation requires recognition of antigen by the TCR and interactions with other molecules on the T cell surface with antigen presenting cells.
• the T-cell responds in two major ways. One is to enlarge and divide, thereby increasing the number of cells that react to the antigen. The other is to secrete lymphokines or cytokines, proteins that directly inhibit the pathogen or that recruit other cells to join in the immune response.
• the cytokine interleukin 2 (IL-2) is a T cell growth factor which plays a pivotal role in the regulation of immune responses.
• T-cells cannot normally respond to IL-2, as these cells do not express detectable high affinity IL-2 receptors on the surface of their cells. Antigenic stimulation is required for the induction of high affinity IL-2 receptor expression, and thus conferral of IL-2 responsiveness. Therefore, the initial activation signals provided by stimulation of the T cell antigen receptor (TCR) , and the costimulatory signal, initiates T cell activation through induction of IL-2 production and IL-2 receptor expression. Subsequent T cell proliferation is driven by the interaction of IL-2 with its IL-2 receptor. If a T-cell receives a signal via the TCR alone, the T-cell becomes anergised or may die (called apoptosis) . If a T-cell receives the co- stimulatory signal alone, the T-cell remains quiescent (or does not respond) .
• TCR T cell antigen receptor
• TCR-mediated induction of protein tyrosine kinase activity results in the tyrosine phosphorylation of many cellular proteins including the TCR zeta chain (Baniyash et al . , J. Biol . Chem.
• PLC gl phospholipase C gl
• CD5 Daavies et al . , Proc . Na tl . Acad . Sci . USA, 89, 6368-6372, 1992
• VCP valosin-containing protein
• ezrin Egerton et al . , EMBO J.
• PLCgl results in its catalytic activation, resulting in the generation of the second messengers of the phosphatidylinositol (PI) pathway.
• PLC cleaves phosphatidylinositol 4, 5-bisphosphate (PIP2) , resulting in the formation of inositol 1, 4, 5-trisphosphate (IP3) and 1, 2-diacylglycerol (DAG) .
• PIP3 inositol 1, 4, 5-trisphosphate
• DAG 2-diacylglycerol
• These molecules function as intracellular second messengers to induce an increase in Ca 2* and activation of PKC, respectively.
• the proto-oncogene Ras is activated, Raf-1 kinase activity is increased and MAPk becomes phosphorylated.
• MAPk activation causes the proto-oncogenes c-fos to form a dimer with c-jun to form the transcriptional complex AP-1.
• the AP-1 complex
• Figure 1 summarizes some of the events associated with TCR activation of the PI pathway that lead to IL-2 gene transcription and IL-2 production.
• Proteases were found to inhibit the kinase cascade which is associated with growth stimulation.
• a factor in this signalling pathway is the ras protein of which aberrant forms are found in 25 to 30% of human tumours.
• Proteases are able to block signals required for the proliferation of T-cells, probably by blocking the tyrosine phosphorylation of proteins including MAP kinase.
• proteases are capable of acting as anti-cancer agents since they will also block the over-production of growth factors such as platelet derived growth factor (PGDF) and epidermal growth factor (EGF) in fibroblasts and epithelial cells.
• PGDF platelet derived growth factor
• EGF epidermal growth factor
• proteases can, in fact, be used either to stimulate or to inhibit cytokine production depending on whether they are used to treat activated cells (such as those already receiving stimuli) , or inactivated (ie quiescent or resting) cells. They thus can be used as immunosuppression agents, e.g. in preventing tissue rejection, or as immunostimulants, e.g. as an adjuvant to a vaccine.
• proteases can also be used to prevent or treat toxic shock by means of their ability to inhibit cytokine production and tyrosine phosphorylation.
• Proteases can also be used to treat allergies .
• Proteases may be administered by a variety of routes including enteral, for example oral, nasal, buccal, or anal administration or parenteral administration for example by intravenous, intramuscular or intraperitoneal injection.
• enteral for example oral, nasal, buccal, or anal administration or parenteral administration for example by intravenous, intramuscular or intraperitoneal injection.
• the oral route is, however, preferred.
• an enteric-protected preparation To assist survival of proteases through the stomach when administered orally, it may be desirable to formulate the enzyme in an enteric-protected preparation.
• Other orally administrable formulations include syrups, elixirs, and hard and soft gelatin capsules, which may also be enteric-coated.
• Dosage of proteases is conventionally measured in Rorer units, FIP units, BTU (bromelain tyrosine units) , CDU
• One Rorer unit of protease activity is defined as- that amount of enzyme which hydrolyses a standardisation casein substrate at pH 7 and 25°C so as to cause an increase in absorbence of 0.00001 per minute at 280nm.
• One FIP unit of bromelain activity is contained in that amount of a standard preparation, which hydrolyses a suitable preparation of casein (FIP controlled) under the standard conditions at an initial rate such that there is liberated per minute an amount of peptide, not precipitated by a specified protein precipitation reagent, which gives the same absorbence as l ⁇ mol of tyrosine at 275nm.
• BTUs, CDUs, GDUs and MCUs are as defined in the literature, as follows:
• One bromelain tyrosine unit is that amount of enzyme which will liberate one micromole of tyrosine per minute under the conditions of the assay (for example, after digestion of an acid denatured haemoglobin substrate at pH 5 and 30°C) .
• the enzyme activity which liberates one milligram (10 "3 g) of amino nitrogen from a standard gelatin solution after 20 minutes digestion at 45°C and at pH 4.5.
• daily dosages of from 50 to 4000 GDU/day is appropriate, for example from 100 to 1000 GDU/day.
• the daily dose may be given in one or more aliquots per day, for example twice, three times or four times a day.
• a particularly preferred dose would be lOmg/kg (giving a dose of 700mg for an average adult human equivalent to 2800 BTU) .
• FIGURE 1 is a diagram illustrating the events associated with T-cell receptor activation of the phosphatidylinositol (PI) pathway which lead to IL-2 gene transcription and IL-2 production;
• PI phosphatidylinositol
• FIGURE 2 shows the effect of bromelain and trypsin on sheep red blood cell (SRBC) antibody responses in mice in vivo
• FIGURE 3 shows the effect of bromelain and trypsin on the proliferation of splenic T cells in vi tro;
• FIGURE 4 shows the effect of bromelain and trypsin treatment on the cell surface expression of (A) CD3e and (B) CD28.
• the Hemolytic Plaque assay was useed to investigate the effect of trypsin and bromelain on antibody production.
• S [ecific details of the assay are desribed in Weir (Handbook of experimental Immunology, 1-4, 4th edn. (1986) Blackwell Scientific Publications, Oxford, UK) .
• Crude bromelain (E. C. 3.4.22.4) ; having activity of 2400 BTU/g and trypsin (E. C. 3.4.21.4) (Sigma Aldrich Ltd) ; having activity of 10,000 BTU/g were used in_the experiments.
• mice Female Balb/c mice (8-10 weeks old) were administered single intravenous injections of proteolytically matched amounts of bromelain or trypsin. Bromelain (doses up to 200 ⁇ g; 480 BTU) or trypsin (doses up to 48 ⁇ g; 480 BTU) was suspended in 0.9% (200 ⁇ l) saline and filter sterilised immediately prior to administration. The dose rate of 480 BTU per injection corresponds to approximately one-fifth of the murine LD50. Mice administered with saline alone were used as controls.
• mice Following administration of protease, the mice were immunised by intraperitoneal injection with sheep red blood cells (SRBC, lOO ⁇ l; 10 7 cells, TCS Biologicals, Buckingham, UK) . Mice used as negative controls were administered with saline alone (lOO ⁇ l) . The mice were sacrificed three days post immunisation, the spleens being removed and splenocytes isolated by filtration through a 100 micron nylon mesh filter. Erythrocytes were lysed at 5 x 10 7 lymphocytes/ml in lysing buffer (140 mM NH 4 C1, 17 mM Tris, pH 7.2) for2 to 5 min. Lysis was terminated by adding tissue culture medium (TCM) .
• TCM tissue culture medium
• Assays were performed in 160 ⁇ l, consisting of 1 x 10 6 splenocytes, 6 x 10 6 SRBC and 1:27 guinea pig complement in RPMI 1640 (GIBCO Laboratories, Grand Island NY) .
• the reaction mix was placed in a chamber created by joining two glass slides together with double sided tape and then sealed with wax. Samples were incubated at 37°C for 1 hour, prior to counting plaque forming cells (PFC) (ie. the number of B-cells producing antibodies specific for SRBC antigen.
• PFC plaque forming cells
• mice treated with bromelain or trypsin produce greater numbers of PFC in comparison with saline-treated controls.
• Mice treated with saline, bromelain or trypsin, and not immunised with SRBC (negative control) produced very few PFC indicating that protease treatment does not cause spontaneous PFC against SRBC (data not shown) .
• TCM tissue culture media
• FCS foetal calf serum
• CD28 mAb (PV-1; a gift from Dr C. June, NMRI, Bethesda, MD) were used to stimulate T cells. All reagents were diluted in TCM, except for immobilised anti-CD3e mAb which was prepared by diluting the mAb in PBS, adding to culture plates to cover the bottom of wells, incubating for 16 h at 4°C and then washing the wells 3 times with PBS. All cells were incubated at 37°C in humidified 5% C0 2 .
• Bromelain and trypsin treatment of splenic T cells caused a significant increase in cell proliferation when cells were stimulated with anti-CD3e mAb or with combined anti- CD3e and anti-CD28 (results shown in figure 3) .
• example 1 Data obtained in example 1 showed that bromelain and trypsin can increase antibody responses when mice are immunised with a T cell dependent antigen. Also, example 2 showed that bromelain and trypsin treatment increased the proliferation of stimulated T cells in vi tro. This data therefore suggests that proteases may induce T cell activation. Proteases have been shown to modify cell surface molecules, therefore we investigated the effect of bromelain and trypsin on CD£e and CD28 molecules (molecules critical for T cell activation) on the surface of murine splenic T cells by flow cytometry.
• FACS fluorescent activated cell sorting
• the binding of mAb to CD3e on the surface of T cells increased when cells were treated with bromelain or trypsin (indicated by the increase in mean fluorescence intensity: profile shifting to the right) as shown in figure 4.
• the increase .observed was a specific event because a control mAb showed no increase in binding above background levels in both protease and PBS treated T cells (data not shown) .

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• 1995
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