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)
• • 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
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • 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
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
  • A61P33/10—Anthelmintics
• • 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

Definitions

• the present invention relates to the use of bromelain in the treatment of a variety of diseases and conditions which are mediated by intracellular signals.
• the invention relates to the use of bromelain in the treatment of diseases and conditions such as cancer and autoimmune diseases and as an immunosuppressive agent.
• bromelain may be used as a vaccine adjuvant.
• 3rc ⁇ elain is the collective name for the proteolytic enzymes found in the tissues of the plant Bromeliaceae.
• 3romelain is a r.ixture of various moieties derived from the stem of the pineapple (Ananas comosu ⁇ ) . It contains at least two proteolytic enzymes but also non-proteolytic enzymes, including an acid phosphatase and a peroxidase; it may also contain amylase and cellulase activity. In addition, various other components are also present.
• ⁇ romelain 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 O-A-9301800, where it is suggested that bromelain works by destroying intestinal receptors for pathogens by proteolysis, and in O-A-8801506 which teaches that bromelain detaches pathogens from intestinal receptors.
• bromelain is also useful in the treatment of non-infectious diarrhoea and this cannot, of course, be explained by the mechanism of action suggested in O-A-9301800.
• 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 ( ⁇ TEC) .
• ⁇ TEC enterotoxigenic strains of E. coli
• Some ⁇ T ⁇ C 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.
• bromelain appears also to affect other intracellular signalling pathways, in particular pathways which are modulated by inositol phosphates, protein kinases and/or protein phosphatases.
• inositol phosphate refers to any phosphorylated inositol molecule, regardless of the degree of phosphorylation cr the positions of the phosphate groups.
• inositol phosphates include phosphatidyl-4, 5-biphosphate (PI? 2 ) and inositol-1,4,5-triphosphate (IP 3 ) .
• 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.
• bromelain is 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 cncogene disrupts the carefully balanced molecular controls on cell proliferation to such an extent that rr.aiignant growth ensues.
• Protein tyrosine kinases such as v-src and the related v-abl protein have proved tc 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 .
• 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). These kinases can integrate signals from multiple membrane receptors.
• PLC protein kinase C
• Raf Raf
• MAP Mitogen-activated protein
• S6 kinase families Cantley et al . , Cell , 64, 281-302, 1991.
• 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; yelin basic protein (MBP) kinase; and RSK I and II) .
• MBP mitogen-activated protein kinases
• 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.
• the mechanism of activation of the MAP kinases is very complex. 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) .
• MEK is in turn regulated through phosphorylation by MAP kinase kinase kinases that include the proto-oncogene product Raf (Anderson et al . , ⁇ iochem. J. , 277, 573-576, 1991) and MEKK, which in turn are regulated by protein kinase C (PKC) .
• PKC protein kinase C
• bromelain is capable cf 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) .
• IL-2 platelet derived growth factor
• IGF insulin like growth factor
• 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 7-ceii growth factor which is required for growth and
• the immune system contains billions of white blood cells or lymphocytes which are divided into two classes, B lymphocytes and T lymphocytes.
• 3-cells function to protect the host from extracellular pathogens and T-cells protect the host from intracellular pathogens.
• 3-cells and T-cells recognise distinct forms cf different antigens using 3-cell receptors (BCR) and T-ceil receptors (TCR) respectively.
• the activation cf 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 ether 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 Ivmphokines 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 ceils. Antigenic stimulation is required for the induction cf 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-ceil 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
• PLC gl phospholipase C gl
• CD5 phospholipase C gl
• VCP valosin-containing protein
• ezrin Egerton ec al. , EMBO J.
• PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) , resulting in the formation of inositol 1,4,5-trisphosphate (I?3) and 1,2-diacylglycerol (DAG) .
• PIP2 phosphatidylinositol 4,5-bisphosphate
• I?3 inositol 1,4,5-trisphosphate
• DAG 1,2-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-onccgenes c-fos to form a dimer with c-jun to form the transcriptional complex AP-1.
• the AP-1 complex binds to elements on the DNA to initiate transcription of IL-2.
• 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.
• Bromelain was 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.
• Bromelain is able to block signals required for the proliferation of T-cells, probably by blocking the tyrosine phosphorylation of proteins including MAP kinase.
• bromelain Because of its ability to block the tyrosine phosphorylation of MAP kinase and other proteins, bromelain is capable of acting as an anti-cancer agent since it 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
• bromelain can, in fact, be used either to stimulate or to inhibit cytokine production depending on whether it is used to treat activated cells (such as those already receiving stimuli) , or inactivated (ie quiescent or resting) cells.. It thus can be used as an immunosuppression agent, e.g. in preventing tissue rejection, or as an immunostimuiant, e.g. as an adjuvant to a vaccine. 3romeiain can also be used to prevent or treat tcxic shock by means of its ability to inhibit cytokine production and tyrosine phosphorylation. Bromelain can also be used to treat allergies.
• bromelain is a mixture of various components. Although it was taught in WO-A- 9400147 that stem bromelain protease is the component of bromelain responsible for the mediation of cyclic nucleotide pathways, it is not clear whether stem bromelain protease is also responsible for the action of bromelain on kinase pathways or whether some other component of the bromelain mixture could be responsible. However, this does not affect the working of the invention since the crude bromelain mixture, at least, is capable of affecting the phosphorylation (or activation) of MAP kinase.
• 3romelain may be administered by a variety cf 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.
• orally administrabie formulations include syrups, elixirs, and hard and soft gelatin capsules, which may also be enteric-coated.
• 3romelain activity is stable over a wide pH range (pH 2- 9) . Therefore, it may not be necessary to enteric- protect ,or enteric-coat) the bromelain from the acid conditions in the stomach. It may, however, be necessary to protect the enzyme from digestion by acid proteases in the gut. 3romelain may therefore, be administered with a buffering agent, for example bicarbonate.
• a buffering agent for example bicarbonate.
• bromelain Dosage of bromelain is conventionally measured in Rorer units, FIP units, ETU (bromelain tyrosine units), CDU (casein digestion units) , GDU (gelatin digestion units) or MCU (milk clotting units) .
• 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.
• FIP unit of bromelain activity is contained in that amount of a standard preparation, which hydroiyses 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 cf tyrosine at 275nm.
• 3TUs, 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 cf 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" : 'g) of amino nitrogen from a standard gelatin solution after 20 minutes digestion at 45°C and at oH 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 n one or more aliquots per day, for example twice, three tines 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 is an immunoblot for the detection of MAP kinase in proteins obtained from the T-cell hybridoma GA15.
• GA15 were stimulated with either calcium ionophore, PMA or ionophore in combination with PMA or mock treated with PBS (unstimulated control) .
• Cells had either been treated with bromelain or with PBS.
• FIGURE 3 is an immunoblot for the detection of MAP kinase. Proteins were obtained from GA15 activated with calcium ionophore in combination with PMA or, PBS treated (unstimulated control) which had either been treated with bromelain or with PBS. Samples were tested for the presence of MAP kinase at timed intervals.
• FIGURE 4 is a plot which shows the appearance over time cf MAP kinase in stimulated T-cells treated with either bromelain or PBS.
• FIGURE 5 is an immunoblot which shows that a polyclonal antibody raised against a highly conserved peptide from MAPk (Erk 1) recognises two proteins of Mr 42000 and 44000.
• FIGURE 6 is an immunoblot with MAPk antibody which indicates that the shifts in electrophoretic mobility normally observed on phosphorylation of protein is partially blocked in cells treated with bromelain.
• FIGURE 7 shows the effect of bromelain treatment on IL-2, L-4 and IFN- ⁇ mRNA accumulation in GA15 cells in vi tro .
• GA15 cells treated with bromelain accumulate less IL-2, IL-4 and IFN- ⁇ mRNA when stimulated with PMA (20 ng/ml) and calcium ionophore A23187 (500 ng/ml) .
• FIGURE 8 shows that splenic T-ceils treated with bromelain produce less IL-2 when stimulated with 2C11 ( ⁇ CD3e) and CD28 ( ⁇ CD28) .
• FIGURE 9 shows the effect of bromelain treatment on IL-2, IL-4 and ⁇ -INF mRNA accumulation in splenic T-cells in vi tro .
• Splenic T-cells treated with bromelain accumulate less IL-2, IL-4 and IFN- ⁇ mRNA when stimulated with immobilised anti-CD3e (4 ⁇ g/ml) and soluble anti-CD28 (10 ⁇ g/ml) mAbs .
• FIGURE 10 shows that bromelain treatment increases the proliferation of splenic T-cells when they are stimulated with immobilised anti-CD3e (4 ⁇ g/ ' mi) and soluble anti-CD28 (10 ⁇ g/ml) mAbs .
• FIGURES 11a and b show that bromelain treatment increases the binding of both anti-CD3e (a) and anti-CD28 (b) mAbs to the cell surface of splenic T- ceiis as indicated by a shift in the FACS profiles to the righ .
• FIGURE 12 shows that bromelain increases the binding of an i-CD3e mAb to the surface of GA15 cells .
• FIGURES 13a and b show immunoblots of tyrosine phcsphorylated proteins in splenic T-cells.
• FIG. 13a shows that bromelain treatment induces protein tyrosine phosphorylation of 56 and 58 kDa proteins.
• Figure 13b shows that bromelain treatment inhibits protein tyrosine phosphorylation of a 16 kDa protein.
• FIGURE 14 shows the effect of bromelain treatment on L-2, IL-4 and IFN- ⁇ mRNA accumulation in GA15 cells in vi tro.
• Cells treated with bromelain accumulate more IL-2, IL-4 and IFN- ⁇ mRNA when stimulated with immobilised anti-CD3e (4 ⁇ g/ml) and soluble anti-CD28 (10 ⁇ g/ml) mAbs.
• FIGURE 15 shows the effect of bromelain on sheep red blood cell (SRBC) antibody responses in vivo.
• SRBC sheep red blood cell
• the ThO cell hybridoma GA15 was used for experiments investigating tyrosine phosphorylation.
• GA15 was generated from the fusion of the thymoma BW5147 with the Th2 clone F4, specific for KLH in association with I-Ab (Fox, Int. Immunol . , 5, 323-330, 1993) .
• Cells were maintained in tissue culture media (TCM) consisting of RPMI 1540 medium (Biofluids, Rockville, MD) supplemented with 10% fetal calf serum (Inovar Biologicals, Gaithersburg, MD) , ⁇ OmM 2-mercaptcethanoi, 4mM glutamine, and 50 ug/ml ' gentamicin.
• TCM tissue culture media
• mice Male C57BL/6NCrlBR mice were purchased from Charles River Laboratories (Wilmington, MA, USA) . Young mice (3 to 4 months old) and aged mice (20 to 26 months old) were used in paired experiments. Mice found to have tumors, visible skin lesions, cr significant pathology were not used.
• female 3alb/c mice were purchased from A. Tuck and son Ltd (UK) . Mice between 6 and 10 weeks of age were used.
• Phorbol esters are structurally related to 1,2-diacylglycerol (DAG) and therefore cause activation of PKC which induces the hyperphosphorylation of Raf-1 (Morrison ec al . , Proc . Na tl . Acad. Sci . USA, 85, 8855-8859, 1988) , as well as the activation cf MAP kinases ' Chung et al . , Mol . Cell . Biol . , 11, 1368-1874, 1991) . Ionophore increases cytopiasmic free calcium in the ceils, which in turn bind caimodulin and PKC.
• DAG 1,2-diacylglycerol
• Phorbol 12, myristate 13-acetate (PMA) and calcium ionophore A23187 were used to stimulate cells.
• Phorbol esters and ionophore treatment of T-lymphocytes act synergistically to mimic the effect of the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), and therefore reproduce many features of TCR stimulation (Truneh et ai. , Nature, 313, 318-320, 1985) such as IL-2 secretion, IL-2 receptor expression, and T cell proliferation.
• DAG diacylglycerol
• IP3 inositol 1,4,5-trisphosphate
• TCM tissue culture media
• FCS heat-inactivated foetal calf serum
• ⁇ rythrocytes were lysed at 5 x 10 7 lymphocytes/ml in iysing buffer (140 mM NH 4 C1, 17 mM Tris, pH 7.2) for 2-5 min.
• TCM Lysis was terminated by adding TCM and T cells were purified by incubation on nylon wool (Polysciences, Warrington, PA) for 1 h at 37°C (Julius et al . , Eur. J. Immunol. 3: 645. (1973)) . T cells were collected in the effluent and contained >90% Thy-1+ and ⁇ 5% MHC class 11+ cells as assessed by flow cytometry.
• mAb monoclonal antibodies
• the monoclonal antibody 2C11 (anti-CD3e chain) was used to cross-link the TCR receptor and mimic the agonistic effects cf antigen peptide/major histocompatabilty (MHO stimulation.
• a co-stimulatory signal was provided through the CD28 molecule by using anti-CD28 mAb (37.51) .
• Antibody ligaticn of the CD28 molecule and cross-linking of the TCR has been demonstrated to initiate specific signal transduction events leading to IL-2 production and T cell proliferation.
• SRBC sheep red blood cells
• TCS Biologicais Buckingham, UK
• anti-CD3e-chain mAb 145-2C11
• Pharmingen San Diego, CA
• anti-CD28 mAb PV-1
• polyclonal hamster IgG control hamster IgG was a generous gift from Dr. C. June (NMRI, Bethesda, MD)
• goat anti-hamster IgG Ab phorbol 12-myristate 13-acetate ;?MA) , calcium ionophore
• GA15 Cell treatment and stimulation.
• GA15 (1 x 10 6 cells) were pre-treated with bromelain (15 ⁇ g/ml or 50 ⁇ g/ml, diluted in phosphate buffered saline, pH 7.4) for 30 min prior to second messenger stimulation. Cells were then washed twice by repeated c ⁇ ntrifugation (1500 rpm, Sorvall RT 6000B refridgerated centrifuge; DuPont) and re-suspension in RPMI. Control cells were treated with PBS vehicle alone.
• Cells were stimulated for various lengths of time with either calcium ionophore (l ⁇ M) , PMA (10 ng/ml) or ionophore and PMA combined. After stimulation the cells were lysed and assessed for tyrosine phosphorylated proteins as described below.
• murine splenic T ceils were cultured in 96-well, flat bottom, microculture plates (Corning, Corning, NY, USA) at 10 5 cells per well.
• Cells were stimulated with immobilized (plate-bound) anti-mouse CD3e mAb (145-2C11) (Leo et al . , Proc . Natl . Acad. Sci . USA. , 84, 1374, 1987) at 100 ⁇ g/ml and soluble anti-mouse CD28 mAb (37.51) (Gross et al . , J. Immunol . , 144, 3201, 1990) at 10 ⁇ g/ml.
• [3H] thymidine for an additional 16 hours.
• Cells were harvested and proliferation assessed by liquid scintillation counting. Units were calculated using recombinant murine IL-2 (Pharmingen, San Diego, CA, USA) as the standard.
• FACS fluorescent activated cell sorting
• RNA recovered from samples in a final reaction volume of 25 ⁇ l was 3 ⁇ g.
• PCRs were performed in duplicate for each sample in a final volume of 25 ⁇ l using 2.5 ⁇ l of reverse transcribed mRNA sample as cDNA template.
• Oligonucleotides specific for IL-2, IL-4, IFN- ⁇ and the housekeeping gene hypoxantine-guanineohosphoribosyl transferase (HPRT) and other components cf the PCRs were as previously described (Svetic et al (1991) supra) .
• PCRs consisted of a denaturation step at 95°C for 1 minute, an annealing step at 55°C for 1 minute and an extension step at 72°C for 2 minutes.
• PCR products were detected by size-fractionating amplified DNA by agarose- gel electrophoresis and transferring the DNA to Hybond N+ nylon membrane according to the manufacturers instructions (Amersham, Buckinghamshire, UK) .
• cytokine mRNA was revealed using cytokine-specific oligonucleotides (Svetic et al (1991) supra) labelled with horse-radish peroxidase ⁇ -IRP) and reacted- with the ECL chemiiuminescence detection system, as described by the manufacturer (Amersham) . Specific signals were recorded on autoradiographic film (Kodak, Rochester, NY) and analysed on a Sharp JX-3F6 computing densitometer (Sharp, Japan) using Phoretix 1-D software (Phoretix International, Newcastle, UK) .
• Cell phosphotyrosine blots were performed as described by Thomas et al . ( Cell , 68, 1031-1040, 1992).
• 3riefly cell 0 lysates from 1 x 10' GA15 cells were prepared by lysing the cells in ice-cold lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 4mM EDTA, 1% Triton X-100, 4 mM sodium orthovanadate, lmM PMSF, 50mM NaF, 10 ⁇ g/ml leupeptin) for 30 min with continual rotation. Lysates were 5 clarified (14,000 xg for 2 min), and suspended in SDS-PAGE sample buffer and boiled for 5 min.
• Immunoblctting of separated proteins for detection cf MAP kinase was conducted using rabbit anti-rat MAP kinase R2 (erkl-CT) polyclonal IgG, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG.
• the anti-MAP kinase antibody used recognises the 42 kda, 43 kda and 44 kda MAP kinases encoded by the erkl gene, mapk gene and mpk gene, respectively.
• Cells were suspended at 5-10 x 10 7 cells/ml in RPMI 1640 at 37°C 5 min prior to stimulation.
• PMA and calcium ionophore A23187 were used at 20 and 500 ng/ml, respectively.
• Cross-linking of CD3e and CD28 mAbs was carried out by incubating T cells on ice for 30 min in the presence of 10 ⁇ g/ml of each mAb. After excess antibodies were washed away, cells were suspended at 5-10 x 10 7 ceils/ml in RPMI 1640 (all at 4°C) . Cross-linking was performed at 37°C with 10 ⁇ g/ml goat anti-hamster IgG. Cells were stimulated for times indicated in figure legends and the text.
• Stimulation was terminated by the addition of ice-cold stop buffer, yielding a final concentration of 0.5% Triton X-100, 25 mM Tris, pH 7.2, 75 mM NaCl, 400 mM ⁇ DTA, 10 mM sodium fluoride, 400 mM sodium crthovanadate, 10 mM sodium pyrophosphate, 74 mg/ml leupeptin, 740 mM PMSF and 74 ⁇ g/ml aprotinin. After lysis at 4 C C, samples were centrifuged at 12,000 rpm (13,200 g) , 4°C for 15 min.
• Postnuclear supernatants were collected and an equal volume of 2 x SDS-PAGE sample buffer '50mM Tris, pH 7, 700 mM 2-ME, 50 % (v/v) glycerol, 2% (w/v) SDS, 0.01% (w/v) bromophenol blue) was added. Proteins were solubilised at 100 °C for 5 min and resolved by SDS-PAGE.
• mice received 3 x 200 ⁇ l intravenous (i.v.) injections of 200 ⁇ g bromelain or and equal volume of 0.9% NaCl (diluent for bromelain) on days 1, 4 and 6 of the experiment. Both treatment groups received either a 100 ⁇ l intraperitoneol (i.p.) injection of 10 7 SRBC or an equal volume of 0.9% NaCl (diluent for SRBC) on day 4 of the experiment. Mice were killed on day 7, their spleens were removed and splenocytes isolated as described in the cell preparation section.
• the number of 3 cells secreting antibodies specific for the SRBC antigen was determined by an assay based on the original method of Jerne and Nordin, described by Weir (1986) . Briefly, assays were performed in 160 ⁇ l, consisting of 5 x 10 s splenocytes, 6 x 10 ri SRBC and 1:27 guinea pig complement in RPMI 1640. 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 h, prior to counting plaque forming cells ⁇ PF (i.e., B cells secreting Ab specific for SRBC) . RESULTS
• Figure 5 shows that a polyclonal antibody raised against a highly conserved peptide from MAPk (Erk 1) recognises two proteins of Mr 42,000 and 44,000 (presumably erk 1 and erk 2, respectively; .
• the 42 kda protein displays an electophoretic mobility similar to the 42 kda phosphctyrosine-containing protein detected in stimulated T- ceils.
• Tl ⁇ e 44 kda band also correlates with a protein which is tyrosine phosphorylated independent of stimulation with PMA plus ionophore.
• a protein cf 48 kda and other low molecular weight proteins were also detected, however as these bands were also detected in control immunoblots with no anti-MAPk addition, reactivity was thought to be non-specific.
• tyrosine phosphorylation when ceils were stimulated with PMA and calcium ionophore A23187.
• bromelain markedly reduced tyrosine phosphorylation of a 42 kDa protein called MAP kinase.
• MAP kinase a 42 kDa protein
• T cells were stimulated for 24 hours with immobilised anti-CD3e mAb in the presence of anti-CD28 mAb, as described above.
• T-ceils were cultured in the absence of antibody or in the presence of anti-CD28 or anti-CD3e alone to serve as controls.
• FIG. 8 shows results from a single experiment.
• T cells cultured in the absence of antibody or with anti-CD28 alone did not produce detectable levels of IL-2 (data not shown) .
• immobilised anti-CD3e alone produced barely detectable levels of 11-2.
• T cells were - * 5
• bromelain might cause reduced cytokine production, but increase responsiveness to growth factors, such as IL-2 and IL-4 (possibly by modifying cell surface receptors for such growth factor or by increasing expression via effects on signalling cascades), in T cells.
• growth factors such as IL-2 and IL-4
• bromelain has previously been demonstrated to remove specific T-celi surface molecules (Hale and Haynes, J. Immunol . , 149:3809-3816 (1992)) we nvesti ⁇ ated if bromelain was removing CD3e and CD28 -53
• T cells were isolated from the spleens of healthy Balb/c mice and stimulated with either PMA and A23187 or immobilised anti-CD3e and soluble anti-CD28 mAbs as described for GA15 ceils above.
• Splenic T ceils were isolated and then cultured for 48 h in TCM containing 5 ng/ml PMA as described by Vandenberghe et al , J. Exp. Med. 175: 951-960 (1992)), prior to stimulation for analysis of tyrosine phosphorylation.
• the reason for this pre-culture in PMA is that increased tyrosine phosphorylation is very difficult to detect in resting T cells (Vandenberghe ec al , (1992) supra) .
• bromelain may activate these cells.
• bromelain caused decreased phosphorylation of proteins, particularly MAP kinase, in GA15 ceils (a T-ceil hybridoma) .
• bromelain treatment of GA15 cells resulted in decreased IL-2, IL-4 and IFN- ⁇ when stimulated with phorbol ester and ionophore.
• bromelain treatment of normal murine splenic T-cells caused a similar reduction in 11-2, IL-4 and IFN- ⁇ when provided with a more physiological stimuli ie, ⁇ CD3e and UCD28 which provides a signal via the T-cell receptor and a co-stimulatory signal via CD28 respectively.
• bromelain could have multiple effects on T ceils (both stimulatory and inhibitory, depending on the ceil type studied) .
• ligands for ceil surface molecules were used to stimulate cells (i.e., anti-CD3e and anti-CD28 mAbs)
• bromelain treatment increased cytokine mRNA accumulation in GA15, but reduced cytokine mRNA in normal splenic T cells, despite causing an increase in proliferation in these cells.
• mice immunised with saline produced very few PFC, regardless of whether they were pre-treated with bromelain cr saline (control) ( Figure 15) .
• bromelain treatment caused increased proliferation cf SRBC-specific T cells (similar to the increased proliferaticn seen in experiments conducted with splenic T-cells in vitro, Figure 10) , resultin- in enhanced T cell help for SRBC-specific Ab production by B cells.
• bromelain was directly stimulating B cells cr some ether cell involved in the antibody response. Regardless of the precise mechanism involved, this result suggests a novel application for bromelain as an adjuvant.
• Bromelain has been reported to inhibit the secretory effects of various second messengers such as cyclic AMP, cyclic GMP and Ca2+ in intestinal cells.
• the mechanism of action of bromelain is unknown, but is thought to act proximal to the accumulation of cyclic nucleotides in the cells.
• bromelain In view of the effects of bromelain on intestinal cells, and the role that second messengers and protein phosphorylation play in these events, we tested whether bromelain could inhibit signal transduction systems in other ceils. Specifically, we examined the effect of bromelain en the inositol pathway and tyrosine phosphorylation required for the production of cytokines-
• bromelain can either inhibit or stimulate tyrosine phosphorylation of proteins when stimulated by phorbol esters and calcium ionophores, and antibodies directed against surface molecules.
• MAPk mitogen activated protein kinase
• MAPk becomes phosphorylated upon stimulation of T-ceils with phorbol esters and calcium ionophores, and is also phosphorylated upon ligation of the T-ceil receptor and a costimulatory molecule.
• a 42 kda protein which corresponds to the literature value of 42 kda is phosphorylated in GA15 cells upon stimulation with phorbol ester and ionophore. This protein was not phosphorylated or phosphorylation was markedly reduced when cells were treated with bromelain.
• MAPk is important for IL-2 production, as it is able to phosphorylate other proteins such as c-Jun, which is required for the initiation of IL-2 transcription. Therefore we tested the ability of bromelain to inhibit IL-2 production in murine splenic T cells. Interestingly, T cells that were pre-treated with bromelain, produced markedly less IL-2 than T-cells that were pre-treated with PBS alone, following stimulation with anti-CD3e mAb and anti CD28 mAb.
• bromelain inhibits tyrosine phosphorylation by inhibiting tyrosine kinase activity, or by stimulating a phcsphatase that results in dephosphorylation of proteins (for example activating CD45) .
• Herbimycin A has been shown to inhibit the early biochemical events of antigen receptor-stimulated T-cell activation and is correlated with inhibition of protein tyrosine kinase activity. Herbimycin, however cannot inhibit effects of phorbol esters and ionophore stimulation.
• bromelain can inhibit effects of T-cell receptor stimulation (demonstrated in antibody ligation experiments) and effects of second messengers (phorbol ester and ionophore) .
• 3romelain has different effects from rapamycin which is also reported to have immunosuppressive properties. Unlike rapamycin, bromelain inhibits tyrosine phosphorylation of MAP kinase (Chung ec al . , Cell 69, 1227-1236, 1992) . Cyclosporin A, another immunosuppresent inhibits calcinuerin, again distinct from the action cf bromelain.
• bromelain has been shown to inhibit tyrosine phosphorylation in T cells, it can therefore be predicted that bromelain too may have potential as an immunosuppressive agent.
• Herbimycin A has also been shown to induce differentiation in a number of cell lines and in one case this has been correlated with inhibition of protein tyrosine kinase activity (Kondo et al . , J. Cell . Biol . , 190, 285-293, 1989) .
• bromelain effects tyrosine phosphorylation patterns in T cells indicates that cellular events resulting from this signalling mechanism could be manipulated by the use of bromelain.
• bromelain could modulate cellular events resulting from signalling mechanisms in a wide range of cells.
• bromelain treatment of T cells could modify cell surface receptors to increase binding to ligands.
• bromelain was only thought to affect T cells by cleaving surface receptors (Hale and Haynes, (1992) supra) . Therefore, we believe that we have found two more mechanisms (in addition to cleaving cell surface molecules) by which bromelain effects T cells (i.e., modifying tyrosine phosphorylation and modifying specific cell surface receptors to increase binding to their physiological ligands) .
• bromelain could be used to modify the following cellular processes
• bromelain may have an application for preventing toxic shock (inflammatory cytokine production by an individual is an important contributor to toxic shock) .
• toxic shock inflammatory cytokine production by an individual is an important contributor to toxic shock
• Tyrosine kinase inhibitors have been demonstrated to inhibit spetic shock in vivo (Novogrodsky et al , Science. 264: 1319-1322 (1994)). Similarly, bromelain may be used to prevent allergic reactions. Inflammatory cytokines and other cellular pro'ducts such as histamine are released from cells following exposure to allergens. The signalling cascades which lead tc secretion of inflammatory products from cells involve tyrosine phosphorylation.
• bromelain can stimulate normal splenic T ceil proliferation (Figure 10) . Given the fact that as a naive T cell proliferates it can differentiate into a specific type of cytokine producing cell, we believe that bromelain may also be able to effect this differentiation process.
• the potential application for using bromelain to stimulate T cell proliferation are the same as for increasing cytokine production. However, in addition to increased T cell proliferation which would lead to more cells capable of producing cytokines, there will also be more cells to provide cell- ⁇ eil interactions, which, in addition to cytokine production, are also a vital component of an immune response.
• Another advantage of promoting cellular differentiation is in leukemias, or T-cell cancers, whereby disease results because of an increased population of undifferentiated T-cells (N.B. we do not anticipate that bromelain would stimulate proliferation of abnormal T cells, as data generated in GA15 cells, a T-cell hybridoma, demonstrate that bromelain inhibits proliferation of these cells) .
• bromelain causing an increase in normal T cell proliferation could be that it prevents programmed cell death (apoptosis) .
• a decrease in cell death would lead to more cells becoming available tc incorporate :' H-TdR (used to measure proliferation) .
• Apoptosis is specific event whereby cells are stimulated to destroy their own DNA and die. It is an essential event in most immune responses (to prevent the accumulation of too many cells) , but can also have immunosuppressive consequences in some instances, such as in HIV infection and ageing (i.e., too many cells die and not enough are left to combat infection)
• bromelain has been shown to modify host signalling pathways (Figure 13a) , and in particular inhibit tyrosine phosphorylation of MAPkinase ( Figure 2) , we believe that another potential application for bromelain could be to inhibit either parasite/pathogen invasion or their survival in cells.

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