WO1993005394A1 - Identifying membranolytic compounds and precursors thereof - Google Patents

Identifying membranolytic compounds and precursors thereof Download PDF

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Publication number
WO1993005394A1
WO1993005394A1 PCT/US1991/006580 US9106580W WO9305394A1 WO 1993005394 A1 WO1993005394 A1 WO 1993005394A1 US 9106580 W US9106580 W US 9106580W WO 9305394 A1 WO9305394 A1 WO 9305394A1
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leu
ome
cells
dipeptide
esters
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PCT/US1991/006580
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French (fr)
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Peter E. Lipsky
Dwain L. Thiele
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Board Of Regents, The University Of Texas System
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Priority to PCT/US1991/006580 priority Critical patent/WO1993005394A1/en
Priority to EP91916121A priority patent/EP0604416A1/en
Publication of WO1993005394A1 publication Critical patent/WO1993005394A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/05—Dipeptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)

Definitions

  • the present invention concerns certain membraneolytic substances, as well as testing for certain cellular effects of dipeptide or dipeptide analog esters or N-substituted dipeptide amides as, for example, in the ablation of cell-mediated immune responses.
  • dipeptide or dipeptide analog esters or N-substituted dipeptide amides are examples of dipeptide or dipeptide analog esters or N-substituted dipeptide amides.
  • L-leucine methyl ester (Leu-OMe) has previously been used as a lysoso otropic agent (Thiele et al . (1983) J. Immunol . , 131:2282-2290; Goldman et al . (1973) J. Biol . Chem . , 254:8914) .
  • the generally accepted lysosomotropic mechanism involved leu-OMe diffusion into cells and into lysosomes, followed by intralysosomal hydrolysis to leucine and methanol. The more highly ionically charged leucine, largely unable to diffuse out of the lysosome was though to cause osmotic lysosomal swelling and rupture.
  • L-amino acid methyl esters have been specifically shown to cause rat liver lysosomal amino acid increases (Reeves (1979) J. Biol. Chem. V 254, pp 8914-8921).
  • Leucine methyl ester has been shown to cause rat heart lysosomal swelling and loss of integrity (Reeves et al. , (1981) Proc. Nat'l. Acad. Sci., V 78, pp 4426-4429).
  • mononuclear phagocytes MP polymorphonuclear leucocytes PMN natural killer cells NK peripheral blood lymphocytes PBL peripheral blood ononuclear cells PBM cytotoxic T-lymphocytes CTL glass or nylon wool adherent cells AC glass or nylon wool non-adherent cells NAC
  • Glycylphenylalanine diazomethane Gly-Phe-CHN 2 Mean survival time MST
  • Natural killer cells are large granular lymphocytes that spontaneously lyse tumor cells and virally-infected cells in the absence of any known sensitization. This cytotoxic activity can be modulated by a host of pharmacologic agents that appear to act directly on NK effector cells.
  • NK activity has been shown to be augmented after exposure to interferons (Gidlund et al. , Nature V 223, p 259), interleukin 2, (Dempsey, et al. (1982) J. Immunol. V 129, p 1314) (Do zig, et al. (1983) J. Immunol. V 130, p 1970), and interleukin 1 (Dempsey et al.. (1982) J.
  • Human peripheral blood mononuclear cells are capable of mediating a variety of cell-mediated cytotoxic functions.
  • spontaneous lysis of tumor cells and virally-infected cells is mediated by natural killer cells (NK) contained within the large granular lymphocyte fraction of human PBM Timonen et al.. (1981) v. J. Exp Med. V 153 pp 569- 582.
  • NK natural killer cells
  • additional cytotoxic lymphocytes capable of lysing a broad spectrum of tumor cell targets can be generated in in vitro cultures (Seeley et al. (1979) J. Immunol. V 123, p 1303; and Grimm et al. (1982) J. Exp. Med.
  • lymphokine activated peripheral blood mononuclear phagocytes are also capable of lysing certain tumor targets (Kleinerman et al. (1984) J. Immunol. V 133, p 4). Following antigen-specific stimulation, cell-mediated lympholysis can be mediated by cytotoxic T lymphocytes (CTL) .
  • CTL cytotoxic T lymphocytes
  • V 127, p 2575 are found on both CTL and NK while the antigen identified by OKM1 is shared by MP and NK (Zarling et al. (1981) J. Immunol. V 127, p 2575; Ortaldo et al. (1981) J. Immunol. V 127, p 2401; Perussia et al. (1983) J. Immunol. V 130, p 2133; and Breard et al. (1980) J.
  • the present invention includes a process for identifying a substance taken up by and deactivating NK or CTL cells.
  • the process comprises the steps of screening candidate substances to determine those which competitively inhibit the uptake or binding by PBL, NK or CTL cells of L-leu-L-leu-OMe or of a substance which itself competitively inhibits L-leu-L-leu-OMe uptake or binding; incubating substances determined to be inhibitory with membrane-enveloped labels and DPPI; determining lysis of the membrane-enveloped labels caused by the incubated inhibitory substances; and lastly identifying substances which are taken up by and deactivate NK or CTL cells as those effectively mediating lysis of membrane-enveloped labels.
  • membrane-enveloped label is that of labeled human erythrocytes, but other types of cells as well as lysosomes may also be used.
  • Those skilled in the art will recognize that a wide variety of appropriately labeled naturally-occurring or artificial membrane structures might serve the same purpose as that described herein for labeled human erythrocytes.
  • dipeptide or dipeptide analog esters or N-substituted amides are preferred candidate substances to be subject to this method.
  • the dipeptides or analogs thereof preferably comprise L-amino acids or structural analogs thereof with hydrophobic side chains. They may be used to deactivate NK or CTL cells.
  • Preferable natural amino acids of such dipeptides or dipeptide analogs are leucine, phenylalanine, valine, isoleucine, alanine, proline, glycine or aspartic acid beta methyl ester.
  • Preferable dipeptides are L leucyl L-leucine, L-leucyl L- phenylalanine, L-valyl L-phenylalanine, L-leucyl L- isoleucine, L-phenylalanyl L-phenylalanine, L-valyl L- leucine, L-leucyl L-alanine, L-valyl L-valine, L- phenylalanyl L-leucine, L-prolyl L-leucine, L-leucyl L- valine, L-phenylalanyl L-valine, L glycyl L-leucine, L- leucyl L-glycine or L-aspartyl beta methyl ester L- phenylalanine.
  • the most preferable dipeptides are glycyl L- phenylalanine, L-leucyl L-leucine, L-leucyl L- phenylalanine, L-valyl L-phenylalanine, L-phenylalanyl L- leucine, L-leucyl L-isoleucine, L-phenylalanyl L- phenylalanine and L-valyl L-leucine.
  • the amide N-substituent or ester alcohol residue of the dipeptide or dipeptide analog derivative is preferably a benzyl or alkyl of up to about four carbon atoms such as propyl, isopropyl, butyl or isobutyl. Larger alkyl groups may be used. Aralkyl or aryl derivatives, for example benzyl and napthyl may be particularly effective.
  • the present invention further involves a method for deactivating NK or CTL cells comprising the step of treating said cells with an aqueous solution comprising a biologically effective level of substances determined by the above method such as a dipeptide or dipeptide analog ester or N-substituted dipeptide or analog amide form consisting essentially of L- ⁇ -amino acids with hydrophobic side chains, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted amide.
  • a dipeptide or dipeptide analog ester or N-substituted dipeptide or analog amide form consisting essentially of L- ⁇ -amino acids with hydrophobic side chains, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted amide.
  • This aqueous solution more preferably comprises a biologically effective level of a dipeptide or dipeptide analog in ester or substituted amide form, said dipeptide or dipeptide analog consisting essentially of at least one of L-leucine, L-phenylalanine, L-valine, L-isoleucine, L-alanine, L-proline, glycine, and L- aspartic acid beta methyl ester, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted amide.
  • the cells being deactivated may be in vitro or may be yet within an animal. In the latter case the animal is parenterally administered a biologically effective amount of the dipeptide or dipeptide analog in ester or substituted amide form.
  • This deactivation of NK and CTL cells may also be adapted as a method for inhibiting bone marrow graft versus host disease comprising the step of contacting the bone marrow cells to be grafted with an aqueous solution comprising a biologically effective level of substances determined by the above method such as a dipeptide or dipeptide analog ester or N-substituted dipeptide or analog amide form consisting essentially of L- ⁇ -amino acids with hydrophobic side chains, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted amide.
  • a biologically effective level of substances determined by the above method such as a dipeptide or dipeptide analog ester or N-substituted dipeptide or analog amide form consisting essentially of L- ⁇ -amino acids with hydrophobic side chains, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted
  • This aqueous solution again more preferably comprises a biologically effective level of a dipeptide or dipeptide analog in ester or substituted amide form, said dipeptide or dipeptide analog consisting essentially of at least one of L-. leucine, L-phenylalanine, L-valine, L-isoleucine, L- alanine, L-proline, glycine, and L-aspartic acid beta methyl ester, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted amide.
  • the biologically effective level should be between about 1 micromolar and about 250 micromolar, depending upon the particular agent being used and its effectiveness.
  • the present invention additionally involves a method of inhibiting the rejection of tissue transplanted into a host.
  • This method comprises the steps of identifying a prospective transplant recipient; and treating the prospective recipient with an aqueous solution comprising a biologically effective level of substances determined by the above method such as a dipeptide or dipeptide analog ester or N-substituted dipeptide or analog amide form consisting essentially of L- ⁇ -amino acids with hydrophobic side chains, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted amide.
  • a biologically effective level of substances determined by the above method such as a dipeptide or dipeptide analog ester or N-substituted dipeptide or analog amide form consisting essentially of L- ⁇ -amino acids with hydrophobic side chains, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted amide.
  • This aqueous solution also more preferably comprises a biologically effective level of a dipeptide in ester or substituted amide form, said dipeptide consisting essentially of at least one of L- leucine, L-phenylalanine, L-valine, L-isoleucine, L- alanine, L-proline, glycine, and L-aspartic acid beta methyl ester, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted amide.
  • the method of the present invention provides for identifying rejection-inhibiting compounds.
  • the biologically effective amount administered is generally between about 10 mg/kg body weight and 300 mg/kg body weight; preferably about 1 X 10 " 4 moles/kg body weight.
  • the aqueous solutions of the present invention include any of those suitable for in vivo administration free of toxins and preferably being of an approximate physiological pH and osmolality.
  • Preferred dipeptide or dipeptide analog esters of the present invention include those formed with an alkaryl alcohol, most preferably benzyl alcohol.
  • alkaryl is used herein to indicate an alkyl group bound in amide or ester linkage to the dipeptide and having an aryl group bound thereto.
  • a particularly preferred dipeptide ester identified by the method of the present invention is L-leucyl-L-leucyl benzyl ester.
  • aralkyl is used herein to indicate an aryl group bound in amide or ester linkage to a dipeptide or dipeptide analog of the present invention and having an alkyl group bound thereto. It is understood that those skilled in the art may make many variations in group substitutions on the alkyl, aryl, aralkyl and alkaryl groups substituents of the present invention and still be within the presently claimed invention.
  • Preferred dipeptides identified by the method of the present invention include L-leucyl, L-leucine, L-leucyl L-phenylalanine, L-valyl L-phenylalanine, L-phenylalanyl L-leucine, L-leucyl L-isoleucine, L-phenylalanyl L- phenylalanine, L-valyl L-leucine, L-leucyl L-alanine, L- valyl L-valine, L-prolyl L-leucine, L-leucyl L-valine, L- phenylalanyl L-valine, glycyl L-leucine, L-leucyl glycine, and L-aspartyl beta methyl ester L- phenylalanine.
  • a more preferred group of dipeptides is L-leucyl L-leucine, L-leucyl L-phenylalanine, L-valyl L- phenylalanine, glycyl L-phenylalanine, L-phenylalanyl L- leucine, L-leucyl L-isoleucine, L-phenylalanyl L- phenylalanine and L-valyl L-leucine.
  • the present invention describes a general method for identifying substances deactivating NK or CTL cells.
  • This general method comprises the step of treating said cells with an aqueous solution comprising a biologically effective level of the identified substance, which competitively inhibits lymphocyte uptake of Leu-Leu-OMe (or of a compound which itself inhibits such uptake) and is polymerized by dipeptidyl peptidase I to form a membranolytic product (shown to be effective by lysis of erythrocytes) .
  • An analogous method for inhibiting bone marrow graft versus host disease may also be so generalized.
  • Such a method comprises the step of contacting bone marrow cells to be grafted with an aqueous solution comprising a biologically effective level of substances determined by the above method, such as a dipeptide or dipeptide analog ester or N-substituted dipeptide or dipeptide analog amide form consisting essentially of L- ⁇ -amino acids with hydrophobic side chains, which competitively inhibits lymphocyte uptake of Leu-Leu-OMe and is polymerized by dipeptidyl peptidase I to form a membranolytic product.
  • substances determined by the above method such as a dipeptide or dipeptide analog ester or N-substituted dipeptide or dipeptide analog amide form consisting essentially of L- ⁇ -amino acids with hydrophobic side chains, which competitively inhibits lymphocyte uptake of Leu-Leu-OMe and is polymerized by di
  • the present invention also includes a process for identifying a substance for inactivation of cells containing dipeptidyl peptidase I (including NK or CTL cells) , the process comprising the steps of testing a compound for inhibition of uptake or binding by PBL of labeled L-Leu-L-Leu-OMe or of a compound itself inhibiting such uptake, and assaying inhibitors to identify those taken up by PBL's and converted by dipeptidyl peptidase I to form a membranolytic product, which product is characterized as being insoluble in 10% aqueous trichloroacetic acid.
  • a membranolytic oligopeptide aryl or alkyl ester or N-aryl substituted oligopeptide amide may be obtained by the present invention by a process comprising identifying a dipeptide or dipeptide analog aryl or alkyl ester or N- aryl substituted dipeptide amide which is converted by dipeptidyl peptidase I to form an oligopeptide or oligopeptide analog aryl or alkyl ester or N-substituted oligopeptide amide having a structure membranolytic to red blood cells, elucidating the structure of the oligopeptide membranolytic product, and preparing a quantity of this product.
  • An alternate process in the present invention for preparing a membranolytic substance comprises the steps of identifying a compound competitively inhibiting the uptake by PBL of L-leu-L-leu-OMe or of a compound which itself inhibits said uptake, ascertaining an identified compound which is converted by dipeptidyl transferase I to a membranolytic substance, structurally defining said membranolytic substance, and preparing a quantity of said structurally defined membranolytic substance.
  • membranolytic substances may be formed by exogenously using dipeptidyl peptidase I to catalyze membranolytic substance formation from hydrophobic amino acid dipeptides.
  • Figure 1 shows results of the assay for competitive inhibition of [ 3 H]-Leu-Leu-OMe uptake by human peripheral blood lymphocytes.
  • Figure 2 shows results of the assay for generation of membranolytic metabolites in sl Cr labeled human erythrocytes.
  • Figure 3A shows a thiopeptide analog of Leu-Leu-OMe
  • Figure 3B shows a schematic structure of leucyl- (N)methyl leucine methyl ester.
  • Figures 4A, 4B and 4C show various examples of dipeptide esters with non-physiological R groups.
  • Figure 5 shows that whereas ablation of NK function during incubation with Leu-OMe can be blocked by lysosomotropic agents, there is a product formed during incubation of Leu-OMe with MP or PMN which has effects on NK function no longer blocked by lysosomal inhibitors.
  • Figure 6 shows Leu-OMe products of PMN in terms of radioactivity and NK suppressive effects of TLC fractions.
  • Figure 7A shows the Cl mass spectrum of TLC fractions with NK toxic activity
  • Figure 7B shows the Cl mass spectrum of synthetic Leu-Leu-OMe.
  • Figures 8A and 8B show the effects of various agents on losses of NK function from MP-depleted lymphocytes.
  • Figure 9 shows the NK-toxicity of various dipeptide esters.
  • Figure 10 shows the loss of NK and MP from PBM incubated with Leu-Leu-OMe at various concentrations.
  • Figure 11 shows the toxicity of various Leu-Leu-OMe concentrations for selected cell types.
  • Figure 12 shows the Leu-Leu-OMe mediated elimination of precursors of cytotoxic T lymphocytes activated NK (A C NK) and NK.
  • Figure 13 shows the sensitivity of activated NK and CTL to treatment with Leu-Leu-OMe.
  • Figure 14 shows the time-dependent uptake of Leu- Leu-OMe by PBL.
  • Figure 15 shows the concentration dependence of Leu- Leu-OMe uptake by PBL.
  • Figure 16 schematically describes the effects upon RBC lysis by Leu-Leu-OMe in the presence ( ⁇ ) and absence (•) of exogenous DPPI.
  • Figures 17A through 17H show RBC lysis as dependent upon the presence ( • +) or absence (-) of DPPI with concentrations gradients of different dipeptide methyl esters.
  • Figure 18 shows the survival of B6D2F1 skin grafts applied to ATXBM, TCD mice in the presence or absence of reconstitution with control or Lue-Leu-OMe treated B6 SpC.
  • Figure 19A shows that Leu-Leu-OMe treatment of B6 donor SpC prevents lethal GVHD in euthymic B6D2F1 recipients
  • Figure 19B shows similar results with thymectomized B6D2F1 recipients.
  • the present invention concerns identifying and testing new compounds useful in ablating either the functions of particular cell types or the cells.
  • the presently described invention relates to the discovery that certain peptide or peptide analog esters and N- substituted amides are cytotoxic to particular cell types.
  • alkyl, aralkyl and aryl esters or amides of dipeptides or dipeptide analogs consisting essentially of ⁇ -amino acids with hydrophobic side chains may function cytotoxically to deactivate natural killer cells (NK) and cytotoxic T lymphocytes (CTL) .
  • NK natural killer cells
  • CTL cytotoxic T lymphocytes
  • hydrophobic as used herein, is meant uncharged in aqueous solution at physiological pH and also as having no hydroxyl, carboxyl or primary amino groups.
  • Treatment of NK or CTL cells with an effective level of a peptide amide or ester consisting essentially of natural or synthetic amino acids with hydrophobic side chains serves to deactivate the cytotoxic functions of said cells.
  • An effective level varies from circumstance to circumstance but generally lies between about 1 micromolar and about 250 micromolar.
  • An effective level for a whole animal dose generally lies between about 100 mg/kg and about 300 mg/kg.
  • Methyl, ethyl and benzyl esters or amides of peptides consisting essentially of natural or synthetic amino acids having hydrophobic side chains have been specifically found to deactivate natural killer cells (NK) or cytotoxic T lymphocytes (CTL) and other alkyl esters of these peptides (or peptide analogs thereof) are confidently predicted to have similar or superior effects.
  • NK natural killer cells
  • CTL cytotoxic T lymphocytes
  • NK or CTL cells Deactivation of NK or CTL cells with substances such as identified peptide esters or amides should increase the success of allogeneic bone marrow transplants by lowering the incidence of graft-versus-host disease (GVHD) and thus lessening the incidence of transplant rejection.
  • GVHD graft-versus-host disease
  • peptide amides or esters consisting essentially of amino acids with hydrophobic side chains
  • NK or CTL are involved in the pathogenesis of disease.
  • organ transplants in general kidney, heart, liver, pancreas, skin, etc.
  • cytotoxic T cells are at least partially responsible for graft rejection (Mayer et al., J. Immunol. V 134, p 258, 1991 and Rosenberg et al. J. Exp. Med. 165, 1296, 1991) .
  • the in vivo administration of peptide esters or amides of the present invention will be of benefit in preventing allograft rejection.
  • the substances identified by the method of the present invention may be of benefit in other spontaneously occurring disease states.
  • a variety of diseases have been classified as "autoimmune diseases” because of the widely accepted belief that they are caused by disorders in the immune system which cause immunologic damage to "self".
  • diseases including primary biliary cirrhosis, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, autoimmune hemolytic anemia, etc.
  • various forms of immunologic damage to selected organs occur.
  • the histologic abnormalities which occur in this case in the liver
  • closely resemble those which occur in GVHD or in rejection of a transplanted liver Fennel, (1981)
  • the peptide esters or amides of the present invention should be usable chemotherapeutic agents for patients with natural killer cell tumors (generally leukemias) , although very few reports of these tumors are found in the literature ( Komiyama et al.. (1982) Blood V 60, p 1428 (1982); Itoh et al. (1983) Blood V 61, p 940; Komiyama et al. (1984) Cancer V 54 p 1547.
  • NK cells can kill normal bone marrow cells (Hansson, et al. (1981) Eur. J. Immunol. V 11, p 8) ; second NK cells inhibit growth of blood cell precursors in vitro (Hansson, et al. (1982) J. Immunol. V 129, p 126; Spitzer et al.: Blood V 63, p 260; Torok-Storb et al. (1982) Nature V 298, p 473; Mangan, et al.
  • NK-like cells with the ability to inhibit the formation of red blood cells have been isolated from patients with aplastic anemia (Mangan, et al. (1982) J. Clin. Invest. V 70, p 1148; and Nogasawa et al. (1981) Blood V 57, p 1025) .
  • recent studies in the mouse indicate that NK cells may function to suppress hemopoiesis in vivo (Holmberg et al. (1984) J. Immunol. V 133, p 2933).
  • an effective level is between about 1 x 10-4 moles/kg and about 1 x 10-2 moles/kg.
  • PBM peripheral blood mononuclear cells
  • PMN peripheral blood cells that penetrated sodium diatrizoate-Ficoll gradients and removing erythrocytes by dextran sedimentation and h potonic lysis as previously outlined by Thiele et al. (1985, J. Immunol. , 134:786-793) .
  • All cell exposures to the amino acids, dipeptides or their methyl esters were carried out by suspending cells in Dulbecco's phosphate buffered saline (PBS) and incubating them at room temperature with the reagent at the indicated concentration and time interval. After incubation, the cells were washed twice with Hanks' balanced salt solution and resuspended in medium RPMI 1640 (Inland Laboratories, Fort Worth, TX) supplemented with 10% fetal bovine serum (Microbiological Associates, Walkersville, MN) for assay of function.
  • PBS Dulbecco's phosphate buffered saline
  • Leu-norLeu-OMe appeared slightly less efficient than Leu-Leu-OMe in the assay of Figure 1 but more efficient in the Figure 2 assay. Thus, it would be expected to have activity similar to but slightly less than norLeu-norLeu-OMe in mediating NK toxicity (Table 2) .
  • norLeu-Leu-OMe and Leu-norVal- OMe were found to be less active than Leu-Leu-OMe in both screening assays, so the finding that they were both less efficient than Leu-Leu-OMe in mediating NK toxicity supports the predictive value of the screening assays.
  • norVal-Leu-OMe an agent that was totally inactive in the screening assay of Figure 1, had no detectable NK toxicity (see Table 2) .
  • MP or PMN prepared as in Example 1 at a concentration of 25 X 10 6 per ml were suspended in PBS and incubated with 25 mM Leu-OMe for 20 minutes at 22°C. Cell suspensions were then centrifuged at 1000 g for 10 minutes and the supernatants harvested and freeze-dried at -70°C, 100 millitorr atmospheric pressure. In some experiments, Leu-OMe-treated MP or PMN were sonicated to increase the yield of the reaction product. Samples were then extracted with methanol for application to thin layer chromatography (TLC) plates (200 micromolar x 20 cm 2 , Analtech, Newark, Delaware) .
  • TLC thin layer chromatography
  • PBM prepared as in Example 1 were incubated with various potential NK toxic agents in the presence or absence of various lysosomal inhibitors for 40 minutes, washed to remove the inhibitor, incubated for 18 hours to permit recovery from any transient inhibition caused by lysosomotropic agents and then tested for NK activity.
  • NK neurotrophic factor
  • 5 mM Leu-OMe ablated all NK activity. This activity of Leu-OMe was largely prevented by chloroquine, NH 4 C1, or Ile-OMe.
  • the products generated by MP or PMN, after exposure to Leu-OMe also completely removed all NK activity from PBM.
  • Inhibitors of lysosomal enzyme function prevent generation of an NK toxic product.
  • PBM (5 X 10 6 /ml) or PMN (25 X 10 6 /ml) preincubated with 25 mM Leu-OMe for 30 minutes were added to cells to be ablated. Cells were incubated with these agents for another 30 minutes at 22°C, then washed and cultured for 18 hours at 37°C before assay of the ability to lyse K562 cells. Data are expressed as percentage of control cytotoxicity observed with an effector:target ratio of 40:1 (results at other E:T were similar) .
  • NK function is mediated by a metabolite of Leu-OMe.
  • PMN 25xl0 6 /ml
  • MP-depleted lymphocytes 2.5xl0 6 cells/ml
  • Samples were considered to contain an NK toxic product when percent specific lysis was less than 25% of control.
  • Figure 6 shows these results.
  • Leu-Leu-OMe When Leu-Leu-OMe was synthesized from reagent grade Leu-Leu, by incubation in methanol hydrochloride, it was found to have TLC mobility identical to NK toxic fractions of MP-Leu-OMe or PMN-Leu-OMe incubation mixtures. Furthermore, its Cl mass spectrum as shown in Figure 7B was identical to that of the 258 molecular weight compound found in these incubation fractions.
  • Leu-Leu-OMe was the product generated by MP or PMN from Leu-OMe that was responsible for the selective ablation of NK function from human lymphocytes.
  • Leu-Leu-OMe was synthesized by addition of Leu-Leu to methanolic HCl.
  • TLC analysis revealed less than 2% contamination of this preparation with leucine, Leu-Leu, or leu-OMe, and Cl mass spectral analysis (Figure 7B) revealed no contaminants of other molecular weights.
  • Figure 7A shows the chemical-ionization Cl mass spectra of TLC fractions with NK toxic activity as described in Figure 6;
  • Figure 7B shows the Cl spectrum of Leu-Leu-OMe synthesized from reagent grade Leu-Leu.
  • Leu-OMe was unique among a wide variety of amino acid methyl esters in its ability to cause MP or PMN dependent ablation of NK cell function from human PBM (Thiele et al . (1985), J. Immunol . 234:786-793) .
  • the identification of Leu-Leu-OMe as the MP-generated metabolite responsible for this phenomenon suggested that either MP/PMN did not generate the corresponding dipeptide methyl esters in toxic amounts from other amino acids, or that Leu-Leu-OMe was unique among dipeptide methyl esters in its toxicity for NK cells. Therefore, experiments were carried out to assess the effect of other dipeptide methyl esters on NK cell function.
  • methyl esters of a variety of dipeptides were synthesized and analyzed for the capacity to deplete NK cell function. Each dipeptide methyl ester was assessed in a minimum of three experiments. As is shown by the results displayed in Figure 9, Leu-Leu-OMe is not the only dipeptide methyl ester which exhibits NK toxicity. When amino acids with hydrophobic side chains were substituted for leucine in either position, the resulting dipeptide methyl ester generally displayed at least some degree of NK toxicity.
  • Leu- Phe-OMe, Phe-Leu-OMe, Val-Phe-OMe, and Val-Leu-OMe produced concentration-dependent ablation of NK function at concentrations comparable to those at which Leu-Leu- OMe was active.
  • the sequence of active amino acids was important, however, as evidenced by the finding that Phe- Val-OMe was markedly less active than Val-Phe-OMe.
  • Leu-Ala-OMe was NK inhibiting, whereas 10-fold greater concentrations of Ala-Leu-OMe had no NK inhibitory effects.
  • the resulting dipeptide methyl esters either had greatly reduced NK toxicity, as in the case of Gly-Leu-OMe or Leu-Gly-OMe, or no observed NK inhibitory effects, as in the case of Leu-Arg-OMe, Leu-Tyr-OMe, Ser-Leu-OMe, Lys- Leu-OMe or Asp-Phe-OMe. Furthermore, when the D- stereoisomer was present in either position of a dipeptide methyl ester, no toxicity was observed for NK cells ( Figure 9) .
  • D-Leu-D-Leu-OMe had no effect on Leu-Leu-OMe mediated NK toxicity although high levels of zinc appeared to inhibit this Leu-Leu-OM, when equal concentrations of Leu-OMe, Val-OMe, or Phe-OMe were added to MP or PMN, the concentrations of Val-Val-OMe generated were 50 to 80% of those found for Leu-Leu-OMe, while Phe- Phe-OMe was detected at only 10-30% of the levels of Leu- Leu-OMe. Dipeptide methyl esters were not generated from D-amino acid methyl esters.
  • Figure 9 shows the NK toxicity of dipeptide methyl esters.
  • MP-depleted lymphocytes were treated with varying concentrations of dipeptide methyl esters as outlined in Figures 8A and 8B. Results are given for the mean ⁇ SEM of at least 3 separate experiments with each compound.
  • Beta methyl aspartyl phenylalanine was prepared by methanolic hydrochloride methylation of aspartyl phenylalanine methyl ester.
  • the NK toxicity of both aspartyl phenylalanine methyl ester and beta methyl aspartyl phenylalanine methyl ester was measured as described for the dipeptide methyl esters in Example 8.
  • Table 3 indicates, when the polar side chain of the aspartyl amino acid dipeptide component is esterified with a methyl group, this being a conversion from relative hydrophilicity to substantial hydrophobicity, NK toxicity becomes apparent.
  • the data in Table 3 indicate that a dipeptide methyl ester comprising synthetic hydrophobic (lipophilic) amino acids may be used to inhibit NK function.
  • Asp-(beta-OMe)-Phe-OMe 100 micromolar 250 micromolar 500 micromolar 1000 micromolar
  • Leu-Leu-OMe or Leu-Phe-OMe were suspended in PBS, pH 7.4. Then individual C3H/HeJ mice (25 gram size) were administered by tail-vein injection either 2.5 x 10- 5 moles (6.5 mg) of Leu-Leu-OMe, 2.5 x 10- 5 moles (7.1 mg) Leu-Phe-OMe, or an equal volume of the PBS diluent, this dose being about lxlO- 3 moles per kg.
  • Leu-Leu OMe and Leu-Phe OMe-treated animals did not the control animals exhibited decreased activity and an apparent increase in sleep. Subsequent to this quiescent period no difference in activity or appearance in the mice was noted.
  • mice Two hours post- injection, the mice were sacrificed and their spleen cells were assayed for NK function in a standard 4 hour assay against YAC-1 tumor targets. In all mice, total cell recovery ranged from lxlO 8 to l.lxlO 8 spleen cells per animal. As noted in Table 4, the control mouse spleen cells exhibited greater killing at 25:1 and 50:1 effector to target cell ratios than did the spleen cells of treated mice at 100:1 and 200:1 E/T, respectively. Thus, Leu-Leu-OMe or Leu-Phe-OMe caused a greater than 75% decrease in splenic lytic activity against YAC-1 tumor targets.
  • Anti-MP monoclonal antibodies (63D3) and anti-NK monoclonal antibodies (leu lib) were obtained from Becton Dickinson Monoclonal Center, Inc., Mountain View, CA.
  • T4 enriched populations of T cells were assessed, however, it was clear that even 1000 micromolar Leu-Leu-OMe caused minimal 51 Cr release from this population. In contrast, when N-SRBC positive cells were depleted of OKT4 positive cells, the remaining T8-enriched population produced high levels of 51 Cr release following exposure to Leu-Leu-OMe.
  • the erythroleukemia line K562 demonstrated no significant 51 Cr release or alteration in subsequent proliferative rate (date not shown) upon exposure to 100 micromolar or lower concentrations of Leu-Leu-OMe. With higher concentrations of Leu-Leu-OMe, modest amounts of 51 Cr release and partial loss of proliferative capacity were observed (data not shown) .
  • a variety of cell types of non-lymphoid, non-myeloid origin including human umbilical vein endothelial cells, the human renal cell carcinoma line, Currie, the human epidermal carcinoma line, HEp-2, and human dermal fibroblasts demonstrated no significant Leu- Leu-OMe induced 51 Cr release. Furthermore, incubation of each of these non—lymphoid cell types with 500 micromolar Leu-Leu-OMe had no discernible effect on subsequent proliferative capacity.
  • HS-Sultan a human plasma cell line (Goldblum et al. (1973) Proc. Seventh Leucocyte Culture Conference, ed by Daguilland, Acad. Press N.Y. pp 15-28) , Daudi, a B lymphoblastoid cell line (Klein et al. (1968) Cancer Res. V 28, p 1300), MoLT-4, an acute lymphoblastic T-cell leukemia line (Monowada et al. (1972) J. Nat'l. Cane. Inst. V 49, p 891), and U-937, a human monocyte-like cell line (Koren et al.
  • UTHSCD Geppert, UTHSCD were serially passaged in culture as well while human umbilical vein endothelial cells (a generous gift of Dr. A. Johnson, UTHSCD) were used after one subculture.
  • Epstein Barr virus (EBV) transformed B lymphoblastoid cell lines JM.6 and SM.4 (kindly provided by Dr. J. Moreno, UTHSCD) and cloned EBV transformed B cell lines SDL-G2 and D8-219 (a generous gift of Drs. L. Stein and M. Dosch, Hospital for Sick Children, Toronto, Canada) were maintained in culture in medium RPMI supplemented with 10% FBS.
  • toxicity of Leu-Leu-OMe for a variety of cell populations was assessed by 51 Cr release.
  • cells were labeled with Na 2 51 Cr0 4 (ICN, Plainview, NY) for 60-90 minutes at 37°C and then washed three times. Cells were then suspended in PBS (2.5xl0 6 /ml) and incubated in microtiter plates, 50 microL/well with indicated concentrations of Leu-Leu-OMe for 15 minutes at room temperature.
  • microtiter wells were seeded with cells (5xlO A /well) and cultured for 24 hours at 37°C.
  • Leu-Leu-OMe does have some minimal toxicity for certain non-cytotoxic lymphoid cells such as EBV transformed B cells and K562 cells. Yet, while it is impossible to exhaustively exclude the possibility that certain non-cytotoxic cells might also be equally sensitive to Leu-Leu-OMe-mediated toxicity, at present the ability to function as a mediator of cell mediated cytotoxicity is the one unifying characteristic of the cell types which are rapidly killed by exposure to Leu-Leu-OMe.
  • Figure 12 shows that incubation with Leu-Leu-OMe eliminates precursors of cytotoxic T lymphocytes (CTL) and activated NK-like cells (AcNK) .
  • CTL cytotoxic T lymphocytes
  • AcNK activated NK-like cells
  • Human peripheral blood lymphocytes were incubated in the presence of [ 3 H] labeled Leu-Leu-OMe at 22°C for varying lengths of time. The incubation mixture was then centrifuged through silicone oil to separate the cells from any unbound or non-internalized [ 3 HLeu-Leu-OMe. As demonstrated in Figure 14, the quantity of cell- associated [ 3 H]Leu-Leu-OMe increased in a linear, time- dependent fashion over the first 30 minutes of incubation. As demonstrated in Table 5, when incubations were performed at temperatures below 4°C, no accumulation of [ 3 H]Leu-Leu-OMe by PBL was observed.
  • the amide derivative of Leu-Leu (L-Leu-L- Leu-NH 2 ) ; the D-stereoisomer containing dipeptide ester, D-Leu-D-Leu-OMe; the amino acid analog Leu-OMe; and the tripeptide analog Leu-Leu-Leu-OMe do not competitively inhibit PBL uptake of L-Leu-L-Leu-OMe.
  • the facilitated transport process utilized by PBL in the uptake of Leu-Leu-OMe appears to be relatively specific for L-stereoisomers of dipeptides or dipeptide esters.
  • lymphocyte subsets were highly purified by fluorescence activated cell sorting and then analyzed for dipeptidyl peptidase I activity, the levels of this enzyme within these cells was noted to vary greatly. Of special note, enzyme levels were highest in NK cells, monocytes (M-phi) and the cytotoxic T cell-enriched T8 cell subset. Furthermore, previously documented sensitivity to the toxic effects of Leu-Leu- OMe (second column. Table 9) was shown to be directly proportional to dipeptidyl peptidase I levels (third column, Table 9) .
  • Dipeptidyl peptidase I is a lysosomal thiol peptidase which has been shown to remove amino terminal dipeptides from proteins.
  • this enzyme at neutral pH, incubation of this enzyme with high concentrations of dipeptide esters or amides has been shown to result in production of higher molecular weight polymerization products with the structure (Rl -R 2 ) n -0R' (J. Biol. Chem. 1952; 195:645-656).
  • R 1 and R2 are amino acids with nonpolar side groups, such products are very hydrophobic and water insoluble (J. Biol. Chem. 1952; 195:645-656).
  • Table 11 shows results of an experiment, where 51 Cr- labeled RBCs were incubated with varying concentrations of the methyl esters of the di-, tetra-, and hexa- peptides of leucine. Disruption of erythrocyte membranes was observed following exposure to the very hydrophobic compound (Leu) 6 -OMe. As detailed in Table 12, the specific inhibitor of DPPI, Gly-Phe-CHN 2 blocks the toxic effects of Leu-Leu- OMe.
  • micro-M Leu-Leu-OMe 60 25.00 micro-M Leu-Leu-OMe 12 50.00 micro-M Leu-Leu-OMe 2 100.00 micro-M Leu-Leu-OMe ⁇ 1 6.25 micro-M Leu-Leu-OBenzyl 62 12.50 micro-M Leu-Leu-OBenzyl ⁇ 1 25.00 micro-M Leu-Leu-OBenzyl 1 250.00 micro-M Leu-Leu-NH 2 71 1.00 micro-M Leu-Leu-NH 2 66
  • Such NK toxic reagents competitively inhibit [ 3 H]Leu- Leu-OMe uptake by human PBL and are therefore likely to be concentrated within lymphocytes by the same facilitated transport mechanism.
  • NK toxic compounds are composed of amino acids with non-polar side groups and are suitable substrates for a DPPI catalyzed polymerization reaction which produces a hydrophobic product that disrupts erythrocyte cell membranes.
  • alkyl, aralkyl and aryl esters or amides of peptides consisting essentially of natural or synthetic amino acids with hydrophobic side chains may function cytotoxically to deactivate natural killer cells (NK) and cytotoxic T lymphocytes (CTL) .
  • NK natural killer cells
  • CTL cytotoxic T lymphocytes
  • hydrophobic as used herein, is meant uncharged in aqueous solution at physiological pH and also as having no hydroxyl, carboxyl or primary amino groups.
  • Example 15 ester or amide derivatives of Leu-Leu or similar dipeptides which contain benzyl, naphthylamine or similar non-polar ring structures should prove to be selectively toxic for cytotoxic lymphocytes at lower concentrations than Leu-Leu-OMe and thus have enhanced clinical efficacy.
  • This example is provided to demonstrate the role of dipeptide alkyl ester, particularly O-alkyl ester (e.g., Leu-Leu-OMe)-sensitive CTL in graft rejection in vivo.
  • the particular in vivo model employed in the present study was the C57BL/6 (B6) mouse model.
  • This particular animal model is also used to demonstrate the use of the referenced dipeptide alkyl esters and O-alkyl esters in diminishing and/or preventing lethal graft vs host disease and graft rejection, most particularly, acute allograft rejection.
  • the experimental design employed in the present study examines whether removal of Leu-Leu-OMe-sensitive, DPPI-enriched CTL is effective in preventing or modulating rejection of Class I + II MHC and multiple non-MHC dispartate skin allografts.
  • Leu-Leu-OMe-treated C57BL6/J (B6) SpC (h-2 b ) are demonstrated herein to be unable to generate lethal GVHD in euthymic or thymectimized B6D2F1 (h-2 b * d ) recipient mice.
  • C57BL/6J (B6) and (C57BL/6xDBA/2)Fl female mice were purchased from the Jackson Laboratory, Bar Harbor, ME.
  • the medium was RPMI 1640 (Hazleton Research Products, Denver, PA) supplemented with 5 mM HEPES, 1 mM sodium pyruvate, 10 " * M 2-mercaptoethanol, penicillin G (200 U/ml) , (gentamicin (10 g/ml) , L-glutamine (0.3 mg/ml) and 10% fetal bovine serum was used for cell cultures.
  • anti-L3T4 GK1.5, 16
  • anti-Lyt2 YTS 169.4, 17
  • the IgG fraction of rabbit anti-mouse thymocyte globulin was purchased from Accurate Chemical and Scientific Corporation, San Diego, CA.
  • anti-Thyl.2 H-13-4, 18
  • anti-L3T4 2B6, 19
  • anti-L3T4 2B6, 19
  • anti-Lyt 2 3.155, 20
  • Bone marrow cells were flushed from femurs and tibias, were suspended in Hanks' balanced salt solution (HBSS) , and were filtered through sterile nylon mesh.
  • Spleen cells SpC were suspended in HBSS, filtered through sterile nylon mesh and then washed. For depletion of T cells, suspensions of cells (40 x 10 6 /ml) were incubated for 30 min.
  • Leu-Leu-OMe was synthesized from leucyl-leucine
  • Thy.2(+) cells were identified by incubation with HO-13-4 culture supernatant followed by staining with Fl- GAMIg and the number of Thyl.2 (+) cells determined by subtraction of the number of cells directly staining with Fl.GAMIg from these staining with Fl.GAMIg after initial incubation with anti-Thyl.2 (HO-13-4). L3T4(+) or
  • Lyt2(+) cells were identified by incubation with GK1.5 or YTS 169.4 followed by staining with fluorescein conjugated F(ab') 2 mouse anti-rat IgG (Jackson Immunoresearch, West Grove, PA) , a secondary antibody with very low levels of direct staining ( ⁇ 1%) of mouse SpC.
  • F(ab') 2 mouse anti-rat IgG Jackson Immunoresearch, West Grove, PA
  • Recipients were maintained on acidified (pH 2) , antibiotic (neomycin, 100 mg/liter, and polymyxin B, 10 mg/liter) H 2 0 for 2 to 3 days before and 7 days after transplantation.
  • antibiotic neomycin, 100 mg/liter, and polymyxin B, 10 mg/liter
  • H 2 0 acidified H 2 0 for 2 to 3 days before and 7 days after transplantation.
  • recipients were irradiated (900 cGy) and 2 to 6 h later were injected via the lateral tail vein with donor cells in 0.5 ml of HBSS.
  • mice were also injected intraperitoneally with 0.5 mg of rabbit anti-thymocyte globulin, a dose 2-fold in excess of that observed to cause >90% depletion of L3T4(+) and Lyt2(+) T cells from euthymic control mice.
  • mice Three to four weeks after completion of in vivo anti-T cell therapy, ATXBM, TCD mice wre reconstituted wth 70 x 10 6 control B6 SpC, 70 x 10 6 Leu-Leu-OMe treated B6 SpC or no SpC.
  • B6D2F1 tail skin in pieces aproximately 4 mm x 10 mm was grafted onto the lateral thoracic wall of recipient mice.
  • the general technique of free skin grafting described by Billingham et al. (1951, J. Exp. Med. , 28:385) was adapted for use in the grafting of skin grafts to the mouse model of the present example, which reference is specifically incorporated herein by reference for this purpose.
  • the grafts were covered with vaseline impregnated gauze and plaster bandages which were removed 8-10 days after grafting. The grafts were observed daily for rejection, which was considered complete when no viable skin was visible.
  • effector T cells were treated ex vivo with the described dipeptide alkyl esters.
  • B6 female mice (5- 6 weeks of age) were serially thymectomized, lethally irradiated, reconstituted with T cell depleted B6 BMC and infused with anti-CD4 and anti-CD8 mAb's as described supra.
  • SpC from such ATXBM, TCD mice were analyzed by flow cytometry for the presence of residual T cells.
  • the spleens of these animals contained less than or equal to 1% CD4(+) or CD8(+) T cells.
  • Long-term reconstitution with CD4(+) and with CD8(+) T cells was achieved when such ATXBM, TCD B6 mice were infused with 70 x 10 6 control or Leu-Leu-OMe treated B6 SpC. (See Table 16)
  • Hemisplenectomies were performed in all animals as a control to verify efficacy of thymectomy and T cell depletion. Only animals without detectable CD4(+) or CD8(+) T cells were used in subsequent skin graft experiments. As shown in the results detailed in Table 17, uniform, rejection of B6D2F1 skin grafts was again observed following transfer of Leu-Leu-OMe treated B6 SpC to such ATXBM pan-T cell depleted hosts. However, such rejection was again significantly delayed in hosts reconstituted with Leu-Leu-OMe treated SpC.
  • mice were sacrificed 100 days after infusion of 70 x 10 6 control B6 SpC, 70 x 10 6 Leu-Leu-OMe treated B6 SpC or cell free medium.
  • mice were thymectomized, irradiated (900 ⁇ Gy) and reconstituted with T cell depleted B6 BMC.
  • mice were injected intraperitoneally on three consecutive days with 200 ⁇ g of anti-L3T4 (GK1.5) and 100 ⁇ g of anti- Lyt2 (YTS 169.4) and on the second day of anti-L3T4 and anti-Lyt 2 treatment with 0.5 mg of rabbit anti- mouse thymocyte globulin.
  • the present example demonstrates a method of inhibiting graft rejection through the administration of particular alkyl esters of dipeptides, most particularly through administration of an O-alkyl ester.
  • the most preferred O-alkyl dipeptide ester is L-leucyl-L-leucine memethyl ester (Leu-Leu-OMe) .
  • the present methods may be used in conjunction with any type of tissue graft to inhibit tissue or whole organ rejection.
  • the prospective transplant recipient is first identified and then prospective transplant recipient is then treated with an effective amount of an alkyl ester of a dipeptide consisting of natural or synthetic L-amino acids with hydrophobic side chains.
  • the present methods are most preferably employed in conjunction with skin grafts.
  • the present disclosure provides a method whereby the rejection of skin and potentially other tissue grafts may be prevented in an animal, for example, of skin grafts in mouse and in human graft recipients.

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Abstract

The present invention includes testing of substances identified by a screening process as likely to be taken up by (and then deactivate) certain cells containing dipeptidyl peptidase I. The process comprises the steps of screening for substances inhibitory for the uptake or binding by PBL, NK or CTL cells of a known inhibitor or of a substance which itself inhibits said known inhibitor; comparing lysis induced in human erythrocytes by screened compounds and known inhibitors; distinguishing among the identified inhibitory substances which are taken up by PBL, NK or CTL cells those likely to cause lysis; and lastly applying to cells effective amounts of distinguished inhibitory substances which are lethal to cells taking them up or which are converted by dipeptidyl peptidase I to membranolytes.

Description

IDENTIFYING MEMBRANOLYTIC COMPOUNDS AND PRECURSORS THEREOF
The present invention concerns certain membraneolytic substances, as well as testing for certain cellular effects of dipeptide or dipeptide analog esters or N-substituted dipeptide amides as, for example, in the ablation of cell-mediated immune responses. For brevity and clarity, many of the terms used herein have been abbreviated and these abbreviations include those shown in Table 1.
L-leucine methyl ester (Leu-OMe) has previously been used as a lysoso otropic agent (Thiele et al . (1983) J. Immunol . , 131:2282-2290; Goldman et al . (1973) J. Biol . Chem . , 254:8914) . The generally accepted lysosomotropic mechanism involved leu-OMe diffusion into cells and into lysosomes, followed by intralysosomal hydrolysis to leucine and methanol. The more highly ionically charged leucine, largely unable to diffuse out of the lysosome was though to cause osmotic lysosomal swelling and rupture. The fate of leu-OMe subjected to rat liver lysosomes was additionally suggested by Goldman et al. (1973) to involve a transpeptidation reaction and a resultant species— "presumably the dipeptide" which was "further hydrolyzed to free amino acids". A subsequent and related paper by Goldman (FEBS (Fed. Europ. Biol. Sci.) Letters V 33, pp 208-212 (1973)) affirmed that non- methylated dipeptides were thought to be formed by lysosomes.
L-amino acid methyl esters have been specifically shown to cause rat liver lysosomal amino acid increases (Reeves (1979) J. Biol. Chem. V 254, pp 8914-8921). Leucine methyl ester has been shown to cause rat heart lysosomal swelling and loss of integrity (Reeves et al. , (1981) Proc. Nat'l. Acad. Sci., V 78, pp 4426-4429).
TABLE 1 Abbreviations
Substance
L-leucine
L-phenylalanine
L-alanine
L-glycine
L-serine
L-tyrosine
L-arginine
L-lysine
L-valine
L-isoleucine
L-proline
L-glutamic acid
L-aspartic acid
L amino acid methyl esters
L amino acid ethyl esters
L amino acid benzyl esters
D-amino acids
D-amino acids methyl esters dipeptides of L-amino acids methyl esters of dipeptide
L amino acids
dipeptide amides
Figure imgf000004_0001
NH, dipeptidyl peptidase-I DPPI
Cell Fraction or Type Symbol mononuclear phagocytes MP polymorphonuclear leucocytes PMN natural killer cells NK peripheral blood lymphocytes PBL peripheral blood ononuclear cells PBM cytotoxic T-lymphocytes CTL glass or nylon wool adherent cells AC glass or nylon wool non-adherent cells NAC
Other Materials phosphate buffered saline PBS thin layer chromatography TLC fluorescence activated cell sorter FACS mixed lymphocyte culture MLC
Miscellaneous dipeptidyl peptidase I DPPI effector:target cell ratio E:T fetal bovine serum FBS
University of Texas Health
Science Center, Dallas, Texas UTHSCD
Standard error of mean SEM probability of significant difference (Student's t-test) P
Graft versus host disease GVHD
Maximum velocity Vmax
Micromolar micro-M Level at which there is a 50% loss of cell function LD50
Trichlσracetic acid TCA
Phenylmethylsulfonyl fluoride PMSF
Glycylphenylalanine diazomethane Gly-Phe-CHN2 Mean survival time MST
Natural killer cells are large granular lymphocytes that spontaneously lyse tumor cells and virally-infected cells in the absence of any known sensitization. This cytotoxic activity can be modulated by a host of pharmacologic agents that appear to act directly on NK effector cells. NK activity has been shown to be augmented after exposure to interferons (Gidlund et al. , Nature V 223, p 259), interleukin 2, (Dempsey, et al. (1982) J. Immunol. V 129, p 1314) (Do zig, et al. (1983) J. Immunol. V 130, p 1970), and interleukin 1 (Dempsey et al.. (1982) J. Immunol. V 129, p 1314), whereas target cell binding is inhibited by cytochalasin B, (Quan, et al. (1982) J. Immunol. V 128, p 1786), dimethyl sulfoxide, 2-mercaptoethanol, and magnesium deficiency (Hiserodt, et al. (1982) J. Immunol. V 129, p 2266).
Subsequent steps in the lytic process are inhibited by calcium deficiency (Quan et al. (1982) J. Immunol. V 128, p 1786, Hiserodt, et al. (1982) J. Immunol. V 129, p 2266), lysosomotropic agents (Verhoef, et al. (1983) J. Immunol. V 131, p 125) , prostaglandin E2 (PGE2 (Roder, et al. (1979) J. Immunol. V 123, p 2785, Kendall, et al. (1980) J. Immunol. V 125, p 2770), cyclic AMP (Roder, et al. (1979) J. Immunol. V 123, p 2785, Katz (1982) J. Immunol. V 129, p 287), lipomodulin (Hattori, et al. (1983) J. Immunol. V 131, p 662) , and by antagonists of lipoxygenase (Seaman (1983) J. Immunol V 131 p 2953). Furthermore, it has been demonstrated that PGE2 and reactive metabolites of oxygen produced by monocytes (MP) or polymorphonuclear leukocytes (PMN) can inhibit NK cell function (Koren, et al. (1982) Mol. Immunol. V 19, p
1341; and Seaman, et al. (1982) J. Clin. Invest. V 69, p 876) .
Previous work by the present applicants has examined the effect of L-leucine methyl ester on the structure and function of human peripheral blood mononuclear cells (PBM) (Thiele, et al . (1983), J". Immunol . , 131 : 2282) .
Human peripheral blood mononuclear cells (PBM) are capable of mediating a variety of cell-mediated cytotoxic functions. In the absence of any known sensitization, spontaneous lysis of tumor cells and virally-infected cells is mediated by natural killer cells (NK) contained within the large granular lymphocyte fraction of human PBM Timonen et al.. (1981) v. J. Exp Med. V 153 pp 569- 582. After lymphokine activation, additional cytotoxic lymphocytes capable of lysing a broad spectrum of tumor cell targets can be generated in in vitro cultures (Seeley et al. (1979) J. Immunol. V 123, p 1303; and Grimm et al. (1982) J. Exp. Med. V 155, p 1823). Furthermore, lymphokine activated peripheral blood mononuclear phagocytes (MP) are also capable of lysing certain tumor targets (Kleinerman et al. (1984) J. Immunol. V 133, p 4). Following antigen-specific stimulation, cell-mediated lympholysis can be mediated by cytotoxic T lymphocytes (CTL) .
While a variety of functional and phenotypic characteristics can be used to distinguish these various types of cytotoxic effector cells, a number of surface antigens and functional characteristics are shared. Thus, the antigens identified by the monoclonal antibodies OKT8 (Ortaldo et al. (1981) J. Immunol. V 127, p 2401; and Perussia et al. (1983) J. Immunol. V 130, p 2133), and OKT11 (Perussia et al. (1983) J. Immunol. V 130, p 2133; and Zarling et al. (1981) J. Immunol. V 127, p 2575) are found on both CTL and NK while the antigen identified by OKM1 is shared by MP and NK (Zarling et al. (1981) J. Immunol. V 127, p 2575; Ortaldo et al. (1981) J. Immunol. V 127, p 2401; Perussia et al. (1983) J. Immunol. V 130, p 2133; and Breard et al. (1980) J.
Immunol. V 124, p 1943. Furthermore, cytolytic activity of both NK and MP is augmented by interferons, (Kleinerman et al. (1984) J. Immunol. V 133, p 4; Gidlund et al. (1978) Nature V 223, p 259; and Trinchieri et al. (1978) J. Exp. Med. V 147, p 1314). Finally, use of metabolic inhibitors has demonstrated some parallels in the lytic mechanism employed by CTL and NK (Quan et al. (1982) J. Immunol. V 128, p 1786; Hiserodt et al. (1982) J. Immunol. V 129, p 1782; Bonavida et al. (1983) Immunol. Rev. V 72, p 119; Podack et al. (1983) Nature V 302, p 442; Dennert et al. (1983) J. Exp. Med. V 157, p 1483; and Burns et al. (1983) Proc. Nat'l. Acad. Sci. V 80, p 7606) .
The present invention includes a process for identifying a substance taken up by and deactivating NK or CTL cells. The process comprises the steps of screening candidate substances to determine those which competitively inhibit the uptake or binding by PBL, NK or CTL cells of L-leu-L-leu-OMe or of a substance which itself competitively inhibits L-leu-L-leu-OMe uptake or binding; incubating substances determined to be inhibitory with membrane-enveloped labels and DPPI; determining lysis of the membrane-enveloped labels caused by the incubated inhibitory substances; and lastly identifying substances which are taken up by and deactivate NK or CTL cells as those effectively mediating lysis of membrane-enveloped labels.
A preferred form of membrane-enveloped label is that of labeled human erythrocytes, but other types of cells as well as lysosomes may also be used. Those skilled in the art will recognize that a wide variety of appropriately labeled naturally-occurring or artificial membrane structures might serve the same purpose as that described herein for labeled human erythrocytes.
Among preferred candidate substances to be subject to this method are dipeptide or dipeptide analog esters or N-substituted amides. The dipeptides or analogs thereof preferably comprise L-amino acids or structural analogs thereof with hydrophobic side chains. They may be used to deactivate NK or CTL cells. Preferable natural amino acids of such dipeptides or dipeptide analogs are leucine, phenylalanine, valine, isoleucine, alanine, proline, glycine or aspartic acid beta methyl ester. Preferable dipeptides are L leucyl L-leucine, L-leucyl L- phenylalanine, L-valyl L-phenylalanine, L-leucyl L- isoleucine, L-phenylalanyl L-phenylalanine, L-valyl L- leucine, L-leucyl L-alanine, L-valyl L-valine, L- phenylalanyl L-leucine, L-prolyl L-leucine, L-leucyl L- valine, L-phenylalanyl L-valine, L glycyl L-leucine, L- leucyl L-glycine or L-aspartyl beta methyl ester L- phenylalanine.
Among preferred analogs of natural amino acids are norleucine and norvaline, while those skilled in the art would recognize many additional analogs and homologs. The most preferable dipeptides are glycyl L- phenylalanine, L-leucyl L-leucine, L-leucyl L- phenylalanine, L-valyl L-phenylalanine, L-phenylalanyl L- leucine, L-leucyl L-isoleucine, L-phenylalanyl L- phenylalanine and L-valyl L-leucine.
The amide N-substituent or ester alcohol residue of the dipeptide or dipeptide analog derivative is preferably a benzyl or alkyl of up to about four carbon atoms such as propyl, isopropyl, butyl or isobutyl. Larger alkyl groups may be used. Aralkyl or aryl derivatives, for example benzyl and napthyl may be particularly effective.
One preferable dipeptide analog has sulfur in place of oxygen in the peptide bond. Because the peptide bond of the dipeptide is not thought essential, other structural dipeptide analogs are believed to be perfectly suitable. The present invention further involves a method for deactivating NK or CTL cells comprising the step of treating said cells with an aqueous solution comprising a biologically effective level of substances determined by the above method such as a dipeptide or dipeptide analog ester or N-substituted dipeptide or analog amide form consisting essentially of L-α-amino acids with hydrophobic side chains, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted amide. This aqueous solution more preferably comprises a biologically effective level of a dipeptide or dipeptide analog in ester or substituted amide form, said dipeptide or dipeptide analog consisting essentially of at least one of L-leucine, L-phenylalanine, L-valine, L-isoleucine, L-alanine, L-proline, glycine, and L- aspartic acid beta methyl ester, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted amide. The cells being deactivated may be in vitro or may be yet within an animal. In the latter case the animal is parenterally administered a biologically effective amount of the dipeptide or dipeptide analog in ester or substituted amide form.
This deactivation of NK and CTL cells may also be adapted as a method for inhibiting bone marrow graft versus host disease comprising the step of contacting the bone marrow cells to be grafted with an aqueous solution comprising a biologically effective level of substances determined by the above method such as a dipeptide or dipeptide analog ester or N-substituted dipeptide or analog amide form consisting essentially of L-α-amino acids with hydrophobic side chains, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted amide. This aqueous solution again more preferably comprises a biologically effective level of a dipeptide or dipeptide analog in ester or substituted amide form, said dipeptide or dipeptide analog consisting essentially of at least one of L-. leucine, L-phenylalanine, L-valine, L-isoleucine, L- alanine, L-proline, glycine, and L-aspartic acid beta methyl ester, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted amide. For in vitro deactivation of natural killer cells or cytotoxic T-lymphocytes, the biologically effective level should be between about 1 micromolar and about 250 micromolar, depending upon the particular agent being used and its effectiveness.
In clinical application, the present invention additionally involves a method of inhibiting the rejection of tissue transplanted into a host. This method comprises the steps of identifying a prospective transplant recipient; and treating the prospective recipient with an aqueous solution comprising a biologically effective level of substances determined by the above method such as a dipeptide or dipeptide analog ester or N-substituted dipeptide or analog amide form consisting essentially of L-α-amino acids with hydrophobic side chains, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted amide. This aqueous solution also more preferably comprises a biologically effective level of a dipeptide in ester or substituted amide form, said dipeptide consisting essentially of at least one of L- leucine, L-phenylalanine, L-valine, L-isoleucine, L- alanine, L-proline, glycine, and L-aspartic acid beta methyl ester, said ester form being an aryl, alkaryl or aralkyl ester and said amide form being a substituted amide. Certainly insofar as such tissue reaction is mediated by NK or CTL cells, the method of the present invention provides for identifying rejection-inhibiting compounds. For clinical treatments involving parenteral administration of substances identified by the method of the present invention, the biologically effective amount administered is generally between about 10 mg/kg body weight and 300 mg/kg body weight; preferably about 1 X 10" 4 moles/kg body weight. The aqueous solutions of the present invention include any of those suitable for in vivo administration free of toxins and preferably being of an approximate physiological pH and osmolality.
Preferred dipeptide or dipeptide analog esters of the present invention include those formed with an alkaryl alcohol, most preferably benzyl alcohol. The term "alkaryl" is used herein to indicate an alkyl group bound in amide or ester linkage to the dipeptide and having an aryl group bound thereto. A particularly preferred dipeptide ester identified by the method of the present invention is L-leucyl-L-leucyl benzyl ester. The term "aralkyl" is used herein to indicate an aryl group bound in amide or ester linkage to a dipeptide or dipeptide analog of the present invention and having an alkyl group bound thereto. It is understood that those skilled in the art may make many variations in group substitutions on the alkyl, aryl, aralkyl and alkaryl groups substituents of the present invention and still be within the presently claimed invention.
Preferred dipeptides identified by the method of the present invention include L-leucyl, L-leucine, L-leucyl L-phenylalanine, L-valyl L-phenylalanine, L-phenylalanyl L-leucine, L-leucyl L-isoleucine, L-phenylalanyl L- phenylalanine, L-valyl L-leucine, L-leucyl L-alanine, L- valyl L-valine, L-prolyl L-leucine, L-leucyl L-valine, L- phenylalanyl L-valine, glycyl L-leucine, L-leucyl glycine, and L-aspartyl beta methyl ester L- phenylalanine. A more preferred group of dipeptides is L-leucyl L-leucine, L-leucyl L-phenylalanine, L-valyl L- phenylalanine, glycyl L-phenylalanine, L-phenylalanyl L- leucine, L-leucyl L-isoleucine, L-phenylalanyl L- phenylalanine and L-valyl L-leucine.
The present invention describes a general method for identifying substances deactivating NK or CTL cells. This general method comprises the step of treating said cells with an aqueous solution comprising a biologically effective level of the identified substance, which competitively inhibits lymphocyte uptake of Leu-Leu-OMe (or of a compound which itself inhibits such uptake) and is polymerized by dipeptidyl peptidase I to form a membranolytic product (shown to be effective by lysis of erythrocytes) .
An analogous method for inhibiting bone marrow graft versus host disease may also be so generalized. Such a method comprises the step of contacting bone marrow cells to be grafted with an aqueous solution comprising a biologically effective level of substances determined by the above method, such as a dipeptide or dipeptide analog ester or N-substituted dipeptide or dipeptide analog amide form consisting essentially of L-α-amino acids with hydrophobic side chains, which competitively inhibits lymphocyte uptake of Leu-Leu-OMe and is polymerized by dipeptidyl peptidase I to form a membranolytic product.
The present invention also includes a process for identifying a substance for inactivation of cells containing dipeptidyl peptidase I (including NK or CTL cells) , the process comprising the steps of testing a compound for inhibition of uptake or binding by PBL of labeled L-Leu-L-Leu-OMe or of a compound itself inhibiting such uptake, and assaying inhibitors to identify those taken up by PBL's and converted by dipeptidyl peptidase I to form a membranolytic product, which product is characterized as being insoluble in 10% aqueous trichloroacetic acid.
A membranolytic oligopeptide aryl or alkyl ester or N-aryl substituted oligopeptide amide may be obtained by the present invention by a process comprising identifying a dipeptide or dipeptide analog aryl or alkyl ester or N- aryl substituted dipeptide amide which is converted by dipeptidyl peptidase I to form an oligopeptide or oligopeptide analog aryl or alkyl ester or N-substituted oligopeptide amide having a structure membranolytic to red blood cells, elucidating the structure of the oligopeptide membranolytic product, and preparing a quantity of this product.
An alternate process in the present invention for preparing a membranolytic substance comprises the steps of identifying a compound competitively inhibiting the uptake by PBL of L-leu-L-leu-OMe or of a compound which itself inhibits said uptake, ascertaining an identified compound which is converted by dipeptidyl transferase I to a membranolytic substance, structurally defining said membranolytic substance, and preparing a quantity of said structurally defined membranolytic substance. In addition, membranolytic substances may be formed by exogenously using dipeptidyl peptidase I to catalyze membranolytic substance formation from hydrophobic amino acid dipeptides.
Figure 1 shows results of the assay for competitive inhibition of [3H]-Leu-Leu-OMe uptake by human peripheral blood lymphocytes. Figure 2 shows results of the assay for generation of membranolytic metabolites in slCr labeled human erythrocytes.
Figure 3A shows a thiopeptide analog of Leu-Leu-OMe, and Figure 3B shows a schematic structure of leucyl- (N)methyl leucine methyl ester.
Figures 4A, 4B and 4C show various examples of dipeptide esters with non-physiological R groups.
Figure 5 shows that whereas ablation of NK function during incubation with Leu-OMe can be blocked by lysosomotropic agents, there is a product formed during incubation of Leu-OMe with MP or PMN which has effects on NK function no longer blocked by lysosomal inhibitors.
Figure 6 shows Leu-OMe products of PMN in terms of radioactivity and NK suppressive effects of TLC fractions.
Figure 7A shows the Cl mass spectrum of TLC fractions with NK toxic activity, and Figure 7B shows the Cl mass spectrum of synthetic Leu-Leu-OMe.
Figures 8A and 8B show the effects of various agents on losses of NK function from MP-depleted lymphocytes.
Figure 9 shows the NK-toxicity of various dipeptide esters.
Figure 10 shows the loss of NK and MP from PBM incubated with Leu-Leu-OMe at various concentrations.
Figure 11 shows the toxicity of various Leu-Leu-OMe concentrations for selected cell types. Figure 12 shows the Leu-Leu-OMe mediated elimination of precursors of cytotoxic T lymphocytes activated NK (ACNK) and NK.
Figure 13 shows the sensitivity of activated NK and CTL to treatment with Leu-Leu-OMe.
Figure 14 shows the time-dependent uptake of Leu- Leu-OMe by PBL.
Figure 15 shows the concentration dependence of Leu- Leu-OMe uptake by PBL.
Figure 16 schematically describes the effects upon RBC lysis by Leu-Leu-OMe in the presence (■) and absence (•) of exogenous DPPI.
Figures 17A through 17H show RBC lysis as dependent upon the presence (•+) or absence (-) of DPPI with concentrations gradients of different dipeptide methyl esters.
Figure 18 shows the survival of B6D2F1 skin grafts applied to ATXBM, TCD mice in the presence or absence of reconstitution with control or Lue-Leu-OMe treated B6 SpC.
Figure 19A shows that Leu-Leu-OMe treatment of B6 donor SpC prevents lethal GVHD in euthymic B6D2F1 recipients, and Figure 19B shows similar results with thymectomized B6D2F1 recipients.
The present invention concerns identifying and testing new compounds useful in ablating either the functions of particular cell types or the cells. The presently described invention relates to the discovery that certain peptide or peptide analog esters and N- substituted amides are cytotoxic to particular cell types.
It has further been found that alkyl, aralkyl and aryl esters or amides of dipeptides or dipeptide analogs consisting essentially of α-amino acids with hydrophobic side chains may function cytotoxically to deactivate natural killer cells (NK) and cytotoxic T lymphocytes (CTL) . By the term "hydrophobic" as used herein, is meant uncharged in aqueous solution at physiological pH and also as having no hydroxyl, carboxyl or primary amino groups.
Treatment of NK or CTL cells with an effective level of a peptide amide or ester consisting essentially of natural or synthetic amino acids with hydrophobic side chains serves to deactivate the cytotoxic functions of said cells. An effective level varies from circumstance to circumstance but generally lies between about 1 micromolar and about 250 micromolar. An effective level for a whole animal dose generally lies between about 100 mg/kg and about 300 mg/kg.
Methyl, ethyl and benzyl esters or amides of peptides consisting essentially of natural or synthetic amino acids having hydrophobic side chains have been specifically found to deactivate natural killer cells (NK) or cytotoxic T lymphocytes (CTL) and other alkyl esters of these peptides (or peptide analogs thereof) are confidently predicted to have similar or superior effects.
Deactivation of NK or CTL cells with substances such as identified peptide esters or amides should increase the success of allogeneic bone marrow transplants by lowering the incidence of graft-versus-host disease (GVHD) and thus lessening the incidence of transplant rejection.
Other clinical uses for the present peptide amides or esters consisting essentially of amino acids with hydrophobic side chains, are other situations where NK or CTL are involved in the pathogenesis of disease. In organ transplants in general (kidney, heart, liver, pancreas, skin, etc.), it is widely accepted that cytotoxic T cells are at least partially responsible for graft rejection (Mayer et al., J. Immunol. V 134, p 258, 1991 and Rosenberg et al. J. Exp. Med. 165, 1296, 1991) . Thus, it is contemplated that the in vivo administration of peptide esters or amides of the present invention will be of benefit in preventing allograft rejection.
It is also contemplated that the substances identified by the method of the present invention may be of benefit in other spontaneously occurring disease states. A variety of diseases have been classified as "autoimmune diseases" because of the widely accepted belief that they are caused by disorders in the immune system which cause immunologic damage to "self". Thus, in a variety of diseases, including primary biliary cirrhosis, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, autoimmune hemolytic anemia, etc. , various forms of immunologic damage to selected organs occur. In some of these diseases, such as primary biliary cirrhosis, the histologic abnormalities which occur (in this case in the liver) closely resemble those which occur in GVHD or in rejection of a transplanted liver (Fennel, (1981),
Pathol.. Annu. V 16 p 289. Thus, it is reasonable that similar mechanisms of cytotoxic lymphocyte damage to liver cells may be occurring, and therefore benefit from therapy with the dipeptide esters or amides of the present invention should also occur in such disease states.
The peptide esters or amides of the present invention should be usable chemotherapeutic agents for patients with natural killer cell tumors (generally leukemias) , although very few reports of these tumors are found in the literature (Komiyama et al.. (1982) Blood V 60, p 1428 (1982); Itoh et al. (1983) Blood V 61, p 940; Komiyama et al. (1984) Cancer V 54 p 1547.
It is contemplated that the peptide esters and amides of the present invention may also be used to treat patients with aplastic anemia and other types of bone marrow dysfunction. This suggestion is based on three sets of observations in human studies: first, NK cells can kill normal bone marrow cells (Hansson, et al. (1981) Eur. J. Immunol. V 11, p 8) ; second NK cells inhibit growth of blood cell precursors in vitro (Hansson, et al. (1982) J. Immunol. V 129, p 126; Spitzer et al.: Blood V 63, p 260; Torok-Storb et al. (1982) Nature V 298, p 473; Mangan, et al. Blood V 63, p 260); and third, NK-like cells with the ability to inhibit the formation of red blood cells have been isolated from patients with aplastic anemia (Mangan, et al. (1982) J. Clin. Invest. V 70, p 1148; and Nogasawa et al. (1981) Blood V 57, p 1025) . Moreover, recent studies in the mouse indicate that NK cells may function to suppress hemopoiesis in vivo (Holmberg et al. (1984) J. Immunol. V 133, p 2933). However, further investigation is desireable before the connection between NK activity and bone marrow dysfunction is considered conclusive. Generally, when the substances identified by the method of the present invention are administered to animals, an effective level is between about 1 x 10-4 moles/kg and about 1 x 10-2 moles/kg.
The following Examples are presented to more fully illustrate preferred embodiments of the present invention as well as uses of substances so identified, and are not intended to limit the invention unless otherwise so stated in the accompanying claims.
Example 1 Cell Preparations and Assays
PBM were separated from heparinized venous blood of healthy donors by centrifugation over sodium diatrizoate- Ficoll gradients (Isolymph, Gallard-Schlesinger Chemical Mfg. Corp., Carle Place, NY). Monocyte-enriched populations ((MP) were prepared from glass adherent cells and MP-depleted lymphocytes from the nonadherent cells remaining after incubation in glass Petri dishes and passage through nylon wool columns as detailed in Rosenberg et al . (1975, J". Immunol . , 122 : 926-831). PMN were collected by resuspending peripheral blood cells that penetrated sodium diatrizoate-Ficoll gradients and removing erythrocytes by dextran sedimentation and h potonic lysis as previously outlined by Thiele et al. (1985, J. Immunol. , 134:786-793) .
All cell exposures to the amino acids, dipeptides or their methyl esters were carried out by suspending cells in Dulbecco's phosphate buffered saline (PBS) and incubating them at room temperature with the reagent at the indicated concentration and time interval. After incubation, the cells were washed twice with Hanks' balanced salt solution and resuspended in medium RPMI 1640 (Inland Laboratories, Fort Worth, TX) supplemented with 10% fetal bovine serum (Microbiological Associates, Walkersville, MN) for assay of function.
Natural killing against K562 target cells was assessed by a 3 hour 51Cr release assay and percent specific lysis calculated as previously described (Thiele et al . , (1985), J. Immunol . , 134:786-793) . Percent of control cytotoxicity was calculated using the formula:
Experimental % specific lysis Control % specific lysis
Example 2
Discovery of Agents with Immunosuppressive Properties Similar to those of Leu-Leu-OMe
Screening assays summarized in Figures 1 and 2 and Table 2 are useful methods of identifying substances such as new dipeptide ester or amide analogs with immunosuppressive properties similar to those previously described for Leu-Leu-OMe (Thiele et al . (1990) J. Exp. Med. 172:183-194; Thiele et al . (1990) Proc. Natl . Acad . Sci . USA 87:83-87. These methods were demonstrated with five non-physiologic dipeptide ester analogs of Leu-Leu- OMe:
L-norleucyl-L-leucine methyl ester (norLeu-Leu-OMe) L-leucyl-L-norleucine methyl ester (Leu-norLeu-OMe) L-norleucyl-L-norleucine methyl ester (norLeu- norLeu-OMe)
L-norvalyl-L-leucine methyl ester (norVal-Leu-OMe) L-leucyl-L-norvaline methyl ester (Leu-norVal-OMe)
These agents were assessed for competitive inhibition of ['H]Leu-Leu-OMe uptake by human PBL's using techniques described in the Methods section and in the footnote to Table 5, p 188 of Thiele et al . (1990, J*. Exp. Med. 172:183-194.) . Results (Figure l) indicate that norVal-Leu-OMe does not competitively inhibit [3H]Leu-Leu-OMe uptake and thus appears not to serve as a good Iigand for the facilitated transporter responsible for uptake of dipeptide derivatives by human lymphocytes. In contrast, all other compounds tested in experiments summarized in Figure 1 are similar to Leu-Leu-OMe in capacity to competitively inhibit [3H]Leu-Leu-OMe uptake by human PBL's. Figure 1 suggests that norVal-Leu-OMe is unlikely to be incorporated by CTL's and therefore is unlikely to mimic the immunosuppressive actions of Leu- Leu-OMe, whereas all other compounds considered appear to be potentially active agents.
Thus, all compounds found to be competitive inhibitors of [^Leu-Leu-OMe uptake by human PBL's were next screened in the assay summarized in Figure 2. In this procedure, aCr labeled human erythrocytes were incubated with varying concentrations of these dipeptide esters in the presence or absence of 8x10"* U/ml of purified bovine DPPI as described in Thiele et al . (1990, ■J. Exp. Med. 172 :183-194) . In the absence of DPPI, 0-2 mM concentrations of these agents did not cause significant erythrocyte lysis. However, as shown in Figure 2, in the presence of DPPI, membranolytic metabolites were generated with varying degrees of efficiency from each of these compounds. In particular, Leu-norLeu-OMe and norLeu-norLeu-OMe were somewhat better substrates, while norLeu-Leu-OMe and Leu-norVal-OMe were significantly inferior to Leu-Leu-OMe for DPPI catalyzed conversion of membranolytic metabolites as measured by efficiency in mediating RBC lysis.
The fact that norLeu-norLeu-OMe, for example, was more active in both of these screening assays than Leu- Leu-OMe suggested that it might also more active in mediating toxicity for human NK cells. In the studies represented in Table 2, each of the synthesized agents (plus Leu-Leu-OMe) was directly tested for human NK cell toxicity; norLeu-norLeu-OMe was indeed found to be superior to Leu-Leu-OMe in this regard.
Analogously, Leu-norLeu-OMe appeared slightly less efficient than Leu-Leu-OMe in the assay of Figure 1 but more efficient in the Figure 2 assay. Thus, it would be expected to have activity similar to but slightly less than norLeu-norLeu-OMe in mediating NK toxicity (Table 2) . Along the same line, norLeu-Leu-OMe and Leu-norVal- OMe were found to be less active than Leu-Leu-OMe in both screening assays, so the finding that they were both less efficient than Leu-Leu-OMe in mediating NK toxicity supports the predictive value of the screening assays. Finally, norVal-Leu-OMe, an agent that was totally inactive in the screening assay of Figure 1, had no detectable NK toxicity (see Table 2) .
TABLE 2
COMPARISON OF THE NK TOXICITY
OF VARIOUS ANALOGS OF LEU-LEU-OME
Compound Toxicity for Human NK Cells
(LD50)*
μM norLeu-norLeu-OMe 25
Leu-Leu-OMe 38
Leu-norLeu-OMe 43
Leu-norVal-OMe 83 norLeu-Leu-OMe 87 norVal-Leu-OMe >500 *LD50 determined from the concentration required to ablate 50% of the cytotoxic activity of human PBL assayed against K562 targets at a 20:1 E/T ratio as previously detailed (PNAS 82:2468).
Taken together, these results support use of the screening assays represented in Figures 1 and 2 for identifying dipeptide ester analogs with immxinosuppressive activity similar or superior to that previously described for Leu-Leu-OMe. For example, since the peptide bond between the first and second amino acid in Leu-Leu-OMe need not be cleaved in the mediation of NK toxicity, alterations of the peptide bond as detailed in Figures 3A and 3B, (i.e. a thiopeptide analog of Leu-Leu- OMe or the use of peptides such as leucyl-N-methyl leucine-methyl ester) may serve to produce an NK-toxic drug of equal or better concentration-dependent activity. As aminopeptidases capable of degrading Leu-Leu-OMe by cleavage of this peptide bond are less likely to degrade such compounds, they are more apt to have a longer in vivo half-life and therefore enhanced efficacy.
Modifications of amino acid R groups which preserve the non-polar nature of the amino acid R groups in dipeptide esters such as Leu-Leu-OMe and the activity of this compound as a substrate for dipeptidyl peptidase I should result in therapeutic agents with enhanced efficacy, since not all peptidases capable of degrading Leu-Leu-OMe may be able to degrade dipeptide esters containing non-physiologic side chains. Examples of some non-physiological dipeptide esters are detailed in Figures 4A, 4B and 4C, where, for simplicity, they are shown as dipeptide methyl esters only. Example 3
General Procedures for Generation,
Purification and Characterization of
L-leucine Methyl Ester and Its Metabolites
MP or PMN (prepared as in Example 1) at a concentration of 25 X 106 per ml were suspended in PBS and incubated with 25 mM Leu-OMe for 20 minutes at 22°C. Cell suspensions were then centrifuged at 1000 g for 10 minutes and the supernatants harvested and freeze-dried at -70°C, 100 millitorr atmospheric pressure. In some experiments, Leu-OMe-treated MP or PMN were sonicated to increase the yield of the reaction product. Samples were then extracted with methanol for application to thin layer chromatography (TLC) plates (200 micromolar x 20 cm2, Analtech, Newark, Delaware) . Following development with chloroform/methanol/acetic acid (19:1:12.5 by volume), 1cm bands were eluted with methanol, dried under nitrogen, and resuspended in 1 ml PBS. Mass spectra were obtained with a Finnegan Model 4021 automated EI/CI,
GC/MS system coupled to an Incos data system. Methane was used as the reagent gas for chemical ionization (CΙ)mass spectral analysis.
Example 4
Lysosomotropic Substances and Formation of NK-toxic Products
The addition of Leu-OMe to human PBM was shown to cause rapid death of MP and NK cells but not T or B lymphocytes (Thiele et al . (1985), J. Immunol . 234:786- 793; Thiele et al . (1983), J. Immunol . 131 : 2282-2290). Amino acid methyl esters are known to be lysosomotropic compounds, and in previous studies it was found that the lysosomal inhibitors, chloroquine and NH4C1, prevented Leu-OMe-induced MP toxicity. To assess whether these agents similarly prevented formation of any NK toxic products, the following experiments were carried out, and the results shown in Figure 5.
PBM (prepared as in Example 1) were incubated with various potential NK toxic agents in the presence or absence of various lysosomal inhibitors for 40 minutes, washed to remove the inhibitor, incubated for 18 hours to permit recovery from any transient inhibition caused by lysosomotropic agents and then tested for NK activity. As can be seen in Figure 5, neither chloroquine, NH4C1, nor Ile-OMe had any substantial permanent effect on NK function. In contrast, 5 mM Leu-OMe ablated all NK activity. This activity of Leu-OMe was largely prevented by chloroquine, NH4C1, or Ile-OMe. The products generated by MP or PMN, after exposure to Leu-OMe also completely removed all NK activity from PBM. In contrast to the effect noted with Leu-OMe, the lysosomal inhibitors did not protect NK cells from the action of this product(s) . Additional experiments indicated that the sonicates of MP or PMN had no effect on NK function in this system whereas the supernatants or sonicates of Leu-OMe treated PMN or MP also depleted NK cells from MP depleted lymphocytes. These results therefore suggest that interaction of Leu-OMe with the lysosomal compartment of MP or PMN produced a product which was directly toxic to NK cells through a mechanism that was no longer dependent on lysosomal processing within the NK cell or an additional cell type.
More particularly, the conditions of the manipulations leading to the results shown in Figure 5 were as follows:
Inhibitors of lysosomal enzyme function prevent generation of an NK toxic product. PBM (5 X 106/ml) or PMN (25 X 106/ml) preincubated with 25 mM Leu-OMe for 30 minutes were added to cells to be ablated. Cells were incubated with these agents for another 30 minutes at 22°C, then washed and cultured for 18 hours at 37°C before assay of the ability to lyse K562 cells. Data are expressed as percentage of control cytotoxicity observed with an effector:target ratio of 40:1 (results at other E:T were similar) .
Example 5 Ablation of NK Function by PMN Produced Leu OMe Product
When the NK toxic properties of MP-Leu-OMe, or PMN- Leu-OMe incubation mixtures were evaluated, it was found that this activity was stable in aqueous solutions for more than 48 hours at 4°C, but labile at 100°C, retarded on Sephadex G-10 columns; dialyzable through 1000 MWCO (molecular weight cut-off) membranes, and could be extracted by chloroform-methanol (3:1, by volume). As shown in Figure 6, when "C-leucine methyl ester was incubated with PMN and the supernatants subsequently separated by TLC, three major peaks of "C activity were found. One of these peaks corresponded to leucine methyl ester itself and one to free leucine while the third represented a new product. This third peak accounted for about 10% of the total "C-labeled material. When MP- depleted lymphocytes were exposed to each TLC fraction, the third peak was found to contain all NK toxic activity. This NK toxic activity not only appeared to be "C labeled but was also ninhydrin positive, suggesting that it was a metabolite which still retained an amino group as well as part of the carbon structure of Leu-OMe. An identical 14C labeled ninhydrin positive product was detected by TLC of MP-Leu-OMe incubation mixture supernatants or sonicates. The production by PMN or MP of this metabolite was inhibited by chloroquine, NH4C1, or Ile-OMe.
Ablation of NK function is mediated by a metabolite of Leu-OMe. PMN (25xl06/ml) were incubated with 25mM C- Leu-OMe for 30 minutes and supernatants harvested for TLC analysis. MP-depleted lymphocytes (2.5xl06 cells/ml) were exposed to varying dilutions of each TLC fraction for 30 minutes, washed and cultured for 2 hours prior to cytotoxicity assay at E:T ratio of 20:1. Samples were considered to contain an NK toxic product when percent specific lysis was less than 25% of control. Figure 6 shows these results.
Example 6
Characterization of
The NK-toxic Metabolite
The nature of the new TLC peak found as described in Example 5 was examined by mass spectroscopy. As shown in Figure 7A, when the TLC-purified, NK-toxic fraction was subjected to mass spectral analysis, results showing peaks at M/2 259 (MN+) , 287 (M+C2H5+) and 299 (M+C3H5+)) indicated the presence of a compound of molecular weight 258. The presence of peaks at M/Z 244 (M+-CH3) and 272 (M+C2H55+-CH3) suggested that this compound contained a methyl ester group. Furthermore, the persistence of peaks corresponding to leucine (MN* •=• 131, M+C2H5 = 159) and leucine methyl ester (MIT = 146, M+C2H5 + = 174) , in spite of careful TLC purification of the NK toxic product from any free leucine or Leu-OMe present in the crude supernatants of the incubation mixtures, suggested that a condensation product of Leu-OMe such as Leu-Leu-OMe (MW258) was present in the NK toxic fraction isolated after incubation of PMN or MP with Leu-OMe. When Leu-Leu-OMe was synthesized from reagent grade Leu-Leu, by incubation in methanol hydrochloride, it was found to have TLC mobility identical to NK toxic fractions of MP-Leu-OMe or PMN-Leu-OMe incubation mixtures. Furthermore, its Cl mass spectrum as shown in Figure 7B was identical to that of the 258 molecular weight compound found in these incubation fractions.
Experiments further confirmed that Leu-Leu-OMe was the product generated by MP or PMN from Leu-OMe that was responsible for the selective ablation of NK function from human lymphocytes. Leu-Leu-OMe was synthesized by addition of Leu-Leu to methanolic HCl. TLC analysis revealed less than 2% contamination of this preparation with leucine, Leu-Leu, or leu-OMe, and Cl mass spectral analysis (Figure 7B) revealed no contaminants of other molecular weights.
Figure 7A shows the chemical-ionization Cl mass spectra of TLC fractions with NK toxic activity as described in Figure 6; Figure 7B shows the Cl spectrum of Leu-Leu-OMe synthesized from reagent grade Leu-Leu.
Example 7 Compounds Lacking NK Toxicity
In the representative experiments shown in Figures 8A and 8B, MP-depleted lymphocytes were exposed to varying concentrations of Leu-Leu-OMe for 15 minutes at room temperature, then washed and assayed for ability to lyse K562 cells. No NK function could be detected in lymphocyte populations exposed to greater than 50 micromolar Leu-Leu-OMe. As previously demonstrated (Thiele et al. (1983), J. Immunol . 232:2282-2300) , exposure of such MP-depleted lymphocyte populations to
100 fold greater concentration of leucine or leu-OMe had no irreversible effect on NK function. Leu-Leu or the D- stereoisomer, D-Leu-D-Leu-OMe, also had no inhibitory effect. When lymphocyte populations exposed to varying concentrations of Leu-Leu-OMe were further analyzed, it was found that exposure to more than 50 micromolar Leu- Leu-OMe resulted in the loss of K562 target binding as well as complete depletion of cells stained by an anti-NK cell monoclonal antibody Leu lib. Thus, the MP-or PMN- generated product of Leu-OMe which is directly toxic for human NK cells is the dipeptide condensation product Leu- Leu-OMe.
The condition of the manipulations resulting in the data leading to Figures 8A and 8B are further detailed as follows: for loss of NK function after exposure to Leu- Leu-OMe, MP-depleted lymphocytes (2.5xl06 cells/ml) were incubated for 15 minutes with the indicated concentrations of leucine containing compounds. Cells were then washed, cultured at 37°C for 2 hours (Expt. 1) or 18 hours (Expt. 2) and then assayed for NK activity. Results are given for E:T ratio of 20:1.
Example 8 NK Ablation by a Variety
Of Dipeptide Methyl Esters
In previously reported studies, Leu-OMe was unique among a wide variety of amino acid methyl esters in its ability to cause MP or PMN dependent ablation of NK cell function from human PBM (Thiele et al . (1985), J. Immunol . 234:786-793) . The identification of Leu-Leu-OMe as the MP-generated metabolite responsible for this phenomenon suggested that either MP/PMN did not generate the corresponding dipeptide methyl esters in toxic amounts from other amino acids, or that Leu-Leu-OMe was unique among dipeptide methyl esters in its toxicity for NK cells. Therefore, experiments were carried out to assess the effect of other dipeptide methyl esters on NK cell function. The methyl esters of a variety of dipeptides were synthesized and analyzed for the capacity to deplete NK cell function. Each dipeptide methyl ester was assessed in a minimum of three experiments. As is shown by the results displayed in Figure 9, Leu-Leu-OMe is not the only dipeptide methyl ester which exhibits NK toxicity. When amino acids with hydrophobic side chains were substituted for leucine in either position, the resulting dipeptide methyl ester generally displayed at least some degree of NK toxicity. In particular, Leu- Phe-OMe, Phe-Leu-OMe, Val-Phe-OMe, and Val-Leu-OMe produced concentration-dependent ablation of NK function at concentrations comparable to those at which Leu-Leu- OMe was active. The sequence of active amino acids was important, however, as evidenced by the finding that Phe- Val-OMe was markedly less active than Val-Phe-OMe. Similarly, Leu-Ala-OMe was NK inhibiting, whereas 10-fold greater concentrations of Ala-Leu-OMe had no NK inhibitory effects. Furthermore, Phe-Phe-OMe was less NK toxic than either Leu-Phe-OMe or Phe-Leu-OMe and Val-Val- OMe was less active than either Leu-Val-OMe or Val-Leu- OMe, yet Val-Phe-OMe was among the most potent of the NK toxic dipeptide methyl esters. Thus, conformational aspects of the dipeptide methyl ester amino acid side chain also seem to be of importance in producing the different levels of observed NK toxicity.
When amino acids with hydrophilic, charged or hydrogen side chains were substituted for leucine, the resulting dipeptide methyl esters either had greatly reduced NK toxicity, as in the case of Gly-Leu-OMe or Leu-Gly-OMe, or no observed NK inhibitory effects, as in the case of Leu-Arg-OMe, Leu-Tyr-OMe, Ser-Leu-OMe, Lys- Leu-OMe or Asp-Phe-OMe. Furthermore, when the D- stereoisomer was present in either position of a dipeptide methyl ester, no toxicity was observed for NK cells (Figure 9) . When unesterified dipeptides were assessed for their effect on NK function, as in the case of Leu-Leu (Figures 8A and 8B) , up to 5 x 10-3 M concentrations of Leu-Phe, Phe-Leu, Val-Leu, and Val-Phe had no effect on NK cell survival or lytic activity.
D-Leu-D-Leu-OMe had no effect on Leu-Leu-OMe mediated NK toxicity although high levels of zinc appeared to inhibit this Leu-Leu-OM, when equal concentrations of Leu-OMe, Val-OMe, or Phe-OMe were added to MP or PMN, the concentrations of Val-Val-OMe generated were 50 to 80% of those found for Leu-Leu-OMe, while Phe- Phe-OMe was detected at only 10-30% of the levels of Leu- Leu-OMe. Dipeptide methyl esters were not generated from D-amino acid methyl esters.
Figure 9 shows the NK toxicity of dipeptide methyl esters. MP-depleted lymphocytes were treated with varying concentrations of dipeptide methyl esters as outlined in Figures 8A and 8B. Results are given for the mean ±SEM of at least 3 separate experiments with each compound.
Example 9 NK Toxicity of an Artificially Hydrophobic Dipeptide Methyl Ester
Beta methyl aspartyl phenylalanine was prepared by methanolic hydrochloride methylation of aspartyl phenylalanine methyl ester. The NK toxicity of both aspartyl phenylalanine methyl ester and beta methyl aspartyl phenylalanine methyl ester was measured as described for the dipeptide methyl esters in Example 8. As the data in Table 3 indicates, when the polar side chain of the aspartyl amino acid dipeptide component is esterified with a methyl group, this being a conversion from relative hydrophilicity to substantial hydrophobicity, NK toxicity becomes apparent. Although yet not as toxically effective as a number of the hydrophobic-type dipeptides in Example 8, the data in Table 3 indicate that a dipeptide methyl ester comprising synthetic hydrophobic (lipophilic) amino acids may be used to inhibit NK function.
TABLE 3
L-ASPARTYL (beta-METHYL ESTER)-L-
PHiπreiALANINE METHYL ESTER IS NK TOXIC WHILE
L-ASPARTYL-L-PH_3^LALANINE METHYL ESTER IS NOT
Preincubation NK Function
% Specific Cytotoxicity
Nil 50.8
Asp-Phe-OMe:
100 micromolar 250 micromolar 500 micromolar 1000 micromolar
Asp-(beta-OMe)-Phe-OMe: 100 micromolar 250 micromolar 500 micromolar 1000 micromolar
Figure imgf000033_0001
Example 10 In Vivo Effects on Cvtotoxic Cell Function
Leu-Leu-OMe or Leu-Phe-OMe were suspended in PBS, pH 7.4. Then individual C3H/HeJ mice (25 gram size) were administered by tail-vein injection either 2.5 x 10-5 moles (6.5 mg) of Leu-Leu-OMe, 2.5 x 10-5 moles (7.1 mg) Leu-Phe-OMe, or an equal volume of the PBS diluent, this dose being about lxlO-3 moles per kg. For 15-30 minutes post-injection, Leu-Leu OMe and Leu-Phe OMe-treated animals but not the control animals exhibited decreased activity and an apparent increase in sleep. Subsequent to this quiescent period no difference in activity or appearance in the mice was noted. Two hours post- injection, the mice were sacrificed and their spleen cells were assayed for NK function in a standard 4 hour assay against YAC-1 tumor targets. In all mice, total cell recovery ranged from lxlO8 to l.lxlO8 spleen cells per animal. As noted in Table 4, the control mouse spleen cells exhibited greater killing at 25:1 and 50:1 effector to target cell ratios than did the spleen cells of treated mice at 100:1 and 200:1 E/T, respectively. Thus, Leu-Leu-OMe or Leu-Phe-OMe caused a greater than 75% decrease in splenic lytic activity against YAC-1 tumor targets.
TABLE 4 Cytotoxic Cell Function
Effector:Target Ratio 25:1 50:1 100:1 200:1
Percent lysis of target cells
Control 8.29 12.88 20.60 29.29
Leu-Leu-OMe 2.37 4.58 7.12 12.77
Leu-Phe-OMe 3.89 4.68 6.91 11.91 Example 11
Differential Sensitivity of
NK and MP Cells to Leu-Leu-OMe
In the experiments depicted in Figure 10, freshly isolated PBM (2.5xl06/ml PBS and lg/1 glucose) were incubated at room temperature with varying concentrations of Leu-Leu-OMe. After a 15 minute exposure to this compound, the cells were washed, incubated for 2 hours at 37°C and then assessed for the percentage of remaining viable cells which were stained by anti-MP or anti-NK monoclonal antibodies. Preincubation with greater than 25-50 micromolar Leu-Leu-OMe led to loss of NK cells. This concentration of Leu-Leu-OMe did not deplete MP from PBM but higher concentrations of Leu-Leu-OMe caused loss of MP. The data is Figure 10 show these results.
Anti-MP monoclonal antibodies (63D3) and anti-NK monoclonal antibodies (leu lib) were obtained from Becton Dickinson Monoclonal Center, Inc., Mountain View, CA. The antibody staining and Fluorescence Activated Cell Sorter (FACS) procedure was that of Rosenberg et al. (1981), J. Immunol . 126:1473. Data are expressed as percent of antibody staining in control cells (mean + SEM, n=4) .
Example 12 Effects of Leu-Leu-OMe On a Variety of Cell Types
While it was clear that a substantial percentage of lymphocytes remained viable following exposure to even 1 mM Leu-Leu-OMe, the finding that disparate cell types such as MP and NK were both susceptible to Leu-Leu-OMe mediated toxicity raised the possibility that this agent was a non-specific cell toxin. Therefore, the series of experiments depicted in Figure 11 was performed to assess other cell types for evidence of toxicity following exposure to Leu-Leu-OMe.
To facilitate screening of multiple cell types for evidence of cell death following exposure to Leu-Leu-OMe, a 51Cr release assay was devised. In preliminary experiments it was noted that 51Cr release from MP- enriched populations exposed to varying concentrations of Leu-Leu-OMe correlated very closely with concentration- dependent loss of anti-MP antibody staining cells from PBM after similar incubation. Following brief exposures to Leu-Leu-OMe at room temperature, the loss of anti-MP antibody staining cells from PBM or the release of 51Cr from MP-enriched populations was always detectable within a 30 to 60 minute period of culture at 37°C and maximal effects were seen within 3 to 4 hours.
Therefore, 51Cr release in a 4 hour assay was used in these experiments to assess toxicity from Leu-Leu-OMe.
As shown in the first graph of Figure 11, when the whole PBM population was exposed to varying concentrations of Leu-Leu-OMe, detectable 51Cr release was observed after exposure to 25 to 50 micromolar Leu-Leu-OMe, but only upon exposure to greater than 100 micromolar Leu-Leu-OMe was the maximal achievable 51Cr release from PBM observed. When MP-enriched adherent cells (AC) were similarly assessed, minimal 51Cr release was observed after exposure to 25-50 micromolar Leu-Leu-OMe whereas upon incubation with higher concentrations of this agent, more 51Cr release from AC was observed than with PBM. When nylon wool non-adherent lymphocytes (NAC) were assessed, small but significant 51Cr release was observed with 25 to 50 micromolar Leu-Leu-OMe. When NAC were exposed to increasing concentrations of Leu-Leu-OMe, greater quantities of 51Cr release were observed. N-SRBC positive cells showed a dose-dependent Leu-Leu-OMe induced 51Cr release pattern indistinguishable from that of NAC. Since both antibody staining (Figure 10) and functional studies (Figures 8A and 8B) have shown that 100 micromolar Leu-Leu-OMe causes maximal depletion of NK, this finding suggested that other lymphocytes were also susceptible to Leu-Leu-OMe toxicity at concentrations greater than 100 micromolar. When T4 enriched populations of T cells were assessed, however, it was clear that even 1000 micromolar Leu-Leu-OMe caused minimal 51Cr release from this population. In contrast, when N-SRBC positive cells were depleted of OKT4 positive cells, the remaining T8-enriched population produced high levels of 51Cr release following exposure to Leu-Leu-OMe.
When cell lines of myeloid or lymphoid origin were similarly assessed, selective toxicity of Leu-Leu-OMe was again observed. The human T cell leukemia line MoLT-4 demonstrated no detectable Leu-Leu-OMe toxicity over a broad concentration range. The human plasma cell lines HS-Sultan and the B lymphoblastoid line Daudi demonstrated no significant 51Cr release or alteration in subsequent proliferative rate (data not shown) after exposure to a broad range of Leu-Leu-OMe concentrations. When the susceptibility of EBV-transformed B cell lines or clones to this agent was assessed, no significant toxicity of less than 250 micromolar Leu-Leu-OMe was seen. However, with higher concentrations of Leu-Leu- OMe, a variable degree of toxicity was seen. Some EBV lines consistently displayed less than 20% 51Cr release even after exposure to lmM Leu-Leu-OMe, while other lines produced 25-35% 51Cr release after exposure to 250 micromolar Leu-Leu-OMe. In contrast, the human cell line U937 was susceptible to concentration-dependent Leu-Leu- OMe toxicity in a pattern indistinguishable from that of the peripheral blood MP with which this cell line shares many phenotypic and functional characteristics. After exposure to more than 250 micromolar Leu-Leu-OMe, extensive 51Cr release was observed and no viable proliferating U937 cell could be detected (data not shown) . Similarly, the erythroleukemia line K562 demonstrated no significant 51Cr release or alteration in subsequent proliferative rate (date not shown) upon exposure to 100 micromolar or lower concentrations of Leu-Leu-OMe. With higher concentrations of Leu-Leu-OMe, modest amounts of 51Cr release and partial loss of proliferative capacity were observed (data not shown) . In contrast, a variety of cell types of non-lymphoid, non-myeloid origin including human umbilical vein endothelial cells, the human renal cell carcinoma line, Currie, the human epidermal carcinoma line, HEp-2, and human dermal fibroblasts demonstrated no significant Leu- Leu-OMe induced 51Cr release. Furthermore, incubation of each of these non—lymphoid cell types with 500 micromolar Leu-Leu-OMe had no discernible effect on subsequent proliferative capacity.
HS-Sultan, a human plasma cell line (Goldblum et al. (1973) Proc. Seventh Leucocyte Culture Conference, ed by Daguilland, Acad. Press N.Y. pp 15-28) , Daudi, a B lymphoblastoid cell line (Klein et al. (1968) Cancer Res. V 28, p 1300), MoLT-4, an acute lymphoblastic T-cell leukemia line (Monowada et al. (1972) J. Nat'l. Cane. Inst. V 49, p 891), and U-937, a human monocyte-like cell line (Koren et al. (1979) Nature V 279, p 891) were obtained from the American Type Culture Collection, Rockville, MD. These lines as well as HEp-2 a human epidermoid carcinoma line (a generous gift of Dr. R. Sontheimer, UTHSCD) ; Currie, a human renal cell carcinoma line (a generous gift of Dr. M. Prager, UTHSCD) ; and K562, a human erythroleukemia line (a generous gift of Dr. M. Bennett, UTHSCD) were maintained in culture in medium RMPI supplemented with 10% FBS. Human dermal fibroblasts (a generous gift of Dr. T. Geppert, UTHSCD) were serially passaged in culture as well while human umbilical vein endothelial cells (a generous gift of Dr. A. Johnson, UTHSCD) were used after one subculture. Epstein Barr virus (EBV) transformed B lymphoblastoid cell lines JM.6 and SM.4 (kindly provided by Dr. J. Moreno, UTHSCD) and cloned EBV transformed B cell lines SDL-G2 and D8-219 (a generous gift of Drs. L. Stein and M. Dosch, Hospital for Sick Children, Toronto, Canada) were maintained in culture in medium RPMI supplemented with 10% FBS.
In some experiments, toxicity of Leu-Leu-OMe for a variety of cell populations was assessed by 51Cr release. In assays where cells obtained from suspension culture were to be used, cells were labeled with Na2 51Cr04 (ICN, Plainview, NY) for 60-90 minutes at 37°C and then washed three times. Cells were then suspended in PBS (2.5xl06/ml) and incubated in microtiter plates, 50 microL/well with indicated concentrations of Leu-Leu-OMe for 15 minutes at room temperature. In assays where cells were obtained from onolayer cultures, microtiter wells were seeded with cells (5xlOA/well) and cultured for 24 hours at 37°C. Cells were then labeled with Na2 51CrO while in adherent culture. Following 51Cr labeling, wells were thoroughly washed and varying concentrations of Leu- Leu-OMe added in 50 microL PBS and the plates incubated for 15 minutes at room temperature.
Following such initial serum-free incubations, 200 microL/well of medium RPMI containing 10% FBS were added and the plates incubated for another 4 hours prior to removal of 100 microliters of supernatant. Radioactivity in the supernatant was measured in an auto-gamma scintillation spectrometer (Packard Instrument Co., Downers Grove, IL) . The percent specific release was calculated from the formula:
% spec, release (rel) = exp. rel fcpm) - spont. rel fcpm) max. rel (cpm) - spont. rel (cpm)
in which maximal release refers to cpm obtained in wells containing 50% lysing agent (American Scientific Products, McGraw Park, IL) and spontaneous release refers to cpm released by cells incubated in control medium in the absence of Leu-Leu-OMe or the lysing agent. Only experiments in which spontaneous release was less than 25% were used for subsequent data interpretation.
While the MP-like tumor line U937 was virtually identical to MP in susceptibility to Leu-Leu-OMe, none of the non-lymphoid, non-myeloid cell lines tested demonstrated such susceptibility to Leu-Leu-OMe mediated toxicity.
The current example demonstrates that at concentrations 10 to 20 fold greater than those at which cytotoxic cells are ablated, Leu-Leu-OMe does have some minimal toxicity for certain non-cytotoxic lymphoid cells such as EBV transformed B cells and K562 cells. Yet, while it is impossible to exhaustively exclude the possibility that certain non-cytotoxic cells might also be equally sensitive to Leu-Leu-OMe-mediated toxicity, at present the ability to function as a mediator of cell mediated cytotoxicity is the one unifying characteristic of the cell types which are rapidly killed by exposure to Leu-Leu-OMe.
In developing the data expressed in Figure 11, cells (2.5 x 106/ml) were exposed to the indicated concentrations of Leu-Leu-OMe for 15 minutes at room temperature, then specific 51Cr release during the next four hours was assessed. Data for the EBV transformed lines JM.6, SDL-G2, D8-219, and SM.4, respectively, are shown in order from top to bottom.
Example 13
Relative Sensitivity of
CTL and NK Cells to Leu-Leu-OMe
Experiments were also designed to assess the relative sensitivity of NK and CTL to Leu-Leu-OMe. In the studies detailed in Figure 12 and 13, cytotoxicity assays were performed over a broad range of E:T ratios and units of lytic activity arising from equal numbers of responding lymphocytes were calculated and compared. As shown in Figure 12, both spontaneous NK and precursors of activated NK were totally eliminated by exposure to 100 micromolar Leu-Leu-OMe while CTL precursors, though diminished, were generally still present at greater than 50% of control levels. Only after exposure to greater than 250 micromolar Leu-Leu-OMe were all CTL precursors eliminated.
Figure 12 shows that incubation with Leu-Leu-OMe eliminates precursors of cytotoxic T lymphocytes (CTL) and activated NK-like cells (AcNK) . Non-adherent lymphocytes (2.5 x 106/ml) were incubated with the indicated concentrations of Leu-Leu-OMe for 15 minutes. Cells were then washed and either placed in mixed lymphocyte culture or assayed for specific lysis of K562 cells (NK) . After 6 day MLC, cells were assayed for specific lysis of allogeneic stimulator lymphoblasts (CTL) or K562 (AcNK) . Data are expressed as percent of control lytic units (mean + SEM, n=6) . When the elimination of CTL and activated NK pre¬ cursors by Leu-Leu-OMe was compared to that of spontaneous NK, the mean Leu-Leu-OMe concentration required to diminish lytic activity by 75% was significantly greater for elimination of CTL precursors (123 + 25 micromolar) than for elimination of precursors of activated NK (50 + 5 micromolar, p 0.05). Both values were also higher than the mean concentration of Leu-Leu- OMe required to diminish spontaneous NK lytic activity by 75% (35 micromolar + 4 micromolar) . Figure 13 shows that, following activation, CTL and AcNK became identical in sensitivity to Leu-Leu-OMe. After 6 day MLC, cells were incubated for 15 minutes with the indicated concentrations of Leu-Leu-OMe, then assayed for CTL or AcNK activity as for Figure 12. Thus, only after MLC activation did CTL display a sensitivity to Leu-Leu-OMe toxicity that was equal to that of NK cells.
Example 14 Mechanism of Leu-Leu-OMe Effects
On Selected Human Cell Populations
Prior examples demonstrated that incubation of mixed lymphoid cell populations with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) results in selective loss of NK cells and precursors of cytotoxic T cells whereas B cell and T helper cell function is relatively preserved. Of note, use of Leu-Leu-OMe to remove donor cytotoxic lymphocytes has been shown to be of benefit in preventing lethal graft-versus-host disease in a murine model of allogeneic bone marrow transplantation. The present example involves elucidation of the mechanism whereby Leu-Leu OMe kills cytotoxic lymphocytes.
Human peripheral blood lymphocytes (PBL) were incubated in the presence of [3H] labeled Leu-Leu-OMe at 22°C for varying lengths of time. The incubation mixture was then centrifuged through silicone oil to separate the cells from any unbound or non-internalized [3HLeu-Leu-OMe. As demonstrated in Figure 14, the quantity of cell- associated [3H]Leu-Leu-OMe increased in a linear, time- dependent fashion over the first 30 minutes of incubation. As demonstrated in Table 5, when incubations were performed at temperatures below 4°C, no accumulation of [3H]Leu-Leu-OMe by PBL was observed. At 37°C, levels of [3H] eu-Leu-OMe accumulation by PBL were increased above those seen at 22°C (see Table 5) . These findings suggested that Leu-Leu-OMe was not simply binding to PBL by an energy (temperature) independent process. The time and temperature dependent increases in cell-associated [3H]labeled Leu-Leu-OMe suggested that this compound was being internalized and retained by PBL.
TABLE 5
TEMPERATURE DEPENDENCE OF r3H1LEU-LEU-OME UPTAKE/BINDING BY LYMPHOCYTES
Expt. Temperature [3H] Leu-Leu-OMe Uptake/Binding (micro-moles/ 106 cells)
0°C 0.05 22°C 0.77
4°C 0.02 22°C 0.31 37°C 0.52
When the concentration dependence of Leu-Leu-OMe uptake by PBL was assessed (see Figure 15) , the quantity of [3H]Leu-Leu-OMe incorporated per unit of time was noted to increase in direct proportion to external concentrations until near maximal uptake was noted with approximately 250-500 micro-M. The findings detailed in Figure 15 indicate the uptake of Leu-Leu-OMe observed after a 15 minute incubation performed at 22°C. The data indicate that under these conditions the Vmax of (maximum velocity) this process is approximately 10-10 moles/minute/106 cells, whereas the Km is approximately 10"* M. These findings indicate that this process is saturable and therefore a facilitated transport mechanism is likely to be involved in the uptake of Leu-Leu-OMe by PBL. As demonstrated by the results displayed in Table 6, [3H]Leu-Leu-OMe uptake by PBL is competitively inhibited not only by excess quantities of unlabeled Leu- Leu-OMe, but also by high concentrations of the dipeptide Leu-Leu and by other esters of Leu-Leu such as Leu-Leu- OBenzyl.
TABLE 6
COMPETITIVE INHIBITION OF [3H]LEU-LEU-OME
UPTAKE BY OTHER DERIVATIVES OF LEU-LEU
Unlabeled % Inhibition of Compound* [3H] Leu-Leu-OMe Uptake+
L-Leu-OMe -10 L-Leu-Leu 44 L-Leu-L-Leu-OMe 72 D-Leu-D-Leu-OMe 7 L-Leu-L-Leu-OBenzyl 87 L-Leu-L-Leu-NH2 8
L-Leu-L-Leu-L-Leu-OMe 6
*250 micro-M+ 10 micro-M
However, the amide derivative of Leu-Leu (L-Leu-L- Leu-NH2) ; the D-stereoisomer containing dipeptide ester, D-Leu-D-Leu-OMe; the amino acid analog Leu-OMe; and the tripeptide analog Leu-Leu-Leu-OMe do not competitively inhibit PBL uptake of L-Leu-L-Leu-OMe. Thus, the facilitated transport process utilized by PBL in the uptake of Leu-Leu-OMe appears to be relatively specific for L-stereoisomers of dipeptides or dipeptide esters.
As detailed in Table 7, competitive inhibition of Leu-Leu-OMe uptake is seen with some but not all dipeptide methyl esters composed of L-stereoisomers of naturally occurring amino acids.
TABLE 7
Figure imgf000045_0001
*250 micro-M + 10 micro-M
As previously reported, (see prior examples and PNAS 1985; 82:2468-2472), incubation of PBL for 15 minutes at 22°C with a variety of dipeptide methyl esters leads to loss of all natural killer cell (NK) function because of the direct toxicity of these compounds for cytotoxic lymphocytes. Concentrations of various dipeptide esters which result in 50% loss of human NK function (LD50) are detailed in the last column of Table 7. Of note, all compounds with significant NK toxicity at concentrations below 250 micro-M appear to be taken up by the same facilitated transport process as evidenced by significant competitive inhibition of [3H] Leu-Leu-OMe uptake. This transport process is not competitively inhibited by some dipeptide esters such as Pro-Leu-OMe or Asp-Leu-OMe. These latter dipeptide esters also exhibit no NK toxicity. Such lack of NK toxicity may be related to lack of accumulation of such agents by NK cells. However, other dipeptide esters such as Leu-Tyr-OMe or Ser-Leu-OMe which display little or no toxicity for NK cells may be, nevertheless, excellent competitive inhibitors of [3H]Leu-Leu-OMe uptake. These findings suggested that characteristics other than capacity for uptake by lymphocytes are likely to be involved in the selective NK toxicity of Leu-Leu-OMe and other dipeptide methyl esters. Whereas uptake by this pathway appears to be necessary for NK cytotoxicity, it is not always sufficient.
Additional experiments, detailed in Table 8, were performed to assess the metabolic fate of [3H]Leu-Leu-OMe within PBL. Cells were incubated with [3H]Leu-Leu-OMe for 15 minutes at 22°C to permit uptake of this compound and then were washed. If the cells were immediately lysed and 10% trichloroacetic acid (TCA) was added to precipitate higher molecular weight cell proteins and nucleic acids, essentially all of the [3H]label remained in the supernatant as anticipated for a small molecular weight peptide which is soluble in 10% TCA. However, as shown in Table 8, if [3H]Leu-Leu-OMe-loaded PBL were incubated at 37°C for 15 to 60 minutes prior to cell lysis, an increasing fraction of the [3H] precipitated in 10% TCA. TABLE 8
[3H]LEU-LEU-OME IS CONVERTED TO A PRODUCT WHICH PRECIPITATES IN THE PRESENCE OF 10% TCA
Duration of Initial Duration of Second [3H]Leu-Leu-OMe
22°C Incubation 37°C incubation Total TCA Prec.
cpm x 10-3
15 minutes 61.4 0.5
15 minutes 24.9 6.8 30 minutes 20.6 12.7 60 minutes 12.7 10.7
In other experiments it was noted that addition of proteinase K (e.g. Protease Type XXVIV from Triterochium album, Sigma Chemical Company, St. Louis, Missouri) to the cell lysate prior to TCA precipitation resulted in loss of all [3H]label from the precipitate. These findings suggested that, within the first hour after [3H]Leu-Leu-OMe uptake by PBL, a significant fraction of this peptide or its amino acid components was incorporated into a higher molecular weight form which was insoluble in 10% TCA.
As detailed in Table 9, when lymphocyte subsets were highly purified by fluorescence activated cell sorting and then analyzed for dipeptidyl peptidase I activity, the levels of this enzyme within these cells was noted to vary greatly. Of special note, enzyme levels were highest in NK cells, monocytes (M-phi) and the cytotoxic T cell-enriched T8 cell subset. Furthermore, previously documented sensitivity to the toxic effects of Leu-Leu- OMe (second column. Table 9) was shown to be directly proportional to dipeptidyl peptidase I levels (third column, Table 9) .
TABLE 9 CELLS WHICH ARE SENSITIVE TO THE TOXIC EFFECTS
OF LEU-LEU-OME HAVE A HIGH CONTENT OF THE LYSOSOMAL THIOL PROTEASE. DIPEPTIDYL PEPTIDASE I CCATHEPSIN C)
Cell Type Leu-Leu-OMe Dipeptidyl Peptidase I Sensitivity (LD50) (nMole-beta Naphthyl/ hr/micro-g protein)*
Figure imgf000048_0001
* The enzyme was assayed in cellular protein at 37°C with l micro-M dithiothreitol with 200 micromolar glycyl L- phenylalanyl-beta napthylamide
Dipeptidyl peptidase I is a lysosomal thiol peptidase which has been shown to remove amino terminal dipeptides from proteins. Alternatively, at neutral pH, incubation of this enzyme with high concentrations of dipeptide esters or amides has been shown to result in production of higher molecular weight polymerization products with the structure (Rl -R2)n-0R' (J. Biol. Chem. 1952; 195:645-656). When R1 and R2 are amino acids with nonpolar side groups, such products are very hydrophobic and water insoluble (J. Biol. Chem. 1952; 195:645-656). As shown by the data displayed in Table 10, incubation of purified bovine dipeptidyl peptidase I (DPPI) at neutral pH with high concentrations of Leu-Leu-OMe results in production of a product which is insoluble in 10% TCA. Of note, much lower extracellular concentrations of Leu- Leu-OMe are required for production of a similar product within NK and T8 cells.
TABLE 10 LEU-LEU-OME IS METABOLIZED (POLYMERIZED) TO A TCA INSOLUBLE PRODUCT BY DIPEPTIDYL PEPTIDASE I (CATHEPSIN C)
Concentration Fractional Conversion of [3H]Leu-Leu-OMe of Leu-Leu-OMe to a product which Precipitates in 10% TCA (micro-M) Intact NK, T8 Cells Purified DPPI
1 <0.1% <0.01%
10 <0.2% 0.03%
100 11.1% 0.08%
250 23.2% ND 1000 ND 20.0%
Since such cells appear to take up and concentrate this compound by a facilitated transport mechanism (Figures 10 and 11) , it is likely that intracellular concentrations of Leu-Leu-OMe comparable to those required for polymerization of this compound by purified DPPI are achieved. These results, therefore, suggest that the 10% TCA insoluble product of Leu-Leu-OMe produced by cytotoxic lymphocytes can be accounted for by the actions of DPPI present within these cells.
The following experiments were carried out to determine whether DPPI could generate a lytic product from Leu-Leu-OMe. In the experiment detailed in Figure 14, 51Cr-labeled human erythrocytes (RBC) were incubated with varying concentrations of Leu-Leu-OMe alone (♦) or in the presence of purified bovine DPPI (■) . As shown by the results displayed in Figure 16, exposure of RBC to either DPPI or Leu-Leu-OMe alone results in no damage to RBC. However, in the presence of higher concentrations of Leu-Leu-OMe and DPPI, RBC lysis occurs. That such damage to erythrocyte cell membranes is likely to be related to production of a higher molecular weight hydrophobic polymer of Leu-Leu-OMe is demonstrated by the results of the experiment detailed in Table 11.
TABLE 11
RED BLOOD CELL LYSIS CAN BE
MEDIATED BY LEU-LEU-LEU-LEU-LEU-LEU-OME
Compound Concentration of 51Cr Release
Added Peptide Ester (micro-M) from RBC
Nil 0 410 + 12
LLOMe 2500 482 ± 121
500 482 ± 57
100 400 + 27
LLLLOMe 500 457 + 41 100 425 ± 24
TiT.TiTiTiTiOMe 500 3 ,531 ± 101
100 6,129 + 400
20 698 + 137
Table 11 shows results of an experiment, where 51Cr- labeled RBCs were incubated with varying concentrations of the methyl esters of the di-, tetra-, and hexa- peptides of leucine. Disruption of erythrocyte membranes was observed following exposure to the very hydrophobic compound (Leu)6 -OMe. As detailed in Table 12, the specific inhibitor of DPPI, Gly-Phe-CHN2 blocks the toxic effects of Leu-Leu- OMe.
TABLE 12
A SPECIFIC INHIBITOR OF
DIPEPTIDYL PEPTIDASE I PREVENTS
LEU-LEU-OME MEDIATED DEPLETION OF CD16(+) LYMPHOCYTES
First Second Cells stained with
Incubation Incubation anti-CD16 anti-CD4 anti-CD8
Figure imgf000051_0001
In the experiments detailed in Figures 17A through 17H, 51Cr-labeled RBC's were exposed to various dipeptide esters or amides in the presence or absence of purified DPPI. The results indicate that DPPI is unable to produce a membrane active metabolite from D-Leu-D-Leu-OMe or from dipeptide esters containing at least one amino acid with a polar side group such as serine or tyrosine. The first observation is probably related to the inability of this enzyme to catalyze transpeptidation of peptide esters containing D-stereoisomers of amino acids. The latter observations are likely to be related to the fact that polymers of Ser-Leu or Leu-Tyr are not hydrophobic and therefore unlikely to enter and disrupt cell membranes.
All of the compounds analyzed in Figures 17A through 17H with the exception of Leu-Leu-NH2 and Leu-Leu-OBenzyl have previously been assessed for NK toxicity (see prior examples and PNAS 1985; 82:2468-2472). In Table 13, results of experiments assessing the effects of these compounds on human NK function are detailed.
TABLE 13
INCUBATION OF MIXED LYMPHOCYTE
POPULATIONS WITH LEUCYL-LEUCINE
BENZYL ESTER RESULTS IN LOSS OF NK FUNCTION
Addition During NK Function Preincubation
% Specific Lysis K562
Nil 66
12.50 micro-M Leu-Leu-OMe 60 25.00 micro-M Leu-Leu-OMe 12 50.00 micro-M Leu-Leu-OMe 2 100.00 micro-M Leu-Leu-OMe <1 6.25 micro-M Leu-Leu-OBenzyl 62 12.50 micro-M Leu-Leu-OBenzyl <1 25.00 micro-M Leu-Leu-OBenzyl 1 250.00 micro-M Leu-Leu-NH2 71 1.00 micro-M Leu-Leu-NH2 66
These results indicate that whereas Leu-Leu-NH2 exhibits no discernible toxicity for NK Cells, exposure to relatively low concentrations of Leu-Leu-OBenzyl ablates human NK activity. Indeed, Leu-Leu-OBenzyl is greater than twofold more potent than Leu-Leu-OMe with respect to the capacity to deplete human PBL of cytotoxic lymphocytes. Table 14
Figure imgf000053_0001
The data summarized in Table 14 indicate that all compounds mediating NK toxicity in assays performed in examples share two characteristics:
1. Such NK toxic reagents competitively inhibit [3H]Leu- Leu-OMe uptake by human PBL and are therefore likely to be concentrated within lymphocytes by the same facilitated transport mechanism.
2. All NK toxic compounds are composed of amino acids with non-polar side groups and are suitable substrates for a DPPI catalyzed polymerization reaction which produces a hydrophobic product that disrupts erythrocyte cell membranes. These studies, therefore, indicate that all dipeptide esters or amides with these characteristics (including Leu-Leu-OBenzyl, see Table 13) are likely to have the same immunosuppressive activities as the dipeptide alkyl esters reported earlier herein.
Furthermore, these studies are the first to demonstrate that dipeptidyl peptidase I levels are selectively increased in cytotoxic lymphocytes. It can therefore be hypothesized that inhibition of this enzyme with Gly-Phe- CHN2 or similar selective dipeptidyl peptidase I inhibitors should alter the function of these cells and therefore may act as an immunosuppressive agent of value in therapy of the same diseases or conditions as proposed for Leu-Leu-OMe or similar agents (albeit by a different mechanism) .
Example 15 NK Toxicity of Peptide N-Substituted Amides
These experiments were designed to assess the NK toxicity of dipeptide amides. In previous studies (see prior Examples and PNAS 82:2468-2472), the present inventors demonstrated that NK toxic effects of Leu-Leu- OMe were only seen when lymphocytes are exposed for 15 minutes at room temperature to Leu-Leu-OMe concentrations in excess of 12.5 micro-M. Whereas Leu-Leu-OBenzyl is a more potent NK toxin than is Leu-Leu-OMe, Leu-Leu-NH2 has no demonstrable NK toxic effects (see Tables 13 and 14 of Example 14) . The experiment detailed in Table 15 demonstrated that whereas Gly-Phe-OMe is a much less potent NK toxin than is Leu-Leu-OMe; and Gly-Phe-NH2, like Leu-Leu-NH2/ has no apparent NK toxic effects; glycyl- phenylalanine-beta-naphthylamide is a very potent NK toxin. TABLE 15
INCUBATION OF MIXED LYMPHOCYTE
POPULATIONS WITH GLYCYL-PHENYLALANINE-
BETA-NAPHTHYLAMIDE RESULTS IN LOSS OF NK FUNCTION
Addition During NK Function Preincubation
% Specific Lysis K562 Nil 40.8
50 micro-M Leu-Leu-OMe 1.1 250 micro-M Gly-Phe-OMe 34.5 500 micro-M Gly-Phe-OMe 10.1
250 micro-M Gly-Phe-NH2 43.9
1 micro-M Gly-Phe-beta-Naphthylamide 52.5
5 micro-M Gly-Phe-beta-Naphthylamide 1.2
20 micro-M Gly-Phe-beta-Naphthylamide 0.6
Additional studies demonstrated that Gly-Phe-beta- naphthylamide competitively inhibits [3H]Leu-Leu-OMe uptake by lymphocytes, whereas simple amide derivatives of Leu-Leu-OMe do not compete for uptake by this facilitated transport mechanism (see Table 14, Example 14) .
Example 16 Other Agents with Immunosuppressive Properties Analogous to Leu-Leu-OMe
It has been found that alkyl, aralkyl and aryl esters or amides of peptides consisting essentially of natural or synthetic amino acids with hydrophobic side chains may function cytotoxically to deactivate natural killer cells (NK) and cytotoxic T lymphocytes (CTL) . By the term "hydrophobic" as used herein, is meant uncharged in aqueous solution at physiological pH and also as having no hydroxyl, carboxyl or primary amino groups.
Both methyl ethyl and benzyl esters or amides of peptides consisting essentially of natural or synthetic amino acids having hydrophobic side chains have been specifically found to deactivate NK or CTL cells, and other alkyl esters of these peptides (or peptide analogs thereof) are confidently predicted to have similar or superior effects.
On the basis of these findings and those contained in Table 15, Example 15, ester or amide derivatives of Leu-Leu or similar dipeptides which contain benzyl, naphthylamine or similar non-polar ring structures should prove to be selectively toxic for cytotoxic lymphocytes at lower concentrations than Leu-Leu-OMe and thus have enhanced clinical efficacy.
Example 17
Ex vivo Dipeptide Alkyl Ester
Treatment in Graft vs Host
Disease and Graft Rejection
This example is provided to demonstrate the role of dipeptide alkyl ester, particularly O-alkyl ester (e.g., Leu-Leu-OMe)-sensitive CTL in graft rejection in vivo. The particular in vivo model employed in the present study was the C57BL/6 (B6) mouse model. This particular animal model is also used to demonstrate the use of the referenced dipeptide alkyl esters and O-alkyl esters in diminishing and/or preventing lethal graft vs host disease and graft rejection, most particularly, acute allograft rejection. The data presented herein employing the particularly defined dipeptide alkyl esters and O- alkyl dipeptide esters demonstrate that T-cell mediated cytotoxicity plays a major, if not essential, role in mediating skin graft or other forms of organ allograft rejection in vivo .
The experimental design employed in the present study examines whether removal of Leu-Leu-OMe-sensitive, DPPI-enriched CTL is effective in preventing or modulating rejection of Class I + II MHC and multiple non-MHC dispartate skin allografts. Leu-Leu-OMe-treated C57BL6/J (B6) SpC (h-2b) are demonstrated herein to be unable to generate lethal GVHD in euthymic or thymectimized B6D2F1 (h-2b*d) recipient mice. Moreover, while transfer of Leu-Leu-OMe-treated B6 SpC to rigorously T cell depleted, thymectomized B6 host mice was sufficient to allow rejection of B6D2F1 skin grafts, the rate of skin graft rejection in these animals was significantly delayed. Thus, the data presented herein indicates that Leu-Leu-OMe sensitive CTL appear to play an active role in acute skin graft rejection.
As the host thymus has been shown to play a major role in the establishment of tolerance to both donor and host alloantigens following bone marrow transplantation, the role of the thymus on the course of GVHD was also examined.
C57BL/6J (B6) and (C57BL/6xDBA/2)Fl female mice were purchased from the Jackson Laboratory, Bar Harbor, ME. The medium was RPMI 1640 (Hazleton Research Products, Denver, PA) supplemented with 5 mM HEPES, 1 mM sodium pyruvate, 10"* M 2-mercaptoethanol, penicillin G (200 U/ml) , (gentamicin (10 g/ml) , L-glutamine (0.3 mg/ml) and 10% fetal bovine serum was used for cell cultures. For in vivo cell depletion, anti-L3T4 (GK1.5, 16) and anti-Lyt2 (YTS 169.4, 17) were purified from hybridoma culture supernatant by ammonium sulfate precipitation and binding to staphylococcal protein A- columns. The IgG fraction of rabbit anti-mouse thymocyte globulin was purchased from Accurate Chemical and Scientific Corporation, San Diego, CA. For in vitro depletion of T cells, anti-Thyl.2 (HO-13-4, 18), anti- L3T4 (2B6, 19) anti-L3T4 (2B6, 19) and anti-Lyt 2 (3.155, 20) were prepared as culture supernatants of hybridoma cells obtained from the American Type Culture Collection, Rockville.
Bone marrow cells (BMC) were flushed from femurs and tibias, were suspended in Hanks' balanced salt solution (HBSS) , and were filtered through sterile nylon mesh. Spleen cells (SpC) were suspended in HBSS, filtered through sterile nylon mesh and then washed. For depletion of T cells, suspensions of cells (40 x 106/ml) were incubated for 30 min. at 4°C with anti-Thyl.2, anti- L3T4 (2B6) and anti-Lyt 2 (3.155) and then rabbit complement (1:8, Pel Freez, Rogers, AR) , previously adsorbed with mouse spleen cells, was added, and cells were incubated at 37°C for an additional 50 minutes. Cells were then pelleted by centrifugation and resuspended in fresh medium an dfresh complement and incubated at 37°C for an additional 50 minutes. Cells were then washed two times in HBSS.
Leu-Leu-OMe was synthesized from leucyl-leucine
(Sigma) as described by Thiele et al. (1985, J. Immunol . , 234:786-793) . Cells were washed and suspended (2.5 to 10 x 106/ml) in PBS and were incubated for 15 min. at room temperature with 250 μM Leu-Leu-OMe. Cells were then washed, resuspended in culture medium, and placed in culture or infused in vivo within 1 hour. For identification of B cells, SpC were stained with fluorescein conjugated F(ab')2 goat anti-mouse immunoglobulin (Fl-GAMIg, Cooper Biomedical, Malvern, PA). Thy.2(+) cells were identified by incubation with HO-13-4 culture supernatant followed by staining with Fl- GAMIg and the number of Thyl.2 (+) cells determined by subtraction of the number of cells directly staining with Fl.GAMIg from these staining with Fl.GAMIg after initial incubation with anti-Thyl.2 (HO-13-4). L3T4(+) or
Lyt2(+) cells were identified by incubation with GK1.5 or YTS 169.4 followed by staining with fluorescein conjugated F(ab')2 mouse anti-rat IgG (Jackson Immunoresearch, West Grove, PA) , a secondary antibody with very low levels of direct staining (<1%) of mouse SpC. Cells were analyzed on a Becton Dickinson FACStar flow cyto eter as described by Thiele et al . (1986, J". Immunol, 236:1038).
Recipients were maintained on acidified (pH 2) , antibiotic (neomycin, 100 mg/liter, and polymyxin B, 10 mg/liter) H20 for 2 to 3 days before and 7 days after transplantation. On the day of transplantation, recipients were irradiated (900 cGy) and 2 to 6 h later were injected via the lateral tail vein with donor cells in 0.5 ml of HBSS.
Adult thymectomy of B6 mice was performed at 5-6 weeks of age by the method of Miller, (1960, Br. J. Cancer, 24:93). Mice were allowed to recover for at least ten days before irradiation (900 cGy) and transplantation with 5 x 106 anti-Thyl.2, anti-L3T4 (2B6) and anti-Lyt2 (3.155) + C treated B6 BMC. In addition, all mice were injected intraperitoneally with 200 g/d of anti-L3T4 (GK1.5) and 100 g/d anti-Lyt2 (YTS169.4) for three consecutive days. These doses of anti-T cell antibodies were 2-fold greater than those at which >95% depletion of L3T4(+) and Lyt2(+) splenic T cells was observed 3 days after completion of this regimen in euthymic, control B6D2F1 mice. In some experiments, on the second day of anti-L3T4 and anti-Lyt2 treatment, mice were also injected intraperitoneally with 0.5 mg of rabbit anti-thymocyte globulin, a dose 2-fold in excess of that observed to cause >90% depletion of L3T4(+) and Lyt2(+) T cells from euthymic control mice.
Three to four weeks after completion of in vivo anti-T cell therapy, ATXBM, TCD mice wre reconstituted wth 70 x 106 control B6 SpC, 70 x 106 Leu-Leu-OMe treated B6 SpC or no SpC. Within 24 hours, B6D2F1 tail skin in pieces aproximately 4 mm x 10 mm was grafted onto the lateral thoracic wall of recipient mice. The general technique of free skin grafting described by Billingham et al. (1951, J. Exp. Med. , 28:385) was adapted for use in the grafting of skin grafts to the mouse model of the present example, which reference is specifically incorporated herein by reference for this purpose. The grafts were covered with vaseline impregnated gauze and plaster bandages which were removed 8-10 days after grafting. The grafts were observed daily for rejection, which was considered complete when no viable skin was visible.
The immunomodulatory effects of Leu-Leu-OMe treatment of effector T cells is demonstrated to alter the course of solid organ graft rejection. In the present study, effector T cells were treated ex vivo with the described dipeptide alkyl esters. B6 female mice (5- 6 weeks of age) were serially thymectomized, lethally irradiated, reconstituted with T cell depleted B6 BMC and infused with anti-CD4 and anti-CD8 mAb's as described supra. Two months after completion of this regimen, SpC from such ATXBM, TCD mice were analyzed by flow cytometry for the presence of residual T cells. As indicated by the results detailed in Table 16, the spleens of these animals contained less than or equal to 1% CD4(+) or CD8(+) T cells. Long-term reconstitution with CD4(+) and with CD8(+) T cells was achieved when such ATXBM, TCD B6 mice were infused with 70 x 106 control or Leu-Leu-OMe treated B6 SpC. (See Table 16)
When B6D2F1 skin grafts were applied to unreconstituted ATXBM, TCD mice, long-term skin graft survival was seen in four of five mice (see Figure 18) . In the single animal in this group in which skin graft rejection was observed, subsequent fluorescence activated cell disorder (FACs) analysis revealed the presence of 12% CD4(+) and 3% CD8(+) cells within the spleen, suggesting that this animal represented a sporadic failure of the thymectomy and/or T cell depletion regimen, as all other animals in this experimental group were found to have less than 1% CD4(+) or CD8(+) T cells. When 70 x 106 control B6 SpC were infused into ATXBM, TCD mice, brisk rejection of B6D2F1 skin grafts by all animals was observed (Figure 18) . When B6 SpC treated with 250 μM Leu-Leu-OMe were infused into ATXBM, TCD mice, rejection of B6D2F1 skin by all mice was again observed. The duration of skin graft survival, however, was significantly prolonged in the recipients of Leu-Leu- OMe treated donor cells relative to that observed in recipients of control cells (p < 0.001 by Mann-Whitney non-parametric analysis) .
It appears that either a CTL depleted, Leu-Leu-OMe treated B6 SpC alone are capable of mediating rejection of B6D2F1 skin grafts, or that Leu-Leu-OMe resistant B6 SpC were providing T helper cell function necessary to generate cytotoxic effector cells from residual T cells or other cells present within ATXBM, TCD mice. In particular, as only irradiation and anti-CD4 and anti-CD8 mAb were employed to deplete T cells from the thymectomized host animals used in this example, Applicants postulate that radio resistant CD4-, CD8- host CTL might be serving as effector cells in skin graft rejection observed in these experiments. Therefore, additional experiments were carried out in which an injection of rabbit anti-mouse thymocyte globulin was added to the host T cell depletion regimen.
Hemisplenectomies were performed in all animals as a control to verify efficacy of thymectomy and T cell depletion. Only animals without detectable CD4(+) or CD8(+) T cells were used in subsequent skin graft experiments. As shown in the results detailed in Table 17, uniform, rejection of B6D2F1 skin grafts was again observed following transfer of Leu-Leu-OMe treated B6 SpC to such ATXBM pan-T cell depleted hosts. However, such rejection was again significantly delayed in hosts reconstituted with Leu-Leu-OMe treated SpC.
Table 16
LONG TERM CELL RECONSTITUTION OF
ATXBM, TCD MICE FOLLOWING TRANSFER
OF CONTROL OR LEU-LEU-OME TREATED SPC
Figure imgf000063_0001
"Results represent means of values obtained in 3 animals within each experimental group. Thymectomized, T cell depleted mice were sacrificed 100 days after infusion of 70 x 106 control B6 SpC, 70 x 106 Leu-Leu-OMe treated B6 SpC or cell free medium.
Table 17
ALLOGENEIC SKIN GRAFT REJECTION IS
SEEN FOLLOWING TRANSFER OF LEU-LEU-OME
TREATED SPC TO ATXBM MICE TREATED WITH
ANTI-L3T4, ANTI-LYT2 AND ANTI-THYMOCYTE GLOBULIN
Figure imgf000064_0001
*B6 host mice were thymectomized, irradiated (900σGy) and reconstituted with T cell depleted B6 BMC. Three weeks after BMC reconstitution, mice were injected intraperitoneally on three consecutive days with 200 μg of anti-L3T4 (GK1.5) and 100 μg of anti- Lyt2 (YTS 169.4) and on the second day of anti-L3T4 and anti-Lyt 2 treatment with 0.5 mg of rabbit anti- mouse thymocyte globulin.
^ouse died with intact skin graft 45 days after skin graft applied.
These results indicate that Leu-Leu-OMe resistant B6 T cells are capable of mediating delayed B6D2F1 skin graft rejection. Transfer of 10 x 106 control B6 SpC into thymectomized B6D2F1 mice (Figure 19A) induced more rapid lethal GVHD than noted in euthymic B6D2F1 recipients of the same number of B6 donor cells (Figure 19B) . Despite the enhanced vulnerability of thymectomized B6D2F1 mice to GVHD, Leu-Leu-OMe treatment of donor B6 SpC prevented lethal GVHD as effectively as was noted with control recipients.
The present example demonstrates a method of inhibiting graft rejection through the administration of particular alkyl esters of dipeptides, most particularly through administration of an O-alkyl ester. The most preferred O-alkyl dipeptide ester is L-leucyl-L-leucine memethyl ester (Leu-Leu-OMe) . The present methods may be used in conjunction with any type of tissue graft to inhibit tissue or whole organ rejection. In such an application, the prospective transplant recipient is first identified and then prospective transplant recipient is then treated with an effective amount of an alkyl ester of a dipeptide consisting of natural or synthetic L-amino acids with hydrophobic side chains.
The present methods, however, are most preferably employed in conjunction with skin grafts. Thus, the present disclosure provides a method whereby the rejection of skin and potentially other tissue grafts may be prevented in an animal, for example, of skin grafts in mouse and in human graft recipients.
* * * * * * * *
The various published literature articles cited in this application are incorporated in pertinent part by reference herein for the reason cited.
Changes may be made in the construction, operation and arrangement of the various elements, steps and procedures described herein without departing from the concept and scope of the invention as defined in the following claims.

Claims

CLAIMS :
1. A method for identifying a substance taken up by and deactivating NK or CTL cells, the process comprising the steps of:
(a) screening candidate substances to determine those which competitively inhibit the uptake by PBL, NK or CTL cells of L-leu-L-leu-OMe or competitively inhibit binding of a substance which itself competitively inhibits L-leu-L- leu-OMe uptake;
(b) incubating substances determined to be inhibitory in step (a) with membrane-enveloped labels and dipeptidyl peptidase I to identify those lysing enveloping membranes; and
(c) identifying substances which are taken up by and deactivate NK or CTL cells as those effectively lysing membrane enveloping the labels.
2. The method of claim 1 wherein membrane-enveloped labels are labeled human erythrocytes.
3. The method of claim 2 wherein candidate substances are dipeptide esters, dipeptide analog esters, dipeptide N-substituted amides or dipeptide analog N-substituted amides consisting essentially of L-α-amino acids with hydrophobic side chains, said esters being alkyl, aryl, alkaryl or aralkyl esters, and said N-substituted amides having N-alkyl, N-aryl, N-alkaryl or N-aralkyl substituents.
4. A method for identifying substances taken up by and deactivating cells containing dipeptidyl peptidase I, the process comprising the steps of:
screening candidate substances to determine those which competitively inhibit the uptake by PBL, NK or CTL cells of Leu-Leu-OMe or competitively inhibits the uptake or binding of a substance which itself competitively inhibits Leu Leu OMe uptake;
incubating substances determined to be inhibitory with internally labeled erythrocytes and dipeptidyl peptidase I to determine those causing erythrocyte lysis; and identifying substances which are taken up by and deactivate cells containing dipeptidyl peptidase I as those inhibitory substances effectively mediating human erythrocyte lysis;
wherein effective amounts of said identified inhibitory substances are taken up by cells and converted by intracellular dipeptidyl peptidase I into inactivating amounts of a membranolytic product.
5. The method of claim 4 wherein the cells containing dipeptidyl peptidase I are NK or CTL cells.
6. The method of claim 4 wherein the L-leu-L-leu-OMe is tritium-labeled.
7. The method of claim 4 wherein candidate compounds comprise dipeptide esters, dipeptide analog esters, dipeptide N-substituted amides or dipeptide analog N- substituted amides consisting essentially of L-α-amino acids with hydrophobic side chains, said esters being aryl, alkaryl or aralkyl esters and said amides being N- aryl or alkyl substituted amides.
8. The method of claim 4 wherein the membranolytic product is a hexapeptide ester, hexapeptide analog ester, N-substituted hexapeptide amide or N-substituted hexapeptide analog amide comprising at least one natural or synthetic amino acid analog having a hydrophobic side chain.
9. The method of claim 4 wherein the membranolytic product is characterized as being insoluble in 10% aqueous trichloroacetic acid.
10. A method for producing a membranolytic substance, the process comprising the steps of:
screening candidate substances to identify those which competitively inhibit uptake by PBL, NK or CTL cells of labeled L-leu-L-leu-OMe or competitively inhibit the uptake or binding of a substance which itself competitively inhibits labeled L-leu-L-leu-OMe uptake;
incubating inhibitory substances with internally labeled human erythrocytes and dipeptidyl peptidase I to identify those substances resulting in erythrocyte lysis; and
treating inhibitory substances causing lysis with dipeptidyl peptidase I to produce a membranolytic product.
11. The method of claim 10 wherein candidate substances comprise dipeptide esters, dipeptide analog esters, or dipeptide N-substituted amides or dipeptide analog N- substituted amides.
12. The method of claim 11 wherein the dipeptide esters, dipeptide analog esters, dipeptide N-substituted amides and dipeptide analog N-substituted amides consist essentially of L-α-amino acids with hydrophobic side chains, said esters being aryl, alkaryl or aralkyl esters, and said amide being N-aryl or N-alkyl substituted amides.
13. The method of claim 10 wherein the membranolytic product is an oligopeptide aryl or alkyl ester, oligopeptide analog aryl or alkyl ester, or an oligopeptide N-aryl substituted amide.
14. A hexapeptide ester, hexapeptide analog ester, N- substituted hexapeptide amide or N-substituted hexapeptide analog amide produced by the method of claim 10 and comprising at least one natural amino acid or amino acid analog having a hydrophobic side chain.
15. A method for preparing a membranolytic substance, the process comprising the steps of:
identifying a substance competitively inhibiting the uptake by PBL, NK or CTL cells of L-leu-L-leu- OMe or competitively inhibiting binding or uptake of a compound which itself competitively inhibits L-leu-L-leu-OMe uptake;
ascertaining an identified inhibitory compound which is converted by dipeptidyl peptidase I to a membranolytic substance;
structurally defining said membranolytic substance; and
preparing a quantity of said structurally defined membranolytic substance.
16. A membranolytic substance produced by the method of claim 10, 13 or 15.
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