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WO1992010198A1 - Agents, compositions et procedes immunotherapeutiques - Google Patents

Agents, compositions et procedes immunotherapeutiques Download PDF

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WO1992010198A1
WO1992010198A1 PCT/AU1991/000563 AU9100563W WO9210198A1 WO 1992010198 A1 WO1992010198 A1 WO 1992010198A1 AU 9100563 W AU9100563 W AU 9100563W WO 9210198 A1 WO9210198 A1 WO 9210198A1
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cells
mop
uva
treatment
treated
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David Marks
Richard Fox
Tom MANDEL
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Johnson & Johnson Research Pty Limited
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    • 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
    • A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A61K31/37—Coumarins, e.g. psoralen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/20—Cellular immunotherapy characterised by the effect or the function of the cells
    • A61K40/22—Immunosuppressive or immunotolerising
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/416—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule

Definitions

  • This invention relates to methods of treatment of immunologically related conditions, and to agents and compositions thereof.
  • the invention relates to a method of treatment of the patient's own lymphocytes, which treatment is carried out extracorporeally.
  • UVA ultraviolet A light 8-MOP 8-methoxypsoralen, dCF deoxycofor ycin, dAdo deoxyadenosine, NOD mouse non-obese diabetic mouse, CTCL cutaneous T-cell lymphoma, PBL peripheral blood lymphocytes, ECPC extracorporeal photochemotherapy, CP cyclophosphamide, Con A Concanavalin A EDTA ethylenediaminotetraacetate, BSS mouse isotonic buffered balanced salt solution; PBL peripheral blood lymphocytes; PBS phosphate buffered saline; %D % double-stranded DNA remaining; DNA SSBs DNA single-strand breaks; MDSS maximum decrease in skin score; S.E. standard error.
  • lymphocytes in particular T cells
  • T cells Treatments to augment the activity of lymphocytes, in particular T cells, have been proposed as a means of treatment for autoimmune disease, and it has been suggested that such treatments result in the generation of 'anti-clonotypic' T cells which inhibit the patient's autoreactive T cells.
  • the procedure has been termed 'T cell vaccination' (Cohen and für, 1988) .
  • the most widely clinically investigated of these procedures is known as photopheresis; this involves exposure of cells to a psoralen, followed by photoactivation with ultraviolet A. See, for example, European Patent Application No. 261648 and Australian Patent Application No. 13584/88, the entire disclosures of which are herein incorporated by reference. It is evident that the following procedures of extracorporeal treatment of lymphocytes are known or suggested to be effective in the treatment of autoimmune disease and/or cancer:
  • Chemical cross linking eg. with formaldehyde or glutaraldehyde; Photoactivated cross-linking using a psoralen and ultraviolet A, optionally with prior activation of lymphocytes using eg. Con A; Activation of lymphocytes eg. with Con A, followed by chemical cross-linking; Irradiation;
  • Infusions of photoirradiated T-cells prevent or ameliorate a number of diseases in animal models of autoimmune disease and transplantation (Knobler and
  • ECPC extracorporeal photochemotherapy
  • Cutaneous T-cell lymphoma is a malignant monoclonal proliferation of T-lymphocytes, usually those of the helper phenotype (Kubler and Edelson, 1986) .
  • the diseases encompassed include Sezary syndrome, mycosis fungoides and various adult T-cell leukemias.
  • Sezary syndrome mycosis fungoides
  • T-cell leukemias There are up to 10,000 new cases per year in the USA; it is more common in males, and is usually diagnosed after the age of 40. It typically remains localized to the skin for a time, then evolves into a nonepidermotropic stage in which there is dissemination and involvement of other organ systems. Extracutaneous spread is associated with refractoriness to treatment and a poor prognosis.
  • CTCL vascular endothelial cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic
  • DNA-damaging agents may kill cells by causing ATP and NAD depletion secondary to poly (ADP-ribosyl)a ion at sites of DNA strand breakage (Berger, 1985) : this might be an important mechanism of the lymphocytotoxicity of CTCL cells treated with ECPC.
  • ADP-ribosyl ADP-ribosyl
  • lymphocytotoxic effect of ECPC in CTCL is mediated by DNA damage, with concomitant stimulation of poly (ADP-ribose) synthetase and depletion of adenine nucleotides in the cells.
  • the treated cells appear programmed to die by apoptosis, with characteristic morphological changes associated with endonucleolytic DNA cleavage.
  • NOD/WEHI mouse was chosen as an animal model, because it provided a well-defined autoimmune disease with clear, easily assessed endpoints (Bescher et ai, 1990; Yasunumi and Bach, 1988; Harada and Makino, 1984).
  • UVA/8-M0P significantly reduces the incidence of diabetes in CP-treated 90-120 day old NOD/WEHI mice.
  • dCF and dAdo were similarly low incidence of diabetes in a group of mice that received splenocytes treated with dCF and dAdo. Both forms of treatment induce apoptosis in these splenocytes.
  • a pharmaceutical preparation for specifically altering the immune response of a mammal comprising mononuclear leukocytes from said mammal, which leukocytes have been treated by extracorporeal incubation with an agent which induces irreversible damage to the cells or cell death.
  • the invention provides a pharmaceutical composition for treatment of an immunologically-mediated condition or a cancer, comprising an effective amount of a preparation according to the first aspect of the invention, together with a pharmaceutically- acceptable carrier.
  • the invention provides a method for prevention or treatment of an immunologically- mediated condition or of a cancer, comprising the step of administering to a patient in need of such treatment an effective quantity of a composition according to the second aspect of the invention.
  • the invention provides a method of preparation of a pharmaceutical preparation according to the invention, comprising the steps of incubating a sample of cells from a patient suffering from an immunologically-mediated condition or a cancer, said sample comprising normal or malignant mononuclear leukocytes, with an agent which induces irreversible damage to the cells or cell death, for a period and under conditions effective to cause irreversible damage to said cells, and optionally washing the cells.
  • Agents suitable for use in the invention are those which damage cellular DNA.
  • Suitable agents for use in the invention include, but are not necessarily limited to antineoplastic or antiviral agents, especially nucleoside analogues, alkylating agents, inhibitors of topisomerase-II, antimetabolites and anthracyclines, and steroids.
  • Two or more active agents may be used in combination.
  • Particularly preferred agents are as follows: 1. An adenosine deaminase inhibitor in combination with an adenosine analogue.
  • Preferred adenosine deaminase inhibitors are deoxycoformycin, EHNA (erythro-9-(3-hydroxy- 2-nonyl)adenine, and 2-chlorodeoxyadenosine, while preferred adenosine analogues are 2'- deoxyadenosine, 3'-deoxyadenosine (cordycepin) , tubercidin and 9-(J-D- arabinofuranosyl)adenine (Ara-A; vidarabine) ;
  • alkylating agents preferred alkylating agents are mafosfamide and 4- hydroperoxycyclo-phosphamide;
  • Antimetabolites a preferred antimetabolite is methotrexate
  • Inhibitors of topoisomerase II; preferred inhibitors are etoposide (VP-16-213) and teniposide (VP-16) , and other derivatives of podophyllotoxin; and 5.
  • Antibiotics which induce DNA strand breakage such as the bleomycins, mitomycin, anthracyclines such as daunorubicin and epirubicin, and gliotoxin.
  • analogues or derivatives of the specific agents named which may also be used, provided that they are effective in inducing cell death. Such effectiveness may be readily established by methods known in the art.
  • the cells which are the target of the extracorporeal treatment will normally have been activated in vivo as part of the disease process.
  • the method of the invention may also optionally include a specific step of activation of the target cells prior to extracorporeal treatment, either by immunization of the subject or by treatment of the cells .in vitro with an agent such as Con A. Such a step would have the advantage of expanding the clone of activated target cells which are to be treated.
  • the cells to be treated are normal or malignant mononuclear leukocytes of ly phocytic or monocytic lineage, preferably lymphoid cells. These cells may be obtained from blood, lymph, bone marrow, or lymphoid tissue. For convenience, the preferred cells to be treated are buffy coat leukocytes.
  • the method of the invention is suitable for the treatment of a condition selected from the group consisting of: autoimmune diseases; cancer; allograft and/or xenograft rejection; graft-versus-host disease; delayed-type hypersensitivity; and allergy, especially autoimmune diseases, and malignancies of the lymphoid system.
  • a condition selected from the group consisting of: autoimmune diseases; cancer; allograft and/or xenograft rejection; graft-versus-host disease; delayed-type hypersensitivity; and allergy, especially autoimmune diseases, and malignancies of the lymphoid system.
  • the treatment of the invention results in the induction of cell death; it is thought that the treatment of the invention induces programmed cell death (apoptosis) rather than necrosis.
  • agents which induce apoptosis are particularly preferred for use in the invention.
  • Such agents induce fragmentation of cellular DNA, and include bleomycin, anthracyclines and gliotoxin.
  • Figure 1 shows % increase in DNA single strand breaks vs. % decrease in cellular (trypan blue) viability.
  • Simple regression analysis yields a R 2 of 0.877.
  • the intercept for the graph is 82.1 (38.9-125.2, 95% confidence limits) and the gradient is -1.03 (-2.12-0.06).
  • Figure 2 shows effects on viability.
  • Figure 3 shows DNA damage in PBL exposed to UVA light (1-5 J/cm 2 ) with or without 8-MOP (300 ng/ml) ; the % increase in DNA single strand breaks was measured 2 hours later fluorometrically. Mean of 2 experiments each performed in duplicate.
  • Figure 4 shows effects on nucleotide content.
  • Figure 5 shows a comparison between photochemo- therapy and treatment with deoxycoformycin plus deoxy- adenosine.
  • A. PBL were exposed to 1 ⁇ M dCF and 0-20 ⁇ M dAdo for 4 hours and corrected viability was determined for the next 72 hours.
  • Figure 6 shows results of an experiment in which mouse splenocytes were exposed to various cytotoxic agents and their corrected viability measured by trypan blue exclusion for the next 48 hours.
  • A. 1 1 J/cm 2 UVA light.
  • 2 1 J/cm 2 UVA light plus 100 ng/ml 8-MOP.
  • B. 1 ⁇ M dCF, 5 ⁇ M dAdo for 4 hours.
  • Results represent the mean of at least 2 separate experiments preferred in duplicate.
  • Figure 7 shows the incidence of diabetes (%) in
  • CP-treated NOD/WEHI mice whose splenocytes were subjected to various treatments.
  • Recipient NOD/WEHI mice were given 350 mg/kg intraperitoneal CP on day 1 and had their plasma glucose measured 14-18 days later. Diabetes was defined as a plasma glucose level of 15 mM or more.
  • UVAR photopheresis system Therakos, King of Prussia, PA, USA
  • This machine is the vehicle for the collection of buffy coat lymphocytes and also contains the clear plastic disposable cassette where lymphocytes are exposed to UVA light.
  • the patients ingested 8-MOP tablets at a dose of 0.6 mg/kg.
  • the patients were venepunctured and connected to the photopheresis machine.
  • the UVA source was switched on to irradiate the buffy coat sample.
  • Treatment policy and assessment of response Patients were treated on two consecutive -days every 4 weeks for 6 months, with treatment frequency modified according to clinical response. Disease progress was monitored by measurement of standardized skin scores, skin biopsies and by regular clinical photographs. Briefly, all skin was graded from 0 to 4, with 4 representing the most severe disease. This was then multiplied by the percentage of total surface area involved so the maximum possible score is 400. Skin score assessments were made before the commencement of therapy and then monthly before each course of ECPC by an experienced dermatologist. Response was defined as a 25% improvement in the skin score, sustainable over a one month period. Complete reassessment of all patients occurred 6 months after starting treatment.
  • Specimens of buffy coat blood were obtained from the first buffy coat specimen and also after UV irradiation was complete, immediately before reinfusion to the patient. About 20 ml of buffy coat blood was underlaid with 5 ml of Ficoll-Hypaque and spun at 450g for 25 minutes. The cloudy mononuclear cell fraction was removed and counted. After washing in phosphate-buffered saline, the lymphocytes were resuspended in RPMI 1640 plus 10% fetal calf serum. After the above handling, the mean composition of the cells (+ S.E.) was: lymphocytes 74 + 9%, granulocytes (including band forms) 22 + 8% and monocytes 3 + 1%.
  • lymphocytes from the buffy coat samples were suspended in RPMI 1640 plus 10% fetal calf serum and incubated at 37°C. Viability was determined by the ability of cells to exclude 0.5% trypan blue. Viability was assessed every 24 for 96 hours after photoirradiation and compared with the 24-96 hour viabilities of the unirradiated buffy coat lymphocytes which were similarly handled.
  • lymphocytes were centrifuged to form a pellet and then processed in triplicate samples according to the fluorometric method of Birnboim and Jevcak (1981) .
  • the percentage of double-stranded DNA remaining (% D) of the first (control) specimen was corrected to 100 and the % D value obtained for the second specimen was expressed as a percentage of this.
  • the increase in DNA SSBs was calculated by subtracting the % D value from 100.
  • the .in vitro processing of lymphocytes from normal human donors and their exposure to 8-MOP did not affect cellular viability; viability was unimpaired at the time of measuring DNA damage.
  • lymphocytes were centrifuged to form a pellet, extracted with ice-cold perchloric acid and their NAD and ATP content were measured by HPLC as previously described (Crescentini and Stocchi, 1984) .
  • the lymphocytes obtained from the unirradiated buffy coat specimen were regarded as having normal (control) NAD and ATP content and the nucleotide content of the lymphocytes from the second specimen was calculated as a percentage of this control value.
  • Fresh human peripheral lymphocytes were obtained by venesection or from normal human volunteers' buffy coat specimens (Victorian Red Cross Blood Bank) which were available within 1 hour of leukapheresis.
  • the 40 ml buffy coat specimen was overlaid onto 5-10 ml of Ficoll-Hypaque and spun at 450g for 25 minutes. Two washes in phosphate-buffered saline were followed by suspension in RPMI 1640 plus 10% fetal calf serum and antibiotics.
  • UV irradiation of lymphocytes or splenocytes UV irradiation of lymphocytes or splenocytes:
  • UV irradiation cells were suspended in culture medium at about 5 x 10 5 cells/ml. 8-MOP was freshly made up from stock solutions (1 mg/ml) and was added to cells shielded from the light for at least 15 minutes before photoirradiation. 5-10 ml aliquots of cell suspension were irradiated in 25 cm 2 plastic tissue culture flasks (Costar, Cambridge, MA, USA) with a Therakos research light box which contained a Photosette-R UVA light assembly mimicking the therapeutic equipment used for humans. Two banks of 6 lamps were located behind windows of transparent glass 10 cm apart. The lamps were operated at the maximum power setting (ten) for at least 10 minutes before experiments. The CSIRO National Measurement Laboratory of Australia calibrated the machine; a 32 second exposure was equivalent to 1 J/cm 2 of energy. The centre of the panel exclusively was used for UV irradiation.
  • Serum 8-MOP levels were measured according to the HPLC method of Puglisi et al (1977) . Blood samples were taken from all patients at hourly intervals during the period of photopheresis, so the peak 8-MOP level and its timing could be determined.
  • Non-obese diabetic mice are non-obese diabetic mice.
  • the non-obese diabetic (NOD) mouse is a model of type I diabetes (Makino et al, 1980; Baxter et al 1990) .
  • a lymphocytic insulitis causes beta cell destruction with consequent hypoinsulinemia and hyperglycemia.
  • NOD/WEHI mice have insulitis, but only a small proportion of susceptible mice (10% females and ⁇ l% males) spontaneously develop diabetes by 150 days of age. It is hypothesized that active suppression mechanisms prevent the progression from insulitis to diabetes.
  • CP usually an immunosuppressive agent
  • NOD/WEHI mice a dose of 300-350 mg/kg increases the incidence of diabetes to nearly 70% within 21 days.
  • the transfer of syngeneic mononuclear cells from prediabetic mice prevents CP-induced diabetes; the same cells from diabetic mice do not affect the incidence of diabetes.
  • CP has an immunological mechanism of action; it does not directly damage beta cells, nor does it cause diabetes in any of several other strains of mice not prone to develop diabetes.
  • mice were maintained in specific pathogen-free conditions before being placed in an open animal room where they were fed commercial food pellets and water ad libitum. All mice were maintained on racks in the same room, with a 12 hour/12 hour light and dark cycle in a constant temperature of 21°C. Overtly unwell mice or mice with evidence of diabetes before treatment were excluded from experiments. CP was freshly dissolved in mouse cell isotonic buffered balanced salt solution (BSS) and injected intraperitoneally into 100-120 day old female mice on day 1 at a dose of 350 mg/kg. This treatment causes diabetes in about 60% of mice 14 days later.
  • BSS mouse cell isotonic buffered balanced salt solution
  • mice Before entry into the study, recipient mice had to be shown to have a random plasma glucose of ⁇ 12 mM. Diabetes was defined as a random plasma glucose of 15 mM or more. Although the incidence of spontaneous diabetes at 90-120 days of age is low ( ⁇ 5%) , in relatively small treatment groups it could be significant; pretreatment glucose measurements eliminated this possibility.
  • mice Syngeneic mouse splenocytes were obtained from diabetic mice of approximately 110 days of age, 14 days after they had been given CP. Mice were killed by exposure to C0 2 and their spleens removed aseptically and placed in ice-cold BSS. The spleen was then processed by routine methods. Splenocytes were suspended in 0.156 M ammonium chloride, 0.1 mM EDTA and 12 mM sodium bicarbonate, pH 7.3, (a red cell lysis buffer) and left on ice for 5 minutes before resuspension in Dulbecco's modified Eagle medium with 5% fetal calf serum. One spleen yielded approximately 10 8 cells whose viability exceeded 80% by trypan blue exclusion. Culture of mouse splenocytes and exposure to DNA-damaging agents:
  • Cells were washed and suspended in Dulbecco's modified Eagle medium with 5% fetal calf serum at a density of 10 6 /ml before exposure, and the cytotoxic drugs were directly added. Cells were then washed in BSS and resuspended in 0.2 ml of this before injection into mice. During cytotoxic exposure, cells were maintained at 37°C in humidified conditions with 7% C0 2 /93% air.
  • Plasma glucose estimations were made with a modified glucose oxidase technique on an autoanalyzer (Beckman Glucose Analyzer 2, Fullerton, CA, USA) .
  • cells were washed twice in RPMI 1640, and then pelleted (200g, 5 minutes) .
  • Cell specimens were fixed in 1% glutaraldehyde in 0.1 M sodium cacodylate, resuspended in 1:1 horse serum:saline for 2 hours (4°C) , then stained with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer.
  • Each slide had several cell clumps taken from different areas in the pellet; cells were scored in several areas in order to avoid possible biases after centrifugation due to the differential density of cells. At least five hundred cells were counted per specimen.
  • Dulbecco's modified Eagle medium and Earle's basic salt solution were from Flow Laboratories (North Ryde, Sydney, Australia) .
  • 8-MOP, dAdo and mitomycin C were from Sigma (St Louis, MO) and cyclophosphamide was from Farmitalia Carlo Erba (Hawthorn, Victoria, Australia) .
  • Deoxycoformycin was a gift from Parke-Davis (Morris Plains, NJ) and mafosfamide (Asta Z 7654) was from Asta Werke (Bielefeld, Germany) .
  • Aqueous lqiuid scintillant and 3 H- NAD(3Ci/mmol) were from Amersham (Bucks, U.K.). All other chemicals were from BDH (Kilsyth, Victoria, Australia) .
  • Examples 1 and 2 are comparative examples designed to investigate the mechanism whereby ECPC exerts its effect.
  • the mean maximum decrease in skin score was 41% (range
  • DNA damage was assessed. Although other forms of DNA damage may have bee induced by the UVA/8-MOP, DNA single strand breaks were assayed by the fluorometric technique. DNA damage was assessed in samples of buffy coat lymphocytes after photoirradiation, just before reinfusion into the patient. The viability of photoirradiated cells at the time of measuring DNA SSBs was the same as that of non- photoirradiated control cells (85%) . The control % double stranded DNA remaining (% D) value for non-photoirradiated lymphocytes was corrected to 100% and the treated lymphocyte sample expressed as a percentage of this.
  • the absolute control % D value for the patients' cells (and for normal PBL) was 79 (range 60 to 97) . All samples assessed had a marked increase in DNA SSBs (Table 1) , but the extent of damage varied considerably between different patients, ranging from 9 to 60%. DNA damage was assessed twice in all patients except number 3. There was less than 15% variation between readings from samples at different time points; only the mean is stated. There was no correlation between the number of malignant cells and the percentage of DNA SSBs.
  • DNA SSBs were measured immediately after photoirradiation by the fluorometric unwinding method 20 (see methods) .
  • the mean peak level was 168 ng/ml and occurred at an average of 2 hours after ingestion. Peak serum 8-MOP levels did not correlate with clinical response, in vitro lymphocytotoxicity or with any of the biochemical parameters associated with cell death.
  • Samples were taken from patients' buffy coat collections before (control) and after photoirradiation, placed in RPMI 1640 plus 10% fetal calf serum, and trypan blue viability measured for the next 96 hours. The results shown represent the mean of nine patient samples from four patients. The mean control cell viability after exposure to 8-MOP and processing was 85%. Many cytotoxic agents that are thought to mediate t h eir cytotoxic effect via poly (ADP-ribosyl)ation have t h eir cytotoxicity reduced (or completely eliminated) by t h e a dd ition of specific poly (ADP-ribose) synthetase inhibitors.
  • the DNA damage for a range of UVA light doses ( 1-5 J/cm 2 ) was studied 2 hours after irradiation (figure 3) .
  • the DNA damaging effect of UVA light on PBL was potentiated by the addition of 8-MOP.
  • the extent of this potentiating effect was dependent on the dose of UVA light and the timing of the assay.
  • Edelson et al ( 1 98 7) reported that the dose of UVA light delivered to their patients' buffy coat lymphocytes was 1-2 J/cm 2 ; we have shown that in vitro this was the dose where 8-MOP maximally potentiated the DNA damage caused by UVA light.
  • UVA/8-MOP-associated DNA damage is associated with adenine nucleotide depletion; our results also suggest that stimulation of poly (ADP-ribosyl)ation and consequent nucleotide depletion are involved in the lymphocytotoxic effect of photoactivated psoralens.
  • Photoirradiation in the presence of 8-MOP caused significantly elevated levels of poly (ADP-ribose) synthetase activity.
  • elevated enzyme activity levels were only found at high doses (10 J/cm 2 ) of UVA light. This may be due to the insensitivity of the assay or may reflect the transient nature of the rise in enzyme activity.
  • Deoxycoformycin and deoxyadenosine lymphocyto ⁇ toxicity has been previously shown to be associated with DNA damage and adenine nucleotide depletion, this is thought to be secondary to stimulation of poly
  • Example 4 Sensitivity of NOD/WEHI mouse splenocytes to UVA light and 8-methoxypsoralen.
  • Freshly harvested mouse splenocytes were exposed to UVA light with or without 100 ng/ml 8-MOP and the -viability measured by trypan blue exclusion. 1 J/cm 2 UVA in the presence of 100 ng/ml 8-MOP caused 100% cell death in 48 hours ( igure 1A) .
  • mice splenocytes The doses of cytotoxic drugs that reproduced the rate of decline in viability caused by exposing mouse splenocytes to 1 J/cm 2 UVA in the presence of 100 ng/ml 8-MOP were identified. Mouse splenocytes were found to be highly sensitive to low doses of cytotoxic agents.
  • Control mouse splenocytes contained 12.4% apoptotic cells: the process of cell harvesting and incubation caused a degree of cell damage.
  • Apoptotic cells were identified by the crescentic argination of their nuclear material to the periphery of the nucleus. The size of apoptotic cells appeared reduced, but the plasma membrane remained intact.
  • mice were given 350 mg/kg intraperitoneal CP. Retroorbital venous blood was sampled between 14 and 18 days later, and the plasma glucose measured. Recipient mice were divided into 5 groups, of which two were control groups. One of the control groups was given no further treatment, in order to determine the control incidence of CP-induced diabetes; the other was given untreated mouse splenocytes. Untreated cells were
  • mice received mouse splenocytes treated with 1 J/cm 2 UVA, 1 J/cm 2 UVA plus 100 ng/ml 8-MOP, or a 4 hour exposure to 1 ⁇ M dCF and 10 ⁇ M dAdo respectively.
  • Mouse splenocytes were injected immediately after cytotoxic exposure.
  • mice There were 139 evaluable mice. Groups were well matched except group E (dCF/dAdo) mice, who received fewer cells than the other 4 groups. There were only small differences between the groups with respect to the ages and initial plasma glucoses of recipient mice. These results are summarized in Table 3.
  • mice received 350 mg/kg CP intraperitoneally on day 1.
  • Splenocytes were injected intravenously at 8, 24 and 48 hours.
  • mice A small number of mice (11/139, 8%) died before the 14-18 day plasma glucose could be measured (Table 3) . Mice were examined daily, and overtly unwell mice had their glucose measured. Our previous experience has shown that CP 350 mg/kg is associated with a low but definite mortality rate. These mice did not reach the defined endpoint, and have been excluded from further analysis. No treatment group had significantly more deaths.
  • mice who developed diabetes in the control (no further treatment) group is consistent with previous reports (Charlton et al, 1989) .
  • the incidence (48%) in the group that received untreated cells was not significantly less (p>0.05).
  • the infusion of UVA/8-MOP-treated splenocytes reduced the incidence to 10% at 14-18 days (p ⁇ 0.01). Further glucose measurements at 21 and 28 days showed that diabetes was prevented, and not just delayed.
  • the UVA-only-treated splenocyte group had more diabetes (4/15, 27%), but this was not statistically significantly different to the incidence in the UVA/8-MOP group (2/20, 10%, p>0.10).
  • mice There were 23 mice in the dCF/dAdo-treated splenocyte group; all were evaluable. This treatment reduced the incidence of diabetes to 14% (p ⁇ 0.01), and appeared to have the same efficacy as UVA/8-MOP (p>0.50). It is noteworthy that this group actually received fewer splenocytes than the other treatment groups.
  • Fluorometric method for rapid detection of DNA strand breaks in human white blood cells produced by low doses of radiation.
  • Makino S. K.Kunimoto, T.Muraoka, Y.Mizushima, K.Katagiri, and Y.Tochino. Breeding of a non-obese diabetic strain of mice. Exp. Anim. (Tokyo) 1980, 29:1-13.

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Abstract

L'invention concerne une préparation pharmaceutique qui modifie de façon spécifique la réponse immunitaire d'un mammifère, comprenant des leucocytes mononucléaires dudit mammifère qui ont été traités par incubation extracorporelle au moyen d'un agent provoquant des lésions irréversibles dans les cellules ou les tuant. L'invention concerne également une composition pharmaceutique et un procédé de traitement d'un état d'origine immunologique ou d'un cancer, ainsi qu'un procédé de préparation de ladite préparation pharmaceutique de l'invention.
PCT/AU1991/000563 1990-12-06 1991-12-05 Agents, compositions et procedes immunotherapeutiques WO1992010198A1 (fr)

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997017079A1 (fr) * 1995-11-08 1997-05-15 Emory University Procede de transplantation de cellules souches hematopoietiques allogeniques sans echec de greffe, et n'entrainant pas de maladie opposant le greffon a l'hote
WO1998007436A1 (fr) * 1996-08-22 1998-02-26 Vasogen Inc. Traitement des maladies auto-immunes
US5773607A (en) * 1991-11-14 1998-06-30 Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai Processes for preparing 2'-deoxy-2'-fluorocoformycin and stereoisomers thereof
US6213127B1 (en) 1996-07-29 2001-04-10 Emory University Methods for treating cancer using allogeneic lymphocytes without graft vs host disease activity
WO2001026605A3 (fr) * 1999-10-11 2002-06-27 Fujisawa Pharmaceutical Co Composition immunosuppressive
US6669965B2 (en) 1992-02-07 2003-12-30 Vasogen Ireland Limited Method of treating atherosclerosis
AT502055B1 (de) * 2005-06-21 2007-11-15 Univ Wien Med Anti tumor medikament
CN117137932A (zh) * 2023-10-18 2023-12-01 中国中医科学院中药研究所 一种用于肿瘤的中药复方制剂及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2012584A (en) * 1978-01-19 1979-08-01 Jaeger K H Process for the manufacture from cells of an immunological active substance complex which acts against malignant growth
GB2048069A (en) * 1979-05-10 1980-12-10 Limburg H Medicinal Preparations for Treating Carcinomas
US4428744A (en) * 1979-12-11 1984-01-31 Frederic A. Bourke, Jr. Method and system for externally treating the blood
US4844893A (en) * 1986-10-07 1989-07-04 Scripps Clinic And Research Foundation EX vivo effector cell activation for target cell killing

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2012584A (en) * 1978-01-19 1979-08-01 Jaeger K H Process for the manufacture from cells of an immunological active substance complex which acts against malignant growth
GB2048069A (en) * 1979-05-10 1980-12-10 Limburg H Medicinal Preparations for Treating Carcinomas
US4428744A (en) * 1979-12-11 1984-01-31 Frederic A. Bourke, Jr. Method and system for externally treating the blood
US4844893A (en) * 1986-10-07 1989-07-04 Scripps Clinic And Research Foundation EX vivo effector cell activation for target cell killing

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5886167A (en) * 1991-11-01 1999-03-23 Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai 2'-deoxy-2'-epi-2'-fluorocoformycin
US5773607A (en) * 1991-11-14 1998-06-30 Zaidan Hojin Biseibutsu Kagaku Kenkyu Kai Processes for preparing 2'-deoxy-2'-fluorocoformycin and stereoisomers thereof
US6669965B2 (en) 1992-02-07 2003-12-30 Vasogen Ireland Limited Method of treating atherosclerosis
US6569467B1 (en) 1992-02-07 2003-05-27 Vasogen Ireland Limited Treatment of autoimmune diseases
US5800539A (en) * 1995-11-08 1998-09-01 Emory University Method of allogeneic hematopoietic stem cell transplantation without graft failure or graft vs. host disease
WO1997017079A1 (fr) * 1995-11-08 1997-05-15 Emory University Procede de transplantation de cellules souches hematopoietiques allogeniques sans echec de greffe, et n'entrainant pas de maladie opposant le greffon a l'hote
US6213127B1 (en) 1996-07-29 2001-04-10 Emory University Methods for treating cancer using allogeneic lymphocytes without graft vs host disease activity
WO1998007436A1 (fr) * 1996-08-22 1998-02-26 Vasogen Inc. Traitement des maladies auto-immunes
WO2001026605A3 (fr) * 1999-10-11 2002-06-27 Fujisawa Pharmaceutical Co Composition immunosuppressive
AT502055B1 (de) * 2005-06-21 2007-11-15 Univ Wien Med Anti tumor medikament
US7981878B2 (en) 2005-06-21 2011-07-19 Medizinische Universitat Wien Tumor treatment with gliotoxin derivatives
CN117137932A (zh) * 2023-10-18 2023-12-01 中国中医科学院中药研究所 一种用于肿瘤的中药复方制剂及其应用
CN117137932B (zh) * 2023-10-18 2024-04-19 中国中医科学院中药研究所 一种用于肿瘤的中药复方制剂及其应用

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