WO1997034639A1 - Animal model for transplantation - Google Patents
Animal model for transplantation Download PDFInfo
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- WO1997034639A1 WO1997034639A1 PCT/AU1997/000185 AU9700185W WO9734639A1 WO 1997034639 A1 WO1997034639 A1 WO 1997034639A1 AU 9700185 W AU9700185 W AU 9700185W WO 9734639 A1 WO9734639 A1 WO 9734639A1
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- tumour
- ketoconazole
- csa
- cancer
- animal model
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
- A61K51/1048—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants the tumor cell determinant being a carcino embryonic antigen
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- 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
- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
- A61K38/13—Cyclosporins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Definitions
- This invention relates to a large animal model of human cancer, in particular in ruminant animals such as sheep which are immunosuppressed by cyclosporin A and ketoconazole and which carry transplanted human or murine tumours, or both.
- the invention also relates to the use of such an animal model in the study of cancer, particularly for evaluating candidates for radio-, chemo- or radiopharmaceutical therapy or radio-immunotherapy.
- the animal model is also useful for radio-imaging of neoplasms or tumours, and for the study of metastasis.
- Radiolabelled monoclonal antibodies against tumour-associated antigens offer a unique potential for targeting radiotherapy to disseminated tumour cells which may ultimately lead to effective treatment of metastatic cancer.
- Radioi munotherapy has been shown to be effective in haematological malignancy, but problems of tumour localisation and penetration have so far prevented successful treatment of solid tumour metastasis.
- it is essential to use animal models of cancer.
- the biodistribution of radiolabelled monoclonal antibodies can only be determined in the intact animal, where the influences of serum protein binding, vascular permeability, interstitial pressure and enzymatic breakdown all affect therapeutic radiation of the target tumour and determine the background irradiation of normal tissues. This essential dosimetry cannot be performed in vitro .
- the immune-incompetent nude mouse, and less commonly, the nude rat, are the only models which are widely used for in vivo study of human tumours. The tumours are usually transplanted subcutaneously in these rodents.
- tumour xenografts in nude animals are disproportionate size of the tumour in relation to the total body weight of the animals, which precludes accurate, predictive pharmacokinetic studies of potential chemotherapeutic and radiopharmaceutical treatments for human cancer, and adversely affects the usefulness of such models for imaging studies.
- a large animal model would be more suitable as a model of cancer and for detailed study of targeted cancer therapy.
- Large animal models of human cancer are not readily available, because of the difficulty of establishing tumours in such hosts; the xenografts usually do not grow or are rejected.
- CsA Cyclosporin A
- CsA has been shown to be effective in preventing transplant rejection in both humans and animals, but its use is often limited by its toxic side-effects (Borel, 1989; Reynolds et al, 1992; Russ, 1992), and by the high concentrations required in order to induce immunosuppression.
- the normal vehicle used, Cremaphor EL can also induce severe toxic side effects.
- Ketoconazole is a synthetic imidazole dioxolane used primarily for the treatment of superficial fungal infections, chronic mucocutaneous candidiasis and genital candidiasis (Bodey, 1992; Breckenridge, 1992; Borelli et al, 1979).
- Ketoconazole indirectly enhances the bioavailability of CsA by inhibiting the hepatic cytochrome P-450 mixed function oxidase system which is primarily responsible for CsA inactivation in vivo (Breckenridge, 1992; First et al, 1991; Wadhwa et al , 1987) . Increased bioavailability reduces the dose of CsA required for therapeutic efficacy, which, in turn, decreases the toxicity associated with its use.
- CsA has been shown to inhibit cell division of both normal and malignant cells in vivo and in vitro (Borel, 1989; Di Padova, 1989; Barbera-Guillem et al, 1988; Kreis and Soricelli, 1979) .
- human and murine T cell lymphomas and leukaemias were found to be sensitive to CsA-induced growth inhibition at doses of 0.5-5 ⁇ g/ml, whereas non-lymphoid cell lines and certain murine B and null cell leukaemias were insensitive to doses of up to lO ⁇ g/ml (Borel, 1989) .
- ketoconazole and CsA concomitant oral administration of ketoconazole and CsA to a mammal produces immunosuppression which allows xenografting of cancer cells or tissues and provides a large animal model for the study of cancer. This is particularly unexpected, in view of the anti-tumour effects of ketoconazole and CsA, and the difficulty of inducing and maintaining immunosuppression with non-toxic doses of CsA.
- a main advantage of the animal model according to the present invention is the cost effectiveness of obtaining and maintaining the animals. No aseptic or sterile conditions are necessary and the animals can be maintained on a normal diet.
- the invention provides an animal model of cancer, comprising a mammal which is immunosuppressed by administration of cyclosporin and ketoconazole, and which carries a tumour xenograft.
- a mammal which is immunosuppressed by administration of cyclosporin and ketoconazole, and which carries a tumour xenograft.
- the mammal is selected from the group consisting of sheep, goats, cattle, pigs or the like. More preferably, the mammal is a sheep.
- the mammal has a plurality of xenografted tumours implanted subcutaneously.
- tumour cells or tumours are transplanted into the host animal using Matrigel as the vehicle .
- Matrigel is a reconstituted basement membrane preparation which facilitates tumour uptake at sites of incubation.
- the invention provides a method of evaluating the efficacy of a putative therapeutic agent against cancer, comprising the step of administering said agent to a ruminant mammal model of the invention.
- the agents which may be tested in this model include but are not limited to i munochemotherapeutic agents, cytokines, chemotherapeutic agents and radiopharmaceuticals, and may also comprise internal or external radioactive agents as well as radiolabelled peptides . Gene therapy may also be evaluated using this mod l.
- the invention provides a method of evaluating the efficacy of a method of radioimaging of tumours or neoplasms, comprising the step of administering a radiolabelled, tumour-specific antibody to the ruminant mammal model of the invention.
- the radiolabelled antibody may be a monoclonal or polyclonal antibody comprising a radiolabel, preferably selected from the group consisting of Technetium-99m, Indium-111, Iodine-13 , Rhenium-186, Rhenium-188, Samarium-153, Lutetium-177, Copper-64, Scandium-47, Yttrium-90.
- a radiolabel preferably selected from the group consisting of Technetium-99m, Indium-111, Iodine-13 , Rhenium-186, Rhenium-188, Samarium-153, Lutetium-177, Copper-64, Scandium-47, Yttrium-90.
- Monoclonal antibodies labelled with therapeutic radionuclides such as Iodine-131, Rhenium-188, Holmium-166, Samarium-153 and Scandium-47, which do not compromise the immunoreactivity of antibodies and are not broken down in vivo, are especially preferred.
- radioactive isotopes are known, and may be suitable for specific applications.
- antibody encompasses fragments and analogues such as Fab, Fv and ScFv, provided that the binding activity is retained.
- Peptide fragments of antibodies are specifically contemplated by the invention.
- the fragments or analogues may be prepared using recombinant DNA methods or by synthetic methods such as solid-phase synthesis.
- the radioimaging may be conducted using Single Photon Emission Computer Tomography (SPECT), Position Emmission Tomography (PET), Computer Tomography (CT) or Magnetic Resonance Imaging (MRI) . Correlative imaging, which permits greater anatomical definition of location of metastases located by radioimmunoimaging, is also contemplated.
- SPECT Single Photon Emission Computer Tomography
- PET Position Emmission Tomography
- CT Computer Tomography
- MRI Magnetic Resonance Imaging
- the invention provides a method of screening of therapeutic radiolabelled peptides directed against tumours, preferably tumour-associated receptors, antigens or ligands or the like.
- Therapeutic radiolabelled peptides such as 90 Yttrium-labelled octreotide or 1:ll Indium-labelled octreotide are contemplated.
- Radiolabelled antibodies to tumour- associated ligands or antigens and therapeutic agents linked to such entities are also within the scope of the invention.
- the invention provides a method of producing a ruminant mammal bearing a tumour xenograft, comprising the step of concomitant administration of CsA and ketoconazole to the mammal.
- ketoconazole is administered in a drench formulation which by-passes the rumen and is absorbed in the abomasum. This provides highly reproducible bioavalability and predictable competitive inhibition of CsA metabolism in the liver.
- the dose of CsA is in the range 2.5 to 3.5 mg per kg administered twice a day and the dose of ketoconazole is 5 to 10 mg per kg administered twice a day.
- lOmg/kg ketoconazole is administered twice a day to maintain trough serum levels of CsA within the range 1000-1500 ng/ml.
- the model system of the invention enables the testing of therapeutic methods directed against primary malignancy or metastatic cancer in a manner which has hitherto not been possible.
- the model is suitable for testing of radiotherapy, immunotherapy (including the use of cytokines), chemotherapy, and gene therapy.
- the model is also useful for testing of targeting or localisation agents, methods of imaging, and methods for monitoring the progress of therapy.
- the invention provides a method of stimulating spontaneous metastasis of tumour cells to a target site such as the liver or lymph nodes, comprising the step of transplanting said cells to a mammal which is immunosuppressed by administration of cyclosporin and ketoconazole and allowing the cells to metatasize in said mammal.
- the invention provides a composition comprising a CsA or cyclosporin-like compounds and ketoconazole or related compounds, together with a pharmaceutically acceptable carrier.
- the invention provides a kit comprising a CsA or cyclosporin-like compound and ketoconazole or a related compound, wherein the ketoconazole or related compound increases the bioavailability of the CsA or cyclosporin-like compound and enhances the establishment of tumour xenografts.
- CsA compounds which the person skilled in the art will recognise as being suitable to improve bioavailability of CsA, such as compounds related to ketoconazole (including, but not limited to, fluconazole) , and calcium channel blockers.
- ketoconazole which bears no chemical structural relationship to CsA, competes with hepatic enzymes which break down CsA.
- Figure 3 shows the pharmacokinetic concentration- time profiles for CsA (3 mg/kg intravenously (iv) in sheep #14 in Example 1, after the first dose m ; day 0) and at steady-state ( ⁇ ; day 18) in the presence of ketoconazole (10 mg/kg orally (po) .
- the solid lines show the log-linear regression fitted line for the last 5 data points.
- Figure 4 is the area under the blood concentration - time curve, AUC, over 24 hours following CsA administration.
- the dose of ketoconazole was 10 mg/kg.
- the full dose of CsA was 5 mg/kg and the half dose was 2.5 mg/kg.
- Asterisks indicate significantly different AUC values relative to a full dose of CsA alone administered iv (p ⁇ 0.05, Dunnett's test).
- Figures 5(a), (b) and (c) show the mitogen- stimulated lymphocyte proliferation responses after CsA (5 mg/kg iv) administration. Individual and mean responses of 5 individuals to ConA (a), PHA (b) and PWM (c) administration are shown as a percentage of the mean change in counts per minute ( ⁇ cpm) of 5 sheep. Interassay coefficients of variation (based on background cpm data from individual sheep assayed on 6 different days) were between 75.7-126.6%.
- Figures 5(d), (e) and (f) show the itogen- stimulated lymphocyte proliferation responses after CsA (5 mg/kg iv) administration with ketoconazole (10 mg/kg). Individual and mean responses of 6 individuals to ConA (d) , PHA (e) and PWM (f) are shown as a percentage of the mean day 0 delta cpm of 6 sheep. Interassay coefficients of variation (based on background cpm data from individual sheep assayed on 6 different days) were between 39-161%.
- Figure 6 shows the ED 50 of CsA and ketoconazole in three tumour cell lines - B16M, HT-29 and SKMEL, represented as tumour cell growth (OD 590nm) in the presence of increasing concentrations of CsA (0-60 ⁇ g/ml) and ketoconazole (0-60 ⁇ g/ml) (log scale) .
- a representative CsA and ketoconazole growth inhibition curve is shown for each cell line.
- Figure 7 shows the isobologram analysis of the effects of combination CsA/ketoconazole on tumour cell growth in vitro .
- the ED 50s of CsA in the presence of 3 sub-optimal concentrations of ketoconazole (no circle) and ketoconazole in the presence of 3 sub-optimal concentrations of CsA (circled) are presented as the FIC for each reagent.
- a representative isobologram is given for each cell line.
- the dotted line depicts the expected shape of the curve if the interaction is additive and is given for comparison.
- Figures 8-15 show the effects of transplantation of various cell lines into sheep immunosuppressed with cyclosporin and ketoconazole.
- Cells were inoculated as suspensions or spheroids with or without Matrigel, and tumour xenografts were examined macroscopically and fixed in formalin:
- Figure 8 is a photograph of SK-melanoma tumour deposit in skin.
- Panel A shows diffuse sheets of malignant cells of "epithelioid” type with abundant amphophilic cytoplasm, vesicular nuclei and variably prominent nucleoli. Moderate nuclear pleomorphism is seen and mitotic figures are easily found. There is no evidence of necrosis and a minor chronic inflammatory cellular exudate is present at the periphery with no significant numbers of tumour infiltrating lymphocytes (H&E stain) .
- Panel B shows a positive granular cytoplasmic staining together with some nuclear staining with the immunoperoxidase preparation S100.
- Panel C is a Mason Fontana preparation which shows no definite pigment deposition.
- Figure 9 is a photograph of SK-melanoma deposit in lymph node.
- Panel A illustrates sections of the lymph node and show extensive replacement of the parenchyma by diffuse sheets of malignant melanoma cells.
- Panel B shows variable mild to moderate predominantly cytoplasmic staining of tumour cells using the S100 preparation.
- Figure 10 (i) is a photograph of skin deposit of adenocarcinoma LS174T.
- Panel A shows well formed ancinar structures lined by tall columnar cells with moderate nuclear pleomorphism in the tumour. Mitotic figures are easily identified and there is mild focal necrosis with no significant inflammatory cellular exudate. Tumour infiltrating lymphocytes are infrequent (H&E) .
- Panel B represents abundant intraluminal PAS positive diastase resistant neutral mucin as seen with the PAS-D preparation.
- Panel C shows immunostaining with Carcinoembryonic antigen. Marked positive, predominantly luminal staining with a lesser degree of granular cytoplasmic staining can be seen.
- Figure 10(ii) is a photograph of HT 29 adenocarcinoma deposit in skin.
- Panel D shows that the tumour is poorly differentiated with little tendency towards acinar formation and is formed by predominantly sheet like arrangements of columnar and cuboidal cells with vesicular nuclei and prominent nucleoli. Mitotic figures are easily identified and there is focal necrosis and some peritumoura1 fibrosis. No significant inflammatory cellular response or tumour infiltrating lymphocytes are seen.
- Panel E shows that a small amount of intraluminal
- PAS positive post diastase neutral mucin is seen in occasional acinar structures present. There are also intracytoplasmic mucin deposits, although this is not marked.
- Panel F represents the immunoperoxidase preparation for CEA antigen which shows mild variable granular cytoplasmic staining with occasional "dot” like intracytoplasmic deposits.
- the LS174T adenocarcinoma is well differentiated with prominent acinar formation, and exhibits marked mucinogenesis and marked positivity with CEA.
- HT29 adenocarcinoma is poorly differentiated with only a mild degree of mucinogenesis and mild variable staining with CEA.
- Figure 12 is a photograph of LS174T adenocarcinoma deposit in the liver.
- Figure 13 is a photograph of LS174T adenocarcinoma deposit in peritoneal wall.
- Panel A shows a tumour deposit with a well differentiated adenocarcinoma having prominent acinar formations lined by tall columnar cells with moderate nuclear pleomorphism and easily identifiable atypical mitoses . No significant necrosis is present and there is a mild peritumoural chronic inflammatory cellular exudate but no significant numbers of tumour infiltrating lymphocytes are seen (H&E stain) .
- Panel B shows abundant intraluminal PAS positive diastase resistant neutral mucin, seen with the PASD preparation.
- Panel C shows immunostaining with Careinoembryonic antigen, leading to marked positive, predominantly luminal staining as well as granular cytoplasmic staining.
- Figure 14 is a photograph of JAM ovarian carcinoma deposit in ovary.
- Tumour giant cells are prominent and there is moderate to marked nuclear pleomorphism with prominent atypical mitoses.
- No definite papillary structures are present and there is a mild peritumoural inflammatory host response and no significant tumour necrosis present.
- Figure 15 is a photograph of JAM ovarian carcinoma in peritoneal wall. There are diffuse sheets of poorly differentiated tumour similar to that described above, associated with a mild to moderate peritumoural chronic inflammatory cellular exudate and some fibrosis. No significant necrosis is seen and only small numbers of tumour infiltrating lymphocytes are present.
- Sheep Mature Suffolk cross and Merino wethers were penned individually but at 4 to a room and were allowed to acclimatise for 10 days in an animal holding facility.
- the ad libitum diet comprised sheep cubes (Glen Forrest Stock Foods, WA) supplemented by rough-cut chaff, hay and water. These animals were used for studying the pharmacodynamics and immunological effects of CsA and ketoconazole, and for implantation of cell lines.
- Cyclosporin A was kindly donated by Sandoz Pharma Pty Ltd (Basel, Switzerland) . The powder was dissolved in ethanol 1.75% v/v and polyethoxylated castor oil cremophor (BASF Chemicals, Melbourne, Australia) 3.25% v/v, then diluted to volume with sterile saline just prior to administration. Ketoconazole was supplied as tablets (Nizoral,
- ketoconazole tablets Prior to administration by the intraperitoneal route, ketoconazole tablets were crushed and dissolved in a minimal volume of methanol before dilution to 50 mg/ml with sterile saline. Before oral administration, crushed ketoconazole tablets were suspended in a drench vehicle which contained silicon dioxide (Ultrasil VN3 colloidal silicon dioxide, Degussa Australia Pty Ltd., Melbourne, Australia) gum xantham
- Fluoxetine hydrochloride was obtained from Eli Lilly Australia Pty, Ltd (West Ryde, NSW) , CsA from Sandoz Australia (North Ryde, NSW) , and ketoconazole from Janssen Cilag. All other chemicals were of analytical reagent grade.
- Silastic catheters were placed to a depth of approximately 20 cm in both external jugular veins of Merino-Cross Dorset ewes under local anaesthesia, two days before drug administration commenced. Catheters were taped/sewn to the lateral surface of the neck, sealed with a three way tap, and protected with an elastic net bandage around the neck. One catheter was used for administration of CsA and the other for venous blood sampling. Catheters were flushed with sterile, heparinised saline twice daily to maintain patency. Experimental Design and Blood Sample Collection Schedule
- CsA pharmacokinetics were studied after the first dose (3 mg/kg iv) and again at steady-state when the animals had been receiving CsA (3 mg/kg iv) and ketoconazole (10 mg/kg po) for 18 days.
- venous blood samples (5ml, anticoagulated with EDTA) were collected at 0.17, 0.33, 0.5, 0.75, 1,2,3,4,6,8,12,15,22,27 and 32 h.
- Blood samples for CsA analysis were obtained twice weekly, just before the morning doses, over the next 18 days.
- CsA in whole blood was measured by enzyme multiplied immunoassay (EMIT ® , Syva Company, Evergreen, CA) as previously described (Dusci et al,1992). Blood samples were then frozen at -20°C and at the end of the experiment were analysed by high performance liquid chromatography (hplc) as previously described (Dusci et al, supra) . Only hplc-derived data were used for subsequent pharmacokinetic analyses.
- the hplc system consisted of a Merck RP Select B Cl ⁇ column (25cm x 4.6 mm id), a mobile phase of 55% acetonitrile in 0.01% v/v H 3 P0 4 and 0.01% w/v NaCl.
- the solvent was pumped at a flow rate of 1.5 ml/min and eluting compounds were detected by their UV absorbence at 210 nm.
- the assay was linear over the range 0.5-33 mg/l with a detection limit of 0.2 mg/l.
- each sheep was fitted with an intrajugular cannula to facilitate the taking of blood samples.
- Cannulae remained in situ for 24 to 48 hours only and were maintained with a solution containing heparin, penicillin and streptomycin and were flushed with sterile saline prior to taking blood samples for pharmacokinetic studies.
- CsA was given by injection into the opposite external jugular vein and subsequent blood samples for lymphocyte culture were obtained by venipuncture from the opposite external jugular vein.
- CsA Assay Levels of CsA in peripheral blood were measured using the CsA Monoclonal Whole Blood fluorescence polarization immunoassay system (Abbott Diagnostics, Abbott Park, IL, USA) and read on a Tdx autoanalyser.
- Lymphocyte Preparation Lymphocytes were prepared from heparinized whole blood collected between 8.30 and 10 am on each test day. The leucocyte-rich buffy coat layer was collected after separation, diluted with phosphate buffered saline (PBS), and applied to Ficoll-hypaque (Pharmacia, North Ryde, NSW) density gradients to obtain lymphocyte preparations. After two low-speed washes with PBS, the lymphocyte preparations were resuspended in RPMI 1640 medium (Flow Laboratories, Australia Biosearch, Karrinyup, Australia) supplemented with 10% foetal calf serum (FCS, Cytosystems, Sydney, Australia) and penicillin (lOO ⁇ /ml) before counting and confirmation of viability. Mitogen Stimulation Assays
- lymphocytes The capacity of lymphocytes to proliferate in response to in vitro stimulation by Phytohaemagglutinin A (PHA; Sigma), Concanavalin A (ConA;Sigma) and Pokeweed Mitogen (PWM; Sigma) was assessed. Lymphocytes were seeded into 96-well flat bottom tissue culture plates (Disposable Products, Sydney, South Australia) at a concentration of IO 5 cells/well. Mitogens (PHA, ConA, and PWM) were added to triplicate wells to obtain concentrations of lO ⁇ g/ml of each mitogen. Plates were incubated at 37°C in 5%C0 2 for 48 hours before labelling with IMBq/well of 3 H-thymidine (Amersham, Melbourne, Australia) .
- lymphocytes The ability of isolated lymphocytes to respond to allogeneic tissue antigen was assessed by mixed lymphocyte culture (MLC) .
- MLC mixed lymphocyte culture
- Lymphocytes obtained as described above from test sheep were adjusted to 2.5xl0 5 /well in 96-well flat bottom tissue culture plates.
- An equal number of lymphocytes isolated from an unrelated untreated donor sheep were subject to 20 Gray (Gy) of X-irradiation and added to triplicate or quadruplicate wells as stimulator cells.
- Irradiated lymphocytes from each individual test sheep were also prepared and set up against homologous responder cells to obtain individual background data. Controls to confirm adequacy of inactivation by X- irradiation were also included in each assay.
- Stimulation indices were determined by dividing the mean cpm of the wells containing test lymphocytes co-cultured with X-irradiated stimulator cells by the mean cpm of the background wells utilizing X-irradiated and non-irradiated homologous cells.
- Lymphocytes prepared as described above were adjusted to 1 to 3xl0 6 /well in 96- well round bottom culture plates (Disposable Products, Sydney, South Australia) and incubated overnight at 4°C with monoclonal antibodies to lymphocyte marker antigens at a final dilution of 1/100.
- a panel of monoclonal antibodies defining ovine lymphocyte surface markers was obtained from the Centre for Animal Biotechnology, The University of Melbourne, Parkville, Australia.
- the monoclonal antibodies used were SBU-LCA (detecting CD45, leucocyte common antigen), SBU-Tl (CD5, all T cells), SBU-T4 pool (CD4, T helper cell subset), SBU-T8 (CD8, T suppressor/cytotoxic subset)and SBU-Tl9 (defining the CD4-CD8-gamma delta T cell subset in sheep, currently of unknown function) .
- B lymphocytes were identified by surface immunoglobulin expression.
- Ketoconazole significantly altered the AUC by reducing CsA clearance, leading to a two-fold increase in AUC after an oral dose of ketoconazole lOmg/kg (p ⁇ 0.05, Dunnett's test; 24,4 d.f.) the AUC following a dose of 2.5mg/kg of CsA with lOmg/kg ketoconazole po was slightly greater than that following 5mg/kg CsA alone, as shown in Figure 4.
- Administration of ketoconazole either ip or po along with a halved dose (2.5mg/kg) of CsA maintained the AUC at a level similar to that achieved with the full dose of 5mg/kg dose of CsA alone.
- Mitogen proliferation data from sheep receiving CsA 5mg/kg alone iv are shown in Figure 5 (a), (b) and (c) .
- the data show that there was a transient suppression of lymphocyte response at 24 hour.
- ketoconazole 5mg/kg and CsA iv 5mg/kg markedly potentiated the depression of lymphoproliferative responses to all mitogens tested ( Figures 5 (d) , (e) and (f)). Normal reactivity to mitogens was regained at 48 hours in sheep, treated with CsA alone. In CsA+ketoconazole treated sheep responses substantially recovered by 48 hours.
- MLC responses were not significantly different from Day 0 at 48 hours, 72 hours or 7 days, (p > 0.05,
- Example 3 Effects of CsA and Ketoconazole on Tumour Cell Growth in vitro Prior to initiating tumour xenograft transplantation in a sheep model, the susceptibility of the tumour xenografts to the growth inhibitory effects of ketoconazole and CsA was determined.
- the tumour cell lines used in this study included a human colon carcinoma, HT-29, a human malignant melanoma, SKMEL, and a murine malignant melanoma, B16M. These tumour types were chosen because they represent common malignancies with high metastatic potential and high morbidity and mortality.
- relatively specific monoclonal antibodies (MoAbs) are available for all of these cell lines (DiMaggio et al, 1990) .
- the experiment was designed to evaluate the growth inhibitory effects of CsA and ketoconazole, used alone or in combination, on the HT-29, SKMEL and B16M tumour cell lines in vitro .
- Tumour cell lines HT-29, SKMEL and B16M were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in RPMI-1640 medium, (Flow Laboratories, Australian Biosearch, Karrinyup, Australia), containing 10% foetal calf serum (FCS, Cytosystems, Sydney, Australia), 2mM L-glutamine, lOOmM sodium pyruvate, lOOmM non-essential amino acids, (all from Aust. Biosearch), and benzylpenicillin (100,000 units/1, Commonwealth Serum Laboratories (CSL), Parkville, Australia) . All tumour cell lines were incubated at
- MTT Assay 3- (4, 5-dimethylthiazo-2-YL) -2, 5-diphenyl- tetrazolium bromide (MTT) was obtained from Sigma Corporation (St. Louis, Mo, USA) .
- the assay was performed according to the method of Mosmann (1983). In general, following incubation of the tumour cells with the CsA and/or ketoconazole (see below), the plates were centrifuged (lOOOg, 5 min) and 10O ⁇ l of the supernatant removed. 20 ⁇ l of MTT (stock at 5mg/ml in PBS) was added to each well and the plates incubated at 37°C for 4 hours.
- ODs optical densities
- Tumour cells were seeded at 5 to 10 x 10 3 /well
- EtOH/Cremophor/saline vehicle vehicle cone, (%/ml) day 3-ED 50 (0.035%) (0.053%) (0.08%) UJ
- Ketoconazole B16M HT-29 SKMEL days in culture ED 50 ( ⁇ g/ml) day 1 22.8 ⁇ 2.8 23.8 ⁇ 2.5 31.0 ⁇ 2.6 day 2 21.2 ⁇ 4.3 18.2 ⁇ 1.8 30.0 ⁇ 1.1 day 3 18.2 ⁇ 3.9 12.7 ⁇ 2.0 28.3 ⁇ 1.7
- EtOH/Methanol vehicle vehicle cone, (%/ml) day 3 - ED 50 (0.06%) (0.035%) (0.85%) a
- the effects of CsA and ketoconazole on tumour cell growth in vitro were determined using the MTT assay.
- the data are presented as the ED 50 (+SD) in ug/ml for each tumour cell line following exposure for 1, 2, or 3 days in culture.
- the vehicle concentration present in the day 3 ED 50 sample is indicated in the parentheses as %/ml. Each tumour cell line was tested 3-5 times.
- tumour cell lines tested were found to be moderately to highly resistant to the growth inhibitory effects of CsA and ketoconazole, demonstrating ED so s at or well above the maximum therapeutic plasma concentration for both reagents, ie. 0.10 ⁇ g/ml (Borel, 1989; Reynolds et al, 1992; Eichenberger et al , 1989a; Figure 6, Table 4) .
- the murine melanoma cell line, B16M was found to be the most susceptible of the three tumour cell lines to CsA, demonstrating an ED 50 of 10.2 ⁇ 3.3 ⁇ g/ml compared to
- ketoconazole was found to be most inhibitory to HT-29 tumour cell growth, demonstrating an ED 50 of 12.7 ⁇ 2.0 ⁇ g/ml compared to 18.2 ⁇ 3.9 ⁇ g/ml and 28.3 ⁇ 1.7 ⁇ g/ml for B16M and SKMEL, respectively ( Figure 6, Table 4).
- CsA vehicle Cremaphor EL/EtOH/saline
- ketoconazole vehicle EtOH/methanol
- Cremphor/EtOH/Saline vehicle at concentrations of 0.1%, and this inhibition has been corrected for in all experiments.
- ketoconazole concentrations determined from the ketoconazole ED 50 for each line
- ketoconazole (0-60 ⁇ /ml) in combination with 3 sub-optimal CsA concentrations were determined and plotted as a fractional inhibitory concentration (FIC) of the ED 50 of CsA, or of ketoconazole alone, which has a designated FIC of 1.
- FIC fractional inhibitory concentration
- HT 29 (ATCC) Colon carcinoma
- Cells were maintained in media recommended by the American Type Culture Collection (ATCC) . Specifically, the cell lines were maintained in 75 cm 2 tissue culture flasks (Costar, USA) in RPMI 1640 (Life Technologies, USA) supplemented with 10% fetal calf serum (FCS) and lOO ⁇ /ml penicillin (CSL, Australia) and for NIH:0VCAR-3, 10% extra FCS and human insulin NIH:OVCAR-3 adenocarcinoma of ovary
- tumours were passaged in nude mice and then transplanted into the immunosuppressed sheep as small solid tumour pieces approximately 2 mm x 2mm diameter.
- a jugular venous catheter was inserted subcutaneously under local anaesthetic, exiting the skin at the back of the neck.
- Twice daily intravenous administration of 3 mg/kg cyclosporin was given via the indwelling jugular vein catheter each day for up to 70 days.
- a drench oral administration of ketoconazole was also given on a daily basis according to standard Murdoch university veterinary techniques in sheep. Parenteral administration of CsA in sheep and concomitant oral ketoconazole had minimal side effects in a controlled environment.
- Haematological, biochemical and CsA assays were performed on a weekly basis on blood obtained directly from the jugular vein.
- CsA determined by trough level assay.
- the typical daily dose of CsA was 6 mg/kg given in an indwelling jugular vein catheter in 2 divided doses given in the morning and evening.
- the cyclosporin may be administered by a continuous infusion pump into the jugular vein to acheive greater control of serum Cs levels and minimize expensive drug use.
- Ketoconazole powder was prepared in a drench formulation as follows:-
- the resulting mixture was stirred for 30 minutes after which 200 ml of warm tap water was added. The mixture was again left to stir for at least one hour, after which the volume was made up to a final total of 720ml. After mixing well, the solution was aliquoted and stored at 4°C. The final concentration of ketoconazole in this solution was 50mg/ml and the dose given to the sheep may be 10 mg/kg or 1 ml of the solution per 5kg sheep weight. Prior to giving the ketoconazole to sheep, the solution should be well mixed to distribute any sedimented powder although care should be taken to avoid frothing or aeration of the solution.
- the basic ingredients of the drench were derived from the standard preparation used for oral administration of medication to ruminants at the School of Veterinary
- ketoconazole ensures abomasal delivery and effectively bypasses the rumen following oral administration to the sheep, and thus rendering the ketoconazole readily bioavailable.
- the daily oral dose of ketoconazole was 20 mg/kg in 2 divided doses, given in the morning and evening.
- CsA assays were performed at the Western Australian Centre for Pathology and Medical Research using the EMIT® Cyclosporin Assays (Syva Co, San Jose, CA, USA) on blood taken immediately before the morning dose. These trough CsA levels were maintained in the optimum range for immunosuppression between 750 and 1500 ng/ml. Trough blood level assays performed at the
- ketoconazole plasma concentration was in the range 2.5 - 3.5 mg/L.
- Lymphocytes were tested on a weekly basis for capacity to respond to a series of mitogens. Lymphocytes were obtained from heparinized blood drawn from the anterior brachial vein by centrifugation and separation on a Ficoll/hypaque gradient. After washing, lymphocytes were dispensed into microlitre trays at 5 x 10 6 celIs/ml with an equal volume (10O ⁇ l) of RPMI containing phytohaemagglutinin, concanavalin A or pokeweed mitogen at 5, 10, and 20 ⁇ g/ml, together with 2-mercapto-ethanol, foetal calf serum and penicillin/streptomycin.
- SI cpm mitogen stimulated cells - cpm background cpm nonstimulated cells - cpm background The functional capacity of lymphocytes was determined weekly.
- lymphocytes prepared as above, was determined by immunofluorescence assay using a range of commercially available ovine monoclonal reagents. After labelling, suspensions were assayed by fluorescence-activated cell sorting. These assays provide an assessment of fluctuations in total B and T lymphocyte populations as well as T cell subjects. The results were expressed as percentages of total lymphocyte numbers.
- Injections were given via a 21G needle as 0.1 to 0.3 ml cells in PBS with or without 0.1 ml Matrigel (Collaborative Biomedical Products, USA) .
- Matrigel Collaborative Biomedical Products, USA
- syringes and needles were pre-chilled on ice, and the needle and syringe held in the injection site until the Matrigel had gelled.
- a minimum of two human tumours was inoculated subcutaneously in each sheep under the bare skin of the inguinal region.
- subcapsular and hepatic in raparenchymal inoculation of at least 2 tumour cell types was performed (one acting as the non-specific control for the labelled monoclonal antibody) .
- Solid tumour pieces were harvested from nude mice and cut into 2 mm x 2 mm cubes for transplantation into the sheep. Each transplantation site corresponded to the location of predilection for each tumour type in human metastasis.
- Skin tumours were measured weekly in two dimensions with calipers excised from the skin at varying times and fixed in formalin for subsequent histological examination. At autopsy 3-6 weeks after tumour cell inoculation appropriate organs and draining lymph nodes were removed and examined macroscopically and fixed in formalin for histology.
- Tissues were also fixed in neutral buffered formalin and processed through alcohol and xylene to paraffin.
- Sections were cut at 4 ⁇ on a rotary microtome, dried at 60°C and stained with Harris haematoxylin (H&E stain) and aqueous eosin on a random access staining machine. These were then coverslipped using a resin mounting medium and viewed under light microscopy.
- Harris haematoxylin H&E stain
- aqueous eosin aqueous eosin
- Antigen demonstration was achieved by a peroxidase conjugated streptavidin staining procedure.
- the primary antibody was first applied to the tissue sections which were then further labelled with a biotinglated link antibody followed by a streptavidin peroxidase enzyme conjugate.
- the bound peroxidase enzyme was then visualised with a diaminobenzidine substrate.
- Tumour xenografts of lcc could be imaged with a gamma camera after administration of suitable gamma- emitting radionuclide labelling of tumour specific monoclonal antibodies.
- suitable gamma- emitting radionuclide labelling of tumour specific monoclonal antibodies were used for quality control of the model to ensure the absence of host reaction and rejection of the tumour xenografts.
- animals were observed for pain and distress by monitoring food and water intake and observing changes in general well-being and presence of teeth grinding. No post-procedural pain was anticipated. If changes to these parameters occurred, the animals were euthanased by intravenous lethabarb. Otherwise, animals were euthanased within 70 days of tumour implantation.
- the human tumours successfully transplanted into the immunosuppressed sheep included the human colon carcinoma cell lines HT-29 and LS 174T, which elaborate CEA, and a human amelanotic melanoma, SKMEL and OVCAR-3 and JAM results are represented in Figures 14 and 15. 97/34639 PC17AU97/00185
- Xenografts of SKMEL remained amelanotic, and showed abundant S100 staining typical of human melanomata.
- human colon tumour pieces or inoculation of cells may be orthotopically implanted ⁇ ubmucosally in the distal colon under sigmoidoscopic control without requirement for abdominal surgery. Such sigmoid tumours may then be monitored endoscopically, and serial biopsies taken as required.
- Matrigel a reconstituted basement membrane matrix preparation (Fridman et al, 1991), was found to facilitate tumour graft acceptance at sites of cell inoculation, particularly for OVCAR NIH3 and JAM human ovarian carcinoma cells orthotopically transplanted into sheep ovaries.
- the uptake of 131 I-A5B7 in LS174T human colon cancer xenografts in the immunosuppressed sheep ranged between 0.014 and 0.035% Dl/g, higher activities being observed in hepatic sites as shown in Table 6.
- This tumour uptake is in accord with that achieved in human colonic tumours studies in patients using 131 I-A5B7 anti-CEA monoclonal antibody, where peak uptake of 0.018% Dl/g was observed at 27 hours after administration of radiolabelled intact antbody (Lane et al, 1994) .
- the SKMEL tumours showed typical morphological charcterisitcs of human melanoma and did not accumlate any of the radiolabelled anti-CEA antibody taken up by the colonic xenografts. Only the melanoma was observed to metastasize from subcutaneous sites of inoculation and
- SKMEL cells were subsequently recovered from the regional pre-stifle lymph node, grown in cell culture and reinoculated subcutaneoulsy in sheep and gave rise to tumours morphologically indistinguishable from those of the primary inouclation.
- the failure of colon cancer to metastasize from subcutaneous sites of inoculation has been observed consistently in nude mice (Fidler, 1990; Kubota, 1994) and orthotopic transplantation has been advocated to maintain the malignant phenotype of human tumour xenografts (Radinsky and Fidler, 1992).
- the subcutaneous route of cell inoculation represents orthotopic transplantation and regional lymph node metastases were demonstrated in our sheep.
- the animal model of the invention will enable us to produce bone marrow metastases of breast cancer by intra-cardiac inoculation of tumour cells, providing a model of bone marrow metastases in breast cancer patients.
- the animal model of the invention will also be useful in the developmental and/or evaluation of novel ligands to permit targetting of therapeutic agents to the tumours. This may be achieved, for example, by radiolabelling of antibodies raised against these ligands (eg. "tumour specific" monoclonal antibodies) with therapeutic radionuclides such as Rhenium-188, Holmium-166 and Samerium-153 without compromising immunoreactivity and without in vivo breakdown of the labelled antibody.
- therapeutic radionuclides such as Rhenium-188, Holmium-166 and Samerium-153
- Bladder carcinoma cells BL-17/0/X1; J82; 5637 in Matrigel are injected into the urothelium of 6-10 sheep via a cystoscope. The sheep are monitored by cystoscopy and small biopsies taken to confirm tumour growth and phenotype.
- the sheep with visible tumours are injected intravesically with Samarium-153 labelled Cl-137, 595 or cytokeratin-8 or labelled isotype control antibody, or with labelled EGF or unlabelled EGF as control. Subsequent cystoscopy is performed 5 and 10 days later to monitor tumour growth. Tumour and surrounding urothelium are examined histologically at autopsy.
- Yttrium-90 labelled octreotide (Novartis) is a somatostatin analogue which targets somatostatin receptors on carcinoid, small cell lung cancer or the like.
- Treatment of glioma by administration of Yttrium- 90 octreotide may also be studied in sheep which have been subjected to orthotransplantation by injection of glioma cells directly into the frontal lobe of the brain.
- the animal model described herein may also be used for direct transplantation of tumours, including human tumours freshly taken from surgical specimens. This has the advantage of preserving the malignant phenotype of the tumour, and the propensity to metastasize when transplanted orthotopically.
- the expression of specific tumour associated antigens which can be lost during passage in cell culture, can also be preserved.
- the unique attribute of the large animal model of human tumours is the ability to measure uptake of radioactivity in tumours and critical normal organs, by quantitative SPECT imaging in vivo and to verify the time course of accumulation of tumour radioactivity by serial biopsies for gamma counting.
- MoAbs are radiolabelled with isotopes such as Iodine-131, Holmium-166, Samarium-153, Rhenium-186, Rhenium-188,
- excisional biopsy and histopathological examination are used for quality control of the model to ensure the absence of host reaction and rejection of the tumour xenografts.
- myeloma cells can be inoculated directly into the marrow of the long bones or spleen in this large animal model.
- the model of the invention may also be used to study the effects of gene therapy or the control of metastasis by a gene of interest.
- cancer suppressor genes such as the p53 gene, the DCC (deleted in colon carcinoma) gene, the metastasis regulating gene, nm23, or any tumour-suppressor or inhibitor gene of interest which can be inserted into a vector such as a viral vector or liposome, may be co-implanted or inoculated with cells as described above.
- genetically manipulated cells containing these genes may be transplanted into the animal model.
- the genes may also be introduced after xenografting and metastasis at the site of the tumour formation. The effects of gene therapy alone, or in combination with the forms of therapy already described, can then be investigated.
- the CsA-immune- suppressed sheep model provides an in vivo system of comparable size and physiology to human patients and allows detailed study of targeted cancer therapy.
- the size of the organs permits tumours which mimic neoplasms in patients presenting with early cancer to be studied under controlled conditions, particularly by organ imaging modalities such as gamma camera SPECT, CT or MRI which are impractical in nude mice or rats.
- tumour specificity and localization in the human cancer cells can be measured directly, and the time relationships examined by serial excisional biopsy and histoche ical or quantitative microautogradiographic examination, or gamma or beta scintillation counting.
- Safe therapeutic application of novel tumour specific radiolabelled monoclonal antibodies requires preclinical delineation of critical organ dosimetry such as can be measured by SPECT imaging of a large animal human tumour model, validated by serial biopsies of major organs for accurate gamma or beta counting.
- the target tumour dosimetry can also be accurately measured by well counting of excisional biopsies to establish the potential efficacy of any radiopharmaceutical prior to embarking upon a clinical trial.
- Computer algorithms can then be developed, and tested, to perform prospective critical organ dosimetry on tracer doses of radioimmunotherapeutic agents, validated by direct measurement in the large animal model as described above, to accurately prescribe a maximum safe tolerated dose to a patient before committing to therapy.
- Butman SM Wild JC, Noland PE, Fagan TC, Finley PR, Hicks
- Ketoconazole A Possible Direct Cytotoxic Effect on Prostate Carcinoma Cells. J of Urology 141:190-191.
- Warhoe KA deNardo SJ
- Wolkov HB Doggett EC
- Kroger LA Kroger LA
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JP09523893A JP2001501446A (en) | 1996-03-21 | 1997-03-21 | Animal models for transplantation |
EP97908085A EP0921822A4 (en) | 1996-03-21 | 1997-03-21 | Animal model for transplantation |
AU20189/97A AU719377B2 (en) | 1996-03-21 | 1997-03-21 | Animal model for transplantation |
US09/155,237 US6538174B2 (en) | 1996-03-21 | 1997-03-21 | Animal model for transplantation |
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AUPN8803 | 1996-03-21 | ||
AUPN8803A AUPN880396A0 (en) | 1996-03-21 | 1996-03-21 | Animal model for transplantation |
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US09/155,237 A-371-Of-International US6538174B2 (en) | 1996-03-21 | 1997-03-21 | Animal model for transplantation |
US10/262,089 Division US6706947B2 (en) | 1996-03-21 | 2002-09-30 | Method for screening for agents against cancer using an immunosuppression animal model |
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US9765119B2 (en) | 2001-10-19 | 2017-09-19 | Aurinia Pharmaceuticals Inc. | Cyclosporine analogue mixtures and their use as immunomodulating agents |
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DE10225932B4 (en) * | 2002-06-11 | 2007-01-11 | Deutsches Krebsforschungszentrum (Dkfz) | Imaging method and apparatus for carrying it out |
US20040268421A1 (en) * | 2003-06-30 | 2004-12-30 | Infraredx, Inc. | Animal model for medical device testing and training using xenografted organ structures such as blood vessels |
CA2465155C (en) | 2004-04-21 | 2008-12-09 | Ibm Canada Limited-Ibm Canada Limitee | Recommendations for intelligent data caching |
JP2006345745A (en) * | 2005-06-15 | 2006-12-28 | Juntendo | Method for creating hepatic carcinoma non-human animal model |
EP1787645A1 (en) * | 2005-11-18 | 2007-05-23 | Institut Curie | New method for treating cancer based on the modulation of the calcineurin and/or the calcineurin/NFAT pathway |
US8263823B2 (en) * | 2008-10-20 | 2012-09-11 | Adimmu Institute Inc. | Immunocompetent xenograft model |
CA2851828A1 (en) | 2011-10-28 | 2013-05-02 | Presage Biosciences, Inc. | Methods for drug delivery |
KR102364713B1 (en) * | 2015-03-27 | 2022-02-22 | 가톨릭대학교 산학협력단 | Producing method of rheumatoid arthritis animal model, AbartaRA, via transplanting rheumatoid arthritis synovium |
KR102240824B1 (en) * | 2018-12-06 | 2021-04-15 | 경북대학교 산학협력단 | Method of preparing animal models for drug screening using cancer cell line speroids and their use |
CN114574442A (en) * | 2022-01-06 | 2022-06-03 | 中国人民解放军空军军医大学 | Human brain glioma cell line and method and application for constructing in-situ transplantation model thereof |
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Non-Patent Citations (15)
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AMERICAN JOURNAL OF NEPHROLOGY, Volume 15, No. 6, 1995, M. SOBH et al., "Coadministration of Ketoconazole to Cyclosporin - Treated Kidney Transplant Recipients: A Prospective Randomized Study", pages 493-499. * |
CHEMICAL ABSTRACTS, Volume 108, No. 21, issued 23 May 1988, KUSUYAMA et al., "Subreral Capsule Assay as a Chemosensitivity Test (v). Experimental Chemotherapy in Cyclosporin A- Treated Mice and Nude Mice", page 29, Abstract 179709m; & GAN TO KAGAKU RYOHO, 15(1), 79-89. * |
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Cited By (3)
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US9765119B2 (en) | 2001-10-19 | 2017-09-19 | Aurinia Pharmaceuticals Inc. | Cyclosporine analogue mixtures and their use as immunomodulating agents |
US10472394B2 (en) | 2001-10-19 | 2019-11-12 | Aurinia Pharmaceuticals Inc. | Cyclosporine analogue mixtures and their use as immunomodulating agents |
USRE48226E1 (en) | 2001-10-19 | 2020-09-29 | Aurinia Pharmaceuticals Inc. | Cyclosporine analogue mixtures and their use as immunomodulating agents |
Also Published As
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AUPN880396A0 (en) | 1996-04-18 |
US6706947B2 (en) | 2004-03-16 |
ZA972501B (en) | 1998-03-20 |
CA2249585A1 (en) | 1997-09-25 |
US20030101466A1 (en) | 2003-05-29 |
US20020147997A1 (en) | 2002-10-10 |
EP0921822A4 (en) | 2002-10-23 |
US6538174B2 (en) | 2003-03-25 |
JP2001501446A (en) | 2001-02-06 |
EP0921822A1 (en) | 1999-06-16 |
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