Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – The nonhuman animal is a model for human disease
Reexamination Certificate
1998-09-21
2003-03-25
Priebe, Scott D. (Department: 1636)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
The nonhuman animal is a model for human disease
C800S008000, C800S010000, C800S014000, C800S015000, C800S016000, C800S017000, C424S093100, C424S093200, C424S093700
Reexamination Certificate
active
06538174
ABSTRACT:
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.
BACKGROUND OF THE INVENTION
At present there is no effective method available for treatment of many solid tumours such as malignant melanoma or cancer of the colon, breast or ovary once the primary tumour has metastasised. 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. Radioimmunotherapy 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.
In order to evaluate therapeutic agents, or methods of imaging tumours, and to study the biological processes taking place in the development and metastasis of solid tumours, 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. The major problem associated with human tumour xenografts in nude animals is the 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.
Similar problems are encountered in orthotopic implantation models, in which human tumours or tumour cells are transplanted or injected into the organ or tissue of origin in recipient immunodeficient athymic mice (Manzotti et al, 1993). Although metastasis of the transplanted tumour is achieved, accurate and reliable data on usefulness of therapeutic agents or methods are still limited by the disproportionate size of the tumour in relation to the total body weight of the mouse.
Therefore, 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.
An animal model which would allow investigation of tumour nodules of a specific size and location, and which would simulate patterns of metastasis in various types of cancer, is particularly desirable. Larger animal models will also permit more effective and accurate evaluation of potential methods of therapy and imaging, and better characterisation of the biological events taking place during development and treatment of such cancers.
One way of inducing acceptance of xenografts is the administration of Cyclosporin A (CsA), a cyclic fungal peptide produced by
Tolypocladium inflatum
Gams. CsA is a neutral cycloundecapeptide with potent immunosuppressive properties (Borel, 1989; Di Padova, 1989; Hess et al, 1988). This antifungal metabolite appears to inhibit both humoral and cellular immune responses by selectively interfering with T-cell activation (Borel, 1989; Di Padova, 1989; Hess et al, 1988). 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.
For example, rabbits given intramuscular injections of CsA at 10 mg/kg suffered from toxic side effects, and became anorexic and developed pneumonia. These effects were only eliminated if larger animals were used, and antibiotic and fluid therapy were instituted together with cyclosporin administration (Liggett et al, 1993). Cats also require high oral doses of CsA in order to accept human tumour xenografts, since intravenous administration is also associated with species-specific Cremaphor-induced vasoconstriction with histamine release and anaphylaxis (Bowers et al, 1991).
However, in sheep, infusion of the castor-oil based vehicle for CsA, Cremaphor EL, is well tolerated (Tresham et al, 1988). There is also no nephrotoxic reaction to intravenous CsA in sheep (Tresham et al, 1990). A recent pharmacokinetic study of CsA administered intravenously to sheep revealed data similar to that reported in human transplant patients (Charles et al, 1993), and no toxic effects were described.
In addition to the toxic effects of CsA, a major disadvantage of this compound is the requirement for daily injections, which is both tedious and expensive and limits the period of time within which animals can be kept for observations (Hu et al, 1994, and de Ward-Siebinga et al, 1994). In all the studies mentioned above, the amount of CsA administered has been more than 10 mg/kg of animal weight.
There has been a single brief report of experiments in which human melanoma tumours have been subcutaneously grown in dogs immune-suppressed by oral CsA (Wiseman et al, 1991). This method, however, also requires high doses of CsA due to its limited bioavailability from oral administration. The intravenous route is precluded by the anaphylactic reaction of dogs to the Cremaphor vehicle in which cyclosporin is dissolved (Bowers et al, 1991).
More recently, several groups have reported the use of ketoconazole in conjunction with CsA as a means of reducing the dose of CsA required in transplant patients to maintain immunosuppression and prevent graft rejection (Gandhi et al, 1992; Butman et al, 1991; First et al, 1991; Wadhwa et al, 1987). 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 vito (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.
Ketoconazole, in addition to its synergism with CSA in the induction and maintenance of immunosuppression, has been reported to exert anti-tumour activity against certain types of cancer (Eichenberger et al, 1989a; Mahler and Denis, 1992). Ketoconazole also acts in synergy with anti-neoplastic drugs (vinblastine, etoposide) to inhibit the growth of human prostate carcinoma cells in vitro (Eichenberger et al, 1989b).
Similarly, 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). Of the cell lines tested, human and murine T cell lymphomas and leukaemias were found to be sensitive to CsA-induced growth inhibition at doses of 0.5-5 &mgr;g/ml, whereas non-lymphoid cell lines and certain murine B and null cell leukaemias were insensitive to
Fremantle Hospital
Fulbright & Jaworski LLP.
Kaushal Sumesh
Priebe Scott D.
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