Title:  Nude mouse model for the growth and treatment of human neurally-derived tumors

United States Patent:  6,362,392

Assignee:  The General Hospital Corporation (Boston, MA)

Filed:  April 10, 2000

A method for treating a neurofibrosarcoma tumor comprising administration of heparin and an angiostatic steroid is disclosed. Animal models for the growth of neurally-derived tumors and for testing therapeutic agents are also provided.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

An animal model has been developed by the inventor for studying the growth of human neurally-derived tumors, and for evaluating the therapeutic efficacy of anti-tumor agents.

By the tenrm "neurally-derived" as used herein is intended tumors which originate in the nervous system and tumors which develop from cells which originate from the embryonic neural crest. Such tumors include, but are not limited to meningiomas, Schwannomas (for example, acoustic neuromas), neurofibrosarcomas, ependymomas, gliomas, and pheochromocytomas.

The animal model of this invention has several advantages including (1) the histologic appearance of the original tumor is retained in the subcapsular site in vivo (2) tumor size and vascularity can be measured serially using an ocular microscope; (3) there is no adipose or fibrous tissue to confound accurate tumor measurement; and (4) tumor take is high. This model system perm itted the development and evaluation of therapeutic regimens for human neurofibrosarcoma, based on the use of angiostatic steroids and heparin, which comprise an additional embodiment of this invention.

This invention is directed to a method for treating, a neurofibrosarcoma tumor in an animal comprising administering an effective amount of a combination of heparin and an angiostatic steroid.

By the term "treating" is intended amelioration or cure of the tumor, which includes the cessation of growth. the regression or disappearance of a detectable solid tumor, or a prevention or diminution in metastasis of the tumor.

The term "substantially associated with" neurofibromatosis means thatthe tumor is one of the pathological manifestations of neurofibromatosis and occurs in a significant percentage of neurofibromatosis patients.

The preferred animal subject of the present invention is a mammal. By the term "mammal" is meant an individual belonging to the class Mammalia. The invention is particularly useful in the treatment of human subjects, although it is intended for veterinary uses as well.

In addition to heparin, included in the scope of the present invention is the use of a heparin fragment or a synthetic heparin substitute in the treatment of neurofibrosarcoma.

The variable activity of different heparin preparations in the inhibition of angiogenesis in combination with steroid is known in the art. Heparin preparations are nonuniform and heterogeneous in composition, molecular size, structure, position ofsubstituents (N-sulfate, O-sulfate, and glucuronic acid), and sequence (Goldgaber, et al. Science 235:877 (1987); Tanzi et al., Science 235:881 (1987); Robakis, N. K et al., Proc. Natl. Accd. Sci. USA 84:4190 (1987)). This heterogeneity is thought to be responsible for various effects observed.

Heparin can be modified, or heparin fragments synthesized by methods known in the art (Choay, J et al., Biochem. Biophys. Res. Comm. 116:492 (1983); van Boeckel, C. A. A. et al., Tetrahedron Lett. 29:803 (1988), both of which references are hereby incorporated by reference).

Preferred heparin fragments include a hexasaccharideora pentasaccharide fragment. Preferred synthetic heparin substitutes comprise cyclodextrins of six to eight glucopyranose units. A more preferred cyclodextrin is .beta.-cyclodextrin tetradecasulfate.

Cyclodextrins are naturally occurring cyclic nonreducing, water-soluble oligosaccharides built up from six to eight glucopyranose units (Bender, M. L. et al., Cyclodextrin Chemistry, Springer Verlag, Berlin, 1978); Saenger, W., Angew. Chem. Int. Ed. Engl. 91:344 (1980); Saenger, W., In: Inclusion Compounds(Atwood, J. L. et al., eds.), Academic Press, New York, 1984, vol. 2, pp. 232-259). (The preceding 3 references and the other references to cyclodextrins, cited below, are hereby incorporated by reference.)

The internal doughnut-shaped molecule provides a hydrophobic cavity at the center and a hydrophilic outer surface. The diameter of the cavity is determined by the number of glucose units that make up the ring (6,7, or 8 units for .alpha.-, .beta.-, .gamma.-cyclodextrins, (respectively). Steroids or other hydrophobic molecules with appropriate structures can form complexes with cyclodextrins (Bergeron, R. J., In: Inclusion Compounds (Atwood, J. L. et al., eds.), Academic Press, New York, 1984, vol. 3, pp. 391-443; Tabushi, I., ibid, pp. 391-443; and Szejti, J., ibid, pp. 331-338). An inclusion complex between hydrocortisone and .beta.-cyclodextrin has been demonstrated (Frank, S. G. et al., J. Pharm. Sci. 72:1215 (1983); Anderson, F. M. et al., Arch. Pharm. Chemi. Sci. Ed. 11:61 (1983); Armstrong, D. W. et al., Anal. Chem. 57:234 (1985)). Cyclodextrins have 18 to 24 hydroxyl units exchangeable for substituents that could increase the hydrophilic and cell-binding activity of the carrier molecule. Various of the .alpha.-, .beta.-, .gamma.-cyclodextrins have angiostatic activity in combination with hydrocortisone and cortexolone (see. Folkman, J. et al., 1989, supra),

The present invention is intended to include all cyclodextrins with angiostatic activity, such as tetrapropoxy-.beta.-cyclodextrin, tetradecamethoxy-.beta.-cyclodextrin, .beta.-cyclodextrin heptasulfate, .beta.-cyclodextrin tetradecasulfate, .alpha.-cyclodextrin dodecasulfate, and .gamma.-cyclodextrin hexadecasulfate. A preferred cyclodextrin is .beta.-cyclodextrin tetradecasulfate.

The term "angiostatic steroid" describes a newly defined class of steroids, based on a particular biological activity, inhibition of angiogenesis. The classification of steroids as glucocorticoids, mineralocorticoids, or as biologically inactive steroids, is inapplicable for identifying angiostatic steroids. Thus, steroid molecules of both known biological classes, and even molecules devoid of known bioactivity, can function as angiostatics (see Crum, R. et al. (1985), supra, which is hereby incorporated by reference). Furthermore, angiostatic activity may be highly concentration-dependent. For example, dexamethasone at 50-60 .mu.g (the optimal angiostatic concentration of hydrocortisone) is not angiostatic in the presence of heparin. A sharp peak of angiostatic activity is detected at 2 .mu.g of dexamethasone (24-fold the activity of hydrocortisone). However, 3 to 200 .mu.g of dexamethasone shows no angiostatic activity.

The preferred angiostatic steroids (natural and synthetic) of this invention include, but are not limited to, hydrocortisone, 11.alpha.-epihydrocotisol, cortexolone, 17.alpha.-hydroxyprogesterone, corticosterone, desoxycorticosterone, testosterone, estrone, dexamethasone, triamcinolone, and 6.alpha.-fluoro-17,21-dihydroxy-16.beta.-methyl-pregna-4,9,(11)-diene-3,20 -dione.

In addition to steroids, the invention includes steroid derivatives, such as those formed metabolically in the liver during steroid inactivation by enzymatic reduction of the 4,5 double bond in the A ring to form the dihydrosteroid derivative. This is further converted into a tetrahydro derivative by the enzymatic reduction of the 3-oxo group to a 3-hydroxyl group. These derivatives are rendered water-soluble during metabolic inactivation by conjugation to glucuronic acid and are subsequently excreted by the kidney, Although considered biologically inactive (Liddle, G. W. et al., Textbook of Endorcrinology, R. H. Williams, Ed., Saunders, Philadelphia, 5th Ed., 1974, p. 244), the dihydro and tetrahydro derivatives retain angiostatic activity in the presence of heparin (Crum et al., supra). The preferred angiostatic steroid derivatives of this invention include dihydro and tetrahydro steroid derivatives.

As used herein, an "effective amount of a combination" is meant to refer to an amount of a combination of heparin, a heparin fragment, or a synthetic heparin substitute with an angiogenic steroid, that is sufficient to cause cessation of growth, regression or disappearance of a tumor, or a diminution in its metastasis, in a subject recipient.

The specific amount of heparin and steroid required by each individual will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. Generally, daily dosages of heparin will be from about 10 to about 50,000 units per kg of body weight. Preferably, from 50 to 2000 units/kg/d, in one or more applications per day, is effective to obtain the desired result. Most preferably, a daily dose from about 70 to about 350 units/kg/day is given. In an alternative approach, the heparin, particularly where formulated in a timed-release form, may be administered less frequently, i.e., every other day or every third day. Depending on the steroid used, the daily dose of steroid will be from about 1 to about 100 mg/kg body weight. Preferably, from about 5 to about 25 mg/kg is effective to obtain the desired results.

Depending on the particular steroid or steroid derivative used, and the particular heparin, heparin fragment, or synthetic heparin substitute used, the effective dose can be varied, as will be apparent to one of skill in the art.

The combination heparin and steroid treatment of the present invention may be administered by any means, routes, or pharmaceutical compositions that achieve their intended purpose. Amounts and regimens for the administration of heparin and any particular steroid can be determined readily by those with ordinary skill in the art. For example, administration may be by parenteral, subcutaneous, intravenous, intramuscular, intrapulmonary, intraperi toneal, intranasal, transdermal, or buccal routes. Alternatively, or concurrently, administration may be by the oral route.

For treatment according to the invention, heparin and the steroid are administered simultaneously, or within a proximity of hours between separate administration of each component of the combination. Thus, for example, a dose of the heparin may be given up to 12 hours before or after a dose of steroid. The preferred timing of administration is simultaneous.

The pharmaceutical composition may be employed in dosage form such as tablets, capsules, powder packets, or liquid solutions, suspensions, or elixirs, for oral administration, or sterile liquid for formulations such as solutions or suspensions for parenteral use. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Carriers or occlusive dressings can be used to increase skin permeability and enhance cutaneous absorption.

The invention also relates to amedicament orpharmaceutical composition comprising heparin and an angiogenic steroid, the medicament being used for treating a neurofibrosarcoma which may be substantially associated with neurofibromatosis.

The invention is also directed to a nude mouse implanted in its renal capsule with a human neurally-derived tumor, and the use of such a mouse as a model to study growth and evaluate treatment of the implanted tumors. Use of nude mice for xenotransplantation of human tumors is well known in the art (Fogh, J. et al. (eds.), The Nude Mouse in Experimental and Clinical Research, New York: Academic Press, Vol. 2, pp. 475-490 (1982), which is hereby incorporated by reference.

Congenitally athymic nude mice of either sex, such as CD-1 nu/nu mice from Charles River Laboratories (Wilmington, Mass.) weighing between about 20 and 30 grams are used. Mice are preferably housed in sterile cages in groups of five, and given autoclaved chow and water ad libitum.

Surgery is preferably performed under sterile conditions. Each mouse is anesthetized by administration of a general anesthetic. For example mice are given an intraperitoneal injection of 0.3 ml of a 4% chloral hydrate solution, and this is supplemented with ether inhalation when necessary (Boyden, A. E. et al., Cancer 48:10-20 (1981)]; (Fingert, H. J. et al., Proc. Natl. Acad. Sci. USA 81:7927-7931 (1984), which references are hereby incorporated by reference).

An oblique incision of about 1-cm length is made in a region of the left flanks which is preferably made aseptic by application of an antiseptic sterilizing solution such as 70% ethanol. Using a stereomicroscope, the kidney capsule is lifted with microforceps, and a tumor implant of about 0.5 to 2 mm in size, either from the surgical specimen or from cell cultures in a fibrin clot, is placed under the renal capsule by means of a needle or trochar of an appropriate gauge to hold the fragment (such as a 19 gauge needle). The implant is mobilized 1 to 2 mm away from the capsule opening to prevent adhesions. Nylon suture, for example of 5.0 gauge, is used for closure in a single layer.

Preparation of a cell pellet or cell cluster from culture for implantation is performed by methods well-known in the art. The cell pellet is suspended in a mixture of, for example, fibrinogen and thrombin and incubated at about 37^oC. for about 5 to 10 minutes to obtain a solid fibrin clot matrix (Fing ert, H. J. et al., supra).

Tumor growth from the implanted fragment, cell pellet or cluster is evaluated by measurement of two perpendicular diameters of the implant using, for example, an ocular micrometer in the eyepiece of a stereomicroscope. Tumor volume is estimated from the formula (volume=length.times.width.times.1/2) (Fingert, H. J. et al., supra).

The same surgical and anesthetic techniques are used to make serial tumor volume measurements, the first being performed upon implantation and the following measurement at about 10 to 21 days after tumor implantation into the subrenal capsule. These times may vary depending upon the type of tumor and its rate of growth as will be appreciated by one of skill in the art. The animals can be reexamined four or five additional times during a three-month period to generate growth curves for each individual tumor.

Tumor vascularity can be graded as follows: grade 0=no visible vessels: grade I=one or two vessels; grade II=three or four vessels; grade III =more than four vessels. Typically, at the end of a study, the animals are humanely sacrificed for histological examination.

Claim 1 of 15 Claims

What is claimed is:

1. A nude mouse comprising a renal capsular implant of a human neurally-derived tumor, wherein tumor cells from said tumor are embedded in a fibrin clot.

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