Multicellular living organisms and unmodified parts thereof and – Method of using a transgenic nonhuman animal in an in vivo...
Reexamination Certificate
1998-09-16
2001-04-17
LeGuyader, John L. (Department: 1633)
Multicellular living organisms and unmodified parts thereof and
Method of using a transgenic nonhuman animal in an in vivo...
C800S009000, C800S008000, C800S021000
Reexamination Certificate
active
06218594
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to the development of a model test system for studying leiomyomas and atherosclerosis treatment modalities of leiomyomas and atherosclerosis.
2. Background Art
Uterine leiomyomas: Leiomyomas (fibroids) are the most common pelvic benign clonal tumors of the uterus originating from myometrial smooth muscle cells (reviews by Barbieri, 1992 and references therein; Tsibris and Spellacy, 1991) that may cause bleeding, a mass, or pelvic pressure (Barbieri and Speroff, 1992). They occur in 25% of women over 35, are a major (30%) cause of hysterectomy. They occur in 20-30% of women over 30 and are therefore an important issue in pregnancy as many women delay childbearing to their late thirties. Leiomyomas are found more frequently in African American than caucasian women (Kjerulff et al, 1993).
Leiomyomas are clonal tumors believed to originate from single myometrial cells (Barbieri and Speroff, 1992; Tsibris and Spellacy, 1991; Rein and Nowak, 1992). They are discrete encapsulated tumors, found submucosally (beneath the endometrium), intramurally (within the myometrium) and subserosally (projecting out of the serosal compartment of the uterus). Some leiomyomas become partially calcified. Although their etiology is unknown it is believed that estrogens, and perhaps progesterone, regulate their development and growth because leiomyomas increase in size during reproductive life and regress after menopause. Estradiol-17&bgr; (E
2
) and progesterone (P
4
) have been implicated in their development and growth. Therapy with agonists (for example Leupron) of gonadotropin releasing hormone (GnRH), which inhibit gonadal function and decrease serum E
2
, results in a 40-60% decrease in leiomyoma size that is reversed when therapy is discontinued (Stewart and Friedman, 1992). It should be noted that Leupron therapy cannot be undertaken for longer than six months due to the risk of osteoporosis.
Karyotypic translocations, rearrangements, breakpoints, mainly in chromosomes 6p, 7q21-31, 12q13-15 and 14q23-24 occur in 20-50% of leiomyomas and bear similarity to those found in lipomas and meningiomas (Kiechle-Schwarz et al, 1991; Pandis et al, 1990; Rein et al, 1991) Leiomyomas have significantly higher levels of E
2
receptors (E
2
R) and P
4
receptors (P
4
R) than normal myometrium throughout the menstrual cycle (Lumsden et al, 1989; Sadan et al, 1987). Therapy with GnRH agonists (GnRHa), which inhibit gonadal function and decrease serum E
2
below 20 pg/ml, result in a 40-60% decrease in the size of leiomyomas (Friedman and Barbieri, 1988). Paradoxically, leiomyomas removed after 3 months of GnRHa therapy had lower E
2
but increased E
2
R, compared to untreated leiomyomas, as determined by both [
3
H]-E
2
displacement assays and immunoassays using monoclonal E
2
R antibodies (Lumsden et al, 1989; Senekjian et al, 1989).
A leiomyoma animal model is needed that resembles, as closely as possible, human uterine leiomyomas. This model would be valuable in unraveling the molecular mechanism(s) by which leiomyomas are regulated in vivo. More importantly, the model would facilitate the design and testing of new therapeutic modalities aimed at retarding leiomyoma growth or even preventing their formation.
Nelson (1937) and Lipschutz and associates (1938; 1939, 1941, 1942) produced tumors on the uterine serosa and the abdominal cavity of guinea pigs after three months exposure to various estrogens. The tumors did not develop if the estrogen treatment was halted, interrupted for a week, or combined with high doses of P
4
.
Female guinea pigs are more susceptible to tumors than males (Rogers and Blumenthal, 1960). Spontaneous uterine leiomyomas occur in 8.4% of older guinea pigs with a mean age of four years, the 80% percentile of their life span (Field et al., 1989; Harkness and Wagner, 1989). In 1981, Fujii et al., (1981) injected E
2
benzoate intramuscularly three times a week, but electron microscopy revealed that the nodules produced after three months of treatment were composed of only fibroblasts. Only when the injections with estrogen continued for a month and were supplemented with weekly injections of 1 mg P
4
did the investigators find nodules composed of cells resembling smooth muscle and decidua cells (Fujii et al., 1981); if a higher dose (3 mg) of P
4
was used, no nodules were formed.
Applicants have previously improved the Lipschutz guinea pig model by exposing animals continuously via a slow release system to pregnancy level serum E
2
(Porter, et al, 1993). These guinea pigs developed leiomyomas mainly on the uterine subserosa and in the anterior abdominal wall. After nine months, numerous abdominal tumors appeared resembling leiomyomatosis peritonealis disseminata which is a very rare disease in humans. However, this model does not provide as close a correlation with the human disease as needed, in particular the appearance of intra-abdominal wall tumors is not seen in humans.
Everitt et al. (1995) and Howe et al. (1995) report that small uterine leiomyomas and leiomyosarcomas were spontaneously formed in Eker rats, as part of a familial renal cancer syndrome, a result of a germline mutation in the tuberous sclerosis gene. It appears that these uterine tumors are similar to the small serosal leiomyomas applicants observed in the E2-only-treated guinea pigs (prior art model). As seen in applicants' prior model numerous small (1-2 mm) or larger cysts located along the blood supply of the horn were seen in the Eker rat tumors. This model again does closely resemble the human leiomyomas as well as being associated with a mutant gene and cancer syndrome which are not seen in the human condition.
Very recently, a transgenic mouse model for uterine leiomyomas was reported by Romagnolo et al. (1996). The formation of leiomyomas in this model still depends on the administration of estrogens. However, preparing and maintaining transgenic animals is expensive and labor intensive. It would be useful to have a model that did not require transgenic manipulations. Also a mouse model does not easily allow for laparoscopic techniques because of the small size of the animal.
There are many animal models for atherosclerosis, from primates, pigs, rabbits to mice, the latter being highly resistant to the disease (Breslow, 1996; the May 3, 1996 issue of Science is devoted to Cardiovascular Medicine). Most, if not all animals, can develop atherosclerosis by dietary manipulation, usually, a high fat diet to induce hypercholesterolemia (Fekete, 1993), by genetic manipulation [transgenic and knockout mice] (Paigen et al., 1994) or exposure to a virus. However, these models do not allow for clarifying yet unexplored mechanisms of pre-atherosclerotic events (Simionescu and Simionescu, 1993) with emphasis on proliferation of vascular smooth muscle cells that become the dominant cell types (Moraghan et al., 1996). It would be useful to have a model where therapy for early pre-atherosclerotic events can be explored.
SUMMARY OF THE INVENTION
According to the present invention, a model test system in guinea pigs for studying leiomyomas and early stage atherosclerosis is disclosed. The model includes ovariectomized young female guinea pigs co-implanted with a continuous slow release device in a preferred embodiment for releasing an estrogenic compound or analogues thereof and for a retinoid compound or analogues thereof. The model test system allows for the testing of pharmaceutical compounds for the treatment of leiomyomas and for atherosclerosis.
The present invention also provides a method of monitoring the effectiveness of the pharmaceutical agent being used to shrink leiomyoma without waiting for the endpoint of tumor shrinkage. The present invention further provides a method of treatment of leiomyomas by treating with antagonists or agonists of the family of nuclear retinoic acid receptors.
REFERENCES:
patent: 4439196 (1984-03-01), Higuchi
patent: 4447224 (1984-05-01), DeCant, Jr. et al.
patent: 4447233 (1984-
Porter Kathy B.
Tsibris John C. M.
Kohn & Associates
LeGuyader John L.
University of South Florida
Wilson Michael C.
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