Strategy and planning for chemopreventive drug development: clinical development plans II.

This is the second publication of Clinical Development Plans from the National Cancer Institute, Division of Cancer Prevention and Control, Chemoprevention Branch and Agent Development Committee. The Clinical Development Plans summarize the status of promising chemopreventive agents regarding evidence for safety and chemopreventive efficacy in preclinical and clinical studies. They also contain the strategy for further development of these drugs, addressing pharmacodynamics, drug effect measurements, intermediate biomarkers for monitoring efficacy, toxicity, supply and formulation, regulatory approval, and proposed clinical trials. Sixteen new Clinical Development Plans are presented here: curcumin, dehydroepiandrosterone, folic acid, genistein, indole-3-carbinol, perillyl alcohol, phenethyl isothiocyanate, 9-cis-retinoic acid, 13-cis-retinoic acid, l-selenomethionine and 1, 4-phenylenebis(methylene)selenocyanate, sulindac sulfone, tea, ursodiol, vitamin A, and (+)-vorozole. The objective of publishing these plans is to stimulate interest and thinking among the scientific community on the prospects for developing these and future generations of chemopreventive drugs.

Age-related neoplastic and degenerative lesions in aging male virgin and ex-breeder ACI/segHapBR rats.

A variety of neoplastic and degenerative lesions were observed in 216 aged male breeder (to 24 months of age) and virgin ACI/segHapBR rats sacrificed from 24 to 40 months of age. The most common neoplasms were pheochromocytomas, pituitary tumors, interstitial cell tumors of the testis, and tumors of the skin and subcutis. Many rats had multiple endocrine tumors. Age-related prostate hyperplasias and tumors were reported previously in these rats. Most of these tumors increased in incidence with advancing age, with few differences between virgins and ex-breeders. Testicular atrophy and tumors were more common in younger ex-breeder rats than in young virgin rats, but tumors reached a similar incidence in older virgin rats. Focal hyperplastic lesions appeared to represent the earliest stages of neoplasia in pituitary and adrenal glands, thyroid, prostate, testis, and liver. Common age-related nonneoplastic degenerative lesions were found in various tissues.

Chemical quantification of unscheduled DNA synthesis in cultured hepatocytes as an assay for the rapid screening of potential chemical carcinogens.

Technical modifications of the quantitative determination of unscheduled DNA synthesis in cultured hepatocytes are described which allow for the rapid identification of potentially carcinogenic chemicals on a large-scale screening basis. The test is based on the biochemical quantification of [methyl-3H]thymidine incorporation into DNA in the presence of hydroxyurea following isolation of nuclei from hepatocytes treated with the agent under study. This procedure ("nuclei procedure") eliminates most of the background radioactivity which otherwise obscures the stimulation of DNA repair synthesis by agents that induce a relatively weak response. By combining the nuclei procedure with a double-labeling technique, test results can be obtained within a few hr after exposure of hepatocytes to the test agents. A test series involving 41 agents confirmed the reliability of the nuclei procedure for the assay of DNA repair synthesis. In addition, chemicals which had yielded conflicting results previously in the autoradiographic hepatocyte DNA repair test, such as 4-acetylaminofluorene, or which had passed unrecognized in previous in vitro tests, such as the potent liver carcinogen methapyrilene hydrochloride, scored clearly positive in our test protocol.

Histogenesis and morphology of naturally occurring prostatic carcinoma in the ACI/segHapBR rat.

The prostates of 201 male virgin or breeder ACI (ACI/segHapBR) rats, 24 to 40 months of age were reviewed histologically, and the genesis of prostatic carcinoma was studied. At 24 months of age, 35 to 45 per cent of the rats had the earliest lesions, intraalveolar atypical hyperplasias. These lesions progressed to intraalveolar cribriform carcinomas which spread along alveoli and ducts. As the tumors enlarged, they became nodular and invaded the capsule or adjacent tissues. By 33 months, 95 to 100 per cent of the rats had intraalveolar prostatic atypical hyperplasias, and 35 to 40 per cent had invasive carcinomas. There was no difference between the incidence of these tumors in virgin or breeder rats. The ACI rat is unique model for studying the etiology and pathogenesis of naturally occurring prostatic cancer.

A nitrogen pressure-shearing apparatus for preparing distinct populations of chromatin fractions.

A method for preparing reproducible chromatin fractions is described. Fragmentation is accomplished by expelling whole chromatin from an enclosed stainless-steel chamber through a restricted orifice utilizing low nitrogen pressure. Sheared chromatin is fractionated into two distinct heavy and light fractions by centrifugation on 5-20% linear sucrose gradient.

Further characterization of intracytoplasmic A particle-associated DNA.

A DNA species with buoyant densities greater than mouse cellular DNA was found associated with intracytoplasmic A particles (CAP) isolated from mouse mammary tumor virus-infected mouse mammary tumors and mouse Leydig cell tumors which produce CAP but no complete mouse mammary tumor virus virions. This DNA species was absent in identically prepared tissue fractions from tumors which did not contain CAP. Treatment of CAP-associated DNA with pancreatic RNase A did not alter the buoyant density although a reduction in apparent molecular weight (broadening of the DNA band at equilibrium) was observed upon analytical equilibrium sedimentation in CsCl. The molecular weight of untreated CAP-associated DNA was estimated to range from 0.8 x 10(6) to 3.1 x 10(6). Base composition analysis showed CAP-DNA to possess an approximate guanine plus cytosine content of 38%. Ninety percent of CAP-associated DNA eluted as single-stranded molecules upon hydroxyapatite column chromatography, a characteristic that accounts in part for its higher buoyant density in neutral CsCl compared to native double-stranded mouse DNA. In two preparations, CAP-DNA had a sedimentation coefficient of 7 to 8S.

Autoradiographic evidence for the association of DNA with intracytoplasmic A-particle inclusions in situ.

Mice with subcutaneous transplants of a Leydig cell tumor were given tritiated thymidine sc proximal to the tumor mass. The labeled tissue was collected and samples for autoradiography were taken. Nuclei and intracytoplasmic. A particle(s) (CAP) were purified from the remainder of the tumor pool. The distribution of radioisotopic grains over electron microscopic autoradiography was semiquantitated and analyzed. Granin were found over CAP inclusions in situ. Comparison of the relative frequency of CAP-associated grains to nuclear grains by probability on a planar area basis revealed a higher frequency of thymidine incorporation into these structures. Isolation and comparison of the DNA(s) associated with CAP and nuclei showed a higher specific activity for CAP-associated DNA than for nuclear DNA, which was in good agreement with the autoradiographic analysis. The DNA associated with the purified CAP possessed a significantly higher buoyant density in CsCl than did the nuclear DNA. These reslult implied that this DNA was present in CAP inclusions in situ.