Characterization of a novel metabolite intermediate of ziprasidone in hepatic cytosolic fractions of rat, dog, and human by ESI-MS/MS, hydrogen/deuterium exchange, and chemical derivatization.

Ziprasidone (Geodone), a novel atypical antipsychotic agent, is recently approved for the treatment of schizophrenia. It undergoes extensive metabolism in preclinical species and humans after oral administration, and only a very small amount of administered dose is excreted as unchanged drug. In vitro studies using human liver microsomes have shown that the oxidative metabolism of ziprasidone is mediated primarily by CYP3A4. However, coadministration of ziprasidone with ketoconazole, a CYP3A4 inhibitor, showed only a modest increase in its exposure. Therefore, in vitro metabolism of ziprasidone was investigated in hepatic cytosolic fractions to further understand its clearance mechanisms in preclinical species and humans. The major metabolite from incubation of ziprasidone in cytosolic fractions of rat, dog, and human was characterized by liquid chromatography-tandem mass spectrometry and found to be the product of reductive cleavage. Derivatization and hydrogen/deuterium exchange were used to deduce that the addition of two hydrogen atoms had occurred at the benzisothiazole moiety. Further studies to determine the enzyme involved in the formation of this metabolite are currently in progress. The identification of this novel metabolite in cytosol has clarified the clearance mechanism of ziprasidone in humans and preclinical species.     

Presence of human papillomavirus DNA and abnormal p53 protein accumulation in lung carcinoma.

BACKGROUND: In some carcinomas inactivation of the tumour suppressor gene product p53, either by point mutation or indirectly by the human papillomavirus (HPV), has been suggested as two alternative routes to malignant transformation. To test this hypothesis in lung tumours, 43 lung carcinomas were analysed by in situ hybridisation and polymerase chain reaction (PCR) for the presence of HPV DNA, and the results were compared with p53 protein immunohistochemical analysis. METHODS: The presence of HPV DNA in lung carcinoma was detected by nucleic acid in situ hybridisation for HPV types 6, 11, 16, 18, 31, and 33 using nonradioactively labelled DNA probes. Polymerase chain reaction (PCR) analysis was performed on all cases showing positive HPV DNA labelling by in situ hybridisation and in an additional 13 negative cases. Abnormal nuclear accumulation of the p53 protein was revealed by immunohistochemistry using the avidin-biotin-peroxidase complex method and a CM-1 polyclonal anti-human p53 antibody and a monoclonal mutation-specific Pab 240 p53 antibody. RESULTS: HPV DNA was found by in situ hybridisation in 13 lung carcinomas (30%). In all these cases subtype-specific HPV DNA could also be detected by PCR. Abnormal p53 protein accumulation was seen in 21 of the 43 carcinomas (49%), of which 18 were HPV negative. Twelve (57%) of the CM-1 positive cases were also positive for the mutation-specific antibody Pab 240. There was an obvious inverse relationship between the presence of papilloma viral DNA and abnormal p53 protein accumulation. CONCLUSIONS: p53 plays an important part in the development of lung carcinomas and, in some cases, HPV may contribute to it by binding and inactivating the p53 protein.     

An optimal three-stage design for phase II clinical trials

A phase II clinical trial in cancer therapeutics is usually a single-arm study to determine whether an experimental treatment (E) holds sufficient promise to warrant further testing. When the criterion of treatment efficacy is a binary endpoint (response/no response) with probability of response p, we propose a three-stage optimal design for testing H₀: p ≤ p₀ versus H₁: p ≥ p₁, where p₁ and p₀ are response rates such that E does or does not merit further testing at given levels of statistical significance (α) and power (1 ? β). The proposed design is essentially a combination of earlier proposals by Gehan and Simon. The design stops with rejection of H₁ at stage 1 when there is an initial moderately long run of consecutive treatment failures; otherwise there is continuation to stage 2 and (possibly) stage 3 which have decision rules analogous to those in stages 1 and 2 of Simon's design. Thus, rejection of H₁ is possible at any stage, but acceptance only at the final stage. The design is optimal in the sense that expected sample size is minimized when p = p₀, subject to the practical constraint that the minimum stage 1 sample size is at least 5. The proposed design has greatest utility when the true response rate of E is small, it is desirable to stop early if there is a moderately long run of early treatment failures, and it is practical to implement a three-stage design. Compared to Simon's optimal two-stage design, the optimal three-stage design has the following features: stage 1 is the same size or smaller and has the possibility of stopping earlier when 0 successes are observed; the expected sample size under the null hypothesis is smaller; stages 1 and 2 generally have more patients than stage 1 of the two-stage design, but a higher probability of early termination under H₀; and the total sample size and criteria for rejection of H₁ at stage 3 are similar to the corresponding values at the end of stage 2 in the two-stage optimal design.     

Failure of in vitro-expanded hyperimmune cytotoxic T lymphocytes to affect survival of mouse embryos in vivo

This study was designed to address the question; does expression of paternal histocompatibility antigens by fetal cells make them susceptible to immune attack in vivo during normal pregnancy? The experimental design was based on the rationale that, if alloantigens are presented by trophoblasts or other fetal cells in a manner which allows accessibility, in vitro-generated immune effector cells of combined helper/cytotoxic phenotype should produce fetal rejection of abortion. Similarly generated effector cells are capable of accelerating skin graft rejection and, when combined with IL-2 in vivo, are capable of causing regression of antigenic, but operationally non-immunogenic, tumors. The alloimmune effector cells generated in vitro during the current study were highly cytotoxic against normal adult target cells, whereas placental cells were completely resistant to cytolysis and fetal cells were only slightly susceptible. Adoptive transfer of effector cells to mice at different stages of gestation had no apparent effect on pregnancy outcome. In vivo administration of IL-2 and/or indomethacin, which expand effector cell numbers in vivo and block PGE2-mediated immune suppression, respectively, failed to potentiate the cellular effect. The data provide additional evidence that paternal histocompatibility antigens are not expressed in a format which allows susceptibility to immune attack during pregnancy. The data are discussed with respect to the role of the trophoblast in protecting developing embryos.     

Radioimmunoassay method for the detection of IgE antibodies specific to alcuronium

A radio-immunoassay (RIA) was used to screen for specific IgE to myorelaxants. Alcuronium was coupled to epoxy-activated Sepharose. Sixteen patients with anaphylaxis to alcuronium (n = 2), gallamine (n = 2) or suxamethonium (n = 12) were studied. The diagnosis was established by intradermal tests (ID), passive cutaneous anaphylaxis tests and human basophil degranulation tests. The amount of non specific label retained by Sepharose-ethanolamine (with sera of patients) and Sepharose-alcuronium (with sera of 11 control subjects) was estimated. The RIA was positive 10/16 (8/14 patients having reacted to a muscle relaxant other than alcuronium). The RIA seemed to be useful in the diagnosis of anaphylaxis to muscle relaxants. Drug-reactive antibodies were specific of the quaternary ammonium radical, which was the common allergenic determinant of all molecules of muscle relaxants. This test accounted for in vitro cross-reactivity, but had no predictive value for the clinical risk of crossed-anaphylaxis. This risk was best assessed by ID; it was positive in three cases. Although it was not possible to compare ID and RIA, the interpretation of which was different, both tests should be recommended for the detection of sensitivity to muscle relaxants.     

Morphological study of some hypothalamic centers in rats after deafferentation of the mediobasal hypothalamus

The volume of the nuclei and nucleoli of certain hypothalamic centers (SON, PVN, SCN, AN, VMN) was determined in control rats and in rats after deafferentation of the mediobasal hypothalamus. Sex differences were found in the parvocellular formations of the control animals: The volumes of nuclei and nucleoli of neurons of AN and VMN, and also of the nucleolus of SCN neurons were larger in females than in males. After deafferentation of the mediobasal hypothalamus the volume of the cell nuclei was increased, especially in hypothalamic formations located outside the isolated zone. This increase was more clearly defined in rats constantly in a state of estrus after the operation. Statistically significant differences between volumes of both nuclei and nucleoli of the cells in subgroups of rats with permanent estrus and with permanent diestrus were found only in the case of SCN. No such differences were found for AN, despite the considerable difference in the constant of luteinizing hormone in the pituitary of the same rats. It is suggested that gonadotropin releasing factors are not produced by the cells of AN and that control over the succession of phases of the sex cycle may be exerted by SCN.Laboratory of Neuroendocrinology, I. M. Sechenov Institute of Evolutionary Physiology and Biochemistry, Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Chernigovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 3, pp. 352–354, March, 1980.