Effect of alosetron on the pharmacokinetics of alprazolam.

Lotronex (alosetron hydrochloride) is a 5-HT3 receptor antagonist indicated for the treatment of irritable bowel syndrome (IBS) in females whose predominant bowel habit is diarrhea. Alosetron is extensively metabolized by multiple cytochrome P450 (CYP) enzymes, including CYP 2C9 and 3A4. Alprazolam is a short-acting benzodiazepine commonly prescribed for the treatment of anxiety disorders and a potential comedication in patients with IBS. Alprazolam is extensively metabolized by CYP3A4. This clinical study was conducted to assess the potential for a metabolic drug interaction between these two CYP3A4 substrates. This was an open-label, randomized, two-period, crossover study in 12 healthy female and male volunteers to determine the effect of concomitant administration of alosetron at the recommended dose of 1 mg p.o. bid on the pharmacokinetics of alprazolam following a single oral 1 mg dose. The results showed no effect of alosetron on the pharmacokinetics of alprazolam. Mean alprazolam AUC was 210 and 202 ng.h/mL in the absence and the presence of alosetron, respectively. Therefore, alprazolam may be safely coadministered with alosetron without the need for dosage adjustment.

Parabolic growth patterns in 96-well plate cell growth experiments.

In preparing for the routine use of the ubiquitous in vitro cell growth inhibition assay for the study of anticancer agents, we characterized the statistical properties of the assay and found some surprising results. Parabolic well-to-well cell growth patterns were discovered, which could profoundly affect the results of routine growth inhibition studies of anticancer and other agents. Four human ovarian cell lines, A2780/WT, A2780/DX5, A2780/DX5B, and A121, and one human ileocecal adenocarcinoma cell line, HCT-8, were seeded into plastic 96-well plates with a 12-channel pipette, without drugs, and grown from 1-5 d. The wells were washed with a plate washer, cells stained with sulforhodamine B (SRB), and dye absorbance measured with a plate reader. Variance models were fit to the data from replicates to determine the nature of the heteroscedastic error structure. Exponential growth models were fit to data to estimate doubling times for each cell line. Polynomial models were fit to data from 10-plate stacks of 96-well plates to explore nonuniformity of cell growth in wells in different regions of the stacks. Each separate step in the assay was examined for precision, patterns, and underlying causes of variation. Differential evaporation of water from wells is likely a major, but not exclusive, contributor to the systematic well-to-well cell growth patterns. Because the fundamental underlying causes of the parabolic growth patterns were not conclusively found, a randomization step for the growth assay was developed.

Modeling of the time-dependency of in vitro drug cytotoxicity and resistance.

For potential clinical extrapolation of in vitro findings, it is of interest to relate the measured effect of an anticancer agent to concentration and exposure time. The Hill model (A. V. Hill, J. Physiol., 40: iv-vii, 1910) is commonly used to describe pharmacodynamic (PD) effects, including drug-induced growth inhibition of cancer cells in vitro. The IC(X)n x T = k relationship, in which IC(X) is the concentration of agent required to reduce cell growth by X%, T is the exposure time, and n and k are estimable parameters, was first applied to bacterial disinfectant action and then was successfully used to model anticancer drug potency as a function of exposure time (D. J. Adams, Cancer Res., 49: 6615-6620, 1989). Our goal was to create a new global PD modeling paradigm to facilitate the quantitative assessment of the growth-inhibitory effect of anticancer agents as a function of concentration and exposure time. Wild-type human ovarian A2780 and ileocecal HCT-8 carcinoma cells and sublines that were resistant to cisplatin (A2780/CP3), doxorubicin (A2780/DX5B), and raltitrexed (RTX) (HCT-8/DW2) were exposed to various anticancer agents, cisplatin, doxorubicin, paclitaxel, trimetrexate, RTX, methotrexate, and AG2034, for periods ranging from 1 to 96 h. Cell growth inhibition was measured with the sulforhodamine B protein dye assay. Patterns of time-dependency of drug potency, slope of the concentration-effect curves, and relative degree of resistance were characterized. Empirical mathematical expressions were built into a global concentration-time-effect model. The global PD model was then fit to the concentration-time-effect data with iteratively reweighted nonlinear regression. Under specific treatment conditions, the examination of the slope and the shape of the concentration-effect curves revealed a large heterogeneity in drug response, e.g., shallow concentration-effect curve or double or triple Hill "roller coaster" concentration-effect curve. These patterns, which were observed at intermediate exposure times in parental and resistant cells for paclitaxel and trimetrexate or only in resistant HCT-8/DW2 cells for RTX, methotrexate, and AG2034, revealed mechanistic insights for the former cases but possible methodological artifacts for the latter cases. The comprehensive PD modeling of the cytotoxic effect of anticancer agents showed that it was possible to modulate drug effect, response heterogeneity, and drug resistance by altering the time of exposure to the agents. This approach will be useful for: (a) describing complex concentration-time-effect surfaces; (b) refining biological interpretations of data; (c) providing insights on mechanisms of drug action and resistance; and (d) generating leads for clinical use of anticancer drugs.

Development of a new quantitative approach for the isobolographic assessment of the convulsant interaction between pefloxacin and theophylline in rats.

PURPOSE: A new mathematical approach was developed to quantify convulsant interaction between pefloxacin and theophylline in rats. METHODS: Animals received each compound separately or in different combination ratios. Infusion was stopped at the onset of maximal seizures. Cerebrospinal fluid (CSF) and plasma samples were collected for HPLC drug determination. The nature and intensity of the pharmacodynamic (PD) interaction between drugs was assessed with a new modeling approach which includes (a) data transformation to create an essentially error-free X-variable and (b) estimation of an interaction parameter a by fitting a nonlinear hyperbolic model to the combination data with unweighted nonlinear regression. RESULTS: Drug disposition to the biophase was linear within the range of administered doses. The estimates of a suggested a Loewe antagonistic interaction between pefloxacin and theophylline at the induction of maximal seizures in rats. Similar intensity of PD interaction was observed at the dose and biophase level (alpha was -0.415 +/- 0.069 and -0.567 +/- 0.079, respectively). CONCLUSIONS: The suitability of the proposed model was assessed by Monte Carlo simulation. This new mathematical approach enabled the characterization of the Loewe antagonistic nature of the PD (convulsant) interaction between pefloxacin and theophylline, whereas previously used methodologies failed to do so.

Implications for clinical pharmacodynamic studies of the statistical characterization of an in vitro antiproliferation assay.

Modeling of nonlinear pharmacodynamic (PD) relationships necessitates the utilization of a weighting function in order to compensate for the heteroscedasticity. The structure of the variance was studied for concentration-effect data generated in an in vitro 96-well plate cell growth inhibition assay, where data are numerous (480 data points per experiment) and replication is easy. From the five candidate models that were considered, the power function S2Y = phi 2Y phi 3, where Y is the sample mean and S2Y is the sample variance, was shown to be the most appropriate to describe the nonuniformity of the variance along the range of measured effect for 253 sets of (Y; S2Y) data. The Hill model was fit to the concentration-effect data with weighted nonlinear regression, where the weights were equal to the reciprocal of the predicted variance. The examination of the distribution of the 253 sets of parameters of the PD model showed that IC50 was lognormally distributed whereas the distribution of gamma was normal. The characterization of the appropriate variance function and concentration-effect function in a simple in vitro experimental setting with a large number of experiments, with each experiment including a large number of data points, will be useful for guiding similar in vitro concentration-effect studies where data are plentiful and for guiding PD modeling in complex clinical settings in which extensive data for model characterization is impossible to obtain.

Combined action of paclitaxel and cisplatin against wildtype and resistant human ovarian carcinoma cells.

PURPOSE: The combination of paclitaxel (PTX) and cisplatin (DDP) shows good clinical efficacy against ovarian cancer. In order to examine the potential cellular basis for this, and provide leads as to how to optimize the combination, we examined the role of sequence of exposure to PTX and DDP on cell growth in vitro. METHODS: Four human ovarian carcinoma cell lines, A121, A2780/WT, A2780/DX5B and A2780/CP3, two human head and neck carcinoma cell lines, A253 and FaDu, and the human ileocecal carcinoma cell line, HCT-8, were treated with PTX + DDP with seven schedules: (A) 96 h exposure to PTX + DDP; (B) 24 h PTX alone, then 72 h PTX + DDP; (C) 4 h DDP alone, then 92 h PTX + DDP; (D) 24 h PTX alone, 4 h DDP alone, then 68 h drug-free; (E) 4 h DDP alone, 24 h PTX alone, then 68 h drug-free; (F) 3 h PTX alone, 1 h DDP alone, then 92 h drug-free; and (G) 1 h DDP alone, 3 h PTX alone, then 92 h drug-free. Each of 66 two-drug experiments included five plates (440 randomly treated wells per experiment). Cell growth was measured by the sulforhodamine B assay. The nature and the intensity of the drug interactions were assessed by fitting a seven-parameter model to data with weighted nonlinear regression, enabling the estimation of an interaction parameter, alpha, with its standard error. RESULTS: Overall there was very little departure from Loewe additivity: 43 experiments showed Loewe additivity, 10 showed Loewe antagonism, and 13 showed slight Loewe synergy. In vitro Loewe synergy was rare, was small when present, and reproducible only for the A121 and HCT-8 cells exposed to schedule D (24 h PTX prior to 4 h DDP). Isobolographic analysis showed complex combined-action surfaces with regions of local Loewe synergy and antagonism. CONCLUSION: It appears unlikely that the good clinical efficacy of the combination is primarily caused by a synergistic interaction at the cellular level.

Effects of mode of inhalation of carbon monoxide and of normobaric oxygen administration on carbon monoxide elimination from the blood.

1. The half-life of carbon monoxide (CO) in blood was studied retrospectively in 26 fire victims and in 19 cases of CO poisoning. Normobaric oxygen therapy was administered via mechanical ventilation in 19 fire victims, and by facial mask to the rest of the casualties. 2. Arterial pH was significantly lower (P < 0.05) and PaO2 significantly greater (P < 0.01) in the mechanically ventilated fire victims compared to the spontaneously breathing fire victims. 3. The blood CO half-lives were 91 +/- 38 min for the 26 fire victims and 87 +/- 40 min for the 19 pure CO intoxications. 4. The blood CO half-lives were 92 +/- 40 min for the 19 mechanically ventilated fire victims and 87 +/- 37 min for the 26 spontaneously breathing subjects. 5. We conclude that the elimination of CO from blood was a slow process with no significant effects on the blood CO half-life of either the cause of the CO poisoning or the mode of normobaric oxygen therapy. These data suggest that enhancement of the elimination of carbon monoxide by normobaric oxygen in both pure CO poisoning and fire victims is more difficult to achieve and more complex than has previously been reported.

Eliciting macroporosity in polyacrylamide and agarose gels with polyethylene glycol.

Preparation of highly porous polyacrylamide has recently been described (Righetti et al., Electrophoresis, 1992, 13, 587-595). In this report we add new observations on the conditions of promoting macroporosity in polyacrylamide gels and extend the possibility of eliciting this phenomenon to agarose matrices by the combined use of polyethylene glycol and glycerol. The process of cluster formation in hot agarose solutions was studied and gel structures were examined by scanning electron microscopy. A definition of macroporosity in gel, related to controlled microsyneresis during gelation, is tentatively proposed. The unexpected influence of acrylamide and agarose concentrations upon the size of macroporous structures in the corresponding gels is revealed.

An objective structured clinical examination for the licentiate: report of the pilot project of the Medical Council of Canada.

The Medical Council of Canada (MCC) administers a qualifying examination for the issuance of a license to practice medicine. To date, this examination does not test the clinical skills of history taking, physical examination, and communication. The MCC is implementing an objective structured clinical examination (OSCE) to test these skills in October 1992. A pilot examination was developed to test the feasibility, reliability, and validity of running a multisite, two-form, four-hour, 20-station OSCE for national licensure. In February 1991, 240 volunteer first- and second-year residents were tested at four sites. The candidates were randomly assigned to one of two forms of the test and one of two sites for two of the four sites. Generalizability analysis revealed that the variance due to form was 0.0 and that due to site was .16 compared with a total variance of 280.86. The reliabilities (inter-station) were .56 and .60 for the two forms. Station total-test score correlations, used to measure station validity, were significant for 38 of the 40 stations used (range .14-.60). The results of the OSCE correlated moderately with the MCC qualifying examination; these correlations were .32 and .35 for the two test forms. Content validity was assessed by postexamination questionnaires given to the physician examiners using a scale of 0 (low) to 10 (high). The physicians' mean ratings were: importance of the stations, 8.1 (SD, 1.8); success of the examination in testing core skills, 8.1 (SD, 1.6); and degree of challenge, 7.8 (SD, 2.1). The results indicate that a full-scale national administration of an OSCE for licensure is feasible using the model developed. Aspects of validity have been established and strategies to augment reliability have been developed.

Focal ectasia of the terminal bowel accompanying low anal deformities.

Three patients were treated at birth for low anal deformities and developed intractable constipation, not imputable to usual causes of rectal inertia. Barium enema, done the first time at ten days of age in one case, revealed focal ectasia of the terminal bowel, with a peculiar "balloon-like" aspect of the rectum. Optimal treatment includes resection of the terminal bowel down to the dentate line. Incidence of this anomaly in association with low anal deformities has been 5% in our series.

Measuring higher cognitive levels by multiple choice questions: a myth?

Available evidence is inconclusive as to the ability of multiple choice items to measure different taxonomic levels of the cognitive domain. The present study analysed the tests of the Examen de Synthèse for the years 1982, 1983 and 1984. Items used in the study were those for which a consensus was reached between three judges and committees for a given taxonomic level. The initial part of the study showed that judges do not classify items at random but according to a mental representation which is individual, personal and relatively stable. In examining results obtained by students, the study failed to show any significant difference in item difficulty or discrimination for items classified as measuring memorization, interpretation of data and problem-solving. Correlations between results (scores and ranks) obtained for items involving memorization and those obtained for items involving higher cognitive levels fail to show that different traits are measured. If further studies corroborate these results, then future efforts should be directed at developing other instruments to measure higher cognitive levels.

Reversion to fetal circulation in congenital diaphragmatic hernia: a preventable postoperative complication.

A review of 26 patients with congenital diaphragmatic hernia, diagnosed in the first 24 hours of life, supports the hypothesis that the postoperative fetal circulation syndrome is an iatrogenic complication, due to the rapid expansion of both lungs, when they are severely hypoplastic. This complication is preventable, when no aspiration of air from the chest cavity is done, and when no tube attached to an underwater seal is inserted. When assisted ventilation is necessary, small volumes at a rapid rate allows satisfactory gaseous exchanges, without pulmonary overinflation.

The surgical separation of pygopagous twins.

A set of female pygopagous twins was successfully separated. Preparation for the main operative procedure and the surgical anatomy encountered are described in details.

Dacron as arterial prosthetic material: nature, properties, brands, fate and perspectives.

Dacron graft prostheses are routinely implanted to overcome impairment of arterial flow to distal tissues. Many different fabric configurations have been developped and evaluated. Unfortunately, after implantation the flow surface of any graft persists as a fibrinous lining. No human specimen has a commonly ingrown extension of tissue exceeding some millimeters in length. Full wall healing and endothelialization of arterial prosthesis is a goal that must be pursued intensively because it seems that the goal is attainable.

Effect of thyroxine on the inactivation of thyrotrophin-releasing hormone by rat and human plasma.

The inactivation of synthetic [3H]thyrotrophin-releasing hormone (TRH) by plasma was studied in rats treated with propylthiouracil (PTU) alone or with PTU and thyroxine. 48 h after the onset of treatment with thyroxine, the capacity of rat plasma to inactivate [3H]TRH was significantly increased. The percentage of deamidation of TRH to TRH-free acid was increased 2-fold after 4 days of administration of thyroid hormone. The inactivation of TRH by plasma from hypothyroid patients was compared to that obtained from hyperthyroid patients. Extraction of human plasma incubated with [3H]TRH, followed by thin-layer electrophoresis, showed that transformation of [3H[TRH into TRH-free acid was 44% higher in plasma from hyperthyroid than from hypothyroid patients (P less than 0-05). These data suggest that the inactivation process of TRH by blood proteins could be an important factor in the regulation of the hypothalamo-hypophyseal-thyroid axis in rat and man.

Addition compounds of oxidizing tocopherol and soybean lecithin.

Evidence is presented for the formation of addition compounds from tocopherol and soybean lecithin, when adsorbed in monolayer on silica from a mixed solution in chloroform and oxidized at 80 C for 72 hr. Tocopherol was present at 7 mol % of the lecithin in the monolayer. The compounds produced are analogous to linoleic acid-tocopherol adducts previously reported by us. UV spectra of the mixed lecithin and tocopherol monolayers while in silica gel slurry in a solvent were obtained by the method previously reported by us for oxidized linoleic acid and tocopherol monolayers. The monolayer spectra show no evidence for tocopherol dimer. A minor amount of quinone is indicated, but the spectrum has a maximum at 287 nm with no other major peaks. The proposed addition compounds have been characterized by transesterification and hydrogenation, UV, IR, thin layer chromatography and gas chromatography retention behavior, mass spectrometry and elemental analysis. IR spectra of twice-chromatographed addition compounds show specific lecithin absorption bands (5.75 µ; 10.3 µ) together with much enhanced 3.4-3.5, 6.8, 7.25 and 9.14 µ peaks, the latter two being characteristic of α-tocopherol. The UV spectrum of the adducts showed λ max (CHCl) 3 287 nm, with shoulders at 276 and 300 nm. Hydrogenation removed the peaks at 287 and 276 nm, leaving a peak at 300 nm similar to that of the linoleic acid-tocopherol adduct. Esters of the adducts prepared by transesterification and hydrogenation were similar by all our chemical, chromatographic and spectral tests to the previously characterized esters of the linoleic acid-tocopherol adduct.