Components in seminal plasma regulating sperm transport and elimination.

Seminal plasma has been suggested to be involved in sperm transport, and as a modulator of sperm-induced inflammation, which is thought to be an important part of sperm elimination from the female reproductive tract. This article reports on recent experiments on the importance of seminal plasma components in sperm transport and elimination. In Experiment 1, hysteroscopic insemination in the presence (n = 3) or absence (n = 3) of 2 ng/mL PGE showed an increased portion of spermatozoa crossing the utero-tubal junction in the presence of PGE in two mares, while no difference was observed between treatments in a third mare. In Experiment 2, whole seminal plasma, heat-treated seminal plasma (90 degrees C for 45 min), and charcoal-treated seminal plasma were added to: (1) sperm samples during opsonization prior to polymorphonuclear neutrophil(s) (PMN)-phagocytosis assays (n = 5); or to (2) phagocytosis assays (n = 5). Opsonization of spermatozoa was suppressed in the presence of whole seminal plasma, compared with samples without seminal plasma (p < 0.05). Charcoal treatment did not remove the suppressive effect of seminal plasma on opsonization, but heat treatment of seminal plasma reduced its suppressive properties (p < 0.05). The addition of whole seminal plasma to opsonized spermatozoa almost completely blocked phagocytosis (p < 0.05). Charcoal treatment did not remove the suppressive effect of seminal plasma. However, heat-treated fractions of seminal plasma removed the suppressive effect of seminal plasma on phagocytosis (p < 0.05). In Experiment 3, viable and non-viable (snap-frozen/thawed) spermatozoa were subjected to in vitro assays for PMN binding and phagocytosis with the following treatments (n = 3): (1) seminal plasma (SP), (2) extender; (3) ammonium sulfate precipitated seminal plasma proteins with protease inhibitor (SPP+); or (4) ammonium sulfate precipitated seminal plasma proteins without protease inhibitor (SPP-). Treatment was observed to impact binding and phagocytosis of viable and non-viable spermatozoa (p < 0.05). SP and SPP+ suppressed PMN-binding and phagocytosis of viable sperm. This effect was also seen, but to a lesser degree, in SPP- treated samples. Non-viable spermatozoa showed less PMN-binding and phagocytosis than live sperm in the absence of SP. The addition of SP promoted PMN-binding and phagocytosis of non-viable spermatozoa. SPP- treated samples also restored PMN-binding of non-viable spermatozoa. The addition of protease inhibitors removed this effect. In Experiment 4, seminal plasma proteins were fractionated based on MW by Sephacryl S200 HR columns (range 5000-250,000 kDa). Fractionated proteins were submitted to sperm-PMN binding assays. A protein fraction <35 kDa suppressed PMN-binding to live and snap-frozen spermatozoa. A greater MW protein fraction appeared to promote binding between PMNs and snap-frozen spermatozoa. While the addition of protease inhibitors was necessary to maintain the protective effect of seminal plasma proteins on viable spermatozoa, the promotive effect of seminal plasma on non-viable spermatozoa appeared to require some protease activity. It was concluded from these experiments that components of seminal plasma play active roles in transportation and survival of viable spermatozoa in the female reproductive tract and in the elimination of non-viable spermatozoa from the uterus.

Hepatic angiosarcoma: detection with computed tomography.

Hepatic angiosarcoma is a rare vascular neoplasm which occurs typically in men aged between 50 and 70 years. The cause is unknown but previous studies have linked several carcinogens to its pathogenesis. A case of advanced multifocal hepatic angiosarcoma with splenic metastasis is presented with brief discussion of the clinical and histological features. Typical CT features and contrast enhancement characteristics are also reviewed.

Predicting drug interactions in vivo from experiments in vitro. Human studies with paclitaxel and ketoconazole.

This study was performed to evaluate whether concomitant treatment with ketoconazole could reduce the clearance of paclitaxel given to ovarian cancer patients. Paclitaxel, 175 mg/m2, was given as a 3-hour continuous intravenous infusion and repeated every 21 days. Initially, ketoconazole, 100 to 1600 mg, was given as a single oral dose 3 hours after paclitaxel. Later, ketoconazole, 200 mg, was given perorally 3 hours before paclitaxel. Plasma drug concentrations were measured by high-pressure liquid chromatography (HPLC), and cytochrome P450 3A (CYP3A) activity was measured with the erythromycin breath test (ERMBT). Ketoconazole did not alter plasma concentrations of paclitaxel or its principal metabolite, 6 alpha-hydroxypaclitaxel. Although there was marked inter- and intrapatient variability in ketoconazole pharmacokinetics, peak plasma concentrations in all but one course were below the 50% inhibitory concentration (IC50) point determined for inhibition of paclitaxel metabolism in vitro. Therefore, paclitaxel and ketoconazole can be coadministered safely without dose adjustments. There was no correlation between ERMBT measurements and serial plasma concentrations of paclitaxel. The erythromycin breath-test measurements did correlate with the corresponding ketoconazole plasma concentrations. The erythromycin breath test is a valuable tool for measuring instantaneous CYP3A activity in vivo. This clinical study confirms the results of prior studies with human-derived materials in vitro, reinforcing the notion that such studies are useful predictors of drug pharmacokinetics and interactions in vivo.

Rifampin and rifabutin and their metabolism by human liver esterases.

1. The main metabolites of rifampin and rifabutin in man are their respective 25 deacetylated derivatives, but the enzyme(s) responsible for these biotransformations are not known. 2. In experiments with human liver slices and human liver microsomes, the 25 deacetylated derivatives of these drugs were the main metabolites observed. Slices and microsomes metabolized rifabutin 3-6-fold faster than rifampin, in agreement with their relative clearance in patients. Rifabutin partitioned into slices more avidly than rifampin. 3. In microsomal incubations, deacetylation did not require NADPH, but the amount of metabolite at the end of incubation was affected by NADPH. With NADPH the amount of 25 deacetyl rifabutin decreased, whereas the amount of 25 deacetyl rifampin increased slightly. A panel of liver microsomes from seven donors showed a 3-4-fold difference in the formation of 25 deacetyl rifabutin or 25 deacetyl rifampin, with strong correlation between the production of the two metabolites (r2 = 0.94). 4. The production of 25 deacetyl rifabutin and 25 deacetyl rifampin by human liver microsomes was not significantly affected by 1 microM 4 chloromercuricbenzoic acid or bis-(4-nitrophenyl) phosphate, but was completely inhibited by 1 microM paraoxon or 1 microM diisopropylfluorophosphate. These results indicate that in man rifampin and rifabutin are deacetylated to their main metabolites by B-esterases.

Small (< or = 3-cm) renal masses: detection with CT versus US and pathologic correlation.

PURPOSE: To determine the sensitivities of computed tomography (CT) and ultrasound (US) for detection and characterization of surgically verified small renal lesions. MATERIALS AND METHODS: Twenty-one patients with von Hippel-Lindau disease or hereditary papillary renal cancer underwent CT and US before partial nephrectomy or enucleation; 205 renal masses were removed (92% were <3 cm). Detection rates and accuracy of CT and US in the characterization of renal morphology were correlated with lesion size. RESULTS: CT and US detection rates for lesions of 0-5 mm were respectively 47% and 0%; 5-10 mm, 60% and 21%; 10-15 mm, 75% and 28%; 15-20 mm, 100% and 58%; 20-25 mm, 100% and 79%; and 25-30 mm, 100% and 100%. Among the lesions 10-35 mm, 80% and 82% were correctly characterized with CT and US, respectively. CONCLUSION: A substantial proportion of lesions under 1 cm were not detected with either modality. Neither CT nor US was superior in the characterization of lesions 3 cm or less. CT and particularly US screening studies in patients with von Hippel-Lindau disease should be interpreted cautiously because missed or mischaracterized small renal lesions are a frequent problem in these patients.

Metabolism of taxol by human and rat liver in vitro: a screen for drug interactions and interspecies differences.

Human liver slices, human liver microsomes, and rat liver microsomes were used to investigate the metabolism of 3H-taxol. The effects of drugs frequently coadministered with taxol and the effects of several cytochrome P450 system probes were studied. In all, 16 compounds were screened. After incubation with liver slices or with microsomal protein, 3H-taxol was converted into several radioactive species resolved by HPLC. There were qualitative and quantitative species differences in the metabolism of taxol. The pattern of metabolism was similar for both human-derived preparations, with 6 alpha-hydroxytaxol being the major metabolite peak. In drug interaction studies performed with human liver microsomes, cimetidine 80 microM, and diphenhydramine 200 microM, had little or no effect on 6 alpha-hydroxytaxol formation. Quinidine, ketoconazole, dexamethasone and Cremophor EL inhibited 6 alpha-hydroxytaxol formation with IC50 values of 36 microM, 37 microM, 16 microM and 1 microliter/ml, respectively, but these concentrations exceed the usual clinical range. Cremophor EL also inhibited microsomal metabolism of taxol, but at 2 microliters/ml it had little or no effect on 6 alpha-hydroxytaxol production by human liver slices. These results suggest that: (1) taxol is metabolized by the cytochrome P450 system; (2) taxol metabolism is different in humans than in rats; (3) taxol metabolism in humans is unlikely to be altered by cimetidine, dexamethasone, or diphenhydramine, drugs regularly coadministered with taxol; (4) taxol metabolism can be indirectly affected by Cremophor EL, the formulation vehicle; (5) taxol metabolism may be altered by concentrations of ketoconazole achievable in humans only at very high doses; and (6) taxol metabolism and drug interaction studies of clinical relevance can be performed in vitro with human liver microsomes and human liver slices, but not with rat liver preparations.

A fatality due to flurazepam.

A fatality attributed to suicidal ingestion of up to 2.2 grams of flurazepam is described. The deceased was a 52-year old female with a history of depression and suicidal attempts. No significant pathology was found at autopsy. Full toxicological analyses detected only flurazepam and metabolites in her tissues. The concentrations of flurazepam in femoral blood, liver, bile, vitreous humor and urine were 5.5 mg/L, 130 mg/kg, 33 mg/L, 1.3 mg/L and 3.3 mg/L, respectively. Analysis of gastric contents showed 600 mg of flurazepam. Desalkylflurazepam was also detected in blood, liver, bile and vitreous, but at much lower concentrations than the parent compound.

Effect of cimetidine, probenecid, and ketoconazole on the distribution, biliary secretion, and metabolism of [3H]taxol in the Sprague-Dawley rat.

Male Sprague-Dawley rats had their bile ducts cannulated and were dosed with [3H]taxol (2 mg/kg, 68-77 microCi/mg) as a continuous intravenous infusion for 6 hr so that the plasma concentrations, tissue distribution, metabolism, and biliary secretion of taxol could be studied. Defining potential drug-drug interactions of taxol with cimetidine (90 mg/kg), probenecid (360 mg/kg), and ketoconazole (50 mg/kg) was motivated by frequent concomitant clinical use or the potential to reduce clearance of taxol so that lower doses could be used. At 6 hr, rats were killed. Samples of blood (plasma), lung, spleen, liver, kidney, heart, skeletal muscle, brain, testes, and fat were obtained. Taxol and metabolites were measured by total radioactivity counting and HPLC separation using on-line radioactivity detection. Concentrations of taxol in plasma increased to 0.19 microM in the control rats and did not reach steady-state by 6 hr. Lung, spleen, liver, and kidneys had the greatest tissue taxol concentrations [4.7-5.7 nmol/g (microM)] and were > 25-fold higher than the simultaneous 6-hr plasma taxol concentration. Taxol concentrations in brain and testes were negligible, 0.06 and 0.07 nmol/g, respectively. Radioactive metabolites were not found in plasma or most tissues. Only liver had appreciable concentrations of taxol and metabolites; however, > 80% of hepatic radioactivity was parent taxol. Through 6 hr of collection, 24% of the dose was secreted in the bile approximately 38% of which was as parent taxol. Cimetidine had no effect on the distribution, metabolism, or elimination of [3H]taxol. Probenecid did not effect tissue distribution or plasma concentrations of taxol.(ABSTRACT TRUNCATED AT 250 WORDS)

Steady-state plasma concentrations and effects of taxol for a 250 mg/m2 dose in combination with granulocyte-colony stimulating factor in patients with ovarian cancer.

Taxol, a natural product initially isolated from the stem bark of the western yew Taxus brevifolia, is undergoing phase II and III evaluation due to its reported activity against a variety of tumors. Previous studies have described correlations between plasma concentrations and toxicity when taxol is given (a) at lower doses, (b) for shorter infusion times, and (c) without granulocyte-colony-stimulating factor. Because the 24-h infusion schedule is most commonly used in current clinical trials, we attempted to correlate steady-state plasma concentrations of taxol achieved with a 24-h continuous i.v. infusion with toxicities and responses. Plasma samples from 48 refractory ovarian cancer patients were obtained 1-2 h prior to the end of the first taxol infusion. Taxol concentrations were measured by high-performance liquid chromatography (HPLC). Interpatient variation of taxol plasma concentrations was small (mean +/- SD, 0.85 +/- 0.21 microM. Total taxol body clearance was 256 +/- 72 ml min-1 m-2 (mean +/- SD). Taxol plasma protein binding was 88.4% +/- 1.3% (mean +/- SD, n = 9). Grade 3-4 hematologic toxicity, mainly leukopenia, occurred in 92% of the patients. The leukopenia was transient and did not warrant a reduction in the dose of taxol. Grade 3-4 nonhematologic toxicity occurred in 8% of the patients. No severe hypersensitivity reaction or grade 3-4 neurotoxicity was observed. Correlations of plasma concentrations and toxicities were not feasible due to the high frequency of hematologic effects and the low frequency of nonhematologic toxicity. The low degree of interpatient variation in plasma concentrations hindered the development of correlations with response.

Effect of suramin on human prostate cancer cells in vitro.

Suramin, a polyanionic compound with known antiparasitic activity, has been shown to be adrenocorticolytic in primates and to have clinical efficacy in the treatment of patients with metastatic prostate cancer refractory to conventional hormonal manipulation. To better characterize the activity of suramin on prostate cancer biology, we studied the effect of the drug on plasma adrenal androgens of patients and on the human prostate adenocarcinoma cell lines PC-3, DU 145 and LNCaP-FGC. Five cancer patients treated with suramin had an approximate 40% decline in circulating androstenedione, dehydroepiandrosterone and dehydroepiandrosterone sulfate levels. The drug inhibited the colony formation in two of the three cell lines at concentrations clinically achievable in humans without excessive drug-related toxicity. The presence of suramin 300 micrograms./ml. partially inhibited the growth stimulatory effect of testosterone and basic fibroblast growth factor, but not that of epidermal growth factor. The cellular concentration of suramin following exposure to a single dose increases linearly over time in each of the cell lines with LNCaP-FGC accumulating the highest levels of the drug; cellular levels of suramin, not androgen or growth factor sensitivity, correlated with the sensitivity to the drug. The concentrations of prostatic acid phosphatase and prostatic specific antigen released by LNCaP-FGC cells in cell culture medium declined in the presence of increasing levels of suramin in a manner which exceeded the decrease in cell number. We conclude that suramin, aside from decreasing circulating androgens through its adrenocorticolytic effect, is also capable exerting a direct inhibitory effect on cell proliferation of prostate cancer cells, and interfere at a cellular level with the growth stimulatory effects of exogenous testosterone and basic fibroblast growth factor.

Induction of thymidylate synthase associated with multidrug resistance in human breast and colon cancer cell lines.

A series of Adriamycin-resistant human breast MCF-7 and human colon DLD-1 cancer cell lines were established by stepwise selection. The concentration of Adriamycin required to inhibit cell proliferation by 50% (IC50) in the parent breast line (MCF-7), Adriamycin-resistant lines (MCF-Ad5 and MCF-Ad10), and a 5-fluorouracil (5-FU)-revertant line (MCF-R) was 0.005, 3.3, 6, and 4.9 microM, respectively. The Adriamycin IC50 value for the resistant colon line (DLD-Ad) was 8.2 microM, 68-fold higher than that for its parent line (DLD-1) (IC50 = 0.12 microM). The MCF-Ad5 and MCF-Ad10 cells were cross-resistant to 5-FU, with respective 5-FU IC50 values of 11.7 and 22.5 microM, or 7.3- and 14-fold less sensitive than their parent MCF-7 (IC50 = 1.6 microM) line. The MCF-R line completely reverted in sensitivity to 5-FU, with an IC50 of 1.7 microM. The resistant DLD-Ad line was 3.5-fold more resistant to 5-FU than was the parent DLD-1 line. Using both the 5-fluoro-2'-deoxyuridine-5'-monophosphate binding and catalytic assays for measurement of thymidylate synthase (TS) activity, there was significantly increased TS activity in the resistant MCF-Ad5 (2.4- and 2.5-fold), MCF-Ad10 (11.5- and 6.8-fold), and DLD-Ad (4.8- and 10.7-fold) lines, for binding and catalytic assays, respectively, compared with their parent MCF-7 and DLD-1 lines. The level of TS in cytosolic extracts, as determined by Western immunoblot analysis, was markedly increased for the resistant MCF-Ad5 (31-fold), MCF-Ad10 (46-fold), and DLD-Ad (52-fold) cells. Measurement of TS mRNA levels by Northern analysis revealed elevation of TS mRNA in the resistant MCF-AD5 (16.7-fold), MCF-Ad10 (31-fold), and DLD-Ad (55-fold) cells. Southern analysis showed that this increase in TS mRNA was not accompanied by any major rearrangements or amplification of the TS gene. Incorporation of 5-FU into the RNA and DNA of the resistant MCF-Ad10 cells was not significantly different, compared with that for parent MCF-7 cells. These studies suggest that exposure of human breast and human colon cancer cells to Adriamycin leads to overexpression of TS, with concomitant development of resistance to 5-FU.

Suramin in adrenal cancer: modulation of steroid hormone production, cytotoxicity in vitro, and clinical antitumor effect.

Suramin, a drug known to have antiparasitic effects, has been previously shown to have adrenocorticolytic activity in primates. We now confirm preferential accumulation of this compound in the normal adrenal gland, evaluate its in vitro effect against two human adrenocortical carcinoma cell lines (SW-13 and NCI-H295), and report the clinical activity of suramin in 17 patients with metastatic adrenocortical carcinoma. Inhibition of colony formation occurred in both adrenal cell lines in vitro at concentrations that are clinically achievable in humans. In addition, suramin concentrations as low as 100 micrograms/mL were able to inhibit glucocorticoid, mineralocorticoid, and androgen production by the NCI-H295 cell line. Of 16 patients with adrenocortical carcinoma now evaluable for tumor response, 2 achieved a partial response, 2 had a minor response, and 5 remained with stable disease for periods ranging from 3-10 months; the remainder progressed. One of 7 patients with excessive steroid hormone production achieved a partial normalization of her steroid levels for the duration of suramin therapy in the setting of radiographic disease stabilization. An additional patient treated off-study for lack of radiographically measurable disease, achieved complete normalization of plasma aldosterone levels. We conclude that suramin preferentially accumulates in adrenal cells, induces cytotoxicity and significant down-regulation of steroid hormone production in vitro, and has some therapeutic efficacy as a single agent in patients with metastatic adrenocortical carcinoma.

65-70 kD protein identified by immunoblotting as the presumptive gastric microsomal autoantigen in pernicious anaemia.

Sera from 20 of 24 patients with pernicious anaemia reacted by immunoblotting with a 65-70 kD protein in canine and rodent gastric mucosal cells enriched for 80-90% parietal cells, and in microsomal preparations derived from these cells. All 20 reactive sera were positive for parietal cell microsomal antibody demonstrated by immunofluorescence. Eighteen parietal cell microsomal antibody-positive sera from patients with unconfirmed pernicious anaemia also reacted with the same 65-70 kD protein. Serum reactivity with the same 65-70 kD protein was not seen with canine and rodent liver cells or with microsomal preparations derived from these cells. Sera from 10 patients with chronic active hepatitis, 10 with scleroderma and 10 with rheumatoid arthritis and 22 healthy persons did not react with the 65-70 kD protein. These results suggest that the 65-70 kD protein is probably the parietal cell microsomal autoantigen. A second antigen of 85-90 kD mol. wt. present only in canine gastric mucosal preparations also correlates with the presence of parietal cell microsomal antibody. However, the contribution of this second antigen to parietal cell microsomal antibody reactivity remains uncertain.

Vimentin intermediate filaments in cultures of human meningiomas.

Monolayer cultures of six human meningiomas and meningeal cells from a human foetus were examined by indirect immunofluorescence with a human autoantibody to intermediate filaments and with a monoclonal antibody to vimentin intermediate filaments. No difference could be demonstrated in the staining of an intricate fibrillar network in cultures of transitional, fibroblastic, psammomatous and sarcomatous meningiomas compared to those of human foetal meninges. Many meningotheliomatous meningioma cells showed staining of distinctive 'whorls' of intermediate filaments, an observation less frequently seen in fetal meningeal cells or in meningiomas of other histological types. Meningioma cells, pretreated with vinblastine, showed staining of rearranged filaments whose conformation and compactness varied from cell to cell. A striking observation frequently seen in transitional and psammomatous meningiomas was the staining of thick intermediate filament 'bands' bridging two contiguous meningioma cells. Immunoblotting experiments confirmed the presence of vimentin intermediate filaments in the cultured meningioma cells.