Gender differences in carbohydrate loading are related to energy intake.

We demonstrated that female endurance athletes did not increase their muscle glycogen concentration after an increase in the dietary carbohydrate intake (58 --> 74%), whereas men did (Tarnopolsky MA, SA Atkinson, SM Phillips, and JD McDougall, J Appl Physiol 78: 1360-1368, 1995). This may have been related to a lower energy or carbohydrate intake by the women or due to an inherent gender difference in glycogen storage capacity. We examined whether well-trained men (n = 6) and women (n = 6) increased muscle glycogen concentration after an increase in both the relative (58 --> 75%) and absolute energy and carbohydrate intake and whether potential gender differences were related to muscle hexokinase enzyme activity. Subjects were randomly allocated to three diets [Hab, habitual; CHO, high carbohydrate (75%); and CHO + E, extra energy + CHO ( upward arrow~34%)] for a 4-day period before a muscle biopsy for analysis of total and pro- and macroglycogen and hexokinase activity. Total glycogen concentration was higher for the men on the CHO and CHO + E trials compared with Hab (P < 0.05), whereas women increased only on the CHO + E trial compared with Hab (P < 0.05). There were no gender differences in the proportion of pro- and macroglycogen or hexokinase activity. A low energy intake may explain the previously reported lower capacity for women to glycogen load compared with men.

Development, characterization and therapy of a disseminated model of childhood neuroblastoma in SCID mice.

PURPOSE: To develop a highly reproducible model of disseminated childhood neuroblastoma in mice to allow secondary evaluation of therapeutics against microscopic disseminated disease. METHODS: CB17/Icr SCID were injected i.v. with 10(3) to 5 x 10(6) human NB-1691 neuroblastoma cells. NB-1691 cells were detected by PCR for synaptophysin and tyrosine hydroxylase in peripheral blood, and bone marrow. Therapeutic studies evaluated topotecan and vincristine as single agents or in combination. Topotecan was administered i.v. daily for 5 days on two consecutive weeks. Courses were repeated every 21 days for three cycles. Vincristine (1 mg/kg) was administered i.v. every 7 days for nine consecutive weeks. Treatment started 11-21 days after tumor cell inoculation. RESULTS: Following injection of > or = 1 x 10(5) cells 100% of mice developed disease. Mice inoculated with 10(7) cells survived a median of 42 days. Survival time was a linear function of the cell inoculum. At autopsy, gross tumor was routinely detected in many organs in particular liver, ovaries, kidneys and adrenals. NB-1691 cells were detected by PCR in peripheral blood, and bone marrow. Immunohistochemical staining showed that lesions were strongly positive for synaptophysin, chromogranin A and negative for leukocyte common antigen. Topotecan (0.6 mg/kg) alone extended median survival from 44 days (controls) to 95 days. When treatment was started 21 days after inoculation of NB-1691 cells, topotecan extended median survival from 39 days (controls) to 91 and 99 days at dose levels of 0.3 and 0.6 mg/kg, respectively. Vincristine (1 mg/kg) extended survival by a median of 9.5 days. In combination with vincristine (1 mg/kg), median survival was increased to 141 days (topotecan 0.6 mg/kg) and 159 days (topotecan 1.0 mg/kg). CONCLUSION: This model of disseminated neuroblastoma is highly reproducible. As this model may more closely simulate childhood disease it may be a valuable adjunct in developing new approaches to advanced stage, poor prognosis neuroblastoma.

Relation between 9-aminocamptothecin systemic exposure and tumor response in human solid tumor xenografts.

9-Aminocamptothecin (9-AC) is a topoisomerase I inhibitor with activity against xenografts from childhood solid tumors; however, clinical trials with this compound have been disappointing, resulting in discontinuation of further development. The objectives of this study were to evaluate the antitumor activity of 9-AC in a panel of pediatric solid tumor xenografts and to relate the 9-AC lactone systemic exposure, defined as area under the concentration time curve (AUC), to the antitumor dose associated with tumor regression in the xenograft model. We evaluated protracted administration of i.v. and oral therapies (daily times 5) for 1, 2, or 3 weeks and for 1 or 3 cycles. The minimum effective dose of 9-AC causing objective regression of advanced tumors was determined for each schedule. 9-AC lactone plasma concentration-time profiles associated with the lowest dose achieving complete and partial responses for each xenograft were then determined for each regimen. Tumors were highly sensitive to 9-AC therapy, but the systemic exposure required for antitumor effect is in excess of that achievable in patients.

Antitumor activity of temozolomide combined with irinotecan is partly independent of O6-methylguanine-DNA methyltransferase and mismatch repair phenotypes in xenograft models.

The activity of temozolomide combined with irinotecan (CPT-11) was evaluated against eight independent xenografts (four neuroblastomas, three rhabdomyosarcomas, and one glioblastoma). In all studies, temozolomide was administered p.o. daily for 5 consecutive days/cycle, found in preliminary studies to be the optimal schedule for administration. Irinotecan was administered i.v. for 5 days for 2 consecutive weeks/cycle. Treatment cycles were repeated every 21 days for a total of three cycles over 8 weeks. In combination, temozolomide and CPT-11 induced complete responses in four neuroblastomas, two rhabdomyosarcomas, and the glioblastoma line. The activity of the combination was significantly greater than the activity of either agent administered alone in four tumor lines. Of interest, the interaction appeared independent of tumor MGMT or mismatch repair phenotype, suggesting that the mechanism of synergy may be independent of O6-methylation by temozolomide. Pharmacokinetic studies indicated no detectable interaction between these two agents. Further, coadministration of CPT-11 appeared to reduce the toxicity of temozolomide in tumor-bearing mice.

Synergy of topotecan in combination with vincristine for treatment of pediatric solid tumor xenografts.

Topotecan and vincristine were evaluated alone or in combination against 13 independent xenografts and 1 vincristine-resistant derivative, representing childhood neuroblastoma (n = 6), rhabdomyosarcoma (n = 5), or brain tumors (n = 3). Topotecan was given by i.v. bolus on a schedule found previously to be optimal. Drug was administered daily for 5 days on 2 consecutive weeks with cycles repeated every 21 days over a period of 8 weeks. Doses of topotecan ranged from 0.16 to 1.5 mg/kg to simulate clinically achievable topotecan lactone plasma systemic exposures. Vincristine was administered i.v. every 7 days at a fixed dose of 1 mg/kg. Given as a single agent, vincristine induced complete responses (CRs) in all mice bearing two rhabdomyosarcomas (Rh28 and Rh30) and some CRs in Rh12-bearing mice (57%) but relatively few CRs (<29%) in other tumors. As a single agent, topotecan induced CR in a low proportion of tumor lines. A dose-response model with a logit link function was used to investigate whether the combination of topotecan and vincristine resulted in greater than expected responses compared with the activity of the agents when administered alone. Only CR was used to evaluate tumor responses. The combination resulted in significantly greater than expected CRs than individual agents in nine tumor lines (four neuroblastoma, three brain tumors, and two rhabdomyosarcomas). Similar event-free (failure) distributions were shown in SJ-GBM2 glioblastoma xenografts, whether vincristine was administered on day 1 or day 5 of each topotecan course. To determine whether the increased antitumor activity with the combination was attributable to a change in drug disposition, extensive pharmacokinetic studies were performed. However, little or no interaction between these two agents was determined. Toxicity of the combination was marked by prolonged thrombocytopenia and decreased hemoglobin. However, approximately 75 and 80% of the maximum tolerated dose of each single agent, topotecan (1.5 mg/kg) or vincristine (1 mg/kg), could be given in combination, resulting in a combination toxicity index of approximately 1.5. These results show that the therapeutic effect of combining topotecan with vincristine was greater than additive in most tumor models of childhood solid tumors, and toxicity data suggest that this can be administered to mice with only moderate reduction in the dose levels for each agent.

Comparison of activation of CPT-11 by rabbit and human carboxylesterases for use in enzyme/prodrug therapy.

Several recent studies have examined the possibility of producing tumor-specific cytotoxicity with various enzyme/ prodrug combinations. The enzymes are targeted to tumor cells either with antibodies (ADEPT, antibody directed enzyme prodrug therapy) or with viruses (VDEPT). The goal of the present study was to identify an appropriate enzyme for use in activating the prodrug 7-ethyl-10-[4-(1-piper-idino)-1-piperidino]carbonyloxycamptothe cin (CPT-11). In this study, we compared the efficiency of CPT-11 metabolism by rabbit and human carboxylesterases in in vitro and in situ assays. Although the rabbit and human enzymes are very similar (81% identical; 86% homologous) and the active site amino acids are 100% identical, the rabbit enzyme was 100-1000-fold more efficient at converting CPT-11 to SN-38 in vitro and was 12-55-fold more efficient in sensitizing transfected cells to CPT-11. In vivo, Rh30 rhabdomyosarcoma cells expressing the rabbit carboxylesterase and grown as xenografts in immune-deprived mice were also more sensitive to CPT-11 than were control xenografts or xenografts expressing the human enzyme. Each of the three types of xenografts regressed when the mice were treated with CPT-11 given i.v. at 2.5 mg of CPT-11/kg/daily for 5 days/week for 2 weeks [(dx5)2] (one cycle of therapy), repeated every 21 days for a total of three cycles. However, following cessation of treatment, recurrent tumors were detected in seven of seven mice bearing control Rh30 xenografts and in two of seven mice bearing Rh30 xenografts that expressed the human enzyme. No tumors recurred in mice bearing xenografts that expressed the rabbit carboxylesterase. We conclude that rabbit carboxylesterase/CPT-11 may be a useful enzyme/prodrug combination.

Effective schedules of exposure of medulloblastoma and rhabdomyosarcoma xenografts to topotecan correlate with in vitro assays.

The camptothecin derivative topotecan has been postulated to mediate its antitumor effect through a drug-induced increase in covalent topoisomerase I-DNA complexes. If this hypothesis is correct, then schedules of exposure to topotecan that maximize the number of topoisomerase I-DNA complexes should produce the greatest cytotoxicity. We identified schedules of exposure to topotecan that maximize levels of complexes in vitro and used these schedules to postulate effective schedules of exposure in vivo in a mouse xenograft model. Unexpectedly, K+-SDS precipitation assays quantitating covalent topoisomerase I-DNA complexes showed that Daoy medulloblastoma and Rh30 rhabdomyosarcoma cells became refractory to drug-induced increases in complexes after an 8-h exposure to 2.5 microM topotecan. In contrast, assays using 10-50 nM topotecan showed that the cells did not become refractory, and more importantly, intermittent exposure to drug increased the level of complexes approximately 2-fold above the maximum level observed after a single drug exposure. The data indicate that continuous exposure to topotecan does not maximize topoisomerase I-DNA complexes and suggest that effective intermittent schedules of exposure to topotecan might be identified. Growth inhibition assays confirmed this hypothesis and showed that growth inhibition by topotecan was extremely schedule dependent in Rh30 cells but not in Daoy cells. Xenograft studies showed that schedules modeled after the in vitro experiments produced complete tumor regressions in mice. Topotecan given daily (0.6-2.2 mg/kg) or every other day (1-3.3 mg/kg) for 2 weeks, repeated every 21 days for three cycles, produced complete regressions of Daoy xenografts; however, daily exposure was required to achieve complete regressions of Rh30 xenografts. We conclude that effective intermittent schedules of exposure to topotecan, based on biochemical parameters, can be identified. The clinical utility of each schedule will depend on the relative antitumor effect compared to the toxic effect on the bone marrow, which usually limits administration of topotecan to patients.

Studies of the efficacy and pharmacology of irinotecan against human colon tumor xenograft models.

Irinotecan, administered i.v. on days 1-5 and 8-12 [(dx5)2 i.v.] has demonstrated significant activity against advanced human tumor xenografts. To explore the feasibility of prolonged oral administration of irinotecan, we compared the efficacy of oral and i.v. irinotecan on the (dx5)2 schedule. We also evaluated oral therapy for 12 consecutive weeks [(dx5)12] at 25 and 50 mg/kg and two consecutive 5-day courses repeated every 21 days for up to four cycles ([(dx5)2]4) at 50 and 75 mg/kg/dose in a series of human colon carcinoma xenograft lines. In addition, we evaluated the effect of a sensitive (HC1) and resistant (ELC2) human colon adenocarcinoma xenograft on irinotecan and SN-38 lactone disposition after administration of irinotecan 10 mg/kg i.v. and 10 and 25 mg/kg p.o. Irinotecan i.v. at 40 mg/kg and oral at 50 and 75 mg/kg on the (dx5)2 schedule had similar activity against the panel of adult colon adenocarcinoma xenografts. Irinotecan given p.o. also demonstrated significant activity against a topotecan-resistant derivative, VRC5/TOPO. Oral administration of 75 mg/kg [(dx5)2]4 and 50 mg/kg (dx5)12 achieved complete response in five of seven xenograft lines evaluated. After i.v. administration, mice bearing HC1 xenografts had 43% greater SN-38 lactone systemic exposure compared to those with ELC2 xenografts and non-tumor-bearing mice. After oral (10 mg/kg) administration, there was a 5-fold higher molar formation of SN-38 lactone compared to i.v. (10 mg/kg) administration in tumor and non-tumor-bearing mice. SN-38 systemic exposure associated with the lowest oral dose (25 mg/kg) achieving complete response for HC1 was 942.6 ng/ml x h. These results emphasize the importance of pharmacokinetic studies as part of tumor response studies in xenograft models.

Altered irinotecan and SN-38 disposition after intravenous and oral administration of irinotecan in mice bearing human neuroblastoma xenografts.

The antitumor activity of irinotecan in vitro primarily results from its hydrolysis by carboxylesterase to the active metabolite SN-38. The present study was conducted to evaluate the effect of human neuroblastoma xenografts on irinotecan and SN-38 disposition after i.v. and oral irinotecan administration. Non-tumor-bearing mice and mice bearing three different human neuroblastoma xenograft lines (NB1691, NB1643, and NBEB) were given irinotecan (10 mg/kg) by short i.v. injection into the tail vein or by oral gavage. Serial plasma samples were obtained, processed to isolate irinotecan and SN-38 lactone, and assayed with a sensitive and specific high-performance liquid chromatography assay. Noncompartmental and compartmental pharmacokinetic analyses were performed. A four-compartment model was used for analysis of irinotecan and SN-38 concentration-time data after i.v. administration. The presence of tumor increased irinotecan systemic exposure (1.2-3.8-fold; P < 0.05) after i.v. and oral administration in mice bearing neuroblastoma xenografts compared to non-tumor-bearing mice. Moreover, SN-38 systemic exposures were higher (1.3-3.8-fold; P < 0.05) in mice bearing human neuroblastoma xenografts as compared to non-tumor-bearing mice, with the greatest effect observed after oral administration of irinotecan. A schematic model is presented to provide a mechanistic basis for our observations. These results emphasize the need to perform preclinical pharmacokinetic studies to evaluate the influence of tumor on drug disposition.

Relationship between topotecan systemic exposure and tumor response in human neuroblastoma xenografts.

BACKGROUND: Topotecan is a topoisomerase I inhibitor with activity against xenografts of childhood solid tumors and established clinical activity against neuroblastoma and rhabdomyosarcoma. We have studied the relationship between systemic exposure to and the antitumor activity of topotecan lactone (the active form of the drug) in the xenograft models. Furthermore, we determined whether the responses seen in these models occur at systemic exposure levels that are tolerable in children. METHODS: Neuroblastoma xenografts derived from the tumors of six different patients were established subcutaneously in immune-deprived mice. Topotecan was administered by intravenous bolus injection 5 days a week for 2 consecutive weeks, repeated every 21 days for three cycles. The minimum daily doses that induced complete responses (CRs) and partial responses (PRs) were determined. Topotecan lactone pharmacokinetic studies were performed in both tumor-bearing and nontumor-bearing mice. RESULTS: The minimum doses associated with CRs and PRs in four of the six neuroblastoma xenografts were 0.61 and 0.36 mg/kg body weight, respectively. The topotecan lactone single-day systemic exposures associated with these doses were 88 and 52 ng x hr/mL, respectively. There was an approximately sixfold difference in topotecan lactone systemic exposure (290 ng x hr/mL versus 52 ng x hr/mL) associated with achieving CRs in the least-sensitive and most-sensitive tumors, respectively. CONCLUSIONS: Neuroblastoma xenografts are highly sensitive to topotecan therapy, and responses in mice are achieved at systemic exposures similar to those that are clinically effective and tolerable in children. These results support the concept of deriving preclinical data relating systemic exposure to antitumor activity in xenograft models. Such data may be valuable in making informed decisions regarding the clinical development of new agents.

Probenecid alters topotecan systemic and renal disposition by inhibiting renal tubular secretion.

Topotecan is primarily eliminated by the kidneys, with 60 to 70% of the dose recovered as topotecan total in the urine. To elucidate the mechanisms of topotecan renal clearance, we evaluated the effect of probenecid on topotecan renal and systemic disposition in mice. Topotecan lactone or hydroxy acid (1.25 mg/kg i.v.) was administered alone or in combination with probenecid (600 or 1,200 mg/kg) given by oral gavage 30 min before and 3 hr after topotecan. Serial blood samples (three mice per time point) and urine samples (five mice per treatment arm) were collected during a 6-hr period. Compared with topotecan alone, coadministration of topotecan lactone or hydroxy acid with probenecid (600 mg/kg) decreased topotecan lactone, total, and hydroxy acid systemic clearance, and total renal clearance. The predominant effect of probenecid was to increase hydroxy acid area under the plasma concentration time curve after administration of topotecan lactone (238.8 vs. 109.9 ng.hr/ml alone, P < .05), or hydroxy acid (1297.2 vs. 355.0 ng.hr/ml alone, P < .05). By inhibiting renal tubular secretion, probenecid decreased renal and systemic clearance which led to an increase in topotecan systemic exposure. These data suggest that probenecid primarily inhibited secretion of the anionic hydroxy acid form, and by direct or indirect mechanisms increased topotecan lactone systemic exposure. Topotecan elimination through renal tubular secretion may have clinical relevance for the use of topotecan in patients with altered renal function.

Randomized trial of endovaginal ultrasound in preterm premature rupture of membranes.

OBJECTIVE: To assess the effect of weekly endovaginal ultrasound on the incidence of maternal infection and the time from rupture to delivery in women with preterm premature rupture of membranes (PROM). METHODS: Women with singleton pregnancies complicated by preterm PROM at 24-34 weeks' gestation were assigned randomly to groups having endovaginal ultrasound or no vaginal sonography. Along with the standard expectant management, the endovaginal-ultrasound group had weekly vaginal probe ultrasound scans. Power analysis based upon expected maternal infection required a sample size of 45 patients in each group. RESULTS: Forty-seven and 45 subjects were assigned to the no-probe and probe groups, respectively. The latency period, defined as days from rupture to delivery, was 9.8 and 11.7 days for the no-probe and probe groups, respectively (95% confidence interval -5.9, 2.1). There were no significant differences in the incidence of chorioamnionitis (28% and 20%), endometritis (6% and 9%), or neonatal infection (17% and 20%). The mean latency period in women who went into spontaneous labor and whose initial cervical length was 3.0 cm or less was 9.4 days, compared with 11.0 days if the cervix exceeded 3.0 cm, a nonsignificant difference. There were three neonatal deaths, all in the probe group and none directly related to infection. CONCLUSIONS: Endovaginal ultrasound in patients whose pregnancies are complicated by preterm PROM does not appear to increase the incidence of maternal infection.