Outcomes from hematopoietic stem cell transplantation following treosulfan-based conditioning: A clinical and pharmacokinetic analysis.

BACKGROUND: The aims of this study are to report our experience with treosulfan-based conditioning regimens for patients with non-malignant hematologic conditions, correlating clinical outcomes at different time points post-transplant with treosulfan exposure (AUC). METHODS: This study was a single-center observational study investigating overall survival (OS), disease-free survival (DFS), and event-free survival (EFS) end-points post-transplant. The consequences of treosulfan AUC with respect to toxicity, correction of underlying disease, and long-term chimerism were also explored using pharmacokinetic analysis. RESULTS: Forty-six patients received 49 transplants with treosulfan and fludarabine-based conditioning between 2005 and 2023. Twenty-four patients also received thiotepa. Donor chimerism was assessed on either whole blood or sorted cell lines at different time points post-transplant. Thirty-nine patients received treosulfan pharmacokinetic assessment to evaluate cumulative AUC, with five infants receiving real-time assessment to facilitate daily dose adjustment. OS, DFS, and EFS were 87%, 81%, and 69%, respectively. Median follow-up was 32.1 months (range 0.82-160 months) following transplant. Lower EFS was associated with patient age (<1 year; p = .057) and lower cumulative treosulfan dose (<42 g/m2; p = .003). Stable donor chimerism in B-cell, NK-cell, and granulocyte lineages at 1-year post-transplant were more prevalent in patients receiving thiotepa conditioning. Two infants required daily dose adjustment to treosulfan to avoid high AUC. CONCLUSIONS: Excellent clinical outcomes and stable chimerism were observed in this patient series. The addition of thiotepa conferred no significant toxicity and trended toward sustained ongoing donor engraftment. Correlating treosulfan AUC with long-term patient outcomes is required.

Polymorphisms of drug-metabolizing enzymes (GST, CYP2B6 and CYP3A) affect the pharmacokinetics of thiotepa and tepa.

AIMS: Thiotepa is widely used in high-dose chemotherapy. Previous studies have shown relations between exposure and severe organ toxicity. Thiotepa is metabolized by cytochrome P450 and glutathione S-transferase enzymes. Polymorphisms of these enzymes may affect elimination of thiotepa and tepa, its main metabolite. The purpose of this study was to evaluate effects of known allelic variants in CYP2B6, CYP3A4, CYP3A5, GSTA1 and GSTP1 genes on pharmacokinetics of thiotepa and tepa. METHODS: White patients (n = 124) received a high-dose regimen consisting of cyclophosphamide, thiotepa and carboplatin as intravenous infusions. Genomic DNA was analysed using polymerase chain reaction and sequencing. Plasma concentrations of thiotepa and tepa were determined using validated GC and LC-MS/MS methods. Relations between allelic variants and elimination pharmacokinetic parameters were evaluated using nonlinear mixed effects modelling (nonmem). RESULTS: The polymorphisms CYP2B6 C1459T, CYP3A4*1B, CYP3A5*3, GSTA1 (C-69T, G-52A) and GSTP1 C341T had a significant effect on clearance of thiotepa or tepa. Although significant, most effects were generally not large. Clearance of thiotepa and tepa was predominantly affected by GSTP1 C341T polymorphism, which had a frequency of 9.3%. This polymorphism increased non-inducible thiotepa clearance by 52% [95% confidence interval (CI) 41, 64, P < 0.001] and decreased tepa clearance by 32% (95% CI 29, 35, P < 0.001) in heterozygous patients, which resulted in an increase in combined exposure to thiotepa and tepa of 45% in homozygous patients. CONCLUSIONS: This study indicates that the presently evaluated variant alleles explain only a small part of the substantial interindividual variability in thiotepa and tepa pharmacokinetics. Patients homozygous for the GSTP1 C341T allele may have enhanced exposure to thiotepa and tepa.

Pharmacokinetics of thiotepa in high-dose regimens for autologous hematopoietic stem cell transplant in Japanese patients with pediatric tumors or adult lymphoma.

PURPOSE: Thiotepa is used in high-dose chemotherapy (HDT) before autologous hematopoietic stem cell transplantation (HSCT) to treat solid tumors and hematological malignancies. This Phase 1 study was conducted to establish the pharmacokinetics (PK) of thiotepa in a Japanese population. METHODS: HDT/HSCT was performed in pediatric patients (≥ 2 years) with solid tumors or brain tumors (thiotepa 200 mg/m2/day IV-infused over 24 h on HSCT Days - 12, - 11, - 5, and - 4 and melphalan 70 mg/m2/day IV-infused over 1 h on Days - 11, - 5, and - 4) and adult patients (≥ 16 years) with malignant lymphoma (thiotepa 200 mg/m2/day 2-h IV-infusion on HSCT Days - 4 and - 3 plus busulfan 0.8 mg/kg 2-h IV-infusion every 6 h from HSCT Days - 8 to - 5). Pharmacokinetics of thiotepa were assessed following initial dose. Safety and efficacy were also evaluated. RESULTS: Nine pediatric and 10 adult patients were enrolled. Mean volume of distribution (Vz) of thiotepa normalized with body surface area (BSA) was lower for pediatric patients (16.4 L/m2) compared with adult patients (26.4 L/m2) as expected due to the higher specific surface area of children. Clearance and biological half-life were similar between pediatric and adult patients. Two serious adverse events (cardiac arrest and pulmonary edema) were observed. Survival rate (Day 100 post-HSCT) was 77.8% (95% CI 36.5-93.9%) for pediatric patients and 100% for adult patients. CONCLUSION: Thiotepa elimination was comparable in pediatric and adult patients with cancer. Lower Vz in pediatric compared with adult patients was expected. HDT with thiotepa prior to autologous HSCT was well tolerated. STUDY REGISTRATION: Japic CTI-163433.

Accuracy, feasibility, and clinical impact of prospective Bayesian pharmacokinetically guided dosing of cyclophosphamide, thiotepa, and carboplatin in high-dose chemotherapy.

PURPOSE: Relationships between toxicity and pharmacokinetics have been shown for cyclophosphamide, thiotepa, and carboplatin (CTC) in high-dose chemotherapy. We prospectively evaluated whether variability in exposure to CTC and their activated metabolites can be decreased with pharmacokinetically guided dose administration and evaluated its clinical effect. EXPERIMENTAL DESIGN: Patients received multiple 4-day courses of cyclophosphamide (1,000-1,500 mg/m2/d), thiotepa (80-120 mg/m2/d), and carbop latin (area under the plasma concentration-time curve 3.3-5 mg x min/mL/d). Doses were adapted on day 3 based on pharmacokinetic analyses of cyclophosphamide, 4-hydroxycyclophosphamide, thiotepa, tepa, and carboplatin done on day 1 using a Bayesian algorithm. Doses were also adjusted before and during second and third courses. Observed toxicity was compared with that in patients receiving standard dose CTC (n = 43). RESULTS: A total of 46 patients (108 courses) were included. For cyclophosphamide, thiotepa, and carboplatin, a total of 39, 58, and 65 dose adaptations were done within courses and 17, 40, and 43 before courses. The precision within which the target exposure was reached improved compared with no adaptation, especially after within-course adaptations (precision for cyclophosphamide, thiotepa, and carboplatin is 19%, 16%, and 13%, respectively); >85% led to an exposure within +/-25% of the target compared with 60% without dose adjustments. Toxicity was similar to that in a reference population, although the incidence of veno-occlusive disease was reduced. CONCLUSIONS: Bayesian pharmacokinetically guided dosing for CTC was feasible and led to a marked reduction in variability of exposure.

Cyclophosphamide and thiotepa with autologous bone marrow transplantation in patients with solid tumors.

Autologous bone marrow transplantation to avoid dose-limiting myelosuppression may allow significant drug dose escalations and exploitation of the linear-log correlation between chemotherapy and tumor cytotoxicity. In a phase I trial of cyclophosphamide and thiotepa (with subsequent addition of melphalan), 23 patients were entered; there were two deaths due to toxic effects. The maximum tolerated dose was 6 g of cyclophosphamide/m2 and 720 mg of thiotepa/m2. No significant dose of melphalan could be added. Stomatitis was dose limiting. Eleven of 20 patients who were able to be evaluated responded. Plasma thiotepa concentrations correlated more closely with toxicity and response than with drug dose level. Continuous infusion cyclophosphamide and thiotepa is an active core regimen for the further design of high-dose combination chemotherapy regimens.

Characterization of nonexchangeable radioactivity in L1210 cells incubated with [14C]thiotepa: labeling of phosphatidylethanolamine.

N,N',N''-Triethylenethiophosphoramide ([14C]thiotepa) accumulation by L1210 cells is a biphasic process. A very rapid initial phase is followed by a much slower second phase that reflects accumulation of radioactivity in a form that is not lost or exchanged when cells are resuspended and incubated in drug-free medium for up to 8 h. In this study we attempted to characterize this nonexchangeable radioactivity. Nuclei (10(7)) isolated from L1210 cells and incubated with [14C]thiotepa did not accumulate 14C during incubations of up to 5 h. Similarly, nuclei isolated from 10(7) L1210 cells that had been shown to accumulate nonexchangeable 14C after incubation with [14C]thiotepa did not show an increase in nuclear-associated 14C. Eighty to 85% of nonexchangeable 14C in L1210 cells incubated with [14C]thiotepa was soluble in ethanol or chloroform:methanol (2:1, v/v), and although most of this cell-associated nonexchangeable 14C was precipitated by trichloroacetic acid, subsequent treatment of that precipitate with methanol solubilized most of the 14C so that only 15 to 20% remained with the final precipitate. When chloroform:methanol-soluble nonexchangeable 14C was analyzed with thin-layer chromatography systems suitable for thiotepa or simple lipids, all radioactivity remained at the origin. In contrast, when analyzed with one- and two-dimensional thin-layer chromatographic systems suitable for complex lipids, all chloroform:methanol-soluble radioactivity was associated with a single lipid spot. This lipid cochromatographed with phosphatidylethanolamine, reacted with ninhydrin but not with 4-(p-nitrobenzyl)pyridine or the Dragendorff choline reagent, and was digested by phospholipases C and D, all of which lead to its identification as phosphatidylethanolamine. This extensive labeling of phosphatidylethanolamine in L1210 cells incubated with [14C]thiotepa can be explained by liberation of [14C]aziridine from [14C]thiotepa, hydrolysis of the [14C]aziridine to [14C]ethanolamine, and incorporation of that radiolabeled material into phosphatidylethanolamine via the normal cellular synthetic pathways for that lipid. This information implies that thiotepa serves, at least in part, as a prodrug for aziridine and has implications as to the mechanism of thiotepa-induced cytotoxicity in that aziridine is a monofunctional alkylating agent incapable of producing interstrand, intrastrand, or protein-DNA cross-links.

N,N',N''-triethylenethiophosphoramide (thio-TEPA) oxygenation by constitutive hepatic P450 enzymes and modulation of drug metabolism and clearance in vivo by P450-inducing agents.

The cancer chemotherapeutic drug N,N',N''-triethylenephosphoramide (thio-TEPA) is oxidatively desulfurated to yield the active metabolite N,N',N''-triethylenephosphoramide (TEPA) in a reaction catalyzed by the phenobarbital-inducible rat liver P450 enzyme IIB1. In the current study, the role of constitutively expressed P450 enzymes in thio-TEPA metabolism was studied using purified P450s, isolated liver microsomes, and intact rats. Metabolism of thio-TEPA (100 microM) to TEPA by uninduced adult female and male rat liver microsomes proceeded at initial rates of 0.10 and 0.28 nmol TEPA formed/min/mg microsomal protein, respectively. Although these rates are low compared to those catalyzed by phenobarbital-induced liver microsomes (3.5 nmol TEPA/min/mg), they are sufficient to contribute to the systemic metabolism of this drug. Thio-TEPA metabolism catalyzed by uninduced female liver microsomes was approximately 70% inhibitable by antibodies selectively reactive with P450 IIC6. For the uninduced male liver microsomes, which exhibit a severalfold higher rate of thio-TEPA metabolism, enzyme activity was only 15-20% inhibitable by these antibodies but was 80-85% inhibited by an anti-P450 IIC6 monoclonal antibody cross-reactive with P450 IIC11, which is expressed only in the males. Consistent with these observations, purified P450s IIC11 and IIC6 both oxidized thio-TEPA in reconstituted systems (turnover, 1.1 and 0.3 min-1 P450-1, respectively, at 100 microM substrate), while several other constitutive hepatic P450s exhibited significantly lower or undetectable activities (turnover, less than or equal to 0.15 min-1 P450-1). Metabolism of thio-TEPA by purified P450 IIC11 was associated with a time-dependent inactivation of the cytochrome analogous to that previously shown to accompany thio-TEPA metabolism catalyzed by P450 IIB1. Depletion of hepatic P450 IIC11 by cisplatin treatment of adult male rats led to a 70% reduction of TEPA formation catalyzed by the isolated liver microsomes, suggesting that cisplatin may influence thio-TEPA pharmacokinetics when these two drugs are given in combination. The extent to which hepatic P450s contribute to thio-TEPA metabolism and clearance in vivo was assessed by monitoring thio-TEPA and TEPA pharmacokinetics in rats that exhibit widely differing rates of microsomal thio-TEPA metabolism, i.e., uninduced female and male rats, and male rats treated with the P450 IIB1 inducers clofibrate and phenobarbital. In accord with the microsomal activities, conversion of thio-TEPA to TEPA was less extensive and thio-TEPA elimination slower in female than in male rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Phase I and pharmacokinetic evaluation of thiotepa in the cerebrospinal fluid and plasma of pediatric patients: evidence for dose-dependent plasma clearance of thiotepa.

A Phase I trial of thiotepa (TT) administered as an i.v. bolus was performed in 19 children with refractory malignancies. The starting dose was 25 mg/m2 with escalations to 50, 65, and 75 mg/m2. Seven additional patients were treated with 8-h infusions at 50 or 65 mg/m2. The maximum tolerated bolus dose was 65 mg/m2. Reversible myelosuppression was the dose-limiting toxicity. The plasma and cerebrospinal fluid (CSF) pharmacokinetic parameters of TT and its major active metabolite tepa (TP) were also evaluated. When the bolus or infusion methods of TT administration were compared, there was little difference observed in any pharmacokinetic parameter for either TT or TP. The plasma disappearance of TT was rapid and biphasic with half-lives of 0.14 to 0.32 and 1.34 to 2.0 h. Dose-dependent pharmacokinetics was demonstrated by steadily declining plasma clearance with increasing TT dose. Clearance values declined from 28.6 liters/m2/h at the 25-mg/m2 dose to 11.9 liters/m2/h at the 75-mg/m2 dose. The half-life of TP was longer than that of TT and ranged between 4.3 and 5.6 h. There was evidence of the saturation of TP production. TT and TP both exhibited excellent penetration into the CSF, producing lumbar and ventricular concentrations which were nearly identical to simultaneous plasma concentrations. In one patient with a Rickham reservoir, the CSF:plasma area under the (concentration x time) curve ratios for TT and TP were 1.01 and 0.95, respectively. The above data indicate that TT can be safely administered to pediatric patients at doses higher than conventionally used. The favorable CSF penetration of TT and TP suggests that Phase II studies of TT be considered in patients with central nervous system tumors.

Phase I study of thiotepa in combination with the glutathione transferase inhibitor ethacrynic acid.

The glutathione transferases comprise a family of isoenzymes, one or more of which are involved in the conjugation of alkylating agents to glutathione (GSH). Increased GSH transferase activity has been shown to underlie acquired resistance to several alkylating agents. Ethacrynic acid inhibits the isoenzymes of GSH transferase with 50% inhibitory concentration values ranging from 0.3 to 6.0 microM and has been shown to restore sensitivity to alkylating agents in drug-resistant animal tumor models. We entered 27 previously treated patients with advanced cancer on a study of ethacrynic acid (25 to 75 mg/m2 p.o. every 6 h for 3 doses) and thiotepa (30 to 55 mg/m2 i.v. 1 h after the second dose of ethacrynic acid). The major toxicity of ethacrynic acid was diuresis, which was observed at every dose level; in addition, severe metabolic abnormalities occurred at 75 mg/m2. At 50 mg/m2, the diuretic effects were manageable. Myelosuppression was the most important effect of the combination. Two of seven courses of ethacrynic acid, 50 mg/m2, and thiotepa, 55 mg/m2, were associated with grade 3 or 4 neutropenia and/or thrombocytopenia. Nausea/vomiting greater than or equal to grade 2 was observed in 16% of courses. GSH transferase activity was assayed spectrophotometrically in the peripheral mononuclear cells of all patients. At each dose level, activity decreased following ethacrynic acid administration, with recovery by 6 h. Administration of ethacrynic acid, 50 mg/m2, resulted in a mean nadir of transferase activity of 37% of control. The pharmacokinetics of thiotepa and its principal metabolite TEPA were studied in 23 patients. The plasma disappearance of thiotepa fit a two-compartment open model with a terminal half-life of approximately 2 h. Plasma TEPA levels peaked at a mean of 2.16 h following thiotepa administration. The harmonic mean terminal half-life of TEPA was 10.4 h, and the TEPA area under the curve (AUC) did not increase with increasing thiotepa dose. The AUC of thiotepa was approximately twice, and the clearance about one-half, of the values obtained in a previous study of single agent thiotepa. The AUC of TEPA was lower than that previously observed. The data suggest that ethacrynic acid inhibits enzymes involved in the metabolic disposition of thiotepa, including its oxidative desulfuration to TEPA. The severity of the platelet toxicity was correlated with the AUC of thiotepa, but not with that of TEPA. This combination of thiotepa and ethacrynic acid will be tested further in Phase II trials.

A search for new metabolites of N,N',N''-triethylenethiophosphoramide.

An attempt was made to unravel the metabolic profile of the alkylating agent N,N',N''-triethylenethiophosphoramide (thioTEPA). thioTEPA and its metabolite N,N',N-triethylenephosphoramide (TEPA) were quantified in urine of treated patients by gas chromatography with selective nitrogen/phosphorous detection. Total alkylating activity was assessed by p-nitrobenzylpyridine reactivity. The total alkylating activity exceeded the amount of thioTEPA and TEPA, indicating the presence of other alkylating metabolites. Solid-phase extraction and liquid-liquid extractions followed by gas chromatography-mass spectrometry analysis revealed the conversion of an aziridinyl function of TEPA into a beta-chloroethyl moiety. This metabolite, N,N'-diethylene-N''-2-chloroethylphosphoramide, was quantified by gas chromatography with selective nitrogen/phosphorous detection and accounted for only 0.69% of the administered dose. Large volumes of urine were concentrated with solid-phase extraction and fractionated with high-performance liquid chromatography. Alkylating activity was determined for each 2-ml fraction and showed the presence of an alkylating compound eluting between 8 and 12 ml. The fractions with alkylating activity were collected, evaporated under a stream of nitrogen at room temperature to dryness, reconstituted in methanol, and subjected to fast atom bombardment-mass spectrometry and fast atom bombardment-tandem mass spectrometry. A new metabolite was found with a molecular mass of 352 Da, the same as that of thioTEPA-mercapturate. thioTEPA-mercapturate is likely the result of glutathione conjugation, after which the glutathione adduct loses two amino acid residues in separate stages. The fragmentation pattern and chromatographic properties of this new metabolite were identical to those of the reference, thioTEPA-mercapturate, which was obtained by incubation of thioTEPA with N-acetylcysteine at pH 11 and 95 degrees C for 30 min. Quantification of thioTEPA-mercapturate was carried out by liquid chromatography-mass spectrometry. The thioTEPA-mercapturate levels in urine accounted for 12.3% of the administered dose and exceeded the amount of TEPA, which was previously assumed to be the main metabolite of thioTEPA. The total excreted amount of thioTEPA and its metabolites accounts for 54-100% of the total alkylating activity, indicating the presence of still other alkylating metabolites.

Phase I/pharmacokinetic reevaluation of thioTEPA.

Because the initial evaluation of N,N',N''-triethylenethiophosphoramide (thioTEPA) preceded the standardized approach to the Phase I trials, uncertainty surrounds the recommended dose. Since it has recently been demonstrated that an almost 100-fold increase in dose can be administered in bone marrow transplant regimens, we conducted a Phase I reevaluation of thioTEPA. ThioTEPA was administered i.v. in 50 ml 5% dextrose in water over 10 min. Twenty-seven patients were entered at doses ranging from 30 to 75 mg/m2. The major toxic effect was myelosuppression; thrombocytopenia greater than or equal to grade 3 occurred in four of seven patients, and leukopenia greater than or equal to grade 3 in two of seven patients at 75 mg/m2. Among eight patients at 65 mg/m2 only two had greater than or equal to grade 3 myelosuppression making this the recommended new phase II dose for the majority of patients. Moderate (grade 2) easily controlled nausea and vomiting was the only other major side effect. There was no alopecia or mucosal or neurological toxicity. Three partial remissions were observed among nine previously treated ovarian cancer patients. Plasma concentrations of thioTEPA and its major active metabolite triethylenephosphoramide (TEPA) were measured by gas chromatography. The half-life of thioTEPA ranged from 51.6 to 211.8 min, and its pharmacokinetics was dose dependent; total body thioTEPA clearance decreased with increasing dose. The half-life of TEPA was considerably longer than that of the parent compound (3.0 to 21.1 h); as a result, the area under the plasma concentration-time curve (AUC) of TEPA was severalfold greater than that of the parent compound. The ratio of TEPA AUC to thioTEPA AUC decreased with increasing dose, suggesting that formation of TEPA is a saturable step in elimination. The AUC and total body clearance of thioTEPA, but not of TEPA, were closely correlated with neutrophil but not platelet toxicity.

Human plasma pharmacokinetics of thiotepa following administration of high-dose thiotepa and cyclophosphamide.

Thiotepa is an established alkylating agent whose pharmacokinetics in standard doses are well defined. In order to ascertain whether dose-dependent variations in pharmacokinetics occur, we have undertaken an analysis of plasma thiotepa levels in 16 patients entered on a phase I-II study of bialkylator chemotherapy. High-dose thiotepa (1.8 to 7.0 mg/kg) and cyclophosphamide (2.5 g/m2) were administered intravenously (IV) on days -6, -4, and -2 followed by autologous marrow reinfusion on day 0. Plasma and urinary thiotepa was assayed by gas chromatography. Biexponential plasma decay curves were seen in ten patients, with a t 1/2 alpha of 10.0 +/- 6.4 minutes, a t 1/2 beta of 174 +/- 61 minutes and a total body clearance of 379 +/- 153 mL/h/kg (mean +/- SD). Six patients displayed monoexponential plasma decay curves with a terminal t 1/2 of 137 +/- 83 minutes and a total body clearance of 440 +/- 195 mL/h/kg. Although there was a trend toward reduced plasma clearance in the three patients treated at the highest dose level, the available data suggest that metabolic clearance mechanisms for thiotepa were not saturated with the doses used in this study. By stepwise regression analysis, linear functions using only 15-minute and four-hour postinfusion plasma levels were derived that correlated closely with area under the plasma concentration X time curves (AUC) (P less than .002). We conclude that high-dose thiotepa results in similar pharmacokinetic values to conventional doses with no apparent dose-dependent variation. The value of specific time points to predict AUC and clearance will require prospective evaluation.

Phase I clinical and pharmacokinetic study of thiotepa administered intraperitoneally in patients with advanced malignancies.

An important subset of malignancies arising in the ovary or digestive organs remains confined to the peritoneal cavity throughout its natural course. These tumors constitute appropriate targets for loco-regional therapy. With this rationale a clinical phase I and pharmacokinetic study of intraperitoneally administered N, N', N'' triethylenethiophosphoramide (thiotepa), an alkylating agent with activity against ovarian carcinoma, was initiated with the objectives of determining the systemic and local toxicities, maximum-tolerated dose, and pharmacokinetic advantage associated with using the drug in this manner. A total of 13 patients received 15 courses of intraperitoneal thiotepa at doses ranging from 30 mg/m2 to 60 mg/m2. The only important systemic toxicity observed was myelosuppression. At 50 mg/m2 two patients developed Eastern Cooperative Oncology Group (ECOG) grade III myelosuppression. At 60 mg/m2, the maximum-tolerated dose, the mean nadir WBC and platelet counts were 2.7 X 10(3)/microliter and 110 X 10(3)/microliter, respectively. There were no instances of vomiting, stomatitis, or alopecia. Pharmacokinetic studies performed in nine patients revealed that thiotepa was rapidly lost from the peritoneal cavity in a biexponential fashion with a mean t1/2 alpha of 0.26 +/- 0.08 hour and a mean t1/2 beta of 2.13 +/- 0.52 hour. Concomitant with the rapid loss of drug from the peritoneal cavity was the rapid rise in drug levels in the plasma, with peak plasma values approaching those associated with intravenous administration. Peritoneal exposure to thiotepa expressed as the area under the curve (AUC)peritoneal fluid was 7 to 34 micrograms/mL X hour. Systemic exposure expressed as the AUCplasma ranged between 0.95 and 7.71 micrograms/mL X hour. The observed pharmacokinetic advantage of intraperitoneal administration calculated as AUCperitoneal fluid/AUCplasma was 4.3 +/- 0.6. This relatively small advantage, combined with our observation of rapid appearance of the active metabolite, tepa, into the plasma argue against an important role for intraperitoneal administration of thiotepa.

Phase I trial of thiotepa in combination with recombinant human granulocyte-macrophage colony-stimulating factor.

PURPOSE: The ability of growth factors to stimulate marrow recovery suggests their potential for use in dose intensification of cytotoxic drugs. We performed a phase I study of the alkylating agent thiotepa in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), with the goal of dose-escalation of thiotepa. Thiotepa was selected based on its capacity for dose escalation to more than 1 g/m2 in the marrow transplantation setting. PATIENTS AND METHODS: The starting dose of thiotepa (75 mg/m2) was the highest dose evaluated in our previous phase I trial. Thirteen patients received 22 courses of thiotepa and GM-CSF. The dose of GM-CSF was 10 micrograms/kg subcutaneously daily in six patients and 5 micrograms/kg in seven patients. RESULTS: Three patients (23%) developed grade 3 to 4 neutropenia on the first course, with a recovery to more than 1000/mm3 in 4.7 days (mean). Recovery was as rapid with the 5 micrograms/kg as it was with the 10 micrograms/kg GM-CSF dose. Thrombocytopenia grade 3 to 4 affected seven of 13 (54%) patients in the first course; counts recovered to more than 50,000/mm3 in a median of 15 days. GM-CSF at either dose did not influence markedly the severity or duration of thrombocytopenia, and did not permit dose escalation of thiotepa. Among the seven patients who received a second cycle of treatment, six of seven experienced grade 3 or 4 thrombocytopenia that lasted a median of 15.5 days. Five had thrombocytopenia that lasted more than 35 days after one to three cycles of treatment. Plasma concentrations of thiotepa and tepa were measured by gas chromatography in eight patients. The plasma elimination of thiotepa fit a two-compartment open model with a harmonic mean terminal half-life of 2.44 hours. The mean total body clearance was 217.9 mL/min/m2, and the mean steady-state volume of distribution (Vdss) was 36.8 L/m2. The half-life of tepa was 7.98 hours, and the ratio of the area under the plasma concentration versus time curve (AUC) of tepa to that of thiotepa was 0.79. CONCLUSIONS: These data were consistent with our previous observations at this dose, and indicated that the severity of toxicity in these patients was not explained by aberrant pharmacokinetic indices. We conclude that, independent of effects on neutropenia, severe and cumulative platelet toxicity precludes further escalation of thiotepa dose despite the use of GM-CSF.

Aprepitant inhibits cyclophosphamide bioactivation and thiotepa metabolism.

BACKGROUND: Patients receiving the highly emetogenic high-dose chemotherapy regimen with cyclophosphamide, thiotepa and carboplatin (CTC) may benefit from the neurokin-1 receptor antagonist aprepitant in addition to standard anti-emetic therapy. As aprepitant has been shown to be a moderate inhibitor of the cytochrome P450 (CYP) 3A4 isoenzyme, its effect on the pharmacokinetics and metabolism of cyclophosphamide and thiotepa was evaluated. Moreover, preliminary results on the clinical efficacy of aprepitant in the CTC regimen are reported. PATIENTS AND METHODS: Six patients were enrolled in a protocol that employed a 4-day course of CTC high-dose chemotherapy with cyclophosphamide (1,500 mg/m2/day), thiotepa (120 mg/m2/day) and carboplatin (AUC 5 mg min/ml/day). Two patients received the tCTC protocol, which comprises two-third of the dose of CTC. In addition to standard anti-emetic therapy, the patients received aprepitant from one day before the start of their course until 3 days after chemotherapy. Blood samples were collected on days one and three of the course and analyzed for cyclophosphamide and its activated metabolite 4-hydroxycyclophosphamide, thiotepa and its main active metabolite tepa. The influence of aprepitant on the pharmacokinetics of cyclophosphamide and thiotepa was analyzed using a population pharmacokinetic analysis including a reference population of 49 patients receiving the same chemotherapy regimen without aprepitant and sampled under the same conditions. The frequency of nausea and vomiting in the six patients receiving CTC was compared with those of the last 22 consecutive patients receiving CTC chemotherapy without aprepitant. Inhibitory activity of aprepitant on cyclophosphamide and thiotepa metabolism was also tested in human liver microsomes. RESULTS: In our patient population, the rate of autoinduction of cyclophosphamide (P=0.040) and the formation clearance of tepa (P<0.001) were reduced with 23% and 33% when aprepitant was co-administered, respectively. Exposures to the active metabolite 4-hydroxycyclophosphamide and tepa were therefore reduced (5% and 20%, respectively) in the presence of aprepitant. In human liver microsomes, the 50% inhibitory concentrations (IC50) of aprepitant for inhibition of cyclophosphamide (IC50=1.3 microg/ml) and thiotepa (IC50=0.27 microg/ml) metabolism were within the therapeutic range. Patients receiving aprepitant experienced less frequently CINV both during and after the CTC course compared with the reference population (nausea 3.7 days vs. 5.8 days, P=0.052; vomiting 0.5 days vs. 4.8 days, P<0.001). CONCLUSION: Aprepitant inhibited both cyclophosphamide and thiotepa metabolism, most probably due to inhibition of the CYP 3A4 and/or 2B6 isoenzymes. The effects of this interaction are, however, small compared to the total variability. Addition of aprepitant may provide superior protection against vomiting in patients receiving the highly emetogenic high-dose CTC chemotherapy.

Dose escalation of N,N',N"-triethylenethiophosphoramide combined with pentoxifylline for advanced breast cancer.

Pentoxifylline potentiates the cytotoxicity of alkylating agents in preclinical models. In this study we sought to define the maximum tolerated dose (MTD) of N,N',N"-triethylenethiophosphoramide (thioTEPA) with pentoxifylline, and to estimate the antitumor response to this combination in previously treated breast cancer patients. Thirty-five previously treated advanced breast cancer patients received 70 cycles (median, 2 cycles/patient; range, 1-6) of 1600 mg of oral sustained release pentoxifylline every 8 h for 4 doses in combination with escalating doses of i.v. bolus thioTEPA 40-65 mg/m2 administered 3 h after the second dose of pentoxifylline. Thrombocytopenia was dose limiting at 65 mg/m2 of thioTEPA, and the MTD was defined as 60 mg/m2. Among 25 patients treated at the MTD, leukopenia was grade 2 in 9 patients (36%), grade 3 in 4 patients (16%), and grade 4 in 2 patients (8%); thrombocytopenia was grade 2 in 3 patients (12%), grade 3 in 4 patients (16%), and grade 4 in 3 patients (12%). No other thioTEPA-related toxicity was observed. Plasma concentrations of thioTEPA, TEPA, and pentoxifylline were measured in 6 patients. The median (range) area under the plasma concentration versus time curve for thioTEPA was 29.4 microM/h (26. 2-40.5) and for TEPA was 16.3 microM/h (9.2-21.7 microM-h). The median (range) maximal plasma concentration of pentoxifylline and major metabolites I, IV, and V were 1.2 microgram/ml (0.2-7.8), 4.0 microgram/ml (0.5-16.4), 0.4 (range 0.1-0.8), and 2.9 (1.1-5.5), respectively. No objective responses were observed among 21 evaluable patients treated at the MTD (95% confidence interval, 0-15%). The combination of pentoxifylline and thioTEPA is well tolerated but not active in previously treated advanced breast cancer patients. Further clinical trials using commercially available oral sustained release pentoxifylline as a modulator of alkylating agents are not warranted.

The extent of chromosomal aberrations induced by chemotherapy in non-human primates depends on the schedule of administration.

We utilized a non-human primate model, the rhesus monkey (Macaca mulatta), to quantitate the extent of chromosomal damage in bone marrow cells following chemotherapy. Thiotepa, etoposide, and paclitaxel were chosen as the chemotherapy agents due to their distinct mechanisms of action. Chromosomal aberrations were quantitated using traditional Giemsa stain. We sought to evaluate the extent to which genotoxicity was dependent on the schedule of administration by giving chemotherapy as either a bolus or a 96 h continuous infusion. Neutropenia and areas under the concentration curve (AUCs) were monitored to ensure comparable cytotoxicity and dose administered. At least 100 metaphases were scored in each marrow sample by an investigator unaware of the treatment history of the animals. All three drugs produced a statistically significant higher percentage of abnormal metaphases following bolus chemotherapy (p<0.0001, p=0.0015 and p<0.0001 for thiotepa, etoposide and paclitaxel, respectively). We conclude that infusional administration of thiotepa, etoposide and paclitaxel is less genotoxic to normal bone marrow cells than is bolus administration. These results suggest infusional regimens may be considered where there are concerns about long-term genotoxic sequelae, including secondary cancer, teratogenicity, or possibly the development of drug resistance. We believe this approach provides a reproducible model in which drugs and eventually, regimens can be compared.

Validation of techniques for the prediction of carboplatin exposure: application of Bayesian methods.

OBJECTIVE: Several methods have been developed for the prediction of carboplatin exposure to facilitate pharmacokinetic guided dosing. The aim of this study was to develop and validate sparse data Bayesian methods for the estimation of carboplatin exposure and to validate other commonly applied techniques, such as the Chatelut formula, the Sorensen limited sampling model, and the Calvert formula, in which glomerular filtration rate was estimated with the Cockcroft-Gault, the Jelliffe, and the recently proposed Wright formulas. METHODS: Complete concentration-time curves were available for a total of 43 patients (45 courses) receiving carboplatin (265 or 400 mg/m2/day) in a 1-hour infusion for 4 consecutive days in combination with thiotepa and cyclophosphamide. A population two-compartment model was developed on an index set of 12 courses. The other 33 courses served as validation set. Bayesian estimates were generated with the population parameters by use of either one or two randomly timed samples or two samples at optimal time points determined with the D-optimality theory. RESULTS: The Bayesian methods provided an accurate and precise prediction of the area under the concentration-time curve (bias <4% and precision <18%). The other formulas (Sorensen model, Chatelut, and Calvert with Jelliffe, Cockcroft-Gault, and Wright) resulted in a precision >18%, whereas the Jelliffe formula and the Sorensen model resulted in a bias >12%. CONCLUSION: The applicability of a Bayesian method for the prediction of the carboplatin exposure by use of one or two samples without the necessity for exact timing of infusion duration and sampling was demonstrated. The Bayesian method may be very instrumental to execute pharmacokinetic guided dosing for carboplatin.

Cellular transport and accumulation of thiotepa.

Because the transport and accumulation of N,N',N''-triethylenethiophosphoramide (thiotepa) by cells has not been characterized, these processes were investigated with [14C]thiotepa and cultured L1210 or freshly obtained human or avian RBCs. The octanol: phosphate-buffered saline (PBS) partition coefficient of thiotepa was 2.4 +/- 0.1 (n = 8). With this value, the permeability coefficient (Ps) for thiotepa was estimated to be between 2.8 x 10(-4) and 1.81 x 10(-3) cm/sec, and the half-life of accumulation of thiotepa by L1210 cells was estimated to be 0.063 to 0.40 seconds. Thiotepa accumulation by cells was measured after incubation of cells with [14C]thiotepa and subsequent harvesting of cells by centrifugation through silicone fluid. Thiotepa accumulation by L1210 cells was biphasic. The initial phase was rapid and essentially complete by 10 seconds. The amount of cell-associated 14C increased linearly with increasing extracellular concentrations of thiotepa or with increasing size of the cell pellet. The absolute amount of cell-associated 14C was consistent with that expected if the [14C]thiotepa had been evenly distributed in the incubation medium and a volume equal to that of the cell pellet had been sampled and counted. This rapid phase of thiotepa accumulation was not slowed when cells were incubated on ice. The second phase of [14C]thiotepa accumulation occurred at a rate much slower than that of the initial phase. This slower phase of drug accumulation was linear for at least 5 hours. The rate of 14C accumulation increased progressively over a range of extracellular thiotepa concentrations from 5 to 100 nmol/mL and could not be saturated under acceptable tissue culture conditions. The slower rate of 14C accumulation was ablated by incubating cells on ice and was reduced by 30% to 50% in the presence of 1mM of sodium azide or 2,4-dinitrophenol. The slow rate of accumulation of 14C reflected summation of a relatively stable or constant amount of exchangeable 14C and an amount of nonexchangeable 14C that increased linearly from almost undetectable levels at the start of the experiment to amounts equal to 64 +/- 11% of total cellular radioactivity after 5 hours. The initial association of [14C]thiotepa with both human and avian RBCs was also very rapid. Avian RBCs also exhibited a slow rate of 14C accumulation that was linear for at least 5 hours but that was 15% to 20% that of L1210 cells.(ABSTRACT TRUNCATED AT 400 WORDS)