Real-time dose adjustment of cyclophosphamide in a preparative regimen for hematopoietic cell transplant: a Bayesian pharmacokinetic approach.

PURPOSE: Dose-related toxicity of cyclophosphamide may be reduced and therapeutic efficacy may be improved by pharmacokinetic sampling and dose adjustment to achieve a target area under the curve (AUC) for two of its metabolites, hydroxycyclophosphamide (HCY) and carboxyethylphosphoramide mustard (CEPM). To facilitate real-time dose adjustment, we developed open-source code within the statistical software R that incorporates individual data into a population pharmacokinetic model. EXPERIMENTAL DESIGN: Dosage prediction performance was compared to that obtained with nonlinear mixed-effects modeling using NONMEM in 20 cancer patients receiving cyclophosphamide. Bayesian estimation of individual pharmacokinetic parameters was accomplished from limited (i.e., five samples over 0-16 hours) sampling of plasma HCY and CEPM after the initial cyclophosphamide dose. Conditional on individual pharmacokinetics, simulations of the AUC of both HCY and CEPM were provided for a range of second doses (i.e., 0-100 mg/kg cyclophosphamide). RESULTS: The results compared favorably with NONMEM and returned accurate predictions for AUCs of HCY and CEPM with comparable mean absolute prediction error and root mean square prediction error. With our method, the mean absolute prediction error and root mean square prediction error of AUC CEPM were 11.0% and 12.8% and AUC HCY were 31.7% and 44.8%, respectively. CONCLUSIONS: We developed dose adjustment software that potentially can be used to adjust cyclophosphamide dosing in a clinical setting, thus expanding the opportunity for pharmacokinetic individualization of cyclophosphamide. The software is simple to use (requiring no programming experience), reads individual patient data directly from an Excel spreadsheet, and runs in less than 5 minutes on a desktop PC.

Rapid quantitation of cyclophosphamide metabolites in plasma by liquid chromatography-mass spectrometry.

A method is described for the quantification of two metabolites of cyclophosphamide, specifically 4-hydroxycyclophosphamide (HCy), and carboxyethylphosphoramide mustard (CEPM). Plasma HCy is derivatized to the phenylhydrazone which is quantitated by LC-MS monitoring the chloride adduct of the derivative. The LLOQ based on material applied to the system is approximately 20 fmol. Plasma CEPM concentration is determined using LC-MS with a deuterated internal standard. Both assays have 50-fold dynamic range and require less than 4h to complete. The development of this rapid analytical method makes it feasible to adjust the dose of cyclophosphamide based on the pharmacokinetic disposition of HCy and CEPM in hopes of decreasing nonrelapse mortality in cancer patients.

Aminotransferase elevations in healthy adults receiving 4 grams of acetaminophen daily: a randomized controlled trial.

CONTEXT: During a clinical trial of a novel hydrocodone/acetaminophen combination, a high incidence of serum alanine aminotransferase (ALT) elevations was observed. OBJECTIVE: To characterize the incidence and magnitude of ALT elevations in healthy participants receiving 4 g of acetaminophen daily, either alone or in combination with selected opioids, as compared with participants treated with placebo. DESIGN, SETTING, AND PARTICIPANTS: A randomized, single-blind, placebo-controlled, 5-treatment, parallel-group, inpatient, diet-controlled (meals provided), longitudinal study of 145 healthy adults in 2 US inpatient clinical pharmacology units. INTERVENTION: Each participant received either placebo (n = 39), 1 of 3 acetaminophen/opioid combinations (n = 80), or acetaminophen alone (n = 26). Each active treatment included 4 g of acetaminophen daily, the maximum recommended daily dosage. The intended treatment duration was 14 days. Main Outcomes Serum liver chemistries and trough acetaminophen concentrations measured daily through 8 days, and at 1- or 2-day intervals thereafter. RESULTS: None of the 39 participants assigned to placebo had a maximum ALT of more than 3 times the upper limit of normal. In contrast, the incidence of maximum ALT of more than 3 times the upper limits of normal was 31% to 44% in the 4 treatment groups receiving acetaminophen, including those participants treated with acetaminophen alone. Compared with placebo, treatment with acetaminophen was associated with a markedly higher median maximum ALT (ratio of medians, 2.78; 95% confidence interval, 1.47-4.09; P<.001). Trough acetaminophen concentrations did not exceed therapeutic limits in any participant and, after active treatment was discontinued, often decreased to undetectable levels before ALT elevations resolved. CONCLUSIONS: Initiation of recurrent daily intake of 4 g of acetaminophen in healthy adults is associated with ALT elevations and concomitant treatment with opioids does not seem to increase this effect. History of acetaminophen ingestion should be considered in the differential diagnosis of serum aminotransferase elevations, even in the absence of measurable serum acetaminophen concentrations.

Metabolism-based cyclophosphamide dosing for hematopoietic cell transplant.

When cyclophosphamide (120 mg/kg) is used for hematopoietic cell transplant, the increased area under the curve of carboxyethylphosphoramide mustard (AUC(CEPM)) is related to liver toxicity and death. We determined the feasibility of dose-adjusting cyclophosphamide to a preset metabolic endpoint (AUC(CEPM), 325 +/- 25 micromol/L.h). In 20 patients blood sampling was done over a 16-hour period after administration of 45 mg/kg cyclophosphamide; AUC(CEPM) from 0 to 16 hours was calculated by noncompartmental analysis. The expected AUC(CEPM) for 0 to 48 hours was estimated, and the second cyclophosphamide dose was determined. The mean second cyclophosphamide dose was 42 mg/kg, and the mean total cyclophosphamide dose was 86 mg/kg (range, 54-120 mg/kg). The mean AUC(CEPM) for the time from 0 to 48 hours was 296 micromol/L.h (95% confidence interval, 275-317 micromol/L.h). A retrospective analysis indicated that AUC(CEPM) could be more accurately predicted by use of a population pharmacokinetic model. We conclude that metabolism-based dosing of cyclophosphamide is feasible and that a lower cyclophosphamide dose does not affect engraftment.

A highly sensitive high-performance liquid chromatography-mass spectrometry method for quantification of fludarabine triphosphate in leukemic cells.

A high-performance liquid chromatography (HPLC)-mass spectrometry (MS) method has been developed for the analysis of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) from biological samples. Quantification is carried out by selected ion monitoring of the parent ion. Baseline separation of the monophosphate (F-ara-AMP) and diphosphate (F-ara-ADP) is achieved using the volatile ion-pairing reagent dimethylhexylamine. This method is selective and sensitive with an on-column detection limit of approximately 50 fmol. It also permits simultaneous monitoring of endogenous adenosine phosphates. The utility of the assay has been demonstrated by the analysis of F-ara-ATP in human leukemic cells after incubation with 9-beta-D-arabinosyl-2-fluoroadenine (F-ara-A) at clinically relevant concentrations.

The effect of clarithromycin, fluconazole, and rifabutin on dapsone hydroxylamine formation in individuals with human immunodeficiency virus infection (AACTG 283).

BACKGROUND: Dapsone hydroxylamine formation is thought to be the cause of the high rates of adverse reactions to dapsone in human immunodeficiency virus (HIV)-infected individuals. Therefore we studied the effect of the commonly coadministered drugs fluconazole, clarithromycin, and rifabutin on hydroxylamine formation in individuals with HIV infection. METHODS: HIV-infected subjects (CD4 + > or =200 cells/mm 3 ) were enrolled in a 2-part (A or B) open-label drug interaction study. In part A, subjects (n = 12) received dapsone (100-mg tablet once daily) alone for 2 weeks and then, in a randomly assigned order, received dapsone and either fluconazole (200 mg daily), rifabutin (300 mg daily), or fluconazole plus rifabutin, each for a 2-week period. Part B (n = 11) was identical to part A except that clarithromycin (500 mg twice daily) was substituted for rifabutin. On the last study day of each 2-week period, plasma and urine were collected over ascorbic acid for 24 hours. RESULTS: In part A, fluconazole decreased the area under the plasma concentration-time curve, percent of dose excreted in 24-hour urine, and formation clearance of the hydroxylamine by 49%, 53%, and 55% (n = 12, P < .05), respectively. This inhibition of in vivo hydroxylamine formation was quantitatively consistent with that predicted from human liver microsomal experiments. Rifabutin had no effect on hydroxylamine area under the plasma concentration-time curve or percent excreted in 24-hour urine but increased formation clearance of the hydroxylamine by 92% (n = 12, P < .05). Dapsone clearance was increased by rifabutin or rifabutin plus fluconazole (67% and 38%, respectively) (n = 12, P < .05) but was unaffected by fluconazole or clarithromycin. In part B, hydroxylamine production was unaffected by clarithromycin but was affected by fluconazole in a manner identical to that in part A. CONCLUSIONS: On the basis of these data and with the assumption that the exposure to the hydroxylamine is a determinant of dapsone toxicity, we predict that coadministration of fluconazole should decrease the rate of adverse reactions to dapsone in persons with HIV infection but that rifabutin and clarithromycin will have no effect. When dapsone is given in combination with rifabutin, dapsone dosage adjustment may be necessary in light of the increase in dapsone clearance.

Diminishing the risk of nonrelapse mortality in hematopoietic stem cell transplantation: Prediction of exposure to the cyclophosphamide metabolite carboxyethylphosphoramide mustard.

OBJECTIVES: Our objectives were (1) to develop a population pharmacokinetic model for cyclophosphamide, 4-hydroxycyclophosphamide, and carboxyethylphosphoramide mustard (a reporter for nonrelapse mortality) in hematopoietic stem cell transplantation patients and (2) to validate a Bayesian approach to dosing. METHODS: In this study 147 patients received intravenous infusions of 60 mg. kg -1. d -1 cyclophosphamide for 2 days, followed by 12 to 14.4 Gy total body irradiation. A population model was developed to fit concentration-time data of cyclophosphamide and metabolites. Bayesian prediction of the area under the curve (AUC) was validated by dividing the data set into an index set (98 patients) and validation set (49 patients). Parameters from the index data set were used as priors. RESULTS: Cyclophosphamide elimination was best described by noninducible and inducible routes producing 4-hydroxycyclophosphamide. Induction was described by a zero-order maximum fold of induction-type increase in enzyme level. The prediction of the AUC of carboxyethylphosphoramide mustard was clinically accurate and precise (mean prediction error = -3.5% and root mean squared prediction error = 12.2%) with data limited to 5 to 6 points obtained in the first 16 hours. However, the AUC of 4-hydroxycyclophosphamide was overestimated (mean prediction error = 16.9%-23.6%). Several alternative models did not improve the result. CONCLUSION: The integrated mechanism-based model describes the pharmacokinetics of cyclophosphamide and carboxyethylphosphoramide mustard. Accurate modeling of 4-hydroxycyclophosphamide is limited by its chemical instability. Exposure to carboxyethylphosphoramide mustard could be accurately and precisely predicted with minimal data obtained over a 16-hour period after the first dose, offering the potential of pharmacokinetically guided dosing to reduce the nonrelapse mortality rate.

The effect of clarithromycin, fluconazole, and rifabutin on sulfamethoxazole hydroxylamine formation in individuals with human immunodeficiency virus infection (AACTG 283).

BACKGROUND: Sulfamethoxazole hydroxylamine formation, in combination with long-term oxidative stress, is thought to be the cause of high rates of adverse drug reactions to sulfamethoxazole in human immunodeficiency virus (HIV)-infected subjects. Therefore the goal of this study was to determine the effect of fluconazole, clarithromycin, and rifabutin on sulfamethoxazole hydroxylamine formation in individuals with HIV-1 infection. METHODS: HIV-1-infected subjects (CD4 + count >/=200 cells/mm 3 ) were enrolled in a 2-part (A and B), open-label drug interaction study (Adult AIDS Clinical Trial Group [AACTG] 283). In part A (n = 9), subjects received cotrimoxazole (1 tablet of 800 mg sulfamethoxazole/160 mg trimethoprim daily) alone for 2 weeks and then, in a randomly assigned order, cotrimoxazole plus either fluconazole (200 mg daily), rifabutin (300 mg daily), or fluconazole plus rifabutin, each for a 2-week period. Part B (n = 12) was identical to part A except that clarithromycin (500 mg twice daily) was substituted for rifabutin. RESULTS: In part A, fluconazole decreased the area under the plasma concentration-time curve (AUC), percent of dose excreted in 24-hour urine, and formation clearance (CL f ) of the hydroxylamine by 37%, 53%, and 61%, respectively (paired t test, P < .05). Rifabutin increased the AUC, percent excreted, and CL f of the hydroxylamine by 55%, 45%, and 53%, respectively ( P < .05). Fluconazole plus rifabutin decreased the AUC, percent excreted, and CL f of the hydroxylamine by 21%, 37%, and 46%, respectively ( P < .05). In part B the fluconazole data were similar to those of part A. Overall, clarithromycin had no effect on hydroxylamine production. CONCLUSIONS: If the exposure (AUC) to sulfamethoxazole hydroxylamine is predictive of sulfamethoxazole toxicity, then rifabutin will increase and clarithromycin plus fluconazole or rifabutin plus fluconazole will decrease the rates of adverse reactions to sulfamethoxazole in HIV-infected subjects.

Transiently altered acetaminophen metabolism after liver transplantation.

BACKGROUND AND OBJECTIVES: Acetaminophen (INN, paracetamol) is metabolized to N-acetyl-p-benzoquinone imine (NAPQI), a hepatotoxic metabolite, predominantly by cytochrome P450 (CYP) 2E1. Alterations in drug metabolism occur after organ transplantation. This study was designed to characterize acetaminophen disposition during the first 6 months after liver transplantation. METHODS: Thirteen liver transplant patients received an oral dose of acetaminophen (500 mg) on days 2, 10, 90, and 180 after transplantation. Serial blood samples were collected for 8 hours, and urine was collected for 24 hours. Liver biopsy specimens were obtained from the donor liver during transplantation (day 0) and on days 10, 90, and 180 after transplantation. RESULTS: There were significant time-dependent changes in acetaminophen metabolism after liver transplantation. When day 2 and day 10 were compared with day 180, the respective mean urinary recovery was 137% and 81% higher for thioether conjugates derived from NAPQI (P =.0002 and P =.01, respectively); 31% and 22% lower for acetaminophen sulfate (P =.0006 and P =.008, respectively); and 22% and 27% lower for acetaminophen glucuronide (P =.05 and P =.004, respectively). Metabolite formation clearances changed in concordance with the fractional urinary recovery. It was surprising that hepatic CYP2E1 content on day 10 after transplantation was only 20% higher, on average, than that found on day 180 (not significant). In contrast, hepatic CYP3A4 content was 984% higher, on average, when tissue from days 10 and 180 was compared after transplantation (P =.007). CONCLUSIONS: Increased recovery of acetaminophen thioether conjugates during the first 10 days after liver transplantation was a result of impaired glucuronidation and sulfation and enhanced NAPQI formation.

The road we must take: multidisciplinary team science.

Translational research is acknowledged to be complex and to require a diverse skill set. Many organizations, particularly academic institutions, have invested in educational programs, facilities, and enhanced resources to encourage translational research. Critically needed, however, is an emphasis on creating and sustaining multidisciplinary research teams. It is through the power of many and a diverse approach to our health care problems that we will realize lasting solutions.

Pharmacodynamics of mycophenolate mofetil after nonmyeloablative conditioning and unrelated donor hematopoietic cell transplantation.

The immunosuppressive drug mycophenolate mofetil (MMF) is used after nonmyeloablative hematopoietic cell transplantation (HCT); however, limited pharmacodynamic data are available. We evaluated plasma concentrations of mycophenolic acid (MPA), the active metabolite of MMF, and outcomes in 85 patients with hematologic malignancies conditioned with fludarabine and 2 Gy total body irradiation followed by HLA-matched unrelated-donor HCT and postgrafting cyclosporine and MMF. The first 38 patients received MMF 15 mg/kg twice daily; the next 47 patients received MMF 3 times daily. MPA pharmacokinetics were determined on days 7 and 21. Comparing the twice-daily and 3-times-daily MMF groups, the mean total MPA concentration steady state (Css) was 1.9 and 3.1 microg/mL; the unbound Css was 18 and 36 ng/mL, respectively (P < .001). Sixteen patients with a total MPA Css less than 3 microg/mL had low (< 50%) donor T-cell chimerism (P = .03), and 6 patients with MPA Css less than 2.5 microg/mL had graft rejection. An elevated unbound Css was associated with cytomegalovirus reactivation (P = .03). There were no significant associations between MPA pharmacokinetics and acute graft-versus-host disease (GVHD) or relapse. We conclude that increased MPA Css's predicted higher degrees of donor T-cell chimerism after unrelated donor nonmyeloablative HCT and suggest that targeting MPA Css's greater than 2.5 microg/mL could prevent graft rejection.

A phase I/II study of mycophenolate mofetil in combination with cyclosporine for prophylaxis of acute graft-versus-host disease after myeloablative conditioning and allogeneic hematopoietic cell transplantation.

Abstract In a phase I/II study, the combination of cyclosporine (CSP) and mycophenolate mofetil (MMF) was investigated as graft-versus-host disease (GVHD) prophylaxis after myeloablative conditioning and hematopoietic cell transplantation from an HLA-matched sibling donor. In phase I, 3 groups, each with 10 or 11 patients, received MMF (15 mg/kg) from day 0 to day 27 at decreasing dose intervals of every 12, 8, and 6 hours to determine a safe and effective total daily dose. At the 45 mg/kg/d dosage level, 4 of 11 patients developed only grade II GVHD, and a concentration at steady state of mycophenolic acid (the active moiety of MMF) consistent with a therapeutic range described for solid-organ transplantation was achieved. There was a suggestion of increased toxicity without improved efficacy at the 60 mg/kg/d dosage level. Accordingly, the 45 mg/kg/d dosage was therefore selected for phase II, and another 15 patients were added to this group from the phase I study (n=26). The concentrations at steady state for this dosage at days 0, 6, 13, 20, and 27 were 2.73, 3.02, 3.20, 2.62, and 2.64 microg/mL, respectively. No toxicities were attributed to MMF at this dose. The median time to engraftment after hematopoietic cell transplantation was 15 days (range, 10-20 days). The incidence of acute GVHD was 62%, which was comparable to a group of historical controls receiving CSP and methotrexate (MTX) for GVHD prophylaxis. Although a significant improvement in the prevention of GVHD was not suggested, compared with CSP and MTX, MMF in combination with CSP could be considered in cases in which MTX is contraindicated.

Effects of ethanol dose and ethanol withdrawal on rat liver mitochondrial glutathione: implication of potentiated acetaminophen toxicity in alcoholics.

Chronic ethanol consumption potentiates acetaminophen (APAP) hepatotoxicity through enhanced NAPQI formation via CYP2E1 induction and selective depletion of mitochondrial glutathione. Because the prevalence of the interaction is extremely low given the use of APAP and the incidence of alcohol abuse, we studied the effects of ethanol dose and ethanol withdrawal on selective mitochondrial glutathione (GSH) depletion and APAP toxicity in liver slices. Rats were fed the Lieber-DeCarli diet containing ethanol (0, 7, 18, 27, and 36% total energy) for 6 weeks. The highest ethanol-containing diet (36% energy as ethanol) was replaced by control diet for 2, 5, 12, and 17 h. Maximal CYP2E1 induction was caused by 36% energy as ethanol diet (2.2-fold, p < 0.05 versus control). The activity and liver protein content returned to the control level 17 h after ethanol withdrawal. The 36% energy as ethanol diet caused maximal mitochondrial GSH depletion (51%, p < 0.05 versus control), which was restored 17 h after ethanol withdrawal (22.0 +/- 4.9 versus 11.7 +/- 1.7 nmol/mg protein of 0 h, p < 0.01). Elevated glutathione S-transferase-alpha release in liver slices (a measure of toxicity) was observed in rats fed 36% energy as ethanol diet (1 mM APAP: 69 +/- 10 versus 3 +/- 1% of control, p < 0.01). Enhanced toxicity disappeared when ethanol dose decreased and when ethanol was removed (7.2% ethanol: 3 +/- 1% and 17 h: 2 +/- 1%, p < 0.01 versus 0 h 36% energy as ethanol). In conclusion, high-dose ethanol potentiated APAP hepatotoxicity via CYP2E1 induction and selective mitochondrial GSH depletion. Mitochondrial GSH depletion quickly reversed when ethanol was withdrawn. The time window for both mechanisms to act in concert is narrow.

Selective mitochondrial glutathione depletion by ethanol enhances acetaminophen toxicity in rat liver.

Chronic alcohol consumption may potentiate acetaminophen (APAP) hepatotoxicity through enhanced formation of N-acetyl-p-benzoquinone imine (NAPQI) via induction of cytochrome P450 2E1 (CYP2E1). However, CYP2E1 induction appears to be insufficient to explain the claimed magnitude of the interaction. We assessed the role of selective depletion of liver mitochondrial glutathione (GSH) by chronic ethanol. Rats were fed the Lieber-DeCarli diet for 10 days or 6 weeks. APAP toxicity in liver slices (% glutathione-S-transferase alpha released to the medium, GST release) and NAPQI toxicity in isolated liver mitochondria (succinate dehydrogenase inactivation, SDH) from these rats were compared with pair-fed controls. Ethanol induced CYP2E1 in both the 10-day and 6-week groups by approximately 2-fold. APAP toxicity in liver slices was higher in the 6-week ethanol group than the 10-day ethanol group. Partial inhibition of NAPQI formation by CYP2E1 inhibitor diethyldithiocarbamate to that of pair-fed controls abolished APAP toxicity in the 10-day ethanol group only. Ethanol selectively depleted liver mitochondrial GSH only in the 6-week group (by 52%) without altering cytosolic GSH. Significantly greater GSH loss and APAP covalent binding were observed in liver slice mitochondria of the 6-week ethanol group. Isolated mitochondria of the 6-week ethanol group were approximately 50% more susceptible to NAPQI (25-165 micromol/L) induced SDH inactivation. This increased susceptibility was reproduced in pair-fed control mitochondria pretreated with diethylmaleate. In conclusion, 10-day ethanol feeding enhances APAP toxicity through CYP2E1 induction, whereas 6-week ethanol feeding potentiates APAP hepatotoxicity by inducing CYP2E1 and selectively depleting mitochondrial GSH.

ABCC2-mediated biliary transport of 4-glutathionylcyclophosphamide and its contribution to elimination of 4-hydroxycyclophosphamide in rat.

Hematopoietic stem cell transplantation patients conditioned with cyclophosphamide (CY) and total body irradiation have substantially greater risk of nonrelapse mortality when plasma area under the concentration-time curve (AUC) of O-carboxyethylcyclophosphoramide mustard (CEPM) is high. The discovery was paradoxical because CEPM is a nontoxic elimination route of the protoxic CY metabolite hydroxycyclophosphamide (HCY). CY was administered to Wistar and TR- rats (a Wistar strain lacking functional ABCC2) at doses of 100 and 200 mg/kg CY, respectively. After either dose, Wistar rats excreted 4-glutathionylcyclophosphamide (GSCY) abundantly in bile; GSCY was absent from bile of TR- rats. Liver AUC(GSCY) was 2- to 2.5-fold greater in TR- than Wistar rats after 100 and 200 mg/kg CY, respectively. Liver AUC(HCY) was 24-46% greater in TR- rats than in Wistar rats after the respective CY doses. Plasma AUC(CEPM) of TR- rats was approximately twice that of Wistar rats after 100 mg/kg, but did not differ between the two strains after 200 mg/kg. Conversely, plasma AUC(HCY) was not different after 100 mg/kg CY, but was 40% greater in TR- rats after 200 mg/kg. The dose dependence of plasma AUC(CEPM) and AUC(HCY) was explained by the concentrations of HCY attained and the in vitro K(m) of aldehyde dehydrogenase and inhibition of aldehyde dehydrogense in TR- rats. We conclude that GSCY is a substrate of ABCC2, and plasma AUC(CEPM) functions as a reporter of liver exposure to HCY and toxins formed from it when HCY concentration is below the K(m) of aldehyde dehydrogenase and the activity is not compromised.

Dose escalation of busulfan with pentoxifylline and ciprofloxacin in patients with breast cancer undergoing autologous transplants.

OBJECTIVE: There is evidence that pentoxifylline (PTX) and ciprofloxacin (Cipro) may protect patients from the effects of chemotherapy and radiation, which could allow further drug dose escalation. This study was conducted to determine whether oral and intravenous (IV) PTX and Cipro permits increased dose levels of oral busulfan (BU) with a fixed dose of IV cyclophosphamide (CY) in patients with breast cancer receiving autologous or syngeneic hematopoetic cell transplantation. METHODS: Sixty-seven patients received PTX and Cipro with CY of 150 mg/kg and escalating doses of BU. The BU dosing began at 15 mg/kg, escalating in 1 mg/kg increments in groups of 4 patients. If no grade 3 or 4 regimen- related toxicities (RRT) were observed, the next 4 patients were treated at a higher dose. RESULTS: Excessive RRT was not observed until BU 21 mg/kg was reached. Two patients at this dose level had RRTs and their BU steady-state concentration (Css) were 1,414 and 1,545 ng/ml. At a BU dose of 20 mg/kg , average BU Css 1,280 ng/ml, 0/4 had RRT. Among 10 patients who had BU Css targeted to 1,350 ng/ml, RRTs occurred in 2 (20%). CONCLUSIONS: In this preliminary study with PTX and Cipro, the maximum tolerated dose of BU that can be given with CY (150 mg/kg) was 20 mg/kg, a BU Css of approximately 1,300 ng/ml. A randomized trial is necessary to determine whether PTX and Cipro reduce the toxicities of this regimen.

Cyclophosphamide metabolism is affected by azole antifungals.

We performed a randomized trial to compare the safety and efficacy of itraconazole with fluconazole in preventing fungal infections in patients undergoing allogeneic stem cell transplantation (SCT). Itraconazole (intravenous 200 mg daily, or oral solution 2.5 mg/kg 3 times daily) and fluconazole (intravenous or oral, 400 mg daily) were administered with the start of conditioning therapy, until at least 120 days after SCT. After enrollment of the first 197 patients, a data and safety monitoring board reviewed potential drug-related toxicities. Patients who received itraconazole developed higher serum bilirubin and creatinine values in the first 20 days after SCT, with highest values in patients who received itraconazole concurrent with cyclophosphamide (CY) conditioning. Analysis of CY metabolism in a subset of patients demonstrated higher exposure to toxic metabolites among recipients of itraconazole compared with fluconazole. These data suggest that azole antifungals, through differential inhibition of hepatic cytochrome P-450 isoenzymes, affect CY metabolism and conditioning-related toxicities.

Cyclophosphamide metabolism, liver toxicity, and mortality following hematopoietic stem cell transplantation.

Liver toxicity caused by high-dose myeloablative therapy leads to significant morbidity after hematopoietic cell transplantation. We examined the hypothesis that liver toxicity after cyclophosphamide and total body irradiation is related to cyclophosphamide through its metabolism to toxins. Cyclophosphamide was infused at 60 mg/kg over 1 to 2 hours on each of 2 consecutive days, followed by total body irradiation. Plasma was analyzed for cyclophosphamide and its major metabolites. Liver toxicity was scored by the development of sinusoidal obstruction syndrome (veno-occlusive disease) and by total serum bilirubin levels. The hazards of liver toxicity, nonrelapse mortality, tumor relapse, and survival were calculated using regression analysis that included exposure to cyclophosphamide metabolites (as the area under the curve). Of 147 patients, 23 (16%) developed moderate or severe sinusoidal obstruction syndrome. The median peak serum bilirubin level through day 20 was 2.6 mg/dL (range, 0.5-41.1 mg/dL). Metabolism of cyclophosphamide was highly variable, particularly for the metabolite o-carboxyethyl-phosphoramide mustard, whose area under the curve varied 16-fold. Exposure to this metabolite was statistically significantly related to sinusoidal obstruction syndrome, bilirubin elevation, nonrelapse mortality, and survival, after adjusting for age and irradiation dose. Patients in the highest quartile of o-carboxyethyl-phosphoramide mustard exposure had a 5.9-fold higher risk for nonrelapse mortality than did patients in the lowest quartile. Engraftment and tumor relapse were not statistically significantly related to cyclophosphamide metabolite exposure. Increased exposure to toxic metabolites of cyclophosphamide leads to increased liver toxicity and nonrelapse mortality and lower overall survival after hematopoietic cell transplantation.

Conditioning with fludarabine and targeted busulfan for transplantation of allogeneic hematopoietic stem cells.

A regimen of busulfan and cyclophosphamide is standard therapy before transplantation of allogeneic hematopoietic stem cells in patients with chronic myelogenous leukemia (CML) or myelodysplastic syndrome (MDS). The clinical trial reported here was undertaken to test the hypothesis that fludarabine can replace cyclophosphamide in this regimen and facilitate donor engraftment with reduced toxicity. The conditioning regimen consisted of 30 mg/m2 intravenous fludarabine daily from day -9 to day -6, and oral busulfan given at 1 mg/kg 4 times a day every 6 hours from day -5 to day -2, with doses adjusted to target plasma levels of 900 +/- 100 ng/mL at steady state. Cyclosporine and methotrexate were used for prophylaxis for graft-versus-host disease. Enrolled were 42 patients with high-risk CML (n = 4) or MDS (n = 38). The median patient age was 52 years (range, 12-65 years). Mobilized blood stem cells were obtained from HLA-compatible siblings (n = 16) or unrelated donors (n = 26). Engraftment was achieved in all patients, and the day-100 regimen-related mortality was 7%. With a median follow-up of 18 months (range, 13-27 months), the probabilities of overall survival, disease-free survival, and nonrelapse mortality were 42.4%, 34.9%, and 24%, respectively. These data indicate that the combination of fludarabine and targeted busulfan is sufficiently immunosuppressive to facilitate engraftment of blood stem cells from HLA-matched siblings and unrelated donors. Based on these encouraging results, further studies of fludarabine and targeted busulfan are warranted in standard-risk patients.