Pharmacokinetics Of Cyclophosphamide Research Articles

Population Pharmacokinetics of Cyclophosphamide in Patients with Thalassaemia Major Undergoing HSCT Shows Body Weight, CYP450, GST and ALDH Polymorphisms as Covariates Explaining Inter-Individual Variation.

Abstract 1182Poster Board I-204Cyclophosphamide (Cy) in combination with busulfan is an important component of myeloablative conditioning regimen used prior to hematopoietic stem cell transplantation (HSCT) for both malignant and non-malignant conditions. We have previously reported up to 20 fold inter-individual variation in the pharmacokinetics (PK) of Cy in patients with beta thalassaemia undergoing HSCT [Blood (ASH Annual Meeting Abstracts), Nov 2004; 104: 99]. PK parameters of Cy have been shown to be associated with regimen related toxicity and outcome of transplant. To explain the basis of the inter-individual variation in Cy PK, we have developed a population PK model. We analyzed the PK of Cy in consecutive children with beta thalassaemia major who received HSCT from HLA identical matched sibling donor at the Christian Medical College, Vellore from 2001 till 2004. A total of 900 cyclophosphamide concentration measurements from 55 patients were included and correlated with age, sex, body weight and 10 polymorphisms in enzymes involved in the metabolism or biotransformation of Cy namely GST A1, M1, T1, P1, CYP2B6, CYP2C9, CYP2C19 and ALDH genes. Non-linear mixed effects modeling analysis was performed with Monolix (version 2.4, www.monolix.org) to investigate the effect of patient covariates on PK, and to estimate the relative magnitude of inter-individual and inter-occasion variability. A two-compartment pharmacokinetic model was used to describe the data. The pharmacokinetic parameters estimated included elimination rate constant and volume (ke (1/hr), V (L or L/kg)), and the inter-compartmental parameters (k12 and k21 (1/hr)). The distribution of the parameters was assumed log-normal. Body weight was the main covariate which explained the largest portion of the IIV (28% and 20% of V and ke IIV, respectively). In addition, the following genotypes showed differences in the pharmacokinetics: GSTP1*B (1.7X higher ke in MUT versus WT or HET; p<0.05), CYP3A4*1B (2X higher ke in HET versus WT; p<0.05), and ALDH1A1*2 (2X higher ke in HET versus WT; p<0.05). We have developed a population PK model for Cy in thalassaemic children by considering morphological and biological covariates, which explains more than 45% and 22% (V and ke IIV, respectively) of the variation in Cy PK in these patients. This model-based algorithm may be used to design and plan targeted dose therapy in this group of pediatric patients and to predict the risk of toxicity and outcome of HSCT. Disclosures:No relevant conflicts of interest to declare.

Influence of Glomerular Filtration Rate on the Pharmacokinetics of Cyclophosphamide Enantiomers in Patients With Lupus Nephritis

The pharmacokinetics of cyclophosphamide (CYC) enantiomers were evaluated in patients with lupus nephritis distributed in 2 groups according to creatinine clearance: group 1 (90.6-144.6 mL/min/1.73 m(2)) and group 2 (42.8-76.4 mL/min/1.73 m(2)). All patients were treated with 0.75 to 1.3 g of racemic CYC as a 2-hour infusion and with 1 mg intravenous midazolam as a drug-metabolizing marker. CYC enantiomers and midazolam concentrations in plasma were measured by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The following differences (Wilcoxon test, P < or = .05) were observed between the (S)-(-) and (R)-(+) enantiomers: AUC(0-infinity) 152.41 vs 129.25 microg.h/mL, CL 3.28 vs 3.89 L/h, Vd 31.38 vs 29.74 L, and t((1/2)) 6.79 vs 5.56 h for group 1 and AUC(0-infinity) 167.20 vs 139.08 microg.h/mL, CL 2.99 vs 3.59 L/h, and t((1/2)) 6.15 vs 4.99 h for group 2. No differences (Mann test, P < or = .05) were observed between groups 1 and 2 in the pharmacokinetic parameters of both enantiomers. No significant relationship was observed between midazolam clearance (2.92-16.40 mL/min.kg) and clearance of each CYC enantiomer. In conclusion, CYC kinetic disposition is enantioselective, resulting in higher exposures of the (S)-(-) enantiomer in lupus nephritis patients, and the pharmacokinetic parameters of both enantiomers are not altered by the worsening of renal condition.

Effect of aprepitant (AP) on cyclophosphamide (CPA) pharmacokinetics (PK) in early breast cancer patients

588 Background: AP, a moderate inhibitor of CYP3A4, is a selective neurokinin 1 receptor antagonist recommended by the ASCO guidelines for prevention of high and some moderately emetogenic chemotherapy regimens, including doxorubicin and CPA (AC). CPA is converted to the active 4-OH metabolite primarily by CYP2B6 (48–57%) and by CYP3A4 (12–18%). CYP3A4 also converts 10% of CPA to the N-dechloroethyl metabolite (DCE), which is responsible for neurotoxicity and nephrotoxicity. Because AP is utilized concurrently with CPA, this creates the potential for drug-drug interaction between AP and CPA. The objective of this study was to evaluate the effect of AP on the PK of CPA, 4-OH, and DCE in patients receiving AC. Methods: This double-blinded, placebo controlled, two-period, crossover study included breast cancer patients receiving regimens containing CPA 600 mg/m2 IV for at least two sequential cycles. Prior to each CPA cycle, patients were randomized to receive either oral AP (125 mg on d 1 prior to chemotherapy and 80 mg on d 2 and 3) or placebo for 3 d. During both cycles, patients were also pretreated with ondansetron 24 mg PO and dex 12 mg PO on d 1 and dex 8 mg PO once on d 2 and 3. Rescue anti-emetic medications were permitted. Serial plasma samples were obtained from 0 to 24 h after administration of CPA in combination with AP or placebo in each patient. CPA, 4-OH, and DCE plasma concentrations were determined using HPLC. Area under the plasma versus conc time curve (AUC) was calculated using WinNonlin. Results: 17 patients completed 2 cycles and were evaluated for PK. Geometric mean AUC data for CPA in combination with AP and placebo are summarized in the table. Conclusions: Based on geometric mean ratios ± 90% CI, concurrent administration of AP with CPA did not result in any significant changes in the 4-OH metabolite AUC, but did result in increased parent CPA and decreased DCE drug exposure levels. Based on these results, it is unlikely that aprepitant has a clinically significant effect on the efficacy of CPA. [Table: see text] [Table: see text]

Pharmacokinetics of Cyclophosphamide and Thiotepa in a Conventional Fractionated High-Dose Regimen Compared With a Novel Simplified Unfractionated Regimen

High-dose alkylating chemotherapy with cyclophosphamide (4000 or 6000 mg/m2) and thiotepa (320 or 480 mg/m2) has commonly been administered in a fractionated regimen over 4 days. A simplified unfractionated regimen would be preferable, especially because cyclophosphamide and thiotepa have been shown to influence the metabolism of each other. However, altering a dose regimen can have a profound effect on the pharmacokinetics of the compounds involved. The aim of this study was to investigate the effect of altering the fractionated administration schedule of the CTC regimen on cyclophosphamide and thiotepa pharmacokinetics. Plasma samples were collected from 124 patients who received a fractionated tiny CTC or CTC regimen of cyclophosphamide (1000 or 1500 mg m(-2) d(-1)), thiotepa (40 or 60 mg/m2 twice daily), and carboplatin (267 or 400 mg m(-2) day(-1)) for 4 days, and 16 patients who received an unfractionated mini CTC regimen of cyclophosphamide (3000 mg/m2 at day 1), carboplatin (400 mg/m2 at days 1 and 2), and thiotepa (250 mg/m2 at day 2). Plasma concentrations of cyclophosphamide and 4-hydroxycyclophosphamide were determined using high-performance liquid chromatography coupled with tandem mass spectrometric detection; plasma concentrations of thiotepa and tepa were determined using gas chromatography. Pharmacokinetics of cyclophosphamide and thiotepa were assessed using nonlinear mixed-effect modeling. The study showed that alteration of a fractionated high-dose regimen into a simplified unfractionated regimen resulted in saturation of thiotepa elimination, with a Vmax of 212 (+/-58) micromol/h and a Km of 13.7 (+/-5.9) microM. This resulted in an increased dose-corrected exposure to thiotepa (13%) and decreased dose-corrected exposure to its metabolite tepa (21%). Elimination of cyclophosphamide was not shown to be saturable. Dose-corrected exposures to cyclophosphamide and its active metabolite 4-hydroxycyclophosphamide were comparable in both regimens. Because the simplified unfractionated mini CTC regimen was more patient-friendly and because overall dose-corrected exposures to cyclophosphamide and thiotepa were not affected to a relevant extent, our data suggest that this unfractionated regimen can be used safely in future studies.

Population Pharmacokinetics of Cyclophosphamide and Metabolites in Children With Neuroblastoma: A Report From the Children's Oncology Group

Cyclophosphamide-based regimens are front-line treatment for numerous pediatric malignancies; however, current dosing methods result in considerable interpatient variability in tumor response and toxicity. In this pediatric population, the authors' objectives were (1) to quantify and explain the pharmacokinetic variability of cyclophosphamide and 2 of its metabolites, hydroxycyclophosphamide (HCY) and carboxyethylphosphoramide mustard (CEPM), and (2) to apply a population pharmacokinetic model to describe the disposition of cyclophosphamide and these metabolites. A total of 196 blood samples were obtained from 22 children with neuroblastoma receiving intravenous cyclophosphamide (400 mg/m2/d) and topotecan. Blood samples were quantitated for concentrations of cyclophosphamide, HCY, and CEPM using liquid chromatography-mass spectrometry and analyzed using nonlinear mixed-effects modeling with the NONMEM software system. After model building was complete, the area under the concentration-time curve (AUC) was computed using NONMEM. Cyclophosphamide elimination was described by noninducible and inducible routes, with the latter producing HCY. Glomerular filtration rate was a covariate for the fractional elimination of HCY and its conversion to CEPM. Considerable interpatient variability was observed in the AUC of cyclophosphamide, HCY, and CEPM. These results represent a critical first step in developing pharmacokinetic-linked pharmacodynamic studies in children receiving cyclophosphamide to determine the clinical relevance of the pharmacokinetic variability in cyclophosphamide and its metabolites.

Influence of polymorphisms of drug metabolizing enzymes (CYP2B6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, GSTA1, GSTP1, ALDH1A1 and ALDH3A1) on the pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide

The anticancer agent, cyclophosphamide, is metabolized by cytochrome P450 (CYP), glutathione S-transferase (GST) and aldehyde dehydrogenase (ALDH) enzymes. Polymorphisms of these enzymes may affect the pharmacokinetics of cyclophosphamide and thereby its toxicity and efficacy. The purpose of this study was to evaluate the effects of known allelic variants in the CYP2B6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, GSTA1, GSTP1, ALDH1A1 and ALDH3A1 genes on the pharmacokinetics of the anticancer agent, cyclophosphamide, and its active metabolite 4-hydroxycyclophosphamide. A cohort of 124 Caucasian patients received a high-dose chemotherapy combination consisting of cyclophosphamide (4-6 g/m2), thiotepa (320-480 mg/m2) and carboplatin (area under the curve 13-20 mg x min/ml) as intravenous infusions over 4 consecutive days. Genomic DNA was analysed using PCR and sequencing. Liquid chromatography-tandem mass spectrometry was used to measure plasma concentrations of cyclophosphamide and 4-hydroxycyclophosphamide. The relationship between allelic variants and the elimination pharmacokinetic parameters noninducible cyclophosphamide clearance (CL(nonind)), inducible cyclophosphamide clearance (CL(ind)) and elimination rate constant of 4-hydroxycyclophosphamide (k(4OHCP)) were evaluated using nonlinear mixed effects modelling. The interindividual variability in the noninducible cyclophosphamide clearance, inducible cyclophosphamide clearance and 4-hydroxycyclophosphamide clearance was 23, 27 and 31%, respectively. No effect of the allelic variants investigated on the clearance of cyclophosphamide or 4-hydroxycyclophosphamide could be demonstrated. This study indicates that the presently evaluated variant alleles in the CYP2B6, CYP2C9, CYP2C19, CYP3A4, CYP3A5, GSTA1, GSTP1, ALDH1A1 and ALDH3A1 genes do not explain the interindividual variability in cyclophosphamide and 4-hydroxycyclophosphamide pharmacokinetics and are, probably, not the cause of the observed variability in toxicity.

Serious haematological toxicity of cyclophosphamide in relation to CYP2B6, GSTA1 and GSTP1 polymorphisms

Cyclophosphamide (CPA) is widely used as an anticancer and immunosuppressive agent in various indications, and the dosage given may vary considerably depending on the disease treated. A recent review [1] has described the pharmacokinetics of CPA, a prodrug of which 70–80% of the administered dose is activated by cytochrome P450 (CYP) to the active alkylating species 4-hydroxycyclophosphamide (4OH-CPA). Several CYPs are responsible for CPA activation, mainly CYP2B6 and, secondarily, CYP2C9, CYP2C19 and CYP3A4/5 (Figure 1) [2]. Systemic exposure to cyclophosphamide metabolites after fixed doses of cyclophosphamide may vary by up to 10-fold between patients [1]. Genetic variants of CYPs involved in CPA metabolism may contribute to its pharmacokinetic variability and genetic polymorphisms of glutathione S-transferases may influence its toxicity. Figure 1 Cyclophosphamide metabolic pathways (adapted from Timm et al.[2]). The mutations (1) CYP2B6516TT, (2) CYP2C9*1/*3, (3) CYP3A5*3/*3, (4) GSTA1*A/*B, and (5) GSTP1*A/*B were identified in the presented case We report a case of a patient who experienced severe toxicity (pancytopenia) with small dosages of CPA (1.60 mg kg−1 day−1). A 25-year-old female patient presenting with extramembranous glomerulonephritis (with tubulointerstitial lesions) began treatment with cyclophosphamide in association with prednisone (40 mg day−1). She also received the following treatments: an angiotensin-converting enzyme inhibitor, an oral anticoagulant, a statin and recombinant erythropoietin. When cyclophosphamide treatment was initiated, creatinine clearance was 32 ml min−1, leucocytes 6500 μl−1, absolute neutrophil count (ANC) 4550 μl−1, platelet count 345 000 μl−1 and haemoglobin 11.6 g dl−1. After 14 days of treatment (day 14), the patient was admitted to hospital because of an intense asthenia accompanied by fever (38.8–39.5°C) which had appeared 78 h previously. The patient had ulcerative pharyngitis with dysphagia, about 20 pyodermitis lesions infected by Staphylococcus epidermidis, total body hair loss and painful diarrhoea. Severe neutropenia (ANC < 100 μl−1) and anaemia (haemoglobin 8.8 g dl−1) were observed, leucocyte and platelet absolute counts were 6000 μl−1 and 178 000 μl−1, respectively. Hepatic enzymes were within normal range. All the treatments were stopped and antibiotherapy was initiated in association with an erythropoiesis-stimulating agent and recombinant granulocyte colony-stimulating factor. At day 20 the platelet count was 34 000 μl−1 and haemoglobin 5.8 g dl−1. At day 21, the patient was apyretic. Aplasia ceased on day 22. Pharyngitis and pyodermitis were cured on day 23 and hairiness was normal 3 months after cyclophosphamide discontinuation. As all the drugs previously used were recommenced without any side-effect and as the clinical symptoms observed caused a CPA overdosage, we therefore suspected an alteration of CPA metabolism or detoxification pathways. Genetic variants of enzymes involved in these pathways were explored. Results were the following: CYP2B6516TT, CYP2C9*1/*3, CYP2C19*1/*1, CYP3A4*1 A/*1 A, CYP3A5*3/*3, GSTA1*A/*B, GSTM1+ (presence of gene), GSTT1+, GSTM3*B/*B and GSTP1*A/*B. The major side-effects observed were aplasia and total body hair loss. They are more likely to be observed when cyclophosphamide is used as a cancer treatment (500–4000 mg m−2 day−1) than for nonmalignant kidney disorders (glomerulonephritis) (100–200 mg m−2 day−1). As leukopenia, thrombocytopenia and anaemia are dose-related, we hypothesized that the aplasia observed in the present patient, despite the low dose of CPA, could be explained by enhanced 4OH-CPA exposure because of increased activation pathway of CPA into 4OH-CPA and less active detoxification pathways of 4OH-CPA and phosphoramide mustard. This was specially as the CLCr was 32 ml min−1 and hepatic function was normal. Indeed, Xie et al.[3] have demonstrated in vivo (patients with haematological malignancies) that the CYP2B6516T variant allele contribution to CPA elimination clearance was about twice that from wild-type gene. Moreover GSTA1*B/*B polymorphism, corresponding to lower detoxification, was correlated with greater survival than GSTA1*A/*B or GSTA1*A/*A in breast cancer therapy with CPA [4]. The detoxification pathway of anticancer drugs was decreased in carriers of GSTP1 variant alleles [5]. Nakajima et al.[6] have demonstrated in vitro that inhibition of GSTP1 enhances the antitumour activity of CPA. The genetic polymorphisms observed in our patient might contribute to decreased detoxification pathways and to increased exposure to 4OH-CPA and could explain the reported adverse effects. Further studies are needed to confirm these results.

Enhancement of intratumoral cyclophosphamide pharmacokinetics and antitumor activity in a P450 2B11-based cancer gene therapy model

The therapeutic utility of cytochrome P450-based enzyme prodrug therapy is well established by preclinical studies and in initial clinical trials. The underlying premise of this gene therapy is that intratumoral P450 expression leads to in situ activation of anticancer P450 prodrugs, such as cyclophosphamide (CPA), with intratumoral accumulation of its activated 4-OH metabolite. In mice bearing 9L gliosarcomas expressing the CPA 4-hydroxylase P450 2B6, enhanced tumor apoptosis was observed 48 h after CPA treatment; however, intratumoral 4-OH-CPA levels were indistinguishable from those of P450-deficient tumors, indicating that the bulk of activated CPA is derived from hepatic metabolism. In contrast, in 9L tumors expressing P450 2B11, a low K(m) CPA 4-hydroxylase, intratumoral 4-OH-CPA levels were higher than in blood, liver and P450-deficient tumors. Intratumoral 4-OH-CPA increased dose-dependently, without saturation at 140 mg kg(-1) CPA, suggesting restricted tumor cell permeation of the parent drug. To circumvent this problem, CPA was administered by direct intratumoral injection, which increased the maximum concentration and area under the curve of drug concentration x time (AUC) of intratumoral 4-OH-CPA by 1.8- and 2.7-fold, respectively. An overall 3.9-fold increase in intratumoral 4-OH-CPA AUC, and in antitumor activity, was obtained when CPA release to systemic circulation was delayed using the slow-release polymer poloxamer 407 as vehicle for intratumoral CPA delivery. These findings highlight the advantage of gene therapy strategies that combine low K(m) P450 prodrug activation enzymes with slow, localized release of P450 prodrug substrates.

Validation of urinary excretion of cyclophosphamide as a biomarker of exposure by studying its renal clearance at high and low plasma concentrations in cancer patients

Cyclophosphamide (CP) is an alkylating agent classified as a human carcinogen. Health care workers handling this drug may be exposed during, e.g., preparation or administration. Cyclophosphamide is readily absorbed by inhalation and by dermal uptake. A biomarker, CP in urine, has frequently been used to assess the occupational exposure to CP, but has not been fully validated. The aim of this study was to investigate if the proportion of the CP dose that is excreted in urine (renal clearance) is constant over different plasma drug concentrations and other pharmacokinetic parameters, e.g., urine flow. Pharmacokinetics of CP were studied in 16 breast cancer patients that were treated with postoperative adjuvant chemotherapy including CP. Plasma and urine from the patients were collected at different occasions up to 12 days after the dose. Urine was collected during 4-h periods and blood was sampled at the end of each period. Analysis of CP was performed by liquid chromatography tandem mass spectrometry. The limit of detection for CP in urine and plasma was 0.01 and 0.02 ng/ml, respectively. The precisions of the developed methods were determined to < or =8%. The administered doses of CP in absolute amounts ranged between 800 and 2,240 mg. Mean renal clearance of CP was 8.6 (confidence interval 6.5-10.7) ml/min and was not significantly dependent of the plasma drug concentration. However, a significant correlation between renal clearance and urine flow was observed. There was a large inter-individual variation in the plasma and urine concentrations even when the same doses were given. Cyclophosphamide in urine can be continued to be used as a biomarker to monitor occupational exposure to CP, however the inter-individual variability of excretion of CP in urine, and its dependency on urine flow must be taken into consideration in future applications.

Cyclophosphamide following Targeted Oral Busulfan as Conditioning for Hematopoietic Cell Transplantation: Pharmacokinetics, Liver Toxicity, and Mortality

The pharmacokinetics of cyclophosphamide (CY) and its metabolites hydroxycyclophosphamide and carboxyethylphosphoramide mustard were determined in 75 patients receiving targeted oral busulfan followed by i.v. CY ( TBU/CY) and in 147 patients receiving i.v. CY followed by total body irradiation (CY/TBI) in preparation for hematopoietic cell transplantation (HCT). In the TBU/CY patients only, the association of the pharmacokinetic data with liver toxicity, relapse, and survival was evaluated. CY was infused at 60 mg/kg/day over 1 or 2 hours on 2 consecutive days; the majority of patients had BU levels targeted to a steady state plasma concentration (Css) of 800-900 ng/mL. Systemic exposure (i.e., area under the concentration-time curve [AUC]) of CY, hydroxycyclophosphamide, and carboxyethylphosphoramide mustard was measured. Liver toxicity was assessed as the development of hepatic sinusoidal obstruction syndrome (SOS). CY metabolism was highly variable and age dependent. TBU/CY-treated patients had lower AUC CY ( P < .0001), higher AUC HCY ( P < .0001), and higher AUC CEPM ( P = .15) than CY/TBI-conditioned patients. Among patients receiving TBU/CY, 17 (23%) developed SOS, and there were no statistically significant associations between the AUC of CY or its metabolites and SOS, nonrelapse mortality, relapse, or survival (all P >.15). In conclusion, CY exhibits conditioning-regimen dependent pharmacokinetics and pharmacodynamics, suggesting that lowering CY doses is unlikely to improve outcomes to TBU/CY. Alternative strategies, such as administering i.v. busulfan or CY before BU, should be explored.

Genetic polymorphisms of CYP2B6 affect the pharmacokinetics/pharmacodynamics of cyclophosphamide in Japanese cancer patients

To evaluate the effects of genetic polymorphisms of drug metabolizing enzymes on the pharmacokinetics of cyclophosphamide and its active metabolite, 4-hydroxycyclophosphamide, and on the pharmacodynamics. One hundred and three Japanese patients with malignant lymphoma or breast cancer treated with cyclophosphamide (500-750 mg/m) participated in this study. The plasma concentrations of cyclophosphamide and 4-hydroxycyclophosphamide were determined by high-performance liquid chromatography, and pharmacokinetic parameters were calculated. The genotypes of CYP2B6, CYP2C19, CYP3A4, CYP3A5, ALDH1A1, GST genes were determined by allele-specific polymerase chain reaction or polymerase chain reaction-restriction-fragment length polymorphism. A large interindividual difference (54-fold) was observed in the area under the curve ratio of 4-hydroxycyclophosphamide/cyclophosphamide calculated as the metabolic index. We first proved that leukocytopenia and neutropenia were significantly (P<0.01) related to the area under the curve of 4-hydroxycyclophosphamide. We found that the homozygotes of CYP2B6*6 (Q172H and K262R) showed significantly (P<0.05) higher clearance and shorter half-life of cyclophosphamide than heterozygotes and homozygotes of CYP2B6*1. The small sample size, however, limited the impact. On the other hand, it was clearly demonstrated that the patients possessing the single nucleotide polymorphisms of the CYP2B6 gene, g.-2320T>C, g.-750T>C (5'-flanking region), g.15582C>T (intron 3), or g.18492T>C (intron 5), had significantly lower area under the curve ratios of 4-hydroxycyclophosphamide/cyclophosphamide, indicating a decreased cyclophosphamide 4-hydroxylation. Of particular importance was the finding that leukocytopenia was significantly related to the single nucleotide polymorphisms g.-2320T>C, g.-750T>C, and g.18492T>C in CYP2B6 gene, which are highly linked. No relationship was observed between the pharmacokinetics of cyclophosphamide or 4-hydroxycyclophosphamide and genetic polymorphisms of the other enzymes. We clarified that the single nucleotide polymorphisms in the promoter region or introns in the CYP2B6 affect the potency of cyclophosphamide activation to 4-hydroxycyclophosphamide. This information would be valuable for predicting adverse reactions and the clinical efficacy of cyclophosphamide.

191: The role of human CYP2B6 polymorphism in the bioactivation of cyclophosphamide using cDNA expressed enzymes

Cyclophosphamide (CPA) is used for the treatment of many tumors and as a part of the conditioning regimen prior to stem cell transplantation. CPA is a prodrug that is activated through cytochrome P450 2B6 (CYP2B6) in the liver. A high degree of inter- and intra-individual variation in CPA pharmacokinetics has been reported. We have investigated the formation of its active metabolite 4-hydroxycyclophosphamide (4-OH-CPA) in vitro using three different mutants of recombinant 2B6. The rate of formation of 4-OH-CPA was correlated to the enzyme activity for different mutations.

Population Pharmacokinetics and Pharmacodynamics of Doxorubicin and Cyclophosphamide in Breast Cancer Patients

To investigate the population pharmacokinetics and pharmacodynamics of doxorubicin and cyclophosphamide in breast cancer patients. Sixty-five female patients with early or advanced breast cancer received doxorubicin 60 mg/m(2) over 15 minutes followed by cyclophosphamide 600 mg/m(2) over 15 minutes. The plasma concentration-time data of both drugs were measured, and the relationship between drug pharmacokinetics and neutrophil counts was evaluated using nonlinear mixed-effect modelling. Relationships were explored between drug exposure (the area under the plasma concentration-time curve [AUC]), toxicity and tumour response. Fifty-nine patients had complete pharmacokinetic and toxicity data. In 50 patients with measurable disease, the objective response rate was 60%, with complete responses in 6% of patients. Both doxorubicin and cyclophosphamide pharmacokinetics were associated with neutrophil toxicity. Cyclophosphamide exposure (the AUC) was significantly higher in patients with at least stable disease (n = 44) than in patients with progressive disease (n = 6; 945 micromol . h/L [95% CI 889, 1001] vs 602 micromol . h/L [95% CI 379, 825], p = 0.0002). No such correlation was found for doxorubicin. Body surface area was positively correlated with doxorubicin clearance; AST and patient age were negatively correlated with doxorubicin clearance; creatinine clearance was positively correlated with doxorubicinol clearance; and occasional concurrent use of carbamazepine was positively correlated with cyclophosphamide clearance. The proposed inhibitory population pharmacokinetic-pharmacodynamic model adequately described individual neutrophil counts after administration of doxorubicin and cyclophosphamide. In this patient population, exposure to cyclophosphamide, as assessed by the AUC, might have been a predictor of the treatment response, whereas exposure to doxorubicin was not. A prospective study should validate cyclophosphamide exposure as a predictive marker for the treatment response and clinical outcome in this patient group.

Il-10 Polymorphism and TGF-β Haplotypes Are Associated with Rejection of Graft in Children with Beta Thalassemia Major Undergoing Matched Related Bone Marrow Transplantation (BMT).

Th2 cytokines, IL-10 and TGF-β have immunoregulatory functions that can affect allograft acceptance. We have evaluated the correlation between IL-10 (-1082, -819, -592) and TGF-β codon 10C>T and 25G>C polymorphisms {(haplotype: TT/GG, TC/GG, high production); (haplotype: TC/GC, CC/GG, TT/GC, intermediate production); (haplotype: CC/GC, CC/CC, TT/CC, TC/CC, low production)}and graft rejection in 129 patients with β thalassaemia major who underwent matched related alloBMT. Patients who expired before day 15 (n=9) and for whom DNA samples was unavailable (n=18) were excluded from this study. None of these patients had graft rejection. Of the 102 patients who were evaluated, 73 (72%) patients received conditioning with Bu (16 mg/kg) / Cy (200 mg/kg) with ALG while 29 (28%) patients received Bu (600 mg/m2) / Cy (200 mg/kg) without ALG. All patients received unmanipulated bone marrow and standard cyclosporine / short course methotrexate regimen as prophylaxis against GVHD. Patients who died before day 21 after transplant (n=2) without achieving ANC >500/mm3 were not evaluated for engraftment. 9 out of 102 patients rejected their graft. Apart from the immunogenetic markers, the effect of other risk factors for rejection like recipient and donor age, sex mismatch, conditioning regimen, cell dose and Lucarelli class were also analyzed. Of these, only increasing donor age was found to mildly increase the risk of rejection (RR=1.06; p=0.019; 95% CI-1.01–1.1). Donor TGF-β haplotypes associated with intermediate or low production and IL-10 polymorphism accounting for low production were found to very significantly increase the risk of graft rejection (Table). TGF-β and IL-10 are essential for both development and the effector function of CD4 regulatory T cells which are important for tolerance induction. Therefore, it is possible that genotypes associated with lower production of these cytokines might contribute to increased graft rejection. We have previously reported that rejection in children with β thalassemia major is influenced by pharmacokinetics of busulfan and cyclophosphamide and genes implicated in their metabolism (BMT 2005 36, 839–845). We conclude that rejection of graft in this condition appears to be a multifactorial event affected not only by pharmacogenetics of the drugs used for conditioning but also by various immunogenetic factors.Risk factorsRelative riskP-value95% CITGFβ haplotypesLow + intermediate6.970.0151.4 – 33.6HighReferenceIL-10 -819 TT5.340.0181.3 – 21.4-819 TC + CCReferenceIL-10 -592 AA5.340.0181.3 – 21.4-592 AC + CCReference

Impact of polymorphisms in the CYP2B6 gene on the pharmacokinetics of cyclophosphamide and thiotepa

13062 Background: Cyclophosphamide and thiotepa are frequently used simultaneously as part of high dose chemotherapy regimens for solid tumours. Although generally well tolerated, severe toxicity may occur. Relationships between pharmacokinetics and toxicity have been identified. Cyclophosphamide and thiotepa are both metabolised by cytochrome P450 (CYP)enzymes, resulting in the active metabolites 4-hydroxycyclophosphamide and tepa respectively. CYP2B6 and CYP3A4 are the major metabolising isozymes for both compounds. In this study we investigated the relationship between polymorphisms of the CYP2B6 gene and the hepatic clearance of cyclophosphamide and thiotepa in order to identify a priori patients at risk for severe toxicity. Methods: CYP2B6*2 (C64T) and variants A785G, C1459T and G516T were identified by PCR and sequencing samples from 82 patients receiving a high dose regimen consisting of cyclophosphamide (4–6 g/m2), thiotepa (320–480 mg/m2) and carboplatin (AUC 13–20 mg*min/ml). Plasma concentrations of thiotepa, cyclophosphamide and metabolites were determined using a validated LC-MS/MS method. Individual pharmacokinetic parameters were obtained using nonlinear mixed effects modelling. Results: A total of 82 patients was included. Not all data of the various variants were available. All investigated variants had an allele frequency of &gt; 3% in the population. While 94% of the patients had the wildtype genotype for CYP2B6*2, 6% were heterozygous. Heterozygous patients for *2 had a lower hepatic clearance of thiotepa compared to wildtype patients (11.7 l*h−1 vs 14.4 l*h−1) (P=0.03). All other allelic variants investigated showed no effect on either hepatic clearance of thiotepa or cyclophosphamide. Conclusions: This study demonstrates that the hepatic clearance of thiotepa is reduced in patients with a heterozygous CYP2B6*2 genotype. Patients with this genotype may have an increased exposure to thiotepa and may, therefore, be at increased risk for organ toxicity. No significant financial relationships to disclose.

Clinical Pharmacology of Cyclophosphamide and Ifosfamide

The oxazaphosphorine cyclophosphamide (CPA) and ifosfamide (IFO) are two commonly used DNA- alkylating agents in cancer chemotherapy. This review highlights the pharmacokinetics and pharmacodynamics of the two important agents. As alkylating agents, CPA and IFO are usually combined with other anticancer drugs in the chemotherapy of solid tumors and hematological malignancies to obtain synergistic or additive anticancer effect due to complementary mechanism of action. Both compounds are prodrugs that are activated via 4-hydroxylation by cytochrome P450s such as CYP2B6 and CYP3A4 to generate alkylating nitrogen mustards (phosphoramide mustard and ifosforamide mustard) and the byproduct acrolein. The resultant mustards can alkylate DNA to form DNA-DNA cross-links, leading to inhibition of DNA synthesis and cell apoptosis. Both CPA and IFO are also inactivated by N-dechloroethylation, resulting in N-dechloroethylated metabolites and the byproduct chloroacetaldehyde. Acrolein is the causative agent for hemorrhagic cystitis, whereas chloroacetaldehyde induces nephrotoxicity and neurotoxicity. Pharmacokinetics of CPA and IFO is markedly influenced by route of administration and duration of treatment, age, comedication, liver and renal function. Large interpatient variability in pharmacokinetics, clinical response rate and toxicity has been observed in cancer patients treated with CPA or IFO. Resistance to CPA or IFO occurs due to decreased activation by CYP3A4 and CYP2B6, increased deactivation of the agents, decreased entry into or increased efflux from tumor cells, increased cellular thiol level, increased DNA repair capacity, and/or deficient apoptotic response to DNA damage. A full understanding of factors affecting the pharmacokinetics, pharmacodynamics, toxicology and pharmacogenetics of CPA and IFO is important to optimize the dose and regimens of CPA and IFO in cancer chemotherapy.

Population Pharmacokinetics of Cyclophosphamide and Its Metabolites 4-Hydroxycyclophosphamide, 2-Dechloroethylcyclophosphamide, and Phosphoramide Mustard in a High-Dose Combination With Thiotepa and Carboplatin

The anticancer prodrug cyclophosphamide (CP) is activated by the formation of 4-hydroxycyclophosphamide (4OHCP), which decomposes into phosphoramide mustard (PM). This activation pathway is inhibited by thiotepa. CP is inactivated by formation of 2-dechloroethylcyclophosphamide (2DCECP). The aim of this study was to develop a population pharmacokinetic model describing the complex pharmacokinetics of CP, 4OHCP, 2DCECP, and PM when CP is administered in a high-dose combination with thiotepa and carboplatin. Patients received a combination of CP (1000-1500 mg/m/d), carboplatin (265-400 mg/m/d), and thiotepa (80-120 mg/m/d) administered in short infusions over 4 days. Twenty blood samples were collected per patient per course. Concentrations of CP, 4OHCP, 2DCECP, PM, thiotepa, and tepa were determined in plasma. Using NONMEM, an integrated population pharmacokinetic model was used to describe the pharmacokinetics of CP, 4OHCP, 2DCECP, and PM, including the already described processes of autoinduction of CP and the interaction with thiotepa. Data were available on 35 patients (70 courses). The pharmacokinetics of CP were described with a 2-compartment model, and those of 4OHCP, 2DCECP, and PM with 1-compartment models. Before onset of autoinduction, it was assumed that CP is eliminated through a noninducible pathway accounting for 20% of total CP clearance, whereas 2 inducible pathways resulted in formation of 4OHCP (75%) and 2DCECP (5%). It was assumed that 4OHCP was fully converted to PM. Induction of CP metabolism was mediated by 2 hypothetical amounts of enzyme whose quantities increased in time in the presence of CP (kenz=0.0223 and 0.0198 hours). Induction resulted in an increased formation of 4OHCP (approximately 50%), PM (approximately 50%), and 2DCECP (approximately 35%) during the 4-day course, and concomitant decreased exposure to CP (approximately 50%). The formation of 2DCECP was not inhibited by thiotepa. Apparent volumes of distribution of CP, PM, and 2DCECP could be estimated being 43.7, 55.5, and 18.5 L, respectively. Exposure to metabolites varied up to 9-fold. The complex population pharmacokinetics of CP, 4OHCP, 2DCECP, and PM in combination with thiotepa and carboplatin has been established and may form the basis for further treatment optimization with this combination.

Effects of polysaccharide peptide (PSP) from Coriolus versicolor on the pharmacokinetics of cyclophosphamide in the rat and cytotoxicity in HepG2 cells

Polysaccharide peptide (PSP), isolated from Coriolus versicolor COV-1, has been shown to restore the immunological effects against cyclophosphamide-induced immuno-suppression, although the mechanism(s) involved remain uncertain. This study investigated the PSP-cyclophosphamide interaction by studying the effects of PSP on the pharmacokinetic of cyclophosphamide in the rat and the effect of PSP on the cytotoxic effects of cyclophosphamide on a cancer cell line (HepG2 cells). In the pharmacokinetic studies in the rat, acute pre-treatment of PSP (4 μmol/kg/day, i.p.) decreased the clearance (CL) of cyclophosphamide by 31%, with a concomitant increase in the area under concentration–time curve (AUC) by 44%, and prolongation of the plasma half-life ( T 1/2) by 43%. Sub-chronic pre-treatment of PSP (2 μmol/kg/day, i.p., 3 days) decreased the CL of cyclophosphamide by 33%, with a concomitant increase in the AUC by 50%, and prolongation of the plasma T 1/2 by 34%. In cytotoxicity studies using HepG2 cells, non-toxic dose of PSP (1–10 μM) enhanced the cytotoxicity of cyclophosphamide. PSP at 10 μM further decreased HepG2 cell viability by 22% compared to when cyclophosphamide was present alone. In summary, PSP enhanced the cytotoxic effect of cyclophosphamide on a cancer cell line in vitro and altered the pharmacokinetics of cyclophosphamide in vivo in the rat. Both of these effects may be beneficial in the use of PSP as an adjunct to cyclophosphamide treatment.

Pharmacogenetics of cyclophosphamide in patients with hematological malignancies

Purpose A high degree of interindividual variation in cyclophosphamide (CPA) pharmacokinetics was reported in certain cancer patient groups. To better understand the mechanisms underlying the variation in CPA metabolism, we have investigated the pharmacokinetics of CPA and its active metabolite 4-hydroxycyclophosphamide (4-OH-CPA) in patients with hematological tumors. The pharmacokinetics of CPA and its active metabolite were related to the genotype of CYP2B6, CYP2C9 and CYP2C19. The influence of liver function on CPA metabolism was also evaluated. Methods: Twenty-nine patients with hematological malignancies (MM, ALL or NHL) treated with a conventional CPA dose (1 g/m 2) were recruited to this study. Blood samples were collected before, during and after CPA treatment. HPLC was used to measure plasma concentrations of CPA and 4-OH-CPA. Patients were genotyped for the CYP2B6 G516T, CYP2C9*2, CYP2C9*3, CYP2C19*2 and CYP2C19*3 alleles. Serum bilirubin levels were measured before the treatment. Data was analyzed individually and by population pharmacokinetic methods, using non-linear mixed effect modeling. Results: The interindividual variability in exposure to CPA, 4-OHCPA and 4-OH-CPA/CPA was 5.8-, 3.3- and 10.3-fold, respectively. A positive correlation between half-lives of CPA and 4-OH-CPA was found while a significant negative correlation between AUCs of CPA and 4-OH-CPA was detected. In the population analysis, the CYP2B6 G516T variant allele contribution to CPA clearance was about twice as the contribution from the wild type gene while the genotype of CYP2C9 and CYP2C19 did not influence clearance. A negative correlation was observed between bilirubin level and CPA bioactivation. Conclusion: This study demonstrates for the first time that the presence of the CYP2B6 G516T mutation increases the rate of 4-OH-CPA formation in patients with hematological malignancies. The liver function prior therapy as assessed by s-bilirubin influences CPA metabolism.

Human Multidrug Resistance Associated Protein 4 Confers Resistance to Camptothecins

The multidrug resistance associated protein (MRP) 4 is a member of the adenosine triphosphate (ATP)-binding cassette transporter family. Camptothecins (CPTs) have shown substantial anticancer activity against a broad spectrum of tumors by inhibiting DNA topoisomerase I, but tumor resistance is one of the major reasons for therapeutic failure. P-glycoprotein, breast cancer resistance protein, MRP1, and MRP2 have been implicated in resistance to various CPTs including CPT-11 (irinotecan), SN-38 (the active metabolite of CPT-11), and topotecan. In this study, we explored the resistance profiles and intracellular accumulation of a panel of CPTs including CPT, CPT-11, SN-38, rubitecan, and 10-hydroxy-CPT (10-OH-CPT) in HepG2 cells with stably overexpressed human MRP4. Other anticancer agents such as paclitaxel, cyclophosphamide, and carboplatin were also included. HepG2 cells were transfected with an empty vehicle plasmid (V/HepG2) or human MRP4 (MRP4/HepG2). The resistance profiles of test drugs in exponentially growing V/HepG2 and MRP4/HepG2 cells were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay with 4 or 48 h exposure time of the test drug in the absence or presence of various MRP4 inhibitors. The accumulation of CPT-11, SN-38, and paclitaxel by V/HepG2 and MRP4/HepG2 cells was determined by validated high-performance liquid chromatography methods. Based on the resistance folds from the MTT assay with 48 h exposure time of the test drug, MRP4 conferred resistance to CPTs tested in the order 10-OH-CPT (14.21) > SN-38 carboxylate (9.70) > rubitecan (9.06) > SN-38 lactone (8.91) > CPT lactone (7.33) > CPT-11 lactone (5.64) > CPT carboxylate (4.30) > CPT-11 carboxylate (2.68). Overall, overexpression of MRP4 increased the IC50 values 1.78- to 14.21-fold for various CPTs in lactone or carboxylate form. The resistance of MRP4 to various CPTs tested was significantly reversed in the presence of dl-buthionine-(S,R)-sulfoximine (BSO, a gamma-glutamylcysteine synthetase inhibitor), MK571, celecoxib, or diclofenac (all MRP4 inhibitors). In addition, the accumulation of CPT-11 and SN-38 over 120 min in MRP4/HepG2 cells was significantly reduced compared to V/HepG2 cells, whereas the addition of celecoxib, MK571, or BSO significantly increased their accumulation in MRP4/HepG2 cells. There was no significant difference in the intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells, indicating that P-glycoprotein was not involved in the observed resistance to CPTs in this study. MRP4 also conferred resistance to cyclophosphamide and this was partially reversed by BSO. However, MRP4 did not increase resistance to paclitaxel, carboplatin, etoposide (VP-16), 5-fluorouracil, and cyclosporine. Human MRP4 rendered significant resistance to cyclophosphamide, CPT, CPT-11, SN-38, rubitecan, and 10-OH-CPT. CPT-11 and SN-38 are substrates for MRP4. Further studies are needed to explore the role of MRP4 in resistance, toxicity, and pharmacokinetics of CPTs and cyclophosphamide.