Simultaneous quantification of pirarubicin, doxorubicin, cyclophosphamide, and vincristine in human plasma of patients with non-Hodgkin's lymphoma by LC-MS/MS method.

Pirarubicin (THP), doxorubicin (DOX), cyclophosphamide (CTX), and vincristine (VCR) are widely used in the treatment of patients with non-Hodgkin's Lymphoma. Herein, a precise and sensitive method was developed for the determination of THP, DOX, CTX and VCR in human plasma by high-performance liquid-chromatography-tandem mass spectrometry (LC-MS/MS). Liquid-liquid extraction was applied to extract THP, DOX, CTX, VCR, and the internal standard (IS, Pioglitazone) in plasma. Agilent Eclipse XDB-C18 (3.0 mm × 100 mm) was utilized and chromatographic separation was obtained in eight minutes. Mobile phases were composed of methanol and buffer (10 mM ammonium formate containing 0.1% formic acid). The method was linear within the concentration range of 1-500 ng/mL for THP, 2-1000 ng/mL for DOX, 2.5-1250 ng/mL for CTX, and 3-1500 ng/mL for VCR. The intra- and inter-day precisions of QC samples were found to be below 9.31 and 13.66%, and accuracy ranged from -0.2 to 9.07%, respectively. THP, DOX, CTX, VCR and the internal standard were stable in several conditions. Finally, this method was successfully utilized to simultaneously determine THP, DOX, CTX and VCR in human plasma of 15 patients with non-Hodgkin's Lymphoma after intravenous administration. Finally, the method was successfully employed in the clinical determination of THP, DOX, CTX, and VCR in patients with non-Hodgkin lymphoma after administration of RCHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone) regimens.

A phase I study of veliparib with cyclophosphamide and veliparib combined with doxorubicin and cyclophosphamide in advanced malignancies.

PURPOSE: Veliparib (V), an oral poly(ADP-ribose) polymerase (PARP) inhibitor, potentiates effects of alkylating agents and topoisomerase inhibitors in preclinical tumor models. We conducted a phase I trial of V with iv cyclophosphamide (C) and V plus iv doxorubicin (A) and C. METHODS: Objectives were to establish the maximum tolerated dose (MTD) of the combinations, characterize V pharmacokinetics (PK) in the presence and absence of C, measure PAR in peripheral blood mononuclear cells (PBMCs) and γH2AX in circulating tumor cells (CTCs). In Group 1, dose escalations of V from 10 to 50 mg every 12 h Days 1-4 plus C 450 to 750 mg/m2 Day 3 in 21-day cycles were evaluated. In Group 2, V doses ranged from 50 to 150 mg every 12 h Days 1-4 with AC (60/600 mg/m2) Day 3 in 21-day cycles. In Group 3, patients received AC Day 1 plus V Days 1-7, and in Group 4, AC Day 1 plus V Days 1-14 was given in 21-day cycles to evaluate effects on γH2AX foci. RESULTS: Eighty patients were enrolled. MTD was not reached for V and C. MTD for V and AC was V 100 mg every 12 h Days 1-4 with AC (60/600 mg/m2) Day 3 every 21 days. V PK appears to be dose-dependent and has no effect on the PK of C. Overall, neutropenia and anemia were the most common adverse events. Objective response in V and AC treated groups was 22% (11/49). Overall clinical benefit rate was 31% (25/80). PAR decreased in PBMCs. Percentage of γH2AX-positive CTCs increased after treatment with V and AC. CONCLUSION: V and AC can be safely combined. Activity was observed in patients with metastatic breast cancer.

Population pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide metabolite in patients with autoimmune glomerulonephritis.

OBJECTIVES: To develop a simultaneous population pharmacokinetic model of cyclophosphamide (CY) and 4-hydroxycyclophosphamide (4-OH) in patients with glomerulonephritis. METHODS: In total, 23 patients participated in a pharmacokinetic evaluation using dense plasma sampling. A population pharmacokinetic model was developed in Monolix Suite 2020R1 that simultaneously describes the kinetics of CY and 4-OH. Several structural and residual error models were evaluated and patient variables were tested as potential covariates. The final model was selected based on visual predictive check and bootstrap. Simulations of plasma concentrations for various doses were conducted. KEY FINDINGS: A model including two compartments for CY and one for 4-OH was found to best describe the data. A proportional error model for both compounds was chosen. The following estimates were found for the main CY pharmacokinetic parameters: total clearance, 13.3 l/h with inter-individual variability (IIV) 32%, and central volume of distribution, 59.8 l with IIV 12%. The metabolite elimination rate constant was 4.3 h-1 with IIV 36% and the proportion of metabolism 64%. Sex was a significant covariate on the central volume of CY, with females exhibiting 25% lower value than males. CONCLUSIONS: A population pharmacokinetic model was developed for CY and 4-OH in patients with autoimmune glomerulonephritis. Simulations using various dose regimens allow for informed dosing before the initiation of therapy.

Phenotyping Study of Cyclophosphamide 4-Hydroxylation in Malay Cancer Patients.

BACKGROUND: Cyclophosphamide (CP) is an anticancer alkylating group (nitrogen mustard) and a prodrug that will be metabolized to form its active metabolite, 4-hydroxycyclophosphamide (4-OHCP). The various enzymes involved in its bioactivation can cause a wide range of CP expression and activity among patients and ultimately affect the metabolism, efficacy and toxicity of this drug. The effectiveness of CP therapy can be determined by 4-OHCP level in dried blood spot (DBS). AIM: The purpose of this study was to conduct the phenotyping of CP 4-hydroxylation rate in Malay cancer patients. METHODOLOGY: Phenotyping study of CP 4-hydroxylation rate to 40 subjects of Malay cancer patients was done based on the value of its bioactivity ratio (4-OHCP to CP levels). RESULTS: The result shown the cyclophosphamide 4-hydroxylation rate of 80% (n=32) subjects as ultrarapid metabolizer (UM) and 20% (n=8) as poor metabolizer (PM). CONCLUSION: Phenotyping study of CP 4-hydroxylation in Malay cancer patients can be conducted by quantifying CP bioactivity ratio (4-OHCP to CP level) in dried blood spot. In majority of Malay cancer patients, cyclophosphamide would be bioactivated through 4-hydroxylation in hepar rapidly as indicated by the high value of the bioactivity ratio or the increased CP clearance and 4-OHCP level.

The Influence of Different Triazole Antifungal Agents on the Pharmacokinetics of Cyclophosphamide.

Background: Cyclophosphamide is one of the most important chemotherapeutic drugs. Known as a widely accepted treatment strategy, chemotherapy may damage the immune function of cancer patients; as a result, invasive fungal infections (IFIs) occur. Triazole antifungal agents are the most acceptable drugs for IFI treatment, especially those infections caused by chemotherapy. Objective: We aimed to investigate the effects of different triazole antifungal drugs, including fluconazole, itraconazole, and ketoconazole, on the pharmacokinetics (PK) of cyclophosphamide. In addition, we also characterize the potential drug-drug interactions (DDIs) between cyclophosphamide and various triazole antifungal drugs. Methods: The necessary pharmacokinetic parameters and physicochemical data were obtained from published studies. Physiologically based pharmacokinetic (PBPK) models were developed and validated in virtual subjects using Simcyp software. The validated PBPK models were used to evaluate potential DDIs between cyclophosphamide and different triazole antifungal agents in cancer patients. Triazole antifungal agents were simulated by oral administration, whereas cyclophosphamide was simulated by intravenous administration. Results: Simulated plasma concentration-time curves of fluconazole, itraconazole, ketoconazole, and cyclophosphamide were in good consistency with the observed profiles. Our results suggested that the pharmacokinetic parameters of cyclophosphamide were increased by various extents when coadministered with different triazole antifungals. The area under the plasma concentration-time curve of cyclophosphamide was increased when combined with fluconazole, itraconazole, or ketoconazole. Conclusions and Relevance: Ketoconazole had the greatest effect on the PK of cyclophosphamide among the 3 triazole antifungals. Our study provides clues that the toxicity and adverse drug reactions that are associated with cyclophosphamide should be closely monitored when coadministered with ketoconazole.

Biomonitoring of pharmacists and nurses at occupational risk from handling antineoplastic agents.

OBJECTIVE: The objective of this study was to evaluate the frequency of genetic lesions in pharmacists and nurses who prepare and/or handle antineoplastic agents and to evaluate whether there are traces of contaminants in the urine of these professionals. METHODS: A total of 59 professionals participated in the study, of which 10 were non-exposed professionals (controls), 25 were pharmacists, and 24 were nurses. KEY FINDINGS: There was a significant increase in genetic damage in lymphocytes and cells of the oral mucosa in both pharmacists and nurses. The levels of cyclophosphamide and ifosfamide were also increased in the urine samples from those individuals. CONCLUSIONS: These results demonstrate the growing need for genetic biomonitoring and biomonitoring of trace antineoplastic agents in the urine of health professionals who prepare and/or handle antineoplastic agents.

Exposure-Toxicity Association of Cyclophosphamide and Its Metabolites in Infants and Young Children with Primary Brain Tumors: Implications for Dosing.

PURPOSE: To characterize the population pharmacokinetics of cyclophosphamide, active 4-hydroxy-cyclophosphamide (4OH-CTX), and inactive carboxyethylphosphoramide mustard (CEPM), and their associations with hematologic toxicities in infants and young children with brain tumors. To use this information to provide cyclophosphamide dosing recommendations in this population. PATIENTS AND METHODS: Patients received four cycles of a 1-hour infusion of 1.5 g/m2 cyclophosphamide. Serial samples were collected to measure cyclophosphamide, 4OH-CTX, and CEPM plasma concentrations. Population pharmacokinetic modeling was performed to identify the patient characteristics influencing drug disposition. Associations between drug exposures and metrics reflecting drug-induced neutropenia, erythropenia, and thrombocytopenia were investigated. A Bayesian approach was developed to predict 4OH-CTX exposure using only cyclophosphamide and CEPM plasma concentrations. RESULTS: Data from 171 patients (0.07-4.9 years) were adequately fitted by a two-compartment (cyclophosphamide) and one-compartment model (metabolites). Young infants (<6 months) exhibited higher mean 4OH-CTX exposure than did young children (138.4 vs. 107.2 µmol/L·h, P < 0.0001). No genotypes exhibited clinically significant influence on drug exposures. Worse toxicity metrics were significantly associated with higher 4OH-CTX exposures. Dosing simulations suggested decreased cyclophosphamide dosage to 1.2 g/m2 for young infants versus 1.5 g/m2 for children to attain similar 4OH-CTX exposure. Bayesian-modeled 4OH-CTX exposure predictions were precise (mean absolute prediction error 14.8% ± 4.2%) and had low bias (mean prediction error 4.9% ± 5.1%). CONCLUSIONS: A 4OH-CTX exposure-toxicity association was established, and a decreased cyclophosphamide dosage for young infants was suggested to reduce toxicity in this population. Bayesian modeling to predict 4OH-CTX exposure may reduce clinical processing-related costs and provide insights into further exposure-response associations.

CNS Penetration of Cyclophosphamide and Metabolites in Mice Bearing Group 3 Medulloblastoma and Non-Tumor Bearing Mice.

PURPOSE: Cyclophosphamide is widely used to treat children with medulloblastoma; however, little is known about its brain penetration. We performed cerebral microdialysis to characterize the brain penetration of cyclophosphamide (130 mg/kg, IP) and its metabolites [4-hydroxy-cyclophosphamide (4OH-CTX) and carboxyethylphosphoramide mustard (CEPM)] in non-tumor bearing mice and mice bearing orthotopic Group 3 medulloblastoma. METHODS: A plasma pharmacokinetic study was performed in non-tumor-bearing CD1- nude mice, and four cerebral microdialysis studies were performed in non-tumor-bearing (M1 and M3) and tumor- bearing mice (M2 and M4). Plasma samples were collected up to 6-hours post-dose, and extracellular fluid (ECF) samples were collected over 60-minute intervals for 24-hours post-dose. To stabilize and quantify 4OH-CTX, a derivatizing solution was added in blood after collection, and either directly in the microdialysis perfusate (M1 and M2) or in ECF collection tubes (M3 and M4). Plasma/ECF cyclophosphamide and CEPM, and 4OH-CTX concentrations were separately measured using different LC-MS/MS methods. RESULTS: All plasma/ECF concentrations were described using a population-based pharmacokinetic model. Plasma exposures of cyclophosphamide, 4OH-CTX, and CEPM were similar across studies (mean AUC=112.6, 45.6, and 80.8 µmol∙hr/L). Hemorrhage was observed in brain tissue when the derivatizing solution was in perfusate compared with none when in collection tubes, which suggested potential sample contamination in studies M1 and M2. Model-derived unbound ECF to plasma partition coefficients (Kp,uu) were calculated to reflect CNS penetration of the compounds. Lower cyclophosphamide Kp,uu was obtained in tumor-bearing mice versus non-tumor bearing mice (mean 0.15 versus 0.22, p=0.019). No differences in Kp,uu were observed between these groups for 4OH- CTX and CEPM (overall mean 0.10 and 0.07). CONCLUSIONS: Future studies will explore potential mechanisms at the brain-tumor barrier to explain lower cyclophosphamide brain penetration in tumor-bearing mice. These results will be used to further investigate exposure-response relationships in medulloblastoma xenograft models.

A high-specificity immunoassay for the therapeutic drug monitoring of cyclophosphamide.

Personalized medicine is pushing forward new diagnostic techniques to aid in controlling drug therapeutic levels and their toxic effects. This study aims to develop a high-throughput screening method for therapeutic drug monitoring (TDM) and occupational exposure of cyclophosphamide (CP), an alkylating agent used as a chemotherapeutic and immunosuppressive drug. In order to achieve this goal, an immunizing hapten that exposes the cyclophosphamide moiety has been designed for the first time. Antibodies produced against this hapten have been used to develop an indirect competitive ELISA for the quantification of CP with high specificity and low cross-reactivity with some metabolites and other anticancer drugs. The assay obtained showed a LOD of 22 ± 6 nM in serum samples, with concentrations much below the blood CP levels of patients treated with the drug. A new tool for the detection and quantification of CP is provided which could be relevant for future pharmacokinetic studies and for therapeutic index improvement.

Accurate Prediction of Initial Busulfan Exposure Using a Test Dose With 2- and 6-Hour Blood Sampling in Adult Patients Receiving a Twice-Daily Intravenous Busulfan-Based Conditioning Regimen.

This study aimed to predict the area under the curve (AUC) of the initial busulfan dose using a test dose with the sparse sampling scheme in adult patients who underwent hematopoietic cell transplant. A test dose of 0.8 mg/kg busulfan was used 2 days before twice-daily intravenous busulfan-based conditioning regimens were administered. The AUC and the clearance (CL) were calculated for both the test dose and the first dose (AUCT , CLT , AUC1, and CL1 ) by noncompartmental analysis. The sparse sampling schemes of the test dose were developed by Bayesian method based on the population pharmacokinetic model. The optimal sparse sampling schemes were determined by evaluating the mean prediction error, the root mean square error, the absolute mean prediction error, and Bland-Altman plot. The mean AUC1 was 7.20 ± 1.48 mg • h/L, which ranged from 4.70 to 9.46 mg • h/L. The AUC1 was below the therapeutic concentration of 7.38 mg • h/L in 45% (9 of 20) of the patients. The CLT of 3.05 ± 0.56 mL/min/kg was not significantly different with the CL1 of 3.03 ± 0.69 mL/min/kg (P = .901). A sampling scheme at 2 and 6 hours after the test dose was developed to predict the AUCT (mean prediction error of 1.64%, root mean square error of 6.17%, and absolute mean prediction error of 4.94%). Additionally, the Bland-Altman plot showed that the 2-sampling scheme provided an acceptably accurate prediction of the AUC1 . A test dose with a 2-sampling scheme was sufficient to personalize the initial busulfan dosing in hematopoietic cell transplant recipients.

Ultrasensitive SERS Substrate for Label-Free Therapeutic-Drug Monitoring of Paclitaxel and Cyclophosphamide in Blood Serum.

Surface-enhanced Raman spectroscopy (SERS) has recently emerged as an innovative tool for therapeutic-drug monitoring (TDM), making it an ideal candidate for personalized treatment. Herein, we report a layer-by-layer (LbL) approach for the fabrication of a highly reproducible hybrid SERS substrate based on graphene oxide (GO)-supported l-cysteine-functionalized starlike gold nanoparticles (SAuNPs). These designed substrates were utilized for TDM of paclitaxel and cyclophosphamide in blood serum. The SAuNPs' efficient binding at the edges of GO creates a better SERS hotspot with enhanced Raman sensitivity because of the spacing of ∼2.28 nm between the SAuNPs. In addition, the hierarchically modified substrate with a self-assembled monolayer of zwitterionic amino acid l-cysteines acts like a brush layer to prevent SERS-hotspot blockages and fouling by blood-serum proteins. The antifouling nature of the substrate was determined quantitatively by a bichinchonic acid assay using bovine-serum albumin (BSA) as a protein model on the l-cysteine SAuNPs@GO hybrid substrate (the test) and a cysteamine SAuNPs@GO substrate (the control). The l-cysteine SAuNPs@GO hybrid exhibited 80.57% lower BSA fouling compared with that of the cysteamine SAuNPs@GO substrate. The SERS spectra were acquired within 20 s, with detection limits of 1.5 × 10-8 M for paclitaxel and 5 × 10-9 M for cyclophosphamide in blood serum. Such sensitivities are 4 times and 1 order of magnitude higher than the currently available sophisticated analytical techniques, which involve high costs with each analysis.

RP-HPLC-UV Method for Estimation of Fluorouracil-Epirubicin-Cyclophosphamide and Their Metabolite Mixtures in Human Plasma (Matrix).

A combination of 5-fluorouracil (FU), epirubicin (EP) and cyclophosphamide (CP) is routinely employed in the treatment of breast cancer. The objective of this study was to develop a reverse phase high-performance liquid chromatography (HPLC)-UV method for simultaneous quantitative analysis of the triple-drug and their metabolites in plasma. RP-HPLC system with a C18 column and a diode array detector was employed. The plasma samples were precipitated with acetonitrile and the supernatant was dried under a flow of nitrogen gas. The mobile phase comprised of two combinations, water (pH 4.0) and methanol (98:2 v/v), and water (pH 4.0):methanol:acetonitrile (70:13:17 v/v/v). The retention times for the compounds were determined and the parameters of validation established in plasma indicated the robustness and reliability. The corresponding HPLC peaks were confirmed using electron spray ionization mass spectrometry. FU and metabolites had a recovery of >93%; EP, epirubicinol and CP were >78% from plasma. Stability at 28-30°C in water (pH 4.0) of FU, 5,6-dihydro-5-fluorouracil and EP were higher followed by CP, EPol, fluorodeoxyuridine and fluorouridine (FUR). Storage of the drug-spiked plasma at -80°C assessed for 72 h showed a small but significant (P < 0.05) change in the recovery of FUR and EP. The method was validated in patient's plasma samples (n = 6).

A sensitive and rapid ultra high-performance liquid chromatography with tandem mass spectrometric assay for the simultaneous quantitation of cyclophosphamide and the 4-hydroxycyclophosphamide metabolite in human plasma.

Analysis of cyclophosphamide (CP) and its metabolite, 4-hydroxycyclophosphamide (4OHCP), in a single assay has the ability to improve sampling techniques benefitting both the patients who are receiving the drug and the clinicians drawing samples. Due to instability in plasma (t1/2 = 4 min), immediate stabilization of 4OHCP with phenylhydrazine is necessary upon sample draw. After stabilization, 4OHCP and the stable CP prodrug concentrations can be analytically measured to elucidate the pharmacokinetics, including half-life and exposure parameters (Cmax and AUC). For this purpose, a sensitive analytical method was developed to measure both the prodrug and active metabolite. A liquid-liquid extraction recovered the analytes prior to analysis with an ultra HPLC-MS/MS. A Thermo Scientific™ Hypersil™ BDS C18, 2.1 × 100 mm, 3.0 µm column was used for compound separation. Mass transitions for CP (m/z 261.0 ➔ 140.0), the internal standard d4-CP (m/z 265.0 ➔ 140.0), 4OHCP (m/z 367.3 ➔ 147.1), and the internal standard AZD7451 (m/z 383.4 ➔ 341.1) were monitored over a calibration range of 34.24-34,240 ng/mL and 3.424-3424 ng/mL for CP and 4OHCP, respectively. Each calibration range proved accurate (<15% deviation) and precise (<15% RSD) for the desired compound. Using this method, CP and 4OHCP plasma levels can be measured in clinical samples from patients receiving this therapy.

Optimization of rituximab for the treatment of DLBCL: increasing the dose for elderly male patients.

Male sex is associated with unfavourable pharmacokinetics and prognosis in elderly patients with diffuse large B-cell lymphoma (DLBCL). We investigated higher rituximab doses for elderly male DLBCL patients. Elderly patients (61-80 years) received 6 cycles CHOP-14 (cyclophosphamide, doxorubicin, vincristine and prednisone at 14-day intervals) and were randomized to 8 cycles rituximab (males 500 mg/m2 , females 375 mg/m2 ) every 2 weeks or according to an upfront dose-dense schedule. In 268 (120 females, 148 males) no difference between the standard and the upfront dose-dense rituximab schedule was found (3-year PFS 72% vs. 74%; OS 74% vs. 77%; P = 0.651). The 500 mg/m2 dose of rituximab for male patients was associated with serum levels and exposure times slightly better than in females and a male/female hazard ratio of 0.9 for progression-free survival (PFS) and 0.8 for overall survival. For elderly males, 500 mg/m2 was not more toxic than 375 mg/m2 rituximab, but improved PFS by 32.5% (P = 0.039), with a trend for a (30%) better overall survival (P = 0.076) in a planned subgroup analysis adjusting for International Prognostic Index risk factors. We conclude that the higher rituximab dose for elderly male patients abrogated the adverse prognosis of male sex without increasing toxicity. In the era of personalized medicine, sex-specific pharmacokinetics and toxicities should be investigated for all drugs where these parameters impact on outcome.

Enantioselectivity in the Metabolism of Cyclophosphamide in Patients With Multiple or Systemic Sclerosis.

The aim of this study was to evaluate the enantioselective pharmacokinetics of cyclophosphamide and its metabolites 4-hydroxycyclophosphamide and carboxyethylphosphoramide mustard in patients with systemic or multiple sclerosis. Patients with systemic sclerosis (n = 10) or multiple sclerosis (n = 10), genotyped for the allelic variants of CYP2C9*2 and CYP2C9*3 and of the CYP2B6 G516T polymorphism, were treated with 50 mg cyclophosphamide/kg daily for 4 days. Serial blood samples were collected up to 24 hours after administration of the last cyclophosphamide dose. Cyclophosphamide, 4-hydroxycyclophosphamide, and carboxyethylphosphoramide enantiomers were analyzed in plasma samples using liquid chromatography-tandem mass spectrometry coupled to chiral column Chiralcel OD-R or Chiralpak AD-RH. Cytokines IL-2, IL-4, IL-6, IL-8, IL-10, IL- 12p70, IL-17, TNF-α, and INT-δ in the plasma samples collected before cyclophosphamide infusion were analyzed by Milliplex MAP human cytokine/chemokine. Pharmacokinetic parameters showed higher plasma concentrations of (S)-(-)-cyclophosphamide (AUC 215.0 vs 186.2 µg·h/mL for multiple sclerosis patients and 219.1 vs 179.2 µg·h/mL for systemic sclerosis patients) and (R)-4-hydroxycyclophosphamide (AUC 5.6 vs 3.7 µg·h/mL for multiple sclerosis patients and 6.3 vs 5.6 µg·h/mL for systemic sclerosis patients) when compared to their enantiomers in both groups of patients, whereas the pharmacokinetics of the carboxyethylphosphoramide metabolite was not enantioselective. Cytokines' plasma concentrations were similar between multiple and systemic sclerosis groups. The pharmacokinetics of cyclophosphamide is enantioselective in patients with systemic sclerosis and multiple sclerosis, with higher plasma concentrations of the (S)-(-)-cyclophosphamide enantiomer due to the preferential formation of the (R)-4-hydroxycyclophosphamide metabolite.

LC-MS/MS method for simultaneous determination of thalidomide, lenalidomide, cyclophosphamide, bortezomib, dexamethasone and adriamycin in serum of multiple myeloma patients.

Multiple myeloma (MM), a malignant neoplastic serum-cell disorder, has been a serious threat to human health. The determination of 6 commonly used drug concentrations, including thalidomide, lenalidomide, cyclophosphamide, bortezomib, dexamethasone and adriamycin, in MM patients was of great clinical interest. Herein, we reported a method for the rapid and simultaneous measurement of the above therapeutics by liquid chromatography-tandem mass spectroscopy (LC-MS/MS) method with solid phase extraction. Analysis was performed on a Waters XBridge(®) BEH C18 column (2.5µm, 2.1 mm×50mm), with formic acid aqueous solution and acetonitrile as the mobile phase at flow rate 0.3mL/min. All analytes showed good correlation coefficients (r>0.996), and LLOQ of thalidomide, lenalidomide, cyclophosphamide, bortezomib, dexamethasone and adriamycin were 4, 2, 2, 2, 2 and 2ng/mL, respectively. The inter- and intra-day precisions and stability were expressed as variation coefficients within 15% and relative error less than 15%. Dilution effect, carryover and incurred sample reanalysis were investigated according to the 2015 edition Chinese Pharmacopoeia guidelines, as US FDA (2013, revision 1) required. The LC-MS/MS based assay described in this article may improve future clinical studies evaluating common therapeutics for MM treatment.

Enantioselective analysis of 4-hydroxycyclophosphamide in human plasma with application to a clinical pharmacokinetic study.

Cyclophosphamide (CY) is one of the most common immunosuppressive agents used in autologous hematopoietic stem cell transplantation. CY is a prodrug and is metabolized to active 4-hydroxycyclophosphamide (HCY). Many authors have suggested an association between enantioselectivity in CY metabolism and treatment efficacy and/or complications. This study describes the development and validation of an analytical method of HCY enantiomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) that can be applied to pharmacokinetic studies, filling this gap in the literature. HCY enantiomers previously derivatized with phenylhydrazine were extracted from 200-µL plasma aliquots spiked with antipyrine as internal standard and a mixture of hexane and dichloromethane (80:20, v/v) was used as the extraction solvent. The derivatized HCY enantiomers were resolved on a Chiracel(®) OD-R column using water:acetonitrile:formic acid (55:45:0.2, v/v) as the mobile phase. No matrix effect was observed and the analysis of HCY enantiomers was linear for plasma concentrations of 5-5000ng of each enantiomer/mL plasma. The coefficients of variation and inaccuracy calculated in precision and accuracy assessments were less than 15%. HCY was stable in human plasma after three successive freeze/thaw cycles, during 3h at room temperature, and in the autosampler at 4°C for 24h after processing, with deviation values less than 15%. The method was applied to evaluate the kinetic disposition of HCY in a patient with multiple sclerosis who was pretreated with intravenous racemic CY for stem cell transplantation. The clinical study showed enantioselectivity in the pharmacokinetics of HCY.

Cyclophosphamide pharmacokinetics and pharmacogenetics in children with B-cell non-Hodgkin's lymphoma.

INTRODUCTION: Variation in cyclophosphamide pharmacokinetics and metabolism has been highlighted as a factor that may impact on clinical outcome in various tumour types. The current study in children with B-cell non-Hodgkin's lymphoma (NHL) was designed to corroborate previous findings in a large prospective study incorporating genotype for common polymorphisms known to influence cyclophosphamide pharmacology. METHODS: A total of 644 plasma samples collected over a 5 year period, from 49 B-cell NHL patients ≤ 18 years receiving cyclophosphamide (250 mg/m(2)), were used to characterise a population pharmacokinetic model. Polymorphisms in genes including CYP2B6 and CYP2C19 were analysed. RESULTS: A two-compartment model provided the best fit of the population analysis. The mean cyclophosphamide clearance value following dose 1 was significantly lower than following dose 5 (1.83 ± 1.07 versus 3.68 ± 1.43 L/h/m(2), respectively; mean ± standard deviation from empirical Bayes estimates; P < 0.001). The presence of at least one CYP2B6*6 variant allele was associated with a lower cyclophosphamide clearance following both dose 1 (1.54 ± 0.11 L/h/m(2) versus 2.20 ± 0.31 L/h/m(2), P = 0.033) and dose 5 (3.12 ± 0.17 L/h/m(2) versus 4.35 ± 0.37 L/h/m(2), P = 0.0028), as compared to homozygous wild-type patients. No pharmacokinetic parameters investigated were shown to have a significant influence on progression free survival. CONCLUSION: The results do not support previous findings of a link between cyclophosphamide pharmacokinetics or metabolism and disease recurrence in childhood B-cell NHL. While CYP2B6 genotype was shown to influence pharmacokinetics, there was no clear impact on clinical outcome.

Genetic markers in CYP2C19 and CYP2B6 for prediction of cyclophosphamide's 4-hydroxylation, efficacy and side effects in Chinese patients with systemic lupus erythematosus.

AIMS: The aim of the study was to investigate the combined impact of genetic polymorphisms in key pharmacokinetic genes on plasma concentrations and clinical outcomes of cyclophosphamide (CPA) in Chinese patients with systemic lupus erythematosus (SLE). METHODS: One hundred and eighty nine Chinese SLE patients treated with CPA induction therapy (200 mg, every other day) were recruited and adverse reactions were recorded. After 4 weeks induction therapy, 128 lupus nephritis (LN) patients continued to CPA maintenance therapy (200-600 mg week(-1)) for 6 months, and their clinical outcomes were recorded. Blood samples were collected for CYP2C19, CYP2B6, GST and PXR polymorphism analysis, as well as CPA and its active metabolite (4-hydroxycyclophosphamide (4-OH-CPA)) plasma concentration determination. RESULTS: Multiple linear regression analysis revealed that CYP2B6 -750 T > C (P < 0.001), -2320 T > C (P < 0.001), 15582C > T (P = 0.017), CYP2C19*2 (P < 0.001) and PXR 66034 T > C (P = 0.028) accounted for 47% of the variation in 4-OH-CPA plasma concentration. Among these variants, CYP2B6 -750 T > C and CYP2C19*2 were selected as the combination genetic marker because these two SNPs contributed the most to the inter-individual variability in 4-OH-CPA concentration, accounting for 23.6% and 21.5% of the variation, respectively. Extensive metabolizers (EMs) (CYP2B6 -750TT, CYP2C19*1*1) had significantly higher median 4-OH-CPA plasma concentrations (34.8, 11.0 and 6.6 ng ml(-1) for EMs, intermediate metabolizers (IMs) and poor metabolizers (PMs), P < 0.0001), higher risks of leukocytopenia (OR = 7.538, 95% CI 2.951, 19.256, P < 0.0001) and gastrointestinal toxicity (OR = 7.579, 95% CI 2.934, 19.578, P < 0.0001), as well as shorter median time to achieve complete remission (13.2, 18.3 and 23.3 weeks for EMs, IMs and PMs, respectively, P = 0.026) in LN patients than PMs (CYP2B6 -750CC, CYP2C19*2*2) and IMs. CONCLUSIONS: Our findings have indicated that genetic markers of drug metabolizing enzymes could predict the 4-hydroxylation, adverse reactions and clinical efficacy of CPA. This is a necessary first step towards building clinical tools that will help assess clinical benefit and risk before undergoing CPA treatment in Chinese SLE patients.

Comparative pharmacokinetics of cyclophosphamide administration alone and combination with vitamin B6 in rats.

A fast and sensitive high performance liquid chromatography coupled with mass spectrometry (LC-MS) method was developed and validated for the determination of cyclophosphamide in rat plasma with and without the combination of vitamin B6. After addition of digoxin used as the internal standard (IS), plasma samples were extracted by protein precipitation with acetonitrile (1:1, v/v), and the analytes were separated by a Kromasil C18 column (150 × 4.6 mm, 5 µm) with a mobile phase of acetonitrile-0.1% formic acid water (40:60, v/v). The detection of the analyte was monitored in positive electrospray ionization by selected ion monitoringmode. The linear range was 0.01-40 µg/mL for cyclophosphamide. The intra- and inter-day precision and accuracy were all <15%. The extraction recoveries and matrix effects of the analyte and IS were all within acceptable range. The selectivity of the method was satisfactory with no endogenous interference. The results for stabilities of cyclophosphamide and IS under various conditions were all within the acceptance criteria. The validated method was successfully applied to evaluate the drug-drug interaction of cyclophosphamide and vitamin B6 in rat plasma. The results showed no differences of pharmacokinetic behaviors between cyclophosphamide administration with and without vitamin B6.