Jamie L. Renbarger

Selective metabolism of vincristine in vitro by CYP3A5

Clinical outcomes of vincristine therapy, both neurotoxicity and efficacy, are unpredictable, and the reported pharmacokinetics of vincristine have considerable interindividual variability. In vitro and in vivo data support a dominant role for CYP3A enzymes in the elimination of vincristine. Consequently, genetic polymorphisms in cytochrome P450 (P450) expression may contribute to the interindividual variability in clinical response, but the contributions of individual P450s and the primary pathways of vincristine metabolism have not been defined. In the present study, vincristine was incubated with a library of cDNA-expressed P450s, and the major oxidative metabolites were identified. CYP3A4 and CYP3A5 were the only P450s to support substantial loss of parent drug and formation of the previously unidentified, major metabolite (M1). The structure of M1, arising as a result of an oxidative cleavage of the piperidine ring of the dihydro-hydroxycatharanthine unit of vincristine, was conclusively established after conversion to suitable derivatives followed by spectroscopic analysis, and a new pathway for vincristine metabolism is proposed. CYP3A5 was more efficient in catalyzing the formation of M1 compared with CYP3A4 (9- to 14-fold higher intrinsic clearance for CYP3A5). The formation of M1 was stimulated (3-fold) by the presence of coexpressed cytochrome b5, but the relative efficiencies of M1 formation by CYP3A4 and CYP3A5 were unaffected. Our findings demonstrate that in contrast to most CYP3A biotransformations, the oxidation of vincristine is considerably more efficient with CYP3A5 than with CYP3A4. We conclude that common genetic polymorphisms in CYP3A5 expression may contribute to the interindividual variability in the systemic elimination of vincristine.

Increased risk of vincristine neurotoxicity associated with low CYP3A5 expression genotype in children with acute lymphoblastic leukemia

This study evaluates the relationship between cytochrome P450 (CYP) 3A5 genotype and vincristine‐induced peripheral neuropathy (VIPN) in children with precursor B cell acute lymphoblastic leukemia (preB ALL). We have shown in vitro that vincristine is metabolized significantly more efficiently by CYP3A5 than by CYP3A4. We also found that vincristine neurotoxicity is less common in African‐Americans (70% express CYP3A5) than in Caucasians. We test the hypothesis that CYP3A5 expressers experience less vincristine neuropathy than do CYP3A5 non‐expressers.

Effect of CYP3A5 expression on vincristine metabolism with human liver microsomes

Vincristine is preferentially metabolized to a secondary amine, M1, by CYP3A5 with a 9- to 14-fold higher intrinsic clearance than CYP3A4 using cDNA-expressed enzymes. The genetically polymorphic expression of CYP3A5 may contribute to interindividual variability in vincristine efficacy and toxicity. The current study quantifies the contribution of cytochromes P450 (P450s), including CYP3A4 and CYP3A5, to vincristine metabolism with a bank of human liver microsomes (HLMs). M1 was the major metabolite formed with HLMs, and selective chemical inhibition of P450s confirmed that CYP3A was the major metabolizing subfamily. The liver tissues were genotyped for low expression alleles, CYP3A5*3,*6, and *7, and the HLMs were phenotyped for CYP3A4 and CYP3A5 expression by Western blot. Testosterone 6β-hydroxylation and itraconazole hydroxylation were used to quantify CYP3A4 activity in the HLMs. For each CYP3A5 high expresser (n = 10), the rate of M1 formation from vincristine due to CYP3A5 was quantified by subtracting the CYP3A4 contribution as determined by linear regression with CYP3A5*3/*3 samples. For CYP3A5 high expressers, the contribution of CYP3A5 to the metabolism of vincristine was 54 to 95% of the total activity, and the rate of M1 formation mediated by CYP3A5 correlated with CYP3A5 protein content (r2 = 0.95). Selective inhibition of CYP3A4 demonstrated that the M1 formation rate with CYP3A5 high expressers was differentially inhibited based on CYP3A4 activity. Using median values, the estimated hepatic clearances were 5-fold higher for CYP3A5 high expressers than low expressers. We conclude that polymorphic expression of CYP3A5 may be a major determinant in the P450-mediated clearance of vincristine.

Effect of race on vincristine-associated neurotoxicity in pediatric acute lymphoblastic leukemia patients.

This report examines the association between race and vincristine‐associated neurotoxicity in pediatric patients with precursor B cell acute lymphoblastic leukemia (preB ALL). Given that in vitro vincristine is metabolized more efficiently by cytochrome P450 (CYP) 3A5 than by CYP3A4 and that 70% African‐Americans (vs. 20% of Caucasians) express CYP3A5, one may hypothesize that African‐Americans metabolize vincristine more efficiently resulting in lower vincristine exposure and associated‐toxicity.

Risk factors for cisplatin-associated ototoxicity in pediatric oncology patients†

Cisplatin is an effective chemotherapy agent against several pediatric malignancies. One of its side effects is irreversible sensorineural hearing damage that is highly variable with a reported incidence of 22–70%. The aim of this study was to evaluate the incidence and identify clinical predictors of cisplatin‐related ototoxicity.

Patterns and severity of vincristine‐induced peripheral neuropathy in children with acute lymphoblastic leukemia

Vincristine, a critical component of combination chemotherapy treatment for pediatric acute lymphoblastic leukemia (ALL), can lead to vincristine‐induced peripheral neuropathy (VIPN). Longitudinal VIPN assessments were obtained over 12 months from newly diagnosed children with ALL (N = 128) aged 1–18 years who received vincristine at one of four academic childrens hospitals. VIPN assessments were obtained using the Total Neuropathy Score‐Pediatric Vincristine (TNS©‐PV), National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE©), Balis© grading scale, and Pediatric Neuropathic Pain Scale©–Five (PNPS©‐5). Of children who provided a full TNS©‐PV score, 85/109 (78%) developed VIPN (TNS©‐PV ≥4). Mean TNS©‐PV, grading scale, and pain scores were low. CTCAE©‐derived grades 3 and 4 sensory and motor VIPN occurred in 1.6%/0%, and 1.9%/0% of subjects, respectively. VIPN did not resolve in months 8–12 despite decreasing dose density. VIPN was worse in older children. Partition cluster analysis revealed 2–3 patient clusters; one cluster (n = 14) experienced severe VIPN. In this population, VIPN occurs more commonly than previous research suggests, persists throughout the first year of treatment, and can be severe.

Dactinomycin and Vincristine Toxicity in the Treatment of Childhood Cancer: A Retrospective Study from the Children’s Oncology Group

Dactinomycin (AMD) and vincristine (VCR) have been used for the treatment of childhood cancer over the past 40 years but evidence‐based dosing guidance is lacking.

The tumor suppressor CDKN3 controls mitosis

A genome-wide screen of phosphatases that control mitosis identified CDKN3, which acts through the CDC2 signaling axis.

Quantification of vincristine and its major metabolite in human plasma by high-performance liquid chromatography/tandem mass spectrometry.

An analytical method using electrospray ionization and high-performance liquid chromatography/tandem mass spectrometry (LC/ESI-MS/MS) was developed to quantify vincristine and M1, the CYP3A-mediated metabolite of vincristine, in human plasma. Vinblastine (internal standard), vincristine, and M1 in plasma were extracted in methylene chloride after acidification with TCAA. The analytes were separated on an Inertsil ODS-3 C18 column (2.1 × 150 mm) with a 5-μm particle size using a gradient elution with a run time of 20 min. The initial mobile phase composition was 0.2% formic acid/water (80:20, v/v) with a final composition of 0.2% formic acid/water (20:80, v/v). Detection was accomplished with multiple reaction monitoring for vinblastine (m/z 406.3→ 271.7), vincristine (m/z 413.2→ 362.2), and M1 (m/z 397.3 → 376.2). At three concentrations of vincristine and M1, the inter-day and intra-day accuracy and precision were within the acceptable limits for validation (106.8 ± 9.6% for intra-day, n = 5 each concentration; 90.9 ± 10.9% for inter-day, n = 4 each concentration). For both vincristine and M1, the concentration limits of quantification and detection were 12 pg/mL and 6 pg/mL, respectively. Stability studies indicated that 80% of M1 degraded in plasma after 15 hours at room temperature (n = 3, high and low QC concentrations). Therefore, short plasma processing times (<30 min) are recommended. The assay was used successfully to quantify vincristine and M1 in pediatric plasma samples up to 24 hours after vincristine administration. Vincristine and M1 concentrations were within the limits of quantification for all patient plasma samples.

Measuring vincristine-induced peripheral neuropathy in children with acute lymphoblastic leukemia

Background: Vincristine-induced peripheral neuropathy (VIPN) is difficult to quantify in children. Objective: The study objective was to examine the reliability, validity, and clinical feasibility of several VIPN measures for use in children with acute lymphoblastic leukemia. Interventions/Methods: Children (n = 65) aged 1 to 18 years receiving vincristine at 4 academic centers participated in the study. Baseline and pre–vincristine administration VIPN assessments were obtained using the Total Neuropathy Score–Pediatric Vincristine (TNS©-PV), the National Cancer Institute Common Terminology Criteria for Adverse Events, the Balis grading scale, and the FACES Pain Scale. The TNS-PV scores (n = 806) were obtained over 15 weeks. Blood was obtained at several time points to quantify pharmacokinetic parameters. Results: Cronbach’s &agr; for a reduced TNS-PV scale was .84. The TNS-PV scores correlated with cumulative vincristine dosage (r = 0.53, P = 0.01), pharmacokinetic parameters (r = 0.41, P = 0.05), and grading scale scores (r range = 0.46–0.52, P = .01). FACES scores correlated with the TNS-PV neuropathic pain item (r = 0.48; P = .01) and were attainable in all ages. A 2-item V-Rex score (vibration and reflex items) was the most responsive to change (effect size = 0.65, P < 0.001). The TNS-PV scores were attainable in 95% of children 6 years or older. Conclusions: The TNS-PV is reliable and valid for measuring VIPN. It is sensitive to change over time (15 weeks) and feasible for use in children 6 years or older. Implications for Practice: The TNS-PV may be a useful tool for assessing vincristine toxicity in children with acute lymphoblastic leukemia.