Cyclophosphamide Enantiomers Research Articles

Chemistry, Cyclophosphamide, Cancer Chemotherapy, and Serendipity: Sixty Years On

Cambridge Dictionary: serendipity | noun | the phenomenon of finding interesting or valuable things by chance.
 The year 2019 marked the 60th anniversary of the approval of cyclophosphamide (CP) as an anticancer by the U.S. Food & Drug Administration in 1959 for the treatment of lymphoma. Between 1959 and 2019 there were ~50,000 publications listed in PubMed that have CP in the title and/or abstract, with these annual numbers showing a continual increase, and over 1,800 such articles in 2019 alone. The discovery of CP is a prime example of serendipity in science, which also applies to key elements of the metabolism and pharmacological basis for the specificity of the cytotoxicity of CP toward cancer cells. Phosphoramide mustard (PM), HO(H2N)P(O)N(CH2CH2Cl)2, the principal metabolite of CP with DNA alkylating activity, was synthesized and reported by Friedman and Seligman in 1954 prior to the discovery of CP. Interestingly, the original drug design premise for synthesizing PM, which was based on elevated phosphamidase enzyme activity in cancer cells proved to be incorrect. While this wrong premise also led to the synthesis of CP, as a six-membered ring cyclic phosphamidase-activated precursor of PM, the actual metabolic conversion of CP to PM was subsequently found to involve a surprisingly complex array of metabolites and metabolic pathways, all completely unrelated to phosphamidase. Although the molecular structure of CP has an asymmetrically substituted, i.e. chiral phosphorus center, the racemic mixture of the Rp and Sp enantiomers of CP was used throughout its initial investigations and subsequent clinical trials despite the involvement of an initial enzyme-mediated metabolic activation step, which could, in principle, be stereoselective for only one of the enantiomers of CP. Stereochemical investigations along those lines were eventually carried out, but the results did not warrant replacement of racemic CP with either enantiomer in the clinic. Amazingly, there are now ~4,000 structural congeners of PM listed Chemical Abstracts, but none have led to an anticancer drug superior to CP. This account provides a synopsis of the key chemistry and stereochemistry investigations that comprise this story of CP, as a remarkable instance of serendipity in science, and my chance involvement in the unfolding of this fascinating story.

Analysis of cyclophosphamide and carboxyethylphosphoramide mustard enantiomers in human plasma and application to clinical pharmacokinetics.

This study describes for the first time a method for the sequential analysis of the enantiomers of cyclophosphamide (CY) and its metabolite carboxyethylphosphoramide mustard (CEPM) in human plasma. The CY and CEPM enantiomers were extracted from plasma using only ethyl acetate and separated on a Chiralpak(®) AD-RH column using a mixture of water:acetonitrile:ethanol (45:30:25, v/v/v) plus 0.1% trifluoroacetic acid as the mobile phase at a flow rate of 0.5mL/min. No matrix effect was observed in the analysis of the enantiomers of both analytes and the analytical method was linear in the range of 0.05-25.0μg and 250-1000ng of each enantiomer/mL plasma. The coefficients of variation and relative errors obtained for the assessment of intra- and interassay precision and accuracy were less than 15%. CY and CEPM were found to be stable in human plasma after three successive freeze/thaw cycles, during storage for 4h at room temperature, and after 24h inside the autosampler at 4°C, with deviations less than 15%. The method was applied to the study of the pharmacokinetics of CY and its metabolite CEPM in patients with multiple sclerosis (n=10) who received a CY pretransplant conditioning regimen for hematopoietic stem cell transplantation. The pharmacokinetic parameters showed plasma accumulation of the (S)-(-)-CY enantiomer (S/R ratio=1.3) and lack of enantioselective exposure to the CEPM metabolite (S/R ratio=1.0).

Pharmacokinetics of cyclophosphamide enantiomers in patients with breast cancer

Adjuvant chemotherapy with cyclophosphamide (CYC) is used for the treatment of breast cancer. CYC is used as a racemic mixture, although preclinical data have demonstrated differences in the efficacy and toxicity of its enantiomers, with (S)-(-)-CYC exhibiting a higher therapeutic index. The present study investigated the enantioselectivity and influence of CYP2B6, CYP2C9, CYP2C19, and CYP3A on the kinetic disposition of CYC in patients with breast cancer. Fifteen patients previously submitted to removal of the tumor and treated with racemic CYC (900 or 1,000mg/m(2)) and epirubicin were included in the study. The in vivo activity of CYP3A was evaluated using midazolam as a marker drug. Serial blood samples were collected up to 24h after administration of the first cycle of CYC. The kinetic disposition of CYC was enantioselective in patients with breast cancer, with plasma accumulation of the (S)-(-)-CYC enantiomer (AUC 195.0 vs. 174.8μgh/mL) due to the preferential clearance of the (R)-(+)CYC enantiomer (5.1 vs. 5.7 L/h). Clearance of either CYC enantiomer did not differ between the CYP2B6, CYP2C9, and CYP2C19 genotypes or as a function of the in vivo activity of CYP3A evaluated by midazolam clearance. The pharmacokinetics of CYC is enantioselective in patients with breast cancer concomitantly treated with epirubicin and ondansetron. Genotyping or phenotyping did not contribute to adjustment of the CYC dose regimen in patients included in this study.

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.

Determination of cyclophosphamide enantiomers in plasma by LC‐MS/MS: Application to pharmacokinetics in breast cancer and lupus nephritis patients

This article describes the enantioselective analysis of cyclophosphamide (CPA) in human plasma using LC-MS/MS. CPA enantiomers were extracted from plasma using a mixture of ethyl acetate and chloroform (75:25, v/v). The enantiomers were separated on a Chiralcel(R) OD-R column, with the mobile phase consisting of a mixture of acetonitrile and water (75:25, v/v) plus 0.2% formic acid. The protonated ions and their respective product ions were monitored using two functions, 261 > 141 for CPA enantiomers and 189 > 104 for the internal standard (antipyrine). Recovery rates were higher than 95% and the quantification limit was 2.5-ng/ml plasma for both enantiomers. The coefficients of variation and the relative errors obtained for the validation of intra- and interassay precision and accuracy were less than 10%. The method was applied for the investigation of the enantioselective pharmacokinetics of CPA in a lupus nephritis patient treated with 1 g CPA infused over 2 h and in a breast cancer patient treated with 0.9 g infused over 1 h. No stereoselectivity in the pharmacokinetic parameters was observed for either patient. Clearance values of 2.63 and 2.93 l/h and of 3.36 and 3.61 l/h for (-)-(S) and (+)-(R)-CPA were obtained for the breast cancer and lupus nephritis patient, respectively.

Pharmacokinetics of (R)- and (S)-cyclophosphamide and their dechloroethylated metabolites in cancer patients.

The complete pharmacokinetics (PK) of (R)- and (S)-cyclophosphamide (CP) and their dechloroethylated (DCE) metabolites have not been reported to date. We collected plasma and urine samples from 12 cancer patients and determined concentrations of both enantiomers of CP and DCE-CP using a chiral GC-MS method. All concentrations of (R)-CP, (S)-CP, (R)-DCE-CP, and (S)-DCE-CP were simultaneously modeled using an enantiospecific compartmental PK model. A population PK analysis was performed. Enantiospecific differences between (R)- and (S)-CP were found for the formation clearance of CP to the DCE metabolites (Clf: 0.25 (R) vs. 0.14 (S) L/h). No difference was found between enantiomers for Cl40H, Cld, Cl(m)R, ClT, or T1/2. In contrast to the adolescent and adult group of patients, a child (6 years old) appeared to have a very different PK and metabolic profile (Bayesian control analysis). Proportions of the (R,S)-CP doses transformed to the (R)-DCE- and (S)-DCE-CP were much higher (R: 25 vs. 1.9%, and S: 38 vs. 3.6%), while formation of active metabolites was much lower (R: 42 vs. 74%, and S: 48 vs. 77%). CP appears to be enantioselectively metabolized to the DCE metabolites. This PK model can evaluate the proportion of a CP dose that is transformed to toxic or active metabolites. It may therefore be used to optimize CP treatment, to identify important drug interactions and/or patients with an abnormal metabolic profile.

High-performance liquid chromatographic determination of the enantiomers of cyclophosphamide in serum

A high-performance liquid chromatographic (HPLC) achiral-chiral coupled assay to measure the serum concentration of the enantiomers of cyclophosphamide is described. The R- and S-enantiomers of cyclophosphamide were quantified using a 5-cm-long C 1 Spherisorb 5-μm column, with switching of the eluent containing racemic cyclophosphamide onto a 10-cm-long α 1, acid glycoprotein column. The limit of determination was 1.25 mg l −1 for each enantiomer and the ratio of the enantiomers over the range 2.5 to 100 mg l −1 was 1. Serum enantiomer concentrations in blood samples taken from patients receiving 0.30 to 0.75 g m −2 of intravenous racemic cyclophosphamide could be measured at least three half-lives post dose. In six patients no significant difference in the clearance of R- and S-cyclophosphamide was found.

Stereoselective pharmacokinetics and metabolism of the enantiomers of cyclophosphamide: Preliminary results in humans and rabbits

[ R(+), S(−)]-Cyclophosphamide [( R, S)-CP] is an anticancer drug, containing a chiral phosphorous atom, which is prepared and used clinically as the racemic mixture. A new high-performance liquid Chromatographic assay suitable for pharmacokinetic studies of CP enantiomers in plasma has been reported recently by this laboratory (Reid et al., Anal Chem 61: 441–446, 1989). Briefly, the assay involves ethyl acetate extraction of CP enantiomers from plasma followed by derivatization to diastereomers in a two-step process utilizing chloral and (+)-naproxen acid chloride. Chromatographic analysis was performed on a reversed phase (ODS) column with detection at 232 nm. In the present study, preliminary results on the applicability of this assay to pharmacokinetic studies are presented. Several rabbits were used to compare the influence of i.p., i.v., and oral routes of administration on the stereoselective disposition of ( R, S)-CP. Following i.p. administration, S-CP was cleared faster than R-CP. Following oral administration, only R-CP was detectable in plasma, while i.v. administration resulted in minor or no stereoselective disposition. These results indicated that there was a marked stereoselective metabolism of the S-CP enantiomer, with the i.p. and oral routes producing the greatest differences due to first-pass metabolism. Incubation of rabbit-liver microsomes with ( R, S)-CP demonstrated that the monooxygenase system can exhibit marked stereoselectivity in its metabolism of CP. The ratio of R-CP to S-CP in the incubation medium increased during the incubation period from 1:1 initially to 4.5:1 after 60 min. The results from the experiments with rabbits indicate that the first-pass metabolism of this drug is highly stereoselective; in contrast, cancer patients who had received ( R, S)-CP as an i.v. infusion showed no stereoselectivity in the elimination of the enantiomers. Pharmacokinetic studies with cancer patients, receiving ( R, R)-CP as an oral dose, are in progress in order to determine if stereoselective first-pass metabolism of this drug also occurs in humans.

Liquid chromatographic determination of cyclophosphamide enantiomers in plasma by precolumn chiral derivatization

On the basis of reactions described in the synthetic literature, a two-step chiral derivatization sequence was developed for the anticancer agent cyclophosphamide (CP). The sequence involves amidoalkylation of CP with anhydrous chloral containing 1% dimethylformamide followed by acylation of the resulting secondary alcohol with a chiral carboxylic acid chloride, (+)-6-methoxy-alpha-methyl-2-naphthaleneacetyl chloride, to form a diastereomeric pair. Derivatized (-)-CP and (+)-CP exhibited retention times of 17.2 and 20.7 min, respectively, when chromatographed on Hypersil ODS with acetonitrile/phosphate buffer (pH 6.8) as the mobile phase. Preparation of the individual diastereomers from enantiomerically pure CP enabled correlation of the chromatographically observed peaks with a particular enantiomer. Various aspects of the overall assay methodology have been systematically investigated (derivatization solvents, temperatures, reaction time, and work-up procedure) and optimized on the scale required for trace analysis in biological fluids. Calibration curves were established for each enantiomer in spiked human plasma over the therapeutically relevant concentration range of 0.99-49.94 micrograms/mL.

Enantiomers of cyclophosphamide and iphosphamide

Enantiomers of cyclophosphamide and iphosphamide

Effects of enantiomeric homogeneity on the in vitro metabolism and in vivo anticancer activity of (+)- and (−)-cyclophosphamide

Separate incubation kinetic measurements for the metabolic “activation” of enantiomerically pure (+)- and (−)-cyclophosphamide (CP) and the racemate of CP by identical phenobarbital (PB)-induced mouse liver microsomal mixed-function oxidase preparations gave, respectively, V max = 13.8 ± 1.0, 20.0 ± 1.5 and 16.3 ± 1.1 μmol bis-2-ehloroethylamine (nor-HN2) equiv./g wet liver/hr, and K m = 0.37 ± 0.02, 0.56 ± 0.04 and 0.45 ± 0.02 mM. The absolute magnitude of the apparent V max kinetic parameter increased by ca. 50 per cent in a subsequent comparative run between (+)- and (−)-CP using a second preparation of the hepatic microsomal ozidase ; however, the relative behaviour of CP enantiomers toward enzymatic “activation” was constant, within experimental error, and revealed that V max −/ V max + = 1.34 ± 0.17 and K m −/ K m + = 1.35 ± 0.14. Removal and quantitative measurement, as a function of time, of free acrolein that is produced by incubation of CP with PB-induced microsomes repeatedly gave a roughly congruous family of “skewed bell-shaped” curves having maxima in the order (±)-CP > (+)-CP > (−)-CP; however, the differences between these acrolein time-course profiles were relatively small ( ca, 10–20 per cent). Repitition of such measurements with freshly prepared mouse liver cytosol and NAD + added to the incubation mixtures of CP and liver microsomes led to comparable suppression of the maxima. Isolation of CP from separate (+)- and (−)-CP incubation mixtures, followed by determination of enantiomeric homogeneity by nuclear magnetic resonance methods, demonstrated that CP is not racemized during in vitro liver microsomal metabolism. Aminal screening data (test/control precentages) for (+)-, (−)-, and (±)-CP activity against mouse L-1210 lymphoid leukemia showed no outstanding differences in therapeutic value. Collectively, these various experimental probes suggest that there is an unusually low degree of biological stereoselectivity associated with the metabolism of CP enantiomers.