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.

Cyclophosphamide exposure assessed with the biomarker phosphoramide mustard-hemoglobin in breast cancer patients: The TailorDose I study

Cyclophosphamide (CPA) dosing by body surface area (BSA, m2) has been questioned as a predictor for individual drug exposure. This study investigated phosphoramide mustard-hemoglobin (PAM-Hb, pmol g−1 Hb) as a biomarker of CPA exposure in 135 female breast cancer patients receiving CPA during three courses based on BSA: 500 mg/m2 (C500 group, n = 67) or 600 mg/m2 (C600 group, n = 68). The inter-individual difference was calculated for both groups by dividing the highest through the lowest PAM-Hb value of each course. The inter-occasion difference was calculated in percentage for each individual by dividing their PAM-Hb value through the group mean per course, and subsequently dividing this ratio of the latter through the previous course. A multivariable linear regression (MLR) was performed to identify factors that explained the variation of PAM-Hb. During the three courses, the inter-individual difference changed from 3.5 to 2.1 and the inter-occasion difference ranged between 13.3% and 11.9% in the C500 group. In the C600 group, the inter-individual difference changed from 2.7 to 2.9 and the inter-occasion difference ranged between 14.1% and 11.7%. The MLR including BSA, age, GFR, and albumin explained 17.1% of the variation of PAM-Hb and was significantly better then the model including only BSA. These factors should be considered when calculating the first dose of CPA for breast cancer patients.

Similar Repair Effects of Human Placenta, Bone Marrow Mesenchymal Stem Cells, and Their Exosomes for Damaged SVOG Ovarian Granulosa Cells

Background. This study is aimed at investigating the repairing effect of mesenchymal stem cells and their exosomes from different sources on ovarian granulosa cells damaged by chemotherapy drugs—phosphoramide mustard (PM). Methods. In this study, we choose bone marrow mesenchymal stem cells (BMSCs) and human placental mesenchymal stem cells (HPMSCs) for research. Then, they were cocultured with human ovarian granulosa cells (SVOG) injured by phosphoramide mustard (PM), respectively. β-Galactosidase staining, flow cytometry, and Western blot were used to detect the changes in the senescence and apoptosis of SVOG cells before and after their coculture with the above two types of MSCs. Subsequently, exosomes from these two types of MSCs were extracted and added to the culture medium of SVOG cells after PM injury to test whether these two types of exosomes played a role similar to that of MSCs in repairing damaged SVOG cells. Results. PM treatment-induced apoptotic SVOG cells were significantly decreased after HPMSCs and BMSCs as compared with control group. After coculturing with these two types of MSCs, PM-treated SVOG cells showed significantly reduced senescence and apoptosis proportions as well as cleaved-Caspase 3 expression, and HPMSCs played a slightly stronger role than BMSCs in repairing SVOG cells in terms of the above three indicators. In addition, the ratios of senescent and apoptotic SVOG cells were also significantly reduced by the two types of exosomes, which played a role similar to that of MSCs in repairing cell damages. Conclusions. The results indicated that BMSCs, HPMSCs, and their exosomes all exerted a certain repair effect on SVOG cells damaged by PM, and consistent repair effect was observed between exosomes and MSCs. The repair effect of exosomes secreted from BMSCs and HPMSCs on the SVOG cells was studied for the first time, and the results fully demonstrated that exosomes are the key carriers for MSCs to play their role.

Ataxia–telangiectasia mutated coordinates the ovarian DNA repair and atresia-initiating response to phosphoramide mustard

Ataxia–telangiectasia-mutated (ATM) protein recognizes and repairs DNA double strand breaks through activation of cell cycle checkpoints and DNA repair proteins. Atm gene mutations increase female reproductive cancer risk. Phosphoramide mustard (PM) induces ovarian DNA damage and destroys primordial follicles, and pharmacological ATM inhibition prevents PM-induced follicular depletion. Wild-type (WT) C57BL/6 or Atm+/− mice were dosed once intraperitoneally with sesame oil (95%) or PM (25 mg/kg) in the proestrus phase of the estrous cycle and ovaries harvested 3 days thereafter. Atm+/− mice spent ~25% more time in diestrus phase than WT. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) on ovarian protein was performed and bioinformatically analyzed. Relative to WT, Atm+/− mice had 64 and 243 proteins increased or decreased in abundance, respectively. In WT mice, PM increased 162 and decreased 20 proteins. In Atm+/− mice, 173 and 37 proteins were increased and decreased, respectively, by PM. Exportin-2 (XPO2) was localized to granulosa cells of all follicle stages and was 7.2-fold greater in Atm+/− than WT mice. Cytoplasmic FMR1-interacting protein 1 was 6.8-fold lower in Atm+/− mice and was located in the surface epithelium with apparent translocation to the ovarian medulla post-PM exposure. PM induced γH2AX, but fewer γH2AX-positive foci were identified in Atm+/− ovaries. Similarly, cleaved caspase-3 was lower in the Atm+/− PM-treated, relative to WT mice. These findings support ATM involvement in ovarian DNA repair and suggest that ATM functions to regulate ovarian atresia.

Novel Sophoridine Derivatives Bearing Phosphoramide Mustard Moiety Exhibit Potent Antitumor Activities In Vitro and In Vivo

Novel mustard functionalized sophoridine derivatives were synthesized and evaluated for their cytotoxicity against of a panel of various cancer cell lines. They were shown to be more sensitive to S180 and H22 tumor cells with IC50 values ranging from 1.01–3.65 μM, and distinctly were more cytotoxic to cancer cells than normal cell L929. In addition, compounds 7a, 7c, and 7e displayed moderate tumor suppression without apparent organ toxicity in vivo against mice bearing H22 liver tumors. Furthermore, they arrested tumor cells in the G1 phase and induced cellular apoptosis. Their potential binding modes with DNA-Top I complex have also been investigated.