Prodrug treosulfan, originally registered for treatment of ovarian cancer, has gained a use in conditioning prior to hematopoietic stem cell transplantation. Treosulfan converts nonenzymatically to the monoepoxide intermediate (EBDM), and then to (2S,3S)-1,2:3,4-diepoxybutane (DEB). The latter alkylates DNA forming mainly (2′S,3′S)-N-7-(2′,3′,4′-trihydroxybut-1′-yl)­guanine (THBG) and (2S,3S)-1,4-bis­(guan-7′-yl)­butane-2,3-diol cross-link (bis-N7G-BD) via the intermediate epoxide adduct (EHBG). It is believed that DNA cross-linking by DEB is a primary mechanism for the anticancer and myeloablative properties of treosulfan, but clear evidence is lacking. Recently, we have proved that EBDM alkylates DNA producing (2′S,3′S)-N-7-(2′,3′-dihydroxy-4′-methylsulfonyloxybut-1′-yl)-guanine (HMSBG) and that free HMSBG converts to EHBG. In this paper, we investigated the kinetics of HMSBG, bis-N7G-BD, and THBG in DNA in vitro to elucidate the contribution of EBDM and DEB to treosulfan-dependent DNA–DNA cross-linking. Calf thymus DNA was exposed to (A) 100 μM treosulfan, (B) 200 μM treosulfan, and (C) DEB at a concentration 100 μM, exceeding that produced by 200 μM treosulfan. Following mild acid thermal hydrolysis of DNA, ultrafiltration, and off-line HPLC purification, the guanine adducts were quantified by LC–MS/MS. Both bis-N7G-BD and THBG reached highest concentrations in the DNA in experiment B. Ratios of the maximal concentration of bis-N7G-BD and THBG to DEB (adduct C max/DEB C max) in experiments A and B were 1.7–3.0-times greater than in experiment C. EHBG converted to the bis-N7G-BD cross-link at a much higher rate constant (0.20 h–1) than EBDM and DEB initially alkylated the DNA (1.8–3.4 × 10–5 h–1), giving rise to HMSBG and EHBG, respectively. HMSBG decayed unexpectedly slowly (0.022 h–1) compared with the previously reported behavior of the free adduct (0.14 h–1), which revealed the inhibitory effect of the DNA environment on the adduct epoxidation to EHBG. A kinetic simulation based on the obtained results and the literature pharmacokinetic parameters of treosulfan, EBDM, and DEB suggested that in patients treated with the prodrug, EBDM could produce the vast majority of EHBG and bis-N7G-BD via HMSBG. In conclusion, EBDM can produce DNA–DNA lesions independently of DEB, and likely plays a greater role in DNA cross-linking after in vivo administration of treosulfan than DEB. These findings compel revision of the previously proposed mechanism of the pharmacological action of treosulfan and contribute to better understanding of the importance of EBDM for biological effects.

Background: Atorvastatin (AT) belongs to cholesterol-lowering agents, commonly used in patients with an increased risk of cardiovascular disease. The drug, as well as its hydroxyl metabolites, exhibit pharmacological activity, and their plasma levels may be helpful in the assessment of the therapeutic effectiveness. Objective: Development and validation of a fast and reproducible RP-HPLC method with UV detection for the simultaneous determination of atorvastatin and its active metabolites, para-hydroxy-atorvastatin (p-OH-AT) and ortho-hydroxy-atorvastatin (o-OH-AT) in human plasma. Methods: Optimal conditions of chromatographic separation of the analytes, as well as rosuvastatin, chosen as an internal standard, were studied. The absorbance of the compounds was measured at λ=248 nm. Validation of the method was performed. The usefulness of the method was confirmed for determination of the analytes in plasma of patients treated with the drug. Results: Total peak separation was achieved at LiChrospher 100 RP-18 column with a mobile phase composed of methanol and water (1:1,v:v) and a flow rate of 1.2 ml/min. The method was linear in the ranges of 0.025 - 1.0 μg/ml for AT, o-OH-AT and p-OH-AT. Intra- and inter-assay precision expressed as relative standard deviation was ≤13% for AT, ≤12% for p-OH-AT and ≤11% for o-OH-AT. Intraand inter-day accuracy of the method, expressed as a relative error, was ≤15%. Conclusion: The elaborated HPLC method is specific, repeatable, reproducible, adequately accurate and precise and fulfills the validation requirements for the bioanalytical method. The method was successfully applied for analysis of atorvastatin and its o-hydroxy metabolite in plasma of patients treated with the drug.