Introduction

N,N',N" -triethylenethiophosphoramide (thioTEPA;Figure 1) is a trifunctional alkylating agent with a broad spectrum of antitumour activity developed in the 1950s. Triethylenethiophosphoramide is adopted as its simplified generic denomination.The drug is now experiencing renewed interest as it appears to be one of the most effective anticancer drugs in high dose regimens. N,N',N" -triethylenethiophosphoramide has been proved to be more stable and also exhibited cytotoxic activity. Antitumour activity has been documented in a broad spectrum of solid tumours. N,N',N" -triethylenethiophosphoramide is registered as antineoplastic for treatment of breast, ovarian and bladder cancer. Interest in N,N',N" -triethylenethiophosphoramide has been renewed by the finding that its dose can be increased dramatically when bone marrow toxicity is not dose-limiting, such as in the bone marrow transplantation setting.[1]

Chemistry[1]

Aziridine or ethylenimine is a cyclic compound consisting of two carbon and one nitrogen atom. It is avolatile liquid with a melting and boiling point of -74 and 57℃, respectively. Aziridine containing compounds can be classified in two groups, (i) activated aziridines; the nitrogen substituent (e.g. carbonyl function) is capable of conjugating with the unshared electrons of the nitrogen and (ii) basic aziridines, without a substituent(Figure 2). N,N',N" -triethylenethiophosphoramide is an activated aziridine with phosphine sulphide as nitrogen substituent, and can undergo degradation reactions as described for suchaziridines.

Degradation products formed during stability studies of N,N',N" -triethylenethiophosphoramide result from ring opening reactions. Stability studies performed in an acidic environment in the presence of chloride show the formation of monochloro, dichloro and trichloroderivatives of N,N',N" -triethylenethiophosphoramide. Hydrolysis experiments with N,N',N" -triethylenethiophosphoramide in acidic environment also resulted in the formation of TEPA, the first identified metabolite of N,N',N" -triethylenethiophosphoramide. Besides the reaction with chloride ions at low pH, substitution with water to form hydroxyl derivatives was also seen in the monochloro and dichloro derivatives of N,N',N" -triethylenethiophosphoramide. Intramolecular alkylation of N,N',N" -triethylenethiophosphoramide at low pH yields a five membered ring, and is converted after hydrolysis in N,N’-diethylene, N”-2-mercaptoethylphosphoramide. Incubation of N,N',N" -triethylenethiophosphoramide in alkaline media resulted in a decrease of N,N',N" -triethylenethiophosphoramide, but no degradation products could be detected. Quinones with an aziridine substituent such as the indoloquinone antitumour agent EO9,are subjected to substitution reactions with hydroxyl ions in alkaline media, resulting in the release of aziridine.

The stability of N,N',N" -triethylenethiophosphoramide in biological samples was also dependent on the pH. In plasma the monochloro derivative of N,N',N" -triethylenethiophosphoramide was formed.Degradation products formed in urine were the monochloro and dichloro derivative of N,N',N"-triethylenethiophosphoramide. Kinetic data show that N,N',N" -triethylenethiophosphoramide in aqueous solutions is most stable between pH 7–11. In plasma at 37°C and physiological pH, N,N',N" -triethylenethiophosphoramide exhibited a half-life of 5 days. In urine at 37°C, N,N',N" -triethylenethiophosphoramide is more rapidly degraded, with a half-life of approximately 16 min at pH 4.0 and 21 h at pH 6.0.[1]

Mechanism of action

N,N′,N′′-triethylenethiophosphoramide is thus a polyfunctional alkylating agent and is capable in forming cross-links with DNA molecules. Formation of interstrand cross-links is seen by incubation of L1210 or MCF-7 cells with N,N′,N′′-triethylenethiophosphoramide. However in previous studies it has also been suggested that N,N′,N′′-triethylenethiophosphoramide functions as a prodrug for aziridine. In this way thioTEPA acts as a cell-penetrating carrier for aziridine, which is released intracellularly after hydrolysis. The released aziridine can react with DNA, resulting in the formation of a stable Gua adduct, imidazole ring opening and DNA chain scission. In the cell, aziridine is hydrolysed to ethanolamine, which is subsequently phosphorylated and incorporated into phosphatidylethanolamine via the normal cellular synthetic pathway for that lipid. The interaction of N,N′,N′′-triethylenephosphoramide (TEPA) with DNA is assumed to be different than that of the parent drug. TEPA produces DNA lesions,which are consistent with that induced by a monofunctional alkylating agent. The two recently identified metabolites of N,N′,N′′-triethylenethiophosphoramide, monochloro TEPA and thioTEPA-mercapturate both possess alkylating activity.[1]

Advances in the Research of N,N′,N′′-triethylenethiophosphoramide

In the 1960s, N,N′,N′′-triethylenethiophosphoramide failed to gain acceptance as a routine adjuvant therapeutic agent for colorectal carcinoma. Survival advantages were only manifested among female patients administered with high-dose regimens, whereas low-dose application yielded no obvious clinical benefit. This trial initiated the exploration of randomized controlled research on postoperative adjuvant chemotherapy for colorectal malignancy, and established essential methodological references for the later clinical application of 5-FU, oxaliplatin and other chemotherapeutic protocols. At present, N,N′,N′′-triethylenethiophosphoramide has been excluded from the standard adjuvant treatment system of colorectal cancer, and is now primarily adopted in the clinical management of breast and ovarian tumors, as well as in high-dose chemotherapy regimens combined with hematopoietic stem cell transplantation.[2]

Studies suggest that CYP2B6 plays an important role in the 4-hydroxylation of cyclophosphamide and that this reaction can be inhibited by N,N′,N′′-triethylenethiophosphoramide. Rae et al. had demonstrated for the first time that thioTEPA is a potentand specific inhibitor of CYP2B6. These findings have important implications. First, the specificity of N,N′,N′′-triethylenethiophosphoramide can be used as a toolto study the activity of CYP2B6 in vitro so that we may be able to further characterize the role of this enzyme in human drug metabolism. Second, the clinical interaction of cyclophosphamide and thioTEPA documented in the literature seems to be mediated by the ability of N,N′,N′′-triethylenethiophosphoramide to inhibit CYP2B6 and underlines the role ofCYP2B6 in cyclophosphamide activation in vivo. Finally, thioTEPA is likely to inhibit the metabolism of agents beyond cyclophosphamide and caution should be used during coadministration with other CYP2B6 substrate drugs.[3]

References

[1] Maanen MJ, Smeets CJ, Beijnen JH. Chemistry, pharmacology and pharmacokinetics of N,N',N" -triethylenethiophosphoramide (ThioTEPA). Cancer Treat Rev. 2000;26(4):257-268. doi:10.1053/ctrv.2000.0170

[2] Holden WD, Dixon WJ, Kuzma JW. The use of triethylenethiophosphoramide as an adjuvant to the surgical treatment of colorectal carcinoma. Ann Surg. 1967;165(4):481-503. doi:10.1097/00000658-196704000-00001

[3] Rae JM, Soukhova NV, Flockhart DA, Desta Z. Triethylenethiophosphoramide is a specific inhibitor of cytochrome P450 2B6: implications for cyclophosphamide metabolism. Drug Metab Dispos. 2002;30(5):525-530. doi:10.1124/dmd.30.5.525