The development of HPLC methods for the determination of methotrexate and doxorubicin metabolites and their application to clinical studies

Farid, Yahya Yahya Zeki (1983) The development of HPLC methods for the determination of methotrexate and doxorubicin metabolites and their application to clinical studies. PhD thesis, University of Glasgow.

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This thesis describes the development of specific assays for two important anti-cancer agents which have been available for some time, methotrexate (MTX) introduced in 1 949 and doxorubicin (ADR) introduced in 1969. Both of these drugs undergo significant Phase I metabolism and since such metabolites may be of pharmacological or toxicological significance the assays developed had to be capable of estimating these as well as the parent drugs. High performance liquid chromatography (HPLC) is an ideal technique for this purpose. The assay developed for MTX and its metabolites employed a liquid/solid extraction which gave reproducibly high recoveries of MTX, its deglutamated metabolites and their 7-hydroxylated derivatives, followed by an HPLC procedure using an anionic ion- pairing agent in the eluant which gave excellent chromatographic efficiency and resolution. This resulted in a highly sensitive and specific assay for all the above components. The assay was applicable to serum and urine and a modified method applicable to in vitro studies was also developed and used for analyses of cells both bacterial and human, and the culture media in which they were grown. Studies using clinical samples showed that the assay was sufficiently sensitive to measure MTX and its metabolites in plasma from patients treated with low-dose oral MTX for psoriasis. High-dose studies were carried out during following IV infusions of MTX in cancer patients. The results obtained gave rise to some new findings concerning MTX handling. In low-dose, intra-individual MTX clearance was highly reproducible, but there was substantial intersubject variation in this and other pharmacokinetic parameters. In particular MTX bioavailability varied between 17 and 120%. The implications of this and the other variations in disposition are considered. In the high-dose studies the kinetics were markedly different from the low-dose studies, most probably due to the presence of other drugs and the possibility of third spaces. Sequential high-dose studies showed that changes in the renal component of clearance was a major factor in accumulation of 7-hydroxy methotrexate (7-OHMTX). Clearances were calculated from both urinary data and plasma data and the results were found to be comparable. A second potential metabolite DAMPA, which had been reported in plasma by Donehower (1979) was detected in some plasma samples following high-dose MTX therapy. The kinetics indicate that the most likely source of this is as an impurity in the infusion fluid rather than as a result of metabolism in man by hepatic bacterial deglutamation. This product was not detected in vitro following incubation of either bacterial or human cell lines with MTX. A third potential metabolite of MTX, 7-OHDAMPA, not previously described in man, was synthesised and characterised in vitro and some evidence obtained as to its presence in urine from patients with DAMPA present in plasma. An HPLC assay for doxorubicin (ADR) was developed which was highly sensitive for ADR and its main Phase I metabolites. The HPLC assay employed a novel separation mechanism which was fully investigated and an explanation proposed. The assay proved suitable for the examination of clinical samples. The effects of gastrointestinal bacteria and human cell lines were investigated and metabolism of these drugs in some cells was found, although the relevance of these findings to cell resistance remains to be ascertained. Similar studies with ADR showed that bacterial cells metabolised the drug but not to any of the known metabolites. These studies indicate that the use of specific methods for measuring metabolites of anti-cancer drugs as well as the parent drug can lead to more information as regards the metabolism and disposition of these drugs than the non-specific tracer methods employed in many reference studies. It is suggested that the application of these and similar methods to clinical studies in patients treated with chemotherapeutic agents may lead to more rational individual dosage regimes giving rise to increased efficiency and reduced toxicity.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: M J Stewart
Keywords: Pharmacology, Analytical chemistry
Date of Award: 1983
Depositing User: Enlighten Team
Unique ID: glathesis:1983-72335
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 24 May 2019 15:12
Last Modified: 24 May 2019 15:12

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