Stabilities of neutral and basic esters of bendamustine in plasma compared to the parent compound: Kinetic investigations by HPLC

• An HPLC method was established to determine neutral and basic bendamustine esters.
• The nitrogen mustard moiety was more stable in human plasma than in buffer.
• Esters being substrates of unspecific esterases were cleaved with half-lives <5 min.
• In human plasma alkyl, morpholinoethyl and α-branched basic esters had t½ > 40 min.
• In murine plasma, t½ of esters was <2 min due to lower protein content and higher esterase activity.

Esters of the cytostatic bendamustine (1), previously demonstrated to be much more potent than the parent compound as antiproliferative agents in vitro, were investigated for stability in buffer and plasma, as well as against porcine liver esterase in the presence of different amounts of albumin using a validated RP-HPLC method with fluorescence detection. The hydrolysis of the nitrogen mustard moiety was retarded (for 1: approximately 130 vs. 11 min) in the presence of plasma proteins. For the derivatives, both cleavage of ester and nitrogen mustard moieties were analyzed. Enzymatic hydrolysis was very fast in the case of 2-pyrrolidino-, 2-piperidino- and 2-(4-methylpiperazino)-ethyl esters, whereas methyl, ethyl, morpholinoethyl and branched 2-pyrrolidinoethyl esters were considerably more stable (half-lives between 41 and 116 min, compared to <5 min). Inhibition by physostigmine indicated unspecific cholinesterases to be involved in the rapid ester cleavage. Due to lower protein content and higher enzymatic activity in murine compared to human plasma, reduced stability of all investigated esters in mouse plasma (t½ <2 min) has to be taken into account with respect to the design of animal studies.

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