Genotoxicity is the capacity of a substance to damage genetic material such as DNA, and thus cause mutations or possibly cancer. Genotoxic impurities need to be avoided in a pharmaceutical product because they can possibly cause serious health consequences even at levels less than standard analytical methods can detect.
It has been argued that most genotoxic reagents/intermediates present in syntheses that have four or more stages prior to API isolation/purification are likely to be deactivated by reaction with other reagents or purged by dissolution in solvents, removed by vacuum distillation or other phase switching operations; however, regulatory reviewers who have a vested interest in emphasizing the seriousness of genotoxins show little sympathy for such arguments. Government reviewers tend predominantly to request quantitative analytical information on the API and/or intermediates possibly combined with the results of spiking experiments (impurity fate analysis) and could well require these in order to demonstrate the absence of carryover of potential genotoxic impurities (PGIs), at a suitably low (TTC) level. Such investigations can be highly resource-intensive and challenging, particularly in respect of developing validated analytical methods for low levels of multiple PGIs. Moreover, limits for certain PGIs may become part of the API specification and so become an ongoing quality-control commitment.
However, concerns usually relate to late-stage operations that involve potential genotoxic impurities PGIs. One EU review raised as a major objection, potential residues of mesityl oxide (4-methyl-3-penten-2-one) in a drug substance crystallized from acetone. Another cited possible traces of alkyl mesylates (methyl, ethyl and isopropyl mesylates) in a mesylate salt drug substance.
Unless the threatened concern about the PGI is identified at the early planning stage, modifying a route that is otherwise the best proposal should be the last resort. Nonetheless, modification of the API synthesis in a way that minimizes PGI levels in the drug substance has been undertaken in some cases; recent published examples pertain to formaldehyde, chloroalkanes and acetamide.
Given the difficulties in obtaining reliable predictions, particularly for aromatic amines, it may be prudent to undertake an Ames assay on any PGI containing an aromatic amine structural alert, if data are not already available in the public domain. Aromatic amines that otherwise make admirable process intermediates because of the multiple options for their purification, should where it is feasible be transformed to more acceptable functionalities early in a process route.
The potential action of small alkyl alkylating agents presents a particularly frequent and acute problem for pharmaceutical products because so many of them in their API salt form are hydrochlorides, methanesulfonates, tosylates and besylates as well as to a much smaller extent, hydrobromides.
These pharmaceutical salts are most often prepared in the final chemical transformation of a sequence and so any PGI has the highest probability of lingering behind in the API.
The best evidence that these concerns about PGIs arising from making the aforementioned pharmaceutical salts are without any foundation is the epidemiological evidence of the health of medicinal and process chemists who have been making and using these salts for years and years without any relative health damage.
The apparent lack of health damage is readily explained since the reactivity of halo compounds in biological systems can be predicted to a significant extent on the basis of their relevant chemical properties such as alkylating potential and susceptibility to hydrolysis. Thus, bromo compounds are expected to be more reactive than chloro compounds. SN1 and SN2 characteristics will determine the nature and the extent of reactivity towards nucleophiles, and steric factors may also play a part in some cases. In the 4-(p-nitrobenzyl)pyridine alkylation assay, alkyl halides generally show negligible activity, methyl methanesulfonate (MMS) being at least 40 times more active than ethyl, propyl or butyl bromide. Allyl bromide appears to be more active (around one-eighth of the activity of MMS although allyl chloride shows minimal activity. Benzyl chloride, while rather active for a chloro compound, is around 20-fold less active than allyl bromide. Owing to their volatility and/or hydrophobicity many alkyl halides show negative results in conventional Ames Salmonella assays, and it is often necessary to employ vapour phase exposure in a closed system (using a desiccator for example) in order to obtain positive results. Most alkyl halides, especially bromides, are Ames positive (using a closed test system if necessary), although 1-chloropropane, 1-chlorobutane and neopentyl bromide are all Ames-negative. As expected, based on their lack of alkylating activity, both chloro- and bromobenzene are Ames-negative. Some unsaturated halo compounds have the potential to be metabolized to form quite active mutagenic molecular species. For example, evidence suggests that oxidative biotransformation of vinyl chloride produces chloroethylene oxide and 2-chloroacetaldehyde as active metabolites. Binding of bromobenzene-3,4-oxide to liver proteins is thought to account for the hepatotoxicity of bromobenzene. The predominant metabolic pathway for simple alkyl halides is halide displacement by glutathione, although some C-hydroxylation reactions may occur.
Rodent bioassay data on alkyl halides strongly suggest that these compounds are either non-carcinogens (1- chlorobutane, bromomethane) or low-potency carcinogens (chloroethane, bromoethane). Both chloroethane and bromoethane produced an increased incidence of a rare type of endometrial tumour in female mice and it seems highly plausible that the carcinogenic effect is caused by a species-/ gender-specific stress-related adrenal overstimulation and excessive corticosteroid production. The rodent carcinogenicity profile for chloroethane (increased incidence of a rare tumour type at an extremely high concentration of 15,000 ppm in one species/gender) is thus much closer to that for a non-genotoxic carcinogen than for a genotoxic carcinogen. Thus, the (feeble) alkylating activity of chloroethane seems largely incidental to its carcinogenic activity, a scenario likely to apply to many other similar alkyl halides. This prediction is strongly supported by the fact that benzyl, ethyl, isopropyl, and trityl bromides were inactive as carcinogens at doses up to 0.83, 12.5, 8.3, and 0.25 mmol/kg, respectively, when administered by single subcutaneous injection to female rats. A number of independent expert assessments are available on halo compounds.
Acceptable/tolerable exposures are expressed in various ways, for example as minimal risk levels (MRLs) by the Agency for Toxic Substances and Disease Registry, or as reference concentrations/doses (RfCs/RfDs) by the U.S. Environmental Protection Agency. There is a clear consensus that chloroethane is less hazardous than the more reactive chloromethane, although recommended safe exposures for the former range from the highly conservative OEHHA value of 150 μg/day to 200 mg/day (10 mg/m3 at an average air intake of 20 m3/day43) based on the EPA IRIS assessment.
Acceptable exposures in the context of genotoxic impurities can also be calculated on the basis of the TD50 values as described above, resulting in PDEs of 1810 and 149 μg/day for chloroethane and bromoethane respectively. A PDE for non-carcinogenic 1-chlorobutane could be determined using ICH Q3C (R3) methodology on the basis of the most appropriate NOAEL in lifetime studies.
Quotes from Org. Process Res. Dev. 2011, 15, 1243–1246
[Compounds can be grouped together that have] strikingly different reactivities, isopropyl chloride and isopropyl mesylate for example. Isopropyl chloride is Ames-negative in assays using standard conditions; testing has to be carried out in a desiccator to obtain a (feebly) positive result; on the other hand, isopropyl mesylate (Swain-Scott s = 0.29) is a potent mutagen in the standard Ames test and gives positive results in several in vivo assays. Hydrolysis half-lives at pH 7.0 and 25 C are 38 days (the same as for chloroethane; TD50 1810 mg/kg/day) and 4.5 h, respectively. Unfortunately, no rodent bioassay data appear to be available for either compound, although 1,2- dichloropropane is Ames-positive in standard assays and has a mouse TD50 of 276 mg/kg/day (negative in the rat). A Risk Specific Dose (RSD) for isopropyl chloride of approximately 37 μg/day (for a 50 kg patient) has been determined by Bercu et al. and Contrera based on QSAR (quantitative structure- activity relationship) techniques using regression analysis of “training sets” of TD50 data. [The choice of compounds in the isopropyl chloride training set could be questioned in that it contained several non-genotoxic polychloro compounds but not 1,2-dichloropropane or chloroethane.] Use of QSAR models to predict genotoxic/carcinogenic potency relies on rule-based techniques (such as DEREK) or statistical techniques (using regression analysis of training data sets), and particularly in relation to carcinogenicity, the latter approach is considered to provide more reliable results. Since halo compounds in particular and other compounds containing “Ashby alerts” are often identified as PGIs, it is interesting to note that in the determination of the TTC limit Kroes et al. classified only two such compounds (5% of the total in the data set) in the lowest potency category (equivalent to 1.5 μg/day), but since the data set is non-transparent it is not possible to identify these two key compounds. [In a prior publication using essentially the same data set, the TD50 for methyl methanesulfonate is listed as 0.178 mg/kg/day, 179 times lower than the true value, and it is unclear whether this miss-transcription was carried forward to the data set used by Kroes et al. in the more “definitive” publication.] In the Kroes publication, no carbamates were classified in the lowest potency category, suggesting that the standard TTC of 1.5 μg/day may be unnecessarily conservative for this structural class.
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