C.J. Soper

Biological indicators for steam sterilization: characterization of a rapid biological indicator utilizing Bacillus stearothermophilus spore‐associated alpha‐glucosidase enzyme

Sterilization is the act or process, physical or chemical, that destroys or eliminates all forms of life, especially micro‐organisms. This term is absolute, i.e. a substance cannot be partially sterile ( Joslyn 1991 ). Due to the difficulty in confirming sterility, a more practical definition of sterility has been adopted, defining sterility as the process by which living organisms are removed or killed to an extent that they are no longer detectable in standard culture media in which they have previously been found to proliferate ( Joslyn 1991 ). In practice, assurance of a low probability of any living micro‐organisms remaining is used as a measure of sterility. Sterility of a particular item can only be confirmed by destructive testing of the item, which is not practical for most purposes.

Studies on the mechanism of microbial N-demethylation of codeine by cell-free extracts of Cunninghamella bainieri

Abstract Cell-free extracts have been prepared from the fungus Cunninghamella bainieri which retain high levels of N -demethylase activity against codeine and other drug molecules. Extraction required both disruption and solubilization, indicating that the N -demethylase enzyme is probably membrane bound. The carbon monoxide-reduced u.v. difference spectrum of the extract suggests the presence of a cytochrome P -450. The effect of specific inhibitors and the generation of formaldehyde during the transformation suggest a mono-oxygenase mediated mechanism. Kinetic studies have shown that N -demethylation is unlikely to occur via the N -oxide intermediate. Instead it is proposed that N -demethylation proceeds via the transient N -hydroxymethyl intermediate.

The screening of some microorganisms for their ability to N-dealkylate drug molecules

SummarySelected microorganisms were screened for their ability to N-dealkylate drug molecules. The compounds studied enabled the investigation of N-alkyl groups in different chemical environments, including alkylaminoalkyl chains, saturated cyclic structures and in amide functions. Transformation products were extracted from the transformation mixtures, derivatised and analysed by gas liquid chromatography (GLC). For the purposes of screening, important transformation products were identified by comparison of their GLC retention data with similar derivatives of authentic standards. N-demethylation was effected by all test strains of the fungus Cunninghamella, except C. elegans, and also by three of the Streptomyces species tested. The Cunninghamella demonstrated the widest spectrum of transformation activity, N-demethylating the alkylaminoalkyl side chains of amitriptyline and chlorpromazine, the N-methylpiperidine function of codeine, and the cyclic amide function of diazepam. The N-demethylation of the latter substrate, which is only slightly water soluble, occurred in surprisingly high yield. There was no evidence that bulky groups such as N-dimethylallyl were celaved and selectivity for N-demethylation rather than O-demethylation was demonstrated when both N-and O-methyl functions were present in the same molecule. Comparisons are made with the mammalian metabolic pathways of the drug compounds studied.

A mutagenesis-enhancing activity of 12-O-tetradecanoylphorbol 13-acetate detected in Chinese hamster ovary cells

Tumour-promoting agents may bring about the completion of multi-step carcinogenesis by acting as enhancers of mutagenesis, recombinogens or clastogens. We report here that the classical mouse skin tumour promoter TPA, although non-mutagenic per se, can enhance the induction of OuaR CHO-K1 cell mutants by MNNG approximately 2-fold. This observation was made at a concentration approaching the compounds aqueous solubility limit which was non-cytotoxic. Mutagenesis enhancement was dependent on TPA being present throughout mutation expression and mutant selection. It was not accompanied by any modification of cell sensitivity to mutagen killing. In the same treatment protocol TPA did not enhance either EMS- or UV-induced mutagenesis. TPA exposure over 2 rounds of cell replication failed to produce an increase in the frequency of SCE in control or mutagen-treated CHO-K1 cultures. Likewise TPA exposure over 1 round of cell replication failed to produce an increase in the frequency of chromosomal aberrations. Apparently TPA is not a recombinogen or clastogen but in the right exposure regime is capable of acting to enhance mutagenesis by certain genotoxic agents, an action which may contribute to tumour promotion.

Biological indicators for low temperature steam and formaldehyde sterilization : effect of variations in recovery conditions on the response of spores of Bacillus stearothermophilus NCIMB 8224 to low temperature steam and formaldehyde

Spores of a Bacillus stearothermophilus strain thought to be a good biological monitor for low temperature steam and formaldehyde (LTSF) sterilization have been subjected to different recovery environments following exposure to relevant LTSF treatments and prior to enumerating the numbers of surviving spores. The recovery treatments essentially consisted of holding samples at a temperature of 90°C for different time periods prior to plating out following exposure to 12 μg ml‐1 formaldehyde at temperatures between 63° and 83°C The data indicate that LTSF‐exposed spores show a marked recovery when kept at 90°C prior to plating out; the extent of the recovery increases with increasing inactivation temperature between 73° and 83°C. The data bring into question the sporicidal activity of LTSF.

Application of 13C-n.m.r. spectroscopy to study the mechanism of N-demethylation of [NMe13C] codeine by cell-free extracts of Cunninghamella bainieri

Abstract Codeine was synthesized with 90% enrichment of the N -methyl group with carbon-13. N -Demethylation of this substrate by cell-free extracts of Cunninghamella bainieri in an n.m.r, tube gave norcodeine and 13 C-labelled formaldehyde. Fourier-transform 13 C-n.m.r. spectroscopy was used to observe the N -demethylation process at selected temperatures. The labelled formaldehyde liberated was trapped with sodium sulphite, and the sulphite adduct, as well as intermediates, were located in the n.m.r, spectrum at each temperature. Intermediate resonances assignable to codeine- N -oxide were not detected during these enzyme-transformation studies. These data suggest that the observed 13 C-n.m.r. signals correspond to the chemically labile carbinolamine intermediate formed during N -demethylation. A methine 13 C signal was not observed. Thus, N -demethylation of codeine by Cunninghamella bainieri occurs by direct C -oxidation and not via an N -oxide intermediate.

MUTAGENICITY OF NN‐DIALKYLAMINOALKYL CHLORIDES

The cyclic aziridinim ion is a powerful alkylating agent of functional nucleophilic groups. The capacity to form this ion and to alkylate DNA is a feature of a number of potent mutagens and carcinogens e.g. ethylenimine, triethylene melamine and the nitrogen mustards. NN-Dialkylaminoalkyl chlorides are widely used for the addition of sidechains to phenothiazines, thioxanthenes and tricyclic antidepressants, and in the synthesis of drugs such as pethidine and methadone. They are stable compounds and may Well Persist through the synthesis and occur as trace contaminants in the final product. It can also be predicted that a number of these compounds may form the aziridinium ion. This work investigates the correlation between mutagenicity and the ability to form the aziridinim ion in a series of NN-Dialkylaminoalkyl chlorides.

The heat resistance of bacterial spores after different vacuum drying treatments.

The resistance of bacterial spores to high temperatures of around 120” has been used in this work as a means of assessing damage induced in the spores by different vacuum drying treatments. The vacuum apparatus used enabled simultaneous measurements to be made, of pressure, sample weight and sample temperature changes, and allowed for correlation between physical measurements and biological response. The effect of different drying treatments was assessed by exposing samples to a constant elevated temperature for different times and estimating the viability. Under all conditions it was found that the log surviving fraction (N/N,)/heating time (t) curves exhibited a shoulder at high survival levels, but were linear below a surviving fraction of 0.1. The linear portion is described by N/N, = ae--kt where “a” is the intercept of the curve with the “y” axis. Two parameters have been used to characterize the response; firstly, the slope of the curve “k”, and secondly, a shoulder constant “s” which is the heating time required to reduce the surviving fraction to 0.1 i.e. s = t, when N/No = 0-1. The usefulness of the constants “k” and “s” in deducing possible lethal mechanismsrelies on them changing with heating temperature in a meaningful way. Therefore the characteristics of the heat response of spores was investigated in aqueous suspension, and also after being subjected to sublimative low vacuum drying, and to high vacuum drying, where additional water is removed by isothermal desorption. Both “k” and “s” were found to be directly related to the heating temperature, and were shown to vary systematically and independently with the drying treatment. When “k” values were treated according to the Arrhenius relationship k = Ae --EafRT it was found that the activation energy for the lethal mechanism (Ea) did not change with different drying treatments, being 155 kJ mol-l (34 k cal mol-l) in all cases. The susceptibility of the spore to these mechanisms, as indicated by the frequency factor (A), was, however, dependent upon the drying treatment, the value being loo0 times smaller after sublimative drying than in aqueous suspension, and 20 times greater after high vacuum than after sublimative drying. A treatment of “s” in a similar way showed that the size of the shoulder decreased with increasing temperature, but that removal of water by sublimative drying caused an increase in “s” by a factor of 1oo0, the value after isothermal drying being 100 times lower than this. A study of the variation of “k” and “s” under different conditions would lead us to doubt that they are representative of one mechanism. Rather we would postulate that “s”, the shoulder constant, represents a lag time during which structural changes occur within the spore that cause it to become susceptible to the lethal mechanism represented by “k”. The effects of different drying treatments have been analysed on this basis. * Present address, School of Pharmacy, City of Leicester Polytechnic, Leicester, U.K. BRITISH PHARMACEUTICAL CONFERENCE 1971 :

Isolation of codeine and norcodeine from microbial transformation liquors by preparative high-performance liquid chromatography

Codeine and norcodeine have been extracted together from a 7-l batch of microbial transformation liquor using an XAD-4 resin column. The extract was divided equally and used to compare normal-phase (NP) and reversed-phase (RP) preparative high-performance liquid chromatography for the recovery of pure codeine and norcodeine. The conditions for each column were optimised for maximum sample throughput with complete resolution of both compounds. The aqueous fractions of codeine and norcodeine from the RP column were also passed through an XAD-4 resin column to extract the compounds finally, whereas the organic solvent fractions from the NP column did not require this step. The RP procedure gave recoveries of 71.25% of codeine (98.6% purity) and 80.69% of norcodeine (97.5% purity) and the NP procedure gave recoveries of 79.20% of codeine (96.7% purity) and 83.97% of norcodeine (97.2% purity). Owing to its superior sample throughput and lower operating costs, the RP procedure is recommmended for routine use.

Procedures for the quantitative analysis of microbial N-dealkylation systems by gas-liquid chromatography

Abstract Gas-liquid chromatography methpods were developed for the determination of transformation products and unaltered substrate in mirobial N -dealkylation studies with drug molecules. the microorganism used was the fungus Cunninghemella echinulata , and the drugs employed included morphine alkaloids, various N -substituted 6,7-benzomorphans, and diazepam, a member of the benzodiazepine group of drugs. Assays based on the GLC system utilizing a non-polar SE-30 stationary phase and employing a derivatization step prior to chromatography were devised. The development of these methods facilitated quantitative studies on microbial N -dealkylation and the relationship between substrate structure and microbial transformation activity.