Proteins control individual patient's response to pharmaceutical medication, be they receptors, transporters or enzymes. These proteins are under the control of genes. The study of these genes and the interplay between multiple genes is pharmacogenomics, with individual genes being termed pharmacogenes. The greatest understanding of pharmacogenetics is of the drug metabolising enzymes, the cytochrome P450s. Almost the entire UK population is likely to have at least one genetic variant that controls these P450s and thus the phenotype for metabolic competence. This means two patients receiving the same medication and dose may have very different responses, from adverse reaction to being ineffective. An individual military person's response to medications can be predicted from their pharmacogenetics, as an example; the response to the commonly prescribed 'pain killers', codeine, tramadol, hydrocodone or oxycodone. These opioids are metabolised into their active forms by the cytochrome 2D6. Four phenotypes classify an individual's metabolic competency: ultra-rapid, extensive, intermediate or poor. A poor metaboliser is at risk of ineffective pain relief from one of the opioids listed, whereas an ultra-rapid metaboliser is at risk of overexposure and subsequent dependency or abuse. In white European populations, the prevalence of the phenotypes is well known and may be used to guide prescribing; however, in other populations such as Nepalese or Pacific Islander the distribution of these phenotypes is unknown. Genotyping provides a framework for the precise treatment of patients and cost-effective use of medication for the UK Armed Forces, as well as potentially providing equity for minority groups.

The bispyridinium oxime HI-6 DMS is in development as an improved therapy for the treatment of patients exposed to organophosphorus nerve agents. The aim of the work described in this paper was to provide nonclinical data to support regulatory approval of HI-6 DMS, by demonstrating efficacy against an oximesensitive agent, GB and an oxime-resistant agent, GD. We investigated the dose-dependent protection afforded by therapy including atropine, avizafone and HI-6 DMS in guinea-pigs challenged with GB or GD. We also compared the efficacy of 30 mg.kg1 of HI-6 DMS to an equimolar dose of the current in-service oxime P2S and the dichloride salt of HI-6 (HI-6 Cl2). In the treatment of GB or GD poisoning there was no significant difference between the salt forms. The most effective dose of HI-6 DMS in preventing lethality following challenge with GB was 100 mg.kg � 1; though protection ratios of at least 25 were obtained at 10 mg.kg � 1. Protection against GD was lower, and there was no significant increase in effectiveness of HI-6 DMS doses of 30 or 100 mg.kg � 1. For GD, the outcome was improved by the addition of pyridostigmine pre-treatment. These data demonstrate the benefits of HI-6 DMS as a component of nerve agent therapy. (c) Crown copyright (2023), Dstl.

S-Carboxymethyl-l-cysteine is a mucolytic agent used as adjunctive therapy in the treatment of respiratory disorders. Various mechanisms of action have been proposed but few studies have attempted to link the required in vitro concentrations with those achieved actually in vivo during clinical therapy. The data from several published studies has been re-analysed by WinNonlin using non-compartmental analysis modelling, Phoenix modelling and Classic PK compartmental modelling for both single (500-1500 mg) and multiple oral administration of the drug. Multiple dose modelling indicated maximum peak concentrations (C-max) ranging from 1.29 to 11.22 mu g/ml and those at steady state (C-ss(av)) from 1.30 to 8.40 mu g/ml. For the standard therapeutic regimen of 3 x 750 mg (2250 mg/day) these values were 1.29-5.22 mu g/ml (C-max) and 1.30-3.50 mu g/ml (C-ss(av)). No accumulation was observed. Hence, only the pharmacodynamic studies reporting significant effects below c.10 mu g/ml were likely to occur in vivo and these were mainly gene-related mechanisms. The majority of events, although demonstrable in vitro, required levels much greater than possible to achieve in the clinical situation. Such unappreciated disregard for in vitro-in vivo 'concentration matching' may lead to erroneous conclusions regarding mechanisms of action for many drugs as well as for S-carboxymethyl-l-cysteine.

Mice that were heterozygous dominant for the enu1 and enu2 mutation in phenylalanine monooxygenase/phenylalanine hydroxylase (PAH) resulted in hepatic PAH assays for S-carboxymethyl-L-cysteine (SCMC) that had significantly increased calculated Km (wild type (wt)/enu1, 1.84-2.12 fold increase and wt/enu2 a 2.75 fold increase in PAH assays). The heterozygous dominant phenotypes showed a significantly reduced catalytic turnover of SCMC (wt/enu1, 6.11 fold decrease and wt/enu2 an 11.25 fold decrease in calculated Vmax). Finally, these phenotypes also had a significantly reduced clearance, CLE (wt/enu1, 13.02 fold and wt/enu2, a 30.80-30.94 fold decrease) The homozygous recessive phenotype (enu1/enu1) was also found to have significantly increased calculated Km (2.16 fold increase), a significantly reduced calculated Vmax (11.35-12.33 fold decrease) and CLE (24.75-25.00 fold decrease). The enu2/enu2, homozygous recessive phenotype had no detectable PAH activity using SCMC as substrate. The identity of the enzyme responsible for the C-oxidation of L-phenylalanine (L-Phe) and the S-oxidation of SCMC in wt/wt (BTBR) mice was identified using monoclonal antibody and selective chemical inhibitors and was found to be PAH. This in vitro mouse hepatic cytosolic fraction metabolism investigation provides further evidence to support the hypothesis that an individual possessing one variant allele for PAH will result in a poor metaboliser phenotype that is unable to produce significant amounts of S-oxide metabolites of SCMC.

Abstract Background The S-oxidation of S-carboxymethyl-L-cysteine has been reported previously to be a biomarker of disease susceptibility in Parkinson’s disease and amyotrophic lateral sclerosis. In the present investigation, the original observations have been extended and confirmed. Methods Meta-analysis of previously published investigations into the S-oxidation polymorphism together with new subject data was evaluated. Results The incidence of the poor metaboliser phenotype (no urinary recovery of S-oxide metabolites) was found to be 3%–7% within healthy and non-neurological disease populations, whereas 38% of the Parkinson’s disease subjects and 39% of the amyotrophic lateral sclerosis group were phenotyped as poor metabolisers. The consequent odds risk ratio of developing Parkinson’s disease was calculated to be 33.8 [95% confidence interval (CI), 13.3–86.1] and for amyotrophic lateral sclerosis was 35.2 (95% CI, 13.0–85.1). Conclusions The possible involvement of the enzyme responsible for this S-oxidation biotransformation reaction, phenylalanine hydroxylase, should be further investigated to elucidate its potential role in the mechanism(s) of toxicity in susceptible individuals displaying these diseases. The “Janus hypothesis,” possibly explaining why phenylalanine hydroxylase is a biomarker of neurodegenerative disease susceptibility, together with the general theme that this concept may apply to many other hitherto unsuspected enzyme systems, is presented.

1. Consistent differences in the proportion of an orally administered dose of S-carboxymethyl-l-cysteine subsequently excreted in the urine as S-oxide metabolites were reported 40 years ago. This observation suggested the existence of inter-individual variation in the ability to undertake the enzymatic S-oxygenation of this compound. Pedigree studies and investigations employing twin pairs indicated a genetically controlled phenomenon overlaid with environmental influences. It was reproducible and not related to gender or age. 2. Studies undertaken in several healthy volunteer cohorts always provided similar results that were not significantly different when statistically analysed. However, when compared to these healthy populations, a preponderance of subjects exhibiting the characteristic of poor sulfoxidation of S-carboxymethyl-l-cysteine was found within groups of patients suffering from various disease conditions. The most striking of these associations were witnessed amongst subjects diagnosed with neurodegenerative disorders; although, underlying mechanisms were unknown. 3. Exhaustive investigation has identified the enzyme responsible for this S-oxygenation reaction as the tetrahydrobiopterin-dependent aromatic amino acid hydroxylase, phenylalanine 4-monooxygenase classically assigned the sole function of converting phenylalanine to tyrosine. The underlying principle is discussed that enzymes traditionally associated solely with intermediary metabolism may have as yet unrecognised alternative roles in protecting the organism from potential toxic assault.

The role that the phase-II reaction, glucuronidation, plays in the biotransformation of endo and xenobiotics is discussed with particular emphasis given to the UGT1A1 isoenzyme. This individual isoenzyme is responsible for both the mono and di-glucuronidation of bilirubin together with the glucuronidation of a number of xenobiotics of clinical interest (irinotecan, belinostat, atazanavir, pegvisomant).The review then discusses the roles that the various allelic variants of the UGT1A1 gene play in bilirubin metabolism and in particular how these allelic variants are involved in the clinical manifestation of the diseases of GS, CN1 and CN2.The review concludes with the roles that the UGT1A1*28 and UGT1A1*6 alleles play in adverse drug reactions (decreased glucuronidation of irinotecan, belinostat, atazanavir, pegvisomant) leading to increased exposure, reduced clearance and neutropenia (irinotecan, belinostat), increased risk for jaundice and hyperbilirubinaemia (atazanavir) and liver toxicity (pegvisomant) before discussing the future role of UGT1A1 in personalised medicine.

ObjectivesTo determine the K-m, V-max, cofactor, activator and inhibitor requirements of human cysteine dioxygenase and S-carboxymethyl-l-cysteine S-oxygenase with respect to both l-Cysteine and S-carboxymethyl-l-cysteine as substrates. MethodsIn vitro human hepatic cytosolic fraction enzyme assays were optimised for cysteine dioxygenase activity using l-Cysteine as substrate and the effect of various cofactors, activators and inhibitors on the S-oxidations of both l-Cysteine and S-carboxymethyl-l-cysteine were investigated. Key findingsThe results of the invitro reaction phenotyping investigation found that although both cysteine dioxygenase and S-carboxymethyl-l-cysteine S-oxygenase required Fe2+ for catalytic activity both enzymes showed considerable divergence in cofactor, activator and inhibitor specificities. Cysteine dioxygenase has no cofactor but uses NAD(+) and NADH(H+) as pharmacological chaperones and is not inhibited by S-carboxymethyl-l-cysteine. S-carboxymethyl-l-cysteine S-oxygenase requires tetrahydrobiopterin as a cofactor, is not activated by NAD(+) and NADH(H+) but is activated by l-Cysteine. Additionally, the sulfydryl alkylating agent, N-ethylmaleimide, activated carboxymethyl-l-cysteine S-oxygenase but inhibited cysteine dioxygenase. ConclusionsHuman hepatic cytosolic fraction cysteine dioxygenase activity is not responsible for the S-oxidation of the substituted cysteine, S-carboxymethyl-l-cysteine.

Although it is believed widely that the various routes of xenobiotic metabolism are now all known and effectively understood, occasionally there emerges a metabolite that signals a novel biotransformation pathway, especially where the xenobiotic may in some way interact with the myriad processes of intermediary metabolism. There are a few reports in the literature where saturated short-chain dicarboxylic acids have been exploited as conjugating agents and these unusual xenobiotic metabolites subsequently excreted intact in the urine.Initially suggested by unpublished observations bolstered by extensive experience of the authors and colleagues in the field of xenobiochemistry, this narrative review has been supplemented by a search of bibliographic databases and the subsequent scrutiny of numerous peer-reviewed research articles. The resultant sparse and widely dispersed information has been examined, analysed and presented in this review.Xenobiotic conjugation with dicarboxylic acids has been demonstrated to occur within several domains of life; microorganisms, plants, invertebrates and mammals. However, considering the number of xenobiotic metabolism investigations that have been undertaken reports of such conjugations are exceedingly rare.Dicarboxylic acid condensation with xenobiotic molecules may occur at nitrogen centres, or more precisely with a primary or secondary amine, that is at nitrogen still possessing a replaceable hydrogen atom. Both aliphatic amines and arylamines may be substrates with many of the free amino groups being formed by previous Ndealkylation reactions. Hopefully, awareness of this metabolic route will be raised and researchers will be enthused to search for this type of conjugate.

The S-oxidation of S-carboxymethyl-l-cysteine has been reported previously to be a biomarker of disease susceptibility in Parkinson's disease and Amyotrophic lateral sclerosis. In this investigation, the original observations have been confirmed with the incidence of the poor metaboliser phenotype (no urinary recovery of S-oxide metabolites) being found to be 3.9% within healthy control population. However, 38.3% of the Parkinson's disease subjects and 39.0% of the Amyotrophic lateral sclerosis group were phenotyped as poor metabolisers. The consequent odds risk ratio of developing Parkinson's disease was calculated to be 15.5 (95% CI 9.5-25.3) and for Amyotrophic lateral sclerosis was 15.2 (95% CI 8.8-26.5). Thus, the possible role of the enzyme responsible for the S-oxidation biotransformation reaction, phenylalanine hydroxylase, must be further investigated to elucidate the mechanism(s) of toxicity in susceptible individuals displaying these diseases. A dual role potentially explaining of the role of phenylalanine hydroxylase as a biomarker of disease susceptibility is presented together with the observation that metabolomics is a possible way forward in the identification of potential pro-toxins/toxins in those individuals phenotyped as poor metabolisers (Controls, Parkinson's disease and Amyotrophic lateral sclerosis subjects).

Background: Although it is believed widely that the various routes of xenobiotic metabolism are now all known and effectively understood, occasionally there emerges a metabolite that signals a novel biotransformation pathway, especially where the xenobiotic may in some way interact with the myriad processes of intermediary metabolism. There are a few reports in the literature where saturated short-chain dicarboxylic acids have been exploited as conjugating agents and these unusual xenobiotic metabolites subsequently excreted intact in the urine. Method: Initially suggested by unpublished observations bolstered by extensive experience of the authors and colleagues in the field of xenobiochemistry, this narrative review has been supplemented by a search of bibliographic databases and the subsequent scrutiny of numerous peer-reviewed research articles. The resultant sparse and widely dispersed information has been examined, analysed and presented in this review. Results: Xenobiotic conjugation with dicarboxylic acids has been demonstrated to occur within several domains of life; microorganisms, plants, invertebrates and mammals. However, considering the number of xenobiotic metabolism investigations that have been undertaken reports of such conjugations are exceedingly rare. Conclusion: Dicarboxylic acid condensation with xenobiotic molecules may occur at nitrogen centres, or more precisely with a primary or secondary amine, that is at nitrogen still possessing a replaceable hydrogen atom. Both aliphatic amines and arylamines may be substrates with many of the free amino groups being formed by previous Ndealkylation reactions. Hopefully, awareness of this metabolic route will be raised and researchers will be enthused to search for this type of conjugate.

Aim: An open-label, phase I dose-escalation trial was performed in adult patients with various solid cancers to identify the maximum tolerated dose (MTD), to assess the safety, pharmacokinetic profile and anti-tumour activity of the new prodrug CAP7.1. The prodrug is converted to its active moiety etoposide via carboxylesterases in selective cells leading to a better tolerability and higher efficacy in therapeutic resistance cells and children with refractory neuroblastoma.Patients and methods: Eligible adult patients with advanced, refractory, solid malignancies received CAP7.1 as intravenous infusion on 5 consecutive days. Doses were escalated in four cohorts consisting of three to six patients, with a starting dose of 45 mg/m(2)/day. Treatment cycles were repeated in 21-day intervals in the absence of disease progression and prohibitive toxicity. The safety, pharmacokinetics and efficacy were evaluated, and the MTD and doselimiting toxicity (DLT) were determined.Results: Nineteen patients were assigned to four CAP7.1 dose cohorts (45, 90, 150 and 200 mg/m(2)/day). CAP7.1 was well tolerated. Haematotoxicity was observed at the two highest dose levels including three DLTs (two febrile neutropenia and one sepsis) only and were reversible with adequate therapy. No organ toxicity was observed. Non-haematological toxicities (mild-moderate) consist mainly of nausea, fatigue, vomiting, pyrexia and alopecia. One partial response and 11 stable diseases were observed as supporting signs of efficacy.Conclusion: MTD of CAP7.1 was reached at the dose of 200 mg/m(2). A favourable safety profile and initial anti-tumour efficacy of CAP7.1 in therapeutic refractory tumours warrant further evaluation in clinical studies. (C) 2017 Elsevier Ltd. All rights reserved.

e15602 Background: The aim of this study was to describe the population pharmacokinietics (PK) of CAP7.1, the prodrug of etoposide, and to describe how changes in total target lesion (TTL) size in biliary tract cancer (BTC) patients depend on treatment with CAP7.1. Methods: Non linear mixed effects modeling with Monolix was used to develop a population PK model. Data for the population PK model was from an open label, dose escalation phase I study and the ongoing randomized, multicenter phase II trial in BTC patients. Data from a total of 39 patients with 434 observations was available for PK modeling. Variability in CAP7.1 plasma concentrations exeeded those of etoposide; for this reason a dose-etoposide PK model was developed without taking into account CAP7.1 concentrations. The individual a posteriori PK model parameter estimates were then used with Simulx to simulate etoposide AUC0-24h , cmax and cmin for each BTC patient and compared with the individual changes in TTL size. Results: Etoposide PK was best described with a two compartmental model with a first order metabolic conversion rate and with linear clearance from the central compartment. The metabolic rate was 0.39 1/h (CV 44%), the clearance 3.2 L/h (CV 56%), the first volume 6.2 L (CV 37%), and the two exchange rates between the two compartments were k12 = 0.53 and k21 = 0.2 1/h. The calculated terminal half life of etoposide was 7.9 h. Linear regression analysis between the simulated AUC0-24h and change in TTL size showed that a higher AUC0-24h was associated with a lower increase in TTL size (R2= 0.172). For an AUC0-24h larger than 100 h ug/mL six out of eight BTC patients had an increase in TTL size of less than 20%. Similarly, a higher cmax (R2= 0.177) or cmin (R2= 0.11) were associated with a larger effect on TTL size although less pronounced for cmin . Conclusions: While the PK of CAP7.1 was variable the dose-etoposide population PK could be well described. The emerging pattern from the data analysis showed that for a higher etoposide AUC0-24h a larger fraction of patients could maintain the increase in TTL size below 20% for the observational period in the phase II study in BTC Clinical trial information: NCT02094560.

1. Incubation of beagle hepatic cytosol, under conditions promoting phenylalanine hydroxylase activity, led to the formation of the sulfoxide derivatives of S-carboxymethyl-L-cysteine, N-acetyl-S-carboxymethyl-L-cysteine, S-methyl-L-cysteine and N-acetyl-S-methyl-L-cysteine. Thiodiglycolic acid was not a substrate. Enzyme kinetic parameters (Km, Vmax) were derived indicating S-carboxymethyl-L-cysteine had the greatest clearance; no enantioselective preference was observed for this S-oxygenation reaction. 2. Following oral administration of S-carboxymethyl-L-cysteine to beagle dogs, the parent substance and its sulfoxide were the only compounds identified in the plasma. Pharmacokinetic data have been obtained indicating that the small amount of sulfoxide formed persisted within the body for longer than the parent material, but that the majority of the ingested dose remained in the administered sulfide form. 3. The sulfide moiety within the muco-regulatory drug, S-carboxymethyl-L-cysteine, is thought to be vital as it acts as a free radical scavenger, resulting in the inactive sulfoxide. Additional extensive enyzme-mediated sulfoxidation would decrease the amount of active sulfide available. In the dog this appears to not be an issue, signalling possible exploitation for therapeutic benefit in treating airway disease.

The extent of sulfoxidation of the drug, S-carboxymethyl-L-cysteine, has been shown to vary between individuals, with this phenomenon being mooted as a biomarker for certain disease states and susceptibilities. Studies in vitro have indicated that the enzyme responsible for this reaction was phenylalanine monooxygenase but to date no in vivo evidence exists to support this assumption. Using the mouse models of mild hyperphenylalaninamia (enu1 PAH variant) and classical phenylketonuria (enu2 PAH variant), the sulfur oxygenation of S-carboxymethyl-L-cysteine has been investigated.Compared to the wild type (wt/wt) mice, both the heterozygous dominant (wt/enu1 and wt/enu2) mice and the homozygous recessive (enu1/enu1 and enu2/enu2) mice were shown to have significantly increased C-max, AUC((0-180 min)) and AUC((0-infinity min)) values (15 -to 20-fold higher). These results were primarily attributable to the significantly reduced clearance of S-carboxymethyl-L-cysteine (13 - to 22-fold lower).Only the wild type mice produced measurable quantities of the parent S-oxide metabolites. Those mice possessing one or more allelic variant showed no evidence of blood SCMC (R/S) S-oxides. These observations support the proposition that differences in phenylalanine hydroxylase activity underlie the variation in S-carboxymethyl-L-cysteine sulfoxidation and that no other enzyme is able to undertake this reaction.

1. Once in a while, during drug metabolism studies, an unusual or unexpected pathway is unearthed.2. Such quirky finds open a refreshing hiatus, providing a departure from the, perhaps now mundane, textbook routes.3. This brief missive draws attention to an interesting anecdote that may be unknown to some and concerns a substituted thioxanthenone drug.

S-carboxymethyl-L-cysteine, the side-chain carboxymethyl derivative of the sulfur-containing amino acid, cysteine, has been known and available for almost 80 years. During this time, it has been put to a variety of uses, but it is within the field of respiratory medicine that, presently, it has found a clinical niche. Early studies indicated that this compound underwent a rather simplistic, predictable pattern of metabolism, whereas later investigations alluded to more subtle interactions with the pathways of intermediary metabolism, as may be expected for an amino acid derivative. In addition, suggestions of polymorphic influences and circadian rhythms within metabolic profiles have emerged. These latter factors may underlie the conflicting reports regarding the therapeutic efficacy of this compound: that it appears to work well in some patients, but has no measurable effects in others. The relevant literature pertaining to the fate of this compound within living systems has been reviewed and a comprehensive précis advanced. Hopefully, this article will serve as a vade mecum for those interested in S-carboxymethyl-L-cysteine and as a catalyst for future research.

The amino acid derivative, S-carboxymethyl-l-cysteine, is an anti-oxidant agent extensively employed as adjunctive therapy in the treatment of human pulmonary conditions. A major biotransformation route of this drug, which displays considerable variation in capacity in man, involves the oxidation of the sulfide moiety to the inactive S-oxide metabolite. Previous observations have indicated that fasted plasma l-cysteine concentrations and fasted plasma l-cysteine/free inorganic sulfate ratios were correlated with the degree of sulfoxidation of this drug and that these particular parameters may be used as endobiotic biomarkers for this xenobiotic metabolism. It has been proposed also that the enzyme, cysteine dioxygenase, was responsible for the drug sulfoxidation. Further in this theme, the degree of S-oxidation of S-carboxymethyl-l-cysteine in 100 human volunteers was investigated with respect to it potential correlation with fasted plasma amino acid concentrations. Extensive statistical analyses showed no significant associations or relationships between the degree of drug S-oxidation and fasted plasma amino acid concentrations, especially with respect to the sulfur-containing compounds, methionine, l-cysteine, l-cysteine sulfinic acid, taurine and free inorganic sulfate, also the derived ratios of l-cysteine/l-cysteine sulfinic acid and l-cysteine/free inorganic sulfate. It was concluded that plasma amino acid levels or derived ratios cannot be employed to predict the degree of S-oxidation of S-carboxymethyl-l-cysteine (or vice versa) and that it is doubtful if the enzyme, cysteine dioxygenase, has any involvement in the metabolism of this drug.