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- What is Already Known about this Subject
- What does this Study Add?
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Biliary secretion is often a major route of elimination of drugs and their metabolites from the body. Knowledge of the biliary disposition of a drug is essential to understanding the relative importance of different routes of metabolism and their relationship to overall clearance . As the clearance of a drug can be modulated by co-administered therapies resulting in clinically significant drug interactions, understanding the drug-related material in bile can help to assess this risk .
There is a wealth of published information illustrating how drug interactions can be attributed to modulation of the clearance mechanism of drugs. The most significant drug interactions in terms of both magnitude and clinical impact are mediated by the cytochrome P450 enzymes (CYPs), i.e. when CYP mediated pathways of metabolism are inhibited by the co-administration of a CYP inhibitor resulting in elevated concentrations of circulating victim drug which may be associated with adverse drug reactions .
The risk of victim drug interactions can be inferred by combining a mechanistic understanding of the enzymes and transporters involved in drug clearance, as determined by in vitro studies, with knowledge of the relative proportion of drug and metabolites excreted in human urine and faeces. Where biliary elimination is a significant route of drug clearance, understanding the composition of drug-related material in the bile is essential to understanding the risk of a victim drug interaction. However, since collection of bile from human subjects is generally recognized to be a complex and invasive process, biliary disposition information is rarely available.
The drug under investigation (GSK1325756, molecular weight 441, partition coefficient [log P] 3.9, see Figure 1) in the current study is intended for the maintenance treatment of chronic obstructive pulmonary disease (COPD). Many COPD patients are elderly and are frequently exposed to many other concomitant medications including some known to cause clinically relevant drug interactions by inhibiting drug metabolizing enzymes and transporters. An understanding of the risks of drug interactions is therefore important in the medical management of COPD patients. An analysis of co-medications has been conducted in the GSK-funded ECLIPSE study (Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints ) which had over 2000 enroled COPD patients and captured patient-reported medication use. This analysis confirmed that CYP3A4 inhibitors are of most concern in this patient population. For example at the time of screening for the study 9% and 6% of patients reported using the CYP3A4 inhibitors, atorvastatin and amlodipine, respectively. Diltiazem and verapamil have been shown to result in the greatest drug interaction due to CYP3A4 inhibition, where an increased plasma exposure of approximately 5-fold has been observed for CYP3A4 metabolized drugs like simvastatin [5, 6]. Understanding the enzymes involved in the metabolic clearance of GSK1325756 is therefore critical to assess the risk of victim drug interactions with co-administered CYP inhibitors.
Figure 1. Structure of GSK1325756 and metabolites identified in human bile captured using the Entero-Test®. Percentage of observed drug related material in pooled bile shown in parenthesis (estimated by semi-quantitative NMR)
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Preclinical excretion and metabolism studies (unpublished data) in the rat indicated that bile is a major elimination route for GSK1325756. In rat bile and urine the major components were parent drug and the O-glucuronidated metabolite (M11, see Figure 1). The metabolism pathways were well predicted using in vitro methodologies in this species i.e. O-glucuronidation was also identified as a major metabolite in rat hepatocytes. The contribution of drug transporters to the elimination of GSK1325756 and its metabolites is currently unknown. Although O-glucuronidation was considered a likely pathway of metabolism in humans, the turnover in human hepatocytes was too low to substantiate this. Furthermore, other human data [unpublished] indicated a potential for oxidative metabolism as GSK1325756 was metabolically unstable when incubated with CYP1A2, 2C9, 2D6 and 3A4 recombinant enzymes and the only observed drug-related material in human blood (in addition to unchanged parent drug) was an oxidative metabolite i.e. there was no evidence of the O-glucuronide metabolite. In human urine, parent drug represented approximately 50% observed drug-related material and the predominant metabolite was the O-glucuronide (M11), which represented approximately 30% of observed drug-related material, with the remaining drug-related material consisting of oxidative metabolites. However, as the total drug-related material in urine accounted for merely 4% of the total administered dose, only a small proportion of the total elimination of GSK1325756 was understood, therefore prompting this investigation of biliary elimination in human subjects.
A simple method for the collection of duodenal bile in human subjects has been previously reported by Guiney et al.  and has demonstrated that the Entero-Test®, a commercially available device, could be used reliably to collect human duodenal bile in order to determine metabolites of the lipid lowering drug simvastatin. The work described herein demonstrates a further application of the Entero-Test® technique to aid the assessment of victim drug interaction risk by characterization of the biliary disposition of GSK1325756.
- Top of page
- What is Already Known about this Subject
- What does this Study Add?
- Competing Interests
Drug–drug interactions (DDIs) are an important consideration in the discovery, development and clinical use of new pharmaceuticals as there are several historical examples of serious harm leading to withdrawal from the market resulting from DDIs caused by inhibition of metabolic clearance (mibefradil  and terfenadine ). Understanding the clinical risk associated with drug interactions is therefore essential when developing new therapeutic agents in order to aid internal decision making and understand the potential impact on clinical safety in a target patient population. Ideally a drug should have minimal interaction liability as a victim otherwise a wide therapeutic index is required to minimize any clinical consequence of increasing drug concentrations. There is also a regulatory requirement to understand drug interaction risks and the FDA  and EMA  recommend that the underlying mechanisms for drug interactions are explored in advance of drug registration. Both regulatory agencies request an understanding of the enzymes involved in metabolic pathways contributing to >25% of a drug's elimination.
A large number of drug interactions have been attributed to the inhibition of CYPs which play a significant role in oxidative drug metabolism. In the CYP superfamily, the CYP3A4 enzyme is responsible for the metabolism of circa 50% of therapeutic drugs and is most frequently implicated in clinical DDIs . Co-administration of a CYP3A4 inhibitor (perpetrator) with a drug whose clearance routes are predominately mediated by CYP3A4 can result in elevated and potentially toxic concentrations of the victim drug . In comparison, drugs that are predominately metabolized by direct conjugation, for example glucuronidation (mediated by uridine 5'-diphospho-glucuronosyltransferase enzymes, UGT), are less sensitive to drug interactions. This can be attributed to the low affinity of substrates to UGT enzymes, the involvement of multiple UGT enzymes with over-lapping specificities and the limited potency of UGT inhibitors [15, 16]. Williams et al.  report that the exposure increases of a glucuronidation substrate rarely exceed 2-fold in the presence of a UGT inhibitor. This is a small magnitude of change compared with CYP3A4 victim drug interactions which can result in over 20-fold increases in the plasma concentration of drugs like midazolam .
In vitro methods to understand the major enzymes and transporters involved in drug elimination are widely implemented. However this information needs to be considered along with knowledge of the parent drug and metabolites eliminated in vivo to enable an accurate assessment of the clinical drug interaction risk . A traditional approach to this involves dosing radiolabelled drug to humans and collecting urine and faeces to assess the routes of excretion and identify the major components. Owing to the considerable investment and enabling studies required to support a human radiolabel study [19, 20], these studies are often conducted too late in clinical development to impact on the co-medication strategy. Also bile is typically not collected in these studies thereby missing a potential key route of drug elimination . Biliary elimination can sometimes be inferred by the drug-related material in faeces. However investigating this latter matrix is often technically challenging and may provide misleading information, e.g. for drugs that are poorly absorbed or those which undergo biliary secretion as conjugates (e.g. glucuronides) which are then subject to hydrolysis by gut microflora.
The human metabolism data obtained in the current study with duodenal bile collection, has significantly contributed to the assessment of the drug interaction risk for GSK1325756. The hypothesis of O-glucuronidation being the major elimination route (based on in vitro and preclinical data) was supported, as indicated by the metabolite M11 representing 80% of the observed drug-related material in bile. Therefore concerns of victim drug interactions are reduced in proposed patient studies. It can be speculated that CYP enzymes are responsible for the production of the oxidative metabolites observed in bile (<15% oDRM) and in urine (<16% oDRM). This is supported by the previous data obtained with CYP recombinant enzymes. However the current data put this oxidative metabolism into context as a likely minor elimination route in vivo. Using extrapolation techniques it can be estimated that contribution of CYP enzymes would need to comprise more than 50% total drug elimination to result in a 2-fold increase in drug exposure with a CYP inhibitor .
Understanding the relative proportion of metabolism and direct secretion of parent drug in bile (mediated by drug transporters ) may further mitigate CYP mediated victim drug interaction risks. This has minimal impact for GSK1325756 as the proportion of parent drug in bile represented only 5% observed drug-related material. The potential for contamination by unabsorbed parent drug on the bile string should be acknowledged. However the estimated ratios of metabolite to parent drug in bile were similar across subjects in this study (data not shown) which tends to support biliary secretion of parent drug rather than contamination by unabsorbed drug. This conclusion is supported by the observation that parent drug was not detected on the strings retrieved without a bile sample. Proving that clearance is predominately via secretion of unchanged parent drug may help mitigate victim drug interaction risk for other drugs, for example where metabolism is mediated primarily by CYP enzymes. Using the Entero-Test® to collect bile following an intravenous dose may be one approach to investigate this, i.e. any drug-related material detected on the string can be directly linked to biliary secretion of parent drug and/or metabolites.
The collection of bile samples was successfully conducted in this clinical study in healthy, elderly volunteers. There were no compliance or adverse events reported with use of the Entero-Test® device and the presence of food less than 3 h before administering the Entero-Test® did not preclude the collection of bile samples in over half the subjects, neither did food interfere with the analysis of the bile samples using spectrometric techniques. As reported previously  there appears to be wide variation in the percentage of gallbladder contraction between subjects and within an individual which may explain the differences in successful pre and post bile collection in this study.
Other potential caveats to the application of the Entero-Test® technique should be considered. It is unclear how changes in bile, pancreatic and intestinal flow may impact on the sample collected and it should be remembered that the bile sample collected represents a snapshot in time of total biliary output, in this instance 7 h post-dose. Therefore there is a risk that the metabolic profile indicated may not represent the complete excretion of drug via bile. To maximize the metabolic relevance of the sample observed in this study, the biliary collection time was optimized to coincide with the approximate half-life of the drug (i.e. 7 h). It is worth noting that this limitation is also common to other bile sampling techniques, e.g. duodenal intubation.
A further potential complication is the phenomenon of entero-hepatic recirculation which may impact on the proportion of drug cleared as glucuronide conjugates . Drugs that are conjugated in the liver and secreted into bile will enter into the intestinal lumen where they may be hydrolyzed by gut microflora, e.g. β-glucuronidase, before de-conjugated drug is reabsorbed into the bloodstream and recycled to the liver. Entero-hepatic recirculation is eventually terminated by the formation of oxidative metabolites or by elimination of parent or metabolite in the faeces or in urine, via the systemic circulation. Taking a bile sample at any point during this process may partially interrupt recycling and the biliary composition may not accurately represent the contribution of a particular human metabolic pathway in the absence of sampling. This could be because less drug-related material may be available for re-conjugation and subsequent biliary secretion or, more significantly, the contribution of oxidative metabolism may be less due to limited recycling through the liver. That said, this situation is unlikely to present when sampling using the Entero-Test® given the very low volumes of bile that are sampled (≤1 ml).
For GSK1325756 the impact of entero-hepatic recirculation in human subjects is thought to be minimal due to the lack of notable and consistent secondary input peaks in the plasma concentration–time profiles, which are often associated with this phenomenon, and the relatively short apparent half-life of 7 h. Therefore the biliary profiles obtained in this study (at 7 h post dosing) are likely to be fairly reflective of the overall contribution of glucuronidation (estimated 80%) to the clearance of GSK1325756 in man and only major changes in this would alter the assessment of drug interaction risk. It can be estimated that the observed contribution of oxidative metabolism would need to increase by >3-fold before any notable impact on GSK1325756 exposure is observed following co-administration with a CYP inhibitor .
This study has demonstrated that for drugs with high biliary secretion, the Entero-Test® technique can be used to obtain bile samples for the evaluation of metabolic routes of clearance. For GSK1325756, which is predominately cleared by glucuronidation, this information has reduced the concern of victim drug interactions with CYP3A4 inhibitors. These data also prompt further mechanistic investigations of GSK1325756 to understand the UGT enzymes involved in glucuronidation and the drug transporters involved in elimination of drug and metabolites. Although further clinical investigations may be a regulatory expectation for drug registration, this study has provided confidence to co-administer GSK1325756 with CYP inhibitors thereby enabling the progression of clinical trials in a COPD patient population.