The aim of the present study was to investigate the gastroretentive capacity of different formulation principles. This was indirectly determined by the absorption behavior of caffeine from the dosage forms. A slow and continuous appearance of caffeine in the saliva of healthy volunteers was used as a parameter for a prolonged gastric retention time. For this purpose, a four-way study was conducted with twelve healthy volunteers using the following test procedures: (1) Effervescent granules with 240 mL of still water administered in fed state, (2) effervescent granules with 20 mL of still water in fed state, (3) extended release (ER) tablet with 240 mL of still water in fed state, and (4) effervescent granules with 240 mL of still water in fasted state. The initial rise of the caffeine concentrations was more pronounced after the intake of the effervescent granules in the fed state compared to that of the ER tablets. However, tmax tended to be shorter in the fed study arms following administration of the ER tablet compared to the granules. Overall, the application of active pharmaceutical ingredients formulated as effervescent granules seems to be a promising approach to increase their gastric residence time after intake in fed state.

Gastrointestinal fluid volumes are a crucial parameter for dissolution and absorption of orally taken medications. Most often 240 mL are used in clinical standard setups. Nonetheless, surveys in patient populations revealed dramatically lower volumes for intake of oral medications in real life and even in some clinical studies reduced fluid volumes are common. These reductions might have serious impact on pharmacokinetics. Thus, it was the aim of this study to compare the gastric emptying of 240 mL and 20 mL of water in 8 healthy volunteers. For investigation of gastric fluid volumes Magnetic Resonance Imaging with strongly T2 weighted sequences was used. Gastric emptying was additionally quantified via caffeine pharmacokinetics measured in saliva. The absolute gastric volumes after intake of 240 mL or 20 mL obviously differed by factor 10 but relative gastric emptying expressed as fraction per time was nearly comparable. Only slighter slower emptying after intake of 20 mL was observed. Salivary caffeine pharmacokinetics representing mass transfer from stomach to small intestine after intake of different volumes did not differ. The absorbed caffeine fraction and emptied gastric volume fraction correlated well after intake of 240 mL, but not after intake of 20 mL, indicating a higher influence of secretion on gastric volume measurements after intake of smaller volumes. Relative gastric emptying as measured with MRI and salivary caffeine method was only slightly delayed, thus transfer of orally administered drug fraction could be comparable even with lower fluid intake as can be seen by comparable caffeine pharmacokinetics. Nonetheless, the considerably reduced volumes might interfere with dissolution and absorption.

Gastric water emptying as a critical parameter for oral drug absorption can be investigated by several imaging techniques or by the interpretation of pharmacokinetics of appropriate substances. Recently introduced salivary caffeine kinetics is a valuable tool, but the required caffeine abstinence limits its applicability. To avoid the caffeine abstinence, stable isotope-labeled caffeine might be used, but the representability and transferability of kinetics for evaluation of gastric emptying must be demonstrated. Thus, salivary caffeine pharmacokinetics were compared for naturally occurring 12C-caffeine and 13C3-caffeine after the administration of water under fasting conditions in six healthy young subjects. For this purpose, an ice capsule containing the two caffeine species was administered with 50 mL tap water. Gastric water emptying was simultaneously quantified using magnetic resonance imaging (MRI). Gastric emptying of 50 mL of water could be successfully evaluated. The salivary caffeine kinetics of 13C3- and 12C-caffeine were nearly congruent and showed good linear correlations in all subjects, with a mean correlation coefficient of 0.96 in pooled data. Thus, the substitution of natural 12C caffeine with stable isotope-labeled 13C3-caffeine offers the opportunity for broader application of the salivary caffeine gastric emptying technique and increases the robustness of the method against environmental contamination with caffeine.

Many orally dosed APIs are bioavailable only when formulated as an enteric dosage form to protect them from the harsh environment of the stomach. However, an enteric formulation is often accompanied with a higher development effort in the first place and the potential degradation of fragile APIs during the coating process. Ready-to-use enteric hard capsules would be an easily available alternative to test and develop APIs in enteric formulations, while decreasing the time and cost of process development. In this regard, Lonza Capsugel® Next Generation Enteric capsules offer a promising approach as functional capsules. The in vivo performance of these capsules was observed with two independent techniques (MRI and caffeine in saliva) in eight human volunteers. No disintegration or content release in the stomach was observed, even after highly variable individual gastric residence times (range 7.5 to 82.5 min), indicating the reliable enteric properties of these capsules. Seven capsules disintegrated in the distal part of the small intestine; one capsule showed an uncommonly fast intestinal transit (15 min) and disintegrated in the colon. The results for this latter capsule by MRI and caffeine appearance differed dramatically, whereas for all other capsules disintegrating in the small intestine, the results were very comparable, which highlights the necessity for reliable and complementary measurement methods. No correlation could be found between the gastric residence time and disintegration after gastric emptying, which confirms the robust enteric formulation of those capsules.

Controlling the time point and site of the release of active ingredients within the gastrointestinal tract after administration of oral delivery systems is still a challenge. In this study, the effect of the combination of small capsules (size 3) and large capsules (size 00) on the disintegration site and time was investigated using magnetic resonance imaging (MRI) in combination with a salivary tracer technique. As capsule shells, Vcaps® HPMC capsules, Vcaps® Plus HPMC capsules, gelatin and DRcaps® designed release capsules were used. The three HPMC-based capsules (Vcaps®, Vcaps® Plus and DRcaps® capsules) were tested as single capsules; furthermore, seven DUOCAP® capsule-in-capsule combinations were tested in a 10-way crossover open-label study in six healthy volunteers. The capsules contained iron oxide and hibiscus tea powder as tracers for visualization in MRI, and two different caffeine species (natural caffeine and 13C3) to follow caffeine release and absorption as measured by salivary levels. Results showed that the timing and location of disintegration in the gastrointestinal tract can be measured and differed when using different combinations of capsule shells. Increased variability among the six subjects was observed in most of the capsule combinations. The lowest variability in gastrointestinal localization of disintegration was observed for the DUOCAP® capsule-in-capsule configuration using a DRcaps® designed release capsule within a DRcaps® designed release outer capsule. In this combination, the inner DRcaps® designed release capsule always opened reliably after reaching the ileum. Thus, this combination enables targeted delivery to the distal small intestine. Among the single capsules tested, Vcaps® Plus HPMC capsules showed the fastest and most consistent disintegration.

The aim of the study was to determine the abomasal emptying rate (AER) of calves suffering from naturally occurring diarrhoea compared with that of healthy calves. Furthermore, the effects of an oral rehydration solution (ORS) mixed into milk replacer on the AER were determined. Acetaminophen absorption test (APAT) was performed to estimate the AER. Sixty Holstein-Frisian calves (age < 14 days) were included in the study and divided into groups as follows: healthy calves (H; n = 16), healthy calves fed with ORS (HORS; n = 14), diarrhoeic calves (D; n = 15) and diarrhoeic calves fed with ORS (DORS; n = 15). For the APAT, the calves were fed 2 L of milk replacer containing 50 mg acetaminophen (AP)/kg body weight. Venous blood samples were collected before and after milk replacer and AP intake in 30-60 min intervals for 12 hr. During the APAT, no significant differences in median maximum acetaminophen concentration (Cmax ) were observed among all groups. Time to reach maximum acetaminophen concentration (Tmax ) in DORS (median 390 min, 25/75 quartiles: 300/480 min) was significantly higher compared with that in H (median: 270 min 25/75 quartiles: 210/315 min) and HORS (median: 300 min (25/75 quartiles: 240/360 min). Non-linear regression revealed that the calculated abomasal half-life (AP t1/2 ) tended to be delayed in DORS (median: 652 min, 25/75 quartiles: 445/795 min, p = .10). The area under the AP curve values (AUC) from 0 to 120 min and 0 to 240 min of the observation period were significantly higher in H than D and DORS. In conclusion, significant differences in the AER indices reflected delayed abomasal emptying in diarrhoeic calves. Furthermore, the hypertonic ORS tended to have an additive delaying impact on the AER, which needs attention for the feeding management of diarrhoeic calves.

The quaternary ammonium compound trospium chloride is poorly absorbed from 2 “absorption windows” in the jejunum and cecum/ascending colon, respectively. To confirm whether intestinal P‐glycoprotein (P‐gp) is involved, a 4‐period, crossover drug interaction study with trospium chloride after intravenous (2 mg) and oral administration (30 mg) without and after comedication of clarithromycin (500 mg), an inhibitor for P‐gp, was initiated in 12 healthy subjects. Pharmacokinetics of trospium was evaluated using gas chromatography–mass spectrometry, noncompartmental evaluation, and pharmacokinetic modeling. Trospium chloride was poorly absorbed after oral administration (absolute bioavailability, ∼8%–10%). About 30% of the bioavailable dose fraction was absorbed from the “narrow window”. Comedication with clarithromycin increased steady‐state distribution volumes by ∼27% (P < .01). Bioavailability was not increased as hypothesized. The geometric mean ratios (90% confidence interval) for area under the plasma concentration–time curve, maximum concentration, and renal clearance accounted for 0.75 (0.56–1.01), 0.64 (0.45–0.89), and 1.00 (0.90–1.13), respectively. The amount of trospium absorbed from the “narrow window” was reduced in all subjects but from the “wider window” in only 9 of them. Bioavailability was strongly predicted by the maximum absorption rate of trospium in the distal “window” (rs2 = 0.910, P < .0001). In conclusion, the P‐gp inhibitor clarithromycin significantly increases distribution volumes but not oral absorption of trospium. The amount absorbed from the “narrow window” was lowered in all subjects. However, the extent of all influences seems not to be of clinical relevance.

Trospium chloride, a muscarinic receptor blocker, is poorly absorbed with different rates from areas in the jejunum and the cecum/ascending colon. To evaluate whether organic cation transporter (OCT) 1, OCT2 and multidrug and toxin extrusion (MATE) 1 and MATE2‐K are involved in pharmacokinetics, competitions with ranitidine, a probe inhibitor of the cation transporters, were evaluated in transfected HEK293 cells. Furthermore, a drug interaction study with trospium chloride after intravenous (2 mg) and oral dosing (30 mg) plus ranitidine (300 mg) was performed in 12 healthy subjects and evaluated by noncompartmental analysis and population pharmacokinetic modeling. Ranitidine inhibited OCT1, OCT2, MATE1, and MATE2‐K with half maximal inhibitory concentration values of 186 ± 25 µM, 482 ± 105 µM, 134 ± 37 µM, and 35 ± 11 µM, respectively. In contrast to our hypothesis, coadministration of ranitidine did not significantly decrease oral absorption of trospium. Instead, renal clearance was lowered by ∼15% (530 ± 99 vs 460 ± 120 mL/min; P < .05). It is possible that ranitidine was not available in competitive concentrations at the major colonic absorption site, as the inhibitor is absorbed in the small intestine and undergoes degradation by microbiota. The renal effects apparently result from inhibition of MATE1 and/or MATE2‐K by ranitidine as predicted by in vitro to in vivo extrapolation. However, all pharmacokinetic changes were not of clinical relevance for the drug with highly variable pharmacokinetics. Intravenous trospium significantly lowered mean absorption time and relative bioavailability of ranitidine, which was most likely caused by muscarinic receptor blocking effects on intestinal motility and water turnover.

Clartihromycin and ranitidine are accepted probe inhibitors in drug/drug interaction studies (DDI) with substrates of the multidrug transporters P-glycoprotein and OCT1. We assayed both drugs in human plasma, urine, and feces using LC–MS/MS with positive mass transition mode (AB Sciex API 2000 with turbo-ion spray) with fexofenadine as an internal standard. After protein denaturation with acetonitrile/water (50:50, v/v), the samples were centrifuged and the supernatants (10 µL) were injected into the chromatographic system (column: Supelco Ascentis® C18, 3 µm, 2.1 × 100 mm). The chromatography was performed with isocratic elution plied using ammonium formate buffer [(5 mM; pH 3.0)/acetonitrile, 40:60, v/v] as mobile phase at a flow rate of 200 μL min−1. The chromatograms were evaluated online with the internal standard method using peak-area-ratios for linear regression analysis weighted by 1/x (x = concentration) for the validation ranges in plasma between 0.005 and 2.0 µg mL−1, and for urine and feces between 0.005 and 10.0 µg mL−1. The method was shown to possess sufficient specificity, accuracy, precision and stability without matrix effects, thereby fulfilling current bioanalytical guidelines. The assay was suitable to simultaneous quantitative analysis of clarithromycin and ranitidine in plasma, urine, and feces of a DDI with trospium chloride to exclude major influence of trospium on the pharmacokinetics of the probe inhibitors. The assay is superior to other methods as it enables for the first time, quantification of the drugs in feces.

BACKGROUND Standard treatment of foals with severe abscessing lung infection caused by Rhodococcus equi using rifampicin and a macrolide antibiotic can be compromised by extensive inhibition and/or induction of drug metabolising enzymes (e.g. CYP3A4) and transport proteins (e.g. P-glycoprotein), as has been shown for rifampicin and clarithromycin. The combination of rifampicin with the new, poorly metabolised gamithromycin, a long-acting analogue of azithromycin and tulathromycin with lower pharmacokinetic interaction potential, might be a suitable alternative. OBJECTIVES To evaluate the pharmacokinetic interactions and pulmonary distribution of rifampicin and gamithromycin in healthy foals, and to investigate the cellular uptake of gamithromycin in vitro. STUDY DESIGN Controlled, four-period, consecutive, single-dose and multiple-dose study. METHODS Pharmacokinetics and lung distribution of rifampicin (10 mg/kg) and gamithromycin (6 mg/kg) were measured in nine healthy foals using LC-MS/MS. Enzyme induction was confirmed using the 4β-OH-cholesterol/cholesterol ratio. Affinity of gamithromycin to drug transport proteins was evaluated in vitro using equine hepatocytes and MDCKII-cells stably transfected with human OATP1B1, OATP1B3 and OATP2B1. RESULTS Rifampicin significantly (P<0.05) increased the plasma exposure of gamithromycin (16.2 ± 4.77 vs. 8.57 ± 3.10 μg × h/mL) by decreasing the total body clearance. Otherwise, gamithromycin significantly lowered plasma exposure of single- and multiple-dose rifampicin (83.8 ± 35.3 and 112 ± 43.1 vs. 164 ± 96.7 μg × h/mL) without a change in metabolic ratio and half-life. Gamithromycin was identified as an inhibitor of human OATP1B1, OATP1B3 and OATP2B1 and as a substrate of OATP2B1. In addition, it was extracted by equine hepatocytes via a mechanism which could be inhibited by rifampicin. MAIN LIMITATIONS Influence of gamithromycin on pulmonary distribution of rifampicin was not evaluated. CONCLUSION The plasma exposure of gamithromycin is significantly increased by co-administration of rifampicin which is most likely caused by inhibition of hepatic elimination.

An enhanced indoleamine 2,3-dioxygenase 1 (IDO1) activity is associated with an increased mortality risk in sepsis patients. Thus, the preventive inhibition of IDO1 activity may be a promising strategy to attenuate the severity of septic shock. 1-methyltryptophan (1-MT) is currently in the interest of research due to its potential inhibitory effects on IDO1 and immunomodulatory properties. The present study aims to investigate the protective and immunomodulatory effects of 1-methyltryptophan against endotoxin-induced shock in a porcine in vivo model. Effects of 1-MT were determined on lipopolysaccharide (LPS)-induced tryptophan (TRP) degradation, immune response and sickness behaviour. 1-MT increased TRP and its metabolite kynurenic acid (KYNA) in plasma and tissues, suppressed the LPS-induced maturation of neutrophils and increased inactivity of the animals. 1-MT did not inhibit the LPS-induced degradation of TRP to kynurenine (KYN)—a marker for IDO1 activity—although the increase in KYNA indicates that degradation to one branch of the KYN pathway is facilitated. In conclusion, our findings provide no evidence for IDO1 inhibition but reveal the side effects of 1-MT that may result from the proven interference of KYNA and 1-MT with aryl hydrocarbon receptor signalling. These effects should be considered for therapeutic applications of 1-MT.

This study investigated abomasal luminal parameters in healthy and diarrheic calves by using a wireless ambulatory capsule (WAC). The acetaminophen absorption test (APAT) was used to determine abomasal emptying rate. Four healthy and five diarrheic female Holstein-Friesian calves (age < 14 days) were included in the study. For APAT, calves were fed 2L of milk replacer containing 50mg acetaminophen/kg body weight, and blood samples were taken during a 12-h period afterward. Concomitantly, a WAC in the abomasum continuously measured luminal pH, pressure, and temperature. Five hours post suckling, intraluminal temperature was significantly higher in diarrheic calves than in healthy calves. Abomasal pH and pressure were not significantly different, but intraluminal pressure was always numerically lower in diarrheic calves. During APAT no significant differences in maximum acetaminophen concentrations (Cmax) and time to reach maximum acetaminophen concentration (Tmax) were observed. Nonlinear regression findings revealed a longer acetaminophen half-time (AAP t1/2) in diarrheic calves compared to healthy calves [564 ± 96min vs. 393 ± 84min, respectively; P = 0.04] and lower area under the concentration curve values (e.g., 60min postprandial AUC60 681 ± 244 (µg∙min)/mL vs. 1064 ± 23 (µg∙min)/mL, respectively; P = 0.04). In conclusion, abomasal luminal conditions were different between diarrheic and healthy calves. Significant differences in APAT reflected a delay in abomasal emptying in diarrheic calves. Impaired abomasal movement may induce enhanced bacterial fermentation processes as indicated by a higher abomasal temperature in diarrheic calves, which should be considered in management of their feeding.

Intramuscular injection of diclofenac is still frequently practiced, although there is ample evidence that the risk of local tissue intolerability is highly underestimated. The aim of this study was to evaluate local toxicity in a patient using magnetic resonance imaging. A patient who gave written informed consent received a medically indicated intramuscular administration of diclofenac 75 mg/2 mL. Simultaneously with magnetic resonance imaging of the depot, a clinical–chemical evaluation and quantification of diclofenac in plasma was performed. A manifold enhancement of the T2-weighted magnetic resonance signal was observed in a muscle area of approximately 60 mL volume, with maximum signal intensity 30 min after injection, the time of maximum diclofenac plasma exposure. Plasma creatine kinase activity was elevated approximately sixfold within 8 h and normalized within 1 week, whereas the magnetic resonance enhancement disappeared within 5 weeks. Interestingly, the patient did not complain about any clinical symptoms at the injection site. Asymptomatic tissue injury after intramuscular injection of diclofenac, caused by intramuscular dosing, can be reliably evaluated by magnetic resonance imaging and should be applied early during the development of parenteral dosage forms. Clinical Trials Registration Number : BB130/16 (Ethics Committee of the University Medicine Greifswald).

The long-acting azalide antibiotic gamithromycin is marketed for intramuscular treatment of bovine and swine infections. Off-label use in foals leads to severe local lesions likely caused by hyperosmolality of the injected solution. We provide evidence from a pharmacokinetic study in 10 warm-blooded healthy foals for intravenous bolus injection of gamithromycin diluted in distilled water to be a safe and well tolerated alternative. By intravenous dosing, markedly higher plasma exposure and better penetration into bronchoalveolar lavage cells but lower distribution into epithelial lining fluid are achieved as after intramuscular or subcutaneous administration. Intravenously injected gamithromycin was tolerated without any adverse drug reactions. The protocols for treatment of equine pulmonary infections caused by Rhodococcus equi should be revised accordingly.

Increased activity of the tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO) is associated with immunological and neurological disorders, and inhibition of its enzyme activity could be a therapeutic approach for treatment of these disorders. The aim of the present study was to establish a large animal model to study the accumulation of the potential IDO inhibitor 1-methyltryptophan (1-MT) in blood and different organs of domestic pigs (Sus scrofa domestica). Because 1-MT has not been previously evaluated in pigs, the pharmacokinetics of a single subcutaneous 1-MT application was investigated. Based on this kinetic study, a profile for repeated 1-MT applications over a period of five days was simulated and tested. The results show that a single administration of 1-MT increases its concentrations in blood, with the maximum concentration being obtained at 12 h. Repeated daily injections of 1‑MT generated increasing plasma concentrations followed by a steady-state after two days. Twelve hours after the final application, accumulation of 1-MT was observed in the brain and other organs, with a substantial variability among various tissues. The concentrations of 1-MT measured in plasma and tissues were similar to, or even higher, than those of tryptophan. Our data indicate that repeated subcutaneous injections of 1-MT provide a suitable model for accumulation of 1-MT in plasma and tissues of domestic pigs. These findings provide a basis for further research on the immunoregulatory functions of IDO in a large animal model.

The present pilot study introduces a method that might give novel insights in drug absorption processes from intramuscularly administered depots. An oily suspension or an aqueous solution of paracetamol (6 mg/kg body mass), prednisolone or its hemisuccinate sodium salt for the aqueous solutions (10mg/kg body mass) or diclofenac (10mg/kg body mass) was injected into the muscle tissue of the hind leg of female Lewis-rats (n=47). For the oily suspensions the micronized particles were suspended in medium-chain triglycerides. The aqueous solutions were buffered to a pH of 7.4 ± 0.5. Polyethylene glycol was added as a cosolvent in the formulations containing paracetamol (acetaminophen) and diclofenac and sodium chloride was added to the aqueous solutions of prednisolone hemisuccinate sodium to achieve nearly isotonic formulations. The formed depot was visualized by magnetic resonance imaging (MRI) and characterized with regard to volume and surface area. A 7 T-small animal scanner was used and T1-weighted and T2-weighted sequences including a fat saturation were performed. Simultaneously blood samples were taken and the drugs were quantitatively analyzed. The water based solvent and the oily dispersion agent were visible in the MRI images without the use of contrast agents. Since a free hand injection mostly led to an application directly into the fascia, resulting in a fast removal of the depot, MRI-guided injection was conducted. Comparing pharmacokinetic data with MRI data it was observed that maximal blood levels occurred before the solvent and the dispersion agent were removed from the muscle tissue. Thus, the drug is not absorbed together with the depot. Furthermore, no correlation was found between the shape of the depot and the rate of absorption. Consequently, a higher surface area or volume of the depot did not result in a faster release or absorption of the drugs from the tested formulations. In contrast to the paracetamol and prednisolone formulations the formulations containing diclofenac led to a massive accumulation of interstitial fluid around the injection area being a sign for an acute local reaction. Histological analysis of the muscle tissue revealed a clear correspondence between the amount of interstitial fluid and the extent of infiltrating lymphocytes and granulocytes indicating a tissue response. In conclusion combining MRI with pharmacokinetic data is a suitable method to gain deeper insights into drug absorption processes from intramuscular depots. Furthermore, MRI offers a great possibility detecting local side effects caused by an intramuscularly applied dosage form. This might be very useful in preclinical phases during the development of new intramuscular formulations.

The clinical efficiency of the highly potent antitumor agent doxorubicin is limited by cardiotoxic effects. In a murine doxorubicin cardiotoxicity model, increased endothelin-1 (ET-1) expression and cardioprotective effects of the dual ET-1 blocker bosentan were demonstrated. To date it is unclear if combined blocking of endothelin A/B receptors is necessary or whether selective inhibition of one of the ET-1 receptors is sufficient for the observed cardioprotection. Therefore, we investigated the impact of dual (bosentan) and single endothelin receptor antagonism through sitaxentan (receptor A blocker) or BQ788 (receptor B blocker) in a murine doxorubicin cardiotoxicity model (C57BL/6N). Simultaneous administration of each endothelin receptor antagonist (ERA) with doxorubicin resulted in a significantly improved hemodynamic performance in comparison to the impaired cardiac function in control mice with bosentan being most effective but closely followed by sitaxentan and also BQ788. This cardioprotection was not caused by diminished doxorubicin levels in heart since the doxorubicin content in cardiac tissue was not altered by ERAs significantly. However, whole transcript expression profiling showed partly different effects of the ERAs on doxorubicin-modulated cardiac gene expression of genes involved in signal transduction (e.g. Stat3, Pim1, Akt1, Plcb2), fibrosis (e.g. Myl4), energy production (e.g. Ant1) or oxidative stress (e.g. Aox1). Furthermore, doxorubicin-mediated gene regulations were verified in the murine cardiomyocyte model HL-1 showing partly reversed expression patterns after co-administration of the ERAs. In summary, our results demonstrate strong cardioprotective effects of blocking ET-1 receptors against the doxorubicin-related cardiomyopathy and provide evidence to potential underlying signaling pathways.

The non-opiate analgesic drug flupirtine was shown in vitro to undergo hydrolysis followed by N-acetylation to form D13223, glucuronidation and conjugation with glutathione to form the stable mercapturic acid derivatives M-424 and M-466. To quantify flupirtine and its metabolites in samples obtained in a clinical study in healthy subjects selected on their genotype of NAT2, UGT1A1 and GSTP1, two LC-MS/MS methods were developed. The validation range for flupirtine and D-13223 in serum was 0.5-500 ng/ml. For urine and feces, the validation ranges for flupirtine and D-13223 were 20-5000 ng/ml and 5.0-5000 ng/ml, respectively. M-424 and M-466 could be quantified in urine between 5.0 and 5000 ng/ml. Free flupirtine and D-13223 were separated from serum, urine and feces with liquid-liquid extraction. For flupirtine and D-13223, the chromatography was performed on a XTerra C18 column isocratically with a mobile phase consisting of ammonium formate buffer (pH 3.5mM) and acetonitrile (50:50; v/v), for M-466 and M-424 a Synergi(®) Fusion-RP column was used and a linear gradient method with water/HCOOH (pH 3) and acetonitrile. The mass spectrometer operated both with electro spray ionization in positive multiple reaction monitoring mode. The developed methods fulfilled the current FDA criteria on bioanalytical method validation for accuracy (error: -16.9 to 11.2%), precision (1.2-13.4%), recovery, stability and matrix effects over the observed analytical range. Thus, the methods were suitable to quantify flupirtine absorption and metabolic disposition in man after single intravenous and oral dosing (100mg) and repeated oral administration (400mg once daily).

AIMS:The rare association of flupirtine with liver injury is most likely caused by reactive quinone diimines and their oxidative formation may be influenced by the activities of N-acetyltransferases (NAT) that conjugate the less toxic metabolite D13223, and by glucuronosyltransferases (UGT) and glutathione S-transferases (GST) that generate stable terminal glucuronides and mercapturic acid derivatives, respectively. The influence of genetic polymorphisms of NAT2, UGT1A1 and GSTP1 on generation of the terminal mercapturic acid derivatives and analgesic effects was evaluated to identify potential genetic risk factors for hepatotoxicity of flupirtine. METHODS:Metabolic disposition of flupirtine was measured after intravenous administration (100 mg), after swallowing an immediate-release (IR) tablet (100 mg) and after repeated administration of modified release (MR) tablets (400 mg once daily 8 days) in 36 selected healthy subjects. Analgesic effects were measured using pain models (delayed onset of muscle soreness, electric pain). RESULTS:Flupirtine IR was rapidly but incompletely absorbed (∼ 72%). Repeated administration of flupirtine MR showed lower bioavailability (∼ 60%). Approximately 12% of bioavailable flupirtine IR and 8% of bioavailable flupiritine MR was eliminated as mercapturic acid derivatives into the urine independent of the UGT1A1, NAT2 and GSTP1 genotype. Carriers of variant GSTP1 alleles showed lower bioavailability but increased intestinal secretion of flupirtine and increased efficiency in experimental pain. Flupirtine was not a substrate for ABCB1 and ABCC2. CONCLUSIONS:Formation of mercapturic acid derivatives is a major elimination route for flupirtine in man. However, the theoretically toxic pathway is not influenced by the frequent polymorphisms of UGT1A1, NAT2 and GSTP1.