Genetic Algebras Associated with ξ(a)-Quadratic Stochastic Operators.

The present paper deals with a class of ξ(a)-quadratic stochastic operators, referred to as QSOs, on a two-dimensional simplex. It investigates the algebraic properties of the genetic algebras associated with ξ(a)-QSOs. Namely, the associativity, characters and derivations of genetic algebras are studied. Moreover, the dynamics of these operators are also explored. Specifically, we focus on a particular partition that results in nine classes, which are further reduced to three nonconjugate classes. Each class gives rise to a genetic algebra denoted as Ai, and it is shown that these algebras are isomorphic. The investigation then delves into analyzing various algebraic properties within these genetic algebras, such as associativity, characters, and derivations. The conditions for associativity and character behavior are provided. Furthermore, a comprehensive analysis of the dynamic behavior of these operators is conducted.

Specific inhibition of the transporter MRP4/ABCC4 affects multiple signaling pathways and thrombus formation in human platelets.

The multidrug resistance protein 4 (MRP4) is highly expressed in platelets and several lines of evidence point to an impact on platelet function. MRP4 represents a transporter for cyclic nucleotides as well as for certain lipid mediators. The aim of the present study was to comprehensively characterize the effect of a short-time specific pharmacological inhibition of MRP4 on signaling pathways in platelets. Transport assays in isolated membrane vesicles showed a concentrationdependent inhibition of MRP4-mediated transport of cyclic nucleotides, thromboxane (Tx)B2 and fluorescein (FITC)- labeled sphingosine-1-phosphate (S1P) by the selective MRP4 inhibitor Ceefourin-1. In ex vivo aggregometry studies in human platelets, Ceefourin-1 significantly inhibited platelet aggregation by about 30-50% when ADP or collagen was used as activating agents, respectively. Ceefourin-1 significantly lowered the ADP-induced activation of integrin aIIbb3, indicated by binding of FITC-fibrinogen (about 50% reduction at 50 mM Ceefourin-1), and reduced calcium influx. Furthermore, pre-incubation with Ceefourin-1 significantly increased PGE1- and cinaciguat-induced vasodilatorstimulated phosphoprotein (VASP) phosphorylation, indicating increased cytosolic cAMP as well as cGMP concentrations, respectively. The release of TxB2 from activated human platelets was also attenuated. Finally, selective MRP4 inhibition significantly reduced both the total area covered by thrombi and the average thrombus size by about 40% in a flow chamber model. In conclusion, selective MRP4 inhibition causes reduced platelet adhesion and thrombus formation under flow conditions. This finding is mechanistically supported by inhibition of integrin aIIbb3 activation, elevated VASP phosphorylation and reduced calcium influx, based on inhibited cyclic nucleotide and thromboxane transport as well as possible further mechanisms.

Chiral Pharmacokinetics and Metabolite Profile of Prolonged-release Ketamine Tablets in Healthy Human Subjects.

BACKGROUND: The anesthetic ketamine after intravenous dosing is nearly completely metabolized to R- and S-stereoisomers of the active norketamine (analgesic, psychoactive) and 2,6-hydroxynorketamine (potential analgesic, antidepressant) as well as the inactive dehydronorketamine. Oral administration favors the formation of 2,6-hydroxynorketamines via extensive presystemic metabolism. The authors hypothesized that plasma exposure to 2,6-hydroxynorketamines relative to the psychoactive ketamine is greater after prolonged-release ketamine tablets than it is after intravenous ketamine. METHODS: Pharmacokinetics of ketamine after intravenous infusion (5.0 mg) and single-dose administrations of 10, 20, 40, and 80 mg prolonged-released tablets were evaluated in 15 healthy white human subjects by means of a controlled, ascending-dose study. The stereoisomers of ketamine and metabolites were quantified in serum and urine by validated tandem mass-spectrometric assays and evaluated by noncompartmental pharmacokinetic analysis. RESULTS: After 40 mg prolonged-release tablets, the mean ± SD area under the concentrations-time curve ratios for 2,6-hydroxynorketamine/ketamine were 18 ± 11 (S-stereoisomers) and 30 ± 16 (R-stereoisomers) compared to 1.7 ± 0.8 and 3.1 ± 1.4 and after intravenous infusion (both P < 0.001). After 10 and 20 mg tablets, the R-ratios were even greater. The distribution volumes at steady state of S- and R-ketamine were 6.6 ± 2.2 and 5.6 ± 2.1 l/kg, terminal half-lives 5.2 ± 3.4 and 6.1 ± 3.1 h, and metabolic clearances 1,620 ± 380 and 1,530 ± 380 ml/min, respectively. Bioavailability of the 40 mg tablets was 15 ± 8 (S-isomer) and 19 ± 10% (R-isomer) and terminal half-life 11 ± 4 and 10 ± 4 h. About 7% of the dose was renally excreted as S-stereoisomers and 17% as R-stereoisomers. CONCLUSIONS: Prolonged-release ketamine tablets generate a high systemic exposure to 2,6-hydroxynorketamines and might therefore be an efficient and safer pharmaceutical dosage form for treatment of patients with chronic neuropathic pain compared to intravenous infusion.

Growth-Inhibitory Effect of Chitosan-Coated Liposomes Encapsulating Curcumin on MCF-7 Breast Cancer Cells.

Current anticancer drugs exhibit limited efficacy and initiate severe side effects. As such, identifying bioactive anticancer agents that can surpass these limitations is a necessity. One such agent, curcumin, is a polyphenolic compound derived from turmeric, and has been widely investigated for its potential anti-inflammatory and anticancer effects over the last 40 years. However, the poor bioavailability of curcumin, caused by its low absorption, limits its clinical use. In order to solve this issue, in this study, curcumin was encapsulated in chitosan-coated nanoliposomes derived from three natural lecithin sources. Liposomal formulations were all in the nanometric scale (around 120 nm) and negatively charged (around -40 mV). Among the three lecithins, salmon lecithin presented the highest growth-inhibitory effect on MCF-7 cells (two times lower growth than the control group for 12 µM of curcumin and four times lower for 20 µM of curcumin). The soya and rapeseed lecithins showed a similar growth-inhibitory effect on the tumor cells. Moreover, coating nanoliposomes with chitosan enabled a higher loading efficiency of curcumin (88% for coated liposomes compared to 65% for the non-coated liposomes) and a stronger growth-inhibitory effect on MCF-7 breast cancer cells.

Combined Application of MRI and the Salivary Tracer Technique to Determine the in Vivo Disintegration Time of Immediate Release Formulation Administered to Healthy, Fasted Subjects.

The process of disintegration is a crucial step in oral drug delivery with immediate release dosage forms. In this work, the salivary tracer technique was applied as a simple and inexpensive method for the investigation of the in vivo disintegration time of hard gelatin capsules filled with caffeine. The disintegration times observed with the salivary tracer technique were verified by magnetic resonance imaging (MRI). After an overnight fast of at least 10 h and caffeine abstinence of minimum 72 h, conventional hard gelatin capsules containing 50 mg caffeine and 5 mg iron oxide were administered to 8 healthy volunteers. For the period of 1 h after capsule intake, subjects were placed in supine position in the MRI scanner, and scans were performed in short time intervals. Each MRI measurement was directly followed by saliva sampling by drooling. Salivary caffeine concentrations were determined by high performance liquid chromatography followed by mass spectrometric detection (LC/MS-MS). The time point of capsule disintegration was determined by visual inspection of the MR images as well as by an increase in the salivary caffeine concentration. The results indicated that the difference in mean disintegration times of the capsules as determined by the two in vivo methods was around 4 min (8.8 min for MRI vs 12.5 min for saliva). All disintegration times determined by the salivary tracer technique were slightly higher. This delay could be explained by the fact that the appearance of caffeine in saliva required drug absorption in the small intestine. Because capsule disintegration happened mainly in the stomach, the exact site of disintegration as well as the processes of gastric mixing and gastric emptying contributed to the delay between the two methods. This work demonstrated the feasibility of the salivary tracer technique to investigate the in vivo disintegration of immediate release dosage forms in a simple and reliable manner.

Topical Mitomycin-C can help as an adjunct to alkaline nasal wash and rifampicin in primary atrophic rhinitis.

PURPOSE: Primary atrophic rhinitis (PAR) is a well-known old disease characterized by a roomy nose and extensive crustations. This study was designed to investigate the effect of topical Mitomycin-C as an adjunct to medical treatment with respect to objective and subjective improvement in patients treated with PAR. MATERIAL AND METHODS: This prospective randomized controlled study was conducted in a tertiary referral hospital in January 2016 and March 2018. Fifty adult patients aged 18 to 45 with PAR were randomly divided into 2 groups. STUDY GROUP: treatment with Mitomycin-C dissolved in an alkaline wash plus rifampicin and control group: only treated with rifampicin and alkaline nasal wash. Subjective scores for the following symptoms: After 12 weeks of treatment, foul smell, anosmia, crusting, epistaxis, and nasal blockade, an objective score of crusting, the status of nasal mucosa, nature of the secretions and condition of nasal cavity were compared between the two groups. RESULTS: The degree of crustations (P < 0.0001) and the severity of epistaxis (P < 0.0001) were significantly improved in patients treated with Mitomycin-C dissolved in an alkaline wash (i.e. the study group), and the secretions returned significantly to normal (P < 0.0001). Both groups had significant improvements in both subjective and objective parameters of the assessment. CONCLUSIONS: In patients with primary atrophic rhinitis, the use of Mitomycin-C dissolved in an alkaline nasal wash as an adjunct to oral rifampicin can produce a beneficial result than rifampicin and alkaline nasal wash alone.

Subcritical Fluid Chromatography at Sub-Ambient Temperatures for the Chiral Resolution of Ketamine Metabolites with Rapid-Onset Antidepressant Effects.

Chiral metabolites of ketamine exerting rapid-onset yet sustained antidepressant effects may be marketed directly in the future, but require chemo- and enantio-selective chromatographic methods for quality assurance and control. The chromatographic behavior of S-/R-ketamine, S-/R-norketamine, S-/R-dehydronorketamine, and (2R,6R)-/(2S,6S)-hydroxynorketamine in supercritical fluid chromatography (SFC) was investigated computationally and experimentally with the aim of identifying problematic pairs of enantiomers and parameters for chiral resolution. Retention on three different polysaccharide-based chiral stationary phases (Lux Amylose-2, i-Amylose-3, and i-Cellulose-5) provided new information on the significance of halogen atoms as halogen bond donors and hydrogen bond acceptors for enantioselectivity, which could be corroborated in silico by molecular docking studies. Modifiers inversely affected enantioselectivity and retention. Methanol yielded lower run times but superior chiral resolution compared to 2-propanol. Lower temperatures than those conventionally screened did not impair phase homogeneity but improved enantioresolution, at no cost to reproducibility. Thus, sub-ambient temperature subcritical fluid chromatography (SubFC), essentially low-temperature HPLC with subcritical CO2, was applied. The optimization of the SubFC method facilitated the chiral separation of ketamine and its metabolites, which was applied in combination with direct injection and online supercritical fluid extraction to determine the purity of pharmaceutical ketamine formulations for proof of concept.

Preparation, Characterization, and Release Kinetics of Chitosan-Coated Nanoliposomes Encapsulating Curcumin in Simulated Environments.

Curcumin, a natural polyphenol, has many biological properties, such as anti-inflammatory, antioxidant, and anti-carcinogenic properties, yet, its sensitivity to light, oxygen, and heat, and its low solubility in water renders its preservation and bioavailability challenging. To increase its bioaccessibility, we fabricated nanoliposomes and chitosan-coated nanoliposomes encapsulating curcumin, and we evaluated the systems in terms of their physicochemical characteristics and release profiles in simulated gastrointestinal mediums. Chitosan-coating enhanced the stability of nanoliposomes and slowed the release of curcumin in the simulated gastrointestinal (GI) environment. This study demonstrates that nanoliposomes and chitosan-coated nanoliposomes are promising carriers for poorly soluble lipophilic compounds with low oral bioavailability, such as curcumin.

Affinity of Ketamine to Clinically Relevant Transporters.

Ketamine is a widely used intravenous anesthetic drug that has also a pronounced analgesic effect. Moreover, one of its metabolites was very recently shown to possess antidepressant activity. Consequently, oral administration of ketamine may become of interest in the future. There is evidence from in vitro data, drug-drug interactions, and the physicochemical properties of the drug that ketamine may be a substrate of drug transporters. Thus, it was the aim of this study to investigate the affinity of ketamine to clinically relevant transporter proteins that are expected to affect its intestinal absorption, distribution, and excretion. Ketamine was shown to be significantly taken up in a time- and concentration-dependent manner by OCT1-3. The affinity to OCT transporters at pH 6.5 (Km ≈ 35-75 µM) was clearly higher than that at pH 7.4. In addition, ketamine permeability was markedly lower at pH 6.5 than at pH 7.4 in a parallel artificial membrane permeability assay (PAMPA). Ketamine showed a low but significant affinity to P-gp at pH 6.5. In contrast to this, we could not detect any transport of ketamine by MATE1/2K. In conclusion, ketamine is a substrate for OCT1-3 and P-gp but is not recognized by MATE1/2K. Considering that ketamine is a lipophilic base that mainly exists as a cationic moiety (>90%) in the intestinal lumen, we conclude that the OCT-mediated cellular uptake as well as P-gp efflux is expected to be only of relevance in the human intestine (i.e., in the case of oral drug administration), where OCT1, OCT3, and P-gp are stably expressed at the apical membrane. On the other side, P-gp is not expected to contribute significantly to tissue (brain) distribution or renal excretion of ketamine.

The Positive Role of Curcumin-Loaded Salmon Nanoliposomes on the Culture of Primary Cortical Neurons.

Curcumin (diferuloylmethane) is a natural bioactive compound with many health-promoting benefits. However, its poor water solubility and bioavailability has limited curcumin’s biomedical application. In the present study, we encapsulated curcumin into liposomes, formed from natural sources (salmon lecithin), and characterized its encapsulation efficiency and release profile. The proposed natural carriers increased the solubility and the bioavailability of curcumin. In addition, various physico-chemical properties of the developed soft nanocarriers with and without curcumin were studied. Nanoliposome-encapsulated curcumin increased the viability and network formation in the culture of primary cortical neurons and decreased the rate of apoptosis.

Pretransplant 4ß-hydroxycholesterol does not predict tacrolimus exposure or dose requirements during the first days after kidney transplantation.

AIMS: The CYP3A metric 4ß-hydroxycholesterol (4ßOHC) has been shown to correlate with tacrolimus steady-state apparent oral clearance (CL/F). Recently, pretransplant 4ßOHC was shown not to predict tacrolimus CL/F after transplantation in a cohort of renal recipients (n = 79). The goal of the current study was determine whether these findings could be validated in a substantially larger cohort. METHODS: In a retrospective analysis of 279 renal recipients, tacrolimus trough concentrations (C0), daily dose, haematocrit and other relevant covariates were registered every day for the first 14 days after transplantation. 4ßOHC and cholesterol were quantified on plasma collected immediately pretransplant using liquid chromatography tandem-mass spectrometry. Patients were genotyped for CYP3A5*1 and CYP3A4*22. RESULTS: A total of 3551 tacrolimus C0 concentrations were registered. In a linear mixed model for the 14-day period, determinants of tacrolimus C0 were CYP3A5 genotype, haematocrit, age and weight (overall R2  = 0.179). Determinants of daily dose were CYP3A5 genotype, age, methylprednisolone dose, tacrolimus formulation, ALT and estimated glomerular filtration rate (overall R2  = 0.242). Considering each of the first 5 days separately, 4ßOHC had a limited effect on tacrolimus C0 on day 3 only (-1.00 ng ml-1 per ln, P = 0.035) but not on any other day, and no effect on dose or C0/dose. During the first 5 days, haematocrit and age, which were previously established as determinants of tacrolimus disposition under steady-state conditions, never explained more than 17.7% of between-subject variability in tacrolimus C0/dose. CONCLUSIONS: The CYP3A metric 4ßOHC cannot be used to predict tacrolimus dose requirements in the first days after transplantation.

Pharmacokinetics and Pulmonary Distribution of Clarithromycin and Rifampicin after Concomitant and Consecutive Administration in Foals.

Drug interactions often result from multiple pharmacokinetic changes, such as after rifampicin (RIF) and clarithromycin (CLA) in the treatment of abscessing lung diseases. Comedication of RIF may interact with CLA disposition by either induction of presystemic elimination processes and/or inhibition of uptake mechanisms because it regulates gene transcription and modulates function of various CYP enzymes, multidrug efflux and uptake transporters for which CLA is a substrate. To distinguish the transcriptional changes from the modulating interaction components upon CLA absorption and pulmonary distribution, we initiated a repeated-dose study in 12 healthy foals with CLA (7.5 mg/kg, p.o., b.i.d.) in comedication with RIF (10 mg/kg, p.o., b.i.d.) given either concomitantly with CLA or consecutively 4 h after CLA. Affinity of CLA to human P-gp, MRP2, and MRP3 and to OCT1, OCT3, and PEPT1 was measured using Sf9-derived inside-out membrane vesicles and transfected HEK293 cells, respectively. ABCB1 (P-gp) induction by RIF and affinity of CLA to equine P-gp were studied using primary equine hepatocytes. Absolute bioavailability of CLA was reduced from ∼40% to below 5% after comedication of RIF in both schedules of administration, and Tmax occurred ∼2-3 h earlier. The loss of bioavailability was not associated with increased 14-hydroxyclarithromycin (14-OH-CLA) exposure. After consecutive dosing, absolute bioavailability and pulmonary penetration of CLA increased ∼2-fold compared to concomitant use. In vitro, CLA showed affinity to human and equine P-gp. Expression of ABCB1 mRNA was upregulated by RIF in 7 of 8 duodenal biopsy specimens and in primary equine hepatocytes. In conclusion, the major undesired influence of RIF on oral absorption and pulmonary distribution of CLA is associated with induction of intestinal P-gp. Consecutive administration to avoid competition with its intestinal uptake transport results in significantly, although not clinically relevant, improved systemic exposure.

COVID-19-induced multisystem inflammatory syndrome in a child with Wilson disease: a case report.

Background: Infection with coronavirus disease 2019 (COVID-19) can progress to the multisystem inflammatory syndrome in children (MIS-C). Patients with liver cirrhosis are at increased risk of complications. Case presentation: We report on a 13-year-old Wilson's disease patient who was referred for liver transplantation because of rapid deterioration in his hepatic condition. After admission, he developed fever, respiratory distress, coronary arteries dilatation on echocardiography, laboratory evidence of inflammation, and positive severe acute respiratory syndrome coronavirus (SARS-CoV-2) PCR. SARS-CoV-2-induced MIS-C was diagnosed. Inspite of aggressive management of MIS-C, progressive deterioration of the respiratory, liver, kidney, and cardiac functions occurred and he passed away. Conclusion: MIS-C is a serious possible complication leading to multiorgan failure and higher death rate especially in cirrhotic children. So, early diagnosis and management with higher level of care by a multidisciplinary team are warranted.

Cardiac Catheterisation Interventions in Neonates and Infants Less Than Three Months.

INTRODUCTION: Pediatric cardiac catheterization interventions become an established way of care for selected patients with congenital heart diseases. Cardiac catheterization for neonates and small infants can be challenging. The indications for diagnostic cardiac catheterization have decreased with the advent of advanced non-invasive imaging modalities. PATIENTS AND METHOD: Between June 2012 and July 2017 patients less than three months who had cardiac catheterization in two centers were reviewed. RESULTS: During the study period, 174 patients underwent interventional cardiac catheterization,83.3% of them had CHD with two-ventricle circulation and 29 patients (16.7%) had single ventricle pathophysiology. Procedures include diagnostic cath, BAS, balloon pulmonary and aortic valvuloplasty, coarctation angioplasty, and stenting procedures. The vascular access depends upon the type of procedure. All except one had general anesthesia. ICU admission was required on 106 patients (62%). Patients were divided according to the type of cardiac lesion (single versus biventricular pathology) as well as according to the type of intervention (stenting and non-stenting procedures). Comparing these groups revealed that: stent procedures and procedures for patients with single ventricle pathologies were performed at an earlier age, with more contrast, fluoro and procedure time than for non-stent procedures and procedures for patients with biventricular pathologies. Complications include transient arrhythmias in most patients, perforation of the RVOT in one and lower limb hypoperfusion in 12 patients. ICU complications include low cardiac output symptoms (LCOS) in 10 (7%), and sepsis in 8. No intra-procedure mortality. The overall survival was 94%. Ten patients died, with one early and 9 late mortality. 60% of the dead patients had PDA stenting. Reintervention varies according to the patient's diagnosis. CONCLUSION: Cardiac catheterization intervention an important modality in the management of neonates and infants with critical CHD. Well planned procedures and team expertise are essential. Stenting procedures and procedures for patients with single ventricles carries higher morbidity and mortality.

Comparison of In Vitro and In Vivo Results Using the GastroDuo and the Salivary Tracer Technique: Immediate Release Dosage Forms under Fasting Conditions.

The fasted state administration of immediate release (IR) dosage forms is often regarded as uncritical since physiological aspects seem to play a minor role for disintegration and drug release. However, recent in vivo studies in humans have highlighted that fasted state conditions are in fact highly dynamic. It was therefore the aim of this study to investigate the disintegration and drug release behavior of four different IR formulations of the probe drug caffeine under physiologically relevant conditions with the aid of the GastroDuo. One film-coated tablet and three different capsule formulations based on capsule shells either made from hard gelatin or hydroxypropylmethyl cellulose (HPMC) were tested in six different test programs. To evaluate the relevance of the data generated, the four IR formulations were also studied in a four-way cross-over study in 14 healthy volunteers by using the salivary tracer technique (STT). It could be shown that the IR formulations behaved differently in the in vitro test programs. Thereby, the simulated parameters affected the disintegration and dissolution behavior of the four IR formulations in different ways. Whereas drug release from the tablet started early and was barely affected by temperature, pH or motility, the different capsule formulations showed a longer lag time and were sensitive to specific parameters. However, once drug release was initiated, it typically progressed with a higher rate for the capsules compared to the tablet. Interestingly, the results obtained with the STT were not always in line with the in vitro data. This observation was due to the fact that the probability of the different test programs was not equal and that certain scenarios were rather unlikely to occur under the controlled and standardized conditions of clinical studies. Nonetheless, the in vitro data are still valuable as they allowed to discriminate between different formulations.

Simultaneous quantification of acidic and basic flupirtine metabolites by supercritical fluid chromatography according to European Medicines Agency validation.

Supercritical fluid chromatography (SFC) holds the potential to become an orthogonal method to HPLC/UHPLC in xenobiotic metabolism studies, due to its outstanding capacity to simultaneously separate highly similar (as HPLC) and physicochemically different analytes (problematic using HPLC). Paucity of guideline-conform validation, however, has been a major obstacle to clinical application of SFC, even in cases where biotransformation yields chemically dissimilar metabolites that require more than one HPLC method for comprehensive analysis. Here, a method based on supercritical fluid chromatography coupled to single quadrupole MS detection was developed to simultaneously quantify the divisive analgesic flupirtine and its acidic and basic metabolites, represented by 4-fluorohippuric acid (4-FHA) and the active metabolite D-13223 respectively, using custom-made synthetic internal standards. Experimental data on the fundamental retention mechanisms under supercritical conditions, indicating the importance of halogen and π-π-bonding for specific retention on polysaccharide-based stationary-phases, is discussed. Compared to previous HPLC methods, the novel method offers higher versatility in terms of the target metabolite range (addressing both acidic and basic metabolites within a singular method), faster analysis (7.5 min), and compliance with green chemistry principles. Validation was performed according to EMA criteria on bioanalytical method validation, demonstrating selectivity, carry-over, calibration curve parameters (LLOQ, range, and linearity), within- and between-run accuracy and precision, dilution integrity, matrix effect and stability. For proof-of-concept, the SFC method was applied to clinical samples of human urine obtained after single intravenous (100 mg), single oral (100 mg), and repeated oral administration (400 mg). Flupirtine, D-13223, and 4-FHA could be quantified, shedding light on the extent of oxidative flupirtine metabolism in humans in the context of the unresolved biotoxification that has led to the withdrawal of specific neuronal KV7 openers.

In vivo characterization of enTRinsic™ drug delivery technology capsule after intake in fed state: A cross-validation approach using salivary tracer technique in comparison to MRI.

The instability of various small molecules, vaccines and peptides in the human stomach is a complex challenge for oral drug delivery. Recently, a novel gastro-resistant capsule - the enTRinsic™ Drug Delivery Technology capsule - has been developed. In this work, the salivary tracer technique based on caffeine has been applied to study the in vivo disintegration of enTRinsic™ capsules in 16 healthy volunteers. In addition, magnetic resonance imaging (MRI) was used to visualize GI transit and to verify the disintegration times determined by using the salivary tracer technique. The enTRinsic™ capsules filled with 50mg of caffeine and 5mg of black iron oxide were administered in the fed state, i.e. 30min after a light meal (500kcal). In the first hour after capsule intake, the subjects were placed in supine position in the MRI scanner and scans were performed in short time intervals. After 1h, the subjects could leave the MRI scanner in between the MRI measurements, which were performed every 15min until disintegration of the capsule was confirmed (maximum observation time: 8h). Saliva samples were obtained simultaneously with MR imaging. Caffeine concentrations in saliva were determined by LC/MS-MS. The starting point of capsule disintegration was determined visually by inspection of the MR images as well as by the onset of salivary caffeine concentrations. In 14 out of 16 subjects, the capsule disintegrated in the small intestine. In one subject, the enTRinsic™ capsule was not emptied from the stomach within the observation time. In another subject, disintegration occurred during gastric emptying in the antropyloric region. In this study, we demonstrated that the enTRinsic™ capsules are also gastro resistant when taken under fed state conditions. Furthermore, we demonstrated the feasibility of using low dose caffeine as a salivary tracer for the determination of the disintegration of an enteric formulation.

Low dose caffeine as a salivary tracer for the determination of gastric water emptying in fed and fasted state: A MRI validation study.

Improving our knowledge about human gastrointestinal physiology and its impact on oral drug delivery is crucial for the development of new therapies and effective drug delivery systems. The aim of this study was to develop an in vivo tool to determine gastric emptying of water by administration of a caffeine as a tracer substance followed by subsequent saliva caffeine analysis. For this purpose, 35 mg of caffeine were given to six healthy volunteers after a 10 h overnight together with 240 mL of tap water either on a fasted stomach or 30 min after the high-caloric, high-fat breakfast recommended for bioavailability/bioequivalence (BA/BE) studies. Caffeine was administered in form of an ice capsule in order to omit the contamination of the oral cavity with caffeine. Parallel to saliva sampling, magnetic resonance imaging (MRI) was applied in order to validate this novel approach. After administration of the ice capsule, MRI measurements were performed every 2 min for the first 20 min followed by further measurements after 25, 30, 35, 40, 50 and 60 min. Saliva samples were collected always 1 min after the MRI measurement in supine position in the MRI scanner and continued for further 240 min. The caffeine concentration in saliva was quantified after liquid-liquid extraction by a validated HPLC/MS-MS method. The obtained MRI data revealed a fast emptying of the co-administered water within 10 to 50 min in the fasted state and likewise in the fed state. Salivary caffeine kinetics showed a Cmax from 150 to 400 ng/mL with a tmax from 20 to 90 min. MRI data were normalized by setting the maximum emptied volume to 100% and the salivary caffeine kinetics were normalized by setting Cmax to 100%. In order to compare the results obtained by the MRI and the saliva method, the normalized data for each volunteer was correlated based on a linear regression. In the fasted state the mean slope for six comparisons was 0.9114 ±â€¯0.1500 and the mean correlation coefficient was 0.912 ±â€¯0.055. In the fed state, a mean slope of 0.8326 ±â€¯0.1630 and a mean correlation coefficient of 0.887 ±â€¯0.047 were obtained. Based on these results, we could show that salivary caffeine concentrations are suitable to describe the emptying of water as a non-caloric liquid from the fasted and the fed stomach. The presented technique provides a straight-forward, inexpensive and noninvasive method to assess gastric emptying of hydrophilic liquids, which can be broadly used in oral biopharmaceutics. Possible applications are the characterization of real-life conditions, specific populations (e.g. elderly people) and the better understanding of the contribution of gastric emptying to pharmacokinetic profiles of orally administered drugs.

Ketamine metabolites with antidepressant effects: Fast, economical, and eco-friendly enantioselective separation based on supercritical-fluid chromatography (SFC) and single quadrupole MS detection.

Increasing evidence accumulates that metabolites of the dissociative anesthetic ketamine contribute considerably to the biological effects of this drug and could be developed as next generation antidepressants, especially for acute treatment of patients with therapy-refractory major depression. Analytical methods for the simultaneous determination of the plethora of hydroxylated, dehydrogenated and/or demethylated compounds formed after administration of ketamine hydrochloride are a prerequisite for future clinical investigations and a deeper understanding of the individual role of the isomers of these metabolites. In this study, we present development and validation of a method based on supercritical-fluid chromatography (SFC) coupled to single quadrupole MS detection that allows the separation of ketamine as well as all of its relevant metabolites detected in urine of healthy volunteers. Inherently to SFC methods, the run times of the novel protocol are four times shorter than in a comparable HPLC method, the use of organic solvents is reduced and we were able to demonstrate and validate the successful enantioselective separation and quantification of R- and S-ketamine, R- and S-norketamine, R- and S-dehydronorketamine and (2R,6R)- and (2S,6S)-hydroxynorketamine isomers differing in either constitution, stereochemistry, or both, in one run. The developed method may be useful in investigating the antidepressant efficacy of ketamine in clinical trials.

Quantitative chiral and achiral determination of ketamine and its metabolites by LC-MS/MS in human serum, urine and fecal samples.

Ketamine (KET) is a widely used anesthetic drug which is metabolized by CYP450 enzymes to norketamine (n-KET), dehydronorketamine (DHNK), hydroxynorketamine (HNK) and hydroxyketamine (HK). Ketamine is a chiral compound and S-ketamine is known to be the more potent enantiomer. Here, we present the development and validation of three LC-MS/MS assays; the first for the quantification of racemic KET, n-KET and DHNK in human serum, urine and feces; the second for the separation and quantification of the S- and R-enantiomers of KET, n-KET and DHNK, and the third for separation and quantification of 2S,6S-hydroxynorketamine (2S,6S-HNK) and 2R,6R-hydroxynorketamine (2R,6R-HNK) in serum and urine with the ability to separate and detect 10 additional hydroxylated norketamine metabolites of racemic ketamine. Sample preparation was done by liquid-liquid extraction using methyl tert-butyl ether. For achiral determination of KET and its metabolites, an isocratic elution with ammonium acetate (pH 3.8; 5mM) and acetonitrile on a C18 column was performed. For the separation of S- and R-enantiomers of KET, n-KET and DHNK, a gradient elution was applied using a mobile phase of ammonium acetate (pH 7.5; 10mM) and isopropanol on the CHIRAL-AGP® column. The enantioselective separation of the HNK metabolites was done on the chiral column Lux®-Amylose-2 with a gradient method using ammonium acetate (pH 9; 5mM) and a mixture of isopropanol and acetonitrile (4:1). The mass spectrometric detection monitored for each analyte 2-3 mass/charge transitions. D4-ketamine and D4-n-KET were used as internal standards. The assays were successfully validated according to current bioanalytical guidelines and applied to a pilot study in one healthy volunteer. Compared to previously published methods, our assays have superior analytical features such as a lower amount of required matrix, faster sample preparation, shorter analytical run time and higher sensitivity (LLOQ up to 0.1ng/ml). Moreover, our assay enables for the first time the enantioselective determination of 2R,6R- and 2S,6S-HNK which were shown to be responsible for the promising antidepressant effects of ketamine.