5-HT6 receptor antagonists. Design, synthesis, and structure-activity relationship of substituted 2-(1-methyl-4-piperazinyl)pyridines.

In this study, we present the discovery and pharmacological characterization of a new series of 6-piperazinyl-7-azaindoles. These compounds demonstrate potent antagonism and selectivity against the 5-HT6 receptor. Our research primarily focuses on optimizing the lead structure and investigating the structure-activity relationship (SAR) of these compounds. Our main objective is to improve their activity and selectivity against off-target receptors. Overall, our findings contribute to the advancement of novel compounds targeting the 5-HT6 receptor. Compound 29 exhibits significant promise in terms of pharmacological, physicochemical, and ADME (Absorption, Distribution, Metabolism, and Excretion) properties. Consequently, it merits thorough exploration as a potential drug candidate due to its favorable activity profile and successful outcomes in a range of in vivo experiments.

How to stop disproportionation of a hydrochloride salt of a very weakly basic compound in a non-clinical suspension formulation.

Our objectives were to stabilize a non-clinical suspension for use in toxicological studies and to develop methods to investigate the stability of the formulation in terms of salt disproportionation. The compound under research was a hydrochloride salt of a practically insoluble discovery compound ODM-203. The first of the three formulation approaches was a suspension prepared and stored at room temperature. The second formulation was stabilized by pH adjustment. In the third approach cooling was used to prevent salt disproportionation. 5 mg/mL aqueous suspension consisting of 20 mg/mL PVP/VA and 5 mg/mL Tween 80 was prepared for each of the approaches. The polymer was used as precipitation inhibitor to provide prolonged supersaturation while Tween 80 was used to enhance dissolution and homogeneity of the suspension. The consequences of salt disproportionation were studied by a small-scale in vitro dissolution method and by an in vivo pharmacokinetic study in rats. Our results show that disproportionation was successfully suppressed by applying cooling of the suspension in an ice bath at 2-8 °C. This procedure enabled us to proceed to the toxicological studies in rats. The in vivo study results obtained for the practically insoluble compound showed adequate exposures with acceptable variation at each dose level.

Metabolism and Mass Balance of the Novel Nonsteroidal Androgen Receptor Inhibitor Darolutamide in Humans.

The biotransformation and excretion of darolutamide were investigated in a phase I study. Six healthy male volunteers received a single dose of 300 mg 14C-darolutamide as an oral solution in the fasted state. Plasma, urine, and feces samples were analyzed for mass balance evaluation by liquid scintillation counting (LSC). Metabolite profiling and identification were determined using liquid chromatography mass-spectrometry with off-line radioactivity detection using LSC. Complete mass balance was achieved, with mean radioactivity recovery of 95.9% within 168 hours (63.4% in urine, 32.4% in feces). The administered 1:1 ratio of (S,R)- and (S,S)-darolutamide changed to approximately 1:5, respectively, in plasma. Darolutamide and the oxidation product, keto-darolutamide, were the only components quantifiable by LSC in plasma, accounting for 87.4% of total radioactivity, with a 2.1-fold higher plasma exposure for keto-darolutamide. Aside from darolutamide, the most prominent metabolites in urine were O-glucoronide (M-7a/b) and N-glucuronide (M-15a/b), as well as pyrazole sulfates (M-29, M-24) and glucuronides (M-21, M-22) resulting from oxidative cleavage of the parent. The darolutamide diastereomers were mainly detected in feces. In vitro assays showed that darolutamide metabolism involves a complex interplay between oxidation and reduction, as well as glucuronidation. Interconversion of the diastereomers involves oxidation to keto-darolutamide, primarily mediated by CYP3A4, followed by reduction predominantly catalyzed by cytosolic reductase(s), with aldo-keto reductase 1C3 playing the major role. The latter reaction showed stereoselectivity with preferential formation of (S,S)-darolutamide. SIGNIFICANCE STATEMENT: The metabolism and excretion of darolutamide in humans revealed that oxidation (CYP3A4) and glucuronidation (UGT1A9, UGT1A1) were the main metabolic routes of elimination. Direct excretion also contributed to overall clearance. The two pharmacologically equipotent diastereomers of darolutamide interconvert primarily via oxidation to the active metabolite keto-darolutamide, followed by reduction predominantly by cytosolic reductase(s). The latter reaction showed stereoselectivity with preferential formation of (S,S)-darolutamide. Data indicate a low drug-drug interaction potential of darolutamide with inducers or inhibitors of metabolizing enzymes.

Pharmacokinetics of Darolutamide, its Diastereomers and Active Metabolite in the Mouse: Response to Saini NK et al. (2020).

BACKGROUND: Saini et al. recently investigated the pharmacokinetics of darolutamide and its diastereomers in vitro and in vivo in Balb/c mice, reporting higher levels of (S,S)-darolutamide than (S,R)-darolutamide following intravenous or oral dosing, and interconversion of (S,R)-darolutamide to (S,S)-darolutamide. OBJECTIVE: To present our in vitro and in vivo studies of darolutamide pharmacokinetics in mice, which contrast with the findings of Saini et al. Methods: Nude male Balb/c mice were orally dosed for 7 days with 25, 50, or 100 mg/kg of darolutamide twice daily. Pharmacokinetic parameters in plasma and tissue samples were assessed by liquid chromatography-tandem mass spectrometry. Metabolism and interconversion of darolutamide and its diastereomers were investigated in cryopreserved Balb/c mouse hepatocytes. Protein binding was determined in plasma samples by equilibrium dialysis. RESULTS: On day 7, Cmax was reached 30 min after the last dose. Rapid formation and greater exposure of keto-darolutamide versus darolutamide were observed. Plasma exposure of (S,R)-darolutamide was 3-5-fold higher than that of (S,S)-darolutamide. The fraction of unbound keto-darolutamide was almost 6-fold lower than for darolutamide. In mouse hepatocytes, the conversion of (S,S)- to (S,R)-darolutamide was observed, but the conversion of (S,R)- to (S,S)-darolutamide was not detectable. Back-formation of keto-darolutamide to both diastereomers occurred at low levels. CONCLUSION: The darolutamide diastereomer ratio changes upon administration in mice and other species due to interconversion through keto-darolutamide. This is not considered clinically relevant since both diastereomers and keto- darolutamide are pharmacologically similar in vitro. Based on the high protein binding of keto-darolutamide, its contribution in vivo in humans is considered low.

First-in-human Phase 1 open label study of the BET inhibitor ODM-207 in patients with selected solid tumours.

BACKGROUND: Bromodomain and extra-terminal domain (BET) proteins are reported to be epigenetic anti-cancer drug targets. This first-in-human study evaluated the safety, pharmacokinetics and preliminary anti-tumour activity of the BET inhibitor ODM-207 in patients with selected solid tumours. METHODS: This was an open-label Phase 1 study comprised of a dose escalation part, and evaluation of the effect of food on pharmacokinetics. ODM-207 was administered orally once daily. The dose escalation part was initiated with a dose titration in the initial cohort, followed by a 3 + 3 design. RESULTS: Thirty-five patients were treated with ODM-207, of whom 12 (34%) had castrate-resistant prostate cancer. One dose-limiting toxicity of intolerable fatigue was observed. The highest studied dose achieved was 2 mg/kg due to cumulative toxicity observed beyond the dose-limiting toxicity (DLT) treatment window. Common AEs included thrombocytopenia, asthenia, nausea, anorexia, diarrhoea, fatigue, and vomiting. Platelet count decreased proportionally to exposure with rapid recovery upon treatment discontinuation. No partial or complete responses were observed. CONCLUSIONS: ODM-207 shows increasing exposure in dose escalation and was safe at doses up to 2 mg/kg but had a narrow therapeutic window. CLINICAL TRIAL REGISTRATION: The clinical trial registration number is NCT03035591.

Predicting the effect of prandial stage and particle size on absorption of ODM-204.

The prediction of absorption properties plays a key role in formulation development when the compound under development shows poor solubility and its absorption is therefore presumed to be solubility limited. In our work, we combined and compared data obtained from in vitro dissolution tests, transit intestinal model studies (TIM-1) and physiologically based pharmacokinetic modelling. Our aim was to determine the ability of these methods to predict performance of poorly soluble lipophilic weak base in vivo. The validity of the predictive methods was evaluated against the in vivo clinical pharmacokinetic (PK) data obtained after administration of the first test formulation, T1. The aim of our study was to utilize the models in evaluating absorption properties of the second test formulation, T2, which has not yet been clinically administered. The compound in the studies was ODM-204, which is a novel, orally administered, investigational, nonsteroidal dual inhibitor of CYP17A1 and androgen receptor. Owing to its physicochemical properties ODM-204 is prone to low or variable bioavailability. The models examined provided congruent data on dose dependent absorption, food effect at a dose of 200 mg and on the effect of API (active pharmaceutical ingredient) particle size on absorption. Our study shows that the predictive tools of in vitro dissolution, TIM-1 system and the PBPK (physiologically based pharmacokinetic) simulation, showed predictive power of different mechanisms of bioavailability and together provided valuable information for decision making.

Absorption, distribution, metabolism and excretion of darolutamide (a novel non-steroidal androgen receptor antagonist) in rats.

1. Darolutamide is a novel selective androgen receptor antagonist consisting of two pharmacologically equipotent diastereoisomers. The absorption, distribution, metabolism and excretion properties of darolutamide in rats are reported.2. Non- or [14C]-labelled darolutamide, its diastereoisomers and major metabolite were studied in intact and bile duct-cannulated rats (oral and intravenous administration), and rat hepatocytes.3. Darolutamide was quickly (1 h to reach maximum plasma concentration) and completely absorbed after oral administration. Absolute bioavailability was high. Keto-darolutamide was the most abundant metabolite in rat hepatocytes and the only major one in plasma. Interconversion between diastereoisomers was observed.4. After oral administration, radioactivity distributed widely and homogeneously. Penetration into brain was low (brain/blood ratio = 0.079). Elimination was rapid from most tissues. Excretion occurred rapidly, and routes were similar irrespective of administration routes. Complete mass balance was reached by 168 h post-dose. Most radioactivity (61-64%) was excreted in faeces, while relevant amounts (30-33%) were also excreted into urine. The main clearance routes were metabolism via oxidative reactions and glucuronidation. After intravenous administration, a relevant extent of the dose (20%) underwent extrabiliary excretion as darolutamide.

Drug-Drug Interaction Potential of Darolutamide: In Vitro and Clinical Studies.

BACKGROUND AND OBJECTIVES: Darolutamide is a novel androgen receptor (AR) antagonist approved for the treatment of nonmetastatic castration-resistant prostate cancer (nmCRPC). Accordingly, the drug-drug interaction (DDI) potential of darolutamide was investigated in both nonclinical and clinical studies. METHODS: In vitro studies were performed to determine the potential for darolutamide to be a substrate, inducer or inhibitor for cytochrome P450 (CYP) isoforms, other metabolizing enzymes and drug transporters. A phase I drug-interaction study in healthy volunteers evaluated the impact of co-administering rifampicin [CYP3A4 and P-glycoprotein (P-gp) inducer] and itraconazole [CYP3A4, P-gp and breast cancer resistance protein (BCRP) inhibitor] on the pharmacokinetics of darolutamide. Two further phase I studies assessed the impact of co-administering oral darolutamide on the pharmacokinetics of midazolam (sensitive CYP3A4 substrate) and dabigatran etexilate (P-gp substrate) and the impact on the pharmacokinetics of co-administered rosuvastatin [a substrate for BCRP, organic anion-transporting polypeptide (OATP)1B1, OATP1B3 and organic anion transporter (OAT)3]. RESULTS: In vitro, darolutamide was predominantly metabolized via oxidative biotransformation catalyzed by CYP3A4 and was identified as a substrate for P-gp and BCRP. The enzymatic activity of nine CYP isoforms was not inhibited or slightly inhibited in vitro with darolutamide, and a rank order and mechanistic static assessment indicated that risk of clinically relevant DDIs via CYP inhibition is very low. In vitro, darolutamide exhibited no relevant induction of CYP1A2 or CYP2B6 activity. Inhibition of BCRP-, P-gp-, OAT3-, MATE1-, MATE2-K-, OATP1B1- and OATP1B3-mediated transport was observed in vitro. Phase I data showed that darolutamide exposure increased 1.75-fold with co-administered itraconazole and decreased by 72% with rifampicin. Co-administration of darolutamide with CYP3A4/P-gp substrates showed no effect or only minor effects. Rosuvastatin exposure increased 5.2-fold with darolutamide because of BCRP and probably also OATPB1/OATPB3 inhibition. CONCLUSIONS: Darolutamide has a low potential for clinically relevant DDIs with drugs that are substrates for CYP or P-gp; increased exposure of BCRP and probably OATP substrates was the main interaction of note.

ODM-203, a Selective Inhibitor of FGFR and VEGFR, Shows Strong Antitumor Activity, and Induces Antitumor Immunity.

Alterations in the gene encoding for the FGFR and upregulation of the VEGFR are found often in cancer, which correlate with disease progression and unfavorable survival. In addition, FGFR and VEGFR signaling synergistically promote tumor angiogenesis, and activation of FGFR signaling has been described as functional compensatory angiogenic signal following development of resistance to VEGFR inhibition. Several selective small-molecule FGFR kinase inhibitors are currently in clinical development. ODM-203 is a novel, selective, and equipotent inhibitor of the FGFR and VEGFR families. In this report we show that ODM-203 inhibits FGFR and VEGFR family kinases selectively and with equal potency in the low nanomolar range (IC50 6-35 nmol/L) in biochemical assays. In cellular assays, ODM-203 inhibits VEGFR-induced tube formation (IC50 33 nmol/L) with similar potency as it inhibits proliferation in FGFR-dependent cell lines (IC50 50-150 nmol/L). In vivo, ODM-203 shows strong antitumor activity in both FGFR-dependent xenograft models and in an angiogenic xenograft model at similar well-tolerated doses. In addition, ODM-203 inhibits metastatic tumor growth in a highly angiogenesis-dependent kidney capsule syngenic model. Interestingly, potent antitumor activity in the subcutaneous syngenic model correlated well with immune modulation in the tumor microenvironment as indicated by marked decrease in the expression of immune check points PD-1 and PD-L1 on CD8 T cells and NK cells, and increased activation of CD8 T cells. In summary, ODM-203 shows equipotent activity for both FGFR and VEGFR kinase families and antitumor activity in both FGFR and angigogenesis models.

IMI - Oral biopharmaceutics tools project - Evaluation of bottom-up PBPK prediction success part 3: Identifying gaps in system parameters by analysing In Silico performance across different compound classes.

Three Physiologically Based Pharmacokinetic software packages (GI-Sim, Simcyp® Simulator, and GastroPlus™) were evaluated as part of the Innovative Medicine Initiative Oral Biopharmaceutics Tools project (OrBiTo) during a blinded "bottom-up" anticipation of human pharmacokinetics. After data analysis of the predicted vs. measured pharmacokinetics parameters, it was found that oral bioavailability (Foral) was underpredicted for compounds with low permeability, suggesting improper estimates of intestinal surface area, colonic absorption and/or lack of intestinal transporter information. Foral was also underpredicted for acidic compounds, suggesting overestimation of impact of ionisation on permeation, lack of information on intestinal transporters, or underestimation of solubilisation of weak acids due to less than optimal intestinal model pH settings or underestimation of bile micelle contribution. Foral was overpredicted for weak bases, suggesting inadequate models for precipitation or lack of in vitro precipitation information to build informed models. Relative bioavailability was underpredicted for both high logP compounds as well as poorly water-soluble compounds, suggesting inadequate models for solubility/dissolution, underperforming bile enhancement models and/or lack of biorelevant solubility measurements. These results indicate areas for improvement in model software, modelling approaches, and generation of applicable input data. However, caution is required when interpreting the impact of drug-specific properties in this exercise, as the availability of input parameters was heterogeneous and highly variable, and the modellers generally used the data "as is" in this blinded bottom-up prediction approach.

IMI - Oral biopharmaceutics tools project - Evaluation of bottom-up PBPK prediction success part 2: An introduction to the simulation exercise and overview of results.

Orally administered drugs are subject to a number of barriers impacting bioavailability (Foral), causing challenges during drug and formulation development. Physiologically-based pharmacokinetic (PBPK) modelling can help during drug and formulation development by providing quantitative predictions through a systems approach. The performance of three available PBPK software packages (GI-Sim, Simcyp®, and GastroPlus™) were evaluated by comparing simulated and observed pharmacokinetic (PK) parameters. Since the availability of input parameters was heterogeneous and highly variable, caution is required when interpreting the results of this exercise. Additionally, this prospective simulation exercise may not be representative of prospective modelling in industry, as API information was limited to sparse details. 43 active pharmaceutical ingredients (APIs) from the OrBiTo database were selected for the exercise. Over 4000 simulation output files were generated, representing over 2550 study arm-institution-software combinations and approximately 600 human clinical study arms simulated with overlap. 84% of the simulated study arms represented administration of immediate release formulations, 11% prolonged or delayed release, and 5% intravenous (i.v.). Higher percentages of i.v. predicted area under the curve (AUC) were within two-fold of observed (52.9%) compared to per oral (p.o.) (37.2%), however, Foral and relative AUC (Frel) between p.o. formulations and solutions were generally well predicted (64.7% and 75.0%). Predictive performance declined progressing from i.v. to solution and immediate release tablet, indicating the compounding error with each layer of complexity. Overall performance was comparable to previous large-scale evaluations. A general overprediction of AUC was observed with average fold error (AFE) of 1.56 over all simulations. AFE ranged from 0.0361 to 64.0 across the 43 APIs, with 25 showing overpredictions. Discrepancies between software packages were observed for a few APIs, the largest being 606, 171, and 81.7-fold differences in AFE between SimCYP and GI-Sim, however average performance was relatively consistent across the three software platforms.

1-Sulfonyl-6-Piperazinyl-7-Azaindoles as potent and pseudo-selective 5-HT6 receptor antagonists.

A series of 1-Sulfonyl-6-Piperazinyl-7-Azaindoles, showing strong antagonistic activity to 5-HT6 receptor (5-HT6R) was synthesized and characterized. The series was optimized to reduce activity on D2 receptor. Based on the selectivity against this off-target and the analysis of the ADME-tox profile, compound 1c was selected for in vivo efficacy assessment, which demonstrated procognitive effects as shown in reversal of scopolamine induced amnesia in an elevated plus maze test in mice. Compound 3, the demethylated version of compound 1c, was profiled against a panel of 106 receptors, channels and transporters, indicating only D3 receptor as a major off-target. Compound 3 has been selected for this study over compound 1c because of the higher 5-HT6R/D2R binding ratio. These results have defined a new direction for the design of our pseudo-selective 5-HT6R antagonists.

TRPA1: a transducer and amplifier of pain and inflammation.

The transient receptor potential ankyrin 1 (TRPA1) ion channel on peripheral terminals of nociceptive primary afferent nerve fibres contributes to the transduction of noxious stimuli to electrical signals, while on central endings in the spinal dorsal horn, it amplifies transmission to spinal interneurons and projection neurons. The centrally propagating nociceptive signal that is induced and amplified by TRPA1 not only elicits pain sensation but also contributes to peripheral neurogenic inflammation through a peripheral axon reflex or a centrally mediated back propagating dorsal root reflex that releases vasoactive agents from sensory neurons in the periphery. Endogenous TRPA1 agonists that are generated under various pathophysiological conditions both in the periphery and in the spinal cord have TRPA1-mediated pro-nociceptive and pro-inflammatory effects. Among endogenous TRPA1 agonists that have been shown to play a role in the pathogenesis of pain and inflammatory conditions are, for example, methylglyoxal, 4-hydroxynonenal, 12-lipoxygenase-derived hepoxilin A3, 5,6-epoxyeicosatrienoic acid and reactive oxygen species, while mustard oil and cinnamaldehyde are most commonly used exogenous TRPA1 agonists in experimental studies. Among selective TRPA1 antagonists are HC-030031, A-967079, AP-14 and Chembridge-5861528. Recent evidence indicates that TRPA1 plays a role also in transition of acute to chronic pain. Due to its location on a subpopulation of pain-mediating primary afferent nerve fibres, blocking the TRPA1 channel is expected to have antinociceptive, antiallodynic and anti-inflammatory effects.

Transient receptor potential ankyrin 1 ion channel contributes to guarding pain and mechanical hypersensitivity in a rat model of postoperative pain.

BACKGROUND: The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed on nociceptive primary afferent nerve fibers. On the distal ending, it is involved in transduction of noxious stimuli, and on the proximal ending (within the spinal dorsal horn), it regulates transmission of nociceptive signals. Here we studied whether the cutaneous or spinal TRPA1 ion channel contributes to mechanical hypersensitivity or guarding, an index of spontaneous pain, in an experimental model of postoperative pain in the rat. METHODS: A skin plus deep-tissue incision was performed under general anesthesia in the plantar skin of one hind paw, after which the incised skin was closed with sutures. Postoperative pain and hypersensitivity were assessed 24-48 h after the operation. Guarding pain was assessed by scoring the hind-paw position. Mechanical hypersensitivity was assessed with a calibrated series of monofilaments applied to the wound area in the operated paw or the contralateral control paw. Chembridge-5861528, a TRPA1 channel antagonist, was administered intaperitoneally (10-30 mg/kg), intraplantarly (10-30 µg), or intrathecally (10 µg) in attempts to suppress guarding and hypersensitivity. RESULTS: Intraperitoneal or ipsi- but not contralateral intraplantar treatment with Chembridge-5861528 reduced mechanical hypersensitivity and guarding in the operated limb. Intrathecal treatment attenuated hypersensitivity but not guarding. Intraplantar Chembridge-5861528 suppressed preferentially mechanical hyperalgesia and intrathecal Chembridge-5861528 tactile allodynia. CONCLUSIONS: The TRPA1 channel in the skin contributes to sustained as well noxious mechanical stimulus-evoked postoperative pain, whereas the spinal TRPA1 channel contributes predominantly to innocuous mechanical stimulus-evoked postoperative pain.

Use of comprehensive screening methods to detect selective human CAR activators.

The so-called human xenosensors, constitutive androstane receptor (hCAR), pregnane X receptor (hPXR) and aryl hydrocarbon receptor (hAhR), participate in drug metabolism and transport as well as in several endogenous processes by regulating the expression of their target genes. While the ligand specificities for hPXR and hAhR are relatively well described, this property of hCAR still remains fairly unclear. Identifying hCAR agonists for drug development and for studying hCAR biology are hindered mainly by the unique properties of the receptor, such as the high constitutive activity and complex signaling network but also by the lack of robust and reliable assays and cellular models. Here, validated reporter assays for these three xenosensors are presented and thereafter used to screen a large set of chemicals in order to find novel selective hCAR ligands. We introduce a novel selective hCAR agonist, FL81, which can be used as a stable positive control in hCAR activity assays. Our established receptor-selective ligand identification methods consisting of supporting biological assays and molecular modeling techniques are then used to study FL81 as well as other discovered ligands, such as diethylstilbestrol, o,p'-DDT, methoxychlor and permethrin, for their ability to specifically activate hCAR and to regulate the CYP enzyme expression and function.

Metabolism of oxycodone in human hepatocytes from different age groups and prediction of hepatic plasma clearance.

Oxycodone is commonly used to treat severe pain in adults and children. It is extensively metabolized in the liver in adults, but the maturation of metabolism is not well understood. Our aim was to study the metabolism of oxycodone in cryopreserved human hepatocytes from different age groups (3 days, 2 and 5 months, 4 years, adult pool) and predict hepatic plasma clearance of oxycodone using these data. Oxycodone (0.1, 1, and 10 µM) was incubated with hepatocytes for 4 h, and 1 µM oxycodone also with CYP3A inhibitor ketoconazole (1 µM). Oxycodone and noroxycodone concentrations were determined at several time points with liquid chromatography-mass spectrometry. In vitro clearance of oxycodone was used to predict hepatic plasma clearance, using the well-stirred model and published physiological parameters. Noroxycodone was the major metabolite in all batches and ketoconazole inhibited the metabolism markedly in most cases. A clear correlation between in vitro oxycodone clearance and CYP3A4 activity was observed. The predicted hepatic plasma clearances were typically much lower than the published median total plasma clearance from pharmacokinetic studies. The data suggests that there are no children-specific metabolites of oxycodone. Moreover, CYP3A activity seems to be the major determinant in metabolic clearance of oxycodone regardless of age group or individual variability in hepatocyte batches.

Determination of permeation resistance distribution in in vitro cell monolayer permeation experiments.

The results of cell monolayer permeation experiments are often affected by concentration dependent cellular processes, such as active transport and metabolism. The rigorous analysis of the concentration dependence of these processes is often limited by the lack of knowledge of the actual concentration at the site of action because the measurement of the local concentration is seldom feasible. However, the local concentrations can be estimated if the rates into and out of a particular location are known. Thus, an insight into the distribution of the permeation resistance in the in vitro cell monolayer permeation experiments would enable the estimation of local concentrations during the permeation experiments and, consequently, a more thorough analysis of the concentration dependent processes involved in the transepithelial transfer of compounds. In this study, a compartmental model was constructed applicable for dissecting the roles of apical and basal side aqueous boundary layers and the cell membranes in the total permeation barrier for weakly acidic and basic compounds. The model was applied for the analysis of the Caco-2 permeation data of three high permeability compounds, propranolol, metoprolol and ibuprofen. Furthermore, the effects of extracellular pH and the agitation conditions on the local concentrations of these compounds were evaluated.

Effects of experimental setup on the apparent concentration dependency of active efflux transport in in vitro cell permeation experiments.

P-Glycoprotein mediated efflux is one of the barriers limiting drug absorption from the intestine. Predictions of the intestinal P-glycoprotein function need to take into account the concentration dependency because high intestinal drug concentrations may saturate P-glycoprotein. However, the substrate binding site of P-glycoprotein lies inside the cells and the drug concentration at the binding site cannot be measured directly. Therefore, rigorous determination of concentration dependent P-glycoprotein kinetics is challenging. In this study, the effects of the aqueous boundary layers, extracellular pH and cellular retention on the apparent saturation kinetics of P-glycoprotein mediated transport of quinidine in an in vitro cell permeation setting were explored. The changes in the experimental conditions caused 1 order of magnitude variation in the apparent affinity to P-glycoprotein (K(m,app)) and a 5-fold difference in the maximum effective P-glycoprotein mediated transport rate of quinidine (V(max,app)). However, fitting the concentration data into a compartmental model which accounted for the aqueous boundary layers, cell membranes and cellular retention suggested that the P-glycoprotein function per se was not altered, it was the differences in the passive transfer of quinidine which changed the apparent transport kinetics. These results provide further insight into the dynamics of the P-glycoprotein mediated transport and on the roles of several confounding factors involved in in vitro experimental setting. Further, the results confirm the applicability of compartmental model based data analysis approach in the determination of active transporter kinetics.

Effects of selective serotonin reuptake inhibitors on timolol metabolism in human liver microsomes and cryo-preserved hepatocytes.

Timolol has been widely used in the treatment of glaucoma. Topically applied, timolol may cause adverse cardiovascular effects due to systemic absorption through the nasolacrimal duct. Timolol is mainly metabolized by cytochrome P450 2D6 (CYP2D6) in the liver. The aim of the present study was to characterize further the metabolism of timolol in vitro. Especially the effect of several drugs such as selective serotonin reuptake inhibitors on the metabolism of timolol was evaluated. In human liver microsomes, four timolol metabolites were identified, in cryo-preserved hepatocytes nine. In both in vitro experiments, the hydroxy metabolite M1 was the main metabolite. The in vivo half-life predicted for timolol was 3.7 hr. in cryo-preserved hepatocytes, corresponding to the half-life of timolol in humans in vivo. Fluoxetine, paroxetine, sertraline, citalopram and fluvoxamine inhibited the formation of M1 in microsomes with IC(50) values of 1.4, 2.0, 3.5, 21 and 20 microM, respectively. In human cryo-preserved hepatocytes, the IC(50) values for fluoxetine, paroxetine and fluvoxamine were 0.7, 0.5 and 5.9 microM, respectively. In conclusion, compounds known to be potent CYP2D6 inhibitors inhibited timolol metabolism in in vitro experiments. The present results strongly suggest that fluoxetine and paroxetine may significantly affect the metabolism of timolol also in vivo and may thus potentiate the adverse cardiovascular effects of topically administered timolol.

Modelling of drug disposition kinetics in in vitro intestinal absorption cell models.

One major prerequisite for an orally administered drug is the ability to cross the intestinal epithelia from intestinal lumen into the blood circulation. Therefore, the absorption potential of molecules is studied early on during the drug development process. Permeation experiments using cultured cell monolayers are one of the most often applied methods to screen and also to predict in more detail the intestinal absorption potential of molecules in preclinical phase. Furthermore, these studies are also used to screen the molecules for transporter interactions as well as for more detailed mechanistic studies of the transfer routes involved. Several mathematical and computational models with complexity varying from simple non-mechanistic single barrier models to mechanistically more detailed compartmental models have been developed to describe the drug disposition during these in vitro permeation experiments. This MiniReview gives an overview of these models and their applications. Also the implications of these models to the prediction of intestinal absorption in vivo are discussed.