Transporter tandems: precise tools for normalizing active transporter in the plasma membrane.

The transport efficiency (TE) describes the performance of a transport protein for a specific substrate. To compare the TE of different transporters, the number of active transporters in the plasma membrane must be monitored, as it may vary for each transporter and experiment. Available methods, like LC-MS quantification of tryptic peptides, fail to discriminate inactive intracellular transporters or, like cell-surface biotinylation followed by affinity chromatography and Western blotting, are imprecise and very laborious. We wanted to normalize active transporters by the activity of a second transporter. A transporter tandem, generated by joining two transporter cDNAs into a single open reading frame, should guarantee a 1 : 1 stoichiometry. Here we created a series of tandems with different linkers between the human ergothioneine (ET) transporter ETT (gene symbol SLC22A4) and organic cation transporter OCT2 (SLC22A2). The linker sequence strongly affected the expression strength. The stoichiometry was validated by absolute peptide quantification and untargeted peptide analysis. Compared with wild-type ETT, the normalized ET clearance of the natural variant L503F was higher (f = 1.34); G462E was completely inactive. The general usefulness of the tandem strategy was demonstrated by linking several transporters with ETT; every construct was active in both parts. Transporter tandems can be used - without membrane isolation or protein quantification - as precise tools for transporter number normalization, to identify, for example, relevant transporters for a drug. It is necessary, however, to find suitable linkers, to check the order of transporters, and to verify the absence of functional interference by saturation kinetics.

Recommendations and feedback from the European Bioanalysis Forum Workshop: 1 year into ICH M10 - keeping our finger on the pulse.

The ICH M10 guideline on bioanalytical method validation and sample analysis is being adopted since 2023. However, and inevitably, some paragraphs or requirements remain ambiguous and are open for different interpretations. In support of a harmonized interpretation by the industry and health authorities, the European Bioanalysis Forum organized a workshop on 14 November 2023 in Barcelona, Spain, to discuss unclear and/or ambiguous paragraphs which were identified by the European Bioanalysis Forum community and delegates of the workshop prior to the workshop. This manuscript reports back from the workshop with recommendations and aims at continuing an open scientific discussion within the industry and with regulators in support of a science-driven guideline for the bioanalytical community and in line with the ICH mission - that is, achieve greater harmonization worldwide to ensure that safe, effective and high-quality medicines are developed and registered in the most resource-efficient manner.

Sorafenib hepatobiliary disposition: mechanisms of hepatic uptake and disposition of generated metabolites.

Sorafenib is an orally active tyrosine kinase inhibitor used in the treatment of renal and hepatocellular carcinoma. This study was designed to establish whether transport proteins are involved in the hepatic uptake of sorafenib and to determine the extent of biliary excretion of sorafenib and its metabolites in human hepatocytes. Initial uptake was assessed in freshly isolated, suspended human hepatocytes in the presence of inhibitors and modulators. [(14)C]Sorafenib (1 µM) uptake at 4°C was reduced by about 61-63% of the uptake at 37°C, suggesting a high degree of passive diffusion. Hepatocyte uptake of [(14)C]sorafenib was not Na(+) dependent or influenced by the organic anion transporter 2 inhibitor ketoprofen. However, initial [(14)C]sorafenib hepatocyte uptake was reduced by 46 and 30% compared with control values in the presence of the organic anion transporting polypeptide inhibitor rifamycin SV and the organic cation transporter (OCT) inhibitor decynium 22, respectively. [(14)C]Sorafenib (0.5-5 µM) uptake was significantly higher in hOCT1-transfected Chinese hamster ovary cells compared with mock cells, and inhibited by the general OCT inhibitor, 1-methyl-4-phenylpryidinium. OCT1-mediated uptake was saturable with a Michaelis-Menten constant of 3.80 ± 2.53 µM and a V(max) of 116 ± 42 pmol/mg/min. The biliary excretion index and in vitro biliary clearance of sorafenib (1 µM) in sandwich-cultured human hepatocytes were low (∼11% and 11 ml/min/kg, respectively). Results suggest that sorafenib uptake in human hepatocytes occurs via passive diffusion, by OCT1, and by organic anion transporting polypeptide(s). Sorafenib undergoes modest biliary excretion, predominantly as a glucuronide conjugate(s).

Immunocapture LC-MS(/MS) assays for biotherapeutic and biomarker proteins: the European Bioanalysis Forum continuing discussions on scientific and regulatory challenges.

The use of LC-MS(/MS) assays to quantify (biotherapeutic or biomarker) proteins is commonplace and well accepted across industry. There is a good understanding on the added value over conventional analytical technologies (i.e., ligand-binding assays). In fact, the impact of combining small- and large-molecule technologies for large-molecule analysis has played a significant part in bringing the bioanalytical communities closer together and building a mutual respect and understanding between scientists. This paper from the European Bioanalysis Forum presents a history of the journey and future perspectives for hybrid assays, with focus on the unanswered scientific questions, including regulatory discussions to be had. Hybrid assays are essentially a combination of ligand-binding assays and MS, and the ICH M10 guideline does not address this approach directly. Decision-based acceptance criteria are still being discussed, and the industry should continue to do so.

In vitro and in vivo P-glycoprotein transport characteristics of rivaroxaban.

Rivaroxaban, an oral, direct factor Xa inhibitor, has a dual mode of elimination in humans, with two-thirds metabolized by the liver and one-third renally excreted unchanged. P-glycoprotein (P-gp) is known to be involved in the absorption, distribution, and excretion of drugs. To investigate whether rivaroxaban is a substrate of P-gp, the bidirectional flux of rivaroxaban across Caco-2, wild-type, and P-gp-overexpressing LLC-PK1 cells was investigated. Furthermore, the inhibitory effect of rivaroxaban toward P-gp was determined. Rivaroxaban exhibited high permeability and polarized transport across Caco-2 cells. Rivaroxaban was shown to be a substrate for, but not an inhibitor of, P-gp. Of a set of potential P-gp inhibitors, ketoconazole and ritonavir, but not clarithromycin or erythromycin, inhibited P-gp-mediated transport of rivaroxaban, with half-maximal inhibitory concentration values in the range of therapeutic plasma concentrations. These findings are in line with observed area under the plasma concentration-time curve increases in clinical drug-drug interaction studies indicating a possible involvement of P-gp in the distribution and excretion of rivaroxaban. In vivo studies in wild-type and P-gp double-knockout mice demonstrated that the impact of P-gp alone on the pharmacokinetics of rivaroxaban is minor. However, in P-gp double-knockout mice, a slight increase in brain concentrations and decreased excretion into the gastrointestinal tract were observed compared with wild-type mice. These studies also demonstrated that brain penetration of rivaroxaban is fairly low. In addition to P-gp, a further transport protein might be involved in the secretion of rivaroxaban.

Design, Synthesis, and Pharmacological Characterization of a Neutral, Non-Prodrug Thrombin Inhibitor with Good Oral Pharmacokinetics.

Despite extensive research on small molecule thrombin inhibitors for oral application in the past decades, only a single double prodrug with very modest oral bioavailability has reached human therapy as a marketed drug. We have undertaken major efforts to identify neutral, non-prodrug inhibitors. Using a holistic analysis of all available internal data, we were able to build computational models and apply these for the selection of a lead series with the highest possibility of achieving oral bioavailability. In our design, we relied on protein structure knowledge to address potency and identified a small window of favorable physicochemical properties to balance absorption and metabolic stability. Protein structure information on the pregnane X receptor helped in overcoming a persistent cytochrome P450 3A4 induction problem. The selected compound series was optimized to a highly potent, neutral, non-prodrug thrombin inhibitor by designing, synthesizing, and testing derivatives. The resulting optimized compound, BAY1217224, has reached first clinical trials, which have confirmed the desired pharmacokinetic properties.

Inhaled agonists of soluble guanylate cyclase induce selective pulmonary vasodilation.

RATIONALE: Nitric oxide-independent agonists of soluble guanylate cyclase (sGC) have been developed. OBJECTIVES: We tested whether inhalation of novel dry-powder microparticle formulations containing sGC stimulators (BAY 41-2272, BAY 41-8543) or an sGC activator (BAY 58-2667) would produce selective pulmonary vasodilation in lambs with acute pulmonary hypertension. We also evaluated the combined administration of BAY 41-8543 microparticles and inhaled nitric oxide (iNO). Finally, we examined whether inhaling BAY 58-2667 microparticles would produce pulmonary vasodilation when the response to iNO is impaired. METHODS: In awake, spontaneously breathing lambs instrumented with vascular catheters and a tracheostomy tube, U-46619 was infused intravenously to increase mean pulmonary arterial pressure to 35 mm Hg. MEASUREMENTS AND MAIN RESULTS: Inhalation of microparticles composed of either BAY 41-2272, BAY 41-8543, or BAY 58-2667 and excipients (dipalmitoylphosphatidylcholine, albumin, lactose) produced dose-dependent pulmonary vasodilation and increased transpulmonary cGMP release without significant effect on mean arterial pressure. Inhalation of microparticles containing BAY 41-8543 or BAY 58-2667 increased systemic arterial oxygenation. The magnitude and duration of pulmonary vasodilation induced by iNO were augmented after inhaling BAY 41-8543 microparticles. Intravenous administration of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), which oxidizes the prosthetic heme group of sGC, markedly reduced the pulmonary vasodilator effect of iNO. In contrast, pulmonary vasodilation and transpulmonary cGMP release induced by inhaling BAY 58-2667 microparticles were greatly enhanced after treatment with ODQ. CONCLUSIONS: Inhalation of microparticles containing agonists of sGC may provide an effective novel treatment for patients with pulmonary hypertension, particularly when responsiveness to iNO is impaired by oxidation of sGC.

Activation of soluble guanylate cyclase reverses experimental pulmonary hypertension and vascular remodeling.

BACKGROUND: Severe pulmonary hypertension is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. Using wild-type and homozygous endothelial nitric oxide synthase (NOS3(-/-)) knockout mice with pulmonary hypertension induced by chronic hypoxia and rats with monocrotaline-induced pulmonary hypertension, we examined whether the soluble guanylate cyclase (sGC) stimulator Bay41-2272 or the sGC activator Bay58-2667 could reverse pulmonary vascular remodeling. METHODS AND RESULTS: Both Bay41-2272 and Bay58-2667 dose-dependently inhibited the pressor response of acute hypoxia in the isolated perfused lung system. When wild-type (NOS3(+/+)) or NOS3(-/-) mice were housed under 10% oxygen conditions for 21 or 35 days, both strains developed pulmonary hypertension, right heart hypertrophy, and pulmonary vascular remodeling, demonstrated by an increase in fully muscularized peripheral pulmonary arteries. Treatment of wild-type mice with the activator of sGC, Bay58-2667 (10 mg/kg per day), or the stimulator of sGC, Bay41-2272 (10 mg/kg per day), after full establishment of pulmonary hypertension from day 21 to day 35 significantly reduced pulmonary hypertension, right ventricular hypertrophy, and structural remodeling of the lung vasculature. In contrast, only minor efficacy of chronic sGC activator therapies was noted in NOS3(-/-) mice. In monocrotaline-injected rats with established severe pulmonary hypertension, both compounds significantly reversed hemodynamic and structural changes. CONCLUSIONS: Activation of sGC reverses hemodynamic and structural changes associated with monocrotaline- and chronic hypoxia-induced experimental pulmonary hypertension. This effect is partially dependent on endogenous nitric oxide generated by NOS3.

Bypassing nonparallelism of a monoclonal antibody ligand-binding assay by employment of alternative assay formats.

Determination of concentration-time profiles in cynomolgus monkeys of a therapeutic monoclonal antibody against a soluble target revealed a substantial discrepancy between a generic anti-human IgG capture/detection and target bridging assay with the target bridging assay leading to dose- and time-dependent underquantification of drug concentrations, lack of parallelism and subsequently different pharmacokinetic parameters. In contrast, plasma levels derived from a target capture and an anti-idiotypic antibody bridging assay were in close concordance with the generic assay and demonstrated parallelism with high precision across several dilutions. The results provide a practical attempt to overcome nonparallelism by employing alternative assay formats utilizing tailored assay reagent combinations in order to obtain unbiased pharmacokinetic data.

Soluble guanylate cyclase activator reverses acute pulmonary hypertension and augments the pulmonary vasodilator response to inhaled nitric oxide in awake lambs.

BACKGROUND: Inhaled nitric oxide (NO) is a potent and selective pulmonary vasodilator, which induces cGMP synthesis by activating soluble guanylate cyclase (sGC) in ventilated lung regions. Carbon monoxide (CO) has also been proposed to influence smooth muscle tone via activation of sGC. We examined whether direct stimulation of sGC by BAY 41-2272 would produce pulmonary vasodilation and augment the pulmonary responses to inhaled NO or CO. METHODS AND RESULTS: In awake, instrumented lambs, the thromboxane analogue U-46619 was intravenously administered to increase mean pulmonary arterial pressure to 35 mm Hg. Intravenous infusion of BAY 41-2272 (0.03, 0.1, and 0.3 mg x kg(-1) x h(-1)) reduced mean pulmonary arterial pressure and pulmonary vascular resistance and increased transpulmonary cGMP release in a dose-dependent manner. Larger doses of BAY 41-2272 also produced systemic vasodilation and elevated the cardiac index. N(omega)-nitro-l-arginine methyl ester abolished the systemic but not the pulmonary vasodilator effects of BAY 41-2272. Furthermore, infusing BAY 41-2272 at 0.1 mg x kg(-1) x h(-1) potentiated and prolonged the pulmonary vasodilation induced by inhaled NO (2, 10, and 20 ppm). In contrast, inhaled CO (50, 250, and 500 ppm) had no effect on U-46619-induced pulmonary vasoconstriction before or during administration of BAY 41-2272. CONCLUSIONS: In lambs with acute pulmonary hypertension, BAY 41-2272 is a potent pulmonary vasodilator that augments and prolongs the pulmonary vasodilator response to inhaled NO. Direct pharmacological stimulation of sGC, either alone or in combination with inhaled NO, may provide a novel approach for the treatment of pulmonary hypertension.

Determination of nifurtimox in dog plasma by stable-isotope dilution LC-MS/MS.

BACKGROUND: Nifurtimox is a 5-nitrofuran derived antiprotozoal drug used to treat diseases caused by trypanosomes including Chagas' disease and sleeping sickness (African trypanosomiasis). Available methods for the determination of nifurtimox in plasma are tedious and of low sensitivity. For the first time, an isotope dilution HPLC/MS/MS method for the sensitive quantitation of nifurtimox down to 10.0 µg/l in plasma is described. RESULTS: Protein precipitation was used for sample preparation. Samples were analyzed on a standard triple quadrupole tandem mass spectrometer. The validated concentration range covers 10.0 µg/l (LLOQ) to 5000 µg/l. Interassay accuracy and precision (%CV) ranged from 98.4 to 101%, and 2.61 to 10.1%, respectively. CONCLUSION: The method consists of very simple sample preparation and provides unmatched sensitivity, high reproducibility and robustness enabling analysis of large sample numbers. Method performance met current guidelines on bioanalytical method validation.

Determination of riociguat and its major human metabolite M-1 in human plasma by stable-isotope dilution LCMS/MS.

BACKGROUND: Riociguat (BAY 63-2521) is an oral NO-independent as well as NO-synergistic stimulator of soluble guanylate cyclase (sGC) for the treatment of pulmonary hypertension. BAY 60-4552 (M-1) is its pharmacologically active major metabolite. An isotope dilution LC-ESI-MS/MS method has been developed and validated for the simultaneous determination of riociguat and M-1 in lithium heparinized human plasma. RESULTS: The validated concentration range covers 0.500 µg/l (LLOQ) to 100 µg/l (ULOQ) for both analytes. Interassay accuracy and precision (%CV) for both analytes ranged from 92.7 to 111% and from 2.61 to 9.89%, respectively. CONCLUSION: The method proved to be selective, specific, sufficiently sensitive, highly reproducible and robust for the analysis of large numbers of samples.

Pharmacokinetics of the soluble guanylate cyclase activator cinaciguat in individuals with hepatic impairment.

Cinaciguat is intended for use in patients with acute decompensated heart failure. The drug is eliminated predominantly via the liver and, therefore, the potential impact of hepatic impairment on cinaciguat pharmacokinetics needs to be determined. This nonrandomized, open-label, observational study investigated the pharmacokinetics of cinaciguat in individuals with mild (Child-Pugh A; n = 8) or moderate (Child-Pugh B; n = 8) hepatic impairment and matched healthy volunteers (n = 16). An exploratory analysis of pharmacodynamic parameters was also conducted. Individuals with mild hepatic impairment and their controls received a single (4-hour) intravenous infusion of 100 µg/h cinaciguat, whereas individuals with moderate hepatic impairment and their controls received 50 µg/h. Cinaciguat was well tolerated and had a favorable safety profile. The most frequent treatment-emergent adverse events were headache (4 participants) and spontaneous penile erection (2 participants). In individuals with mild hepatic impairment, only minor increases in plasma cinaciguat concentrations and no significant differences in pharmacodynamic parameters were observed, compared with controls. Individuals with moderate hepatic impairment had a substantially higher cinaciguat exposure than controls. This higher exposure was associated with more pronounced vasodilatation. This study demonstrates that in individuals with mild hepatic impairment, individual dose adaptation may not be required.

Volume to dissolve applied dose (VDAD) and apparent dissolution rate (ADR): tools to predict in vivo bioavailability from orally applied drug suspensions.

Low solubility of drug candidates generated in research contributes to their elimination during subsequent development due to insufficient oral bioavailability (BA) of crystalline compound. Therefore, the purpose of the study was to identify critical in vitro solubility and dissolution parameter that would predict critical in vivo dissolution by means of in vitro-in vivo correlation. Thermodynamic solubility and apparent dissolution rate (ADR) were determined using the shake-flask method and mini-flow-through-cell, respectively. Oral BA studies in rats and humans were conducted from drug solution and suspension/tablets. Relative BA was calculated using F(rel) [%]=AUC(suspension)/AUC(solution)*100, representing a measure of in vivo dissolution. Roughly, F(rel) rat >50% translates into F(rel) human of >90%. Both, ADR and log volume to dissolve applied dose (VDAD), when plotted against F(rel) rat, revealed certain threshold levels, (ADR, ∼150-200 µg of compound dissolved under respective assay conditions; VDAD, ∼100-500 ml/kg) which translate into F(rel) in rats of >50%. Thus, assuming that F(rel)>50% in rats is indicative of sufficient in vivo dissolution in humans after oral application, drugs should exhibit a VDAD of ∼100-500 ml/kg or less in aqueous media to avoid insufficient or varying drug absorption.

A novel PDE2A reporter cell line: characterization of the cellular activity of PDE inhibitors.

We report here the generation and pharmacological characterization of a phosphodiesterase 2A (PDE2A) reporter cell line. Human PDE2A was stably transfected in a parental cell line expressing the atrial natriuretic peptide (ANP) receptor and the cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the biosensor for intracellular cGMP. In this reporter cell line, cGMP levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the CNG channel. By using different PDE inhibitors, we could show that our PDE2A reporter assay specifically monitors PDE2A inhibition with high sensitivity. In the absence of ANP stimulation, the PDE2A selective inhibitors EHNA, BAY 60-7550 and PDP did not increase basal luminescence levels in this experimental setting. However, in combination with ANP, these inhibitors stimulated luminescence signals and induced leftward shifts of ANP concentration-response curves. Similar results were obtained when using IBMX, trequinsin and dipyridamole, which inhibit PDE2A nonselectively with lower potency. PDP, the most potent PDE2A inhibitor known to date, was found to exhibit much lower cellular activity as anticipated from its biochemical PDE2A inhibitory activity. By cellular uptake and transport studies we could show that PDP's cell permeability is low and that the compound is a substrate for an efflux transporter. Other PDE inhibitors including vinpocetine, milrinone, rolipram, sildenafil, zaprinast, BRL 50481 and BAY 73-6691 did not stimulate luminescence signals on our PDE2A reporter cell line. The results imply that this novel PDE2A reporter assay provides an efficient, high throughput means for the identification and characterization of PDE2A inhibitors.

Characterization of substrates and inhibitors for the in vitro assessment of Bcrp mediated drug-drug interactions.

PURPOSE: In vitro assessment of drug candidates' affinity for multi-drug resistance proteins is of crucial importance for the prediction of in vivo pharmacokinetics and drug-drug interactions. To have well described experimental tools at hand, the objective of the study was to characterize substrates and inhibitors of Breast Cancer Resistance Protein (BCRP) and P-glycoprotein (P-gp). METHODS: Madin-Darbin canine kidney cells overexpressing mouse Bcrp (MDCKII-Bcrp) were incubated with various Bcrp substrates, or a mixture of substrate and inhibitor to either the apical (A) or basolateral (B) compartment of insert filter plates. Substrate concentrations in both compartments at time points t = 0 h and t = 2 h were determined by LC-MS/MS, and respective permeation coefficients (Papp) and efflux ratios were calculated. RESULTS: The Bcrp inhibitor Ko143 blocked topotecan and ABZSO transport in a concentration-dependent manner. P-gp inhibitors ivermectin, LY335979, PSC833, and the P-gp/Bcrp inhibitor ritonavir did not influence Bcrp mediated topotecan transport, however, blocked ABZSO transport. Additionally, neither was ABZSO transport influenced by topotecan, nor topotecan transport by ABZSO. CONCLUSIONS: Data suggest different modes of substrate and inhibitor binding to Bcrp. In order to not overlook potential drug-drug interactions when testing drug candidates for inhibitory potential towards Bcrp, distinct Bcrp probe substrates should be used.

Design and synthesis of potent and selective azaindole-based Rho kinase (ROCK) inhibitors.

Rho kinase plays a pivotal role in several cellular processes such as vasoregulation, making it a suitable target for the treatment of hypertension and related disorders. We discovered a new compound class of Rho kinase (ROCK) inhibitors containing a 7-azaindole hinge-binding scaffold tethered to an aminopyrimidine core. Herein we describe the structure-activity relationships elucidated through biochemical and functional assays. The introduction of suitable substituents at the 3-position of the bicyclic moiety led to an increase in activity, which was required to design compounds with favorable pharmacokinetic profile. Azaindole 32 was identified as a highly selective and orally available ROCK inhibitor able to cause a sustained blood pressure reduction in vivo.