Assessment of the potential of the MET inhibitor tepotinib to affect the pharmacokinetics of CYP3A4 and P-gp substrates.

Tepotinib is a highly selective, potent, mesenchymal-epithelial transition factor (MET) inhibitor, approved for the treatment of non-small cell lung cancer harboring MET exon 14 skipping alterations. The aims of this work were to investigate the potential for drug-drug interactions via cytochrome P450 (CYP) 3A4/5 or P-glycoprotein (P-gp) inhibition. In vitro studies were conducted in human liver microsomes, human hepatocyte cultures and Caco-2 cell monolayers to investigate whether tepotinib or its major metabolite (MSC2571109A) inhibited or induced CYP3A4/5 or inhibited P-gp. Two clinical studies were conducted to investigate the effect of multiple dose tepotinib (500 mg once daily orally) on the single dose pharmacokinetics of a sensitive CYP3A4 substrate (midazolam 7.5 mg orally) and a P-gp substrate (dabigatran etexilate 75 mg orally) in healthy participants. Tepotinib and MSC2571109A showed little evidence of direct or time-dependent CYP3A4/5 inhibition (IC50 > 15 µM) in vitro, although MSC2571109A did show mechanism-based CYP3A4/5 inhibition. Tepotinib did not induce CYP3A4/5 activity in vitro, although both tepotinib and MSC2571109A increased CYP3A4 mRNA. In clinical studies, tepotinib had no effect on the pharmacokinetics of midazolam or its metabolite 1'-hydroxymidazolam. Tepotinib increased dabigatran maximum concentration and area under the curve extrapolated to infinity by 38% and 51%, respectively. These changes were not considered to be clinically relevant. Tepotinib was considered safe and well tolerated in both studies. The potential of tepotinib to cause clinically relevant DDI with CYP3A4- or P-gp-dependent drugs at the clinical dose is considered low. Study 1 (midazolam): NCT03628339 (registered 14 August 2018). Study 2 (dabigatran): NCT03492437 (registered 10 April 2018).

Cladribine as a Potential Object of Nucleoside Transporter-Based Drug Interactions.

Cladribine is a nucleoside analog that is phosphorylated in its target cells (B and T-lymphocytes) to its active triphosphate form (2-chlorodeoxyadenosine triphosphate). Cladribine tablets 10 mg (Mavenclad®), administered for up to 10 days per year in 2 consecutive years (3.5-mg/kg cumulative dose over 2 years), are used to treat patients with relapsing multiple sclerosis. Cladribine has been shown to be a substrate of various nucleoside transporters (NTs). Intestinal absorption and distribution of cladribine throughout the body appear to be essentially mediated by equilibrative NTs (ENTs) and concentrative NTs (CNTs), specifically by ENT1, ENT2, ENT4, CNT2 (low affinity), and CNT3. Other efficient transporters of cladribine are the ABC efflux transporters, specifically breast cancer resistance protein, which likely modulates the oral absorption and renal excretion of cladribine. A key transporter for the intracellular uptake of cladribine into B and T-lymphocytes is ENT1 with ancillary contributions of ENT2 and CNT2. Transporter-based drug interactions affecting absorption and target cellular uptake of a prodrug such as cladribine are likely to reduce systemic bioavailability and target cell exposure, thereby possibly hampering clinical efficacy. In order to manage optimized therapy, i.e., to ensure uncompromised target cell uptake to preserve the full therapeutic potential of cladribine, it is important that clinicians are aware of the existence of NT-inhibiting medicinal products, various lifestyle drugs, and food components. This article reviews the existing knowledge on inhibitors of NT, which may alter cladribine absorption, distribution, and uptake into target cells, thereby summarizing the existing knowledge on optimized methods of administration and concomitant drugs that should be avoided during cladribine treatment.

Review of Transporter Substrate, Inhibitor, and Inducer Characteristics of Cladribine.

Cladribine is a nucleoside analog that is phosphorylated in its target cells (B- and T-lymphocytes) to its active adenosine triphosphate form (2-chlorodeoxyadenosine triphosphate). Cladribine tablets 10 mg (Mavenclad®) administered for up to 10 days per year in 2 consecutive years (3.5-mg/kg cumulative dose over 2 years) are used to treat patients with relapsing multiple sclerosis. The ATP-binding cassette, solute carrier, and nucleoside transporter substrate, inhibitor, and inducer characteristics of cladribine are reviewed in this article. Available evidence suggests that the distribution of cladribine across biological membranes is facilitated by a number of uptake and efflux transporters. Among the key ATP-binding cassette efflux transporters, only breast cancer resistance protein has been shown to be an efficient transporter of cladribine, while P-glycoprotein does not transport cladribine well. Intestinal absorption, distribution throughout the body, and intracellular uptake of cladribine appear to be exclusively mediated by equilibrative and concentrative nucleoside transporters, specifically by ENT1, ENT2, ENT4, CNT2 (low affinity), and CNT3. Renal excretion of cladribine appears to be most likely driven by breast cancer resistance protein, ENT1, and P-glycoprotein. The latter may play a role despite its poor cladribine transport efficiency in view of the renal abundance of P-glycoprotein. There is no evidence that solute carrier uptake transporters such as organic anion transporting polypeptides, organic anion transporters, and organic cation transporters are involved in the transport of cladribine. Available in vitro studies examining the inhibitor characteristics of cladribine for a total of 13 major ATP-binding cassette, solute carrier, and CNT transporters indicate that in vivo inhibition of any of these transporters by cladribine is unlikely.

Phase 1 study in healthy participants of the safety, pharmacokinetics, and pharmacodynamics of enpatoran (M5049), a dual antagonist of toll-like receptors 7 and 8.

This study evaluated the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of single and multiple oral doses of enpatoran (formerly named M5049), a new toll-like receptor (TLR) 7 and 8 dual antagonist, and the effect of food on a single dose in healthy participants. In this single phase 1, randomized (3:1), double-blind, placebo-controlled study, 96 participants received single and multiple ascending oral doses of enpatoran. Participants in single-dose cohorts received one dose of enpatoran (1, 3, 9, 25, 50, 100, or 200 mg) or placebo using a sentinel dosing strategy. Multiple-dose cohorts received enpatoran (9, 25, or 200 mg once daily, or 25 or 50 mg twice daily) or placebo for 14 days. Safety, tolerability, PK, and PD (ex vivo-stimulated cytokine secretion) were assessed in both parts. The effect of food was assessed in an open-label, one-way crossover study in the 25 mg single-dose cohort. Single- and multiple-oral doses of enpatoran up to 200 mg were well tolerated and no significant dose-limiting adverse events or safety signals were observed under fasting or fed conditions. PK parameters were linear and dose-proportional across the dose range evaluated, with a slightly delayed absorption and lower peak concentration observed at 25 mg with food. Exposure-dependent inhibition of ex vivo-stimulated interleukin-6 secretion was observed, with maximum inhibition at 200 mg. Enpatoran was well tolerated at doses up to 200 mg. Further investigation of enpatoran is warranted as a potential treatment for diseases driven by TLR7/8 overactivation, such as systemic lupus erythematosus and COVID-19 pneumonia.

On functions of cholinesterases during embryonic development.

Expression of cholinesterase (ChE) activity during phases of embryonic development is a general phenomenon in embryonic tissues. To elucidate the role(s) of ChEs during embryonic development, one line of research followed the assumption of a primitive muscarinic system involved in morphogenesis (Hohmann et al., 1995). This means that ChE functioning during development fits into the classical cholinergic neurotransmitter system: acetylcholine (ACh), as a signal, binds to ACh receptors and then is degraded by acetylcholinesterase (AChE) as the terminating enzyme. However, this is just one of the possible mechanisms. The other line of research was driven by evidence for noncholinergic functions of ChE proteins (AChE and butyrylcholinesterase [BChE]). There is accumulating data that other sites on AChE could exert nonclassical roles related to cell differentiation, neurite outgrowth, and adhesion.

Lamina formation in the Mongolian gerbil retina (Meriones unguiculatus).

Retinae of nocturnal rodents, such as mice and rats, are almost exclusively rod-dominated. The gerbil, in contrast, shows active periods during day and night and uses both rod- and cone-based vision. However, its retina has not been studied in detail, except for one developmental study analysing its prenatal period (Wikler et al. 1989). Here, the formation of the laminar structure of the gerbil retina was studied from birth until late adult stages. At birth, the retina consisted of a wide neuroblastic layer, with 30% of cells still dividing, a rate decreasing to nearly zero by P6. Shortly after birth, segregation of a ganglion cell layer began. All retinal layers reached their final size around P20, as determined from DAPI-stained cryosections. Muller glial cells developed their typical structure from P1 onwards, e.g. announcing an outer plexiform layer (OPL) at P5, as analysed by the Ret-G7 and glutamine synthetase antibodies. The analyses of the inner retina were performed by antibodies to calretinin (CR) and calbindin (CB). CR is expressed in ganglion cells followed by amacrine cells from P1 onwards; their processes formed four subbands in the inner plexiform layer (IPL) and appeared sequentially after P5 until P20. CB stained a subtype of horizontal cells with their processes into the OPL from P14 onwards. The rod-specific antibody rho4D2 announced photoreceptors at P4, showing signs of outer segments from P10 onwards. The study shows that the formation of all retinal layers in the gerbil occurs postnatally. This and the fact that the gerbil retina is not exclusively rod-dominated could render the gerbil a valuable model for in vitro studies of retinogenesis in rodents.

Cell-by-cell reconstruction in reaggregates from neonatal gerbil retina begins from the inner retina and is promoted by retinal pigmented epithelium.

For future retinal tissue engineering, it is essential to understand formation of retinal tissue in a 'cell-by-cell' manner, as can be best studied in retinal reaggregates. In avians, complete laminar spheres can be produced, with ganglion cells internally and photoreceptors at the surface; a similar degree of retinal reconstruction has not been achieved for mammals. Here, we have studied self-organizing potencies of retinal cells from neonatal gerbil retinae to form histotypic spheroids up to 15 days in culture (R-spheres). Shortly after reaggregation, a first sign of tissue organization was detected by use of an amacrine cell (AC)-specific calretinin (CR) antibody. These cells sorted out into small clusters and sent unipolar processes towards the centre of each cluster. Thereby, inner cell-free spaces developed into inner plexiform layer (IPL)-like areas with extended parallel CR(+) fibres. Occasionally, IPL areas merged to combine an 'inner half retina', whereby ganglion cells (GCs) occupied the outer sphere surface. This tendency was much improved in the presence of supernatants from retinal pigmented cells (RPE-spheres), e.g. cell organization and proliferation was much increased, and cell death shortened. As shown by several markers, a perfect outer ring was formed by GCs and displaced ACs, followed by a distinct IPL and 1-2 rows of ACs internally. The inner core of RPE spheres consisted of horizontal and possibly bipolar cells, while immunostaining and RT-PCR analysis proved that photoreceptors were absent. This shows that (1) mammalian retinal histogenesis in reaggregates can be brought to a hitherto unknown high level, (2) retinal tissue self-organizes from the level of the IPL, and (3) RPE factors promote formation of almost complete retinal spheres, however, their polarity was opposite to that found in respective avian spheroids.

Impaired formation of the inner retina in an AChE knockout mouse results in degeneration of all photoreceptors.

Blinding diseases can be assigned predominantly to genetic defects of the photoreceptor/pigmented epithelium complex. As an alternative, we show here for an acetylcholinesterase (AChE) knockout mouse that photoreceptor degeneration follows an impaired development of the inner retina. During the first 15 postnatal days of the AChE-/- retina, three major calretinin sublaminae of the inner plexiform layer (IPL) are disturbed. Thereby, processes of amacrine and ganglion cells diffusely criss-cross throughout the IPL. In contrast, parvalbumin cells present a nonlaminar IPL pattern in the wild-type, but in the AChE-/- mouse their processes become structured within two 'novel' sublaminae. During this early period, photoreceptors become arranged regularly and at a normal rate in the AChE-/- retina. However, during the following 75 days, first their outer segments, and then the entire photoreceptor layer completely degenerate by apoptosis. Eventually, cells of the inner retina also undergo apoptosis. As butyrylcholinesterase (BChE) is present at a normal level in the AChE-/- mouse, the observed effects must be solely due to the missing AChE. These are the first in vivo findings to show a decisive role for AChE in the formation of the inner retinal network, which, when absent, ultimately results in photoreceptor degeneration.