The aim of the study was to compare the pharmacokinetics (PK) and pharmacodynamics (PD) of T-glu (GP40321, test drug), and reference insulin glulisine in a hyperinsulinemic-euglycemic clamp procedure. During this study, 34 healthy male volunteers underwent the hyperinsulinemic-euglycemic clamp procedure following subcutaneous 0.3 U/kg injection of T-glu or reference insulin glulisine in a randomized, double-blind, crossover study. Plasma glucose levels were monitored every 5 minutes for 8 hours. Glucose infusion rate adjustment was based on the blood glucose measurements. Evaluation of PD was performed using the glucose infusion rate values, while PK was calculated using insulin concentrations measured via enzyme-linked immunosorbent assay. The study results showed that the 90% CI for the geometric mean ratios of primary PK and PD of T-glu and reference insulin glulisine were within 80%-125% comparability limits, and that the safety profiles were comparable. PK, PD, and safety similarity of T-glu and reference insulin glulisine was demonstrated.

Parkinson's disease is the second most common neurodegenerative pathology. Due to the limitations of existing therapeutic approaches, novel anti-parkinsonian medicines with non-dopamine mechanisms of action are clearly needed. One of the promising pharmacological targets for anti-Parkinson drug development is phosphodiesterase (PDE) 10A. The stimulating motor effects of PDE10A inhibition were detected only under the conditions of partial dopamine depletion. The results raise the question of whether PDE10A inhibitors are able to restore locomotor activity when dopamine levels are very low. To address this issue, we (1) developed and validated the rat model of acute severe dopamine deficiency and (2) tested the action of PDE10A inhibitor MP-10 in this model. All experiments were performed in dopamine transporter knockout (DAT-KO) rats. A tyrosine hydroxylase inhibitor, α-Methyl-DL-tyrosine (αMPT), was used as an agent to cause extreme dopamine deficiency. In vivo tests included estimation of locomotor activity and catalepsy levels in the bar test. Additionally, we evaluated the tissue content of dopamine in brain samples by HPLC analysis. The acute administration of αMPT to DAT-KO rats caused severe depletion of dopamine, immobility, and catalepsy (Dopamine-Deficient DAT-KO (DDD) rats). As expected, treatment with the L-DOPA and carbidopa combination restored the motor functions of DDD rats. Strikingly, administration of MP-10 also fully reversed immobility and catalepsy in DDD rats. According to neurochemical studies, the action of MP-10, in contrast to L-DOPA + carbidopa, seems to be dopamine-independent. These observations indicate that targeting PDE10A may represent a new promising approach in the development of non-dopamine therapies for Parkinson's disease.

Abstract Aims The pharmacodynamic (PD) similarity between GP40141, a proposed romiplostim biosimilar, and reference romiplostim was evaluated. Pharmacokinetics and safety were also assessed. Methods In this phase 1, randomized, double‐blind, single‐dose, crossover comparative study with an adaptive design, 56 healthy male volunteers were randomized 1:1 to receive a 3 ug × kg−1 subcutaneous dose of GP40141 and reference romiplostim. The PD similarity between GP40141 and the reference romiplostim was determined using the standard equivalence criteria (80%–125%) for the area under the platelet count‐time curve from time 0 to the time of the last sampling for PD (AUCplt) and the maximum observed platelet count (Pmax). Results GP40141 and the reference romiplostim exhibited similar PD profiles. 90% CI for the geometric mean ratios for the primary PD parameters (AUCplt, Pmax) for GP40141 (T) and the reference romiplostim (R) were fully contained within the predefined equivalence limits of 80%–125%: 98.13%–102.42% for AUCplt and 97.56%–105.80% for Pmax. The pharmacokinetic profiles of GP40141 and the reference romiplostim were well described. No adverse events were observed during the clinical trial after the administration of GP40141 and the reference romiplostim. Conclusion This study demonstrates the PD similarity of GP40141 to the reference romiplostim. Both treatments had comparable safety profiles (NCT05652595)

The results of preclinical studies of romiplostim analogue GP40141 are presented. The effect of a cell proliferation, phosphorylation of the TPO receptor and JAK2 phosphorylation were studied in the presence of romiplostim and in the presence of GP40141 in a cell line of mice (Mus musculus) lymphoblasts with stable expression of human TPO receptor 32D-hTPOR clone 63. Binding to the TPO receptor and to the neonatal Fc receptor (FcRn) was examined for both romiplostim and the developed analogue. In Sprague-Dawley rats, the dynamics of platelet count after the administration of romiplostim or GP40141 were determined. The pharmacokinetics of romiplostim and GP40141, as well as the dynamics of platelet count, were studied in cynomolgus monkeys. The serum concentrations of romiplostim were determined using a modified colorimetric enzyme-linked immunosorbent assay (ELISA). The data obtained allow us to assert the similarity of the biological action of Nplate® and GP40141.

Biphasic insulin aspart 30 is a premixed formulation containing a soluble fraction of insulin aspart (30%) and a protamine‐crystallized fraction (70%) that was developed to combine the rapid‐acting and prolonged advantages of commercially available insulins. The aim of this bioequivalence study was to compare the pharmacokinetics (PKs) of GP‐bi‐asp and Novo‐bi‐asp, and evaluate the pharmacodynamic (PD) properties as well as the safety of these drugs in the hyperinsulinemic euglycemic clamp (HEC) procedure. This was a phase 1, randomized, double‐blind, 2‐sequence, 2‐period crossover study. Thirty‐four male volunteers who met the inclusion criteria underwent the HEC procedure following a single subcutaneous injection of 0.4 IU/kg of either GP‐bi‐asp or Novo‐bi‐asp in the abdomen. After the treatment, the subjects’ plasma glucose levels were monitored for 24 hours and the glucose infusion rate (GIR) was adjusted to maintain the target blood glucose level. The PD parameters were calculated using GIR values. Insulin aspart concentrations were measured in blood plasma using validated ELISA assays to evaluate the PK parameters of the investigated drugs. The 90% confidence intervals for the geometric mean ratios of PK (C ins and AUC ins–T ) parameters of Gp‐bi‐asp and Novo‐bi‐asp were close to 100% and within the 80%‐125% limits for establishing bioequivalence. The safety profiles of both drugs were also comparable.

Insulin aspart is a short-acting insulin analogue that is used to control postprandial glycemia levels in diabetic patients. The aim of this clinical trial was to compare the pharmacokinetics and pharmacodynamics of GP40071 (GP-Asp) and NovoRapid Penfill (Novo-Asp) in a hyperinsulinemic euglycemic clamp (HEC). This trial was conducted as a part of a GP40071 biosimilar clinical development program. This was a phase I randomized, double-blind, two-period crossover study. Twenty-six healthy male volunteers aged 18 to 45 years who met the inclusion criteria underwent the procedure of an HEC following a single subcutaneous injection of 0.3 IU/kg of either GP-Asp or Novo-Asp into the abdomen. After doses, plasma glucose levels were monitored every 5 minutes for 8 hours. The adjustment of the glucose infusion rate (GIR) was based on the blood glucose measurements. The GIR values were used to evaluate the PD profiles of the studied drugs. Regular blood sampling was performed during the study to obtain sufficient pharmacokinetic data. The 90% confidence intervals for the geometric mean ratios of the pharmacokinetic (AUC(ins.0-t), C-ins.max) and pharmacodynamic (GIR(max), AUC(GIR0-t)) parameters of GP-Asp were within the 80%-125% comparability limits. The safety profiles of the drugs were also comparable. Bioequivalence, similar PD, and safety of GP-Asp and Novo-Asp were demonstrated.

This paper presents an analysis of data from a comparative study of biosimilarity in terms of pharmacokinetics and pharmacodynamics in healthy volunteers using a hyperinsulinemic euglycemic clamp for reference and test biphasic insulin aspart 30 (BIAsp 30). As a result of the study, one of the secondary pharmacodynamic (PD) endpoints did not satisfy the classical criterion of 80%-125% (the lower limit for PD parameter area under the glucose infusion rate-time curve [ AUC GIR 0 - t ${\rm{AUC}}_{{\rm{GIR}}_{0 - {\rm{t}}}}$ ] turned out to be 79.5%). The main hypothesis explaining this result is that the sample size is insufficient to conduct a PD test with 90% statistical power, since the sample size has been calculated based on the coefficient of variation (CV) of pharmacokinetic (PK) parameters. To test this hypothesis, population PKPD (popPKPD) modeling and subsequent simulations of the required number of PD profiles were used. Two popPKPD models were constructed (a one-compartment double simultaneous absorption model for PK and an effect compartment Emax model for PD) to describe the PKPD data of reference and test insulins. As a result, using real data along with model-based simulation data, a biosimilarity test for PD was performed, and the lower limit for AUC GIR 0 - t ${\rm{AUC}}_{{\rm{GIR}}_{0 - {\rm{t}}}}$ became 82.6%, while the CV decreased from 31.7% to 24.1%. Thus, popPKPD modeling and simulations have been shown to be effective in interpreting and supporting the results of clinical biosimilarity trials.

Background and Purpose: Phosphodiesterases (PDEs) are a family of enzymes, which hydrolyze cAMP and cGMP. PDE10A is expressed mainly in the medium spiny neurons of the striatum that provides an opportunity to modulate the movement control pathways of the basal ganglia: “direct” (D1 receptor-dependent) and “indirect” (D2 receptor-dependent). Thus, inhibition of PDE10A can functionally mimic the action of both D1 receptor agonists and D2 receptor antagonists, although much less attention has been paid to the assessment of D1 receptor agonist-like effects. The purpose of the present study was (1) to confirm the motor stimulatory effects of PDE10A inhibitors and (2) to test whether these effects are subject to the development of tolerance. Experimental Approach: The ability of single or repeated (5 or 10 days) administration of selective PDE10A inhibitors, MP-10 (0,3-5 mg/kg) and RO5545965 (0.1-0.9 mg/kg), to stimulate locomotor activity was assessed in rats after single tetrabenazine challenge (3 mg/kg). The study was pre-registered on PreclinicalTrials.eu. Key Results: PDE-10A inhibition exerted paradoxical motor stimulatory properties in a dose-dependent manner. However, repeated administration of PDE10A inhibitors led to a reduction of their effects. Conclusion and Implications: PDE-10A inhibition produces a paradoxical increase in motor activity in animals with low dopamine tone. After repeated administration of PDE-10A inhibitors, these effects disappeared. The development of tolerance similar to that previously observed for D1-receptor agonists may limit the potential clinical use of the stimulatory effects of PDE10A inhibitors. Further studies aimed at analyzing the molecular mechanisms of this tolerance are warrant

It is important to understand the features of the interaction of drug components with body receptors and obtain data on its distribution in various administration routes in recommended doses in order for its usage in clinical practice to be safe and effective.PURPOSE:To investigate in vitro the interactions of a drug consisting of water-soluble polypeptide fractions produced on animal retina with a wide range of receptor targets, and to assess its biodistribution in the organs of laboratory animals.MATERIAL AND METHODS:The biodistribution of the radioactively marked drug in different organs and tissues of laboratory mice in various routes of administration was studied at the National Research Centre «Kurchatov Institute». Evaluation of the ligand-receptor interaction of the drug was carried out in the laboratory at Eurofins Pharma Discovery Services by the method of competitive radioligand binding.RESULTS:A significant effect of the interaction of the polypeptide drug was revealed with different subtypes of glutamate receptors: AMPA, NMDA, and mGluR1. As a result of an in vivo test, we have obtained biodistribution data of the drug for intravenous, intramuscular and parabulbar administration, and the dynamics of drug accumulation in the tissues of the brain and eyes.CONCLUSION:According to the study results, the peptide drug binds to receptors associated with the loss of retinal ganglion cells. Interaction with these receptors potentially provides the test subject with neuroprotective effect. The content dynamics of the studied drug in the blood of animals depends on the route of administration and the amount of drug administered. At the time point of 0.5 hours for intravenous and intramuscular administration in the dose of 1.7 mg/kg, the studied drug has sufficiently high bioavailability in the tissues of the brain and eye. The data suggest that the main route of excretion of the studied drug is through kidneys.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

This study was the first to compare the neuroprotective activity of Cerebrolysin®, Actovegin® and Cortexin® in rodent models of acute and chronic brain ischemia. The neuroprotective action was evaluated in animals with acute (middle cerebral artery occlusion) or chronic (common carotid artery stenosis) brain ischemia models in male rats. Cortexin® (1 or 3 mg/kg/day), Cerebrolysin® (538 or 1614 mg/kg/day) and Actovegin® (200 mg/kg/day) were administered for 10 days. To assess the neurological and motor impairments, open field test, adhesive removal test, rotarod performance test and Morris water maze test were performed. Brain damage was assessed macro- and microscopically, and antioxidant system activity was measured in brain homogenates. In separate experiments in vitro binding of Cortexin® to a wide panel of receptors was assessed, and blood-brain barrier permeability of Cortexin® was assessed in mice in vivo. Cortexin® or Cerebrolysin® and, to a lesser extent, Actovegin® improved the recovery of neurological functions, reduced the severity of sensorimotor and cognitive impairments in rats. Cortexin® reduced the size of necrosis of brain tissue in acute ischemia, improved functioning of the antioxidant system and prevented the development of severe neurodegenerative changes in chronic ischemia model. Radioactively labeled Cortexin® crossed the blood-brain barrier in mice in vivo with concentrations equal to 6-8% of concentrations found in whole blood. During in vitro binding assay Cortexin® (10 μg/ml) demonstrated high or moderate binding to AMPA-receptors (80.1%), kainate receptors (73.5%), mGluR1 (49.0%), GABAA1 (44.0%) and mGluR5 (39.7%), which means that effects observed in vivo could be related on the glutamatergic and GABAergic actions of Cortexin®. Thus, Cortexin, 1 or 3 mg/kg, or Cerebrolysin®, 538 or 1614 mg/kg, were effective in models acute and chronic brain ischemia in rats. Cortexin® contains compounds acting on AMPA, kainate, mGluR1, GABAA1 and mGluR5 receptors in vitro, and readily crosses the blood-brain barrier in mice.

OBJECTIVE:To compare the effects of cortexin, cerebrolysin and actovegin on memory impairment, cerebral circulation and morphological changes in the hippocampus of rats with chronic brain ischemia.MATERIAL AND METHODS:The study was conducted using male rats with chronic brain ischemia caused by stenosis of the common carotid arteries by 50%. Animals received cortexin (0,3; 1 or 3 mg/kg), cerebrolysin (0,8; 2,5 or 7,5 ml/kg) and actovegin (5 ml/kg) in two 10-day courses with 10 days of treatment break. The severity of cognitive impairment was evaluated using the Morris water maze, passive and active avoidance tests. Cerebral circulation using laser flowmetry and brain hippocampus structures were studied in the end of treatment.RESULTS:Cognitive impairment in animals with chronic brain ischemia was accompanied by the development of pathological changes in the CA1 and CA4 regions of the hippocampus. Administration of cortexin (1 and 3 mg/kg) and cerebrolysin (2.5 and 7.5 ml/kg) to rats with chronic brain ischemia had almost no effect on cerebral blood flow, but contributed to the improvement in memory formation and retrieval processes in the Morris water maze. The treatment effect was comparable for both drugs and persisted after 10 days of treatment break. Morphological assessment showed a decrease in the severity of pathological changes in the hippocampal regions.CONCLUSION:The course-administration of cortexin and cerebrolysin lead to a decrease in the severity of memory impairment and pathomorphological changes in the hippocampus in rats with chronic brain ischemia.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Trace amine-associated receptor 1 (TAAR1) is a widely recognized new perspective target for the neuropsychiatric pharmacological treatment. Despite a growing number of studies investigating TAAR1 role in the animal models of different pathologies, information of TAAR1 agonists impact on executive cognitive functions is limited. The goal of the present study was to evaluate the activity of highly selective partial TAAR1 agonist RO5263397 on various executive cognitive functions. The results of the present study demonstrated that the pretreatment with RO5263397 was able to increase attention and decrease cognitive flexibility in rats. The analysis of the RO5263397 action on impulsivity demonstrated that the TAAR1 activation failed to affect premature responding but was able to slightly modify impulsive choice. Problem solving was resistant to the pharmacological intervention.

Genetic and physiological studies have implicated the striatum in bipolar disorder (BD). Although Glycogen synthase kinase 3 beta (GSK3β) has been suggested to play a role in the pathophysiology of BD since it is inhibited by lithium, it remains unknown how GSK3β activity might be involved. Therefore we examined the functional roles of GSK3β and one of its substrates, CRMP2, within the striatum.Using CRISPR-Cas9 system, we specifically ablated GSK3β in the striatal neurons in vivo and in vitro. Sholl analysis was performed for the structural studies of medium spiny neurons (MSNs) and amphetamine-induced hyperlocomotion was measured to investigate the effects of gene ablations on the mania-like symptom of BD.GSK3β deficiency in cultured neurons and in neurons of adult mouse brain caused opposite patterns of neurite changes. Furthermore, specific knockout of GSK3β in the MSNs of the indirect pathway significantly suppressed amphetamine-induced hyperlocomotion. We demonstrated that these phenotypes of GSK3β ablation were mediated by CRMP2, a major substrate of GSK3β.Amphetamine-induced hyperlocomotion only partially recapitulate the symptoms of BD. It requires further study to examine whether abnormality in GSK3β or CRMP2 is also involved in depression phase of BD. Additionally, we could not confirm whether the behavioral changes observed in GSK3β-ablated mice were indeed caused by the cellular structural changes observed in the striatal neurons.Our results demonstrate that GSK3β and its substrate CRMP2 critically regulate the neurite structure of MSNs and their functions specifically within the indirect pathway of the basal ganglia network play a critical role in manifesting mania-like behavior of BD. Moreover, our data also suggest lithium may exert its effect on BD through a GSK3β-independent mechanism, in addition to the GSK3β inhibition-mediated mechanism.

Trace amine-associated receptor 1 (TAAR1) has emerged as a promising target for addiction treatments because it affects dopamine transmission in the mesolimbic pathway. TAAR1 is involved in the effects of addictive drugs, such as amphetamines, cocaine and ethanol, but the impact of TAAR1 on the effects of nicotine, the psychoactive drug responsible for the development and maintenance of tobacco smoking, has not yet been studied. This study was performed to investigate the possible modulatory action of TAAR1 on the effects of nicotine on locomotor behaviors in rats and mice. Pretreatment with the TAAR1 agonist RO5263397 dose-dependently decreased nicotine-induced hyperlocomotion in rats habituated to locomotor boxes, prevented the development of nicotine sensitization and blocked hypermotility in nicotine-sensitized rats at the highest tested dose (10 mg/kg). The lack of TAAR1 failed to affect the effects of nicotine on the locomotion of mutant mice. Based on the results of the present study, TAAR1 activation attenuates the locomotion-stimulating effects of nicotine on rats. These results further support the previously proposed hypothesis that TAAR1 is a promising target for the prevention and treatment of drug addiction. Further studies aimed at analyzing the effects of TAAR1 agonists on animal models of nicotine addiction are warranted.

Trace Amine Associated Receptor 1 (TAAR1) is a novel pharmacological target. TAAR1 are well-documented to play a modulatory role in the dopaminergic system. In spite of a growing number of studies of TAAR1 effects, little is still known about the behavioral pharmacology of TAAR1 ligands, including effects of repeated TAAR1 agonist administration. The present study appears to be the first that estimated the action of TAAR1 agonists on schedule-induced polydipsia, a type of adjunctive behavior, which is considered to be useful for evaluating certain aspects of obsessive-compulsive and related disorders (OCD) and schizophrenia. Our results have demonstrated that the wide range of RO5263397, the highly selective partial TAAR1 agonist, doses (1-10 mg/kg) attenuated the polydipsia induced by two different schedules of food delivery in rats. The effect remained unchanged for the 7 days of repeated treatment. However, the highest tested doses of RO5263397 (6 and 10 mg/kg) decreased the vertical locomotor activity of the animals and the volume of water intake of thirsty rats following the acute treatment. Also, though, the repeated RO5263397 administration is exhibited to diminish the volume of consumed water and weight of rats without SIP, on the other hand, the tolerance was observed to these drug effects. In general, the RO5263397 decreases specifically the adjunctive drinking and this effect is maintained with repeated drug administration without the development of tolerance. The interpretation of these results as an evidence for the RO5263397 anticompulsive-like action, however, should be taken with caution because the drug also influenced the drinking behavior and only weakly affected the other parameters of SIP used to reveal the potential anticompulsive-like effects of drugs.

In clinical practice the treatment of psychiatric diseases usually requires long-term drug administration, but the effects of many psychiatric medications decrease upon repeated exposure, a phenomenon described as ‘tolerance’ [1]. Our previous study has shown that acute administration of partial Trace Amine-Associated Receptor 1 (TAAR1) agonist (RO5263397) attenuated adjunctive water drinking in schedule-induced polydipsia (SIP) [2], a successful model of compulsive behavior [3]. The present study was aimed to evaluate the anticompulsive activity of RO5263397 following repeated administration and analyze RO5263397 effects on the levels of monoamines and their metabolites in the striatum and frontal cortex. To induce SIP, 16 Wistar rats were habituated to operant chambers and pellet feeding to fixed-time 60-second (FT-60 sec) schedule of food-pellet reinforcement. After the acquisition of stable adjunctive drinking behaviour, the animals were divided into two equal subgroups. The different subgroups were injected with either 12 mg/kg of RO5263397 or vehicle every 12 hours (9:00 and 21:00) during 7 consecutive days. The drug dose and the schedule of injections were chosen in concordance with a previously published information about pharmacokinetic of RO5263397 [4]. Thus, the serum concentration of RO5263397 was higher than 0.67 μg/ml (maximal concentration after i.p. administration of RO5263397 in dose 3 mg/kg which decreased SIP in our previous experiments [2]) for 14 hours in a day. To analyze monoamines tissue content high-performance liquid chromatography (HPLC) was used. HPLC measurements were carried out as described before [5]. Striatum and frontal cortex tissues were dissected from the rats subchronically treated with RO5263397 and levels of norepinephrine, dopamine, serotonin and their metabolites were evaluated. Before the start of the treatment there was no difference between the subgroups in water consumption in home cages and operant boxes (U-tests, Ps>0.05). Two-way repeated measures ANOVA showed that the treatment with RO5263397 significantly reduced SIP (F(1.13)=17.865, P<0.001), but the repeated administration did not decrease this effect (F(3.32)=1.023, P=0.39). Bonferroni’s post hoc test revealed that the pretreatment with RO5263397 attenuated SIP in all days of the treatment (Ps<0.05). There were no effects of the treatment with RO5263397 to water drinking in home cages (F(1.14)=0.539, P=0.48). At the same time, repeated treatment with RO5263397 significantly decreased dopamine metabolism and increased norepinephrine and serotonin levels in the striatum and prefrontal cortex (with exception of norepinephrine) (t- and U-tests, Ps<0.05). Thus, these findings indicate that the repeated administration did not influence SIP ameliorating effect of RO5263397 and further support previously proposed view that TAAR1 is promising target for the treatment of disorders accompanied by compulsive behavior. Moreover, HPLC results suggest that the mechanisms of the TAAR1 agonist anticompulsive activity might involve the modulation of not only dopamine but also other catecholamine systems. Certainly, additional studies aimed at analysis of neurochemical profile of activity of TAAR1 agonists are warranted.

Liver iron deposits are frequent in viral chronic liver disease. Expression of Ki-67 is strongly associated with tumor cell proliferation and growth. Metallothioneins (MTs) play a protective role via scavenging of reactive oxygen species and participate in cell proliferation and apoptosis. The current study investigated the relations between hepatic iron load, Ki-67 and MT in chronic hepatitis C (CHC) and hepatocellular carcinoma (HCC).Prussian blue stain and immunohistochemical staining for Ki-67 and MT antibodies were applied on 80 core liver biopsies and specimens (45 with CHC, 15 with cirrhosis on top of CHC and 20 with HCC on top of CHC) and 5 normal controls.Significant higher iron overload and Ki-67 expression were detected in HCC and cirrhosis compared to CHC; higher CHC stages of fibrosis and grades of necro-inflammation compared to lower ones; and higher grades of HCC compared to lower grades. Insignificant higher MT expression was detected in CHC and cirrhosis compared to HCC; higher CHC stages of fibrosis and grades of necro-inflammation compared to lower ones; and in lower grades of HCC compared to higher grades.Ki-67 expression associated with progression of CHC disease and prognosis of HCC, while MT was down-regulated in HCC. So both Ki-67 and MT can be used as prognostic and predictive markers in HCC and may be promising molecular candidate for diagnosis and treatment of HCC.