Research Results in Pharmacology

From PDC to integral quantitative metrics: an innovative approach to assessing adherence to combination pharmacotherapy in outpatient practice

2026-02-18T19:01:34+03:00

Introduction: Current methods for assessing adherence to combination therapy based on aggregated measures (e.g., PDC, proportion of days covered) poorly capture treatment intensity and sustainability over time. We attempted to describe adherence trajectories for combination therapy using integral metrics. Aim: To develop a set of integral metrics analogous to key pharmacokinetic (PK) parameters – area under the concentration-time curve (AUC, area under the curve), maximum concentration (C_max), and time to maximum concentration (T_max) – to assess adherence to combination therapy as time-based exposure on the PDC methodology among outpatients.

Materials and Methods: As a specific example and a substrate for method development, we used results from a retrospective pharmacoepidemiologic cohort study of patients with heart failure (HF) who had experienced an acute myocardial infarction. Data were extracted from the Unified Medical Information and Analytical System of Moscow (Russia). Three patients with a follow-up duration of ≥24 months were randomly selected. We analyzed benefit-covered electronic prescription fills for key HF therapy classes and calculated adherence for each class using PDC across four half-year intervals. We constructed a stepwise trajectory of combination therapy as the function N(t), where N is the number of therapy classes for which the patient was covered at a given time point based on dispensed supply (accounting for days’ supply and overlaps), and t is follow-up time. By analogy with the “concentration-time” curve, we considered N(t) as a dynamic “therapy class coverage curve” and used it to calculate PK-analog adherence metrics: exposure to actually dispensed combination therapy, AUC_N(t)(class-months); normalized AUC_norm(0-1); C_maxN(t) (number of classes); and T_maxN(t) (months). In addition, we set a target threshold of N(t)≥3, determined the time to first attainment, T_optN(t), and calculated the time (or proportion of time) with N(t)≥k, denoted as T_N(t)≥k. We also quantified therapy “losses” between intervals based on a decrease in the modal value of N(t) and failure to reach the threshold over the follow-up period.

Results: AUC_N(t) values were 26.0, 87.37, and 36.67 class-months, and AUC_norm values were 0.27, 0.74, and 0.39 in patients with internal IDs 13, 39, and 110, respectively. The proportion of time with N(t)≥3 was 0.079, 0.96, and 0.23 (1.9, 22.9, and 5.5 months), and the modal N(t) values by half-year corresponded to patterns of 2→0, 3→3→4→4, and 3→2→1→0. The numeric profiles were consistent with the stepwise N(t) trajectories and reflected three distinct adherence patterns.

Conclusion: The proposed PK-associated approach enabled quantification of complex adherence trajectories for combination therapy in HF by representing them as an interpretable set of integral numeric metrics. These metrics characterize time-based exposure to combination therapy while accounting for adherence. The results support the conceptual validity of the method and may serve as a basis for further studies of its prognostic value.

Morphometric visualization, analysis, and assessment of atherosclerotic lesions in an apolipoprotein E–deficient mouse model (ApoE−/−)

2026-01-13T14:13:45+03:00

Introduction: The improvement of diagnostic methods in atherosclerosis is aimed at accurately determining the stage of the pathological process and eliminating subjective interpretation of data. Implementation of this approach increases the reliability of comparative assessment of the effectiveness of therapeutic and interventional strategies and reduces statistical variability within experimental studies.

Materials and Methods: The study was performed on 20 male ApoE−/− mice (40 weeks old) divided into groups receiving a standard diet or a Western diet. After anesthesia, PBS perfusion was performed; the heart with the aorta was excised and fixed in 10% formalin; the aorta was cleaned and stained with Oil Red O. The aorta was longitudinally opened, photographed (GelDoc), and quantitatively analyzed using a Python script. Data are presented as mean ± SD; statistical significance was assessed using Student’s t-test at p < 0.05.

Results and Discussion: Thus, the use of the Oil Red O staining method in combination with automated image analysis in Python enables a transition from subjective assessment to precise quantitative measurement of the area of atherosclerotic lesions. This approach increases the reliability of screening therapeutic interventions in experimental models and enhances the statistical power of the study.

Conclusion: In this study, an original approach to morphometric visualization, analysis, and quantitative assessment of atherosclerotic lesions of the vascular wall in an apolipoprotein E–deficient mouse model (ApoE−/−) is proposed and validated for the first time.

Evaluation of pharmacological correction of hypoxic-ischemic encephalopathy sequelae using peptide erythropoietin analogs in a mouse model of mild and moderate hypoxic-ischemic encephalopathy during the late remodeling phase

2026-01-10T09:33:36+03:00

Introduction: Hypoxic-ischemic encephalopathy (HIE) is an early-life brain injury that remains a leading cause of long-term neurodevelopmental deficits, including cerebral palsy, epilepsy, and cognitive-behavioral impairment. Because persistent neuroinflammation and progressive neurodegeneration contribute to delayed outcomes, targeting EPOR-CD131 with nonerythropoietic erythropoietin derivatives represents a promising strategy to modulate neurovascular unit function and limit secondary injury beyond the acute therapeutic window.

Materials and Methods: CD-1 mice underwent neonatal hypoxia–ischemia at postnatal day 9 (modified Rice–Vannucci) and were stratified 3 h later into mild or moderate injury using laser speckle imaging (RFLSI-ZW), then allocated to treatment groups. Starting on day 7 post-injury, EPO (5000 IU/kg) and Epobis (20 mg/kg) were administered subcutaneously once daily and Ara-290 (30 µg/kg) intraperitoneally twice daily for 7 days, after which exploratory behavior and caffeine-induced neurostimulation responses were evaluated.

Results: In this experimental study, Epobis and Ara-290 normalized mild HIE hyperlocomotion in the open field, whereas EPO mainly improved anxiety-like behavior and only Epobis increased peripheral time toward intact levels. In the caffeine challenge, all treatments reversed paradoxical suppression in mild HIE, and Epobis most effectively reduced caffeine-induced hyperreactivity and restored baseline activity in moderate HIE.

Conclusion: Epobis and Ara-290 normalized mild HIE hyperlocomotion in the open field, whereas Epobis provided the most comprehensive behavioral correction and EPO mainly improved anxiety-related measures. All treatments restored a physiological caffeine response in mild HIE, and Epobis most effectively improved behavior and reduced caffeine-induced hyperreactivity in moderate HIE, supporting further preclinical and subsequent clinical evaluation.

Evaluation of the neuroprotective effects of synthetic erythropoietin derivatives in a mouse model of mild and moderate hypoxic-ischemic encephalopathy

2025-12-24T22:08:17+03:00

Introduction: Hypoxic-ischemic encephalopathy (HIE) is a leading cause of neonatal brain injury and long-term neurodevelopmental impairment, and current therapies only partially prevent adverse outcomes. Although recombinanterythropoietin is neuroprotective via EPOR–CD131 (βcR), its use is limited by hematopoietic side effects, motivating evaluation of non-hematopoietic peptide analogs such as Ara-290 and Epobis in HIE.

Materials and Methods: Neonatal hypoxia-ischemia was induced in 9-day-old CD-1 mice (n = 204) using a modified Rice–Vannucci model, and animals were stratified into mild and moderate severity groups 3 hours later by laser speckle contrast imaging (RFLSI-ZW) before assignment to treatment. EPO (5000 IU/kg) and Epobis (20 mg/kg) were administered subcutaneously every 24 hours and Ara-290 (30 µg/kg) intraperitoneally every 12 hours for 7 days, after which motor-coordination performance, macroscopic lesion volume, and expression of IL-4, IL-1b, IL-6, and TNF-α were assessed.

Results: In both mild and moderate HIE, Epobis provided the most consistent neuroprotection, improving motor performance and neurological outcomes and being the only treatment to significantly reduce macroscopic lesion volume. EPO produced moderate functional benefits, whereas Ara-290 showed a less stable efficacy profile, with limited or absent effects in several behavioral and morphological endpoints.

Conclusion: In mild HIE, Epobis showed the strongest neuroprotection, improving motor-coordination performance, reducing neurological deficits, and producing the greatest reduction in macroscopic lesion volume, whereas EPO and Ara-290 had only moderate effects. In moderate HIE, Epobis and EPO most consistently improved motor outcomes, but only Epobis significantly reduced lesion volume and neurological symptom severity and produced the most pronounced immunomodulation.

Correction by neuroglutam and succicard of age-associated impairments in accelerated aging in rats with diabetes

2025-12-14T17:23:19+03:00

Introduction: The study aimed to investigate the impact of a long-term diabetic state on the development of accelerated aging and to evaluate the efficacy of therapy with metformin, neuroglutam, and succicard in correcting associated metabolic, behavioral, and molecular impairments.

Materials and Methods: A diabetes model was induced in rats by combined administration of streptozotocin (50 mg/kg) and nicotinamide (110 mg/kg). The total number of animals involved in the experiment was 50 individuals (25 males, 25 females). The animals were divided into 5 groups: intact, control (diabetic), and groups receiving a 4-week course of metformin (400 mg/kg), neuroglutam (26 mg/kg), or succicard (50 mg/kg). A comprehensive assessment was performed, including behavioral tests (open field, elevated cross maze, novel object recognition, forced swimming with a load, rotarod), metabolic status (oral glucose tolerance test, OGTT), and Klotho protein levels (ELISA).

Results: Diabetes induced anxiety-like behavior, cognitive decline, impaired motor coordination, and a significant decrease in Klotho protein levels. Therapy with metformin and succicardsignificantly increased Klotho protein levels and improved metabolic parameters. Neuroglutam exhibited pronounced nootropic and antidepressant-like effects. All drugs demonstrated differential effects on various aspects of behavioral activity.

Conclusion: The study demonstrates the potential of using metformin (400 mg/kg), neuroglutam (26 mg/kg), and succicard (50 mg/kg) to correct the manifestations of accelerated aging in diabetic rats with daily administration for 4 weeks. Metformin and succicard showed the most pronounced geroprotective potential. The results justify further investigation of combination therapy for correcting age-associated impairments.

The C3435T genetic polymorphism of the ABCB1 (MDR1) gene as a predictor of antihypertensive efficacy of losartan monotherapy in newly diagnosed arterial hypertension

2025-11-27T12:43:40+03:00

Introduction: Losartan, a selective angiotensin II receptor type 1 antagonist, is transported across cell membrane mediated by P-glycoprotein, the product of the ABCB1 (also known as MDR1) gene. The level of expression of this transporter protein is characterized by significant individual variability, caused by genetic factors. Well-known polymorphisms C3435T and C1236T in the ABCB1 gene potentially could affect a functional activity of the P-glycoprotein, thus modulating the pharmacokinetic parameters and clinical efficacy of the substrates of this transporter, including losartan. Therefore, the aim of this research is to indicate the correlation between common SNPs C3435T and C1236T of the MDR1 gene and the efficacy of 6-week losartan monotherapy course in patients with a newly diagnosed arterial hypertension (AH).

Materials and Methods: The study included 34 patients (70.6% women, mean age 48.3 ± 7.4 years). All participants were given losartan 100 mg/day for 6 weeks. Genotyping for C3435T and C1236T polymorphisms was made by using allele-specific PCR with electrophoretic detection. The efficacy of the therapy was evaluated by the reduction of systolic (SBP) and diastolic (DBP) blood pressure from the reference level.

Results: Patients with CT or TT genotypes for the C3435T polymorphism showed a statistically meaningful, greater reduction in SBP (11.8% ± 9.7) compared to homozygous CC genotype (”wild” type; 6.7% ± 9.6; p=0.03). No significant differences were found in SBP reduction for the C1236T polymorphism (p=0.07). Changes in DBP did not correlate with either of the studied polymorphisms.

Conclusions: The C3435T genetic polymorphism of the MDR1 gene is a potential predictor of the efficacy of losartan antihypertensive therapy. The carriers of the T allele (CT/TT genotypes) demonstrate more expressed hypotensive response, which may be caused by modulation of tissue distribution of the drug or its interaction with endogenous systems (ouabain) regulating blood pressure. The obtained data highlights the importance of a pharmacogenetic approach for personalizing AH treatment.

The effect of the azolo-triazine derivative AB-19 on the development of diabetic cardiomyopathy in rats

2025-11-13T13:10:27+03:00

Introduction: Cardiovascular diseases, including cardiomyopathies associated with endothelial dysfunction and impaired protein glycation, are the predominant cause of mortality in diabetes mellitus. This article presents data on the identification of functional and structural changes occurring during the development of diabetic cardiomyopathy and investigates the cardioprotective effects of the anti-glycating agents – the azolo-triazine derivative AB-19 and aminoguanidine.

Materials and Methods: Diabetic cardiomyopathy was modeled using streptozotocin 45 mg/kg I.V. on 60 male Sprague-Dawley rats. Aminoguanidine was investigated at the dose of 50 mg/kg and compound AB-19 – 20 mg/kg once daily. Observations were conducted over a period of 12 weeks. Blood glucose levels and glycated hemoglobin concentration were monitored. Upon completion of the diabetic cardiomyopathy induction period, the following were studied: endothelioprotective properties, cardiac contractile activity, solubility of tail tendon collagen and morphological examinations of the heart and myocardial blood vessels.

Results: The oral administration of AB-19 (20 mg/kg) and aminoguanidine (50 mg/kg) to animals with experimental diabetes mellitus resulted in a 17% reduction in blood HbA1c levels compared to that in the diabetic control rats. This treatment also limited the increase in AGEs in the blood by 51% and 39%, improved the solubility of collagen by 42% and 51%, respectively, restored endothelium-dependent vascular reactivity, and attenuated manifestations of left ventricular diastolic dysfunction associated with myocardial hypertrophy and fibrosis in diabetic animals. These findings were morphologically corroborated: animals treated with AB-19 and aminoguanidine exhibited a reduction in perivascular connective tissue, a decrease in collagen fibers in the myocardium, and a lower expression of AGEs and RAGE in IHC analysis using primary antibodies against AGEs and RAGE compared to the diabetic control group.

Conclusion: Compound AB-19 (20 mg/kg once daily) attenuates functional and structural manifestations of diabetic cardiomyopathy.

Effect of a new acetylaminohexanic acid derivative on wound healing and wound blood supply in mice with a mutation in the Agouti gene with inhibition of NOS isoform expression

2025-11-11T12:00:41+03:00

Introduction: Metabolic disorders, particularly diabetes mellitus, can lead to the formation of chronic wounds, slowing healing processes. A key factor impeding wound healing is the decreased production of endogenous nitric oxide (NO) due to a hyperglycemic microenvironment. Nitric oxide (NO) is a key signaling molecule produced by three different isoforms of nitric oxide synthase (NOS) – iNOS, eNOS, and nNOS – which regulate all phases of skin wound healing.

Materials and Methods: The effect of N-acetyl-6-aminohexanoic acid derivative in the form of a 5% carbopol-based hydrogel dosage form (carbopol 940) with 2-ethyl-6-methyl-3-hydroxypyridinium N-acetyl-6-aminohexanoate (LHT 2-18) on wound healing was studied during blockade of eNOS and iNOS nitric oxide synthase synthesis by intraperitoneal administration of a non-selective NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) at a dose of 25 mg/kg/day and aminoguanidine at a dose of 50 mg/kg/day, respectively. The studies were conducted on 30 Agouti viable yellow (Avy/a) mice weighing 42.9±0.12 g, which indicates the development of obesity. The degree of wound healing and wound blood flow were assessed on days 3, 7, 14, and 21. Wound healing was assessed using the gravimetric method. Expression levels of eNOS, iNOS, and VEGF mRNA were analyzed using quantitative PCR.

Results: Topical application of 5% hydrogel based on the LHT 2-18 complex improves the rate of wound healing in Avy/a mice when administered L-NAME and aminoguanidine and local blood flow indices, increases the level of mRNA expression of the eNOS and VEGF genes and reduces the level of iNOS.

Conclusion: The identified regenerative effect of the LHT 2-18 complex is of interest for further study with the aim of developing an effective agent that stimulates reparative regeneration of wounds, including those caused by metabolic damage.

Proteins with the HAEE tetrapeptide motif as potential targets for beta-amyloid

2025-11-04T13:17:02+03:00

Introduction: Beta-amyloid (Aβ) is involved in numerous physiological and pathophysiological processes and is one of the key players in the pathogenesis of Alzheimer’s disease. Aβ interacts with the 35-HAEE-38 site of the α4 subunit of the α4β2 nicotinic acetylcholine receptor. The synthetic tetrapeptide HAEE effectively inhibits the aggregation of endogenous Aβ. HAEE specifically binds to the 11-EVHH-14 site of Aβ both in the absence and presence of zinc ions, leading to the formation of stable complexes. We hypothesized that the HAEE motif could represent a universal binding site for Aβ within the human proteome.

Materials and Methods: To test this hypothesis, a large-scale search for all amino acid sequences containing the HAEE motif in the human (Homo sapiens) proteome was performed using our in-house PepString server (http://pepstring.eimb.ru/). The conservation of the identified sites was analyzed across jawed vertebrates using BLAST. Protein localization and structural features were determined based on data from UniProt, PDB, and AlphaFold.

Results: We identified 85 proteins (including 200 isoforms) containing the HAEE motif. Of these, 26 proteins are membrane proteins, including receptors, ion channels, and transporters (e.g., CACNA1B, MRS2, SLC15A2), and 59 are intracellular proteins, mostly nuclear transcription factors (including 13 zinc finger proteins). Structural analysis revealed that the HAEE motif is often located within functionally important domains, such as cytoplasmic loops of transmembrane proteins or DNA-binding domains.

Conclusion: Given that Aβ acts as an extracellular ligand and can also penetrate various intracellular compartments, all identified proteins with the HAEE motif are considered potential physiological and pathophysiological targets for Aβ. The most promising candidates are proteins whose HAEE sites are structurally similar to that in α4β2-nAChR and/or coordinate zinc ions. These findings enhance our understanding of the molecular mechanisms of Aβ function and open new avenues for the search of therapeutic targets in AD.

Platform for preclinical screening of neuroprotective strategies: a clinically relevant ICH model and standardized functional test battery

2025-11-01T08:20:36+03:00

Introduction: Intracerebral hemorrhage (ICH) is among the most severe forms of stroke. Whereas traditional hemorrhagic stroke models reproduce hemorrhage within the striatum (specifically the putamen), localization of the hematoma with involvement of the internal capsule and globus pallidus may better recapitulate clinically relevant sensorimotor deficits due to more extensive basal ganglia involvement and damage to dense clusters of white matter tracts.

Materials and Methods: Male CD1 mice (3–4 months, 30–35 g) were assigned to intact control (n=15) or ICH (n=15) groups. ICH was induced by stereotaxic injection of 30 μL autologous blood into the right cerebral hemisphere (2.3 mm lateral, 0.2 mm caudal to bregma, depths 3.0 and 3.5 mm). Neurological status was assessed using the modified Garcia scale, limb placement tests, horizontal and vertical pole tests, inverted grid test, corner test, and open field test at baseline and on days 1, 3, 7, 14, 21, and 28.

Results and Discussion: The model showed high reproducibility with 33–40% acute mortality. All ICH mice developed contralateral sensorimotor-pyramidal deficits with predominant forelimb involvement. Garcia scores declined from 21 to 14.83±1.72 on day 1 with gradual but incomplete recovery by day 28 (18 [18;18.5]). Forelimb placement showed >50% acute deficit with a nonlinear recovery pattern, whereas hindlimb deficits appeared later (days 3–7) and persisted to day 21. The modified Garcia scale, forelimb placement, modified horizontal bar, and open field tests were most sensitive to functional impairment and recovery.

Conclusion: Stereotaxic autologous blood injection into the basal ganglia (putamen and globus pallidus) with involvement of the internal capsule in mice reproduces clinically relevant, reproducible sensorimotor deficits with persistent functional impairment, making this model suitable for preclinical testing of neuroprotective strategies.