


Vol 29, No 3 (2024)
Original Study Articles
Pharmacokinetics of pertuzumab biosimilar compared with the original drug in cynomolgus monkeys
Abstract
BACKGROUND: Developing biosimilar monoclonal antibodies for treating malignant diseases is a critical task. RPH-051 (pertuzumab) is a biosimilar drug, a recombinant humanized monoclonal antibody targeting type 2 human epidermal growth factor receptors. At the stage of non-clinical development, studies of pharmacokinetics of biosimilar vs. original drug product in a relevant animal species, provide important information on the comparability of drugs.
AIM: To evaluate the pharmacokinetics of the biosimilar product RPH-051 in comparison with the original drug after a single intravenous injection in a cynomolgus monkey model.
MATERIALS AND METHODS: The study was conducted in 8 cynomolgus monkeys (Macaca fascicularis) divided into 2 groups, each including 2 females and 2 males. The first group received the biosimilar RPH-051 (international nonproprietary name is pertuzumab, AO R-Pharm, Russia), the second group was treated with the comparator Perjeta® (international nonproprietary name is pertuzumab). The drugs were administered as a single intravenous dose of 50 mg/kg. Blood samples were collected before dosing, then at 1 min, 1, 2, 4, 8, 24 h, and on days 3, 7, 10, 14, 21 and 28 post-dosing. Quantitative analysis of pertuzumab serum levels was performed using enzyme-linked immunosorbent assay (ELISA) with spectrophotometric detection in the visible range of the spectrum.
RESULTS: The method of quantitative assessment of serum level of pertuzumab using (ELISA) in cynomolgus monkeys was developed. The method was validated against the following parameters: selectivity, minimum required dilution, lower limit of quantification, calibration range, accuracy and precision (within the analytical run and between runs), specificity, linearity (possibility) of sample dilution, parallelism, and stability of the analyte in serum.
Comparability of the drugs in the main pharmacokinetic parameters was established. The mean Cmax and AUClast values of RPH-051 and of the comparator were 1684.34 and 1405.34 μg/mL (Cmax); 265,282.18 and 280,168.92 (μg/mL)×h (AUClast), respectively.
CONCLUSION: Comparability between the pharmacokinetic profiles of the biosimilar RPH-051 and the originator’s drug was demonstrated. In the course of the study, no adverse clinical events, animal body weight changes, or injection site reactions associated with the tested drugs were observed.



Cytotoxicity of curcumin-loaded nanoparticles based on amphiphilic poly-N-vinylpyrrolidone derivatives in 2D and 3D in vitro models of human ovarian adenocarcinoma
Abstract
BACKGROUND: Nanocarriers based on biocompatible polymers are a promising delivery tool for biologically active substances and drugs, in particular antitumor agents. Curcumin, a polyphenol, is known to possess pleiotropic therapeutic effects, including antitumor activity. The antitumor potential of curcumin has been shown in various tumor types, including ovarian adenocarcinoma. However, its lipophilic properties and very low bioavailability limits its use. Incorporating curcumin into nanocarriers enhances its delivery options and expands its potential as an antitumor agent.
AIM: To produce curcumin-loaded polymeric nanoparticles based on amphiphilic poly-N-vinylpyrrolidone derivatives and its copolymers with acrylic acid, explore their accumulation in the tumor cells; evaluate in vitro cytotoxicity in 2D (monolayer cell culture) and 3D (tumor spheroids) models of human ovarian adenocarcinoma.
MATERIALS AND METHODS: The polymers of the amphiphilic poly-N-vinylpyrrolidone derivatives and its copolymers with acrylic acid were obtained using radical polymerization. Emulsion method was used to obtain polymeric nanoparticles. Accumulation of nanoparticles in tumor cells was assessed using flow cytometry (for monolayer culture) or fluorimetric analysis (for spheroids). Cytotoxicity was studied in 2D and 3D models obtained of the human ovarian adenocarcinoma cell line OVCAR-3 using 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay).
RESULTS: The effective accumulation of curcumin-loaded polymeric nanoparticles in both monolayer culture cells and tumor spheroids was demonstrated. Curcumin-loaded nanoparticles exhibited high-level cytotoxicity in the 2D model of human ovarian adenocarcinoma cells OVCAR-3 (IC50 up to 137±9 μg/mL) and a moderate, although significant cytotoxic effect in a 3D in vitro model. Meanwhile, nanoparticles not loaded with curcumin did not show any cytotoxic activity regardless of their composition or of the additional modification, i.e. with the use of maleimide functional groups.
CONCLUSION: These data can provide a foundation for further studies to assess the safety and in vivo antitumor activity of curcumin-loaded nanoparticles based on amphiphilic poly-N-vinylpyrrolidone derivatives.



Viral microRNA in HPV16-associated cervical cancer: expression, diagnostic potential, and biological functions
Abstract
BACKGROUND: Cervical cancer (CC) is the fourth most common cancer among women worldwide in terms of incidence and mortality. High-risk human papillomaviruses (HPVs) are etiologic factor of CC in more than 90% of cases, with type 16 HPV (HPV16) revealed in >50% of cancers. Dysregulation of expression of viral oncogenes E6 and E7 is the main cause of malignant transformation in infected cervical epithelial cells. The mechanisms of impaired expression of these genes are still underexplored. The dysfunction of viral microRNAs may be among the underlying factors.
AIM: To analyze the expression of HPV16-associated microRNA-H1 and microRNA-H2 in cervical cancer specimens, evaluate the correlation of their expression to viral load and overall patient survival, and analyze in silico their potential viral and cellular targets.
MATERIALS AND METHODS: The expression of HPV16 microRNA-H1 and HPV16 microRNA-H2 was evaluated in the real-time polymerase chain reaction. With this purpose, small RNAs were isolated from 36 specimens of HPV16-positive squamous cell carcinomas of the cervix. Further, the viral load was assessed after calculating the value of HPV16 DNA copies per cell. The association between microRNA expression and the viral load was evaluated using the nonparametric Spearman’s correlation coefficient. Kaplan-Meier curves were plotted to analyze the dependence of 5-year overall survival on the level of viral microRNA expression. The miRanda algorithm and online services mirDB, MR-microT and TargetScan Custom 5.2 were used for in silico search of theoretical microRNA targets.
RESULTS: MicroRNA-H1 expression was revealed in 33 of 38 specimens (86.8%), microRNA-H2 was detected in 37 of 38 specimens (97.4%) of HPV16-positive cervical cancer. There was a positive correlation between both microRNA-H1 (r=0.36, p=0.042) and microRNA-H2 (r=0.51, p=0.001) expression and HPV16 viral load. Higher level of expression of viral microRNA-H1 and microRNA-H2 tended to correlate with better overall patient survival. The theoretical microRNA-H1 (E7, E2, E5, L2 and URR) and microRNA-H2 (E1, E2, E5, L2, L1, URR) targets in the HPV16 genome were identified in silico, as well as theoretical cellular targets indicating possible regulation of cellular signaling pathways by means of viral microRNAs, both controlling normal viral cycle and promoting tumor transformation.
CONCLUSION: The results of this study demonstrate promising further investigation of the functions of viral microRNAs in relation with the infectious process and virus-induced malignant transformation, and their potential importance in the diagnosis of HPV16-associated cancers.



Expression profile of cellular microRNAs and their potential role in cervical cancer
Abstract
Background. Cervical cancer (CC) is the fourth leading cause of cancer-related morbidity and mortality among women. Despite the established etiologic factor of cervical cancer, especially high-risk human papillomavirus (HPV, human papillomavirus) and available vaccine prophylaxis, the issue persists both in understanding the mechanism of HPV-associated carcinogenesis and in developing new approaches for diagnosis and treatment of cervical cancer. Epigenetic regulation of gene expression by means of microRNAs plays an important role in the pathogenesis of cervical cancer. Therefore, the search for new differentially expressed microRNAs in order to reveal new mechanisms of HPV-associated transformation of tumor cells is of high priority.
AIM: To explore microRNAs with differentiated expression in the cervical cancer tissue and to assess the functional potential of their detection in silico.
MATERIALS AND METHODS: The spectrum of microRNAs characterizing by modified expression in the tumor tissue of HPV16-positive squamous cell carcinomas of the cervix was determined using NGS sequencing (Next Generation Sequencing) of tumor tissue and of apparently unchanged adjacent epithelium obtained with the use of microdissection. Potential target genes of the investigated microRNAs were identified using MirTargetLink, Tarbase v9.0, and LinkedOmics services. Gene Set Enrichment Analysis was performed using Metascape. The relations between the level of microRNA expression and clinical SCC features (according to TCGA data, CESC sample) were searched using USCS Xena service.
RESULTS: NGS-sequencing of paired HPV16-positive specimens of tumor tissue and normal cervical tissue resulted in the identification of 42 differentially expressed microRNAs. Specifically, the levels of 22 microRNAs in the tumor tissue were higher than in the apparently normal adjacent epithelium, while 20 microRNAs demonstrated lower levels in the tumor specimens. Analysis of the potential targets of significant microRNAs revealed multiple functional gene categories, potentially involved in carcinogenesis as well as an association with clinical features. We showed that increased expression levels of microRNA-20b in the tumor tissue correlated with the risk of distant metastases, whereas the lower levels of microRNA-218-1 and microRNA -218-2 were associated with unfavorable prognosis of disease. With regard to microRNA-363, -615, and -769, their increase in cervical cancer was described for the first time, and the potential targets and signaling pathways associated with THEIR EXPRESSION LEVELS WERE IDENTIFIED.
CONCLUSION: The search for differentially expressed microRNAs in cervical cancer has revealed a spectrum of microRNAs with potentially important role in the process of malignant transformation and persistence of tumor phenotype. In addition to microRNAs demonstrating functional characteristics described in the world literature, we found microRNAs that play an unknown role in cervical cancer. In this relation, the potentially relevant targets were identified that could be helpful in understanding the mechanisms of carcinogenesis. The data obtained may form the basis for the development of new approaches to the diagnosis and therapy of SCC.



Evaluation of biodistribution of Riboplatin, a Pt(IV) double prodrug based on cisplatin, using fluorescent visualization
Abstract
BACKGROUND: Developing new platinum-based antitumor agents with improved efficacy and reduced toxicity remains a critical challenge. One promising approach is the synthesis of Pt(IV) prodrugs, such as precursor complexes of cisplatin and its analogs, which release active Pt(II) directly into the intracellular environment of malignant cells. To determine the optimal administration route for these new platinum-based drugs in vivo, it is essential to study their acute toxicity and biodistribution in vital organs.
AIM: To evaluate the tolerability, acute toxicity, biodistribution, and accumulation in breast tumors of Riboplatin and its liposomal formulation.
MATERIALS AND METHODS: The study was conducted at Moscow Pedagogical State University in female BALB/c mice (healthy or bearing transplanted breast adenocarcinoma EMT-6) and in immunodeficient BALB/c nude mice (bearing human breast adenocarcinoma SK-BR-3). The fluorescence imaging and inductively coupled plasma mass spectrometry were used.
RESULTS: A single dose (up to 48 mg/kg) of either water solution or liposomal formulation of Riboplatin did not cause any decrease of body weight in mice. The animals tolerated the dose 48 mg/kg of the drug, whereas the body weight decreased by less than 5% within 3 days. The distribution of Riboplatin and of its liposomal formulation over the body organs was assessed in BALB/c nude mice using fluorescence imaging and inductively coupled plasma mass spectrometry. Both forms provided Riboplatin delivering to EMT-6 tumor. Riboplatin in the form of water solution was predominantly excreted through liver and kidney, while the liposomal formulation lewd to drug accumulation in the spleen.
CONCLUSION: Riboplatin is a Pt(IV) prodrug with good tolerability, reduced toxicity compared with cisplatin, with effective accumulation in malignant breast tumors. The opportunity of assessing Riboplatin biodistribution in vivo in EMT-6 and SK-BR-3 tumor lines using fluorescence imaging has been demonstrated. Significant Riboplatin accumulation in EMT-6 tumors suggests the possibility of riboflavin-specific uptake.



Comparative evaluation of antitumor effects of methionine-γ-lyase in in vitro 2D and 3D human tumor models
Abstract
BACKGROUND: Promising antitumor drug screening results obtained from monolayer cultures are often poorly reproduced in the in vivo models. Using clinically relevant 3D in vitro human tumor models, such as spheroids, provides a more reliable framework for evaluating antitumor activity. This is particularly important when the drug’s mechanism of action targets cellular metabolism.
AIM: To conduct a comparative study of the antitumor activity of methionine-γ-lyase (MGL) in 2D and 3D human tumor models.
MATERIALS AND METHODS: Human fibroblast cell culture and tumor cell lines, e.g. MCF7 human breast cancer, HCT-116 colorectal cancer, PANC-1 human pancreatic cancer, Huh7 liver cancer, and LNCaP human prostate cancer were used to evaluate MGL cytotoxicity. Cultural plates with low-adhesive coating were used to produce the spheroids. Cell survival was assessed using the resazurin test.
RESULTS: The spheroid model showed higher cell survival after 72-hour MGL exposure compared with the monolayer culture model in all tested cultures. Fibroblasts demonstrated the lowest sensitivity to MGL exposure in both 2D and 3D culture models, with IC50=2.2 and 9.1 IU/mL, respectively. The rapidly proliferating PANC-1 and HCT-116 cells showed the highest sensitivity to MGL in 2D and 3D models: IC50=0.23 and 1.5 IU/mL; IC50=0.83 and 1.43 IU/mL, respectively.
CONCLUSION: The effect of MGL on cell survival in the spheroid systems is less pronounced than in monolayers. The viability of cells exposed to survival MGL in the spheroid culture system is independent of spheroid size or growth rate. Despite the maximum cytotoxic effect being observed in the fast-growing spheroid model of colon cancer HCT-116, and the lowest in the model of slowly dividing fibroblasts, this dependence was not so obvious for other cell types. Overall, the spheroid model has proven useful for testing the specific activity of enzyme-based antitumor drugs, as far as it overcomes the excessive intrinsic sensitivity observed in monolayer cancer models.



Vessel visualization and inhibition of tumor growth after injection of nanosystems based on magnetic nanoparticles modified by serum albumin with free radical approach
Abstract
BACKGROUND: Nanosystems based on magnetic nanoparticles (MNPs) of iron oxides coated with human serum albumin (HSA) have a set of unique characteristics that make their use promising in the diagnosis and treatment of tumors.
AIM: To investigate, using in vitro and in vivo models, the possibilities of using the systems we developed based on MNPs magnetite, modified by HSA using the free radical method, for contrasting tumors and slowing their growth.
MATERIALS AND METHODS: The stability and integrity of the albumin coating formed on synthesized nanoparticles as a result of protein adsorption and fixation of adsorbed albumin molecules on nanoparticles due to modification of albumin by the action of a hydroxyl radical formed in a Fenton-like reaction was controlled by a change in the apparent optical density by 450 nm with the addition of immunoglobulin G. After in vitro confirmation of the contrasting properties and possible consequences of the contact of MNPs and nanosystems with blood by agglutination test, nanosystems containing MNPs and HSA were injected intraarterially into tumors implanted in rats at a dose of 20–60 μg per animal and the contrasting properties were studied in vivo using radiography and computed tomography. The effect of nanosystems on the tumor was assessed by the tumor growth index (TGI) and by the results of a pathomorphological study.
RESULTS: To obtain nanosystems, joint incubation of MNPs and HSA was carried out for 24 hours in the presence of hydrogen peroxide, then subjected to magnetic separation. The stability and integrity of the protein coatings were confirmed with the addition of immunoglobulin G. In vitro studies have shown that the resulting nanosystem preparation in an aqueous medium with a MNPs concentration of 200 μg/ml does not lead to agglutination of blood cells and has a pronounced contrast. Vascular contrast in vivo, recorded 30 minutes after intraarterial administration, persisted for 14 days of follow-up. The tolerability study did not reveal any adverse side effects. With the introduction of nanosystems, a significant inhibition of tumor growth was noted compared with the control groups (p ≤0.05). Thus, tumors without the introduction of nanosystems reached a volume of 95,726.9±38,040.3 mm3 during the observation period — the TGI was 11±4.5. In the groups of rats treated with nanosystems at a dose of 20 micrograms of MNPs, tumors grew to 49,801±6011.2 mm3 (TGI 4.7±0.5), at a dose of 40 μg of MNPs — to 54,670.2±17 983.4 mm3 (IPO 5.5±1.4), at a dose of 60 μg of MNPs — to 43,342.5±14,637.2 mm3 (TGI 4.5±1.3).
CONCLUSION: The steady contrast of tumor vessels with the introduction of nanosystems based on MNPs and HSA and their cytotoxic effect on the tumor is shown, which gives reason to consider the nanosystems developed by us promising for tumor theranostics.



In vivo antitumor effects of bis-benzimidazole derivatives in mouse melanoma and lung cancer models
Abstract
BACKGROUND: The development of new antitumor agents on the basis of benzimidazoles opens new opportunities for treatment of the malignant tumors, including those refractory to conventional therapies. The benzimidazole derivatives demonstrate wide spectrum of biological activities, specifically antineoplastic effects, and high-level cytotoxicity toward human’s tumor cell lines. The exploration of antineoplastic effects of new synthetic benzimidazole derivatives in in vivo models of tumor growth will help the development of the effective doses and cancer treatment regimens with the use of such compounds.
AIM: To assess the antineoplastic effects of the benzimidazole derivatives — monomeric compound MB2Py(Ac) and dimeric compound DB2Py(3) — in the continuous cellular models of Lewis lung carcinoma (LLC) and melanoma B16 in mice.
MATERIALS AND METHODS: In vivo assessment of antineoplastic effects was performed in the continuous mouse models of Lewis lung carcinoma (LLC) and B16 melanoma after a single intravenous injection of MB2Py(Ac) and DB2Py(3). Irinotecan was used as a comparator drug. Antitumor effects were measured via standard parameters, such as tumor growth inhibition (TGI%) and tumor growth rate (TGR).
RESULTS: The study compounds DB2Py(3) and MB2Py(Ac) used in the study doses and regimen demonstrated mild antitumor effect in the model of murine solid tumors (TGI <50%). In melanoma B16 model, the maximum tumor growth inhibition was 15% and 38.5%, respectively, for MB2Py(Ac) and DB2Py(3). In the lung carcinoma model, the mild effect was observed, e.g. 8% for MB2Py(Ac) and 23.4% for DB2Py(3). The effect of the comparator drug, irinotecan, was more expressive, although short-term. In particular, TGI achieved 52.5% for melanoma B16 and 34.5% for Lewis lung carcinoma (LLC). In general, melanoma B16 was more sensitive to the effects of both bis-benzimidazoles and irinotecan, compared to the lung carcinoma LLC.
CONCLUSION: This study shows that the dimeric compound DB2Py(3) was the most promising among three study substances in the models of melanoma and lung carcinoma in mice. Nevertheless, its high toxicity imposes the necessity of further optimization for the clinical use. The substance MB2Py(Ac) demonstrated lowest efficacy, although it may be also explored in the modified treatment regimens.



Reviews
The claudin family of proteins in the pathogenesis and treatment of malignancies: current insights and future prospects
Abstract
This review presents data concerning claudins, the proteins of tight junctions, and their role in the pathogenesis and therapy of malignancies. Particular attention is paid to the variability in claudin expression levels, their intracellular localization in tumors, and the prognostic significance of these variations in cancer. The review highlights the role of claudins in metastatic spread, invasion, and tumor cell resistance to antitumor drug therapy. Moreover, the potential of claudins as targets for novel diagnostic and treatment methods for malignant neoplasms is also discussed.


