Nanocarriers overcoming biological barriers induced by multidrug resistance of chemotherapeutics in 2D and 3D cancer models.

Multidrug resistance (MDR) is currently a big challenge in cancer therapy and limits its success in several patients. Tumors use the MDR mechanisms to colonize the host and reduce the efficacy of chemotherapeutics that are injected as single agents or combinations. MDR mechanisms are responsible for inactivation of drugs and formbiological barriers in cancer like the drug efflux pumps, aberrant extracellular matrix, hypoxic areas, altered cell death mechanisms, etc. Nanocarriers have some potential to overcome these barriers and improve the efficacy of chemotherapeutics. In fact, they are versatile and can deliver natural and synthetic biomolecules, as well as RNAi/DNAi, thus providing a controlled release of drugs and a synergistic effect in tumor tissues. Biocompatible and safe multifunctional biopolymers, with or without specific targeting molecules, modify the surface and interface properties of nanocarriers. These modifications affect the interaction of nanocarriers with cellular models as well as the selection of suitable models for in vitro experiments. MDR cancer cells, and particularly their 2D and 3D models, in combination with anatomical and physiological structures of tumor tissues, can boost the design and preparation of nanomedicines for anticancer therapy. 2D and 3D cancer cell cultures are suitable models to study the interaction, internalization, and efficacy of nanocarriers, the mechanisms of MDR in cancer cells and tissues, and they are used to tailor a personalized medicine and improve the efficacy of anticancer treatment in patients. The description of molecular mechanisms and physio-pathological pathways of these models further allow the design of nanomedicine that can efficiently overcome biological barriers involved in MDR and test the activity of nanocarriers in 2D and 3D models of MDR cancer cells.

The Effect of 4-(Dimethylamino)phenyl-5-oxopyrrolidines on Breast and Pancreatic Cancer Cell Colony Formation, Migration, and Growth of Tumor Spheroids.

A series of hydrazones, azoles, and azines bearing a 4-dimethylaminophenyl-5-oxopyrrolidine scaffold was synthesized. Their cytotoxic effect against human pancreatic carcinoma Panc-1 and triple-negative breast cancer MDA-MB-231 cell lines was established by MTT assay. Pyrrolidinone derivatives 3c and 3d, with incorporated 5-chloro and 5-methylbenzimidazole fragments; hydrazone 5k bearing a 5-nitrothien-2-yl substitution; and hydrazone 5l with a naphth-1-yl fragment in the structure significantly decreased the viability of both cancer cell lines. Compounds 3c and 5k showed the highest selectivity, especially against the MDA-MB-231 cancer cell line. The EC50 values of the most active compound 5k against the MDA-MB231 cell line was 7.3 ± 0.4 µM, which were slightly higher against the Panc-1 cell line (10.2 ± 2.6 µM). Four selected pyrrolidone derivatives showed relatively high activity in a clonogenic assay. Compound 5k was the most active in both cell cultures, and it completely disturbed MDA-MB-231 cell colony growth at 1 and 2 µM and showed a strong effect on Panc-1 cell colony formation, especially at 2 µM. The compounds did not show an inhibitory effect on cell line migration by the 'wound-healing' assay. Compound 3d most efficiently inhibited the growth of Panc-1 spheroids and reduced cell viability in MDA-MB-231 spheroids. Considering these different activities in biological assays, the selected pyrrolidinone derivatives could be further tested to better understand the structure-activity relationship and their mechanism of action.

Anticancer Activity of Sunitinib Analogues in Human Pancreatic Cancer Cell Cultures under Normoxia and Hypoxia.

Pancreatic cancer remains one of the deadliest cancer types. It is usually characterized by high resistance to chemotherapy. However, cancer-targeted drugs, such as sunitinib, recently have shown beneficial effects in pancreatic in vitro and in vivo models. Therefore, we chose to study a series of sunitinib derivatives developed by us, that were proven to be promising compounds for cancer treatment. The aim of our research was to evaluate the anticancer activity of sunitinib derivatives in human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 under normoxia and hypoxia. The effect on cell viability was determined by the MTT assay. The compound effect on cell colony formation and growth was established by clonogenic assay and the activity on cell migration was estimated using a 'wound healing' assay. Six out of 17 tested compounds at 1 µM after 72 h of incubation reduced cell viability by 90% and were more active than sunitinib. Compounds for more detailed experiments were chosen based on their activity and selectivity towards cancer cells compared to fibroblasts. The most promising compound EMAC4001 was 24 and 35 times more active than sunitinib against MIA PaCa-2 cells, and 36 to 47 times more active against the PANC-1 cell line in normoxia and hypoxia. It also inhibited MIA PaCa-2 and PANC-1 cell colony formation. Four tested compounds inhibited MIA PaCa-2 and PANC-1 cell migration under hypoxia, but none was more active than sunitinib. In conclusion, sunitinib derivatives possess anticancer activity in human pancreatic adenocarcinoma MIA PaCa-2 and PANC-1 cell lines, and they are promising for further research.

Synthesis and In Vitro Evaluation as Potential Anticancer and Antioxidant Agents of Diphenylamine-Pyrrolidin-2-one-Hydrazone Derivatives.

The title compounds were synthesized by the reaction of 5-oxo-1-(4-(phenylamino)phenyl)pyrrolidine-3-carbohydrazide with various aldehydes bearing aromatic and heterocyclic moieties and acetophenones, and their cytotoxicity was tested via MTT assay against human triple-negative breast cancer MDA-MB-231, human melanoma IGR39, human pancreatic carcinoma Panc-1, and prostate cancer cell line PPC-1. Furthermore, the selectivity of compounds towards cancer cells compared to fibroblasts was also investigated. Four compounds were identified as the most promising anticancer agents out of a series of pyrrolidinone-hydrazone derivatives bearing a diphenylamine moiety. These compounds were most selective against the prostate cancer cell line PPC-1 and the melanoma cell lines IGR39, with EC50 values in the range of 2.5-20.2 µM against these cell lines. In general, the compounds were less active against triple-negative breast cancer MDA-MB-231 cell line, and none of them showed an inhibitory effect on the migration of these cells. In the 'wound healing' assay, N'-((5-nitrothiophen-2-yl)methylene)-5-oxo-1-(4-(phenylamino)phenyl)pyrrolidine-3-carbohydrazide was identified as the most promising derivative that could be further developed as an antimetastatic agent. N'-(5-chloro- and N'-(3,4-dichlorobenzylidene)-5-oxo-1-(4-(phenylamino)phenyl)pyrrolidine-3-carbohydrazides most efficiently reduced the cell viability in IGR39 cell spheroids, while there was no effect of the investigated pyrrolidinone-hydrazone derivatives on PPC-1 3D cell cultures. Antioxidant activity determined via FRAP assay of N'-(1-(4-aminophenyl)ethylidene)-5-oxo-1-(4-(phenylamino)phenyl)pyrrolidine-3-carbohydrazide was 1.2 times higher than that of protocatechuic acid.

The Effect of Beta Adrenoreceptor Blockers on Viability and Cell Colony Formation of Non-Small Cell Lung Cancer Cell Lines A549 and H1299.

Beta adrenoblockers are a large class of drugs used to treat cardiovascular diseases, migraines, glaucoma and hyperthyroidism. Over the last couple of decades, the anticancer effects of these compounds have been extensively studied. However, the exact mechanism is still not known, and more detailed studies are required. The aim of our study was to evaluate the anticancer activity of beta adrenoblockers in non-small cell lung cancer cell lines A549 and H1299. In order to find the relationship with their selectivity to beta adrenoreceptors, selective (atenolol, betaxolol, esmolol, metoprolol) and non-selective (pindolol, propranolol and timolol) beta blockers were tested. The effect on cell viability was evaluated by MTT assay, and the activity on cell ability to form colonies was tested by clonogenic assay. The type of cell death was evaluated by cell double staining with Hoechst 33342 and Propidium iodide. The most active adrenoblockers against both tested cancer cell lines were propranolol and betaxolol. They completely inhibited lung cancer cell colony formation at 90% of the EC50 (half-maximal effective concentration) value. Most tested compounds induced cell death through apoptosis and necrosis. There was no correlation established between beta adrenoblocker anticancer activity and their selectivity to beta adrenoreceptors.

The roles of carbonic anhydrases IX and XII in cancer cell adhesion, migration, invasion and metastasis.

The main function of carbonic anhydrases (CAs) in cancer cells is the pH regulation through a conversion of H2 O and CO2 to H+ and HCO3 - . However, the data of in vitro and in vivo studies have demonstrated that transmembrane isoforms of CA IX and CA XII are involved in various steps of cancer cell migration, invasion and metastasis. According to literature, inhibition of these CAs can affect the expression of multiple proteins. Some scientific groups have reported the possible interactions between CA IX and E-cadherin-catenin system, CA IX and integrins, CA IX, CA XII and ion transporters, which all are highly involved in cell-to-cell adhesion, the formation of membrane protrusions and focal adhesions. Nevertheless, CA IX and CA XII have a high impact on tumour growth and metastases formation. The data discussed in this review are quite recent. It highly support the role of CA IX and CA XII in various cancer metastasis processes through their interactions to other invasion proteins. Nevertheless, all findings show the great potential of these CAs in the context of research and application in clinical use.

Evaluation of N-aryl-ß-alanine derivatives as anticancer agents in triple-negative breast cancer and glioblastoma in vitro models.

Synthesis of ß-amino acid derivatives containing hydrazone and azole moieties is described. For this purpose, the appropriate hydrazide was treated with aromatic aldehydes, ketones and phenyl iso(thio)cyanates to obtain the desired outcome. The synthesized target compounds were evaluated for their anticancer properties. The assay displayed 3,3'-((2,6-diethylphenyl)azanediyl)bis(N'-(benzylidene)propanehydrazide) to possess the convincing anticancer effect against triple-negative breast cancer cells in vitro. To further study the anticancer properties of compounds containing a hydrazone moiety in breast cancer, series of previously and newly prepared dihydrazones were investigated. It was determined that derivatives with the bis(N'-(4-bromobenzylidene) fragment in the structure are exclusively cytotoxic to cancer cells. The most active compounds against both cell lines were those containing electron withdrawing 4-BrPh or 4-ClPh moieties, together with either chlorine, bromine or iodine groups in para position of phenyl ring. Selected two representative compounds showed migrastatic activity in MDA-MB-231 cell line, where both of them reduced the growth of breast cancer and glioblastoma cell 3D cultures and inhibited cell colony formation. 2009 Elsevier Ltd. All rights reserved.

Nanomedicine to target multidrug resistant tumors.

Nanomedicine employs nanotechnologies to develop innovative applications, and more specifically nano-objects in the field of human health, through exploitation of the physical, chemical and biological properties of materials at the nanoscale. The use of nanovehicles capable of transporting and releasing the active therapeutic payload into target cells, particularly in the case of cancer or inflammatory diseases, can also enhance diagnosis. Therefore, nanomedicines improve the benefit/risk ratio of drugs by increasing their bioavailability, selectivity, and efficacy in the target tissue, while reducing the necessary doses and hence diminishing untoward toxicity to healthy tissues. Overcoming multidrug resistance (MDR) to antitumor agents is a central goal of cancer research and therapeutics, making it possible to treat these diseases more accurately and effectively. The adaptability of nanomedicines e.g. modulation of their components, surface functionalization, encapsulation of various active therapeutics as well as the possibility of combining several treatments using a single nanoparticle platform, are characteristics which are perfectly poised to address classical chemoresistance, a major obstacle towards curative cancer therapy. In this review, we discuss an assortment of nanomedicines along with those that should be developed in order to surmount cancer MDR; these include exosomes, natural compounds, lipid nanocapsules, prodrug self-assemblies, and gold nanoparticles.

3D Tumor Spheroid Models for In Vitro Therapeutic Screening of Nanoparticles.

The anticancer activity of compounds and nanoparticles is most often determined in the cell monolayer. However, three-dimensional (3D) systems, such as tumor spheroids, are more representing the natural tumor microenvironment. They have been shown to have higher invasiveness and resistance to cytotoxic agents and radiotherapy compared to cells growing in 2D monolayer. Furthermore, to improve the prediction of clinical efficacy of drugs, in the past decades, even more sophisticated systems, such as multicellular 3D cultures, closely representing natural tumor microenvironment have been developed. Those cultures are formed from either cell lines or patient-derived tumor cells. Such models are very attractive and could improve the selection of tested materials for clinical trials avoiding unnecessary expensive tests in vivo. The microenvironment in tumor spheroids is different, and those differences or the interaction between several cell populations may contribute to different tumor response to the treatment. Also, different types of nanoparticles may have different behavior in 3D models, depending on their nature, physicochemical properties, the presence of targeting ligands on the surface, etc. Therefore, it is very important to understand in which cases which type of tumor spheroid is more suitable for testing specific types of nanoparticles, which conditions should be used, and which analytical method should be applied.

Inhibition of Carbonic Anhydrase IX Suppresses Breast Cancer Cell Motility at the Single-Cell Level.

Protein Carbonic Anhydrase IX (CA IX), which is expressed in various hypoxic solid tumors in order to maintain proper pH, is also related to cancer cell adhesion, invasion, and metastasis processes. Here, we investigated whether CA IX inhibition by a highly CA IX selective agent benzenesulfonamide VD11-4-2 triggers changes in individual cell motility. We seeded breast cancer cells on an extracellular matrix-coated glass-bottomed dish and in a microfluidic device with a gradient flow of epidermal growth factor (EGF), tracked individual cell movement, calculated their migration speeds, and/or followed movement direction. Our results showed that the inhibitor VD11-4-2 decreased the speed of CA IX positive breast cancer cells by 20-26% while not affecting non-cancerous cell migration. The inhibitor suppressed the cell migration velocity increment and hindered cells from reaching their maximum speed. VD11-4-2 also reduced CA IX, expressing cell movement towards the growth factor as a chemoattractant. Such a single cell-based migration assay enabled the comprehensive investigation of the cell motility and revealed that VD11-4-2 shows the ability to suppress breast cancer cell migration at a lower concentration than previously tested CA IX inhibitors.

Novel N-Substituted Amino Acid Hydrazone-Isatin Derivatives: Synthesis, Antioxidant Activity, and Anticancer Activity in 2D and 3D Models In Vitro.

A series of novel mono and bishydrazones each bearing a 2-oxindole moiety along with substituted phenylaminopropanamide, pyrrolidin-2-one, benzimidazole, diphenylmethane, or diphenylamine fragments were synthesized, and their anticancer activities were tested by MTT assay against human melanoma A375 and colon adenocarcinoma HT-29 cell lines. In general, the synthesized compounds were more cytotoxic against HT-29 than A375. 3-((4-Methoxyphenyl)(3-oxo-3-(2-(2-oxoindolin-3-ylidene)hydrazinyl)propyl)amino)-N'-(2-oxoindolin-3-ylidene)propanehydrazide and (N',N‴)-1,1'-(methylenebis(4,1-phenylene))bis(5-oxo-N'-(2-oxoindolin-3-ylidene)pyrrolidine-3-carbohydrazide) were identified as the most active compounds against HT-29 in 2D and 3D cell cultures. The same compounds showed the highest antioxidant activity among the synthesized compounds screened by ferric reducing antioxidant power assay (FRAP). Their antioxidant activity is on par with that of a well-known antioxidant ascorbic acid.

Pharmacological Potential and Chemical Composition of Crocus sativus Leaf Extracts.

Crocus sativus L. (saffron) has been traditionally used as a food coloring or flavoring agent, but recent research has shown its potent pharmacological activity to tackle several health-related conditions. Crocus sp. leaves, and petals are the by-products of saffron production and are not usually used in the medicine or food industries. The present study was designed to determine the chemical composition of the water and ethanolic extracts of C. sativus leaves and test their cytotoxic activity against melanoma (IGR39) and triple-negative breast cancer (MDA-MB-231) cell lines by MTT assay. We also determined their anti-allergic, anti-inflammatory, and anti-viral activities. HPLC fingerprint analysis showed the presence of 16 compounds, including hydroxycinnamic acids, xanthones, flavonoids, and isoflavonoids, which could contribute to the extracts' biological activities. For the first time, compounds such as tectoridin, iristectorigenin B, nigricin, and irigenin were identified in Crocus leaf extracts. The results showed that mangiferin (up to 2 mg/g dry weight) and isoorientin (8.5 mg/g dry weight) were the major active ingredients in the leaf extracts. The ethanolic extract reduced the viability of IGR39 and MDA-MB-231 cancer cells with EC50 = 410 ± 100 and 330 ± 40 µg/mL, respectively. It was more active than the aqueous extract. Kaempferol and quercetin were identified as the most active compounds. Our results showed that Crocus leaves contain secondary metabolites with potent cytotoxic and antioxidant activities.

In Search of Advanced Tumor Diagnostics and Treatment: Achievements and Perspectives of Carbonic Anhydrase IX Targeted Delivery.

The research of how cells sense and adapt the oxygen deficiency has been recognized as worth winning a Nobel Prize in 2019. Understanding hypoxia-driven molecular machinery paved a path for novel strategies in fighting hypoxia-related diseases including cancer. The oxygen depletion inside the tumor provokes HIF-1 dependent gene and protein expression which helps the tumor to survive. For this reason, tumor related molecules are in the spotlight for scientists developing anticancer agents. One such target is carbonic anhydrase IX (CA IX)-a protein located on the outer cell membrane of most hypoxic tumor cells. This offers the opportunity to exploit it as a target for delivery of cytotoxic drugs, dyes, or radioisotopes to cancer cells. Therefore, researchers investigate CA IX specific small molecules and antibodies as tumor-targeting moieties in nanosystems and conjugates which are expected to overcome the limitations of some existing diagnostic and treatment strategies. This review covers the vast majority of CA IX-targeted systems (nanoparticle and conjugate based) for both therapeutic and imaging purposes published up to now. Furthermore, it shows their stage of development and gives an assessment of their clinical translation possibilities.

Inhibitory Monoclonal Antibodies and Their Recombinant Derivatives Targeting Surface-Exposed Carbonic Anhydrase XII on Cancer Cells.

Monoclonal and recombinant antibodies are widely used for the diagnostics and therapy of cancer. They are generated to interact with cell surface proteins which are usually involved in the development and progression of cancer. Carbonic anhydrase XII (CA XII) contributes to the survival of tumors under hypoxic conditions thus is considered a candidate target for antibody-based therapy. In this study, we have generated a novel collection of monoclonal antibodies (MAbs) against the recombinant extracellular domain of CA XII produced in HEK-293 cells. Eighteen out of 24 MAbs were reactive with cellular CA XII on the surface of live kidney and lung cancer cells as determined by flow cytometry. One MAb 14D6 also inhibited the enzymatic activity of recombinant CA XII as measured by the stopped-flow assay. MAb 14D6 showed the migrastatic effect on human lung carcinoma A549 and renal carcinoma A498 cell lines in a 'wound healing' assay. It did not reduce the growth of multicellular lung and renal cancer spheroids but reduced the cell viability by the ATP Bioluminescence assay. Epitope mapping revealed the surface-exposed amino acid sequence (35-FGPDGENS-42) close to the catalytic center of CA XII recognized by the MAb 14D6. The variable regions of the heavy and light chains of MAb 14D6 were sequenced and their complementarity-determining regions were defined. The obtained variable sequences were used to generate recombinant antibodies in two formats: single-chain fragment variable (scFv) expressed in E. coli and scFv fused to human IgG1 Fc fragment (scFv-Fc) expressed in Chinese Hamster Ovary (CHO) cells. Both recombinant antibodies maintained the same specificity for CA XII as the parental MAb 14D6. The novel antibodies may represent promising tools for CA XII-related cancer research and immunotherapy.

Antioxidant and Anticancer Activity of Novel Derivatives of 3-[(4-Methoxyphenyl)amino]propane-hydrazide.

Series of novel 3-[(4-methoxyphenyl)amino]propanehydrazide derivatives bearing semicarbazide, thiosemicarbazide, thiadiazole, triazolone, triazolethione, thiophenyltriazole, furan, thiophene, naphthalene, pyrrole, isoindoline-1,3-dione, oxindole, etc. moieties were synthesized and their molecular structures were confirmed by IR, 1H-, 13C-NMR spectroscopy and mass spectrometry data. The antioxidant activity of the synthesized compounds was screened by DPPH radical scavenging method. The antioxidant activity of N-(1,3-dioxoisoindolin-2-yl)-3-((4-methoxyphenyl)amino)propanamide and 3-((4-methoxyphenyl)amino)-N'-(1-(naphthalen-1-yl)-ethylidene)propanehydrazide has been tested to be ca. 1.4 times higher than that of a well-known antioxidant ascorbic acid. Anticancer activity was tested by MTT assay against human glioblastoma U-87 and triple-negative breast cancer MDA-MB-231 cell lines. In general, the tested compounds were more cytotoxic against U-87 than MDA-MB-231 cell line. 1-(4-Fluorophenyl)-2-((5-(2-((4-methoxyphenyl)amino)ethyl)-4-phenyl-4H-1,2,4-triazol-3-yl)thio)ethanone has been identified as the most active compound against the glioblastoma U-87 cell line.

Qualitative and Quantitative Analysis of Ukrainian Iris Species: A Fresh Look on Their Antioxidant Content and Biological Activities.

The major groups of antioxidant compounds (isoflavonoids, xanthones, hydroxycinnamic acids) in the rhizome methanol extracts of four Ukrainian Iris sp. (Iris pallida, Iris hungarica, Iris sibirica, and Iris variegata) were qualitatively and quantitatively analyzed using HPLC-DAD and UPLC-MS/MS. Gallic acid, caffeic acid, mangiferin, tectoridin, irigenin, iristectorigenin B, irisolidone, 5,6-dihydroxy-7,8,3',5'-tetramethoxyisoflavone, irisolidone-7-O-ß-d-glucopyranoside, germanaism B, and nigricin were recognized by comparing their UV/MS spectra, chromatographic retention time (tR) with those of standard reference compounds. I. hungarica and I. variegata showed the highest total amount of phenolic compounds. Germanaism B was the most abundant component in the rhizomes of I. variegata (7.089 ± 0.032 mg/g) and I. hungarica (6.285 ± 0.030 mg/g). The compound analyses showed good calibration curve linearity (r2 > 0.999) and low detection and quantifications limit. These results validated the method for its use in the simultaneous quantitative evaluation of phenolic compounds in the studied Iris sp. I. hungarica and I. variegata rhizomes exhibited antioxidant activity, as demonstrated by the HPLC-ABTS system and NRF2 expression assay and anti-inflammatory activity on respiratory burst in human neutrophils. Moreover, the extracts showed anti-allergic and cytotoxic effects against cancer cells. Anti-coronavirus 229E and lipid formation activities were also evaluated. In summary, potent antioxidant marker compounds were identified in the examined Iris sp.

Relationship between Antioxidant and Anticancer Activity of Trihydroxyflavones.

Plant polyphenols have been highlighted not only as chemopreventive, but also as potential anticancer substances. Flavones are a subclass of natural flavonoids reported to have an antioxidant and anticancer activity. The aim of our study was to evaluate antioxidant and anticancer activity of seventeen trihydroxyflavone derivatives, including apigenin (API) and baicalein (BCL). Also, we wanted to find out if there is a correlation between those two effects. Cell growth inhibition testing was carried out using MTT assay in three different human cancer cell lines: lung (A549), breast (MCF-7) and brain epithelial (U87). Antioxidant activity was determined by the DPPH radical scavenging method. Thirteen trihydroxyflavones possessed anticancer activity against at least one tested cancer cell line. They were more active against the MCF-7 cell line, and the lowest activity was determined against the U87 cell line. The majority of compounds inhibited cancer cell growth at EC50 values between 10-50 µM. The most active compound was 3',4',5-trihydroxyflavone 7, especially against A549 and MCF-7 cell lines. The correlation between anti-proliferative and antioxidant activity was only moderate, and it was determined for A549 and U87 cancer cell lines. The most important fragment for those two effects is the ortho-dihydroxy group in ring B. CONCLUSIONS: Trihydroxyflavones demonstrated anticancer activity. Further and more detailed studies should to be carried out to estimate the structure-activity relationship of these compounds.

APPLICATION OF DRY HAWTHORN (CRATAEGUS OXYACANTHA L.) EXTRACT IN NATURAL TOPICAL FORMULATIONS.

There is a great potential for a semi-solid preparation for topical application to the skin that would use materials of natural origin not only as an active substance but also as its base. The aim of this research was to model semisolid preparations containing hawthorn extract and to determine the effect of their bases (carriers) on the release of active components from experimental dosage forms, based on the results of the in vitro studies of the bioactivity of hawthorn active components and ex vivo skin penetration studies. The active compounds of hawthorn were indentified and quantified by validated HPLC method. The antimicrobial and anti-radical activity of dry hawthorn extract were evaluated by methods in vitro. The penetration of active substances into the full undamaged human skin was evaluated by method ex vivo. Natural topical composition was chosen according to the results of release of active compounds. Release experiments were performed with modified Franz type diffusion cells. B.ceieus was the most sensitive bacteria for the hawthorn extract. Extract showed antiradical activity, however the penetration was limited. Only traces of hyperoside and isoquercitrin were founded in epidermis. Protective topical preparation with shea butter released 41.4-42.4% of active substances. Four major compounds of dry hawthorn extract were identified. The research showed that extract had antimicrobial and antiradical activity, however compounds of hawthorn stay on the surface of the undamaged human skin. Topical preparation containing beeswax did not release active compounds. Beeswax was identified as suspending agent. Topical preparations released active compounds when shea butter was used instead of beeswax.

MODELING AND BIOPHARMACEUTICAL EVALUATION OF SEMISOLID SYSTEMS WITH ROSEMARY EXTRACT.

Scientific literature provides a great deal of studies supporting antioxidant effects of rosemary, protecting the body's cells against reactive oxygen species and their negative impact. Ethanol rosemary extracts were produced by maceration method. To assess biological activity of rosemary extracts, antioxidant and antimicrobial activity tests were performed. Antimicrobial activity tests revealed that G+ microorganisms are most sensitive to liquid rosemary extract, while G-microorganisms are most resistant to it. For the purposes of experimenting, five types of semisolid systems were modeled: hydrogel, oleogel, absorption-hydrophobic ointment, oil-in-water-type cream and water-in-oil-type cream, which contained rosemary extract as an active ingredient. Study results show that liquid rosemary extract was distributed evenly in the aqueous phase of water-in-oil-type system, forming the stable emulsion systems. The following research aim was chosen to evaluate the semisolid systems with rosemary exctract: to model semisolid preparations with liquid rosemary extract and determine the influence of excipients on their quality, and perform in vitro study of the release of active ingredients and antimicrobial activity. It was found that oil-in-water type gel-cream has antimicrobial activity against Staphylococcus epidermidis bacteria and Candida albicans fungus, while hydrogel affected only Candida albicans. According to the results of biopharmaceutical study, modeled semisolid systems with rosemary extract can be arranged in an ascending order of the release of phenolic compounds from the forms: water-in-oil-type cream < absorption-hydrophobic ointment < Pionier PLW oleogel < oil-in-water-type eucerin cream < hydrogel < oil-in-water-type gel-cream. Study results showed that oil-in-water-type gel-cream is the most suitable vehicle for liquid rosemary extract used as an active ingredient.

Monoclonal antibodies raised against 167-180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity.

Abstract Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application.