Silver Complexes of Miconazole and Metronidazole: Potential Candidates for Melanoma Treatment.

Melanoma, arguably the deadliest form of skin cancer, is responsible for the majority of skin-cancer-related fatalities. Innovative strategies concentrate on new therapies that avoid the undesirable effects of pharmacological or medical treatment. This article discusses the chemical structures of [(MTZ)2AgNO3], [(MTZ)2Ag]2SO4, [Ag(MCZ)2NO3], [Ag(MCZ)2BF4], [Ag(MCZ)2SbF6] and [Ag(MCZ)2ClO4] (MTZ-metronidazole; MCZ-miconazole) silver(I) compounds and the possible relationship between the molecules and their cytostatic activity against melanoma cells. Molecular Hirshfeld surface analysis and computational methods were used to examine the possible association between the structure and anticancer activity of the silver(I) complexes and compare the cytotoxicity of the silver(I) complexes of metronidazole and miconazole with that of silver(I) nitrate, cisplatin, metronidazole and miconazole complexes against A375 and BJ cells. Additionally, these preliminary biological studies found the greatest IC50 values against the A375 line were demonstrated by [Ag(MCZ)2NO3] and [(MTZ)2AgNO3]. The compound [(MTZ)2AgNO3] was three-fold more toxic to the A375 cells than the reference (cisplatin) and 15 times more cytotoxic against the A375 cells than the normal BJ cells. Complexes of metronidazole with Ag(I) are considered biocompatible at a concentration below 50 µmol/L.

Cervical microbiota dysbiosis associated with high-risk Human Papillomavirus infection.

High-risk Human Papillomavirus (HR-HPV) genotypes, specifically HPV16 and HPV18, pose a significant risk for the development of cervical intraepithelial neoplasia and cervical cancer. In the multifaceted cervical microenvironment, consisting of immune cells and diverse microbiota, Lactobacillus emerges as a pivotal factor, wielding significant influence in both stabilizing and disrupting the microbiome of the reproductive tract. To analyze the distinction between the cervical microbiota and Lactobacillus-dominant/non-dominant status of HR-HPV and non-infected healthy women, sixty-nine cervical swab samples were analyzed, included 44 with HR-HPV infection and healthy controls. All samples were recruited from Human Papillomavirus-based cervical cancer screening program and subjected to 16s rRNA sequencing analysis. Alpha and beta diversity analyses reveal no significant differences in the cervical microbiota of HR-HPV-infected women, including 16 and 18 HPV genotypes, and those with squamous intraepithelial lesion (SIL), compared to a control group. In this study we identified significantly lower abundance of Lactobacillus mucosae in women with HR-HPV infection compared to the control group. Furthermore, changes in bacterial diversity were noted in Lactobacillus non-dominant (LND) samples compared to Lactobacillus-dominant (LD) in both HR-HPV-infected and control groups. LND samples in HR-HPV-infected women exhibited a cervical dysbiotic state, characterized by Lactobacillus deficiency. In turn, the LD HR-HPV group showed an overrepresentation of Lactobacillus helveticus. In summary, our study highlighted the distinctive roles of L. mucosae and L. helveticus in HR-HPV infections, signaling a need for further research to demonstrate potential clinical implications of cervical microbiota dysbiosis.

Amino acid derivative of probenecid potentiates apoptosis-inducing effects of vinblastine by increasing oxidative stress in a cancer cell-specific manner.

Many chemotherapeutic drugs suffer from multidrug resistance (MDR). Efflux transporters, namely ATP-binding cassettes (ABCs), that pump the drugs out of the cancer cells comprise one major reason behind MDR. Therefore, ABC inhibitors have been under development for ages, but unfortunately, without clinical success. In the present study, an l-type amino acid transporter 1 (LAT1)-utilizing derivative of probenecid (PRB) was developed as a cancer cell-targeted efflux inhibitor for P-glycoprotein (P-gp), breast cancer resistant protein (BCRP) and/or several multidrug resistant proteins (MRPs), and its ability to increase vinblastine (VBL) cellular accumulation and apoptosis-inducing effects were explored. The novel amino acid derivative of PRB (2) increased the VBL exposure in triple-negative human breast cancer cells (MDA-MB-231) and human glioma cells (U-87MG) by 10-68 -times and 2-5-times, respectively, but not in estrogen receptor-positive human breast cancer cells (MCF-7). However, the combination therapy had greater cytotoxic effects in MCF-7 compared to MDA-MB-231 cells due to the increased oxidative stress recorded in MCF-7 cells. The metabolomic study also revealed that compound 2, together with VBL, decreased the transport of those amino acids essential for the biosynthesis of endogenous anti-oxidant glutathione (GSH). Moreover, the metabolic differences between the outcomes of the studied breast cancer cell lines were explained by the distinct expression profiles of solute carriers (SLCs) that can be concomitantly inhibited. Therefore, attacking several SLCs simultaneously to change the nutrient environment of cancer cells can serve as an adjuvant therapy to other chemotherapeutics, offering an alternative to ABC inhibitors.

Flavonol and A-type procyanidin-rich extracts of Prunus spinosa L. flower exhibit anticoagulant activity through direct thrombin inhibition, but do not affect platelet aggregation in vitro.

Background: Blackthorn flower (Prunus spinosa L.) is a traditional herbal remedy recommended for treating cardiovascular diseases (CVDs). Aim: This in vitro study investigates the effects of flavonol and A-type procyanidin-rich blackthorn flower extracts on the hemostatic system, including the blood plasma coagulation cascade and platelet aggregation. Methods: Six distinct extracts, characterized through various techniques, including LC-MS/MS, were assessed at in vivo-relevant levels (1-50 µg/mL) for their antithrombotic activity. The thrombin, prothrombin, and activated partial thromboplastin times were measured. Additionally, the thrombin enzymatic activity was tested using the chromogenic substrate S-2238 and fibrinogen as the physiological substrate of the enzyme. To gain insights into the mechanism of action, the interactions between the primary extracts' constituents, their potential metabolites, and thrombin were examined in silico. The computational analyses were complemented by in vitro experiments and circular dichroism spectroscopy. The platelet aggregation in human platelet-rich plasma was assessed after ADP or collagen stimulation. Furthermore, the extracts' biocompatibility was tested on human peripheral blood mononuclear cells (PBMCs) and red blood cells (RBCs). Results: The extracts slightly prolonged the prothrombin and thrombin times and effectively inhibited the thrombin's enzymatic activity, reducing its amidolytic and proteolytic functions at 50 µg/mL by 91.2% and 74.8%, respectively. In silico molecular docking demonstrated a strong binding affinity of the examined polyphenols and their metabolites to thrombin. Most analytes bound exclusively within the enzyme active site; however, afzelin, kaempferitrin, and procyanidin A2 revealed the affinity to additional binding sites, including exosite I. The structure-activity relationship of flavonols as thrombin inhibitors was studied in vitro. Circular dichroism spectroscopy confirmed that the interactions between thrombin and the compounds (even at 1 µg/mL) induce alterations within the α-helices' secondary structure, resulting in noticeable changes in the enzyme's CD spectrum. On the other hand, the extracts did not influence platelet aggregation. Eventually, their cellular biocompatibility with PBMCs and RBCs was confirmed. Conclusion: The extracts directly inhibit thrombin, a critical serine protease in hemostasis and a prime anticoagulant drug target, and do not exhibit antiplatelet effects. This study enhances the knowledge of the biological activity of blackthorn flowers and supports their traditional use in CVDs.

Metformin derivatives - Researchers' friends or foes?

Metformin has been used for ages to treat diabetes mellitus due to its safety profile and low cost. However, metformin has variable pharmacokinetics in patients, and due to its poor oral absorption, the therapeutic doses are relatively high, causing unpleasant gastrointestinal adverse effects. Therefore, novel derivatives of metformin have been synthesized during the past decades. Particularly, after the mid-2000 s, when organic cation transporters were identified as the main metformin carriers, metformin derivatives have been under intensive investigation. Nevertheless, due to the biguanide structure, derivatives of metformin have been challenging to synthesize. Moreover, the mechanisms of metformin's action are not fully understood to date, and since it has multifunctional properties, the interests have switched to re-purposing for other diseases. Indeed, metformin derivatives have been demonstrated in many cases to be more effective than metformin itself and have the potential to be used in different diseases, including several types of cancers and neurodegenerative diseases. On the other hand, the pleiotropic nature of metformin and its derivatives can also create challenges. Not all properties are fit for all diseases. In this review, the history of the development of metformin-like compounds is summarized, and insights into their potential for future drug discovery are discussed.

Substituent effects of sulfonamide derivatives of metformin that can dually improve cellular glucose utilization and anti-coagulation.

Metformin, the most frequently prescribed medicine for the management of type 2 diabetes, has been shown to reduce cardiovascular events in diabetic patients in pre-clinical and clinical studies. The present work reports the design, synthesis, and biological assessment of the impact of six benzenesulfonamide biguanides on various aspects of hemostasis, cell function, red blood cell integrity (RBC), and their ability to uptake glucose in human umbilical endothelial cells (HUVECs). It was found that all synthesized o- and m-benzenesulfonamides, particularly derivatives with nitro (3) and amino groups (4), are characterized by a good safety profile in HUVECs, which was further confirmed in the cellular integrity studies. The biguanide analogues with methoxy group (1, 2) and an amino substituent (5, 6) significantly increased glucose utilization in HUVECs, similarly to the parent drug. Intriguingly, compounds 1, 3, and 6 favourably influenced some of the coagulation parameters. Furthermore, derivative 3 also slowed the process of fibrin polymerization, indicating more beneficial anti-coagulant properties than metformin. None of the novel metformin analogues interact strongly with the erythrocyte lipid-protein bilayer. Our findings indicate that derivative 3 has highly desirable anti-coagulant properties, and compounds 1 and 6 have potential dual-action activity, including anti-hyperglycaemic properties and anti-coagulant activity. As such, these derivatives can be used as lead molecules for further development of anti-diabetic agents with a beneficial effect on hypercoagulability.

Trends in development and quality assessment of pharmaceutical formulations - F2α analogues in the glaucoma treatment.

The ocular delivery route presents a number of challenges in terms of drug administration and bioavailability. The low bioavailability following topical ophthalmic administration shows that there is a clear need for in-depth research aimed at finding both more efficacious molecules and formulations precisely targeted at the site of action. Continuous technological development will eventually result in improved bioavailability, lower dosages, reduced toxicity, fewer adverse effects, and thus better patient compliance and treatment efficacy. Technological development, as well as increasingly stringent quality requirements, help stimulate analytical progress. This is also clearly evident in the case of medicinal products used in the treatment of glaucoma, which are the subject of this review. Impurity profiling of PGF2α analogues, either in the pure substance or in the finished formulation, is a crucial step in assessing their quality. The development of specific, accurate and precise stability-indicating analytical methods for determining the content and related substances seems to be an important issue in relation to this tasks. A total of 27 official and in-house analytical methods are presented that are used for the analysis of latanoprost, travoprost and bimatoprost. The conditions for chromatographic separation with UV or MS/MS detection and the available results obtained during method validation are described. In addition, several aspects are discussed, with particular emphasis on the instability of the analogues in aqueous solution and the phenomenon of isomerism, which affects a potentially large number of degradation products.

Long-Acting Injectable Antipsychotics-A Review on Formulation and In Vitro Dissolution.

Long-acting injectable (LAI) neuroleptics constitute an effective therapeutical alternative for individuals suffering from persistent mental illness. These injectable pharmaceuticals help patients manage their condition better and improve long-term outcomes by preventing relapses and improving compliance. This review aims to analyse the current formulation aspects of LAI neuroleptics, with particular emphasis on analysis of drug release profiles as a critical test to guarantee drug quality and relevant therapeutical activity. While there is no officially approved procedure for depot parenteral drug formulations, various dissolution tests which were developed by LAI manufacturers are described. In vitro dissolution tests also possess a critical function in the estimation of the in vivo performance of a drug formulation. For that reason, thorough inspection of the in vitro-in vivo correlation (IVIVC) is also discussed.

Current approaches to facilitate improved drug delivery to the central nervous system.

Although many pharmaceuticals have therapeutic potentials for central nervous system (CNS) diseases, few of these agents have been effectively administered. It is due to the fact that the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSF) restrict them from crossing the brain to exert biological activity. This article reviews the current approaches aiming to improve penetration across these barriers for effective drug delivery to the CNS. These issues are summarized into direct systemic delivery and invasive delivery, including the BBB disruption and convection enhanced delivery. Furthermore, novel drug delivery systems used at the nanoscale, including polymeric nanoparticles, liposomes, nanoemulsions, dendrimers, and micelles are discussed. These nanocarriers could contribute to a breakthrough in the treatment of many different CNS diseases. However, further broadened studies are needed to assess the biocompatibility and safety of these medical devices.

L-type Amino Acid Transporter 1 Utilizing Ferulic Acid Derivatives Show Increased Drug Delivery in the Mouse Pancreas Along with Decreased Lipid Peroxidation and Prostaglandin Production.

Oxidative stress and pathological changes of Alzheimer's disease (AD) overlap with metabolic diseases, such as diabetes mellitus (DM). Therefore, tackling oxidative stress with antioxidants is a compelling drug target against multiple chronic diseases simultaneously. Ferulic acid (FA), a natural antioxidant, has previously been studied as a therapeutic agent against both AD and DM. However, FA suffers from poor bioavailability and delivery. As a solution, we have previously reported about L-type amino acid transporter 1 (LAT1)-utilizing derivatives with increased brain delivery and efficacy. In the present study, we evaluated the pharmacokinetics and antioxidative efficacy of the two derivatives in peripheral mouse tissues. Furthermore, we quantified the LAT1 expression in studied tissues with a targeted proteomics method to verify the transporter expression in mouse tissues. Additionally, the safety of the derivatives was assessed by exploring their effects on hemostasis in human plasma, erythrocytes, and endothelial cells. We found that both derivatives accumulated substantially in the pancreas, with over a 100-times higher area under curve compared to the FA. Supporting the pharmacokinetics, the LAT1 was highly expressed in the mouse pancreas. Treating mice with the LAT1-utilizing derivative of FA lowered malondialdehyde and prostaglandin E2 production in the pancreas, highlighting its antioxidative efficacy. Additionally, the LAT1-utilizing derivatives were found to be hemocompatible in human plasma and endothelial cells. Since antioxidative derivative 1 was substantially delivered into the pancreas along the previously studied brain, the derivative can be considered as a safe dual-targeting drug candidate in both the pancreas and the brain.

Increased/Targeted Brain (Pro)Drug Delivery via Utilization of Solute Carriers (SLCs).

Membrane transporters have a crucial role in compounds' brain drug delivery. They allow not only the penetration of a wide variety of different compounds to cross the endothelial cells of the blood-brain barrier (BBB), but also the accumulation of them into the brain parenchymal cells. Solute carriers (SLCs), with nearly 500 family members, are the largest group of membrane transporters. Unfortunately, not all SLCs are fully characterized and used in rational drug design. However, if the structural features for transporter interactions (binding and translocation) are known, a prodrug approach can be utilized to temporarily change the pharmacokinetics and brain delivery properties of almost any compound. In this review, main transporter subtypes that are participating in brain drug disposition or have been used to improve brain drug delivery across the BBB via the prodrug approach, are introduced. Moreover, the ability of selected transporters to be utilized in intrabrain drug delivery is discussed. Thus, this comprehensive review will give insights into the methods, such as computational drug design, that should be utilized more effectively to understand the detailed transport mechanisms. Moreover, factors, such as transporter expression modulation pathways in diseases that should be taken into account in rational (pro)drug development, are considered to achieve successful clinical applications in the future.

The Influence of Selected Antipsychotic Drugs on Biochemical Aspects of Alzheimer's Disease.

The aim of this study was to assess the potency of selected antipsychotic drugs (haloperidol (HAL), bromperidol (BRMP), benperidol (BNP), penfluridol (PNF), pimozide (PIM), quetiapine (QUET) and promazine (PROM)) on the main pathological hallmarks of Alzheimer's disease (AD). Binary mixtures of donepezil and antipsychotics produce an anti-BuChE effect, which was greater than either compound alone. The combination of rivastigmine and antipsychotic drugs (apart from PNF) enhanced AChE inhibition. The tested antipsychotics (excluding HAL and PNF) significantly reduce the early stage of Aß aggregation. BRMP, PIM, QUET and PROM were found to substantially inhibit Aß aggregation after a longer incubation time. A test of human erythrocytes hemolysis showed that short-term incubation of red blood cells (RBCs) with QUET resulted in decreased hemolysis. The antioxidative properties of antipsychotics were also proved in human umbilical vein endothelial cells (HUVEC); all tested drugs were found to significantly increase cell viability. In the case of astrocytes, BNP, PNF, PIM and PROM showed antioxidant potential.

Structural Comparison of Sulfonamide-Based Derivatives That Can Improve Anti-Coagulation Properties of Metformin.

Due to its high efficiency, good safety profile, and potential cardio-protective properties, metformin, a dimethyl biguanide, is the first-line medication in antihyperglycemic treatment for type 2 diabetic patients. The aim of our present study was to assess the effects of eight new sulfonamide-based derivatives of metformin on selected plasma parameters and vascular hemostasis, as well as on endothelial and smooth muscle cell function. The compounds with an alkyl chain (1-3), trifluoromethyl substituent (4), or acetyl group (5) significantly elevated glucose utilization in human umbilical endothelial cells (HUVECs), similarly to metformin. Our novel findings showed that metformin analogues 1-3 presented the most beneficial properties because of their greatest safety profile in the WST-1 cell viability assay, which was also proved in the further HUVEC integrity studies using RTCA DP. Compounds 1-3 did not affect either HUVEC or aortal smooth muscle cell (AoSMC) viability up to 3.0 mM. Importantly, these compounds beneficially affected some of the coagulation parameters, including factor X and antithrombin III activity. In contrast to the above-mentioned metformin analogues, derivatives 4 and 5 exerted more profound anticoagulation effects; however, they were also more cytotoxic towards HUVECs, as IC50 values were 1.0-1.5 mM. In conclusion, the chemical modification of a metformin scaffold into sulfonamides possessing alkyl substituents results in the formation of novel derivatives with potential bi-directional activity including anti-hyperglycemic properties and highly desirable anti-coagulant activity.

Current Chemical, Biological, and Physiological Views in the Development of Successful Brain-Targeted Pharmaceutics.

One of the greatest challenges with successful pharmaceutical treatments of central nervous system (CNS) diseases is the delivery of drugs into their target sites with appropriate concentrations. For example, the physically tight blood-brain barrier (BBB) effectively blocks compounds from penetrating into the brain, also by the action of metabolizing enzymes and efflux transport mechanisms. However, many endogenous compounds, including both smaller compounds and macromolecules, like amino acids, sugars, vitamins, nucleosides, hormones, steroids, and electrolytes, have their peculiar internalization routes across the BBB. These delivery mechanisms, namely carrier-mediated transport and receptor-mediated transcytosis have been utilized to some extent in brain-targeted drug development. The incomplete knowledge of the BBB and the smaller than a desirable number of chemical tools have hindered the development of successful brain-targeted pharmaceutics. This review discusses the recent advancements achieved in the field from the point of medicinal chemistry view and discusses how brain drug delivery can be improved in the future.

Sulfonamide metformin derivatives induce mitochondrial-associated apoptosis and cell cycle arrest in breast cancer cells.

Metformin, an oral anti-diabetic drug, has attracted scientific attention due to its anti-cancer effects. This biguanide exerts preventive effects against cancer, and interferes with cancer-promoting signaling pathways at the cellular level. However, the direct cytotoxic or anti-proliferative effect of the drug is observed at very high concentrations, often exceeding 5-10 mM. This paper presents the synthesis of eight novel sulfonamide-based biguanides with improved cellular uptake in two breast cancer cell lines (MCF-7 and MDA-MB-231), and evaluates their effects on cancer cell growth. The synthesized sulfonamide-based analogues of metformin (1-5) were efficiently taken up in MCF-7 and MDA-MB-231 cells, and were characterized by stronger cytotoxic properties than those of metformin. Generally, compounds were more effective in MCF-7 than in MDA-MB-231. Compound 2, with an n-octyl chain, was the most active molecule with IC50 = 114.0 µmol/L in MCF-7 cells. The cytotoxicity of compound 2 partially results from its ability to induce early and late apoptosis. Increased intracellular reactive oxygen species (ROS) production and reduced mitochondrial membrane potential suggest that compound 2 promotes mitochondrial dysfunction and activates the mitochondrial-associated apoptosis-signaling pathway. In addition, compound 2 was also found to arrest cell cycle in the G0/G1 and G2/M phase and significantly inhibit cancer cell migration. In conclusion, this study supports the hypothesis that improved transporter-mediated cellular uptake of potential drug molecule is accompanied by its increased cytotoxicity. Therefore, compound 2 is a very good example of how chemical modification of a biguanide scaffold can affect its biological properties and improve anti-neoplastic potential.

Ganciclovir and Its Hemocompatible More Lipophilic Derivative Can Enhance the Apoptotic Effects of Methotrexate by Inhibiting Breast Cancer Resistance Protein (BCRP).

Efflux transporters, namely ATP-binding cassette (ABC), are one of the primary reasons for cancer chemoresistance and the clinical failure of chemotherapy. Ganciclovir (GCV) is an antiviral agent used in herpes simplex virus thymidine kinase (HSV-TK) gene therapy. In this therapy, HSV-TK gene is delivered together with GCV into cancer cells to activate the phosphorylation process of GCV to active GCV-triphosphate, a DNA polymerase inhibitor. However, GCV interacts with efflux transporters that are responsible for the resistance of HSV-TK/GCV therapy. In the present study, it was explored whether GCV and its more lipophilic derivative (1) could inhibit effluxing of another chemotherapeutic, methotrexate (MTX), out of the human breast cancer cells. Firstly, it was found that the combination of GCV and MTX was more hemocompatible than the corresponding combination with compound 1. Secondly, both GCV and compound 1 enhanced the cellular accumulation of MTX in MCF-7 cells, the MTX exposure being 13-21 times greater compared to the MTX uptake alone. Subsequently, this also reduced the number of viable cells (41-56%) and increased the number of late apoptotic cells (46-55%). Moreover, both GCV and compound 1 were found to interact with breast cancer resistant protein (BCRP) more effectively than multidrug-resistant proteins (MRPs) in these cells. Since the expression of BCRP was higher in MCF-7 cells than in MDA-MB-231 cells, and the cellular uptake of GCV and compound 1 was smaller but increased in the presence of BCRP-selective inhibitor (Fumitremorgin C) in MCF-7 cells, we concluded that the improved apoptotic effects of higher MTX exposure were raised mainly from the inhibition of BCRP-mediated efflux of MTX. However, the effects of GCV and its derivatives on MTX metabolism and the quantitative expression of MTX metabolizing enzymes in various cancer cells need to be studied more thoroughly in the future.

Incorporation of Sulfonamide Moiety into Biguanide Scaffold Results in Apoptosis Induction and Cell Cycle Arrest in MCF-7 Breast Cancer Cells.

Metformin, apart from its glucose-lowering properties, has also been found to demonstrate anti-cancer properties. Anti-cancer efficacy of metformin depends on its uptake in cancer cells, which is mediated by plasma membrane monoamine transporters (PMAT) and organic cation transporters (OCTs). This study presents an analysis of transporter mediated cellular uptake of ten sulfonamide-based derivatives of metformin in two breast cancer cell lines (MCF-7 and MDA-MB-231). Effects of these compounds on cancer cell growth inhibition were also determined. All examined sulfonamide-based analogues of metformin were characterized by greater cellular uptake in both MCF-7 and MDA-MB-231 cells, and stronger cytotoxic properties than those of metformin. Effective intracellular transport of the examined compounds in MCF-7 cells was accompanied by high cytotoxic activity. For instance, compound 2 with meta-methyl group in the benzene ring inhibited MCF-7 growth at micromolar range (IC50 = 87.7 ± 1.18 µmol/L). Further studies showed that cytotoxicity of sulfonamide-based derivatives of metformin partially results from their ability to induce apoptosis in MCF-7 and MDA-MB-231 cells and arrest cell cycle in the G0/G1 phase. In addition, these compounds were found to inhibit cellular migration in wound healing assay. Importantly, the tested biguanides are more effective in MCF-7 cells at relatively lower concentrations than in MDA-MB-231 cells, which proves that the effectiveness of transporter-mediated accumulation in MCF-7 cells is related to biological effects, including MCF-7 cell growth inhibition, apoptosis induction and cell cycle arrest. In summary, this study supports the hypothesis that effective transporter-mediated cellular uptake of a chemical molecule determines its cytotoxic properties. These results warrant a further investigation of biguanides as putative anti-cancer agents.

Hemocompatible L-Type amino acid transporter 1 (LAT1)-Utilizing prodrugs of perforin inhibitors can accumulate into the pancreas and alleviate inflammation-induced apoptosis.

Cytolytic pore-forming protein, perforin, has been associated with autoimmune destruction of pancreatic ß-cells in type 1 diabetes mellitus (T1DM) once released from CD8+ T cells. Curiously, perforinopathy has also been implicated in numerous brain diseases. Therefore, inhibitors of perforin have been in demand with targeted delivery in mind. l-Type amino acid transporter 1 (LAT1) is known to be expressed in both the above-mentioned target tissues, in the pancreas as well as in the brain. Thus, in the present study, the distribution of two LAT1-utilizing prodrugs of investigational perforin inhibitors into the pancreas was explored after intraperitoneal (i.p., 30 µmol/kg) bolus injection to mice. The effects of prodrug 1 were also studied in lipopolysaccharide (LPS)-induced in vitro (50 µg/mL) and in vivo (250 µg/kg x 3 days) apoptosis and pancreatitis models by determining the cellular apoptotic levels with human umbilical vein endothelial cells (HUVEC) and pancreatic caspase-3/-7 activity in mice. Furthermore, the biocompatibility of prodrug 1 was explored in human plasma and towards red blood cells. According to the results, both prodrugs were accumulated more effectively into the pancreas than their parent drugs (in addition to the brain that has been previously reported). Prodrug 1 (30 µmol/kg) also decreased the pancreatic caspase-3/-7 activity (52%) and with 2.5 µM concentration, the number of early and late apoptotic cells (32-53%). Since prodrug 1 was also found to be hemocompatible and not affecting human plasma hemostasis or inducing hemolysis of erythrocytes at the concentration <50 µM, it can be considered biocompatible in systemic circulation and ready to be studied in the future as a dual-acting drug candidate (in the pancreas and brain) in diseases like T1DM with neurodegenerative comorbidities.

Molecular characteristics supporting l-Type amino acid transporter 1 (LAT1)-mediated translocation.

l-Type amino acid transporter 1 (LAT1) is an interesting protein due to its peculiar expression profile. It can be utilized not only as a carrier for improved or targeted drug delivery, e.g., into the brain but also as a target protein by which amino acid supply can be restricted, e.g., from the cancer cells. The recognition and binding processes of LAT1-ligands, such as amino acids and clinically used small molecules, including l-dopa, gabapentin, and melphalan, are today well-known. Binding to LAT1 is crucial, particularly when designing the LAT1-inhibitors. However, it will not guarantee effective translocation across the cell membrane via LAT1, which is a definite requirement for LAT1-substrates, such as drugs that elicit their pharmacological effects inside the cells. Therefore, in the present study, the accumulation of known LAT1-utilizing compounds into the selected LAT1-expressing cancer cells (MCF-7) was explored experimentally over a time period. The differences found among the transport efficiency and affinity of the studied compounds for LAT1 were subsequently explained by docking the ligands into the human LAT1 model (based on the recent cryo-electron microscopy structure). Thus, the findings of this study clarify the favorable structural requirements of the size, shape, and polarity of the ligands that support the translocation and effective transport across the cell membrane via LAT1. This knowledge can be applied in future drug design to attain improved or targeted drug delivery and hence, successful LAT1-utilizing drugs with increased therapeutic effects.