Transporter Protein Expression of Corneal Epithelium in Rabbit and Porcine: Evaluation of Models for Ocular Drug Transport Study.

The transcorneal route is the main entry route for drugs to the intraocular parts, after topical administration. The outer surface, the corneal epithelium (CE), forms the rate-limiting barrier for drug permeability. Information about the role and protein expression of drug and amino acid transporter proteins in the CE is sparse and lacking. The aim of our study was to characterize transporter protein expression in rabbit and porcine CE to better understand potential drug and nutrient absorption after topical administration. Proteins, mainly Abc and Slc transporters, were characterized with quantitative targeted absolute proteomics and global untargeted proteomics methods. In the rabbit CE, 24 of 48 proteins were detected in the targeted approach, and 21 of these were quantified. In the porcine CE, 26 of 58 proteins were detected in the targeted approach, and 20 of these were quantified. Among these, 15 proteins were quantified in both animals: 4f2hc (Slc3a2), Aqp0, Asct1 (Slc1a4), Asct2 (Slc1a5), Glut1 (Slc2a1), Hmit (Slc2a13), Insr, Lat1 (Slc7a5), Mct1 (Slc16a1), Mct2 (Slc16a7), Mct4 (Slc16a3), Mrp 4 (Abcc4), Na+/K+-ATPase, Oatp3a1 (Slco3a1), and Snat2 (Slc38a2). Overall, the global proteomics results supported the targeted proteomics results. Organic anion transporting polypeptide Oatp3a1 was detected and quantified for the first time in both rabbit (1.4 ± 0.4 fmol/cm2) and porcine (11.1 ± 5.3 fmol/cm2) CE. High expression levels were observed for L-type amino acid transporter, Lat1, which was quantified with newly selected extracellular domain peptides in rabbit (48.9 ± 11.8 fmol/cm2) and porcine (37.6 ± 11.5 fmol/cm2) CE. The knowledge of transporter protein expression in ocular barriers is a key factor in the successful design of new ocular drugs, pharmacokinetic modeling, understanding ocular diseases, and the translation to human.

Sodium-Dependent Neutral Amino Acid Transporter 2 Can Serve as a Tertiary Carrier for l-Type Amino Acid Transporter 1-Utilizing Prodrugs.

Membrane transporters are the key determinants of the homeostasis of endogenous compounds in the cells and their exposure to drugs. However, the substrate specificities of distinct transporters can overlap. In the present study, the interactions of l-type amino acid transporter 1 (LAT1)-utilizing prodrugs with sodium-coupled neutral amino acid transporter 2 (SNAT2) were explored. The results showed that the cellular uptake of LAT1-utilizing prodrugs into a human breast cancer cell line, MCF-7 cells, was mediated via SNATs as the uptake was increased at higher pH (8.5), decreased in the absence of sodium, and inhibited in the presence of unselective SNAT-inhibitor, (α-(methylamino)isobutyric acid, MeAIB). Moreover, docking the compounds to a SNAT2 homology model (inward-open conformation) and further molecular dynamics simulations and the subsequent trajectory and principal component analyses confirmed the chemical features supporting the interactions of the studied compounds with SNAT2, which was found to be the main SNAT expressed in MCF-7 cells.

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.

Pharmacoproteomics of Brain Barrier Transporters and Substrate Design for the Brain Targeted Drug Delivery.

One of the major reasons why central nervous system (CNS)-drug development has been challenging in the past, is the barriers that prevent substances entering from the blood circulation into the brain. These barriers include the blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), blood-cerebrospinal fluid barrier (BCSFB), and blood-arachnoid barrier (BAB), and they differ from each other in their transporter protein expression and function as well as among the species. The quantitative expression profiles of the transporters in the CNS-barriers have been recently revealed, and in this review, it is described how they affect the pharmacokinetics of compounds and how these expression differences can be taken into account in the prediction of brain drug disposition in humans, an approach called pharmacoproteomics. In recent years, also structural biology and computational resources have progressed remarkably, enabling a detailed understanding of the dynamic processes of transporters. Molecular dynamics simulations (MDS) are currently used commonly to reveal the conformational changes of the transporters and to find the interactions between the substrates and the protein during the binding, translocation in the transporter cavity, and release of the substrate on the other side of the membrane. The computational advancements have also aided in the rational design of transporter-utilizing compounds, including prodrugs that can be actively transported without losing potency towards the pharmacological target. In this review, the state-of-art of these approaches will be also discussed to give insights into the transporter-mediated drug delivery to the CNS.

Exploring the Biochemical Foundations of a Successful GLUT1-Targeting Strategy to BNCT: Chemical Synthesis and In Vitro Evaluation of the Entire Positional Isomer Library of ortho-Carboranylmethyl-Bearing Glucoconjugates.

Boron neutron capture therapy (BNCT) is a noninvasive binary therapeutic modality applicable to the treatment of cancers. While BNCT offers a tumor-targeting selectivity that is difficult to match by other means, the last obstacles preventing the full harness of this potential come in the form of the suboptimal boron delivery strategies presently used in the clinics. To address these challenges, we have developed delivery agents that target the glucose transporter GLUT1. Here, we present the chemical synthesis of a number of ortho-carboranylmethyl-substituted glucoconjugates and the biological assessment of all positional isomers. Altogether, the study provides protocols for the synthesis and structural characterization of such glucoconjugates and insights into their essential properties, for example, cytotoxicity, GLUT1-affinity, metabolism, and boron delivery capacity. In addition to solidifying the biochemical foundations of a successful GLUT1-targeting approach to BNCT, we identify the most promising modification sites in d-glucose, which are critical in order to further develop this strategy toward clinical use.

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.

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.

Comparison of Experimental Strategies to Study l-Type Amino Acid Transporter 1 (LAT1) Utilization by Ligands.

l-Type amino acid transporter 1 (LAT1), expressed abundantly in the brain and placenta and overexpressed in several cancer cell types, has gained a lot of interest in drug research and development, as it can be utilized for brain-targeted drug delivery, as well as inhibiting the essential amino acid supply to cancer cells. The structure of LAT1 is today very well-known and the interactions of ligands at the binding site of LAT1 can be modeled and explained. However, less is known of LAT1's life cycle within the cells. Moreover, the functionality of LAT1 can be measured by several different methods, which may vary between the laboratories and make the comparison of the results challenging. In the present study, the usefulness of indirect cis-inhibition methods and direct cellular uptake methods and their variations to interpret the interactions of LAT1-ligands were evaluated. Moreover, this study also highlights the importance of understanding the intracellular kinetics of LAT1-ligands, and how they can affect the regular function of LAT1 in critical tissues, such as the brain. Hence, it is discussed herein how the selected methodology influences the outcome and created knowledge of LAT1-utilizing compounds.

Hemocompatible LAT1-inhibitor can induce apoptosis in cancer cells without affecting brain amino acid homeostasis.

Increased amounts of amino acids are essential for cancer cells to support their sustained growth and survival. Therefore, inhibitors of amino acid transporters, such as L-type amino acid transporter 1 (LAT1) have been developed. In this study, a previously reported LAT1-inhibitor (KMH-233) was studied for its hemocompatibility and toxicity towards human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (AoSMCs). Furthermore, the cytotoxic effects against human breast adenocarcinoma cells (MCF-7) and its ability to affect mammalian (or mechanistic) target of rapamycin (mTOR) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling were evaluated. Moreover, the effects of this inhibitor to modulate LAT1 function on the cell surface and the brain amino acid homeostasis were evaluated after intraperitoneal (i.p.) administration of LAT1-inhibitor (23 µmol/kg) in mice. The results showed that LAT1-inhibitor (KMH-233) is hemocompatible at concentrations below 25 µM and it does not affect coagulation in plasma. However, it can reduce the total protein amount of mTOR and NF-κB, resulting in increased apoptosis in LAT1-expressing cancer cells. Most importantly, the inhibitor did not affect mouse brain levels of L-Leu, L-Tyr or L-Trp or modulate the function of LAT1 on the MCF-7 cell surface. Therefore, this inhibitor can be considered as a safe but effective anti-cancer agent. However, due to the compensative mechanism of cancer cells for their increased amino acid demand, this compound is most effective inducing apoptosis when used in combinations with other chemotherapeutics, such as protease inhibitor, bestatin, as demonstrated in this study.

Formulating of the sustained release of Tebuconazole pesticide using chitosan aerogel reinforced NFC/CaCO3 nanocomposite.

Chitosan-based aerogels were fabricated through utilizing of nanofibrillated cellulose (NFC)/CaCO3 composites. Chitosan aerogel and extra three aerogels loaded different concentrations of NFC/CaCO3 were investigated to explore their release efficiency of Tebuconazole pesticides. Results obtained from ATR-FTIR showed a remarkable decline of the characterized chitosan hydroxyl group peak prolonging with appearance of new peaks assigned to the inclusion of inorganic calcium element. Also, SEM images showed chitosan aerogel with regular porous structure increased by incorporation with of NFC/CaCO3 nanocomposite, while EDS affirmed the presence of calcium element rather pristine chitosan aerogel. In addition to this, the physical characterizations showed significant improvement in swelling properties for aerogels incorporated NFC/CaCO3 nanocomposite at low ratios. Chitosan aerogel reinforced NFC/CaCO3 nanocomposite exhibited benefit on loading and release efficiency of Tebuconazole. All samples showed accessibility to column release method with fastest release at low slow rate 2 mL/min as giving chance for diffusion and solubility of ingredient, while release increase as heat increase as result of pore expansion. In conclusion, chitosan aerogels incorporated calcium carbonate showed better-sustained release of Tebuconazole pesticides than pristine chitosan aerogel. The produced aerogels loaded NFC/CaCO3 nanocomposite could be promising for controlled release of pesticides at water-streams in agriculture sector.

Decoration of viscose fibers with silver nanoparticle-based titanium-organic framework for use in environmental applications.

To effectively remove pharmaceuticals, nitroaromatic compounds, and dyes from wastewater, an efficient multifunctional material was created based on silver nanoparticles (Ag) and MIL-125-NH2 (MOF) immobilized on viscose fibers (VF) as a support substrate. Firstly, silver nanoparticles (Ag) were immobilized on the surface of viscose fibers (VF) via in situ synthesis using trisodium citrate (TSC) as a reducing agent to create (VF-Ag). Then, VF and VF-Ag were decorated with the titanium metal-organic framework MIL-125-NH2 (MOF) to create VF-MOF and VF-Ag-MOF. The influence of VF-Ag, VF-MOF, and VF-Ag-MOF on the sonocatalytic or sonophotocatalytic degradation of sulfa drugs was investigated. The results show that VF-Ag-MOF showed excellent sonocatalytic and sonophotocatalytic activity towards the degradation of sulfa drugs compared to VF-Ag and VF-MOF. Furthermore, sonophotodegradation showed a dramatic enhancement in the efficiency of degradation of sulfa drugs compared to sonodegradation. The sonophotodegradation degradation percentage of sulfanilamide, sulfadiazine, and sulfamethazine drugs in the presence of VF-Ag-MOF was 65, 90, and 95 after 45 min of ultrasonic and visible light irradiation. The catalytic activity of VF-Ag, VF-MOF, and VF-Ag-MOF was evaluated through the conversion of p-nitrophenol (4-NP) to p-aminophenol (4-AP). The results demonstrate that VF-Ag-MOF had the highest catalytic activity, followed by VF-Ag and VF-MOF. The conversion percentage of 4-NP to 4-AP was 69%. The catalytic or photocatalytic effects of VF-Ag, VF-MOF, and VF-Ag-MOF on the elimination of methylene blue (MB) dye were investigated. The results demonstrate that VF-Ag-MOF showed high efficiency in removing the MB dye through the reduction (65%) or photodegradation (71%) after 60 min. VF-Ag-MOF composites structure-activity relationships represent that doping within silver NPs enhanced the photocatalytic activity of MIL-125-NH2, which could be explained as follows: (i) Due to the formation of a Schottky barrier at the junction between MIL-125-NH2 and Ag NPs, the photogenerated electrons in the conduction band of MIL-125-NH2 were supposed to be quickly transferred to the valence band of the Ag NPs, and subsequently, the electrons were transferred to the conduction band of Ag NPs. This considerable electron transferring process, which is reported as Z scheme heterojunction, can efficiently suppress the recombination of electron/hole pairs in VF-Ag-MIL-125-NH2 composites. (ii) Sufficient separation between the photogenerated charge carriers (holes and electrons) and avoiding their recombination enhanced the photocatalytic activity of composites.

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.

Physiologically based pharmacokinetics modeling and transporter proteomics to predict systemic and local liver and muscle disposition of statins.

Statins are used to reduce liver cholesterol levels but also carry a dose-related risk of skeletal muscle toxicity. Concentrations of statins in plasma are often used to assess efficacy and safety, but because statins are substrates of membrane transporters that are present in diverse tissues, local differences in intracellular tissue concentrations cannot be ruled out. Thus, plasma concentration may not be an adequate indicator of efficacy and toxicity. To bridge this gap, we used physiologically based pharmacokinetic (PBPK) modeling to predict intracellular concentrations of statins. Quantitative data on transporter clearance were scaled from in vitro to in vivo conditions by integrating targeted proteomics and transporter kinetics data. The developed PBPK models, informed by proteomics, suggested that organic anion-transporting polypeptide 2B1 (OATP2B1) and multidrug resistance-associated protein 1 (MRP1) play a pivotal role in the distribution of statins in muscle. Using these PBPK models, we were able to predict the impact of alterations in transporter function due to genotype or drug-drug interactions on statin systemic concentrations and exposure in liver and muscle. These results underscore the potential of proteomics-guided PBPK modeling to scale transporter clearance from in vitro data to real-world implications. It is important to evaluate the role of drug transporters when predicting tissue exposure associated with on- and off-target effects.

Specific transport of temozolomide does not override DNA repair-mediated chemoresistance.

Temozolomide (TMZ) a DNA alkylating agent, is the standard-of-care for brain tumors, such as glioblastoma multiforme (GBM). Although the physicochemical and pharmacokinetic properties of TMZ, such as chemical stability and the ability to cross the blood-brain barrier (BBB), have been questioned in the past, the acquired chemoresistance has been the main limiting factor of long-term clinical use of TMZ. In the present study, an L-type amino acid transporter 1 (LAT1)-utilizing prodrug of TMZ (TMZ-AA, 6) was prepared and studied for its cellular accumulation and cytotoxic properties in human squamous cell carcinoma, UT-SCC-28 and UT-SCC-42B cells, and TMZ-sensitive human glioma, U-87MG cells that expressed functional LAT1. TMZ-AA 6 accumulated more effectively than TMZ itself into those cancer cells that expressed LAT1 (UT-SCC-42B). However, this did not correlate with decreased viability of treated cells. Indeed, TMZ-AA 6, similarly to TMZ itself, required adjuvant inhibitor(s) of DNA-repair systems, O6-methylguanine-DNA methyl transferase (MGMT) and base excision repair (BER), as well as active DNA mismatch repair (MMR), for maximal growth inhibition. The present study shows that improving the delivery of this widely-used methylating agent is not the main barrier to improved chemotherapy, although utilizing a specific transporter overexpressed at the BBB or glioma cells can have targeting advantages. To obtain a more effective anticancer prodrug, the compound design focus should shift to altering the major DNA alkylation site or inhibiting DNA repair systems.

Altering distribution profile of palbociclib by its prodrugs.

Palbociclib, a cyclin-dependent kinase (CDK) 4/6 inhibitor, is currently used clinically for treating hormone receptor-positive and human epidermal growth factor receptor 2 negative breast cancer. Additionally, it has the potential to be utilized in the treatment of various tumors, including malignant glioblastoma. Previous research has indicated that palbociclib is a substrate for two efflux transporters, P-glycoprotein (P-gp; MDR1) and breast cancer-resistant protein (BCRP), which restrict the brain exposure of palbociclib. In the present study, our objective was to alter the brain distribution pattern of palbociclib by creating and assessing two novel prodrugs through in vitro, in situ, and in vivo evaluations. To this end, we synthesized two prodrugs of palbociclib by attaching it to the tyrosine promoiety at the para- (PD1) and meta-(PD2) position via a carbamate bond. We hypothesized that the prodrugs could bypass efflux transporter-mediated drug resistance by leveraging the l-type amino acid transporter (LAT1) to facilitate their transport across the blood-brain barrier (BBB) and into cancer cells, such as glioma cells that express LAT1. The compounds PD1 and PD2 did not show selective binding and had limited inhibitory effects on LAT1 in three cell lines (MCF-7, U87-MG, HEK-hLAT1). However, PD1 and PD2 demonstrated the ability to evade efflux mechanisms, and their in vitro uptake profiles were comparable to that of palbociclib, indicating their potential for effective cellular transport. In in situ and in vivo studies, brain uptake was not significantly improved compared to palbociclib, but the pharmacokinetic profiles showed encouraging enhancements. PD1 exhibited a higher AUCbrain/plasma ratio, suggesting safer dosing, while PD2 showed favorable long-acting pharmacokinetics. Although our prodrug design did not significantly improve palbociclib brain delivery due to the potential size limitation of the prodrugs, the study provides valuable insights for future prodrug development and drug delivery strategies targeting specific transporters.

Fungi associated with ornamental plants in some Nurseries in Al-Qurayyat, Jouf region, Saudi Arabia.

Background and Objectives: Fungi communities are important soil components as decomposers and plant symbionts, and they play an important part in natural ecological and biogeochemical processes. In this study, isolation and identification of terrestrial and zoosporic fungi were detected. Materials and Methods: Sixty-seven fungal species from thirty-four genera were isolated from 45 soil samples obtained randomly from nurseries in Al-Qurayyat, Jouf reagon, Saudi Arabia using the soil dilution technique on glucose-Czapek's agar medium, cellulose-Czapek's agar, and Potato dextrose agar medium.Authentic fungus manuals were then used to identify and characterise the mycoflora. Results: A total of 46 fungal species belonging to 22 terrestrial fungal genera were recovered on glucose-Czapek's agar, 38 species belonging to 20 terrestrial fungal genera were recovered on cellulos-Czapek's agar and 27 fungal species belonging to 15 terrestrial fungal genera were recovered on PDA medium while 12 species belonging to 7 genera zoosporic fungal genera were discovered. Conclusion: The most common terrestrial fungal genera were Aspergillus, Penicillium, Fusarium, Trichoderma, Acremonium, and Cladosopium while in zoosporic fungus. Allomyces was the most prevalent, followed by Achlya and Pythium.

Self-crosslinked polyvinyl alcohol/cellulose nanofibril cryogels loaded with synthesized aminophosphonates as antimicrobial wound dressings.

Microbial infection is the most common obstacle in the wound healing process, leading to wound healing impairment and complications and ultimately increasing morbidity and mortality. Due to the rising number of pathogens evolving resistance to the existing antibiotics used for wound care, alternative approaches are urgently required. In this study, α-aminophosphonate derivatives as antimicrobial agents were synthesized and incorporated into self-crosslinked tri-component cryogels composed of fully hydrolyzed polyvinyl alcohol (PVA-F), partially hydrolyzed polyvinyl alcohol (PVA-P), and cellulose nanofibrils (CNFs). Initially, the antimicrobial activity of four α-aminophosphonate derivatives against selected skin bacterial species was tested and their minimum inhibitory concentration was determined based on which the most effective compound was loaded into the cryogels. Next, the physical and mechanical properties of cryogels with various blending ratios of PVA-P/PVA-F and fixed amounts of CNFs were assessed, and drug release profiles and biological activities of drug-loaded cryogels were analyzed. Assessment of α-aminophosphonate derivatives showed the highest efficacy of a cinnamaldehyde-based derivative (Cinnam) against both Gram-negative and Gram-positive bacteria compared to other derivatives. The physical and mechanical properties of cryogels showed that PVA-P/PVA-F with a 50/50 blending ratio had the highest swelling ratio (1600%), surface area (523 m2 g-1), and compression recoverability (72%) compared to that with other blending ratios. Finally, antimicrobial and biofilm development studies showed that the cryogel loaded with a Cinnam amount of 2 mg (relative to polymer weight) showed the most sustained drug release profile over 75 h and had the highest efficacy against Gram-negative and Gram-positive bacteria. In conclusion, self-crosslinked tri-component cryogels loaded with the synthesized α-aminophosphonate derivative, having both antimicrobial and anti-biofilm formation properties, can have a significant impact on the management of uprising wound infection.

Targeting Glial Cells by Organic Anion-Transporting Polypeptide 1C1 (OATP1C1)-Utilizing l-Thyroxine-Derived Prodrugs.

OATP1C1 (organic anion-transporting polypeptide 1C1) transports thyroid hormones, particularly thyroxine (T4), into human astrocytes. In this study, we investigated the potential of utilizing OATP1C1 to improve the delivery of anti-inflammatory drugs into glial cells. We designed and synthesized eight novel prodrugs by incorporating T4 and 3,5-diiodo-l-tyrosine (DIT) as promoieties to selected anti-inflammatory drugs. The prodrug uptake in OATP1C1-expressing human U-87MG glioma cells demonstrated higher accumulation with T4 promoiety compared to those with DIT promoiety or the parent drugs themselves. In silico models of OATP1C1 suggested dynamic binding for the prodrugs, wherein the pose changed from vertical to horizontal. The predicted binding energies correlated with the transport profiles, with T4 derivatives exhibiting higher binding energies when compared to prodrugs with a DIT promoiety. Interestingly, the prodrugs also showed utilization of oatp1a4/1a5/1a6 in mouse primary astrocytes, which was further supported by docking studies and a great potential for improved brain drug delivery.

Aminopeptidase B can bioconvert L-type amino acid transporter 1 (LAT1)-utilizing amide prodrugs in the brain.

A prodrug approach is a powerful method to temporarily change the physicochemical and thus, pharmacokinetic properties of drugs. However, in site-selective targeted prodrug delivery, tissue or cell-specific bioconverting enzyme is needed to be utilized to release the active parent drug at a particular location. Unfortunately, ubiquitously expressed enzymes, such as phosphatases and carboxylesterases are well used in phosphate and ester prodrug applications, but less is known about enzymes selectively expressed, e.g., in the brain and enzymes that can hydrolyze more stable prodrug bonds, such as amides and carbamates. In the present study, L-type amino acid transporter 1 (LAT1)-utilizing amide prodrugs bioconverting enzyme was identified by gradually exploring the environment and possible determinants, such as pH and metal ions, that affect amide prodrug hydrolysis. Based on inducement by cobalt ions and slightly elevated pH (8.5) as well as localization in plasma, liver, and particularly in the brain, aminopeptidase B was proposed to be responsible for the bioconversion of the majority of the studied amino acid amide prodrugs. However, this enzyme hydrolyzed only those prodrugs that contained an aromatic promoiety (L-Phe), while leaving the aliphatic promoeities (L-Lys) and the smallest prodrug (with L-Phe promoiety) intact. Moreover, the parent drugs' structure (flexibility and the number of aromatic rings) largely affected the bioconversion rate. It was also noticed in this study, that there were species differences in the bioconversion rate by aminopeptidase B (rodents > human), although the in vitro-in vivo correlation of the studied prodrugs was relatively accurate.

Enhanced drug delivery by a prodrug approach effectively relieves neuroinflammation in mice.

AIMS: Neuroinflammation is a prominent hallmark in several neurodegenerative diseases (NDs). Halting neuroinflammation can slow down the progression of NDs. Improving the efficacy of clinically available non-steroidal anti-inflammatory drugs (NSAIDs) is a promising approach that may lead to fast-track and effective disease-modifying therapies for NDs. Here, we aimed to utilize the L-type amino acid transporter 1 (LAT1) to improve the efficacy of salicylic acid as an example of an NSAID prodrug, for which brain uptake and intracellular localization have been reported earlier. MAIN METHODS: Firstly, we confirmed the improved LAT1 utilization of the salicylic acid prodrug (SA-AA) in freshly isolated primary mouse microglial cells. Secondly, we performed behavioural rotarod, open field, and four-limb hanging tests in mice, and a whole-brain proteome analysis. KEY FINDINGS: The SA-AA prodrug alleviated the lipopolysaccharide (LPS)-induced inflammation in the rotarod and hanging tests. The proteome analysis indicated decreased neuroinflammation at the molecular level. We identified 399 proteins linked to neuroinflammation out of 7416 proteins detected in the mouse brain. Among them, Gps2, Vamp8, Slc6a3, Slc18a2, Slc5a7, Rgs9, Lrrc1, Ppp1r1b, Gnal, and Adcy5/6 were associated with the drug's effects. The SA-AA prodrug attenuated the LPS-induced neuroinflammation through the regulation of critical pathways of neuroinflammation such as the cellular response to stress and transmission across chemical synapses. SIGNIFICANCE: The efficacy of NSAIDs can be improved via the utilization of LAT1 and repurposed for the treatment of neuroinflammation. This improved brain delivery and microglia localisation can be applied to other inflammatory modulators to achieve effective and targeted CNS therapies.