Novel biotinylated lipid prodrugs of acyclovir for the treatment of herpetic keratitis (HK): transporter recognition, tissue stability and antiviral activity.

PURPOSE: Biotinylated lipid prodrugs of acyclovir (ACV) were designed to target the sodium dependent multivitamin transporter (SMVT) on the cornea to facilitate enhanced cellular absorption of ACV. METHODS: All the prodrugs were screened for in vitro cellular uptake, interaction with SMVT, docking analysis, cytotoxicity, enzymatic stability and antiviral activity. RESULTS: Uptake of biotinylated lipid prodrugs of ACV (B-R-ACV and B-12HS-ACV) was significantly higher than biotinylated prodrug (B-ACV), lipid prodrugs (R-ACV and 12HS-ACV) and ACV in corneal cells. Transepithelial transport across rabbit corneas indicated the recognition of the prodrugs by SMVT. Average Vina scores obtained from docking studies further confirmed that biotinylated lipid prodrugs possess enhanced affinity towards SMVT. All the prodrugs studied did not cause any cytotoxicity and were found to be safe and non-toxic. B-R-ACV and B-12HS-ACV were found to be relatively more stable in ocular tissue homogenates and exhibited excellent antiviral activity. CONCLUSIONS: Biotinylated lipid prodrugs demonstrated synergistic improvement in cellular uptake due to recognition of the prodrugs by SMVT on the cornea and lipid mediated transcellular diffusion. These biotinylated lipid prodrugs appear to be promising drug candidates for the treatment of herpetic keratitis (HK) and may lower ACV resistance in patients with poor clinical response.

Honokiol inhibits HIF pathway and hypoxia-induced expression of histone lysine demethylases.

Hypoxia-inducible-factor (HIF)-mediated expression of pro-angiogenic genes under hypoxic conditions is the fundamental cause of pathological neovascularization in retinal ischemic diseases and cancers. Recent studies have shown that histone lysine demethylases (KDMs) play a key role in the amplification of HIF signaling and expression of pro-angiogenic genes. Thus, the inhibitors of the HIF pathway or KDMs can have profound therapeutic value for diseases caused by pathological neovascularization. Here, we show that hypoxia-mediated expression of KDMs is a conserved process across multiple cell lines. Moreover, we report that honokiol, a biphenolic phytochemical extracted from Magnolia genus which has been used for thousands of years in the traditional Japanese and Chinese medicine, is a potent inhibitor of the HIF pathway as well as hypoxia-induced expression of KDMs in a number of cancer and retinal pigment epithelial cell lines. Further, treating the cells with honokiol leads to inhibition of KDM-mediated induction of pro-angiogenic genes (adrenomedullin and growth differentiation factor 15) under hypoxic conditions. Our results provide an evidence-based scientific explanation for therapeutic benefits observed with honokiol and warrant its further clinical evaluation for the treatment of pathological neovascularization in retinal ischemic diseases and cancers.

Transfected MDCK cell line with enhanced expression of CYP3A4 and P-glycoprotein as a model to study their role in drug transport and metabolism.

The aim of this study was to characterize and utilize MDCK cell line expressing CYP3A4 and P-glycoprotein as an in vitro model for evaluating drug-herb and drug-drug of abuse interactions. MDCK cell line simultaneously expressing P-gp and CYP3A4 (MMC) was developed and characterized by using expression and activity studies. Cellular transport study of 200 µM cortisol was performed to determine their combined activity. The study was carried across MDCK-WT, MDCK-MDR1 and MMC cell lines. Similar studies were also carried out in the presence of 50 µM naringin and 3 µM morphine. Samples were analyzed by HPLC for drug and its CYP3A4 metabolite. PCR, qPCR and Western blot studies confirmed the enhanced expression of the proteins in the transfected cells. The Vivid CYP3A4 assay and ketoconazole inhibition studies further confirmed the presence of active protein. Apical to basal transport of cortisol was found to be 10- and 3-fold lower in MMC as compared to MDCK-WT and MDCK-MDR1 respectively. Higher amount of metabolite was formed in MMC than in MDCK-WT, indicating enhanced expression of CYP3A4. Highest cortisol metabolite formation was observed in MMC cell line due to the combined activities of CYP3A4 and P-gp. Transport of cortisol increased 5-fold in the presence of naringin in MMC and doubled in MDCK-MDR1. Cortisol transport in MMC was significantly lower than that in MDCK-WT in the presence of naringin. The permeability increased 3-fold in the presence of morphine, which is a weaker inhibitor of CYP3A4. Formation of 6ß-hydroxy cortisol was found to decrease in the presence of morphine and naringin. This new model cell line with its enhanced CYP3A4 and P-gp levels in addition to short culture time can serve as an invaluable model to study drug-drug interactions. This cell line can also be used to study the combined contribution of efflux transporter and metabolizing enzymes toward drug-drug interactions.

Hypoxia induced expression of histone lysine demethylases: implications in oxygen-dependent retinal neovascular diseases.

Hypoxia inducible factor (HIF) plays a critical role in cellular adaptation to hypoxia by regulating the expression of essential genes. Pathological activation of this pathway leads to the expression of pro-angiogenic factors during the neovascularization in cancer and retinal diseases. Little is known about the epigenetic regulations during HIF-mediated transcription and activation of pro-angiogenic genes in oxygen-dependent retinal diseases. Here, we show that hypoxia induces the expression of a number of histone lysine demethylases (KDMs) in retinal pigment epithelial cells. Moreover, we show that the expression of pro-angiogenic genes (ADM, GDF15, HMOX1, SERPE1 and SERPB8) is dependent on KDMs under hypoxic conditions. Further, treating the cells with a general KDM inhibitor blocks the expression of these pro-angiogenic genes. Results from these studies identify a new layer of epigenetic transcription regulation under hypoxic conditions and suggest that specific inhibitors of KDMs such as JMJD1A can be a new therapeutic approach to treat diseases caused by the hypoxia induced neovascularization in cancer and retinal diseases.

Uptake and Permeability Studies to Delineate the Role of Efflux Transporters.

Chemotherapy is one of the major therapeutic interventions in oncology. Despite numerous advances and intensive research, a large number of patients acquire multidrug resistance (MDR) and no longer respond to chemotherapy. Efflux transporters play a predominant role in mediating MDR. Cellular accumulation (uptake) and permeability studies serve as invaluable methods to detect drug efflux/transport mechanism. These methods are generally performed on transfected cells (e.g., MDCKII-MDR1, MDCKII-MRP2, and MDCKII-BCRP) or cells expressing high amount of intrinsic efflux transporters (Caco-2) utilizing specific inhibitors as positive controls. This chapter presents a method of performing uptake and permeability studies, including the preparation of various buffers required for the study.

Functional characterization and molecular identification of vitamin C transporter (SVCT2) in human corneal epithelial (HCEC) and retinal pigment epithelial (D407) cells.

PURPOSE: The main goal of this study is to investigate the existence of sodium-dependent vitamin C transport system (SVCT2) and to define time-dependent uptake mechanism and intracellular regulation of ascorbic acid (AA) in human corneal epithelial (HCEC) and human retinal pigment epithelial (D407) cells. METHODS: Uptake of [(14)C] AA was studied in HCEC and D407 cells. Functional aspects of [(14)C] AA uptake were studied in the presence of different concentrations of unlabeled AA, pH, temperature, metabolic inhibitors, substrates and structural analogs. Molecular identification of SVCT2 was examined with reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Uptake of [(14)C] AA was observed to be sodium, chloride, temperature, pH and energy-dependent in both cell lines. [(14)C] AA uptake was found to be saturable, with Km values of 46.14 ± 6.03 and 47.26 ± 3.24 µM and Vmax values of 17.34 ± 0.58 and 31.86 ± 0.56 pmol/min/mg protein, across HCEC and D407 cells, respectively. The process is inhibited by structural analogs (L-AA and D-Iso AA) but not by structurally unrelated substrates (glucose and PAHA). Ca(++)/calmodulin and protein kinase pathways play an important role in modulating uptake of AA. A 626 bp band corresponding to a vitamin C transporter (SVCT2) has been identified by RT-PCR analysis in both the cell lines. CONCLUSION: This research article reports regarding the ascorbic acid uptake mechanism, kinetics and regulation by sodium dependent vitamin C transporter (SVCT2) in HCEC and D407 cells. Also, SVCT2 can be utilized for targeted delivery in enhancing ocular permeation and bioavailability of highly potent ophthalmic drugs.

Aqueous nanomicellar formulation for topical delivery of biotinylated lipid prodrug of acyclovir: formulation development and ocular biocompatibility.

PURPOSE: The objective of this study was to develop a clear, aqueous nanomicellar formulation and evaluate its in vitro ocular biocompatibility as a novel carrier for topical ocular delivery of biotinylated lipid prodrug for the treatment of herpetic keratitis. METHODS: Micellar formulation of Biotin-12Hydroxystearic acid-acyclovir (B-12HS-ACV) was prepared by solvent evaporation/film hydration method with two nonionic surfactants, vitamin E TPGS and octoxynol-40. The optimized formulation was characterized for various parameters including micelle size, polydispersity index (PDI), and zeta-potential and in vitro prodrug release. Human corneal epithelial cells (HCECs) were employed for studying the cytotoxicity of the formulation. Further, mRNA expression levels of various cytokines were also studied with quantitative real-time PCR (qPCR). RESULTS: Average size was 10.46±0.05 nm with a PDI of 0.086 for blank nanomicelles, and 10.78±0.09 nm with a PDI of 0.075 for prodrug-loaded nanomicelles. Both unloaded and prodrug-loaded nanomicelles had low negative zeta potential. Prodrug encapsulation efficiency of mixed nanomicelles was calculated to be ∼90%. Transmission electron microscopy analysis revealed that nanomicelles were spherical, homogenous, and devoid of aggregates. B-12HS-ACV release from nanomicelles was slow with no significant burst effect. Results show a sustained release of the prodrug from nanomicelles over a period of 4 days. Neither the blank formulation nor the prodrug-loaded micellar formulation demonstrated any cytotoxic effects. Further, incubation of HCECs with blank and prodrug-loaded nanomicellar groups did not significantly alter the expression levels of IL-1ß, IL-6, IL-8, IL-17, TNF-α, and IFN-γ. CONCLUSIONS: In summary, a topical clear, aqueous nanomicellar formulation comprised of vitamin E TPGS and octoxynol-40 loaded with 0.1% B-12HS-ACV was successfully developed. B-12HS-ACV-loaded nanomicelles are small in size, spherical, and homogenous, without any aggregates. The micellar formulations were perfectly transparent similar to pure water. Ocular biocompatibility studies indicated that mixed nanomicelles were nontoxic and noninflammatory to corneal epithelial cells. Therefore, nanomicellar technology represents a promising strategy for the delivery of biotinylated lipid prodrugs of ACV.

Role of membrane transporters and metabolizing enzymes in ocular drug delivery.

Ocular disorders can significantly lower a patient's quality of life. Centers for Disease Control and Prevention's Vision Health Initiative have estimated that the number of people affected by age-related ocular diseases may be doubled in the United States by 2030. Although availability of newer therapeutics has improved the prognosis of ocular diseases, poor ocular bioavailability still remains a major concern. Combinations of pharmacodynamic and pharmacokinetic barriers have been known to determine the amount of drug delivered to the target tissue. However, presence of membrane transporters and metabolizing enzymes pose a significant challenge to ocular drug disposition. Scientific literature confirms the expression of efflux/ATP-binding cassette transporters, influx/solute carrier transporters and several metabolic enzymes including oxidoreductases, hydrolases and transferases in different ocular tissues. Therefore, this review article describes the anatomical features of the eye and various barriers regulating ocular drug disposition. Differential expression of membrane transporters and metabolizing enzymes in normal and diseased states are briefly discussed. Further, the significance of transporter- metabolism interplay in ophthalmic drug design and various ocular drug delivery strategies are also outlined.

Binary and ternary combinations of anti-HIV protease inhibitors: effect on gene expression and functional activity of CYP3A4 and efflux transporters.

BACKGROUND: The purpose of this study is to identify the effect of binary and ternary combinations of anti-HIV protease inhibitors (PIs) on the expression of metabolizing enzyme (CYP3A4) and efflux transporters [multidrug resistance-associated protein 2 (MRP2), P-glycoprotein (P-gp) and breast cancer resistant protein (BCRP)] in a model intestinal cell line (LS-180). METHODS: LS-180 cells were treated with various combinations of PIs (amprenavir, indinavir, saquinavir and lopinavir), and the mRNA expression levels of metabolizing enzyme and efflux transporters were measured using quantitative reverse transcription polymerase chain reaction. The alteration of gene expression was further correlated to the expression of nuclear hormone receptor PXR. Uptake of fluorescent and radioactive substrates was carried out to study the functional activity of these proteins. Cytotoxicity and adenosine triphosphate (ATP) assays were carried out to measure stress responses. RESULTS: Binary and ternary combinations of PIs appeared to modulate the expression of CYP3A4, MRP2, P-gp and BCRP in a considerable manner. Unlike the individual PIs, their binary combinations showed much greater induction of metabolizing enzyme and efflux proteins. However, such pronounced induction was not observed in the presence of ternary combinations. The observed trend of altered mRNA expression was found to correlate well with the change in expression levels of PXR. The gene expression was found to correlate with activity assays. Lack of cytotoxicity and ATP activity was observed in the treatment samples, suggesting that these alterations in expression levels were probably not stress responses. CONCLUSIONS: In the present study, we demonstrated that combinations of drugs can have serious consequences toward the treatment of HIV infection by altering their bioavailability and disposition.

Clinically relevant drug-drug interactions between antiretrovirals and antifungals.

INTRODUCTION: Complete delineation of the HIV-1 life cycle has resulted in the development of several antiretroviral drugs. Twenty-five therapeutic agents belonging to five different classes are currently available for the treatment of HIV-1 infections. Advent of triple combination antiretroviral therapy has significantly lowered the mortality rate in HIV patients. However, fungal infections still represent major opportunistic diseases in immunocompromised patients worldwide. AREAS COVERED: Antiretroviral drugs that target enzymes and/or proteins indispensable for viral replication are discussed in this article. Fungal infections, causative organisms, epidemiology and preferred treatment modalities are also outlined. Finally, observed/predicted drug-drug interactions between antiretrovirals and antifungals are summarized along with clinical recommendations. EXPERT OPINION: Concomitant use of amphotericin B and tenofovir must be closely monitored for renal functioning. Due to relatively weak interactive potential with the CYP450 system, fluconazole is the preferred antifungal drug. High itraconazole doses (> 200 mg/day) are not advised in patients receiving booster protease inhibitor (PI) regimen. Posaconazole is contraindicated in combination with either efavirenz or fosamprenavir. Moreover, voriconazole is contraindicated with high-dose ritonavir-boosted PI. Echinocandins may aid in overcoming the limitations of existing antifungal therapy. An increasing number of documented or predicted drug-drug interactions and therapeutic drug monitoring may aid in the management of HIV-associated opportunistic fungal infections.

Hypoxia-inducible factor-1 (HIF-1): a potential target for intervention in ocular neovascular diseases.

Constant oxygen supply is essential for proper tissue development, homeostasis and function of all eukaryotic organisms. Cellular response to reduced oxygen levels is mediated by the transcriptional regulator hypoxia-inducible factor-1 (HIF-1). It is a heterodimeric complex protein consisting of an oxygen dependent subunit (HIF-1α) and a constitutively expressed nuclear subunit (HIF-1ß). In normoxic conditions, de novo synthesized cytoplasmic HIF-1α is degraded by 26S proteasome. Under hypoxic conditions, HIF-1α is stabilized, binds with HIF-1ß and activates transcription of various target genes. These genes play a key role in regulating angiogenesis, cell survival, proliferation, chemotherapy, radiation resistance, invasion, metastasis, genetic instability, immortalization, immune evasion, metabolism and stem cell maintenance. This review highlights the importance of hypoxia signaling in development and progression of various vision threatening pathologies such as diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration and glaucoma. Further, various inhibitors of HIF-1 pathway that may have a viable potential in the treatment of oxygen-dependent ocular diseases are also discussed.

Recent patents and emerging therapeutics for HIV infections: a focus on protease inhibitors.

The inclusion of protease inhibitors (PIs) in highly active antiretroviral therapy has significantly improved clinical outcomes in HIV-1-infected patients. To date, PIs are considered to be the most important therapeutic agents for the treatment of HIV infections. Despite high anti-HIV-1 potency, poor oral bioavailability of PIs has been a major concern. For achieving therapeutic concentrations, large doses of PIs are administered, which results in unacceptable systemic toxicities. Such severe and long-term toxicities necessitate the development of safer and potentially promising PIs. Recently, considerable attention has been paid to the development of newer compounds capable of inhibiting wild-type and resistant HIV-1 protease. Some of these PIs have displayed potent HIV-1 protease inhibitory activity. In this review, we have made an attempt to provide an overview on clinically approved and newly developing PIs, and related recent patents in the development of novel PIs.

Mechanisms of drug resistance in cancer chemotherapy: coordinated role and regulation of efflux transporters and metabolizing enzymes.

Cancer remains one of the major leading causes of death worldwide. Acquisition of multidrug resistance (MDR) remains a major impediment to successful chemotherapy. As the name implies, MDR is not limited only to one drug but often associated to structurally and functionally unrelated chemotherapeutics. Extensive research and investigations have identified several mechanisms underlying the development of MDR. This process of drug resistance is considered to be multifactorial including decreased drug accumulation, increased efflux, increased biotransformation, drug compartmentalization, modification of drug targets and defects in cellular pathways. In the first part of the review, these pharmacokinetic and pharmacodynamic mechanisms have been described in brief. Although the pathways can act independently, they are more often intertwined. Of the various mechanisms involved, up-regulation of efflux transporters and metabolizing enzymes constitute a major resistance phenotype. This review also provides a general biological overview of important efflux transporters and metabolizing enzymes involved in MDR. Further, synergistic action between efflux transporters and metabolizing enzymes leading to MDR could possibly arise due to two different factors; overlapping substrate specificity and coordinated regulation of their expression. The expression of efflux transporters and metabolizing enzymes is governed by nuclear receptors, mainly pregnane X receptor (PXR). The pharmacological role of PXR and advances in the development of PXR antagonists to overcome MDR are outlined.

Biotin uptake by T47D breast cancer cells: functional and molecular evidence of sodium-dependent multivitamin transporter (SMVT).

The objective of this study was to investigate functional and molecular evidence of carrier mediated system responsible for biotin uptake in breast cancer (T47D) cells and to delineate mechanism of intracellular regulation of this transporter. Cellular accumulation of [3H] biotin was studied in T47D and normal mammary epithelial (MCF-12A) cells. Reverse transcription polymerase chain reaction (RT-PCR) was carried out to confirm the molecular expression of sodium dependent multivitamin transporter (SMVT) in T47D cells. Quantitative real time PCR analysis was also performed to compare the relative expression of SMVT in T47D and MCF-12A cells. [3H] biotin uptake by T47D cells was found to be concentration dependent with K(m) of 9.24 µM and V(max) of 27.34 pmol/mg protein/min. Uptake of [3H] biotin on MCF-12A cells was also found to be concentration dependent and saturable, but with a relatively higher K(m) (53.10 µM) indicating a decrease in affinity of biotin uptake in normal breast cells compared to breast cancer cells. [3H] biotin uptake appears to be time-, temperature-, pH- and sodium ion-dependent but independent of energy and chloride ions. [3H] biotin uptake was significantly inhibited in the presence of biotin, its structural analog desthiobiotin, pantothenic acid and lipoic acid. Concentration dependent inhibition of biotin uptake was evident in the presence of valeric acid which possesses free carboxyl group and biocytin and NHS biotin which are devoid of free carboxyl group. No significant inhibition was observed in the presence of structurally unrelated vitamins (ascorbic acid, folic acid, nicotinic acid, thiamine, pyridoxine and riboflavin). Modulators of PTK, PKC and PKA mediated pathways had no effect, but uptake in presence of calmidazolium (calcium-calmodulin inhibitor) was significantly inhibited. [3H] biotin uptake in the presence of calmidazolium was found to be saturable with a K(m) and V(max) values of 13.49 µM and 11.20 pmol/mg protein/min, respectively. A band of SMVT mRNA at 774 bp was identified by RT-PCR analysis. Quantitative real time PCR confirmed higher expression of SMVT in T47D cells relative to MCF-12A cells. All these studies demonstrated for the first time the functional and molecular expression of sodium dependent multivitamin transporter (SMVT), a specific carrier-mediated system for biotin uptake, in human derived breast cancer (T47D) cells. The present study also indicated that cancer cells could import more vitamin compared to normal breast cells possibly for maintaining high proliferative status. We investigated the likelihood of selecting this cell line (T47D) as an in vitro cell culture model to study biotin-conjugated anti-cancer drugs/drug delivery systems.

Molecular expression and functional activity of efflux and influx transporters in hypoxia induced retinal pigment epithelial cells.

A decrease in tissue oxygen levels (aka hypoxia) mediates a number of vascular retinal diseases. Despite introduction of novel therapeutics, treatment of retinal disorders remains challenging, possibly due to complex nature of hypoxia signaling. To date, the differential effect of hypoxia on expression of efflux and influx transporters in retinal cells has not been studied. Therefore, the objective of this study was to delineate molecular and functional expression of membrane transporters in human retinal pigment epithelial (RPE) cells cultured under normoxic and hypoxic conditions. Quantitative real time polymerase chain reaction (qPCR), ELISA and immunoblot analysis were performed to examine the RNA and protein expression levels of transporters. Further, functional activity was evaluated by performing the uptake of various substrates in both normoxic and hypoxic conditions. qPCR analysis showed elevated expression of efflux transporters (P-glycoprotein, multidrug resistant protein 2, breast cancer resistant protein) and influx transporters (folate receptor-α, cationic and neutral amino acid transporter, sodium dependent multivitamin transporter) in a time dependent manner. Immunoblot analysis further confirmed elevated expression of breast cancer resistant protein and sodium dependent multivitamin transporter. A decrease in the uptake of efflux transporter substrates (digoxin, lopinavir and abacavir) and enhanced uptake of influx transporter substrates (arginine, folic acid and biotin) in hypoxia relative to normoxia further confirmed elevated expression of transporters, respectively. This study demonstrates for the first time that hypoxic conditions may alter expression of efflux and influx transporters in RPE cells. These findings suggest that hypoxia may further alter disposition of ophthalmic drugs.

Molecular and functional characterization of riboflavin specific transport system in rat brain capillary endothelial cells.

Riboflavin is an important water soluble vitamin (B2) required for metabolic reactions, normal cellular growth, differentiation and function. Mammalian brain cells cannot synthesize riboflavin and must import from systemic circulation. However, the uptake mechanism, cellular translocation and intracellular trafficking of riboflavin in brain capillary endothelial cells are poorly understood. The primary objective of this study is to investigate the existence of a riboflavin-specific transport system and delineate the uptake and intracellular regulation of riboflavin in immortalized rat brain capillary endothelial cells (RBE4). The uptake of [3H]-riboflavin is sodium, temperature and energy dependent but pH independent. [3H]-Riboflavin uptake is saturable with K(m) and V(max) values of 19 ± 3 µM and 0.235 ± 0.012 pmol/min/mg protein, respectively. The uptake process is inhibited by unlabelled structural analogs (lumiflavin, lumichrome) but not by structurally unrelated vitamins. Ca(++)/calmodulin and protein kinase A (PKA) pathways are found to play an important role in the intracellular regulation of [3H]-riboflavin. Apical and baso-lateral uptake of [3H]-riboflavin clearly indicates that a riboflavin specific transport system is predominantly localized on the apical side of RBE4 cells. A 628 bp band corresponding to a riboflavin transporter is revealed in RT-PCR analysis. These findings, for the first time report the existence of a specialized and high affinity transport system for riboflavin in RBE4 cells. The blood-brain barrier (BBB) is a major obstacle limiting drug transport inside the brain as it regulates drug permeation from systemic circulation. This transporter can be utilized for targeted delivery in enhancing brain permeation of highly potent drugs on systemic administration.

Molecular expression and functional activity of sodium dependent multivitamin transporter in human prostate cancer cells.

Nutrient transporters expressed on cell membrane have been targeted for enhancing bioavailability of poorly permeable drugs. Sodium dependent multivitamin transporter (SMVT) is once such carrier system, utilized for improving drug targeting to specific tissues. Therefore, the main objective of this study is to characterize SMVT in human derived prostate cancer cells (PC-3). Reverse transcription polymerase chain reaction (RT-PCR) analysis has provided product band at 774 bp, specific to SMVT. The mechanism and intracellular regulation of [3H]-biotin is also studied. [3H]-biotin uptake is found to be time and concentration dependent with K(m) and V(max) values of 19±2 µM and 23±1 pmol/min/mg protein, respectively. The uptake process is saturable in micromolar concentration range but linear in nanomolar concentration range. [3H]-biotin uptake shows significant sodium, temperature, pH and energy dependency. The process is strongly inhibited by unlabeled biotin and structural analogs such as desthiobiotin, pantothenate, lipoate and valeric acid. Intracellular regulatory pathways such as Ca(2+)/calmodulin and PKC pathway but not PTK pathway appears to play an important role in modulating [3H]-biotin uptake. This study for the first time confirms the molecular expression of SMVT and demonstrates that SMVT, responsible for biotin uptake is functionally active in PC-3 cells.

Sodium dependent multivitamin transporter (SMVT): a potential target for drug delivery.

Sodium dependent multivitamin transporter (SMVT; product of the SLC5A6 gene) is an important transmembrane protein responsible for translocation of vitamins and other essential cofactors such as biotin, pantothenic acid and lipoic acid. Hydropathy plot (Kyte-Dolittle algorithm) revealed that human SMVT protein consists of 635 amino acids and 12 transmembrane domains with both amino and carboxyl termini oriented towards the cytoplasm. SMVT is expressed in various tissues such as placenta, intestine, brain, liver, lung, kidney, cornea, retina and heart. This transporter displays broad substrate specificity and excellent capacity for utilization in drug delivery. Drug absorption is often limited by the presence of physiological (epithelial tight junctions), biochemical (efflux transporters and enzymatic degradation) and chemical (size, lipophilicity, molecular weight, charge etc.) barriers. These barriers may cause many potential therapeutics to be dropped from the preliminary screening portfolio and subsequent entry into the market. Transporter targeted delivery has become a powerful approach to deliver drugs to target tissues because of the ability of the transporter to translocate the drug to intracellular organelles at a higher rate. This review highlights studies employing SMVT transporter as a target for drug delivery to improve bioavailability and investigate the feasibility of developing SMVT targeted drug delivery systems.

Functional and molecular aspects of biotin uptake via SMVT in human corneal epithelial (HCEC) and retinal pigment epithelial (D407) cells.

Sodium-dependent multivitamin transporter (SMVT) is a vital transmembrane protein responsible for translocating biotin and other essential cofactors such as pantothenate and lipoate. Unlike primary cultures of corneal and retinal pigment epithelial (RPE) cells, immortalized cells can be subcultured many times, yet maintain their physiological properties. Hence, the purpose of this study was to delineate the functional and molecular aspects of biotin uptake via SMVT on immortalized human corneal epithelial (HCEC) and RPE (D407) cells. Functional aspects of [(3)H] biotin uptake were studied in the presence of different concentrations of unlabeled biotin, pH, temperature, metabolic inhibitors, ions, substrates, structural analogs and biotinylated prodrug (Biotin-Acyclovir (B-ACV)). Molecular identity of SMVT was examined with reverse transcription-polymerase chain reaction. Biotin uptake was found to be saturable in HCEC and D407 cells with K (m) of 296.2 ± 25.9 and 863.8 ± 66.9 µM and V (max) of 77.2 ± 2.2 and 308.3 ± 10.7 pmol/mg protein/min, respectively. Uptake was found to be pH, temperature, energy, and sodium-dependent. Inhibition of biotin uptake was observed in the presence of structural analogs and specific substrates. Further, uptake was lowered in the presence of B-ACV indicating the translocation of biotinylated prodrug by SMVT. A distinct band at 774 bp confirmed the molecular existence of SMVT in both the cells. This study shows for the first time the functional and molecular presence of SMVT in HCEC and D407 cells. Therefore, these cell lines may be utilized as in vitro models to study the cellular translocation of biotin-conjugated prodrugs.

Targeted lipid based drug conjugates: a novel strategy for drug delivery.

A majority of studies involving prodrugs are directed to overcome low bioavailability of the parent drug. The aim of this study is to increase the bioavailability of acyclovir (ACV) by designing a novel prodrug delivery system which is more lipophilic, and at the same time site specific. In this study, a lipid raft has been conjugated to the parent drug molecule to impart lipophilicity. Simultaneously a targeting moiety that can be recognized by a specific transporter/receptor in the cell membrane has also been tethered to the other terminal of lipid raft. Targeted lipid prodrugs i.e., biotin-ricinoleicacid-acyclovir (B-R-ACV) and biotin-12hydroxystearicacid-acyclovir (B-12HS-ACV) were synthesized with ricinoleicacid and 12hydroxystearicacid as the lipophilic rafts and biotin as the targeting moiety. Biotin-ACV (B-ACV), ricinoleicacid-ACV (R-ACV) and 12hydroxystearicacid-ACV (12HS-ACV) were also synthesized to delineate the individual effects of the targeting and the lipid moieties. Cellular accumulation studies were performed in confluent MDCK-MDR1 and Caco-2 cells. The targeted lipid prodrugs B-R-ACV and B-12HS-ACV exhibited much higher cellular accumulation than B-ACV, R-ACV and 12HS-ACV in both cell lines. This result indicates that both the targeting and the lipid moiety act synergistically toward cellular uptake. The biotin conjugated prodrugs caused a decrease in the uptake of [(3)H] biotin suggesting the role of sodium dependent multivitamin transporter (SMVT) in uptake. The affinity of these targeted lipid prodrugs toward SMVT was studied in MDCK-MDR1 cells. Both the targeted lipid prodrugs B-R-ACV (20.25 ± 1.74 µM) and B-12HS-ACV (23.99 ± 3.20 µM) demonstrated higher affinity towards SMVT than B-ACV (30.90 ± 4.19 µM). Further, dose dependent studies revealed a concentration dependent inhibitory effect on [(3)H] biotin uptake in the presence of biotinylated prodrugs. Transepithelial transport studies showed lowering of [(3)H] biotin permeability in the presence of biotin and biotinylated prodrugs, further indicating a carrier mediated translocation by SMVT. Overall, results from these studies clearly suggest that these biotinylated lipid prodrugs of ACV possess enhanced affinity towards SMVT. These prodrugs appear to be potential candidates for the treatment of oral and ocular herpes virus infections, because of higher expression of SMVT on intestinal and corneal epithelial cells. In conclusion we hypothesize that our novel prodrug design strategy may help in higher absorption of hydrophilic parent drug. Moreover, this novel prodrug design can result in higher cell permeability of hydrophilic therapeutics such as genes, siRNA, antisense RNA, DNA, oligonucleotides, peptides and proteins.