Aswani Dutt Vadlapudi

Approaches for Enhancing Oral Bioavailability of Peptides and Proteins

Oral delivery of peptide and protein drugs faces immense challenge partially due to the gastrointestinal (GI) environment. In spite of considerable efforts by industrial and academic laboratories, no major breakthrough in the effective oral delivery of polypeptides and proteins has been accomplished. Upon oral administration, gastrointestinal epithelium acts as a physical and biochemical barrier for absorption of proteins resulting in low bioavailability (typically less than 1-2%). An ideal oral drug delivery system should be capable of (a) maintaining the integrity of protein molecules until it reaches the site of absorption, (b) releasing the drug at the target absorption site, where the delivery system appends to that site by virtue of specific interaction, and (c) retaining inside the gastrointestinal tract irrespective of its transitory constraints. Various technologies have been explored to overcome the problems associated with the oral delivery of macromolecules such as insulin, gonadotropin-releasing hormones, calcitonin, human growth factor, vaccines, enkephalins, and interferons, all of which met with limited success. This review article intends to summarize the physiological barriers to oral delivery of peptides and proteins and novel pharmaceutical approaches to circumvent these barriers and enhance oral bioavailability of these macromolecules.

Novel Strategies for Anterior Segment Ocular Drug Delivery

Research advancements in pharmaceutical sciences have led to the development of new strategies in drug delivery to anterior segment. Designing a new delivery system that can efficiently target the diseased anterior ocular tissue, generate high drug levels, and maintain prolonged and effective concentrations with no or minimal side effects is the major focus of current research. Drug delivery by traditional method of administration via topical dosing is impeded by ocular static and dynamic barriers. Various products have been introduced into the market that prolong drug retention in the precorneal pocket and to improve bioavailability. However, there is a need of a delivery system that can provide controlled release to treat chronic ocular diseases with a reduced dosing frequency without causing any visual disturbances. This review provides an overview of anterior ocular barriers along with strategies to overcome these ocular barriers and deliver therapeutic agents to the affected anterior ocular tissue with a special emphasis on nanotechnology-based drug delivery approaches.

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.

Novel Nanomicellar Formulation Approaches for Anterior and Posterior Segment Ocular Drug Delivery

One of the most challenging areas of pharmaceutical research is ocular drug delivery. The unique anatomy and physiology of the eye impedes drug permeation to deeper ocular tissues. Nanosized carrier systems such as nanoparticles, liposomes, suspensions, dendrimers, and nanomicelles are being explored for ocular drug delivery. In this review, we have focused on application of emerging nanomicellar carrier systems in ocular drug delivery. Nanomicelles are nanosized vesicular carriers formed from amphiphilic monomer units. Surfactant and polymeric micellar nanocarriers provide an amenable means to improve drug solubilization, develop clear aqueous formulations and deliver drugs to anterior and posterior ocular tissues. Nanomicelles due to their amphiphilic nature encapsulate hydrophobic drugs and aid in drug delivery. Various methods are employed to develop nanosized micellar formulations depending upon the physicochemical properties of the drug. Nanomicellar carriers appear to be promising vehicles with potential applications in ocular drug delivery. In this review, we attempted to discuss about the progress in ocular drug delivery research using nanomicelles as carriers from the published literature and issued patents. Also, with regards to ocular static and dynamic barriers which prevent drug permeation, a brief discussion about nanomicelles, types of nanomicelles, their methods of preparation and micellar strategy to overcome ocular barriers, delivering therapeutic levels of drugs to anterior and posterior ocular tissues are discussed.

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.

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-1β). In normoxic conditions, de novo synthesized cytoplasmic HIF-1α is degraded by 26S proteasome. Under hypoxic conditions, HIF-1α is stabilized, binds with HIF-1β 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.

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.

Nanomicelles: an emerging platform for drug delivery to the eye

Delivery of pharmacologically active drugs at therapeutic concentrations to the target tissues with minimal/no toxicity to the healthy ocular tissues still remains a significant challenge for ocular pharmacologists. The complex anatomy and physiology of the eye restricts drug entry to the desired site of action, thus rendering it a highly protected organ

Interaction of gatifloxacin with efflux transporters: A possible mechanism for drug resistance

The purpose of the study is to screen the interactions of fourth generation fluoroquinolone-gatifloxacin with efflux pumps, i.e., P-gp, MRP2 and BCRP. Mechanism of gatifloxacin interaction with efflux transporters may explain its acquired resistance. Such clarification may lead to the development of strategies to overcome efflux and enhance its bioavailability at target site. This process will aid in the reduction of dose volume, further eliminating the chances of systemic toxicity from topical gatifloxacin eye drops. MDCK cell lines transfected with the targeted efflux transporters were used for this study. [(14)C] Erythromycin was selected as a model substrate for P-gp and MRP2 whereas Hoechst 33342 was employed as a substrate for BCRP. Uptake and transport studies of these substrates were performed in the presence of gatifloxacin to delineate its interaction with efflux transporters. Further the efflux ratio in the presence of gatifloxacin was calculated from bidirectional transport studies. The concentration of [(14)C] erythromycin and Hoechst 33342 was measured using scintillation counter and fluorescence plate reader, respectively. A concentration dependent inhibition effect in the presence of gatifloxacin was revealed on [(14)C] erythromycin uptake. The efflux ratio (BL-AP/AP-BL) of substrates was found to approach unity at higher gatifloxacin concentrations. Increased concentration of gatifloxacin did not elevate uptake of Hoechst 33342. All these studies were validated with known inhibitors as positive control. Uptake and transport studies support the hypothesis that gatifloxacin is a substrate for P-gp, MRP2 but not for BCRP. Possible interactions of gatifloxacin with P-gp and MRP2 may be a possible mechanism for acquired resistance of gatifloxacin. This information can be further extended to design prodrugs or formulations in order to prevent development of acquired resistance and improve therapeutic efficacy with its reduction in side effects.

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.