Ravinder Earla

Development and validation of a fast and sensitive bioanalytical method for the quantitative determination of glucocorticoids--quantitative measurement of dexamethasone in rabbit ocular matrices by liquid chromatography tandem mass spectrometry.

A sensitive, selective, accurate and robust LC-MS/MS method was developed and validated for the quantitative determination of glucocorticoids in rabbit ocular tissues. Samples were processed by a simple liquid-liquid extraction procedure. Chromatographic separation was performed on Phenomenex reversed phase C18 gemini column (50mmx4.6mm i.d.,) with an isocratic mobile phase composed of 30% of acetonitrile in water containing 0.1% of formic acid, at a flow rate 0.2mL/min. Dexamethasone (DEX), prednisolone (PD) and hydrocortisone (HD) were detected with proton adducts at m/z 393.20-->355.30, 361.30-->147.20 and 363.20-->121.0 in multiple reaction monitoring (MRM) positive mode respectively. Finally, 50microL of 0.1% novel DEX mixed micellar formulation was topically administered to a rabbit eye and concentrations were measured. The method was validated over a linear concentration range of 2.7-617.6ng/mL. Lower limit of quantitation (LLOQ) of DEX and PD was measured in the concentration range of 2.7 and 11.0ng/mL respectively. The resulting method demonstrated intra and inter-day precision within 13.3% and 11.1% and accuracy within 19.3% and 12.5% for DEX and PD, respectively. Both analytes were found to be stable throughout freeze-thaw cycles and during bench top and postoperative stability studies (r(2)>0.999). DEX concentrations in various ocular tissue samples i.e., aqueous humor, cornea, iris ciliary body, sclera and retina choroid were found to be 344.0, 1050.07, 529.6, 103.9 and 48.5ng/mg protein respectively. Absorption of DEX after topical administration from a novel aqueous mixed micellar formulation achieved therapeutic concentration levels in posterior segment of the rabbit eye.

Novel nanoparticulate gel formulations of steroids for the treatment of macular edema.

PURPOSE This article describes the development and characterization of PLGA nanoparticles of dexamethasone (DEX), hydrocortisone acetate (HA), and prednisolone acetate (PA) suspended in thermosensitive gels indicated for the treatment of macular edema (ME). METHODS Nanoparticles were prepared by oil-in-water (O/W) emulsion and dialysis methods using PLGA 50:50 and PLGA 65:35. These particles were characterized for entrapment efficiency, size distribution, surface morphology, crystallinity, and in vitro release. Further, ex vivo permeation studies of DEX in suspension and nanoparticulate formulations were carried out across the rabbit sclera. RESULTS Entrapment efficiencies of DEX, HA, and PA were found to be lower with the dialysis method. O/W emulsion/solvent evaporation technique resulted in higher entrapment efficiencies, that is, 77.3%, 91.3%, 92.3% for DEX, HA, and PA, respectively. Release from nanoparticles suspended in thermosensitive gels followed zero-order kinetics with no apparent burst effect. Ex vivo permeability studies further confirmed sustained release of DEX from nanoparticles suspended in thermosensitive gels. CONCLUSIONS These novel nanoparticulate systems containing particles suspended in thermosensitive gels may provide sustained retina/choroid delivery of steroids following episcleral administration.

Molecular Expression and Functional Evidence of a Drug Efflux Pump (BCRP) in Human Corneal Epithelial Cells

Purpose: Breast Cancer Resistance Protein (BCRP) belongs to the family of efflux transporters involved in drug efflux leading to drug resistance. The objective of this study was to explore physical barriers for ocular drug absorption and to verify the presence and possible role of BCRP as a barrier for ocular drug resistance. Methods: Transfected human corneal epithelial cells (SV40-HCEC) wereselectedas an in vitro model for corneal epithelium with MDCKII-BCRP as positive control. [3H]-Mitoxantrone ([3H]-MTX), which is a proven substrate for organic anion transporter like BCRP, was selected as a model drug for functional expression studies. Fumetremorgin C (FTC), a known specific inhibitor for BCRP and GF120918, an inhibitor for BCRP and P-gp, were added to inhibit BCRP-mediated efflux. PGP-4008, a specific inhibitor of P-gp was used to delineate the contribution of P-gp. The mRNA extracted from cells was used for RT-PCR analysis and gene expression. Membrane fractions of SV40-HCEC and MDCKII-BCRP were used for immunoprecipitation followed by Western blot analysis. Results: Efflux was inhibited significantly in the presence of FTC and GF120918. Dose-dependent inhibition of efflux by BCRP was noticed in SV40-HCEC and MDCKII-BCRP in the presence of FTC and GF120918, and the efflux was ATP-dependent. The metabolic inhibitor, 2,4-DNP, significantly inhibited efflux. No pH-dependent efflux was noticed except at pH 5.5. RT-PCR analysis indicated a unique and distinct band at ∼ 429 bp, corresponding to BCRP in SV40-HCEC and MDCKII-BCRP cells. Western Blot analysis indicated a specific band at ∼ 70 kDa in the membrane fraction of SV40-HCEC and MDCKII-BCRP cells. Conclusions: We have demonstrated the expression of BCRP in human corneal epithelial cells and, for the first time, demonstrated its functional activity leading to drug efflux. RT-PCR and Western blot analysis further confirmed this finding.

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.

Effect of short term and chronic administration of Sutherlandia frutescens on pharmacokinetics of nevirapine in rats.

Sutherlandia frutescens (sutherlandia), an African herbal supplement was recommended by the South African Ministry of Health for the treatment of AIDS patients. However, no reports yet exist delineating the effect of sutherlandia on pharmacokinetics of antiretroviral agents. Therefore, this investigation aimed at screening the effects of short term and chronic exposure of sutherlandia on oral bioavailability and pharmacokinetics of nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor, in Sprague Dawley rats. NVP (6 mg/kg) was administered orally alone (control) and with co-administration of sutherlandia; short term (12 mg/kg single dose) and long term (12 mg/kg, once a day for 5 days). No significant difference in the pharmacokinetic parameters of NVP was found upon short-term co-administration of Sutherlandia. However, there was a 50% decrease (p<0.05) in the AUC and C(max) values of NVP after 5 days of chronic exposure with Sutherlandia. In addition, quantitative RT-PCR studies demonstrated a 2-3-fold increase in the hepatic and intestinal mRNA expression of CYP3A2, relative to vehicle control. To further confirm, if this could translate into a clinically relevant pharmacokinetic interaction in patients, we tested this hypothesis employing LS-180 cells as an in vitro induction model for human CYP3A4. Ninety-six hours post treatment, similar to positive control rifampicin (25 μM), sutherlandia extract (300 μg/mL) resulted in elevated m-RNA expression levels and functional activity of CYP3A4 (human homologue of rodent CYP3A2) in LS-180 cells. Taken together, these results suggest that a potential drug-herb interaction is possible when NVP is co-administered with S. frutescens, although this hypothesis still remains to be investigated in a clinical setting.

Nanomicellar Topical Aqueous Drop Formulation of Rapamycin for Back-of-the-Eye Delivery

The objective of this study was to develop a clear, aqueous rapamycin-loaded mixed nanomicellar formulations (MNFs) for the back-of-the-eye delivery. MNF of rapamycin (0.2%) was prepared with vitamin E tocopherol polyethylene glycol succinate (TPGS) (Vit E TPGS) and octoxynol-40 (Oc-40) as polymeric matrix. MNF was characterized by various parameters such as size, charge, shape, and viscosity. Proton nuclear magnetic resonance (1H NMR) was used to identify unentrapped rapamycin in MNF. Cytotoxicity was evaluated in human retinal pigment epithelial (D407) and rabbit primary corneal epithelial cells (rPCECs). In vivo posterior ocular rapamycin distribution studies were conducted in male New Zealand white rabbits. The optimized MNF has excellent rapamycin entrapment and loading efficiency. The average size of MNF was 10.98 ± 0.089 and 10.84 ± 0.11 nm for blank and rapamycin-loaded MNF, respectively. TEM analysis revealed that nanomicelles are spherical in shape. Absence of free rapamycin in the MNF was confirmed by 1H NMR studies. Neither placebo nor rapamycin-loaded MNF produced cytotoxicity on D407 and rPCECs indicating formulations are tolerable. In vivo studies demonstrated a very high rapamycin concentration in retina-choroid (362.35 ± 56.17 ng/g tissue). No drug was identified in the vitreous humor indicating the sequestration of rapamycin in lipoidal retinal tissues. In summary, a clear, aqueous MNF comprising of Vit E TPGS and Oc-40 loaded with rapamycin was successfully developed. Back-of-the-eye tissue distribution studies demonstrated a very high rapamycin levels in retina-choroid (place of drug action) with a negligible drug partitioning into vitreous humor.

Enhanced Nicotine Metabolism in HIV-1–Positive Smokers Compared with HIV-Negative Smokers: Simultaneous Determination of Nicotine and its Four Metabolites in Their Plasma Using a Simple and Sensitive Electrospray Ionization Liquid Chromatography–Tandem Mass Spectrometry Technique

Smoking is approximately three times more prevalent in HIV-1–positive than HIV-negative individuals in the United States. Nicotine, which is the major constituent of tobacco, is rapidly metabolized mainly by cytochrome P450 (CYP2A6) to many metabolites. In this study, we developed a simple, fast, and sensitive electrospray ionization liquid chromatography–tandem mass spectrometry method using a strong cation solid phase extraction, and determined the concentration of nicotine and its four major metabolites (cotinine, nornicotine, norcotinine, and trans-3′-hydroxycotinine) in the plasma of HIV-1–positive and HIV-negative smokers. The multiple reaction monitoring transitions for nicotine, cotinine, trans-3′-hydroxycotinine, nornicotine, norcotinine, nicotine-d4, and cotinine-d3 were selected at mass-to-charge ratios of 163.3/117.1, 177.5/80.3, 193.2/80.1, 149.5/132.3, 163.4/80.3, 167.3/121.4, and 180.3/101.2, respectively. The lower limit of quantitation for nicotine and its metabolites was 0.53 ng/ml, which is relatively more sensitive than those previously reported. The concentration of nicotine was detected 5-fold lower in HIV-1–positive smokers (7.17 ± 3.8 ng/ml) than that observed in HIV-negative smokers (33.29 ± 15.4 ng/ml), whereas the concentration of the metabolite nornicotine was 3-fold higher in HIV-1–positive smokers (6.8 ± 2.9 ng/ml) than in HIV-negative smokers (2.3 ± 1.2 ng/ml). Although it was statistically nonsignificant, the concentration of the metabolite cotinine was also higher in HIV-1–positive smokers (85.6 ± 60.5 ng/ml) than in HIV-negative smokers (74.9 ± 40.5 ng/ml). In conclusion, a decrease in the concentration of nicotine and an increase in the concentration of its metabolites in HIV-1–positive smokers compared with HIV-negative smokers support the hypothesis that nicotine metabolism is enhanced in HIV-1–positive smokers compared with HIV-negative smokers.

An LC–MS/MS method for concurrent determination of nicotine metabolites and the role of CYP2A6 in nicotine metabolite-mediated oxidative stress in SVGA astrocytes

BACKGROUND Nicotine is known to generate oxidative stress through cytochrome P450 2A6 (CYP2A6)-mediated metabolism in the liver and other organs, including macrophages. This study has been designed to examine the role of CYP2A6 in nicotine metabolism and oxidative stress in SVGA cells, an immortalized human astrocyte cell line. METHODS SVGA astrocytes were treated with 1 μM nicotine, followed by determination of mRNA and protein levels of several CYPs using quantitative RT-PCR and western blot analyses, respectively. Quantitation of nicotine and the nicotine metabolites, cotinine and nicotine-derived nitrosamine ketones (NNK), was performed using an LC-MS/MS method. The generation of reactive oxygen species (ROS) was measured using flow cytometry. RESULTS Nicotine significantly upregulated mRNA and protein expression of the most abundantly expressed CYPs in SVGA astrocytes, CYP2A6 and CYP1A1. To characterize the metabolism of nicotine in astrocytes, a highly sensitive LC-MS/MS method was developed which is capable of quantifying very low concentrations of nicotine (0.3 ng/mL), cotinine and NNK (0.11 ng/mL). The LC-MS/MS results showed that nicotine is steadily metabolized to cotinine and NNK from 0.5 to 4h. Finally, we showed that nicotine initially causes an increase in ROS formation which is then gradually decreased, perhaps due to the increase in superoxide dismutase level. Nicotine metabolism and ROS formation by CYP2A6 were further confirmed by using tryptamine, a selective inhibitor of CYP2A6, which significantly lowered the levels of cotinine and NNK and inhibited ROS formation. CONCLUSIONS CYP2A6 plays a key role in nicotine metabolism and oxidative stress in astrocytes, and this has implications in nicotine-associated brain toxicity.

Ocular microdialysis: a continuous sampling technique to study pharmacokinetics and pharmacodynamics in the eye

The unique anatomy and physiology of the eye present many challenges to the successful development and delivery of ophthalmic drugs. Any therapeutic strategy developed to control the progression of anterior and posterior segment diseases requires continuous monitoring of effective drug concentrations in the relevant ocular tissues and fluids. Ocular microdialysis has gained popularity in recent years due to its ability to continuously monitor drug concentrations and substantially reduce the number of animals needed. The intrusive nature of ocular microdialysis experimentation has restricted these studies to animal models. This review article intends to highlight various aspects of ocular microdialysis and its relevance in examining the disposition of drugs in the anterior and posterior segments.

Novel Biotinylated Lipid Prodrugs of Acyclovir for the Treatment of Herpetic Keratitis (HK): Transporter Recognition, Tissue Stability and Antiviral Activity

ABSTRACTPurposeBiotinylated 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.MethodsAll the prodrugs were screened for in vitro cellular uptake, interaction with SMVT, docking analysis, cytotoxicity, enzymatic stability and antiviral activity.ResultsUptake 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.ConclusionsBiotinylated 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.