Thirumurthy Velpandian

Evaluation of pharmacological activities and assessment of intraocular penetration of an ayurvedic polyherbal eye drop (Itone™) in experimental models

BackgroundThe polyherbal eye drop (Itone™) is a mixture of aqueous distillates of nineteen traditionally used ingredients that sum up to impart potency to the formulation and make it a useful adjunct in various ocular pathologies. However, as there have been no controlled experimental studies accounting to the above claim, therefore, the present study was designed to evaluate the polyherbal formulation (PHF) for antiangiogenic, anti-inflammatory, anticataract, antioxidant and cytotoxicity in addition to the evaluation of intraocular penetration of PHF in rabbit eyes using LC-MS/MS.Materials and methodsAntiangiogenic activity of the PHF was evaluated using in ovo chick chorio-allantoic membrane (CAM) assay and in vivo cautery induced corneal neovascularization assay in rats. Anticataract potential was evaluated using steroid induced cataract in developing chick embryos, sodium selenite induced cataract in rat pups and galactose induced cataract in rats. The antioxidant activity was evaluated using di-phenyl picryl hydrazyl (DPPH) radical scavenging assay. Anti-inflammatory activity was evaluated in vitro using inhibition of LTB4 formation in human WBCs and in vivo using carrageenan induced paw edema assay in rats. The cytotoxicity was evaluated against HeLa cancer cell lines using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore evaluation of the intraocular penetration of the PHF was carried out in rabbit eyes via aqueous humor paracentesis and further analysis using LC-MS/MS.ResultsPHF significantly inhibited VEGF induced proliferation of new blood vessels in CAM assay and inhibited the cautery induced corneal neovascularization in rats. Additionally, PHF showed noticeable delay in the progression of cataract in the selenite and galactose induced cataract models whereby the PHF treated lenses were graded for stages II and III respectively. However, the PHF did not show any anticataract activity in the hydrocortisone induced cataract model. Moreover, PHF exhibited anti-inflammatory activity whereby it showed 39.34% inhibition of LTB4 formation and significantly inhibited carrageenan induced paw edema in rats. Eight compounds of PHF viz. camphor, casticin, curcumin-II, quercetin, rosmarinic acid, γ-terpinene, β-pinene and dipentene exhibited transcorneal penetration in rabbit eyes.ConclusionThe significant antiangiogenic and anti-inflammatory activities evinced by the PHF merits further investigation for ocular neovascular and inflammatory diseases in humans.

Development and validation of a new high-performance liquid chromatographic estimation method of meloxicam in biological samples.

A simple, HPLC method was developed to estimate meloxicam (COX-2 inhibitor) using piroxicam as the internal standard. The mobile phase containing methanol, acetonitrile and an aqueous solution of diammonium hydrogenorthophosphate (50 mM) in the ratio of 4:1:5 was pumped at the rate 1 ml/min. Lichrocart RP-18 (125 x 4 mm) was used as an analytical column and the analytes were detected at 364 nm using a UV detector. Acidified plasma samples were extracted with chloroform, evaporated to dryness, reconstituted in the mobile phase and then a volume of 10 microl of the prepared sample was injected in the column. The retention time of meloxicam and piroxicam was found to be 2.7 and 1.9, respectively. This method showed an accuracy of 102.3% at 0.52 microg/ml and was capable of detecting a minimum concentration of 0.029 microg/ml meloxicam from biological samples. The analytical method was successfully utilized for estimating meloxicam in biological samples.

Comparison of Analgesic and Anti-Inflammatory Activity of Meloxicam Gel with Diclofenac and Piroxicam Gels in Animal Models: Pharmacokinetic Parameters after Topical Application

Meloxicam, a non-steroidal anti-inflammatory drug, is a preferential inhibitor of cyclooxygenase-2 and has demonstrated potent analgesic and anti-inflammatory activity after oral administration. The present work was carried out to elucidate the anti-inflammatory and analgesic activity of a newer topical gel formulation of meloxicam (1% w/w gel) and compare it with 0.5% w/w piroxicam and 1% w/w diclofenac gels in experimental animal models. The study was also extended to determine the pharmacokinetic profile of a newer formulation of meloxicam gel after topical application on depilated skin of rats. The anti-inflammatory activities of meloxicam, piroxicam and diclofenac gels were compared using carrageenan-induced acute paw oedema and complete Freund’s adjuvant-induced chronic paw oedema in rats. Meloxicam gel showed increased protection against inflammation as compared to piroxicam and diclofenac gels. Acetic acid-induced writhing and formalin-induced phase I and phase II pain models were used to compare their analgesic activity. Meloxicam gel showed significant protection in formalin-induced phase II pain whereas its analgesic activity was less as compared to diclofenac and piroxicam gels in writhing test and formalin-induced phase I pain. The pharmacokinetic studies showed peak plasma drug concentration (Cmax) of 48.48 ± 6.57 µg/ml at 2 h (Tmax) after topical application of 500 mg of meloxicam gel formulation. The area under the curve as calculated from 0 to 6 h was found to be 114.18 ± 4.23 and 194.13 ± 3.78 µg·h/ml for 0 to ∝. The results indicate that topical preparation of meloxicam could be an effective alternative to diclofenac and piroxicam gels in inflammatory conditions and its associated pain with the possibility of less systemic side-effects.

Evaluation of Umbilical Cord Serum Therapy in Acute Ocular Chemical Burns

PURPOSE To evaluate the role of umbilical cord serum therapy in cases of acute ocular chemical burns. METHODS In a double-blind prospective randomized controlled clinical study, 33 eyes of 32 patients with acute ocular chemical burns of grade III, IV, and V severity were randomized into three groups: umbilical cord serum (n = 12), autologous serum (n = 11), and artificial tears (0.5% HPMC+0.3% glycerin; n = 10). In addition, all eyes received standard medical therapy. The parameters evaluated were pain score, size, and area of epithelial defect, extent of limbal ischemia, corneal clarity, and symblepharon formation. The patients were followed up at day 1, 3, 7, 14, and 21 and at the end of months 1, 2, and 3. RESULTS Mean time to complete epithelialization was 21.16 ± 26.81, 56.6 ± 35.5, and 40.13 ± 35.79 days in cord serum, autologous serum, and artificial tears groups respectively (P = 0.02). By day 21, the mean percentage decrease in epithelial defect diameter was 94.63 ± 11.99 with cord serum compared with 53.17 ± 34.81 and 64.22 ± 42.43 with autologous serum and artificial tears, respectively (P = 0.01). By month 3, the extent of limbal ischemia with cord serum showed a mean percentage decrease of 73.43 ± 25.51 compared with 35.64 ± 25.60 and 43.71 ± 28.71 with autologous serum and artificial tears, respectively (P = 0.008). More patients had clear corneas with cord serum compared with autologous serum and artificial tears (P = 0.048). No significant difference was seen between the groups with regard to symblepharon formation (P = 0.07). CONCLUSIONS Umbilical cord serum therapy is more effective than autologous serum eye drops or artificial tears in ocular surface restoration after acute chemical injuries. (www.controlled-trials.com number, ISRCTN08131903.).

Comparative Evaluation of Topical versus Intrastromal Voriconazole as an Adjunct to Natamycin in Recalcitrant Fungal Keratitis

OBJECTIVE To compare the efficacy of topical voriconazole and topical natamycin with that of intrastromal voriconazole and topical natamycin in patients with recalcitrant fungal keratitis. DESIGN Randomized clinical trial. PARTICIPANTS Forty eyes of 40 patients with fungal keratitis (positive smear or culture results or both) larger than 2 mm, involving up to two thirds of the stromal depth, and not responding to topical natamycin therapy for 2 weeks were recruited. INTERVENTION The patients were randomized to receive either topical 1% voriconazole therapy (n = 20) or intrastromal injections of voriconazole 50 μg/0.1 ml (n = 20). The patients in both groups continued topical natamycin 5% every 4 hours until the ulcer healed. MAIN OUTCOME MEASURES Primary outcome measure was best spectacle-corrected visual acuity (BSCVA) 3 months after intervention, and secondary outcome measures were time to healing and the size of the scar. RESULTS The patients in both groups had comparable baseline parameters. The mean BSCVA after treatment was 1.295 ± 0.5 logarithm of the minimum angle of resolution (logMAR) units in the topical group and 1.692 ± 0.29 logMAR units in the intrastromal group. The visual acuity after treatment was significantly better in the topical voriconazole group (P = 0.008). Nineteen patients receiving topical voriconazole and 16 patients who were given intrastromal voriconazole healed with therapy. CONCLUSIONS Topical voriconazole seems to be a useful adjunct to natamycin in fungal keratitis not responding to topical natamycin. Intrastromal injections did not offer any beneficial effect over topical therapy.

Analgesic efficacy and pharmacokinetics of topical nimesulide gel in healthy human volunteers: double-blind comparison with piroxicam, diclofenac and placebo

AbstractObjective: The present study was conducted to compare the analgesic efficacy of a new topical gel formulation of nimesulide (10 mg of pure drug) with that of placebo, diclofenac and piroxicam gels (10 mg of pure drug) in three parallel groups in a double-blinded, randomized fashion with vehicle placebo. The analgesic activity of nimesulide was subsequently correlated with its pharmacokinetic profile. Methods: The drugs were applied on a fixed marked area on the skin of the right forearm. Pain stimulus was administered using a modification of the Hollander method, before and at 15, 30, 60, 120 min and 240 min post-treatment. The pain experienced by the subjects was ranked separately on the visual analogue scale (VAS) and the ten-point category scale. Antinociception induced by the treatments was evaluated through the placebo-related ratings (PRR) and total pain relief (TOTPAR) analysis. The plasma concentration of nimesulide was estimated using high-performance liquid chromatography (HPLC). Results: Nimesulide exhibited better efficacy than diclofenac, piroxicam and placebo. It demonstrated faster onset of action in concordance with earlier studies. Peak analgesic effect was observed at 120 min post-treatment, which correlated with the pharmacokinetic profile of the drug in gel formulation. In this study, diclofenac was found to be superior to piroxicam though both drugs exhibited peak analgesic effect at 60 min post-treatment. In the modified Hollander method, a good correlation was found between the ten point category scale and the VAS, indicating that it may serve as a sensitive and reliable method for the screening of analgesic drugs. Conclusion: The superior analgesic activity of nimesulide (as gel formulation), correlating with its pharmacokinetic profile, indicates that the topical route of administration may be a safe and effective alternative to the presently used oral and rectal routes.

Serum rifampicin levels in patients with tuberculosis - Effect of P-glycoprotein and CYP3A4 blockers on its absorption

AbstractObjective: To identify patients with tuberculosis (TB) showing poor bioavailability for rifampicin and to delineate the role of possible factors such as overexpression of intestinal cytochrome P450 3A4 (CYP3A4) and P-glycoprotein (PGP) in the drug’s bioavailability by administering known blockers. Patients, design and setting: 77 clinically proven TB patients were included in this nonblinded, randomised, comparative trial carried out at one centre at the All India Institute of Medical Sciences, New Delhi, India. Interventions: The concentrations of rifampicin and its active metabolite, 25-desacetylrifampicin (DRMP), were measured in blood samples of the 77 TB patients at 0, 1, 2 and 4 hours after their usual morning rifampicin dose. Of these, 19 patients showing the lowest area under the concentration-time curve values from 0 to 4 hours after administration (AUC0–4) were selected and pretreated with a single dose of either verapamil (80mg) or itraconazole (200mg) as both PGP and CYP3A4 blockers 1 hour prior to rifampicin administration. Rifampicin and DRMP concentrations were estimated using high performance liquid chromatography in all serum samples collected at the same timepoints. Main outcome measures and results: A statistically significant increase (p <0.05) was found both in the serum levels of rifampicin at 2 hours and in the AUC0–4 values (158% and 84%, respectively) after pretreatment with verapamil. However, an increase in the levels of rifampicin was found to be insignificant on pretreatment with itraconazole. The estimated levels of DRMP also supported these results. Conclusions: The increase in rifampicin levels on administration of a PGP/ CYP3A4 blocker suggests a pivotal role for PGP/CYP3A4 in the absorption of rifampicin in patients with TB, which may be responsible for lower or variable levels of rifampicin.

Intraocular penetration of antimicrobial agents in ophthalmic infections and drug delivery strategies

Conventionally, antimicrobial drugs developed and approved for systemic infections are re-investigated for ocular infections. However, developing a new antimicrobial agent with good intraocular penetration by considering the anatomical and physiological constraints exerted by the barriers of eye is not a popularly perceived strategy. For the last three to four decades much emphasis has been placed on drug delivery systems to enhance the ocular penetration of antimicrobial agents. In order to compare ocular drug delivery strategies for ocular infections, the existing studies and methods were revisited using an extensive literature search. The present analysis also encompasses the scientific outcomes of endophthalmitis studies to interpret the intraocular penetration data of various antimicrobial agents and their requirements before and after the onset of inflammation. This article critically analyses the systemic and topical drug delivery methods adopted for antimicrobial use and their applicability to the newer class of antimicrobial agents, thus giving space for further developments. This review emphasizes the requirement of stage-by-stage insights about ocular infections, the need for an eye-specific antimicrobial agent and the inevitability of an appropriate drug delivery approach to revolutionize future therapy.

Correlation of plasma anti-tuberculosis drug levels with subsequent development of hepatotoxicity.

OBJECTIVES To compare the free and total plasma drug concentrations of rifampicin (RMP), isoniazid and pyrazinamide in subjects with or without anti-tuberculosis drug-induced hepatotoxicity (DIH). METHODS A total of 110 tuberculosis (TB) patients were administered daily anti-tuberculosis treatment and were prospectively followed for the development of DIH. Plasma drug levels were measured at 0, 1, 2 and 4 h on days 1, 7 and 14 of treatment. Plasma drug levels in 15 patients who developed DIH (cases) were compared with 95 patients who did not (controls). RESULTS Female sex, body mass index < 17 kg/m(2) and baseline serum albumin < 4 g/dl predicted risk of DIH on univariate analyses. Free and total plasma RMP levels (Cmax and AUC0-4) on days 1, 7 and 14 were significantly higher in cases compared to controls and predicted development of DIH. Day 7 total RMP Cmax and AUC0-4 were higher in cases (mean 26.73, standard deviation [SD] 5.72 and 47.58, SD 33.10) than in controls (7.87, SD 10.95 and 14.01, SD 10.69, respectively). CONCLUSIONS Plasma RMP levels were higher in cases than in controls and independently predicted subsequent development of DIH. The Cmax of Day 7 total RMP level (cut-off 12.50 mg/l) predicted subsequent development of DIH in 93.3% of the patients.

Toxicological investigation of surface engineered fifth generation poly (propyleneimine) dendrimers in vivo

Dendrimers are three dimensional polymers, nanoscopic in size, most widely explored in the field of drug delivery in recent times. In order to establish these polymers as controlled and targeted drug delivery systems, they should be non-toxic, biocompatible and biodegradable. The purpose of the present study is to investigate the toxicological profile of fifth generation poly (propyleneimine) dendrimers (PPI) and some of its surface engineered derivatives. Functionalized PPI dendrimers (TPPI, MPPI and TuPPI) were synthesized to mask the primary amino groups responsible for the positive charge and associated toxicity. Each polymer is administered in three different doses: 2.5 mg/kg, 25 mg/kg and 250 mg/kg (i.e., low, intermediate and high dose) to Wister rats, and blood as well as tissue samples were collected after 24 h and 15 days. Decrease in RBC count and hemoglobin content after 24 h, in case of animals administered with PPI suggests hemolytic activity of PPI. Significant increase in SGOT, SGPT and LDH indicates that PPI causes severe damage to the membranes of the various tissues of the body, especially that of the liver leading to the leakage of these marker enzymes in blood. Sections of liver of animals administered with PPI showed signs of tissue degeneration after 24 h. No signs of toxicity were observed in case of animals administered with functionalized PPI. Neither PPI nor its surface engineered derivatives showed any signs of immunogenicity. It can be concluded that functionalization of dendrimers leads to drastic reduction of toxicity and increases biocompatibility.