Nihar Ranjan Biswas

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.

Endogenous cortisol profile in patients with central serous chorioretinopathy

AIM To study the endogenous cortisol levels in patients with central serous chorioretinopathy (CSCR). METHODS Endogenous cortisol levels in urine and plasma were determined in 30 patients with acute CSCR and compared with 30 age and sex matched controls. RESULTS The mean values of the 8 am plasma cortisol (29.97 μg/dl v 18.76 μg/dl), 11 pm plasma cortisol (22.03 μg/dl v 13.06 μg/dl), and 24 hour urine cortisol (11.01 mg/24 h v 7.39 mg/24 h) revealed significantly higher values in the patient group (p<0.001). CONCLUSIONS Increased levels of endogenous cortisol are present in patients with CSCR.

Evaluation of umbilical cord serum therapy for persistent corneal epithelial defects

Aims: To evaluate umbilical cord serum therapy as a means of promoting the healing of persistent corneal epithelial defects. Methods: Umbilical cord serum or autologous serum drops were used to promote the healing of persistent epithelial defects. The study design was a prospective randomised controlled clinical trial. 60 eyes of 59 patients were divided into two groups, 31 in the cord serum group and 29 in the autologous serum control group. Epithelial defects measuring at least 2 mm in linear dimension resistant to conventional medical management were included. Serial measurements of the size of the epithelial defects—namely, two maximum linear dimensions perpendicular to each other, and the area and perimeter was done at start of therapy and follow up days 3, 7, 14, 21. Rate of healing of the epithelial defects were measured as percentage decrease from the baseline parameter at each subsequent follow up. The data were analysed by the non-parametric Wilcoxon rank sum test using STATA 7.0. Results: The median percentage decrease in the size of the epithelial defect was significantly greater in the cord serum group at days 7, 14 and 21 (p<0.05) when measured in terms of the area and perimeter. A greater number of patients showed complete re-epithelialisation with umbilical cord serum (n = 18) than with autologous serum (n = 11) (Pearson χ = 0.19). None of the patients reported any side effects or discomfort with either treatment. Conclusions: Umbilical cord serum leads to faster healing of the persistent corneal epithelial defects refractory to all medical management compared to autologous serum.

Mycoplasma pneumoniae and its role in asthma

Mycoplasma pneumoniae (M pneumoniae), primarily recognised as a causative agent of community-acquired pneumonia has recently been linked to asthma. An infection with M pneumoniae may precede the onset of asthma or exacerbate asthma symptoms. Chronic infection with M pneumoniae has been suspected to play a part in some patients with asthma. The role of immunoglobulin E-related hypersensitivity and induction of T helper type 2 immune response leading to inflammatory response in M pneumoniae-infected patients with asthma have also been proposed. Use of macrolides in reducing asthma symptoms only in M pneumoniae-infected patients supports the use of macrolides in patients with asthma having M pneumoniae infection. As macrolides are both antimicrobial and anti-inflammatory drugs, the therapeutic role of their biphasic nature in reducing asthma symptoms needs further attention in clinical research.

Role of 0.02% polyhexamethylene biguanide and 1% povidone iodine in experimental Aspergillus keratitis.

Purpose. To determine the efficacy of 0.02% polyhexamethylene biguanide and 1% povidone iodine in experimental Aspergillus keratitis. Methods. Aspergillus fumigatus keratitis was induced by corneal intrastromal injection of spores in 24 healthy rabbits that were randomly divided into four groups of six rabbits each. Drugs used were 5% natamycin (standard antifungal), 0.02% polyhexamethylene biguanide (PHMB) (test drug), 1% povidone iodine (test drug), and 0.5% hydroxypropylmethyl cellulose (HPMC) (control). Results. The average healing times of the ulcer were 21.5 ± 3.08 days with 5% natamycin, 27.8 ± 2.28 days with 0.02% PHMB, 36.4 ± 2.57 days with 1% povidone iodine, and 38.2 ± 4.74 days with 0.5% HPMC. While no corneal perforations occurred with natamycin treatment, one perforation was noted with PHMB, three perforations were noted with povidone iodine, and five perforations were noted with controls. Conclusion. Polyhexamethylene biguanide (0.02%) is a moderately effective drug for experimental Aspergillus keratitis, but 1% povidone iodine is not effective.

Animal Study on the Effects of Catalin on Aftercataract and Posterior Capsule Opacification

Purpose: To find out the role of Catalin in the prevention of posterior capsular opacification (PCO), we undertook this experimental study in rabbits. Methods: Twenty rabbits (10 for the Catalin group and 10 for the placebo group) were operated on for extracapsular clear lens extraction in an aseptic environment. In all cases, capsulorrhexis of 8 mm diameter was carried out. Both the test drug and placebo were given at a dose of 4 times/day from the first postoperative day for 8 weeks. Common drugs in both groups were topical corticosteroid, antibiotic and cycloplegic drops. Periodic slitlamp examinations and photographic documentations were carried out to find any evidence of aftercataract. At the end ot 8 weeks, histopathological examination was carried out to document any evidence of aftercataract. Result: Overall, evidence of aftercataract was seen in 6 rabbits in the control group and 3 in the Catalin group; the degree of PCO was higher in the placebo group. Conclusion: We found that Catalin played some role in preventing PCO.

Evaluation of the Modulation of P-glycoprotein (P-gp) on the Intraocular Disposition of its Substrate in Rabbits

Purpose: To evaluate the functional role of P-gp and ocular tissue distribution of intravitreally injected Rhodamine-123 (Rho-123) in the presence of P-gp specific blocker (GF 120918) in normal as well as rifampicin-fed rabbits using microdialysis and direct sampling technique. Methods: Intravitreal pharmacokinetics of Rho-123 were conducted in male New Zealand albino rabbits. Direct sampling and microdialysis were employed to study the disposition of Rho-123 in normal as well as rifampicin-fed conditions. Control animals received Rho-123 at the concentration of 350 ng in PBS (0.05 ml) intravitreally, and the blocker-treated group received GF 120918 intravenously at the dose of 3.5 mg/kg 30 min prior to intravitreal injection of Rho-123. In case of direct sampling, four eyes were enucleated at different time points, and ocular tissues and humors were stored at –86°C until analysis by HPLC with fluorescence detection. Results: In direct sampling, the blocker group showed significant increase (2.6 fold) in the mean vitreous concentration of Rho-123. Other tissues like ret-choroid, iris, and cornea also showed significant increase in their mean concentration. Microdialysis did not significantly predict the changes observed with direct sampling. Rifampicin-fed rabbits showed a vitreous pharmacokinetic profile comparable with non-fed (control) animals, and the pharmacokinetic parameters were unaffected by the blocker pretreatment. Conclusion: Intravenously injected blocker significantly altered the ocular disposition of intravitreally injected P-gp substrate. Rifampicin pretreatment did not upregulate P-gp transporters of the retina to the extent to affect the intravitreal kinetics of Rho-123 significantly.

Effect of fluoroquinolones on the expression of matrix metalloproteinase in debrided cornea of rats

Matrix metalloproteinases (MMPs) are implicated in regenerative and healing processes in corneal injuries. Based upon reports that topical fluoroquinolones (FQs) may cause perforations during corneal healing by modulating MMPs, this study evaluated the comparative effects of commercially available FQs eye drops on the expression of MMP-2 and MMP-9 in the cornea after ethanol injury. Uniform corneal epithelial defects were created using 70% ethanol in the right eye of the rats (n = 6). The groups studied were (I) sham, (II) normal saline with benzalkonium chloride (NS-BKC), (III) norfloxacin 0.3%, (IV) ciprofloxacin 0.3%, (V) lomefloxacin 0.3%, (VI) sparfloxacin 0.3%, (VII) gatifloxacin 0.3%, and (VIII) moxifloxacin 0.5%. Each treatment was instilled six times/day up to 48 h and rats were sacrificed using excess of anesthesia. The corneas were excised to study the expression of MMP-2 and MMP-9 using gelatin zymography and real-time PCR. All the FQs significantly increased the expression of MMP-2 and MMP-9 as compared to the sham and NS-BKC-treated group. NS-BKC did not show a significant effect on MMPs expression compared to the sham group. Among the studied FQs, ciprofloxacin was observed to exhibit maximal induction of MMP-2 and MMP-9, whereas lomefloxacin exhibited an equivocal effect on both MMP-2 and MMP-9 expression. Findings of the present study demonstrate that topical application of FQs may induce the expression of MMP-2 and MMP-9 in debrided corneal epithelium and, therefore, may delay corneal wound healing. Thus, it can be concluded that selecting a FQ for ophthalmic use having minimal effect on MMPs may impact wound healing in injured or vulnerable cornea.

Evaluation of the Impact of P-Glycoprotein (P-gp) Drug Efflux Transporter Blockade on the Systemic and Ocular Disposition of P-gp Substrate

PURPOSE The impact of P-glycoprotein (P-gp) blockade on the intravenous (i.v.) pharmacokinetics of rhodamine-123 (Rho-123), and the subsequent effect on its disposition in ocular and nonocular tissues, was studied by using rabbits. METHODS Three (3) control rabbits received only an i.v. bolus dose of Rho-123 (1.52 mg/kg). Three (3) blocker-pretreated rabbits received an i.v. dose of GF120918 (3.5 mg/kg) 30 min before the i.v. bolus of Rho-123. The plasma concentration of Rho-123 at different time points was subjected to a pharmacokinetic compartmental analysis, using WinNonlin (Scientific Consultants, Lexington, KY). For tissue-distribution study, a drug treatment similar to the i.v. kinetic study was followed by having 5 rabbits in each group. The animals were sacrificed at 30 min with an excess of anesthesia. Plasma and tissues samples were analyzed by using a validated high-performance liquid chromatographic IV method with a fluorescent detector. RESULTS The method validated was sensitive enough to estimate Rho-123 up to 1.94 ng/mL in plasma. I.v. Rho-123 data fitted well into the three-compartment model, and P-gp blocker treatment changed it into a two-compartment model. The P-gp blockade significantly increased the mean tissue concentrations in the lungs and spleen, whereas the rise in mean tissue levels in the heart, liver, and kidney and in all ocular tissues were found to be statistically insignificant. CONCLUSIONS Increasing the ocular concentration of systemically given drugs may not be possible with the degree of P-gp blockade achieved when using GF120918 at the studied concentration after an i.v. administration.

Development and validation of a highly sensitive LC-MS/MS method for organic cation transporter (OCT) substrate tetraethylammonium (TEA) in rabbits.

Tetraethylammonium is widely used as a probe in organic cation transporters studies. A simple, highly sensitive, and specific method using direct protein precipitation was developed using Hydrophilic Interaction Liquid Chromatography coupled with positive electrospray ionization tandem mass spectrometry for the determination of tetraethylammonium (TEA) in rabbit plasma. Isocratic separation was achieved using a ZIC-HILIC column with acetonitrile and 5mM ammonium acetate in the ratio of 8:2 containing 0.1% formic acid. Acquisition was performed in multiple reaction monitoring mode with the transitions: m/z 130→100 and 130→86 for TEA and m/z 276.1→142.2 for internal standard (homatropine). This method was validated to determine selectivity, linearity, sensitivity, precision, accuracy, recovery and stability. A good linearity was found within a range of 1.53-784.6 ng/mL. The above method has been demonstrated for its capability to estimate the plasma levels of TEA after its topical instillation in rabbit eyes. This method provides an accurate, precise and sensitive tool for determining TEA levels for transporter studies.