Validated LC-MS/MS Method for Simultaneous Quantitation of SAFit-1 and SAFit-2 in Mice Plasma: Application to a Pharmacokinetic Study.

SAFit-1 and SAFit-2 are selective FKBP51 (FK506-binding protein 51) ligands. In this paper, we present the development and validation data of an LC-MS/MS method for the simultaneous quantitation of SAFit-1 and SAFit-2 in mice plasma as per FDA regulatory guideline. SAFit-1 and SAFit-2 along with internal standard were extracted from mice plasma using liquid-liquid extraction method. Chromatographic resolution of SAFit-1, SAFit-2 and the internal standard (warfarin) was achieved on an X-Terra phenyl column using 0.2% formic acid:acetonitrile (20:80, v/v) as an eluent, which was delivered at a flow-rate of 0.9 mL/min. The MS/MS ion transitions monitored were m/z 748.4→420.4, 803.7→384.3 and 309.2 →163.2 for SAFit-1, SAFit-2 and the internal standard, respectively. The linearity range was 2.45-2446 ng/mL for both SAFit-1 and SAFit-2. The intra- and inter-day accuracy and intra- and inter-day precision were in the range of 0.90-1.07 and 2.38-10.8%, respectively for SAFit-1; 0.97-1.15 and 0.23-12.5%, respectively for SAFit-2. Both SAFit-1 and SAFit-2 were found to be stable in stability studies (up to three freeze-thaw cycles and for long-term at -80°C for 30 days) and processed (bench-top for 3 h and in in-injector for 16 h) samples. The application of the validated method was shown in a pharmacokinetic study in mice.

Design and synthesis of a novel series of cyanoindole derivatives as potent γ-secretase modulators.

The discovery, design and synthesis of a new series of GSMs is described. The classical imidazole heterocycle has been replaced by a cyano group attached to an indole nucleus. The exploration of this series has led to compound 26-S which combined high in vitro and in vivo potency with an acceptable drug-like profile.

Novel KV7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue contraction.

Neuronal voltage-gated potassium channels, KV7s, are the molecular mediators of the M current and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we report novel small molecule KV7 openers that demonstrate anti-seizure activities in electroshock and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The anti-seizure activity was determined to be proportional to the unbound concentration in the brain. KV7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of these channels may cause localized undesired effects. Therefore, the impact of individual KV7 isoforms was investigated in human detrusor tissue using a panel of KV7 openers with distinct activity profiles among KV7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in detrusor tissue, yet a compound that has significantly reduced activity on homomeric KV7.4 did not reduce detrusor contraction. This may suggest that the homomeric KV7.4 channel plays a less significant role in bladder contraction and further investigation is needed.

Assessment of Drug Usage Pattern in Patients Treated for Vernal Keratoconjunctivitis Attending a Tertiary Eye Care Centre in Eastern India: A Cross-Sectional Study.

INTRODUCTION: Vernal keratoconunctivitis is a recurrent, seasonal, allergic condition affecting children and adolescents in warmer regions worldwide. OBJECTIVE: To assess the drug usage pattern for the management of vernal keratoconjunctivitis (VKC). MATERIALS & METHODS: This was a 12 weeks long hospital based cross sectional study conducted in the outpatient unit of the department of ophthalmology of a teaching hospital. It included all consecutive patients diagnosed with VKC who satisfied the inclusion criteria. Patients underwent clinical examination and the severity of their disease was graded. Their prescriptions were scanned for duration of therapy, total number of medications, route of administration, the frequency of dosage and change in medications if any, generic names of drugs being prescribed or not and all demographic parameters were recorded in a suitable record form. A p value of <0.05 was considered to be statistically significant. RESULTS: 248 patients were enrolled in this study of whom the majority were male (172). The average age of the patients was 8.27 years (SD ± 3.02 years). The mean duration of disease was 15 months (SD ± 9.13 years). The greatest number of children belonged to severity grade 1 (27.82%). The total number of drugs prescribed in this study was 583 with an average of 2.26 drugs per encounter. The commonest prescribed drugs were topical anti-allergics (26.75%) followed closely by lubricants (25.73%) and topical steroids (21.42%). CONCLUSION: Anti allergics and lubricants are the mainstays in the management of VKC. The current study is reflective of this. Clearer guidelines need to be formulated for the better and rational management of VKC.

Validated LC-MS-MS Method for Determination of SF0034 in Mice Plasma: Application to a Pharmacokinetic Study in Mice.

A rapid and sensitive assay method has been developed and validated using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode for the estimation of SF0034 in mice plasma. The assay procedure involves a simple protein precipitation of SF0034 and tolbutamide (internal standard, IS) from mice plasma. Chromatographic separation was performed on an Atlantis dC18 column using an isocratic mobile phase comprising 0.2% formic acid:acetonitrile (10:90, v/v) at a flow rate of 0.60 mL/min. The total run time was 2.5 min. For mass spectrometric detection, the multiple reaction monitoring was used and ion transitions monitored were m/z 322 → 248 for SF0034 and 271 → 155 for IS. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. A calibration curve was constructed in the range of 2.08-2,078 ng/mL. The intra- and inter-day precision was in the range of 1.06-14.4% and 7.16-11.7%, respectively. This novel method has been applied to a pharmacokinetic study in mice.

Penetrating ocular fish hook injury: a case report.

BACKGROUND: Fishing is a popular rural recreational activity. Fortunately, penetrating ocular injuries with fish hooks are rare. These injuries are usually caused by fish hook prongs penetrating the ocular tissues. We report a rare case of penetrating fish hook injury to the globe and its successful surgical management. CASE: A 12- year-old female child was referred to the causality with a fish hook embedded in her right eye. She sustained the injury while fishing with her father. The fish hook had penetrated the globe obliquely. The hook was removed via its entrance wound under general anesthesia. Subsequently the child developed traumatic cataract which was operated with a final visual outcome of 6/12. CONCLUSION: Visual prognosis can be profoundly affected by the initial management. Prompt surgical intervention as done in our case is recommended to prevent significant visual loss. Keywords Fishhook; penetrating ocular injury; traumatic cataract.

Bilateral central serous chorio-retinopathy in pregnancy presenting with severe visual loss.

INTRODUCTION: Bilateral central serous chorio-retinopathy is an unusual presentation. OBJECTIVE: To report a case of pregnancy-induced bilateral central serous chorio-retinopathy. CASE: A thirty-year-old woman complaining of reduced vision in both the eyes was referred for ophthalmoscopic evaluation in her third trimester of pregnancy. The best-corrected visual acuity was 6/60 in the right eye and finger counting at four meters in the left. The funduscopic examination revealed serous macular detachment with white sub-retinal exudates in both the eyes. Optical coherence tomography sections through the macula demonstrated serous elevation of the retina and retinal pigment epithelial detachment in both the eyes. The serous macular detachment resolved and the patient recovered to the visual acuity of 6/6 in both the eyes after twelve weeks of delivery. The OCT showed resolution of the sub-retinal fluid and disappearance of the sub-macular exudate in the right eye, but the resolution was partial in the left eye. CONCLUSION: Pregnancy is a risk factor for CSCR and it can present with severe bilateral visual diminution.

Micronucleus assay in buccal smears of breast carcinoma patients.

The aim of this study is to compare micronucleus assay in buccal smear of breast carcinoma patients versus normal benign cases as control group. In this prospective study, we selected a total 32 patients of carcinoma of breast and 49 patients of benign breast lesions diagnosed in fine needle aspiration cytology (FNAC). Acridine orange stain was done on buccal smears of these cases and micronucleus (MN) scoring was performed in 40× 0bjective in a fluorescent microscope. The MN score was expressed as positivity per 1,000 cells. The MN scoring in buccal smear was compared in malignant and benign breast cases. In fluorescence microscope, the micronucleus was detected as round orange shaped small intracytoplasmic structure around the nucleus. The mean MN scores in buccal smears of benign and carcinoma cases were 0.5014 ± 0.45768 and 2.1938 ± 1.08656 cases respectively. Independent sample Student's t test showed significantly high MN score in buccal smear of the cancer patients (P < 0.001). Micronucleated cells are significantly increased in buccal cells of the breast carcinoma cases. The increased number of MN in buccal smears raises the possibility that the genetic damage in breast cancer patients is generalized. In future, MN scoring could be used as biomonitoring of DNA damage and in early detection of high risk cases of carcinoma of breast.

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.

Micronucleus and its applications.

Micronucleus (MN) is a small additional nucleus and is readily identifiable by light microscopy. Biologically, micronuclei are the chromosome fragments or whole chromosomes that lag behind at anaphase during nuclear division. MN occurs due to genetic damage of the cell and the MN scoring is the indicator of the genetic damage. Therefore MN scoring can be used in various clinical setting such as to supervise genotoxicity, biomonitoring of diseases, screening of preneoplastic diseases and identification of high risk patients. In this brief review, the morphology, etiology, estimation, and applications of MN have been discussed.

Isolated idiopathic right ventricular dilated cardiomyopathy.

Isolated idiopathic right ventricular dilated cardiomyopathy is rare and is a diagnosis by exclusion. There is a distinct male predominance. The usual clinical presentations are syncope, ventricular tachycardia, left bundle branch block on ECG and right heart failure. Diagnosis is usually established based on the clinical and laboratory parameters. Confirmation of the same is done by studying the pathological features of the heart in an endomyocardial biopsy or at post-mortem. Herein we report a case of this rare entity which was diagnosed clinically and was further confirmed at autopsy.

Micronucleus in atypical squamous cell of undetermined significance.

The aim of this study was to explore the role of micronucleus (MN) scoring in distinguishing the smears of atypical squamous cell of undetermined significance (ASCUS) with reactive outcome versus ASCUS with cervical intraepithelial lesions (CIN) outcome. In this retrospective study, there were 53 cases of ASCUS diagnosed on cervical cytology smear and of which 30 cases showed reactive changes (group 1) and 23 cases showed CIN (group 2) on follow up histology. MN score of group 1 was compared with group 2 in conventional Papanicolaou's stained smear. The micronucleated cell per 1,000 epithelial cells in oil immersion magnification (100× objective) was counted by two observers and expressed as MN score. The data were compared in the two groups. The mean MN score was 2.8667 ± 2.20866 in group 1 and 8.3478 ± 6.44987 in group 2 cases. The Student's t-test showed significant difference of MN score in group 2 compared to group 1 (P < 0.0001). MN score may be helpful in identifying the true CIN cases that are mislabeled as ASCUS on cervical smear. In future, MN score can be used as an additional biomarker in cervical cancer screening.

Development and characterization of nanoparticulate formulation of a water soluble prodrug of dexamethasone by HIP complexation.

The objective of this study was to develop and characterize a nanoparticulate-based sustained release formulation of a water soluble dipeptide prodrug of dexamethasone, valine-valine-dexamethasone (VVD). Being hydrophilic in nature, it readily leaches out in the external aqueous medium and hence partitions poorly into the polymeric matrix resulting in minimal entrapment in nanoparticles. Hence, hydrophobic ion pairing (HIP) complexation of the prodrug was employed with dextran sulphate as a complexing polymer. A novel, solid in oil in water emulsion method was employed to encapsulate the prodrug in HIP complex form in poly(lactic-co-glycolic acid) matrix. Nanoparticles were characterized with respect to size, zeta potential, crystallinity of entrapped drug and surface morphology. A significant enhancement in the entrapment of the prodrug in nanoparticles was achieved. Finally, a simple yet novel method was developed which can also be applicable to encapsulate other charged hydrophilic molecules, such as peptides and proteins.

Micronucleus in cervical intraepithelial lesions and carcinoma.

AIMS AND OBJECTIVES: To score and compare micronucleus (MN) in the whole spectrum of cervical lesions including normal, inflammatory, abnormal squamous cell of undetermined significance (ASC-US), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL) and invasive cancer (IC) and to evaluate the role of MN as a biomarker in different pre-neoplastic and neoplastic lesions. MATERIALS AND METHODS: A total of 224 slides, comprised of normal (40), inflammatory (40), ASC-US (30), LSIL (38), HSIL (30) and IC (46), were studied. All the cases of HSIL, IC and ASC-US had histopathology. The LSIL, normal and inflammatory smears were again reviewed by 2 experienced cytopathologists independently. Two observers separately and independently counted the number of micronucleated cells per 1,000 of epithelial cells in oil immersion magnification (×100 objective) which was expressed as MN score per 1,000 cells. RESULTS: The mean MN scores±SD in normal, inflammatory, ASC-US, LSIL, HSIL and IC cases of cervical lesions were 1.02±1.59, 0.4250±0.71208, 2.87±2.21, 4.7368±5.62179, 21.30±17.18 and 18.50±9.54, respectively. MN scores of IC and HSIL were significantly high compared to the normal (p<0.000), the inflammatory (p<0.000), the ASC-US (p<0.000) and to the LSIL (p<0.000) group (analysis of variance test). LSIL showed significant difference with the normal (p=0.043), the inflammatory (p=0.019), the HSIL (p<0.000) and the IC (p<0.000) group but not with the ASC-US (p=0.342) group. CONCLUSIONS: MN scoring on the epithelial cells of cervix could be used as a biomarker in cancer screening. This is an easy, simple, reliable, reproducible and objective test which can be performed on routinely stained smears.

Fractal dimension of cervical intraepithelial lesions on cytology smear.

OBJECTIVE: To explore the role of fractal dimension (FD) in distinguishing normal and different grades of cervical intraepithelial lesions. STUDY DESIGN: We selected 15 cases each of normal samples and low-grade intraepithelial lesions (LSIL) and 14 cases of high-grade intraepithelial lesions (HSIL). All HSIL cases were histopathology proven. The LSIL diagnosis was verified by two cytopathologists. In each case at least 10 abnormal cells were selected in LSIL and HSIL cases and 10 normal cells in normal smears. A total of 202, 157, and 184 cells were selected from the normal, LSIL, and HSIL groups, respectively. The FD was measured in nucleus of each cell by box counting FD method with the help of appropriate software. RESULTS: The mean FD of the normal, LSIL, and HSIL nucleus was 1.2425 +/- 0.09188, 1.3504 +/- 0.6766, and 1.4379 +/- 0.08830, respectively. An analysis of variance test showed significant difference ofFD among the three groups (p < 0.0001). CONCLUSION: FD may be used as an additional factor to distinguish the dysplastic cells in the cervical cytology smear, which may be useful in automated screening in future.

Vitreal pharmacokinetics of biotinylated ganciclovir: role of sodium-dependent multivitamin transporter expressed on retina.

PURPOSE: The objective of this study was to investigate the role of sodium-dependent multiple vitamin transporter (SMVT) on Biotin-Ganciclovir (biotin-GCV) uptake on both human retinal pigmented epithelium cell line (ARPE-19) and rabbit retina. Study also aims to delineate the vitreal pharmacokinetics of biotin-GCV. METHOD: ARPE-19 was employed to study the in vitro uptake experiments. New Zealand white albino rabbits were used to study in vivo retinal uptake and vitreal pharmacokinetics following intravitreal administration of biotin-GCV. In vitro uptake kinetics of [3H] biotin was determined at various initial concentrations. Competitive inhibition studies were conducted in the presence of unlabelled biotin, desthiobiotin, pantothenic acid, and lipoic acid. Various other uptake studies were performed to functionally characterize the transporter. To provide the molecular evidence of this transporter, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) studies were also conducted. In vivo retinal/choroidal uptake studies were carried out with New Zealand albino rabbits. Unconscious animal ocular microdialysis studies were performed in order to evaluate intravitreal pharmacokinetics of GCV and Biotin-GCV. RESULTS: Uptake of [3H] biotin into ARPE-19 was linear over 7 min, and found to be saturable with K(m) of 138.25 muM and Vmax of 38.85 pmol/min/mg protein. Both pantothenic acid and lipoic acid decreased significantly in uptake of biotin in the concentration-dependent manner. Uptake of biotin into ARPE-19 was found to be temperature, energy, and Na+ dependent but Cl(-)independent. Further, RT-PCR studies identified a band exhibiting presence of hSMVT on ARPE-19. Biotin-GCV is recognized by SMVT system present on the ARPE-19 and rabbit retina. Vitreal Pharmacokinetics profile reveals that most of the parameters were not significantly different for GCV and Biotin-GCV. However, use of Biotin-GCV may result in sustain levels of regenerated GCV in vitreous. CONCLUSIONS: SMVT was identified and functionally characterized on ARPE-19 cells. Further, Biotin-GCV shares this transport system. Vitreal pharmacokinetics of the conjugate was determined in unconscious rabbit model.

Synthesis, metabolism and cellular permeability of enzymatically stable dipeptide prodrugs of acyclovir.

The objective of this study was to synthesize and evaluate novel enzymatically stable dipeptide prodrugs for improved absorption of acyclovir. l-Valine-l-valine-acyclovir (LLACV), l-valine-d-valine-acyclovir (LDACV), d-valine-l-valine-acyclovir (DLACV) and d-valine-d-valine-acyclovir (DDACV) were successfully synthesized. The uptake and transport studies were conducted on a Caco-2 cell line. Buffer stability and metabolism of the prodrugs in Caco-2, rat intestine and liver homogenates were studied. Structure and purity of the all compounds were confirmed with LC-MS/MS and NMR spectroscopy. Uptake and transport of [(3)H] glycylsarcosine was inhibited by all prodrugs except DDACV. DLACV and DDACV exhibited no measurable degradation in Caco-2 homogenate. Except DDACV other three prodrugs were hydrolyzed in rat intestine and liver homogenates. The order of permeability across Caco-2 was LDACV>LLACV>DDACV>DLACV. A linear correlation between the amount of prodrug transported and over all permeability of acyclovir was established. This study shows that the incorporation of one d-valine in a dipeptide did not abolish its affinity towards peptide transporters (PEPT). Moreover, it enhanced enzymatic stability of prodrug to a certain extent depending on the position in a dipeptide conjugate. This strategy improved both the cellular permeability and the amount of intact prodrug transported which would enable targeting the nutrient transporters at blood ocular barrier (BOB).