Pharmacokinetic Analysis of Gatifloxacin and Dexamethasone in Rabbit Ocular Biofluid Using a Sensitive and Selective LC-MS/MS Method.

Bacterial keratitis (BK) is an infection that causes inflammation of the cornea and, if severe, can result in blindness. Topical fluoroquinolones combined with corticosteroids have been shown to be useful in the treatment of BK. A rapid, selective, and sensitive bioanalytical method for simultaneous quantification of Gatifloxacin (GAT) and Dexamethasone (DEX) has been developed and validated using tandem mass spectrometry (LC-MS/MS). Optimal separation was accomplished in under 5 min using an Agilent Zorbax C18 column (100 mm × 4.6 mm, 3.5 µm). The mobile phase was composed of a blend of 0.2% formic acid in triple distilled water and methanol with a flow rate of 0.65 mL/min in isocratic mode. GAT and DEX were detected in positive electrospray ionization multiple reaction monitoring mode (MRM), and the retention time was found to be at 1.64 and 2.93 min, respectively. The linearity of GAT and DEX was found to be in the range of 1.56-400 ng mL-1 with good precision and accuracy. The method was validated according to USFDA regulatory guidelines. The validated method was effectively utilized for preclinical pharmacokinetic analysis of GAT and DEX in rabbit tear fluid following the topical application of a commercial formulation.

Antitubercular evaluation of dihydropyridine-triazole conjugates: design, synthesis, in vitro screening, SAR and in silico ADME predictions.

This study investigates the potential of click chemistry for the development of novel anti-tuberculosis agents. A targeted library of 1,4-dihydropyridine-1,2,3-triazole conjugates was synthesized and evaluated for their in vitro activity against Mycobacterium tuberculosis H37Ra using the resazurin microtiter assay (REMA). Among the synthesized derivatives, compounds J10, J11, J14, J22 and J23 demonstrated significant antimycobacterial activity. These compounds exhibited low MIC values ranging from 6.24 to 6.64 µg mL-1, highlighting their promising potential as lead compounds for further developing novel tuberculosis therapeutics. In addition to the promising in vitro activity, structure-activity relationship (SAR) analysis revealed that electron-withdrawing groups on the aryl-substituted ring of the dihydropyridines (J10-J24), a triazole with an unsubstituted aryl ring or with electron-donating groups (methyl or methoxy), and a geminal dimethyl group are essential structural features for the observed antitubercular activity. Furthermore, in silico ADME (absorption, distribution, metabolism, and excretion) parameters and pharmacokinetic studies supported the potential of these conjugates for oral bioavailability. These findings collectively highlight the 1,4-dihydropyridine-1,2,3-triazole scaffold as a promising platform for developing novel orally active anti-tuberculosis drugs.

Polymers and their engineered analogues for ocular drug delivery: Enhancing therapeutic precision.

Ocular drug delivery is constrained by anatomical and physiological barriers, necessitating innovative solutions for effective therapy. Natural polymers like hyaluronic acid, chitosan, and gelatin, alongside synthetic counterparts such as PLGA and PEG, have gained prominence for their biocompatibility and controlled release profiles. Recent strides in polymer conjugation strategies have enabled targeted delivery through ligand integration, facilitating tissue specificity and cellular uptake. This versatility accommodates combined drug delivery, addressing diverse anterior (e.g., glaucoma, dry eye) and posterior segment (e.g., macular degeneration, diabetic retinopathy) afflictions. The review encompasses an in-depth exploration of each natural and synthetic polymer, detailing their individual advantages and disadvantages for ocular drug delivery. By transcending ocular barriers and refining therapeutic precision, these innovations promise to reshape the management of anterior and posterior segment eye diseases.

Recent Insights into the Etiopathogenesis of Diabetic Retinopathy and Its Management.

Purpose: Diabetic retinopathy (DR) is a microvascular retinal disease associated with chronic diabetes mellitus, characterized by the damage of blood vessels in the eye. It is projected to become the leading cause of blindness, given the increasing burden of the diabetic population worldwide. The diagnosis and management of DR pose significant challenges for physicians because of the involvement of multiple biochemical pathways and the complexity of ocular tissues. This review aims to provide a comprehensive understanding of the molecular pathways implicated in the pathogenesis of DR, including the polyo pathway, hexosamine pathway, protein kinase C (PKC), JAK/STAT signaling pathways, and the renin-angiotensin system (RAS). Methods: Academic databases such as PubMed, Scopus, Google Scholar and Web of Science was systematically searched using a carefully constructed search strategy incorporating keywords like "Diabetic Retinopathy," "Molecular Pathways," "Pharmacological Treatments," and "Clinical Trials" to identify relevant literature for the comprehensive review. Results: In addition to activating other inflammatory cascades, these pathways contribute to the generation of oxidative stress within the retina. Furthermore, it aims to explore the existing pharmacotherapy options available for the treatment of DR. In addition to conventional pharmacological therapies such as corticosteroids, antivascular endothelial growth factors, and nonsteroidal anti-inflammatory drugs (NSAIDs), this review highlights the potential of repurposed drugs, phyto-pharmaceuticals, and novel pipeline drugs currently undergoing various stages of clinical trials. Conclusion: Overall, this review serves as a technical exploration of the complex nature of DR, highlighting both established and emerging molecular pathways implicated in its pathogenesis. Furthermore, it delves into the available pharmacological treatments, as well as the promising repurposed drugs, phyto-pharmaceuticals, and novel drugs currently being evaluated in clinical trials, with a focus on their specific mechanisms of action.

Concurrent determination of anti-microbial and anti-inflammatory drugs in lachrymal fluid and tissue by LC-MS/MS: A potential treatment for microbial keratitis and its PK-PD evaluation.

Unforeseen surfacing of microbial keratitis (MKT) over the years has led to a requisite for promising treatment strategy involving combination of antifungal and antibacterial agents. Subsequently, symptoms associated with MKT including inflammation and watery eyes require treatment with anti-inflammatory agents. Thus, a requirement of functional clinical treatment strategy involving combination of anti-inflammatory corticosteroids (Betamethasone) with antifungal polyene (Amphotericin B, AmB) and antibacterials macrolide (Azithromycin, AZT) and aminoglycoside (Neomycin, NEO). In the ensuing pursuit, a sensitive and fast simultaneous LC-MS/MS method of four drastically different analytes in rabbit tear fluid and cornea was developed and validated as per US-FDA guidelines. The gradient LC set-up was used with C18 column and flow rate of 0.55 mL/min along with short run time of 7 min. The calibration curves showed good linearity over the concentration range of 0.07-300 ng/mL, 1.00-400 ng/mL, 3.00-600 ng/mL and 8.00-900 ng/mL for AZT, AmB, NEO and BEM respectively. The bioanalytical method requires only 10 µL of ocular sample and analytes were extracted with fast protein precipitation with acidic methanol. Finally, the developed method was validated for selectivity, linearity (r2 > 0.99), precision, accuracy, matrix effects, and stability. PK-PD indices and dosing frequency was predicted using Phoenix WinNonlin Software, based on single dose ocular pharmacokinetics and MIC values of AmB, AZT and NEO. According to the PK-PD simulation, S. aureus and E. coli required 6 and 12 instillations of AZT per 24 h, respectively whereas 12 instillation of NEO requires per 24 h for S. aureus. The result suggests that to minimize antimicrobial resistance; drug, dose and dosing schedule depend upon the pathogen as well as the strain.

Trends in Formulation Approaches for Sustained Drug Delivery to the Posterior Segment of the Eye.

The eye, an intricate organ comprising physical and physiological barriers, poses a significant challenge for ophthalmic physicians seeking to treat serious ocular diseases affecting the posterior segment, such as age-related macular degeneration (AMD) and diabetic retinopathy (DR). Despite extensive efforts, the delivery of therapeutic drugs to the rear part of the eye remains an unresolved issue. This comprehensive review delves into conventional and innovative formulation strategies for drug delivery to the posterior segment of the eye. By utilizing alternative nanoformulation approaches such as liposomes, nanoparticles, and microneedle patches, researchers and clinicians can overcome the limitations of conventional eye drops and achieve more effective drug delivery to the posterior segment of the eye. These innovative strategies offer improved drug penetration, prolonged residence time, and controlled release, enhancing therapeutic outcomes for ocular diseases. Moreover, this article explores recently approved delivery systems that leverage diverse polymer technologies, such as chitosan and hyaluronic acid, to regulate drug-controlled release over an extended period. By offering a comprehensive understanding of the available formulation strategies, this review aims to empower researchers and clinicians in their pursuit of developing highly effective treatments for posterior-segment ocular diseases.

Hesperidin: Enrichment, forced degradation, and structural elucidation of potential degradation products using spectral techniques.

RATIONALE: Hesperidin (HES) is a well-known citrus bioflavonoid phyto-nutraceutical agent with polypharmacological properties. After 2019, HES was widely used for prophylaxis and COVID-19 treatment. Moreover, it is commonly prescribed for treating varicose veins and other diseases in routine clinical practice. Pharmaceutical impurities and degradation products (DP) impact the drug's quality and safety and thus its effectiveness. Therefore, forced degradation studies help study drug stability, degradation mechanisms, and their DPs. This study was performed because stress stability studies using detailed structural characterization of hesperidin are currently unavailable in the literature. METHODS: In the HES enrichment method crude HES was converted to its pure form (98% purity) using column chromatography and then subjected to forced degradation under acid, base, and neutral hydrolyses followed by oxidative, reductive, photolytic, and thermal stress testing (International Conference on Harmonization guidelines). The stability-indicating analytical method (SIAM) was developed to determine DPs using reversed-phase high-performance liquid chromatography (C18 column with methanol and 0.1% v/v acetic acid in deionized water [70:30, v/v] at 284 nm). Further, structural characterization of DPs was performed using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy. In addition, in silico toxicity predictions were performed using pKCSM and DataWarior freeware. RESULTS: HES was found to be susceptible to acidic and basic hydrolytic conditions and yielded three DPs in each, which were detected using designed SIAM. Of six DPs, three were pseudo-DPs (short lived), and the remaining were characterized using LC-MS/MS and NMR spectroscopy. The tentative mechanism of the formation of proposed DPs was explained. The proposed DPs were found inactive from in silico toxicity predictions. CONCLUSIONS: Hesperidin was labile under acidic and basic stress conditions. The potential DPs were characterized using LC-ESI-MS/MS and NMR spectral techniques. The proposed mechanism of formation was hypothesized. In addition, to identify and characterize the DPs, a SIAM, which has broad biomedical applications, was successfully developed.

Preclinical Pharmacokinetics and CYP Modulation Activity of Chebulinic Acid: A Potent Molecule Against Metabolic Disease.

BACKGROUND: Chebulinic acid (CA) is an active constituent of Terminalia chebula fruits with therapeutic potential against multiple metabolic diseases, including dementia, benign prostate hyperplasia, and osteoporosis. OBJECTIVE: The present work intends to explore the preclinical pharmacokinetics, including the absolute bioavailability of CA and its influence on the gene expression of cytochrome P450 enzymes in the liver. METHODS: Quantifying CA and probe drugs in vitro samples and preclinical serum samples of male SD rats were performed using LC-MS/MS. The influence of CA on the hepatic CYPs and their gene expression was analyzed in rat liver by quantitative real-time polymerase chain reaction. RESULTS: The plasma protein binding was found to be 84.81 ± 7.70 and 96.34 ± 3.12, blood-to-plasma ratio of 0.62 ± 0.16 and 0.80 ± 0.23 at 1 µM and 10 µM concentrations, respectively. Again, the absolute oral bioavailability of CA at 100 mg/kg was found to be 37.56 ± 7.3%. The in-vivo pharmacokinetic profile of probe drugs revealed CA to have significant inducing effects on CYP1A2, 2C11, 2D2, and 2E1 after 14 days, which correlates to both in-vitro rat microsomal data and gene expression results. CONCLUSION: Altogether, pharmacokinetic parameters reveal CA to have an affinity to distribute across different extravascular tissues and induce rat liver CYP enzymes.

LC-MS/MS based quantification of steroidal biomarkers in polycystic ovary syndrome induced rats.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder that causes reproductive hormones imbalance, missed periods, infertility and distributed steroidogenesis. Reportedly, during PCOS, the endogenous levels of P4 (Progesterone), 17OHP4 (17-α hydroxy progesterone), and T4 (Testosterone) were significantly altered. Thus, quantification of steroid biomarkers involved in the steroidogenesis pathway of PCOS, such as P4, 17OHP4, and T4, holds significant importance. One important drawback of current methods is steroid metabolome traceability. Without adequate traceability, the findings of these techniques will be less reliable for identifying P4, 17OHP4, and T4. These methods also need a high sample size, especially for the most important biomarker that initiates steroidogenesis. To address these challenges, we require a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for steroid biomarker analysis. Herein the present work, using validated LC-MS/MS, PCOS biomarkers were measured and compared between normal control rats and PCOS-induced rats before and after analyte administration. The experiment utilized an isocratic separation method employing an analytical C18 column. The mobile phase consisted of acetonitrile (ACN) and aqueous 0.1% formic acid (FA) in a ratio of 90:10 (v/v). The plasma samples were processed with protein precipitation (PPT) followed by the liquid-liquid extraction (LLE) method. The lower limit of quantification (LLOQ) was 0.5 ng/mL in plasma. According to USFDA criteria, the method's systematic validation took into account linearity (r2 > 0.99), accuracy and precision of intra- and inter-batch measurements, stability, biomarker recovery (60-85%) and matrix effect (<± 15%), all of which were determined to be within range ( ± 15%). The pharmacokinetic data showed that, as compared to normal rats, PCOS-induced animals had significantly higher Cmax values for 17OHP4 and T4 (∼2 fold), while lower Cmax values for P4 (∼2 fold). The present work is novel and provides scientific information to explore systematic processes involved in steroidogenesis and boost clinical applicability for PCOS therapy.

Preclinical pharmacokinetics of 4-hydroxy isoleucine using LC-MS/MS: a potential polycystic ovary syndrome phytopharmaceutical therapeutics.

Aim: To study the preclinical pharmacokinetics of 4-hydroxy isoleucine (4-HIL) targeted for polycystic ovary syndrome. Methodology: The quantitative bioanalysis of 4-HIL in different biological matrices in female Sprage-Dawley rats using LC-MS/MS. Results: At 50 mg/kg, 4-HIL had 56.8% absolute oral bioavailability. It was quickly absorbed and distributed in various tissues in order of small intestine > kidney > ovary > spleen > lung > liver > heart > brain after oral administration. Moreover, 11.07% of 4-HIL was recovered in urine and feces within 72 h. Conclusion: 4-HIL levels in vital organs were found safe, as per tissue distribution results. Hence, 4-HIL could be used as promising therapeutics for management of polycystic ovary syndrome.

A QbD-led simple and sensitive RP-UHPLC method for simultaneous determination of moxifloxacin, voriconazole, and pirfenidone: An application to pharmaceutical analysis.

A novel, quick and precise RP-UHPLC analytical method for the simultaneous determination of moxifloxacin (MFX), voriconazole (VCZ) and pirfenidone (PIR) was developed and validated according to the International Conference on Harmonization guidelines using a QbD-driven response surface Box-Behnken design. The developed method was validated considering the selectivity, sensitivity, linearity, accuracy-precision, robustness, stability, limit of detection and limit of quantification, respectively. Resolution between MFX, VCZ and PIR was achieved using a gradient elution protocol against a Waters Symmetry Shield C18 column (150 × 4.6 mm2 , 5 µm) using an Agilent 1290, Infinity II series LC system. The method was applied to quantitatively estimate proprietary and in-house prepared pharmaceutical topical ophthalmic formulations containing MFX, VCZ and PIR at wavelength (λmax ) of 296, 260 and 316 nm. The method is sensitive enough to detect up to 0.1 ppm of analytes in the formulation. The method was further exploited to study and identify the possible degradation products of the analytes. The proposed chromatographic method is simple, economical, reliable and reproducible. In conclusion, the developed method could be applicable for routine quality control analysis of single or combined MFX, VCZ and PIR-containing units or bulk dosage forms in pharmaceutical industries and research organizations working on drug discovery and development.

Preclinical pharmacokinetics, CYP phenotyping, and tissue distribution study of novel anti-breast cancer candidate S-011-1559.

S-011-1559 is a tyrosine-derived novel benzoxazine CDRI molecule targeted to the oestrogen-related receptor (ER-α/ß) modulator in breast cancer. To explore the pharmacokinetics of S-011-1559, a selective and sensitive bioanalytical method using LC-MS/MS was established and validated in different biological matrices of female rats.Blood-to-plasma ratio and plasma protein binding (PPB) of S-011-1559 were found to be <1 and >97% in both rats and humans, respectively. The human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG) binding was found in the range of > 68 to 45% and >14% respectively. Half-life and intrinsic clearance by microsomal stability study were found to be 28.83 min and 0.05 mL/min/mg in rats, 78.35 min and 0.036 mL/min/mg in humans, respectively. The IC50 value of S-011-1559 against CYP isoforms was revealed to moderately inhibit CYP2D6 by a reversible non-competitive mechanism.Tissue distribution of S-011-1559 on single intravenous injection at 2 mg/kg was found in the order of C lungs > C mammary gland > C spleen > C heart > C kidney > C liver > C brain.The data from the present study provides crucial information about S-011-1559 for further development as a novel potential drug candidate in modulating ER-α/ß receptors of lung and breast neoplasia.