Sustained insulin treatment restoring metabolic status, body weight, and cognition in an anorexia nervosa-like animal model in mice.

INTRODUCTION: Anorexia Nervosa (AN) is a psycho-socio-biological disease characterized by severe weight loss as result of dieting and hyperactivity. Effective treatments are scarce, despite its significant prevalence and mortality. AN patients show lower basal insulin levels and increased metabolic clearance, leading to weight loss, cognitive deficits, and hormonal imbalances. Low-dose polymer insulin could potentially reverse these effects by restoring brain function, reducing fear of weight gain, encouraging food intake, and restoring fat depots. This study evaluates an insulin delivery system designed for sustained release and AN treatment. METHODS: AN-like model was established through dietary restriction (DR). On days 1-25, mice were on DR, and on days 26-31 they were on ad libitum regimen. An insulin-loaded delivery system was administered subcutaneously (1% w/w insulin). The impact of insulin treatment on gene expression in the hippocampus (cognition, regulation of stress, neurogenesis) and hypothalamus (eating behavior, mood) was assessed. Behavioral assays were conducted to evaluate motor activity and cognitive function. RESULTS: The delivery system demonstrated sustained insulin release, maintaining therapeutic plasma levels. Diet restriction mice treated with the insulin delivery system showed body weight restoration. Gene expression analysis revealed enhanced expression of CB1 and CB2 genes associated with improved eating behavior and cognition, while POMC expression was reduced. Insulin-polymer treatment restored cognitive function and decreased hyperactivity in the AN-like model. CONCLUSION: The PSA-RA-based insulin delivery system effectively restores metabolic balance, body weight, and cognitive function in the AN model. Its ability to steadily release insulin makes it a promising candidate for AN treatment."

Peptide and Protein Stereocomplexes.

Stereocomplexation in peptides and proteins is a fascinating phenomenon arising from their inherent stereoisomerism. Peptides and proteins, with their three-dimensional helical structures, exhibit stereoselectivity and form intertwined complexes when complementary left- and right-handed structures are mixed together. Stereocomplexation provides an unprecedented opportunity to impart some valuable biological, chemical, and physical properties in peptide and protein polymeric platforms that can be employed in various applications such as catalysis and drug delivery and to improve the stability of these therapeutics. However, exploration of stereocomplexation in peptides and proteins remains limited. We report on a comprehensive understanding of stereocomplexation in peptides and proteins, compiling existing reports, discussing its implications, and highlighting its role in different applications, aiming to inspire further research and advancements in this direction.

Technology Transfer of a Validated RP-HPLC Method for the Simultaneous Estimation of Andrographolide and Paclitaxel in Application to Pharmaceutical Nanoformulation.

Many analytical methods are reported for simultaneous estimation of pharmaceutical dosages form. However, only a few are reproducible at an industrial scale. The proposed research aims to establish a technology transfer (TT) protocol between two laboratories (Lab-X, originator) with binary and (Lab-Y, receiver) with quaternary high-performance liquid chromatography (HPLC) system. Thus, utilizing reverse-phase HPLC (RP-HPLC), a robust, sensitive and repeatable analytical method has been developed, validated and TT between two laboratories for simultaneous estimation of Andrographolide (AG) and Paclitaxel (PTX). The method has been developed on a Phenomenex Luna C18 column (150 x 4.6, 5) sustained at 40°C and validated under the International Conference on Harmonisation (ICH) Q2 (R1) regulatory guideline and TT USP chapter 1224. The mobile phase consisted of MilliQ (pH = 3) and a combination of acetonitrile and methanol (1:1) in the ratio 50:50 with a flow rate of 0.45 mL/min, linear gradient elution in both labs. The AG and PTX were detected on the PDA detector at 224 and 227 nm wavelength with retention time of 4.5 ± 0.34 and 8.2 ± 0.02 min and limit of detection was found 0.028 ± 0.004 µg/mL, and 0.028 ± 0.0007 µg/mL, whereas limit of quantification as 0.086 ± 0.011 µg/mL and 0.088 ± 0.0014 µg/mL respectively in both labs. Throughout, this approach we have proved that proposed method is repeatable in both labs, and it can be used to quantify AG and PTX in developed pharmaceutical nano-formulations.

Ratiometric codelivery of Paclitaxel and Baicalein loaded nanoemulsion for enhancement of breast cancer treatment.

The most prevalent clinical option for treating cancer is combination chemotherapy. In combination therapy, assessment and optimization for obtaining a synergistic ratio could be obtained by various preclinical setups. Currently, in vitro optimization is used to get synergistic cytotoxicity while constructing combinations. Herein, we co-encapsulated Paclitaxel (PTX) and Baicalein (BCLN) with TPP-TPGS1000 containing nanoemulsion (TPP-TPGS1000-PTX-BCLN-NE) for breast cancer treatment. The assessment of cytotoxicity of PTX and BCLN at different molar weight ratios provided an optimized synergistic ratio (1:5). Quality by Design (QbD) approach was later applied for the optimization as well as characterization of nanoformulation for its droplet size, zeta potential and drug content. TPP-TPGS1000-PTX-BCLN-NE significantly enhanced cellular ROS, cell cycle arrest, and depolarization of mitochondrial membrane potential in the 4T1 breast cancer cell line compared to other treatments. In the syngeneic 4T1 BALB/c tumor model, TPP-TPGS1000-PTX-BCLN-NE outperformed other nanoformulation treatments. The pharmacokinetic, biodistribution and live imaging studies pivoted TPP-TPGS1000-PTX-BCLN-NE enhanced bioavailability and PTX accumulation at tumor site. Later, histology studies confirmed nanoemulsion non-toxicity, expressing new opportunities and potential to treat breast cancer. These results suggested that current nanoformulation can be a potential therapeutic approach to effectively address breast cancer therapy.

Enhanced apoptosis and mitochondrial cell death by paclitaxel-loaded TPP-TPGS1000-functionalized nanoemulsion.

Background: The present research was designed to develop a nanoemulsion (NE) of triphenylphosphine-D-α-tocopheryl-polyethylene glycol succinate (TPP-TPGS1000) and paclitaxel (PTX) to effectively deliver PTX to improve breast cancer therapy. Materials & methods: A quality-by-design approach was applied for optimization and in vitro and in vivo characterization were performed. Results: The TPP-TPGS1000-PTX-NE enhanced cellular uptake, mitochondrial membrane depolarization and G2M cell cycle arrest compared with free-PTX treatment. In addition, pharmacokinetics, biodistribution and in vivo live imaging studies in tumor-bearing mice showed that TPP-TPGS1000-PTX-NE had superior performance compared with free-PTX treatment. Histological and survival investigations ascertained the nontoxicity of the nanoformulation, suggesting new opportunities and potential to treat breast cancer. Conclusion: TPP-TPGS1000-PTX-NE improved the efficacy of breast cancer treatment by enhancing its effectiveness and decreasing drug toxicity.

HPLC method for simultaneous estimation of paclitaxel and baicalein: pharmaceutical and pharmacokinetic applications.

Aim: A novel HPLC method was developed and validated for the simultaneous estimation of paclitaxel (PTX) and baicalein (BAC). Materials & methods: The analytes were resolved in a C18 column using the aqueous solution of formic acid (0.10% v/v) and MeOH (30:70 v/v). Results: The developed method was found to be linear over the concentration ranges 0.039-10 µg/ml and 0.019-10 µg/ml for PTX and BAC, respectively. The lower limits of quantification obtained were 0.042 µg/ml and 0.361 µg/ml for PTX and BAC, respectively. Conclusion: The developed method was found to be precise and accurate as per the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use guidelines, for simultaneous estimation of PTX and BAC, having an application in formulation development and bioanalytical studies.

Effective uptake of folate-functionalized ethionamide-loaded hybrid system: targeting alveolar macrophages.

Aim: To assess the targeting ability of hybrid nanosystems functionalized with folate. It also aimed to reduce stomach intolerance by substituting the oral route for parenteral delivery. Method: The nanosystems, prepared by nanoprecipitation technique, utilized a one-step method to prepare nanoparticles followed by surface functionalization through adsorption. The prepared nanosystems underwent physical characterization, in vitro and in vivo evaluations. Result: The nanosystems were effective in targeting the alveolar macrophages. Ethionamide was released from the formulation over 5 days. Fourier-transform infrared results proved the structural characteristics, and the positive charge further improved the targeting efficacy on the functionalized system. Conclusion: The hybrid formulation improved the release characteristics. Reduction in dosing frequency due to prolonged release improves compliance with the dosage regimen.

Validated HPLC-UV Method for Simultaneous Estimation of Paclitaxel and Doxorubicin Employing Ion Pair Chromatography: Application in Formulation Development and Pharmacokinetic Studies.

Though paclitaxel (PTX) and doxorubicin (DOX) are amongst the most widely used and investigated drug pair for combination chemotherapy but surprisingly, not a single validated HPLC-UV method is available to analyze PTX and DOX simultaneously. So, herein a HPLC-UV method is developed and validated for the same, filling an indispensable gap in the literature. As these two moieties have characteristically different polarities, resolving them under the common chromatographic conditions is a challenging task. Herein, the principle of ion pair chromatography is utilized to resolve these two moieties on a C18 column employing an isocratic mobile phase comprised of acetonitrile and octane sulfonic acid buffer (67 : 37) and detected simultaneously at 231 nm using a UV detector only. The retention time is 4.4 and 7.2 min for PTX and DOX, respectively, with a total analysis time of less than 10 minutes, suitable for the formulation development and research, while LOQ is less than 0.066 µg/ml for both the drugs, suitable for the therapeutic drug monitoring at preclinical and clinical research setup. To substantiate the applicability of the developed method, a nanoformulation coloaded with PTX and DOX was designed and analyzed using the developed protocol. The method is also applied successfully to study the plasma kinetic profile of both the moieties simultaneously in Balb/c mice. Further, the method is validated as per the ICH guidelines fulfilling the unmet need of a validated analytical tool to simultaneously estimate PTX and DOX. Moreover, the results suggest that the principal of common ion chromatography demonstrated here can also be applied further for the simultaneous chromatographic separation of other polar and nonpolar moieties too. Consequently, the reported method surely will advance the toolset required for the precision-based combination chemotherapy.

An Injectable In Situ Depot-Forming Lipidic Lyotropic Liquid Crystal System for Localized Intratumoral Drug Delivery.

To address the need for localized chemotherapy against unresectable solid tumors, an injectable in situ depot-forming lipidic lyotropic liquid crystal system (L3CS) is explored that can provide spatiotemporal control over drug delivery. Although liquid crystals have been studied extensively before but their application as an injectable intratumoral depot system for locoregional chemotherapy has not been explored yet. The developed L3CS in the present study is a low-viscosity injectable fluid having a lamellar phase, which transforms into a hexagonal mesophase depot system on subcutaneous or intratumoral injection. The transformed depot system can be preprogrammed to provide tailored drug release intratumorally, over a period of one week to one month. To establish the efficacy of the developed L3CS, doxorubicin is used as a model drug. The drug release mechanism is studied in detail both in vitro and in vivo, and the efficacy of the developed system is investigated in the murine 4T1 tumor model. The direct intratumoral injection of the L3CS provided localized delivery of doxorubicin inside the tumor and restricted its access within the tumor only for a sustained period of time. This led to an over 10-fold reduction in tumor burden, reduced cardiotoxicity, and a significant increase in the median survival rate, compared to the control group. The developed L3CS thus provides an efficient strategy for localized chemotherapy against unresectable solid tumors with a great degree of spatial and temporal control over drug delivery.

Appraisal of fluoroquinolone-loaded carubinose-linked hybrid nanoparticles for glycotargeting to alveolar macrophages.

There is a curious case in Alveolar macrophages (AM), the frontline defence recruits that contain the spread of all intruding bacteria. In response to Mycobacterium tuberculosis (M.tb), AM either contain the spread or are modulated by M.tb to create a region for their replication. The M.tb containing granulomas so formed are organised structures with confined boundaries. The limited availability of drugs inside AM aid drug tolerance and poor therapeutic outcomes in diseases like tuberculosis. The present work proves the glycotargeting efficiency of levofloxacin (LVF) to AM. The optimised formulation developed displayed good safety with 2% hemolysis and a viability of 61.14% on J774A.1 cells. The physicochemical characterisations such as Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) proved that carubinose linkage was accomplished and LVF is entrapped inside carubinose-linked hybrid formulation (CHF) and hybrid formulation (HF) in amorphous form. The transmission electron microscopy (TEM) images revealed a core-shell structure of HF. The particle size of 471.5 nm estimated through dynamic light scattering (DLS) is enough to achieve active and passive targeting to AM. The nanoparticle tracking analysis (NTA) data revealed that the diluted samples were free from aggregates. Fluorescence-activated cell sorting (FACS) data exhibited excellent uptake via CHF (15 times) and HF(3 times) with reference to plain fluorescein isothiocyanate (FITC). The pharmacokinetic studies revealed that CHF and HF release the entrapped moiety LVF in a controlled manner over 72 h. The stability studies indicated that the modified formulation remains stable over 6 months at 5 ± 3℃. Hence, hybrid systems can be efficiently modified via carubinose to target AM via the parenteral route.

Cyclodextrin based bone regenerative inclusion complex for resveratrol in postmenopausal osteoporosis.

Recent preclinical studies have shown that resveratrol (RSV), is a promising remedy for osteoporosis owing to its estrogenic, anti-inflammatory, and antioxidant properties. However, RSV has met limited success due to its poor oral bioavailability and inefficient systemic delivery. In this study, we prepared the inclusion complex of RSV with sulfo-butyl ether ß-cyclodextrin (SBE-ß-CD) to enhance the aqueous solubility of RSV. The in-silico docking studies and Physico-chemical characterization assays were performed to understand the interaction of RSV inside the SBE-ß-CD cavity. The in vivo safety assessment of RSV-SBE-ß-CD inclusion complex (R-CDIC) was performed in healthy Wistar rats. The efficacy of the inclusion complex against postmenopausal osteoporosis was further investigated in ovariectomized (OVX) rat model. The alteration in the bone micro-architectural structure was evaluated by microcomputed tomographic scanning, serum biochemical estimations, biomechanical strength and histopathological investigation. Administration of RSV-SBE-ß-CD inclusion complex was found to be safe and significantly improved micro-architectural deterioration induced by estrogen withdrawal. Results of bone morphometry and biomechanics study further emboldened the efficacy claim of the RSV-SBE-ß-CD complex. Thus, the present study demonstrated the efficacy of the RSV-SBE-ß-CD inclusion complex for treating osteolytic degradation in osteoporosis.

Paclitaxel-Loaded Colloidal Silica and TPGS-Based Solid Self-Emulsifying System Interferes Akt/mTOR Pathway in MDA-MB-231 and Demonstrates Anti-tumor Effect in Syngeneic Mammary Tumors.

A solid self-emulsifying drug delivery system (SEDDS) of paclitaxel (PTX) was developed that could enhance its oral bioavailability and neutralize other niggles associated with conventional delivery systems of PTX. TPGS-centered SEDDS containing PTX was optimized by Box-Behnken experimental design and then formulated as fumed colloidal silica-based solid SEDDS microparticles (Si-PTX-S-SEDDS). AFM analysis exhibited round-shaped microparticles of approximately 2-3 µM diameter, whereas after reconstitution, particle size measurement showed nanoemulsion droplets of 30.00 ± 2.00 nm with a zeta potential of 17.38 ± 2.88 mV. Si-PTX-S-SEDDS displayed improved efficacy proven by reduced IC50 of 0.19 ± 0.03 µM against MDA-MB-231 cells and a 45.83-fold higher cellular uptake in comparison to free PTX. Molecular mechanistic studies showed mitochondria-mediated intrinsic pathway of apoptosis following Akt/mTOR pathway, which is accompanied by survivin downregulation. Rhodamine 123 assay and chylomicron flow blocking studies revealed P-gp inhibition potential and lymphatic uptake of Si-PTX-S-SEDDS, responsible for over 4-fold increment in oral bioavailability compared to PTX administered as Taxol. In vivo anti-tumor studies in syngeneic mammary tumor model in SD rats revealed higher efficacy of Si-PTX-S-SEDDS as evident from significant reduction in tumor burden. In total, the developed Si-PTX-S-SEDDS formulation was found as an appropriate option for oral delivery of PTX.

Effect of rutin on retinal VEGF, TNF-α, aldose reductase, and total antioxidant capacity in diabetic rats: molecular mechanism and ocular pharmacokinetics.

PURPOSE: The current study was conducted to explore the potential of rutin in preventing sight-threatening diabetic retinopathy. METHODS: Wistar albino rats (either sex) weighing 200-225 g were intraperitoneally injected with 45 mg/kg streptozotocin (pH 4.5). Rats having blood glucose ≥ 300 mg/dL were divided into two groups (n = 8; each group). Group I served as diabetic control and received normal saline p.o. Group II received rutin 50 mg/kg p.o. for 24 weeks. At the end of 24 weeks, retinal fundus and fluorescein imaging were done, rats were killed, and retinal biochemical assessments were conducted. Moreover, ocular pharmacokinetics of rutin was assessed in the normal rats after a single oral dose of 50 mg/kg. RESULTS: Rutin treatment significantly (p < 0.001) lowered retinal vascular endothelial growth factor, tumor necrosis factor-α, and aldose reductase. Rutin treatment significantly (p < 0.001) elevated the levels of total antioxidant capacity of the retinas. Fundus examination of rutin-treated group showed significantly lower tortuosity index and normal fluorescein angiography. Rutin was detected in the retina as well as in aqueous humor of normal rats. CONCLUSION: Rutin treatment significantly arrested the biochemical disturbances of diabetic retinopathy. The distribution of orally ingested rutin in ocular tissues further substantiate its site-specific action.

Cardioprotective effects of rutin via alteration in TNF-α, CRP, and BNP levels coupled with antioxidant effect in STZ-induced diabetic rats.

Diabetic cardiomyopathy (DCM) is a dreadful complication of diabetes responsible for 80 % mortality in diabetic patients, but unfortunately its pharmacotherapy is still incomplete. Rutin is a naturally occurring flavonoid having a long history of use in nutritional supplements for its action against oxidative stress, inflammation, and hyperglycemia, the key players involved in the progression of DCM, but remains unexplored for its role in DCM. This study was conducted to address this lacuna. It was performed in 4-week-old Streptozotocin-induced (45 mg/kg) diabetic rats for a period of 24 weeks to mimic the cardiotoxic effect of chronic hyperglycemia in diabetic patient's heart and to investigate the effect of rutin (50 mg/kg/day) in ameliorating these effects. Heart of the diabetic rats showed altered ECG parameters, reduced total antioxidant capacity, increased inflammatory assault, and degenerative changes. Interestingly, rutin treatment significantly ameliorated these changes with decrease in blood glucose level (p > 0.001), % HbA1c (p > 0.001) and reduced expression of TNF-α (p < 0.001), CRP (p < 0.001), and BNP (p < 0.01) compared to diabetic control rats. In addition, rutin provided significant protection against diabetes associated oxidative stress (p < 0.05), prevented degenerative changes in heart, and improved ECG parameters compared to diabetic control rats. The heart-to-body weight ratio was significantly reduced in rutin treatment group compared to diabetic control rats (p < 0.001). In conclusion, this study implicates that oxidative stress and inflammation are the central players involved in the progression of DCM and rutin ameliorates DCM through its antioxidant and anti-inflammatory actions on heart.

Clinical biomarkers and molecular basis for optimized treatment of diabetic retinopathy: current status and future prospects.

Diabetic retinopathy is a highly specific microvascular complication of diabetes and a leading cause of blindness worldwide. It is triggered by hyperglycemia which causes increased oxidative stress leading to an adaptive inflammatory assault to the neuroretinal tissue and microvasculature. Prolonged hyperglycemia causes increased polyol pathway flux, increased formation of advanced glycation end-products, abnormal activation of signaling cascades such as activation of protein kinase C (PKC) pathway, increased hexosamine pathway flux, and peripheral nerve damage. All these changes lead to increased oxidative stress and inflammatory assault to the retina resulting in structural and functional changes. In addition, neuroretinal alterations affect diabetes progression. The most effective way to manage diabetic retinopathy is by primary prevention such as hyperglycemia control. While the current mainstay for the management of severe and proliferative diabetic retinopathy is laser photocoagulation, its role is diminishing with the development of newer drugs including corticosteroids, antioxidants, and antiangiogenic and anti-VEGF agents which work as an adjunct to laser therapy or independently. The current pharmacotherapy of diabetic retinopathy is incomplete as a sole treatment option in view of limited efficacy and short-term effect. There is a definite clinical need to develop new pharmacological therapies for diabetic retinopathy, particularly ones which would be effective through the oral route and help recover lost vision. The increasing understanding of the mechanisms of diabetic retinopathy and its biomarkers is likely to help generate better and more effective medications.