Newly synthesized acridone derivatives targeting lung cancer: A toxicity and xenograft model study.

AKT is one of the overexpressed targets in nonsmall cell lung cancer (NSCLC) and plays an important role in its progression and offers an attractive target for the therapy. The PI3K/AKT/mTOR pathway is upregulated in NSCLC. Acridone is an important heterocycle compound which treats cancer through various mechanisms including AKT as a target. In the present work, the study was designed to evaluate the safety profile of three acridone derivatives (AC-2, AC-7, and AC-26) by acute and repeated dose oral toxicity. In addition to this, we also checked the pAKT overexpression and its control by these derivatives in tumor xenograft model. The results from acute and repeated dose toxicity showed these compounds to be highly safe and free from any toxicity, mortality, or significant alteration in body weight, food, and water intake in the rats. In the repeated dose toxicity, compounds showed negligible variations in a few hematological parameters at 400 mg/kg. The histopathology, biochemical, and urine parameters remained unchanged. The xenograft model study demonstrated AC-2 to be inhibiting HOP-62 induced tumor via reduction in p-AKT1 (Ser473) expression significantly. In immunofluorescence staining AC-2 treated tissue section showed 2.5 fold reduction in the expression of p-AKT1 (Ser473). Histopathology studies showed the destruction of tumor cells with increased necrosis after treatment. The study concluded that AC-2 causes cell necrosis in tumor cells via blocking the p-AKT1 expression. The findings may provide a strong basis for further clinical applications of acridone derivatives in NSCLC.

Phage Display Identified Novel Leydig Cell Homing Peptides for Testicular Targeting.

Conventional drug delivery methods to treat testicular disorders face various challenges, which could be circumvented by using targeted drug delivery. Testicular cell targeting ligands, such as Leydig cell homing peptides, would be an excellent choice to achieve the targeted delivery of drugs to the testis. In this study, Leydig cell homing peptides (LCHPs), LCHP1 and LCHP2, were identified via in vitro, followed by in vivo biopanning of a phage display peptide library and next-generation sequencing. Both of the LCHPs were validated in vitro for their specific Leydig cell and in vivo testis targeting potential. Furthermore, molecular targets of the LCHP1 and LCHP2 were identified using affinity purification mass spectrometry (APMS). The LCHP1 and LCHP2 are able to specifically target Leydig cells of the testis and undergo cell internalization as well as target the testis at the in vivo level, hence providing an opportunity to be utilized as a potential ligand for drug delivery to the testis.

Development and Characterization of Modified Chitosan Lipopolyplex for an Effective siRNA Delivery.

Cytotoxicity, speedy degradation, and limited cellular absorption are the foremost features influencing the successful delivery of RNAs. Chitosan (Cs) is a polymer that offers an advantage due to its bio-compatibility and biodegradable nature, making it an ideal polycationic vector for delivering siRNA. In this study, chitosan has been modified with arginine in order to increase its encapsulation of siRNA and improve cellular absorption. It was discovered that arginine and guanidino moieties could transport through membranes of cells and play an important part in membrane permeability. FTIR and 13C NMR were used to characterize the complex. These chitosan-arginine (CsAr) siRNA complexes are further encapsulated in anionic DPPC/cholesterol liposomes to combine the effects of liposome-chitosan-arginine complexes called lipopolyplexes (LCAr). Formed LCAr were investigated for their lipid/CsAr-siRNA ratios, size, zeta-potential, heparin, and serum RNase stability by agarose gel retardation, and cell uptake efficiency compared to their "parent" polyplexes. Results revealed complete lipidation of CsAr-siRNA polyplexes at lipid mass ratio 10 resulting in lipopolyplexes in the 120 to 230nm range. Polyplex entrapped ~70% of siRNA, whereas lipidation increases siRNA encapsulation to ~95%. Developed LCAr showed ~4 times less hemolytic potential as compared to the parent polyplexes at the highest siRNA dose. The CsAr-siRNA and its lipid-coated form showed enhanced cellular association as compared to the marketed Lipofectamine 2000 proving its effectiveness in siRNA delivery. CsAr-liposome conjugation is simple and safe, and serves as a robust carrier for gene transport in physiological situations without compromising transfection efficacy.

Analysis of RNA Interference Targeted Against Human Antigen R (HuR) to Reduce Vascular Endothelial Growth Factor (VEGF) Protein Expression in Human Retinal Pigment Epithelial Cells.

VEGF-A or vascular endothelial growth factor-A is an important factor in enabling neovascularization and angiogenesis. VEGF-A is regulated transcriptionally as well as post transcriptionally. Human antigen R (HuR) belonging to the embryonic lethal abnormal vision (ELAV) family is a key regulator promoting stabilization of VEGF-A mRNA. In this research we investigate, whether HuR targeted RNA interference would enable the reduction of the VEGF-A protein in human retinal pigment epithelial cells (ARPE-19) in-vitro, in normoxic conditions. Three siRNA molecules with sequences complementary to three regions of the HuR mRNA were designed. The three designed siRNA molecules were individually transfected in ARPE-19 cells using Lipofectamine™2000 reagent. Post-transfection (24 h, 48 h, 72 h), downregulation of HuR mRNA was estimated by real-time polymerase reaction, while HuR protein and VEGF-A protein levels were semi-quantitatively determined by western blotting techniques. VEGF-A protein levels were additionally quantified using ELISA techniques. All experiments were done in triplicate. The designed siRNA could successfully downregulate HuR mRNA with concomitant decreases in HuR and VEGF-A protein. The study reveals that HuR downregulation can prominently downregulate VEGF-A, making the protein a target for therapy against pathological angiogenesis conditions such as diabetic retinopathy.

Identification and characterization of phage display-selected peptides having affinity to Peste des petits ruminants virus.

Phage display is a well-established technique used for selecting novel ligands having affinity to a plethora of targets including proteins, viruses, whole bacterial and mammalian cells as well as lipid targets. In the present study, phage display technology was used to identify peptides having affinity to PPRV. The binding capacity of these peptides was characterized through various formats of ELISA using phage clones, linear and multiple antigenic peptides. The whole PPRV was used as an immobilized target in a surface biopanning process using a 12-mer phage display random peptide library. After five rounds of biopanning, forty colonies were picked and amplified followed by DNA isolation and amplification for sequencing. Sequencing suggested 12 different clones expressing different peptide sequence Phage-ELISA was performed using all 12 phage clones. Results indicated that four phage clones i.e., P4, P8, P9 and P12 had a specific binding activity to PPR virus. Linear peptides displayed by all 12 clones were synthesized using solid phase peptide synthesis and subjected to virus capture ELISA. No significant binding of the linear peptides with PPRV was evident which may be due to loss of conformation of linear peptide after coating. When the four selected phage clones displayed peptide sequences were synthesized in Multiple antigenic peptide (MAP) format and used in virus capture ELISA, the results indicated significant binding of PPRV to the MAPs. It may be due to increased avidity and/or better projection of binding residues in 4-armed MAPs as compared to linear peptides. MAP-peptides were also conjugated on gold nanoparticles (AuNPs). Visual colour change from wine red to purple was observed on addition of PPRV in MAP-conjugated AuNPs solution. This colour change may be attributable to the networking of PPRV with MAP -conjugated AuNPs resulting in aggregation of AuNPs. All these results supported the hypothesis that the phage display selected peptides were capable of binding to the PPRV. The potential of these peptides to develop novel diagnostic or therapeutic agents remains to be investigated.

Liposome-polyethylenimine complexes for the effective delivery of HuR siRNA in the treatment of diabetic retinopathy.

Diabetic retinopathy (DR) is a vision-impairing complication of diabetes, damaging the retinal microcirculatory system. Overexpression of VEGF (vascular endothelial growth factor) is implicated in the pathogenesis of DR. Human antigen R (HuR) is an RNA-binding protein that favorably regulates VEGF protein expression by binding to VEGF-encoding mRNA. Downregulating HuR via RNA interference strategies using small interfering RNAs (siRNAs) may constitute a novel therapeutic method for preventing VEGF protein overexpression in DR. Delivery of siRNAs to the cellular cytoplasm can be facilitated by cationic peptides or polymers and lipids. In this study, a cationic polymer (polyethylenimine (PEI)) and lipid nanoparticles (liposomes) were co-formulated with siRNA to form lipopolyplexes (LPPs) for the delivery of HuR siRNA. LPPs-siRNA were analyzed for size, zeta potential, serum stability, RNase stability, heparin stability, toxicity, and siRNA encapsulation efficiency. Cellular uptake, downregulation of the target HuR (mRNA and protein), and associated VEGF protein were used to demonstrate the biological efficacy of the LPPs-HuR siRNA, in vitro (human ARPE-19 cells), and in vivo (Wistar rats). In vivo efficacy study was performed by injecting LPPs-HuR siRNA formulations into the eye of streptozotocin (STZ)-induced diabetic rats after the development of retinopathy. Our findings demonstrated that high retinal HuR and VEGF levels observed in the eyes of untreated STZ rats were lowered after LPPs-HuR siRNA administration. Our observations indicate that intravitreal treatment with HuR siRNA is a promising option for DR using LPPs as delivery agents.

Identification and validation of Sertoli cell homing peptides as molecular steering for testis targeted drug delivery.

The testicle, an organ privileged with immunity because of Blood-Testis Barrier (BTB), poses a major impediment to developing and delivering drugs to the testes. These problems can be prevented by targeting testicular cells using specific ligands, such as homing peptides. This is the first study to demonstrate the successful selection of Sertoli cell homing peptides using a phage display peptide library. The identification of peptides is performed with Sanger sequencing and high-throughput NGS. The Sertoli cell and testis targeting potential of the SCHP1 and SCHP2 was confirmed using confocal microscopy and flow cytometry of the FITC-labelled peptides and in vivo bio-distribution of the corresponding Cy5.5-tagged peptides. Secondary structures were predicted in the setting of different polarity by circular dichroism. The results suggest that SCHP1 and SCHP2 can effectively target Sertoli cells. In vivo bio-distribution in mouse models indicated significantly higher uptake of SCHP1 and SCHP2 by testes compared with the heart, brain, and spleen. SCHP1 and SCHP2 can be adopted as molecular steering for targeted male contraceptive delivery, treatment of testicular cancer, and male infertility. Further development of the peptides into peptidomimetics may increase their stability, and information on the molecular targets of these peptides may reveal their therapeutic potential.

Evaluation of Pharmacokinetics, Biodistribution, and Antimalarial Efficacy of Artemether-Loaded Polymeric Nanorods.

Artemether oily injection is recommended for the treatment of severe malaria by the intramuscular route. The major limitations of the artemisinin combination therapy are erratic absorption from the injection site and high dosing frequency due to a very short elimination half-life of the drug. Advanced drug delivery systems have shown significant improvement in the current malaria therapy; the desired drug concentration within infected erythrocytes is yet the major challenge. Recently, we have reported the fabrication of artemether-loaded polymeric nanorods for intravenous malaria therapy which was found to be biocompatible with THP-1 monocytes and rat erythrocytes. The objective of the present study was the evaluation of pharmacokinetics, biodistribution, and antimalarial efficacy of artemether-loaded polymeric nanorods. Scanning electron microscopy and confocal microscopy studies revealed that both nanospheres and nanorods were adsorbed onto the surface of rat erythrocytes after an incubation of 10 min. After intravenous administration to rats, artemether nanorods showed higher plasma concentration and lower elimination rate of artemether when compared with nanospheres. The biodistribution studies showed that, at 30 min, the liver concentration of DiR-loaded nanospheres was higher than that of DiR-loaded nanorods after intravenous administration to BALB/c mice. The in vitro schizont inhibition study showed that both nanorods and nanospheres exhibited concentration-dependent parasitic inhibition, wherein at lower concentrations (2 ppm), nanorods were more effective than nanospheres. However, at higher concentrations, nanospheres were found to be more effective. Nanorods showed higher chemosuppression on day 5 and day 7 than nanospheres and free artemether when studied with the Plasmodium berghei mouse model. Moreover, the survival rate of P. berghei infected mice was also found to be higher after treatment with artemether nanoformulations when compared with free artemether. In conclusion, polymeric nanorods could be a promising next-generation delivery system for the treatment of malaria.

Targeted delivery of ursolic acid and oleanolic acid to lungs in the form of an inhaler for the management of tuberculosis: Pharmacokinetic and toxicity assessment.

INTRODUCTION: Ursolic acid (UA) and oleanolic acid (OA) are triterpenoids. They are used to treat numerous diseases, including tuberculosis. Combinations of these drugs provide new insight into the management of tuberculosis. The major obstacle is the effective delivery of these drugs to the lungs, which are mainly affected due to M. tuberculosis. A metered-dose inhaler (MDI) was developed to address this issue containing UA and OA, followed by in-vitro and in-vivo evaluation. METHODS: In the present study, MDI formulation was prepared by incorporating UA and OA at the dose level of 120 µg/ml in each actuation. In-vitro evaluation of this MDI formulation was performed to ensure its suitability to deliver UA and OA preciously. With prior approval of IAEC, a pharmacokinetic and acute inhalation toxicity study was conducted using MDI on Wistar rats. RESULTS: The pharmacokinetic study showed an increased biological half-life of UA (9.23±0.104 h) and OA (8.93±0.166 h) in combination therapy. In-vivo toxicity study demonstrated no adverse effects on body weight and vital organs in the treatment group compared with the control group. Histopathology examination of these essential organs showed no abnormalities. Mild alternation in the biochemical and hematological parameters was observed. However, these alterations did not affect the overall health of the animals. CONCLUSION: The present study documents a detailed study for the safety and pharmacokinetics of UA and OA in-vivo for their advanced application in tuberculosis disease.

FSHR antagonists can trigger a PCOS-like state.

Over the recent years, FSHR has become an important target for development of fertility regulating agents, as impairment of FSH-FSHR interaction can lead to subfertility or infertility. In our previous study, we identified a 9-mer peptide (FSHß (89-97)) that exhibited FSHR antagonist activity. The histopathological and biochemical observations indicated, in addition to FSHR antagonism, a striking resemblance to a PCOS-like state. These observations led us to hypothesize that use of FSHR antagonists can trigger a PCOS-like state. In the present study, to validate this hypothesis, we performed qRT-PCR validation using ovarian tissue samples from our previous study. Expression of three genes known to be differentially expressed in PCOS was evaluated and found to be similar to the PCOS state. To further test the hypothesis, theoretical simulations were carried out by using the human menstrual cycle model available in the literature. Model simulations for FSHR antagonism were indicative of increased testosterone levels, increased ratio of luteinizing hormone/follicle stimulating hormone, and stockpiling of secondary follicles, which are typical characteristics of PCOS. The findings of this study will be relevant while reviewing the utility of FSHR antagonists for fertility regulation and reproductive medicine.Abbreviations: FSH: Follicle-stimulating hormone; FSHR: Follicle-stimulating hormone receptor; cAMP: Cyclic adenosine 3'5' monophosphate; PKA: Protein kinase A; PI3K: Phosphoinositide 3-kinase; PKB: protein kinase B; ERK1/2: Extracellular signal-regulated protein kinase 1/2; MAPK: Mitogen-activated protein kinases; T: testosterone; E2: estradiol; PCOS: Polycystic ovarian syndrome; LH: luteinizing hormone; Lhcgr: luteinizing hormone/choriogonadotropin receptor; CYP17A1: cytochrome P450 family 17 subfamily A member 1; Inhba: inhibin subunit beta A; qRT-PCR: Real-Time quantitative reverse transcription polymerase chain reaction; FSHß: Follicle-stimulating hormone ß subunit; Ct: Cycle threshold; Rn18s: Rattus norvegicus 18S ribosomal RNA.

Exposure to Atrazine through gestation and lactation period led to impaired sexual maturation and subfertility in F1 male rats with congenital deformities in F2 progeny.

Several scientific reports suggest perturbed reproductive and developmental defects associated with environmental exposure to Atrazine (ATR). ATR has been associated with altered endocrine and reproductive functioning in-vivo exposed during the critical window of development. Thus, the present study investigates the effect of ATR exposure on F1-F2 male progeny exposed through gestation and lactation. F0 dams administered with ATR at doses 2, 10, 70, and 100 mg/kg b. wt/day from gestation day 6 to postnatal day 21. The F1 male rats were monitored for sexual maturation and subjected to fertility assessment on PND75. Delayed testicular descent was observed in 10, 70, and 100 mg/kg b. wt/day ATR dose with significantly lower serum testosterone, sperm count, and motility with testicular defects in F1 male. Expression of Androgen receptor (AR), Estrogen receptors (ER α and ER ß), StAR, Aromatase, and INSL-3 were upregulated at all doses indicating estrogenic/anti-androgenic activity of ATR. Fertility assessment revealed subfertility in F1 males with high (%) pre- and post-implantation loss at 10, 70, and 100 mg/kg b. wt/day dose as compared to control. Further, F2 fetuses exhibited congenital disabilities viz. decreased weight, crown-rump length, and anogenital distance with several other morphological deformities. To conclude, ATR exerted estrogenic and/or anti-androgenic activity with fetotoxic effects through the male germline.

Central residues of FSHß (89-97) peptide are not critical for FSHR binding: Implications for peptidomimetic design.

In our previous study, we had identified a 9-mer peptide (FSHß (89-97)) derived from seat belt loop of human FSHß and demonstrated its ability to function as FSHR antagonist in vivo. Structure analysis revealed that the four central residues 91STDC94 within this peptide may not be critical for receptor binding. In the present study, 91STDC94 residues were substituted with alanine to generate ΔFSHß 89-97(91STDC94/AAAA) peptide. Analogous to the parent peptide, ΔFSHß 89-97(91STDC94/AAAA) peptide inhibited binding of iodinated FSH to rat FSHR and reduced FSH-induced cAMP production. The peptide could impede granulosa cell proliferation leading to reduction in FSH-mediated ovarian weight gain in immature female rats. In these rats, peptide administration further downregulated androgen receptor and estrogen receptor-alpha expression and upregulated estrogen receptor-beta expression. The results indicate that substitution of 91STDC94 with alanine did not significantly alter FSHR antagonist activity of FSHß (89-97) peptide implying that these residues are not critical for FSH-FSHR interaction and can be replaced with non-peptidic moieties for development of more potent peptidomimetics.

Bivalirudin and sirolimus co-eluting coronary stent: Potential strategy for the prevention of stent thrombosis and restenosis.

Localized drug delivery with sustained elution characteristics from nanocarrier coated stents represents a viable therapeutic approach to circumvent concerns related to coronary stent therapy. We fabricated a Sirolimus (SRL) and Bivalirudin (BIV) releasing nanoparticles (NPs) coated stent for concurrent mitigation of vascular restenosis and acute stent thrombosis. SRL NPs were prepared by nanoprecipitation method whereas the BIV vesicles were generated using hydrophobic ion pair approach followed by micellization phenomenon. MTT assay and confocal microscopic analysis indicated superior anti-proliferative activity and higher cellular uptake of SRL NPs into human coronary artery smooth muscle cells, respectively. DSC and ATR-FTIR techniques confirmed the formation of complex between BIV and phosphatidylglycerol via some weak physical interactions. More than 2 fold rise in log P value was obtained for DSPG-BIV at 3:1 M ratio compared with native BIV solution. The SAXS analysis indicated formation of oligolamellar vesicles of DSPG-BIV complex which was preferentially entrapped into lipophilic lamellae of vesicles. APTT, PT, and TT tests revealed that the BIV vesicles caused significant prolongation of clotting time compared to native BIV solution. The SEM analysis showed uniform and defect free stent coating. In vitro release study demonstrated that SRL and BIV were eluted in a sustained manner from coated stents.

Ethyl ferulate, a lipophilic phenylpropanoid, prevents diabetes-associated renal injury in rats by amelioration of hyperglycemia-induced oxidative stress via activation of nuclear factor erythroid 2-related factor 2.

Diabetic nephropathy affects approximately 20%-40% of diabetes patients worldwide and is the leading cause of end-stage renal failure. Oxidative stress has been identified as a major causative factor in the development and progression of diabetic nephropathy; Nuclear factor erythroid 2-related factor 2 (Nrf2) activation protects the body against oxidative stress by induction of antioxidant enzymes. The renoprotective effect of ethyl ferulate was investigated in diabetes-induced renal injury. Ethyl ferulate was administered orally at three doses (50 mg/kg, 75 mg/kg, and 100 mg/kg). Metformin (500 mg/kg, p.o.) was used as a standard. Ethyl ferulate treatment decreased serum advanced glycation end products, glycosylated hemoglobin (HbA1c) levels, renal oxidative stress, tumor necrosis factor-α (TNF-α) level, and kidney hypertrophy index. It restored serum lipid profile, biomarkers of renal function, and mitigated histopathological signs of renal damage. Immunohistochemistry demonstrated higher Nrf2 protein levels in kidney sections of ethyl ferulate-treated rats. These findings suggest that ethyl ferulate ameliorated hyperglycemia-induced oxidative stress by increasing renal Nrf2 levels, thereby preventing diabetes-induced kidney injury. In conclusion, the present study endorses the usefulness of Nrf2 activators, such as ethyl ferulate, as adjuvant therapy for preventing the diabetic nephropathy. PRACTICAL APPLICATIONS: Ethyl ferulate (ethyl-3-hydroxyl-4-methoxycinnamate), a phenylpropanoid, is a naturally occurring ethyl ester of ferulic acid and is widely present in plants and especially grains, such as rice and maize. Our study has highlighted the renoprotective effect of ethyl ferulate in preventing diabetes-associated renal injury. The observed effect of ethyl ferulate is due to amelioration of diabetes-induced oxidative stress and inflammation, by activation of the Nrf2 pathway. These results indicate the potential of ethyl ferulate as a nutraceutical or adjuvant therapy in prevention of diabetic nephropathy.

A 5-mer peptide derived from hinge region of hFSHR can function as positive allosteric modulator in vivo.

Interaction of follicle stimulating hormone (FSH) with its cognate receptor (FSHR) is critical for maintaining reproductive health. FSHR has a large extracellular domain (ECD), composed of leucine rich repeats (LRRs) and hinge region, a transmembrane domain (TMD) and a short C-terminal domain (CTD). In this study, we have identified a short peptidic stretch in the hinge region (hFSHR(271-275)), through extensive computational modeling, docking and MD simulations, that is capable of independently interacting with the extracellular loops of FSHR(TMD). In vitro studies revealed that FSHR(271-275) peptide increased binding of [125I]-FSH to rat Fshr as well as FSH-induced cAMP production. Administration of FSHR(271-275) peptide in immature female rats significantly increased FSH-mediated ovarian weight gain and promoted granulosa cell proliferation. In summary, the results demonstrate that the synthetic peptide corresponding to amino acids 271-275 of hFSHR-hinge region stimulates FSH-FSHR interaction and behaves as positive allosteric modulator of FSHR. The study also lends evidence to the existing proposition that hinge region maintains the receptor in an inactive conformation in the absence of its ligand by engaging in intramolecular interactions with extracellular loops of TMD.

Nose-to-Brain Delivery of Diazepam from an Intranasal Aqua-Triggered In-Situ (ATIS) Gelling Microemulsion: Monitoring Brain Uptake by Microdialysis.

BACKGROUND AND OBJECTIVES: An innovative intranasal aqua-triggered in-situ (ATIS) gel is a polymer-free in-situ gelling microemulsion which gels instantaneously on contact with minute quantities of water to form a mucoadhesive gel. The objective of the study was to develop ATIS diazepam (ATIS-diazepam) as an alternative to the injection for epileptic emergencies and evaluate its brain uptake and nose-to-brain targeting efficiency in rats. METHODS: ATIS-diazepam (1 mg/100 µL) was prepared and characterized for in vitro formulation characteristics. An LC-MS/MS method was developed and validated for the bioanalysis of diazepam. In vivo studies for pharmacokinetics, brain uptake and nasal irritation of intranasal ATIS-diazepam were conducted in rats. Brain uptake was investigated with brain microdialysis, a highly sensitive technique enabling quantification of free drug, which correlates to efficacy. RESULTS: ATIS-diazepam exhibited globule size < 200 nm, low viscosity, negative zeta potential and good stability. A significant increase in mucoadhesion was exhibited by ATIS-diazepam following the addition of a small quantity of water. ATIS-diazepam showed burst release in pH 6.4 with 50% diazepam release in ~ 10 min, which was sustained over 1 h. The absolute bioavailability was ~ 50% with both intranasal free-diazepam and ATIS-diazepam. Intranasal administration of ATIS-diazepam revealed immediate absorption with rapid and high brain extracellular fluid concentration compared to intravenous free-diazepam solution. The estimated direct transport potential and drug targeting efficiency of intranasal ATIS-diazepam was significantly higher (2-fold) than intranasal free-diazepam solution, which was attributed to the mucoadhesive and microemulsion properties of ATIS-diazepam. The nasal irritation study revealed the safety of ATIS-diazepam compared to free-diazepam solution. CONCLUSION: Intranasal ATIS-diazepam showed promise of higher direct nose-to-brain targeting, better safety and hence has an immense implication in the treatment of epileptic emergencies.

Long-Acting Efavirenz and HIV-1 Fusion Inhibitor Peptide Co-loaded Polymer-Lipid Hybrid Nanoparticles: Statistical Optimization, Cellular Uptake, and In Vivo Biodistribution.

The objective of the present study was to develop long-acting efavirenz (Efa)-enfuvirtide (Enf) Co-loaded polymer-lipid hybrid nanoparticles (PLN) with improved intracellular delivery to target T-cells and macrophage cells located in multiple human immunodeficiency virus sanctuaries. The Box-Behnken design was utilized to optimize three high-risk factors, namely, Efa amount, sonication time for primary emulsion, and sonication time for aqueous nanodispersion obtained from preliminary studies. Lyophilized Efa-Enf Co-loaded PLN using trehalose elicited spherical morphology, drug amorphization on incorporation, and absence of drug-excipient interaction. In vitro release studies revealed an sustained release of both the drugs from PLN with the differential release profile. Efa-Enf Co-loaded PLN exhibited low hemolytic, platelet and leukocyte aggregation as well as low cytotoxicity in Jurkat E6.1 T-cells and U937 macrophage cells. Circular dichroism spectra confirmed the presence of an α-helix form of Enf after encapsulation in PLN. Coumarin-6-loaded PLN exhibited enhanced cellular uptake in Jurkat E6.1 T-cells and U937 macrophage cells in comparison to free coumarin-6, as evidenced by fluorescence microscopy and flow cytometry. In vivo biodistribution studies after intravenous administration of near-infrared dye-loaded PLN (surrogate for Efa-Enf PLN) revealed non-uniform distribution within 2 h in the order of spleen ≥ liver > lymph node > thymus > lungs > female reproductive tract (FRT) > heart > kidneys > brain. However, subcutaneous administration caused non-uniform biodistribution after 3 days, eliciting a long-acting slow release from the injection site depot until day 5 in the infection-spread site (lymph nodes and FRT), reservoir sites (liver and spleen) and the difficult-to-access site (brain). Furthermore, it presents a vital illustration of the available tissue-specific drug concentration prediction from simulated surrogate PLN.

Identification and in vivo validation of a 9-mer peptide derived from FSHß with FSHR antagonist activity.

FSH-FSHR interaction is critical for folliculogenesis, spermatogenesis and progression of several cancers. Therefore, FSHR is an attractive target for fertility regulation and cancer therapeutics. Based on homology and structural analysis of hFSH-FSHR(ECD) complex, a minimal continuous stretch within FSHß seat-belt loop (FSHß (89-97)) was identified to be crucial for FSHR interaction. The ability of FSHß (89-97) peptide to neutralize FSHR activity was evaluated by a panel of in vitro and in vivo experiments. The synthetic peptide significantly inhibited binding of [125I]-FSH to rat Fshr as well as FSH-induced cAMP production. In immature rats, FSHß (89-97) peptide administration reduced FSH-mediated increase in ovarian weight. The peptide inhibited transition of follicles from pre-antral to antral stage and hindered the cell cycle progression of granulosa cells beyond G0/G1 phase. In adult rats, administration of the peptide inhibited estradiol synthesis and significantly perturbed folliculogenesis.

Mechanistic evaluation of Ursolic acid against rotenone induced Parkinson's disease- emphasizing the role of mitochondrial biogenesis.

Parkinson's disease (PD) is an age associated, progressive and a second most common neurodegenerative disease. It is caused due to degeneration of dopaminergic neurons in substantia nigra (SN). Various studies implicate mitochondrial dysfunction, oxidative stress, altered degradation of misfolded proteins in PD pathogenesis. Ursolic acid (UA), a natural pentacyclic triterpenoid carboxylic acid, is reported to possess a number of biological activities viz. anti-oxidant, anti-inflammatory etc. The focus of our study was to assess the neuroprotective potential of UA against the rotenone induced pathophysiological alterations. In this study rats were subjected to stereotaxic bilateral injection of rotenone (12 µg/µl) in SN. Further, they were treated per-orally with UA (5 and 10 mg/kg) for 30 days. During the study, neurobehavioral tests comprising Rota-rod, Open field and Barnes maze (BMT) were conducted. At the end of 30 days, the antioxidant (Reduced glutathione, superoxide dismutase, catalase and lipid peroxidation), inflammatory (TNF-α) parameters, mitochondrial complex I, mitochondrial biogenesis (MB) and immunohistochemical analysis (TH positive neurons, Glial Fibrillary Acidic Protein (GFAP)) was performed. The results exhibited significant amelioration in the motor deficits by UA which can be attributed to the protection of TH positive neurons from degeneration. A significant improvement in the cognitive function due to UA was observed in BMT. Biochemically, the oxidative stress and inflammation triggered by rotenone was significantly diminished by UA. It also significantly obviated the complex I inhibition and promoted MB. The preliminary results thus firmly advocate the neuroprotective potential of UA to prevent rotenone induced neurotoxicity in rats.

Cystic fibrosis transmembrane conductance regulator-related male infertility: Relevance of genetic testing & counselling in Indian population.

BACKGROUND & OBJECTIVES: Due to limited information available on the frequency and spectrum of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene mutations in congenital bilateral absence of vas deferens (CBAVD) in Indian population, it is difficult to provide accurate genetic counselling to couples. The present study was undertaken to investigate the spectrum and frequency of CFTR gene mutations in Indian men with CBAVD and to determine the female CF carrier status. METHODS: Direct DNA sequencing of the CFTR gene was carried out in eighty CBAVD men, their female partners and fifty controls from the general population. Pathological significance of the identified novel CFTR gene variants was carried out using in silico tools. Appropriate genetic counselling was provided to the couples prior to intracytoplasmic sperm injection (ICSI). RESULTS: A significant association was observed for CFTR gene variants in Indian CBAVD men versus controls (odds ratio: 12.1; 95% confidence interval: 4.8-30.4; P<0.0001). A total of 20 CFTR gene variants were identified in 53 CBAVD men. Eight novel missense CFTR gene variants (L214V, A238P, E379V, L578I, F587L, L926W, R1325K and R1453Q); two novel splice-site gene variants (c.1-30C>G and IVS1+2T>G) and ten previously reported mutations (R75Q, c.1210-12[5], F508del, A309G, R334W, I444T, R668C, R709X, A1285V and Q1352H) were detected in CBAVD men. The novel and reported CFTR gene mutations were L926W (2.5%, P=0.26), R1453Q (2.5%, P=0.26), F508del (8.75%, P=0.03) and c.1210-12[5] (42.5%, P=0.002). A total of 13 (16.2%) female partners were found to be a CF carrier. Nine couples had a risk of transmitting mutant CFTR allele to the offspring. INTERPRETATION & CONCLUSIONS: The heterogeneous spectrum of CFTR gene in Indian population suggests the necessity of screening CBAVD men and female partners for accurate genetic counselling prior to undergoing ICSI.