There is a need to establish consensus for harmonization in antiretroviral (ARV) therapy (ART) switch treatment strategy and address the dilemma that exists in terms of subpar immune response to therapy or an immunologic deterioration while on therapy. The purpose of this review is to identify the factors that contribute to ARV treatment failure, such as insufficient dosage, drug interactions, poor adherence, drug resistance, and poor medication absorption. It is crucial to adopt a more efficient strategy to address this challenging dilemma. After ARV treatment failure, the aim of therapy is virologic suppression, which targets plasma viral load below the limits of detection as assessed by very sensitive tests with lower limits of quantification of 20 to 75 RNA copies/ml. The therapeutic objectives when complete virologic suppression is not possible, should be to maintain or restore immunologic function, stop the progression of the clinical illness, and minimize the emergence of new drug resistance that could further restrict the options for ARV drugs. Treatment history and drug-resistance testing, including the findings of previous and ongoing resistance tests, should be considered while selecting ARV regimens. Hence, the treatment approach postARV failure can be personalized based on clinical, immunologic, virologic, or as a mix of the three domains on a case-to-case basis. The evaluation of projected ARV activity should be based on treatment history and previous resistance test findings.

Background: In patients with diabetes, the endothelial function precedes and predicts the risk of cardiovascular disorders. Current treatment approaches fail to decrease unwanted cardiovascular outcomes in these patients. The blood levels of microRNA-122 (miR-122) elevated in patients with diabetes and correlated with risk and severity of cardiovascular disorders. Therefore, targeting miR-122 with efficient and safer inhibitors could be a promising approach. The objective of this study was to investigate the effectiveness of gamma peptide nucleic acid (γPNA)-based miR-122 inhibitor in preventing the development of endothelial dysfunction in pre-diabetic mice. Methods: To test the effectiveness of γPNA technology in inhibiting miR-122, we synthesized γPNA-miR-122 inhibitor (γP-122-I) and γPNA-scrambled control (γP-SC). The purity and molecular weight of γP-122-I and γP-SC were assessed by HPLC and MALDI spectrometry, respectively. The binding and affinity of γP-122-I were determined by gel-shift assay and thermal denaturation. The insulin-resistance (induced by high-fat-diet, HFD) mice were injected with γP-122-I (5 mg/kg/day, i.p., 6 weeks). miR-122 expression was measured by real-time PCR. The safety of γP-122-I was evaluated by biochemical and histological analysis of vital organs (liver & kidney). The vascular endothelial function was assessed by determining endothelium-dependent and independent relaxation of phenylephrine-induced precontracted aortic rings. Results were expressed as mean ± s.e.m. Results: The HPLC of γPNA oligomers show that both γP-122-I and γP-SC were >95% pure. The molecular weight of γP-122-I (m/z: 9227.4) and γP-SC (m/z: 9232.7) were confirmed by MALDI spectrometry. γP-122-I strongly binds with miR-122 and forms a stable complex compared to commercially available miR-122 inhibitor. A comprehensive safety analysis of γP-122-I in mice shows that neither it affected liver and kidney function nor their morphology during acute and chronic exposure, respectively. Next, HFD-fed mice show higher serum and aortic miR-122 levels, endothelial dysfunction, and inflammation, which were reversed by γP-122-I treatment. Conclusions: γP-122-I inhibits the development of impaired endothelial function in pre-diabetic mice without any evidence of toxicity

ABSTRACT Background: Antiretroviral therapy (ART) is the cornerstone for the treatment of human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS). Our study aimed to compare the impact of early versus delayed access to ART over clinical and immunological outcomes in HIV-positive adults. Methods: The prospective, randomized, open-label study was conducted for nine months, and comprised HIV-positive adults who presented to the ART center. Patients who presented early in their course of disease with baseline cluster of differentiation (CD) 4 count ≥350/mm3 were recruited in the early arm and in the late arm, if <350/mm3. The primary objectives were to evaluate disease progression in terms of the Centers for Disease Control and Prevention (CDC) stages, functional status, and opportunistic infections. Statistical analysis was done by applying an unpaired t-test, analysis of variance (ANOVA), Chi-square test, and Kaplan–Meier analysis with a P value <0.05 as significant at a 95% confidence interval. Results: A total of 134 HIV-positive patients meeting eligibility criteria were randomized. All patients including 60 in the early and 74 in the late arm received tenofovir + lamivudine + efavirenz (TLE). There was a significant difference in CDC stages and immunological status at baseline and post ART initiation (p-value < 0.001). TB-HIV co-infections were significantly (p-value = 0.006) more in late arm. Conclusion: The study suggests CD4 counts at ART initiation, as the most important factor in predicting post-treatment recovery in terms of clinical and immunological outcomes.

Since their inception, biomaterial-based systems have played a central role in various biomedical applications. However, there are still gaps in knowledge about their in vivo fate which limits their applications. Understanding these gaps is the key to developing next generation materials that are easy to translate to a clinical setting. In this chapter, we will discuss a few examples of widely used nanocarriers (NCs) with known immune interactions and clearance, and their success in preclinical and clinical models. We will further discuss certain barriers to translation and how to overcome them.

Peptide nucleic acids (PNAs) are nucleic acid analogs with superior hybridization properties and enzymatic stability than deoxyribonucleic acid (DNA). In addition to gene targeting applications, PNAs have garnered significant attention as bio-polymer due to the Watson-Crick -based molecular recognition and flexibility of synthesis. Here, we engineered PNA amphiphiles using chemically modified gamma PNA (8 mer in length) containing hydrophilic diethylene glycol units at the gamma position and covalently conjugated lauric acid (C12) as a hydrophobic moiety. Gamma PNA (γPNA) amphiphiles self-assemble into spherical vesicles. Further, we formulate nano-assemblies using the amphiphilic γPNA as a polymer via ethanol injection-based protocols. We perform comprehensive head-on comparison of the physicochemical and cellular uptake properties of PNA derived self- and nano-assemblies. Small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) analysis reveal ellipsoidal morphology of γPNA nano-assemblies that results in superior cellular delivery compate to the spherical self-assembly. Next, we compare the functional activities of γPNA self-and nano-assemblies in lymphoma cells via multiple endpoints, including gene expression, cell viability, and apoptosis-based assays. Overall, we establish that γPNA amphiphile is a functionally active bio-polymer to formulate nano-assemblies for a wide range of biomedical applications.

Ethnopharmacological relevance: Rheumatoid arthritis is a chronic inflammatory disease of joints. Dysoxylum binectariferum Hook.f (Family: Meliaceae) is a Indian medicinal plant which is traditionally being used to heal inflammation of joints. Aim of the study: This work was aimed to carry out chemical standardization, in-vitro/in-vivo validation, oral pharmacokinetics and formulation development of anti-arthritic botanical lead, the rohitukine-enriched fraction of D. binectariferum. Materials and methods: The rohitukine-enriched fraction of D. binectariferum was standardized using four chemical markers and was checked for microbial load, heavy metal content, aflatoxins and pesticides. Its in-vitro inhibitory effect on the lipopolysaccharide (LPS) induced production of pro-inflammatory cytokines TNF-alpha and IL-6 was studied in THP-1 cells. The in-vivo anti-arthritic activity was investigated in collagen-induced arthritis model in DBA/1J mice. The sustained release capsule formulation was developed and characterized for physicochemical and pharmacokinetic properties. Results: Rohitukine and schumaniofioside A were found to be major chemical constituents of the botanical lead. The rohitukine-enriched fraction of D. binectariferum significantly reduced the production of both pro-inflammatory cytokines TNF-alpha and IL-6 ( > 50% inhibition at 3.12 mu g/mL) in THP-1 cells. In LPS-treated wild-type mice model, the rohitukine-enriched fraction at 200 mg/kg (PO, QD) completely reduced serum TNF-alpha levels. In transgenic mice model (collagen-induced arthritis in DBA/1J mice), rohitukine-enriched fraction at 100 mg/kg (PO, QD) dose has resulted in > 75% reduction of TNF-alpha/IL-6 serum levels, 68% reduction in anti-mouse type II collagen IgG1 antibody levels, decreased joint proteoglycan loss and reduced paw edema in DBA/1J mice. The sustained release capsule formulation of rohitukine-enriched fraction showed sustained-release of rohitukine over the period of 24 h, and resulted in an improved plasma-exposure of rohitukine in SD rats. Conclusions: The data presented herein demonstrated anti-arthritic potential of rohitukine-enriched fraction of D. binectariferum and this study will serve as the benchmark for further research on this botanical lead and developed sustained release capsule formulation.

Objective: Bergenia ciliata (Haw.) Sternb. is used in the Indian traditional system of medicine to treat various ailments including rheumatism and to heal wounds. The objective of the present study was to perform a preclinical characterization of the B. ciliata-based botanical extract IIIM-160. Methods: IIIM-160 was chemically standardized and analyzed for heavy metal content, aflatoxins, pesticides and microbial load. The in vitro and in vivo efficacies were determined in suitable models of inflammation, arthritis and nociception. An acute oral toxicity study was performed in Swiss albino mice. A suitable oral formulation was developed and characterized. Results: Bergenin was found to be the major component (9.1% w/w) of IIIM-160. The botanical lead displayed inhibition of lipopolysaccharide-induced production of proinflammatory cytokines in THP-1 cells, with selectivity toward interleukin-6 (IL-6) and had an excellent safety-window. It showed anti-inflammatory, anti-arthritic and antinociceptive activity in animal models and was not toxic at oral doses up to 2 g/kg in Swiss-albino mice. The gastroretentive, sustained-release capsule formulation showed sustained-release of the bergenin over the period of 24 h, resulting in improved plasma-exposure of bergenin in Sprague-Dawley rats. Conclusion: The dual-activity of IL-6 inhibition and antinociception marks the suitability of IIIM-160 for treating rheumatoid arthritis. This study will serve as the benchmark for further research on this botanical formulation. (C) 2019 Shanghai Changhai Hospital. Published by Elsevier B.V. All rights reserved.

IIIM-290, a semisynthetic derivative of natural product rohitukine, is an orally bioavailable Cdk inhibitor, efficacious in the xenograft models of colon, pancreatic, and leukemia cancer. Its low aqueous solubility (∼8.6 μg/mL) could be one of the reasons for achieving optimal in vivo efficacy relatively at a higher dose. Being a nitrogenous compound, salt formation was envisaged as one of the ideal approaches to enhance its solubility and dissolution profile. Thus, herein, a solubility-guided miniaturized 96-well plate salt screening protocol was devised for identification of the suitable salt form of this preclinical candidate. The solubility-guided strategy has resulted in the identification of hydrochloride as the most favorable counterion, resulting in 45-fold improvement in aqueous solubility. The HCl salt was then scaled up at a gram size and characterized using 1H and 13C NMR, scanning electron microscopy, powder X-ray diffraction, Fourier-transform infrared, and differential scanning calorimetry studies. The HCl salt displayed enhancement in the in vitro dissolution profile as well as improved plasma exposure in the pharmacokinetic study. The oral administration of the IIIM-290·HCl salt in BALB/c mice resulted in >1.5-fold improvement in areas under the curve, Cmax, and half-life. The prepared salt also did not alter its cyclin-dependent kinase (Cdk)-2 and Cdk-9 inhibition activity. This biopharmaceutically improved lead has a potential to investigate further in preclinical studies. The solubility-guided salt screening strategy implemented herein could be utilized for other preclinical leads.

Bergenin is a unique C-glycoside natural product possessing anti-inflammatory and anti-arthritic activity. It is hydrophilic molecule and stable under acidic conditions however is unstable at neutral-basic pH conditions. The rate of degradation is directly proportional to the increase in pH which might be one of the reasons for its low oral bioavailability. Thus, herein our objective was to improve its stability using prodrug strategy. Various ester and ether prodrugs were synthesized and studied for lipophilicity, chemical stability and enzymatic hydrolysis in plasma/esterase. The stability of synthesized prodrugs was evaluated in buffers at different pH, in biorelevant media such as SGF, SIF, rat plasma and in esterase enzyme. All prodrugs displayed significantly improved lipophilicity compared with bergenin, which was in accordance with the criteria of drug-like compounds. Acetyl ester 4a2 appeared to be the most promising prodrug as it remained stable at gastric/intestinal pH and was completely transformed to the parent compound bergenin in plasma as desired for an ideal prodrug. The data presented herein, will help in designing stable prodrugs of unstable molecules with desired physicochemical properties in structurally similar chemotypes.

The structure of alpha-napthoflavone (ANF), a potent inhibitor of CYP1A1 and CYP1B1, mimics the structure of chalcones. Two potent CYP1B1 inhibitors 7k (DMU2105) and 6j (DMU2139) have been identified from two series of synthetic pyridylchalcones. They inhibit human CYP1B1 enzyme bound to yeast-derived microsomes (Sacchrosomes™) with IC50 values of 10 and 9 nM, respectively, and show a very high level of selectivity towards CYP1B1 with respect to the IC50 values obtained with CYP1A1, CYP1A2, CYP3A4, CYP2D6, CYP2C9 and CYP2C19 Sacchrosomes™. Both compounds also potently inhibit CYP1B1 expressed within 'live' recombinant yeast and human HEK293 kidney cells with IC50 values of 63, 65, and 4, 4 nM, respectively. Furthermore, the synthesized pyridylchalcones possess better solubility and lipophilicity values than ANF. Both compounds overcome cisplatin-resistance in HEK293 and A2780 cells which results from CYP1B1 overexpression. These potent cell-permeable and water-soluble CYP1B1 inhibitors are likely to have useful roles in the treatment of cancer, glaucoma, ischemia and obesity.

The 66-kDa Src homology 2 domain-containing protein (p66Shc) is a master regulator of reactive oxygen species (ROS). It is expressed in many tissues where it contributes to organ dysfunction by promoting oxidative stress. In the vasculature, p66Shc-induced ROS engenders endothelial dysfunction. Here we show that p66Shc is a direct target of the Sirtuin1 lysine deacetylase (Sirt1), and Sirt1-regulated acetylation of p66Shc governs its capacity to induce ROS. Using diabetes as an oxidative stimulus, we demonstrate that p66Shc is acetylated under high glucose conditions and is deacetylated by Sirt1 on lysine 81. High glucose-stimulated lysine acetylation of p66Shc facilitates its phosphorylation on serine 36 and translocation to the mitochondria, where it promotes hydrogen peroxide production. Endothelium-specific transgenic and global knockin mice expressing p66Shc that is not acetylatable on lysine 81 are protected from diabetic oxidative stress and vascular endothelial dysfunction. These findings show that p66Shc is a target of Sirt1, uncover a unique Sirt1-regulated lysine acetylation-dependent mechanism that governs the oxidative function of p66Shc, and demonstrate the importance of p66Shc lysine acetylation in vascular oxidative stress and diabetic vascular pathophysiology.

In the present study, a novel series of 3-pyrimidinylazaindoles were designed and synthesized using a bioinformatics strategy as cyclin-dependent kinases CDK2 and CDK9 inhibitors, which play critical roles in the cell cycle control and regulation of cell transcription. The present approach gives new dimensions to the existing SAR and opens a new opportunity for the lead optimizations from comparatively inexpensive starting materials. The study led to the identification of the alternative lead candidate 4ab with a nanomolar potency against CDK2 and CDK9 and potent antiproliferative activities against a panel of tested tumor cell lines along with a better safety ratio of ∼33 in comparison to reported leads. In addition, the identified lead 4ab demonstrated a good solubility and an acceptable in vivo PK profile. The identified lead 4ab showed an in vivo efficacy in mouse triple-negative breast cancer (TNBC) syngeneic models with a TGI (tumor growth inhibition) of 90% without any mortality growth inhibition in comparison to reported leads.

Crocus sativus, commonly known as saffron or Kesar, is used in Ayurveda and other folk medicines for various purposes as an aphrodisiac, antispasmodic, and expectorant. Previous evidence suggested that Crocus sativus is linked to improving cognitive function in Alzheimer's disease (AD) patients. The aim of this study was to in vitro and in vivo investigate the mechanism(s) by which Crocus sativus exerts its positive effect against AD. The effect of Crocus sativus extract on Aβ load and related toxicity was evaluated. In vitro results showed that Crocus sativus extract increases the tightness of a cell-based blood-brain barrier (BBB) model and enhances transport of Aβ. Further in vivo studies confirmed the effect of Crocus sativus extract (50 mg/kg/day, added to mice diet) on the BBB tightness and function that was associated with reduced Aβ load and related pathological changes in 5XFAD mice used as an AD model. Reduced Aβ load could be explained, at least in part, by Crocus sativus extract effect to enhance Aβ clearance pathways including BBB clearance, enzymatic degradation and ApoE clearance pathway. Furthermore, Crocus sativus extract upregulated synaptic proteins and reduced neuroinflammation associated with Aβ pathology in the brains of 5XFAD mice. Crocin, a major active constituent of Crocus sativus and known for its antioxidant and anti-inflammatory effect, was also tested separately in vivo in 5XFAD mice. Crocin (10 mg/kg/day) was able to reduce Aβ load but to a lesser extent when compared to Crocus sativus extract. Collectively, findings from this study support the positive effect of Crocus sativus against AD by reducing Aβ pathological manifestations.

Rohitukine is a chromone alkaloid isolated from an Indian medicinal plant Dysoxylum binectariferum. This natural product has led to the discovery of two clinical candidates (flavopiridol and P276-00) for the treatment of cancer. Herein, for the first time we report an efficient protocol for isolation and purification of this precious natural product in a bulk-quantity from leaves (a renewable source) of D. binectariferum (>98% purity) without use of chromatography or any acid–base treatment. Despite of the fact that this scaffold has reached up to clinical stage, particularly for leukemia; however the antileukemic activity of a parent natural product has never been investigated. Furthermore, rohitukine has never been studied for cyclin-dependent kinase (Cdk) inhibition, kinase profiling and for its experimental physicochemical properties. Thus, herein, we report in vitro cytotoxicity of rohitukine in a panel of 20 cancer cell lines (including leukemia, pancreatic, prostate, breast and CNS) and 2 normal cell lines; kinase profiling, Cdk2/9 inhibition, and physicochemical properties (solubility and stability in biological medias, pKa, LogP, LogD). In cytotoxicity screening, rohitukine displayed promising activity in HL-60 and Molt-4 (leukemia) cell lines with GI50 of 10 and 12μM, respectively. It showed inhibition of Cdk2/A and Cdk9/T1 with IC50 values of 7.3 and 0.3μM, respectively. The key interactions of rohitukine with Cdk9 was also studied by molecular modeling. Rohitukine was found to be highly water soluble (Swater=10.3mg/mL) and its LogP value was −0.55. The ionization constant of rohitukine was found to be 5.83. Rohitukine was stable in various biological media’s including rat plasma. The data presented herein will help in designing better anticancer agents in future.

We describe the identification of 7-(prolinol-N-yl)-2-phenylamino-thiazolo[5,4-d]pyrimidines as a novel chemotype of non-nucleoside partial agonists for the A2A adenosine receptor (A2AAR). Molecular-modeling indicated that the (S)-2-hydroxymethylene-pyrrolidine could mimic the interactions of agonists' ribose, suggesting that this class of compounds could have agonistic properties. This was confirmed by functional assays on the A2AAR, where their efficacy could be associated with the presence of the 2-hydroxymethylene moiety. Additionally, the best compound displays promising affinity, selectivity profile, and physicochemical properties.