Highly purified charge variants of a proposed biosimilar to Omalizumab: impact on in vitro potency and stability under thermal stress.

Biosimilars are highly complex and similar biological drugs are developed with different manufacturing processes which are not similar to originator manufacturing process. Due to this, biosimilar products inherently have quality differences in comparison to innovator molecule which may be related to size, charge and glycosylation. Despite these differences they are supposed to demonstrate similar behaviour in safety and efficacy profile to the reference product and these differences should not be clinically meaningful. Charge variants are one of the critical quality attributes and sources of heterogeneity. In this study, highly purified charge variants cluster (acidic, main peak and basic) of biosimilar product of Xolair were assessed for their impact on in vitro potency and stability at different thermal stress conditions (2-8 °C and - 20 °C). The study data indicating purified charge variants (> 90%) have no impact on in vitro potency and are stable at different thermal stress conditions up to a week.

Charge variants of proposed biosimilar to Omalizumab: Isolation, purification and analysis by HPLC methods.

Omalizumab (Xolair) is a humanized monoclonal antibody derived by recombinant DNA technology. It binds specifically to immunoglobulin E (IgE) which plays a major role in allergic reaction by releasing histamine and other inflammatory factors from mast cells. Omalizumab binds circulatory IgE with high affinity and prevents from its binding to mast cell receptor. Charge variants are one of the critical quality attributes (CQAs) in biological drug development and sources of heterogeneity which needs to be considered in biosimilarity assessment. In this study, biosimilar product of Xolair was expressed in mammalian cell culture process in laboratory to isolate charge variants (acidic, main peak and basic). Different charge variants were isolated from intermediate purified biosimilar product of Xolair. Isolated charge variants were purified with preparative cation exchange chromatography technique and characterized with different analytical tools includes size exclusion chromatography (SEC-HPLC) and cation exchange chromatography (CEX-HPLC). Purity of acidic, main peak and basic variants was 99.58%, 99.98% and 98.64% respectively as per SEC-HPLC and according to CEX-HPLC purity was 94.25%, 95.58% and 91.33% respectively. The study data indicates that isolated charge variants were purified with desired purity and can be further used for process characterization, in vitro potency and in vivo kinetics studies.

Vitamin-H Channeled Self-Therapeutic P-gp Inhibitor Curcumin-Derived Nanomicelles for Targeting the Tumor Milieu by pH- and Enzyme-Triggered Hierarchical Disassembly.

An effective nanocarrier-mediated drug delivery to cancer cells primarily faces limitations like the presence of successive drug delivery barriers, insufficient circulation time, drug leakage, and decreased tumor penetration capacity. With the aim of addressing this paradox, a self-therapeutic, curcumin-derived copolymer was synthesized by conjugation with PEGylated biotin via enzyme- and acid-labile ester and acetal linkages. This copolymer is a prodrug of curcumin and self-assembles into ∼150-200 nm-sized nanomicelles; it is capable of encapsulating doxorubicin (DOX) and hence can be designated as self-therapeutic. pH- and enzyme-responsive linkages in the polymer skeleton assist in its hierarchical disassembly only in the tumor microenvironment. Further, the conjugation of biotin and poly(ethylene glycol) (PEG) imparts features of tumor specificity and improved circulation times to the nanocarrier. The dynamic light scattering (DLS) analysis supports this claim and demonstrates rapid swelling and disruption of micelles under acidic pH. UV-vis spectroscopy provided evidence of an accelerated acetal degradation at pH 4.0 and 5.0. The in vitro release studies revealed a controlled release of DOX under acidic conditions and curcumin release in response to the enzyme. The value of the combination index calculated on HepG2 cells was found to be <1, and hence, the drug pair curcumin and DOX acts synergistically for tumor regression. To prove the efficiency of acid-labile linkages and the prodrug strategy for effective cancer therapy, curcumin-derived polymers devoid of sensitive linkages were also prepared. The prodrug stimuli-responsive nanomicelles showed enhanced cell cytotoxicity and tumor penetration capability on HepG2 cells as well as drug-resistant MCF-7 cell lines and no effect on normal NIH/3T3 fibroblasts as compared to the nonresponsive micelles. The results were also supported by in vivo evidence on a hepatocellular carcinoma (HCC)-induced nude mice model. An evident decrease in MMP-2, MMP-9, and α-fetoprotein (AFP), the biomarkers specific to tumor progression, was observed along with metastasis upon treatment with the drug-loaded dual-responsive nanomicelles. These observations corroborated with the SGOT and SGPT data as well as the histoarchitecture of the liver tissue in mice.

Colonic Microflora Protagonist of Liver Metabolism and Gut Permeability: Study on Mice Model.

Alteration of gut microflora results in a metabolic imbalance in the liver. In the present study, we investigate the reversal potential of alteration of the colonic microflora via improving metabolism balance and regulating the altered tight junction of the intestinal tract. Animals were fed with high sugar diet to mimic the onset of the pathophysiological conditions of diabetes. Following induction, animals were divided into two reversal groups i.e., crude cefdinir and colon-specific formulated cefdinir, to alter the gut microflora. In the present study, we have tried to quantify the microbial content via metagenome analysis to provide an actual picture of the alteration and subsequent reversal. Expression of mRNA of junctional protein and parameters involved in liver metabolism was determined using qPCR. Results indicated direct effect of altered composition of gut microflora on the gut permeability and metabolic alteration. Metagenomic analysis showed least evenness and richness in the HSD group whereas antibiotic-treated groups showed reversal of microflora towards control group with increased richness, evenness and decreased distance on PCoA plot. This changes in gut microflora composition changes expression of metabolic markers and thus insulin sensitivity. Targeting colonic microflora to have a reversal effect on T2D pathogenesis, found to have a positive impact on liver metabolic state with improved permeability markers of gut with SCFA alteration. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01032-x.

Short Chain Fatty Acids, pancreatic dysfunction and type 2 diabetes.

The microbiota living in gut influence the immune response, metabolism, mood and behavior. The diet plays a pivotal role in maintaining healthy gut microbiota composition and its fermentation leads to production of Short Chain Fatty Acids (SCFAs) mainly acetate, propionate and butyrate. During pancreatic dysfunction, insulin mediated suppression of glucagon is impaired leading to uncontrolled glucose production by liver and state of hyperglycemia. Insulin and glucagon balance is as important as insulin sensitivity which is reduced during Type 2 Diabetes (T2D). Glucagon like peptide-1 (GLP1) produced by Intestinal epithelial cells regulates insulin and glucagon secretion directly via GLP1 receptor on pancreatic cells or via nervous system. But half-life period of GLP1 is very short i.e. about 2 min, after which it is cleaved and inactivated. SCFAs are well documented to induce GLP1 but its direct effect on pancreatic dysfunction has not been reported. This review opens a new avenue to study the role of SCFAs as treatment to pancreatic dysfunction and T2D.

Crucial residues in falcipains that mediate hemoglobin hydrolysis.

Falcipain-2 (FP2) and falcipain-3 (FP3) constitute the major hemoglobinases of Plasmodium falciparum. Previous biochemical and structural studies have explained the mechanism of inhibition of these enzymes by small molecules. However, a residue-level protein-protein interaction (PPI) with its natural macromolecular substrate, hemoglobin is not fully characterized. Earlier studies have identified a short motif in the C-terminal of FP2, an exosite protruding away from the active site, essential for hemoglobin degradation. Our structural and mutagenesis studies suggest that hemoglobin interacts with FP2 via specific interactions mediated by Glu185 and Val187 within the C-terminal motif, which are essential for hemoglobin binding. Since FP3 is also a major hemoglobinase and essential for parasite survival, we further demonstrate its interactions with hemoglobin. Our results suggest that Asp194 of FP3 is required for hemoglobin hydrolysis and residue-swap experiments confirmed that this position is functionally conserved between the two hemoglobinases. Residues involved in protein-protein interactions constitute important targets for drug-mediated inhibition. Targeting protein-protein interactions at exosites may likely be less susceptible to emergence of drug resistance and thus is a new field to explore in malaria.

Current scenario and future strategies to fight artemisinin resistance.

Despite several setbacks in the fight against malaria such as insecticide and drug resistance as well as low efficacy of available vaccines, considerable success in reducing malaria burden has been achieved in the past decade. Artemisinins (ARTs and their combination therapies, ACTs), the current frontline drugs against uncomplicated malaria, rapidly kill plasmodial parasites and are non-toxic at short exposures. Though the exact mode of action remains unclear, the endoperoxide bridge, indispensable for ART activity, is thought to react with heme released from hemoglobin hydrolysis and generate free radicals that alkylate multiple protein targets, thereby disrupting proteostasis pathways. However, rapid development of ART resistance in recent years with no potential alternatives on the horizon threaten the elimination efforts. The Greater Mekong Subregion in South-East Asia continues to churn out mutants resistant to multiple ACTs and detected in increasingly expanding geographies. Extensive research on ART-resistant strains have identified a potential candidate Kelch13, crucial for mediating ART resistance. Parasites with mutations in the propeller domains of Plasmodium falciparum Kelch13 protein were shown to have enhanced phosphatidylinositol 3-kinase levels that were concomitant with delayed parasite clearance. Current research focused on understanding the mechanism of Kelch13-mediated ART resistance could provide better insights into Plasmodium resistome. This review covers the current proposed mechanisms of ART activity, resistance strategies adopted by the parasite in response to ACTs and possible future approaches to mitigate the spread of resistance from South-East Asia.

Correction to: Current scenario and future strategies to fight artemisinin resistance.

Author Atul Yadav would like to present his name as Atul only to be the same with his previous publications. The original article has been corrected.

ERK1/2 activated PHLPP1 induces skeletal muscle ER stress through the inhibition of a novel substrate AMPK.

Nutritional abundance associated with chronic inflammation and dyslipidemia impairs the functioning of endoplasmic reticulum (ER) thereby hampering cellular responses to insulin. PHLPP1 was identified as a phosphatase which inactivates Akt, the master regulator of insulin mediated glucose homeostasis. Given the suggestive role of PHLPP1 phosphatase in terminating insulin signalling pathways, deeper insights into its functional role in inducing insulin resistance are warranted. Here, we show that PHLPP1 expression is enhanced in skeletal muscle of insulin resistant rodents which also displayed ER stress, an important mediator of insulin resistance. Using cultured cells and PHLPP1 knockdown mice, we demonstrate that PHLPP1 facilitates the development of ER stress. Importantly, shRNA mediated ablation of PHLPP1 significantly improved glucose clearance from systemic circulation with enhanced expression of glucose transporter 4 (GLUT-4) in skeletal muscle. Mechanistically, we show that endogenous PHLPP1 but not PP2Cα interacts with and directly dephosphorylates AMPK Thr172 in myoblasts without influencing its upstream kinase, LKB1. While the association between endogenous PHLPP1 and AMPK was enhanced in ER stressed cultured cells and soleus muscle of high fat diet fed mice, the basal interaction between PP2Ac and AMPK was minimally altered. Further, we show that PHLPP1α is phosphorylated by ERK1/2 at Ser932 under ER stress which is required for its ability to interact with and dephosphorylate AMPK and thereby induce ER stress. Taken together, our data position PHLPP1 as a key regulator of ER stress.

In vivo evaluation of Eclipta alba extract as anticancer and multidrug resistance reversal agent.

The present study investigates the anticancer and multidrug resistance (MDR) reversal potential of hydro-alcoholic Eclipta alba extract (EAE) through in vivo experiments. Diethylnitrosamine (DEN) and 2-acetylaminofluorene (AAF) were used for liver cancer induction in animal model, whereas for MDR induction, AAF was used. The level of antioxidant enzymes was studied in serum along with biochemical parameters. Cancer and MDR-induced liver cells have higher levels of reactive oxygen species (ROS) and, in turn, are responsible for the maintenance of the cancer phenotype. Treatment with EAE declines the ROS level and revealed the ROS scavenging properties. Alfa feto protein levels were found to increase significantly in cancer-induced animals confirming induction and progression of liver cancer, EAE treatment was found to bring back the altered levels within normal range indicating the therapeutic effect of plant extract over liver cancer. Zymogram showed the inhibition of MMPs and RT-PCR analysis revealed that the mRNA expression of nuclear factor-kB was markedly decreased upon EAE treatment. Further, our results showed that EAE could significantly inhibit mdr1 gene encode P-glycoprotein expression. Our data suggest that EAE is a novel anticancer and potent MDR reversal agent and may be a potential adjunctive agent for tumor chemotherapy.

Composition and interaction of maternal microbiota with immune mediators during pregnancy and their outcome: A narrative review.

The connection between maternal microbiota and infant health has been greatly garnered interest for therapeutic purposes. The early resident microbiota perpetually exhibits much more flexibility as compared to that of the adults, and therefore, constant need of understanding the infant as well as maternal microbiota and their implications however has increased. In this review, we focus mainly on the diversity of overall maternal microbiota including the gut, vaginal, colostrum microbiota and how inflammatory markers fluctuate throughout the normal pregnancy as well in pregnancy with complications. The maternal body undergoes a cascade of physiological changes including hormonal, immunological and metabolic events to support the fetal development. These changes at the time of pregnancy have been correlated with alteration in the composition and diversity of maternal microbiota. Along with alteration in microbiome, the levels of circulatory cytokines fluctuate by complex network of inflammation, in order to prevent the fetal allograft throughout the pregnancy. The dynamic relationship of gut microbiota with the host and its immune system allows one to have greater insights of their role in pregnancy and newborn's health. Emerging evidence suggests that the vertical transmission of bacterial community from mother to newborn may begin in-utero which contributes in developing the immune system and infant gut microbiota.

Understanding the role of gut microfloral bifidobacterium in cancer and its potential therapeutic applications.

Gut microbiota research has gained a tremendous amount of attention from the scientific community because of its contribution to gut homeostasis, human health, and various pathophysiological conditions. The early colonizer of the human gut, i.e., bifidobacteria, has emerged as an efficient probiotic in various diseased conditions, including cancer. This review explores the pros and cons of Bifidobacterium in various malignancies and various therapeutic strategies. We have illustrated the controversial role of bifidobacteria participating in various malignancies as well as described the current knowledge regarding its use in anticancer therapies. Ultimately, this article also addresses the need for further extensive research in elucidating the mechanism of how bifidobacteria is involved and is indirectly affecting the tumor microenvironment. Exhaustive and large-scale research is also required to solve the controversial questions regarding the involvement of bifidobacteria in cancer research.

Easily injectable gelatin-nonanal hydrogel for endoscopic resectioning of gastrointestinal polyps.

The use of submucosal injection is crucial for satisfactory submucosal elevation in the early resection of flat polyps originating from the gastrointestinal tract (GIT). Injectable hydrogels derived from natural polypeptides are attractive candidates due to their excellent biocompatibility and easy gelation properties. However, most of the reported hydrogels are not the class of catheter delivery materials due to quick gelation, high inherent viscosity, and injection clogging. This study presents a novel injectable shear-thinning hydrogel platform of small molecules (nonanal) modified gelatin polymer, which offers a promising submucosal injection for effective removal of polyps from GIT. Physicochemical characterizations of hydrogel demonstrate the suitable features as an effective submucosal injection, including shear thinning property, self-assembly, methylene blue dye encapsulation, flow behavior, stability, syringeability (18 G, 21 G, and 24 G needles) and fibrous morphology. Ex vivo investigations of developed submucosal formulation on goat intestines demonstrate the enhanced visibility of cushions and the ability to produce stable, long-lasting cushions of about 8.07 mm up to ∼60 min of submucosal injection. The rapid blood clotting behavior of hydrogel was observed in about 120 s without compromising hemocompatibility with the hemolysis of about 3.77 % only. In vitro biocompatibility of the hydrogel was also verified using the HepG2 and nHDF cells. In vivo study depicts desirable biocompatibility, a non-toxic organ profile, and optimal cushion height in mice models. Studies established the foundation of novel submucosal fluid to improve the therapeutic outcomes of early resection for gastrointestinal polyps.

Evaluation of hydro-alcoholic extract of Eclipta alba for its multidrug resistance reversal potential: an in vitro study.

The development of multidrug resistance (MDR) causes problems in the chemotherapy of human cancer. The present study was designed to evaluate and establish the role of Eclipta alba as MDR reversal agent using multidrug resistant hepatocellular carcinoma cell line (DR-HepG2). To develop DR-HepG2, hepatocellular carcinoma cell line (HepG2) was transfected with 2-Acetylaminofluorene (AAF) and Aflatoxin B1 (AFB). Cytotoxic effects of the Eclipta alba hydroalcoholic extract (EAE) and standard anti-ancer drug Doxorubicin (DOX) were determined in DR-HepG2 and the parental cells HepG2 using MTT assay. The expression level of MDR1 gene and P-glycoprotein (P-gp) level was analyzed by RT-PCR and western blotting. From the present investigation, it was found that EAE (10 and 20 µg/ml) could significantly inhibit cell proliferation in DR-HepG2 whereas DOX (0.5 µg/ml) could not because of enhancement effect of MDR1/P-gp. This study demonstrated for the first time the antiproliferative activities of EAE in multidrug resistant DR-HepG2 cells. The findings revealed that Eclipta alba components are effective inhibitors of MDR1/P-gp.

Isolation and characterization of probiotic properties of Lactobacilli isolated from rat fecal microbiota.

The objective of the present study was to characterize lactobacilli isolates from the feces of male Wistar rats. Various physiological features of the candidate probiotic isolates were preliminarily investigated, including tolerance to simulated gastric juice and bile salts, antimicrobial activity, antibiotic susceptibility and in vitro aggregation. Based on their morphological and biochemical characteristics, four potential probiotic isolates (CS2, CS3, CS4, and CS7) were screened. The isolates showed good tolerance to stimulated gastric juice and bile salts. CS4 and CS7 exhibited strong antibacterial activities against the pathogens tested as assessed in neutral pH culture supernatants. All lactobacilli isolates were susceptible to all the tested antibiotics, except vancomycin. Moreover, the isolate CS4 and CS7 were found to possess stronger cell surface traits such as hydrophobicity, auto-aggregation and co-aggregation capacity. In addition, CS4 and CS7 had greater ß-galactosidase activities than the others. Biochemical tests and 16S rRNA gene sequencing confirmed that CS2, CS3, CS4 and CS7 are Lactobacillus intestinalis PJ2, L. sakei PJ3, L. helveticus PJ4, and L. plantarum PJ7, respectively. Based on the obtained results, L. helveticus PJ4 and L. plantarum PJ7 are ideal in vitro probiotic candidates and require further in vivo evaluation.

Gut microbiome and type 2 diabetes.

Dietary patterns with excess caloric have shaped a complex metabolic disorders like type 2 diabetes (T2D). T2D involves complications in the metabolism of glucose, lipid, cholesterol and their storage. Along with the metabolic dysregulation, systemic inflammation is also the reason for Insulin Resistance and T2D. The importance of gut microbiota has recently been highlighted. It establishes a link between dietary patterns and the types of bacteria that overgrow and modify fermentation bi-products such as SCFA, secondary bile acids, and mucosal immune cells. These changes have a direct impact on the liver's metabolism and immune system. As a result, using Pre-Pro-biotics to manage microbiota can assist overcome or lessening disease symptoms. Antibiotics are currently employed to produce a germ-free environment or to eradicate specific types of bacteria in order to better understand the role of microflora. This chapter covers the basics of good bacteria, as well as the mechanisms that they work on.

The Combinatorial Effect of Acetate and Propionate on High-Fat Diet Induced Diabetic Inflammation or Metaflammation and T Cell Polarization.

High-fat diet (HFD) alters the gut microbiota and its fermentation products mainly acetate, propionate, and butyrate. Butyrate is well studied as a regulator of host metabolism and inflammation while acetate and propionate still need to be studied. Therefore, we aim to decipher the role of acetate and propionate alone and in combination in HFD-induced diabetic mice. HFD was given to mice for 4 months followed by treatment of butyrate, acetate, and propionate as well as acetate + propionate in combination for 1 month. Diabetic outcome was confirmed by evaluating fasting glucose, lipid profile, oral glucose tolerance test, % HbA1c, fasting insulin, and glucagon. To check the immune response, spleen and mesenteric lymph node-specific T cell polarization and serum cytokine profile were studied. HFD-fed mice showed increased body weight and diabetic characteristics while treatment with acetate and propionate regulated their levels in a healthy manner similar to butyrate. In HFD-fed mice, Th1 and Th17 cells were increased while Treg cells were decreased along with increased pro-inflammatory cytokines and decreased IL-10 in serum. The T cell polarization and cytokine profile was reversed by the treatment of acetate and propionate alone and in combination. Acetate reduced the levels of IL-1ß and IL-6 and acetate + propionate reduced IL-6 more significantly than butyrate. Although, we did not find any synergistic effect in combination group, the results were better compared with acetate, propionate, and butyrate. In conclusion, acetate + propionate effectively reduced inflammation and improved insulin sensitivity in HFD-induced diabetic mice.

In vitro antibacterial activity in seed extracts of Manilkara zapota, Anona squamosa, and Tamarindus indica.

Extracts prepared from seeds of Manilkara zapota, Anona squamosa, and Tamarindus indica were screened for their antibacterial activity by disc diffusion and broth dilution methods. Acetone and methanol extracts of T. indica seeds were found active against both gram-positive and gram-negative organisms. MIC values of potent extracts against susceptible organisms ranged from 53-380 µg/mL. Methanol extract of T. indica and acetone extract of M. zapota seeds were found to be bactericidal.

Bacteriocin PJ4 from probiotic lactobacillus reduced adipokine and inflammasome in high fat diet induced obesity.

One of the characteristic features of obesity is increased body weight and accumulation of adipose tissue. It is associated with low grade inflammation and gut dysbiosis. Probiotics and its products could be an ideal strategy to prevent or treat diabetes. In the present study, animals were induced obesity by providing them with high fat diet. Three purified bacteriocins i.e., DT24, PJ4 and TSU4, previously isolated and purified from various probiotic strains, were given as treatment strategies, following the induction of obesity. Upon the completion of the study, animals were sacrificed and were checked for their tissue expression of inflammatory mediators and adipokines. Serum hormone and cytokines analysis were performed to check their inflammatory state. Treatment with purified bacteriocin DT24 did not show any therapeutic effect in any of the parameter studied. Bacteriocin TSU4 on the other hand showed better reversal compared to DT24. Bacteriocin PJ4 showed the most promising results by reversing all the altered parameters significantly. It significantly reversed all the biochemical, immunological in terms of serum cytokines as well as altered morphological characteristics. PJ4 can be further explored to determine its mode of action. The anti-microbial proteins or to be more specific, bacteriocins, which shows broad spectrum efficacy, could be a better alternative in modulating gut microflora for the treatment of obesity and diabetes characteristics. The efficacy of bacteriocin PJ4 may also be due to the source of the host of Lactobacillus.

Carbon nanotube embedded cyclodextrin polymer derived injectable nanocarrier: A multiple faceted platform for stimulation of multi-drug resistance reversal.

A combination of cocktail chemotherapy (CCT), photothermal therapy (PTT) and inhibition of angiogenesis was investigated as an effective approach to combat major challenges of multidrug resistance and non-targeted drug delivery encountered in conventional cancer therapy. An injectable nanocarrier was developed through functionalization of carbon nanotubes (CNTs) with rationally modified carbohydrate (ß-Cyclodextrin-CD) derived pH and thermo responsive polymer. Embedding CNT to CD polymer offers a nanocarrier which effectively demonstrated CCT, high NIR triggered photothermal efficiency, anti-angiogenic potential for selective tumor homing as well as enhanced multi-drug resistance (MDR) reversal with minimal toxic effects on normal cells. The simultaneously loading with curcumin and doxorubicin hydrochloride exhibited synergistic effect for triggering antitumor effect in vitro and demonstrated down regulation of growth factors associated with angiogenesis ex-ovo. In-vivo studies ascertained that the nanocarrier synthesized with the rational for MDR reversal can lead to enhanced cancer cell death via multiple approaches.