Photostability Issues in Pharmaceutical Dosage Forms and Photostabilization

Photodegradation is one of the major pathways of the degradation of drugs. Some therapeutic agents and excipients are highly sensitive to light and undergo significant degradation, challenging the quality and the stability of the final product. The adequate knowledge of photodegradation mechanisms and kinetics of photosensitive therapeutic entities or excipients is a pivotal aspect in the product development phase. Hence, various pharmaceutical regulatory agencies, across the world, mandated the industries to assess the photodegradation of pharmaceutical products from manufacturing stage till storage, as per the guidelines given in the International Conference on Harmonization (ICH). Recently, numerous formulation and/or manufacturing strategies has been investigated for preventing the photodegradation and enhancing the photostability of photolabile components in the pharmaceutical dosage forms. The primary focus of this review is to discuss various photodegradation mechanisms, rate kinetics, and the factors that influence the rate of photodegradation. We also discuss light-induced degradation of photosensitive lipids and polymers. We conclude with a brief note on different approaches to improve the photostability of photosensitive products.     

Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect

The excluded volume effect (EVE) rules all life processes. It is created by macromolecules that occupy a given volume thereby confining other molecules to the remaining space with large consequences on reaction kinetics and molecular assembly. Implementing EVE in fibroblast culture accelerated conversion of procollagen to collagen by procollagen C-proteinase (PCP/BMP-1) and proteolytic modification of its allosteric regulator, PCOLCE1. This led to a 20-30- and 3-6-fold increased collagen deposition in two- and three-dimensional cultures, respectively, and creation of crosslinked collagen footprints beneath cells. Important parameters correlating with accelerated deposition were hydrodynamic radius of macromolecules and their negative charge density.     

Homodimerization Controls the Fibroblast Growth Factor 9 Subfamily's Receptor Binding and Heparan Sulfate-Dependent Diffusion in the Extracellular Matrix

Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20''s receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.Fibroblast growth factor (FGF) signaling plays pleiotropic roles throughout the life spans of mammalian organisms, ranging from germ cell maturation, mesoderm induction, body plan formation, and organogenesis during embryonic development to serum phosphate homeostasis and glucose, bile acid, lipid, and cholesterol metabolism in the adult (3, 23, 27, 28, 57, 60, 62). The diversity of FGF signaling is underscored by virtue of the fact that aberrant FGF signaling leads to a wide array of human diseases, including skeletal and olfactory/reproductive syndromes, phosphate wasting disorders, and cancer (16, 60, 67). Recent data also implicate dysregulated FGF signaling in the etiology of neurodegenerative disorders, such as major depressive disorder and Parkinson''s disease (10, 63, 64).Based on pairwise sequence homology and phylogeny, the 18 bona fide mammalian FGFs (FGF1 to FGF10 and FGF16 to FGF23) are divided into six subfamilies (45). Five FGF subfamilies have high-to-moderate affinity for pericellular heparan sulfate (HS) glycosaminoglycans and thus diffuse locally within tissues to act in a paracrine fashion, whereas the poor affinity of the FGF19 subfamily for HS enables this subfamily to act in an endocrine manner (28, 38). All FGFs share a core homology region of about 120 amino acids, which fold into 12 antiparallel β strands (β1 to β12) that are arranged into three sets of four-stranded β sheets (β-trefoil fold) (39). The globular FGF core domain is flanked by highly divergent N- and C-terminal extensions, which are the principal regions responsible for the different biology of FGFs.FGFs exert their diverse actions by binding and activating FGF receptors (FGFRs) in an HS-dependent fashion (51, 53, 69). There are four distinct mammalian FGFR genes (FGFR1 to FGFR4), each coding for a single-pass transmembrane tyrosine kinase receptor whose ectodomain consists of three immunoglobulin-like domains (D1 to D3) connected by flexible linkers and whose intracellular domain contains the conserved tyrosine kinase domain flanked by the juxtamembrane (JM) and C-terminal regions (38). The 210-amino-acid-long D2-D3 segment of the ectodomain is both necessary and sufficient for ligand binding (20, 51, 52, 58, 70).FGF signaling is tightly regulated by spatial and temporal expression of ligands, receptors, HS cofactors, and most critically by means of FGF-FGFR binding specificity. The tissue-specific alternative splicing in the D3 domain of FGFR1 to FGFR3 is the main mechanism by which FGF-FGFR binding specificity is regulated. This splicing event gives rise to epithelial “b” isoforms (FGFR1b to FGFR3b) and mesenchymal “c” isoforms (FGFR1c to FGFR3c) (24, 25, 47, 68), which differ from one another at the primary sequences of their key ligand binding regions and thus in their FGF binding specificity/promiscuity profiles. Most FGFs are also expressed in either epithelial or mesenchymal tissues and exhibit specificity for FGFR isoforms expressed in the opposite tissues. This results in the establishment of a bidirectional signaling loop between the epithelium and mesenchyme that is essential for organogenesis and tissue homeostasis. It is well established that FGF7 and FGF10, which are expressed exclusively in the mesenchyme, activate specifically FGFR2b to mediate mesenchymal-to-epithelial signaling in the lung, prostate, and lacrimal, mammary, and salivary glands (19, 29, 35, 36, 59). Several lines of genetic and biochemical evidence suggest that the members of the FGF9 subfamily, which includes FGF9, FGF16, and FGF20, convey the reciprocal signaling from the epithelium to the mesenchyme. In the prostate, the epithelial-specific FGF9 has been shown to activate mesenchymal FGFR3c isoforms (25). In the heart, FGF9, FGF16, and FGF20 in the epicardium and endocardium stimulate myocardial proliferation and differentiation in vivo, acting redundantly through FGFR1c and FGFR2c (32). Analysis of FGF9-deficient mice has identified FGF9 as a reciprocal epithelial-to-mesenchymal signal required for morphogenesis of the lung, cecum, small intestine, and inner ear (14, 49, 65, 71). In addition, studies in zebra fish show that FGF16 and FGF20 are apical ectodermal ridge factors that are required for pectoral fin bud outgrowth and, in general, for cell proliferation and differentiation of the mesenchyme (41, 66).In light of the key role of the FGF9 subfamily in tissue homeostasis, it is essential to investigate the molecular mechanisms by which the activity of this subfamily is regulated. Our previous structural and in vitro studies of FGF9 showed that homodimerization masks FGF9''s key receptor binding sites, suggesting that ligand dimerization may autoinhibit FGF9''s biologic activity (50). In this report, we show that, like FGF9, FGF20 also homodimerizes in the crystal and in solution. Characterization of the dimer interface mutations in vitro and in living cells demonstrates that ligand homodimerization autoinhibits FGF9 and FGF20 signaling by suppressing both receptor binding and HS-dependent diffusion in the extracellular matrix (ECM). Our study is the first to implicate ligand dimerization as an autoregulatory mechanism in growth factor bioactivity.     

vRhyme enables binning of viral genomes from metagenomes

Genome binning has been essential for characterization of bacteria, archaea, and even eukaryotes from metagenomes. Yet, few approaches exist for viruses. We developed vRhyme, a fast and precise software for construction of viral metagenome-assembled genomes (vMAGs). vRhyme utilizes single- or multi-sample coverage effect size comparisons between scaffolds and employs supervised machine learning to identify nucleotide feature similarities, which are compiled into iterations of weighted networks and refined bins. To refine bins, vRhyme utilizes unique features of viral genomes, namely a protein redundancy scoring mechanism based on the observation that viruses seldom encode redundant genes. Using simulated viromes, we displayed superior performance of vRhyme compared to available binning tools in constructing more complete and uncontaminated vMAGs. When applied to 10,601 viral scaffolds from human skin, vRhyme advanced our understanding of resident viruses, highlighted by identification of a Herelleviridae vMAG comprised of 22 scaffolds, and another vMAG encoding a nitrate reductase metabolic gene, representing near-complete genomes post-binning. vRhyme will enable a convention of binning uncultivated viral genomes and has the potential to transform metagenome-based viral ecology.     

Activating cryptic biosynthetic gene cluster through a CRISPR–Cas12a-mediated direct cloning approach

Direct cloning of biosynthetic gene clusters (BGCs) from microbial genomes facilitates natural product-based drug discovery. Here, by combining Cas12a and the advanced features of bacterial artificial chromosome library construction, we developed a fast yet efficient in vitro platform for directly capturing large BGCs, named CAT-FISHING (CRISPR/Cas12a-mediated fast direct biosynthetic gene cluster cloning). As demonstrations, several large BGCs from different actinomycetal genomic DNA samples were efficiently captured by CAT-FISHING, the largest of which was 145 kb with 75% GC content. Furthermore, the directly cloned, 110 kb long, cryptic polyketide encoding BGC from Micromonospora sp. 181 was then heterologously expressed in a Streptomyces chassis. It turned out to be a new macrolactam compound, marinolactam A, which showed promising anticancer activity. Our results indicate that CAT-FISHING is a powerful method for complicated BGC cloning, and we believe that it would be an important asset to the entire community of natural product-based drug discovery.     

Social convergence of gut microbiomes in vampire bats

The ‘social microbiome’ can fundamentally shape the costs and benefits of group-living, but understanding social transmission of microbes in free-living animals is challenging due to confounding effects of kinship and shared environments (e.g. highly associated individuals often share the same spaces, food and water). Here, we report evidence for convergence towards a social microbiome among introduced common vampire bats, Desmodus rotundus, a highly social species in which adults feed only on blood, and engage in both mouth-to-body allogrooming and mouth-to-mouth regurgitated food sharing. Shotgun sequencing of samples from six zoos in the USA, 15 wild-caught bats from a colony in Belize and 31 bats from three colonies in Panama showed that faecal microbiomes were more similar within colonies than between colonies. To assess microbial transmission, we created an experimentally merged group of the Panama bats from the three distant sites by housing these bats together for four months. In this merged colony, we found evidence that dyadic gut microbiome similarity increased with both clustering and oral contact, leading to microbiome convergence among introduced bats. Our findings demonstrate that social interactions shape microbiome similarity even when controlling for past social history, kinship, environment and diet.     

Evaluation of bioelectronics sensor compared to other diagnostic test in diagnosis of Johne's disease in goats

A bioelectronics sensor has been developed and it is evaluated for the diagnosis of paratuberculosis in goats. Initially hematite nanoparticles were prepared and using this nanoparticles as core, electrically active polyaniline coated magnetic (EAPM) nanoparticles are synthesized from aniline monomer (made electrically active by acid doping). These EAPM nanoparticles were fabricated with rabbit anti-goat IgG for the detection of goat antibodies on the capture pad. The protoplasmic antigen of Mycobacterium avium subspecies paratuberculosis (MAP) immobilized onto the capture pad will detect the antibody against MAP in the goat sera samples. This bound goat antibody will be detected by the anti-goat IgG previously bound to EAPM. Upon detection the EAPM nanoparticles bridges an electric circuit between the silver electrodes, flanking the capture membrane. The electrical conductance, caused by EAPM, was measured as direct charge transfer between the electrodes. Testing of the biosensor with known Johne's disease (JD) positive and negative serum samples gave significant difference in the electrical conductance value. Further the efficacy of this biosensor was compared with other serological tests like agar gel immunodiffusion (AGID) and absorbed ELISA using field sera. Out of 265 goat sera tested, positive results recorded were; AGID 36 (13.59%), bioelectronics sensor 49 (19.14%), and absorbed ELISA 51 (19.25%). This biosensor was also compared in live animals using intradermal Johnin test and nested PCR (detecting mycobacterial DNA in feces) in 65 animals. Of which, positive results recorded in animals were; Johnin test 21 (32%), biosensor 26 (40%) and fecal PCR detected mycobacterial DNA in 28 (43%) animals. Though the nanobioelectronics sensor was slightly less sensitive (not statistically significant) compared to absorbed ELISA and fecal nested PCR for mycobacterial DNA but it was simple to perform in field conditions and requires less time. The speed of detection and the equipment involved would support its application toward the various point-of-care opportunities aimed at control and management of Johne's disease in goats.     

Role of TSC1 in physiology and diseases

Since its initial discovery as the gene altered in Tuberous Sclerosis Complex (TSC), an autosomal dominant disorder, the interest in TSC1 (Tuberous Sclerosis Complex 1) has steadily risen. TSC1, an essential component of the pro-survival PI3K/AKT/MTOR signaling pathway, plays an important role in processes like development, cell growth and proliferation, survival, autophagy and cilia development by co-operating with a variety of regulatory molecules. Recent studies have emphasized the tumor suppressive role of TSC1 in several human cancers including liver, lung, bladder, breast, ovarian, and pancreatic cancers. TSC1 perceives inputs from various signaling pathways, including TNF-α/IKK-β, TGF-β-Smad2/3, AKT/Foxo/Bim, Wnt/β-catenin/Notch, and MTOR/Mdm2/p53 axis, thereby regulating cancer cell proliferation, metabolism, migration, invasion, and immune regulation. This review provides a first comprehensive evaluation of TSC1 and illuminates its diverse functions apart from its involvement in TSC genetic disorder. Further, we have summarized the physiological functions of TSC1 in various cellular events and conditions whose dysregulation may lead to several pathological manifestations including cancer.

     

The role of phosphoenolpyruvate carboxykinase in neuronal steroidogenesis under acute inflammation

Phosphoenolpyruvate carboxykinase (PEPCK) is a key gluconeogenic enzyme found in many tissues throughout the body including brain. In the present study, we have investigated the effect of bacterial lipopolysaccharide (LPS) on PEPCK and its role in neuronal steroidogenesis. Adult female albino rats were administered LPS (5 mg/kg body weight) to induce acute inflammation. LPS administration resulted in a significant increase of PEPCK mRNA expression with concomitant increase in mRNA levels of steroidogenic acute regulatory (StAR) protein and other steroidogenic enzymes including 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD) and aromatase in brain tissue. Further, the inhibition of PEPCK expression by glipizide significantly decreased the mRNA expression of steroidogenic proteins and concurrently increased the mRNA levels of proinflammatory cytokines under LPS administration. The results of this study suggest a novel finding that PEPCK may have an important role in neuronal steroidogenesis; which serves as an adaptive response under inflammation.