Objective: This study aimed to improve drug mixability and drug uniformity in personalized medicine low-dose tablet by developing a stable, easily mixable, drug-adsorbed filler. Methods: The research involved adsorbing drug onto filler by using three solvents and drug-to-excipient ratios of 1:50 and 1:75. The drug adsorbed fillers, were analyzed for drug content uniformity and flow properties, which are crucial for accurate dosing and manufacturing of low dose tablet. Results: Formulations T1, T2 and T3 showed similar flow properties, including bulk and tapped densities, Carr's indices, and Hausner ratios. T1 had better flowability with a lower angle of repose (23.97 degrees) compared to T2 (35.42 degrees), T3 (49 degrees), and T4 (39 degrees) and it also had higher drug uniformity (99.89%, 99.54%, 97.12%, 96.83%). Tablet evaluations of TS1, TS2, TS3 and TS4 met standard criteria for weight variation, friability, and hardness criteria, with TS1 showing a quicker disintegration time (2:58 min), indicating faster dissolution and potentially better bioavailability. Dissolution tests showed both exceeded 85% drug release within 30 min, with TS1 achieving a higher release (99.98), suggesting more efficient drug release. Conclusion: The drug-adsorbed filler premix technique effectively ensures drug content uniformity and improves low-dose drug mixing, contributing to the development of safe, efficient low-dose pharmaceuticals.

SCARF1 (Scavenger receptor class F member 1, SREC-1 or SR-F1) is a type I transmembrane protein that recognizes multiple endogenous and exogenous ligands such as modified low-density lipoproteins (LDL) and is important for maintaining homeostasis and immunity. But the structural information and the mechanisms of ligand recognition of SCARF1 are largely unavailable. Here we solve the crystal structures of the N-terminal fragments of human SCARF1, which show that SCARF1 forms homodimers and its epidermal growth factor (EGF)-like domains adopt a long-curved conformation. Then we examine the interactions of SCARF1 with lipoproteins and are able to identify a region on SCARF1 for recognizing modified LDLs. The mutagenesis data show that the positively charged residues in the region are crucial for the interaction of SCARF1 with modified LDLs, which is confirmed by making chimeric molecules of SCARF1 and SCARF2. In addition, teichoic acids, a cell wall polymer expressed on the surface of gram-positive bacteria, are able to inhibit the interactions of modified LDLs with SCARF1, suggesting the ligand binding sites of SCARF1 might be shared for some of its scavenging targets. Overall, these results provide mechanistic insights into SCARF1 and its interactions with the ligands, which are important for understanding its physiological roles in homeostasis and the related diseases.

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract Alkaloids are important bioactive molecules throughout the natural world, and in many animals they serve as a source of chemical defense against predation. Dendrobatid poison frogs bioaccumulate alkaloids from their diet to make themselves toxic or unpalatable to predators. Despite the proposed roles of plasma proteins as mediators of alkaloid trafficking and bioavailability, the responsible proteins have not been identified. We use chemical approaches to show that a ~50 kDa plasma protein is the principal alkaloid-binding molecule in blood of poison frogs. Proteomic and biochemical studies establish this plasma protein to be a liver-derived alkaloid-binding globulin (ABG) that is a member of the serine-protease inhibitor (serpin) family. In addition to alkaloid-binding activity, ABG sequesters and regulates the bioavailability of 'free' plasma alkaloids in vitro. Unexpectedly, ABG is not related to saxiphilin, albumin, or other known vitamin carriers, but instead exhibits sequence and structural homology to mammalian hormone carriers and amphibian biliverdin-binding proteins. ABG represents a new small molecule binding functionality in serpin proteins, a novel mechanism of plasma alkaloid transport in poison frogs, and more broadly points toward serpins acting as tunable scaffolds for small molecule binding and transport across different organisms. Editor's evaluation Poison frogs contain alkaloids in their plasma that make them toxic or unpalatable to predators, but how these animals avoid damage to themselves from their own defenses is not well understood. This valuable study identifies an alkaloid-binding protein, a member of the serpin superfamily, in the plasma of poison frogs that may explain how these animals are able to sequester a diverse array of alkaloids. With a convincing series of experiments, the authors advance our knowledge of the roles of serpins in animal ecophysiology. https://doi.org/10.7554/eLife.85096.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Alkaloids are nitrogenous small molecules that play important ecological and physiological roles throughout nature, one of which is mediating predator–prey interactions. Species across many taxa, including plants, insects, marine invertebrates, and vertebrates, have co opted alkaloids as chemical defenses, methods for hunting, and pheromone signals. Some of the most potent alkaloid toxins, including batrachotoxin (BTX), saxitoxin (STX), and tetrodotoxin (TTX), act specifically by affecting voltage-gated ion channels, leading to disruption of nerve and muscle cells (Wright, 2001; Wang et al., 2007; Efimova et al., 2020). In the blue-ringed octopus, Hapalochlaena lunulata, TTX is used to paralyze prey (Asakawa et al., 2019), while in the pufferfish Takifugu niphobles it also acts as a pheromone (Matsumura, 1995), and in the California newt Taricha torosa it is a defense against predation (Bucciarelli et al., 2014). Other less-potent alkaloids also play important roles in predator–prey interactions. For example, Lepidoptera insects (butterflies and moths) and Coleoptera beetles sequester pyrrolizidine alkaloids from plants for predation, defense, and production of pheromones (Hartmann et al., 1999; Cogni et al., 2012). Although the identities of these alkaloids are well documented, less is known about the physiological mechanisms that allow animals to produce, sequester, and autoresist these small molecules. Identifying and characterizing proteins that interact with alkaloids in ecological contexts allow us to better understand how animal physiology has coevolved with alkaloids. Despite the important ecological and physiological roles of alkaloids in animals, the molecular mechanisms involved in alkaloid production, transport, and resistance have been elusive and typically focused on a single alkaloid or specific structural class of alkaloids. In grasshoppers and moths, passive absorption of pyrrolizidine alkaloids is followed by conversion into a non-toxic form by hemolymph flavin-dependent monooxygenase, allowing the insects to avoid autointoxication (Wang et al., 2012). In some beetle species, ATP-binding cassette transporters actively pump pyrrolizidine alkaloids into reservoir defensive glands (Strauss et al., 2013). In vertebrates, the proteins that allow for the sequestration of alkaloids without autotoxicity are unclear with the exception of previous work with tetrodotoxin (TTX) and saxitoxin (STX). The pufferfish saxitoxin- and tetrodotoxin-binding protein (PSTBP) was originally identified in the plasma of Fugu pardalis (Yotsu-Yamashita et al., 2001), and is thought to play a role in the transport of TTX and STX to the site of bioaccumulation in the liver and ovaries in many pufferfish species (Yotsu-Yamashita et al., 2018). The soluble protein saxiphilin has been proposed as a toxin sponge for STX in various species of amphibians (Abderemane-Ali et al., 2021), although it remains unclear whether these species come into contact with STX in nature and whether saxiphilin acts as the predominant STX transporter in the plasma of frogs. While these insights have advanced our understanding of toxin physiology, studies in vertebrates have been narrowly focused on a few potent alkaloids with high-specificity binding proteins. The field is lacking a deeper molecular understanding of how certain species are able to accumulate multiple structurally diverse alkaloids for chemical defense. Some species of frogs sequester a remarkable diversity of dietary alkaloids onto their skin as a chemical defense. This trait has independently evolved in several frog families, including Dendrobatidae in Central and South America and Mantellidae in Madagascar. Over 500 compounds have been found on the skin of Dendrobatidae frogs, with some alkaloids sourced from ants, mites, millipedes, and beetles (Daly et al., 2002; Daly et al., 2005; Saporito et al., 2009). Within dendrobatids, alkaloid-based chemical defenses have evolved independently at least three times (Santos et al., 2003; Summers, 2003), where non-toxic species do not uptake alkaloids onto their skin even when they are present in the diet (Daly et al., 1994; Caldwell, 1996; Darst et al., 2005; Toft, 1980). Well-studied poison frog alkaloids include pumiliotoxins (PTX), which targets sodium and potassium ion channels (Vandendriessche et al., 2008; Daly et al., 1990), and decahydroquinolines (DHQ), which affect nicotinic acetylcholine receptors (Okada et al., 2021). Epibatidine was first identified in the genus Epipedobates and specifically binds certain nicotinic receptors, leading it to be proposed as an analgesic alternative to morphine (Spande et al., 1992). Although there is limited research into the mechanisms of sequestration and autoresistance of alkaloids in poison frogs (Abderemane-Ali et al., 2021; Caty et al., 2019; Alvarez-Buylla et al., 2022; O'Connell et al., 2021; Tarvin et al., 2017), it is likely this process involves alkaloid transport through circulation for these dietary compounds to end up in skin storage glands. Based on the extensive work on plasma small molecule transport in mammals, one might expect that proteins like albumin, which is an abundant and promiscuous small molecule binder in the blood (Peters, 1995; Baker, 2002; Czub et al., 2020), or vitamin transporters (Haddad et al., 1993; Hall, 1975; Kanai et al., 1968), which are able to interact with diet derived molecules, might be involved in alkaloid sequestration in poison frogs. In this study, we tested the hypothesis that poison frogs have an alkaloid-binding protein in the plasma and aimed to uncover its functional role and evolutionary significance. We predicted this protein would bind a range of poison frog alkaloids and would be present in frogs that are chemically defended in nature, but not in undefended species. Results An alkaloid-like photoprobe identifies a binding protein in poison frog plasma We used a biochemical strategy to directly test for alkaloid binding in the plasma of different species of poison frogs. To do this, we obtained a UV crosslinking probe with an indolizidine functional group that shares structural similarity to the poison frog alkaloid pumiliotoxin 251D (PTX) (Figure 1A, functional group highlighted in blue). Upon UV irradiation, the diazirine (green) enables protein crosslinking and the subsequent probe–protein complex can be conjugated to a fluorophore for gel-based visual analysis or biotin for streptavidin enrichment. Application of this photocrosslinking approach outside of mammalian systems has been remarkably limited, and in frogs has been limited to studying neuromuscular receptors (Kiefer et al., 1970; Shinozawa et al., 1987). We found the PTX-like photoprobe shows binding activity within the plasma of three species of dendrobatid poison frogs, Oophaga sylvatica, Dendrobates tinctorius, and Epipedobates tricolor (Figure 1B). In these species, this binding activity was restricted to a few bands in the 50–60 kDa range. Plasma from a non-toxic dendrobatid poison frog (Allobates femoralis), a mantellid poison frog (Mantella aurantiaca), the cane toad (Rhinella marina), and humans showed no binding activity with the photoprobe (Figure 1B). We further tested whether the presence of alkaloids would compete off photoprobe binding. In O. sylvatica, photoprobe binding resulted in two bands that showed competition by the addition of PTX, decahydroquinoline (DHQ), or epibatidine (epi), but not with nicotine (Figure 1C). In D. tinctorius, the photoprobe showed a two-band binding pattern, where the bottom band was competed by PTX and there was slight competition of both bands with DHQ and epibatidine, but no competition with nicotine (Figure 1D). In E. tricolor plasma two bands were observed, and these were more faint in the presence of PTX or DHQ, but not epibatidine or nicotine (Figure 1E). In both O. sylvatica and D. tinctorius, competition occurred when PTX was 10-fold higher in concentration than the photoprobe (Figure 1—figure supplement 1, Figure 1—figure supplement 2). We conclude from these photocrosslinking experiments that plasma binding of alkaloids in three species of chemically defended dendrobatid poison frogs is mediated by a ~50–60 kDa plasma protein. Figure 1 with 2 supplements see all Download asset Open asset Alkaloid-like photocrosslinking probes show binding and competition in poison frog plasma. (A) Structures of alkaloid-like photocrosslinking probe and alkaloids tested, with the functional group in blue, the diazirine group in green, and the terminal alkyne in yellow. In (B–E), the top images show the TAMRA signal, which visualizes photoprobe binding, and the bottom images show coomassie staining of the same gel to assess total protein concentration in each well. (B) Plasma from different species (Oophaga sylvatica – Os, Dendrobates tinctorius – Dt, Epipedobates tricolor – Et, Allobates femoralis – Af, Rhinella marina – Rm, and humans – Hs, from left to right) shows different plasma photoprobe-binding activity and competition. Orange lines on phylogeny indicate independent evolutionary origins of chemical defense in Dendrobatidae and Mantellidae, with the number representing the number of times the phenotype arose along that branch. (C) Oophaga sylvatica plasma shows crosslinking, and competition with pumiliotoxin (PTX), decahydroquinoline (DHQ), and epibatidine (epi), but not nicotine (nic). (D) Dendrobates tinctorius plasma shows crosslinking and competition with PTX, slight competition with DHQ and epi, and no competition with nic. (E) Epipedobates tricolor plasma shows crosslinking and competition with PTX and DHQ, but not with epi or nic. Figure 1—source data 1 Raw data for the gels shown in Figure 1. https://cdn.elifesciences.org/articles/85096/elife-85096-fig1-data1-v2.zip Download elife-85096-fig1-data1-v2.zip Proteomic analysis identifies an alkaloid-binding globulin To identify the alkaloid-binding protein found in the plasma assays, we used O. sylvatica plasma to perform a pulldown and gel-punch proteomics on three conditions: no photoprobe (negative control), photoprobe only (positive control), and photoprobe with PTX competitor (Figure 2A). A biotin handle, instead of the fluorophore used above, was chemically added to the photoprobe for the enrichment of proteins using streptavidin beads. We used an untargeted proteomics approach to quantify and compare these enriched fractions using a proteome reference created from the O. sylvatica genome. Plasma from five O. sylvatica individuals was pooled to ensure sufficient quantity of protein for comparison. On average, 3876 unique peptides were found per sample, mapping to 433 O. sylvatica proteins (Figure 2B). The most highly abundant protein in the photoprobe condition had an average number of peptide spectral counts of 1224.5 and was competed off in the photoprobe with PTX condition by 64% (Figure 2B), resembling background levels (Figure 2C). This protein is annotated as serine-protease inhibitor A1 (serpinA1), which encodes for the protein alpha-1-antitrypsin (A1AT). As our subsequent experiments demonstrate this protein functions as an alkaloid binding and sequestration protein, we refer to it hereafter as 'alkaloid-binding globulin' (ABG). Mapping the 72 unique peptides onto the protein sequence of ABG showed full coverage across the protein, excluding the signal peptide, which in other serpins is cleaved during secretion (Figure 2—figure supplement 1). In comparison to ABG, albumin showed high abundance but no competition (Figure 2B, D). We conclude that ABG functions as a major alkaloid-binding protein in poison frog plasma. Figure 2 with 1 supplement see all Download asset Open asset Proteomics identifies a serpinA1-like protein as the main pumiliotoxin (PTX)-binding protein in Oophaga sylvatica plasma. (A) Streptavidin blot of the proteins pulled down from O. sylvatica plasma across the three conditions: no photoprobe, photoprobe, and photoprobe plus competitor PTX. (B) Quantitative proteomics output in terms of percent competition defined as 100% − average spectral counts in the photoprobe + PTX condition divided by average spectral counts in the photoprobe only condition. Average was taken across two replicates. SerpinA1-like protein and albumin are annotated. (C) The number of spectral counts across conditions for the serpinA1-like protein, each replicate is shown individually. (D) The number of spectral counts across conditions for the albumin protein, each replicate is shown individually. Figure 2—source data 1 Raw data for the blot and proteomics shown in Figure 2. https://cdn.elifesciences.org/articles/85096/elife-85096-fig2-data1-v2.zip Download elife-85096-fig2-data1-v2.zip Structural predictions of ABG show binding pocket similarities to mammalian hormone carriers The identification of ABG as the principal alkaloid-binding protein in plasma was unexpected, as plasma binding of small molecules is commonly mediated by albumin. Nevertheless, in mammals, members of the serpinA family function as carriers of lipophilic hormones, providing plausibility to the hypothesis that frog serpin protein family members may also bind small molecules. Therefore, we sought further structural insights into ABG using protein structure predictions and molecular docking simulations to examine if this protein has a predicted binding pocket for small molecules. Using AlphaFold to predict the structure of the full protein sequence without the signal peptide resulted in a high confidence structure (Figure 3A). We then compared it to the structures of serpinA6/corticosteroid-binding globulin (CBG, Figure 3B; Klieber et al., 2007), biliverdin-binding serpin (BBS, Figure 3C; Manoilov et al., 2022), serpinA1/alpha-1-antitrypsin (A1AT, Figure 3—figure supplement 1; Kim et al., 2001), and serpinA7/thyroxine-binding globulin (TBG, Figure 3—figure supplement 2; Qi et al., 2011). The AlphaFold prediction for O. sylvatica ABG (OsABG) demonstrated a conserved structural element of three alpha helices backed by a set of beta sheets, which is the small molecule binding pocket in CBG, BBS, and TBG (black boxes, Figure 3A–C, Figure 3—figure supplement 1, Figure 3—figure supplement 2), and also exists in the non-small molecule binding A1AT (black box, Figure 3—figure supplement 1). When a molecular docking simulation was run with the whole OsABG protein as the search space and PTX as the ligand, the highest affinity binding site was in the same binding pocket defined by this structural motif (Figure 3D). Although the overall structural components of the binding pockets show similarities across OsABG, CBG, BBS, and TBG, the individual amino acids coordinating small molecule binding differ across proteins (Figure 3D–F, Figure 3—figure supplement 2, Figure 3—figure supplement 3). These results offer a structural explanation for PTX binding by ABG and highlight the homology between ABG and other small molecule binding serpins. Figure 3 with 3 supplements see all Download asset Open asset Predicted alkaloid-binding globulin (ABG) structure and binding pocket resembles that of other small molecule binding serpins. (A) AlphaFold structure predicted with the protein sequence of the Oophaga sylvatica ABG, with color representing model confidence and predicted binding pocket based on molecular docking simulation indicated with a black box. (B) Crystal structure for rat SerpinA6/corticosteroid-binding globulin (CBG), with the cortisol molecule shown in orange (PDB# 2V95). (C) Crystal structure for tree frog Boana punctata biliverdin-binding serpin (BBS), with biliverdin shown in orange (PDB# 7RBW). (D) Close-up of predicted binding pocket of pumiliotoxin (PTX) in O. sylvatica ABG, with residues proximal to PTX highlighted in magenta. The structure of PTX is indicated on the top right. (E) Close-up of cortisol binding in CBG (PDB# 2V95), with proximal residues highlighted in magenta. Cortisol structure is displayed on the top right. (F) Close-up of biliverdin binding in BBS (PDB# 7RBW), with some proximal residues highlighted in magenta. Biliverdin structure is shown on the top right. Figure 3—source data 1 Raw data for the structure prediction shown in Figure 3. https://cdn.elifesciences.org/articles/85096/elife-85096-fig3-data1-v2.zip Download elife-85096-fig3-data1-v2.zip Recombinant expression recapitulates binding activity of different ABG proteins To confirm ABG-binding activity in vitro and compare across different species, we recombinantly expressed and purified O. sylvatica ABG (OsABG), and its closest homolog from the D. tinctorius and E. tricolor transcriptomes (DtABG and EtABG, respectively). The resulting purified protein doublet is due to post-translational glycosylation differences, as OsABG has two predicted N-glycosylation sites (Gupta and Brunak, 2002) and the doublet disappears when treated with a glycosylase enzyme (Figure 4—figure supplement 1). As expected, purified OsABG recapitulated the binding and competition seen with the plasma, where the photoprobe was most fully competed by the presence of PTX, and also competed off by DHQ and epibatidine, but not nicotine (Figure 4A). The competition activity with the purified protein was noticeable at a ratio of one to one photoprobe to PTX (Figure 4—figure supplement 2). Purified DtABG and EtABG required higher concentrations of protein to see a signal and showed much weaker photoprobe binding, which was competed off by the presence of PTX and DHQ for both DtABG (Figure 4B) and EtABG (Figure 4C). Together these results confirm the plasma findings that ABG is a multi-alkaloid-binding protein with different specificities and affinities across poison frog species, and that OsABG alone is sufficient to recapitulate the crosslinking activity observed in the plasma. Figure 4 with 3 supplements see all Download asset Open asset Recombinant expression and binding pocket mutants confirm plasma-binding activity and binding pocket predictions. (A) Photoprobe crosslinking and competition with different compounds of 10 μg recombinantly expressed and purified OsABG recapitulates the binding activity seen in the plasma (Figure 1C). (B) Photoprobe crosslinking with 20 μg recombinantly expressed Dendrobates tinctorius alkaloid-binding globulin (ABG) shows crosslinking, and competition with pumiliotoxin (PTX) and decahydroquinoline (DHQ). (C) Photoprobe crosslinking with 80 μg recombinantly expressed Epipedobates tricolor ABG shows crosslinking, and competition with PTX and DHQ. (D) Alignment of protein sequence of proteins homologous to OsABG across species shows conservation of certain amino acids. Coloring of amino acids is based on the RasMol 'amino' coloring scheme, which highlights amino acid properties. (E) Potential binding residues were identified from the molecular docking simulation. Five different mutants were made based on specific amino acids in the binding pocket, with either a combination of four different alanine substitutions (m1 – yellow and teal residues, and m2 – yellow and green residues) or a single substitutions at D383 (m3), Y36F (m4), or S374A (m5). P8TX is shown in magenta. Oxygen atoms on the molecules are highlighted in red, nitrogen in blue. (F) Quadruple binding pocket mutants (m1 and m2) lose binding activity of the photoprobe, single amino acid substitutions (m3, m4, and m5) show reduced photoprobe binding and retained competition with PTX. Figure 4—source data 1 Raw data for the gels shown in Figure 4. https://cdn.elifesciences.org/articles/85096/elife-85096-fig4-data1-v2.zip Download elife-85096-fig4-data1-v2.zip Given the predicted binding pocket from the molecular docking simulations, and the differences in binding activity of the ABG proteins in different poison frog species, we used a sequence (Figure 4D) and predicted structure (Figure 4E) informed approach to mutate residues that might coordinate alkaloid binding in the hypothesized pocket. We identified six residues with proximity to the docked PTX molecule that might have important binding activity: Y36, S268, D273, W276, S374, and D383 (Figure 4E). Mutating different sets of these binding residues in the OsABG sequence led to a disruption of binding and competition. The combined mutations of Y36A, W276A, S374A, D383A (m1) and Y36A, S268A, D273A, D383A (m2) disrupted binding to the photoprobe completely (Figure 4F). The single-point mutations of D383A (m3), Y36F (m4), and S374A (m5) weakened photoprobe binding significantly, to the point of being nearly undetectable (Figure 4F) in comparison to the wild-type protein. All single-point mutations retained the ability to compete photoprobe binding with PTX (Figure 4F). These results demonstrate that mutating residues in the binding pocket identified through molecular docking disrupts binding activity of OsABG, providing biochemical evidence that the structurally predicted binding pocket of ABG indeed is the relevant binding site for PTX. Furthermore, we have identified a set of residues that are necessary for PTX binding with high affinity, showing that the plasma-binding activity is coordinated by specific amino acids in OsABG. OsABG sequesters free PTX in solution with high affinity Previous work has described small molecule binding serpins and their important role in regulating the pool of free versus bound ligands in circulation (Chan et al., 2013; Pemberton et al., 1988; Gardill et al., 2012; Lewis et al., 2005). We hypothesized that OsABG might play a similar role for alkaloids in the poison frog plasma. To test this, we examined both the direct binding of OsABG for PTX and its ability to regulate the pool of bioavailable alkaloids in solution. Using microscale thermophoresis (MST) we found that wild-type OsABG binds PTX with greater affinity than bovine serum albumin (BSA) or OsABG mutant 3 (Figure 5A). OsABG mutant 3, D383A, had similar binding affinity for PTX as BSA (Figure 5A). To test the ability of OsABG to sequester alkaloids in vitro, we used a 3-kDa molecular weight cutoff centrifuge filter to separate the 'bound' and 'free' PTX (Figure 5B), which we then quantified by liquid chromatography–mass spectrometry (LC–MS) We found that in the presence of OsABG, the amount of 'free' PTX is dramatically reduced, while that of nicotine is not (Figure 5C). These direct binding assay results show that OsABG is able to bind PTX with high affinity, and therefore may regulate the amount of free PTX in solution. Regulation of bioavailable pools of PTX in circulation may have downstream consequences on sequestration, transcription, and signaling throughout the organism. Figure 5 Download asset Open asset OsABG sequesters free pumiliotoxin (PTX) in solution. (A) Microscale thermophoresis (MST) of labeled OsABG with PTX finds that binding is of higher affinity than that of OsABG mutant 3 (D383A) and bovine serum albumin (BSA). (B) A 3-kDA molecular weight cut off (MWCO) centrifuge filter was used to separate 'free' versus 'bound' alkaloids in solutions with and without OsABG present, to later be quantified with liquid chromatography–mass spectrometry (LC–MS). (C) The percent of 'free' PTX 251D (purple) dropped when OsABG was present, however the amount of 'free' nicotine (gray) remained unchanged by the presence of OsABG. Figure 5—source data 1 Raw data for the microscale thermophoresis (MST) and liquid chromatography–mass spectrometry (LC–MS) data shown in Figure 5. https://cdn.elifesciences.org/articles/85096/elife-85096-fig5-data1-v2.zip Download elife-85096-fig5-data1-v2.zip OsABG is highly expressed in wild frogs and binds ecologically relevant toxins We next sought to better understand the functional role of OsABG in a context relevant to the ecology and physiology of poison frogs. O. sylvatica frogs were collected across three different locations in Ecuador (Figure 6A). Tissue RNA sequencing revealed that OsABG mRNA is expressed very highly in the liver compared to other tissues (Figure 6—figure supplement 1). Hierarchical clustering of all unique serpinA genes in the genome shows that OsABG is most closely related to two other serpinA1 genes, Os4677 and Os4682 (Figure 6B). The liver expression of OsABG is higher than all other serpinA genes, and is higher than the expression of albumin in the liver (Figure 6B). Field-collected O. sylvatica frog skin contains alkaloids from many different classes, with 33% of the summed alkaloid load being histrionicotoxins, followed by 22% in 5,8-indolizidines, 15% in 3,5-indolizidines, 13% in 5,6,8-indolizidines, and 10% in DHQ (Figure 6C). Further crosslinking experiments with purified OsABG found that it also binds a histrionicotoxin-like base ring structure (HTX), indolizidine (indol), and shows slight competition by a toxin mixture created from wild frog skin extracts (Figure 6D). To understand the tissue distribution of OsABG, we created a custom anti-OsABG antibody and stained O. sylvatica intestines, skin, and liver (Figure 6E, F, Figure 6—figure supplement 2). We found OsABG staining signal along the deeper layers of the intestinal mucosa and along the inner dermal layers underlying the skin granular glands (Figure 6E). When the anti-OsABG antibody was pre-incubated with purified OsABG protein prior to staining, the signal was lost (Figure 6F). Together, these data characterize the expression profile and distribution of OsABG and show that it is capable of binding other alkaloid classes that are found in wild frogs. Figure 6 with 3 supplements see all Download asset Open asset OsABG is expressed in the liver and binds ecologically relevant alkaloids. (A) Wild Oophaga sylvatica were collected across three locations in Ecuador, n = 10 per location. (B) The liver expression level of OsABG was higher than that of other members of the serpinA family found in the genome, and of albumin. (C) Dorsal skin alkaloids fell into nine different classes, with the size of the circle representing the averaged percent of total skin alkaloid load. (D) Photoprobe binding with recombinantly expressed OsABG was competed by pumiliotoxin (PTX), decahydroquinoline (DHQ), epi, a histrionicotoxin-like compound (HTX), and indolizidine ring without R groups (indol), and slightly by a mixture of skin toxins from the wild specimens (TM). Photoprobe binding was not competed by nicotine (nic) or cortisol (cort). (E) Custom anti-OsABG antibody staining (magenta) in the skin and intestines, with actin stain (blue) and 4',6-diamidino-2-phenylindole (DAPI, shown in white). (F) Pre-incubation of anti-OsABG with purified OsABG protein in the skin and intestines shows loss of OsABG staining, indicating specific staining activity. White bars represent 50 μm. Figure 6—source data 1 Raw data for the gene expression, gels, and immunohistochemistry shown in Figure 6. https://cdn.elifesciences.org/articles/85096/elife-85096-fig6-data1-v2.zip Download elife-85096-fig6-data1-v2.zip Discussion ABG represents a new small molecule binding functionality for a member of the serpin family, with a structurally conserved binding pocket similar to mammalian hormone carriers and BBS. This provides evidence for convergent evolution of serpin proteins for the binding and transport of small molecules across taxa and physiological roles. Most serpin proteins are known for their protease inhibitory activity, ho

Traditional drug delivery methods aimed for a consistent or sustained medication output to maximize treatment efficacy while minimizing side effects. These dosage forms release medications in a controlled or varied manner. Illnesses are treated by administering drugs to patients in a variety of traditional dose patterns. All these dosage patterns should always be administered monotonously for retaining the drug concentration in a therapeutically effective spectrum. Chronotherapeutics, a type of drug delivery system, has become increasingly important in the treatment of chronic diseases in recent years. Today's environment necessitates chronopharmaceutical formulations that increase patient compliance, optimize medicine distribution at the target site, and minimize side effects to reduce mortality rates. A mechanism in which a medicament has been distributed rapidly after a specified lag interval or time gap in compliance with the circadian rhythm of sickness conditions is known as pulsative drug release. Pulsatile medication delivery is becoming more prevalent these days. The main benefit of this method of the medication delivery system is that the substance is only aired when it is required. Because of this, the risk of developing drug resistance, which is common in both preparations for both conventional and sustained release, is minimized. In addition, certain anticancer medications are quite hazardous. In both traditional and sustained release therapy, these medicines cause serious complications. There are now a plethora of FDA-approved chronotherapeutic medications on the market. This treatment is most useful when a long-term effect is just not necessary and medications are harmful. The most important aspect of this formulation's development is determining the circadian rhythm or an appropriate criterion that would set off the drug's release.

Artificial intelligence is the leading branch of technology and innovation. The utility of artificial intelligence in the field of medicine is also remarkable. From drug discovery and development to introducing products to the market, artificial intelligence can play its role. As people age, they are more prone to be affected by eye diseases around the globe. Early diagnosis and detection help minimize the risk of vision loss and provide a quality life. With the help of artificial intelligence, the workload of humans and manmade errors can be reduced to an extent. The need for artificial intelligence in the area of ophthalmic is also significant. In this review, we elaborated on the use of artificial intelligence in the field of pharmaceutical product development, mainly with its application in ophthalmic care. AI in the future has a high potential to increase the success rate in the drug discovery phase has already been established. The application of artificial intelligence for drug development, diagnosis, and treatment is also reported with the scientific evidence in this paper.

Cardiovascular disease is a chronic multifactorial health complication that is either directly or indirectly associated with pathophysiological mechanisms, including pro-oxidation, pro-inflammation, vascular and endothelial dysfunction, impaired platelet function, thrombosis, and others. The therapeutic options to circumvent cardiovascular complications include several phytomedicines, including green tea polyphenols. However, while many experimental and clinical studies report distinct mechanisms by which the polyphenols of green tea elicit a beneficial role in cardiometabolic health, the translation and applications of green tea polyphenols in clinics have yet to gain their optimal use on the broader population. This review critically appraises the various reported mechanisms of green tea polyphenols in modulating cardio-metabolic health and associated phyto-genomic challenges. Further, our review highlights the probability of gene polymorphic associated therapeutic variations in individuals using green tea for cardio-metabolic effects and the necessity to personalize green tea for clinical use, thereby improvising the risk-benefit ratio.

In recent decades, nanomedicines have become a major interest in pharmaceutical research, presenting new challenges for the scientific community, industry and regulators. There is a significant need for scientific and technological methods to be rapidly developed to address unmet medical requirements, increasing human health and life quality. Massive advancements in the domains of biomaterials and nanotechnology have spurred their usage as promising tools to overcome substantial disadvantages, primarily related to the nonspecific effects of traditional treatment methods. On the other hand, herbs have been studied since ancient times for their numerous medicinal uses. Despite their benefits, phytomedicines have numerous disadvantages, including toxicity, poor bioavailability, stability issues and patient compliance. Researchers introduced herbal nanoformulations to address these limitations. These formulations have shown the interaction between nanotechnology and herbal products and are also patented. Since nanotechnology slowly impacts every element of life, there is a growing worry about human safety. As a result, the current moment necessitates the establishment of nanomaterial regulations. This paper strives to compile different herbal nanoformulations and has provided an overview of the regulatory aspects of various regulatory bodies on nanotechnology.

A time-adjustable pulsatile release system containing atenolol as an active pharmaceutical agent was developed for bedtime dosage administration and release of medicine in the early morning to manage elevated blood pressure. The system contained an immediate release (IR) core, a sustained-release (SR) plug, and a mucoadhesive cup layer and it was designed by the cup and core technique. The immediate-release layer was composed of atenolol, croscarmellose sodium, microcrystalline cellulose (MCC), sorbitol, talc, and magnesium stearate. The SR plug was composed of hydroxyl propyl methylcellulose (HPMC), polyvinyl pyrrolidine (PVP), MCC, Lactose, talc, and magnesium stearate. The mucoadhesive cup contains polycaprolactone and mucoadhesive polymer chitosan. Bilayer tablets were punched by applying different compressive forces. Based on the Release data of the drug from each layer F5 and S5 were selected for the preparation of the bilayer tablet. Cup composition containing 3% of chitosan provided the highest mucoadhesion time up to 10h. Pulsatile delivery of the drug from the formulation was observed at 300 min after the administration of the drug. The constructed pulsatile delivery systems were compared to that of commercially available atenolol IR tablet. The comparison demonstrated that the formulation is suitable for the intended chronopharmaceutical delivery of antihypertensive drugs. Keywords: Chronopharmaceuticals, Pulsatile Drug Delivery, Hypertension, Mucoadhesive Polymer, Atenolol

Peptidylarginine deiminase-4 (PAD4) is a calcium-dependent enzyme that catalyzes the conversion of arginine into citrulline of macromolecules in the body. It governs several processes including apoptosis, innate immunity (Netosis), and pluripotency. Dysregulated PAD4 plays a vital role in the occurrence and development of Rheumatoid arthritis (RA). Therefore, PAD4 is considered a promising target for diagnosing and treating RA. Over the last few years research has been carried out on PAD4 inhibitors. When administered it circulates to the entire body and inhibits PAD4 causing immunosuppression which may lead to infection. A growing number of studies have demonstrated infiltration and differentiation of monocytes and macrophages into the inflamed synovium, inducing overexpression of PAD4 levels in the inflamed joints. To overcome the above-mentioned critical issues, the targeted drug delivery systems inhibit PAD4 at the inflamed site. This review provides an update on the PAD4 inhibitors and emerging advanced drug delivery systems for the treatment of RA. Finally, we concluded that active targeting of PAD4 inhibitors to inflamed joints via hybrid nanocarriers provided an improved therapeutic efficacy, minimized extra synovial toxicity, and prevent the occurrence of inflammation in RA.

Nutraceuticals are essential for healthcare which is an alternative medicine that has gained popularity in recent years. Nutraceuticals consist of nutrients, herbals, and dietary supplements, which make them useful in preserving and promoting health, fighting illness, and improving overall quality of life. Its success or failure will be determined by its rapid expansion, research advances, lack of standards, marketing enthusiasm, quality assurance, and regulations. Nutraceuticals have been used in different regions under different names/categories. however, globally there are no stringent pharmaceutical standards for nutraceutical health products till date, but slowly regulators are paying attention on it. Nutraceuticals can be broadly classified according to it clinical significance, source and therapeutic effects. Nutraceuticals and functional foods have grown to be a multibillion-dollar business worldwide in recent years and personalization is the emerging approach to deliver the best therapeutic effect in future. This review carries extensive information about nutraceutical history, classification, regulatory aspects and industrial perspective.

: Advances in personalized medicine are currently impacting improvement in the field of metabolic diseases and patient care. The current innovations in integrating nanotechnology and nanobiotechnology tools in pharmaceutical formulation development have proven the effectiveness of xenobiotics for the diagnosis, treatment, and cure of various metabolic diseases. The implementation of nanomedicines for the treatment of metabolic diseases has served the advantage of overcoming the limitation of bioavailability, selectivity and specificity, biological barriers, and toxicity. Simultaneously, the hybridization of drug molecules and nanomaterials builds promising effective tools for the same. While on the other hand, the development in omics sciences has further supported the detection, diagnosis, and treatment of various metabolic disease conditions. Therapy and analysis of metabolic diseases in asymptomatic patients can be facilitated whereas, harsh complications in diagnosis and disease progression can be avoided by the use of molecular metabolic and genetic biomarkers, biosensor miniatures, and transducers. Therefore, a combination of personalized medicine and nanotechnology gives rise and serves an ultimate goal of predicting, preventing, and treating metabolic diseases. The current article reviews the interdisciplinary nature of personalized medicine, nanotechnology, and nanobiotechnology to employ a safe, efficient, stable, cost-effective futuristic approach for individualized treatment strategies and challenges in the application of personalized medicines for metabolic diseases.

Screening of Plectranthus amboinicus against COVID-19 — in silico approachMeenaxi M. Maste, Akash Saxena

Personalized medicine or precision medicine is a preventive standard that splits people into distant groups with pharmaceutical accord, method, mediation and products are being made to measure to the entity case based on their forecast response or hazard of infection. The Promise of Personalized Medicine, “therapy with the right drug at the right dose in the right patient” is a description of how personalized medicine will affect the future of treatment. A form of a patient's heredity abnormality can guide the choice of drugs or treatment custom to physicians that will diminish the harmful side effects or ensure the more successful outcomes. Personalized medicine reduces the cost of drugs, adverse drug reactions to patients and Increasing patient compliance with treatment. As Fluticasone and cyclophosphamide has high inter-individual variability, genetic polymorphism and population pharmacokinetics was studied, in-order to provide optimum dose for all individuals. Genetic Polymorphism associated with fluticasone was ABCB1 and CYP3A4*22, whereas genetic polymorphism associated with cyclophosphamide was CYP2B6 and GSTP1. Population Pharmacokinetics of fluticasone was done in different places using the co-factors such as genetic polymorphism, Alcohol consumption, weight, Height, Age and Smoking, whereas population pharmacokinetics of cyclophosphamide was done using co-factors such as Genetic polymorphism, Age, Body weight, Liver dysfunction. From the information obtained from population pharmacokinetics, a personalized chart for fluticasone and cyclophosphamide was constructed. In this study, genetic polymorphism and population pharmacokinetics of Fluticasone and Cyclophosphamide towards Personalized medicine was carried out.

Facial Skin is elegant and the ordinary soaps make it to lose texture and make it dry. Facewash is a mild cleanser acts without producing any harshness to skin. The purpose of facewash is to proclaim the cleansing, antiwrinkle, anti-acne, moisturizing and enhance the fairness of skin and thus skin look young and energetic The present form of facewashes available in market were gel and cream state, which should be packed in large collapsible tube or plastic containers in turn difficult for the consumer to carry during their travel and being this facewash are aqueous in nature, it needs preservative to maintain the stability. The objectives of this present work is to overcome the drawbacks of synthetic facewashes and to prepare herbal facewash tablet. Facewash tablets are formulated to minimize the cost, packaging size and the utilization of harmful preservatives and easily portable. Facewash tablet was prepared with various concentration of ingredients and evaluated for parameters like pH, Irritability, Hardness, Friability, Thickness, Foaming capacity and Accelerated Stability. The present study revealed that the formulated batch (F6) produces an excellent foams and produce a fine facewash.

Low dose mixing and its optimization process parameters are facing more challenges. Moreover, minor variation in a dose will tend to significantly have a huge impact on the products' performance including safety and efficacy of the dosage form. Hence, the characterization in every stage of product development of a low dose compound is mandatory. For this purpose, glimepiride and metformin in the ratio of 1:500 was formulated into tablet deciphering a fixed-dose combination product. The tablet was prepared by wet granulation method and optimized by time-dependent random mixing. Confocal Raman microscopy was employed for the present work for evaluation of compatibility studies, in-process and post-compression parameters. Further, the microscopical evaluation was done for the particle scout analysis. The obtained results indicated that the powder blend was compatible and provided uniform mixing in 20 min. Microscopical evaluation has shown that glimepiride still existed in the amorphous state even after compression. Moreover, the glimepiride area in the single-layer was found to be 0.06%. With this, we propose that confocal Raman microscopy plays the promising role in the detectability of the glimepiride. Therefore, in future, the layer by layer analysing can be opted to study the entire spatial arrangement of the API and excipients, which may tend to have a better understanding of dissolution and their related parameters.

Cancer is one of the life-taking diseases worldwide and among cancer-related death; colorectal cancer is the third most. Though conventional methods of treatment are available, multidrug resistance and side effects are predominant. Physicians and scientists are working side by side to develop an effective medicament, which is safe and cost-effective. However, most failures are obtained when focused on the clinical perspective. This review mainly brings out the correlation between the curcumin and its use for the mitigation of colorectal cancer, the use of curcumin as a chemotherapeutic agent, chemosensitizer, and in a combination and synergistic approach. The pharmacokinetics and pharmacodynamics properties of curcumin and its formulation approach helps in giving an idea to develop new approaches for the treatment of colorectal cancer using curcumin.

Orlistat is a tetrahydrolipstatin, which inhibits both pancreatic and gastric lipase enzymes in the gut and prevents the absorption of dietary fats in the intestine. Therefore, it is widely used as an anti-obesity medication to control and manage body weight in obese patients worldwide. Despite this, the mounting number of clinical studies report that orlistat treatment induces adverse effects like pancreatitis, hepatic failure, kidney stone, acute oxalate nephropathy, and others. Although various factors could mediate the adverse effects of orlistat, emerging shreds of evidence suggest that genetic polymorphism-associated factors could be the primary reason for the cause. Hence, in this review, we first collated the most critical clinical adverse effects caused by orlistat and then appraised the susceptible genetic polymorphism that could further worsen the adverse effects of orlistat. Nonetheless, this review warrants similar conceptual approaches to understand orlistat's adverse effects in the selective genetic polymorphism and improve the therapeutic outcome of orlistat.

Coronavirus disease-19 (COVID-19), a devastating respiratory illness caused by SARS-associated coronavirus-2 (SARS-CoV-2), has already affected over 64 million people and caused 1.48 million deaths, just 12 months from the first diagnosis. COVID-19 patients develop serious complications, including severe pneumonia, acute respiratory distress syndrome (ARDS), and or multiorgan failure due to exaggerated host immune response following infection. Currently, drugs that were effective against SARS-CoV are being repurposed for SARS-CoV-2. During this public health emergency, food nutraceuticals could be promising prophylactic therapeutics for COVID-19. Curcumin, a bioactive compound in turmeric, exerts diverse pharmacological activities and is widely used in foods and traditional medicines. This review presents several lines of evidence, which suggest curcumin as a promising prophylactic, therapeutic candidate for COVID-19. First, curcumin exerts antiviral activity against many types of enveloped viruses, including SARS-CoV-2, by multiple mechanisms: direct interaction with viral membrane proteins; disruption of the viral envelope; inhibition of viral proteases; induce host antiviral responses. Second, curcumin protects from lethal pneumonia and ARDS via targeting NF-κB, inflammasome, IL-6 trans signal, and HMGB1 pathways. Third, curcumin is safe and well-tolerated in both healthy and diseased human subjects. In conclusion, accumulated evidence indicates that curcumin may be a potential prophylactic therapeutic for COVID-19 in the clinic and public health settings.

The benefit of personalized medicine is that it allows the customization of drug therapy - maximizing efficacy while avoiding side effects. Genetic polymorphisms are one of the major contributors to interindividual variability. Currently, the only gold standard for applying personalized medicine is dose titration. Because of technological advancements, converting genotypic data into an optimum dose has become easier than in earlier years. However, for many medications, determining a personalized dose may be difficult, leading to a trial-and-error method. On the other hand, the technologically oriented pharmaceutical industry has a plethora of smart drug delivery methods that are underutilized in customized medicine. This article elaborates the genetic polymorphisms of tacrolimus as case study, and extensively covers the diagnostic and therapeutic technologies which aid in the delivery of personalized tacrolimus treatment for better clinical outcomes, thereby providing a new strategy for implementing personalized medicine.