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Venkata Raveendra Pothineni,1,* Mansi B Parekh,1,* Mustafeez Mujtaba Babar,1 Aditya Ambati,2 Peter Maguire,1 Mohammed Inayathullah,1 Kwang-Min Kim,1 Lobat Tayebi,3 Hari-Hara SK Potula,1 Jayakumar Rajadas1,4 1Biomaterials and Advanced Drug Delivery, Stanford Cardiovascular Pharmacology Division, Cardiovascular Institute, School of Medicine, Stanford University, Palo Alto, CA, USA; 2Center for Sleep Sciences and Medicine, Department of Psychiatry and Behavioral Sciences, School of Medicine, Stanford University, Palo Alto, CA, USA; 3Department of Developmental Sciences, Marquette University School of Dentistry, Milwaukee, WI, USA; 4Department of Bioengineering and Therapeutic Sciences, Schools of Pharmacy and Medicine, University of California San Francisco, San Francisco, CA, USA *These authors contributed equally to this work Background: Lyme disease accounts for >90% of all vector-borne disease cases in the United States and affects ~300,000 persons annually in North America. Though traditional tetracycline antibiotic therapy is generally prescribed for Lyme disease, still 10%–20% of patients treated with current antibiotic therapy still show lingering symptoms.Methods: In order to identify new drugs, we have evaluated four cephalosporins as a therapeutic alternative to commonly used antibiotics for the treatment of Lyme disease by using microdilution techniques like minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). We have determined the MIC and MBC of four drugs for three Borrelia burgdorferi s.s strains namely CA8, JLB31 and NP40. The binding studies were performed using in silico analysis.Results: The MIC order of the four drugs tested is cefoxitin (1.25 µM/mL) > cefamandole (2.5 µM/mL), > cefuroxime (5 µM/mL) > cefapirin (10 µM/mL). Among the drugs that are tested in this study using in vivo C3H/HeN mouse model, cefoxitin effectively kills B. burgdorferi. The in silico analysis revealed that all four cephalosporins studied binds effectively to B. burgdorferi proteins, SecA subunit penicillin-binding protein (PBP) and Outer surface protein E (OspE).Conclusion: Based on the data obtained, cefoxitin has shown high efficacy killing B. burgdorferi at concentration of 1.25 µM/mL. In addition to it, cefoxitin cleared B. burgdorferi infection in C3H/HeN mice model at 20 mg/kg. Keywords: Lyme disease, Borrelia burgdorferi, antimicrobials, penicillin-binding proteins

Venkata Raveendra Pothineni,1 Dhananjay Wagh,1 Mustafeez Mujtaba Babar,1 Mohammed Inayathullah,1 David Solow-Cordero,2 Kwang-Min Kim,1 Aneesh V Samineni,1 Mansi B Parekh,1 Lobat Tayebi,3 Jayakumar Rajadas1 1Biomaterials and Advanced Drug Delivery Laboratory, Stanford Cardiovascular Pharmacology Division, Cardiovascular Institute, Stanford University School of Medicine, Palo Alto, 2Chemical & Systems Biology, Stanford University School of Medicine, Stanford, CA, 3Department of Developmental Sciences, Marquette University School of Dentistry, Milwaukee, WI, USA Abstract: Lyme disease is the most common zoonotic bacterial disease in North America. It is estimated that >300,000 cases per annum are reported in USA alone. A total of 10%–20% of patients who have been treated with antibiotic therapy report the recrudescence of symptoms, such as muscle and joint pain, psychosocial and cognitive difficulties, and generalized fatigue. This condition is referred to as posttreatment Lyme disease syndrome. While there is no evidence for the presence of viable infectious organisms in individuals with posttreatment Lyme disease syndrome, some researchers found surviving Borrelia burgdorferi population in rodents and primates even after antibiotic treatment. Although such observations need more ratification, there is unmet need for developing the therapeutic agents that focus on removing the persisting bacterial form of B. burgdorferi in rodent and nonhuman primates. For this purpose, high-throughput screening was done using BacTiter-Glo assay for four compound libraries to identify candidates that stop the growth of B. burgdorferi in vitro. The four chemical libraries containing 4,366 compounds (80% Food and Drug Administration [FDA] approved) that were screened are Library of Pharmacologically Active Compounds (LOPAC1280), the National Institutes of Health Clinical Collection, the Microsource Spectrum, and the Biomol FDA. We subsequently identified 150 unique compounds, which inhibited >90% of B. ­burgdorferi growth at a concentration of

Dhananjay Wagh,* Venkata Raveendra Pothineni,* Mohammed Inayathullah, Song Liu, Kwang-Min Kim, Jayakumar Rajadas Biomaterials and Advanced Drug Delivery Laboratory, Stanford Cardiovascular Pharmacology Division, Cardiovascular Institute, Stanford University School of Medicine, Palo Alto, CA, USA *These authors contributed equally to this work Abstract: Borrelia burgdorferi, the causative agent of Lyme disease, utilizes manganese (Mn) for its various metabolic needs. We hypothesized that blocking Mn transporter could be a possible approach to inhibit metabolic activity of this pathogen and eliminate the infection. We used a combination of in silico protein structure prediction together with molecular docking to target the Borrelia metal transporter A (BmtA), a single known Mn transporter in Borrelia and screened libraries of FDA approved compounds that could potentially bind to the predicted BmtA structure with high affinity. Tricyclic antihistamines such as loratadine, desloratadine, and 3-hydroxydesloratadine as well as yohimbine and tadalafil demonstrated a tight binding to the in silico folded BmtA transporter. We, then, tested borreliacidal activity and dose response of the shortlisted compounds from this screen using a series of in vitro assays. Amongst the probed compounds, desloratadine exhibited potent borreliacidal activity in vitro at and above 78 µg/mL (250 µM). Borrelia treated with lethal doses of desloratadine exhibited a significant loss of intracellular Mn specifically and a severe structural damage to the bacterial cell wall. Our results support the possibility of developing a novel, targeted therapy to treat Lyme disease by targeting specific metabolic needs of Borrelia. Keywords: Lyme disease, BmtA, Borrelia burgdorferi, desloratadine, Bac Titer-Glo assay

Inflammatory skin disease is characterized by a pathologic interplay between skin cells and immunocytes and can result in disfiguring cutaneous lesions and systemic inflammation. Immunosuppression is commonly used to target the inflammatory component; however, these drugs are often expensive and associated with side effects. To identify previously unidentified targets, we carried out a nonbiased informatics screen to identify drug compounds with an inverse transcriptional signature to keratinocyte inflammatory signals. Using psoriasis, a prototypic inflammatory skin disease, as a model, we used pharmacologic, transcriptomic, and proteomic characterization to find that benzamil, the benzyl derivative of the US Food and Drug Administration-approved diuretic amiloride, effectively reversed keratinocyte-driven inflammatory signaling. Through three independent mouse models of skin inflammation (Rac1 G12V transgenic mice, topical imiquimod, and human skin xenografts from patients with psoriasis), we found that benzamil disrupted pathogenic interactions between the small GTPase Rac1 and its adaptor NCK1. This reduced STAT3 and NF-κB signaling and downstream cytokine production in keratinocytes. Genetic knockdown of sodium channels or pharmacological inhibition by benzamil prevented excess Rac1-NCK1 binding and limited proinflammatory signaling pathway activation in patient-derived keratinocytes without systemic immunosuppression. Both systemic and topical applications of benzamil were efficacious, suggesting that it may be a potential therapeutic avenue for treating skin inflammation.

In the present study, we explored the development of a novel noninvasive liposomal drug delivery material for use in intranasal drug delivery applications in human diseases. We used drug entrapment into liposomal nanoparticle assembly to efficiently deliver the drugs to the nasal mucosa to be delivered to the brain. The naturally occurring flavonoid 7,8-dihydroxyflavone (7,8-DHF) has previously been shown to have beneficial effects in ameliorating Parkinson's disease (PD). We used both naturally occurring 7,8-DHF and the chemically modified form of DHF, the DHF-ME, to be used as a drug candidate for the treatment of PD and l-DOPA induced dyskinesia (LID), which is the debilitating side effect of l-DOPA therapy in PD. The ligand-protein interaction behavior for 7,8-DHF and 6,7-DHF-ME was found to be more effective with molecular docking and molecular stimulation studies of flavonoid compounds with TrkB receptor. Our study showed that 7,8-DHF delivered via intranasal route using a liposomal formulation ameliorated LID in hemiparkinsonian mice model when these mice were chronically administered with l-DOPA, which is the only current medication for relieving the clinical symptoms of PD. The present study also demonstrated that apart from reducing the LID, 7,8-DHF delivery directly to the brain via the intranasal route also corrected some long-term signaling adaptations involving ΔFosB and α Synuclein in the brain of dopamine (DA) depleted animals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Innate immunity, the first line of defense against pathogens, relies on efficient elimination of invading agents by phagocytes. In the co-evolution of host and pathogen, pathogens developed mechanisms to dampen and evade phagocytic clearance. Here, we report that bacterial pathogens can evade clearance by macrophages through mimicry at the mammalian anti-phagocytic "don't eat me" signaling axis between CD47 (ligand) and SIRPα (receptor). We identified a protein, P66, on the surface of Borrelia burgdorferi that, like CD47, is necessary and sufficient to bind the macrophage receptor SIRPα. Expression of the gene encoding the protein is required for bacteria to bind SIRPα or a high-affinity CD47 reagent. Genetic deletion of p66 increases phagocytosis by macrophages. Blockade of P66 during infection promotes clearance of the bacteria. This study demonstrates that mimicry of the mammalian anti-phagocytic protein CD47 by B. burgdorferi inhibits macrophage-mediated bacterial clearance. Such a mechanism has broad implications for understanding of host-pathogen interactions and expands the function of the established innate immune checkpoint receptor SIRPα. Moreover, this report reveals P66 as a novel therapeutic target in the treatment of Lyme Disease.

Repurposing pharmaceuticals is a technique used to find new, alternate clinical applications for approved drug molecules. It may include altering the drug formulation, route of administration, dose or the dosage regimen. The process of repurposing medicines starts with screening libraries of previously approved drugs for the targeted disease condition. If after an the initial in silico, in vitro or in vivo experimentation, the molecule has been found to be active against a particular target, the molecule is considered as a good candidate for clinical trials. As the safety profile of such molecules is available from the previous data, significant time and resources are saved. These advantages of drug repurposing approach make it especially helpful for finding treatments for rapidly evolving conditions including bacterial infections. An ever-increasing incidence of antimicrobial resistance, owing to the mutations in bacterial genome, leads to therapeutic failure of many approved antibiotics. Repurposing the approved drug molecules for use as antibiotics can provide an effective means for the combating life-threatening bacterial diseases. A number of drugs have been considered for drug repurposing against bacterial infections. These include, but are not limited to, Auranofin, Closantel, and Toremifene that have been repurposed for various infections. In addition, the reallocation of route of administration, redefining dosage regimen and reformulation of dosage forms have also been carried out for repurposing purpose. The current chapter addresses the drug discovery and development process with relevance to repurposing against bacterial infections., (Copyright © 2024. Published by Elsevier Inc.)

The CRISPR-Cas9 method has revolutionized the gene editing. Epigenetic changes, including DNA methylation, RNA modification, and changes in histone proteins, have been intensively studied and found to play a key role in the pathogenesis of human diseases. CRISPR-While the utility of DNA and chromatin modifications, known as epigenetics, is well understood, the functional significance of various alterations of RNA nucleotides has recently gained attention. Recent advancements in improving CRISPR-based epigenetic modifications has resulted in the availability of a powerful source that can selectively modify DNA, allowing for the maintenance of epigenetic memory over several cell divisions. Accurate identification of DNA methylation at specific locations is crucial for the prompt detection of cancer and other diseases, as DNA methylation is strongly correlated to the onset as well as the advancement of such conditions. Genetic or epigenetic perturbations can disrupt the regulation of imprinted genes, resulting in the development of diseases. When histone code editors and DNA de-/ methyltransferases are coupled with catalytically inactive Cas9 (dCas9), and CRISPRa and CRISPRi, they demonstrate excellent efficacy in editing the epigenome of eukaryotic cells. Advancing and optimizing the extracellular delivery platform can, hence, further facilitate the manipulation of CRISPR-Cas9 gene editing technique in upcoming clinical studies. The current chapter focuses on how the CRISP/ Cas9 system provides an avenue for the epigenetic modifications and its employability for human benefit., (Copyright © 2024. Published by Elsevier Inc.)

The drug discovery and development (DDD) process greatly relies on the data available in various forms to generate hypotheses for novel drug design. The complex and heterogeneous nature of biological data makes it difficult to utilize or gather meaningful information as such. Computational biology techniques have provided us with opportunities to better understand biological systems through refining and organizing large amounts of data into actionable and systematic purviews. The drug repurposing approach has been utilized to overcome the expansive time periods and costs associated with traditional drug development. It deals with discovering new uses of already approved drugs that have an established safety and efficacy profile, thereby, requiring them to go through fewer development phases. Thus, drug repurposing through computational biology provides a systematic approach to drug development and overcomes the constraints of traditional processes. The current chapter covers the basics, approaches and tools of computational biology that can be employed to effectively develop repurposing profile of already approved drug molecules., (Copyright © 2024. Published by Elsevier Inc.)

Cancer has been a primary contributor to morbidity and mortality worldwide. With an increasing trend of incidence and prevalence of cancer, progress has also been made in its treatment, starting from radiation and chemotherapy to immunotherapy and gene therapy. CRISPR-Cas technique, a promising gene editing tool, has been employed in cancer research for novel treatment regimens, identification of therapeutic targets, and unraveling the genetic mechanisms behind oncogenesis. CRISPR-based genome editing helped in identifying the roles of specific genetic factors linked to treatment resistance, metastasis, and cancer development. CRISPR allows the discovery of genes and treatment options through specifically interrupting tumor activators or activating tumor suppressor genes in cancer cells. Advancements in CRISPR technology, especially the use of immune cells like chimeric antigen receptor (CAR) T cells, has the potential to revolutionize personalized cancer treatment by precisely targeting and killing cancer cells. Furthermore, reactivating tumor suppressor genes makes cancer cells more susceptible to chemotherapy or immunotherapy. CRISPR-mediated genome editing can, hence, help to overcome resistance to traditional cancer treatments. The current manuscript covers that how is the CRISPR technology propelling revolutionary development in the field of cancer research, providing advance perspectives on the molecular causes of the disease and creating new lines for the development of more precise and potent cancer therapies., (Copyright © 2024. Published by Elsevier Inc.)

Mucopermeating nanoformulations can enhance mucosal penetration of poorly soluble drugs at their target site. In this work, thiolated chitosan (TCS)-lithocholic acid (LA) nanomicelles loaded with β-carotene, a safe phytochemical with anticancer properties, were designed to improve the pharmaceutical and pharmacological drug profile. The TCS-LA nanomicelles were characterized by FTIR to confirm the presence of the thiol group that favors skin adhesion, and to corroborate the conjugation of hydrophobic LA with hydrophilic CS to form an amphiphilic polymer derivative. Their crystalline nature and thermal behavior were investigated by XRD and DSC analyses, respectively. According to DLS and TEM, their average size was <300 nm, and their surface charge was +27.0 mV. β-carotene entrapment and loading efficiencies were 64 % and 58 %, respectively. In vitro mucoadhesion and ex vivo mucopenetration analyses further corroborated the potential of the nanoformulation to deliver the drug in a sustained manner under conditions mimicking cancer micro-environment. Anticancer studies in mice demonstrated that the loaded nanomicelles delayed skin cancer growth, as revealed by both morphological and biochemical parameters. Based on the results obtained herein, it can be concluded that drug-loaded TCS-LA is a novel, stable, effective and safe mucoadhesive formulation of β-carotene for the potential treatment of skin cancer., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Gradient scaffolds are isotropic/anisotropic three-dimensional structures with gradual transitions in geometry, density, porosity, stiffness, etc., that mimic the biological extracellular matrix. The gradient structures in biological tissues play a major role in various functional and metabolic activities in the body. The designing of gradients in the scaffold can overcome the current challenges in the clinic compared to conventional scaffolds by exhibiting excellent penetration capacity for nutrients & cells, increased cellular adhesion, cell viability & differentiation, improved mechanical stability, and biocompatibility. In this review, the recent advancements in designing gradient scaffolds with desired biomimetic properties, and their implication in tissue regeneration applications have been briefly explained. Furthermore, the gradients in native tissues such as bone, cartilage, neuron, cardiovascular, skin and their specific utility in tissue regeneration have been discussed in detail. The insights from such advances using gradient-based scaffolds can widen the horizon for using gradient biomaterials in tissue regeneration applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Purpose: Alternate formulation strategies need to be devised for improving the absorption and bioavailability of drug molecules administered through the intravaginal route. Enhancing the coating of vaginal mucosa can aid the achievement of this goal. The aim of the current study is to develop a mucoadhesive formulation having adequate adhesiveness, spreading, and viscosity profiles that can ensure good tissue absorption of adapalene upon intravaginal application. Method: A combination of mucoadhesive agents has been employed, including Carbopol-934, HPMC K-15M, and xanthan gum, in varying ratios to formulate five different gels. Furthermore, a cost-effective UV-spectroscopic analytical method was developed to quantify the amount of adapalene in tested samples, both of in vitro and in vivo origin. The analytical method was validated for different parameters, including specificity, linearity, range, accuracy, precision, and ruggedness. The modified USP-II apparatus was used for dissolution studies, while in vivo pharmacokinetic validation was performed in a murine model. Result: Of all the tested formulations, on the basis of the rheo-mechanical attributes, ACX3 performed better than the rest, including the commercially available intravaginal reference product. ACX3 had an average adhesion time of 12 min and a spread diameter of 37 mm. It showed 35 mm as average distance travelled by the diluted sample for leakage assessment. The analytical method developed for the adapalene muco-adhesive gel was within the range for all the validation parameters. For further evaluating the performance of the formulation, dissolution studies were conducted in simulated vaginal conditions which showed 94.83% of drug release within 5 minutes, while on completion of 30 min, it was measured to be 92.90%. Moreover, approximately 67% of the administered drug was recovered after 5 min of administration as evaluated through tissue recovery procedures in mice. Conclusion: The study aided in development of a formulation which can enhance the muco-adhesion of the drug molecule, resulting in an improved pharmacokinetic profile. Moreover, it established an efficient assay method which can be employed for in vitro and in vivo quantification of adapalene in simulated and physiological fluids., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Afzaal, Shahiq-uz-Zaman, Saeed, Hamdani, Raza, Gul, Babar and Rajadas.)

BACKGROUNDHyaluronan (HA), an extracellular matrix glycosaminoglycan, has been implicated in the pathophysiology of COVID-19 infection, pulmonary hypertension, pulmonary fibrosis, and other diseases, but is not targeted by any approved drugs. We asked whether hymecromone (4-methylumbelliferone [4-MU]), an oral drug approved in Europe for biliary spasm treatment that also inhibits HA in vitro and in animal models, could be repurposed as an inhibitor of HA synthesis in humans.METHODSWe conducted an open-label, single-center, dose-response study of hymecromone in healthy adults. Subjects received hymecromone at 1200 (n = 8), 2400 (n = 9), or 3600 (n = 9) mg/d divided into 3 doses daily, administered orally for 4 days. We assessed safety and tolerability of hymecromone and analyzed HA, 4-MU, and 4-methylumbelliferyl glucuronide (4-MUG; the main metabolite of 4-MU) concentrations in sputum and serum.RESULTSHymecromone was well tolerated up to doses of 3600 mg/d. Both sputum and serum drug concentrations increased in a dose-dependent manner, indicating that higher doses lead to greater exposures. Across all dose arms combined, we observed a significant decrease in sputum HA from baseline after 4 days of treatment. We also observed a decrease in serum HA. Additionally, higher baseline sputum HA levels were associated with a greater decrease in sputum HA.CONCLUSIONAfter 4 days of exposure to oral hymecromone, healthy human subjects experienced a significant reduction in sputum HA levels, indicating this oral therapy may have potential in pulmonary diseases where HA is implicated in pathogenesis.TRIAL REGISTRATIONClinicalTrials.gov NCT02780752.FUNDINGStanford Medicine Catalyst, Stanford SPARK, Stanford Innovative Medicines Accelerator program, NIH training grants 5T32AI052073-14 and T32HL129970.

Tissue repair and healing remain among the most complicated processes that occur during postnatal life. Humans and other large organisms heal by forming fibrotic scar tissue with diminished function, while smaller organisms respond with scarless tissue regeneration and functional restoration. Well-established scaling principles reveal that organism size exponentially correlates with peak tissue forces during movement, and evolutionary responses have compensated by strengthening organ-level mechanical properties. How these adaptations may affect tissue injury has not been previously examined in large animals and humans. Here, we show that blocking mechanotransduction signaling through the focal adhesion kinase pathway in large animals significantly accelerates wound healing and enhances regeneration of skin with secondary structures such as hair follicles. In human cells, we demonstrate that mechanical forces shift fibroblasts toward pro-fibrotic phenotypes driven by ERK-YAP activation, leading to myofibroblast differentiation and excessive collagen production. Disruption of mechanical signaling specifically abrogates these responses and instead promotes regenerative fibroblast clusters characterized by AKT-EGR1., (© 2021. The Author(s).)