Pyruvate kinase (PK), a key ATP-generating enzyme in glycolysis, is a target for novel sickle cell disease (SCD) therapies. Enhancing PK activity lowers 2,3-diphosphyglycerate (2,3-DPG), increases adenosine triphosphate (ATP), and may prevent red blood cell (RBC) sickling. Townes and Berkeley SCD mouse models are commonly used for the development of novel drugs for SCD, but differ from humans in 2,3-DPG and ATP levels, which could be related to underlying differences in PK properties. This study revealed important distinctions with humans (SCD vs healthy controls), such as similar PK/hexokinase (HK) ratios between sickling and non-sickling mouse models and significantly lower PK thermostability in mice. We additionally investigated the effect of a novel RBC PK activator, compound A, on PK properties and sickling tendency in these mice in order to assess SCD mouse model suitability. Results showed that a single dose of compound A led to an increased affinity of PK for phosphoenolpyruvate, a significant increase in PK/HK ratio and a decrease of 2,3-DPG levels. Together, these results offer detailed characterization in the PK properties of two commonly used SCD mouse models, and provide insight into the mode of action of PK activator therapy in SCD mice models.

This report details the discovery path of GBT021601 (osivelotor) (16), a novel, small molecule, sickle hemoglobin (HbS) polymerization inhibitor. Following a streamlined testing funnel with cassette dosing in rat pharmacokinetic (PK) studies, we identified this next-generation HbS polymerization inhibitor, which had improved PK properties compared with the first-in-class drug, voxelotor (1). GBT021601 has ∼4.8-fold greater exposure and a ∼3.5-fold longer half-life in rats compared with voxelotor. In a murine model of sickle cell disease (SCD), GBT021601 treatment resulted in an increase in hemoglobin oxygen affinity, a reduction in sickling of red blood cells (RBCs), and an increase in both RBC half-life and hemoglobin levels not seen with voxelotor preclinically. The improved half-life and exposure appear to translate to similar levels of HbS occupancy at lower doses than voxelotor, thus reducing treatment burden. GBT021601 is being investigated in a phase 2/3 clinical trial for the treatment of patients with SCD (NCT05431088).

Introduction : Sickle cell disease (SCD) is caused by a single nucleotide mutation in the β-globin gene resulting in sickle hemoglobin (HbS). Red blood cell (RBC) deoxygenation leads to HbS polymerization, RBC sickling and subsequent chronic hemolysis, anemia, vascular endothelial cell activation, vaso-occlusion, and ischemia. SCD is associated with extensive morbidity and markedly reduced survival. GBT021601 is an investigational, second-generation HbS polymerization inhibitor that reduces RBC sickling and anemia, and improves both O 2 delivery and consumption in chronically treated Townes Sickle Cell mice that harbor 2 HbS alleles (SS). Objectives of this 2-part study were to (1) characterize the effects of GBT021601 administered ad libitum on compound exposure and pharmacodynamics (PD), (2) examine the impact of long-term (>24 weeks) treatment with GBT021601 on % reticulocytes and overall survival (OS) of Townes SS mice, and (3) assess the utility of early changes in % reticulocytes as a predictive biomarker of survival in Townes SS mice treated with GBT021601. Methods: In Part 1, 24 male Townes SS mice 8-10 weeks old were split into 3 cohorts (n=8/group) and for 3 weeks were fed standard chow containing 0% (control), 0.05% (low dose), or 0.2% (high dose) of GBT021601. Body weight and food consumption were measured weekly. Mice were bled daily on days 10-21 to determine RBC half-life and on days 10 and 21 for analysis of compound exposure. On day 22 mice were euthanized. Whole blood was collected for analyses of compound exposure, % reticulocytes, Hb levels, RBC numbers, RBC half-life, and RBC deformability. In Part 2, 5-week-old Townes mice (AA and SS genotype; 50% male) were fed either standard chow (AA [n=57] and SS mice [n=54]) or chow containing 0.05% or 0.2% of GBT021601 (SS mice only; n=55 each). Body weight and food consumption were measured weekly. Blood was collected at 1, 14, 31, 43, 55, and 67 weeks of treatment for compound exposure analysis and % reticulocytes. Mice were euthanized when the humane end-point criteria were met or after 72 weeks of treatment, and whole blood was collected for compound exposure and % reticulocyte analyses. Results: GBT021601 in chow was well tolerated during both study parts; food consumption and body weight were unaffected. Outcomes in Part 1 confirmed the same compound exposure/PD trends as mice orally gavaged with GBT021601 as observed by Dufu et al. (Br J Haematol 2023); Hb levels, RBC numbers, and RBC half-life increased, RBC deformability improved, and % reticulocytes decreased. Long-term dosing of GBT021601 in the chow of Townes SS mice revealed similar compound exposure and % reticulocytes as our 3-week dosing study (Part 1) and Dufu et al. In Part 2, Townes SS mice fed GBT021601 chow, achieving compound exposures after 1 week of dosing as depicted in Figure 1, had a longer median survival vs control Townes SS mice (60 vs 56 weeks). In control Townes SS mice, % reticulocytes increased during the first 14 weeks of the study and then plateaued, whereas in Townes SS mice fed GBT021601 there was a dose-dependent sustained reduction in % reticulocytes compared with control Townes SS mice. Reductions in % reticulocytes after 1 week of dosing were greater in Townes SS mice achieving a higher compound exposure (Figure 1), a trend sustained after 14 and 31 weeks of treatment. OS increased in Townes SS mice of the same sex achieving a ≤30% reduction in % reticulocytes vs Townes SS mice with % reticulocytes >30% after 1 week of treatment; P=0.00048 (Figure 2). Multivariate survival analysis, including % reticulocytes at 1 week (categorical variable of ≤30% or >30%) and sex as predictors confirmed significant association of >30% reticulocytes with reduced survival (hazard ratio=1.48, P=0.027 using Cox proportional hazard). Conclusions:Longer-term GBT021601 treatment as part of chow was well tolerated by Townes SS mice, and compound exposure was comparable with studies of a shorter duration of treatment. Early compound exposure was predictive of a long-term survival benefit in Townes SS mice. Early reductions in % reticulocytes were more likely in Townes SS mice with a high GBT021601 exposure and were predictive of a long-term survival benefit. Overall, our findings support the use of GBT021601 to reduce % reticulocytes and improve OS in Townes SS mice and suggest the clinical relevance of early reductions in % reticulocytes as a biomarker to predict long-term survival.

The pathophysiologic mechanism of sickle cell disease (SCD) involves polymerization of deoxygenated haemoglobin S (HbS), leading to red blood cell (RBC) sickling, decreased RBC deformability, microvascular obstruction, haemolysis, anaemia and downstream clinical complications. Pharmacological increase in the concentration of oxygenated HbS in RBCs has been shown to be a novel approach to inhibit HbS polymerization and reduce RBC sickling and haemolysis. We report that GBT021601, a small molecule that increases HbS-oxygen affinity, inhibits HbS polymerization and prevents RBC sickling in blood from patients with SCD. Moreover, in a murine model of SCD (SS mice), GBT021601 reduces RBC sickling, improves RBC deformability, prolongs RBC half-life and restores haemoglobin levels to the normal range, while improving oxygen delivery and increasing tolerance to severe hypoxia. Notably, oral dosing of GBT021601 in animals results in higher levels of Hb occupancy than voxelotor and suggests the feasibility of once-daily dosing in humans. In summary, GBT021601 improves RBC health and normalizes haemoglobin in SS mice, suggesting that it may be useful for the treatment of SCD. These data are being used as a foundation for clinical research and development of GBT021601.

Chronic pharmacologically increased hemoglobin affinity for oxygen in sickle cell disease mice alleviated hematological consequences of sickle cell disease, increasing RBC half-life, hematocrit, and hemoglobin concentration, while also decreasing reticulocyte count. Additionally, chronically increased hemoglobin affinity for oxygen significantly improved survival as well as cortical tissue oxygenation in sickle cell disease mice during hypoxia, suggesting that oxygen delivery and utilization is improved by increased hemoglobin affinity for oxygen.

Sickle cell disease (SCD) is a genetic disorder caused by inheritance of two alleles bearing a single nucleotide change in the β globin gene coding sequence. The pathophysiologic mechanism of SCD involves polymerization of intracellular hemoglobin S (HbS) following deoxygenation in the microvasculature, leading to decreased red blood cell (RBC) deformability, morphologic sickling of RBCs, decreased RBC survival, microvascular obstruction, and clinical complications (Bunn H, N Engl J Med, 1997). Voxelotor, a hemoglobin S polymerization inhibitor recently approved for the treatment of SCD, is an allosteric modifier of Hb that increases the proportion of oxygenated Hb in all RBCs. In clinical studies in subjects with SCD, voxelotor has demonstrated that doses of up to 1500 mg daily achieved Hb modifications of ~27%, was well tolerated, and resulted in reduced hemolytic anemia (Howard J et al., Blood, 2019; Vichinsky E et al., N Engl J Med, 2019). GBT021601 is a potent second generation HbS polymerization inhibitor with the potential to achieve even higher Hb modification in subjects with SCD at lower doses and therefore with less pill burden. To evaluate its effect on the pathophysiology of SCD, GBT021601 was administered at 20, 40, 75, and 150 mg/kg QD via oral dosing for 21 days in Townes SCD mice (SS mice). At steady state, GBT021601-treated SS mice achieved Hb occupancies (at Cmin) of 6%, 12%, 21%, and 29% corresponding to 20, 40, 75, and 150 mg/kg doses, respectively. This resulted in a dose-dependent reduction in p50 of SS mouse blood from ~28 mm Hg (vehicle-treated) to 11 mm Hg at the highest dose tested. Consequently, GBT021601 significantly reduced in vivo circulating sickled cells and ex vivo sickling of SS mouse blood under hypoxic conditions (20 mmHg). Consistent with its anti-polymerization activity, GBT021601 reduced hemolysis as demonstrated by 1) an increase in hemoglobin of up to 6.7 g/dL (Figure 1A), thereby achieving the normal range for wildtype mice, and 2) a reduction in the percentage of reticulocytes of up to 58% relative to vehicle-treated SS mice (Figure 1B). Additionally, GBT021601 treatment increased the percentage of mature RBCs and reduced the percentage of mitochondria-containing RBCs indicating an overall improvement in RBC health. Consistent with improved hemoglobin, reduced hemolysis, and improved RBC health, GBT021601 dose-dependently increased RBC half-life from 1.3 days in vehicle-treated SS mice to 7.4 days in SS mice treated with 150 mg/kg GBT021601 (Figure 1C). Furthermore, treatment of SS mice with GBT021601 for 21 days led to a significant reduction in spleen weight, indicating an improvement in splenic blood flow and organ function. Together, these data demonstrate that GBT021601, a next generation HbS polymerization inhibitor, normalized hemoglobin, reduced hemolysis and significantly improved RBC health, RBC half-life and organ function in a mouse model of SCD. These data support clinical development of GBT021601 as a potential best-in-class HbS polymerization inhibitor for SCD patients. Disclosures Dufu: Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Alt:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Strutt:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tang:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liao-Zou:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yuan:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cathers:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Oksenberg:Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Mobilization of hematopoietic stem and progenitor cells (HSPCs) has become increasingly important for hematopoietic cell transplantation. Current mobilization approaches are insufficient because they fail to mobilize sufficient numbers of cells in a significant fraction of patients and are biased toward myeloid immune reconstitution. A novel, single drug mobilization agent that allows a more balanced (myeloid and lymphoid) reconstitution would therefore be highly favorable to improve transplantation outcome. In this present study, we tested commercially available IL-33 molecules and engineered novel variants of IL-33. These molecules were tested in cell-based assays in vitro and in mobilization models in vivo. We observed for the first time that IL-33 treatment in mice mobilized HSPCs and common myeloid progenitors more efficiently than clinical mobilizing agents granulocyte colony-stimulating factor (G-CSF) or AMD3100. We engineered several oxidation-resistant IL-33 variants with equal or better in vitro activity. In vivo, these variants mobilized HSPCs and, interestingly, also hematopoietic stem cells, common lymphoid progenitor cells, and endothelial progenitor cells more efficiently than wild-type IL-33 or G-CSF. We then engineered an IL-33-Fc fusion molecule, a single dose of which was sufficient to significantly increase the mobilization of HSPCs after 4 days. In conclusion, our findings suggest that long-acting, oxidation-resistant IL-33 may be a novel approach for HSPC transplantation. IL-33-mobilized HSPCs differ from cells mobilized with G-CSF and AMD3100, and it is possible that these differences may result in better transplantation outcomes.

Sickle Cell Disease (SCD) is characterized by hemolytic anemia, vaso-occlusion, and progressive end-organ damage. The underlying mechanism of SCD is the polymerization of sickle hemoglobin (HbS) that occurs when sickle erythrocytes (SS RBCs) are partially deoxygenated in microcirculation, leading to SCD pathophysiologic features. One of the most devastating complications of SCD occurs in the central nervous system (CNS), where overt stroke or repeated silent cerebral infarcts lead to significant physical and neurocognitive consequences. In SCD, the brain's response to insufficient oxygen (O2) delivery is balanced by increased blood flow to preserve O2 supply. However, the systemic endothelial dysfunction in SCD limits the capacity for vascular regulatory and compensatory changes to preserve appropriate tissue oxygenation, especially in tissues with high O2 demand like the brain. Low hemoglobin (Hb) levels and increased cerebral blood flow (CBF) are associated with increased stroke risk, suggesting that anemia-induced tissue hypoxia is an important factor contributing to subsequent morbidity in SCD patients. Voxelotor (GBT440) is a small molecule, HbS polymerization inhibitor being developed by Global Blood Therapeutics (GBT) for the treatment of SCD. By addressing the underlying mechanism of SCD, voxelotor has the potential to be disease-modifying and alleviate the clinical manifestations of SCD. Mechanistically, voxelotor increases Hb-O2 affinity and delays the transition from oxyHb to deoxyHb under hypoxic conditions. This study assessed the impact of a pharmacologically mediated increase in Hb-O2 affinity on brain tissue oxygenation under both normoxic and hypoxic conditions in Townes transgenic sickle mice (SCD mice). Two compounds that increase the Hb-O2 affinity with similar potency, voxelotor and an analog to voxelotor, GBT1118, were considered for the study. The target for Hb occupancy with test compounds was ≥30% based on the therapeutic target occupancies observed with voxelotor in clinical studies. The effects of increased Hb-O2 affinity on brain tissue oxygenation were assessed directly with O2-specific microelectrodes in a cranial window and indirectly with hypoxyprobe staining (pimonidazole) of brain tissue. Unique to this SCD model, the targeted Hb occupancy (≥30%) could not be consistently achieved by voxelotor. In contrast, repeat oral dosing of GBT1118 at 200 mg/kg/day for 2 weeks in SCD mice achieved steady state concentrations of 802 ± 81 µM (mean ± SD; n=5), corresponding to a Hb occupancy of 44 ± 5%. Consequently, GBT1118 decreased the p50 (partial pressure of O2 at which Hb is 50% saturated) values of SCD mouse blood from 39 ± 0.8 mmHg (Vehicle-dosed) to 21 ± 1.6 mmHg (GBT1118-dosed). While we could not achieve the desired Hb occupancy (≥30%) with voxelotor in this model, the Hb occupancy and change in p50 achieved with GBT1118 afforded us the opportunity to ask whether significantly increasing Hb-O2 affinity affects brain O2 tension. Measurements of cortical O2 tension (pO2) showed no difference in pO2 values under normoxia (21% O2) (Figure A), and slightly higher pO2 values under hypoxia (10% O2) (Figure B) for GBT1118-dosed SCD mice compared with vehicle-dosed SCD mice. Collectively across all brain tissues, the GBT1118-induced increase in Hb-O2 affinity reduced tissue hypoxia in SCD mice under hypoxia as measured by pimonidazole staining (Figure C). Together, these results indicate that a pharmacological increase of Hb-O2 affinity does not decrease cortical tissue pO2 in SCD mice and may reduce brain hypoxia under hypoxic conditions. Figure Disclosures Dufu: Global Blood Therapeutics: Employment, Equity Ownership. Lucas:Global Blood Therapeutics: Research Funding. Muller:Global Blood Therapeutics: Research Funding. Williams:Global Blood Therapeutics: Research Funding. Zhang:Global Blood Therapeutics: Employment, Equity Ownership. Rademacher:Global Blood Therapeutics: Employment, Equity Ownership. Alt:Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Cabrales:Global Blood Therapeutics: Research Funding.

Previous studies have led to opposing hypotheses about the requirement of intermolecular disulfide exchange in the binding of the iron regulatory peptide hepcidin to its receptor ferroportin. To clarify this issue, we used the diaminodiacid approach to replace the disulfide bonds in hepcidin with non-reducible thioether bonds. Our results implied that disulfide exchange is not required for the interaction between hepcidin and ferroportin. This theory is further supported by our development of biologically active minihepcidins that do not show activity dependence on cysteine.

Mobilization and collection of peripheral blood hematopoietic stem and progenitor cells (HSPC) has become increasingly important for hematopoietic cell transplantation. Granulocyte-colony stimulating factor (G-CSF) alone or in combination with the CXCR4-inhibitor AMD3100 are the most common mobilization approaches. These are however limited since they (a) fail to mobilize a sufficient number of cells in a significant fraction of patients, and (b) are biased towards myeloid immune reconstitution. A novel, single drug mobilization agent that results in a more balanced - myeloid and lymphoid - reconstitution would therefore be highly favorable to improve transplantation outcome. Interleukin (IL)-33 has recently been described as a potent mobilizer of HSPCs with long-term reconstitution potential. IL-33 is an intracellular protein that is believed to be enzymatically activated, released upon cell death, and inactivated upon oxidation. Whereas mobilization of HSPCs by G-CSF has been linked, at least in-part, to modulating the CXCR4-CXCL12 axis, the mobilizing function of IL-33 has been attributed to a different chemokine, CCL7. Here we asked whether IL-33 mobilizes HSPCs better than current clinical mobilizing agents, G-CSF and/or AMD3100, and how the quality of the mobilized cells compares. By flow cytometry and colony-forming assays, we observed for the first time that IL-33 treatment in mice mobilized Lin- Sca-1+ c-kit+ (LSK) cells and common myeloid progenitors (CMP/CFU-GEMMs) more efficiently than G-CSF or AMD3100 alone, and that IL-33 acts synergistically with both drugs. Next we engineered IL-33 variants lacking native cysteine residues, thereby rendering these variants resistant to oxidative inactivation. These variants had equal or better in vitro activity (a) in cell-based reporter assays and (b) in inducing CCL7 protein production by primary human umbilical vein endothelial cells (HUVECs). In vivo, these variants mobilized LSK cells and, interestingly also common lymphoid progenitor (CLP) and endothelial progenitor cells, more efficiently than wild-type IL-33 or G-CSF. We then engineered a one-armed IL-33-Fc fusion molecule comprising a single oxidation-resistant IL-33 fused to an Fc domain. Interestingly, a single dose of this oxidation-resistant IL-33-Fc variant was sufficient to significantly increase the mobilization of LSK, CLP, and endothelial progenitor cells, whereas oxidation-resistant IL-33 or G-CSF were not. In conclusion, our findings suggest that a single dose of long-acting, oxidation-resistant IL-33 may allow efficient mobilization of high-quality HSPCs including CLP cells, and may thus represent a novel mechanism to improve bone marrow transplantation. All authors were employed by Bayer at the time the studies were conducted. Disclosures Alt: Bayer: Equity Ownership; Global Blood Therapeutics: Employment, Equity Ownership. Schopies: Bayer: Employment. Liu: Global Blood Therapeutics: Employment. Kauser: Global Blood Therapeutics: Employment, Equity Ownership. Bringmann: Cerus Corporation: Employment. Thompson: Bayer: Employment. Yuan: Bayer: Employment.

Recurrent, painful vaso-occlusive events are one of the most debilitating complications of sickle cell disease (SCD). The process involves occlusion of blood flow through small vessels by heterogeneous cell clusters comprised of red blood cells (RBCs), neutrophils, and platelets. Activated endothelial cells localize and facilitate vaso-occlusion. The central triggering event in the process is hemoglobin S (HbS) polymerization and the consequent damage to RBCs. Many recent clinical efforts to prevent vaso-occlusive events have focused on downstream events mediated by inflammatory molecules. The present work however studies the triggering event by assessing the direct impact of allosteric modulators of hemoglobin oxygen (Hb-O2) affinity on vaso-occlusion in a murine model of SCD.

The main objective of this investigation was to study the feasibility of developing a vaginal bioadhesive microbicide using a SRI’s proprietary two-polymer gel platform (SR-2P). Several formulations were prepared with different combinations of temperature-sensitive polymer (Pluronic® F-127) and mucoadhesive polymer (Noveon® AA-1), producing gels of different characteristics. Prototype polymeric gels were evaluated for pH, osmolality, buffering capacity, and viscosity under simulated vaginal semen dilutions, and bioadhesivity using ex vivo mini pig vaginal tissues and texture analyzer. The pH of the polymeric gel formulations ranged from 5.1 to 6.4; the osmolality varied from 13 to 173 mOsm. Absolute viscosity ranged from 513 to 3,780 cPs, and was significantly reduced (1.5- to 3-fold) upon incubation with simulated vaginal and semen fluid mixture. Among the tested gels (indicated in the middle row as a molar ratio of a mixture of Noveon vs. Pluronic), only SR-2P retained gel structure upon dilution with simulated fluids and mild simulated coital stress. The pH of the SR-2P gel was maintained at about 4.6 in simulated vaginal fluid and also showed high peak force of adhesion in mini pig vaginal tissue. Furthermore, SR-2P gel caused no or only minimal irritation in a mouse vaginal irritation model. The results of this preliminary study demonstrated the potential application of SR-2P gel as a vaginal microbicide vehicle for delivery of anti-HIV agents.

Thiamine (vitamin B1) deficiency, associated with a variety of conditions, including chronic alcoholism and bariatric surgery for morbid obesity, can result in the neurological disorder Wernicke's encephalopathy (WE). Recent work building upon early observations in animal models of thiamine deficiency has demonstrated an inflammatory component to the neuropathology observed in thiamine deficiency. The present, multilevel study including in vivo magnetic resonance imaging (MRI) and spectroscopy (MRS) and postmortem quantification of chemokine and cytokine proteins sought to determine whether a combination of these in vivo neuroimaging tools could be used to characterize an in vivo MR signature for neuroinflammation. Thiamine deficiency for 12days was used to model neuroinflammation; glucose loading in thiamine deficiency was used to accelerate neurodegeneration. Among 38 animals with regional brain tissue assayed postmortem for cytokine/chemokine protein levels, three groups of rats (controls+glucose, n=6; pyrithiamine+saline, n=5; pyrithiamine+glucose, n=13) underwent MRI/MRS at baseline (time 1), after 12days of treatment (time 2), and 3h after challenge (glucose or saline, time 3). In the thalamus of glucose-challenged, thiamine deficient animals, correlations between in vivo measures of pathology (lower levels of N-acetyle aspartate and higher levels of lactate) and postmortem levels of monocyte chemotactic protein-1 (MCP-1, also known as chemokine ligand 2, CCL2) support a role for this chemokine in thiamine deficiency-related neurodegeneration, but do not provide a unique in vivo signature for neuroinflammation.

OBJECTIVES Over-the-counter access to an inexpensive, effective topical microbicide could reduce the transmission of HIV and would increase women's control over their health and eliminate the need to obtain their partners' consent for prophylaxis. Chronic infection with herpes simplex virus 2 (HSV-2), also known as human herpes virus 2, has been shown to facilitate HIV infection and speed the progression to immunodeficiency disease. Our objective is to develop a drug formulation that protects against both HSV-2 and HIV infection and adheres to the vaginal surface with extended residence time. METHODS We developed a formulation using two approved antiviral active pharmaceutical ingredients, aciclovir and tenofovir, in a novel bioadhesive vaginal delivery platform (designated SR-2P) composed of two polymers, poloxamer 407 NF (Pluronic(®) F-127) and polycarbophil USP (Noveon(®) AA-1). The efficacy of the formulation to protect from HSV-2 infection was tested in vitro and in vivo. In addition to its efficacy, it is essential for a successful microbicide to be non-irritating to the vaginal mucosa. We therefore tested our SR-2P platform gel in the FDA gold-standard microbicide safety model in rabbits and also in a rat vaginal irritation model. RESULTS Our studies indicated that SR-2P containing 1% aciclovir and 5% tenofovir protects (i) Vero cells from HSV-2 infection in vitro and (ii) mice from HSV-2 infection in vivo. Our results further demonstrated that SR-2P was not irritating in either vaginal irritation model. CONCLUSIONS We conclude that SR-2P containing aciclovir and tenofovir may be a suitable candidate microbicide to protect humans from vaginal HSV-2 infection.

AIDS Research and Human RetrovirusesVol. 30, No. S1 Novel Formulations, Agents and MicrobicidesFree AccessDevelopment of a Novel Bioadhesive Microbicide Gel Formulation for Prophylactic Protection against HIV and HSV-2Gita N. Shankar, Gaurav Bhatia, and Carsten AltGita N. ShankarSRI International, Pharmaceutical Sciences, Menlo Park, CA, United StatesSearch for more papers by this author, Gaurav BhatiaSRI International, Pharmaceutical Sciences, Menlo Park, CA, United StatesSearch for more papers by this author, and Carsten AltPalo Alto Institute for Research and Education, Palo Alto, CA, United StatesSearch for more papers by this authorPublished Online:30 Oct 2014https://doi.org/10.1089/aid.2014.5286.abstractAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail P15.04Background: Over-the-counter access to an inexpensive topical microbicide could reduce transmission of human immunodeficiency virus (HIV) and would increase women's control over their health and eliminate the need to obtain their partners' consent for prophylaxis. Chronic infection with herpes simplex virus-2 (HSV-2) has been shown to facilitate HIV infection and speed the progression to disease. Our objective is to develop a drug formulation that adheres to the vaginal surface for extended time, while protecting against both HIV and HSV-2. We present here development studies of a novel bioadhesive vaginal delivery platform “SR-2P” which is composed of two FDA-approved polymers, acting as a depot for two approved antiviral drugs, acyclovir and tenofovir.Methods: Prototype gels were developed and evaluated for pH, osmolality, buffering capacity and viscosity under simulated vaginal semen dilutions, and bioadhesivity using minipig vaginal tissues. Release profile of drug loaded SR-2P was performed through porcine vaginal mucosa. Vaginal irritation studies were performed in mice and rabbits.Results: Results show that SR-2P retains its structure and bioadhesivity upon dilution in presence of simulated fluids under stress conditions. The in vitro release profile of SR-2P demonstrated that ∼40% tenofovir and ∼38% acyclovir was retained in the porcine vaginal mucosa, in the presence of vaginal semen fluids even after 6 hours of contact time. SR-2P caused minimal irritation in a mouse and a rabbit vaginal model. Our results indicate that SR-2P containing acyclovir and tenofovir protects Vero cells from infection with HSV-2 in vitro and mice from HSV-2 in vivo.Conclusions: This preliminary study demonstrates that SR-2P gel could be used as a vaginal microbicide for prophylaxis against vaginal HIV and HSV-2. Research reported in this publication was supported by NIAID/NIH Award No.AI098658. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.FiguresReferencesRelatedDetailsCited byThermosensitive hydrogels a versatile concept adapted to vaginal drug delivery15 November 2017 | Journal of Drug Targeting, Vol. 26, No. 7 Volume 30Issue S1Oct 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Gita N. Shankar, Gaurav Bhatia, and Carsten Alt.Development of a Novel Bioadhesive Microbicide Gel Formulation for Prophylactic Protection against HIV and HSV-2.AIDS Research and Human Retroviruses.Oct 2014.A142-A142.http://doi.org/10.1089/aid.2014.5286.abstractPublished in Volume: 30 Issue S1: October 30, 2014PDF download

The Duffy antigen/receptor for chemokines, DARC, belongs to the family of atypical heptahelical chemokine receptors that do not couple to G proteins and therefore fail to transmit conventional intracellular signals. Here we show that during experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, the expression of DARC is upregulated at the blood-brain barrier. These findings are corroborated by the presence of a significantly increased number of subcortical white matter microvessels staining positive for DARC in human multiple sclerosis brains as compared to control tissue. Using an in vitro blood-brain barrier model we demonstrated that endothelial DARC mediates the abluminal to luminal transport of inflammatory chemokines across the blood-brain barrier. An involvement of DARC in experimental autoimmune encephalomyelitis pathogenesis was confirmed by the observed ameliorated experimental autoimmune encephalomyelitis in Darc(-/-) C57BL/6 and SJL mice, as compared to wild-type control littermates. Experimental autoimmune encephalomyelitis studies in bone marrow chimeric Darc(-/-) and wild-type mice revealed that increased plasma levels of inflammatory chemokines in experimental autoimmune encephalomyelitis depended on the presence of erythrocyte DARC. However, fully developed experimental autoimmune encephalomyelitis required the expression of endothelial DARC. Taken together, our data show a role for erythrocyte DARC as a chemokine reservoir and that endothelial DARC contributes to the pathogenesis of experimental autoimmune encephalomyelitis by shuttling chemokines across the blood-brain barrier.

Inflammatory bowel diseases, primarily Crohn's disease and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal tract with unknown etiology. The majority of current therapeutic agents focus on controlling proinflammatory molecules. The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been described as a potential immunomodulator for inflammatory bowel diseases. In this study, we asked whether the small molecule N/OFQ antagonist (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB612111) would inhibit the development of dextran sodium sulfate-induced colitis in C57BL/6 mice. Inhibition of the N/OFQ receptor (NOP) by SB612111 significantly ameliorated the clinical disease course in these animals, as indicated by reduced fecal bleeding, improved recovery from diarrhea and weight loss, and a reduction in histopathological alterations. In addition, the inflammatory response in the colon was diminished, as demonstrated by reduced cytokine protein and messenger RNA expression for CXCL1/keratinocyte-derived chemokine, interferon-γ, interleukin-1β, interleukin-6, and tumor necrosis factor-α, some of which are known targets for the treatment of this devastating disease. Our results strongly support a role for the receptor-ligand pair NOP-N/OFQ in the pathogenesis of colitis. We conclude that inhibition of NOP receptors with small molecule inhibitors may constitute a novel, urgently needed approach for the treatment of inflammatory bowel diseases.

B cells infiltrate the skin in many chronic inflammatory diseases caused by autoimmunity or infection. Despite potential contribution to disease, skin-associated B cells remain poorly characterized. Using an ovine model of granulomatous skin inflammation, we demonstrate that B cells increase in the skin and skin-draining afferent lymph during inflammation. Surprisingly, skin B cells are a heterogeneous population that is distinct from lymph node B cells, with more large lymphocytes as well as B-1-like B cells that coexpress high levels of IgM and CD11b. Skin B cells have increased MHC class II, CD1, and CD80/86 expression compared with lymph node B cells, suggesting that they are well-suited for T cell activation at the site of inflammation. Furthermore, we show that skin accumulation of B cells and Ab-secreting cells during inflammation increases local Ab titers, which could augment host defense and autoimmunity. Although skin B cells express typical skin-homing receptors, such as E-selectin ligand and α-4 and β-1 integrins, they are unresponsive to ligands for chemokine receptors associated with T cell homing into skin. Instead, skin B cells migrate toward the cutaneously expressed CCR6 ligand CCL20. Our data support a model in which B cells use CCR6-CCL20 to recirculate through the skin, fulfilling a novel role in skin immunity and inflammation.

B cells infiltrate skin in many chronic inflammatory diseases caused by autoimmunity or infection. Despite their potential to contribute to disease processes, skin-associated B cells remain poorly characterized. Here, we find in an ovine granuloma model of cutaneous inflammation, B cells increase during the chronic phase of inflammation in both relative and absolute number in the skin and skin-draining afferent lymph. Surprisingly, these B cells are a heterogeneous population that is phenotypically distinct from the B cell population in lymph nodes with more ‘innate-like’ B cells co-expressing high levels IgM and CD11b. Furthermore, skin B cells have increased MHCII and CD80/86 expression compared to lymph node B cells, suggesting that they are well-suited for T cell activation at the site of inflammation. Additionally, unlike co-isolated T cells, skin B cells are not responsive to ligands of chemokine receptors typically associated with skin homing, implying that they utilize alternative chemokine receptors to migrate to the skin. In summary, our data show that a population of B cells recirculates through uninflamed and inflamed skin, fulfilling a so far unappreciated role in skin immunity.

Angiogenesis is one of the major processes controlling growth and metastasis of tumors. Angiogenesis inhibitors have been targeted for the treatment of various cancers for more than 2 decades. We have developed a novel class of steroidal compounds aimed at blocking the angiogenic process in cancerous tissues. Our lead compound, SR16388, is a potent antiangiogenic agent with binding affinity to estrogen receptor-α (ER-α) and -β (ER-β) at the nanomolar range. This compound inhibited the proliferation of human microvascular endothelial cells (HMVEC) and various types of human cancer cells in vitro. SR16388 inhibited embryonic angiogenesis as measured in the chick chorioallantoic membrane (CAM) assay. The blood vessel density in the CAM was greatly reduced after the embryos were treated with 3 μg/CAM of SR16388 for 24 h. SR16388 at a dose of 2 μM prevented tube formation in Matrigel after HMVEC cells were treated for 8 h. In a modified Boyden chamber assay, SR16388 inhibited the migration of HMVECs by 80% at 500 nM. Using a novel in vivo Fibrin Z-chamber model, we demonstrated that SR16388 at a single daily oral dose of 3 mg/kg for 12 days significantly inhibited the granulation tissue (GT) thickness and the microvessel density of the GT as compared to control. More importantly, SR16388 down-regulated the pro-angiogenic transcription factors, hypoxia inducible factor 1α (HIF-1α) and signal transducer and activator of transcription 3 (STAT3) in non-small cell lung cancer (NSCLC) cells. Together, these effects of SR16388 can lead to the reduction of vascularization and tumor growth in vivo.