An exploratory, randomised, placebo-controlled, 14 day trial of the soluble guanylate cyclase stimulator praliciguat in participants with type 2 diabetes and hypertension.

AIMS/HYPOTHESIS: Praliciguat (IW-1973), a soluble guanylate cyclase stimulator, amplifies nitric oxide signalling. This exploratory trial investigated the safety, tolerability, pharmacokinetic profile and pharmacodynamic effects of praliciguat in individuals with type 2 diabetes and hypertension. METHODS: This Phase IIA, double-blind, placebo-controlled trial investigated praliciguat in 26 participants with type 2 diabetes and hypertension on stable glucose- and BP-lowering therapies. Participants were randomly allocated in a 3:5:5 ratio to three groups: placebo (n = 6), praliciguat 40 mg once daily for days 1-14 (n = 10), or praliciguat 20 mg twice daily for days 1-7 then 40 mg once daily for days 8-14 (n = 10). Assessments were made in clinic and included treatment-emergent adverse events, pharmacokinetics, metabolic variables, 24 h BP and heart rate, platelet function, reactive hyperaemia index (RHI) and plasma biomarkers. Participants, the sponsor, the investigator and clinic study staff (except designated pharmacy personnel) were blinded to group assignment. RESULTS: Participants treated for 14 days with praliciguat had least-square mean change-from-baseline differences vs placebo (95% CI) of -0.7 (-1.8, 0.4) mmol/l for fasting plasma glucose, -0.7 (-1.1, -0.2) mmol/l for total cholesterol, -0.5 (-1.0, -0.1) mmol/l for LDL-cholesterol, -23 (-56, 9) for HOMA-IR in those not being treated with insulin, and -5 (-10, 1) mmHg and 3 (-1, 6) beats/min for average 24 h mean arterial pressure and heart rate, respectively. Apart from one serious adverse event (SAE; upper gastrointestinal haemorrhage), praliciguat was well tolerated. Praliciguat did not affect platelet function or RHI. Among exploratory biomarkers, plasma levels of asymmetric dimethylarginine decreased in praliciguat vs placebo recipients. CONCLUSIONS/INTERPRETATION: In participants with type 2 diabetes and hypertension on standard therapies, over 14 days praliciguat was well tolerated, except for a single SAE, and showed positive trends in metabolic and BP variables. These results support further clinical investigation of praliciguat. TRIAL REGISTRATION: ClinicalTrials.gov NCT03091920. FUNDING: This trial was funded by Cyclerion Therapeutics.

Praliciguat inhibits progression of diabetic nephropathy in ZSF1 rats and suppresses inflammation and apoptosis in human renal proximal tubular cells.

Praliciguat, a clinical-stage soluble guanylate cyclase (sGC) stimulator, increases cGMP via the nitric oxide-sGC pathway. Praliciguat has been shown to be renoprotective in rodent models of hypertensive nephropathy and renal fibrosis. In the present study, praliciguat alone and in combination with enalapril attenuated proteinuria in the obese ZSF1 rat model of diabetic nephropathy. Praliciguat monotherapy did not affect hemodynamics. In contrast, enalapril monotherapy lowered blood pressure but did not attenuate proteinuria. Renal expression of genes in pathways involved in inflammation, fibrosis, oxidative stress, and kidney injury was lower in praliciguat-treated obese ZSF1 rats than in obese control rats; fasting glucose and cholesterol were also lower with praliciguat treatment. To gain insight into how tubular mechanisms might contribute to its pharmacological effects on the kidneys, we studied the effects of praliciguat on pathological processes and signaling pathways in cultured human primary renal proximal tubular epithelial cells (RPTCs). Praliciguat inhibited the expression of proinflammatory cytokines and secretion of monocyte chemoattractant protein-1 in tumor necrosis factor-α-challenged RPTCs. Praliciguat treatment also attenuated transforming growth factor-ß-mediated apoptosis, changes to a mesenchyme-like cellular phenotype, and phosphorylation of SMAD3 in RPTCs. In conclusion, praliciguat improved proteinuria in the ZSF1 rat model of diabetic nephropathy, and its actions in human RPTCs suggest that tubular effects may contribute to its renal benefits, building upon strong evidence for the role of cGMP signaling in renal health.

Rationale and design for a multicenter, randomized, double-blind, placebo-controlled, phase 2 study evaluating the safety and efficacy of the soluble guanylate cyclase stimulator praliciguat over 12 weeks in patients with heart failure with preserved ejection fraction (CAPACITY HFpEF).

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is a significant cause of morbidity and mortality worldwide. Exercise intolerance is the main symptom of HFpEF and is associated with a poor quality of life and increased mortality. Currently, there are no approved medications for the treatment of HFpEF. Praliciguat (IW-1973), a novel soluble guanylate cyclase stimulator that may help restore deficient nitric oxide-soluble guanylate cyclase-cyclic guanosine 3',5'-monophosphate signaling, is being investigated for the treatment of patients with HFpEF. METHODS: CAPACITY HFpEF is a phase 2, multicenter, randomized, double-blind, placebo-controlled, parallel-group trial designed to evaluate the safety and efficacy of praliciguat over 12 weeks in approximately 184 patients with HFpEF. Eligible patients must have evidence supporting clinical HFpEF and at least 2 of the following 4 conditions associated with NO deficiency: diabetes/prediabetes, hypertension, obesity, and age >70 years. The primary efficacy end point is the change from baseline in peak VO2 by cardiopulmonary exercise test (CPET). Secondary end points include the change from baseline in 6-minute walk test distance and the change in ventilatory efficiency on CPET, as well as number of CPET responders. Other exploratory end points include changes in echocardiographic parameters, New York Heart Association functional classification, cardiac events, blood and urine biomarkers pathophysiologically relevant to heart failure, and patient-reported outcomes including Kansas City Cardiomyopathy Questionnaire. CONCLUSIONS: The CAPACITY HFpEF trial will provide data on short-term safety and efficacy of praliciguat on peak exercise capacity, as well as multiple secondary end points of submaximal functional capacity, patient-reported outcomes, and biomarkers.

Effect of Praliciguat on Peak Rate of Oxygen Consumption in Patients With Heart Failure With Preserved Ejection Fraction: The CAPACITY HFpEF Randomized Clinical Trial.

Importance: Heart failure with preserved ejection fraction (HFpEF) is often characterized by nitric oxide deficiency. Objective: To evaluate the efficacy and adverse effects of praliciguat, an oral soluble guanylate cyclase stimulator, in patients with HFpEF. Design, Setting, and Participants: CAPACITY HFpEF was a randomized, double-blind, placebo-controlled, phase 2 trial. Fifty-nine sites enrolled 196 patients with heart failure and an ejection fraction of at least 40%, impaired peak rate of oxygen consumption (peak V̇o2), and at least 2 conditions associated with nitric oxide deficiency (diabetes, hypertension, obesity, or advanced age). The trial randomized patients to 1 of 3 praliciguat dose groups or a placebo group, but was refocused early to a comparison of the 40-mg praliciguat dose vs placebo. Participants were enrolled from November 15, 2017, to April 30, 2019, with final follow-up on August 19, 2019. Interventions: Patients were randomized to receive 12 weeks of treatment with 40 mg of praliciguat daily (n = 91) or placebo (n = 90). Main Outcomes and Measures: The primary efficacy end point was the change from baseline in peak V̇o2 in patients who completed at least 8 weeks of assigned dosing. Secondary end points included the change from baseline in 6-minute walk test distance and in ventilatory efficiency (ventilation/carbon dioxide production slope). The primary adverse event end point was the incidence of treatment-emergent adverse events (TEAEs). Results: Among 181 patients (mean [SD] age, 70 [9] years; 75 [41%] women), 155 (86%) completed the trial. In the placebo (n = 78) and praliciguat (n = 65) groups, changes in peak V̇o2 were 0.04 mL/kg/min (95% CI, -0.49 to 0.56) and -0.26 mL/kg/min (95% CI, -0.83 to 0.31), respectively; the placebo-adjusted least-squares between-group difference in mean change from baseline was -0.30 mL/kg/min ([95% CI, -0.95 to 0.35]; P = .37). None of the 3 prespecified secondary end points were statistically significant. In the placebo and praliciguat groups, changes in 6-minute walk test distance were 58.1 m (95% CI, 26.1-90.1) and 41.4 m (95% CI, 8.2-74.5), respectively; the placebo-adjusted least-squares between-group difference in mean change from baseline was -16.7 m (95% CI, -47.4 to 13.9). In the placebo and praliciguat groups, the placebo-adjusted least-squares between-group difference in mean change in ventilation/carbon dioxide production slope was -0.3 (95% CI, -1.6 to 1.0). There were more dizziness (9.9% vs 1.1%), hypotension (8.8% vs 0%), and headache (11% vs 6.7%) TEAEs with praliciguat compared with placebo. The frequency of serious TEAEs was similar between the groups (10% in the praliciguat group and 11% in the placebo group). Conclusions and Relevance: Among patients with HFpEF, the soluble guanylate cyclase stimulator praliciguat, compared with placebo, did not significantly improve peak V̇o2 from baseline to week 12. These findings do not support the use of praliciguat in patients with HFpEF. Trial Registration: ClinicalTrials.gov Identifier: NCT03254485.

Pharmacological Characterization of IW-1973, a Novel Soluble Guanylate Cyclase Stimulator with Extensive Tissue Distribution, Antihypertensive, Anti-Inflammatory, and Antifibrotic Effects in Preclinical Models of Disease.

Soluble guanylate cyclase (sGC), a key signal-transduction enzyme, increases the conversion of guanosine-5'-triphosphate to cGMP upon binding of nitric oxide (NO). Endothelial dysfunction and/or reduced NO signaling have been implicated in cardiovascular disease pathogenesis and complications of diabetes and have been associated with other disease states and aging. Soluble guanylate cyclase (sGC) stimulators are small-molecule drugs that bind sGC and enhance NO-mediated cGMP signaling. The pharmacological characterization of IW-1973 [1,1,1,3,3,3-hexafluoro-2-(((5-fluoro-2-(1-(2-fluorobenzyl)-5-(isoxazol-3-yl)-1H-pyrazol-3-yl) pyrimidin-4-yl)amino)methyl)propan-2-ol], a novel clinical-stage sGC stimulator under clinical investigation for treatment of heart failure with preserved ejection fraction and diabetic nephropathy, is described. In the presence of NO, IW-1973 stimulated sGC in a human purified enzyme assay and a HEK-293 whole cell assay. sGC stimulation by IW-1973 in cells was associated with increased phosphorylation of vasodilator-stimulated phosphoprotein. IW-1973, at doses of 1-10 mg/kg, significantly lowered blood pressure in normotensive and spontaneously hypertensive rats. In a Dahl salt-sensitive hypertension model, IW-1973 significantly reduced blood pressure, inflammatory cytokine levels, and renal disease markers, including proteinuria and renal fibrotic gene expression. The results were affirmed in mouse lipopolysaccharide-induced inflammation and rat unilateral ureteral obstruction renal fibrosis models. A quantitative whole-body autoradiography study of IW-1973 revealed extensive tissue distribution and pharmacokinetic studies showed a large volume of distribution and a profile consistent with predicted once-a-day dosing in humans. In summary, IW-1973 is a potent, orally available sGC stimulator that exhibits renoprotective, anti-inflammatory, and antifibrotic effects in nonclinical models.

Pharmacokinetics, mass balance, tissue distribution, metabolism, and excretion of praliciguat, a clinical-stage soluble guanylate cyclase stimulator in rats.

The pharmacokinetics (PK), metabolism, excretion, mass balance, and tissue distribution of [14 C]praliciguat were evaluated following oral administration of a 3-mg/kg dose in Sprague-Dawley rats and in a quantitative whole-body autoradiography (QWBA) study conducted in male Long-Evans rats. Plasma Tmax was 1 hour and the t1/2 of total plasma radioactivity was 23.7 hours. Unchanged praliciguat accounted for 87.4%, and a minor metabolite (N-dealkylated-praliciguat) accounted for 7.6% of the total radioactivity in plasma through 48 hours (AUC0-48 ). Tissues with the highest exposure ratios relative to plasma were liver, intestines, adrenal gland, and adipose, and those with the lowest values were seminal vesicle, blood, CNS tissues, lens of the eye, and bone. Most of the [14 C]praliciguat-derived radioactivity was excreted within 48 hours after oral administration. Mean cumulative recovery of the administered radioactivity in urine and feces over 168 hours was 3.7% and 95.7%, respectively. Unchanged praliciguat was not quantifiable in urine or bile of cannulated rats; however, based on the total radioactivity in these fluids, a minimum of approximately 82% of the orally administered dose was absorbed. [14 C]Praliciguat was metabolized via oxidative and glucuronidation pathways and the most abundant metabolites recovered in bile were praliciguat-glucuronide and hydroxy-praliciguat-glucuronide. These results indicate that praliciguat had rapid absorption, high bioavailability, extensive tissue distribution, and elimination primarily via hepatic metabolism.

Effects of the Soluble Guanylate Cyclase Stimulator Praliciguat in Diabetic Kidney Disease: A Randomized Placebo-Controlled Clinical Trial.

BACKGROUND AND OBJECTIVES: Impaired nitric oxide signaling through soluble guanylate cyclase has been implicated in the pathophysiology of diabetic kidney disease. Praliciguat, a soluble guanylate cyclase stimulator that amplifies nitric oxide signaling, inhibited kidney inflammation and fibrosis in animal models. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: In a phase 2 trial, 156 adults with type 2 diabetes, eGFR 30-75 ml/min per 1.73 m2, and urine albumin-creatinine ratio 200-5000 mg/g treated with renin-angiotensin system inhibitors were randomly allocated 1:1:1 to placebo, 20 mg praliciguat, or 40 mg praliciguat daily for 12 weeks. The primary efficacy and safety outcomes were change from baseline to weeks 8 and 12 in urine albumin-creatinine ratio and treatment-emergent adverse events, respectively. Other outcomes assessed were 24-hour ambulatory BP and metabolic parameters. RESULTS: Of 156 participants randomized, 140 (90%) completed the study. The primary efficacy analysis demonstrated a mean change from baseline in urine albumin-creatinine ratio of -28% (90% confidence interval, -36 to -18) in the pooled praliciguat group and -15% (-28 to 0.4) in the placebo group (difference -15%; -31 to 4; P=0.17). Between-group decreases from baseline to week 12 for praliciguat versus placebo were seen in mean 24-hour systolic BP (-4 mm Hg; -8 to -1), hemoglobin A1c (-0.3%; -0.5 to -0.03), and serum cholesterol (-10 mg/dl; -19 to -1). The incidence of treatment-emergent adverse events was similar in the pooled praliciguat and placebo groups (42% and 44%, respectively). Serious adverse events, events leading to study drug discontinuation, and events potentially related to BP lowering were reported at higher frequency in the 40-mg group but were similar in 20-mg and placebo groups. CONCLUSIONS: Praliciguat treatment for 12 weeks did not significantly reduce albuminuria compared with placebo in the primary efficacy analysis. Nonetheless, the observed changes in urine albumin-creatinine ratio, BP, and metabolic variables may support further investigation of praliciguat in diabetic kidney disease. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: A Study to Evaluate the Soluble Guanylate Cyclase (sGC) Stimulator IW-1973 in Diabetic Nephropathy/Diabetic Kidney Disease as Measured by Albuminuria, NCT03217591.

A Randomized, Placebo-Controlled, Multiple-Ascending-Dose Study to Assess the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of the Soluble Guanylate Cyclase Stimulator Praliciguat in Healthy Subjects.

Nitric oxide (NO)-soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) signaling is central to the regulation of several physiological processes, including blood flow and inflammation. Deficient NO signaling is implicated in multiple diseases. sGC stimulators are small molecules that enhance sGC activity, particularly in combination with NO. In a randomized, placebo-controlled phase 1 study, the safety, tolerability, pharmacokinetics, and pharmacodynamics of multiple ascending doses of the sGC stimulator praliciguat were assessed in 44 healthy adults. Four cohorts of 11 subjects (8 praliciguat, 3 placebo) received once-daily praliciguat for 14 days before up-titrating for 7 days (treatment sequences: 15/30 mg, 20/40 mg, 30/40 mg, and weight-based). All doses were tolerated. No serious or severe adverse events (AEs) were reported. The most common AEs in praliciguat recipients were headache and symptoms consistent with blood pressure (BP) lowering/vasodilation. There were no laboratory, vital sign, electrocardiographic, or platelet function findings indicative of a safety concern. Pharmacokinetics were dose proportional, with an effective half-life of 24-37 hours, supporting once-daily dosing. Praliciguat produced dose-related increases in plasma cGMP consistent with stimulation of sGC. Repeated once-daily dosing showed sustained decreases in BP. Results support evaluation of praliciguat for the treatment of conditions associated with deficient NO signaling.

PRO 2000 elicits a decline in genital tract immune mediators without compromising intrinsic antimicrobial activity.

OBJECTIVE: Vaginal microbicides should protect against infection without disrupting the mucosal environment or its mediators of host defense. The objective of this study was to examine the effect of 14 daily applications of 0.5% PRO 2000 or placebo gel on mediators of mucosal immunity and intrinsic antimicrobial activity. DESIGN AND METHODS: A randomized, prospective, double-blind, placebo-controlled study was conducted among 24 healthy, abstinent women. Levels of cytokines, chemokines, defensins, and other protective factors and intrinsic antimicrobial activity were determined in cervicovaginal lavage samples collected on study days 0, 7, 14, and 21. RESULTS: No increase in pro-inflammatory cytokines was observed. Rather cytokines and protective factors including interleukin (IL)-1 receptor antagonist, immunoglobulins and human beta-defensin 2 were lower in the drug compared with the placebo group. All of the mediators returned towards baseline on day 21. Women who were cycling had lower levels of most proteins on study days 7 and/or 14 compared with women on oral contraceptives; however, the magnitude of decline was greater in women who received PRO 2000 compared with placebo gel. The reduction in protective factors was not associated with a loss in the intrinsic anti-viral (HIV or herpes simplex virus) activity or anti-bacterial activity (Escherichia coli or Staphylococcus aureus). CONCLUSION: In contrast to experience with nonoxynol-9, PRO 2000 did not trigger an inflammatory response in cervicovaginal secretions. There was a modest reduction in mucosal immune mediators, but this loss was not associated with a reduction in intrinsic antimicrobial activity.

Direct measurement of in-vivo vaginal microbicide levels of PRO 2000 achieved in a human safety study.

OBJECTIVES: To directly measure the cervico-vaginal lavage (CVL) and plasma PRO 2000 concentrations achieved by vaginal dosing. DESIGN: A sub-study of a prospective randomized, double-blind phase 1 trial of a candidate vaginal microbicide, PRO 2000 gel. METHODS: Thirty-six sexually abstinent women self-administered 4% PRO 2000 gel, 0.5% PRO 2000 gel or placebo gel twice on day 0 and then once daily for a further 12 days. RESULTS: There was no evidence of systemic absorption of PRO 2000. PRO 2000 concentrations in CVL exceeded 25 microg/ml in all women in both the 4 and 0.5% groups at 2 h post-first dose, and in 10 of 12 of the women in the 4% gel group compared with five of 12 of women in the 0.5% group at 12 h post-seventh dose. Single use of both 4 and 0.5% PRO 2000 gels was therefore associated with levels of PRO 2000 in CVL that would be capable of preventing HIV infection in vitro, although the 4% gel gave a greater margin of excess. Levels substantially in excess of the target concentration were present 12 h after repeated dosing in twice as many 4% gel recipients compared with 0.5% gel recipients. CONCLUSIONS: Both PRO 2000 gel strengths provided satisfactory in-vivo HIV inhibitory concentrations. However, our observations show that higher concentrations of PRO 2000 are likely to provide a greater margin of potential efficacy in the context of sexual intercourse provided safety issues are equivalent for differing concentrations of the agent.

Safety and tolerability of vaginal PRO 2000 gel in sexually active HIV-uninfected and abstinent HIV-infected women.

OBJECTIVES: To evaluate once or twice daily vaginal exposure to 2 and 4% PRO 2000 Gel, a naphthalene sulfonate polymer microbicide, in sexually active HIV-uninfected women to determine the highest tolerated frequency and concentration combination, and to assess this in sexually abstinent HIV-infected women. METHODS: Sixty three women from Providence, Philadelphia, Durban and Johannesburg were enrolled after being screened to exclude pre-existing illnesses and were instructed to use the product once or twice daily for 14 intermenstrual days. They underwent colposcopy prior to product use and after 14 days of product use, with a pelvic examination at day 7. RESULTS: The product was well tolerated, with no serious adverse events, even though 73% of the participants had at least one adverse experience: 82% of these were classified as mild, and over 90% of the findings and symptoms were localized to the genital tract. Women who used the 4% gel twice daily tended to have more adverse events than all the other groups. Three participants did not complete the study; one because of Herpes simplex virus cervicitis, the second because of epithelial disruption, and the third because she became pregnant. The remaining participants adhered to the study protocol and indicated that they would use the product if it were shown to be effective. CONCLUSIONS: PRO 2000 Gel was safe and well tolerated in sexually active HIV-uninfected and sexually abstinent HIV-infected women, enabling the product to be considered for evaluation in efficacy trials.

The acceptability of an investigational vaginal microbicide, PRO 2000 Gel, among women in a phase I clinical trial.

PURPOSE: Vaginal microbicides will provide a woman-initiated prevention strategy that could substantially reduce rates of HIV infection. The acceptability of microbicides will greatly influence the use and, hence, effectiveness of such products. In this study, the acceptability of an investigational microbicide, PRO 2000 Gel (Indevus Pharmaceuticals, Inc., Lexington, MA), was assessed, and women's opinions about microbicides and their potential for real world use were gathered. METHODS: Quantitative and qualitative data were collected from 30 U.S. and 33 South African women. All sexually active HIV-uninfected women and all sexually abstinent HIV-infected women participating in this phase I clinical trial stated in a survey that they would use PRO 2000 Gel if they had reason to be concerned about HIV and the product were available. Qualitative data, however, provided insight into the nuances of acceptability ratings. Women rated product safety, ease of use, and positive effects on sexual pleasure among the most important characteristics of acceptable microbicides. RESULTS: Opinions regarding product leakage, contraceptive capability, and the ability to be used without partners noticing, as well as characteristics of the product itself, varied substantially based on the context of sex and perceptions of risk within each individual woman's life. CONCLUSIONS: As microbicide development continues and the first investigational products move into efficacy trials, the needs and preferences of those women who constitute the potential users of microbicides become paramount. Providing woman-initiated microbicides that are safe, easy to use, and pleasurable will be key to the impact these products will have on the AIDS epidemic worldwide.

Postcoital bioavailability and antiviral activity of 0.5% PRO 2000 gel: implications for future microbicide clinical trials.

BACKGROUND: The pharmacokinetics and pharmacodynamics of vaginal microbicides are typically assessed among sexually abstinent women. However, the physical act of sex may modulate gel distribution, and preclinical studies demonstrate seminal plasma interferes with the antiviral activity of several microbicides. This study compared the biological activity and concentration of PRO 2000 in cervicovaginal lavage (CVL) collected in the absence or following coitus. METHODS: CVL samples were collected from ten heterosexual couples at baseline, after sex, after a single dose of 0.5% PRO 2000 gel and sex, and after gel application without sex. The impact of CVL on HIV-1 infection of TZM-bl cells and HSV-2 infection of CaSki cells was monitored by luciferase and plaque assay, respectively. PRO 2000 concentrations were measured by fluorescence. RESULTS: CVL collected after PRO 2000 application significantly inhibited HIV-1 and HSV-2 (p = 0.01). However, the antiviral activity was reduced following sex and no significant protective effect was observed in postcoital CVL obtained in the presence compared to the absence of PRO 2000 for HIV (p = 0.45) or HSV-2 (p = 0.56). Less PRO 2000 was recovered in postcoital CVL, which, in conjunction with interference by seminal plasma, may have contributed to lower antiviral activity. CONCLUSIONS: Postcoital responses to PRO 2000 differ from precoital measures and the results obtained may provide insights into the clinical trial findings in which there was no significant protection against HIV-1 or HSV-2. Postcoital studies should be incorporated into clinical studies before embarking on large-scale efficacy trials.

The candidate sulfonated microbicide, PRO 2000, has potential multiple mechanisms of action against HIV-1.

PRO 2000 is a polyanionic compound under development as a topical antimicrobial gel for the potential prevention of HIV-1 transmission. It has been shown that PRO 2000 binds to HIV-1 gp120 and interferes with virus attachment to and/or fusion with CD4(+) T cells. Here, we demonstrate that PRO 2000 interacts not only with viral gp120 but also with CD4 and CXCR4 receptors on the cell surface. Minor or no effects were noticed on DC-SIGN and on CCR5. PRO 2000 dose-dependently inhibited the interaction of CXCL12 with the CXCR4 receptor as demonstrated with CXCL12(AF647)-labeled binding, CXCL12-induced calcium signaling, CXCR4 internalization and chemotaxic assays. These CXCR4 antagonistic properties of PRO 2000 are a potential additional mechanism of action that could explain the observation that PRO 2000 is described to be more active against X4 viruses than R5 viruses. The cellular activation potential and inflammatory properties of PRO 2000 were also examined in PBMCs. PRO 2000 had minor effects on the expression level of several cellular activation markers and enhanced the production of a small number of cytokines/chemokines, as determined by the Bio-Plex human cytokine 27-plex assay system. PRO 2000 showed less mitogenic and stimulatory activity than cyanovirin-N, but careful monitoring for potential side-effects is still advised.

The differential binding and activity of PRO 2000 against diverse HIV-1 envelopes.

OBJECTIVE: PRO 2000 is a polyanionic microbicide that binds directly to the glycoprotein 120 (gp120) envelope protein to inhibit HIV-1 entry. We studied the breadth of PRO 2000 activity against HIV-1 derived from recently transmitted R5 viruses. We also investigated the interaction of this compound with X4 and R5 HIV-1 envelope glycoproteins using an epitope-mapping strategy. METHODS: The anti-HIV activity of PRO 2000 against subtype B and C Env-pseudotyped viruses was assessed in saline and cervicovaginal lavage fluid. Competitive binding assays were performed with X4 and R5 monomeric and virus-associated gp120. RESULTS: PRO 2000 was found to be active against recently transmitted subtype B and C viruses tested in vitro, however, at 1 microg/mL in saline, activity against subtype C was decreased compared with subtype B. Epitope mapping using anti-V3 region antibodies showed that PRO 2000 binds to the V3 region of monomeric and virus-associated X4 gp120 with a higher affinity than to V3 of R5 gp120. In contrast, the interaction of PRO 2000 with the CD4-binding site was similar for both X4 and R5 monomeric and virus-associated gp120. CONCLUSIONS: PRO 2000 has significant activity against recently transmitted viruses, although some activity is lost at low concentrations. Epitope binding studies suggest that this broad activity is due to direct and indirect interactions with multiple gp120 sites rather than V3 binding alone.

PRO 2000 gel inhibits HIV and herpes simplex virus infection following vaginal application: a double-blind placebo-controlled trial.

BACKGROUND: Microbicides used to prevent the transmission of human immunodeficiency virus (HIV) are advancing to clinical trials on the basis of activity observed in vitro and in animal models. However, no data demonstrate activity of microbicides after application in humans. This study was designed to determine the antiviral activity in cervicovaginal lavage (CVL) samples collected after intravaginal application of 0.5% PRO 2000 gel (Indevus). METHODS: A randomized, double-blind study was conducted to assess the anti-HIV and anti-herpes simplex virus (HSV) activity of PRO 2000 in CVL samples obtained at screening (48 hours before) and 1 hour after application of study or placebo gel. HeLa cells or human macrophages were inoculated with CVL samples spiked with replication-defective HIV containing a luciferase indicator gene and pseudotyped with an R5 envelope. Human cervical epithelial cells were inoculated with CVL samples and challenged with HSV-2(G), and the virus titer was then determined. RESULTS: CVL samples obtained after application of PRO 2000 gel significantly inhibited HIV and HSV infection by at least 1000-fold, compared with CVL samples obtained at screening (P < .001). There were no differences in cytokine levels between the drug and placebo groups. CONCLUSIONS: PRO 2000 gel (0.5%) is sufficiently bioavailable and retains substantial antiviral activity after intravaginal application. This strategy provides a mechanism for testing the efficacy of a microbicide before embarking on large-scale clinical trials.

ACIDFORM inactivates herpes simplex virus and prevents genital herpes in a mouse model: optimal candidate for microbicide combinations.

The acidic vaginal milieu is presumed to inactivate pathogens but is neutralized by semen. This notion fostered the development of acid-buffering products, such as ACIDFORM (developed by Program for Topical Prevention of Conception and Disease, Rush University, and licensed by Instead), as microbicides. However, the extent and mechanism of protective activity provided by buffering gels is not known. Exposure of herpes simplex virus (HSV) to pH 4.5 or lower irreversibly inactivated HSV and reduced HSV yields by at least 90%; exposure to pH 5.0 had little or no effect. Pretreatment of HSV-2 with pH 3.5-4.5 triggered proteolysis, disrupting the HSV particle and resulting in a reduction in binding and invasion. ACIDFORM protected 21 (81%) of 26 mice from genital herpes, compared with 3 (12%) of 25 mice who received a placebo gel. ACIDFORM retained significant activity if mice were challenged with HSV delivered in seminal fluid. These findings suggest that ACIDFORM offers considerable protection against HSV and may be an optimal candidate for developing combination microbicides.

Candidate sulfonated and sulfated topical microbicides: comparison of anti-human immunodeficiency virus activities and mechanisms of action.

Poly(styrene 4-sulfonate), cellulose sulfate, polymethylenehydroquinone, and PRO 2000 are sulfated or sulfonated polymers (SPs) under development as topical microbicides. They are presumed to work through similar mechanisms of action, although to date there has been no extensive comparison of their anti-human immunodeficiency virus activities. To determine whether any of these candidate microbicides offers a potential advantage, their in vitro activities, mechanisms of action, stabilities in biological secretions, and toxicities were compared. All four compounds were found to be active against X4, R5, and dualtropic primary isolates and against X4 and R5 laboratory-adapted strains in CD4+ T cells, macrophages, and single-coreceptor cell lines. Our single-cycle experiments using pseudotyped virus suggest that all four SPs function at the binding and entry stages of the viral life cycle but differ in degree of postentry effect. Surface plasmon resonance analyses demonstrate that SPs bind to X4 and R5 monomeric glycoprotein 120 with similar high binding affinities. When mixed with cervicovaginal lavage fluid, SPs maintain inhibitory activity at concentrations achievable in formulations.

Recurring conformation of the human immunodeficiency virus type 1 gp120 V3 loop.

The crystal structure of the human immunodeficiency virus type 1 (HIV-1) neutralizing, murine Fab 83.1 in complex with an HIV-1 gp120 V3 peptide has been determined to 2.57 A resolution. The conformation of the V3 loop peptide in complex with Fab 83.1 is very similar to V3 conformations seen previously with two other neutralizing Fabs, 50.1 and 59.1. The repeated identification of this same V3 conformation in complex with three very different, neutralizing antibodies indicates that it is a highly preferred structure for V3 loops on some strains of the HIV-1 virus.

Candidate topical microbicides bind herpes simplex virus glycoprotein B and prevent viral entry and cell-to-cell spread.

Topical microbicides designed to prevent acquisition of sexually transmitted infections are urgently needed. Nonoxynol-9, the only commercially available spermicide, damages epithelium and may enhance human immunodeficiency virus transmission. The observation that herpes simplex virus (HSV) and human immunodeficiency virus bind heparan sulfate provided the rationale for the development of sulfated or sulfonated polymers as topical agents. Although several of the polymers have advanced to clinical trials, the spectrum and mechanism of anti-HSV activity and the effects on soluble mediators of inflammation have not been evaluated. The present studies address these gaps. The results indicate that PRO 2000, polystyrene sulfonate, cellulose sulfate, and polymethylenehydroquinone sulfonate inhibit HSV infection 10,000-fold and are active against clinical isolates, including an acyclovir-resistant variant. The compounds formed stable complexes with glycoprotein B and inhibit viral binding, entry, and cell-to-cell spread. The effects may be long lasting due to the high affinity and stability of the sulfated compound-virus complex, as evidenced by surface plasmon resonance studies. The candidate microbicides retained their antiviral activities in the presence of cervical secretions and over a broad pH range. There was little reduction in cell viability following repeated exposure of human endocervical cells to these compounds, although a reduction in secretory leukocyte protease inhibitor levels was observed. These studies support further development and rigorous evaluation of these candidate microbicides.