Genistein for the Reduction of Adverse Cardiovascular Events (GRACE)

A. Rationale Despite the gains in life expectancy afforded by effective antiretroviraltherapy (ART), people living with HIV (PLHIV) continue to exhibit persistent immuneactivation, immune dysfunction and chronic inflammation, all of which contribute to anelevated risk of cardiovascular disease (CVD) [1‐5]. To address this excess risk, thereis an ongoing need for safe adjunctive therapies capable of reducing inflammation,particularly because conventional anti‐inflammatory agents can cause adverse effects orinteract with ART [6‐9].. B. Background CVD in PLHIV on ART The World Health Organization estimates that thereare 41 million PLHIV with 32 million ( 77%) receiving ART and an additional 1.5 millionmore cases predicted to arise each year(https://www.who.int/teams/global‐hiv‐hepatitis‐and‐stis‐programmes/hiv/strategic‐information/hiv‐data‐and‐statistics). PLHIV on ART represent a state of virologic controlwithout full immunologic or inflammatory normalization, characterized by persistentimmune activation, chronic inflammation, and multisystem dysregulation all contributingto increased risk of CVD [1‐5, 10‐14]. This excess risk is not fully explained bytraditional CVD risk factors, such as hypertension, dyslipidemia, diabetes, smoking,obesity, sedentary lifestyle, unhealthy diet and psychosocial stress. Additionally, ARTsthemselves are attributed to increased risk of hypertension, dyslipidemia and diabetes[2]. Adding to this complexity, the treatment of dyslipidemia with statins‐‐that have theadded benefits of pleiotropic anti‐inflammatory off‐targets‐‐does not reduce the CVD riskto levels to those of an unaffected population [2] and may increase the incidence ofdiabetes [7, 15], myalgia, muscle weakness or myopathy [7].In PLHIV, paramount among non‐traditional CVD risk factors is chronic low grade systemicinflammation, characterized by persistently elevated inflammatory and immune activationmarkers, notably IL 6, TNF α, IL 1β, C reactive protein, D dimer, sCD14, and sCD163 [3,16, 17]. These abnormalities are driven by multiple overlapping mechanisms, includingresidual HIV transcription, gut associated tissue damage with microbial translocation,chronic co infections, and incomplete immune reconstitution [18]. Gut barrier dysfunctiontriggered by early HIV is hypothesized to be the potential underlying cause for ongoingimmune dysfunction despite effective ARTs (Fig 1). Growing evidence that gut microbiomeassociated metabolites regulate gene expression profiles of immune cells linked toinflammation [19]. Sustained immune activation of T cells (e.g., CD38⁺HLA DR⁺),monocytes, and innate immune pathways persists despite undetectable viral load [20].Immune dysregulation remains, accompanied by reduced naïve T cell pools, immunesenescence, and immune exhaustion. Persistent monocyte and macrophage activation has beendirectly linked to coronary plaque burden and vascular inflammation, reinforcing theconnection between immune activation and CVD [1, 2].These inflammatory and immune abnormalities converge on endothelial dysfunction, acentral and early event in CVD pathogenesis. In PLHIV, chronic inflammation and ARTexposure impair nitric oxide signaling, increase oxidative stress, and promote arterialstiffness [13]. Endothelial dysfunction is further exacerbated by comorbid exposures andconditions common in this population. Cannabis use, for example, is four times moreprevalent among PLHIV [21] and has been associated with vascular inflammation, oxidativestress, and impaired endothelial dependent vasodilation [22‐24]. Chronic pain states, inan estimated 25% to 80% of PLHIV on ART, similarly contribute through sustainedsympathetic activation and neuroimmune signaling, which impair vascular repair mechanisms[25]. Collectively, these interrelated mechanisms drive the increased burden of CVD andother non AIDS comorbidities in PLHIV receiving ART.Genistein as a therapeutic for PLHIV at risk of CVD Non immunosuppressive adjunctivetherapeutic strategies that safely target upstream inflammation and endothelialdysfunction represent a critical unmet need to reduce CVD in PLHIV. Genistein, anaturally occurring soy derived isoflavone consumed in human diets for millennia,exhibits multi modal anti inflammatory, antioxidant, and vasoprotective properties (Fig2) [26]. Mechanistically, genistein inhibits key pathways activated in treated HIV: NFκB‐dependent transcription, pro inflammatory cytokine signaling (Fig 3), inducible nitricoxide synthase activity, and reactive oxygen species generation. Importantly, genisteindoes not induce global immune suppression and has demonstrated a favorable safetyprofile.Anti‐Inflammatory properties of genistein Inflammation, while normally a protectivebiological response triggered by harmful stimuli, can become harmful when persistentand/or systemic.Inhibition of NF κB signaling. At the molecular level, genistein inhibits NF κB primarilyby suppressing IκB kinase activity, preserving IκBα stability, and preventing nucleartranslocation of the NF κB p65 subunit [27]. This action occurs downstream of receptorengagement but upstream of transcriptional activation, resulting in broad suppression ofinflammatory gene expression rather than selective cytokine blockade. Through thismechanism, genistein attenuates one of the central drivers of persistent inflammation.Modulation of JAK/STAT and cytokine amplification. Genistein dampens JAK/STAT signalingby reducing JAK phosphorylation and STAT nuclear localization, particularly within IL6‐STAT3‐driven inflammatory loops [26]. By interfering at this level, genistein limitsthe systemic propagation and stabilization of inflammation rather than its initialtriggering. This distinguishes its effects from cytokine specific biologics (e.g. IL1ßantibody therapeutics) and aligns it more closely with network oriented anti inflammatorystrategies.Effects on MAPK signaling and stress responses. Experimental models demonstrate thatgenistein reduces phosphorylation of p38 and JNK, thereby attenuating MAPK dependentinflammatory amplification and AP 1 transcriptional activity [28]. This mechanism isparticularly relevant to stress responsive and senescence linked inflammation, suggestinga role for genistein in mitigating pathways associated with inflammation and aging,inflammaging, rather than acute immune activation.COX‐2 Inhibition. Cyclooxygenase‐2 (COX‐2) is part of a metabolic pathway thatsynthesizes pro‐inflammatory prostaglandins. Genistein inhibits COX‐2 which leads to thereduction of inflammation and pain [29, 30].Antioxidant properties of genistein Oxidative stress, the imbalance between free radicalproduction and antioxidant defenses, can lead to cell and tissue damage. It is thought toplay a critical role in cardiovascular health. Through redox normalization, genisteinindirectly suppresses NFκB and MAPK activation while reducing DNA damage and senescencesignaling [31]. Genistein acts as a direct antioxidant by scavenging and neutralizingfree radicals to prevent their cellular damage [31] and by activating the Nrf2 pathwayand upregulating antioxidant gene expression [32].Genistein and the endothelium Vascular endothelium dysfunction is a pathologicalcondition characterized by the disruption of the balance between vasodilation andvasoconstriction. It is considered an early indicator in the development ofatherosclerotic CVD. In addition to its anti inflammatory effects, genistein directlytargets endothelial dysfunction which is relevant in PLHIV, perhaps to a higher degree inthose with long term cannabis exposure. As a CB1 receptor antagonist, genistein cancompetitively block delta 9 tetrahydrocannabinol (THC) signaling [24], potentiallymitigating cannabis associated vascular inflammation and endothelial impairment.Together, these properties position genistein as a biologically plausible and clinicallyrelevant intervention to address the persistent inflammatory and vascular abnormalitiesunderlying CVD risk in PLHIV receiving ART.Genistein and the microbiome While genistein is known to be pleiotropic, new evidencesuggests genistein modifies the intestinal microbiome positively. Many of the biomarkersassociated with cardiometabolic health in our assessments and their physiologicalactivity could also be explained by changes in circulating lipopolysaccharide (LPS) andother inflammatory cytokines regulated as a result of intestinal microbiota changepotentially caused by genistein, indicating the monitoring the microbiome duringgenistein treatment necessary. The elevation of LPS concentration and low‐grade chronicinflammation has been associated with mechanisms of the change in intestinal permeabilityand strongly associated with underlying cardiometabolic disease risk and progression[33]. It has been demonstrated that dysbiosis of the gut microbiota can alter the gutpermeability, and several factors can contribute to modifications of the gut microbiota,such as intestinal infections by pathogens, the use of antibiotics, or dietary factorsleading to the development of metabolic endotoxemia [34]. Akkermansia muciniphila (phylumVerrucomicrobia) is a mucin‐degrading bacterium believed to contribute to intestinalhealth and glucose homeostasis and improved insulin sensitivity due to reduction in thecirculating concentration of LPS [35]. Mice treated with genistein show a selectiveincrease in Akkermansia muciniphila, leading to a reduction of metabolic endotoxemia andinsulin sensitivity [36‐39]. Furthermore, clinical trials in obese subjects show thatconsumption of genistein over two months (50mg/day) modifies gut microbiota taxonomy,remarkably increasing the Verrucomicrobia phylum[35]. This selective increasemodification of the intestinal microbiota was attributed to the improvement in insulinsensitivity in the patients tested and concomitant reduction in metabolic endotoxemia,increase in AMPK phosphorylation, and expression of genes involved in fatty acidoxidation in skeletal muscle. We expect that genistein will alter the gut microbiome byincreasing the presence of the Verrucomicrobia phylum. In PLHIV on ART, gut dysbiosislikely due to microbial translocation plays a central role in maintaining systemicinflammation and cardiovascular risk. Genistein has demonstrated the ability to modulatethe microbiome, reduce inflammatory metabolite production, and shift gut barrier functiontoward a healthier phenotype. Monitoring the gut microbiome during genistein interventionwill uncover mechanistic pathways, act as a predictive biomarker of efficacy, and guidepotential additional therapeutics.Given these potential multimodal actions, there is a clear rationale to explore theefficacy of genistein in PLHIV on ART through well‐structured clinical protocols.Genistein's side effect profile is considered safe, and it has been consumed as asupplement for many centuries. A critical challenge, however, lies in its lowbioavailability. To overcome the low bioavailability, we are proposing a phase I clinicaltrial to explore the safety and efficacy of higher doses of genistein.C. Safety Considerations. The cumulative evidence from extensive clinical studiessupports the clinical safety of Genistein (see Investigator's Brochure). We have beenapproved for higher doses up to 1500mg per day in heart failure patients (GASPAR:Genistein In Transthyretin Receptor Amyloid Cardiomyopathy, WREM 122758, ReDA:13179,Appendix 1). The safety concerns for PLHIV have been examined and summarized forover‐the‐counter (OTC) medications (Table 1), prescription medications (Table 2), ART forPLHIV (Table 3) and in the context of cannabis use which is statistically higher in thispopulation.D. Specific Objectives The systemic inflammation in PLHIV is well established as well asthe limitations of therapies, notably the undesirable side effects of currentanti‐inflammatory medications. This study aims to investigate the efficacy of genisteinin countering the systemic inflammation underlying CVD risk and chronic pain in PLHIV,including those whose vascular dysfunction may be exacerbated by cannabis use. Theprimary objectives are to evaluate genistein's impact on inflammatory and cardiometabolicbiomarkers, as well as vascular function and pain level. This study also incorporates twoexploratory objectives: an investigation on whether the microbiome is a key modulator ofinflammatory markers and the use of induced pluripotent stem cells (iPSCs) derived fromparticipant blood samples to assess cellular mechanisms influenced by genistein. Thiscomplementary approach will provide mechanistic insight into genistein's therapeuticpotential in PLHIV.Objective 1: Investigate whether genistein attenuates systemic inflammation associatedwith CVD in PLHIV.Objective 2: Investigate whether the vascular dysfunction found in PLHIV is attenuated bygenistein.Objective 3: Investigate whether genistein attenuates pain, chronic or otherwise, inPLHIV.Objective 4: Investigate whether genistein in PLHIV alters the microbiome to reducelevels of inflammation.Exploratory Objective: An optional sub‐study will incorporate the use of inducedpluripotent stem cells (iPSCs) derived from participant blood samples to assess cellularmechanisms influenced by genistein to gain further insight into genistein's therapeuticpotential in PLHIV.