Drug-drug interactions in HIV-infected patients receiving chemotherapy.

INTRODUCTION: Coadministration of antiretrovirals and anti-cancer medications may present many complex clinical scenarios. This is characterized by the potential for drug-drug interactions (DDIs) and the challenges that arise in patient management. In this article, we investigate the potential for DDIs between antiretrovirals, including protease inhibitors (PIs), non-nucleoside reverse transcriptase inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs), integrase strand transfer inhibitors (INSTIs), and anti-cancer medications. AREAS COVERED: PubMed, Google Scholar, and Clinicaltrials.gov were searched for relevant articles in April 2024. Our review highlights PIs and NNRTIs as particularly prone to DDIs with anticancer agents, with implications for efficacy and toxicity of concomitant cancer therapy. We explain the mechanisms for interactions, emphasizing the significance of pharmacokinetic effects and enzyme induction or inhibition. We discuss clinical challenges encountered in the management of patients receiving combined ART and cancer therapy regimens. EXPERT OPINION: Data are lacking for potential DDIs between antiretroviral and anti-cancer agents. While some interactions are documented, others are theoretical and based on the pharmacokinetic properties of the medications. Awareness of these interactions, inter-collaborative care between healthcare providers, and standardized treatment guidelines are all crucial for achieving optimal treatment outcomes and ensuring the well-being of patients with HIV/AIDS and cancer comorbidities.

Substances of abuse and their effect on SAR-CoV-2 pathogenesis.

Following the emergence of SARS-CoV-2, various reports suggest that there has been a significant increase in substance abuse due to social distancing and related issues. Several reports have suggested the impact of chronic substance use on individuals' physiological and psychological health. Therefore, there is a need to know the impact of SARS-CoV-2 on persons with substance use disorders. Individuals with substance use disorders are the most vulnerable groups and are at a high risk of SARS-CoV-2 infection due to their already existing health issues associated with substance use. This review discusses some of the molecular and systemic/organic effects chronic substance use such as alcohol, nicotine, marijuana (cannabis), opioids, methamphetamine, and cocaine have on SARS-CoV-2 infectivity and its potential cause for worsened disease outcomes in persons with substance use disorder. This will provide healthcare providers, public health policies, and researchers with the needed knowledge to address some of the many challenges faced during the Covid-19 pandemic to facilitate treatment strategies for persons with substance use disorders.

Drug-drug interactions between COVID-19 therapeutics and antiretroviral treatment: the evidence to date.

INTRODUCTION: With new effective treatments for SARS-CoV-2, patient outcomes have greatly improved. However, new medications bring a risk of drug interactions with other medications. People living with HIV (PLWH) are at particular risk for these interactions due to heightened risk of immunosuppression, polypharmacy, and overlap in affected organs. It is critical to identify drug interactions are a significant barrier to care for PLWH. Establishing a better understanding of the pharmacologic relationships between COVID-19 therapies and antiretrovirals will improve patient-centered care in COVID-19. AREAS COVERED: Potential drug-drug interactions between Human Immunodeficiency Virus (HIV) and COVID-19 treatments are detailed and reviewed here. The mechanisms seen in these interactions include alterations in metabolic enzymes, drug transporters, pharmacoenhancement, and organ toxicities. We also review the limitations and solutions that can be used to combat drug-drug interactions between these two disease states. EXPERT OPINION: While current drug interactions are relatively mild between HIV and COVID-19 therapies, improvements in identifying these beforehand must take place as new therapies are approved. Antiretroviral therapy (ART) is essential in PLWH and must be maintained when treating COVID-19. As advancements in care occur, there is the possibility that newly approved drugs may have additional unknown interactions.

Expanding therapeutic options: lenacapavir + bictegravir as a potential treatment for HIV.

INTRODUCTION: Treatment for people with HIV/AIDS has radically evolved since the introduction of the first antiretrovirals. One newly approved antiretroviral is lenacapavir, which targets the viral capsid. Lenacapavir is currently approved as a therapeutic addition for subjects who are treatment-experienced, and who have developed resistance to multiple antiretrovirals. It is available both as a daily oral tablet and a once every 6-month subcutaneous injection. It is currently undergoing clinical trials in combination with the integrase inhibitor bictegravir as a dual therapy option, both for treatment experienced and treatment naïve individuals. AREAS COVERED: We reviewed published articles, conference proceedings, and clinical trial databases to assess the current status of the research into lenacapavir and bictegravir. While the clinical trials are ongoing, with little published data to date, this combination shows promise for the treatment of both treatment experienced and naïve patients. We review the studies relevant to the pharmacokinetic/pharmacodynamic properties of the drugs. EXPERT OPINION: The new combination with bictegravir will be beneficial for treatment experienced patients, as it represents a dual therapy modality with high barriers of resistance. As a therapy for treatment naïve patients, its use is likely more niche, as other combinations are available.

Senescent macrophages alter fibroblast fibrogenesis in response to SARS-CoV-2 infection.

SARS-CoV-2 has, since its emergence in 2019, become a global pandemic. Disease outcomes are worsened in older patients who are infected. The causes for this is multifactorial, but one potential cause for this disparity is increased rates of cellular senescence in older individuals, particularly in immune cells. Cellular senescence, the accumulation of factors resulting in cell growth arrest and apoptosis resistance, increases as individuals age. In immune cells, senescence is associated with increased inflammation, and alterations in immune response. We utilized a co-culture system consisting of senescent or non-senescent macrophages directly cultured with fibroblasts, and infected with SARS-CoV-2. We assessed the expression of collagen and fibronectin, important molecules in the extracellular matrix, as well as a number of fibrogenic factors. We observed that infection with SARS-CoV-2 induced collagen production in co-cultures with senescent, but not non-senescent macrophages. Fibronectin expression was decreased in both co-culture conditions. While significant results were not observed, concentrations of other fibrogenic molecules were consistent with the collagen results. These data demonstrate that senescence in macrophages alters the production of fibrotic molecules from fibroblasts in a SARS-CoV-2 infection model. As collagen and fibronectin expression are generally directly correlated, this suggests that senescence dysregulates fibrogenesis in response to infection with SARS-CoV-2. There is a need to further investigate the mechanisms for these changes.

An evaluation of long-acting cabotegravir + rilpivirine for the treatment of virologically suppressed adults living with HIV.

INTRODUCTION: HIV is a global disease that has seen significant improvements in care over the past decades. Despite improvements, treatments for maintaining suppression are complex for patients and include two to three oral medications. The approval of intramuscular cabotegravir (CAB) and rilpivirine (RPV) offers a new therapeutic modality with the opportunity of a longer dosing frequency. The data from recent trials including FLAIR and ATLAS have shown non-inferiority in treatment based on the current standard of care. This approval has the potential to simplify patient medication regimens, while maintaining virologic suppression in HIV-1 patients. AREAS COVERED: Cabotegravir + rilpivirine's recent approval for the treatment of HIV and its significant impact it may have on people living with HIV. EXPERT OPINION: Cabotegravir + rilpivirine is a long-acting injectable that can be used for patients who want to reduce the frequency antiretroviral administration. CAB+RPV allows for virologic suppression with monthly or less often administration, but comes with a significant price point, although injection site reactions may limit utility for many patients.

The Importance of Tissue Sanctuaries and Cellular Reservoirs of HIV-1.

PURPOSE OF REVIEW: There have been significant developments in the treatment of people living with HIV-1/AIDS with current antiretroviral therapies; however, these developments have not been able to achieve a functional or sterilizing cure for HIV-1. While there are multiple barriers, one such barrier is the existence of pharmacological sanctuaries and viral reservoirs where the concentration of antiretrovirals is suboptimal, which includes the gut-associated lymphoid tissue, central nervous system, lymph nodes, and myeloid cells. This review will focus on illustrating the significance of these sanctuaries, specific barriers to optimal antiretroviral concentrations in each of these sites, and potential strategies to overcome these barriers. RECENT FINDINGS: Research and studies have shown that a uniform antiretroviral distribution is not achieved with current therapies. This may allow low-level replication associated with low antiretroviral concentrations in these sanctuaries/reservoirs. Many methods are being investigated to increase antiretroviral concentrations in these sites, such as blocking transporting enzymes functions, modulating transporter expression and nanoformulations of current antiretrovirals. While these methods have been shown to increase antiretroviral concentrations in the sanctuaries/reservoirs, no functional or sterilizing cure has been achieved due to these approaches. SUMMARY: New methods of increasing antiretroviral concentrations at the specific sites of HIV-1 replication has the potential to target cellular reservoirs. In order to optimize antiretroviral distribution into viral sanctuaries/reservoirs, additional research is needed.

Metformin Suppresses Monocyte Immunometabolic Activation by SARS-CoV-2 Spike Protein Subunit 1.

A hallmark of COVID-19 is a hyperinflammatory state associated with severity. Monocytes undergo metabolic reprogramming and produce inflammatory cytokines when stimulated with SARS-CoV-2. We hypothesized that binding by the viral spike protein mediates this effect, and that drugs which regulate immunometabolism could inhibit the inflammatory response. Monocytes stimulated with recombinant SARS-CoV-2 spike protein subunit 1 showed a dose-dependent increase in glycolytic metabolism associated with production of pro-inflammatory cytokines. This response was dependent on hypoxia-inducible factor-1α, as chetomin inhibited glycolysis and cytokine production. Inhibition of glycolytic metabolism by 2-deoxyglucose (2-DG) or glucose deprivation also inhibited the glycolytic response, and 2-DG strongly suppressed cytokine production. Glucose-deprived monocytes rescued cytokine production by upregulating oxidative phosphorylation, an effect which was not present in 2-DG-treated monocytes due to the known effect of 2-DG on suppressing mitochondrial metabolism. Finally, pre-treatment of monocytes with metformin strongly suppressed spike protein-mediated cytokine production and metabolic reprogramming. Likewise, metformin pre-treatment blocked cytokine induction by SARS-CoV-2 strain WA1/2020 in direct infection experiments. In summary, the SARS-CoV-2 spike protein induces a pro-inflammatory immunometabolic response in monocytes that can be suppressed by metformin, and metformin likewise suppresses inflammatory responses to live SARS-CoV-2. This has potential implications for the treatment of hyperinflammation during COVID-19.

An evaluation of ledipasvir + sofosbuvir for the treatment of chronic hepatitis C infection.

Introduction: Hepatitis C (HCV) is viral disease with a global impact. Over the last 10 years, the treatment of this disease has evolved. Treatment guidelines have evolved to adopt new medications for HCV. These drugs have shown efficacy over 90% throughout the class as well as a better safety profile than the previous recommended pharmacotherapy. Dual-therapy DAAs emerged with FDA approval of Ledipasvir/Sofosbuvir (LDV/SOF) in 2014.Areas Covered: LDV/SOF is a dual-therapy option for chronic HCV patients (>6 months of infection) in select genotypes. This article reviews the studies relevant to the pharmacokinetic/pharmacodynamic properties of these drugs as well as its trials leading to approval.Expert opinion: LDV/SOF is included in the AASLD/IDSA guidelines for the treatment of HCV genotypes 1a and 1b with or without cirrhosis and genotype 4 without cirrhosis with an evidence and recommendation rating of IA. Genotype 4 with cirrhosis and genotypes 5 and 6 carry a Class IIa level B recommendation. The combination is not FDA approved for genotypes 2 and 3. Single-pill regimens, like LDV/SOF, are important to maintain the quality of life of children and other special populations infected with HCV by shortening treatment regimens, avoiding complex pill regimens, and eliminating injection therapies.

A Narrative Systematic Literature Review: A Focus on Qualitative Studies on HIV and Medication-Assisted Therapy in the United States.

Over the last two decades, the United States (U.S.) has experienced an opioid crisis that has had a significant negative societal and economic impact. Due to the high utilization of opioids in Persons Living with HIV and AIDS (PLWHA), there is a need for a qualitative literature review that presents opioid-use related problems in this population. This study aims to present and identify a thematic overview of the qualitative manuscripts on PLWHA who take opioid medications in the U.S., with a focus on perceptions surrounding medication assisted therapy. The systematic literature search was performed in December 2019. Four databases were searched: PubMed/MEDLINE, Scopus, Web of Science, and Cumulative Index to Nursing & Allied Health Literature (CINAHL). A total of 5348 results were exported from databases into EndNote x9, and duplicates were removed for a total of 3039 unique abstracts to screen. The records were imported into Rayyan, an online platform designed to expedite the screening process. Three authors screened titles and abstracts and determined 19 articles that would be screened in full text. On 9 April 2020, it was determined that eight articles would be included for review. The analysis of the eight manuscripts that fit the inclusion and exclusion criteria revealed barriers and facilitators to medication assisted therapy (MAT) in PLWHA. This review communicates or describes the story of PLWHA who might have delayed access to HIV healthcare providers and the commencement of antiretroviral therapy. In the literature, several studies have focused on the role of physicians in prescribing and addressing the medication regimens but none of the studies examined the role of pharmacists in access to care in this population. Therefore, further research is needed for a better understanding of the social aspects of taking opioid medications in PLWHA and the role of pharmacists within the continuum of care.

Suppression of HIV-1 Viral Replication by Inhibiting Drug Efflux Transporters in Activated Macrophages.

BACKGROUND: Ethanol has been shown to increase oxidative stress, drug efflux transporter expression, and promote HIV progression. Macrophages, which express drug efflux transporters, serve as an essential sanctuary site for HIV. The antiretroviral drug lopinavir, a protease inhibitor, is a substrate of the drug efflux transporters P-glycoprotein and multidrug resistance-associated protein 1. The NF-κB signaling pathway is associated with inflammation and drug efflux transporter expression. OBJECTIVE: To examine the effects of ethanol on drug efflux transporters and HIV replication of macrophages and develop strategies to increase the efficacy of the protease inhibitor. METHODS: The expression of PGP and MRP1 was examined with western blot. The NF- κB inhibition was assessed with nuclear western blot. LC-MS/MS and p24 ELISA were used to assess intracellular LPV and viral replication. RESULTS: Ethanol at 40mM slightly increased drug efflux transporter PGP and MRP1 expression in activated macrophages. IKK-16, an NF- κB inhibitor, counteracted the increased transporter expression caused by ethanol exposure. MK571, an MRP1 inhibitor, and IKK-16 significantly increased intracellular LPV concentration with or without ethanol treatment. MK571 significantly increased LPV efficacy in suppressing viral replication with or without ethanol treatment. A decreasing trend and a significant decrease were observed with IKK-16+LPV treatment compared with LPV alone in the no ethanol treatment and ethanol treatment groups, respectively. CONCLUSION: In activated macrophages, inhibiting drug efflux transporter MRP1 activity and reducing its expression may represent a promising approach to suppress viral replication by increasing intracellular antiretroviral concentrations. However, different strategies may be required for ethanolrelated vs. untreated groups.

Opioid Use Disorders in People Living with HIV/AIDS: A Review of Implications for Patient Outcomes, Drug Interactions, and Neurocognitive Disorders.

The opioid epidemic has had a significant, negative impact in the United States, and people living with HIV/AIDS (PLWHA) represent a vulnerable sub-population that is at risk for negative sequela from prolonged opioid use or opioid use disorder (OUD). PLWHA are known to suffer from HIV-related pain and are commonly treated with opioids, leading to subsequent addictive disorders. PLWHA and OUD are at an increased risk for attrition in the HIV care continuum, including suboptimal HIV laboratory testing, delayed entry into HIV care, and initiation or adherence to antiretroviral therapy. Barriers to OUD treatment, such as medication-assisted therapy, are also apparent for PLWHA with OUD, particularly those living in rural areas. Additionally, PLWHA and OUD are at a high risk for serious drug-drug interactions through antiretroviral-opioid metabolic pathway-related inhibition/induction, or via the human ether-a-go-go-related gene potassium ion channel pathways. HIV-associated neurocognitive disorders can also be potentiated by the off-target inflammatory effects of opioid use. PLWHA and OUD might require more intensive, individualized protocols to sustain treatment for the underlying opioid addiction, as well as to provide proactive social support to aid in improving patient outcomes. Advancements in the understanding and management of PLWHA and OUD are needed to improve patient care. This review describes the effects of prescription and non-prescription opioid use in PLWHA.

Extracellular Vesicles in Viral Replication and Pathogenesis and Their Potential Role in Therapeutic Intervention.

Extracellular vesicles (EVs) have shown their potential as a carrier of molecular information, and they have been involved in physiological functions and diseases caused by viral infections. Virus-infected cells secrete various lipid-bound vesicles, including endosome pathway-derived exosomes and microvesicles/microparticles that are released from the plasma membrane. They are released via a direct outward budding and fission of plasma membrane blebs into the extracellular space to either facilitate virus propagation or regulate the immune responses. Moreover, EVs generated by virus-infected cells can incorporate virulence factors including viral protein and viral genetic material, and thus can resemble noninfectious viruses. Interactions of EVs with recipient cells have been shown to activate signaling pathways that may contribute to a sustained cellular response towards viral infections. EVs, by utilizing a complex set of cargos, can play a regulatory role in viral infection, both by facilitating and suppressing the infection. EV-based antiviral and antiretroviral drug delivery approaches provide an opportunity for targeted drug delivery. In this review, we summarize the literature on EVs, their associated involvement in transmission in viral infections, and potential therapeutic implications.

An update on drug-drug interactions between antiretroviral therapies and drugs of abuse in HIV systems.

INTRODUCTION: While considerable progress has been made in the fight against HIV/AIDS, to date there has not been a cure, and millions of people around the world are currently living with HIV/AIDS. People living with HIV/AIDS have substance abuse disorders at higher rates than non-infected individuals, which puts them at an increased risk of drug-drug interactions. AREAS COVERED: Potential drug-drug interactions are reviewed for a variety of potential drugs of abuse, both licit and illicit. These drugs include alcohol, cigarettes or other nicotine delivery systems, methamphetamine, cocaine, opioids, and marijuana. Potential interactions include decreased adherence, modulation of drug transporters, or modulation of metabolic enzymes. We also review the relative incidence of the use of these drugs of abuse in People living with HIV/AIDS. EXPERT OPINION: Despite considerable improvements in outcomes, disparities in outcomes between PLWHA who use drugs of abuse, vs those who do not still exist. It is of critical necessity to improve outcomes in these patients and to work with them to stop abusing drugs of abuse.

Differential packaging of inflammatory cytokines/ chemokines and oxidative stress modulators in U937 and U1 macrophages-derived extracellular vesicles upon exposure to tobacco constituents.

Smoking, which is highly prevalent in HIV-infected populations, has been shown to exacerbate HIV replication, in part via the cytochrome P450 (CYP)-induced oxidative stress pathway. Recently, we have shown that extracellular vesicles (EVs), derived from tobacco- and/or HIV-exposed macrophages, alter HIV replication in macrophages by cell-cell interactions. We hypothesize that cigarette smoke condensate (CSC) and/or HIV-exposed macrophage-derived EVs carry relatively high levels of pro-oxidant and pro-inflammatory cargos and/or low levels of antioxidant and anti-inflammatory cargos, which are key mediators for HIV pathogenesis. Therefore, in this study, we investigated differential packaging of pro- and anti-inflammatory cytokines/chemokines and pro- and anti-oxidant contents in EVs after CSC exposure to myeloid cells (uninfected U937 and HIV-infected U1 cells). Our results showed that relatively long to short exposures with CSC increased the expression of cytokines in EVs isolated from HIV-infected U1 macrophages. Importantly, pro-inflammatory cytokines, especially IL-6, were highly packaged in EVs isolated from HIV-infected U1 macrophages upon both long and short-term CSC exposures. In general, anti-inflammatory cytokines, particularly IL-10, had a lower packaging in EVs, while packaging of chemokines was mostly increased in EVs upon CSC exposure in both HIV-infected U1 and uninfected U937 macrophages. Moreover, we observed higher expression of CYPs (1A1 and 1B1) and lower expression of antioxidant enzymes (SOD-1 and catalase) in EVs from HIV-infected U1 macrophages than in uninfected U937 macrophages. Together, they are expected to increase oxidative stress factors in EVs derived from HIV-infected U1 cells. Taken together, our results suggest packaging of increased level of oxidative stress and inflammatory elements in the EVs upon exposure to tobacco constituents and/or HIV to myeloid cells, which would ultimately enhance HIV replication in macrophages via cell-cell interactions.

Evaluating emtricitabine + rilpivirine + tenofovir alafenamide in combination for the treatment of HIV-infection.

Introduction: Antiretroviral therapy (ART) is recommended for all people who are living with HIV to suppress viral load and to stop the progression and transmission of HIV-1. Fixed-dose combinations of antiretrovirals largely reduce pill burden.Areas covered: The authors first provide an overview of the use of non-nucleoside reverse transcriptase inhibitor (NNRTI) based therapy in HIV care. They then summarize the properties of each drug in the fixed-dose combination of tenofovir alafenamide/emtricitabine/rilpivirine/(TAF/FTC/RPV). The efficacy and safety of each component and the combination as a whole are reviewed: FTC is non-inferior to lamivudine (3TC) at assessed dosages; TAF was non-inferior to tenofovir disoproxil fumarate (TDF); the viral efficacy of RPV is non-inferior with EFV at the assessed dosage; TAF/FTC/RPV is non-inferior in efficacy but shows less of a decline in bone mineral density and renal function compared to TDF/FTC/RPV. Finally, adverse effects and drug-drug interaction data with FTC/RPV/TAF are discussed.Expert opinion: TAF/FTC/RPV can be used as an initial regimen for people living with HIV whose HIV RNA <100,000 copies/ml and CD4 cell count > 200 cells/mm3 when INSTI-based regimens are not a treatment option. Future antiretroviral therapy development may focus on dual therapy-based regimens containing RPV, particularly as long-acting formulations.

Proteomic Profiling of Exosomes Derived from Plasma of HIV-Infected Alcohol Drinkers and Cigarette Smokers.

Abuse of alcohol and tobacco could exacerbate HIV pathogenesis by transferring materials through exosomes (small nanovesicles). Exosomes present a stable and accessible source of information concerning the health and/or disease status of patients, which can provide diagnostic and prognostic biomarkers for myriad conditions. Therefore, we aimed to study the specific exosomal proteins that are altered in both HIV-infected subjects and alcohol/tobacco users. Exosomes were isolated from plasma of the following subjects: a) HIV-negative subjects (healthy), b) HIV-positive subjects (HIV), c) HIV-negative alcohol drinkers (drinkers), d) HIV-negative tobacco smokers (smokers), e) HIV-positive drinkers (HIV + drinkers), and f) HIV-positive smokers (HIV + smokers). Quantitative proteomic profiling was then performed from these exosomes. Sixteen proteins were significantly altered in the HIV group, ten in drinkers, four in HIV + drinkers, and fifteen in smokers compared to healthy subjects. Only one protein, fibulin-1 (FBLN1), was significantly altered in HIV + smokers. Interestingly, hemopexin was not significantly altered in drinkers or HIV patients but was significantly altered in HIV + drinkers. Further, our study is the first to show properdin expression in plasma exosomes, which was decreased in HIV + smokers and HIV + drinkers compared to HIV patients. The present findings suggest that hemopexin and properdin show potential as markers for physiological effects that may arise in HIV-infected individuals who abuse alcohol and tobacco. Graphical abstract This study presents a proteomic analysis of plasma-derived exosomes from HIV-infected alcohol drinkers and smokers. Among the proteins altered due to drug-abuse, hemopexin and properdin were of highest significance. These proteins can be potential biomarkers for co-morbid conditions associated with drug abuse in HIV-patients.

Nanotechnology approaches for delivery of cytochrome P450 substrates in HIV treatment.

Introduction: Antiretroviral therapy (ART) has led to a significant reduction in HIV-1 morbidity and mortality. Many antiretroviral drugs (ARVs) are metabolized by cytochrome P450 (CYP) pathway, and the majority of these drugs are also either CYP inhibitors or inducers and few possess both activities. These CYP substrates, when used for HIV treatment in the conventional dosage form, have limitations such as low systemic bioavailability, potential drug-drug interactions, and short half-lives. Thus, an alternative mode of delivery is needed in contrast to conventional ARVs. Areas covered: In this review, we summarized the limitations of conventional ARVs in HIV treatment, especially for ARVs which are CYP substrates. We also discussed the preclinical and clinical studies using the nanotechnology strategy to overcome the limitations of these CYP substrates. The preclinical studies and clinical studies published from 2000 to February 2019 were discussed. Expert opinion: Since preclinical and clinical studies for prevention and treatment of HIV using nanotechnology approaches have shown considerable promise in recent years, nanotechnology could become an alternative strategy for daily oral therapy as a future treatment.

Azithromycin Polarizes Macrophages to an M2 Phenotype via Inhibition of the STAT1 and NF-κB Signaling Pathways.

Azithromycin is effective at controlling exaggerated inflammation and slowing the long-term decline of lung function in patients with cystic fibrosis. We previously demonstrated that the drug shifts macrophage polarization toward an alternative, anti-inflammatory phenotype. In this study we investigated the immunomodulatory mechanism of azithromycin through its alteration of signaling via the NF-κB and STAT1 pathways. J774 murine macrophages were plated, polarized (with IFN-γ, IL-4/-13, or with azithromycin plus IFN-γ) and stimulated with LPS. The effect of azithromycin on NF-κB and STAT1 signaling mediators was assessed by Western blot, homogeneous time-resolved fluorescence assay, nuclear translocation assay, and immunofluorescence. The drug's effect on gene and protein expression of arginase was evaluated as a marker of alternative macrophage activation. Azithromycin blocked NF-κB activation by decreasing p65 nuclear translocation, although blunting the degradation of IκBα was due, at least in part, to a decrease in IKKß kinase activity. A direct correlation was observed between increasing azithromycin concentrations and increased IKKß protein expression. Moreover, incubation with the IKKß inhibitor IKK16 decreased arginase expression and activity in azithromycin-treated cells but not in cells treated with IL-4 and IL-13. Importantly, azithromycin treatment also decreased STAT1 phosphorylation in a concentration-dependent manner, an effect that was reversed with IKK16 treatment. We conclude that azithromycin anti-inflammatory mechanisms involve inhibition of the STAT1 and NF-κB signaling pathways through the drug's effect on p65 nuclear translocation and IKKß.