Introduction

Obesity is an increasing concern among people living with HIV (PWH). Several studies have reported an increasing prevalence of being overweight and obese in PWH, and have demonstrated that weight gain occurs in many PWH after initiating antiretroviral therapy (ART) [1–4]. Factors associated with weight gain in PWH include demographic factors (such as sex and race), HIV disease-related factors (such as disease stage and viral load), and ART-associated factors (specific antiretroviral drugs) [2, 3, 5–9]. These observations have led to several non-exclusive mechanistic hypotheses for ART-associated weight gain, including a mirroring of societal trends, a return-to-health effect of ART, improved tolerability of ART regimens, and off-target effects of antiretroviral drugs.

Among the antiretroviral drugs, the integrase strand transfer inhibitors (INSTIs) have specifically been associated with weight gain in studies of treatment-naïve PWH and in PWH switching to INSTI-based therapy [2, 7, 8, 10]. Whether this association is causative is unknown, and no mechanism to explain the association has been demonstrated. Clinical data on the effect of INSTIs on appetite has not been reported to date. An off-target effect of INSTIs has been hypothesized as a potential mechanism, based on data discussed in the European Products Assessment Report for the INSTI dolutegravir (DTG), which states that DTG can inhibit the binding of endogenous ligand to the human MC4R in vitro [11].

The regulation of body weight is a complex, integrated process linking peripheral signals of energy stores to homeostatic responses. Key centers in the brain provide overarching control of processes that regulate food intake (via satiation and appetite, and hedonic mechanisms) and energy metabolism [12]. The prevailing overview of the central control of food intake highlights the role of the hypothalamic melanocortin system, whereby peptides derived from the precursor protein proopiomelanocortin (POMC) inhibit feeding behavior via their agonistic action on central melanocortin-3 and -4 receptors (MC3R, MC4R) [13]. Conversely, blockade of MC4R by the agouti-related protein (AgRP) increases feeding [14], and complete loss of MC3R or MC4R in mice is associated with increased food intake and concomitant obesity [15, 16]. Mutations in MC4R that render the receptor less- or non-responsive to POMC-derived peptides are commonly associated with human obesity [17]. Thus, the notion that modulation of the melanocortin system can influence food intake and body weight homeostasis is supported by rodent and clinical evidence from both genetic and pharmacological paradigms.

In this study, we have investigated the potential for approved INSTIs to interfere with endogenous ligand binding to MC4R thereby potentially providing a plausible explanation for the clinical body weight gain noted. Specifically, cellular functional assays were performed to delineate potential antagonistic or agonist effects of the following INSTIs: bictegravir (BIC), dolutegravir (DTG), cabotegravir (CAB), raltegravir (RAL), and elvitegravir (EVG). Comparisons of antagonism or agonism in the cellular assays (IC₅₀ values) to clinical C_max at the recommended dosages are provided.

Materials and methods

Results

The objective of an initial screening study was to evaluate the potential for various INSTIs to interfere with ligand binding to the MC4R in an in vitro biochemical assay. Specifically, binding was calculated as % inhibition of the binding of a radioactively labeled ligand (NDP-α-MSH), specific for MC4R, at a fixed concentration of each INSTI (100 μM). Any result showing an inhibition greater than 50% was considered to represent a significant effect and followed-on by determination of IC₅₀ values using a multi-dose concentration curve. The initial biochemical binding assay at a single 100 μM concentration showed a range from 55% to 91% binding inhibition for the five INSTIs evaluated (S1 Table). Specifically, in this study DTG showed 91% inhibition at 100 μM compared to the previously reported inhibition binding value of 64% at 10 μM [11]. Given the significant binding for all compounds assayed (>50%), IC₅₀ values were determined, and ranged from 0.46 μM to 78 μM.

To further evaluate the biochemical findings in a more physiologically relevant system, we performed cell-based in vitro MC4R antagonism and agonism assays. Validity and specificity of the assays were established with the concurrent inclusion of MC4R agonist or antagonist positive controls (Table 1). Positive controls for antagonism included AgRP or HS024 or SHU9119, with potent effects at relatively low concentrations in alignment with historical data (per vendor confirmation). Positive controls for agonism included the endogenous agonist αMSH, MTII, and NDP-α-MSH which likewise showed selective results. Overall, concurrent positive controls for antagonism or agonism confirmed the assays performed as anticipated.

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Table 1. Summary of MC4R agonism and antagonism assay results and clinical C_max margins for INSTIs.

https://doi.org/10.1371/journal.pone.0229617.t001

INSTIs evaluated in the functional cellular in vitro assay for potential antagonistic or agonistic effects are listed in Table 1. Exposure margins comparing the in vitro assay to the human clinical exposures were determined by dividing the IC₅₀ values by the clinical C_max (unbound; protein-free) values based upon maximum approved clinical dose (S2 Table). It is noted that for BIC, DTG, and CAB, the highest concentration of INSTI evaluated was limited by compound solubility in the cellular buffer solution. However, overall, no agonistic effects were observed at any concentration of INSTIs evaluated; IC₅₀ values were not able to be determined up to the highest concentrations tested. As a result, exposure margins for MC4R agonism based upon IC₅₀ values were greater than 160- to 13,000-fold versus the clinical C_max (unbound) values. For the antagonism assay, IC₅₀ values were calculated for DTG (69.4 μM) or EVG (2.75 μM) resulting in C_max margins of 1120- or 120-fold, respectively. The remaining INSTIs evaluated, BIC, CAB, and RAL, were tested up to maximum dose concentrations of 100, 30, or 300 μM (based upon solubility for BIC or CAB), respectively, and IC₅₀ values were unable to be determined, resulting in C_max margins of greater than 2900-, 1200-, or 160-fold, respectively. Fig 1 further depicts the considerable margin between IC₅₀ calculations and C_max from the clinical dose for antagonistic effects.

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Fig 1. Depiction of MC4R antagonism assay results and clinical C_max margins for INSTIs.

Lines depict the substantial difference between the clinical dose C_max value (protein-free) and the reported EC₅₀ value in the MC4R antagonism assay for each INSTI evaluated.

https://doi.org/10.1371/journal.pone.0229617.g001

Discussion

Due to the potential off-target liability of MC4R for body weight gain cited for DTG, and observed body weight gain in patients receiving INSTIs, a number of INSTIs were evaluated for potential interaction with MC4R in cellular functional antagonist or agonist assays. Overall, our data demonstrate that all tested INSTIs have the ability to antagonize the MC4R receptor to some degree, but importantly, inhibition occurs only at drug concentrations substantially greater than the therapeutic plasma concentrations of each drug, ranging from 160-fold to >13,000-fold greater than unbound C_max. Furthermore, the relative IC₅₀ values for the INSTIs tested are inconsistent with their relative risk for clinical weight gain. For instance, DTG is associated with greater weight gain than EVG [18], however EVG was approximately 25-fold more potent in MC4R antagonism than DTG in our experiments. These findings suggest that it is unlikely that MC4R antagonism contributes to the weight gain associated with INSTI use.

There are several limitations to our findings. Our data are derived from an in vitro assay, and thus do not recreate the physiological milieu in which a potential interaction between INSTIs and MC4R would occur in vivo. For instance, because MC4R is localized primarily to the hypothalamus, the ratio of MC4R IC₅₀ to plasma free drug concentrations may not be physiologically relevant. However, studies using cerebrospinal fluid INSTI concentration as a surrogate for central nervous system penetration have documented CSF drug levels equal to or less than free plasma levels, making it unlikely that more free drug is available for interaction with MC4R in the central nervous system [19–21]. It is noted that following daily oral administration with BIC in repeat dose toxicity studies, no effects on body weight gain in mice (4 weeks), rats (26 weeks), or cynomolgous monkeys (39 weeks) were reported with AUC plasma exposure levels at 16- to 30-fold the BIC plasma exposure at the clinical dose of 50 mg in Biktarvy^®. While we do not know what fractional antagonism of MC4R is needed to induce weight gain, the dose-response relationships we have observed make it unlikely that the INSTI concentrations achieved in clinical practice would induce physiologically relevant antagonism of MC4R.

The regulation of body weight can also be modified via manipulation of other signaling pathways independent of MC4R. In nonclinical models, activation of the melanin-concentrating hormone receptor-1 [22], neuropeptide-Y receptors [23, 24] or ghrelin receptor [25] leads to weight gain. Whether INSTIs are able to activate these receptors is unknown. Furthermore, other MC4R-independent pathways are pharmacologically utilized to promote weight loss. One example is the glucagon-like peptide-1 (GLP-1) pathway, wherein synthetic peptide agonists of the GLP-1 receptor drive weight loss and are currently approved for the treatment of diabetes and obesity [26]. In nonclinical models, blockade or loss of GLP-1 receptors does not promote weight gain [27], however it remains a possibility that INSTIs may affect the GLP-1 receptor or its ligands in a clinical setting to the extent it may increase body weight. These hypotheses remain to be directly tested. In summary, our findings demonstrate a class-wide ability of INSTIs to bind and antagonize MC4R only at very high drug concentrations, making the INSTI-MC4R interaction implausible as a molecular mechanism to explain INSTI-associated weight gain. Further work is needed to understand potential explanations for the observed association between INSTI use and weight gain.

Supporting information