Pharmacokinetics of Antiretroviral Drugs in Older People Living with HIV: A Systematic Review.

BACKGROUND AND OBJECTIVE: The life expectancy of people living with HIV (PLWHIV) has significantly improved in recent decades, mostly due to antiretroviral (ARV) therapy. Aging can affect the pharmacokinetics of drugs and, as a consequence, increase the risk of drug interactions and toxicity that may impact treatment. The aim of this study was to carry out a systematic review of the literature on the effect of aging on ARV pharmacokinetics. METHODS: Searches were performed in the BVS, EMBASE and PUBMED databases until November 2022. All studies available in English, Spanish and Portuguese investigating the pharmacokinetics of ARV approved by the US Food and Drug Administration (FDA) from 2005 to 2020 were selected. Peer-reviewed publications were included if they met all criteria: adults (≥ 18 years of age) living with or without HIV; report any pharmacokinetic parameter or plasma concentration of at least one of the following ARVs: tenofovir alafenamide fumarate (TAF); doravirine (DOR), rilpivirine (RIL) and etravirine (ETR); darunavir (DRV), tipranavir (TPV) and fostemsavir (FTR); dolutegravir (DTG), raltegravir (RAL), bictegravir (BIC) and elvitegravir (EVG); maraviroc (MVC); ibalizumab (IBA); cobicistat (COBI). Pharmacokinetic parameters were reported stratified per age group: young adults (aged 18-49 years) or older (age ≥ 50 years) and all studies were evaluated for quality. The review protocol was registered in the PROSPERO database (registration number CRD42021236432). RESULTS: Among 97 studies included, 20 reported pharmacokinetic evaluation in older individuals (age ≥ 50 years). Twenty five percent of the articles were phase I randomized clinical trials with HIV-negative participants and non-compartmental pharmacokinetic analysis presenting the parameters area under the curve (AUC) and peak drug concentration (Cmax). Seven age-stratified studies evaluated BIC, ETR, DRV, DTG, DOR and RAL. We found publications with discordant results for ETR and DTG pharmacokinetics in different age groups. DRV exposure was highly variable but modestly increased in aging PLWHIV. In contrast, no influence of age on BIC, DOR and RAL exposure was observed. A variability in pharmacokinetic parameters could be observed for the other ARVs (TAF and MVC) in different age groups. CONCLUSION: Exposure to DRV increases modestly with age, while exposure to BIC, DOR and RAL appears to be unaffected by age. As the available evidence to confirm a potential effect of aging on ARV pharmacokinetics is limited, further studies are necessary.

4-Oxoquinoline Derivatives as Antivirals: A Ten Years Overview.

4-Oxoquinoline derivatives constitute an important family of biologically important substances, associated with different bioactivities, which can be synthesized by different synthetic methods, allowing the design and preparation of libraries of substances with specific structural variations capable of modulating their pharmacological action. Over the last years, these substances have been extensively explored by the scientific community in efforts to develop new biologically active agents, with greater efficiency for the treatment of a variety of diseases. Viral infections have been one of the targets of these studies, although to a lesser extent than other diseases such as cancer and bacterial infections. Nevertheless, the literature provides examples that corroborate with the fact that these substances may act on different pharmacological targets in different viral pathogens. This review provides a compilation of some of the major studies published in recent years showing the discovery and/or development of new antiviral oxoquinoline agents, highlighting, whenever possible, their mechanisms of action.

Modulation of CD4 T cell function via CD6-targeting.

In recent years molecules involved on the immune synapse became successful targets for therapeutic immune modulation. CD6 has been extensively studied, yet, results regarding CD6 biology have been controversial, in spite of the ubiquitous presence of this molecule on virtually all CD4 T cells. We investigated the outcome of murine and human antibodies targeting CD6 domain 1. We found that CD6-targeting had a major impact on the functional specialization of CD4 cells, both human and murine. Differentiation of CD4 T cells towards a Foxp3+ Treg fate was prevented with increasing doses of anti-CD6, while Th1 polarization was favoured. No impact was observed on Th2 or Th17 specialization. These in vitro results provided an explanation for the dose-dependent outcome of in vivo anti-CD6 administration where the anti-inflammatory action is lost at the highest doses. Our data show that therapeutic targeting of the immune synapse may lead to paradoxical dose-dependent effects due to modification of T cell fate.

Design, Synthesis and Antileishmanial Activity of Naphthotriazolyl-4- Oxoquinolines.

BACKGROUND: Leishmaniasis is a neglected public health problem caused by several protozoanspecies of the genus Leishmania. The therapeutic arsenal for treating leishmaniasis is quite limited, raising concerns about the occurrence of resistant strains. Furthermore, most of these drugs were developed more than 70 years ago and suffer from poor efficacy and safety and are not well adapted to the needs of patients. Therefore, research on novel natural or synthetic compounds with antiparasitic activity is urgently needed. In this paper, we evaluated the effect and the mechanism of action of naphthotriazolyl-4-oxoquinolines on promastigotes and intracellular amastigotes of Leishmania amazonensis. MATERIALS AND METHODS: The naphthotriazolyl-4-oxoquinoline derivatives were obtained in good to moderate yields via the [3+2] cycloaddition reaction between 1,4-naphtoquinone and azido-4- oxoquinoline derivatives. HMPA at 100°C was established as the best solvent and temperature condition for this reaction. The structures of the compounds were confirmed by spectral analyses (infrared spectroscopy, one- and two-dimensional ¹H and ¹³C NMR spectroscopy, and high-resolution mass spectrometry). The compounds exhibited promising activities with IC50 values ranging from 0.7 to 2.0 µM against intracellular amastigotes of Leishmania amazonensis. The most selective compound was the Npentyl- substituted derivative, which showed a Selectivity Index (SI) of 8.6, making it less toxic than pentamidine (SI 4.5). RESULTS: Our results demonstrated that all compounds, except the N-propyl-substituted derivative, induce ROS production by parasites early in the culture. As a proof of concept, we demonstrated that the most selective compound was able to interfere with sterol biosynthesis in L. amazonensis. CONCLUSION: The naphthotriazolyl-4-oxoquinoline derivatives were obtained in good to moderate yields. These conjugates have potent in vitro antileishmanial activity involving at least two different mechanisms of action, making them promising lead compounds for the development of new therapeutic alternatives for leishmaniasis.

A Novel Naphthotriazolyl-4-oxoquinoline Derivative that Selectively Controls Breast Cancer Cells Survival Through the Induction of Apoptosis.

BACKGROUND: Breast cancer is a major cause of death among women worldwide. Treatment for breast cancer involves the surgical removal of cancer tissue, followed by chemotherapy. Although the treatment is efficient, especially when the cancer is detected early, recurrence is common and is often resistant to the previous treatment. Therefore, a constant search for efficient and novel drugs for the treatment of breast cancer is mandatory. Recently, triazole derivatives have shown promising effects against different types of cancer, revealing these molecules as putative anticancer drugs. EXPERIMENTAL: We have synthesized a series of naphthotriazolyl-4-oxoquinoline derivatives and tested their activity against a human breast cancer cell line. Among the compounds tested, we identified a molecule that killed the human breast cancer cell line MCF-7 with minimal effects on its noncancer counterpart, MCF10A. This effect was seen after 24 hours of treatment and persisted for additional 24 hours after treatment withdrawal. After 1 hour of treatment, the compound, here named 12c, promoted a decrease in cell glucose consumption and lactate production. Moreover, the cells treated with 12c for 1 hour showed diminished intracellular ATP levels with unaltered mitochondrial potential and increased reactive oxygen species production. Additionally, apoptosis was triggered after treatment with the drug for 1 hour. All of these effects are only observed with MCF-7 cells, and not MCF10A. These data show that 12c has selective activity against breast cancer cells and is a potential candidate for a novel anticancer drug. RESULTS AND CONCLUSION: The naphthotriazolyl-4-oxoquinoline derivatives were obtained in good to moderate yields, and one of them, 12c, exhibited strong and selective antitumor properties. The antitumor mechanism involves inhibition of glycolysis, diminished intracellular ATP levels, induction of ROS production and triggering of apoptosis. These effects are all selective for cancer cells, since noncancer cells are unaffected, and these effects can only be attributed to the whole molecule, as different pharmacophoric groups did not reproduce these effects.

Route of Antigen Presentation Can Determine the Selection of Foxp3-Dependent or Foxp3-Independent Dominant Immune Tolerance.

It has been shown that dominant tolerance, namely in transplantation, requires Foxp3+ regulatory T cells. Although most tolerance-inducing regimens rely on regulatory T cells, we found that induction of tolerance to proteins in aluminum hydroxide can be achieved in Foxp3-deficient mice using nondepleting anti-CD4 Abs. This type of tolerance is Ag specific, and tolerant mice retain immune competence to respond to unrelated Ags. We demonstrated with chicken OVA-specific TCR-transgenic mice that the same tolerizing protocol (CD4 blockade) and the same target Ag (OVA) achieves Foxp3-dependent transplantation tolerance to OVA-expressing skin grafts, but Foxp3-independent tolerance when the Ag is provided as OVA-aluminum hydroxide. In the latter case, we found that tolerance induction triggered recessive mechanisms leading to elimination of effector cells and, simultaneously, a dominant mechanism associated with the emergence of an anergic and regulatory CTLA-4+IL-2lowFoxp3- T cell population, where the tolerance state is IL-10 dependent. Such Foxp3-independent mechanisms can improve the efficacy of tolerance-inducing protocols.

In vivo delivery of peptides and Toll-like receptor ligands by mannose-functionalized polymeric nanoparticles induces prophylactic and therapeutic anti-tumor immune responses in a melanoma model.

We hypothesized that the co-entrapment of melanoma-associated antigens and the Toll-like receptor (TLR) ligands Poly(I:C) and CpG, known to be Th1-immunopotentiators, in mannose-functionalized aliphatic polyester-based nanoparticles (NPs) could be targeted to mannose receptors on antigen-presenting cells and induce anti-tumor immune responses. High entrapment efficiencies of antigens and immunopotentiators in 150nm NPs were obtained. The co-entrapment of the model antigen ovalbumin and the TLR ligands was crucial to induce high IgG2c/IgG1 ratios and high levels of IFN-γ and IL-2. Mannose-functionalization of NPs potentiated the Th1 immune response. The nanoparticulate vaccines decreased the growth rate of murine B16F10 melanoma tumors in therapeutic and prophylatic settings. The combination of mannose-functionalized NPs containing MHC class I- or class II-restricted melanoma antigens and the TLR ligands induced the highest tumor growth delay. Overall, we demonstrate that the multifunctional properties of NPs in terms of targeting and antigen/adjuvant delivery have high cancer immunotherapeutic potential.

Development of functionalized nanoparticles for vaccine delivery to dendritic cells: a mechanistic approach.

AIM: Produce biodegradable nanoparticles to target antigen-presenting cells (APCs) and evaluate their potential to be used as a vaccine delivery system. MATERIALS & METHODS: Untargeted PEGylated poly(d,l-lactic-co-glycolide)-based nanoparticles and mannose-grafted nanoparticles were formulated and physicochemically characterized. Immortalized and primary APCs were used to study nanoparticle internalization patterns. The endocytic pathways and intracellular trafficking followed by nanoparticles were also investigated. RESULTS & DISCUSSION: Nanoparticles displayed mannose residues available for binding at the nanoparticle surface. Different nanoparticle internalization patterns by immortalized and primary APCs were verified. Macropinocytosis, clathrin-mediated endocytosis, caveolin- and lipid raft-dependent endocytosis are involved in nanoparticles internalization. Nanoparticles demonstrate both endolysosomal and cytosolic localizations and a tendency to accumulate nearby the endoplasmic reticulum. CONCLUSION: The developed nanoparticles might drive antigens to be presented through MHC class I and II molecules to both CD8(+) and CD4(+) T cells, favoring a complete and coordinated immune response.

The fate of CD4+ T cells under tolerance-inducing stimulation: a modeling perspective.

Non-depleting anti-CD4 monoclonal antibodies (MAbs) induce long-term dominant tolerance mediated by regulatory T cells in several animal models of transplantation, allergy and autoimmunity. However, despite many studies on tolerance induction following CD4 blockade, the consequences of this intervention on T-cell kinetics are still unknown. Mathematical models have been useful to understand lymphocyte dynamics, estimating rates of proliferation and cell death following an intervention. Using the same strategy, we found that CD4(+) T cells activated in vitro in the presence of non-depleting anti-CD4 MAbs are prevented from undergoing optimal proliferation and show a higher frequency of apoptosis. Although the changes are small, during the course of a proliferative response, they lead to very distinct final levels of cell numbers. The importance of these mechanisms, predicted by the mathematical model, was validated by showing that lck-driven Bcl-x(L) transgenic mice, bearing T cells resistant to apoptosis, fail to become tolerant to skin grafts following CD4-blockade. Our data show that, in addition to induction of regulatory T cells, CD4 blockade has a marked effect in the effector T-cell pool by the combined action of hindering proliferation while favoring apoptosis. It is, therefore, the combination of all those mechanisms that leads to stable tolerance.

Adjuvant facilitates tolerance induction to factor VIII in hemophilic mice through a Foxp3-independent mechanism that relies on IL-10.

Current treatment of hemophilia consists of the administration of recombinant clotting factors, such as factor VIII (FVIII). However, patients with severe hemophilia can mount immune responses targeting therapeutically administered FVIII through inhibitory immunoglobulins that limit treatment efficacy. Induction of immune tolerance to FVIII in hemophilia has been extensively studied but remains an unmet need. We found that nondepleting anti-CD4 monoclonal antibodies (mAbs) are effective in inducing long-term tolerance to FVIII in different strains of hemophilic mice. Tolerance induction was facilitated when anti-CD4 mAbs were administered together with FVIII adsorbed in an adjuvant (alum). The observed state of tolerance was antigen specific, with mice remaining immune competent to respond to different antigens. Importantly, we found that following immunization with FVIII, the primed cells remained susceptible to tolerance induction. Studies with Foxp3-deficient and interleukin 10 (IL-10)-deficient mice demonstrated that the underlying tolerance mechanism is Foxp3 independent but requires IL-10. Our data show that an adjuvant, when administered together with a tolerizing agent such as nondepleting anti-CD4, can facilitate the induction of long-term tolerance to recombinant proteins, possibly not only in hemophilia but also in other diseases that are treated with potentially immunogenic therapeutics.

T cell apoptosis and induction of Foxp3+ regulatory T cells underlie the therapeutic efficacy of CD4 blockade in experimental autoimmune encephalomyelitis.

The pathogenesis of multiple sclerosis requires the participation of effector neuroantigen-specific T cells. Thus, T cell targeting has been proposed as a promising therapeutic strategy. However, the mechanism underlying effective disease prevention following T cell targeting remains incompletely known. We found, using several TCR-transgenic strains, that CD4 blockade is effective in preventing experimental autoimmune encephalopathy and in treating mice after the disease onset. The mechanism does not rely on direct T cell depletion, but the anti-CD4 mAb prevents the proliferation of naive neuroantigen-specific T cells, as well as acquisition of effector Th1 and Th17 phenotypes. Simultaneously, the mAb favors peripheral conversion of Foxp3(+) regulatory T cells. Pre-existing effector cells, or neuroantigen-specific cells that undergo cell division despite the presence of anti-CD4, are committed to apoptosis. Therefore, protection from experimental autoimmune encephalopathy relies on a combination of dominant mechanisms grounded on regulatory T cell induction and recessive mechanisms based on apoptosis of neuropathogenic cells. We anticipate that the same mechanisms may be implicated in other T cell-mediated autoimmune diseases that can be treated or prevented with Abs targeting T cell molecules, such as CD4 or CD3.

Regulation of the germinal center reaction by Foxp3+ follicular regulatory T cells.

Follicular helper T (T(FH)) cells participate in humoral responses providing selection signals to germinal center B cells. Recently, expression of CXCR5, PD-1, and the transcription factor Bcl-6 has allowed the identification of T(FH) cells. We found that a proportion of follicular T cells, with phenotypic characteristics of T(FH) cells and expressing Foxp3, are recruited during the course of a germinal center (GC) reaction. These Foxp3(+) cells derive from natural regulatory T cells. To establish the in vivo physiologic importance of Foxp3(+) follicular T cells, we used CXCR5-deficient Foxp3(+) cells, which do not have access to the follicular region. Adoptive cell transfers of CXCR5-deficient Foxp3(+) cells have shown that Foxp3(+) follicular T cells are important regulators of the GC reaction following immunization with a thymus-dependent Ag. Our in vivo data show that Foxp3(+) follicular T cells can limit the magnitude of the GC reaction and also the amount of secreted Ag-specific IgM, IgG1, IgG2b, and IgA. Therefore, Foxp3(+) follicular regulatory T cells appear to combine characteristics of T(FH) and regulatory T cells for the control of humoral immune responses.

Sub-optimal CD4+ T-cell activation triggers autonomous TGF-ß-dependent conversion to Foxp3+ regulatory T cells.

Classical in vitro Treg conversion assays, which rely on optimal T-cell activation in the presence of exogenous TGF-ß, induce Foxp3 expression at a frequency far above that which is observed in vivo in Treg-dependent models of oral or transplantation tolerance. We have found that suboptimal murine T-cell activation in vitro results in induction of Foxp3 expression, in the absence of exogenous TGF-ß, at a frequency similar to that which we found in vivo upon anti-CD4-induced transplantation tolerance. We show that TCR triggering with either low-dose anti-CD3 or low-dose agonist peptide, as well as down-modulation of the TCR signal with non-depleting anti-CD4, promotes TGF-ß production by T cells, an event that precedes Foxp3 expression and is Foxp3 independent. These findings support the view that sub-immunogenic regimens lead to dominant tolerance as a result of T-cell intrinsic properties.

Modulation of IL-17 and Foxp3 expression in the prevention of autoimmune arthritis in mice.

BACKGROUND: Rheumatoid Arthritis (RA) is a chronic immune mediated disease associated with deregulation of many cell types. It has been reported that different T cell subsets have opposite effects in disease pathogenesis, in particular Th17 and Treg cells. METHODOLOGY AND FINDINGS: We investigated whether non-depleting anti-CD4 monoclonal antibodies, which have been reported as pro-tolerogenic, can lead to protection from chronic autoimmune arthritis in SKG mice--a recently described animal model of RA--by influencing the Th17/Treg balance. We found that non-depleting anti-CD4 prevented the onset of chronic autoimmune arthritis in SKG mice. Moreover, treated mice were protected from the induction of arthritis up to 60 days following anti-CD4 treatment, while remaining able to mount CD4-dependent immune responses to unrelated antigens. The antibody treatment also prevented disease progression in arthritic mice, although without leading to remission. Protection from arthritis was associated with an increased ratio of Foxp3, and decreased IL-17 producing T cells in the synovia. In vitro assays under Th17-polarizing conditions showed CD4-blockade prevents Th17 polarization, while favoring Foxp3 induction. CONCLUSIONS: Non-depleting anti-CD4 can therefore induce long-term protection from chronic autoimmune arthritis in SKG mice through reciprocal changes in the frequency of Treg and Th17 cells in peripheral tissues, thus shifting the balance towards immune tolerance.