DISCUSSION
LATE EFFECTS
The BAF complex is an important molecular regulator of HIV-1 latency and offers a new yet clinically unstudied pharmacological target for latency reversal. In this randomized controlled trial, we demonstrated that BAF complex inhibition by pyrimethamine induces cellular HIV-1 transcription and reversal of latency in cART-suppressed PLWH. A comparable effect on the reservoir by pyrimethamine was also observed when VPA was added as partner LRA. Thus, the combination treatment failed to show synergistic or additive effects. Furthermore, interventional treatment adjustments occurred most frequently in PLWH randomized to the combinatorial arm, although the number and grade of the reported AE were in line with the ones observed in the individual monotherapy arms. We cannot exclude the possibility of physician- or participant-directed biases in the combinatorial arm where both the treating physician and the participant were aware of the higher drug burden. However, we believe that this trial, as the first randomized controlled clinical study on combinations of LRA, underlines that testing combinatorial LRA approaches is feasible. It also highlights the need to monitor for drug tolerability and provides a safety benchmark for future studies combining LRAs in the clinical HIV-1 cure trial field.The most important finding is the identification of pyrimethamine as an effective drug to reverse HIV-1 latency in PLWH. It acts through a novel mechanism of action that we unraveled first in in vitro and ex vivo studies and now successfully moved into clinical practice (With regard to VPA, we optimized the dosing and used an acid-resistant formulation for a more direct gastrointestinal absorption unlike the previous studies where underdosing or underexposure might have been the cause of the observed discrepant effects of VPA as LRA (Strategies that use combinatorial approaches of LRAs could result in higher efficacy in reactivating HIV-1 latency (To put our clinical study and the role of pyrimethamine as an LRA in the context of HIV-1 cure clinical studies, we compared the effect of pyrimethamine on HIV-1 latency with other LRAs tested in PLWH. First, the effect of pyrimethamine on HIV-1 transcription did not appear to augment with multiple dosing. This is in contrast to observations made in studies with other LRAs from the HDACi class and disulfiram, where the mean effect on cellular HIV-1 transcription increased with sequential dosing during treatment (In our study, pyrimethamine alone led to a twofold increase in levels of CA US HIV-1 RNA at the time point where all participants had received their allocated dose. While this effect is rather modest, it is comparable to recent reports using other classes of LRAs. The HDACi VPA did not result in an increase in CA US HIV-1 RNA in this trial, as opposed to what has been observed with other HDACi such as vorinostat, romidepsin, and panobinostat (The assay we developed and validated to measure pyrimethamine plasma levels turned out to be critical since, unexpectedly, the combination arm had lower pyrimethamine plasma levels with a concomitant lower induction of various BAF target genes. Given that both pyrimethamine and VPA are highly protein-bound, a plausible explanation for this effect could be attributed to plasma protein-protein competition dynamics, although further research needs to be conducted to support this statement. We identified a similar mechanism previously where VPA affected dolutegravir levels (This proof-of-concept study has strengths and limitations. The study’s main strength is the inclusion of a control arm and a combination arm that allowed us to assess the specific effect of pyrimethamine alone and in combination with an HDACi. Regarding limitations, although not exceeding predefined safety criteria, the combinatorial arm faced a disproportionate amount of treatment adjustments before the end of the intervention period. This influenced our ability to draw solid conclusions on potential late LRA effects combination therapy might have. However, the absence of an effect with VPA alone together with the absent synergism or additive effects in PLWH on the combination of drugs makes potential late effects less likely. Our sampling strategy for this pilot favored having many time points for the primary end point over blood quantity for in-depth analyses. Leukapheresis was practically not possible. This turned out to be a challenging strategy in some cases, especially in individuals with less blood CD4In summary, inhibition of the BAF complex by pyrimethamine resulted in modest but significant reversal of HIV-1 latency in PLWH immediately after the first dose and persisted during treatment course. Our data are supportive of the BAF inhibitor pyrimethamine as a novel drug option in the LRA arsenal, which has widespread use in the clinic, excellent safety profile, and favorable pharmacological features, including its excellent brain penetration that offers potential for penetration in HIV-1 reservoir sanctuary sites (