HIV Protease Inhibitors Lopinavir Research Articles

Lopinavir and ritonavir act synergistically with azoles against Candida auris in vitro and in a mouse model of disseminated candidiasis

The emergence of Candida auris has created a global health challenge. Azole antifungals are the most affected antifungal class because of the extraordinary capability of C. auris to develop resistance against these drugs. Here, we used a combinatorial therapeutic approach to sensitize C. auris to azole antifungals. We have demonstrated the capability of the HIV protease inhibitors lopinavir and ritonavir, at clinically relevant concentrations, to be used with azole antifungals to treat C. auris infections both in vitro and in vivo. Both lopinavir and ritonavir exhibited potent synergistic interactions with the azole antifungals, particularly with itraconazole against 24/24 (100%) and 31/34 (91%) of tested C. auris isolates, respectively. Furthermore, ritonavir significantly interfered with the fungal efflux pump, resulting in a significant increase in Nile red fluorescence by 44%. In a mouse model of C. auris systemic infection, ritonavir boosted the activity of lopinavir to work synergistically with fluconazole and itraconazole and significantly reduced the kidney fungal burden by a 1.2 log (∼94%) and 1.6 log (∼97%) CFU, respectively. Our results urge further comprehensive assessment of azoles and HIV protease inhibitors as a novel drug regimen for the treatment of serious invasive C. auris infections.

Current Potential Therapeutic Approaches against SARS-CoV-2: A Review.

The ongoing SARS-CoV-2 pandemic is a serious threat to public health worldwide and, to date, no effective treatment is available. Thus, we herein review the pharmaceutical approaches to SARS-CoV-2 infection treatment. Numerous candidate medicines that can prevent SARS-CoV-2 infection and replication have been proposed. These medicines include inhibitors of serine protease TMPRSS2 and angiotensin converting enzyme 2 (ACE2). The S protein of SARS-CoV-2 binds to the receptor in host cells. ACE2 inhibitors block TMPRSS2 and S protein priming, thus preventing SARS-CoV-2 entry to host cells. Moreover, antiviral medicines (including the nucleotide analogue remdesivir, the HIV protease inhibitors lopinavir and ritonavir, and wide-spectrum antiviral antibiotics arbidol and favipiravir) have been shown to reduce the dissemination of SARS-CoV-2 as well as morbidity and mortality associated with COVID-19.

The protease inhibitor lopinavir, boosted with ritonavir, as treatment for COVID-19: a rapid review.

The HIV protease inhibitor lopinavir, boosted with ritonavir, has been used off-label to treat COVID-19. We aimed to synthesize the clinical evidence for lopinavir/ritonavir as a treatment for COVID-19. We performed a rapid review by searching databases including PubMed, GoogleScholar, medRxiv, ClinicalTrials.gov and the Cochrane COVID-19 Study Register, for COVID-19 studies comparing outcomes between patients who did and did not receive lopinavir/ritonavir. The quality of evidence was assessed using the GRADE criteria. We identified five completed randomized controlled trials (RCTs) and 14 retrospective cohort studies. Two large RCTs of 5,040 and 2,771 hospitalized adults with COVID-19 found no evidence that lopinavir/ritonavir influenced the primary outcome of mortality, or secondary outcomes including progression to mechanical ventilation or time to discharge. Results remained similar in all sub-group analyses including by age, gender, baseline ventilation and time since symptom onset. The three smaller RCTs (n=86-199) also found no evidence of a benefit in the primary outcomes of time to clinical improvement or time to viral clearance. The 14 observational studies included between 50 and 415 participants, and were limited by a lack of adjustment for potential confounding variables. The majority of these studies found no evidence that lopinavir/ritonavir was associated with improved mortality or other clinical outcomes, although results regarding viral clearance were mixed. Good evidence from large clinical trials does not support using lopinavir/ritonavir to treat COVID-19 amongst hospitalized patients.

Altered Plasmodium falciparum Sensitivity to the Antiretroviral Protease Inhibitor Lopinavir Associated with Polymorphisms in pfmdr1.

The HIV protease inhibitor lopinavir inhibits Plasmodium falciparum aspartic proteases (plasmepsins) and parasite development, and children receiving lopinavir-ritonavir experienced fewer episodes of malaria than those receiving other antiretroviral regimens. Resistance to lopinavir was selected in vitro over ∼9 months, with ∼4-fold decreased sensitivity. Whole-genome sequencing of resistant parasites showed a mutation and increased copy number in pfmdr1 and a mutation in a protein of unknown function, but no polymorphisms in plasmepsin genes.

Lopinavir Resistance Classification with Imbalanced Data Using Probabilistic Neural Networks.

Resistance to antiretroviral drugs has been a major obstacle for long-lasting treatment of HIV-infected patients. The development of models to predict drug resistance is recognized as useful for helping the decision of the best therapy for each HIV+ individual. The aim of this study was to develop classifiers for predicting resistance to the HIV protease inhibitor lopinavir using a probabilistic neural network (PNN). The data were provided by the Molecular Virology Laboratory of the Health Sciences Center, Federal University of Rio de Janeiro (CCS-UFRJ/Brazil). Using bootstrap and stepwise techniques, ten features were selected by logistic regression (LR) to be used as inputs to the network. Bootstrap and cross-validation were used to define the smoothing parameter of the PNN networks. Four balanced models were designed and evaluated using a separate test set. The accuracies of the classifiers with the test set ranged from 0.89 to 0.94, and the area under the receiver operating characteristic (ROC) curve (AUC) ranged from 0.96 to 0.97. The sensitivity ranged from 0.94 to 1.00, and the specificity was between 0.88 and 0.92. Four classifiers showed performances very close to three existing expert-based interpretation systems, the HIVdb, the Rega and the ANRS algorithms, and to a k-Nearest Neighbor.

HIV protease inhibitor Lopinavir induces apoptosis of primary effusion lymphoma cells via suppression of NF-κB pathway

Primary effusion lymphoma (PEL) is a non-Hodgkin lymphoma that occurs predominantly in patients with advanced AIDS. In this study, we examined the effect of HIV protease inhibitors, Lopinavir (LPV), Ritonavir (RTV) and Darunavir (DRV) on PEL cell lines in vitro and in vivo. LPV and RTV, but not DRV induced caspase-dependent apoptosis and suppressed NF-κB activity by inhibiting IKK phosphorylation in PEL cells. In a PEL xenograft mouse model, LPV significantly inhibited the growth and invasion of PEL cells. These results suggest that LPV may have promise for the treatment and prevention of PEL, which occurs in HIV/AIDS patients.

The effect of antiretrovirals on Plasmodium falciparum liver stages

HIV and malaria overlap geographically, but the full impact of different antiretrovirals on malaria remains poorly understood. We examined the antimalarial activity of the HIV protease inhibitors lopinavir and saquinavir and the non-nucleoside reverse transcriptase inhibitor nevirapine on Plasmodium falciparum liver stages. Our results demonstrate that the HIV PI lopinavir inhibits liver stage parasites at clinically relevant concentrations, that is, at drug levels achieved in HIV-infected patients on standard dosing regimens. Because drugs that inhibit liver stages target parasites when they are present in lower numbers, these results might have implications for eradication efforts.

Lopinavir shows greater specificity than zinc finger ejecting compounds as a potential treatment for human papillomavirus-related lesions

Non-surgical, antiviral treatment options are desirable for HPV-related lesions within the genitourinary and upper digestive tract. We compared the toxicity of three zinc finger-ejecting (ZFE) compounds (4,4-dithiodimorpholine, azodicarbonamide, and diamide) to the HIV protease inhibitor lopinavir using HPV-positive SiHa, CaSki, HeLa, ME180, and HPV-negative C33A cervical carcinoma cell lines as well as primary human foreskin keratinocytes (PHFKs). Colorimetric growth assays revealed selective toxicity when treated with lopinavir. All carcinoma cell lines, except CaSki, were sensitive to 20 μM lopinavir whereas primary PHFKs were highly resistant. In contrast, 4,4-dithiodimorpholine was uniformly toxic to all cells tested while azodicarbonamide and diamide showed no effect at all. It is concluded that lopinavir may be an attractive candidate to treat pre-cancerous and cancerous HPV-positive lesions.

Niveau de preuve du suivi thérapeutique pharmacologique du lopinavir

The HIV protease inhibitor lopinavir presents a wide inter-individual variability related to liver and intestinal metabolism involving CYP3A. Published studies were analyzed to establish whether there is evidence that therapeutic drug monitoring of lopinavir could improve patient care. In naïve or pretreated HIV-infected patients, no relationship could be evidenced between virological efficacy and trough lopinavir concentration, most likely because concentrations are above inhibitory concentrations. Although data are limited, patients with elevated triglycerides and cholesterol had trough lopinavir concentrations >8000 ng/mL. These data suggest that the level of evidence of interest of lopinavir therapeutic drug monitoring is may be recommended in some situations such as children, pregnant women, pretreated patients if the number of mutations is <5, when coadministration with drug with metabolizing enzyme inducing properties is warranted and toxicity.

Age-Related Changes in Plasma Concentrations of the HIV Protease Inhibitor Lopinavir

The advent of highly active antiretroviral therapy in the treatment of HIV disease has substantially extended the lifespan of individuals infected with HIV resulting in a growing population of older HIV-infected individuals. The efficacy and safety of antiretroviral agents in the population are important concerns. There have been relatively few studies assessing antiretroviral pharmacokinetics in older patients. Thirty-seven subjects aged 18-30 years and 40 subjects aged 45-79 years, naive to antiretroviral therapy, received lopinavir/ritonavir (400/100) bid, emtricitibine 200 mg qd, and stavudine 40 mg bid. Trough lopinavir concentrations were available for 44 subjects, collected at 24, 36, and 96 weeks. At week 24, older age was associated with higher lopinavir trough concentrations, and a trend was observed toward older age being associated with higher lopinavir trough concentrations when all time points were evaluated. In the young cohort, among subjects with two or more measurements, there was a trend toward increasing intrasubject trough lopinavir concentrations over time. Using a nonlinear, mixed-effects population pharmacokinetic model, age was negatively associated with lopinavir clearance after adjusting for adherence. Adherence was assessed by patient self-reports; older patients missed fewer doses than younger patients (p = 0.02). No difference in grade 3-4 toxicities was observed between the two age group. Older patients have higher trough lopinavir concentrations and likely decreased lopinavir clearance. Age-related changes in the pharmacokinetics of antiretroviral drugs may be of increasing importance as the HIV-infected population ages and as older individuals comprise an increasing proportion of new diagnoses.

Lopinavir Up-Regulates Expression of the Antiviral Protein Ribonuclease L in Human Papillomavirus-Positive Cervical Carcinoma Cells

We have previously shown that the HIV protease inhibitor lopinavir has selective toxicity against human papillomavirus (HPV)-positive cervical carcinoma cells via an unknown mechanism. SiHa cervical carcinoma cells were stably transfected with the proteasome sensor vector pZsProSensor-1 to confirm lopinavir inhibits the proteasome in these cells. The Panorama Xpress profiler 725 antibody array was then used to analyse specific changes in protein expression in lopinavir-treated versus control untreated SiHa cells followed by PCR and western blotting. Colorimetric growth assays of lopinavir-treated E6/E7 immortalised versus control human keratinocytes were performed. Targeted small interfering RNA gene silencing followed by growth assay comparison of lopinavir-treated/untreated SiHa cells was also used. Lopinavir induced an increase in the fluorescence of pZsProSensor-1 transfected SiHa cells, indicative of proteasomal inhibition. Ribonuclease L (RNASEL) protein was shown to be up-regulated in lopinavir-treated SiHa cells, which was confirmed by PCR and western blot. Targeted silencing of RNASEL reduced the sensitivity of SiHa cells to lopinavir. Selective toxicity against E6/E7 immortalised keratinocytes versus control cells was also seen with lopinavir and was associated with up-regulated RNASEL expression. These data are consistent with the toxicity of lopinavir against HPV-positive cervical carcinoma cells being related to its ability to block viral proteasome activation and induce an up-regulation of the antiviral protein RNASEL. This is supported by the drug's selective toxicity and up-regulation of RNASEL in E6/E7 immortalised keratinocytes combined with the increased resistance to lopinavir observed in SiHa cells following silencing of RNASEL gene expression.

Enzymatic and structural analysis of the I47A mutation contributing to the reduced susceptibility to HIV protease inhibitor lopinavir

Lopinavir (LPV) is a second-generation HIV protease inhibitor (PI) designed to overcome resistance development in patients undergoing long-term antiviral therapy. The mutation of isoleucine at position 47 of the HIV protease (PR) to alanine is associated with a high level of resistance to LPV. In this study, we show that recombinant PR containing a single I47A substitution has the inhibition constant (K(i) ) value for lopinavir by two orders of magnitude higher than for the wild-type PR. The addition of the I47A substitution to the background of a multiply mutated PR species from an AIDS patient showed a three-order-of-magnitude increase in K(i) in vitro relative to the patient PR without the I47A mutation. The crystal structure of I47A PR in complex with LPV showed the loss of van der Waals interactions in the S2/S2' subsites. This is caused by the loss of three side-chain methyl groups due to the I47A substitution and by structural changes in the A47 main chain that lead to structural changes in the flap antiparallel beta-strand. Furthermore, we analyzed possible interaction of the I47A mutation with secondary mutations V32I and I54V. We show that both mutations in combination with I47A synergistically increase the relative resistance to LPV in vitro. The crystal structure of the I47A/I54V PR double mutant in complex with LPV shows that the I54V mutation leads to a compaction of the flap, and molecular modeling suggests that the introduction of the I54V mutation indirectly affects the strain of the bound inhibitor in the PR binding cleft.

Lopinavir impairs protein synthesis and induces eEF2 phosphorylation via the activation of AMP‐activated protein kinase

HIV anti-retroviral drugs decrease protein synthesis, although the underlying regulatory mechanisms of this process are not fully established. Therefore, we investigated the effects of the HIV protease inhibitor lopinavir (LPV) on protein metabolism. We also characterized the mechanisms that mediate the effects of this drug on elongation factor-2 (eEF2), a key component of the translational machinery. Treatment of C2C12 myocytes with LPV produced a dose-dependent inhibitory effect on protein synthesis. This effect was observed at 15 min and was maintained for at least 4 h. Mechanistically, LPV increased the phosphorylation of eEF2 and thereby decreased the activity of this protein. Increased phosphorylation of eEF2 was associated with increased activity of its upstream regulators AMP-activated protein kinase (AMPK) and eEF2 kinase (eEF2K). Both AMPK and eEF2K directly phosphorylated eEF2 in an in vitro kinase assay suggesting two distinct paths lead to eEF2 phosphorylation. To verify this connection, myocytes were treated with the AMPK inhibitor compound C. Compound C blocked eEF2K and eEF2 phosphorylation, demonstrating that LPV affects eEF2 activity via an AMPK-eEF2K dependent pathway. In contrast, incubation of myocytes with rottlerin suppressed eEF2K, but not eEF2 phosphorylation, suggesting that eEF2 can be regulated independent of eEF2K. Finally, LPV did not affect PP2A activity when either eEF2 or peptide was used as the substrate. Collectively, these results indicate that LPV decreases protein synthesis, at least in part, via inhibition of eEF2. This appears regulated by AMPK which can act directly on eEF2 or indirectly via the action of eEF2K.

Plasma Concentrations of the HIV-Protease Inhibitor Lopinavir are Suboptimal in Children Aged 2 Years and Below

Lopinavir/ritonavir (LPV/r) has been licensed for the treatment of HIV-infected children >6 months in the US and >2 years in the EU. Limited LPV paediatric pharmacokinetic data are available. We studied LPV pharmacokinetics to determine whether the recommended dose (230/57.5 mg/m2 twice daily) results in optimal LPV exposure in all age groups. Virological efficacy was a secondary objective. HIV-1-infected children who started treatment with LPV/r and two nucleoside reverse transcriptase inhibitors underwent a 12-h pharmacokinetic curve. LPV plasma concentrations were determined with a validated HPLC method with UV detection. If Cmin was <1.0 mg/l LPV/r dose was increased by 33%. Plasma trough levels were drawn subsequently. HIV-1 RNA was followed-up until week 48. A total of 23 children were included (seven girls; 16 boys), with a median (range) age of 5.6 (0.4-13.2) years. Mean (+/-SD) AUC0-12h, Cmax and Cmin of LPV were 75.3 (+/-33.7) mg/l.h, 9.33 (+/-3.27) mg/l and 3.68 (+/-2.48) mg/l, respectively, which is similar to previously published data. Interindividual variability was large. Cmin was inadequate in 7/23 children. Significantly more children <2 years had inadequate Cmin compared with children >2 years. Dose increase to +/-300/75 mg/m2 LPV/r led to Cmin >1.0 mg/l. The studied regimen provided excellent viral suppression for naive and pretreated patients. Mean LPV pharmacokinetic parameters in these HIV-infected children are similar to published data, but exposure is significantly reduced in children <2 years. Prospective pharmacokinetic studies using 300/75 mg/m2 LPV/r in this age population are urgently warranted.

Monitoring of lopinavir and ritonavir in peripheral blood mononuclear cells, plasma, and ultrafiltrate using a selective and highly sensitive LC/MS/MS assay

For the determination of the HIV protease inhibitors lopinavir and ritonavir in human plasma, plasma ultrafiltrate, and peripheral blood mononuclear cells (PBMCs) a highly sensitive and selective method has been developed, validated, and applied to samples of a healthy volunteer. BD Vacutainer ® CPT™ and Amicon Centriplus ® centrifugal filter devices were used for separation of PBMCs and for ultrafiltrate generation, respectively. After liquid/liquid-extraction extracts were chromatographed isocratically within 6 min on a Jupiter Proteo column. The drugs were quantified using 2H 5-saquinavir as internal standard and electrospray tandem mass spectrometry in the selected reaction monitoring mode. Limits of quantification for both analytes were 4.0 ng/mL in plasma, 0.2 ng/mL in ultrafiltrate, and 0.1 ng/cell pellet (∼3 × 10 6 cells) in PBMCs. The calibration ranges were linear over more than three logs with an over-all accuracy varying between 98.7% and 111.5% and an over-all precision ranging from 6.2% to 14.0% (SD batch-to-batch). After a regular oral dose of Kaletra ® (400 mg lopinavir, 100 mg ritonavir) analyte concentrations were detectable over a full dosing interval in plasma, ultrafiltrate, and PBMCs. The method is well suited for monitoring of free and total plasma, and intracellular lopinavir/ritonavir concentrations in samples from clinical trials.

Metabolism and Disposition of the HIV-1 Protease Inhibitor Lopinavir (ABT-378) Given in Combination with Ritonavir in Rats, Dogs, and Humans

The objective of this study was to examine the metabolism and disposition of the HIV protease inhibitor lopinavir in humans and animal models. The plasma protein binding of [14C]lopinavir was examined in vitro via equilibrium dialysis technique. The tissue distribution of radioactivity was examined in rats dosed with [14C]lopinavir in combination with ritonavir. The metabolism and disposition of [14C]lopinavir was examined in rats, dogs, and humans given alone (in rats only) or in combination with ritonavir. The plasma protein binding of lopinavir was high in all species (97.4-99.7% in human plasma), with a concentration-dependent decrease in binding. Radioactivity was extensively distributed into tissues, except brain, in rats. On oral dosing to rats, ritonavir was found to increase the exposure of lopinavir-derived radioactivity 13-fold. Radioactivity was primarily cleared via the hepato-biliary route in all species (>82% of radioactive dose excreted via fecal route), with urinary route of elimination being significant only in humans (10.4% of radioactive dose). Oxidative metabolites were the predominant components of excreted radioactivity. The predominant site of metabolism was found to be the carbon-4 of the cyclic urea moiety, with subsequent secondary metabolism occurring on the diphenyl core moiety. In all the three species examined, the primary component of plasma radioactivity was unchanged lopinavir (>88%) with small amounts of oxidative metabolites. Lopinavir was subject to extensive metabolism in vivo. Co-administered ritonavir markedly enhanced the pharmacokinetics of lopinavir-derived radioactivity in rats, probably due to inhibition of presystemic and systemic metabolism, leading to an increased exposure to this potent HIV protease inhibitor.

Novel Lopinavir Analogues Incorporating Non‐aromatic P‐1 Side Chains — Synthesis and Structure—Activity Relationships.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Novel lopinavir analogues incorporating non-Aromatic P-1 side chains—Synthesis and structure–Activity relationships

The HIV protease inhibitor Lopinavir has a pseudosymmetric core unit incorporating benzyl groups at both P-1, P-1′ positions. A series of analogues incorporating non-aromatic side chains at the P-1 position were synthesized and the structure–activity relationships explored.