Ritonavir Research Articles

Impact of Simulated Intestinal Fluids on Dissolution, Solution Chemistry, and Membrane Transport of Amorphous Multidrug Formulations.

The solution behavior and membrane transport of multidrug formulations were herein investigated in a biorelevant medium simulating fasted conditions. Amorphous multidrug formulations were prepared by the solvent evaporation method. Combinations of atazanavir (ATV) and ritonavir (RTV) and felodipine (FDN) and indapamide (IPM) were prepared and stabilized by a polymer for studying their dissolution (under non-sink conditions) and membrane transport in fasted state simulated intestinal fluid (FaSSIF). The micellar solubilization by FaSSIF enhanced the amorphous solubility of the drugs to different extents. Similar to buffer, the maximum achievable concentration of drugs in combination was reduced in FaSSIF, but the extent of reduction was affected by the degree of FaSSIF solubilization. Dissolution studies of ATV and IPM revealed that the amorphous solubility of these two drugs was not affected by FaSSIF solubilization. In contrast, RTV was significantly affected by FaSSIF solubilization with a 30% reduction in the maximum achievable concentration upon combination to ATV, compared to 50% reduction in buffer. This positive deviation by FaSSIF solubilization was not reflected in the mass transport–time profiles. Interestingly, FDN concentrations remain constant until the amount of IPM added was over 1000 μg/mL. No decrease in the membrane transport of FDN was observed for a 1:1 M ratio of FDN-IPM combination. This study demonstrates the importance of studying amorphous multidrug formulations under physiologically relevant conditions to obtain insights into the performance of these formulations after oral administration.

Патогенез COVID-19

В статье на основании литературных источников представлены современные данные об основных патогенетических особенностях коронавирусной инфекции, связанной с вирусом SARS-CoV-2, вызвавшим в 2019 году, по определению Всемирной организации здравоохранения, пандемию. В литературном обзоре подробно освещены процессы связывания вируса SARS-CoV-2 с рецептором клеток человека, которые экспрессируют ангиотензинпревращающий фермент 2 (АСЕ2), а также интернализация, репликация вируса и высвобождение новых вирионов из инфицированной клетки, которые поражают таргетные органы (легкие, пищеварительный тракт, сердце, центральную нервную систему и почки) и индуцируют развитие местного и системного воспалительного ответа. Описаны существующие способы медикаментозного воздействия, препятствующие инфицированию человека вирусом SARS-CoV-2. Выделены основные эпидемиологические моменты инфицирования вирусом SARS-CoV-2, указывающие на преимущественное поражение пожилых людей и чаще лиц мужского пола в связи с более высоким уровнем экспрессии АСЕ2, в большей степени в альвеолоцитах, чем у лиц женского пола. Продемонстрированы механизмы развития ответной реакции врожденной и адаптивной иммунной системы макроорганизма на инфицирование вирусом SARS-CoV-2. Представлены терапевтические стратегии, связанные с влиянием на различные этапы жизнедеятельности вируса SARS-CoV-2: интернализацию — использование солютабных доменов S-белка, антител против S-белка, одноцепочечного вариабельного фрагмента антител к АСЕ2 или ингибирования гликозилирования клеточных рецепторов, блокирования взаимодействия S-протеина вируса SARS-CoV-2 с протеином ACE2 и подавления интернализации вируса за счет назначения препаратов хлорохин и гидроксихлорохин; репликацию — ингибирование вирусной РНК-зависимой РНК-полимеразой и применение фавипиравира, ненуклеозидного противовирусного препарата триазавирина, антиретровирусных препаратов (лопинавира в сочетании с ритонавиром), нелфинавира, рибавирина, галидесивира, умифеновира, ингибиторов химотрипсиноподобной протеазы (цинансерина, флавоноидов) и папаиноподобной протеазы. Вышеперечисленные терапевтические методы в ближайшем будущем будут направлены на предупреждение развития и лечение как острого респираторного дистресс-синдрома, так и состояний, обусловленных поражением других таргетных органов при COVID-19.

Novel Bruton's Tyrosine Kinase inhibitor remibrutinib: Drug-drug interaction potential as a victim of CYP3A4 inhibitors based on clinical data and PBPK modeling.

Remibrutinib, a novel oral Bruton’s Tyrosine Kinase inhibitor (BTKi) is highly selective for BTK, potentially mitigating the side effects of other BTKis. Enzyme phenotyping identified CYP3A4 to be the predominant elimination pathway of remibrutinib. The impact of concomitant treatment with CYP3A4 inhibitors, grapefruit juice and ritonavir (RTV), was investigated in this study in combination with an intravenous microtracer approach. Pharmacokinetic (PK) parameters, including the fraction absorbed, the fractions escaping intestinal and hepatic first‐pass metabolism, the absolute bioavailability, systemic clearance, volume of distribution at steady‐state, and the fraction metabolized via CYP3A4 were evaluated. Oral remibrutinib exposure increased in the presence of RTV 4.27‐fold, suggesting that remibrutinib is not a sensitive CYP3A4 substrate. The rich PK dataset supported the building of a robust physiologically‐based pharmacokinetic (PBPK) model, which well‐described the therapeutic dose range of 25–100 mg. Simulations of untested scenarios revealed an absence of drug‐drug interaction (DDI) risk between remibrutinib and the weak CYP3A4 inhibitor fluvoxamine (area under the concentration‐time curve ratio [AUCR] <1.25), and a moderate effect with the CYP3A4 inhibitor erythromycin (AUCR: 2.71). Predictions with the moderate and strong CYP3A4 inducers efavirenz and rifampicin, suggested a distinct remibrutinib exposure decrease of 64% and 89%. Oral bioavailability of remibrutinib was 34%. The inclusion of an intravenous microtracer allowed the determination of all relevant remibrutinib PK parameters, which facilitated construction of the PBPK model. This will provide guidance on the selection or restriction of comedications and prediction of DDI risks.

Cellulose derivatives as effective recrystallization inhibitor for ternary ritonavir solid dispersions: In vitro-in vivo evaluation

Amorphous solid dispersions (ASDs) are regarded as one of the most promising techniques for poorly-soluble active pharmaceutical ingredients (API). However, the thermodynamic instability of ASDs at supersaturated state makes them easy to recrystallize in aqueous media. In this study, ritonavir (RTV) was selected as a model drug for evaluating the solubility enhancement and recrystallization inhibition effect of various cellulose derivatives and the combinations of them with typical surfactants. Combination of HPMCAS-HF/SLS was filtrated for preparing ternary RTV solid dispersions (RTV SD) via solvent evaporation method. RTV SD exhibited enhanced dissolution manner, while the oral bioavailability of RTV SD was equivalent with the Reference Standard Norvir® but increased significantly compared to the ternary physical mixture. Thus, the ternary SD system might be promisingly employed as efficient drug delivery system for RTV, while the HPMCAS-HF/SLS combination could be recommended as effective excipient for fabricating steady solid dispersions loading poorly soluble API.

A review on recent electroanalytical methods for the analysis of antiviral COVID-19 drugs

Currently, there are no specific drugs for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, designated as coronavirus disease 2019 (COVID-19). Several therapeutic options including antiviral, antithrombotic, immunosuppressive, and anti-rheumatic drugs are researched all over the world. Analytical methods are needed in every step of innovation, research, development, and manufacturing process of pharmaceuticals, therefore new analytical methods for pharmaceuticals are developed and validated increasingly over time. In this review, recent reports on electroanalytical techniques for the determination of selected COVID-19 drugs, favipiravir (FAV), remdesivir (REM), lopinavir (LOP) / ritonavir (RIT), and hydroxychloroquine (HCQ) were emphasized. Electroanalysis of antiviral active pharmaceutical ingredients carried out at various modified or non-modified electrodes by cyclic voltammetry (CV), linear sweep voltammetry (LSV), differential pulse voltammetry (DPV), and square wave voltammetry (SWV) were compiled from the literature. The effects of supporting electrolyte and pH on the current and potential of the analytical signal were evaluated. Scan rate results obtained by the CV method showed whether the redox process of the drug active ingredient diffusion or adsorption controlled at the electrode used in the selected solvent-supporting electrolyte and pH systems. Linearity range and the limit of detection (LOD) of applied electroanalytical methods were compared by combining the results obtained from drug active ingredients given in references.

Development of Spray-Dried Cyclodextrin-Based Pediatric Anti-HIV Formulations.

The human immunodeficiency virus (HIV) impacts up to 37 million people globally, of which 1.8 million are children. To date, there is no cure for HIV, although treatment options such as antiretroviral therapy (ART) are available. ART, which involves a patient taking a combination of antiretrovirals, is being used to treat HIV clinically. Despite the effectiveness of ART, there is currently no palatable pediatric formulation to treat HIV in children, which has hindered patient compliance and overall treatment efficacy. In addition, anti-HIV therapeutics are often poorly water-soluble, and hence have poor bioavailability. In the present study, we developed a pediatric-friendly formulation for anti-HIV therapeutics with improved dissolution characteristics of the therapeutic agents. Lopinavir (LPV) and ritonavir (RTV), available as FDA-approved fixed-dose combination products, were chosen as model ART drugs, and the formulation and processing parameters of spray-dried cyclodextrin (CD)-based LPV and RTV complexes were studied. Results showed that the spray-dried complexes exhibited enhanced dissolution profiles in comparison to pure drugs, particularly spray-dried β-CD complexes, which showed the most favorable dissolution profiles. This current formulation with enhanced dissolution and taste-masking ability through the use of cyclodextrin has the potential to address the unmet need for the development of suitable pediatric formulations.

Development of In Situ Self-Assembly Nanoparticles to Encapsulate Lopinavir and Ritonavir for Long-Acting Subcutaneous Injection.

Most antiretroviral medications for human immunodeficiency virus treatment and prevention require high levels of patient adherence, such that medications need to be administered daily without missing doses. Here, a long-acting subcutaneous injection of lopinavir (LPV) in combination with ritonavir (RTV) using in situ self-assembly nanoparticles (ISNPs) was developed to potentially overcome adherence barriers. The ISNP approach can improve the pharmacokinetic profiles of the drugs. The ISNPs were characterized in terms of particle size, drug entrapment efficiency, drug loading, in vitro release study, and in vivo pharmacokinetic study. LPV/RTV ISNPs were 167.8 nm in size, with a polydispersity index of less than 0.35. The entrapment efficiency was over 98% for both LPV and RTV, with drug loadings of 25% LPV and 6.3% RTV. A slow release rate of LPV was observed at about 20% on day 5, followed by a sustained release beyond 14 days. RTV released faster than LPV in the first 5 days and slower than LPV thereafter. LPV trough concentration remained above 160 ng/mL and RTV trough concentration was above 50 ng/mL after 6 days with one subcutaneous injection. Overall, the ISNP-based LPV/RTV injection showed sustained release profiles in both in vitro and in vivo studies.

Functionalized graphene oxide tablets for sample preparation of drugs in biological fluids: Extraction of ritonavir, a HIV protease inhibitor, from human saliva and plasma using LC–MS/MS

In this work, graphene oxide based tablets (GO-Tabs) were prepared by applying functionalized GO as a thin layer on a polyethylene substrate. The graphene oxide was functionalized with amine groups (-NH2 ) by Poly (ethylene glycol) bis(3-aminopropyl) terminated (GO-NH2 -PEG-NH2 ). The functionalized GO-Tabs were used for extraction of ritonavir in human saliva samples. The analysis of ritonavir in plasma and saliva samples was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gradient LC system with MS/MS in the positive ion mode (ESI+) was used. The transitions m/z 721→269.0 and m/z 614→421 were used for the ritonavir and the internal standard indinavir, respectively. In this study, the determination of the HIV protease inhibitor ritonavir in human saliva samples was carried out using functionalized GO-Tab and LC-MS/MS and the method was validated. Standard calibration curve for plasma and saliva samples was constructed from 5.0 to 2000 nmol L-1 . The limit of detection was 0.1 nmol L-1 and the limit of quantification was 5.0 nmol L-1 in both plasma and saliva matrices. The intra- and inter-assay precision values were found to be between 1.5 and 5.8%, and the accuracy values ranged from 88.0% to 108% utilizing saliva and plasma samples. The extraction recovery was over 80%, and the presented functionalized GO-Tabs could be re-used for more than ten extractions without deterioration in recovery.

Development, Characterization and Comparative Phar-macokinetic Evaluation of Ritonavir Nanosuspension, with a Model Nanoemulsion and Coarse Suspension for Improved Oral Delivery

Ritonavir (RV) is an antiretroviral drug, classified as BCS class II pharmaceutical active.It has limited bioavailability due to poor aqueous solubility and first pass metabolism. The purpose of this investigation was to develop optimal nanosuspension and to compare with a model nanoemulsion of RV for improved oral delivery. DSC studies showed good compatibility of excipients with drug. Nanosuspension was prepared by high pressure homogenization, while nanoemulsion was prepared by hot homogenization followed by ultrasonication. All prepared RV formulations were characterized and optimal system was selected and in vivo evaluated. Nanosuspension (F1) formulation containing 0.5% SLS showed homogeneity with least particle size was optimized and compared with coarse (powder) suspension. SEM studies on lyophilized nanosuspension revealed the absence of needle shaped drug crystals, indicating the loss of crystallinity. Prepared a model lipid nanoemulsion. It was having the size, PDI, ZP, EE and assay of 193.14 ± 12.66nm, 0.311 ± 0.04, -27.6 ± 1.184mV, 90.79 ± 0.319% and 99.57 ± 1.25% respectively. Next, a comparative pharmacokinetic study of RV nanosuspenison with respect to lipid nanoemulsion and coarse suspension was performed in male wistar rats. The Cmax of nanosuspension was significantly more when compared to that of NE or coarse drug suspension. The tmax was similar in case of both nanodelivery systems and significantly less when compared to that of coarse suspension. About 1.36 and 1.27 fold improvements in relative bioavailability (BA) of ritonavir via lipid nanoemulsion and nanosuspension were found when compared to coarse suspension. The study results revealed nominal but nonsignificant difference in oral bioavailability for the two nanodelivery systems. Taken together, this study confirmed the potential of nanosuspension and nanoemulsion systems in improving the bioavailability of ritonavir.

The effects of secreted aspartyl proteinase inhibitor ritonavir on azoles‐resistant strains of Candida albicans as well as regulatory role of SAP2 and ERG11

Background Candida albicans, the main human fungal pathogen, can cause fungal infection and seriously affect people's health and life. This study aimed to investigate the effects of ritonavir (RIT) on C. albicans and the correlation between SAP2 as well as ERG11 and drug resistance.ResultsSecreted aspartyl proteinases (Saps) activities and pathogenicity of C. albicans with different drug resistance were measured. M27‐A4 broth microdilution method was used to analyze the drug sensitivity of RIT combined with fluconazole (FCA) on C. albicans. After that, SAP2 and ERG11 mutations were examined by polymerase chain reaction (PCR) and sequencing, and quantitative real‐time PCR was utilized to determine the expression of the two genes. By analyzing pz values, the Saps activity of cross‐resistant strains was the highest, followed by voriconazole (VRC)‐resistant strains, FCA‐resistant strains, itraconazole (ITR)‐resistant strains, and sensitive strains. The pathogenicity of C. albicans in descending order was as follows: cross‐resistant strains, VRC‐resistant strains, ITR‐resistant strains, FCA‐resistant strains, and sensitive strains. With the increase of RIT concentrations, the Saps activity was gradually inhibited. Drug sensitivity results showed that there was no synergistic effect between RIT and FCA. Additionally, no gene mutation sites were found in SAP2 sequencing, and 17 synonymous mutations and 6 missense mutations occurred in ERG11 sequencing. Finally, the expression of SAP2 and ERG11 was significantly higher in the resistant strains compared with the sensitive strains, and there was a positive liner correlation between SAP2 and ERG11 messenger RNA expression (r = .6655, p < .001).ConclusionThese findings may help to improve our understanding of azole‐resistant mechanisms of C. albicans and provide a novel direction for clinical therapeutics of C. albicans infection.

Novel pulsed oxygen plasma mediated surface hydrophılizatıon of ritonavır for the enhancement of wettability and solubility

The pulsed oxygen plasma treatment (POPT) of the ritonavir (RTV) powder/compacts was investigated as a novel possible physical technique for improving wettability, intrinsic dissolution rate (IDR), and, consequently, solubility of the poorly water-soluble RTV drug. Induction of POPT for 30 min enhanced 5.87-folds apparent solubility and 1.95-folds IDR at 24 h, attributed to RTV's surface hydrophilization. The POPT RTV exhibited zero contact angle compared to RTV suggesting improved wettability (untreated RTV = 74° and 30 min after POPT = 0°). The ATR-FTIR showed a broad peak of –OH group around 3300 cm -1, and XPS showed an increase in surface oxygen concentration from 9 % to 12 % aided through POPT lead to improved wettability of RTV. Moreover, the increased negative zeta potential was observed due to surface hydroxylation on the POPT RTV, resulting in increased surface polarity and RTV's solubility. It was found that there was no substantial thermal or physical alteration after POPT, as evident from DSC, SEM, and XRD studies. This study successfully demonstrated the impact of POPT on improvement in the wettability, solubility, and IDR of hydrophobic drugs RTV.

Management of Antiretroviral Therapy with Boosted Protease Inhibitors-Darunavir/Ritonavir or Darunavir/Cobicistat.

A major challenge in the management of antiretroviral therapy (ART) is to improve the patient’s adherence, reducing the burden caused by the high number of drugs that compose the treatment regimens for human immunodeficiency virus positive (HIV+) patients. Selection of the most appropriate treatment regimen is responsible for therapeutic success and aims to reduce viremia, increase the immune system response capacity, and reduce the incidence rate and intensity of adverse reactions. In general, protease inhibitor (PI) is one of the pillars of regimens, and darunavir (DRV), in particular, is frequently recommended, along with low doses of enzyme inhibitors as cobicistat (COBI) or ritonavir (RTV), by the international guidelines. The potential of clinically significant drug interactions in patients taking COBI or RTV is high due to the potent inhibitory effect on cytochrome CYP 450, which attracts significant changes in the pharmacokinetics of PIs. Regardless of the patient or type of virus, the combined regimens of DRV/COBI or DRV/RTV are available to clinicians, proving their effectiveness, with a major impact on HIV mortality/morbidity. This study presents current information on the pharmacokinetics, pharmacology, drug interactions, and adverse reactions of DRV; it not only compares the bioavailability, pharmacokinetic parameters, immunological and virological responses, but also the efficacy, advantages, and therapeutic disadvantages of DRV/COBI or DRV/RTV combinations.

Effect of drug-coformer interactions on drug dissolution from a coamorphous in mesoporous silica

In this study, the molecular state of ritonavir (RTN)-saccharin (SAC) coamorphous incorporated into mesoporous silica by solvent evaporation and the effect of SAC on the RTN dissolution from mesopores were investigated. The amorphization of RTN-SAC was confirmed as a halo pattern in powder X-ray diffraction measurements and a single glass transition event in the modulated differential scanning calorimetry (MDSC) curve. 13C solid-state NMR spectroscopy revealed a hydrogen bond between the thiazole nitrogen of RTN and the amine proton of SAC. The glass transition of the RTN-SAC coamorphous in mesoporous silica was not found in the MDSC curve, indicating that RTN and SAC were monomolecularly incorporated into the mesopores. Solid-state NMR measurements suggested that the co-incorporation of SAC into the mesopores decreased the local mobility of the thiazole group of RTN via hydrogen bond formation. The RTN-SAC 1:1 coamorphous in mesoporous silica retained the X-ray halo-patterns after 30 d of storage, even under high temperature and humidity conditions. In the dissolution test, the RTN-SAC 1:1 coamorphous in mesoporous silica maintained RTN supersaturation for a longer time than the RTN amorphous in mesoporous silica. This study demonstrated that the drug-coformer interaction within mesoporous silica can significantly improve drug dissolution.

Hyaluronic Acid-Targeted Stimuli-Sensitive Nanomicelles Co-Encapsulating Paclitaxel and Ritonavir to Overcome Multi-Drug Resistance in Metastatic Breast Cancer and Triple-Negative Breast Cancer Cells

Active targeting and overcoming multi-drug resistance (MDR) can be some of the important attributes of targeted therapy for metastatic breast cancer (MBC) and triple-negative breast cancer (TNBC) treatment. In this study, we constructed a hyaluronic acid (HA)-decorated mixed nanomicelles-encapsulating chemotherapeutic agent paclitaxel (PTX) and P-glycoprotein inhibitor ritonavir (RTV). HA was conjugated to poly (lactide) co-(glycolide) (PLGA) polymer by disulfide bonds (HA-ss-PLGA). HA is a natural ligand for CD44 receptors overexpressed in breast cancer cells. Disulfide bonds undergo rapid reduction in the presence of glutathione, present in breast cancer cells. The addition of RTV can inhibit the P-gp and CYP3A4-mediated metabolism of PTX, thus aiding in reversing MDR and sensitizing the cells toward PTX. An in vitro uptake and cytotoxicity study in MBC MCF-7 and TNBC MDA-MB-231 cell lines demonstrated the effective uptake of the nanomicelles and drug PTX compared to non-neoplastic breast epithelium MCF-12A cells. Interestingly, in vitro potency determination showed a reduction in mitochondrial membrane potential and reactive oxygen species in breast cancer cell lines, indicating effective apoptosis of cancer cells. Thus, stimuli-sensitive nanomicelles along with HA targeting and RTV addition can effectively serve as a chemotherapeutic drug delivery agent for MBC and TNBC.

Development of nanoparticle-based orodispersible palatable pediatric formulations

Despite a collaborative effort towards developing suitable oral drug products for pediatrics over the past decade, appropriate pediatric dosage forms have remained lacking due to special considerations in dose flexibility, swallowability, palatability, and safety of excipients for pediatrics. The present research aims to develop a nanoparticle-based orodispersible pediatric drug delivery platform to improve oral bioavailability and taste of poorly water-soluble and unpalatable therapeutics. Two Biopharmaceutics Classification System II/IV compounds lopinavir (LPV) and ritonavir (RTV) with unpleasant taste were chosen as the model compounds. LPV and RTV Eudragit® E PO nanoparticles (NP) were prepared using a nanoprecipitation method and their key quality attributes and taste-masking effect were evaluated. Moreover, in vitro dissolution testing was conducted at simulated gastrointestinal pH conditions. The in vivo bioavailability of the developed NP formulations was assessed using a rat model. Following the formulation optimization, over 98% encapsulation efficiency was obtained for both LPV and RTV NP and both drugs remained amorphous in its respective NP. LPV/RTV NP combination (4/1, w/w) showed comparable in vitro dissolution to that of the commercial LPV/RTV tablet (Kaletra®). In addition, the taste-masking effect of the developed NP formulations was confirmed by an E-tongue study. The lyophilized LPV and RTV NP were completely dispersible in water within 7 sec and remained stable at 4 ± 2 °C over three months. Lastly, the pharmacokinetic study demonstrated that the LPV/RTV NP combination (4/1, w/w) had improved oral bioavailability compared to Kaletra® and their corresponding raw drug powders. The results demonstrated a novel nanoparticle-based orodispersible platform that is capable of improving oral bioavailability and taste of poorly water-soluble and unpalatable therapeutics for pediatric use.

Efavirenz, atazanavir, and ritonavir disrupt sarcoplasmic reticulum Ca2+ homeostasis in skeletal muscles

While muscle fatigue, pain and weakness are common co-morbidities in HIV-1 infected people, their underlying cause remain poorly defined. To this end, we evaluated whether the common antiretroviral drugs efavirenz (EFV), atazanavir (ATV) and ritonavir (RTV) could be a contributing factor by pertubating sarcoplasmic reticulum (SR) Ca2+ cycling. In live-cell imaging, EFV (6.0 μM), ATV (6.0 μM), and RTV (3.0 μM) elicited Ca2+ transients and blebbing of the plasma membranes of C2C12 skeletal muscle myotubes. Pretreating C2C12 skeletal muscle myotubes with the SR Ca2+ release channel blocker ryanodine (50 μM), slowed the rate and amplitude of Ca2+ release from and reuptake of Ca2+ into the SR. EFV, ATV and RTV (1 nM - 20 μM) potentiated and then displaced [3H] ryanodine binding to rabbit skeletal muscle ryanodine receptor Ca2+ release channel (RyR1). These drugs at concentrations 0.25–31.2 μM also increased and or decreased the open probability of RyR1 by altering its gating and conductance. ATV (≤5 μM) potentiated and >5μM inhibited the ability of sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA1) to hydrolyze ATP and transport Ca2+. RTV (2.5–31.5 μM) dose-dependently inhibited SERCA1-mediated, ATP-dependent Ca2+ transport. EFV (0.25–31.5 μM) had no measurable effect on SERCA1's ability to hydrolyze ATP and transport Ca2+. These data support the notion that EFV, ATV and RTV could be contributing to skeletal muscle co-morbidities in PLWH by modulating SR Ca2+ homeostasis.

Protease Inhibitor Anti-HIV, Lopinavir, Impairs Placental Endocrine Function.

Protease Inhibitors (PI e.g., ritonavir (RTV) and lopinavir (LPV)) used to treat pregnant mothers infected by HIV induce prematurity and endocrine dysfunctions. The maintenance of pregnancy relies on placental hormone production (human Chorionic Gonadotrophin (hCG) and progesterone (P4)). Those functions are ensured by the villous trophoblast and are mainly regulated by the Unfolded Protein Response (UPR) pathway and mitochondria. We investigated, in vitro, if PI impair hCG and P4 production and the potential intracellular mechanisms involved. Term villous cytotrophoblast (VCT) were cultured with or without RTV or LPV from 6 to 48 h. VCT differentiation into syncytiotrophoblast (ST) was followed measuring hCG and P4 secretion. We evaluated the expression of P4 synthesis partners (Metastatic Lymph Node 64 (MLN64), cholesterol side-chain cleavage (P450SCC), Hydroxy-delta-5-Steroid Dehydrogenase and 3 Beta-and steroid delta-isomerase 1 (HSD3B1)), of mitochondrial pro-fusion factors (Mitofusin 2 (Mfn2), Optic Atrophy 1 (OPA1)) and of UPR factors (Glucose-Regulated Protein 78 (GRP78), Activating Transcription Factor 4 (ATF4), Activating Transcription Factor 6 (ATF6), spliced X-box Binding Protein 1 (sXBP1)). RTV had no significant effect on hCG and P4 secretion, whereas lopinavir significantly decreased both secretions. LPV also decreased P450SCC and HSD3B1 expression, whereas it increased Mfn2, GRP78 and sXBP1 expression in ST. RTV has no effect on the endocrine placenta. LPV impairs both villous trophoblast differentiation and P4 production. It is likely to act via mitochondrial fusion and UPR pathway activation. These trophoblastic alterations may end in decreased P4 levels in maternal circulation, inducing prematurity.

Simultaneous quantification of Darunavir and Ritonavir in human plasma and pharmacokinetic study by LC MS/MS

The combination of Darunavir (DRV) and Ritonavir (RTV) at a dose of 800/100mg has exhibit durable efficacy in both untreated and treated HIV positive patients with no observed DRV resistance-associated mutations (RAMs) and the RTV improves the pharmacokinetic profile of DRV by enhancing its bioavailability. Hence a sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method has been developed and validated for the simultaneous quantification of Darunavir (DRV) and Ritonavir (RTV) in human plasma. The chromatographic separation has been accomplished on Thermo Hypersil Gold (50×4.6mm, 3μ) analytical column using isocratic elution using 0.1% Formic Acid buffer solution/Acetonitrile (50:50, v/v) at a flow rate of 0.6 mL/min. The linearity of the method ranged 150.000 ng/mL to 15000.000 ng/mL &amp; 10.000 ng/mL to 3000.00 ng/mL for DRV &amp; RTV respectively, using 100 μL of plasma. The method was completely validated for its sensitivity, selectivity, linearity, accuracy and precision, recovery, matrix effect, stability, and dilution integrity. The absolute recovery for DRV ranged from 79.12 to 72.71 % while for RTV it ranged from 63.39% to 66.99 %. For DRV-D9 and RTV-D6 the recovery rates are 94.19 and 80.05% respectively. The method exhibit good intra-day and inter-day precision with low % CV of less than 5.0% at each quality control level for both the analytes. The developed method has been applied successfully for pharmacokinetic study in healthy humans by oral administration of Darunavir/Ritonavir tablets 400/50 mg (dose; 02x400/50 mg) in 77 healthy male volunteers under fed condition.

Multidomain drug delivery systems of β-casein micelles for the local oral co-administration of antiretroviral combinations

The antiretroviral (ARV) cocktailrevolved the treatment of the human immunodeficiency virus (HIV) infection. Drug combinations have been also tested to treat other infectious diseases, including the recentcoronavirus disease 2019 (COVID-19) outbreak. To simplify administration fixed-dose combinationshave been introduced, however, oral anti-HIV therapy still struggles with low oral bioavailability of many ARVs.This work investigated the co-encapsulation of two clinically relevant ARV combinations,tipranavir (TPV):efavirenz (EFV) anddarunavir (DRV):efavirenz (EFV):ritonavir (RTV),within the core of β-casein (bCN) micelles. Encapsulation efficiency in both systems was ~100%. Cryo-transmission electron microscopy and dynamic light scattering of the ARV-loaded colloidaldispersions indicatefull preservation of the spherical morphology, and x-ray diffraction confirm that the encapsulated drugs are amorphous. To prolong the physicochemical stabilitythe formulations were freeze-driedwithout cryo/lyoprotectant, and successfully redispersed, with minor changes in morphology.Then, theARV-loaded micelles were encapsulated within microparticles of Eudragit® L100, which prevented enzymatic degradation and minimized drug release under gastric-like pH conditionsin vitro. At intestinal pH, the coating polymer dissolved and released the nanocarriers and content. Overall, our results confirm the promise of this flexible and modular technology platform for oral delivery of fixed dose combinations.

Evaluation of Caco-2 cell permeability of ritonavir nanosuspensions

Background and Aims: Poor aqueous solubility limits drug absorption through intestinal mucosa. Nanosuspensions with nanometer range particle size provides enhanced aqueous solubility and hence permeability. The objective of this study was to investigate the cytotoxicity and in vitro cell permeability through human adenocarcinoma (Caco-2) cells of ritonavir (RTV) nanosuspensions. Methods: The Microfluidization method was used to prepare nanosuspensions. Particle size (PS), polydispersity index (PI) and zeta potential (ZP) values were measured as characterization. MTT test was applied to evaluate the cytotoxic effect. Caco-2 cell lines were used for transport studies with RTV coarse powder, physical mixtures and nanosuspension. Results: Approximately 600 nm PS, 0.4 PDI and 22 mV ZP values were observed for nanosuspensions. The sample groups showed no cytotoxicity on the cell lines in any RTV concentration. However, significant cytotoxic effect was determined in groups with high amounts of sodium dodecyl sulfate. The transported RTV in nanosuspension formulation enhanced by 5.3- fold and 1.5-fold in comparison with RTV coarse powder and physical mixture, respectively. Rate of the transportation and also the amount of the transported RTV were improved with nanosuspension formulation. Conclusion: Particle size reduction of RTV into nanometer size and preparing nanosuspension system was found effective to obtain enhanced cell permeability.