Simultaneous quantification of seven repurposed COVID-19 drugs remdesivir (plus metabolite GS-441524), chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin by a two-dimensional isotope dilution LC-MS/MS method in human serum.

BACKGROUND: The present COVID-19 pandemic has prompted worldwide repurposing of drugs. The aim of the present work was to develop and validate a two-dimensional isotope-dilution liquid chromatrography tandem mass spectrometry (ID-LC-MS/MS) method for accurate quantification of remdesivir and its active metabolite GS-441524, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in serum; drugs that have gained attention for repurposing in the treatment of COVID-19. METHODS: Following protein precipitation, samples were separated with a two-dimensional ultra-high performance liquid chromatography (2D-UHPLC) setup, consisting of an online solid phase extraction (SPE) coupled to an analytical column. For quantification, stable isotope-labelled analogues were used as internal standards for all analytes. The method was validated on the basis of the European Medicines Agency bioanalytical method validation protocol. RESULTS: Detuning of lopinavir and ritonavir allowed simultaneous quantification of all analytes with different concentration ranges and sensitivity with a uniform injection volume of 5 µL. The method provided robust validation results with inaccuracy and imprecision values of ≤ 9.59 % and ≤ 11.1 % for all quality controls. CONCLUSION: The presented method is suitable for accurate and simultaneous quantification of remdesivir, its metabolite GS-441525, chloroquine, hydroxychloroquine, lopinavir, ritonavir, favipiravir and azithromycin in human serum. The quantitative assay may be an efficient tool for the therapeutic drug monitoring of these potential drug candidates in COVID-19 patients in order to increase treatment efficacy and safety.

Pharmacokinetics and Safety of Zidovudine, Lamivudine, and Lopinavir/Ritonavir in HIV-infected Children With Severe Acute Malnutrition in Sub-Saharan Africa: IMPAACT Protocol P1092.

BACKGROUND: Severe acute malnutrition (SAM) may alter the pharmacokinetics (PK), efficacy, and safety of antiretroviral therapy. The phase IV study, IMPAACT P1092, compared PK, safety, and tolerability of zidovudine (ZDV), lamivudine (3TC), and lopinavir/ritonavir (LPV/r) in children with and without SAM. MATERIALS AND METHODS: Children living with HIV 6 to <36 months of age with or without World Health Organization (WHO)-defined SAM received ZDV, 3TC, and LPV/r syrup for 48 weeks according to WHO weight band dosing. Intensive PK sampling was performed at weeks 1, 12, and 24. Plasma drug concentrations were measured using liquid chromatography tandem mass spectrometry. Steady-state mean area under the curve (AUC0-12h) and clearance (CL/F) for each drug were compared. Grade ≥3 adverse events were compared between cohorts. RESULTS: Fifty-two children were enrolled across 5 sites in Africa with 44% (23/52) female, median age 19 months (Q1, Q3: 13, 25). Twenty-five children had SAM with entry median weight-for-height Z-score (WHZ) -3.4 (IQR -4.0, -3.0) and 27 non-SAM had median WHZ -1.0 (IQR -1.8, -0.1). No significant differences in mean AUC0-12h or CL/F were observed (P ≥ 0.09) except for lower 3TC AUC0-12h (GMR, 0.60; 95% CI, 0.4-1.0; P = 0.047) at week 12, higher ZDV AUC0-12h (GMR, 1.52; 1.2-2.0; P = 0.003) at week 24 in the SAM cohort compared with non-SAM cohort. Treatment-related grade ≥3 events did not differ significantly between cohorts (24.0% vs. 25.9%). CONCLUSION: PK and safety findings for ZDV, 3TC, and LPV/r support current WHO weight band dosing of syrup formulations in children with SAM.

Plasma Concentrations and Safety of Lopinavir/Ritonavir in COVID-19 Patients.

BACKGROUND: Although the efficacy of lopinavir/ritonavir has not been proven, it has been proposed as an off-label treatment for COVID-19. Previously, it has been reported that the plasma concentrations of lopinavir significantly increase in inflammatory settings. As COVID-19 may be associated with major inflammation, assessing the plasma concentrations and safety of lopinavir in COVID-19 patients is essential. METHODS: Real-world COVID-19 data based on a retrospective study. RESULTS: Among the 31 COVID-19 patients treated with lopinavir/ritonavir between March 18, 2020 and April 1, 2020, higher lopinavir plasma concentrations were observed, which increased by 4.6-fold (interquartile range: 3.6-6.2), compared with the average plasma concentrations in HIV. Lopinavir concentrations in all except one patient were above the upper limit of the concentration range of HIV treatment. Approximately one to 5 patients prematurely stopped treatment mainly because of an ADR related to hepatic or gastrointestinal disorders. CONCLUSIONS: Lopinavir plasma concentrations in patients with moderate-to-severe COVID-19 were higher than expected, and they were associated with the occurrence of hepatic or gastrointestinal adverse drug reactions. However, a high plasma concentration may be required for in vivo antiviral activity against SARS-CoV-2, as suggested by previous studies. Therefore, in the absence of adverse drug reaction, lopinavir dosage should not be reduced. Caution is essential because off-label use can be associated with a new drug safety profile.

Safety of hydroxychloroquine and darunavir or lopinavir in COVID-19 infection.

BACKGROUND: Combination therapy with hydroxychloroquine and darunavir/ritonavir or lopinavir/ritonavir has been suggested as an approach to improve the outcome of patients with moderate/severe COVID-19 infection. OBJECTIVES: To examine the safety of combination therapy with hydroxychloroquine and darunavir/ritonavir or lopinavir/ritonavir. METHODS: This was an observational cohort study of patients hospitalized for COVID-19 pneumonia treated with hydroxychloroquine and darunavir/ritonavir or lopinavir/ritonavir. Clinical evaluations, electrocardiograms and the pharmacokinetics of hydroxychloroquine, darunavir and lopinavir were examined according to clinical practice and guidelines. RESULTS: Twenty-one patients received hydroxychloroquine with lopinavir/ritonavir (median age 68 years; 10 males) and 25 received hydroxychloroquine with darunavir/ritonavir (median age 71 years; 15 males). During treatment, eight patients (17.4%) developed ECG abnormalities. Ten patients discontinued treatment, including seven for ECG abnormalities a median of 5 (range 2-6) days after starting treatment. All ECG abnormalities reversed 1-2 days after interrupting treatment. Four patients died within 14 days. ECG abnormalities were significantly associated with age over 70 years, coexisting conditions (such as hypertension, chronic cardiovascular disease and kidney failure) and initial potential drug interactions, but not with the hydroxychloroquine concentration. CONCLUSIONS: Of the patients with COVID-19 who received hydroxychloroquine with lopinavir or darunavir, 17% had ECG abnormalities, mainly related to age or in those with a history of cardiovascular disease.

Abnormal laboratory findings and plasma concentration monitoring of lopinavir and ritonavir in COVID-19.

It is not known whether the adverse events (AEs) associated with the administration of lopinavir and ritonavir (LPV/r) in the treatment of COVID-19 are concentration-dependent. In a retrospective study of 65 patients treated with LPV/r and therapeutic drug monitoring (TDM) for severe forms of COVID-19 (median age: 67; males: 41 [63.1%]), 33 (50.8%) displayed a grade ≥2 increase in plasma levels of hepatobiliary markers, lipase and/or triglycerides. A causal relationship between LPV/r and the AE was suspected in 9 of the 65 patients (13.8%). At 400 mg b.i.d., the plasma trough concentrations of LPV/r were high and showed marked interindividual variability (median [interquartile range]: 16,600 [11,430-20,842] ng/ml for lopinavir and 501 [247-891] ng/ml for ritonavir). The trough lopinavir concentration was negatively correlated with body mass index, while the trough ritonavir concentration was positively correlated with age and negatively correlated with prothrombin activity. However, the occurrence of abnormal laboratory values was not associated with higher trough plasma concentrations of LPV/r. Further studies will be needed to determine the value of TDM in LPV/r-treated patients with COVID-19.

Comparative Antiviral Efficacy of Viral Protease Inhibitors against the Novel SARS-CoV-2 In Vitro.

The recent outbreak of novel coronavirus pneumonia (COVID-19) caused by a new coronavirus has posed a great threat to public health. Identifying safe and effective antivirals is of urgent demand to cure the huge number of patients. Virus-encoded proteases are considered potential drug targets. The human immunodeficiency virus protease inhibitors (lopinavir/ritonavir) has been recommended in the global Solidarity Trial in March launched by World Health Organization. However, there is currently no experimental evidence to support or against its clinical use. We evaluated the antiviral efficacy of lopinavir/ritonavir along with other two viral protease inhibitors in vitro, and discussed the possible inhibitory mechanism in silico. The in vitro to in vivo extrapolation was carried out to assess whether lopinavir/ritonavir could be effective in clinical. Among the four tested compounds, lopinavir showed the best inhibitory effect against the novel coronavirus infection. However, further in vitro to in vivo extrapolation of pharmacokinetics suggested that lopinavir/ritonavir could not reach effective concentration under standard dosing regimen [marketed as Kaletra®, contained lopinavir/ritonavir (200 mg/50 mg) tablets, recommended dosage is 400 mg/10 mg (2 tablets) twice daily]. This research concluded that lopinavir/ritonavir should be stopped for clinical use due to the huge gap between in vitro IC50 and free plasma concentration. Nevertheless, the structure-activity relationship analysis of the four inhibitors provided further information for de novel design of future viral protease inhibitors of SARS-CoV-2.

Effect of Systemic Inflammatory Response to SARS-CoV-2 on Lopinavir and Hydroxychloroquine Plasma Concentrations.

Coronavirus disease 2019 (COVID-19) leads to inflammatory cytokine release, which can downregulate the expression of metabolizing enzymes. This cascade affects drug concentrations in the plasma. We investigated the association between lopinavir (LPV) and hydroxychloroquine (HCQ) plasma concentrations and the levels of the acute-phase inflammation marker C-reactive protein (CRP). LPV plasma concentrations in 92 patients hospitalized at our institution were prospectively collected. Lopinavir-ritonavir was administered every 12 hours, 800/200 mg on day 1 and 400/100 mg on day 2 until day 5 or 7. HCQ was given at 800 mg, followed by 400 mg after 6, 24, and 48 h. Hematological, liver, kidney, and inflammation laboratory values were analyzed on the day of drug level determination. The median age of study participants was 59 (range, 24 to 85) years, and 71% were male. The median durations from symptom onset to hospitalization and treatment initiation were 7 days (interquartile range [IQR], 4 to 10) and 8 days (IQR, 5 to 10), respectively. The median LPV trough concentration on day 3 of treatment was 26.5 µg/ml (IQR, 18.9 to 31.5). LPV plasma concentrations positively correlated with CRP values (r = 0.37, P < 0.001) and were significantly lower when tocilizumab was preadministered. No correlation was found between HCQ concentrations and CRP values. High LPV plasma concentrations were observed in COVID-19 patients. The ratio of calculated unbound drug fraction to published SARS-CoV-2 50% effective concentrations (EC50) indicated insufficient LPV concentrations in the lung. CRP values significantly correlated with LPV but not HCQ plasma concentrations, implying inhibition of cytochrome P450 3A4 (CYP3A4) metabolism by inflammation.

Pharmacokinetics of lopinavir/ritonavir oral solution to treat COVID-19 in mechanically ventilated ICU patients.

BACKGROUND: The combination lopinavir/ritonavir is recommended to treat HIV-infected patients at the dose regimen of 400/100 mg q12h, oral route. The usual lopinavir trough plasma concentrations are 3000-8000 ng/mL. A trend towards a 28 day mortality reduction was observed in COVID-19-infected patients treated with lopinavir/ritonavir. OBJECTIVES: To assess the plasma concentrations of lopinavir and ritonavir in patients with severe COVID-19 infection and receiving lopinavir/ritonavir. PATIENTS AND METHODS: Mechanically ventilated patients with COVID-19 infection included in the French COVID-19 cohort and treated with lopinavir/ritonavir were included. Lopinavir/ritonavir combination was administered using the usual adult HIV dose regimen (400/100 mg q12h, oral solution through a nasogastric tube). A half-dose reduction to 400/100 mg q24h was proposed if lopinavir Ctrough was >8000 ng/mL, the upper limit considered as toxic and reported in HIV-infected patients. Lopinavir and ritonavir pharmacokinetic parameters were determined after an intensive pharmacokinetic analysis. Biological markers of inflammation and liver/kidney function were monitored. RESULTS: Plasma concentrations of lopinavir and ritonavir were first assessed in eight patients treated with lopinavir/ritonavir. Median (IQR) lopinavir Ctrough reached 27 908 ng/mL (15 928-32 627). After the dose reduction to 400/100 mg q24h, lopinavir/ritonavir pharmacokinetic parameters were assessed in nine patients. Lopinavir Ctrough decreased to 22 974 ng/mL (21 394-32 735). CONCLUSIONS: In mechanically ventilated patients with severe COVID-19 infections, the oral administration of lopinavir/ritonavir elicited plasma exposure of lopinavir more than 6-fold the upper usual expected range. However, it remains difficult to safely recommend its dose reduction without compromising the benefit of the antiviral strategy, and careful pharmacokinetic and toxicity monitoring are needed.

Assessment of lamivudine, zidovudine, lopinavir, and ritonavir plasma levels in HIV-positive pregnant women: Drug monitoring application to improve patient safety.

Simultaneous therapeutic drug monitoring (TDM) of combination antiretroviral therapy (cART) is critical during pregnancy in order to improve clinical follow-up, monitor viral load, and patient adherence to treatment.A modified simple and fast ultra-high performance liquid chromatography coupled with tandem mass spectrometry and electrospray ionization (UPLC-ESI-MS/MS) method was developed and validated according to national and international guidelines for the simultaneous determination of lamivudine (LMV), zidovudine (ZDV), lopinavir (LPV), and ritonavir (RTV) concentrations in 100-µL plasma sample of Human Immunodeficiency Virus (HIV)-positive pregnant women. Protein precipitation using 0.1% formic acid in cold acetonitrile was used for sample preparation. The chromatographic separation was achieved with a run-time of 3.0 minutes and 3-µL injection on an ethylene bridged hybrid C18 column (2.1 µm × 50 mm, 1.7 µm), under gradient conditions using acetonitrile and formic acid (0.1%).The chromatographic method was used to analyze 10 plasma samples from 8 HIV pregnant women as a clinical patient routinely follow-up by applying TDM criteria.The protonated precursor/product ion transitions for LMV (230.18/112.08), ZDV (268.22/127.10), LPV (629.55/447.35), and RTV (721.50/296.20) were recorded in multiple-reaction-monitoring (MRM) mode. The calibration curve was linear in the range of 50-3,000, 75-4,500, 250-15,000, and 25-1,500-ng/mL for LMV, ZDV, LPV, and RTV, respectively. The range of accuracy was 97.2% to 100.1% and precision 3.4% to 12.7%. The method showed specificity and matrix effect values of < 15%. Minimum absolute recovery percentages (%CV) were 90.5 (5.4), 90.8 (5.0), 95.4 (3.5), and 93.7 (6.9), for LMV, ZDV, LPV, and RTV, respectively. Drug concentrations in patient samples had high inter-individual variability with %CV of 91.98%, 77.54%, 53.80%, and 92.16% for ZDV, LMV, LPV, and RTV, respectively. Two of the 8 patients showed no adherence due to the absence of Protease Inhibitors (PIs) levels in plasma.This technique demonstrated to be effective in therapeutic drug monitoring and is intended to be used in population pharmacokinetics specifically for HIV-positive pregnant women.

Development and validation of a cost-effective and sensitive bioanalytical HPLC-UV method for determination of lopinavir in rat and human plasma.

A simple, sensitive and cost-effective HPLC-UV bioanalytical method for determination of lopinavir (LPV) in rat and human plasma was developed and validated. The plasma sample preparation procedure includes a combination of protein precipitation using cold acetonitrile and liquid-liquid extraction with n-hexane-ethyl acetate (7:3, v/v). A good chromatographic separation was achieved with a Phenomenex Gemini column (C18 , 150 mm × 2.0 mm, 5 µm) at 40°C with gradient elution, at 211 nm. Calibration curves were linear in the range 10-10,000 ng/mL, with a lower limit of quantification of 10 ng/mL using 100 µL of plasma. The accuracy and precision in all validation experiments were within the criteria range set by the guidelines of the Food and Drug Administration. This method was successfully applied to a preliminary pharmacokinetic study in rats following an intravenous bolus administration of LPV. Moreover, the method was subsequently fully validated for human plasma, allowing its use in therapeutic drug monitoring (TDM). In conclusion, this novel, simple and cost-efficient bioanalytical method for determination of LPV is useful for pharmacokinetic and drug delivery studies in rats, as well as TDM in human patients.

Pharmacokinetics of plasma lopinavir and ritonavir in tuberculosis-HIV co-infected African adult patients also receiving rifabutin 150 or 300 mg three times per week.

BACKGROUND: To evaluate the pharmacokinetic of plasma lopinavir (LPV) and ritonavir (RTV) when co-administered with three times weekly (TPW) rifabutin (RBT) at a dose of either 150 or 300 mg in African tuberculosis (TB) and HIV co-infected adult patients. METHODS: This is a pharmacokinetic study conducted in Ouagadougou among patients treated with a standard dosage of LPV/RTV 400/100 mg twice daily and RBT 150 mg TPW (arm A = 9 patients) or rifabutin 300 mg TPW (arm B = 7 patients) based regimens. Patients were recruited from the Bogodogo and Kossodo district hospitals in Ouagadougou from May 2013 to December 2015. Study inclusion criteria were that the patients were between 18 and 60 years of age, HIV-1 infected with pulmonary tuberculosis confirmed or suspected. Subsequent blood samples for pharmacokinetic monitoring were collected at 1, 2, 3, 4, 6, 8 and 12 h after combined drug ingestion for plasma drug monitoring using HPLC/MS assays. RESULTS: The medians LPV Cmax and Tmax were respectively, 20 µg/mL and 4 h for the RBT 150 mg group (arm A) and 7.7 µg/mL and 3 h for the RBT 300 mg group (arm B). The AUC0-12 of LPV was 111.8 µg h/mL in patients belonging to arm A versus 69.9 µg/mL for those in arm B (p = 0.313). The C0 of LPV was lower than 4 µg/mL in three patients receiving RBT 300 mg. Of note, the RTV plasma concentrations were nearly halved among patients on RBT 300 mg compared to those on lower RBT doses. The AUC0-12 of RTV in arm A was 12.7 µg h/mL versus 6.6 µg h/ml in arm B (p = 0.313). CONCLUSION: In our study, the pharmacokinetic of LPV and RTV was found to be highly variable when coadministrated with RBT 150 mg or 300 mg three times per week. There is a need for specific large study to verify clinical and virological effects of this variation, especially when coadministrated with RBT of 300 mg TPW, and to prevent viral resistance in response to under-dosing of LPV. Trial registration PACTR201310000629390. Registered 28 October 2013, http://www.pactr.org/.

Semi-quantification of HIV-1 protease inhibitor concentrations in clinical samples of HIV-infected patients using a gold nanoparticle-based immunochromatographic assay.

Individual drug concentration data can be a valuable tool for the clinical management of antiretroviral therapy (ART) for the treatment of HIV infection. High performance liquid chromatography (HPLC) based assays are currently the gold standard for drug measurement but its high cost and requirement of technical expertise limits its widespread use. Simpler user-friendly and inexpensive detection assays are needed. A novel immunochromatographic (IC) strip test to detect HIV-1 protease inhibitors (PIs) was fabricated by combining the proteolysis activity of HIV-protease (PR) and an immunochromatographic reaction. The PIs-IC strip cut-off to detect lopinavir (LPV) concentrations was set at 1,000 ng mL-1. We evaluated this novel PIs-IC strip for the semi-quantification of HIV PIs in plasma samples collected from healthy subjects and HIV-infected patients receiving antiretroviral treatment with and without LPV. LPV plasma drug levels were quantified by HPLC and evaluated (blinded to the HPLC results) using the PIs-IC strip. Results of plasma samples tested using the PIs-IC strip were available within 5 min. Using the PIs-IC strip test the accuracy, specificity and sensitivity were 97.8%, 97.1%, and 100%, respectively, compare to the gold-standard assay, to detect LPV in human plasma samples. This novel PIs-IC strip test could be used as a simple tool for the rapid monitoring of PIs levels in HIV-infected patients, although further clinical evaluation is needed.

Nanocapsules embedded in microparticles for enhanced oral bioavailability and efficacy of Lopinavir as an anti-AIDS drug.

Lopinavir (LPV), an efficient drug for HIV infection treatment, was incorporated into biodegradable PLGA nanocapsules (NCs) embedded in microparticles (MCPs) using the spray-drying technique in an attempt to bypass the P-gp efflux and protect the drug from CYP3A pre-systemic metabolism without ritonavir (RTV). SEM observations confirmed the formation of NCs and their entrapment in the MCPs. LPV-loaded NCs and free LPV were released from the MCPs at pH of 7.4 as evidenced by in vitro release studies. Results obtained from rat studies showed a two-fold higher bioavailability of LPV following oral administration of the optimal formulation than Kaletra®, the marketed drug, showing that when properly entrapped, LPV can be effectively protected from CYP degradation in the gut as well as from the liver following systemic absorption. It was also shown that serum derived from rats following LPV oral administration in two formulations and Kaletra® significantly decreased the multiplication of HIV-1 in cultured SupT1 cells. Furthermore, the LPV formulations markedly restricted the titre of infectious HIV-1 production compared with Kaletra® confirming the improved antiviral activity of LPV delivered in the rat blood circulation by the nanocapsules embedded in microparticle formulations.

Efavirenz and Lopinavir Levels in HIV-Infected Women and Their Nursing Infants, in Mali.

Limited data are currently available on antiretroviral pharmacokinetics in breast milk (BM) and in breastfed infants' blood. To explore these parameters in patients in Mali, we measured plasma antiretroviral levels in human immunodeficiency virus (HIV)-infected mothers and their breastfed infants over 6 months. We specifically analyzed the concentrations of efavirenz (EFV) and lopinavir (LPV) in the plasma of mothers living with HIV and their breastfed infants. Blood samples were collected at delivery and at month 1, 3, and 6 postpartum. EFV and LPV concentrations were measured by liquid chromatography-tandem mass spectrometry. HIV-1 RNA load was measured by Abbott M2000RT RealTime System at delivery and 6 months postpartum for mothers, and at 3 and 6 months postbirth for infants. The median duration of antiretroviral therapy at study inclusion was 57 months [interquartile range (IQR), 0-168 months]. The median EFV ratios of infant plasma/maternal plasma (MP) were 0.057 at month 1, 0.072 at month 3, and 0.048 at month 6. During the study period, the median BM/MP ratio of EFV was 1.16 (IQR, 0.96-20.62), which corresponds to a relative infant dose of 2.46% of the recommended weight-adjusted pediatric EFV dose at month 6. The apparent infant clearance of EFV was 0.146 l/h per kilogram at month 6. The LPV concentrations in the plasma of all infants were undetectable. No drug-related adverse reaction or toxicity was observed in any of the infants. The two women who presented a viral load of >50 copies/ml at month 6 had undetectable plasma drug concentrations at the same period. This study showed that breastfed infants received a low level of EFV but not LPV from their treated mothers.

Using dried blood spots to facilitate therapeutic drug monitoring of antiretroviral drugs in resource-poor regions.

Objectives: We evaluated whether dried blood spots (DBS) are suitable to monitor combined ART when samples are collected in rural Tanzania and transported over a long distance to a specialized bioanalytical laboratory. Methods: Plasma and DBS samples were collected in Tanzania from study patients treated with nevirapine, efavirenz or lopinavir. In addition, plasma, whole blood and DBS samples were obtained from a cohort of HIV patients at the site of the bioanalytical laboratory in Switzerland. DBS samples were analysed using a fully automated LC-MS/MS method. Results: Comparison of DBS versus plasma concentrations of samples obtained from the bridging study in Switzerland indicated an acceptable bias only for nevirapine (18.4%), whereas for efavirenz and lopinavir a pronounced difference of -47.4% and -48.1% was found, respectively. Adjusting the DBS concentrations by the haematocrit and the fraction of drug bound to plasma proteins removed this bias [efavirenz +9.4% (-6.9% to +25.7%), lopinavir +2.2% (-20.0% to +24.2%)]. Storage and transportation of samples from Tanzania to Switzerland did not affect the good agreement between plasma and DBS for nevirapine [-2.9% (-34.7% to +29.0%)] and efavirenz [-9.6% (-42.9% to +23.8%)]. For lopinavir, however, adjusted DBS concentrations remained considerably below [-32.8% (-70.4% to +4.8%)] corresponding plasma concentrations due to decay of lopinavir in DBS obtained under field conditions. Conclusions: Our field study shows that the DBS technique is a suitable tool for therapeutic drug monitoring in resource-poor regions; however, sample stability remains an issue for certain analytes and therefore needs special consideration.

Long-Acting Profile of 4 Drugs in 1 Anti-HIV Nanosuspension in Nonhuman Primates for 5 Weeks After a Single Subcutaneous Injection.

Daily oral antiretroviral therapy regimens produce limited drug exposure in tissues where residual HIV persists and suffer from poor patient adherence and disparate drug kinetics, which all negatively impact outcomes. To address this, we developed a tissue- and cell-targeted long-acting 4-in-1 nanosuspension composed of lopinavir (LPV), ritonavir, tenofovir (TFV), and lamivudine (3TC). In 4 macaques dosed subcutaneously, drug levels over 5 weeks in plasma, lymph node mononuclear cells (LNMCs), and peripheral blood mononuclear cells (PBMCs) were analyzed by liquid chromatography-tandem mass spectrometry. Plasma and PBMC levels of the active drugs (LPV, TFV, and 3TC) were sustained for 5 weeks; PBMC exposures to LPV, ritonavir, and 3TC were 12-, 16-, 42-fold higher than those in plasma. Apparent T1/2z of LPV, TFV, and 3TC were 219.1, 63.1, and 136.3 h in plasma; 1045.7, 105.9, and 127.7 h in PBMCs. At day 8, LPV, TFV, and 3TC levels in LNMCs were 4.1-, 5.0-, and 1.9-fold higher than in those in PBMCs and much higher than in plasma. Therefore, 1 dose of a 4-drug nanosuspension exhibited persistent drug levels in LNMCs, PBMCs, and plasma for 5 weeks. With interspecies scaling and dose adjustment, this 4-in-1 HIV drug-combination could be a long-acting treatment with the potential to target residual virus in tissues and improve patient adherence.

Mechanism-based pharmacokinetic (MBPK) models describe the complex plasma kinetics of three antiretrovirals delivered by a long-acting anti-HIV drug combination nanoparticle formulation.

Existing oral antiretroviral (ARV) agents have been shown in human studies to exhibit limited lymph node penetration and lymphatic drug insufficiency. As lymph nodes are a reservoir of HIV, it is critical to deliver and sustain effective levels of ARV combinations in these tissues. To overcome lymph node drug insufficiency of oral combination ARV therapy (cART), we developed and reported a long-acting and lymphocyte-targeting injectable that contains three ARVs-hydrophobic lopinavir (LPV) and ritonavir (RTV), and hydrophilic tenofovir (TFV)-stabilized by lipid excipients in a nanosuspension. A single subcutaneous (SC) injection of this first-generation formulation of drug combination nanoparticles (DcNPs), named TLC-ART101, provided persistent ARV levels in macaque lymph node mononuclear cells (LNMCs) for at least 1 week, and in peripheral blood mononuclear cells (PBMCs) and plasma for at least 2 weeks, demonstrating long-acting pharmacokinetics for all three drugs. In addition, the lymphocyte-targeting properties of this formulation were demonstrated by the consistently higher intracellular drug concentrations in LNMCs and PBMCs versus those in plasma. To provide insights into the complex mechanisms of absorption and disposition of TLC-ART101, we constructed novel mechanism-based pharmacokinetic (MBPK) models. Based upon plasma PK data obtained after single administration of TLC-ART101 (DcNPs) and a solution formulation of free triple-ARVs, the models feature uptake from the SC injection site that respectively routes free and nanoparticle-associated ARVs via the blood vasculature and lymphatics, and their eventual distribution into and clearance from the systemic circulation. The models provided simultaneous description of the complex long-acting plasma and lymphatic PK profiles for all three ARVs in TLC-ART101. The long-acting PK characteristics of the three drugs in TLC-ART101 were likely due to a combination of mechanisms including: (1) DcNPs undergoing preferential lymphatic uptake from the subcutaneous space, (2) retention in nodes during lymphatic first-pass, (3) subsequent slow release of ARVs into blood circulation, and (4) limited extravasation of DcNP-associated ARVs that resulted in longer persistence in the circulation.

Pharmacokinetics and Drug-Drug Interactions of Lopinavir-Ritonavir Administered with First- and Second-Line Antituberculosis Drugs in HIV-Infected Children Treated for Multidrug-Resistant Tuberculosis.

Lopinavir-ritonavir forms the backbone of current first-line antiretroviral regimens in young HIV-infected children. As multidrug-resistant (MDR) tuberculosis (TB) frequently occurs in young children in high-burden TB settings, it is important to identify potential interactions between MDR-TB treatment and lopinavir-ritonavir. We describe the pharmacokinetics of and potential drug-drug interactions between lopinavir-ritonavir and drugs routinely used for MDR-TB treatment in HIV-infected children. A combined population pharmacokinetic model was developed to jointly describe the pharmacokinetics of lopinavir and ritonavir in 32 HIV-infected children (16 with MDR-TB receiving treatment with combinations of high-dose isoniazid, pyrazinamide, ethambutol, ethionamide, terizidone, a fluoroquinolone, and amikacin and 16 without TB) who were established on a lopinavir-ritonavir-containing antiretroviral regimen. One-compartment models with first-order absorption and elimination for both lopinavir and ritonavir were combined into an integrated model. The dynamic inhibitory effect of the ritonavir concentration on lopinavir clearance was described using a maximum inhibition model. Even after adjustment for the effect of body weight with allometric scaling, a large variability in lopinavir and ritonavir exposure, together with strong correlations between the pharmacokinetic parameters of lopinavir and ritonavir, was detected. MDR-TB treatment did not have a significant effect on the bioavailability, clearance, or absorption rate constants of lopinavir or ritonavir. Most children (81% of children with MDR-TB, 88% of controls) achieved therapeutic lopinavir trough concentrations (>1 mg/liter). The coadministration of lopinavir-ritonavir with drugs routinely used for the treatment of MDR-TB was found to have no significant effect on the key pharmacokinetic parameters of lopinavir or ritonavir. These findings should be considered in the context of the large interpatient variability found in the present study and the study's modest sample size.

Impact of Single Nucleotide Polymorphisms on Plasma Concentrations of Efavirenz and Lopinavir/Ritonavir in Chinese Children Infected with the Human Immunodeficiency Virus.

Single nucleotide polymorphisms (SNPs) in the genes that encode the cytochrome P450 (CYP) drug metabolizing enzymes and drug transporters have been reported to influence antiretroviral drug pharmacokinetics. Although primarily metabolized by CYP2B6 and -3A, efavirenz (EFV) and lopinavir/ritonavir (LPV/r) are substrates of P-glycoprotein and the solute carrier organic (SLCO) anion transporter, respectively. We investigated the association between SNPs and efavirenz (EFV) or lopinavir/ritonavir (LPV/r) concentrations in Chinese children infected with the human immunodeficiency virus (HIV). Genotyping was performed on CYP2B6 516G→T, -1459C→T, and -983T→C, ABCB1 3435C→T, and SLCO1B1 521T→C in 229 HIV-infected Chinese pediatric patients (age range 4.0 to 17.5 yrs). Plasma concentrations of EFV and LPV/r were measured using validated high-performance liquid chromatography coupled with the mass spectrum method among 39 and 69 children who received EFV- and LPV/r-containing regimens, respectively. The frequencies of CYP2B6 516G→T in the study participants were 71%, 25%, and 4% for the G/G, G/T, and T/T genotypes, respectively. Among the children under therapeutic drug monitoring, 21% and 39% experienced EFV and LPV concentrations, respectively, above the upper threshold of the therapeutic window. CYP2B6 516G→T was significantly associated with EFV concentrations (p<0.001). Older children (older than 10 yrs) were more likely to have significantly higher EFV concentrations than the younger ones (p=0.0314). CYP2B6 genotyping and EFV concentration monitoring may help optimize antiretroviral therapy in pediatric patients who initiate an EFV-based regimen.