Quadruple isotope dilution gas chromatography-mass spectrometry after simultaneous derivatization and spraying based fine droplet formation liquid phase microextraction method for the accurate and sensitive quantification of chloroquine phosphate in human serum, urine and saliva samples at trace levels.

This study presents an accurate and precise analytical strategy for the determination of chloroquine phosphate at trace levels in human body fluids (urine, serum, and saliva). Simultaneous derivatization-spraying based fine droplet formation-liquid phase microextraction (SD-SFDF-LPME) method was used to derivatize and preconcentrate the analyte prior to gas chromatography-mass spectrometry (GC-MS) measurements. Acetic anhydride was employed as derivatizing agent in this study. After optimizing the SD-SFDF-LPME method, the limit of detection (LOD) and limit of quantitation (LOQ) were found to be 0.16 and 0.53 mg/kg, respectively. Quadruple isotope dilution (ID4) was coupled to the SD-SFDF-LPME method in order to alleviate matrix effects and promote accuracy/precision of the method. Chloroquine acetamide-d3 was firstly synthesized in our research laboratory and used as the isotopic analogue of the analyte in the ID4 experiments. Superior percent recovery results (99.4% - 101.0%) with low standard deviation values were obtained for the spiked samples. This validated the developed SD-SFDF-LPME-ID4-GC-MS method as highly accurate and precise for the determination of chloroquine phosphate at trace levels. In addition, the isotopic analogue of the analyte was obtained via the acetamide derivative of the analyte, which is an alternative to obtain isotopic analogues of organic compounds that are not accessible or commercially available.

Development and validation of an LC-MS/MS method for determination of hydroxychloroquine, its two metabolites, and azithromycin in EDTA-treated human plasma.

BACKGROUND: Hydroxychloroquine (HCQ) and azithromycin (AZM) are antimalarial drugs recently reported to be active against severe acute respiratory syndrome coronavirus- 2 (SARS-CoV-2), which is causing the global COVID-19 pandemic. In an emergency response to the pandemic, we aimed to develop a quantitation method for HCQ, its metabolites desethylhydroxychloroquine (DHCQ) and bisdesethylchloroquine (BDCQ), and AZM in human plasma. METHODS: Liquid chromatography tandem mass spectrometry was used to develop the method. Samples (20 µL) are extracted by solid-phase extraction and injected onto the LC-MS/MS system equipped with a PFP column (2.0 × 50 mm, 3 µm). ESI+ and MRM are used for detection. Ion pairs m/z 336.1→247.1 for HCQ, 308.1→179.1 for DHCQ, 264.1→179.1 for BDCQ, and 749.6→591.6 for AZM are selected for quantification. The ion pairs m/z 342.1→253.1, 314.1→181.1, 270.1→181.1, and 754.6→596.6 are selected for the corresponding deuterated internal standards (IS) HCQ-d4, DHCQ-d4, BDCQ-d4, and AZM-d5. The less abundant IS ions from 37Cl were used to overcome the interference from the analytes. RESULTS: Under optimized conditions, retention times are 0.78 min for BDCQ, 0.79 min for DHCQ, 0.92 min for HCQ and 1.87 min for AZM. Total run time is 3.5 min per sample. The calibration ranges are 2-1000 ng/mL for HCQ and AZM, 1-500 ng/mL for DHCQ and 0.5-250 ng/mL for BDCQ; samples above the range are validated for up to 10-fold dilution. Recoveries of the method ranged from 88.9-94.4% for HCQ, 88.6-92.9% for DHCQ, 88.7-90.9% for BDCQ, and 98.6%-102% for AZM. The IS normalized matrix effect were within (100±10) % for all 4 analytes. Blood samples are stable for at least 6 hr at room temperature. Plasma samples are stable for at least 66 hr at room temperature, 38 days at -70°C, and 4 freeze-thaw cycles. CONCLUSIONS: An LC-MS/MS method for simultaneous quantitation of HCQ, DHCQ, BDCQ, and AZM in human plasma was developed and validated for clinical studies requiring fast turnaround time and small samples volume.

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.

The extent of chloroquine underdosing in adult patients with malaria by Plasmodium vivax from an endemic area of the Brazilian Amazon basin.

OBJECTIVES: To evaluate the extent of chloroquine underdosing and to measure the concentrations of chloroquine and desethylchloroquine in adult patients with P. vivax malaria in the Brazilian Amazon basin. METHODS: Prospective study of cases in male adult patients with malaria by Plasmodium vivax treated with a total dose of 1500 mg chloroquine over three days and a short course of primaquine. Patients were weighed at admission, and the dose per mg/kg was determined. Blood samples were collected at 24 and 168 h after enrolment, and the concentrations of chloroquine and desethylchloroquine were measured in plasma by high-performance liquid chromatography with fluorescence detection. RESULTS: Of 61 patients were included in the study, and 60% received a total dose of chloroquine below 25 mg/kg. Plasma chloroquine concentrations ranged from 90 to 184 ng/ml and from 175 to 827 ng/ml at 24 and 168 hours. For desethylchloroquine, the values ranged from 32 to 144 ng/ml and from 90 to 440 ng/ml at 24 and 168 h. There were no significant correlations between the plasma levels of chloroquine and the doses administered (mg/kg) at 24 and 196 h. Similar results were found for desethylchloroquine. CONCLUSION: There is widespread suboptimal dosing of chloroquine that is probably due to the dosing regimen based on patient age, which reduces the drug exposure with a possible influence on parasite clearance.

OBJECTIFS: Evaluer l'impact du sous-dosage de la chloroquine et mesurer les concentrations de chloroquine et de déséthylchloroquine chez les patients adultes atteints de paludisme à P. vivax dans le bassin de l'Amazonie brésilienne. MÉTHODES: Nous avons mené une étude prospective de cas chez des patients adultes de sexe masculin atteints de paludisme par Plasmodium vivax traités avec une dose totale de 1500 mg de chloroquine sur trois jours et une courte cure de primaquine. Les patients ont été pondérés à l'admission et la dose par mg/kg a été déterminée. Des échantillons de sang ont été prélevés 24 et 168 heures après l'enrôlement dans l'étude, et les concentrations de chloroquine et de déséthylchloroquine ont été mesurées dans le plasma par chromatographie liquide à haute performance avec détection par fluorescence. RÉSULTATS: Un total de 61 patients a été inclu dans l'étude et 60% ont reçu une dose totale de chloroquine en dessous de 25 mg/kg. Les concentrations plasmatiques de chloroquine variaient de 90 à 184 ng/ml et de 175 à 827 ng/ml à 24 et 168 heures. Pour la déséthylchloroquine, les valeurs variaient de 32 à 144 ng/ml et de 90 à 440 ng/ml à 24 et à 168 heures. Il n'y avait pas de corrélation significative entre les taux plasmatiques de chloroquine et les doses administrées (mg/kg) à 24 et à 168 heures. Des résultats similaires ont été trouvés pour la déséthylchloroquine. CONCLUSION: Il existe un dosage sous-optimal répandu de chloroquine qui est probablement dû au schéma posologique basé sur l'âge du patient, ce qui réduit l'exposition au médicament avec une influence possible sur la clairance des parasites.

Concentration-dependent mortality of chloroquine in overdose.

Hydroxychloroquine and chloroquine are used extensively in malaria and rheumatological conditions, and now in COVID-19 prevention and treatment. Although generally safe they are potentially lethal in overdose. In-vitro data suggest that high concentrations and thus high doses are needed for COVID-19 infections, but as yet there is no convincing evidence of clinical efficacy. Bayesian regression models were fitted to survival outcomes and electrocardiograph QRS durations from 302 prospectively studied French patients who had taken intentional chloroquine overdoses, of whom 33 died (11%), and 16 healthy volunteers who took 620 mg base chloroquine single doses. Whole blood concentrations of 13.5 µmol/L (95% credible interval 10.1-17.7) were associated with 1% mortality. Prolongation of ventricular depolarization is concentration-dependent with a QRS duration >150 msec independently highly predictive of mortality in chloroquine self-poisoning. Pharmacokinetic modeling predicts that most high dose regimens trialled in COVID-19 are unlikely to cause serious cardiovascular toxicity.

Hydroxychloroquine and chloroquine are closely-related drugs used for the treatment of malaria and rheumatological conditions, such as lupus. Laboratory tests have indicated that these drugs could also be used against the virus that causes COVID-19. Given the urgent need, these drugs have been fast-tracked into large-scale clinical trials, bypassing the usual stages that would provide estimates for suitable dosage. The dosage is a critical factor in a clinical trial: too low and the drug will not have an effect, too high and the side effects may counteract any potential benefits. Laboratory tests suggest that higher doses of chloroquine or hydroxychloroquine are needed for treating COVID-19 compared to malaria or lupus. However, there are concerns about the high doses used in some trials, as the drugs can have lethal side effects. Indeed, chloroquine has been used extensively in suicide attempts, particularly in France. To address these concerns, Watson et al. set out to determine the highest dosage of chloroquine (and thus of hydroxychloroquine, approximately) that does not cause unacceptable side effects. First, data was analysed regarding the concentration of chloroquine in the blood of 302 patients who had intentionally overdosed on the drug, since this concentration is tightly correlated with their risk of death. Watson et al. used a statistical model to calculate the maximal chloroquine concentration in a person's blood associated with a one per cent risk of death. This is taken to be the threshold above which any potential benefit of chloroquine treatment would be outweighed by the possibility of lethal toxicity. Watson et al. also estimated the relationship between chloroquine concentrations and changes in electrocardiogram patterns, which record the electrical activity of the heart. This makes it possible to determine whether a high dose of chloroquine has led to dangerous levels in the blood. Using a mathematical model of how chloroquine is metabolised, Watson et al. predicted that most of the trials that tested chloroquine as a treatment for COVID-19 did not reach the calculated threshold concentration. An exception was the CloroCovid-19 trial in Brazil, which was stopped early because people in the higher dosage group suffered more heart problems and died in greater numbers than those in the lower dosage group. Two large randomised trials, RECOVERY and SOLIDARITY, have shown no benefit of hydroxychloroquine or chloroquine in the treatment of COVID-19, changing clinical practice worldwide. Both of these trials used high doses resulting in higher hydroxychloroquine or chloroquine concentrations than normally observed in the treatment of malaria or rheumatological conditions. The results from Watson et al demonstrate that the lack of benefit seen in these two large clinical trials is not due to the drug dosage being too high.

Safety, Pharmacokinetics, and Activity of High-Dose Ivermectin and Chloroquine against the Liver Stage of Plasmodium cynomolgi Infection in Rhesus Macaques.

Previously, ivermectin (1 to 10 mg/kg of body weight) was shown to inhibit the liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (50% inhibitory concentration [IC50], 10.42 µM) and hypnozoites (IC50, 29.24 µM) in primary macaque hepatocytes when administered as a high dose prophylactically but not when administered in radical cure mode. The safety, pharmacokinetics, and efficacy of oral ivermectin (0.3, 0.6, and 1.2 mg/kg) with and without chloroquine (10 mg/kg) administered for 7 consecutive days were evaluated for prophylaxis or radical cure of P. cynomolgi liver stages in rhesus macaques. No inhibition or delay to blood-stage P. cynomolgi parasitemia was observed at any ivermectin dose (0.3, 0.6, and 1.2 mg/kg). Ivermectin (0.6 and 1.2 mg/kg) and chloroquine (10 mg/kg) in combination were well-tolerated with no adverse events and no significant pharmacokinetic drug-drug interactions observed. Repeated daily ivermectin administration for 7 days did not inhibit ivermectin bioavailability. It was recently demonstrated that both ivermectin and chloroquine inhibit replication of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro Further ivermectin and chloroquine trials in humans are warranted to evaluate their role in Plasmodium vivax control and as adjunctive therapies against COVID-19 infections.

A Sensitive and Optimized HPLC-FLD Method for the Simultaneous Quantification of Hydroxychloroquine and Its Two Metabolites in Blood of Systemic Lupus Erythematosus Patients.

Quantification of hydroxychloroquine (HCQ) and its two metabolites desethylchloroquine and desethylhydroxychloroquine in human blood can provide insight into the pharmacokinetic/pharmacodynamic characteristics of HCQ for the treatment of systemic lupus erythematosus (SLE), which is crucial for the optimization of the therapy. A simple, sensitive and optimized high performance liquid chromatography with fluorescence detection method has been developed and validated for the simultaneous determination of HCQ and its two metabolites in human blood. After addition of internal standard chloroquine, the blood sample was deproteinized with 2-fold acetonitrile and separated on an YMC-Triart C18 column (250 × 4.6 mm, 5 µm) with a mobile phase of 20 mM sodium phosphate buffer solution containing 0.25% triethylamine (pH 8.0)-acetonitrile (60:40, v/v). The analytes were detected by using fluorescence detection at an excitation and emission wavelength of 337 and 405 nm, respectively. The method was linear over the range of 3-3000 ng/mL for all three analytes and the chromatographic run time was 9 min. The values for intra- and inter-day precisions were ranged from 1.3 to 7.3. This method was successfully applied to quantify the concentrations of HCQ and its two metabolites in blood of 92 SLE patients.

Population Pharmacokinetics and Pharmacodynamics of Chloroquine in a Plasmodium vivax Volunteer Infection Study.

Chloroquine has been used for the treatment of malaria for > 70 years; however, chloroquine pharmacokinetic (PK) and pharmacodynamic (PD) profile in Plasmodium vivax malaria is poorly understood. The objective of this study was to describe the PK/PD relationship of chloroquine and its major metabolite, desethylchloroquine, in a P. vivax volunteer infection study. We analyzed data from 24 healthy subjects who were inoculated with blood-stage P. vivax malaria and administered a standard treatment course of chloroquine. The PK of chloroquine and desethylchloroquine was described by a two-compartment model with first-order absorption and elimination. The relationship between plasma and whole blood concentrations of chloroquine and P. vivax parasitemia was characterized by a PK/PD delayed response model, where the equilibration half-lives were 32.7 hours (95% confidence interval (CI) 27.4-40.5) for plasma data and 24.1 hours (95% CI 19.0-32.7) for whole blood data. The estimated parasite multiplication rate was 17 folds per 48 hours (95% CI 14-20) and maximum parasite killing rate by chloroquine was 0.213 hour-1 (95% CI 0.196-0.230), translating to a parasite clearance half-life of 4.5 hours (95% CI 4.1-5.0) and a parasite reduction ratio of 400 every 48 hours (95% CI 320-500). This is the first study that characterized the PK/PD relationship between chloroquine plasma and whole blood concentrations and P. vivax clearance using a semimechanistic population PK/PD modeling. This PK/PD model can be used to optimize dosing scenarios and to identify optimal dosing regimens for chloroquine where resistance to chloroquine is increasing.

Doses of chloroquine in the treatment of malaria by Plasmodium vivax in patients between 2 and 14 years of age from the Brazilian Amazon basin.

BACKGROUND: A total dose of chloroquine of 25 mg/kg is recommended by the World Health Organization (WHO) to treat malaria by Plasmodium vivax. In several endemic areas, including the Brazilian Amazon basin, anti-malarial drugs are dispensed in small plastic bags at a dosing regimen based on age. This practice can lead to suboptimal dosing of the drug, which can impact treatment outcomes. The aim of the present study was to estimate the extent of sub-dosing of chloroquine in children and adolescents with vivax malaria using an age-based dose regimen, in addition to investigating the influence of age on the plasma concentrations of chloroquine and desethylchloroquine. METHODS: A study of cases was conducted with male patients with a confirmed infection by P. vivax, ages 2 to 14 years, using a combined regimen of chloroquine and primaquine. Height, weight and body surface area were determined at admission on the study. The total dose of chloroquine administered was estimated based on the weight and on the body surface area of the study patients. Chloroquine and desethylchloroquine were measured on Day 7 in each patient included in the study by a high-performance liquid chromatographic method with fluorescence detection. RESULTS: A total of 81 patients were enrolled and completed the study. The median age was 9 years (2-14 years). All patients presented negative blood smears at 42 days follow-up. The total dose of chloroquine ranged from 13.1 to 38.1 mg/kg. The percentage of patients with a total dose of the drug below 25 mg/kg ranged from 29.4 to 63.6%. The total dose of chloroquine administered based on BSA ranged from 387 to 1079 mg/m2, increasing with age. Plasma chloroquine concentrations ranged from 107 to 420 ng/ml, increasing with age. For desethylchloroquine, the plasma concentrations ranged from 167 to 390 ng/ml, with similar values among age-groups. CONCLUSION: The data demonstrated the widespread exposure of children and adolescents to suboptimal doses of chloroquine in the endemic area investigated.

Development and Validation of a Fast Ultra-High Performance Liquid Chromatography-Fluorescent Method for the Quantification of Hydroxychloroquine and Its Metabolites in Patients With Lupus.

BACKGROUND: Hydroxychloroquine (HCQ) is approved for the treatment of systemic lupus erythematosus (SLE). Therapeutic drug monitoring of HCQ is necessary to detect nonadherence and to improve treatment efficacy in patients with SLE. Liquid chromatographic-tandem mass spectroscopy and high performance liquid chromatography (HPLC)-fluorescent methods are currently used to measure whole blood concentrations of HCQ and its 2 main metabolites desethylhydroxychloroquine and desethylchloroquine in patients with SLE. This study reports the development and validation of an ultra-HPLC (U-HPLC) method with fluorescence detection for the simultaneous quantification of HCQ and its metabolites in whole blood. METHODS: After adding chloroquine (internal standard) to the samples, a single-step protein precipitation and a subsequent filtration were used for blood sample preparation. Analytes were separated under isocratic elution on a U-HPLC RP18 column with a total run time of 7 minutes. The mobile phase consisted of piperazine buffer (46.4 mM, pH = 9.8) and acetonitrile (68:32, vol/vol), which was delivered at a flow rate of 0.4 mL/min. Fluorescence excitation and emission wavelengths were 335 and 390 nm, respectively. Assay performance parameters were evaluated per FDA bioanalytical guidelines. RESULTS: The calibration curve was linear from 125 to 4000 ng/mL for HCQ. The lower limit of quantification was 10 ng/mL for all analytes. For HCQ, desethylchloroquine, and desethylhydroxychloroquine, accuracies and imprecisions ranged from -7.90% to 7.85% and 1.14% to 8.78%, respectively. CONCLUSIONS: A sensitive, accurate, and fast U-HPLC-fluorescent method was validated and successfully applied to quantify whole blood concentrations to perform therapeutic drug monitoring of HCQ in pediatric and adult lupus patients.

Recrudescence, Reinfection, or Relapse? A More Rigorous Framework to Assess Chloroquine Efficacy for Plasmodium vivax Malaria.

Background: Plasmodium vivax resistance to chloroquine (CQ) has been reported worldwide, although the World Health Organization clinical drug efficacy studies protocol does not permit classification of patient outcomes. Methods: We enrolled 40 patients with P. vivax malaria in northeastern Cambodia, where >17% treatment failures were previously reported. Patients were treated with CQ (30 mg/kg) and followed for 2 months, with frequent clinical examination and capillary blood sample collection for microscopy, molecular parasite detection and genotyping, and drug concentration measurements. Reinfections were prevented by relocating patients to a transmission-free area. Results: P. vivax parasites were eliminated in all patients by day 3. Genomic analyses revealed that all clones in polyclonal infections were cleared at the same rate, indicating their equal susceptibility to CQ. CQ blood concentrations were below the therapeutic level in all recurrent infections (24 of 40 patients), which were efficiently cleared by a second course of CQ treatment. Genotyping (128 SNPs barcode) and sequences of entire parasite genome (Whole-Genome Sequencing, Illumina) indicated that two thirds (6 of 8) of the recurrent parasites resulted from heterologous relapses whose 50% are from by sibling/recombinant clones. Conclusions: No evidence of CQ resistance was observed. Our data suggest that P. vivax antimalarial drug resistance is likely overestimated and that the current guidelines for clinical drug studies of P. vivax malaria need to be revised.

Chloroquine-Primaquine Therapeutic Efficacy, Safety, and Plasma Levels in Patients with Uncomplicated Plasmodium vivax Malaria in a Colombian Pacific Region.

In Colombia, published studies for the treatment of uncomplicated Plasmodium vivax malaria with chloroquine-primaquine (CQ-PQ) are scarce. The aim of the study was to evaluate the therapeutic efficacy and safety profile of this combination. A clinical trial was performed in adults with uncomplicated P. vivax malaria using the 28-day World Health Organization validated protocol. Patients received supervised antimalarial treatment and the primary efficacy end point was the clinical and parasitological response. Safety was assessed through adverse events surveillance, and plasma levels of antimalarial drugs were measured. A total of 77 patients were included. Adequate clinical and parasitological response rate diagnosed by thick blood smear examination was achieved in 72 of 73 patients (98.6%) with a complete 28-day follow-up. There were two parasitological therapeutic failures (TFs) (2.9%) on day 28, established by polymerase chain reaction among 68 patients, one of them with a positive film. No adverse events were detected. After completing the antimalarial treatment, all patients reached adequate plasma concentrations of CQ and desethylchloroquine (DECQ), with medians of 302.9 and 104.0 ng/mL, respectively. Uncomplicated P. vivax malaria treatment with CQ-PQ standard treatment was effective and safe in the study population; TFs were not associated with low plasma levels of CQ and DECQ.

Effect of Antiretroviral Therapy on Plasma Concentrations of Chloroquine and Desethyl-chloroquine.

The effect of antiretroviral therapy (ART) on chloroquine and desethyl-chloroquine plasma concentrations was evaluated in clinical trial participants. Concentrations did not differ among participants receiving protease inhibitor-based ART (n = 9), efavirenz-based ART (n = 15), or other ART (n = 8) and those not receiving ART (n = 31). Efavirenz seemed to inhibit chloroquine desethylation.

Hydroxychloroquine: balancing the need to maintain therapeutic levels with ocular safety: an update.

PURPOSE OF REVIEW: Antimalarial drugs including chloroquine, its less toxic quinolone-derivative hydroxychloroquine (HCQ), and quinacrine have become cornerstones in the treatment of autoimmune diseases including systemic lupus, rheumatoid arthritis, sarcoidosis, and Sjogren syndrome; cutaneous disorders, antiphospholipid syndrome, and have recently been employed at higher dioses in oncology. Benefits include anti-inflammatory effects, protection against thrombosis, and improved control of hyperglycemia and hyperlipidemia. In general, both the therapeutic advantages and the toxic effects of the drugs correlate with the dose and the duration of therapy. Here we summarize the current literature regarding the administration and the safety profile of HCQ in management of rheumatologic disease and focus on the most recent revised American Academy of Ophthalmology (AAO) guidelines for prevention and detection of hydroxychloroquine retinopathy to help guide therapeutic decision-making for patients. RECENT FINDINGS: The risk of antimalarial-induced retinal toxicity is better predicted by calculating the daily dosage based on 5 mg/kg total body weight rather than 6.5 mg/kg lean body weight and reducing dosage in patients with risk factors such as renal failure. The risk of retinal toxicity after 5 years is substantially increased even when these guidelines are followed; hence dose reduction is appropriate with long-term use. Newer techniques provide improved detection of early signs of retinal damage. These advances are reflected in the revised AAO guidelines 2016, which are in part based on the retrospective study by Melles and Marmor of HCQ toxicity. SUMMARY: The most important changes in practice guidelines include dose calculation based on total body weight, dose reduction after long-term use, and intensified screening with techniques including optical coherence tomography (OCT) after 5 years.

Health status and concomitant prescription of immunosuppressants are risk factors for hydroxychloroquine non-adherence in systemic lupus patients with prolonged inactive disease.

Background/objective The objectives of this paper are to assess the extent of and the factors associated with hydroxychloroquine (HCQ) non-adherence in systemic lupus erythematosus (SLE) patients with prolonged inactive disease and to investigate relationships between blood HCQ concentration and quality of life (QoL). Methods Consecutive SLE patients, in remission for at least one year and taking a stable dose of HCQ were investigated. At study entry (T0) and six months later (T6) a blood venous sample was taken to measure whole blood concentration of [HCQ] and desethylchloroquine ([DCQ]). Moreover, at T0 each patient completed validated questionnaires assessing QoL, disability, anxiety, depression and visual analogue scales for fatigue, pain, general health (GH), and self-assessment of disease activity. Results Eighty-three patients with a median [HCQ] of 327 ng/ml were enrolled. At T0, 24 (29%) were defined as non-adherent ([HCQ] < 100 ng/ml). At multiple logistic regression analysis the physical summary of SF-36 ( p = 0.038), and the concomitant use of immunosuppressants ( p = 0.010) were independently associated with non-adherence. A significant increase of HCQ adherence was observed at T6 ( p < 0.05). Conclusions A better health status and the concomitant prescription of immunosuppressants represent risk factors for HCQ non-adherence in SLE patients in remission. Monitoring HCQ levels might represent an important opportunity to improve adherence.

Optimal antimalarial dose regimens for chloroquine in pregnancy based on population pharmacokinetic modelling.

Despite extensive use and accumulated evidence of safety, there have been few pharmacokinetic studies from which appropriate chloroquine (CQ) dosing regimens could be developed specifically for pregnant women. Such optimised CQ-based regimens, used as treatment for acute malaria or as intermittent preventive treatment in pregnancy (IPTp), may have a valuable role if parasite CQ sensitivity returns following reduced drug pressure. In this study, population pharmacokinetic/pharmacodynamic modelling was used to simultaneously analyse plasma concentration-time data for CQ and its active metabolite desethylchloroquine (DCQ) in 44 non-pregnant and 45 pregnant Papua New Guinean women treated with CQ and sulfadoxine/pyrimethamine or azithromycin (AZM). Pregnancy was associated with 16% and 49% increases in CQ and DCQ clearance, respectively, as well as a 24% reduction in CQ relative bioavailability. Clearance of DCQ was 22% lower in those who received AZM in both groups. Simulations based on the final multicompartmental model demonstrated that a 33% CQ dose increase may be suitable for acute treatment for malaria in pregnancy as it resulted in equivalent exposure to that in non-pregnant women receiving recommended doses, whilst a double dose would likely be required for an effective duration of post-treatment prophylaxis when used as IPTp especially in areas of CQ resistance. The impact of co-administered AZM was clinically insignificant in simulations. The results of past/ongoing trials employing recommended adult doses of CQ-based regimens in pregnant women should be interpreted in light of these findings, and consideration should be given to using increased doses in future trials.

Evaluation of pharmacokinetics of single-dose chloroquine in malnourished children with malaria- a comparative study with normally nourished children.

OBJECTIVES: Studies on antimalarial kinetics in children or adults who are undernourished or malnourished are both limited and have yielded conflicting results. The present study was carried out with the objectives of evaluating the pharmacokinetics of single dose chloroquine and its metabolite desethylchloroquine in children who were undernourished and compare them with children who were normally nourished. METHODS: Children of either gender between the ages of 5 and 12 years, smear positive for P. vivax malaria and classified either as well nourished or undernourished were included. Undernourishment was adjudged based on the Indian Academy of Pediatrics (IAP) classification of protein energy malnutrition [PEM] which in turn was based on Khadilkar's growth charts. All participants received 10 mg/kg on the first day followed by 10 mg/kg on Day 2 and 5 mg/kg on Day 3 along with supportive treatment. Blood samples for the levels of chloroquine [CQ] and desethylchloroquine [DECQ] were collected at 0, 0.5, 1, 2 4, 8, 12, 24, 48, 72 hours and 14 days after the first dose and levels assessed by High Performance Liquid Chromatography. RESULTS: A total of 12 children who were normally nourished and 13 who were undernourished were studied. Wide inter-individual variability was seen in the levels of both drug and metabolite in both groups of patients. However, the differences in Cmax, AUC 0-inf, Clearance, half life and Vd between the two groups were not significantly different. DISCUSSION: Our results indicate that dosage requirement is unlikely to be needed for chloroquine in undernourished children with uncomplicated P. vivax malaria.

Development and validation of an HILIC-MS/MS method by one-step precipitation for chloroquine in miniature pig plasma.

BACKGROUND: Quantification of polar compounds such as chloroquine by revered-phase LC is a challenge because of poor retention and silanol interactions with stationary phase. Strong ion-pairing reagents added to mobile phases to improve reversed-phase retention and improve peak shape can be harmful for MS. RESULTS: This new approach provides a rapid and sensitive method for the detection of chloroquine using hydrophilic interaction LC coupled to MS/MS (HILIC-MS/MS). Ammonium formate and formic acid were added to mobile phase to attain good peak shapes and the salified chloroquine as well retained in an HILIC column. Linearity, intra- and inter-day precision, accuracy, recovery, matrix effect and stability were evaluated during the validation process. CONCLUSION: The validated method has been successfully used in a PK study in miniature pigs, and paves way for future development.

An HPLC method with diode array detector for the simultaneous quantification of chloroquine and desethylchloroquine in plasma and whole blood samples from Plasmodium vivax patients in Vietnam, using quinine as an internal standard.

A sensitive, simple method for quantification of chloroquine (CQ) and desethylchloroquine (MCQ) in whole blood and plasma from Plasmodium vivax patients has been developed using HPLC with diode array detection (DAD). Solid-phase extraction on Isolute-96-CBA was employed to process 100 µL of plasma/whole blood samples. CQ, MCQ and quinine were separated using a mobile phase of phosphate buffer 25 mm, pH 2.60-acetonitrile (88:12, v/v) with 2 mm sodium perchlorate on a Zorbax SB-CN 150 × 4.6 mm, 5 µm column at a flow rate of 1.2 mL/min, at ambient temperature in 10 min, with the DAD wavelength of 343 nm. The method was linear over the range of 10-5000 ng/mL for both CQ and MCQ in plasma and whole blood. The limit of detection was 4 ng/mL and limit of quantification was 10 ng/mL in both plasma and blood for CQ and MCQ. The intra-, inter- and total assay precision were <10% for CQ and MCQ in plasma and whole blood. In plasma, the accuracies varied between 101 and 103%, whereas in whole blood, the accuracies ranged from 97.0 to 102% for CQ and MCQ. The method is an ideal technique with simple facilities and instruments, bringing about good separation in comparison with previous methods. © 2016 The Authors Biomedical Chromatography Published by John Wiley & Sons Ltd.