Identification of Potential Antiviral Inhibitors from Hydroxychloroquine and 1,2,4,5-Tetraoxanes Analogues and Investigation of the Mechanism of Action in SARS-CoV-2.

This study aimed to identify potential inhibitors and investigate the mechanism of action on SARS-CoV-2 ACE2 receptors using a molecular modeling study and theoretical determination of biological activity. Hydroxychloroquine was used as a pivot structure and antimalarial analogues of 1,2,4,5 tetraoxanes were used for the construction and evaluation of pharmacophoric models. The pharmacophore-based virtual screening was performed on the Molport® database (~7.9 million compounds) and obtained 313 structures. Additionally, a pharmacokinetic study was developed, obtaining 174 structures with 99% confidence for human intestinal absorption and penetration into the blood-brain barrier (BBB); posteriorly, a study of toxicological properties was realized. Toxicological predictions showed that the selected molecules do not present a risk of hepatotoxicity, carcinogenicity, mutagenicity, and skin irritation. Only 54 structures were selected for molecular docking studies, and five structures showed binding affinity (ΔG) values satisfactory for ACE2 receptors (PDB 6M0J), in which the molecule MolPort-007-913-111 had the best ΔG value of -8.540 Kcal/mol, followed by MolPort-002-693-933 with ΔG = -8.440 Kcal/mol. Theoretical determination of biological activity was realized for 54 structures, and five molecules showed potential protease inhibitors. Additionally, we investigated the Mpro receptor (6M0K) for the five structures via molecular docking, and we confirmed the possible interaction with the target. In parallel, we selected the TopsHits 9 with antiviral potential that evaluated synthetic accessibility for future synthesis studies and in vivo and in vitro tests.

A Review of Modifications of Quinoline Antimalarials: Mefloquine and (hydroxy)Chloroquine.

Late-stage modification of drug molecules is a fast method to introduce diversity into the already biologically active scaffold. A notable number of analogs of mefloquine, chloroquine, and hydroxychloroquine have been synthesized, starting from the readily available active pharmaceutical ingredient (API). In the current review, all the modifications sites and reactivity types are summarized and provide insight into the chemistry of these molecules. The approaches include the introduction of simple groups and functionalities. Coupling to other drugs, polymers, or carriers afforded hybrid compounds or conjugates with either easily hydrolyzable or more chemically inert bonds. The utility of some of the compounds was tested in antiprotozoal, antibacterial, and antiproliferative assays, as well as in enantiodifferentiation experiments.

[The Inhibiting Effect of Autophagy Inhibitor ROC-325 on Multiple Myeloma].

OBJECTIVE: To investigate the effects of autophagy inhibitor ROC-325 and its combination with bortezomib on the proliferation, apoptosis and autophagy of multiple myeloma cell lines. METHODS: Multiple myeloma cells were treated with ROC-325 at different concentration. The cell proliferation was detected by CCK-8. Apoptosis was determined by Caspase-3/7 and Caspase-9 activity assays. Autophagy was detected by monodansylcadaverine staining. The apoptosis-related proteins (PARP and Caspase-3) and autophagy-related proteins (P62, Beclin-1, and LC3A/B) were analyzed by Western blot. The combined effect with bortezomib on bortezomib-resistant cell line was detected by CCK-8. RESULTS: ROC-325 inhibited the proliferation of RPMI 8226, RPMI 8226-BTZ100, U266 and IM9 cells in a dose-dependent manner (r=-0.8275, r=-0.9079, r=-0.9422, r=-0.9305), the 72 h IC50 values were 2.795, 4.020, 5.432 and 4.755 µmol/L, respectively. The activity assays of Caspase-3/7 and Caspase-9 showed that their relative activity was increased gradually in proportion to the drug concentration with the statistically significant difference (r=0.9648, r=0.9377, r=0.9318; r=0.9087, r=0.9431, r=0.8914). MDC staining results showed that the number of autophagic vacuoles increased with the rise of ROC-325 concentration (r=0.9565, r=0.9373, r=0.9233). ROC-325 could increase the expression of apoptosis-related proteins (PARP and Caspase-3) and autophagy-related proteins (P62 and LC3-Ⅱ/LC3-Ⅰ), but decrease the expression of Beclin-1 detected by Western blot. The CCK-8 assay showed that ROC-325 combined with bortezomib had synergistic effect on the inhibition of drug resistant cell line RPMI 8226-BTZ100. CONCLUSION: ROC-325 can inhibit the proliferation, induce the apoptosis of myeloma cells through the mitochondrial pathway, inhibit the autophagy of myeloma cells by affecting the fusion of autophagosomes and lysosomes, and overcome bortezomib resistance by the combination of ROC-325 with bortezomib.

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.

Analogue discovery of safer alternatives to HCQ and CQ drugs for SAR-CoV-2 by computational design.

COVID-19 outbreak poses a severe health emergency to the global community. Due to availability of limited data, the selection of an effective treatment is a challenge. Hydroxychloroquine (HCQ), a chloroquine (CQ) derivative administered for malaria and autoimmune diseases, has been shown to be effective against both Severe Acute Respiratory Syndrome (SARS-CoV-1) and SARS-CoV-2. Apart from the known adverse effects of these drugs, recently the use of CQ and HCQ as a potential treatment for COVID-19 is under flux globally. In this study, we focused on identifying a more potent analogue of HCQ and CQ against the spike protein of SAR-CoV-2 that can act as an effective antiviral agent for COVID-19 treatment. Systematic pharmacokinetics, drug-likeness, basicity predictions, virtual screening and molecular dynamics analysis (200 ns) were carried out to predict the inhibition potential of the analogous compounds on the spike protein. This work identifies the six potential analogues, out of which two compounds, namely 1-[1-(6-Chloroquinolin-4-yl) piperidin-4-yl]piperidin-3-ol and (1R,2R)-2-N-(7-Chloroquinolin-4-yl)cyclohexane-1,2-diamine interact with the active site of the spike protein similar to HCQ and CQ respectively with augmented safety profile.

The Inhibiting Effect of Autophagy Inhibitor ROC-325 on Multiple Myeloma / 中国实验血液学杂志

OBJECTIVE@#To investigate the effects of autophagy inhibitor ROC-325 and its combination with bortezomib on the proliferation, apoptosis and autophagy of multiple myeloma cell lines.@*METHODS@#Multiple myeloma cells were treated with ROC-325 at different concentration. The cell proliferation was detected by CCK-8. Apoptosis was determined by Caspase-3/7 and Caspase-9 activity assays. Autophagy was detected by monodansylcadaverine staining. The apoptosis-related proteins (PARP and Caspase-3) and autophagy-related proteins (P62, Beclin-1, and LC3A/B) were analyzed by Western blot. The combined effect with bortezomib on bortezomib-resistant cell line was detected by CCK-8.@*RESULTS@#ROC-325 inhibited the proliferation of RPMI 8226, RPMI 8226-BTZ100, U266 and IM9 cells in a dose-dependent manner (r=-0.8275, r=-0.9079, r=-0.9422, r=-0.9305), the 72 h IC@*CONCLUSION@#ROC-325 can inhibit the proliferation, induce the apoptosis of myeloma cells through the mitochondrial pathway, inhibit the autophagy of myeloma cells by affecting the fusion of autophagosomes and lysosomes, and overcome bortezomib resistance by the combination of ROC-325 with bortezomib.

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.

Molecular Insights into the MAPK Cascade during Viral Infection: Potential Crosstalk between HCQ and HCQ Analogues.

The mitogen-activated protein kinase (MAPK) pathway links the cell-surface receptors to the transcription machinery, transducing the extracellular signals into several outputs, which may also adapt the host defense mechanism to viral attacks. The Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) that causes the COrona VIrus Disease 2019 (COVID-19) has infected upwards of nearly 70 million people and worldwide has claimed more than 1,600,000 deaths. So far, there continues to be no specific treatment for this novel coronavirus-induced disease. In the search to control the global COVID-19 pandemic, some eastern and developing countries have approved a variety of treatments with controversial efficacy, among which is the use of the antimalarial hydroxychloroquine (HCQ). Interestingly, prior data had indicated that the HCQ/CQ could influence the MAPK cascade. The main aim of this review is to address molecular mechanisms, beyond drugs, that can be helpful against viral infection for this and future pandemics. We will highlight (1) the contribution of the MAPK cascade in viral infection and (2) the possible use of MAPK inhibitors in curbing viral infections, alone or in combination with HCQ and quinoline analogues. We are convinced that understanding the molecular patterns of viral infections will be critical for new therapeutical approaches to control this and other severe diseases.

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.

Factors Related to Blood Hydroxychloroquine Concentration in Patients With Systemic Lupus Erythematosus.

OBJECTIVE: To identify factors associated with blood concentrations of hydroxychloroquine (HCQ) and its major metabolite, N-desethylhydroxychloroquine (DHCQ), in patients with systemic lupus erythematosus (SLE; lupus) receiving long-term oral HCQ treatment. METHODS: SLE patients who had been taking HCQ for more than 3 months were recruited. Various clinical characteristics, laboratory values, and SLE Disease Activity Index (SLEDAI) scores were examined. The concentrations of HCQ and DHCQ ([HCQ] and [DHCQ]) were measured by liquid chromatography mass spectrometry, and the relationship between [HCQ], [DHCQ], and [HCQ]:[DHCQ] ratio to various factors was investigated. RESULTS: In total, 189 SLE patients receiving long-term HCQ treatment were included in the analysis. The median (interquartile range [IQR]) [HCQ] was 515 (IQR 353-720) ng/ml, the median [DHCQ] was 417 (IQR 266-591) ng/ml, and the median [HCQ]:[DHCQ] ratio was 1.3 (range 1.0-1.7). [HCQ] was closely associated with [DHCQ] (r = 0.81, P < 0.0001). The weight-adjusted oral HCQ dose was strongly associated with both [HCQ] (P < 0.001) and [DHCQ] (P < 0.001). Time since last dose was associated with [HCQ] (P < 0.001). No statistically significant association was found between renal function or smoking and [HCQ] or [DHCQ]. Use of additional immunosuppressants increased both [HCQ] and [DHCQ] after adjusting for possible confounders (P = 0.04 and P = 0.03, respectively). The lower SLEDAI score was significantly related to higher [HCQ], after adjusting for age, sex, weight-adjusted HCQ dose, time since last dose, number of other immunosuppressants, and smoking status (P = 0.007). CONCLUSION: Various factors affected blood levels of [HCQ], [DHCQ], or the [HCQ]:[DHCQ] ratio of SLE patients receiving long-term oral HCQ treatment. Notably, higher [HCQ] was associated with a lower SLEDAI score in our typical outpatient clinic population with lupus.

Disruption of Autophagic Degradation with ROC-325 Antagonizes Renal Cell Carcinoma Pathogenesis.

Purpose: Although autophagy plays important roles in malignant pathogenesis and drug resistance, there are few clinical agents that disrupt this pathway, and the potential therapeutic benefit of autophagy inhibition remains undetermined. We used medicinal chemistry approaches to generate a series of novel agents that inhibit autophagic degradation.Experimental Design: ROC-325 was selected as a lead compound for further evaluation. Comprehensive in vitro and in vivo studies were conducted to evaluate the selectivity, tolerability, and efficacy of ROC-325 in preclinical models of renal cell carcinoma (RCC) with HCQ serving as a comparator. Markers of autophagy inhibition and cell death were evaluated in tumor specimens.Results: ROC-325 exhibited superior in vitro anticancer effects compared with the existing autophagy inhibitor hydroxychloroquine (HCQ) in 12 different cancer cell lines with diverse genetic backgrounds. Focused studies of the mechanism of action and efficacy of ROC-325 in RCC cells showed that drug treatment induced hallmark characteristics of autophagy inhibition, including accumulation of autophagosomes with undegraded cargo, lysosomal deacidification, p62 stabilization, and disruption of autophagic flux. Subsequent experiments showed that ROC-325 antagonized RCC growth and survival in an ATG5/7-dependent manner, induced apoptosis, and exhibited favorable selectivity. Oral administration of ROC-325 to mice bearing 786-0 RCC xenografts was well tolerated, was significantly more effective at inhibiting tumor progression than HCQ, and inhibited autophagy in vivoConclusions: Our findings demonstrate that ROC-325 has superior preclinical anticancer activity compared with HCQ and support the clinical investigation of its safety and preliminary efficacy in patients with RCC and other autophagy-dependent malignancies. Clin Cancer Res; 23(11); 2869-79. ©2016 AACR.

Association of Polymorphisms of Cytochrome P450 2D6 With Blood Hydroxychloroquine Levels in Patients With Systemic Lupus Erythematosus.

OBJECTIVE: To evaluate associations of genetic polymorphisms in cytochrome P450 (CYP) isoforms 2D6, 3A5, and 3A4 with blood concentrations of hydroxychloroquine (HCQ) and its metabolite, N-desethyl HCQ (DHCQ), in patients with systemic lupus erythematosus (SLE). METHODS: SLE patients taking HCQ for >3 months were recruited and were genotyped for 4 single-nucleotide polymorphisms in CYP2D6*10, CYP3A5*3, and CYP3A4*18B. Blood HCQ and DHCQ concentrations ([HCQ] and [DHCQ]) were measured and their association with corresponding genotypes was investigated. RESULTS: A total of 194 patients were included in the analysis. CYP2D6*10 polymorphisms (rs1065852 and rs1135840) were significantly associated with the [DHCQ]:[HCQ] ratio after adjustment for age, sex, dose per weight per day, and SLE Disease Activity Index score (P = 0.03 and P < 0.01, respectively). In adjusted models, the [DHCQ]:[HCQ] ratio was highest in patients with the G/G genotype of the CYP2D6*10 (rs1065852) polymorphism and lowest in those with the A/A genotype (P = 0.03). Similarly, the [DHCQ]:[HCQ] ratio was highest in patients with the C/C genotype of the CYP2D6*10 (rs1135840) polymorphism and lowest in those with the G/G genotype (P < 0.01). The CYP2D6*10 (rs1065852) polymorphism was significantly related to the [DHCQ] (P = 0.01). However, the polymorphisms of CYP3A5*3 and CYP3A4*18B did not show any significant association with the [HCQ], [DHCQ], or [DHCQ]:[HCQ] ratio. CONCLUSION: Our study showed that the [DHCQ]:[HCQ] ratio was related to CYP2D6 polymorphisms in Korean lupus patients taking oral HCQ. CYP polymorphisms may explain why there is wide variation in blood HCQ concentrations. The role of an individual's CYP polymorphisms should be considered when prescribing oral HCQ.

Development, validation and clinical application of a LC-MS/MS method for the simultaneous quantification of hydroxychloroquine and its active metabolites in human whole blood.

A rapid, sensitive and specific method using liquid chromatography coupled to tandem mass spectrometry was developed for the simultaneous quantification of hydroxychloroquine (HCQ) and its three major metabolites in human whole blood. The assay, using a sample volume of 100µL, was linear in a dynamic 25-2000ng/mL range (R(2)>0.99) for all four compounds and suitable for the determination of elevated HCQ concentrations up to 20,000ng/mL, after appropriate sample dilution. Inter- and intra-assay precisions were <18.2% and accuracies were between 84% and 113% for any analyte. No matrix effects were observed. The assay was successfully applied to a blood sample obtained from one poisoned patient following a massive HCQ self-ingestion resulting in an estimated concentration of 19,500ng/mL on hospital admission. In this patient, HCQ metabolites were identified and quantified at 1123, 465 and 91ng/mL for monodesethylhydroxychloroquine, desethylchloroquine and bisdesethylchloroquine, respectively. Further investigations are still required to assess the usefulness of the simultaneous measurement of blood concentrations of HCQ and its three active metabolites for monitoring HCQ treatment and managing HCQ poisoning.

Growth inhibitory and differentiation effects of chloroquine and its analogue on human leukemic cells potentiate fetal hemoglobin production by targeting the polyamine pathway.

Elevated arginase activity has been implicated in several pathological conditions in sickle cell disease (SCD) and other inflammatory disorders. Recently, we showed that chloroquine (CQ), an anti-malarial and anti-rheumatoid drug, displays a competitive mode of inhibition on sickle erythrocyte arginase. However, the effects of CQ and its analogue, hydroxychloroquine (HCQ) on erythroid differentiation leading to induced fetal hemoglobin (Hb F) production is unknown. In the present study, we obtained evidence of the anti-proliferative and differentiation effects of CQ and HCQ at pharmacologically attainable concentrations. This differentiation effect was linked to a dose-dependent inhibition of arginase activity and induced hemoglobinization, as Hb F synthesis was increased by 3.4- and 3.2-fold for CQ or HCQ, respectively. Treatment of K562 cells with lipopolysaccharide (LPS) or 8-bromo-cAMP (Br-cAMP) failed to reverse the inhibitory effects of CQ or HCQ on arginase activity. Indeed, the combination of Br-cAMP with CQ in LPS-treated cells resulted in a significant enhancement of Hb F and total hemoglobin production. Further, we showed that CQ or HCQ maximally stimulated intracellular cGMP levels by 6.6- and 3.0-fold at 6 and 3h, respectively, as demonstrated by immunosorbent assay. However, co-treatment of K562 cells with CQ or HCQ in the presence of inhibitors of sGC-PKG-pathways reduced Hb F stimulation, suggesting the possible involvement of the sGC-PKG pathway. This is the first evidence demonstrating the capacity of anti-rheumatoid drugs to modulate the arginine-pathway and result in the enhancement of Hb F production, and thus may provide a paradigm for targeted therapy of hemoglobinopathies and other inflammation-related disorders.

Blood concentrations of hydroxychloroquine and its desethyl derivative correlate negatively with the percentage of CD45RO+ cells among CD4+ lymphocytes in hydroxychloroquine-treated lupus patients.

The objective of the study was to investigate the influence of the blood concentrations of hydroxychloroquine ([HCQ]) and its derivative desethylhydroxychloroquine ([DHCQ]) on lymphocyte activation or differentiation in HCQ-treated lupus patients. We studied the correlations between [HCQ], [DHCQ], and the frequency of various lymphocyte subsets in 58 HCQ-treated lupus patients (mean HCQ dose: 4.93 +/- 1.58 mg/kg/day; mean duration of the disease: 122 +/- 64 months). [HCQ] and [DHCQ] were determined by high-performance liquid chromatography (HPLC). Lymphocyte markers were studied by flow cytometry using monoclonal anti-CD3, -CD4, -CD8, -CD25, -DR, -CD45RA, -CD45RO, -CD19, -CD38, and -CD86 antibodies. sIL2-R serum concentrations were measured by enzyme-linked immunosorbent assay (ELISA). [HCQ] and [DHCQ] were 599.9 ng/mL (median: 529.5; range: 55-1935) and 353.43 (median: 286 ng/mL; range: 118-1090). In a multiple regression analysis, [HCQ] and [DHCQ] were associated with the HCQ prescribed dose in mg/kg/day (P = 0.0002 and P = 0.03) and with compliance to the treatment (P = 0.004 and P = 0.03). We found a negative correlation between [HCQ], [DHCQ], and the CD45RO+ cell frequency among CD3+CD4+ cells (P = 0.03 and P = 0.007, respectively). Other lymphocyte subset markers (LSMs) and sIL2-R concentrations were not significantly associated with [HCQ] or [DHCQ]. In the multiple regression analysis, CD45RO+ expression was negatively influenced by [HCQ] (P = 0.005), and positively influenced by smoking habits (P = 0.005) and age (P = 0.005). Similar results were found in the multivariate model including [DHCQ]. Disease activity and taking more than 10 mg/day of corticosteroids or an immunosuppressive drug did not influence CD45RO+ expression. Lupus patients had less CD3+CD4+CD45RO+ cells than controls (P = 0.03). In lupus patients, HCQ and DHCQ may alter the generation or the blood circulation of CD4+CD45RO+ lymphocytes in a concentration-dependent pattern.

Capillary electrophoretic chiral separation of hydroxychloroquine and its metabolites in the microsomal fraction of liver homogenates.

A rapid, selective, and low-cost chiral capillary electrophoretic method was developed for the simultaneous analysis of hydroxychloroquine (HCQ) and its three chiral metabolites: desethylchloroquine (DCQ), desethylhydroxychloroquine (DHCQ), and bisdesethylchloroquine (BDCQ) in the microsomal fraction of liver homogenates. After liquid-liquid extraction using toluene as extracting solvent, the drug and metabolites were resolved on a fused-silica capillary (50 microm ID, 50 cm total length, and 42 cm effective length), using 100 mmol/L of Tris/phosphate buffer, pH 9.0 containing 1% w/v sulfated-beta-CD and 30 mg/mL hydroxypropyl-beta-CD. Detection was carried out at 220 nm. The extraction procedure was efficient in removing endogenous interferents, and low values (

[Analysis of 925 patients on long-term hydroxychloroquine or chloroquine treatment: results of ophthalmological screening]. / Surveillance ophtalmologique des patients sous APS au long cours: analyse d'une population de 925 patients.

PURPOSE: The aim of this 4-Year study was to analyze the population referred to our laboratory for the visual follow-up of hydroxychloroquine or chloroquine treatment. MATERIAL AND METHODS: We received 925 patients: 78% female, 22% male. For each patient, a 13-item criteria was filled out and regular exams were performed. The pathologies were divided in to 4 groups: rheumatoid polyarthritis (P), lupus (L), sarcoidosis (S), others (O). RESULTS: The pathologies were distributed as follows: 48% "P", 29% "L", 3% "S", 1% "P + L", 19% "O". Of these patients, 19% had less than 1 Year of treatment, 73% 1-10 Years and 8% more than 10 Years. The screening exposed no retinal intoxications but 3% presented pre-clinical intoxication (PCI) and 80% were allowed to continue their treatment. The most important statistical results were: 1) a significant relation between the PCI and the duration of the treatment (p<0.001); 2) a non-significant relation between the PCI and the daily dose (p=0.417); and 3) a significant relation between PCI and the cumulative dose (p=0.003). CONCLUSION: The results shows the advantage of the ERG in screening to prevent anti-malarial retinal toxicity. This study confirms that the cumulative dose seems to be more important than the daily dose, but we agree with the international consensus to respect a daily dose under 6.5 mg/kg/d. The results also demonstrated that, with this large and diversified population, there is a need for prospective and multi-centric studies. With the above results, international standards should be established in order to obtain the most efficient screening for each category of patient.

Hydroxychloroquine concentration-response relationships in patients with rheumatoid arthritis.

OBJECTIVE: A dose-response relationship for hydroxychloroquine (HCQ), in terms of the proportion of patients achieving the Paulus 20% criteria for improvement, had previously been observed in patients with rheumatoid arthritis (RA) receiving a 6-week loading regimen of 400, 800, or 1,200 mg HCQ daily. This present retrospective analysis was performed to investigate possible relationships between the blood HCQ and HCQ-metabolite concentrations and measures of efficacy and toxicity. In addition, we sought to ascertain whether further investigation of HCQ/HCQ-metabolite levels might lead to testing of one of these substances as a new antirheumatic drug. METHODS: Patients with active RA (n = 212) began a 6-week, double-blind trial comparing 3 different doses of HCQ at 400, 800, or 1,200 mg/day, followed by 18 weeks of open-label HCQ treatment at 400 mg/day. Patients were repeatedly evaluated for treatment efficacy and toxicity. Blood samples were available from 123 patients for analysis of HCQ, desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylchloroquine (BDCQ) levels using high-performance liquid chromatography. Achievement of the modified Paulus 20% improvement criteria for response in RA was used as the primary efficacy parameter. Spontaneously reported adverse events were categorized and analyzed as toxicity outcome variables. The relationship between response (efficacy and toxicity) and drug levels was evaluated using logistic regression analysis. RESULTS: The subset of patients with blood concentration data was equivalent to the larger study population in all demographic and outcome characteristics. The mean HCQ, DHCQ, and DCQ elimination half-lives were 123, 161, and 180 hours, respectively. There was a positive correlation between the Paulus 20% improvement criteria response and blood DHCQ concentrations during weeks 1-6 (P < 0.001). A potential relationship between ocular adverse events and BDCQ levels was found (P = 0.036). Logistic regression analysis of adverse events data showed that adverse gastrointestinal events were associated with higher HCQ levels (P = 0.001-0.021) during weeks 1, 2, and 3. CONCLUSION: There is a weak, but predictable, relationship between blood DHCQ concentrations and efficacy of treatment with HCQ. In addition, there is an association between gastrointestinal adverse events and elevated blood HCQ concentrations. Further investigation of these relationships is warranted to see if DHCQ may be introduced as a new antirheumatic drug.