Osimertinib Covalently Binds to CD34 and Eliminates Myeloid Leukemia Stem/Progenitor Cells.

Osimertinib is a third-generation covalent EGFR inhibitor that is used in treating non-small cell lung cancer. First-generation EGFR inhibitors were found to elicit pro-differentiation effect on acute myeloid leukemia (AML) cells in preclinical studies, but clinical trials yielded mostly negative results. Here, we report that osimertinib selectively induced apoptosis of CD34+ leukemia stem/progenitor cells but not CD34- cells in EGFR-negative AML and chronic myeloid leukemia (CML). Covalent binding of osimertinib to CD34 at cysteines 199 and 177 and suppression of Src family kinases (SFK) and downstream STAT3 activation contributed to osimertinib-induced cell death. SFK and STAT3 inhibition induced synthetic lethality with osimertinib in primary CD34+ cells. CD34 expression was elevated in AML cells compared with their normal counterparts. Genomic, transcriptomic, and proteomic profiling identified mutation and gene expression signatures of patients with AML with high CD34 expression, and univariate and multivariate analyses indicated the adverse prognostic significance of high expression of CD34. Osimertinib treatment induced responses in AML patient-derived xenograft models that correlated with CD34 expression while sparing normal CD34+ cells. Clinical responses were observed in two patients with CD34high AML who were treated with osimertinib on a compassionate-use basis. These findings reveal the therapeutic potential of osimertinib for treating CD34high AML and CML and describe an EGFR-independent mechanism of osimertinib-induced cell death in myeloid leukemia. SIGNIFICANCE: Osimertinib binds CD34 and selectively kills CD34+ leukemia cells to induce remission in preclinical models and patients with AML with a high percentage of CD34+ blasts, providing therapeutic options for myeloid leukemia patients.

Identification of SARS-CoV-2 m6A modification sites correlate with viral pathogenicity.

It has recently been found that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) m6A modifications can affect viral replication and function. However, no studies to date have shown a correlation between SARS-CoV-2 m6A modifications and viral pathogenicity. In this study, we analyzed m6A modification in 2,190,667 SARS-CoV-2 genomic RNAs. m6A modifications of SARS-CoV-2 from different lineages, causing mild or severe COVID-19 and showing breakthrough for different vaccines were analyzed to explore correlations with viral pathogenicity. The results suggested that the presence of more m6A modifications in the SARS-CoV-2 N region (positive strand) correlates with weaker pathogenicity. In addition, we identified three m6A modification sites correlating with weak pathogenicity (924 in ORF1ab, 15,659 in ORF1ab, 28,288 in N, 28,633 in N and 29,385 in N, 29,707 in 3'UTR) and one with strong pathogenicity (74 in 5'UTR). These results provide new information for understanding the prevalence of SARS-CoV-2 and controlling the virus.

A two-stage genome-wide association study identifies novel germline genetic variations in CACNA2D3 associated with radiotherapy response in nasopharyngeal carcinoma.

BACKGROUND: Radiotherapy (RT) is the standard treatment for nasopharyngeal carcinoma (NPC). However, due to individual differences in radiosensitivity, biomarkers are needed to tailored radiotherapy to cancer patients. However, comprehensive genome-wide radiogenomic studies on them are still lacking. The aim of this study was to identify genetic variants associated with radiotherapy response in patients with NPC. METHODS: This was a large­scale genome-wide association analysis (GWAS) including a total of 981 patients. 319 individuals in the discovery stage were genotyped for 688,783 SNPs using whole genome-wide screening microarray. Significant loci were further genotyped using MassARRAY system and TaqMan SNP assays in the validation stages of 847 patients. This study used logistic regression analysis and multiple bioinformatics tools such as PLINK, LocusZoom, LDBlockShow, GTEx, Pancan-meQTL and FUMA to examine genetic variants associated with radiotherapy efficacy in NPC. RESULTS: After genome-wide level analysis, 19 SNPs entered the validation stage (P < 1 × 10- 6), and rs11130424 ultimately showed statistical significance among these SNPs. The efficacy was better in minor allele carriers of rs11130424 than in major allele carriers. Further stratified analysis showed that the association existed in patients in the EBV-positive, smoking, and late-stage (III and IV) subgroups and in patients who underwent both concurrent chemoradiotherapy and induction/adjuvant chemotherapy. CONCLUSION: Our study showed that rs11130424 in the CACNA2D3 gene was associated with sensitivity to radiotherapy in NPC patients. TRIAL REGISTRATION NUMBER: Effect of genetic polymorphism on nasopharyngeal carcinoma chemoradiotherapy reaction, ChiCTR-OPC-14005257, Registered 18 September 2014, http://www.chictr.org.cn/showproj.aspx?proj=9546 .

C1Q labels a highly aggressive macrophage-like leukemia population indicating extramedullary infiltration and relapse.

Extramedullary infiltration (EMI) is a concomitant manifestation that may indicate poor outcome of acute myeloid leukemia (AML). The underlying mechanism remains poorly understood and therapeutic options are limited. Here, we employed single-cell RNA sequencing on bone marrow (BM) and EMI samples from a patient with AML presenting pervasive leukemia cutis. A complement C1Q+ macrophage-like leukemia subset, which was enriched within cutis and existed in BM before EMI manifestations, was identified and further verified in multiple patients with AML. Genomic and transcriptional profiling disclosed mutation and gene expression signatures of patients with EMI that expressed high levels of C1Q. RNA sequencing and quantitative proteomic analysis revealed expression dynamics of C1Q from primary to relapse. Univariate and multivariate analysis demonstrated adverse prognosis significance of C1Q expression. Mechanistically, C1Q expression, which was modulated by transcription factor MAF BZIP transcription factor B, endowed leukemia cells with tissue infiltration ability, which could establish prominent cutaneous or gastrointestinal EMI nodules in patient-derived xenograft and cell line-derived xenograft models. Fibroblasts attracted migration of the C1Q+ leukemia cells through C1Q-globular C1Q receptor recognition and subsequent stimulation of transforming growth factor ß1. This cell-to-cell communication also contributed to survival of C1Q+ leukemia cells under chemotherapy stress. Thus, C1Q served as a marker for AML with adverse prognosis, orchestrating cancer infiltration pathways through communicating with fibroblasts and represents a compelling therapeutic target for EMI.

Mitochondrial oxidative phosphorylation is dispensable for survival of CD34+ chronic myeloid leukemia stem and progenitor cells.

Chronic myeloid leukemia (CML) are initiated and sustained by self-renewing malignant CD34+ stem cells. Extensive efforts have been made to reveal the metabolic signature of the leukemia stem/progenitor cells in genomic, transcriptomic, and metabolomic studies. However, very little proteomic investigation has been conducted and the mechanism regarding at what level the metabolic program was rewired remains poorly understood. Here, using label-free quantitative proteomic profiling, we compared the signature of CD34+ stem/progenitor cells collected from CML individuals with that of healthy donors and observed significant changes in the abundance of enzymes associated with aerobic central carbonate metabolic pathways. Specifically, CML stem/progenitor cells expressed increased tricarboxylic acid cycle (TCA) with decreased glycolytic proteins, accompanying by increased oxidative phosphorylation (OXPHOS) and decreased glycolysis activity. Administration of the well-known OXPHOS inhibitor metformin eradicated CML stem/progenitor cells and re-sensitized CD34+ CML cells to imatinib in vitro and in patient-derived tumor xenograft murine model. However, different from normal CD34+ cells, the abundance and activity of OXPHOS protein were both unexpectedly elevated with endoplasmic reticulum stress induced by metformin in CML CD34+ cells. The four major aberrantly expressed protein sets, in contrast, were downregulated by metformin in CML CD34+ cells. These data challenged the dependency of OXPHOS for CML CD34+ cell survival and underlined the novel mechanism of metformin. More importantly, it suggested a strong rationale for the use of tyrosine kinase inhibitors in combination with metformin in treating CML.

Complex analysis of the personalized pharmacotherapy in the management of COVID-19 patients and suggestions for applications of predictive, preventive, and personalized medicine attitude.

AIMS: Coronavirus disease 2019 (COVID-19) is rapidly spreading worldwide. Drug therapy is one of the major treatments, but contradictory results of clinical trials have been reported among different individuals. Furthermore, comprehensive analysis of personalized pharmacotherapy is still lacking. In this study, analyses were performed on 47 well-characterized COVID-19 drugs used in the personalized treatment of COVID-19. METHODS: Clinical trials with published results of drugs use for COVID-19 treatment were collected to evaluate drug efficacy. Drug-to-Drug Interactions (DDIs) were summarized and classified. Functional variations in actionable pharmacogenes were collected and systematically analysed. "Gene Score" and "Drug Score" were defined and calculated to systematically analyse ethnicity-based genetic differences, which are important for the safer use of COVID-19 drugs. RESULTS: Our results indicated that four antiviral agents (ritonavir, darunavir, daclatasvir and sofosbuvir) and three immune regulators (budesonide, colchicine and prednisone) as well as heparin and enalapril could generate the highest number of DDIs with common concomitantly utilized drugs. Eight drugs (ritonavir, daclatasvir, sofosbuvir, ribavirin, interferon alpha-2b, chloroquine, hydroxychloroquine (HCQ) and ceftriaxone had actionable pharmacogenomics (PGx) biomarkers among all ethnic groups. Fourteen drugs (ritonavir, daclatasvir, prednisone, dexamethasone, ribavirin, HCQ, ceftriaxone, zinc, interferon beta-1a, remdesivir, levofloxacin, lopinavir, human immunoglobulin G and losartan) showed significantly different pharmacogenomic characteristics in relation to the ethnic origin of the patient. CONCLUSION: We recommend that particularly for patients with comorbidities to avoid serious DDIs, the predictive, preventive, and personalized medicine (PPPM, 3 PM) strategies have to be applied for COVID-19 treatment, and genetic tests should be performed for drugs with actionable pharmacogenes, especially in some ethnic groups with a higher frequency of functional variations, as our analysis showed. We also suggest that drugs associated with higher ethnic genetic differences should be given priority in future pharmacogenetic studies for COVID-19 management. To facilitate translation of our results into clinical practice, an approach conform with PPPM/3 PM principles was suggested. In summary, the proposed PPPM/3 PM attitude should be obligatory considered for the overall COVID-19 management. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13167-021-00247-0.

HE4 and eIF3a Expression Correlates with Surgical Outcome and Overall Survival in Ovarian Cancer Patients with Secondary Cytoreduction.

For recurrent ovarian cancer (ROC), secondary cytoreductive surgery (SCS) is recommended as one optional treatment. However, little is known about the expression and clinical significance of biomarkers during SCS. Human epididymis protein 4 (HE4) is a clinical biomarker for ovarian cancer. Eukaryotic translation initiation factor 3a (eIF3a) is investigated extensively as a potential biomarker for malignancy. The purpose of this study was to investigate the expressions of HE4 and eIF3a at SCS, as well as their associations with surgical outcome and survival in ROC patients. Immunohistochemistry was performed to determine the expressions of HE4 and eIF3a in ovarian tumors taken from both initial and secondary cytoreductive surgery of 35 ROC patients. eIF3a levels were significantly increased at SCS, compared to those at initial cytoreductive surgery (ICS), while HE4 levels were similar. Both HE4 and eIF3a expressions were associated with surgical outcome, in terms of residual tumor. For ICS, patients with high HE4 expression achieved a higher incidence of optimal cytoreduction than those with low HE4 expression (81.0% vs. 33.3%, P = 0.015). A similar result happened in SCS, indicated by higher incidence of no residual tumor in patients with high HE4 expression (76.4% vs. 44.4%, P = 0.046). And high HE4 expression at SCS was more likely to enhance surgical outcome of SCS (77.8% vs. 29.4%, P = 0.038). Therefore, high HE4 expression at either surgery is a predictor of better overall survival (OS) (P = 0.011 and 0.002). Furthermore, patients with an elevated total score (TS) of HE4 between the two surgeries tended to have prolonged OS, compared to those with a non-elevated TS of HE4 (P = 0.076). For eIF3a, initial eIF3a expression was associated with secondary residual tumor (P = 0.035), and the difference in eIF3a expression between the two surgeries correlated with OS (P = 0.052). The expressions of HE4 and eIF3a in tumor specimens correlated with surgical outcome and predicted OS in ROC patients with SCS, thus meriting further investigation.

Downregulation of RIF1 Enhances Sensitivity to Platinum-Based Chemotherapy in Epithelial Ovarian Cancer (EOC) by Regulating Nucleotide Excision Repair (NER) Pathway.

BACKGROUND/AIMS: Rap1 interacting factor 1 (RIF1) was deemed to be involved in replication timing regulation and DNA damage response. However, little is known about the role of RIF1 in malignancies. Thus, this study aimed to investigate whether the expression of RIF1 is relevant to the response of epithelial ovarian cancer (EOC) patients to cisplatin chemotherapy and its underlying mechanism. METHODS: Immunohistochemistry was used for detecting the expression of RIF1 in 72 human ovarian cancer tissues followed by association analysis of RIF1 expression with patients' responses to platinum-based chemotherapy. The survival analysis of ovarian patients based on platinum chemotherapy was analyzed using online databases. RNA interference of RIF1 was carried out in OVCAR3 and A2780 cell lines, to determine the effect of lacking RIF1 expression on cellular responses to cisplatin by using MTS assay. The nucleotide excision repair (NER) capacity of these cells was assessed by using host-cell reactivation and UV sensitivity assay. Western Blot analysis was carried out to determine the effect of RIF1 on the proteins of NER and apoptosis signaling pathway by using RIF1 knockdown cells. BALB/c nude mice model was used for detection of response to cisplatin in vivo. RESULTS: RIF1 expression was significantly associated with the response of ovarian patients to platinum-based chemotherapy (P< 0.01). In cohorts from online databases, high expression of RIF1 was associated with higher mortality of EOC patients based on platinum chemotherapy (P < 0.01). RIF1 knockdown increased sensitivity to cisplatin in EOC in vitro and in vivo. Deletion of RIF1 impaired the NER activity by inhibiting the NER proteins in ovarian cancer cells. Besides, knockdown of RIF1 enhanced cisplatin-induced apoptosis. CONCLUSIONS: RIF1 plays an important role in regulating the expression of NER proteins, which in turn contributes to cellular response to cisplatin and EOC patients' response to platinum-based chemotherapy. RIF1 knockdown also promotes cisplatin-induced apoptosis. RIF1 may serve as a novel biomarker for predicting platinum-based chemosensitivity and the prognosis of EOC patients.

Increased HE4 mRNA Expression Correlates with High Level Of eIF3a mRNA And Better Survival in Women with Epithelial Ovarian Cancer.

Human epididymis protein 4 (HE4) is one of the most promising biomarkers for epithelial ovarian cancer (EOC). The majority of previous studies utilized the serum level or tissue protein expression of HE4 based upon immunohistochemistry (IHC) to evaluate the role of HE4 in the diagnosis, prognosis, and surveillance of EOC, but very little is known about HE4 mRNA expression. Eukaryotic translation initiation factor 3a (eIF3a) is implicated in oncogenesis and has been investigated extensively as a potential biomarker for malignancy. We previously reported a positive correlation between IHC expressions of eIF3a and HE4 in EOC. In the present study, we performed RT-PCR to determine mRNA expressions of HE4 and eIF3a in 30 normal ovarian tissues, 45 benign, 20 borderline and 94 malignant ovarian tumors. The association of HE4 and eIF3a mRNA expressions with clinicopathological characteristics and patient survivals was investigated. IHC was also performed in the same participants to investigate the correlation between mRNA and protein levels of HE4. HE4 mRNA level was found to be 48.42 ± 74.55 (mean ± SD, range: 0.01-343.99), significantly higher in primary EOC than in the borderline tumor, benign tumor, and normal ovarian tissue (P<0.001). The cutoff value was 13.99 for HE4 to discriminate malignant from benign tumors at 68.1% sensitivity and 93.0% specificity. By Spearman's correlation test, HE4 mRNA expression was indicated to positively correlate with serum CA125 level (r=0.530, P<0.001). Higher HE4 mRNA expression was associated with decreased frequency of lymph node metastasis (P=0.038) and better overall survival (OS) (P=0.007) in primary EOC. Multivariable analysis showed an independent prognostic value of the relative mRNA level of HE4 greater than one for OS (Hazard Ratio, 0.069, 95%CI, 0.009-0.530, P=0.010). eIF3a mRNA expression in women with primary EOC was 0.95 ± 1.19 (mean ± SD, range: 0.06-7.46), which was in a positive linear correlation with HE4 mRNA expression (r=0.310, P=0.002). In the present study, the HE4 mRNA level was unparalleled with IHC expression of HE4 (P>0.05). Collectively, our study revealed that increased HE4 mRNA expression correlates with high level of eIF3a mRNA and better survival in women with EOC, which calls for further investigations.

CD44v6 promotes ß-catenin and TGF-ß expression, inducing aggression in ovarian cancer cells.

A high expression of CD44v6 has been reported in numerous malignant cancers, including stomach, prostate, lung and colon. However, the pathological role and the regulatory mechanisms of CD44v6 have yet to be elucidated. In the present study, the expression levels of CD44v6 were shown to be significantly higher in ovarian cancer tissues, as compared with adjacent normal tissues. Furthermore, the upregulated expression levels of CD44v6 were correlated with disease recurrence and poor survival in patients. The expression of CD44v6 was knocked down in the CAOV3 ovarian cell line, by transfection of a specific small hairpin RNA. The present study showed a correlation between the aggression, viability, invasion and migration of the ovarian cancer cells, with the expression of CD44v6. In addition, the expression of CD44v6 was positively correlated with the expression levels of ß­catenin and tumor growth factor­ß, which indicates that the effects of CD44v6 on ovarian cancer cell aggression may be mediated by these two signaling pathways. In conclusion, the present study provides a novel insight into the association between CD44v6 expression and ovarian cancer. CD44v6 may provide a novel target for the prognosis and treatment of ovarian cancer.

TRPM7 is required for ovarian cancer cell growth, migration and invasion.

Our previous study demonstrated that the melastatin-related transient receptor potential channel 7 (TRPM7) was highly expressed in ovarian carcinomas and its overexpression was significantly associated with poor prognosis in ovarian cancer patients. However, the function of TRPM7 in ovarian cancer is mostly unknown. In this study, we examined the roles of TRPM7 in ovarian cancer cell proliferation, migration and invasion. We found that short hairpin RNA interference-mediated silence of TRPM7 significantly inhibited cell proliferation, colony formation, migration and invasion in multiple ovarian cancer cell lines. Mechanistic investigation revealed that silence of TRPM7 decreased phosphorylation levels of Akt, Src and p38 and increased filamentous actin and focal adhesion number in ovarian cancer cells. Thus, our results suggest that TRPM7 is required for proliferation, migration and invasion of ovarian cancer cells through regulating multiple signaling transduction pathways and the formation of focal adhesions.

Determinants of the over-anticoagulation response during warfarin initiation therapy in Asian patients based on population pharmacokinetic-pharmacodynamic analyses.

UNLABELLED: To clarify pharmacokinetic-pharmacodynamic (PK-PD) factors associated with the over-anticoagulation response in Asians during warfarin induction therapy, population PK-PD analyses were conducted in an attempt to predict the time-courses of the plasma S-warfarin concentration, Cp(S), and coagulation and anti-coagulation (INR) responses. In 99 Chinese patients we analyzed the relationships between dose and Cp(S) to estimate the clearance of S-warfarin, CL(S), and that between Cp(S) and the normal prothrombin concentration (NPT) as a coagulation marker for estimation of IC50. We also analyzed the non-linear relationship between NPT inhibition and the increase in INR to derive the non-linear index λ. Population analyses accurately predicted the time-courses of Cp(S), NPT and INR. Multivariate analysis showed that CYP2C9*3 mutation and body surface area were predictors of CL(S), that VKORC1 and CYP4F2 polymorphisms were predictors of IC50, and that baseline NPT was a predictor of λ. CL(S) and λ were significantly lower in patients with INR≥4 than in those with INR<4 (190 mL/h vs 265 mL/h, P<0.01 and 3.2 vs 3.7, P<0.01, respectively). Finally, logistic regression analysis revealed that CL(S), ALT and hypertension contributed significantly to INR≥4. All these results indicate that factors associated with the reduced metabolic activity of warfarin represented by CL(S), might be critical determinants of the over-anticoagulation response during warfarin initiation in Asians. TRIAL REGISTRATION: ClinicalTrials.gov NCT02065388.

Effect of probenecid on pharmacokinetics and tolerability of olmesartan in healthy chinese volunteers.

BACKGROUND: Olmesartan is an angiotensin II receptor antagonist and is effective and well tolerated in the treatment of arterial hypertension. Probenecid is a well-established hypouricemic agent for the treatment of hyperuricemia and gout. OBJECTIVE: The goal of this study was to examine the impact of coadministration of probenecid on the pharmacokinetic parameters and tolerability of olmesartan in healthy volunteers. METHODS: In a randomized, open-label, 2-way crossover study, 12 volunteers received 2 oral treatments (olmesartan alone or olmesartan plus probenecid) separated by 4 days. Blood samples were obtained for a 48-hour pharmacokinetic evaluation after drug administration. Tolerability was assessed by monitoring vital signs and laboratory tests before and after administration of the study drug. RESULTS: Pharmacokinetic parameters were evaluated in 6 male and 6 female healthy volunteers (mean age, 22 [range, 20-25] years]; weight, 56.0 [range, 51.0-60.0] kg). Probenecid coadministration increased olmesartan Css-av, AUC0→∞, and AUC0-48 by 40%, 50%, and 50%, respectively (P = 0.018, 0.000, 0.000, respectively), but there was no statistical significance for Tmax, t1/2, Css-max, and Css-min between olmesartan plus probenecid and olmesartan alone (P = 0.697, 0.053, 0.521, and 0.734, respectively). No serious adverse event (AE) was reported during the study. The proportion of volunteers with AEs in the olmesartan plus probenecid period (5 of 12 [42%]) was higher than that in the olmesartan-alone period (1 of 12 [8%]). All of the AEs during the olmesartan plus probenecid period were abnormal routine urine test results. The AE in olmesartan-alone period was dizziness. All AEs were classified as mild and considered to be at least possibly related to treatment. All volunteers recovered from the AEs by 2 weeks after the end of the study. CONCLUSIONS: Probenecid increases the exposure speed of olmesartan by increasing the AUC0-48, AUC0→∞, and Css-av. The combined treatment of olmesartan medoxomil with probenecid may increase the occurrence of genitourinary side effects. ClinicalTrials.gov identifier: NCT01907373.

Overexpression of TRPM7 is associated with poor prognosis in human ovarian carcinoma.

BACKGROUND: The melastatin-related transient receptor potential 7 channel (TRPM7) is a nonselective cation channel that has been shown to promote tumor metastasis and progression. In this study, we determined the expression of TRPM7 in ovarian carcinomas and investigated its possible prognostic value. MATERIALS AND METHODS: Samples were collected from 138 patients with ovarian cancer. Expression of TRPM7 was assessed by real-time PCR and immunohistochemistry, expressed with reference to an established scoring system and related to clinical pathological factors. Kaplan-Meier survival analysis was applied to estimate disease-free survival (DFS) and overall survival (OS). Univariate and multivariate cox regression analyses were performed to correlate TRPM7 expression levels with DFS and OS. RESULTS: TRPM7 was highly expressed in ovarian carcinoma and significantly associated with decreased disease-free survival (DFS: median 20 months vs. 42 months, P=0.0002) and overall survival (OS: median 27 months vs. 46 months, P<0.001). CONCLUSION: Overexpression of TRPM7 expression is significantly associated with poor prognosis in patients with ovarian cancer.

Validation of a liquid chromatography-electrospray ionization-tandem mass spectrometry method for determination of all-trans retinoic acid in human plasma and its application to a bioequivalence study.

A sensitive, reliable and specific LC-MS-MS method was developed and validated for the identification and quantitation of all-trans retinoic acid (ATRA) in human plasma. Acitretin was used as the internal standard (IS). After liquid-liquid extraction of 500 µL plasma with methyl tert-butyl ether (MTBE), ATRA and the IS were chromatographed on a HyPURITY C18 column (150 mm×2.1 mm, 5 µm) with the column temperature set at 40 °C. The mobile phase was consisted of 40% phase A (MTBE-methanol-acetic acid, 50:50:0.5, v/v) and 60% phase B (water-methanol-acetic acid, 50:50:0.5, v/v) with a flow rate of 0.3 mL/min. The API 4000 triple quadrupole mass spectrometer was operated in multiple reaction monitoring (MRM) mode via the positive electrospray ionization interface using the transition m/z 301.4→123.1 for ATRA and m/z 326.9→177.1 for IS, respectively. The calibration curve was linear over the range of 0.45-217.00 ng/mL (r≥0.999) with a lower limit of quantitation (LLOQ) of 0.45 ng/mL. The intra- and inter-day precisions values were below 8% relative standard deviation and the accuracy was from 98.98% to 106.19% in terms of relative error. The validated method was successfully applied in a bioequivalence study of ATRA in Chinese healthy volunteers.

The relative bioavailability and fasting pharmacokinetics of three formulations of olmesartan medoxomil 20-mg capsules and tablets in healthy Chinese male volunteers: An open-label, randomized-sequence, single-dose, three-way crossover study.

BACKGROUND: Olmesartan medoxomil is an angiotensin II-receptor antagonist used in the treatment of hypertension. It is a prodrug and is converted to the pharmacologically active compound on de-esterification by arylesterase in the gastrointestinal tract. OBJECTIVE: This study investigated the relative bioavailability and fasting pharmacokinetic properties of olmesartan after single doses of a 20-mg test tablet, a 20-mg test capsule, and a commercially available 20-mg reference tablet in healthy Chinese male volunteers. The study was conducted to satisfy Chinese State Food and Drug Administration regulatory requirements for approval of a generic formulation of olmesartan medoxomil. METHODS: This study had an open-label, randomized-sequence, single-dose, 3-treatment, 3-period crossover design. Healthy volunteers were randomly assigned in a 1:1:1 ratio to receive a single 20-mg dose of the test tablet, test capsule, or reference tablet, each administered after a 12-hour overnight fast, followed by a 1-week washout period and administration of the alternate formulation. Blood samples were obtained at baseline and at 0.5, 1, 1.5,2,2.5,3,4,6,8,12,24,36, and 48 hours after dosing. Tolerability was assessed based on vital signs and laboratory values obtained before and after administration of study drug. The formulations were assumed to be bioequivalent if the 90% CIs for the log-transformed ratios of C(max), AUC(0-t), and AUC(0-∞) were within the predetermined equivalence range (70%-143% for C(max); 80%-125% for AUC(0-t) and AUC(0-∞)), as established by the Chinese State Food and Drug Administration. RESULTS: Twenty-one healthy male subjects (mean age, 21 years [range, 18-25 years]; weight, 62.1 kg [range, 54.0-80.0 kg]) were enrolled in and completed the study. No period or sequence effect was observed. The mean AUC(0-∞) values for the test tablet, test capsule, and reference tablet were 3993 (1070), 3567 (850), and 3849 (872) ng/mL/h, respectively. The 90% CIs for the log-transformed ratios of test tablet to reference tablet for C(max), AUC(0-48), and AUC(0-∞) were 103.9 to 124.9, 94.0 to 111.5, and 94.4 to 111.7, respectively (all, P = NS). The corresponding 90% CIs for the log-transformed ratios of test capsule to reference tablet were 90.8 to 109.2, 84.9 to 107.9, and 85.1 to 100.7 (all, P = NS). Ten adverse events were reported during the study; 7 subjects complained of pain during blood sampling, and 3 had a blocked venous catheter. No treatment-related adverse events were reported or observed. CONCLUSIONS: In this single-dose crossover study in healthy Chinese male volunteers, the test and reference formulations of olmesartan medoxomil 20-mg capsules and tablets met the regulatory criteria for assuming bioequivalence. The 3 formulations were well tolerated.

Effect of the CYP2C9*3 allele on lornoxicam metabolism.

BACKGROUND: We investigated whether the CYP2C9 genotypes would affect lornoxicam metabolism in healthy volunteers. METHODS: Twelve healthy volunteers who had been genotyped for CYP2C9 gene were selected to participate in our study. After 8 mg lornoxicam was taken, blood samples were drawn from 0 to 36 h. The plasma concentrations of lornoxicam and 5'-hydroxylornoxicam were determined by HPLC method. 5'-hydroxylornoxicam was purified from rabbits'urine by semi-preparative HPLC. RESULTS: Lornoxicam and 5'-hydroxylornoxicam both exhibit CYP2C9 genotype-dependent pharmacokinetic profiles. The area under the plasma concentration-time curve (AUC) of lornoxicam increased by 60 +/- 9.78% (P <0.05) and the AUC of 5'-hydroxylornoxicam decreased by 65 +/- 11.75% (p <0.001) in heterozygous CYP2C9*1/*3 subjects (n=6) compared with CYP2C9*1/*1 group (n=6). t1/2 value of lornoxicam and 5'-hydroxylornoxicam prolonged by 39 +/- 8.35% and curtailed by 59 +/- 6.83% respectively in CYP2C9*1/*3 subjects. But no significant differences in Tmax of lornoxicam and 5'-hydroxylornoxicam were observed between these 2 genotypes. In addition, for the first time we exploit the purification method for 5'-hydroxylornoxicam from rabbits' urine. CONCLUSION: The CYP2C9*3 allele significantly affected the metabolism of lornoxicam. The pharmacokinetic parameters of both lornoxicam and 5'-hydroxylornoxicam were significantly different between these 2 genotypes.

Gender specific association of CYP2C9*3 with hyperlipidaemia in Chinese.

AIMS: To investigate the association of CYP2C9*3 and *6 with hyperlipidaemia in Chinese. METHODS: Four hundred and seventy-six Chinese participated in the study, including 211 uncomplicated hyperlipidaemic patients and 265 healthy controls. PCR-RFLP was used to identify CYP2C9*3 and *6. RESULTS: CYP2C9*6 was not detected in this study. The allelic frequency of CYP2C9*3 was 0.039 (95% CI 0.022, 0.056). A nonsignificant difference existed in CYP2C9*3 frequencies between males and females (P = 0.605, OR = 1.194, 95% CI 0.610, 2.336), patients and controls (P = 0.063, OR = 0.506, 95% CI 0.244, 1.049) in the total population. However, in the female group, CYP2C9*3 frequency in patients with hyperlipidaemia was significantly lower than that in controls (P < 0.0001, OR = 0.062, 95% CI 0.008, 0.476). CONCLUSIONS: The association of CYP2C9*3 with hyperlipidaemia was specific for females in this Chinese population.

Ile118Val genetic polymorphism of CYP3A4 and its effects on lipid-lowering efficacy of simvastatin in Chinese hyperlipidemic patients.

OBJECTIVES: To determine the frequencies of CYP3A4 alleles (CYP3A4*4,*5 and *6) in Chinese hyperlipidemic patients and to observe the impact of CYP3A4*4 (Ile118Val) genetic polymorphism on the lipid-lowering effects of simvastatin and on the activity of CYP3A4. METHODS: From hospitalized and non-hospitalized patients, 211 unrelated hyperlipidemic patients were recruited for genotyping. CYP3A4 genotypes were determined by means of polymerase chain reaction and restriction fragment length polymorphism analysis. Of the non-hospitalized hyperlipidemic patients, 8 with CYP3A4*1/*1 and 8 with CYP3A4*1/*4 genotypes were selected to be treated with 20 mg simvastatin daily for 4 weeks. Serum triglycerides (TG), cholesterol (CHO) and low-density lipoprotein (LDL) levels were determined using an automated analyzer (Hitachi 747, Boehringer Mannheim, Mannheim, Germany). CYP3A4 activity was determined by the ratio of 6-hydroxycortisol to free cortisol (6-OHC/FC) in the morning spot urine with a high-throughput liquid chromatography-tandem mass spectrometry method. RESULTS: Of 211 subjects, 14 (allele frequency 3.32%) were heterozygous for CYP3A4*4 (Ile118Val). Nevertheless, no subjects with a CYP3A4*5 or CYP3A4*6 allele or homozygous for CYP3A4*4 were identified. The ratio of 6beta-OHC/FC was 9.9 +/- 13.7 and 56.6 +/- 35.7 in subjects with the Ile118Val variant (n = 8) and in CYP3A4 wild-type subjects (n = 8), respectively (P = 0.0039). After oral intake of simvastatin 20 mg daily for 4 weeks, the change of serum lipids in CYP3A4*1/*1 and CYP3A4*1/*4 groups showed a significant difference, with a mean decrease in triglycerides and total cholesterol of 38.1 +/- 7.6% versus 25.1 +/- 8.3% (P = 0.034) and of 35.8 +/- 9.6% versus 22.0 +/-20.4% (P = 0.0015) (means +/- SD), respectively. We found no statistically significant difference in the reductions of LDL between subjects carrying the *1 and *4 genotypes (29.0 +/- 7.4% versus 36.8 +/- 8.8%, P = 0.0721). CONCLUSIONS: The allele frequency of CYP3A4*4 was 3.32% among the hyperlipidemic patients from the Chinese mainland. CYP3A4*4 was an allelic variant related to a functional decrease of CYP3A4 activity, and *4 expression seemed to increase the lipid-lowering effects of simvastatin.

CYP2C9 allele variants in Chinese hypertension patients and healthy controls.

BACKGROUND: Cytochromes P450 (CYP) 2C9 are polymorphic enzymes which catalyze a wide spectrum of drugs. It is also responsible for the metabolism of arachidonic acid into EETs. EETs are known to be a vasoactive substance and play an important role in a hypertensive episode. Whether the genetic polymorphism of CYP2C9 will affect the vasoactive effect and consequently affect hypertension formation is still unknown. We investigated the association of CYP2C9*2, CYP2C9*3 and CYP2C9*6 with hypertension. METHODS: Two hundred and thirty-nine hypertension patients and 265 healthy controls participated in our study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to identify CYP2C9*2, CYP2C9*3 and CYP2C9*6. RESULTS: CYP2C9*2 and CYP2C9*6 were not detected in our study. The allelic frequency of CYP2C9*3 was 0.015 in hypertension patients in our study. In healthy controls, the allelic frequency of CYP2C9*3 was 0.049. Significant difference existed in CYP2C9*3 frequency between hypertension patients and healthy controls (0.015 for hypertension patients vs. 0.049 for healthy controls; chi2 = 9.728, P < 0.005, OR = 0.277, 95% CI: 0.118-0.651). Also, gender-dependent difference was observed. In females, CYP2C9*3 frequency of hypertension patients was significantly lower than that of healthy controls (chi2 = 11.513, P < 0.001, OR = 0.113, 95% CI: 0.026-0.500). CONCLUSION: To our knowledge, this is the first report on CYP2C9 frequencies in hypertension patients. Our study implied that CYP2C9*3 had a secondary protective effect in females, which may be useful for studying hypertension pathogenesis and therapeutics.