Determination of mycophenolic acid in human plasma by ultra performance liquid chromatography tandem mass spectrometry

A simple, sensitive and high throughput ultra performance liquid chromatography tandem mass spectrometry method has been developed for the determination of mycophenolic acid in human plasma. The method involved simple protein precipitation of MPA along with its deuterated analog as an internal standard （IS） from 50 mL of human plasma. The chromatographic analysis was done on Acquity UPLC C18 （100mm×2.1mm,1.7μm） column under isocratic conditions using acetonitrile and 10 mM ammonium formate, pH 3.00 （75：25, v/v） as the mobile phase. A triple quadrupole mass spectrometer operating in the positive ionization mode was used for quantitation. In-source conversion of mycophenolic glucuronide metabolite to the parent drug was selectively controlled by suitable optimization of cone voltage, cone gas flow and desolvation temperature. The method was validated over a wide concentration range of 15-15000 ng/mL. The mean extraction recovery for the analyte and IS was 〉95%. Matrix effect expressed as matrix factors ranged from 0.97 to 1.02. The method was successfully applied to support a bioequivalence study of 500 mg mycophenolate mofetil tablet in 72 healthy subjects.

Clinical mycophenolic acid monitoring in liver transplant recipients

In liver transplantation, the efficacy of mycophenolate mofetil(MMF) has been confirmed in clinical trials and studies. However, therapeutic drug monitoring for mycophenolic acid(MPA) has not been fully accepted in liver transplantation as no long-term prospective study of concentration controlled vs fixed-dose prescribing of MMF has been done. This review addressed MPA measurement, pharmacokinetic variability and reasons of this variation, exposure related to acute rejection and MMF-associated side effects in liver transplant recipients. Limited sampling strategies to predict MPA area under the concentration-time curve have also been described, and the value of clinical use needs to be investigated in future. The published data suggested that a fixed-dosage MMF regimen might not be suitable and monitoring of MPA exposure seems helpful in various clinical settings of liver transplantation.

Pharmacokinetics of mycophenolic acid in Chinese kidney transplant patients

To assess the influence of cyclosporin A (CsA) and tacrolimus (FK506) on mycophenolic acid (MPA) and correlation analysis of the pharmacokinetic parameters and patient characteristics, clinical outcome in Chinese kidney transplant recipients, the pharmacokinetics of 1000 mg mycophenolate mofetil (MMF) twice daily was measured by high-performance liquid chro- matography (HPLC). PKS (Pharmaceutical Kinetics Software) 1.0.2 software package was used for the calculation of pharma- cokinetic parameters. The mean Cmax, tmax, and AUC(0?12)were (21.88±10.52) μg/ml, (1.20±0.95) h, and (52.546±13.215) μg·h/ml, respectively. The level of AUC(0?12) in the FK506 group was significantly higher than that in the CsA group. MPA appeared not to be affected by renal function. MPA AUC(0?12) showed statistically significant difference according to the patient’s gender.

Effects of Mycophenolic Acid on High Glucose-induced Expression of TGF-β and CTGF in Mesangial Cells

The effects of mycophenolic acid （MPA） on high glucose-induced expression of transforming growth factor-β （TGF-β） and connective tissue growth factor （CTGF） in mesangial cells （MC） were investigated. Rat MC were cultured in the presence of different concentrations of MPA （1.0 and 10.0 μmol/L） or MPA plus high glucose for 72 h. The expression of TGF-β and CTGF was detected by Western blot. The results showed that high glucose could induce the expression of TGF-β and CTGF in MC, but MPA could inhibit this effects. MPA did not influence the expression of TGF-β and CTGF in normal glucose. It was concluded that MPA might prevent the progression of diabetic nephropathy by inhibiting the expression of TGF-β and CTGF in MC.

Influence of organic anion transporting potypeptide(SLCO1B1 and SLCO1B3)genetic polymorphisms on mycophenolic acid in Chinese kidney transplantation patients

Objective To analyze the relationship between genetic polymorphisms of organic anion transporting polypeptide ( SLCO1B1 and SLCO1B3) and mycophenolic acid ( MPA) pharmacokinetics in Chinese kidney transplant recipients. Methods Gene mutations ( SLCO1B3 T334G,SLCO1B1 A338G) were detected in 68 recipi-

A simple capillary zone electrophoresis method for the determination of mycophenolic acid in human plasma

Mycophenolic acid （MPA）, an immunosuppressive drug, was determined in human plasma by a simple capillary zone electrophoresis （CZE） method. A bare fused-silica capillary with an internal diameter （i.d.） of 75/am and an effective length of 50 cm was used for the separation. A 200 }aL plasma sample was deproteinized with 400 μL acetonitrile, and the supematant was directly injected into the capillary after a water plug at a pressure of 0.5 psi for 10 s. Boric acid （H3BO3, 200 mmol/L） solution adjusted to a pH of 8.60 with 1 mol/L sodium hydroxide （NaOH） solution was selected as the background electrolyte （BGE） through a uniform design. Calibration curve established on each day was linear over the MPA concentration range of 0.625-30.0 Bg/mL in human plasma with the correlation coefficient （r） larger than 0.9997. The limit of detection （LOD） was 0.200 ~tg/mL. Intra- and inter-day precisions at three concentrations within the calibration range were less than 4.45%. Plasma samples collected from 14 renal transplant recipients treated with the prodrug mycophenolate mofetil （MMF） were analyzed successfully by both the developed CZE technique and a validated high-performance liquid chromatography-tandem mass spectrometry （HPLC-MS/MS） technique. The mean absolute and relative differences between the concentrations measured by the two methods were -0.17 and -0.20%, respectively. In conclusion, the CZE method described here was validated to be accurate, precise, and economical for the clinical quantification of MPA.

Nonlinear relationship between enteric-coated mycophenolate sodium dose and mycophenolic acid exposure in Han kidney transplantation recipients

The aim of the research was to investigate the pharmacokinetics(PK) of enteric-coated mycophenolate sodium(EC-MPS) by quantification of the active metabolite of mycophenolic acid(MPA)after multiple escalating oral doses in Han kidney transplant recipients. A total of 28 Han postoperative kidney transplant recipients were given a multiple-dose of 540, 720 or 900 mg of EC-MPS two times a day in combination with tacrolimus for 6 days. Blood specimens were collected at each time point from0 to 12 h after EC-MPS administration. MPA plasma concentrations were measured by UPLC–UV. The relationship between the EC-MPS dose and its PK parameters was assessed. In the range from 540 to900 mg, C_(max) and AUC_(0–12h) did not increase with dose escalation. The AUC_(0–12h), C_(max), C_0 and T_(max) for the 540 720 and 900 mg doses were not significantly different, respectively(P 40.05). AUC_0–12 h and C_(max) were increased less than proportionally with increasing EC-MPS dose levels. Inter-individual variability in AUC_(0–12h), C_(max) and C_0 were considerable. Nonlinear PK relationships were found from the doses of 540–900 mg of EC-MPS.

Penicacids E-G, three new mycophenolic acid derivatives from the marine-derived fungus Penicillium parvum HDN17-478

Three new mycophenolic acid derivatives, penicacids E-G(1-3), together with three known analogues, mycophenolic acid(4), 4′-hydroxy-mycophenolic acid(5) and mycophenolic methyl ester(6), were isolated from a marine-derived fungus Penicillium parvum HDN17-478 from a South China Sea marine sediment sample. The structures of compounds 1-3 were elucidated by HRMS, NMR, and Mosher’s method. Among them, compounds 1 and 2 were the first examples of mycophenolic acid analogs with a double bond at C-3′/C-4′ position. The cytotoxicity of 1-6 was evaluated against the HCT-116, BEL-7402, MGC-803, SH-SY5 Y, HO-8910 and HL-60 cell lines, and compounds 4 and 6 showed potent cytotoxicity with IC50 values ranging from 1.69 to 12.98 μmol·L–1.

Mycophenolic Acid Synergizing with Lipopolysaccharide to Induce Interleukin-1β Release via Activation of Caspase-1

Background： The previous study showed that mycophenolic acid （MPA） synergizing with lipopolysaccharide （LPS） promotcd interleukin （IL）-1β release, but the mechanism is unclear. This study aimed to investigate the nlechanism ofMPA synergizing with LPS to induce IL-1β release. Methods： Undiluted human blood cells, THP- I human rnyeloid leukemia mononuclear cells （THP- 1 ） cells, or monocytes were stimulated with L PS and treated with or without M PA, and the supernatant IL-1β was detected by enzyme-linked immunosorbent assay. The m RN A levels of IL- 1β were detected by real-time quantitative polymerase chain reaction. The intracellular protein levels of nuclear factor kappa B （NF-κ） phospho-p65 （p-p65）, precursor interleukin-1β （pro-IL-1β）, NOD-like receptor pyrin domain containing-3 （NLRP3）, and cysteine aspartic acid-specific protease-1 （caspase-1 ） p20 in THP-1 cell were measured by Western blot. Results： The MPA alone failed to induce IL-1β, whereas MPA synergized with LPS to increase IL-1β in a dose-dependent manner （685.00 ± 20.00 pg/ml in LPS ＋ 5 μmol/L MPA group, P =0.035; 742.00 ± 31.58 pg/ml in LPS ＋ 25 μmol/L MPA group, P = 0.017：1000.00 ± 65.59 pg/ml in LPS ＋ 75 μmol/L MPA group, P = 0.024： versus 408.00 ± 35.50 pg/ml in LPS group）. MPA alone has no effect on the IL-1β mRNA expression, LPS induced the expression of IL-1β mRNA 2761 fold, and LPS ＋ MPA increased the IL-1β expression 3018 fold, which had the same effect with LPS group （P = 0.834）. MPA did not affect the intracellular NF-κB p-p65 and pro-IL-1β protein levels but activated N LRP3 inflammasonae. Ac-YVAD-cmk blocked the activation ofcaspase-1 and subsequently attenuated IL- 1β secretion （ 181.00 ± 45.24 pg/ml in LPS ＋ M PA ＋ YVAD group vs. 588.00 ± 41.99 pg/ml in LPS ＋ M PA group, P= 0.014）. Conclusions： Taken together, MPA synergized with LPS to induce IL- 1β release via the activation of caspase- 1, rather than the enhanced production ofpro-IL-1β. These findings suggested that patients immunosuppressed with mycophenolate mofetil may have overly activated caspase- 1 during infection, which might contribute to a more sensitive host defense response to invading germs.

Mycophenolic acid derivatives from cultures of the mushroom Laetiporus sulphureu

AIM: To investigate the chemical constituents of the cultures of Laetiporus sulphureus(Bull.) Murrill. METHOD: Compounds were isolated and purified by various chromatographic techniques. The structure of the new compound was determined by interpretation of MS and 1D-, 2D-NMR spectroscopic data, while the known compounds were identified by comparison of their data with those reported. RESULTS: Three mycophenolic acid derivatives, 6-((2E, 6E)-3, 7-dimethyldeca-2, 6-dienyl)-7-hydroxy-5-methoxy-4-methylphtanlan-1-one(1), 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl)-5, 7-dihydroxy-4-methylphtanlan-1-one(2), and 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl)-7-hydroxy-5-methoxy-4-methylphtanlan-1-one(3) were isolated. CONCLUSION: Among them, compound 1 was new, and compound 2 exhibited moderate cytotoxicity against HL-60, SMMC-7721, A-549, and MCF-7 cells, with IC50 values of 39.1, 31.1, 27.4, and 35.7 μmol·L-1, respectively.

Conventional therapy for moderate to severe inflammatory bowel disease: A systematic literature review

BACKGROUND Despite the advent of biological drugs, conventional therapy continues to be used in moderate to severe inflammatory bowel disease(MS-IBD). This study hypothesized that as a standard of treatment and the primary alternative to biologics, conventional therapy should present robust effectiveness results in IBD outcomes.AIM To investigate the effectiveness of conventional therapy for MS-IBD.METHODS A systematic review with no time limit was conducted in July 2017 through the Cochrane Collaboration, MEDLINE, and LILACS databases. The inclusion criteria encompassed meta-analyses, systematic reviews, randomized clinical trials, observational and case-control studies concerning conventional therapy in adult patients with MS-IBD, including Crohn's disease(CD) and ulcerative colitis(UC). Corticosteroids(prednisone, hydrocortisone, budesonide, prednisolone,dexamethasone), 5-aminosalicylic acid(5-ASA) derivatives(mesalazine and sulfasalazine) and immunosuppressants [azathioprine(AZA), methotrexate(MTX), mycophenolate, cyclosporine, tacrolimus, 6-mercaptopurine(6-MP)] were considered conventional therapy. The exclusion criteria were sample size below50; narrative reviews; specific subpopulations(e.g., pregnant women,comorbidities); studies on postoperative IBD; and languages other than English,Spanish, French or Portuguese. The primary outcome measures were clinical remission(induction or maintenance), clinical response and mucosal healing. As secondary outcomes, fecal calprotectin, hospitalization, death, and surgeries were analyzed. The quality of the evidence was assessed using the Grading of Recommendations Assessment, Development and Evaluation criteria.RESULTS The search strategy identified 1995 citations, of which 27 were considered eligible(7 meta-analyses, 20 individual studies). For induction of clinical remission, four meta-analyses were selected(AZA and 6-MP showed no advantage over placebo,MTX or 5-ASA in CD; MTX showed no statistically significant difference versus placebo, 6-MP, or 5-ASA in UC; tacrolimus was superior to placebo for UC in two meta-analyses). Only one meta-analysis evaluated clinical remission maintenance, showing no statistically significant difference between MTX and placebo, 5-ASA, or 6-MP in UC. AZA and 6-MP had no advantage over placebo in induction of clinical response in CD. Three meta-analyses showed the superiority of tacrolimus vs placebo for induction of clinical response in UC. The clinical response rates for cyclosporine were 41.7% in randomized controlled trials(RCTs) and 55.4% in non-RCTs for UC. For induction of mucosal healing,one meta-analysis showed a favorable rate with tacrolimus versus placebo for UC. For secondary outcomes, no meta-analyses specifically evaluated fecal calprotectin, hospitalization or death. Two meta-analyses were retrieved evaluating colectomy rates for tacrolimus and cyclosporine in UC. Most of the twenty individual studies retrieved contained a low or very low quality of evidence.CONCLUSION High-quality evidence assessing conventional therapy in MS-IBD treatment is scarce, especially for remission maintenance, mucosal healing and fecal calprotectin.

Initial steroid-free immunosuppression after liver transplantation in recipients with hepatitis c virus related cirrhosis

AIM:Steroids can increase hepatitis C virus(HCV) replication.After liver transplantation(LTx),steroids are commonly used for immunosuppression and acute rejection is usually treated by high steroid dosages.Steroids can worsen the outcome of recurrent HCV infection.Therefore, we evaluated the outcome of HCV infected liver recipients receiving initial steroid-free immunosuppression. METHODS:Thirty patients undergoing LTx received initial steroid-free immunosuppression.Indication for LTx included 7 patients with HCV related cirrhosis.Initial immunosuppression consisted of tacrolimus 2×0.05mg/kg.d po and mycophenolate mofetil(MMF)2×15mg/kg.d po.The tacrolimus dosage was adjusted to trough levels in the target range of 10-15μg/L during the first 3 mo and 5-10μg/L thereafter.Manifestations of acute rejection were verified histologically. RESULTS:Patient and graft survival of 30 patients receiving initial steroid-free immunosuppression was 86% and 83% at 1 and 2 years.Acute rejection occurred in 8/30 patients, including 1 HCV infected recipient.All HCV-infected patients had HCV genotype Ⅱ(lb).HCV seropositivity occurred within the first 4 mo after LTx.The virus load was not remarkably increased during the first year after LTx.Histologically,grafts had no severe recurrent hepatitis. CONCLUSION:From our experience,initial steroid-free immunosuppression does not increase the risk of acute rejection in HCV infected liver recipients.Furthermore,none of the HCV infected patients developed serious chronic liver diseases.It suggests that it may be beneficial to avoid steroids in this particular group of patients after LTx.

Current aspects of renal dysfunction after liver transplantation

The development of chronic kidney disease(CKD)after liver transplantation(LT)exerts a severe effect on the survival of patients.The widespread adoption of the model for end-stage liver disease score strongly impacted CKD incidence after the procedure,as several patients are transplanted with previously deteriorated renal function.Due to its multifactorial nature,encompassing pre-transplantation conditions,perioperative events,and nephrotoxic immunosuppressor therapies,the accurate identification of patients under risk of renal disease,and the implementation of preventive approaches,are extremely important.Methods for the evaluation of renal function in this setting range from formulas that estimate the glomerular filtration rate,to non-invasive markers,although no option has yet proved efficient in early detection of kidney injury.Considering the nephrotoxicity of calcineurin inhibitors(CNI)as a factor of utmost importance after LT,early nephroprotective strategies are highly recommended.They are based mainly on delaying the application of CNI during the immediate postoperativeperiod,reducing their dosage,and associating them with other less nephrotoxic drugs,such as mycophenolate mofetil and everolimus.This review provides a critical assessment of the causes of renal dysfunction after LT,the methods of its evaluation,and the interventions aimed at preserving renal function early and belatedly after LT.

Mycophenolate mofetil vs cyclophosphamide therapy for patients with diffuse proliferative lupus nephritis

OBJECTIVE: To make an open label prospective trial for comparing the therapeutic effects of mycophenolate mofetil (MMF) vs cyclophosphamide (CYC) pulse therapy on patients with diffuse proliferative lupus nephritis (DPLN). METHODS: Forty-six patients with biopsy proven active DPLN were enrolled in this study. Twenty-three patients were given MMF orally at a dosage of 1.0 - 1.5 g/d (MMF Group). Another 23 cases received conventional intermittent CYC pulse therapy (CYC Group). Supplemental steroid treatment was offered in the same manner to both groups. The age, sex distribution and severity of renal damage were matched in two groups. Therapeutic effects were evaluated at the end of six-month treatment. Fifteen patients in the MMF Group and 12 patients in the CYC Group had repeated renal biopsy at that time. RESULTS: MMF therapy was more effective in reducing proteinuria and hematuria. A 50% reduction of urinary protein and urinary red blood cell excretion from baseline value in 69.6% and 91.3% patients in the MMF Group, while only 47.8% and 65.2% in the CYC Group. MMF was more effective in inhibiting autoantibody production (especially anti-dsDNA antibody) and in decreasing serum cryoglobulin levels. Pathologically, the MMF group showed more markedly reduction in glomerular immune deposits with less glomerular necrosis, and less microthrombi, less crescent formation and vascular changes in the repeated renal biopsy as compared with the CYC group. Adverse reactions related to the treatment included gastrointestinal symptoms 26.1% and 43.5% in the MMF and CYC Groups respectively, infection 17.4% in the MMF group and 30.4% in the CYC group. CONCLUSION: MMF was more effective in controlling the clinical activity of DPLN and renal vascular lesions as compared with CYC pulse therapy in a 6 month follow-up study.