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The Application of Inosine 5 -Monophosphate Dehydrogenase pharmaceuticals Article 0 The Application of Inosine 5 -Monophosphate Dehydrogenase Activity Determination in Peripheral Blood Mononuclear Cells for Monitoring Mycophenolate Mofetil Therapy in Children with Nephrotic Syndrome Joanna Sobiak 1,* , Alicja Jó´zwiak 1, Honorata Wzi˛etek 1, Jacek Zachwieja 2 and Danuta Ostalska-Nowicka 2 1 Department of Physical Pharmacy and Pharmacokinetics, Poznan University of Medical Sciences, 60-781 Pozna´n,Poland; [email protected] (A.J.); [email protected] (H.W.) 2 Department of Pediatric Nephrology and Hypertension, Poznan University of Medical Sciences, 60-572 Pozna´n,Poland; [email protected] (J.Z.); [email protected] (D.O.-N.) * Correspondence: [email protected]; Tel.: +48-61854-6435 Received: 15 July 2020; Accepted: 16 August 2020; Published: 18 August 2020 Abstract: In pediatric nephrotic syndrome, recommended mycophenolic acid (MPA) pharmacokinetics are higher than those for transplant recipients. In MPAtherapeutic monitoring, inosine-50-monophosphate dehydrogenase (IMPDH) activity may be useful. Wemodified the method established for renal transplant recipients and determined IMPDH activity in peripheral blood mononuclear cells (PBMCs) from healthy volunteers and children (4–16 years) with nephrotic syndrome treated with mycophenolate mofetil (MMF). From children, four blood samples were collected, and MPA concentrations were also determined. IMPDH activity was calculated using xanthosine monophosphate (XMP) normalized with adenosine monophosphate (AMP), both determined with the HPLC-UV method. The modified method was accurate, precise, and linear for AMP and XMP within 0.50–50.0 µmoL/L. Mean IMPDH activity in volunteers was 45.97 6.24 µmoL s 1 moL 1 AMP, whereas for children, the values were variable ± · − · − and amounted to 39.23 27.40 µmoL s 1 moL 1 AMP and 17.97 15.24 µmoL s 1 moL 1 AMP ± · − · − ± · − · − before the next MMF dose and 1 h afterward, respectively. The modified method may be applied to IMPDH activity determination in children with nephrotic syndrome treated with MMF. IMPDH activity should be determined after one thawing of PBMCs due to the change in AMP and XMP concentrations after subsequent thawing. For children, the lowest IMPDH activity was observed concomitantly with the highest MPA concentration. Keywords: mycophenolic acid; inosine-50-monophosphate dehydrogenase activity; pediatric patients; HPLC method; therapeutic drug monitoring; nephrotic syndrome; pharmacokinetics; pharmacodynamics 1. Introduction Immunosuppressive drugs aim to decrease immune system activity and inhibit inflammatory processes. The ideal immunosuppressant should act selectively on immune system reactions and consequently present low toxicity. So far, no immunosuppressant is devoid of adverse effects, as these drugs act on quickly proliferating cells. One of the most frequently administered drugs after solid organ transplantation is mycophenolate mofetil (MMF), the active form of which, mycophenolic acid (MPA), inhibits the activity of inosine 50-monophosphate dehydrogenase (IMPDH) [1,2]. MMF is more effective, reduces the severity of graft rejection episodes, and influences the overall survival of patients and, therefore, has replaced azathioprine [3]. Pharmaceuticals 2020, 13, 200; doi:10.3390/ph13080200 www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2020, 13, 200 2 of 14 MPA inhibits IMPDH activity expressed in activated T and B lymphocytes. IMPDH is responsible for de novo purine synthesis and depends on nicotinamide adenine dinucleotide (NAD+). IMPDH catalyzes the oxidation of inosine-50-monophosphate (IMP) to xanthosine-50-monophosphate (XMP), which subsequently changes into guanosine-50-monophosphate (GMP) and converts to guanosine-50- triphosphate (GTP) and deoxyguanosine triphosphate (dGTP). GTP and dGTP are essential nucleotides for DNA and RNA synthesis [4,5]. As a consequence, by inhibiting IMPDH activity in a selective, reversible, and noncompetitive manner, MPA leads to a decrease in guanine nucleotide synthesis and reduces cell proliferation. This mechanism is responsible for MPA’s favorable anticancer, immunosuppressive, and antiviral activity [6]. Significant inter- and intrapatient variability of MPA pharmacokinetics provides the basis for therapeutic drug monitoring (TDM) in the case of MMF; however, its importance is still being discussed [1]. In the literature, studies of MPA pharmacokinetics concern mainly children after transplantation [7–11]. However, in our previous study [12], in children with nephrotic syndrome, we observed higher trough MPA concentration (Ctrough) and area under the time-concentration curve (AUC) if compared to recommended Ctrough and AUC for transplant recipients [8]. This observation was later described also by other authors [13,14]. Determining IMPDH activity may serve as the pharmacodynamic marker of MPA as, according to the literature, determining IMPDH activity before transplantation should help MMF dose individualization [15]. Although this approach is promising [16–18], studies concerning IMPDH activity in relation to MPA concentrations are limited [15,19–23]. Moreover, according to our knowledge, there are no studies including IMPDH activity in children with nephrotic syndrome. Due to the difference in MPA pharmacokinetics between children with nephrotic syndrome and renal transplant recipients, and the suggestions for introducing MPA TDM in children with nephrotic syndrome, we decided to verify whether the IMPDH activity values in children with nephrotic syndrome are within the range of that reported in the literature for other subjects. To achieve this aim, firstly, we modified and validated the HPLC-UV method of Glander et al. [24]. Secondly, we determined the IMPDH activity in peripheral blood mononuclear cells (PBMCs) from healthy volunteers. Thirdly, we determined the IMPDH activity in PBMCs and plasma MPA for children with nephrotic syndrome treated with MMF and compared the results with the literature data. 2. Results 2.1. The Modifications of the IMPDH Activity Determination Method For determining IMPDH activity in PBMCs, we introduced some changes to Glander et al.’s study [24]. We checked a few method parameters: mobile phase (pH 5.2 and 5.6; buffer:methanol proportions of 92:8 and 90:10), NAD+ concentrations in the incubation buffer (0.5 and 1.0 mmoL/L), PBMC incubation time (120, 150, and 180 min), detection wavelength (254 and 260 nm), and the stability after freeze–thaw cycles. Initially, we used bovine serum albumin to prepare the incubation buffer for calibration curves, as adenosine monophosphate (AMP) and XMP occur in PBMCs, and blank samples of PBMCs cannot be used for calibration. However, we found an interfering peak around AMP retention time if bovine serum albumin was included. Therefore, we decided to use HPLC-grade water without bovine serum albumin for preparing the incubation buffer for calibration curves (Figure1a,b). To separate the analytes precisely, we checked different pHs of the mobile phase buffer (5.6 and 5.2). We observed the best separation and symmetry of peaks at pH 5.2 (Figure1c,d). Therefore, pH 5.2 was used throughout the rest of the studies. We checked the influence of different NAD+ concentrations (0.5 and 1.0 mmoL/L) on the AMP and XMP chromatographic peak appearances (Figure1e,f). PharmaceuticalsPharmaceuticals2020 2020, ,13 13,, 200 x FOR PEER REVIEW 3 of3 of14 14 7 mAU mAU6 5 4 min 3 2 6.708 1 0 024681012 -1 024681012 min -2 min (a) (b) mAU AMP mAU AMP 8 XMP 8 XMP 7 pH = 5.2 6 pH = 5.6 6 5 4 4 3 2 2 0 1 024681012 0 -2 -1 024681012min min (c) (d) 8 8 mAU mAU 6 6 4 4 interfering peak interfering peaks 2 2 0 0 02468101214 02468101214 -2 -2 min min (e) (f) Figure 1. The HPLC chromatograms of (a) incubation buffer with bovine serum albumin (mobile Figure 1. The HPLC chromatograms of (a) incubation buffer with bovine serum albumin (mobile phase buffer pH 5.2), (b) incubation buffer without bovine serum albumin (mobile phase buffer pH 5.2), phase buffer pH 5.2), (b) incubation buffer without bovine serum albumin (mobile phase buffer pH c µ µ ( 5.2),) 10 (moLc) 10/L μ ofmoL/L adenosine of adenosine monophosphate monophosphate (AMP) and (AMP) 10 moL and/L of10 xanthosine μmoL/L monophosphateof xanthosine µ µ (XMP)monophosphate at mobile (XMP) phase buat ffmobileer pH phase of 5.2, buffer (d) 10 pHmoL of 5.2,/L of(d) AMP 10 μmoL/L and 10 of moLAMP/ Land of XMP10 μmoL/L at mobile of phaseXMP buat ffmobileer pH ofphase 5.6, (buffere) incubation pH of 5.6, buff (ere) containingincubation 0.5 buffer mmoL containi/L nicotinamideng 0.5 mmoL/L adenine nicotinamide dinucleotide + + (NADadenine), anddinucleotide (f) incubation (NAD bu+), ffander containing(f) incubation 1.0 buffer mmoL containing/L NAD . 1.0 mmoL/L NAD+. Better symmetry and less blur were observed with 1.0 mmoL/L NAD+ in the incubation buffer. Better symmetry and less blur were observed with 1.0 mmoL/L NAD+ in the incubation buffer. However,However, concomitantly, concomitantly, the the analysis analysis revealed revealed the th interferinge interfering peak peak around around the the retention retention time time of AMP. of + ForAMP. NAD For NADat a+ concentration at a concentration of 0.5 of 0.5 mmoL mmoL/L,/L, two two peaks peaks were were observed, observed, whereas for for 1.0 1.0 mmoL/L mmoL/ L + NADNAD+,, oneone peakpeak appeared. We We verified verified whether whether any any change change in in HPLC HPLC analysis analysis parameters parameters (detection (detection wavelength 260 nm instead of 254 nm; mobile phase composition 92:8 instead of 90:10, v/v) could help Pharmaceuticals 2020, 13, 200 4 of 14 reduce the peak surface area. We found the smallest peak surface area along with the best AMP and XMP peaks’ resolutions for detection wavelength 254 nm, and the mobile phase ratio of mixing 90:10 (v/v). Therefore, these conditions were used during HPLC analysis along with NAD+ 1 mmoL/L for sample preparation. The interfering peak surface area was reproducible; thus, the AMP surface area was reduced by the surface area of the interfering peak in the incubation buffer after each PBMC sample analysis.
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