MOLECULAR & CELLULAR BIOLOGY ANIMAL CELLS AND SYSTEMS 2019, VOL. 23, NO. 3, 155–163 https://doi.org/10.1080/19768354.2019.1614091 Pharmacokinetic, metabolic stability, plasma protein binding and CYP450s inhibition/induction assessment studies of N-(2-pyridylmethyl)-2- hydroxiymethyl-1-pyrrolidinyl-4-(3-chloro-4-methoxy-benzylamino)-5- pyrimidine-carboxamide as potential type 5 phosphodiesterase inhibitors Haijun Qu, Xiaoxiao Hu, Xiaoli Shi, Chuan Wang, Longyuan Wang and Guoping Wang Department of Pharmacy, Aﬃliated Hospital of Qingdao University, Qingdao, People’s Republic of China ABSTRACT ARTICLE HISTORY Received 29 October 2018 N-(2-pyridylmethyl)-2-hydroxiymethyl-1-pyrrolidinyl-4-(3-chloro-4-methoxy-benzylamino)-5- Revised 27 April 2019 pyrimidine-carboxamide (NHPPC) is a new potential of type 5 phosphodiesterase (PDE5) inhibitors, Accepted 28 April 2019 synthesized from the avanaﬁl analogue for the treatment of erectile dysfunction. The targets of this article were to assess plasma protein binding, liver microsomal metabolic stability, inhibition and KEYWORDS induction on cytochrome P450 isozymes and the pharmacokinetics of NHPPC. Equilibrium Plasma protein binding; dialysis technique was applied to determine Plasma protein binding (PPB) and NHPPC was metabolic stability; evaluated in male Sprague–Dawley rats and Beagle dogs in vivo pharmacokinetic. The NHPPC cytochrome p450 inhibition was highly bound to plasma proteins in rats, dogs and human tested and the mean values for and induction; pharmacokinetics; type 5 PPB rate were 96.2%, 99.6% and 99.4%, respectively. After in vitro liver microsomes incubated for phosphodiesterase inhibitors 60 min, the percent remaining of NHPPC was 42.8%, 0.8% and 42.0% in rats, dogs and human, respectively. In vitro intrinsic clearance was found to be 0.0233, 0.1204 and 0.0214 mL/min/mg protein in rat, dog and human liver microsomes of NHPPC, respectively. NHPPC showed no signiﬁcant inhibitory eﬀects on major CYP450 enzymes, and had no signiﬁcant induction potential on CYP1A2 and CYP3A4. Following oral administration in rats and dogs, t was 6 and max 0.5 h, respectively. The clearance for NHPPC was 1.19 and 1.46 L/h/kg in rats and dogs, respectively. And absolute bioavailability in rat and dog were approximately 34.5% and 53.1%, respectively. These results showed that NHPPC has a good development prospect. 1. Introduction therapeutic drugs have not yet met with therapeutic pur- poses, and there are some adverse reactions. Therefore, N-(2-pyridylmethyl)-2-hydroxiymethyl-1-pyrrolidinyl-4- the novel highly selective PDE5 inhibitors with strong (3-chloro-4-methoxy-benzylamino)-5-pyrimidine-carbox- eﬃcacy and fast onset of action have great signiﬁcance amide (NHPPC, Figure 1), a potential of type 5 phosphodi- and economic value, and improve the safe use of drugs esterase inhibitors, was avanaﬁl modiﬁed structural to prevent adverse reactions. Compared with other analog. Avanaﬁl is a type 5 phosphodiesterase (PDE5) similar drugs, avanaﬁl provide a rapid onset of action inhibitor approved for erectile dysfunction (ED) by the with long-term eﬃcacy in treating ED (Limin et al. 2010; FDA on 27 April 2012. According to relevant literature Belkoﬀ et al. 2013). But the most common adverse reac- (Zhao et al. 2012; Belkoﬀ et al. 2013), avanaﬁlis eﬀective tions reported in drug’s instructions are headache, dizzi- and safe for ED. NHPPC may be eﬀective in treating ED ness, cold symptoms, ﬂushing, nasal congestion, back and reduce side eﬀects through further optimization. pain and visual impairment. It can also have more The preliminary eﬃcacy experiment of rabbit model in severe side eﬀects such as: change or loss of vision and/ vivo showed that NHPPC has stronger than avanaﬁl. or hearing, priapism, chest or stomach pain. Ananaﬁlas This data cannot be published publicly for the time new high selectivity PDE5 inhibitors, analogs of its struc- being, and an article will be written in the future. It is note- ture will be potential biological activity of drug molecules. worthy that ED is widespread complaint, aﬀecting 150 It may be that we need to discover and develop new com- million men worldwide (Selvin et al. 2007). PDE5 inhibitors pounds, which can improve the safe use of drugs to including avanaﬁl, tadalaﬁl, vardenaﬁl and sildenaﬁl have prevent adverse reactions. So in the early research been widely accepted as ﬁrst-line therapy for ED (Doggrell stage, we need to conduct a series of in vivo and in vitro 2005; Yuan et al. 2013). But these conventional CONTACT Longyuan Wang firstname.lastname@example.org Department of Pharmacy, Aﬃliated Hospital of Qingdao University, Haier Road No. 59, Laoshan District, Qingdao 266003, Shandong, People’s Republic of China © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 156 H. QU ET AL. dog liver microsomes were purchased from BD Gentest (Woburn, MA, USA). Collagenated 48-well plate was obtained by BD Biocoat. HPLC-grade acetonitrile and methanol were purchased from Merck (Darmstadt, Germany). Deionized water was obtained from a Milli-Q plus water puriﬁcation system (Milli-Q, Millipore Corp., MA, USA). All other chemicals and reagents used were of either HPLC or analytical grade. All LC analysis was carried out on a liquid chromato- graphic system (Kyoto, Japan) consisting of Shimadzu model LC-20A pump, an autosampler (SIL-20ACHT, UFLC), a column oven (CTO-20A), and an on-line degas- ser (DGU-20A3). The autosampler temperature was main- Figure 1. Chemical structure of NHPCC.# tained at 4 °C. studies including plasma protein binding (PPB) (Clark and 2.2. Animals and dose formulation Grootenhuis 2002; Vande and Giﬀord 2003), metabolic Sprague–Dawley (SD) rats (180–220 g, 8 weeks) were pur- stability (Xu et al. 2002; Baranczewski et al. 2006; Zhang chased from Beijing Vital River Laboratory Animal Tech- et al. 2007)and a simple pharmacokinetic (PK) model. nology Co., Ltd. (Beijing, China), and Beagle dogs (age: 8 months, body weight: 6-9 kg) were purchased from 2. Materials and methods Beijing Marshall Biotechnology Co., Ltd. The room was controlled and monitored for humidity (40%–70%) and 2.1. Chemicals, reagents and instruments temperature (20 °C–25 °C) with 10–20 air changes/h, N-(2-pyridylmethyl)-2-amino-4-(3-chloro-4-methoxy- and a 12-h light/dark cycle. All animals were fasted for benzylamino)-5-pyrimidine-carboxamide (NHPPC) and 12 h before experiment with water ad libitum and the the internal standard (IS) with purities of >95%, varde- feed was provided 4 h post-dose. The food and water naﬁl, were obtained from MedChemExpress (Shanghai, were certiﬁed to adhere to the national standards China). The positive control drug (propafenone and GB14924.3–2010 and GB14925–2010. For preparation of warf) were obtained from Sigma Chemical Co. (St. Louis, standards and quality controls, blank plasma was col- MO, USA). Pharmaceutical grade dimethyl sulfoxide lected from adult healthy SD rats and Beagle dogs. All (DMSO, purity >99.98%) was supplied by Zhonglan animal procedures were conducted according to the Insti- Industry Co., Ltd. (Shanghai, China). Pharmaceutical tutional Animal Care and Use Committee (IACUC) for grade Hydroxypropyl-beta-cyclodextrin (HP-β-CD, purity animal experimentation, and the protocol was approved >99.9%) was purchased from Shandong Binzhou by the Animal Ethics Committee of Qingdao University. Zhiyuan Biotechnology Co. Ltd. (Shangdong, China). Solution for the intravenous (IV) and oral (PO) dosing Control human, Sprague–Dawley rat and Beagle dog formulation were prepared freshly before dosing as plasma (EDTA-K2 anti-coagulant) was purchased from follows: appropriate amount of NHPPC was weighted Bioreclamation (Hicksville, NY, USA) for plasma protein accurately and dissolved in appropriate volume of binding experiments. Potassium dihydrogen phosphate, DMSO, and followed by adding appropriate volume of dipotassium hydrogen phosphate and sodium hydroxide 6%HP-β-CD in aqueous solution while vortexing. Then were obtained from Sinopharm Chemical Reagent Co., the resulting solution, containing 2% DMSO and 98% Ltd. (Shanghai, China). Equilibrium dialysis membranes 6%HP-β-CD in aqueous solution, was ﬁltered by with a molecular mass cut-oﬀ of 0.8–14 kDa were pur- 0.22 µm membrane. The ﬁnal concentration of NHPPC chased from Viskase (Darien, USA). HPLC-grade formic was 1.0 mg/mL. The IV dose volume was 1 mL/kg for acid (FA), DMSO, isocitric acid (IA), magnesium dichloride rats and dogs, and the PO dose volume was 2 mL/kg (MgCl ), β-Nicotinamide adenine dinucleotide phos- for rats and 1 mL/kg for dogs. phate (NADP) and isocitric dehydrogenase (IDH), henace- tin, diclofenac, S-mephenytoin, dextromethorphan, 2.3. Preparation of working solutions Midazolam, testosterone, α-Naphtoﬂavone, sulfaphena- zole, N-3-benzylnirvanol, quinidine, ketoconazole, ome- The NHPPC and vardenaﬁl (internal standard, IS) stock prazole and rifampin were purchased from Sigma- solutions of 1 mg/mL were prepared in an appropriate Aldrich (Steinheim, Germany). Pooled human, rat, and volume of DMSO for standard and quality control ANIMAL CELLS AND SYSTEMS 157 samples. The NHPPC and the positive control drug (pro- 95% to 5% B, 2.3–3.0 min, isocratic elution with 5% pafenone and warf) stock solutions of 10 mM were pre- B. The column temperature was maintained at 35 °C pared in an appropriate volume of DMSO for plasma and the injection volume at 10 μL. Mass spectrometric protein binding and metabolic stability studies. The detection was performed on API4000 Qtrap mass spec- working solutions of NHPPC were obtained by serial trometer with an electrospray ionization (ESI) interface dilution using acetonitrile: H O (1:1, v/v) mixture as a operating in positive ion mode, was manufactured by diluent. The ﬁnal concentrations of NHPPC working sol- Applied Biosystems (Toronto, Canada). The MS/MS ution was 20, 50, 100, 250, 500, 1000, 2000, 5000, system was operated at unit resolution in the multiple 10,000, and 20,000 ng/mL for standard calibration, and reaction monitoring (MRM) mode, and the monitored 40, 2000, and 16,000 ng/mL for quality control samples. transitions were m/z 483.1→375.2 for NHPPC, m/z The IS stock solution was diluted to 100 ng/mL by step- 489.4→151.3 for IS, m/z 309.0→163.1 for warf and m/z wise dilution of the stock solution with acetonitrile. 342.4→116.2 for propafenone, respectively. The mass spectrometry conditions were as follows: CAD: medium, CUR: 20 psi, Gas1: 55 psi, Gas2: 55 psi, ISV: 5000 V, TEM: 2.4 Preparation of standard calibration and 600°C, EP: 10 V, CXP: 12, and dwell time: 100 ms. Com- quality control samples pound parameters like DP and CE were optimized at The calibration curve standard samples were prepared by 100 and 40 V for NHPPC, 120 and 60 V for IS, 48 and spiking 450 μL of blank rat or dog plasma with 50 μLof 15 V for warf and 100 and 50 V for propafenone, respect- each NHPPC working solution to yield. The ﬁnal concen- ively. Analyst 1.6.1 from AB SCIEX was used for output- trations for the NHPPC standard samples were 2, 5, 10, ting raw chromatograms and generating concentration 25, 50, 100, 250, 500, 1000 and 2000 ng/mL. Quality results. control (QC) samples for NHPPC were prepared at three diﬀerent concentrations (4, 200 and 1600 ng/mL) in the 2.7. Plasma protein binding (PPB) study same way as the plasma samples for calibration. Stock sol- utions were stored at –4°C until analysis. The phosphate buﬀer (100 mM) and sodium hydroxide solution (1.0 M) were prepared in deionized water. The stability in plasma was evaluated at 0.2 and 1.0 μMof 2.5. Preparation of samples NHPPC and the positive control warf for each species The samples including plasma of calibrators and QCs, by incubating at 37°C for 6 h before proceeding with plasma protein binding, metabolic stability study and PPB study. Non-speciﬁc binding potential of NHPPC unknown samples of PK studies were processed by and warf to dialysis membrane was assessed by spiking simple protein precipitation with acetonitrile. The them into phosphate buﬀer and incubating for 6 h. unknown samples were thawed at room temperature. NHPPC and warf were stable in plasma for 6 h and A50 μL aliquot from each sample was pipetted into cen- showed no non-speciﬁc binding to dialysis membrane. trifuge tubes, followed by adding 200 μL of the IS PPB was determined by equilibrium dialysis method. working solution and vortexed for 5 min. The mixture An aliquot (150 μL) of NHPPC spiked plasma and phos- was centrifuged at 12,000 rpm for 5 min at 4°C. A 50 phate buﬀer (pH 7.4) was added into the donor side aliquot of each separated supernatant was transferred and the receiver side of each designated well. The into a clean 96-well plate which was contained 150 μL plate was covered with adhesive sealing ﬁlm to of water. Then an aliquot of 10 μL of each samples was prevent evaporation and placed in a water-bath shaker vortexed for 5 min and injected onto LC-MS/MS for kept at 37°C for 6 h with a rate of 50 rpm. The cross- analysis. species assessment of bound fraction was conducted with 6 h equilibration in rat, dog, and human plasma at NHPPC and warf concentrations of 0.2 and 1.0 μM, 2.6. LC-MS/MS conditions respectively. At the end of 6 h, aliquots were taken Analytes and IS peaks were carried out on an Agilent XDB from each side of each well (n = 3) and kept in a −20°C C column (2.1 × 50 mm, i.d.; 5 μm, Agilent, CA, USA) freezer prior to analysis. The stability, device recovery The mobile phase consisted of (A) 0.1% FA in water and PPB were calculated using the following equations: and (B) 0.1% FA in acetonitrile at a ﬂow rate 0.5 mL/ 6h Remaining at 6 h (%) = × 100%, min. The gradient elution program was as follows: 5% 0h B as initial mobile phase, 0–0.3 min, 0.3–0.8 min, linear C + C Donor Receiver gradient from 5% to 95% B; 0.8–2.0 min, isocratic Device recovery (%) = × 100%, elution with 95% B; 2.0–2.3 min, linear gradient from 6h 158 H. QU ET AL. 2.9 Inhibition and induction on CYP isozymes C − C Donor Receiver PPB (%) = × 100%. C The stock solutions of the probe substrates phenacetin Donor (20.0 mM), diclofenac (20.0 mM), S-mephenytoin (30 mM), dextromethorphan (10.0 mM), midazolam (10.00 mM) and testosterone (50.0 mM) were prepared 2.8 In vitro metabolic stability study using methanol, while α-Naphtoﬂavone (3.0 mM), sulfa- phenazole (3.0 mM), N-3-benzylnirvanol (3.0 mM), quini- The phosphate buﬀer (pH 7.4, 100 mM) and (300 mM) dine (3.0 mM), ketoconazole (3.0 mM) and NHPPC were prepared in deionized water. NADP and IA were dis- (10.0 mM) were prepared using DMSO, and diluted solved in phosphate buﬀer and MgCl solution and then with phosphate buﬀer (PB, 100 mM, pH 7.4) before added 20 μL IDH (18 units/mg protein) and mixed. added into the incubation system. The organic solvents NHPPC (1 mM) and positive control drug were prepared in the ﬁnal incubate were no more than 0.20%. The incu- by dilution using DMSO and then the NHPPC and posi- bation medium was 100 mM phosphate buﬀered saline, tive control drug working solution (50 μM) were and the ﬁnal incubation volume was 200 μL containing obtained by dilution using methanol: H O (1:1, v/v) 25 pmol/mL human liver microsomes CYP enzyme, mixture. Pooled human, rat and dog liver microsomes probe substrate, 1 mM NADPH and NHPPC at 3.00 μM. were added to phosphate buﬀer pH7.4 to a concen- The reactions were initiated after 5 min pre-incubation tration 20 mg/mL. NHPPC was incubated with liver at 37°C and were terminated by the addition of 400 μL microsomes. The incubation solution of the ﬁnal reaction ice-cold acetonitrile. All incubations were conducted in conditions contained 50 mM potassium phosphate triplicate, and the samples were stored at −80°C until buﬀer (pH 7.4), 0.5 mg protein/mL microsomes, 3 mM analysis. Calculation of IC50 was performed by using fol- MgCl , 2 mM NADPH and 1 μM NHPPC or positive lowing equations, where C is concentration of test control drug in a ﬁnal volume of 100 μL. In sample and compounds. positive control groups, NADPH was added into the incu- bation solutions. In the negative control group, water (the replacement of NADPH) was added into the incu- C×(100−%inhibiton at C) bation solutions. Samples were incubated aerobically in IC50= ,(suppose Hill slope = 1) %inhibition a shaking water bath at 37°C and the reaction was termi- nated by adding 300 μL of ice cold acetonitrile (contain The hepatocytes were seeded on a collagenated 48- 100 ng/mL IS) at diﬀerent interval 0, 5, 10, 20, 30 and well plate for 24 h in an incubator at 37°C (moisturized, 60 min. While positive control and negative control with 5% CO ). The hepatocytes were then incubated were incubated aerobically in a shaking water bath at with the test article thereafter. The stock solutions of 37°C for 0 and 60 min, respectively. NHPPC and control omeprazole (100 mM), rifampin (20.0 mM), testosterone concentrations were determined by LC-MS/MS at each (200 mM), phenacetin (100 mM) and NHPPC (50.0 mM) time point (n = 3). Calculation of t and CL was per- 1/2 int were prepared in DMSO, and were diluted using the formed by using following equations, where K is the cell culture medium. The DMSO content in the ﬁnal sol- −1 elimination rate constant (min ) and Q is hepatic utions was 0.10%, the medium containing 0.10% DMSO blood ﬂow (mL/min/kg). was used as the blank control. The cells were co-incu- 0.693 bated with the positive control inducers or NHPPC for t = 1/2 K 72 h and during the incubation the medium was changed every day. After the incubation, the medium CL =V × K int,in vitro D was discarded and replaced with 200 μL phenol red- Volume of reaction mixture(mL) free culture medium containing 100 μM phenacetin V = . mg of protein per reation (CYP1A2 probe substrate) or 200 μM testosterone (CYP3A4 probe substrate). After the incubation in the Hepatic intrinsic clearance was estimated by using the presence of the probe substrates at 37°C for 1 h, the scaling factors and calculated by using the following supernatants were collected and stored at −80°C until equation (Lave et al. 1997). analysis. All incubations were also conducted in triplicate. CL =CL × mg microsomal protein per liver int,in vivo int The generated metabolites of the CYP1A2 and CYP3A4 ×g liver per kg body weight, probe substrates were analyzed by LC/MS/MS methods. The induction eﬀects of the positive control inducers CL × Q CL , H int,in vivo H int C = , E = . int,H H and NHPPC were assessed by the amount of metabolites CL + Q Q int,in vivo H H formed in the induced and uninduced hepatocytes. The ANIMAL CELLS AND SYSTEMS 159 percent induction of positive control was calculated pharmacokinetic parameters (for example: K , AUC , 10 0–t using the following equation. AUC , C , T , t , CL, V ,MRT , etc.) were ana- 0-∞ max max 1/2 ss 0–∞ lyzed based on a non-compartmental model and using Percent induction of positive control(%) Pharsight WinNonlin version 6.3 (St. Louis, USA). The (A − A ) × 100% t n area under the concentration time curve (AUC and 0–t = , A − A p n AUC ) was calculated by linear trapezoidal rule. The 0–∞ natural logarithms (Ln) of the fraction remaining where A = test sample activity, A = negative control t n against incubation times (min) was plotted and the activity and A = positive control activity. ﬁrst-order rate constant of turnover is determined by a linear regression model. The ﬁnal results (for example: 2.10. In vivo pharmacokinetic (PK) study the mean, standard deviation, the remaining, device recovery, k , bioavailability, etc.) were electronically For the iv experiment, male SD rats (n = 3) were given a exported into Excel 7.0 (Redmond, USA) for data proces- single dose of NHPPC (1 mg/kg) over 1 min by dorsal sing and formatting. venous arch of foot. Approximately 200 µL blood samples were collected at 5, 15, 30 min and 1, 2, 4, 6, 8 and 24 h post-dose via tail vein into heparinized 3. Results (coated with 0.1% heparin in saline) tubes. For the oral experiments, the male rats (n = 3) were given a single 3.1 Plasma protein binding dose of 2 mg/kg. Then the blood samples were collected NHPPC was highly bound (>95%) to rats, dogs and at 10, 30 min and 1, 2, 4, 6, 8 and 24 h post-dose. Plasma humans plasma proteins. The PPB rates of NHPPC in was obtained after centrifugation at 8000 rpm for 6 min rats were assessed to be 95.7% and 96.7% at 0.2 and (4°C) within 10 min of blood-drawing and stored at –70°C 1.0 µM, respectively. The PPB rates of NHPPC in dogs until they were analyzed. were assessed to be 99.2% and 99.9% at 0.2 and For the iv experiment, male Beagle dogs (n = 3) were 1.0 µM, respectively. The PPB rates of NHPPC in given a single dose of NHPPC (1 mg/kg) over 1 min by humans were assessed to be 99.3% and 99.5% at 0.2 forelimb saphenous vein. Approximately 500 µL blood and 1.0 µM, respectively. The plasma stability tests of samples were collected at 5, 15, 30 min and 1, 2, 4, 6, 8 NHPPC at 0.2 and 1.0 µM, respectively, at 37°C showed and 24 h post-dose via small saphenous vein into hepar- that NHPPC was stable for 6 h in the rats (100.9%), inized (coated with 0.1% heparin in saline) tubes. For the dogs (100.0%) and human (100.7%) plasma, with ≥99% oral experiments, the male rats (n = 3) were given a NHPPC remaining after the 6 h incubation. In addition, single dose of 1 mg/kg. Then the blood samples were non-speciﬁc binding was not observed with membrane collected at 10, 30 min and 1, 2, 4, 6, 8 and 24 h post- or equilibrium device. All data of PPB studies of NHPPC dose. Plasma was obtained after centrifugation at and the positive control warf were summarized in 8000 rpm for 6 min (4°C) within 10 min of blood- Tables 1 and 2. drawing and stored at –70°C until they were analyzed. The bioavailability (BA %) was calculated using the formula below: 3.2 In vitro metabolic stability AUC (PO) × Dose (IV) inf F% = × 100. NHPPC incubated with microsomes without NADPH AUC (IV) × Dose (PO) inf regenerating system for 60 min, and the percent remain- ing of NHPPC was 102.4%, 98.4% and 99.4% in rat, dog and human liver microsomes, respectively. After incu- 2.11. Data analysis bated for 60 min in the presence of NADPH, the Data analysis including analyte/IS was acquired using percent remaining of NHPPC was 42.8%, 0.8% and Analyst version 1.6.1 software (AB SCIEX). All 42.0% in rat, dog and human liver microsomes Table 1 . Stability of NHPPC and warf in plasma after 6 h incubation at 37°C. Mean drug remaining % (n =3) NHPPC Warf Concentration (µM) Rat Dog Human Rat Dog Human 0.2 100.3 ± 1.93 99.6 ± 0.53 99.8 ± 1.37 99.2 ± 0.93 99.9 ± 1.46 99.0 ± 0.50 1 101.5 ± 1.26 100.4 ± 0.98 101.7 ± 1.50 99.7 ± 1.09 100.2 ± 2.05 99.6 ± 0.87 Mean 100.9 ± 1.01 100.0 ± 1.24 100.7 ± 1.23 99.5 ± 0.82 100.1 ± 1.87 99.3 ± 1.08 160 H. QU ET AL. Table 2. The PPB and recovery of NHPPC and warf in plasma determined using an equilibrium dialysis method. Mean PPB % (n = 2) or mean device recovery % (n =3) NHPPC Warf Concentration (µM) Rat Dog Human Rat Dog Human 0.2 95.7 ± 0.26 99.2 ± 0.12 99.3 ± 0.20 94.8 ± 0.36 95.1 ± 0.20 99.2 ± 0.23 1 96.7 ± 0.21 99.9 ± 0.38 99.5 ± 0.16 95.4 ± 0.23 95.3 ± 0.13 99.8 ± 0.29 Mean PPB 96.2 ± 0.18 99.6 ± 0.26 99.4 ± 0.13 95.1 ± 0.18 95.2 ± 0.04 99.5 ± 0.19 Mean recovery 98.6 ± 0.13 95.7 ± 0.30 101.2 ± 0.02 107.9 ± 0.03 100.7 ± 0.49 107.0 ± 0.25 (Figure 2), respectively. After incubated for 60 min, the no inhibition on CYP1A2, CYP2C9, CYP2C19, CYP2D6 percent remaining of the positive control propafenone and CYP3A4. were <0.1%, 0.2% and 0.1% in rat, dog and human liver The changes of CYP1A2 and CYP3A4 activities in microsomes, respectively. The positive control was human primary hepatocytes of three batches after co- employed in order to ensure that microsomes and incu- incubation with NHPPC at 0.10, 1.00 and 10.0 μM were bation conditions were appropriate to assess liver micro- investigated to evaluate the induction potential of somal metabolic stability studies. The results indicated NHPPC on CYP enzymes. Test compounds would be con- that the incubation system was normal. The determined sidered as inducers when the percent induction of posi- parameters are K , t ,CL ,CL and regression co- tive control ≥40% (US FDA 2000). The results showed e 1/2 int, ivtro H eﬃcient (R ) are shown in Table 3. that the percent induction of NHPPC after the treatment at 0.10, 1.00 or 10.0 μM was less than 40% of that from the positive control for both CYP1A2 and CYP3A4; suggesting NHPPC had no induction on CYP1A2 and 3.3 Inhibition and induction CYP3A4 in human primary hepatocytes. The changes of the human liver microsomes CYP isozyme activities after the co-incubation with NHPPC (3 μM) and the corresponding probe substrates were 3.4 In vivo pharmacokinetics investigated and the IC50 values were calculated to The plasma concentration–time proﬁles of NHPPC after a assess the inhibition potential of NHPPC on CYP iso- single IV and a single PO in rats and dogs were showed in zymes. The enzyme activities were reﬂected by the formed amount of the metabolites of the probe sub- Table 3. Parameters of NHPPC in rat, dog and human liver strates. According to relevant literature (White 2000; microsomes. Yan and Caldwell G 2001), the results showed that the CL CL int, ivtro H IC50 values of NHPPC were greater than 100 μM for −1 2 Species K (min ) t (min) R (mL/min/mg) (mL/min/kg) e 1/2 CYP1A2, CYP2C19, CYP2D6, CYP3A4 (midazolam Rat 0.0163 42.5 0.8434 0.0233 27.5 based) and CYP3A4 (testosterone based), while Dog 0.0843 8.2 0.9905 0.1204 25.3 Human 0.0150 46.2 0.9134 0.0214 9.80 21.9 μM for CYP2C9. The results suggested NHPPC had Figure 2. Time-dependent metabolic depletion proﬁles of NHPPC in rat, dog and human liver microsomes (LM) for: the reaction with NADPH and control without NADPH (% amount remaining vs. incubation time). ANIMAL CELLS AND SYSTEMS 161 Figures 3 and 4 and the pharmacokinetic parameters were BA was credible. From Cl and CL , the int, in vivo int, in vitro calculated and summarized in Table 4. After IV at 1 mg/kg, result manifested that there was in vivo and in vitro corre- the total plasma CL of NHPPC was similar values for both lation in rat. species (1.19 L/h/kg in rats and 1.46 L/h/kg in dogs). The steady state volume of distribution (V ) was higher in ss 4. Discussion rats (6.50 L/kg) than in dogs (1.68 L/kg), so the terminal plasma elimination t was 4.55 h in rats and 1.33 h in 1/2 The results of PPB indicated that only a very small dogs, respectively. After PO, NHPPC was rapidly absorbed amount of unbound NHPPC was available in blood for with a T between 0.5 and 1.0 h in dogs, while T was max max its therapeutic action. The percentage of drug-plasma 6 h in rats. The t values in rats and dogs were 4.02 and 1/2 protein binding was measured in vitro to provide a 1.57 h for PO. NHPPC absolute bioavailability (BA) in rats basis for estimating potential free drug concentrations and dogs were approximately 34.5% and 53.1%, respect- in vivo. According to the ranking criteria for clearance ively. The elimination rate constant (K ) of NHPPC after 10 in diﬀerent species (Houston 1994; Kerns and Di 2008), PO was consistent with that after IV and dose design NHPPC was high clearance in dog and middle clearance were very similar for both species. It indicated that the in rat and human. In general industry practice, Figure 3. The plasma concentration–time proﬁles of NHPPC after a single intravenous and oral dose in rats. Figure 4. The plasma concentration–time proﬁles of NHPPC after a single intravenous and oral dose in dogs. 162 H. QU ET AL. Table 4. Pharmacokinetic parameters of NHPPC in plasma after a single intravenous dose and a single oral dose in rats and dogs (each value represents the mean ± SD of three animals). Rout/Dose K t CL V AUC AUC MRT 10 1/2 ss 0–t 0-∞ 0-∞ −1 Species (mg/kg) (hr ) (hr) (L/hr/kg) (L/kg) (hr*ng/mL) (hr*ng/mL) (hr) Rat IV: 1 0.15 ± 0.00 4.55 ± 0.11 1.19 ± 0.25 6.50 ± 1.00 847 ± 195 866 ± 201 5.50 ± 0.36 Dog IV: 1 0.52 ± 0.03 1.33 ± 0.06 1.46 ± 0.09 1.68 ± 0.08 679 ± 38.5 685 ± 40.0 1.15 ± 0.04 Species Rout/Dose K t T C AUC AUC MRT BA 10 1/2 max max 0–t 0-∞ 0-∞ −1 (mg/kg) (hr ) (hr) (hr) (hr*ng/mL) (hr*ng/mL) (hr*ng/mL) (hr) (%) Rat PO: 2 0.17 ± 0.02 4.02 ± 0.39 6.00 52.6 ± 12.9 583 ± 135 593 ± 137 7.80 ± 0.79 34.5 ± 7.91 Dog PO: 1 0.45 ± 0.06 1.57 ± 0.22 0.50 144 ± 35.3 351 ± 96.1 364 ± 97.4 2.54 ± 0.15 53.1 ± 14.2 compounds demonstrate high metabolic instability if the Disclosure statement remaining is less than 30%. Based on drug–drug inter- No potential conﬂict of interest was reported by the authors. action evaluation, NHPPC is expected to have no or low possibility in CYP450-mediated drug–drug inter- References action in humans. The elimination rate constant (K )of NHPPC after PO was consistent with that after IV and Baranczewski P, Stanczak A, Sundberg K, Svensson R, Wallin A, dose design were very similar for both species. It indi- Jansson J, Garberg P, Postlind H. 2006. Introduction to in vitro estimation of metabolic stability and drug interactions cated that the BA was credible. of new chemical entities in drug discovery and development. Pharmacol Rep. 58:453–472. Belkoﬀ LH, McCullough A, Goldstein A, Goldstein I, Jones L, 5. Conclusions Bowden CH, DiDonato K, Trask B, Day WW. 2013. An open- label, long-term evaluation of the safety, eﬃcacy and toler- The science of preclinical drug development is a risk-based ability of avanaﬁl in male patients with mild to severe erectile exercise that extrapolates nonhuman safety and eﬃcacy dysfunction. Int J Clin Pract. 67:333–341. information to a potential human outcome. So in the Clark DE, Grootenhuis PD. 2002. Progress in computational present work, the potential of NHPPC was investigated in methods for the prediction of ADMET properties. Curr Opin preclinical evaluation of drug metabolism and pharmacoki- Drug Discov Dev. 5:382–390. netics. NHPPC was highly bound to plasma proteins. From Doggrell SA. 2005. Comparison of clinical trials with sildenaﬁl, vardenaﬁl and tadalaﬁl in erectile dysfunction. Expert Opin the in vitro microsomal incubation studies, the result Pharmacother. 6:75–84. showed high metabolic instability and there is no corre- Houston JB. 1994. Utility of in vitro drug metabolism data in lation in vitro and in vivo study and the good correlation predicting in vivo metabolic clearance. Biochem appears to have been observed in rats. According to the Pharmacol. 47:1469–1479. reported criteria (IC50 > 10 μM categorized as weak inhi- Kerns EH, Di L. 2008. Disposition, metabolism, and safety. In: Drug-like Properties: Concepts, structure design and bition, 1 μM< IC50<10 μM categorized as medium inhi- methods. Part 3. Oxford: Elsevier. bition and IC50 < 1 μM categorized as potent inhibition), Lave TH, Dupin S, Schmitt C, Chou RC, Jaeck D, Coassolo PH. NHPPC had little inhibition on CYP1A2, CYP2C9, 1997. Integration of in vitro data into allometric scaling CYP2C19, CYP2D6 and CYP3A4. The results using human to predict hepatic metabolic clearance in man: application primary hepatocytes showed that NHPPC had no induction to 10 extensively metabolized drugs. J Pharm Sei. 86:584– eﬀect on CYP1A2 or CYP3A4. Based on the data, NHPPC is 590. Limin M, Johnsen N, Wayne JGH. 2010. Avanaﬁl, a new rapid- safe and unlikely to cause any clinically signiﬁcant herb- onset phosphodiesterase 5 inhibitor for the treatment of drug interactions and thus cause the occurrence of erectile dysfunction. Expert Opin Invest Drugs. 19:1427– adverse drug reactions in humans when co-adminstered with substrates of the ﬁve CYPs. NHPPC was found to be Selvin E, Burnett AL, Platz EA. 2007. Prevalence and risk a rapidly absorbed, low clearance and good oral bioavail- factors for erectile dysfunction in the US. Am J Med. 120:151–157. ability compound in dogs. While the result showed that US FDA (2012-2-1). Guidance for industry: drug interaction NHPPC has a longer plasma half-life in rats. In addition, studies-study design, data analysis, implications for dosing, this is the ﬁrst report on plasma protein binding, metabolic and labeling recommendations [EB/OL]. www.fda.gov/ stability and pharmacokinetic of NHPPC and provide downloads/DrugsGuidanceComplianceRegulatoryInformation/ important leads for studying PDE5 inhibitors. Guidances/UCM292362.pdf. Vande WH, Giﬀord E. 2003. ADMET in silico modelling: towards prediction paradise? Nat. Rev. Drug Discov. 2:192–204. Acknowledgments White RE. 2000. 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Curr TY, Moon KH, Chung WS, et al. 2012.Eﬃcacy and safety of Top Med Chem. 5:403–425. avanaﬁl for treating erectile dysfunction: results of a multi- Yuan JQ, Zhang RJ, Yang ZY, Lee J, Liu YL, Tian JH. 2013. centre, randomized, double-blind, placebo-controlled trial. Comparative eﬀectiveness and safety of oral BJU Int. 110:1801–1806.
Animal Cells and Systems
– Taylor & Francis
Published: May 4, 2019
Keywords: Plasma protein binding; metabolic stability; cytochrome p450 inhibition and induction; pharmacokinetics; type 5 phosphodiesterase inhibitors