Accepted Manuscript Title: Plasma protein binding, pharmacokinetics, tissue distribution and CYP450 biotransformation studies of fidarestat by ultra high performance liquid chromatographyhigh resolution mass spectrometry Author: Roshan M. Borkar Murali Mohan Bhandi Ajay P. Dubey Prajwal P. Nandekar Abhay T. Sangamwar Sanjay K. Banerjee R. Srinivas PII: DOI: Reference:

S0731-7085(14)00499-3 http://dx.doi.org/doi:10.1016/j.jpba.2014.10.008 PBA 9759

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received date: Revised date: Accepted date:

24-6-2014 6-10-2014 8-10-2014

Please cite this article as: R.M. Borkar, M.M. Bhandi, A.P. Dubey, P.P. Nandekar, A.T. Sangamwar, S.K. Banerjee, R. Srinivas, Plasma protein binding, pharmacokinetics, tissue distribution and CYP450 biotransformation studies of fidarestat by ultra high performance liquid chromatography- high resolution mass spectrometry, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.10.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Highlights

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First bioanalytical method has been developed and validated for fidarestat

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

9.5% of the free form of the drug may be available for the pharmacological action.

5



CYP1A2 and CYP2D6 appeared to be the key enzymes for the biotransformation.

6



It is a weak inhibitor of CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4

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

It may not alter the pharmacokinetic and clearance of other co-administered drug.

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1 2 3

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N

O

9

F

CYP450 Inhibition

OH

O

O

F

HLMs

Biotransformation

N

O

CYP1A2 & CYP2D6

O

H N

11

H

90

(a)

1000

29 30 31

Concentration(ng/mL)

40 30 20

0

1

2

3

4

5

Time (h)

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0

1

2

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Time (h)

800 18

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15

12 9 6 3 0

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ain Br

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Tissue concentration (ng/mg of tissue)

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800

Fidarestat concentration (ng/mL)

1200

Unconjugated Fidarestat Total Fidarestat

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50

0

0

1200

27

60

10

400

200

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26

600

Tissue Distribution

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18

H

Unbound drug

Concentration (ng/mL)

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(b)

80

Pharmacokinetic 800

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Total plasma concentration (bound+unbound)

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N

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Plasma protein binding, pharmacokinetics, tissue distribution and CYP450 biotransformation studies of fidarestat by ultra high performance liquid chromatographyhigh resolution mass spectrometry

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Roshan M. Borkar1,4, Murali Mohan Bhandi1, Ajay P. Dubey2, Prajwal P. Nandekar3,

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Abhay T. Sangamwar3, Sanjay K. Banerjee4*† and R. Srinivas1, 2* 1

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National Centre for Mass Spectrometry, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500 007, India 2

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Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research (NIPER), Balanagar, Hyderabad, 500037, India

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National Institute of Pharmaceutical Education and Research (NIPER), Sector 67, S.A.S. Nagar (Mohali), 160 062, Punjab, India 4

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Medicinal Chemistry and Pharmacology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500 007, India

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6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

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Telephone Number

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Fax Number

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[email protected] (R. Srinivas)

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*Corresponding author :

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[email protected] (S K Banerjee)

+91-40-27193122 : +91-40-27193156

†Present

Address: Drug Discovery Research Center, Translational Health Science and Technology Institute, Gurgaon, 122016, India

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Abstract: Fidarestat, an aldose reductase inhibitor, has been used for the treatment of the diabetic

3

associated complications such as retinopathy, neuropathy, and nephropathy. To better understand

4

the metabolism and pharmacokinetics of fidarestat, we have evaluated plasma protein binding,

5

pharmacokinetics, tissue distribution of the drug and its conjugated metabolites and CYP450

6

biotransformation by liquid chromatography-high resolution mass spectrometry. Effective

7

chromatographic separation of fidarestat and hydrochlorothiazide (IS) in rat plasma and tissues

8

was achieved on Hypersil gold C-18 column in an isocratic elution mode. For detection, a high-

9

resolution Orbitrap mass spectrometer with heated electrospray ionization inlet in the negative ion

10

mode was used. High-resolution extracted ion chromatograms for each analyte were obtained by

11

processing the full-scan MS mode with 5 ppm mass tolerance. The impact of plasma protein

12

binding with the drug and conjugated metabolites of the drug on pharmacokinetics has been

13

determined. The study indicated that 9.5% of free form of fidarestat may be pharmacologically

14

active and the Cmax for free fidarestat was found to be 80.30±6.78 ng/mL. The AUC0–t and

15

AUC0–∞ were found to be 185.46±32 and 195.92±15.06 ng h/mL, respectively. Among tissues,

16

the maximum observed distribution was found to be in kidney followed by liver and heart.

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Docking experiments and in vitro CYP450 reaction phenotyping revealed that two CYP1A2 and

18

CYP2D6 are involved in the phase-I metabolism of fidarestat. Oxidative deamination and N/O

19

glucuronidation are the major phase I and phase II metabolites, respectively. In vitro CYP450

20

inhibition assay of fidarestat for drug-drug interaction showed weak inhibition and may not alter

21

pharmacokinetics, distribution or clearance of other co-administered drug.

22

Keywords: Cytochrome P450, protein binding, UPLC/ESI/HRMS method, drug-drug and drug-

23

herb interaction.

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1. Introduction

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In spite of advances made by modern medicine, there is no single drug to prevent the

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development and progression of many of the hidden complications such as neuropathy,

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retinopathy, nephropathy associated with the diabetes. Diabetic neuropathy, a common

5

complication in 8–26% of diabetic patients, is a degenerative disorder activated by persistent

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hyperglycemia [1]. .

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Aldose reductase is the rate limiting enzyme in the polyol pathway and has a wide variety

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of physiological and pathological role in diabetic vascular and neural complications [2]. Among

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several aldose reductase inhibitors (ARIs) that have been developed over the years, fidarestat got

10

widespread attention. It prevents diabetic retinopathy and cataracts by reducing oxidative stress

11

and VEGF [3]. Recent studies have suggested that fidarestat inhibits aldose reductase enzyme

12

which have pathophysological role in the disease unrelated to hyperglycemia such as

13

lipopolysaccharide-induced acute kidney injury and allergen-induced airway inflammation [4-5].

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Aldose reductase contributes to oxidative stress associated with cerebral ischemic injury,

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suggesting that inhibition of aldose reductase by fidarestat protects animals against cerebral

16

ischemic injury [6].

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Plasma proteins such as albumin and alpha-1 acid glycoprotein binds to drug and limits

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the availability of free drug concentration in plasma to act on its biological targets such as

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enzyme, receptor transporter etc. The ratio of free and plasma protein bound drugs will have an

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impact on pharmacokinetics, pharmacodynamic and safety margins of the compound [7].

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Glucuronidation and sulfation are the major metabolic pathways for detoxification of many drugs

22

and xenobiotics. Acylglucuronides and N-glucuronides have been linked with idiosyncratic

23

adverse drug reactions and bladder cancer, respectively [8]. Therefore, in drug metabolism and

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toxicology studies, it is necessary to quantify such glucuronide metabolites in biological samples.

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It is essential to study plasma protein binding, pharmacokinetics, tissue distribution and 5 Page 5 of 39

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cytochrome P450 biotransformation behavior of fidarestat to know its pharmacodynamic effect,

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but it was not reported in the scientific literature. Cytochrome P450 monoxygenase (CYPs), flavin-containing monoxygenase (FMOs)

4

aldehyde oxidases, xanthine oxidases and monoamine oxidases can play a role in the metabolic

5

clearance of the drug. Recently more stringent requirements have been proposed by the FDA

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(Food and Drug Administration) [9] and European Medicines Agency (EMA) [10], emphasizing

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the importance to identify the metabolic pathway of lead compound for understanding the drug-

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drug interaction (DDI).

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High resolution mass spectrometry (HRMS) has been wieldy used for structural

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identification and characterization of drug metabolites because of the elemental compositions

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provided by the HRMS of target ions, especially for small molecules with molecular weight less

12

than 600 Da. In HRMS, selectivity of the target ion for quantification can be improved by

13

narrowing the extraction window. Full scan quantitative analysis by Orbitrap provides satisfactory

14

mass accuracy, sensitivity and sufficient to achieve the necessary chemical specificity. Whereas,

15

low resolution MS instruments apply MS/MS methods to obtain the specificity of target ions for

16

routine quantification analysis.

17

measurement has been increasingly used to quantify small molecules within preclinical

18

pharmacokinetics samples in three previous reports [11-13]. In the present work, we explore the

19

use of Orbitrap high resolution mass spectrometry to develop plasma protein binding assay and

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determination of fidarestat and its glucuronide/sulphate metabolites in rat plasma. Further, study

21

is extended to quantify fidarestat in rat tissues and CYPs biotransformation studies to predict

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DDI.

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Over the years, LC/MS in combination with accurate mass

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2. Material and methods

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2.1 Chemicals and regents.

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The working standard of fidarestat is a gift samples from Symed Laboratories, Hyderabad, India.

4

HPLC-grade acetonitrile and methanol used in the present study were purchased from Merck

5

(Mumbai, India) and used without further purification. Water was purified by using a Millipore

6

Milli-Q plus purification system (Millipore Corp., Bedford, MA, USA).Trifluoroacetic acid was

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purchased from Sigma-Aldrich, Co. St. Louis, MO, USA. All other chemicals used in the

8

method were of analytical grade. β–glucuronidase (GLU) from Helix Pomatia, β-Nicotinamide

9

adenine dinucleotide phosphate (NADPH) reduced tetra (cyclohexylammonium) salt, warfarin,

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human liver microsomes (HLMs), human liver S9 fraction, Uridine 5′-diphosphoglucuronic acid

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trisodium salt

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adenosynlmethionine (SAM) were purchased from Sigma-Aldrich. Tacrine, diclofenac,

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dextromethorphan, midazolam and S-mephenytoin, ketoconazole, sulphafenazole, N-3-

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Benzylnirvanol, quinidine and α-naphthoflavone were procured from NIPER-Hyderabad, India.

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2.2 Instrumentation

16

Analysis was carried out on U-HPLC instrument (Thermo Scientific Accela™, Thermo Fisher

17

Scientific, San Jose, USA)) equipped with a quaternary pump, a de-gasser, a diode-array detector,

18

an auto sampler and a column compartment. Mass spectrometric analysis was carried out on an

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Orbitrap high-resolution mass spectrometer (Exactive™, Thermo Fisher Scientific, Bremen,

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Germany) equipped with a heated electrospray ionization (ESI) source and used for all HRMS

21

analysis. The data acquisition was under the control of Xcalibur software. The extracted ion

22

chromatograms (EICs) were generated by extracting a small range (e.g. ±5-10 ppm) centered on

23

the exact m/z of each analyte.

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Equilibrium dialysis was performed using rapid equilibrium dialysis (RED) Device, Pierce

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Biotechnology, Thermo Scientific (Rockford, IL USA). The RED device consists of single use

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3′-phosphate

5′-phosphosulfate

(PAPS) and S-

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base plate made of Teflon and the Inserts. Each Insert is comprised of two side-by-side chambers

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separated by an O-ring- sealed vertical cylinder of dialysis membrane (Molecular Weight Cut Off

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-8,000 KD). Assay incubation was done in the Thermomixer eppendorf, Hamburg, Germany.

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Evaporation of samples was done on ScanVac speed Vacuum Concentrator (mas-tek Instruments

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Co. Hyderabad, India).

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2.3 Animals

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All animal experiments were undertaken with the approval of Institutional Animal Ethical

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Committee of Indian Institute of Chemical Technology (IICT), Hyderabad. Female Sprague–

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Dawley rats (200–250 g) were purchased from the Teena Biolab, Hyderabad, India. The animals

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were housed in BIOSAFE, an animal quarantine facility of IICT, Hyderabad, India. The animal

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house was maintained at temperature 22±2°C with relative humidity of 50±15% and 12 h

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dark/light cycle. The animals were fasted for 12 h prior to pharmacokinetic study. Six female

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Sprague–Dawley rats with similar weight and age were utilized to carry out the present

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pharmacokinetic study to reduce the variation of drug concentration in plasma.

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3. Quantification of fidarestat in rat plasma and tissues.

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3.1 Preparation of stock, working solutions and Internal Standard (IS)

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The stock solution of fidarestat was prepared in methanol: water (1:1). Serial dilutions were

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made, using this stock solution, to prepare the primary aliquots of fidarestat in methanol: water

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(1:1) for calibration curve and quality control (QC) samples. Similarly, stock solution of 1 mg/mL

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of hydrochlorothiazide (IS) was also prepared in methanol, further diluted with methanol: water

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(1:1) to prepare working solution containing a concentration of 10 ng/mL.

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3.2 Preparation of calibration and quality control (QC) samples

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5 µL of primary aliquot of fidarestat was spiked in 90 µL of blank K-3 EDTA rat plasma to yield

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calibration curve samples ranging from 0.1-2000 ng/mL for fidarestat. Similarly, low, middle and

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high QC samples (LQC, MQC and HQC) at three different levels were prepared independently at

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concentrations of 20 ng/mL (LQC); 800 ng/mL (MQC); 1400 ng/mL (HQC) for fidarestat. For

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brain and kidney tissues 5 µL of primary aliquot of fidarestat was spiked in 90 µL of pre-

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homogenized blank brain and kidney tissue in phosphate buffer solution (pH- 7.4) (100 mg/mLof

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tissues) to yield calibration curve samples ranging from 0.4-400 ng/mL. Similarly, quality control

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samples (2, 100, 400 ng/mL) were prepared. Blank plasma sample, tissues sample and zero

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samples were also prepared and analyzed. All the stock solutions were stored at 0-4oC for further

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use.

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3.3. Extraction of fidarestat from plasma and tissues samples

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To an aliquot of 100 µL of plasma and tissues samples (calibration standard and test sample), 5

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µL of IS solution was added at a concentration of 10 ng/mL, followed by 50 µL of 0.1 %

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ammonia. This mixture was thoroughly mixed. To this mixture, 850 µL of tert- butyl methyl ether

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(TBME) was added. The mixture solutions were kept in shaker for 10 min and then supernatant

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layer were taken into another eppendorf tube. Evaporation of supernatant was done on ScanVac

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speed Vacuum Concentrator and 100 µL of methanol- water (1:1) was added. Only 10 µL

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aliquots of sample solution were injected into LC/MS system for analysis.

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3.4. UPLC/ESI/HRMS conditions

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The separation of fidarestat and IS from endogenous substances was achieved using Hypersil gold

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C-18 column (50 mm x 2.1 mm I.D.; particle size 3 µm, Thermo Fisher Scientific, Madison,

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USA), C18 HQ105 (10 x 2.1mm) guard column and mobile phase consisting of a mixture of

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acetonitrile-methanol-water (pH adjusted to 3.7 by 0.1 % trifluoroacetic acid) (71:14:15) in an

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isocratic elution mode. The flow rate of the mobile phase was 0.2 mL/min, the column

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temperature at 25°C and the injection volume was 10 μL. Compound dependent parameters and

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instrumental parameters were optimized by infusing neat solutions of fidarestat and the IS

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separately by using a syringe pump. The typical operating source conditions for MS scan in

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negative ion ESI mode were optimized as follows sheath gas flow rate 20; Aux gas flow rate 5;

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Spray voltage 4.50 kV; Capillary temperature 250 °C; Capillary voltage -65.00 V; Tube lens

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voltage -95.0 V; Skimmer voltage -26.00 V and Heater temperature 100°C. The theoretical

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monoisotopic m/z of each [M-H] was derived from the elemental composition. The full-scan

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mode across m/z 250–350 that include fidarestat and hydrochlorothiazide and EICs (m/z

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theoretical ±5 ppm) was used. For quantification, EICs of [M-H] at m/z 278.05771 for fidarestat

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and m/z 295.95622 for hydrochlorothiazide with a 5 ppm range centered on the exact m/z value

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were generated (Fig.1).

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3.5 Bioanalytical Method Validations

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The method was validated according to U.S. Food and Drug Administration (FDA) guidelines for

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the validation of the bioanalytical method (US-FDA 2001) [14].

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3.5.1 Specificity and selectivity

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The specificity and selectivity of the method were investigated by the screening analysis of six

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individual blank rat plasma, blank rat tissues, spiked plasma and spiked tissues samples. Two

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other blank plasma and tissues samples containing an IS concentration of 10.0 ng/mL were also

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tested for interference. Each blank sample was tested for exclusion of endogenous interference at

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the retention times of fidarestat and IS.

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3.5.2 Linearity and Lower limit of quantification (LLOQ)

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The linearity of the method was evaluated by using six calibration standards over a calibration

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range of 0.1-2000 ng/mL in rat plasma and 0.4-400 17 ng/mg in rat brain, kidney, and heart and

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liver tissues. LLOQ was determined as the lowest concentration of analytes with % CV

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(coefficient of variation) not exceeding 20% and accuracy in the range of 80-120%.

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3.5.3 Precision and accuracy

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Intra- and inter-batch precision and accuracy of the developed method were investigated by

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analyzing QC samples at four different concentrations for six replicates. The precision of the

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method was determined by % CV and accuracy was evaluated by recovery. As per US-FDA

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guidelines the acceptable limit of % CV was < 20% for LLOQ whereas acceptance criteria of ≤

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15% for LQC, MQC and HQC. The accuracy was calculated as percent difference in mean value

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of the observed concentration and nominal concentrations of QC samples.

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3.5.4 Extraction recovery and matrix effect

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The extraction recovery and matrix effects were investigated at LQC, MQC and HQC

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concentration levels. Extraction recovery of fidarestat in rat plasma and tissues were evaluated by

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comparing peak area ratios of fidarestat to IS of an extracted sample (n = 6) to the standard

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analytes solution of same concentration. To study matrix effect, initially blank plasma and tissue

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samples were processed followed by spiking of the analyte and IS to the post processed samples.

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Further, aqueous solutions of analyte and IS of the same concentrations were prepared and

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analyzed [12]. The matrix factor was calculated using the equation, [mean peak area ratio of

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processed sample]/ [mean peak area ratio of aqueous sample]. Matrix effect should be in the range

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of 0.8-1.2.

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3.5.5 Dilution Integrity and carry over effect

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To determine the dilution integrity test, the concentrations were extended beyond the upper limit

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of the calibration range or less than validated samples. The plasma and tissues samples containing

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fidarestat at a concentration of 3000 ng/mL and 600 ng/mL, respectively, were prepared. The

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plasma and tissue samples were diluted with two fold and four fold the original concentration

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using blank plasma and tissue samples. Dilution integrity was determined by calculating the

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concentration of diluted samples whose % CV not exceeding 15%.

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Carry over effect was determined by successive injections of one blank aqueous sample, aqueous

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upper limit of quantification (ULOQ) sample, extracted blank plasma sample and extracted

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ULOQ plasma and tissue samples. Carry-over should be ≤20% of response of the mean extracted

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LLOQ samples and ≤5% of the response of the extracted IS samples.

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3.5.6 Stability studies

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To evaluate the stability of fidarestat in rat plasma and tissues samples, six replicates of low and

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high quality control samples were analyzed. Post preparative stability was carried out at 10°C for

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24 h. The bench top and long-term stability were analyzed at ambient temperature 25°C for 8 h,

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and -80°C for 25 days, respectively. For freeze–thaw stability, quality control samples were

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stored at -20°C and thawed at room temperature every 24 h. After three freeze-thaw cycles,

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quality control samples were analyzed. The stability was assessed by comparing with nominal

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concentration of the analyte at 0h and the mean percentage changes are within the acceptance

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criteria of±10%.

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4. Application of method

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4.1. Determination of unbound fidarestat using RED Device

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The standard stock solutions of fidarestat and warfarin were prepared separately in methanol at a

15

concentration of 10mM. Working stock solutions, 10μM of both fidarestat and warfarin were

16

prepared separately in plasma. An aliquot of 300μL of plasma containing fidarestat was added to

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the red chambers of RED device insert, while 500μL of phosphate buffer saline (PBS; pH 7.4) to

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the other chamber. The RED device was covered with Immunoware sealing tape and was

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incubated at 37°C while shaking at 550 rpm for 5 hours. The samples were matrix matched with

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opposite matrix after incubation. The percentage unbound of fidarestat was compared with

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standard compound warfarin prepared by substituting the sample with standard solution which

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was run in parallel. The percent of plasma unbound fraction was calculated by the following

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equations: % Unbound = 100 *FC / TC

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Where, FC = Free compound concentration as determined by the calculated concentration on the

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buffer side of the membrane

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TC = Total compound concentration as determined by the calculated concentration on the plasma

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side of the membrane

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4.2. Pharmacokinetic study

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The plasma samples from six adult female Sprague dawley rats were analyzed for quantification

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of fidarestat with the validated method described above. After fasting for 12h, rats were orally

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administered with fidarestat in saline through gavage of 2 mg/kg dose. Blood samples were

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collected at 0.08, 0.16, 0.33, 0.50, 1, 1.30, 2, 2.30, 3 and 4h, vortexed thoroughly and centrifuged

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at 5000 rpm for 20 min at 4°C. Plasma was collected and stored at -20°C until analysis.

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4.3. Quantification of glucuronides metabolites

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In the present study, quantification of total fidarestat in plasma and tissues were measured by an

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enzymatic hydrolysis which is an indirect approach. GLU cleaved the glucuronides metabolites

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back to their parent aglycone. The free aglycone is quantified according to its calibration curve.

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To an aliquot of 50 µL plasma, 30 µL of GLU from Helix Pomatia containing some companion

14

of sulfatase and 420 µL of Tris*HCl buffer (50mM, pH 7.4) were added and incubated for 2h

15

at 37°C. After incubation, the samples were centrifuged at 20,000×g at 4°C for 10 min. To the

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supernatant, 5 µL of 50 ng/mL of IS and 50 µL of 0.1% ammonia was added followed by 500 µL

17

TMBE. The mixture solutions were kept in shaker for 10 min and then supernatant layer was

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taken into another eppendorf tube. Evaporation of supernatant was done on ScanVac speed

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Vacuum Concentrator and 100 µL of methanol: water (1:1) was added. Only 10 µL aliquots of

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sample solution were injected into LC/MS system for analysis. Similarly quantification of

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glucuronide metabolites was done in 5 mg/kg of kidney, brain, heart and liver tissues.

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4.4. Tissue distribution study in rat brain, kidney heart and liver tissues

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In tissue distribution study, three rats (200-250g) in each group were orally administered

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fidarestat (2 mg/kg). Rats were then sacrificed at 1.30h after dosing. Organs like brain, kidney,

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heart and liver were removed, washed with normal saline, blotted dry with filter paper and then

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accurately weighed. The tissues were homogenized in saline solution (Approx. 100 mg/mL) and

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the homogenates were stored at−20°C until analysis.

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4.5. Structure-based Fidarestat-Cytochrome P450 (CYP450) docking analysis

4

The crystal structures of the CYP450 isoform were retrieved from the Protein Data Bank (PDB ID

5

1TQN for CYP3A4, solved at 2.05 Å resolution [15]; PDB ID 1OG5 for CYP2C9, solved at 2.55

6

Å resolution [16-17]; PDB ID 4GQS for CYP2C19 solved at 2.87 Å resolution; PDB ID 2F9Q for

7

CYP2D6, solved at 3.00 Å resolution [18-19] and PDB ID 2HI4 for CYP1A2, solved at 1.95 Å

8

resolution [20]. The 3D structures of proteins were prepared using protein preparation wizard

9

included in Schrodinger suite 9.0.02. The centroid of co-crystallized ligand was used for protein

10

active site grid definition with a grid size of 30 Å. The molecular docking of ligand fidarestat in

11

mentioned CYPs was performed using Glide molecular docking program included in Schrodinger

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suite 9.0.02 [21]. The previously validated molecular docking parameters were used for docking

13

exercise [22]. Molecular docking was performed with extra precision using OPLS (2001) force

14

field [23]. Scaling factor of 0.8 with partial cut-off of 0.25 and Coulomb- Van der Waals cut-off

15

of 50kcal/mol were used. The best-docked structure for each ligand was selected on the basis of

16

Glide XP docking score.

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4.6. Phase I metabolism study of fidarestat

18

Incubation of fidarestat in HLMs was performed in duplicates. The standard preincubation

19

mixture (final volume, 330 µL) consisted of microsomes (150 µL, 0.28 mg/mL) protein

20

concentration), 170 µL of 100 mM phosphate buffer solution (PBS, pH 7.4) and 1 µM fidarestat.

21

The mixture was pre-incubated for 10 min at 37°C. The cofactor solution of 2.5 mM NADPH was

22

prepared. Blank samples without cofactor were prepared by adding 65 µL of preincubation

23

mixture into 35 µL of PBS buffer solution and incubated for 60 min at 37°C . Incubation of zero

24

min samples was performed with the cofactor, in which 17.5 µL of cofactor was added into 32.5

25

µL of preincubation mixture. To the reaction mixture of blank and zero min samples, 5 µL of IS

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14 Page 14 of 39

solution was added at a concentration of 10 ng/mL. The mixture was thoroughly mixed and 50 µL

2

of 0.1% ammonia followed by 500 µL of TBME was added. In the remaining preincubation

3

mixtures, 124 µL of 2.5 mM cofactor was added and incubated for 60 min at 37°C to prepare

4

incubation mixture. At each time interval of 15, 30 45 and 60 min 50 µL incubation mixture were

5

withdrawn and 5 µL of IS solution was added at concentration of 10 ng/mL, followed by 50 µL of

6

0.1% ammonia. The mixture was thoroughly mixed. To these, 500 µL of TBME ether was added.

7

The samples were kept in shaker for 10 min and then supernatant layer were taken into another

8

eppendorf tube. Evaporation of supernatant was done on ScanVac speed Vacuum Concentrator

9

and 100 µL of methanol: water (1:1) was added. Only 10 µL aliquots of sample solution were

10

injected into LC/MS system for analysis. A series of incubations were also performed with

11

previously inactivated HLMs. The CYP450 proteins were inactivated by heating microsomes at

12

60°C for 10 min. The extent of fidarestat metabolism was measured as percentage of control

13

activity.

14

4.7 Phase II conjugation metabolism study of fidarestat

15

Incubation of fidarestat in S9 fraction was performed at 10μM in duplicates. The S9 fraction (3.33

16

mg/mL protein concentration) was prepared in 100 mM potassium buffer solution (pH 7.4). The

17

mixture was preincubated at 37°C for 10 min. To initiate the reaction, cofactor UDPGA (2 mM),

18

NADPH (1 mM), PAPS (1 mM) and alamethicin (25 μg/mL) were added to give a 330-μl final

19

volume. Blank incubations were performed without cofactors. The final reaction mixture was

20

incubated for 60 min and terminated by adding 200 μl of ice cold acetonitrile containing IS of 20

21

ng/mL. Only 10 µL aliquots of sample solution were injected into UPLC/ESI/HRMS system for

22

analysis. In brief, Agilent XDB C-18 column (150 mm x 4.6 mm I.D.; particle size 5 µm) and

23

mobile phase comprising of a mixture of 10 mM ammonium acetate-acetonitrile (70:30) in

24

an

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isocratic elution

mode

was

used. The phase II metabolites were predicted using

15 Page 15 of 39

1

UPLC/ESI/MS and accurate mass measurements. EICs of [M+H] + ions of phase II metabolites

2

with a 10 ppm range centered on the exact m/z value were generated.

3

4.8. CYP Phenotyping by Chemical inhibition assay

5

The incubations were performed in duplicates. The standard preincubation mixture consisted of

6

microsomes (150 µL, 0.28 mg/mL) protein concentration and 170 µL of 100 mM phosphate

7

buffer solution (PBS, pH 7.4) and 2.5mM of NADPH as cofactor. Selective inhibitor

8

Ketoconazole (selective inhibitor of CYP3A4, final concentration 3 µM) [24], sulphafenazole

9

(selective inhibitor of CYP2C9, final concentration 3 µM) [25], N-3-Benzylnirvanol (selective

10

inhibitor of CYP2C19, final concentration 3 µM), quinidine (selective inhibitor of CYP2D6, final

11

concentration 3 µM) [26-27] and α-naphthoflavone (selective inhibitor of CYP1A2, final

12

concentration 3 µM) [28] were added individually to preincubation mixtures. The final

13

concentration of 1mM fidarestat was added to reaction mixture. The reaction mixtures containing

14

enzymatic sources, PBS buffer, cofactor NADPH and inhibitors were warmed at 37°C for 10 min

15

prior to above. The rate of decomposition of fidarestat in presence of specific inhibitor were

16

expressed as percentage of fidarestat remained at different time points relative to the amount of

17

fidarestat present in incubation mixture at zero time point.

18

4.9. In Vitro CYP inhibition evaluation of fidarestat in HLMs

19

Inhibition experiments were performed in HLMs to determine IC50 values of ketoconazole for

20

CYP3A4 catalyzed midazolam to 1-hydroxy midazolam, α-naphthoflavone for CYP1A2

21

catalyzed tacrine to 1-hydroxy tacrine, N-3-benzylnirvanol for CYP2C19 catalyzed S-

22

mephenytoin to 4-hydroxy mephenytoin, quinidine for CYP2D6 catalyzed dextromethorphan to

23

dextrorphan and sulphafenazole CYP2C9 catalyzed diclofenac to 4-hydroxy diclofenac.

24

Incubation mixtures (200 µL) contained potassium phosphate buffer (100 mM), HLMs (0.22 mg/

25

mL), standard inhibitors (concentration between 0.01 µM to 3 µM), and substrates (final

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16 Page 16 of 39

concentration of 5 µM tacrine, 5 µM diclofenac, 5 µM dextromethorphan, 2 µM midazolam and

2

30 µM S-mephenytoin based on their Km values) were preincubated for 10 min at 37°C before

3

the reactions were initiated by adding NADPH (1 mM, final concentration). The reactions were

4

terminated by adding 200 µL cold ACN containing 10 ng/mL metoprolol as IS. The samples were

5

vortexed and centrifuged at 4000 rpm for 20 min. The supernatant from each sample was

6

transferred to a separate vial for UPLC/ESI/HRMS analysis. In brief, Hypersil gold C-18 column

7

(50 mm x 2.1 mm I.D.; particle size 3 µm,), C18 HQ105 (10 x 2.1mm) guard column and mobile

8

phase consisting of a mixture of 0.1 % formic acid-acetonitrile (30:70) in an isocratic elution

9

mode was used to measure 1-OH tacrine, 4-OH mephenytoin, 1-OH midazolam, dextrorphan and

10

4-OH diclofenac. The metabolites were measured in presence of their selective standard inhibitor.

11

UPLC/ESI/HRMS EICs of 1-OH tacrine (Retention time (Rt) =1.2 min, m/z 215.1182, 1.5 ppm),

12

4-OH mephenytoin (Rt =0.9, m/z 235.1104, -0.4 ppm), 1-OH midazolam (Rt =1.4 min, m/z

13

342.0814, 3.0 ppm), dextrorphan (Rt =1.3 min, m/z 258.1852, -0.2 ppm) and 4-OH diclofenac

14

(Rt =1.7 min, m/z 312.0190, 0.5 ppm) are shown in figure S3-1 and S3-2 (Supplementary data).

15

The amount of substrates metabolite in each sample (relative to negative control samples) was

16

plotted versus concentration of inhibitor present. The method was validated by incubating specific

17

CYP inhibitors with the substrates. A sigmoid-shaped curve was fitted to the data and IC50s

18

calculated using GrapPad Prism software.

19

For in vitro CYP inhibition, the incubations were performed in duplicates. Fidarestat (10 µM) or

20

selective inhibitor ketoconazole (selective inhibitor of CYP3A4, final concentration 3 µM),

21

sulphafenazole (selective inhibitor of CYP2C9, final concentration 3 µM), N-3-benzylnirvanol

22

(selective inhibitor of CYP2C19, final concentration 3 µM), quinidine (selective inhibitor of

23

CYP2D6, final concentration 3 µM) and α-naphthoflavone (selective inhibitor of CYP1A2, final

24

concentration 3 µM) were added to the 158 µL of HLMs (0.28 mg/mL protein concentration).

25

Pooled substrates of tacrine (CYP1A2 substrate, final concentration 5 µM), diclofenac (CYP2C9

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17 Page 17 of 39

substrate, final concentration 5 µM), dextromethorphan (CYP2D6 substrate, final concentration 5

2

µM), midazolam (CYP3A4 substrate, final concentration 2 µM) and S-mephenytoin (CYP2C19

3

substrate, final concentration 30 µM) were prepared and 20 µL each was added to the HLMs. The

4

samples were vortexed and preincubated at 37°C for 5 min. To initiate the reaction, 20 µL of pre

5

warmed 10mM NADPH solution was added to the reaction mixture and then incubated at 370C

6

for 10 min. The reaction was terminated by adding 200 µL cold ACN and 5 µL of metoprolol (IS)

7

at concentration of 10ng/mL to the reaction mixture. . After centrifugation at 4,000 rpm for 20

8

min, 10 µL aliquots of sample solution were injected into UPLC/MS system for analysis. A series

9

of incubation were also performed in absence of specific CYP450 inhibitors and fidarestat. The %

10

activity of substrates main metabolite was measured in presence of specific CYP450 inhibitors

11

and fidarestat by UPLC/ESI/HRMS method. The % activity of metabolites, free of inhibitors and

12

fidarestat was defined as 100%. The IC50 value of fidarestat was calculated by the percent

13

inhibition of each CYPs enzyme.

14

5. Results and discussion

15

5.1 Method Development

16

The separation of analytes and IS from endogenous substance was tested on Hypersil gold C-18

17

column (50 mm x 2.1 mm I.D.; particle size 3 µm) with different mobile phases comprising

18

different buffers such as formic acid, ammonium acetate, ammonium formate with various

19

combinations of methanol and acetonitrile to optimize peak shape response. Thus the

20

method was optimized with mobile phase consisting a mixture of acetonitrile- methanol- water

21

(pH adjusted to 3.7 by 0.1 % trifluoroacetic acid) (71:14:15) in an isocratic elution mode. The

22

flow rate of the mobile phase was 0.2 mL/min, the column temperature at 25°C and the injection

23

volume was 10 μL. The optimized ESI source conditions were used as described above.

24

5.2 Method validation

25

5.2.1 Specificity and selectivity

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1

The chromatograms showed no significant interference around the Rt of fidarestat and

2

hydrochlorothiazide in drug free rat plasma samples.

3

hydrochlorothiazide were 0.90 and 0.80 min, respectively. Representative chromatograms of

4

blank plasma, blank plasma spiked with IS, blank plasma spiked with fidarestat and rat plasma

5

samples are shown in Fig. 2. Chromatograms of blank samples and zero samples of tissue are

6

shown in Fig. S-1 and S-2 respectively (Supplementary data).The UPLC/ESI/HRMS spectrum of

7

plasma spiked with fidarestat and rat plasma samples show [M-H]- ion at m/z 278.05771 (2.063

8

ppm) with an elemental composition of C12H10FN3O4.

9

5.2.2 Linearity and lower limit of quantification

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The calibration data were analyzed by linear least-square regression analysis. The calibration

11

curve exhibited a good linearity between peak area ratios and the concentrations of analytes with

12

a mean correlation coefficient, r2 of 0.9997 for fidarestat in rat plasma, while r2 of 0.9996; r2 of

13

0.9995 ; r2 of 0.9989 and r2 of 0.9990 for fidarestat in brain, kidney, heart and liver tissues,

14

respectively. The LLOQ of fidarestat was 0.1 ng/mL in rat plasma and 0.417 ng/mg in rat brain,

15

kidney, heart and liver tissues sample.

16

5.2.3 Precision and accuracy

17

The intra and inter day precisions for fidarestat ranged from 3.96% to 8.21%, 3.95% to 12.35%

18

2.52% to 13.10%, 5.01% to 13.15% and 4.44% to 8.40% in rat plasma, brain tissue, kidney tissue,

19

heart and liver respectively. The accuracy ranged from 86.87 % to 92.68% for fidarestat in

20

plasma, 86.59% to 96.46% in brain 87.67% to 95.04% in kidney, 80.97% to 94.33% in heart and

21

88.80% to 94.56 % in liver. Data of assay precision (% CV) and accuracy (% recovery) were

22

within the acceptable limits (Table S1, Supplementary data).

23

5.2.4 Extraction recovery and matrix effect

24

The extraction recovery of fidarestat in plasma, brain, kidney, heart and liver tissue was found to

25

be >70 % (Table S2, Supplementary data). The recovery of IS was>80%. The matrix factor was

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0.91–1.08 (LQC) and 0.88–1.08 (HQC) in plasma; 0.89–1.01 (LQC) and 0.86–1.01 (HQC) in

2

brain; 0.89–1.01 (LQC) and 0.89–1.10 (HQC) in kidney; 0.88–1.03 (LQC) and 0.87–1.11 (HQC)

3

in heart and 0.93–1.16 (LQC) and 0.91–1.15 (HQC) in liver. These results indicate that there was

4

no endogenous interference in the quantification of analytes.

5

5.2.5 Dilution test and carry -over effect

6

The percentage accuracies of six replicates of 2- and 4-fold dilutions were within 85–115% of

7

their nominal concentrations. The CVs for both 2- and 4-fold dilutions were less than 10% for

8

fidarestat. No carry-over effect was observed at the retention times of fidarestat and IS.

9

5.2.6 Stability

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Bench-top stability was carried out on storage of samples at ambient temperature (25°C) for 10 h;

11

the mean percentage change of analytes in plasma was less than 8.5% from their nominal

12

concentrations. The mean percentage changes of fidarestat in plasma during three freeze–thaw

13

cycles were within the acceptance limits. The analytes were stable at 10°C for 24 h in the

14

autosampler (post preparative stability) and at -80°C for 25 days (long-term stability). The mean

15

% changes of fidarestat in rat plasma and in tissues samples during auto sampler, short term, long

16

term and three freeze thaw cycles were within the acceptance limit. The stability data were given

17

in Table S3 (Supplementary data).

18

5.3 Application of validated method

19

5.3.1 Determination of plasma protein binding of fidarestat using RED Device

20

The degree of plasma protein binding determines the fraction of the total plasma concentration of

21

a drug bound to plasma proteins. The drug in bound form to plasma protein is not available for

22

interaction with its biological targets such as enzyme, receptor transporter etc. At pharmacological

23

drug concentration of 10μM of warfarin, a highly protein bound standard drug, the percent bound

24

(±SD) was 99.68% (±0.06) and % CV is 0.19. The determined value of the percent bound (±SD)

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of fidarestat was 90.47 (±0.28) and % CV is 0.03. The 9.5% of free form of fidarestat may be

2

pharmacologically active.

3

5.3.2 Quantification of total (bound and unbound) and unbound fidarestat

4

The validated method was utilized to measure concentration of total (bound and unbound)

5

fidarestat to protein and unbound fidarestat in plasma after administration of a single oral dose.

6

The major pharmacokinetic parameter of total fidarestat was calculated by a non compartmental

7

analysis with Kinetica 2000 software (version 3.0). As shown in Fig. 3, both total (bound+

8

unbound) was measurable after 20 min dosing indicating rapid absorption of the compound. The

9

maximum plasma concentration (Cmax) of total fidarestat, time at which the concentration reached

10

the maximum (Tmax) and the terminal half-life (t1/2) were found to be 845.18±71.47 ng/mL, 1h and

11

1.29±0.23h, respectively. The plasma concentration–time curve from 0 h to the last measurable

12

concentration (AUC0–t) and area under plasma concentration–time curve from 0 h to infinity

13

(AUC0–∞) for total fidarestat were found to 2064.4±582.20 and 2414.55±642.54 ng h/mL,

14

respectively. The Cmax for unbound fidarestat was found to be 80.30±6.78 ng/mL. The AUC0–t

15

and AUC0–∞ were found to be 185.46±32 and 195.92±15.06 ng h/mL, respectively. Mean plasma

16

concentrations of total fidarestat and unbound fidarestat to protein after oral route versus time

17

were depicted in Fig.3.

18

5.3.3 Tissue distribution study of unconjugated and total fidarestat in rat brain, kidney, heart

19

and liver tissue

20

Quantification of conjugated metabolites in plasma and tissue involves treatment of samples with

21

GLU (type H-1, from H. pomatia) containing some companion of sulfatase, which acts as a

22

secondary enzyme for the hydrolysis of glucuronidates. In such case, the total drug (conjugated

23

plus unconjugated) obtained by GLU is the summation of the hydrolysis results from both

24

glucuronides and sulfates. The concentration of unconjugated fidarestat in brain, kidney, heart

25

liver tissue and plasma at 1.30h after single dose to rat was found to be 0.86±0.35 ng/mg,

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13.16±1.65 ng/mg, 1.34±0.02 ng/mg, 1.61±0.09 ng/mg and 670.50 ±71.50 ng/mL, respectively

2

(Fig. 4). There was a wide tissue distribution of fidarestat in rat at 1.30h after oral administration.

3

The observed distribution consequence in fidarestat concentration was as follow: kidney > liver >

4

heart > brain. The high distribution in kidney confirms that the kidney is the primary excretory

5

organ for fidarestat. Meanwhile, fidarestat found in brain implied that it can cross the blood–

6

brain barrier.

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Total fidarestat (conjugated plus unconjugated) concentration in brain, kidney, heart, liver

8

tissue and plasma after β-glucuronidase enzymatic hydrolysis was found to be 1.04±0.17 ng/mg,

9

17.83±1.75 ng/mg, 1.78±0.10 ng/mg, 2.47±0.21 ng/mg of tissue and 1226.62±61.45 ng/mL,

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respectively.

11

LC/ESI/HRMS EICs of glucuronidation/sulphation assay of fidarestat shows the protonated

12

metabolite M-1 at m/z 456.10606 ([M + H]+; 2.5 ppm) with an elemental composition of

13

C18H19FN3O10, eluted at 2.7 min (Fig.5). This data reveal that that there is an increase in the m/z

14

value by 176 Da (C6H8O6), suggesting the N-glucuronidation of fidarestat which is supported by

15

accurate mass measurement. It can be noted that N-glucuronidation can occur at one of the ring

16

nitrogens of imidazolidine-2, 4-dione part of fidarestat or oxidative deaminated phase I metabolite

17

of fidarestat.

18

glucuronidation occurs in the former. The [M + H] + ion of the protonated metabolite M-2 (Rt=3.3

19

min) at m/z 457.08891(0.9 ppm) with an elemental composition of C18H18FN2O11 (Fig 5) clearly

20

indicates that it may be formed by O-glucuronidation of oxidative deaminated fidarestat. The

21

sulphate conjugated metabolite was found to absent.

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However, elemental composition of protonated M1 clearly indicates that

22

For many drugs glucuronidation is the major metabolic pathway of detoxification.

23

Although the glucuronidation is usually considered as a deactivation reaction, electrophilic acyl

24

glucuronides showed chemical reactivity towards plasma and tissue proteins which resulted into 22 Page 22 of 39

idiosyncratic adverse drug reactions [29]. Similarly, N-glucuronides releases the toxic aglycone

2

inside the bladder after deconjugation and causes bladder cancer [8]..Among tissues, N-

3

glucuronide metabolite was found in liver, kidney, heart and brain while O-glucuronide

4

metabolite was found only in liver and kidney. Oxidative deamination, N-glucuronidation and O-

5

glucuronidation of fidarestat were found in plasma samples. Glucuronidation in plasma can be

6

cleaved back to the active parent compound and may increase the drug concentration in tissue on

7

long term use.

8

5.3.4 CYP450 Biotransformation studies of fidarestat

9

Computer assisted docking analysis was performed by Glide docking program to study the

10

binding affinity between ligand fidarestat and each CYP450 isozymes. As shown in Fig.6 (A), the

11

docking score of CYP1A2, CYP2D6, and CYP3A4 showed significantly good docking

12

performance. The molecular interactions between fidarestat and the three CYP450s (CYP1A2,

13

CYP2D6, and CYP3A4) are shown in Fig. 6 (B-D). Fidarestat anchored in the binding site of

14

CYP1A2 through hydrogen bond interaction with the side chain of Gly316. In addition, the

15

residues Phe226 and Phe260 produced π-π stacking interactions with fidarestat, which contributed

16

to a tight binding and stable placement of ligand in CYP1A2 active site cavity. In the fidarestat-

17

CYP2D6 docking pattern, hydrogen bonding interaction was observed between fidarestat and the

18

residue Ser217. The substrate binding cavity of CYP3A4 was lined by the residues Arg212,

19

Ile369 and Ala370 and formed hydrogen bond interaction with fidarestat. These results indicate

20

that the three CYP450 (CYP1A2, CYP2D6, and CYP3A4) isoenzymes might have good binding

21

affinity toward fidarestat hence primarily responsible for their major metabolism.

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22

The docking experiment was confirmed with biochemical assay. Fidarestat was incubated

23

with HLM for 60 min at 37°C with and without cofactor NADPH. The extent of fidarestat

24

metabolism was determined by calculating the percentage remaining amount of fidarestat at 15,

25

30, 45 and 60 min by liquid chromatography high resolution mass spectrometry. The result was 23 Page 23 of 39

1

shown in Fig. 7(A). Approximately 50% of fidarestat undergo metabolism at 60 min. Incubated

2

fidarestat with HLMs shows oxidative deamination of fidarestat as major metabolite. The

3

deprotonated metabolite at m/z 279.04033 was eluted at 2.5 min (Fig.8).

4

composition of m/z 279.04033 (C12H9FN2O5) has been confirmed by accurate mass measurements

5

having error of -3.02 ppm.

6

5.3.5 CYP reaction Phenotyping by Chemical inhibition assay

7

Chemical inhibition assay were carried out in the HLMs reaction system for the CYP1A2,

8

CYP2C19, CYP2C9, CYP2D6 and CYP3A4 mediated fidarestat biotransformation, respectively.

9

Fidarestat was treated with HLMs for 60 min in the presence or absence of specific CYP450

10

isoenzymes inhibitors. The effects of the CYP450 inhibitors on fidarestat metabolism in HLMs

11

reaction were shown in Fig. 7. HLMs reaction at 0 min was defined as control sample. The

12

reaction activity of control sample was defined as 100% for the comparison with that of the

13

samples at 15, 30, 45 and 60 min with CYP450 inhibitors. Metabolism of fidarestat was decreased

14

significantly (p< 0.05) at 60 min in presence of CYP2D6 inhibitor, quinidine (Fig.7 (B)) but not

15

with sulphafenazole, N-3-Benzylnirvanol and Ketoconazole inhibitors of CYP2C9, CYP2C19 and

16

CYP3A4 isoenzymes, respectively. α- naphthoflavone, inhibitor of CYP1A2 isoenzyme

17

decreased significantly (p< 0.05) at 60 min the metabolism of fidarestat by about 40% (Fig.7

18

(D)). Fidarestat anchored in the binding site of CYP3A4 through hydrogen bond interaction with

19

residues Arg-212, Ile-369 and Ala-370 and showed good docking performance but docking

20

experiment was not corroborated with biochemical assay. Ketoconazole, a specific CYP3A4

21

inhibitor showed very weak effect on the inhibition of fidarestat metabolism compared to

22

CYP1A2 and CYP2D6 inhibitor. The results showed the role of the two CYP450 isoenzymes in

23

the fidarestat phase-I metabolism, and were consistent with those from computer docking study.

24

The inhibition rate of each of the inhibitors for CYP1A2 and CYP2D6 which involved in the

25

metabolism of fidarestat was around 40%.

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The elemental

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5.3.6 In Vitro CYP450 inhibition evaluation of fidarestat in HLMs for DDI

2

Inhibition of the CYP450 enzymes is undesirable because of the potential risk of side effect due

3

to DDI. Overcoming this challenge in drug discovery the in vitro characterization of DDI is

4

optimized. This in-vitro study may revealed more information of drug clearance which may help

5

to predict the DDI effect in human. In-vitro CYP inhibition evaluation of fidarestat assay was

6

carried out in the HLMs reaction system for the CYP1A2, CYP2C19, CYP2C9, CYP2D6 and

7

CYP3A4 enzymes respectively. Tacrine (CYP1A2 substrate), diclofenac (CYP2C9 substrate),

8

dextromethorphan (CYP2D6 substrate), midazolam (CYP3A4 substrate) and S-mephenytoin

9

(CYP2C19 substrate) were treated HLMs in presence of their specific inhibitors and fidarestat.

10

The method was validated by incubating specific CYP inhibitors with the substrates. A sigmoid-

11

shaped curve was fitted to the data and IC50s calculated using GrapPad Prism software. Inhibition

12

(%) curves obtained from these experiments are shown in Fig.9. The best-fit log IC50 value

13

determined from the data is displayed on each graph. The % activity of substrates metabolite of

14

midazolam, tacrine, S-mephenytoin, dextromethorphan and diclofenac were found to be 7.73%,

15

22.85%, 9.24%, 3.76% and 21.58%, respectively at 3 µM in presence of their particular inhibitor.

16

The effects of the CYP inhibitors and fidarestat on the % activity of substrates main metabolite

17

were shown in Fig. 10. The % activity of metabolites, free of inhibitors and fidarestat was defined

18

as 100%.The IC50 (µM) value > 10 µM, 1-10 µM and 10 µM, a weak inhibitor. The result showed

21

the weak inhibition of fidarestat on major CYP450 enzymes.

22

6. Conclusions

23

The method reported here is the first UPLC/ESI/HRMS quantitative assay for the determination

24

of fidarestat in rat plasma and tissues followed by phase I and phase II biotransformation study.

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Pharmacokinetic study displaced rapid absorption and elimination of fidarestat. The

2

pharmacokinetics and pharmacodynamic properties of drugs are largely functions of the

3

reversible binding of drug to plasma or serum proteins. Only 9.5% of the free form of fidarestat

4

may be available for the pharmacological action. In tissue distribution studies, the highest level in

5

kidney demonstrated that kidney might be the primary excretion organ of fidarestat. Furthermore,

6

our data indicated that fidarestat can cross the blood–brain barrier. Phase II conjugation

7

metabolism study and GLU hydrolysis of plasma and tissue samples indicated presence of

8

conjugated fidarestat metabolites. Docking experiment and CYP450 reaction Phenotyping by

9

Chemical inhibition assay revealed CYP1A2 and CYP2D6 appeared to be the key enzymes for

10

the phase I metabolism. Hence fidarestat may require special attention if used along with the drug

11

or herb which is an inhibitor of CYP1A2 or CYP2D6 to avoid the complications due to the

12

increased bioavailability of fidarestat. Fidarestat is a weak inhibitor of major drug metabolizing

13

enzymes such as CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4. Fidarestat may not alter

14

the pharmacokinetics, metabolism and distribution or clearance of other co-administered drug.

15

Fidarestat may not alter the pharmacokinetics, metabolism and distribution or clearance of other

16

co-administered drug.

17

Acknowledgement

18

The authors thank Dr M. Lakshmi Kantam, Director, IICT, Hyderabad and Dr Ahmed Kamal,

19

Project Director, NIPER, Hyderabad for facilities and their cooperation. Financial support was

20

provided by the Ramalingaswami Fellowship fund (S.K.B.) from the Department of

21

Biotechnology, CMET (CSC0110) and AARF (CSC0406) projects. R.M.B and M.M.B are

22

thankful to CSIR, New Delhi, for awarding Senior Research Fellowship and Junior Research

23

Fellowship, respectively. A. D and P.P are thankful to the Ministry of Pharmaceuticals, New

24

Delhi, for providing Research Fellowship.

25

References:

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1

26 Page 26 of 39

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Resolution.J Biol Chem 279 (2004), 35630--35637. 17. P.A. Williams, J. Cosme, A. Ward, H.C. Angove, D. Matak Vinković, H. Jhoti Crystal

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structure of human cytochrome P450 2C9 with bound warfarin. Nature 424 (2003), 464-468.

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18. P. Rowland, F.E. Blaney, M.G. Smyth, J.J.Jones, V.R. Leydon, A.K. Oxbrow, C.J. Lewis,

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M.G. Tennant, S. Modi, D.S. Eggleston, R.J. Chenery A.M. Bridges, Crystal structure of

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human cytochrome P450 2D6.J Biol Chem 281(2006),7614-7622.

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19. R.L. Reynald, S. Sansen, C.D. Stout, E.F. Johnson Structural characterization of human

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Biol.Chem. 287(2012), 44581-44591

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Adaptations for the Oxidation of Polycyclic Aromatic Hydrocarbons Exhibited by the

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Structure of Human P450 1A2. J Biol Chem. 282(2007), 14348-14355.

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18

Metabolites: Toxicological and Analytical Implications. Ther Drug Monit 25(2003),1-16

19 20 21 22 23 24 25

30 Page 30 of 39

Figure(s)

278.05771

100 90

H O

O

70

F

N

50

O

40 30 20

0

H

255

260

265

270

40 30 20 10 0

290

295

300

250

260

270

280

290

H N

Cl O

te

Ac ce p

50

Relative Abundance

60

285

d

(B)

90

70

280

295.95622

100

80

275 m/z

M

250

an

10

Relative Abundance

O

H N

cr

60

H

us

Relative Abundance

80

N

ip t

(A)

H2 N

300 m/z

310

S O

320

NH

S O

O

330

340

350

Figure 1: Negative ion ESI-HRMS spectra of (A) fidarestat (m/z 278.05771) and (B) hydrochlorothiazide (m/z 295.95622)

Page 31 of 39

MA: 21370

90

80

80

50 40 30

40 30 20

10

10 0 1.0

1.5

2.0

(C)

3.0 0.0 100

2.5 Time (min)

90

11494988

Relative Abundance

70

50 40 30

0.5

1.0

1.5

100

40 30 20 10

(E)

2.0

Ac ce p

50

3.0

17783296

70 60

0 0.0

2.5 Time (min)

50 40 30

10 3.0

0 0.0

1.0

1.5

100

2.5 Time (min)

3.0

(F)

313168 90

Relative Abundance

0 0.0

te

10

60

2.0

2.5 Time (min)

20

d

20

70

0.5

1.5

M

60

80

(D)

1.0

80

80

90

2.0

0.5

an

0.5

100

Relative Abundance

50

20

0 0.0

Relative Abundance

60

cr

60

70

us

70

187774

(B)

ip t

15889

90

90

MA: 391184

100

(A)

Relative Abundance

Relative Abundance

100

80 70 60 50 40 30

0.5

1.0

1.5

2.0

Time (min)

2.5 Time (min)

3.0

0.0

0.5

1.0

1.5

2.0

Time (min)

2.5 Time (min)

3.0

Figure 2: Extracted ion chromatograms of fidarestat at (A) LLOQ 0.1 ng/mL (B) LQC 20 ng/mL (C) MQC 800 ng/mL (D) HQC 1400 ng/mL; (E) IS at 10 ng/mL and (F) blank

Page 32 of 39

Total plasma concentration (bound+unbound) 90

(a)

1000

600

70 60 50

ip t

Concentration(ng/mL)

Unbound drug

800

40 30 20

cr

10

400

0 0

1

2

3

4

5

Time (h)

200

0 0

1

2

3

4

5

an

Time (h)

us

Concentration (ng/mL)

(b)

80

M

Figure 3: Mean plasma concentration time profile of fidarestat (a) total plasma concentration of drug (bound+unbound) (b) Unbound drug in plasma. All values are mean ± standard error mean (SEM)

18

15

15

12

12

9

9

6

6

3

3

0

ain Br

y ne Kid

Tissue concentration (ng/mg of tissue)

800

te

18

Unconjugated Fidarestat Total Fidarestat

Ac ce p

Fidarestat concentration (ng/mL)

800

1200

d

1200

0 art He

er Liv

a sm Pla

Figure 4: Concentrations of unconjugated fidarestat and total fidarestat (conjugated + unconjugated) in plasma and tissues at 1.30h after single oral administration in rats. All values are mean ± SEM

Page 33 of 39

100

90

ip t

80

70 60 50 40

70 60 50

cr

R e la t iv e A b u n d a n c e

40 30

10

20

0 455.5

456.0

456.5 m/z

10 0 0.0

0.5

100

1.0

1.5

2.0

(B)

90

30

70 60 50 40 30 20 10

20

0 456.4

456.6

Ac ce p

10

0 0.0

3.5

4.0

4.5

M-2

RT: 3.37

d

40

80

te

50

90 R e la tiv e A b u n d a n c e

60

3.0

M

80

2.5 Time (min)

457.08933 0.91936 ppm

100

70

us

20

30

an

Relative Abundance

RT: 2.74

90

80

Relative Abundance

M-1

456.09752 C 18 H 19 O 10 N 3 F = 456.10490 456.10606 -16.16816 ppm

(A) 100

0.5

1.0

456.8

1.5

457.0 m/z

2.0

457.2

457.4

2.5 Time (min)

3.0

3.5

4.0

4.5

Figure 5: Extracted ion chromatograms of (A) N-glucuronidation metabolite of fidarestat (M-1; m/z 456.10606; 2.5 ppm) eluted at 2.7 min (B) O-glucuronidation of oxidative deaminated fidarestat (M-2; m/z 457.08933; 0.9 ppm) eluted at 3.3 min

Page 34 of 39

-7

A

B

-5

ip t

-4 -3 -2

cr

Docking Score between CYP450 and fidarstat

-6

-1

us

0

Ac ce p

C

te

d

M

an

9 D6 C9 A4 A6 A2 C1 P3 P2 P1 P2 P2 P2 CY CY CY CY CY CY

D

Figure 6: Interaction between CYP450 and fidarestat were analyzed with docking score generated from Schrodinger software analysis. (A) CYP450 isoenzymes with docking scores were labeled with the score value. The 3-D structural docking patterns between fidarestat and CYP1A2 (B) CYP3A4 (C) CYP2D6 (D) were generated.

Page 35 of 39

(A)

(B)

Fidarestat

Fidarestat + CYP2D6 inhibitor

100

80

80

% Remaining (Fidarestat)

40

20

60

ip t

60

40

0

0

(C)

0

15

30

45

0

60

(D)

Time (min)

100

60

15

40

45

0 0

60

15

15

30

Time (min)

45

45

60

(F) 100

Fidarestat + CYP2C19 inhibitor % Remaining (Fidarestat)

Ac ce p 0

30

Time (min)

Fidarestat + CYP3A4 inhibitor

100

% Remaining (Fidarestat)

60

Time (min)

(E)

0

30

te

0

d

0

20

80

20

20

40

60

M

40

60

45

an

% Remaining (Fidarestat)

% Remaining (Fidarestat)

Fidarestat + CYP2C9 inhibitor

80

30

Time (min) Fidarestat + CYP1A2 inhibitor

100

80

15

cr

20

us

(% Remaining (Fidarestat)

100

80

60

40

20

0

60

0

15

30

45

60

Time (min)

Figure 7: Identification of CYP450 isoenzymes that transform fidarestat: (A) Fidarestat was incubated with HLMs for 60 min at 37°C with cofactor NADPH. In the chemical inhibition assay, (B) quinidine for CYP2D6 inhibition (C) sulphafenazole for CYP2C9 (D) αnaphthoflavone was for CYP1A2 (E) ketoconazole for CYP3A (F) N-3-Benzylnirvanol for CYP2C19. The enzyme catalyzing activity of sample free of inhibitors (control) was defined as 100%. All values are mean ± SEM

Page 36 of 39

RT: 2.54

100 90

(A)

70

ip t

60 50 40 30

cr

Relative Abundance

80

20

0 0.0

0.5

1.0

1.5

2.0

2.5 3.0 Time (min)

279.04033 -16.07172 ppm

(B)

90 80

4.0

4.5

5.0

70 60

M

Relative Abundance

3.5

an

100

us

10

50 40 30

10 0 274

276

278

te

272

280 m/z

282

284

286

288

290

Ac ce p

270

d

20

Figure 8: (A) Extracted ion chromatograms of oxidative deamination metabolite of fidarestat eluted at 2.5 min (B) Negative ion ESI-HRMS spectra of oxidative deamination metabolite of fidarestat (m/z 279.04033; 3.02 ppm)

Page 37 of 39

α -Napthoflavone (CYP1A2)

k e to c o n a zo le (C Y P3A4 )

% Inhibition

% Inhibition

75 55 35 15 -5 -2

-1

0

1

Log concentration (µM)

-1.0

REP 1 0.01612

EC50

N -3 B e nz ylnirv inol (C Y P 2C 19)

-0.5

0.0

REP 2 0.01710

Q u in id in e ( C Y P 2 D 6 )

110

95

(D)

100

(C) % Inhibition

75 55

90 80

M

% Inhibition

-1.5

an

EC50

-2.0

Log concentration (µM)

REP 2 0.1521

REP 1 0.1597

(B)

us

-25 -3

95 90 85 80 75 70 65 60 55 50 -2.5

ip t

(A)

cr

95

35 15 -5

70 60 50

d

40

-25 -3

-2

-1

0

30 -3

1

-2

te

REP 1 0.2734

REP 2 0.2740

Ac ce p

EC50

-1

0

1

L o g c o n c e n t r a t io n ( µ M )

Log concentration (µM)

REP 1 0.03918

EC50

REP 2 0.03778

S u lp h a f e n a z o le ( C Y P 2 C 9 )

90

(E)

% Inhibition

70 50 30 10

-1 0

-3

-2

-1

0

1

L o g c o n c e n t r a t io n ( µ M ) EC50

REP 1 0.5464

REP 2 0.5775

Figure 9: NADPH-dependent IC50 of standard inhibitors (A) Ketoconazole for CYP3A4 catalyzed midazolam to 1-hydroxy midazolam; (B) α-naphthoflavone for CYP1A2 catalyzed tacrine to 1-hydroxy tacrine; (C) N-3-Benzylnirvanol for CYP2C19 catalyzed mephenytoin to 4hydroxy mephenytoin; (D) Quinidine for CYP2D6 dextromethorphan to dextrorphan; (E) Sulphafenazole CYP2C9 catalyzed diclofenac to 4-hydroxy diclofenac in HLMs.

Page 38 of 39

C o n tro l I n h ib it o r s F id a r e s t a t

120

ip t

100

60

cr

% Activity

80

us

40

0 -T a

c r in

e

O

eph H -M

en y

to in -M OH

id a z

o la m

D ex

to rp

han

-D OH

ic lo

fen a

c

M

OH

an

20

Ac ce p

te

d

Figure 10: CYP450 inhibition potential of fidarestat with respect to specific inhibitors: αnaphthoflavone was used for the inhibition of tacrine to form main metabolite 1-hydroxy tacrine by CYP1A2; N-3-Benzylnirvanol for mephenytoin to 4-hydroxy mephenytoin by CYP2C19; ketoconazole for midazolam to 1-hydroxy midazolam by CYP3A4; quinidine for dextromethorphan to dextrorphan by CYP2D6 and sulphafenazole for diclofenac to 4-hydroxy diclofenac by CYP2C9. The % activity of metabolites, free of inhibitors and fidarestat was defined as 100%. All values are mean ± SEM.

Page 39 of 39