Development of a method for in vitro investigation of CYP3A activity in human liver microsomes

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investigation of CYP3A activity in human liver microsomes

Vilde Fjell

Thesis submitted for the degree of Master of Pharmacy

45 credits

Section for Pharmacology and Pharmaceutical Biosciences

Department of Pharmacy

Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO

May 2021

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Development of a method for in vitro investigation of CYP3A activity in

human liver microsomes

by

Vilde Fjell

Thesis submitted for the degree of Master of Pharmacy

Section for Pharmacology and Pharmaceutical Biosciences

Department of Pharmacy

Faculty of Mathematics and Natural Sciences

UNIVERSITY OF OSLO

May 2021

Supervisors:

Professor Hege Christensen

Sec. for Pharmacology and Pharmaceutical Biosciences, Department of Pharmacy, UiO PhD student Markus H. Hovd

Sec. for Pharmacology and Pharmaceutical Biosciences, Department of Pharmacy, UiO Associate professor Ida Robertsen

Sec. for Pharmacology and Pharmaceutical Biosciences, Department of Pharmacy, UiO

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Title: Development of a method for in vitro investigation of CYP3A activity in human liver microsomes

Vilde Fjell

http://www.duo.uio.no/

Print: Reprosentralen, Universitetet i Oslo

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Forord

Denne masteroppgaven ble utført ved Avdeling for farmasøytisk biovitenskap, under veiledning av professor Hege Christensen, PhD student Markus H. Hovd, og førsteamanuensis Ida Robertsen. Arbeidet ble påbegynt i august 2019, og avsluttet i mai 2020.

Først og fremst vil jeg gi en stor takk til Hege Christensen for fantastisk veiledning, konstruktive tilbakemeldinger, smittende positivitet, og all støtte underveis. Du har virkelig gjort dette til året lærerikt og minneverdig, og har lært meg utrolig mye. Tusen takk, Ida, for all hjelp, og kunnskap du har bidratt med under utformingen av denne oppgaven. En spesiell takk må også rettes til Markus som har vært så involvert i dette prosjektet, og i tillegg har hjulpet til med alle utfordringer som har måtte oppstå underveis. Du har lært meg utrolig mye nytt som jeg kommer til å ta med meg videre. Tusen takk til Kine for masse positivitet, og gode innspill. Eline, tusen takk for alt du har bidratt med på lab dette året. Tusen takk til Grete som har hjulpet til når problemer har oppstått på MS’en. Tusen takk til hele PK-gruppa for et minnerikt år som jeg aldri kommer til å glemme, dere har gjort det til en glede å dra til Gydas!

Jeg vil rette en stor takk til mine medstudenter Ole Martin Drevland, og Tine Herlofsen for alle gode diskusjoner, løpeturer, og avsporinger som jeg ikke kunne vært foruten. Uten dere hadde ikke dette året vært det samme. Tusen takk til «Bestegjengen» for å hjulpet meg gjennom studiet.

Til slutt vil jeg takke familie og venner for all støtten gjennom studiet.

Oslo, mai 2021 Vilde Fjell

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List of abbreviations

1-OH-MDZ 1’-hydroxy-midazolam AUC Area under the curve CAR Chimeric antigen receptor CI Confidence interval

CL Clearance

CLint Clearance intrinsic Cmax Maximal concentration CV Coefficient of variation

CYP Cytochrome P450

d5 Deuterated (tracer)

EMA European Medicines Agency

F Bioavailability

FAD Flavin adenine dinucleotide FMN Flavin mononucleotide HIM Human intestinal microsomes HIV Human immunodeficiency virus HLM Human liver microsomes

IL Interleukin

IQR Inter quartile range

Km Michaelis-Menten constant LLOQ Lower limit of quantitation

MDZ Midazolam

mRNA Messenger ribonucleic acid

MS Mass spectrometer

NADPH Nicotinamide adenine dinucleotide phosphate POR NADPH cytochrome P450 oxidoreductase PXR Pregnane X receptor

QC Quality control

R Coefficient of correlation RA Rheumatoid arthritis

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VII SD Standard deviation

SN Signal-noise

TNF Tumor necrosis factor

UHPLC Ultra-high-performance liquid chromatography ULOQ Upper limit of quantitation

Vmax Maximal velocity

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Abstract

Introduction: Variation in drug metabolizing enzymes is an important factor regarding pharmacokinetic variability. The cytochrome P450 (CYP) superfamily accounts for the metabolism of approximately 75% of metabolically cleared drugs. CYP3A4 is the most abundant CYP enzyme in the liver and intestine, and variability CYP3A-activity is hence important with regard to the interindividual variability. The aim of the thesis is to develop a method for in vitro investigation of CYP3A-activity in pooled human liver microsomes (HLMs) with midazolam as probe drug.

Method: Validation of a new sample preparation method for quantification of 1’-hydroxy- midazolam by UHPLC-MS/MS was performed according to the EMA guidelines on bioanalytical method validation. Subsequently the method was applied in assays to determine the optimal incubation time in studies with pooled HLMs. This was performed by incubation of HLMs with midazolam for an increased amount of time (0-20 minutes) to find the linear range. The activity assays were also set up on VICTOR^™ Nivo^® to investigate if the use of its automatic dispenser to dispense HLMs and stop reagent would have an impact on the variability and be suitable for future CYP3A-activity assays in HLMs and human intestinal microsomes (HIMs) from individual patients.

Results: The sample preparation method fulfilled the EMA requirements for bioanalytical method validation with acceptable accuracy and precision. An incubation time of 5 minutes was adequate for the enzymatic reaction to remain in the linear range of metabolite formation throughout the experiments. The use of VICTOR™ Nivo® to dispense HLMs and formic acid gave results comparable with other studies, with Km-values of 3.2-9.1 µM and Vmax-values in the range of 58.3-151.4 pmol/min/mg protein. Between parallel variation obtained with the automatic dispenser unit had a coefficient of variation of 8.7%.

Conclusion: An UHPLC-MS/MS sample preparation method was developed for the quantification of 1’-hydroxy-midazolam obtained from in vitro CYP3A-activity assays. The method showed high accuracy and precision and was applied to determine metabolite formation in samples from the CYP3A-activity assays performed on VICTOR™ Nivo®. The method showed low between parallel imprecision, and the addition of temperature control and shaking makes it advantageous to automate the in vitro incubation. Some adjustments are necessary prior to further application in CYP3A-activity investigations in individual HLMs and HIMs.

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Sammendrag

Introduksjon: Variasjon i legemiddelmetaboliserende enzymer er en viktig faktor med tanke på farmakokinetisk variabilitet. Cytokrom P450 (CYP) superfamilien står for metabolismen av omtrent 75% av alle legemidler som metabolsk fjernes fra kroppen. Av CYP-enzymene har CYP3A4 den største anrikningen i både lever og tarm, og variabilitet i CYP3A4-aktivitet er derav viktig med tanke på interindividuell variabilitet. Hensikten med oppgaven var å utvikle en metode for å utforske CYP3A-aktivitet in vitro i humane levermikrosomer (HLM) med midazolam som probelegemiddel.

Metode: Valideringen av en ny prøveopparbeidelsesmetode for kvantifisering av 1’-hydroxy- midazolam på UHPLC-MS/MS ble utført etter EMAs retningslinjer for bioanalytisk metodevalidering. Deretter ble analysemetoden anvendt i forsøk for bestemmelse av optimal inkubasjonstiden i kommersielle HLM. Bestemmelse av inkubasjonstid ble utført ved å inkubere HLM med midazolam ved økende tid (0-20 minutter) for å finne det lineære området for metabolittdannelse. Aktivitetsforsøkene ble videre flyttet til VICTOR™ Nivo® for å undersøke om bruken av den automatiske dispenseren til å tilsette HLM og reagens for å stoppe den enzymatiske reaksjonen (maursyre) ville påvirke variabiliteten, og om den var egnet til å bruke i fremtidige CYP3A-aktivitetsmålinger i HLM og humane intestinale mikrosomer (HIM) fra individuelle pasienter.

Resultater: Prøveopparbeidelsesmetoden oppfylte EMAs krav for bioanalytisk metodevalidering med tilfredsstillende nøyaktighet og presisjon. For at produktdannelsen skulle være i det lineære området under in vitro inkubasjonen, ble det observert at en inkubasjonstid på 5 minutter var tilstrekkelig. Bruk av VICTOR^™ Nivo^® til å tilsette HLM og maursyre ga resultater sammenliknbare med andre studier med Km-verdier i området 3.2-9.1 µM, og Vmax-verdier var i området 58.3-151.4 pmol/min/mg protein. Variasjon mellom parallellene ved bruk av den automatiske dispenseringsenheten gav en variasjonskoeffisient på 8.7%.

Konklusjon: En prøveopparbeidelsesmetode for kvantifisering av 1’-hydroxy-midazolam på UHPLC-MS/MS i prøver fra in vitro CYP3A-aktivitetsmålinger ble utviklet. Metoden viste god nøyaktighet og presisjon, og ble brukt til å bestemme metabolittdannelse i prøver fra CYP3A- aktivitetsforsøkene utført i VICTOR^™ Nivo^®. Metoden viste høy presisjon mellom parallellene, noe som i tillegg til temperaturkontroll og risting gjør det fordelaktig å automatisere in vitro inkubasjonen. Noen justeringer bør gjøres før metoden for CYP3A-aktivitetsmåling kan i anvendes på individuelle HLM, eller HIM.

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Adapted figure from Anchour et al.(12) ... 2 Figure 2: The abundance distribution of intestinal cytochrome P450 (CYP) enzymes where the percentage is based on the total immunoquantified amount of CYP enzymes. Adapted figure from Paine et al. (13) ... 3 Figure 3: FF is the fraction neither lost in feces, nor metabolized in the intestines. Fraction FF

enters the gut lumen, and FG is the fraction escaping metabolism there, and/or transport back to the intestinal tract. FG will reach the portal vein, and the fraction escaping metabolism by the liver is FH which enter the systemic circulation. The bioavailability is the product of all these processes: F=FF x FG x FH. ... 4 Figure 4: (L) A visualization of NADPH cytochrome P450 oxidoreductase (POR) where the flavogroups, FAD and FMN, mediate the electron transfer from NADPH to the CYP enzyme.

(R) The CYP catalytic cycle illustrating the multiple steps necessary for oxidation of a drug molecule (D-H). POR is central as a redox partner to transfer electrons to the CYP-reaction.

Adapted figure from Rang et al. (9) ... 5 Figure 5: Midazolam and its metabolites 1'-hydroxy-midazolam, 4'-hydroxy-midazolam, and 1'-4'-dihydroxy-midazolam. Estimated amounts of the administered dose secreted as the respective components are 75%, 3%, and 1%. Adapted figure from Heizmann et al. (73). .... 11 Figure 6: A flow chart showing the steps in for subcellular fractionation of a liver biopsy. 1) A liver biopsy is taken from a study subject. 2) The biopsy is weighted and cut. 3) The liver biopsy and a homogenization buffer is added to a tube. 4) A homogenizer, e.g. Potter- Elvehjem, is used to make the final homogenate which is further centrifuged. 5)The homogenate is centrifuged at 1000 G for 10 minutes to form pellet 1 (P1)( containing e.g.

nuclei, plasma membrane and unbroken cells), and supernatant 1 (S1). 2) S1 is then

centrifuged at 3000 G for 10 minutes to form P2 (containing e.g. heavy mitochondria), and S2. 3) S2 is centrifuged at 10 000 G for 20 minutes to form P3 (containing e.g. mitochondria, and lysosomes), and S3. 4) S3 is centrifuged at 100 000 G for 40 mins to form P4 containing vesicles of the rough and smooth endoplasmic reticulum (microsomes)and the formed HLMs are further used in activity assays. Adapted from Rickwood D. (84). ... 13 Figure 7: (1) To prepare for analysis 100 µL of calibrator and QC solutions, and 20 µL of formic acid were added to a 96-well plate. (2) The plates containing the incubates (80 µL midazolam buffer and 20 µL HLMs, and 20 µL formic acid), and calibrators and QC samples were added 200 µL acetonitrile. (3) To a Vanquish plate, 80 µL of mobile phase A was transferred to the respective wells. (4) Supernatant from both 96-well plates were transferred to the Vanquish plate in a volume of 20 µL to the wells containing 80 µL mobile phase A.

Adhesive aluminum foil was used to cover the microplate prior to analysis. (5) The samples were analyzed by UHPLC-MS/MS. ... 23 Figure 8: A flow chart following the steps of the initial CYP3A-activity assay. 1) At first 170 µL of midazolam (MDZ) buffer solution were transferred to a microplate after a scheme. 2) The microplate and the human liver microsomes (HLMs) were preincubated at 37ºC for 5-10 minutes in a water bath with shaking. 3) The time was started at the first addition of HLMs to the wells with MDZ-buffer. 4) The microplate now containing MDZ-buffer and HLMs were incubated in a water bath at 37ºC, with shaking and was taken out after 7 minutes. 5) At target time, 7.5 minutes, 150 µL ice cold acetonitrile containing internal standard was added to the wells as stop reagent, and protein precipitant. ... 26 Figure 9:Boxplot showing dispersion in detected internal standard signal heights, 1’-hydroxy- midazolam-d5 and midazolam-d6), by the use of different pipetting methods. The box defines the interquartile range (IQR), the line in the box indicates the median, and the whisker shows the lower and upper 25% of the observations. ... 33

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XIII Figure 10:Boxplot showing variation in internal standard, 1’-hydroxy-midazolam-d5 and midazolam-d6, by 3 dilution factors (125, 200, and 500). The signal height was normalized by the relationship between signal height and mean signal height for each dilution. The box defines the interquartile range (IQR), the line in the box indicates the median, and the whisker shows the lower and upper 25% of the observations. ... 34 Figure 11: The two chromatograms at the top shows the peak detection for 1’-hydroxy-

midazolam, and 1’-hydroxy midazolam-d5 at 0.79, and 0.78 minutes respectively after

injection of the 1’hydroxy-midazolam calibrator at the upper limit of quantitation (ULOQ, 10 µM). The chromatograms at the bottom shows the peaks for the injection of a blank sample after the ULOQ. ... 36 Figure 12: The chromatograms display the peaks for 1’-hydroxy midazolam (1’-OH

midazolam), and 1’-hydroxy-midazolam-d5 (1-OH midazolam d5) after injection of the lower limit of quantitation (LLOQ 0.004 µM) of 1’-hydroxy-midazolam. ... 37 Figure 13:Example of a calibration curve for 1’-hydroxy-midazolam from the validation assays with affiliated linear equation and correlation coefficient (R²). For this particular validation assay the R² was 1.0, and the linear equation was y=0.548453x – (9.4201 x 10^-6)x². ... 38 Figure 14: Velocity curves fitted with nonlinear regression with Michaelis Menten (L) and substrate inhibition (R) model. The formation velocity of 1’-hydroxy-midazolam (1-OH MDZ) (pmol/min/unit protein) is plotted against added midazolam concentration ([MDZ]) (µM). The unit of measure is stated as per “unit protein” as the determination of total protein was missing. ... 40 Figure 15: Formation of 1’-hydroxy midazolam (1-OH MDZ) as a function of incubation time (0, 1, 2, 3, 4, 5, 6, 7 minutes) after incubation of midazolam buffer at three concentrations (0.25 µM, 2.5 µM, and 25 µM) with HLMs in two parallels for each incubation time. ... 41 Figure 16: Formation of 1’-OH midazolam plotted against incubation time (0.5-20 minutes) after incubation of HLMs, and midazolam-buffer with the concentrations 1 µM, 10 µM, and 100 µM in two parallels for each incubation time. ... 41 Figure 17: Velocity curve showing the formation of the metabolite 1’-OH midazolam as a function of midazolam (MDZ) concentration. Nonlinear regression with substrate inhibition model gave the best curve fit. ... 43 Figure 18: Lineweaver-Burk plot (L) made from plotting 1 over the formation velocity of 1’- hydroxy-midazolam (1-OH MDZ) (pmol/min/mg protein^-1) against 1 over the midazolam (MDZ) concentration (µM^-1). The linear regression gave the equation y=0.0543x + 0.0067 which was used to determine Km and Vmax. Michaelis-Menten plot (R) for the 4 lowest

concentrations. ... 44 Figure 19: Velocity curve showing the formation of 1’-hydroxy-midazolam (1-OH MDZ) as a function of midazolam (MDZ) concentration added to the HLM-incubate. Curves are shown for three different assays: A (n=3), B (n=3), and C (n=1). Nonlinear regression with

Michaelis-Menten fit without substrate inhibition was applied. The bottom left is plot A and B, but without correction for total protein. ... 45

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1 Introduction

1.1 Pharmacokinetic variability

Pharmacokinetics describes the relationship between the administered dose of drug and its systemic exposure over time (1). The systemic exposure, often referred to as area under the curve (AUC), is determined by bioavailability, dose, and clearance (CL) (1). Patients are highly variable considering the dose required to achieve the same systemic exposure and drug response. Therefore, a tailoring of the drug regime to the individual patient is often desired to ensure an effective and harmless therapy (1, 2). Variation in drug metabolizing enzymes is an important factor regarding interindividual variability in pharmacokinetics (1).

1.1.1 Drug metabolism

The elimination of a drug from the body may occur by excretion of changed or unchanged drug, or by metabolism of the parent drug to more water-soluble metabolites. Drugs may be excreted by different routes such as through the kidneys, secretion into bile, with feces, or by breath (1, 3). Approximately 75% of drugs are cleared from the systemic circulation by metabolism (4).

Whilst as many as 25-30% of all drugs used in humans have renal excretion of unchanged drug as their primary route of elimination. Hence the kidneys are regarded as the main organ of excretion (3). Drug metabolism take place in different tissues like the kidneys, lungs, blood, and the gastrointestinal wall (1). Yet the organ responsible for a majority of all drug metabolism is the liver (1). Metabolic reactions are referred to as phase I and phase II reactions. Oxidation, reduction and hydrolysis are representative examples of phase I reactions, while conjugation, such as glucuronidation, sulfation, and acetylation, are examples of phase II reactions. A drug usually undergoes a phase I reaction and is either excreted or undergo a phase II reaction with conjugation of a hydrophilic moiety. Some drugs are directly metabolized in phase II reactions (1).

Cytochrome P450 (CYP) enzymes

The cytochrome P450 (CYP) superfamily accounts for the metabolism of approximately 75%

of metabolically cleared drugs, and is hence regarded as the major catalyst of phase I reactions (4). Until now, 57 functional genes encoding CYP enzymes are discovered (5). The accumulations of enzymes most crucial for drug metabolism are greatest in the liver, and the

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intestine (6). Some expression also occur in the brain, placenta, kidneys and lungs (7). CYP enzymes are membrane bound haem proteins located in the smooth endoplasmic reticulum (8).

CYP superfamily are further classified into families and subfamilies by their differing amino acid sequence which provides distinct, but often overlapping substrate specificities (9). Only a fraction of the known CYP enzymes is responsible for the metabolism of the majority of drugs, and xenobiotics. These belong to CYP family 1, 2, and 3 (10, 11). The abundance of the respective CYP enzymes in the liver and intestines have been reported by Anchour et al. (Figure 1) and Paine et al. respectively (Figure 2) (12, 13). Despite the relatively low amount of CYP enzymes in the intestine compared with the liver, intestinal CYP3A plays a major role in drug metabolism (7). Most CYP enzymes have functions in the biosynthesis of endogenous compounds such as steroid hormones, prostaglandins, bile acids etc. (11). An example is the bioactivation of vitamin D3 by 1- and 25- hydroxylation carried out by CYP27A1 and -27B1 in the liver and the kidneys respectively (14, 15). CYP3A4 enzymes are involved in the metabolism of 𝜔 fatty acids, leukotrienes, and eicosanoids (16).

Figure 1: The relative abundance distribution of hepatic cytochrome P450 (CYP) enzymes where the percentage is based on the total immunoquantified amount of CYP enzymes. Adapted figure from Anchour et al.(12)

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3 1.1.2 Bioavailability

The fraction of administered drug reaching systemic circulation is called the bioavailability (Figure 3) (1). Drug metabolizing enzymes are known contributors who affect the fraction reaching systemic circulation (1). If a drug is administered orally there are multiple sites of potential drug loss before it is absorbed, and variability in drug metabolizing enzymes can contribute to a variable bioavailability. Drugs may be lost by decomposition or metabolism in the gastrointestinal tract and the fraction that is neither lost in feces nor decomposed, FF, is the fraction that enters the gastrointestinal tissue. In the gut lumen the drug may be metabolized, transported back to the intestinal lumen, or both. The fraction escaping this, FG, will reach the portal vein. The next obstacle is the liver, and the fraction that escapes extraction in the liver, FH, reaches the systemic circulation (1). The bioavailability (F) of a drug will be influenced by both the first-pass metabolism encompassing both intestinal, and hepatic metabolism which in turn reduces FG, and FH (7). To illustrate the importance of intestinal CYP3A on the bioavailability, a study by Lown et al. showed the effect of ingesting the calcium-channel- blocker felodipine, a CYP3A4 substrate, with a well-known CYP3A4 inhibitor, grapefruit juice.

The results demonstrated a 225% increase in the maximum observed concentration (Cmax) and

Figure 2: The abundance distribution of intestinal cytochrome P450 (CYP) enzymes where the percentage is based on the total immunoquantified amount of CYP enzymes. Adapted figure from Paine et al. (13)

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a 116% increase in AUC (17). A similar study has been conducted for midazolam where an increase of 52% and 46% was seen for the AUC and bioavailability, respectively (18).

1.2 CYP catalytic cycle

Oxidative metabolism of drug molecules by CYP enzymes require a substrate/drug (D), molecular oxygen, NADPH, and the presence of a redox partner such as NADPH cytochrome P450 oxidoreductase (POR), or cytochrome b5 (8, 19). The net reaction is addition of one oxygen atom to the drug to form a hydroxylated product (9). The monooxygenation reaction requires a steady supply of electrons derived from NADPH to metabolize drug molecules. The microsomal CYP enzymes mainly receive electrons from POR, but cytochrome b5 can also function as a redox partner for some CYP enzymes such as CYP3A4 (20). POR is a microsomal flavoprotein embedded in the membrane of the endoplasmic reticulum. The two groups in POR (FMN and FAD), mediate the transfer of electrons derived from reduction of NADPH to the heme group in the CYP enzymes (Figure 4) (11). To utilize the derived electrons to oxygenate a drug molecule the CYP enzyme perform a catalytic cycle. The CYP enzyme containing Fe³⁺

couple with a drug molecule (D-H) and receives an electron from POR which then reduces the iron to Fe²⁺and combines with molecular oxygen (Fe²⁺-O2). A proton and an electron from

Figure 3: FF is the fraction neither lost in feces, nor metabolized in the intestines. Fraction FF

enters the gut lumen, and FG is the fraction escaping metabolism there, and/or transport back to the intestinal tract. FG will reach the portal vein, and the fraction escaping metabolism by the liver is FH which enter the systemic circulation. The bioavailability is the product of all these processes: F=FF x FG x FH.

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5 POR, or cytochrome b5 forms Fe²⁺-OOH-D-H-complex. This combines with another proton to yield water and a (FeO)³⁺-D-H complex. (FeO)³⁺ extracts a hydrogen atom from DH and that forms a couple of short-lived free radicals. At last the hydroxylated product is liberated from the CYP enzyme (Figure 4) (9).

1.3 CYP3A subfamily

The majority of CYP-mediated drug oxidations are carried out by members of the CYP3A family (4). The enzyme plays a role in metabolizing 30-50% of all drugs (10, 11). There are four members of the human CYP3A subfamily: 3A4, 3A5, 3A7, 3A43 (21-25). CYP3A7 has been isolated from fetal liver tissue, and it seems like it may only be expressed in fetal liver, adult endometrium, and in the placenta (23, 26). The more unfamiliar CYP3A43 enzyme is expressed in liver, kidneys, prostate, and testis (24).

1.3.1 CYP3A4

The catabolism of several endogenous steroids (e.g. testosterone, progesterone, and cortisol) and drugs (e.g. tacrolimus, opioids, benzodiazepines) depend on metabolism by CYP3A4 (11).

Even though all individuals somehow express this important enzyme, the variability reported for microsomal CYP3A4 content in adult liver is considerable, more than 100 fold (11). The

Figure 4: (L) A visualization of NADPH cytochrome P450 oxidoreductase (POR) where the flavogroups, FAD and FMN, mediate the electron transfer from NADPH to the CYP enzyme. (R) The CYP catalytic cycle illustrating the multiple steps necessary for oxidation of a drug molecule (D-H). POR is central as a redox partner to transfer electrons to the CYP-reaction. Adapted figure from Rang et al. (9)

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average contribution of CYP3A4 to the total CYP content is estimated to be approximately 30%

and 80% in the liver and the intestines respectively (13, 27). Even though the overall share of CYP3A4 is greater in the intestine than in liver, the amount of CYP3A4 in the intestine is calculated to be less than 1% of that in the liver (28).

1.3.2 CYP3A5

CYP3A5 is a CYP3A isoenzyme which is polymorphically expressed in only 10-30% of the Caucasian population (21, 29, 30). Genetic polymorphism is defined as the occurrence of two or more alleles of a specific gene (31). CYP3A5 is expressed in the human liver, intestine, and kidney (21, 32, 33). There is an 85% similarity between the amino acid sequence of CYP3A5 and 3A4. Still there has been reported some differences regarding substrate- and regioselectivity between the two enzymes (11). This specific CYP enzyme has shown a somehow different substrate specificity and lower catabolic rate compared to CYP3A4 for most substrates, with some exceptions (30). In the metabolism of vincristine the catalytic efficacy was 9-14 fold higher for CYP3A5 compared to CYP3A4 (34). In an in vitro study examining the affinity and catabolic rate of CYP3A4 and CYP3A5 on tacrolimus metabolism, CYP3A5 showed a 64%

higher catalytic efficacy for metabolism compared to CYP3A4 (35).

1.4 Interindividual variability

Large interindividual variability has been observed in meta-analyses performed by both Rowland-Yeo et al. and Anchour et al. (12, 36). For CYP3A4 the mean abundance is reported to be 93 pmol/mg protein in liver, but the variability in relation to the mean is 81%, for CYP3A5 the values are a mean of 17 pmol/g in liver with a variation of 185% which implies a large variation between individuals in the expression of these enzymes (12). Reasons for the variability in response to the same drug dose may be genetics, gender, age, disease, drugs given simultaneously, and environmental factors (1).

1.4.1 Genetic variability

Mutations in CYP coding genes can give rise to both decreased, and increased enzyme activity which in turn can affect the pharmacokinetics of drugs. The activity of CYP2D6, CYP2C9, CYP2C19, and CYP3A5 are known to be greatly influenced by their genetic variation (37).

The calculated genetic contribution to CYP3A4 variability is 90% (38). Despite multiple studies trying to explain this heritability no genetic factors have been able to explain the considerable

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7 genetic component (39). One of the possible contributors to some of the genetic variability is the CYP3A4*22 allele (40). This variant allele has a frequency of 5-7% in Caucasians. Livers from CYP3A4*1/*1 (wild-type) carriers showed a 1.7-fold higher CYP3A4 mRNA-expression compared with CYP3A4*22 carriers. When considering its effect on activity the hydroxylation of testosterone is shown to be 2.4 times higher in wild type compared to CYP3A4*22 carriers.

The presence of a CYP3A4*22 allele can explain 12% of the observed variability in CYP3A4 (41).

On the other hand, CYP3A5 is highly polymorphic. Only expressors of at least one CYP3A5*1 allele will polymorphically express CYP3A5 whilst individuals carrying CYP3A5*3/*3 (wild type) will not. It is shown that in Caucasian and African carriers of the CYP3A5*1 allele that CYP3A5 accounts for as much as 50% of the total hepatic CYP3A content (29). Attempts have been made to quantify the share of people who express CYP3A5 with variable results. It has been reported that only a fraction of Caucasians express appreciable amounts, approximately 5-10% (11). Yet, an allelic frequency of about 60% is reported in Africans, and African Americans which implies an ethnic diversity regarding the CYP3A5*3 allelic frequency (29).

The presence of a functional allele could make a big contribution for the metabolism of drugs that is preferentially metabolized by CYP3A5, and in individuals who express low levels of CYP3A4 (11).

1.4.2 NADPH cytochrome P450 oxidoreductase

NADPH cytochrome P450 oxidoreductase (POR) is essential for the monooxygenase activity of the CYP enzymes. In addition, POR is essential for multiple oxygenase enzymes e.g., heme- oxygenase, 7-dehydrocholesterole reductase, and squalene monooxygenase. A significant correlation is seen between POR expression and monooxygenase activity of multiple CYP enzymes (11).

Mutations in POR are shown to affect the monooxygenase activity of CYP3A4. POR*28 is the most sequenced variant with an allelic frequency on 19-37% depending on the ethnicity of the study population. This mutation causes alterations in the electron binding moiety of POR (42).

Individuals carrying this allele has shown a 20-40% reduction in CYP3A4-activity in terms of metabolism of midazolam and testosterone. A 75% reduction in CYP3A4-activity has been reported with the A287P mutation, and the alleles, C569Y and V608F have been reported to cause 65-85% loss of CYP3A4 activity (43).

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1.4.3 Sex and age

The influence of sex and age on CYP-metabolism have been extensively studied, but with so far unconclusive results. The overall trend suggest that women have increased CYP3A4- activity, CYP3A4 hepatic content, and levels of CYP3A4 mRNA compared to men. In contradictory an increased bioavailability of midazolam and verapamil has been observed in women compared to men (44). Clearance of zolpidem is mainly mediated by CYP3A4, and a positive correlation is seen between serum testosterone in men, and zolpidem clearance which suggest that testosterone is a contributor to the age reliant changes in zolpidem clearance.

Whether these findings are related to CYP-activity in general, or CYP3A-activity explicitly was not concluded (45). It is hypothesized that the observed sex-dependent variation may be caused by variability in the activity of P-glycoprotein (PGP). Expression of PGP is shown to be greater in men compared to women, and studies have shown a higher clearance in women for PGP- and CYP3A4-substrates, compared to CYP3A4 substrates only (46).

Age is a contributing factor to variability in pharmacokinetic parameters. Infants (∼0-2 years) have an increased gastric pH, decreased intestinal transit time, and decreased intestinal permeability all of which can affect the pharmacokinetics of a drug (47). The CYP enzymes are not readily studied in such young patients, but there has been reported different developmental patterns for different CYP enzymes (48). CYP3A7 exhibits high activity in fetal life, but its activity is diminished during early infancy, unlike CYP3A4 which matures more slowly and reach adult levels in late infancy (49, 50). On the other hand, elderly has been found to have a decreased drug clearance. This is not likely caused by decreased activity nor expression of CYP enzymes, but rather drug-drug-interactions, decreased liver-, and kidney function (44). A hypothesis is that co-medication may have greater influence on clearance of drugs by CYP3A than age and sex (11).

1.4.4 Environmental factors

The body is exposed to a number of environmental factors such as drugs, foods, herbal medicines, dietary supplements etc. Some of these may lead to altered drug metabolism. A well- known problem is drug-drug-interactions which are a result of comedication that changes the effect of the drugs by potentiation or attenuation (51). A pharmacokinetic interaction may cause phenoconversion by induction, or suppression of e.g., CYP enzymes. This may lead to a genotype-phenotype mismatch which in turn makes it difficult to predict the patients drug response (52). Itraconazole and erythromycin are known inhibitors of CYP3A4 (53, 54).

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9 Nevonen et al. reported that itraconazole inhibit CYP3A4 mediated metabolism of simvastatin (53). Healthy volunteers were given a single dose of 40 mg simvastatin after a four-day treatment with 200 mg itraconazole, which resulted in a greater than 10-fold increase in AUC and Cmax (53). A study performed by Olkkola et al. presented a 4-fold increase in midazolam AUC as well as a prolonged half-life from 2.4 to 5.7 hours after an oral dose of 15 mg midazolam subsequently to a seven day treatment with 500 mg erythromycin three times a day (54). In contrast, rifampin is a potent inducer of both hepatic and intestinal CYP3A4. A study showed a 96-98% decreased AUC for orally administered midazolam in patients using rifampin (55). Not only drugs can alter drug metabolism. Grapefruit is a well-known foodstuff that inhibits intestinal CYP3A4 which will alter the bioavailability of CYP3A4 substrates (56). The coadministration of the herbal medicine St. John’s wort and the CYP3A4 substrate indinavir is shown to be undesirable since the induction of CYP3A4 by St. John’s wort led to a 57%

decrease in indinavir AUC (57).

1.4.5 Disease

A variety of diseases may have impact on drug metabolism. Conditions that raise the concentration of proinflammatory cytokines, such as inflammatory diseases and infections, have been reported to suppress the biosynthesis of various CYP enzymes. Conditions associated with raised levels of proinflammatory cytokines are rheumatoid arthritis (RA), liver disease, chronic kidney disease, diabetes, and HIV (58). The major contributors to suppression of CYP enzymes are believed to be interleukin (IL)-1, IL-6, and tumor necrose factor (TNF)-α, which have all shown to downregulate CYP3A4 mRNA expression (59). In patients with RA the systemic exposure of simvastatin was decreased by 57% one week after injection of the IL-6 inhibitor tocilizumab which is consistent with the hypothesis that IL-6 and CYP-activity are inversely related (60). Erythromycin clearance in advanced cancer patients is shown to be inversely correlated with IL-6 plasma concentrations (61). In patients with HIV a high plasma TNF-α concentration has been negatively correlated with CYP3A4 activity (62). A study in patients with type 2 diabetes (T2D) showed a 38% reduction in CYP3A4 activity compared to nondiabetic patients (63).

Obesity is another condition associated with low-grade inflammation. It is suggested that this low-grade inflammation decrease the expression of pregnane X receptor (PXR) and chimeric

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10

antigen receptors (CAR) which in turn decrease expression of certain CYP enzymes (64).

Obesity is also associated with altered physiological processes such as accelerated gut wall permeability and gastric emptying and increased cardiac output and liver blood flow (65). The oral bioavailability of midazolam has been shown to be increased in obese individuals compared to nonobese (60% vs. 28%). It is postulated that this is due to a decreased intestinal CYP3A4 activity, or increased blood flow and/or permeability to the intestine (66). A significantly lower CYP3A4 activity is reported in obese patients. Meanwhile CYP2E1 activity is augmented compared with nonobese (67). A study, using midazolam as a probe, was able to demonstrate an inverse correlation between body weight and CYP3A activity with a 5%

reduction in activity with 10% weight gain. The same study pointed to a positive correlation between IL-6 and body weight (68).

1.5 Midazolam as a probe drug for CYP3A

To determine enzyme activity, enzyme-specific probe drugs can be used to determine total systemic clearance of the probe drug (69). An ideal drug probe should have a clearance directly correlated with the intrinsic clearance by a specific liver enzyme (69). Midazolam is a short acting benzodiazepine that is extensively used as a CYP3A probe drug to determine hepatic and intestinal CYP3A activity (69). A desirable property of midazolam is that the hydroxylation of midazolam is believed to be mediated almost exclusively by CYP3A-isoforms (14, 15).

Another property that makes it a useful probe is that the cellular uptake of midazolam is unaffected by any currently known transport mechanisms, and the probable cellular uptake is by diffusion (69). The use of midazolam as a CYP3A probe drug in vivo has some limitations due to its variable extraction ratio, and high plasma protein binding (70). Despite of this the drug holds other desirable features such as rapid, and nearly complete absorption across the intestinal lumen after oral administration, a short plasma half-life, and little to no side effects after subtherapeutic dosing. Overall, this makes midazolam a useful probe to study CYP3A activity in vivo and in vitro (16, 17).

1.5.1 Midazolam pharmacokinetics

Midazolam is absorbed completely following oral administration (71). After absorption midazolam is mainly metabolized by hepatic CYP3A (72, 73). When administered orally midazolam undergoes first-pass metabolism by intestinal and hepatic CYP3A (74). To determine the bioavailability it would be necessary to administer an oral dose, and an I.V. dose

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11 (75). Midazolam is metabolized to three major metabolites: 1’-hydroxy-midazolam, 4’- hydroxy-midazolam, and 1’-4’-dihydroxy-midazolam (Figure 5) (76). The 1’- and 4’- hydroxylation of midazolam are performed both CYP3A4 and CYP3A5. The latter has shown a higher catalytic efficiency to 1’-hydroxylation of midazolam compared with CYP3A4 (77).

About 75% of the administered dose is excreted as 1’-hydroxy-midazolam conjugated with glucuronic acid. This metabolite has shown some pharmacologically active properties, but less than unaltered midazolam. The metabolites 4’-hydroxy-midazolam, and 1’-4’-dihydroxy- midazolam are formed to less extent, 3% and 1% respectively. Immediately following their formation, the primary metabolites are conjugated with glucuronic acid to form a pharmacologically inactive product. Hence, they have no significance as active components.

The main pathway of midazolam excretion is renal (90%) while 2-10% of the administered drug is excreted with the feces (76). In vitro the formation of 1’-hydroxy-midazolam has demonstrated to be suspectable to substrate inhibition as studies has shown declining reaction velocity at higher concentrations (73, 78).

Figure 5: Midazolam and its metabolites 1'-hydroxy-midazolam, 4'-hydroxy-midazolam, and 1'-4'-dihydroxy-midazolam. Estimated amounts of the administered dose secreted as the respective components are 75%, 3%, and 1%. Adapted figure from Heizmann et al. (73).

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12

1.6 Determination of CYP-activity in vitro

In vitro models are used to quantitatively predict metabolic clearance in e.g. liver or intestines (79). Examples of models used in hepatic drug metabolism assays are recombinant enzyme systems, liver microsomes, hepatocytes, and tissue slices (80). While liver microsomes may be prepared from both animal and human tissue, human liver microsomes (HLMs) are most extensively used (80, 81). Microsomes are subcellular fractions of the cells endoplasmic reticulum (ER) prepared by homogenization followed by subcellular fractionation (82). The same procedure may be used to prepare human intestinal microsomes (HIM) (83). Liver microsomes contain CYP enzymes, and are frequently used to investigate CYP-mediated drug metabolism (81).

To prepare HLMs or HIMs, the first step is homogenization of the tissue sample in a homogenization medium to prepare a homogenate. This can be achieved by using homogenizers such as the Dounce-, Potter-Elvehjem, or Townsen & Mercer homogenizer (84). The different homogenizers utilize the rotation and movement of the pestle to make a suspension, through the void between the pestle and a glass outer vessel (Figure 6). The efficiency and reproducibility of the homogenization depend on number of strokes of the pestle, thrust of the pestle, rotation speed, pestle clearance, and the amount of material (84). After a homogenate is made, centrifugation is used to isolate the desired organelle by its size and density. Sequential centrifugations at increasing speeds may be used to form pellets of material with decreasing sedimentation rates (Figure 6) (85). In turn this makes it possible to differentiate the larger cellular components (e.g. nuclei) from the smaller ones (e.g. endoplasmic reticulum and mitochondria), thus microsomes can be isolated. The sedimentation rate used to differentiate between the organelles can be described by Stokes law (Equation 1) (84). The sedimentation rate (s) is a product of the radius of the particle (rp), the density of the particle (ϱp), the density of the medium (ϱm), centrifugal force (g), and the viscosity of the liquid (𝜂). Sedimentation rate will increase with the square of the particle radius, and the difference in density between the particles and the medium. In contrast it will decrease with the viscosity of the medium(84).

Equation 1: Stokes law where the sedimentation rate (s) is a product of of the radius of the particle (rp), the density of the particle (ϱp), the density of the medium (ϱm), centrifugal force (g), and the viscosity of the liquid (𝜂).

𝑠 = 2𝑟_!^" (𝜚_!− 𝜚_#+𝑔 9𝜂

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Figure 6: A flow chart showing the steps in for subcellular fractionation of a liver biopsy. 1) A liver biopsy is taken from a study subject. 2) The biopsy is weighted and cut. 3) The liver biopsy and a homogenization buffer are added to a tube.

4) A homogenizer, e.g. Potter-Elvehjem, is used to make the final homogenate which is further centrifuged. 5)The homogenate is centrifuged at 1000 G for 10 minutes to form pellet 1 (P1)( containing e.g. nuclei, plasma membrane and unbroken cells), and supernatant 1 (S1). 2) S1 is then centrifuged at 3000 G for 10 minutes to form P2 (containing e.g.

heavy mitochondria), and S2. 3) S2 is centrifuged at 10 000 G for 20 minutes to form P3 (containing e.g. mitochondria, and lysosomes), and S3. 4) S3 is centrifuged at 100 000 G for 40 mins to form P4 containing vesicles of the rough and smooth endoplasmic reticulum (microsomes)and the formed HLMs are further used in activity assays. Adapted from Rickwood D. (84).

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1.7 Aim of the study

The overall aim was to determine in vitro CYP3A-activity in HLMs. Validation of a sample preparation method to use for quantification of 1’-hydroxy-midazolam by UHPLC-MS/MS is a necessity to quantify the metabolite formation in the activity assays, hence such a method has to be developed. This method is thereafter going to be used in development of a method for in vitro investigation of CYP3A-activity in pooled human liver microsomes (HLMs) with midazolam as probe drug. The intention is to use the method for CYP3A activity studies in individual HLMs/HIMs prepared by homogenization and subcellular fractionation from patient livers/intestines.

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15

2 Materials and methods

2.1 Materials

2.1.1 Chemicals

Chemicals Supplier

1’-hydroxy-midazolam Toronto Research Chemicals

1’-hydroxy-midazolam-d5 Toronto Research Chemicals

Acetonitrile hypergrade for LC-MS Merck, Darmstadt, Germany

Ammonia (NH3) Merck, Darmstadt, Germany

Bio-Rad Protein Assay Dye Reagent Concentrate Bio-Rad Laboratories Inc, California, USA

cOmplete™, mini, EDTA-free, Protease inhibitor

cocktail Merck, Darmstadt, Germany

Corning® Gentest™ human liver microsomes,

pooled Corning, Woburn, MA, USA

EDTA (Ethylenediaminetetraacetic acid) Merck, Darmstadt, Germany

Formic acid (HCOOH) VWR, Pennsylvania, USA

Hepes Sigma-Aldrich, St. Louis, MO, USA

Magnesium sulfate (MgSO4) Merck, Darmstadt, Germany Methanol hypergrade for LC-MS (MeOH) Merck, Darmstadt, Germany

Midazolam Toronto Research Chemicals,

Canada

Midazolam-d6 Toronto Research Chemicals,

Canada

NADPH Sigma-Aldrich, St. Louis, MO, USA

Nitrogen gas (N2) AGA Progas A/S, Oslo, Norway

Sodium hydroxide (NaOH) Merck, Darmstadt, Germany

Sucrose Merck, Darmstadt, Germany

Sulfuric acid (H2SO4) Merck, Darmstadt, Germany

Trismabase Sigma-Aldrich, St. Louis, MO, USA

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16

2.1.2 Equipment

Equipment Producer

Aluminum foil seals for 96-well plates VWR, Pennsylvania, USA Biosan Multi Bio RS-24, sample mixer Biosan, Riga, Latvia

Combitips advanced® Eppendorf AG, Hamburg, Germany

Cryotube™ Vials, 1.8 mL Thermo Scientific, Waltham, USA Dri-block® DB-3D, sample concentrator Techne, Staffordshire, UK

Freezer, -20ºC Bosch

Finnpipette Thermo Scientific, Waltham, USA

Hulamixer™ Sample Mixer Thermo Scientific, Waltham, USA

MegaBlock® 96-well plate Sarstedt AG & Co., Nümbrecht, Germany Megafuge™ 16R centrifuge Thermo Scientific, Waltham, MA

Meterlab® PHM210 Radiometer analytical, Villeurbanne,

France

Microtest plate, 96 well Sarstedt AG & Co., Nümbrecht, Germany

Milli-Q Integral 3 Millipore A/S, Norge

Ministar silverline, Microcentrifuge VWR International, Leuven, Belgium

Multipette® M4 Eppendorf, Hamburg, Germany

Optifit Tip, 0.5-200 µL Sartorius AG, Goettingen, Germany Optima MAX ultracentrifuge 130000 rpm Beckman Coulter, Fullerton, CA, USA Phoenix Control 2, fume hood Phoenix Control

Pipette tips Sarstedt AG & Co., Nümbrecht, Germany

Potter-Elvehjem homogenizer IKA Eurostar 20, Wilmington, USA SafeSeal tube 1.5 mL/ 2mL Sarstedt AG & Co., Nümbrecht, Germany

Scalpel sterile Swann-Morton Limited, Sheffield, England

Semimicro balance CPA225D Sartorius AG, Goettingen, Germany Screw cap tube, 15 mL/50 mL Sarstedt AG & Co., Nümbrecht, Germany Ultrasonic bath Sonorex RK100 Bandelin, Berlin, Germany

Vanquish 96-well plate VWR, Pennsylvania, USA

VICTOR® Nivo™ Multimode microplate

reader Perkin Elmer, Finland

Vortex-Genie 2 Scientific Industries, New York, USA

Water bath Julabo SW22 Julabo gmbH, Seelbach, Germany

TSQ Altis Thermo scientific, Waltham, MA

Vanquish Flex Binary UHPLC system Thermo scientific, Waltham, MA

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17 Accucore Vanquish S18⁺

1.5 µM, 5.0x2.1 mm column Thermo scientific, Waltham, MA

2.1.3 Software

Analytical software Xcalibur (Version 4.1, Thermo Scientific)

Statistical software R (Version 3.6.3)

Microsoft Excel (Version 16.44)

Enzyme kinetics GraphPad Prism (Version 8.3.0)

2.1.4 Solutions

Mobile phase A: 10 mM ammonium formate, pH 3, 5% acetonitrile

Chemicals Volume

Ammonia 25% 150 µL

Formic acid 98% 384 µL

Acetonitrile 50 mL

MilliQ-water 950 mL

Mobile phase B: 90% acetonitrile, 10% MeOH

Chemicals Volume (mL)

MeOH 100

Acetonitrile 900

200 mM Tris-H2SO4

A 200 mM Tris-H2SO4 solution was made by weighing of 1.21 g of trismabase (Semimicro balance CPA225D, Sartourious, Germany). The trismabase was then transferred to a beaker where 40 mL of MilliQ-water was added. The pH of the trismabase-solution was measured (Meterlab® PHM210, Radiometer Analytical, France) and pH-adjusted with H2SO4 to pH target at 7.4. The trismabase-H2SO4 solution was transferred to a 50 mL volumetric flask and remaining MilliQ-water was added. Chemicals and amounts are listed in Table 1.

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Table 1: The chemicals, and the amounts used to make a 200 mM Tris-H2SO4 solution.

Chemical Amount

Trismabase 1.21 g

MilliQ-water 50 mL

20 mM MgSO4

A 20 mM MgSO4 solution was made by weighing in 0.246 g of MgSO4 (Semimicro balance CPA225D, Sartourious, Germany). The MgSO4 solution was then transferred to a beaker where 40 mL of MilliQ-water was added to solve the MgSO4. Thereafter the solution was transferred to a 50 mL volumetric flask, and the remaining MilliQ-water was added. The final concentration of MgSO4 was 20 mM. Chemicals and amounts are listed in Table 2.

Table 2:The chemicals, and amounts used to make a 20 mM MgSO4 solution.

MgSO4 0.246 g

MilliQ-water 50 mL

10 mM NADPH

A 10 mM NADPH solution was made by weighing in more than 0.01166 g of NADPH in a 1.8 mL vial. The vial was used instead of a ship because of the small amount, and the static character of NADPH. Thereafter an appropriate volume of MilliQ-water was added to gain a concentration of 10 mM by using Equation 1. The 10 mM NADPH solution was put on ice after preparation. Chemicals and amounts are listed in Table 3.

Equation 1: The equation used to determine the amount of MilliQ-water (MilliQ) to add to the weighed in NADPH to gain a concentration of 10 mM.

𝑦 µ𝐿 𝑀𝑖𝑙𝑙𝑖𝑄 = 1000 µ𝐿

8.33 𝑚𝑔 𝑥 𝑋 𝑚𝑔 𝑤𝑒𝑖𝑔ℎ𝑒𝑑 𝑁𝐴𝐷𝑃𝐻

Table 3: The chemicals and amounts used to make a 10 mM NADPH solution.

NADPH ≥ 0.01166 g

MilliQ-water q.s.

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19 Buffer solution

The buffer solution was made by adding 4200 µL of 200 mM Tris-H2SO4 to a tube. Thereafter 1140 µL of 10 mM NADPH-, and 180 µL of MgSO4-solution was added. The final concentrations are listed in Table 4. The solution was further used to dissolve evaporated midazolam, and the midazolam buffer solution was used for the incubation of microsomes.

Table 4: The amounts used, and the final concentrations of the previously made 20 mM MgSO4-, 10 mM NADPH-, and 200 mM Tris-H2SO4 solutions to make the buffer solution.

Chemical Amount (µL) Final concentration (mM)

20 mM MgSO4 180 0.5

10 mM NADPH 1140 1.6

200 mM Tris-H2SO4 4200 118

Midazolam buffer solution used to incubate HLMs

Midazolam buffer solution was made by transferring the given volumes of 0.1 mg/mL, 0.01 mg/mL, and 0.001 mg/mL midazolam stock solution in methanol listed in Table 5 to Eppendorf tubes. The Eppendorf tubes were placed on a heat block at 60ºC and evaporated to dryness before reconstitution in 300 µL of the buffer solution (Table 4). It was then placed on a sample mixer (Biosan Multi Bio RS-24, Riga, Latvia) for 10 minutes to ensure complete dissolution.

Table 5: The volume of the midazolam stock solutions (0.1 mg/mL, 0.01 mg/mL, and 0.001 mg/mL) evaporated for each concentration level used in the activity assay. The evaporated substance was later dissolved in 300 µL of buffer solution to make the midazolam buffer solution.

Stock solution Level (µM) Volume (µL) Midazolam

0.1 mg/mL

100 100

50 50

25 25

Midazolam 0.01 mg/mL

10 100

8 80

5 50

2.5 25

Midazolam 0.001 mg/mL

1 100

0.5 50

0.25 25

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2.2 Methods

In this thesis, a new ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) sample preparation method was developed in order to determine 1’- hydroxy-midazolam concentration in samples from in vitro CYP3A-activity assay. The method development was based on a previously developed and validated UHPLC-MS/MS method for determination of midazolam and 1’-hydroxy-midazolam in plasma (86), and performed prior to the development of the method for in vitro investigation of CYP3A-activity in pooled HLMs.

As both the matrix and the concentration range differed from the previous validated method in plasma (86), considerable modification in the sample preparation was necessary.

2.3 Quantification of 1’-hydroxy-midazolam

2.3.1 Initial sample preparation

In the initial development, 50 µL of the calibrators (midazolam, and 1’-hydroxy-midazolam, (Table 6) in methanol solution were added to Eppendorf tubes before evaporated to dryness in the presence of nitrogen gas and 60ºC on a heat block. The samples were then reconstituted in 150 µL 200 mM Tris-H2SO4 buffer solution (Table 1). Subsequently, 100 µL of the calibrators, and QC-samples were transferred to a 96-well plate, and 150 µL ice cold acetonitrile containing internal standard (3.0 µM midazolam-d6, and 0.3 µM 1’-hydroxy-midazolam-d5) was added as precipitant. The same was applied to the 96-well plate containing the incubates from the activity assay. The 96-well plates were centrifuged at 4000 rpm for 10 minutes at 4ºC. Then 20 µL supernatant from both plates were transferred to a Vanquish plate containing 80 µL mobile phase A. Lastly the plate was covered with adhesive aluminum foil and stored at -20ºC until the analysis at the UHPLC-MS/MS, method is described in 2.3.2. The MS was set to detect both midazolam, and 1’-hydroxy-midazolam enabling investigation of both substrate loss and metabolite formation. In the development of the sample preparation, no microsomes were added as this would bias the analysis.

Table 6: Concentrations of midazolam (MDZ), and 1’-hydroxy-midazolam (1-OH) in the respective calibrators (1-10).

Calibrator 1 2 3 4 5 6 7 8 9 10

[MDZ] (µM) 0.195 0.391 0.781 1.563 3.125 6.25 12.5 25 50 100 [1-OH] (µM) 0.02 0.039 0.078 0.156 0.313 0.625 1.25 2.5 5 10

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21 2.3.2 UHPLC-MS/MS

The unknown samples were analyzed using a validated ultra-high-performance liquid chromatography-tandem tandem mass spectrometry (UHPLC-MS/MS) method (86). The system consisted of a Vanquish UHPLC coupled to an Altis triple quadrupole mass spectrometer (Thermo-Fisher, Waltham, MA). The samples were injected with a volume of 1.0 µL to a Accucore Vanquish C18, 2.1 x 50 mm reverse phase column with a particle size of 1.5 µM (Thermo-Fisher, Waltham, MA). Mobile phases were delivered in a gradient flow rate of 0.4 mL/min. Mobile phase B started at 30% before switching to 95% at 1.4 minutes, and back to 30% at 2.65 minutes. Thereafter it equilibrated for 1.85 minutes before the next injection (Table 7). The retention time for 1’-hydroxy-midazolam and the internal standard (1’-hydroxy- midazolam-d5) was about 0.8 minutes. Total run time was 4.5 minutes.

Table 7:Mobile phase composition and flow over time during the gradient elution used to detect 1’-hydroxy- midazolam.

Time (min) Mobile phase B (%) Mobile phase A (%) Flow (mL/min)

0 30 70 0.4

1.4 95 5 0.4

2.65 30 70 0.4

4.5 30 30 0.4

2.3.3 Variation in internal standard and analyte signal height

Based on the results from the initial sample preparation adjustments were required to improve the sample preparation. Initially, variability in signal heights of the internal standards, 1’- hydroxy-midazolam-d5 and midazolam-d6, were observed. Application of different pipettes to add the acetonitrile with internal standard were tested in order to investigate if any clear differences in variability was observed between the techniques. A dispensing pipette, multichannel pipette, and individual pipetting was used to add 150 µL of acetonitrile with internal standard to the calibrators in the 96-well-plate.

Another approach was to investigate if a lower signal height for the highest midazolam calibrator would result in a less variable response of midazolam-d6. This was carried out by serial dilution of calibrators dispensed at the Vanquish plate by applying 7 divergent dilution factors in the range of 5 to 4000 (5, 25, 125, 500, 1000, 2000, and 4000). Degree of dilution was aimed at a signal height ≤ 6.5 x 10⁶ for the upper limit of quantitation (ULOQ), but still an

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acceptable signal height for the lower limit of quantitation (LLOQ) at least 5 times the signal to noise (SN) ratio. Depending on the results from the experiment, the best fit dilution factor was tried out in a separate assay.

2.3.4 1’-Hydroxy-midazolam as the only analyte

In the above mentioned assays the MS was set to quantify both midazolam and its metabolite 1’-hydroxy-midazolam. Attempts were made to make it possible to detect both substances with less variability, but with no satisfying results. In the following assays midazolam was excluded, thus only 1’-hydroxy-midazolam was quantified. Midazolam-d6 was also removed from the acetonitrile with internal standard. The detection of 1’-hydroxy-midazolam would give information about metabolite formation, which was adequate to determine enzyme kinetic parameters. The calibrator range of 1’-hydroxy-midazolam were changed accordingly, initially to 0.05-50 µM, thereafter to 0.039-20 µM, and lastly to 0.001-10 µM. The latter was further used as this is the concentration range that can be expected at the aim of < 10% substrate loss when a concentration of 100 µM midazolam is added to the incubate. Since the 1’-hydroxy- midazolam concentrations in the calibrator curve was in the range 0.001-10 µM a dilution factor of 200 was not necessary. Dilution factors of 20, 10, and 5 were applied in the sample preparation of calibrators, and QCs to investigate which dilution factor that gave an acceptable signal height and SN-ratio of 1’-hydroxy-midazolam.

2.3.5 Final sample preparation

The steps of the final sample preparation used for the unknown samples, calibrators, and QCs are shown in Figure 7. Protein precipitation was used to prepare the unknown samples for analysis, and ice cold acetonitrile containing internal standard (1.0 µM 1’-hydroxy-midazolam- d5) was used as precipitant. In the final sample preparation, 60 µL of calibrators and QC- samples (1’-hydroxy-midazolam) in methanol solution were added to an Eppendorf tube before evaporated to dryness in the presence of nitrogen gas and 60ºC on a heat block. The samples were then reconstituted in 120 µL 120 mM Tris-H2SO4 buffer solution. Thereafter, 100 µL of calibrators- and QC-samples in buffer were transferred to a 96-well plate, and 20 µL of formic acid was added. Thereafter, 200 µL of acetonitrile was added to the microplate containing the incubate from the activity assay (80 µL midazolam-buffer, 20 µL HLMs, and 20 µL 1.2 M formic acid), and to the wells on the 96-well plate containing calibrators and QC samples.

Adhesive aluminum foil was used to cover the plates. Subsequently the plates were freezed at

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23 -20ºC for 60 minutes. The plates were then centrifuged at 4000 rpm, for 10 minutes at 4 ºC before the two plates were merged on a single Vanquish plate. Mobile phase A was added in a volume of 80 µL to the Vanquish plate prior to transfer of 20 µL of supernatant. Lastly, adhesive aluminum foil was used to cover the plate before it was freezed at -20ºC until analysis. The adhesive aluminum foil was removed before the plate was put in the autosampler. A validated UHPLC-MS/MS method, as described in 2.3.2, was applied to analyze the samples.

2.3.6 Validation

Validation of the sample preparation applied for quantification of 1’-hydroxy-midazolam by UHPLC-MS/MS was based on the EMA guideline on bioanalytical method validation (87).

Calibrators and QC-samples were prepared as described in 2.3.5.

Figure 7: (1) To prepare for analysis 100 µL of calibrator and QC solutions, and 20 µL of formic acid were added to a 96-well plate. (2) The plates containing the incubates (80 µL midazolam buffer and 20 µL HLMs, and 20 µL formic acid), and calibrators and QC samples were added 200 µL acetonitrile. (3) To a Vanquish plate, 80 µL of mobile phase A was transferred to the respective wells. (4) Supernatant from both 96-well plates were transferred to the Vanquish plate in a volume of 20 µL to the wells containing 80 µL mobile phase A. Adhesive aluminum foil was used to cover the microplate prior to analysis. (5) The samples were analyzed by UHPLC-MS/MS.

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24

Carry over

Carry over was analyzed by injecting a blank sample of mobile phase A after the highest calibrator with a concentration of 10.0 µM 1’-hydroxy-midazolam (ULOQ). To meet the requirements set by the EMA guidelines carry over had to be ≤ 20% of the lower limit of quantitation (LLOQ) for the analyte and ≤ 5% for the internal standard.

Lower limit of quantitation

The lower limit of quantitation (LLOQ) is considered being the lowest calibrator standard and is examined by looking at the signal-noise (SN) ratio, accuracy, and precision at an analyte concentration of 0.004 µM. The LLOQ is the lowest concentration that can be quantified with acceptable accuracy and precision of ± 20% in the method. In addition, the SN-ratio should be at least 5 by comparison with a blank sample.

Calibration curve

A calibration curve was applied to quantify the concentrations of 1’-hydroxy-midazolam in unknown samples. The curve was made using 10 concentrations of 1’-hydroxy-midazolam in the range of 0.001-10 µM (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2.5, 5, 10 µM). The choice of calibration model built upon evaluation of 5 separate assays of calibrators and QC-samples.

Emphasis was placed on the coefficient of variation (%CV), and the percentage of QCs that failed (% QC fail). These were evaluated for linear and quadratic curve fit with either equal, 1/X, or 1/X² weighing, and with or without forcing the curve through origo. The calibration model that gave the lowest total %CV and % QC fail was chosen. Back-calculated values within 85-115%, and 80-120% for the LLOQ, of nominal values were accepted.

Accuracy and precision

Inter- and intraday accuracy and precision were determined for the method. This was used to decide the between-day precision (interday), and the within-day repeatability (intraday). The QC samples used in the validation were of the concentrations 0.004, 0.04, 0.4, and 4.0 µM 1’- hydroxy-midazolam. These concentrations were used both for the intra- (n = 5), and interday procedures (n = 3). One serie of the intraday, and a total of 4 individual series performed at 3 separate days for the interday were performed.

Development of a method for in vitro investigation of CYP3A activity in human liver microsomes

investigation of CYP3A activity in human liver microsomes

Vilde Fjell

Thesis submitted for the degree of Master of Pharmacy

45 credits

Section for Pharmacology and Pharmaceutical Biosciences

Department of Pharmacy

Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO

Development of a method for in vitro investigation of CYP3A activity in

human liver microsomes

Vilde Fjell

Thesis submitted for the degree of Master of Pharmacy

Section for Pharmacology and Pharmaceutical Biosciences

Department of Pharmacy

Faculty of Mathematics and Natural Sciences

UNIVERSITY OF OSLO

Forord

List of abbreviations

Abstract

Sammendrag

Table of contents

1 Introduction

1.1 Pharmacokinetic variability

1.2 CYP catalytic cycle

1.3 CYP3A subfamily

1.4 Interindividual variability

1.5 Midazolam as a probe drug for CYP3A

1.6 Determination of CYP-activity in vitro

1.7 Aim of the study

2 Materials and methods

2.1 Materials

2.2 Methods

2.3 Quantification of 1’-hydroxy-midazolam