Mediation of in Vitro Cytochrome P450 Activity by Common

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Mediation of in vitro cytochrome P450 activity by common pharmaceutical excipients Philip Martin, Marco Giardiello, Tom O McDonald, Steven P Rannard, and Andrew Owen Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp400175n • Publication Date (Web): 23 May 2013 Downloaded from http://pubs.acs.org on June 3, 2013

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Molecular Pharmaceutics

Mediation of in vitro cytochrome P450 activity by common pharmaceutical excipients Philip Martin,1 Marco Giardiello,2 Tom O. McDonald,2 Steven P. Rannard2,3,* and Andrew Owen1,3,* 1

Department of Molecular and Clinical Pharmacology, University of Liverpool, Block H, 70

Pembroke Place, Liverpool, L69 3GF, UK 2

Department of Chemistry, University of Liverpool, Crown Street, L69 3BX, UK

3

MRC Centre for Drug Safety Science, University of Liverpool, Liverpool, L69 3GE.

* Andrew Owen. Department of Molecular and Clinical Pharmacology, University of Liverpool, Block H, 70 Pembroke Place, Liverpool, L69 3GF, UK. Tel:+44(0)1517948211. Fax: +44(0)1517945656. Email: [email protected] * Steven . P. Rannard. Department of Chemistry, University of Liverpool, Crown Street, L69 3BX, UK. Tel:+44(0)1517943501. Fax: +44(0)7945656. Email: [email protected]

ACS Paragon Plus Environment

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ABSTRACT Polymers and surfactants are commonly used as excipients in oral formulations and are generally considered to be inert. However, relatively few studies have assessed their interaction with enzymes involved in the absorption, distribution, metabolism and elimination of drugs. These excipients are generally thought to be inactive but relatively few studies have assessed their interaction with enzymes involved in the absorption, distribution, metabolism and elimination of drugs. We have investigated the impact of twenty three commonly used excipients (ten polymers and thirteen surfactants) on seven cytochrome P450 (CYP450) isoforms using baculosomederived CYP450 enzymes across a range of concentrations. Time-course fluorescent readings were then taken to generate IC50 (inhibition) or EC50 (activation) values for excipient effects on CYP450 activity. All excipients had an observable effect activity of at least one CYP450 isoform with the majority of excipients altering substrate metabolism of at least 57% of CYP450s studied. In addition, most excipients were capable of inhibiting and increasing activity of several different CYP450 isoforms. Although the majority of these effects required concentrations outside those achievable therapeutically (>100µM), almost 20% were seen at concentrations below 100µM and these results indicate that several excipients have the potential to modify the pharmacokinetics of administered drugs.

KEYWORDS: Metabolism, Formulation, Excipients, Inactive ingredients, Clearance, Pharmacokinetics


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INTRODUCTION Polymers and surfactants are extensively added to oral and parenteral formulations to enable active pharmaceutical ingredient (API) formulation and are generally regarded as inert. The large variety of materials that are used span multiple functional groups, molecular weights, polydisperisities, ionic charge and molecular architecture and increasing evidence has shown that some common excipients may actually influence the absorption, disposition and subsequent elimination of active pharmaceutical ingredients (APIs).1 Indeed, recent studies have shown that some surfactants can affect intestinal efflux by interacting with transport systems such as the ATP-binding cassette (ABC) transporters, specifically ABCB1, commonly known as Pglycoprotein (P-gp).2 Many new drugs have poor aqueous solubility and various formulation strategies with polymers and surfactants are utilised to aid dissolution3 and improve oral bioavailability.4 However, some surfactants can alter the composition and characteristics of the gastrointestinal (GI) fluid

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and molecular dispersion in the GI tract is essential for facilitating

absorption of drugs across biological membranes.6 In addition to affecting permeation through biological membranes, certain excipients may also affect

metabolic

enzymes

(e.g.

cytochrome

P450;CYP450) enzymes

and

modulate

bioavailability of drugs.7 CYP450s are membrane-bound enzymes and are estimated to contribute to the metabolism of approximately 75% of all marketed drugs8 (Figure 1). Although there are approximately 60 human CYP450 homologues,9 an estimated, 90% of therapeutic drugs are metabolised by CYP2E1, CYP3A4, CYP3A5, CYP2C9, CYP2C19,


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Figure 1. Schematic of the entrance of drug molecules into a cell and metabolism by cytochrome P450 enzymes within the endoplasmic reticulum. CYP1A2 and / or CYP2D6.10 These homologues vary in their organ distribution and activity, are predominantly expressed in liver but are also present in the small intestine (reducing drug bioavailability), lungs, placenta, and kidneys.11 However, enzymes of the CYP3A family constitute more than 70% of CYP450 content within the small intestine.12 Indeed, CYP3A4 plays a significant role in overall first-pass metabolism of orally administered drugs,13 alone metabolising more than 50% of these drugs.14 CYP3A5 is also found in the liver and intestinal mucosa and extraheptic tissues15 and although there is significant discordance for levels of


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expression (between 2 and 60% of total hepatic CYP3A content

16 17

compounds with greater

inhibitory potential for CYP3A4 compared with CYP3A5 indicates that CYP3A5 may have a significant role in drug metabolism particularly with drugs known to inhibit CYP3A4.18 Conversely, research has shown that CYP2E1 metabolises a wide variety of therapeutic compounds, including possible procarcinogens, carcinogens and protoxants such as acetaminophen.19 Some compounds can also induce CYP2E1 protein expression, significantly elevating oxidative stress and resulting in production of metabolites capable of redox cycling, increasing target organ damage and potential tumorogenesis.20 Therefore, substances that inhibit or enhance CYP450 activity can alter the rate of metabolism of drugs and lead to either a decrease in efficacy or an increase in bioavailability. Therefore, inhibition or induction of CYP450 enzymes is recognised as a major cause of drug-drug interactions seen clinically. However, the effects of commonly used excipients on CYP450 activity have not been characterised in detail. With the increasing study of new materials for drug delivery vehicles, including block copolymer micelles,21 and vesicles,22 dendrimers,23 degradable polymer particles,24 lipid nanoparticles,25 and polymer pro-drugs,26 the understanding of the effect of materials used to carry drugs is growing in importance and is often ignored. The aim of this study was to assess the effects of 23 different pharmaceutical excipients, commonly used in drug formulation, on the activity of the major CYP450 isoforms. The materials were chosen to ensure variation in molecular characteristics and included polymers and surfactants with varying charge, molecular weight and architecture.


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EXPERIMENTAL METHODS Materials. All chemicals were purchased from Sigma-Aldrich, UK, unless otherwise stated. Polymers and surfactants used include (molecular weights of small molecule surfactants shown to nearest 1 g/mol); poly(ethylene glycol) (PEG, 1000 g/mol), poly(ethylene oxide)80-blockpoly(propylene oxide)27-block-poly(ethylene oxide)80 (Pluronic® F68, MW = 8400 g/mol), poly(ethylene oxide)101-block-poly(propylene oxide)56-block-poly(ethylene oxide)101 (Pluronic® F127, MW = 12600 g/mol), poly(vinyl alcohol)–graft-poly(ethylene glycol) copolymer (KollicoatTM, MW = 45000 g/mol), poly(vinyl alcohol) (80% hydrolysed PVA, MW = 9500 g/mol), poly(vinyl pyrrolidone) (PVP K30, MW = 40000 g/mol), hydroxypropyl cellulose (HPC, MW = 80000 g/mol), hydroxypropylmethyl cellulose (HPMC, MW = 10000 g/mol), hydrolysed gelatin (HG, MW = 1980 g/mol), sodium carboxymethyl cellulose (NaCMC, MW = 90000 g/mol), sodium deoxycholate (NaDC, MW = 414 g/mol), sodium caprylate (NaCap, MW = 166 g/mol), α-tocopherol poly(ethylene glycol) succinate (Vit-E-PEG, MW = 1000 g/mol), Sisterna 11 (sucrose stearate, MW = 608 g/mol), Sisterna 16 (sucrose palmitate, MW = 580 g/mol), sodium 1,4-bis(2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate (AOT, MW = 444 g/mol)), poly(ethyleneoxide)35 modified castor oil (Cremophor EL, MW = 2500 g/mol), polyethylene glycol15-hydroxystearate (Solutol® HS 15, MW = 345 g/mol), poly(ethyleneoxide)20 sorbitan monolaurate (Tween® 20, MW = 1230 g/mol), poly(ethyleneoxide)20 sorbitan monooleate (Tween® 80, MW = 1300 g/mol), poly(ethylene glycol) hexadecyl ether (Brij 58, MW = 1124 g/mol), alkyl(C12-16) dimethylbenzylammonium chloride (Hyamine®, MW = 448 g/mol) and cetyl trimethylammonium bromide (CTAB, MW = 364 g/mol).


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Cytochrome P450 fluorescent assay. The effect of excipients on CYP450 activity was determined using the Vivid® CYP450 Screening Kit (Invitrogen, UK) as per manufacturer’s instructions and the assay constituents and conditions can be seen in Table 1. Briefly, 50µl of Master Pre-Mix (4850µl of Vivid® CYP450 reaction buffer (2x) and 100µl of Regeneration System plus 50µl of either CYP2E1, CYP3A4, CYP3A5, CYP2C9, CYP2C19, CYP1A2 or CYP2D6) was added to each well of a 96 well plate (NunclonTM, Denmark). 50µl of 20000 µM working stock solution of each of the 23 excipients tested (10 polymers: PEG, F68, F127, Kollicoat, PVA, PVP, HPC, HPMC, HG, or NaCMC and 13 surfactants: NaDC, NaCap, Vit-EPEG, Sisterna 11, Sisterna 16, AOT, Cremophor, Solutol HS, Tween 20, Tween 80, Brij 58, Hyamine or CTAB) were separately added to each individual well to give a range of final concentrations 10000, 1000, 250, 500, 100, 10, 1, 0.1, 0.01 or 0µM. Finally, 10µl of fluorescent substrate consisting of 885µl of Vivid® CYP450 reaction buffer, 15µl of reconstituted substrate and 100µl of NADP+, was added to the samples and incubated for 2 minutes. Fluorescent readings were then taken every 5 minutes to endpoint (30 minutes) at ambient temperature using a Fluoro4 Tecan Genosis plate reader (Magellan, Austria) with excitation and emission wavelengths appropriate to each Vivid® substrate as follows: Vivid® Red substrates (excitation 530 nm, emission 585 nm, and cutoff 570 nm), Vivid® Green substrates (excitation 485 nm, emission 530 nm, and cutoff 515 nm) and Vivid® Blue substrates (excitation 409 nm, emission 460 nm, and cutoff 45 nm). Data analysis. Data from 3 separate experiments conducted in duplicate were corrected to baseline against appropriate test compound vehicle control and assay specific acetonitrile concentration (Table 1). IC50 (inhibition) and EC50 (activation) data were then generated using GraphPad Prism 3.0 statistical analysis software.


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CYP450 Baculosome

Vivid Substrate

CYP2E1

2E1 Blue

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Fluorescence Assay Conditions CYP450 Vivid Substrate Acetonitrile from Vivid Substrate (nM) (µM) (%) 10

5

Buffer (mM)

0.5

50

CYP3A4

3A4 Red

5

5

0.1

100

CYP3A5

3A5 Green

10

5

0.5

50

CYP2C9

2C9 Green

10

2

0.1

50

CYP2C19

2C19 Blue

5

10

0.1

50

CYP1A2

1A2 Blue

5

3

0.15

100

CYP2D6

2D6 Blue

10

10

0.5

100

Table 1: Baculosome derived cytochrome P450 enzyme and specific fluorescent probe substrate concentrations


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RESULTS CYP450 enzyme activity was determined as either an IC50 (half maximal inhibitory concentration) or EC50 (half maximal effective concentration) indicating inhibition or activation respectively from the addition of either polymer or surfactant. A summary of the effects of 10 different polymers commonly used in drug formulation on the activity of 7 CYP450 isoforms known to be responsible for the metabolism and detoxification of numerous APIs (CYP2E1, CYP3A4, CYP3A5, CYP2C9, CYP2C19, CYP1A2 and CYP2D6) is shown in Table 2 and a graphical representation for polymers exhibiting an IC50 or EC50

Mediation of in Vitro Cytochrome P450 Activity by Common

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