Impact of Gut Microbiota-Mediated Bile Acid Metabolism on the

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Article

The impact of gut microbiota-mediated bile acid metabolism on the solubilization capacity of bile salt micelles and drug solubility Elaine F. Enright, Susan A. Joyce, Cormac G.M. Gahan, and Brendan T. Griffin Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b01155 • Publication Date (Web): 10 Feb 2017 Downloaded from http://pubs.acs.org on February 12, 2017

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Molecular Pharmaceutics

The impact of gut microbiota-mediated bile acid metabolism on the solubilization capacity of bile salt micelles and drug solubility Elaine F. Enright1,2, Susan A. Joyce1,3, Cormac G.M. Gahan1,2,4, Brendan T. Griffin1,2* 1. 2. 3. 4.

APC Microbiome Institute, University College Cork, Cork, Ireland School of Pharmacy, University College Cork, Cork, Ireland School of Biochemistry & Cell Biology, University College Cork, Cork, Ireland School of Microbiology, University College Cork, Cork, Ireland

*Corresponding author: Brendan T. Griffin, School of Pharmacy, University College Cork, Ireland. Telephone number: +353 21 4901657, Fax number: +353 21 4901656, Electronic address: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

In recent years, the gut microbiome has gained increasing appreciation as a determinant of the health status of the human host. Bile salts that are secreted into the intestine may be biotransformed by enzymes produced by the gut bacteria. To date, bile acid research at the host-microbe interface has primarily been directed toward effects on host metabolism. The aim of this work was to investigate the effect of changes in gut microbial bile acid metabolism on the solubilization capacity of bile salt micelles and consequently intraluminal drug solubility. Firstly, the impact of bile acid metabolism, mediated in vivo by the microbial enzymes bile salt hydrolase (BSH) and 7α-dehydroxylase, on drug solubility was assessed by comparing the solubilization capacity of (a) conjugated vs deconjugated and (b) primary vs secondary bile salts. A series of poorly water-soluble drugs (PWSDs) were selected as model solutes on the basis of an increased tendency to associate with bile micelles. Subsequently, PWSD solubility and dissolution was evaluated in conventional biorelevant simulated intestinal fluid containing host-derived bile acids, as well as in media modified to contain microbial bile acid metabolites. The findings suggest that deconjugation of the bile acid steroidal core, as dictated by BSH activity, influences micellar solubilization capacity for some PWSDs; however, these differences appear to be relatively minor. Contrastingly, the extent of bile acid hydroxylation, regulated by microbial 7αdehydroxylase, was found to significantly affect the solubilization capacity of bile salt micelles for all 9 drugs studied (p < 0.05). Subsequent investigations in biorelevant media containing either the trihydroxy bile salt sodium taurocholate (TCA) or the dihydroxy bile salt sodium taurodeoxycholate (TDCA) revealed altered drug solubility and dissolution. Observed differences in biorelevant media appeared to be both drug- and amphiphile (bile salt/lecithin) concentration-dependent. Our studies herein indicate that bile acid modifications occurring at the host-microbe interface could lead to alterations in the capacity of intestinal bile salt micelles to solubilize drugs, providing impetus to consider the gut microbiota in the drug absorption process. In the clinical setting, disruption of the gut microbial ecosystem, through disease or antibiotic treatment, could transform the bile acid pool with potential implications for drug absorption and bioavailability.

KEYWORDS: microbiota, bile acid metabolism, bile micelle, solubility, solubilization capacity, poorly water-soluble drug, pharmacokinetics, biorelevant media

ABBREVIATIONS: API, active pharmaceutical ingredient; BCS, biopharmaceutics classification system; BSH, bile salt hydrolase, bSR, biorelevant solubilization ratio; CDCA, chenodeoxycholate; CMC, critical micelle concentration; D0, dose number; DCA, deoxycholate; FaSSIF, fasted state simulated intestinal fluid; FeSSIF, fed state simulated intestinal fluid, HPLC, high performance liquid chromatography; LCA, lithocholate; LogP, octanol/water partition coefficient; OA, oleic acid/oleate; PSA, polar surface area, PWSDs, poorly water-soluble drugs; SE, solubilization enhancement; SR, solubilization ratio; TCA, taurocholate; TDCA; taurodeoxycholate; Tm, melting temperature;

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INTRODUCTION

Recently, the intricate cohabitation of man and microbe has gained increasing appreciation as a determinant of the health status of the human host, shedding new insights into disease progression and affording novel opportunities to develop microbiome therapeutics.1 Moreover from an oral drug delivery perspective, the gut microbiota, specifically the bacterial communities populating the gastrointestinal tract, has been shown to influence drug metabolism and to potentially modulate therapeutic outcomes.2 However to date, a limited number of studies have explored the effect of the gut microbiota on drug absorption.3, 4 Elucidating the possible mechanisms by which the gut microbiota might affect or contribute to altered drug absorption in vivo is thus warranted. Previous work conducted within our research group identified bacterial bile acid modification as a regulator of lipid metabolism and host weight gain.5 We therefore hypothesised that gut microbial modification of the bile acid pool may broadly affect the intraluminal solubilization of drugs whose intestinal absorption is solubility and/or dissolution rate limited. Given that poor aqueous solubility often correlates with high inter-patient variability in drug absorption, it can be theorised that gut microbial perturbation, and consequently disturbance of the bile acid pool, could account for altered pharmacokinetic profiles for drugs dependent on bile solubilization. Understanding the factors underpinning such pharmacokinetic variability is of value in an industry and healthcare system progressively adopting a personalized medicine approach. The accelerating advent of modern pharmaceuticals characterised by poor aqueous solubility provides renewed impetus to consider the intraluminal factors affecting the bioavailability of Biopharmaceutics Classification System (BCS) class II and IV drugs.6, 7 For instance, it is now increasingly apparent that the solubilization capacity of innate solubilizers, such as bile salts, as well as concomitantly administered dietary lipids, must be considered to more accurately predict the intestinal solubility of poorly watersoluble drugs (PWSDs).8, 9 As solubilization is a prerequisite to partitioning the aqueous boundary layer adjacent to the enterocytes, association of PWSDs within bile micelles is a key step in their absorption process. The postprandial increase in bile secretion into the intestine, can further enhance the solubilization of PWSDs, leading to the “food effect” phenomenon.10 On this premise, we theorised that alterations in the gut microbiota, precisely the capacity of the microbial enzymes bile salt hydrolase (BSH) and 7α-dehydroxylase in modifying the composition of the bile acid pool, might correspondingly affect the intra-luminal solubility of PWSDs. Human primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesised from cholesterol by the hepatocytes of the liver.11 Prior to hepatic secretion, these free primary bile acids are conjugated to glycine (predominately) or taurine, after which the term “bile salt” is more correctly ascribed in light of augmented hydrophilicity. Thereafter, the bile acid pool size and composition is transformed by the gut microbiota in the distal small intestine and colon (Figure 1). Firstly, BSH catalyses the deconjugation of taurine/glycine-conjugates, liberating the bile acid from its taurine/glycine appendage. Regenerated free primary bile acids are substrates for further microbial metabolism, 7αdehydroxylation, yielding the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) from CA and CDCA, respectively. All of these microbial bile acid species can enter the portal blood for enterohepatic circulation to the liver where conjugation to taurine or glycine can occur. Considering previous reports that modifications of the bile acid steroidal system can result in profound changes in physicochemical properties and solubilization capacities for cholesterol, a similar alteration in lipophilic drug solubilization capacity can be rationalised.12 Whilst previous studies have investigated the drug solubilization capacity of different bile salt micelles (as reviewed by Wiedmann and Kamel13), to the

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best of our knowledge no study has correlated these differences to the metabolizing capacity of the gut microbiota. The primary focus of this research was, therefore, to independently assess the effect of both microbial processes, explicitly deconjugation and dehydroxylation of the bile acid steroidal structure, by comparing the apparent solubility of a selection of PWSDs in various bile salt solutions. In vivo, dietary lipids are also involved in PWSD solubilization and absorption.14 Accordingly, in an attempt to elucidate whether any observed differences could persist under more representative conditions of the intestinal milieu, comparisons of apparent solubility were also made in bile salt: fatty acid mixed micelles. The absorptive process is not solely influenced by solubility but rather a complex interplay between solubility, dissolution rate and intestinal permeation. In this respect, a secondary objective of this work was to investigate whether microbial bile acid metabolism could affect drug solubility and dissolution in biorelevant media. The first biorelevant media to simulate the fasted human intestine (fasted state simulated intestinal fluid, FaSSIF), that is a media designed to consider factors beyond pH and buffering capacity, was put forward by Galia and Dressman et al. and remains the most widely utilised in vitro intestinal fluid model.15 This media and subsequent versions thereof (for example, FaSSIF-V2) contain the bile salt sodium taurocholate. Similarly, this bile salt was employed in the ensuing fed state simulated intestinal fluids FeSSIF and FeSSIF-V2.15, 16 Whilst taurocholate provides an adequate model for the average patient population, in whom primary bile salts dominate, there is increasing evidence that the bile acid pool is altered in disease states and with the administration of therapeutic drugs.17-20 Conventional FaSSIF media was therefore altered, by equimolar substitution with TDCA, to simulate microbial bile acid metabolism. Since PWSDs are more likely, as compared to their high solubility counterparts, to exhibit a food effect, the impact of microbial bile acid metabolism on the propensity for food dependent solubility was investigated.10 To facilitate this latter study, solubility assessments were thus additionally performed under both simulated pre- and post-prandial conditions. Given the intent of in vitro solubility and dissolution studies to gauge in vivo performance in patient populations, advancing knowledge of the potential factors which may underlie inter-individual variability in drug-response is of value. Finally therefore, this paper affords an insight into the possibility of simulating the intraluminal intestinal fluid of patient cohorts exhibiting altered primary and secondary bile acid signatures. Herein, methods to screen drugs likely to display altered intestinal solubility in disease states characterised by or coinciding with an altered microbiome are explored.

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Figure 1. Chemical structures of host-derived taurocholic acid, and microbe-generated cholic acid and deoxycholic acid. Microbial bile acid metabolism results in a progressive increase in the hydrophobic character of the bile acid skeleton, as indicated by a reduction in predicted hydrophilic-lipophilic balance (HLB) values. Through the process of enterohepatic recirculation, these microbial bile acid metabolites can be reabsorbed from the intestine and (re-)conjugated to taurine/glycine in the liver. The predicted HLB value for taurodeoxycholic acid (structure not shown) is 4.86. Chemical structures and predicted HLB values were obtained using MarvinSketch version 17.1.30.0, ChemAxon (http://www.chemaxon.com).

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EXPERIMENTAL SECTION

Materials. Sodium taurocholate hydrate (≥ 95%), cholic acid sodium salt hydrate (≥ 96%), sodium taurodeoxycholic acid hydrate (≥ 95%), sodium oleate (≥ 82% fatty acids, as oleic acid), potassium phosphate monobasic (≥ 98%), sodium phosphate dibasic (≥ 99%), sodium phosphate monobasic (≥ 99%), sodium chloride (≥ 99.5%), sodium hydroxide BioXtra (≥ 98%) pellets (anhydrous), hydrococortisone (≥ 98%) and progesterone (≥ 99%) were sourced from Sigma-Aldrich Ireland. Celecoxib, danazol, fenofibrate, felodipine, ketoconazole, nifedipine and phenytoin were purchased from Kemprotec Ltd. (UK). Lipantil® Micro 67mg hard capsules were procured from BGP Products Ireland Limited. Lipoid E PCS was obtained from Lipoid GmbH (Ludwigshafen, Germany). Hard gelatin capsules (size 0) were sourced from Capsugel (Coni-Snap®). Filtropur S 0.45 syringe filters were procured from Sarstedt. All solvents (acetonitrile, methanol and dichloromethane) were of HPLC grade and were purchased from Sigma-Aldrich Ireland. Drug solubility in bile salt micelles. Solubility studies were performed in triplicate by adding an excess of the model drug to 3 mL of phosphate buffer (pH 6.8) containing either 20 mM bile salt alone (simple micelles) or in combination with 5 mM sodium oleate (mixed micelles). A concentration of 20 mM was selected to ensure all bile salts were utilized at levels exceeding their reported critical micelle concentration (CMC) ranges (TCA: 4-5 mM, CA: 11-13 mM and TDCA: 2-3 mM).13 Sample vials were screw capped, vortexed to aid dispersion and subjected to standard shake flask conditions at 37 °C. 1 mL volumes were withdrawn at 24 hr intervals and transferred to 1.5 mL Eppendorf tubes. Samples were subsequently centrifuged at 13000 rpm for 13 minutes (Hermle z233M-2 fixed angle rotor centrifuge, HERMLE Labortechnik GmbH, Wehingen, Germany). Clear supernatant was then pipetted into clean tubes and the centrifugation cycle was repeated to ensure separation from any residual solid phase drug. The resulting secondary supernatant, deemed clear of precipitate, was subject to HPLC analysis. Biorelevant solubility and dissolution studies. FaSSIF and FeSSIF media were freshly prepared in compliance with previously described methods employing dichloromethane.15 Modifications of these media, to simulate microbial bile acid metabolism, consisted exclusively of substitution of the bile salt component, sodium TCA, with sodium TDCA at equivalent molarity (altered versions herein referred to as FaSSIFSecondary and FeSSIFSecondary, Table 1). Dissolution studies of Lipantil Micro® 67mg hard capsules and gelatin capsules containing 200mg pure ketoconazole API were performed in triplicate using USP type II paddle apparatus (37 ± 0.5 °C, 75 rpm) in 500 mL of biorelevant FaSSIF or FaSSIFSecondary media. Capsules containing fenofibrate or ketoconazole were placed dissolution in vessels attached to wire sinkers. 4 mL samples withdrawn at 5, 10, 15, 20, 30, 45, 60, 90 and 120 minutes were filtered through 0.45 µm membrane filters, discarding the first 3 mL to ensure adequate priming. At each time point, the sample volume was replenished through the addition of fresh blank media (pre-heated to 37 °C). The percent drug release at each time point was determined by HPLC. Solubility studies in FaSSIF, FeSSIF, FaSSIFSecondary and FeSSIFSecondary were conducted for all study PWSDs as outlined for phosphate buffer. Subsequently, the solubility of fenofibrate, progesterone (neutral compounds), celecoxib (a weakly acidic drug) and ketoconazole (a weakly basic drug) as a function of increasing bile and lecithin concentrations (constant 4:1 ratio) was investigated in FaSSIF blank buffer (phosphate buffer pH 6.5). The apparent solubility of these 4 compounds was further

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determined in FaSSIF blank buffer containing 3 mM and 20 mM TCA or TDCA, that is, lecithin was omitted. Table 1. Composition of biorelevant simulated intestinal fluid (FaSSIF, FeSSIF) and modified versions simulating microbial bile acid metabolism (FaSSIFSecondary and FeSSIFSecondary)

FaSSIF Bile Salt (mM)

Sodium TCA Sodium TDCA

Egg phosphatidylcholine (lecithin) (mM) Sodium hydroxide (NaOH) (g) Sodium dihydrogen phosphate (NaH2PO4.H2O) (g) Sodium chloride (NaCl) (g) Glacial Acetic Acid (g) Purified water qs. pH

FaSSIFSecondary

3

FeSSIF

FeSSIFSecondary

15 3

15

0.75

0.75

3.75

3.75

0.348

0.348

4.04

4.04

3.438

3.438

6.186

6.186

11.874

11.874

8.65

8.65

1000 mL 5

1000 mL 5

1000 mL 6.5

1000 mL 6.5

Analytical methods – HPLC The apparent solubility of all model PWSDs in bile salt containing solutions, as well as in biorelevant media, was determined by HPLC with UV detection (Agilent Technologies 1200 series, Agilent Technologies, Santa Clara, Ca.) in accordance with the conditions outlined in Table 2. Clear secondary supernatant was diluted 1 in 3 or 1 in 10 with drug-specific HPLC mobile phase to facilitate suitable peak areas. An injection volume of 10 µL was utilised for all assays, with the exception of fenofibrate for which a 20 µL injection volume was employed. The stationary phase consisted of a Kinetex 5 µm XBC18, 250 × 4.6 mm reversed phase column (Phenomenex Inc., Macclesfield, UK). Agilent EZChrom Elite was used to process all chromatographic data. Apparent solubility was determined using calibration curves based on ≥ 6 known standards, with coefficients of determination (r2) of at least 0.99. The percent release of fenofibrate and ketoconazole in FaSSIF and FaSSIFSecondary media at each time point was determined by diluting 100 µL of filtrate 1 in 10 with acetonitrile (fenofibrate) or methanol (ketoconazole) and analysing API concentration according to the HPLC methods outlined in Table 2.

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Table 2. HPLC conditions to determine the apparent solubility of model drugs in various bile salt solutions

Drug

Stationary phase

Mobile phase

Injection volume

Celecoxib

Kinetex 5 µm XB-C18, 250 x 4.6 mm Kinetex 5 µm XB-C18, 250 x 4.6 mm Kinetex 5 µm XB-C18, 250 x 4.6 mm Kinetex 5 µm XB-C18, 250 x 4.6 mm Kinetex 5 µm XB-C18, 250 x 4.6 mm Kinetex 5 µm XB-C18, 250 x 4.6 mm Kinetex 5 µm XB-C18, 250 x 4.6 mm

Acetonitrile/water (70:30 v/v)

Danazol

Felodipine

Fenofibrate

Hydrocortisone

Ketoconazole

Nifedipine

Phenytoin

Progesterone

Kinetex 5 µm XB-C18, 250 x 4.6 mm Kinetex 5 µm XB-C18, 250 x 4.6 mm

Wavelength detection (nm) 254

Adapted from [Ref]

10 µL

Flow rate (ml/min) 1


10 µL

1

285

22


10 µL

1

243

23


20 µL

1

286

24

Methanol/water (60:40 v/v)

10 µL

1

248

25

Methanol/acetonitrile (75:25 v/v)

10 µL

1

254

26

Acetonitrile/ water/methanol (45:30:25 v/v, pH 4 with 1M HCL) Acetonitrile/water (50:50 v/v)

10 µL

1

235

27

10 µL

1

200

28

Methanol/water (80:20 v/v)

10 µL

1

254

25

21

Surface tension measurements. The surface tension of TDCA- and TCA- containing buffered solutions with and without lecithin were measured using the Attension Theta Optical Tensiometer (Attension Biolin Scientific Tietäjäntie 2 fin–02130 Espoo, Finland), calibrated by prior measurements with deionised water (72.8 mN/m). Surface tension was determined over a bile salt concentration range of 0-30 mM by serial dilution of 30 mM stocks. All measurements were based on a 10 µL droplet size and were performed at ambient temperature. Surface tension data are presented as mean ± standard deviation (n = 3).

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Biorelevant solubilisation ratio (bSR). The ratio of fed to fasted state apparent solubility was determined for each compound in conventional biorelevant media proposed by Galia and Dressman et al. 15 (eq. (1)29) and modified biorelevant media containing the secondary bile salt TDCA to simulate microbial bile acid metabolism (eq. (2)). bSR =

ୗూ౛౏౏౅ూ ୗూ౗౏౏౅ూ

bSR ୗୣୡ୭୬ୢୟ୰୷ =

(1) ୗూ౛౏౏౅ూ ౏౛ౙ౥౤ౚ౗౨౯ ୗూ౗౏౏౅ూ ౏౛ౙ౥౤ౚ౗౨౯

(2)

Dose number (D0) determination. A dose number (D0) was calculated for all drugs in biorelevant FaSSIF and FaSSIFSecondary media according to the equation proposed by Amidon et al. (eq. (3)). 30 D

଴ ୀ

୑బ ( ௏బ ଡ଼஼ೞ )

(3)

Where M0 is the typical oral dose (µg), V0 is the initial gastric volume available (set to 250mL) and CS is the determined apparent solubility (µg/ml). It can be inferred from a D0 of less than 1 that a given dose is completely soluble in the available fluid volume, whilst the converse is true for a D0 greater than 1. The larger the D0 the greater the volume of fluid required for maximal solubilization. Comparative D0 in FaSSIF and FaSSIFSecondary media were used to explore the impact of microbial bile acid metabolism on PWSDs displaying solubility limited absorption. Statistical analysis. All experimental data (with the exception of fold differences) are presented as the mean ± standard deviation (n = 3). A two-tailed, independent samples t-test assuming normal distribution and equal variance was employed for the assessment of statistical significance in the solubilization capacity of comparative bile salt-containing solutions (conjugated vs deconjugated, primary vs secondary). A p value < 0.05 indicated that solubilization variances were statistically significant. Fold differences in bile salt solubilization capacities were determined from the ratio of apparent solubility in comparative phosphate buffer or biorelevant solutions. Fold differences are presented as the mean value ± the standard error of the fold difference (SEFD). The SEFD for these ratios was calculated according to equation (4)31; SE ଶ SE ଶ SE୊ୈ = FD × ඨ ଶ୅ + ଶ୆ S୅ S୆

(4)

Where; FD is the mean fold difference in PWSD solubility in solution A versus solution B. SA, SB, SEA and SEB represent the mean apparent solubility (S) and standard errors (SE) for comparative solutions A and B. All statistical analyses were conducted using Microsoft Excel 2013 and GraphPad Prism 5 (San Diego, California).

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RESULTS

1. Impact of gut microbiome derived enzyme activity on drug solubilization by bile salt micelles Bile salts secreted into the intestinal lumen are substrates for microbial BSH which cleaves (sulfo-) amino acid linkages, liberating the free bile acid and taurine/glycine moieties. The solubilization capacities of the host derived conjugated bile acid TCA and the deconjugated microbial metabolite CA were thus compared to assess the impact of BSH activity on the solubility of PWSDs. The apparent solubility of a range of PWSDs in sodium TCA (conjugated) versus sodium CA (deconjugated) micelles is presented in Figure 2 (a). Of the 9 drugs assessed, 5 displayed a statistically significant (p < 0.05) apparent solubility difference in conjugated versus deconjugated simple micellar systems. The magnitude of differential solubilization was drug-specific and in instances where a solubility distinction was noted, deconjugation generally reduced the solubilization capacity of bile salt micelles. In the intestinal lumen, the microbial enzyme 7α-dehydroxylase produces the secondary bile acid deoxycholic acid (DCA) through the metabolism of CA. To examine the effect of this microbial biotransformation, the solubilization capacities of TCA and TDCA were compared. Comparative apparent solubility measurements in TCA and TDCA indicate that the extent of bile acid hydroxylation can significantly alter the solubilization capacity of bile salt micelles (p < 0.05 for all PWSDs, Figure 2 (b)). Similar to the observed impact of microbial deconjugation, the magnitude of this dehydroxylation mediated effect appeared to be drug-specific. However, microbial bile acid dehydroxylation had a more pronounced and generalized effect on micellar solubilization capacity than deconjugation. For example, the solubilization capacity of secondary bile salt micelles was up to 4.6, 3.4 and 3.2 fold higher than primary salt bile micelles for fenofibrate, ketoconazole and progesterone, respectively (Figure 2 (c)). The surface tension of buffered solutions of TCA and TDCA were assessed to afford additional insight into the observed common solubilization enhancement (SE) of TDCA micelles. TDCA micellar solutions displayed a reduced surface tension relative to TCA micellar solutions (Figure 3). This most likely reflects the greater lipophilic character, and resultantly lower critical micelle concentration (CMC), of TDCA.12 The magnitude of solubilization enhancement in simple micelles composed of TDCA as compared to TCA appeared to be drug-specific (Figure 2 (c)). In an effort to investigate this drug specific SE effect, relationships between SE and physicochemical properties of the drugs were assessed. A plot of TDCA/TCA solubilization ratio as a function of predicted logP revealed a strong positive trend (Figure 4 (a), r2 = 0.7955), that is, SE was greater with increasing PWSD lipophilicity. Tm was additionally found to be correlated with SE, with a general tendency toward reduced TDCA/TCA SR with increasing Tm (Figure 4 (b), r2 = 0.5258). A plot of SE and PSA generally revealed that PWSDs with lower PSAs exhibited greater SE in the more hydrophobic microbe-derived bile salt TDCA (Figure 4 (c)). This latter correlation between SE and PSA is weak (r2 = 0.2964), indicating that the ability of PWSDs to partition in to the more hydrophobic TDCA micelle is best predicted according to lipophilic descriptors, such as LogP.

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(a)

*

Log Solubility (µg/ml)

1000

Conjugated (TCA) Deconjugated (CA)

*

100

**

**

**

10

Fe K e nof to ibra c Pr on te og az es ole te Ce ron le e co Da xib n H Felo azo yd l ro dip c o ine rti Ph son en e N y to ife in di pi ne

1

(b) ***

Log Solubility (µg/ml)

1000 ***

*** ***

100 ***

Primary (TCA) Secondary (TDCA)

*** ***

*

***

10

Fe Ke nof to ibra c Pr on te og az es ole te C ron el ec e o D xib a Fe naz H yd lod ol ro ip co ine rt i Ph son en e N yto ife in di pi ne

1

(c) Fold Difference in Solubilization Capacity

5

Conjugated (TCA)/Deconjugated (CA) Secondary (TDCA)/Primary (TCA)

4 3 2 1 0

Fe Ke nof to ibra c Pr on te og az es ole te C ron el ec e o Da xib n a Hy Felo zo dr di l oc pin or e ti Ph son en e N yto ife in di pi ne

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Solubilization capacity of; (a) conjugated (TCA) versus deconjugated (CA) and (b) primary (TCA) versus secondary (TDCA) bile salt micelles for a selection of PWSDs (n = 3, mean ± SD). Figure 2. (c) Fold differences in bile salt solubilization capacity as a function of microbial deconjugation and dehydroxylation (n = 3, mean ± SE).

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75 Surface Tension (mN/m)

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TCA

70 65

TDCA

60 55 50 45 40 35 0

10 20 30 Bile Salt Concentration (mM)

40

Figure 3. Surface tension of sodium TCA and TDCA solutions containing 0 to 30 mM bile salt (n=3, mean ± SD for all data points).

Table 3. Molecular weight (MW), octanol/water partition coefficient (LogP), melting temperature (Tm) and polar surface area (Å2) of the study PWSDs

Drug

MW (g/mol)a

LogPb

Tm (°C)

Polar (Å2)b

Celecoxib Danazol Felodipine Fenofibrate Hydrocortisone Ketoconazole Nifedipine Phenytoin Progesterone

381.4 337.5 384.3 360.8 362.5 531.4 346.3 252.3 314.5

4.01 3.46 3.44 5.28 1.28 4.19 1.82 2.15 4.15

163.5 32 225.0 31 145.0 31 81.5 33 220.0 34 146.0 31 173.0 34 286.0 34 121.0 31

77.98 46.26 64.63 52.60 94.83 69.06 110.45 58.20 34.14

a

Martindale: The Complete Drug Reference

b

DrugBank https://www.drugbank.ca/ (ChemAxon predicted properties)

12


surface

area

Page 13 of 29

(a)

SE (TDCA/TCA)

5

r2 = 0.7955

4 3 2 1 0 0

2

4

6

LogP

(b)

SE (TDCA/TCA)

5

r2 = 0.5258

4 3 2 1 0 0

100 200 300 Melting Temperature (°C)

400

(c) 5 SE (TDCA/TCA)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


r2 = 0.2964

4 3 2 1 0 0

50

100

150

PSA (Å2)

Figure 4. The solubility enhancement of the study PWSDs in TDCA versus TCA micelles (SE (TDCA/TCA)) as a function of; (a) the octanol/water partition coefficient (predicted values, Table 3), (b) melting temperature and (c) polar surface area (predicted values, Table 3).

13



2. Impact of gut microbiome derived enzyme activity on drug solubilization by bile salt micelles in the presence of dietary fatty acids To explore whether effects observed in simple bile salt micelles would persist under more physiologically relevant mixed micelle conditions, the impact of microbial bile acid metabolism was assessed in the presence of the fatty acid sodium oleate. In general, deconjugation of the cholate steroidal ring did not appreciably impact the solubilization capacity of mixed micelles (Figure 5). Microbial bile acid dehydroxylation was determined to have the most notable effect on the solubilization capacity of mixed micelle systems, although this was drug-specific and reduced up to approximately two fold compared to simple micelles. Collectively these data demonstrate, under more physiologically relevant mixed bile salt micellar conditions, that gut microbial enzymatic activity, and in particular 7α-dehydroxylase, can affect solubilization capacity.

Conjugated & OA/Deconjugated & OA Secondary & OA/Primary & OA

Fold Difference in Solubilization Capacity

2.0 1.5 1.0 0.5

o Fe naz o Pr nofi le og br H es ate yd t e ro ro co ne rti Ph son en e C yto el in ec o D xib an Ni a z f e ol d Fe ipin lo e di pi ne

0.0

Ke to c

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 29

Figure 5. Fold difference in the solubilization capacity of bile salt micelles for a selection of PWSDs in the presence of the fatty acid sodium oleate (OA) (n = 3, mean ± SE). Bile salts; conjugated TCA, deconjugated CA, secondary TDCA and primary TCA.

14


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3. Dissolution of PWSDs in biorelevant media simulating microbial mediated bile acid metabolism Biorelevant media containing the secondary bile acid TDCA (herein referred to as FaSSIFSecondary) was developed to explore the effect of microbial bile acid metabolism on drug dissolution. The hydroxylation state of the bile salt component of fasted biorelevant media was determined to affect the dissolution (as assessed by terminal percent released) of a sample of PWSDs. Fenofibrate and ketoconazole were selected as their solubilization, in both simple and mixed micelle systems (Figure 2 (c) and Figure 5), was found to be most sensitive to the extent of bile acid hydroxylation. The cumulative percent release of fenofibrate after 2 hours was 7.37 ± 0.001% and 4.62 ± 0.001% in FaSSIF (containing TCA) and FaSSIFSecondary, respectively (Figure 6 (a)). In the context of microbial bile acid metabolism, the in vitro dissolution of fenofibrate was thus negatively impacted, in contrast to what was expected based on apparent solubility measurements in simple and mixed micelles. At the terminal 2 hour time point, the percent release of ketoconazole in FaSSIF and FaSSIFSecondary was 5.27 ± 0.12% and 10.48 ± 0.14%, respectively (Figure 6 (b)). The effect of microbial bile acid metabolism on the dissolution behaviour of ketoconazole was hence determined to concur with previously observed apparent solubility effects in simple and mixed micelles. Drug dissolution in trihydroxy and dihydroxy bile salt containing-biorelevant media was resultantly determined to be compound-specific. (a) FaSSIF

% Fenofibrate Release

100

FaSSIFSecondary

80 15 10 5 0 0

20

40

60 80 Time (min)

100

120

(b) % Ketoconazole Release

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FaSSIF

100

FaSSIFSecondary

80 15 10 5 0 0

20

40

60 80 Time (min)

100

120

Figure 6. (a) Dissolution profile of fenofibrate in FaSSIF containing sodium TCA, and FaSSIFSecondary containing sodium TDCA and (b) dissolution profile of ketoconazole in FaSSIF and FaSSIFSecondary. (n=3, mean ± SD for all data points).

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In an attempt to decipher the unexpected effect of microbial 7α-dehydroxylation on the in vitro dissolution of fenofibrate, the apparent solubilities of fenofibrate and ketoconazole were determined in FaSSIF and FaSSIFSecondary media. Apparent solubility measurements of fenofibrate in these media were consistent with the observed effects on dissolution, that is, fenofibrate was solubilized to a greater extent in the host derived primary bile acid TCA in comparison to the microbial metabolite TDCA (10.46 ± 0.27 µg/ml and 5.40 ± 0.02 µg/ml in FaSSIF and FaSSIFSecondary, respectively). The apparent solubility of ketoconazole in these simulated fasted fluids was similarly reflective of observed dissolution trends (19.31 ± 0.66 µg/ml and 46.11 ± 4.92 µg/ml in FaSSIF and FaSSIFSecondary).

4. Exploring altered drug solubilization capacity as a function of increasing bile salt concentration The concentration of bile salts in the intestine increases postprandially. The effect of microbial bile acid metabolism on micelle solubilization capacity was thus assessed under simulated fasted (FaSSIF, FaSSIFSecondary) and fed (FeSSIF, FeSSIFSecondary) state conditions. In concurrence with the literature for BCS class II drugs, solubility was enhanced in media mimicking the fed state intestine (FeSSIF, FeSSIFSecondary) as compared to the fasted state (FaSSIF, FaSSIFSecondary) (Table 5), an effect attributable to the greater concentration of bile salts and lecithin.10 The effect of microbial bile acid metabolism on the apparent solubility of PWSDs in fasted and fed state biorelevant media appears to be drug-specific (Table 5). Interestingly for some compounds, such as fenofibrate, celecoxib and progesterone, opposing effects of bile acid metabolism on apparent solubility were noted at bile concentrations typical of the fasted (3 mM FaSSIF/FaSSIFSecondary) versus fed (15 mM FeSSIF/FeSSIFSecondary) states (Table 5). For example, the solubility of fenofibrate was significantly enhanced in FaSSIF as compared to FaSSIFSecondary (10.46 ± 0.27 µg/ml and 5.40 ± 0.02 µg/ml, respectively), indicating that bile acid dehydroxylation reduces the solubilization capacity of fasted state biorelevant media for fenofibrate. Contrastingly, solubility in FeSSIF was relatively comparable to that determined in FeSSIFSecondary (26.53 ± 0.94 µg/ml and 31.87 ± 2.00 µg/ml, respectively), suggesting that in the fed state microbial bile acid metabolism would not appreciably affect micellar solubilization capacity for fenofibrate. Celecoxib similarly displayed a greater apparent solubility in FaSSIF than FaSSIFSecondary (30.17 ± 0.21 µg/ml and 17.21 ± 0.56 µg/ml, respectively), whilst comparable solubilization was observed in both fed media (95.83 ± 1.82 µg/ml and 102.56 ± 0.65 µg/ml). In contrast, the degree of bile acid hydroxylation, and therefore microbial bile acid metabolism, did not affect progesterone solubility in fasted media (22.01 ± 0.91 µg/ml and 24.10 ± 0.16 µg/ml in FaSSIF and FaSSIFSecondary, respectively). However, progesterone solubility was significantly increased in FeSSIFSecondary (109.36 ± 2.80 µg/ml) relative to FeSSIF (66.41 ± 2.29 µg/ml), signifying that microbial bile acid metabolism could potentially facilitate enhanced progesterone solubilization under fed state conditions. A series of apparent solubility studies in progressively increasing concentrations of bile and lecithin (fixed 4:1 ratio, Figure 7) is consistent in concluding that the effect of bile acid dehydroxylation on PWSD solubility in biorelevant media is not only drug-specific, but is also governed by bile acid concentration. In order to gain a greater appreciation of the possible factors responsible for the observed drug-specific effects of bile salt dehydroxylation on PWSD solubility in biorelevant media, further investigations were undertaken. Firstly, the surface tension of solutions containing pre- and post-prandial concentrations of TDCA or TCA in the presence of lecithin (4:1 ratio) were determined. Considering the lower surface tension of TDCA: lecithin-, relative to TCA: lecithin-, containing media (Figure 8), the solubilization capacity of the former would be expected to be superior. Subsequently, the apparent solubilities of

16


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fenofibrate, celecoxib, progesterone and ketoconazole were determined in media containing TDCA and TCA, at both lower (3 mM) and higher (20 mM) concentrations of bile in the absence of lecithin. Under these conditions, all four compounds were solubilized to a greater extent in media containing TDCA, as compared to TCA (Figure 7). The presence of lecithin in biorelevant media is thus likely to contribute to the observed drug-specific effect of microbial bile acid metabolism on drug solubility.

(a) TCA:Lecithin (4:1)

40

TDCA:Lecithin (4:1) TCA

30

TDCA

20 10 0 0

5

(c)

10 15 Bile (mM)

20

TCA

100

TDCA

50

0 0

TDCA:Lecithin (4:1)

150

TCA TDCA

100 50 0 10 15 Bile (mM)

TCA:Lecithin (4:1) TDCA:Lecithin (4:1)

5

(d) TCA:Lecithin (4:1)

5

150

25

200

0

Celecoxib Solubility (µg/ml)

50

20

25

Ketoconazole Solubility (µg/ml)

Fenofibrate Solubility (µg/ml)

(b)

Progesterone Solubility (µg/ml)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


10 15 Bile (mM)

20

25

300

TCA:Lecithin (4:1) TDCA:Lecithin (4:1) TCA TDCA

200

100

0 0

5

10 15 Bile (mM)

20

25

Figure 7. Apparent solubility of fenofibrate (a), celecoxib (b), progesterone (c) and ketoconazole (d) in FaSSIF blank buffer (phosphate buffer pH 6.5) with varying concentrations of sodium TCA or TDCA with/without lecithin (bile salt: lecithin ratio 4:1) (n = 3, mean ± SD for all data points).

17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Surface Tension (mN/m)


75 70 65

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TCA:lecithin (4:1) TDCA:lecithin (4:1)

60 55 50 45 40 35 0

10 20 30 Bile Salt Concentration (mM)

40

Figure 8. Surface tension of sodium TCA and TDCA solutions containing lecithin (bile salt: lecithin ratio 4:1) (n = 3, mean ± SD for all data points).

5. Microbial mediated bile acid dehydroxylation: impact on drugs displaying solubility limited oral absorption Dose: solubility ratios provide an indication of whether the volume of gastrointestinal fluid is likely to be sufficient to dissolve the administered dose of a drug.7 Forecasted dose: solubility ratios exceeding 250 mL, otherwise interpreted as a dose number (D0) > 1, theoretically suggest suboptimal fluid volumes, that is, the persistence of solid phase drug in the lumen and incomplete absorption. By comparing dose numbers calculated with solubility measurements in fasted state biorelevant media containing primary (FaSSIF) and secondary (FaSSIFSecondary) bile salts, we theoretically assessed the impact of the microbial 7α-dehydroxylase on the solubility limited absorption of the study PWSDs. For 4/9 study drugs, microbial 7α-dehydroxylase activity was determined to alter the fasted state D0 to an extent (≥ 20%) deemed to be of potential clinical significance.35 Based on these assessments, celecoxib, danazol and fenofibrate are theoretically predicted to exhibit exacerbated solubility limited absorption in the fasted state as a consequence of microbial bile acid metabolism. Contrastingly under these same conditions, an improvement in the solubility limited absorption of ketoconazole is expected. In the case of felodipine whilst the D0 varied by > 20% between the fasted media, the impact was concluded to be negligible since complete dissolution is anticipated irrespective of the specific bile salt present (D0 < 1 under all conditions).

18


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Table 4. The impact of microbial 7α-dehydroxylase activity on drug dose number (D0)

Model Drug

Typical Oral Dose (mg)

D0 FaSSIF

D0 FaSSIFSecondary

Impact on D0

Celecoxib Danazol Fenofibrate Ketoconazole Felodipine Hydrocortisone Nifedipine Phenytoin Progesterone

200 200 200 200 5 10 5 200 100

26.51 149.33 76.48 41.42 0.53 0.12 1.79 24.26 18.18

46.49 185.52 148.15 17.35 0.71 0.12 1.91 24.84 16.60

↑ ↑ ↑ ↓ ↔ ↔ ↔ ↔ ↔

6. Microbial mediated bile acid dehydroxylation: impact on PWSD absorption in the fed versus fasted state The impact of microbial 7α-dehydroxylation of the cholate steroidal structure on the propensity of PWSDs to display altered food-dependent solubility was investigated. Observed differences in drug solubilization ratios (SR) in FeSSIFSecondary/FaSSIFSecondary and FeSSIF/FaSSIF suggests that microbial bile acid metabolism could potentially influence the “food effect” (altered drug bioavailability in the presence of food) characteristic of some medicines (Table 5 and Figure 9). Considering a deviation of ≥ 20% as a measure of possible clinical consequence,35 7α-dehydroxylase activity could, at least theoretically, increase the positive food effect of fenofibrate, celecoxib and progesterone. Contrastingly, 7αdehydroxylation would appear to reduce the fed/fasted solubilization ratio of nifedipine.

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Table 5. Apparent solubility and solubilization ratios of model PWSDs in conventional and modified biorelevant media Model Drug

FaSSIF Solubility (µg/ml) ± SD

FeSSIF Solubility (µg/ml) ± SD

FaSSIFSecondary Solubility (µg/ml) ± SD

FeSSIFSecondary Solubility (µg/ml) ± SD

FeSSIF/FaSSIF SR ± SE

FeSSIFSecondary/ FaSSIFSecondary SR ± SE

Celecoxib

30.17 ± 0.21

102.6 ± 0.65

17.21 ± 0.56

95.83 ± 1.82

3.40 ± 0.02

5.57 ± 0.12

Danazol

5.36 ± 0.37

23.66 ± 0.57

4.31 ± 0.1

22.63 ± 0.43

4.42 ± 0.19

5.25 ± 0.09

Felodipine

37.57 ± 2.09

233.6 ± 7.83

28.34 ± 0.68

157.7 ± 5.12

6.22 ± 0.23

5.56 ± 0.11

Fenofibrate

10.46 ± 0.27

26.53 ± 0.94

5.40 ± 0.02

31.87 ± 2.00

2.54 ± 0.06

5.90 ± 0.21

Hydrocortisone

342.3 ± 10.95

497.4 ± 15.66

343.8 ± 16.43

566.3 ± 20.93

1.25 ± 0.03

1.34 ± 0.04

Ketoconazole

19.31 ± 0.66

467.9 ± 9.31

46.11 ± 4.92

919.3 ± 23.05

24.23 ± 0.55

19.93 ± 1.26

Nifedipine

11.17 ± 0.18

54.24 ± 0.97

10.45 ± 0.54

40.10 ± 1.47

4.85 ± 0.07

3.84 ± 0.14

Phenytoin

32.97 ± 1.34

47.17 ± 0.84

32.20 ± 1.00

53.15 ± 0.81

1.43 ± 0.03

1.65 ± 0.03

Progesterone

22.01 ± 0.91

66.41 ± 2.29

24.10 ± 0.16

109.4 ± 2.80

3.05 ± 0.08

4.54 ± 0.03

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FeSSIF/FaSSIF FeSSIFsecondary/FaSSIFsecondary

**

Fe Biorelevant Solubilization Ratio no fib C rat e Pr le e og co es xib te N ron ife e di pi n Ke Da e n to a co zo na l Ph zo en le Fe yto H yd lod in ro ip co ine rti so ne

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30 20 10

8

*

6 4 2 0

Figure 9. Comparative fed/fasted state solubilization ratios (SR) in conventional (FeSSIF/FaSSIF) and modified (FeSSIFSecondary/FaSSIFSecondary) simulated intestinal media (n = 3, mean ± SE). * indicates a ≥ 20% difference in the fed/fasted SR, ** indicates SR differences are < 20%.

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DISCUSSION

The effect of gut microbe mediated bile acid metabolism on PWSD solubility Despite a widespread appreciation of the role of bile salts in the solubilization and absorption of PWSDs in vivo, a paucity of data exists on the generation, that is, the host or microbe origin of, and contribution of specific bile salts to these processes.15, 36 As BCS class II compounds are characterised by low solubility and high permeability, we focussed our efforts on elucidating potential microbial effects on drug solubility and dissolution, rate limiting factors in the absorption of these drugs in vivo. The effect of microbial bile acid metabolism on drug solubilization, that is the ability of the microbiota to deconjugate and dehydroxylate the core steroid structure, was mechanistically assessed through a series of apparent solubility experiments. Firstly, to investigate the effect of liberating the taurine constituent of conjugated bile acids, by the activity of the microbial enzyme BSH, the solubilization capacities of sodium TCA and sodium CA were compared at equivalent molarity. TCA was chosen due to its use in current gold standard biorelevant media and given its reduced propensity for precipitation (lower pKa) in comparison to other common conjugated bile salts such as glycocholate (GCA).15, 37, 38 Furthermore, tauro- versus glyco- conjugation of the bile acid steroidal ring has not been shown to extensively affect drug solubilization.26 The data herein suggest that microbial bile acid deconjugation activity can influence the solubilization capacity of bile micelles. The findings indicate that this effect is drug-specific and not generalizable to all PWSDs. In instances where a disparity in solubility was observed, solubility was generally greater in micellar solutions of the conjugated bile salt TCA in comparison to CA, its deconjugated counterpart (Figure 2 (a)). The CMC of both TCA and CA are expected to be similar because the hydrophilicity of the amide group of taurine, which would increase the CMC, is nullified by the methylene groups of this same steroidal appendage, resulting in a marginal reduction in the CMC overall.39 Considering the latter, solubilization capacity differences observed for conjugated versus deconjugated simple bile micelles cannot be definitively assigned to the hydrophilic-lipophilic balance (HLB) of individual bile acids. In mixed micellar systems with sodium oleate, CA conjugation status was similarly noted to affect the solubilization capacity for some PWSDs. Further work is required to elucidate this drug-specific effect, which may be related to the intrinsic capacity of specific drug molecules to self-associate or to interact with micelles.40, 41 Our findings indicate that microbial bile acid dehydroxylation can significantly impact the solubilization capacity of bile micelles. In simple buffered micellar systems, the degree of bile acid hydroxylation was found to significantly influence solubilization capacity (Figure 2 (b)). The solubilization capacity of the dihydroxy bile salt TDCA was superior to the trihydroxy bile salt TCA for all PWSDs. Solubilization capacity was thus found to be positively correlated with the hydrophobicity of specific bile salt amphiphiles and, as demonstrated, consequently inversely related to surface tension lowering properties, since removal of a hydroxyl group from TCA yields the more hydrophobic TDCA molecule. This inverse relationship between micelle solubilization capacity for PWSDs and the hydrophilic-lipophilic balance (HLB) of bile salts is consistent with previous observations for the innate lipophilic molecule cholesterol.12 Furthermore as reported for cholesterol, the greater hydrophobicity of TDCA, relative to TCA, is likely to resultantly increase bile micelle size and aggregation number, and therefore enhance PWSD solubilization.12

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In an effort to explore the likely physicochemical characteristics underlying the observed “drug-specific” solubility enhancement in TDCA versus TCA micelles, the relationship between solubilization ratios and various physicochemical descriptors was examined. Previous studies have reported strong correlations between drug solubilization in bile salt micelles and logP, prompting investigations into the utility of this descriptor for the prediction of SE in microbe- versus host- derived bile salts (SE TDCA/TCA).13, 42 A good correlation was correspondingly noted between SE TDCA/TCA and logP (r2 = 0.7955). Recently, it has been reported that drug solubility in pure lipids is related to its melting point43; this physicochemical property was therefore assessed for its ability to predict the partitioning of PWSDs into TDCA versus TCA micelles. A trend was observed between SE TDCA/TCA and Tm, indicating that SE is likely to be inversely correlated to the PWSD melting temperature (r2 = 0.5258). Considering our dataset, it appears that the extent improvement in PWSD solubility in different bile salt micelles could be reasonably approximated on the basis of LogP. The addition of the fatty acid sodium oleate resulted in similar observations, although a deviation from the general trend was noted for felodipine and nifedipine (Figure 5). Whilst in vivo the conjugated taurine sidechain must first be cleaved by BSH prior to CA functioning as a substrate for 7α-dehydroxylase, the gelation tendency of sodium deoxycholate (DCA) precluded the preparation of buffered micellar solutions and TDCA was thus utilised. The observed gelation property of DCA in buffered solutions reflective of physiological pH values is consistent with previous reports in the literature.44 Furthermore, studies in unbuffered aqueous solutions indicate that the solubilization capacity of DCA is comparable to TDCA (in house data). However to eliminate any potential contribution of conjugation to solubilization capacity, and hence more accurately simulate the in vivo bile acid metabolism process, assessments in buffered solutions were made with TCA and TDCA (respectively substituted for cholate and deoxycholate). Collectively in both simple and mixed micellar systems, microbial bile acid metabolism was demonstrated to affect the solubilization capacity of bile micelles for PWSDs. These studies therefore suggest that perturbation of the gut microbiota and thus microbial enzymatic activity, at extremes of overgrowth and suppression, could result in modifications of the bile acid pool and potentially culminate in altered drug solubilization in situ. In particular, the impact of microbial 7α-dehydroxylation revealed a markedly generalized increase in the solubilization capacity of bile micelles. Clearly from a molecular perspective, the hydrophobicity of the bile acid steroid nucleus is the main determinant of solubilization capacity in simple micelle systems. Because drug dissolution is closely related to solubility, we hypothesised that observed solubilization differences would translate to discrete in vitro dissolution profile characteristics. Conducting dissolution studies of PWSDs in the fasted state constitutes a “worst case” simulation scenario due to the limited availability of bile and phospholipids, such that any potential improvement in solubility is more likely to be of clinical relevance. In this respect, dissolution studies were performed in media containing bile acid and lecithin concentrations reflective of the in vivo fasted state (3 mM and 0.75 mM, respectively 15). The dissolution of formulations of fenofibrate and ketoconazole (devoid of excipients which could considerably augment solubility) were shown to be affected by the hydroxylation state of the bile acid steroidal structure. As expected on the basis of assessments in simple bile salt micelles (Figure 2 (b)), ketoconazole release was greater in biorelevant media containing 3 mM of the secondary bile salt TDCA in comparison to the primary bile salt TCA. Contrastingly and unexpectedly however, fenofibrate release was greater in media containing TCA in comparison to TDCA, indicating that microbial bile acid metabolism negatively affects the in vitro dissolution of fenofibrate. The hydroxylation status of the bile acid steroid nucleus was thus found to influence drug dissolution in biorelevant media in a drug-specific

23



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Page 24 of 29

manner. The opposing effect of bile acid metabolism on the dissolution of these drugs can, however, be explained by their apparent solubility in FaSSIF and FaSSIFSecondary media (Table 5). In light of the drug-specific fasted state solubility and dissolution findings for fenofibrate and ketoconazole, apparent solubility measurements for all study PWSDs were conducted in conventional and modified biorelevant media reflecting the fasted and fed intestine. The hydroxylation status of the bile salt component was similarly found to influence the biorelevant pre-prandial solubility of the other PWSDs in a drug-specific fashion. This drug-specific effect was unexpected on the basis of surface tension determinations, which revealed that the surface tension of TDCA containing biorelevant media was lower than that containing TCA. Furthermore opposing trends in the solubilization capacities of TCA and TDCA in the presence of lecithin, for some drugs such as celecoxib and fenofibrate, under fasted and fed state conditions was unexpected, revealing that the directional, that is the increased or reduced solubilization, effect of steroidal dehydroxylation was not only drug-specific but also amphiphile (bile/lecithin) concentration dependent (Table 5). Apparent solubility studies in progressively increasing concentrations of bile salt and lecithin, reflective of the fasting and prandial intestinal range, support the conclusion that the effect microbial bile acid metabolism in mixed micellar systems of bile salt and lecithin is governed by both the specific drug and the amphiphile concentration (Figure 7). These findings further highlight the need for investigations, at a molecular level, of the interactions between APIs and bile salts, particularly in the presence of additional amphiphiles such as lecithin, as is the case in biorelevant media.45 To the best of our knowledge, this study is the first attempt to challenge the concept of biorelevant media from the perspective of the metabolising capacity of the microbiota. Whilst this research has focused on the equimolar substitution of primary and secondary bile salts, it is reasonable to envisage the future development of media proportionately reflective of the bile acid pool of disease states, such as cirrhosis or crohn’s disease.46, 47 Future work might also reveal that protracted antibiotic use is not only of significance in the setting of drug-drug interactions from the perspective of effects on host metabolism (enzyme induction/inhibition), but also for other pharmacokinetic processes through indirect effects on the microbiota. Recent studies in this regard have concluded that dysbiosis, by virtue of altered drugmetabolizing enzyme and transporter expression, may affect host pharmacokinetics.48 The results herein indicate that variations in the microbiome might additionally impact the absorption kinetics of medicines whose solubility is affected by the bile acid hydroxylation state.

Gut Microbe mediated bile acid dehydroxylation: impact on drugs displaying solubility limited oral absorption The dose number (D0), that is the ratio of an oral dose to the maximum quantity that can dissolve in 250mL, was calculated for each PWSD. Whilst the dose number concept proposed by Amidon et al. was initially founded on aqueous solubility measurements, biorelevant solubility has more recently been substituted as an optimised oral absorption model input.8, 9, 31, 49 Comparative biorelevant D0 values were thus used to investigate the potential contribution of microbe mediated bile acid metabolism to altered fasted state absorption of solubility limited drugs. A variation of at least 20 percent was regarded as potentially clinically important, in line with regulatory guidance concerning bioequivalence.35 The D0 of 4/9 PWSDs were duly determined to be affected by bile salt dehydroxylation (Table 4). Conceivably, the absorption of celecoxib, danazol and fenofibrate could be adversely affected in vivo under fasting conditions characterised by a preponderance of secondary bile salts, for example in a subgroup of patients with cholesterol gallstones.50, 51 Contrastingly, the fasted state absorption of ketoconazole would

24


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theoretically be predicted to be enhanced, relative to the general population, in individuals who accumulate high levels of DCA through the enterohepatic bile acid pool. These theoretical extrapolations are, however, based on extremes of primary and secondary bile acid substitutions and in this respect caution is advised in their interpretation.

Biorelevant solubilization ratios (bSR) as a predictor of potential gut microbe mediated alteration of fed versus fasted state bioavailability The ratio of drug solubility in FeSSIF and FaSSIF media, denoted “bSR” (biorelevant solubilization ratio) has previously been proposed as a crude indicator of potential food effects.29 Whilst other factors, such as gastrointestinal transit time, direct dietary-medicine interactions, pharmaceutical excipient effects and altered first pass clearance must be considered, this ratio affords a rapid high throughput preliminary estimate of possible food effects. 4/9 of the study PWSDs displayed a potentially clinically relevant difference (≥ 20%) in bSR in media containing primary (TCA) versus secondary (TDCA) bile salts. For population subsets accumulating high levels of secondary bile acids, we postulate that concomitant food intake could enhance the luminal solubilization, and therefore potentially alter the bioavailability, of certain drugs such as fenofibrate, celecoxib, progesterone (Figure 9). The opposite is theoretically expected in the case of nifedipine (Figure 9). Whilst this data set is too limited to draw parallels with the in vivo environment, it is supportive of the hypothesis that gut microbial activity may potentially affect the absorption of PWSDs.

CONCLUSIONS

Overall, it has been demonstrated through a series of experiments of advancing physiological relevance and theoretical extrapolations that gut microbial enzymatic activity could potentially affect the solubilization capacity of bile micelles, the apparent solubility and consequently the bioavailability of PWSDs displaying solubility limited absorption. Both conjugation and, in particular, the degree of hydroxylation of the bile acid structure can affect the solubilization capacity of bile micelles and therefore potentially the apparent solubility of drugs in the intestinal lumen. The varying solubilization enhancement of sodium TCA, CA and TDCA for the study drugs may be attributed to the structural features and therefore the HLB of these distinct bile acids, as well as the intrinsic capacity of some drugs to undergo self-micellization or to differentially associate with bile micelles.40, 52 Comparisons of drug dissolution in conventional biorelevant fasted state simulated intestinal fluid (primary bile salt component) and modified media representative of microbe-derived secondary bile acid species revealed altered, drug-specific profiles which correlated with solubility measurements. Theoretically, therefore, altered intestinal proportions of primary and secondary bile acids, as a consequence of disease, aging, or therapeutic intervention, could contribute to altered rates or extents of solubilization for drugs displaying solubility limited absorption. Whilst, future work is necessary to decipher whether these differences could persist practically in vivo; these findings provide additional evidence that the microbiome, in addition to the host, has the potential to affect drug pharmacokinetics. Gut microbial dysbiosis, by virtue of its postulated effects on bile acid metabolism and drug solubilization, may thus contribute to inter-patient pharmacokinetic variability in the clinical setting.

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ACKNOWLEDGEMENTS

Elaine Enright is a recipient of a Government of Ireland Postgraduate Scholarship from the Irish Research Council (grant number GOIPG/2015/3261). The authors acknowledge the funding of the APC Microbiome Institute by the Science Foundation of Ireland Centres for Science, Engineering and Technology (CSET) programme (Grant Number SFI/12/RC/2273).

ABSTRACT GRAPHIC

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Impact of Gut Microbiota-Mediated Bile Acid Metabolism on the

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