Rapid Throughput Solubility Screening Method for BCS Class II Drugs in Animal GI Fluids and Simulated Human GI Fluids Using a 96‐well Format

DRUG DISCOVERY INTERFACE Rapid Throughput Solubility Screening Method for BCS Class II Drugs in Animal GI Fluids and Simulated Human GI Fluids Using a 96-Well Format JEREMY GUO,1 PAUL A. ELZINGA,2 MICHAEL. J. HAGEMAN,2 JAMES N. HERRON1 1

Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112

2

Bristol Myers Sqibb, Princeton, New Jersey

Received 6 October 2005; revised 13 April 2007; accepted 15 April 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21041

ABSTRACT: A rapid solubility-screening assay was developed with a focus on Biopharmaceutic Classification Scheme (BCS) class II drug solubility in animal and simulated human gastrointestinal (GI) fluids. The assay enables biologically promising drug leads to be evaluated for solubility limitations earlier in the drug development process, minimizes GI fluid needs, and produces in vitro solubility information with potential in vivo implications. A number of BCS II drugs were dissolved in DMSO at
40 mM, and robotically distributed to a 96-well plate. The DMSO was evaporated and drugs were equilibrated with selected GI fluids, both fed and fasted states. After equilibration, precipitated wells were subjected to HPLC analysis. A spreadsheet calculated solubility automatically from HPLC output. Intra-day, inter-day, and inter-plate reproducibility were within 15% RSTD for the tested drugs with the primary source of variability being injection precision of our injector system. The reported solubility from screening assays was well correlated with literature data (r2 ¼ 0.80) with a slope of 0.86 and (r2 ¼ 0.99) with a slope of 0.89. This screening assay converts conventional solubility measurements to a 96-well format for increased throughput (>12 samples/h), reduces fluid needs, and minimizes drug consumption. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:1427–1442, 2008

Keywords: solubility; gastrointestinal; dissolution; in vitro/in vivo correlations (IVIVC); BCS

INTRODUCTION Biopharmaceutical properties such as gastric emptying, gastrointestinal (GI) transit, dosage Abbreviations used: BCS biopharmaceutic classification scheme Jeremy Guo’s present address is 1201 Amgen Ct. West, Seattle, Washington, 98119. Correspondence to: Jeremy Guo (Telephone: 425-444-0301; Fax: 206-217-0491; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 97, 1427–1442 (2008) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association

form disintegration, drug particle dissolution, membrane permeability, drug solubility, and chemical stability directly and indirectly affect extent of oral drug absorption.1–4 Oral drug absorption can also be limited by factors such as drug product decomposition in the GI tract, inefficient transport across the gut wall in the apical to basal direction, and metabolism or elimination en route to systemic circulation.5,6 The dynamic nature of the GI environment requires that drug release and uptake be completed within a designed time frame. It is well recognized

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that drug solubility plays a pivotal role in dissolution and absorption.2,4,7 The availability of in vivo solubility data during early drug development could provide valuable insights for drug selection, formulation, and predicting oral drug absorption, but obtaining such information is very challenging due to problems associated with using human GI fluids. Such problems include limited resources, institutional review board concerns, and inherent molecular and biological variations, thus making routine in vivo assays impractical during the lead identification stage. For these reasons, considerable research effort has focused on developing in vitro GI fluids that mimic in vivo conditions.2,7–12 Developing such mimics is complex because drug dissolution in GI fluid is both dynamic and highly individualized. GI fluid composition and interactions with formulation components vary considerably from site to site within the GI tract, and following food intake.12 Several different fluid mimics have been investigated, many of them containing bile acid mixed micelles to mimic both fed and fasted states.2,8,12 A method allowing quick evaluation of a number of different media could provide greater insight into solubility and its role in drug absorption, thereby providing a solubility map across a number of possible mimics. The screening method described herein allows multiple fluids to be examined and compared to in vivo canine and porcine fluids. The Biopharmaceutic Classification Scheme (BCS) proposed by Amidon et al.13 correlates drug absorption criteria to drug solubility and permeability in the GI tract. The scheme categorizes drug leads into four basic groups according to their solubility properties and abilities to penetrate the GI mucosa. Our research focuses on BCS class II drugs (poorly water soluble but highly permeable) because dissolution rate is most certainly the principal limitation to their oral absorption. In vitro media simulating human GI fluids are formulated based upon available information on GI physiology and the composition of the GI contents.8,10,12 A number of publications relate in vivo drug solubility to in vitro solubility data obtained in GI fluid mimics.2,7,9–11,14–16 For instance, the solubility of danazol (a BCS class II drug) in human GI fluid is comparable to its mean solubility in simulated GI fluids containing bile salts, provided that lecithin is added to the bile salt mixture (bile salt mixtures without lecithin underestimate in vivo danazol solubility).15,16 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008

Bile salts solubilize drug molecules through micelle formation. Addition of lecithin lowers the CMC, which effectively increases the number of micelles, leading to increased solubility. Thus, in vivo solubility largely depends on the composition of the GI fluid mimic, which includes surfactant concentration, pH, buffer capacity, ionic strength and any other participating solubilizing agents. A meaningful predictive relationship between in vitro and in vivo solubilities can be drawn when simulated GI fluids are formulated to closely mimic human GI conditions. Food-induced effects on drug solubility should also be considered in formulating human GI fluid mimics. Food typically enhances the absorption of poorly water-soluble drugs. This enhancement results from drug interaction with food components and the biochemical and physiological changes of the GI tract from fasted to fed states. The secretion of additional bile salts along with phospholipids, cholesterol, triglycerides, pigments, and ions can all modify drug solubilization leading to dissolution rate and bioavailability changes with BCS class II drugs.4 On average, bile salt concentrations in the duodenum and upper jejunum are approximately two to three times higher postprandially, as compared to fasting conditions.3,15–17 The increased bile salt concentration upon food intakes subsequently leads to higher solubility, and consequently enhanced in vivo solubility. For example, danazol exhibits an in vitro solubility of 0.79 mg/mL at 3.75 mM bile salt concentration (fasting conditions) that increases dramatically to 6.1 mg/mL at 15 mM bile salt concentration (fed condition).2 Similarly the dissolution rate increases from 0.3 to 1.8  103 mg/mL/min.4 These observations correlate to increased bioavailability parameters such as plasma area under curve (AUC) and Cmax in vivo where fed state condition triggered a threefold increase in plasma AUC (204  125 to 639  259 ng h/mL).2–4,15 Halofantrine, another BCS class II drug, has a solubility of only 2.1 mg/mL with a dissolution rate of 0.0061  103 mg/mL/min at 3.75 mM bile salt concentration, but solubility increases to greater than 2000 mg/mL with a dissolution rate at 1.2243  103 mg/mL/min at 15 mM bile salt concentration.18 Plasma AUC increases from 3.9  2.6 to 11.3  3.5 mg h/L following a food intake.4,18 Therefore, food presence in the GI environment can significantly impact solubility, dissolution rate, and consequently bioavailability of drugs, particularly BCS class II drugs, and DOI 10.1002/jps

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should be adequately simulated in GI fluid mimics. Solubility evaluations using GI fluids (either simulated or from animal sources) need to be fast and inexpensive, with minimal drug and media requirements, while producing data of sufficient quality for decision making. Screening assays for physiochemical parameters have become to play an increasingly important role in addressing this unmet need. We describe a 96-well format solubility assay designed specifically for screening BCS class II drugs in simulated human intestinal fluids. The 96-well format offers substantially increased throughput and flexibility, minimizes resources, and enables automation at early development stage, thereby allowing the examination of more permutations of media conditions.

MATERIALS AND METHODS Materials Table 1 lists the BCS class II drugs2–4,7,11,13,15,17,19 investigated in this study and their relevant physical properties. All drugs were purchased from Sigma (St. Louis, MO). Primary literature citations for simulated human GI fluids are given below in Simulated Human GI Fluids Preparation. Several fluids were investigated including a simulated intestinal bile salts-lecithin mixture (SIBLM), a medium simulating fasted state conditions in the small intestine, a medium simulating fed state conditions in the duodenum, a perfusion buffer for simulating fasted conditions, and a perfusion buffer for simulating fed conditions. The following ingredients were purchased from Sigma: trifluoroacetic acid, taurocholic acid, taurochendeoxycholic acid, deoxycholic acid, glycochenodeoxycholic acid, sodium glycocholate, glycodeoxycholic acid, glycochendeoxycholic acid, propyl gallate, and sodium chloride. Solvents included water, acetonitrile, and DMSO pur-

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chased from EM Science (Darmstadt, Germany). Dog GI fluids, fasted, were obtained from Covance Inc. (Madison, WI) and pig GI fluid from Sinclair Research Center (Columbia, MO).

Equipment A Biomek 2000 laboratory automation workstation from Beckman Instruments (Fullerton, CA) was used in liquid dispensing. Drug concentration assays were performed by HPLC using an Agilent model 1100 (Foster City, CA) equipped with binary pump (G1312A), autosampler (G1313A), column compartment (G1316A), and photodiode array detector (G1315A). A Gilson model 215 liquid handler (Middleton, WI) was used to inject samples directly from a 96-well plate. DMSO solvent was evaporated by centrifugal evaporation, Genevac Technologies, model HT-4 (New York, NY). Titer plate shaker purchased from Lab-Line Instruments (Dubuque, IA) accompanied by coated parylene stir bars purchased from V&P Scientific (San Diego, CA) were used in equilibration. Multiscreen 96-well 0.4 mm filter plates (model MAHVN4550) purchased from Millipore (Billerica, MA) were used for filtration. Sample vials (12 mm  32 mm) were obtained from VWR (West Chester, PA), vial caps and 6 mm inserts from Kimble Chromatography (Vineland, NJ), P250 and P20 pipette tips from Beckman Instruments Inc. and/or Molecular Bioproducts Inc. (San Diego, CA). Cushion pads were fabricated from silicone rubber sheet (ca. 3-mm thick) and sized to fit underneath the 96-well plates.

Drug Plate Preparation Drugs were dissolved at high concentration (typically 10 mM) in DMSO. Drug stocks, depending on desired final concentration (typically 2.5 mM of drug at equilibrium), were

Table 1. BCS Class II Drugs Investigated and Their Relevant Physiochemical Properties

Name Triamcinolone2 Phenytoin17 Griseofulvin4,13,17 Danazol2,3,7,11,15 Mefenamic acid11,19 Betamethasone2 DOI 10.1002/jps

Molecular Formula

Molecular Weight

Aqueous Solubility (mg/mL)

Log p

Relevant pKa

Melting Point (8C)

C21H27FO6 C15H12N2O2 C17H17ClO6 C22H27NO2 C15H15NO2 C22H29FO5

394.4 252.3 352.2 337.46 241.3 392.5

158 27.1 29.9 1.05 0.5 63

1.03 1.92 2.18 4.53 5.3 1.97

— 8.1 9 — 4.2 —

270 296 220 225 230 232

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robotically (Biomek) distributed into 96-well flat bottom polystyrene Costar 3595 plates (Corning, NY). DMSO was then evaporated (Genevac, typical run time
2 h). Only solid drug remained in the wells after evaporation, although the exact form (amorphous or crystalline) was not determined.

Perfusion Buffer for Simulated Fed Condition (PBSFe) Stock solution consisted of propyl gallate (1 mg/ mL), NaH2PO4  H2O (18.4 mg/mL), sodium taurocholate (10.69 mg/mL), oleic acid (3 mg/ mL), monolein (1 mg/mL), lecithin (3.67 mg/mL), NaOH, final pH adjusted to 6.5; osmolarity was 280–300 mOsm.8,12

Simulated Human GI Fluids Preparation Simulated Intestinal Bile Salts-Lecithin Mixture (SIBLM) The stock bile salt mixture consisted of sodium glycocholate (0.030 M), sodium glychenodesoxycholate (0.030 M), sodium glycodesoxycholate (0.015 M), sodium taurocholate (0.010 M), sodium taurochenodes-oxycholate (0.010 M), sodium taurodesoxycholate (0.005 M), and sodium chloride (0.050), prepared in a 1.0 L volume.20 The working solution (SIBM) was prepared by adding the stock bile salts mixture (400 mL) to sodium phosphate buffer pH 6.4 and adjusting the volume to 1000 mL, 0.15 M with respect to both sodium and phosphate concentration. SIBLM was made by mixing 400 mL of the SIBM with lecithin (0.011 M) and sodium phosphate buffer, reaching a final pH of 6.4 and adjusting the volume to 1000 mL (0.15 M with respect to sodium and phosphate ion concentration). Medium Simulating Fasted State Conditions in the Small Intestine (FaSSIF) Stock solution consisted of KH2PO4 (0.029 M), sodium taurocholate (5 mM), lecithin (1.5 mM), and KCl (0.22 M), NaOH, final pH adjusted to 6.8; osmolarity was 280–310 mOsm.9,10 Medium Simulating Fed State Conditions in the Duodenum (FeSSIF) Stock solution consisted of acetic acid (0.144 M), Na taurocholate (15 mM), lecithin (4 mM), KCl (0.19 M), NaOH, final pH adjusted to 5.0; osmolarity was 485–535 mOsm.9,10

Animal GI Fluid Preparation Canine and porcine GI fluids posed significant filtration problems in the 96-well plate format because solids in the fluids can foul 0.4 mm pore 96-well filter plates. Thus, fluids were clarified before solubility experiments were performed. In particular, fluids collected from animals’ small intestines were centrifuged at 8000 rpm for 20 min to remove large solids. Aliquots of supernatant were collected and transferred to a 10-mL syringe with a 0.4 mm size Millipore filter. Supernatants were manually filtered with filters changed as needed to generate the stock solutions for further solubility studies. All the simulated and animal fluids were robotically transferred to the DMSO evaporated drug stock plate. This procedure was not expected to significantly affect the overall drug solubility because macromolecules that could impact overall solubility (e.g., protein, bile salts, and lipids) are typically less than 0.4 mm in size.

Material Minimization Usually 150 mL of solution was transferred to each well for HPLC analysis. Cushion pads were placed beneath the 96-well plates (allowing injection needles to immerse deeper into the wells) to reduce the volume necessary per well to get reproducible sample pull, hence decreasing the amount of animal fluids used in each experiment. Using the same HPLC method, sample peaks from wells containing 150 mL in volume but no cushions were compared with wells with less volume but cushion pads underneath.

Perfusion Buffer for Simulated Fasted Conditions (PBSFa) Stock solution consisted of sodium taurocholate (2.69 mg/mL), propyl gallate (1 mg/mL), NaH2PO4  H2O (18.4 mg/mL), NaOH, final pH adjusted to 6.5; osmolarity was 280–300 mOsm.8,12 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008

Equilibration Small stirring bars were added to each well of the drug plate, which was then sealed with aluminum seals purchased from Beckman and placed on a shaker. The solid drug in each well was DOI 10.1002/jps

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equilibrated with its corresponding GI fluid for 24 h with agitation (assumed to be sufficient for equilibration). After equilibration, the pH of each solution was measured and compared with the pH of GI fluids without drugs to verify adequate buffer capacity of the biofluids. Equilibrated samples were then filtered through a 0.4 mm pore 96-well filtration plate into a round bottom 96-well Falcon plate purchased from Becton Dickinson Labware (Franklin Lakes, NJ). Drug solutions were diluted as necessary and the diluted drug plate was sealed with aluminum seals, ready for HPLC analysis.

HPLC Method Optimization A generic HPLC method was developed that could separate the BCS class II drugs listed above (see Tab. 1) from interferences in the media fluids. The method used a short C8 (Zorbax, 2.1 mm  50 mm) column and preguard column (Zorbax). The flow rate was 1 mL/min, with a gradient from 95% mobile phase A (H2O with 0.1% TFA) to 95% mobile phase B (acetonitrile with 0.01% TFA) over 3 min, followed by constant 95% B for 45 s, and then re-equilibration at 95% A for 30 s. Total assay time was about 5 min. Detection utilized a photodiode array (Agilent) utilizing routine detection at 214, 230, 254, 280, and 295 nm. A standard curve consisting of 5 points (1 mg/mL, 0.25 mg/ mL, 0.0625 mg/mL, 0.0156 mg/mL, 0 mg/mL) was constructed (linear regression forced through the origin) and used to determine the solution concentration of the sample. The sample concentration was determined from each peak’s area and the slope obtained from the standard curve.

RESULTS AND DISCUSSION Solubility PreScreening Using Turbidity Measurements A triage method was used to differentiate soluble compounds from those with poor solubility so that the more time-consuming HPLC assay was only performed on the latter. Turbid wells were subject to HPLC analysis following filtration, and nonturbid wells were reported as having solubility greater than 4 mg/mL (depends on loading in given study). Given the flexibility of this 96-well format, the assay could easily be adjusted to expand the dynamic range of the assay by changing drug load per well or altering turbidity DOI 10.1002/jps

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threshold of the assay. On a typical plate, rows A through H contain eight types of simulated human GI mimics and animal GI fluids, and columns 1–12 contain 12 drugs that are being evaluated in these GI fluids.

Materials Minimization Quantities of drug candidates are often very limited during the early phase of drug development. The 96-well plate solubility assay was designed to glean as much physiochemical information as possible from limited materials (both candidate drug and GI fluids) using available equipment. A simple, yet effective approach was to elevate the plates on the HPLC liquid handler platform so that the aspiration needle drew fluid near the bottom of the well. Using two shim pads beneath the plate afforded as much as a 60% reduction in GI fluid needed. Without the shim pads, a well volume of 150 mL of GI fluids was required for proper aspiration, although the assay only used 75 mL at equilibration stage. As Figure 1 shows, even small volumes (e.g., 50 mL) may be sufficient, but 75 mL per well was deemed to be a safe and sufficient choice.

DMSO Effects on Overall Drug Solubility As described in Materials and Methods section, DMSO was used as a carrier solvent for spotting drug solution into each well of the multi-well plate. Even though drug plates were then evaporated for 2 h, questions remained as to whether all DMSO had evaporated and whether residual amounts of DMSO would affect the overall solubility of the drug in biological fluids. The mass of a 96-well plate decreases as DMSO evaporates over time. Figure 2 shows a timedependent plate mass profile for both a highly soluble drug (antipyrine) and a poorly soluble drug (danazol). After 2 h, most of the DMSO had evaporated, leaving less than 1.5 mL DMSO per well on average remaining for both highly watersoluble drug and poorly water-soluble drug. This would result in less than 2% DMSO for an addition of only 150 mL of media during equilibration. Two additional hours of evaporation did not further reduce the remaining DMSO. Hence, 2-h evaporation time was selected as the routine evaporation time. Mass analysis measured residual DMSO, but could not directly assess DMSO effects, if any, on JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008

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Figure 1. Minimization of biofluid usage. The minimal volume of biofluid required for HPLC injection was investigated by elevating a 96-well plate with shim pads. Conservatively, sample volume could be reduced in half from 150 to 75 mL.

overall drug solubility in the selected GI fluids. Although cosolvent concentrations lower than 2% were not expected to significantly impact solubility, any existent cosolvent effects should be more

Figure 2. Residual DMSO following evaporation. One hundred fifty microliters of DMSO solution containing a soluble drug (10 mM antipyrine) were added to each well on a 96-well flat bottom polystyrene plate and then evaporated leaving solid for equilibration with acid solution. Wells were weighed at 30 min intervals over a 4-h evaporation period, converted to volume with a density close to that of 1 g/mL. The experiment was then repeated with a poorly soluble drug (10 mM danazol) less than 0.7% (v/v) of DMSO was retained regardless of drug solubility after 2 h. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008

pronounced with lipophilic drugs. Therefore, residual DMSO effects on Danazol solubility (log p 4.5) were assessed by adding different amounts of DMSO to the wells after equilibration, thus generating a DMSO concentration profile versus drug solubility. DMSO was directly added to buffer and a medium simulating fasted conditions containing danazol. Figure 3 shows that the addition of DMSO had minimal effect on the overall drug solubility up to
3%. This was higher than the 2% that was predicted from the indirect DMSO measurement. Therefore, the residual DMSO after evaporation was not expected to have significant effect on drug’s overall solubility, certainly none of importance in a screening assessment.

Filtration Effect on Solubility Canine and porcine GI fluids could not be used directly in solubility assays because they contained solids that could block the 0.4 mM filters used in the 96-well plates. Such solids were removed by centrifugation at relative low speed (2862g) to remove large solids; supernatants were then collected and filtered sequentially DOI 10.1002/jps

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Figure 3. DMSO effects on drug solubility. Danazol has a very low intrinsic solubility. It was equilibrated in FaSSIF with increasing concentrations of DMSO. HPLC measurement showed that danazol’s intrinsic solubility in the buffer solution was much lower than that in FaSSIF. FaSSIF provided additional solubilization to the drug molecule. Although the 0% DMSO starting solubility is different in buffer and fasted medium, the addition of DMSO did not have any significant effect on the overall solubility until 3% DMSO v/v was added.

through 10 and 5 mm filters to remove particulates that could clog the 0.4 mm filters normally used before HPLC analysis. The impact of filtration on drug solubility was evaluated by comparing solubility in unfiltered and filtered animal GI fluids for two BCS class II drugs (triamcinolone and mefenamic acid). Figure 4A compares triamcinolone solubility in unfiltered and filtered canine GI fluids from three different animals and porcine GI fluids from two different animals; while Figure 4B compares mefenamic acid solubility in the same fluid series. Mean solubility values and standard deviations were determined from either duplicate or triplicate measurements. Paired t-tests (two-tailed) assuming equal variances were performed to determine whether solubility differences before and after filtration were significant. In addition, a paired t-test for means was performed to compare the average prefiltration solubility (average of all solubility measurements determined from GI fluids from a given species) to the average postfiltration solubility (average of all measurements determined for a given species). None of these t-tests were significant to a level of p  0.05, thus we concluded that filtration effects on drug solubility were negligible. As can be seen from Figure 4, the inherent interanimal variations within the aniDOI 10.1002/jps

mal fluids can easily mask any potential filtration effects. For example, a modest change in GI fluid pH from 6.8 in canine 1–7.5 to canine 3 resulted in over fivefold difference in the solubility of mefenamic acid, an ionizable drug with pKa of 4.2. Published data on mefenamic acid have shown its aqueous solubility improved from less than 0.5 mg/mL at pH 5.0 to over 25 mg/mL at pH 6.5.19 Because pH variations are inherent with animal GI fluids, we recommend controlling this variability by standardizing ordering procedure and processing protocol, using a drug panel as controls to characterize fluids, and adjusting the fluid pH to a preset value (preferably 6.5).

Reproducibility The same drugs were evaluated in intra-plate, inter-plate, intra-day, and inter-day validation studies in the selected simulated and animal fluids. Four representative BCS class II drugs were chosen based upon drug intrinsic solubility and pKa values. For example, danazol has very low solubility at 1.05 mg/mL but no pH effects; mefenamic acid’s solubility is 0.5 mg/mL at low pH but increases dramatically at higher pH due to a pKa value of 4.2; phenytoin has moderate JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008

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Figure 4. Filtration effects on overall drug solubility in animal GI fluids. Plot A: Triamcinolone; Plot B: Mefenamic acid. Unfiltered: Both drugs were equilibrated for 24 h in unfiltered canine GI fluids from three different animals and unfiltered porcine GI fluids from two different animals. After equilibration, supernatants were collected through a serial filtration using 10 mL BD syringes, and subjected to HPLC measurement. Filtered: Both drugs were equilibrated for 24 h in filtered canine and porcine GI fluids from the same animals, and then measured by HPLC without any additional filtration. Reported solubility values are the mean and standard deviation of either duplicate or triplicate measurements for each animal GI fluid. Paired t-tests compared unfiltered and filtered solubilities for each animal GI fluid; none of the comparisons were significant except for mefenamic acid in one of the porcine animal GI fluids (Porcine 1).

solubility at 27.1 mg/mL, but its weak acid pKa value of 8.06 is not a factor over the investigated pH range; griseofulvin has a solubility at 29.9 mg/ mL and expected to have no pH effect on solubility. These four drugs were used to demonstrate that assay reproducibility was independent of drug specificity. Table 2 summarized the relative standard deviations obtained for each drug in different types of GI fluids by varying independent parameters, plate, and time. The variability of results from intra-day, intra-plate (wherein the same standards were used) was reasonably consistent JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008

with the inherent variability of the HPLC autoinjection system for 96-well format, which was shown in independent studies to be as high as 7– 8% (data not shown). The intra-plate, inter-day solubility would really only be relevant if portions of a given plate were run on different days. The intra-plate, inter-day variabilities were similar to that obtained for inter-plate, inter-day, with variabilities as high as 20%, but generally running around 10–15% or less. For example, phenytoin (moderate solubility, no pH effects) exhibited a maximum of 15% variability in both intra-day and inter-day assessments (Fig. 5). The DOI 10.1002/jps

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Table 2. Variation Obtained for Selected BCS II Drugs

Drug Danazol Cs ¼ 1.05 mg/mL

Mefenamic acid Cs ¼ 0.5 mg/mL

Phenytoin Cs ¼ 27.1 mg/mL

Griseofulvin Cs ¼ 29.9 mg/mL

Fluid Types

RSTDa, Intra-plate, Intra-day

RSTDa, Intra-plate, Inter-day

RSTDa, Inter-plate, Inter-day

Phosphate SIBLMb Medium simulated fasted Medium simulated fed Perfusion buffer for fasted Perfusion buffer for fed Phosphate SIBLMb Medium simulated fasted Medium simulated fed Perfusion buffer for fasted Perfusion buffer for fed Phosphate SIBLMb Medium simulated fasted Medium simulated fed Perfusion buffer for fasted Perfusion buffer for fed Phosphate SIBLMb Medium simulated fasted Medium simulated fed Perfusion buffer for fasted Perfusion buffer for fed

N/A 5.7% 15.5% 5.3% N/A 6.2% 9.2% 3.9% 5.6% 4.7% 3.3% 3.4% 9.3% 8.8% 9.7% 7.2% 10.0% 9.5% 2.0% 5.4% 6.7% 13.1% 7.1% 5.4%

N/A 3.0% 15.5% 12.7% 16.7% 6.2% 20.0% 14.1% 11.3% 10.6% 13.0% 14.3% 9.0% 12.9% 16.5% 11.2% 11.6% 10.6% 15% 3.6% 6.8% 16.5% 8.2% 13%

N/A 5.6% 19.6% 15.6% 18.0% 7.1% 27.2% 14.2% 17.4% 13.5% 8.7% 14.2% 7.1% 9.5% 9.6% 13.4% 6.9% 7.8% 8.9% 7.5% 9.7% 7.2% 10% 9.5%

a

RSTD, relative standard deviation. SIBLM, simulated intestinal bile salts-lecithin mixture.

b

variabilities for less soluble drugs such as danazol and mefenamic acid were slightly higher. The intrinsic solubility is so low that the sensitivity of the system plays an increasing role in variability. Still, this level of reproducibility is acceptable for solubility screening during early drug development. Assay Accuracy Data generated by the 96-well format screening assay for the solubilities of six BCS class II drugs (danazol, triamcinolone, mefenamic acid, griseofulvin, phenytoin, betamethasone) in both fastedand fed-state GI fluid mimics (FaSSIF and FeSSIF, described by Dressman et al.9,10) were compared to existing literature data using correlation plots (Fig. 6).2,9,10,15–17,19 Although the drugs tested were all classified BCS class II drugs, their aqueous solubility values ranged from less than 1 mg/mL to several hundred mg/mL. The correlation plot for FaSSIF9,10 (Fig. 6A) exhibited slope and correlation coefficient (R2) values of 0.864 and 0.792, respectively, while that for DOI 10.1002/jps

FeSSIF9,10 (Fig. 6B) exhibited slope and R2 values of 0.887 and 0.996, respectively. We attribute the lower R2 value observed for the fasted state to the fact that three of the six reported solubility values were not experimentally determined, but rather obtained from a model developed by Mithani et al., in which only bile salt solubilizing capacity was considered.2,9,10,15–17,19 Solubilizing contributions from lecithin were not incorporated in this model, although they are included in the simulated GI mimic recipes used in our studies. Experimentally determined data were available for all six drugs in fed state and correlated well to our values measured by the 96-well screening assay. Based on these results, we concluded that our 96-well format assay was a viable approach for solubility screening of BCS class II drugs in GI fluid mimics. Solubility Comparisons between Fasted and Fed States Figure 7 and Table 3 compare drug solubility in the fasted- and fed-state GI fluid mimics (FaSSIF vs. FeSSIF) described by Dressman et al.9,10 Each JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008

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Figure 5. Phenytoin’s solubility in various simulated GI fluids with variations of plate and time. Reproducibility was investigated on an intra-day, inter-day, intra-plate, and intra-day basis for the BCS class II drug phenytoin, showing that variability in various simulated GI fluids was around 15%.

of the six aforementioned BCS class II drugs was examined. A paired t-test (two-tailed) indicated that statically significant solubility differences were observed for danazol, phenytoin, griseofulvin, and mefenamic acid were ( p < 0.05), while the difference observed for triamcinolone was marginally significant ( p ¼ 0.07). A t-test was not performed for betamethasone because N ¼ 1, though its solubility values were comparable to phenytoin’s. Solubility differences for all drugs except mefenamic acid were consistent with available published data, which suggest that the apparent solubility increases with bile salt concentration.2,15,19 For example, triamcinolone (log p ¼ 1.03) has a solubility of 144 mg/mL at 3.75 mM bile salt concentration, and increases to 165 mg/mL at 15 mM bile salt concentration.2 This is consistent with data shown in Figure 7 where triamcinolone has a solubility of 142 mg/mL in fasted conditioned media and 199 mg/mL in fed conditioned media. Solubility differences are more pronounced with lipophilic drugs such as danazol (log p ¼ 4.53). Danazol’s solubility increases from 2 mg/mL at 1 mM bile salt to 12 mg/mL at just 4 mM bile concentration.15 The 96-well screening assay found danazol’s solubility to increase almost fivefold from 5 to 15 mM bile salt concentration. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008

Although mefenamic acid appears to be an exception to the above rule, its higher solubility in FaSSIF is due to a pH effect (see below). Figure 8A compares drug solubility in fastedstate GI fluid mimics described by Dressman et al. (FaSSIF)9,10 and Carey et al. (PBSFa).8,12 Five different BCS class II drugs (danazol, triamcinolone, phenytoin, griseofulvin, and mefenamic acid) were examined. For each drug, FaSSIF versus PBSFa solubility was compared using a paired t-test with equal variances. Statistically significant ( p  0.05) differences were only observed for danazol and phenytoin. In both cases, solubility was higher in FaSSIF than in PBSFa. The presence of lecithin in FaSSIF could have contributed to the increased solubilization capacity in comparison to PBSFa that lacks lecithin. Lecithin was found to provide additional solubilization power when used in combination with bile salts.15 The lecithin effect on solubility is probably more prevalent with highly lipophilic drugs such as danazol and phenytoin than relatively less lipophilic drugs, triamcinolone, griseofulvin, and mefenamic acids. Figure 8B compares drug solubility in fed-state GI fluid mimics described by Dressman et al. (FeSSIF)9,10 and Carey et al. (PBSFe).8,12 The five BCS class II drugs examined in Figure 8A were DOI 10.1002/jps

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Figure 6. A and B: Solubility values of BCS class II drugs determined from screening assays correlate well with reported solubility values. Several BCS class II drugs with varying intrinsic solubility (10–200 mg/mL) were equilibrated in either FaSSIF or FeSSIF. Equilibrated supernatants were collected and subjected to HPLC measurements. The solubility values measured in FaSSIF were plotted against solubility values reported in the literature9,10 in A; the solubility values measured in FeSSIF were plotted against solubility values reported in the literature9,10 in B.

used in this set of comparisons as well. For each drug, FeSSIF versus PBSFe solubility was compared using a paired t-test with equal variance. Statistically significant differences ( p  0.05) were observed for mefenamic acid, danazol, and phenytoin. We attribute mefenamic acid’s higher solubility in PBSFe to its pKa value (4.2) because the pH of the solution often has a greater effect on solubility than other solubilization mechanisms as discussed in two paragraphs below. The solubilities of both danazol and phenytoin were higher in PBSFe than in FeSSIF. PBSFe contains lecithin along with additional lipophilic components, which increase drug solubility. Although statistical significant solubility differences were found between the Dressman et al.9,10 and Carey et al. recipes8,12 for some of the drugs, DOI 10.1002/jps

the differences were probably more dependent on the lipophilicity of the drug molecules than the mediums. From an early stage screening perspective, it is probably easier to use the Dressman et al. recipes rather than the Carey et al. recipes because of the simplicity of the former recipes. If needed, solubility determinations of lipophilic drugs in the perfusion buffers can be easily accommodated by the increased capacity of the 96-well format-screening assay. As mentioned previously, mefenamic acid has a lower solubility in FeSSIF than in FaSSIF (Fig. 7). Conversely, it has a higher solubility in PBSFe than in PBSFa (Fig. 8). Mefenamic acid’s pKa value (4.2) is much lower than that of any of the other BCS class II drugs investigated in this study. Furthermore, FeSSIF has a pH value of 5.0 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008

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Figure 7. Drug solubility in fasted and fed state conditions. Six BCS class II drugs (danazol, triamcinolone, phenytoin, griseofulvin, betamethasone, and mefenamic acids) with varying intrinsic solubility (10–200 mg/mL) were equilibrated in either FaSSIF or FeSSIF. Equilibrated supernatants were collected and measured by HPLC. All solubility measurements were performed in triplicate except for betamethasone where N ¼ 1. Solubilities in FaSSIF and FeSSIF were compared for each BCS class II drug using a paired t-test. Statistically significant ( p  0.05) comparisons are denoted with an asterisk.

while all the other simulated GI fluids (FaSSIF, PBSFa, and PBSFe) have pH values of 6.5. This pH difference has a significant effect on solubility and a much greater solubilization effect than those exerted by the bile salts and lipids. For example, mefenamic solubility in citrate buffer at pH 5 is much lower than its solubility in phosphate buffer at pH 7. Thus, three factors should be considered in designing drug solubility screens in GI fluids: the drug-solubilizing components of GI fluid; GI fluid pH; and the drug’s intrinsic pKa value. The effect of each factor on overall solubility can readily be evaluated using the flexible capabilities of the 96-well plate-

screening format, such evaluations should be much quicker and less material-intensive than more traditional solubility measurements. In summary, the marked differences between the fasted and fed states indicate that the increased concentration of bile salts and addition of lecithin clearly increases solubilization of BCS class II drugs. Hence, both the fasted and fed states should be investigated in solubility studies. While it is reasonable to assume that large differences in fed versus fasted state solubilities may result in an in vivo food effect, the absence of such differences does not necessarily imply that being the single contributing factor.

Table 3. Solubility Improvement by FeSSIF and FaSSIF GI Fluids

Compounds Triamcinolone Phenytoin Griseofulvin Danazol Mefenamic acid Betamethasome

Solubility Improvement FastedSolu: =AqueousSolu:

Solubility Improvement FastedSolu: =AqueousSolu:

Solubility Improvement FedSolu: =FastedSolu:

0.90 2.57 0.77 21.15 119.91 0.95

1.25 3.84 2.11 104.12 47.22 1.83

1.39 1.49 2.74 4.92 0.39 1.93

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Figure 8. Drug solubility in GI fluids mimicking fed and fasted conditions. BCS class II drug solubility was compared in different recipes of simulated human GI fluids. Five different BCS class II drugs (danazol, triamcinolone, phenytoin, griseofulvin, and mefenamic acids) were examined. Plot A compares the solubility of each drug in two different fasted-state GI fluid mimics (FaSSIF vs. PBSFa). Plot B compares the solubility of each drug in two different fed-state GI fluild mimics (FeSSIF vs. PBSFe). All solubility determinations were performed in triplicate. The statistical significance for each comparison was evaluated with a paired t-test. Significant comparisons ( p  0.05) are denoted with an asterisk.

Solubility Comparisons between Simulated Human GI Mimics and Animal Fluids BCS class II drug solubility in FaSSIF was compared to that in pooled canine and porcine GI fluids (obtained under fasted conditions). Figure 9A compares five BCS class II drugs (danazol, triamcinolone, phenytoin, griseofulvin, mefenamic acid) between FaSSIF and canine GI fluid, while Figure 9B compares the same drugs between FaSSIF and porcine GI fluid. A paired t-test (two-tailed) showed that all comparisons gave significant differences in solubility ( p  0.05), except for danazol in canine GI fluid. Solubility in animal GI fluids were generally DOI 10.1002/jps

higher than in FaSSIF, which may be due to additional and/or higher concentrations of endogenous solubilizing agents in the animal GI fluids. Such endogenous solubilizing agents may include additional types of bile salts, lipids, and other macromolecules. For instance, the Dressman et al. recipes contained relatively low concentrations of bile salt, sodium taurocholate, and lecithin, resulting in a lower solubilizing capacity than observed for the Carey et al. recipes or SIBLM (data not shown), which both contained higher concentrations or additional types of bile salts and lecithin. Animal GI fluids usually possess an inherent variability even from lot to lot as shown previously in Figure 4A and B, and drug solubility in animal JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008

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Figure 9. Solubility comparison between fasted state condition human GI mimic and Canine GI fluid (A) and porcine GI fluid (B). Five BCS class II drugs (danazol, griseofulvin, triamcinolone, phenytoin, mefenamic acid) were equilibrated in FaSSIF, pooled fasted-state canine GI fluid, or pool fasted-state porcine GI fluid. Equilibrated supernatants were then collected and measured by HPLC. For each drug, solubility was compared between FaSSIF and pooled canine GI fluid in Plot A, and between FaSSIF and pooled porcine GI fluid in Plot B. All solubility determinations were performed in triplicate. The statistical significance for each comparison was evaluated with a paired t-test. Significant comparisons ( p  0.05) are denoted with an asterisk.

GI fluids may be as much as five times higher than simulated GI fluids in some cases. The inherent lot-to-lot variability of animal fluids makes routine solubility screening experiments and data analysis more complicated than simpler simulated GI fluids. Given that the animal GI fluids usually are composed of more ingredients than the simpler simulated GI fluids, the solubilization mechanisms of animal fluids to BCS class II drugs may be different from the simple simulated GI fluids, thus making solubility characterizations much more difficult. The complex nature of the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 97, NO. 4, APRIL 2008

animal GI fluids, however, is similar to actual physiological conditions of human GI fluids as both are dynamic and highly individualized. Therefore, the solubility in animal GI fluids does present a reference point for all the simulated GI fluids in the absence of human GI fluids. Given the increased capability offered by the 96-well format, we recommend inclusion of animal GI fluids in routine screening assays. Use of animal fluids as reference points become especially beneficial if solubilization mechanisms can be well characterized. DOI 10.1002/jps

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Solubility information obtained from the 96well format-screening assay is used to compare the different human GI mimics. Drugs exhibiting similar solubilities across all the examined human GI mimics are probably not very sensitive to the composition or pH of such fluids, hence the choice of one GI fluid over other is not significant. In fact, all the BCS class II drugs except mefenamic acid exhibited similar solubilities in the Dressman et al. recipes and the Carey et al. recipes, indicating that any of these was a suitable mimic. For mefenamic acid, however, the mimic’s pH value had greater impact on solubility than its composition did because of the drug’s low pKa value. Drugs that are insensitive to the composition of the mimics are termed ‘‘low specificity drugs.’’ The choice of either using the Dressman et al. recipes or the Carey et al. recipes is dependent on the lipophilicity of the drug, and unlikely to significantly impact solubility or dissolution of such drugs. For the purpose of developing a routine 96-well format assay while returning acceptable values that mimic the real human GI environment, either the Dressman et al. or the Carey et al. biofluid recipes are sufficient.

CONCLUSIONS The new 96-well format biofluid screening assay described herein was used to estimate the solubility of six different BCS class II drugs in a number of human GI fluid mimics and actual biological fluids, with increased throughput and minimal drug material consumption over conventional assays. The 96-well format offers the flexibility of screening i drugs, in j fluids, under k conditions as long as ijk ¼ 96, and can readily be expanded to 384- and 1536-well formats as well. Small well volume (75–150 mL) reduces biofluid cost by 10-fold and only 4–5 mg of drug is needed per plate. Data generated with this screening method agreed well with literature data. Assay variability (15%) is largely limited by the HPLC autoinjector used in these studies and can likely be reduced with alternative injector systems. Although human GI fluid are rarely available for screening experiments at the early state of pharmaceutical development, animal GI fluids are suitable substitutes as they represent the dynamic and complex nature of the human GI fluids. Animal fluids can be used routinely, as long as proper controls are performed for variabilities in parameters such as pH values and bile salt DOI 10.1002/jps

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concentration. While it makes good sense to run both fed and fasted GI fluid mimics, there is also some value in running the fluids at identical pH to eliminate pH effects for ionizable drugs. The flexibility of the 96-well format can easily accommodate multiple conditions such as constant pH and fed versus fasted to be performed concurrently.

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DOI 10.1002/jps