In Vivo Dog Intestinal Precipitation of Mebendazole: A Basic BCS

Sep 6, 2012 - Department of Pharmacy, Uppsala University, Box 580, S-751 23 Uppsala, Sweden. ‡ Department of .... Materials Science and Engineering:...
0 downloads 8 Views 852KB Size
Download PDF
Article pubs.acs.org/molecularpharmaceutics

In Vivo Dog Intestinal Precipitation of Mebendazole: A Basic BCS Class II Drug Sara Carlert,† Pernilla Åkesson,‡ Gunilla Jerndal,§ Lennart Lindfors,‡ Hans Lennernas̈ ,† and Bertil Abrahamsson*,‡ †

Department of Pharmacy, Uppsala University, Box 580, S-751 23 Uppsala, Sweden Department of Global Medicines Development, AstraZeneca R&D, S-431 83 Mölndal, Sweden § Department of Drug Metabolism and Pharmacokinetics, AstraZeneca R&D, S-431 83 Mölndal, Sweden ‡

ABSTRACT: The purpose of this study was to investigate in vivo intestinal precipitation of a model drug mebendazole, a basic BCS class II drug, using dogs with intestinal stomas for administration or sampling. After oral administration of a solution with an expected intestinal supersaturation of approximately 20 times the solubility, the measured supersaturation in dog intestinal fluid (DIF) was up to 10 times and, on average, only 11% of the given dose was retrieved as solid drug in the collected fluid from the stoma. The drug was rapidly absorbed with >90% of the total systemic exposure reached within three hours after duodenal administration of a solution. In silico absorption modeling showed that in vivo data were reasonably well described by a nonprecipitating solution. An in vitro model of precipitation in DIF predicted that the intestinal concentration of dissolved mebendazole would be less than 1/5 of the initial concentration within 10 min at concentrations comparable to in vivo. It was concluded that intestinal precipitation did not have any major influence on mebendazole absorption. The extent of precipitation was overpredicted in vitro given the in vivo absorption rate, and further work is needed to identify in vitro factors that could enable more accurate in vivo predictions of intestinal precipitation from solutions. KEYWORDS: precipitation, gastrointestinal, in vitro/in vivo correlations (IVIVC), absorption, biopharmaceutics classification system, in silico prediction

■

INTRODUCTION Active pharmaceutical ingredient (API) characteristics and formulation approaches creating supersaturated drug solutions in the intestines are frequently used to improve dissolution and absorption of low solubility compounds. Intestinal supersaturation is also likely to occur for basic, low solubility drugs due to the pH increase from the acidic environment in the stomach, where the drug has a high solubility to the neutral pH of the proximal small intestine which leads to a lowered equilibrium solubility of the drug. However, supersaturation carries a risk of subsequent drug precipitation which could lead to a reduced and highly variable absorption rate, bioavailability and erratic plasma exposure−time curves for BCS class II or IV substances. We have previously investigated the accuracy of traditional in vitro tests for prediction of small intestinal precipitation of a basic BCS class II substance.1 The orally administered BCS class II model substance was according to in vitro experiments expected to precipitate rapidly at high clinical doses after emptying into the small intestine. However, no effects of precipitation could be detected from clinical drug plasma concentration data, thus indicating that employed in vitro tests overpredicted the in vivo rate of precipitation in the human small intestine from a supersaturated solution. A similar finding © 2012 American Chemical Society

was made in a recent study measuring in vivo precipitation in humans by retrieving duodenal fluid via a two-lumen tube, where it was concluded that in vivo duodenal precipitation of ketoconazole and dipyridamole was limited and overestimated in previous in vitro studies.2 Bergman et al. have also attempted to verify differences in pharmacokinetic parameters (mono- vs biphasic postabsorptive plasma concentration time phases) due to precipitation/dissolution of a BCS class II substance, gefitinib.3 The approach included solid phase determination of retrieved samples of human intestinal fluid using the Loc-iGut method, but no difference in either solid phase or precipitation/dissolution could be detected to explain the deviations in pharmacokinetic behavior. Thus, the extensive precipitation obtained in simple in vitro methods only including a pH change step seems not to correspond well to the in vivo situation. There is still a need for more mechanistically oriented in vivo studies to better understand influence of intestinal drug precipitation on the drug absorption allowing development and validation of in vivo predictive methods. Received: Revised: Accepted: Published: 2903

April 20, 2012 September 4, 2012 September 6, 2012 September 6, 2012 dx.doi.org/10.1021/mp300224h | Mol. Pharmaceutics 2012, 9, 2903−2911

Molecular Pharmaceutics

Article

Table 1. Study Design of the Four Different Treatment Groups

a

study

no. of dogs

size (kg)

dose

administration route

sampling

concn (μM)

vol

T1 T2 T3 T4

3 3 4 4

21−36 21−36 30−32 30−32

0.097 μmol/kg 2.4 μmol/kg 24 μmol 3.0 μmol

oral oral idb iv

jejunum + iva jejunum + iv iv iv

52 1300 1200 300

1.9 mL/kg 1.9 mL/kg 20 mL 10 mL

Intravenous. bIntraduodenal.

In vivo precipitation rate in the small intestine will be affected by the degree of supersaturation, which will be influenced by the highly dynamic GI factors such as gastric emptying, secretion, effective permeability of the drug, drug solubilization by bile acid micelles, drug dissolution and reabsorption of water in the intestine. In addition, the nucleation in the GI tract may also be influenced by the constituents in the intestinal fluids beyond simple changes of supersaturation level, and the hydrodynamic conditions created by the gut wall motility may also be of importance. As a consequence of this complexity, it is hardly possible to accurately predict drug concentration in the intestinal lumen and the influence of other physiological conditions on the nucleation by relatively simple in vitro tests. One promising approach to overcome some of these problems that has been tested lately is to combine in vitro data with in silico models that capture many aspects of the intestinal dynamics of importance for the absorption process.1,4−6 This approach may develop into models that are able to at least qualitatively but ultimately quantitatively predict effects of drug precipitation on absorption rate in the upper small intestine. However, in order to better understand and predict small intestinal supersaturation and precipitation, direct identification and quantification of relevant intestinal contents are needed. In this study dogs equipped with jejunal stomas were used allowing retrieval and analysis of intestinal fluid. Dogs with duodenal stomas were also used to allow direct administration to the small intestine bypassing the stomach, thereby removing the effect of gastric emptying on the variability in plasma pharmacokinetics within and between animals. In order to further simplify and focus interpretation of in vivo results in our study, we have administered the model drug as a solution to exclude influence of the initial dissolution process from any particle and ensure that any in vivo effect on plasma concentration profile is due to precipitation. Complementary in vitro experiments were performed to characterize drug precipitation properties in intestinal fluid. The model substance used in this study was the BCS class II API mebendazole (pKa = 3.5;7 log P = 2.88), an anthelmintic drug introduced to the market in the 1970s. There have been extensive reports on the poor solubility and low bioavailability of 0.6−2% for mebendazole at therapeutic dose (100 mg),9−11 and also on how to increase the dissolution rate and/or solubility of the substance.8,12−15 Mebendazole’s main properties as a model compound are the solubility limited absorption and physicochemical properties that enable high supersaturation of the substance during proximal small intestinal conditions, which could lead to a rapid crystallization rate of supersaturated solutions. The ratio between amorphous and intrinsic crystalline drug solubility could be predicted to be in excess of 100,16 given the high melting point (>190 °C),17,18 reported enthalpy of melting18 and low solubility.12,19 The objective of this study was to investigate in vivo precipitation of a basic BCS class II drug, mebendazole, in the

small intestine, and its influence on absorption rate and plasma exposure of the parent drug.

■

MATERIALS AND METHODS Chemicals. Mebendazole and verapamil were purchased from Sigma (St. Louis, MO, USA). Hydrochloric acid, HCl, concentrated 37% (Merck, Darmstadt, Germany), sodium hydroxide, NaOH, pellets (Scharlau, Barcelona, Spain, and Merck), sodium chloride, NaCl (Scharlau), sodium dihydrogenphosphate monohydrate, NaH2PO4·H2O (Merck), N,Ndimethylacetamide, DMA (99+%, Aldrich, St. Louis, MO, USA), ammonium acetate, CH3COONH4 (Scharlau), triethylamine, TEA (Sigma), formic acid (Scharlau), acetonitrile, ACN (Fisher Chemical, Loughborough, U.K.), methanol (Merck), taurocholic acid, TCA (Acros Organics, New Jersey, USA), taurochenodeoxycholic acid, TCDCA (Sigma), taurodeoxycholic acid, TDCA (Sigma), cholic acid, CA (Sigma), and PEG400 (Clariant, Muttenz, Switzerland) were used as received. Test Fluids for Solubility and in Vitro Precipitation Studies. Simulated gastric fluid, SGF, was prepared according to USP 32-NF 2720 without pepsin. Dog intestinal fluid (DIF) was collected and pooled from 3 Labradors, one female and two male, age 3.5−7 years, weight 26−35 kg, surgically equipped with midjejunum stomas. The dogs were fasted overnight with access to water at all times. At the day of the intestinal fluid sampling, 75 mL of water was administered orally by the use of an orogastric tube. DIF was collected from the midjejunum stomas using plastic tubing for the duration of three hours. The untreated DIF was then stored at −70 °C. The sampling of DIF was approved by the Animal Ethics Committee in Gothenburg, approval number 173-2006. DIF was characterized in terms of pH and content of bile acids in the form of TCA, TCDCA, TDCA and CA. All measurements were made at room temperature. Preparation of samples for bile acid analysis was made by mixing DIF with cold methanol at a ratio of 1:9, centrifuging the sample at 13680 relative centrifugal force (rcf) 22 °C for 12 min and further diluting the sample with 60:40 (v/v) methanol/ deionized water. The concentrations of bile acids were measured using a previously described novel and selective method21 with minor differences in use of column and detection method. The column used was an XBridge C18 column (150 × 3.0 mm, 3.5 μM; Waters, Milford, MA, USA), and charged aerosol detection (CAD) was here used for the detection of bile acids. Formulations. Aqueous solutions of mebendazole used in the dog trials were prepared by adding a stock solution of mebendazole in DMA to an aqueous solution of 0.025 M HCl. The DMA solution contained 4:1 molar equiv of HCl:mebendazole. The percentage of DMA in the final solution was 3% (v/v). The low amount of DMA was not expected to significantly alter the solubility of the drug in the final solution. The solutions were filtered with a 0.2 μm hydrophilic PTFE 2904

dx.doi.org/10.1021/mp300224h | Mol. Pharmaceutics 2012, 9, 2903−2911

Molecular Pharmaceutics

Article

filter (Dismic-13HP; Advantec, Dublin, CA, USA) and further diluted before analysis. The solutions had concentrations of mebendazole in the range of 0.052−1.3 mM. The pH of the solution was approximately 2.5. The DMA stock solution was also used for in vitro experiments with DIF. An additional formulation containing 0.68 mM mebendazole in a cosolvent solution containing 30:70 (v/v) PEG400/ deionized water was included for intravenous administration in dogs. In Vivo Studies. All studies were approved by the Animal Ethics Committee of Gothenburg, approval number 173-2006. The in vivo studies were performed on Labrador dogs surgically equipped with a duodenal or jejunal stoma. The details of the different study groups in the trials are summarized in Table 1. The first two studies included three dogs with jejunal stomas. The treatment of the dogs prior to drug administration was similar to the treatment before DIF sampling previously described above, but at the day of the trial, the dogs were pretreated with 10 mL of 0.1 M HCl administered orally by the use of an orogastric tube approximately 30 min prior to administration of drug solution in order to lower the pH in the dog stomach, which has been established as a reliable method to obtain a consistent acidic gastric pH in dogs similar to humans (pH < 3) for a duration of at least 1.5 h.22 A drug solution with mebendazole of either 52 μM or 1.3 mM at a dose of 0.097 and 2.4 μmol/kg, respectively, was then administered in the orogastric tube, and the tube was subsequently rinsed with 10 mL of 0.1 M HCl. All solutions were administered at room temperature. The two concentrations were selected such that the lower would not provide any significant intestinal supersaturation and thereby act as nonprecipitating reference administration. The higher level was selected to generate a high degree of supersaturation in the small intestine primarily due to the change of pH from acid to neutral. The study was a crossover design with one low dose (0.097 μmol/kg) and two high doses (2.4 mmol/kg) over a two week period with at least 48 h between administrations. Stomach pH in two dogs was measured in study T1 with a digital pH meter inserted into a gastric fistula for the duration of the study (3 h), which confirmed a low gastric pH for the majority of the sampling time (pH < 3 for at least 1.5 h). DIF was collected from the jejunal stomas for a time period of three hours. Sampling was made at room temperature. The samples of DIF were centrifuged at 2000 rcf at 37 °C for 10 min as soon as a 15 mL plastic tube was filled, or a time period of 15 min passed without further volume being retrieved from the jejunum. The supernatant and pellet were separated for future determination of supersaturation and precipitation of mebendazole in the samples and stored at −70 °C. Supernatant samples of DIF were later thawed, and pH of the DIF was measured using a pH meter (PHM 240, Radiometer Medical, Brønshøj, Denmark). Sampling from the supernatant was made after homogenization of the sample. The sample was then diluted at a ratio of at least 1:1 with ACN + 10 mM formic acid. The samples were centrifuged at room temperature, 9500 rcf for 7 min. In samples where high concentrations were expected, further dilution was made with 50% ACN. Sampling was if possible made in duplicate. Concentration determination of mebendazole in DIF was made using the method for DIF described in the following section. Blood samples of 2 mL were collected from a foreleg or neck vein into heparin tubes every fifteen minutes up to 1 h, and subsequent samples (in total 10) were taken regularly up to 24

h. The total blood volume was less than 2.5% of the dog’s total blood volume. The blood samples were centrifuged at 2100 rcf, and plasma was transferred to a Cryovial tube and immediately frozen at −70 °C until analysis of the content of mebendazole could be made. Studies T3 and T4 utilized four male Labrador dogs equipped with duodenal stomas. The prior treatment of the dogs was identical to the treatment before the mebendazole study using jejunal sampling of DIF. These dogs were also pretreated with 10 mL of 0.1 M HCl orally 30 min prior to administration of drug solution. The drug solution was then administered either intravenously directly into a foreleg vein as a reference dose, or directly into the duodenum stoma through a plastic tube in order to accurately assess the absorption and bioavailability of an orally administered solution at a concentration that would be expected to generate intestinal precipitation due to the pH change. The volume of the isotonic intravenous administration was 10 mL with a total dose of 6.8 μmol that was administered as a bolus injection during 2 min. The volume of the duodenal administration was 20 mL with a dose of 24 μmol. Each animal received the duodenal solution twice and the intravenous solution once over a two week period with at least 48 h between drug administrations. All solutions were administered at room temperature. No jejunal sampling was made in studies T3 and T4. Blood samples of 2 mL from the intravenous study were collected from a foreleg vein into heparin tubes at 0, 2, 5, 10, 20, 30, 40, 50, 60 min, and subsequent samples were taken regularly up to 8 h. Blood samples of 2 mL from the duodenal study were collected from a foreleg vein into heparin tubes every ten minutes up to 1 h, and subsequent samples were taken regularly up to 8 h. The total blood volume was less than 2.5% of the dog’s total blood volume. The samples were centrifuged at approximately 2100 rcf, and plasma was transferred to a Cryovial tube and immediately frozen at −18 °C. Solubility of mebendazole and concentration of bile acids in a selected number of supernatants were also determined. Bile acid content was measured using the previously described method in Test Fluids for Solubility and in Vitro Precipitation Studies. Solubility in the supernatants was determined by incubating the samples at 37 °C for 3 h, and centrifuging the samples for 10 min at 2000 rcf at 37 °C. The resulting supernatants were diluted with 70% ACN, further centrifuged for 5 min at 2000 rcf at 37 °C and analyzed for mebendazole content. The pellet of the DIF samples was diluted with organic solvent to dissolve mebendazole, and it was then further diluted and centrifuged at 9500 rcf for 7 min before determination of mebendazole content with HPLC−UV or HPLC−MS (see Assays of Mebendazole) for samples with too low drug concentration for UV determination. Sampling was made in duplicate. The centrifuged pellet contained a small volume of fluid, and the dissolved amount of drug in this fluid was included in the measured amount of solid mebendazole in the samples. Assays of Mebendazole. Concentration determinations of mebendazole in all fluids excluding blood plasma were made using the same HPLC method with ultraviolet (UV) or mass spectrometry (MS) detection. The wavelength used for UV detection was 254 nm. The separation was made on an XTerra RP8 column (100 × 3.9 mm, 3.5 μM; Waters, Milford, MA, USA). A binary gradient was used to elute the substance, and the solutions used were as follows: (A) ACN/deionized water 2905

dx.doi.org/10.1021/mp300224h | Mol. Pharmaceutics 2012, 9, 2903−2911

Molecular Pharmaceutics

Article

Solubility of Mebendazole. In some in vitro precipitation experiments, additional sampling was made after 24 h for determination of equilibrium solubility of precipitated mebendazole. An additional solubility experiment was made in blank fasted state simulated intestinal fluid (FaSSIF without sodium taurocholate and lecithin1) at pH 6.5 in order to determine the ratio between the solubility of mebendazole with and without micelles. These data are needed for in silico calculations described in more detail below. Solubility determination was made in triplicate. The pH was recorded at the end point with a pH meter (PHM 240, Radiometer Medical, Brønshøj, Denmark). Solid State Determination of Mebendazole by X-ray Powder Diffraction (XRPD). Precipitate from experiments made in DIF and blank FaSSIF where the precipitation had proceeded for 4 h before sampling were analyzed with XRPD24 on moist samples after centrifugation to determine the solid phase of mebendazole. The samples were applied on spinning zero background sample holders of silicon crystal. Using a Bruker D8 Advance theta-2 theta diffractometer with a position sensitive detector (PSD), LynxEye (Bruker AXS, Inc., Madison, WI, USA), the sample was irradiated with X-rays generated by a copper tube operated at 30 kV and 50 mA with a wavelength of 1.5406 Å. Automatic variable divergence slits were used. A step size of 0.02° with an integration time of 30 or 76 s was used. The analysis was repeated until crystalline peaks could be visible through the background noise of the water and was repeated on dry samples. Data Analyses. Supersaturation. Supersaturation, σ, in the jejunal samples was determined from the concentration of the drug in solution, C, and the solubility of the drug in the media, S.

20:80 (v/v) + 10 mM formic acid; (B) ACN + 10 mM formic acid. The quantification limit of the UV detection was 0.1 μM with injection volume 10 μL (noise × 10, RSD < 3%), and the quantification limit using MS was 0.003 μM with injection volume 5 μL (noise × 10, RSD < 10%). The concentration of mebendazole in blood plasma was determined with a HPLC−MS method. The proteins in the dog plasma samples were first precipitated by addition of cold acetonitrile containing an internal standard, and after centrifugation, the supernatant was diluted with 0.2% formic acid and an aliquot was separated on a Hypurity C18 column (2.1 × 50 mm, 5 μm; Thermo Electron Corp., Beverly, USA) A binary gradient was used to elute the substance, from 7% to 90% ACN in 0.2% formic acid from 0.2 to 2.2 min, and then held at 90% ACN for 1.5 min. Mebendazole and the internal standard verapamil were detected after electrospray ionization by mass spectrometry in MRM mode on a Micromass Quattro Micro instrument (Waters, Milford, MA, USA). The quantification limit of the method was 0.003 μM. In Vitro Studies To Determine Intestinal Precipitation. A simple one-step in vitro precipitation method was used in this study in order to use DIF as a medium in this in vitro intestinal model based on our previously reported method.1 Since DIF cannot be diluted without altering the resulting intestinal media, no formulation solution could be added. Instead, 102 μL of DMA stock solution was added directly to 10 mL of DIF, leading to similar DMA and drug concentrations as in the in vivo trials. The in vitro precipitation experiments were made in conical 25 mL flasks. The flask was placed in a water bath (Clifton shaking bath, model NE5-10D, Bennett Scientific Ltd., Newton Abbot, U.K.) at 37 °C and shaken at approximately 85 strokes/min with a one-way distance of 2 cm. In vitro precipitation in DIF was studied at three different drug concentrations where the initial concentrations of mebendazole in the intestinal fluid were 0.067, 0.17 and 0.44 mM, respectively. The span of concentrations was chosen to include a nonprecipitating concentration up to the expected intestinal concentration of mebendazole in the jejunal study at the higher dose. Additional experiments at 0.17 and 0.44 mM were also made in DIF without shaking the sample in order to investigate differences in precipitation rate due to hydrodynamic conditions. In these experiments the total volume was 5 mL. The pH in the test medium was maintained 5.4−5.7 for experiments with DIF. The pH of the dog jejunal fluid is usually considered to be higher,23 but the difference in pH was not expected to significantly change the solubility of mebendazole since virtually all substance is expected to be neutral above pH 5.4 (pKa = 3.5.) Samples of the fluid were collected after 2, 10, 20, 40, 60, 90 and 120 min with an eppendorf pipet. All precipitation experiments were performed in duplicate. Samples of DIF were immediately centrifuged at 2000 rcf at 37 °C for 10 min. The supernatant was diluted with organic solvent to ensure no further precipitation of mebendazole, and for protein precipitation. Samples were centrifuged at 2000 rcf at room temperature for 5 min to remove precipitated proteins, and the samples were analyzed with HPLC−UV as described in Assays of Mebendazole. Filtrating the undiluted DIF samples with a 0.2 μm hydrophilic PTFE filter (Dismic-13HP; Advantec, Dublin, CA, USA) or using a second centrifugation did not reduce the measured concentration in samples, indicating that all particles had sedimented in the initial centrifugation at 2000 rcf, and that further centrifugation did not induce precipitation.

σ=

C S

(1)

Pharmacokinetic Data Analysis. Pharmacokinetic calculations of the plasma concentration−time profile from the duodenal study were made using WinNonlin Professional 5.2.1 (Pharsight, St. Louis, MO, USA). A two-compartmental intravenous model was used for determination of the macro constants A, B, α and β, describing the intravenous plasma concentration−time curves for each dog: C = A e−ατ + B e−βτ

(2)

The disposition parameters were then used for determination of the bioavailability of mebendazole over time using numerical deconvolution25 of the plasma concentration−time data following the duodenal and jejunal single dose administrations. Individual data was used in the former case whereas average iv data was used in the latter case since iv administration was not given to the same dogs as included in the jejunal study. Noncompartmental calculations of AUCτ, the area under the plasma concentration−time curve up to the last plasma sampling point, was made by the log−linear trapezoidal rule, and together with the dose, bioavailability was determined, F=

AUCid dose iv AUCiv dose id

(3)

In Silico Simulation of Mebendazole Plasma Concentrations. Computer simulations of mebendazole plasma concentration−time profiles expected in the dog study with duodenal administration of solution were performed with the 2906

dx.doi.org/10.1021/mp300224h | Mol. Pharmaceutics 2012, 9, 2903−2911

Molecular Pharmaceutics

Article

ACAT model described by Agoram et al.26 in order to further estimate influence of intestinal drug precipitation on systemic drug absorption. The settings for the simulations are described in Table 2. Two different scenarios are simulated, one where no

Both values of Peff,dog were used in the in vivo simulation of dog absorption profile since the two models of human jejunal permeability predicted very different values of Peff,human.

■

RESULTS Mebendazole Polymorph Determination in Precipitates. The diffractograms of precipitates from in vitro precipitation experiments are shown in Figure 1. The

Table 2. Summary of Parameters Used in Computer Simulation of Absorption Time Profile of Mebendazole in Dog Following Duodenal Single Dose Administration parameters physiological intestinal radius (cm)a body weight (kg) stomach transit (min)b small intestinal transit (min)c small intestinal fluid volume (mL)c small intestinal pH colon absorption off physicochemical properties Mw (g/mol) density (g/cm3)d diffusion rate (×10−9 cm2/s)e S0 (μM) SDIF (μM) pharmacokinetic parameters Peff, low (×10−4 cm/s)f Peff, high (×10−4 cm/s)g plasma clearance (L/h) vol of central compartment (l) k12 (1/h) k21 (1/h) 1st pass extraction (%)h

1 30 0.01 109 118 6.2−6.75

295.3 1.3 0.75 5 24

Figure 1. X-ray powder diffractogram (XRPD) of mebendazole precipitated from dimethylacetamide (DMA) solution in dog intestinal fluid (DIF) and from simulated gastric fluid (SGF) solution in buffer (blank FaSSIF).

7.9 24.5 120 26.8 4.9 3.1 75

diffractograms for the precipitates in DIF and water both displayed the most significant peaks for identification of polymorph C (Figure 1).18 No conformational change was seen on dry samples. Solubility of Mebendazole and Bile Acid Content of DIF. The mean bile acid concentration in a pooled sample from the three dogs with jejunal stoma was 6.6 mM. Selected samples from the supernatant of individual jejunal samples in the dog in vivo precipitation studies showed an interval of bile acid concentrations ranging from 1.8 to 23 mM. The mean solubility (±SD) of mebendazole in DIF at 37 °C (24 ± 3 μM) appeared to be stable over a 20 h period. The solubility of mebendazole in selected individual samples from the dog in vivo jejunal precipitation study was in the range of 9 to 84 μM. pH of individual samples was not considered to have a significant effect on solubility. The mean solubility (±SD) of mebendazole in blank FaSSIF was 5.2 ± 0.2 μM. In Vivo Studies. Oral Administration with Jejunal and Plasma Drug Sampling. The concentration of mebendazole in supernatant of sampled DIF versus time after administration for the individual dogs in the jejunal study is presented in Figure 2. The results showed that the low dose created minimal supersaturation in the dog small intestine, and no intestinal precipitation was expected. At the higher dose, supernatant concentrations of 130−370 μM during the first hour indicated significant supersaturation in all three dogs. An additional determination of the solubility of mebendazole in a selected number of separate individual jejunal samples was made since bile acid content in the samples was highly variable. The highest supersaturation levels (σ) obtained in individual samples were approximately 10 in both dog U and dog G, and approximately 5 in dog R. The supersaturation levels detected clearly showed that there was a potential for intestinal precipitation.

a

Approximated dog intestinal radius from human intestinal radius and species weight difference.27 bShort transit due to simulation of duodenal administration. cAdapted from Gastroplus version 3.0. d Calculated from chemical formula.28 eCalculated from Mw.29 f Calculated from model 1a and eq 4.30 gCalculated from model 1b and eq 4.31 hEstimated from F in duodenal study.

precipitation of mebendazole was expected from the solution, and one where precipitation was instantaneous in the duodenum, leading to a suspension of mebendazole particles exemplified by a particle size of 1 μm. The residence time in the stomach was set to be infinitely small in the simulation, since the drug substance was administered directly to the duodenum. The concentration of bile acids, influencing the total solubility of drug in the intestinal fluid and the supersaturation of drug, was constant in the duodenum and jejunum. No colonic absorption was included in the simulation model applied. Pharmacokinetic parameters were estimated from the intravenous WinNonlin modeling described in Pharmacokinetic Data Analysis. The human effective jejunal permeability Peff,human were estimated from two computational models correlating polar surface area (PSA), log D and hydrogen bond donors (HBD) to human effective jejunal permeability (Ka or Peff,human).30,31 The two models with the best correlation to fraction absorbed in human were used here,32 and the result was then scaled to dog Peff by the following correlation (dog results unpublished results).33,34 Peff,dog = 3.52Peff,human − 0.116

(4) 2907

dx.doi.org/10.1021/mp300224h | Mol. Pharmaceutics 2012, 9, 2903−2911

Molecular Pharmaceutics

Article

Figure 2. Individual intestinal concentration in DIF over time in dog study with jejunal collection of fluid: (circle) low dose of mebendazole (concentration 52 μM in administered solution) (T1), no supersaturation expected (square) and (downward triangle) high dose of mebendazole (concentration 1.3 mM in administered solution) (T2), individual trials. Dog G was not available for the second administration of the high dose. The dark and light gray lines represent the fraction of the total bioavailabile amount over time in the respective trials, deconvoluted from mean iv plasma exposure curves in other dogs. Time dependent bioavailability in the trials was converted to the fraction of the total bioavailable amount over time.

absorbed from the stomach. Mean (SD) absolute bioavailability was 41 (±21) % after oral administration of the high dose. Duodenal Administration of Mebendazole. The bioavailability of mebendazole solution after administration of the high dose (T3) solution was 25% (±14%). The individual bioavailability of mebendazole was below 70%, but over 90% of that was absorbed within the first three hours (Figure 4).

The amount of mebendazole found in the centrifugation pellet from the DIF sample from midjejunum was variable, but showed that the drug precipitation was negligible after administration of the low dose, or that formed particles had redissolved in the upper jejunum (Figure 3). The measured

Figure 3. Individual percentage of dose collected from jejunum in solution (light gray) and as solid (black) drug in low (1) and high (2 + 3) dose.

Figure 4. Individual bioavailability of mebendazole at different times obtained by deconvolution from plasma concentrations of mebendazole in studies with intravenous and duodenal administration of drug solution (n = 8).

small amount of drug precipitated from the low dose was in part explained by drug dissolved in the residual fluid in the centrifuged pellet. At the higher dose, some solid drug was found in all dog intestinal samples, on average 11% of the total dose administered. About 70% of the fluid administered was recovered through the intestinal fistula in both the low and high dose experiments. The fraction of the total bioavailable amount of mebendazole over time was assessed from plasma drug concentrations following the higher dose (T2) by using the intravenous dose as a reference (Figure 2). Plasma drug concentrations at the lower dose were below the detection limit of the drug assay. One interesting observation at the high oral dose was the coincidence of onset of drug absorption and appearance of fluid at midjejunum for dog G. This implies that the reason for the late sampling of jejunal fluid and lag time prior to onset of drug absorption is most likely explained by delayed gastric emptying since practically nothing is absorbed prior to the first sampling event. This experimentally demonstrates that basic drug is not

The mean bioavailability and plasma concentration−time profile from the dog in vivo study with administration of solution directly into the duodenum (T3) was simulated using two different scenarios. Administration of a nonprecipitating solution and administration of a suspension with 1 μm mebendazole particles were simulated and are shown in Figure 5 together with the average in vivo plasma concentration curve. The absorption time profile simulation of the nonprecipitating solution was a reasonably good model for experimental data indicating that precipitation did not have any major effect on the extent of in vivo drug absorption. However, the slower absorption rate in the in vivo study compared to the prediction during the second half of the absorption process may be attributed to redissolution of precipitated drug. In Vitro Intestinal Precipitation Model. The in vitro precipitation rate in DIF was extremely fast, regardless of level 2908

dx.doi.org/10.1021/mp300224h | Mol. Pharmaceutics 2012, 9, 2903−2911

Molecular Pharmaceutics

Article

This was also supported by solid state drug analysis of precipitates from in vitro experiments with supersaturated concentrations of mebendazole in DIF where solid mebendazole was detected in crystalline form as polymorph C. In addition, this in vitro precipitation was happening almost instantly at drug concentrations corresponding to the expected levels in the initial small intestine in the in vivo experiments. Drug supersaturation was detected in the jejunal samples following oral administration from all dogs at up to 5−10 times higher drug luminal concentrations than the equilibrium solubility. In general, supersaturation was measurable in midjejunum up to approximately 90 min (Figure 2). The jejunal fluid after oral administration of a low dose contained 1.8−4.0% of the total dose as solid drug whereas at the high dose more solid mebendazole was detected (3−28% of total dose). The high level of supersaturation that could be maintained in the proximal small intestine and the limited amount of drug precipitation is surprising considering the almost immediate precipitation obtained in DIF at similar concentrations in the in vitro tests. Similar findings of relatively small amounts of drug precipitation were obtained in a recent in vivo study by Psachoulias et al. of administration of solutions of two basic BCS II substances, dipyridamole and ketoconazole, to the antrum of the subjects’ stomach. The solutions of the highest doses were expected to provide approximately 5 and 17 times supersaturation, respectively, upon entry into the small intestines, given the volume administered and the median value of drug substance solubility in intestinal fluid. The detected precipitation in that study was less than 7% for dipyridamole and less than 16% for ketoconazole.2 The limited influence of intestinal precipitation on intestinal drug absorption was also supported by the deconvolution analysis of the plasma concentrations of mebendazole over time (i.e., bioavailability) obtained after administration of a solution directly to duodenum (Figure 5). Supersaturation levels in the proximal small intestine of about 40 times were expected in this experiment, still the bioavailability following intestinal administration agreed with the estimated bioavailable fraction vs time profile expected from administration of a nonprecipitating solution. This result is comparable to our previous study in human, where intestinal precipitation of a BCS II substance was expected based on in vitro experiments, but no significant effects were seen in vivo on rate and extent of absorption of that BCS class II drug either.1 A common property of these two compounds (AZD0865 and mebendazole) and the two compounds dipyridamole and ketoconazole investigated by Psachoulias et al. is the ability to form highly supersaturated solutions, originated from the large difference in amorphous and crystalline solubility. This can be estimated from differential scanning calorimetry data (DSC) as a function of the melting temperature and enthalpy of melting. The relatively high melting points (dipyridamole 168 °C,35 ketoconazole 150 °C36 and AZD0865 246 °C1) and high enthalpy of melting (dipyridamole 45 kJ/mol,35 ketoconazole 55 kJ/mol,36 AZD0865 54 kJ/mol1) indicate that strong molecular bonds form in the crystals. Thus, even for drugs with very favorable crystallization properties such as ability to form supersaturated solutions and strong intermolecular crystal forces, significant supersaturation can be maintained in the small intestine. This leads to limited influence of crystalline precipitation on small intestinal absorption for BCS class II basic drugs with high melting point and enthalpy of melting once particles are fully dissolved.

Figure 5. Mean bioavailability of mebendazole vs time: (●) from experimental data of the duodenal administration of a solution (n = 8); () from simulation of a nonprecipitating solution administered to duodenum, Peff = 24.5; (− − −) from simulation of a nonprecipitating solution, Peff = 7.9; (−·−·−) from simulation of a suspension administered to duodenum, Peff = 24.5; (- - - - - -) from simulation of a suspension, Peff = 7.9.

of supersaturation, which is illustrated in Figure 6 where the supersaturation levels at different times are shown. There was

Figure 6. Mean (±SD) supersaturation (σ) over time of drug dissolved in DIF after testing three different concentrations with initial supersaturation of mebendazole in DIF of (circle) 2.4, (square) 7.2, (tilted square) 18.9. Filled markers represent stirred system, and open markers represent unstirred system.

no reduction in precipitation rate when the system was not stirred. The highest supersaturation levels in vivo, approximately 10 according to the jejunal study, could not be duplicated in vitro, since the highest measured supersaturation in vitro in DIF was 4.2.

■

DISCUSSION Intestinal supersaturation and precipitation of mebendazole, a basic BCS class II drug, were investigated in vivo in dogs with jejunal or duodenal stomas (T1−T3). Drug solutions, expected to achieve jejunal supersaturation of up to approximately 20 times the solubility, were administered either orally combined with sampling from the jejunum (T1, T2) or directly into the duodenum through an intestinal stoma (T3). Since the apparent amorphous solubility was estimated to be much higher than the initial intestinal lumen concentrations, any precipitation was expected to provide crystalline drug form. 2909

dx.doi.org/10.1021/mp300224h | Mol. Pharmaceutics 2012, 9, 2903−2911

Molecular Pharmaceutics

Article

Notes

However, for drugs with poor small intestinal permeability, e.g., BCS class IV drugs, supersaturation levels can be maintained for a longer period due to the slower removal of dissolved drug from the intestinal lumen, and therefore the risk that intestinal crystallization might affect both rate and extent of intestinal absorption is increasing. Another situation that provides more favorable conditions for precipitation than in the current study is when the drug is administered as a solid and is incompletely dissolved when emptied into the small intestine. In this case, if supersaturation has been reached, nucleation is not required as a prerequisite for precipitation but crystal growth on remaining particles will be an immediate process following crystal growth kinetics, as recently indicated for the BCS class IV drug nelfinavir.6 The precipitation rate would also be affected by the lowered supersaturation levels when drug particles are undissolved, but it has been shown for the model drug bicalutamide that the rate of particle growth of undissolved particles is much faster than nucleation of particles from a solution.37 There is thus still a need to develop more quantitative predictive models for the influence of intestinal precipitation to be used in a more rational future pharmaceutical development. The novel data in this report has the potential to provide a basis for a successful development and validation of such methods. More in vivo data on precipitation especially for compounds where nucleation plays a clear role for drug absorption would be desirable to further strengthen the basis for development of predictive methods. A remaining question to resolve is to identify the critical factors in the design of an in vitro test to capture critical characteristics for nucleation and crystal growth. One possible factor could be the hydrodynamic conditions where previous studies have shown differences of several hours in maintaining supersaturation dependent on intensity of stirring.38 However, the results from the current nonstirred in vitro experiments (Figure 6) showed no significant difference from the stirred results, despite the fact that stirring is known to increase the crystallization rate substantially.39 The reason for this could be due to specific substance characteristics (possible indifference to stirring), but it could also be a sensitivity to the manner of initial mixing of the two phases creating extreme local supersaturation with subsequent instant precipitation not being representative of the in vivo situation when the drug enters the small intestine.

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We would like to thank Anders Carlsson for technical support with analysis of bile acids and other analysis issues. We would also like to thank David Elmqvist for assistance with XRPD measurements and Petra Delavaux and Helena Douglasson for assistance with in vivo studies.

■

(1) Carlert, S.; Palsson, A.; Hanisch, G.; Von Corswant, C.; Nilsson, C.; Lindfors, L.; Lennernas, H.; Abrahamsson, B. Predicting intestinal precipitation-A case example for a basic BCS class II drug. Pharm. Res. 2010, 27, 2119−2130. (2) Psachoulias, D.; Vertzoni, M.; Goumas, K.; Kalioras, V.; Beato, S.; Butler, J.; Reppas, C. Precipitation in and supersaturation of contents of the upper small intestine after administration of two weak bases to fasted adults. Pharm. Res. 2011, 28, 3145−3158. (3) Bergman, E.; Forsell, P.; Persson, E. M.; Knutson, L.; Dickinson, P.; Smith, R.; Swaisland, H.; Farmer, M. R.; Cantarini, M. V.; Lennernas, H. Pharmacokinetics of gefitinib in humans: The influence of gastrointestinal factors. Int. J. Pharm. 2007, 341, 134−142. (4) Takano, R.; Takata, N.; Saito, R.; Furumoto, K.; Higo, S.; Hayashi, Y.; MacHida, M.; Aso, Y.; Yamashita, S. Quantitative analysis of the effect of supersaturation on in vivo drug absorption. Mol. Pharmaceutics 2010, 7, 1431−1440. (5) Sugano, K. Computational oral absorption simulation of free base drugs. Int. J. Pharm. 2010, 398, 73−82. (6) Shono, Y.; Jantratid, E.; Dressman, J. B. Precipitation in the small intestine may play a more important role in the in vivo performance of poorly soluble weak bases in the fasted state: Case example nelfinavir. Eur. J. Pharm. Biopharm. 2011, 79, 349−356. (7) Diaz, D.; Bernad Bernad, M. J.; Gracia-Mora, J.; Escobar Llanos, C. M. Solubility, 1H-NMR, and molecular mechanics of mebendazole with different cyclodextrins. Drug Dev. Ind. Pharm. 1999, 25, 111−115. (8) Nielsen, L. S.; Slok, F.; Bundgaard, H. N-alkoxycarbonyl prodrugs of mebendazole with increased water solubility. Int. J. Pharm. 1994, 102, 231−239. (9) Charoenlarp, P.; Waikagul, J.; Muennoo, C.; Srinophakun, S.; Kitayaporn, D. Efficacy of single-dose mebendazole, polymorphic forms A and C, in the treatment of hookworm and Trichuris infections. Southeast Asian J. Trop. Med. Public Health 1993, 24, 712− 716. (10) Dawson, M.; Braithwaite, P. A.; Roberts, M. S.; Watson, T. R. The pharmacokinetics and bioavailability of a tracer dose of [3H]mebendazole in man. Br. J. Clin. Pharmacol. 1985, 19, 79−86. (11) Dawson, M.; Watson, T. R. The effect of dose form on the bioavailability of mebendazole in man. Br. J. Clin. Pharmacol. 1985, 19, 87−90. (12) Chiba, Y.; Kohri, N.; Iseki, K.; Miyazaki, K. Improvement of dissolution and bioavailability for mebendazole, an agent for human echinococcosis, by preparing solid dispersion with polyethylene glycol. Chem. Pharm. Bull. 1991, 39, 2158−2160. (13) Brusau, E. V.; Cami, G. E.; Narda, G. E.; Cuffini, S.; Ayala, A. P.; Ellena, J. Synthesis and characterization of a new mebendazole salt: Mebendazole hydrochloride. J. Pharm. Sci. 2008, 97, 542−552. (14) Daniel-Mwambete, K.; Torrado, S.; Cuesta-Bandera, C.; PonceGordo, F.; Torrado, J. J. The effect of solubilization on the oral bioavailability of three benzimidazole carbamate drugs. Int. J. Pharm. 2004, 272, 29−36. (15) Nielsen, L. S. Improved peroral bioavailability of mebendazole in rabbits by administration of various N-alkoxycarbonyl derivatives of mebendazole. Int. J. Pharm. 1994, 104, 175−179. (16) Lindfors, L.; Forssen, S.; Skantze, P.; Skantze, U.; Zackrisson, A.; Olsson, U. Amorphous drug nanosuspensions. 2. Experimental determination of bulk monomer concentrations. Langmuir 2006, 22, 911−916.

■

CONCLUSIONS In conclusion, emerging data indicates that supersaturation and precipitation have a limited effect on small intestinal absorption of BCS class II basic drugs being completely dissolved in the stomach prior to entry into the small intestine. This is very much in contrast to estimates from traditional in vitro and physicochemical models, which predict significant precipitation. Thus, there is a remaining need to develop more predictive methods not exaggerating influence of precipitation and to relate these solid state properties in the context of small intestinal permeability. This would also benefit from additional mechanistic in vivo data for drugs with even more challenging properties with respect to precipitation.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Department of Global Medicines Development, AstraZeneca R&D, S-431 83 Mölndal, Sweden. Phone: +46317761262. Fax: +46317763817. E-mail: [email protected]. 2910

dx.doi.org/10.1021/mp300224h | Mol. Pharmaceutics 2012, 9, 2903−2911

Molecular Pharmaceutics

Article

(17) De Villiers, M. M.; Terblanche, R. J.; Liebenberg, W.; Swanepoel, E.; Dekker, T. G.; Song, M. Variable-temperature X-ray powder diffraction analysis of the crystal transformation of the pharmaceutically preferred polymorph C of mebendazole. J. Pharm. Biomed. Anal. 2005, 38, 435−441. (18) Costa, J.; Fresno, M.; Guzman, L.; Igual, A.; Oliva, J.; Vidal, P.; Perez, A.; Pujol, M. Polymorphic forms of mebendazole: Analytical aspects and toxicity. Cir. Farm. 1991, 49, 415−424. (19) Liebenberg, W.; Dekker, T. G.; Lotter, A. P.; De Villiers, M. M. Identification of the mebendazole polymorphic form present in raw materials and tablets available in South Africa. Drug Dev. Ind. Pharm. 1998, 24, 485−488. (20) The United States Pharmacopeia (USP 32); United States Pharmacopeial Convention, Inc.: Rockville, 2008. (21) Persson, E. M.; Nilsson, R. G.; Hansson, G. I.; Lofgren, L. J.; Liback, F.; Knutson, L.; Abrahamsson, B.; Lennernas, H. A clinical single-pass perfusion investigation of the dynamic in vivo secretory response to a dietary meal in human proximal small intestine. Pharm. Res. 2006, 23, 742−751. (22) Polentarutti, B.; Albery, T.; Dressman, J.; Abrahamsson, B. Modification of gastric pH in the fasted dog. J. Pharm. Pharmacol. 2010, 62, 462−469. (23) Kalantzi, L.; Persson, E.; Polentarutti, B.; Abrahamsson, B.; Reppas, C. Canine intestinal contents vs. simulated media for the assessment of solubility of two weak bases in the human small intestinal contents. Pharm. Res. 2006, 23, 1373−1381. (24) Kitaigorodsky, A. I. Molecular Crystals and Molecules; Academic Press: New York, 1973. (25) Langenbucher, F. Numerical convolution/deconvolution as a tool for correlating in vitro with in vivo drug availability. Pharm. Ind. 1982, 44, 1166−1172. (26) Agoram, B.; Woltosz, W. S.; Bolger, M. B. Predicting the impact of physiological and biochemical processes on oral drug bioavailability. Adv. Drug Delivery Rev. 2001, 50, S41−S67. (27) Knutson, T.; Fridblom, P.; Ahlstrom, H.; Magnusson, A.; Tannergren, C.; Lennernas, H. Increased Understanding of Intestinal Drug Permeability Determined by the LOC-I-GUT Approach Using Multislice Computed Tomography. Mol. Pharmaceutics 2009, 6, 2−10. (28) Cao, X.; Leyva, N.; Anderson, S. R.; Hancock, B. C. Use of prediction methods to estimate true density of active pharmaceutical ingredients. Int. J. Pharm. 2008, 355, 231−237. (29) Avdeef, A. Leakiness and Size Exclusion of Paracellular Channels in Cultured Epithelial Cell Monolayers-Interlaboratory Comparison. Pharm. Res. 2010, 27, 480−489. (30) Linnankoski, J.; Makela, J. M.; Ranta, V.; Urtti, A.; Yliperttula, M. Computational prediction of oral drug absorption based on absorption rate constants in humans. J. Med. Chem. 2006, 49, 3674− 3681. (31) Winiwarter, S.; Bonham, N. M.; Ax, F.; Hallberg, A.; Lennernas, H.; Karlen, A. Correlation of human jejunal permeability (in vivo) of drugs with experimentally and theoretically derived parameters: A multivariate data analysis approach. J. Med. Chem. 1998, 41, 4939− 4949. (32) Linnankoski, J.; Ranta, V.; Yliperttula, M.; Urtti, A. Passive oral drug absorption can be predicted more reliably by experimental than computational models - Fact or myth. Eur. J. Pharm. Sci. 2008, 34, 129−139. (33) Personal communication: Amidon, G. L. Yamashita, S. Dog Peff results for a number of pharmaceutical drugs. (34) Sun, D.; Lennernas, H.; Welage, L. S.; Barnett, J. L.; Landowski, C. P.; Foster, D.; Fleisher, D.; Lee, K.-.; Amidon, G. L. Comparison of human duodenum and Caco-2 gene expression profiles for 12,000 gene sequences tags and correlation with permeability of 26 drugs. Pharm. Res. 2002, 19, 1400−1416. (35) Adhiyaman, R.; Basu, S. K. Crystal modification of dipyridamole using different solvents and crystallization conditions. Int. J. Pharm. 2006, 321, 27−34. (36) Viseras, C.; Ismail Salem, I.; Rodriguez Galan, I. C.; Cerezo Galan, A.; López Galindo, A. The effect of recrystallization on the

crystal growth, melting point and solubility of ketoconazole. Thermochim. Acta 1995, 268, 143−151. (37) Lindfors, L.; Forssen, S.; Westergren, J.; Olsson, U. Nucleation and crystal growth in supersaturated solutions of a model drug. J. Colloid Interface Sci. 2008, 325, 404−413. (38) Veintemillas-Verdaguer, S.; Osuna Esteban, S.; Herrero, M. A. The effect of stirring on sodium chlorate crystallization under symmetry breaking conditions. J. Cryst. Growth 2007, 303, 562−567. (39) Myerson, A. S.; Ginde, R. Crystals, Crystal Growth, and Nucleation; In Handbook of Industrial Crystallization, 2nd ed.; Myerson, E. S., Ed.; Butterworth-Heinemann: Woburn, 2002; pp 52−53.

2911

dx.doi.org/10.1021/mp300224h | Mol. Pharmaceutics 2012, 9, 2903−2911