Investigation of the Rat Model for Preclinical Evaluation of pH

Aug 20, 2013 - Joseph W. Lubach,* Jacob Z. Chen, Jonathan Hau, Jose Imperio, Melis Coraggio, Lichuan Liu, and Harvey Wong. Genentech, Inc., 1 DNA Way ...
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Investigation of the Rat Model for Preclinical Evaluation of pHDependent Oral Absorption in Humans Joseph W. Lubach,* Jacob Z. Chen, Jonathan Hau, Jose Imperio, Melis Coraggio, Lichuan Liu, and Harvey Wong Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States ABSTRACT: Many pharmaceutically active compounds are weak electrolytes and are ionizable in the pH range experienced throughout the gastrointestinal tract. Changes in protonation state due to pH changes in the gut can have dramatic effects on solubility, dissolution, and permeation through biological barriers. Preclinical assessment of the pHdependence of oral absorption is critical for compounds possessing pH-dependent solubility. Here we examine pHdependent solubility and oral exposure in rat for three model compounds, dasatinib, ketoconazole, and mefenamic acid. Dasatinib and ketoconazole are both weak bases, while mefenamic acid is a carboxylic acid. The effects of gastric pH modulators, pentagastrin and famotidine, were investigated in rat PK studies to assess the applicability of using the rat to evaluate the risk of pH-dependent oral exposure for ionizable compounds. Dasatinib showed similar exposure between control and pentagastrin-pretreated groups, and 4.5-fold lower AUC in famotidine-pretreated rats. Ketoconazole showed a 2-fold increase in AUC in pentagastrin-treated rats relative to control, and 4.5-fold lower AUC in famotidine treated rats, relative to the pentagastrin group. Mefenamic acid showed highly similar exposures among control, pentagastrin-pretreated, and famotidine-pretreated groups. The rat model was shown to be useful for compounds displaying pH-dependent solubility and oral absorption that may be affected by gastric pH modulators. KEYWORDS: pH-dependent solubility, ketoconazole, dasatinib, mefenamic acid, oral exposure, bioavailability, gastrointestinal pH

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INTRODUCTION Many commercial and investigational medicines contain ionizable functional groups and display pH-dependent solubility, thus raising the possibility of pH-dependent exposure following an oral dose.1−7 The oral absorption of ionizable compounds is dependent on factors such as the acid dissociation constant (pKa), intrinsic solubility, permeability, dose, prandial state, disease state, and comedications. Weakly basic compounds typically have good aqueous solubility at low pH (pH ≪ pKa), while weak acids are more water-soluble at higher pH (pH ≫ pKa). However, the human gastrointestinal (GI) tract is inherently a dynamic environment, and drugs experience a wide range of pH conditions following oral dosing. Compounds first enter the acidic environment of the stomach, with a typical pH of 1−2, and then proceed through the intestinal tract, where the pH generally ranges from 5 to 7.8,9 These pH values can vary significantly from subject to subject and from day to day within a subject, and also vary greatly depending on if the subject is fed or fasted.10−16 Patients may also be taking pH-modifying medications such as proton pump inhibitors (PPI), histamine H2-receptor antagonists, or simple antacids, which can reduce exposure of basic compounds or increase exposure of acidic compounds.17−21 For all these reasons it is generally of great interest to gauge the effects and risk of pH-dependent absorption as early as possible in drug discovery and development. © 2013 American Chemical Society

The tetrapeptide pentagastrin is well-known to stimulate acid secretion in mammals and is widely used in the pharmaceutical industry to ensure acidity and reduce intersubject variability in canine pharmacokinetic (PK) studies.22−27 The pentagastrintreated dog is a common model for investigating human dissolution and absorption in the pharmaceutical industry. A key study by Zhou and co-workers sought to develop this canine model to study pH-dependent absorption of weakly the basic compounds ketoconazole and dipyridamole.24 The authors used in vitro dissolution experiments in conjunction with in vivo studies using pentagastrin-pretreated and famotidine-pretreated dogs. In these studies, dogs treated with pentagastrin had gastric pH values of 2−3, while H2 antagonist famotidine pretreatment resulted in gastric pH levels of 5−7.5. Ketoconazole, a weak base, showed a 4-fold increase in AUC with pentagastrin pretreatment compared to the control group, and AUC decreased 7-fold following famotidine pretreatment. Dipyridamole also displayed marked pH-dependSpecial Issue: Impact of Physical Chemical Drug-Drug Interactions from Drug Discovery to Clinic Received: Revised: Accepted: Published: 3997

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secretion and reduce potential interanimal variation, whereas the H2 blocker famotidine was used to reduce acid secretion. Gastric pH in rat was measured in fasted control rats as well as in pentagastrin and famotidine pretreated animals to characterize the time course of effects on stomach pH (data partially presented as an abstract at the 2006 AAPS National Meeting).40 Pharmacokinetic studies were then conducted for each model compound under different gastric pH conditions (partially presented as an abstract at the 2011 AAPS National Meeting).41 Using rat as a model for pH-dependent absorption may help move these studies earlier in development, as rat studies require far less compound than canine studies, and are generally much less expensive. This rat model could serve to derisk or confirm pH-dependent absorption and potentially aid decision-making as to which compounds to move forward among a series of analogous candidate molecules.

ent exposure in this study, with a 4.5-fold increase in AUC following pentagastrin pretreatment, and a 2-fold decrease in AUC with famotidine pretreatment. In a more recent study, Fancher and co-workers further examined the canine model for studies on pH-dependent absorption.27 They too used both pentagastrin and famotidine pretreatments to use the canine model preclinically to aid in candidate selection and differentiation, and to help assess various formulation and/or prodrug strategies. Little work has been reported on evaluating rats as a preclinical model for pH-dependent absorption studies. However, numerous reports exist on pentagastrin effects in rat, and a number of other studies have investigated food and GI tract pH effects using rats.28−33 Synnerstad, Johansson, and co-workers examined the mechanism and effects of acid secretion on gastric mucosa integrity, as well as the importance of endogenous bicarbonate as a protective buffering agent.34,35 Chu and co-workers have investigated the effects of pentagastrin and the PPI omeprazole on acid secretion rates in the rat stomach mucosa, confirming that pentagastrin enhances surface acidity while omeprazole effectively eliminated surface acidity.36 In another study, omeprazole was found not to impact bioavailability of vinpocetine, although food strongly enhanced the absorption of this drug.37 Sherwood et al. investigated the effects of acid secretion on gastric antibiotic transfer, and found that metronidazole transfer increased with acid secretion but declined with omeprazole present, with similar effects observed for clarithromycin.38 In a study looking to control gastric acid secretion in rat, Decktor and co-workers observed the effects of histamine H2 antagonists ranitidine and famotidine, as well as the PPI omeprazole.39 The authors found that each acid secretion inhibitor resulted in hypergastrinemia, with omeprazole showing the most prolonged response. Here we aim to investigate the use of rats as an in vivo model for pH effects on oral absorption. Two basic compounds, dasatinib and ketoconazole, and one acidic compound, mefenamic acid, were used as tools for these studies (Figure 1). Pentagastrin was used as pretreatment to induce gastric acid

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EXPERIMENTAL SECTION Materials. Male Sprague−Dawley rats (300−400 g) were ordered from Charles River, Hollister, CA. Pentagastrin, famotidine, mefenamic acid (MFA), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO). Ketoconazole (KET) was obtained from Enzo Life Sciences, Inc. (Farmingdale, NY). Dasatinib (DAS) free base was purchased from LC Laboratories (Woburn, MA), and dasatinib 100 mg tablets were manufactured by Bristol-Myers Squibb (New York, NY). Size 9 hard gelatin capsules were purchased from Capsugel (Greenwood, SC). Acetic acid was purchased from Mallinckrodt Chemicals (St. Louis, MO); phosphoric acid and sodium borate decahydrate were purchased from J.T. Baker (Phillipsburg, NJ); sodium acetate trihydrate and acetonitrile (ACN) were purchased from EMD Chemicals (Gibbstown, NJ); and boric acid, dibasic sodium phosphate, and 0.1 N HCl were purchased from Acros Organics (Geel, Belgium). Aqueous buffers for solubility measurements contained 20 mM each phosphate, acetate, and borate. pH−Solubility Profiles. Excess DAS, KET, and MFA powder was added separately to 2 mL aliquots of aqueous phosphate/acetate/borate buffers (20 mM each) with pH values ranging from 2 to 9. Crystal forms of each compound were assessed with powder X-ray diffraction and Raman spectroscopy to ensure that the commercialized crystal forms were used (data not shown). Samples were allowed to equilibrate at 37 °C with continuous stirring for 96 h. Following the incubation period, 1 mL of each sample was transferred to a polypropylene centrifuge tube and centrifuged at 13000 rpm for 10 min. pH measurements were recorded using a Thermo Orion 4 Star pH meter (Thermo Scientific, Waltham, MA). The supernatant was then sampled and diluted appropriately using 10/45/45 DMSO/ACN/H2O, followed by HPLC analysis (Agilent 1100 series, Santa Clara, CA) to determine concentrations. Rat Stomach pH Assessment. Rats were fasted overnight for ∼16 h. Pretreatment groups were administered either pentagastrin (0.25 mg/kg sc) or famotidine (10 mg/kg iv) to modulate gastric acidity. After pretreatment, the anesthetized rat (ketamine 60−80 mg/kg +10−15 mg/kg xylazine ip cocktail) was placed on its back with its tail toward the investigator. The abdominal cavity was opened with a V-cut made through the skin and abdominal wall starting at the base of the abdomen and proceeding diagonally across on each side to end up at the dorsolateral edges of the thorax. The flap of skin was moved onto the chest wall and the stomach was

Figure 1. Chemical structures of model compounds with their corresponding pKa values. 3998

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Table 1. Time Course of Gastric pH in Rats Following Pentagastrin or Famotidine Pretreatmenta post-treatment time (h) treatment group b

control pentagastrin famotidine a

1

2

3

6

1.9 ± 0.9 (n = 12) 1.9 ± 0.3 (n = 8) 3.9 ± 0.7 (n = 7)

2.4 ± 0.8 (n = 4) 4.7 ± 1.0 (n = 4)

1.3 ± 0.1 (n = 5) 5.2 ± 0.8 (n = 6)

2.9 ± 0.5 (n = 6) 5.0 ± 0.5 (n = 6)

Values presented are mean ± SEM. bStomach pH in fasted rats assumed to be relatively constant; time course was not studied.

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RESULTS AND DISCUSSION Gastric pH Measurements in Rat. Table 1 shows the results of gastric pH measurements in fasted male Sprague− Dawley rats in control, pentagastrin-pretreated, and famotidinepretreated groups. The control group confirmed that fasted rats have highly acidic gastric juice, with a pH around 2. Pentagastrin pretreatment did not show a marked impact on gastric pH, presumably because the fasted state pH was already quite low. As expected, famotidine pretreatment showed a sustained elevation of gastric pH to 5.0 ± 0.5 out to 6 h postdose. This indicates that compounds exhibiting pHdependent solubility in this pH range may have significantly impacted oral absorption if histamine H2 antagonists like famotidine are coadministered to reduce acidity in the stomach. pH-Dependent Solubility of Dasatinib and Oral Exposure in Rat. Dasatinib is a weak base and is wellknown to exhibit pH-dependent solubility.4,43−45 Figure 2

located. An incision on the stomach wall was made, and stomach pH was measured with a Thermo Orion 525 A+ pH meter (Thermo Scientific, Waltham, MA). Rats were sacrificed (ketamine/xylazine, 0.2−0.5 mL iv) for gastric pH measurements at 1, 2, 3, and 6 h postdose of pentagastrin or famotidine. Gastric fluid pH was measured immediately following anesthesia to minimize the possibility of the anesthetic treatment perturbing the pH of gastric fluid. Formulations. Pretreatments of pentagastrin and famotidine were formulated in 10% PEG 400 in normal saline and 50 mM TRIS, pH 6.4, respectively. Dasatinib tablets were ground in a mortar and pestle and subsequently filled into size 9 capsules containing 3.4 ± 0.1 mg of dasatinib. Neat ketoconazole was filled directly into size 9 capsules containing 14 ± 0.2 mg each. Neat mefenamic acid was filled directly into size 9 capsules containing 17 ± 0.2 mg each. Rat doses were chosen based on the relevant human doses normalized to differences in gastric fluid volume, considering an average rat gastric fluid volume of 3.4 mL, compared to human at 50 mL.42 This approach was taken so as to achieve similar concentrations in gastric fluid between human and rat. Pharmacokinetic Studies. For each model drug, DAS, KET, and MFA, rats were divided into three groups of five precannulated rats per group (Charles River Laboratories, Wilmington, MA). The control group was treated only with model drug (DAS, KET, or MFA) dosed orally in a single size 9 capsule. The other two groups were pretreated with either pentagastrin (0.25 mg/kg sc) or famotidine (10 mg/kg iv via a femoral vein cannula) 2 h prior to administration of the capsules. Animals were fasted ∼16 h prior to po dosing and kept in a single house after surgery. All rats were fasted ∼16 h prior to po capsule dosing, were fed 4 h after dosing, and were euthanized 24 h after po dosing. Blood samples (0.2 mL) were collected via jugular vein cannula (or tail vein in the event of cannula failure) at 0.033, 0.083, 0.25, 1, 2, 4, 6, 8, and 24 h. Blood sample volume was replaced with equal volume of 0.9% saline. Blood samples (0.2 mL each) were placed in Becton Dickinson (Franklin Lakes, NJ) EDTA Microtainer tubes. Tubes were inverted several times to disperse the anticoagulant and then kept chilled on ice until centrifugation within 30 min of collection. Whole blood was centrifuged at 11000 rpm for 5 min at 4 °C using a 30-well fixed-angle rotor on a 580R Eppendorf Brinkman centrifuge (Hamburg, Germany). Plasma was harvested and transferred to a 1.2 mL polypropylene Costar tube (96 well format) and kept frozen on dry ice container until all samples were collected. All plasma samples then were stored at −70 °C (±10 °C) until thawed for LC/MS/MS analysis. Animals were euthanized with ketamine/xylazine cocktail, 0.2−0.5 mL iv after completion of blood sample collection. Plasma concentrations were quantified using LC/MS/MS. Pharmacokinetic parameters for ketoconazole were determined by noncompartmental methods using WinNonlin software (Pharsight, St. Louis, MO).

Figure 2. pH−solubility profile of dasatinib in aqueous buffer at 37 °C.

shows the pH−solubility curve of DAS measured at 37 °C, in the pH range most relevant to oral absorption. The structure of DAS contains two basic nitrogen atoms with pKa values of 6.8 and 3.1 (Figure 1). The nitrogen with a pKa of 6.8 is the most physiologically relevant, as this site considerably modulates DAS solubility as its protonation state changes in the pH environments experienced throughout the GI tract. This nitrogen becomes fully protonated at pH conditions ∼2 log units below the pKa, or around pH 4.8. The nitrogen pKa of 3.1 also adds solubility at very acidic conditions experienced in a normal fasting stomach (pH 1−2), as it is fully protonated around pH 1.1. DAS solubility was measured to be 49.6 mg/mL in 0.1 N HCl (pH ∼1), where both basic nitrogen atoms are expected to be protonated. Solubility drops drastically as pH increases to about 5, where the extremely low intrinsic solubility of neutral DAS begins to dominate the overall solubility. Above pH 6.5 DAS was undetectable in these studies. 3999

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Table 2. Pharmacokinetic Parameters of Dasatinib in Rat Following a Single 3.4 mg Po Dose with or without Pentagastrin or Famotidine Pretreatment (n = 5/Group)

a

group

pretreatment

dasatinib dose (mg/rat)

Cmax (μg/mL)

AUC0−∞ (μg·h/mL)

1 2

none pentagastrin

3.4 3.4

0.133 ± 0.097 0.076 ± 0.065

0.421 ± 0.266 0.297 ± 0.220

3

famotidine

3.4

0.024 ± 0.017

0.094 ± 0.066

rel changea Cmax ↓43% AUC0−∞ ↓29% Cmax ↓82% AUC0−∞ ↓78%

Percent change relative to control group 1.

conclude that DAS oral exposure in rat relies on dissolution at low stomach pH values with subsequent intestinal absorption. This agrees well with the clinical observation that DAS exposure is reduced by 61% (AUC) when famotidine is administered 10 h prior to DAS dosing4 (Table 5). Dosing famotidine at least 2 h after the DAS dose can mitigate this interaction. pH-Dependent Solubility of Ketoconazole and Oral Exposure in Rat. Ketoconazole, like DAS, is dibasic and has similar pKa values of 6.5 and 2.9 (Figure 1). Not surprisingly, it also displays a pH−solubility profile similar to DAS (Figure 4),

The high solubility at low pH indicates that DAS is likely completely dissolved in the stomach, provided that gastric pH is below ∼4. However, as the drug moves through the intestinal tract and pH increases to 5−7, concentrations in the gut can rapidly become supersaturated and precipitation is possible. For this reason, compounds with basic pKa values similar to DAS are at a high risk of intestinal precipitation, which can reduce or prevent systemic absorption. However, with adequate epithelial permeability, absorption may occur before the compound precipitates. Due to inter- and intrasubject variability in physiologic conditions, this can sometimes lead to undesirable PK variability. Whenever possible, precautions should be taken to reduce the chance of precipitation and control variability through the dosage form, prandial state, and consideration of comedications. Table 2 and Figure 3 show the results of a DAS rat PK study conducted on three groups using either no pretreatment,

Figure 4. pH−solubility profile of ketoconazole in aqueous buffer at 37 °C.

characteristic of a weakly basic molecule. KET has excellent solubility in 0.1 N HCl (37.4 mg/mL), though this rapidly drops down to 7 to show an increase in MFA dissolution and exposure. Famotidine only serves to raise rat gastric pH to around 5 (Table 1) and, thus, should not affect MFA dissolution and exposure, which is indeed what we observed in these studies. In human studies reported by Neuvonen and co-workers, magnesium hydroxide administration resulted in an earlier Tmax and 2−3-fold greater Cmax and a marginal 1.4-fold increase in AUC.5,17 This likely indicates that the mineral antacid magnesium hydroxide raised stomach pH enough to afford gastric dissolution of MFA in humans. Contrarily, the authors observed that sodium bicarbonate coadministration had no effect on the exposure of the structurally similar fenamate, tolfenamic acid. In addition, aluminum hydroxide slowed the Tmax and reduced Cmax, but showed no effect on AUC of tolfenamic acid.17 Table 5 summarizes published work on pH-dependent oral absorption in humans for the three model compounds used in our studies. Obviously there are no pentagastrin-pretreated human studies, but patients taking antacids, PPIs, or H2 blockers are very common and present a widely acknowledged drug−drug interaction for ionizable compounds. Human

gastric pH modulator

2 h after DAS 10 h before DAS

Maalox

2 h before DAS

Maalox

concomitant

omeprazole

6−8 h before KET

omeprazole + Coca-Cola

6−8 h before KET (omep) concomitant (cola) concomitant

cimetidine

mefenamic acid

dosing scheme

famotidine famotidine

85 mg of Mg(OH)2

concomitant

425 mg of Mg(OH)2

concomitant

1700 mg of Mg(OH)2

concomitant

results no effect Cmax ↓63% AUC0−12 ↓ 61% Cmax ↑26% no effect on AUC0−12 Cmax ↓58% AUC0−12 ↓ 55% Cmax ↓81% AUC0−∞ ↓ 81% Cmax ↓41% AUC0−∞ ↓ 37% Cmax ↓>90% AUC0−12 ↓ >90% Cmax ↑43% AUC0−1 ↑37% AUC0−∞ ↓4% Cmax ↑99% AUC0−1 ↑ 160% AUC0−∞ ↑ 10% Cmax ↑125% AUC0−1 ↑ 187% AUC0−∞ ↑ 36%

ref 4 4

4

4

2

2

21

17

17

17

studies on DAS showed that famotidine taken 10 h prior to a DAS dose showed significant reductions in both Cmax and AUC, however, if the famotidine dose is taken 2 h after DAS, it has no effect on absorption of that dose.4 In the same study, patients taking Maalox (Al(OH)3/Mg(OH)2) 2 h before DAS had no effect on AUC and only a minor elevation in Cmax, but significant reductions in Cmax and AUC when taking the antacid concomitantly with DAS. In a study on ketoconazole absorption, when the PPI omeprazole was administered 6−8 h before KET, both Cmax and AUC were reduced by 81%.2 However, the omeprazole-induced reductions in exposure were somewhat offset by concurrent ingestion of the acidic beverage Coca-Cola (Table 5). When the H2 antagonist cimetidine was administered concomitantly with KET, greater than 90% reductions in Cmax and AUC were observed.21 Mefenamic acid data in humans taking PPIs or H2 blockers has not been reported to the best of our knowledge, but the antacid 4002

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(6) Derendorf, H.; VanderMaelen, C. P.; Brickl, R.-S.; MacGregor, T. R.; Eisert, W. Dipyridamole bioavailability in subjects with reduced gastric acidity. J. Clin. Pharmacol. 2005, 45 (7), 845−850. (7) Russell, T. L.; Berardi, R. R.; Barnett, J. L.; O’Sullivan, T. L.; Wagner, J. G.; Dressman, J. B. pH-Related changes in the absorption of dipyridamole in the elderly. Pharm. Res. 1994, 11 (1), 136−43. (8) Dressman, J. B. Comparison of canine and human gastrointestinal physiology. Pharm. Res. 1986, 3 (3), 123−31. (9) Lui, C. Y.; Amidon, G. L.; Berardi, R. R.; Fleisher, D.; Youngberg, C.; Dressman, J. B. Comparison of gastrointestinal pH in dogs and humans: implications on the use of the beagle dog as a model for oral absorption in humans. J. Pharm. Sci. 1986, 75 (3), 271−4. (10) Carver, P. L.; Fleisher, D.; Zhou, S. Y.; Kaul, D.; Kazanjian, P.; Li, C. Meal composition effects on the oral bioavailability of indinavir in HIV-infected patients. Pharm. Res. 1999, 16 (5), 718−724. (11) Fleisher, D.; Li, C.; Zhou, Y.; Pao, L.-H.; Karim, A. Drug, meal and formulation interactions influencing drug absorption after oral administration: clinical implications. Clin. Pharmacokinet. 1999, 36 (3), 233−254. (12) Singh, B. N.; Malhotra, B. K. Effects of food on the clinical pharmacokinetics of anticancer agents: Underlying mechanisms and implications for oral chemotherapy. Clin. Pharmacokinet. 2004, 43 (15), 1127−1156. (13) Gu, C.-H.; Li, H.; Levons, J.; Lentz, K.; Gandhi, R. B.; Raghavan, K.; Smith, R. L. Predicting effect of food on extent of drug absorption based on physicochemical properties. Pharm. Res. 2007, 24 (6), 1118− 1130. (14) Lentz, K. A. Current methods for predicting human food effect. AAPS J. 2008, 10 (2), 282−288. (15) Lentz, K. A.; Quitko, M.; Morgan, D. G.; Grace, J. E., Jr.; Gleason, C.; Marathe, P. H. Development and validation of a preclinical food effect model. J. Pharm. Sci. 2006, 96 (2), 459−472. (16) Daneshmend, T. K.; Warnock, D. W.; Ene, M. D.; Johnson, E. M.; Potten, M. R.; Richardson, M. D.; Williamson, P. J. Influence of food on the pharmacokinetics of ketoconazole. Antimicrob. Agents Chemother. 1984, 25 (1), 1−3. (17) Neuvonen, P. J.; Kivisto, K. T. Effect of magnesium hydroxide on the absorption of tolfenamic and mefenamic acids. Eur. J. Clin. Pharmacol. 1988, 35 (5), 495−501. (18) Decktor, D. L.; Ciccone, P. E. Effects of famotidine vs antacid on gastric acidity: onset of action and symptom relief. JAMA, J. Am. Med. Assoc. 1996, 276 (11), 873−4. (19) Shi, S.; Klotz, U. Proton pump inhibitors: an update of their clinical use and pharmacokinetics. Eur. J. Clin. Pharmacol. 2008, 64 (10), 935−951. (20) Kaniwa, N.; Ogata, H.; Aoyagi, N.; Ejima, A. The bioavailability of flufenamic acid from aluminum flufenamate tablet and flufenamic acid capsule, and the influence of food and aluminum hydroxide gel. J. Pharmacobio-Dyn. 1982, 5 (3), 187−92. (21) Blum, R. A.; D’Andrea, D. T.; Florentino, B. M.; Wilton, J. H.; Hilligoss, D. M.; Gardner, M. J.; Henry, E. B.; Goldstein, H.; Schentag, J. J. Increased gastric pH and the bioavailability of fluconazole and ketoconazole. Ann. Intern. Med. 1991, 114 (9), 755−7. (22) Akimoto, M.; Nagahata, N.; Furuya, A.; Fukushima, K.; Higuchi, S.; Suwa, T. Gastric pH profiles of beagle dogs and their use as an alternative to human testing. Eur. J. Pharm. Biopharm. 2000, 49 (2), 99−102. (23) Knupp, C. A.; Shyu, W. C.; Morgenthien, E. A.; Lee, J. S.; Barbhaiya, R. H. Biopharmaceutics of didanosine in humans and in a model for acid-labile drugs, the pentagastrin-pretreated dog. Pharm. Res. 1993, 10 (8), 1157−64. (24) Zhou, R.; Moench, P.; Heran, C.; Lu, X.; Mathias, N.; Faria, T. N.; Wall, D. A.; Hussain, M. A.; Smith, R. L.; Sun, D. pH-Dependent Dissolution in Vitro and Absorption in Vivo of Weakly Basic Drugs: Development of a Canine Model. Pharm. Res. 2005, 22 (2), 188−192. (25) Polentarutti, B.; Albery, T.; Dressman, J.; Abrahamsson, B. Modification of gastric pH in the fasted dog. J. Pharm. Pharmacol. 2010, 62 (4), 462−469.

magnesium hydroxide has been shown to significantly increase MFA Cmax and AUC0−1 in a dose-dependent manner.17

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CONCLUSIONS In summary, we have presented validation work on using the male Sprague−Dawley rat as a model for studying pHdependent absorption in pharmaceutical research and development. Both control and pentagastrin-treated rats show gastric pH values of approximately 2, with pentagastrin clearly inducing acid secretion and boosting exposure with model compound ketoconazole. Gastric acid secretion was inhibited by pretreating animals with famotidine, a histamine H2 antagonist, raising stomach pH to ∼5. In the cases of dasatinib and ketoconazole, both weakly basic compounds, famotidine markedly reduced oral exposure. This clearly demonstrates the validity of the rat model for studying pH-dependent exposure on basic compounds. Famotidine pretreatment did not raise gastric pH enough to show an effect on mefenamic acid exposure, indicating that exposure would likely be unaffected by gastric pH modulators that raise stomach pH to no greater than 5. While the primary acidic moieties in drugs are carboxylic acids and sulfonamides, there is a wide array of basic amine and heterocyclic functionality in investigational druglike compounds with a wide range of pKa values. These factors all need to be investigated at some point in development, and typically earlier is preferred rather than later. This model may also be particularly useful in investigations of pH effects on a wider range of doses, especially when the human dose is unknown as is typically the case in the discovery setting. The rat may serve to be a useful model to study oral absorption of ionizable compounds in drug discovery and early development, and help make decisions on which compounds to advance deeper in the pipeline.

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AUTHOR INFORMATION

Corresponding Author

*1 DNA Way, MS 432a, South San Francisco, CA 94080. Phone: (650) 225-5072. Fax: (650) 467-2179. E-mail: lubach. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge Kirsten Messick, Shannon Stainton, Michelle Schweiger, and the Genentech In Vivo Studies group for their valuable assistance in these studies.

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REFERENCES

(1) Lin, J. H.; Chen, I. W.; Vastag, K. J.; Ostovic, D. pH-Dependent oral absorption of L-735,524, a potent HIV protease inhibitor, in rats and dogs. Drug Metab. Dispos. 1995, 23 (7), 730−5. (2) Chin, T. W. F.; Loeb, M.; Fong, I. W. Effects of an acidic beverage (Coca-Cola) on absorption of ketoconazole. Antimicrob. Agents Chemother. 1995, 39 (8), 1671−5. (3) Brass, C.; Galgiani, J. N.; Blaschke, T. F.; Defelice, R.; O’Reilly, R. A.; Stevens, D. A. Disposition of ketoconazole, an oral antifungal, in humans. Antimicrob. Agents Chemother. 1982, 21 (1), 151−8. (4) Eley, T.; Luo, F. R.; Agrawal, S.; Sanil, A.; Manning, J.; Li, T.; Blackwood-Chirchir, A.; Bertz, R. Phase I study of the effect of gastric acid pH modulators on the bioavailability of oral dasatinib in healthy subjects. J. Clin. Pharmacol. 2009, 49 (6), 700−709. (5) Neuvonen, P. J.; Kivisto, K. T. Enhancement of drug absorption by antacids. An unrecognized drug interaction. Clin. Pharmacokinet. 1994, 27 (2), 120−8. 4003

dx.doi.org/10.1021/mp400283j | Mol. Pharmaceutics 2013, 10, 3997−4004

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dx.doi.org/10.1021/mp400283j | Mol. Pharmaceutics 2013, 10, 3997−4004