A rabbit model for sublingual drug delivery: Comparison with human pharmacokinetic studies of propranolol, verapamil and captopril

A Rabbit Model for Sublingual Drug Delivery: Comparison with Human Pharmacokinetic Studies of Propranolol, Verapamil and Captopril MANISHA M. DALI, PAUL A. MOENCH, NEIL R. MATHIAS, PAUL I. STETSKO, CHRISTOPHER L. HERAN, RONALD L. SMITH Bristol-Myers Squibb Pharmaceutical Research Institute, One Squibb Drive, New Brunswick, New Jersey 08903-191

Received 29 June 2004; revised 17 November 2004; accepted 3 December 2004 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20312

ABSTRACT: A rabbit model for investigating sublingual drug absorption was established yielding results consistent with clinical data reported in the literature. Using propranolol as a model compound the effect of formulation and dosing variables was explored as a means to characterize the limiting parameters of this model. In addition, verapamil and captopril were selected as reference compounds to compare this model to sublingual absorption in humans. Rabbits were dosed sublingually and systemic absorption was measured over time. Sublingual absorption of propranolol was dependent on dosing solution pH and volume. Intra-oral spray device did not affect the overall exposure compared to instillation using a syringe. Despite species and dosing regimen differences the relative bioavailabilities of propranolol and verapamil were very similar in rabbits and humans. In contrast, captopril absorption from the sublingual cavity of rabbits was low and did not agree with that observed in man. Here we report a sublingual rabbit model of drug delivery and its potential utility in preclinical development of intraoral dosage forms. ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:37–44, 2006

Keywords:

rabbit model; buccal; sublingual; formulation; absorption; bioavailability

INTRODUCTION The oral cavity has been investigated as an alternative route of delivery for therapeutic small molecules and macromolecules having low oral bioavailability due to poor gastrointestinal (GI) absorption, GI instability, and/or susceptibility to first-pass metabolism.1–3 Primary considerations for systemic intra-oral drug delivery are potency (typically dose
5 mg), acceptable taste, reasonable saliva solubility, and membrane permeability. As with drug absorption Correspondence to: Manisha M. Dali (Telephone: 732-2276442; Fax: 732-227-3990; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 95, 37–44 (2006) ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association

across other mucosal barriers, fundamental factors that influence intra-oral absorption are pKa and pH for ionizable drugs, drug solubility in the saliva, partitioning into the mucosal membrane, diffusion across the epithelial lining, and residence time at the site of absorption.4,5 To demonstrate the feasibility of delivering a drug via the intra-oral route, suitable in vitro and/ or in vivo models must be used to assess delivery potential in clinical studies. Commonly used methods for assessing intra-oral drug delivery include, epithelial cell culture models, diffusion cell studies using isolated human or animal tissue, in situ and in vivo animal models. Stratified squamous epithelial cell culture models for buccal permeability studies have been described.6–9 While these cultures provide useful

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

37

38

DALI ET AL.

information on epithelial permeability under controlled conditions, their simplicity precludes understanding complex issues such as clearance from the site of delivery by salivation and systemic clearance from the circulation. In vitro diffusion and in situ perfusion studies using animal oral mucosa are also common, but are subject to many of the same limitations inherent in cell culture systems.10 Thus, suitable animal models are necessary to demonstrate the feasibility and practicality of bringing an alternate route delivery medicine to patients. Comparative review of several species demonstrate that nonkeratinized oral mucosa dissected from dogs, rabbits, pigs, and Rhesus monkeys are acceptable models, yielding permeability values similar to those found for humans.11–15 However, anatomical composition of animal tissue such as extent of keratinization and mucosal thickness pose a significant barrier to intra-oral absorption that impact its correlation to that in humans. For example, the heavily keratinized rat or hamster oral mucosa tends to underestimate absorption from the non-keratinized human oral mucosa.2,11,16 Various in vivo models for intra-oral drug delivery studies have been evaluated.17 Rabbit and dog are generally regarded as suitable animal models since the oral cavity of both are histologically similar to man.17–19 The thickness of the rabbit mucosa (600 mm) is comparable to humans and offers adequate surface area for experimental work. Moreover, the ease of handling and cost factor makes the rabbit more attractive. A significant difference between the oral cavities of rabbits and humans is the degree to which the buccal cheek pouch epithelium is keratinized, with rabbits being more so than humans. As the sublingual mucosa of both the rabbit and human is non-keratinized, delivery to the rabbit sublingual cavity presents an opportunity to correlate intra-oral absorption in man. Here we describe a rabbit model of intra-oral drug delivery. First, the delivery parameters: the effect of dosing solution volume, dosing solution pH, and method of delivery, that is, solution spray versus instillation were characterized. Second, systemic absorption was compared to sublingually administered drug in human. Two weak bases (propranolol and verapamil) and 1 weak acid (captopril), for which clinical sublingual absorption data is available, were used as model compounds.20–23 Pharmacokinetic findings are presented and compared to those published for human subjects. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

MATERIALS AND METHODS Materials Propranolol HCl, captopril, verapamil HCl, and glycine were purchased from Sigma Chemical Co. (St. Louis, MO). All other chemicals were reagent grade. Male vascular access ported (VAP) New Zealand rabbits (3 kg) were obtained from Covance (Denver, PA). Intra-Oral Studies in Rabbits Effect of pH on Intra-Oral Absorption of Propranolol Propranolol formulations at pH 5.0, 6.4, 7.4 (0.15M Mcllvaine’s buffer containing 48.49 mM citric acid monohydrate and 103.05 mM sodium phosphate dibasic), and pH 9.0 (0.05M GlycineNaOH buffer containing 50 mM glycine and 8.8 mM NaOH) each containing 10% (v/v) propylene glycol and 5% (v/v) ethanol co-solvents were prepared at 41.0 mM propranolol HCl concentration. The formulations were freshly prepared prior to dosing. The dosing solution (0.5 mL, 2 mg/kg) was administered under the rabbit tongue using a needleless tuberculin syringe (n ¼ 3). It was expected that following sublingual dosing, most of the intra-oral cavity will be exposed to the dosing solution. Holding the rabbit head in the upright position for 30 seconds post administration minimized dose swallowing. In a crossover study, designed to determine the contribution of swallowed fraction of the dose (if any) to the plasma propranolol levels seen following intra-oral dosing, the dosing solution (0.5 mL, 2 mg/kg) at pH 9.0 was administered by oral gavage. Blood samples (1.5 mL) were collected from ear vein at predose, 5, 10, 15, 20, 30, and 60 min in heparinized vacutainers, centrifuged, and the plasma stored at 808C until HPLC analysis. Effect of Dose Volume A 41.0 mM propranolol HCl solution in GlycineNaOH buffer (pH 9.0) containing 10% (v/v) propylene glycol and 5% (v/v) ethanol was instilled sublingually (n ¼ 3) at dosing volumes of 100 mL (4.06 mmole dose), 250 mL (10.14 mmole dose), and 500 mL (20.28 mmole dose). Blood samples (1.5 mL) were collected as a function of time, centrifuged, and the plasma stored at 808C until HPLC analysis.

RABBIT MODEL FOR SUBLINGUAL DRUG DELIVERY

Effect of Dosing Device A 41.0 mM propranolol HCl solution in GlycineNaOH buffer (pH 9) containing 10% (v/v) propylene glycol and 5% (v/v) ethanol was either instilled sublingually (n ¼ 3) using a 1 mL needleless tuberculin syringe or sprayed using a spray device (no. 6958, actuator no. 44421, screw closure GCMI 18/415, Pfeiffer of America, Princeton, NJ) at a fixed dosing volume of 250 mL. Blood samples (1.5 mL) were collected as a function of time, centrifuged, and the plasma stored at 808C until HPLC analysis. Sublingual Pharmacokinetic of Propranolol, Verapamil, and Captopril in Rabbits Three groups of VAP New Zealand White rabbits (three animals/group) were used in this study. Animals were anesthetized for approximately 3– 5 min by inhalation of isoflurane with a precision vaporizer administered to effect. To eliminate vehicle effects, all dosing solution were aqueous in nature: Propranolol HCl in sterile H2O at 30 mg/mL (101.4 mM, pH 7.5) concentration, verapamil HCl in sterile H2O at 30 mg/mL (61.0 mM, pH 5) concentration, and 40 mg/mL (184.0 mM, pH 7) concentration of captopril in PBS were administered by sublingual instillation at a dose of 1 mg/kg. Blood samples were collected at predose, 5, 10, 15, 20, 30, 60, 90, and 120 min in heparinized vacutainers, centrifuged, and the plasma stored at 808C until HPLC analysis. Pharmacokinetic parameters were calculated using Kinetica Software (InnaPhase Corp., Philadelphia, PA). Bioavailabilities were calculated based on intravenous (IV) data in rabbits. (%F ¼ (AUCSL/AUCIV) (doseIV/doseSL)  100).

Analytical Methods Propranolol HPLC Analysis Timolol maleate was added as an internal standard to all plasma samples and standards, and vortexed for 10 seconds. An equal volume of 0.25M aqueous sodium carbonate solution (pH 12.0) was added to each sample and standard and vortexed. Propranolol was extracted into 1.5 mL of ethyl acetate by vortexing for 30 seconds. The organic phase was separated by centrifugation at 14,000 rpm for 10 min at 48C and evaporated to dryness under a stream of nitrogen at room temperature. The residue was reconsti-

39

tuted in 300 mL of mobile phase (10 mM ammonium acetate with 0.1% acetic acid: Acetonitrile: Methanol (56: 28: 16 v/v)) for injection onto a 150  3.9 mm Nova-Pak C18 (4 mm) (Waters Corp., Milford, MA) column maintained at 308C. The HPLC system consisted of Waters Alliance 2690 module, model 470 fluorescence detector at excitation wavelength of 238 nm and emission at 360 nm for the detection of propranolol and model 996 photodiode array detector (PDA) at 290 nm for timolol. The Injection volume was 25 mL and retention times for timolol and propranolol under isocratic conditions (flow rate of 1.25 mL/min) were 1.7 and 4.0 min respectively. Verapamil HPLC Analysis A 100 mL aliquot of plasma sample was vortexed with 100 mL of 90% acetonitrile, 10 mM ammonium acetate with 0.1% acetic acid. Samples were then centrifuged at 14,000 rpm for 10 min at 48C to precipitate plasma proteins. A 10 mL sample of supernatant fluid was injected onto a YMC Pro C18 column (2.5 mm, 2.1  50 mm). The mobile phase consisted of 10 mM phosphate buffer (pH 7.4) and acetonitrile gradient at a flow rate of 0.3 mL/min. Verapamil was detected using a Waters 470 fluorescence detector set at an excitation/emission wavelength of 276/316 nm. Captopril Analysis A 100 mL aliquot of plasma sample was mixed with 10 mL of a 1 mg/mL solution of Timolol (50:50 MeOH:10 %Acetic Acid), 10 mL of a 20 mM solution of dithiothreitol, and 10 mL of HNO3 and incubated at 378C for 30 min. Ethyl acetate (200 mL) was added to 100 mL samples, vortexed and centrifuged at 10,000 rpm for 10 min at 48C. The supernatant was evaporated to dryness under a stream of N2. The samples were resuspended in 50 mL of mobile phase containing 10 mM Ammonium acetate/0.1% Acetic Acid:Methanol (90:10), and analyzed on a Waters Xterra MS C18, 2.5mm, 2.1  50 mm column. The LC system was a Waters 2790 separations module linked to a ThermoQuest Finnigan LCQ Duo Mass Spectrometer. Captopril was trapped at m/z 217.9 (positive ion mode, ESI, collision energy 28%) and a daughter ion of 115.9 was isolated for quantitation. Timolol was trapped at m/z 317.1 and a daughter ion of 171.9 (positive ion mode, ESI, collision energy 32%) was isolated for quantitation. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

40

DALI ET AL.

RESULTS Effect of pH Following intra-oral dosing of propranolol solutions at different pH, high plasma propranolol levels were obtained as early as 5 min post dosing (Figure 1). The extent of sublingual absorption of propranolol increased with pH as a function of unionized fraction of the drug. The Cmax of propranolol after intra-oral dosing of a 6 mg dose was 30  5 ng/mL, 38  8 ng/mL, 46  5 ng/mL, and 78  4 ng/mL at pH 5.0, 6.4, 7.4, and 9.0 respectively. The Tmax at pH 5.0 was 30 min with the absorption rate constant (Ka) of  0.09/min. The Tmax ranged from 5–10 min at pH 6.4–9.0 with no significant difference in the Ka (0.3/min). AUC0–20 min were 335  50 ng  min/mL, 420  7 ng  min/mL, 670  100 ng  min/mL and 1150  105 ng  min/mL from the formulations at pH 5.0, 6.4, 7.4, and 9.0, respectively (Figure 2). The extent of absorption due to swallowing was determined from plasma concentration time curve following intra-gastric dosing of propranolol at pH 9.0. Two out of three animals did not show detectable plasma propranolol levels for up to 60 min post dosing. In one animal, propranolol was detected only 20 min post dosing (AUC0–60 min ¼ 1773 ng  min/mL). Therefore, plasma levels detected within the first 20 min post intra-oral dosing were due to absorption of propranolol from the oral cavity and not the gastrointestinal (GI) tract (Figure 3).

Figure 2. Area under the propranolol plasma concentration-time curve as a function of pH.

administered dose (Table 1). Based on these results, dosing volume was fixed at a maximum of 250 mL for further experiments.

Effect of Dosing Device After dosing from a syringe, Cmax was 50 ng/mL and Tmax was 5 min. The plasma concentrationtime profile and hence AUC0–60 min were comparable from both dosing methods (Figure 4).

Sublingual Pharmacokinetic of Propranolol, Verapamil, and Captopril in Rabbits

Intra-oral absorption of propranolol at pH 9.0 was linear up to a dosing volume of 250 mL. Both Cmax and AUC0–20 min were directionally higher with

Sublingual dosing studies were done at clinically acceptable pH rather than at pH 9 as reported here. Following sublingual administration, systemic absorption of two basic drugs (propranolol and verapamil) but not the weak acid (captopril) was comparable between humans and rabbits (Figure 5 and Table 2). The absolute bioavailability for propranolol was 50% and

Figure 1. Effect of pH on the intra-oral absorption of propranolol in rabbits. Dose was instilled sublingually at 2 mg/kg.

Figure 3. Plasma concentration-time profile following intra-oral and intra-gastric dosing of propranolol at pH 9.0, n ¼ 3.

Effect of Dose Volume

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

RABBIT MODEL FOR SUBLINGUAL DRUG DELIVERY

41

Table 1. Effect of Dosing Volume on Intra-Oral Absorption of Propranolol at pH 9.0 Dose Volume (mL) 100 250 500

Dose (mg) 1.2 3 6

Cmax (ng/mL) 22  5 51  18 79  4

Tmax (min)

AUC 0–20 (ng  min/mL)

AUC 0–60 (ng  min/mL)

20  5 50 10  0

284  55 841  212 1309  62

970  165 2200  115 3200  240

Values are expressed as mean  SEM (n ¼ 3).

42.7 %(20.9 SD) in humans and rabbits, respectively. Only a typical plasma profile (n ¼ 1) from which to extract data was available in the literature, therefore a standard error could not be calculated for human propranolol. The bioavailability for sublingual verapamil was 57.2% (41.7 SD) and 42.3% (16.8 SD) for humans and rabbits, respectively. The absolute bioavailability of sublingual captopril in rabbits was very low (4% 1.8 SD).

DISCUSSION This article describes a convenient, reproducible, and physiologically relevant rabbit model suitable to assess the sublingual absorption potential of drug substances for clinical development. Absorption across the rabbit sublingual mucosa is dependent on the dosing solution pH, concordant with the pH-partition theory. The dosing volume, method of delivery (based on device), and formulation or dosage form composition are delivery variables that can play a role in assessing the preclinical feasibility of sublingual delivery. In comparing sublingual absorption in rabbits with that in humans, we found that the weak bases, propranolol and verapamil, were in better agreement than was the weak acid captopril within the limited window of absorption.

Figure 4. Effect of dosing device on intra-oral absorption of propranolol at pH 9.0.

The extent of propranolol absorption following intra-oral dosing of solutions at different pH, increased with pH as a function of unionized fraction of the drug (Figure 1). These findings are in accordance with the pH partition theory,24 published data in humans,4,25,26 and permeability data obtained using hamster cheek pouch in vitro.27 At pH 9.0, about 30% of propranolol is in the neutral/unionized form (pKa ¼ 9.2 at 258C) and is thus favored for transport across the epithelial bilayer as compared to ionized species. In contrast, propranolol is almost completely ionized at pH 5.0. Drug absorption through oral mucosa was greater, more rapid and the relative bioavailability (AUC0–20 min) was three-fold higher at pH 9.0 than at pH 5.0 (Figure 2). Differentiating between intra-oral absorption from that due to swallowing is critical to the interpretation of results. To this end, the gastric absorption profile of propranolol was plotted against that of intra-oral administration to yield an intra-oral ‘‘absorption window.’’ As expected, intra-oral absorption for propranolol was rapid (Tmax 5–15 min.) while gastric absorption was delayed by as much as 20–60 min post-dose. Therefore, plasma levels detected within the first 20 min post intra-oral dosing were entirely due to absorption from the oral cavity and not the GI tract (Figure 3). Intra-oral dosing is limited by the size of the oral cavity. Given the relatively small size of the rabbit oral cavity, we tested the effect of dosing volume on intra-oral absorption of propranolol. Absorption was found to deviate from linearity at a dose volume greater than 250 mL (Table 1). Deviation from linearity at higher dose volumes is likely due to swallowing and subsequent GI absorption. Clearly, the volume of the dose administered must be considered in designing sublingual instillation and/or ‘‘swish and spit’’ clinical protocols. The effect of dosing device on the intra-oral absorption of propranolol was also investigated. Absorption of propranolol from solution delivered using either syringe instillation or a spray device was equivalent. Taken together, the data suggests JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

42

DALI ET AL.

that intra-oral drug absorption is determined by the dose, more specifically fraction of the drug in unionized form, and not by the method of dosing. The potential of a spray to deposit the dose over a larger surface area might yield increased rate and magnitude of absorption. Given that our study restricted the site of administration to the nonkeratinized tissue in the sublingual cavity, the effect of increased surface area was not explored. Despite significant differences in the formulations and dosing protocols employed in the sublingual rabbit and human studies20–23,28 sublingual absorption of the weak bases propranolol and verapamil was comparable to that in human (Figure 5 and Table 2). Propranolol (pKa 9.2, logP 3.2) and verapamil (pKa 8.6, logP 3.8) were readily absorbed in rabbits. In rabbits, the Cmax was higher commensurate with a higher dose, while the Tmax was lower compared to that in humans. On the other hand, captopril (pKa 4.3, logP 1.4) bioavailability in rabbits was low (4%) with a delayed Tmax. In the absence of a published human sublingual bioavailability, calculated as a fraction of IV exposure, extrapolating sublingual exposure of 85% relative to per oral route,22 yields a estimated sublingual bioavailability value of 60%. The oral bioavailability of captopril is reported to be 75% in Physicians Desk Reference. The low plasma concentrations at the early time points together with delayed Tmax at 90 min suggests captopril may have limited absorption from the oral cavity of rabbits, and that contribution from the swallowed fraction of the weak acid increases as it experiences the acidic environment of the stomach and duodenum. It is likely, that the physiochemical properties of captopril and the

Figure 5. Plasma concentration-time profiles for sublingual propranolol, captopril, and verapamil in humans and rabbits. Human data reference: aKates20; bBerk et al.21; cMcElnay et al.22

Table 2. Comparison of Human and Rabbit Pharmacokinetics Following Sublingual Administration of Propranolol, Verapamil and Captopril Propranolol

SL dose (mg/kg) Dosing pH Cmax (ng/mL) Tmax (min) AUC0–20(ng  min/mL) %F

Verapamil

Captopril

Humana

Rabbit

Humanb

Rabbit

Humanc

Rabbit

0.7 6 11.5 60 1107 50

1 7 75.4 (28.8) 5 3930 (1177) 42.7 (20.9)

0.6 n/a 72.3 (46.4) 90 n/a 57.2 (41.7)

1 7.0 149.9 (60.3) 12.5 5947 (2364) 42.3 (16.8)

0.2 7 88.7 (39.0) 40 6511 (268)* 60%**

1 7 63.0 (38.9) 90 3983 (1857) 5 (1.8)

Values are mean  SD, n ¼ 3–5. Tmax, median value. Human data reference: aKates20; bBerk et al.21; cMcElnay et al.22 *Human captopril AUC is 0–180 min. **Human sublingual captopril bioavailability estimated as 85% relative to oral. n/a, not available. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

RABBIT MODEL FOR SUBLINGUAL DRUG DELIVERY

barrier properties of the sublingual mucosa do not favor absorption in rabbits. In addition to evaluating intra-oral absorption feasibility of acidic and basic drugs, the effect of formulation variables and various dosage forms can also be evaluated in the rabbit model. The use of short-acting anesthesia in the dosing protocol allows the evaluation of these dosing options without compromising the interpretation of results. For example, when adjusted for dose and volume, the propranolol PK profile in isoflurane treated animals agrees with that found with untreated conscious rabbits. However, long acting anesthesia does impact the pharmacokinetics of sublingually administered drugs by altering salivary clearance, sublingual absorption, distribution, metabolism, elimination, and/ or hemodynamic effects.29

CONCLUSION An in vivo rabbit model for investigating sublingual absorption of drug substances was established using propranolol as a reference compound with results comparable with those reported in the literature. Although differences might be expected between dosing conditions and data obtained in humans versus rabbits, the relative bioavailabilities of propranolol and verapamil were very similar suggesting that the rabbit model may be suitable for assessing intra-oral drug delivery in early development.

REFERENCES 1. Motwani JG, Lipworth BJ. 1991. Clinical pharmacokinetics of drug administered buccally and sublingually. Clin Pharmacokinet 21:83–94. 2. Squier CA, Wertz PW. 1996. Structure and function of the oral mucosa and implications for drug delivery. In: Rathbone MJ, editor. Oral mucosal drug delivery. New York: Marcel Dekker, Inc. pp 1– 26. 3. Squier CA, Johnson NW. 1975. Permeability of oral mucosa. Br Med Bull 31:169–175. 4. Beckett AH, Triggs EJ. 1967. Buccal absorption of basic drugs and its application as an in vivo model of passive drug transfer through lipid membranes. J Pharm Pharmacol 19:41S. 5. Beckett AH, Moffat AC. 1971. The buccal absorption of some barbiturates. J Pharm Pharmacol 23:15–18.

43

6. Audus KL, Tavakoli-Saberi MR, Zheng H, Boyce EN. 1992. Chlorhexidine effects on membrane lipid domains of human buccal epithelial cells. J Dent Res 71:1298–1303. 7. Audus KL. 1996. Buccal epithelial cell cultures as a model to study oral mucosal drug transport and metabolism. In: Rathbone MJ, editor. Oral mucosal drug delivery. New York: Marcel Dekker, Inc. pp 101–119. 8. MacCallum DK, Lillie JH, Jepsen A, ArenholtBindslev D. 1987. The culture of oral epithelium. Int Rev Cytol 109:313–330. 9. Tavakoli-Saberi MR, Audus KL. 1989. Cultured buccal epithelium: An in vitro model derived from the hamster pouch for studying drug transport and metabolism. Pharm Res 6:160–166. 10. Zhang H, Robinson JR. 1996. In vitro methods for measuring permeability of the oral mucosa. In: Rathbone MJ, editor. Oral mucosal drug delivery. New York: Marcel Dekker, Inc. pp 85–100. 11. Jepsen A. 1974. An in vitro model of an oral keratinizing squamous epithelium. Scand J Dent Res 82:144–146. 12. Garren KW, Topp EM, Repta AJ. 1989. Buccal absorption. III. Simultaneous diffusion and metabolism of an aminopeptidase substrate in the hamster cheek pouch. Pharm Res 6:966–970. 13. Garren KW, Repta AJ. 1989. Buccal drug absorption. II: In vitro diffusion across the hamster cheek pouch. J Pharm Sci 78:160–164. 14. Siegel IA, Izutsu KT, Watson E. 1981. Mechanisms of non-electrolyte penetration across dog and rabbit oral mucosa in vitro. Arch Oral Biol 26:357–361. 15. Lesch CA, Squier CA, Cruchley A, Williams DM, Speight P. 1989. The permeability of human oral mucosa and skin to water. J Dent Res 68:1345– 1349. 16. Squier CA, Fejerskov O, Jepsen A. 1978. The permeability of a keratinizing squamous epithelium in culture. J Anat 126:103–109. 17. Rathbone MJ, Purves R, Ghazali FA, Ho PC. 1996. In vivo techniques for studying the oral mucosal absorption characteristics of drugs in animals and humans. In: Rathbone MJ, editor. Oral mucosal drug delivery. New York: Marcel Dekker, Inc. pp 121–156. 18. Chen SY. 1970. Comparison of the fine structure of the mucosa of cheek and hard palate in the rabbit. M.S.Thesis, University of Illinois Medical Center. 19. Alvares OF, Meyer J. 1971. Current concepts of the histology of the oral mucosa. In: Squier CA, Meyer J, editors. Current concepts of the histology of the oral mucosa. Springfield, IL: Charles C. Thomas. 97p. 20. Kates RE. 1977. Absorption kinetics of sublingually administered propranolol. J Med 8:393–402. 21. Berk SI, Beckman K, Hoon TJ, Hariman RJ, Hu D, Siegel FP, Bauman JL. 1992. Comparison of the

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

44

22.

23.

24.

25.

DALI ET AL.

pharmacokinetics and electrocardiographic effects of sublingual and intravenous verapamil. Pharmacotherapy 12:33–39. McElnay JC, Al Furaih TA, Hughes CM, Scott MG, Nicholls DP. 1996. A pharmacokinetic and pharmacodynamic evaluation of buffered sublingual captopril in patients with congestive heart failure. Eur J Clin Pharmacol 49:471–476. Al Furaih TA, McElnay JC, Elborn JS, Rusk R, Scott MG, McMahon J, Nicholls DP. 1991. Sublingual captopril—a pharmacokinetic and pharmacodynamic evaluation. Eur J Clin Pharmacol 40: 393–398. Maitani Y, Coutel-Egros A, Obata Y, Nagai T. 1993. Prediction of skin permeabilities of diclofenac and propranolol from theoretical partition coefficients determined from cohesion parameters. J Pharm Sci 82:416–420. Schurmann W, Turner P. 1978. A membrane model of the human oral mucosa as derived from buccal absorption performance and physicochemical prop-

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

26.

27.

28.

29.

erties of the beta-blocking drugs atenolol and propranolol. J Pharm Pharmacol 30:137–147. Achari R, Beckett AH. 1982. Buccal uptake, urinary excretion, and physicochemical properties of zipeprol and its N-dealkylated products. Biopharm Drug Dispos 3:203–209. Coutel-Egros A, Maitani Y, Veillard M, Machida Y, Nagai T. 1992. Combined effects of pH, cosolvent and penetration enhancers on the in vitro buccal absorption of propranolol through excised hamster cheek pouch. Inter J Pharm 84:117– 128. McElnay JC, Al Furaih TA, Hughes CM, Scott MG, Elborn JS, Nicholls DP. 1995. The effect of pH on the buccal and sublingual absorption of captopril. Eur J Clin Pharmacol 48:373–379. Moench PA, Heran CL, Stetsko PI, Mathias NR, Wall DA, Hussain MA, Smith RL. 2003. The effect of anesthesia on the pharmacokinetics of sublingually administered verapamil in rabbits. J Pharm Sci 92:1735–1738.