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Toward Successful Cyclodextrin-Based Solubility-Enabling Formulations for Oral Delivery of Lipophilic Drugs: Solubility-Permeability Tradeoff, Biorelevant Dissolution, and the Unstirred Water Layer Noa Fine-Shamir, Avital Beig, Moran Zur, David Lindley, Jon Miller, and Arik Dahan Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 15 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017

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Molecular Pharmaceutics

Toward Successful Cyclodextrin Based SolubilityEnabling Formulations for Oral Delivery of Lipophilic Drugs: Solubility-Permeability Tradeoff, Biorelevant Dissolution, and the Unstirred Water Layer Noa Fine-Shamir1, Avital Beig1, Moran Zur1, David Lindley2, Jonathan M Miller2,3, and Arik Dahan1,*

1

Department of Clinical Pharmacology, School of Pharmacy, Faculty of Health

Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel

2

AbbVie Inc, North Chicago, Illinois 60064

3

Current Address: Vertex Pharmaceuticals Inc., 50 Northern Ave, Boston, MA 02210

*

Corresponding Author: Department of Clinical Pharmacology, School of

Pharmacy, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O.Box 653, Beer-Sheva 84105, Israel. Tel: +972-8-6479483. Fax: +972-8-6479303. E-mail address: [email protected]

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ABSTRACT:

The purpose of this work was to investigate key factors dictating the success/failure of cyclodextrin-based solubility-enabling formulations for oral delivery of lowsolubility drugs. We have studied the solubility, the permeability, and the solubilitypermeability interplay, of the highly lipophilic drug danazol, formulated with different levels (8.5, 10, 20 and 30%) of the commonly used hydroxypropyl-βcyclodextrin (HPβCD), accounting for the biorelevant solubilization of the drug along the gastrointestinal tract (GIT), the unstirred water layer (UWL) adjacent to the GI membrane, and the overall absorption. HPβCD significantly increased danazol solubility, and decreased the drugs' permeability, in a concentration-dependent manner. These Peff results were in good correlation (R2=0.977) to literature rat AUC data of the same formulations. Unlike vehicle without HPβCD, formulations containing 8.5% HPβCD and above were shown to successfully dissolve the drug dose during the entire biorelevant dissolution experiment. We conclude that CD-based solubility-enabling formulations should contain the minimal amount of CD sufficient to dissolve the drug dose throughout the GIT, and not more than that; excess CD does not provide solubility gain but cause further permeability loss, and the overall absorption is then impaired. Moreover, a significant UWL effect was revealed in danazol intestinal permeability, and accounting for this effect allowed an excellent prediction of the solubility-permeability tradeoff vs. % HPβCD. Overall, this work assessed the contribution of each individual step of the absorption cascade to the success/failure of HPβCD-based formulation, allowing a more mechanistic development process of better solubility-enabling formulations.


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KEYWORDS: Cyclodextrins; drug absorption, intestinal permeability, low solubility, oral drug delivery, solubility-enabling formulations, solubility-permeability interplay



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1.

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INTRODUCTION Low aqueous solubility is a central challenge in todays' oral drug delivery 1-3.

Various solubility-enabling formulations allow tackling this obstacle efficiently

4-5

;

however, while the apparent solubility of the drug may be significantly increased, the apparent permeability may concomitantly decrease. This solubility-permeability tradeoff was shown for formulations containing cyclodextrins

6-7

, surfactants

8-9

,

hydrotropy 10 and cosolvents 11-12. Since the solubility and the permeability together are the two key factors dictating oral drug absorption 13-14, the overall absorption of the drug becomes unpredicted due to this solubility-permeability tradeoff 15-16.

The formulation development process has to account for the dynamic environment that the drug product is exposed to throughout its gastrointestinal tract (GIT) journey, e.g. pH changes, residence times, and fluid volumes/dilutions. A good formulation will allow a solution mediated phase transformation or supersaturation of the drug dose throughout the entire travel along the GIT, while an inferior one will fail to solubilize the drug dose in this dynamic surroundings, and drug precipitation will occur

17-18

. Different in-vitro biorelevant dissolution methods have been

developed throughout the years, including systems with "gastric" and "intestinal" compartments

19-20

, two-phase dissolution apparatus

21-22

, the gastrointestinal

simulator system 23-26, the dynamic lipolysis model for lipid-based formulations 27-29, and the dilution-dissolution approach

30-31

. These (and other) in-vitro models have

shown significant success in predicting the in-vivo ability of a given formulation to retain the drug dose in solution (or supersaturation) in the dynamic GIT environment. 4 ACS Paragon Plus Environment

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Specifically for cyslodextrin-based solubility-enabling formulations, a tremendous improved solubility can be certainly achieved, that may also be maintained throughout the entire GIT travel, however, this advantage comes with the price of concomitant permeability decrease, that hamper the chances of the formulation to improve the overall absorption of the drug. Since cyclodextrins anable apparent solubility increase by hosting the lipophilic molecule inside their hydrophobic cavity, the free fraction of the drug dose available for absorption may decrease, explaining the apparent permeability decrease. Both simulations and experimental data demonstrated that cyclodextrins can improve, have no effect, or impair drug absorption when administered as a physical mixture with a lipophilic drug

32-36

. Defining the different criteria that may allow achieving the maximal

potential of cyclodextrin-based solubility enabling formulations represents an important unmet need.

The purpose of this work was to investigate key factors that may dictate the success/failure of cyclodextrin-based solubility-enabling formulations for oral delivery of low-solubility drugs. We have studied the highly lipophilic drug danazol, formulated with different levels of the commonly used hydroxypropyl-β-cyclodextrin (HPβCD), accounting for the solubility-permeability interplay, biorelevant solubilization of the drug along the gastrointestinal tract (GIT), the unstirred water layer (UWL) adjacent to the GI membrane, and the overall absorption.

The effects of the formulations on the solubility, in-vivo permeability, and biorelevant dissolution of the drug, were investigated, and were correlated with relevant literature in-vivo absorption data 37. The results were mechanistically 5 ACS Paragon Plus Environment


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analyzed, and together with experiments of the UWL effect, an excellent prediction of the solubility-permeability tradeoff was enabled. Since formulation development is often an empirical process that is based on trial and error, this work aimed to assess the contribution of each individual step of the absorption cascade to the overall success/failure of a given formulation, allowing a more mechanistic a-priori development of better solubility-enabling formulations.


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2.

MATERIAL AND METHODES

2.1.

Materials

Danazol was purchased from Carbosynth Ltd. (Compton Berkshire, UK). 2hydroxypropyl-β-cyclodextrin (HPβCD) was purchased from Glentham life sciences Ltd (Corsham, UK). SIF powder was obtained from Biorelevent.com Ltd. (London, UK). MES buffer, KCl, NaCl and DMSO were obtained from Sigma Chemical Co. (St. Louis, MO). Acetonitrile and water (Merck KGaA, Darmstadt, Germany) were UPLC grade. All other chemicals were of analytical reagent grade.

2.2.

Solubility Experiments

The solubility experiments for danazol with HPβCD were conducted according to a previously described method 38-39. Excess amounts of danazol were added to number of glass test tubes containing MES buffer solution (pH 6.5) of different HPβCD concentrations (0, 1, 2.5, 5, 8.5, 10, 20, and 30 % w/v). The intrinsic solubility of danazol in MES buffer was determined from the samples without any HPβCD. The test tubes were placed in a 100 rpm shaking water bathes at room temperature (25 °C) or 37 °C for 48 h. After 48 hours the vials were centrifuged (14,000 rpm) for 15 min, filtered, and immediately assayed for drug content by UPLC.

2.3.

Rat Jejunal Perfusion 7 ACS Paragon Plus Environment


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The protocol used for all animal experiments was approved by the BenGurion University of the Negev Animal Use and Care Committee (Protocol IL-08-012015). Animals were housed and handled according to the Ben-Gurion University of the Negev Unit for Laboratory Animal Medicine Guidelines. Male ~300 g Wistar rats (Harlan, Israel) were used for all studies. Rats were fasted overnight (12 h) before each experiment, with free access to water. Animals were randomly assigned to the different experimental groups.

Single-pass intestinal perfusion (SPIP) studies were carried out according to previously reported protocol 40-41. Rats were anesthetized (1 mL/kg ketamine:xylazine solution) and placed on a 37 °C pad (Harvard Apparatus Inc., Holliston, MA). About 10-20 cm proximal jejunal segment was cannulated on two ends, and was thoroughly rinsed with 37 °C normal saline solution. Danazol solutions were prepared with different concentrations of HPβCD (0, 1, 8.5, 10, 20, and 30 % w/v) in MES buffer (pH 6.5), and were perfused through the intestinal segment at a flow rates of 0.2 mL/min (Watson-Marlow 205S, Wilmington, MA). To assess the UWL effect on danazol intestinal permeability, similar studies with flow rates of 2, 3, 4, or 4.5 mL/min were also carried out 42. To ensure steady-state conditions, the test solutions were first perfused for 60 min, followed by an additional 60 min, during which samples were collected at 5-10 min intervals 43. Samples were centrifuged (14,000 rpm) for 15 min and immediately assayed for danazol content (UPLC). The effective permeability (Peff) through the rat jejunum was determines by the equation 44

:


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Pୣ୤୤

Cᇱ −Qln( ᇱ୭୳୲ ) C ୧୬ = 2πRL

where Q is the perfusion flow rate (0.2-4.5 mL/min), C'out/C'in is the ratio of danazol outlet vs. inlet concentrations adjusted for water flux using the gravimetric method 45

, R is the radius of the intestinal segment (0.2 cm), and L is the exact length of the

perfused intestinal segment as was measured at the endpoint of each experiment (10-20 cm).

2.4.

In vitro pH-Dilution Dissolution studies

A biorelevant in vitro pH-dilution dissolution method was used, aiming to mimic physiological conditions in the different segments throughout the GIT journey. The method has been shown to be successful in simulating drug dissolution from a given formulation while traveling along the GIT 30-31.

Danazol dose in the different formulations (600 μL) was first diluted into 1M HCl (pH 4.0) at a dilution factor of 1:0.66. Sample vials were agitated for 15 min, using 100 rpm shaking water bath at 37 °C. Then, the vials were further diluted with FaSSIF (Biorelevant.com ltd, London, UK) at a dilution factor of 1:0.5 and continued to be agitated for another 15 min. Further FaSSIF dilutions were carried out with a dilution factor of 1:1 (agitation time of 30 min), and then twice 1:1 dilution every 60 min, for a total agitation time of 3 hours. Samples (100 μL) were taken at a set time



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points throughout the experiment; the samples were centrifuged (14,000 rpm) for 20 min. filtered, and immediately assayed for drug content (UPLC). The comparison of solubilized drug (obtained from the UPLC results) and total drug content (calculated from the initial dose and the subsequent dilutions) provides the insight into the solubilization performance of the formulations during the GIT travel.

2.5.

True membrane permeability

Determination of the true membrane permeability of the drug is important for the understanding of the solubility permeability interplay. To reveal the role of the UWL vs. the membrane in the overall permeability of the drug, we used the above mentioned single-pass perfusion method. Danazol solution (with 0% CD content) was perfused under five different flow rates: 0.2, 2, 3, 4, and 4.5 mL\min, and permeability was calculated. If the UWL is a barrier to the intestinal absorption, higher flow rates will result in higher Peff. Otherwise, the effective permeability should remain steady regardless of the flow rate 42, 46.

2.6.

Permeability predictions

The central quasi-equilibrium analysis of the effect of increased apparent solubility via complexation with cyclodextrins on apparent membrane permeability has been described in detail previously 6, 7, 33, 42. Briefly, the apparent membrane


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permeability (Pm) dependence on drug apparent aqueous solubility (Saq) can be written as:

P୫ =

ܲ௠(଴) × ܵ௔௤(଴) ܵ௔௤

where Pm(0) is the intrinsic membrane permeability of the free drug and Saq(0) is the intrinsic aqueous solubility of the drug in the absence of cyclodextrins.

2.7.

Ultra performance liquid chromatography (UPLC)

The concentration of danazol in the solution was determined by Waters UPLC H-Class system equipped with photodiode array detector and Empower software. Waters sunFireTM C18 3.5 μm 4.6 × 100 mm column was used. The detection wavelength was 285 nm. A gradient mobile phase consisting of 15:85 going to 6:94 and then back to 15:85 (v/v) of water:acetonitrile (both with 0.1% TFA) over 6 min was used. The flow rate was 0.5 mL/min, and the injection volumes ranged from 20 to 100 μL.

2.8.

Statistical analysis

Solubility studies were performed in triplicates, while each group of the animal studies consisted of 4 rats. Values are expressed as means ± standard deviation (SD). To determine statistically significant differences among the experimental groups, the nonparametric Kruskal-Wallis test was used for multiple 11 ACS Paragon Plus Environment


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comparisons, and the two-tailed nonparametric Mann-Whitney U-test for two-group comparison, where appropriate. p

Toward Successful Cyclodextrin Based Solubility ... - ACS Publications

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