Enzyme Based Acceleration of Absorption of Hydrophobic

DOI: 10.1021/mp400272m. Publication Date (Web): August 12, 2013. Copyright © 2013 American Chemical Society. *Phone: 612-624-6164. Fax: 612-626-2125 ...
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Prodrug/Enzyme Based Acceleration of Absorption of Hydrophobic Drugs: An in Vitro Study Mamta Kapoor† and Ronald A Siegel*,†,‡ †

Departments of Pharmaceutics and ‡Biomedical Engineering, University of Minnesota, Minneapolis, Minnesota 55455, United States S Supporting Information *

ABSTRACT: Poor water solubility of APIs is a key challenge in drug discovery and development as it results in low drug bioavailability upon local or systemic administration. The prodrug approach is commonly utilized to enhance solubility of hydrophobic drugs. However, for accelerated drug absorption, supersaturated solutions need to be employed. In this work, a novel prodrug/enzyme based system was developed wherein prodrug and enzyme are coadministered at the point of absorption (e.g., nasal cavity) to form in situ supersaturated drug solutions for enhanced bioavailability. A combination of fosphenytoin/alkaline phosphatase was used as a model system. Prodrug conversion kinetics were evaluated with various prodrug/enzyme ratios at pH 7.4 and 32 °C. Phenytoin permeation rates were determined at various degrees of supersaturation (S = 0.8−6.1), across confluent Madin Darby canine kidney II-wild type monolayers (a nasal epithelium model), with prodrug and enzyme spiked into the apical chamber. Membrane intactness was confirmed by measuring transepithelial electrical resistance and inulin permeability. Fosphenytoin and phenytoin concentrations were analyzed using HPLC. Results indicated that a supersaturated solution could be formed using such prodrug/enzyme systems. Drug absorption increased proportionately with increasing degrees of supersaturation; this flux was 1.5−6 fold greater than that for the saturated phenytoin solution. The experimental data fitted reasonably well to a two compartment pharmacokinetic (PK) model with first order conversion of prodrug to drug. This prodrug/enzyme system markedly enhances drug transport across the model membrane. Applied in vivo, this strategy could be used to facilitate drug absorption through mucosal membranes when absorption is limited by solubility. KEYWORDS: poor water solubility, rapid absorption, supersaturation, MDCK membrane, prodrug, enzymatic conversion

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INTRODUCTION Numerous drugs and drug candidates suffer from low aqueous solubility, limiting their bioavailability when administered orally, intravenously, or by other parenteral routes. On the other hand, low aqueous solubility is often correlated with high potency at the site of action and with the ability of the drug to cross lipidic membranes. Prodrug approaches, where the native hydrophobic drug is derivatized to a bioavailable hydrophilic form that can be converted by endogenous enzymes to the native drug, have been utilized in attempts to “rescue” water insoluble drug candidates or to enhance the usefulness of established drugs.1,2 In this communication, we provide in vitro data suggesting a new drug delivery strategy based on prodrug conversion, in which a water-soluble prodrug and its converting enzyme are codelivered at a parenteral point of administration such as the nasal or buccal mucosa. Enzymatic conversion produces drug in concentrations exceeding the drug’s thermodynamic solubility, or saturation level. Given enough time, the drug will crystallize and lose its bioavailability. However, if the supersaturated drug can cross the mucosal membrane quickly enough, as a result of its high thermodynamic activity, then crystallization will be bypassed. We envision that this strategy will be particularly © 2013 American Chemical Society

useful when fast action is required, for example, in preventing or responding rapidly to Status Epilepticus (SE) or other cerebral conditions such as migraine. In support of this principle, we have developed an in vitro model in which fosphenytoin, a water-soluble phosphoester prodrug of the antiepileptic phenytoin is introduced on the apical side of (nasal epithelium model) Madin-Darby canine kidney-wild type (MDCKII-wt) membranes, along with a small amount of the converting enzyme alkaline phosphatase. Native phenytoin’s aqueous solubility is very low,3 so it will cross the membrane very slowly. However, when phenytoin is rapidly produced by enzymatic conversion, its concentration can exceed its thermodynamic solubility by several-fold, and it therefore should cross the membrane more rapidly. This principle, carried over to nasal delivery, could be useful for treating neurological emergencies, including SE. Received: Revised: Accepted: Published: 3519

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Figure 1. (a) Prodrug conversion rate as a function of fosphenytoin (prodrug) concentration with 0.4 U/mL alkaline phosphatase enzyme. Symbols represent the experimental data, and the regression curve is fitted to the Michaelis−Menten equation. (b) Fosphenytoin (prodrug) disappearance kinetics as a function of enzyme concentration (U/mL) with fixed initial fosphenytoin concentration. Curves represent data fitted to eq 2. (c) Phenytoin (drug) appearance rate as a function of enzyme concentration. These reactions were performed in assay buffer, pH 7.4 at 32 °C in an orbital shaker. Mean ± SD n = 3.

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EXPERIMENTAL SECTION Materials. Fosphenytoin disodium, phenytoin (HPLC grade), tolbutamide (internal standard), trifluoroacetic acid (HPLC grade), alkaline phosphatase from bovine intestinal mucosa (MW ∼ 160 kDa), and chemicals used for “assay buffer” preparation were purchased from Sigma. Scintillation cocktail (ScintiSafe Econol), HPLC grade acetonitrile and water, were purchased from Fisher Scientific. Dulbecco’s modified Eagle’s medium (DMEM), antibiotics, and fetal bovine serum (FBS) were purchased from Invitrogen. 14Cinulin (specific activity 1−3 μCi/g) was purchased from American Radiolabeled Chemicals, Inc. Madin-Darby canine kidney wild type cells (MDCKII-wt) cells were generously provided by Dr. Alfred Schinkel (The Netherlands Cancer Institute, Amsterdam). HPLC Method Development and Validation. Concentrations of fosphenytoin and phenytoin were determined by HPLC (Beckman Coulter SYSTEM GOLD: solvent module 126, autosampler 508, and UV detector 166, attached to a computer with 32.0 Karat software, version 5.0). For chromatographic separation, the stationary phase was a Zorbax XDB Eclipse C18 (50 × 4.1 mm, 3.5 μm particle size) analytical column attached behind a Zorbax XDB Eclipse C18 (12.5 × 4.1 mm, 5.0 μm particle size) guard column. The mobile phase was acetonitrile/water (30:70 v/v) with 0.1% v/v trifluoroacetic acid (TFA) as the ion-pairing reagent. Pump flow rate was 1 mL/min with run time 10 min. Samples were diluted appropriately in the mobile phase containing 7.4 μM tolbultamide as the internal standard. Then, 50 μL of sample was injected onto the column and UV absorbance was detected at 210 nm. Drug concentrations were obtained from peak area ratios (drug peak area divided by the area of internal standard

obtained from the same injection) using calibration curves prepared with standard drug solutions. A typical HPLC chromatogram for phenytoin, fosphenytoin, and the tolbutamide standard is shown in Figure S1, and the HPLC method validation is summarized in Table S1 (see the Supporting Information). Equilibrium Solubility. An amount of 10 mg of phenytoin was added to a 5 mL glass vial containing 2 mL of assay buffer, pH 7.4 (122 mM NaCl, 25 mM NaHCO3, 10 mM glucose, 10 mM HEPES, 3 mM KCl, 1.2 mM MgSO4, 1.4 mM CaCl2, and 0.4 mM K2HPO4). The vials were placed in a shaker incubator at different temperatures (28, 32, and 37 °C). After 48 h, drug suspension in the vials was centrifuged at 13 000g for 20 min. Using a 0.2 μm syringe filter, the supernatant was filtered into a fresh glass vial and analyzed using HPLC. The experiments were performed in triplicate. Preparation of Supersaturated Solutions. Supersaturated phenytoin solutions were prepared by incubating the enzyme with appropriate molar concentrations of prodrug (equivalent to their respective phenytoin concentrations upon complete conversion) in assay buffer, pH 7.4 at 32 °C. Considering rapid conversion of prodrug to drug (at optimal enzyme concentration), the degree of supersaturation, S, was calculated using the formula: S=

initial molar concentration of prodrug molar concentration of phenytoin in its saturated state

Evaluation of Enzyme Kinetics. Enzymatic conversion of fosphenytoin (prodrug, 12.3 mM stock) to phenytoin (drug) was carried out using alkaline phosphatase (enzyme, 14.34 U/ mL or 12 μM stock) in assay buffer, pH 7.4. For prodrug activation, appropriate volumes from stock solutions of enzyme 3520

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Figure 2. (a) Permeability of phenytoin across MDCKII-wt monolayer from its saturated solution (symbols). The curve represents the data fitted to eq 3. (b) Accumulation rate (on the basal side of monolayer) of phenytoin (symbols) produced from prodrug-enzyme mixtures prepared with various initial prodrug concentrations (μM). S represents the corresponding degree of supersaturation. Curves represent predictions from eq 4. (c) Initial phenytoin flux at different S values obtained from data (symbols) in (b). (d) Concentration−time profile for fosphenytoin-enzyme reaction (S = 6.1, cenz = 0.6 U/mL) on the apical side of MDCKII-wt membrane. Horizontal (red) line represents phenytoin saturation level (cd,sat). (e) Phenytoin amount produced from prodrug-enzyme mixture (S = 6.1, cenz = 0.6 U/mL) in apical compartment (symbols) compared to predicted values (solid line) obtained using eq 5. These experiments were performed in assay buffer, pH 7.4 at 32 °C using 12-well Transwell plates. Mean ± SD n = 3.

buffer (1 mL) placed in the basal chamber. The transwell plate was placed at 32 °C in an orbital shaker at 60 rpm. Aliquots were withdrawn from the apical side (quenched with methanol) and the basal side at various time points and analyzed for drug and prodrug using HPLC. Fosphenytoin, phenytoin, enzyme, buffer, untreated cells and blank filters were used as controls.

and prodrug were diluted in prewarmed assay buffer (0.9 mL final volume) to obtain the desired concentrations. From these solutions, 0.1 mL aliquots were immediately separated into 2 mL glass vials, closed, and kept at 32 °C (approx. temperature of nasal epithelium) in an orbital shaker (Shellab, Cornelius, Oregon) at 60 rpm. At each time point (0, 5, 10, 15, 30, 45, and 60 min), one vial was withdrawn and 0.9 mL of methanol was added to quench the enzymatic reaction. Samples were analyzed for prodrug and drug by HPLC. Buffer only and prodrug alone (no enzyme) were used as negative controls. Cell Culture. MDCKII-wt cells were cultured in DMEM supplemented with 10% (v/v) FBS and antibiotics (100 mg/ mL streptomycin, 100 U/mL penicillin, and 250 ng/mL amphotericin B). Cells were grown in T-25 flasks incubated at 37 °C, in a 5% CO2 atmosphere. At confluency, the cells were trypsinized and seeded at 2 × 105 cells/mL in a 12-well Transwell plate (0.4 μm pore size, polyester, Corning). Medium was replaced every second day until a cell monolayer was observed (∼4 days). MDCKII-wt cells with passages between 20 and 30 were used. Evaluation of Monolayer Integrity. Monolayer integrity was determined by transepithelial electrical resistance (TEER) measurements and inulin permeability studies, as described in the Supporting Information. Membrane Permeability. Fosphenytoin (different concentrations) and alkaline phosphatase (fixed concentration) were spiked into the apical side (0.2 mL) of the MDCKII-wt monolayer membrane (in Transwell), with drug-free assay

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RESULTS Equilibrium Solubility. Phenytoin solubility at pH 7.4 and 32 °C was found to be 126.5 ± 5.6 μM. Solubility was unaffected by a few degrees of change in temperature (28 and 37 °C). Enzyme Kinetics. To determine the enzyme’s kinetic parameters, initial conversion rates were measured for varying concentrations of prodrug, with enzyme concentration fixed at 0.4 U/mL. Figure 1a shows the amount of conversion as a function of prodrug concentration (cp) after 10 min. Data were well fit by the Michaelis−Menten equation, V=

Vmaxc p KM + c p

(1)

with KM = 827.8 ± 81.6 μM (SEM) and Vmax = 51.1 ± 1.8 μM/ min. Further studies were carried out with different enzyme concentrations but at fixed initial prodrug concentration, c0p = 586 μM. As expected and shown in Figure 1b and c, conversion of prodrug to drug was accelerated with increasing enzyme concentration, cenz. 3521

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cap(0), in μM, along with the ratio S = cap(0)/cd,sat, which represents the degree of supersaturation that the solution would attain if all of the prodrug was immediately converted to drug. By convolving the models for conversion (eq 2) and permeation/distribution (eq 3), we arrive (Supporting Information) at a prediction for drug accumulation on the basal side:

Since the initial prodrug concentrations were all appreciably below KM, we took the liberty to characterize the conversions as pseudo-first-order, with prodrug concentration kinetics, c p(t ) = c p0 e−kconvt ,

t>0

(2)

where kconv = (Vmax/KM)cenz = kcatcenz, with kcat = 1.73 × 105 min−1. Curves in Figure 1b represent back fits of eq 2 to the data. Complete conversion of prodrug to drug is confirmed by Figure 1c. Membrane Permeability. The MDCKII-wt (monolayer) membranes were tested for permeability to both drug and prodrug. Each of these molecules was spiked on the apical side of the monolayer membrane at or below its saturation, with no added enzyme. Accumulation was measured on the basal side. Taking into account distribution of drug into both the basal and apical sides, results were fitted by the equation, dosex −( 1 + 1 )CL t cxb = [1 − e Va Vb x ], Va + Vb

t>0

cdb(t )

⎡ dose p ⎢ −k t = ⎢1 − e conv − kconv Va + Vb ⎢ ⎣ ⎤ 1 1 e−kconvt − e−( Va + Vb )CLdt ⎥ ⎥ 1 1 ⎥ + CL − k d conv ⎦ Va Vb

(

)

(4)

Curves calculated on this basis are plotted with data in Figure 2b, and agreement between predictions and measurements is reasonable. Notably, accumulation rates (flux) are proportional to S (Figure 2c) and these were 1.5- to 6-fold greater (at S ≥ 2) than the flux obtained with saturated phenytoin solution (Figure 2a). Mass balance considerations, in which drug in the cell monolayer is regarded as negligible, lead to the following expression (Supporting Information) for drug concentration on the apical side:

(3)

where x refers to drug (d) or prodrug (p), cxb is the concentration (μg/mL) on the basal side, Va and Vb are the volumes of the apical and basal sides, respectively, and CLx is the membrane’s clearance (permeability-area product) to x. As shown in Figure 2a, drug accumulated in the basal compartment according to eq 3, with CLd = 0.0833 ± 0.0051 mL/h. Without enzyme, prodrug did not appear on the basal side, although drug was detected on both the apical and basal sides. This observation is consistent with prodrug being charged and hydrophilic/lipophobic, while drug is hydrophobic/lipophilic. This drug, which must have been converted by endogenous enzyme, appeared very slowly, with less than 30% conversion after 3 h. This is due to a scarce amount of alkaline phosphatase in the apical (luminal) side of the MDCKII cell membrane.4 We therefore neglect prodrug permeation and endogenous conversion in the following analysis. In the final set of experiments, prodrug was dosed into the apical compartment in the presence of enzyme (0.6 U/mL). Conversion of prodrug to drug on the apical side (by exogenous enzyme) was followed by drug permeation across the membrane to the basal side, as diagrammed in Figure 3. Results obtained with a series of prodrug concentrations are shown as symbols in Figure 2b. The label represents initial molar concentration of prodrug introduced into the apical side,

⎡ ⎢ dose Vb p −k t cda(t ) = kconv ⎢1 − e conv + Va + Vb ⎢ Va ⎣ ⎤ 1 1 e−kconvt − e−( Va + Vb )CLdt ⎥ ⎥ 1 1 ⎥ + CL − k d conv Va Vb ⎦

(

)

(5)

The data obtained for prodrug conversion (prodrug at S = 6.1, cenz = 0.6 U/mL) on the apical side is represented by Figure 2d along with a horizontal line corresponding to cd,sat. At this value of S, drug produced on the apical side exists in the supersaturated state for a significant period, leading to faster transport by the mechanism under study compared with administration of a saturated drug solution. If instead drug were to crystallize on the apical side when its concentration exceeded its solubility limit, then the rate of accumulation of drug on the basal side would exhibit a ceiling independent of the apical prodrug dose, contrary to observation. In addition, no turbidity of the apical side was detected, consistent with absence of crystal growth. The data in Figure 2d for phenytoin concentration in the apical side was compared to predictions based on eq 5. As seen from Figure 2e, the observed phenytoin concentrations are slightly lower than predictions. This could be due to a slight alteration in membrane permeability (shown by relatively low TEER, Figure S3, Supporting Information), causing faster drug transport. Several controls were run. Details are presented in the Supporting Information. First, it was shown that the presence of enzyme on the apical side did not alter transport of drug when the latter was administered apically (Figure S2). Second, TEER studies demonstrated that membrane integrity was not

Figure 3. Schematic representation of a typical transwell representing apical (top) and basal (bottom) compartments separated by MDCKIIwt monolayer membrane. Prodrug conversion via the enzyme (Enz) on the apical side produces the drug that permeated through membrane into the basal side. Drug is considered to be distributed between apical and basal sides. 3522

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compromised by the enzyme or by prodrug at low (S = 0.8) or high concentrations (S = 6.1), while at high prodrug concentrations (S = 6.1) with 0.6 U/mL enzyme concentration, there is statistical evidence for minor compromise of intercellular tight junctions (phenytoins apparent permeability coefficient was unaffected) (Figure S3). However, the TEER value with this treatment was over the (lowest acceptable) limit of 60 ohms/cm2.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 612-624-6164. Fax: 612-626-2125. E-mail: siege017@ umn.edu. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the AHC Faculty Research Development Program at the University of Minnesota for research funding (grant #180311406-21287-3672675, PI G. Georg). We also thank Prof. William Elmquist for providing lab facilities to perform cell studies, Shruthi Vaidyanathan and Dr. Raj Mittapalli for providing training in the Transwell experiments, and Prof. Karunya Kandimalla for providing the HPLC equipment. Discussions with Profs. Gunda Georg, James Cloyd, and Edward Patterson are gratefully acknowledged.

DISCUSSION AND CONCLUSIONS

Supersaturation has long been proposed as a means to improve the bioavailability of low solubility, high permeability (BCS Class II) drugs.5,6 Formulating drug as a high solubility crystalline polymorph or as an amorphous solid has been studied as a means to achieve at least temporary supersaturation in the GI tract.7−9 Solid dispersion of drug in a glassy polymer by spray drying10−12 or by quenching a hot melt13−15 of drug in polymer has also been studied. Kinetic stability of these drug dosage forms must be demonstrated under a variety of storage conditions. Alternatively, one may wish to create a supersaturated drug solution at the point of administration. Rapid change in drug activity can be brought about by sudden change in solvent environment16−18 or by rapid change in pH.19,20 If these changes are made at or just before contact with the site of absorption, and if the now supersaturated solution permeates the absorbing membrane faster than it can crystallize, then significant improvement in bioavailability and absorption rate is expected. For drugs that need to be fast acting, these properties are desirable. For example, Hou and Siegel demonstrated that adding water to a saturated diazepam-in-water/glycofurol solution drove diazepam into a supersaturated state, which was stable long enough to cross synthetic membranes several fold faster than saturated diazepam.21 A limited clinical pharmacokinetic study provided evidence for rapid absorption of supersatured diazepam administered intranasally, but the formulation was intolerable to the human subjects.22 The present study suggests that supersaturation and increased rate and degree of absorption can be achieved in aqueous solution by enzymatic conversion from a highly watersoluble prodrug. The phenytoin/fosphenytoin/alkaline phosphatase/MDCKII-wt system was chosen to demonstrate the principle because the prodrug is commercially available, the enzyme reaction is well understood, and because MDCKII-wt is often used as a model for nasal mucosa.21,23 Applications of this principle to other fast acting low solubility drugs might also be contemplated. Confirmation of this principle in vivo is an obvious next step, but there may be certain challenges, including endogenous species that interfere with the prodrug/ enzyme reaction, and poor formulation retention at the site of administration.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

HPLC chromatogram for fosphenytoin, phenytoin, and internal standard (tolbutamide) (Figure S1), HPLC validation parameter (Table S1), phenytoin flux in the presence or absence of enzyme (Figure S2), %TEER of MDCKII monolayer in the presence of various treatments (Figure S3), derivation of eqs 4 and 5, and methods for TEER measurement and inulin permeability. This material is available free of charge via the Internet at http://pubs.acs.org. 3523

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(15) Zheng, X.; Yang, R.; Tang, X.; Zheng, L. Part I: Characterization of Solid Dispersions of Nimodipine Prepared by Hot-melt Extrusion. Drug Dev. Ind. Pharm. 2007, 33 (7), 791−802. (16) Davis, A. F.; Hadgraft, J. Effect of supersaturation on membrane transport: 1. Hydrocortisone acetate. Int. J. Pharm. 1991, 76 (1−2), 1− 8. (17) Iervolino, M.; Raghavan, S. L.; Hadgraft, J. Membrane penetration enhancement of ibuprofen using supersaturation. Int. J. Pharm. 2000, 198 (2), 229−238. (18) Santos, P.; Watkinson, A. C.; Hadgraft, J.; Lane, M. E. Enhanced permeation of fentanyl from supersaturated solutions in a model membrane. Int. J. Pharm. 2011, 407 (1−2), 72−77. (19) Zhang, J.; Sun, M.; Fan, A.; Wang, Z.; Zhao, Y. The effect of solute-membrane interaction on solute permeation under supersaturated conditions. Int. J. Pharm. 2013, 441 (1−2), 389−394. (20) Hsieh, Y.-L.; Ilevbare, G. A.; Van Eerdenbrugh, B.; Box, K. J.; Sanchez-Felix, M. V.; Taylor, L. S. pH-Induced Precipitation Behavior of Weakly Basic Compounds: Determination of Extent and Duration of Supersaturation Using Potentiometric Titration and Correlation to Solid State Properties. Pharm. Res. 2012, 29 (10), 2738−2753. (21) Hou, H.; Siegel, R. A. Enhanced permeation of diazepam through artificial membranes from supersaturated solutions. J. Pharm. Sci. 2006, 95 (4), 896−905. (22) Ivaturi, V. D.; Riss, J. R.; Kriel, R. L.; Siegel, R. A.; Cloyd, J. C. Bioavailability and tolerability of intranasal diazepam in healthy adult volunteers. Epilepsy Res. 2009, 84 (2), 120−126. (23) Charlton, S. T.; Davis, S. S.; Illum, L. Evaluation of bioadhesive polymers as delivery systems for nose to brain delivery: In vitro characterisation studies. J. Controlled Release 2007, 118 (2), 225−234.

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