Evaluation of in vitro Models for Screening Alkaline ...dmd.aspetjournals.org/content/dmd/early/2009/04/16/dmd.108.0262… · The highest amount of ALP was detected in rat intestinal

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Evaluation of in vitro Models for Screening Alkaline

Phosphatase Mediated Bioconversion of Phosphate Ester

Prodrugs

Haodan Yuan, Na Li and Yurong Lai

Department of Pharmacokinetics, Dynamics, and Drug Metabolism, Pfizer Global

Research & Development, St. Louis Laboratories, Pfizer Inc, St. Louis, MO

DMD Fast Forward. Published on April 16, 2009 as doi:10.1124/dmd.108.026245

Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on April 16, 2009 as DOI: 10.1124/dmd.108.026245

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Running title: In vitro models for phosphate prodrug conversion.

Corresponding authors:

Yurong Lai

Department of Pharmacokinetics, Dynamics, and Drug Metabolism, Pfizer Global

Research & Development, St. Louis Laboratories, Pfizer Inc, St. Louis, MO

[email protected];

Number of text pages:

Number of tables: 1

Number of Figures: 5

Number of references: 19

Number of words in Abstract: 216

Number of words in Introduction: 664

Number of words in Discussion: 767

Abbreviations: Caco-2, human colon carcinoma cell; MDCK, Madin-Darby Canine

Kidney Cells; ALP, Alkaline phosphatase.


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ABSTRACT:

Generating a phosphate prodrug is one of the common approaches for

circumventing poor solubility issues of a parent drug. Alkaline phosphatase level was

determined in rat intestine mucosa scraps, Caco-2 cell and MDCK cell to characterize in

vitro models for alkaline phosphatase mediated phosphate prodrug conversion. In

addition, fosphenytoin and fosfluconazole were used as probe prodrugs to evaluate the

models. The highest amount of ALP was detected in rat intestinal mucosa scraps, while

alkaline phosphatase in 5-day cultured MDCK cells was minimal. As anticipated,

alkaline phosphatase levels correlated with the parent drug conversion, and the shortest

cleavage half-life (t1/2) was observed in rat mucosa scraps and MDCK cells showed the

slowest conversion. Furthermore, the polarized conversion for the prodrugs was observed

in Caco-2 monolayer cells, suggesting the polarized localization of alkaline in

differentiated Caco-2 cells. The rate of alkaline phosphatase mediated conversion was

prodrug concentration dependent with Michaelis-Menten constants of 1160 μM and 351

μM for fosphenytoin and fosfluconazole, respectively, determined in Caco-2 cells. The

results revealed that while the intestinal mucosa scraps reserved the highest ALP

activities and demonstrated as a promising in vitro tool for screening the bioconversion of

phosphate prodrug, Caco-2 monolayers could provide the predictive information of

bioconversion and further offer the capability in characterizing the permeability of

prodrug and parent drug.


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INTRODUCTION

Poor aqueous solubility could be a serious barrier for the successful delivery of a

therapeutic candidate for both oral and parenteral administrations. For oral delivery,

drugs with poor aqueous solubility often show low or erratic bioavailability, such as large

pill size or/and certain food and water restrictions are required. In parenteral use, the

drug candidates with poor water solubility often requires a large injection volume for

dissolving the drug mass and longer injection time, which may cause side effects, such as

local irritation at the injection site or erratic systemic delivery due to the possible

crystallization at the local administrative site. Phosphate prodrugs are one of the effective

and most commonly used approaches employed to overcome these issues in drug delivery

(Fleisher et al., 1996). By introducing an ionizable phosphate group to the parent drug

molecule, phosphate prodrugs are usually highly water soluble. Most importantly

phosphate prodrugs are readily cleaved by alkaline phosphatase (ALP), an enzyme

widely distributed in plasma and variety of tissues, to their parent drugs (Fleisher et al.,

1985; Fleisher et al., 1996). This approach has been successfully used for a number of

oral and parenterally administered drug candidates on the market (de Jong et al., 1997).

Due to the introduction of ionic and polar features, a phosphate prodrug usually

has inherent low permeability than its parent drug molecule. There is abundant ALP

expressed on intestine epithelial membrane cells, therefore, when orally administrated, a

phosphate prodrug is rapidly cleaved to its parent drug before the parent drug is absorbed

into the systemic circulation (Fleisher et al., 1985; Amidon et al., 1995; Kasim et al.,

2004). To achieve the maximal absorption, it is ideally required that the poorly soluble

parent drug possesses high permeability, known as class II compounds in the


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biopharmaceutical classification system (BCS) (Amidon et al., 1995; Fleisher et al., 1996;

Kasim et al., 2004) . The anti-viral drug, fosamprenavir, serves as an example of a

phosphate prodrug converted its parent drug, amprenavir, by ALP located in intestine

epithelial membranes. The highly permeable parent drug, amprenavir, is subsequently

rapidly absorbed (Becker and Thornton, 2004; Furfine et al., 2004). However, there are

many phosphate prodrugs which fail to achieve an acceptable oral profile, so the

phosphate prodrug approach still remains a challenge for providing an oral delivery

option. Therefore, efficient in vitro screening tools for phosphate prodrug bioconversion

and absorption assessment are needed at an early stage of drug discovery in the

pharmaceutical industry.

In in vivo, ALP is broadly distributed in plasma, the brush broader membrane of

gastrointestinal tract and other soft tissues. ALP is also used as a marker of cell

differentiation and polarity in in vitro cell cultures, as epithelial cells have demonstrated

an asymmetric distribution of ALP on their luminal surface (Lai et al., 2002). Human

colon carcinoma cell line (Caco-2) is spontaneously differentiated and forms monolayer

structure that highly mimics the intestine epithelium when cultured in vitro. ALP levels in

Caco-2 cells are considered to be similar to that in human small intestine (Pinto et al.,

1983). Therefore, the Caco-2 cell monolayer has been utilized for the evaluation of

phosphate prodrug bioconversion and permeation (Heimbach et al., 2003). However,

long-time culture of Caco-2 cell to achieve the differentiated characteristics limited the

application of this model as a high throughput tool for the drug screening. Recently,

Madin-Darby Canine Kidney (MDCK) cells have become an alternative to Caco-2 cells

for high throughput screening of cell permeability of new chemicals entities, because it


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requires less time to differentiate to form the monolayer morphology. Although ALP has

been detected as a marker enzyme for differentiation of MDCK cells as well (Lai et al.,

2002), the efficiency of ALP mediated bioconversion of phosphate prodrugs in this cell

model remains unknown. In this study, we investigated ALP mediated conversion of

probe phosphate prodrugs, fosphenytoin and fosfluconazole, in rat intestine mucosa

scraps, Caco-2 cells and MDCK cells. The results will provide the information for the in

vitro model selection in phosphate drug screening.

MATERIALS AND METHODS

Chemicals and Reagents

Fosfluconazole and fosphenytoin (Figure 1) were synthesized internally by Pfizer

Global Research and Development. HPLC grade acetonitrile and water was purchased

from Burdick & Jackson (Muskegon, MI) and EMD Chemicals, Inc (Gibbstown, NJ),

respectively. The ALP quantification kit was purchased from Sigma-Aldrich (St. Louis,

MO). Dulbecco's Modified Eagle Medium (DMEM), minimum essential medium

(MEM), fetal bovine serum (FBS), non-essential amino acids, Glutamax, sodium

pyruvate, gentamicin, L-glutamine and Hank’s balanced salt solution (HBSS) were

purchased from Invitrogen (Carlsbad, CA).

Rat Intestine Mucosa Scrap Preparation

Immediately after excising intestines from male Sprague-Dawley rats, a segment

of about 1 meter small intestine was placed in an ice cold stainless dish and then washed

with phosphate-buffered saline (PBS, pH 7.4) in the presence of phenylmethylsulphonyl

fluoride (PMSF, 1mM) for 1 min. Subsequently, mucosal layers were gently scraped off


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with a glass slide. The scrap was dipped in the buffer containing PMSF and protease

inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). The mucosa scrap was

dissolved in 22 mL of lysis buffer (Sigma Aldrich, St. Louis, MO). This solution was

used for the ALP protein and total protein level measurement. The Pfizer Institutional

Animal Care and Use Committee reviewed and approved the animal use in these studies.

The animal care and use program is fully accredited by the Association for Assessment

and Accreditation of Laboratory Animal Care, International.

Quantification of ALP in Caco-2, MDCK Cells, Rat Intestine Mucosa Scraps

The homogenate of rat intestine mucosa scrap were prepared as described above.

Caco-2 cells and MDCK were harvested at the day of cellular conversion assay and then

lysed in proper amount of cell lysis buffer (Sigma Aldrich, St. Louis, MO) containing

1mM phenylmethylsulphonyl fluoride (PMSF) and complete protease inhibitors cocktail

(Roche Diagnostics, Indianapolis, IN). The total protein concentration of individual

lysate was determined by BCA protein assay kit (Piece Biotechnology). ALP activity was

measured using ALP fluorescence detection kit (Sigma Aldrich, St. Louis, MO),

according to manufacture suggested protocol. Ten μl of the samples were incubated with

the ALP substrate, 4-methylumbeffiferyl phosphate disodium salt in the presence of the

dilution buffer and fluorescent assay buffer in the assay kit. Due to the large dynamic

range of ALP in different biological samples, the cell lysates were proper diluted to fit the

calibration curve. In addition, to minimize the matrix effect, the calibration curve was

prepared in the same cell lysis buffer at a range of concentration from 0.0125 to 1 ng/µl.

The fluorescence converted by ALP was determined at 360 nm excitation and 440 nm

emission using a spectrophotometer (SAFIRE, TECAN, Research Triangel Park, NC).


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The amount of ALP was determined by plotting in the standard curve prepared by control

enzyme in the assay kit, and normalized by the amount of total protein in each individual

samples.

Bioconversion Rate of Phosphate Prodrugs in Rat Intestine Mucosa Scraps

An aliquot of 200 µL of mucosa scraps lysate solution was mixed with 100mM

phosphate buffer (PH7.4) to a final volume at 1 ml. The concentration of the test

compounds (fosphenytoin and fosfluconazole) was 10 μM. The incubation media was

pre-warmed at 37ºC before the reaction was initiated by addition of the tested

compounds. An aliquot of 100 μL was collected from the incubation vial at the time

points: 0min, 5 min, 10 min, 20 min, 30min, 45min and 60min, and transferred to 96-well

plate, in which 100uL acetonitrile was pre-filled to terminate the reaction. The samples

were diluted 5 folds with acetonitrile containing 1 μM tolbutamide as an analytical

internal standard. The samples were centrifuged at 4000 rpm for 5 min to precipitate

protein. The supernatant was transferred to a new 96-well plate for concentrations

analysis by LC-MS/MS.

Bioconversion and Transwell Transport of Prodrugs in Caco-2 Cells

Caco-2 cells (ATCC, VA) were maintained in DMEM with 10% FBS, 1% non-

essential amino acids, 1% glutamax, 1 mM sodium pyruvate and 0.06 mg/ml gentamicin.

The cells were seeded at a density of 1×105 cells/cm2 in 24-well transwell plates

(Millipore, Billerica, MA) and cultured for 21 to 25 days before assay. Transepithelial

electrical resistance values were measured to ensure that tight junctions are formed

(>=600 ohms/cm2, Millicell-ERS; Millipore). After the transwell filter was washed three

times with HBSS (pH 7.4), each prodrug (10µM) was applied to the donor side (either


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apical or basal chamber) to initiate the bioconversion and subsequent transport in Caco-2

monolayer. The incubation was maintained at 37°C for 2 h under gentle shaking

(Precision Scientific, Winchester, VA). A 25 µl Aliquots was collected from both the

apical (A) and basal (B) sides at indicated time points, and compound concentrations

were determined by LC-MS/MS. The extent of permeation (Papp) was generated for both

A→B and B→A transport. Both parental compounds and prodrugs were monitored. The

concentration dependent bioconversions mediated by ALP were also conducted to

investigate the kinetics of bioconversion.

ALP Mediated Prodrug Bioconversion in MDCK Cells

MDCK cells were maintained in DMEM-α media supplemented with 10% FBS,

100 units penicillin and 100 μg/ml streptomycin, 1% L-glutamine and 1% NEAA. The

cells were grown on Millipore 24-well transwell plates for 4-5 days before transport

assay. Similar to Caco-2 cells, the transport and bioconversion assays were conducted in

live cells and initiated by applying the testing compounds to the donor chamber (either

apical or basal chamber). The following procedures were similar to Caco-2 assays

described above.

LC-MS/MS Analysis

An API 4000 triple quadrupole mass spectrometer (AB/MDS-Sciex, Concord,

Ontario, Canada) with a Turboionspray (TIS) interface operated in positive ionization

mode was used for the multiple reactions monitoring (MRM) LC-MS/MS analysis. An

AD20 quaternary micro pump (Shimadzu, USA) and a Agilent Zorbax Extend C18

(50mm×2.1 mm, 3.5µm particle size) column (Santa Clara, CA, USA) were used for the

chromatographic separation. The autosampler was a HTS-PAL from Leap Technologies


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(Carrboro, NC, USA). The mobile phases were water containing 0.1% formic acid (A)

and acetonitrile containing 0.1% formic acid (B). Chromatographic separation was

achieved by maintaining 90% A for 0.5 min isocratically and ramped to 90% B in 3.5

min, holding for 0.5 min followed by dropping to 10% B in 0.2 min and holding at 10%

B another 1.3 min before the next injection. The total run time was 6 min. The flow rate

was maintained at 0.4 mL/min . The mass spectrometric conditions were optimized for

fosphenyotin, phenytoin, fosfluconazole, fluconazole and tolbutamide (internal standard).

The following precursor→product ion transitions were used for multiple reactions

monitoring (MRM): fosfluconazole, m/z 385→79; fosphenytoin, m/z 363→237;

fluconazole, 307 → 220; phenytoin, m/z 253 → 182; and tolbutamide, m/z 272→155,

respectively.

Data Analysis

The concentration-time profiles were fitted to a one compartment model using

equation: At =A0e-kt, where A0 and At represent the prodrugs level at time zero and at time

t. k is the first-order elimination rate constant and t1/2 was calculated as 0.693/k. Data

were representative of a minimum of two experiments performed on different days with

different batches of cells or scrap preparations. The Michaelis-Menten constant for

prodrug conversions were estimated by the equation:

][

][max

SK

SVV

m +=

where V is the apparent linear initial rate, [S] the initial substrate concentration, Vmax is

the maximum bioconversion rate, and Km is the Michaelis-Menten constant.


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RESULTS

ALP in Intestinal Mucosa Scraps, MDCK and Caco-2 Cells

In the present study, we have evaluated the ALP amount in the in vitro models

that might potentially be employed for the screening of ALP mediated phosphate prodrug

bioconversion. As described above, the ALP levels in rat intestinal mucosa scraps, Caco-

2 and MDCK cells were quantified by an ALP fluorescence detection kit. The results are

shown in Figure 2. The ALP in rat intestine scraps exhibited the highest level among the

tested models. The ALP level detected in Caco-2 monolayers cultured for 21 days was

about 10-fold less, compared to the rat intestinal mucosa scraps. The 5-day cultured

MDCK monolayers expressed the least amount of ALP compared to other models.

Bioconversion of Fosphenytoin and Fosfluconazole in Intestinal Mucosa Scraps,

MDCK and Caco-2 Cells

Fosphenytoin and fosfluconazole were used as probe substrates to assess the

bioconversion of phosphate prodrugs. Initially, the disappearance of the phosphate

prodrugs was monitored during the incubation. As anticipated based on ALP levels, the

intestinal mucosa scraps demonstrated the shortest half-lives for the cleavage of

fosphenytoin and fosfluconazole, compared to MDCK and Caco-2 monolayers. The

overall clearance rate of both probe substrates was correlated with the amount of ALP

detected in the individual model. In addition, we observed that the disappearance of

fosphenytoin was faster than fosfluconazole in all tested models (Table 1). The apparent

half-life for fosphenytoin bioconversion in rat mucosa scraps was 1.2 mins (Table 1). In

contrast, a flat disappearance slope was found for fosfluconazole when incubating with

intestinal mucosa scraps, compared to bioconversion of fosphenytoin (Figure 3a). More


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flat disappearance slopes were observed in MDCK and Caco-2 monolayers for the

cleavage of both probe substrates (Figure 3b,c). The apparent half-life for fosfluconazole

bioconversion in intestinal mucosa scraps was 10 minutes (Table 1), while it was stable

in Caco-2 and MDCK cell monolayers with the half-lives 31 and 83 minutes,

respectively. The results revealed that there are at least two factors, chemical

structure/properties and ALP expression, determining the bioconversion of phosphate

prodrugs in in vitro systems.

The Phosphate Prodrug Bioconversion and Transport in Caco-2 Monolayer

Caco-2 cells are spontaneously differentiated after reaching confluence when

being cultured on a transwell membrane. The cells form polarized monolayers and

express ALP activity at their apical membranes in the levels similar to those found in the

human intestine (Pinto et al., 1983). In the present study, the ALP dependent

bioconversions of fosphenytoin and fosfluconazole in Caco-2 cells were also examined.

To investigate the polarized bioconversion and the transwell transport of phosphate

prodrugs in Caco-2 monolayer, 10 μM fosfluconazole or fosphenytoin was dosed either

in the apical or basal compartment in transwell plates. As shown in Figure 4, both

prodrugs were efficiently cleaved in the apical compartment following a 2-hour

incubation. The converted parent drugs, phenytoin and fluconazole, were detected in the

dosing chamber as well as in receiver chamber (Figure 4). In contrast, the bioconversions

for both prodrugs were minimal or undetectable when dosing in the basal chamber. The

results suggested the ALP mediated bioconversion occurred on apical side of Caco-2

monolayers. However, the prodrugs were not detectable in the receiver compartment

irrespective of dosing in the apical or basal chamber, corroborate the poor permeability of


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phosphate prodrugs. To further investigate the kinetics of ALP mediated bioconversion,

the concentration dependent ALP mediated bioconversions were conducted to determine

Michaelis-Menten Constant (Km) of prodrug bioconversion in Caco-2 monolayers. As

shown in Figure 5, the saturation curves of fosphenytoin and fosfluconazole with the

concentration increase were found. The estimated Km values of fosphenytoin and

fosfluconazole were 1160 µM and 357 μM, respectively.

DISCUSSION

Alkaline phosphatases are phosphatidylinositol-linked membrane hydrolase

enzymes responsible for removing phosphate moiety from many type of molecules. At

least four genes that encode distinct ALP proteins occur in different tissues: intestinal,

placental, placental-like and liver/bone/kidney alkaline phosphatases (Henthorn et al.,

1988). The ALP level in blood represents the total amount of alkaline phosphatases

released from these tissues. Based on the concept of in vivo bioconversion of alkaline

phosphatases, phosphate ester prodrugs are designed to successfully overcome drug

delivery problems that may compromise the therapeutic utility of a potential drug

(Boucher, 1996; Becker and Thornton, 2004). Through a direct incorporation of a

phosphate moiety into the hydroxyl or amine functionalities of a parent drug, for

instance, to form a phosphomonoester or attaching it to the parent drug via a chemical

linker phosphate prodrug gains sufficient aqueous solubility, adequate chemical stability

and undergoes quantitative in vivo bioconversion to the pharmacologically active drug.

The chemical linkers were basically used to enhance the enzymatic conversion due to the

steric hindrance. The linkage was made through the nitrogen on the indole moiety of the


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parent drug such as the prodrug of phenytoin, fosphenytoin (Luer, 1998). A methylene

spacer is also a very common linker that is used for the prodrug design. Such prodrugs

release the parent via a two-step conversion process, an ALP mediated dephosphorylation

followed by a fast spontaneous chemical breakdown of the hemiacetal intermediate

(Figure 1). However, in terms of bioconversion mediated by ALPs, not all phosphate

prodrugs undergo the desired rapid bioconversion to release the parent drug. For

example, reports have shown that phosphomonoester of secondary and tertiary alcohols

undergo slower rates of enzymatic conversion in vitro, while phosphomonoesters are

better substrates for ALP than phosphoramidates (Kearney and Stella, 1992; Safadi et al.,

1993; Vyas et al., 1993). Therefore, in vitro/ in vivo models for evaluating bioconversion

are potentially of great value at early stage of drug discovery.

In the present study, two probe compounds were selected as model compounds to

study the in vitro bioconversion. One is the fosfluconazole (Figure 1) (Diflucan® from

Pfizer Inc.), in which the phosphoryl group is linked on the tertiary alcohol of fluconazole.

Unlike the parent compound, fosfluconazole is more highly water-soluble (>300mg/mL

vs 1 mg/mL) and chemically stable in the solid state and in aqueous solution

(Yamreudeewong et al., 1993; Bentley et al., 2002). Another substrate used in present

study is fosphenytoin, in which the phosphoryl group is attached to NH group on the

indole ring of phenytoin through a self-cleavable linker, the hydroxymethyl group (Figure

1). With the hydroxymethyl linker, fosphenytoin conversion is fast and efficient in vivo

(8.1 minutes), while fosfluconazole is hydrolyzed by alkaline phosphatases in blood and

tissues with the t1/2 of 1.5 to 2.5 hours (Boucher, 1996; Sobue et al., 2004). Coupled with

the confirmation of ALP level, we concluded that clearance rate of phosphate prodrugs in


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an in vitro model can reflect the chemical structure/properties. Rat intestinal mucosa

scraps reserved ALP activities demonstrated as a promising model for in vitro phosphate

prodrug screening. In addition, as the preparation was isolated directly from tissues and

could be pooled and stored at freezer for repeatedly using, the bioconversion data

obtained from the model could offer the predictive information in in vivo situation.

Because of the similarity of the differentiated monolayer formed by Caco-2 cells

with human intestinal mucosa, Caco-2 cells have been widely used to predict drug

absorption in human. In the present study, Caco-2 cell showed the efficient bioconversion

for probe phosphate prodrugs (Figure 4). Simultaneously, the permeability of prodrug

and parent drug molecules could also be determined. Recently, due to labor savings on

cell maintenance, MDCK cells show promise as an alternative approach for permeability

screening for predicting the in vivo absorption and has been gradually accepted by the

pharmaceutical industry. However, the 5-day grown MDCK cells produced only minimal

amount of alkaline phosphatase, therefore, MDCK monolayer was not be able to

distinguish bioconversion rates related to diversified chemical spaces and might not be a

suitable tool for phosphate prodrug screening.

In conclusion, the intestinal mucosa scraps reserved the highest ALP activities

and demonstrated as a promising in vitro tool for screening the bioconversion of

phosphate prodrug. MDCK shows the promising on labor saving as a predictive tool for

drug absorption, however, it might not replace Caco-2 cells for the investigation of

phosphate prodrugs due to lack of efficient ALP mediated bioconversion. The special

value of Caco-2 cell model is that Caco-2 cells might not only provide the predictive


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information of bioconversion, but be able to characterize the permeability of the prodrug

and the parent drug.


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ACKNOWLEDGEMENT

We would like to thank Mrs. Kathy Sampson for the help with culturing the Caco-

2 cells. We would like to thank Dr. Timothy G. Heath for valuable scientific discussions.


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Boucher BA (1996) Fosphenytoin: a novel phenytoin prodrug. Pharmacotherapy 16:777-791.

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LEGENDS FOR FIGURES

Figure 1. Structures and bioconversion pathways for fosfluconazole to fluconazole (top)

and fosphenytoin to phenytoin (bottom).

Figure 2. Alkaline phosphatase levels in rat intestine mucosa scraps, Caco-2 cells and

MDCK cells.

Figure 3. The Bioconversion rate of fosphenytoin and fosfluconazole in (a) rat mucosa

scraps, (b) Caco-2 cells and (c) MDCK cells. Percentage activity remainings of the

prodrug probes, fosphenytoin and fosfluconazole, were monitored by LC-MS/MS.

Figure 4. Bioconversion of fosphenytoin and fosfluconazole in Caco-2 cells after 120 min

incubation. The prodrugs (10µM) were applied either on the apical or basal compartment

of a transwell plate. The parental drugs and prodrugs were monitored by LC-MS/MS.

Figure 5. Michaelis-Menten Plots for (a) fosphenytoin and (b) fosfluconazole

bioconversion to parent drugs mediated by ALP. The experiments were conducted in

Caco-2 cells. The incubation time was 10 min at 37˚C. The generation of parent drugs

was monitored.


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Table 1. Bioconversion half-life (t1/2) (min) of phosphatase prodrug in rat mucosa scraps,

MDCK cells and Caco-2 cells.

Rat Intestine Mucosa Scraps

Caco-2 MDCK

Fosphenytoin 1.2±0.041 18±4.6 31±6.8

Fosfluconazole 10±3.4 39±19 83±47


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Evaluation of in vitro Models for Screening Alkaline ...dmd.aspetjournals.org/content/dmd/early/2009/04/16/dmd.108.0262… · The highest amount of ALP was detected in rat intestinal