Selection of development candidates based on in vitro permeability measurements

advanced

drug delivery reviews Advanced Drug

Selection of development Chao-Pin

Delivery

Reviews 23

( 1997)

47-62

candidates based on in vitro permeability measurements

Lee”‘“, Remco L.A. de Vruehb, Philip L. Smith”

Received 25 June 1996: accepted 2X July

1996

Abstract

Based on the desire to develop orally active drug candidates, approaches have been evaluated to identify molecules with characteristics which not only allow them to he potent at their specific receptor but also to have the appropriate membrane properties to allow absorption across the cells lining the gastrointestinal tract. The Ussing chamber approach has been instrumental in advancing our understanding of the mechanisms involved in drug transport and is also being employed to screen large numbers of compounds for selection of clinical candidates. From these types of studies, it has been demonstrated that synthesis of an amino acid prodrug of the nucleoside antiviral, acyclovir, provides an approach for enhancing uptake across the apical cell membrane with subsequent intracellular hydrolysis and release of the parent molecule across the basolateral membrane. It is these types of approaches which should provide a more rapid and rational method for identification of clinical candidates in the future. Kqwordc:

Intestinal

permeability;

Absorption:

Transport;

Oligopeptide

transporter;

Apical

recycling;

Bioavailability;

Acyclovir

Contents

48 48 48 1.2. In vitro approaches ........................................................................................................................................................... 48 I .2.l. Solubility.. ............................................................................................................................................................. 4Y 12.2. pKa .......................................................................................................................................... I .2.3. Intestinal permeability .................................................................................................................................... so so Applications ...................................................................................................................................................................... so 2. I. In vitro/in vivo correlations.. .......................................................................................................................................... 52 2.2. Mechanistic studies ........................................................................................................................................................ 53 2.3. Formulation evaluation ..................................................................................................................................................... .. 54 2.4. EL aluatton ot metahohsm ................................................................................................................................................. 55 Case history.. .......................................................................................................................................................................... sx Conclusion5 ............................................................................................................................................................................ SY Acknowledgments . . . . . . . . . . . . . ..__.__.....................................................................................................,.......................,,,,,................. .,... .._..__....__. s9 References ..__.... . . . . . . . . . . . . . . ..__..........................................................................................................................,,.,,,, Introduction ............................................................................................................................................................................ I .I. In viva models.. ...............................................................................................................................................................

“Corresponding

Author

0169-409X/97/$32.00 Copyright PI/ SOl69-409X(96)00425-.5

0

1997 Elsevier Science B.V. All rights reserved

1. Introduction Based on ease of administration and hence enhanced patient compliance, the oral route of administration continues to be the preferred route for delivery of drugs. Due to the increase in time and resources associated with drug development. pharmaceutical companies are working to implement strategies for reducing the time required to identify and enter a new molecule onto the market. For many years, the process for oral drug discovery involved identification of the most active molecule for a particular target by the medicinal chemists. Once identified, this molecule was put into the development program. There are a number of drawbacks to this approach with the biggest obstacle being that many of the molecules which were put into development using this process had neither the physicochemical (e.g. sofubility, dissolution rate, chemical stability) nor biological (e.g. membrane permeability. enzymatic stability. pharmacokinetic profile) properties required for a rapid development program. Thus, formulation scientists and clinical pharmacologists were often called on to perform ‘magic’ with these molecules. This approach can result in long formulation development time for molecules and often in prolonged clinical trials due ttt low and variable bioavailability.

Rational design of molecules with appropriate characteristics for oral absorption requires that discovery scientists consider a number of features of the compound: ( I ) chemical and enzymatic stability: (2) appropriate physiochemical properties as discussed above; (3) transepithelial permeability; and (4) clearance mechanisms such as hepatic first pass effects. Often, the features of a molecule which impal-t

Scheme I.

enhanced stability, solubility. permeability or reduced hepatic clearance detract from a molecules inherent activity. However, it is becoming increasingly obvious that a molecule with more appropriate properties for oral absorption can often progress through development more rapidly. Thus, to understand the reasons for poor oral bioavailability, it is necessary to first evaluate the various barriers to drug absorption. The first step in evaluating molecules for oral absorption is to determine stability in the intestinal lumen. Molecules which are acid unstable or susceptible to enzymatic degradation may need to be formulated appropriately to protect them during transit to the site(s) of absorption in the gastrointestinal tract. The next step in the process of intestinal absorption is transit across the epithelial cells lining the gastrointestinal tract. Approaches to determine intestinal absorption in man have been described [I]. However, these techniques are resource intensive and due to the complexity involved in measuring intestinal absorption (e.g. portal blood sampling), these methods provide only a measure of disappearance from the intestinal lumen 1I]. As reported by Amidon and coworkers [ 11, a correlation has been generated between human jejunal permeability and fraction absorbed in man. Due to the limitations described above. animal models have been employed to generate intestinal absorption data. Whole animal studies with potential development candidates can provide information relevant to a number of the issues including enzymatic stability, intestinal absorption and hepatic tirst pass effects (see Stewart et al., this issue). Specialized animal models can be utilized to determine whether pool bioavailability is related to intestinal absorption or to [irat pass clearance [2,X1. However, from whole animal studies, it is not possible to identify reasons ~OI-low absorption from the gastrointestinal tract and. thus. chemical strategies to overcome poor intestinal absorption cannot be formulated on a rational basis.

A variety of approaches for predicting membrane permeability have been investigated. Physicochemcal approaches such as measurement of log P have been proposed as a method for selecting development candidates with appropriate membrane per-

C. Lee et al. I Advanced

Drug Deliwry

Reviews 23 (1997)

49

47-62

?_

Apical ‘1 L”mi”a’

Fig. I. Human oral bioavailability coefticient. log P.

vs. octanol/water

partition

meability. However, as shown in Fig. 1, there is no correlation between log P and oral bioavailability in man. Among the reasons for this lack of correlation are the presence of multiple pathways for intestinal absorption (e.g. transcellular vs paracellular, Fig. 2), carrier-mediated transport processes such as the oligopeptide transporter [4-61 and P-glycoprotein (Fig. -3) (7-101 or clearance mechanisms for molecules such as 5-fluorouracil and verapamil. Aqueous solubility/dissolution rate is another physicochemical property that has been hypothesized

Fig. 2. Routes and mechanisms for the transport of drugs and delivery systems across cellular barriers. The paracellular route is a passive. diffusional transport pathway taken by small, hydrophilic molecules (e.g. mannitol) which can pass through the tight junctions between adjacent epithelial cells ( I ). Transcellular routes include: carrier-mediated active transport of orally active p-lactama, ACE inhibitors and di/tripeptides (2a); passive diffusion of lipophilic molecules across cell membranes (2b): receptormediated transcytosis of surface-bound ligands such as intrinstic factor-cobalamin complexes, which are transported within vesicles (2~); and uptake of the products of fat digestion into the cell, followed by their reesterification and assembly into lipoprotein particles within the endoplasmic reticulum/Golgi apparatus and exocytotic release at the basolateral membrane (2d). Reprinted with permission [3X].

Fig. 3. Model for the apical efflux mechanism in intestinal epithelial cells - the apical (luminal, mucosal) surface of the intestinal epithelial cells has a transporter which is similar to the multidrug resistance transporter (MDR) or P-glycoprotein in tumor cells. Hydrophobic molecules (e.g. cyclosporin A, vinblastine, verapamil) have been shown to be recycled at the apical cell membrane of the human intestinal cell line, Caco-2 and intestinal tissues in vitro.

to influence oral bioavailabiltiy of drug molecules. An example was reported by Eldred et al. [ 1I] in a series of benzamidine containing fibrinogen receptor antagonists (Scheme 1). When the highly potent compounds 1 and 2 were dosed in the marmoset, low oral activity was observed. The lack of oral activity of these molecules was assumed to be due to poor absorption which in turn was claimed to be the result of the high basicity and their poor aqueous solubility. Oral activity was increased by introduction of a central piperazine ring (3), possibly as a result of improved solubility. However, no further studies were performed to confirm these conclusions. 1.22. pKcd Membrane permeability of lipophilic molecules with ionizable groups is normally dependent on the acidity or basicity (pK,) of the ionizable groups [ 121. Thus, it has been proposed that molecules which have a pK, close to the pH of the gastrointestinal lumen will have enhanced absorption compared to the ionized species. Approaches to eliminate charge in the structure without sacrificing biological activity (e.g. prodrug approaches or the use of isosteres to replace the ionizable group) are the most common ways to improve oral absorption of ionizable lipophilic molecules. One example is in the area of indane containing endothelin receptor antagonists (see Ellens et al., this issue). The pH dependent absorption of SB 217242 clearly demonstrates that the closer the pH is to the pK,, of the ionizable

group, the higher the percentage of the unionizable group and also the higher the rate of absorption. Another example is in the area of benzodiazepine containing GPIIb/llIa antagonists [ 131. As shown in Fig. 4, removal of both the negative and positive charges from the zwitterionic molecule 4 by esterihcation of the acidic group and elimination of the amidino group gave compound 6 which has the highest rabbit ileal permeability in this series. However, in the case of peptidomimetic agents such as the cephalosporins, the presence of acidic and basic functional groups in the molecule is required for optimal intestinal absorption [4,5,14]. Thus, prediction of intestinal absorption based on physicochemical properties is not a reliable approach for rational drug design. 1.23. Intestinal prrmubilit~ To overcome the limitations of whole animal studies and physicochemical approaches, many laboratories have developed in vitro techniques for determining intestinal epithelial permeability of molecules (e.g. Caco-2 cells grown as confluent monolayers, intestinal tissues). In comparison to in vivo studies, in vitro permeability studies provide several advantages: ( 1) requirement of less compound for evaluation; (2) relatively easier to conduct than absorption studies which may require complicated surgery and maintenance of surgically prepared

Fig. 4. In vitro permeabilities containing GPllb/IlIa

of a serves of henwdiazepine-

antagonists in rabbit ileum. Compound 6

with no charge at physiologic pH has the highest ileal permeahillty in this series.

animals; (3) more rapid and require less animal usage even when intestinal tissues are being employed since a number of variables can be evaluated in a single study: (4) allow for evaluation of mechanisms involved in transport; (5) provide information about metabolism of molecules which occurs during transport: (6) aid in formulation design and development of structure/transport relationships; and (7) provides samples for analysis which do not contain plasma proteins and can thus be analyzed more easily. This article will focus on the use of permeability measurements employing in vitro techniques to aid in the design of oral drug candidates. The in vitro techniques described in this article resulted from the early work of Ussing and coworkers who developed these systems for the measurement of ion transport across frog skin [1.5-201. During the past half century, these techniques have been applied to the study of the mechanisms and regulation of epithelial ion transport and drug transport in a variety of tissues including the intestine 121-261, skin 1271, lung [28], trachea [29J, buccal mucosa 1.301, nasal mucosa 13 1,321, and cultured epithelial cells grown as confluent monolayers 15,3X34].

2. Applications

Use of in vitro systems for studying drug absorption provides a number of advantages over conventional in vivo studies for rapidly identifying optimal drug candidates for entry into development. The utility of these systems is based on the ease with which they can be established in a laboratory, lack of a complex biological matrix (e.g. plasma, serum or whole blood) from which molecules are analyzed, rapid turn around of results to guide the medicinal chemists, ability to evaluate non-active molecules to provide information critical for development of structure/absorption relationships, and ability to evaluate transport mechanisms and metabolic pathways influencing drug permeation across an epithelial barrier. Although these in vitro systems do provide information regarding the metabolism and transport of molecules across the intestinal epithelium, they do not directly correlate with oral bioavailability. This is

C. Lee et al. I Advanced Drug Delivery Reviews 23 (1997) 47-62

necessarily the case since oral bioavailability is dependent on a number of factors including first hepatic clearance and solubility/dissolution rate. This point is illustrated in Fig. 5 which shows that although there is a general trend for molecules with greater permeability to have higher bioavailability, there are examples such as verapamil and Sfluorouracil which have lower bioavailabilities than predicted from their permeability values. This is related to their metabolism and clearance mechanisms. Thus, from Fig. 5, it can be seen that molecules which have high permeability will not necessarily have high bioavailability but molecules with low permeability will always have low bioavailability. Therefore, from this correlation it is possible to eliminate molecules with low permeability values from further in vivo evaluation. The use of confluent monolayers of cultured cells (e.g. Caco-2 cells) and/or intestinal tissues for studying drug transport has been established in a large number of academic as well as industrial laboratories around the world. Although these systems are relatively easy to establish for measuring epithelial or endothelial permeability, the major issue for generating meaningful data with these systems is to establish validation criteria which can be monitored regularly to identify problems [22]. Validation criteria include determination of transepithelial resistance and/or permeability of marker molecules as

a measure of cell monolayer or tissue integrity, evaluation of the permeability of marker molecules for appropriate transporters including the oligopeptide transporter (e.g. cephalexin), nutrient transporters (e.g. glucose, amino acids), P-glycoprotein (e.g. vinblastine) and establishment of permeability values for passive permeability markers which traverse the cellular pathway (e.g. acetaminophen, benzodiazepines). Establishment of these values in a particular laboratory is important since it has been demonstrated that although the relative transport rates/ permeabilities for molecules may be the same from different laboratories, the absolute values can vary significantly [3.5]. The reasons for these variations may be related to the particular subclone of epithelial cells being used, differences in incubation buffers (e.g. pH, ionic strength) or other as yet undefined laboratory specific differences. These reference data are essential for establishing selection criteria for screening molecules. With an increasing confidence in these in vitro systems for selecting molecules with appropriate transport characteristics for high oral bioavailability, there has been a greater emphasis on increasing the capacity of these systems. Presently, there are approaches being developed to automate drug transport studies in an attempt to meet the increased demands which are being generated by combinatorial chemistry approaches [ 361.

100

80

20

0 .ooi

.Ol

.l

1

Heal permeability (cm/h)

Fig. 5. Human oral bioavailability

51

vs. rabbit ileal permeability

In determining structural features which may restrict passage across an epithelial barrier and to aid in establishment of structure/transport relationships. it is often helpful to be able to dissect the molecule and evaluate the permeability of its components. This has been accomplished, for example. with a series of benzodiazepines [ 131. As shown in Fig. 4, removal of the charged amidino group of the Arg side-chain mimetic of 4 enhanced the transport rate across rabbit ileum by more than 200 fold. Removing the negative charge of the Asp side-chain mimetic by making an ethyl ester enhanced the transport rate by only 2-fold. These results suggest that the charge on the Arg side-chain mimetic plays an important role in determining the permeability of these non-peptide fibrinogen receptor antagonists. Fig. 6 shows the effect of size and lipophilicity of a series of benzodiazepine-containing non-peptide fibrinogen receptor antagonists. Changing the 4-N substituent on the benzodiazepine ring from the larger and more lipophilic phenethyl group ( 10) to :I smaller and less lipophilic methyl group (7) results in a IO-fold increase in ileal permeability. The mucosal (m)-to-serosal (s) and s-to-m transport rates are not significantly different for these two antagonists. Furthermore, the ileal permeabilities of these

antagonists are significantly higher than their distal colonic permeability. These results suggest that the Lwitterionic molecules, 10 and 7, follow a passive paracellular transport route and that the size of these molecules determines their rate of transport/absorption.

With in vitro studies, it is possible to evaluate both the m-to-s and s-to-m transport of a molecule. Since the transport of drugs may occur by a variety of mechanisms 1381 and these mechanisms may vary with the location of the molecule within the gastrointestinal tract (e.g. duodenum vs ileum or small vs large intestine). it is imperative for optimal drug design that the system employed for evaluation of drug absorption provide the flexibility to evaluate these mechanisms in various segments of intestine. The in vitro Ussing chamber approach or moditications of this approach provide the required flexibility 120,39,40]. From previous studies, the utility of intestinal tissue for evaluating differences in segmental absorption between molecules has been demonstrated. A comparison of cephalexin transport in the small and large intestine demonstrates several aspects of the information which can be obtained from in vitro studies 1261. From Fig. 7, it can be seen that the n-to-l; flux of cephalexin in the small intestine occurs by a mechanism which is enhanced as the pH of the mucoaal bathing solution is reduced from 7.4 to 5.5. However. the s-to-m flux of cephalexin and mannitol permeability are not altered when the ~m~osal bathing solution pH is reduced from 7.4 to

Fig. 7. pH dependence (mucod

Flp. h.

Rabhlt

ileal

pernlrahilitir\ of ;I wirs

contazning

GPIIh/IIIa

substituent

at the 4pwition

the ileal permeability.

antagonisr.

In this wk.

of the hewodiaqkvz

of bewodwep~nethe smaller

the

ring. the hlghrt

wrowl

pH constant

\r1-o~ill-to-111tIcos~l hsrh) Ruxes acre\, means z

I S.E.for

pH varies between 5 and 7.4 with

at pH 7.3)

cB ) crphalexin rahhit

ileum

tour animals.

OF nluCosUI-tO-\er(~\iII

(A)

(solid bars) and manmtol and chd Reprinted

colon.

and (open

Results

with permission

are 126).

C. Lee rf rd. / Advunced

Drug Delivery

5.5. Furthermore, from Fig. 7 it can be seen that cephalexin transport is less in the distal colon than in the small intestine and that a reduction in the mucosal bathing solution pH in the distal colon does not alter the transport of cephalexin. An additional feature of cephalexin transport in the small intestine, demonstrated by intestinal tissue studies, was the inhibition of this transport process by the inhibitor of Na’ /K+-ATPase, ouabain, and by the Na+/H’ exchange inhibitor, amiloride (Fig. 8). Together, these results have been used to support a model for absorption of cephalexin as well as other drugs via the oligopeptide transporter which exists in the small but not the large intestine and functions normally to ensure the efficient absorption of di- and tri-peptides [4]. Similar types of studies have demonstrated that amino acid transport occurs via a carrrier-mediated mechanism which exists in the small but not the large intestine [41]. In comparison to the active transport of cephalexin and arginine discussed above, there are numerous examples of molecules which are transported by passive mechanisms and thus there are only small or no differences in their m-to-s and s-to-m permeabilities or in their permeabilities in the large and small intestine ( 137,411 and Ellens et al., this issue). From Fig. 9, it can be seen that in both rabbit ileum and the human intestinal epithelial cells, Caco-2, grown as confluent monolayers, unidirectional fluxes, of diazepam and mannitol were identical suggesting

Rrvir,va

I.? (1997)

A

Diazepam M-S

0

Mannitol M-S

A

Diszepam S-M

l

Mannitol S-M

30

0

5.3

47-Q

60

90

120

IS0

180

Minutes

B

30 25 -

0 0 .

.

DiazepamM-S Mannitol M-S Diazepam S-M MannitoiS-M

20 1s 10 5o-. 30

0

60

Fig. Y. Mucosal-to-aerosal

90 Minutes

120

I

1

1.50

180

(open symbols) and serosal-to-mucosal

(close symbols) fluxes of diazepam and mannitol in rabbit ileum (A) (triangle for diaLepam and circles for mannitol) and human mtestinal cell monolayer, Caco-2

(B) (square for diazepam and

circles for mannitol).

L._t L.h that both transported

diazepam and mannitol are passively across the intestinal membrane.

2.J. Formulation

control

ouabain

Experimental

amiloride

Conditions

Fig. 8. Effects of serosal ouabain (0.1 mM) or luminal amiloride

(I

mM) on cephalexin (solid) and mannitol (open) fluxes in rabbit

ileum with pH 7.4 in both bathing solutions. Results are means t

I

S.E. for three nmmals. Reprinted with permission [26].

evaluation

In vitro permeability studies have also been beneficial in elucidating the potential mechanisms involved in formulation approaches designed to enhance oral absorption. It has been proposed that enhancers may provide a strategy for overcoming low oral bioavailability and a number of laboratories have investigated this approach [42-461. Effects of enhancers on morphology, nutrient and ion transport

and drug permeability of the intestinal mucosa have been evaluated with in vitro techniques in several laboratories 147-531. From these studies. morphologic changes in the intestinal epithelium have been demonstrated including transient denudation of the villus region (Fig. 10) resulting in increased permeability to ions and drugs (481 and a reduced nutrient absorptive capacity (Fig. 1 1) due to loss of active carrier mechanisms [47-49.52.531. As shown in Fig. 12 Fig. 13. following removal of the enhancer, rapid reversal of the epithelial damage to regain barrier function is observed [48,49,52,53].

The Ussing chamber technique has also been employed to evaluate intestinal metabolism by a number of investigators [ 54-631. Recently, interest in intestinal metabolism has focused on the activity of cytochrome P45O’s (CYP) in the intestine and their role in detoxification and limiting drug absorp tion. Kolars and coworkers 1641 have reported that CYP3A-related proteins are present throughout the

gastrointestinat tract including the gastric parietal cells, pericentral hepatocytes and ductular cells of the pancreas. In addition to CUP, the multidrug resistance (P-glycoprotein) efflux mechanism is expressed in the intestine and these two mechanisms appear to act in concert to increase the efficiency of xenobiotic metabolism. Schuetz and coworkers [6S 1 have reported that the human colon carcinoma cell lines LS 180/ADSO and LS 180/WT constitutively express both P-glycoprotein and CYP3A4 and that these proteins are upregulated by a number of drugs including rifampicin. phenobarbital, clotrimazole, reserpine and isosafrole while midazolan and nifedipine only upregulate P-glycoprotein. Thus, for a number of agents, it appears that P-glycoprotein and CYP3A4 are important modulators of drug absorption and drug/drug interactions. Further support for the role of CYP3A and P-glycoprotein in limiting intestinal metabolism has been provided by studies with cyclosporin A in Caco-2 cells [62]. In their study, Can and coworkers [62] provided evidence for an enhancement of cyclosporin metabolism due to the action of P-glycoprotein. From these

C. Lee et (11. I Advanced

Drug Drlil’er? Reviewx 2.7 (lY97)

z

120-

E

loo-

O"

80-

=

60-

8 E

95

47-62

4020-

wash Fig. 13. Reversibility

of CapMul

rabbit distal colon. CapMul mucosal

chamber

tissues). After

at time

Minute MCM-induced

MCM

(O-I %)

zero (60-120

IO min, CapMul

change in Rt in was added to the

min after

was removed

mounting

by rinsing the

mucosal chamber with 100 mL of mucosal buffer solution without CapMul MCM.

Data shown are means t I S.E. for S-IO

tissues

from five animals. Reprinted with permission [4X].

CapMul

I I.

Fig.

MCM

Change in 15L in rabbit ileum upon addition of mucosal

leucine (5 mM)

and glucose (IO mM),

and serosal PGE,

(0.01

mM) in the presence of different concentrations of CapMul MCM

results, it is evident that in vitro transport studies will be instrumental in advancing our understanding of intestinal mechanisms responsible for limiting absorption and strategies for avoiding these mechanisms.

in the mucosal chamber. Data shown are means t I S.E. for S-IO tissues from five animals, Response to L-Leucine

(wide cross

hatched); response to glucose (solid); response to PGE,

(narrow

3. Case history

cross hatched). Reprinted with permission 1481.

5 z s 0 z S _:

200looO-

-lOO-

-2+-----

50

0

wash

Fig. 12. Reversibility

of CapMul MCM-induced

rabbit distal colon. CapMul mucosal

chamber

tissues). After

100

at time

MCM

(O-l%)

zero (60-120

IO min, CapMul

150

Minute changes in I_ in was added to the min after

was removed

mounting

by rinsing the

mucosal chamber with 100 mL of mucosal buffer solution without CapMul

MCM.

Data shown are means i- I S.E. for 5-10

from five animals. Reprinted with permission [48].

tissues

Acyclovir [9-(2-hydroxyethoxymethyl)guanine] is an agent used for the treatment of infections caused by herpes viruses [66]. The low oral bioavailability of acyclovir has limited its usefulness (67-691. It has been reported that the oral bioavailability of acyclovir is highly variable and species dependent [67-691. It ranges from 75.3+ 1.3% in dogs to 3.7?0.5% in rhesus monkey. Poor water and lipid solubility of acyclovir (solubility in water of 0.13% and an octanol to water partition coefficient of 0.0 18) may contribute to its low gastrointestinal absorption [68,69]. The existence of a saturable, carrier mediated process in the oral absorption of acyclovir by mice, rats and dogs has been proposed based on a decline in the fraction of dose absorbed with increasing doses. On the other hand, the low oral bioavailabiltiy observed in man may simply be a function of poor membrane permeability due to the low partition coefficient of acyclovir. Meadows and Dressman [70] have investigated the uptake mechanisms of acyclovir by determining the uptake of acyclovir

both in vitro via an intestinal ring method and in vivo by an in situ single-pass perfusion technique in rats. Acyclovir uptake was linear in the concentration range 0.0 l-5 mM in vitro. Use of 2.4-dinitrophenol (DNP). ouabain or K’ substitituted buffer did not reduce the rate of acyclovir uptake. The in situ single-pass perfusion method yielded an intestinal wall permeabiltiy of 0.2. which did not vary consistently with increasing concentration. &perfusion of acyclovir with DNP did not decrease but slightly increased the wall permeability. In order to further elucidate the absorption mechanisms of acyclovir, we have measured both the m-to-s and s-to-m fluxes of acyclovir in vitro using

0.04

1

M”COSd-tO-SWO**l

Fig. 14. Mucosal-to-~ero\aI

and \ero~al-to-t~~ucos111 pel-meab!litic\

of acyclovir

in rabbit ileum.

S-10

tssues

from three animals.

Fig.

IS. Mucosnl-to-serohnl

permeabilitiea

solutions

mean-tS.E.M.

Data shown are meanh il

(tilled

across Caco- 2 cell were

buffered

S.E. fat

bars) and ~erOlrill-to-rnuCohitl

(open bars) of acyclovir

esters (0.X mM) bathing

SWOS~l4bM”C06~1

(0.X mM) monolayer~

at pH

7.4.

of at least three dekrminationv.

and rth Valyl tit 37°C.

Reults

arc

Both the

rabbit ileum (Fig. 14). Acyclovir showed low m-to-s transport consistent with its low oral bioavailability in man. Interestingly, the s-to-m flux of acyclovir is 5-fold higher than its m-to-s flux at the concentration employed. These results together with prior in vivo results 1701 suggest that acyclovir absorption occurs predominantly by passive diffusion. These results further suggest that acyclovir may be a substrate for apical recycling by the intestinal epithelial cells. In an attempt to increase oral absorption, prodrugs of acyclovir were prepared and investigated [ 7 1,721. Recently. synthesis of a series of amino acid esters of acyclovir were reported and evaluated in rats and man 173.741. Oral administration of amino acid esters of acyclovir increased urinary excretion of acyclovir in rats. No prodrugs could be detected in the urine, indicating that these esters are subjected to extensive hydrolysis in vivo. The L-valinyl ester of acyclovir produced the greatest increase in urinary excretion (60%) while the D-valinyl ester resulted in a urinary excretion of only 7%. These results indicate that the increase in intestinal absorption of acyclovir esters is not due to a change in physicochemical properties (i.e. aqueous solubility) and is stereospecific suggesting that a carrier-mediated transport mechanism may be involved. The mechanisms involved in transepithelial transport of valine esters of acyclovir have been investigated 1751 employing the intestinal cell line. Caco-2. Caco-2 cells have been shown to express carriermediated transport processes for amino acids and oligopeptides (76.77 1. Both the m-to-s and the s-to-m fluxes of acyclovir and its valyl esters were determined (Fig. IS). For D-\,alyl-acyclovir, no difference between the two fluxes was observed. The s-to-m flux of acyclovil and D-val-acyclovir were similar. However, the s-tom Rux of' acyclovir was three fold greater than the n-to-s Hux of acyclovir. These results suggest that transport of acyclovir and D-val-acyclovir occurs mainly by passive diffusion through the intestinal rpithelium. The n-to-s flux of the L-valyl ester of acyclovir is greater than the m-to-s fluxes of the D-valyl ester and acyclovir itself ( = 7-fold). Furthermore, the finding that the m-to-s flux of L-val-acyclovir is c
Sl

C. Lee uf al. I Advanced Drug Deliver? Reviews 23 (1997) 47-62

0

500

1000

1500

2ooo

[L-val-aeyclovir], Fig.

16.Concentration

dependence of L-Val-acyclovir

according to the equation: J, = Jz,,C/(K

2500

3oocl

3500

w

fluxes across Caco-2 cells at 37°C. The calculated mucosal-to-serosal fluxes were fit

,,,,,,r,,, L + C) + KFC. The first component of the equation represents the carrier-mediated

flux, the

second component represents the passive permeability. J,. is the total flux. C is the donor side concentration, J,,,,_ is the maximum flux of the transport carrier. K ,,,,i,., 11 is the concentration at which half maximal transport is observed and K, is the passive permeability constant, The serosal-to-mucosal

flux increased linearly with increasing concentrations, suggesting passive permeability.

The calculated fluxes were fit

according to the equation: J,. = KXC. Both curves had a coefficient of correlation greater than 0.99. Each point represents the meaniS.E.M. of three determinations.

<


+

I..“&,

+ Gh

Sd,

Fig. 17. The mucosal-to-serosal permeabilities of L-Val-acyclovir (0.8 mM) Gly-Sar

in the presence or absence of L-Valine

(20 mM)

(20 mM)

or

in the donor side chamber of Caco-2 cells at

37°C. Results are the mean5S.E.M.

of three determinations.

bathing solution is greater than 90% after completion of the transport study. Thus, differences in transport rates of these esters cannot be explained by differences in stability. However, following transport in the m-to-s or s-to-m direction. the receiver side contained = 90% and = 60% acyclovir, respectively. These results indicate that ester hydrolysis occurs within the epithelial cells. Further support for the conclusion that transport of L-val-acyclovir occurs by a carrier-mediated mechanism is provided by the finding that the m-to-s transport of L-val-acyclovir is a saturable function of concentration (concentration for half-maximal transport = 292255 PM, the maximal transport passive rate = 15.751.3 nmol/h*cm’ and permeability = 0.003 cm/h) while the s-to-m flux is

B

Y $

3 “’ 8

8

IX.

c;

6

A

Fig.

..

B

9

Inhibition

cephalexin

(2.5 mM).

of

1‘Hlcephalexin

acyclovir

(0.5

(0.1

mM)

mM)

uptake

bq

and Valyl esters of

acyclovir (0.5 mM). The inhlbition I expreahed as a percentage (11 the

control

mean?S.E.M.

uptake

(pmolimg

protein/min).

Results

are

the

of three determinations.

a linear function of concentration (permeability = 0.003 cm/h) (Fig. 16). For comparison, the average permeability of mannitol in these studies is 0.002 cm/h. Based on its structure, L-Val-acyclovir may interact with and be transported by an amino acid transporter or by the oligopeptide transporter (4.74,78-801. Involvement of these carriers was investigated through competition studies, employing L-W or Gly-Sar as inhibitors. From the results presented in Fig. 17, it can be seen that L-Val did not inhibit L-Val-acyclovir transport at the concentrations employed (L-Val = 20 mM: L-Val-ayclovir = 0.8 mM) while Gly-Sar (20 mM) significantly reduced L-Val-acyclovir transport. These results implicate the dipeptide transporter as the carrier involved in absorption of L-Val-acyclovir. Further support for this conclusion is provided by uptake studies with cephalexin. a substrate for the dipeptide transporter 179). As shown in Fig. 18,

L-Val-acyclovir at a concentration of only 0.5 mM has an inhibitory effect on 13H]cephalexin uptake comparable to that of 25 mM cephalexin. whereas both D-Val-acyclovir and acyclovir (0.5 mM) do not show inhibitory effects. At 20 mM concentration, L-Val-acyclovir inhibited cephalexin uptake by = 88c/c. Transport by the oligopeptide transporter is proposed to be dependent on a pH gradient 141. However, the introduction of a pH-gradient did not increase the transport of L-Val-acyclovir as would be expected (Fig. 191. Lack of pH dependence of L-Valacyclovir transport may indicate that there is a novel transporter present in the apical cell membrane. Alternatively, the lack of pH dependence may result from use of a saturable substrate concentration. This latter hypothesis would suggest that pH-dependent transport by the oligopeptide carrier is only present at low substrate concentrations and becomes indistinct at saturating concentrations. Further studies are ongoing to elucidate the mechanisms involved in the transport of L-Val-acyclovir. These results indicate that regardless of the uptake mechanism, it is possible to design prodrugs which enhance membrane permeability and are hydrolyzed within the intestinal epithelial cells to present the parent drug to the portal circulation. Such a prodrug avoids complications associated with identifying pharmacokinetic and pharmacodynamic properties of both the parent and prodrug.

4. Conclusions The drug discovery/development process is becoming increasingly focused on reducing the time required to enter molecules into the market. To accomplish this reduction in discovery/development time. novel approaches are being employed to determine not only activity but also absorption, pharmacokinetic and metabolic profiles for potential drug candidates earlier in the process. In this article, applications of in vitro transport have been described with particular reference to use of the Ussing chamber technique. Based on the success that these types of studies have demonstrated, this approach is gaining further support across the industry. To meet the demands of combinatorial chemistry approaches,

C. Lee et al. I Advunced

Drug

Drliwr-y

Rev&s

pH 7.4 - pH 7.4

2.3 (1997) 47-61

59

pH 6.0 - pH 7.4

Fig. 19. Mucosal-to-serosal permeabihty of L-Val-acyclovir (0.8 mM) across Caco-2 cells at 37°C. The mucosal bathing solutions were buffered at pH 6.0 or 7.4, whereas the serosal bathing solutions were buffered at pH 7.4. Results are the mean5S.E.M. of three determinations.

in vitro methods will need to be modified to increase capacity thereby allowing more rapid screening. As additional studies are completed and databases are established, predictions for absorption based on structure/transport relationships may become possible. Until that time, novel techniques such as multiple molecule analysis following addition of several candidates to a single tissue will help to increase capacity and advance our understanding of the mechanisms involved in drug transport.

Acknowledgments The authors would like to acknowledge Ping-Yang Yeh, Frederick Ryan, Gary Marks, Joelle Burgess and Pradip Bhatnagar for their contributions to the completion of this manuscript.

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