Designing Prodrugs for the hPEPT1 Transporter - American Chemical

Chapter 5

Designing Prodrugs for the hPEPT1 Transporter

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Hyo-kyung Han and Gordon L . Amidon College of Pharmacy, The University of Michigan, Ann Arbor, MI 48109-1065

Prodrug delivery has generally utilized a passive membrane transport to enhance cellular membrane permeability and, hence, uptake. This, however, generally enhances uptake into all cells. An alternative strategy, that we have developed, focuses on enhancing the uptake via cellular transporters, in particular the hPEPT1 peptide transporter found in mucosal epithelial cells. We have designed a variety of prodrugs that utilize this transporter for uptake and shown that these antiviral prodrugs, both in vitro and in vivo, show saturation and competition characteristics of carrier-mediated transport via the hPEPT1 transporter. These results indicate that design of drugs for specific membrane transporters can achieve enhanced membrane permeability and drug efficacy.

Intestinal drug absorption and first-pass drug metabolism have been the main concern in oral drug delivery to assure sufficient oral bioavailability for the adequate therapeutic effect of drugs. Among many attempts to overcome the pharmaceutical and pharmacokinetical barriers causing low oral bioavailability, the chemical approach using drug derivatization offers the highest flexibility and has been recognized as an important means of producing better pharmaceuticals. The prodrug approach which is a chemical approach using reversible derivatives could be useful to enhance the intestinal absorption of polar drugs. Whereas the classical prodrug design utilizes lipophilic derivatives to increase the passive membrane permeability, 'targeted-prodrug design' utilizes the membrane transporters for polar nutrients such as amino acids, nucleosides or peptide transporters (1-3). This type of'targeted prodrug design' requires considerable knowledge of the particular carrier system including

46

© 2000 American Chemical Society

Park and Mrsny; Controlled Drug Delivery ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

47 their molecular and functional characteristics. Recently, the advances in gene cloning and controlled gene expression techniques in mammalian cells can be used to elucidate the molecular nature of carrier proteins and make possible more rational design of'targeted prodrugs'. Compared to other transporters, peptide transporters have broad substrate specificity (4, 5) and could be a good target for chemical modification to improve oral drug absorption of polar drugs (6-8). Peptide transporters have been extensively studied to optimize the structural modification of drugs and their substrate specificity has been discussed in major reviews (1, 9-11). However, recently, novel findings on the substrate specificity of peptide transporters (12-20), strongly suggest the reevaluation of structural requirements for these transporters. In this manuscript, we will briefly summarize the peptidyl substrates of peptide transporters and then mainly discuss the new structural features for peptide transporters focused on our new findings with amino acid ester prodrugs and other non-peptide substrates.

PEPTIDYL SUBSTRATES O F PEPTIDE

TRANSPORTERS

In addition to the endogenous peptides, various therapeutic drugs are known as substrates of peptide transporters. As shown in Fig. 1, β-lactam antibiotics, angiotensin-converting enzyme inhibitors, renin inhibitors and bestatin are wellknown substrates for peptide transporters (2, 4, 21) and possess peptide-like chemical structures with a peptide bond, an α-amino group and a C-terminal carboxyl group. Substitution of N-terminal α-amino group or C-terminal carboxyl group of the peptidyl substrates greatly influences the affinity for the peptide transport system ( 10, 22, 23). Blocking of these groups reduces the affinity to a significant extent but they still can be recognized as substrates of peptide transporters. For example, several βlactam antibiotics (e.g., cefixime, cefdinir) and ACE inhibitors (e.g., captopril, enalapril, quinapril, benazepril) have been shown to be transported via the intestinal peptide transport system even though they do not have an N-terminal α-amino group (24-26). Also, thyrotropin releasing hormone (TRH) and some renin inhibitors which do not have a free C-terminal carboxyl group are reported to be transported by the peptide transporters (27, 28). Therefore, an N-terminal α-amino group and a Cterminal carboxyl group do not appear to be critical requirements for the peptide transporters although modification of these groups generally diminishes the substrate affinity to the transporters. Several studies have indicated that the intestinal and renal peptide transporters are stereoselective (29-31). Peptides containing L-amino acids interact with the peptide transporter with greater affinity than do peptides containing D-amino acids. The same is true with peptidomimetic drugs, which are substrates for the peptide transporters. In addition, a D-amino acid at the N-terminal end of a peptide may have more effect on transport than one at the carboxyl terminal end (32). On the other hand, until recently, the presence of a peptide bond in the substrate has been considered as a prerequisite for the recognition by peptide transporters. However, recent findings on the non-peptidyl substrates of peptide transporters such

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ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

48 (a) Di-/Tripeptides 0

R

Ο

2

Η

I

R;

Η 2 Ν γΠ^ >Γγ Ν Ν

H

R,

H

Ο Ο

COOH

τ

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3

Tripeptide

Dipeptide

(b) β-Lactam antibiotics ÇOOH CH

COOH HO-

Ο

V - N ^ V ^

NH

â,

s V

2

H

3

3

H

H

Cephradine

H

Cefadroxil

ÇOOH CH=CH

H N-

2

2

! NH

2

H

V

Ampicillin (c) ACE inhibitors

CH CH OOC 3

CH

2

H

H

\y

3

OCH

H

S

2

Cetixime

COOH

,COOH

Enalaprii

SH

y^ -r

cooH

Lisinopril

N

CH X/

Captopril

3

(d) Miscellaneous

CH

u

3 o

^Ç^j

Renin inhibitor (S 863390) Thrombin inhibitor

OH

H ccr 3

NH ô 2

C00HCH3

XH CH

3

3

Bestatin

Fig. 1; Peptidyl substrates ofpeptide transporters

as Aφharmenine A (12, 13), amino acid ester prodrugs of acyclovir and AZT (14, 15), and 4-aminophenylacetic acid (16) (Fig. 2), strongly challenge the obligatory


49 need for a peptide bond. Therefore, in the following section, the recent studies on the non-peptidyl substrates will be discussed in detail.

(a) Amino acid ester prodrugs of acyclovir and AZT ο

Τ Ι> *ûx}

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2

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υ

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g

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glycyl ester of acyclovir L-valyl ester of acyclovir (Valacyclovir)

L

.

v a

,,

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o

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A

Z

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J

(b) 4-aminophenylacetic acid

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(c) Arphamenine A

nh

2

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cooh — n

f

Fig. 2: Non-peptidyl substrates ofpeptide transporters

NON-PEPTIDYL SUBSTRATES O F PEPTIDE TRANSPORTERS

Amino acid ester prodrugs of acyclovir and zidovudine In our present study, two nucleoside antiviral drugs, acyclovir (ACV) and Zidovudine (AZT) were converted to the amino acid ester prodrugs such as L-valyl esters of ACV and AZT (L-Val-ACV and L-Val-AZT), D-valyl ester of ACV (D-ValACV) and glycyl ester of ACV (Gly-ACV). The intestinal absorption mechanism of these amino acid ester prodrugs was characterized in three different experimental systems; in situ rat perfusion model, CHO/hPEPTl cells and Caco-2/hPEPTl cells.


50 Intestinal membrane permeability studies in the in s/Yw-perfused rat jejunum indicated that the membrane permeabilities (P ) of glycyl ester (Gly-ACV) and Lvalyl ester prodrugs (L-Val-ACV and L-Val-AZT) were three to ten fold higher than those of the parent drugs (ACV and AZT) but there was no differences in P values between D-valyl ester prodrug (D-Val-ACV) and acyclovir (ACV) (Table I). Furthermore, transport of the prodrugs was concentration dependent supporting a carrier-mediated transport mechanism (14). Studies with competitive inhibitors of the intestinal peptide transporter, cephalexin and various dipeptides, significantly reduced the permeability of L-Val-ACV, while the free amino acid, L-valine, had no effect. In addition, L-Val-AZT and Gly-ACV strongly inhibited the transport of the L-ValACV, indicating a common transport pathway (14). These results suggest that the peptide transporter is primarily responsible for the transport of these amino acid ester prodrugs across the apical membrane of the intestinal epithelial cell. w

w

Table I: Wall permeability of amino acid ester prodrugs and their parent drugs in rats (0.01 mM, Mean + SE) Drugs ACV L-Val-ACV D-Val-ACV Gly-ACV AZT L-Val-AZT

Ν 4 6 4 4 6 6

P (x W'\ cm/sec) 1.3 + 0.2 13.5 + 3.1 1.9 + 0.3 6.6 + 1.3 1.7 + 0.3 6.5 + 0.6 w

Data are from reference 14.

A peptide carrier mediated membrane transport of the amino acid esters was confirmed by competitive uptake studies with stably transfected Chinese Hamster Ovary (CHO) cells overexpressing the hPEPTl transporter. The amino acid ester prodrugs showed strong inhibition of the uptake of a standard peptide substrate GlySar and their IC values were similar to that of Gly-Sar while lower than those for cephradine and enalapril (14). Thus, these non-peptide amino acid-nucleoside esters display surprisingly good affinity for the hPEPTl transporter. We also studied the stability and uptake characteristics of these prodrugs in Caco-2/hPEPTl cells. After 30-min incubation, the hydrolysis of amino acid ester prodrugs was less than 10 % in the supernatant, while it was above 95 % inside the cells except for D-Val-ACV which was relatively stable against the enzymatic hydrolysis (15). These data indicate that L- amino acid ester prodrugs are rapidly converted to the parent drugs by the intracellular hydrolysis following the apical membrane transport. In addition to the inhibition effect on the cellular uptake of L-Val-ACV by peptidyl substrates (15), the membrane transport mechanism of these prodrugs was further supported by the 50


51 enhanced cellular uptake of L-Val-ACV following the overexpression of hPEPTl transporters in the Caco-2 cells (Table II).

Table 11: Initial uptake rate of L-Val-ACV in the untransfected Caco-2 cells and the transfected Caco-2/hPEPTl cells (Mean + SD) Ν Caco-2 cells Caco-2/hPEPTl ceils

4

6

2

Uptake rate (pmol/min/cm ) 13.9 + 3.2 63.8 + 9.2

Data are from reference 15.

The results from three different model systems were very consistent in all cases and can be summarized as follows. First, amino acid ester prodrugs significantly (three to ten fold) increase membrane permeability compared to their parent drugs; second, the L-configuration of amino acid showed more favorable membrane transport and faster reconversion to the parent drug than the D-configuration, and; third, the intestinal absorption of amino acid ester prodrugs is peptide transporter-mediated, even though there is no peptide bond in their structures. Recently, other research groups independently reported the peptide transportermediated absorption mechanism of valacylovir (L-Val-ACV) (17-20). However, distinguished from other studies, our present study examined several amino acid ester prodrugs as well as valacyclovir and demonstrates that the hPEPTl transporter can recognize various amino acid progroups with stereoselectivity and also the nucleoside component can be variable. The present study demonstrates that amino acid ester prodrugs can be a good approach to targeting a peptide transporter for improving oral drug absorption of polar nucleoside analogs.

Other non-peptidyl substrates Daniel et al. (12) has shown that Arphamenine A, a Arg-Phe analogue without a peptide bond (the peptide bond (CONH) is replaced by a ketomethylene function (COCH )), could be a potent inhibitor for peptide transporters in the renal brush border membrane vesicles and subsequently, Enjoh et al. (13) demonstrated that Arphamenine A is transepithelially transported by a peptide transporter in Caco-2 cells. 4-Aminophenylacetic acid (4-APAA), a small totally non-peptidyl drug, has been shown to interact with a proton-coupled oligopeptide transporter by Temple et al. (16). 4-APAA transport across the rat intact intestine was stimulated 18-fold by luminal acidification (to pH 6.8) and using renal brush border membrane vesicles and Xenopus oocytes expressing PepTl, 4-APAA was shown as a substrate for 2


52 translocation by peptide transporters. These studies on the non-peptidyl substrates of peptide transporters further support the suggestion that a peptide bond is not prerequisite for recognition by peptide transporters.

CONCLUSION

The previously proposed substrate specificity on peptide transporters has been challenged by the recent new findings on non-peptidyl substrates and needs to be reevaluated with more direct evidence at the molecular level. It is still difficult to clearly visualize the structure-transport relationship on peptide transporters, even though these transporters have been extensively studied for several decades. Furthermore, the basolateral membrane transport of dipeptides seems to be different from the apical membrane transport, adding to the complexity of characterizing the minimal structural requirements for peptide transporters. However, as shown in our studies on the amino-acid ester prodrugs, 'targeted-prodrug design' utilizing the peptide transporters can be an efficient strategy to improve oral drug absorption of polar drugs. For rational prodrug design to develop the efficient oral drug delivery systems, the structure-transport relationship of peptide transporters needs to be further investigated at the molecular level.

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Designing Prodrugs for the hPEPT1 Transporter - American Chemical

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