Oral absorption of β-lactams by intestinal peptide transport proteins

advanced

drug deliiry reviews ELSEVIER

Advanced

Drug Delivery

Reviews 23 (1997) 63-76

Oral absorption of p-lactams by intestinal peptide transport proteins Anne H. Dantzig” &l/x

Resnrrc~h

Labotwtorirs,

Eli Lilly cmd Compuny,

Received

IO May 1996: accepted

Indinnupoli.s,

IN 46260424.

USA

28 July 1996

Abstract

The oral p-lactams arc an extremely well-absorbed class of antibiotics. They share a common uptake mechanism with small peptides for entry into the enterocyte and exit through the basolateral membrane. Transport proteins involved in the absorption of these antibiotics have been identified. Three strategies were employed to identify proteins important for the proton dependent uptake of p-lactams. First, a 127-kDa (5lactam transporter in rabbit intestinal brush border membranes was identified by photoaffinity labeling, protein purification and reconstitution. Second, an immunological approach resulted in the identification of HPT-I, a 120.kDa protein in apical membranes of human intestinal Caco-2 cells. The hpr- I cDNA confers the ability to transport both cephalexin and bestatin in a proton-dependent fashion. Third, functional cloning of poly(A)’ RNA of rabbit intestine identified a PepTI protein that cotransports protons with small peptides, P-lactams, and angiotensin-converting enzyme inhibitors. The kidney also expresses a transporter, PepT2. with high homology to PepTI. The expression and regulation of the intestinal transporters is reviewed. The usefulness of these transport proteins to the discovery of new oral therapeutics is discussed. Keywords:

P-Lactam;

Antibiotics;

Peptide; Oral; Absorption;

Transport;

Intestine

Contents 64 1. Introduction ............................................................................................................................................................................ 64 2. Transcellular transport of p-lactams across the intestinal enterocyte ............................................................................................ 66 3. Identification of intestinal (5lactam/peptide transporters ............................................................................................................ 3. I. Purification and reconstitution of it p-lactam transporter ...................................................................................................... 66 1.2. Clonmg and chamcterization of a protein associated with intestinal p-lactam transport. IWl- I .............................................. 66 67 3.2. I. Conserved xquences with cadherin superfamily ....................................................................................................... 68 3.2.2. Proteins related to HPT- I ....................................................................................................................................... 68 3.3. Clonmg and characterization of the intestinal peptide transporter PepT 1.. ............................................................................. 6X 3.3.1. Identification of PepTl ........................................................................................................................................... 6X 3.3.2. Homologh of rabbit PepTl ...................................................................................................................................... 3.3.3. Sequence comparison with other peptide transporters ...............................................................................................70 70 3.3.4. Similarity of intestinal PepTl to Irenal PepT2 .......................................................................................................... 71 4. Regulation of P-lactam transport ............................................................................................................................................. 71 4. I. Expression along the GI tract .......................................................................................................................................... 71 4.2. The effect or diet and developmental changes .................................................................................................................... 72 4.3. Direct modulation of transporter activity ............................................................................................................................ 72 4.4. Indirect modulation of tl-ansporter activity.. ....................................................................................................................... 72 5. Speciea differences .................................................................................................................................................................. 73 6. Importance to drug delivery ..................................................................................................................................................... 73 References .................................................................................................................................................................................. “Corresponding

author. Tel.:

0169-409X/97/$32.00 Copyright fll SOl69-409X(96)00426-7

+ 1 3 I7 2769083; 0

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l997 Elsevier Science B.V. All rights reserved

1. Introduction The p-lactams are a class of pharmacologically important antibiotics that include penicillins, cephalosporins, and most recently the carbacephalosporins (Fig. 1). These antibiotics are tripeptide mimetics that contain a p-lactam ring, two peptide bonds and a free carboxylic acid group. There are many examples of p-lactams that are well absorbed orally (Fig. 2), as well as others that are not. Penicillin was the first p-lactam discovered and was orally absorbed. The first orally absorbed cephalosporin and carbacephalosporin were cephalexin and ioracarbef, respectively. in general, the orai cephaiosporin antibiotics are nearly completely absorbed from the gastrointestinal tract. Absorption is much higher than would be predicted based upon their hydrophobicity. Early studies with cephalexin indicated that the drug was taken up by an undescribed intestinal transporter that was inhibited by small peptides [ 1,2]. Subsequent studies confirmed that these drugs share a common uptake mechanism with small peptides. Nevertheless, cephalosporins may be administered with food, which results in only a short delay in their oral absorption with little effect on their total drug absorption [3,4]. Apparently, the gastrointestinal tract has a tremendous capacity for the absorption of peptides and p-lactams. Since their discovery, many studies have been conducted to determine the transport mechanisms responsible for the oral absorption of p-lactams. In the past two years, genes encoding proteins important in p-lactam and peptide transport have been identified. Several reviews have recently been pub-

Cephalospom A,-

CONH

Carbacephakxpann R,-

CONH 7

’ 2 Nci? R2

0Q co;

Fig.

I.

General

structures uf

pemcdhns,

cephaloaporma

and

carbacephalosporins. Each is a trlpeptide mimetic and contains a four-membered

p-lactam

boxylic acid group.

ring. two pepttde bonds. and one car-

lished that describe different aspects of intestinal P-lactamipeptide transport [5-121. The present review is not intended to be comprehensive, but will discuss recent developments in the identification of proteins responsible for p-lactam transport at the biochemical and molecular levels and their regulation. Gaining an understanding of the basic mechanisms and properties of these transport proteins will enable the design of new therapeutics that are able to utilize these mechanisms for oral absorption.

2. Transcellular transport the intestinal enterocyte

of p-Iactams

across

Because p-lactams are hydrophillic they do not readily diffuse across cellular membranes; consequently, transporters exist that facilitate their entry (Fig. 3). The intestinal enterocyte is located in an acidic luminal microenvironment [ 13 3. Transport of p-lactams into the enterocyte is via an intestinal p-lactam transporter which is a proton- and energydependent mechanism that permits p-lactams to be concentrated intracelluiarly against a gradient [ 14). The properties of the peptide/p-lactam transport mechanism(s) have been extensively studied over the years using intestinal segments, membrane vesicle preparations, intestinal enterocytes and intestinal cell lines. Salient features have been defined for the transport mechanism. The transporter is a symporter and cotransports proton(s) with the substrate. Uptake is dependent on an inwardly directed proton-gradient and on a membrane potential. The carrier mediates the uptake of small peptides, p-lactams, angiotensinconverting enzyme (ACE) and renin inhibitors [ 151. Peptides competitively inhibit the uptake of p-lactams and vice versa. The substrate specificity has not been well defined; however, smaller peptides are substrates (di- and tripeptides). The binding affinity is higher for p-lactams with the naturally occurring I.- than o-isomer. A free carboxylic acid group appear< to be required for compounds to be a substrate I IO]. The transporter takes up p-lactams (Fig. 2) that exist as zwitterions such as the aminocephalosporins, as well as cephalosporins that exist as anions (e.g., cefixime and ceftibuten) [16,17]. Once concentrated within the intestinal enterocyte, p-lactams must ultimately exit the basolateral surface

65

A.H. Dantzig I Advanced Drug Delivery Reviews 23 (1997) 63-76

R2 co2-

penicilfin R Amoxicillin

Rl

HO

%

Cephalexin

Cyclacillin

-CH,

Cephadroxii HO

TV-+

-CH3

3 Benzylpenicillin

,-, o-

CH-

-CH,

-CH=CH2

Cefiibuten ‘H,N ~+$~i2c02_

-H

VsDorin Loracarbef

-Cl

Fig. 2. Structures of oral p-lactam antibiotics discussed in this review. Each compound is shown in its ionized form. Cefixime and ceftibuten are dianonic containing two carboxyl groups and an aminothiazole group. The others except benzylpenicillin exist predominately as zwitterions in the acidic microenvironment of the intestinal lumenal.

into the blood stream. Much less is known about the passage of compounds through the basolateral membrane than through the apical membrane. Studies by Dryer et al. indicate that a peptide glycylsarcosine (Gly-Sar) transporter resides in basolateral membrane vesicle preparations that is proton-dependent and sensitive to thiol reagents [18]. Using human intestinal Caco-2 cells as a model system for an intact intestinal epithelium, transcellular studies of Gly-Sar suggest that a peptide carrier is located on each surface and is proton-dependent [19]. Several p-lactams (cephalexin, cephradine, loracarbef) and bestatin have been demonstrated to undergo vectorial transport across an intact Caco-2 monolayer by uptake via the apical peptide transporter into the cell and exit by a specific basolateral carrier [20-221. The apical and basolateral transporters appear to have different affinities for cephradine [20]. Studies of loracarbef suggested that the basolateral carrier may be proton-independent [21]. These studies sug-

gest that proton-dependent peptide transport carrier are located on each cellular membrane and that they are capable of transporting both peptides and plactams into and out of the cell. Whether the transporters are distinct from one another or are related transporters with somewhat different properties when expressed in different environments remains to be elucidated. Multiple transporters appear to exist on the basolateral surface [23]. In addition, the rate limiting step for transcellular transport of cephalexin and other p-lactams appears to be the departure from the cell by the basolateral transporter(s) [24,25]. Thus, the most important step for transcellular transport of p-lactams appears to be the entry and accumulation intracellularly within the enterocyte, where it is sequestered initially and prevents potential loss down the gastrointestinal tract. Subsequent exit from the cell and entry into the blood stream is driven by the availability of the drug and occurs much more slowly.

66

H+

R-lactam

N

Fig. 3. Diagram of an intestinal enterocyte. The entrl-ocytc 1, a polarizd

cell wth

transporters asymmetrically

distributed

be-

~ween the apical and basolateral surfaces. The apical surface hn\ the microvilli that express a proton-dependent p-lactam transporter along with a Na

/H

’ antiporter.

has a proton-dependent Na’ /K’-ATPase.

During

The basolateral surface also

f?-lactam transporter and expreaws transcellular

transport, p-lactam

the anti-

biotics enter the cell via the apical transporter and accumulate intracellularly

before exiting via the basolateral transporter(s).

3. Identification transporters

of intestinal

P-lactam/peptide

In the last few years, several barked on the identification of transporters. Different strategies ployed. These different approaches the identification of more than one role in p-lactam/peptide transport. 3.1. Purijication trunsporter

and reconstitution

groups have emintestinal peptide have been emhave resulted in protein playing a

cf u p-lactarn

The earliest approach was to identify the protein that bound p-lactams with photoaffinity labels. Kramer et al identified a = 127-kDa glycoprotein in intestinal brush border membranes that was covalently labeled with radiolabeled p-lactam analogs as [3H]benzylpenicillin and [‘Hlazidosuch cephalexin [261 and reviewed by Kramer et al. [ I 11. Photoaffinity labeling was reduced by the presence

of I>-carnosine, cephalexin and cehxime and not by amino acids. This indicated that the same protein might be responsible for the transport of both Lwitterionic and anionic P-lactams [27]. Agents that modify histidine (diethylpyrocarbonate) or tyrosine (N-acetylimidazole) inhibited labelling indicated the involvement of these residues in the binding of these photoaffinity labels and transport of oral p-lactams 126,281. By contrast, thiol-reactive reagents had no apparent effect on transport. These studies suggest that a histidine residue of the transporter protein serves as a proton donor-acceptor pair that facilitates the translocation of p-lactams across the membrane [ 11,291. The = 127-kDa protein was subsequently purified using wheat germ lectin affinity chromatography followed by fast protein liquid chromatograpy and cation-exchange chromotrography and reconstituted into liposomes [30]. The reconstituted rabbit intestinal protein preferentially transported I)cephalexin and not t_-cephalexin [ 3 11. Subsequently, a Y- to 1%amino acid portion of the protein was sequenced; however, the sequence of this fragment has not been published. in a recent review of his work, Kramer indicated that this sequence does not correspond to sequences contained within the pro teins encoded by the peptide transporter-related genes to be discussed next [ 111. This suggests that another gene remains to be identified that is involved in the translocation of p-lactams in the intestine. .?.Z. Cloning and characteriztrtion c$ a protein ussoriuted with intestinal p-lactcrm transport, HPT- I An immunological approach has demonstrated the involvement of a protein, named HPT-1 (for human peptide transport), in intestinal p-lactam transport. Dantzig and coworkers developed monoclonal antibodies that inhibited cephalexin uptake into human intestinal Caco-2 cells when these antibodies were incubated with the apical surface of differentiated cells 1.321. The antibody bound to a = 120-kDa glycoprotein in Caco-2 membranes. lmmunohistochemical studies of normal human tissues indicated that the protein was expressed along the gastrointestinal tract. duodenum, jejunum, ileum, colon and pancreatic ducts, but not by the kidney, liver, or lung. Using the antibody to follow the expression of the protein. hpt- I cDNA was identified. When expressed

protein is related to members of the superfamily of cadherin genes that are important in calcium-dependent cell-cell adhesion [33). Although HPT-1 is only 20-30% similar to most members of this family; HPT- 1 possesses several highly conserved extracellular elements that are putative calcium binding sites as well as four cysteine residues near the transmembrane region (Fig. 4). The most highly conserved elements of the cadherin superfamily are a His-Ala-Val (HAV) adhesion recognition sequence in the amino-terminal region and the cytoplasmic portion corresponding to the carboxy terminal that are required for cell-cell adhesion. Both of these elements are missing in HPT- 1. The HAV recognition site is replaced with an Ala-Ala-Leu (ALL) sequence and the cytoplasmic region has been trun-

in Chinese hamster ovary cells, the protein conferred the ability to take up cephalexin and bestatin by a proton-dependent mechanism. Uptake was inhibited by excess cephalexin or the dipeptide, glycylproline, and was also inhibited by thiol-reactive agents. These results were identical to that obtained for cephalexin uptake and bestatin into Caco-2 cells [201. .?.2. I. Conserved sequences with cadherin supe$atnily The hpt-1 cDNA encodes a protein of 832 amino acids corresponding to a = 92-kDa protein with several potential N-glycosylation sites and contained only two putative transmembrane (TM) domains, one of which appears to be a leader sequence. This

1 1

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481 480 420

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541 540 48C

FGVKYNASSFAKFTLIVTDVEAPQFSQHVFQAKVSEDVAIGTKVGNVTAK~PEGL~~SY N.IE S.E.T __.._. V.V.P.QI...N T N.IE.......S.E.T.X....“.“.P.QI...N....T.......T...i(.....TV..

781 780

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LI

”

HPT-1 LI-Cadherin RPT-I

t

T...R

___..

T”..

HPT-1 LI-Cadherin

HPT-1 LI-Cadherin

Fig. 4. Comparison of predicted amino acid sequences for HPT-I single amino acid letter abbreviations.

Identical

and rat homologs, LI-cadherin

622 827

and RPT-I

Amino acids are mdlcated by

residues are indicated with dots. Hyphens represent raps introduced to optimize the

alignment. Residue numbers are indicated on the left. The region AAL

(denoted with an A) was changed from the highly conserved

sequence HAV present in cadherins. The V indicates potential glycoaylation sites. The boxed sequences and the circled cysteine residues represent conserved regions which are highly conserved in the cadherin superfamily. The single transmembrane domain is denoted with a M.

cated from = 1.50 - typical of most cadherns - to only 24 amino acids. Since the protein lacks multiple TMs typically present in transport carriers, the role of the protein in transport is unknown. HPT-1 may self-associate to form a multimeric structure with transport activity or associate with another protein to form a fully functional p-lactam transporter. HPT-1 has been proposed to act as a regulatory protein [S] or as a component of a hetero-oligomer with PepTl

171. 3.2.2. Proteins related to HPT- I Recently, proteins related to HPT-1 have been identified that appear to form a subgroup of the cadherin superfamily in that they have a severely truncated cytoplasmic carboxy-terminus. A rat homolog of HPT-1 has been identified as the PCR product rpt-1 protein (Fig. 4) and as the cDNA as the liver-intestine (Ll-) cadherin protein [ 34,351. The RPT-1 protein sequence and the Ll-cadherin show 99% identity to each other; RPT-1 may be a truncated version of the LI-cadherin. Ll-cadherin is specifically expressed in both the intestine and the liver on the basolateral surfaces of these cells, and appears to function in intercellular calcium-dependent adhesion. LI-cadherin exhibits 79% identity to HPT-1 (Fig. 4) 1351. Unlike LI-cadherin, HPT-1 has not been detected in the liver and appears to be located on the apical surface of Caco-2 cells [32]. The rp-1 mRNA is expressed in the small intestine and its regulation is discussed below. Another member of this cadherin subgroup is the rabbit kidney-specific Ksp-cadherin, that has 29% identity to HPT-I and LI-cadherin, and is expressed exclusively in the kidney [36]. This protein was partially purified by disulfonic stilbene affinity chromatography and is believed to be a component of the renal Na+/HCO, cotransporter. Thus. two members of the cadherin superfamily subgroup appear to be associated with transport processes in epithelial cells. _7._7.Cloning und charuc‘terizution peptide transporter PepTI

of the i~ltrstincll

Several laboratories embarked on the identification of the gene for the intestinal peptide transporter by functionally cloning [37-391. By this approach, intestinal poly(A)+ RNA was prepared and injected into a recipient cell, the Xen0pu.s laevis oocyte;

transport activity was typically observed several days later. This approach would be expected to permit the identification of transporters present on both the apical and basolateral cell surfaces. _3..3.I. Ident$fication of PepTI Leibach and coworkers were the first to identify a cDNA for a rabbit intestinal peptide transporter, PepTl 1401 that transported Gly-Sar. Hybrid depletion of rabbit small intesitne poly(A)+ RNA with antisense oligonucleotides before injection blocked the stimulation of Gly-Sar uptake. This suggested the presence of only one intestinal transporter for Gly-Sar in rabbit intestine. The cDNA encodes a protein containing 707 amino acids with 12 putative TMs with a very large extracellular domain (Fig. 5). The gene contains sites for phosphorylation by protein kinase C (PKC) and protein kinase A (PKA). The substrate specificity of PepTl was examined by a voltage-clamp analysis by holding the potential and determining current responses by the addition of test substrates. A number of dipeptides and tripeptides elicited strong responses as well as three p-lactams examined, cephradine, cefadroxil and cyclacillin. High stringency Northern analysis of RNA from rabbit tissues showed the expresion of PepTl in the small intestine and liver, low levels in the kidney and brain and even lower but detectable levels by in situ hybridization in the colon 1411. Independently, the rabbit PepTl was functionally expressed in frog oocytes based upon its ability to take up cefadroxil 1421. Cefadroxil uptake via the carrier was dependent on the membrane potential and inhibited by oral p-lactams and ACE inhibitors, enalapril and captopril. Moreover, captopril was transported by PepTl. When expressed in oocytes, PepTl showed a strong preference for the uptake of L-cephalexin over I)cephalexin in sharp contrast to the properties of the reconstituted = 127-kDa transporter described by Kramer and coworkers (3 I,43 1. .i..?.?. Homologs of‘ r&bit PepTI Both the rat and human homologs of PepTl were subsequently cloned and encode a protein that has a high degree of identity (77% and 81%, respectively) to the rabbit transporter [44-471 (Fig. 5) and has no homology with HPT-1 (Fig. 6). When the rat PepTl was expressed in Xenol’us oocytes, the uptake of ceftibuten (anion) and cephradine (zwitterion) was

A.H. Dant:ig

I Advunced

Drug

Delivrrx

Revkws

2.3 (1997)

69

6_3- 76

me Rabbit human

I-

+h43+

Rat

PabbiL human

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. . . . ..*

... .

..

--M5-

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Rabbit human

t------6-4 lSl:fPILRVWCGIHSaQACYPLAFGYP~VALIVFV[ICSC~KKFQ~NIMC~~CI 18l:..Mv........vI(.........I..I.. .S....II........K . . ..LS.."... 181: .n..........K.........................,,.....K........,.... .. ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . ..

I------7-

Rat

ubbit hum."

Rat

Rabbit hum&"

Rat Rabbit hwMll

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......... . . ..AL.................. .....................................

.......

..F...L ................ f... .* f.... ..

361&F---Mg-

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P

Rat_ Rabbit human

~~~:~sQT~F~~FDMOLT~I~VSSPCSPCYP~~AHEFEPCHRH~Q

*at Rabbit human

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*at Rabbit human

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Rabbit human

.... .... .* ..

Fig. 5. Comparison of amino acid sequences of rat, rabbit, and human PepTl. Single letter symbols are used to denote amino acids in the sequences, dots represent identical amino acids, asterisks represent sequence conserved in all three sequences, and hyphens represent gaps to optimize alignment of residues. Residue numbers are indicated on the left. The V above a letter indicates an asparagine with a potential for glycosylation. The 12 predicted transmembrane regions (Ml to M 12) are denoted above those regions. Two boxed sequences correspond to consensus sequences (ERFSYYG and ALGTGG) present in other nonmammalian transporters. The circled S represents PKC site and the boxed T represents PKA site. (Reprinted from Biockimica et Biophysics Actcc. Vol. 1305, Miyamoto et al. [47 1, Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide transporter, pp. 34-38 (1996), with kind permission of Elsevier Science - NL, Sara Burgerhartstratt 25, 1055 KV Amsterdam, The Netherlands.)

stimulated by an inwardly directed proton gradient and was inhibited by dipeptides [46]. The affinity of the carrier was 14-fold higher for ceftibuten than for cephradine and the maximal velocity was only a 4-fold only lower for cetibuten than cephradine. This

clearly demonstrates that the PepTl transporter can translocate differently charged substrates. Two-microelectrode voltage-clamp studies of Gly-Sar uptake by the human PepTl indicate that the transporter is dependent on membrane potential and that the

and p-lactams. Further elucidation should give insights into their precise role in the translocation of these substrates.

i 2 0

e

2

-2

EE 0

hpt-1

200

400 Acd Residue

Ammo

2

600

800

.1..3.4.

Sirnilurity

qf

irztrstinal

PepTI

to wnd

PqT2

t

I

200

0

Amino

Fig. 6. Hydrophobicit)

Aad

400 Resrdue

600

analysis 01 two human protein.\ responsible

for p-lactam transport. Plots are shown for HPT-I PepT

I

using :I Kytr

and Doolittle

vale.

putative membrane spanning region tn HPT-I indicate putative trananembrane Krwcwrh

a

and the numbers

spanning regions (TM

in the human peptide transporter hPepT1. ~~/wrnic~ci/ r/rid Bioplf~.kt/

and for human

The arrow indicate\

(Modified

I -TM

I2

I

from Rir~-

Cr~t,lr,llr/lic,trrio,ls. Vol. 2 Ih. bq

Erickson et al. [ 341. Regional cxpres\ion and dietary regulation ol rat small intestinal peptide pp. 249-2.57

(1995).with

and nmino acid transporter mRNAa. kind permissmn from Academic Pre\\

Inc.. Orlando, FL).

Hill coefficient for H. is one 1481. There appears to be a sequential. ordered binding of the proton and substrate with the proton binding first. Taken together, these studies indicate that the PepTl transporter takes up a wide variety of peptides, @-lactams, and ACE-inhibitors. An understanding of the basic mechanism by which substrates are transported is now being defined.

Comparision of sequences of a number of protondependent oligopeptide transporters from eukaryotes and prokaryotes and other homologous transporters for other substrates have shown a common topology of 12 TMs as well as two highly conserved regions located in between TM2 and TM3 and within TM5 [49]. This family is referred to as POT (for protondependent oligopeptide transporters). In addition, comparison of sequences of roughly SO peptide transporters that included POT family members and members of the ABC (ATP-binding cassette) superfamily (such as CFTR and MDR genes) have shown a highly conserved motif, FYXXINXGSL, that occurs in the fourth or fifth TM of each protein [50]. This group of transporters is named PTR (for peptide transporters). These highly conserved regions are clearly implicated as having important functional roles in the proton-dependent transport of peptides

The reabsorption of peptides from the glomerulal filtrate in the kidney has been shown to be mediated by peptide transporters, one with a high affinity and another with low affinity [51-531. The human kidney expresses two peptide transporter genes, PepTl and a closely related gene, PepT2 [40,54,55]. The human PepT2 was identified from a human kidney cDNA library using a probe prepared to the 5’ end coding region of the rabbit intestinal peptide transporter cDNA [55,.56]. The full length cDNA encodes a 729-amino acid protein with a predicted molecular weight of = X2 kDa. PepT2 shares significant homology with PepTI with 50% identity and 70% similarity: however, PepT2 is 21 amino acids Iongel than PepT I. Like human PepTl, PepT2 also has I2 putative TM domains; similarity is much higher in these domains than the large extracellular loops. Human PepT2 also has sites for glycosylation, sites for PKC-dependent phosphorylation and lacks sites for PKA-dependent phosphorylation. The properties of human PepTl and human PepT2 were compared after transient expression in HeLa cells with vaccinia virus expression system 1541. Inhibition of Gly-Sar uptake by cefadroxil and cyclacillin indicated that the affinity of the human PepTl for these substrates is in the mM range compared to that of the human PepT2 which is in the FM range. The PepTl transporter has a higher affinity for cyclacillin than cefadroxil: whereas. the PepT2 transporter has a higher affinity for cefadroxil than cyclacillin. This clearly demonstrates that the relative affinities for the intestinal and renal transporters are quite different even though the two nucleotide sequences are 70% similar. Alterations present in PepT2 have conferred higher affinity for substrates and have also altered its preference of substrates from that of the intestinal transporter. The rabbit high affinity peptide transporter PepT2 was also cloned based upon its ability to transport cefadroxil. Using a photoaffinity probe of cefadroxil, a glycoprotein of 107 kDa was identified [57]. By target analysis, the functional molecular mass of the cefadroxil transport carrier was determined to be

A.H. Dmrzig

I Advanced

Drug Deliveyv

= 414 kDa, suggesting the renal PepT2 functions as an oligomer [58]. This has clear implications for the functional unit of the intestinal PepTl transporter existing as a multimeric protein.

4. Regulation

of p-lactam

transport

There are several mechanisms by which the absorption of B-lactams and peptides by B-lactam transporters may be regulated in the intestine. Regulation may result from different expression along the GI tract that may be influenced by diet and age of the animal. The activity of the transporters may be modulated by mechanisms that act directly on the transport protein itself or indirectly through proteins that influence the activity of the transporter.

4.1. Expression

along the GI tract

Efforts have been made to determine the sites for maximal absorption of nutrients and drugs along the small intestine. Studies examining the uptake of carnosine indicated uptake was greatest in the jejunum of rats (591. By contrast, the uptake of the B-lactams, cephalexin and cephradine, is higher in in the duodenum than in the jejunum or ileum when measured in membrane vesicle preparations or in intact enterocytes [60,61]. The transport of [“Hlcephalexin is equivalent for jejunum and ileum of rabbit intestine mounted in Ussing chambers with the same lumenal pH [62]. When absorption was examined along the axis of the villus, drug accumulation was highest in enterocytes towards the tip of the villus and not detected in stem cells of the undifferentiated crypt region, suggesting that expression of the transporter may be enhanced as cells migrate to the tip of the villus [61]. Expression of rabbit PepTl mRNA correlates reasonably well with these observations [41]. PepTl mRNA expression is higher in the duodenum and jejunum and is somewhat lower in the ileum [40,41]. PepT 1 mRNA is absent in cells of the lower-mid crypt region, present in cells close to the crypt-villus junction and maximally expressed in enterocytes near the tip of both the jejunum and ileum villi and declined in cells as they approach the tip of the duodenum villus.

Reviews 2.3 (1997)

71

63- 76

4.2. The effect of diet and developmental

changes

The expression of enzymes and transporters is also regulated in response to dietary stimuli and developmental changes [63,64]. Rodents maintained on a high protein diet absorb more of the peptide, L-carnosine, than those on a low protein diet 1591. Recently, Erickson and coworkers examined the effect of low and high protein diets on the expression of peptide transport proteins, rPepT1 and RPT- 1, in the rat small intestine [34]. Expression of mRNA for both transport proteins was evenly distributed along the longitudinal axis of the small intestine in rats fed a low protein diet. A high protein diet upregulated the expression of both proteins by 1.5- to 2-fold in the middle and distal intestine. In addition a high affinity glutamate transporter, EAAC 1, was upregulated while no effect was observed on the expression of the neutral/dibasic transporter D, /rBAT, indicating selectivity in the regulation of transporters. Developmental changes have also been observed for the expression of PepTl in the rat jejunum. Expression is highest at birth and declines with age [47]. This may indicate an important role of this transporter in the absorption of peptides from a diet consisting of a protein-rich milk source. Thus when more dietary peptides are available, the capacity of the intestine apparantly increases to enhance absorption of these nutrients. The increase observed in the distal portion of the intestine, presumably reduces the amount of nutrients/drugs that might otherwise pass through the body. The regulation of peptide transport has also been studied in human intestinal Caco-2 cells as a function of nutrients in the growth medium and the differentiation of these cells into enterocytes. The transport of Gly-Sar is maximal in Caco-2 cells at confluence and declines subsequently; while transport of cephalexin and ceftibuten is maximal 14-days postconfluence [16,65,66]. Loracarbef uptake is intluenced by both the feeding schedule of the cells and the amount of time the cells are allowed to differentiate into enterocytes, similar to the differentiation of the crypt cell in the villus [67]. Expression of the transporter was not coordinately regulated with prolidase, an intracellular peptidase important in the degradation of certain transported peptides into amino acids. Differences in the expression of the transport mechanisms versus metabolism may pro-

vide a window of opportunity for the transcellular transport of intact peptides to occur with minimal degradation. This points to an inherent need for the presence of basolateral transporter carrier(s) for the exit of these nutrients from the cell. Since Caco-2 cells express both human PepTl and HPT- 1, they will permit a detailed study of the expression of these transport proteins [32,65 I. 4.J. Direct modulation

oj’ transporter

activity

The activity of the peptide transport protein may be regulated directly. The cloning of PepTl indicated the potential for protein phosphorylation by both PKC and PKA in the rabbit and rat forms and by only PKC in the human form. Using the human intestinal Caco-2 cell line, Gly-Sar transport was significantly inhibited by agents known to elevate cellular CAMP levels and by ones that stimulate PKC 1651. These effects could be blocked by staurosinhibitior of PKC. non-specific porine, a chelerythrine, a selective inhibitor of PKC as well as H-89, a selective inhibitor of PKA, even though the putative site for phosphorylation is apparently absent in the human form [65,68]. In addition, activity was inhibited by treatment of Caco-2 cells with Cholera toxin, E. coli heat-labile enterotoxin, forskolin and isobutylmethylxanthine which elevate cellular CAMP [68]. Inhibition of transport was due to a reduction in the maximal velocity (y,,,,) with no apparent change in the affinity (K,,). Inhibition did not result from inhibition of protein synthesis or a decline in the proton gradient. These studies provide evidence that the activity of the human intestinal PepTl transporter is under the direct regulatory control of PKC and that PKC may modulate the activity of the protein. 4.4. Indirect modulation

of transporter

activit)

The Na’/H+-antiporter is believed to play an important role in the maintenance of the luminal pH microenvironment of the enterocyte 169,701. Consequently, the uptake of peptides and p-lactams are thought to be indirectly coupled to the sodium gradient that exists across the brush border by the Na+ /H+-antiporter (Fig. 3) (701. Clinical studies examining the effect of the coadministration of the Nat /H’-antiporter inhibitor, amiloride, with amoxicillin reduced the oral bioavailability of this p-lactam

by 270/o, indicating a role for the antiporter in the regulation of /3-lactam absorption [71]. This reduction in bioavailability was also correlated with a decrease in potasssium renal excretion, suggesting a regulatory function for Na’/K’-ATPase for controlling the intracellular sodium concentration necessary for Na /H +-antiporter activity [72]. Coadministration of the calcium channel blocker, nifedipine enhanced the oral bioavailability of amoxicillin by 42% and cefixime by 71% without altering their distribution or elimination [73,74]. This dramatic effect was not due to an increased blood flow and may result from regulation of the Na+/H+-antiporter and/or the peptide transporter by calcium-dependent pathways, such as PKC [74]. These studies suggest an important role of the Nai /H+-antiporter in the regulation of p-lactam uptake in man.

5. Species differences @lactam antibiotics were discovered initially for their antibiotic activity and subsequently tested for their oral bioavailability. In order to select compounds most likely to be orally absorbed, oral absorption is usually assessed in two or more species prior to clinical evaluation in man. The animal species have included rodents (mice, rats), dogs, and monkeys. Researchers have noted differences in the oral absorption of antibiotics between species although little has been published on these findings. Compounds which are not moderately well absorbed in one or more of these species generally are not evaluated in man. Thus, recent clinically used oral p-lactams must be orally absorbed in man and other species in order to be marketed. Because of this paradigm, it is not surprising that studies of transport mechanisms for marketed oral p-lactam antibiotics using in vitro systems have shown little differences between species. Studies by Sugawara et al using intestinal brush border membrane vesicles from rat, rabbit and man with oral p-lactam antibiotics have demonstrated that species differences do exist however [75]. They examined the uptake of cephradine and ceftibuten and inhibition of each other’s uptake and by analogs. They concluded that the rabbit was not a good model for the absorption of p-lactams in humans. Moreover, it has been proposed that the rabbit intestine has several P-lactam carriers. This

A.H. Dantzig

I Advanced

Drug

has been supported by differences observed in the thermal sensitivity of the uptake of cefixime and cephalexin and in the inhibition and trans-stimulation of cephradine uptake by enalapril in rabbit intestinal brush border vesicles [27,76]. Thus, differences between species may occur due to differences in the individual transport carriers as well as the existence of multiple transport carriers with overlapping specificities. The recent cloning of genes responsible for Blactam transport have clearly demonstrated that there is a high degree of similarity between homologs from different species [44-461 (Figs. 4 and 5). Even so, some differences have been observed in the properties of PepTl. For example, the rabbit transporter but not the human transporter may be regulated by PKA. When PepTl was expressed in XUIO~LLS oocytes from rabbit and from rat, the affinity for cephradine was 1 l-fold higher for rabbit (K, of 1.1 mM) than for rat (K, of 12.4 mM) [42,46]. Whether substantial differences exist in other substrate affinities for intestinal transporters from different species remains to be determined.

6. Importance

to drug delivery

The identification of transport proteins, and their cDNA, that are responsible for the absorption of B-lactams and peptidyl mimetics provides research tools with which to answer fundamental questions about the multiplicity of transport carriers, transport mechanisms and substrate specficities. By understanding the properties of individual transporters, it should be possible to identify binding pockets for substrates and the residues with which substrates interact within the transport protein. This information can be used to rationally design drugs that may utilize these absorption pathways for improved oral absorption. By incorporation of this information into studies of structure-activity-relationships (SAR) for the target activity of interest, medicinal chemists will be able to develop structure-transport-activity-relationships (STAR) for optimizing absorption properties. The study of human transporters, in particular, will permit the discovery of agents that are most likely to succeed clinically and diminish the reliance on other animal models for the evaluation of compounds orally. Knowledge of the specificity of the

Delivery

Reviews 2.3 (1997)

73

63-76

transporters involved in intestinal absorption along with the specificity of transporters involved in excretion may ultimately permit the design of therapeutics which are not only absorbed orally but possess longer half-lives [ 1 l]. Thus, the fundamental understanding of individual transport mechanisms should greatly speed the discovery and development of new orally absorbed pharmaceutical therapeutics.

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[91 Tsuji, A. (1995) Intestinal absorption of B-lactam antibiotics. In: Taylor M.D., Amidon G.L.. Eds., Peptide-Based Dtug Design. Controlling Transport and Metabolism. Washington, DC: American Chemical Society, pp. IOI- 134. [101 Sadee. W., Drubbisch, V. and Amidon, G.L. ( 1995) Biology of membrane transport proteins. Pharm. Res. 12, 1823- 1837. S. [I 11 Kramer, W., Girbig, F., Gutjahr, J. and Kowalewski, ( 1995) The intestinal oligopeptide transporter. Molecular characterization and substrate specificity. In: Taylor M.D., Amidon G.L., Eds., Peptide-Based Drug Design. Controlling Transport and Metabolism. Washington. DC: American Chemical Society, pp. 150- 179. [I21 Meredith. D. and Boyd, C.A.R. (1995) Oligopeptide transport by epithelial ceils. J. Membr. Biol. 145, I-12. [I31 Lucas. M.L., Schneider, W.. Haberich, F.J. and Blair, J.A. ( 1975) Direct measurement by pH-microelectrode of the pH microclimate in rat proximal jejunum. Proc. R. Sot. Lond. B 192, 39-48. [I41 Ganapathy, V. and Leibach. F.H. ( 1991) Proton-coupled solute transport in the animal cell plasma membrane. Curr. Opin. Cell Biol. 3. 695-701.

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