OLIGOPEPTIDE TRANSPORTERS

Abstract: The present invention relates to di- and tripeptides and their use as pro-moieties to facilitate the transmembrane transport of drugs, in particular of small molecular weight drugs and more particularly poorly absorbed small molecular weight drugs.

Description: Qliαopeptide Transporters FIELD OF THE INVENTION The present invention relates to di- and tripeptides and their use as pro-moities to facilitate the transmembrane transport of drugs, in particular of small molecular weight drugs and more particular poorly absorbed small molecular weight drugs BACKGROUND OF THE INVENTION The oligopeptide transporter is expressed in mammalian small intestinal epithelia and a number of biomembranes, and is instrumental in the process of ammo acid absorption via active uptake of digested di- and tripeptides from the gastrointestinal lumen ( 1 , 2) This transporter is energized by a Na τ - independent H * -gradιent, which is generated within the acid microclimate located adjacent to the apical surface of jejunal microvilh (2, 3) As opposed to transport systems specific for single ammo acids and sugars, the oligopeptide transporter has an immense number of potential di- and tripeptide substrates (4) Various types of peptidomimetics have been shown to serve as efficient substrates for the oligopeptide transporter, including β-lactam antibiotics (5-7) , angiotensm converting enzyme inhibitors (8) , and rennm inhibitors (6) , yet, free ammo acids or intact tetrapeptides are not substrates for this transporter ( 1 , 2) Due to the fact that it displays such a broad affinity for a variety of structurally similar molecules, the oligopeptide transporter has attracted significant attention within the field pharmaceutics for its potential use as a drug delivery vehicle (9, 1 0) . In both human ( 1 1 ) and rabbit (1 2) small intestine, a protein has been identified as one which is primarily responsible for di- and tripeptide absorption via a H + cotransport mechanism. Interestingly, these human and rabbit H peptide cotransporters exhibit an extremely high degree of cross-species homology ( 1 1 ) . Although it has yet to be fully characterized, a functionally similar version of the oligopeptide transporter is expressed and utilized by the human adenocarcmoma cell line, Caco-2, once the monolayer is fully differentiated and polarized (3, 5, 7, 1 3) . Several groups have described the valuable utility of conducting both uptake and transport experiments using confluent Caco-2 monolayers as a model system for screening potential oligopeptide transporter substrates (3, 5, 7, 1 3-1 5); thus, it may be possible to extrapolate information from these types of in vitro studies in order to predict the absorption of various compounds across the mammalian gastrointestinal tract (7, 1 5) . From the perspective of drug delivery, it may be possible to exploit substrates for the oligopeptide transporter to function as pro-moieties, thus enhancing the bioavailability of poorly absorbed drug compounds. This type of rational drug design has been suggested previously (9, 1 0) ; yet, to date, the use of a hydrolysis-resistant substrate in this manner has not been demonstrated One conceivable tactic would be to introduce a D-configured ammo acid within a dipeptide in efforts to circumvent the metabolic lability inherent to naturally occurring dipeptides ( 1 0, 14, 1 6) . Several studies have demonstrated that the oligopeptide transporter displays a preferential affinity for L/L-configured dipeptides as opposed to L/D, D/L, and especially D/D combinations ( 1 0, 14, 1 7) . The structural requirements of peptides and peptidomimetics influencing their ability to interact with the oligopeptide transporter have been investigated, e.g. , cychzation ( 1 4), N-termmal α-amιno modification (4, 8) and assessment of hydrophobic potential (4, 1 5) ; but, it has not yet been determined if covalent side-chain modification of one of the ammo acids in a linear di- or tripeptide results in a loss of affinity for the oligopeptide transporter. According to the present invention it has surprisingly been found that esteπfication of the aspartic acid in D-Asp-Ala does not interfere with its ability to inhibit the uptake of [ 14 C]glycylsarcosιne ([ 14 C]Gly-Sar) , an enzymatically stable dipeptide having a high affinity for the oligopeptide transporter expressed in Caco-2 monolayers ( 1 6) MATERIALS AND METHODS [ u C]glycylsarcosιne ([' 4 C]Gly-Sar, 60 mCi/mmol) and [ ,4 C]Mannιtol (56 mCi/mmol) were purchased from Amersham International (Buckinghamshire, U. K.) . Fetal bovine serum (FBS) , Dubeicco's Modified Eagle's Medium (D-MEM) , 100x non-essential ammo acids (NEAA), solution, trypsin (0.25%)-EDTA (1 mM), penicillin (1 0,000 U/ml) and streptomycin ( 1 0,000 μg/ml) solution, and Hank's Balanced Salt Solution (HBSS) were purchased from Life Technologies (Roskilde, Denmark) 2-(N-morpholιno)ethanesulfonιc acid (MES), N-2-hydroxyethylpoperazιne-N'-2-ethanesulfonιc acid (HEPES), and Bovine Serum Albumin (BSA) were purchased from Sigma (St. Louis, MO, USA) . Various dipeptides, amino acids, Boc-D-Asp(OtBu)-0H DCHA, Boc-D- Asp(OcHex)-OH, Boc-D-Asp(OBzl)-OH, and H-Ala-OtBu were purchased from Bachem Feinchemikalien AG (Bubendorf, Switzerland) . HObt was purchased from Fluka, (Buchs, Switzerland) , and TBTU was purchased from NovaBiochem (Laufeifmgen, Switzerland) . All solvents were obtained from Riedel-de Haen (Seelze, Germany; all analytical grade solvents used for HPLC analysis were obtained from Gerner & Seusen (Copenhagen, Denmark) ; and the Ultima Gold scintillation fluid used was purchased from Packard (Gronmgen, The Netherlands) . CELL CULTURE Caco-2 epithelial cells were obtained from the ATCC (Rockville, MD), and were used between passages 21 and 40. They were seeded into tissue culture treated TranswellsTM (4.7 cm 2 , 0.4 / pore size, Costar Corp., Cambridge, MA) at a density of 1 0 5 cells/cm 2 Cells were maintained in a humidified 5% C0 2 in air atmosphere at 37°C, and were cultured in Dubelco's Minimal Essential Medium (D-MEM) supplemented with 1 0% fetal bovine serum (FBS), penicillin/streptomycin ( 1 00 U/ml and 1 00 /vg/ml, respectively), 1 % L-glutamine, and 1 % NEAA. The growth media was replaced every other day, and apical and basal volumes were maintained at 1 .5 ml and 2.5 ml, respectively. Confluent cell monolayers were obtained 14-1 7 days post-inoculation, with each well demonstrating a transmembrane electrical resistance (TEER) of between 500- 600 ohms. cm 2 as measured by an epithelial voltohmmeter (EVOM, World Precision Instruments, West Haven, CT) . Cell monolayers could be maintained at a constant resistance for at least 7 days without suffering leakage between apical and basal compartments (as determined by [ 14 C]Mannιtol flux) or a loss in TEER The total amount of protein on each Transwell filter was calculated using the Lowry method to be 0 42 mg/cm 2 UPTAKE AND TRANSPORT EXPERIMENTS Uptake experiments were performed in pH-adjusted HBSS supplemented with 0.05% BSA. Prior to conducting uptake experiments, Caco-2 monolayers were first rinsed and then incubated with HBSS (apical media = 0.05% BSA, 1 0 mM Mes, pH 6.0; basal media = 0.05 % BSA, 1 0 mM Hepes, pH 7.4) for 1 5 mm at 37°C under a 5% C0 2 atmosphere in order to equillibrate the cells to the change in pH gradient Next, [ 14 C]Gly-Sar (0.5 μCi), and in certain wells, inhibitors of various concentrations were added concomitantly to the apical media of the Caco-2 Transwells Following either a 1 5 or 1 20 mm incubation period, buffer was removed from both the apical and basal chambers and the cells were washed four times with ice-cold HBSS, pH 7. Following this washing step, the entire polycarbonate membrane was cut off of the Transwell support and placed into a scintillation vial, scintillation fluid was added, and then the cell-associated radioactivity was counted via liquid scintillation spectrometry Transport experiments were performed similarly, but following the incubation period the basolateral media was removed and analyzed for the presence of either [ 1 C]Gly- Sar or [ , 4 C]Mannιtol (to assess paracellular permeabililty) . Synthesis of D-Asp-Ala, D-Asp(cHex)-Ala, and D-Asp(BZ)-Ala Boc-D-Asp(OtBu)-OH: Boc-D-Asp(OtBu)-OH was liberated from its DCHA salt by standard procedure yielding a colourless oil which was used without further purification Boc-D-Asp(OtBu)-Ala-OtBu: 1 .23 g Boc-D-Asp(OtBu)-OH (4.25 mmol) was dissolved in 20 ml fresh distilled DMF at room temperature together with 0.574 g HObt (4.25 mmol) and 1 .365 g TBTU (4.25 mmol) The mixture was then stirred at room temperature for 5 mm before adding 0.772 g H-Ala-OtBu (4.25 mmol) and 1 .648 g DIPEA ( 1 2.75 mmol) . The resulting mixture was stirred for 2 hr at room temperature and then evaporated to dryness. The remainder was dissolved in 25 ml ethyl acetate and then extracted with 3 x 20 ml 10% NaHC0 3 in water, 3 x 20 ml 5 % acetic acid in water v/v. The ethyl acetate phase was dried over Na 2 S0 4 and then evaporated under reduced pressure, resulting in a colourless oil. The oil was used directly without further purifica¬ H-D-Asp-Ala-OH: The oil was dissolved in 20 ml DCM and 20 ml concentrated TFA was added and the mixture stirred for 2 hr at room temperature. The solvent was then evaporated under reduced pressure and the resulting oil was extracted with 3 x 1 0 ml ether, which turned the oil into 600 mg crystals, 70% in overall yield. The purity was verified by TLC and HPLC and was found to be better than 98%. The product was identified by ' H-NMR H-D-Asp(OcHex)-Ala-OH: H-Asp(OcHex)-Ala-OH was prepared using the above described procedure resulting in a 75% overall yield (91 0 mg) . The purity was verified by TLC and HPLC and was found to be better than 98%, and the product was identified by ' H-NMR. H-D-Asp(OBzl)-Ala-OH: H-Asp(OBzl)-Ala-OH was prepared using the above described procedure resulting in a 59% overall yield (735 mg) . The purity was verified by TLC and HPLC and was found to be better than 98%, and the product was identified by 1 H-NMR. RESULTS pH-Dependence of [ 14 C]Gly-Sar Uptake and Transport In order to insure that H + -dependent, carrier-mediated uptake was the mechanism by which [ 14 C]Gly-Sar was internalized and transported by the Caco-2 cells, the pH-dependency of [' 4 C]Gly-Sar uptake into and transport across the monolayers was ascertained Previous studies using the Caco-2 model have shown that optimal conditions for substrate uptake via the apical oligopeptide transporter require an apical pH of 6.0 and a basolateral pH of 7.4 ( 1 3) , thus, these conditions were maintained for all controls in our experiments As is shown in Fig. 1 , the amount of uptake and transport of [ 1 C]Gly-Sar following a 2 hr incubation was 3.01 ± 0.1 1 and 0.98 ± 0.06 pmol/mg protein/mm, respectively. When the apical pH was raised to 7 4, the degree of [ 14 C]Gly-Sar uptake and transport was significantly less, i.e. , 1 .72 ± 0.1 0 and 0.51 ± 0.1 3 pmol/mg protein/mm, respectively. For the transport experiments, the values are corrected for [ 1 C]Mannιtol flux across the monolayers, which was 0.06 ± 0.02 for the pH 6 and 0.1 1 ± 0.02 pmol/mg/min for the pH 7.4 experiments. [ 1 C]Gly-Sar Uptake via the Oligopeptide Transporter is Inhibited by Various Dipeptides The affinities of a variety of dipeptides and ammo acids for the oligopeptide transporter, as determined by their relative ability to inhibit the uptake of [ 14 C]Gly-Sar into Caco-2 monolayers, were assessed (Fig. 2) The two tested L/L-configured dipeptides, Gly-Sar and Gly-Pro, inhibit the apical uptake of [1 4C]Gly-Sar by > 90%. Four dipeptides having a D-configured ammo acid in either the first or second position, i.e., D-Leu-Tyr, D-Val-Asp, Gly-D-Glu, and Gly-D-Asp, were also tested for their relative affinities for the oligopeptide transporter. These D/L- or L/D-configured dipeptides demonstrate a reduced affinity for the oligopeptide transporter relative to the L/L-configured dipeptides, nonetheless, they were all able to inhibit [ 1 C]Gly-Sar uptake by > 75 % As an additional control, two L-ammo acids, L-Tyr and L-Phe, were tested for their ability to inhibit [ 14 C]Gly-Sar uptake in an identical manner, and as is shown in Fig. 2, they were both unable to inhibit [ 14 C]Gly-Sar uptake to any significant degree. D-Asp-Ala, D-Asp{cHex)-Ala, and D-Asp(BZ)-Ala Inhibit [ 14 C]Gly-Sar Uptake D-Asp-Ala, D-Asp(cHex)-Ala, and D-Asp(BZ)-Ala (all 20 mM in the apical media) were tested in order to assess their respective abilities to inhibit the apical uptake of [ 14 C]Gly-Sar into Caco-2 monolayers during either a 1 5 or a 1 20 mm experiment. As is shown in Fig. 3, the extent of inhibition of [ , C]gly-sar uptake is > 95 % for all three compounds following 1 5 mm of competitive inhibition, and > 80% following 1 20 mm of competitive inhibition. Following several [ ,4 C]Gly-Sar apical uptake inhibition experiments using various concentrations of each compound as a competitive inhibitor, the IC 50 values for D-asp-ala, D- asp(cHex)-ala, and D-asp(BZ)-ala were calculated and found to be 4.37 ± 0.02, 4.23 ± 0.08, and 3.20 ± 0.33 mM, respectively. D-Asp(cHex)-Ala and D-Asp(BZ)-Ala Stability during Uptake Experiments Using HPLC, the stabilities of D-Asp(cHex)-Ala and D-Asp(BZ)-Ala under experimental conditions were calculated. HPLC analysis of the apical media of Caco-2 cells containing the β-carboxyl modified D-Asp-Ala compounds demonstrates that both D-Asp(cHex)-Ala and D-Asp(BZ)-Ala are 1 00% stable under these conditions for up to 5 hr. The apparent permeability of D-Asp(cHex)-Ala and D-Asp(BZ)-Ala was also calculated. In order to insure that an equil brous mixture was attained, samples from the aqueous phase were submitted for HPLC analysis at both 8 and 24 hr. These experiments were performed in triplicate and the partition coefficient (P) was calculated using Equation 1 : Equation 1 P = (C, - C w )/C w x V w /V 0 where C, and C w represent the concentration of D-Asp(cHex)-Ala and D-Asp(BZ)- Ala in the aqueous buffer phase before and after distribution, respectively; V w represents the volume of the aqueous phase; and V 0 is the volume of the octanol phase The log P values for D-Asp(cHex)-Ala and D-Asp(BZ)-Ala were calculated to be -0.1 7 ± 0.01 and 0.1 5 ± 0.09, respectively; these values represent the means of N = 3 experiments ± S.D. D-Asp-Ala could not be detected via HPLC due to its lack of retention on the column used Thus, its log P values could not be determined. There was no significant difference in the concentration of either compound in the aqueous phase 8 and 24 hr after rotation was initiated, indicating that equilibrium was indeed reached after 8 hr No degradation products were seen in the chromatogram following 24 hr of rotation; thus, both D-Asp(cHex)-Ala and D-Asp(BZ)-Ala are completely stable for the duration of the uptake experiments. REFERENCES: 1 D. M . Matthews and S. A. Adibi. Intestinal absorption of peptides. Physiol. Rev 55. 537-608 ( 1 976) . 2. V. Ganapathy and F. H. Leibach. Is intestinal peptide transport energized by a proton gradient? Am. J. Physiol 249: G 1 53-G 1 60 ( 1 985) 3 D. T Thwaites, C. D. A. Brown, B. H Hirst, and N. L. Simmons. Transepi- thelial glycylsarcosme transport in intestinal Caco-2 cells mediated by expression of H + -coupled carriers at both apical and basal membranes. J. Biol. Chem 268. 7640-7642 ( 1 993) . 4 H. Daniel, E. L. Morse, and S. A Adibi. Determinants of substrate affinity for the olιgopeptιde/H + symporter in the renal brush border membrane. J. Biol. Chem. 267( 1 4) : 9565-9573 ( 1 992) . 5. A. H. Dantzig and L. Bergm. Uptake of the cephalospoπn, cephalexm, by a dipeptide transport carrier in the human intestinal cell line, Caco-2. Biochim. Biophys. Acta. 1027: 21 1 -21 7 ( 1 990) . 6. W. Kramer, F Girbig, U. Gutjagr, H. W. Kleemann, I . Leipe, J. Urbach, and A. Wagner. Interaction of renm inhibitors with the intestinal uptake system for oligopeptide and B-lactam antibiotics. Bioceim. Biophys. Acta 1 027 * 25-30 ( 1 990) . 7. C H. Gochoco, F M. Ryan, J. Miller, P. L. Smith, and I. J. Hildago Uptake and transepithehal transport of the orally adsorbed cephalosporm cephalexm in the human entest al cell line, Caco-2. Int. J. Pharm. 104: 187-202 (1994). 8. M. Hu and G. L. Amidon. Passive and carrier-mediated intestinal absorption components of Captopπl. J. Pharm. Sci. 77:1007-1011 (1988). 9. P W. Swaan, M. C. Stehouwer, R. I. C. Blok, and J. J. Tukker. Prodrug approach using the intestinal peptide carrier. Pharm. Res. 10:S-295 (1993). 10. P. L. Smith, E. P. Eddy, C.-P. Lee, and G. Wilson. Exploitation of the intestinal oligopeptide transporter to enhance drug absorption. Drug Delivery 1: 103-111 (1993). 11. R. Liang, Y.-J. Fei, P. D. Prasad, S Ramamoorthy, J. Jan, T L Yang-Feng, M. A. Hediger, V. Ganapathy, and F. H. Leibach. Human intestinal H + /peptιde cotransporter. JBC 270:6456-6463 (1995). 12. Y.-J. Fei, Y. Kanai, S. Nussberger, V. Ganapathy, F.H. Leibach, M. F. Romero, S. K. Singh, W. F. Boron, and M. A. Hediger. Experession cloning of a mammalian proton-coupled oligopeptide transporter. Nature 368: 563-566 (1994). 13. H Saito and K.-l. Inui. Dipeptide transporters in apical and basolateral membranes of the human intestinal cell line Caco-2 Am. J. Physiol 265:G289-G294 (1993). 14. I. J. Hidalgo, P. Bhatnagar, C.-P. Lee, J. Miller, G. Cucullino, and P.L. Smith. Structural requirements for interaction with the ohgopeptide transporter in Caco-2 cells. Pharm. Res. 12(2): 317-319 (1995). 15. E. P. Eddy, C. Wood, J. Miller, G. Wilson, and I. J. Hidalgo. A comparison of the affinities of depeptides and antibiotics for the dι-/trιpeptιde transporter in Caco-2 cells. Int. J. Pharmaceutics 115: 79-86 (1995) 16. J. P -F. Bai and G. L. Amidon. Structural specificity of mucosal-cell transport and metabolism of peptide drugs: Implication for oral peptide drug delivery. Pharm. Res. 9(8): 969-978 (1992). 17. D. T. Thwaites, B. H. Hirst, and N. L. Simmons. Substrate specificity of the di/tπpeptide transporter in human intestinal epithelia (Caco-2): Identification of substrates that undergo H+ coupled absorption. Br. J. Pharmacol. 113: 1050-1056 (1994).

Claims: CLAIMS 1 . A compound of general formula I W 1 W 2 W 3 H 2 N-A 1 - A 2 - (A 3 ) p -COOH wherein p is 0 or 1 ; A 1 , A 2 and A 3 are natural or modified amino acids, when p is 1 , at least one of W < , W 2 and W 3 are a residue of a pharmaceutically useful agent and the others are hydrogen, when p is 0, W^ is a residue of a pharmaceutically useful agent and W 2 is hydrogen or both W^ and W 2 are pharmaceutically useful agents. 2. A compound of formula I wherein A 1 , A 2 or A 3 intended for linkage of the pharmaceutically useful agents are selected from the group consisting of Asp, Glu, Asn, Gin, Lys, Orn, Cys, Tyr, Thr and Ser. 3. A compound of formula I wherein A^ or A 2 are natural amino acids in their D or L configuration or modified amino acids. 4. A compound according to claim 1 wherein k ' * , A 2 or A 3 are D-Asp or MeGly. 5. A compound according to claim 1 wherein at least one of W^ , W 2 and W 3 are a residue of a poorly absorbed pharmaceutically useful agent and the others are hydrogen. 6. A compound of formula I wherein p is 0 and A ^ and A 2 is selected from the group consisting of L-Asp-L-Ala, L-Lys-L-Ala, L-Cys-L-Ala, L-Ala-L-Asp, L-Ala-L- Lys, L-Ala-L-Cys, L-Asp-L-Gly, L-Lys-L-Gly, L-Cys-L-Gly, L-Gly-L-Asp, L-Gly-L-Lys, L- Gly-L-Cys, L-Asp-L-MeGly, L-Lys-L-MeGly, L-Cys-L-MeGly, L-MeGly-L-Asp, L- MeGly-L-Lys, L-MeGly-L-Cys, D-Asp-L-Ala, D-Lys-L-Ala, D-Cys-L-Ala, D-Ala-L-Asp, D-Ala-L-Lys, D-Ala-L-Cys, D-Asp-L-Gly, D-Lys-L-Gly, D-Cys-L-Gly, L-Asp-D-Ala, L- Lys-D-Ala, L-Cys-D-Ala, L-Ala-D-Asp, L-Ala-D-Lys, L-Ala-D-Cys, L-Gly-D-Asp, L-Gly- D-Lys r L-Gly-D-Cys, L-Orn-L-Ala, L-Ala-L-Om, L-Orn-L-Gly, L-Gly-L-Orn, L-Orn-L- MeGiy, L-MeGly-L-Orn, D-Orn-L-Aia, D-Ala-L-Orn, D-Orn-L-Gly, L-Orn-D-Ala, L-Ala- D-Orn, L-Gly-D-Orn. 7. A peptide of the general formula II: H 2 N-A '-A 2 -(A 3 ) p -COOH wherein p, A 1 A 2 and A 3 are defined as in claim 1 . 8. The use of a compound of the general formula II H 2 N-A 1 -A 2 -(A 3 ) p -COOH wherein p, A 1 A 2 and A 3 are defined as in claim 1 , for transport of pharmaceutically useful agents. 9. The use according to claim 8 wherein the pharmaceutically useful agent is poorly absorbed in its free form. 1 0. The use according to claim 8 wherein the pharmaceutically useful agent is selected from Fluorouracil or 6-Mercaptopurιne

Inventors: FROEKJAER SVEB, LARSEN BJARNE DUE, STEFFANSEN INGE-BENTE DALSGAAR, TAUB MITCHELL ETHAN, FROEKJAER, SVEB, LARSEN, BJARNE, DUE, STEFFANSEN, INGE-BENTE, DALSGAARD, TAUB, MITCHELL, ETHAN
Applicants: FROEKJAER SVEB, LARSEN BJARNE DUE, NOVONORDISK AS, STEFFANSEN INGE BENTE DALSGAAR, TAUB MITCHELL ETHAN, FROEKJAER, SVEB, LARSEN, BJARNE, DUE, NOVO NORDISK A/S, STEFFANSEN, INGE-BENTE, DALSGAARD, TAUB, MITCHELL, ETHAN
Application year: 1997
Main classification: C07K5
IPC codes: A61K47, C07K5