Controlled Drug Delivery - ACS Publications - American Chemical

Chapter 4

Prodrug Strategies to Improve the Oral Absorption of Peptides and Peptide Mimetics 1

Ronald T. Borchardt and Binghe Wang

2

Downloaded by UNIV LAVAL on July 11, 2016 | http://pubs.acs.org Publication Date: May 15, 2000 | doi: 10.1021/bk-2000-0752.ch004

1Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66047 2Departmentof Chemistry, North Carolina State University, Raleigh, NC 27695

Recently, our laboratories have introduced the concept of preparing cyclic prodrugs of peptides and peptide mimetics as a means of modifying their physicochemical properties sufficiently to allow them to permeate biological barriers (i.e., intestinal mucosa). These cyclization strategies employ new chemical linkers that were designed to be susceptible to esterase metabolism, leading to a cascade of chemical reactions that result in release of the peptide or peptide mimetic. These cyclic prodrug strategies have been applied to opioid peptides and RGD peptide mimetics to stabilize them to metabolism and/or to improve their intestinal mucosal permeation.

In recent years, many structurally diverse peptides and peptide mimetics possessing a broad spectrum of pharmacological effects have been synthesized by medicinal chemists (1). Many of these peptides and peptide mimetics have unique therapeutic indications that warrant their clinical development. However, in many cases, the clinical development of these molecules has been prevented because of their unfavorable biopharmaceutical properties, which include (i) metabolic lability and (ii) poor permeation of cell membranes (e.g., intestinal mucosa) (2,3). The problem of metabolic lability by hydrolytic pathways (e.g., peptidases) has, for all practical purposes, been resolved by medicinal chemists through structural manipulation of the peptide (i.e., introducing peptidase-resistant bonds) to produce peptide mimetics (2,3). Until recently, medicinal chemists have had less success in manipulating the structures of peptides and peptide mimetics to achieve good permeation of cell membranes (i.e., intestinal mucosa) while still retaining high affinity for the macromolecular target (2,3). Permeation of peptides and peptide mimetics across the intestinal mucosa can occur via the paracellular pathway (pathway A, Figure 1) or transcelluiar pathway

36

© 2000 American Chemical Society

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

37


(pathway Β, Figure 1). In general, hydrophilic peptides (e.g., opioid peptides) and peptide mimetics (e.g., RGD peptide mimetics) are restricted to the paracellular pathway, which consists of aqueous pores (average size in the small intestine, approx. 7-9 Â) created by the cellular tight junctions (3). These aqueous pores restrict peptide and peptide mimetic permeation based on size and charge (4). Hydrophilic peptides and peptide mimetics, whose permeation is restricted to the paracellular pathway, typically exhibit oral bioavailabilities of 50%) because they serve as substrates for the oligopeptide trans porter (pathway C, Figure 1) (5).

Basolaleral

Figure 1. Pathways ofpeptide mimetic transport across the intestinal mucosa: A, passive diffusion via paracellular route; B, passive diffusion via transcellular route; C, transporter-facilitated (e.g., oligopeptide transporter); D, transportrestricted (e.g., efflux transporters). In contrast to hydrophilic molecules, hydrophobic peptides, peptide mimetics or prodrugs of these molecules that lack charge and exhibit low hydrogen bonding potential can traverse the intestinal mucosa by passive diffusion via the transcellular pathway. Previously, it was thought that the enzymatic barrier to peptide transport consisted only of brush border membrane and cytoplasmic peptidases (6). Recently, however, it was shown that a specific isozyme (3A4) of cytochrome P450 plays an important role in the metabolism of peptides (e.g., cyclosporin) and peptide mimetics (e.g., HIV protease inhibitors) (7,8). In addition, the intestinal mucosa has been shown to contain efflux transporters [e.g., multidrug resistance protein (MDR1 in humans, also called P-glycoprotein)] that restrict transcellular permeation of hydro philic peptides (e.g., cyclosporin) and peptide mimetics (e.g., HIV protease inhibi tors) (8,9), as well as hydrophobic prodrugs of peptides and peptide mimetics (10). To succeed in developing orally bioavailable peptides and peptide mimetics, it will be necessary to incorporate structural features into the molecules that will opti mize their pharmacological (e.g., high receptor affinity) as well as their biopharmaceutical (e.g., intestinal mucosal permeation) properties. As an alternative approach, unfavorable biopharmaceutical properties of peptides and peptide mimetics can be


38 transiently modified using prodrug strategies. In general, prodrug strategies applied to peptides and peptide mimetics have focused on modification of a single functional group (e.g., N-terminal amino group or C-terminal carboxyl group) (3,11,12). More recently, our laboratories have introduced the concept of preparing cyclic prodrugs of peptides (e.g., opioid peptides) and peptide mimetics (e.g., RGD peptide mimetics) as a way to modify their physicochemical properties sufficiently to overcome their undesirable biopharmaceutical properties (e.g., poor intestinal mucosal permeation). Our progress to date on this strategy is the subject of this chapter.


Results and Discussion

Chemical Linkers Used to Prepare Cyclic Prodrugs From a membrane permeation perspective, there are several possible advantages to considering cyclization strategies for making prodrugs of peptides and peptide mimetics (11,13). These include: (i) derivatization of key functional groups (e.g., N-terminal amino and C-terminal carboxyl groups) would reduce the overall charge of the molecule; (ii) cyclization could lead to the creation of unique solution structures that reduce the hydrogen bonding potential of the molecule; and (iii) cyclization could lead to a reduction in the size of the molecule. Key to the success of these cyclic prodrug strategies was the identification of chemical linkers that were susceptible to esterase metabolism, leading to a cascade of chemical reactions and resulting in release of the therapeutic peptide or peptide mimetic (Figure 2). Recently, Borchardt's laboratory has described synthetic methodologies for linking the N-terminal amino group to the C-terminal carboxyl group of peptides using an acyloxyalkoxy promoiety (Figure 2A) (14,15) or a 3-(2 -hydroxy-4',6'-dimethylphenyl)-3,3,

Figure 2. Cyclic prodrug strategies. Panel A, acyloxyalkoxy-based prodrug; pane B, phenylpropionic acid-based prodrug; Panel C, coumarinic acid-based prodrug


39


dimethylpropiortic acid promoiety (Figure 2B) (16,17). In a similar manner, Wang's laboratory (18-20) has used coumarinic acid (Figure 2C) as a chemical linker to make cyclic prodrugs of peptides. Using a model hexapeptide, Borchardt's laboratory showed that an acyloxyalkoxy cyclic prodrug (21) and a phenylpropionic acid prodrug (22) were converted to the parent peptide more rapidly in human blood than in physiological buffer, supporting the involvement of esterases in their bioconversion. In addition, in trans port studies conducted using Caco-2 cell monolayers, an in vitro model of the intes tinal mucosa, both cyclic prodrugs were shown to permeate at least 70 times better than the parent hexapeptide (21,22). The enhanced permeation of the acyloxylalkoxy cyclic prodrug could be explained by formation of unique solution structures that reduced the hydrogen bonding potential of the molecule (23).

Applications to Opioid Peptides Recently, our laboratories have applied the cyclic prodrug strategies shown in Figure 2 to opioid peptides. [Leu ]-enkephalin (H-Tyr-Giy-Gly-Phe-Leu-OH) and its metabolically stable analog DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) were chosen as model opioid peptides because many of the structural features of these molecules (e.g., free N-terminal amino and C-terminal carboxyl groups) that bestow upon them affinity and specificity for the opioid receptors also endow these molecules with undesirable physicochemical properties (e.g., charge, hydrophilicity, high hydrogen bonding potential) that limit their permeation via the transcellular route. Consistent with the enzymatic/chemical pathway of degradation of the coumarinic acid-based cyclic prodrugs shown in Figure 2C, the [Leu ]-enkephalin and DADLE prodrugs using this linker were shown to release the parent peptides more rapidly in biological media containing esterase activity than in pH 7.4 Hanks' balanced salt solution (HBSS) (24,25). When [Leu ]-enkephalin was applied to the apical (AP) side of Caco-2 cell monolayers, no detectable levels of this opioid peptide were observed in the basolateral (BL) side of the monolayer (25). A maximum apparent permeability (Papp) value of 0.31 χ 10" cm/s was calculated based on the limit of detection of our analytical method for [Leu ]-enkephalin. In contrast, DADLE, when applied to the AP side of the Caco-2 monolayer, was relatively stable and could be detected on the BL side (Ρ,φρ = 7.8 ± 0.7 χ 10" cm/s). When the coumarinic acid-based cyclic prodrugs were applied to the AP side of Caco-2 monolayers, significant quantities of these cyclic prodrugs were detected on the BL side of the monolayer (25). Based on the estimated P^p value for [Leu ]-enkephalin and the determined P^p value of the corresponding [Leu ]-enkephalin cyclic prodrug, the prodrug was shown to be approx. 665-fold more able to permeate the cell monolayer than was the opioid peptide itself. In comparison, the cyclic prodrug of DADLE was approx. 31-fold more able to permeate the monolayer than was DADLE itself. Characterization of the membrane interaction potentials (log k ' ^ ) of the cyclic prodrugs and the opioid peptides by immobilized artificial membrane (IAM) chromatography revealed that 5

5

5

8

5

8

5

5



40 the cyclic prodrugs had significantly higher tendency to partition into the artificial membrane than did the opioid peptides (24). Therefore, a good correlation was observed between this physicochemical property (log k ' ^ ) and the cell permeation characteristics of the prodrugs. In fact, the membrane interaction potentials of the coumarinic acid-based cyclic prodrugs were sufficiently favorable to allow these prodrugs to traverse the monolayer via the transcellular pathway without interacting with polarized efflux transporters (e.g., MDR1) (25). In contrast, the opioid peptides themselves could only permeate via the paracellular pathway. In an attempt to understand how the solution structures of the coumarinic acidbased cyclic prodrugs influenced their passive diffusion, conformational studies using spectroscopic techniques (2D-NMR, CD) and molecular dynamics were per formed (26). These studies indicated that the cyclic prodrugs had compact and rigid secondary structures composed of β-turns that are partially stabilized by formation of intramolecular hydrogen bonds. The existence of these well-defined secondary structures in the cyclic prodrugs, particularly the presence of intramolecular hydro gen bonds, correlates well with the ability of these prodrugs to partition into an artificial membrane and to permeate the Caco-2 cell monolayers. Like the coumarinic acid-based cyclic prodrugs described above, the phenylpropionic acid-based cyclic prodrugs were shown to release [Leu ]-enkephalin and DADLE by enzymatic hydrolysis of the ester promoiety to form an intermediate that would then undergo lactonization to release the parent peptides (24,27). When the phenylpropionic acid-based cyclic prodrugs of [Leu ]-enkephalin and DADLE were applied to the AP side of Caco-2 cell monolayers, no significant degradation by chemical or enzymatic (peptidases, esterases) processes was observed (27). How ever, both cyclic prodrugs showed excellent cell permeation characteristics. For example, based on the estimated P^p value for [Leu ]-enkephalin (see above) and the determined P^p value of the corresponding phenylpropionic acid-based [Leu ]enkephalin cyclic prodrug, the prodrug was approx. 1680-fold more able to permeate the cell monolayer than was the opioid peptide itself. In comparison, the cyclic pro drug of DADLE was approx. 72-fold more able to permeate the monolayer than was DADLE. Characterization of the membrane interaction potentials (log k ' ^ ) of the cyclic prodrugs by LAM chromatography revealed that the cyclic prodrugs had sig nificantly higher tendency to partition into the artificial membrane than did the opioid peptides (24). Therefore, a good correlation was observed between this physicochemical property (log k^A^) and the actual cell permeation characteristics of the prodrugs. Similar to those of the coumarinic acid-based prodrugs, the membrane interaction potentials of the phenylpropionic acid-based cyclic prodrugs were suffi ciently favorable to allow these prodrugs to traverse the monolayer via the transcellu lar pathway without interacting with polarized efflux transporters (e.g., MDRl) (27). To better understand their lipophilic character and their ability to permeate cell membranes, we conducted conformational studies on these phenylpropionic acidbased cyclic prodrugs using lD-and 2D-NMR and CD (26). These studies revealed that the phenylpropionic acid-based cyclic prodrugs exhibited more well-defined, rigid and compact secondary structures composed of β-turns than did the opioid peptides. The formation of the intramolecular hydrogen bonds, as well as the incor5

5

5

5


41 poration of the lipophilic phenylpropionic acid promoiety into the relatively hydrophobic peptide sequence, may account for the ability of the prodrugs to partition into an artificial membrane as determined by IAM chromatography. Furthermore, the existence of these well-defined secondary structures in cyclic prodrugs, particularly the presence of intramolecular hydrogen bonds, correlates well with their membrane interaction potential and their ability to permeate the Caco-2 cell monolayers. Like the coumarinic acid-based and phenylpropionic acid-based cyclic prodrugs described above, the acyloxyalkoxy cyclic prodrugs were shown to rapidly release [Leu ]-enkephalin and DADLE by an esterase-catalyzed reaction (10,28). Because of the physicochemical properties (e.g., high membrane interaction potential, low hydrogen bonding potential) of the acyloxyalkoxy-based cyclic prodrugs (28), it was expected that these cyclic prodrugs of [Leu ]-enkephalin and DADLE would have favorable permeation characteristics across Caco-2 cell monolayers. This hypothesis was based on our earlier observations with the phenylpropionic acid- and coumarinic acid-based cyclic prodrugs of the same opioid peptides. These cyclic prodrugs exhibited favorable physicochemical properties (e.g., log k ' ^ ) that correlated well with their excellent Caco-2 permeation characteristics (see discussion above). However, it should be noted that, while these phenylpropionic acid- and coumarinic acid-based cyclic prodrugs of [Leu ]-enkephalin and DADLE had favorable log k'j^M values, they were not shown to be substrates for apically polarized efflux systems in Caco-2 cells, which could have restricted their transcellular permeation. Therefore, it was very surprising to observe that the acyloxyalkoxy-based cyclic prodrugs of [Leu ]-enkephalin and DADLE exhibited P^p values across Caco-2 cell monolayers in the AP-to-BL direction that were approximately 200-300 times lower than the values observed previously with the phenylpropionic acid- and coumarinic acid-based cyclic prodrugs of the same opioid peptides (10,25,27). In fact, the acyloxyalkoxy-based cyclic prodrug of DADLE had a P^p A P - B L value approx. four times lower than that of DADLE itself, in spite of having a membrane interaction potential (log k ' ^ ) approx. four times higher (10). However, an explanation for the lower permeation of acyloxyalkoxy-based cyclic prodrugs across Caco-2 cells became obvious when the P ^ values in the BL-to-AP direction for these prodrugs were determined. The much higher P ^ _ values compared to the P^p AP-ÎO-BL values for cyclic prodrugs ([Leu ]-enkephalin prodrug, 36 times greater; DADLE prodrug, 52 times greater) suggest that these acyloxyalkoxy-based prodrugs are substrates for apically polarized efflux systems (10). Further evidence in support of this hypothesis was obtained by conducting competition experiments using cyclosporin, a known inhibitor of P-glycoprotein (10). The substrate activity of the acyloxyalkoxy-based cyclic prodrugs of [Leu ]enkephalin and DADLE for efflux systems in Caco-2 cells versus the nonsubstrate activity of the phenylpropionic acid- and coumarinic acid-based cyclic prodrugs of the same opioid peptides is very intriguing. Based on their membrane interaction potentials (log k'iAM) as measured by their abilities to partition into an immobilized artificial membrane, the acyloxyalkoxy-based prodrugs had the least favorable membrane partition characteristics (low log k'ïAM values) (28) and the phenylpropionic acid-based prodrugs had the most favorable membrane partition characteristics (high 5


5

5

5

BL4o

AP

5

5



42 log k'ïAM values) (24). These results would suggest, based on what is known about the substrate activity of P-glycoprotein, that the phenylpropionic acid- and coumarinic acid-based prodrugs would be more likely to be substrates for Pglycoprotein than the acyloxyalkoxy-based prodrugs. Obviously, these predictions were wrong because neither the phenylpropionic acid- nor the coumarinic acid-based prodrugs were substrates for efflux systems (e.g., P-glycoprotein), whereas the acyloxyalkoxy-based prodrugs were excellent substrates. These results suggest that, for this series of compounds, the membrane interaction potential alone was not a good predictor of substrate activity for P-glycoprotein. One possible difference between the acyloxyalkoxy-based prodrugs and the phenylpropionic acid- and coumarinic acid-based prodrugs is their solution conformations. While the phenylpropionic acid- and coumarinic acid-based prodrugs exist in well-defined solution conformations with significant secondary structure (e.g., β-turns) (26), the acyloxyalkoxybased prodrugs have two major conformers that exist in equilibrium as determined by NMR (29). These conformers arise from the cis-trans isomerization of the car bamate junction between the promoiety and the peptide. Therefore, it may be pos sible that the acyloxyalkoxy-based prodrugs in the lipid environment of the cell membrane adopt structures more conducive to interactions with P-glycoprotein. Alternately, the acyloxyalkoxy linker itself cannot be rule out at this time as the structural feature recognized by P-glycoprotein.

Application to RGD Peptide Mimetics Our laboratories have also applied the coumarinic acid-based cyclic prodrug strategy to RGD peptide mimetics. For these studies, we used MK-383 (4c, tirofiban) and several of its analogs (4a, 4b) (30). The corresponding cyclic prodrugs la-c were designed to be sensitive to esterases and were shown to undergo bioconversion as illustrated in Figure 3 (31). The cell transport characteristics of the RGD peptide mimetics (4a-b) and the corresponding cyclic prodrugs (la-b) were determined in Caco-2 cell monolayers (32). The apparent membrane permeability of RGD analogs 4a and 4b were determined to be 3.94 ± 0.05 χ 10~ and 3.88 ± 0.10 χ 10" cm/s, respectively. The coumarinic acid-based prodrugs la and lb exhibited apparent membrane permeabilities that were approximately sixfold (2.42 ± 0.29 χ 10 cm/s) and fivefold (1.90 ± 0.21 χ 10" cm/s) higher than their corresponding RGD analogs, respectively (32). These data indicate that the coumarinic acid-based prodrug strategy can indeed be used to improve the ability of such RGD analogs to permeate membranes. However, the magnitude of the improvement was less than what we observed with a cyclic prodrug of a metabolically stable opioid peptide, DADLE, which was 31 times more able to permeate than was the peptide itself (25). One possible explanation for this difference is that intramolecular hydrogen bond forma tion in the cyclic prodrug of DADLE could help to improve membrane permeation (26), while similar intramolecular hydrogen bonds cannot be formed with the cyclic 7

7

-6

6


43 prodrugs la,b of the RGD peptide mimetics because of the lack of the regularly spaced amide bonds. OOC

Ν NH < A Si


NHR « · R«-60z(3u 4b Re-CXHCtfefePti

W

NHSO^Bu

Figure 3. Esterase-sensitive cyclic prodrugs of RGD analogs. The ultimate objective of our prodrug research with RGD peptide mimetics is to identify an orally bioavailabie derivative that exhibits in vivo efficacy. Therefore, the ex vivo pharmacological effect of one prodrug (lc) was evaluated after oral adminis tration in dog (Binghe Wang, David C. Sane, Guy L. Wheeler and Che-Ping Cheng, unpublished data). In a very preliminary study using a single beagle dog, the prodrug was administered using a gelatin capsule. Blood samples drawn at different intervals were examined for indications of altered platelet aggregation patterns due to the administration of the prodrug. In this particular experiment the prodrug of tirofiban (lc), was used because it is a potent antiplatelet agent and is known to be orally unavailable (33). Significant and prolonged antiplatelet activity after oral administration of prodrug lc was observed. In contrast, administration of an equiva lent dose of MK-383 to the same dog had a minimal effect on ADP-induced platelet aggregation. However, it must be emphasized that these results are preliminary. More studies will be needed to further confirm the observed effects.

Conclusion The results of our studies have shown that cyclization of linear peptides produces a dramatic effect on their metabolic stability and their ability to permeate a biological barrier (e.g., intestinal mucosa). Therefore, the chemical linkers described in this chapter can be used to prepare cyclic prodrugs of therapeutically important peptides (e.g., opioid peptides) and peptide mimetics (e.g., RGD peptide mimetics) so as to transiently alter their physicochemical properties, thereby potentially improving their membrane permeation. However, because these prodrugs are designed to partition significantly into the lipid biiayer, they will be exposed to polarized efflux systems and cytochrome P-450-3A4 that could restrict their overall cell permeation. Cur rently, we have the knowledge to predict the physicochemical properties that would


44 optimize the partitioning of a peptide or peptideomimetie prodrug into a lipid bilayer. However, we lack the knowledge to predict which structural features will induce substrate activity for apically polarized efflux systems and cytochrome P4503A4.

Acknowledgments Financial support from the American Heart Association (BW), the Presbyterian Health Foundation (BW), United States Public Health Service (DA09315) (RTB), and the Costar Corporation (RTB) is gratefully acknowledged.


References th

1. Proceedings of the 15 American Peptide Symposium; Tarn, J. P.; Kaumaya, P. T. P., Eds.; Kluwer Academic Publisher: Dordrecht, The Netherlands, 1998. 2. Peptide-Based Drug Design: Controlling Transport and Metabolism; Taylor, M . D.; Amidon, G. L., Eds.; American Chemical Society: Washington, DC, 1995. 3. Pauletti, G. M.; Gangwar, S.; Siahaan, T. J.; Aube, J.; Borchardt, R. T. Adv. Drug Del. Rev. 1997, 27, 235. 4. Pauletti, G.M.;Okumu, F. W.; Borchardt, R. T. Pharm. Res. 1997,14, 164. 5. Tsuji, A. In Peptide-Based Drug Design: Controlling Transport and Metabo lism; Taylor, M . D.; Amidon, G. L., Eds.; American Chemical Society: Washington, DC, 1995, pp. 101-117. 6. Krishnamoorthy, R.; Mitra, A. K. In Peptide-Based Drug Design: Controlling Transport and Metabolism; Taylor, M . D.; Amidon, G. L., Eds.; American Chemical Society: Washington, DC, 1995, pp. 47-.68 7. Thummel, Κ. E.; Wilkinson, G. R. Annu. Rev. Pharmacol. Toxicol 1998, 38, 389. 8. Watkins, P. B. Adv. DrugDel.Rev. 1997,27,161. 9. Wacher, V. J.; Salphati, L.; Benet, L. Z. Adv. DrugDel.Rev. 1996,20,99. 10. Bak, Α.; Gudmundsson, O. S.; Friis, G. J.; Siahaan, T. J.; Borchardt, R. T. Pharm. Res. 1999,16, 24. 11. Gangwar, S.; Pauletti, G. M.; Wang, B.; Siahaan, T. J.; Stella, V. J.; Borchardt, R. T. Drug Discovery Today 1997, 2, 148. 12. Oliyai, R. Adv. DrugDel.Rev. 1996,19,275. 13. Shan, D.; Nicolaou, M . G.; Borchardt, R. T.; Wang, B. J. Pharm.Sc.1997, 86, 765. 14. Gangwar, S.; Pauletti, G. M.; Siahaan, T. J.; Stella, V. J.; Borchardt, R. T. J. Org. Chem. 1997, 62, 1356.



45 15. Gangwar, S.; Pauletti, G. M.; Siahaan, T. J.; Stella, V. J.; Borchardt, R. T. In Peptidomimetics Protocols; Kazmierski, W. M . Ed.; Humana Press: Totowa, NJ, 1998, pp. 37-59. 16. Wang, B.; Gangwar, S.; Pauletti, G. M . ; Siahaan, T. J.; Borchardt, R. T. J. Org. Chem. 1997, 62, 1363. 17. Wang, B.; Gangwar, S.; Pauletti, G.; Siahaan, T. J.; Borchardt, R. T. In Peptidomimetics Protocols; Kazmierski, W. M . Ed.; Humana Press: Totowa, NJ, 1998, pp. 53-69. 18. Wang, B.; Zhang, H.; Wang, W. Bioorg. Med. Chem. Lett. 1996, 6, 945. 19. Wang, B.; Wang, W.; Zhang, H.; Shan, D.; Smith, T. D. Bioorg. Med. Chem. Lett. 1996, 6, 2823. 20. Wang, B.; Shan, D.; Wang, W.; Zhang, H.; Gudmundsson, O. In Peptido mimetics Protocols; Kazmierski, W. M . Ed.; Humana Press: Totowa, NJ, 1998, pp. 71-85. 21. Pauletti, G. M . ; Gangwar, S.; Okumu, F. W.; Siahaan, T. J.; Stella, V. J.; Borchardt, R. T. Pharm. Res. 1996,13, 1615. 22. Pauletti, G. M.; Gangwar, S.; Wang, B.; Borchardt, R. T. Pharm. Res. 1997, 14, 11. 23. Gangwar, S.; Jois, S. D. S.; Siahaan, T. J.; Vander Velde, D. G.; Stella, V. J.; Borchardt, R. T. Pharm. Res. 1996,13, 1657. 24. Wang, B.; Nimkar, K.; Wang, W.; Zhang, H.; Shan, D.; Gudmundsson, O.; Gangwar, S.; Siahaan, T. J.; Borchardt, R. T. J. Peptide Res. 1999, 53, 370. 25. Gudmundsson, O.; Pauletti, G. M . ; Wang, W.; Shan, D.; Zhang, H.; Wang, B.; Borchardt, R. T. Pharm. Res. 1999,16,7. 26. Gudmundsson, O.; Jois, S. D. S.; Vander Velde, D. G.; Siahaan, T. J.; Wang, B.; Borchardt, R. T. J. Peptide Res. 1999, 53, 383. 27. Gudmundsson, O.; Nimkar, K.; Gangwar, S.; Siahaan, T. J.; Borchardt, R. T. Pharm. Res. 1999,16, 16. 28. Bak, Α.; Siahaan, T. J.; Gudmundsson, O.; Gangwar, S.; Friis, G. J.; Borchardt, R T. J. Peptide Res. 1999, 53, 393. 29. Gudmundsson, O.; Vander Velde, D. G.; Jois, S. D. S.; Bak, Α.; Siahaan, T. J.; Borchardt, R. T. J. Peptide Res. 1999, 53, 403. 30. Hartman, G. D.; Egbertson, M . S.; Halczenko, W.; Laswell, W. L.; Duggan, M . E.; Smith, R L.; Naylor, A. M.; Manno, P. D.; Lynch, R. J.; Zhang, G.; Chang, C. T.-C.; Gould, R. J. J. Med. Chem. 1992, 32,4640. 31. Wang, B.; Wang, W.; Camenisch, G. P.; Elmo, J.; Zhang, H.; Borchardt, R. T. Chem. Pharm. Bull. (Tokyo) 1999, 47, 90. 32. Camenisch, G. P.; Wang, W.; Wang, B.; Borchardt, R. T. Pharm. Res. 1998,15, 1174. 33. Peerlinck, K.; De Lepeleire, I.; Goldberg, M.; Farrell, D.; Barrett, J.; Hand, E.; Panebianco, D.; Deckmyn, H.; Vermylen, J.; Arnout, J. Circulation 1993, 88, 1512.


Controlled Drug Delivery - ACS Publications - American Chemical

Recommend Documents