Abstract
Purpose. To synthesize a series of reversible fatty acid-desmopressin (DDAVP) conjugates and to study their structure-activity relationship as anti-diuretic drugs.
Methods. Seven fatty acid conjugates of DDAVP were prepared using various reversible lipidization reagents as described in our previous reports. All products were purified by acid precipitation and/or size-exclusion chromatography. Reversed-phase HPLC was used to evaluate their purity and lipophilicity. The anti-diuretic efficacy of these fatty acid conjugates was assessed in vasopressin-deficient Brattleboro rats. Four selected conjugates, i.e., DPA, DPH, DPD and DPP (acetic, hexanoic, decanoic, and palmitic acid conjugate, respectively), along with DDAVP itself were used in Caco-2 cell uptake studies and their degradation and the regeneration of active DDAVP were investigated using an in vitro liver slice metabolic system coupled with a HPLC assay.
Results. All fatty acid-DDAVP conjugates were more lipophilic than DDAVP as examined by HPLC analyses. When cysteine was used as the linker, the capacity index (k′, a measure of lipophilicity) of the conjugates was linearly correlated with the number of carbons in the fatty acid chain. The anti-diuretic activity of the conjugates was correlated with the length of the fatty acid chain, with C10 as the minimal requirement for possessing the enhanced anti-diuretic activity. Among the seven fatty acid conjugates, palmitic acid conjugate was the most potent DDAVP derivative. Removal of carboxyl group from the cysteine linker completely abolished the enhancement of the activity. The extent of cellular uptake also positively correlated with the lipophilicity of the conjugates. The metabolism of DDAVP, DPH, DPD, and DPP by liver slices all followed first order kinetics with half-life of 0.30, 0.01, 0.06 and 3.44 hr, respectively. The degradation rates of DPH and DPD in the liver slice incubation were much faster than that of DDAVP and therefore an accumulation of regenerated DDAVP in the media was observed. In contrast, DPP was metabolized much slower than DDAVP and, consequently, no significant accumulation of regenerated DDAVP could be detected.
Conclusion. Conjugation of DDAVP with fatty acids increased the lipophilicity and the anti-diuretic activity of this peptide drug. The anti-diuretic activity of lipidized DDAVP was dependent on the chain length of the fatty acid, as well as the structure of the linker in the conjugate. The preservation and enhancement of the in vivo anti-diuretic activity of the conjugates is most likely due to a combination of an improved pharmacokinetic behavior and a concurrent regeneration of active DDAVP in tissues.
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REFERENCES
D. J. Cada. (Exec. Ed.) Drug Facts and Comparisons (54th ed), Wolters Kluwer Co., St. Louis, Missouri, 2000 pp. 349–351.
H. Weingartner, P. Gold, J. C. Ballenger, S. A. Smallberg, R. Summers, D. R. Rubinow, R. M. Post, and F. K. Goodwin. Effects of vasopressin on human memory functions. Science 211: 601–603 (1981).
I. Vavra, A. Machova, V. Holecek, J. H. Cort, M. Zaoral, and F. Sorm. Effect of a synthetic analogue of vasopressin in animals and in patients with diabetes insipidus. Lancet 1:948–952 (1968).
M. Manning, L. Balaspiri, and J. Moehring. Synthesis and some pharmacological properties of deamino [4-threonine, 8-Darginine] vasopressin and deamino [8-D-arginine] vasopressin highly potent and specific antidiuretic peptides, and [8-Darginine] vasopressin and deamino-arginine-vasopressin. J.Med.Chem. 19:842–845 (1976).
J. Wang, D. Shen, and W-C Shen. Preparation, purification and characterization of a reversibly lipidized desmopressin with potentiated anti-diuretuc activity. Pharm.Res. 16:1674–1679 (1999).
H. Ekrami, A. R. Kennedy, and W. C. Shen. Water-soluble fatty acid derivatives as acylating agents for reversible lipidization of polypeptides. FEBS Lett. 371:283–286 (1995).
H. W. Sokol and H. Valtin. The Brattleboro Rat. Ann., New York Academy of Science, New York, 1982.
C. Q. Xia, J. Wang, and W. C. Shen. Hypoglycemic effect of insulin-transferrin conjugate in streptozotocin-induced diabetic rats. J.Pharmcol.Exp.Ther. 295:594–600 (2000).
P. C. McConahey and F. J. Dixon. Radiation of proteins by the use of the chloramine-T method. Methods Enzymol. 70:221–247 (1980).
S. J. Weber, T. J. Abbruscato, E. A. Brownson, A. W. Lipkowski, R. Polt, A. Misicka, R. C. Haaseth, H. Bartosz, V. J. Hruby, and T. P. Davis. Assessment of an in vitro blood-brain barrier model using several [Met5]enkephalin opioid analogs. J.Pharmcol.Exp.Ther. 266:1649–1655 (1993).
C. Martorell, A. C. Calpena, J. M. Poblet, and J. Freixas. Influence of the chromatographic capacity factor (log k') as an index of lipophilicity in the antibacterial activity of a series of 6-fluoroquinolones. J.Chromatogr.A 655:177–184 (1993).
M. Calvani, L. Critelli, G. Gallo, F. Giorgi, G. Gramiccioli, M. Santaniello, N. Scafetta, M. O. Tinti, and F. De Angelis. LCarnitine esters as “soft”, broad-spectrum antimicrobial amphiphiles. J.Med.Chem. 41:2227–2233 (1998).
E. Yodoya, K. Uemura, T. Tenma, T. Fujita, M. Murakami, A. Yamamoto, and S. Muranishi. Enhanced permeation of tetragastrin across the rat intestinal membrane and its reduced degradation by acylation with various fatty acids. J.Pharmacol.Exp.Ther. 271:1509–1513 (1994).
H. Bundgaard and J. Mø ss. Prodrugs of peptides. 6. Bioreversible derivatives of thyrotropin-releasing hormone (TRH) with increased lipophilicity and resistance to cleavage by the TRHspecific serum enzyme. Pharm.Res. 7:885–892 (1990).
V. P. Chekhonin, I. A. Ryabukhin, Y. A. Zhirkov, I. A. Kashparov, and T. B. Dmitriyeva. Transport of hydrophobized fragments of antibodies through the blood-brain barrier. NeuroReport 7:129–132 (1995).
H. Asada, T. Douen, Y. Mizokoshi, T. Fujita, M. Murakami, A. Yamamoto, and S. Muranishi. Stability of acyl derivatives of insulin in the small intestine: Relative importance of insulin association characteristics in aqueous solution. Pharm.Res. 11:1115–1120 (1994).
L. Honeycutt, J. Wang, H. Ekrami, and W. C. Shen. Comparison of pharmacokinetic parameters of a polypeptide, the Bowman-Birk protease inhibitor (BBI), and its palmitic acid conjugate. Pharm.Res. 13:1373–1377 (1996).
E. Yodoya, K. Uemura, T. Tenma, T. Fujita, M. Murakami, A. Yamamoto, and S. Muranishi. Enhanced permeation of tetragastrin across the rat intestinal membrane and its reduced degradation by acylation with various fatty acids. J.Pharmacol.Exp.Ther. 271:1509–1513 (1994).
F. Al-Obeidi, V. J. Hruby, N. Yaghoubi, M. M. Marwan, and M. E. Hadley. Synthesis and biologic activities of fatty acid conjugates of a cyclic lactam α-melanotropin. J.Med.Chem. 35:118–123 (1992).
M. Hashimito, K. Takada, Y. Kiso, and S. Muranishi. Synthesis of palmitoyl derivatives of insulin and their biologic activities. Pharm.Res. 6:171–176 (1989).
Y. Ando, M. Inoue, T. Utsumi, Y. Morino, and S. Araki. Synthesis of acylated SOD derivatives which bind to the biomembrane lipid surface and dismutate extracellular superoxide radicals. FEBS Lett. 240:216–220 (1988).
J. Markussen, S. Havelund, P. Kurtzhals, A. S. Andersen, J. Halstrom, E. Hasselager, U. D. Larsen, U. Ribel, L. Schaffer, K. Vad, and I. Jonassen. Soluble, fatty acid acylated insulins bind to albumin and show protracted action in pigs. Diabetologia 39:281–288 (1996).
L. B. Knudsen, F. P. Nielsen, P. O. Huusfeldt, N. L. Johansen, K. Madsen, F. Z. Pedersen, H. Thogersen, M. Wilken, and H. Agerso. Potent derivatives of glucagons-like-1 with pharmacokinetic properties suitable for once daily administration. J.Med.Chem. 43:1664–1669 (2000).
S. R. Myers, F. E. Yakubu-Madus, W. T. Johnson, J. E. Baker, T. S. Cusick, V. K. Williams, F. C. Tinsley, A. Kriauciunas, J. Manetta, and V. J. Chen. Acylation of human insulin with palmitic acid extends the time of action of human insulin in diabetic dogs. Diabetes 46:637–642 (1997).
W. C. Shen, J. Wang, and D. Shen. Reversible lipidization for the delivery of peptide and protein drugs. In S. Frokjaer, L. Christrup, and P. Krogsgaard-Larsen (eds), Peptide and Protein Drug Delivery, Munksgaard, Copenhagen, 1998 pp. 397–408.
C. J. Porter and S. A. Charman. Lymphatic transport of proteins after subcutaneous administration. J.Pharm.Sci. 89:297–310 (2000).
C. J. H. Porter. Drug delivery to the lymphatic system. Crit.Rev.Ther.Drug Carrier Syst. 14:333–393 (1997).
P. E. Thorpe, P. M. Wallace, P. P. Knowles, M. G. Relf, A. N. Brown, G. J. Watson, R. E. Knyba, E. J. Wawrzynczak, and D. C. Blakey. New coupling agents for the synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vitro. Cancer Res. 47:5924–5931 (1987).
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Wang, J., Wu, D. & Shen, WC. Structure-Activity Relationship of Reversibly Lipidized Peptides: Studies of Fatty Acid-Desmopressin Conjugates. Pharm Res 19, 609–614 (2002). https://doi.org/10.1023/A:1015397811161
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DOI: https://doi.org/10.1023/A:1015397811161