Abstract
Purpose. The objective of this study is to investigate the pathways and kinetics of degradation of deslorelin, pGlu1-His2-Trp3-Ser4-Tyr5-D-Trp6-Leu7-Arg8-ProNHEt9 (Des1-9), in a human airway epithelial cell line (Calu-1).
Methods. The degradation of deslorelin in membrane and cytosolic fractions of Calu-1 cells was studied at 37°C up to 24 h. The degradation products were separated using HPLC and identified by amino acid analysis, sequencing, and mass spectrometry. The rate constants for deslorelin degradation and the formation of degradation products were determined by fitting the concentration vs. time data to pharmacokinetic models using WinNonlinTM. The effect of enzyme inhibitors, captopril, phosphoramidon, and disodium EDTA on deslorelin degradation was also assessed.
Results. Des1-3, Des4-9, and Des5-9 were the deslorelin fragments detected in the membrane fraction. Apart from these degradation products, Des5-7 was also detected in the cytosolic fraction. The deslorelin degradation was 8.5 times faster in the cytosolic fraction compared to the membrane fraction. The disappearance of deslorelin and the kinetics of degradation products could be explained by simple 2 compartment iv bolus model and 1 compartment absorption model, respectively. EDTA and captopril decreased deslorelin degradation in the membrane and cytosolic fractions.
Conclusions. Deslorelin is initially cleaved at the 3-4 bond in the membrane and cytosolic fractions, possibly by a metalloendopeptidase and/or angiotensin converting enzyme, with the degradation being more rapid in the cytosol.
Similar content being viewed by others
REFERENCES
W. Vale, C. Rivier, M. Brown, and J. Rivier. Clinical Endocrinology, 5th Suppl., I. McIntyre (eds.), 2615, Blackwell Scientific Publications, Oxford, 1976.
J. K. Christiansen. The facts about fibroids. Presentation and latest management options. Postgrad. Med. 94:129-137 (1993).
U. B. Kompella. Protein drug delivery. In S. Wu-Pong and Y. Rojanasakul (eds.), Biopharmaceutical Drug Design and Development, Humana Press, New Jersey, 1999 pp. 239-274.
V. H. Lee, A. Yamamoto, and U. B. Kompella. Mucosal penetration enhancers for facilitation of protein and peptide drug absorption. CRC. Crit. Rev. Ther. Drug Carrier Syst. 8:91-192 (1991).
E. Krondahl, A. Tronde, S. Eirefelt, H. Forsma-Bruce, G. Ekström, U. H. Bengtsson, and H. Lennernäs. Regional differences in bioavailability of an opiod tetrapeeptide in vivo in rats after administration to the respiratory tract. Peptides 23:479-488 (2002).
K. N. Koushik, N. Bandi, and U. B. Kompella. Interaction of [D-Trp6, Des-Gly10]. LHRH ethylamide with hydroxypropyl-beta-cyclodextrin (HPβCD): thermodynamics of interaction and protection from degradation. Pharm. Dev. Technol. 6:595-605 (2001).
J. B. Houston and K. E. Kenworthy. In vitro-in vivo scaling of CYP kinetic data not consistent with the classical Michealis Menten model. Drug Metab. Dispos. 28:246-254 (2000).
M. C. Schmidt, W. Rubas, and H. P. Merkle. Nasal epithelial permeation of thymotrinan (TP3) versus thymocartin (TP4): Competitive metabolism and self-enhancement. Pharm. Res. 17:222-228 (2000).
U. B. Kompella and B. A. Dani. Metabolism of [Des-Gly10, D-Trp6] LHRH ethylamide in rabbit nasal tissue. Life Sci. 58:2201-2207 (1996).
U. B. Kompella and B. A. Dani. Metabolism of [Des-Gly10, D-Trp6] LHRH ethylamide in the rabbit conjunctiva. J. Ocul. Pharmacol. Ther. 13:163-170 (1997).
B. A. Dani and U. B. Kompella. Inhibition of Corneal Metabolism of Deslorelin by EDTA and ZnCl2. Drug Dev. Ind. Pharm. 24:11-17 (1998).
C. Botti, E. Seregni, S. Menard, P. Collini, E. Tagliabue, M. Campiglio, B. Vergani, C. Ghirelli, P. Aiello, S. Pilotti, and E. Bombardieri. Two novel monoclonal antibodies against the MUC4 tandem repeat reacting with an antigen overexpressed by lung cancer. Int. J. Biol. Markers 15:312-320 (2000).
N. Bandi and U. B. Kompella. Budesonide reduces multidrug resistance-associated protein 1 expression in an airway epithelial cell line (Calu-1). Eur. J. Pharmacol. 437:9-17 (2002).
C. J. Molineaux, A. Lasdun, C. Michaud, and M. Orlowski. Endopeptidase-24.15 is the primary enzyme that degrades leutinizing hormone releasing hormone both in vitro and in vivo. J. Neurochem. 51:624-633 (1988).
M. Brudel, U. Kertscher, H. Berger, and B. Mehlis. Liquid chromatographic-mass spectrometric studies on the enzymatic degradation of gonadotrophin releasing hormone. J. Chromatogr. A 661:55-60 (1994).
A. Lasdun, S. Reznik, C. J. Molineaux, and M. Orlowski. Inhibition of Endopeptidase 24.15 slows the in vivo degradation of leutinizing hormone releasing hormone. Am. Pharmacol. Exp. Ther. 251:439-447 (1989).
U. Kertscher, M. Brudel, B. Mehlis, J. Sandow, and H. Berger. Pathways of degradation of buserelin by rat kidney membrane. J. Pharmacol. Exp. Ther. 273:709-715 (1995).
G. Halmos and A. V. Schally. Changes in subcellular distribution of pituitary receptors for luteinizing hormone-releasing hormone (LH-RH) after treatment with the LH-RH antagonist cetrorelix. Proc. Natl. Acad. Sci. USA 99:961-965 (2002).
H. L. Jackman, F. Tan, D. Schraufnagel, T. Dragović, B. Dezsö, R. P. Becker, and E. G. Erdö. Plasma membrane-bound and lysosomal peptidases in human alveolar macrophages. Am. J. Respir. Cell Mol. Biol. 13:196-204 (1995).
X. Yang, Y. Rojanasakul, L. Wang, J. Y. C. Ma, and J. K. H. Ma. Enzymatic degradation of luteinizing hormone releasing hormone (LHRH)/ [D-Ala6]-LHRH in lung pneumocytes. Pharm. Res. 15:1480-1484 (1998).
K. Han, J. S. Park, Y. B. Chung, M. J. Lee, D. C. Moon, and J. R. Robinson. Identification of enzymatic degradation products of leutinizing hormone releasing hormone (LHRH)/[D-Ala6] LHRH in rabbit mucosal homogenates. Pharm. Res. 12:1539-1544 (1995).
J. G. W. Wenzel, K. S. Sreebalaji, K. Koushik, C. Navarre, S. H. Duran, C. H. Rahe, and U. B. Kompella. Pluronic F127 gel formulations of deslorelin and GnRH reduce drug degradation and sustain drug release and effect in cattle. J. Control. Release 85:51-59 (2002).
H. Berger, N. Heinrich, E. Albrecht, U. Kertscher, J. Oehlke, M. Bienert, H. Schafer, I. Baeger, and B. Mehlis. Gonadotrophin-releasing hormone (GnRH) analogs: relationship between their structure proteolytic inactivation and pharmacokinetics in rats. Regul. Pept. 33:299-311 (1991).
D. S. Auld. Removal and replacement of metal ions in metallopeptidases. Methods Enzymol. 248:228-242 (1995).
M. P. Hedger, D. M. Robertson, S. J. Tepe, C. A. Browne, and D. M. de Kretser. Degradation of luteinizing hormone releasing hormone (LHRH) and an LHRH agonist by the rat testis. Mol. Cell. Endocrinol. 46:59-70 (1986).
D. W. Cushman, G. S. Cheung, E. F. Sabo, and M. A. Ondetti. Design of potent competitive inhibitors of angiotensin-converting enzyme. Carboxyalkanoyl and mercaptoalkanoyl amino acids Biochemistry 16:5484-5491 (1977).
P. Leblanc, E. Pattou, A. L'Heritier, and C. Kordon. Some properties of peptidasic activity bound to the anterior pituitary membranes. Biochem. Biophys. Res. Commun. 96:1457-1465 (1980).
B. Spencer-Dene, P. Thorogood, S. Nair, A. J. Kenny, M. Harris, and B. Henderson. Distribution of and a putative role for, the cell surface neutral metallo-endopeptidases during mammalian craniofacial development. Development 120:3213-3226 (1994).
A. J. Kenny and J. Ingram. Proteins of the kidney microvillar membrane. Purification and properties of the phosphoramidon-insensitive endopeptidase (“endopeptidase-2”) from rat kidney. Biochem. J. 245:515-524 (1987).
S. L. Stephenson and A. J. Kenny. The metabolism of neuropeptides: Hydrolysis of peptides by the phophoramidon-insensitive rat kidney enzyme ‘endopeptidase-2’ and by rat microvillar membranes. Biochem. J. 255:45-51 (1988).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Koushik, K., Sunkara, G., Gwilt, P. et al. Pathways and Kinetics of Deslorelin Degradation in an Airway Epithelial Cell Line (Calu-1). Pharm Res 20, 779–787 (2003). https://doi.org/10.1023/A:1023489620394
Issue Date:
DOI: https://doi.org/10.1023/A:1023489620394