Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.678202
Title: Design and characterisation of novel GnRH analogues conjugated to hapten carriers
Author: Ratcliffe, Karen Elizabeth
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2003
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Abstract:
GnRH analogues are extremely useful pharmacological agents, both in the investigation of the hypothalamic-pituitary axis and in the manipulation of gonadotropins for the treatment of reproductive cancers, polycystic ovarian syndrome, endometriosis and precocious puberty, to name but a few. Most GnRH analogues are based on the substitution of the decapeptide sequence of GnRH with unnatural amino acids to modify receptor binding affinity, receptor activation and to decrease proteolysis. Nevertheless these peptides tend to be rapidly cleared from the circulation and in most cases have to be administered by injection. The aim of this study was to modify GnRH agonists and antagonists to enhance half-life by conjugation to a steroid, thereby conferring plasma protein binding affinity and also to enhance oral absorption by further conjugation a carrier molecule to achieve oral absorption. [DLys6]GnRH analogue-progesterone conjugates were designed and synthesized and the products were analysed by HPLC and mass spectrometry. The most successful method of conjugation was with N,N'- dicyclohexylcarbodiimide (DCC) in the presence of hydroxybenzotriazole (HOBt). The pharmacological properties of five GnRH antagonist-21- hydroxyprogesterone 21-hemisuccinate conjugates were analysed (conjugates A, B, C, D, and E). The five conjugates were shown to bind to mammalian GnRH receptors in whole cell binding assays. The IC50 values of conjugates A, B, C and E were not significantly different (108 ± 22nM, 105 ± 27nM, 134 ± 26nM and 104 ± 7nM respectively), but the IC50 of conjugate D was significantly lower at 8390 ± 936nM (p < 0.001, STT). The conjugates were analysed for the ability to inhibit mammalian GnRH-stimulated inositol phosphate production as a measure of GnRH receptor antagonism. Conjugates A and B had the lowest IC50 values at 97 ± 40nM and 76 ± 17nM (p > 0.05, STT), conjugates C and E were significantly less able to inhibit IP production (p < 0.01, STT), with IC50 values of 5580 ± 127nM and 16,000 ± 5190nM, whereas conjugate D did not show inhibition of IP production. None of the conjugates showed any evidence of partial agonism. Plasma protein binding was measured in pregnant guinea pig plasma containing high levels of the progesterone-specific plasma protein PBG. All five conjugates were able to inhibit [3H]progesterone binding to plasma proteins, but with higher ED50 values than progesterone itself (p < 0.05, STT). All antagonist-progesterone conjugates were found to activate the progesterone receptor, as measured by synthesis of chloramphenicol acetyltransferase (CAT) by a CAT reporter gene linked to the progesterone receptor in stably transfected T47D cells. The conjugates induced a similar level of activation to progesterone. Conjugate A inhibited gonadal steroid production in the marmoset. In the female marmoset, l.Omg and 0.5mg conjugate A prematurely terminated the luteal phase when administered subcutaneously on day 8 postovulation. In the male marmoset, 0.5mg conjugate A inhibited testosterone concentrations for at least 72h after subcutaneous injection, whereas with the same dose of the unmodified peptide, testosterone concentrations were not significantly different from baseline levels within 24h of injection (p > 0.05, STT). The duration of action of conjugate A was also compared to the unmodified peptide in an ovarectomized adult macaque. There are a number of ways to improve the oral absorption of peptides. Vitamin B12 is absorbed through the GIT by complexing with intrinsic factor, a large molecular weight protein and the entire vitamin B12-IF complex is internalised by the intrinsic factor-cobalamin receptor. Therefore the design and synthesis of GnRH antagonist-steroid-vitamin B12 molecules was attempted and discussed. In conclusion, novel GnRH analogue-progesterone conjugates have been designed, synthesized and shown to be fully bifunctional in vitro with respect to the GnRH receptor, plasma protein binding and progesterone receptor activation. A GnRH antagonist-progesterone conjugate was active in vivo in a marmoset model, inhibiting gonadal steroid production for significantly longer than the unconjugated peptide. These novel molecules demonstrate that the pharmacokinetics of a peptide drug can be significantly enhanced by conjugation to steroid molecules to improve halflife and in addition, the concept that further modification may be used to increase oral absorption.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.678202  DOI: Not available
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