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Title: The effects of cyclophosphamide and its derivatives on cyclic nucleotide metabolism
Author: Hunter, Gary James
ISNI:       0000 0001 3584 6768
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 1981
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(1) Three methods for the production of an "activated" (if-hydroxylated or if-hydroperoxylated) derivative of cyclophosphamide were investigated: (i) Photooxidation of cyclophosphamide by polymer-bound Rose Bengal was unsuccessful. No products could be detected by high performance liquid chromatography and no change in alkylating activity during incubation was observed. (ii) Incubation of cyclophosphamide with rat liver microsomes followed by deproteinisation with ethanol to produce 4-ethoxycyclophosphamide was partially successful. The product was purified by flash chromatography but was too unstable for further concentration and characterisation. (iii) Ozonisation of cyclophosphamide produced both 4-hydroperoxycyclo- phosphamide and 4-ketocyclophosphamide. The latter was easily purified by fractional crystallisation and the former by flash chromatography. Both of these derivatives were used in the enzyme studies in this project. (2) The compounds used in these enzyme studies were : cyclophosphamide, ^-hydroperoxycyclophosphamide, 4-ketocyclophosphamide, phosphoramide mustard and nitrogen mustard. All cyclophosphamide derivatives were fully characterised and checked for purity by melting point determination, nuclear 1 13 magnetic resonance spectroscopy (1H and 13C), infrared and mass spectroscopy and high performance liquid chromatography. Their behaviour in thin layer chromatographic systems and in a chemical assay for alkylating activity was also measured. (3) The spontaneous production of the primary cyclophosphamide metabolite, 4-hydroxycyclophosphamide from 4-hydroperoxycyclophosphamide in aqueous solution was demonstrated by changes in alkylating activity and thiolbinding properties of the product. (4) Normal rat hepatic plasma membranes were prepared and the adenylate cyclase activity characterised. The effect of cyclophosphamide derivatives on basal (Mg2+ ions present) and glucagon and fluoride-stimulated activity was investigated. Only if-hydroperoxycyclopho6pharaide was found to produce any significant difference in enzyme activity and inhibited basal activity by 50% at a concentration of 5 mM. Glucagon stimulation was totally abolished and fluoride stimulation only partially inhibited at this concentration. (5) Guanylate cyclase of normal rat hepatic cytosol was characterised and the effects of cyclophosphamide derivatives on its activity was investigated. Phosphoramide mustard was found to stimulate 40% at 30 mM concentration) and ^-hydroperoxycyclophosphamide to inhibit (50% at 30 mM concentration) the basal (Mn2+ ions present) activity of the enzyme. All derivatives were found to inhibit the carcinogen (N-methyl-N'-nitro-N- nitrosoguanidine)-stimulated guanylate cyclase activity with varying effectiveness. Using a carcinogen concentration of 40 uM, 50% inhibition of enzyme activity was observed with 4-hydroperoxycyclophosphamide (1.97 mM), phosphoramide mustard 16.72 mM), 4-ketocyclophosphamide (17.70 mM), cyclophosphamide (18.20 mM) and nitrogen mustard ( >30 mM). (6) The possible mechanisms of action of cyclophosphamide derivatives on the cyclase enzymes and possible relevance of these results to cyclophosphamide chemotherapy and its mode of action are discussed.
Supervisor: Not available Sponsor: Science Research Council (Great Britain)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP Physiology ; RC0254 Neoplasms. Tumors. Oncology (including Cancer)