Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Cyclopentanohydrophenanthrene ring system doai
Reexamination Certificate
1998-10-08
2002-07-16
Criares, T J (Department: 1617)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Cyclopentanohydrophenanthrene ring system doai
Reexamination Certificate
active
06420353
ABSTRACT:
The present invention relates to novel uses for 7&agr;-hydroxy-substituted steroids, to a process for preparing such steroids and to novel steroids so produced.
In particular the invention relates to the use of cytochromes of the cytochrome P450 family designated Cyp7b to effect 7&agr;-hydroxylation of certain 3&bgr;-OH steroids so as to produce a 7&agr;-hydroxy-substituted steroids. Certain of the 7&agr;-hydroxy-substituted steroids so produced, as well the corresponding 7-oxo derivatives, are novel and form further aspects of the invention. The invention also relates to uses of these steroids, to uses of Cyp7b enzymes and to uses of novel macromolecular species, e.g. antibodies and DNAs, which are biologically related to the Cyp7b enzymes.
Cytochromes P450 are a diverse group of heme-containing mono-oxygenases (termed CYP's; see Nelson et al., DNA Cell Biol. (1993) 12, 1-51) that catalyse a variety of oxidative conversions, notably of steroids but also of fatty acids and xenobiotics. While CYP's are most abundantly expressed in the testis, ovary, placenta, adrenal and liver, it is becoming clear that the brain is a further site of CYP expression. Several CYP activities or mRNA's have been reported in the nervous system but these are predominantly of types metabolizing fatty acids and xenobiotics (subclasses CYP2C, 2D, 2E and 4). However, primary rat brain-derived glial cells have the capacity to synthesize pregnenolone and progesterone in vitro. Mellon and Deschepper, Brain Res. (1993), 629, 283-292(9) provided molecular evidence for the presence, in brain, of key steroidogenic enzymes CYP11A1 (scc) and CYP11B1 (11&bgr;) but failed to detect CYP17 (c17) or CYP11B2 (AS). Although CYP21A1 (c21) activity is reported to be present in brain, authentic CYP21A1 transcripts were not detected in this tissue.
Interest in steroid metabolism in brain has been fuelled by the finding that adrenal- and brain-derived steroids (neurosteroids) can modulate cognitive function and synaptic plasticity. For instance, pregnenolone and steroids derived from it are reported to have memory enhancing effects in mice. However, the full spectrum of steroid metabolizing CYP's in brain and the biological roles of their metabolites in vivo has not been established.
Many aspects of brain function are modulated by steroids. Intracellular receptors for glucocorticoids (cortisol, corticosterone) are particularly abundantly expressed in the hippocampus (1), a brain region that plays a key role in specific aspects of memory formation, and which is an early and prominent target for dysfunction and damage in Alzheimer's disease (AD). While glucocorticoids regulate learning and memory, mood and neuroendocrine control, chronic glucocorticoid excess compromises neuronal activity, synaptic plasticity and eventually survival, particularly in the hippocampus. These findings prompted the suggestion that glucocorticoid-mediated neurotoxicity might underpin some age-related brain disorders, including AD, in which plasma cortisol levels are markedly elevated (2).
Conversely, dehydroepiandrosterone (DHEA), the most abundant steroid product of the human adrenal cortex, has been proposed to protect against disorders of the aging brain (3). Plasma levels of DHEA often show a striking age-associated decline which correlates with loss of cognitive function (4). In rodents, injection of DHEA or its sulfate into limbic structures improves post-training memory and enhances synaptic plasticity (5). DHEA and glucocorticoids thereby appear to exert inverse effects upon memory function and synaptic plasticity, and DHEA has been advocated as an endogenous ‘anti-glucocorticoid’. However, despite considerable circumstantial evidence to support this contention, there is no evidence for a direct interaction between DHEA and glucocorticoid signalling pathways in neurons.
Neurosteroidogenesis has been reported in isolated rat retina (8) and brain (9). In addition to the production of pregnenolone and DHEA from cholesterol, a variety of novel steroids are made in brain extracts or cultured brain cells, including 20&agr;-dehydropregnenolone, 7&agr;-hydroxy derivatives of pregnenolone and DHEA, progesterone, and both 3&agr;- and 3&bgr;-hydroxy-5&agr;-pregnan-20-one (reviewed in Ref. 7). Androgens are also modified, particularly through the action of aromatase and a 5&agr;-reductase (reviewed in Ref. 10). However, the specific enzymes responsible for these and other transformations in the central nervous system have not been well characterized.
As referred to above, several Cyps are present in the central nervous system (11-22). Activities or mRNAs corresponding to key steroidogenic enzymes (23-25), in addition to Cyp19 (aromatase) have been detected. Furthermore, mRNAs encoding the non-Cyp hydroxysteroid dehydrogenases (HSD) 3&agr;-HSD, 3&bgr;-HSD and 11&bgr;-HSD have been reported in the central nervous system (25, 27-29).
To investigate regulation of brain function, studies reported in copending International Patent Application No PCT/GB95/02465, published as WO 96/12810, and in Stapleton et al (J. Biol. Chem. 270, 29739-1995, Dec. 15, 1995), focused on the hippocampus, a brain region important in learning and memory. A copy of the specification of International Patent Application No PCT/GB95/02465 has been filed with the priority documents filed in respect of this specification.
That copending application, PCT/GB95/02465, describes and claims novel cytochrome P450 proteins designated Hct-1. These Hct-1 proteins have now been named as Cyp7b by the Committee on Standardized Cytochrome P450 Nomenclature and the name Cyp7b will be used in this application.
The Cyp7b enzyme shares 39% sequence identity to hepatic cholesterol 7&agr;-hydroxylase (Cyp7a) and lesser but significant homology with other steroidogenic Cyps. The postulated steroidogenic domain (30,31), found in many of these enzymes, is present in both Cyp7a and Cyp7b. Cyp7b mRNA is predominantly expressed in rodent brain, particularly in the hippocampus, unlike Cyp7a, which is liver-specific (31-33 and EP0648840 A2).
The present inventors have now investigated the substrate specificity of Cyp7b and found that Cyp7b catalyses the introduction of a hydroxyl group at the 7&agr; position in steroid substrates, particularly 3&bgr;-hydroxy steroids. Cytochromes Cyp7b are thus steroid hydroxylase enzymes having 7&agr;-specificity. The ability to produce 7&agr;-hydroxylated steroids is of major commercial importance, because such steroids are of particular use in the manufacture of pharmaceuticals (either as drugs per se or as intermediates), and in the manufacture of test kits and assays for pathological conditions associated with the presence of abnormal levels of endogenous enzyme, substrate or product.
The abbreviation “DHEA” will be used herein to designate dehydroepiandrosterone, thus 7&agr;-hydroxy-DHEA designates 7&agr;-hydroxydehydroepi-androsterone.
The present inventors have identified substrate/product pairs associated with Cyp7b, particularly DHEA/7&agr;-hydroxy-DHEA (7-HD), pregnenolone/7&agr;-hydroxy-pregnenolone (7-HP) and &bgr;-estradiol/7&agr;-hydroxy-&bgr;-estradiol (7-HE). They have also determined that DHEA concentration in brain tissue declines with age, whereas the concentrations of other brain steroids do not, and determined that the ageing process may be associated with deficits in certain steroids and also with deficits in the concentration of Cyp7b itself. It is also believed that one of the products produced by Cyp7b mediated reactions, namely 7&agr;-hydroxy dehydroepiandrosterone, plays an important role in the operation of the immune system. Because 7&agr;-hydroxy-DHEA is believed to be made substantially only in the brain, the inventors hypothesize that senescence may be due to a deficit in brain-produced 7&agr;-hydroxy-DHEA as well as in other steroids found in the brain such as DHEA, pregnenolone and 7&agr;-hydroxy-pregnenolone.
The present inventors have now further determined that one of the specific properties of the 7&agr;-hydroxy-sub
Best Ruth
Lathe Richard
Leckie Caroline McKenzie
Rose Kenneth Andrew
Seckl Jonathan Robert
BTG International Limited
Criares T J
Vanderhye Nixon
LandOfFree
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