A comparison of the oxidations of diclofenac with microsomes of yeasts expressing various human liver cytochromes P450 showed that P450 2C9 regioselectively led to 4'-hydroxy diclofenac (4'-OHD) whereas P450 3A4 only led to 5-hydroxy diclofenac (5-OHD). P450 2C19, 2C18, and 2C8 led to the simultaneous formation of 4'-OHD and 5-OHD (respective molar ratios of 1.3, 0.37, and 0.17), and P450 1A1, 1A2, 2D6, and 2E1 failed to give any detectable hydroxylated metabolite under identical conditions. P450 2C9 was found to be much more efficient for diclofenac hydroxylation than all the other P450s tested (k(cat)/K(M) of 1.6 min(-1) microM(-1) instead of 0.025 for the second more active P450), mainly because of markedly lower K(M) values (15 +/- 8 instead of values between 170 and 630 microM). Oxidation of diclofenac with chemical model systems of cytochrome P450 based on iron porphyrin catalysts exclusively led to the quinone imine derived from two-electron oxidation of 5-OHD, in an almost quantitative yield. Two derivatives of diclofenac lacking its COO(-) function were then synthesized; their oxidation by recombinant human P450 2Cs always led to a major product coming from their 5-hydroxylation. Substrate 2, which derives from reduction of the COO(-) function of diclofenac to the CH(2)OH function, was studied in more detail. All the P450s tested (1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6, and 3A4) almost exclusively led to its 5-hydroxylation. P450s of the 2C subfamily were found to be the most efficient catalysts for this reaction, with k(cat)/K(M) values between 0.2 and 1.6 min(-1) microM(-1). Oxidation of 2 with an iron porphyrin-based chemical model of cytochrome P450 also led to a product derived from the oxidation of 2 at position 5. These results show that oxidation of diclofenac and its derivative 2, either with chemical model systems of cytochrome P450 or with recombinant human P450s, generally occurs at position 5. This position, para to the NH group on the more electron-rich aromatic ring of diclofenac derivatives, is thus, as expected, the privileged site of reaction of electrophilic, oxidant species. The most spectacular exception to this chemoselective 5-oxidation of diclofenac derivatives was found for oxidation of diclofenac itself with P450 2C9 (and P450 2C19 and 2C18 to a lesser extent), which only led to 4'-OHD. A likely explanation for this result is a strict positioning of diclofenac in the P450 2C9 active site, via its COO(-) function, to completely orientate its hydroxylation toward position 4', which is not chemically preferred. P450 2C19, 2C18, and 2C8 would not lead to such a strict positioning as they give mixtures of 4'-OHD and 5-OHD. The above results show that diclofenac derivatives are interesting tools to compare the active site topologies of human P450 2Cs.
The insulin-like peptide human relaxin-2 was identified as a hormone that, among other biological functions, mediates the hemodynamic changes occurring during pregnancy. Recombinant relaxin-2 (serelaxin) has shown beneficial effects in acute heart failure, but its full therapeutic potential has been hampered by its short halflife and the need for intravenous administration limiting its use to intensive care units. In this study, we report the development of long-acting potent single-chain relaxin peptide mimetics. Modifications in the B-chain of relaxin, such as the introduction of specific mutations and the trimming of the sequence to an optimal size, resulted in potent, structurally simplified peptide agonists of the relaxin receptor Relaxin Family Peptide Receptor 1 (RXFP1) (e.g., 54). Introduction of suitable spacers and fatty acids led to the identification of single-chain lipidated peptide agonists of RXFP1, with subnanomolar activity, high subcutaneous bioavailability, extended half-lives, and in vivo efficacy (e.g., 64).
Immune-related drug responses are one of the most common sources of idiosyncratic toxicity. A number of organs may be the target of such reactions; however, this review concentrates mostly on the liver. Drug-induced hepatitis is generally divided into two categories: acute hepatitis in which the drug or a metabolite destroys a vital target in the cell; immunoallergic hepatitis in which the drug triggers an adverse immune response directed against the liver. Their clinical features are: a) low frequency; b) dose independence; c) typical immune system manifestations such as fever, eosinophilia; d) delay between the initiation of treatment and onset of the disease; e) a shortened delay upon rechallenge; and f) occasional presence of autoantibodies in the serum of patients. Such signs have been found in cases of hepatitis triggered by drugs such as halothane, tienilic acid, dihydralazine and anticonvulsants. They will be taken as examples to demonstrate the recent progress made in determining the mechanisms responsible for the disease. The following mechanisms have been postulated: 1) the drug is first metabolized into a reactive metabolite which binds to the enzyme that generated it; 2) this produces a neoantigen which, once presented to the immune system, might trigger an immune response characterized by 3) the production of antibodies recognizing both the native and/or the modified protein; 4) rechallenge leads to increased neoantigen production, a situation in which the presence of antibodies may induce cytolysis. Toxicity is related to the nature and amount of neoantigen and also to other factors such as the individual immune system. An effort should be made to better understand the precise mechanisms underlying this kind of disease and thereby identify the drugs at risk; and also the neoantigen processes necessary for their introduction into the immune system. An animal model would be useful in this regard.
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