The process of enzymatic reduction was responsible for one of the greatest advances in human medicine-and a significant change in how drug metabolism was perceived. The history began in 1863 with E. Lautemann who had been fascinated by the ease with which quinic acid could be reduced to benzoic acid in the test tube and decided to examine its metabolism in humans. He and two of his colleagues each ingested 8 g of calcium quinate and collected their urines. They found conjugated benzoic acid in all three urine samples.1
Felix Hoppe-Seyler published the first study indicating the ability of the body to reduce nitro groups to the corresponding amines.2 He isolated indoxylsulfate following administration of o-nitrophenylpropiolic acid. The initial reaction in the conversion was presumed to be the reduction of the nitro group to a primary amine. In 1893, Karplus published an account of the conversion of picric acid (2,4,6 trinitrophenol) to 2-amino-4,6 dinitrophenol a reaction that had been postulated earlier by A.Rymsza.3
It was the research of Gerhard Domagk (pictured lower left) which energized the world of drug metabolism, and yielded a product with benefits for all of humanity.4 In 1932, Domagk, working in the laboratories of I.G.Farbenindustrie, directed a series of experiments aimed at finding an agent that would treat streptococcal infections. A series of sulfonamide compounds prepared by Mietzsch and Klarer, were administered to mice 1.5 hours after infection. All of the animals dosed with Prontosil (2'-4'-diaminoazobenzene-4'-sulfonamide) survived while all the controls died. The results were initially published in 1935 and led to the first effective treatment for acute bacterial infection. Domagk was awarded the Nobel Prize in Medicine in 1939.
In France, Trefouel and coworkers found that the diazo link of Prontosil was not required for therapeutic efficacy and speculated that the active principle may be formed by reduction of the diazo bond yielding p-aminobenzenesulfonamide.5 A similar reduction of an azo dye had been reported in 1911 by Sisley and Porcher.6
The definitive proof came from A.T.Fuller working in Colebrook's laboratory in London.7 Fuller provided the analytical evidence that aminobenzenesulfonamide was present in the urine and blood after dosing with Prontosil. The following method, which was typical of the era, can make us thankful for modern analytical methods:
The urine was acidified and decolorised, and the sulphanilamide precipitated as a mercury salt, which was removed and decomposed with hydrogen sulphide. The mercury-free solution was evaporated to dryness, and sulphanilamide separated from other substances by repeated extractions with acetone. It was finally purified by crystallising three times from water. It then melted at 162-3°C. A sample of sulphanilamide and a mixture of this with the recovered substance also melted at 162-3°C, showing that the two substances were identical. The acetyl compounds from both melted at 214-5°C.
Colebrook and Kenny had expanded the early German clinical trials and provided definitive data on the curative properties of Prontosil. Colebrook and Kenny's accounts on the treatment of 26 additional cases of puerperal fever includes this graphic depiction of the break in fever and clearance of the cell culture:8
Ref.: 1E.Lautemann, Ann. Chem. Pharm., 125:9 (1863) 2F.Hoppe-Seyler, Z.Physiol.Chem. 7:718 (1882) 3J.P.Karplus Z.Clin.Med.22:210 (1893) 4Domagk, G. Deut.med.Woch.lxi:250 (1935) 5J.Trefouel, J.Trefouel, F.Nitti, and D.Bovet, C.R.Seances Soc.Biol., 120:756 (1935) 6P.Sisley and C.Porcher,C.R.Acad.Sci (Paris),152:1062 (1911). 7Colebrook, L. and Kenny, M. Lancet I:1279 (1936) 8Colebrook, L and Kenny, M. Lancet I:1319 (1936)
Pictures: Courtesy of Munchener medizinische Wochenschrift
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