Transition from pharmaceutical to environmental research.

Drug Metabolism Reviews

ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20

History of Drug Metabolism: Discoveries of the Major Pathways in the 19th Century A. Conti & M. H. Bickel To cite this article: A. Conti & M. H. Bickel (1977) History of Drug Metabolism: Discoveries of the Major Pathways in the 19th Century, Drug Metabolism Reviews, 6:1, 1-50, DOI: 10.3109/03602537708993764 To link to this article: http://dx.doi.org/10.3109/03602537708993764

Published online: 22 Sep 2008.

Submit your article to this journal

Article views: 19

View related articles

Citing articles: 11 View citing articles

Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=idmr20 Download by: [University of Toronto Libraries]

Date: 12 November 2016, At: 21:51

DRUG METABOLISM REVIEWS, 6(1), 1-50 (1977)

History of Drug Metabolism: Discoveries of the Major Pathways in the 19th Century* A. CONTI and M. H. BICKEL Medizinisch-chemisches Institut University of B e r n e Berne, Switzerland

.. ................................... 2 It. GLYCINE CONJUGATION ............ ................. 4 III. OXIDATION ................................. ........ 11 IV. SULFURIC ACID CONJUGATION ... ...., . ,.... .. ..... . 20 I. INTRODUCTION

V. REDUCTION

........................................

25

............. ...... 31 VII. ACETIC ACID CONJUGATION ............ ..... .. .... .. 36 WI. GENERAL COMMENTS . ...... ........... ........ ..... 39 IX. SUMMARY-TABLES O F K E Y DATES .................. 4 3 A. Glycine Conjugation ... ........................... 43 B . Oxidation ........................................ 43 C . Sulfuric Acid Conjugation .. .. ..... , . . .... ... ...... 44 VI. GLUCURONIC ACID CONJUGATION.

D. Reduction

.......................................

44

*Based upon D r . Conti's doctoral thesis (written in German) which received the Henry E . S i g e r i s t award b y the Swiss Society of the History of Medicine in 1975. 1 Copyright 0 1977 hy Marcel Dekker. Inc All Rights Reserved Neither this work nor any part may be reproduced or transmitted in any form or by any means. electronic or mechanical. including photocopying. microfilming. and recording. or by any information storage and retrieval system. without permission in writing from the publisher

CONTI AND BICKEL

2

E. Glucuronic Acid Conjugation F. Acetic Acid Conjugation

......................

........................... References ..........................................

44 45 45

I. INTRODUCTION

Biochemistry has a history of breathtaking successes and in addition has contributed immensely to the progress of medicine during the past hundred years. Yet the history of biochemistry, unlike that of other biomedical sciences, has attracted surprisingly little attention. One of the reasons is that biochemistry is rather young compared with, say, anatomy o r physiology. The very problem of biochemistry was nonexistent before 1750, and only in the second half of the 19th century did it become a respectable science. Although early biochemistry was strongly associated with organic chemistry, little help is available from the well-covered history of chemistry, since chemists used to regard biochemistry either a s a chemical specialty of lower standing o r else as a topic within the sphere of medicine. One of the very few attempts at a comprehensive history of biochemistry is Geschichte der physiologischen Chemie by Lieben [l], written in 1935. A new monumental work i s being written by Florkin [2, 31. Lieben's book, although invaluable as a source of information, is an example of how difficult it is to portray the development of a complex science like biochemistry without sacrificing either the chronology of its authors o r of i t s problems o r even both. Thus the representation of a singled-out author o r problem is a much easier task which may still, and even in greater clarity, give the reader an idea of the development and the spirit of early biochemistry. This work is an attempt at representing the history of the metabolism of foreign compounds. The problem of this topic emerged once scientists had realized that the living organism is a machinery performing chemical reactions, and that synthetic compounds were becoming available in increasing numbers. How, then, would the o r ganism deal with compounds that were neither nutrients nor body constituents? This new line of biochemical research originated in the first half of the 19th century and indeed became an important aspect to many of the pioneers of biochemistry. It is remarlable that all major pathways of drug metabolism were discovered and verified d u r ing the 19th century: oxidation, reduction, and the major conjugation reactions. Close interactions existed between these studies and general biochemistry as well as neighboring disciplines like physiology and particularly pharmacology. Buchheim, the very founder of modern pharmacology, as early as 1859 made the following statement:

HISTORY OF DRUG METABOLISM

3

In order to understand the actions of drugs i t is an absolute necessity to have knowledge of the transformations they undergo in the body. It is obvious that we must not judge drugs according to the form and amount administered, but rather according to the form and amount which actually is eliciting the action. [4] What Buchheim had anticipated became one of the chief reasons for the pharmacologists' interest in drug metabolism and for its amazing development in the course of the 20th century. A history of the early stage of drug metabolism would be justified by the m e r e fact that many of the foremost old-day biochemists, chemists, physiologists and pharmacologists were heavily involved, among them: Wohler, Liebig, Naunyn, Nencki, Baumann, Jaffe, Bunge, Schmiedeberg. Most of their activities took place in Germany, which was the major stage of early biochemistry. The present review is restricted to the 19th century which not only contains the roots of drug metabolism research but also sees it come to a certain completion. Emphasis is on the history of the discoveries of the major pathways, with one section for each of the following reactions : glycine conjugation, oxidation, sulfuric acid conjugation, reduction, and conjugations with glucuronic acid and with acetic acid. This sequence was chosen for reasons of chronology and context of individual discoveries. Each section describes the history of the discovery from the first observations to the final proof. In addition to developing the subject, which is based on the key publications, attempts have also been made at drawing attention to the scientific background of the respective periods. All quotations have been translated from the German originals. Thereby the language has been moderately modernized. A concluding section deals with some aspects of the history of ideas and with practical problems of biochemical research in the 19th century. Finally, cables of key dates and events were the only conceivable form of a summary for this type of work. The following references may be helpful to the reader who seeks access to the original literature on the history of drug metabolism. Friinkel [51 and particularly Heffter (6, 7 3 a r e the authors of the first and most helpful reviews written shortly after 1900. Other authors have dealt with the topic in our time. Williams [S], in his standard book Detoxication Mechanisms, gives a short survey of the history of his subject matter. This coverage was later extended by Smith and Williams [93. Some reflections have been written by Butler [lo'], and Bickel [ll], in a book on Nencki (1847-1901), has devoted one chapter to drug metabolism.

CONTI AND BICKEL

4

n.

GLYCINE CONJUGATION

The flrst compound to be investigated with respect to its fate in the body was benzoic acid. Being the prototype for the study of glycine conjugation, i t will also be the only example followed in this section. The discovery of the biotransformation of benzoic acid is a long story of numerous, often contradictory, observations dating back to the 18th century and gaining shape in the 1840s. Benzoic acid was used therapeutically in the 18th century a s a stimulant of the respiratory system and a s a tonic. The problem of the presence of benzoic acid in urine was discussed by Thompson l i 2 3 in 1802. He was referring to Scheele who had evaporated urine, eliminated the salt component, and eventually obtained "benzoic acid" by sublimation of the residue. According to Hallwachs [ 131, Rouelle in 1773 was the first to mention the presence of "benzoic acid" in cow's and later in camel's urine. These observations were cited by Berzelius: Rouelle found that from cow's urine which had been concentrated by evaporation, hydrochloric acid would precipitate an acidic salt which he compared with benzoic acid. [ 14 3 Fourcroy and Vauquelin [15] in 1799 also observed an acid in the urine of herbivorous animals which they thought to be "benzoic acid. W e purposely use quotation marks to indicate the likely incorrectness of all these observations. Proust 1161 a s early as 1801 believed the acid found in urine to be similar to rather than identical with henzoic acid. The first researcher who s e t up an experiment to specifically investigate the fate of a foreign compound was Wohler [17] in 1824. He gave a dog half a drachm of benzoic acid in its food and then isolated from the urine a n acid which crystallized in prismatic needles and had the general properties of benzoic acid. According to Wohler this experiment proved that the compound absorbed in the gastrointestinal tract reached the urine unchanged. Only in 1829 did Liebig 118 1discover the compound he named hippuric acid because he had isolated it from equine urine, and, in contrast to benzoic acid, it contained nitrogen. Further investigations convinced Liebig that hippuric acid contained a benzoic acid moiety which was bound to a nitrogenous compound. Retrospectively, it becomes evident that Wohler and his predecessors were dealing with the then unknown Nppuric acid which they mistakenly thought to be benzoic acid. Erdmann [19], who also succeeded in isolating hippuric acid from equine urine, was surprised to I'


5

find this acid mainly in saddle horses, whereas farm horses mainly excreted benzoic acid. This observation was ascribed to different feeding, while Dumas [201 preferred the explanation that benzoic acid would be formed as an artifact during the isolation procedure. It was not until 1845 that the structure of hippuric acid was established when Dessaignes [21, 22 1 boiled it with inorganic acids which made it split into its components, benzoic acid and glycine: QCQ-NH-CH~-COOH

Hippuric acid

+

~

C

O

Q

Benzoic acid

H+

NH~-CH~-CQOH

Glycine

Dessaignes was also the first to perform a chemical synthesis of hippuric acid in vitro by heating benzoic acid and glycine in a sealed tube for 12 h r at 1GO"C [223. An empirical formula could now be established beyond doubt. This exemplifies that in the 19th century work on metabolic processes w a s highly dependent on progress made in organic chemistry and particularly analytical methodology. A decisive contribution to the problem of the fate of benzoic acid in the human body was made in 1841 by Ure [23], who knew that in the meantime hippuric acid had also been found in man [24]. Administering benzoic acid to himself and to volunteers, Ure succeeded in isolating large quantities of hippuric acid from the urine collections. He thus must be given the credit of being the first one to discover a biotransformation of a foreign compound. Since he also observed a concomitant decrease of uric acid, he concluded that this nitrogen compound contributed to the formation of hippuric acid and he therefore also suggested the use of benzoic acid for the treatment of gout. After Liebig had discovered hippuric acid, Wohler [25], in a letter of 1830 to his friend, speculated on the transformation of benzoic acid into hippuric acid during digestion. H i s pupil, Keller [26], felt challenged by Ure's results, and with an experiment on himself was able to confirm that benzoic acid was metabolized to hippuric acid. In contrast to Ure's finding, however, the excretion of uric acid was not diminished by the administration of benzoic acid. Finally, Liebig [27] found that hippuric acid is also a normal urinary product in humans consuming a mixed diet. The first investigations focused upon the localization of glycine conjugation were carried out by Kiihne and Hallwachs [281 in 1857. They must have been the first to study the localization of a biosynthetic process and to use a foreign compound for the purpose. Less than a decade earlier, WGhler and Frerichs 1293 discussed the

CONTI AND BICKEL

6

problem of the localization of a metabolic process but dismissed it in view of the unsilrmountable difficulties. Kiihne and Hallw'achs, however, very clearly stated their questions:

Is the ingested benzoic acid after its absorption transformed to hippuric acid by the action of the blood, and does this process therefore occur in the whole vascular system? O r else, is benzoic acid rather transformed by the action of bile, i.e., already in the intestinal lumen o r within the hepatic vessels? [Z8

1

The authors realized that the first question could only be answered by administering benzoic acid intravenously, Another problem was the proper choice of the animal species to be used. Hallwachs and Kiihne had observed that certain herbivorous species excreted hippuric acid without being treated with benzoic acid. Thus only carnivorous species would be useful, and even with those the diet should be strictly controlled to avoid vegetable components. In this case a serious source of e r r o r had been recognized and avoided in time. This was not the rule in those days, and therefore false results and conclusions, often to be perpetuated for decades, were frequent. In a first series of experiments K ~ n and e Hallwachs injected 20 dogs intravenously with benzoic acid without, however, detecting more than traces of hippuric acid in the urine. They therefore switched to test their "bile hypothesis" which had the following background. Strecker (301 had found that bile contains the two bile acids, taurocholic and glycocholic acid. Since no glycine could be isolated from blood a t that time, glycocholic acid was considered to be the only source of glycine in the body. In order to eliminate this source, the bile ducts of dogs were cannulated according to the technique introduced by Schwann [31] and Blondlot [32]. In their new series of experiments K b and Hallwachs demonstrated that animals with and without bile fistulae were transforming ingested benzoic acid to hippuric acid and thus they proved that the presence of bile in the gut was not a prerequisite for the formation of hippuric acid. Based on the presumption that bile acids are produced in the liver, they hypothesized that hippuric acid is formed within the liver by using the glycine moiety of glycocholic acid. This hypothesis was to be further tested by suppressing the liver function. Indeed, Kuhne and Hallwachs in 1857 seem to have been the first to introduce such a technique by ligating the afferent and efferent liver vessels in cats. The technique was adopted and improved by Eck in 1577 who introduced the classical portocaval shunt then called Eckls fistula.

HISTORY O F DRUG METABOLISM

7

In the urine of cats with ligated hepatic blood vessels, K i h e and Hallwachs were not able to detect hippuric acid. Yet they did not draw the conclusion that the liver was the site of this synthesis since they recognized that the portal ligation also blocked the reabsorption of bile acids from the gut. Therefore, they injected cats intravenously with a mixture of benzoic acid and bile. The finding that hippuric acid only was excreted in these animals led to the conclusion that the presence of glycocholic acid in blood was essential to the conjugation of benzoic acid. At this stage the authors no longer took into consideration that their results had been collected with two species, dogs and cats, which might have influenced their reasoning in the light of the findings of Strecker [333 and Hoppe-Seyler r341 who reported that dog bile had the estreme composition of 100% taurocholic acid and 0% glycocholic acid. Why was this important information unknown to Kuhne and Hallwachs? Although there was no flood of literature like in our times, not only the language but also the diffusion of scientific journals was national. In many instances this situation prevented interaction between researchers. In a third series of experiments, benzoic acid and a smaller ainount of bile were injected into cats and resulted in the excretion of benzoic acid in addition to hippuric acid. To Kiihne and Hallwachs this finding was proof that the formation of hippuric acid was dependent on the presence of bile acids, a condition met in the hepatic vascular system exclusively. Once they knew both the composition of hippuric acid and the site of its formation, they felt ready for the venture of reproducing the biosynthesis of this compound in vitro by incubation of benzoic acid with bile at 32OC. The authors must have been rather perplexed by the total absence of hippuric acid: It is hard to see what specific conditions exist in the organism. Their ignorance usually precludes artificial simulations of physiological processes. [ 2 8 ] This was the situation when 19 years later Bunge and Schmiedeberg [35] made another approach toward the same problem. In the introduction of their publication they state that: Many organic substances, when ingested, are hydrolytically split in the digestive tract: fat is split into glycerine and fatty acids, starch into sugar. These processes are not puzzling to a chemist, because they can be simulated in vitro. However, the situation is different with certain chemical processes occurring with absorbed compounds within the

CONTI AND BICKEL

8

.

tissues.. .The mechanism of the synthetic reactions (e. g., the reaction of benzoic acid and glycine to form hippuric acid) is absolutely puzzling, because in vitro they can only be reproduced under conditions which do not exist in the organism. r35 1

Thus biochemical reactions were subdivided into hydrolyses , which were easy to simulate and to understand, and into still mysterious biosyntheses. The very discrepancy between the fast-growing a r t of the synthetic chemists on one hand and the total lack of knowledge concerning biosyntheses on the other, was a stimulus to investigate the latter, whereby the glycine conjugation of a foreign compound became an important model. The conflict of the laws of cheniistry existing in the test tube and in living organisms was, in fact, one of the great challenges for the early biochemists. More than their predecessors Kuhne and Hallwachs [2R], who indicated the liver vessels as the site of hippuric acid formation, and Meissner and Shepard [361, for whom it was the kidney, Bunge and Schmiedeberg were aware of the difficulties and requirements of their project. The experiments were carefully planned, and the chemical methodology was given full attention: The fact that previous investigations on hippuric acid forniation in the body have not produced unequivocal results is mainly due to the use of insufficient methods for the detection of hippuric acid in tissues and body fluids. It was o u r prime task to develop a method which was absolutely certain to detect even minute amounts of hippuric acid and which was not disturbed by the other constituents of the animal body. [35 1

Again, the localization of the process was their first target. A f t e r injections of,both benzoic acid and glycine into operated dogs, hippuric acid was detected in the blood of an animal with ligated hepatic vessels, but not in another one whose renal vessels were ligated. This was a first hint that the kidneys might be involved in this biosynthesis. In intact animals the blood contained both benzoic and hippuric acid, whereas the urine contained exclusively hippuric acid. Dogs with ligated ureters showed higher blood concentrations of hippuric acid. Bunge and Schmiedeberg were not yet convinced that the Mdney was the sole site of hippuric acid synthesis. In addition, they realized that their ligation of hepatic vessels resulted in serious disturbances of the hemodynamic situation. They therefore developed a technique

HISTORY O F DRUG MGTABOLISM

9

of total hepatectomy which became famous 10 years later under the authorship of Minkowski. The first animals to be hepatectomized by Bunge and Schmiedeberg were frogs. Not only were they still able to produce hippuric acid when benzoic acid was injected with glycine, but they also did so when benzoic acid was injected alone. This observation disproved the theory that the liver was the origin of the glycine moiety of hippuric acid. Still, Bunge and Schmiedeberg were not happy with their unphysiological technique of total organ extirpation. They therefore adopted the technique of artificial perfusion of isolated organs which had been developed by Ludwig and Schmidt [37] in 1868. In their first experiment Bunge and Schmiedeberg perfused a kidney for 8 h r with blood containing benzoic acid and glycine. Hippuric acid appeared in h t h blood and urine. This success led to the new question, namely, whether glycine which was added to the blood participates in hippuric acid formation, o r whether the glycine moiety of hippuric acid is formed from some constituents of blood o r kidneys and combines with benzoic acid in statu nascendi. c353

This problem had just been investigated in 1875 by Spengel [38 1 in Nencki's laboratory who gave an intact dog 3 g of benzoic acid in food and found equal amounts of benzoic and hippuric acid in the 24h r urine. If, however, the same dog was given 5 g of benzoic acid together with the same amount of glycine, no unconjugated benzoic acid appeared in the urine. This demonstrated that the dog was able to use exogenous glycine for the conjugation of benzoic acid. The same observation was made in man by Hoffmann [39] in 1877. On the other hand, Bunge and Schmiedeberg were wondering about being unable to verify these results, thereby overlooking that their experimental conditions (i.v. and 3 . 5 h r urine instead of p.0. and 24 h r ) were different in essential details. It was another advantage of the perfusion of isolated organs that this technique eliminated pharmacokinetic factors which were much neglected a t that time. Further experiments showed that after several hours of perfusion of a kidney with drug-free blood no hippuric acid was formed, whereas the addition of benzoic acid into the perfusion system of the other kidney of the same dog led to the formation of hippuric acid. Its amount, however, was much lower than with concomitant addition of glycine. Again, new and important questions came up. Was the biosynthetic ability due to the organ as such, to its cells, o r even to chemical constituents of the cells? By perfusing kidneys 5 h r post-mortem o r after a 48-hr storage in an ice

10

CONTI AND BICKEL

chest, the authors were still able to detect the formation of hippuric acid. On the other hand, fresh calf kidneys, extensively homogenized in a mortar and incubated with benzoic acid and glycine, did not yield hippuric acid, This result was confirmed by Koch [401 in Pfluger's laboratory. If, however, he only mildly minced the kidneys with a meat grinder, hippuric acid was formed. The more Koch homogenized the tissue in his grinder, the less hippuric acid did he obtain. He concluded that these experiments demonstrate that hippuric acid is synthesized by the vital functions of the individual intact cells when exposed to benzoate and glycine in oxygenated blood. [4Ol What, then, was the role of oxygen in glycine conjugation? Bunge and Schmiedeberg realized that this problem was well suited for study with their perfusion technique. When they used saline o r plasma instead of whole blood as a perfusion medium, hippuric acid was detectable in traces only. They concluded that red cells play an important role in glycine conjugation, but that very likely they serve a s m e r e c a r r i e r s of oxygen. Hoffmann [393, also a member of Schmiedeberg's team, modified that experiment by using whole blood whose oxygen had been displaced by carbon monoxide. No hippuric acid was formed by the perfused Iddney. But then this could have been due to lack of oxygen as well as to a toxic effect of carbon monoxide. Another approach tried by H o f h a n n was the use of quinine instead of CO. Why quinine? Studies carried out by Binz [41, 423 and by Scharrenbroich [437 had shown that low concentrations of quinine block the motility of white blood cells and protozoa. Since all vital functions seemed to disappear, Hoffmann concluded that quinine had a specific cytotoxic effect. H i s use of quinine is of particular interest, since this may have been the first attempt to influence the metabolism of a drug by a second o r "tool" drug. Hoffmann indeed was successful in demonstrating that with increasing amounts of added quinine the amount of hippuric acid formed was decreasing. He thereby also indicated the possibility of metabolic interactions between drugs, one of the problems to be rediscovered a hundred years later! Glycine conjugations of compounds other than benzoic acid a r e mentioned in the first half of the following section.


11

III. OXIDATION Ure [44] and Erdmann and Marchand [45, 461 in 1842 were the first to indicate that acids other than benzoic acid a r e also excreted as hippuric acid. Being interested in the fate of cinnamic acid, the latter authors gave 5 to G g of the compound in syrup to six volunteers. The urine of all volunteers then disclosed a compound which was likely to be hippuric acid, although the glycine conjugate of cinnamic acid could not be excluded [47]. This was one of the many instances in which early biochemists approached a problem without sound knowledge of the underlying organic chemistry and without adequate analytical inethodology. Even though Erdmann and Marchand were well aware of their equivocal result, they were eager to draw attention to the possibility of an oxidation-conjugation sequence in the metabolism of a foreign compound according to the (modern) reaction scheme: ~ C H = C H - C O O H IO C O O H

Cinnamic acid

Benzoic acid

+

0CO -G 1y

Hippuric acid

This hypothesis of oxidative processes in drug metabolism was confirmed in 1848 by Wohler and Frerichs [291 who administered cinnamic acid to dogs and indeed detected hippuric acid as the urinary product. The same product was also isolated in considerable amount from the urine of dogs and rabbits which were fed oil of bitt e r almonds (benzaldehyde). Based on their sound analytical methodology, Wohler and Frerichs must therefore be given credit of having discovered the metabolic oxidation of benzaldehyde to benzoic acid and with having formulated the reaction sequence of oxidation and subsequent glycine conjugation. In the same work [29] these authors also made a noteworthy statement on the importance of metabolic studies with foreign compounds for physiology and biochemistry at large: The transformations which substances undergo in the animal body between their ingestion and excretion in the urine, a r e of considerable interest to physiology and biochemistry, because we thereby may expect to gain insight into the chemical forces which a r e acting in the organism and particularly in blood, and which a r e responsible for the metabolic processes. In particular this is the case when compounds are

12

CONTI AND BICKEL studied whose chemical constitution and properties are well h w n and whose metabolic transformation thus allows conclusions with respect to the cause giving rise to metabolism. Obviously, in order to obtain general results along these. lines, extensive and time-consuming series of experiments will be required and particular difficulties will have to be overcome. C29 3

A new impulse came from studies on the volatile constituents of urine, carried out in 1851 by Staedeler [48], who isolated a compound he believed to be phenol. H e was surprised to find a compound known a s a poison in the urine of healthy humans and animals. Based on a report by Schlieper [49], who had considered phenol an oxidation product, Staedeler speculated on the formation of phenol by metabolic oxidation of aromatic precursors. Although Staedeler's observation was to be disproved, his hypothesis became one of the starting points for the famous investigations of Schultzen and Naunyn C50] in 1867. These authors were interested in the fate of therapeutically used hydrocarbons such as benzene, which was prescribed as an intestinal disinfectant. They first developed a more sensitive method for the detection of phenol by adding ferric chloride to the distillate of urine, which resulted in a dark violet color. Whereas this test was negative with urine of untreated animals and humans, phenol was detected in patients, normal humans, and dogs treated with benzene. These results led Schultzen and Naunyn to the following conclusion:

Thus, it seems that benzene during its passage through the organism undergoes a simple oxidation which leads to the formation of phenol. If this result can be adequately verified, it would be of great interest since it has never been possible so f a r to obtain simple derivatives by direct oxidation of benzene. [501 A s has been mentioned, the biosynthesis of hippuric acid surprised its investigators because the reaction in vitro would progress under extreme conditions only. Another, even more puzzling situation was now introduced by the hydroxylation of benzene, a reaction unheard of in organic chemistry. The latest attempts a t chemical oxidation of benzene by Carius [51] resulted in the formation of formic acid, benzoic acid, and phthalic acid, but not phenol. Another problem in the investigation of Schultzen and Naunyn remained unsolved: Only a small fraction of the ingested benzene was excreted as phenol. What, then, happened to the remainder? They


13

speculated that either phenol was further oxidized in the body o r else that benzene was also excreted by the lungs. Both hypotheses would later be proved correct. Having discovered the metabolic hydroxylation of benzene, Schultzen and Naunyn were now eager to study and compare the fate of benzene derivatives. Toluene, the first compound of a series, was found to be oxidized in the sidechain rather than in the aromatic nucleus, thus forming benzoic acid which was then conjugated with glycine: O C H 3

Toluene

-

O C O O H

j

Benzoic acid

#-CO-G

1y

Hippuric acid

Thi finding, which was w n confirmed by Nencki (521, demonstrated still another foreign compound to be ultimately excreted as hippuric acid. Many further instances of glycine conjugations were soon to be discovered, Another model compound used by Schultzen and Naunyn in their systematic investigation was o-xylene. This compound was recovered in the urine of dogs and humans as an acid, identical to toluric acid, which had been described by Hofmann 1533 and Kraut (54, 551 as the urinary glycine conjugation product of toluic acid. &!:OH

o -Xylene

o-Toluic acid

j

d3 CO-Gly

Toluric acid

Again, Schultzen and Naunyn had discovered an oxidation-conjugation sequence. In addition, they stressed the fact that only one of the two sidechains attached to the benzene ring was oxidized and conjugated. Working along the same lines, Nencki (523 (Fig. 1)was wondering which of two sidechains of different sizes would be metabolically attacked. He cited his consideration of a recent chemical study of Fittig and K6nig [5G] who showed that only the longer of two sidechains was oxidized by boiling nitric acid. This chemical finding gave Nencki the idea to use p-cymene as a substrate. In 1872 Nencki and Ziegler [573 were able to detect cumic acid in the urine of dogs and humans given 3-g doses of the hydrocarbon. Thus, contrary to nitric acid, biological oxidation attacked the smaller of the two sidechains. Apparently the acid metabolite was not conjugated with glycine but instead was excreted unchanged, an observation which had also been made by H o k a n n [531, Kraut [553, and Buchheim [581 who found cumic acid to be excreted unchanged. Later on, however,

CONTI AND BICKEL

14

... 1

FIG. 1. Marcell NencM, 1847-1901. Berlin, Berne, St. Petersburg.

Jacobsen C591,after giving a dog 11g of cymene, was able to detect cuminuric acid, the glycine conjugate:

p-Cymene

Cumic acid

Cuminuric acid


15

A s a further step, Nencki [ G O ] then studied the fate of a benzenoid hydrocarbon with three sidechains, mesitylene, which again was found to be oxidized in only one of the sidechains:

Mesitylene

Mesitylene carboxylic acid

Mesitylenuric acid

Another study in this field was carried out by Schultzen and Graebe [ G l l who felt disturbed about the conflicting reports concerning glycine conjugation of aromatic acids. Thus Bertagnini [G23 ingested small amounts of nitrobenzoic acid and, noticing no adverse effects, increased the dose to a total of G g, after which he was able to detect nitmhippuric acid in his urine. This was confirmed in the dog by Jaff6 [G3!. In a later study Bertagnini cG41 reported that salicylic acid given to humans is excreted partly unchanged and partly as its glycine conjugate, salicyluric acid. In contrast, Wohler and Frerichs [29], working with dogs, found unchanged salicylic acid only. Bertagnini c641 also found that anisic acid did not undergo glycine conjugation. Similarly, Beilstein and Schlun [653 reported chlorobenzoic acid, and Berg [ G G ] coumaric acid, to be excreted unchanged. All these results prevented Schultzen and Graebe from discerning a rule for the glycine conjugation of aromatic acids. At the same time these authors also had their doubts about the methodology used by some of their predecessors and therefore decided to repeat certain experiments. They selected cinnamic acid, chlorobenzoic acid, and anisic acid, which they ingested. Their urine collections were found to contain hippuric acid, chlorohippuric acid, and anisuric acid, respectively.

Anisic acid

Anisuric acid

CONTI AND BICKEL

16

From their experiments, Schultzen and Graebe drew the conclusions

that c a r b x y l groups attached to the aromatic nucleus a r e directly conjugated with glycine, whereas in aryl aliphatic acids the sidechain is first oxidized and only subsequently does the newly formed carboxyl group undergo glycine conjugation. Thus all aromatic acids are eventually transformed to glycine conjugates. Although the series of compounds investigated at the time was too small to allow the establishment of such a rule, glycine conjugation had definitely been shown to be a biochemical principle rather than an isolated reaction of benzoic acid. A b a l experiment carried out by Schultzen and Graebe was aimed at elucidating the metabolic fate of the aromatic dicarboxylic acid, phthalic acid, since doubly glycine-conjugated acids would have been a novelty. Unfortunately, the small amounts of isolated urinary metabolite precluded elementary analysis and establishment of the empirical formula. Another unsuccessful attempt at identifylng the metabolite of phthalic acid was made by Nencki, who was doing his thesis work as a medical student under Schultzen's guidance. In this thesis of 1870, entitled "Oxidations of Aromatic Compounds in the Animal Body" 1521, Nencki not only formulated the rules of oxidation and glycine conjugation, which he himself was to enrich and improve by his lifework, but he also characterized the potential of drug metabolism studies : There can be no doubt that chemical groups and their homol o p e s not only show an analogous behavior toward certain chemical reagents, but also, if within limits, toward the animal organism. By studying the metabolic fate of chemical substmces, one will on one hand be able to establish laws allowing predictions on the fate of new compounds, and on the other hand gain increasing insight into the organism as a 'tchemical agent. I t Of course, sound la-iowledge of the chemical structure of compounds is a prerequisite to the study of their metabolic transformations. c527 A decade after the discovery of the metabolic oxidation of benzene to phenol by Schultzen and Naunyn, Munh- rG71 expressed renewed surprise about this reaction which could not be performed in the test tube even using the strongest o.xidmts. Two reasons caused him to repeat these experiments: first his conviction that this was a question of fundamental importance in the light of biological oxidation, and second the availability of an improved method for the determination of phenol


17

as tribromophenol, introduced by Landolt rG8 7. Munk and volunteers ingested 20 drops of benzene. Although the experiment was performed according to a carefully prepared protocol, Munk was not able to detect phenol in the urine. Only in the light of sulfuric acid conjugation could this dilemma be solved in the same year. However, Munk correctly interpreted part of the fate of benzene in his comment: Part of the benzene is excreted unchanged as a gas from the stomach. During the first 8 h r after ingestion we were frequently annoyed by hiccups which had the exquisite smell and taste of coal t a r and therefore were rich in benzene vapors. We do not know whether benzene is also excreted by the lungs, a s suggested by Schultzen and Naunyn, nor, most important, what happened to the remaining benzene. 1671 So f a r , studies on the metabolism of foreign compounds were motivated by scientific curiosity. Nencki and Sieber rG9-711 in the early 1880s may have been the first ones to think of clinical applications. Typically, they associated a particular metabolic disorder with a chemical model system. The metabolic disorder in diabetes w a s characterized by the carbohydrates being excreted without having undergone combustion. Thus the underlying defect was believed to be a n insufficient oxidation capacity of the body. At the same time Nencki and Sieber were dealing with a new and fascinating chemical system, able to oxidize glucose to lactate by simply incubating the substrate at 37°C with strong alkali in the presence of oxygen for 24 hr. Working with this so-called Radziszewski system, they made the important observation that benzene was oxidized to phenol and, furthermore, that this oxidation was suppressed in the presence of glucose. Thus a competition of the two substrates for oxygen was assumed, and this principle was then applied to the organism. If, in the body, glucose and benzene would compete for a common olddation system, then the oxidation capacity of the organism could be tested by measuring the phenol formed from a dose of benzene. In contrast to biological substrates, benzene is not degraded to COz and H20,and its oxidation product, phenol, is still an aromatic compound which can easily be detected and determined. Actually, Nencki in his extensive studies on the fate of aromatic compounds made use of the stable benzene ring as a label, much as isotopically labeled molecules were used almost a century later. In order to use the benzene test" to measure oxidation capacity under pathological conditions in patients, the constancy and reproducibility of phenol formation was carefully studied in healthy rabbits,

18

CONTI AND BICKEL

dogs, and humans. Indeed, in all three species standard doses of benzene led to comparable amounts of f r e e and conjugated urinary phenol, independent of diuresis and nutritional state. Nencki and Sieber were now convinced that the benzene test was superior to the then used determination of urinary lactate as a n indicator of the "intensity of vital combustion. ' I The flrst patient on whom the benzene test was performed was a diabetic woman. She was given an oral dose of 6 g of benzene, and urine was collected fbr 4 days and analyzed ibr the presence of phenol. The fact that its amount was comparable with that of healthy persons was interpreted as an indication that diabetes was not due to a deficient oxidation capacity of the organism. In a patient with splenic leukemia, the benzene test resulted in decreased amounts of phenol. Since, however, in this case even under lactate treatment the patient's urine remained free of lactate, Nencki and Sieber were inclined to assume hypoxia to be due to low red cell counts rather than to an impaired oxidation capacity of the tissues. On the other hand, Schultzen and Riess [72] and Quincke [73! had reported that patients with phosphor~ poisoning excreted lactate but had normal hemoglobin values. Thus this kind of poisoning was selected as a model of likely oxidation deficiency by Nencki and Sieber. F o r obvious reasons the benzene tests were now performed on animals. They clearly showed a decrease of phenol excretion which corresponded to the degree and duration of phosphorus poisoning. Drug metabolism studies were not only carried into the realms of diagnostics and etiology, but also played a role in the long and tedious process of understanding the mechanism of biological oxidation. Up to the 1870s the oxidation of nutrients and body constituents was believed to take place in the blood. One of the first attempts at explaining why and how oxygen in blood was able to oxidize otherwise stable substrates was based on Schiinbein's discovery of ozone and its oxidative power in 1840. In 1862 Schmidt [741 postulated the presence of ozone in blood to explain the oxidations. This hypothesis was supported by Schtinbein c753 on the grounds that ozone is formed by atmospheric electricity and thus must be present in air. Another hypothesis was expressed by Clausius "761 who postulated oxygen splitting by some biological process, thus Eorming a mixture of 0 2 , 0 3 , and easily available 0 , as the actual oxidant. A totally different explanation was offered in Traube's work Theory of Enzyme Effects [.;? 3 in 1858. This author postulated body constituents being able to bind and transfer oxygen much like catalysts. As an analogy he mentioned alcohol which is oxidized by oxygen only in the presence of highly dispersed platinum as an apparent oxygen carrier. The time was not


19

ready for Traube’s remarkably modern theory. On the other hand, it became clear by 1870 that ozone was not a biological constituent. What remained of the ozone theory was the concept of nascent olrygen. Thus Binz “781 differentiated between 0,, which is taken up by the lungs and transported by the blood to the tissues on the one hand, and 0 , , which is formed in the tissues from 0,. Binz [78] and Pfluger [793 were the ones who in 1872 showed that biological oxidation talces place in the solid tissues rather than in blood. Still another oxidation theory was postulated by the eminent and most influential biochemist, HoppeSeyler [SO’] in 1877. His theory originated from observations in the fields of fermentation and putrefaction, and also from the discovery that benzene could be oxidized to phenol by oxygen in the presence of hydrogen adsorbed to palladium. According to Hoppe-Seyler’s explanation, nascent hydrogen of the palladium surface would react with one oxygen atom of 0 2 , thereby forming nascent oxygen which in turn would oxidize benzene. Fascinated by the oxidation of benzene to phenol in the body, Hoppe-Seyler and his students went so f a r as to postulate the formation of nascent hydrogen in the tissues as the actual source of nascent oxygen. This hydrogen theory was never accepted by Nencki. On the other hand, he and his colleague Kaufmann [Sl] were still intrigued by the similarities between oxidations in the animal body and by ozone, even though they accepted the view that ozone was not a physiological agent. Indeed, Nencki and Giamsa [ S Z ] were able to obtain phenol by blowing ozonized oxygen through benzene in the absence of any catalyst. At the same time Radziszewski [83] introduced the already mentioned model system which oxidized substrates in the presence of oxygen and strong alkali. Studying the question of how molecular oxygen is split in the tissues, and experimenting with the Radziszewski system, Nencki and Sieber [69, 703 investigated the possible role of alkali in living cells, which they dismissed. Rather, they developed Traube’s [773 hypothesis and also indicated the interior of tissue cells as the site of biological oxidation: Many elcperiments have shown that protein of the living protoplasm is able to strongly absorb oxygen. The reason for this affinity must therefore be sought in the molecular architecture of that protein.. .It must be concluded that the oxidizing function of cells consists in their formation of easily oxidizable, strongly reducing substances, the major one being the protoplasm itself, which is made up of a labile protein molecule. This protein molecule must be oxidizable by molecular oxygen much like cuprous oxide o r

.

20

CONTI AND BICKEL benzaldehyde and at the same time must form atomic oxygen which in turn oxidizes cellular substrates which are not oxidizible by molecular oxygen. As C B I ~be seen, the questions to be answered will first of all depend on progress in chemistry, such aa the discovery of new compounds which a r e oxidizable by molecular oxygen, the understanding of the reactions involved, the study of the composition of the protein molecule with respect to its fragments of cleavage. Until we shall be able to represent the mechanism of oxidation in an animal cell by a simple chemical equation, all these difficult questions will have to be answered by chemical experimentation. [70 3

Thus Nencki in 1880, at a time when terms like "protein" o r "enzyme" were extremely vague, anticipated the principle of respiration chain enzymes. Obviously, the oxidation of benzene and other foreign compounds had helped a great deal.

lV. SULFURIC ACID CONJUGATION A s has been mentioned before, Munk LG71 in 1876 speculated on the fate of the major part of ingested benzene that did not appear as urinary phenol. He quoted Buliginsky L841 who in 1567 had reported a compound in bovine and equine urine which, upon heating with dilute acids, was split and formed phenol. This observation had been confirmed in the meantime by Hoppe-Seyler 1853. Apparently these authors were not aware of the compound, phenylsulfuric acid, which had been synthesized by Staedeler [a61 from phenol and sulfur trioxide in 1867. Since phenol in urine was only obtained after acid hydrolysis, Munk [67] assumed this compound to be bound to an unknown component, and he named the hypothetical compound ' I phenol-forming substance. ' I After ingesting varying amounts of benzene, Munk was able to detect a direct relationship between benzene dose and phenol-forming substance excreted. H e thereby confirmed his hypothesis that a considerable part of benzene is transformed into phenol which is bound to an unhown constituent and that this indicates another type of conjugation in addition to the well-known glycine conjugation. Munk's phenol-forming substance was not the only example of an unidentified hydrolyzable constituent of urine. An indigo-forming substance (indican) was found in urine by various authors, and Baumann [ 8 7 ] (Fig. 2) in 1876 detected a catechol-forming substance in the urine of horses and humans. In the same year he performed an


FIG. 2. Eugen Baumann, 1846-1896. Berlin, Freiburg.

21

Tiibingen, Strassburg,

analysis of the components of the indigo-forming substance, showing that: if purified indigo-forming substance is split with acetic o r hydrochloric acid, one always obtains a certain amount of sulfuric acid which had not been present in the solution betherefore seems to be a conjufore.. .This substance gated sulfuric acid whose carbon-containing atomic complexes have not yet been identified. [88]

.

...

Further investigations led Baumann to the conclusion that sulfuric acid conjugates a r e regular constituents of human, canine, and particularly equine urine, In the same year and still under the influence of his teacher, Hoppe-Seyler, Baumann tried to connect his discovery of sulfuric acid conjugation to Munk's reported phenol-forming

CONTI AND BICKEL

22

substance [SS]. From horses' urine he isolated a considerable amount of phenol-forming substance which he was then able to split into phenol and sulfuric acid by acid hydrolysis [89 1. Thereupon he verified his flnding by administering phenol to volunteers. While their urine did not contain the unchanged compound, large amounts of phenol and sulfuric acid were obtained upon hydrolysis. Sulfuric acid conjugation of catechol was also observed. Thus conjugation with sulfuric acid began to emerge aa a biochemical principle. Bauxnann also succeeded in identifying the chemical structure of the phenol-forming substance and of sulfuric acid conjugates in general, Elementary analyses yielded the empirical formula CBH5KS04. He realized that this formula could apply either to one of the three iso-

' 'S40K

meric phenol-sulfonic acids C H [OH acid C&5\S04.

or else to phenylsulfuric

Each of the sulfonic acid structures would contain

a phenolic hydroxyl group which would react with alkyl halide to form an ether. Since this could not be achieved, Baumann concluded that the conjugate was an ether of phenol and sulfuric acid. He then once more went back to the metabolism of benzene, this time by measuring not only urinary phenol but also sulfuric acid [90]. This resulted in the remarkable observation that the ingestion of benzene led to a 10- to 15-fold increase of total urinary sulfuric acid as determined after hydrolysis. B a u m m pl] also succeeded in identifying "taurinic acid, '!which had been found in urine and described by Staedeler [48], as the sulfuric acid conjugate of p-cresol. This finding was confirmed in 1877 by Baumann and Herter 1921 in a study involving a series of phenols, phenol homologs, and aromatic compounds. Some, but not all, of the latter were metabolize and excreted as sulfuric acid conjugates. Among them was indole, which was hydroxylated to indoxyl and excreted as indoxyl sulfate (indican), the indigo-forming substance mentioned above. Of particular interest was the fate of aniline, which Wohler and Frerichs [29] in 1848 could not find in the urine after oral administration. This had been confirmed in the meantime by Schuchardt [93] and Sonnenkalb [943. Baumann and Herter [92] found aniline to be hydroxylated to p-aminophenol and conjugated to p-aminophenyl sulfate, which was soon to be confirmed by Schmiedeberg C951.

7

N

Aniline

H

-j ~

N

H~ ~

~

p-Aminophenol

-+ O H NH

-O-S03H

p-Aminophenyl sulfate


23

The studies of Baumann and Herter made it clear that, as with glycine conjugation, sulfuric acid conjugation w a s often preceded by oxidation of the drug. In contrast to these authors, however, Schmiedeberg [9G] in 1881 postulated an inverse sequence: #H

+ 0 + H2S0,+ I DO-S03H

+ H20

+O

O

H + H2S04

The question was settled by an elegant argumentation in a paper of Nencki and Sieber [71]. In phosphorus poisoning the oxidation of benzene to phenol waa blocked, whereas the sulfuric acid conjugation of administered phenol was the same as before poisoning. This proved the oxidation-conjugation sequence to be the correct one. Another problem studied by Baumann and Herter [ 9 2 ] was the fate of hydroxybenzoic acids. These compounds were promising models for the study of conjugation processes since they contained both an acidic group, likely to conjugate with glycine, and a phenolic group, likely to conjugate with sulfuric acid. In agreement with Bertagnini [MI,the ortbo isomer, salicylic acid, was conjugated with glycine. With the m- and p-isomers, however, only a small part was conjugated with glycine, whereas the major part appeared as the sulfuric acid conjugate. It was the first example of compounds able to use two alternative conjugation pathways. This surprising finding brought up the idea of a possible competition of the two conjugating agents for these substrates and, furthermore, that the formation of a conjugate molecule would inhibit its reaction with the alternate conjugating agent. Experimentally, Baumann and Herter then tried to increase one of the conjugating agent pools by administering either glycine or sulfate. Contrary to their expectations, the excretion of either conjugate was not increased and the attempt at shifting the reaction equilibrium within the organism had failed. Still, the approach was remarkable. A quantitative study on the conjugation of phenol was carried out by Schaffer [ 973 in Nencki's laboratory in 1878. He administered phenol to dogs, collected their urine for several days, and determined the phenylsulfuric acid excreted. In all animals he thereby recovered only two-thirds of the dose, a result soon confirmed by Tauber [98] and Auerbach [99]. Like Schultzen and Naunyn and Munk, Schaffer thus was once more confronted with the question about the fate of the unrecovered compound. The oxidation of benzene to phenol might serve as an indication that phenol underwent further hydroxylation to an unknown product which presumably would also be excreted as a sulfuric acid conjugate. H i s theory for an

CONTI AND BICKEL

24 _____~

~~

~

experimental approach was the following. When a dog was given a dose of phenol, the excess sulfuric acid in the urine should correspond with the excreted phenol moiety. Any further excess would then indicate that in addition to phenol another substance had been formed and excreted a s sulfate conjugate. Such a further excess was indeed detected by Schaffer, thus suggesting the formation of an additional sulfuric acid conjugate. This result was examined in the light of two observations made by Salkowski who found that after intestinal ligation the urine contained sulfuric acid in excess of indican and phenol [loo], and furthermore, that phenol-poisoned rabbits excrete oxalic acid [ l o l l . However, Schaffer was not able to detect an increase in oxalic acid in the urine of phenol-treated dogs and thus dismissed the hypothesis of oxalic acid as a phenol metabolite. The problem was followed by Baumann and Preusse b023 who, in analogy to the hydroxylation of benzene to phenol, postulated a hydroxylation of phenol to catechol and hydroquinone. The sulfate conjugates of these oxidation products were regarded a s Schaffer's missing phenol metabolites. Baumann's and Preusse's hypothesis was soon proved right by Nencki and Giacosa [lo31 in 1880. A dog was given a large dose of benzene p e r 0s and by simultaneous cutaneous administration. Catechol could indeed be detected in the dog's urine. Studies in man again yielded catechol and, in addition, trace amounts of hydroquinone. Thus Schultzen and Naunyn's hypothesis of a further oxidation of phenol eventually proved to be correct. Nenclci and Giacosa, however, were aware that, starting from benzene, a considerable proportion still escaped detection. Their explanation for this fact was incomplete intestinal absorption and excretion of the unchanged compound by the lungs. A t that point benzene was the first foreign compound that had been shown to form a s many as half a dozen metabolites by stepwise metabolism along oxidative and conjugative pathways:

C atechol

H ydroquinone


25

V. REDUCTION The first reduction of a foreign compound was discovered 21 years after the first oxidation. It was a quick discovery by one man who, in 1863, had knowledge of the metabolism of benzoic acid and who had improved analytical and physiological techniques at his disposal. Speculating on chemical similarities between quinic acid and benzoic acid, Lautemanu [lo41 succeeded in reducing the former to the latter by using hydrogen iodide. Once more, an analogy between chemical reagents and the organism as a chemical agent was considered: The fact that quinic acid was so easily reduced to benzoic acid in the test tube suggested investigating whether the former compound was also reduced in the animal body and the resulting benzoic acid excreted a s hippuric acid. 11043 The author and two volunteers ingested 8 g of calcium quinate and collected their urines which were subsequently analyzed. All three specimens did contain hippuric acid. This finding was soon confirmed by Meissner and Shepard [36] in humans, rabbits, and goats; however, in contrast to Mattschersky [105], not in dogs. A t that time both the empirical formula for quinic acid, CeH,(OH)*(C02H), and its sixmembered ring structure were known. HO

H O B o H HO

COOH

Quinic acid

)

O C O O H

+

OCO-Gly

-bH20

Benzoic acid

Hippuric acid

The studies on quinic acid metabolism were carried on in 1879 by Stadelmann [106]. His goal was to quantify the metabolic reaction by means of hippuric acid determination according to Bunge and Schmiedeberg and, furthermore, to clarify the species differences mentioned above. In all cases he carefully determined hippuric acid excretion in his animals before and after quinic acid administration. In agreement with Meissner and Shepard [36], dogs given 2 to 3 g of quinic acid showed no increase in hippuric acid excretion. In contrast, rabbits, selected as a herbivorous species, in all cases excreted excess hippuric acid. Two important observations were emphasized by Stadelmann: The excess hippuric acid was far from representing

CONTI AND BICKEL

26

the quinic acid doses ingested, and its appearance in the urine occurred 24 to 48 h r after ingestion only. The first observation was confirmed much later by Forater 1107 1 and Hupfer [lo8 1 who, after ingesting a total of up to 100 g of quinic acid, recovered some 20% of the dose as urinary hippuric acid. The lack of quinic acid reduction in the dog was confirmed by Schmidt [log] who, on the other hand, found an increase in sulfuric acid after urinary hydrolysis, indicating an unlmown sulfuric acid conjugate. Stadelmann then investigated the localization of quinic acid reduction much as Schmiedeberg, whose disciple he was. had done in the case of glycine conjugation. A reduction in the blood was unlikely in view of the remarkable time lapse from ingestion of quinic acid to excretion of hippuric acid. Still, the substrate was injected into the jugular vein of rabbits. As expected, no increase in hippuric acid occurred. Reduction in the gastrointestinal tract was therefore more likely. The question here was in which part the reaction might occur, and whether o r not under the influence of a secretion product. The 5rst experiments were aimed at the stomach as a potential r e duction site. However, pylorus ligation in rabbits did not result in increased hippuric acid after quinic acid administration; neither did incubation with pancreas homogenate for 18 hr a t 37°C. At this point Stadelmann gave up further experimentation and offered a hypothesis which later proved right: The site of metabolism of quinic acid is in all likeliness the intestine, although we have not succeeded in furnishing the appropriate experimental evidence.. .The retarded excretion suggests that reduction of quinic acid occurs slowly, presumably in the distal p a r t of the intestine.. .Whether the transformation in the lower intestine is part of the putrefactive reduction processes, o r whether it is due to the action of glandular secretions, cannot be decided yet, although the former alternative seems to be more likely. [lo63

.

.

Stadelmann, too, was harassed by the problem of low metabolite recovery. Even though he was working with a sensitive method for the determination of hippuric acid, the urine of his rabbits yielded only a fraction of the administered dose. Since, however, there was no method for the detection of quinic acid a t that time, he speculated that the major part of a dose was excreted as unchanged quinic acid. Quinone was another foreign compound to be reduced in the body. The first experiments were carried out by Wohler and Frerichs [29]


27

in 1848. These authors were not able to detect quinone in the urine of dogs dosed with 0.5 to 1.0 g of the compound which, in an e r a not used to drug metabolic reactions, was most surprising to them. Only in 1892 was this investigation resumed by Schulz [ l l O ] who found that urine after ingestion of quinone had a dark brown-green color and that after boiling with hydrochloric acid a compound was obtained which reduced silver nitrate and was likely to be hydroquinone. Indeed, S. Cohn [ 1113 succeeded in isolating crystalline hydroquinone which had been excreted as its sulfuric acid conjugate:

Quinone

Hydroquinone

Hydroquinone sulfate

The last two decades of the 19th century also led to the discovery of nitro reduction. The first example was indicated by HoppeSeyler [:1123 who found that o-nitrophenylpropiolic acid was metabolized and excreted as indoqlsulfuric acid. This was interpreted as an initial reduction followed by decarboxylation to indoxyl and sulfuric acid conjugation. The last reaction had already been described by Baumann and Herter [92]. ,C-COOH

NO,z o-Nitrophenylpropiolic acid

Indoxylic acid

N H I

J. H

Indoxyl

H

Indoxyl sulfate

Another instance of reductive metabolism of nitro groups was the metabolic transformation of picric acid (2,4,6-trinitrophenol) to picramic acid (2-amim-4,6-dMtrophenol). This reaction was postulated by Rymsza [_113] and established by Karplus [1141. The nitro reductions of nitrobenzaldehydes were of particular interest. Sieber and Smirnow [1151 reported that in dogs the three isomers of nitrobenzaldehyde were oxidized to the corresponding nitrobenzoic acid isomers which then usually were conjugated to nitrohippuric acids. R. Cohn [l16], who was also dealing with the

CONTI AND BICKEL

28

metabolism of nitrobenzaldehydes, observed that rabbits given the mand p-isomers of that compound excreted an unknown product in addition to the nitrobenzoic and nitrohippuric acid isomers. Cohn succeeded in identifying the new metabolite as acetylaminobenzoic acid. Whereas he first assumed the reduction to take place in the intestine, he later also obtained the metabolite in rabbit urine after subcutaneous administration of nitrobenzaldehyde and thus concluded that this nitro reduction was a truly endogenous metabolic reaction [ 1173. Cohn was the first to demonstrate that two functional groups in a single molecule could be metabolically attacked s o that such drugs undergo two separate pathways:

i" N02-@X0 \

N O ~ ~ C O O -+ H

N O2 G C O - G l Y

1

I [..2-#-COOH]

+

CH3-CO-NHeCOOH

Among the highlights of early pharmacology and drug metabolism is the saga of chloral hydrate. This compound was discovered in 1831 by Liebig, but only in 1861 was it studied with respect to its pharmacological properties by Buchheim. Based on the formation of chloroform and formic acid upon heating chloral hydrate with potassium hydroxyde, Buchheim thought he had found a drug able to release acid in the body and thus become useful in counteracting alkalosis. Animal and self-experimentation with the drug then disclosed the strong hypnotic action of chloral hydrate, which Buchheim ascribed to the metabolic formation of chloroform. Unfortunately, he did not publish these results which therefore remained in obscurity until Kobert [ 1181 and others were able to quote from his posthumous works. For this reason Liebreich [I191 who, 8 years after Buchheim, worked and published on chloral hydrate, is given credit for discovering its hypnotic action. Indeed, his discovery was made without lcnowledge of Buchheim's work and was the starting point for the introduction of this new synthetic hypnotic. Liebreich, too, postulated that the drug's hypnotic action i s due to chloroform which is formed by alkaline constituents of the blood. Loebisch in 1888 [120] praised Liebreich not only for the introduction of a most successful drug, but also for being the inaugurator of modern experimental pharmacology which synthesizes drugs based on chemical and biochemical howledge, tests their ac-


29

tions in animals, and introduces them into therapy with a circumscribed indication. This statement, to be sure, characterized new trends in pharmacology rather than Liebreich in particular, who had introduced chloral hydrate and propagated the chloroform theory, but was unable to prove it. Even after giving high doses of chloral hydrate, the urine of his animals was devoid of chloroform. The only positive statement was his impression that the animals' expiration might contain chloroform. In the followipg years a host of authora tried to obtain evidence for o r against the chloroform theory. Personne [121] reported detecting chloroform and formic acid in the blood of chloralized animals, although most French investigators denied Liebreich's theory. Others, like Hammarsten [1223 and Rajewsky [1231, were unable to detect chloroform in blood o r expired air. Kiilz [124] reported having found chloroform, and Bouchut [125] chloral hydrate in urine. Tomaszewicz [126] added to the confusion by reporting the presence of chloral hydrate and the absence of chloroform. This was the state of affairs in 1875 when the classical paper of von Mering (Fig. 3) and Musculus [127] appeared: There is hardly a drug which in recent years was consumed in such large amounts as chloral hydrate, and on whose mechanism of action so much controversy exists. .These contradictory reports above all incited us to investigate the fate of chloral hydrate in the organism. [127 1

..

Von Mering and Musculus found no chloroform in the expired air of chloralized animals. In the urine of humans who had ingested 5 to 6 g of the drug daily for some time, small amounts of unchanged drug, but no chloroform o r formic acid was detected. This was not the end but rather the beginning of their investigation which next disclosed that the above urine was levorotatory without containing albumin. Tedious work eventually resulted in the isolation of the levorotatory compound which was an acid called urochloralic acid by von Mering and M u s d u s . It was believed to originate from the reaction of an u n h o w n metabolite of chloral hydrate with an endogenous compound. The same compound was isolated two years later by Falck [128 1 who also believed it to be a metabolite of chloral hydrate and who predicted that the discovery of urochloralic acid would mark the end of Liebreichs's chloroform theory. After several investigators had not succeeded in preparing urochloralic acid in amounts sufficient for analytical work, Kulz [129] by 1880 made another most remarkable contribution. By boiling urochloralic acid from the urine of chloralized dogs with hydrochloric acid, he obtained a chlorinated compound

30

CONTI AND BICKEL

FIG. 3. Josef von Mering, 1849-1906. Strassburg, Halle. and a dextrorotatory, strongly reducing acid. The latter proved to correspond to glucuronic acid which had just been discovered by Schmiedeberg and Meyer (1301. By administering single doses of 20 to 25 g of chloral hydrate to dogs, Kulz (1311 succeeded in isolating enough urochloralic acid to perform elementary analysis, The empirical formula, C B H , ~ C ~ ~ even O , , though obtained with two different methods, unfortunately was not correct and created some confusion. Convinced of the correctness of both the formulas of urochloralic and glucuronic acid, K d z postulated the unconjugated metabolite of chloral hydrate to be C2H,C130. He adhered to that formula, although he was aware that Garzarolli-Thurnlaclch (1321 had obtained


31

trichloroethanol (C2H3C130), a likely candidate for the unconjugated chloral hydrate metabolite, by reducing the drug with ethyl zinc. Von Mering and Musculus [127 J, too, had indicated a wrong formula for urochloralic acid in their work of 1875; however, they had emphasized the formula to be preliminary, since the purity of their preparation was not satisfactory. In 1882, the same year Kiilz published his incorrect findings, von Mering 033, 1343 had reached his goal of correctly identifying urochloralic acid and its aglycone resulting from acid hydrolysis:

Urochloralic acid

Trichloroethanol

Glucuronic acid

Von Mering found the properties of his aglycone to fully correspond to trichloroethanol as obtained in the test tube by Garzarolli-Thurnlackh, and thus urochloralic acid to be trichloroethyl glucuronide. He particularly emphasized that trichloroethanol was the primary metabolite of chloral hydrate: With the glucuronides known so far, the corresponding aglycones originated in the organism by oxidation of the ingested compound. In our case, however, the aglycone (trichloroethyl alcohol) resulted from reduction. This is why urochloralic acid deserves particular interest. L134 1 Thus, 13 years after the introduction ,of chloral hydrate as a hypnotic and after Liebreich's chloroform theory, the dmg had eventually been shown to undergo metabolic reduction and subsequent glucuronic acid conjugation. A final contribution was made by Kiilz [135] in 1884 by demonstrating the formation of urochloralic acid from ingested trichloroethanol in rabbits. This unequivocally proved von Mering's findings.

VI.

GLUCURONIC ACID CONJUGATION

It has been mentioned in the previous section that in 1875 von Mering and Musculus [127] in the course of their work on the fate of chloral hydrate isolated urochloralic acid, an apparently conjugated metabolite, which subsequently was characterized as a glucuronide by Kulz [129]. These discoveries, however, were only the final stages of the process, which over three decades gradually disclosed the mechanism of glucuronic acid conjugation.

CONTI A N D BICKEL

32

This development started with Indian yellow, a dye obtained from the urine of cows fed on mango leaves. Its main constituent, euxanthic acid, waa suspected by Erdmann L136 1 in 1846 to be a conjugated compound, since it formed a new acid upon hydrolysis. Schmid [137] [mew that Indian yellow waa produced from the urine of camels fed on the fruits of Mangostana mangifer. In 1855 he therefore analyzed both euxarithic acid and what he called mangostin, a yellow pigment from the mango h i t . Obtaining similar empirical formulas for mangostin and euxanthic acid, Schmid speculated that the latter compound might be formed in the camel from the former: I t is readily conceivable that mangostin is transformed to ewanthic acid by the vital process.. .If euxanthic acid is treated with concentrated sulfuric acid and the resulting solution poured into water, then, as is known, ewanthone precipitates. The flltrate then easily reduces copper oxide dissolved in potassium hydroxide, whereas neither euanthic acid nor euxanthone have reducing properties. c1371

.

Thus the filtrate was shown to contain a substance that had been part of euxanthic acid, thereby demonstrating that this compound was a conjugate. In addition, a first chemical property of the new conjugating agent had been discovered. Although Schmid did not mention whether he was familiar with glycine conjugation, this is strongly suggested by his cxperimental procedure. After Schmid's paper had appeared, several other investigators dealt with euxanthone and proposed a number of empirical formulas. Real progress was not achieved before Baeyer [138] in 1870 gave the first correct formula for euxanthone and verified Schmid's findings, including the copper oxide-reducing conjugating agent. Based on accurate analyses, he then was able to indicate the correct relationship b e b e e n euxanthone and e u a n t h i c acid, which in Baeyer's empirical formulation and in modern structural formulas reads: C19H16010

COOH

+

n

HZo = nu

v Euxanthic acid (Indian yellow)

C6H,007

+

C13H804

COOH

0

OH

OH Glucuronic acid

Euxanthone

HISTORY OF DRUG METASOLISM

33

Baeyer then tried to characterize the unknown conjugating agent, C6H10O7, obtained by hydrolysis of ewanthic acid. In addition to its reducing power he was aware of Laurent's observation that upon heating, euxanthic acid decomposes into euxanthone, carbon dioxide, and water. Both the reduction and the type of decomposition were reminiscent of sugars. Furthermore, the empirical formula of the conjugating agent almost corresponded to that of saccharic acid, C,H,,O,. Based on these pieces of evidence, Baeyer proposed that the conjugating agent was kind of a saccharic acid and thus euxanthic acid had to be considered as a glycoside of saccharic acid with euxanthone When von Mering and Musculus [127] in 1875 isolated urochloralic acid as a metabolite of chloral hydrate in human urine, they were mainly interested in the chlorine-containing moiety of the molecule. Yet they also contributed to the characterization of the conjugating agent by comparing the conjugate, urochloralic acid, with the parent drug. Thus, in agreement with Baeyer and Schmid, urochloralic acid was found to be considerably acidic and able to reduce boiling alkaline copper solution and silver and bismuth oxides. Furthermore, the conjugate possessed levorotatory optical activity and, upon boiling with potassium hydroxide, turned brown and developed a smell of caramel. Von Mering and Musculus concluded from these findings that urochloralic acid could be split into a chloral hydrate derivative (which later turned out to be trichloroethanol) and an endogenous, sugarlike compound. Being familiar with glycine conjugation, they were convinced that they were dealing with an unknown type of conjugation. They also mentioned having found other substances forming reducing and levorotatory urinary metabolites such as morphine and crotonchloral hydrate, thereby suggesting that all these drugs undergo a common conjugation reaction. When dogs or rabbits were poisoned with nitrobenzene, Ewald [139] found that their urines contained a reducing compound which he believed to be sugar. He therefore suggested the toxicity of nitrobenzene to be due to diabetogenic action. This, however, was disproved by von Mering [140] who demonstrated that the urine of nitrobenzene-treated rabbits contained a levorotatory compound rather than dextromtatory glucose. Using the same model, Jaff6 [1411 in 1878 was not able to find optical activity and reducing power in all his experiments. He questioned whether the urinary metabolite was a derivative of nitrobenzene and offered the hypothesis that the metabolite originated from nitrotoluene, a possible contaminant of commercial nitrobenzene. For a number of years Jaff6 had been investigating the toxicology and metabolism of the three isomers of nitrotoluene. He was particularly curious to know whether all isomers were

.

CONTI AND BICKEL

34

metabolized like toluene, or else, whether their different chemical properties were reflected in different metabolic pathways. In a first paper, Jaff6 [as'] reported that besides p-nitrotoluene, the urine contained both p-nitrobenzoic acid and its glycine conjugate, p-nitrohippuric acid, Four years later c1411 he stated that, in contrast to the p-isomer, o-nitrotoluene in the dog resulted in only 10% of o-nitrohippuric acid, the major part being a more complex conjugate. Due to certain similarities with urochloralic acid, Jaffe' called his conjugate uronitrotoluic acid. Starting out from a correct empirical formula of the conjugate, and based on the assumed analogy with the pisomer a~ well a s a sugarlike conjugating agent, Jaffe flrst proposed Reaction I. However, neither nitrobenzoic acid nor a hexose could be identified after hydrolysis. Rather, the aglycone was found to be nitrobenzyl alcohol. This then led to the correct Reaction II:

Uronitrotoluic acid

I1 (correct): C 1 3 H t ~ N 0 9 Uronitrotoluic acid

Nitrobenzoic acid +

H2O

-,C7H7N03 Nitrobenzyl alcohol

Sugar + C6H1007

Yhgar"

Jaffe'now paid particular attention to the conjugate, C 6 H l o 0 7 , which he cautiously called "hypothetical acid. ' I Like some of his predecessors, he characterized it a s a reducing dextrorotatory compound which, upon heating, developed a caramel-like smell. These were obviously properties of carbohydrates. However, sugar could be ruled out due to the empirical formula a s well as to the observation that the compound did not undergo fermentation by yeast. Jaffe' concluded that the conjugating agent was an oxidation product of sugar, probably formed by transformation of an -CH20H into a -COOH group. By these correct conclusions Jaffe'indeed came very close to glucuronic acid. Camphor was still another drug forming a metabolite which was composed of a camphorlike and a sugarlike moiety. This observation was made by Wiedemann [142] who boiled the urine of camphortreated dogs with dilute acids. He speculated that the metabolite might be a nitrogen-containing glycosidic acid. This study was resumed by Schmiedeberg (Fig. 4) and Meyer [ 1301 who, in 1879, succeeded in solving the problem of the sugarlike conjugating agent. They reproduced the formation of the conjugated camphor metabolite in dogs as well a s the hydrolysis of the conjugate. With relative ease


35

FIG. 4. Oswald Schmiedeberg, 1838-1921. Dorpat, Strassburg.

they were able to extract and purify the camphorlike aglycone which they characterized a s C,,,H ,602,a dextrorotatory secondary alcohol; i.e., an oxidation product of camphor with an additional alcohol group. This primary metabolite was therefore named campherol. More difficulties were encountered by Schrniedeberg and Meyer during the identification of the conjugating agent. They were the first to eventually succeed in isolating a small amount of the pure conjugating acid. This then allowed detailed investigation of the compound which led to the following conclusions: There can be no doubt that this acid is a direct derivative of glucose. This is suggested by the formula, the reduction of alkaline copper solution, the dextrorotation of the plane of polarization, and the results of oxidation experiments which exclude the participation of an aromatic nucleus in its formation, Based on the composition of this acid, its structure is intermediate between gluconic acid and saccharic acid..

..

CONTI AND BICKEL

36

According to Fittig's views on the structure of glucose, the acid must be given the formula (CH'0H'4

CHO [COOH..

.

Arbitrarily we call the compound glucuronic acid. [130] Thus Schmiedeberg and Meyer established the metabolism of camphor as a primary oxidation to campherol, followed by glucuronic acid conjugation which resulted in the formation of camphoglucuronic acid. Finally, Schmiedeberg and Meyer also speculated on the origin of glucuronic acid in the organism. They strongly suspected that their discovery allowed insight into the mechanism of glucose oxidation. Since acids had been postulated a s early intermediary metabolites of glucose, glucuronic acid was a likely metabolite which, by conjugating with campherol, had escaped further breakdown and combustion. Thus experiments with foreign compounds like camphor could also be useful in investigating processes of intermediary metabolism.

VII. ACETIC ACID CONJUGATION The finding of acetylaminobenzoic acid as a metabolite of nitrobenzaldehyde [ U G ] has been reported in the section on metabolic reductions. The actual discovery of acetic acid conjugation took place s i x years earlier, in 1887, when Jaffe and R. Cohn [143 3 described what still is known a s a most unusual metabolic reaction involving acetic acid. Based on chemical similarities of benzene and iuran, the authors investigated the fate of furfural in dogs and rabbits. The urine of the animals contained a-furoic acid (called pyromucic acid, C5H,03), furoylglycine (pyromucuric acid), and a third acid metabolite. The latter could be split by hydrolysis into glycine and a nitrogen-free acid with the empirical formula C7H603. A compound of the same empirical formula had been obtained ten years earlier by Baeyer [144] after boiling furfural and sodium acetate for 8 hr. He correctly called the product furfuracrylic acid. Jaffe'and Cohn carefully compared their nitrogen-free acid with Baeyer's furfuracrylic acid and found identity of all chemical properties tested. This was further confirmed by administering the acid to rabbits which then excreted the glycine conjugate. Thus furfural in the organism must have undergone a synthetic reaction with acetic acid, thereby forming furfuracrylic acid, which subsequently was conjugated with glycine. Jaffe' and Cohn expressed their surprise about this unheard of


37

metabolic reaction which was thought to shed new light on biosynthetic processes at large. Their results on the metabolism of furfural can be formulated as follows:

/” ~

C

H

Furfural

~

P C O O H

O a-Furoic acid

j

V C O - G l y

Furoylglycine

L Furfuracrylic acid

Obviously, this discovery justified further investigation with additional aldehydes. R. Cohn [llS ] used a-thiophene aldehyde, the thio analog of furfural, as another substrate to demonstrate the biosynthetic process with acetic acid. The urine of rabbits and dogs treated with the substrate contained thiophenuric acid, the glycine conjugate of cu-thiophenic acid. However, i t did not contain thienylacrylic acid, the expected analog of furfuracrylic acid. On the other hand, injection of synthetic thienylacrylic acid resulted in the excretion of oLthiophenic acid without unchanged compound in the urine of a rabbit, thus indicating the metabolic cleavage rather than the biosynthesis of thienylacrylic acid: CO-Gly

2 / Thiophenuric acid P C H O

a-Thiophene aldehyde

QCH=CH-COOH

Thienylacrylic acid

~ C O O H+

C H ~ O H

a-Thiophenic acid

R. Cohn was so convinced of the formation of thienylacrylic acid that he explained its lack in the urine by its rapid breakdown into cu-thiophenic acid. Actually, by discovering the metabolic formation of furfuracrylic acid from furfural, R. Cohn had come across an atypical biosynthesis with acetic acid. This made him believe acetic acid conjugation to be a reaction between an aldehyde and the methyl group of

CONTI AND BICKEL

38

acetic acid. His stubborness in testing suitable aldehydes eventually led him to discover the typical acetic acid conjugation of amines: Atypical:

R-CHO

+ CH,-COOH

Typical:

R-NH,

+ HOOC-CH3 + R-NH-CO-CH3

+R-CH=CH-COOH

Nitrobenzaldehydes became the crucial substrates for R. Cohn's discovery of the typical acetic acid conjugation. Again, the first experiments were disappointing; using o-nitrobenzaldehyde, R. Cohn, like Sieber and Smirnow [115], found o-nitrobenzoic acid in the urine, but no conjugate whatsoever. A dog given m-nitrobenzaldehyde excreted m-nitrohippuric acid without acetic acid conjugate. However, using rabbits instead of dogs, R. Cohn obtained an unknown acid with an empirical formula C g H g N 0 3 . Hydrolytic cleavage of this metabolite yielded m-aminobenzoic acid and acetic acid. The underlying metabolic process was correctly interpreted by R. Cohn: Concerning the formation of acetylaminobenzoic acid from nitrobenzaldehyde in the animal body, one must assume that nitrobenzoic acid, which is primarily formed, is reduced to aminobenzoic acid in the long intestine of the rabbit, and that the latter compound is absorbed and finally in some organ meets acetic acid: CaH,

+ CH,.COOH + CBH4[NH.CO.CH, COOH

+

HzO

...

I have demonstrated in the above communication that syntheses with acetic acid play an important role in the animal body. Acetic acid is thus frequently involved in metabolic reactions, even though it usually escapes detection due to its rapid oxidation. [ 116 1 A s has been mentioned in the section entitled 'Tteduction," R. Cohn [ 117 3 later found that the nitro reduction took place in organs rather than in the intestinal lumen. Thus R. Cohn's work was not only a remarkable contribution to the history of drug metabolism but, as demonstrated by the end of the above quotation, it also introduced acetate. one of the most important intermediary metabolites, into biochemistry.


39

VIIL GENERAL COMMENTS This work is restricted to the discoveries of the major metabolic pathways within the history of drug metabolism in the 19th century. Since the main events described in Sections II to W a r e summarized below, this section attempts to discuss and comment upon a number of individual aspects. At times, this is deliberately done from the viewpoint of modern biochemistry. The previous sections deal with the history of drug metabolism o r xenobiochemistry during the period 1840 to 1900. What was achieved during that period of early biochemistry in our particular field? Above all i t brought about knowledge of those metabolic pathways of xenobiotics which still today a r e considered the most important ones: oxidation, reduction, and conjugations with glycine, sulfuric acid, glucuronic acid, and acetic acid. The wealth of individual studies and results published during that period was quite remarkable, but this particular aspect is beyond the purpose of this work. Suffice it therefore to mention Heffter's [7] monograph of 1905, which reviews the fate in the organism of over 400 organic compounds and lists no less than 600 references. It characterizes the time and the stage of development of biochemistry when Heffter uses the classification of his compounds according to the venerable Beilstein Handbook, when foreign to the body" is used synonymously with "foreign to the urine, when many compounds like fatty'acids, amino acids, and other intermediary metabolites a r e treated a s foreign compounds. Dealing with 19th century drug metabolism, one should bear in mind that important dimensions like enzymology, chemical reaction mechanisms, pharmacokinetics , and cell biology were introduced in the course of the 20th century. The fact that 19th century biochemistry was preenzymological is of particular importance. This precluded a real distinction between chemistry o r test tube chemistry on the one hand and biochemistry a s a chemistry of selected compounds occurring in organisms and determined by genetically coded enzymes on the other hand. Still, within that frame, biochemistry, which started at a near-zero level of knowledge around 1800, became a remarkable success story in the course of its first century. The factors limiting its progress were chiefly the respective state of analytical and physiological techniques. Many brilliantly conceived projects were doomed to failure for lack of sufficiently sensitive analytical methods. Who were the investigators who decided to study the metabolism of foreign compounds, and what was their motivation? By training, I'

40

CONTI AND BICKEL

they were chemists as well a s physiologists, pharmacologists, and clinical physicians. Accordingly, their outlooks and approaches varied a t times. Clinically relevant problems were relatively rare at that time. The same holds for the interest in foreign compounds as drugs, since the amount and importance of synthetic drugs was still modest and the notion of environmental contaminants wm nonexistent. Thus it was biochemistry which mainly motivated research in the field. The discovery of the biotransformation of benzoic acid to hippuric acid was a major event since it suggested that metabolic pathways and processes could be investigated by means of simple foreign compounds. This was a tempting alternative to the use of the complex nutrients and body constituents, which in most instances were poorly characterized. Many investigators using a chemical approach dealt with the organism a s a chemical reagent which allowed them to study new reactions. Most researchers used the same type of basic methodology in ord e r to obtain infomation on the biochemical fate of a drug. The compound was administered per 0s to a laboratory animal o r to a volunteer, and the urine was qualitatively analyzed for unchanged drug o r its metabolite. Very rarely were blood o r tissue samples analyzed, since the available methods lacked the sensitivity for the detection of the small amounts of drug which had been diluted in the large volume of the organism. In most cases the doses used in an experiment were limited because of the unpleasant o r toxic effects of the compounds. A most striking feature was the habit of self-experimentation, performed by many investigators. No less impressive a r e the "heroic" doses used, which often were in the multigram range. Many critical remarks can be made with respect to the a r t of conducting experiments in those days. The following by no means is meant to be a critique of the pioneers, underprivileged in terms of information and technical facilities, but rather as a means of characterizing their time and their situation. For example, the methods used often p r e cluded the detection of several metabolites of a drug, and only allowed the detection of what had been expected o r suspected. Most e.xperiments were carried out in vivo, i. e., in an extremely complex and largely udaown system. Pharmacokinetic processes like absorption, distribution, tissue uptake, extrarenal excretion, though closely associated with metabolism, were frequently neglected, Also neglected were species differences; in fact, many well-conceived experiments were carried out in unsuitable species, o r else results were briskly extrapolated to other species. Quantitative determinations, if at all considered, were hampered by poor accuracy and sensitivity of m a lytical methods. Decisive control experiments were omitted at times; e.g., not all hippuric acid determinations were checked against hippuric


41

acid produced from endogenous aromatic compounds. Recoveries often comprised a minor fraction of the dose, which then led to the conclusion that the remaining drug had been degraded to carbon dioxide and water. Certain researchers were very quick in making a grandiose biological principle out of one o r a few experimental results. Finally, it took biochemists the whole 19th century to quit thinking in terms of organic chemical test tube reactions, However, a number of works were produced in drug metabolism, which in terms of conception, quality, and impact were far ahead of their time and must be regarded a s pacemakers of modern biochemistry. These achievements a r e highly respectable in view of the I'preenzymological" e r a and the primitive equipment of the laboratories, Many of the important discoveries made in xenobiochemistry were aiming at o r have solved complex and difficult problems. It has been mentioned that the conjugating agent glucuronic acid was gradually unraveled by a number of investigators over the period 1846 to 1879, and that its basic structure was established long before the complete structure and the metabolism of glucose was known. In addition to a host of simple metabolic reactions, multiple pathways of xenobiotics were also studied o r even provoked with suitable substrates. Of particular importance was the successful elucidation of reaction sequences like oxidation conjugation, which has become the backbone of xenobiochemistry. Within series of aromatic or other compounds, scores of individual substances were studied so as to allow the establishment of rules on their metabolic fate. The popularity of aromatic compounds in the field of drug metabolism was due to their easy recognizability and to the fact that, except for aromatic amino acids, they hardly occur in the organism and a r e not metabolized to the extent of rupturing the benzene ring. Therefore, the benzene ring was used a s a metabolically stable label and experiments were conceived which a r e reminiscent of those with radioactive labels performed in o u r time. Experimental failures often were transformed into provocative and stimulating hypotheses. For example, hydrolyses like digestive processes, but not biosyntheses like hippuric acid formation, could easily be stimulated in vitro. This dilemma provided a strong stimulus to develop in vitro systems and thereby to study the conditions prevailing in vivo. In the case of the above biosyntheses, this may even have been a last opportunity to reflect on the 'lvis vitalis.'l Another type of experiment which did not yield the expected answer was the attempt a t influencing what was supposed to be a competition of conjugation reactions. But again, this stimulated the conception of limited pools of endogenous conjugating agents, like glucuronate and sulfate. The use of h o w l edge in drug metabolism to improve pharmacodynamic properties of

42

CONTI AND BICKEL

drugs anticipated certain aspects of what in the second half of the 20th century became known as molecular pharmacology. Furthermore, certain investigations of the localization of xenobiochemical processes must be called spectacular a t a time which hardly had abandoned the notion of the blood as the site of general metabolism. Some of these localization studies were performed with new and bold techniques whereby biochemical processes were explored under well-defined conditions of physiological alterations. Among these new techniques were the bile fistula which eliminated a secretion, nephrectomy which eliminated an organ, the isolated perfused organ eliminating undesired influences of the whole organism, the ligated liver, and the portocaval shunt which was then called Eck's fistula. Among the achievements of the time there was even the use of 'I tool" drugs which were given to alter physiological parameters for the study of a test drug. There can be no doubt that studies on the metabolism of foreign compounds, which in the 19th century was a modest speciality, has made major contributions to general biochemistry. For example, the formation of hippuric acid from glycine and benzoic acid was the first example of a biosynthetic process and thus introduced a concept of fundamental importance. Similar to this conjugation process s the oxidation of xenobiotics like benzene could only be simulated outside the o r ganism by using chemical tricks, such a s ozone as an oxidant. Studies along that line strongly stimulated interest in the general process of biological oxidation, and it was precisely the use of foreign compounds which provided a promising tool for further investigation. Thus there is a direct line leading from benzene oxidation to the clinically used "benzene test" and the ozone theory to the concept of active oxygen and eventually to the hypothesis of labile, autoxidizable cell proteins as catalysts for the oxidation of endogenous and foreign compounds. These efforta have also accelerated the rise of the concept of tissues rather than blood a s metabolic sites. The study of metabolic reductions then demonstrated that, in addition to tissues, the intestinal lumen was still another site of xenobiochemical reactions. The conjugation reactions with glycine, glucuronic acid, and acetic acid were all discovered with foreign compounds. It was only later that these processes were found to be part of the intermediary metabolism of endogenous substrates. Many of the discoveries made in the field of drug metabolism in those days had their consequences in general biochemistry o r pharmacology a t a much later stage. A s an example, Nencki's and Salkowski's studies on the sidechain oxidation of aromatic compounds resulted in Knoop's discovery, forty years later, of the principle of beta-oxidation of fatty acids. Similarly, further studies on acetylation of drugs led to the discovery of coenzyme A in 1951.


43

Finally, 19th century investigators recognized and dealt with several problems of drug metabolism which still are studied today and still pose considerable difficulties despite the immense increase in knowledge and research technology. M. SUMhIARY-TABLES O F KEY DATES

A. 1773 1801 1824 1829 1830 1835 1841 1842 1844 1845 1857 1863/66 1875 1876 1877 1879

Glvcine Coniuaation*

Rouelle: Detection of tlBlrin urine of cows. Scheele, Proust: "Bll in human urine. lrBttnot identical with B ? Wohler: B in dogs excreted a s unchanged "B. Liebig: 'rBfffrom equine urine contains nitrogen. New compound called hippuric acid. Wohler: Hypothesis of biotransformation of B to H. Lelunann: H in urine of a diabetic patient. U x : Biotransformation experiment in humans shows B to be excreted as H. Keller: Confirmation of B 3 H in humans. Liebig: H also a normal constituent of human urine. Dessaignes: Identification as benzoylglycine and synthesis of H. Kiihne, Hallwachs: H formation in vivo from B and glycocholic acid. Site: hepatic vascular system? Meissner, Shepard: H formation in kidneys? Spengel: Exogenous Gly used for H formation in vivo. Bunge, Schmiedeberg: H formation localized in kidneys. Hoffmann: CO and quinine inhibit H formation in isolated perfused liver. Koch: H formation in kidney homogenate.

--

B. Oxidation

1842 1842 1848

U x : Cinnamic acid excreted a s hippuric acid. Erdmann, Marchand: Other hippuric acid forming substrates. Primary oxidation? Wohler, Frerichs: Benzaldehyde excreted a s hippuric acid. Oxidation-glycine conjugation sequence.

*B = benzoic acid; H = hippuric acid, lrBtlhippuric acid believed to be benzoic acid.

CONTI AND BICKEL

44

1851 1867

1870-90 1876 1880ff

Staedeler: Hypothesis of benzene oxidation to phenol. Schultzen, Naunyn, Graebe: Fate of aromatic compounds. Benzene +phenol, toluene + hippuric acid, xylene + toluric acid, etc. Aromatic hydroxylations and glycine conjugations. Nencki: Elaboration of aromatic xenobiochemistry Rules. Metabolically stable benzene ring as marker. Munk: Benzene oxidation as a tool for studies in biological oxidation. Nencki, Sieber: Benzene test for metabolic oxidation capacity. Theories of biological oxidation (active oxygen, autoxidizable cell proteins).

.

--

C. Sulfuric Acid Conjugation

1867 1876 1876

1877

1878/80 1881 1882

Buliginsky: Appearance of phenol in hydrolyzed urine. Mu&: - "Phenol-forming substance" as benzene metabolite. New type of conjugation? Baumanu: %dip-forming substance" on hydrolysis yields sulfuric acid. Ether sulfates as conjugates of xenobiotics. Baumann, Herter: Sulfuric acid conjugations of phenols. Hydroxybenzoic acids as substrates for conjugation with glycine and/or sulfuric acid. Schaffer; Baumann, Preusse; Nencki, Giacosa: Phenol 3 hydroxyphenols + sulfates. Schmiedeberg: Hypothesis of conjugation-oxidation sequence. Nencki: Proof of oxidation-conjugation sequence. D. Reduction

1863 1875/82 1879 1882/92 1892/93

Lautemann: Quinic acid excreted as hippuric acid. P r i mary reduction. Von Mering, Musculus; Kiilz: Chloral hydrate reduced to trichloroethanol and conjugated to "urochloralic acid. ' I Stadelmann: Quinic acid reduction in intestinal lumen. Hoppe-Seyler; Karplus; R. Cohn: Reduction of nitro to amino compounds. Localization in tissues. Schulz, S. Cohn: Quinone + hydroquinone + sulfate.

-

--

--

E. Glucuronic Acid Conjugation 1846

Erdmann: Euxanthic acid (Indian yellow) is a conjugate with an acid moiety.


18 55 1870 1875

1878

1879

45

Schmid: Hydrolysis of ewanthic acid yields euxanthone and a reducing conjugant. Baeyer: Euxanthic acid conjugating agent is C6H,,0,. Glycoside-like conjugate ? Von Mering, Musculus: Urochloralic acid conjugating agent is acidic, reducing, sugarlike compound with optical activity. JS': o-Nitrobenzyl alcohol conjugated with acidic, reducing, dextrorotatory, nonfementable, oxidized sugar derivative. Schmiedeberg, Meyer: Isolation and identification of the CHO conjugating agent for camphor: (CHOH), COOH, called glucuronic acid.

{

F. Acetic Acid Conjugation 1887

1893

Jaffg, R. Cohn: -

Furfural forms glycine conjugate of furfuracrylic acid, a reaction product with acetic acid (R4H-COOH) R. Cohn: m- and p-Nitrobenzaldehyde excreted as Nacetylaminobenzoic acid. Sequence: aldehyde oxidation, nitro reduction, acetic acid conjugation of amino group. REFERENCES

t 11 121

131 [4 1 I51

[61 [71 [81

F. Lieben, Geschichte d e r physiologischen Chemie, Deuticke, Leipzig, 1935. M. Florkin, "A History of Biochemistry, I t in Comprehensive Biochemistry, Vol. 30 (M. Florkin and E. H. Stotz, eds.), Elsevier, Amsterdam, 1972. Ref. 2, Vol. 3 1 (1975). R. Buchheim, Lehrbuch der Arzneimittellehre, Voss, Leipzig, 1859, p. 19. S. FrBnkel, Die Arzneimittelsynthese auf Grundlage d e r Beziehungen zwischen chemischem Aufbau und Wirkung, Springer, Berlin, 1901, pp. 105-129. More detailed in 3rd ed., 1912, pp. 49-58, 159-206, 817-823. A. Heffter, Ergeb. Physiol., 2, 95 (1903). A. Heffter, Rid., A, 184 (1905). R. T. W i l l i a m , Detoxication Mechanisms, 2nd ed., Chapman & H a l l , London, 1959, pp. 13-22.

CONTI AND BICKEL

46

R. L. Smith and R. T , Williams, "History of the Discovery of the Conjugation Mechanisms, in Metabolic Conjugation and Metabolic Hydrolysis, Vol. 1 (W. H. Fishman, ed.), Academic, New York, 1970, p. 1. T. C. Butler, Ann. N. Y. Acad. Sci., 179, 502 (1971). M. H. Bickel, Marceli Nencki 1847-1901, Huber, Bern 1972, pp. 33-53. T. Thompson, A System of Chemistry, Vol. 4, Edinburgh, 1802, p. 4-47, W. Hallwachs, Ann. Chem. Pharm., 105, 207 (1858). , J. Berzelius, Lehrbuch der Chemie, Vol. 9, 3rd ed., Dresden, 1840, p. 425. A. F. de Fourcroy and L. N. Vauquelin, Ann. Chim. Phys.,

31,

1261 [ 271 [281

63 (1799).

L. Proust, IbiJ., 36, 273 (1801). F. Wohler, Tiedemann's Z . Physiol., 1,142 (1824). J. Liebig, Poggendorff's Ann. Phys. Chem., 11, 389 (1829). 0. L. Erdmann, J. Prakt. Chem., 13,422 (1838). J. A. Dumas, Ann. Chtm. Phys., 57, 3 3 1 (1837). V. Dessaignes, C. R. Acad. Sci. (Paris), 2, 1224 (1845). V. Dessaignes, J. Pharm. (Paris), 32, 14 (1857). A. Ure, Pharm. J. Transact., 1, 24 (1841). C. G. Lehmmn, J. Prakt. Chem., 2, 113 (1835). F. Wohler, quoted by J. Berzelius, Lehrbuch der Chemie, Vol. 4, Dresden, 1831, p. 376. W. Keller, Ann. Chem. Pharm., g, 108 (1842). J. Liebig, 0 , 1 6 1 (1844). W. Kuhne and W. Hallwachs, Virchow's Arch. Pnthol. Anat.,

c.,

12,

386 (1857).

F. Wohler and F. T. Frerichs, Ann. Chem. Pharm.,

65, 335

(1848).

A. Strecker, Ibid., 65, 1, 130 (1848); 67, l ( 1 8 4 8 ) . T. Schwann, duois-Reymond's Arch. Anat. Physiol.,

1844,

127.

N. Blondlot, M6m. SOC.Sci. (Nancy), (1851). A. Strecker, Ann. Chem. Pharm., 70, 149 (1849). F. Hoppe-Seyler, J. Prakt. Chem., 283 (1863). G. Bunge and 0 . Schmiedeberg, Naunyn-Schmiedeberg's Arch. E.q. Pathol. Pharmakol., 6, 233 (1876). G. Meissner and C. U. Shecard, Untersuchungen uber das Entstehen der Hippursiiure im tierischen Organismus, Hannover, 1866. C. Ludwig and A . Schmidt, Arb. Physiol. Anstalt Leipzig, 1868, 1.

e,


47

L. Spengel, "Ueber den chemischen Vorgang bei d e r Bildung d e r Hippursaure im Tierkbrper, 'I Thesis, Bern, 1875. A. Hoffmann, Naunyn-Schmiedeberg's Arch. Exp. Pathol. Pharmaliol., 1, 233 (1877). W. Koch, Pfluger's Arch. Physiol., 2, 64 (1879). C. Binz, Arch. Mikrosk. Anat., 3, 383 (1867). C. Binz, Virchow's Arch. Pathol. Anat., 46, 129 (1869). Scharrenbroich, "Das Chinin als Antiphlogisticum, ' I Thesis, Bonn, 1867. A. Ure, quoted by J. Liebig, Ann. Chem. Pharm., 44, 345 ( 1842). 0. L. Erdmann and R. F. Marchand, B i d . , 44,344 (1842). 0.L. Erdmann and R. F. Marchand, T P r z , Chem., 2, 491 (1842). 0.L. Erdmann and R. F. Marchand, E., 2, 307 (1845). G. Staedeler, Ann. Chem. Pharm., 77, 1 7 (1851). A. Schlieper, E . ,2, l(1846). 0.Schultzen and B. Naunyn, du Bois-Reymond's Arch. Anat. Physiol., - -1867, 349. L. Carius, Am. Chem. Pharm., 148,50 (1868). M. Nencki, du Bois-Reymond's Arch. Anat. Physiol., 1870, 399. A. W. H o h a n n , Ann. Chem. Pharm., 74,342 (1850). C . Kraut, Thesis, Gottingen, 1854. C. Kraut, Ann. Chem. Pharm., 98, 360 (1856). R. Fittig and J. Konig, g., 144,277 (1867). M. Nencki and E. Ziegler, BerrDtsch. Chem. Ges., 5 , 749 (1872). R. Buchheim, Arch. Physiol. Heilkunde, rN.F.1 1, 122 (1857). 0. Jacobsen, Ber. Dtsch. Chem. Ges., 12, 1512 (i879). M. Nencki, Naunyn-Schmiedeberg's Arch. Exp. Pathol. Pharmakol., 1, 420 (1873). 0.Schultzen and C . Graebe, du Bois-Reymond's Arch. Anat. Physiol., 1867, 166. C. Bertagnini, Ann. Chem. Pharm., 78, 100 (1851). M. Jaff6, Ber. Dtsch. Chem. as.,?, 248 (1873). C. Bertagnini, Ann. Chem. Pharm., 97, 248 (1856). F. Beilstein and F. Schlun, 133,239 (1865). C. Berg, "De nonnullarum materiarum in urinam transitu disquisitiones, 'I Thesis, Dorpat, 1858. I. Munk, Pfliiger's Arch. Physiol., 12, 142 (1876). H. Landolt, Ber. Dtsch. Chem. Ges7-l. 770 (1871). M. Nencki and N. Sieber, J. Prakt. Chem., 3, l(1882). M. Nencki and N. Sieber, 2, 498 (1881).

w.,

=.,

48

CONTI AND BICKEL M. Nencki and N. Sieber, PfliigerfsArch. Physiol., 2,319 (1883). 0. Schultzen and L. Riess, Ann. Charig-Krankenh., (Berlin), 15, 57 (1869). Quincke, Virchowfs Arch. Pathol. Anat., 2,537 (1872). A. Schmidt, Ueber Ozon im Blute, Dorpat, 1862; Hiimatologische Studien, Dorpat, 1865. C. F. Schanbein, -Sitzungsber. K. Akad. Wiss. (Munich), 1, 274 (1863). R. Clausius, Poggendorff's Ann. Phys. Chem., 250 (1864). M. Traube, Theorie d e r Fermentwirkungen, Berlin, 1858. C. Binz, Berl. Klin. Wochenschr., 9, 638 (1872). E. P€lLger, Pfliiger's Arch. Physiol., 6, 43 (1872); 10, 251 (1875). F. Hoppe-Seyler, Z. Physiol. Chem., I, 396 (1877). C. K a u h a n n , J. Prakt. Chem., g,79 (1878). M. Nencki and P. Giacosa, Z. Physiol. Chem., 1, 330 (1880). B. Radziszewski, Ann. Chem. Pharm., 203, 305 (1880). A. Buliginsky, Hoppe-Seyler's Med. -Chem. Untersuchungen, -2, 234 (1867). F. Hoppe-Seyler, Pfluger's Arch. Physiol., 5, 470 (1872). G. Staedeler, Ann. Chem. Pharm., 3,295 (1867). E. Baumann, PEuger's Arch. Physiol., G3 (1876). E. Baumann, l2, 69 (1876). E. Baumann, Ber. Dtsch. Chem. Ges., 2, 54 (1876). E. Baumann, Pfliiger's Arch. Physiol., 13, 285 (1876). E. Baumann, Ber. Dtsch. Chem. Ges., 2, 1389 (1876). E. Baumann and E. Herter, 2. Physiol. Chem., 244 (1877 B. Schuchardt, Arch. Pharm., 156, 144 (1861). Sonnenkalb, Anilin und Anilinfaxben, Leipzig, 1864. 0. Schmiedeberg, Naunyn-Schmiedebergfs Arch. E,v.Pathol Pharmakol., 5, 1 (1878). 0. Schmiedeberg, 288 (1881). F. Schaffer, J. Prakt. Chem., 1_8, 282 (1878). E. Tauber, Z. Physiol. Chem., 2, 366 (1878). A. Auerbach, Virchow's Arch.'Pathol. Anat., 7 7 , 226 (1879). E. Sallcowski, g., 409 (1878). E. Sallowski, Pfliiger's Arch. Physiol., 5, 335 (1572). E. Baumann and C . Preusse, -Z . Physiol. Chem., 3, 156 (1879). M. Nencki and P. Giacosa, E.,4, 325 (1880). E. Lautemann, Ann. Chem. Pharm., 125, 9 (1863).

121,

-

-

~

E.,

12,

A,

u., s,

z,


49

P. Mattschersky, Virchow's Arch. Pathol. Anat., 28, 538 (18 63). E. Stadelmann, Naunyn-Schmiedeberg's Arch. Exp. Pathol. Pharmakol. , lo, 317 (1879). W. Forster, Thesis, Breslau, 1900. F. Hupfer, Z. Physiol. Chem., 37, 302 (1903). J. Schmidt, Biochem. Zentralbl., 3, 568 (1905). 0.Schulz, "Untersuchungen uber die Wirking des Chinons und einiger Chinonderivate, Thesis, Rostock, 1892. 11111 S. Cohn, "Ueber das Verhalten des Chinons im tierischen Organismus, '' Thesis, Konigsberg, 1893. F. Hoppe-Seyler, 2. Physiol. Chem., 1, 178 (1882). A. Rymsza, "Ein Beitrag zur Toxikologie der Pikrinsaure, ' I Thesis, Dorpat, 1889. J. P. Karplus, Z . Klin. Med., 22, 210 (1893). N. Sieber and A. Smirnow, Monatsh. Chem., 8, 88 (1887). R. Cohn, Z . Physiol. Chem., 2, 274 (1893). R. Cohn, $, 133 (1894). R. Kobert, Lehrbuch der Pharmakotherapie, Stuttgart, 1897, p. 462. t 1191 0.Liebreich, Das Chloralhydrat, ein neues Hypnoticum und A&theticum, Berlin, 1869. W. F. Loebisch, Die neueren Arzneimiltel, 3rd ed., Vienna, 1888. J. Personne, C. R. Acad. Sci. (Paris), 979 (1869). 0.Hammarsten, Upsala L'dcarefiiren. Forehandl., 5, 424. A. Rajewsky, Centralbl. Med. Wissensch., 5, 211 (1870). E. Kulz, Sitzungsber. Ges. Befiird. Naturwiss. (Marburg), 2, (1872). M. Bouchut, C. R. Acad. Sci. (Paris), E, 966 (1869). A. Tomaszewicz, Pfliiiger's Arch. Physiol., 2, 35 (1874). J. von Mering and F. MUBCU~US, Ber. Dtsch. Chem. Ges., 8, 662 (1875). C. P. Falck, Dtsch. Z . Prakt. Med., 23, 64 (1877). E. Kiilz, Centralbl. Med. Wissensch., 19, 337 (1881). 0.Schmiedeberg and H. Meyer, Z. Physiol. Chem., 3, 422 (1879). E. Kulz, Pfluger's Arch. Physiol., 28, 506 (1882). K. Garzar-CheG. Pharm., 210, 63 (1881). vohMering, Ber. Dtsch. Chem. Ges., 15, 1019 (1882). J. von Mering, Z. Physiol. Chem., 6, 480 (1882). E. Kulz, 2. Biol., 2, 157 (1884).

w.,

e,

i.

CONTI AND BICKEL

50

0. L. Erdmann, J . Prakt. Chem., 33, 190 (1844); 3 7 , 385 (1846). W. Schmid, Ann. Chem. Phann., 3, 83 (1855). A. Baeyer, E d . , 155, 257 (1870). C. A. Ewald, Centralbl. Med. Wissensch., Ll, 819 (1873); Jahresber. Thierchem., 3, 313 (1873). J. von Mering, Centralbl. Med. Wissemch., 13,945 (1875); Jahresber. Thierchem., 5, 61 (1875). M. Jaff6, Z. Physiol. Chem., 2, 47 (1878). C Wiedemann, Naunyn-Schmiezeberg's Arch. Exp. Pathol. Pharmakol., 6, 216 (1877). M. Jaffe'andR. C o b , Ber. Dtsch. Chem. Ges., 2311 (1887). A. Baeyer, B i d . , 10, 355 (1877).

.

z,

- -

GI Research Path: From Academia to the Pharmaceutical Industry.

industrial chemicals.

[Key points in clinical pharmaceutical education and research, as seen from pharmaceutical consultation clinics].

Future pharmaceutical research: the need to look beyond science.

The transferability from rat subacute 4-week oral toxicity study to translational research exemplified by two pharmaceutical immunosuppressants and two environmental pollutants with immunomodulating properties.

Approaches to modelling the human immune response in transition of candidates from research to development.

Proteomics in Pharmaceutical Research and Development.

In vivo NMR in pharmaceutical research.

Incorrect conclusions about unpublished pharmaceutical research.

Proteomics in pharmaceutical research and development.

Pharmaceutical research and the world's health.

Three propositions for improved science communication from environmental research.

A transition in fetal alcohol syndrome research: the shift from animal modeling to human intervention.

From subjects to experts--on the current transition of patient participation in research.

Nutrition and physical activity during the transition from adolescence to adulthood: further research is warranted.

Environmental impact assessment of pharmaceutical prescriptions: Does location matter?

Hidden assumptions in environmental research.

Environmental health research and regulation.

Transition from haploidy to diploidy.

Current Research and Opportunities to Address Environmental Asbestos Exposures.

Elementary School Children Contribute to Environmental Research as Citizen Scientists.

Community-based approaches to environmental health research around the globe.

The health care transition research consortium health care transition model: a framework for research and practice.

Cardiovascular drug discovery: a perspective from a research-based pharmaceutical company.