P A R T I11

ABSORPTION, EXCRETION, ACETYLATION AND DISTRIBUTION

I N MAN

C H A P T E R 1 0

Previous Investiga,tions

T t is obvious that a rational system for the administration of different sulfanilamide derivatives cannot be developed without accurate knowledge concerning the absorption, excretion and dis- tribution of each separate sulfanilamide compound. Not until 1937, when adequate methods for the quantitative determination of sulfanilamide and its derivatives were worked out (209, 98), was i t possible to gain information on these points. Since then a large amount of research has been done in this field, both in animals and in man.

Marsliall et al. (209, 210, 208, 207) showed how sulfanilamide was absorbed and eliminated. Their observations were confirmed and extended by several authors, mainly Long and Bliss (177), and Stewart and his co-workers (295). Sulfanilamide administered per 0s was found to be rapidly absorbed, the blood concentr a t' ion reaching its maximum within two or three hours and then sinking gradually to zero twenty-four hours later. The compound diffuses out easily, occurring in all the tissues of the body in practically the same concentration as in the blood. About 10 to 20 per cent of the sulfanilamide in the blood occurs in conjugated (acetylated) form. The excretion takes place almost exclusively through the kidneys, about half in free form and half in acetylated form; the rate of excretion depends not only on the concentration in the blood but also on the degree of the diuresis; 90 to 95 per cent of the given amount is recoverable in the urine.

EARLIER WORK 71

Marshall et al. (209) showed that, in the dog, sulfanilamide was excreted in unchanged form in the urine, while in other mammals it was excreted partly in the free form and partly as a conjugated compound. The conjugated compound was shown to be N*-acetylsulfanilamide in man and in the rabbit (208).

The other sulfanilamide derivatives examined have also been shown to occur as a free and a conjugated compound in the blood and urine of humans and other mammals. I n the case of a few substances, like sulfapyridine (297) sulfathiazole (306), and sulfaguanidine (206), the conjugated compounds have been iden- tified as the N*-acetyl derivatives. Harris and Klein (115) showed in in vitro experiments that when fresh slices of rabbit liver tissue were immersed in sulfanilamide solutions some of the sulfanilamide changed to the acetyl form. Stewait et al. (296) demonstrated by experimenting on the rabbit that the acetylation took place in the liver. Van Winkle and Cutting (307) confirmed this fact in experiments on cats and showed that in the case of sulfapyridine extrahepatic acetylation may also occur.

Using deuterium as an index, Bernhard (14 a) showed that orally administered acetic acid participates in the acetylation of the amino group in sulfanilamide as well as in p-aminobenzoic acid. Bernhard and Steinhauser (14 b) showed later that amino acids foreign to the body are acetylated in a similar manner. They stated that the acetylation takes place directly with acetic acid to a great part, and consequently that acetic acid in the food or formed during the metabolism, from pyruvic acid for example, participates to a large extent in the acetylation of the amino group in the benzene ring and also in the acetylation of the amino group in amino acids foreign to the body. Du Vigneud et al. (56 a) concluded, aster a study of the acetylation of l-phenyl- aminobutyric acid by means of the isotopes of nitrogen and hydrogen, that when direct acetylation comes into question the possibility of acetic acid being the acetylating agent cannot be ruled out, even though its source may be pyruvic acid.

James (130 a) found that mice receiving sodium acetate along with sulfanilamide excreted more acetyl derivative than mice receiving the same dosage of sulfanilamide alone. The simul- taneous administration of the acetate also reduced the acute toxicity. The interpretation that acetylation is a detoxicating mechanism is hard to reconcile with the observations (208, 130 a)

72 ABSORPTION, EXCRETION, DISTRIBUTION

that the acetyl compounds are decidedly more toxic than the parent drugs. James (130 b) came t o the conclusion after further experimentation that the toxicity of sulfanilamide and sulfa- pyridine was due to the withdrawal of acetate precursors and to lowering of the carbon dioxide capacity of the blood. Acetate given at the same time rectifies this defect. He also observed a fall in blood sugar when sulfanilamides were given. This was due, he said, to glucose being an acetate precursor; when acetate was given the fall was prevented.

Xcudi et al. (277) observed an increase in the excretion of glycuronic acid during sulfapyridine medication and calculated that up to 40 per cent of the free form of sulfapyridine excreted into the urine was coupled with glycuronic acid. If glycuronic acid is administered simultaneously with the sulfanilamide, i t prevents the conjugation of sulfanilamide (214). No inhibition is caused by ascorbic acid, glycine or cystine.

The absorption and excretion of sulfapyridine in man was first described by Long et al. (180) and Baines and Wien (6). Their observations were verified on the whole by several authors (297, 265, 236, 27, 85, 303, 243, 147, 90, 91, 301) and were amplified and elucidated by Taylor et al. (303, 301) and myself (90, 91). It was shown that sulfapyridine given per 0s is absorbed less and more slowly than sulfanilamide. The blood concentration reaches its maximum within four to five hours and then decreases slowly. The drug passes easily into the tissues of the body, where it occurs in practically the same concentration as in the blood. Large amounts of acetylsulfapyridine occur in the blood and urine, the quantity varying greatly from case to case, but as a rule 30 per cent of the total sulfapyridine occurs in the conjugated form in the blood. The compound is more slowly excreted through the kidneys than sulfanilamide. As a rule, 60 to 70 per cent of the administered dose is found in the urine.

At about the same time, and independently of one another, Reinhold and his co-workers (244), Long et al. (181), Gsell (110) and I (86) described the absorption and excretion of sul- fathiazole in man. Our results were soon verified (281, 282, 8, 229) and complemented in later publications (256, 90, 91, 301). Administered by mouth, sulfathiazole is more quickly and com- pletely absorbed than sulfapyridine, resembling sulfanilamide in this respect. The blood concentration reaches its maximum within two to four hours and then sinks more rapidly than in the

EARLIER WORK 73

case of sulfapyridine. The compound diffuses freely over into the pleura and ascitic fluid while the amount in the cerebrospinal fluid equals only about 25 per cent of the simultaneous amount in the blood. I (91) and Strauss et al. (301) demonstrated, indepen- dently of each other, the unequal distribution of sulfathiazole between red cells and plasma, the concentration being much higher in the latter. The drug is less acetylated both in the blood and urine than sulfapyridine, about 20 per cent occurring as acetyl- sulfathiazole in the blood. It is more rapidly excreted through the kidneys than sulfapyridine, 80 to 90 per cent of the adminis- tered dose being found in the urine.

The absorption and excretion of sulfamethylthiazole in man was described simultaneously by myself (90, 91) and Strauss et al. (301). Our observations were confirmed by Nissen and his co- workers (28, 229, 65) and others. Sulfamethylthiazole given by mouth is absorbed more slowly than sulfathiazole but more rapidly than sulfapyridine. The blood concentration reaches its maximum in three to four hours and then drops slowly. Sulfamethylthiazole is distributed almost exclusively to the plasma. It is more acety- lated than sulfathiazole but less than sulfapyridine. It is excreted through the kidneys a t about the same rate as sulfapyridine. Fifty to 70 per cent of the given dose may be recovered in the urine.

Marshall et al. (206) and later I myself (94) showed that sul- faguanidine given by mouth is absorbed from the human gas- trointestinal tract to a considerably lesser extent than sulfapyri- dine and sulfathiazole. Only 15 to 53 per cent of the dose given is recovered from the urine. Sulfaguanidine is fairly rapidly absorbed, maximum blood levels being attained within two to four hours. The compound diffuses over easily into the tissues and fluids of the body, where, except for the cerebrospinal fluid, it occurs in nearly the same concentration as in the blood. It is acetylated to about the same extent as sulfapyridine, about 30 to 40 per cent being present as conjugated compound in the blood. It is rapidly eliminated with the urine. My (94) results indicated that sulfa- guanidine is more rapidly excreted than either sulfanilamide or sulfathiazole.

Vonkennel, Kimmig and Korth (310, 309) showed that sul- famethylthiodiazole is rapidly absorbed and excreted in man, a fact confirmed by Andersen et al. ( 3 ) and myself (96). After oral administration it reaches its highest level in the blood within two hours, and then decreases rapidly, only a trace remaining

6-424084

’ 74 ABSORPTION, EXCRETION, DISTRIBUTION

eight hours later (96). In the blood, it is present almost solely in the plasma and a small amount passes into the cerebrospinal fluid (96). It is only slightly acetylated. It is rapidly excreted through the kidneys, being almost entirely eliminated in ten hours. Ninety-three to 101 per cent of the given dose may be recovered from the urine.

Sulfaethylthiodiazole is fairly rapidly absorbed but i t is excre- ted slowly (310, 309, 145). Like sulfamethylthiodiazole, i t is only slightly acetylated.

The absorption and excretion of sulfapyrimidine in man was first described by Plummer and Ensworth (235). The results of these authors were substantiated and extended by other authors (245, 234, 257). After oral administration, sulfapyriniidinc is fairly slowly absorbed; the blood level attains its maximum after four to six hours, and then drops slowly. It diffuses rapidly over into all the tissues of the body, where it is distributed like sulfapyridine. Ten to 20 per cent occurs in the acetylated form in the blood, and 30 to 40 per cent in the urine. It is excreted slowly through the kidneys. About 75 per cent of the dose atliiiinistered may be recovered from the urine.

Schmid and Sauberman (260) found that N1-dimethylacrylyl- sulfanilamide, given by mouth, was relatively slowly absorbed, the blood concentration attaining its maximum after three or four hours. It was also slowly eliminated through the kidneys, and about 60 per cent of the given dose could be recovered from the urine.

Much of the data just given is based on perfunctory and inade- quate research. The plan of the experiments and likewise the sizes of the doses administered vary from author to author. It is not possible, therefore, to compare the absorption, excretion and distribution of a large number of sulfanilamide derivatives on the basis of previous studies, and so far no author has made such a comparison on the basis of his own results.

In the experimental part of this communication, the decisive importance of the blood and tissue concentration of the drug to the end therapeutic results was pointed out. The concentration in the blood is deterniined entirely by how the drug is absorbed, excreted and distributed in the body. Only on the basis of thorough and comparative studies on these conditions, can a rational plan of dosage be worked out for each sulfanilamide derivative.

In the present investigation, the absorption, excretion, acetyla-

METHODS 75

tion and distribution in m a n of sulfanilamide a n d nine of its N1-derivatives were examined in a uniform manner for all t h e compounds and t h e results t hen compared. The results i n t h e case of a few of t h e substances have already been briefly reported (85, 86, 90, 91, 94, 96).

C H A P T E R 1 1

Experillien tnl Procedure

Methods for Deterniinntion of Siilhnil.zmide Drugs Most methods used for the determination of sulfanilaniide and its

derivatives are based on the presence of a free, primary amino group in the 1)-position. The amino group is diazotized and the diazo com- pound formed coupled with an aromatic amine, producing an azo dye which is then mrasurwl colorirnctrirally. Marshall and his co-workers (209) introduccd this principle for the determination of sulfanilaniide derivatives and worked out a mcthoci on its basis. Other procedures (273, 261, 155) have been described, but none of them have proved satisfactory (64).

A whole series of different methods based on the diazotization prin- ciple of Marshall have bcen described (98, 238, 143, 118, 151, 279, 85, fj4, 56). Most of them are niodifications of Marshall’s method. Mar- shall and his ro-workers have made repeated improvements on their method (200, 201, 211) and it was finally perfected by Bratton and Marshall (26). Eldahl, Joensen and Vehrmehren (64) recently made a critical analysis of the different methods and described a micromethod of their own.

The Method Employed I have used my modification of Marshall’s method (85). This modi-

fication has been tested and employed by other authors (28, 229, 112).

SoIutions required 1 ) *4 solution of trichloracetic acid containing 15 Gm. dissolved in water and

diluted to 100 cc. 2 ) An aqueous solution of sodium nitrite (Schering-Kahlbaum) containing one

p u n p r hundrcd cubic centimeters. 3) An nqnrous solution of sulfamic acid (Schcring-Kahlhaum), containing

0.2 Bm. pcr hundred cubic centimeters. 1) An aqueous solution of N-ethyl-2-nuphthylamine hydrochloride containing

0.j Qm. per hundred cubic centimeters. Thls solution should be kept in a dark bottle.

5 ) An aqueous solution of saponin (Schcring-ICahlbaum) containing 0.5 Gm. per liter.

6) A four times normal solution of hydrochloric acid. 7) Methanol.