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logical activity. The manner in which Triscoline is handled by the kidney was
studied in collaboration with Drs. R. W, Berliner and J. Orlo'ff (Laboratory of
Kidney and Electrolyte Metabolism) , and it was found to be secreted by kidney
tubules at the same rate as p-amino-hippuric acid but by a different mechan-
ism. Further studies of the manner in which the kidney excretes basic organic
compounds will be carried on.

Localization in Tissues. It is becoming evident that few drugs would
exert a significant effect were it not for the storage depots of the body.
Most drugs with a long duration of action are characterized by extensive and
reversible localization in some tissue depot. This protects drugs by with-
drawing them from body fluids and thus slowing their rates of biotransforma-
tion and urinary excretion.

Drugs are localized in a number of ways. One way is by deposition in
body fat. We found that the Dibenamine type drugs, agents of potential use in
hypertension, have a long duration of action due to their extensive localiza-
tion in fat from which they are only slowly released, Acetylmethadol, an
analgesic agent which produces an effect of considerable duration, owes this
property to its extensive fat localization. This drug is said to be too toxic
for human use, but it has provided a clue toward the development of a long
lasting analgesic. The intravenous barbiturates such as Pentothal and Evipal
metabolize slowly and are short acting not because they are rapidly metabo-
lized, as thought, but because they have a high affinity for fat. They
rapidly diffuse from brain and other tissues into body fat. This explains
their usefulness in operative procedures of short duration, but their limited
applicability in operative procedures of long duration. These findings have
led to a more rational use of Pentothal as well as directing attention toward
the need for a more unstable intravenous anesthetic.

Fui'ther work is designed to define other depots, probably protein in
nature, which localize drugs. In particular. Dr. G. M. Tomkins is studying
reversible combination between nucleoproteins and certain organic bases such
as the antimalarial drug atabrine, which accounts for the extensive tissue
localization of these compounds.

Metabolic Transformation. The duration of action of a drug is limited
by its stability in the body. Little is known about the relationship between
the structure of drugs and their stability in the body or about the biochemi-
cal mechanisms which inactivate them. However, it is becoming more and more
apparent that drug biotransformations can be described in terms of a rela-
tively few biochemical mechanisms. We have studied two of these in some
detail.



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(1) Dealkylatlon. Many drugs are alkylamines . We have fouad that the
body almost invariably detaches the alkyl groups from the nitrogen atom; for
instance, with ephedrine, aminopyrine, methylamphetamine, Regitine, and
Demerol. The biochemical system responsible is in liver and has been par-
tially purified. Preliminary results suggest that a common enzyme is involved
whether the dealkylation involves a methyl^ ethyl, or butyl group.

SKF, a relatively nontoxic substance with little or no pharmacological
action, was found to inhibit the dealkylation system both in vitro and in the
intact animal. It also inhibits the metabolism of various barbiturates
which are inactivated by oxidation of the side chain. This suggests that
dealkylation is also an oxidative mechanism. Pharmacologically, the action of
SKF is reflected in a dramatic prolongation of the pharmacological effects of
various narcotic analgesics (alkylamines) and barbiturates when animals are
pretreated with the compound. It is planned to purify the dealkylation and
side-chain oxidation systems and to use SKF as a tool in unraveling their com-
mon characteristics and their normal role in the body economy.

(2) Oxidation of Aromatic Compounds. A large number of aromatic drugs,
e.g., acetanillde, aniline, theophylline, salicylic acid, and amphetamine, are
oxidized in the body to the corresponding phenols. We have found that a model
system consisting of ascorbic acid and Fe++ also oxidizes these drugs to yield
the identical products formed in the body. Presumably the model system oper-
ates by introducing positively charged hydroxyl (OH"^) groups into the aromatic
ring. It is probable that a similar type of reaction occurs in vivo, i.e.,
what has been termed oxidation is in reality the substitution of a hydroxyl
group for a hydrogen on the benzene ring. We have shown that ascorbic acid
also plays a role in this reaction in vivo, since guinea pigs depleted of as-
corbic acid have an impaired ability to oxidize drugs. Ascorbic acid also
plays some role in the metabolism of other types of drugs, since rats given'
chloretone and barbiturates tremendously increase their synthesis and excre-
tion of the vitamin. Nothing is known about the chemical role of ascorbic
acid in vivo, and this work provides a wedge in this direction.

Although drugs are hydroxylated by a nonspecific biochemical mechanism,
there appears to exist more specific mechanisms for the hydroxylation of nor-
mally occurring substrates. For instance, phenylanine and tryptophan are
oxidized to phenols by quite specific hydroxylation mechanisms. These new
types of enzymes are being exhaustively studied to find out how the body in-
troduces hydroxyl groups.

Our work has turned up a marked species variation in the biotransfor-
mation of drugs which is both q.ualltative and quantitative. Ordinarily,
potential therapeutic agents are first screened for activity and toxicity in
animals. Species variation makes it difficult to carry over results into man.
A comparative study of biochemical mechanisms which inactivate drugs has been
undertaken. The results of this study should aid in the choice of suitable
animals for screening the activity and toxicity of various classes of drugs.
Parallel studies will evaluate the influence of age and sex on drug
metabolism.

Development of New Compounds for Pharmacological and Clinical Studies.
Investigators: John Burns, Murray Weiner, Arthur Schulert, Bert La Due and
Bernard B. Brodie.



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Anticoagulants. In an attempt to develop a synthetic substitute for
Heparin, a number of sulfuric acid and sulfonic acid esters, mainly dyes,
were tested In vitro for their effect on the clotting mechanism. Active com-
pounds were found but none of them had appreciable activity in the intact
animal. We are still looking for a Dicumarol type drug, less capricious in
its biological behavior. Dr. H. B. Wood and Dr. E. C. Horning (Laboratory of
Chemistry of Natural Products) have synthesized some anticoagulants of the
Dicumarol type. An active compound is being studied.

Barbiturates. Pentothal is too stable in the body to be short-acting
after sustained administration. In collaboration with E. R. Sq.uibb, we have
developed Rapidal, an intravenous anesthetic. For comparable depth of
anesthesia, subjects awake sooner and with considerably less postanesthetic
somnolence than after Pentothal. This product is now being given extensive
clinical trial.

In collaboration with Eli Lilly, the potentialities of the N-alkyl
thiobarbiturates are being investigated. Preliminary studies with these com-
pounds show that the recovery time after large doses is considerably shorter
and postoperative somnolence much less than following Pentothal.

Drug potentiating agents. In collaboration with Smith, Kline, and
French, and Dr. Wood and Dr. Horning, derivatives of SKF are being screened.
This type of compound while almost devoid of pharmacological activity, mark-
edly prolongs the action of analgesics and anticonvulsants by blocking their
metabolic transformation. These derivatives will also be tested against
malaria, since SKF shows some antimalarial activity.

Tyrosine Oxidation. Investigator: Bert La Du.

Further studies on the intermediary metabolism of tyrosine have out-
lined the intermediates in its breakdown to fumaric and acetoacetic acids.
Partial purification of the enzyme system involved iii one of the steps, the
oxidation of p-hydroxphenylpyruvic acid, has been obtained. This reaction
appears to require ascorbic acid. This may be in^iortant since a definite
chemical role for ascorbic acid has not been tied down. Further purifica-
tion of this enzyme system is in progress.

Biological Significance of Choline. Investigators: Bert La Due and
Bernard B. Brodle.

Difficulties met in the method for estimating free choline in plaama
have finally been resolved. The procedure is being applied to studies of
the metabolism and synthesis of choline in vivo . There appears to be a
mechanism for maintaining constant the plaema choline level. Even after
large intravenous or oral doses of choline to animals the plasma choline
concentration rapidly returns to the pretreatment level. After the injec-
tion of choline labelled with C-'-^ in the ethanol moiety, only a trace of
radioactivity appears in urine and only a fraction in the respired air in
2k hours. This indicates that the choline is probably transformed to rela-
tively stable compounds. The nature of these products is being investigated.

Biogenesis of Hormones. Investigators: Elwood Titus, Sidney
Udenfriend, Carroll Clark, and Jack Cooper.



- 52 -

A prerequisite to an over- all study of cardiovascular disease is
knowledge of the mechanisms by which the hody elaborates the hormones which
mediate the activities of the cardiovascular system. It is obvious that
physiological factors or chemical agents affecting the formation of these
substances will markedly alter physiological function. The purpose of this
prdgram is to determine which precursors the body uses in the synthesis of
three such agents, adrenaline, noradrenaline, and serotonin, and to study
the enzymes involved in the synthetic reactions.

We have found that adrenaline and noradrenaline is formed from
phenylalanine, which via tyrosine, goes through a series of chemical changes
to yield the adrenalines. Normally, the turn-over of adrenaline in the
adrenal gland is extraordinarily slow. One of the intermediate enzymes,
dihydroxyphenylalanine decarboxylase, has been considerably purified. Work
is continuing to establish the other compounds and enzymes in the chain of
synthetic reactions leading to adrenaline.

The formation of serotonin from tryptophan has been demonstrated to
occur by way of a new metabolic pathway involving the formation of a hitherto
unknown amino acid, 5-hydroxytryptophan. An enzyme catalyzing the decarboxyl-
ation of the latter compound to serotonin has been isolated from kidney in
partially purified form. Other enzymes involved in the synthesis will be
studied, and the fate of serotonin in the body will be followed.

Studies with Butazolidln, a Non-Steroid Anti- Inflammatory Agent.
Investigators: John Burns, Murray Weiner, Theodore Chenkin and Bernard B.
Brodie. In collaboration with Dr. E. Lowman, Institute for Rehabilitation,
and Dr. J. M. Steele, Goldwater Memorial Hospital.

The implications of a simple, nonsteroidal compound for the treatment
of rheumatoid arthritis, gout and other inflammatory diseases are profound
and for this reason Butazolidln has merited a detailed study. In patients
with severe rheumatoid arthritis with evidence of joint inflammation, etc.,
we found the drug exerted definite anti-inflammatory effects and increase in
joint motion comparable to cortisone. In patients in whom the drug had no
effect (i.e., analgesia only) cortisone also usually elicited no effect.
Butazolidln, as does cortisone, causes a marked retention of sodium, chloride,
and water. It also causes some retention of potassium, whereas cortisone and
ACTH cause an excretion of this ion. Unlike cortisone and ACTH, Butazolidln
does not affect the concentration of sodium and potassium in saliva. These
results suggest a different mechanism in its effect on electrolyte excretion.
Unlike ACTH, Butazolidln does not increase excretion of ketosteroids, depress
circulatory eosinophils, or affect carbohydrate metabolism. This suggests
that Butazolidln does not exert its effects through the pituitary- adrenal
axis. Butazolidln has a number of side-effects causing discontinuance of
therapy in 25 percent of patients. Certain of these are similar to those
produced by cortisone, such as edema, congestive heart failure, and reactiva-
tion of peptic ulcers. Other side effects axe nausea, vomiting, and derma-
titis. Unlike cortisone or ACTH, it does not produce hormonal effects.

The chemical fate of the drug has been studied and several metabolites
isolated. These will be tested to assay the part they play in the activity
and toxicity of the drug.



- 53 -

Butazolidin is probably a landmark in medicine since it is the first
nonsteroidal compound to have definite cortisone- like activity in a number of
inflammatory diseases. It is not the final answer, however, as a compound
with less side effects is needed. We have instituted a collaborative effort
with Geigy Pharmaceuticals, the National Institute of Arthritis and Metabolic
Diseases (Dr. J. J. Bunim) , and Goldwater Memorial Hospital (Dr. J. M. Steele)
to develop a better anti-inflammatory agent.

Laboratory of Chemistry of Natural Products

Evan C. Horning is chief of this laboratory. Research projects include
studies on the isolation and identification of materials of biochemical inter-
est from natural sources, and fundamental studies on the structure and reac-
tions of organic compounds related to life processes. Joint projects with the
Laboratory of Chemical Pharmacology involve the study of drug action and drug
metabolism through the synthesis of active agents or metabolic products.

Investigations of Physiologically Active Material from Plant Sources.
Study of plant materials affecting the cardiovascular system is in progress.
This work is concerned with a search for physiologically active agents, which
are usually alkaloids or glycosides, but which may also fall into other chemi-
cal classifications, and it involves the isolation of active agents and the
determination of their structure. Pharmacological and, in some cases, clini-
cal, studies are correlated with the chemical studies. The work is being
carried on with the cooperation of the Bureau of Plant Industry of the U. S.
Department of Agriculture and with the pharniacological and clinicsil labora-
tories of the National Heart Institute. The materials selected for study are
those for which physiological activity has been reported, or which are botani-
cally related t'o plant materials of reported ax:tlvlty.

Reports of the influence of plant materials on the cardiovascular sys-
tem are frequently incomplete and conflicting. It is particularly difficult
to evaluate reports of hypotensive activity, yet this is one of the most im-
portant areas of work in this field. Crataegus oxyacantha has been used in
the United States and In Europe in extract form as a hypotensive agent, but a
clinical investigation under Dr. Luther Terry (Clinic of General Medicine and
Experimental Therapeutics) showed no evidence of activity and chemical studies
were therefore not undertaken. Currently, several other materials which are
reported to depress blood pressure or to have adrenolytic activity are under
investigation. A new crystalline alkaloid has been isolated from Vinca minor ,
a common trailing evergreen vine, as a consequence of old reports that Vinca
species contain alkaloids which depress blood pressure. A method of isolation
has been developed for the alkaloid, and its structure is studied by Dr. S. M
Goodwin. It has been-knt5wn for many years that plants of the Ericaceae fam-
ily, including the mountain laurel and native rhododendron species, contain a
potent active principle which affects the cardiovascular system. Previous
reports on the structure and pharmacological activity of the active agent are

^"^""^^l f'.^""^ ^^^ ^^^""^ ''^ ^"""^^ °'^ly ^s ^^ i"?'"^^ material. From sample
of Phododendron maximum obtained in Virginia, Dr. H. B. Wood Isolated for the
Itrrh^^^«? T^ crystalline glycoside which is active in very small amounts.
Its chemical structure is under investigation, and larger samples are beins
?^T ^°Lf n""'"^ pharmacological evaluation. Plants of the Amaryllis
family are widely distributed over the world, but only two sub-groups, one

iZToll: C Tit f r/'^f .r'^^ '° ^^'^^^ ^°^*^^- appreciSle'l^ounts of
alkaloids The alkaloids of the Japanese species show a relatively low order
Of physiological activity, but those from the African species aremuch morf



- 3k -
potent. Boophane distlchia , the "gif bol" of South Africa, was once used in
the preparation of arrow poisons, and it is a member of the Amaryllis family.
It has been found by Dr. V. L. Stromberg that there is a major new component
in the alkaloid fraction of the African species which is not present in the
Japanese species, and work is in process on the isolation of this component.
Studies on the structure of the alkaloids gelsemlne, aspidosperimine, and
pereirine were carried out by Dr. Patrick. Other alkaloid fractions which
. have been isolated, but about which very little is known at present, include
those from Cephalotaxus sp. and from Cassis marginata . In all of these cases
the pharmacological examination is not complete. The Cephalotaxus problem
is under investigation by Dr. H. Lloyd.

In general, these problems require the testing, by chemical or pharma-
cological methods, of plant materials or plant extracts. The next step is
the isolation of a sufficient quantity of the active agent or agents for chemi-
cal study and for more detailed pharmacological study. Intensive chemical work
is then usually required to determine the structure of the active compound. At
the present time the preliminary chemical examinations are being carried out
under the direction of Dr. V. L. Stromberg, and the isolation work for the
group is under the direction of Dr. M. S. Fish.

These investigations will be continued and additional problems will be
brought under intensive study. The materials which will be examined include
Ornithogalum thyrsoides (South Africa), Picrasma crenata (Brazil), Urginea
altissima (South Africa) emd several genera in the Ameryllidaceae, Apocyana-
ceae, and Liliaceae families.

Studies on the Structure and Properties of Molecular Complexes.
Molecular coB^slexes have for many years been one of the least well understood
groups of organic chemical materials. These substsinces in some cases are
relatively labile combinations existing in the solid state, but which are dis-
sociated in solution; in other cases the complex structure exists in the
liquid phase but not in the solid phase, and in some cases the stability of a
complex may approach that of a compound, and may show no evidence of dissocia-
tion in the solid state or in solution. The importance of these substances
lies in the fact that they often may be intermediates in irreversible chemical
reactions, with a consequent lowering of the activation energy.

The most recent and notable development in this field was the announce-
ment last year of a new quantum-mechanical theory of structure for charge-
transfer complexes by Dr. R. S. Mulliken of the University of Chicago. At the
same time in this Laboratory an investigation of one class of charge-transfer
complexes, those related to quinhydrone, was under way. This class was chosen
for study because of its relationship to energy- transfer systems in biological
reactions in which one-electron oxidation reduction is known to occur. This
work was carried out by Dr. Stromberg, with contributions by Dr. H. Lloyd,
Dr. M. S. Fish, and Dr. G. N. Walker. Several new conclusions about the
structure of a number of organic conipounds emerged from this work, but perhaps
the most significant result from a biochemical point of view is the conclusion
that the intermediate semiquinone in one-electron oxidation-reduction is a
molecular complex.

Studies of Drug Metabolism. The project on intravenous anesthetics
under Dr. Brodle indicated that Pentothail is converted into carboxylic acid on
oxidation in humans. The proposed structure for the metabolite was confirmed



- 55 -

by its synthesis by Dr. H. B. Wood. Further contributions to the study of
barbiturates were made by Dr. Lloyd and Dr. Stromberg. The project on the
metabolism of anticoagulants of the Tromexan and Dicoumarol type was aided by
Dr. Wood, and a new active anticoagulant was prepared for study.

Investigations of the Structure and Reactions of Organic Compounds.
These studies were primarily concerned with aspects of fundamental organic
chemistry, where the compounds and the reactions were of interest because of
biological implications. Investigations were carried out of the reactions of
polyphosphoric acid, a linear plymeric anhydride related in structure to the
polyphosphate chain frequently found in biological systems. A new method for
accomplishing the Beckmann rearrangement, together with new information about
these reactions, resulted from the work of Dr. Stromberg and Dr. Lloyd.
Dr. Kissman developed a new indole synthesis, and Dr. G. N. Walker studied
the cycllzation of benzylsuccinic acids. The synthesis and rearrangement of
quinols was studied by Dr. Goodwin in order to substantiate the mechanism of
the rearrangement which occurs during the metabolic transformation of
tyroside to homogentisic acid. An extensive investigation of ozonides and
peroxy conqpounds was carried out by Dr. Witkop, Dr. Goodwin, Dr. Kissman, and
Dr. Patrick. The compounds investigated were oxidation products of anils,
phenylhydrozones, alloxan, various indoles, and derivatives of maleic and
fumaric acids.

Synthesis of Compounds of Biological Interest. A cooperative program
between Dr. B. Witkop and A. Ek resulted in the synthesis of a number of 5-
and 7-liydroxytryptamlnes, including serotonin (5-hydrocytryptamlne) .

A synthesis of compounds related to podophyllotoxin was undertaken by
Dr. G. N. Walker, and the desired products containing the entire carbon-
oxygen skeleton of podophyllotoxin were obtained.

Laboratories of Cellular Physiology and Metabolism (Combined Report)

Activities of these two laboratories (Christian B. Anfinsen, Chief)
are considered in this report as a single, loosely integrated unit, as the
small size of the Metabolism group relative to that of the Laboratory of
Cellular Physiolosr and of other laboratories in the Heart Institute neither
requires nor justifies separate summary. The increasing research effort as
well as the considerably augmented staff and Clinical Center housing plans
of the Laboratory of Metabolism will, however, in the coming few months, lead
to administrative and spatial separation of the combined groups.

The research activities of the Laboratory of Metabolism are primarily
concerned with basic biochemical and physiological studies on metabolic
diseases and diseases involving metabolic disorders (e.g., atherosclerosis,
diabetes, nephrosis, thyroid disease, etc.). Direct clinical application is,
of course, impossible at present because of the unavailability of convenient
hospital facilities. It is planned to expajid the pjresent efforts in such a
direction in the new Clinical Center quarters, and professional personnel
here and arriving later have been recruited with such expansion in mind.

The staff and facilities of the Laboratory of Cellular Physiology, on
the other hand, constitute an ideal counterpart to the former group since
problems of a particularly technical or biochemically involved sort can be
studi.ed collaboratively with full advantage being taken of the specialized



- 56 -

instrumentation and training of the Cellular Physiology group. The latter
group is primarily interested in the fundamental biochemical mechanisms of
cell metaholism with emphasis on energy production and utilization of such
energy for biosynthetic processes.

Over-all research in both laboratories is primarily concerned with a
limited number of major biological phenomena, but a flux of exploratory work
is constantly in progress for the purpose of delimiting new and possibly
important areas for study, and for the examination of side developments of
major programs which appear to merit special attention.

Laboratory of Metabolism

Studies on the Metabolic Defect in Atherosclerosis. Professional
staff: R. K. Brown, Edwin Boyle, Joseph Bradgon, Howard Eder, and C. B.
Anfinsen.

Earlier studies by Gofman and his co-workers at the University of



Online LibraryNational Institutes of Health (U.S.)Annual report : National Institutes of Health (Volume 1952) → online text (page 7 of 29)