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m5 NATR-EURETIC Aswan 0F DT~327

Thesis fey the Degree of M. 8.,
MICHIGAN STATE UNWERSWY’
BERNARD TERRY
1968

(“gin

 

ABSTRACT
THE NATRIURETIC ACTION OF DT-327
(N-[2', 6' - dimethyl piperidyl - (1')]
3 - sulfamyl - 4 - chlorobenzamide)

by Bernard Terry

DT—327 is a new natriuretic-diuretic agent whose
chemical structure is related to the benzothiadiazines and
furosemide. The nature of the chemical structure of
DT-327 was the stimulus to compare its natriuretic action
with that of furosemide and the thiazides. The
natriuretic activity of this drug was studied following
intravenous administration in the anesthetized dog.

DT-327 produced a prompt increase in urine flow and
excretion of sodium, potassium and chloride following
intravenous administration with no significant alteration
in inulin or PAH clearance. Marked diuresis continued
for at least 90 minutes following a single intravenous
injection of l mg/Kg. When increasing doses of DT-327
were administered during mannitol diuresis, the natriuretic
response observed was dose-related between 0.01 and 1 mg/Kg.

Infusion of DT-327 into the left renal artery at
0.051ug/Kg/min. resulted in a significant ipsilateral
increase in the excretion of sodium within 5 minutes with—
out affecting the rate of glomerular filtration. A slight
but delayed contralateral response was also noted, occurring
10-15 minutes after the ipsilateral response. Systemic

administration of probenecid inhibited both the ipsilateral

Bernard Terry

and contralateral responses. The data demonstrated that
DT-327-induced natriuresis was the result of a direct
action on the kidney. The inhibition of this response

by probenecid indicated that either cellular and/or

luminal concentration, rather than plasma concentration, was
a prerequisite to the natriuretic activity of this compound.

At a dose capable of producing a significant
natriuresis, DT-327 did not alter renal blood flow and by
implication did not affect renal vascular resistance.

This is in contrast to furosemide which has been shown to
decrease resistance and subsequently increase renal blood
flow.

Alterations of plasma pH did not appear to affect the
natriuretic response to DT-327 in the dog. The small
increase in bicarbonate excretion produced by DT—327 in
normal animals was enhanced during metabolic alkalosis,
thus indicating some carbonic anhydrase inhibition by the
drug. There appeared to be no alteration in the chloruretic
response following DT-327 administration in rats or dogs
pretreated with saline or bicarbonate. This weak carbonic
anhydrase inhibitory activity observed with DT-327 appeared
to be similar to that observed by others with the more
lipid soluble thiazide derivatives.

DT-327, like the thiazides, decreased free water
clearance without altering negative free water clearance
indicating an action on the distal nephron. This is in

contrast to furosemide which has been reported to decrease

Bernard Terry

negative free water clearance. In stOp-flow eXperiments,
DT-327 produced a significant increase in the distal
sodium minimum, thereby confirming a distal locus of
action.

During maximal natriuresis produced by hydrochloro-
thiazide, DT-327 had no effect on sodium excretion. Con-
versely, following a maximally effective dose of DT-327,
hydrochlorothiazide was without effect. However, these
animals were still capable of responding to furosemide.
Thus, it appeared that DT-327, although chemically not a
thiazide, could produce a relatively Specific blockade of
the action of a thiazide diuretic. It is concluded that
the natriuretic action of DT-327 is not unique but closely

resembles the thiazides.

THE NATRIURETIC ACTION OF DT-327
by

Bernard Terry

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Pharmacology

1968

ACKNOWLEDGEMENTS

I would like to gratefully acknowledge the valuable
assistance, encouragement and guidance offered by Dr. J. B.
Hook through the course of this investigation.

I wish to thank Dr. T. M. Brody, Dr. G. L. Gebber,
and Dr. D. A. Reinke and Dr. R. M. Daugherty for their
helpful assistance in the preparation of this thesis.

I also wish to thank the excellent technical assistance
of Mrs. Barbara Michaluk, Miss Jo Ann Nelson, Miss Kathleen
Tinsley, Miss Charlene D. Benjamin and James L. Ecker.

The diuretic agents employed in this investigation
were supplied by the manufacturers. I would like to thank
the following for their generous supply of drugs: Sandoz
Pharmaceuticals, Inc., for DT-327; Hoechst Pharmaceuticals
for furosemide; Merck, Sharp and Dohme for hydrochloro-

thiazide and probenecid.

ii

TABLE OF CONTENTS

Page
ACKNOWLEDGEMENTS. . . . . . . . . . . . . . . . . . . . ii
TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . iii
TABLE OF TABLES . . . . . . . . . . . . . . . . . . . . iv
TABLE OF FIGURES. . . . . . . . . . . . . . . . . . . . v
INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . 1
METHODS . . . . . . . . . . . . . . . . . . . . . . . . 22

The Effect of Intrarenal Infusion of DT-327. . . . . 23
Effects of DT-327 on Renal Hemodynamics. . . . . . . 24
Effects of Acid- Base Balance on the Natriuretic

Activity of DT- 327 . . . . . . . . . . . . . 24
Effects of DT- 327 on Free Water and Negative

Free Water Clearance . . . . . . . . . . . . 26
StoP-Flow Analysis of the Tubular Site of

Action of DT— 327 . . . . . . . . . . . . . . . 27
Effect of DT- 327 During Maximal Thiazide and

Furosemide Natriuresis . . . . . . . . . . . . . . 28

RESULTS 0 O O O O O O O O O C O O O O O O O O O O O O O 30

The Effect of Intrarenal Infusion of DT-327. . . . . 30
Effects of DT-327 on Renal Hemodynamics. . . . . . . 31
Effects of Acid- Base Balance on the Natriuretic

Activity of DT- 327 . . . . . . . . . . . . . . . 32
Effects of DT- 327 on Free Water and Negative

Free Water Clearance . . . . . . . . . . . . . . . 33
Stop-Flow Analysis of the Tubular Site of

Action of DT- 327 . . . . . . . . . . . . . . . 33
Effect of DT-327 During Maximal Thiazide and

Furosemide Natriuresis . . . . . . . . . . . . . . 35

DISCUSSION. 0 O O O O O O O O O O O O O O O O O O O O O 36
SUMMARY 0 O O O O O O O O O O O O O O O O O O O O O O O 45

REFERENCES 0 O O O O O O O O O O O O O O O O O O O O O O 47

iii

TABLE OF TABLES
Table Page

1 Protocol of a typical experiment
demonstrating the dose-response
relationShip Of DT‘327 o o o o o o o o o o o o o 51

2 Protocol of a typical eXperiment
demonstrating the renal actions of
DT-327 o o o o o c o o o o o o o o o o o o o o o 52

3 Effects of DT-327 (l mg/Kg, i.v.) on
bicarbonate excretion . . . . . . . . . . . . . 53

4 Effects of DT-327 (l mg/Kg, i.v.) during
saline diuresis . . . . . . . . . . . . . . . . 54

5 Effects of DT-327 (1 mg/Kg, i.v.) during
metabolic acidosis . . . . . . . . . . . . . . . 55

~6 Effects of DT-327 (l mg/Kg, i.v.) during
metabolic alkalosis . . . . . . . . . . . . . . 56

7 Effects of furosemide (5 mg/Kg, i.v.)
during maximal DT-327 natriuresis . . . . . . . 57

iv

Figure

10

11

TABLE OF FIGURES

Chemical structure of hydrochlorothiazide,
furosemide, DT-327, and chlorthalidone . . . .

Effects of increasing doses of DT-327 on
sodium excretion . . . . . . . . . . . . . . .

Effects of DT-327 (l mg/Kg, intravenously)
on sodium excretion in dog during mannitol
diureSis I O O O O O O O O O O O O O O O O O 0

Effect of probenecid on the natriuretic
action 0f DT-327 o o o o o o o o o o o o o o 0

Effect of DT-327 on renal blood flow in
the anesthetized dog . . . . . . . . . . . . .

Effect of increasing doses of DT-327 on sodium
and chloride excretion in rats orally loaded
with sodium chloride or sodium bicarbonate
(25m1/Kg)..................

The relationship of free water clearance

(C ) to urine volume during hypotonic
sagige infusion (control) compared to that
following administration of DT-327 (l mg/Kg,
intravenously) in 5 dogs . . . . . . . . . . .

The relationship of negative free water
clearance (TcH ) to osmolar clearance (Cosm)
during hypertoggc saline infusion (control)
compared to that following the administration
of DT-327 (l mg/Kg, intravenously) in four
dogs . . . . . . . . . . . . . . . . . . . . .

Stop-flow analysis of the tubular site of
action of DT-327 . . . . . . . . . . . . . . .

Effect of DT-327 (1 mg/Kg, intravenously)
during maximal thiazide diuresis . . . . . . .

Effect of hydrochlorothiazide (l mg/Kg,
intravenously) during maximal DT-327
diuresis . . . . . . . . . . . . . . . . . . .

Page

59

61

63

65

67

69

71

73

75

77

79

INTRODUCTION

It has been about fifty years since Vogl observed the
diuretic effect of a new organic mercurial compound,
novasurol, in a patient who had received it as an anti—
syphilitic agent (Vogl, 1950). Since that fortunate
incident, the deve10pment of diuretic therapy has proceeded
rapidly. Today, there are a large number of drugs avail-
able whose administration results in the increased
excretion of water by the kidneys. It has been traditional
to define a diuretic as a drug that increases the flow of
urine. In current pharmacological thinking however, the
primary purpose of a diuretic is to rid the body of
electrolytes, primarily sodium. Increased electrolyte
excretion reduces extracellular fluid volume and results
in the elimination of edema. Thus, the most effective
agents in the removal of fluid from the body would be those
drugs that increase the excretion of sodium. Such drugs
are referred to as natriuretic agents. The term saluretic
often appears in the literature referring to the action of
furosemide or that of the thiazides. Saluresis refers to
the excretion of sodium chloride. Kaluretic and chloruretic
agents refer to drugs that increase the excretion of
potassium and chloride respectively. Thus, as in other
disciplines, advancement in renal pharmacology has brought
with it a more precise nomenclature to explain certain

phenomena.

2

A new experimental natriuretic agent, DT-327
(N—[2', 6' - dimethyl piperidyl - (1')] 3 - sulfamyl -

4 - chlorobenzamide) was shown to have a chemical structure
similar to other natriuretics but different enough to
warrant more specific studies. Investigations were carried
out in an attempt to more carefully define the natriuretic
activity of this compound. This report describes these
investigations and the results obtained.

To provide background information concerning this
study, a review of the available information concerning
Other natriuretic-diuretic agents is in order. The
following pages serve as a brief review of the literature
pertaining to the major classes of diuretics. Much of
the information presented and techniques described were
utilized as background information for this investigation.

Inorganic mercury compounds (e.g., calomel) were
utilized as diuretics as early as the 16th century in the
treatment of "dropsy." It was not until Vogl's demonstra-
tion in 1919 of the diuretic action of the antisyphilitic
organomercurial, novasurol, that organic mercurials found
their place in clinical medicine (Vogl, 1950).

Following Vogl's discovery many attempts were made to
elucidate the nature of the mercurial induced diuretic
activity. The classical renal transplant experiments of
Govaerts (1928) were responsible for determining that the
target organ for organic mercurials is the kidney. He

administered an organic mercurial to a dog and during

3

maximal diuresis removed the kidney and placed it in the
neck of another dog which had received no drug. The
transplanted kidney continued to produce large amounts of
urine. No diuresis occurred, however, from a kidney which
was transplanted from a dog which did not receive drug
treatment into the neck of a dog which had been pretreated
with an organic mercurial. Therefore, he concluded the
target organ of the organic mercurials was the kidney.

Govaerts' findings were later confirmed by Bartram
(1932). By injecting mercurial diuretics directly into
the renal circulation of one kidney he established the
renal action of the mercurial. With low doses he obtained
only an ipsilateral diuretic response. Higher doses pro-
duced a response from the contralateral kidney. It was
concluded that mercury in low doses was removed from the
blood and bound in the kidney where it exerted its effect.
When the dose of the drug was increased the capacity of the
kidney to bind the drug was at a maximum. The excess drug
reached the systemic circulation and exerted an effect
on the contralateral kidney. It was also noted that the
contralateral kidney was unable to produce urine at these
high doses. This was due to the fact that at high doses,
mercury is nephrotoxic.

Pitts (1958) conducted an experiment similar to that

203 labeled chlormerodrin into

of Bartram. He injected Hg
one renal artery and observed a diuretic effect first on
the ipsilateral kidney and later an increased flow from the

contralateral kidney.

4

Once it had been established that the organic
mercurials acted directly on the kidney, much work was done
in trying to elucidate the site and mode of action. The
literature is extensive in this area but even at present
relatively little is known regarding the mechanism of the
natriuretic activity of these compounds. It was demon-
strated that a mercurial diuresis was not dependent upon
changes in filtration rate or renal plasma flow but was the
result of inhibiting the reabsorption of certain ions
(Blumgart 25 31., 1935; Farnsworth, 1946). Some
investigators believed that the saluresis produced by
mercury was due to a decreased distal reabsorption of
chloride (Farnsworth, 1946; Pitts, 1958). Mudge and Weiner
(1958) demonstrated, in salt retaining patients, that the
increase in chloride excretion often exceeds that of the
increase in sodium excretion and that a hypochloremic
alkalosis results with prolonged mercury therapy. This
was interpreted as an action of the organic mercurial on
the reabsorption of the chloride ion. However, micro-
puncture studies conducted by Giebisch (1958) have shown
that the intratubular potential produced by active sodium
transport is diminished by a mercurial. Such findings
indicate that mercury inhibits active sodium reabsorption
and the increased chloride excretion is merely secondary
to the sodium effect. Giebisch's findings tend to
substantiate a theory set forth by Berliner (1958).

Berliner stated that mercury inhibits sodium transport

5
proximally. The excess sodium chloride delivered to the
distal area would be handled in the following fashion.
Some of the excess sodium would be exchanged with potassium
or hydrogen through a distal exchange mechanism. The
chloride ion, on the other hand, would remain in the lumen,
thus explaining the greater increase in ch10ride ion
excretion over that of sodium ion excretion. With prolonged
administration, the use of mercury can lead to a
hypochloremic alkalosis (Axelrod and Pitts, 1952). It has
also been reported that acidosis potentiates the action of
mercurials (Ethridge gt 31., 1936) and alkalosis renders
the mercurials ineffective. Thus, the mercurials are
self-limiting as they produce metabolic alkalosis with
prolonged use.

With the advent of more SOphisticated techniques
others have attempted to explain the site of action of the
organic mercurials. The literature contains much con-
flicting data. Vander gt 31. (1958) and White and Rolf
(1963) have stOp-flow data that indicates a proximal site
of action. Recently, however, Orloff (1966) has pointed
out some of the pitfalls in the interpretation of stop-
flow results especially if the diuretic produced a pattern
that would indicate a proximal action.

Other techniques have been utilized to determine site
of action of diuretics. One such method is that of the
free« water clearance technique. The concept of free

water clearance is best eXplained in terms of the functional

6
anatomy of the kidney and has been reviewed by Gottschalk
(1964). In the proximal portion of the nephron,
reabsorption of solutes is isosmotic with respect to plasma.
Thus, the concentration of fluid passing through the
proximal nephron does not change and is isosmotic with
plasma when it reaches the loop of Henle. The cells lining
the descending limb of the loop of Henle are freely per-
meable to both solutes and water. However, in the ascend-
ing limb, the cells of the tubule are impermeable to
water. Sodium is actively transported out of the
ascending limb resulting in the delivery of a hyposmotic
fluid into the distal portion of the nephron. In the
distal areas, certain distinct processes can occur
(reabsorption, sodium-potassium exchange, hydrogen ion
secretion, etc.) resulting in a decreased concentration of
total solutes but with relatively little change in volume
occurring until the fluid enters the collecting duct. In
the presence of antidiuretic hormone (ADH), water in the
collecting duct diffuses back into the medullary
interstitium due to the high sodium content (resulting
from sodium transport out of the ascending limb of the
loop of Henle) producing a concentrated or hyperosmotic
urine. Without antidiuretic hormone, the collecting duct
is relatively impermeable to water resulting in the
excretion of a dilute or hyposmotic urine. Thus dilution
or concentration of the urine is determined by events
occurring within the 100p of Henle and in the collecting

duct.

Free water clearance may be defined as osmotically
unobligated water or the amount of water excreted in excess
of the volume necessary to maintain the solutes of the
urine isosmotic with respect to plasma. Free water
clearance is eXpressed in ml/min. and is obtained by
subtracting the osmolar clearance (the number of milli—
liters of plasma cleared of osmotically active solutes per
minute) from the volume (ml/min.) of urine excreted.

Agents that act as a single anatomical site in the
nephron can be eXpected to alter free water clearance in
a predictable manner. Once a maximum water diuresis is
obtained by the infusion of hyposmotic saline or dextrose,
the drug in question is administered and the free water
clearance is determined and compared to that obtained under
control conditions. One other point should be emphasized.
Normally, the two major sites of water reabsorption within
the nephron are in the proximal area and the collecting
duct. By creating a water diuresis, it can be assumed that
the amount of ADH acting at the collecting duct is
negligible. Thus, there is only one major site of water
reabsorption, that being the proximal portion of the
nephron where reabsorption is isosmotic with respect to
plasma. Now, what is the effect of natriuretic agents
that act at different sites along the nephron on free water
clearance? If a drug were to act solely on the proximal
convoluted tubule to prevent the reabsorption of sodium,

one would observe an increased delivery of filtrate to the

8

loop of Henle. Some of this increased filtrate would be
reabsorbed in the distal portions of the nephron but the
excess water that accompanied this increased solute out of
the proximal nephron would be excreted since water
reabsorption in the collecting duct is negligible (no
ADH). Thus, one would observe an increase in free water
clearance following such a drug. In contrast, drugs that
act solely in the distal tubule to inhibit sodium
reabsorption would tend to increase the amount of solute
excreted in the urine and thus decrease free water
clearance. If a drug were to act solely on the ascending
limb of the loop of Henle to inhibit sodium reabsorption,
one would expect to see an increase in electrolyte
excretion and therefore a decrease in free water clearance.
It should be emphasized that the free water clearance
technique is only able to differentiate agents that act
proximally as compared to distally. With this technique
one cannot differentiate between an effect on the distal
nephron and the ascending 100p of Henle. Thus, if a drug
were to decrease free water clearance, it then would be
necessary to perform a negative free water clearance study.

Negative free water clearance eXperiments have also
been utilized to elucidate the site of action of
diuretics. During hydropenia, there is maximum ADH
activity, and the urine is more concentrated than the
plasma. The amount of water needed to render the urine

isosmotic with respect to plasma is referred to as the

9
negative free water clearance. It actually is a reflec-
tion of water reabsorption out of the collecting duct.
Negative free water clearance is eXpressed in ml/min. and
is obtained by subtracting the urinary volume (ml/min.)
from the osmolar clearance (m1/min.).

During hydrOpenia, one measures the negative free
water clearance before and after drug administration to
see if a significant change has occurred. As noted before,
this technique is primarily used to differentiate between
a site of action on the ascending limb of the loop of Henle
and the distal nephron. The elaboration of negative free
water is dependent on the reabsorption of sodium in the
medullary portion of the ascending limb. To test the
effects of diuretics on negative free water clearance,

a normal relationship between negative free water and
osmolar clearance is established. Negative free water
clearance increases with increasing osmolar clearance
under control conditions. Since the rate at which solute
free water is removed from the collecting duct must be
dependent to a major extent on the rate at which sodium is
transported from the ascending limb of the 100p of Henle
into the medullary interstitium, it follows that impair-
ment of solute transport in this segment diminishes the
ability of the urine to become concentrated. Thus,
negative free water clearance would decrease. Seldin
(1966) has reported such a decrease in negative free water

clearance follows furosemide and ethacrynic acid, agents

10

that are believed to act on the ascending limb. If, on
the other hand, an agent acted distally to prevent
reabsorption of sodium, no change in negative free water
clearance would be observed compared to that of control.
Since there is no alteration of the concentration within
the medullary interstitium, the urine is still able to
become concentrated and the effect observed on negative
free water clearance is merely that which occurs when the
osmolar clearance increases.

Results from free water clearance studies on the
organomercurials had to be reinterpreted when it was
realized that theophylline which is found in combination
with many organic mercurial preparations was, in part,
the reason for the increased free water clearance
observed (Farah and Miller, 1962). It has been shown
that theophylline is a proximally acting diuretic
(Walker 35 31., 1937) and this could eXplain the increase
in free water clearance that was observed. Miller and
Riggs (1961) using organomercurials without theophylline
found no change in free water clearance. This would
tend to indicate a site of action on the loop of Henle
or the distal tubule in addition to an effect on the prox-
imal tubule. Utilizing negative free water clearance
technique Lambie and Robson (1960) found that under
conditions of acidosis the mercurials could decrease
negative free water thus acting to reduce the concen-

trating capacity of the kidney. This points to a site of

11

action on the ascending limb of the loop of Henle. It

now appears that, under the prOper conditions, the organic
mercurials might act along the entire tubule (Seldin EE.El°'
1966). This latter theory would help eXplain much of the

conflicting data in the literature.

Carbonic Anhydrase Inhibitors

 

 

The development of carbonic anhydrase inhibitors was
an outgrowth of investigations with sulfanilamide.

Pitts 25 El- (1949) traced the metabolic acidosis produced
by sulfanilamide to an inhibition of the acidification of
the urine. This observation combined with the knowledge
that this compound was an inhibitor of carbonic anhydrase
led to the idea that other inhibitors of carbonic anhydrase
might be effective diuretics.

According to the most generally accepted concept,
bicarbonate reabsorption is mediated through an exchange
of intracellulary produced hydrogen ions for reabsorbed
sodium ions. Carbonic anhydrase catalyzes the intra-
cellular reaction of carbon dioxide and water to form
carbonic acid which breaks down into hydrogen ions and
bicarbonate ions. An adequate supply of hydrogen ions
is thus generated to take part in this exchange process.
The hydrogen ion is secreted into the lumen and can react
with filtered bicarbonate to form water and carbon dioxide.
The bicarbonate formed intracellulary from the breakdown
of carbonic acid diffuses back into the blood stream

(Pitts, 1963).

12

The importance of the enzyme carbonic anhydrase in
the process of bicarbonate reabsorption has been estab-
lished in the rat, dog and man by the use of carbonic
anhydrase inhibitors. Berlinger (1952) has shown in the
dog as much as 50% of the filtered bicarbonate can be
excreted in the urine after carbonic anhydrase inhibition
by acetazolamide. Since the major part of the filtered
bicarbonate is reabsorbed in the proximal tubule (Clapp
22.3l'r 1963) it seems reasonable that inhibition of
carbonic anhydrase would result in a decreased bicarbonate
reabsorption by the proximal tubule.

Unlike the mercurials, which result in a hypochloremic
alkalosis, the carbonic anhydrase inhibitors lead to a
hyperchloremic acidosis due to the enchanced bicarbonate
excretion (Berliner 35 31., 1951). Like the mercurials,
the carbonic anhydrase inhibitors are self-limiting. They
result in the production of a metabolic acidosis which
decreases the amount of bicarbonate filtered thus reducing
the effectiveness of these agents as diuretics (Pitts 32 31.,
1949). The use of carbonic anhydrase inhibitors in
intermittent therapy will probably potentiate the action
of the mercurials due to the production of metabolic

acidosis.

The Thiazides

 

The thiazides were discovered during the search for
improved diuretics similar in structure to the sulfonamide,
acetazolamide. Novello and Sprague (1957) synthesized

chlorothiazide and Beyer (1958) reported on the diuretic

13

activity of this compound. Since then chlorothiazide has
become the prototype of a series of heterocyclic sulfonamides
possessing diuretic prOperties. The discovery of
chlorothiazide was the result of an attempt to increase
carbonic anhydrase activity by the addition of two func-
tional sulfonamide groups. However, it was noted by Beyer
(1958) that this benzothiadiazine compound increased
chloride excretion, an action not attributed to former car-
bonic anhydrase inhibitors. Newer compounds chemically
related to chlorothiazide have demonstrated a significant
chloruretic activity but reduced effectiveness as carbonic
anhydrase inhibitors. Longemann 33 El. (1959) reported
that benzothiadiazine compounds substituted on the
sulfonamide nitrogen were completely devoid of carbonic
anhydrase inhibitory activity yet still retained the
capacity to produce a significant chloruresis as well as

a saluresis. It seems clear that this increase in sodium
and chloride excretion is not entirely due to the carbonic
anhydrase inhibitory activity of these compounds.

In fairly large doses, chlorothiazide has been shown
to increase the excretion of sodium, chloride, potassium,
and bicarbonate (Beyer, 1958). A maximally effective dose
of 10 mg/Kg affects approximately 10% of filtered
sodium and chloride (Pitts 35 $1., 1958). The new
derivatives are more potent on a milligram basis but they
do not increase the maximum reSponse observed with

chlorothiazide (Beyer and Baer, 1961).

l4

Beyer (1958) also noted that alterations in plasma pH
did not affect the natriuretic response to chlorothiazide.
This was indeed a distinct advantage over the mercurials
and carbonic anhydrase inhibitors. Beyer also observed
that during conditions of acidosis, the excretion of
chloride was greatly enhanced compared to that of bicar—
bonate, whereas during conditions of alkalosis, bicarbonate
was more affected. One might conclude from the above
observations that carbonic anhydrase inhibition occurs
when the bicarbonate levels in the plasma are elevated, but
this in itself is not a primary requisite for the estab-
lishment of a natriuresis.

The thiazides, as a group, are rapidly absorbed from
the gastrointestinal tract and excreted in the urine by a
process involving active tubular secretion (Beyer, 1958).
Chlorothiazide is secreted by a renal mechanism similar
to the one used by paraaminohippurate, (PAH), phenol red
and other organic acids. Beyer (1958) was able to block
the diuretic action of a minimal effective dose of
chlorothiazide by pretreatment with probenecid. Chloro-
thiazide can also inhibit the accumulation of PAH in
renal slices of the rabbit (Essig, 1961). Thus, it would
appear that an active secretory process is responsible,
in part, for the entrance of the drugs into the renal
cells.

Kessler and his associates (1959) and Vander 33 31.

(1958), using the stOp-flow method in dogs, concluded that

15
the major effect of chlorothiazide was on the proximal
tubule. However, Earley (1961) and others utilizing free
water clearance have reported no alteration in negative
free water clearance following thiazide administration.
These results would indicate a distal action of the drug.
Orloff (1966) pointed out certain problems of interpretation
of a stOp-flow pattern eSpecially if the results indicate
that the diuretic is acting on the proximal tubule.
Inasmuch as the proximal samples move past the distal
areas of the nephron prior to collection, small alterations
in distal function could be reflected in proximal samples.
A second difficulty has to do with continuing filtration
during the period of stasis. Filtration cannot cease
entirely during the period of obstruction since if fluid
is reabsorbed to any extent, it should be replaced by new
filtrate. A third difficulty is known as the so-called
"smearing" of the concentration profile since tubules of
different length contribute to Specific samples. Any
given sample of urine will contain fluid from more than
one nephron area. The first samples may be an admixture
of urine from the pelvis and collecting ducts, the next
from the collecting ducts and distal tubules and so on.
The technique of step-flow is certainly not without serious
fault and its use in the localization of the diuretic
action of a specific agent leaves much to be desired.
Thus, considering the disadvantages in the stOp-flow
technique and also the data obtained utilizing free water

and negative free water clearances, it would appear that

16

the major site of action of the thiazides is the distal
tubule.

In an attempt to distinguish the diuretic action of
the thiazides from the mercurials Pitts 33 31. (1958)
performed an additive study. It was observed that
chlormerodrin was capable of producing its natriuretic
response during maximal natriuresis produced by chloro-
thiazide. Conversely, chlorothiazide produced its effect
during maximal mercurial natriuresis. Since either agent
was capable of eliciting an additive response during
maximal natriuresis produced by the other, it was reasoned
that they must be acting by different mechanisms or at
different sites along the nephron. Pitts gt El-
also observed an additive response between chlorothiazide
and acetazolamide, thus again indicating that the mech-
anism of action of the thiazides is not entirely due to

its carbonic anhydrase inhibitory activity.

Ethacrynic Acid

 

In 1962 Schultz gt 31. (1962) reported a class of
diuretic compounds, the a, £3, unsaturated ketone
derivatives of aryloxyacetic acids. One of the compounds,
ethacrynic acid, has been the subject of extensive
experimentation and clinical evaluation. Chemically, it
is quite different than previously know diuretics.

Baer 35 31. (1964) and Beyer (1965) have described the
saluretic activity of this unique compound. Like the

organomercurials, ethacrynic acid results in an enhanced

l7

excretion of sodium and chloride. Bicarbonate excretion
is unchanged or may actually decrease in contrast to the
carbonic anhydrase inhibitors and some of the thiazides
which increase the excretion of bicarbonate. Ethacrynic
acid is quite potent and the magnitude of natriuresis is
equal to or greater than the mercurials, affecting
approximately 18% of filtered sodium. Unlike carbonic
anhydrase inhibitors, ethacrynic acid is effective in the
presence of acidosis. It has a rapid onset of action and
peak effects occur within 20 minutes in contrast to the
1-2 hours with mercurials. Finally, during maximal effects
of either organomercurial or thiazides, ethacrynic acid
produces a further increase in sodium excretion. However,
during maximal mercurial diuresis, the increase in sodium
excretion that occurred when ethacrynic acid was adminis-
tered was less than additive. Also, during an acid
potentiated mercurial diuresis, ethacrynic acid produced
no further response. Such data indicates that perhaps
the mode of action or the site of action or both of
ethacrynic acid is similar to the mercurials under acidotic
conditions.

Beyer (1965) reported that ethacrynic acid abolishes
the renal medullary sodium gradient, which would imply an
action on the ascending limb of the 100p of Henle. His
stOp-flow data suggests that this agent acts all along the
nephron. Goldberg gt gt. (1964) and Barley and Friedler

(1964) utilized free water and negative free water

l8

clearance techniques to demonstrate inhibition of sodium
reabsorption on the ascending limb of the 100p of Henle.
Both the free water and negative free water clearance
decreased. However, since these techniques represent the
algebraic sum of events that occur along the entire nephron,
a site of action of ethacrynic acid on the proximal

nephron cannot be ignored (Seldin,1966). This proximal
action would certainly help to explain the results of

Beyer (1965) which indicate that ethacrynic acid is

capable of increasing urine flow to nearly 40% of GFR.
Giebisch and Wendhaser (1964) demonstrated that approx-
imately 70% of filtrate is normally reabsorbed in the
proximal nephron. Excretion of 40% of the filtrate by
ethacrynic acid would tend to indicate that the proximal
tubule is being affected. Thus, it appears that ethacrynic
acid, like the mercurials, acts along the entire tubule

with a substantial effect on the loop of Henle.

Furosemide

 

Furosemide, a new and extremely potent diuretic-
saluretic agent, has been the subject of much investigation
in the last few years. Structurally, it is similar to the
benzothiadiazines but compared to hydrochlorothiazide,
furosemide is capable of eliciting a greater saluretic
response (Timmerman gt gt., 1964). Their studies in both
rat and man indicate that not only did furosemide produce
a greater maximal response than the thiazides but also was

more rapid in onset and shorter in duration.

19

Studies conducted in the attempt to elucidate the
localization of the site of action of furosemide have proved
to be quite interesting. Suki SE.E£° (1964) reported that
furosemide impairs the concentrating ability of the kidney
which implies an inhibition of sodium reabsorption along
the ascending limb of the loop of Henle. Suki gt_gt.
also observed that furosemide increased urine flow to
approximately 40% of GFR. This clearly points to an action
on the proximal tubule. However, free water clearance
decreased which would not be expected if an agent acted
solely on the proximal tubule. This decrease in free
water clearance could be eXplained however, if furosemide
acted to inhibit sodium reabsorption in an area distal to
the proximal tubule. Negative free water clearances were
performed and a decrease was observed indicating an action
on the ascending limb of the loop of Henle. It was
therefore concluded that furosemide inhibited sodium
reabsorption primarily in the proximal tubule and ascending
limb of the loop of Henle. Hook and Williamson (1965a)
reported the abolishment of the renal medullary sodium
gradient with furosemide indicating an action on the
ascending limb.

Additive studies have also been conducted to compare
the action of furosemide with other known diuretics. Hook
and Williamson (1965b) showed that the natriuretic action
of furosemide and ethacrynic acid were not additive. They

also observed that the administration of furosemide during

20

maximal mercurial natriuresis resulted in an increase in
sodium excreted. However, during an acid-potentiated
mercurial natriuresis, furosemide did not produce an
increase in sodium excretion (Hook and Williamson, 1966b).
Since the natriuretic activity of furosemide is not altered
during acidosis,(Hook and Williamson, 1965c), it would
appear that during the acidotic state of the action of the
mercurials might be extended to the ascending limb of the
loop of Henle.

The effect of furosemide on renal hemodynamics has
important clinical significance. The organic mercurials
have been shown to increase renal vascular resistance
(Vargas and Cafruny, 1962) and produce a transient fall in
GFR (Farah, 1952). Studies with furosemide indicate a
rapid response in patients with severely impaired renal
function (Jachnecke gt gt., 1964). They observed that
furosemide increased the clearance of PAH and inulin,
particularly in patients with depressed renal function.
Hook and Williamson (1966ap using an electromagnetic
flowmeter, reported that furosemide and ethacrynic acid
reduced renal vascular resistance in contrast to the
thiazides and the organomercurials which increased renal
vascular resistance. Since one of the major clinical uses
of saluretic agents is in the management of edema secondary
to congestive heart failure, a condition often character-
ized by an increased renal vascular resistance, the ability

of furosemide and ethacrynic to decrease renal vascular

21
resistance and thus increase renal blood flow might be
the reason why these agents are effective in patients
which do not respond to either mercurial or thiazide
therapy.

Other natriuretic agents are in use today like the
spironolactones, chlorthalidone, and others, each
possessing certain advantages and disadvantages. Due to the
disadvantages, none of these natriuretic agents are
considered to be ideal. Inasmuch as diuretic therapy is
becoming more widespread in the treatment of various
pathological conditions, the search is continuing for new
and better agents.

DT-327 has been shown to be an orally effective
natriuretic-diuretic in man and laboratory animals (Gold,
1965). Although chemically related, in part to diuretics
like hydrochlorothiazide, furosemide and chlorthalidone
by virture of its substituted benzyl sulfamyl group, the
structure of DT-327 on the whole is different because of
its side chain (figure 1). Even though the chemical
structure of furosemide closely resembles the thiazides it
is a much more efficacious natriuretic agent (Hook and
Williamson, 1965b). It was, therefore, of interest to
determine if this side chain modification in the structure
of DT—327 resulted in an agent whose natriuretic action
more closely resembles furosemide or, like chlorthalidone,

more closely resembles the thiazides.

METHODS

Mongrel dogs of either sex weighing from 8-18 Kg
were anesthetized with pentobarbital sodium (30 mg/Kg,
intravenously) and tracheotomized. Carotid blood pressure
was monitored using a Statham arterial pressure transducer
and an Offner direct writing oscillograph. A femoral
artery and vein were cannulated with polyethylene tubing
to facilitate withdrawal of blood samples and infusion
of fluids respectively. Both ureters were eXposed by a
small midline incision and were cannulated with polyethylene
tubing. Unless otherwise indicated urine from both kidneys
was collected in single graduated cylinders. All collec-
tion periods were of 10 minute duration. Blood samples
were obtained at 20 minute intervals. Inulin (0.2%) was
added to all infusions to monitor the rate of glomerular
filtration. In some experiments para-aminohippurate
(0.4%) was also added to the infusion so that estimations
of renal plasma flow could be made.

Preliminary experiments were performed to determine
both a dose response curve and duration of action of DT—327.
Control diuresis was produced by infusing 5% mannitol into
a femoral vein at 0.3 ml/Kg/min. A dose response curve
was obtained by intravenously injecting increasing doses
of DT—327, each successive dose being given when the
diuretic response from the previous dose had reached a

plateau. To determine the duration of action, a single dose

22

23
of DT-327 (l mg/Kg) was administered, intravenously. Urine
was collected for a total of 90 minutes at 10 minute

intervals.

The Effect gt Intrarenal Infusion gt DT-327

 

 

The following investigation was performed for a variety
of purposes. A series of experiments were conducted to
verify the assumption that the target organ of DT-327 is
the kidney. The second purpose was to observe if the
natriuretic activity was due to the drug per se or the
result of some metabolic alteration. Finally, it was of
interest to determine whether probenecid inhibited the
natriuretic activity of DT-327, thus providing information
as to whether the plasma, tubular or luminal concentration
of this drug was responsible for its action.

The left renal artery was isolated retrOperitoneally
through a flank incision and a 21-gauge needle was intro-
duced as close to the aorta as possible. A slow (1 ml/min.)
saline infusion insured patency of this system. In order
to insure an adequate rate of urine flow, these experiments
were conducted during an osmotic diuresis produced by the
intravenous infusion of 5% mannitol (0.5 ml/Kg/min.).

Urine was collected every 5 minutes and blood samples
withdrawn from the femoral artery every 10 minutes. After
the rate of urine flow had stabilized, several control
urine samples were collected. The saline infusion into
the renal artery was then replaced with a solution con-

taining sufficient DT-327 to deliver 0.5/ug/Kg/min. Two

24

animals were treated with 30 mg/Kg of probenecid intra-

venously and then the above procedure was repeated.

Effects gt DT-327 22 Renal Hemodynamics

 

Since certain natriuretic agents like furosemide and
ethacrynic acid have been reported to increase renal blood
flow without any significant alteration in the clearance
of inulin, (Hook and Williamson, 1966a, it was of inter-
est to determine the effects of DT-327 on renal blood flow.

The left kidney was approached retrOperitoneally
through a flank incision and the ureter was cannulated.
Systemic hydration was attained by infusing 0.9% sodium
chloride (0.5 mg/Kg/min.) throughout the experiment. The
left renal artery was isolated and flow was measured
directly using an electromagnetic flow meter (Carolina
Medical Electronics) with a probe placed on the renal
artery. DT-327 (l mg/Kg) was administered intravenously
and the effects on renal blood flow were monitored on an
oscillograph. At the end of the experiment, the renal
artery was cannulated and blood was collected in a

graduated cylinder for the purpose of calibration.

Effects gt.Acid—Base Balance 22 the Natriuretic Activity

 

 

9; DT-327

The natriuretic activity of the mercurials and the
carbonic anhydrase inhibitors have been reported to be

affected by changes in acid-base balance (Ethridge gt gt.,

25
1936; Pitts gt gt., 1949). Since such changes occur in
pathological conditions, it was of interest to determine
if the natriuretic activity of DT-327 was likewise
altered by changes in plasma pH.

In order to determine the effect of DT-327 during
metabolic acidosis and alkalosis, acid-base balance was
altered by a slight modification of the method of Farah
gt gt. (1959). To induce systemic acidosis, mongrel dogs
were pretreated with ammonium chloride (100 mEq/day) for
3 days prior to the eXperiment. Hydrochloric acid (0.08N)
was infused intravenously at the rate of 6 ml/min/square
meter of body surface (in 0.9% sodium chloride) prior to
and throughout the eXperiment. When plasma pH reached
7.2, DT-327 (l mg/Kg) was administered intravenously.
Blood samples were drawn under oil and pH was recorded
anaerobically. To induce systemic alkalosis, animals
were pretreated with sodium bicarbonate (100 mEq/day) for
3 days prior to the experiment. A 0.25N sodium bicarbonate
infusion replaced the 0.08N hydrochloric acid infusion.
The animals were considered to be alkalotic when plasma pH
reached 7.5.

The influence of metabolic alkalosis on the electro-
lyte excretion of DT—327 was determined in another species
of animals using the assay procedure of Lipschitz gt gt.
(1943). Male Sprague-Dowley rats, 250-300 g, were given
an oral load of either 0.9% sodium chloride or 1.0% sodium

bicarbonate, 25 ml/Kg, at the beginning of the eXperiment.

26
Various concentrations of DT-327 (0.01 mg/Kg, 0.1 mg/Kg,
or 1.0 mg/Kg) were dissolved in the oral load. For urine
collection the rats were placed in metabolism cages and
urine was collected over a 3 hour period. At the end of the
collection period, the animals were killed by cervical
dislocation,the bladders removed and their contents com-
bined with the previously collected urine. Urine samples

were analyzed for sodium, potassium and chloride.

Effects gt DT—327 gg Free Water and Negative Free Water

 

Clearance

 

To more carefully delineate the tubular areas
affected by DT-327, studies were conducted utilizing the
free water clearance techniques. The following procedure
was employed in our free water clearance studies. Dogs
were hydrated by the infusion of 500 ml of 6% dextran in
0.9% sodium chloride at a rate of l ml/Kg/min. This served
the purpose of expanding blood volume and limiting the
endogenous release of antidiuretic hormone (ADH). A 2.5%
glucose solution was then infused at l ml/Kg/min. When
the urine osmolality was below 100 milliosmoles, and the
urine volume relatively constant, the diuretic (l mg/Kg)
was administered intravenously and urine collected for
40 minutes.

To observe the effect of DT—327 on negative free water
clearance, the following procedure was employed. Mongrel
dogs were deprived of food and water 24 hours prior to the

experiment. An injection of vasopressin in oil (Pitressin

27
tannate, 5 units) was given intramusculary the night before
surgery. The animals were anesthetized and prepared
surgically as in the preceding experiments. An infusion of
2% sodium chloride containing aqueous Pitressin (100 mU/Kg/
hr.) was administered at 0.5 ml/Kg/min. and maintained
throughout the experiment. After urine flow had stablized,
DT-327 (l mg/Kg) was injected intravenously and urine was

collected for 40 minutes.

Stop-Flow Analysis gt the Tubular Site gt Action gt DT-327

 

 

The technique of stop-flow analysis (Malvin gt gt.,
1958) with a slight modification (Sullivan and Pirch, 1965)
was also employed to elucidate the site of action of DT-327.
Although the stop-flow technique has certain limitations
as mentioned in the introduction, it can serve as a useful
tool especially if the drug under investigation acts
primarily on the distal tubule.

The left ureter was exposed by a flank incision and
catheterized. Twenty percent mannitol in a 1% sodium
chloride solution containing 0.8% inulin and 0.08% PAH
was infused at a rate of 10 ml/min. to establish a diuresis.
Two grams of sodium chloride were injected intravenously
approximately 10 minutes prior to the occlusion of the
left ureter. When urine flow was adequate, two 5-minute
control urine samples were collected, blood samples being
taken for each control urine period. The ureter was then
occluded for 4 minutes, a blood sample being taken at the

end of the first 2-minutes of occlusion. At the end of the

28
4 minute occlusion, the urine was collected serially in
small graduated tubes. The same procedure was repeated
after the administration of DT—327 (l mg/Kg, intravenously)

and the two stop-flow patterns were then compared.

Effect gt DT-327 During Maximal Thiazide and Furosemide

 

Natriuresis

 

To further classify DT—327 as to site of action, an
additive study with hydrochlorothiazide and furosemide was
employed using the method described by Pitts gt gt. (1958).
Such a technique, as described in the introduction, has
proved to be quite useful in determining the site or mech-
anism of action of various diuretic agents.

Saline (0.9%) was infused intravenously at 0.8 ml/Kg/
min. for about 1 1/2 hours prior to and throughout the
eXperiment. After equilibration, maximal thiazide
saluresis was produced by the administration of 3 mg/Kg of
hydrochlorothiazide intravenously; a maintenance dose of
3 mg/Kg/hr. was then added to the infusion. When urine
flow had plateaued, l mg/Kg of DT-327 was injected intra-
venously. Urine was then collected for 40 minutes. In
another series of animals, the order of drugs administered
was reversed in an attempt to determine if hydrochloro-
thiazide was additive in animals undergoing a DT-327
saluresis. Maximal DT-327 saluresis was produced by the
administration of 5 mg/Kg and a maintenance infusion of
5 mg/Kg/hr. When urine flow had reached a plateau,

3 mg/Kg of hydrochlorothiazide was injected intravenously.

29
The above procedure was repeated substituting 5 mg/Kg of
furosemide for hydrochlorothiazide.

Inulin was determined by the method of Schreiner
(1950) and PAH by the method of Smith gt gt. (1945).
Sodium and potassium were determined by flame photometry
(Instrumentation Laboratories, internal standard flame
photometer) and chloride was determined with a Buchler-
Cotlove Chloridometer. Bicarbonate was determined by
simple titration. Osmolalities were determined with an
Advanced Osmometer. The data obtained were analyzed
statistically using the student "t" test (Snedecor, 1956).
The 0.05 level of probability was used as the criterion

of significance.

RESULTS

The diuretic response to DT-327 is dose related. The
protocol of an individual eXperiment is illustrated in
table 1 and the results of 4 animals are summarized in
figure 2. When electrolyte excretion was plotted against
increasing doses of DT-327, increased sodium, chloride, and
potassium excretion was observed. No significant alteration
in the clearance of inulin or PAH occurred. The increased
cation excretion was not completely balanced by increased
chloride excretion indicating that another anion was
probably being affected. The greater effect of DT-327 on
sodium excretion relative to potassium excretion is
illustrated by the slopes of their respective dose response
curve (figure 2).

The natriuretic effect of a single intravenous injec-
tion of l mg/Kg of DT-327 is illustrated in figure 3.
Maximum sodium excretion, as well as the other variables
measured, occurred in the first 10-minute interval following
DT—327 injection. A substantial effect was still present

90 minutes after drug administration.

The Effect gt Intrarenal Infusion gt DT-327

 

 

Infusion of DT-327 (0.5.ug/Kg/min.) into the left
renal artery increased the rate of urine flow and sodium
excretion from the left kidney. A slight contralateral
response from the right kidney was also noted appearing

approximately 10-15 minutes after the response observed

30

31

on the ipsilateral side. The protocol of a typical
eXperiment is shown in table 2 and the results from 4
animals in figure 4. Urine flow and sodium excretion
from the left kidney increased during the first S-minutes
of DT-327 infusion and continued to rise during the remain-
ing drug periods. Upon withdrawal of the DT—327 infusion,
both urine flow and sodium excretion declined. A slight
increase in urine flow and sodium excretion appeared from
the right kidney after approximately 15 minutes of drug
infusion but appeared to be markedly less in magnitude
than the increase observed from the left kidney. The
response noted from the right kidney did not decrease as
quickly as did that from the left kidney once the drug
infusion was discontinued.

The effect of 30 mg/Kg of probenecid on the natriuresis
induced by DT-327 is illustrated in figure 4. Following
probenecid administration, the natriuresis produced by

DT-327 was greatly reduced e3pecially from the left kidney.

Effects gt DT-327 gg Renal Hemodynamics

 

Studies concerning the effect of DT-327 on renal blood
flow were conducted in 3 animals and the results of a typi-
cal blood flow eXperiment are illustrated in figure 5. Mean
blood pressure (BP) before DT-327 administration was
100 mm/Hg and renal blood flow (RBF) was approximately
210 ml/min. Following the intravenous injection of 1 mg/Kg
of DT-327, there was no demonstrable alteration in either

renal blood flow or mean blood pressure. However, urinary

32
volume increased from control values of 0.30 ml/min. to
1.17 ml/min. following drug administration. Sodium
excretion likewise increased from control values of
20.nEq/min. to 196,M£q/min. This increased sodium excretion
is comparable to changes observed in other eXperiments
(figure 2, 3). There was no alteration in the clearance

of inulin.

Effects gt Acid-Base Balance gg the Natriuretic Activity

 

 

gt DT-327

When injected intravenously into dogs receiving a
saline infusion, DT-327 (1 mg/Kg) produced a marked
natriuretic-saluretic response (figure 2, table 3, 4). An
increase in potassium and bicarbonate excretion was also
observed, the magnitude of the latter, compared to the
sodium and chloride increase, being quite small. As
illustrated in table 5, 1 mg/Kg of DT-327 administered
_during metabolic acidosis produced an increase in sodium,
chloride, and potassium excretion. The increase in chloride
excretion was slightly greater than that of the increase
in sodium excretion. No apparent change in the clearance
of inulin occurred. During metabolic alkalosis, l mg/Kg
of DT-327 resulted in an increased excretion of sodium
chloride, potassium and bicarbonate (table 6). The
increase in bicarbonate excretion appeared to be greater
than that observed in the dogs receiving saline infusion.
However, due to the small number of animals studied, no

statistical comparison could be made.

33
The effect of increasing doses of DT-327 on sodium and
chloride excretion in rats orally loaded with saline or
sodium bicarbonate is illustrated in figure 6. The increase
in chloride excretion seemed to parallel quite closely the
increase in sodium excretion in both the saline and

bicarbonate treated rats.

Effects gt DT-327 gg Free Water and Negative Free Water

 

 

Clearance

 

A significant decrease in free water clearance was
observed with a dose of l mg/Kg of DT-327. Figure 7
illustrates the results of plotting free water clearance
against urinary volume. In a total of 5 animals, free
water clearance decreased from a control value of 9.4 I
0.6 (S.E.) ml/min. to 6.1 t 0.5 ml/min. It is apparent
that the diuretic reduced the maximal capacity to form
free water to about 70% of that obtained in control
animals at comparable rates of delivery.

The effect of DT-327 on negative free water clearance
is illustrated in figure 8. Negative free water clearance
is plotted against osmolar clearance. No significant
difference in negative free water clearance was observed

between control values and values obtained with DT-327.

Stop-Flow Anatysis gt the Tubular Site gt Action gt DT—327

 

 

The concentrating pattern of a typical stOp-flow
experiment before and after DT-327 administration is
illustrated in figure 9. The urine/plasma (U/P) ratios

of sodium and potassium, each factored by the corresponding

34
U/P ratio of inulin (to account for water reabsorption) are
plotted against the accumulated urine volumes. In the
control, or non-drug stop-flow pattern, the U/P ratios of
sodium during free flow were approximately 0.35. After
the release of occlusion there was a fall in these ratios,
the lowest value reached being 0.05. This ratio represen-
ted that sample acted upon by a maximum number of distal
tubules and is referred to as the distal minimum. Those
samples that represented the loop of Henle and proximal
tubules showed a gradual increase in the U/P ratios of
sodium eventually establishing a plateau at the original
free flow ratio of 0.35. The U/P ratios of potassium
obtained from samples after the occlusion did not seem to
vary significantly from the ratios obtained during free
flow, an average value being 0.10.

Following administration of DT-327, there appeared to
be a significant alteration in the stOp-flow pattern. The
low U/P ratio of sodium achieved by the distal tubule
during the control stop-flow procedure was not present,
the lowest ration being 0.14 as compared to the control
ratio of 0.05. In a total of 4 animals, this increase in
the distal minimum following DT—327 administration proved
to be statistically significant.. A mean increase in the
U/P ratio for sodium of 0.056 t 0.021 (S.E.) was significant
(p 0.05 "one tailedf). The remaining portion of the stop-
flow pattern with DT—327 appeared to be identical with the
control stOp-flow pattern concerning the U/P ratios of

sodium. The U/P ratios for potasSium following DT-327

35

administration increased in the distal area of the tubule,
a maximum value of 0.30 being obtained as compared to

0.12 from the control stOp-flow pattern.

Effects gt DT-327 During Maximal Thiazide and Furosemide

 

Natriuresis

 

During administration of a maximally effective dose
of hydrochlorothiazide, DT-327 (l mg/Kg) did not produce
saluretic and diuretic responses significantly greater than
those produced by hydrochlorothiazide alone (figure 10).
A small but significant decrease in potassium excretion
occurred. It should be noted that this decrease in
potassium excretion was quite small in most cases only
amounting to a few qu/min. thus creating doubt as to its
practical significance. During maximal DT-327 diuresis
intravenous administration of hydrochlorothiazide (3 mg/Kg)
did not significantly affect any of the variables measured
(figure 11). However, animals were capable of responding
to other agents. In 4 animals receiving a maximally
effective dose of DT-327, furosemide (5 mg/Kg) produced a
further increase in sodium and chloride excretion (table 7).
This indicates that the blockade of hydrochlorothiazide by
DT-327 was, most probably, a specific effect and not some

non—specific blockade of all natriuretic agents.

DISCUSSION

Intravenous administration of DT-327 resulted in a
rapid onset of action, producing a significant increase in
urine volume and the excretion of sodium, potassium, and
chloride (figure 2, 3, table 1). The magnitude of response
was related to the dose, and an effect was present with
a dose as low as 0.01 mg/Kg (figure 2, table 1). With a
supramaximal dose of DT-327 (5 mg/Kg), the drug appeared
to be equi-effective with hydrochlorothiazide (figure 7).
The duration of action of DT—327 (figure 3) appeared to be
similar to that of the thiazides, a substantial effect
being present 90 minutes after drug administration.

DT-327 infusion (0.5.ug/Kg/min.) into the renal artery
resulted in an ipsilateral increase in sodium excretion
(figure 4, table 2). The contralateral natriuresis observed
was delayed in onset and only a small fraction of the
response of the ipsilateral kidney. This observation
verified the assumption that the kidney is the target organ
for the natriuretic effect of DT-327. Inasmuch as the
ipsilateral increase in sodium excretion occurred within
the first S-minutes of drug infusion, it can be concluded
that the drug per se acted directly on the kidney and that
transport into the systemic circulation and metabolism of
the parent compound was not necessary for natriuresis to
occur. The lipid solubility of DT-327 offers a possible
eXplanation for the contralateral response that occurred.
Most likely some of the drug was reabsorbed from the urine

36

37

of the ipsilateral kidney and reached the contralateral
kidney via the systemic circulation. Such an explanation
would account for the time lag noted before observing this
contralateral response. A tubular site of action within

the kidney is also indicated by these results as DT-327 did
not have any significant effect of the glomerular filtration
rate.

Since DT-327 is an organic acid, one could speculate
that DT-327 would be actively secreted by the tubular cells.
Probenecid, a competitive inhibitor of the secretory
transport for organic acids (Weiner gt gt., 1960), was used
to block the tubular transport of DT-327 i2.!i!2° A large
dose (30 mg/Kg) was administered intravenously to insure
a significant blockade of this transport system. This
dose has been shown by others to have no demonstrable
effect on renal function other than to block organic anion
transport (Weiner gt gt., 1960; Hook and Williamson, 1965c).
As illustrated in figure 4, the natriuretic response to
DT—327 after probenecid administration was greatly reduced.
Probenecid has been reported to have a similar effect on
the natriuresis produced by chlorothiazide (Beyer and Baer,
1961). Thus, it appears that DT-327 is transported to a
great extent by tubular secretion and that cellular or
luminal concentration rather than plasma concentration of
the drug appears to be critical for the natriuretic response
to occur.

The organomercurials have been shown to increase renal

vascular resistance (Vargas and Cafruny, 1962) and produce

38
a transient fall in glomerular filtration rate (Farah,
1952). It has been shown that patients with severely
impaired renal function do not respond well to the organo-
mercurials (Reubi and Cottier, 1961). In contrast, Hook
and Williamson (1966a) have reported that saluretic doses
of furosemide significantly decrease renal vascular
resistance. They also indicated that hydrochlorothiazide
has no significant effect on renal vascular resistance
although it tended to increase it. If renal blood flow
is diminished due to some pathological state, it is con-
ceivable that increased vascular resistance produced by
the mercurials, for example, would be great enough to
limit blood flow to the point of masking the saluretic
response. Therefore, it was of interest to observe the
response of DT-327 on renal vascular resistance and compare
this to the response of other natriuretic agents.

The effect of DT-327 on renal hemodynamics is illus-
trated in figure 5. It was apparent that the drug at a
dose capable of producing a significant natriuresis did
not alter renal blood flow and consequently did not affect
renal vascular resistance. The clearance of inulin was
likewise unaltered following DT-327 administration. Thus,
it appeared that an increase in blood flow or glomerular
filtration rate was not necessary for a natriuretic
response to occur. DT-327, like the thiazides and the
organomercurials, would therefore appear to have definite
'1imitations in the management of edema secondary to con-

gestive heart failure which is usually characterized by an

39
increase vascular resistance.

When compared to normal animals (table 4) the natriuretic
response to DT-327 did not seem to be altered by the exis-
tance of metabolic acidosis or alkalosis. However, there
did appear to be a slight alteration in the nature of the
anion excreted with changes in the acid-base balance. During
systemic acidosis, administration of DT-327 produced a
greater increase in chloride excretion than sodium excretion
(table 5). In our preliminary experiments with normal
animals, it was noted that following DT-327 administration,
the increase in sodium excretion was not completely
balanced by the increase in chloride excretion suggesting
that DT—327 might have an effect on another anion, perhaps
bicarbonate. Some tg{ytttg carbonic anhydrase inhibitory
activity has been observed (Trapold, l967).and our results
tend to confirm this in the intact animal. In dogs
undergoing a saline diuresis, administration of DT-327
produced a small but significant increase in bicarbonate
excretion (table 3, 4). During alkalosis, an injection of
DT—327 resulted in an even greater increase in bicarbonate
excretion (table 6) which would be eXpected if a diuretic
possessed carbonic anhydrase inhibitory activity. However,
it should be noted that the magnitude of this increase in
bicarbonate excretion did not appear to decrease the effect
of the drug on chloride excretion. Additional studies
conducted in rats orally loaded with saline or sodium

bicarbonate tend to confirm the above finding as illustrated

40

in figure 6. There appeared to be no significant alteration
in the chloruretic response following DT-327 administration
between rats pretreated with saline or bicarbonate. It

has been reported by Baer gt gt. (1959) that the thiazides
possess carbonic anhydrase inhibitory activity. It was
also noted that the enhanced bicarbonate excretion produced
by hydrochlorothiazide during alkalosis was of a lesser
magnitude than that produced by an equivalent dose of
chlorothiazide. Such differences appear to be related to
the greater inhibitory effect of chlorothiazide on carbonic
anhydrase. Beyer and Baer (1961) reported that as the
lipid solubility of a thiazide increases, its carbonic
anhydrase inhibitory activity decreases. Likewise, DT—327
being extremely lipid soluble, possesses some carbonic
anhydrase activity but not to such a degree as to signifi-
cantly alter the nature of the major anion affected.

In an attempt to delineate the tubular areas affected
by DT-327, the free water clearance technique was utilized.
Free water clearance has been defined as osmotically
unobligated water (Pitts, 1963). In the absence of anti-
diuretic hormone, the collecting duct is relatively
impermeable to water resulting in the excretion of a dilute
urine. If a drug were to act only on the proximal tubule
to inhibit isosmotic reabsorption, the excess solute
retained within the nephron would be accompanied by an
osmotically equivalent amount of water. The excess solute
would be partially reabsorbed in the distal nephron,

resulting in an increase free water clearance. If, on the

41
other hand, a drug acted distally to the site of isosmotic
reabsorption of water to prevent reabsorption of electro-
lytes, the excess solute delivered to the collecting duct
and later excreted would result in a decreased free water
clearance. The thiazides decrease free water clearance,
and this has been attributed to a distal site of action
(Earley gt gt., 1961; Seldin gt gt., 1966). Results with
DT-327 also show a significant depression of free water
clearance suggesting a distal locus of action for this
compound (figure 7). Negative free water clearance
(TCHZO), measured during maximal ADH activity is a measure
of renal concentrating capacity and results from sodium
reabsorption out of the ascending limb of the loop of
Henle (Seldin gt gt., 1966). Inasmuch as DT-327 had no
demonstrable effect on negative free water clearance
(figure 8), an effect on the loop of Henle was considered
unlikely.

In an attempt to confirm the data obtained from free
water and negative free water clearance studies which
indicated a distal site of action for DT-327, eXperiments
were performed utilizing the stOp-flow technique. As
discussed in the introduction, the interpretation of a
stop—flow pattern is highly questionable if the data
indicate an effect on the proximal tubule because urine
from this tubular segment has to pass through the distal
tubule after the occlusion period is terminated. Since

this is not the case with stop-flow samples collected from

42

the distal nephron, stop-flow patterns which suggest that
a drug inhibits reabsorption of sodium in the distal tubule
are not considered as questionable as those which suggest
drug inhibition of sodium in the proximal tubule.

Examination of the stop-flow pattern before and after
DT-327 administration clearly indicated that a major site
of action is the distal nephron. The results of an eXperi-
ment in which an animal was subjected to a slight modifica-
tion of the stop-flow technique are illustrated in
figure 9. This animal was given 2 g of sodium chloride
prior to occlusion thus preventing electrolyte depletion
during the mannitol diuresis. In 4 animals the distal
minimum of sodium, or the U/P ratio that represents the
tubular area in the distal nephron where maximum reabsorp—
tion of sodium normally occurs, was significantly increased
following DT-327 administration. Since sodium is not
secreted along the tubular nephron, such data suggested
that inhibition of sodium reabsorption had occurred.
Likewise, the U/P ratio of potassium also increased in the
distal nephron after an injection of DT-327. This would
be eXpected especially if sodium reabsorption was
inhibited in this tubular area. More sodium would now be
available to exchange with intracellular potassium, this
being prOposed as the major mechanism of potassium
secretion (Pitts, 1963). A similar stOp-flow pattern to
that of DT-327 has been reported to occur with the
thiazides (Cafruny and Ross, 1962). Thus, the effect of

DT-327 on free water and negative free water clearances

43
relate quite well with the stOp-flow data indicating
that a major site of action of this drug is the distal
nephron.

Pitts gt gt. (1958) demonstrated that chlormerodrin
was capable of producing its natriuretic response during
maximal natriuresis produced by chlorothiazide. Conversely,
chlorothiazide produced its effect during maximal
mercurial natriuresis. Since either agent was capable of
eliciting an additive response during maximal natriuresis
produced by the other, it was reasoned that they must be
acting by different mechanisms or at different sites along
the nephron. By the same reasoning, lack of a response
to a given agent during maximal natriuresis produced by
another has been interpreted to indicate that the two
agents act by the same mechanism or at the same site
(Hook and Williamson, 1965b). During maximal hydrochloro-
thiazide natriuresis (figure 10) DT—327 was without effect
except for a significant depression of potassium excretion
which was probably due to a general deterioration of the
animals. During maximal DT-327 natriuresis, hydrochloro-
thiazide was without effect (figure 11). Using the
reasoning outlined above, it could be assumed that the two
agents were acting by the same mechanism and at the same
tubular site. However, Small and Cafruny (1967) demon-
strated the dangers of concluding that lack of addition of
maximally effective doses of two agents implies that two

drugs are acting by a similar mechanism. It is possible

44
that two drugs would not be additive if they acted at
different steps in the same biochemical sequence or if they
differed in the affinity for the same biochemical receptor.
Nevertheless, the ability of DT-327 to decrease free water
clearance without altering negative free water clearance
in addition to the lack of addition with hydrochlorothiazide
gives ample support to the prOposition that both act at the
same site along the nephron.

In maximally effective doses furosemide ismore
efficacious than the thiazides (Hook and Williamson, 1965b)
and by implication more efficacious than DT-327. Further-
more, in four animals furosemide was capable of increasing
sodium excretion during maximal DT-327 natriuresis whereas
hydrochlorothiazide was not (table 5). Thus, even though
the precise biochemical site cannot be defined, it is
apparent that DT-327 produced a Specific blockade of the
action of the thiazide. Furosemide also has the ability
to decrease negative free water clearance (Seldin gt gt.,
1966) and to decrease renal vascular resistance (Hook and
Williamson, 1966a) both properties not shared by DT-327 or
the thiazides. It is concluded, therefore, that the
natriuretic activity of DT-327 more closely resembles the

thiazides than furosemide.

S UMMARY

The diuretic response to DT-327 is dose related. The
intravenous administration of DT-327 resulted in the in-
creased excretion of sodium, chloride, and potassium, the
responses observed being dose related. Maximum sodium
excretion, as well as other variables measured, occurred
in the first lO-minute interval following the administra-
tion of a maximal effective dose of DT-327 (l mg/Kg).

Infusion of DT-327 into the left renal artery resulted
in a significant ipsilateral increase in the excretion of
sodium within 5 minutes without affecting the rate of
glomerular filtration. A Slight contralateral response
was also noted occurring 10-15 minutes after the ipsilateral
response. At a dose capable of producing a significant
natriuresis, DT-327 did not alter renal blood flow and
thus did not affect renal vascular resistance.

Alterations of plasma pH with hydrochloric acid and
sodium bicarbonate did not appear to affect the natriuretic
response to DT-327. Increased bicarbonate excretion did
occur during metabolic alkalosis following drug administra-
tion indicating some carbonic anhydrase inhibitory activity,
but this did not seem to affect the increase in chloride
excretion observed.

DT-327 decreased free water clearance without altering
negative free water clearance indicating an action on the
distal nephron. The diuretic produced a significant
increase in the distal minimum of the stop-flow pattern

45

46
again indicating a distal site of action. During maximal
DT-327 natriuresis, hydrochlorothiazide was without effect,
and likewise during maximal hydrochlorothiazide natriuresis,
DT-327 was without effect. However, during maximal DT-327
natriuresis, furosemide was capable of producing an
increased effect. Thus, results from free water and
negative free water clearance studies along with the stOp-
flow, renal hemodynamics, and additive studies all seem to
indicate that the natriuretic activity of DT-327 more

closely resembles the thiazides than furosemide.

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50

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1963.

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TABLE 7

Effects of furosemide (5 mg/Kg, i.v.) during
maximal DT-327 natriuresisa

 

Electrolyte Excretion

 

.qu/min.
Na K Cl
DT-327 1200 112 1179
DT-327 plus furosemide 1978 154 2093
Mean Difference 778b 42b 914b
(t 3.3.) (48) (17) (15)

 

aElectrolyte excretion during the two lO-minute
collection periods prior to furosemide were averaged
as were the data from the two lO-minute collection
periods following furosemide. Values in the table

represent the means of 4 animals.

bSignificant difference (P .05).

57

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Figure 2:

Effect of increasing doses of DT-327 on sodium
excretion. Increasing doses were administered
intravenously when the diuretic response from
the previous dose had plateaued. Bars indicate
means (t standard error) of comparable clearance
periods from four experiments.

FIGURE 2

I
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Electrolyte

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61

Figure 3:

Effects of DT-327 (1 mg/kg, intravenously) on
sodium excretion in the dog during mannitol
diuresis. Bars indicate means (i standard
error) of comparable clearance periods from
five eXperiments.

FIGURE 3

440

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63

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65

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67

Figure 6:

Effect of increasing doses of DT-327 on sodium
and chloride excretion in rats orally loaded with
sodium chloride or sodium bicarbonate (25 ml/Kg).
The solid line represents those rats orally
loaded with sodium chloride while the broken line
represents those rats orally loaded with sodium
bicarbonate. One of three doses of DT-327

(0.01 mg/Kg, 0.1 mg/Kg, or 1.0 mg/Kg) was
administered to either the sodium chloride or
sodium bicarbonate oral load. The excretion data
obtained from twelve rats were averaged. The
verticle bars represent standard errors.

0-
C)
C)

400

200

200

FIGURE 6

400 600 800
A No (A) Eq/min.)

69

1000

 

1200

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hmmtso mo cowumnumHSHEpm mcfizoammm umnu ou pmummfiou Aaouusoov conSmSw wcflamm
cacouomwn mswnsm mESHo> means on A UV museummao Hmpmz comm mo mfinmcofiumamu mna Nb musmflm

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_

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u 0
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1 312325230 52; SE

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71

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mcw3oHH0m umnu on Umnmmeoo Aaouucoov co«msmcw mcwamm cacouummmn mcwuzo Ramouv
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0 DT-327

F
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l2

l0

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73

Figure 9:

Stop-flow analysis of the tubular site of action
of DT—327. DT-327 (1 mg/Kg) was administered
intravenously thirty.minutes before the second
occlusion. The closed circles represent the
control stOp-flow pattern and the open circles
represent the stOp—flow pattern after administra-
tion of DT-327. The arrow indicating maximum

PAH represents the stOp—flow samples where
maximum para-aminohippurate (PAH) secretion
occurred. U/P = (Urine/Plasma).

FIGURE 9

0.4
0.3 o o
HALL o
U/P in
0.2 o 0 o
o o o o o . . O.
. . °.°:"°:°.°°°:°°°.°:°°
0.1 . . o o . ”.0
4 minuto
occlusion ‘
- PAH
maximum
0.4 Q
i 3 o 0.. 00
o . 0
o 0.0. O
m 0° 0 o o
U/Pin . .3; . °
0 . o .
0.2 o .0 o
0.] o ’
O
. o
O O
O
O 5 IO )5 20

FREE FLOW ACC UMU lAT ED URINE VOL. (Ml)

75

O
25
FREE now

Figure 10:

Effect of DT-327 (l mg/Kg, intravenously)

during maximal thiazide diuresis. The excretion

data obtained during two lO-minute periods

after DT-327 were averaged. Bars represent

means (+ standard error) of four eXperimentS.
indicates clearance of inulin. The decrease

iEN potassium excretion was statistically

Significant.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

EXCRETION

 

Figure 11:

Effect of hydrochlorothiazide (3 mg/Kg, intra-
venously) during maximal DT—327 diuresis. The
excretion data obtained during two 10-minute
periods prior to and three lO-minute periods
after DT-327 were averaged. Bars represent
means (+ standard error) of four eXperiments.

CIN indicates clearance of inulin.

 

 

 

 

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HQ

L.

600 *-

 

I000
800

y Eq/min.

 

400
200 r

1N0

EXCRETED

 

79

MICHIGAN STATE UNIVERSITY LIBRARIES

3 1293 03

75 0916

1