The pharmacology of hydrochlorothiazide (Esidrix™), a new, orally effective sulfonamide diuretic

TOXICOLOGY

AND APPLIED

PHARMACOLOGY

The Pharmacology A New, Orally W. E. Research

BARRETT, Department,

R. A.

1, 333-349

(1959)

of Hydrochlorothiazide Effective Sulfonamide H.

RUTLEDGE,

CUBA

PhurmaceuticaI

Received

February

SHEPPARD, Products

AND Inc.,

(Esidrix’“), Diuretic A. J.

Summit,

PLUMMER New

Jersey

2, 1959

The search for a potent, orally effective, and clinically useful diuretic drug has been going on for many years with only moderate success. The organomercurial drugs have been looked upon as being not only potentially toxic but requiring frequent parenteral administration for optimal effects. Their irritant action on the gastrointestinal tract when given orally is well recognized. In a recent review of the chemistry of diuretic drugs, Sprague (1958) has summarized the relationship of the chemical structure of a series of compounds to diuretic activity. Among this group of compounds was a series of sulfonamides, the most active being 6-chloro-7-sulfamyl-1,2,4benzothiadiazine-1 ,l -dioxide with the generic name chlorothiazide (Diuril). The mechanism of action has been described by Beyer (1958). It is the purpose of this presentation to describe the activity of an orally effective sulfonamide diuretic, 6-chloro-3,4,-dihydro-7-sulfamyl2H-1,2,4-benzothiadiazine-l,l-dioxide, with the generic name hydrochlorothiazide (Esidrix). The chemistry of this compound has recently been reported by destevens et al. (1958) and has the following structural formula :

‘: Cl / N\CH, “POzS a\ s’LH METHODS

A.

Canine Diuretic Experiments Six mongrel dogs were used in these experiments. These animals have been employed in various diuretic experiments for more than a year. At 333

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ET

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the start of each experiment the dogs received a subcutaneous injection of 100ml of 0.9% saline. Immediately before the saline injection they received the compound orally by capsule. Urine was collected by catheterization at intervals of 2, 4, and 6 hours after the administration of the drug. Urinary sodium and potassium concentrations were measured by means of a Baird flame photometer and expressed as milliequivalents of sodium and potassium excreted in 6 hours or in each of the three successive 2-hour intervals. Urinary chloride analyses were carried out by a polarographic method developed by Allen (1958), and the concentration was expressed in the same units as for sodium and potassium. The results are expressed as the average volume of urine in milliliters and average milliequivalents of electrolyte excreted per dog in the 6-hour period or for each 2-hour interval. Saline control experiments were obtained prior to the drug experiments and also at intervals during the course of the drug studies which were performed at weekly intervals. During a control or drug experiment, water and food were withheld from the dogs. Both hydrochlorothiazide and chlorothiazide were administered orally in a graduated dosage schedule in which the interval between successive doses varied by a factor of four. The relative potencies of these compounds in promoting water and electrolyte excretion were determined by a two by three or two by two factorial assay in randomized blocks, since all of the dogs received each of the various doses of each drug. B.

General Pharmacology

1. Eflect 071 the canine cardiovascruhzr system. Mongrel dogs were anesthetized with pentobarbital sodium, and the anesthetic state was maintained by an intravenous drip of 60 pg/kg/min of pentobarbital sodium administered in a total volume of 0.5 ml/min. The blood pressure was recorded from the femoral artery by means of an Anderson (1941) glass capsule manometer. Respiration was recorded from the cannulated trachea by means of an Anderson (1953) glass inspiratory manometer. All drugs were administered intravenously into the femoral vein. Hydrochlorothiazide was dissolved in warm water in a concentration of 0.5% solution which was prepared immediately prior to injection. 2. Direct cardiac .efiects. a. The direct cardiac activity of hydrochlorothiazide was studied on the isolated feline heart preparation. The drug was dissolved in the perfusion fluid and perfused through the heart at concentrations of 1, 10, and 100 pg/ml.

HYDROCHLOROTHIAZIDE-AN

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335

DIURETIC

3. Effect on gastrointestinal sys.&m. a. Hydrochlorothiazide was studied for its anticholinergic, antihistaminic, and spasmolytic activity on the isolated ileum of the guinea pig suspended in the isolated glass tissue baths described by Anderson .et aZ. ( 1949). b. The effect of this drug on gastric secretion was studied in unanesthetized dogs having surgically prepared gastric fistulas. Histamine diphosphate in a total dose of 1.5 mg subcutaneously was employed as the standard stimulus of gastric secretion. The hydrochlorothiazide was administered intravenously in a dose of 1.0 or 3.0 mg/kg 30 minutes before the injection of the histamine. Gastric secretion was collected every 15 minutes for 2 hours, and the free hydrochloric acid was determined by titration with 0.01 N sodium hydroxide. The milligrams of free acid were calculated for each 15minute interval and these results were plotted graphically. C. Hydrochlorothiazide was studied in anesthetized dogs each of which had a surgically prepared Thiry-Vella loop for its effect on the smooth muscle of the ileum. In addition, the effect of hydrochlorothiazide on the responses of the ileum to intravenously administered histamine and methacholine were also obtained. C.

Urivze Excretion Studies

The urinary excretion of hydrochlorothiazide was determined in a &hour period in the same dogs which were employed in the diuretic studies after oral doses of 1.25 and 5.0 mg/kg. For the analysis, the urine was hydrolyzed with 10 N NaOH for 30 minutes at 83°C and the product of the hydrolysis was determined by a modification of the method of Bratton and Marshall (1939). In other experiments the pH of the urine was measured at each of the 2-hour collection periods. D.

T.otiity

The intravenous LDsO of hydrochlorothiazide and of chlorothiazide was determined in rats.’ Because of relatively low solubility in aqueous solutions, a 105% solution was prepared in 80% N,N-dimethylacetamide and 20% water. Ten female rats were employed at each dose level. Doses were administered via the tail vein over a 30-second interval. The mortality that ensued during the 48 hours following the administration of the drug was considered in the estimation of the LDZo values. Solutions 1 The

toxicity

studies

were

performed

by Dr.

A. E. Earl

and

his associates.

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ET

AL.

of the vehicle were administered as a control in the same volumes as employed for each dose level of the LDhO study. The oral LDc0 for hydrochlorothiazide was also determined in rats, using a 20% solution of the drug in 0.1 N sodium hydroxide. RESULTS

A.

Oral Diuretic Activity

in the Dfog

Table 1 summarizes the results obtained in these experiments. It will be TABLE COMPARISON

OF THE TION

URINE

1

BY Dam

EXCRETION

OF HYDROCHLOROTHIAZIDE

FOLLOWING AND

THE

ORALADMINISTRA-

CHLOROTEIAZIDE

Average milliliters of urine excreted per dog per 6 hours

Oral dose/kg (4

Hydrochlorothiazide

Control 0.02 0.04 0.08 0.16 0.3 1 1.25

Chlorothiazide

51.9

51.9

61.5 67.6

-

77.9 77s

48.8 55.5 87.7 96.5

105.1

126.3

noted that the minimally effective oral dose of hydrochlorothiazide was 0.02 mg/kg. Chlorothiazide at a dose of 0.16 mg/kg, eight times the minimally effective dose of hydrochlorothiazide, still failed to evoke a diuretic response in these same dogs. The relative potencies of the two compounds were calculated for water, sodium, potassium, and chloride excretion and are presented in Table 2. TABLE A COMPARISON

OF THE AND

Pa*sIWter

Water excretion Na excretion K excretion Cl excretion

RELATIVE

CHLOROTTIIAZIDE

2

POTENCIES

OF HYDROCHLOROTHIAZIDE

AS DIURETIC

DRUGS

Relative potency

Confidence limits at p = 0.05

6.3

2.7-10.9

5.0

1.9-16.3

4.4

1.6-13.3

9.4

5.8-18.8

HYDROCHLOROTHIAZIDE-AN

ORAL

337

DIURETIC

Hydrochlorothiazide was found to be 6.3 times as potent as chlorothiazide with respect to urine excretion, 5.0 times as potent in terms of sodium excretion, 4.4 times as potent in terms of potassium excretion, and 9.4 times as potent with respect to chloride excretion. In order to compare the duration of action, the water, sodium, potassium, and chloride excretion values were calculated for three successive 2-hour periods. These results are presented in Figs. l-4. Examination of these graphs indicates that hydrochlorothiazide is not only more potent, I

I m

CONTROL

m

HYOROCHLOROTHlAZlDE

0

DIURIL

: -E~IDRIX=SU-5879

z I .

i

60 i

%ose~O.OEmg/kg

.

/

i I I I I

Do**= 0.31mg/kg

: I I

Dose:1.25

mdkg

i

2-4

4- 6

O-2 TI

tn

darn

cdminirhrrd

omlly

IME 2-4

4-6

o-2

2-4

4-6

: IN HO

Comparison of the duration of the diuretic effect in dogs of hydrochlorothiazide and chlorothiazide after oral doses of 0.08, 0.31, and 1.25 mg/kg. Each column represents the average total urine excreted per dog for three successive 2-hour intervals. FIG.

1.

but possesses a greater duration of action as well. At the 0.08 mg/kg dose level, chlorothiazide failed to evoke a diuretic response at any of the 2-hour intervals. As the dose was increased, the longer duration of action of hydrochlorothiazide resulted in a marked diuretic effect even at the third 2-hour (4-6 hour) interval. Chlorothiazide regularly showed a reduction in diuretic activity at the 2-to-4 hour interval, compared with the O-to-2 hour interval, and failed to exhibit any diuretic effect at the 4-to-6 hour period with the doses employed. A comparison of the urinary excretions of sodium, potassium, and chloride after the administration of the two drugs revealed the same general excretory pattern as was

338

W.

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E.

BARRETT

ET

AL.

CONTROL

; I HYOROCHLOROTHlAZlOE-ESIDRIX’SU-5879 I DIUAIL :

Im I2

: : : :

*Dor.=O.OEIma/kg

: I

IO-

Doro=Odl mg/kg

Dor.L2Smg/kg

:

6-

B b w” E

64-

1

e-

2-4

o-2

4-6

o-2

2-4

4-6

o-2

TIME IN HOURS

FIG. 2. Comparison of the duration of the natriuretic effect in dogs of hydrochlorothiazide and chlorothiazide after oral doses of 0.08, 0.31, and 1.25 mg/kg. Each column represents the average total milliequivalents of sodium excreted per dog for three successive 2-hour intervals. m

CONTROL

m

HYDROCHLOROTHIAZIDE-&DRIX:SU-SS79 DIURIL : I

lo

3.oc

: : : :

:

0orr:1.25mg/kg

:

i

o-2

2-4

4-6

o-2

2-4

4-6

o-2

2-4

4-6

TIME IN HOURS *all doll

adminiM)urdomlly

FIG. 3. Comparison of the duration of the kaliuretic effect in dogs of hydrochlorothiazide and chlorothiazide after oral doses of 0.08, 0.31, and 1.25 mg/kg. Each column represents the total milliequivalents of potassium excreted per dog for three successive 2-hour intervals.

HYDROCHLOROTHIAZIDE-AN

ORAL

339

DIURETIC

observed for water excretion, with hydrochlorothiazide exhibiting the greater duration of action when both drugs were administered at the same dose level. This was especially striking in respect to the urinary excretion of chloride.

I I .o

I

m

CONTROL

m

HVOROCHLOROTHlAZlOE - ESIDRIX= SU-5.679

0

CHLOROTHIAZIDE - DIURIL

lO.O-

s.ooowOalmg/kg SO-

0 f

5.0

4.0

3.0

2.0

I .o

2-4

4-6

o-2

z-4

4-6

o-2

2-4

4-6

TIME IN HOURS

FIG. 4. Comparison of the chloruretic effect in dogs of hydrochlorothiazide and chlorothiazide after oral doses of 0.08, 0.31, and 1.25 mg/kg. Each column represents the total milliequivalents of chloride excreted per dog for three successive Z-hour intervals.

B.

General Pharmacology 1. Cardiovascular system. When hydrochlorothiazide was administered intravenously in doses up to 9.0 mg/kg it did not produce a significant decrease in the mean arterial blood pressure in twelve separate experiments.

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The compound was also studied in the anesthetized dog for any effect on the responses evoked by I-epinephrine, l-norepinephrine, acetylcholine, and histamine. It appeared to cause a slight decrease in the pressor response elicited by I-norepinephrine but exhibited no constant effect against the vascular responses following the injection of Lepinephrine and acetylcholine. However, the depressor response to histamine was potentiated 1-2 hours after the intravenous administration of 9.0 mg/kg of hydrochlorothiazide in six of twelve experiments (Fig. 5). A bradycardia developed in approximately 5’0% of the dogs l-3 hours after administration of the drug. The intravenous administration of 9.0 mg/kg of hydrochlorothiazide after prior injection of a nonhypotensive dose (300 yg/kg) of hydralazine, produced a hypotensive response of 6-25 mm Hg (average, 16 mm Hg) in the mean arterial blood pressure. This type of hypotensive effect is illustrated in Fig. 6. When this dose of hydralazine was administered intravenously 2-3 hours after a previous injection of hydrochlorothiazide, a hypotensive response resulted. The average decline in the mean arterial pressure amounted to 21 mm Hg (Fig. 7). 2. Dtiect cardiac eflects. Concentrations of 1.0 and 10.0 ug/ml of hydrochlorothiazide produced no effect on the isolated perfused feline heart. A concentration of 100 pg/ml caused a slight decrease in the amplitude of the contraction. The inotropic activity of I-epinephrine and I-norepinephrine was not altered in these experiments. 3. Gastrointestinal tract. Hydrochlorothiazide in the doses employed did not prevent contraction evoked by acetylcholine or histamine on the isolated guinea pig ileum. It would appear, therefore, that this drug has no significant anticholinergic or antihistaminic activity. Intravenous administration of hydrochlorothiazide, 1.O mg/kg, produced neither a stimulation of the motility of the intestine nor a decrease in the tone of the smooth muscle (ileum) in a dog with a Thiry-Vella loop. At this dose the compound definitely augmented the stimulatory effect of histamine and appeared to decrease the duration of the stimulatory response induced by methacholine (Fig. 8). A study of the effect of hydrochlorothiazide on gastric secretion was carried out in the unanesthetized dog with a surgically prepared gastric fistula in which gastric secretion was induced by the subcutaneous administration of histamine. Hydrochlorothiazide per se did not stimulate gastric secretion when administered intravenously at a dose of 3.0 mg/kg

HYDROCHLOROTHL‘UIDE-AN

ORAL

341

DIURETIC

30 minutes before the> injection of histamine. In some experiments the drug caused an initial slight increase in the gastric secretory response induced by histamine, which was followed by a decreased sensitivity of EFFECT

OF ESIDREX

(SU-5679)

RESPONSES,

OF THE

ON THE

1 -Epinephrine -E 1 -Norepinephrine=NE Dosed kg W)

200

roi

Ii:27

-

DOG TO;

HirtaminwH AcetyichoiinvA 10-9-56

L L

I 60 -‘urc -I

Tlmo

CARDIOVASCULAR

ANESTHETIZED

Ii:41

Ii:33

Ii:46

Ii:56

12:oo 200 160 120

40 E ‘PLg

A

NE

5p9

‘pLe

F ‘W

5M

A =tQ

2t29

2:37

2149

0

n

NC

‘PJJ

II09

1x5

FIG. 5. Potentiation in an anesthetized dog

I:24

1129

of the cardiovascular depressor treated with hydrochlorothiazide.

response

to injected

2:56 histamine

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ET

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the gastric mucosa to subsequent injections of histamine for several days after the original dose of hydrochlorothiazide (Fig. 9A). In other experiments, no initial increased sensitivity to histamine was observed, but, nevertheless, a decreased response of the gastric mucosa to the subsequent stimulatory action of histamine was noted (Fig. 9B). Anesthetized Dog-Pentoborbitol L-Epinephrine =E L-Norepinephrine =NE Doses/ kg W.)

Sodium Hydrochlorothiazidr = Hy Hydralarinr=H 9-E-58

‘Femorol Blood Pressure

Respirotlon

10:37

200-e-~

IO:41

10: 57

II:18

I I:24

II:33

-

160 120-m, 00 40 0

rime

FIG. 6. hypotensive

-

II 42

Hypotension produced dcse of hydralazine.

12:oo

by

hydrochlorothiazide

l2:15

4:02

administered

after

a non-

HYDROCHLOROTHIAZIDE-AN

C.

ORAL

343

DIURETIC

Urinary Excretion Study

The results obtained from the study of the urinary excretion of hydrochlorothiazide by dogs are summarized in Table 3. Six hours after the oral administration of 1.25 mg/kg of the compound, an average of 38.2% Hydrochlorothiozide,9.0mg./Kg injected 3% hr before Hydmlozine Hydrolozine=H Doses/kg IV 200m=H.R. 160 BP

I 20

mHg 60

RESP.

212

1 3oop9

236

HR H 160 1 300/Q FIG. 7. Hypotension produced by hypotensive dose of hydrochlorothiazide. failed to produce a hypotension when

11-5-56

HR I60

hydralazine when administered Thii dose of bydralazine given alone.

after a non(300 pg/kg)

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ET

AL.

of the total dose was excreted in the urine. After an oral dose of 5.0mg/kg, an average of 9.0% of the total dose administered was excreted in the urine. Thiry-Vallo

Loop

Anesthesia Dose/

kg

Sodium

(I.‘.‘.)

Drugs-SLJ-5979 Recording

Dog

- Pentobarbitol

Hydrachlorothiozldo

Hitiaminr,

Mecholyl

from ileum of small intestine

IO-ID-m

Hirtomine

I.Omdkg

l.O/&kglY.

I.V.

l-M-84

HR-92

After Drug

Mecholyl !ZO&kg

Histoml’nr l*O~/kg

I.V.

L.

FIG. 8. Effect dog. Potentiation

of

hydrochlorothiazide of the histamine

on response

the by

IY

small intestine hydrochlorothiazide.

of

the

anesthetized

HYDROCHLOROTHIAZIDE-AN

ORAL

34.5

DIURETIC

Measurement of the pH of the urine at three successive 2-hour intervals after oral administration of 0.3 1 mg/kg of hydrochlorothiazide and 1.25 mg/kg chlorothiazide revealed that the pH of the urine after chlorothiazide was more alkaline (pH = 8.70, 8.70, 8.83) for each of the 2-hour collection periods than after the administration of hydrochlorothiazide (pH = 6.61, 6.52, 8.69). D.

Toxicity Studies

“I I’

The relative_insol_ubility of hydrochlorothiazide in water necessitated the employment of a different vehicle as the solvent. With the high doses

-Histamine

Control

Rasponse

- - Histamine msponse (M hour after Hydrochlorothiadda 20-

...**-* Histamine response 4 doys oftr HydrocMorothiorido

.4 CR \ ‘5Tl IO

?

\

I'

5

I \

\

I’

\

\

I’

L

=-2

15

30

45

60 TIME

FIG.

as total

9.

Effect milligrams

of hydrochlorothiazide of free HCl per

I

75

90

IO6

120

IN MINUTES

on canine 15 minutes.

gastric

secretion.

Results

plotted

346

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ET

AL.

required in these studies in rats to attain an LDj,, value, the vehicle itself produced toxic effects. Thus it was not possible to obtain a precise LDSO. The estimated intravenous LDSO in rats was 350 mg/kg for hydrochlorothiazide and 200mg/kg for chlorothiazide. The oral LDjo of hydrochlorothiazide in rats was found to be approximately 2.75 g/kg. TABLE URINARY

Dog “0. 4

36

80

139

EXCRETION

Oral d&kg (wd

3

OF HYDROCHLOROTEIAZIDE Milligrams o-2 hours

IN

THE

excreted

2-4 hours

44 hours

1.25

1.73

1.25

0.00

5.00

2.4

1.3

0.9

1.25

1.93

1.39

0.38

5.0

2.4

1.4

0.6

1.25

2.28

6.3

3.22

5 .oo

1.9

5.3

0.4

1.25

0.43

1.83

1.98

5.00

0.9

0.8

3.2

DOG Per cent excreted 04 houra 22.1 8.4 34.1 8.6 65.2 9.9 31.4 8.9

DISCUSSION

A comparative study of the absolute potencies of these compounds in the dog has revealed that hydrochlorothiazide is more than eight times as potent as chlorothiazide. Absolute potency is expressed as the difference between the threshold doses of each drug. Relative potency was computed for the following four parameters: water, sodium, potassium, and chloride excretion. For each of these parameters hydrochlorothiazide proved to be more potent than chlorothiazide. A major factor in the quantitative advantage of hydrochlorothiazide as a diuretic is its prolonged duration of action. As the oral dose of the drug was increased, the intensity of the diuretic response as measured in the first 2 hours was enhanced, but the duration of the diuretic response of hydrochlorothiazide was increased to an even greater extent. Meier st al. (1954) has emphasized the inherent practical advantage when a pharmacologic agent increases the duration of the effect rather than the intensity of the initial response as the dosage is increased. One of the striking differences in action between these two compounds is the significantly greater urinary excretion of chloride following the oral administration of hydrochlorothiazide (Fig. 4). The major anion excreted was the chloride ion. With an increase in the oral dosage the duration of the increased urinary excretion of the chloride ion also in-

HYDROCHLOROTHIAZIDE-AN

ORAL

DIURETIC

347

creased. This increased chloride excretion is further reflected in the fact that the urinary pH is far less alkaline. Thus hydrochlorothiazide would be expected to cause less disturbance in acid-base balance at the clinical level. Hydrochlorothiazide has been found to possess but one-ninth the carbonic anhydrase-inhibiting activity of chlorothiazide in vitro (Sheppard, unpublished data). This is consistent with the observation that it evokes a more marked chloruretic response than does chlorothiazide. The exact mechanism by which the drug is eliminated by the kidney is not known. However, the difference in relative potencies cannot be explained on the basis of a difference in rates of urinary excretion, since both compounds exhibited similar urinary excretion time patterns. Renzi et #al. (1959) have recently found that hydrochlorothiazide is much more potent than chlorothiazide in terms of water, sodium, and potassium excretion in the rat. Thus the results obtained in two different species of animals by different techniques are in agreement. From the results obtained from the studies on the antihypertensive properties of hydrochlorothiazide it would appear that this drug will not produce hypotension in normotensive individuals. The fact that hydrochlorothiazide potentiated the hypotensive effect of hydralazine, evoked a hypotensive response per se after nonhypotensive doses of hydralazine and potentiated the cardiovascular depressor response to intravenously administered histamine, suggests that it may increase the responsiveness of the dog to endogenous histamine. This activity may account for its augmentative effect with hydralazine. The increased responsiveness to endogenous histamine may be secondary to electrolyte alterations brought about by hydrochlorothiazide. It does not appear probable that the hypotensive effect evoked by hydralazine, administered after a prior injection of hydrochlorothiazide, was due in part to a dehydration of the animal-a dehydration produced by this potent diuretic drug-because each of the dogs received a constant infusion of a small dose of pentobarbital dissolved in saline (see Methods). The volume of saline administered amounted to 30 ml/hr. Hydrochlorothiazide per se did not produce a stimulation of the small intestine of the dog, nor did it cause a stimulation of the gastric secretory mucosa. It appeared, in fact, to decrease the gastric secretory response induced by histamine. No gastrointestinal disturbances were noted with any of the orally administered doses of hydrochlorothiazide employed in the canine diuretic studies.

348

W.

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BARRETT

Based on the acute studies in rats, be less toxic than chlorothiazide.

ET

AL.

hydrochlorothiazide

was found

to

Hydrochlorothiazide has been found to he a potent, orally eff&ive diuretic drug. This activity combined with its cardiovascular inkractions with hydralazine suggests that it may have an important clinical application in the treatment of hypertension. SUMMARY 1. On the basis of comparative minimally effective diuretic doses, hydrochlorothiazide was eight times as active as chlorothiazide, and on the basis of dose-

response effects, it was 6.3 times more potent. In terms of the relative potencies for influencing each of the four parameters selected, the excretion of water, sodium, potassium, and chloride, hydrochlorothiazide was found to predominate over chlorothiazide. In addition, this drug is characterized by a long duration of action. 2. The major anion excreted by the dogs thiazide was chloride rather than bicarbonate,

under thus

the influence of hydrochloroyielding a less alkaline urine.

3. It was found that hydrochlorothiazide increased or potentiated the activity of the antihypertensive drug, hydralazine, which in turn augmented the antihypertensive properties of hydrochlorothiazide. The hypotensive response elicited by histamine was also enhanced by hydrochlorothiazide in 50% of the experiments. 4. Hydrochlorothiazide did not stimulate it produce a stimulation of gastric secretion, secretory response produced by histamine. 5. toxic

In acute toxicity than chlorothiazide.

studies

6. It has been suggested that of dogs to endogenous histamine. latory effects.

in

rats,

the but

small rather

intestine tended

hydrochlorothiazide

hydrochlorothiazide

This activity

may

of the dog, nor did to reduce the gastric was

increase

found

to

be

less

the responsiveness

could account in part for its circu-

REFERENCES (1958). Personal communication from CIBA Chemical Laboratories. F. F. (1941). A glass-capsule manometer for recording the blood pressure. J. Lab. Clin. Med. 26, 1520-1521. ANDERSON, F. F. (1953). Quantitative volumetric respiratory recorder. Arch. intern. phermacodynamie therap. 94, 460-461. ANDERSON, F. F., BARRETT, W. E., and CRAVER, B. N. (1949). A multiple bath arrangement for studying the responses of isolated tissues. Arch. Snntern. pharmacodynamie therap. 18, 544-547. BEYER, K. H. (1958). The mechanism of action of chlorothiazide. Ann. N. Y. Acad. Sci. 71, 363-379. BRATTON, A. C., and MARSHALL, E. K., JR. (1939). A new coupling component for sulfanilamide determination. J. Biol. Chem. 126, 537-550. DESTEVENS, G., WERNER, L. H., HALAMANDARIS, A., and RICCA, S., JR. (1958). Dihydrobenzothiadiazine dioxides with potent diuretic effect. Ezperientiu 14, 463.

ALLEN,

M.

ANDERSON,

HYDROCHLOROTHIAZIDE-AN

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DIURETIC

349

MEIER, R., BEIN, H. J., GROSS,F., and TRIPOD, J. (1954). Pharmacodynamic components of hypotensors and their significance to the hypotensive effect. Presented at the 3rd International Congress for Internal Medicine, Stockholm, Sweden, September, 1954. Acta Med. Sound. 164, Suppl. 312, pp. 165-175. RENZI, A. A., CHART, J. J., and GAUNT, R. (1959). Sulfonamide compounds with high diuretic activity. Toxicol. Appl. Pharmacol. 1, 406-416. SHEPPARD,H. Unpublished data. SPRAGUE,J. M. (1958). The chemistry of diuretics. Ann. N. Y. Acad. Sci. 71, 328-343.