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Studies on a New 1, 5-Benzothiazepine Derivative (CRD-401)
STUDIES
ON A NEW 1,5-BENZOTHIAZEPINE DERIVATIVE (CRD-401) VI. EFFECTS ON RENAL BLOOD FLOW AND RENAL FUNCTION Isao YAMAGUCHI, Katsuo IKEZAWA, Toshikazu TAKADA and Akio KIYOMOTO Pharmacological
Research
Laboratory,
Toda, Accepted
Saitama, January
Tanabe
Seiyaku
Co.,Ltd.,
Japan 16,
1974
Abstract -Effects of a new coronary vasodilator, CRD-401, on renal blood flow and renal function were investigated in anesthetized dogs. Renal arterial or systemic administration of the compound produced an increase in renal blood flow. The increase in renal blood flow induced by CRD-401 was not affected by pre-treatment with propranolol, atropine or dephenhydramine. Renal arterial administration of CRD-401 exhibited an antagonistic effect on the vasoconstricting action of angioten sin-II, whereas it had no effect on that of epinephrine. When infused continuously into the renal artery, CRD-40l increased the urine flow and sodium excretion as well as renal blood flow in all the conditions of fluid loading tested. The glomerular filtration rate was enhanced under saline loading but not under water diuresis. CRD 401 caused an increase in free water clearance. When the renal blood flow was kept constant with an aortic clamp during CRD-401 infusion, sodium excretion was signifi cantly increased but no change was observed in glomerular filtration rate and PAH clearance. The results show that CRD-401 induced natriuresis was not entirely dependent upon the renal hemodynamic changes caused by the compound. By the stop-flow method, it was shown that the ratio of [urine sodium to plasma sodium] to [urine creatinine to plasma creatinine] increased in the distal portion of the nephrun by CRD-401 infusion. The present results indicate that the natriuretic action of' CRD-401 is not only due to the changes in renal hemodynamics but it also may be ascribable to a direct effect of CRD-410 on the sodium reabsorption in the distal part of the nephron. A new benzothiazepine
derivative,
ethyl]-2-(p-methoxyphenyl)-I, has been shown blood
to have a vasodilating
flow without
augmenting
effect of the drug is assumed In addition pound
to the coronary
produced
d-3-acetoxy-cis-2,
5-benzothiazepin-4(5H)-one action
myocardial
and
3-dihydro-5-[(dimethylamino) hydrochloride
to produce
oxygen
an increase
in renal
activity,
an increase
consumption
to he direct action on coronary vasodilating
(CRD-40I)
(2).
blood flow and exerted
in coronary
The vasodilating
and femoral
it was also reported
(1),
blood vessels (3).
briefly that the com
influence
upon
renal func
tion (2, 4-6). In the present experiments, esthetized
dogs.
netic flowmeter amined
Firstly,
and secondly,
by clearance
also investigated.
the renal action of CRD-401
the renal vasodilating
technique.
was studied
action
was investigated
effects on renal function
and electrolyte
The mechanism
of diuretic
action
in detail in an
using electromag excretion
were ex
of the compound
was
I.
Renal r•asoclilatin,raction Male mongrel dogs weighing 15 to 18kg were anesthetized with sodium pentobar bital (P.B.) (35 mg'kg, .v.). The left renal artery was exposed through a flank incision and a probe connected to a squarewave electromagnetic flowmeter (Nihon Kohden, MF 25) was placed around the renal artery. Blood pressure was monitored from the femoral artery with a pressure transducer. Both renal blood flow (RBF) and blood pressure were recorded simultaneously on an ink-writing oscillograph. The renal vasodilating actions of drugs were estimated by measuring the maximal increase in the blood flow measured from the pre-administration level. Drugs were administered into the renal artery through a catheter that terminated with a 22 gauge needle inserted into the artery, or into the cannu lated fermoral vein. In order to investigate the mechanism of vasodilation by CRD-401, an inhibitor such as adrener-*c E~blocker (propranolol), anticholinergics (atropine) or antihistamine (di phenhydramine) was injected into the femoral vein through a cannula 3 to 10 min prior to administration of CRD-401. In experiments where interaction of CRD-401 with a vasoconstrictor such as angio tensin-II or epinephrine was studied, the vasoconstrictor was injected into the renal artery before and during the continuous infusion of CRD-401. 2. Renal Jllnctinl (1) Clearance e.tperintents Male mongrel dogs weighing 16 to 20 kg were anesthetized with P.B. (35 nlgkg, i.v.). The trachea was cannulated and the animal was ventilated by an artificial respirator with room air. Both ureters were exposed through a small abdominal midline incision and catheters were introduced into the renal pelvis. The femoral artery and vein were can nulated for sampling blood and loading saline or glucose solution, respectively. The left renal artery was exposed through a flank incision and a probe was placed around the ar tery. RBF and blood pressure were measured by the methods mentioned above. A 22 gauge needle connected to a polyethylenecatheter was inserted into the renal artery through which saline was infused continuously with an infusion pump at a rate of 0.3 ml,/min. Drugs were dissolved into saline. Clearance studies were performed under one of three conditions of fluid load: a) a priming load of 200 nil of isotonic saline and the continuous infusion of saline at a rate of 2 ml min (small saline load), b) a priming load of 400 ml of 6, dextran and a constant infusionof 2.5 ° glucose solution at a rate of 9.5 ml/min (water dieresis) and c) an initial load of 400 nil of isotonic saline and a continuous infusionof iso tonic saline at a rate of 5.5 ml' min (mild saline load). Glomerular filtration rate (GFR; was determined by measuring inulin or creatinine clearance. Clearance of para-ami nohipptn-icacid (PAH) was measured simultaneously with RBF, determined by an elec. tromagnetic llowmeter. Inulin, or creatinine and PAH were dissolved in the infusion SO. lotions mentioned above and given into the femoral vein. Each priming and sustaining close was 1.8 ~-,10 min and 20-25 mg,rain for inulin, 2.5 g/ 10min and 15-20 mg/min for
creatinine, and 200 mg/10 min and 2-3 nag/min for PAH, respectively. After urine flow had become stabilized, periodic collections of urine and blood were started. Several u rine samples serving as controls were collected during saline infusion into the renal artery. The saline infusion was then replaced with infusion of a saline containing CRD-401 and urine samples were collected. Each collection period was 5 or 10min. Blood samples were drawn at the mid-point of each urine collection period. (2) Aortic clamp experiments In order to examine the effect of increased RBF produced by CRD-401 on the na triuretic response, urinary sodium excretion rate was determined under conditions where the change in RBF caused by the continuous infusion of CRD-401 was prevented by means of an aortic clamp (7). A variable resistant clamp was placed around the aorta just prox imal to the left renal artery of the anesthetized dogs. The renal perfusion pressure was estimated through the catheter in the left femoral artery and the systemic blood pressure was monitored from the carotid artery. A mild saline load was performed as mentioned previously. Isotonic saline was infused continuously into the renal artery at a rate of 0.3 ml/min throughout the experiment and CRD-401 administration was performed by adding the drugs to the infusate (10 fig/kg/min). When the rate of urine flow became constant, two 10 min collections of urine were carried out and blood samples were drawn at the mid point of each urine collection period. Following collection of control samples of urine and blood, CRD-401 was infused into the renal artery and the renal perfusion pressure was reduced by way of the aortic clamp so as to eliminate the increment in RBF caused by CRD-401 and to keep it constant at the control level before CRD-401 administration. Under these conditions, two to three samples of urine were collected in 5 or 10 min periods and blood samples were drawn at the mid-point of each period. Control experiments were run for each animal by clamping the aorta to give the same decrement in renal perfusion pressure as observed during CRD-401 infusion. Urine and blood samples were collected as described above. (3) Stopflow experiments The site of action of CRD-401 diuresis along the nephron was examined in dogs by stop-flow technique (8). Osmotic diuresis was established by an intravenous infusion of 20 mannitol in isotonic saline at a rate of 9.5 ml/min. Creatinine and PAH were added to the infusion solution. Experiments were divided into two periods: an initial control period and subsequent experimental period during the continuous infusion of CRD-401 into the renal artery. After a high and stable rate of urine flow (5 to 10ml/min) had been obtained, urine samples were collected in two 5 min periods and blood samples were drawn at the mid-point of each urine collection. A clamp was used to block off the ureter for 4 min, during which time a blood sample was drawn. After the release of the clamp, 30 serial urine sample of 0.5 to 0.8 nil in volume were collected. The constant infusion of CRD-401 into the left renal artery was then started and the same procedureswere repeated. 3. /l iralrsis Sodium and potassium concentrations in plasma and urine were determined using an
Evans flame photometer.
Concentrations
urine were determined
by the methods
al. (1 1) respectively.
Osmolality
Ramsay's 4.
method
of inulin, creatinine
of Davison
of plasma
and PAH
in plasma
and
et al (9), Peters (10) and Hamburger
and urine
was measured
et
cryoscopically
by
(12).
Drugs The drugs used in the present experiments
(Iwaki
Seiyaku),
propranolol
(I.C.I.),
(Tanabe
Seivaku),
mine hydrochloride Univ.),
epinephrine
hydrochloride
Kugita et al. (I) of Organic
Chemistry
were as follows:
atropine
sulfate
angiotensin-II (Sankyo Research
papaverine
hydrochloride
Seiyaku),
diphenhydra
(Tanabe
(Institute
Seiyaku). Laboratory
for Protein
CRD-401 of Tanabe
was
Research,
Osaka
synthesized
Seiyaku
Co.,
by Ltd..
RESULTS 1.
Renal vasoililating
el/eel
(1) Effect on renal blood flow CRD-401, when injected into the renal artery, produced an increase in RBF.
With
increasing doses of the drug, greater increase in RBF was obtained, while the systemic blood pressure remained unaltered (Fig. I-A). When papaverine was injected into the renal ar tery, it also produced a dose-related increase in RBF. However, the time course of the change in RBF produced by papaverine was somewhat different from that produced by CRD-401 (Figg.1-B). CRD-101 caused only an increase in the blood flow, while papave rine, in most cases, gave a biphasic response, i.e., an increase followed by a decrease.
From
the dose-response curve in Fig. I-A, it was concluded that renal vasodilating potency of
Fi
Fiu. 2. Effect of i.v. injection of CRD-401 on renal blood flow. BP-blood pressure, RBF=renal blood flow.
CRD-401 was more than three times that of papaverine. CRD-401 also produced about 10'/',o'increase in RBF, when given intravenously at a dose of 100beg/kg(Fig. 2). (2) Efcct of inhibitors on iwsodilating action of CRD-401 The increase in RBF after the renal arterial administration of CRD-401 was not affected by prior injection of propranolol (0.5 mg/kg), atropine (1 mg/kg) or diphenhydra mine (5 mg/kg). Each inhibitor alone also exerted no effect upon the RBF and blood pressure. (3) Effect of CRD-40/ on the r'asoconstrictingaction of angiotensin-11and cpincphrine Angiotensin-Il and epinephrine were injected into the renal artery before and during the renal arterial infusion of CRD-401 (10 1-ig/kg/min). It can be seen in Fig. 3 that the vasoconstricting action of angiotensin-II was significantlysuppressed by CRD-401, where as that of epinephrine was not.
FIG. 3. Effect of CRD-401 on the vasoconstrictor action of angiotensin-II and epinephrine in anesthetized dogs. Each vasoconstrictor was injected into the renal artery during the renal arterial infusion of CRD-40I (10 ug/kg/min) .
?.
Renal firrrcliorr (1)
Clearance
caper imenls
Effects of CRD-401 tinuous
infusion
Ft(-;.
on renal function
technique
and urine formation
under a mild saline load.
4. Dose-response relationship between meters of the renal function. CRD-401 was infused continuously -.UV increase increase Each
the into
were studied
The results are shown
dose
of
the
renal
CRD-401
and
by the con in Fig. 4 and
various
para
artery.
--increase in urine flow, ~ UN.,V--increase in sodium excretion, "'.U,;V in potassium excretion, _C,,, -increase in inulin clearance, --CII %iI in PAH clearance, !1,RBF=-increase in renal blood flow. column represents the mean SE during the infusion of CRD-401.
T.AIftr
1.
Effect
CRD-401 was infused into saline load. UV= urine flow, C;,,-inulin flow, U..,;,v sodium excretion, N -. 4 ,, : P. 0.05
the
of
CRD-401
renal
clearance,
artery C, qtr
UI,V-potassium
on
renal
function.
of
anesthetized
PA 11 clearance, excretion,
dogs RBF
13P--blood
under
a mild
=renal pressure.
blood
Table 1. When CRD-401 was infused at a rate of 1 fag/kg/min, it did not produce any significant change in all the parameters measured, although urine flow and sodium excre tion tended to increase. in all the parameters.
At a rate of 10 1o/kg/min, however, it caused significant increase Average increases in urine flow, and in sodium and potassium ex
cretion rates were 3.5, 2.6 and 1.9 fold of the respective control values. PAH clearance were increased by 16",
Both RBF and
The increase in inulin clearance was 11.30/.
At
the highest close (30 lag/kg/min) tested, CRD-401 caused further increases in urine flow and sodium excretion, i.e., 5.1 and 3.7 fold of the control respectively, although the blood pressure was slightly decreased. creased by 16.3, 27.2 and 29.5 The effects of CRD-401 under a small saline load.
Clearances of inulin and PAH, and RBF were also in respectively.
on renal function
were compared
The data are shown
in Table 2.
renal artery at a rate of 10 ,cag/kg/min, caused statistically sodium
excretion,
paverine
clearances
and
ble, whereas significant,
PAH
clearance
than CRD-401.
inulin clearance by papaverine
CRD-401
and
2.
into the
increases in urine flow,
On the other
hand, the pa
smaller increases in urine flow, sodium
The change though
infused
in RBF was hardly discerni
the degree was small but statistically
infusion.
The effect of CRD-401
TABLE
was decreased,
CRD-401,
significant
of inulin and PAH, and RBF.
infusion at a rate of 100 ,reg/kg/rnin caused
excretion
with those of papaverine
(10 dig/kg/min)
Effects
papaverine
under a small saline load. N==6 K: P. 0.05
of
CRD-401
were
infused
(See legend
on free water
and
into
papaverine
the renal
for Table
1)
clearance
on
renal
artery
was determined
function.
of
anesthetized
dogs
with
water diuresis.
Results are summarized
in urine flow, sodium PAH clearance (2)
excretion,
in Table 3.
osmolar
were also increased
significantly,
produced
prompt increases
and free water clearance.
but creatinine
clearance
RBF and
was not.
Aortic clamp experiments
Aortic
clamp
experiments
were done on 5 mongrel
Fig. 5 shows a representative
record of the experiments
was infused into the renal artery A typical record
obtained
experiment
Table 4 summarizes
sure on renal function
with and without
ing CRD-40l by means
infusion,
of the aortic
excretion
was reduced magnitude
the renal clamp.
necessary
the effects of reduction
perfusion
significantly
the increased
in renal perfusion
by approx.
which
flow caused
absence
of the reduction of CRD-401
in perfusion
follo«ing
aortic
pressure clamping.
upon
renal
(See legend
by CRD-401,
blood
flow in the
for Fig. 5)
pres dur
and
pressure
corresponds
FiG. 5. Prevention of CRD-401-induced increase in renal blood flow by clamping the aorta just above the renal artery. CBP==carotid blood pressure, RPP=renal perfusion pressure, RBF==renal blood flow.
Fi ;. 6. Effect
is
25 mmHg
PAH clearances
When the perfusion
25 mmHg, blood
clamp.
To keep RBF constant
inulin and
(Table 4-B).
by about
(10 /_eg/kg/min)
of the aortic clamp
was reduced
these conditions,
of CRD-401
for preventing
infusion.
pressure
a mild saline load.
by an aortic
in the presence
CRD-401
Under
were not altered
in the absence
dogs under
in which CRD-401
while the RBF was kept constant
from control
also shown in Fig. 6.
sodium
CRD-40l
clearance
to the RBF
TABLE 4. Effect without
of reduction
in renal
perfusion
Control experiments were carried out with renal artery at a rate of 10 pgjkg/min. A : during B : during
pressure
on
renal
function
with
and
infusion
into
the
CRD-401.
CRD-401 CRD-401
infusion infusion
saline
(0.3 ml;'min)
without aortic clamp with aortic clamp.
(normal
positive
control).
C : control with aortic clamp. UN1V=sodium excretion, C,n=inulin clearance, CPAx=PAH clearance, RBF= renal blood flow, RPP=renal perfusion pressure, which was equivalent to the femoral blood pressure distal N--5 * : P<0.05
to the clamp.
TABLE 5. Effect of CRD-401 of the rhAn ,e in renal
1) Data
taken
from
Table
4-C.
2) Data N==5
taken from ~` : P<0.05
Table
4-B.
and the clearances significantly clamp 4.
(Table
experiments
A paired
difference
mount
of sodium
(3)
on
(See legend
sodium
4-C).
Table
5 shows various
with and without between
during
absence
4)
but sodium excretion
parameters
obtained
was reduced
from
infusion
the aortic from
that despite the absence
of inulin and PAH,
CRD-401
in the
These values were derived
these values indicated
in RBF, clearances excreted
CRD-401.
excretion
for Table
of inulin and PAH were not altered,
comparison
nificant
excreted
(10 pg/kg/min) hPmndvnamirc
and perfusion
was significantly
of a sig
pressure, greater
Table
than
the a that
in the control. Stop-/10w experiments
Fig. 7 shows the urinary stop-flow PAl-1 and creatinine
during
patterns
the infusion
The ratio of urine to plasma concentrations
for the change
of saline (control) of sodium,
in concentration or CRD-401
potassium
of sodium,
(10 /ag/kg/min).
and PAH were all cor
FIG. 7. Effect of CRD-401
on the urinary
stop-flow
patterns
in the anesthetized
dog.
PNa!PCr=ratio of(urine sodium toplasma sodium) to (urine creatinine to plasma
creatinine).
PK/PCr_ ratioof (urine potassium toplasma potassium) to(urine creati nine to plasma creatinine).
PPAH! PCr=ratio of (urine PAH toplasma PAH) to(urine creatinine to plasma
creotinine).
PCr- ratioofurine creatinine toplasma creatinine. rected
by dividing
be seen,
the sodium
the
distal
segment
tire
nephron.
by
the pattern
of the
ratio
of
urine
to plasma
concentrations
was
elevated
by
CRD-401
over
nephron,
while
the
potassium
pattern
the
of creatinine. region was
As can
corresponding
elevated
over
to the en
DISCUSSION In the present experiments, effects of CRD-401 on renal blood flow and renal function were examined in anesthetized dogs. When injected into the renal artery, CRD-401 caused a dose-related increase in RBF without affecting the systemic blood pressure.
This renal vasodilating action was not af
fected by the pre-treatment with inhibitors such as propranolol, atropine or diphenhydra mine. This fact suggests that the vasodilating action is due to its direct action on the re nal blood vessels, and this conclusion is compatible with the results obtained in other ves sels (3). Whencompared with the directly acting vasodilator, papaverine, CRD-401 exhibited a longer-lasting renal vasodilation than papaverine and did not produce such a hiphasic
change
in the blood
with papaverine
flow as papaverine.
it was decreased.
reported
by Baer et al (13).
response
between
produced
and
that there are differences
that
CRD-401
but not by epinephrine,
against
increased
GFR,
caused by papaverine
while
has been
in vasodilating
papaverine.
it was observed
by angiotensin-II
non-competitively
CRD-401
in GFR
These facts suggest
CRD-401
In the renal artery,
Furthermore,
The decrease
the constriction
caused
suppressed
although
the vasoconstriction
the compound
by various
antagonized
kinds of spasmogens
in vitro
(14). The renal arterial excretion altered
and
RBF under
by CRD-401
These facts indicate dependent
upon
A variety simultaneous
infusion
of CRD-401
all the fluid loading
in water diuresis, that the diuretic
the increase
of vasodilators
and Friedler
and natriuretic
have been shown
of the diuresis
produced
(20) have suggested
lation.
of this increase
As a result
This increased
to cause
the GFR
was not
significantly
in saline loading.
of CRD-401
were not entirely
natriuresis
infusion
in capillary cortical
was accompanied
were likely responsible
perfusion volume
sodium
bility that CRD-401
induced
natriuresis
during
resistance
Early and al
to the capillary
pressure,
the cortical
circu
volume
is
is believed to increase the pressure
reabsorption.
for the natriuresis,
in free water clearance
Concerning
pressure
Since the natriuresis
with an increase in RBF,
In fact, the increase
with the
of vasodilators,
reduces precapillary
of an existing perfusion
interstitial
associated
into the renal artery (15-19).
by renal arterial
upon the tubules and results in decreased by CRD-401
However,
actions
that vasodilation
transmission
dynamics
conditions.
in GFR.
lows more complete increased.
in an increase in urine flow, sodium
while it was increased
increase in RBF when administered
the mechanism
duced
resulted
changes
as suggested CRD-401
is based upon
pro
in renal
hemo
by Early and Friedler.
infusion
the change
supports
in renal
the possi
hemodynam
ics (21). Willis et al. investigated induced
changes
in renal
the correlation
hemodynamics
between
by placing
the natriuresis a clamp
and the vasodilator
around
the aorta
and thus
preventing the drug-induced increase in RBF (7, 21-23). They reported that the natri uresis induced by acetylcholine (7), bradykinin (21) or histamine (22) was entirely depend ent upon the increase in RBF, while that induced
by aminophylline
its direct action on renal tubules.
whether
by renal arterial variable
resistant
tightened
enough
clamp
of CRD-401
was placed
to prevent
at the pre-treatment
ter measured
(Table
CRD-401
5).
dependent
possesses
around
the aorta
despite
These facts indicate upon
the changes
a direct effect on tubular
The result of the stop-flow experiments
proximal
of CRD-401,
the absence
produced
to the renal
artery
and
When the RBF was kept
there was a statistically
signifi
of the effect on any other parame
that the natriuresis in renal sodium
indicates
due to
upon the increase in RBF, a
increase in RBF.
level after administration
in sodium excretion
or not the natriuresis
was dependent
the drug-induced
cant increase
was not entirely
To determine
administration
(23) was partly
produced
hemodynamics
by CRD-401
and suggest
that
reabsorption.
that CRD-40l
suppressed
the sodium
reabsorption in the distal nephron. A similar result was also obtained by Abe et al (5). If changes in renal hemodynamics affect only proximal sodium reabsorption (24, 25), suppression of sodium reabsorption in the distal nephron by CRD-401 observed in the stop-flow experiments may be the result of the direct effect of CRD-401 on the renal tubules as indicated by the aortic clamp experiments. Thus, the natriuretic action of CRD-401 is not only due to the changes in renal hemodynamics, but also may be ascribable to the direct effect of CRD-401 on the sodium reabsorption in the distal part of the nephron. Acknowleclgenients: We wish to thank Professor Emeritus H. Kumagai and Profes sor F. Sakai, University of Tokyo, Dr. K. Abe, Director of PharmacologicalResearch La boratory,Tanabe Seiyaku Co., Ltd., and Dr. H. Nakajima for helpful advice and encour agement. Thanks are also due to Mr. M. Ogawa, M. Sato and T. Endo for excellent technical assistance. REFERENCES1 ) 2)
KUGIFA, H., INOUE, H., IKEZAKI,M. AND TAKEO,S.: Chem. Pharm. Bull. 18, 2028 (1970) SATO,M., NAGAO,T., YAMAGUCHI,I., NAKAJIMA,H. AND KIYOMOTO,A.: Arzneim.-Forsch. 21, 1338 (1971) 3) NAGAO,T., SATO. M., NAKAJIMA,H. AND KIYOMOTO,A.: Japan. J. Pharmacol. 22, 1 (1972) 4) TAKADA,T., YAMAGUCHI,I., IKEZAWA,K. AND KIYOMOTO, A.: Folia pharmacol. japon. 67, 82p (1971) (in Japanese) 5) ABE, Y., OKAHARA,T. AND YAMAMOTO,K.: Japan. Cire. J. 36, 1002 (1972) 6) IKEZAWA, K., YAMAGUSHI,I. AND KIYOMOTO,A.: Folia pharmacol. japon. 69, 11p (1973) (in Japanese) 7) WILLIS, L.R., LUDENS,J.H. AND WILLIAMSON, H.E.: Proc. Soc. exp. Biol. Med. 128, 1069 (1968) 8) MALVIN, R.L., WILDE, W.S. AND SULLIVAN,L.P.: Am. J. Physiol. 194, 135 (1958) 9) DAVIDSON,W.O. AND SACKNER,M.A.: J. Lab. clip. Med. 62, 351 (1963) 10) PETERS: J. biol. Chem. 46, 179 (1942) 11) HAMBURGER,J., RYCKEWAERT,DUIZEND AND ARGANT: Ann. Biol. Clin. 6, 358 (1948) 12) RAMSAY,J. AND BROWN, R.: J. scient. Instrum. 32, 372 (1965) 13) BAER, P.G., NAVAR, L.G. AND GUYTON,A.C.: Am. J. Physiol. 219, 619 (1970) 14) NAGAO,T., SATO,M., IWASAWA,Y., TAKADA,T., ISHIDA,R., NAKAJIMA,H. ANDKIYOMOTO, A.: Japan. J. Paamacol. 22, 471 (1972) 15) VANDER, A.J.: Am. J. Physiol. 206, 492 (1964) 16) WILLIAMS,R.L., PEARSON,J.E., JR. AND CARTER, M.K.: J. Pharmac. exp. Ther. 147, 32 (1965) 17) MCDONALD, R.H., JR., GOLDBERG,L.L, MCNAY, J.L. AND TUTTLE, E.P., JR.: J. Clin. In est. 43, 1116 (1964) v 18) WEBSTER,M.L. AND GLIMORE,J.P.: Am. J. Physiol. 206, 714 (1964) 19) MEYER, M.B. AND GOLDBERG,L.I.: Fed. Proc. 24, 258 (1965) 20) EARLY, L.E. AND FRIEDLER,R.M.: J. clin. Invest. 45, 542 (1966) 21) WILLIS, L.R., LUDENS,J.H. AND WILLIAMSON,H.E.: Am. J. Physiol. 217, 1 (1969) 22) O'BRIEN, K.P. AND WILLIAMSON,H.E.: Europ. J. Pharmacol. 16, 385 (1971) 23) LUDENS, J.H., WILLIS, L.R. AND WILLIAMSON,H.E.: Arch. init. Pharmacodyn. Thér. 185. 274 (1970) 24) DIRKS, J.H. AND SEELY,J.F.: Clin. Res. 15, 478 (1967) 25) HAYSLETT,J.P., DOMOTO,D.T., KASHGARIAN,M. AND EPSTEIN,F.H.: Am. J. Physiol. 218, 880 (1970)
ON A NEW 1,5-BENZOTHIAZEPINE DERIVATIVE (CRD-401) VI. EFFECTS ON RENAL BLOOD FLOW AND RENAL FUNCTION Isao YAMAGUCHI, Katsuo IKEZAWA, Toshikazu TAKADA and Akio KIYOMOTO Pharmacological
Research
Laboratory,
Toda, Accepted
Saitama, January
Tanabe
Seiyaku
Co.,Ltd.,
Japan 16,
1974
Abstract -Effects of a new coronary vasodilator, CRD-401, on renal blood flow and renal function were investigated in anesthetized dogs. Renal arterial or systemic administration of the compound produced an increase in renal blood flow. The increase in renal blood flow induced by CRD-401 was not affected by pre-treatment with propranolol, atropine or dephenhydramine. Renal arterial administration of CRD-401 exhibited an antagonistic effect on the vasoconstricting action of angioten sin-II, whereas it had no effect on that of epinephrine. When infused continuously into the renal artery, CRD-40l increased the urine flow and sodium excretion as well as renal blood flow in all the conditions of fluid loading tested. The glomerular filtration rate was enhanced under saline loading but not under water diuresis. CRD 401 caused an increase in free water clearance. When the renal blood flow was kept constant with an aortic clamp during CRD-401 infusion, sodium excretion was signifi cantly increased but no change was observed in glomerular filtration rate and PAH clearance. The results show that CRD-401 induced natriuresis was not entirely dependent upon the renal hemodynamic changes caused by the compound. By the stop-flow method, it was shown that the ratio of [urine sodium to plasma sodium] to [urine creatinine to plasma creatinine] increased in the distal portion of the nephrun by CRD-401 infusion. The present results indicate that the natriuretic action of' CRD-401 is not only due to the changes in renal hemodynamics but it also may be ascribable to a direct effect of CRD-410 on the sodium reabsorption in the distal part of the nephron. A new benzothiazepine
derivative,
ethyl]-2-(p-methoxyphenyl)-I, has been shown blood
to have a vasodilating
flow without
augmenting
effect of the drug is assumed In addition pound
to the coronary
produced
d-3-acetoxy-cis-2,
5-benzothiazepin-4(5H)-one action
myocardial
and
3-dihydro-5-[(dimethylamino) hydrochloride
to produce
oxygen
an increase
in renal
activity,
an increase
consumption
to he direct action on coronary vasodilating
(CRD-40I)
(2).
blood flow and exerted
in coronary
The vasodilating
and femoral
it was also reported
(1),
blood vessels (3).
briefly that the com
influence
upon
renal func
tion (2, 4-6). In the present experiments, esthetized
dogs.
netic flowmeter amined
Firstly,
and secondly,
by clearance
also investigated.
the renal action of CRD-401
the renal vasodilating
technique.
was studied
action
was investigated
effects on renal function
and electrolyte
The mechanism
of diuretic
action
in detail in an
using electromag excretion
were ex
of the compound
was
I.
Renal r•asoclilatin,raction Male mongrel dogs weighing 15 to 18kg were anesthetized with sodium pentobar bital (P.B.) (35 mg'kg, .v.). The left renal artery was exposed through a flank incision and a probe connected to a squarewave electromagnetic flowmeter (Nihon Kohden, MF 25) was placed around the renal artery. Blood pressure was monitored from the femoral artery with a pressure transducer. Both renal blood flow (RBF) and blood pressure were recorded simultaneously on an ink-writing oscillograph. The renal vasodilating actions of drugs were estimated by measuring the maximal increase in the blood flow measured from the pre-administration level. Drugs were administered into the renal artery through a catheter that terminated with a 22 gauge needle inserted into the artery, or into the cannu lated fermoral vein. In order to investigate the mechanism of vasodilation by CRD-401, an inhibitor such as adrener-*c E~blocker (propranolol), anticholinergics (atropine) or antihistamine (di phenhydramine) was injected into the femoral vein through a cannula 3 to 10 min prior to administration of CRD-401. In experiments where interaction of CRD-401 with a vasoconstrictor such as angio tensin-II or epinephrine was studied, the vasoconstrictor was injected into the renal artery before and during the continuous infusion of CRD-401. 2. Renal Jllnctinl (1) Clearance e.tperintents Male mongrel dogs weighing 16 to 20 kg were anesthetized with P.B. (35 nlgkg, i.v.). The trachea was cannulated and the animal was ventilated by an artificial respirator with room air. Both ureters were exposed through a small abdominal midline incision and catheters were introduced into the renal pelvis. The femoral artery and vein were can nulated for sampling blood and loading saline or glucose solution, respectively. The left renal artery was exposed through a flank incision and a probe was placed around the ar tery. RBF and blood pressure were measured by the methods mentioned above. A 22 gauge needle connected to a polyethylenecatheter was inserted into the renal artery through which saline was infused continuously with an infusion pump at a rate of 0.3 ml,/min. Drugs were dissolved into saline. Clearance studies were performed under one of three conditions of fluid load: a) a priming load of 200 nil of isotonic saline and the continuous infusion of saline at a rate of 2 ml min (small saline load), b) a priming load of 400 ml of 6, dextran and a constant infusionof 2.5 ° glucose solution at a rate of 9.5 ml/min (water dieresis) and c) an initial load of 400 nil of isotonic saline and a continuous infusionof iso tonic saline at a rate of 5.5 ml' min (mild saline load). Glomerular filtration rate (GFR; was determined by measuring inulin or creatinine clearance. Clearance of para-ami nohipptn-icacid (PAH) was measured simultaneously with RBF, determined by an elec. tromagnetic llowmeter. Inulin, or creatinine and PAH were dissolved in the infusion SO. lotions mentioned above and given into the femoral vein. Each priming and sustaining close was 1.8 ~-,10 min and 20-25 mg,rain for inulin, 2.5 g/ 10min and 15-20 mg/min for
creatinine, and 200 mg/10 min and 2-3 nag/min for PAH, respectively. After urine flow had become stabilized, periodic collections of urine and blood were started. Several u rine samples serving as controls were collected during saline infusion into the renal artery. The saline infusion was then replaced with infusion of a saline containing CRD-401 and urine samples were collected. Each collection period was 5 or 10min. Blood samples were drawn at the mid-point of each urine collection period. (2) Aortic clamp experiments In order to examine the effect of increased RBF produced by CRD-401 on the na triuretic response, urinary sodium excretion rate was determined under conditions where the change in RBF caused by the continuous infusion of CRD-401 was prevented by means of an aortic clamp (7). A variable resistant clamp was placed around the aorta just prox imal to the left renal artery of the anesthetized dogs. The renal perfusion pressure was estimated through the catheter in the left femoral artery and the systemic blood pressure was monitored from the carotid artery. A mild saline load was performed as mentioned previously. Isotonic saline was infused continuously into the renal artery at a rate of 0.3 ml/min throughout the experiment and CRD-401 administration was performed by adding the drugs to the infusate (10 fig/kg/min). When the rate of urine flow became constant, two 10 min collections of urine were carried out and blood samples were drawn at the mid point of each urine collection period. Following collection of control samples of urine and blood, CRD-401 was infused into the renal artery and the renal perfusion pressure was reduced by way of the aortic clamp so as to eliminate the increment in RBF caused by CRD-401 and to keep it constant at the control level before CRD-401 administration. Under these conditions, two to three samples of urine were collected in 5 or 10 min periods and blood samples were drawn at the mid-point of each period. Control experiments were run for each animal by clamping the aorta to give the same decrement in renal perfusion pressure as observed during CRD-401 infusion. Urine and blood samples were collected as described above. (3) Stopflow experiments The site of action of CRD-401 diuresis along the nephron was examined in dogs by stop-flow technique (8). Osmotic diuresis was established by an intravenous infusion of 20 mannitol in isotonic saline at a rate of 9.5 ml/min. Creatinine and PAH were added to the infusion solution. Experiments were divided into two periods: an initial control period and subsequent experimental period during the continuous infusion of CRD-401 into the renal artery. After a high and stable rate of urine flow (5 to 10ml/min) had been obtained, urine samples were collected in two 5 min periods and blood samples were drawn at the mid-point of each urine collection. A clamp was used to block off the ureter for 4 min, during which time a blood sample was drawn. After the release of the clamp, 30 serial urine sample of 0.5 to 0.8 nil in volume were collected. The constant infusion of CRD-401 into the left renal artery was then started and the same procedureswere repeated. 3. /l iralrsis Sodium and potassium concentrations in plasma and urine were determined using an
Evans flame photometer.
Concentrations
urine were determined
by the methods
al. (1 1) respectively.
Osmolality
Ramsay's 4.
method
of inulin, creatinine
of Davison
of plasma
and PAH
in plasma
and
et al (9), Peters (10) and Hamburger
and urine
was measured
et
cryoscopically
by
(12).
Drugs The drugs used in the present experiments
(Iwaki
Seiyaku),
propranolol
(I.C.I.),
(Tanabe
Seivaku),
mine hydrochloride Univ.),
epinephrine
hydrochloride
Kugita et al. (I) of Organic
Chemistry
were as follows:
atropine
sulfate
angiotensin-II (Sankyo Research
papaverine
hydrochloride
Seiyaku),
diphenhydra
(Tanabe
(Institute
Seiyaku). Laboratory
for Protein
CRD-401 of Tanabe
was
Research,
Osaka
synthesized
Seiyaku
Co.,
by Ltd..
RESULTS 1.
Renal vasoililating
el/eel
(1) Effect on renal blood flow CRD-401, when injected into the renal artery, produced an increase in RBF.
With
increasing doses of the drug, greater increase in RBF was obtained, while the systemic blood pressure remained unaltered (Fig. I-A). When papaverine was injected into the renal ar tery, it also produced a dose-related increase in RBF. However, the time course of the change in RBF produced by papaverine was somewhat different from that produced by CRD-401 (Figg.1-B). CRD-101 caused only an increase in the blood flow, while papave rine, in most cases, gave a biphasic response, i.e., an increase followed by a decrease.
From
the dose-response curve in Fig. I-A, it was concluded that renal vasodilating potency of
Fi
Fiu. 2. Effect of i.v. injection of CRD-401 on renal blood flow. BP-blood pressure, RBF=renal blood flow.
CRD-401 was more than three times that of papaverine. CRD-401 also produced about 10'/',o'increase in RBF, when given intravenously at a dose of 100beg/kg(Fig. 2). (2) Efcct of inhibitors on iwsodilating action of CRD-401 The increase in RBF after the renal arterial administration of CRD-401 was not affected by prior injection of propranolol (0.5 mg/kg), atropine (1 mg/kg) or diphenhydra mine (5 mg/kg). Each inhibitor alone also exerted no effect upon the RBF and blood pressure. (3) Effect of CRD-40/ on the r'asoconstrictingaction of angiotensin-11and cpincphrine Angiotensin-Il and epinephrine were injected into the renal artery before and during the renal arterial infusion of CRD-401 (10 1-ig/kg/min). It can be seen in Fig. 3 that the vasoconstricting action of angiotensin-II was significantlysuppressed by CRD-401, where as that of epinephrine was not.
FIG. 3. Effect of CRD-401 on the vasoconstrictor action of angiotensin-II and epinephrine in anesthetized dogs. Each vasoconstrictor was injected into the renal artery during the renal arterial infusion of CRD-40I (10 ug/kg/min) .
?.
Renal firrrcliorr (1)
Clearance
caper imenls
Effects of CRD-401 tinuous
infusion
Ft(-;.
on renal function
technique
and urine formation
under a mild saline load.
4. Dose-response relationship between meters of the renal function. CRD-401 was infused continuously -.UV increase increase Each
the into
were studied
The results are shown
dose
of
the
renal
CRD-401
and
by the con in Fig. 4 and
various
para
artery.
--increase in urine flow, ~ UN.,V--increase in sodium excretion, "'.U,;V in potassium excretion, _C,,, -increase in inulin clearance, --CII %iI in PAH clearance, !1,RBF=-increase in renal blood flow. column represents the mean SE during the infusion of CRD-401.
T.AIftr
1.
Effect
CRD-401 was infused into saline load. UV= urine flow, C;,,-inulin flow, U..,;,v sodium excretion, N -. 4 ,, : P. 0.05
the
of
CRD-401
renal
clearance,
artery C, qtr
UI,V-potassium
on
renal
function.
of
anesthetized
PA 11 clearance, excretion,
dogs RBF
13P--blood
under
a mild
=renal pressure.
blood
Table 1. When CRD-401 was infused at a rate of 1 fag/kg/min, it did not produce any significant change in all the parameters measured, although urine flow and sodium excre tion tended to increase. in all the parameters.
At a rate of 10 1o/kg/min, however, it caused significant increase Average increases in urine flow, and in sodium and potassium ex
cretion rates were 3.5, 2.6 and 1.9 fold of the respective control values. PAH clearance were increased by 16",
Both RBF and
The increase in inulin clearance was 11.30/.
At
the highest close (30 lag/kg/min) tested, CRD-401 caused further increases in urine flow and sodium excretion, i.e., 5.1 and 3.7 fold of the control respectively, although the blood pressure was slightly decreased. creased by 16.3, 27.2 and 29.5 The effects of CRD-401 under a small saline load.
Clearances of inulin and PAH, and RBF were also in respectively.
on renal function
were compared
The data are shown
in Table 2.
renal artery at a rate of 10 ,cag/kg/min, caused statistically sodium
excretion,
paverine
clearances
and
ble, whereas significant,
PAH
clearance
than CRD-401.
inulin clearance by papaverine
CRD-401
and
2.
into the
increases in urine flow,
On the other
hand, the pa
smaller increases in urine flow, sodium
The change though
infused
in RBF was hardly discerni
the degree was small but statistically
infusion.
The effect of CRD-401
TABLE
was decreased,
CRD-401,
significant
of inulin and PAH, and RBF.
infusion at a rate of 100 ,reg/kg/rnin caused
excretion
with those of papaverine
(10 dig/kg/min)
Effects
papaverine
under a small saline load. N==6 K: P. 0.05
of
CRD-401
were
infused
(See legend
on free water
and
into
papaverine
the renal
for Table
1)
clearance
on
renal
artery
was determined
function.
of
anesthetized
dogs
with
water diuresis.
Results are summarized
in urine flow, sodium PAH clearance (2)
excretion,
in Table 3.
osmolar
were also increased
significantly,
produced
prompt increases
and free water clearance.
but creatinine
clearance
RBF and
was not.
Aortic clamp experiments
Aortic
clamp
experiments
were done on 5 mongrel
Fig. 5 shows a representative
record of the experiments
was infused into the renal artery A typical record
obtained
experiment
Table 4 summarizes
sure on renal function
with and without
ing CRD-40l by means
infusion,
of the aortic
excretion
was reduced magnitude
the renal clamp.
necessary
the effects of reduction
perfusion
significantly
the increased
in renal perfusion
by approx.
which
flow caused
absence
of the reduction of CRD-401
in perfusion
follo«ing
aortic
pressure clamping.
upon
renal
(See legend
by CRD-401,
blood
flow in the
for Fig. 5)
pres dur
and
pressure
corresponds
FiG. 5. Prevention of CRD-401-induced increase in renal blood flow by clamping the aorta just above the renal artery. CBP==carotid blood pressure, RPP=renal perfusion pressure, RBF==renal blood flow.
Fi ;. 6. Effect
is
25 mmHg
PAH clearances
When the perfusion
25 mmHg, blood
clamp.
To keep RBF constant
inulin and
(Table 4-B).
by about
(10 /_eg/kg/min)
of the aortic clamp
was reduced
these conditions,
of CRD-401
for preventing
infusion.
pressure
a mild saline load.
by an aortic
in the presence
CRD-401
Under
were not altered
in the absence
dogs under
in which CRD-401
while the RBF was kept constant
from control
also shown in Fig. 6.
sodium
CRD-40l
clearance
to the RBF
TABLE 4. Effect without
of reduction
in renal
perfusion
Control experiments were carried out with renal artery at a rate of 10 pgjkg/min. A : during B : during
pressure
on
renal
function
with
and
infusion
into
the
CRD-401.
CRD-401 CRD-401
infusion infusion
saline
(0.3 ml;'min)
without aortic clamp with aortic clamp.
(normal
positive
control).
C : control with aortic clamp. UN1V=sodium excretion, C,n=inulin clearance, CPAx=PAH clearance, RBF= renal blood flow, RPP=renal perfusion pressure, which was equivalent to the femoral blood pressure distal N--5 * : P<0.05
to the clamp.
TABLE 5. Effect of CRD-401 of the rhAn ,e in renal
1) Data
taken
from
Table
4-C.
2) Data N==5
taken from ~` : P<0.05
Table
4-B.
and the clearances significantly clamp 4.
(Table
experiments
A paired
difference
mount
of sodium
(3)
on
(See legend
sodium
4-C).
Table
5 shows various
with and without between
during
absence
4)
but sodium excretion
parameters
obtained
was reduced
from
infusion
the aortic from
that despite the absence
of inulin and PAH,
CRD-401
in the
These values were derived
these values indicated
in RBF, clearances excreted
CRD-401.
excretion
for Table
of inulin and PAH were not altered,
comparison
nificant
excreted
(10 pg/kg/min) hPmndvnamirc
and perfusion
was significantly
of a sig
pressure, greater
Table
than
the a that
in the control. Stop-/10w experiments
Fig. 7 shows the urinary stop-flow PAl-1 and creatinine
during
patterns
the infusion
The ratio of urine to plasma concentrations
for the change
of saline (control) of sodium,
in concentration or CRD-401
potassium
of sodium,
(10 /ag/kg/min).
and PAH were all cor
FIG. 7. Effect of CRD-401
on the urinary
stop-flow
patterns
in the anesthetized
dog.
PNa!PCr=ratio of(urine sodium toplasma sodium) to (urine creatinine to plasma
creatinine).
PK/PCr_ ratioof (urine potassium toplasma potassium) to(urine creati nine to plasma creatinine).
PPAH! PCr=ratio of (urine PAH toplasma PAH) to(urine creatinine to plasma
creotinine).
PCr- ratioofurine creatinine toplasma creatinine. rected
by dividing
be seen,
the sodium
the
distal
segment
tire
nephron.
by
the pattern
of the
ratio
of
urine
to plasma
concentrations
was
elevated
by
CRD-401
over
nephron,
while
the
potassium
pattern
the
of creatinine. region was
As can
corresponding
elevated
over
to the en
DISCUSSION In the present experiments, effects of CRD-401 on renal blood flow and renal function were examined in anesthetized dogs. When injected into the renal artery, CRD-401 caused a dose-related increase in RBF without affecting the systemic blood pressure.
This renal vasodilating action was not af
fected by the pre-treatment with inhibitors such as propranolol, atropine or diphenhydra mine. This fact suggests that the vasodilating action is due to its direct action on the re nal blood vessels, and this conclusion is compatible with the results obtained in other ves sels (3). Whencompared with the directly acting vasodilator, papaverine, CRD-401 exhibited a longer-lasting renal vasodilation than papaverine and did not produce such a hiphasic
change
in the blood
with papaverine
flow as papaverine.
it was decreased.
reported
by Baer et al (13).
response
between
produced
and
that there are differences
that
CRD-401
but not by epinephrine,
against
increased
GFR,
caused by papaverine
while
has been
in vasodilating
papaverine.
it was observed
by angiotensin-II
non-competitively
CRD-401
in GFR
These facts suggest
CRD-401
In the renal artery,
Furthermore,
The decrease
the constriction
caused
suppressed
although
the vasoconstriction
the compound
by various
antagonized
kinds of spasmogens
in vitro
(14). The renal arterial excretion altered
and
RBF under
by CRD-401
These facts indicate dependent
upon
A variety simultaneous
infusion
of CRD-401
all the fluid loading
in water diuresis, that the diuretic
the increase
of vasodilators
and Friedler
and natriuretic
have been shown
of the diuresis
produced
(20) have suggested
lation.
of this increase
As a result
This increased
to cause
the GFR
was not
significantly
in saline loading.
of CRD-401
were not entirely
natriuresis
infusion
in capillary cortical
was accompanied
were likely responsible
perfusion volume
sodium
bility that CRD-401
induced
natriuresis
during
resistance
Early and al
to the capillary
pressure,
the cortical
circu
volume
is
is believed to increase the pressure
reabsorption.
for the natriuresis,
in free water clearance
Concerning
pressure
Since the natriuresis
with an increase in RBF,
In fact, the increase
with the
of vasodilators,
reduces precapillary
of an existing perfusion
interstitial
associated
into the renal artery (15-19).
by renal arterial
upon the tubules and results in decreased by CRD-401
However,
actions
that vasodilation
transmission
dynamics
conditions.
in GFR.
lows more complete increased.
in an increase in urine flow, sodium
while it was increased
increase in RBF when administered
the mechanism
duced
resulted
changes
as suggested CRD-401
is based upon
pro
in renal
hemo
by Early and Friedler.
infusion
the change
supports
in renal
the possi
hemodynam
ics (21). Willis et al. investigated induced
changes
in renal
the correlation
hemodynamics
between
by placing
the natriuresis a clamp
and the vasodilator
around
the aorta
and thus
preventing the drug-induced increase in RBF (7, 21-23). They reported that the natri uresis induced by acetylcholine (7), bradykinin (21) or histamine (22) was entirely depend ent upon the increase in RBF, while that induced
by aminophylline
its direct action on renal tubules.
whether
by renal arterial variable
resistant
tightened
enough
clamp
of CRD-401
was placed
to prevent
at the pre-treatment
ter measured
(Table
CRD-401
5).
dependent
possesses
around
the aorta
despite
These facts indicate upon
the changes
a direct effect on tubular
The result of the stop-flow experiments
proximal
of CRD-401,
the absence
produced
to the renal
artery
and
When the RBF was kept
there was a statistically
signifi
of the effect on any other parame
that the natriuresis in renal sodium
indicates
due to
upon the increase in RBF, a
increase in RBF.
level after administration
in sodium excretion
or not the natriuresis
was dependent
the drug-induced
cant increase
was not entirely
To determine
administration
(23) was partly
produced
hemodynamics
by CRD-401
and suggest
that
reabsorption.
that CRD-40l
suppressed
the sodium
reabsorption in the distal nephron. A similar result was also obtained by Abe et al (5). If changes in renal hemodynamics affect only proximal sodium reabsorption (24, 25), suppression of sodium reabsorption in the distal nephron by CRD-401 observed in the stop-flow experiments may be the result of the direct effect of CRD-401 on the renal tubules as indicated by the aortic clamp experiments. Thus, the natriuretic action of CRD-401 is not only due to the changes in renal hemodynamics, but also may be ascribable to the direct effect of CRD-401 on the sodium reabsorption in the distal part of the nephron. Acknowleclgenients: We wish to thank Professor Emeritus H. Kumagai and Profes sor F. Sakai, University of Tokyo, Dr. K. Abe, Director of PharmacologicalResearch La boratory,Tanabe Seiyaku Co., Ltd., and Dr. H. Nakajima for helpful advice and encour agement. Thanks are also due to Mr. M. Ogawa, M. Sato and T. Endo for excellent technical assistance. REFERENCES1 ) 2)
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