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EFFECT OF ANTIBIOTICS ON THE URETER MOTOR ACTIVITY
EFFECT
OF
ANTIBIOTICS
G. BENZI,
ON
THE
E. ARRIGONI,
URETER
P. PANCERI,
F. BERTE'
MOTOR
ACTIVITY
R. PANZARASA,
and A. CREMA
Department of Pharmacology,
University of Pavia, Pavia, Italy
Accepted February
5, 1973
Abstract-The effect of certain antibiotics on the ureteral tone and motility has been studied in vitro and in situ in guinea-pigs and dogs. Tetracycline and rolitetracycline induced a constriction, while ampicillin, isoxazolyl penicillins, gentamicin, aminosidin, spiramycin and chloramphenicol induced a relaxation of the ureter. In vivo, the re sponses were independent of both bladder activity and systemic effects related to the antibiotic flowing into the ureter and eventually absorbed from it. The anti biotics acted directly on the ureteral smooth muscle; factors influencing the action were: (a) the free or conjugated form of the antibiotic flowing through the ureter; (b) pH of the urine; (c) time of contact; (d) time lag. Antibiotics
have been used extensively
in the treatment
the kidneys being the main route by which a great number the body
in both
vancement tone
modified
has been made
and
motility
other hand,
and unchanged
of the urinary
it is known
that antibiotics
This report
presents
on the tone and motility
The experiments
experimental
were performed
The antibiotics
sidin sulphate,
spiramycin
base); ampicillin
adipate
sodium
(the amounts
penicillanic (different
acid). samples
39.4), doxycycline (7.4;
findings
with the indicated (6.8;
on the action
oxacillin
percentage
sodium, in terms
of certain
biliary
antibiotics and dogs.
gentamicin
sulphate,
amino
in terms of the
in terms of n(-)-6-(a-amino
cloxacillin
sodium,
dicloxacillin
of 3-phenyl-5-methyl-4-isoxazolyl
also with the following
of epiderivative):
tetracycline
tetracyclines (7.4; 25.7;
(0.8; 7.2; 18.8; 41.2) and rolitetracycline
of which are expressed
in terms
of the base.
and the 4-epiderivatives
to the methods
of Lodi et a]. (9) and Gyanchandani
the degradation
products
was calculated
On the
muscle of the intes
of which are expressed
were performed
of the tetracyclines
on
and in the dog both in vitro and
of which are expressed
17.6; 37.4), mepicycline
30.2; 47.7), the amounts
tion and determination
of the ureter.
(7), of the extrahepatic
chloramphenicol,
(the amounts
acid);
little ad
AND METHODS
of which are expressed
The experiments
that
antibiotics
both in vitro and in situ, in guinea-pigs
D(-)
sodium (the amounts
a-phenylacetamido)-penicillanic
particularly
in the guinea-pig
used were:
surprising
can interfere with the smooth
of the ureter
tract infections, are excreted from
of the excreted
tract (2, 6), of the uterus
MATERIALS in situ.
It is rather
of the action
tract musculature,
tinal tract (1-5), of the bronchial tract (8).
forms.
in the knowledge
of urinary
of antibiotics
et al. (10).
as a percentage
The separa
were made according The concentration
of the total tetracycline.
of
Experiments in vitro Ureters were removed from 40 adult guinea-pigs of both sexes and from 62 adult male and female mongrel dogs. These were carefully dissected and set up in a 20 ml or 50 ml organ-bath containing Tyrode solution gassed with 95% oxygen and 5 car bon dioxide; temp. was 37.2_0.2'C and pH was buffered to 7.2±0.05. The longitudinal movements of the ureter were recorded by a strain-gauge with isotonic transducer; the lever exerted a resting tension of 0.5 to 1.5 g on the tissue and the magnification was 10 20 times. The action of the antibiotics was evaluated on the ureter in normal condition or after stimulation by barium chloride (10 to 50 ,cag/mlplus 0.1 beg/ml of atropine sulphate), hista mine (2.5 to 10.0 ig/ml) or serotonin (2.5 to 10.0pg/ml). For the construction of the dose-response curves, the activity of the antibiotics was taken as the change in the recorded area of barium chloride induced movements during a 30 min period (tetracyclines)or 20 min period (other antibiotics) of contact before and after antibiotic addition. The tested antibiotics were added to the bath 2 min (tetracyclines) or 10 min (other antibiotics) before the barium chloride administration. The ED,, and the slope function of the lines, with 95% confidence limits, were evaluated according to Litchfield and Wilcoxon (11). Experiments in vivo Studies were performed on 260 adult female guinea-pigs and 56 mongrel dogs of both sexes. The guinea-pigs were anaesthetized with urethane (1 -/kg i.p.) and chlora lose (20 mg/kg i.p.). The dogs were sedated with urethane (0.4 g/kg i.p.) and anaesthesia was induced by chloralose (100 mg/kg i.v.) or by nitrous oxide or cyclopropane in closed circuit. The animals were given artificial ventilation. Arterial blood pressure was mea sured from a cannula inserted into a carotid (guinea-pig) or femoral (dog) artery. After laparotomy a cannula was inserted into the renal pelvis to perfuse the ureter by Tyrode solution with and without antibiotics. The pH of the Tyrode solution was generally ad justed to 6.6-0.05, but in some experiments the pH was buffered to 7.8;0.05 or 5.5 0.05. In guinea-pigs, a draining tube was inserted into the fundus vesicae; in dogs, an urethral catheter was inserted. The flow into the ureters was measured for a 30 min period immediatelyafter the beginning of the change induced by antibiotics, compared with the flow during the 30 min period before the addition of the antibiotics. The ureter was either normal or activated by perfusing harium chloride (50 to 400 ,mg/'ml). The and the slope function of the lines, with 95''„ confidencelimits, were evaluated according to Litch field and Wilcoxon (11). In some experiments, prior to addition of an antibiotic to the solution flowing through the ureter, the animals were pretreated with: atropine sulphate (1-2 mg/kg s.c.), chlor pheniramine maleate (1-2 mg/kg s.c.), methysergide maleate (25-50 /1g/kg s.c.), cypro heptadine hydrochloride (150-3001g/kg i.v.), dibenamine hydrochloride (2-4 mg/kg i.v.), o(-) INPEA (4-8 mg/kg i.v.), or hexamethonium bromide (100-200/-tg/kg i.v.). In a series of experiments it was possible to evaluate in situ the flow through the ureter pulled out from the ureter-bladder junction and therefore free from urinary vesica inter
ference. In 22 mongrel Rectangular
dogs electrodes
pulses,
of 0.2-1
were placed
cosec duration
quency of 5 to 10 shocks/sec
around
the hypogastric
and 5-20 V strength
for 20 sec, before
and after
or pelvic nerves.
were applied
the addition
at a fre
of the antibiotics
into the renal pelvis. Cross-urine-perfusion Studies donor
preparation
were carried
animal
on 8 groups
of 3 beagle
bitches.
was given the tested antibiotic
i.v.; another
donor
tion i.v. During
out
4-8 hr, urine samples
tying the bladder
at each collection
sured in both ureters; the urine collected
the control
in a random
treated
animal;
Linear
Graphical
Representations
Dixon procedure:
This method
(b) the other
tives concentrations
of the combined
order: ureter
(Dixon
of
barium
was evaluated
response
induced
with
by per
This method
animal.
by tetracyclines. as a function
of the tested
of the effect on the ureteral
concentration
chloride.
flow was mea
of the effect is expressed
(contained
at two fixed concentrations
procedure:
emp
both the ureters
the placebo-treated
at two fixed concentrations
the reciprocal
of the tested tetracyclines
solu
to study the extent to which the 4-epideriva
12), the reciprocal
we expressed
tetracycline)
concentrations
The response
one
(a) one ureter with the urine from the antibiotic
was applied
of the 4-epiderivatives
concentration
animal.
and completely
the urinary
by perfusing
with the urine from
drug B concentration,
In our procedures
Linewcearer-Bark
In the recipient
could affect the pharmacodynamic
In the Dixon procedure
of the tested
period.
group,
was given Tyrode
by catheterizing
flow was obtained
from the placebo-treated
fusing the recipient
a function
were obtained
In each
in a constant
of barium
was used to evaluate on the
ureter
Quantitative
drug
A.
musculature
as
concentration
chloride. the effect of a constant
stimulated
relationships
in vitro with varying between
drug
A
and
receptor:
can be rearranged
The reciprocal
(Lineweaver-Burk,
of the pharmacodynamic
of the drug concentration. action
13) as:
between
barium
effect is expressed
A similar procedure
chloride
and the members
as a function
was applied
of the reciprocal
in the
of a homologous
case
of the
inter
series of tetracyclines.
RESULTS 1)
Pharnlacodyna,nic Studies
aminosidin,
action
in guinea-pigs ampicillin,
in vitro of the spasm
of the antibiotics and
isoxazolyl
dogs
demonstrated
penicillins
by agonist (barium
in situ of the flow through
the ureter,
on ureter
and
chloride, either
that
spiramycin histamine,
normal
chloramplien induce
or serotonin)
or activated
icol, gentamicin,
both
an inhibition
and an increase
by agonist.
The ED,,
TABLE1. ED50 and slope function of the line (S), with 95% confidence limits, of the myolytic action of some antibiotics tested by dog isolated ureter in vitro and by guinea-pig ureter in situ normal or activated by barium chloride.
(a) ED50=antibiotic concentration (pg/ml in the Tyrode solution of the bath) reducing by 50% the contracting action of barium chloride (30=10 ,ug/,nl) during 20 min period of contact. (b) ED50 =antibiotic concentration (pg/ml in the solution flowing through the ureter) inducing an increase of the intraureteral flow equal to 50% of the maximal flow, as evaluated during the control condition. (c) ED50=antibiotic concentration (pg/ml in the solution flowing through the ureter) inducing an increase of the intraureteral flow equal to 50°%of the maximal flow, as evaluated during the control condition. The antibiotics were tested during the local action of barium chloride (200 pg/ml). (d) S==fold change in dose required to produce a unit standard deviation change in response along the line ; thus S=antilog s=antilog 1/b, where b and s are respectively the slope constant and standard deviation of a line relating log dose of antibiotic, and probit per cent effect. on the ureter, order
normal
of potency
or activated
in the myolytic
by barium activity
chloride,
gentamicin;,>aminosidin;~spiramycin-ampicillin. action
was affected:
and rolitetracycline
(a) by the antibiotic increased
higher the 4-epiderivative ride (Fig. 2). tetracycline,
concentration,
On the contrary, oxytetracycline,
As
concerns
used: mepicycline
the contracting
muscle (Fig. 1); (b) by the percentage
is summarized
in Table
was chloramphenicol>isoxazolyl
action
the
antagonized,
of barium
of the 4-epiderivatives
chloride
and chlortetracycline
(Table
tetracyclines,
on ureter smooth used:
action of barium
differences
their
while tetracycline
in the tetracycline
the greater the stimulating
there were no significant
1 ; the
penicillins>
among
the chlo
activities
of
2).
The in vivo addition of antibiotics into the solution flowing through the ureter in duced no effect on the response to electrical stimulation of the hypogastric or pelvic nerves, due to the predominant contraction of the bladder muscle which was unaffected by the antibiotics flowing from ureter-bladder junction into the urinary bladder.
FIG. 1. Dog ureter in vitro. Lineweaver-Burk plots calculated : (a) for the effect of barium chloride in the absence of tetracycline (WT) ; and (b) for the effect of a constant concentration (11.25 x 10' M) of various tetracyclines (A mepicyc line ; • tetracycline ; f,] rolitetracycline) introduced into the bath 2 min before the barium chloride. Abscissa, the reciprocals of barium chloride molar con centration (1/M). Ordinate, the reciprocals of the effect (100/per cent of maximal contraction assumed equal to 100). Plotted points represent average values in four preparations for tetracyclines plus barium chloride, and in sixteen preparations for barium chloride alone. The concentrations of 4-epiderivatives contained in the various tetracyclines were the following : mepicycline=18.8% ; tetracycline = 25.7% ; rolitetracycline = 30.2%.
FIG. 2. Dog ureter in vitro. Dixon straight lines calculated for the effect of two fixed concentrations of barium chloride (A,=7.22 x 10' M ; A,=2.88 x 10-4 M) expressed as a function of the 4-epiderivatives concentration contained in a constant concentration (11.25 x 10' M) of the tetracyclines tested, introduced into the bath 2 min before the barium chloride. Abscissa, % concentration of the 4-epiderivatives. Ordinate, the reciprocals of the effect (100/% contraction). Plotted points represent average values in four preparations.
TABLE2. Dog isolated ureter. EDSOand slope function of the line (S), with 95% confidence limits, of three different tetracyclines, the 4-epiderivative concentration being quite equal in the preparations (from 23.2 to 29.4°%).
(a) ED50 antibiotic concentration (ug/ml) reducing ureteral flow during 30 min period of contact. (b) S as in Table 1.
by 50°
the intra
2) Mode of pharmacodynaniicaction of antibiotic on the ureter Responses in situ of the ureter to antibiotics flowing through the ureter were inde pendent of the ureter-bladder junction activity, as they were present even when the ureter was freed from urinary musculature. The action was also independent of any systemic effects related to the antibiotic eventually absorbed from ureter into the bloodstream and distributed throughout the body, as the urinary flow into the other ureter was unaffected during the antibiotic action on the ureter being tested. The direct action of the antibiotics on the extrarenal urinary tract smooth muscle was supported by: (a) the failure of cholinoceptor, adrenoceptor, histamine, 5-hydroxy tryptamine and ganglion blocking agents to prevent the in vivo action of tetracycline and rolitetracycline, (h) the ability of gentamicin, ampicillin, isoxazolyl penicillins,aminosidin, and chloramphenicol to remove in situ the ureteral spasm caused locally by barium chlo ride, histamine and serotonin.
3) Factors influencingantibiotic pharinaco(lvnamicaction 3.1) Free or con/ugatedform of antibiotics into the urine This factor has been exemplifiedby the activity of chloramphenicol and its metabolites on the extrarenal urinary smooth muscle by the cross-urine preparation in the dog. In fact, in a variety of animal species, including man, about 90% of an administered dose of chloramphenicol can be recovered from the urine in 24 hr principally in the form of inactive metabolic products which retain the arylnitrogroup intact. Less than 10% is free chloramphenic , 10119represents drug in which the aromatic nitrogroup hasactive been reduced to an aminogroup, and the greater portion of the excreted antibiotic is a therapeutically inactive conjugation product with glucuronic acid (14). In the present study, the action of the urine from chloramphenicol-treateddogs was similar to the action of the urine from placebo-treated dogs added to the antibiotic at a concentration equal to the microbiologicalactivity of the urine from the chloramphenicol treated dogs. Assuming that less than 10%, of the administered dose is excreted un changed (as determined by microbiological assay) and that about 90% is excreted
unchanged and conjugated with glucuronic acid (as determined by chemical assay for total nitro compounds), it is possible to conclude that the conjugated antibiotic does not produce any effecton the ureter smooth muscle, the binding of antibiotic with glucuronic acid probably preventing the penetration of the drug to its muscular sites of action. 3.2)
The pH of the urine
FIG. 3. Guinea-pig
The pharmacodyn_imic antibiotics
action
can be pH-dependent,
of the as indi
cated in Fig. 3 for ampicillin,
dicloxacillin,
and mepicycline.
of both am
The
ED,,
line (MPC). concentration,
increased
the urinary pH. Actually,
the
ampicillin), ionized
The explanation antibiotics
No
of penetration
into
as it was scantly
substance
such as mepicycline,
ionization
character
under the same conditions,
flow value.
Abscissa,
urinary
(as
mepicycline)
mucosa is due to the ratio for
is greatly
ionized
affected
passed
from
environment.
across
the mucosa
its passage
unionized/
molecules.
Therefore
by the pH of the
readily
low-pH
poorly
or acids (as
to biophase
the acid Conversely,
with regard being
urine.
urine
to
a basic to its high
greater
from
the
urine.
On the other hand, tic-site of action"
presuming
binding,
the pH of the biophase lies the contact
the interaction
could both he influenced
active
that ampicillin,
in the ionized
will depend on the degree of ionization. of the target site attract
nic the groups drugs
sites.
form.
Thus
and in which the antibiotic itself.
in the biophase, attraction
a basic grouping
There
was phar
is the force for
and the interaction
The lower the pH, the more the cationic
On the contrary, a
by
are dissolved
If electrostatic
ampicillin.
for mepicycline,
groups
it can be presumed
and the lower the pH, the less anio
will be, and the less they will be able to attract (15), Rocha
extent
in the biophase
by the pH of the biophase
must contain
by Se-re
in the "antibio
to a considerable
after distribution
with the target site, this must contain
that the site of action
forces are implicated
can be influenced
in which the basic or acid antibiotics
fore it was possible to suppose macodynamically
that electrostatic
the interaction
with the acid or basic target
and the site of binding
for other
ED5o -antibiotic solution flowing
both the drug and the receptor.
bases
is assumed
in this
passed
of
the urinary
the biophase
ionized
pH
pH.
acid such as ampicillin
biophase
The
of
active transport
In this way an organic
alkaline
the
Ordinate, in the
the maximal
is that the pH influences
having
the degree of passage across
molecules.
the extent
with the decrease
in situ.
through the ureter, inducing an increase of the intraureteral flow equal to 50% of
picillin and dicloxacillin decreased with the decrease of the pH, while the ED., of mepicycline
ureter
dependence of the ED5o of dicloxacillin (DCL), ampicillin (AMP) and mepicyc
and Silva (16), and
the antibiotic,
as indicated
Ariens and Simonis
(17).
FIG. 4. Guinea-pig ureter in situ. Time lag of dicloxacillin, gentamicin and amino sidin in inducing a change of the intraureteral flow. Ordinate, time lag in min. Abscissa, the intraureteral antibiotics concentration in beg/ml.
3.3)
Time lag As indicated
delay situations
in Fig.
4,
in the onset
there
were
of antibiotic
pharmacodynamic action after addition into the urine of what has been evaluated to be an effective
dose of the drug.
The
time for onset of action was concentration dependent,
the
greater
the concentration,
the lower the time lag. delays in the onset on the ureter include the passage across time for diffusion
The
reasons
for
of effect of antibiotic the time it takes
the ureteral
FIG. 5. Guinea-pig ureter in situ. The time of contact dependence of the EDSO of
for
mucosa,
the
aminosidin
into the reactive tissues,
and the time it takes for the pharmacody namic 3.4)
effects to reach
an evaluable
level.
uniformity
in the
time-action
relationships
dose-response, a general
description
Nevertheless the
ureter
so that
than
the
time
teral
of the change
flow induced
for antibiotics
to exemplify of
Ordinate,
in intraure
by the antibiotic.
of
of the phenomenon
a function
the beginning
of
the formulation
it is possible was
in those
gentamicin.
flow equal to 50% of the maximal flow value. Abscissa, three subsequent periods of contact with the antibiotic : min from
Time of contact There is less general
and
ED50 -antibiotic concentration, in the solution flowing through the ureter, in ducing an increase of the intraureteral
for gentamicin of
contact,
also has been and
difficult
aminosidin
as indicated
that
in Fig.
to develop.
the
action
on
5.
DISCUSSION Not
all
mentation
pharmacodynamic
can
the
pharmacodynamic
are
mostly
peutic
doses
effects
be extrapolated
included
concentrations within
of antibiotics.
produced
to humans.
the For
In
by any
of antibiotics
range
of the
example,
antibiotics case,
in extrarenal
urinary
the urinary
in
it seems
levels active
lower-animal useful
urinary
occurring
tract
in man
concentration
experi
to remark
that
of animals after
thera
of aminosidin
on guinea-pig's ureter in situ ranges from 100 to 1600,ag/ml; on the other hand, in man the peak level of the antibiotic varies from 1300 to 2100beg/mlin the 4 hr urine fractions collected after i.m. administration of a therapeutic dose of 500 mg of aminosidin (18). Likewise, chloramphenicol pharmacodynamic intraureteral active concentrations on nor mal or activated ureter in situ of guinea-pigrange from 4.0 to 45.5 '"g/ml; after oral ad ministration of 1.5 g of antibiotic to humans the urinary concentration varies from 70 to 210 ag/ml in 24 hr urine specimens by microbiological assay (14). Therefore, the pre sent experimentaldata can be used to predict in humans qualitative and quantitative effects that may be expected from an antibiotic, although there is no set of rules of different dis tribution and elimination rates, and of different rates of antibiotic metabolism. In any case it is possible to predict the difficultyof the studies in humans because the response of the urinary smooth muscle is dependent on the urinary levels of the antibiotic used, on the time lag and time of contact, on the presence in the urine in free or conju gated form, etc. Evaluations on humans are even more difficult as the time lag, the phar macodynamic effect etc. are a function of the urinary level, and this parameter changes during the antibiotic urinary elimination as a function of time. The finding that the pharmacodynamic response to the antibiotics is pH-dependent is particularly important during inflammatory processes of the urinary tract which induce changes of the physicochemical properties of the urine and of the ureteral mucosa with consequent modification of the passage of antibiotics across the mucosa. REFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 18)
POPOVICI,G.G., MOISA,L., NEGOITA,M., MANOILA,V., BOTEZ,E. AND GUMENI,N.: Archs. int. Pharmacodyn. Ther. 154, 374 (1965) MATSUZAKI,A.: Japan J. Antibiotics 21, 274 (1968) KUDRYASHOVA, A.Y.: Antibiotiki 14, 501 (1969) GUITI, N. AND KERMANI,M.H.: Chemotherapy 15, 65 (1970) JOB, D.D.: Am. J. Pliysiol. 220, 299 (1971) BENZI, G., BERTE', F., BERMUDEZ, E. AND ARRIGONI,E.: J. Pharm. Sci. 59, 1198 (1970) Popovici, G.G., NEGOITA,M., MOISA,L., MANOILA,V., HAFNER,R. AND BOTEZ,E.: Archs. int. Pharmacodyn. Thér. 166, 20 (1967) BENZI, G., ARRIGONI,E., FRIGO, G.M., FERRARA,A., PANCERI,P., BERTE',F. AND CREMA, A.: Digestion 7, 54 (1972) LODI, L., MEINARDI,G. AND ROSSI, E.: II Farmaco, Ed. Pr. 24, 759 (1969) GYANCHANDANI, N.D., MCGILVERAY,I.J. ANDHUGHES,D.W.: J. Pharm. Sci. 59, 224 (1970) LITCHFIELD,J.T. Jr. AND WILCOXON,F.: J. Pharmacol. exp. Ther. 96, 99 (1949) DIXON, M.: Biochem. J. 55, 170 (1953) LINEWEAVER,H. AND BURK, D.: J. Am. Chem. Soc. 56, 658 (1934) GLAZKO, A.J., WOLF, L.M., DILL, W.A. AND BRATTON,A.C. Jr.: J. Pharmacol. exp. Ther. 96, 445 (1949) SEGRE, G.: Archs. int. Pharmacodyn. Thér. 124, 53 (1960) ROCHA e SILVA, M.: Archs. int. Pharmacodyn. Thér. 128, 355 (1960) ARIENS, E.J. AND SIMONIS,A. M.: Archs. int. Pharmacodyn. Thér. 141, 309 (1963) CASSANO,A., NUZZOLO, L. AND RAVETTA,M.: Clin. Terapeutica 17, 307 (1959)
OF
ANTIBIOTICS
G. BENZI,
ON
THE
E. ARRIGONI,
URETER
P. PANCERI,
F. BERTE'
MOTOR
ACTIVITY
R. PANZARASA,
and A. CREMA
Department of Pharmacology,
University of Pavia, Pavia, Italy
Accepted February
5, 1973
Abstract-The effect of certain antibiotics on the ureteral tone and motility has been studied in vitro and in situ in guinea-pigs and dogs. Tetracycline and rolitetracycline induced a constriction, while ampicillin, isoxazolyl penicillins, gentamicin, aminosidin, spiramycin and chloramphenicol induced a relaxation of the ureter. In vivo, the re sponses were independent of both bladder activity and systemic effects related to the antibiotic flowing into the ureter and eventually absorbed from it. The anti biotics acted directly on the ureteral smooth muscle; factors influencing the action were: (a) the free or conjugated form of the antibiotic flowing through the ureter; (b) pH of the urine; (c) time of contact; (d) time lag. Antibiotics
have been used extensively
in the treatment
the kidneys being the main route by which a great number the body
in both
vancement tone
modified
has been made
and
motility
other hand,
and unchanged
of the urinary
it is known
that antibiotics
This report
presents
on the tone and motility
The experiments
experimental
were performed
The antibiotics
sidin sulphate,
spiramycin
base); ampicillin
adipate
sodium
(the amounts
penicillanic (different
acid). samples
39.4), doxycycline (7.4;
findings
with the indicated (6.8;
on the action
oxacillin
percentage
sodium, in terms
of certain
biliary
antibiotics and dogs.
gentamicin
sulphate,
amino
in terms of the
in terms of n(-)-6-(a-amino
cloxacillin
sodium,
dicloxacillin
of 3-phenyl-5-methyl-4-isoxazolyl
also with the following
of epiderivative):
tetracycline
tetracyclines (7.4; 25.7;
(0.8; 7.2; 18.8; 41.2) and rolitetracycline
of which are expressed
in terms
of the base.
and the 4-epiderivatives
to the methods
of Lodi et a]. (9) and Gyanchandani
the degradation
products
was calculated
On the
muscle of the intes
of which are expressed
were performed
of the tetracyclines
on
and in the dog both in vitro and
of which are expressed
17.6; 37.4), mepicycline
30.2; 47.7), the amounts
tion and determination
of the ureter.
(7), of the extrahepatic
chloramphenicol,
(the amounts
acid);
little ad
AND METHODS
of which are expressed
The experiments
that
antibiotics
both in vitro and in situ, in guinea-pigs
D(-)
sodium (the amounts
a-phenylacetamido)-penicillanic
particularly
in the guinea-pig
used were:
surprising
can interfere with the smooth
of the ureter
tract infections, are excreted from
of the excreted
tract (2, 6), of the uterus
MATERIALS in situ.
It is rather
of the action
tract musculature,
tinal tract (1-5), of the bronchial tract (8).
forms.
in the knowledge
of urinary
of antibiotics
et al. (10).
as a percentage
The separa
were made according The concentration
of the total tetracycline.
of
Experiments in vitro Ureters were removed from 40 adult guinea-pigs of both sexes and from 62 adult male and female mongrel dogs. These were carefully dissected and set up in a 20 ml or 50 ml organ-bath containing Tyrode solution gassed with 95% oxygen and 5 car bon dioxide; temp. was 37.2_0.2'C and pH was buffered to 7.2±0.05. The longitudinal movements of the ureter were recorded by a strain-gauge with isotonic transducer; the lever exerted a resting tension of 0.5 to 1.5 g on the tissue and the magnification was 10 20 times. The action of the antibiotics was evaluated on the ureter in normal condition or after stimulation by barium chloride (10 to 50 ,cag/mlplus 0.1 beg/ml of atropine sulphate), hista mine (2.5 to 10.0 ig/ml) or serotonin (2.5 to 10.0pg/ml). For the construction of the dose-response curves, the activity of the antibiotics was taken as the change in the recorded area of barium chloride induced movements during a 30 min period (tetracyclines)or 20 min period (other antibiotics) of contact before and after antibiotic addition. The tested antibiotics were added to the bath 2 min (tetracyclines) or 10 min (other antibiotics) before the barium chloride administration. The ED,, and the slope function of the lines, with 95% confidence limits, were evaluated according to Litchfield and Wilcoxon (11). Experiments in vivo Studies were performed on 260 adult female guinea-pigs and 56 mongrel dogs of both sexes. The guinea-pigs were anaesthetized with urethane (1 -/kg i.p.) and chlora lose (20 mg/kg i.p.). The dogs were sedated with urethane (0.4 g/kg i.p.) and anaesthesia was induced by chloralose (100 mg/kg i.v.) or by nitrous oxide or cyclopropane in closed circuit. The animals were given artificial ventilation. Arterial blood pressure was mea sured from a cannula inserted into a carotid (guinea-pig) or femoral (dog) artery. After laparotomy a cannula was inserted into the renal pelvis to perfuse the ureter by Tyrode solution with and without antibiotics. The pH of the Tyrode solution was generally ad justed to 6.6-0.05, but in some experiments the pH was buffered to 7.8;0.05 or 5.5 0.05. In guinea-pigs, a draining tube was inserted into the fundus vesicae; in dogs, an urethral catheter was inserted. The flow into the ureters was measured for a 30 min period immediatelyafter the beginning of the change induced by antibiotics, compared with the flow during the 30 min period before the addition of the antibiotics. The ureter was either normal or activated by perfusing harium chloride (50 to 400 ,mg/'ml). The and the slope function of the lines, with 95''„ confidencelimits, were evaluated according to Litch field and Wilcoxon (11). In some experiments, prior to addition of an antibiotic to the solution flowing through the ureter, the animals were pretreated with: atropine sulphate (1-2 mg/kg s.c.), chlor pheniramine maleate (1-2 mg/kg s.c.), methysergide maleate (25-50 /1g/kg s.c.), cypro heptadine hydrochloride (150-3001g/kg i.v.), dibenamine hydrochloride (2-4 mg/kg i.v.), o(-) INPEA (4-8 mg/kg i.v.), or hexamethonium bromide (100-200/-tg/kg i.v.). In a series of experiments it was possible to evaluate in situ the flow through the ureter pulled out from the ureter-bladder junction and therefore free from urinary vesica inter
ference. In 22 mongrel Rectangular
dogs electrodes
pulses,
of 0.2-1
were placed
cosec duration
quency of 5 to 10 shocks/sec
around
the hypogastric
and 5-20 V strength
for 20 sec, before
and after
or pelvic nerves.
were applied
the addition
at a fre
of the antibiotics
into the renal pelvis. Cross-urine-perfusion Studies donor
preparation
were carried
animal
on 8 groups
of 3 beagle
bitches.
was given the tested antibiotic
i.v.; another
donor
tion i.v. During
out
4-8 hr, urine samples
tying the bladder
at each collection
sured in both ureters; the urine collected
the control
in a random
treated
animal;
Linear
Graphical
Representations
Dixon procedure:
This method
(b) the other
tives concentrations
of the combined
order: ureter
(Dixon
of
barium
was evaluated
response
induced
with
by per
This method
animal.
by tetracyclines. as a function
of the tested
of the effect on the ureteral
concentration
chloride.
flow was mea
of the effect is expressed
(contained
at two fixed concentrations
procedure:
emp
both the ureters
the placebo-treated
at two fixed concentrations
the reciprocal
of the tested tetracyclines
solu
to study the extent to which the 4-epideriva
12), the reciprocal
we expressed
tetracycline)
concentrations
The response
one
(a) one ureter with the urine from the antibiotic
was applied
of the 4-epiderivatives
concentration
animal.
and completely
the urinary
by perfusing
with the urine from
drug B concentration,
In our procedures
Linewcearer-Bark
In the recipient
could affect the pharmacodynamic
In the Dixon procedure
of the tested
period.
group,
was given Tyrode
by catheterizing
flow was obtained
from the placebo-treated
fusing the recipient
a function
were obtained
In each
in a constant
of barium
was used to evaluate on the
ureter
Quantitative
drug
A.
musculature
as
concentration
chloride. the effect of a constant
stimulated
relationships
in vitro with varying between
drug
A
and
receptor:
can be rearranged
The reciprocal
(Lineweaver-Burk,
of the pharmacodynamic
of the drug concentration. action
13) as:
between
barium
effect is expressed
A similar procedure
chloride
and the members
as a function
was applied
of the reciprocal
in the
of a homologous
case
of the
inter
series of tetracyclines.
RESULTS 1)
Pharnlacodyna,nic Studies
aminosidin,
action
in guinea-pigs ampicillin,
in vitro of the spasm
of the antibiotics and
isoxazolyl
dogs
demonstrated
penicillins
by agonist (barium
in situ of the flow through
the ureter,
on ureter
and
chloride, either
that
spiramycin histamine,
normal
chloramplien induce
or serotonin)
or activated
icol, gentamicin,
both
an inhibition
and an increase
by agonist.
The ED,,
TABLE1. ED50 and slope function of the line (S), with 95% confidence limits, of the myolytic action of some antibiotics tested by dog isolated ureter in vitro and by guinea-pig ureter in situ normal or activated by barium chloride.
(a) ED50=antibiotic concentration (pg/ml in the Tyrode solution of the bath) reducing by 50% the contracting action of barium chloride (30=10 ,ug/,nl) during 20 min period of contact. (b) ED50 =antibiotic concentration (pg/ml in the solution flowing through the ureter) inducing an increase of the intraureteral flow equal to 50% of the maximal flow, as evaluated during the control condition. (c) ED50=antibiotic concentration (pg/ml in the solution flowing through the ureter) inducing an increase of the intraureteral flow equal to 50°%of the maximal flow, as evaluated during the control condition. The antibiotics were tested during the local action of barium chloride (200 pg/ml). (d) S==fold change in dose required to produce a unit standard deviation change in response along the line ; thus S=antilog s=antilog 1/b, where b and s are respectively the slope constant and standard deviation of a line relating log dose of antibiotic, and probit per cent effect. on the ureter, order
normal
of potency
or activated
in the myolytic
by barium activity
chloride,
gentamicin;,>aminosidin;~spiramycin-ampicillin. action
was affected:
and rolitetracycline
(a) by the antibiotic increased
higher the 4-epiderivative ride (Fig. 2). tetracycline,
concentration,
On the contrary, oxytetracycline,
As
concerns
used: mepicycline
the contracting
muscle (Fig. 1); (b) by the percentage
is summarized
in Table
was chloramphenicol>isoxazolyl
action
the
antagonized,
of barium
of the 4-epiderivatives
chloride
and chlortetracycline
(Table
tetracyclines,
on ureter smooth used:
action of barium
differences
their
while tetracycline
in the tetracycline
the greater the stimulating
there were no significant
1 ; the
penicillins>
among
the chlo
activities
of
2).
The in vivo addition of antibiotics into the solution flowing through the ureter in duced no effect on the response to electrical stimulation of the hypogastric or pelvic nerves, due to the predominant contraction of the bladder muscle which was unaffected by the antibiotics flowing from ureter-bladder junction into the urinary bladder.
FIG. 1. Dog ureter in vitro. Lineweaver-Burk plots calculated : (a) for the effect of barium chloride in the absence of tetracycline (WT) ; and (b) for the effect of a constant concentration (11.25 x 10' M) of various tetracyclines (A mepicyc line ; • tetracycline ; f,] rolitetracycline) introduced into the bath 2 min before the barium chloride. Abscissa, the reciprocals of barium chloride molar con centration (1/M). Ordinate, the reciprocals of the effect (100/per cent of maximal contraction assumed equal to 100). Plotted points represent average values in four preparations for tetracyclines plus barium chloride, and in sixteen preparations for barium chloride alone. The concentrations of 4-epiderivatives contained in the various tetracyclines were the following : mepicycline=18.8% ; tetracycline = 25.7% ; rolitetracycline = 30.2%.
FIG. 2. Dog ureter in vitro. Dixon straight lines calculated for the effect of two fixed concentrations of barium chloride (A,=7.22 x 10' M ; A,=2.88 x 10-4 M) expressed as a function of the 4-epiderivatives concentration contained in a constant concentration (11.25 x 10' M) of the tetracyclines tested, introduced into the bath 2 min before the barium chloride. Abscissa, % concentration of the 4-epiderivatives. Ordinate, the reciprocals of the effect (100/% contraction). Plotted points represent average values in four preparations.
TABLE2. Dog isolated ureter. EDSOand slope function of the line (S), with 95% confidence limits, of three different tetracyclines, the 4-epiderivative concentration being quite equal in the preparations (from 23.2 to 29.4°%).
(a) ED50 antibiotic concentration (ug/ml) reducing ureteral flow during 30 min period of contact. (b) S as in Table 1.
by 50°
the intra
2) Mode of pharmacodynaniicaction of antibiotic on the ureter Responses in situ of the ureter to antibiotics flowing through the ureter were inde pendent of the ureter-bladder junction activity, as they were present even when the ureter was freed from urinary musculature. The action was also independent of any systemic effects related to the antibiotic eventually absorbed from ureter into the bloodstream and distributed throughout the body, as the urinary flow into the other ureter was unaffected during the antibiotic action on the ureter being tested. The direct action of the antibiotics on the extrarenal urinary tract smooth muscle was supported by: (a) the failure of cholinoceptor, adrenoceptor, histamine, 5-hydroxy tryptamine and ganglion blocking agents to prevent the in vivo action of tetracycline and rolitetracycline, (h) the ability of gentamicin, ampicillin, isoxazolyl penicillins,aminosidin, and chloramphenicol to remove in situ the ureteral spasm caused locally by barium chlo ride, histamine and serotonin.
3) Factors influencingantibiotic pharinaco(lvnamicaction 3.1) Free or con/ugatedform of antibiotics into the urine This factor has been exemplifiedby the activity of chloramphenicol and its metabolites on the extrarenal urinary smooth muscle by the cross-urine preparation in the dog. In fact, in a variety of animal species, including man, about 90% of an administered dose of chloramphenicol can be recovered from the urine in 24 hr principally in the form of inactive metabolic products which retain the arylnitrogroup intact. Less than 10% is free chloramphenic , 10119represents drug in which the aromatic nitrogroup hasactive been reduced to an aminogroup, and the greater portion of the excreted antibiotic is a therapeutically inactive conjugation product with glucuronic acid (14). In the present study, the action of the urine from chloramphenicol-treateddogs was similar to the action of the urine from placebo-treated dogs added to the antibiotic at a concentration equal to the microbiologicalactivity of the urine from the chloramphenicol treated dogs. Assuming that less than 10%, of the administered dose is excreted un changed (as determined by microbiological assay) and that about 90% is excreted
unchanged and conjugated with glucuronic acid (as determined by chemical assay for total nitro compounds), it is possible to conclude that the conjugated antibiotic does not produce any effecton the ureter smooth muscle, the binding of antibiotic with glucuronic acid probably preventing the penetration of the drug to its muscular sites of action. 3.2)
The pH of the urine
FIG. 3. Guinea-pig
The pharmacodyn_imic antibiotics
action
can be pH-dependent,
of the as indi
cated in Fig. 3 for ampicillin,
dicloxacillin,
and mepicycline.
of both am
The
ED,,
line (MPC). concentration,
increased
the urinary pH. Actually,
the
ampicillin), ionized
The explanation antibiotics
No
of penetration
into
as it was scantly
substance
such as mepicycline,
ionization
character
under the same conditions,
flow value.
Abscissa,
urinary
(as
mepicycline)
mucosa is due to the ratio for
is greatly
ionized
affected
passed
from
environment.
across
the mucosa
its passage
unionized/
molecules.
Therefore
by the pH of the
readily
low-pH
poorly
or acids (as
to biophase
the acid Conversely,
with regard being
urine.
urine
to
a basic to its high
greater
from
the
urine.
On the other hand, tic-site of action"
presuming
binding,
the pH of the biophase lies the contact
the interaction
could both he influenced
active
that ampicillin,
in the ionized
will depend on the degree of ionization. of the target site attract
nic the groups drugs
sites.
form.
Thus
and in which the antibiotic itself.
in the biophase, attraction
a basic grouping
There
was phar
is the force for
and the interaction
The lower the pH, the more the cationic
On the contrary, a
by
are dissolved
If electrostatic
ampicillin.
for mepicycline,
groups
it can be presumed
and the lower the pH, the less anio
will be, and the less they will be able to attract (15), Rocha
extent
in the biophase
by the pH of the biophase
must contain
by Se-re
in the "antibio
to a considerable
after distribution
with the target site, this must contain
that the site of action
forces are implicated
can be influenced
in which the basic or acid antibiotics
fore it was possible to suppose macodynamically
that electrostatic
the interaction
with the acid or basic target
and the site of binding
for other
ED5o -antibiotic solution flowing
both the drug and the receptor.
bases
is assumed
in this
passed
of
the urinary
the biophase
ionized
pH
pH.
acid such as ampicillin
biophase
The
of
active transport
In this way an organic
alkaline
the
Ordinate, in the
the maximal
is that the pH influences
having
the degree of passage across
molecules.
the extent
with the decrease
in situ.
through the ureter, inducing an increase of the intraureteral flow equal to 50% of
picillin and dicloxacillin decreased with the decrease of the pH, while the ED., of mepicycline
ureter
dependence of the ED5o of dicloxacillin (DCL), ampicillin (AMP) and mepicyc
and Silva (16), and
the antibiotic,
as indicated
Ariens and Simonis
(17).
FIG. 4. Guinea-pig ureter in situ. Time lag of dicloxacillin, gentamicin and amino sidin in inducing a change of the intraureteral flow. Ordinate, time lag in min. Abscissa, the intraureteral antibiotics concentration in beg/ml.
3.3)
Time lag As indicated
delay situations
in Fig.
4,
in the onset
there
were
of antibiotic
pharmacodynamic action after addition into the urine of what has been evaluated to be an effective
dose of the drug.
The
time for onset of action was concentration dependent,
the
greater
the concentration,
the lower the time lag. delays in the onset on the ureter include the passage across time for diffusion
The
reasons
for
of effect of antibiotic the time it takes
the ureteral
FIG. 5. Guinea-pig ureter in situ. The time of contact dependence of the EDSO of
for
mucosa,
the
aminosidin
into the reactive tissues,
and the time it takes for the pharmacody namic 3.4)
effects to reach
an evaluable
level.
uniformity
in the
time-action
relationships
dose-response, a general
description
Nevertheless the
ureter
so that
than
the
time
teral
of the change
flow induced
for antibiotics
to exemplify of
Ordinate,
in intraure
by the antibiotic.
of
of the phenomenon
a function
the beginning
of
the formulation
it is possible was
in those
gentamicin.
flow equal to 50% of the maximal flow value. Abscissa, three subsequent periods of contact with the antibiotic : min from
Time of contact There is less general
and
ED50 -antibiotic concentration, in the solution flowing through the ureter, in ducing an increase of the intraureteral
for gentamicin of
contact,
also has been and
difficult
aminosidin
as indicated
that
in Fig.
to develop.
the
action
on
5.
DISCUSSION Not
all
mentation
pharmacodynamic
can
the
pharmacodynamic
are
mostly
peutic
doses
effects
be extrapolated
included
concentrations within
of antibiotics.
produced
to humans.
the For
In
by any
of antibiotics
range
of the
example,
antibiotics case,
in extrarenal
urinary
the urinary
in
it seems
levels active
lower-animal useful
urinary
occurring
tract
in man
concentration
experi
to remark
that
of animals after
thera
of aminosidin
on guinea-pig's ureter in situ ranges from 100 to 1600,ag/ml; on the other hand, in man the peak level of the antibiotic varies from 1300 to 2100beg/mlin the 4 hr urine fractions collected after i.m. administration of a therapeutic dose of 500 mg of aminosidin (18). Likewise, chloramphenicol pharmacodynamic intraureteral active concentrations on nor mal or activated ureter in situ of guinea-pigrange from 4.0 to 45.5 '"g/ml; after oral ad ministration of 1.5 g of antibiotic to humans the urinary concentration varies from 70 to 210 ag/ml in 24 hr urine specimens by microbiological assay (14). Therefore, the pre sent experimentaldata can be used to predict in humans qualitative and quantitative effects that may be expected from an antibiotic, although there is no set of rules of different dis tribution and elimination rates, and of different rates of antibiotic metabolism. In any case it is possible to predict the difficultyof the studies in humans because the response of the urinary smooth muscle is dependent on the urinary levels of the antibiotic used, on the time lag and time of contact, on the presence in the urine in free or conju gated form, etc. Evaluations on humans are even more difficult as the time lag, the phar macodynamic effect etc. are a function of the urinary level, and this parameter changes during the antibiotic urinary elimination as a function of time. The finding that the pharmacodynamic response to the antibiotics is pH-dependent is particularly important during inflammatory processes of the urinary tract which induce changes of the physicochemical properties of the urine and of the ureteral mucosa with consequent modification of the passage of antibiotics across the mucosa. REFERENCES 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13) 14) 15) 16) 17) 18)
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