J. vet. Phamacol. T h ~ a p15,42 . 1 4 3 2 , 1992.
Effect of water deprivation on absorption (oral, intramuscular) and disposition of ampicillin in sheep M. OUKESSOU* & P. L. T O U T A I N t *Department of Physiology and Therapeutics, Hassan I1 Agronomic and Veterinary Institute, Rabat, Morocco and tEcole Nationale VCterinaire, Department of Physiopathology, 23 chemin des Capelles, 31076 Toulouse Cedex, France
Oukessou, M., Toutain, P.L. Effect of water deprivation an absorption (oral, intramuscular) and disposition of ampicillin in sheep. J. vet. Phannacol. Therup. 15, 421432. T h e effects of a 72 h water deprivation on the absorption -intramuscular (im.) and oral and disposition of ampicillin, inulin and para-aminohippuric acid (PAH) were investigated in six sheep. After intravenous (i.v.) administration of ampicillin sodium (10 mglkg), the water deprivation decreased slightly the initial volume of distribution (0.082 k 0.033 vs. 0.055 f 0.030 I/kg) but not the steady state volume of distribution. T h e plasma clearance was significantly decreased (6.21 k 1.94 vs. 3.90 +. 1.92 ml/min/kg) and the mean residence time (MRT) was increased from 22.25 k 4.91 to 33.36 k 8.16 min. After i.m. administration of ampicillin sodium (20 mg/kg), ampicillin concentrations were systematically higher after a 3-day period of water deprivation than during the control period but the muscular absorption rate was not modified. After oral administration of ampicillin trihydrate ( 1 g in loto) plasma concentrations were much lower and more persistent than after an i.m. administration and the systemic availability remained low whatever the hydration status. Influences of water deprivation on ampicillin disposition were linked to adaptation of renal function as assessed by inulin and PAH clearances. T h e therapeutic relevance of the results are discussed for a better definition of dosage regimens for sheep reared in arid environments. P . L. Touhin, ENVT, Department of Physiopalhology, 23 chetnin des Capelles, 3 1076 Toulouse Cedex, France.
I N T R O D JCTION In hot and arid environments, possible excessive insensible heat loss and water deprivation are factors which must be taken into account when selecting a dosage regimen. In sheep, dehydration may have many physiological consequences including loss of body weight, reduction of food intake, modification of water spaces with a reduction of the extracellular sector, increase of plasma protein concentration, reduction of renal filtration etc.
(MacFarlane, 1964). Potentially all these factors individually or collectively can modify the process of absorption at the administration site (digestive tract, muscle), distribution and drug elimination. Currently, few experimental data exist on the influence of hydration status on drug disposition despite the fact that various disease conditions such as polyuria and diarrhoea are concerned by disturbance of the water balance. In the rat, after a 4-day water deprivation, the plasma and tissue gentamicin concentrations were increased and 42 1
422 M. Oukessou @ P . L. Toukzin the elimination rate of the drug was decreased leading to a reduction of 22 and 40% of the central volume of distribution and of the plasma clearance respectively (Lecompte et al., 1981). Siniilarly, water deprivation in rat increased the biological half-life of salicylic acid by 72% (Bakar & Niazi, 1983). In domestic species, there is to our knowledge no information on the effect of water deprivation on drug kinetics and the purpose of the present study was to investigate the effect of a 72 h water deprivation on the absorption (im. and oral) and disposition of ampicillin in sheep. A 72 h deprivation was selected as it represents a frequent situation in Moroccan pastoral husbandry. MATERIALS A N D M E T H O D S Animals
Six adult Sardi ewes, weighing 32-57 kg were used. During the experiments, they were placed in individual metabolism cages. T h e ambient temperature was controlled at 20 2°C.
*
solved in distilled water just before administration. Intramuscular administrations of ampicillin sodium (20 mg/kg) were made deep in the lumbar muscle at a single site with a 40*11 mm needle. Oral administration of ampicillin trihydrate (1 g per animal) was made after mixing ampicillin powder with 40 ml of tap water; the suspension was administered with an appropriate syringe placed on the tongue. T h e same technique was used to administer glucose (0.3 g k g ) for the oral glucose test. Blood samples (6 ml) were collected from the other jugular vein using heparinized vacutainers (Becton Dickinson vacutainer system, Becton Dickinson and Co., France) before drug administration (time 0) and at 2, 4, 8,15,30,60,120,240,360,480,600 and 1440 min after the i.v., i.m. and oral ampicillin administrations. After inulin and PAH administration, samples were collected at the same times u p until 300 min. Samples were collected before and 5, 10, 20, 30, 60, 90, 120, 180, 240 and 300 min after oral glucose administration. All blood samples were centrifuged within 30 min. Plasma was stored at -25°C until assay.
Drugs and les! articles
Expa'mental design
T h e following drugs were used: Ampicillin sodium (Ampivet, Arkovet, Ciba-Geigy, France), Ampicillin trihydrate (Totapen, Laboratoires Rhbne Poulenc Pharma, Casablanca, Morocco), inulin (Sigma, St Louis, MO. USA), para-aminohippuric acid (Sigma) and glucose (30% solution, Laprophan S.A., Casablanca, hlorocco).
In a first set of experiments, the animals had a free access to water. First, all sheep received inulin and PAH by the i.v. route. They were then allocated to groups of two and received ampicillin by i.v.. i.m. and oral route according to a three-period cross-over design. Each administration was separated by a washout period of 7-10 days. In a second set of experiments, animals were deprived of water for 72 h but had free access to food. T h e ewes were allocated to 2 groups of 3. First, they received ampicillin by i.m. or peroral (p.0.) at the E n d h following the withdrawal of water according to a two-period crossover design. Each administration was separated by a washout period of 1 week. After the i.m. or p.0. administration animal has no access to water for a supplementary time of 24 h. Then, at least 1 week later, the animal received according to a two-period crossover design either an i.v. administration of ampicillin or an i.v. administration of the
Drug udministruLiom and sample collection
intravenous (i.v.) administrations of ampiciilin sodium (10 mg/kg) were made via an indwelling catheter placed in a jugular vein jusr before the experimental session. Lyophilisrd ampicillin was dissolved in its commercial solvent just before administration. A mixture of inulin (40 mgikg) and para-aminohippuric acid (PAH) (20 mg/kg) was administered intravenously via an indwelling jugular catheter within 30 s. Inulin and PAH were dis-
Ampicillin in sheep 423
inulin/PAH mixture. For the i.v. studies, water deprivation was obtained and maintained as for the i.m. or p.0. administration. I n a last experiment, the influence of water deprivation o n the spontaneous closure of the oesophageal groove was assessed by drenching sheep with a 30% glucose solution in normal condition or after a 72 h water deprivation. Analytical techniques
Plasma ampicillin levels were measured within 1 week, of sample collection with a microbiological assay procedure using Sarcdna luteu ATCC 934 1 as test organism (Arret et al., 1971). Each sample was measured in duplicate. T h e level of quantitation was 0.005 pg/ ml. Standard dose response curves were obtained using buffer ampicillin solution (Ampicillin, anhydrous, Sigma) diluted in normal sheep plasma. T h e standard curves were constructed the day of sample collection and kept in the same conditions as the samples until analysis. All samples from a given experiment were run in the same assay. Plasma inulin concentrations were measured using a colorimetric method. T h e level of quantitation was 4 pglml. PAH plasma concentrations were determined by the use of a high performance liquid chromatographic technique (adapted from Prueksaritanont el al., 1984). Briefly deproteinisation was obtained by mixing 200 pl of plasma with 300 pl of perchloric acid 0.33 N. T h e mixture was vortexed for 15 s. and centrifuged at 5400 g for 10 min. The supernatant (20 pl) was injected into a Lichrosorb C18reversed phase column (particle size 10 pm). A variable wavelength detector (Kontron, Paris, France) operated at 254 nm and an integrator (Merck, Paris, France) were used. T h e mobile phase was a acetic mixture of acetonitrile (5%)and a WOO acid solution (95%).A flow rate of 1 ml/min was maintained using an isocratic pump (Kontron, Paris, France). T h e mean coefficient of extraction was 86.6%.T h e level of quantitation was 0.2 pg/ml. Phurmacokinetic analysis Compartmental analysis. T h e data were ana-
lysed using a programme for non-linear regression analysis (Yamaoka et al., 1981). Initial estimates were calculated by linearregression methods (Gilbaldi & Perrier, 1982). Plasma concentrations of ampicillin were fitted to equation 1: C(,) = YI exp (-Alt)
+ Y~exp(-A2t)
(1)
where C(,) represents ampicillin plasma concentration at time t; Y1 and Y2 are coefficients and 11 and A2 a r e exponents. For non-linear regression analysis, the data points were weighted according to equation 2.
where Wi is the weight and pi is the fitted value of the ith observation. T h e relevant pharmacokinetic parameters (elimination half-time, volume of central compartment, steady state volume of distribution . . .) were calculated according to the classic equations associated with compartmental analysis (Gilbaldi & Perrier, 1982). Non compartmental analysis. For ampicillin, inulin and PAH, areas under t h e plasma concentration-times curve AUC(o-,,,u,, were calculated using the linear trapezoidal rule without extrapolation to infinity i.e. from time 0 to the time corresponding to the last measured concentration (0-clad).T h e mean residence time (MRT(%r/u,,,)),which is the mean time for a molecule of ampicillin, inulin and PAH to transit through the body, was calculated by the linear trapezoidal rule from time 0 to the time corresponding to Cjart.T h e mean absorption time (MAT), which is the mean time for a molecule of ampicillin to be absorbed after oral or i.m. administration was calculated according to equation 3:
In Equation 3, MRTi.,, or MRTp,,. is the mean residence time of ampicillin after i.m. or oral administration respectively and MRTi,.,, is the mean residence time after i.v. administration. M A T under normal conditions or after water deprivation were calculated using the
424 M . Oukessou U
P. L. Touiain
MRT, ., obtained under normal conditions or after water deprivation respectively. T h e body clearance for ampicillin, inulin and PAH were calculated using equation 4: D
c1 =
parisons between treatment (water deprivation vs. non deprivation) were performed using a unilateral paired student's t-test. Regression analysis were carried out to assess the relationship between inulin, o r PAH and ampicillin selected kinetic parameters.
(4) A UC(~ 0.05). T h e plasma clearance
1
c 0 .C
c,
ml L
CI
c Q)
u l
c
0
0
1
0
60
120
240
360
480
600
T i m e (min 1 FIG. 1. Mean plasma concentration o f ampicillin (pg/ml) vs. time (min) after the i.v. administration of ampicillin sodium at a dose of 10 rng/kg in five or six ewes under normal conditions ( 0 - 0 ) or after a 3-day period of water deprivation (0-0).
Ampicillin in sheep
425
1 o2
.u
c Q) u
t
(s:
lo 0
60
120
240
360
480 600 Time (min )
FIG. 2. Mean plasma concentration of ampicillin (pg/ml) vs. time (min) after the i.ni. administration of ampicillin sotliuni at a dose of 20 mg/kg in six ewes under normal conditions ( 0 - 0 ) or after a 3-day period of water deprivation (0-0).
(6.21 k 1.94 vs. 3.90 k 1.92 ml/min/kg) was significantly decreased for P = 0.056. T h e MRT was significantly increased by water deprivation (22.25 f 4.91 vs. 33.36 f 8.16 min, P < 0.05). T h e individual values of the different parameters are given in Table I. T h e inulin water spaces were not significantly modified by water deprivation (Vc: 0.069 f 0.015 vs. 0.063 f 0.029 Vkg; Vss: 0.145 f 0.063 vs. 0.156 f 0.086 I/kg). T h e inulin clearance, which is an indicator of glomerular filtration rate was not significantly decreased by water deprivation (2.08 f 0.57 vs. 1.77 f 0.91 ml/min/kg, P > 0.05) but the effective renal plasma flow (ERPF), as estimated by the PAH clearance, was significantly reduced by water deprivation (14.30 f 3.63 vs. 11.38 k 2.68 ml/midkg, P < 0.05). Individual data are given in Table 11. Figure 2 gives the mean plot of plasma concentrations (kg/ml) for ampicillin as a function of time (min) after a single intrarnuscular administration of ampicillin sodium in the six ewes under normal conditions and after a 3-day period of water deprivation. Visual inspection of the figures indicates clearly that plasma ampicillin concentrations
were increased after water deprivation. This is confirmed by the significant increase of AUC (2746 _+ 624 vs. 4599 ?I610 pg.min/ml, P < 0.05); M A T were similar before and after water deprivation (70.5 k 54.6 vs. 65.3 f lG.5 min, P > 0.05) suggesting that the miiscular absorption rate was not modified by water deprivation. Similarly, the mean systemic availability was not affected by water deprivation (83.2 k 23.8 vs. 97.5 f 80.7%.P > 0.05). Individual data are given in Table 111. Figure 3 gives the mean plot of plasma concentrations (pg/ml) vs. tinle (min) for ampicillin after administration of anipicillin trihydrate by oral route in the six ewes in normal conditions and after a 3-day period of water deprivation. Visual inspection indicates that under both conditions, the plasma concentrations were much lower and more persistent than after an i.m. administration and that water deprivation increased plasma ampicillin concentrations. T h e AUC was multiplied by a factor of 3.5 (64.4 k 20.4 vs. 227.6 f 83.3 pg. min/ml, P < 0.05) by water deprivation. T h e systemic availability remains low whatever the hydric status (2.07 f 1.06 vs. 4.13 f 2.66%,P < 0.05) suggesting no or limited closure of
97.04 21.19 0.107 0.0208 33.35 28.05 0.084 0.155 0.250 4.93 2025.9
N
’r
P
150.8 80.9 2.12 10.23 0.1 15 0.033 0.0194 0.0077 35.62 89.39 35.51 25.03 0.065 0.109 0.074 0.220 0.420 0.268 2.17 7.67 1302.4 4605.0
N
V P
N
X
P
N
Y P
N
2
I’
80.6 281.4 104.3 98.1 115.9 248.9 177.5 490.7 1.67 15.24 55.85 13.15 0.169 19.25 33.20 31.76 0.403 0.072 0.103 0. I 3 6 0.233 0.083 0.989 0.303 0.0 I 8 0.0089 0.0059 0.0 174 0.0258 0.0093 0.0207 0.0151 38.17 74.32 77.78 117.1 1 39.69 26.83 33.50 45.90 14.43 43.27 25.2 I 21.51 19.28 37.02 21.85 29.17 0.090 0.094 0.018 0.124 0.052 0.033 0.067 0.035 0.175 0.154 0.075 0.161 0.267 0.119 0.109 0.292 0.1 14 0.328 1.70 0.689 0.284 0.189 0.388 0.237 6.55 4.70 5.78 4.74 1.75 9.44 4.84 4.42 1729.5 1573.3 1058.9 226 I .8 2127.3 2065.5 2 109.2 5700.2
N
W
*
k 10.63
& 68.3
*
I’
228.6 f 162.5 22.42 f 22.84 k 0.081 0.346 & 0.388 & 0.0067 0.01 17 f 0.0043 34.34 65.42 k 21.50 f 4.91 33.36 k 8.16 f 0.034 0.055 k 0.030 f 0.040 0.172 f 0.103 & 0.583 0.348 & 0.214 6.21 2 1.94 1.92 3.!)0 1725.5 & 452.2 3241.1 rt 1804.8 130.5 15.29 0.141 0.0184 47.43 22.25 0.082 0.156 0.520
N
Means f SD
Y I and Y, are pre-exponential constants, 1 and 2 are the exponential for the biexponential equation describing the plasma level-time curve. fp2 2: plasma half-life; difference not significant (P > 0.05); M R T mean residence time: significant difference (P < 0.05); vc: volume of the central compartment: significant difference (P < 0.05); V,,: steady state volume of distribution: difference not significant (P > 0.05); C1: plasma clearance: significant difference (P = 0.056); AUC: Area under the plasma level time curve: significant difference (P =0.058). MRT, C1, and AUC were calculated with the trapezoidal rule without extrapolation to infinity.
Parameters (units)
Ewes
T A B L E 1 . I’harniacokinetic parameters describing the disposition kinetics ofanipicillin in plasma after an intravenous administration of ampicillin sodium (10 rng/kg) i n six ewes in normal ( N ) condition and after a 3 days water deprivation (P)
3’
$
Y 0
N
T
N P
N
W
P
0.067 0.076 0.089 0.062 0.023 0.116 0.129 0.113 0.131 0.100 2.04 1.51 3.30 2.09 0.71 12.75 11.45 11.72 11.19 9.68
P
V
0.097 0.273 2.93 20.90
N
X N
P
0.101 0.062 0.045 0.331 0.115 0.125 2.07 1.57 1.34 15.85 14.79 8.43
P
Y
0.058 0.116 1.80 12.37
N
2
0.053 0.132 1.19 9.86
P
+
P 0.069 zk 0.015 0.063 k 0.029 0.145 k 0.063 0.156+0.086 1.77 0.91 2.08 k 0.57 14.30 k 3.63 11.38 k 2.68
N
Means k SD
V 0.05);Vrr: steady stale volume of the distribution for i n u h ( P > 0.05); ClIN: plasma clearance for inulin (P < 0.05); ClpAH:plasma clearance Ior PAH ( P < 0.05).
v,, (Vkg)
V , (Vkg)
0.058 0.109 CllN(ml/min/kg) 2.57 C l p A H (ml/min/kg) 15.11
Parameters (units)
Ewes
T A B L E I I . Selected pharmacokinetic parameters describing the disposition of inulin (40 mg/kg) and PAH (20 mg/kg) in six ewes in normal (N) conditions and after a 3-day water deprivation (P)
3'
49.6 21.5 60.8 8.0 3096 76.4 -
N
114.7 47.0 4.0 5519 136.2
P
164.0 139.0 9.7 60.0 2264 86.9 -
N
v
107.7 72.2 44.4 8.0 2579 28.0 99.0
I'
54.3 29.1 59.1 15.0 3439 99.4 -
N
W
97.2 75.3 58.7 15.0 7068 224.6 "4.3
P
70.6 56.2 35.6 8.0 2444 115.4 -
N
X
89.6 46.3 33.1 15.0 3560 78.7 168. I
I' 56.4 37.1 32.4 15.0 1914 45.0 -
N
Y
86.4 49.4 62.2 4.0 5068 122.7 119.1
P
161.2 139.7 17.5 60.0 3318 78.G -
N
Z
112.8 83.6 24.7 15.0 3798 33.3 90.0
P
92.7 f 54.6 70.4 f 54.6 35.9 k 20.9 27.7 f 25.2 2746k624 8 3 . 6 k 23.8 -
N
P
65.3 f 16.5 45.0 f 14.4 10.1 f 5.5 4 5 9 9 k 1610 9 7 . 5 f 80.7 136.1 k 43.5
101.4 k 12.1
Means k SD
hINT: niriin residence tinie; hIAE nitmi absorptioli tinie. C,,,,, observed maxinial concentration. I,,,., observed time for C,,,,.; A UC: Area under the plasma level tinie curve. MRT and A UC were calculated by [lie trapezoidal rule witliout extrapolation to infinity. The systemic bioavailability was calculated or the i.v. AUC obtained in nornial condition (F2). Water deprivation has no taking into account either the i.v. AUC obtained after water deprivation (F,) significant influence (P > 0.05) on MRT, hfAT, C,,,,,, and F , . AUC and F2 were significantly increased (P < 0.02).
MRT (rnin) MAT (min) C,,,.,, W m l ) t,,,,, ( n W AUC(pg.niin/iril) I.'I (%) I.'2 (%)
Parameters (uniis)
T
Ewes
1'A I3 L E I I I . Selected pliarmacokinetic parameters describing the disposition of ampicillin in plasma aftei- a n intraniuscular adniinistration of anipicilliii sotliuni (20 mg/kg) in six ewes in nornial ( N ) condition and alter a 3-day water deprivation (1')
P
W
r\3
554.2 526.1 73.7 1.82 -
N
T
6.53
-
612.5 264.7
P
448.2 423.2 85.9 3.30 -
N
V
394.3 358.8 241.8 2.62 9.30
P 190.5 165.3 61.4 1.77 -
N
W
494.4 472.5 95.6 3.04 2.76
P 289.0 274.6 71.2 3.36 -
N
X
415.5 372.2 215.4 4.80 10.20
P
-
552.8 533.5 68.2 1.60
N
Y
742.8 705.8 349.2 8.45 8.20
P
-
631.0 609.5 26.0 0.61
N
Z
511.0 481.8 199.3 1.75 4.72
P 444.3 422.0 64.4 2.07
2 20.5 k 1.06 -
k 2.85
f 83.3 f 2.66
k 139.0
k 130.5
k 171.6
k 170.9
P
N 528.4 478.2 227.6 4.13 6.94
Means k SD
M R T mean residence time; M A T : mean absorption time; AUC: area under the plasma level time curve normalized for a standard dose of 20 mg/kg. The systemic bioavailability was calculated taking into account either the i.v. AUC obtained after water deprivation ( F , ) or the i.v. A UC obtained in normal condition (F2).MRT and AUC were calculated by trapezoidal rule without extrapolation to infinity. Water deprivation has no influence on MRT and MAT, but significantly increased AUC (P < 0.04) and F2 (P < 0.01).
FP(%)
FI (%I
MAT(min) AUC(pg.rnin/rnl)
MRT (min)
Parameters (units)
Ewes
T A B L E I V. Selected pharmacokinetic parameters describing the disposition of ampicillin in plasma after an oral administration of ampicillin trihydrate ( 1 g per animal) in six ewes in normal (N) condition and after a 3-day water deprivation (P)
(D
k2
A
siF
h
F s.
h
a.
3.
b 3
430 M. Oukessou U P. L. Toulain
0.3 n c
E \
u (D
L
0.2
0.1
0
6 0 120
240
360
480 600 Time (min )
FIG. 3. Mean plasma concentration of ampicillin (pghl) vs. time (min) after the oral administration of ampicillin trihydrate at a dose o f 1 g in tofo in six ewes under normal conditions ( 0 - 0 ) or after a 3-day period o f water deprivation (0-0).
the esophageal groove during the ampicillin drenching. This is consistent with the absence of hyperglycemic response to an oral glucose test which was observed before and after water deprivation as evaluated by the oral glucose test. T h e AUC of plasma glucose calculated for the first 5 h after a glucose challenge were 18469 k 800 and 20743 2 2814 mg.mid100 mi before and after water deprivation respectively (P> 0.05). T h e MRT of ampicillin (444 k 171.6 min in normal conditions and 528 k 130.5 min after water deprivation) were not significantly different (P > 0.05) but much were longer than after an im. administration (P < 0.05). This is explained by a MAT which is about six times longer after an oral administration (422 k 170.9 min in normal conditions and 478.2 k 139.0 min after water deprivation) than after an i.m. administration. Individual values are given in Table IV.
DISCUSSION
When sheep are raised in a dry hot environment and when herdsmen are obliged to haul water from wells, water is generally not offered each day but more often every 2 or 3
days. From a therapeutic point of view it is relevant to know if this kind of husbandry is able to influence d r u g disposition. T h e physiological effects of water deprivation in sheep are rather complex as they depend on other factors including breed and external temperature. In the present experiment, the Sardi breed which was used is well known for its adaptation to arid environments and the experiment was carried out at a relatively low temperature (20 k 2"). Consequently, a 3-day water deprivation was unlikely to produce dramatic physiological changes. In terms of water compartment, inulin space which is considered as equivalent to the extracellular volume (Caudino & Levitt, 1949) was not modified by a 3-day water deprivation (Vss of about 15% of body weight) and was identical to that reported in lamb (Devaskar el al., 1985). Similarly, the initial volume of distribution of inulin (V, = 6-776 of body weight) which is approximately equal to the plasma volume was not modified by the water deprivation. It can thus be concluded that the 3 days water deprivation had a minimal effect on extracellular water spaces; this explains why a water deprivation under our experimental conditions had no or minimal effect on volumes of distribution of- ampicillin. O n the
Ampicillin in sheep 431
other hand, plasma ampicillin concentrations were systematically higher after a 3-day period of water deprivation than during the control period. This can only be linked to an adaptation of the renal function with both a reduction of renal filtration as suggested by the reduction (non statistically significant) of inulin clearance, and of the ERPF (as demonstrated by the significant reduction of the PAH clearance). Such adaptation has already been reported in dehydrated sheep (MacFarlane, 1964). Renal elimination of unchanged ampicillin appeared to be the major route of elimination (Prescott & Baggot, 1988). In the present experiment, the plasma ampicillin clearance was higher than glomerular filtration rate (i.e., inulin clearance) suggesting active secretion of ampicillin by the kidney. This explains that water deprivation by reducing the ERPF, also reduced the elimination of ampicillin and increased the MRT of ampicillin in the body by about 50%. After i.m. administration, neither the mean absorption time nor the systemic bioavailability was modified by water deprivation indicating that physiological processes implicated in muscular resorption (local blood flow) were not affected by water deprivation. After i.m. administration, large variations in pharmacokinetic parameters between individual and varying in opposite direction were observed. Similarly, a higher than 200% bioavailability was calculated for one of the sheep. We have no consistent explanation to offer for this: a partial explanation should be that a 3-day dehydration is not a reproductible status leading to within and between sheep variations. After oral administration, the plasma concentration in the control ewe was low. This was expected, as ampicillin arriving in the rumen must be largely inactivated as has been observed by others in calves (Thompson & Black, 1978). After a water deprivation, the oral administration of ampicillin still led to a low systemic availability but the bioavailability was significantly higher than in control conditions. The effect of water deprivation is probably due to a partial esophageal groove closure causing a rumen bypass and a direct delivery of a fraction of the dose to the abomasum. It is well demonstrated that in ruminants the closure of the oesophageal groove is facilitated by thirst
(Monnig & Quin. 1933). In goats, a 48-h water deprivation induced groove closure during drinking (Mikhall el al., 1988). Nevetherless, in the present experiment, most of the ampicillin dose was probably directed to the rumen explaining both a relatively low availability and a mean residence time (600 min) corresponding to a generally reported mean transit time from the rumen to the abomasum (Faichney, 1984). T h e ultimate objective of this experiment was a better definition of dosage regimens for sheep reared in arid environments. First, it can be concluded that the oral route is unable to provide therapeutically significant ampicillin plasma concentrations even after a water deprivation despite the fact it has been reported that water deprivation facilitates the reflex closure of the oesophageal groove. Second, the intramuscular (i.m.) route o f , administration maintains its performance in terms of absorption rate and provides an effective ampicillin concentration. If a germ with a minimum inhibitory concentration (MIC) of 0.5 pg/ml, is considered, water deprivation increases the duration of an effective plasma concentration by 26%; the corresponding figure for an organism with a MIC of 1 pg/ml is 24%. Third, it can be suggested that when water deprivation occurs without producing a severe dehydration, the water spaces are not modified, allowing betalactam antibiotics to have the same local distribution as in control conditions. Finally, the only difference between control and deprived sheep concerns the renal elimination which is slowed down thus increasing the residence time of the antibiotic in the body.
ACKNOWLEDGMENTS This work was supported by a grant from International Foundation for Science (B/ 11 10-2). T h e authors thank Mr M. Ablouh, and Mr S. Suilahi, for their technical assistance.
REFERENCES Arret, B., Johnson, D.P.& Kirshbaum, A. (1971) Outline of details for microbiological assays of
432 M . Oukessou &3 P. L. Toutain antibiotics: Second revision. Journal of Phannaceufical Sciences, 60, 1689-1694. Bakar, S.K. & Niazi, S. (1983) Effect of water deprivation on aspirin disposition kinetics. Journal of Pharmaceutical Sciences, 72, 1030-1034. Devaskar. S.U., Devaskar, U.P. & Kleinman, L.I. (1985) Inulin space studies in fetal sheep. Deuelopmenfal Pharmacology and Therapeufics, 8; 55GO. Faichney. G.J. (1984) The kinetics of particulate matter in the rumen. In Control of Digestion and Metabolism in Ruminan&. Eds Milligan, L.P. Grovum, W.L. & Dobson, A. pp. 173-195. Englewood Cliffs, New Jersey. Guadino, M. & Levitt, M.F. (1949) Inulin space as a measure of extracellular fluid. American Journal Physiology, 157, 387-393. Gilbaldi, M. & Perrier. D. (1982) Pharmucokinetics. 2nd edn. Marcel Dekker Inc.. New York. Lam. F.C., Hung, C.T. & Perrier, D.G. (1985) Estimation of variance for harmonic mean halflives. Journal of Pharmaceutical Sciences, 74, 229231. Lecompte, J., Dumont, L., Hill, J.. Du Souich, P. & Lelorier. J. (1981) Effect of water deprivation and rehydration on gentamicin disposition in the rat. The journal of Pharmacology and Expm'mental Therapeutics. 218, 23 1-236. MacFarlane, W . V . (1964) Tenesfrial atrinlaLc in dty heat: ungulates. In Handbook of Phjsiology. Eds Visscher, M.B., Hastings, A.B., Pappeheimer,
J.R. & Rahn, H. Section 4: Adaptation to the environment. pp. 509-539. American Physiological Society, Washington, DC. Mikhail, M., Brugere. H., Le Bars, H. & Colvin, H.W. (1988) Stimulated esophageal groove closure in adult goats. Atnm'canJouriud of Veten'naq Research, 49, 1713-1715. Monnig, H.O. & Quin, J.I. (1933) Studies on the alimentary tract of the merino sheep in South Africa. I. Investigation into the physiology of deglutition. Ondersteloort Journal of V e t e r i n q Science, 1, 117-133. Prescott, J.F. & Baggot, J.D. (1988) Anfitnicrobtal fhernpy in ueterinuty Medicine. Blackwell Scientific Publications, Oxford. Prueksaritanont, T., Chen, M. & Chiou, W.L. ( 1 984) Simple and microhigh-performance liquid chromatographic method for simultaneous determination of p-aminohippuric acid and iothalamat in biological fluids. Journal of Chroniatograplry, 306, 89-97. Thompson, S.M.R. & Black, W.D. (1978) A study of influence of the method of oral administration of ampicillin upon plasma drug levels in calves. CanadianJournal of Cornparalive Medicine, 42,255259. Yamaoka, K., Tanigawara, Y., Nakagawa, T. & Uno, T. (1981) A Pharmacokinetic analysis program (MULTI) for microcomputer. Jounial u/ Pharitiacobiody,iainics, 4, 879-885.
Effect of water deprivation on absorption (oral, intramuscular) and disposition of ampicillin in sheep M. OUKESSOU* & P. L. T O U T A I N t *Department of Physiology and Therapeutics, Hassan I1 Agronomic and Veterinary Institute, Rabat, Morocco and tEcole Nationale VCterinaire, Department of Physiopathology, 23 chemin des Capelles, 31076 Toulouse Cedex, France
Oukessou, M., Toutain, P.L. Effect of water deprivation an absorption (oral, intramuscular) and disposition of ampicillin in sheep. J. vet. Phannacol. Therup. 15, 421432. T h e effects of a 72 h water deprivation on the absorption -intramuscular (im.) and oral and disposition of ampicillin, inulin and para-aminohippuric acid (PAH) were investigated in six sheep. After intravenous (i.v.) administration of ampicillin sodium (10 mglkg), the water deprivation decreased slightly the initial volume of distribution (0.082 k 0.033 vs. 0.055 f 0.030 I/kg) but not the steady state volume of distribution. T h e plasma clearance was significantly decreased (6.21 k 1.94 vs. 3.90 +. 1.92 ml/min/kg) and the mean residence time (MRT) was increased from 22.25 k 4.91 to 33.36 k 8.16 min. After i.m. administration of ampicillin sodium (20 mg/kg), ampicillin concentrations were systematically higher after a 3-day period of water deprivation than during the control period but the muscular absorption rate was not modified. After oral administration of ampicillin trihydrate ( 1 g in loto) plasma concentrations were much lower and more persistent than after an i.m. administration and the systemic availability remained low whatever the hydration status. Influences of water deprivation on ampicillin disposition were linked to adaptation of renal function as assessed by inulin and PAH clearances. T h e therapeutic relevance of the results are discussed for a better definition of dosage regimens for sheep reared in arid environments. P . L. Touhin, ENVT, Department of Physiopalhology, 23 chetnin des Capelles, 3 1076 Toulouse Cedex, France.
I N T R O D JCTION In hot and arid environments, possible excessive insensible heat loss and water deprivation are factors which must be taken into account when selecting a dosage regimen. In sheep, dehydration may have many physiological consequences including loss of body weight, reduction of food intake, modification of water spaces with a reduction of the extracellular sector, increase of plasma protein concentration, reduction of renal filtration etc.
(MacFarlane, 1964). Potentially all these factors individually or collectively can modify the process of absorption at the administration site (digestive tract, muscle), distribution and drug elimination. Currently, few experimental data exist on the influence of hydration status on drug disposition despite the fact that various disease conditions such as polyuria and diarrhoea are concerned by disturbance of the water balance. In the rat, after a 4-day water deprivation, the plasma and tissue gentamicin concentrations were increased and 42 1
422 M. Oukessou @ P . L. Toukzin the elimination rate of the drug was decreased leading to a reduction of 22 and 40% of the central volume of distribution and of the plasma clearance respectively (Lecompte et al., 1981). Siniilarly, water deprivation in rat increased the biological half-life of salicylic acid by 72% (Bakar & Niazi, 1983). In domestic species, there is to our knowledge no information on the effect of water deprivation on drug kinetics and the purpose of the present study was to investigate the effect of a 72 h water deprivation on the absorption (im. and oral) and disposition of ampicillin in sheep. A 72 h deprivation was selected as it represents a frequent situation in Moroccan pastoral husbandry. MATERIALS A N D M E T H O D S Animals
Six adult Sardi ewes, weighing 32-57 kg were used. During the experiments, they were placed in individual metabolism cages. T h e ambient temperature was controlled at 20 2°C.
*
solved in distilled water just before administration. Intramuscular administrations of ampicillin sodium (20 mg/kg) were made deep in the lumbar muscle at a single site with a 40*11 mm needle. Oral administration of ampicillin trihydrate (1 g per animal) was made after mixing ampicillin powder with 40 ml of tap water; the suspension was administered with an appropriate syringe placed on the tongue. T h e same technique was used to administer glucose (0.3 g k g ) for the oral glucose test. Blood samples (6 ml) were collected from the other jugular vein using heparinized vacutainers (Becton Dickinson vacutainer system, Becton Dickinson and Co., France) before drug administration (time 0) and at 2, 4, 8,15,30,60,120,240,360,480,600 and 1440 min after the i.v., i.m. and oral ampicillin administrations. After inulin and PAH administration, samples were collected at the same times u p until 300 min. Samples were collected before and 5, 10, 20, 30, 60, 90, 120, 180, 240 and 300 min after oral glucose administration. All blood samples were centrifuged within 30 min. Plasma was stored at -25°C until assay.
Drugs and les! articles
Expa'mental design
T h e following drugs were used: Ampicillin sodium (Ampivet, Arkovet, Ciba-Geigy, France), Ampicillin trihydrate (Totapen, Laboratoires Rhbne Poulenc Pharma, Casablanca, Morocco), inulin (Sigma, St Louis, MO. USA), para-aminohippuric acid (Sigma) and glucose (30% solution, Laprophan S.A., Casablanca, hlorocco).
In a first set of experiments, the animals had a free access to water. First, all sheep received inulin and PAH by the i.v. route. They were then allocated to groups of two and received ampicillin by i.v.. i.m. and oral route according to a three-period cross-over design. Each administration was separated by a washout period of 7-10 days. In a second set of experiments, animals were deprived of water for 72 h but had free access to food. T h e ewes were allocated to 2 groups of 3. First, they received ampicillin by i.m. or peroral (p.0.) at the E n d h following the withdrawal of water according to a two-period crossover design. Each administration was separated by a washout period of 1 week. After the i.m. or p.0. administration animal has no access to water for a supplementary time of 24 h. Then, at least 1 week later, the animal received according to a two-period crossover design either an i.v. administration of ampicillin or an i.v. administration of the
Drug udministruLiom and sample collection
intravenous (i.v.) administrations of ampiciilin sodium (10 mg/kg) were made via an indwelling catheter placed in a jugular vein jusr before the experimental session. Lyophilisrd ampicillin was dissolved in its commercial solvent just before administration. A mixture of inulin (40 mgikg) and para-aminohippuric acid (PAH) (20 mg/kg) was administered intravenously via an indwelling jugular catheter within 30 s. Inulin and PAH were dis-
Ampicillin in sheep 423
inulin/PAH mixture. For the i.v. studies, water deprivation was obtained and maintained as for the i.m. or p.0. administration. I n a last experiment, the influence of water deprivation o n the spontaneous closure of the oesophageal groove was assessed by drenching sheep with a 30% glucose solution in normal condition or after a 72 h water deprivation. Analytical techniques
Plasma ampicillin levels were measured within 1 week, of sample collection with a microbiological assay procedure using Sarcdna luteu ATCC 934 1 as test organism (Arret et al., 1971). Each sample was measured in duplicate. T h e level of quantitation was 0.005 pg/ ml. Standard dose response curves were obtained using buffer ampicillin solution (Ampicillin, anhydrous, Sigma) diluted in normal sheep plasma. T h e standard curves were constructed the day of sample collection and kept in the same conditions as the samples until analysis. All samples from a given experiment were run in the same assay. Plasma inulin concentrations were measured using a colorimetric method. T h e level of quantitation was 4 pglml. PAH plasma concentrations were determined by the use of a high performance liquid chromatographic technique (adapted from Prueksaritanont el al., 1984). Briefly deproteinisation was obtained by mixing 200 pl of plasma with 300 pl of perchloric acid 0.33 N. T h e mixture was vortexed for 15 s. and centrifuged at 5400 g for 10 min. The supernatant (20 pl) was injected into a Lichrosorb C18reversed phase column (particle size 10 pm). A variable wavelength detector (Kontron, Paris, France) operated at 254 nm and an integrator (Merck, Paris, France) were used. T h e mobile phase was a acetic mixture of acetonitrile (5%)and a WOO acid solution (95%).A flow rate of 1 ml/min was maintained using an isocratic pump (Kontron, Paris, France). T h e mean coefficient of extraction was 86.6%.T h e level of quantitation was 0.2 pg/ml. Phurmacokinetic analysis Compartmental analysis. T h e data were ana-
lysed using a programme for non-linear regression analysis (Yamaoka et al., 1981). Initial estimates were calculated by linearregression methods (Gilbaldi & Perrier, 1982). Plasma concentrations of ampicillin were fitted to equation 1: C(,) = YI exp (-Alt)
+ Y~exp(-A2t)
(1)
where C(,) represents ampicillin plasma concentration at time t; Y1 and Y2 are coefficients and 11 and A2 a r e exponents. For non-linear regression analysis, the data points were weighted according to equation 2.
where Wi is the weight and pi is the fitted value of the ith observation. T h e relevant pharmacokinetic parameters (elimination half-time, volume of central compartment, steady state volume of distribution . . .) were calculated according to the classic equations associated with compartmental analysis (Gilbaldi & Perrier, 1982). Non compartmental analysis. For ampicillin, inulin and PAH, areas under t h e plasma concentration-times curve AUC(o-,,,u,, were calculated using the linear trapezoidal rule without extrapolation to infinity i.e. from time 0 to the time corresponding to the last measured concentration (0-clad).T h e mean residence time (MRT(%r/u,,,)),which is the mean time for a molecule of ampicillin, inulin and PAH to transit through the body, was calculated by the linear trapezoidal rule from time 0 to the time corresponding to Cjart.T h e mean absorption time (MAT), which is the mean time for a molecule of ampicillin to be absorbed after oral or i.m. administration was calculated according to equation 3:
In Equation 3, MRTi.,, or MRTp,,. is the mean residence time of ampicillin after i.m. or oral administration respectively and MRTi,.,, is the mean residence time after i.v. administration. M A T under normal conditions or after water deprivation were calculated using the
424 M . Oukessou U
P. L. Touiain
MRT, ., obtained under normal conditions or after water deprivation respectively. T h e body clearance for ampicillin, inulin and PAH were calculated using equation 4: D
c1 =
parisons between treatment (water deprivation vs. non deprivation) were performed using a unilateral paired student's t-test. Regression analysis were carried out to assess the relationship between inulin, o r PAH and ampicillin selected kinetic parameters.
(4) A UC(~ 0.05). T h e plasma clearance
1
c 0 .C
c,
ml L
CI
c Q)
u l
c
0
0
1
0
60
120
240
360
480
600
T i m e (min 1 FIG. 1. Mean plasma concentration o f ampicillin (pg/ml) vs. time (min) after the i.v. administration of ampicillin sodium at a dose of 10 rng/kg in five or six ewes under normal conditions ( 0 - 0 ) or after a 3-day period of water deprivation (0-0).
Ampicillin in sheep
425
1 o2
.u
c Q) u
t
(s:
lo 0
60
120
240
360
480 600 Time (min )
FIG. 2. Mean plasma concentration of ampicillin (pg/ml) vs. time (min) after the i.ni. administration of ampicillin sotliuni at a dose of 20 mg/kg in six ewes under normal conditions ( 0 - 0 ) or after a 3-day period of water deprivation (0-0).
(6.21 k 1.94 vs. 3.90 k 1.92 ml/min/kg) was significantly decreased for P = 0.056. T h e MRT was significantly increased by water deprivation (22.25 f 4.91 vs. 33.36 f 8.16 min, P < 0.05). T h e individual values of the different parameters are given in Table I. T h e inulin water spaces were not significantly modified by water deprivation (Vc: 0.069 f 0.015 vs. 0.063 f 0.029 Vkg; Vss: 0.145 f 0.063 vs. 0.156 f 0.086 I/kg). T h e inulin clearance, which is an indicator of glomerular filtration rate was not significantly decreased by water deprivation (2.08 f 0.57 vs. 1.77 f 0.91 ml/min/kg, P > 0.05) but the effective renal plasma flow (ERPF), as estimated by the PAH clearance, was significantly reduced by water deprivation (14.30 f 3.63 vs. 11.38 k 2.68 ml/midkg, P < 0.05). Individual data are given in Table 11. Figure 2 gives the mean plot of plasma concentrations (kg/ml) for ampicillin as a function of time (min) after a single intrarnuscular administration of ampicillin sodium in the six ewes under normal conditions and after a 3-day period of water deprivation. Visual inspection of the figures indicates clearly that plasma ampicillin concentrations
were increased after water deprivation. This is confirmed by the significant increase of AUC (2746 _+ 624 vs. 4599 ?I610 pg.min/ml, P < 0.05); M A T were similar before and after water deprivation (70.5 k 54.6 vs. 65.3 f lG.5 min, P > 0.05) suggesting that the miiscular absorption rate was not modified by water deprivation. Similarly, the mean systemic availability was not affected by water deprivation (83.2 k 23.8 vs. 97.5 f 80.7%.P > 0.05). Individual data are given in Table 111. Figure 3 gives the mean plot of plasma concentrations (pg/ml) vs. tinle (min) for ampicillin after administration of anipicillin trihydrate by oral route in the six ewes in normal conditions and after a 3-day period of water deprivation. Visual inspection indicates that under both conditions, the plasma concentrations were much lower and more persistent than after an i.m. administration and that water deprivation increased plasma ampicillin concentrations. T h e AUC was multiplied by a factor of 3.5 (64.4 k 20.4 vs. 227.6 f 83.3 pg. min/ml, P < 0.05) by water deprivation. T h e systemic availability remains low whatever the hydric status (2.07 f 1.06 vs. 4.13 f 2.66%,P < 0.05) suggesting no or limited closure of
97.04 21.19 0.107 0.0208 33.35 28.05 0.084 0.155 0.250 4.93 2025.9
N
’r
P
150.8 80.9 2.12 10.23 0.1 15 0.033 0.0194 0.0077 35.62 89.39 35.51 25.03 0.065 0.109 0.074 0.220 0.420 0.268 2.17 7.67 1302.4 4605.0
N
V P
N
X
P
N
Y P
N
2
I’
80.6 281.4 104.3 98.1 115.9 248.9 177.5 490.7 1.67 15.24 55.85 13.15 0.169 19.25 33.20 31.76 0.403 0.072 0.103 0. I 3 6 0.233 0.083 0.989 0.303 0.0 I 8 0.0089 0.0059 0.0 174 0.0258 0.0093 0.0207 0.0151 38.17 74.32 77.78 117.1 1 39.69 26.83 33.50 45.90 14.43 43.27 25.2 I 21.51 19.28 37.02 21.85 29.17 0.090 0.094 0.018 0.124 0.052 0.033 0.067 0.035 0.175 0.154 0.075 0.161 0.267 0.119 0.109 0.292 0.1 14 0.328 1.70 0.689 0.284 0.189 0.388 0.237 6.55 4.70 5.78 4.74 1.75 9.44 4.84 4.42 1729.5 1573.3 1058.9 226 I .8 2127.3 2065.5 2 109.2 5700.2
N
W
*
k 10.63
& 68.3
*
I’
228.6 f 162.5 22.42 f 22.84 k 0.081 0.346 & 0.388 & 0.0067 0.01 17 f 0.0043 34.34 65.42 k 21.50 f 4.91 33.36 k 8.16 f 0.034 0.055 k 0.030 f 0.040 0.172 f 0.103 & 0.583 0.348 & 0.214 6.21 2 1.94 1.92 3.!)0 1725.5 & 452.2 3241.1 rt 1804.8 130.5 15.29 0.141 0.0184 47.43 22.25 0.082 0.156 0.520
N
Means f SD
Y I and Y, are pre-exponential constants, 1 and 2 are the exponential for the biexponential equation describing the plasma level-time curve. fp2 2: plasma half-life; difference not significant (P > 0.05); M R T mean residence time: significant difference (P < 0.05); vc: volume of the central compartment: significant difference (P < 0.05); V,,: steady state volume of distribution: difference not significant (P > 0.05); C1: plasma clearance: significant difference (P = 0.056); AUC: Area under the plasma level time curve: significant difference (P =0.058). MRT, C1, and AUC were calculated with the trapezoidal rule without extrapolation to infinity.
Parameters (units)
Ewes
T A B L E 1 . I’harniacokinetic parameters describing the disposition kinetics ofanipicillin in plasma after an intravenous administration of ampicillin sodium (10 rng/kg) i n six ewes in normal ( N ) condition and after a 3 days water deprivation (P)
3’
$
Y 0
N
T
N P
N
W
P
0.067 0.076 0.089 0.062 0.023 0.116 0.129 0.113 0.131 0.100 2.04 1.51 3.30 2.09 0.71 12.75 11.45 11.72 11.19 9.68
P
V
0.097 0.273 2.93 20.90
N
X N
P
0.101 0.062 0.045 0.331 0.115 0.125 2.07 1.57 1.34 15.85 14.79 8.43
P
Y
0.058 0.116 1.80 12.37
N
2
0.053 0.132 1.19 9.86
P
+
P 0.069 zk 0.015 0.063 k 0.029 0.145 k 0.063 0.156+0.086 1.77 0.91 2.08 k 0.57 14.30 k 3.63 11.38 k 2.68
N
Means k SD
V 0.05);Vrr: steady stale volume of the distribution for i n u h ( P > 0.05); ClIN: plasma clearance for inulin (P < 0.05); ClpAH:plasma clearance Ior PAH ( P < 0.05).
v,, (Vkg)
V , (Vkg)
0.058 0.109 CllN(ml/min/kg) 2.57 C l p A H (ml/min/kg) 15.11
Parameters (units)
Ewes
T A B L E I I . Selected pharmacokinetic parameters describing the disposition of inulin (40 mg/kg) and PAH (20 mg/kg) in six ewes in normal (N) conditions and after a 3-day water deprivation (P)
3'
49.6 21.5 60.8 8.0 3096 76.4 -
N
114.7 47.0 4.0 5519 136.2
P
164.0 139.0 9.7 60.0 2264 86.9 -
N
v
107.7 72.2 44.4 8.0 2579 28.0 99.0
I'
54.3 29.1 59.1 15.0 3439 99.4 -
N
W
97.2 75.3 58.7 15.0 7068 224.6 "4.3
P
70.6 56.2 35.6 8.0 2444 115.4 -
N
X
89.6 46.3 33.1 15.0 3560 78.7 168. I
I' 56.4 37.1 32.4 15.0 1914 45.0 -
N
Y
86.4 49.4 62.2 4.0 5068 122.7 119.1
P
161.2 139.7 17.5 60.0 3318 78.G -
N
Z
112.8 83.6 24.7 15.0 3798 33.3 90.0
P
92.7 f 54.6 70.4 f 54.6 35.9 k 20.9 27.7 f 25.2 2746k624 8 3 . 6 k 23.8 -
N
P
65.3 f 16.5 45.0 f 14.4 10.1 f 5.5 4 5 9 9 k 1610 9 7 . 5 f 80.7 136.1 k 43.5
101.4 k 12.1
Means k SD
hINT: niriin residence tinie; hIAE nitmi absorptioli tinie. C,,,,, observed maxinial concentration. I,,,., observed time for C,,,,.; A UC: Area under the plasma level tinie curve. MRT and A UC were calculated by [lie trapezoidal rule witliout extrapolation to infinity. The systemic bioavailability was calculated or the i.v. AUC obtained in nornial condition (F2). Water deprivation has no taking into account either the i.v. AUC obtained after water deprivation (F,) significant influence (P > 0.05) on MRT, hfAT, C,,,,,, and F , . AUC and F2 were significantly increased (P < 0.02).
MRT (rnin) MAT (min) C,,,.,, W m l ) t,,,,, ( n W AUC(pg.niin/iril) I.'I (%) I.'2 (%)
Parameters (uniis)
T
Ewes
1'A I3 L E I I I . Selected pliarmacokinetic parameters describing the disposition of ampicillin in plasma aftei- a n intraniuscular adniinistration of anipicilliii sotliuni (20 mg/kg) in six ewes in nornial ( N ) condition and alter a 3-day water deprivation (1')
P
W
r\3
554.2 526.1 73.7 1.82 -
N
T
6.53
-
612.5 264.7
P
448.2 423.2 85.9 3.30 -
N
V
394.3 358.8 241.8 2.62 9.30
P 190.5 165.3 61.4 1.77 -
N
W
494.4 472.5 95.6 3.04 2.76
P 289.0 274.6 71.2 3.36 -
N
X
415.5 372.2 215.4 4.80 10.20
P
-
552.8 533.5 68.2 1.60
N
Y
742.8 705.8 349.2 8.45 8.20
P
-
631.0 609.5 26.0 0.61
N
Z
511.0 481.8 199.3 1.75 4.72
P 444.3 422.0 64.4 2.07
2 20.5 k 1.06 -
k 2.85
f 83.3 f 2.66
k 139.0
k 130.5
k 171.6
k 170.9
P
N 528.4 478.2 227.6 4.13 6.94
Means k SD
M R T mean residence time; M A T : mean absorption time; AUC: area under the plasma level time curve normalized for a standard dose of 20 mg/kg. The systemic bioavailability was calculated taking into account either the i.v. AUC obtained after water deprivation ( F , ) or the i.v. A UC obtained in normal condition (F2).MRT and AUC were calculated by trapezoidal rule without extrapolation to infinity. Water deprivation has no influence on MRT and MAT, but significantly increased AUC (P < 0.04) and F2 (P < 0.01).
FP(%)
FI (%I
MAT(min) AUC(pg.rnin/rnl)
MRT (min)
Parameters (units)
Ewes
T A B L E I V. Selected pharmacokinetic parameters describing the disposition of ampicillin in plasma after an oral administration of ampicillin trihydrate ( 1 g per animal) in six ewes in normal (N) condition and after a 3-day water deprivation (P)
(D
k2
A
siF
h
F s.
h
a.
3.
b 3
430 M. Oukessou U P. L. Toulain
0.3 n c
E \
u (D
L
0.2
0.1
0
6 0 120
240
360
480 600 Time (min )
FIG. 3. Mean plasma concentration of ampicillin (pghl) vs. time (min) after the oral administration of ampicillin trihydrate at a dose o f 1 g in tofo in six ewes under normal conditions ( 0 - 0 ) or after a 3-day period o f water deprivation (0-0).
the esophageal groove during the ampicillin drenching. This is consistent with the absence of hyperglycemic response to an oral glucose test which was observed before and after water deprivation as evaluated by the oral glucose test. T h e AUC of plasma glucose calculated for the first 5 h after a glucose challenge were 18469 k 800 and 20743 2 2814 mg.mid100 mi before and after water deprivation respectively (P> 0.05). T h e MRT of ampicillin (444 k 171.6 min in normal conditions and 528 k 130.5 min after water deprivation) were not significantly different (P > 0.05) but much were longer than after an im. administration (P < 0.05). This is explained by a MAT which is about six times longer after an oral administration (422 k 170.9 min in normal conditions and 478.2 k 139.0 min after water deprivation) than after an i.m. administration. Individual values are given in Table IV.
DISCUSSION
When sheep are raised in a dry hot environment and when herdsmen are obliged to haul water from wells, water is generally not offered each day but more often every 2 or 3
days. From a therapeutic point of view it is relevant to know if this kind of husbandry is able to influence d r u g disposition. T h e physiological effects of water deprivation in sheep are rather complex as they depend on other factors including breed and external temperature. In the present experiment, the Sardi breed which was used is well known for its adaptation to arid environments and the experiment was carried out at a relatively low temperature (20 k 2"). Consequently, a 3-day water deprivation was unlikely to produce dramatic physiological changes. In terms of water compartment, inulin space which is considered as equivalent to the extracellular volume (Caudino & Levitt, 1949) was not modified by a 3-day water deprivation (Vss of about 15% of body weight) and was identical to that reported in lamb (Devaskar el al., 1985). Similarly, the initial volume of distribution of inulin (V, = 6-776 of body weight) which is approximately equal to the plasma volume was not modified by the water deprivation. It can thus be concluded that the 3 days water deprivation had a minimal effect on extracellular water spaces; this explains why a water deprivation under our experimental conditions had no or minimal effect on volumes of distribution of- ampicillin. O n the
Ampicillin in sheep 431
other hand, plasma ampicillin concentrations were systematically higher after a 3-day period of water deprivation than during the control period. This can only be linked to an adaptation of the renal function with both a reduction of renal filtration as suggested by the reduction (non statistically significant) of inulin clearance, and of the ERPF (as demonstrated by the significant reduction of the PAH clearance). Such adaptation has already been reported in dehydrated sheep (MacFarlane, 1964). Renal elimination of unchanged ampicillin appeared to be the major route of elimination (Prescott & Baggot, 1988). In the present experiment, the plasma ampicillin clearance was higher than glomerular filtration rate (i.e., inulin clearance) suggesting active secretion of ampicillin by the kidney. This explains that water deprivation by reducing the ERPF, also reduced the elimination of ampicillin and increased the MRT of ampicillin in the body by about 50%. After i.m. administration, neither the mean absorption time nor the systemic bioavailability was modified by water deprivation indicating that physiological processes implicated in muscular resorption (local blood flow) were not affected by water deprivation. After i.m. administration, large variations in pharmacokinetic parameters between individual and varying in opposite direction were observed. Similarly, a higher than 200% bioavailability was calculated for one of the sheep. We have no consistent explanation to offer for this: a partial explanation should be that a 3-day dehydration is not a reproductible status leading to within and between sheep variations. After oral administration, the plasma concentration in the control ewe was low. This was expected, as ampicillin arriving in the rumen must be largely inactivated as has been observed by others in calves (Thompson & Black, 1978). After a water deprivation, the oral administration of ampicillin still led to a low systemic availability but the bioavailability was significantly higher than in control conditions. The effect of water deprivation is probably due to a partial esophageal groove closure causing a rumen bypass and a direct delivery of a fraction of the dose to the abomasum. It is well demonstrated that in ruminants the closure of the oesophageal groove is facilitated by thirst
(Monnig & Quin. 1933). In goats, a 48-h water deprivation induced groove closure during drinking (Mikhall el al., 1988). Nevetherless, in the present experiment, most of the ampicillin dose was probably directed to the rumen explaining both a relatively low availability and a mean residence time (600 min) corresponding to a generally reported mean transit time from the rumen to the abomasum (Faichney, 1984). T h e ultimate objective of this experiment was a better definition of dosage regimens for sheep reared in arid environments. First, it can be concluded that the oral route is unable to provide therapeutically significant ampicillin plasma concentrations even after a water deprivation despite the fact it has been reported that water deprivation facilitates the reflex closure of the oesophageal groove. Second, the intramuscular (i.m.) route o f , administration maintains its performance in terms of absorption rate and provides an effective ampicillin concentration. If a germ with a minimum inhibitory concentration (MIC) of 0.5 pg/ml, is considered, water deprivation increases the duration of an effective plasma concentration by 26%; the corresponding figure for an organism with a MIC of 1 pg/ml is 24%. Third, it can be suggested that when water deprivation occurs without producing a severe dehydration, the water spaces are not modified, allowing betalactam antibiotics to have the same local distribution as in control conditions. Finally, the only difference between control and deprived sheep concerns the renal elimination which is slowed down thus increasing the residence time of the antibiotic in the body.
ACKNOWLEDGMENTS This work was supported by a grant from International Foundation for Science (B/ 11 10-2). T h e authors thank Mr M. Ablouh, and Mr S. Suilahi, for their technical assistance.
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