Species dependent gentamicin pharmacokinetics and nephrotoxicity in the young horse

FUNDAMENTAL AND APPLIEDTOXICOLOGY 3:448-457 (1983)

Species Dependent Gentamicin Pharmacokinetics and Nephrotoxicity in the Young Horse J. EDMOND RIVIERE*, GORDON L. COPPOC, EDWARD J. HINSMAN, WILLIAM W. CARLTON, and DOUGLAS S. TRAVER Departments of Veterinary Physiology and Pharmacology, Veterinary Clinics, Veterinary Anatomy, and Veterinary Microbiology, Pathology and Public Health, SchooI of Veterinary Medicine, Purdue University, West Lafayette, IN ,17907

A B S TRA C T Species Dependent Gentamicin Pharmacokinetics and Nephrotoxicity in the Young Horse. Riviere, J.E., Coppoc, G.L., Hinsman, E.J., Carlton, W.W. and Traver, D.S. (19i63). Fundam. A p p L Toxicol. 3:448-457. Gentamicin pharlnacokinetics and nephrotoxic potential were evaluated in twelve 2 to 3 month-old horses. Whereas recent evidence in our clinic indicated that young horses may be especially susceptible to gentamicin nephrotoxicity, young rabbits and rats are usually resistant. Gentamiein (4.5 mg/kg) was given by rapid intravenous injection, Serum gentamicin concentrations over a 13-hour period were fitted to an open, twoc o m p a r t m e n t , pharmacokinetie model. Subsequently, the same horses were divided into groups of 3 horses each. Each group received 0, 2.2, 4.4 or 8.8 mg gentamicin/kg, intramuscularly, every 12 hours.for 15 days. Renal function was monitored. Peak and trough gentamicin concentrations were monitored daily. Renal sections were collected for histopathoiogie and electron microscopic examination. "lhe (mean =t= SD) serum halflife was 194 =l= 37 minutes, total body clearance (CI,) was 1.65 =t= 0.79 m L / m i n / k g and volume of distribution at steady state (Vd(~,l) was 30.6 :t: 9.4 L / 1 0 0 kg. Decreased renal function, as detected by elevated serum urea nitrogen or creatinine concentrations, was detected only in the two youngest foals (including animals in both the 4.4 and 8.8 m g / k g dose groups). The trough serum gentamicin concentrations o f these 2 horses increased over time. These horses had the lowest CI~ and V d ~ in the intravenous stud]t. Morphologic changes were seen in kidneys 0fall treated horses and were similar to those occurring with gentamicin toxicity in other species. Our results support the clinical impression that very young horses may be more susceptible than adult horses, and adults of other species, to gentamiein nephrotoxicity. INTRODUCTION Gentamicin is an aminoglycoside antibiotic which causes nephrotoxiciW in numerous animal species including man, dog, rat and rabbit (Ginsbur9 el a/., 1976; Houghton e t a / . , 1976; Reiner el al., i 978; Kaloyanides and Pastoriza-Munoz, 1980), Susceptibility differs between species and within strains of a species. Adult rats are less susceptible to genta-

micin nephrotoxicity than are.humans and dogs (Black et al., 1963; Falco e t a / . , 1969; Fillastre e t a / . , 1974; Reiner e t a / . , 1978). Recent clinical reports indicate that young horses may be more susceptible to gentamicin nephrotoxicity than the young of other species (Tobin, 1979; Riviere et al., 1982). A pharmacokinetic study of gentamicin in adult horses has indicated a m e a n s e r u m elimination half-life of 152 minutes (Pedersoli et al., 1980), which is longer than other species. Mean elimination half-lives are reported to be 30-60 minutes in rats (Barza et al., 1978; Soberon et aL, 1979; Marre et al.. 1980; Lecompte el aL, 1981); 70 to 150 minutes in man (Meyers and Hirschman, 1972; R e g a m e y el al,, 1973; Simon el al., 1973; Lode et al., 1975; Pechere and Dugal, 1979; Chung et al., 1980); 61 to 75 minutes in dogs (Baggot 1977; Riviere and Coppoc, 1981 ; Riviere et al., 1981 a); 50 to 63 minutes in rabbits (Frame eta/., 1977; Huang el a/., 1979); and 85 to 111 minutes in sheep (Ziv and Sulman, 1974: Wilson el al., 1981 ). The volume of distribution of gentamicin in these studies ranged from 15 to 30 L/IO0 kg. Similar pharmacokinetic studies have not been conducted in young horses. Young rats (Marre et al., 1980) and rabbits (Chonko et el., 1979) have been found to be relatively resistant to gentamicin nephrotoxicity. The horse may be a sensitive species in this respect and, therefore, might serve as a useful animal model to investigate aminoglycoside nephrotoxicosis.

TABLE 1 Pharmacokinetic Parameters for Gentamicin After Administration of a Single Intravenous Bolus (4.5 mglkg) to Nine Horses

Parameter C° (,uglmL) A (pglraL) alpha (rain-l) B (/ag/mL) beta (min-1) T 112 (beta) (rain) k,i (rain"~) V~ (LllOO kg) Vd (ss) (LlloO kg) Clu (mLIrnin/kg) Body weight (kg) Total protein (gldL) Hematocrit (%)

*Currcnt Address:t,aboratoryof Pharmacologyand Toxico!ogy.Schoolof Vcterinary Medicin,. NorthCarolina State Unlversity.Raleigh.NC 27650: Send Reprint Requeststo: Dr. J.E. Rivierc. Laboratoryof Pharmacologyand Toxicology. ,5~:hoolof Veterinary Medicine. North Carolina State University. Raleigh. NC 27650. Copyright19~3oSocietyofToxicology 448

Fundam. Appl. Toxieol. (3)

Mean + SD 34.5 4- ,5.6 26.9 -f- 15.6 0.0275 ± 0.0127 7.64- 3.1 0.0037 + 0.0008 194 -.t:37 0.0121 4- 0.0085 15.2 + 5.6 30.6 --t-9.4 1.65 + 0.79 110 • 23 6.4 4- 0.4 33 4- 4

September/October, 1983

GENTAMICIN IN THE YOUNG HORSE

Serum Gentamlcin {pg/mt)

METttODS Cp " Ae- ( t + Be-~t

160.0

Animals Twelve clinically healthy foals ranging in weight from 82 to 145 kg, two to three months of age respectively, were used in this study. Animals were housed in individual stalls and maintained on a hay and mixed grain diet. Routine hematology and blood chemistries were performed on peripheral blood samples.

[

Intravenous study

0,1

T

0

~

150

,o °, % ~

~

,

=

300

,

,

450

600

,

750

Time (rain) FIG. 1. T w o c o m p a r t m e n t o p e n model used to describe the data. T h e data points r e p r e s e n t . l e a n s ± SD for foals at the respective time periods. T h e curves w e r e d r a w n using the c o m b i n e d estim a t e s of pharmacokinetic p a r a m e t e r s calculated individually for each foal.

This study was designed to characterize gentamicin pharmacokinetics in young horses and to evaluate renal function and morphology after a two-week course of intramuscular drug administration at one-half, one and two times the norr~al clinical dose.

Nine horses were given gentamicin sulfate (Schering Corp., Kenilworth, NJ) at a close of 4.5 mg/kg as a rapid intravenous injection. Blood samples were collected through the jugular vein at approximaately O, 15, 20, 30, 40, 50 min, and 1,1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 12 and 13 hr after dosing. Duplicate serum samples were assayed for gentamicin using a double antibody gentamicin ~2.s;radioimmunoassay procedure (New England Nuclear Corp., North Billerica, MA). Radioactivity was measured with an automated gamma counter (Beckman Instruments, Inc., Fullerton, CA)and concentrations were determined by reducing these raw data on a digital computer using a legit-log transformation in a least squares linear regression model (Lewis and Nelson, 1977). The resulting gentamicin serum concentrations were fit for each horse to a two-compartment, open, pharmacokinetic model of the form Cp = Ae"" + Be "l" where Cp is the concentration of drug at time t, A and B are intercept terms, alpha is the distribution rate constant and beta is the elimination rate constant. An initial exponential "stripping" procedure (CSTRIP, Sedman and Wagner, 1976) was used to generate parameter estimates for final solution using a nonlinear least squares regression program (SPSS-NONLINEAR, Madison Academic Computing Center, University of Wisconsin) (Nie et el., 1 975). Error sums of

TABLE ,2 L a b o r a t o r y D a t a f o r H o r s e s O v e r C o u r s e of I n t r a m u s c u l a r Gentamicin Administration Serum Creatinine (mg/dL) Dose Group 2.2

mglkg"

4.4 mglkg

8.8 mglkg

Control

Serum Urea Nitrogen (mgldL)

Horse

Day 0

Day 14

Day 0

Day 14

A 13 C

1.2 1.2 1.0

1.0 1.1 0.8

13 22 29

15 19 17

Mean :i: SD

1.1 4. 0.0

1.0 + 0.2

21 4. 8

17 ± 2

D E F

1.4 1.0 1.8

0.8 1.0 7.2 b

19 20 21

10 13 121 h

Mean 4- SD

1,4 4- 0.4

3.0 4- 3.6

20 4- 1

48 + 63

G H

1.5 1.5

1.0 0.9

1

I..5

2.7 b

21 17 12

13 10 32 b

Mean d- SD

1.4 + 0.2

1.5 + 1.0

17 4- 4

18 4- 12

J K L

1.1 1.2 1.4

1.1 1.0 1.2

22 22 27

22 22 16

Mean ~ SD

1.2 + 0.2

1.1 + 0.1

24 :l: 3

20 + 4

=Doses administered every 12 hours. b

Regression coefficient of concentration versus clay significantly greater than zero (p < 0.05L

Fundamental and Applied Toxicology

(3) 9-10/83.

449

RIVIERE, COPPOC, HINSMAN, CARLTON AND TRAVER TABLE 3

Individual Scores for Histological Changes in Kidneys of Horses in This Study Horse

Interstitial Nephritis

Tubular Casts

A

+h

+

0

0

0

B

0

0

+

0

0

C

o

+

+

+

+

4.4 mglkg

D E F

0 + 0

0 + +

++ ++ +

+ + +++

++ + +

8.8 mglkg

G H

1

0 0 ++

+ + ++

+++ ++ ++

+ ++ ++

+ ++ +++

]

0

0

0

0

0

K L

0 0

0 0

0 0

0 0

0 0

Dose Group 2.2 mglkg"

Control

Hyaline Droplet

Tubular

Tubular,

Change

Necrosis

Regeneration

"Dose administered every 12 hours. ~'0 : no change, + = slight, + + = mild, + + + = moderate.

squares, r" values, and residual plot analyses were used to evaluate goodness of fit. Parameters determined were then used to calculate the pharmacokinetic rate constants (ko~)the volume of the central compartment (V~), the volume of distribution of steady state (Vd~.~)), the body Clearance (Cl~d, the betaphase elimination half-life ( t l / 2 ) . and the concentration of drug at time zero (CO) (Gibaldi and Perrier, 1975; Wagner, 1 975, 1976). Vdt..~ was selected as the volume of distribution estimate because it is independent of elimination processes (Riegelman et al., 1968; Jusko and Gibaldi, 1972).

Intramusc.ular study Twelve foals, three per dose group, were given gentamicin intramuscularly at 0, 2.2, 4.4 and 8.8 mg/kg every 12 hours for 1 4 days. Control animals were given 10 mL of gentamicin vehicle every 1 2 hours. Serum samples were collected each morning for gentamicin analysis immediately prior to (trough) and one hour after (peak) drug administration. Renal function

FIG: 2; Renal cortical tubules with "hyaline droplet" degeneration. H ~ E. X560 (High dose g r o u p ) . 450

was evaluated daily by monitoring serum creatinine and serum u'rea nitrogen concentrations as well as by performing routine urinalysis (pH, protein, glucose, and blood by dipstick; specific gravity). These data were subjected to least squares linear regression analysis to detect trends over time in individual animals. Likewise, daily peak and trough gentamicin serum concentratidns and elimination rate constants were analyzed by linear regression. The daily elimination rate constant (min "z) was calculated using the following equation: Elimination rate constant (rain z) = In Peak (/~g/mL) - In Trough (/~g/mL) Time Interval (min) The time interval averaged 660 minutes.

Postmortem evaluation At the termination of the experiment, horses were killed w i t h sodium pentobarbital and necropsied. A visual examina-

FIG. 3. Renal cortex illustrating tubular cellular degenerat'ion and necrosis, T u b u l a r regeneration and interstitial cellular infiltration. H & E. M350 (High dose group),

Fundam. Appl. Toxicol. (3)

September/October, 1983

GENTAMICIN IN THE YOUNG HORSE

'~

.. ti~--.i.'~:.~,<,

: ;~t ~ ~i:.,~,tl~:~, tit i(.7~0'~8

FIG. 4. Renal cortical tubules with cellular degeneration and necrosis. H & E. X3SO (High dose group).

~

~

~

~*

, ~ ~: •

.

~.'" .|,,,.~'- ~.. !~."*~'; '

.*:

"~

..

.

~

~i.

:' -~ .... "¥~¢:~t•'.~"~ ,,1

•

',s;-.:=. ; ~ . : .

FIG..5. Typical appearance of the proximal tubule epithelium from a control horse illustrating some cytosomes without myeloid figures. X6250. tion of all organ systems for gross lesions was performed. Renal tissue samples were collected and prepared in the following manner: For light microscopy, tissue was fixed in 10% (vol/vol) neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin, These histological sections were then examined in a blind fashion (WWC) quantitating the degree of interstitial nephritis, tubular casts, hyaline droplet changes, tubular necrosis and tubular regeneration. For transmission electron microscopy, the kidney was removed quickly and perfused through the renal artery with phosphate buffered 2% (vol/vol) formaldehyde and 2.5% (vol/ vol) glutaraldehyde using gravity flow (Bulger et ~/.. 1979). The entire kidney was then placed in this fixative overnight. Sam[)les of cortex that appeared to be well-fixed were post-fi-~ed in 2% (vol/vol) osmium tetroxide in 0.08 M phosphate buffer (pH 7.3) and embedded in Epon 812. Thick sections were cut at approximately 1 pm and stained with methylene blue azure II. Grids were cut from 3 blocks from each horse and stained with uranyl acetate-lead acetate. Then at least 2 blocks from each horse were examined on a transmission electron microscope (JEOL IOOCX, Tokyo, Japan) at approximately 3000 diameters. The first four positively identified proximal tubules were photographed. Observations on glomerulio distal tubules and FundamentAland Applied Toxicology

(3) 9-10/83

FIG. 0. Proximal tubule illust ratin,~ cells with increased numbers of cytose~resomes on either ~ide of a less electron dense epithelial cell. The lighter cell may be a regenerating cell, ×0500 (Higi~ dose ~roup).

FIG. 7. Typical appearance of cytose~resomqs containing myeloid figures in proximal tubular epithelium of treated horses. X I 0 400 (High dose group).

interstitial cells were recorded. Pictures from each animal were examined blindly (EJH) for changes in the ultrastructure of the proximal tubular epithelium.

RES UL TS All horses used in this study had normal clinicaJ pathologic parameters before administration of gentamicin.

Intravenous study The pharmacokinetic parameters defining gentamicin disposition in 9 horses after a rapid intravenous injection of 4.5 mg gentamicin/kg did not reveal any remarkable features other than the relatively iong serum beta:phase half-life (Table 1). The biexponential semilogarithmic decay of gentamicin concentration in serum (Figure 1) was plotted using mean values for A, alpha, B and beta obtained in individual horses. The actual serum concentrations plotted in Figure 1. are mean values for 9 horses.

Intramuscular study Before gentamicin was given (day O) all values for serum creatinine and serum urea nitrogen concentrations were within normal limits for horses (Gelsa, 1979; Knudsen, 1959). 451

RIVIERE, C O P I ' O C , H I N S M A N , C A R L T O N A N D "FRAVER

TABLE 4 Mean Peak and Trough Gentamicin Serum Concentrations and Calculated Elimination Rate Constants After Intramuscular Drug Administration Mean Serum Concentration ± SD" Dose Group 2.2 m g l k g '

•1,4 m g l k g

8.8 m r l k g

ttorse

Peak

Elimination Rate Constant ± SD t'

Trough

A B C

6.0 .1. 2.5 0.4 + 1.8 8.2 4- 2..5

0.70 + 0.4 ! 0.52 ± 0.28 0.47 ± 0.17

0.0032 ± 0.0005 0.0038 ± 0.0007 0.0042 ± 0.000.5

Me,H~ -t- SE

0.7 f- 1.3

0.50 ± 0.10

0.0037 .1. 0.0003

D E F

11.5.1.2.3 13.6 4. 2.9 155;' .1. 0.3

0.80+0.15 1.01 .1. 0.48 2.72 .1. 3.0o d

0.00424-0.0010 0.0038 4- 0.0005 0.0029 .1. 0.0010 ~

M e a n .1. SE

13.o .1. 2.2

1.54 :J::0.71

0.0030 ± 0.0005

C; tl I

28.2 • 3.5 23.8 ± 5.4

2.5,8-t- 1.53 1.29 ± 0.52

0.00384" 0.000o 0.0043+ 0.0004

24.0 ± 0.5

9.82 -i-4.80'd

0,0017 ± 0.0010

Me,H'I .1. SE

25.5 4- 2.9

4.50 .1. 1.32

0.0033 ± 0.0004

"Peak and trough serum concentrations over days 1-15 (12.-15data points per ,~nlmal) (pglmL). i.fl(mi n i) c.Hculated from daily pe,~kand trough serum concentrations. Mean ± S E for all 9 horses was 0.0035 + 0.0002. "Doses , s d m i n i s t e r e d ew, ry 12 h o u r s .

" R e g r e s s i o n coefficient of In ¢oncenir.~tion v e r s u s day significantly different t h a n zero (p < 0.05).

~Regression coefficient

of elimination rate c o n s t a n t v e r s u s day significantly d i f f e r e n t t h a n zero (p < 0.05).

Serum urea nitrogen and serum creatinine concentrations were not elevated in any group after the course of gentamicin administration (day 14,,Table 2), However, in horse F (4.4 mg/kg dose group) and horse I (8.8 mg/kg dose group), both values were increased (Table 2). Only in these two horses were the linear regression coefficients for gentamicin concentration versus day greater than zero (P < 0.05), indicating a trend toward decreasing renal function with time, Horses F and I had decreased urinary specific gravity (1.07 and 1.01, respectively) and increased urinary protein (100 and 300 mg/dL, respectively) compared with their pretreatment values. Similar changes were not found in other horses. Pathoanatomic alterations were present in the horses of all three treatment groups given gentamicin, but were minimal in those given the lowest dose (Table 3). The alterations affecting the cortical tubules were those of a toxic tubular nephrosis and included tubular cell degeneration and necrosis, tubular protein and cellular casts, and tubular cell regeneration (Figures 2 to 4). One animal in each dose group had interstitial nephritis characterized by multiple, variably sized collections of mononuclear inflammatory cells in the interstitium (Figure 3). Transmission electron microscopy revealed numerous lysosomes (cytosomes) in the proximal tubule cells of all animals (Figure 5). They were more numerous in the treated animals. Cytosomes containing myeloid figures (cytosegresomes) were found only in the gentamicin treated animals (Figures 6 and 7). There w a s a reduction in the height of the proximal tubular epithelium i n the most severely affected animals (horses F and I). The microvilli of some tubular cells were absent except near cell borders: These coUld have been either cells which had lost 452

microvilli or cells that were regenerating and had not yet formed a complete border. Some cells with a lighter cytoplasm, little rough endoplasmic reticulum, and few mitochondria were observed and thought to be regenerating cells (Figure 6). The ultrastructural findings supported the results as reported for light microscopic examination of these tissues. Changes were not seen in the glomeruli o'r distal tubules. Peak serum gentamicin concentrations were directly related to dose (Table 4, Figure 8), increasing from (mean + SE) 6.7 + 1.3 p g / mL at 2.2 mg/kg to 25.5 + 2.9 p g / mL at 8.8 mg/kg (P < 0.001 ). Trough concentrations were likewise related to dose, increasing from 0.56 + 0.16 p g / m L at 2.2 mg/kg to 4.56 + 1.32 p g / m L at 8.8 mg/kg (P < 0.05). However, mean group values are misleading because horses F and I had relatively higher trough concentrations than the other horses in the same dose groups. Additionally, the linear regression coefficient of the natural logarithm of the trough concentration versus day was greater than zero (P < 0.05) only in horses F and I, indicating a trend for trough concentratic ns to increase over time in these two horses (Figure 8). No trends in peak concentrations were detected (P > 0.05). Only in animal I was the initial trough concentration (Day 2) elevated (9.8 pg/mL). All other initial trough concentrations, regardless of dose, were less than 2.5 pg/mL. The daily elimination rate constant (mean :1:SE), based on peak-trough values for all 9 horses was 0.0035 + 0.0002, rain -I, a value essentially identical to the corresponding beta calculated from the intravenous data, 0.0037 + 0.0008 min q. The linear regression coefficient of elimination rate constant versus day was only different than zero ( P < 0.05) in horse F, indicating a trend toward decreasing drug elimination with Fundam. AopL ToxicoL (3)

September~October, 1983

@

II m m =2 Q,

=2

g

Ig =3 II

,11

8.

16-

o

_

o o

,

4-.-~_ &

_

,

4

.

&

O - - - - 0 Foa~A o, oFo~l, • ..... ~ Foal C

. ,

,

DAY

8

.

.

I

,

12

I

,

I

,

0

16

-

"l

"

~

7

,

~

"1

4

I

--.-~-~_~__..

,

O-----O FoalD o 0F~IE ~ ...... • Foal F

I

,

I

DAY

8

.

,

4.4 mg/k9

I

,

o

12

I

~

,

I v

,

16

I

.....4k

~

~

0

~

I

,

4

I

,

o

8. . . . . - 4 F o a l ,

"''-'--~ ,~ o • ~. ~- • • . _-

s

I

,

.-"

0

o ~

DAY

8

I

,

I

,

-~

s

12

I

,

~ ~ ~ _ _~ _ ~ o_~ ~ ~ - " ° Z

8.8 mg/kg

A

~

O

I

,

6.A

18

i

_ _ -8 "-0

FIG. 8. Daily peak (1 hour after morning injection) and trough (immedia rely prior to morning iniection ) plasma gentamicin concentrations in the foals during the 15 days of twice daily intramuscular injections of gentamicin. Note that the points plotted on day 0 and day 16 are calculated interceptr

Z

E

r~

~S

5, 32 "~ z

2.2 mg/kg

0

..< O C Z

nl

.--t

Z

~r

CI rrl Z >--t

RIVIERE, COI'POC, HINSMAN, CARLTON AND TRAVER

time in this animal. Horse I had a very low initial elimination rate constant (0.0017 ± 0.0010 rain ~) which did not decrease further over time. The pharmacokinetic parameters of horses F and I were analyzed separately from the other 7 horses (Table 5). Both horses had decreased CI, and k,j compared to the group mean. Animal F (4.4 mg/kg) also had lower V~, Vd¢,,~and an increased C'~. In order to explore the relationship between the phar macekinetic parameters in all 9 horses and the initial clinical parameters in each horse, a Pearson correlation analysis (Nie eta/., 1975) was conducted. Parameters in Table 5 were compared to the initial weight, packed cell volume, total serum protein, serum urea nitrogen and serum creatinine. The only significant correlation coefficient (P < 0.05) was between k.,= and weight (r = = 0.62, p = 0.037). A marginal correlation (P < O. 10) was observed between weight and CI, (r'~ = 0.51, p.= 0.078). This analysis indicated that low body weight, and therefore, young age, was directly correlated with decreased ko=and CIit. Animals F and I had the lowest weight and lowest values of k,= and CI=~, DISCUSSION

The results of this study indicate that young horses may be more susceptible to gentamicin nephrotoxicity than adult horses and other species. All horses given gentamicin had pathoanatomic alterations consistent ,with gentamicin induced nephrotoxicity. Two of 6 horses (F and I) given relatively low doses of the drug developed renal insufficiency manifested by elevated serum creatinine and serum urea nitrogen concentrations, decreased urine specific gravity, increased urine protein excretion, and morphological evidence of proximal renal tubular nephrosis. All 9 horses had normal renal function at the onset of the study indicated by normal serum creatinine and serum urea nitrogen concentrations. These findings are characteristic of gentamicin induced toxic nephropathy in all animal species thus (at investigated, however, they occurred earlier and at lower total daily doses than in other animals. Histological alterations of the severity seen in this study have been observed in adult rats only at daily doses of 20 to 40 mg/kg (Kosek eta/., 1974; Houghton et al., 1976; Cuppage et al.. 1977) or in adult dogs at doses of 10 to 30 mg/kg (Cronin et aL, 1980; Spangler et aL, 1980). There are no reports of controlled gentamicin toxicity studies in adult horses, but nephrofox(costs was not observed in horses treated with 9 m g / k g / d a y for up to 24 days (Beech et al., 1977; Harem, 1978). Administration of 9 mg of gentamicin/kg/day for 7 days to adult sheep in our laboratory did not produc~ histological evidence of tubular nephrosis or elevation of serum creatinine or serum urea nitrogen concentrations (unpublished observations). Interstitial nephritis similar to that seen in three of the horses in this study has been reported in humans receiving gentamicin (Saltissi et aL, 1979; Bell and Thomson, 1980; Kourilsky eta/., 1982). The ultrastructural changes seen in these horses at all doses, notably the cytosomes with myeloid figures in the proximal tubule cells, are similar to those seen in rats (Houghton et al., 1976; Kosek et al., 1974; Vera-Roman et al., 1975; Luft et a/., 1975), dogs (Spangler et aL, 1980; Riviere et al., 1981 b) and man (Acchiardo and Murphy, 1977; Houghton et ale 1978), The mean peak and trough concentrationsfor each dosegroup given intramuscular injections are directly related to dose. The trough concentrations of horses F and I, which developed r e n a l dysfunction manifested by altered clinical ,$$4

TABLE 5 S e l e c t e d P h a r m a c o k i n e t i c P a r a m e t e r s for

Gentamicin Disposition Tabulated According to Occurrence of Nephrotoxiclty ~ Non-toxic

n

C~ (IsglmL~ T 112 (fl) {rain) k,l (rain ~) V¢ (L/t00 kg) Vd (ss) (LI100 kg) Chl (mLIminlkg) Weight (kg)

Toxic

Mean

+ SD

M e a n (values) L'

7 33.6 192 0.0132 15.8 32..0 1.~1 118

:i: 6.8 .t- 41 .'+"00085 ~ 6.0 _.+ 9.5 ± 0.82 =t=20

2 37.7 (4S.2, 27.1) 204 (195, 213) 0.0085 [0.0092, 0.0078) 13.1 (9.4, 16.8) 2.5.9 (18.4, 33.4) 1.09 (0.~,7, 1.31) 84 (82, 80)

"Single intravenous bolus of gentamici:~ (4.5 mglkg) administered. t'Anim,fls F and I.

chemistry values, increased over time. The increase in trough concentrations would be expected as a result of drug accumulation secondary to decreased glomerular filtration, a situation similar to that occurring in nephrotoxic human patients (Scher~tag, 1980). The high trough concentrations in Horse I, which was in the high.dose group, are consistent with the fact that it,had the lowest mean apparent elimination rate constant of the 9 horses studied. A two compartment pharmacokinetic model was used to describe gentamicin disposition. An extended, slow, terminal elimination phase with a half-life of approximately 150 hr has been detected in experiments conducted over 2 to 3 weeks and is roughly correlated with the well-documented renal cortical accumulation of gentamicin observed in all species studied (Luft and Kleit, 1974; Kahlmeter and Kamme, 1975; Schentag and Jusko, 1977; Whelton et aL, 1978; Huang et aL, 1979~. The slow elimination phase is especially pronounced in prenephrotoxic patients due to a propensity for such patients to accumulate drug (Colburn et a/., 1978; Schentag et aL, 1979). This results in a slight increase~n trough concentrations over time, however, the rise in trough levels in horses F and I is greater than can be explained by this hypothesis and is probably secondary to decreased drug clearance. The pharmacokinetic parameters of gentamicin disposition are similar to those reported for adult horses by Pedersoli et al. (1980) except for a slightly prolonged half-life, slightly increased t:learance and increased Vd(,: in the present study. Pedersoli et al. (1980) published the following parameter values for adult horses;t1/2= 152_+ 20 rain. Ch~= 1 , 1 5 ± O , 1 2 m L / m i n / k g ; a n d Vd(,,~ = 24.3 ± 2.7 L / I O 0 kg). When comparing drug elimination in various species, it is important to consider CIB and Vd rather than focussing only on t l / 2 because the former parameters represent two distinct physiological processes as shown by the following equation (Gibaldi and Perrier. 1975): 11/2 = 0.693 x Vd CI. For example, a decrease in CIB associated with a corresponding decrease in Vd would not change t l / 2 : Large differences in phvsio-pathoiogically important parameters could be missed if one comgared only t l / 2 of drugs. In t h i s light, the horse Fundam, Appl.Toxicoi. (3)

SejTtember/ October, 1983

GENTAMICIN IN THE YOUNG HORSE would appear to have a lower drug clearance (mL/min/kg), (CIB = 1.65 ± O.79, current study and CIH= 1.15 ± O. 12, Pedersoli et al., 1980) due to a lower glomerular filtration rate (1.5 ± 1.7 m L / m i n / k g ) , (Knudsen, 1959; Gelsa, 1979) which results in a prolonged t l / 2 compared to rats, dogs, and rabbits. The CI=~for rats (Lecompte et al., 1981 ), dogs (Baggot, 1977; Riviere and Coppoc, 1981; Rivere et al., 1981 a), and rabbits (Huang et al., 1979) ranges from 3 to 8 mL/min/kg, values very close to the glomerular filtration rate in each respective species. These findings are consistent with allometric principles. (Boxenbaum, 1982) The volume of distribution in all of these species, including the horse, is similar. In contrast, sheep which also have a low CIB (1.6 + 0.4 m L / m i n / k g , Wilson et al., 1981) and low glomerular filtration rate (2.5 m L / m i n / k g , Rabinowitz et al., 1977) do not have a prolonged half-life because o~ a relatively small volume of distribution (19.4 ± 5,9 L/1 O0 kg, Wilson et al., 1981 ) which masks the low clearance. The magnitude of these pharmacokinetic parameters must also be interpreted as a function of age. The slightly increased Vd(=,~and t 1 / 2 compared to adults is consistent with pharmacokinetic studies of gentamicin disposition in man (Milner, 1974; Siber et al., 1975; McCracken et al., 1977) and rats (Marre et al., 1980), It is probable that the seven young horses in the present study which did not have clinical chemistry findings indicative of renal insufficiency, had mature renal function, because CI, was comparable to glomerular filtration rate of normal adults. As in other young animals, only the volume of distribution was incri~ased which resulted an an increased half-life. In the two horses (F and I) which exhibited signs of nephrotoxicity, CIB was lower than the group mean indicating decreased renal function. Very young animals have immature renal function (Jose et aL, 1971; Kleinman and Reuter, 1973; Aperia and Herin, 1975). Using a three compartment pharmacokinetic analysis, Assael et al. (1980) demonstrated that newborns have lower values of gentamicin body clearance and steady state volumes of distribution than do infants and children. It is plausible that in very young horses, i.e. two months old, 82 to 86 kg, the pharmacokinetic profile is one of a low CIB and normal to contracted Vd~.,), two factors which could potentiate nephrotoxicity (Cronin, 1979; Kaloyanides and Pastoriza-Munoz, 1980). Horse F in the present study had very low values of CIB and k~ and a contracted volume of distribution, as reflected in decreased V¢, Vdc,,~ and increased C°. Horse I had a relatively normal V~, Vd(,,~ and COcompared to the other horses, but had a decreased CIB and k~l. All seven other horses had relatively high CIB and volume of distribution compared to adults (Pedersoli et al., 1980) and did not exhibit clinical signs of nephrotoxicity. The Pearson correlation analysis supports this observation because CIB and k~ was correlated with body weight for all 9 horses. These seven "'young" horses probably had mature renal function compared to the 2 month old horses and thus would be analogous to young children rather than neonates. Studies involving maturation of renal function in the horse have not been conducted to test this hypothesis. Young.rabbits, dogs and rats are generally resistant to aminoglycoside nephrotoxicosis compared to adults of the same species (Chonko et al., 1979, Marre et al.,1980, Cowan et al., 1980). This relative resistance apparently also occurs in human infants (McCracken eta/., 1971; Milner, 1974; Mehta, 1981 ). Marre et al. (1980) attributed this decreased toxic threshold to an observed deficiency of the young rat's renal cortex

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to concentrate and accumulate aminoglycosides, a factor often correlated with nephrotoxic potential. In support of this concept, Cowan et al. (1980) detected lesser amounts of gentamicin in puppy kidneys compared to adults due to a redistribution of blood flow away from outer cortical nephrons in neonatal animals. The reason for the increased sensitivity of all young horses in the present study to gentamicin-induced histopathologic renal lesions is not known, however, adult species differences in susceptibility to gentamicin nephrotoxicity exist. There may also be species differences in the susceptibility of young animals. This conclusion supports the clinical evidence of gentamicin nephrotoxicity observed in 2 to 10 week old foals (Tobin, 1979; Riviere e t a / . , 1982). In these clinical reports, all horses had evidence of bacterial infections. It is possible that the kidney of young horses is inherently more sensitive to toxic nephropathy from ger~tamicin. The intrarenal pattern of drug distribution could be unique or equine proximal tubules may be more sensitive. Tile distribution of nephrons between the inner and outer renal cortex may be different in horses. Alternatively, extrarenal systemic factors may potentiate the expression of renal dysfunction in the foal: This area needs further study. The importance of the interstitial nephritis to the pathogenesis of the renal insufficiency is not known, but it may be contributory to the nephropathy. In conclusion, the results of this study indicate that species differences in gentamicin pharmacokinetics exist. In addition, young horses appear to be more susceptible to gentamicin toxicity than the young of other species and thus may serve as a useful model in which to explore the toxicokinetics of gentamicin. A C I ( N O WL E D G E M E N TS

This project was supported in part by AES Project No. 76014. Gentamicin was generously supplied by Schering Corp. (Kenilworth, NJ). Published as paper no. 8843, Agriculture Experiment Station, Purdue University, West Lafayette, IN. We wish to think Mr. Michael P, Carver for technical assistance. REFERENCES

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