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Anticoccidial and Tolerance Studies in the Chicken with Two 6-Amino-9-(substituted benzyl)purines
Anticoccidial and Tolerance Studies in the Chicken with Two 6-Amino-9-(substituted benzyl)purines B. M. MILLER, E. C. McMANUS, G. OLSON, K. D. SCHLEIM, A. A. VAN IDERSTINE, D. W. GRAHAM, J. E. BROWN and E. F. ROGERS Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey 07065 (Received for publication April 20, 1977)
Poultry Science 56:2039-2044, 1977 INTRODUCTION 6-Amino-9-(substituted benzyl)purines are a n e w chemical class of c o m p o u n d s possessing b r o a d - s p e c t r u m anticoccidial activity (Lira et al., 1 9 7 4 ) . A large n u m b e r of analogs of this class of c o m p o u n d was synthesized and tested for anticoccidial efficacy (Lira et al., 1 9 7 7 , Unpublished d a t a ) . T w o c o m p o u n d s , 6-amino9-(2,6-dichlorobenzyl)purine ( L - 6 2 8 , 9 1 4 ) and 6-amino-9-(2-chloro-6-fluorobenzyl)purine (MK-302) 1 showed promise on t h e basis of broad-spectrum efficacy a n d p o t e n c y in t h e initial assays. It is t h e purpose of this paper t o present t h e results of efficacy and tolerance studies c o m p a r i n g these t w o c o m p o u n d s . METHODS Battery Efficacy. T h e efficacies of MK-302 and L - 6 2 8 , 9 1 4 were c o m p a r e d in b a t t e r y trials utilizing sex-separated H u b b a r d X H u b b a r d chickens o b t a i n e d from a commercial h a t c h e r y . A t 2 weeks of age t h e birds were individually weighed and 5 birds from t h e m i d d l e of t h e weight distribution were r a n d o m l y assigned t o each e x p e r i m e n t a l pen. There were t w o replicates assigned per drug t r e a t m e n t per test and usually a larger n u m b e r of pens of noninfected and infected, n o n m e d i c a t e d controls. Only
1
ARPOCOX-Merck & Co., Inc.
male birds were used in these studies with t h e e x c e p t i o n of tests with E. tenella strain D P - 6 3 1 , where only female chickens were used. T h e birds were housed in stainless steel wire-floored cages in t e m p e r a t u r e and lightcontrolled r o o m s . T h e basal ration was a commercial u n m e d i c a t e d chick starter diet (Pennfield Corporation, Lancaster, Pa.). Feed and water were supplied w i t h o u t restriction during b o t h t h e pre-experimental and experim e n t a l periods. T h e medicated diets were prep a r e d s h o r t l y before t h e start of t h e e x p e r i m e n tal period by blending t h e test c o m p o u n d i n t o a small p o r t i o n of t h e basal diet forming a p r e m i x which was t h e n a d d e d t o t h e a p p r o p r i a t e a m o u n t of basal diet and mixed. T h e birds were placed o n t h e medicated diets 2 4 hr. prior t o infection. Cocccidial infections were i n d u c e d b y administering a 1.0 ml. suspension of sporulated oocysts directly i n t o the b i r d ' s c r o p . T h e i n o c u l u m was 50,000—75,000 o o c y s t s per bird in test with E. tenella or E. necatrix, 100,000 oocysts per bird in tests with E. brunetti or E. maxima and 2 0 0 , 0 0 0 oocysts per bird in tests with E. acervulina t y p e . T h e d u r a t i o n of t h e e x p e r i m e n t following inoculation was 7 days for E. acervulina t y p e species, 7—8 days for tests with E. maxima a n d E. brunetti and 8 days for tests with E. tenella or E. necatrix. A s u m m a r y of t h e coccidial strains used in these studies is presented in Table 1. All strains were relatively r e c e n t field isolates which had been
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ABSTRACT Initial assays of 6-amino-9-(2-chloro-6-fluorobenzyl) purine (MK-302) and 6-amino-9-(2,6-dichlorobenzyl)purine (coded L-628,914) showed potential as anticoccidial agents on the basis of broad-spectrum activity and safety. In battery efficacy studies, dietary levels of 60 to 70 p.p.m. and above MK-302 and 45 to 60 p.p.m. L-628,914 proved to have excellent broad-spectrum anticoccidial activity in chickens given heavy exposure to virulent field isolates of coccidia. Eight-week floor-pen tolerance trials showed that the maximum tolerated diet concentration (MTC) of MK-302 was approximately 95 p.p.m. while the MTC of L-628,914 was approximately 60 p.p.m. Dietary relationships (p.p.m. MK-302:p.p.m. L-628,914 for equivalent effects) derived from the efficacy and tolerance results were 1.2:1 and 1.6:1 respectively and clearly demonstrated a higher therapeutic ratio for MK-302.
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B. M. MILLER, E. C. MCMANUS, G. OLSON, K. D. SCHLEIM, A. A. VAN IDERSTINE, D. W. GRAHAM, J. E. BROWN AND E. F. ROGERS TABLE 1 .—Test strains of coccidia Identification
Origin
B. E. E. E. E. E. E. E. E.
DP-667 DP-620 DP-669 DP-631 DP-259 DP-702 FS-24 DP-677 DP-541
Australia Puerto Rico Australia Louisiana Spain Maryland Alabama Maryland Texas
tenella tenella tenella tenella necatrix acervulina t y p e acervulina t y p e maxima brunetti
maintained in the laboratory. Several criteria were used to evaluate the efficacy of the drugs. These included observations and records on mortality rate, growth, severity of gross lesions and number of oocysts produced. To obtain estimates of oocyst production, ceca (tests with E. tenella) or 1/2 of a two-day collection of feces (for tests with the intestinal species including E. necatrix) were homogenized in a blender and appropriate aliquots were diluted, placed in a hemocytometer and oocysts counted. For each group of birds these several parameters were summarized into one number by use of the "anticoccidial index". The general equation for this index is: (% Survival + % Relative Weight Gain) (Lesion Index + Oocyst Index) = I The anticoccidial index for infected, medicated groups indicates the overall relative anticoccidial activity for the compounds tested. The index for the infected, nonmedicated groups
TABLE 2.-Prophylactic
efficacy of L-628,914 and MK-302 against E. tenella
S t r a i n s : D P - 6 6 7 , DP-620, DP-669, DP-631
% p.p.m.
No. birds
Mortality
Rel. % w t . gain
Avg. lesion score
Avg. n o . oocysts/ bird X 10 6
Anticoccidial index
50 60 60 70 80 90
120 119 50 60 60 60 60 20
0 30 0 0 0 0 0 0
100 56 87 85 97 93 92 94
0.0 3.6 0.8 0.3 0.7 0.2 0.0 0.0
0.0 37.2 8.8 1.3 9.9 1.1 0.0 0.1
200 50 170 181 179 190 192 194
Dietary m e d i c a t i o n Compound N o n e (noninfected) N o n e (infected) L-628,914 MK-302
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Species
indicates the severity of the infection. Its description was made by McManus et al., 1968. In order for the oocyst component of the index to best measure the clinical disease, it is necessary to avoid excessive infection because in very heavy infections, the oocyst count of infected, nonmedicated groups is sharply reduced ("overcrowding"). Furthermore, suboptimally medicated groups showing relatively good weight gain and clinical condition may have oocyst counts greatly exceeding those of infected, nonmedicated controls. (Barwick and Casorso, 1970; Williams, 1973; Reid, 1975). A good example is shown in Table 6, where the groups medicated with the two lower levels of L-628,914 show a higher oocyst leakage than the infected, nonmedicated control groups. These phenomena can be regulated by controlling the size of the inoculum to obtain maximum pathology in infected, nonmedicated controls. Conditions other than coccidiosis which affect the weight gain parameter of the anticoccidial index need to be recognized. An example. is found in Table 2 where L-628,914 depressed weight gain at both levels tested. Tolerance. For a comparison of the toxicity of these two compounds, two 8-week floor-pen trials were conducted with noninfected chickens. Day-old sex-separated Hubbard X Hubbard chickens were obtained from a commercial hatchery for these trials. The birds were housed in masonry broiler units each containing 20 pens measuring 50 square feet per pen. Each pen housed 50 birds and contained a gas brooder, one automatic waterer and one barreltype feeder. The houses were ventilated with time- and temperature-controlled exhaust fans. In each of two identical tests, 20 pens were
BENZYL PURINE ANTICOCCIDIALS TABLE 3 .—Prophylactic efficacy ofL-628,914
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and MK-302 against E. necatrix
Strain: DP-259
% No. birds
Mortal ity
Rel. % wt. gain
Avg. lesion score
Avg. n o . oocysts/ bird X 10 6
Anti-
Dietary medication p.p.m. Compound None (noninfected) None (infected) L-628,914
20 20 10 10 10 10 10
0 5 0 0 0 0 0
100 76 91 76 98 92 88
0.0 2.1 0.2 0.0 0.1 0.0 0.0
0.0 3.0 0.1 1.4 0.0 0.0 0.0
200 110 188 158 197 192 189
MK-302
TABLE 4.—Prophylactic efficacy ofL-628,914
%
Dietary medication Compound Strain: DP-702 None (noninfected) None (infected) L-628,914 MK-302
Strain: FS-24 None (noninfected) None (infected) L-628,914 MK-302
and MK-302 against E. acervulina type Avg.
p.p.m.
birds
ity
Rel. % wt. gain
15 30 45 30 45 60
15 25 10 10 10 10 10 10
0 0 0 0 0 0 0 0
100 87 89 105 97 88 107 105
0.0 2.8 2.5 1.9 0.5 2.4 1.0 0.1
50 60 60 70 80
10 10 10 10 10 10 10
0 0 0 0 0 0 0
100 92 82 82 95 98 91
0.0 2.5 0.0 0.0 0.0 0.0 0.0
No.
TABLE 5.—Prophylactic efficacy ofL-628,914
score
Avg. no. oocysts/ bird X 10 6
0.0
157.4 205.8 42.8 11.2 202.8 14.8 0.6
0.0
263.1 0.0 0.0 0.0 0.0 0.0
Antiindex
200 119 124 175 189 124 193 204
200 127 182 182 195 198 191
and MK-302 against E. maxima
Strain: DP-677
%
Dietary medication Compound None (noninfected) None (infected) L-628,914 MK-302
Avg.
p.p.m.
birds
ity
Rel. % wt. gain
15 30 45 30 45 60
10 10 10 10 10 10 10 10
0 10 0 0 0 0 0 0
100 50 82 98 90 95 93 93
No.
score 0.0 2.4 2.0 0.9 0.2 0.5 0.1 0.4
Avg. no. oocysts/ bird X 10 6 0.0
14.3 10.0 1.2 0.0 1.6 0.0 0.0
Antiindex 200 76 134 186 188 186 192 189
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50 60 60 70 80
index
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B. M. MILLER, E. C. MCMANUS, G. OLSON, K. D. SCHLEIM, A. A. VAN IDERSTINE, D. W. GRAHAM, J. E. BROWN and E. F. ROGERS TABLE 6.—Prophylactic efficacy ofL-628,914 and MK-302 against E. brunetti Strain: DP-541
% No. birds
Mortal ity
Rel. % wt. gain
Avg. lesion score
None (noninfected) None (infected) L-628,914
10 10 10 10 10 10 10 10
0 0 0 0 0 0 0 0
100 56 93 108 95 95 103 100
0.0 3.0 1.6 0.7 0.7 0.1 0.2 0.1
MK-302
15 30 45 30 45 60
grouped into four blocks of 5 pens, two blocks of males and two blocks of females, and the treatments were randomly assigned to pens within blocks. The test rations were prepared in the same manner as previously described for the battery efficacy trials. The average initial weight of the birds was recorded and pen weights were obtained at 3, 5 and 8 weeks on experiment. Feed consumption was recorded at each of the weighing intervals.
RESULTS AND DISCUSSION
Efficacy. In initial efficacy studies with this chemical class of compounds, E. tenella was the most difficult species to control. Therefore, the most detailed comparisons of efficacies between MK-302 and L-628,914 were made with strains of E. tenella. Six assays, three with female birds and three with male birds, with two replicates per medication level, provided data for a good approximation of the relative activities of the two compounds. Four strains
Avg. no. oocysts/ bird X 10 6
Antiindex 200 86 137 161 187 154 196 196
0.0
16.4 40.3 20.8 0.4
22.5 2.0 1.2
of E. tenella were used in the six assays. Table 2 shows averaged values of the several parameters measured and clearly demonstrates that the strains were virulent. All medication levels tested prevented mortality and resulted in dramatic improvements in the other parameters. Treatment with 60 p.p.m. MK-302 was essentially equal in potency to 50 p.p.m. L-628,914 and treatment with 70 p.p.m. MK-302 was close in potency to 60 p.p.m. L-628,914. Levels of L-628,914 higher than 60 p.p.m. were not tested because of depressing effects on weight gains. Only 80 p.p.m. MK-302 completely controlled the infection based on all parameters. Weight gains of pens fed 60, 70 and 80 p.p.m. MK-302 were statistically significantly higher (P<0.005) than those fed 60 p.p.m. L-628,914 and numerically superior to those fed 50 p.p.m. L-628,914. The results of tests with E. necatrix, E. acervulina type, E. maxima and E. brunetti show that 60 p.p.m. MK-302 and above provided good control of these species (Tables 3—6). The infections with E. maxima and E.
TABLE 7.—Weight gain and feed efficiency of broiler chickens fed MK-302 or L-628,914 after 8 weeks of medication
period 0 to 8 weeks
Cone .in feed, p.p.m. 0
50 60 75 100 150 200
Weight gain
Feed/gilin
MK-302
L-628,914
1971.8 g. +3.4%
1971.8g. +0.8%
-5.6% -26.8% -51.0%
-9.6% -33.1%
MK-302 2.12 g. /g-3.1% +1.5% +27.8% +52.5%
L-628,914 2.12 g./g. -2.7% +0.3% -1.0% +34.9%
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Dietary medication p.p.m. Compound
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BENZYL PURINE ANTICOCCIDIALS +10
+ 10
100
100
•I o
-'0
CT
-20
I%
-30
«
-40
I
-50
o Q.
,
„
-10
0= MK-302 • = L-628,914
-20
I
r
p
~sP5\V
\\\
\ 8. \ • \ \ > \
0=MK-302 628,914
-30 -40
•
\\
\
-60 0
\ 50
60
75
100
J150
2 0l_ 0
300
RP.M. Drug in Diet
50 60
75
100
150
v 200
P.P.M. Drug in Diet
FIG. 1. Effect of feeding graded levels of L-628,914 and MK-302 on growth of chickens from 0 to 3 weeks of age in floor pens. Each dot or open circle represents the relative percent weight gain of one pen of 50 chickens. The horizontal solid line represents the average weight gain of 8 pens of control chickens (100% = 418 + 10 g.) and the horizontal dashed lines represent the 0.95 probability confidence interval for the control line. The dose response curves for the two compounds are represented by the sloping lines (L-628,914 = — a n d MK-302 = ) with the dashed lines representing the 0.95 probability confidence intervals. The dose response curve for L-628,914 represents 16 pens of chickens fed graded levels of L-628,914 ranging from 50 p.p.m. to 100 p.p.m. in the diet. The equation for this response curve is Y = 209.11 -126.28(X). The dose response curve for MK-302 represents 12 pens of birds fed levels of MK-302 ranging from 100 p.p.m. to 200 p.p.m. in the diet. The equation for this curve is Y = 226.73 —123.08(X). In these equations, X equals the log of p.p.m. drug in the diet and Y equals the relative percent weight gain.
FIG. 2. Effect of feeding graded levels of L-628,914 and MK-302 on growth of chickens from 0 to 5 weeks of age in floor pens. Each dot or open circle represents the relative percent weight gain of one pen of 50 chickens. The horizontal solid line represents the average weight gain of 8 pens of control chickens (100% = 1058 ± 16 g.) and the horizontal dashed lines represent the 0.95 probability confidence interval for the control line. The dose response curves for the two compounds are represented by the sloping lines (L-628,914 = and MK-302 = ) with the dashed lines representing the 0.95 probability confidence intervals. The dose response curve for L-628.914 represents 16 pens of chickens fed graded levels of L-628,914 ranging from 50 p.p.m. to 100 p.p.m. in the diet. The equation for this response curve is Y = 237.99 -140.02(X). The dose response curve for MK-302 represents 12 pens of birds fed levels of MK-302 ranging from 100 p.p.m. to 200 p.p.m. in the diet. The equation for this curve is Y = 286.94 -150.50(X). In these equations, X equals the log of p.p.m. drug in the diet and Y equals the relative percent weight gain.
brunetti were particularly severe as indicated b y relative weight gains of 50% and 56%, respectively, in t h e infected, n o n m e d i c a t e d c o n t r o l groups. In s u m m a r y t h e efficacy ratio b e t w e e n MK-302 and L - 6 2 8 , 9 1 4 was 1.2:1 in E. tenella infections and greater t h a n this for intestinal infections. Tolerance. T w o 8-week floor-pen trials utilizing n o n i n f e c t e d chickens fed graded levels of b o t h c o m p o u n d s were c o n d u c t e d t o define differences in tolerance u n d e r m o r e practical conditions. T h e final average weight gain and feed efficiency d a t a from these e x p e r i m e n t s are presented in Table 7. Graphic p r e s e n t a t i o n s of these growth d a t a are shown in Figures 1—3. T h e g r o w t h depression curves a t 3 weeks of age s h o w t h a t t h e m a x i m u m tolerated c o n c e n t r a t i o n (MTC) of
MK-302 was 7 0 p . p . m . and t h e MTC of L - 6 2 8 , 9 1 4 was a p p r o x i m a t e l y 4 5 p . p . m . T h e 5-week data s h o w t h a t t h e MTC for M K - 3 0 2 increased t o a p p r o x i m a t e l y 8 0 p . p . m . a n d t h e MTC for L-628,914 increased t o 50 p.p.m. T h e 8-week data s h o w a further increase t o approxim a t e l y 95 p . p . m . and 6 0 p.p.m., respectively. A comparison of t h e g r o w t h depression curves presented in Figures 1—3 s h o w t h a t t h e MTC of MK-302 is a p p r o x i m a t e l y 1.5 t i m e s t h e MTC of L-628,914. T h e ratio b e t w e e n t h e MTC's for these t w o c o m p o u n d s remains relatively cons t a n t over t h e period from 3 t o 8 weeks of age. However, t h e MTC for each c o m p o u n d increases with t h e age of the birds. If it is assumed t h a t t h e rate of t h e c h i c k e n ' s m e t a b o lism d o e s n ' t change dramatically during this period of g r o w t h , t h e increased tolerance w o u l d
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x
-60
-50
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B. M. MILLER, E. C. MCMANUS, G. OLSON, K. D. SCHLEIM, A. A. VAN IDERSTINE, D. W. GRAHAM, J. E. BROWN AND E. F. ROGERS
be expec t e d since feed intake per kg. of b o d y weight is inversely related t o bird size. ACKNOWLEDGMENT T h e a u t h o r s express appreciation t o Dr. T h o m a s B. Farver, Biometrics Research Departm e n t , a n d Dr. J a m e s L. Cox, Animal Science Research, for assistance in t h e statistical analyses.
REFERENCES Barwick, M. W., and D. R. Casorso, 1970. Coccidiostat activity. Down to Earth 26:17—20. Lire, E. P., W. M. Barker and R. C. McCrae, 1974. 6-Amino-9-(substituted benzyl) purines and their N 1 oxides. U.S. Patent 3,846,426. November 5,
1974. McManus, E. C , W. C. Campbell and A. C. Cuckler, 1968. Development of resistance to quinoline coccidiostats under field and laboratory conditions. J. Parasitol. 54:1190-1193. Reid, W. M., 1975. Relative value of oocyst counts in evaluating anticoccidial activity. Avian Dis. 19:802-811. Williams, R. B., 1973. Effects of different infection rates on the oocyst production of Eimeria acervulina or Eimeria tenella in the chicken. Parasitol. 67:279-288.
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 30, 2015
300 P.P.M. Drug in Diet
FIG. 3. Effect of feeding graded levels of L-628,914 and MK-302 on growth of chickens from 0 to 8 weeks of age in floor pens. Each dot or open circle represents the relative percent weight gain of one pen of 50 chickens. The horizontal solid line represents the average weight gain of 8 pens of control chickens (100% = 1972 + 24 g.) and the horizontal dashed lines represent the 0.95 probability confidence interval for the control line. The dose response curves for the two compounds are represented by the sloping lines (L-628,914 = and MK-302 = — ) with the dashed lines representing the 0.95 probability confidence intervals. The dose response curve for L-628,914 represents 16 pens of chickens fed graded levels of L-628,914 ranging from 50 p.p.m. to 100 p.p.m. in the diet. The equation for this response curve is Y = 234.04 -132.49(X). The dose response curve for MK-302 represents 12 pens of birds fed levels of MK-302 ranging from 100 p.p.m. to 200 p.p.m. in the diet. The equation for this curve is Y = 293.1 - 1 4 8 . 6 K X ) . In these equations, X equals the log of p.p.m. drug in the diet and Y equals the relative percent weight gain.
Poultry Science 56:2039-2044, 1977 INTRODUCTION 6-Amino-9-(substituted benzyl)purines are a n e w chemical class of c o m p o u n d s possessing b r o a d - s p e c t r u m anticoccidial activity (Lira et al., 1 9 7 4 ) . A large n u m b e r of analogs of this class of c o m p o u n d was synthesized and tested for anticoccidial efficacy (Lira et al., 1 9 7 7 , Unpublished d a t a ) . T w o c o m p o u n d s , 6-amino9-(2,6-dichlorobenzyl)purine ( L - 6 2 8 , 9 1 4 ) and 6-amino-9-(2-chloro-6-fluorobenzyl)purine (MK-302) 1 showed promise on t h e basis of broad-spectrum efficacy a n d p o t e n c y in t h e initial assays. It is t h e purpose of this paper t o present t h e results of efficacy and tolerance studies c o m p a r i n g these t w o c o m p o u n d s . METHODS Battery Efficacy. T h e efficacies of MK-302 and L - 6 2 8 , 9 1 4 were c o m p a r e d in b a t t e r y trials utilizing sex-separated H u b b a r d X H u b b a r d chickens o b t a i n e d from a commercial h a t c h e r y . A t 2 weeks of age t h e birds were individually weighed and 5 birds from t h e m i d d l e of t h e weight distribution were r a n d o m l y assigned t o each e x p e r i m e n t a l pen. There were t w o replicates assigned per drug t r e a t m e n t per test and usually a larger n u m b e r of pens of noninfected and infected, n o n m e d i c a t e d controls. Only
1
ARPOCOX-Merck & Co., Inc.
male birds were used in these studies with t h e e x c e p t i o n of tests with E. tenella strain D P - 6 3 1 , where only female chickens were used. T h e birds were housed in stainless steel wire-floored cages in t e m p e r a t u r e and lightcontrolled r o o m s . T h e basal ration was a commercial u n m e d i c a t e d chick starter diet (Pennfield Corporation, Lancaster, Pa.). Feed and water were supplied w i t h o u t restriction during b o t h t h e pre-experimental and experim e n t a l periods. T h e medicated diets were prep a r e d s h o r t l y before t h e start of t h e e x p e r i m e n tal period by blending t h e test c o m p o u n d i n t o a small p o r t i o n of t h e basal diet forming a p r e m i x which was t h e n a d d e d t o t h e a p p r o p r i a t e a m o u n t of basal diet and mixed. T h e birds were placed o n t h e medicated diets 2 4 hr. prior t o infection. Cocccidial infections were i n d u c e d b y administering a 1.0 ml. suspension of sporulated oocysts directly i n t o the b i r d ' s c r o p . T h e i n o c u l u m was 50,000—75,000 o o c y s t s per bird in test with E. tenella or E. necatrix, 100,000 oocysts per bird in tests with E. brunetti or E. maxima and 2 0 0 , 0 0 0 oocysts per bird in tests with E. acervulina t y p e . T h e d u r a t i o n of t h e e x p e r i m e n t following inoculation was 7 days for E. acervulina t y p e species, 7—8 days for tests with E. maxima a n d E. brunetti and 8 days for tests with E. tenella or E. necatrix. A s u m m a r y of t h e coccidial strains used in these studies is presented in Table 1. All strains were relatively r e c e n t field isolates which had been
2039
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 30, 2015
ABSTRACT Initial assays of 6-amino-9-(2-chloro-6-fluorobenzyl) purine (MK-302) and 6-amino-9-(2,6-dichlorobenzyl)purine (coded L-628,914) showed potential as anticoccidial agents on the basis of broad-spectrum activity and safety. In battery efficacy studies, dietary levels of 60 to 70 p.p.m. and above MK-302 and 45 to 60 p.p.m. L-628,914 proved to have excellent broad-spectrum anticoccidial activity in chickens given heavy exposure to virulent field isolates of coccidia. Eight-week floor-pen tolerance trials showed that the maximum tolerated diet concentration (MTC) of MK-302 was approximately 95 p.p.m. while the MTC of L-628,914 was approximately 60 p.p.m. Dietary relationships (p.p.m. MK-302:p.p.m. L-628,914 for equivalent effects) derived from the efficacy and tolerance results were 1.2:1 and 1.6:1 respectively and clearly demonstrated a higher therapeutic ratio for MK-302.
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B. M. MILLER, E. C. MCMANUS, G. OLSON, K. D. SCHLEIM, A. A. VAN IDERSTINE, D. W. GRAHAM, J. E. BROWN AND E. F. ROGERS TABLE 1 .—Test strains of coccidia Identification
Origin
B. E. E. E. E. E. E. E. E.
DP-667 DP-620 DP-669 DP-631 DP-259 DP-702 FS-24 DP-677 DP-541
Australia Puerto Rico Australia Louisiana Spain Maryland Alabama Maryland Texas
tenella tenella tenella tenella necatrix acervulina t y p e acervulina t y p e maxima brunetti
maintained in the laboratory. Several criteria were used to evaluate the efficacy of the drugs. These included observations and records on mortality rate, growth, severity of gross lesions and number of oocysts produced. To obtain estimates of oocyst production, ceca (tests with E. tenella) or 1/2 of a two-day collection of feces (for tests with the intestinal species including E. necatrix) were homogenized in a blender and appropriate aliquots were diluted, placed in a hemocytometer and oocysts counted. For each group of birds these several parameters were summarized into one number by use of the "anticoccidial index". The general equation for this index is: (% Survival + % Relative Weight Gain) (Lesion Index + Oocyst Index) = I The anticoccidial index for infected, medicated groups indicates the overall relative anticoccidial activity for the compounds tested. The index for the infected, nonmedicated groups
TABLE 2.-Prophylactic
efficacy of L-628,914 and MK-302 against E. tenella
S t r a i n s : D P - 6 6 7 , DP-620, DP-669, DP-631
% p.p.m.
No. birds
Mortality
Rel. % w t . gain
Avg. lesion score
Avg. n o . oocysts/ bird X 10 6
Anticoccidial index
50 60 60 70 80 90
120 119 50 60 60 60 60 20
0 30 0 0 0 0 0 0
100 56 87 85 97 93 92 94
0.0 3.6 0.8 0.3 0.7 0.2 0.0 0.0
0.0 37.2 8.8 1.3 9.9 1.1 0.0 0.1
200 50 170 181 179 190 192 194
Dietary m e d i c a t i o n Compound N o n e (noninfected) N o n e (infected) L-628,914 MK-302
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 30, 2015
Species
indicates the severity of the infection. Its description was made by McManus et al., 1968. In order for the oocyst component of the index to best measure the clinical disease, it is necessary to avoid excessive infection because in very heavy infections, the oocyst count of infected, nonmedicated groups is sharply reduced ("overcrowding"). Furthermore, suboptimally medicated groups showing relatively good weight gain and clinical condition may have oocyst counts greatly exceeding those of infected, nonmedicated controls. (Barwick and Casorso, 1970; Williams, 1973; Reid, 1975). A good example is shown in Table 6, where the groups medicated with the two lower levels of L-628,914 show a higher oocyst leakage than the infected, nonmedicated control groups. These phenomena can be regulated by controlling the size of the inoculum to obtain maximum pathology in infected, nonmedicated controls. Conditions other than coccidiosis which affect the weight gain parameter of the anticoccidial index need to be recognized. An example. is found in Table 2 where L-628,914 depressed weight gain at both levels tested. Tolerance. For a comparison of the toxicity of these two compounds, two 8-week floor-pen trials were conducted with noninfected chickens. Day-old sex-separated Hubbard X Hubbard chickens were obtained from a commercial hatchery for these trials. The birds were housed in masonry broiler units each containing 20 pens measuring 50 square feet per pen. Each pen housed 50 birds and contained a gas brooder, one automatic waterer and one barreltype feeder. The houses were ventilated with time- and temperature-controlled exhaust fans. In each of two identical tests, 20 pens were
BENZYL PURINE ANTICOCCIDIALS TABLE 3 .—Prophylactic efficacy ofL-628,914
2041
and MK-302 against E. necatrix
Strain: DP-259
% No. birds
Mortal ity
Rel. % wt. gain
Avg. lesion score
Avg. n o . oocysts/ bird X 10 6
Anti-
Dietary medication p.p.m. Compound None (noninfected) None (infected) L-628,914
20 20 10 10 10 10 10
0 5 0 0 0 0 0
100 76 91 76 98 92 88
0.0 2.1 0.2 0.0 0.1 0.0 0.0
0.0 3.0 0.1 1.4 0.0 0.0 0.0
200 110 188 158 197 192 189
MK-302
TABLE 4.—Prophylactic efficacy ofL-628,914
%
Dietary medication Compound Strain: DP-702 None (noninfected) None (infected) L-628,914 MK-302
Strain: FS-24 None (noninfected) None (infected) L-628,914 MK-302
and MK-302 against E. acervulina type Avg.
p.p.m.
birds
ity
Rel. % wt. gain
15 30 45 30 45 60
15 25 10 10 10 10 10 10
0 0 0 0 0 0 0 0
100 87 89 105 97 88 107 105
0.0 2.8 2.5 1.9 0.5 2.4 1.0 0.1
50 60 60 70 80
10 10 10 10 10 10 10
0 0 0 0 0 0 0
100 92 82 82 95 98 91
0.0 2.5 0.0 0.0 0.0 0.0 0.0
No.
TABLE 5.—Prophylactic efficacy ofL-628,914
score
Avg. no. oocysts/ bird X 10 6
0.0
157.4 205.8 42.8 11.2 202.8 14.8 0.6
0.0
263.1 0.0 0.0 0.0 0.0 0.0
Antiindex
200 119 124 175 189 124 193 204
200 127 182 182 195 198 191
and MK-302 against E. maxima
Strain: DP-677
%
Dietary medication Compound None (noninfected) None (infected) L-628,914 MK-302
Avg.
p.p.m.
birds
ity
Rel. % wt. gain
15 30 45 30 45 60
10 10 10 10 10 10 10 10
0 10 0 0 0 0 0 0
100 50 82 98 90 95 93 93
No.
score 0.0 2.4 2.0 0.9 0.2 0.5 0.1 0.4
Avg. no. oocysts/ bird X 10 6 0.0
14.3 10.0 1.2 0.0 1.6 0.0 0.0
Antiindex 200 76 134 186 188 186 192 189
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 30, 2015
50 60 60 70 80
index
2042
B. M. MILLER, E. C. MCMANUS, G. OLSON, K. D. SCHLEIM, A. A. VAN IDERSTINE, D. W. GRAHAM, J. E. BROWN and E. F. ROGERS TABLE 6.—Prophylactic efficacy ofL-628,914 and MK-302 against E. brunetti Strain: DP-541
% No. birds
Mortal ity
Rel. % wt. gain
Avg. lesion score
None (noninfected) None (infected) L-628,914
10 10 10 10 10 10 10 10
0 0 0 0 0 0 0 0
100 56 93 108 95 95 103 100
0.0 3.0 1.6 0.7 0.7 0.1 0.2 0.1
MK-302
15 30 45 30 45 60
grouped into four blocks of 5 pens, two blocks of males and two blocks of females, and the treatments were randomly assigned to pens within blocks. The test rations were prepared in the same manner as previously described for the battery efficacy trials. The average initial weight of the birds was recorded and pen weights were obtained at 3, 5 and 8 weeks on experiment. Feed consumption was recorded at each of the weighing intervals.
RESULTS AND DISCUSSION
Efficacy. In initial efficacy studies with this chemical class of compounds, E. tenella was the most difficult species to control. Therefore, the most detailed comparisons of efficacies between MK-302 and L-628,914 were made with strains of E. tenella. Six assays, three with female birds and three with male birds, with two replicates per medication level, provided data for a good approximation of the relative activities of the two compounds. Four strains
Avg. no. oocysts/ bird X 10 6
Antiindex 200 86 137 161 187 154 196 196
0.0
16.4 40.3 20.8 0.4
22.5 2.0 1.2
of E. tenella were used in the six assays. Table 2 shows averaged values of the several parameters measured and clearly demonstrates that the strains were virulent. All medication levels tested prevented mortality and resulted in dramatic improvements in the other parameters. Treatment with 60 p.p.m. MK-302 was essentially equal in potency to 50 p.p.m. L-628,914 and treatment with 70 p.p.m. MK-302 was close in potency to 60 p.p.m. L-628,914. Levels of L-628,914 higher than 60 p.p.m. were not tested because of depressing effects on weight gains. Only 80 p.p.m. MK-302 completely controlled the infection based on all parameters. Weight gains of pens fed 60, 70 and 80 p.p.m. MK-302 were statistically significantly higher (P<0.005) than those fed 60 p.p.m. L-628,914 and numerically superior to those fed 50 p.p.m. L-628,914. The results of tests with E. necatrix, E. acervulina type, E. maxima and E. brunetti show that 60 p.p.m. MK-302 and above provided good control of these species (Tables 3—6). The infections with E. maxima and E.
TABLE 7.—Weight gain and feed efficiency of broiler chickens fed MK-302 or L-628,914 after 8 weeks of medication
period 0 to 8 weeks
Cone .in feed, p.p.m. 0
50 60 75 100 150 200
Weight gain
Feed/gilin
MK-302
L-628,914
1971.8 g. +3.4%
1971.8g. +0.8%
-5.6% -26.8% -51.0%
-9.6% -33.1%
MK-302 2.12 g. /g-3.1% +1.5% +27.8% +52.5%
L-628,914 2.12 g./g. -2.7% +0.3% -1.0% +34.9%
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 30, 2015
Dietary medication p.p.m. Compound
2043
BENZYL PURINE ANTICOCCIDIALS +10
+ 10
100
100
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-'0
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-20
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-30
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-40
I
-50
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-10
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-30 -40
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-60 0
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60
75
100
J150
2 0l_ 0
300
RP.M. Drug in Diet
50 60
75
100
150
v 200
P.P.M. Drug in Diet
FIG. 1. Effect of feeding graded levels of L-628,914 and MK-302 on growth of chickens from 0 to 3 weeks of age in floor pens. Each dot or open circle represents the relative percent weight gain of one pen of 50 chickens. The horizontal solid line represents the average weight gain of 8 pens of control chickens (100% = 418 + 10 g.) and the horizontal dashed lines represent the 0.95 probability confidence interval for the control line. The dose response curves for the two compounds are represented by the sloping lines (L-628,914 = — a n d MK-302 = ) with the dashed lines representing the 0.95 probability confidence intervals. The dose response curve for L-628,914 represents 16 pens of chickens fed graded levels of L-628,914 ranging from 50 p.p.m. to 100 p.p.m. in the diet. The equation for this response curve is Y = 209.11 -126.28(X). The dose response curve for MK-302 represents 12 pens of birds fed levels of MK-302 ranging from 100 p.p.m. to 200 p.p.m. in the diet. The equation for this curve is Y = 226.73 —123.08(X). In these equations, X equals the log of p.p.m. drug in the diet and Y equals the relative percent weight gain.
FIG. 2. Effect of feeding graded levels of L-628,914 and MK-302 on growth of chickens from 0 to 5 weeks of age in floor pens. Each dot or open circle represents the relative percent weight gain of one pen of 50 chickens. The horizontal solid line represents the average weight gain of 8 pens of control chickens (100% = 1058 ± 16 g.) and the horizontal dashed lines represent the 0.95 probability confidence interval for the control line. The dose response curves for the two compounds are represented by the sloping lines (L-628,914 = and MK-302 = ) with the dashed lines representing the 0.95 probability confidence intervals. The dose response curve for L-628.914 represents 16 pens of chickens fed graded levels of L-628,914 ranging from 50 p.p.m. to 100 p.p.m. in the diet. The equation for this response curve is Y = 237.99 -140.02(X). The dose response curve for MK-302 represents 12 pens of birds fed levels of MK-302 ranging from 100 p.p.m. to 200 p.p.m. in the diet. The equation for this curve is Y = 286.94 -150.50(X). In these equations, X equals the log of p.p.m. drug in the diet and Y equals the relative percent weight gain.
brunetti were particularly severe as indicated b y relative weight gains of 50% and 56%, respectively, in t h e infected, n o n m e d i c a t e d c o n t r o l groups. In s u m m a r y t h e efficacy ratio b e t w e e n MK-302 and L - 6 2 8 , 9 1 4 was 1.2:1 in E. tenella infections and greater t h a n this for intestinal infections. Tolerance. T w o 8-week floor-pen trials utilizing n o n i n f e c t e d chickens fed graded levels of b o t h c o m p o u n d s were c o n d u c t e d t o define differences in tolerance u n d e r m o r e practical conditions. T h e final average weight gain and feed efficiency d a t a from these e x p e r i m e n t s are presented in Table 7. Graphic p r e s e n t a t i o n s of these growth d a t a are shown in Figures 1—3. T h e g r o w t h depression curves a t 3 weeks of age s h o w t h a t t h e m a x i m u m tolerated c o n c e n t r a t i o n (MTC) of
MK-302 was 7 0 p . p . m . and t h e MTC of L - 6 2 8 , 9 1 4 was a p p r o x i m a t e l y 4 5 p . p . m . T h e 5-week data s h o w t h a t t h e MTC for M K - 3 0 2 increased t o a p p r o x i m a t e l y 8 0 p . p . m . a n d t h e MTC for L-628,914 increased t o 50 p.p.m. T h e 8-week data s h o w a further increase t o approxim a t e l y 95 p . p . m . and 6 0 p.p.m., respectively. A comparison of t h e g r o w t h depression curves presented in Figures 1—3 s h o w t h a t t h e MTC of MK-302 is a p p r o x i m a t e l y 1.5 t i m e s t h e MTC of L-628,914. T h e ratio b e t w e e n t h e MTC's for these t w o c o m p o u n d s remains relatively cons t a n t over t h e period from 3 t o 8 weeks of age. However, t h e MTC for each c o m p o u n d increases with t h e age of the birds. If it is assumed t h a t t h e rate of t h e c h i c k e n ' s m e t a b o lism d o e s n ' t change dramatically during this period of g r o w t h , t h e increased tolerance w o u l d
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x
-60
-50
2044
B. M. MILLER, E. C. MCMANUS, G. OLSON, K. D. SCHLEIM, A. A. VAN IDERSTINE, D. W. GRAHAM, J. E. BROWN AND E. F. ROGERS
be expec t e d since feed intake per kg. of b o d y weight is inversely related t o bird size. ACKNOWLEDGMENT T h e a u t h o r s express appreciation t o Dr. T h o m a s B. Farver, Biometrics Research Departm e n t , a n d Dr. J a m e s L. Cox, Animal Science Research, for assistance in t h e statistical analyses.
REFERENCES Barwick, M. W., and D. R. Casorso, 1970. Coccidiostat activity. Down to Earth 26:17—20. Lire, E. P., W. M. Barker and R. C. McCrae, 1974. 6-Amino-9-(substituted benzyl) purines and their N 1 oxides. U.S. Patent 3,846,426. November 5,
1974. McManus, E. C , W. C. Campbell and A. C. Cuckler, 1968. Development of resistance to quinoline coccidiostats under field and laboratory conditions. J. Parasitol. 54:1190-1193. Reid, W. M., 1975. Relative value of oocyst counts in evaluating anticoccidial activity. Avian Dis. 19:802-811. Williams, R. B., 1973. Effects of different infection rates on the oocyst production of Eimeria acervulina or Eimeria tenella in the chicken. Parasitol. 67:279-288.
Downloaded from http://ps.oxfordjournals.org/ at Northern Arizona University on May 30, 2015
300 P.P.M. Drug in Diet
FIG. 3. Effect of feeding graded levels of L-628,914 and MK-302 on growth of chickens from 0 to 8 weeks of age in floor pens. Each dot or open circle represents the relative percent weight gain of one pen of 50 chickens. The horizontal solid line represents the average weight gain of 8 pens of control chickens (100% = 1972 + 24 g.) and the horizontal dashed lines represent the 0.95 probability confidence interval for the control line. The dose response curves for the two compounds are represented by the sloping lines (L-628,914 = and MK-302 = — ) with the dashed lines representing the 0.95 probability confidence intervals. The dose response curve for L-628,914 represents 16 pens of chickens fed graded levels of L-628,914 ranging from 50 p.p.m. to 100 p.p.m. in the diet. The equation for this response curve is Y = 234.04 -132.49(X). The dose response curve for MK-302 represents 12 pens of birds fed levels of MK-302 ranging from 100 p.p.m. to 200 p.p.m. in the diet. The equation for this curve is Y = 293.1 - 1 4 8 . 6 K X ) . In these equations, X equals the log of p.p.m. drug in the diet and Y equals the relative percent weight gain.