Use of azalide antibiotic compositions for treating or preventing a bacterial or protozoal infection in mammals

Methods for treating or preventing bacterial or protozoal infections in mammals by administering a single dose of an antibiotic composition comprising a mixture of azalide isomers and a pharmaceutically acceptable vehicle are disclosed. Methods for increasing acute or chronic injection-site toleration in mammals by administering a single dose of antibiotic compositions comprising a mixture of azalide isomers and a pharmaceutically acceptable vehicle are also disclosed. A combination comprising: an antibiotic composition comprising a mixture of azalide isomers, a pharmaceutically acceptable carrier, and instructions for use in a single-dose administration is also disclosed.

EXAMPLE 1 Synthesis of N-(n-propyl) isomer II. To a 2 L erlenmeyer flask was added desmethylazithromycin (190.5 g, 259.2 mmol), methylene chloride (572 mL), and magnesium sulfate (38 g). The mixture was stirred for 10 minutes then filtered into a 5 L round bottom flask. Additional methylene chloride (2285 mL) was added and the solution cooled to 0-5° C. CBZ-CI (58.4 mL) was then added over 10 minutes. The reaction stirred at −0° C. for 6 hrs then at ambient temperature overnight. HPLC analysis indicated the presence of residual starting material such that the reaction was re-cooled to ˜0° C. and additional CBZ-CI (19.5 mL) was added in a single portion. The reaction stirred for 5.5 hrs at 0° C. then for 2.5 hrs at ambient temperature. TLC indicated a complete reaction. The reaction was quenched with saturated aqueous sodium bicarbonate (953 mL) and the phases separated. The organic phase was dried over magnesium sulfate, then filtered and concentrated to afford the compound of formula (III): 4 To a 5 L round bottom flask containing the compound of formula (III) (225.3 g) in methylene chloride (901 mL) and DMSO (450 mL) at −65° C. was added trifluoroacetic anhydride (82.4 mL). The temperature was maintained at ˜60° C. throughout the addition which was complete in 9 minutes. The reaction was stirred at −65 to −70° C. for 20 minutes. The reaction was quenched with triethylamine (145 mL) then stirred at −60° to −65° C. for 20 minutes. To the reaction mixture was then added water (1127 mL) over 3 minutes, at which point the temperature rose to −2° C. The reaction mixture was stirred for 10 minutes and the phases were allowed to separate. The organic phase was washed with water, (675 mL) then with saturated aqueous sodium chloride (675 mL). The organic phase was dried over magnesium sulfate then filtered and organic solvents removed by distillation. MTBE was added and distilled to remove all traces of methylene chloride and DMSO. Additional MTBE was added to a total volume of 3380 mL. Dibenzoyl-D-tartaric acid monohydrate (87.8 g) in MTBE (1126 mL) was added to form a thick slurry. The mixture was heated to reflux and stirred overnight. After cooling to ambient temperature, the solids were collected on a Buchner funnel and rinsed with MTBE. The solids were dried in a drying oven at 40° C. to afford 258.3 g of the dibenzoyl tartrate salt of the compound of formula (IV): 5 To a 3 L round bottom flask was added methylene chloride (800 mL) and the dibenzoyl tartrate salt of the compound of formula (IV) (188 g). Water (400 mL) and potassium carbonate (45.5 g) were added and the mixture stirred at ambient temperature for 5 minutes. The organic phase was separated, then washed with water (250 mL) and dried over magnesium sulfate. Drying agent was removed by filtration, and the resultant solution evaporated under a stream of nitrogen to a final volume of 623 mL to afford a free-base ketone. To a 5 L round bottom flask was added THF (623 mL) and trimethylsulfonium bromide (74.7 g). The resultant slurry was cooled to −10° C. and potassium tert-butoxide (54.4 g) added. The reaction mixture was stirred for 10 minutes at −10° C. then cooled to −70° C. over 5 minutes. A solution of the free-base ketone was added over 11 minutes, keeping the temperature between −60 and −650° C. HPLC indicated the reaction was complete after 90 minutes. The reaction was quenched at −60° C. using a solution of ammonium chloride (315 g) in water (1800 mL). The temperature rose to −5° C. during the quench. The reaction mixture was warmed to 5-10° C., and the phases separated. The organic phase was dried over sodium sulfate then filtered and concentrated to afford the compound of formula (V), (117.4 g) as a yellow foam. HPLC indicated a purity of 61.4% by peak area. 6 To a solution of the compound of formula (V) (275 g, 312 mmol) in dry methanol (2.75 L) was added potassium iodide (518 g, 3.12 mol) and n-propylamine (250 mL, 3.04 mol). The mixture was stirred overnight at 45° C. TLC indicated a complete reaction. The reaction was concentrated on a rotary evaporator and the residue partitioned between water (2.5 L) and methylene chloride (2.5 L). The pH of the aqueous phase was adjusted to 6.7 using 3N aqueous HCl. The extraction was repeated one additional time. Combined aqueous phases were combined with fresh methylene chloride (1.5 L) and the pH of the aqueous phase adjusted to 8.5 using solid potassium carbonate. The phases were separated and the aqueous phase re-extracted twice with additional methylene chloride. Combined organic phases were dried over sodium sulfate, then filtered. The filtrate was concentrated on a rotary evaporator to afford a beige foam (230 g). Purification of the foam was effected on a slurry-packed silica gel column using 19/3 (v/v) hexanes-diethylamine as the mobile phase. In this manner, 125 g of crude product afforded 72 g N-(n-propyl) isomer I as a white, amorphous foam. N-(n-propyl) isomer I was dissolved in acetonitrile (0.5 L) at ambient temperature. Deionized water (1 L) was then added, which caused precipitation. Additional acetonitrile (0.5 L) was then added to afford a homogenous solution which was stirred at ambient temperature for 30 hrs. HPLC analysis indicated the formation of a new component that comprised &square;20% total peak area. Organic solvent was removed on a rotary evaporator. Potassium carbonate (30 g) was added to the aqueous residue followed by methylene chloride (0.3 L). The mixture was shaken and the lower organic phase removed. Two additional extractions (2×0.3 L) were also performed. Combined organic phases were dried over sodium sulfate, then filtered and the resultant solution concentrated to a dry foam (˜10 g). The resultant mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II was dissolved in a mixture of methylene chloride and 19/3 (v/v) hexanes-diethylamine, and placed on a slurry-packed silica gel column, then eluted with the 19/3 system. The eluant was switched to 19/6 hexanes-diethylamine in fraction 56. Fraction 9-17 were combined and concentrated to a dry foam which contained only unreacted starting material. Fractions 52-72 were combined and concentrated, and contained N-(n-propyl) isomer II (79% purity by HPLC). 
 Example 2 Table 1 below shows the effect of pH, temperature, acid type, and concentration of N-(n-propyl) isomer I on the equilibration reaction rate and on levels of major impurities following equilibration. Replicated experiments (data not shown) demonstrated reproducibility of results. The equilibrium ratio of N-(n-propyl) isomer I and N-(n-propyl) isomer II (about 90%±4% to about 10%±4%, respectively) was consistent for all experiments. Analysis of the data indicates that pH and temperature have a significant effect on the time required for equilibration. Without being bound by any theory, lower equilibration temperatures or lower pH values generally result in substantially longer equilibration times. Equilibration time can also depend on, inter alia, the concentration of starting material, and the type and concentration of the acid used. N-(n-propyl) isomer I at a concentration of up to about 300 mg per mL of composition was heated to a temperature of about 40° C. to about 80° C. in the presence of one or more acids at a concentration of about 0.2 mmol to about 1.0 mmol per mL of mixture and with a sufficient quantity of hydrochloric acid to achieve a pH of about 6.5 to about 7.5 for up to about 20 hours to produce an equilibrium mixture of isomers that is about 95%-98% pure. Equilibration kinetic parameters and impurity levels for equilibration of N-(n-propyl) isomer I and N-(n-propyl) isomer II were determined as a function of pH, equilibration temperature, type of acid, and N-(n-propyl) isomer I concentration and are listed in Table 1. Known methods, including high performance liquid chromatography (“HPLC”), nuclear magnetic resonance spectroscopy (“NMR”), gas chromatography (“GC”), mass spectrometry (“MS”), liquid chromatography/mass spectrometry (“LC/MS”), GC/MS, and thin layer chromatography (“TLC”), can be used to identify the impurities. “DS” refers to N-(n-propyl) isomer I prior to equilibration and is included for comparison. Equilibrium mixtures of isomers were prepared and assayed as follows. 40 mL of solution were prepared in each of experiments 1A-11 A, and each solution was divided into 1 mL aliquots prior to heating in order to more easily monitor equilibration at different time points. 20 mL of solution were prepared in each of experiments 12B-24B, and each solution was divided into 0.7 mL aliquots prior to heating. 100 mL of solution were prepared in each of experiments 25C-28C, 200 mL of solution were prepared in each of experiments 29C-30C, and equlibration was monitored from 0.5 mL aliquots removed from the solutions. 60 mL of solution were prepared in each of experiments 31D-33D and 35D-41D, 170 mL of solution were prepared in experiment 34D, and equilibration was monitored from 0.5 mL aliquots removed from the solutions. From 7,200 mL to 54,000 mL of solution were prepared in each of experiments 42E-46E, and equilibration was monitored by removing from 2 mL to 5 mL aliquots from the solutions. From 35 mL to 50 mL of solution were prepared in each of experiments 47F-50G, and each solution was divided into 1 mL aliquots prior to heating. Water was added to the appropriate container, followed by the type and amount of acid listed in column 4 of Table 1. The term “qs” preceding the acid type refers an amount of the acid sufficient to achieve the pH listed in column 2. Where 0.1 M citric or tartaric acid was used, hydrochloric acid was also added in a quantity sufficient to obtain the pH listed in column 2. Where an acid concentration is recited in column 4 (e.g., “0.1 M citric”), this is the concentration of acid in a solution having an equilibrated mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II present in a concentration of 100 mg/mi. The mixture of water and acid was stirred until all of the acid was dissolved (about 5 minutes or less for smaller volumes, and about 20 minutes for larger volumes). N-(n-propyl) isomer I was added slowly and in small portions to avoid clumping, and the resulting mixture was stirred vigorously until dissolved (less than 30 minutes for smaller volumes, and about 60-120 minutes for larger volumes). After dissolution of N-(n-propyl) isomer I, the pH of the resulting solution was measured. If the pH was lower than the pH listed in column 2, it was raised to the pH listed in column 2 with 10% sodium hydroxide. If the pH was higher than the pH listed in column 2, it was lowered with the appropriate acid(s). For each experiment, the solution was heated at the temperature noted in column 3 until an equilibrium mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II was obtained, as determined by one of the HPLC assays described below. In some experiments, mixtures were heated for a period of time longer than required for equilibration (percentages greater than 100% in column 8) to determine the effects of prolonged heat on the degree of impurity. To monitor equilibration, reaction mixture aliquots were assayed by HPLC at various times during equilibration. For the majority of equilibration experiments shown in Table 1, aliquots were diluted with 40 mM potassium phosphate buffer (pH 6.0) to a concentration of approximately 0.5 mg of N-(n-propyl) isomer I and N-(n-propyl) isomer II per mL total sample volume and subjected to chromatography using an Asahipak ODP-50, 5 &mgr;m, 250×4.0 mm column (40% acetonitrile/35% methanol/25% 40 mM potassium phosphate; pH 8.5 mobile phase; flow rate 0.7 mL/minutes; room temperature) on an HP 1090 Liquid Chromatograph equipped with an external Applied Biosystems 783A Programmable Absorbance Detector. Peaks were detected by monitoring ultraviolet absorption at 210 nm. For the remaining equilibration experiments shown in Table 1 (experiments 31 D-46E), aliquots were diluted with 20% acetonitrile/50% methanol/30% 50 mM potassium phosphate (pH 5.5) to a concentration of 1.0 mg of N-(n-propyl) isomer I and N-(n-propyl) isomer II per mL of total sample volume and subjected to chromatography using a YMC Pro-Pack C 18 , 3 &mgr;m, 50×2.0 mm column (20% acetonitrile/50% methanol/30% 50 mM potassium phosphate; pH 7.0 mobile phase; flow rate 0.5 mL/minutes; room temperature) on an HP 1090 Liquid Chromatograph with internal UV Detector. Peaks were detected by monitoring ultraviolet absorption at 210 nm. Relative amounts of N-(n-propyl) isomer I and N-(n-propyl) isomer II were determined by taking the ratio of their relative chromatogram-peak areas. Under the above HPLC conditions, N-(n-prdpyl) isomer I has a retention time of approximately 13-23 minutes, and N-(n-propyl) isomer II has a relative retention time (“RRT”) of approximately 0.8 to 0.9. By “RRT” is meant a retention time relative to that of N-(n-propyl) isomer I under the above-described HPLC conditions. The purity of equilibrated samples in Table 1 was determined using HPLC according to one of three procedures. In experiments 1A-24B, 48F, and 50G, aliquots were diluted with 25 mM potassium phosphate buffer (pH 5.5) to a concentration of 1.25 mg of N-(n-propyl) isomer I and N-(n-propyl) isomer II per mL total sample volume and assayed using an Eclipse XDB-C 8 , 5 &mgr;m, 250×4.6 mm column (22% acetonitrile/58% methanol/20% 25 mM potassium phosphate; pH 8.0 mobile phase; flow rate 0.6 mL/minutes; room temperature) on a Waters Alliance 2690 Separation Module with BAS CC-5/LC-4C Amperometric Detector. Peaks were detected electrochemically with one electrode at &plus;0.70 V, a second electrode at &plus;0.88 V, and a range of 0.5 &mgr;A. In experiments 25C-41 D, aliquots were diluted with 50 mM citric acid (pH 5.5) to a concentration of 0.25 mg of the mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II per mL of total sample volume and assayed using a YMC Pro-Pack C 18 , 3 &mgr;m, 150×4.6 mm column (70% methanol/30% 50 mM phosphate; pH 7.0 mobile phase; flow rate 1 mL/minutes; room temperature) on the Waters Alliance system. Peaks were detected electrochemically with only one electrode at &plus;0.90 V. In experiments 42E-43E, aliquots were diluted with 50 mM citric acid (pH 5.5) to a concentration of 0.25 mg of the mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II per mL of total sample volume and assayed using a YMC Pro-Pack C 18 , 3 &mgr;m, 150×4.6 mm column (70% methanol/30% 50 mM phosphate; pH 7.0 mobile phase; flow rate 1 mL/minute; room temperature) on an HP 1090 Liquid Chromatograph with BAS CC-5/LC-4C Amperometric Detector. Peaks were detected electrochemically with only one electrode at &plus;0.90 V. The percentage of the equilibrium mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II (column 9) and impurities (column 10) relative to the assayed sample was determined using the areas under the peaks in the chromatograms. Some of the detected impurities were: a descladinose azalide (its RRT being approximately 0.26 on an Eclipse XDB-C 8 column), an acetaldehyde insertion product (its RRT being approximately 1.75 on an Eclipse XDB-C 8 column), and a formaldehyde insertion product (its RRT being approximately 1.6 on an Eclipse XDB-C 8 column). The descladinose azalide has the structure: 7 The acetaldehyde insertion product has the structure: 8 The formaldehyde insertion product has the structure: 9 The descladinose azalide, the acetaldehyde insertion product, and the formaldehyde insertion product, and pharmaceutically acceptable salts thereof, have antibiotic properties and are useful as antibiotic agents. The experiments of groups A and B (identified by the letter following the experiment number) in Table 1 were performed to determine the effects of pH, temperature, type of acid, concentration of acid, and N-(n-propyl) isomer I concentration on equilibration. The experiments of group C in Table 1 illustrate the effects of pH and temperature on equilibration. The experiments of group D in Table 1 illustrate the effects of pH, temperature, and acid concentration on equilibration. The experiments of group E in Table 1 illustrate a preferred method of equilibration, that is, at a pH of about 7.0, an equilibration temperature of about 70° C., and N-(n-propyl) isomer I concentration of about 250 mg/mL. Experiments in group F tested the effects of alternate acids and equilibration temperatures, and experiment G was performed in the presence of 50% propylene glycol co-solvent. Results of these experiments indicate that, even under a variety of conditions, equilibration of the mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II consistently results in the formation of from about 90%±4% of N-(n-propyl) isomer I and about 10%±4% of N-(n-propyl) isomer II. Equilibration temperature and pH appear to have the largest effect on equilibration rate, with higher temperatures generally leading to faster rates, even with higher concentrations of N-(n-propyl) isomer I. In most cases, however, longer equilibration times resulted in higher concentration of impurities, and therefore, optimal equilibration conditions are those leading to relatively high equilibration rates, i.e., that form the equilibrium mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II in 1-3 hours. 2 TABLE 1 Initial N-(n-propyl) % of N-(n- Equilibration isomer I propyl) time to % Equilibrium Experiment temperature concentration isomer II at equilibration % of time to mixture of % Number & Group pH (° C.) acid (mg/mL) equilibration (hrs) equilibration isomers Impurities DS 98.39 1.61 1A 6.3 65 qs citric 112 12.2 5.1 100 96.09 3.91 150 94.03 5.97 2A 6.0 80 qs phosphoric 75 11.6 2 100 94.57 5.43 370 86.71 13.29 3A 7.0 70 qs citric 75 11.5 1.4 100 96.36 3.64 640 89.62 10.38 4A 7.0 50 qs citric 150 11.5 10.9 100 97.76 2.24 200 96.94 3.06 5A 6.0 60 qs phosphoric 150 11.2 12.9 100 96.52 3.48 170 95.18 4.82 6A 6.0 60 qs citric 75 12 3 17.6 100 96.15 3.85 170 94.95 5.05 7A 6.0 80 qs citric 150 11.9 2.2 100 96.15 3.85 160 95.20 4.80 8A 7.0 50 qs phosphoric 75 11.4 7.2 100 97.79 2.21 150 96.47 3.53 9A 7.0 70 qs phosphoric 150 12.1 1.2 100 97.66 2.34 160 96.70 3.30 10A 6.5 65 qs citric 112 11.3 3.3 100 96.63 3.37 190 94.64 5.36 11A 6.5 65 qs citric 112 12.4 3.6 100 96.87 3.13 200 94.76 5.24 12B 7.0 70 qs citric 150 12.5 1.4 100 94.33 5.67 150 94.00 6.00 13B 7.25 65 0.1 M citric/ 225 11.2 3.3 100 94.55 5.45 qs HCl 150 94.26 5.74 14B 7.25 65 qs citric 225 11.2 2.5 100 94.75 5.25 150 94.17 5.83 15B 7.0 70 0.1 M citric/ 300 11.0 3.6 100 93.08 6.92 qs HCl 150 92.74 7.26 16B 7.5 70 0.1 M citric/ 150 11.6 1.4 100 94.98 5.02 qs HCl 150 94.82 5.18 17B 7.0 60 qs citric 300 11.4 4.9 100 93.93 6.07 150 93.80 6.20 18B 7.5 60 qs citric 150 12.1 2.6 100 94.00 6.00 150 93.89 6.11 19B 7.5 60 0.1 M citric/ 300 11.3 4.5 100 93.89 6.11 qs HCl 150 93.78 6.22 20B 7.5 70 qs citric 300 11.3 1.5 100 93.88 6.12 150 93.65 6.35 21B 7.0 60 0.1 M citric/ 150 12.3 4.1 100 94.31 5.69 qs HCl 150 94.27 5.73 22B 7.25 65 0.1 M citric/ 225 11.7 3.0 100 94.51 5.49 qs HCl 150 94.24 5.76 23B 7.25 65 citric 225 12.3 2.3 100 94.23 5.77 150 94.10 5.90 24B 7 0 70 citric 300 11.4 2.2 100 94.43 5.57 150 93.91 6.09 25C 7.5 75 0.1 M citric/ 250 11.8 1.3 100 98.59 1.41 qs HCl 26C 7.5 65 0.1 M citric/ 250 10.5 3.0 100 98.78 1.22 qs HCl 27C 6.5 75 0.1 M citric/ 250 n/a 4.0 85 98.74 1.26 qs HCl 28C 6.5 65 0.1 M citric/ 250 n/a 9.9 50 98.91 1.09 qs HCl 29C 7.0 70 0.1 M citric/ 250 11.1 1.8 100 98.90 1.10 qs HCl 30C 7.0 70 0.1 M citric/ 250 11.3 2.2 100 98.91 1.09 qs HCl 31D 7.0 70 0.125 M 250 10.2 2.6 100 — — citric/ qs HCl 32D 7.0 70 0.125 M 250 10.1 2.8 100 — — citric/ qs HCl 33D 7.0 70 0.125 M citric/ 250 10.2 2.6 100 qs HCl 34D 7.0 70 0.175 M citric/ 250 10.5 2.5 100 qs HCl 35D 7.0 70 0.1 M citric/ 250 10.6 2.2 100 qs HCl 36D 7.5 75 0.1 M citric/ 250 11.2 1.0 100 qs HCl 37D 7.5 65 0.1 M citric/ 250 11.1 1.9 100 qs HCl 38D 7.0 70 0.1 M citric/ 250 10.8 2.4 100 qs HCl 39D 8.0 70 0.1 M citric/ 250 11.1 1.1 100 qs HCl 40D 6.5 75 0.1 M citric/ 250 10.6 1.9 100 qs HCl 41D 6.5 65 0.1 M citric/ 250 10.8 5.8 100 qs HCl 42E 7.0 70 0.1 M citric/ 250 11.6 1.1 100 96.00 4.00 qs HCl 43E 7.5 70 0.1 M tartaric/ 250 12.0 2.0 100 94.90 5.10 qs HCl 44E 7.0 70 0.1 M 250 13.6 1.4 100 — — citric/ qs HCl 45E 6.8 70 0.1 M 250 12.7 1.1 100 — — citric/ qs HCl 46E 6.9 70 0.1 M 250 12.4 1.3 100 — — citric/ qs HCl 47F 7 70 qs citric 250 10.8 1.4 100 94.78 5.22 48F 7 70 0.1 M 250 11.5 7.7 100 — — tartaric/ qs HCl 49F 7.4 39 qs 1 11.1 6.7 100 — — phosphori c 50G 7 70 qs citric 75 9.4 2.7 100 — — 
 EXAMPLE 3 The stability of compositions comprising an equilibrium mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II stored at 50° C. for 12 weeks and stabilized with co-solvent is shown in Table 2 below. The results indicate that the compositions containing no co-solvent are significantly less stable than compositions containing co-solvent in an amount of from about 250 to about 500 mg per mL of the composition (experiments 1A-11B). Compositions having a pH of about 5.4 and containing propylene glycol in an amount of from about 450 to about 550 mg per mL of the composition are the most stable. Other co-solvents may be used to stabilize the compositions (experiments 1E-2E); however, propylene glycol is preferred. As shown in Table 2, stability is dependent on pH, and it can also be dependent on type and quantity of acid used, and concentration of the equilibrated mixture of isomers. These compositions were prepared as follows. After heating to the desired temperature (column 2) and allowing the mixture of water, acid, and N-(n-propyl) isomer I to equilibrate for the time shown in column 3, equilibrium mixtures of isomers were allowed to cool to room temperature. When the mixtures reached room temperature, the appropriate amount of the desired co-solvent was added (column 6). The percentage of co-solvent shown in column 6 is a weight-to-volume percentage (e.g., 50% PG is 500 mg propylene glycol per mL of pharmaceutical composition). If an antioxidant or a preservative was used, the appropriate amounts were added (columns 8 and 9). The pH of the solution was measured and adjusted to the value in column 5 by adding one or more acids and/or 10% w/w sodium hydroxide. The volumes of the resulting solutions were then adjusted by adding water. The compositions were filtered through a 0.2 micron sterilizing filter. Vials were filled in a laminar-flow hood, and the vial head space was flushed with the appropriate gas mixture (column 10) before sealing. Equilibration and purity were monitored using HPLC as described above in Example 2. The stability of stabilized, equilibrated compositions sealed in glass vials was determined after storage for 12 weeks at 50° C. The effects of concentration of the equilibrium mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II, pH, co-solvent amount and type, type and concentration of acid, exposure to air, presence of preservatives, and presence of antioxidants were monitored. Results are shown in Table 2. Experiments 1A-3A were performed to monitor the effect of equilibrium mixture concentration on stability. Experiments 2A, 6A, and 7A were performed to monitor the effect of pH on stability. Experiments 2A, 4A, and 5A show the effect of co-solvent amount on stability, and experiments 3A and 8A show the effect of using citric acid alone, as opposed to mixtures of citric and phosphoric acid, for obtaining an acidic pH. Experiments 1B-11B show the effects of pH and propylene glycol (“PG”) co-solvent on stability. Experiments 1C and 2C show the effect of using tartaric acid alone, as opposed to a mixture of tartaric and hydrochloric acid, for obtaining an acidic pH. Experiments 9B-11B and 3C show the effects of a preservative on stability of the mixture, and experiments 9B-11B, 4C, and 5C show the effects of an antioxidant on stability of the mixture. Experiments 6C and 7C show the effects of using a mixture of tartaric and hydrochloric acid or a mixture of citric and hydrochloric acid on stability. Experiments 1D-12D show the effects of different amounts of monothioglycerol (“MTG”) antioxidant and different degrees of oxygen exposure on stability. Experiments 4D-6D and 13D-18D demonstrate the effects of pH of the composition and acid concentration on stability. Results of these experiments indicate that after storage for 12 weeks at 50° C., the equilibrated compositions that contain at least 50% propylene glycol and have a pH ranging from about 5.2 to about 5.5 retain greater than 93% of the initial concentration of the equilibrium mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II. The highest level of impurities was found in a composition having no co-solvent (experiment 4A). Accordingly, the presence of co-solvent surprisingly and unexpectedly limits the amount of impurities. High levels of impurities were found after 12 weeks in compositions having less than 40% co-solvent and a pH of less than 5.0. The concentration of the acid also affects stability of the pharmaceutical compositions. Compositions with relatively low concentrations of acid (about 20 mM) and a pH of about 5.4 show the greatest stability after storage. However, low acid concentrations result in low buffer strength, which leads to fluctuating pH and may lead to a relatively high degree of impurity under other time or temperature conditions. 3 TABLE 2 Concentration of equilibrium mixture of % at 12 weeks and N-(n-propyl) 50° C. Experiment Equil. Equil. isomer I and N-(n-propyl) Co-solvent Acid N-(n- N-(n- number temp time isomer II in pharmaceutical type and concentration Preser- Antioxidant Head-space propyl) propyl) % Equilibrium Mixture of and Group (° C.) (hrs) comp. (mg/mL) pH amount on (M) vative (mg/mL) Filler isomer II isomer I Isomers % Impurities 1A 60 16 10 .0 50% PG qs citric — — N 2 12.33 0 48 92.81 7 19 2A 60 16 30 0 50% PG qs citric — — N 2 12.09 7 88 89.97 10 03 3A 60 16 100 .0 50% PG qs citric — — N 2 11.95 6 13 88 08 11 92 4A 60 16 30 .0 — qs citric — — N 2 12 78 6 44 79.22 20 78 5A 60 16 30 .0 25% PG qs citric — — N 2 13 07 5 50 88.57 11 43 6A 60 16 30 .5 50% PG qs citric — — N 2 10 71 6 71 87.42 12 58 7A 60 16 30 5 50% PG qs citric — — N 2 11 81 0 34 92.15 7 85 8A 60 24 100 .0 50% PG 0 23 M — — N 2 11 80 4 27 86 07 13 93 citric/ qs H 3 PO 4 1B 70 1 5 100 .0 25% PG qs citric — — N 2 10 90 9 50 90.40 9 60 2B 70 1 5 100 0 50% PG qs citric — — N 2 9 10 3 20 92 30 7 70 3B 70 1 5 100 5 25% PG qs citric — — N 2 10.70 1 40 92.10 7 90 4B 70 1.5 100 .5 50% PG qs citric — — N 2 9.50 3 80 93 30 6 70 5B 70 1 5 100 25 20% PG qs citric — — N 2 10 90 9 70 90.60 9 40 6B 70 1 5 100 25 55% PG qs citric — — N 2 9.30 4 40 93.70 6 30 7B 70 1 5 100 75 37.5% PG qs citric — — N 2 9.70 9 40 89.10 10.90 8B 70 1.5 100 .75 37 5% PG qs citric — — N 2 9.90 2 70 92.60 7 40 9B 70 1.5 100 .25 37 5% PG qs citric — — N 2 9.50 3.10 92.60 7.40 10B 70 1.5 100 .25 37.5% PG qs citric — — N 2 9.90 1.70 91 60 8.40 11B 70 1.5 100 .25 37.5% PG qs citric — — N 2 10.20 2.20 92 40 7 60 1C 70 1.5 100 5.25 37.5% PG qa tartaric — — N 2 10 10 3.40 93.50 6.50 2C 70 1.5 100 5.25 37.5% PG 0.1 M tartaric/ — — N 2 10.00 4.30 94.30 5.70 qa HCl 3C 70 1.5 100 5.25 37.5% PG qs citric phenol — N 2 10.40 9.60 100.0 — 4C 70 1.5 100 5.25 37.5% PG qs citric — 5 N 2 9 90 3.00 92.90 7.10 5C 70 1.5 100 5.25 37.5% PG qs citric — 5 N 2 9.90 3.00 92.90 7.10 Propyl gallate 6C 70 1.5 100 5.50 50% PG 0.1 M tartaric/ — 5 N 2 8.40 8.20 96.60 3.40 qs HCl MTG 7C 70 1.5 100 5.50 50% PG 0.1 M citric/ — 5 N 2 8.40 8.80 97.20 2.80 qs HCl MTG 1D 70 1.5 100 5.40 50% PG 0.1 M citric/ — 10 air 9.02 7.40 96.42 3.58 qs HCl MTG 2D 70 1.5 100 5.40 50% PG 0.1 M citric/ — 10 5% O 2 9.03 7.40 96.43 3 57 qs HCl MTG 3D 70 1 5 100 5.40 50% PG 0.1 M citric/ — 5 air 9 07 7.17 96.24 3.76 qs HCl MTG 5D 70 1 5 100 5.40 50% PG 0.1 M citric/ — 5 10% O 2 9 14 7.38 96.52 3.48 qs HCl MTG 6D 70 1.5 100 5.40 50% PG 0.1 M citric/ — 5 10% O 2 9.14 7.44 96 58 3.42 qs HCl MTG 7D 70 1.5 100 .40 50% PG 0 1 M citric/ — 5 mM MTG 5% O 2 9 20 8 96 36 3 64 qs HCl 7 16 8D 70 1.5 100 .40 50% PG 0.1 M citric/ — 5 1% O 2 9.20 8 96 34 3 66 qs HCl MTG 7 14 9D 70 1.5 100 40 50% PG 0.1 M citric/ — 2.5 air 9 19 8 96 36 3 64 qs HCl MTG 7 17 10D 70 1 5 100 .40 50% PG 0.1 M citric/ — 2 5 5% O 2 9.24 8 96.38 3 62 qs HCl MTG 7 14 11D 70 1.5 100 .40 50% PG 0 1 M citric/ — — air 9 18 8 96 12 3 88 qs HCl 6 94 12D 70 1 5 100 .40 50% PG 0 1 M citric/ — — 5% O 2 9.18 8 96 10 3 90 qs HCl 6 92 13D 70 1 5 100 .70 50% PG 0 1 M citric/ — 5 10% O 2 9 18 8 96 14 3 86 qs HCl MTG 6 96 14D 70 1 5 100 .10 50% PG 0.1 M citric/ — 5 10% O 2 9 20 8 96 25 3 75 qs HCl MTG 7 05 15D 70 1 5 100 40 50% PG 0.05 M citric/ — 5 10% O 2 9.21 8 96.65 3 35 qs HCl MTG 7 44 16D 70 1 5 100 .70 50% PG 0 025 M citric/ — 5 10% O 2 9.11 8 96 62 3 38 HCl MTG 7 51 17D 70 1 5 100 .10 50% pg 0.025 M citric/ — 5 10% O 2 9 11 8 95 90 4 10 qs HCl MTG 6 79 18D 70 1 5 100 5.40 50% PG 0.025 M citric/ — 5 10% O 2 9 07 87.86 96 93 3 07 qs HCl MTG 1E 30 5.0 50% qs citric — — air 99 7 glycerol formal 2E 30 5 0 50% N- qs citric — — air 94.9 methyl 2- pyrrolidone 
 EXAMPLE 4 Fifty-two liters of an injectable pharmaceutical composition containing 100 mg of equilibrium mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II per mL of composition were prepared as follows. 16.584 kg of Water for Injection (USP grade) sparged with nitrogen (NF grade) was added to a stainless steel compounding vessel and agitation was begun. Nitrogen was also used as an overlay to reduce oxygen exposure of the solution in the compounding vessel during manufacture. Approximately 1 kg of anhydrous citric acid (USP grade) was added to the water and the resulting mixture was agitated until the acid dissolved. 1.511 kg of a 10% (w/w) solution of hydrochloric acid (NF grade) in water (USP grade) was subsequently added to the mixture. 5.357 kg of a mixture containing approximately 97% of N-(n-propyl) isomer I and N-(n-propyl) isomer II (in a ratio of about 99:1) and 3% of one or more impurities was slowly added to the agitating mixture and was allowed to dissolve. The pH of the resulting solution was adjusted to 7.0&plus;0.5 by adding 0.224 kg of a 10% (w/w) solution of hydrochloric acid in water. Equilibration of N-(n-propyl) isomer I and N-(n-propyl) isomer II was achieved by heating the solution to 70° C. &plus;10° C. for 105 minutes. Once equilibration was complete, as determined using HPLC, the solution was allowed to cool to 25° C.±10° C., and 26.008 kg of propylene glycol (USP grade) was added to the agitating mixture. After the propylene glycol was completely mixed in, 0.26 kg of monothioglycerol (NF grade) was added to the solution, and the pH was readjusted to 5.4±0.3 by adding 2.349 kg of 10% (w/w) hydrochloric acid in water. The final volume was adjusted to 52.015 liters by adding 1.843 kg of water. The resulting composition contained 100 mg of the equilibrium mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II per mL of composition, 500 mg per mL of propylene glycol, citric acid at a concentration of 0.1 M, and monothioglycerol at a concentration of 5 mg/mL of composition. The composition was filtered through a 6 micron pre-filter and then through a 0.2 micron final sterilizing filter, which was sterilized by moist-heat autoclaving for 60 minutes at 121° C. and tested for integrity using the pressure-hold method both prior to sterilization and after filtration. 20 mL flint type I serum glass vials (Wheaton Science Products, Millville, N.J.) were sterilized and depyrogenated in a dry heat tunnel at 250° C. for 240 minutes. 20 mm 4432/50 gray chlorobutyl siliconized stoppers (The West Company, Lionville, Pa.) were depyrogenated by washing and were sterilized by moist-heat autoclaving for 60 minutes at 121° C. Each of 2,525 vials was filled under sterile conditions with 20 mL of the resulting composition plus 0.6 mL overfill (20.6 mL/vial is 2.06 g/vial unit potency of pharmaceutical composition at 100 mg/mL of equilibrium mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II based on an actual drug substance lot potency of 97.1%), the vial head spaces were flushed with nitrogen, and the vials were sealed with the stoppers and overseals (20 mm aluminum seals, product &num; 5120-1125, The West Company, Lionville, Pa.). 
 EXAMPLE 5 From about 0.125 mL to about 0.5 mL of a pharmaceutical composition having a pH of 5.4 and containing the equilibrium mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II present in an amount of 100 mg per mL of the pharmaceutical composition, where 100 mg per mL is the “potency-actual” number; citric acid present in an amount of 0.1 mmol per mL of the pharmaceutical composition; hydrochloric acid present in an amount of 19.58 mg of the concentrated acid (36-38% by weight potency) per mL of the pharmaceutical composition; sodium hydroxide present in an amount of 0.09 mg of a 1.0 M sodium hydroxide solution per mL of the pharmaceutical composition; sodium hydroxide present in an amount of 0.09 mg of a 10 M sodium hydroxide solution per mL of the pharmaceutical composition; propylene glycol present in an amount of 501.25 mg per mL of the pharmaceutical composition; and water present in an amount of 418.20 mg per mL of the pharmaceutical composition were administered to swine infected with Pasteurella multocida in order to determine its therapeutic efficacy. By Apotency-actual&commat; number, as used herein, is meant the actual mg per mL of the substantially pure mixture or equilibrium mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II present in the pharmaceutical composition. Fifty clinically normal and healthy pigs having a consistent body weight of approximately 10 kg were selected from a pool of 60 animals. Selected animals (10 per treatment) were randomly assigned to treatment and sorted into pens accordingly. On day 0, each animal was inoculated endotracheally with 25 mL of Pasteurella multocida challenge culture. Each lot of animals was injected intramuscularly with a single dose of one of the following solutions approximately 1 hour post-inoculation: (1) about 1.5 mL of sterile 0.9% sodium chloride (saline); (2) about 0.5 mL of 25 mg/mL danofloxacin at a dose of 1.25 mg/kg of body weight; (3) about 0.125 mL of the pharmaceutical composition at a dose of 1.25 mg/kg of body weight; (4) about 0.25 mL the pharmaceutical composition at a dose of 2.5 mg/kg of body weight; or (5) about 0.5 mL of the pharmaceutical composition at a dose of 5 mg/kg of body weight. Only danofloxacin was re-administered on each of the following two days. All other treatments were given in a single dose injection. Temperatures and illness scores were recorded 6 hours post-challenge and once daily beginning at 24 hours post-challenge. Animals that developed severe pneumonia (i.e., illness score of 4) were euthanized and listed as a mortality. Animals that died during the course of the experiment were necropsied. Their lungs were removed and examined grossly for pneumonic lesions. An estimate of the percentage of affected lung tissue was determined and recorded. On day 5 post-challenge, all surviving animals were euthanized and necropsied as described above. Assessment of efficacy was determined based upon comparison of mean daily illness scores, temperatures and lung-lesion scores. Differences between treatments for mean daily rectal temperatures and illness scores were evaluated by a repeated measures analysis of variance. Differences between mean lung-lesion scores for each treatment were evaluated using a factorial analysis of variance procedure. A comparison of mortality rates between treatments was performed using Chi-Square analysis and Fisher's Exact test. The disease challenge in this study was relatively severe. Six hours post-challenge, the pigs were depressed, cyanotic and showed signs of dyspnea. Rectal temperatures were elevated in all treatment groups. The overall mortality rate for the study was 18% (9/50 pigs). Calculations of mean daily rectal temperatures indicated no statistically significant differences in these values among treatment groups. Although temperatures were elevated in all groups 6 hours post-challenge, the mean daily temperatures for all treatment groups remained within normal ranges. Animals treated with the pharmaceutical composition at 5 mg/kg of body weight or at 2.5 mg/kg of body weight, or with danofloxacin, displayed statistically significant (p<0.05) reductions in mean daily illness scores (about 2) compared to the mean daily illness scores of animals injected with saline (about 3). No significant differences were observed when comparing animals treated with three doses of danofloxacin to animals treated with a single dose of the pharmaceutical composition at 5 mg/kg of body weight or 2.5 mg/kg of body weight. Comparisons of the three treatments with the pharmaceutical composition indicated that pigs treated with the pharmaceutical composition at 5 mg/kg of body weight displayed statistically significant (p<0.05) lower clinical illness scores than pigs treated with the pharmaceutical composition at 1.25 mg/kg of body weight. No significant differences in illness scores were seen between pigs treated with the pharmaceutical composition at 5 mg/kg or the pharmaceutical composition at 2.5 mg/kg of body weight. The effects of the various treatments upon mortality rates and lung-lesion scores are summarized in Table 3, below. Mortality rates ranged from 0% to 40% in the treated animals. Forty percent (4/10) of the animals in the saline control group died of pneumonia between 48-72 hours post-challenge. There was one death in the group treated with 2.5 mg/kg of body weight of the pharmaceutical composition and 4 deaths (40%) in the group treated with 1.25 mg/kg of body weight of the pharmaceutical composition. No deaths occurred in the groups treated with danofloxacin or 5 mg/kg of body weight of the pharmaceutical composition. The mean lung-lesion score for the saline control pigs was 44%. Pigs treated with danofloxacin, or 2.5 mg/kg of body weight or 5 mg/kg of body weight of the pharmaceutical composition showed statistically significant (p<0.05) reductions in mean lung-lesion scores when compared to the saline controls. When comparing treated animals, animals treated with danofloxacin, or 2.5 mg/kg of body weight or 5 mg/kg of body weight of the pharmaceutical composition displayed statistically significant (p<0.05) reductions in mean lung lesion scores when compared to animals treated with 1.25 mg/kg of body weight of the pharmaceutical composition. 4 TABLE 3 Treatment Mean Lung Lesion Score (intramuscular injection) Mortality (%) saline 4/10 (40%) 44.0 (1.5 ml) Danofloxacin 0/10  (0%) 1.9 (1.25 mg/kg) Pharmaceutical composition 0/10  (0%) 3.8 (5 mg/kg) Pharmaceutical composition 1/10 (10%) 6.6 (2.5 mg/kg) Pharmaceutical composition 4/10 (40%) 29.2 (1.25 mg/kg) 
 EXAMPLE 6 From about 1.25 mL to about 5.0 mL of a pharmaceutical composition having a pH of 4.9 and containing a mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II in a ratio of from about 95% to about 99% of N-(n-propyl) isomer I and from about 1% to about 5% of N-(n-propyl) isomer II present in an amount of 200 mg per mL of the pharmaceutical composition, where 200 mg per mL is the “potency-actual” number; citric acid present in an amount of 85.09 mg per mL of the pharmaceutical composition; propylene glycol present in an amount of 253.40 mg per mL of the pharmaceutical composition; and water present in an amount of 541.46 mg per mL of the pharmaceutical composition were administered to calves with naturally occurring bacterial bovine respiratory disease. Two hundred and thirteen calves (average weight of 200 kg) were purchased and co-mingled for approximately 2-3 days at the assembly point and transported approximately 1,000 miles by truck for delivery at a veterinary facility. No anti-bacterial treatment was given at any time during acquisition or pre-study handling. Upon arrival, the animals were unloaded into receiving pens, ear-tagged and provided with access to water and forage material. The animals were vaccinated with BOVISHIELD 4&plus;L5 vaccine containing modified live viruses IBR, PI, BVD and BRSV, and a bacterin containing 5 servovars against Leptospira (Pfizer Animal Health). In addition, they were treated with the anti-parasitic agent DECTOMAX (Pfizer Animal Health) and implanted with a growth promotant (SYNOVEX-C, Syntex Laboratories). Beginning on the day after arrival, all animals were observed daily for clinical signs consistent with bovine respiratory disease. Individual animals exhibiting clinical signs of acute respiratory disease were selected (pulled) and their rectal temperatures were recorded. The selection criteria for inclusion in the study were a clinical presentation consistent with acute respiratory disease (i.e., illness score greater than or equal to 1 and less than 4) and pyrexia (rectal temperature greater than or equal to 104.0° F.) Once selected, animals were randomly allotted to one of five treatment groups using a randomized block allotment. All treatments were equally represented in each test pen (2 animals/treatment/pen). Each lot of animals was injected subcutaneously with a single dose of one of the following solutions on the day the selection criteria were met: (1) about 6.6 mL of sterile 0.9% sodium chloride (saline); (2) about 6.6 mL of MICOTIL 300 at a dose of 10 mg/kg of body weight; (3) about 1.25 mL of the pharmaceutical composition at a dose of 1.25 mg/kg of body weight; (4) about 2.5 mL of the pharmaceutical composition at a dose of 2.5 mg/kg of body weight; or (5) about 5 mL of the pharmaceutical composition at a dose of 5 mg/kg of body weight. All solutions were administered in a single-dose subcutaneous injection. During the post-treatment observation period, no further medication was administered. Temperatures and illness scores were recorded daily for all animals for 14 days post-treatment. Beginning 48 hours post-treatment, animals that exhibited an illness score of greater than or equal to 1 and a temperature of 104.0° F. were identified as re-pulls at the time of data analysis. Animals that developed severe pneumonia (i.e., illness score of 4) were euthanized and listed as a mortality. Animals that died during the course of the experiment were weighed and necropsied. Their lungs were removed and examined grossly for pneumonic lesions. An estimate of the percentage of affected lung tissue was determined and recorded. If possible, lung samples from a typically diseased area were collected from all animals for bacteriologic culture. On day 14, all surviving animals were euthanized. Animals were necropsied, and their lungs were assessed grossly for lesions as described above. Lung samples from all animals were collected for bacteriologic culture. The performance of the animals was assessed by evaluating individual weight gains. Each animal was weighed on days 7 and 14. Assessment of efficacy was determined based upon analysis of mean daily illness scores, temperatures and lung-lesion scores. The proportion of successful responders in each treatment on day 14 was determined as the initial number of animals per treatment minus the number of mortalities and re-pulls. A comparison between treatment groups of the proportion of animals within each group exhibiting an illness score of 0 (normal) or greater than or equal to 1 on day 14 was evaluated using Chi-Square analysis and Fisher's Exact test. Differences in temperature and weight gain between treatments were evaluated using a repeated-measures ANOVA. The comparisons of mortality rates and responder rates between treatment groups were also performed using Chi-square analysis and Fisher's Exact test. The outbreak of respiratory disease in this natural-disease study was extremely severe. The mortality rate for the saline controls was 75%. The mean lung-lesion score of the saline controls was 38.4%. The time course of the onset of clinical signs of disease was typical of that normally observed in a commercial feedyard with calves of this age and background. Calculations of mean daily rectal temperatures indicated statistically significant (p<0.01) reductions in mean daily rectal temperatures in all treatment groups when compared to the saline controls. Temperatures in the treated groups remained lower than those of the saline controls through day 7 of the study. Animals treated with either 2.5 mg/kg or 5 mg/kg of the pharmaceutical composition exhibited significantly (p<0.01) lower mean daily rectal temperatures than did animals treated with MICOTIL. Temperature responses of animals treated with 1.25 mg/kg of the pharmaceutical composition were similar to those of the MICOTIL controls. Therapeutic treatment of animals with either MICOTIL or the pharmaceutical composition at any dose level resulted in significant (p<0.01) reductions in mean daily illness scores compared to the saline controls. When comparing these treatments, calves treated with the pharmaceutical composition at 2.5 mg/kg displayed significantly (p<0.05) decreased mean daily illness scores compared to MICOTIL-treated calves. Calves treated with the pharmaceutical composition at either 1.25 mg/kg or 5 mg/kg displayed mean daily illness scores that were similar to calves treated with MICOTIL. Re-pull rates, mortality and lung-lesion score data are summarized in Table 4, below. Seventy-five percent of the saline controls met the re-pull criteria in this study. Administration of MICOTIL or the pharmaceutical composition at 1.25 mg/kg resulted in reductions in the incidence of re-pulls (55% and 40%, respectively) relative to the saline controls. In contrast, re-pull rates for animals treated with either 2.5 mg/kg or 5 mg/kg of the pharmaceutical composition were significantly (p<0.01) lower than that of the saline controls. Re-pull rates for animals treated with 2.5 mg/kg of the pharmaceutical composition were significantly lower than those observed with MICOTIL. Re-pull rates for animals treated with either 1.25 mg/kg or 5 mg/kg of the pharmaceutical composition were reduced relative to MICOTIL. Fifteen of twenty (75%) saline control calves succumbed to pneumonia during the course of the study. Administration of MICOTIL resulted in a significant (p<0.01) reduction in the number of mortalities (25%) relative to the saline controls. Significant (p<0.01) reductions in mortality relative to the saline controls were also observed for all three groups of animals treated with the pharmaceutical composition. Comparative mortality rates were significantly (p<0.05) lower for animals treated with the pharmaceutical composition administered at 5 mg/kg relative to MICOTIL-treated calves. The two lower doses of the pharmaceutical composition provided reductions in mortality relative to MICOTIL. The mean lung-lesion score of the saline treated calves was 38.4%. Animals treated with either MICOTIL or the pharmaceutical composition at any dose level exhibited significant (p<0.01) reductions in mean lung-lesion scores relative to the saline controls. The pharmaceutical composition administered at either 2.5 mg/kg or 5 mg/kg provided reductions in mean lung-lesion scores relative to MICOTIL. Lung-lesion scores for animals treated with 1.25 mg/kg of the pharmaceutical composition were similar to those for animals treated with MICOTIL. 5 TABLE 4 Treatment (subcutaneous injection) Re-Pull Rate Mortality Rate Lung-Lesion Score Saline 15/20 (75%) 15/20 (75%) 38.4% (6.6 mL) MICOTIL 11/20 (55%) 5/20 (25%) 18.0% (10 mg/kg) Pharmaceutical 8/20 (40%) 1/20  (5%) 14.0% Composition (1.25 mg/kg) Pharmaceutical 2/20 (10%) 1/20  (5%) 8.6% Composition (2.5 mg/kg) Pharmaceutical 6/20 (30%) 0/20  (0%) 8.9% Composition (5 mg/kg) The proportion of responders for each treatment was calculated by subtracting the number of mortalities and re-pulls from the initial number of animals per treatment. Responder rates are summarized in Table 5. Twenty-five percent of the animals treated with MICOTIL met the responder criteria. Responder rates for animals treated with either 2.5 mg/kg or 5 mg/kg of the pharmaceutical composition were significantly (p<0.01 and p<0.05, respectively) improved relative to the MICOTIL treated animals. The responder rate for animals treated with 1.25 mg/kg of the pharmaceutical composition was greater than that observed for MICOTIL-treated animals. Clinically healthy calves were defined as those with an illness score of zero on day 14 (Table 5). In this study, only one of the saline controls was clinically healthy on day 14. Therapeutic administration of MICOTIL provided an increase in the number of healthy animals on day 14. The proportion of animals that were characterized as clinically healthy on day 14 in each of the treatments with the pharmaceutical composition was significantly (p<0.05) greater than the proportion in the saline control group. Similarly, the proportion of clinically healthy animals in all of the pharmaceutical composition treatment groups was greater than the proportion of clinically healthy animals in the MICOTIL group 6 TABLE 5 Proportion of Clinically Treatment Responder Rates Healthy Animals Saline 3/20 (15%) 1/20  (5%) (6.6 mL) MICOTIL 5/20 (25%) 4/20 (20%) (10 mg/kg) Pharmaceutical composition 12/20 (60) 9/20 (45%) (1.25 mg/kg) Pharmaceutical composition 17/20 (85%) 8/20 (40%) (2.5 mg/kg) Pharmaceutical composition 14/20 (70%) 8/20 (40%) (5 mg/kg) Table 6, below, summarizes the effects of therapeutic treatment upon 7- and 14-day weight gains. Animals treated with either MICOTIL or with the pharmaceutical composition exhibited significantly (p<0.01) increased average daily gains at both days 7 and 14 relative to the saline controls. Animals treated with either 2.5 mg/kg or 5 mg/kg of the pharmaceutical composition exhibited improved weight gains relative to animals treated with MICOTIL. Animals treated with 1.25 mg/kg of the pharmaceutical composition exhibited similar weight gains to those treated with MICOTIL. 7 TABLE 6 Average Daily Weight Gain Average Daily Weight Gain Treatment Over 7 Days (kg/day) Over 14 Days (kg/day) Saline −1.18 0.36 (6.6 mL) MICOTIL 0.60 0.78 (10 mg/kg) Pharmaceutical 0.71 0.77 composition (1.25 mg/kg) Pharmaceutical 1.00 1.20 composition (2.5 mg/kg) Pharmaceutical 1.20 1.35 composition (5 mg/kg) 
 EXAMPLE 7 From about 1.25 mL to about 5 mL of a pharmaceutical composition having a pH of 6.0 and containing a mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II in a ratio of from about 95% to about 99% of N-(n-propyl) isomer I and from about 1% to about 5% of N-(n-propyl) isomer II present in an amount of 200 mg per mL of the pharmaceutical composition, where 200 mg per mL is the “potency-actual” number; citric acid present in an amount of 60.00 mg per mL of the pharmaceutical composition; propylene glycol present in an amount of 251.01 mg per mL of the pharmaceutical composition; and water present in an amount of 569.00 mg per mL of the pharmaceutical composition were administered to calves at a high risk for developing bacterial bovine respiratory disease. Two hundred and twenty-two calves (average weight of 200 kg) were purchased, co-mingled for approximately 2 days at the assembly point and transported approximately 1,000 miles by truck for delivery at a veterinary facility. No anti-bacterial treatment was given at any time during acquisition or pre-study handling. Upon arrival, the animals were unloaded into receiving pens, ear-tagged and provided with access to water and forage material. All animals were vaccinated with BOVISHIELD 4&plus;L5 vaccine containing modified live viruses IBR, PI, BVD and BRSV, and a bacterin containing 5 servovars against Leptospira (Pfizer Animal Health). In addition, they were treated with the anti-parasitic agent DECTOMAX (Pfizer Animal Health). On the day after arrival (day 0), the clinical condition of each animal was evaluated and an illness score recorded. On the day of allotment (day 0), animals that exhibited signs of fatigue, including mild depression or lack of rumen fill, in the absence of clinical signs of disease did not qualify for an illness score of greater than or equal to 1. Animals that exhibited an illness score of less than or equal to I and a body temperature of less than 104.0° F. were selected for inclusion in the study. Once selected, animals were randomly allotted to one of five treatment groups (20 calves per group) using a systematic randomized block allotment. The first ten animals selected were assigned to the first pen. Subsequent animals were assigned to pens in groups of ten until all pens were full. Each pen contained one or more animals form each treatment group. Each animals weight, body temperature and illness score were recorded prior to treatment on day 0. Each lot of animals was injected subcutaneously with a single dose of one of the following solutions within the first 30 hours after arrival: (1) about 6.6 mL of sterile 0.9% sodium chloride (saline); (2) about 6.6 mL of MICOTIL; (3) about 1.25 mL of the pharmaceutical composition at a dose of 1.25 mg/kg of body weight; (4) about 2.5 mL of the pharmaceutical composition at a dose of 2.5 mg/kg of body weight; or (5) about 5 mL of the pharmaceutical composition at a dose of 5 mg/kg of body weight All solutions were administered in a single dose injection. Acute injection-site toleration observations were made at 24 and 48 hours post-injection. Temperatures and illness scores were recorded daily for all animals. Animals that exhibited an illness score of greater than or equal to 1 and a temperature of greater than or equal to 104° F. were identified as morbid (pulls) at the time of data analysis. Animals that developed severe pneumonia (i.e., illness score of 4) were euthanized and listed as a mortality. Animals that died during the course of the experiment were weighed and necropsied. Their lungs were removed and examined grossly for pneumonic lesions. An estimate of the percentage of affected lung tissue was determined and recorded. If possible, lung samples from a typically diseased area of the lung were collected from all animals for bacteriologic culture. On day 14, all surviving animals were euthanized and necropsied, and lung samples were assessed grossly for lesions and collected for bacteriologic culture as described above. The performance of the animals was assessed by evaluating individual weight gains. Each animal was weighed on days 7 and 14. Assessment of efficacy was determined based upon comparison of mean daily illness scores, temperatures and lung-lesion scores. The proportion of successful responders in each treatment on day 14 was determined as the initial number of animals per treatment minus the number of mortalities and pulls. A comparison between treatment groups of the proportion of animals within each group exhibiting an illness score of 0 (normal) or greater than or equal to 1 on day 14 was evaluated using Chi-Square analysis and Fisher's Exact test. Differences in temperature and weight gain between treatments were evaluated by a repeated measures ANOVA. A comparison of mortality, morbidity and responder rates between treatments was also performed using Chi-Square analysis and Fisher's Exact test. The outbreak of respiratory disease in this natural-disease study was moderately severe. The morbidity rate for the saline controls was 60%, and 25% of these animals died of acute pneumonia. The mean lung-lesion score of the saline controls was 24.3%. The time course of the onset of clinical signs of disease was typical of that normally observed in a commercial feedyard with calves of this age and background. Statistically significant (p<0.01) reductions in mean daily rectal temperatures were seen in all treatment groups when compared to the saline controls. Temperatures in the treated groups remained lower than those of the saline controls throughout day 10 of the study. Animals treated with any of the three doses of the pharmaceutical composition exhibited statistically significant (p<0.01) lower mean daily rectal temperatures than did animals treated with MICOTIL. The magnitude of the differences was greatest for animals treated with either 2.5 mg/kg or 5 mg/kg of the pharmaceutical composition. Metaphylactic treatment of calves with either MICOTIL or the pharmaceutical composition resulted in significant (p<0.01) reductions in mean daily illness scores compared to the saline controls. When comparing the antibiotic treatments, calves treated with 5 mg/kg of the pharmaceutical composition exhibited statistically significant (p<0.01) decreases in mean daily illness scores compared to animals treated with MICOTIL. Illness score responses of animals treated with either 1.25 mg/kg or 2.5 mg/kg of the pharmaceutical composition were similar to those of calves treated with MICOTIL. Morbidity rates, mortality rates and lung-lesion score data are summarized in Table 7, below. In this moderately severe natural-infection study, the saline controls exhibited 60% morbidity. All antibiotic treatments exhibited significant (p<0.05) reductions in morbidity relative to the saline controls. Animals treated with the pharmaceutical composition exhibited numerical reductions in morbidity relative to the MICOTIL controls; however, none of the differences were statistically significant. Six of twenty (30%) saline control calves succumbed to bronchopneumonia during the course of the study. Administration of MICOTIL resulted in a reduction in the number of mortalities relative to the saline controls. Significant (p<0.05) reductions in mortality relative to the saline controls were observed for all three groups of animals treated with the pharmaceutical composition. The mean lung-lesion score for the saline control calves was 24.3%. Animals treated with MICOTIL or the pharmaceutical composition exhibited significant (p<0.01) reductions in mean lung-lesion scores relative to the saline controls. Calves treated with the pharmaceutical composition at 5 mg/kg exhibited significantly (p<0.05) lower lung-lesion scores than did animals treated with MICOTIL. Animals treated with either 1.25 mg/kg or 2.5 mg/kg of the pharmaceutical composition exhibited reductions in mean lung-lesion scores relative to calves treated with MICOTIL. 8 TABLE 7 Treatment Morbidity Rate Mortality Rate Lung-lesion Score Saline 12/20 (60%) 6/20 (30%) 24.3% (6.6 mL) MICOTIL 5/20 (25%) 1/20  (5%) 10.4% (10 mg/kg) Pharmaceutical 1/20  (5%) 0/20  (0%) 3.4% composition (1.25 mg/kg) Pharmaceutical 3/20 (15%) 0/20  (0%) 5.3% composition (2.5 mg/kg) Pharmaceutical 2/20 (10%) 0/20  (0%) 2.0% composition (5 mg/kg) The proportion of responders for each treatment was calculated by subtracting the number of mortalities and pulls from the initial number of animals per treatment. Responder rates are summarized in Table 8. Differences in the relative responder rates observed for the various treatments were similar to differences described above for morbidity rates. Clinically healthy calves were defined as those with an illness score of zero on day 14. In this study, only one of the saline controls was clinically healthy on day 14. A significantly (p<0.01) greater proportion of the animals treated with either MICOTIL or the pharmaceutical composition were observed to be clinically healthy on day 14 relative to the saline controls. Similarly, a greater proportion of the animals treated with any of the doses of the pharmaceutical composition were determined to be more clinically healthy than of those treated with MICOTIL. However, these differences were not statistically significant (p>0.05). 9 TABLE 8 Proportion of Clinically Treatment Responder Rates Healthy Animals Saline 8/20 (40%) 1/20  (5%) (6.6 mL) MICOTIL 15/20 (75%) 10/20 (50%) (10 mg/kg) Pharmaceutical composition 19/20 (95%) 14/20 (70%) (1.25 mg/kg) Pharmaceutical composition 17/20 (85%) 13/20 (65%) (2.5 mg/kg) Pharmaceutical composition 18/20 (90%) 14/20 (70%) (5 mg/kg) Table 9 summarizes the effects of metaphylactic treatment upon 7- and 14-day weight gains. Animals treated with either MICOTIL or the pharmaceutical composition exhibited significantly (p<0.05) increased average daily gains at both days 7 and 14 relative to the saline controls. Weight-gain responses for the various antibiotic treatments were similar. 10 TABLE 9 Average Daily Weight Gain Average Daily Weight Gain Treatment Over 7 Days (kg/day) Over 14 Days (kg/day) Saline 0.21 0.46 (6.6 mL) MICOTIL 1.15 0.94 (10 mg/kg) Pharmaceutical 1.09 1.20 composition (1.25 mg/kg) Pharmaceutical 0.96 1.00 composition (2.5 mg/kg) Pharmaceutical 1.55 1.25 composition (5 mg/kg) Acute injection sites were examined at 24 and 48 hours and assessments were made using the following scale: 0-no affected area (swelling/inflammation) observed; 1&equals;small-affected area (swelling/inflammation) less than 6 inches in diameter; 2&equals;medium-affected area (swelling/inflammation) 6-8 inches in diameter; 3&equals;large-affected area (swelling/inflammation) greater than 8 inches in diameter; 4&equals;extreme-affected area (swelling/inflammation) greater than 8 inches and/or radiating into the brisket or causing lameness. Grades were assigned depending upon the size and extent of the acute affected area. The 24 and 48 hour assessments are summarized in Table 10. In this study, the statistical significance of differences in the proportion of animals within each treatment scoring of greater than or equal to 2 at 24 hours post-injection was evaluated. There were no statistically significant differences between treatments. However, the number of abnormal injection sites was greater for animals treated with MICOTIL than for animals treated with the pharmaceutical composition. 11 TABLE 10 24 hr assessment 48 hr assessment Treatment 0 1 2 3 4 0 1 2 3 4 Saline (6.6 mL) 100% 0% 0% 0% 0% 100% 0% 0% 0% 0% MICOTIL (10 mg/kg) 80% 15% 5% 0% 0% 95% 5% 0% 0% 0% Pharmaceutical Composition 100% 0% 0% 0% 0% 100% 0% 0% 0% 0% (1.25 mg/kg) Pharmaceutical composition 100% 0% 0% 0% 0% 100% 0% 0% 0% 0% (2.5 mg/kg) Pharmaceutical composition 100% 0% 0% 0% 0% 100% 0% 0% 0% 0% (5 mg/kg) 
 EXAMPLE 8 From about 0.5 mL to about 2 mL of a pharmaceutical composition having a pH of 6.1 and containing a mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II in a ratio of from about 95% to about 99% of N-(n-propyl) isomer I and from about 1% to about 5% of N-(n-propyl) isomer II present in an amount of 50 mg per mL of the pharmaceutical composition, where 50 mg per mL is the “potency-actual” number; citric acid present in an amount of 15.00 mg per mL of the pharmaceutical composition; propylene glycol present in an amount of 250.13 mg per mL of the pharmaceutical composition; and water present in an amount of 734.43 mg per mL of the pharmaceutical composition were administered to pigs at a high risk for developing an Actinobacillus pleuropneumoniae infection. One hundred and thirty clinically healthy pigs having an average body weight of approximately 10 kg were purchased, identified with an ear tag and acclimated to the study site 2 days before the study began. On day -1, all animals were weighed and 100 animals were selected for consistency of body weight (about 10 kg) and lack of signs of clinical abnormalities. Selected animals (20 per treatment) were randomly assigned to treatment and sorted into individual pens. A group of 25 additional animals were randomly assigned as seeder pigs (5 per treatment). On day 0, animals were injected intramuscularly with a single dose of one of the following solutions: (1) about 1.5 mL of sterile 0.9% sodium chloride (saline); (2) about 0.5 mL of 25 mg/mL danofloxacin at a dose of 1.25 mg/kg of body weight; (3) about 0.5 mL of the pharmaceutical composition at a dose of 2.5 mg/kg of body weight; (4) about 1 mL the pharmaceutical composition at a dose of 5 mg/kg of body weight; or (5) about 2 mL of the pharmaceutical composition at a dose of 10 mg/kg of body weight. Only danofloxacin was re-administered on each of the following two days. All other treatments were administered in a single dose injection. Concurrently, on day 0, the 25 seeder pigs were challenged with 3 mL/nare of Actinobacillus pleuropneumonia challenge culture. Five infected seeder animals were added to each pen of 20 test animals. Test animals and seeder pigs were co-mingled. Seeder pigs that died during the study were removed from the pens. At 48 hours post-challenge, surviving seeder pigs were removed from treatment pens and euthanized. Temperatures and illness scores were recorded daily. Animals that died during the course of the experiment were necropsied. The lungs were removed and examined grossly for pneumonic lesions. An estimate of the percentage of affected lung tissue was determined and recorded. Animals with a lung-lesion score of greater than or equal to 5% were considered morbid. On day 7, all surviving animals were euthanized. Animals were necropsied and lungs removed and examined grossly for pneumonic lesions. Assessment of efficacy was determined based upon comparison of mean daily illness scores, temperatures and lung-lesion scores. Differences between treatments for mean daily rectal temperatures and illness scores were evaluated by a repeated-measures analysis of variance. A comparison between treatment groups of the proportion of animals within each group exhibiting an illness score of 0 (normal) or greater than or equal to 1 on day 7 were evaluated using Chi-Square analysis and Fisher's Exact test. Comparisons of morbidity (greater than or equal to 5% lung-lesion score) and mortality rates between treatment groups were performed using Chi-Square analysis and Fisher's Exact test. Eighty percent of the seeder pigs died of pneumonia within 24 hours of challenge, indicating adequate exposure of the test animals to the bacterial pathogen. Temperatures in the saline-treated pigs began to rise on day 1 post-exposure and remained significantly elevated throughout the duration of the study compared to the pharmaceutical composition- and danofloxacin-treated groups. Mean daily rectal temperatures for the pharmaceutical composition-treated groups at 5 mg/kg and 10 mg/kg were significantly (p<0.05) lower than the danofloxacin-treated pigs. Initial reductions in temperature occurred in pigs treated with the pharmaceutical composition at 2.5 mg/kg compared to pigs treated with danofloxacin. However, differences in mean daily rectal temperatures between these two treatments were not statistically significant (p>0.05). Statistically significant (p<0.05) elevations in mean daily illness scores were seen in the saline-treated pigs when compared to danofloxacin- and pharmaceutical composition-treated animals. However, there were no differences in mean daily illness scores between the danofloxacin- and pharmaceutical composition-treated pigs. A comparison between treatment groups of the proportion of animals within each group exhibiting an illness score of 0 (normal) or greater than or equal to 1 on day 7 showed no differences among any of the treated groups. Data summarizing the morbidity and mortality rates are presented in Table 11. Morbidity criteria were established from pigs having a mean lung-lesion score of greater than or equal to 5%. A statistically significant (p<0.05) increase in morbidity rate was seen in the saline control group in this study compared to the danofloxacin- and pharmaceutical composition-treated pigs. However, there were no differences in morbidity rates between the danofloxacin- and pharmaceutical composition-treated pigs. 12 TABLE 11 Treatment Proportion of Morbid Pigs Saline (1.5 mL) 13/20 (65%) Danofloxacin (1.25 mg/kg) 6/20 (30%) Pharmaceutical composition (2.5 mg/kg) 6/20 (30%) Pharmaceutical composition (5 mg/kg) 1/20  (5%) Pharmaceutical composition (10 mg/kg) 5/20 (25%) The effects of the various treatments upon mortality rates and lung-lesion scores are summarized in Table 12, below. The mean lung-lesion score for the saline control pigs was 22.2%. Pigs treated with danofloxacin and the pharmaceutical composition showed statistically significant (p<0.05) reductions in mean lung-lesion scores when compared to the saline controls. However, there were no statistically significant (p<0.05) differences in mean lung-lesion scores between the danofloxacin- and pharmaceutical composition-treated pigs. 13 TABLE 12 Treatment Mortality Mean Lung-lesion Score Saline 2/20 (10%) 22.2% (1.5 mL) Danofloxacin 0/20  (0%) 4.8% (1.25 mg/kg) Pharmaceutical composition 0/20  (0%) 4.6% (2.5 mg/kg) Pharmaceutical composition 0/20  (0%) 0.6% (5 mg/kg) Pharmaceutical composition 0/20  (0%) 3.1% (10 mg/kg) 
 EXAMPLE 9 From about 3 mL to about 6 mL of a pharmaceutical composition having a pH of 5.4 and containing an equilibrium mixture of N-(n-propyl) isomer I and N-(n-propyl) isomer II present in an amount of 100 mg per mL of the pharmaceutical composition, where 100 mg per mL is the potency-actual number; citric acid present in an amount of 0.1 mmol per mL of the pharmaceutical composition; hydrochloric acid present in an amount of 19.58 mg of the concentrated acid (36-38% by weight potency) per mL of the pharmaceutical composition; sodium hydroxide present in an amount of 0.09 mg of a 1.0 M sodium hydroxide solution per mL of the pharmaceutical composition; sodium hydroxide present in an amount of 0.09 mg of a 10 M sodium hydroxide solution per mL of the pharmaceutical composition; propylene glycol present in an amount of 501.25 mg per mL of the pharmaceutical composition; and water present in an amount of 418.20 mg per mL of the pharmaceutical composition were administered to calves challenged with 2 mL of a coccidia challenge culture containing 125,000 sporulated oocysts with a species percent count of 93% Eimeria bovis, 4% Eimeria auburnenis and 3% Eimeria zuernii coccidia oocysts. Sixty naive calves weighing approximately 110-125 kg were purchased from local dairies, weighed, identified by ear tag, and observed for general health assessments. Animals considered physically abnormal, undersized or moribund on arrival were excluded from the study. Calves were housed in five holding pens (12 animals/pen). Calves were held for 7 days prior to challenge in order to acclimate them to the facility. Prior to challenge, calves were excluded from the study at the discretion of the investigator. On days-6,-4 and -2 pre-challenge, fecal samples were obtained for semi-quantitative oocyst counts. On day-4 pre-challenge, oocysts, if present, were speciated. On day 8 post-arrival (study day 0), calves were inoculated orally with the Eimeria culture. Beginning on day 1, temperatures were determined and recorded at approximately the same time each day for the duration of the study. Attitude, hydration and fecal consistency scores were evaluated daily. Post-challenge, fecal samples were collected on days 2, 4, 6, 8 and 10. Oocysts were speciated on day 10 post-challenge. On day 10 post-challenge, fifty animals were randomly allotted to one of five treatment groups using a randomized block allotment. Treatments were equally represented in each pen. Animals were injected subcutaneously with a single dose of one of the following solutions: (1) about 4 mL of sterile 0.9% sodium chloride (saline); (2) about 4 mL of 300 mg/mL MICOTIL at a dose of 10 mg/kg of body weight; (3) about 6 mL of the pharmaceutical composition at a dose of 5 mg/kg of body weight; (4) about 3 mL of the pharmaceutical composition at a dose of 2.5 mg/kg of body weight; or animals were drenched dosed orally with (5) about 2 oz. of amprolium in a 9.6% oral solution at a dose of 10 mg/kg of body weight. Only amprolium was re-administered on each of the following four days. All other treatments were given in a single dose injection. Fecal samples were examined semi-quantitatively post-treatment for shedding of coccidia oocysts on days 12, 14, 16 and 18. Beginning on day 19 and continuing through day 28, daily fecal samples were evaluated for semi-quantitative counts. Speciation of shed oocysts was performed on days 19-21, 23, 26 and 28. Calves that died during the course of the study or that were euthanized due to a moribund condition associated with clinical coccidiosis were counted as mortalities. Mortalities were necropsied and gross findings were recorded. At the termination of the study on day 28, all remaining animals were weighed, euthanized and examined post-mortem. Assessment of drug efficacy was determined based upon analysis of mean daily clinical scores, temperature and oocyst shedding. Differences in clinical scores and temperature between different treatments were evaluated by repeated measures ANOVA. Differences in weight gain were determined by factorial ANOVA. Comparisons of mortality rates and oocyst shedding between treatments was performed using Chi-Square analysis and Fisher's Exact test. At day 19 post-challenge, oocyst shedding was detected. Mean daily rectal temperatures for each treatment remained in the normal range during the duration of the study. No significant differences (p>0.05) between treatment groups were detected. Clinical score assessments included scores for fecal consistency, hydration and attitude. Attitude and fecal scores indicated that calves treated with MICOTIL, amprolium or the pharmaceutical composition at either dose level responded favorably to treatment, compared to the saline control calves. Increases in fecal scores, hydration scores and attitude scores corresponded to the time of detectable shedding of oocysts (day 19). Animals treated with amprolium, MICOTIL, or the pharmaceutical composition at either dose level displayed statistically significant reductions (p<0.05) in mean daily fecal consistency scores compared to the saline treated calves. The increased fecal scores occurred 2-3 days prior to shedding of oocysts and remained elevated throughout the 28-day study. No differences were detected upon comparison of calves treated with amprolium, MICOTIL or pharmaceutical composition. Calves treated with amprolium displayed statistically significant reductions (p<0.05) in mean daily hydration scores compared to the saline treated calves. No differences in hydration scores were seen between calves treated with amprolium, MICOTIL or pharmaceutical composition. Treatment of calves with amprolium, MICOTIL or the pharmaceutical composition at either dose level resulted in significant reductions (p<0.05) in mean daily attitude scores compared to the saline controls. The differences in attitude scores were noted between the amprolium and saline treated calves at the time of peak oocyst shedding. Animals treated with MICOTIL or the pharmaceutical composition at either dose level exhibited numerical reductions in attitude scores relative to the saline control calves during the last seven days of the study. No significant differences (p>0.05) were seen between the MICOTIL, amprolium or pharmaceutical composition treatment groups. Mortality rates are summarized in Table 13. Five calves died due to coccidiosis in this study. Three calves died on day 23 post-challenge and two calves died on day 28 post-infection. Two animals died in each of the saline and MICOTIL treatment groups. One animal in the amprolium treated group died during the course of the study. There were no mortalities among animals treated with the pharmaceutical composition. There were no statistically significant (p>0.05) differences in mortality rates among the non-saline-treated animals. 14 TABLE 13 Treatment Mortality Saline (6 mL) 2/10 (20%) Amprolium (2 oz.) 1/10 (10%) MICOTIL (10 mg/kg) 2/10 (20%) Pharmaceutical composition 0/10 (0%) (2.5 mg/kg) Pharmaceutical composition 0/10 (0%) (5 mg/kg) Table 14 summarizes the effects of treatments upon weight gains. Positive average daily gains were seen in all treatment groups. Increases in weight gain were seen in calves treated with the pharmaceutical composition and with amprolium compared to animals in the saline and MICOTIL treatment groups. MICOTIL- and saline-treated animals responded similarly when assessing the 21-day average daily gains. However, no statistical differences in weight gain were seen among the non-saline-treated groups. 15 TABLE 14 21-day Average Daily Weight Gain Treatment (kg) Saline (6 mL) 0.30 Amprolium (2 oz.) 0.60 MICOTIL (10 mg/kg) 0.21 Pharmaceutical composition 0.45 (2.5 mg/kg) Pharmaceutical composition 0.44 (5 mg/kg) Eimeria oocyst shedding was monitored prior to challenge and post-challenge. Oocyst shedding was first detectable on day 19 post-challenge. In this study, statistically significant (p<0.05) increases in oocyst shedding were seen in saline-treated animals when compared to the MICOTIL-, amprolium- and pharmaceutical composition-treated animals. Also, MICOTIL-treated animals displayed statistically significant (p<0.05) increases oocyst shedding compared to animals treated with amprolium. However, no statistically significant (p>0.05) differences in oocyst shedding were seen when comparing the MICOTIL- and amprolium-treated calves to calves treated with either dose of the pharmaceutical composition. In this study, 40-100% of the animals in the saline control group were consistently shedding oocysts on days 19, 20, 21, 23, 26 and 28 post-challenge. Animals treated with amprolium, MICOTIL or the pharmaceutical composition displayed decreased oocyst shedding compared to the saline controls. In this study, E. bovis accounted for approximately 60-100% of the shed oocysts per sample. E. auburnenis and E. zuernii accounted for approximately 10-40% of the shed oocysts per sample. There was an apparent increase in the shedding of E. zuemii oocysts on day 28 post-challenge, which corresponded to a decrease in shedding of E. bovis oocysts. However, over the entire monitored shedding period, none of the compounds tested appeared to significantly alter the speciation profiles of the shed oocysts. At necropsy, the majority of the animals displayed gross pathology consistent with a moderate to severe coccidial infection. In this study, calves from all treatment groups showed signs of hemorrhagic ilietis and colitis. Fourteen percent of the calves in this study (7/50) displayed no gross pathology at necropsy. However, the shed oocysts from calves in each of the treatment groups suggested some level of coccidia infection in these animals. The present invention is not to be limited in scope by the specific embodiments disclosed in the Examples, which are intended as illustrations of a few aspects of the invention. Any embodiments which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the appended claims. All references disclosed herein are hereby incorporated by reference in their entirety.