NEOVE APRAMYCIN SOLUBLE POWDER ANDAPRABIOPHAR
Ashraf El-Komy & Mohamed Aboubakr
1 Professor and Head of Pharmacology Department, Faculty of Veterinary Medicine, Benha
University, Egypt.
2 Assistant Professor of Pharmacology, Faculty of Veterinary Medicine, Benha University,
Egypt.
ABSTRACT
The present study was designed to assess the comparative bio-
equivalence of Neove Apramycin Soluble Powder® and
Aprabiophar® in healthy broiler chicken after intramuscular injection
(IM) of both products in a dose of 10 mg apramycin/kg b.wt. Twenty
four broiler chickens were divided into two groups. The first group was
designed to study the pharmacokinetics of Neove Apramycin Soluble
Powder®, while the 2nd group was designed to study the
pharmacokinetics of Aprabiophar®. Each broiler chicken in both
groups was IM injected with 10 mg apramycin/kg b.wt. Blood samples
were obtained from the wing vein and collected immediately before
and at 0.08, 0.16, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours after a single IM
injection. The disposition kinetics of Neove Apramycin Soluble Powder® and
Aprabiophar® following IM injection of 10 mg apramycin /kg b.wt, revealed that the
maximum blood concentration of apramycin [Cmax] were 9.95 and 9.67 μg/ml and attained at
[tmax] of 2.33 and 2.35 hours, respectively. In conclusion: Aprabiophar® is bioequivalent to
Neove Apramycin Soluble Powder® since the ratios of Cmax, AUC0-24 and AUC0-∞ (T/R) was
0.97, 0.96 and 0.96 respectively. These are within the bioequivalence acceptance range.
Aprabiophar® and Neove Apramycin Soluble Powder® are therefore bioequivalent and
interchangeable.
INTRODUCTION
The bioavailability and bioequivalence studies play an important role in determining therapeutic efficacy to register the generic drug products according to the Food and Drug Administration (FDA) regulations (Chen et al., 2001). Bioavailability is defined as the rate and extent to which an active drug ingredient is absorbed and becomes available at the site of drug action. In case of bioequivalence it is defined as statistically equivalent bioavailability between two products at the same molar dose of the therapeutic moiety under similar experimental conditions (Chen et al., 2001; Toutain and Bousquet-Melou, 2004). The drug products are said to be bioequivalent if they are pharmaceutical equivalents or pharmaceutical alternatives and if their rate and extent of absorption do not show a significant differences statistically according to the FDA regulations (Chen et al., 2001). Apramycin is a bactericidal aminocyclitol broadspectrum antibiotic primarily prescribed for medication of systemic and enteric infections caused by Gram-negative bacteria in a variety of animals species. It acts by irreversible binding to the 30S ribosomal subunit thereby inhibiting protein synthesis. It is active against many Gram-negative bacteria (E. coli, Pseudomonas spp., Salmonella spp., Klebsiella spp., Proteus spp., Pasteurella spp., Treponema hyodysenteriae and Bordetella bronchiseptica). In addition, it is also active
Pathogenic Escherichia coli cause intestinal and extra-intestinal diseases in human and
animals. Avian pathogenic Escherichia coli cause a variety of infections in poultry, generally
referred as colibacillosis (Barnes et al, 2003). One of the most frequently encountered
clinical manifestations of colibacillosis in poultry is respiratory origin colisepticemia, which
is one of the principal causes of economic loss in the poultry industry. In poultry flocks,
antibacterial agents are widely used to control E. coli and Salmonella infections. However,
the extensive use of antibacterial agents enhances the chance for selection of resistant
bacterial isolates (Schwarz et al., 2001; Khoshkhoo and Peighambari, 2005).
Neomycin is the general term for a mixture of antibiotics obtained from cultures of
Streptomyces fradiae (Waksman and Lechevalier, 1949). Two active components, which
are isomeric, of the mixture have been identified as neomycin B and neomycin C. The
neomycins belong to the aminoglycoside class of antibiotics, form salts with organic or
inorganic acids, and are sold, usually as a sulfate salt, as neomycin sulfate. Neomycin is
water soluble and is stable in animal feeds. Neomycin is also classed as broad spectrum as it
is effective against Gram-positive and Gram-negative bacterial species (Waksman et al.,
1950). Neomycin is an aminoglycoside antibiotic. Neomycin has a mechanism of action and
spectrum of activity (primarily gram negative aerobes) similar to the other aminoglycosides,
but in comparison to either gentamicin or amikacin, it is significantly less effective against
several species of gram negative organisms, including strains of Klebsiella, E. coli and
Pseudomonas. Neomycin is poorly absorbed from the gastrointestinal tract of birds and a low
acute toxicity after oral administration. It is often added to medicated poultry feeds.
Neomycin has been widely used in the animal industry (Kitchen and Waksman, 1955) since
its introduction in 1949. Neomycin use continues in cattle, swine, and goats. Neomycin was
removed from use in turkeys and chickens after a National Academy of Science/National Research Council Drug Efficacy Study Implementation Program review in 1971 reported a
lack of controlled studies to support efficacy and safety in these species.
Administration of antibiotics is the most common and fast way for treating of APEC infection
in broiler chickens, but the major problem associated with the treatment is the development
of drug resistant strains to the most commonly used drugs (Vandemaele et al., 2002). Hence,
it’s necessary to search for new therapeutic agents to control this infection and to define the
most effective route of administration.
Therefore, the purpose of the present study was to determine the efficacy of neomycin
(Neobiotic® and Anticin)® in the treatment of E.coli infection in the broiler chickens after
oral administration of 11 mg neomycin sulphate/kg b.wt.
The aim of this study is to evaluate bioequivalence of two apramycin formulation (Neove
Apramycin Soluble Powder® and Aprabiophar®) after IM injection of a single dose in
broiler chickens
against Staphylococcus spp.
and Mycoplasma spp (Ryden and Moore, 1977; Walton, 1978; Theys et al., 1983; Ziv et
al., 1985; Freidlin et al., 1985). It is generally poorly absorbed from gastrointestinal tract of
animals (Thomson et al., 1991) and active in vitro against Salmonella spp. and Escherichia
coli strains that are resistant to neomycin and dihydrostreptomycin (Theys et al., 1983; Ziv
et al., 1985; Freidlin et al., 1985). Oral and parenteral preparations of apramycin are
commercially available in many countries.
The pharmacokinetic profile of apramycin has been extensively investigated in many animal
species; in turkey (Freidlin et al., 1985), in calves (Ziv et al., 1985; Shiha, 1987), in sheep,
rabbits, chickens and pigeons (Lashev et al., 1992), in Japanese quail (Lashev and
Mihailov, 1994), in lactating cows, ewes and goats, (Ziv et al., 1985), in chickens (Donos,
1982; Afifi and Ramadan, 1997; Lashev, 1998), in turkeys roosters and hens (Haritova et
al., 2004). The large-scale use of the orally administered aminocyclitols such as apramycin
for treatment of enteric infectious diseases caused by Salmonella spp. and E. coli in poultry.
MATERIALS AND METHODS - Drugs
Neove Apramycin Soluble Powder® was obtained from Neove Pharma Company, Australia (it was used as reference product) and Aprabiophar® was obtained from Boston Company, Egypt (it was used as test product). Both products contains 550 mg/g apramycin (as sulphate). 2.2. Broiler Chickens and Experimental Design Twenty four healthy broiler chickens (30 days old and weighing 1.60 – 1.85 kg) were obtained from Benha private poultry farm, Egypt. They were kept individually in cages, within a ventilated, heated room (20˚C), and 14 hours of day light. They received a standard commercial ration free from any antibiotics before starting the experiment to insure complete clearance of any anti-bacterial substances from their bodies. Water was offered ad-libitum. 2.3. Bioequivalence Study Broiler chickens were used to study the bio-equivalence of Neove Apramycin Soluble Powder® and Aprabiophar® after IM injection. Broiler chickens were divided into two groups. The 1st group (12 broiler chickens) was used to study the pharmacokinetics of Neove Apramycin Soluble Powder®. The 2nd group (12 broiler chickens) was used to study the pharmacokinetics of Aprabiophar®. Broiler chickens in the 1st group were IM injected in right thigh muscle with Neove Apramycin Soluble Powder® in a dose of 10 mg apramycin/kg b.wt, while broiler chickens in the 2nd group were IM injected in right thigh muscle with Aprabiophar® in a dose of 10 mg apramycin.
Blood Samples
Blood samples were obtained from the wing vein (1 ml) and collected in test tubes
immediately before and at 0.08, 0.16, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after a single IM
injection (groups 1 and 2). Samples were centrifuged at 3000 rpm for 10 minutes and the
obtained sera were used for the estimation of apramycin concentration. The serum samples
were stored at −20˚C until drug assay.
Analytical Procedure
The concentration of apramycin in serum samples was estimated by a standard
microbiological assay using Bacillus subtilis ATCC 6633 as test micro-organism (Bennett et
al., 1966). The medium was prepared by dissolving 9.5 g Mueller–Hinton agar in 250 ml
distilled water in a 0.5 l flat-bottomed flask, which was autoclaved for 20 min. After cooling
to 50oC in a water bath, 0.4 ml of the diluted suspension of reference organism was added to
the media. Six wells, 8 mm in diameter were cut at equal distances in standard Petri dishes
containing 25 ml seeded agar. The wells were filled with 100 μl of either the test samples or
apramycin standards. The plates were kept at room temperature for 2 h before being
incubated at 37oC for 18 h. Zones of inhibition were measured using micrometers, and the
apramycin concentrations in the test samples were calculated from the standard curve.
Negative control samples showed no bacterial inhibition, indicating no intrinsic antibacterial
activity of the samples.
Pharmacokinetics analysis
Serum concentrations of apramycin versus time data obtained during the study were utilized
for calculating various pharmacokinetic variables using a compartmental and non-
compartmental analysis using computerized program, WinNonline 4.1 (Pharsight, USA).
The peak concentrations, Cmax and time to peak, Tmax were obtained from the serum
concentration-time data directly. The areas under the serum concentration of apramycin time
curves from time 0 to the last sample collected (AUC0-24) were calculated using linear
trapezoidal method (Baggot, 2001). While AUC0-∞ was derived from AUC0-24 + AUC24-∞,
where AUC24-∞ = C24/ß. For bioequivalence evaluation, the ratios of Cmax (T/R), AUC0-24
(T/R) and AUC0-∞ (T/R) were calculated. Values within the bioequivalence acceptable range
at 90% confidence interval, 0.80 – 1.25 were considered for accepting the null hypothesis of
bioequivalence between the reference and the test brands (EMEA, 2002, 2006).
RESULTS
The mean serum concentrations of apramycin in Neove Apramycin Soluble Powder® and
Aprabiophar® following IM injection of 10 mg apramycin/kg b.wt, in broiler chickens are
shown in (Table 1 and Figure 1).
The mean pharmacokinetic parameters of apramycin in Neove Apramycin Soluble Powder®
and Aprabiophar® after IM injection of 10 mg apramycin/kg b.wt, in broiler chickens are
shown in (Table 2).
The disposition kinetics of apramycin in Neove Apramycin Soluble Powder® and
Aprabiophar® following IM injection of 10 mg apramycin/kg b.wt, revealed that the
maximum blood concentration [Cmax] were 9.95 and 9.67 μg/ml and attained at [Tmax] of 2.33
and 2.35 hours, respectively. The mean ratio of Cmax and AUC of the reference and tested
formulations were within bioequivalence range and summarized in Table 3. All the
experimental chickens remained healthy during and after the study.