bromhexine on chicken

Bromhexine and its main metabolite, ambroxol, (trans-4-[(2-amino-3.5-dibromobenzil) amino] cyclohexanol hydrochloride) have been widely used therapeutically in the treatment of lung diseases because of their mucolytic properties, as well as their ability to stimulate the release of surfactants substances, standardize the production of mucus and facilitate expectoration. Recent studies have shown they have antioxidant and anti-inflammatory benefits which have been attributed to their property of inactivating free radicals; also it has been shown in vitro that they stimulate cytokine release (Gibbs et al., 1999), which promotes neutrophils chemotaxis and sodium absorption by the lung epithelium.

Bromhexine can be administered in combination with antimicrobial agents in the treatment of respiratory infections as it causes disruption on the mucopolysaccharides of bronchial secretion, thus improving bronchial penetration of antimicrobial drugs. Escoula et al. (1981) found that after intramuscular administration of bromhexine, an increase in spiramycin bioavailability in nasal secretions of bovines was achieved. This remarkable effect stimulated administration of combinations among bromhexine and antimicrobials for the treatment of various infectious diseases of the respiratory tract. Later, Bergogne (1985) and Martin et al. (1993) confirmed the same behavior when finding that bromhexine hydrochloride increased oxytetracycline concentration inside secreted mucus.

Bromhexine is absorbed reaching maximum concentration in plasma after one hour (Tmax), with an absorption half-life of 25 minutes. It undergoes a first pass of 75 to 80% and its absolute bioavailability is if 20 to 25%. Bromhexine is bound to plasma proteins (95 to 99%), a high volume of distribution. Its accumulation is higher in the lungs than in plasma. Disposal half-life may be about 13 to 40 hours, depending on the species.

Use of bromhexine is approved by intramuscular or oral administration in calves, pigs and chickens at 0.5 mg/kg/day for 5 consecutive days. Though EMEA Committee for veterinary products did not establish an MRL for bromhexine (EMEA, 1998a), a withdrawal of 24-48 h after last administration is recommended, given a toxicological ADI of 0.3 mg/person. Its use in hens producing eggs for human consumption is not recommended.

Enrofloxacin is an antimicrobial agent developed in late 1980 to be used exclusively in veterinary medicine. It is a second generation fluoroquinolone with bactericidal activity against Enterobacteriaceae, other Gram-negative bacteria and some Gram-positive cocci (Martinez et al. 2006; Otero et al., 2001a, 2001b). Fluoroquinolones damage bacterial DNA and alter the super-coiling due to inhibition of DNAgyrase activity (Prescott, 2000). In some bacterial species such as E. coli, the major site of action is at DNAgyrase while in others, such as S. aureus, is at topoisomerase IV. As these enzymes have somewhat different functions, it is likely that bacteria differ in their responses according to which is the point of action.

Its therapeutic and prophylactic use is approved in chickens and other domestic species. Infectious processes caused by mycoplasma, colibacilli and Pasteurella, are often treated with enrofloxacin (Martinez et al., 2006).

After oral application, enrofloxacin is well absorbed and distributed at tissue level, to be excreted in urine and feces at high concentrations (Prescott, 2000). It is metabolized in the liver, generating its main metabolite, ciprofloxacin (EMEA, 1998b). Ciprofloxacin also has bactericidal activity and is a compound approved for use in human medicine. In most animal species, enrofloxacin has a high volume of distribution, being much higher than that achieved by beta-lactams and aminoglycosides. It is mainly concentrated in saliva, nasal secretion, mucosa, epithelium and bronchial secretion, as well as in the liver and urinary tract. Penetrates well into lung tissue, lining fluid and alveolar macrophages, resulting in higher concentrations than in serum.

Based on above, our hypothesis is that concentrations of enrofloxacin in bronchial lavage of broiler chickens treated with an enrofloxacin-bromhexine combination are superior to those determined in broilers treated with enrofloxacin alone. For this, we developed a model for washing birds' bronchi and air sacs in order to determine their antimicrobial secretions levels.

120 chickens of 4 weeks of age were used. Animals were conventionally kept and fed, with access to balanced food and water "ad libitum". Food was controlled throughout the trial period, to be certain there were no substances with antimicrobial power. Experimental animals were divided into two groups:

Group 1: Comprised of 60 broilers that were treated for 5 days with a formulation based on enrofloxacin-bromhexine (Bromeflox^®, Cevasa S.A.) at 10 mg/kg and 0.5 mg/kg of live weight, respectively, orally administrated with drinking water.

Group 2: Comprised of 60 broilers that were treated for 5 days with a formulation based on enrofloxacin without bromhexine through drinking water, at the same dosage as group 1.

Animals were sacrificed by pentobarbital anesthesia and bleeding. At the time of slaughter we proceeded to trachea channeling, air sacs emptying and two washings of bronchi and air bags with a total volume of 40 mL of saline solution in each animal. Animals were sacrificed in groups of 8 at the following intervals: 24, 72, 120 h (during treatment) and 126, 132, 138 and 144 hours post-first drug administration, this is, 6, 12, 18 and 24 h post-last dosing.

Samples were perfectly labeled and stored at -20°C until microbiological testing.

Equivalent concentrations of enrofloxacin in samples extracted were determined by microbiological method (cylinder plate technique) using Bacillus subtilis BGA Merck (10⁷ CFU/mL) as control strain. We placed 100 μL/120mL of agar (8.3 x 10 ³ CFU/mL). We used plates of 25 x 25 charged with 120 mL of culture medium inoculated in monolayer. We put 49 stainless steel cylinders in each plate, using a previously established design. Standards (200 uL) were seeded in duplicate and each problem sample (200 uL) was seeded in quadruplicate. Quantification methodology of enrofloxacin in the various fluids tested was validated by analyzing the following parameters: linearity, specificity, precision, accuracy and quantification limit for each biological matrix.

Microbiological method was linear between 0.04 and 0.62 mg/mL enrofloxacin equivalents. Quantification limit obtained was 0.08 mg/mL in plasma and detection was 0.04 mg/mL. Percentage recovery was 84.62 ± 10.47% with a CV of 12.37%.

In Fig.1 we can see average concentrations obtained in each fluid after administration of both formulations.

ENR maximum plasma concentration obtained after administration of combined ENR/Bromhexine was higher than that achieved after administration of ENR alone (0.77 mg/mL vs 0.63 mg/mL, P = 0.0332) and was achieved later (Tmax 105.75 vs 90 h). Though no statistically significant difference was found when comparing the areas under plasma concentration curve related to time (AUC) between both groups, a significant difference were found in the mean residence time (MRT 88.14 vs 80.66 h, P = 0.0214). In bronchial lavage major differences were found after administration of Bromeflox combination, as we obtained a greater Cmax (0.90 vs 0.49 mg/mL,  P= 0.0344) more rapidly (Tmax of 24 h vs 58.29 h, P = 0.0082), with greater bioavailability (AUC 59.50 vs 36.03 mg* h/mL, P = 0.0457), but the mean residence time was lower (MRT 55.10 vs. 81.66 h, P= 0.0139). Relevant pharmacokinetic parameters obtained after analyzing plasma disposition curves and at bronchial lavage, together with statistical comparison between the two formulations, are presented in Table I. Recall that fluoroquinolones belong to the group of antimicrobial with concentration-dependent bactericidal action, so the main efficacy predictor is the parameter PK/PD Cmax/MIC> 8-10. Reported MIC₉₀ for the majority of avian pathogens is 0.06 µg/mL (Sanjib et al., 2005), so in this study we would obtain a Cmax/MIC₉₀ ratio of 15, i.e. the addition of bromhexine would maximize ENR effectiveness.

Figure 1. Enrofloxacin plasma profile and in bronchial lavage after administration of bromhexine-enrofloxacin combination and after administration of enrofloxacin only with respect to time.

Table 1. Statistical comparison between plasma pharmacokinetic parameters and in bronchial lavage obtained after administration of ENR alone vs Bromeflox ^® (Test t)

Based on results obtained we can conclude that Bromeflox is a formulation with certain advantages over formulas based on enrofloxacin alone, as bromhexine facilitates antimicrobial penetration into the airways, achieving higher concentrations in a faster way. Addition of bromhexine can achieve a Cmax/MIC ratio greater than 10, thus maximizing ENR efficiency.

Bergogne B, Berthelot G, Kafe HP, Doournovo P. 1985. Influence of a fluidifying agent (bromhexine) on the penetration of antibiotics into respiratory secretions. Int. J. Clin. Pharmacol. Res. 5(3):341-344.

EMEA. 1998a. The European Agency for the Evaluation of Medicinal Products, Veterinary Medicines Evaluation Unit. EMEA/MRL/503/98-FINAL URL:http://www.ema.europa.eu/docs/
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EMEA. 1998b. The European Agency for the Evaluation of Medicinal Products, Veterinary Medicines Evaluation Unit. URL:http://www.ema.europa.eu/docs/en_GB/document_library/
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Otero JL, Mestorino N, Errecalde J. 2001b. Enrofloxacina: Una Fluoroquinolona de uso exclusivo en veterinaria. Parte II: Farmacocinética y toxicidad. Analecta Veterinaria 21:42-49.

Prescott JF. 2000. In Antimicrobial Therapy in Veterinary Medicine, 3^rd edn. Eds Prescott J, Baggot D, Walter R. Iowa State University Press, Ames, IA.

Sanjib I, Chandana, Pritam M Mohan B. 2005. Pharmacokinetics studies of enrofloxacin in Yak after intramuscular administration. Ir.J.Pharmacol.Therap. 4:91-94.