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ISSN : 1976-7447(Print)
ISSN : 2287-7363(Online)
Journal of Biomedical Research Vol.14 No.1 pp.8-12
DOI : https://doi.org/10.12729/jbr.2013.14.1.8

Comparison of blood concentration for oral administration of micronized and non-micronized amoxicillin in Sprague-Dawley rats

Hu Jang Lee6*, Yong-Jae Ryu1#, Chun-Nam Cha2#, Eun-Ah Yu3, Eun-Kee Park4, Beom Jun Lee5, Suk Kim6
6Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Chinju 600-701, Korea
1Food and Drug Analysis Section, Chungcheongbuk-do Institute of Health and Environment, Cheongwon 363-951, Korea
2Engineering Research Institute, Department of Industrial Systems Engineering, Gyeongsang National University, Chinju 600-701, Korea
3Tongyeong National Quarantine Station, Ministry of Health & Welfare, Tongyeong 650-110, Korea
4Department of Medical Humanities and Social Medicine, College of Medicine, Kosin University, Busan 602-703, Korea
5College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Korea
Received 8 Jan. 2013, Revised 6 Mar. 2013, Accepted 11 Mar. 2013

Abstract

Amoxicillin, a well-known antibiotic, has a broad spectrum against gram-negative and gram-positive bacteria. This experiment was conducted in order to investigate the effect of micronized and non-micronized amoxicillin prepared using different comminution techniques on change in blood concentration of rats. Forty adult male Sprague Dawley rats (6~7 weeks of age, body weight 128.3 ± 10.7 g) were randomly allocated to two treatment groups: micronized amoxicillin (MA) group treated with micronized amoxicillin trihydrate powder (particle size, over 90% of 10 μm), non-micronized amoxicillin (NMA) group treated with non-micronized amoxicillin trihydrate powder (particle size, over 70% of 100 μm), given 480 mg/kg body weight once daily for four days. The results showed a significant increase in serum concentration in the MA group on days 3 and 4, compared to the NMA group (P<0.05). In particular, serum concentration of the MA group on day 4 was increased almost two times that of the NMA group. The results indicate that due to the increase of the drug’s oral bioavailability, higher serum concentration would be achieved with the micronized amoxicillin trihydrate than with the non-micronized drug.

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Introduction

 Amoxicillin is a β-lactam antibiotic that has a broad in vitro spectrum against gram-negative and gram-positive bacteria, as well as good absorption and penetration into tissues [1-3]. Also, various hydrated forms of amoxicillin, including monohydrate, dihydrate, and trihydrate, have been reported and amoxicillin which the trihydrate is the most stable hydrated form [4, 5]. Amoxicillin trihydrate show high activity against the major respiratory tract pathogens in pigs and against some digestive tract pathogens [6].

 Moreover, amoxicillin achieves high lung tissue concentration and has good penetration to bronchial secretions and bronchial mucosa both in healthy pigs and infected pigs [7]. The main routes of administration of antibiotics are intravenous and oral route. Practically, administration of amoxicillin formulation in feed and drinking water is widely used to control bacterial infection in pigs. However, water medication appears to be less suitable than in-feed medication because the stability of the drug in aqueous solution is poor and medicated water must be freshly prepared [8]. The oral administration route is especially useful because of its efficacy and easy handling. However, the oral bioavailability of amoxicillin in pigs and rats was 28~33 and 44%, respectively [7, 8].

In general, micronization of a compound enhances the dissolution rate due to the increase in surface are available to the dissolving medium, and the increase of the dissolution rate usually results in more rapid and complete absorption [9]. 

 To achieve the increase of the bioavailability, a new micronized amoxicillin (≤ 10 μm) for in-feed medication was developed using the fluidized-bed jet milling [10]. The fluidized-bed jet milling is an effective tool for the micronization of pharmaceutical compounds, because it is not limited for feed size, heat sensitivity of material, or abrasive characteristics. Moreover, this can be controlled particle sizes and distributions [10-12].

 Up to now, no studies have been done on the effect of amoxicillin prepared by using different comminution techniques on change in the blood concentration of rats. In this study, the time course alteration of amoxicillin concentration in blood was observed after oral administration of micronized and non-micronized amoxicillin on rats.

Materials and Methods

Animals and experimental design

 Forty adult male specific and pathogen-free Sprague Dawley (SD) rats, of age 6 to 7 weeks old, with body weight between 123 to 132 g were used in the study. The animals were obtained from SAMTAKO Bio Korea Co., Ltd. (Yongin, Korea). The care and handling of the experimental animals are according to the test guidelines issued by the National Veterinary Research and Quarantine Service [13]. The study design and the use of the experimental animals were reviewed and approved by the Institutional Animal Care and Use Committee of Gyeongsang National University (GNU-LA-2013-03) in Korea.

 The experimental animals were housed individually in cages for the duration of the study. All the experimental animals were kept in one room and maintained under temperature and humidity of 27 ± 2°C and 65.85 ± 6.76%, respectively. Fluorescent lighting was turned on at 06:00 hr and off at 18:00 hr to provide a 12-hr light/dark cycle. SD rats were fed with certified rodent food (SAMTAKO Bio Korea, Korea) and drinking water was available ad libitum throughout the study. The experimental animals were acclimatized for 7 days before the commencement of the study, and they were labeled appropriately.

Drugs and treatments

 The reference product non-micronized amoxicillin trihydrate powder (NMA, Amocillin, 20%) and the micronized amoxicillin trihydrate powder (MA, Amophen, 20%) were supplied by Dae Han New Pharm (Seoul, Korea). Particle size was determined according to Evans [14], and micronized and non-micronized amoxicillin trihydrate powders were composed with over 90% of 10 μm and over 70% of 100 μm, respectively.

 The SD rats were randomly divided into two experimental groups (n=20 each). According to the manufacture’s guidelines, micronized and non-micronized amoxicillin trihydrate at the concentration of 480 mg/kg body weight were orally administered daily one time to both control and experimental group for four days.

 At every 24 hours after treatment, five rats of each group were sacrificed by withdrawing blood from the heart under diethyl ether anesthesia. Serum was obtained by centrifugation at 1,000 ×g, for 15 min at 4°C and stored at −80°C pending assay.

Serum amoxicillin assay

 Serum amoxicillin trihydrate was measured by the high-performance liquid chromatography (HPLC) with UV detection according to a modified procedure derived from that of Matar [15]. After addition of 20 μl internal standard (cefadroxil, 40 μg/ml) to 200 μl serum and centrifugation (15,000 ×g, 15 min), the superficial fluid was analyzed in isocratic mode with a Shiseido Capcell Pak UG 120 C18  (5 μm particle size, 250 × 4.6 mm I.D.) (Kyoto, CA, Japan). The mobile phase was 75 mM potassium dihydrogen phosphate buffer solution with methanol (90:10, v/v) (pH 3.0). Detection was done by ultraviolet radiation at 228 nm, and retention time was about 11.5 min. The detection limit was 0.05 mg/L with a coefficient of variation of 5%.

Standard curve and recovery rate

 A working standard solution of amoxicillin (100 μg/ml was added in appropriate volumes to seven volumetric flasks (10-ml capacity) and volumes completed to 10-ml with drug-free serum of rat to provide calibration standards of 0.5, 1.0, 2.0, 4.0, 5.0, 10.0 and 20.0 μg/ ml. Aliquots of these standards were distributed into 1.5 ml Eppendorf polypropylene micro-centrifuge tubes in volumes of 50 μl and stored at −70°C.

 Amoxicillin recoveries from serum of rat were determined by spiking drug-free serum of rat with known amounts of the drug to achieve amoxicillin concentrations of 1, 3 and 5 μg/ml. The spiked samples were processed and analyzed with the above procedure. The relative recovery was calculated by comparing the concentrations obtained from the drug-supplemented serum with actual added amounts. The recovery of spiking drug samples was obtained by comparing the observed peak areas obtained from the processed standard samples to direct injections of standard aqueous solutions prepared at concentrations which represented 100% recovery.

Statistical analysis

 Data were analyzed by one-way analysis of variance (ANOVA) followed by two-tailed Student’s t-test when the ANOVA yielded statistically significant differences (P<0.05). All the statistical analyses were performed using the Stat View J-5.0 program (SAS Institute Inc., Cary, NC, USA) on a Macintosh computer. All data were expressed as the mean ± standard deviation (SD).

Results

Calibration curve of amoxicillin

 The working standard calibration curve was drawn by plotting the known amoxicillin concentrations (0.5, 1.0, 2.0, 5.0, 10.0, 20.0 μg/ml) prepared from the stock solution by dilution against the average peak area (Fig. 1). The calibration curve showed good linearity (r2>0.9995) within the test ranges. The limit of detection (LOD) for amoxicillin (S/N = 3:1) was about 0.05 mg/L. The extraction procedure of amoxicillin from serum of rats used in this study was a modification of that described for purification from human plasma by Matar [15]. The HPLC method adopted proved to be very sensitive and suitable for the quantitative analysis of amoxicillin in serum of SD rat.

Fig. 1. The amoxicillin calibration curve from 0.05 to 20 μg/ml or amoxicillin. Plotting the peak area vs. all measured sequences gave a regression line: y = 9,499x + 1,607.3 with a linear correlation coefficient of 0.99952.

Recovery rate

 Recoveries of amoxicillin from fortified serum were shown in Table 1. A recovery test was used to evaluate the accuracy of this method. The test was performed by adding standard stock of amoxicillin into drug-free serum of rat with the concentration of 0.5, 2.5, and 5 μg/ml. The mixture was extracted and analyzed using the aforementioned method in triplicate. The average recoveries were estimated by the following formula: recovery (%) = (amoxicillin found / amoxicillin added to drug-free serum sample) × 100. The overall recoveries laid between 86.6% and 89.7%. HPLC-UV chromatogram of rat serum spiked with 5 μg/ml amoxicillin was shown in Fig. 2.

Table 1. Recoveries of amoxicillin from fortified drug-free serum samples

Fig. 2. Chromatogram of rat serum spiked with 5 μg/ml amoxicillin in HPLC with UV detection at 228 nm wavelength of UV radiation wavelength. HPLC condition; column, Shiseido Capcell Pak UG 120 C18 (5 μm particle size, 250 × 4.6 mm I.D.), mobile phase, 75 mM potassium dihydrogen phosphate-methanol (90:10). The meaning of ISTD abbreviation is internal standard, and cefadroxil was used as ISTD in this study.

Serum amoxicillin assay

 Table 2 shows changes in serum amoxicillin concentrations in MA and NMA on days 1, 2, 3, and 4 after administration. The concentration of serum amoxicillin in MA significantly increased compared with that in NMA at day 3 and 4 after administration. Especially, the level of serum amoxicillin in SD rats administered with MA at day 4 was almost two times higher level than that in SD rats treated with NMA.

Table 2. Changes of serum amoxicillin concentration in rat during the experimental period

Discussion

 Amoxicillin is bactericidal in action and acts through the inhibition of biosynthesis of cell wall mucopeptide of susceptible organisms, and is usually orally administered because it is well absorbed from the gastro-intestinal tract in animals [16]. However, the oral bioavailability of amoxicillin in pigs was reported about 25% [17].

 Micronization technique is a very promising approach to enhance dissolution rate and bioavailability of poor water-soluble drugs. A reduction in particle size improves the dissolution rate as a result of increased surface area in contact with the aqueous medium [18].

 The present study was conducted to investigate the serum concentration of amoxicillin trihydrate in SD rat orally administered with micronized and non-micronized amoxicillin trihydrate. After administration of micronized and non-micronized amoxicillin trihydrate to SD rats, serum concentration of amoxicillin trihydrate was analyzed by HPLC. Li et al. [19] was validated a HPLC-UV detector for the simultaneous determination of amoxicillin and ranitidine in rat plasma and the recovery of amoxicillin at the level of 0.5 and 5.0 μg/ml was 88.0 and 87.4%, respectively. In addition, Du et al. [20] developed a sensitive and simple HPLC and analyzed amoxicillin in mouse serum, and then the mean recoveries of amoxicillin were 87 %. In the present study, the recoveries were slightly higher or similar with the results of the above researches.

Chang et al. [21] investigated the micronization of sulfamethoxazole using the supercritical anti-solvent precipitation method and reported that the dissolution rate of the micronized sulfamethoxazole in a simulated intestinal fluid was significantly enhanced compared to that of non-micronized sulfamethoxazole. Furthermore, Kraml et al. [22] observed that a 0.5-g dose of micronized griseofulvin, antifungal antibiotic produced serum levels nearly equivalent to those obtained following a 1.0-g dose of non-micronized griseofulvin. In addition, Voelker and Hammer [23] reported that the micronized aspirin tablet was a very rapid dissolving aspirin formulation showing a substantial improvement of time to maximum plasma concentration compared to regular aspirin tablets. In the present study, the concentration of amoxicillin in MA group treated micronized amoxicillin at day 4 was increased almost two times compared to that in NMA group treated with non-micronized amoxicillin trihydrate, which was similar to the results of the above researches. At day 3 and 4 after administration of amoxicillin trihydrate, the level of amoxicillin in MA group was higher than that in NMA group, because the performance of particle size reduction was resulted in very fine particles and a larger surface area leading to an increase dissolution rate and bioavailability of drug [24, 25]. 

In conclusion, the micronization of amoxicillin trihydrate may improve higher oral bioavailability of the drug than its normal form. 

Acknowledgements

 This study was supported by a grant from Dae Han New Pharm (Seoul, Korea).

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