Question

Give me at least 6 in vivo and in vitro bioassays about amoxicillin. Give me the original source (at least 6) and briefly explain each bioassay. FDA approval.

Answer 1

Some in vivo models such as implanted fibrin clots or dial ysis sacks allow the diffusion of antimicrobial agents into the site of infection but can limit cellular and humoral defense.

Granulomatous or fibrous tissues are formed as a result of the implantation of porous or hollow devices. These models may be valuable for determining the capacities of antibiotics to penetrate a specific site.)

Many different factors are known to affect antimicrobial efficacy in vivo. Some of these factors, such as the infectious microorganisms, the site of infection, the intrinsic activity of an antimicrobial agent, and its pharmacokinetic behavior, have been recognized and investigated in experimental infection tion models.2) When the antibacterial activity, pharmacokinetics and pharmacodynamics of an agent are considered, the treatment time should be more specifically defined as the time during which the concentration of the agent is greater than the mini mum inhibitory concentration (MIC) of the pathogen at the site of infection. In fact, the MIC should be considered in relation to tissue concentration, and not in relation to blood serum concentration, because the development of an infection tion generally requires superficial adherence to some biologi cal barriers. Furthermore, most bacteria have no pathogenic effects on body fluids; they are found in such fluids as a re sult of diffusion from the infection site, contamination by in instrumentation or due to tissue rupture.

Few infection models utilize endogenous infection in ex experimental chemotherapy because the administration of an infectious agent, usually in a large inoculum, is required to establish a reproducible infection in animals. This large in oculum may be needed to overcome host defenses, but may result in a fulminant course of infection owing to the imme diate introduction of large numbers of infectious organisms.

The disadvantages of animal infection models include un naturalness, possibility of fulminant infections and an artifi cial mode of infection. Chronic or progressive infections may result in an ever-increasing pain level owing to continuous deterioration of the host. The pain and suffering of the ani

mals should be minimized.) Amoxicillin is a widely prescribed aminopenicillin, mainly administered orally. Ninety percent of the adminis metered dose is absorbed without molecular modification.)

It provides serum concentration ranging from 7.6 to 10.8 ug/ml when 500 mg/p.o. is used;$9) 15.1 ug/ml when 15.4 mg/kg/p.o. is used, used. 10) and 14.5 ug/ml when 40 mg/kg/p.o. is The amoxicillin has plasmatic protein binding ranging from 17 to 20%. Protein binding has no clinical importance if less than 70% of an antimicrobial agent is bound.') Food does not interfere with either absorption or plasmatic con centration. 10) Mercury nitrate titration, 2) microbiological method, iodo metric assay, optical method, 5) spectrophotometric assay, 4) and HPLC assay are some of the possible methods, cur rently used to quantify amoxicillin. A comparison between the HPLC and microbiological methods showed no differ 15,16) inches.

Amoxicillin is metabolized into penicilloic acid to a lim ited extent, which is then excreted in urine. About 60% of an oral dose of amoxicillin is excreted, in an unchanged form, in the urine in 6h by glomerular filtration and tubular secre tion. The penicilloic acid has no antimicrobial activity.

In the present experimental study we assessed the utility of an animal model in predicting tissue concentration of antimi microbial agents in induced granulomatous tissue.

Histological technique showed no qualitative differences among granulomatous tissue of the control or treated groups.

The findings of the 7-d groups showed a fibrous capsule in volving the sponge that was delimited very well. Fibroblasts, mesenchymal cells and capillary formation was noted in large scale. After 14 d we found more fibrous tissue involving the sponge, but with a reduced number of cells in the periphery and cell proliferation at the center of the tissue. Macrophages were observed at 21 d of tissue formation. At 28 d the granu lomatous tissue was completely formed, showing fibrous fibers involving fewer cells and blood vessels in the center of the tissue.

Regression Line The detection limits used in the regress sion line were 8.8 mm (obtained with 0.03 µg) to 36.2 mm (obtained with 15.0 ug). Figure 1 shows the regression line.

The data were plotted with computer software (Microsoft Excel 97 for Windows) using the equation y=4.2009 X Ln(x)+25.083 or Zone (mm)=4.2009XLN(ug of antimicro bail agent)+25.083 with an R-squared value of 0.994.

It was possible to calculate the antimicrobial concentration in serum and granulomatous tissue by using the mean inhibi tion zone diameter calculated and the wet weight for each

group.

Tissue and Blood Figure 2 shows the mean tissue and blood serum concentrations obtained with the equation con version for all study periods. The results of inhibition zone diameter for all study periods of the control group were zero.

Analysis of variance did not show significant differences among the groups in inhibition zone diameter or in wet weight when the same dose was considered.

In vitro

The antibacterial activity of amoxicillin is illus trated in Fig. 2. The drug was especially active against group A beta-hemolytic streptococci, pneumococci, and penicillin G-susceptible S.

aureus. Only 28% of isolates of S. aureus resistant to 50 µg of penicillin G per ml were susceptible to amoxicillin at this concentration or less, and none were susceptible to less than 12.5 ug/ml.

Seventy-six per cent of P. mirabilis isolates were susceptible to 1.56 ug or less of amoxicillin per ml, but 20% were resistant to 12.5 ug/ml or more.

Fifty-seven per cent of E. coli isolates were sus ceptible to 6.25 mg/ml or less, but most of the remaining isolates were resistant to 50 ug/ml or more. Only a few of the other gram-negative bacilli were sensitive to amoxicillin.

d

The activity of amoxicillin was compared to that of ampicillin and cephalothin

Amoxicillin was slightly more active than ampicillin and considerably more active than cephalothin against group A beta-hemolytic streptococci and pneumococci. Cephalothin was the most active antibiotic against S. aureus, and inhibited all pen icillin G-susceptible and resistant isolates at a concentration of 0.78 ug/ml or less. Amoxicillin

and ampicillin were similar in activity against Proteus spp. and more active than cephalothin.

Ampicillin was slightly more active than amoxi cillin against E. coli and considerably more active against members of the Klebsiella-Enterobacter Serratia group.

Serum levels obtained after oral administration of 125 mg and 250 mg of amoxicillin are shown in Fig. 4. The mean peak serum level after the 125-mg dose was achieved at 1 hr and was 2.30 ug/ml. A mean serum level of 0.47 µg/ml was present at 4 hr. The mean peak serum level after the 250-mg dose occurred between 1 and 2 hr and was 3.43 ug/ml. A mean serum level of 1.45 ug/ml was present at 4 hr.

A comparison was made between serum levels

achieved after oral administration of 500 mg of amoxicillin and 500 mg of ampicillin (Fig. 5).

With amoxicillin, the mean peak serum level oc cured at 2 hr and was 6.75 ug/ml. With ampicil lin, the mean peak serum level occurred at 3 hr and was 2.28 ug/ml. Between 1 and 3 hr, the dif ferences in the serum levels were statistically sig nificant.

Mean serum levels obtained after the oral ad ministration of 1 g of amoxicillin with and with out probenecid. The mean peak serum level obtained when the drug was given alone was 9.90 ug/ml, and a mean serum level of 1.11 ug/ml was still present at 6 hr. When amoxicillin was given with probenecid, the mean peak serum level obtained was 16.04 µg/ml. The

mean serum levels at 8 and 12 hr were 1.30 ug/ml and 0.30 ug/ml, respectively.

The peak serum level in different individuals occurred from 30 min to 4 hr after drug adminis tration. Considering the highest serum level achieved in each subject with the different doses of amoxicillin, the means were 2.40 µg/ml with 125 mg, 4.69 ug/ml with 250 mg, 7.34 ug/ml with 500 mg, 10.74 ug/ml with 1 g, 17.41 ug/ml with 1 g of amoxicillin plus probenecid, and 2.68 ug/ml with 500 mg of ampicillin.

The urinary excretion of the antibiotics was de termined during the first 6 hr of each study (Ta ble 1). The urinary concentrations varied consid erably depending on the amount of urine excreted.

The mean proportion of amoxicillin excreted in the urine with each dose varied between 70 and 78%.