Effect of Treatment of Prulifloxacin Induced Arthropathy in Young Rats

About Authors: Rekha. S^1*
1*Department of Pharmacology, Mother Theresa Postgraduate and Research Institute of Health Sciences, Indira Nagar, Gorimedu, Puducherry 605 006.
Kavimani. S^1
1Professor and Head, Department of Pharmacology, Mother Theresa Postgraduate and Research Institute of Health Sciences, Puducherry.

Reference ID: PHARMATUTOR-ART-1069

Summary
Objective
The present study was undertaken to further evaluate the adverse effect potential of Prulifloxacin to induce arthropathy in rats with reference to its duration of treatment.

Methods
Groups of one month old young rats were administered Prulifloxacin intraperitonially 200 mg/kg and 400 mg/kg doses for 15 and 30 days respectively. Control group received normal saline. At the end of treatment
period, animals in each group were subjected to serum alkaline phosphatase estimation. The serum alkaline phosphatase assay was carried out by using alkaline phosphatase reagent kit.

Introduction
Prulifloxacin was developed by Nippon Shinyaku Co. and in-licensed by Optimer for development and commercialization in the U.S. It is approved for a broad range of indications in Japan and in parts of the European Union. In clinical trials, prulifloxacin appeared as effective as ciprofloxacin, co-amoxiclav or pefloxacin in the treatment of bronchitis exacerbations or lower urinary tract infections. It was tolerated as well as ciprofloxacin. Prulifloxacin has been approved for use in Japan.  In the United States, it is undergoing phase III clinical trials for the treatment of traveler's diarrhea (Medical news today 2008).
There are a number of reports of fluoroquinolone adverse effects especially the arthropathy induced in young rats and dogs (Schluter G 1989). One out of 30 children receiving high dose of ciprofloxacin have been found to develop reversible arthritis (Raeburn J A et al., 1987). These findings have restricted the use of this potent agent in pediatric patients. However its use in many children with serious pseudomonas infection in cases of fibrocystic lung disease was without much serious side effects (Schaad U B et al., 1997). The present study was undertaken to further evaluate the adverse effects of prulifloxacin with reference to arthropathy in young rats by administering high dose for different duration to find out the minimum duration for which prulifloxacin can be administered safely and to know the extent to which arthropathic changes. The serum alkaline phosphatase is used to check the possibility of bone disease or liver disease.

Materials and Methods
Thirty days old rats of either sex (weight 25±5 Gms) were used for this study. They were divided into three groups, five in each group. Group I –control, which received intraperitonially 0.5 ml normal saline. Group II and Group III – were administered prulifloxacin intraperitonially 200 mg and 400 mg/kg doses for 15 and 30 days respectively. The serum alkaline phosphatase assay was carried out by using alkaline phosphatase reagent kit (Accucare alkaline phosphate kit-SLR).

Assay Procedure for Alkaline Phosphatase
Based on that of Bessey et al., in which the rate of formation of the yellow colour of  p-nitrophenol (p-NP) produced by hydrolysis of p-nitrophenylphosphate (p-NPP) in alkaline solution is measured spectrophotometrically at 405nm and 37°C.

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Alkaline phosphatase    
p-NPP + H2Oz→p-NP + Pi

Specimen Collection and Storage
• Non-haemolyzed serum (plasma should not be used since anticoagulants inhibit alkaline phosphatase activity) was used for this study.
• Serum samples were stored at 2-8°C and assay was carried out within two days.

Procedure
1. Reagent was reconstituted according to instructions.
2. 1.0 ml of reagent was pipette out into appropriate tubes and prewarm at 37°C for five minutes.
3. Spectrophotometer was adjusted to zero with water at 405nm.
4. 0.025ml (25µl) of sample was added to reagent, mix and incubated at 37°C for one minute.
5. After one minute absorbance was recorded. Return tube to 37°C. Repeat readings every minute for the next two minutes.
6. Calculated the average absorbance difference per minute (Δabs. /min).
7. The ΔAbs. /min. multiplied by the factor 2720 (see Calculation) gives results in IU/L.

Calculation
One international Unit (IU/L) is defined as the amount of enzyme that catalyzes the transformation of one micromole of substrate per minute under specified conditions.

(IU/L) = ΔAbs./Min. x 1000 x 1.025/15.07 x 1 x .025 = ΔAbs. /min. x 2720

Where ΔAbs. /Min. = Average absorbance change per minute
1000 = Conversion of IU/ml to IU/L
1.025 = Total reaction volume (ml)
15.07 = Millimolar absorptivity of ρ-Nitrophenol
0.025 = Sample Volume (ml)
1 = Light path in cm (adapted from accucare alkaline phosphate kit-SLR)

Result
The effects of duration of treatment of prulifloxacin induced arthropathy in young rats were given in table I.  In these study one month old young rats, treated with prulifloxacin 200 mg/kg showed decreased serum alkaline phosphatase level at 15^th and 30^th day. At 15^th day of prulifloxacin 400 mg/kg showed increased the serum alkaline phosphatase level.

Discussion
The result suggested that there was no evidence of prulifloxacin induced arthropathy in young rats when treated up to 15 and 30 days at a dose of 200 mg/kg. But there was an increase in serum alkaline phosphatase level at a dose of 400 mg/kg of prulifloxacin at 15 th day of treatment. A decreased serum alkaline phosphatase may be due to Zinc, folic acid, Vitamin C and B6 deficiency or Celiac disease. Ciprofloxacin was reported for an increase in serum alkaline phosphatase level which was proportionate to the duration of therapy.  However the values returned to normal when it was repeated 15 days after stopping the therapy indicating the changes were transient and reversible. The less marked arthropathic changes observed in ciprofloxacin compared to many of the earlier studies could be attributed to the well balanced diet with adequate magnesium content (2.29 gms/kg of pellet diet) was used. There are reports that magnesium deficiency produces arthropathic changes in young animals and also found to potentiate the arthropathy induced by fluoroquinolones (Mohanasundaram J et al., 2001 and Forster C et al., 1998).

Table I: Serum alkaline phosphatase level in prulifloxacin treated young rats for different duration