PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES

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1 PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES DISPOSITION KINETICS AND IN VITRO PLASMA PROTEIN BINDING OF ENROFLOXACIN FOLLOWING SINGLE DOSE INTRAPERITONEAL ADMINISTRATION IN ALBINO RATS Nitesh Kumar * Department f Veterinary Pharmaclgy and Txiclgy, Cllege f Veterinary Science and A. H., Mhw (M. P.), India. ABSTRACT Pharmackinetics f enrflxacin (Frtius - Virbac Animal Health India Pvt. Ltd., Mumbai, India) was studied in five albin rats fllwing intraperitnial (i.p.) administratin (5 mg/kg). Estimatin f enrflxacin in plasma was analyzed by micrbilgical assay technique (cylinder plate diffusin methd) using E. cli (ATCC 25922) as the test rganism. Kinetic parameter f enrflxacin was calculated by using ne-cmpartment pen mdel. Therapeutic cncentratin f 0.12 g/ml was maintained up t 72 h and the drug was als detectable up t 72 h. Absrptin half life (t 1/2 Ka), eliminatin half life (t 1/2 ), mean residence time (MRT), Vlume f distributin during area under curve (Vd area ) and ttal bdy clearance (Cl B ) f h, h, h, l/kg and ml/kg/min, respectively were btained fr enrflxacin in albin rats. The in vitr prtein binding percent f enrflxacin in albin rats ranged frm 37% t 44% with average f %. The AUC/MIC 90 fr a successful clinical/micrbilgical utcme f > was achieved at MIC 90 f 0.25 µg/ml and ptimal C p /MIC (>10) was attained at MIC 90 f µg/ml in albin rats. The highest enrflxacin MIC 90 f bacteria that fulfills the minimum AUC/MIC rati (>100) fr the present dsage regimen is 0.25 µg/ml. T achieve the ptimal C p /MIC (>10), the present dsing regimen wuld allw an MIC 90 f µg/ml fr Gram-negative and Gram-psitive pathgens. Fr maintaining therapeutic cncentratin f g/ml, a lading dse (D*) f arund 3 mg/kg and maintenance dse (D 0 ) f 2 mg/kg may be used at the dsage interval (τ) f 72 h fr treating systemic infectins in albin rats. Key wrds: Dispsitin kinetics, in vitr plasma prtein binding, enrflxacin, albin rats. INTRODUCTION Enrflxacin [1-cyclprpyl-7- (4-ethyl-1-piperazinyl) - 6- flur - 1, 4 dihydr 4 x 3- quinline carbxylic acid] is a recent flurinated quinlne carbxylic acid derivatives develped exclusively fr veterinary use [1]. It effectively penetrates all rgans IC VALUE

2 and tissues and the distributin pattern is mre r less similar in all species. In different species, enrflxacin is de-ethylated t ciprflxacin in viv [2], which is als a ptent antimicrbial agent used in human medicine [3]. Bth enrflxacin and ciprflxacin are bactericidal at very lw cncentratins fr a brad spectrum f gram-negative and grampsitive bacteria as well as mycplasmas [4]. Pharmackinetic studies f enrflxacin in healthy albin rats was carried ut t btain the detailed pharmackinetic data and s as t derive apprpriate dsage regimen f enrflxacin t treat varius systemic infectins. MATERIALS AND METHODS Experimental animals Five clinically healthy adult albin rats f either sex weighing between 200 t 250 g bdy weights were used. The rats were kept in clny cage and maintained under hygienic cnditin in the department f veterinary pharmaclgy and txiclgy and prvide standard rdent diet as well as ad libitum clean drinking water with the help f rdent drinking bttle. All animals were kept under the same experimental cnditins with natural day and night cycle. The animals did nt receive any drug treatment befre the study. The study was apprved by instutinal ethical cmmittee f Cllege f Veterinary Science and Animal Husbandry, Mhw, Madhya Pradesh, India. Dsage frms Enrflxacin (Frtius - 10%) - an injectable single applicatin cmmercial preparatin cntaining enrflxacin in cncentratin f 100 mg/ml marketed by Virbac Animal Health India Pvt. Ltd., Mumbai, India was used in the present study. Enrflxacin (5 mg/kg, i.p.) was given in each f the five albin rats. Experimental design The animals were individually weighed immediately befre administering the drug in rder t determine the precise dse. Enrflxacin (5 mg/kg) was injected intraperitneally in each albin rats and bld samples (apprx.0.5 ml) were withdrawn frm the infra rbital sinus thrugh an inner medial canthus f eye int heparinised glass centrifuge tubes befre and at 0.083, 0.25, 0.50, 0.75, 1, 1.5, 2, 4, 6, 8, 12, 24, 48 and 72 h pst drug administratin. Bld samples were centrifuged at 3000 r.p.m. fr 15 min at rm temperature in rder t separate plasma and kept at - 4ºC until analysis, which was usually dne n the day f cllectin. IC VALUE

3 Analytical methd The plasma samples were assayed by a standard micrbilgical assay technique [5] using Escherichia cli (ATCC 25922) as the test rganism. The six wells, 8 mm in diameter, were cut at equal distances int a ( mm) petriplate cntaining 25 ml f seeded agar. The test rganism was grwn in nutrient brth fr 1/2 t 1 hur at 37 C until the grwth was seen (turbid by naked eye). Enrflxacin assay plates were flded with the brth cntaining the rganism and excess brth was drained ut after sme time. The plates were dried in the incubatr at 37 C fr a perid f abut an hur. The wells were filled with the test samples and/r an enrflxacin standard slutin (prepared frm a cmmercial slutin). Standard curve f enrflxacin was prepared in pled antibacterial-free plasma. The standards and samples were tested in triplicate. The plates were kept at rm temperature fr 2 h befre being incubating at 37 C fr 18 h. The mean inhibitin zne diameters were measured and the cncentratins in the plasma samples were calculated frm the standard curve. The standards were included in each assay plate in rder t cmpensate fr any plate-t-plate variatins. There was a linear relatinship between the zne f inhibitin and the lgarithm f the plasma enrflxacin cncentratin with a crrelatin cefficient f The reprducibility f this methd was excellent and the inter-assay variability was < 5%. The limit f quantitatin was 0.04 μg/ml. A standard curve was cnsidered acceptable if the quality cntrl samples were within 15% f the nminal cncentratin. Pharmackinetic analysis The plasma cncentratin-time prfile f enrflxacin pltted n a semilgarithmic scale after intraperitneal administratin in each animal was used t establish varius dispsitin kinetic determinants and mean kinetic variables were btained by averaging the variables calculated fr individual animals. The lg plasma drug cncentratins versus time prfile fr each rat were analyzed accrding t the cmputed least square regressin technique. The kinetic parameters were calculated manually by applying ne-cmpartment pen mdel as described [6]. The cncentratin f the drug in plasma at any time is btained by the fllwing frmula (Eqn. 1): Cp = Be -β.t - A'e Ka.t (Eqn. 1) IC VALUE

4 where, Cp is the plasma level f enrflxacin at time t and e represents the base f natural lgarithm, A' and B are the extraplated zer-time intercepts f the absrptin and eliminatin phases, respectively, Ka and β are the absrptin and eliminatin rate cnstants, respectively. The ultimate bjective f present dispsitin study was t cmpute dsage regimen f enrflxacin in albin rats fr the treatment f infectius diseases. Apprpriate lading (D*) and maintenance (D 0 ) dse fr maintaining minimum therapeutic cncentratin (C p min = MIC) f 0.125, 0.25 and 0.5 g/ml at desired dsage interval (τ) f 24, 48 and 72 h were als derived [7] as per the fllwing equatin (Eqn. 2 & 3): D* = C p (min). Vd area. (e. τ ) (Eqn. 2) D 0 = C p (min). Vd area. (e. τ - 1) (Eqn. 3) Prtein binding Drug cncentratin f 1, 2, 3, 4 and 5 µg/ml were added t the plasma f albin rats and binding f enrflxacin was determined in vitr based n the diffusin f free antibitic int the agar medium [8]. The cncentratin f enrflxacin in phsphate buffer and plasma were estimated by micrbilgical assay. The differences in the diameters f the inhibitin znes between the slutins f the drugs in the buffer and plasma samples were calculated. Pharmacdynamic efficacy / Efficacy predictrs Clinical and micrbilgical utcmes f enrflxacin therapy can be predicted by the site f infectin and in terms f pharmackinetic pharmacdynamic (PK/PD) relatinships based n C max : MIC and the AUC : MIC rati [9]. The pharmacdynamic efficacy f enrflxacin was determined by calculating C p / MIC 90 and AUC/MIC ratis fllwing i.p. administratin f drugs. In rder t calculate the PK/PD efficacy predictrs hypthetical MIC values were used. T he minimum therapeutic cncentratin (MIC 90 ) v a lu e f e nr f l x a c in f r d i f f e r e nt s p e c ie s f micrrganisms ranged between t 1.0 µg/ml [10]. Keeping in mind the synergistic effect f the bdy immune system and ther in viv factrs as well as t cver mst f the susceptible rganisms, the MIC 90 f 0.125, 0.25 and 0.5 g/ml are taken int cnsideratin. In the IC VALUE

5 present investigatin, a MIC 90 f μg/ml f enrflxacin was taken int cnsideratin fr discussin. RESULTS Clinical examinatin f all animals befre and after each trial did nt reveal any abnrmalities. N serius adverse events f enrflxacin were bserved thrughut the study. The plasma cncentratins (µg/ml) f enrflxacin at different time interval in albin rats fllwing single intraperitneal administratin f enrflxacin (5 mg/kg, b.wt.) were depicted in Figure 1. Enrflxacin cncentratins were present in plasma frm t 72 h. The mean peak plasma cncentratin f enrflxacin was bserved g/ml at time intervals f 1 h and plasma cncentratins gradually declined up t 72 h. The minimum therapeutic cncentratin ( g/ml) f enrflxacin was maintained thrughut frm t 72 h. Figure 1 Plasma cncentratins (µg/ml) f enrflxacin in albin rats The values f different kinetic parameters f enrflxacin such as the absrbtin rate cnstant (Ka) and absrbtin half - life (t 1/2 Ka) f enrflxacin was nted t be h -1 and h, respectively and the eliminatin rate cnstant ( ), eliminatin half life (t 1/2 ), area under curve (AUC), area under first mment curve IC VALUE

6 (AUMC), mean residence time (MRT), vlume f distributin at area under curve (Vd area ) and ttal bdy clearance (Cl B ) f enrflxacin were depicted in Table 1. Table 1: shws kinetic parameters f enrflxacin in albin rats calculated by ne cmpartment pen mdel fllwing single intraperitneal (i.p.) 5 mg/kg b.wt. PK parameters (Unit) Enrflxacin A (µg/ml) 0.49 ± 0.05 B (µg/ml) 0.82 ± 0.01 C 0 p (µg/ml) 1.31 ± 0.04 Ka (h -1 ) ± 0.05 t 1/2 Ka (h) 5.83 ± 1.24 β (h -1 ) ± t 1/2 β (h) ± 2.43 AUC (µg/ml.h) ± 4.39 AUMC (µg/ml.h 2 ) ± MRT (h) ± 2.48 K 12 (h -1 ) ± 0.01 K 21 (h -1 ) ± 0.04 Kel (h -1 ) ± Fc ± 0.01 T P ± 0.03 Vd C (L/kg) 3.83 ± 0.14 Vd B (L/kg) 6.08 ± 0.07 Vd area (L/kg) 6.76 ± 0.30 Vd SS (L/kg) 5.21 ± 0.17 CI B (ml/kg/min) 1.87 ± 0.18 Where, A = zer time intercept fr distributin phase; B = zer time intercept fr eliminatin phase; C 0 p ( g/ml) = theretical zer time cncentratin (A+B); Ka = absrptin rate cnstant; t 1/2 Ka = absrptin half life; = eliminatin rate cnstant; t 1/2 IC VALUE

7 = eliminatin half life; AUC = ttal area under plasma drug cncentratin curve; AUMC = area under first mment curve; MRT = mean residential time; K 12 = rate cnstant f drug transfer frm central cmpartment t peripheral cmpartment; K 21 = rate cnstant f drug transfer frm peripheral t central cmpartment; Kel = rate cnstant f drug eliminatin frm central cmpartment; Fc = fractin f drug available fr eliminatin frm central cmpartment; T P = apprximate tissue t plasma cncentratin rati; Vd area = apparent vlume f distributin; Vd SS = vlume distributin at steady state; Cl B = ttal bdy clearance. The dsage regimen t maintain the different levels f therapeutic cncentratins in plasma fr i.p. rute in albin rats at different dsage intervals are presented in Table 2. Fr maintaining C P min f g/ml, the lading dses (D*) were calculated t be and mg/kg while maintenance dses (D 0 ) were calculated t be and mg/kg at dsage interval (τ) f 48 and 72 h, respectively. Like wise, D*s and D 0 s were derived fr maintaining C P min f 0.25 and 0.5 g/ml at τ f 48 and 72 h (Table 2). Table 2: shws calculated dsage regimens f enrflxacin in albin rats fr intraperitneal rute. C p min (µg/ml) τ (h) Dse (mg/kg) Enrflxacin D* 1.87 ± 0.16 D ± D* 2.80 ± 0.30 D ± D 0 D* D ± ± D* 5.60 ± ± D 0 D* 7.50 ± ± D* ± 1.19 D ± 1.05 Where, D* = Lading dse, D 0 = Maintenance dse, τ = Dsage interval, C p min = Minimum therapeutic cncentratin in plasma (MIC). IC VALUE

8 The individual PK/PD ratis fr the assumed MIC 90 values fr enrflxacin are summarized in Table 3. The threshld AUC/MIC 90 fr a successful clinical/micrbilgical utcme f > was achieved at MIC 90 f 0.25 µg/ml and ptimal C p /MIC (>10) was attained at MIC 90 f µg/ml in albin rats. The highest enrflxacin MIC 90 f bacteria that fulfills the minimum AUC/MIC rati (>100) fr the present dsage regimen is 0.25 µg/ml. T achieve the ptimal C p /MIC (>10), the present dsing regimen wuld allw an MIC 90 f µg/ml fr Gram-negative and Gram-psitive pathgens. Table 3: depicts efficacy predictrs ( C p /MIC and AUC/MIC) estimated fr enrflxacin in albin rats using different MIC values. MIC (µg/ml) MIC 0.25 (µg/ml) MIC 0.5(µg/ml) C p /MIC AUC/MIC Fr calculatins the applied values were: C p = 1.31 µg/ml, AUC = g/ml.h in albin rats. DISCUSSION In the current investigatin, we measured the residual cncentratins f enrflxacin in the plasma f albin rats using the micrbial inhibitin test. The reasns that we selected the biassay are: (a) the biassay measures the ttal activity, which culd be mre useful fr pharmacdynamic evaluatins than HPLC [11] ; (b) cngruent results between the data determined by the micrbilgical assay and thse determined by HPLC [12] ; and (c) the biassay methd is precise, reprducible, and des nt require specialized equipment r txic slvents [13]. Fr these reasns, the applicatin f micrbilgical assay fr measuring enrflxacin cncentratin is suitable [14]. The minimum inhibitry cncentratin (MIC 90 ) values f enrflxacin fr different species f micrrganisms ranged between t 1.0 g/ml [10]. Hence, in the present investigatin, the dsage regimen f enrflxacin was calculated at three different therapeutic levels (0.125, 0.25 and 0.5 g/ml) and at tw dsage intervals (48 and 72 h). IC VALUE

9 As cmpared t the present study, a shrter t 1/2 f h in gat [15], 2.5 h [16] & 2.19 h [17] in rabbit and 2.4 h [18] & 3 h [19] in dg were reprted after i.v. administratin f enrflxacin. Hwever, a very lwer t 1/2 f h (enrflxacin) and h (ciprflxacin) [20], h [21] and 1.7 h [22] were nted in cws after i.v. administratin f enrflxacin. A very lwer t 1/2 f h was nted in buffal bulls [23] and very lwer value f 0.74 h (enrflxacin) and 1.38 h (ciprflxacin) in gats were nted [24]. The much higher t 1/2 f h btained in the present study indicate cmparatively the much slwer remval f enrflxacin frm the bdy f albin rats as cmpared t abve nted species. This is further supprted by very lwer value f rate cnstant f drug eliminatin frm central cmpartment (Kel) btained fr enrflxacin in albin rats ( h -1 ). A vlume f distributin f 0.6 L/kg [20,21] and 1 L/kg [22] in cws, L/kg in buffal bulls [23], 2 L/kg [25], t L/kg [26] and 2.3 L/kg [27] in hrses, L/kg in fals [28], L/kg in sheep [29] and L/kg in gats [15] were reprted while a very high vlume f distributin (Table 2) was nted in the present study. This may suggest that enrflxacin may be distributed well in bdy tissues and fluids in albin rats than all abve nted species, which may als be beneficial fr treating systemic micrbial infectins in albin rats. Cl B values f enrflxacin were nted t be ml/kg/min in the present study. Mre r less similar Cl B values f 1.73 ml/kg/min in fals [28] and ml/kg/min in hrses [26] were nted fr enrflxacin. Hwever, a very lnger Cl B value f 27.1 ml/kg/min in dgs [18], ml/kg/min in rabbits [17], ml/kg/min in sheep [29] and ml/kg/min in gats [15] were btained after i.v. administratin f enrflxacin. Variatin amng species, breed, sex, age and different methds fr estimatin f drugs may cntribute t the wide discrepancies in kinetic parameters as suggested by varius wrkers [30]. The majr bjective f the present study was t calculate and mdify the dsage regimen f enrflxacin. Enrflxacin may be rutinely used at the dse rate f 2.8 mg/kg as lading dse (D*) and 2 mg/kg as maintenance dse (D 0 ) fr maintaining MIC IC VALUE

10 f g/ml at the dsage interval (τ) f 72 h fr treating septicemia and any ther systemic infectins in albin rats. Fr treating severe infectins (MIC = 0.25 and 0.5 g/ml), the suggested D* and D 0 may be used (Table 3). The in vitr prtein binding percent f enrflxacin in albin rats ranged frm 37% t 44% with average f %. The extent f binding f enrflxacin t the plasma prteins f albin rats (41.23 %) in the present study was in accrdance t the crrespnding values f 36-45% fr enrflxacin [22] in cattle, 24-38% fr levflxacin in man [31] and 17.0 ± 1.2% in calves fr levflxacin [32] as well as 26% fr danflxacin [33], % fr rbiflxacin in hrses [34] and 34.5% fr mxiflxacin in lactating ewes [35] have been recrded. The drug was mainly bund t albumin, and the binding was fully reversible. This lw degree f prtein binding will nt inhibit distributin f the drug t the interstitial fluid (the site f actin fr mst antibacterial drugs), and interstitial fluid drug cncentratins are expected t equal the plasma cncentratins [34]. There is general cnsensus that the clinical and micrbilgical utcmes f flurquinlne treatment are favurable and selectin f a mutant subppulatin is preventable if an AUC/MIC and a C max, /MIC f 10 are achieved in Gramnegative infectins [36,37]. Fr Gram-psitive pathgens, the minimum required C max, /MIC is als 10, while the ptimum AUC/MIC target values are still a tpic f debate [36]. An AUC/MIC f is claimed t be ptimal in numerus studies perfrmed mainly in in vitr r animal mdels [38]. Other studies cnducted n different patient ppulatins suggested a minimum AUC/MIC f t achieve a favurable utcme and t avid develpment f resistance regardless f whether the rganism is Gram-psitive r Gramnegative [37]. Cnsidering the AUC/MIC 90 and C p /MIC 90 ratis btained in the present study, it can be stated that enrflxacin administered intraperitneally in the dsing schedule applied is efficacius against bacteria with MIC 90 values under µg/ml in albin rats. The high value f AUC/MIC 90 (371.52) and C p /MIC 90 (10.48) btained in the present study, prvides supprt fr excellent clinical and bacterilgical efficacy f enrflxacin in albin rats. In agreement with the present results, a C max /MIC rati f mre than 10 has been reprted fllwing subcutaneus administratin f bth IC VALUE

11 danflxacin and enrflxacin in calves [39]. The AUC/MIC rati was higher in present study than the values f 76.6 reprted fr levflxacin administered intramuscularly in calves [32] and fr ther flurquinlnes, 40.7 fr marbflxacin in cws [40]. Hwever, it is necessary t nte that the numerical values f AUC/MIC 90 and C max /MIC 90 used as a surrgate marker t predict ptimal dsage, have been generated in experimental infectins in labratry animals r in human clinical trials [41]. CONCLUSION In cnclusin, the fact that general adverse reactins were nt bserved in any albin rats and favurable pharmackinetic prperties f enrflxacin, such as lng halflife and high biavailability with wide penetratin int different bdy fluids and tissues frm bld, were fund. Based n the calculated AUC/MIC 90 and C p /MIC 90, a dsage f 3 mg/kg culd be effective in albin rats MIC 90 > 0.125µg/ml fllwing intraperitneal rute. ACKNOWLEDGEMENT The authr is thankful t Virbac Animal Health India Pvt. Ltd., Mumbai, India fr prviding gift samples f enrflxacin (Frtius ). REFERENCES 1. Altreuther P: Data n chemistry and txiclgy f Baytril. Veterinary Medical Review 1987; 2: Tyczkwska K, Hedeen KM, Aucin DP and Arnsn AL: High perfrmance liquid chrmatgraphic methd fr the simultaneus determinatin f enrflxacin and its primary metablite ciprflxacin in canine serum and prstatic tissue. Jurnal f Chrmatgraphy 1989; 493: Bergan T, Dalhff A and Rhwedder R: Pharmackinetics f ciprflxacin. Infectin 1988; 16: Hper DC and Wlfsn JS: Flurquinlne antimicrbial agents. New England Jurnal f Medicine 1991; 7: Aerts MML, Hgenbm AC and Brinkman UATh: Analytical strategies fr the screening f veterinary drugs and their residues in edible prducts. Jurnal f Chrmatgraphy B 1995; 667: IC VALUE

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15 36. Kiem S and Schentag JJ: Relatinship f minimal inhibitry cncentratin and bactericidal activity t efficacy f antibitics fr treatment f ventilatr-assciated pneumnia. Sem Resp Crit Care M. 2006; 27: Frrest A, Nix DE, Ballw CH, Gss TF, Birmingham MC and Schentag JJ: Pharmacdynamics f intravenus ciprflxacin in seriusly ill patients. Antimicrbial Agents Chemtherapy 1993; 37: Lister PD: Impact f AUC/MIC ratis n the pharmacdynamics f the des-f (6) quinlne garenxacin (BMS ) is similar t ther flurquinlnes, Jurnal f Antimicrbial and Chemthrapy 2003; 51: TerHune TN, Skgerbe TL, Shstrm VK and Weigel DJ: Cmparisn f pharmackinetics f danflxacin and enrflxacin in calves challenged with Mannheimia haemlytica. American Jurnal f Veterinary Research 2005; 66: Schneider M, Valle M, Wehrle F and Bisrame B: Pharmackinetics f marbflxacin in lactating cws after repeated intramuscular administratins and pharmacdynamics against mastitis islated strains. Jurnal f Dairy Science 2004; 87: Tuitan PL and Lees P: Integratin and mdelling f pharmackinetic and pharmacdynamic data t ptimize dsage regimens in veterinary medicine. Jurnal f Veterinary Pharmaclgy and Therapeutics 2004; 27: Fr Crrespndence: Dr. Nitesh Kumar Assistant Prfessr and I/C Head Department f Pharmaclgy and Txiclgy Cllege f Veterinary Science and A. H., Mhw (M. P.), India Mb. N Fax N drnitesh_kumar01@yah.c.in IC VALUE