The pharmacokinetics of phenylbutazone and its interaction with dexamethasone in chicks

1. The type of pharmacological interaction between phenylbutazone and dexamethasone is synergism.
2. There is a rise in phenylbutazone plasma concentration when given simultaneously with dexamethasone.
3. Dexamethasone modifies the phenylbutazone pharmacokinetics which improves the treatment efficiency in the chicks as a model.

Introduction

Phenylbutazone is one of the non-steroidal anti-inflammatory drugs (abbreviated as NSAIDs) that serve multiple usages for veterinary medicine as an antipyretic, anti-inflammatory, and analgesic (1-3). These properties result from the non-selective mode of action via direct inhibition of cyclooxygenase isoforms which decrease the synthesis of prostaglandin (PG), especially the PGE₂ type (from the arachidonic acid precursor). The latter is considered a chemical mediator that plays a crucial role in inducing fever, inflammation as well as pain sensation (4-7). Phenylbutazone is a well-known NSAIDs member characterized by a high protein bound to albumins ≥99% and its induction of the microsomal enzymes responsible for the Phase-I metabolism of the drugs (6,7). Dexamethasone, on another hand, is a corticosteroid drug that is like an in vivo hormone in being synthesized by the adrenal gland. It is often used to relieve inflammatory conditions with weak analgesic and antipyretic action in comparison to NSAIDs (8). Dexamethasone acts by inhibiting the phospholipase A₂ enzyme which is responsible for converting the membrane phospholipids to arachidonic acid (the precursor of PG synthesis), thus inhibiting the PG synthesis by indirect modulatory action (9,10). As a result, dexamethasone and NSAIDs are used concomitantly due to their possible synergistic interaction against lowering the PG synthesis (11). Because of the proposed pharmacological interaction between phenylbutazone and dexamethasone on PG biosynthesis, the present aim of the study is to investigate the influence of dexamethasone on phenylbutazone plasma levels and pharmacokinetics as well as their pharmacological interaction.

Materials and methods

Laboratory animals

Broiler chicks of both genders from a local hatchery, aged seven to fourteen days and weighing between 90-130 g, were used in the study. The chicks were well-cared for in a temperature range of 29-33 °C with proper lighting. The floor litter was of wood shreds and the chicks had free allowable water and food.

Preparation of medications

Phenylbutazone (20%, Interchemie, Holland) and dexamethasone (0.2% VAPCO, Jordan) were prepared in a saline solution to have the desired concentration and to be given by intraperitoneal (IP) administration as 5 ml/kg of injection volume.

Ethics

The Scientific Council of the College of Veterinary Medicine, University of Mosul has approved the study protocols on experimental animals.

The pharmacological interaction between phenylbutazone and dexamethasone

The analgesic ED_50s, of phenylbutazone and dexamethasone, were given separately and assessed for each drug by the up-and-down technique (12). The first given dosage of phenylbutazone and dexamethasone was at 7 and 2 mg/kg, IP, respectively. The chicks were tested using an electro-stimulator (Harvard device, USA) before and after 30 minutes of treatment with each medication. The pain symptoms were indicated by a distress call in the chick model (13-19). The dosage for both medications was lowered or increased by 2 and 0.5 mg/kg of the initial dosage administered according to the presence and absence of analgesia (13-19). Subsequently, the ED₅₀ values of phenylbutazone and dexamethasone combination (at a ratio of 1:1) were measured in the chicks via the isobolographic analysis (20,21). The first dosage of phenylbutazone and dexamethasone was at 5.60 and 0.63 mg/kg, IP, and was tested with the previously mentioned technique. The dosages of both medications were lowered or increased by 25% at 1.4 and 0.16 mg/kg from their initial doses according to the previously mentioned effect.

Isobolographic analysis between phenylbutazone and dexamethasone

On the y and x-axes, the ED₅₀ values of phenylbutazone (5.60 mg/kg, IP) and dexamethasone (0.63 mg/kg, IP) are pointed by a direct line of representation to have the isobolographic investigation among the ED₅₀s doses of phenylbutazone and dexamethasone individually. The analysis described as the mark underneath the line is considered synergism although the mark is over directed to antagonism. The Y symbol marked as an interaction index can be obtained using the next formula [da / Da + db / Db], in which Da and Db refer to the analgesic ED₅₀ of phenylbutazone and dexamethasone given alone whereas da and db stand for their combined ED₅₀s (Table 1). When Y value is less than 1, it specifies synergistic.  Antagonistic interaction is referred to if Y value is greater than 1 (20,21).

Assessment of phenylbutazone plasma concentration: influence of dexamethasone

The first group received phenylbutazone at 11.20 mg/kg, IP, whereas the second received phenylbutazone (11.20 mg/kg, IP) plus dexamethasone (1.26 mg/kg, IP) injections. For both groups, blood samples were obtained from the jugular vein at different times of 0.25, 0.5, 1, 2 and 4 for 24 hours. The plasma was procured by adding heparin anticoagulant (B. Braun Medical Inc, USA) to blood samples at a ratio of 1:10 and centrifuging for 15 minutes at 3000 rpm (Chalice, UK). Finally, the plasma samples were frozen (-18°C for 3 days) before being analyzed by a UV-visible spectrophotometric apparatus (Lovibond, Germany) (22).

Preparing the buffered permanganate solution (BPS)

The BPS was used in the analysis. It was set by dissolving 15.2 g of Na₂HPO₄.12 H₂O, 1.6 g of NaOH, and 1 g of potassium permanganate in 100 ml of distilled water in a graded flask for daily fresh preparation, and the pH was adjusted to 12.4 by addition of HCl or NaOH (22).

Phenylbutazone standards preparation

The phenylbutazone standards were prepared at 25, 50, 100, 200, 400, and 800 µg/ml concentrations by diluting phenylbutazone with BPS of pH 12.4 to get the necessary phenylbutazone concentration (22). In contrast to the BPS blank, the solution was examined by a spectrophotometer apparatus (314 nm of wavelength). As a calibration curve, the linear regression formula of phenylbutazone standards was utilized. For the two groups of chicks that received phenylbutazone or phenylbutazone and dexamethasone, the concentration of phenylbutazone in plasma samples could be measured as follows: y= a + b x [coefficient of determination (R²) = 0.9469], it is to be noted that y specifies the absorbance of samples and a indicates the intercept 0.0766, b is the slope of a calibration curve 0.0012, and x is the phenylbutazone concentration of the plasma which is unidentified as shown in Figure 1.

Figure 1: The absorbance (314 nm) of the phenylbutazone standards at 25, 50, 100, 200, 400, and 800 µg/ml as represented in a calibration curve

Liquid-liquid extraction of phenylbutazone

The plasma samples were extracted using a simple, approved, and reliable liquid-liquid extraction technique for phenylbutazone (22). A 0.5 ml (of 1 Molar) HCl and 5 ml of n-heptane were added to 0.5 ml of samples with the cover application. The tube was shaken for 30 min, and then centrifuged at 3000 rpm for 5 min. 4 ml of n-heptane upper phase was transferred to another glass tube containing 3 ml of 0.5 molars NaOH. The tube was shaken for 5 min discarding the n-heptane (upper phase). Following the previously mentioned procedure, 2.5 ml of the aqueous phase was added to a previously prepared 5 ml of BPS in another glass tube. Water bathing was applied to the tubes at 65 ^OC for 5 min, and then the tubes were cooled. A 2 ml of n-heptane was added and shaken for 20 min to the content of the previous step with centrifugation. The n-heptane phase was transferred to another glass tube and stored for 3 hours at room temperature. In contrast to the blank consisting of n-heptane, the final solution was detected using a spectrophotometer device at 314 nm.

Influence of dexamethasone administration on the pharmacokinetics of phenylbutazone

A PKSolver tool was used in this study to derive the phenylbutazone pharmacokinetic characteristics whether given separately or simultaneously with dexamethasone using a non-compartmental pharmacokinetic model (23). The characteristics of pharmacokinetics include AUC_0-∞ (µg.h/ml), AUMC_0-∞ (µg.h²/ml), K_el (0.693/t_1/2β) (h^-1), C_max (µg/ml), T_max (h), t_1/2β (h), MRT (AUMC/AUC) (h), V_ss [dose× AUMC/(AUC)²] (L/kg), and the total Cl (dose/AUC) (L/h/kg). The percentages conducted for these parameters were increased or decreased in the two clusters injected with phenylbutazone separately or simultaneously with dexamethasone.

Statistics

The averages of the two groups in the parametric analysis were analyzed and correlated by using the unpaired student T-test (24). When p is less than 0.05, the level is deemed significant.

Results

Isobolographic analysis of phenylbutazone and dexamethasone

The analgesic ED₅₀ value of phenylbutazone only was 5.60 mg/kg, IP, and the analgesic ED₅₀ value of dexamethasone alone was 0.63 mg/kg, IP, respectively, as shown in Table 1. When phenylbutazone and dexamethasone were administered jointly at 1:1 from their ED₅₀s, the analgesic ED₅₀ values were 1.76 and 0.19 mg/kg, IP, correspondingly (Table 2).

Pharmacological interaction among phenylbutazone and dexamethasone

The interaction index (Y symbol) is smaller than 1, indicating that the type of pharmacological interaction among phenylbutazone and dexamethasone is synergistic as shown in Table 2 and Figure 2.

Table 1: Analgesic ED₅₀s of phenylbutazone and dexamethasone given separately  

Table 2: Isobolographic analysisbetween phenylbutazone and dexamethasone

^* ED₅₀ estimated= xf + kd

In these tables, X means analgesia whereas O indicates no analgesic effect. The variables were recorded pre and post 30 minutes of phenylbutazone and dexamethasone treatment. In addition, ⁺Da and Db specify the ED₅₀ for phenylbutazone and dexamethasone only, while da and db are their adjunct ED₅₀, correspondingly. The resultant interaction index which is 1 specifies an antagonistic interaction.

Figure 2: Isobolographic study of phenylbutazone and dexamethasone's analgesic interaction. The ED₅₀ value of phenylbutazone (5.60 mg/kg, IP) is represented by the y-axis, while the ED₅₀s of dexamethasone (0.63 mg/kg, IP) are represented by the x-axis. For both medications (1.76 and 0.19 mg/kg, IP for phenylbutazone and dexamethasone, respectively), the triangle point reflects 1:1 of ED₅₀s combinations. The triangle point suggests that phenylbutazone and dexamethasone have a synergistic interaction.

Phenylbutazone plasma concentration is given separately or as a mixture with dexamethasone

When phenylbutazone is combined with dexamethasone, the plasma concentration of phenylbutazone increases significantly, compared to the plasma concentration when phenylbutazone is given alone. In the present study, the plasma concentration of phenylbutazone (11.20 mg/kg, IP) was measured at various periods of 0.25, 0.5, 1, 2, 4, and 24 hours as 39.83, 66.17, 48.00, 35.30, 26.50 and 13.33 µg/ml. Phenylbutazone and dexamethasone (11.20 and 1.26 mg/kg, IP, correspondingly) raised plasma concentration by 43, 426, 339, 293, 147 and 276 % to 57.00, 384.17, 210.67, 138.67, 65.50 and 50.10 µg/ml, respectively as shown in Table 3.

Table 3: Phenylbutazone plasma levels alone or as a mixture with dexamethasone in the chicks

Numbers are shown as the mean ± SE of five chicks/ assessed time. (^*) differs significantly as of the phenylbutazone group at p less than 0.05. Phenylbutazone injected 11.20 mg/kg, IP separately or simultaneously with dexamethasone (1.26 mg/kg, IP). (⁺ %) is the influence of dexamethasone on plasma concentration of phenylbutazone= phenylbutazone plus dexamethasone - phenylbutazone alone / phenylbutazone alone × 100.

Phenylbutazone pharmacokinetics with and without dexamethasone

When phenylbutazone is given alone, it shows the pharmacokinetic characteristics as AUC_0-∞ 867.71 µg.h/ml, AUMC _0-∞ 20699.03 µg.h²/ml, K_el 0.04 h^-1 and C_max 66.17 µg/ml, while dexamethasone increases these parameters when given with phenylbutazone by 196, 140, 50 and 426 % to be 2570.03 µg.h/ml, 49660.79 µg.h²/ml, 0.06 h^-1 and 348.17 µg/ml, respectively.  

The other pharmacokinetic variables of phenylbutazone when given alone involved t_1/2β 17.22 h, MRT 23.53 h, V_ss 0.32 L/kg, and Cl 0.01 L/h/kg was reduced with dexamethasone administration by 33,18, 78 and 60 % to be 11.59 h, 19.32 h, 0.07 L / kg and 0.004 L/h/kg, correspondingly (Table 4).

Table 4: Pharmacokinetic characteristics of phenylbutazone alone or with dexamethasone in the chicks

Phenylbutazone was injected at 11.20 mg/kg, IP separately or simultaneously with dexamethasone (1.26 mg/kg, IP). The PK Solver tool was used to get non-compartmental model of pharmacokinetics. (^* %) is the influence of dexamethasone on plasma concentration of phenylbutazone= phenylbutazone plus dexamethasone - phenylbutazone alone / phenylbutazone alone × 100.

Discussion

The aim of the study is to investigate the effect of dexamethasone on the plasma concentration of phenylbutazone along with its pharmacokinetic characteristics. In addition, the putative pharmacological interaction in the chick model is studied as shown via the isobolographic study.The values of ED₅₀s for phenylbutazone and dexamethasone in combination are found to be lower in this study when compared to their values, implying an increase in the analgesic effectiveness necessary to elicit the pharmacological effect in half of the laboratory chicks utilized as the experimental model. The isobolographic analysis is a useful pattern for detecting the analgesic interaction between two medications (20,21). As the study reveals, measuring their interaction index shows a synergism of pharmacological interaction between phenylbutazone and dexamethasone as concluded over assessing the interaction index assembled as a Y symbol. The elevation in plasma concentration and variation in the various pharmacokinetic characteristics is attributed to the phenylbutazone when given with dexamethasone at the same time (AUC and AUMC). This is reported by this study for the first time. It is an additional significant key to increasing the pharmacological efficiency of phenylbutazone and dexamethasone. The change in the phenylbutazone pharmacokinetic profile is attributed to a rise in the plasma levels of the free drug due to the direct competition at the protein binding sites on albumins which directly affects the apparent volume of distribution of phenylbutazone and dexamethasone. It can be ascribed to the contention at the protein binding sites on albumins as one of the phenylbutazone and dexamethasone characteristics (99% protein bound) (6,7), the latter being a highly protein-bound drug of 60.5% as found in another study (25). This increases the amount of phenylbutazone-free drug allowable to access the binding sites on the receptors which are responsible for enhancing the therapeutic efficacy of phenylbutazone with possible subsequent toxicity and altering its safety. In addition, dexamethasone has a direct influence on other important pharmacokinetic variables such as absorption, metabolism, and excretion, and on other factors like half-life, mean residence time, elimination rate constant, and the clearance of the medication as illustrated in the findings. Other studies have found that dexamethasone also improves the efficacy of other medicines used to induce postoperative analgesia, such as opioids (25).

Conclusions

The findings of the present study reveal that there is a synergism as a type of pharmacological interaction between phenylbutazone and dexamethasone. The study also shows a change in the phenylbutazone pharmacokinetics as well as its plasma concentration which improves the therapeutic efficiency of phenylbutazone in chicks.

Acknowledgments

The manuscript is a part of the M.Sc. thesis of the first author. The authors express gratitude to the College of Veterinary Medicine, University of Mosul for the facilities provided.

Conflict of interest

The authors declare there is no conflict of interest.