Drug Interaction Effect of Ketoprofen and Dexamethasone With Thiopental: A Pharmacokinetics and Anesthetics Study in Male Dogs

There is a growing concern about drug interactions on the pharmacokinetic parameters of anesthetics. The aim of this study was to evaluate the pharmacokinetic effects of thiopental anesthesia under the administration of ketoprofen and dexamethasone in the male dogs. 21 adult healthy male stray dogs weighing 21-23 kg were randomly divided into three groups. The control group received normal saline (0.2 ml/kg) 5 minutes before intravenous administration of thiopental (17 mg/kg), the T1 group received ketoprofen (2.2 mg/kg) 5 minutes before thiopental, and the T2 group received dexamethasone (0.2 mg/kg) 5 minutes before thiopental. After anesthesia, clinical parameters of anesthesia, heart rate, respiration rate and electrocardiography were measured. Serum sample was also used to assay thiopental concentration using HPLC method, and then thiopental pharmacokinetic parameters were calculated. Changes in rate of heart and respiration were signicant intra-group differences 5 and 10 minutes after anesthesia.In addition, in the ketoprofen group, premature atrial contractions (PACs) were seen after induction of anesthesia with thiopental. Recovery time parameters showed a signicant difference between T1 and control groups (P<0.05). Elimination and half-life of thiopental in the T1 group compared to the control and T2 groups showed a signicant decrease and increase, respectively. In addition, the distribution volume of thiopental in T1 showed a signicant increase compared to other groups. However, thiopental clearance in T1 and T2 groups did not show a signicant decrease compared to control (P> 0.05). It can be concluded that dexamethasone can be used as an anti-inammatory drug combination with thiopental in comparison with ketoprofen.


Introduction
Since the advent of the science of surgery, the use of safe methods to reduce pain and complications of surgery has been considered. Balanced and safe anesthesia is one of the most important needs of a successful and uncomplicated surgery (Forman and Chin, 2008). Anesthesia is performed by various methods such as injection and inhalation methods, and the choice of anesthesia depends on a wide range of factors, including the patient's condition and the type of surgery performed by the surgeon.
Despite the widespread use of inhaled anesthesia in veterinary medicine and the bene cial bene ts of its use, the use of injectable anesthesia is still necessary (Tranquilli et al., 2013, Wagner andHellyer, 2000).
The use of injectable anesthesia increases the induction and depth of anesthesia, which is very effective in cases of emergency anesthesia and eld surgery (Wagner and Hellyer, 2000). In addition to, so far, no anesthetic has been provided alone that can provide all aspects of ideal and balanced anesthesia without any side effects, and each patient with special physiological and pathological conditions needs a special anesthesia diet and special care (Zambouri, 2007). Therefore, in most cases to minimize the side effects of anesthetics and establish stability in the patient's vital parameters, a combination of anesthesia methods and a combination of complementary drugs with anesthetics is used (Franks and Lieb, 1990). The concomitant use of multiple drugs can target several therapeutic goals or can focus a common action upon one single goal or disease (Brown et al., 2018).
The positive effects of drug interactions include reducing the required dose of the drug while producing the same effect and reducing its toxicity and the incidence and extent of side effects, minimizing the rate of drug resistance and producing a selective and synergistic effect on the target (Palleria et al., 2013).
However, drug-drug interactions (DDIs) as a part of clinical pharmacology investigates the risk of drug interactions to predict the consequences of combined drug use, and it can alter pharmacokinetic parameters and thus drug performance (Palleria et al., 2013, Marín et al., 2020. It is of interest to many researchers and physicians (Moradinejade et al., 2018, Raeeszadeh andRajaian, 2020). During anesthesia, due to receiving various drugs, DDIs are more likely to occur (Palleria et al., 2013). Thiopental sodium as a very fast-acting barbiturate is mainly used in combination with analgesics or muscle relaxants to induce general anesthesia in short-term surgery with minimal painful stimulation (Atalay and Tomak Leman, 2017, Çakırtekin et al., 2015). Ketoprofen is a propionic acid derivative and a nonsteroidal anti-in ammatory drug (NSAID) used with thiopental. Its anti-in ammatory effect is weaker than other NSAIDs (Fosse et al., 2011). In addition to ketoprofen, dexamethasone can be also used with thiopental to induce general anesthesia (Ahmad et al., 2013, Al Katheeri et al., 2004. Dexamethasone exerts its antiin ammatory effects by preventing the accumulation of in ammatory cells in the area of in ammation, inhibiting phagocytosis and releasing enzymes responsible for in ammation, inhibiting the production and release of chemical mediators of in ammation (Al Katheeri et  Considering the importance of recognizing and controlling drug interactions and examining their effect on clinical conditions and pharmacodynamic properties of anesthetics and the widespread use of analgesics and anti-in ammatory drugs before or during anesthesia, the present study was to investigate the effect of ketoprofen and dexamethasone drug interaction on changes in thiopental pharmacokinetic factors.

Materials And Methods
Animals and study design All stages of this study in accordance with the "Guidelines for the care and use of research animals" approved by the the Ethics Committee of Kurdistan University of Medical Sciences with the code IR.MUK. REC. 1399.156 were performed. In the present study, a randomized controlled clinical intervention, 21 adult healthy male stray dogs weighing 23-21 kg were used. After the initial physical examination, hematological parameters such as mean corpuscular volume (MCV), cell blood count (CBC), hemoglobin (HB), hematocrit (HCT), Mean corpuscular hemoglobin concentration (MCHC) and biochemical parameters including Aspartate transaminase (AST), Alanine transaminase (ALT), total protein, albumin, urea and creatinine were performed using standard methods to con rm the health of the animals. One week after adaptation to the experimental conditions, the animals were randomly divided into three equal groups as followed: Control group (C) animals receiving 0.1 ml/kg normal saline ve minutes prior administration of thiopental sodium (5%) with a dosage of 17 mg/kg.sodium intravenously.
In all groups, the time of induction of anesthesia and recovery time, which included the time of disappearance of eyelid re ex, return time of eyelid re ex, animal sitting time, standing time, unbalanced walking time and normal walking time were evaluated. Respiratory rate and heart rate were measured before thiopental sodium injection and at 0, 5, 10, 20, 30, 45, 60, 90 and 120 minutes after injection 8.
Blood samples were taken from animals at different times to evaluate thiopental pharmacokinetic parameters. After separation of serum by centrifugation (3000rmp, 10min), thiopental concentration was measured by high-performance liquid chromatography (HPLC) method. Then the concentration diagram was drawn in terms of time and its different pharmacokinetic parameters were calculated in all the groups (Raeeszadeh and Rajaian, 2013).

Measurement of thiopental concentration by HPLC method
The standard stock solution was prepared by dissolving 800 mg of thiopental in 100 ml of methanol. This solution was diluted with HPLC grade water (1:1) to precipitate the protein. 100 μl of ltered plasma was mixed with 200 μl of protein precipitating solution (100 ml of methanol with 0.5 g of zinc sulfate and 1 ml of ethylene glycol) for 10 seconds and then 200 μl of 10% trichloroacetic acid solution was added. The solution is then mixed again for 20 seconds and centrifuged at 4500 g for 5 minutes. After centrifugation, 20 μl of supernatant was prepared for injection in HPLC column. Acetonitrile was measured at 0.8 ml/min and acetic acid at 1% as the mobile phase at a ow rate of 1.2 ml/min with a wavelength of 260 nm (Raeeszadeh and Rajaian, 2013). Clinical parameters of return from anesthesia including eyelid re ex return, animal sitting, standing time, unbalanced walking time and normal walking time were also measured (Malayeri and Bakhtiari, 2018).

Data analysis
Data were reported as mean ± standard error of mean (Mean ± SEM). Data were analyzed by one-way analysis of variance (ANOVA) and to compare the mean of parametric data between the groups Tukey's postdoc test were used. Repeated measures ANOVA was used to analyze the data of pharmacodynamic parameters, heart rate and respiratory rate at the desired times. All statistical analyzes of the data were performed by SPSS (version 23) and p<0.05 was considered as a signi cant level.

Results
The results showed that the hematological and biochemical parameters of the studied groups were in the normal range (Table 1). Mean ±SEM of white and red blood cell counts were estimated to be 12.2±1.2 Í10 3 (meq/l) and 6.34±2.8 Í10 6 (meq/l), respectively. Hemoglobin levels were also estimated in the normal range (16.3±2 g/dl). Blood indices related to the volume and content of hemoglobin and red blood cells were estimated in the normal range. In this study, liver enzymes, albumin, creatinine and blood urea were measured to ensure liver and kidney function. Serum levels of enzymes AST and ALT were 24.5±15.2 and 41±15.8, respectively, and creatinine and urea levels were 12 and 45, respectively, indicating the health and proper function of these two tissues. The serum level of albumin and total protein was in the normal range.
According to Fig. 1, the heart rate before the start of the study and at the moment of injection, i.e., 0 minutes did not show a signi cant change among the studied groups. At 5, 10, 20, 30, 45, 60, 90 and 120 minutes after induction of anesthesia, the trend of data change was decreasing and there was no statistically signi cant difference in the studied groups (P> 0.05). The results showed that during the study time, heart rate in all groups signi cantly decreased compared to 5 minutes after the start of the study. In order to monitor changes in heart rate and possible arrhythmias, all animals before, at the moment of anesthesia and after that underwent ECG. In the ketoprofen group, premature atrial contractions (PACs) were seen after induction of anesthesia with thiopental, which indicates an arrhythmia caused by this drug interaction (Fig. 2), while no erythema was observed in other groups except bradycardia. According to Figure 3, the number of breaths between the studied groups before the start of the study and at minute 0 did not show a signi cant change (P> 0.05). Changes in respiratory rate in all groups during the study at the target times compared to the time before the start of the study and 10 minutes after induction of anesthesia were signi cantly reduced.
As shown in Table 2, the return time of palpebral re ex, sitting time, standing time, unbalanced walking time and normal walking time, there was a signi cant difference in group T1 compared to control, however, the differences between group T2 and control were not signi cant. (P <0.05). In the ketoprofen group, the recovery time was longer than the control group and the dexamethasone group.
All values are presented as mean ±standard error of mean (Mean ± SEM). Mismatched letters in columns indicate signi cant statistical differences. * P <0.05 The results of changes in thiopental concentration in control and experimental groups are shown in Figure 4. Table 3 shows the iodine weight and changes in pharmacokinetic parameters of the tested animals in different groups. The weight of animals in the control and experimental groups did not show a statistically signi cant difference (P = 0.76). Thiopental elimination rate in control group, T1 and T2 were 0.72±0.006, 0.61±0.004 and 0.71±0.005 h -1 , respectively. The reduction of drug elimination coe cient in T1 group compared to control was signi cant. The thiopental half-life was increased in experimental groups 1 and 2 compared to the control group. This increase was signi cant in control group 1 compared to the control (P = 0.045). the volume of thiopental distribution in group T1 was 3.419±0.026 and in control group and T2 were 2.81±0.038 and 2.74±0.021 L h -1 kg -1 , respectively. This increase in distribution volume was signi cant in T1 group compared to control and T2 groups. Thiopental clearance decreased in T1 and T2 groups compared to control, however, this decrease was not statistically signi cant (P> 0.05).

Discussion
It is important to understand the pharmacokinetic changes (PK) of drugs to minimize the required dose of drugs and their potential harm (Billard, 2015). This feature allows physicians and veterinarians to control the concentration of the drug at the site of action under conditions such as anesthesia. It also helps to achieve ideal surgical and uncomplicated recovery by titrating anesthesia according to the patient's needs (Billard, 2015). Due to the weak analgesic effects of thiopental, the use of analgesic and antiin ammatory compounds such as ketoprofen and dexamethasone could improve anesthesia and uncomplicated recovery after surgery (Vardanyan and Hruby, 2016).
The results of this study also showed that ketoprofen injection after induction of anesthesia by thiopental was seen in six of the seven dogs premature atrial contractions (PACs), the results that were not seen in the control and dexamethasone groups. Also, further evaluation of the electrocardiogram parameters in this group showed that this arrhythmia disappeared during the recovery period and completely disappeared after anesthesia, which in fact indicates an drug interaction in this group. PACs are early contractions that occur in the atrial myocardium and do not originate in the atrial sinus node (SA node) (Conen et al., 2012). PACs are also commonly referred to as atrial premature complexes (APCs), premature supraventricular complexes, premature supraventricular beat, and premature atrial beat. This phenomenon can be caused by a combination of medical diseases, structural abnormalities and drugs (Krasteva et al., 2006). The mechanism of this type of arrhythmia following ketoprofen administration is not well understood and requires a cardiovascular pharmacological study. However, we know that Ketoprofen is one of the propionic acid class of nonsteroidal anti-in ammatory drugs (NSAIDs) with analgesic and antipyretic effects. It acts by inhibiting cyclooxygenase-1 and -2 (COX-1 and COX-2) enzymes reversibly, which decreases production of proin ammatory prostaglandin precursors (Kantor, 1986).
In this study, we investigated the drug interaction of ketoprofen and dexamethasone with thiopental in healthy male dogs to improve anesthesia and uncomplicated recovery after surgery. In this study, we evaluated the anesthetic and pharmacokinetic parameters of thiopental along with changes in heart rate and respiratory rate after injection of ketoprofen and dexamethasone. The results of hematological and biochemical parameters of liver and kidney in studied animals and physical examinations con rm the relative health of animals in this study. Liver and kidney have also normal function. Therefore, it can be concluded that and the changes in clinical parameters and thiopental kinetics can be attributed to drug interaction conditions.
The results of recovery parameters in thiopental anesthesia showed a signi cant increase in the group receiving ketoprofen compared with dexamethasone. The mean heart rate in the experimental groups was higher than the control group. Except for a normal decrease in respiration rate during anesthesia, it did not show any signi cant change among the controlled and treated groups compared to before. ECGs taken from animals did not show cardiac arrhythmias, which may indicate some of the absence of side effects of this interaction at clinically prescribed doses. The evaluated pharmacokinetic parameters of thiopental showed a signi cant decrease in thiopental elimination coe cient and an increase in its halflife with ketoprofen. The volume of thiopental distribution in the ketoprofen group showed a signi cant increase compared to the control and dexamethasone groups, however, the decrease in thiopental clearance in the experimental groups was not signi cant compared to the control group.
To interpret these results, we rst discuss how to select the doses of drugs used in this study. The In these studies, the pharmacokinetic parameters of each of these drugs have been well studied. Due to the fact that the volume of distribution depends on the amount of cardiac output and the amount of binding to plasma proteins (Buur et al., 2009), and on the other hand, ketoprofen and dexamethasone in the previous studies caused an increase in heart rate (Margolis et al., 1987, Smith et al., 2010. Therefore, we can point to the competitive effects of thiopental in the presence of ketoprofen for binding to plasma proteins, which has increased the free form of the drug and thus increased its volume of distribution (Buur et al., 2009). Horie et al., (2009) in a pharmacokinetic study reported that the distribution of ketoprofen in tissues is very low and has the highest concentration in plasma (Horie et al., 2009). The results of our study showed an increase in thiopental half-life in the presence of ketoprofen and this increase in half-life was due to a decrease in thiopental drug elimination. Increasing the half-life of thiopental has also reduced its clearance in drug interaction conditions. In fact, this suggests that the a nity of thiopental to bind to plasma proteins is greater than that of ketoprofen. Burga et al. (1997) reported a 53% plasma protein bond for ketoprofen under exogenous conditions and stated that the compound could cause mutations in the binding site and drug interaction conditions with other drugs (Borgå and Borgå, 1997). Accordingly, the results of this study indicate drug interaction and thiopental binding site shift in the presence of ketoprofen. The concentration of thiopental in serum decreased at zero time and its distribution volume were increased. Increasing the free form of thiopental in the conditions of ketoprofen drug interaction causes its distribution in other tissues, especially adipose tissue, and this leads to an increase in the volume of thiopental distribution. The results of this study were in line with the study of Borgå and Borgå (1997) (Borgå and Borgå, 1997) and Raeeszadeh and Rajaian (2013) (Raeeszadeh and Rajaian, 2013) on the interaction of phenylbutazone with thiopental. Signi cant reduction of thiopental elimination and increase of thiopental half-life and consequently increase of its recovery time in comparison with dexamethasone and control group, indicates thiopental redistribution from tissues to blood in this interference (Heuberger et al., 2013, Smith et al., 2010, Gibaldi et al., 1978. Single dose of dexamethasone before surgery can control nausea and vomiting and to some extent pain after surgery (De Oliveira et al., 2011, Fujii andItakura, 2010). It was recommended that the use of dexamethasone in surgery at doses higher than 0.1 controls the postoperative pain and reduces the use of opioid analgesics (De Oliveira et al., 2011, Fujii andItakura, 2010). Considering the pharmacokinetic and clinical changes of dexamethasone drug interaction with thiopental, the controlled effects of this drug interaction and the analgesic and anti-in ammatory effects of dexamethasone and the control of anesthesia side effects, it can be suggested that dexamethasone be used instead of NSAIDs and opioids. We know that increasing the recovery time in conditions of thiopental drug interaction with ketoprofen and severe weakening of the respiratory center is very common in the simultaneous use of opioids (Boom et al., 2012). However, in completing this study, the use of dexamethasone and thiopental in combination under anesthesia and its analgesic and anti-in ammatory effects in clinical surgery is recommended.

Conclusion
Drug interaction of thiopental with ketoprofen by reducing the elimination and increasing the volume of distribution causes an increase in recovery time from thiopental anesthesia. However, dexamethasone at a dose of 0.2 mg/kg with insigni cant changes in the pharmacokinetic parameters of thiopental and its anesthetic parameters, can be a good option during surgery and anesthesia with thiopental.     Figure 1 Changes in heart rate in the studied groups before and after anesthesia. All values are presented as mean ± standard error of mean (Mean ± SEM). * is a signi cant difference within the group. C: Control group. T1: Treatment group 1. T2: Treatment group 2.

Figure 2
A typical sample of electrocardiogram (ECG) taken from animals in control and treatment groups before and 5 minutes afer thiopental injection.
Page 16/16 Figure 3 Changes in respiratory rate in the studied groups before and after induction of anesthesia. All values are presented as mean ± standard error of mean (Mean ± SEM). # indicates a signi cant statistical difference within the group Figure 4 Changes in thiopental concentrations in studied groups by HPLC method.