Effects of hydrogen peroxide-induced oxidative stress on the plasma concentration and pharmacokinetics of ketorolac in chicks

1. Hydrogen peroxide-inducedoxidative stress increased the analgesic efficacy of ketorolac in the chickens.
2. Hydrogen peroxide-inducedoxidative stress modifies the plasma concentration of ketorolac in the chickens.
3. Hydrogen peroxide-inducedoxidative stress alters the pharmacokinetics of ketorolac in the chickens. 

Introduction

Ketorolac is considered one of the most famous agents that belong to the first generation of non-steroidal anti-inflammatory drugs (NSAIDs), which have a therapeutic benefit for preventing nociception thus producing analgesia and its effect on lowering the pyresis and its anti-inflammatory action (1-3). Ketorolac’s effects inside the body were attained through the reversible and non-selective reduction of the cyclooxygenase (both the inducible and housekeeping enzymes), breaking-down arachidonic acid conversion to chemical mediator prostaglandins responsible for fever, pain, and inflammation production (1,4-6). Ketorolac has benefits as an analgesic to achieve moderate and severe nociception. In contrast, it has limited adverse effects rendering it an excellent, effective, inexpensive, and economical analgesia compared to the opioid analgesics (for example, morphine and tramadol), which resulted in serious adverse effects such as life-threatening respiratory depression, and addiction (7,8). The pharmacological mechanism of ketorolac at the non-opioid targets diminishes the potential risk of simultaneous adverse effects like hemodynamic imbalance, respiratory depression, and centrally acting adverse effects (9,10). Ketorolac is also valuable for use as a postoperative analgesic because of the inflammation and pain produced by surgical operation (9,11). Otherwise, ketorolac is extensively bound more than 99% to albumins (plasma proteins) and has a small volume of distribution comparable with those of other NSAIDs (11-13).

Many stressful conditions, such as chemical (for example H₂O₂) and physical (like heat) stressful factors are well-known to induce modifications in the pharmacological drug response (14-16). H₂O₂ has formerly identified as changes in the sedative effect of diazepam (17,18) and xylazine (19), modifying the anesthetic action of ketamine in the chicks, which is supposed to have numerous adverse effects in the stressed animals (20). Besides modifying the pharmacological effects of xylazine and diazepam efficacy (20), and thiopental anesthesia (21), which is particularly important for the drugs with a narrow margin of safety. H₂O₂-inducesOS through elevating the reactive O₂ species and decreasing the antioxidant capacity, therefore, raising the free radicals components that cooperate and alter the viable functions of the cells, especially the receptor targets accountable for pharmacodynamics and pharmacokinetics of the administered drugs to the stressed animals (22-24), and also can destruct the blood-brain barrier (25,26) therefore altering the drug distribution and concentration at the specified tissues.

Due to ketorolac having fewer adverse effects with multiple practical therapeutic effects, like analgesic, antipyretic, and anti-inflammatory, the study aimed to use ketorolac and determine the effect of OS induced by H₂O₂ at the plasma concentration and pharmacokinetics of ketorolac in the chicks model.

Materials and methods

Lab animals and preparation of drugs

The broiler chicks of 7-14 day-old were cast-off in the experiments delivered from a local chick hatchery. The mean bodyweight of the chick was 72-110 g, and they were well-kept at 29-36^○C, with constant lighting. The experimental chicks have been permitted water and food freely. Ketorolac (3% Ketorolac trometamol, Spain) doses were adjusted by a 0.9% sodium chloride intended to be administered intramuscularly (IM).

Ethics

The methods, experimental design, and experimental chicks have been authenticated through the scientific board employed at the University of Mosul, College of Veterinary Medicine.

Induction of OS by using H₂O₂

At one day old, the chicks were separated randomly into the control (H₂O) group (supplied tap water) while the remaining H₂O₂ group of chicks had supplied with daily H₂O₂ (Scharlau, Spain) as 0.5% in the consumed water (17-19). On days 7^th, 10^th, and 14^th of chickens, the chicks were subjected to blood collection to obtain plasma using heparin (1:10 v/v). The plasma was then measured by a specialized kit (Solarbio, China) of total antioxidant status (Catalog No. BC1310) in the H₂O₂ group of chicks compared to the H₂Ogroup of chicks (27,28). The age of OS confirmed in the chickens will be used in the following experiments in this study.

Analgesic ED₅₀ in the H₂O and H₂O₂ groups of chicks

The analgesic ED₅₀ of ketorolac was done by using the up and down method (29) for the H₂O and H₂O₂ chick groups to choose the ketorolac dose, which will be used in the following experiments. The first ketorolac dose was at 13 mg/kg, IM (30) for both groups of chicks. The increase or reduce the dose was at 3 mg/kg. The analgesia was obtained using the electro stimulator apparatus (Harvard apparatus, USA) (30-34). Ketorolac injection, before and after 30 minutes, the distress call indicated nociception was recorded for each chick separately. Ketorolac, then considered to have an analgesic effect by the voltage increases at post-injection comparable to the volts noted pre-injection appointed as X symbol (34,35). In contrast, if not, the symbol marked as O. The following formula was applied to determine the effect of the OS on ketorolac’s analgesia:

% OS effect on ketorolac's ED₅₀= ED₅₀ of H₂O group - ED₅₀ of H₂O₂ group / ED₅₀ of H₂O group × 100.

Plasma concentration of ketorolac for the H₂O and H₂O₂ chicks

One single dose of ketorolac at 14 mg/kg, IM, (which resembles the total dose of ketorolac determined in the above groups of the preceding experiment), to H₂O and H₂O₂ chicks. The blood was collected from five chicks for each measured times of 0.25, 0.5, 1, 2, 4, and 24 hours from the control and stressed chicks’ groups. At that moment, adding the heparin anticoagulants (Braun Inc., USA) at a 1: 10 v/v to the tubes containing blood, then centrifuged (3000 rpm for at least 15 minutes) (Chalice, UK) to obtain the plasma that was frozen awaiting spectrophotometric analysis for three days (36).

Spectrophotometric analytical procedure

Preparing and calibrating the ketorolac standards

The standards were made of 6, 12, 24, 48, 96, and 192 µg/ml of ketorolac diluted in acidic methanol. Then, the absorbance of optical density was determined using spectrophotometry (Lovibond, Germany) at a wavelength of 319 nm (36). The absorbance of samples was determined against the blank sample consisting of acidic methanol alone. The standard curve was used to estimate the ketorolac concentration in the plasma. The calibration curve equation for ketorolac standards revealed a determination coefficient that is R² as 0.9858 (Figure 1). The concentration of ketorolac in plasma was then estimated for the H₂O and H₂O₂ groups: y = a + b x (y = 0.2827 + 0.0087 x) (Figure 1); y means the optical density of ketorolac at the plasma measured at 319 nm; a is the intercept; b is the slope of calibration curve while x is the ketorolac plasma concentration.

Figure 1: Ketorolac's standards of 6, 12, 24, 48, 96, and 192 µg/ml with their absorbance of 319 nm

Extraction with an estimation of ketorolac at the plasma

The extract of ketorolac from the plasma samples was made from a simple, acknowledged, and specific procedure for estimating ketorolac concentration in the plasma (37). The ketorolac concentration was measured at different measured times of 0.25, 0.5, 1, 2, 4, and 24 hours after ketorolac injection for both the control and stressed chicks. The method is illustrated by adding 1 ml of precipitating agent 10% trichloroacetic acid to 1 ml of plasma and vortexes for 2 min. Furthermore, the final solution was then centrifuged (3000 rpm for 10 min). The supernatant was used for spectrophotometric examination (with 319 nm of wavelength) using the cuvette to determine the optical density for every anonymous plasma sample. The blank solution was made of 10% trichloroacetic acid.

Pharmacokinetic profile of ketorolac in the H₂O and H₂O₂ chicks

The pharmacokinetics profile of ketorolac was estimated using the non-compartmental model of measuring in the H₂O and H₂O₂ chicks and using a program named PKSolver (38). The parameters of pharmacokinetics included the AUMC_0-∞ (µg.h²/ ml), AUC_0-∞ (µg.h/ ml), C_max (µg/ ml), K_el (0.693 / t_1/2β)(h^-1), T_max (h), MRT (AUMC /AUC)(h), t_1/2β (h), V_ss [dose. AUMC /(AUC)²](L /kg), and Cl (dose / AUC)(L/h/kg). Following percentages of rising or decreasing the ketorolac pharmacokinetics profile were estimated in the H₂O chicks and then compared to the pharmacokinetic parameters in the H₂O₂ chicks to determine the changes in the pharmacokinetics to the influence of OS.

Statistical evaluation

The means of two groups of parametric data were analyzed and compared using the unpaired student T-test. The one-way analysis of variance was employed for comparing the means of three parametric groups (39,40). Significance deemed to all data as P < 5%.

Results

Induction of OS with H₂O₂

Table 1 shows a significant decrease in the total antioxidant status and subsequent occurrence of OS in the H₂O₂ group of chicks compared to the H₂O group on days 7^th, 10^th, and 14^th of chickens treated with H₂O₂ by 39, 29, and 41%, respectively.

Table 1: Measuring the Total Antioxidant Status in the H₂Oand H₂O₂ chickens

Numbers denoted mean ± Std Err. of concentration in µmol/ml for six chickens per group. The freshwater was supplied for the control (H₂O) group; H₂O₂ (0.5%) was added for the H₂O₂ group (day 1 to day 14 of chicks’ age). *Different significantly from the H₂O chicks (P < 5 %). % inhibition= H₂O - H₂O₂ / H₂O ×100.

Analgesic ED₅₀ in the H₂O and H₂O₂ groups of chicks

The ketorolac analgesic effect has changed with amplification in the H₂O₂ group through assessing the ketorolac analgesia ED₅₀. The ED₅₀ was 7.79 mg/kg, IM in the H₂O group whereas it decreased by 16% of the H₂O₂ chicks because of H₂O₂- induced OS to 6.58 mg/kg, IM as in table 2.

Table 2: ED₅₀ of ketorolac in the H₂O and H₂O₂ chicks

^*X is the analgesia while O is no analgesic effect

Plasma concentration of ketorolac for the H₂O and H₂O₂ chicks

A significant increase was observed for the ketorolac plasma concentration in the H₂O₂ chicks group associated with the H₂O group measured except 0.25 and 24 h after ketorolac administration. The plasma concentration of ketorolac in the control group 14 mg/kg, i.m. assessed at multiple times of measurements 0.25, 0.5, 1, 2, 4, and 24 hours were 110.38, 181.46, 66.24, 13.08, 10.11, and 4.12 µg/ml. The plasma concentration of ketorolac in the H₂O₂ chicks was elevated to 136.45, 250.88, 102.03, 39.13, 19.55, and 6.55 µg/ml by 24, 38, 54, 199, 93, and 59 %, correspondingly showed in table 3 and figure 2.

Table 3: Ketorolac plasma concentration in the H₂O and H₂O₂ chicks

Numbers are mean ± SE for 5 chicks in the assessed time. Ketorolac administered at 14 mg/kg, IM. ^*Differ significantly from the H₂O group (p < 0.05). % Influence of OS on ketorolac plasma concentration = H₂O₂ – H₂O / H₂O × 100.

Figure 2: Ketorolac plasma concentration in the H₂O and H₂O₂ chicks

Pharmacokinetics profile of ketorolac in the H₂O and H₂O₂ chicks

Administration of ketorolac in the H₂O group of chicks elucidate the pharmacokinetic parameters included AUC_0-∞ 400.72 µg.h /ml, AUMC_0-∞ 5251.36 µg. h²/ml, C_max 181.46 µg/ml, K_el 0.049 h^-1, T_max 0.5 h, MRT 13.10 h, t_1/2β 14.03 h, V_ss 0.71 L /kg and Cl 0.035 L/h/kg. The AUC_0-∞, AUMC_0-∞, C_max, and K_el values of the H₂O₂ chicks treated with ketorolac were raised to 636.90, 6231.07, 250.88, and 0.070 by 59, 19, 38, and 43%, respectively whereas other pharmacokinetic parameters included MRT, t_1/2β, V_ss and Cl were reduced to became 9.78, 9.84, 0.31, and 0.022 by 25, 30, 56 and 37% respectively compared to the H₂O group (Table 4).

Table 4: Pharmacokinetic parameters of ketorolac in the H₂O and H₂O₂ chicks

Ketorolac administered at 14 mg/kg, IM. Pharmacokinetic variables were estimated by using the non-compartment model and measured by usage of the program of PKSolver. % impact of OS at ketorolac pharmacokinetic parameters = H₂O₂ – H₂O / H₂O × 100

Discussion

The goal designed for this study was composed of administering ketorolac and determination of the impact of H₂O₂-induced OS on the ketorolac plasma concentration and pharmacokinetics in the chicks. Ketorolac is considered one of the most famous agents that belonging to the NSAIDs which have a therapeutic benefit for preventing nociception, thus producing analgesia as reported in this study, besides its effect on lowering the pyresis and its anti-inflammatory action (1-3). Ketorolac’s effects inside the body, which causes analgesia, were attained through the reversible and non-selective reduction of the cyclooxygenases, both the inducible and housekeeping enzymes, therefore breaking-down arachidonic acid conversion to chemical mediator prostaglandins responsible for fever, pain, and inflammation production (1,3).

This trial used H₂O₂ as a known powerful oxidant to induce OS in various animals as experimental models (17-19). Total Antioxidant status is a valuable key indicating the occurrence of OS in the body (36), which is used here in this study to indicate at which day of treatment with H₂O₂ was OS occur, and based on these days, the subsequent experiments are conducted.

The reasons ascribed to the amplification in the ketorolac effects (as noticed here through rising the ketorolac plasma concentration) are called the H₂O₂-inducesOS through elevating the reactive O₂ species and decreasing the antioxidant capacity. Therefore, rising the free radicals’ components that cooperate and alter the viable functions of the cells, especially the receptor targets (like COX) (14-16) and binding sites of ketorolac which are enrolled in pharmacodynamics (efficacy and affinity) of ketorolac.

H₂O₂ can destruct the cytochrome P₄₅₀ enzymatic system postponing the ketorolac elimination and increasing its plasma concentration. The damage it causes is projected on the exact ketorolac protein-bound sites on the plasma proteins (albumins). This is because ketorolac is extensively bound with more than 99% of albumins. Ketorolac also has a low distribution volume to the target site of action compared with other NSAIDs (6,14-16) reported from rising off the pharmacokinetic elements like AUC, AUMC, C_max and K_el.It decreases the other crucial elements like MRT, t_1/2β, V_ss and total Cl which are all responsible for modifying ketorolac’s pharmacokinetic property and subsequently improving ketorolac’s pharmacological effects are following the previous study (13).

Conclusion

The results showed that the H₂O₂-inducedOS amplified the analgesic action of ketorolac in a chick model; besides its modification of the plasma concentration and pharmacokinetics of ketorolac, it recommended that the dose of ketorolac modified as a therapeutic regimen was prepared in the stressed animals.

Acknowledgments

Many greetings and appreciation to the University of Mosul / Veterinary Medicine College for the tools used to accomplish this research.

Conflict of Interest

The authors declare there is no conflict of interest.