Evaluation of the advantages of orphenadrine in anaesthesia caused by ketamine in mice

1. Orphenadrine has many advantages on anesthesia by ketamine
2. Orphenadrine produces a muscle relaxant effect during anesthesia, unlike ketamine
3. Orphenadrine overcomes the body reflexes that are persistent in ketamine
4. The pharmacological interaction between orphenadrine and ketamine was synergistic.

Introduction

Anesthesia is a physiological condition that generates a regulated, temporary lack of sensation or consciousness for veterinary or medical applications (1,2). The ideal anesthesia depends on the concurrent treatment of diverse medications to achieve balanced anesthesia. The classic anesthetic state comprises essential drugs formed from hypnotics, painkillers, and muscle relaxants. It is mainly the analgesic support of this triad that has become increasingly reinforced by accessory medicines (3). Ketamine is an injectable anesthetic that is widely used in humans, especially in pediatric and veterinary surgery (4); it produces dissociative anesthesia and good analgesic, which has minimal respiratory depression and potent sympathetic effects on the cardiovascular system that are characterized by increases in inotropic and chronotropic and blood pressure (5,6), because of its psychotic side effects and inability to relax skeletal muscles (7,8) adequately, the limited use of ketamine can be treated by discover balanced ideal anesthesia to decrease the required doses needed for each drug while still producing the desired clinical effect (9). An anticholinergic drug called orphenadrine is primarily used to treat Parkinson's disease to reduce some of its bothersome symptoms, mainly resting tremors (10,11). Orphenadrine is a medication that blocks muscarinic receptors (12), histaminic H1 receptors (13), N-methyl-D-aspartate (NMDA) receptors (13), voltage sodium channels, and potassium channels (14). Furthermore, it has an inhibitory effect on norepinephrine and dopamine reuptake (15). Orphenadrine applies unspecific antagonistic activity at the phencyclidine binding site of NMDA receptors, one of the subtypes of glutamate receptors (16). Orphenadrine has many therapeutic uses, such as muscle relaxant by central mechanism (17), pain killer mainly in combination with NSAIDs (18), and has activity in the treatment of Parkinson's disease (19). Our main aim of this study was to evaluate the possible effect of the coadministration of orphenadrine and ketamine on the quality of the anesthetic state. We put a hypothesis that the administration of orphenadrine might counteract muscle rigidity made by ketamine.

We assessed the effects of orphenadrine on onset, duration, and recovery in mice anesthetized with ketamine in addition to an evaluation of body reflexes and determined the type of interaction with ketamine at a level of analgesic activity.

Materials and methods

Ethical approval

Ethical approval for the study was obtained after the Institutional Animal Care Committee formed in the College of Veterinary Medicine at the University of Mosul, which is consistent with the principles of international ethics in dealing with animals, reviewed the application submitted to it by us on 15/3/2023 with the code UM.VET.2023.006.

Animals

Male albino mice weighing 35-40 grams were used in this study, and they were purchased and bred in the laboratory animal house in the College of Veterinary Medicine, University of Mosul. The mice were adapted in specially manufactured plastic cages for mice and allowed free access to water and food in a minimum pathogen-free laboratory animal room (12/12-h light/dark cycle, 24±2°C; relative humidity: 56%±2%). All experiments were performed between 8:00 a.m. and 2:00 p.m. to diminish the confounding effects of circadian timing on the experimental results. It is important to remember that the mice's cages have been washed once a week by changing the soiled bedding and cleaning down the remaining cages.

Drugs

Orphenadrine citrate injection 30mg/ml Teva®, and ketamine (10% INJ, Dutch Farm.) was further diluted in a saline solution to gain the recommended doses of the drugs. The administration volume of each drug was 10mg/ml body weight administered intraperitoneal.

Study design

Eighteen male albino mice were distributed into three equal groups: the first group was injected with ketamine 100mg/kg intraperitoneal (IP); the second group was administered with ketamine 100mg/kg and orphenadrine 10mg/kg IP simultaneously, the third group was administered with ketamine 100mg/kg and orphenadrine 20mg/kg simultaneously. The dose of ketamine and orphenadrine in our study was chosen based on previous studies (20,21). The intraperitoneal injection was accomplished adjacent to the midline near the umbilicus with a 25-gauge needle inserted at a 45° angle to the belly wall in the lower left guardant of the belly.

Anesthetic activity evaluation

After drug injection, the mouse was retained alone in a crate with a sawdust floor until loss of righting reflex (defined as incapability to revert to ventral recumbency) was achieved. This interval was known as the onset of anesthesia (Table 1). The pedal withdrawal reflex (assessed by pinching the tail and the metacarpal region of the hind foot between the finger and the thumb) was monitored every 5 minutes. The interval between the loss of the righting reflex and the return to the righting reflex is known as the duration of anesthesia, and the interval between the return to the righting reflex is known as recovery (Table 1) (22). Muscle tone was assessed by gently touching the caudal thigh, lumber, and temporal muscles for the presence or absence state with the tip of a finger (23). The corneal reflex was assessed by touching the cornea of a mouse with a cotton swab and noting if the mouse blinks or shakes its head (24).

Table 1: Anesthetic activity evaluation

Evaluation of analgesic activity

Using the tail immersion test (25), the median analgesic doses of orphenadrine and ketamine alone or in combination were calculated using the up-and-down method (26). The tail immersion test was carried out by holding the mouse and immersing its tail in a water bath at a temperature of 55-56°C; then, we calculated the time required to retract the tail in seconds with a stopwatch. The tail immersion test was conducted after 30 minutes of intraperitoneal injection of the drugs (27,28). The first dose of orphenadrine was 10mg/kg and ketamine 3mg/kg, whereas, in combination, the first doses of orphenadrine and ketamine were 7 and 1.5mg/kg, respectively (0.5:05 of their individual ED₅₀ values). The interaction index was determined using the following equation, were Y=(da/Da)/(db/Db). da: ED₅₀ of orphenadrine in combination with ketamine. db: ED_{50 of} ketamine in combination with orphenadrine. Da: ED₅₀ of orphenadrine alone. Db: ED₅₀ of ketamine alone (29-31). The resulting number indicates the type of interaction between the two drugs. If the number is less than one, the interaction is considered synergistic. If the number is greater than one, the interaction is considered antagonistic. If the number is equal to one, the interaction is additive. The analgesic doses of our drugs were chosen from previous studies (30,32).

Statistical analysis

The data obtained from the anesthesia experiment were analyzed using a statistical program (SPSS), where the results were analyzed using a one-way analysis of the variance test and least significant difference tests. The data are expressed as the mean ± standard error, and the values were considered statistically significant when the probability (P) was less than or equal to 0.05.

Results

Comparison of anesthetic effects

Mice in all groups lost their righting reflex in 55-109 seconds; there was a significant dose-dependent decline in the onset time in the group injected with orphenadrine 20 mg/kg plus ketamine compared to the group injected with orphenadrine 10 mg/kg and ketamine alone. There was a significant dose-dependent rise in the period of anesthetic time in the group injected with orphenadrine 20 mg/kg plus ketamine compared to the group injected with orphenadrine 10 mg/kg and ketamine alone. There was a significant decrease in recovery time among groups treated with orphenadrine in comparison with a group of ketamine alone. All mice recovered from anesthesia and were returned to the home cage following treatment (Table 2).

Administration of the anesthetic dose of ketamine alone caused death in 33% of mice, whereas this dose caused the presence of tail pinch, corneal, and toe pinch reflexes at 100%, and the muscle tone appeared at 100%. The depth of anesthesia appeared to be more typical in the groups treated with orphenadrine and ketamine (Table 3). Table 3 reveals that the analgesic ED_50s for orphenadrine and ketamine were 13.4 and 2.4mg/kg, respectively. However, the coadministration of orphenadrine and ketamine at a static ratio (50%:50%) of their separate ED₅₀ value were 4.3 and 0.8mg/kg, respectively (Table 4). Calculation of the interaction index revealed that the coadministration of orphenadrine and ketamine had a synergistic index on initiating analgesia in our laboratory animals. The interaction index (Y) was 0.65 between both drugs (an index of < 1 indicated synergism).

Table 2: The anesthetic effect of coadministration of orphenadrine and ketamine

Table 3: The effect of coadministration of orphenadrine and ketamine on the depth of anesthesia

The value represented as (mean ± SD) for 6mice per groups. *Significantly (P< 0.05) different from corresponding values from group 1. a Significantly (P< 0.05) different from corresponding values from group 2.

Table 4: The interaction of analgesic effect of orphenadrine-ketamine coadministration

X= Death. O= alive.

Discussion

Mice and rats are widespread laboratory animals and are often available to veterinarians for assessment and medical management (33). Anesthesia in pet animals is essential for many surgical and diagnostic procedures, and it is related to advanced perioperative hazards in rodents and rabbits compared to canine and feline (33-35). There are many advantages to injectable anesthesia, such as needing no more instruments, administration, and dosing are very easily educated and calculated (23). The mixture of ketamine and xylazine is one of the most commonly used anesthetic regimens in rats and mice (8,36,37). Bradycardia and hypotension are the main side effects of xylazine/ketamine in mice (36), cows (38), sheep, and goats (39). The primary objective of our study was to evaluate a new mixture of anesthetics, orphenadrine, and ketamine to reduce the side effects of ketamine/xylazine. The main side effects of using ketamine alone were muscle stiffness, the presence of body reflexes during anesthesia, and the adverse effects of xylazine on laboratory animals and ruminants.

One of the most essential clinical uses of orphenadrine is muscle relaxation, owing to the multiple mechanisms through which it works on the body. Therefore, this pharmacological action affects the quality of anesthesia when used with ketamine, which lacks the most important element of ideal anesthesia: the relaxation of skeletal muscles. Muscle relaxation is necessary when performing surgical operations, as the surgical incision of the skin and muscles during the operation is easier with relaxed muscles through a small incision and then pulling the skin and muscles with special tools, furthermore, it’s very important. It is a fundamental process during abdominal closure (40). Thus, wound healing was faster. One of the critical things we noticed in mice anesthetized with a combination of orphenadrine and ketamine is the disappearance of body reflexes, which may constitute an obstacle to the surgeon when choosing ketamine alone. Anesthesia mixtures of xylazine and ketamine are common in veterinary medicine, especially in small animals such as dogs and cats, and laboratory animals such as rodents (41), hamsters (42), guinea pigs (43), and rabbits (44); however, ruminants are very sensitive to xylazine, and their doses must be calculated carefully because of their side effects (45). Therefore, our study aimed to evaluate the efficacy of orphenadrine as a substitute for xylazine in combination with ketamine. To the best of our knowledge, no study has evaluated the combination of anesthesia orphenadrine with ketamine in humans or animals. Our findings indicated that the administration of orphenadrine decreased the onset of anesthesia and increased the duration of anesthesia with ketamine in mice; the expected hypothesis for these effects may be that the two drugs have similar mechanisms of action on NMDA receptors. The NMDA receptor is an ionotropic receptor that permits electrical impulses to be transferred between neurons in the brain and the spinal cord neurons. The NMDA receptor must be open for electrical signals to pass; glycine and glutamate have an agonist activity on the NMDA receptor to maintain keep it open. Ketamine and orphenadrine are non-competitive antagonists of NMDA receptors (16). The anesthetic effect of ketamine was not attributed to antagonistic activity on NMDA receptors only, but it acts on muscarinic receptors, opioid receptors, monoaminergic receptors, and voltage-sensitive calcium ion channels. Ketamine, unlike other general anesthetic agents, does not affect GABA receptors (46). After evaluating the anesthetic action of the mixture of orphenadrine and ketamine, we decided to evaluate the analgesic action of this mixture, as pain relief is one of the aspects of ideal anesthesia. The mechanism of action of these two drugs is via the same NMDA receptor. Orphenadrine is used as a painkiller alone or in combination with paracetamol (47) and diclofenac (18). A molecular study refers to a new mechanism action of the analgesic effect of orphenadrine that inhibits diverse subtypes of voltage-gated sodium channels at recommended doses, including the Nav1.7, Nav1.8, and Nav1.9 channel subtypes that are primarily involved in pain (13). Pain, inflammation, and some toxicological and behavioral studies on laboratory animals are trustworthy and their scientific findings can be relied upon (48-51). Further studies should be conducted in dogs and cats.

Conclusions

In conclusion, administering orphenadrine with ketamine has many advantages; it produces safe, smooth, and surgical anesthesia, and the unwanted effects of ketamine can disappear with orphenadrine. Orphenadrine and ketamine has a synergistic analgesic effect.

Acknowledgments

We (authors) extend our thanks and gratitude to the Deanship of the College of Veterinary Medicine at the University of Mosul and the Presidency of the Physiology, Biochemistry, and Pharmacology department for their care of the research needs.

Competing interests

The authors declare no competing interests.