JM-1232(-) and propofol, a new combination of anesthetics with short-acting and non-cumulative preferable properties

Background Drug interactions are significant in anesthesiology because drug combinations can potentially possess novel properties. The pharmacological advantages of a new combination of the benzodiazepine receptor agonist JM-1232(-) and propofol were investigated in mice. Methods Male adult mice were administered JM-1232(-) or propofol or combinations of the two drugs intravenously. Loss of the righting reflex was evaluated as achieving hypnosis, and the time until recovery of the reflex was measured as hypnosis time. After determining the ED50, doses double and triple the ED50 of propofol were injected with JM-1232(-) to compare hypnosis time. The injections were repeated four times, and the hypnosis times were compared. Flumazenil was administered separately immediately after the last dose was injected.


Results
The ED50 values ([95% confidence interval]) for hypnosis were 3.76 [3.36-4.10] for JM-1232(-) and 9.88 [8.03-11.58] mg kg-1 for propofol. Co-administration of 0.05 and 0.1 mg kg-1 JM-1232(-) reduced the ED50 values of propofol to 1.76 [1.21-2.51] and 1.00 [0.46-1.86] mg kg-1, respectively. The drug combination for hypnosis produced a supraadditive interaction. Hypnosis time was significantly shorter in the groups given the mixtures compared to each hypnotic administered alone. After repeated injections, hypnosis time with the mixtures showed smaller prolongation than that with the hypnotic alone. Flumazenil completely restored the recovery time after anesthesia.
Conclusions The combination of JM-1232(-) and propofol showed a supra-additive interaction, and the reduced hypnotic dose contributed to a faster recovery even after multiple injections. Background 3 JM-1232(-) is a newly developed isoindoline derivative and potential anesthetic with a short duration of action 1,2 . JM-1232(-) is water soluble and is highly potent. Although the molecular structure of JM-1232(-) is different from classical and typical benzodiazepines, JM-1232(-) enhances synaptic inhibition by modulating benzodiazepine binding sites on γaminobutyric acid A receptors, similar to benzodiazepine derivatives 3 . The pharmacological parameters of JM-1232(-) might be suitable for supporting general anesthesia and intensive care drugs 4 . Moreover, JM-1232(-) has been administered to humans as "MR04A3," and demonstrated favorable and acceptable profiles in a preclinical trial 5 .
Propofol is one of the most popular intravenous anesthetics in daily clinical practice 6 . The characteristics of propofol, including rapid onset and prompt recovery, make it an appropriate drug for general anesthesia. However, a long-lasting infusion of propofol might lead to prolongation of its effect and delay recovery 7 . The dose of propofol required to induce anesthesia can be reduced by a series of pre-medications 8,9 . Some of these drugs, such as benzodiazepine derivatives, significantly enhance the hypnotic activity of propofol 10,11 . Thus, co-administration of JM-1232(-) could reduce the required dose of propofol. Moreover, the reduced propofol dose might lead to faster recovery.
In the current study, the interaction between JM-1232(-) and propofol was evaluated in an in vivo investigation using mice. The anesthesia and recovery profiles were investigated after repeated injections of the drug mixture, which simulated prolonged infusion. Finally, the animals were administered flumazenil to assess its antagonistic effects after longlasting anesthesia.

Methods 4
After obtaining approval from the Ethics Committee for Animal Experiments at our institution (Final registration number: 26401), all experiments were performed in an animal laboratory. Male adult Deutsch-Denken-Yoken (ddY, closed colony) mice weighing 38 to 45 g (SLC Japan, Nagoya, Japan) were used. The animals were maintained on a 12/12-h light/dark cycle and fed ad libitum before the experiments. All experiments were conducted between 10 a.m. and 4 p.m. The mice were examined three times at most and had a recovery period of more than 7 days.
The mice were set in a transparent animal holder to place a 24 G plastic IV cannula (SurFlo, Terumo, Tokyo, Japan) into the tail vein. After confirming venous catheterization by checking the backflow of blood, another customized injection needle connected to a micro-syringe was set into the plastic cannula, and the prepared material was quickly injected over 2-3 s. If the injection was irregular and incomplete, such as being resistant or showing extravasation, the mouse was omitted from the study. Mice were released from the animal holder and individually evaluated for hypnosis on a flat table by another observer. The criterion for hypnosis was loss of the righting reflex, occurring <10 s after the start of the injection 12, 13 . When hypnosis was observed, the mice were gently placed in the lateral decubitus position until spontaneous recovery to the upright position, which was defined as the end of hypnosis. The time from the start of drug injection to the end of hypnosis was defined as hypnosis time. Hypnosis time was recorded in the laboratory room and was verified by another blinded technical assistant using recorded movies of the experiments on another day. The animals were killed by carbon dioxide inhalation after the final experiments.
All solutions were mixed with the same volume of diluent and administered intravenously.
Injection volume was set at 10 ml kg -1 in experiment 1 and at 5 ml kg -1 in experiments 2 and 3. The experimental doses of JM-1232(-), propofol, and the drug mixtures were calculated based on past and preliminary experiments 12, 13 ( Table 1). The ED 50 of propofol was first tested, and then other combinations were tried until all animals in the group showed the same response (hypnosis or not). After the same injections were performed as in experiment 2, the last (fourth) injection of the hypnotic drug was immediately followed by administration of 0.2 mg kg -1 flumazenil (5 ml kg -1 ). Each group consisted of six animals. Anesthesia time after the injection was measured and was compared with the results of experiment 2.
After the experiments, all animals were killed with inhalation of pure carbon dioxide more than 5 min following to the regulation of our institute.
The sample size of the study was determined following a previous investigation 12, 13 . To analyze the 50% effective dose (ED 50 ) and the 95% confidence intervals (CIs) for loss of the righting reflex, we determined the number of animals that lost the righting reflex from the total that received an assigned pharmacological treatment and correlated the results with the probability of their being under hypnosis using nonlinear least-squares logistic regression. The results for the required propofol dose for each group are presented as the ED 50 and 95% CI.
Hypnosis time is expressed as mean ± standard deviation. Analysis of variance (ANOVA) was used to compare the hypnosis time among groups, and the Newman-Keuls post hoc multiple-comparison test was used when ANOVA showed a significant difference (P < 0.05). All calculations were performed using a statistical software package (SPSS 24, IBM Japan, Tokyo, Japan).

Results
The rate of successful injections was 85%. Although a few animals showed signs of temporal respiratory depression (hypopnea) immediately after the injection, there was no animal death during the study. The limitations of the current study should be addressed. We could not predict the precise properties of the interaction; thus, the dose of the drugs and the sample size of the study were determined following the results of our previous investigation with extrapolation 13,23 .
Diazepam is a popular benzodiazepine, but a lipophilic agent and insoluble in water, whereas midazolam is a water soluble benzodiazepine. However, our preliminary experiments showed that the potency of midazolam in mice is very low, which is similar to the results of Kilpatrick et al. 24 Thus, the highly effective JM-1232(-) was chosen as the supplemental drug for co-administration with propofol in the current investigation. Coadministration of midazolam decreases the time to achieve hypnosis without delaying emergence during short-term propofol anesthesia in daily clinical settings 10,23 . Not only JM-1232(-) but also other benzodiazepines might well produce similar results to those seen in the present study. However, JM-1232(-) has a short duration of action, which makes it more suitable for use as a supplementary drug 25 .
Another limitation was that we only evaluated the dose required to achieve hypnosis and the hypnosis time. Due to technical problems, the blood concentration of each drug was not determined. An electroencephalographic analysis might be useful to compare the synergistic effects of the drugs from a pharmacodynamic point of view 26,27 . Therefore, further investigation is required. Consent to participate is not applicable for the study.

Consent for publication
Not applicable for the study.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests

Funding
The part of the current investigation was funded by Grant-in-Aid for Scientific Research, Japan Society for the Promotion of Science, 25462902. The funder officialy gave the endowment after the review of the study protocol as competitive funds.

Authors' contributions
ST performed the experiment, writing the manuscript.
MH helped to writing and correction of the manuscript and conducting the study.
NM helped and directed study.
MS organized the study and writing the manuscript.
TT performed the experiment and writing the manuscript.
YUA conducted the study design, performed the experiments and data analysis, and writing the current manuscript.
ASB directed the study and revised the manuscript.
MO organized the study and examined pharmacological procedures.
All authors have read and approved the manuscript.  The ratios of responders to total number of animals (n = 6) are expressed as the percentage (%).

Supplementary Files
This is a list of supplementary files associated with the primary manuscript. Click to download.