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The Efficacy and Safety of the Novel Peripheral Analgesic Isovaline as an Adjuvant to Propofol for General Anesthesia and Conscious Sedation: A Proof-of-Principle Study in Mice

Whitehead, Ryan A. PhD*; Schwarz, Stephan K. W. MD, PhD, FRCPC*†; Asiri, Yahya I. BSc*; Fung, Timothy BSc*; Puil, Ernest PhD*; MacLeod, Bernard A. MD, FRCPC*

doi: 10.1213/ANE.0000000000000996
Anesthetic Pharmacology: Research Report

BACKGROUND: The combination of propofol and an opioid analgesic is widely used for procedural sedation, as well as total IV anesthesia. However, opioids produce respiratory depression, a primary cause of death due to these agents. We recently reported on the antinociceptive actions of isovaline, a small nonbiogenic amino acid that does not readily cross the blood-brain barrier and acts on peripheral γ-aminobutyric acid type B receptors. Here, we explored the possibility that isovaline may be an effective and safe alternative to opioids as an adjunct to propofol for producing anesthesia.

METHODS: With approval from our Animal Care Committee, we conducted an in vivo study in adult female CD-1 mice using Dixon’s “up-and-down” method for dose assessment. Animals received intraperitoneal saline, propofol, isovaline, fentanyl, or coadministration of propofol with isovaline or fentanyl. We assessed hypnosis by a loss of righting reflex and immobility by an absence of motor response to tail clip application. General anesthesia was defined as the presence of both hypnosis and immobility. We assessed conscious sedation as a decrease in time on a rotarod. The maximal dose without respiratory rates of <4 per minute, apnea, or death was defined as the maximal tolerated dose.

RESULTS: Either isovaline or fentanyl coadministered with propofol at its half-maximal effective dose (ED50) for hypnosis produced general anesthesia (isovaline ED50, 96 mg/kg [95% confidence interval {CI}, 88–124 mg/kg]; fentanyl ED50, 0.12 mg/kg [95% CI, 0.08–3.5 mg/kg]). Propofol produced hypnosis (ED50, 124 mg/kg [95% CI, 84–3520 mg/kg]) but did not block responses to tail clip application. Neither isovaline nor fentanyl produced hypnosis at doses which produced immobility (isovaline ED50, 350 mg/kg [95% CI, 286–1120 mg/kg]; fentanyl ED50, 0.35 mg/kg [95% CI, 0.23–0.51 mg/kg]). Isovaline at its analgesic ED50, coadministered with a subhypnotic dose of propofol (40 mg/kg), did not exacerbate propofol-induced deficits in rotarod performance. The median maximal tolerated dose of fentanyl coadministered with the hypnotic ED50 of propofol was 11 mg/kg (95% CI, 8–18 mg/kg). Isovaline at a maximal deliverable (soluble) dose of 5000 mg/kg produced no apparent respiratory depression or other adverse effects.

CONCLUSIONS: The novel analgesic, isovaline, coadministered with propofol, produced general anesthesia and conscious sedation in mice. The margin of safety for propofol-isovaline was considerably higher than that for propofol-fentanyl. This study's results show that propofol-based sedation and general anesthesia can be effectively and safely produced by replacing the conventional opioid component with a brain-impermeant peripherally acting γ-aminobutyric acid type B receptor agonist. The results provide proof of the principle of combining a peripheral analgesic with a centrally acting hypnotic to produce general anesthesia. This principle suggests a novel approach to clinical general anesthesia and conscious sedation.

From the *Department of Anesthesiology, Pharmacology & Therapeutics, The University of British Columbia, Vancouver, British Columbia, Canada; and Department of Anesthesia, St. Paul’s Hospital, Vancouver, British Columbia, Canada.

Ryan A. Whitehead, PhD, is currently affiliated with Department of Anesthesiology and Critical Care Medicine and Department of Neurosciences, The University of New Mexico, Albuquerque, New Mexico.

Accepted for publication July 14, 2015.

Funding: Support was provided, in part, by the Dr. Jean Templeton Hugill Endowment for Anesthesia Memorial Fund, the Canadian Institutes for Health Research, the Canada Foundation for Innovation, the UBC Faculty of Medicine, and the Canadian Anesthesiologists’ Society.

Conflict of Interest: See Disclosures at the end of the article.

Reprints will not be available from the authors.

Address correspondence to Bernard A. MacLeod, MD, FRCPC, Department of Anesthesiology, Pharmacology &Therapeutics, The University of British Columbia, 2176 Health Sciences Mall, Vancouver, British Columbia, Canada V6T 1Z3. Address e-mail to bernard.macleod@gmail.com.

General anesthesia consists of a loss of consciousness ([LOC]; hypnosis) and immobility in response to noxious stimuli. Propofol (2, 6-diisopropylphenol) produces hypnosis by modulating central nervous system (CNS) inhibition mediated by γ-aminobutyric acid (GABA) at GABAA receptors.1 Although the hypnosis induced by propofol is accompanied by some degree of analgesia,2 propofol alone is unable to produce immobility to surgical stimuli in rodents, except at doses that produce intolerable side effects.3 The combination of propofol and an opioid analgesic, such as fentanyl or derivative, is widely used to produce total IV anesthesia (TIVA) and conscious sedation. A disadvantage of opioid use is respiratory depression, a primary cause of death due to opioid administration.4 This is of particular concern during IV sedation with other CNS depressants (especially propofol).5,6 A decreased risk of respiratory depression could result in an increased margin of safety for propofol-based sedation and anesthesia. A peripherally acting analgesic with little or no ability to penetrate the CNS could provide a therapeutic alternative to mitigate the adverse effects of opioids.

We recently showed that isovaline (2-aminoisobutyric acid), a blood-brain barrier-impermeant amino acid, produces analgesia in rodents7,8 and inhibits neurons in nociceptive thalamus by activating metabotropic GABAB receptors.9 The pain assays included allodynia induced by prostaglandin E2 peripheral sensitization and formalin-induced nocifensive behaviors.7,8 Isovaline produces analgesia, at least in part, by activating GABAB receptors in the periphery, without the adverse CNS effects of the GABAB agonist baclofen or opioids (e.g., sedation or respiratory depression).8 A combination of propofol acting at GABAA and isovaline acting at GABAB receptors suggests the possibility of synergism in the attainment of general anesthesia.10

As isovaline has neither been tested nor been approved for human use, we conducted a proof-of-principle study in mice to test whether coadministration of isovaline with propofol produces surgical general anesthesia and conscious sedation. We now report on the effects of isovaline versus fentanyl on murine responses to tail clip application (loss of response to tail clip [LRTC]), a widely validated measure of surgical analgesia that has been used extensively in determination of the minimum alveolar concentration of inhalational anesthetics.11 We also determined drug doses for LOC to assess hypnosis and conducted safety assessments of isovaline versus fentanyl as the analgesic adjuvant for general anesthesia.

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METHODS

Animals

With approval from the institutional Animal Care Committee (The University of British Columbia, Vancouver, British Columbia, Canada), we conducted an in vivo study with 114 adult female CD-1 mice according to the guidelines of the Canadian Council on Animal Care. The animals weighed 20 to 25 g and were housed in The University of British Columbia Animal Resource Unit. Mice were kept at controlled room temperature (21°C) and humidity (55%), housed in groups of 4 per cage with a 12-hour light/dark cycle, and had free access to food and water. Naive mice were used for the experiments.

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Drugs

Propofol (Diprivan) was purchased from AstraZeneca (Mississauga, Ontario, Canada), fentanyl citrate from McNeil Laboratories Ltd. (Don Mills, Ontario, Canada), and RS-isovaline hydrochloride from Biofine International Inc. (Vancouver, British Columbia, Canada). Saline (0.9% NaCl) was used as control and for dilution of fentanyl and isovaline. All drugs were injected intraperitoneally (IP).

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Experimental Design and Procedures

Mice were gently restrained for injection.12 After injection, mice were placed in separate cages for observation, video recording, and testing. All restraints and injections were performed by the same experimenter. A blinded experimenter recorded the experimental results. Subsequently, the observations were reassessed through video analysis by an additional independent, blinded observer. Because of the binary nature of the “up-and-down” method (see below), sequences were to be discarded if there were disagreement regarding the response of an animal in the sequence. However, no such instances occurred.

Each animal received a single injection of saline, propofol, isovaline, or fentanyl, or pretreatment with either isovaline or fentanyl, followed by a single injection of propofol. Propofol was injected 15 minutes before experimental end points. Isovaline was injected 1 hour before assessment (and 45 minutes before propofol injection), whereas fentanyl was coinjected with propofol. For efficacy and safety assessments of the agents and combinations, we used Dixon’s up-and-down method (see Statistical Analysis later).13 We determined the half-maximal effective doses (ED50s) for hypnosis and immobility. Each animal was used only once.

For assessment of surgical general anesthesia, we determined drug doses to produce LOC (hypnosis)14 and immobility.11 Hypnosis was defined as an inability of a supine mouse to right itself to sternal recumbency (loss of righting reflex [LRR]) and the absence of a response to light touch. Immobility as a surrogate of surgical analgesia was defined by LRTC.11 A standard alligator clip was applied 1 cm from the base of the tail. A purposeful, directed response of the mouse’s head toward its tail followed by physical contact with the clip served as our binary model for determining nociceptive response. This movement in response to tail clamping is well established and effectively demonstrated in minimum alveolar concentration studies of inhalational anesthetics.11 General anesthesia was defined as the presence of both LOC and LRTC without intolerable side effects. The ED50 for LOC of propofol was administered with isovaline or fentanyl to determine the incidence of LRTC. The respective ED50s for LRTC of isovaline or fentanyl were coadministered with varying doses of propofol to determine the ED50 for LOC of the combination using Dixon’s up-and-down method.13

For the assessment of conscious sedation, we used the rotarod latency assay.14,15 A custom built rotarod with 3.3-cm rod diameter and 50-cm fall height onto a soft surface to prevent injury was used for the study. Mice were trained on the rotarod rotating at 12 rpm 3 times daily with a 5-minute intertrial interval for 10 consecutive days before the test. Each trial had a maximum cutoff time of 60 seconds. Mice that were unable to remain on the rod for a minimum of 10 seconds by the third day of training were excluded from the study. On the test day, 15 minutes before the test, animals randomly received an IP injection of either 40 mg/kg propofol, 350 mg/kg isovaline, 40 mg/kg propofol with 350 mg/kg isovaline, 40 mg/kg propofol with 0.35 mg/kg fentanyl, or saline. Each mouse was tested in triplicate with a 1-minute intertrial interval on the rod rotating at 12 rpm with a maximum cutoff time of 60 seconds, and the latency to fall was recorded. The sedative dose of propofol was defined as the minimal dose that significantly decreased the time on the rotarod.

For safety assessments, we determined the maximal tolerated doses (MTDs) of isovaline and fentanyl in the presence of the ED50 for LOC of propofol. MTD was defined as the maximal dose that did not produce a respiratory rate of <4 per minute, multiple apneic periods, or death. Respiratory rates were determined by visual observation and verified from video recordings. As determinations of lethal doses are no longer acceptable by the Animal Care Committee of our institution, we used the up-and-down method and MTD.16,17 The maximal soluble concentration of isovaline at an acceptable volume for IP injection was 5000 mg/kg.

We determined therapeutic indices of propofol/isovaline and propofol/fentanyl by dividing the ED50 of general anesthesia by the MTD50. After experiments, animals were euthanized by anesthetic overdose with isoflurane, followed by cervical dislocation.

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Statistical Analysis

Similar to our previous studies,17 we used Dixon’s up-and-down method13 (c.f. above) to determine ED50s with their 95% confidence intervals (CIs) for LRR and LRTC. For the present experiments, test series sequences, sample sizes, and ED50s were determined by AOT425StatPgm software (developed for the U.S. Environmental Protection Agency by Westat [Rockville, MD]), with a sigma (estimated log ED50 SD) of 0.15. This protocol is an implementation of Dixon’s method, which allows accurate determination of ED50s but with a significant reduction in the number of animals.18 In brief, this method stipulates that the choice of an initial dose is a rough estimate of the ED50 for the measurement of interest. Starting doses and intervals were selected according to literature estimates on analgesic or hypnotic efficacy for isovaline, fentanyl, and propofol.3,7,8 If there is no response to the initial dose, then the subsequent dose is increased by a defined (log) dosing increment.13 If there is an effect at the initial dose, then the subsequent dose is decreased by an amount equivalent to the dosing increment. This procedure is repeated until the stopping criteria are met, provided in the present study by AOT425StatPgm.19 Statistical significance equivalent to P < 0.05 was assessed based on the absence of overlapping 95% CIs and derived from Dixon’s maximum likelihood estimations.19

Conscious sedation data were recorded, graphed, and assessed for differences to the control cutoff value of 60 seconds using 1-sample t tests. Data passed a test for normality (the D’Agostino and Pearson omnibus normality test) and were presented as mean and 95% CI. These analyses were performed with the use of Prism software, version 6 (GraphPad, La Jolla, CA).

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RESULTS

Table 1

Table 1

As summarized in Table 1, we found that propofol produced hypnosis but not surgical analgesia. Isovaline or fentanyl produced surgical analgesia but not hypnosis. The combination of propofol with either fentanyl or isovaline produced surgical anesthesia.

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Loss of Consciousness

Table 2

Table 2

Table 2 shows the results of the respective up-and-down test series used to determine the ED50s of propofol, fentanyl, and isovaline for LOC. Propofol produced LOC without respiratory depression at an ED50 of 124 mg/kg (95% CI, 84–3520 mg/kg; n = 5). Fentanyl on its own did not produce LOC before the occurrence of unacceptable respiratory depression or death. Fentanyl at its ED50 for LRTC (0.35 mg/kg) did not significantly change propofol’s ED50 for LOC from 124 to 160 mg/kg (95% CI, 124–175 mg/kg). The ED50 of fentanyl for LRR was 114 mg/kg (95% CI, 97–124 mg/kg; n = 5). Unlike mice that received hypnotic doses of propofol, animals treated with fentanyl at this ED50 displayed hyperlocomotor responses to light touch, indicating an absence of LOC from a practical standpoint. Isovaline alone did not produce LOC, even at a maximal deliverable (soluble) dose of 5000 mg/kg. Fentanyl did not produce LOC without unacceptable side effects.

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Loss of Response to Tail Clip

Propofol when administered alone was unable to produce LRTC, as animals became moribund without achieving LRTC. Fentanyl produced LRTC at an ED50 of 0.35 mg/kg (95% CI, 0.23–0.51 mg/kg; n = 6). Isovaline produced LRTC (ED50, 350 mg/kg; 95% CI, 286–1120 mg/kg; n = 9). Table 3 shows the results of the respective up-and-down test series used to determine the ED50s of propofol, fentanyl, and isovaline for LRTC. In summary, either isovaline or fentanyl alone produced LRTC, whereas propofol alone did not produce LRTC without intolerable side effects.

Table 3

Table 3

Table 4

Table 4

Because of the wide CI, we were unable to determine whether the coadministration of isovaline at its ED50 for LRTC reduced propofol’s ED50 for LOC (c.f. above; n = 5). The wide 95% CI in Table 4 was computed by the AOT425StatPgm software suite (0 to >20,000 mg/kg). Similarly, coadministration of fentanyl at its ED50 for LRTC did not significantly affect propofol’s ED50 for LOC (160 vs 124 mg/kg; 95% CI, 124–175 mg/kg, n = 10). Neither isovaline nor fentanyl changed the ED50 of propofol to produce unconsciousness.

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General Anesthesia

Table 5

Table 5

The coadministration of propofol and isovaline, as well as propofol and fentanyl at appropriate doses, produced general anesthesia. The ED50 for LRTC of isovaline, when coadministered with the ED50 for LOC of propofol (c.f. above), was reduced to 96 mg/kg (95% CI, 88–124 mg/kg; n = 6). The ED50 of fentanyl for LRTC, when coadministered with the ED50 for LOC of propofol, was reduced to 0.12 mg/kg (95% CI, 0.08–3.5 mg/kg; n = 5). Table 5 summarizes results of the up-and-down test series.

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Conscious Sedation

Figure 1

Figure 1

The dose of propofol for conscious sedation was preselected as a minimal sedating dose,14 which produced a significant decrease in rotarod latency. Propofol at 40 mg/kg (approximately one-fourth of the hypnotic dose) significantly reduced the mean time on the rotarod to 39 seconds (95% CI, 25–54 seconds; n = 8) from saline (60 seconds, cutoff; n = 6) without producing unconsciousness. Isovaline at its ED50 for LRTC did not significantly reduce mean rotarod time (57 seconds, 95% CI, 51–63 seconds; n = 6) compared with control (P = 0.24). The combinations of propofol with either isovaline or fentanyl (350 or 0.35 mg/kg) significantly reduced rotarod time from 60 to 37 seconds for propofol + isovaline (95% CI, 23–50 seconds; n = 8, P = 0.0048) and to 36 seconds for propofol + fentanyl (95% CI, 14–57 seconds; n = 8, P = 0.03). The addition of either isovaline or fentanyl to the conscious sedating dose of propofol produced no significant difference in mean rotarod time compared with propofol alone. Figure 1 provides a graphic summary of the rotarod latency assay results.

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Maximal Tolerated Dose

Table 6

Table 6

The MTD50 for fentanyl coadministered with propofol at its ED50 for LOC was 11 mg/kg (95% CI, 8–18 mg/kg; n = 15). This finding is consistent with recent reports of LD50s in mice, where fentanyl was administered alone at 26 mg/kg IP20 and 62 mg/kg PO.21 As the MTD50 neared, respirations became shallow and were dominated by thoracic breathing. As noted earlier,7 water solubility of isovaline limits an accurate determination of MTD. Coadministration of isovaline at a maximal soluble concentration in an acceptable volume for a mouse (5000 mg/kg) with propofol at the ED50 for LOC produced no changes in respiration and heart rate or adverse effects (Table 6). Therefore, no MTD could be determined. The safety of propofol-isovaline, demonstrated by the ratio of the MTD to the ED50 for general anesthesia (>5000/96), was greater than twice that of propofol-fentanyl (11/0.12).

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DISCUSSION

These studies in mice demonstrate that the novel analgesic, isovaline, when coadministered with propofol produced general anesthesia and conscious sedation. Although the combination of isovaline and propofol resulted in LOC and immobility to noxious stimuli, propofol alone was not analgesic. Isovaline alone or in combination with propofol did not produce respiratory depression nor did it exaggerate propofol’s effects on the CNS. The combination of fentanyl and propofol also resulted in general anesthesia. However, opioids are associated with a risk of severe respiratory depression.22 Our experiments showed that in principle, an improved margin of safety is achievable by combining peripherally acting isovaline with a centrally acting hypnotic for TIVA and procedural sedation.

Isovaline coadministered with propofol produced general anesthesia without altering propofol’s ED50 for hypnosis or producing additional CNS side effects. Like isovaline, fentanyl did not modify the propofol dose requirement, indicating that the cerebrospinal effects of fentanyl did not make a major contribution to the hypnotic state. However, the wide CI associated with the ED50 values suggests caution in drawing this inference. Alone, isovaline, even at a maximal dose, did not produce apparent side effects or an LOC. Similar to our previous studies,7,8 we found little or no evidence that isovaline produced CNS effects. This is consistent with the inability of radiolabeled isovaline to appreciably enter into the brain.23 Although isovaline has anticonvulsant effects during seizure conditions induced by 4-aminopyridine or pilocarpine,24,25 such effects may result from induced alterations in blood-brain barrier permeability.26 In summary, isovaline is a prototype of an unconventional class of peripherally acting analgesics that avoid the risk of CNS side effects.8

Systemic administration of isovaline produced surgical analgesia, defined by immobility or loss of purposeful response toward a tail clip applied to the base of the animal’s tail.11 The ED50 for isovaline to block tail clip responses was consistent with its potency in the formalin-induced pain model.7 Coadministration of propofol at a hypnotic dose, with isovaline or fentanyl, significantly increased their analgesic potency. This suggests that propofol modifies the sensitivity of neurons to the antinociceptive actions of isovaline and fentanyl.

Isovaline and baclofen have antinociceptive effects. Both agents interact with GABAB receptors.8 Baclofen, which crosses into the CNS, is more potent than isovaline, but the resulting sedation and respiratory depression severely limit its use.27 Because isovaline’s peripheral GABAB action is distinct from that of a μ-opioid agonist, synergistic analgesia is possible. Recently, we have shown that isovaline activates another class of analgesic receptors—group II metabotropic glutamate receptors.28 This provides an additional reason to expect heightened analgesia with isovaline combined with an opioid. Synergism between isovaline and an opioid would provide systemic super-additive analgesia without increasing undesirable side effects.10

General anesthesia is composed of hypnotic and analgesic components. Propofol is commonly used in conjunction with an opioid for surgical general anesthesia because propofol alone produces little or no surgical analgesia.2,3 In the present experiments, propofol did not block motor responses to tail clip, an established model of surgical analgesia11 (except at doses that caused intolerable respiratory depression). Conscious sedation induced by propofol decreased the time a mouse could stay on a rotating rod. Coadministration of analgesic doses of isovaline or fentanyl with a sedating dose of propofol did not further reduce time on the rotarod. These findings suggest that isovaline may be useful in TIVA and procedural sedation. Such application may be appropriate when considering the hazardous situation where opioid-induced respiratory depression is delayed and dissociated from the degree of analgesia.29

An exact figure for the therapeutic index of isovaline in the presence of a hypnotic dose of propofol could not be obtained because isovaline did not produce adverse effects, even at the maximal administrable dose. The MTD of isovaline was at least 50 times the ED50 for analgesia. The MTD50 for fentanyl divided by the ED50 for analgesia was approximately one-half of the ratio for isovaline. This improvement in the therapeutic index reflected differences in respiratory depression because of isovaline and fentanyl.

The complexity of perioperative care and risks of surgery are increasing in the present era of an aging and multimorbid patient demographic. To maintain the excellent patient safety and risk reduction trend of modern anesthesiology, perioperative approaches and anesthetic drugs must continue to evolve. Current approaches combining opioid analgesics with hypnotics are effective, but respiratory depression and other adverse effects associated with opioids complicate their use.30 Adding drugs that can selectively produce analgesia with minimal risk to current multimodal anesthesia would improve safety. Because the potency of volatile anesthetics is higher for hypnosis than for surgical analgesia, they are often used with opioid analgesics.31 Opioid respiratory depression could be reduced by the addition of a peripherally restricted analgesic. Opioids produce hyperalgesia, complicating postoperative pain control. This results in progressively increasing opioid requirements and adverse effects (for which tolerance does not develop). Because isovaline does not have significant CNS effects, adding it to balanced anesthesia would decrease the requirements for opioids and reduce opioid-induced hyperalgesia.

Difficulty in extrapolating the present results from mice to humans is an obvious limitation of this study. However, rodent models of anesthesia are highly reproducible and have been validated for predictive efficacy in humans. Another consideration was the wide CIs observed on estimation of propofol’s ED50 for LOC in the presence of isovaline. This results from limitations of the AOT425StatPgm software. However, no dose of propofol alone produced general anesthesia. The estimated ED50 for LOC of propofol in other mouse studies was within approximately 20% of our estimated ED50s.32 Our estimated ED50 in combination with either isovaline or fentanyl produced general anesthesia without significant side effects, demonstrating that the addition of isovaline to propofol can produce safe general anesthesia.

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CONCLUSIONS

The combination of propofol’s actions on CNS neurons and isovaline’s actions on peripheral nociceptive neurons8 resulted in general anesthesia. The anesthesia in mice was similar to that of fentanyl with propofol. Administering an analgesic dose of isovaline or fentanyl to a conscious mouse receiving propofol did not increase sedation. This study demonstrates that propofol-based general anesthesia and sedation can be effectively and more safely produced by substituting a peripherally acting GABAB agonist for the conventional centrally acting opioid. A propofol and isovaline combination may improve clinical anesthesia.

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DISCLOSURES

Name: Ryan A. Whitehead, PhD.

Contribution: This author helped to develop the study rationale and design, conceived the experimental groupings, collected the data, analyzed the data, and co-wrote the manuscript.

Attestation: Ryan A. Whitehead has reviewed the study data and data analysis, attests to their integrity, and approved the final manuscript.

Conflicts of Interest: None.

Name: Stephan K. W. Schwarz, MD, PhD, FRCPC.

Contribution: This author contributed to the development of the study rationale, revised the data analysis plan, helped conduct statistical analyses, and co-wrote the manuscript.

Attestation: Stephan K. W. Schwarz approved the final manuscript.

Conflicts of Interest: None.

Name: Yahya I. Asiri, BSc.

Contribution: This author helped to conduct the study, assisted with data analysis, and co-wrote the manuscript.

Attestation: Yahya I. Asiri has reviewed the study data and data analysis, attests to their integrity, and approved the final manuscript.

Conflicts of Interest: None.

Name: Timothy Fung, BSc.

Contribution: This author helped to conduct the study, co-wrote the manuscript, and assisted with data analysis.

Attestation: Timothy Fung has reviewed the study data and data analysis, attests to their integrity, and approved the final manuscript.

Conflicts of interest: None.

Name: Ernest Puil, PhD.

Contribution: This author helped develop the study rationale and design, and co-wrote the manuscript.

Attestation: Ernest Puil approved the final manuscript.

Conflicts of Interest: Ernest Puil is the co-holder of a patent on the use of isovaline as an analgesic and Chief Executive Officer of TherExcell Pharma (Vancouver, British Columbia, Canada), developing isovaline as a clinical analgesic.

Name: Bernard A. MacLeod, MD, FRCPC.

Contribution: This author developed the study rationale and experimental design, and co-wrote the manuscript.

Attestation: Bernard A. MacLeod has reviewed the study data and data analysis, attests to their integrity, and approved the final manuscript.

Conflicts of Interest: Bernard A. MacLeod is the co-holder of a patent on the use of isovaline as an analgesic and a major shareholder, as well as member of the board of directors of TherExcell Pharma (Vancouver, British Columbia, Canada), developing isovaline as a clinical analgesic.

This manuscript was handled by: Markus W. Hollmann, MD, PhD, DEAA.

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