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.
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.
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.
Maximal Tolerated Dose
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).
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.
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.
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.
1. Nishikawa K, Kubo K, Obata H, Yanagawa Y, Saito S. The influence of manipulations to alter ambient GABA concentrations on the hypnotic and immobilizing actions produced by sevoflurane, propofol, and midazolam. Neuropharmacology. 2011;61:172–80
2. Pavlin DJ, Coda B, Shen DD, Tschanz J, Nguyen Q, Schaffer R, Donaldson G, Jacobson RC, Chapman CR. Effects of combining propofol and alfentanil on ventilation, analgesia, sedation, and emesis in human volunteers. Anesthesiology. 1996;84:23–37
3. Alves HC, Valentim AM, Olsson IA, Antunes LM. Intraperitoneal propofol and propofol fentanyl, sufentanil and remifentanil combinations for mouse anaesthesia. Lab Anim. 2007;41:329–36
4. Dahan A, Aarts L, Smith TW. Incidence, reversal, and prevention of opioid-induced respiratory depression. Anesthesiology. 2010;112:226–38
5. Bailey PL, Pace NL, Ashburn MA, Moll JW, East KA, Stanley TH. Frequent hypoxemia and apnea after sedation with midazolam and fentanyl. Anesthesiology. 1990;73:826–30
6. Miner JR, Heegaard W, Plummer D. End-tidal carbon dioxide monitoring during procedural sedation. Acad Emerg Med. 2002;9:275–80
7. MacLeod BA, Wang JT, Chung CC, Ries CR, Schwarz SKW, Puil E. Analgesic properties of the novel amino acid, isovaline. Anesth Analg. 2010;110:1206–14
8. Whitehead RA, Puil E, Ries CR, Schwarz SKW, Wall RA, Cooke JE, Putrenko I, Sallam NA, MacLeod BA. GABA(B) receptor-mediated selective peripheral analgesia by the non-proteinogenic amino acid, isovaline. Neuroscience. 2012;213:154–60
9. Cooke JE, Mathers DA, Puil E. R-Isovaline: a subtype-specific agonist at GABA(B)-receptors? Neuroscience. 2012;201:85–95
10. Tallarida RJ. Revisiting the isobole and related quantitative methods for assessing drug synergism. J Pharmacol Exp Ther. 2012;342:2–8
11. Eger EI II, Xing Y, Laster M, Sonner J, Antognini JF, Carstens E. Halothane and isoflurane have additive minimum alveolar concentration (MAC) effects in rats. Anesth Analg. 2003;96:1350–3
12. Hurst JL, West RS. Taming anxiety in laboratory mice. Nat Methods. 2010;7:825–6
13. Dixon WJ. The up-and-down method for small samples. J Am Stat Assoc. 1965;60:967–78
14. Nadeson R, Goodchild CS. Antinociceptive properties of propofol: involvement of spinal cord gamma-aminobutyric acid(A) receptors. J Pharmacol Exp Ther. 1997;282:1181–6
15. Hayes AG, Tyers MB. Determination of receptors that mediate opiate side effects in the mouse. Br J Pharmacol. 1983;79:731–6
16. Lipnick RL, Cotruvo JA, Hill RN, Bruce RD, Stitzel KA, Walker AP, Chu I, Goddard M, Segal L, Springer JA. Comparison of the up-and-down, conventional LD50, and fixed-dose acute toxicity procedures. Food Chem Toxicol. 1995;33:223–31
17. Cheung HM, Lee SM, MacLeod BA, Ries CR, Schwarz SKW. A comparison of the systemic toxicity of lidocaine versus its quaternary derivative QX-314 in mice. Can J Anaesth. 2011;58:443–50
18. Lichtman AH. The up-and-down method substantially reduces the number of animals required to determine antinociceptive ED50 values. J Pharmacol Toxicol Methods. 1998;40:81–5
19. OECD Guideline for Testing of Chemicals. Acute Oral Toxicity – Up-and-Down-Procedure.Adopted. December 7, 2001
20. Vuckovic S, Prostran M, Ivanovic M, Dosen-Micovic L, Savic Vujovic K, Vucetic C, Kadija M, Mikovic Z. Pharmacological evaluation of 3-carbomethoxy fentanyl in mice. Pharmaceuticals. 2011;4:233–43
21. Higashikawa Y, Suzuki S. Studies on 1-(2-phenethyl)-4-(N-propionylanilino)piperidine (fentanyl) and its related compounds. VI. Structure–analgesic activity relationship for fentanyl, methyl-substituted fentanyls and other analogues. Forensic Toxicol. 2008;26:1–5
22. Shook JE, Watkins WD, Camporesi EM. Differential roles of opioid receptors in respiration, respiratory disease, and opiate-induced respiratory depression. Am Rev Respir Dis. 1990;142:895–909
23. Shiba K, Mori H, Hisada K. Analogues of alpha-aminoisobutyric acid with various alkyl groups as potential tumour imaging agents; effects of chain length on tumour and normal tissue specificity. Nucl Med Commun. 1989;10:751–8
24. Yu W, Smith AB, Pilitsis J, Shin DS. Isovaline attenuates epileptiform activity and seizure behavior in 4-aminopyridine treated rats. Epilepsy Res. 2014;108:331–5
25. Yu W, Smith AB, Pilitsis JG, Shin DS. Isovaline attenuates generalized epileptiform activity in hippocampal and primary sensory cortices and seizure behavior in pilocarpine treated rats. Neurosci Lett. 2015;599:125–8
26. Persidsky Y, Ramirez SH, Haorah J, Kanmogne GD. Blood-brain barrier: structural components and function under physiologic and pathologic conditions. J Neuroimmune Pharmacol. 2006;1:223–36
27. Mehta AK, Ticku MK. Baclofen induces catatonia in rats. Neuropharmacology. 1987;26:1419–23
28. Asseri KA, Puil E, Schwarz SKW, MacLeod BA. Group II metabotropic glutamate receptor antagonism prevents the antiallodynic effects of R-isovaline. Neuroscience. 2015;293:151–6
29. Smydo J. Delayed respiratory depression with fentanyl. Anesth Prog. 1979;26:47–8
30. Chu LF, Angst MS, Clark D. Opioid-induced hyperalgesia in humans: molecular mechanisms and clinical considerations. Clin J Pain. 2008;24:479–96
31. Sonner JM, Antognini JF, Dutton RC, Flood P, Gray AT, Harris RA, Homanics GE, Kendig J, Orser B, Raines DE, Rampil IJ, Trudell J, Vissel B, Eger EI II. Inhaled anesthetics and immobility: mechanisms, mysteries, and minimum alveolar anesthetic concentration. Anesth Analg. 2003;97:718–40
© 2015 International Anesthesia Research Society
32. Irifune M, Takarada T, Shimizu Y, Endo C, Katayama S, Dohi T, Kawahara M. Propofol-induced anesthesia in mice is mediated by gamma-aminobutyric acid-A and excitatory amino acid receptors. Anesth Analg. 2003;97:424–9