Antinociceptive effects of morphine and naloxone in mu-opioid receptor knockout mice transfected with the MORS196A gene.
BACKGROUND
Opioid analgesics such as morphine and meperidine have been used to control moderate to severe pain for many years. However, these opioids have many side effects, including the development of tolerance and dependence after long-term use, which has limited their clinical use. We previously reported that mutations in the mu-opioid receptors (MOR) S196L and S196A rendered them responsive to the opioid antagonist naloxone without altering the agonist phenotype. In MORS196A knock-in mice, naloxone and naltrexone were antinociceptive but did not cause tolerance or physical dependence. In this study we delivery this mutated MOR gene into pain related pathway to confirm the possibility of in vivo transfecting MORS196A gene and using naloxone as a new analgesic agent.


METHODS
The MOR-knockout (MOR-KO) mice were used to investigate whether morphine and naloxone could show antinociceptive effects when MORS196A gene was transfected into the spinal cords of MOR-KO mice. Double-stranded adeno-associated virus type 2 (dsAAV2) was used to deliver the MORS196A-enhanced green fluorescence protein (EGFP) gene by microinjected the virus into the spinal cord (S2/S3) dorsal horn region. Tail-flick test was used to measure the antinociceptive effect of drugs.


RESULTS
Morphine (10 mg/kg, s.c.) and naloxone (10 mg/kg, s.c.) had no antinociceptive effects in MOR-KO mice before gene transfection. However, two or three weeks after the MOR-S196A gene had been injected locally into the spinal cord of MOR-KO mice, significant antinociceptive effects could be induced by naloxone or morphine. On the other hand, only morphine but not naloxone induced significant tolerance after sub-chronic treatment.


CONCLUSIONS
Transfecting the MORS196A gene into the spinal cord and systemically administering naloxone in MOR-KO mice activated the exogenously delivered mutant MOR and provided antinociceptive effect without causing tolerance. Since naloxone will not activate natural MOR in normal animals or humans, it is expected to produce fewer side effects and less tolerance and dependence than traditional opioid agonists do.
Chen
Shiou-Lan
SL
Department of Psychiatry, College of Medicine and Hospital, National Cheng Kung University, Tainan, Taiwan.
Ma
Hsin-I
HI
Han
Jun-Ming
JM
Lu
Ru-Band
RB
Tao
Pao-Luh
PL
Law
Ping-Yee
PY
Loh
Horace H
HH
D019943:Amino Acid Substitution; D000700:Analgesics / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D004361:Drug Tolerance; D055879:Gene Knock-In Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009020:Morphine / Q000494:pharmacology*; D009021:Morphine Dependence; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009270:Naloxone / Q000494:pharmacology*; D017450:Receptors, Opioid, mu / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013116:Spinal Cord / Q000378:metabolism; D014162:Transfection
D000700:Analgesics; D050505:Mutant Proteins; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D009270:Naloxone; D009020:Morphine
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