Mechanism(s) Underlying Toxicity and the Development of Tolerance to Methamphetamine Diana G. Wilkins, PhD. Center for Human Toxicology University of Utah Salt Lake City, Utah
Methamphetamine METH, ice, crank or speed; Schedule II stimulant Various routes of administration
How is METH Used? Dependent upon route of administration; 50 100 mg/episode; as much as 1-2 g in a binge (i.e., very high doses) Effect persists 6-8 hours
How is METH Used? Immediately after inhalation or i.v. administration, user experiences brief flash or rush Snorting or oral administration produces euphoria, but not an intense rush; effects last as long as 12 hours Snorting produces effects within 3-5 minutes; oral administration within 15 minutes Tolerance to euphoric effects occurs within minutes
METH Trends 25000 20000 Mentions 15000 10000 5000 Methamp Amphet 0 1998 1999 2000 2001 2002
METH Lab Seizures Source: US Department of Justice
Why is METH Abused? Long duration of action (especially relative to cocaine) Inexpensive, easy to manufacture
What are METH s Adverse Effects? After initial rush, often agitation and perhaps violent behavior Extremely addicting Severe craving Hyperthermia Weight loss
What Does METH Do? What happens at the synapse after a single dose of METH?
Brain Regions of Interest * * *
Neuronal Morphology
Model of a Dopaminergic Synapse
Model of a Serotonergic Synapse
Synaptosomes (P2) Homogenize Centrifuge 1000 x g Resuspend Centrifuge S1 @ 22,000 x g Osmotic Lysis And Centrifugation @ 100,000 x g Vesicles (P3) Membranes (P3)
Mechanism of Action Causes release of dopamine via reversal of the dopamine transporter Causes some release of serotonin and norepinephrine as well
What Does METH Do? What happens after multiple doses of METH (neurotoxic regimen)?
Neurotoxicity A neurotoxic regimen of METH: - Causes long-term reduction in TH and TPH enzyme activities (Koda and Gibb, 1973; Hotchkiss and Gibb 1980; Bakhit and Gibb, 1980) Hotchkiss and Gibb, 1980
Neurotoxicity A neurotoxic regimen of METH: - Causes long-term depletion of dopamine in striatum and other brain regions (Kogan and Gibb, 1976; Morgan and Gibb,1980; Ricaurte et al., 1980) Morgan and Gibb, 1980
Neurotoxicity A neurotoxic regimen of METH: - Causes hyperthermia that is linked to depletion in dopamine. Blocking hyperthermia attenuates the dopamine deficits (Bowyer et al., 1992; 1993; 1994 ) Bowyer et al., 1994
Neurotoxicity A neurotoxic regimen of METH: - Increases free radical formation in striatum (Giovanni et al., 1995; Fleckenstein et al. 1999)
Neurotoxicity A neurotoxic regimen of METH: - Decreases DA transporter re-uptake (Kokoshka et al., 1998; Metzger et al., 2000)
Adverse Effects Persistent dopaminergic deficits McCann et al., J. Neurosci. 1998
Neurotoxicity A neurotoxic regimen of METH: - Decreases VMAT2 uptake (Brown et al., 2000)
Neurotoxicity A neurotoxic regimen of METH: - Demonstrates age-dependent changes for DAT uptake and TH (Kokoshka( et al., 2000)
Neurotoxicity A neurotoxic regimen of METH: - Demonstrates age-dependent differential responses in METH concentrations. Kokoshka et al., 2000
Neurotoxicity A neurotoxic regimen of METH: - Causes a redistribution of synaptic vesicles (Riddle et al., 2002; 2003) 1200 1000 800 600 400 200 0 Synaptosome Membrane Vesicle * 1000 1800 800 1500 600 1200 900 400 600 200 300 0 Saline METH Saline METH Saline METH 0 *
Dopaminergic Synapse : Neurotoxicity
Neurotoxicity: Important Concepts METH causes persistent dopaminergic deficits Hyperthermia contributes to deficits Reactive oxygen species contribute to deficits METH causes trafficking of vesicles
What Does METH Do? What happens when multiple doses of METH are given at frequent intervals?
Tolerance Tolerance is defined as a diminished response to a drug after repeated doses. - Can be behavioral, pharmacokinetic, etc. - For this presentation, tolerance will be defined in terms of striatal dopamine and serotonin (outcome markers).
Tolerance Protocols
Tolerance A tolerance-inducing METH regimen: - Attenuates the depletion in striatal dopamine and serotonin observed after a neurotoxic regimen Treatment SAL Pretxt + METH Challenge METH PreTxt + METH Challenge (2.5-7.5 mg/kg/day) Dopamine 38.5 + 5.0* % of control 70.7 + 1.6* # % of control 5-HT 23.7 + 5.6* % of control 63.6 + 4.0* # % of control Schmidt et al., 1985
What Does METH Do? So, integrating what the lab is exploring in neurotoxicity,, what happens during tolerance with respect to: - Temperature? - Persistence of tolerance? - Alterations in distribution of drug? - Effects on transporters? - Reactive oxygen species?
Tolerance A tolerance-inducing METH regimen: - Attenuates the dopamine depletion after a neurotoxic regimen given 24 hours after pre- treatment Dopamine Concentrations in Striatum DA (ng/mg protein) 140 120 100 80 60 40 20 0 SS MS SM MM * Mean +/- S.D. METH Pretxt: : 5 escalating s.c.. doses of 2.5, 5.0, 7.5 mg/kg @ 6-hr 6 intervals METH Challenge: : 5 s.c.. doses of 12.5 mg/kg @ 4-hr 4 intervals
Tolerance A tolerance-inducing METH regimen: - Attenuates the hyperthermia observed during a neurotoxic regimen Temp 0 C 42 41 40 39 38 37 36 35 Temp Data 4-30-03 Tolerance Experiment Ptx with 5x2.5, 5, 7.5 @ 6 Hr Int Challenge with 5x12.5 mg/mg @ 4 Hr Int * * * -5 0 5 10 15 20 Time (hrs) * * * * SS MS SM MM Dosing
Tolerance A tolerance-inducing METH regimen: - With this protocol, does not alter the brain concentration of METH METH Concentrations in Three Brain Regions 4-30-03 Tolerance Experiment 10 Meth (n g /m g) 8 6 4 2 SM MM 0 Frontal Cortex Hippocampus Striatum
Tolerance A tolerance-inducing METH regimen: - Attenuates deficits in several brain regions 1 week after challenge STRIATUM STRIATUM HIPPOCAMPUS FRONTAL CORTEX 200 6 9 6 DOPAMINE (pg /µg protein) 160 120 80 40 SEROTONIN (pg /µg protein) 4 3 * * 5 2 1 SEROTONIN (pg /µg protein) 8 7 6 5 4 3 2 1 * * SEROTONIN (pg /µg protein) 5 4 3 2 1 * SALINE/SALINE SALINE/METH METH/SALINE METH/METH 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 Davis et al., 2003 (In Press)
Tolerance A tolerance-inducing METH regimen: - No significant alteration in DAT re-uptake of dopamine DA Uptake (fmol / mg protein) 2 1.6 1.2 0.8 0.4 0 SALINE/SALINE SALINE/METH METH/SALINE METH/METH
Tolerance A tolerance-inducing METH regimen: - Decreases the attenuation of VMAT2 dopamine uptake (function of VMAT) Vesicular DA Uptake (fmol / mg protein) 250 200 150 100 50 * * + * # * 0 SALINE/SALINE SALINE/METH METH/SALINE METH/METH
Tolerance A tolerance-inducing METH regimen: - Attenuated the redistribution of VMAT2 within nerve terminal Non-membrane-associated fraction Band Density (Arbitrary Units) 200000 150000 100000 50000 200000 150000 100000 50000 * * 200000 150000 100000 50000 200000 150000 100000 50000 ** 0 SALINE/SALINE METH/SALINE 0 SALINE/SALINE SALINE/METH 0 METH/SALINE METH/METH 0 SALINE/METH METH/METH
Dopaminergic Synapse : Tolerance
Tolerance: Important Concepts Development of tolerance is dependent on the dosing paradigm Tolerance protocols attenuate persistent dopaminergic deficits Temperature and tolerance are linked Tolerance protocols appear to alter trafficking of vesicles
Acknowledgements Kamisha Johnson-Davis*, B.S. Jonathan Danaceau,, PhD.* Jayme Day, B.S., Brad Charles, B.S., Cassie Deering,, B.S. and Stacy Smeal,, B.S. Annette Fleckenstein, PhD. and Laboratory NIDA Program Project Grant No. DA 13367