cross tolerance & cross dependency

Cross Tolerance & Cross Dependency Between Opioids & Cannabinoids

in isolated tissues in cholestasis model

By M.H.Farjoo

Neuroscience research center & pharmacology Dep.Shahid Beheshti University Of Medical Science

Abbreviations

• GPI = Guinea pig ileum • MVD = Mouse vas deferens• ACEA = Arachidonyl chloroethylamide

(cannabinoid agonist)

Contents

Why this topic ?

Goals

Tolerance

Dependency

Cross tolerance and cross dependency

Methods & material

Results for tolerance

Results for dependency

Discussion & conclusion

Acknowledgements

Why this topic ?

• It was feasible in Iran!!• Drug abuse is a major

problem in our country• Possible applications for

drug abuse is worth the cost and time

• Cholestasis is a common problem in Iran.

• Cholestasis is a quite valid model for research on opioids and it is a well characterized model in our research centers.

• Cannabinoids are under intensive research all over the world.

Goals

• Evaluation of occurrence of tolerance and/or dependency to cannabinoid and/or opioid in GPI and/or MVD in sham/cholestatic group

• Evaluation of occurrence of cross tolerance and/or cross dependency between cannabinoid and opioid (and vice versa) in GPI and/or MVD in sham/cholestatic group

Definition of Tolerance

• Reduction in sensitivity to an agent following repeated exposure

• Decrease in agonist effect during a time course and/or significant shift to right of Dose-Response curve

Classification of Tolerance

• Homologous : Specific to the agonists of the same system– Within seconds to minutes (Desensitization) un-coupling of R

and G-protein (with or without internalization) , high dose of agonist

– Within hours : alteration in AC

• Heterologous : Extends to agonists of other systems– Within days, down regulation of sodium pump & partial

depolarization , gene modulation

Mechanism of Tolerance

• Not well understood• VERY complex • Contradictory data!!• Best studied in opioids and cannabinoids

(GPCRs)• Mechanisms of tolerance to any agent can be

absolutely specific!!

Dependency

• Definition:– Absolute requirement for the agent to maintain

normal physiological function

• Classification:– Physical: presence of withdrawal reactions– Psychic: presence of a “drug craving” component

Cross Tolerance

• Definition– Tolerance to an exposed agonist elicits tolerance to other

NON exposed agonists

• Classification– Within agonists of one system (or subtype receptors of one

system)– Between agonists of two (or more) system

Cross Dependency

• Definition– Withdrawal syndrome elicited by antagonist of an

exposed agent can also be precipitated by antagonists of NON exposed agents

• Classification– Within antagonists of one system– Between antagonists of two (or more) system

Mechanism

Exact mechanism of cross tolerance & cross dependency is:

UNKNOWN !

Methods and Materials

Drugs & Animals

• Morphine (temad)• Naloxone• ACEA (sigma)• AM 251 (sigma)• phenylephrine

• Male mice (pasteur and razi institute)

• Male Guinea pig (pasteur institute)

Method for tolerance

1. Animal euthanized by least possible pain

2. Target tissue isolated and prepared

3. Isolated tissue was suspended and stretched in organ bath (with/without drug) for adaptation

4. Tissue stimulated by electrical impulses

5. Result of stimulation registered and analyzed

Method for dependency

1. Animal euthanized by least possible pain2. Target tissue isolated and prepared3. Isolated tissue was suspended and stretched in organ

bath with drug for adaptation and tolerance development

4. Tissue stimulated by relevant agonist (phenylephrine for MVD & acetyl choline for GPI) to elucidate maximal response

5. Relevant antagonist is added and response measured6. Degree of dependency (tension ratio) is calculated

Results(for tolerance)

IC50 of morphine in Intact MVD(n = 6-8)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

110%

0.E+00 1.E-09 3.E-09 1.E-08 3.E-08 1.E-07 3.E-07 1.E-06 3.E-06

Molarity of drug

perc

ent

of in

hibi

tion

IC50=1.62 E-07

IC50 of morphine in Intact MVD after incubation with 0.5 IC50 for 2 hr (n = 6-8)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

110%

0.E+00 1.E-09 3.E-09 1.E-08 3.E-08 1.E-07 3.E-07 1.E-06 3.E-06

Molarity of drug

prec

ent

of in

hibi

tion

IC50 of morphine in Intact MVD after incubation with 1 IC50 for 2 hr (n = 6-8)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

110%

0.E+00 1.E-09 3.E-09 1.E-08 3.E-08 1.E-07 3.E-07 1.E-06 3.E-06

Molarity of drug

pert

cent

of

inhi

biti

on

IC50 of morphine in Intact MVD after incubation with 2 IC50 for 4 hr (n = 6-8)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

110%

0.E+00 1.E-09 3.E-09 1.E-08 3.E-08 1.E-07 3.E-07 1.E-06 3.E-06

Molarity of drug

perc

ent

of in

hibi

tion

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

110%

0.E+00 1.E-09 3.E-09 1.E-08 3.E-08 1.E-07 3.E-07 1.E-06 3.E-06

Molarity of drug

perc

ent o

f inh

ibiti

onIC50 of morphine in Intact MVD after incubation

with 4 IC50 for 2 hr (n = 6-8)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

110%

0.E+00 1.E-09 3.E-09 1.E-08 3.E-08 1.E-07 3.E-07 1.E-06 3.E-06 1.E-05

Molarity of drug

perc

ent o

f inh

ibiti

onIC50 of morphine in cholestatic MVD 4 days after

cholestasis (n = 6-8)

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

110%

0.E+00 1.E-09 3.E-09 1.E-08 3.E-08 1.E-07 3.E-07 1.E-06 3.E-06 1.E-05

Molarity of drug

perc

ent o

f inh

ibiti

onIC50 of morphine in cholestatic MVD 5 days after

cholestasis (n = 6-8)

Degree of tolerance in MVDs incubated with different IC50s of morphine

Degree of tolerance

IC50 (molar)Animal or tissue

2.7164.40 E- 075 days Cholestasis

4.9908.09 E- 074 days Cholestasis

0.2173.52 E- 08Intact, 4 IC50-2hr

1.3242.15 E- 07Intact, 2 IC50-4hr

1.6052.60 E- 07Intact, 2 IC50-2hr

1.7652.86 E- 07Intact, 1 IC50-2hr

11.62 E- 07Intact, 0.5 IC50-2hr

 11.62 E- 07Intact, No incubation

Degree of tolerance in MVDs incubated with ACEA

Degree of toleranceIC50 (molar)Animal or tissue

1.6791.78E-075 day Cholestasis

1.3841.47E-07Intact-2 IC50-4hr

11.06E-07Intact

Degree of tolerance in GPIs incubated with different IC50s of morphine

Degree of tolerance

IC50 (molar)Animal or tissue

0.551.59E-085 day Cholestasis

0.922.66E-08Intact-4 IC50-2hr

1.724.99E-08Intact-2 IC50-2hr

1.002.90E-08Intact

Results(for dependency)

• In intact MVDs which have been incubated with 2 and 4 IC50 of morphine for 2 hr, naloxone (10-5 and 10-3 molar) neither elicits any contraction nor it augments contractions caused by electrical stimulation

• The above mentioned issue holds true in 4 and 5 days cholestatic MVDs

Discussion & conclusion

It appears that morphine has behaved unexpectedly!!!

Why?

??

??

?

??

? ? ?

?

??

It appears that morphine has behaved unexpectedly!!!

Why?

No body exactly knows !!!

We know that: 1

• Opioid agonists such as morphine have been found to exert excitatory and inhibitory receptor-mediated effects at low and high doses, respectively (powell et al J Pharmacol Exp Ther. 2002)

• morphine exerted biphasic effects on clonic seizure threshold with anticonvulsant effect at lower and proconvulsant effect at a higher doses (Dehpour et al. Neuropharmacology. 2004)

• Systemic administration of ultra-low doses of naltrexone potentiated the anticonvulsant effect of morphine (Dehpour et al, Neuroscience. 2004)

• Significant potentiation of analgesic effects of opioids can be achieved by ultra-low doses of opioid receptor antagonists (Dehpour et al, Neuroscience. 2004)

We know that: 2

We know that: 3

• alpha2-Adrenoceptors play a dual role in the anticonvulsant effects of morphine. (Dehpour et al, Epilepsia. 2002)

• Ultra-low doses of opioid antagonists which inhibit the excitatory effects, inhibit the development of tolerance/physical dependence (powell et al J Pharmacol Exp Ther. 2002)

We know that: 4

• Large doses of spinal morphine have produced a distinctive paradoxical algesic response (Michael et al, Biopolymers. 2005)

• The paradoxical effects of opioid antagonists on pain sensitivity are thought to result from a bimodal G protein-coupled µ-opioid receptor. Its activity produces excitatory effects in response to ultra-low doses of agonist and inhibitory effects in response to high doses (Crain and Shen, 1995 , 1998)

• Systemic injection of heroin or morphine produced a rebound hyperalgesia after the antinociceptive effect was terminated (Michael et al, Biopolymers. 2005)

• many patients developed hyperesthesia and allodynia after continuous morphine administration (Michael et al, Biopolymers. 2005)

We know that: 5

conclusion

Morphine (and cannabinoids?) does not always behave as we guess !!!