Amino acids -- treatment for chronic pain

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Information about Amino acids -- treatment for chronic pain
Health & Medicine

Published on January 15, 2009

Author: nightning

Source: slideshare.net

Amino acids – treatment for chronic pain? Cheryl Chung, Jan 15, 2009

In the beginning…

Timeline Isovaline Not found in biosphere Non-biogenic Sep 28, 1969 Murchison Meteorite Sep 28, 1969 Timeline

Isovaline

Not found in biosphere

Non-biogenic

Timeline Search for novel analgesics Investigated isovaline & cyclic analogue ACBC (1-amino cyclobutane carboxylic acid) Found they blocked acute allodynia & responses to pain Year 2006 MacLeod’s Lab Year 2006 Sep 28, 1969 Timeline

Search for novel analgesics

Investigated isovaline & cyclic analogue ACBC (1-amino cyclobutane carboxylic acid)

Found they blocked acute allodynia & responses to pain

Why study amino acids? Amino acids are endogenous inhibitory neurotransmitters, e.g. Glycine GABA Act on pentameric ligand-gated Cl - channels Regulate nociception Lack of receptor type specificity leads to toxicity Betz & Laube 2006

Amino acids are endogenous inhibitory neurotransmitters, e.g.

Glycine

GABA

Act on pentameric ligand-gated Cl - channels

Regulate nociception

Lack of receptor type specificity leads to toxicity

Isovaline & ACBC – analogues of glycine & GABA Glycine ACBC (1-amino cyclobutane carboxylic acid) GABA (gamma- amino butyric acid) Isovaline

Glycine

ACBC (1-amino cyclobutane carboxylic acid)

GABA (gamma- amino butyric acid)

Isovaline

Timeline Test amino acids in models of chronic pain Suppress paw licking response in 2 nd phase of formalin foot model Alleviate pain responses & allodynia in osteoarthritis model Present Project Goals Present Timeline Year 2006 Sep 28, 1969

Test amino acids in models of chronic pain

Suppress paw licking response in 2 nd phase of formalin foot model

Alleviate pain responses & allodynia in osteoarthritis model

Osteoarthritis (OA) A degenerative joint disease Most commonly found in weight-bearing joints Characterized by loss of articular cartilage, subchondral remodeling & joint misalignment Movement of joint produces pain WHO 40% of people over age 70 have the condition in their knees

A degenerative joint disease

Most commonly found in weight-bearing joints

Characterized by loss of articular cartilage, subchondral remodeling & joint misalignment

Movement of joint produces pain

WHO

40% of people over age 70 have the condition in their knees

Experiments

3 part study Maximum tolerated dose (MTD) study Dose ranging studies in “formalin foot” model Effectiveness study in osteoarthritis (OA) model (using ED 50 from 2 )

Maximum tolerated dose (MTD) study

Dose ranging studies in “formalin foot” model

Effectiveness study in osteoarthritis (OA) model (using ED 50 from 2 )

1. Maximum tolerated dose study

1. Maximum tolerated dose Purpose: establish MTD of isovaline in rats Animal: Sprague-Dawley ♀ rats (dose doubling until drug not tolerated) Signs of Toxicity: BP & HR (40% changes from baseline) Sedation Respiratory depression (40% decrease in breathing rate)

Purpose: establish MTD of isovaline in rats

Animal: Sprague-Dawley ♀ rats (dose doubling until drug not tolerated)

Signs of Toxicity:

BP & HR (40% changes from baseline)

Sedation

Respiratory depression (40% decrease in breathing rate)

1. MTD study – Method & Protocol Permanent cannula inserted into carotid artery Subdermal ECG leads – at right shoulder and left abdomen 2h recovery period after surgery Monitor BP, ECG, breathing rate in conscious animal Administered isovaline s.c. (injection vol <1mL) Continue monitoring until readings return to baseline values (>1 h post drug)

Permanent cannula inserted into carotid artery

Subdermal ECG leads – at right shoulder and left abdomen

2h recovery period after surgery

Monitor BP, ECG, breathing rate in conscious animal

Administered isovaline s.c. (injection vol <1mL)

Continue monitoring until readings return to baseline values (>1 h post drug)

1. MTD study - Results Tested 500 – 2000mg/kg isovaline (s.c.) Using a total of 6 animals Very minor changes in Heart rate Blood pressure ECG Breathing rate No signs of sedation

Tested 500 – 2000mg/kg isovaline (s.c.)

Using a total of 6 animals

Very minor changes in

Heart rate

Blood pressure

ECG

Breathing rate

No signs of sedation

1. MTD study – An anecdote Rat 6, 2000mg/kg racemic isovaline

1. MTD study - Mean arterial pressure

1. MTD study - Heart rate

1. MTD study - Breathing rate

2. Dose ranging studies

2. Dose ranging studies Purpose: determine effective analgesic dose of isovaline & ACBC Method: “ formalin foot” model “ up-and-down” method Animals: SD rats (7 animals total) CD-1 mice (11 animals total) Test for analgesia: decrease of total paw licking time (in the 2 nd phase of response to formalin) by 40% from control.

Purpose: determine effective analgesic dose of isovaline & ACBC

Method:

“ formalin foot” model

“ up-and-down” method

Animals:

SD rats (7 animals total)

CD-1 mice (11 animals total)

Test for analgesia:

decrease of total paw licking time (in the 2 nd phase of response to formalin) by 40% from control.

2. DR Studies – Up & down method ED 50 estimate via the up-and-down method (Dixon 1965) ( - ) ( - ) ( - ) ( + ) ( + ) ED50 Est. range of ED50 Dose 1 Dose 2 Dose 3 Dose 4 Dose 5

ED 50 estimate via the up-and-down method (Dixon 1965)

2. DR studies - Formalin foot test -5 drug TIME (min) formalin 20 5 10 15 25 35 40 30 -20 Acclimatization

2. DR studies - Results Time course similar to previous results Isovaline ED 50 = 740±20mg/kg ACBC ED 50 = 88±9mg/kg Finding ED 50 for inhibition of phrase II formalin foot paw licking response using the “up and down” method

Time course similar to previous results

Isovaline ED 50 = 740±20mg/kg

ACBC ED 50 = 88±9mg/kg

3. Osteoarthritis Model

3. Osteoarthritis model Purpose: Assess analgesic effects of isovaline & ACBC in a chronic pain model Method : Induction of osteoarthritis with monosodium iodoacetate injection (MIA) Randomized blinded testing with controls in treated animals Pharmacological dosing & evaluation Day 3, 14, 28

Purpose:

Assess analgesic effects of isovaline & ACBC in a chronic pain model

Method :

Induction of osteoarthritis with monosodium iodoacetate injection (MIA)

Randomized blinded testing with controls in treated animals

Pharmacological dosing & evaluation

Day 3, 14, 28

3. OA model - Protocol Treatment groups s.c. injection, n = 9 per group Saline control Morphine 6mg/kg Sodium diclofenac 30mg/kg Isovaline ACBC Behavioral tests Change in weight bearing (incapacitance tester) Tactile allodynia (von Frey hair stimulation) Measurement of knee swelling

Treatment groups

s.c. injection, n = 9 per group

Saline control

Morphine 6mg/kg

Sodium diclofenac 30mg/kg

Isovaline

ACBC

Behavioral tests

Change in weight bearing (incapacitance tester)

Tactile allodynia (von Frey hair stimulation)

Measurement of knee swelling

3. OA model - Induction of osteoarthritis Anaesthetize rat using sevoflurane MIA injection Right knee flex at 90 ° MIA (2mg in 25µL of sterile saline) injected through infrapatellar ligament into joint

Anaesthetize rat using sevoflurane

MIA injection

Right knee flex at 90 °

MIA (2mg in 25µL of sterile saline) injected through infrapatellar ligament into joint

OA Model – Objective evaluation of pain Use of body weight distribution as a surrogate of pain The incapacitance tester Industry accepted method in assessment of the effectiveness of analgesics in OA pain Dual channel weight averager Arthritis induce imbalance in distribution Drugs can restore normal weight bearing

Use of body weight distribution as a surrogate of pain

The incapacitance tester

Industry accepted method in assessment of the effectiveness of analgesics in OA pain

Dual channel weight averager

Arthritis induce imbalance in distribution

Drugs can restore normal weight bearing

3. OA model - Incapacitance tester

OA model - Incapacitance tester

OA model – Allodynia test Change in paw withdrawal threshold to stimulation with von Frey hairs ↑ pressure applied using stiffer hair Until paw lifted or licking response ellicited Compare left & right paw thresholds Drugs used for treating chronic pain reverse allodynia

Change in paw withdrawal threshold to stimulation with von Frey hairs

↑ pressure applied using stiffer hair

Until paw lifted or licking response ellicited

Compare left & right paw thresholds

Drugs used for treating chronic pain reverse allodynia

OA model – Tactile allodynia

OA model - Effects of conventional analgesics Fernihough et al. 2004

OA model – Preliminary data N = 5 / time pt Error in SD

N = 5 / time pt

Error in SD

Take home messages Isovaline and ACBC are structural analogues of glycine and GABA ACBC is a known glycine B receptor partial agonist Mechanism of action for isovaline under investigation Isovaline very well tolerated in rat Up to max soluble dose of 2000mg/kg Both are analgesics as tested by the “formalin foot” model ACBC is more potent than isovaline (~10 fold difference in ED 50 )

Isovaline and ACBC are structural analogues of glycine and GABA

ACBC is a known glycine B receptor partial agonist

Mechanism of action for isovaline under investigation

Isovaline very well tolerated in rat

Up to max soluble dose of 2000mg/kg

Both are analgesics as tested by the “formalin foot” model

ACBC is more potent than isovaline (~10 fold difference in ED 50 )

Acknowledgements Co-supervisors Dr. Bernard MacLeod Dr. David Mathers Tormentor Dr. Michael Walker Special thanks to: Dr. Daegeun Jeon Harry Chang Photographs NASA The Hugill Centre for Anaesthesia & Analgesia University Graduate Fellowship

Co-supervisors

Dr. Bernard MacLeod

Dr. David Mathers

Tormentor

Dr. Michael Walker

Special thanks to:

Dr. Daegeun Jeon

Harry Chang

Photographs

NASA

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