Amino acids –treatment for chronic

pain? Cheryl Chung, Jan 15, 2009

2

In the beginning…

3

Sep 28, 1969 Murchison Meteorite

Timeline

Isovaline Not found in biosphere Non-biogenic

Sep 28, 1969

Timeline

4

Year 2006 MacLeod’s Lab

Timeline

Search for novel analgesics Investigated isovaline & cyclic analogue

ACBC (1-amino cyclobutane carboxylic acid)

Found they blocked acute allodynia & responses to pain

Year 2006Sep 28, 1969

Timeline

5

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

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Isovaline & ACBC – analogues of glycine & GABA

a) Glycine

b) ACBC (1-amino cyclobutane carboxylic acid)

c) GABA (gamma- amino butyric acid)

d) Isovaline

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Present Project Goals

Timeline

Test amino acids in models of chronic pain Suppress paw licking response in 2nd

phase of formalin foot model Alleviate pain responses & allodynia in

osteoarthritis model

Present

Timeline

Year 2006Sep 28, 1969

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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

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Experiments

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3 part study

1. Maximum tolerated dose (MTD) study

2. Dose ranging studies in “formalin foot” model

3. Effectiveness study in osteoarthritis (OA) model (using ED50 from 2)

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1. Maximum tolerated dose study

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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)

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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)

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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

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1. MTD study – An anecdote

Rat 6, 2000mg/kg racemic isovaline

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1. MTD study - Mean arterial pressure

Change in Mean Arterial Pressure

-20 0 20 40 60 80-50

0

50

100500mg/kg1000mg/kg

1500mg/kg

2000mg/kg

Time after injection (min)

% c

han

ge

MA

P f

rom

bas

elin

e

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1. MTD study - Heart rate

Change in Heart Rate

-20 0 20 40 60 80-50

-25

0

25

50500mg/kg1000mg/kg

1500mg/kg

2000mg/kg

Time after injection (min)

% c

han

ge

HR

fro

m b

asel

ine

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1. MTD study - Breathing rate

Change in Breathing Rate

-20 0 20 40 60 80-50

-25

0

25

50

1000mg/kg

1500mg/kg

2000mg/kg

Time after injection (min)

% c

han

ge

BR

fro

m b

asel

ine

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2. Dose ranging studies

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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 2nd phase of

response to formalin) by 40% from control.

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2. DR Studies – Up & down method ED50 estimate via the up-and-down method

(Dixon 1965)

ED50

Est. range of ED50

Dose 1Dose 2Dose 3Dose 4

Dose 5( - )

( - )( - )

( + ) ( + )

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2. DR studies - Formalin foot test

-5 drug

TIME (min)formalin

205 10 15 25 35 4030-20

Acclimatization

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2. DR studies - Results

Time course similar to previous results Isovaline ED50 = 740±20mg/kg ACBC ED50 = 88±9mg/kg

Finding ED50 for inhibition of phrase II formalin foot paw licking responseusing the “up and down” method

Isovaline

1 2 3 4 5 6 7 80

500

600

700

0

ED50

Test animals

Do

se (

mg

/kg

)

ACBC

2 4 6 8 10

10

46

53

61

70

80

92

106

O < 60% inhibition of licking responsefrom control

> 60% inhibition of licking responsefrom control

0

ED50

Test animals

Do

se (

mg

/kg

)

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3. Osteoarthritis Model

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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

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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

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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

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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

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3. OA model - Incapacitance tester

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OA model - Incapacitance tester

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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

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OA model – Tactile allodynia

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OA model - Effects of conventional analgesics

Fernihough et al. 2004

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OA model – Preliminary data

N = 5 / time pt Error in SD

Knee diameter of MIA treated animals

7 14 21 28-1

0

1

2

3

4

5

time after MIA injection (days)ri

gh

t kn

ee -

lef

t kn

ee (

mm

)

Body weight distribution for MIA treated animals

Non arth

ritic

Day 3

Day 1

4

Day 2

80

20

40

60

dif

fere

nc

e i

n w

eig

ht

(le

ft -

rig

ht

in g

ram

s)

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Take home messages

1. Isovaline and ACBC are structural analogues of glycine and GABA

ACBC is a known glycineB receptor partial agonist Mechanism of action for isovaline under investigation

2. Isovaline very well tolerated in rat Up to max soluble dose of 2000mg/kg

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

ACBC is more potent than isovaline (~10 fold difference in ED50)

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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