anaesthetic activity. blocking of the sciatic nerve in the

REPORT NO. CD-98/6 169FD

ANAESTHETIC ACTIVITY.BLOCKING OF THE SCIATIC NERVEIN THE ANAESTHETISED RAT.

TEST SUBSTANCES: RACEMlC IQB-9302

(+)-IQB-9302

(-)-IQB-9302

REPORT NO. CD-98/6 169FD- -

ANAESTHETIC ACTIVITY. BLOCKING OF THE SCIATIC NERVE IN

ANAESTHETISED RAT.

TEST SUBSTANCES: RACEMIC IQB-9302, (+)-IQB-9302, (-)-IQB-9302

Head Pharmacodynamics Section: Antonia Arañó

Biologist

Study Director: Lluis Perez

Biologist

Scientist involved: Oscar Ciervo

Biologist

Sponsor: INSTITUTO DE INVESTIGACION Y

DESARROLLO QUIMICO BIOLOGICO, S.A.

c/ Tebas, 20

28230-LAS ROZAS

(Madrid)

Tel.: 34 91 63 160 26

Fax : 34 91 63 165 03

Sponsor’s Monitoring Scientist:

Study Location:

Typed by: N. Perombelon

Date Report Issued: ‘i

Dr Alvaro Galiano

CENTRO DE INVESTIGACION Y

DESARROLLO APLICADO, S.A.L.

Centro Industrial Santiga

c/Argenters, 6

08130-SANTA PERPETUA DE MOGODA

Barcelona

Spain

Tel.: 34 93 719 03 61

Fax: 34 93 718 96 67

1.

REPORT NO. CD-9816 169FDA:ygXV=: -_

ANAESTHETIC ACTIVITY. BLOCKING OF THE SCIATIC NERVE IN TH#-

ANAESTHETISED RAT.


No. pages in Report: 49

This Study was carried out according to the Good Laboratory Practice regulations publishedby the OECD (OECD Principles of Good Laboratory Practice, C (81) 30 (Final), Paris, 12May, 198 1. Annex II), and adopted by the EEC (now EU) according to Directive 87/l 8/EECof 18 December 1986 and in Spain by Real Decreto 822/1993, of 28th May.

No circumstances which could affect the reliability of the data obtained in the Study were recorded.

Head Pharmacodynamics Section:

Study Director:

The results presented in this Report refer only to the sample(s) received and tested, as indicated in thecorresponding section.

This Report may not be partially reproduced without the prior written consent of Centro de Investigación yDesarrollo Aplicado, S.A.L.

II.

QUALITY ASSURANCE UNIT (QAU)

Inspection of Study no. CD-98/6169FD

This Study was carried out according to the Good Laboratory Practice regulations published by the

OECD (OECD Principles of Good Laboratory Practice, C (81) 30 (Final), Paris, 12th May, 1981.

Annex 2) and adopted by the EEC (now EU) according to Directive 87/18/EEC of 18th

December 1986 and in Spain by Real Decreto 822/1993, of 28th May.

The inspection dates are as follows:

03.JUN.98 ISOLATION OF SCIATIC NERVE.

DISSECTION OF THE GASTROCNEMIUS

MUSCLE. STIMULATION AND

ADMINISTRA TION

04.JUN.98 FORMULATION

03 JUL.98 FINAL REPORT

QAU

INSPECTION

NUMBER

14792 08.MAY.98

14936 05.JUN.98

14942

15074

REPORTS TO

MANAGEMENT

05.JUN.98

07 SEP.98

A. Flores

Quality Assurance Unit

Date

III.

CD-98/6169FD

CONTENTS

IDENTIFICATION SHEET .................................................................................................... I

SIGNATURES ...................................................................................................................... II

QAU STATEMENT ............................................................................................................. III

CONTENTS ....................................................................................................................... IV

SUMMARY.. ....................................................................................................................................V

1. OBJECTIVE ......................................................................................................................3

2. JUSTIFICATION ...............................................................................................................3

3. IDENTIFICATION OF THE TEST SUBSTANCES ........................................................ .3

4. TEST FACILITIES .......................................................................................................... .4

5. STUDY DATES.. ............................................................................................................. .4

6. MATERIAL ...................................................................................................................... 4

6.1. Animals ................................................................................................................................. 4

6.2. Equipment .................................................................................................................. 4

6.3. Reagents ............................................................................................................ 5

7. METHOD ................................................................................................................ 5

7.1. Animal identification and housing ........................................................................ .5

7.2. Experimental design............................................................................................ 6

8. EVALUATION OF THE RESULTS ................................................................................ 7

9. ARCHIVES ...................................................................................................................... 8

10. STANDARD OPERATING PROCEDURES .................................................................. 8

11. PROTOCOL DEVIATIONS ........................................................................................... 9

12. RESULTS ...................................................................................................................... 9

13. CONCLUSIONS ............................................................................................................10

14. REFERENCES.. ..................................................................................................... 12

TABLES AND FIGURES .......................................................................................................13

IV.

REPORT NO. CD-98/6169FD

ANAESTHETIC ACTIVITY. BLOCKING OF THE SCIATIC NERVE IN THE

ANAESTHETISED RAT.


SUMMARY

The aim of this Study was to determine the local anaesthetic activity of three substances:

RACEMIC IQB-9302, (+)-IQB-9302 and (-)-IQB-9302 when administered topically to the sciatic

nerve of the rat.

BUPIVACAINE at the concentrations of 0.l%, 0.25% and 0.5%, was used as the reference

substance. The test substances, RACEMIC IQB-9302, (+)-IQB-9302 and (-)-IQB-9302, were

also tested at these concentrations (0.1%, 0.25% and 0.5%).

Overall, considering the administration of all of the substances at all of the concentrations tested,

there was marked, local anaesthetic activity observed in the experimental model used (blocking of

the sciatic nerve of the rat).

Therefore and in view of the results obtained for the area under the curve, the treatment groups in

which the anaesthetic activity was greatest, in descending order, were 0.5% RACEMIC IQB-9302,

0.25% and 0.5% (-)-IQB-9302.

Lower activities were observed in the groups treated with 0.1% (+)-IQB-9302, 0.5%, 0.25% and

0.1% BUPIVACAINE, 0.25% and 0.5% (+)-IQB-9302. The lowest activities were observed in

the groups administered with 0.1% RACEMIC IQB-9302, 0.1% (-)-IQB-9302 and 0.25%

RACEMIC IQB-9302.

At the concentrations of 0.25% and 0.5%, the effect of the administration of BUPIVACAINE was

practically maximal 20 to 30 minutes post-administration, while at the concentration of 0.1% the

maximal activity was reached 80 minutes after administration. For the three concentrations of

BUPIVACAINE tested, the recovery of the nervous response was similar.

1.

CD-98/6 169FD

For the test substance RACEMIC IQB-9302 administered at 0.5%, the maximal effect was

observed 10 minutes after administration while at the concentration of 0.25%, it was reached 80

minutes afterwards. At the concentration of 0.l%, a complete blockage of the sciatic nerve was

not obtained and the maximal activity achieved was reached later than those obtained with the

higher concentrations.

The administration of the test substance (+)-IQB-9302 did not manage to block completely the

sciatic nerve and the maximum effect was observed at the concentration of 0.1%.

For the test substance (-)-IQB-9302 at the concentrations of 0.25% and 0.5%, complete blockage

of the sciatic nerve was obtained 20 minutes after administration. The administration of this

substance at the concentration of 0.1% did not obtain complete blockage of the sciatic nerve and

the maximal activity observed occurred later.

In conclusion and in view of the calculations of the area under the curve, the results obtained

demonstrate a local anaesthetic activity for the test substances (-)-IQB-9302 and RACEMIC

IQB-9302 greater than that of BUPIVACAINE.

2.

REPORT NO. CD-98/6169FD

ANAESTHETIC ACTIVITY. BLOCKING OF THE SCIATIC NERVE IN THE

ANAESTHETISED RAT.


1.

2.

OBJECTIVE

The aim of this Study was to determine the local anaesthetic activity of the substances:

RACEMIC IQB-9302, (+)-IQB-9302 and (-)-IQB-9302 on the sciatic nerve of the rat and

compare it to that of BUPIVACAINE, one of the most potent local anaesthetics in current

clinical use.

JUSTIFICATION

The use of local anaesthesia in the clinical context

topical anaesthesia by conduction and by infiltration.

can be subdivided into superficial or

Conduction anaesthesia is achieved by the local injection of the anaesthetic, in the vicinity

of the nerve. The anaesthetic diffuses throughout the nervous trunk and anaesthetises the

innervated area, inhibiting the conduction of nervous impulses throughout the nerve.

A experimental model used frequently to study conduction anaesthesia is blocking of the

sciatic nerve of the rat (1) (2).

The rat was used because it is the species specified in the previously published experimental

method.

3. IDENTIFICATION OF THE TEST SUBSTANCES

The identity, quality, concentration and purity of the test substances RACEMIC IQB-9302,

(+)-IQB-9302 and (-)-IQB-9302, supplied for this Study are the responsibility of the

Sponsor.

3.

CD-98/6 169FD

4

5

6.

On 8th April 1998, Centro de Investigación y Desarrollo Aplicado, S.A.L. received 3 vials of

the test substances RACEMIC IQB-9302, (+)-IQB-9302 and (-)-IQB-9302, supplied by

LEBSA and on 3rd April 1998, received one vial containing one gram of the reference

substance BUPIVACAINE, supplied by INIBSA.

A sample of each test substance will be kept in the Centro de Investigación y Desarrollo

Aplicado, S.A.L. archives for a period of 5 years starting from the date of issue of the Final

Report, or until its expiry date.

TEST FACILITIES

This Study was carried out in the Pharmacology Department laboratories at Centro de

Investigación y Desarrollo Aplicado, S.A.L., Centro Industrial Santiga, c/Argenters 6,

08130-Santa Perpetua de Mogoda, Barcelona., Spain.

STUDY DATES

5.1. Protocol accepted: 24th May 1998

5.2. Arrival of test and reference substances: 3rd and 8th April 1998

5.3. Arrival of animals: 20th and 27th May 1998

5.4. Experimental work started: 25th May 1998

5.5. Experimental work ended: 9th June 1998

5.6. Final Report issued : See page I

MATERIAL

6.1. Animals

65 male Wistar rats, weighing 273-364 g at the start of the assay and bred by CHARLES

RIVER S. A., were supplied by CRIFFA, S. A.

6.2. Equipment

- Polygraph LECTROMED Mod. Multitrace 4P

- High-gain amplifier LECTROMED Mod. 524 1

- Isometric transducer PANLAB Mod. UFI

4.

CD-98/6 169FD

- Electrical Stimulator SRI Mod. 6053

- Digital thermometer PANLAB Mod. 33 l-02

- Temperature probe YSI Mod. 423

- Thermostatic bath B. BRAUN Mod. Thermomix MM

- Chronometer HANHART Mod. Controller

- Balance SARTORIUS Mod. L22OOS

- Automatic pipette GILSON Mod. P-100

- Automatic pipette NICHIRYO Mod. 5000DG

- Electric shaver OSTER Mod. Golden 15

6.3. Reagents

- Urethane (SIGMA, Batch no. 20H0481)

- Physiological saline (B. BRAUN MEDICAL, S.A., Batch no. M061A43A)

- 0.1 N Hydrochloric acid (NORMASOLV, Batch no. 16129)

7. METHOD

7.1. Animal Identification and housing

The animals were identified by markings on the tail and were housed in groups of 5 in

Makrolon cages (47.6 x 22.7 x 14.5 cm) with fixed floor and sawdust bedding.

Each cage had a label stating the Study code number, Study Director’s name and

animal numbers, sex and their arrival dates.

All the rats were offered free access to the dried, pelleted standard rat diet UAR A04C,

(Usine d’Alimentation Rationnelle, 9 1360-Villemoisson sur Orge, France) batches

71203 and 80327.

The water, supplied by Compañia de Aguas de Sabadell, S.A., was provided to the

animals ad libitum. The water is periodically analyzed for possible contaminants.

5.

CD-98/6 169FD

Temperature in the animal room was maintained at 22 +/-3°C and the relative humidity

at 60 +/- 10%, occasionally reaching values of 40%.

Lighting was provided using a 12-hour light (7:00 to 19:00 hours) and 12-hour dark

cycle every 24 hours.

7.2. Experimental design

The animals underwent an acclimatization period of at least 5 days before the experimental

work was started.

They were allocated, at random, to 13 test groups each consisting of 5 animals. The

test groups were as follows:

Group

ABCDEFGHIJKLM

Treatment

CONTROLBUPIVACAINEBUPIVACAINEBUPIVACAINE

RACEMIC IQB-9302RACEMIC IQB-9302RACEMIC IQB-9302

(+)-IQB-9302(+)-IQB-9302(+)-IQB-9302(-)-IQB-9302(-)-IQB-9302(-)-IQB-9302

Concentration

(%)

0.10.250.50.1

0.250.50.1

0.250.50.1

0.250.5

Colour

WhiteYellow

Yellow/BlackYellow/Red

BlueBlue/BlackBlue/Red

GreenGreen/BlackGreen/Red

RedRed/BlackRed/Red

On the day of the assay, each animal was anaesthetised intraperitoneally with urethane

(1 g/kg, 10 mL/kg).

Each animal was then placed in a prone position on a thermostatic board with

circulating water system at 37”C, to maintain its body temperature.

6.

CD-98/6 169FD

The position of the sciatic nerve in one of the rear legs was determined and a length of

1 cm was exposed. An electrode was attached to the nerve and to the stimulator, A

small cotton swab was placed on the nerve. The gastrocnemius muscle was dissected

and the distal end connected to an isometric transducer using a suture. The isometric

transducer was connected to an instrument to record the readings.

The sciatic nerve was stimulated with an impulse of 1.5 V and amplitude of 30

milliseconds and the resulting contraction by the gastrocnemius muscle was recorded.

After a stabilization period of 5 minutes (basal reading), the cotton swab was

impregnated with 100 uL of the test solution. The response of the gastrocnemius

muscle to the electrical stimulation of the sciatic nerve was recorded every 10 minutes

for a period of 2 hours after administration of the test solution.

At the end of this period, the cotton swab was washed with physiological saline and the

recovery of the nervous response was recorded.

8. EVALUATION OF THE RESULTS

A basal reading was made and thereafter readings were taken every 10 minutes for the 2

hours after administration of the test substance and then after washing the swab, to evaluate

the recovery of the nervous response.

The absolute values were converted to percentages (relative values) to take into account

the different basal values. The area under the curve (AUC) of the contraction of the

gastrocnemius muscle was also calculated, according to Simpson’s method (3).

The mean, standard deviation (S.D.) and standard error of the mean (S.E.M.) were

calculated for each reading taken and treatment group (4).

7.

CD-98/6 169FD

9.

10.

The means of the different groups were compared using a one-way analysis of variance (4).

Where there were statistically significant differences, the Duncan-Kramer test for the

comparison of means was applied (5) (6).

Where this test is used, each mean value is assigned a letter and these letters are placed in

ascending order (lowest to highest). The diierence between two means which are not

underlined by the same line is statistically significant and the difference between two means

which are underlined by the same line is not statistically significant.

The percentage of anaesthetic activity was calculated for each of the treatments given using

the following formula:

% Anaesthetic activity =AUCc - AUCT x 100

AUCc

where AUCT is the area under the curve for the different treatment groups and AUCc is the

area under the curve for the Control group.

ARCHIVES

All the material related to the Study, including Study notebooks, results obtained and all other

relevant documentation will be kept in a suitable filing system for at least 5 years according to

the Centro de Investigation y Desarrollo Aplicado, S.A.L. Standard Operating Procedures.

No material relating to the Study will be disposed of without the written consent of the Sponsor.

STANDARD OPERATING PROCEDURES

All operations were carried out according to the Centro de Investigación y Desarrollo

Aplicado, S.A.L. Standard Operating Procedures.

8.

CD-98/6 169FD

11.

12.

PROTOCOL DEVIATIONS

The following deviations from the experimental protocol were detected in the course of the

Study:

- the bodyweight of the animals used in the assay were between 273 and 364 g, a range

slightly higher than that indicated in the protocol.

- the relative humidity of the animal room in which the animals were kept was 60 +/- 10%,

reaching values of 40% occasionally, a range slightly greater than that specified in the

experimental protocol.

Although these values are distinct from those specified in the experimental protocol, these

deviations did not influence the course of the Study nor alter its results.

RESULTS

The absolute overall results obtained are shown in Table no. 1 and in the following page, in the

annex to Table no. 1, the statistical evaluation. The relative global results are shown in Table

no. 2.

The absolute individual results are presented in Tables nos. 3 to 15 and the Figures nos. 1 to 4

and the individual values relative to their basal values in the Tables nos. 16 to 28 and the

Figures nos. 5 to 8.

No statistically significant differences were observed using the one-way analysis of variance for

the comparison of the means of the basal values of the different treatment groups.

For the following treatments, 10 minutes after their administration, there were statistically

significant differences between the corresponding treatment groups and the Control group

(Duncan-Kramer test, p<0.05): 0.25% and 0.5% BUPIVACAINE, 0.5% RACEMIC

IQB-9302, 0.5% (+)-IQB-9302 and 0.25% and 0.5% (-)-IQB-9302.

9.

CD-98/6 169FD

13.

From 20 minutes after administration and onwards, including the recovery period, all the

treatment groups were significantly different from the Control group (Duncan-Kramer test,

p < 0.05).

The percentages of recovery for BUPIVACAINE at the concentrations of 0. l%, 0.25% and

0.5% were 25.6%, 33.1% and 42.1%, respectively and the percentages of inhibition were

82.3%, 84.1% and 90.0%, respectively.

The percentages of recovery for RACEMIC IQB-9302 at the concentrations of 0. l%, 0.25%

and 0.5% were 10.2%, 18.0% and 4.5%, respectively and the percentages of inhibition were


The percentages of recovery for (+)-IQB-9302 at the concentrations of 0.l%, 0.25% and

0.5% were 66.5%, 58.2% and 24.6%, respectively and the percentages of inhibition were


The percentages of recovery for (-)-IQB-9302 at the concentrations of 0.l%, 0.25% and 0.5%

were 60.3%, 22.3% and 11.4%, respectively and the percentages of inhibition were 76.8%,

95.8% and 94.1%, respectively.

CONCLUSIONS

The local anaesthetic activity of RACEMIC IQB-9302, (+)-IQB-9302 and (-)-IQB-9302

were determined in the experimental model used: blocking of the sciatic nerve of the rat.

Under our experimental conditions, marked local anaesthetic activity was observed for all the

treatments given at all of the concentrations tested.

Therefore and according to the results obtained for the area under the curve, the treatment

groups in which the anaesthetic activity was greatest, in descending order, were 0.5%

RA CEMIC IQB-9302, 0.25% and 0.5% (-)-IQB-9302.

10.

CD-98/6 169FD

Lower activities were observed in the groups treated with 0.1% (+)-IQB-9302, 0.5%, 0.25%

and 0.1% BUPIVACAINE, 0.25% and 0.5% (+)-IQB-9302. The lowest activities were

observed in the groups administered with 0.1% RACEMIC IQB-9302, 0.1% (-)-IQB-9302

and 0.25% RACEMIC IQB-9302.

At the concentrations of 0.25% and 0.5%, the effect of the administration of BUPIVACAINE

was practically maximal 20 to 30 minutes post-administration, while at the concentration of

0.1% the maximal activity was reached 80 minutes after administration. For the three

concentrations of BUPIVACAINE tested, the recovery of the nervous response was similar.

For the test substance RACEMIC IQB-9302 administered at 0.5%, the maximal effect was

observed 10 minutes after administration while at the concentration of 0.25%, it was reached

80 minutes afterwards. At the concentration of 0.1%, a complete blockage of the sciatic nerve

was not obtained and the maximal activity achieved was reached later than those obtained with

the higher concentrations.

The administration of the test substance (+)-IQB-9302 did not manage to block completely the

sciatic nerve and the maximum effect was observed at the concentration of 0.1%.

For the test substance (-)-IQB-9302 at the concentrations of 0.25% and 0.5%, complete

blockage of the sciatic nerve was obtained 20 minutes after administration. The administration

of this substance at the concentration of 0.1% did not obtain complete blockage of the sciatic

nerve and the maximal activity observed occurred later than those obtained at the higher

concentrations.

In conclusion and in view of the calculations of the area under the curve, the results obtained

demonstrate a local anaesthetic activity for the test substances (-)-IQB-9302 and RACEMIC

IQB-9302 greater than that of BUPIVACAINE.

11

CD-98/6 169FD

14. REFERENCES

(1)

(2)

(3)

(4)

(5)

(6)

Pharmacological Experiments on Intact Preparations. The Staff of the Department of

Pharmacology, University of Edinburgh. E&S LIVINGSTONE. Edinburgh and

London (1970).

Anatomy of the Laboratory Rat. Rudolf Hebel and M. W. Stromberg.. The Williams

& Wilkins Company. Baltimore (1976).

Manual of Pharmacological Calculations with Computer Programs. Ronald J.

Tallarida and Rodney B. Murray. Springer-Verlag New York Inc. (198 1).

STEEL, R.G.D., TORRIE, J.H. Principles and Procedures of Statistics. McGraw-

Hill Book Company, Inc. N.Y. (1960)

DUNCAN, D.B. Multiple range and multiple F test. Biometrics, 11, 1 (1955).

HARTER H.L. Critical values for Duncan’s new multiple range test. Biometrics,

l6, 671 (1960).

12.

anaesthetic activity. blocking of the sciatic nerve in the

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