WO2002093163A1

WO2002093163A1 - Test method for pain evaluation

Info

Publication number: WO2002093163A1
Application number: PCT/DK2002/000312
Authority: WO
Inventors: Rigmor Jensen; Lars Bendtsen; Messoud Ashina; Jes Olesen; Claus Mathiesen; Alexander Norup Nielsen
Original assignee: Head Explorer A/S
Priority date: 2001-05-16
Filing date: 2002-05-14
Publication date: 2002-11-21

Abstract

The present invention relates to a method for inducing a muscle pain and/or tenderness, a myofascial pain, and/or a central sensitization in a test animal. The method comprises the intramuscolar infusion of one or more endogenous substances, selected from bradykinin, serotonin, histamine and prostaglandin E2. The invention also relates to a method for measuring the effect of a test substance in the treatment, prevention or alleviation of myofascial pain and/or a disease associated with central sensitization in a subject.

Description

TEST METHOD FOR PAIN EVALUATION

The present invention relates to a method for inducing a muscle pain and/or tenderness, a myofascial pain, and/or a central sensitization in a test animal. The invention also relates to a method for measuring the effect of a test substance in the treatment, prevention or alleviation of myofascial pain and/or a disease associated with central sensitization in a subject.

BACKGROUND ART

Pain related to the musculoskeletal system is very prevalent in the general population. The pathophysiological mechanisms underlying myofascial pain and tenderness may include: a) sensitization of peripheral myofascial nociceptors, b) sensitization of second order neurons at the spinal / trigeminal level, or c) altered central modulation of the nociceptive activity.

Tension-type headache (TTH) is an extremely prevalent disorder since 78 % of the population has experienced this type of headache - and 3 % are suffering from chronic tension-type headache (CTTH) with symptoms every or every other day. The increased pericranical myofascial tenderness by palpation in TTH is positively correlated to frequency of headache and is the most prominent abnormal finding in patients with TTH. A characterisation of the neurobiologic basis of human muscle pain may therefore be of clinical importance in TTH as well in other myofascial pain disorders. Several human experimental models of localized myofascial pain have been developed, but only few studies used endogenous substances to induce muscle pain (Jensen et al., Peptides, 11 :1127-1132 (1990), Babenko et al., Pain, 82:1-8 (1999), and Ernberg et al., Pain 85:31-39 (2000)). These studies have reported that bolus injections of endogenous substances induced a brief pain of mild intensity and a decrease of mechanical pain thresholds.

The short duration is a distinct disadvantage. Thus, there is a strong need for a reliable in vivo (animal or human) model of protracted muscle pain and tenderness, such as myofascial pain and tenderness.

SUMMARY OF THE INVENTION

According to the invention it has now been found that intramuscular infusion of one or more endogenous substances selected from bradykinin, serotonin, histamine and prostaglandin E₂ in a muscle of a test animal induces a muscle pain and/or tenderness. Therefore, such an induced pain and/or tenderness can be used as a model for in vivo evaluation of pain, such as myofascial pain, and/or central sensitization.

Thus, in a first aspect, the invention relates to a method for inducing a muscle pain and/or tenderness, a myofascial pain, and/or a central sensitization in a test animal, which method comprises:

• intramuscular infusion of one or more endogenous substances selected from bradykinin, serotonin, histamine and prostaglandin E₂ in a muscle of the test animal.

In a second aspect, the invention relates to a method for measuring the effect of a test substance in the treatment, prevention or alleviation of myofascial pain and/or a disease associated with central sensitization in a subject.

In a third aspect, the invention relates to the use of a compound identified in above method.

In a fourth aspect, the invention relates to a drug development method. Other objects of the invention will be apparent to the person skilled in the art from the following detailed description and examples.

DETAILED DISCLOSURE OF THE INVENTION

In a first aspect, the invention provides a method for inducing a muscle pain and/or tenderness in a test animal, which method comprises:

In one embodiment, the invention provides a method for inducing a myofascial pain in a test animal, which method comprises:

• intramuscular infusion of one or more endogenous substances selected from bradykinin, serotonin, histamine and prostaglandin E₂ in a facial or neck muscle of the test animal.

In a further embodiment, the invention provides a method for inducing a central sensitization in a test animal, which method comprises:

In a further aspect, the invention provides a method for measuring the effect of a test substance in the treatment, prevention or alleviation of myofascial pain in a subject, which method comprises the following steps:

• inducing a pain and/or tenderness in a facial or neck muscle of a test animal; • administering to the test animal an amount of the test substance;

• measuring the effect of the test substance on the pain and/or tenderness induced.

In a still further aspect, the invention provides a method for measuring the effect of a test substance in the treatment, prevention or alleviation of a disease associated with central sensitization in a subject, which method comprises the following steps:

• inducing a central sensitization by inducing a pain and/or tenderness in a facial or neck muscle of a test animal; • administering to the test animal an amount of the test substance;

• measuring the effect of the test substance on the central sensitization induced. In a further aspect, the invention provides a drug development method, which comprises the identification of a compound by screening test substances according to any one of the methods as described above. In a still further aspect, the invention provides the use of a compound identified by the any one of the methods as described above as being able to reduce the pain and/or tenderness, or a pharmaceutically acceptable salt or a prodrug thereof for the manufacture of a medicament for the treatment, prevention or alleviation of myofascial pain. In a further aspect, the invention provides a method for the treatment, prevention, or alleviation of myofascial pain in a subject comprising administering to said subject a therapeutically effective amount of a compound identified by any one of the methods as described above as being able to reduce the pain and/or tenderness, or a pharmaceutically acceptable salt or a prodrug of said compound. In one embodiment, the endogenous substances are prostaglandin E₂and one or more of the substances bradykinin, serotonin, and histamine. In a special embodiment the endogenous substances are prostaglandin E₂and one of the substances bradykinin, serotonin, and histamine. In a further special embodiment the the endogenous substances are prostaglandin E₂and two of the substances bradykinin, serotonin, and histamine. In a still further special embodiment the endogenous substances is a combination of all four substances prostaglandin E₂, bradykinin, serotonin, and histamine. The concentration of each of the endogenous substances bradykinin, serotonin, and histamin in the infusion is from about 1 to about 10,000 nmol per ml, preferably from about 10 to about 1000 nmol per ml, most preferred from about 50 to about 300 nmol per ml. The concentration of the endogenous substance prostaglandin E₂ in the infusion is from about 0.02 to about 200 nmol per ml, preferably from about 0.2 to about 20 nmol per ml, most preferred from about 1 to about 4 nmol per ml. In a special embodiment, the infusion contain (per ml) from about 50 to about 200 nmol of bradykinin, from about 75 to about 300 nmol of serotonin, from about 75 to about 300 nmol of histamin, and from about 1 to about 4 nmol of prostaglandin E₂.

In a second embodiment, the muscle of the test animal is a facial or neck muscle, such as a frontal muscle, a masseter muscle, a temporal muscle, a sternocleidomastoid muscle, a splenius capitis muscle, or a trapezius muscle. In a special embodiment, the muscle of the test animal is a trapezius muscle.

In a further embodiment, the test animal is a rodent, such as a rat. In a still further embodiment, the test animal is a human being.

In a special embodiment, the test animal is a human being and the measuring of the effect of the test substance on the pain and/or tenderness is performed by assessment of pain intensity, pain quality, local muscle tenderness and/or total tenderness.

In a further special embodiment, the test animal is a rodent and the measuring of the effect of the test substance on the central sensitization is performed by electrophysiological measures and/or behavioural testings. Examples of electrophysiological measures are measurement of EEG, field potentials and single unit recordings. An example of behavioural testing is the von Frey hair test.

In a further embodiment, the disease associated with central sensitization is tension-type headache, such as episodic tension type headache or chronic tension type headache.

The intramuscular infusion is performed by continous infusion or a combination of a bolus injection and continuous infusion. In one embodiment the intramuscular administration is performed by continuous infusion.

The infusion rate of the infusion depends on the test animal and the muscle used. For infusion in the trapezius muscle in a human being, the infusion rate is from about 0.01 to about 1 ml per min, preferably from about 0.05 to about 0.2 ml per min. For infusion in the trapezius muscle in a rodent, the infusion rate is from about 1 to about 100 μl per min, preferably from about 5 to about 20 μl per min.

The infusion period depends on the test animal and the muscle used. Typically, the infusion period is between 1 and 120 mins, preferably between 5 and 90 mins, most preferably between 10 and 60 mins.

The subject to be treated according to this invention is a living body, preferably a mammal, most preferably a human, in need for such treatment.

The test substance of which the effect on the induced pain and/or tenderness or central sensitization is to be measured may be administered before, during or after inducing the pain and/or tenderness or central sensitization. Pharmaceutical Compositions

While a chemical compound as identified by the method according to the invention for use in therapy may be administered in the form of the raw chemical compound, it is preferred to introduce the active ingredient, optionally in the form of a physiologically acceptable salt, in a pharmaceutical composition together with one or more adjuvants, excipients, carriers, buffers, diluents, and/or other customary pharmaceutical auxiliaries.

In a preferred embodiment, the invention provides pharmaceutical compositions comprising the chemical compound of the invention, or a pharmaceutically acceptable salt or derivative thereof, together with one or more pharmaceutically acceptable carriers therefor, and, optionally, other therapeutic and/or prophylactic ingredients, know and used in the art. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof. The pharmaceutical composition of the invention may be administered by any convenient route which suit the desired therapy. Preferred routes of administration include oral administration, in particular in tablet, in capsule, in drage, in powder, or in liquid form, and parenteral administration, in particular cutaneous, subcutaneous, intramuscular, or intravenous injection. The pharmaceutical composition may be prepared by the skilled person using standard and conventional techniques appropriate to the desired formulation. When desired, compositions adapted to give sustained release of the active ingredient may be employed.

Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).

The following examples will illustrate the invention further, however, they are not to be construed as limiting.

EXAMPLES

Example 1

Inducing a muscle pain and/or tenderness in a human being

Test persons

Five healthy test persons (3 males and 2 females) with a mean age of 26 years (23 - 33 years) participated in the study. All test persons underwent a physical and neurological examination prior to participation in the study. Exclusion criteria's were: a history of more than one episode of tension-type headache per month; migraine; allergy; serious somatic or psychiatric disorders; use of any kind of daily medication (except for oral contraceptives); excessive use of analgesics (> 2 g of aspirins or equivalent daily); pregnancy or breast-feeding; abnormal physical or neurological examination. The test persons were not allowed to take analgesics or any other medication 24 hours prior to examination.

Infusions

The infusions were given in a standardized anatomical point at the center of the descending part of the trapezius muscle midway between the processus spinosus of the seventh cervical vertebra and the acromion. The infusions were administered by a syringe pump (Graseby 3400) using a 5 ml plastic syringe (CODAN Medical ApS). A tube (Medex, Simonsen & Weel) was connected from the syringe to the disposable EMG-needle (27 G and 25 mm, Judex). The needle was connected to an electromyograph (Counterpoint, Dantec, Copenhagen) to ensure intramuscular infusion. An infusion rate of 1 ml over 10 minutes was used.

Preparation

The doses of the 4 substances in the combination were: Bk 92 nmol, 5-HT 156 nmol, His 140 nmol, and PGE₂ 1.95 nmol per 1 ml. All substances were prepared under sterile conditions as well as pH and microbiological tested by The Central Pharmacy, Copenhagen Community Hospital Services.

Design

Each test person received one infusion of combinations of active substances. The trial was designed as a randomized, double-blinded, and placebo-controlled trial. Each experiment was initiated with an infusion in the right shoulder, followed by an infusion in the left shoulder. The sessions were separated by at least 2 weeks.

Assessment The recordings were performed by the same observer (a trained technician, HA) throughout the study. The test persons were placed in a dental chair in a calm and temperate room.

Pain intensity. Spontaneous pain intensity was scored on a 100 mm Visual Analogue Scale (VAS) where 0 corresponded to no pain and 100 mm to the worst imaginable pain (Price et al. 1983). The pain intensity after infusion of the combination of substances was recorded at baseline and every minute until 5 minutes after stop of infusion, then every 5 minutes until 30 minutes, and at 45 and 60 minutes after start of infusion.

Pain quality. Pain quality was scored on a Danish version of the McGill Pain Questionnaire (Drewes et al. 1993) at 15 (for the period: 0-15 minutes) and 60 minutes (for the period: 15-60 minutes) after start of infusions. Pain rating index (PRI (R)) (0-15 and 15 - 60 minutes) was calculated as outlined by Melzack (1975). Local muscle tenderness. Local tenderness was recorded using palpation with a fixed pressure of 160 arbitrary units corresponding to moderate pressure intensity by means of a palpometer (Bendtsen et al. 1994). The method has previously been described and was demonstrated to improve the reliability of palpation (Bendtsen et al. 1995). Local tenderness was assessed at the infusion site on the trapezius muscle on VAS prior to infusion and 15, 30, 45, and 60 minutes after start of infusion. Total tenderness. Tenderness of pericranial myofascial tissues was recorded according to the Total Tenderness Scoring system (Langemark and Olesen 1987), which has previously proved to be reliable (Bendtsen et al. 1995). Eight pairs of muscles and tendon insertions (masseter, temporal, frontal, sternocleidomastoid and trapezius muscles, coronoid and mastoid processus and neck muscle insertions) were palpated. Tenderness was scored on a 4-point (0 - 3) scale at each location (local tenderness score) and values from left and right sides were summated to a total tenderness score (TTS) (maximum possible score = 48). TTS was measured prior to infusion and 60 minutes after start of infusion.

Pressure pain detection thresholds. An electronic pressure algometer (Somedic AB, Stockholm, Sweden) was used to measure pressure pain detection thresholds (PPDTs) at the dorsum of the second finger (middle phalanx) and at a fixed point at the anterior part of the temporal muscle at the non-dominant side (Bendtsen et al. 1996). A circular stimulation probe (0.50 cm²) and a pressure loading rate of 22 kPa/s (1 kPa = 10³ N/m²) were used and the PPDT-value was reported as the mean of 5 consecutive measurements. PPDT was recorded prior to infusion and 30 and 60 minutes after start of infusion. Other parameters. Wheal and flare and the anatomic localization of local pain and referred pain were noted during the observation time (60 minutes). The presence of tenderness in shoulder or neck region, local or referred pain, headache or intake of medication was recorded 24 hours after the infusions.

Data analysis and statistics Results are presented as medians and quartiles. Wilcoxon ^'s Signed Ranks Test was used comparing active substance and placbo for the following: the area under the pain and local tenderness curves (VAS curves) (AUCpain and AUC τ (0-60 minutes) (Matthews et al. 1990); the maximum pain (P_max) and maximum local tenderness (LT_max) scores (VAS peak); and the duration of pain and duration of tenderness (pain offset -pain onset, tenderness offset -tenderness _onSet). Likewise were the AUC_pai_n , AUCLT , P_max, and LTmax between different doses of PGE₂ and ATP. To assess changes in TTS and PPDTs, the differences between the pre-treatment value and each of the post-treatment values were calculated for active substances and placebo before compared by Wilcoxon's Signed Ranks test. Pain quality was presented as words used by 60 % or more of the participants and the pain-rating index (PRI (R)) was presented as median-values, and scores for active substances and placebo were compared by Wilcoxon's Signed Ranks test. Five percent was accepted as level of significance. All data were analyzed with SPSS ^®software, version 10.0 (SPSS Inc., USA).

Results

Pain intensity and quality. Pain quality is shown in Table I. The AUC_pain and the Pmax were significantly increased after infusion of the combination compared to placebo (P = 0.04, in both cases). Also the duration of pain was significantly longer after infusion of active substance compared to placebo (P = 0.04). Tenderness. The AUCLT and the LT_max were significantly increased after infusion of the combination compared to placebo (P = 0.04, in both cases). Duration of tenderness after infusion of the combination was also longer compared to placebo (P = 0.04). TTS did not show any difference after infusion of active substance compared to placebo. PPDTs increased significantly in the period from 0 - 30 minutes both on the finger (P = 0.04) and in the temporal region (P = 0.04) after infusion of the combination compared to placebo, whereas the PPDTs were normalized 60 min after infusion.

Table I. Pain and tenderness after infusion of the combination of bradykinin (92 nmol), serotonin (156 nmol), histamine (140 nmol), and prostaglandin E₂ (1.95 nmol) or placebo into the trapezius muscle of 5 healthy test persons. Combination / placebo

Number of test persons AUCpain (min x mm) 544 / 22 *

(373-967 / 0-145)

Pmax (VAS mm) 37 /5 *

(19-45 / 0-23) Pain-duration (min) >51 / 5 * .

(26->51 / 0-22)

McGill

-0-15 min: -words chosen by > 60 % Drilling, Hot, Sore, Taut, Nagging

-PRI(R) 16 / 3 (10-21/ 0-13)

-15-60 min:-words chosen by > 60 % -PRI(R) 9 / 0 (2-15 / 0)

AUCLT (min x mm) 660 / 0 (142-1237 / 0-150) LTmax (VAS mm) 19 / 0 (4-38 / 0-6) Tenderness-duration (min) >52 / 0 (>52 / 0-45)

Median values with ranges are given within brackets. Abbreviations: AUC: Area Under the Curve (VAS, min x mm), P_maχ'- Maximum pain score (VAS peak, mm), U_msκ: Maximum local tenderness score (VAS peak, mm), PRI (R): Pain Rating Index. *: P < 0.05

Discussion

The combination of the four substances, bradykinin, serotonin, histamine, and prostaglandin E₂, produced a more pronounced and a more prolonged pain and tenderness than shown in previous studies using a combination of 2 substances given as bolus injections (Jensen et al. 1990b, Babenko et al. 1999). PGE₂ seems to make a difference with respect to the production of pain and tenderness as the combination of 4 substances produced more pain than combinations of 2 or 3 substances. Generalized tenderness as reflected by The Total Tenderness Scores showed no significant changes in any of the present experiments indicating that the tenderness in other pericranial muscles was not affected by this local experimental model. In the present example it has been demonstrated that a combination of Bk, 5- HT, His, and PGE₂ are capable of inducing moderate prolonged muscle pain and tenderness and thereby represents a valuable experimental model of muscular pain. The present model has several features that are advantageous compared to previous models of myofascial pain: a) use of naturally occurring substances or combinations already documented to play a role in pain; b) infusion of substances rather than injecting them in order to mimic clinical situations; c) reliably scoring pain and tenderness by palpometry.

Example 2

Method for measuring of nociceptive input induced central sensitization

General animal preparation Male Wistar rats (200-350 g) are anaesthetized with mebumal (50 mg/kg i.p.). Catheters are inserted in the femoral artery for recording of arterial blood pressure, and in the femoral vein. Rats are tracheotomised and artificially ventilated. The head is placed in a stereotactic headholder, and the cranial bone is removed over the sensory cortex (-2 to -4 mm posterior to bregma and 2 to 4 mm lateral to the midline). A bipolar stimulation electrode is positioned on the exposed surface of the cervical part of trapezius muscle. The muscle is electrically stimulated (0.2 ms duration 0.2-0.8 mA), which results in a small muscle twitch. A recording electrode is inserted contra lateral to the stimulation electrode in 300-500 μm depth at coordinates 2.8 mm posterior to bregma and 3.1 mm lateral to the midline for recording of somatosensory evoked extracellular field potentials. For injection of local irritants a needle (diameter 0.5 mm) is inserted 3 mm into the cervical part of the trapezius muscle 1.2 cm caudal to the stimulation electrode. The needle is connected to a syringe, and used for intramuscular infusion of irritants.

Protocol

Evoked field potentials, electroencephalography (EEG) and/or single unit recordings are recorded during a 10 min control period, with the injection needle positioned in the muscle. The irritant combination (comprising the combination of bradykinin, serotonin, histamine and prostaglandin E₂; saline (0.9%) in control rats) is infused 0.5-3.0 μl/min for periods of 10 min separated by 20 min. After the last irritant injection the test substance is injected intraperitonally. Control animals are injected with saline. The amplitude of the field is calculated as an average of 100 stimulations.

Claims

CLAIMS:

1. A method for inducing a muscle pain and/or tenderness in a test animal, which method comprises: • intramuscular infusion of one or more endogenous substances selected from bradykinin, serotonin, histamine and prostaglandin E in a muscle of the test animal.

2. A method for inducing a myofascial pain in a test animal, which method comprises:

3. A method for inducing a central sensitization in a test animal, which method comprises:

4. The method according to any one of claims 1 -3, wherein the endogenous substances are prostaglandin E₂and one or more of the substances bradykinin, serotonin, and histamine.

5. The method according to any one of claim 1 -4, wherein the test animal is a rodent or a human being.

6. A method for measuring the effect of a test substance in the treatment, prevention or alleviation of myofascial pain in a subject, which method comprises the following steps:

• inducing a pain and/or tenderness in a facial or neck muscle of a test animal;

• administering to the test animal an amount of the test substance;

7. A method for measuring the effect of a test substance in the treatment, prevention or alleviation of a disease associated with central sensitization in a subject, which method comprises the following steps: • inducing a central sensitization by inducing a pain and/or tenderness in a facial or neck muscle of a test animal;

• administering to the test animal an amount of the test substance;

• measuring the effect of the test substance on the central sensitization induced.

8. The method according to claims 6 or 7, wherein the pain and/or tenderness is induced by intramuscular infusion of one or more endogenous substances selected from bradykinin, serotonin, histamine and prostaglandin E₂.

9. The method according to claim any one of claims 6-8, wherein the endogenous substances are prostaglandin E₂and one or more of the substances bradykinin, serotonin, and histamine.

10. The method according to any one of claims 6-9, wherein the test animal is a human being and the measuring of the effect of the test substance on the pain and/or tenderness is performed by assessment of pain intensity, pain quality, local muscle tenderness and/or total tenderness.

11. The method according to any one of claims 6-9, wherein the test animal is a rodent and the measuring of the effect of the test substance on the central sensitization is performed by electrophysiological measures and/or behavioural testing.

12. A drug development method, which comprises the identification of a compound by screening test substances according to any one of the claims 6-11.

13. The use of a compound identified by the method according to any one of the claims 6-11 as being able to reduce the pain and/or tenderness, or a pharmaceutically acceptable salt or a prodrug thereof for the manufacture of a medicament for the treatment, prevention or alleviation of myofascial pain.

14. A method for the treatment, prevention, or alleviation of myofascial pain in a subject comprising administering to said subject a therapeutically effective amount of a compound identified by the method according to any one of the claims 6-11 as being able to reduce the pain and/or tenderness, or a pharmaceutically acceptable salt or a prodrug of said compound.