A simple cold pressure technique for the evaluation of analgesic drugs in healthy subjects.

AIM: An experimental pain model which is sensitive and reproducible would be a useful pharmacological tool both for existing and new drugs. The aim of the present study was to establish a simple and reliable method of producing experimental pain which can be used for screening of analgesic agents.
MATERIALS AND METHODS: The method was standardized by recording pain threshold and pain tolerance values in 24 healthy volunteers. Reproducibility of the test procedure was evaluated by recording the pain threshold and pain tolerance values by a single observer on two sessions (inter-day reproducibility), and second observer in one session (inter-observer reproducibility), separately. Validity of the model was further tested by evaluating the analgesic effect of tramadol in 12 healthy volunteers.
RESULTS: Cold pain model was found to produce low variability with coefficient of variation less than 15%. Inter-observer and inter-day reproducibility was very good as shown by Bland - Altman plot with most of the values within ± 2SD. Analgesic activity by Tramadol was statistically different from placebo (P < 0.05).
CONCLUSION: The newly developed pain model offers a stable and sensitive method for the early assessment of analgesic activity.

RCT Entities:   Population Interventions Outcomes

Introduction

The validity of experimental pain, particularly in assessing analgesic agents, has been a challenging and contentious issue among clinicians and researchers. Historically, it has been argued that experimental pain is qualitatively different from pain associated with injury or disease.[1] While it is recognized that the effectiveness of an analgesic intervention must be assessed in the circumstances for which it is intended, there is a strong argument that the assessment of dose–response, evaluation of optimal dosage, and comparison of relative efficacy with known substances are best conducted with a sample of healthy volunteers who are as homogeneous as possible.[2] Assessment of the analgesic effect of an agent in an experimental pain model permits a level of control not possible in a clinical pain setting and is an ideal approach for the systematic comparison of a range of dose ratios.

The cold pressure technique, first used by Hines[3] and studied by Wolf and Hardy[4] was finally established for analgesic evaluation by Wolff et al.[5] The cold pressor test, a provocative test developed by Hines, is considered to be a good indicator of future hypertension.[6] However, with the cold pressure technique, a lack of standardization is revealed with substantial variation in both equipment and methodology used.[7] The present study aimed to design the cold pressor study with the use of cold pressor equipment that ensures a precise and constant temperature to ensure replicable and reliable results. Further validation of this experimental model was carried-out by demonstrating the analgesic effect of tramadol.

Materials and Methods

The cold pressor equipment was designed and developed by the Department of Clinical Pharmacology and Therapeutics, Nizam's Institute of Medical Sciences, Hyderabad.

Study Participants

Twenty four healthy participants (8 females) aged 23-33 years, with a Body Mass Index (BMI) 20.1-25.9 kg/m2 were studied. The volunteers were given a short explanation of the purpose of the research and a description of the procedure to be followed. They were further given a description of any reasonably foreseeable risks and discomforts. Written consent to participate was obtained from each volunteer. The study was approved by the Institutional Ethics Committee on Research Involving Human Beings.

Cold Stimulation Technique

A deflated blood pressure cuff was placed on the non-dominant arm of the subject. After 30 seconds rest, subject placed his hand upto the wrist into the warm-water bath (at 35 ± 0.5 °C) for exactly 2 minutes. Fifteen seconds before transferring the forearm into the cold water bath (at 1 ± 0.5 °C with constant water circulation), a blood pressure cuff was inflated to 20 mm Hg below the diastolic blood pressure. Subject placed non-dominant forearm upto the wrist in a fixed position with the fingers wide apart into the cold water bath and were instructed to indicate when the subject started to feel sensation of pain (Pain threshold), and when the subject felt an intolerable sensation of pain (Pain tolerance). The subject was then asked to put his hand immediately into the warm water bath, at this time the cuff pressure was released and constant flexion and extension movements were made for 20 seconds. An electronic digital clock is used to record the reaction time in seconds. This entire cold stimulation procedure was repeated three times with 5 minutes interval between the tests. The mean of the three readings was noted and considered for analysis.

Measurements

All experiments were performed with the subjects in sitting position, after good overnight sleep and at least 8 hours of fasting.

Reproducibility

Each subject participated in three experimental sessions separated by intervals of four to five days. The method reproducibility across sessions and subjects was evaluated by recording the pain threshold and pain tolerance values by a single observer on two sessions (inter-day reproducibility) and second observer on one session (inter-observer reproducibility) separately.

Effect of Tramadol

Twelve healthy male participants aged 23-37 years, with a BMI 21.6-26.0 kg/m2 were studied. Participants were randomly assigned to receive either placebo or tramadol 50 mg at morning session after light breakfast, according to the crossover design. There was a 1-week washout period between the drug and placebo phases. Randomization of the sequence of placebo and active treatment periods was performed by a pharmacist who had no contact with the volunteers or the experimenter. On the day of experiment, procedure as described earlier to elicit pain was carried out. Pain threshold and pain tolerance values in seconds were recorded at baseline (0 minutes) and then at 30, 60, 120 and 180 minutes after drug administration.

Sedation Scoring

During the application of the study drugs, a sedation score (0 = awake, 1 = tired, 2 = asleep but arousable, 3 = non-arousable) was obtained at every 10 minutes. All side effects were noted.

Statistical Analysis

The data on pain threshold and pain tolerance values was recorded in seconds and presented as mean (±S Standard Deviation, D) and coefficient of variation (CV). Bland – Altman Plotting was performed for the assessment of method reproducibility.[8] The relative (positive or negative) difference between each pair of measurement was plotted against the mean of the pair to make sure that no obvious relation appeared between the estimated values of mean and difference. The Bland – Altman analysis was done to compare the values of pain threshold time obtained by two observers separately. Similarly, the comparisons were also made to confirm the reproducibility by analyzing the pain threshold time values obtained on two sessions. The paired student t-test was used to compare the difference within the group and between the two groups, value of P < 0.05 was considered to indicate statistical significance. Statistical analyses were performed using the Graphpad PRISM software 4 (Graphpad software Inc. San Diego, California, United States of America).

Results

Results of the method standardized refer to a group of 24 healthy volunteers (mean age, 28 ± 4.8 years; height, 169.2 ± 7 cm; weight, 66.1 ± 11.4 kg and BMI, 23 ± 2.9 kg/ m2). Subjects included in the study were free from the interfering factors for a cold stimulation test such as Raynauds syndrome, rheumatoid arthritis, scleroderma, systemic lupus erythromatosus, gangrenous fingers and use of nicotine and caffeine. Subjects with a pain tolerance of <15 sec or >120 sec were excluded from the study. The results of cold stimulation test are shown in Table 1. The relationship and Bland-Altman Plot comparing inter-day and inter-observer measurements are shown in Figure 1a–d. In the Bland – Altman Plot of inter-day measurement of pain threshold and pain tolerance [Figure 1a and b], there was no significant difference in the values for reproducibility reported between the sessions and range of most of the values was within mean ± 2SD. Similarly, there was good reproducibility between the differences among two observers shown as Bland – Altman Plot [Figure 1c and d] for pain threshold, and same was observed with pain tolerance, showing less variation in reproducibility with most of the points lying within mean ± 2SD.

Table 1

Pain threshold and pain tolerance values for the experimental pain stimuli

Figure 1

(a and b) Bland – Altman plot showing session 1 and session 2 differences in easurements of pain threshold and pain tolerance values. (c and d) Bland – Altman plot showing observer 1 and observer 2 differences in measurements of pain threshold and pain tolerance values

To confirm the validity of the method, tramadol was used as a reference analgesic. Twelve healthy male volunteers (mean age: 30.1 ± 6.3 yr; height: 168.5 ± 4.2 cm; weight: 67.9 ± 7.2 kg; and BMI: 23.8 ± 2.2 kg/m2) completed the study. No significant difference in pain threshold and pain tolerance was noted at baseline between placebo and tramadol (P > 0.05). Mean values are shown in Table 2a and b. Figure 2a and b display mean change from baseline pain threshold and tolerance. We noted a significant increase in pain threshold and pain tolerance time at 120 and 180 minutes. 3a and b displays the difference in area under the curve (AUC) for pain threshold and pain tolerance between placebo and tramadol. Mild sedation was seen in some volunteers, but it never exceeded sedation score of 1.

Table 2a

Pain threshold values (seconds) in placebo and tramadol treated subjects across all time points (mean ± Standard Error of Mean)

Table 2b

Pain tolerance values (seconds) in placebo and tramadol treated subjects across all time points (mean ± Standard Error of Mean)

Figure 2

(a and b) Showing mean (± Standard Error of Mean) % change from baseline pain threshold and pain tolerance values for placebo and tramadol

Figure 3

Displays the mean (± Standard Error of Mean) of the difference in area under curve (AUC) between % change from baseline pain threshold and pain tolerance values for placebo and tramadol

Discussion

The present study describes an experimental pain technique in humans that is sensitive to tramadol at doses known to be effective in acute pain. It is simple to perform, cost effective and requires few personnel. We have maintained a constant temperature of 1 ± 0.5 °C for cold water bath, and 35 ± 0.5 °C for warm water bath. An important methodologic feature in achieving consistency throughout each cold immersion is circulation of water. Without this, heat is able to build up around the hand, making longer tolerance possible. Studies using water circulation have a lower average tolerance range (28.7-119.8 sec) than those using uncirculated water (37.1-190.3 sec).[7] In our study, a constant water circulation was maintained during immersion. The pressure used in the sphygmomanometer was 20 mm Hg below the diastolic pressure, sufficient to produce a temporary occlusion of the venous return during stimulation, avoiding normal loss of heat and adaptive reactions.

One well known index of accuracy of a method is the Coefficient of Variance (CV) (CV = ± SD/mean) and a CV < 10% is considered to be the hallmark of a good assay for a subjective phenomenon.[9] In the present study, we observed a satisfactory CV of 13.4 and 14.5% for pain threshold and pain tolerance values. Also, there was no significant difference in the reproducibility between observers and the sessions, and most of the values ranged within the mean (± 2SD) of the Bland – Altman Plot. Reproducibility is an important factor in the testing of analgesics, where it is necessary to repeat the pain stimulation several times during active and placebo treatments. Earlier studies aimed at presenting data of thermal pain, assessed and reported inter-session repeatability employing methods based on standard statistical techniques.[8]

Response to pain stimulus is highly subjective and varies from subject to subject necessitating proper sample size estimation for evaluation of an analgesic drug on human participants. For method optimization and standardization, we included 24 healthy human volunteers in our experiment. Trials involving experimental pain often use small sample sizes as the variation of the outcome measures is less than the traditional clinical trials. Trials with fewer than 10-12 subjects are hard to test statistically and findings become questionable. However, it has been shown that in experimental models with a high reproducibility, a sample size <10 is powered to show the effect of analgesics.[10]

The major criticism of experimental pain techniques is short duration of exposure to the stimuli, which differs from clinical pain. The ability of this method to discriminate tramadol from placebo was attributed to the ‘tonic’ nature of the stimulus. However, it is probably irrelevant whether the experimental pain stimulus is delivered as ‘phasic’ or ‘tonic’. It is more important that the stimulus reaches sufficient intensity to produce pain sensation (due to activity in the unmyelinated C nociceptors), because it is the latter sensation that is reliably attenuated by both non-opioid and opioid analgesics.[11] However, the high cold pain intensity as used in the present study could be difficult to reduce by drugs with another pharmacological mechanism than opioids and sodium channel blockers.[12] It is suggested that the cold pressor test is sensitive for detecting statistically significant opioid-mediated analgesic effects.[13]

In the present study, we have utilized the crossover design to compare the analgesic effect of tramadol and placebo in healthy subjects and single investigator performed all the pain assessments. In general parallel studies give a weaker statistical power than a cross-over design, demanding larger sample size.[14] In case of cross-over studies it is important to maintain the same investigator in all the pain assessments, as the gender and appearance of the investigator can influence the pain rating of the volunteers.[15] The sensitivity of a given experimental model for detecting analgesia is affected by the method used to measure this pain. Hence, good sensitivity of a model is obtained by using a pain assessment method that is reliable in producing data with modest variance (noise). In an earlier study, the value of objective pain assessment was shown since the effect of tramadol was found only on evoked brain potentials and not on pain ratings.[16] It is however important to note, that although evoked potentials can be a sensitive measure of nociceptive process, they only measure a single dimension of pain. Pain is a multidimensional sensation and this is reflected better in the subjective pain measure. This limits the translation of analgesic effect on evoked brain potentials into effect on clinical pain measures. In the present study, we have used quantitative sensory testing as a functional test which provides a reliable assessment of changes in the pain threshold and pain tolerance values. Also, thermal Quantitative Sensory Testing (QST) (heat and cold) allows a distinction between predominantly C-fibre activity and A-delta fiber activity. Sensitivity of the experimental pain model will also depend on the number of measurements used for a single assessment. In our study, three measurements were used and an average of three was considered for a single assessment.

The study duration of the drug was based on a consideration of t-max. Thus, tramadol which has a t max of ~3 hours was tested for 3 hours. There is strong evidence that for most analgesics, clinical analgesia is not a direct function of drug concentration but that of pharmacologic effect (analgesia), consistent with a role of an endogenous substance. The kinetic profile is necessary to determine when it is optimal to perform the pain tests, bearing in mind that bad timing of the pain testing can jeopardize an otherwise well-designed trial. For opioids it is particularly important to remember that they often need to cross the blood-brain barrier and enter the central nervous system to have an analgesic effect. This causes a lag-time to the onset of analgesia. The study design should consider these different lag-times for different opioids. In the present study, considering the t-max of the tramadol, we have performed all pain assessments at 30, 60, 120 and 180 minutes post-drug.

In the present study, the subjects were given light breakfast, as oral administration of tramadol with food does not significantly affect its rate or extent of absorption. Also, fasting may introduce additional stress for the subjects. When opioids are applied in experimental pain sedation, it is particularly troublesome as it can affect the pain scoring. In our study, mild sedation was seen in some volunteers, but it never exceeded the sedation score of 1. There are few limitations in the present study. This is an acute, controlled painful experience. Given the artificial nature of the experimental procedures, the outcomes may have limited practical utility. However, several studies have shown the relevance of using experimental pain induction procedures in order to predict clinical pain.[17-20] It is also not known whether non-opiate analgesics will show analgesia in cold pressure technique.