Diphenhydramine produces local cutaneous analgesia in
response to dorsal skin noxious stimuli in the rat
Yu-Wen Chen1,2, Ph.D., Jann-Inn Tzeng3,4, M.D., M.S., Ting-Yun Chen1, M.S., Jhi-Joung Wang2, M.D., Ph.D., Yu-Chung Chen5, M.S., Ching-Hsia Hung6,*, Ph.D.
1 Department of Physical Therapy, Graduate Institute of Rehabilitation Science, China Medical University, Taichung, Taiwan
2 Department of Medical Research, Chi-Mei Medical Center, Yong Kang, Tainan City, Taiwan
3 Department of Anesthesiology, Chi-Mei Medical Center, Yong Kang, Tainan City, Taiwan
4 Department of Food Sciences and Technology, Chia Nan University of Pharmacy and Sciences, Jen-Te, Tainan City, Taiwan
5 Division of Physical Therapy, Department of Physical Medicine and Rehabilitation, Cheng Hsin General Hospital, Taipei, Taiwan
6 Institute & Department of Physical Therapy, National Cheng Kung University, Tainan, Taiwan
Conflicts of interest: There is no conflict of interests for all authors.
Running Head (Limit 6 words): local cutaneous analgesia of diphenhydramine
Yu-Wen Chen and Yu-Chung Chen contributed equally to this work.
*Corresponding author:
Ching-Hsia Hung, Ph.D.
National Cheng Kung University, Department of Physical Therapy, No.1 Ta-Hsueh Road, Tainan, Taiwan Phone: 886-6-2353535 ext 5939 FAX: 886-6-2370411
Email: [email protected]
Although diphenhydramine has been shown to produce longer duration of spinal block than lidocaine, few studies disclose its skin infiltrative anesthesia when
compared with a long-lasting local anesthetic, bupivacaine. The purpose of this study was to investigate whether diphenhydramine elicited cutaneous analgesia in
comparison with bupivacaine. After inhibition of cutaneous trunci muscle reflex via subcutaneous injection of drugs in rats, we examined the local anesthetic effect of diphenhydramine and bupivacaine as infiltrative cutaneous analgesia in a dose-dependent fashion. We showed that diphenhydramine, as well as bupivacaine displayed a dose-dependent cutaneous analgesia in response to dorsal cutaneous noxious stimuli. The relative potency (50% effective dose) was bupivacaine (0.023 [0.013–0.035] %) > diphenhydramine (0.078 [0.068–0.091] %) (P < 0.001). On an equipotent basis, diphenhydramine had a similar duration of action to bupivacaine. Neither local injection of saline nor intraperitoneal administration of a large dose of diphenhydramine or bupivacaine produced cutaneous analgesia (data not shown). We conclude that diphenhydramine is less potent than bupivacaine at producing cutaneous analgesia. At equipotent doses for infiltrative cutaneous analgesia, the duration of action of diphenhydramine is equal to that of bupivacaine.
cutaneous analgesia; dose-response curve
INTRODUCTION
Diphenhydramine is a first generation antihistamine possessing sedative,
(Editor change - Diphenhydramine is a first generation antihistamine possessing sedative, antiemetic and hypnotic properties, and is mainly used to treat
allergies) [1, 2]. Furthermore, diphenhydramine has been used as a local anesthetic successfully when allergies to other local anesthetic drugs exist [3]. Recently, there
has been a growing body of evidence that supports diphenhydramine’s significant
local anesthetic effects [2, 4-6]. For instance, it has been shown that diphenhydramine produces spinal anesthesia and is even more potent than lidocaine [4]. Therefore, the clinical relevance of diphenhydramine effect warrants further investigation.
Blockade of voltage-gated Na+ channels is a key feature of local anesthetics that accounts for production of infiltrative cutaneous analgesia, peripheral neural
blockade, and spinal/epidural anesthesia [7, 8]. Because diphenhydramine has been shown to block Na+ channels [9, 10], it may have a local anesthetic effect, for instance, in the production of infiltrative cutaneous anesthesia. Skin infiltrative anesthesia is an acceptable choice for the management of surgical anesthesia and postoperative pain, because it is relatively free of side effects [11]. However, to the best of our knowledge, the duration of local anesthesia with diphenhydramine following subcutaneous injection in comparison with bupivacaine has not been reported. The aim of this study was to evaluate the local/cutaneous anesthetic effect of diphenhydramine and compare it with the long-acting local anesthetic (bupivacaine)
using a rat model of subcutaneous injection.
MATERIALS AND METHODS
Animals
Male Sprague-Dawley rats (205-255 g) were purchased from the National Laboratory Animal Center (Taipei, Taiwan) and kept in the animal housing facilities
at China Medical University, with controlled humidity (50% relative humidity), room temperature (21 degrees centigrade), a 12-hour (6:00 am to 6:00 pm) light-dark cycle, and unlimited access to food and water.The experimental protocol was approved via the Institutional Animal Care and Use Committee of China Medical University (Taiwan), and conformed to the recommendations and policies of the International Association for the Study of Pain (IASP).
Drugs
Diphenhydramine HCl and bupivacaine HCl were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All drugs were freshly prepared (weight/volume percent) and dissolved in 0.9% NaCl (saline) before studies. Experimental protocols
Three studies were designed and performed. In study 1, the potencies of
diphenhydramine (0.175, 0.088, 0.073, 0.058, 0.029%) and bupivacaine (0.091, 0.076, 0.037, 0.021, 0.014%) as infiltrative cutaneous analgesia were evaluated (n=8 rats for each drug). The cutaneous analgesic effect of 0.175% diphenhydramine was
compared with that of 0.091% bupivacaine. Subcutaneous injection of saline (vehicle) produced no cutaneous analgesia. In study 2, on an equipotent basis (25% effective dose [ED25], ED50, and ED75), the sensory block duration of diphenhydramine was compared with that of bupivacaine. In study 3, to rule out the possibility of systemic effect of drugs, one group (n=8 rats for each drug) received subcutaneous injection of
saline combined with intraperitoneal injection of each drug (diphenhydramine or bupivacaine) at a dose of 2xED75.
Infiltrative cutaneous analgesia
The hair on the dorsal surface of the rats’ thoracolumbar region (10×6 cm2) was mechanically removed on the day before subcutaneous injection. Subcutaneous injection of drug was performed as reported previously [12, 13]. In brief, the drug in 0.6 mL saline was injected subcutaneously using a 30-gauge needle in unanesthetized rats at the dorsal surface of the thoracolumbar region. Subcutaneous injection
exhibited a circular skin wheal, approximately 2 cm in diameter. The wheal was marked with ink within one minute after subcutaneous injection. In order to decrease the numbers of experimental animals used, the back of each rat was further divided into left and right parts, either of which, after a washout period of 1 week, received one drug injection.
For consistency, an experienced investigator, who was blinded to the identity of the injected drugs, was responsible for assessing cutaneous analgesia. Infiltrative skin analgesia produced by various agents was evaluated by the cutaneous trunci muscle reflex (CTMR), characterized by the reflex movement of the skin over the back elicited by twitches of the lateral thoracispinal muscle in response to local dorsal cutaneous stimulation [14, 15]. The cut end of an 18-gauge needle (a fresh regular
bevel needle) was affixed to a Von Frey filament (No.15; Somedic Sales AB,
Stockholm, Sweden) to produce a standardized noxious punctate mechanical stimulus (19±1 g) without producing tissue damage. After observing an animal’s normal reaction to stimuli applied on the contralateral side and outside the wheal, we applied six stimuli at six different points within each wheal, with a frequency of 1-2 Hz, and scored the number to which the rat failed to react [16]. Each drug’s cutaneous
analgesic effect was evaluated quantitatively as the number of times the stimuli failed to elicit a response (CTMR). For example, the absence of any response after 6 stimuli was defined as complete nociceptive block (100% of possible effect; 100% PE), which was calculated as follows:
% PE = ((number of stimuli that provoked no response)/6) 100%
The maximum blockade in a time course of cutaneous analgesia with drugs was described as the percent of maximal possible effect (% MPE). Neurobehavioral testing was applied 5 min before injection of drugs to confirm normal responses, 2 min after injection, then every 5 min after injection for the first 30 min and every 10-15 min thereafter, until the CTMR fully recovered from the block (no more than 3 h). Each drug’s duration of action was defined as the time from drug injection (i.e., time=0) to full recovery of CTMR (no analgesic effect, i.e. 0% PE).
After rats were injected with drugs (n = 8 for each drug) subcutaneously, response curves by the % MPE of each dose of each drug were constructed. The dose-response curves were then fitted by SAS Nonlinear (NLIN) Procedures (version 9.1, SAS Institute, Cary, NC), and the values of ED50, defined as the dose that caused 50% cutaneous analgesia, were obtained [17, 18]. The ED25 and ED75 of drugs were
obtained by using the same curve-fitting (SAS NLIN Procedures) that was used to derive the ED50 [19]. Additionally, the area under the curve (AUC) of
nociceptive/sensory blockades of drugs was estimated by Kinetica version 2.0.1 (InnaPhase Corporation, Philadelphia, PA).
Statistical Analysis
Data are presented as mean ± S.E.M. or ED25, ED50, and ED75 values with 95% confidence interval (95% CI). The differences in potencies (ED50s in Table 1) between medications were evaluated using one-way analysis of variance (ANOVA) and Tukey's honestly significant difference (HSD) test for paired comparisons. In the control groups, a one-way ANOVA followed by the Dunnett test was used to evaluate the effects of medications. The differences in Table 2 between drugs were evaluated by 2-sided Student t test with unequal variances. The differences in durations (Fig. 3) among agents were evaluated by two-way ANOVA followed by pairwise Tukey's HSD test. A statistical software, SPSS for Windows (version 17.0, SPSS, Inc,
Chicago, IL, USA), was used, and a p value less than 0.05 was considered statistically significant.
RESULTS
Diphenhydramine is less potent than bupivacaine as infiltrative cutaneous analgesia
Diphenhydramine, as well as the local anesthetic bupivacaine dose-dependently produced cutaneous analgesia in rats (Fig. 1). The lower doses were tested first, and then larger doses were given until comparable doses of each drug were obtained. The ED25, ED50, and ED75 of drugs (diphenhydramine and bupivacaine) are shown in Table 1. On the ED50 basis, the relative potency of these 2 drugs was found to be
bupivacaine > diphenhydramine (P < 0.001, Fig.1 and Table 1).
Diphenhydramine 0.175 % and bupivacaine 0.091% elicit a similar duration of action as infiltrative cutaneous analgesia
Due to the similarities of those figures, only the figures obtained from 0.175% diphenhydramine, 0.091% bupivacaine, and saline (vehicle) are shown in Fig. 2. Diphenhydramine (0.175%) displayed 100% sensory/nociceptive blockade (100%
MPE) with duration of action of 96 ± 5 min (Fig. 2 and Table 2). Bupivacaine
(0.091%) exhibited 100% sensory/nociceptive blockade with duration of action of 101 ± 10 min (Fig. 2 and Table 2). Subcutaneous injection of saline (vehicle) produced no cutaneous analgesia (Fig. 2). By comparison, there were no significant differences between diphenhydramine and bupivacaine for the % MPE, complete blockade time, time to full recovery, and AUCs (Table 2).
Diphenhydramine and bupivacaine at equianalgesic doses exhibit a similar duration of cutaneous analgesia
On an equipotent basis (ED25, ED50, and ED75), the blockade duration caused by diphenhydramine was similar to that caused by bupivacaine (Fig. 3) as infiltrative cutaneous analgesia. Consistent with our previous studies, intrathecal
diphenhydramine produced more potent and longer spinal block duration than lidocaine in rats [4]. Also, subcutaneous administration of saline combined with intraperitoneal injection of diphenhydramine or bupivacaine (2ED75) elicited no
cutaneous analgesia, sedation or loss of motor activity (data not shown). In this study, all rats recovered completely after subcutaneous drug injection.
DISCUSSION
In this report, we demonstrated that diphenhydramine produced a
dose-dependent local anesthetic effect as infiltrative cutaneous analgesia in rats. Another finding in this work revealed that diphenhydramine was less potent (50% effective dose) than bupivacaine at producing cutaneous analgesia, and that diphenhydramine elicited similar analgesic duration to bupivacaine at equipotent doses.
Infiltration anesthesia can be accomplished by administering the local anesthetic solution subcutaneously across the nerve path and is often performed for dental and minor surgical procedures [20, 21]. In this experiment, diphenhydramine displayed
Additionally, it has been known that local anesthetics elicit neural blockade by
inhibiting the sodium currents in the nervous tissues through the voltage-gated sodium channels [8, 22]. Because diphenhydramine blocked sodium channels of sensory neurons [9, 10], it produced dermal local anesthesia [6], topical anesthesia [5], spinal anesthesia [4], and infiltration analgesia of skin.
Our resulting data depicted that diphenhydramine and bupivacaine elicited dose-dependent cutaneous analgesia. (Editor change - Bupivacaine had almost 3 to
4-fold higher potency) Bupivacaine had almost 3.4-4-folds higher potency than
diphenhydramine as skin infiltrative anesthesia. Interestingly, the previous studies reported from this laboratory have indicated that diphenhydramine shows more potent and longer spinal block duration when compared with lidocaine [4]. Furthermore, the injection of lesser diphenhydramine dosage with venous occlusion could be
considered as an alternative to larger lidocaine dosage in suppressing the prevalence of pain induced through injection of propofol into forearm veins [23]. In this present study we found that systemic (intraperitoneal) administration of a large dose of diphenhydramine exhibited no cutaneous analgesia (data not shown). This result supported our finding that diphenhydramine elicited local/cutaneous analgesia.
Infiltrative analgesia of skin is one of the available choices for management of surgical analgesia and postoperative pain, because it is relatively free of side effects
[11, 24]. There are few cases where ultrashort local anesthesia is needed, and for these they use 2-chloroprocaine clinically. For this reason, we examined diphenhydramine and bupivacaine at equianalgesic doses. Our results demonstrated that the duration caused by diphenhydramine was similar to that caused by bupivacaine on an equipotent basis (ED25, ED50 and ED75) (Fig. 3). The histological study for nerve toxicity and systemic toxicity should be tested in the future before the possible use of diphenhydramine as a local anesthetic in humans.
In summary, we showed that diphenhydramine elicits local/skin analgesia following cutaneous nociceptive stimuli, is less potent than bupivacaine, and has a similar duration of action. Diphenhydramine has been in use for local anesthesia for some 40 years [25], and, indeed, H1 antagonists were first suggested for this use (topically) in dental anesthesia in the 1940's. Although it is not a replacement for local anesthetics, diphenhydramine may have a value in producing adequate skin analgesic effect after subcutaneous injection.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the financial support provided for this study by the National Science Council (NSC 101-2918-I-039-001; NSC 101-2815-C-039-019-B) of Taiwan.
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Table 1. The 50% effective dose (ED50), ED25, and ED75 of diphenhydramine and bupivacaine for skin infiltrative analgesia.
Drug ED25 ( 95% CI ) ED50 ( 95% CI ) ED75 ( 95% CI )
Diphenhydramine 0.052 (0.044 – 0.062) 0.078 (0.068 – 0.091) 0.101 (0.093 – 0.112) Bupivacaine 0.015 (0.007 – 0.025) a 0.023 (0.013 – 0.035) a 0.045 (0.036 – 0.056) a
The ED50 of drugs (%) were obtained from Figure 1. CI = confidence interval. The potency of drugs (ED50) was bupivacaine > diphenhydramine. The symbol (a) indicates P < 0.001 when compared with diphenhydramine.
Table 2. The percent of maximal possible effect (%MPE), duration of action, and area under curves (AUCs) of 0.175% diphenhydramine and
0.091% bupivacaine as infiltrative cutaneous analgesia.
%MPE Complete Blockade Time
(min)
Time to Full Recovery
(min) AUCs (%min)
Diphenhydramine 100 ± 0 24 ± 6 96 ± 5 5557 ± 524
Bupivacaine 100 ± 0 29 ± 7 101 ± 10 6644 ± 710
Values are expressed as mean S.E.M. For each group of the time course study, n=8 rats. Of note, all of the rats exhibited complete sensory blockade (100%MPE) after injection. There are no significant differences between these two groups.
Dose ( )
0.01 0.1%
M
PE
(
m
ax
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0 20 40 60 80 100 Bupivacaine DiphenhydramineFig. 1.
Time (min)
0
15
30
45
60
75
90 105 120 135 150
%
PE
(
po
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ib
le
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0
20
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Bupivacaine (0.09%)
Diphenhydramine (0.18%)
Saline
Fig. 2.
ED ( effective dose )
25 50 75F
ul
l R
ec
ov
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y
T
im
e
(
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0 15 30 45 60 Bupivacaine Diphenhydramine Diphenhydramine = BupivacaineFig. 3.
FIGURE LEGENDS
Fig. 1. Dose-response curves of cutaneous analgesia after subcutaneous injection of
diphenhydramine and bupivacaine (5 doses in each drug) in rats. Values are expressed as mean S.E.M. For each testing point of the dose-dependent curves, n=8 rats.
Fig. 2. Time courses of cutaneous analgesia after subcutaneous injection of 0.175%
diphenhydramine and 0.091% bupivacaine in rats. The saline (vehicle) group elicited no cutaneous analgesia. Values are expressed as mean ± S.E.M. Each testing point of the time course study contained eight rats.
Fig. 3. Full recovery time (duration) of drug effect as infiltrative cutaneous analgesia
at equivalent doses of ED25, ED50, and ED75 (n = 8 at each testing point). Values are mean ± S.E.M. The difference in duration was evaluated using 2-way ANOVA followed by pairwise Tukey's HSD test.
