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Pharmacologic modulation of the whealing response to histamine in human skin: Identification of doxepin as a potent in vivo inhibitor
Original articles Pharmacologic modulation of the whealing response to histamine in human skin: identification of doxepin as a potent in vivo inhibitor T i m o t h y J. Sullivan, M.D. Dallas, Tex.
Tricyclic antidepressants are known to exert potent in vitro Ha antihistaminic effects. Doxepin, a heterocyclic variant of amitriptyline, is a particularly effective Hi-antihistamine in vitro. In this stud), we examined the ability of doxepin to inhibit histamine-induced whealing reactions in human skin. Percutaneous introduction of histamine into skin on the volar surface of the forearm provoked a whealing response that was linearly related to the concentration of histamine from O.O002M to 1.0M histamine. An average increase in wheal diameter of 2.39 mm (95% confidence interval of 2.19 to 2.60) was observed for each lO-foM increase in the histamine concentration above threshold. Day-to-day shifts in slopes of dose-response curves in 41 subjects were minimal. Day-to-day changes in the concentrations of histamine required to provoke whealing responses equivalent to responses on the first day of testing averaged 1.8-fold (+-0.6 SE). Single doses of 5, 10, or 25 mg of doxepin increased the concentrations of histamine required to provoke responses equivalent to control reactions 5.2 (+0.9 SE), 18.4 (+3.3 SE) and 82.2-fold ( +37.7 SE), respectively, when reactions were measured 4 hr after drug administration. A randomized double-blind, placebo-controlled study of the effects of five doses of 5 mg of doxepin administered at 12-hr intervals confirmed the effectiveness of doxepin. The blinded stud>' also demonstrated that the amount of histamine required to p,rovoke a response equivalent to the control response increased from 2.5-fold (+0.8 SE) after one dose to 17.2-foM (+-4.7 SE) after three doses and to 22.2-fold (+-5.9 SE) after five doses. These experiments demonstrate that doxepin is a potent antihistamine in man and suggest that this agent, related antidepressants, or compounds derived from tricyclic antidepressant structures may be useful in the clinical regulation of histamine-mediated reactions. (J ALLERGY CLtN IMMUNOL 69:260, 1982.)
Tricyclic antidepressants were originally developed as H,-antihistamines.' Since the first such compounds offered no clear advantages over existing H1 inhibitors,
From the Allergy and Clinical ImmunologySection, Departments of Internal Medicine and Microbiology, University of Texas Health Science Center at Dallas. Supported by a grant from the Southwest Medical Foundation and U.S.P.H.S. grant K07-AI00414-04. Received for publication Aug. 19, 1981. Accepted for publication Nov. 24, 1981. Reprint requests to: Timothy J. Sullivan, M.D., Allergy and Clinical Immunology Section, Departmentof Internal Medicine, University of Texas Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235. Vol. 69, No. 3, pp. 260-267
the antidepressant effects of this family of molecules were pursued. Examination of the in vitro HIantihistaminic properties of the resulting antidepressant agents has revealed that many of the tricyclic antidepressant agents now in clinical use are extraordinarily potent Hi-antihistamines. z These agents were assessed for their abilities to block histamine-induced H, receptor-mediated accumulation of cyclic 3 ' , 5 ' guanosine monophosphate in cultured murine neuroblastoma cells.2 The in vitro potencies of these agents varied, but the most potent agent approved for human use, doxepin, was approximately 775-fold more potent as an H, inhibitor than diphenhydramine, on a molar basis. 2 If the H~-antihistaminic properties of tricyclic antidepressants approach this degree of po-
0091-6749/82/030260+08500.80/09 1982 The C. V. Mosby Co.
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tency in man, these drugs could be extremely useful antiallergic agents. Tricyclic antidepressants in doses effective against depression achieve blood levels of approximately 1 /xM, 3' 4 some 10-fold higher levels than conventional Hi-antihistamines appear to achieve.:" " In the experiments summarized in this article, the ability of doxepin to modify histamine-induced whealing responses in human skin was examined. A simple method for determining dose-response relationships for histamine-induced whealing was developed and characterized. Doxepin was found to be a potent antihistamine in this in vivo human system.
the long axis of the forearm and 90 degrees to the long axis. Crossed diameters of each wheal (measured to the nearest 0.5 mm) were averaged, and replicate values were averaged to give the mean response. Similar methods are used by many clinicians in the diagnosis of allergic diseases/ The areas of the histamine-induced wheals were calculated from the average diameters. Areas of wheals were also estimated by a tape-transfer method. Wheals were outlined in ink. Clear cellophane adhesive tape was pressed onto the site and then transferred, with adherent ink, to a piece of weighing paper. The region defined by tile ink border was cut out and weighed. The weight of the paper and tape is proportional to the area of the lesion.
METHODS Sources of materials
Doxepin and filler were removed from the manufacturer's capsule. This material was weighed, mixed thoroughly, and placed into opaque red gelatin capsules in quantities sufficient to prepare capsules containing 5, 10, or 25 mg of doxepin. Any empty space in the capsules was filled with lactose. Other capsules were prepared that contained only lactose. The contents of the doxepin and placebo capsules were very similar in appearance and could not be distinguished by visual inspection.
Histamine dihydrochloride and lactose were obtained from Sigma Chemical Co., St. Louis, Mo.; 50% glycerine in saline as a diluent was from Hollister-Steir, Spokane, Wash.; 0.22-/z filters for sterilization of reagents were from Millipore, Bedford, Mass.; doxepin as Sinequan (10-mg or 25-mg capsules), generic chlorpheniramine (4 mg); and empty red opaque gelatin capsules were from the Parkland Memorial Hospital pharmacy; and MULTITEST devices were purchased from Lincoln Laboratories, Decatur, I11.
Patient population Subjects were 21 yr of age or older, with no known systemic diseases, and were recruited from medical center personnel. They were taking no medication (except birthcontrol pills) and had no known illnesses that could have been complicated by any of the test substances. Women who were pregnant or who were lactating were excluded. Informed consent was obtained according to the rules and advice of this University's Human Experimentation Committee.
Skin-test methods Histamine dihydrochloride was dissolved in 50% glycerine in normal saline, the pH was adjusted to 7.4, and solutions containing 1M, 10-'M, 10-"M, and 10-ZM histamine were prepared. Histamine solutions were stored at - 2 0 ~ C for up to 7 days. Prick tests were performed by placing a drop of the test solution on the skin of the forearm and then pricking the underlying skin with a 26-gauge needle without inducing bleeding/ MULTITEST devices are sterile, disposable-plastic, multiple test-head applicators. Test heads are 2 by 2 mm square clusters of 2 mm long tines in three rows of three tines. Eight heads are available on each device in two rows separated by 3 cm. The four heads in each row are separated by 2 cm. MULTITEST procedures involved loading 0.01-ml samples of test solutions on each of the eight positions. The apparatus was pressed firmly against the skin of the arm without inducing bleeding.~ Tests with diluent alone acted as negative controls. Results were determined precisely 15 min after application by measuring the diameters of the reactions in
Preparation of doxepin and placebo
Double-blind testing protocol A table of random numbers was used to develop a group of sequences of placebo and drug treatment trials. From these a group of 10 sequences were selected that tested both drug and placebo at least once in five trials. Five subjects, three men and two women from 27 to 40 yr of age, were assigned to a specific sequence of trials using a table of random numbers. The individual controlling the assignments of trials also distributed the capsules. Unmarked vials containing five capsules were given to the test subjects on a Monday morning. The test subjects and the individual assessing histamine responsiveness had no knowledge of the contents of the capsules until after completion of the study. Histamine-induced whealing responses in test subjects were measured at 8:00 A.M. on a Monday. Doxepin or placebo was administered at 9:00 A.M. of the same day. Four additional doses were administered at 12-hr intervals. Histamine responsiveness was assessed 4, 28, and 52 hr after the first dose. All testing was performed by the same person. The subjects avoided additional drug therapy for the remainder of the week. One course of therapy was tested each week until the subjects had been tested while taking both drug and placebo.
Statistical analysis A group of interactive statistical programs was used in a DEC-10 computer system at the Biometrics Section, Medical Computing Resources Center, at this institution. Mean, standard deviation, standard error, and 95% confidence intervals were determined using the SIMPLE program. The significance of differences between groups of observations was tested by Student's t test and Welch's approximation to the Student's t test for unequal variances with the NGROUP
262 Sullivan
J. ALLERGY CLIN. IMMUNOL. MARCH 1982
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FIG. 1. Reproducibility of histamine-induced whealing responses in an individual subject. Control and histamine tests were performed in duplicate. A, Mean (-+SD) of the diameters of wheals induced by the indicated concentrations of histamine on 14 different days. The correlation coefficient of the regression curve defined by these points was r = 0.999, Sy 9 x = 0.32 (ref. 9a). B, These same whealing responses are expressed as the mean (-+SD) of the area of the wheal. The correlation coefficient for the linear regression curve for this data was r = 0.987, Sy. x = 2.78.
program. Linear regression equations were generated, evaluated, and compared by the REGRES program. Doseresponse curves generated in the presence of a competitive inhibitor are parallel but occur at higher agonist concentrations. A conventional index of the degree of inhibition is the increase in agonist concentration required to produce a response equivalent to a control response. In the present study we compared the X-intercept values--the lowest concentrations of histamine capable of causing a wheal. RESULTS Assessment
of histamine-induced
whealing
The MULTITEST method of introducing test substances into the skin was selected because control solutions usually produce no wheal, histamine induces virtually circular reactions, tests are applied simultaneously, the procedure requires little time, and patients experience little discomfort. 8 Whealing responses to histamine provoked by the needle-prick method were similar in magnitude and were not significantly more reproducible than those obtained with the MULTITEST method (not shown). Preliminary experiments indicated that histamine in concentrations ranging from 10-3M to 1M provoked progressively larger wheals. Concentrations above 1M frequently induced irregular wheals with pseudopods or satellite wheals. Since erythematous reactions were irregular and inconsistent and are indirect manifestations of the action of histamine, 9 only whealing reactions are presented in this article. As illustrated in Fig. 1, A, the whealing responses t o various concentrations of histamine were linearly
related to the average diameter of the wheals. When the areas of the wheals, calculated from these diameters, were plotted against histamine concentration, the slope of the curve was not constant (Fig. 1, B). When the areas of wheals were estimated by the tapetransfer method rather than by calculation, a bowed curve similar to that in Fig. 1, B, was obtained (data not shown). As summarized in the legend to Fig. 1, the diameters of wheals described a more nearly linear relationship to histamine concentration. Because we wished to estimate the degree of drug-induced parallel shift in dose curves, the histamine-induced whealing responses were expressed as the diameter of the wheals. Variations among replicates and among measurements by different observers were detected, but the magnitude of this variation was acceptably low. The coefficients of variation averaged 6.0% for 1M histamine, 10.1% for 10-1M, 32.3% for 10-2M and 45.5% for 10-3M histamine when seven replicate tests at each concentration were performed on one subject on three occasions. Variation among measurements taken by two different observers averaged 5.1% of the mean of both sets of observations at 1M histamine, 4.4% at 10-~M, 11.2% at 10-ZM, and 10.5% at 10-ZM in three sets of paired readings, using one constant observer, three different comparative observers, and eight replicates at each concentration. No significant differences in whealing were observed when tests were performed on the volar surfaces of the right or left forearms or on the lateral aspects of the right or left arms (p > 0.30; data from seven ob-
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Doxepin inhibits histamine wheals
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FIG. 2. Histamine-induced whealing responses in normal individuals. Means (-+SEM) of wheal diameters observed in 25 normal subjects after stimulation by the indicated concentrations of histamine are presented. Whealing responses were determined in duplicate as described in Methods. Spearman's Rho = 1.000; Kendall's Tau = 1.000; Pearson's r = 1.000. The probability that the slope is greater than 0 is p < 0.001. These data were described by equation Y = 9.165 + 2.485 Log10 X. The X intercept was 2.05 x 10-4M histamine.
servations at each site in two patients). The histaminecontaining solutions appeared to be stable for at least 1 wk, as assessed by the diameters of wheals induced (data not shown). New solutions were prepared every 7 days. Dose-response curves measured on consecutive days were consistent when assessed with duplicate tests for each concentration of histamine. As illustrated in Fig. l, the histamine responses measured in a single individual in the morning on 14 different days varied over a relatively narrow range. The slopes of these curves averaged a 2.28-mm increase in diameter per 10-fold increase in histamine concentration with a 95% confidence interval of 2.10 to 2.48 mm per 10fold increase. The threshold concentrations (the lowest concentration of histamine causing a wheal) were calculated to average 4.2 x 10-4M, with a 95% confidence interval of 2.8 to 7.5 x 10-4M. Shifts in threshold concentrations significantly different from the threshold observed on the initial day were observed on four of these 14 days (p < 0.05). No significant change in slope was detected. When five dose-response curves obtained from testing between
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FIG. 3. Effect of doxepin on histamine-induced whealing in an individual subject. Responses to histamine were measured 4 hr after no treatment (o), oral administration of 5 mg of doxepin (o), oral administration of 10 mg of doxepin (•) at 8:00 A.M. The data are presented a s t h e mean (• of 12 control observations, five observations after 5 mg of doxepin, and three observations after 10 mg of doxepin.
8:00 and 10:00 A.M. on different days were compared with each other and with five curves obtained from responses between l:00 and 3:00 P.M. on the same 5 days all in the same subject, no significant difference was noted (p > 0.30, not shown). Three additional individuals studied on 3 to 5 consecutive days demonstrated a similar degree of reproducibility of the dose-response curves. Dose-response curves obtained between 8:00 and 10:00 A.M. were not significantly different from each other or from curves obtained from responses provoked between 1:00 and 3:00 P.M. in these three participants (not shown). The concentrations of histamine selected for testing clearly defined a dose-response relationship in each of 25 normal subjects (Fig. 2). The slope of this curve was a 2.49-mm increase in wheal diameter per 10fold increase in histamine concentration (___0.03 SE; 95% confidence interval 2.34 to 2.63). Correlation coefficients for the regression curves that described the individual respqnses averaged 0.986 (_+0.006 SD). The threshold for whealing calculated from these curves averaged 2.0 x 10-~4M, with a 95% confidence interval of 9.7 x 10-ZMto 4.3 x 10-4M. These results indicated that human skin 'whealing responses to histamine could be assessed with reason-
264 Sullivan
J. ALLERGY CLIN. IMMUNOL. MARCH 1982
TABLE I. Modulation of the whealing response to histamine by single doses of doxepin or chlorpheniramine Ratio of Threshold concentration after treatment 1" Threshold concentration before treatment Treatment*
Mean • SEM
Range
nt
None Chlorepheniramine (12 mg) Doxepin 5 mg 10 mg 25 mg
1.8 + 0.6 3.0 _+ 0.4
0.4 - 6.7 2.4 - 3.7
41 3
5.2 --+ 0.9 18.4 - 3.3 82.2 _+ 37.7
0.7 - 12.5 5.0 - 44.6 11.2 - 316
14 16 8
*The test agent was administered orally at 9:00 A.M. after control skin testing at 8:00 A.M. Skin whealing responses were retested at 1:00 P.M. *Linear regression lines describing skin-test responses were calculated as described in Methods. No significant differences in slopes were detected. The lowest concentration of histamine capable of provoking a wheal was termed the threshold concentration. Alterations in histamine responsiveness were calculated by determining the ratio of threshold concentrations after treatment to threshold concentrations before treatment. *Number of subjects examined. In subjects receiving no treatment, observations on two or more days were compared.
able precision by means of a MULTITEST device to apply duplicate tests with histamine in concentrations ranging from 10-aM to 1M. The degree of reproducibility of measurements of responses and the reproducibility of the responses themselves indicated thatthis approach could be used to assess pharmacologic modulation of histamine-induced wheaiing in human skin. Since wheals induced by 10-3M histamine (1.6- to 4.2-mm wheals) were difficult to measure accurately and since regression curves defined by IM, 10-1M, and 10-2M, histamine were not significantly different from those defined by inclusion of the 10-3M histamine point, the 10-3M histamine concentration was dropped from the testing procedure. This permitted duplicate tests with 1M, 10-'M, and 10-2M histamine and diluent control with a single MULTITEST device.
Effects of pharmacologic agents on histamine-induced whealing Doxepin caused a significant dose-related inhibition of histamine-induced whealing (Table I, Fig. 3). A single dose of 25 mg of doxepin increased the concentration of histamine required to produce responses equivalent to control responses an average of 82-fold (Table I) when responses were measured 4 hr after ingestion of the drug. The magnitude of the shift in the histamine dose-response curve decreased with decreasing doxepin dose, but significant effects (5.2fold shift) were detected after a single 5-mg dose (Table I). The magnitude of doxepin effects on histamine-induced whealing were variable from one patient to the next (Table I) but were relatively consis-
tent when individual patients were tested on more than one occasion (Fig. 3). Chlorpheniramine was examined for comparison to doxepin. Measurements taken 2 hr after a single 12-mg dose of chlorpheniramine indicated a threefold shift in the dose-response curve (Table I). As illustrated in Fig. 3, doxepin caused a doserelated parallel displacement of the histamine doseresponse curve to the right, as assessed by an analysis of variance of the slopes (F = 87.3, p < 0.001). Newman-Keuls multiple comparisons of the slopes indicated no differences among the control, 5-mg doxepin, and 10-mg doxepin regression lines. 10 Comparison of the intercepts of these curves by NewmanKeuls multiple comparisons indicated that each of the regression lines was significantly different from the others. Similar results were obtained in series of determinations on two other subjects. Each of the eight subjects taking 25 mg of doxepin reported significant sedation. Patients receiving 10 mg reported noticeable but mild sedation. A 5-mg dose of doxepin did not produce noticeable effects.
Double-blind, placebo-controlled evaluation of doxepin effects To be certain that the measurement of histamineinduced wheals was not influenced by the expectations of the observer and that the responses were reproducible when the subjects were not aware of the nature of test substances, a double-blind evaluation was performed. The predictable mild sedative properties of doxepin in doses of 10 mg or higher every 12 hr would have permitted the subjects to distinguish
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Doxepin inhibits histamine wheals 265
T A B L E U. Double-blind evaluation of the effect of doxepin on whealing responses to histamine 9 Threshold concentration after treatment Ratio or . . . . . Threshold concentration before treatment Subject
Treatment*
4 hrt
28 hr
52 hr
T. J.S.
Placebo Doxepin Placebo Doxepin Placebo Doxepin Placebo Doxepin Placebo Doxepin Placebo Doxepin
1.1 3.0 0.5 4.5 1.5 3.5 1.6 1.3 1.2 0.7 1.2 (+_0.2) 2.5 (---0.8)
1.2 23.4 1.1 5.2 6.5 20.9 1,1 29.4 0.7 7.3 2.1 (+_ 1.1) 17.2 (+_4.7)
4.8 36.3 0.7 i2.3 6.7 26.3 1.1 31.6 0.4 4.7 2.7 ( - 1.3) ?.2.2 (-+5.9)
S.R. T. R.S. M.W. R.T. All subjects
*The double-blind protocol used is described in detail in Methods. Doses were administered every 12 hr for a total of five doses. Each subject received one or more courses of treatment with doxepin (5 mg) and with placebo. tWhealing responses w e r e a s s e s s e d at 8:00 A.M. and the first dose was administered at 9:00 A.M. on a Monday. Skin whealing r e s p o n s e s were assessed 4, 28, and 52 hr after the first dose. The results of multiple courses of drug and of placebo were averaged. The responses for all five subjects were expressed as the mean (+_ SEM).
placebo from drug. A 5-mg dose was selected to optimize the blind feature of the study at the price of using a less effective dose. A preliminary study indicated that when a 5-mg dose of doxepin was administered every 12 hr, the drug effect increased over the first 24 hr and then remained constant for at least 3 days. Preliminary studies also demonstrated that skin whealing responses returned to control levels within 2 days after five doses of 5 mg of doxepin administered at 12-hr intervals (not shown). A double-blind, placebo-controlled study of doxepin effects was then performed with 5 mg of doxepin or placebo administered every 12 hr for five doses. Doxepin was a highly effective inhibitor of histamine-induced whealing (Table II). Relatively little effect was noted 4 hr after a single 5-mg dose of doxepin, but after three doses an average of 17.2-fold more histamine was required to provoke dose-related whealing equivalent to control responses. The average effect measured after five doses was not significantly higher than that detected after three doses. Each of the five subjects demonstrated significant doxepin inhibition of whealing responses after three and five doses (p < 0.05). The slopes of the curves in the drug and placebo treatment periods were not significantly different. Statistically significant shifts in threshold (p < 0.05) were observed during courses of placebo treatment in two subjects, T. J. S. (52hr) and T. R. S. (28 and 5 2 hr).
DISCUSSION
The results of these experiments lead to two principal new insights. First, a simple method for assessing dose-related human skin whealing responses to histamine was defined. The variability among observations taken on different occasions and variability among subjects were measured and found to be small. Second, the development and characterization of this method permitted us to perform a double-blind study that demonstrated the marked antihistaminic properties of doxepin in man. Studies of wheals elicited by introducing histamine into human skin have been performed for nearly 70 yr. Eppinger 11 described the wheal-and-flare-inducing properties of histamine in 1913. By the time of the publication of Lewis' still remarkable monograph 12 on human skin blood vessels and their physiology in 1927, the whealing response was understood to result at least partially from vasodilation and increased capillary permeability2' t2 The flare response was recognized as a secondary neural reflex response provoked by histamine2 The synthesis of competitive inhibitors of HI receptors by Bovet and Staub ~3 in t937 and the evolution of this class of agents led to renewed interest in human cutaneous whealing, now as an in vivo model for assessing the clinical efficacy of antihistamines. ~4-~ The magnitude of the wheal was found to be linearly related to the log of the histamine dose over a 1000-fold or greater range of doses. Conventional H1 antagonists were reported to cause parallel displacements of these dose-response
266 Sullivan
curves to higher histamine dose ranges. 15, 16 Recent studies indicate that human cutaneous whealing responses are mediated primarily by H I receptors, but a measurable minor contribution by cimetidine-inhibitable H2 receptors is also present. 17, 18 This study demonstrates that percutaneous administration of histamine with a MULTITEST device is a simple, rapid, reproducible method that permits the measurement of dose-related histamine responses in vivo in an acceptably precise manner. This method minimizes the requirement for skill that is known to be essential for reproducible testing by intradermal or needle-prick methods. 18 The simultaneous application of stimuli facilitates measurement of responses at a consistent time after administration. The speed, simplicity, and reproducibility of this method are advantages over prior methods, but careful introduction of histamine by any method is likely to be adequate to define dose-response relationships. 15.16, 18 Most investigators have expressed histamine responses as the area of the wheal o r f l a r e , 14-16 but the results of this inquiry were more accurately described as a function of the diameter of the wheal (Fig. 1). The complexity of the events taking place in the course of whealing 12' 19 precludes a priori predictions of the most precise measurement of dose-related reactions. In radial immunodiffusion reactions, for example, the antigen concentration is usually related to the diameter of the expanding ring. 2~ The antigen concentration is usually related to the square of the diameter of the ring when the ring stops expanding, zl Histamine-induced whealing responses measured 15 min into their evolution appear to be analogous to the expanding ring of radial immunodiffusion reactions. Earlier studies have demonstrated a degree of variability among histamine responses measured on individuals at different times of day, 16" 22 but there has been an implicit assumption that responses at the same hour on different days are directly comparable.14-16, 2.~ The results of this study demonstrate that histamine dose-response curves remain parallel in individual subjects but can vary over as much as a sevenfold range during a 3-day period of observation (Table II). Although the variation from day to day usually fell within much narrower boundaries (Fig. 1), this observation emphasizes the need to consider the variance from day to day, not just the variance in a single control observation, when histamine responses after drug treatment are compared with control responses. The reasons for significant intrinsic variation in histamine responsiveness are currently unknown. This issue deserves careful attention as a possible source of clinical variation during allergic or other diseases. To assess the antihistaminic properties of doxepin,
J. ALLERGY CLIN. IMMUNOL. MARCH 1982
we designed a study that would permit assessment of intrinsic variation in histamine responsiveness as well as doxepin effects in a randomized double-blind protocol. As illustrated in Fig. 3 and Table II, doxepin caused a dose-related inhibition of histamine-induced whealing responses. The effect of doxepin (5 mg) administered at 12-hr intervals increased over 48 hr to an average 22-fold shift in the histamine dose-response curve. These changes were statistically significant when compared with responses during placebo treatment. No double-blind, placebo-controlled studies of tricyclic antidepressant inhibition of human responses to histamine are available for quantitative comparison. However, controlled studies of some conventional Hi-antagonists have been reported. Two studies indicate that multiple doses of 4 mg of chlorpheniramine 23 or 8 mg of chlorpheniramine 17 cause threefold to sixfold shifts to the right of histamine dose-response curves. A recent study by Nathan et al. 24 detected a similar degree of inhibition after single or multiple doses of 8 mg of chlorpheniramine. Inhibition of high-dose histamine effects assessed 2 hr after a single dose of chlorpheniramine was nearly identical to that observed after multiple doses in their study. The threefold shift detected 4 hr after single 12-mg doses of chlorpheniramine in this study (Table I) are in agreement with these previous reports. Multiple doses of 25 mg of hydroxyzine administered orally were reported to inhibit histamine-induced whealing, 25 but the drug apparently caused nonparallel displacement of the dose-response curve, making quantitative comparisons difficult. The available data indicate that doxepin is considerably more potent than chlorpheniramine. Potency relative to other agents remains to be established in controlled trials. Multiple doses of 25 mg of doxepin would be expected to shift histamine dose-response curves considerably more than the 82fold observed after one dose (Table II). The precise means by which doxePin suppresses histamine-induced vascular responses is not certain. Some tricyclic antidepressants exert H1 and H2 antihistaminic actions as well as anticholinergic and other effects.2" 26, 27 A direct competitive H1 blocking effect or Hi plus H2 blocking effect appear to be the most likely explanations, since the drug caused a dose-related parallel displacement of the histamine close-response curve to higher histamine concentrations. Doxepin or related drugs may be found to have a role in the treatment of clinical allergic diseases. Preliminary clinical studies in this laboratory indicate that doxepin may be useful in the management of chronic urticaria and allergic rhinitis. Use of this particular agent is limited to some extent by the observa-
VOLUME 69 NUMBER 3
tion that n o n d e p r e s s e d s u b j e c t s m a y e x p e r i e n c e d r o w s iness at doses o f 10 to 25 m g t w i c e daily. A t d o s e s c o m m o n l y u s e d to treat d e p r e s s i o n (25 m g orally three t i m e s daily or 75 m g at b e d t i m e , or h i g h e r ) , m o s t n o r m a l s u b j e c t s w o u l d b e e x p e c t e d to note significant d r o w s i n e s s . L i q u i d f o r m s o f d o x e p i n are a v a i l a b l e , h o w e v e r , that p e r m i t s i m p l e a d j u s t m e n t o f d o s a g e . I d e n t i f i c a t i o n o f the p o t e n t a n t i h i s t a m i n i c properties o f d o x e p i n indicates that tricyclic a n t i d e p r e s s a n t s s h o u l d be e x a m i n e d c a r e f u l l y for t h e i r r e l a t i v e p o t e n c i e s as a n t i h i s t a m i n e s . T h e use o f d o x e p i n or related agents m a y m a r k e d l y i m p r o v e o u r ability to s u p p r e s s Ha-rec e p t o r - m e d i a t e d effects o f h i s t a m i n e in m a n . 1 gratefully acknowledge the assistance of Dr. Gary N. Gross in recruiting volunteers, and the expert technical assistance of Ms. Susan Robinson and Mr. Edward Lewis. I would like to state my appreciation for the unusual level of cooperation the volunteers contributed to this study. REFERENCES 1. Kuhn R: The imipramine story, in Ayd FJ Jr, Blackwell B, editors: Discoveries in biological psychiatry. Philadelphia, 1970, J.B. Lippincott Co., pp. 205-217. 2. Richelson E: Tricyclic antidepressants and histamine HI receptors. Mayo Clin Proc 54:669, 1979. 3. Friedel RO, Raskind MA: Relationship of blood levels of Sinequan to clinical effects in the treatment of depression in aged patients, in Mendels J, editor: Sinequan (doxepin HC1). A monograph of recent clinical studies. Amsterdam, 1975, Excerpta Medica, pp. 51-53. 4. Ziegler VE, Clayton PJ, Biggs JT: A comparison study of amitriptyline and nortriptyline with plasma levels. Arch Gen Psychiatry 34:607, 1977. 5. Tozzi S, Roth FE, Tabachnick IA: Pharmacology of azatadine, a potential anti-allergy drug. Agents Actions 4:264, 1974. 6. Yacobi A, Stoll RG, Chao GC, Carter JE, Baaske DM, Kamath BL, Amann AH, Lai C-M: Evaluation of sustainedaction chlorpheniramine-pseudoephedrine dosage form in humans. J Pharm Sci 69:1077, 1980. 7. Norman PS: Skin testing, in Rose NR, Friedman H, editors: Manual of clinical immunology. Washington, D. C., 1976, American Society for Micriobiology, pp. 585-589. 8. Murphree JT, Kniker WT: Correlation of immediate skin test responses to antigens introduced by multitest and intracutaneous routes. Ann Allergy 43:279, 1979. 9. Thomas L, Grant RT: Vascular reactions of the skin to injury. Part II. The liberation of a histamine-like substance in injured skin: the underlying cause of factitious urticaria and of wheals produced by burning: and observations upon the nervous control of certain skin reactions. Heart 11:109, 1924.
D o x e p i n inhibits histamine w h e a l s
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9a. O'Brien PC, Shampo MA: Statistics fcr clinicians. 7. Regression. Mayo Clin Proc 56:452, 1981. 10. Zar JH: Biostatistical analysis. Englewood Cliffs, N. J., 1974, Prentice Halt, Inc., pp. 230-233. 11. Eppinger H: Uber eine eigentumliche Hautreaktion, hervorgerufen durch Ergamin. Wien Med Wochenschr 43:1414, 1913. 12. Lewis T: The blood vessels of the human skin and their responses. London, 1927, Shaw and Sons, pp. 1-322. 13. Bovet D, Staub A-M: Action protectrice des ethers phenoliques au cours de l'intoxication histaminique. Compt Rend Soc Biol 125:547, 1937. 14. Bain WA: The quantitative comparison of histamine antagonists in man. Proc R Soc Med 42:615, 1949. 15. Bain WA: The evaluation of drugs in man, with special references to antihistaminics. Analyst 76:573, 195 t. 16. Fowle ASE, Hughes DTD, Knight GJ: The evaluation of histamine antagonists in man. Eur J Clin Pharmacol 3:215, 1971. 17. Marks R, Greaves MW: Vascular reactions to histamine and compound 48/80 in human skin: suppression by histamine Hzreceptor blocking agent. Br J Clin Pharmacol 4:367, 1977. 18. Harvey RP, Schocket AL: The effect of H1 and H2 blockade on cutaneous histamine response in man. J ALLERGY CLIN IMMUNOL65:136, 1980. 19. Beaven MA: Histamine: its role in physiological and pathological processes. Monogr Allergy. 13:1, 1979. 20. Fahey JL, McKelvey EM: Quantitative determination of serum immunoglobulins in antibody-agar plate~;. J lmmunol 94:84, 1965. 21. Mancini G, Carbonara AD, Heremans JF: lmmunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2:235, 1965. 22. Lee RE, Smolensky MH, Leach CS, McGovern JP: Circadian rhythms in the cutaneous reactivity to histamine and selected antigens, including phase relationship to urinary cortisol excretion. Ann Allery 38:231, 1977. 23. Galant SP, Bullock J, Wong D, Maibach HI: The inhibitory effect of antiallergy drugs on allergen and histamine induced wheal and flare response. J ALLERGY CLIN IMMUNOL51:11, 1973. 24. Nathan RA, Segall N, Schocket AL: A comparison of the actions of H1 and H2 antihistamines on histamine-induced bronchoconstriction and cutaneous wheal response in asthmatic patients. J ALLERGYCLIN IMMUNOL67:171, 1981. 25. Harvey RP, Schocket AL: The effect of H1 and H2 blockade on cutaneous histamine response in man. l ALLERGY CLIN IMMUNOL65:136, 1980. 26. U'Prichard DC, Greenberg DA, Sheehan PP, Snyder SH: Tricyclic antidepressants: therapeutic properties and affinity for alpha-noradrenergic receptor binding sites in the brain. Science 199:197, 1978. 27. Green JP, Maayani S: Tricyclic antidepressant drugs block histamine H~ receptor in brain. Nature 269:163, 1977.
Tricyclic antidepressants are known to exert potent in vitro Ha antihistaminic effects. Doxepin, a heterocyclic variant of amitriptyline, is a particularly effective Hi-antihistamine in vitro. In this stud), we examined the ability of doxepin to inhibit histamine-induced whealing reactions in human skin. Percutaneous introduction of histamine into skin on the volar surface of the forearm provoked a whealing response that was linearly related to the concentration of histamine from O.O002M to 1.0M histamine. An average increase in wheal diameter of 2.39 mm (95% confidence interval of 2.19 to 2.60) was observed for each lO-foM increase in the histamine concentration above threshold. Day-to-day shifts in slopes of dose-response curves in 41 subjects were minimal. Day-to-day changes in the concentrations of histamine required to provoke whealing responses equivalent to responses on the first day of testing averaged 1.8-fold (+-0.6 SE). Single doses of 5, 10, or 25 mg of doxepin increased the concentrations of histamine required to provoke responses equivalent to control reactions 5.2 (+0.9 SE), 18.4 (+3.3 SE) and 82.2-fold ( +37.7 SE), respectively, when reactions were measured 4 hr after drug administration. A randomized double-blind, placebo-controlled study of the effects of five doses of 5 mg of doxepin administered at 12-hr intervals confirmed the effectiveness of doxepin. The blinded stud>' also demonstrated that the amount of histamine required to p,rovoke a response equivalent to the control response increased from 2.5-fold (+0.8 SE) after one dose to 17.2-foM (+-4.7 SE) after three doses and to 22.2-fold (+-5.9 SE) after five doses. These experiments demonstrate that doxepin is a potent antihistamine in man and suggest that this agent, related antidepressants, or compounds derived from tricyclic antidepressant structures may be useful in the clinical regulation of histamine-mediated reactions. (J ALLERGY CLtN IMMUNOL 69:260, 1982.)
Tricyclic antidepressants were originally developed as H,-antihistamines.' Since the first such compounds offered no clear advantages over existing H1 inhibitors,
From the Allergy and Clinical ImmunologySection, Departments of Internal Medicine and Microbiology, University of Texas Health Science Center at Dallas. Supported by a grant from the Southwest Medical Foundation and U.S.P.H.S. grant K07-AI00414-04. Received for publication Aug. 19, 1981. Accepted for publication Nov. 24, 1981. Reprint requests to: Timothy J. Sullivan, M.D., Allergy and Clinical Immunology Section, Departmentof Internal Medicine, University of Texas Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75235. Vol. 69, No. 3, pp. 260-267
the antidepressant effects of this family of molecules were pursued. Examination of the in vitro HIantihistaminic properties of the resulting antidepressant agents has revealed that many of the tricyclic antidepressant agents now in clinical use are extraordinarily potent Hi-antihistamines. z These agents were assessed for their abilities to block histamine-induced H, receptor-mediated accumulation of cyclic 3 ' , 5 ' guanosine monophosphate in cultured murine neuroblastoma cells.2 The in vitro potencies of these agents varied, but the most potent agent approved for human use, doxepin, was approximately 775-fold more potent as an H, inhibitor than diphenhydramine, on a molar basis. 2 If the H~-antihistaminic properties of tricyclic antidepressants approach this degree of po-
0091-6749/82/030260+08500.80/09 1982 The C. V. Mosby Co.
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tency in man, these drugs could be extremely useful antiallergic agents. Tricyclic antidepressants in doses effective against depression achieve blood levels of approximately 1 /xM, 3' 4 some 10-fold higher levels than conventional Hi-antihistamines appear to achieve.:" " In the experiments summarized in this article, the ability of doxepin to modify histamine-induced whealing responses in human skin was examined. A simple method for determining dose-response relationships for histamine-induced whealing was developed and characterized. Doxepin was found to be a potent antihistamine in this in vivo human system.
the long axis of the forearm and 90 degrees to the long axis. Crossed diameters of each wheal (measured to the nearest 0.5 mm) were averaged, and replicate values were averaged to give the mean response. Similar methods are used by many clinicians in the diagnosis of allergic diseases/ The areas of the histamine-induced wheals were calculated from the average diameters. Areas of wheals were also estimated by a tape-transfer method. Wheals were outlined in ink. Clear cellophane adhesive tape was pressed onto the site and then transferred, with adherent ink, to a piece of weighing paper. The region defined by tile ink border was cut out and weighed. The weight of the paper and tape is proportional to the area of the lesion.
METHODS Sources of materials
Doxepin and filler were removed from the manufacturer's capsule. This material was weighed, mixed thoroughly, and placed into opaque red gelatin capsules in quantities sufficient to prepare capsules containing 5, 10, or 25 mg of doxepin. Any empty space in the capsules was filled with lactose. Other capsules were prepared that contained only lactose. The contents of the doxepin and placebo capsules were very similar in appearance and could not be distinguished by visual inspection.
Histamine dihydrochloride and lactose were obtained from Sigma Chemical Co., St. Louis, Mo.; 50% glycerine in saline as a diluent was from Hollister-Steir, Spokane, Wash.; 0.22-/z filters for sterilization of reagents were from Millipore, Bedford, Mass.; doxepin as Sinequan (10-mg or 25-mg capsules), generic chlorpheniramine (4 mg); and empty red opaque gelatin capsules were from the Parkland Memorial Hospital pharmacy; and MULTITEST devices were purchased from Lincoln Laboratories, Decatur, I11.
Patient population Subjects were 21 yr of age or older, with no known systemic diseases, and were recruited from medical center personnel. They were taking no medication (except birthcontrol pills) and had no known illnesses that could have been complicated by any of the test substances. Women who were pregnant or who were lactating were excluded. Informed consent was obtained according to the rules and advice of this University's Human Experimentation Committee.
Skin-test methods Histamine dihydrochloride was dissolved in 50% glycerine in normal saline, the pH was adjusted to 7.4, and solutions containing 1M, 10-'M, 10-"M, and 10-ZM histamine were prepared. Histamine solutions were stored at - 2 0 ~ C for up to 7 days. Prick tests were performed by placing a drop of the test solution on the skin of the forearm and then pricking the underlying skin with a 26-gauge needle without inducing bleeding/ MULTITEST devices are sterile, disposable-plastic, multiple test-head applicators. Test heads are 2 by 2 mm square clusters of 2 mm long tines in three rows of three tines. Eight heads are available on each device in two rows separated by 3 cm. The four heads in each row are separated by 2 cm. MULTITEST procedures involved loading 0.01-ml samples of test solutions on each of the eight positions. The apparatus was pressed firmly against the skin of the arm without inducing bleeding.~ Tests with diluent alone acted as negative controls. Results were determined precisely 15 min after application by measuring the diameters of the reactions in
Preparation of doxepin and placebo
Double-blind testing protocol A table of random numbers was used to develop a group of sequences of placebo and drug treatment trials. From these a group of 10 sequences were selected that tested both drug and placebo at least once in five trials. Five subjects, three men and two women from 27 to 40 yr of age, were assigned to a specific sequence of trials using a table of random numbers. The individual controlling the assignments of trials also distributed the capsules. Unmarked vials containing five capsules were given to the test subjects on a Monday morning. The test subjects and the individual assessing histamine responsiveness had no knowledge of the contents of the capsules until after completion of the study. Histamine-induced whealing responses in test subjects were measured at 8:00 A.M. on a Monday. Doxepin or placebo was administered at 9:00 A.M. of the same day. Four additional doses were administered at 12-hr intervals. Histamine responsiveness was assessed 4, 28, and 52 hr after the first dose. All testing was performed by the same person. The subjects avoided additional drug therapy for the remainder of the week. One course of therapy was tested each week until the subjects had been tested while taking both drug and placebo.
Statistical analysis A group of interactive statistical programs was used in a DEC-10 computer system at the Biometrics Section, Medical Computing Resources Center, at this institution. Mean, standard deviation, standard error, and 95% confidence intervals were determined using the SIMPLE program. The significance of differences between groups of observations was tested by Student's t test and Welch's approximation to the Student's t test for unequal variances with the NGROUP
262 Sullivan
J. ALLERGY CLIN. IMMUNOL. MARCH 1982
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FIG. 1. Reproducibility of histamine-induced whealing responses in an individual subject. Control and histamine tests were performed in duplicate. A, Mean (-+SD) of the diameters of wheals induced by the indicated concentrations of histamine on 14 different days. The correlation coefficient of the regression curve defined by these points was r = 0.999, Sy 9 x = 0.32 (ref. 9a). B, These same whealing responses are expressed as the mean (-+SD) of the area of the wheal. The correlation coefficient for the linear regression curve for this data was r = 0.987, Sy. x = 2.78.
program. Linear regression equations were generated, evaluated, and compared by the REGRES program. Doseresponse curves generated in the presence of a competitive inhibitor are parallel but occur at higher agonist concentrations. A conventional index of the degree of inhibition is the increase in agonist concentration required to produce a response equivalent to a control response. In the present study we compared the X-intercept values--the lowest concentrations of histamine capable of causing a wheal. RESULTS Assessment
of histamine-induced
whealing
The MULTITEST method of introducing test substances into the skin was selected because control solutions usually produce no wheal, histamine induces virtually circular reactions, tests are applied simultaneously, the procedure requires little time, and patients experience little discomfort. 8 Whealing responses to histamine provoked by the needle-prick method were similar in magnitude and were not significantly more reproducible than those obtained with the MULTITEST method (not shown). Preliminary experiments indicated that histamine in concentrations ranging from 10-3M to 1M provoked progressively larger wheals. Concentrations above 1M frequently induced irregular wheals with pseudopods or satellite wheals. Since erythematous reactions were irregular and inconsistent and are indirect manifestations of the action of histamine, 9 only whealing reactions are presented in this article. As illustrated in Fig. 1, A, the whealing responses t o various concentrations of histamine were linearly
related to the average diameter of the wheals. When the areas of the wheals, calculated from these diameters, were plotted against histamine concentration, the slope of the curve was not constant (Fig. 1, B). When the areas of wheals were estimated by the tapetransfer method rather than by calculation, a bowed curve similar to that in Fig. 1, B, was obtained (data not shown). As summarized in the legend to Fig. 1, the diameters of wheals described a more nearly linear relationship to histamine concentration. Because we wished to estimate the degree of drug-induced parallel shift in dose curves, the histamine-induced whealing responses were expressed as the diameter of the wheals. Variations among replicates and among measurements by different observers were detected, but the magnitude of this variation was acceptably low. The coefficients of variation averaged 6.0% for 1M histamine, 10.1% for 10-1M, 32.3% for 10-2M and 45.5% for 10-3M histamine when seven replicate tests at each concentration were performed on one subject on three occasions. Variation among measurements taken by two different observers averaged 5.1% of the mean of both sets of observations at 1M histamine, 4.4% at 10-~M, 11.2% at 10-ZM, and 10.5% at 10-ZM in three sets of paired readings, using one constant observer, three different comparative observers, and eight replicates at each concentration. No significant differences in whealing were observed when tests were performed on the volar surfaces of the right or left forearms or on the lateral aspects of the right or left arms (p > 0.30; data from seven ob-
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Doxepin inhibits histamine wheals
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~0
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FIG. 2. Histamine-induced whealing responses in normal individuals. Means (-+SEM) of wheal diameters observed in 25 normal subjects after stimulation by the indicated concentrations of histamine are presented. Whealing responses were determined in duplicate as described in Methods. Spearman's Rho = 1.000; Kendall's Tau = 1.000; Pearson's r = 1.000. The probability that the slope is greater than 0 is p < 0.001. These data were described by equation Y = 9.165 + 2.485 Log10 X. The X intercept was 2.05 x 10-4M histamine.
servations at each site in two patients). The histaminecontaining solutions appeared to be stable for at least 1 wk, as assessed by the diameters of wheals induced (data not shown). New solutions were prepared every 7 days. Dose-response curves measured on consecutive days were consistent when assessed with duplicate tests for each concentration of histamine. As illustrated in Fig. l, the histamine responses measured in a single individual in the morning on 14 different days varied over a relatively narrow range. The slopes of these curves averaged a 2.28-mm increase in diameter per 10-fold increase in histamine concentration with a 95% confidence interval of 2.10 to 2.48 mm per 10fold increase. The threshold concentrations (the lowest concentration of histamine causing a wheal) were calculated to average 4.2 x 10-4M, with a 95% confidence interval of 2.8 to 7.5 x 10-4M. Shifts in threshold concentrations significantly different from the threshold observed on the initial day were observed on four of these 14 days (p < 0.05). No significant change in slope was detected. When five dose-response curves obtained from testing between
10-3
tO-Z
tO -4
l
40o
HISTAMINE (M)
FIG. 3. Effect of doxepin on histamine-induced whealing in an individual subject. Responses to histamine were measured 4 hr after no treatment (o), oral administration of 5 mg of doxepin (o), oral administration of 10 mg of doxepin (•) at 8:00 A.M. The data are presented a s t h e mean (• of 12 control observations, five observations after 5 mg of doxepin, and three observations after 10 mg of doxepin.
8:00 and 10:00 A.M. on different days were compared with each other and with five curves obtained from responses between l:00 and 3:00 P.M. on the same 5 days all in the same subject, no significant difference was noted (p > 0.30, not shown). Three additional individuals studied on 3 to 5 consecutive days demonstrated a similar degree of reproducibility of the dose-response curves. Dose-response curves obtained between 8:00 and 10:00 A.M. were not significantly different from each other or from curves obtained from responses provoked between 1:00 and 3:00 P.M. in these three participants (not shown). The concentrations of histamine selected for testing clearly defined a dose-response relationship in each of 25 normal subjects (Fig. 2). The slope of this curve was a 2.49-mm increase in wheal diameter per 10fold increase in histamine concentration (___0.03 SE; 95% confidence interval 2.34 to 2.63). Correlation coefficients for the regression curves that described the individual respqnses averaged 0.986 (_+0.006 SD). The threshold for whealing calculated from these curves averaged 2.0 x 10-~4M, with a 95% confidence interval of 9.7 x 10-ZMto 4.3 x 10-4M. These results indicated that human skin 'whealing responses to histamine could be assessed with reason-
264 Sullivan
J. ALLERGY CLIN. IMMUNOL. MARCH 1982
TABLE I. Modulation of the whealing response to histamine by single doses of doxepin or chlorpheniramine Ratio of Threshold concentration after treatment 1" Threshold concentration before treatment Treatment*
Mean • SEM
Range
nt
None Chlorepheniramine (12 mg) Doxepin 5 mg 10 mg 25 mg
1.8 + 0.6 3.0 _+ 0.4
0.4 - 6.7 2.4 - 3.7
41 3
5.2 --+ 0.9 18.4 - 3.3 82.2 _+ 37.7
0.7 - 12.5 5.0 - 44.6 11.2 - 316
14 16 8
*The test agent was administered orally at 9:00 A.M. after control skin testing at 8:00 A.M. Skin whealing responses were retested at 1:00 P.M. *Linear regression lines describing skin-test responses were calculated as described in Methods. No significant differences in slopes were detected. The lowest concentration of histamine capable of provoking a wheal was termed the threshold concentration. Alterations in histamine responsiveness were calculated by determining the ratio of threshold concentrations after treatment to threshold concentrations before treatment. *Number of subjects examined. In subjects receiving no treatment, observations on two or more days were compared.
able precision by means of a MULTITEST device to apply duplicate tests with histamine in concentrations ranging from 10-aM to 1M. The degree of reproducibility of measurements of responses and the reproducibility of the responses themselves indicated thatthis approach could be used to assess pharmacologic modulation of histamine-induced wheaiing in human skin. Since wheals induced by 10-3M histamine (1.6- to 4.2-mm wheals) were difficult to measure accurately and since regression curves defined by IM, 10-1M, and 10-2M, histamine were not significantly different from those defined by inclusion of the 10-3M histamine point, the 10-3M histamine concentration was dropped from the testing procedure. This permitted duplicate tests with 1M, 10-'M, and 10-2M histamine and diluent control with a single MULTITEST device.
Effects of pharmacologic agents on histamine-induced whealing Doxepin caused a significant dose-related inhibition of histamine-induced whealing (Table I, Fig. 3). A single dose of 25 mg of doxepin increased the concentration of histamine required to produce responses equivalent to control responses an average of 82-fold (Table I) when responses were measured 4 hr after ingestion of the drug. The magnitude of the shift in the histamine dose-response curve decreased with decreasing doxepin dose, but significant effects (5.2fold shift) were detected after a single 5-mg dose (Table I). The magnitude of doxepin effects on histamine-induced whealing were variable from one patient to the next (Table I) but were relatively consis-
tent when individual patients were tested on more than one occasion (Fig. 3). Chlorpheniramine was examined for comparison to doxepin. Measurements taken 2 hr after a single 12-mg dose of chlorpheniramine indicated a threefold shift in the dose-response curve (Table I). As illustrated in Fig. 3, doxepin caused a doserelated parallel displacement of the histamine doseresponse curve to the right, as assessed by an analysis of variance of the slopes (F = 87.3, p < 0.001). Newman-Keuls multiple comparisons of the slopes indicated no differences among the control, 5-mg doxepin, and 10-mg doxepin regression lines. 10 Comparison of the intercepts of these curves by NewmanKeuls multiple comparisons indicated that each of the regression lines was significantly different from the others. Similar results were obtained in series of determinations on two other subjects. Each of the eight subjects taking 25 mg of doxepin reported significant sedation. Patients receiving 10 mg reported noticeable but mild sedation. A 5-mg dose of doxepin did not produce noticeable effects.
Double-blind, placebo-controlled evaluation of doxepin effects To be certain that the measurement of histamineinduced wheals was not influenced by the expectations of the observer and that the responses were reproducible when the subjects were not aware of the nature of test substances, a double-blind evaluation was performed. The predictable mild sedative properties of doxepin in doses of 10 mg or higher every 12 hr would have permitted the subjects to distinguish
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Doxepin inhibits histamine wheals 265
T A B L E U. Double-blind evaluation of the effect of doxepin on whealing responses to histamine 9 Threshold concentration after treatment Ratio or . . . . . Threshold concentration before treatment Subject
Treatment*
4 hrt
28 hr
52 hr
T. J.S.
Placebo Doxepin Placebo Doxepin Placebo Doxepin Placebo Doxepin Placebo Doxepin Placebo Doxepin
1.1 3.0 0.5 4.5 1.5 3.5 1.6 1.3 1.2 0.7 1.2 (+_0.2) 2.5 (---0.8)
1.2 23.4 1.1 5.2 6.5 20.9 1,1 29.4 0.7 7.3 2.1 (+_ 1.1) 17.2 (+_4.7)
4.8 36.3 0.7 i2.3 6.7 26.3 1.1 31.6 0.4 4.7 2.7 ( - 1.3) ?.2.2 (-+5.9)
S.R. T. R.S. M.W. R.T. All subjects
*The double-blind protocol used is described in detail in Methods. Doses were administered every 12 hr for a total of five doses. Each subject received one or more courses of treatment with doxepin (5 mg) and with placebo. tWhealing responses w e r e a s s e s s e d at 8:00 A.M. and the first dose was administered at 9:00 A.M. on a Monday. Skin whealing r e s p o n s e s were assessed 4, 28, and 52 hr after the first dose. The results of multiple courses of drug and of placebo were averaged. The responses for all five subjects were expressed as the mean (+_ SEM).
placebo from drug. A 5-mg dose was selected to optimize the blind feature of the study at the price of using a less effective dose. A preliminary study indicated that when a 5-mg dose of doxepin was administered every 12 hr, the drug effect increased over the first 24 hr and then remained constant for at least 3 days. Preliminary studies also demonstrated that skin whealing responses returned to control levels within 2 days after five doses of 5 mg of doxepin administered at 12-hr intervals (not shown). A double-blind, placebo-controlled study of doxepin effects was then performed with 5 mg of doxepin or placebo administered every 12 hr for five doses. Doxepin was a highly effective inhibitor of histamine-induced whealing (Table II). Relatively little effect was noted 4 hr after a single 5-mg dose of doxepin, but after three doses an average of 17.2-fold more histamine was required to provoke dose-related whealing equivalent to control responses. The average effect measured after five doses was not significantly higher than that detected after three doses. Each of the five subjects demonstrated significant doxepin inhibition of whealing responses after three and five doses (p < 0.05). The slopes of the curves in the drug and placebo treatment periods were not significantly different. Statistically significant shifts in threshold (p < 0.05) were observed during courses of placebo treatment in two subjects, T. J. S. (52hr) and T. R. S. (28 and 5 2 hr).
DISCUSSION
The results of these experiments lead to two principal new insights. First, a simple method for assessing dose-related human skin whealing responses to histamine was defined. The variability among observations taken on different occasions and variability among subjects were measured and found to be small. Second, the development and characterization of this method permitted us to perform a double-blind study that demonstrated the marked antihistaminic properties of doxepin in man. Studies of wheals elicited by introducing histamine into human skin have been performed for nearly 70 yr. Eppinger 11 described the wheal-and-flare-inducing properties of histamine in 1913. By the time of the publication of Lewis' still remarkable monograph 12 on human skin blood vessels and their physiology in 1927, the whealing response was understood to result at least partially from vasodilation and increased capillary permeability2' t2 The flare response was recognized as a secondary neural reflex response provoked by histamine2 The synthesis of competitive inhibitors of HI receptors by Bovet and Staub ~3 in t937 and the evolution of this class of agents led to renewed interest in human cutaneous whealing, now as an in vivo model for assessing the clinical efficacy of antihistamines. ~4-~ The magnitude of the wheal was found to be linearly related to the log of the histamine dose over a 1000-fold or greater range of doses. Conventional H1 antagonists were reported to cause parallel displacements of these dose-response
266 Sullivan
curves to higher histamine dose ranges. 15, 16 Recent studies indicate that human cutaneous whealing responses are mediated primarily by H I receptors, but a measurable minor contribution by cimetidine-inhibitable H2 receptors is also present. 17, 18 This study demonstrates that percutaneous administration of histamine with a MULTITEST device is a simple, rapid, reproducible method that permits the measurement of dose-related histamine responses in vivo in an acceptably precise manner. This method minimizes the requirement for skill that is known to be essential for reproducible testing by intradermal or needle-prick methods. 18 The simultaneous application of stimuli facilitates measurement of responses at a consistent time after administration. The speed, simplicity, and reproducibility of this method are advantages over prior methods, but careful introduction of histamine by any method is likely to be adequate to define dose-response relationships. 15.16, 18 Most investigators have expressed histamine responses as the area of the wheal o r f l a r e , 14-16 but the results of this inquiry were more accurately described as a function of the diameter of the wheal (Fig. 1). The complexity of the events taking place in the course of whealing 12' 19 precludes a priori predictions of the most precise measurement of dose-related reactions. In radial immunodiffusion reactions, for example, the antigen concentration is usually related to the diameter of the expanding ring. 2~ The antigen concentration is usually related to the square of the diameter of the ring when the ring stops expanding, zl Histamine-induced whealing responses measured 15 min into their evolution appear to be analogous to the expanding ring of radial immunodiffusion reactions. Earlier studies have demonstrated a degree of variability among histamine responses measured on individuals at different times of day, 16" 22 but there has been an implicit assumption that responses at the same hour on different days are directly comparable.14-16, 2.~ The results of this study demonstrate that histamine dose-response curves remain parallel in individual subjects but can vary over as much as a sevenfold range during a 3-day period of observation (Table II). Although the variation from day to day usually fell within much narrower boundaries (Fig. 1), this observation emphasizes the need to consider the variance from day to day, not just the variance in a single control observation, when histamine responses after drug treatment are compared with control responses. The reasons for significant intrinsic variation in histamine responsiveness are currently unknown. This issue deserves careful attention as a possible source of clinical variation during allergic or other diseases. To assess the antihistaminic properties of doxepin,
J. ALLERGY CLIN. IMMUNOL. MARCH 1982
we designed a study that would permit assessment of intrinsic variation in histamine responsiveness as well as doxepin effects in a randomized double-blind protocol. As illustrated in Fig. 3 and Table II, doxepin caused a dose-related inhibition of histamine-induced whealing responses. The effect of doxepin (5 mg) administered at 12-hr intervals increased over 48 hr to an average 22-fold shift in the histamine dose-response curve. These changes were statistically significant when compared with responses during placebo treatment. No double-blind, placebo-controlled studies of tricyclic antidepressant inhibition of human responses to histamine are available for quantitative comparison. However, controlled studies of some conventional Hi-antagonists have been reported. Two studies indicate that multiple doses of 4 mg of chlorpheniramine 23 or 8 mg of chlorpheniramine 17 cause threefold to sixfold shifts to the right of histamine dose-response curves. A recent study by Nathan et al. 24 detected a similar degree of inhibition after single or multiple doses of 8 mg of chlorpheniramine. Inhibition of high-dose histamine effects assessed 2 hr after a single dose of chlorpheniramine was nearly identical to that observed after multiple doses in their study. The threefold shift detected 4 hr after single 12-mg doses of chlorpheniramine in this study (Table I) are in agreement with these previous reports. Multiple doses of 25 mg of hydroxyzine administered orally were reported to inhibit histamine-induced whealing, 25 but the drug apparently caused nonparallel displacement of the dose-response curve, making quantitative comparisons difficult. The available data indicate that doxepin is considerably more potent than chlorpheniramine. Potency relative to other agents remains to be established in controlled trials. Multiple doses of 25 mg of doxepin would be expected to shift histamine dose-response curves considerably more than the 82fold observed after one dose (Table II). The precise means by which doxePin suppresses histamine-induced vascular responses is not certain. Some tricyclic antidepressants exert H1 and H2 antihistaminic actions as well as anticholinergic and other effects.2" 26, 27 A direct competitive H1 blocking effect or Hi plus H2 blocking effect appear to be the most likely explanations, since the drug caused a dose-related parallel displacement of the histamine close-response curve to higher histamine concentrations. Doxepin or related drugs may be found to have a role in the treatment of clinical allergic diseases. Preliminary clinical studies in this laboratory indicate that doxepin may be useful in the management of chronic urticaria and allergic rhinitis. Use of this particular agent is limited to some extent by the observa-
VOLUME 69 NUMBER 3
tion that n o n d e p r e s s e d s u b j e c t s m a y e x p e r i e n c e d r o w s iness at doses o f 10 to 25 m g t w i c e daily. A t d o s e s c o m m o n l y u s e d to treat d e p r e s s i o n (25 m g orally three t i m e s daily or 75 m g at b e d t i m e , or h i g h e r ) , m o s t n o r m a l s u b j e c t s w o u l d b e e x p e c t e d to note significant d r o w s i n e s s . L i q u i d f o r m s o f d o x e p i n are a v a i l a b l e , h o w e v e r , that p e r m i t s i m p l e a d j u s t m e n t o f d o s a g e . I d e n t i f i c a t i o n o f the p o t e n t a n t i h i s t a m i n i c properties o f d o x e p i n indicates that tricyclic a n t i d e p r e s s a n t s s h o u l d be e x a m i n e d c a r e f u l l y for t h e i r r e l a t i v e p o t e n c i e s as a n t i h i s t a m i n e s . T h e use o f d o x e p i n or related agents m a y m a r k e d l y i m p r o v e o u r ability to s u p p r e s s Ha-rec e p t o r - m e d i a t e d effects o f h i s t a m i n e in m a n . 1 gratefully acknowledge the assistance of Dr. Gary N. Gross in recruiting volunteers, and the expert technical assistance of Ms. Susan Robinson and Mr. Edward Lewis. I would like to state my appreciation for the unusual level of cooperation the volunteers contributed to this study. REFERENCES 1. Kuhn R: The imipramine story, in Ayd FJ Jr, Blackwell B, editors: Discoveries in biological psychiatry. Philadelphia, 1970, J.B. Lippincott Co., pp. 205-217. 2. Richelson E: Tricyclic antidepressants and histamine HI receptors. Mayo Clin Proc 54:669, 1979. 3. Friedel RO, Raskind MA: Relationship of blood levels of Sinequan to clinical effects in the treatment of depression in aged patients, in Mendels J, editor: Sinequan (doxepin HC1). A monograph of recent clinical studies. Amsterdam, 1975, Excerpta Medica, pp. 51-53. 4. Ziegler VE, Clayton PJ, Biggs JT: A comparison study of amitriptyline and nortriptyline with plasma levels. Arch Gen Psychiatry 34:607, 1977. 5. Tozzi S, Roth FE, Tabachnick IA: Pharmacology of azatadine, a potential anti-allergy drug. Agents Actions 4:264, 1974. 6. Yacobi A, Stoll RG, Chao GC, Carter JE, Baaske DM, Kamath BL, Amann AH, Lai C-M: Evaluation of sustainedaction chlorpheniramine-pseudoephedrine dosage form in humans. J Pharm Sci 69:1077, 1980. 7. Norman PS: Skin testing, in Rose NR, Friedman H, editors: Manual of clinical immunology. Washington, D. C., 1976, American Society for Micriobiology, pp. 585-589. 8. Murphree JT, Kniker WT: Correlation of immediate skin test responses to antigens introduced by multitest and intracutaneous routes. Ann Allergy 43:279, 1979. 9. Thomas L, Grant RT: Vascular reactions of the skin to injury. Part II. The liberation of a histamine-like substance in injured skin: the underlying cause of factitious urticaria and of wheals produced by burning: and observations upon the nervous control of certain skin reactions. Heart 11:109, 1924.
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