Br. J. clin. Pharmac. (1991), 31, 471-475
ADONIS 030652519100089 Q
Skin irritation induced by topically applied timolol K. KUBOTA1, E. KOYAMA' & K. YASUDA2 'Division of Clinical Pharmacology, Clinical Research Institute, National Medical Center, Tokyo and 2Tokyo Research Center of Clinical Pharmacology, Tokyo, Japan
1 Erythema induced by topically applied timolol, a non-selective ,B-adrenoceptor blocker, was assessed in six male volunteers. Intensity of erythema developed on the inner surface of the left forearm where timolol free base was applied for 10 h was measured by a visual score, laser doppler flowmeter and reflectance spectrophotometry. Plasma timolol concentrations collected from the left and right arms were also measured. 2 The mean values for blood volume and blood flow per unit volume of tissue both of which were assessed by a laser doppler flowmeter, haemoglobin index measured by reflectance spectrophotometry, and magnitude of erythema graded by a visual score significantly increased after the application of an acrylic co-polymer adhesive patch containing 20% (w/v) timolol free base. 3 Plasma timolol concentrations collected from the left antecubital vein were 2.4 to 10.7 times greater than those from the right arm and had significant correlations (r, = 0.55 to 0.76) with the parameters indicating the extent of erythema developed where a patch containing timolol was applied. 4 The inter-individual variation of timolol was attributed to that of the diffusivity of timolol through skin rather than that of the skin reactivity to topically applied timolol because the plasma timolol concentrations drawn from the left arm in the subjects who did not develop erythema were very low. 5 Provided that the timolol concentrations in plasma collected from the left antecubital vein are roughly proportional to the flux values of timolol from skin to blood, it would be difficult to give a sufficient amount of timolol to expect systemic effect(s) with no concurrent erythema because erythema develops unless drug flux is unacceptably low.
Keywords timolol transdermal skin
,-adrenoceptor blocker
percutaneous absorption
Introduction
al., 1988). Though this local side effect is reported to be mild or minimal for some P-adrenoceptor blockers including timolol (Cargill et al., 1986; Vlasses et al., 1985), the skin irritation probably hampers a wide clinical usage of transdermal application of ,-adrenoceptor blockers. Because timolol does cause irritation only when the free base form is applied as a nearly saturated solution in rat, more detailed studies are necessary to evaluate further if the irritation can be limited by the use of a timolol formulation at a low thermodynamic activity (Cargill et al., 1986). In this article, the relationship was studied between the magnitude of skin irritation caused by topically applied timolol and drug concentration in the blood which has just perfused the skin where timolol
Several ,-adrenoceptor blockers are known to penetrate human skin and show systemic ,B-adrenoceptor blocking effects (Spieker et al., 1989; Vlasses et al., 1985; Wellstein et al., 1986). Many 3-adrenoceptor blockers undergo extensive first-pass metabolism in the liver, resulting in wide individual variations in plasma levels when given orally (Riddell et al., 1987). Thus, the transdermal route for administration of a ,B-adrenoceptor blocker is certainly one worth investigating. However, a transdermal route for the administration of 1B-adrenoceptor blockers has not been widely accepted in clinical practice. Transdermal application of ,-adrenoceptor blockers is reported to cause local irritation in human beings and animals (de Mey et al., 1989a,b; Spieker et
Correspondence and present address: Dr Kiyoshi Kubota, Department of Dermatology, University of California, San Francisco, School of Medicine, Box 0989, Surge 110, San Francisco, CA, 94143-0989, USA
471
472
K. Kubota, E. Koyama & K. Yasuda
was applied. This is because when this relationship is scrutinized, it may indicate if it is possible to reduce the skin irritation while a sufficient amount of timolol to produce systemic effect(s) is absorbed.
Methods
Subjects and drug The study held in the Tokyo Research Center of Clinical Pharmacology (Takata-no-baba, Tokyo, Japan), where phase I studies of various newly developed drugs are mainly conducted, was approved by the Ethics Committee of Tokyo (Chairman, Professor C. Ibukiyama, Tokyo College of Medicine). Six volunteers (subjects 1 to 6, aged 36 to 51 years with a mean of 46.5 years and weighing 49.5 to 66.9 kg with a mean of 59.0 kg) agreed to participate in the study after receiving full explanation and giving written consent. They were judged to be in good health on the basis of history, physical examination, routine laboratory data (complete haemogram, urinalysis, serum creatinine, transaminases, alkaline phosphatase, total bilirubin, total cholesterol, blood glucose, total serum protein, electrolyte) and electrocardiogram. No subject had a history of asthma. Each of four therapeutic timolol patches (2.5 x 6 cm2) containing 20% (w/v) timolol free base in acrylic co-polymer, 40 ,um in thickness (Kubota et al., 1990), was applied on the same site on the inner surface of the left forearm near the wrist. Each timolol patch contained 12 mg of timolol free base. Four placebo patches containing no timolol were also applied consecutively on the same site of the right forearm symmetrically to the timolol patch. An area, 4 cm proximal to the placebo patch, was used as a control. Each patch was applied for 2.5 h, then removed and erythema developed beneath the patch was evaluated. Next timolol and placebo patches were applied 0.5 h after the removal of the previous timolol and placebo patches, respectively. Evaluation of effects The study was conducted on the same day for all six volunteers in the room where the temperature and relative humidity were conditioned at 26 ± 1° C and 71 ± 2%, respectively. The evaluation of magnitude of erythema and measurements of blood pressure and heart rate were executed just before the application of active and placebo patches (at 0 h), after the removal of each patch (at 2.5, 5.5, 8.5 and 11.5 h) as well as at 14, 26 and 29 h. Blood pressure and heart rate were measured in duplicate after subjects had been supine for 5 min and after standing for 2 min with an automatic sphygmomanometer (Japan Colin BP 203-N, Komaki City, Japan). Erythema was evaluated by a visual score, laser doppler flowmeter and reflectance spectrophotometry at least 10 min after the removal of the patch. Erythema was graded by a dermatologist according to a visual score modified from that by Frosch & Kligman (1979) as: (0) no erythema; (0.5) slight spotty erythema; (1) weak diffuse erythema; (2) moderate, uniform erythema and (3) intense redness. A He-Ne laser doppler flowmeter (ALF 2100, Advance,
Tokyo, Japan) was employed to measure blood volume (mass) per unit volume of tissue expressed by an arbitrary unit as well as blood flow rate expressed by a unit, ml min-1 100 g-1 (Cao et al., 1989; Yamaguchi, 1989). The blood volume and blood flow were measured in triplicate for each of the active patches, placebo patches and control sites. An index of haemoglobin concentration (IHB) expressed by an arbitrary unit was recorded in duplicate for each of the three sites by a reflectance spectrophotometry (Tissue Spectrum Analyzer, TS-200, Sumitomo electric industries, Osaka, Japan). An IHB defined as the difference of absorbances between 569 nm (green light) and 650 nm (red light) (Leung et al., 1987) was employed to evaluate the extent of erythema, because an 'erythema index' (Diffey et al., 1984; Feather et al., 1982) has been defined as the difference between the absorbances at wave lengths corresponding to green and red lights. Sample collection Blood samples (10 ml) were collected from the left antecubital vein exactly 30 s after the application of a rubber tourniquet at 0 (pre), 3, 6, 9, 12, 14, 26 and 29 h. Venous blood samples (10 ml) were also collected from the right arm at 0 (pre), 12, 14, 26 and 29 h. Plasma was separated immediately and stored at -20° C until
analyzed. Drug analyses Plasma timolol concentrations were measured using the h.p.l.c. method by a Hitachi L-6000 pump (Tokyo, Japan) equipped with a Hitachi L-4000 u.v. detector. To 1-2 ml plasma, 100 ,u 4 M sodium hydroxide, 10 ,ug metoprolol-(+) tartrate, as internal standard, and 5 ml dichloromethane were added and vortexed for 180 s. After centrifugation for 5 min at 1500 g, the organic phase was evaporated to dryness. The residue was reconstituted with 100 ,ul of mobile phase and 50 to 75 RI of the solution was injected onto the chromatograph. The h.p.l.c. column, mobile phase and other chromatographic conditions were the same as previously described (Kubota et al., 1990). The absolute recoveries of timolol and internal standard in plasma were more than 97%. The detection limit was 0.3 ng ml-' when 2 ml of plasma sample was used and a 75 pI aliquot was injected onto the chromatograph. At 5 ng ml-' of plasma timolol concentration, the coefficient of variation (c.v.) in the within-day study was 1.7% (n = 9), while that in the dayto-day study was 7.5% (n = 7).
Statistical analysis All data were expressed as mean ± s.e. mean. Statistical significances were calculated by one-way analysis of variance (ANOVA) for blood pressure and heart rate measured at t = 0 to 29 h. The difference in the time courses among the three skin sites where active, placebo and no patches were applied was tested by two-way ANOVA for each of the changes in blood volume (mass), blood flow and IHB from the respective values at t = 0, which are defined as AMass, AFlow and AIHB, respectively. That for visual score was also tested by two-way
473
Skin irritation induced by topically applied timolol ANOVA. When there was a statistically significant difference in the time courses among three skin sites for either of AMass, AFlow, AIHB and visual score, the analysis of significance of differences of the time courses between two skin sites (i.e. timolol vs control sites, timolol vs placebo sites, and placebo vs control sites) were conducted by the Tukey Tmethod. The correlation between the plasma concentration collected from the left antecubital vein vs either of AMass, AFlow, AIHB and visual score was tested using Spearman's rank correlation coefficient, rs, as well as the simple linear correlation coefficient, r. P < 0.05 was considered statistically significant.
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Figure 2 Time courses of the mean ( s.e. mean, n 6) change in blood volume (mass) per unit volume of tissue (arbitrary unit) from that at t = 0, AMass, measured at the sites where the active (0), placebo (@) and no (C) patches were applied (a), change in blood flow, AFlow (b), change in the index of haemoglobin concentration (arbitrary unit), AIHB (c), and visual score (d).
The resting supine and upright blood pressure and heart rate did not change significantly throughout the study (ANOVA). No erythema developed in subjects 1 and 2 by a visual score. In subjects 3 to 6, erythema graded as weak (1) eventually developed on the left forearm where the active patch containing timolol was applied. In subjects 3 and 4, erythema assessed by a visual score where active, placebo and no patches were applied was developed also on the right forearm where the placebo statistically significant for all of AMass (P < 0.05), patch was applied. However, in subjects 5 and 6, no AFlow (P < 0.05), AIHB (P < 0.01) and visual score erythema was visually recognized except for the active (P < 0.01) (ANOVA). Using the Tukey T method, for patch site. No erythema was graded as moderate (2) or AMass, the difference between timolol and control sites (P < 0.01) and that between placebo and control sites more. Erythema was confined to the area where the active or placebo patch was applied and not accompanied (P < 0.01) were significant. For AFlow, that between by oedema or any other effect. No symptoms developed placebo and control sites (P < 0.05) was significant. For AIHB and visual score, that between timolol and placebo in any subject. After the removal of the patches, erythema eventually disappeared in 48 h without sites (P < 0.01) and that between timolol and control sites (P < 0.01) were significant. There was a significant sequelae. correlation between the plasma concentration collected timolol s.e. mean) plasma In Figure 1, the mean (± concentrations collected from the left and right arms are from the left antecubital vein and one of AMass, AFlow, AIHB and visual score (r, = 0.670, 0.760, 0.549 and shown. The mean plasma timolol concentrations from the left antecubital vein were 2.4 to 10.7 times higher 0.708, respectively; P < 0.001; r = 0.574, 0.708, 0.485 and 0.642, respectively; P < 0.01; Figure 3). The median than those from the right arm and had a great interindividual variation (coefficient of variation, c.v. 160%). The time courses of the mean (± s.e. mean)avf ;,,., 4^. ;b .4A t. .~ c fi values for AMass, AFlow, AIHB and visual score are shown in Figure 2, where those for the active patch site were greater than the placebo and control sites. The difference of those values among the three skin sites 0
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Time (h) Figure 1 Time courses of the mean (± s.e. mean, n = 6) plasma timolol concentration collected from the antecubital vein of the left arm (0) where the timolol patch was applied and the right arm (OJ) where the placebo patch was applied.
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Figure 3 Relation between the plasma timolol concentration collected from the left arm where the active patch containing timolol free base was applied and one of (a) change in blood volume (mass) per unit volume of tissue (arbitrary units) from that at t = O, i\Mass, (b) change in blood flow AvFlow, (c) change in index of haemoglobin concentration (arbitrary units), AIHB and (d) visual score. Symbols, 0, A, *, A, O and-l represent subjects 1 to 6, respectively.
474
K. Kubota, E. Koyama & K. Yasuda
plasma timolol concentration corresponding to erythema graded as slightly spotty (0.5) was 3.0 ng ml-', whereas that graded as weak diffuse (1.0) was 12.4 ng ml-' (Figure 3d). The correlation between visual score vs one of AMass, AFlow and AIHB was also significant (rs = 0.731, 0.772 and 0.731, respectively; P < 0.001; r = 0.692, 0.674 and 0.704, respectively; P < 0.001).
Discussion Blood flow measured by a laser doppler flowmeter (Nilsson et al., 1982) and 'erythema index' by a reflectance spectrophotometry (Diffey et al., 1984; Feather et al., 1982) have been considered to be the quantitative expres sions of the extent of erythema. Because the visual score was significantly correlated with AMass, AFlow and AIHB, those values could also be regarded as the quantitative expression of the intensity of erythema in this study. The logarithm of plasma concentration collected from the left antecubital vein and those changes seem to have the linear correlation in Figure 3. In other words, in Figure 3, there seems to be no specific 'threshold' plasma concentration which is critical for the development of erythema. It is of note that all the timolol concentrations in plasma drawn from the left antecubital vein of subjects 1 and 2 designated by open circles and triangles, respectively in Figure 3, were less than 5 ng ml-'. Thus, the reason why no erythema developed in these two subjects is explained merely by the fact that the plasma concentra tions from the left antecubital vein were very low in these subjects, rather than the fact that those concentrations did not reach some critical 'threshold' value, neither had they low reactivity to topically applied timolol. The great interindividual variation of timolol permeation was also noted in the previous in vivo study where 30 to 60 mg of timolol free base was given to six volunteers (Vlasses et al., 1985). According to Cargill et al. (1986), the timolol-induced erythema may be caused by the alkaline nature of timolol free base, its ,B-adrenoceptor blocking effect, or both. Though the data shown in Figure 3 do not offer any direct evidence, we may suggest that the former is a likely mechanism. This is because the plasma timolol concentration collected from the left arm in Figure 3 could be reflected by the extent of the derangement from the physiological pH in the local tissue. On the other hand, most 3-adrenoceptor blockers cause vasoconstric tion and blanching of skin rather than erythema (Hornqvist et al., 1984), even though a 3-adrenoceptor blocker is able to augment skin reactivity where the
chemical mediator(s) are released from dermal mast cells by the challenge of antigen (Shereff et al., 1973) or when histamine is given exogenously (Lamkin et al., 1976). It should be noted that erythema observed in this study occurs in a modest range of dose absorbed. The peak plasma concentrations in the systemic circulation (collected from the right arm) were lower than the detection limit of 6.9 ng ml-1 in six subjects in this study where 15 cm2 of skin was used. On the other hand, those were approximately 10 to 50 ng ml-' in the previous study (Vlasses et al., 1985) where 25 cm2 of skin was used. When the plasma concentrations from the left antecubital vein are considered to be roughly proportional to the drug flux from skin to local circulation, the results shown in Figure 3 indicate that timolol flux should be much smaller than its possible maximum (unit activity) flux (Flynn & Stewart, 1988; Kubota & Yamada, 1990) to inhibit the development of erythema completely. In this laboratory, in an in vitro study using excised human skin, this maximum flux or that from the saturated timolol aqueous solution was measured to be near 30 ,ug cm-2 h-1 (unpublished data) and that from acrylic copolymer was calculated to be near 45 ,ug cm-2 h-1 (Kubota & Yamada, 1990). If a daily dose of 10 mg timolol, the minimum oral daily dose (Frishman, 1982), is to be absorbed through skin using a flux, 4 ,ug cm-2 h-1, which is approximately one tenth of the possible attainable maximum flux, timolol should be applied over an area of 100 cm2. Because erythema developed in a wide range of the timolol concentrations collected from the left arm in this study (Figure 3), erythema at least graded as slight (0.5) will likely develop at the flux, 4 ,ug cm-2 h-1. On the other hand, the area, 100 cm2, may be considered near or more than the maximum acceptable area. Thus, it seems difficult to provide sufficient amounts of timolol to deliver the desired systemic effect(s) with no concurrent erythema. However, more extensive studies where timolol is applied on various sites other than the forearm (e.g. the abdomen, chest and back) are necessary to draw the final conclusion whether a ,-adrenoceptor blocker can be topically administered without skin irritation because it is well appreciated that there is regional variation in certain skin reactions including allergic and irritant ones (Maibach & Prystowsky, 1977; Lahti & Maibach, 1984; Van der Valk & Maibach, 1989). Similarly, more studies are necessary to know whether erythema graded as slight to weak in this study and graded as mild or minimal in the previous studies (Cargill etal., 1986; Vlasses et al., 1985) should exclude transdermal application as an alternate route of administration of ,B-adrenoceptor blockers.
References Cao, W. H., Inanami, O., Sato, A. & Sato, Y. (1989). Stimulation of the septal complex increases local cerebral blood flow in the hippocampus in anesthetized rats. Neurosci. Lett., 107, 135-140. Cargill, R., Engle, K., Rork, G. & Caldwell, L. J. (1986). Systemic delivery of timolol after dermal application: transdermal flux and skin irritation potential in the rat and dog. Pharm. Res., 3, 225-229.
de Mey, C., Enterling, D., Ederhof, M., Wesche, H. & Osterwald, H. (1989a). Transdermal delivery of mepindolol and propranolol in normal man: 1st communication: study design, clinical and pharmacodynamic aspects. Arzneim. Forsch., 39, 1505-1508. de Mey, C., Meineke, I., Enterling, D., Rehbock, C. & Osterwald, H. (1989b). Transdermal delivery of mepindolol and propranolol in normal man: 2nd communication:
Skin irritation induced by topically applied timolol pharmacokinetic and neuro-endocrine aspects. Arzneim. Forsch., 39, 1508-1512. Diffey, B. L., Oliver, R. J. & Farr, P. M. (1984). A portable instrument for quantifying erythema induced by ultraviolet radiation. Br. J. Dermatol., 111, 663-672. Feather, J. W., Ryatt, K. S., Dawson, J. B., Cotterill, J. A., Barker, D. J. & Ellis, D. J. (1982). Reflectance spectrophotometric quantification of skin colour changes induced by topical corticosteroid preparations. Br. J. Dermatol., 106, 437-444. Flynn, G. L. & Stewart, B. (1988). Percutaneous drug penetration: choosing candidates for transdermal development. Drug Dev. Res., 13, 169-185. Frishman, W. H. (1982). Atenolol and timolol, two new systemic ,B-adrenoceptor antagonists. New Engl. J. Med., 306, 1456-1462. Frosch, P. J. & Kligman, A. M. (1979). The soap chamber test: a new method for assessing the irritancy of soaps. J. Am. Acad. Dermatol., 1, 35-41. Hornqvist, R., Back, 0. & Henriksson, R. (1984). Adrenoceptor-mediated responses in human skin studied by iontophoresis. Br. J. Dermatol., 111, 561-566. Kubota, K., Yamada, T., Ogura, A. & Ishizaki, T. (1990). A novel differentiation method of vehicle models for topically applied drugs: application to a therapeutic timolol patch. J. pharm. Sci., 79, 179-184. Kubota, K. & Yamada, T. (1990). Finite dose percutaneous drug absorption: theory and its application to in vitro timolol permeation. J. pharm. Sci., 79, 1015-1019. Lahti, A. & Maibach, H. I. (1984). An animal model for nonimmune contact urticaria. Toxicol. appl. Pharmac., 76, 219-224. Lamkin, N., Lieberman, P., Shereff, R., Rosenberg, E. W. & Robinson, H. (1976). Effect of beta adrenergic stimulation and blockade on cutaneous reactivity to histamine. J. Allergy clin. Immunol., 57, 449453. Leung, F. W., Slodownik, E., Jensen, D. M., Van Deventer, G. M. & Guth, P. H. (1987). Gastroduodenal mucosal hemodynamics by endoscopic reflectance spectrophotometry. Gastrointest. Endosc., 33, 284-288.
475
Maibach, H. I. & Prystowsky, S. D. (1977). Glutaraldehyde (pentanedial) allergic contact dermatitis. Arch. Dermatol., 113, 170-171. Nilsson, G. E., Otto, U. & Wahlberg, J. E. (1982). Assessment of skin irritancy in man by laser doppler flowmetry. Contact Dermatitis, 8, 401406. Riddell, J. G., Harron, D. W. G. & Shanks, R. G. (1987). Clinical pharmacokinetics of P-adrenoceptor antagonists: an update. Clin. Pharmacokin., 12, 305-320. Shereff, R. H., Harwell, W., Lieberman, P., Rosenberg, E. W. & Robinson, H. (1973). Effect of beta adrenergic stimulation and blockade on immediate hypersensitivity skin test reactions. J. Allergy clin. Immunol., 52, 328-333. Spieker, C., Vetter, H., Liedtke, R., Zidek, W. & Vetter, W. (1988). Transdermal ,B-blocker therapy in essential hypertension. Am. J. Hypertension, 1, 199S-200S. Spieker, C., Vetter, W., Zidek, W. & Vetter, H. (1989). Evaluation of the therapeutic effect of transdermal 1blocker therapy in patients with essential hypertension. Arzneim. Forsch., 39, 1512-1514. Van der Valk, P. G. M. & Maibach, H. I. (1989). Potential for irritation increased from the wrist to the cubital fossa. Br. J. Dermatol., 121, 709-712. Vlasses, P. H., Ribeiro, L. G. T., Rotmensch, H. H., Bondi, J. V., Loper, A. E., Hichens, M., Dunlay, M. C. & Ferguson, R. K. (1985). Initial evaluation of transdermal timolol: serum concentration and 13-blockade. J. cardiovasc. Pharmac., 7, 245-250. Wellstein, A., Kuppers, H., Pitschner, H. F. & Palm, D. (1986). Transdermal delivery of bupranolol: pharmacodynamics and beta-adrenoceptor occupancy. Eur. J. clin. Pharmac., 31, 419422. Yamaguchi, T. (1989). Continuous monitoring of gastroduodenal mucosal hemodynamics in rats by laser-doppler flowmetry and reflectance spectrophotometry. Gastro-
enterologia Japonica, 24, 619-625.
(Received 28 June 1990, accepted 30 October 1990)
ADONIS 030652519100089 Q
Skin irritation induced by topically applied timolol K. KUBOTA1, E. KOYAMA' & K. YASUDA2 'Division of Clinical Pharmacology, Clinical Research Institute, National Medical Center, Tokyo and 2Tokyo Research Center of Clinical Pharmacology, Tokyo, Japan
1 Erythema induced by topically applied timolol, a non-selective ,B-adrenoceptor blocker, was assessed in six male volunteers. Intensity of erythema developed on the inner surface of the left forearm where timolol free base was applied for 10 h was measured by a visual score, laser doppler flowmeter and reflectance spectrophotometry. Plasma timolol concentrations collected from the left and right arms were also measured. 2 The mean values for blood volume and blood flow per unit volume of tissue both of which were assessed by a laser doppler flowmeter, haemoglobin index measured by reflectance spectrophotometry, and magnitude of erythema graded by a visual score significantly increased after the application of an acrylic co-polymer adhesive patch containing 20% (w/v) timolol free base. 3 Plasma timolol concentrations collected from the left antecubital vein were 2.4 to 10.7 times greater than those from the right arm and had significant correlations (r, = 0.55 to 0.76) with the parameters indicating the extent of erythema developed where a patch containing timolol was applied. 4 The inter-individual variation of timolol was attributed to that of the diffusivity of timolol through skin rather than that of the skin reactivity to topically applied timolol because the plasma timolol concentrations drawn from the left arm in the subjects who did not develop erythema were very low. 5 Provided that the timolol concentrations in plasma collected from the left antecubital vein are roughly proportional to the flux values of timolol from skin to blood, it would be difficult to give a sufficient amount of timolol to expect systemic effect(s) with no concurrent erythema because erythema develops unless drug flux is unacceptably low.
Keywords timolol transdermal skin
,-adrenoceptor blocker
percutaneous absorption
Introduction
al., 1988). Though this local side effect is reported to be mild or minimal for some P-adrenoceptor blockers including timolol (Cargill et al., 1986; Vlasses et al., 1985), the skin irritation probably hampers a wide clinical usage of transdermal application of ,-adrenoceptor blockers. Because timolol does cause irritation only when the free base form is applied as a nearly saturated solution in rat, more detailed studies are necessary to evaluate further if the irritation can be limited by the use of a timolol formulation at a low thermodynamic activity (Cargill et al., 1986). In this article, the relationship was studied between the magnitude of skin irritation caused by topically applied timolol and drug concentration in the blood which has just perfused the skin where timolol
Several ,-adrenoceptor blockers are known to penetrate human skin and show systemic ,B-adrenoceptor blocking effects (Spieker et al., 1989; Vlasses et al., 1985; Wellstein et al., 1986). Many 3-adrenoceptor blockers undergo extensive first-pass metabolism in the liver, resulting in wide individual variations in plasma levels when given orally (Riddell et al., 1987). Thus, the transdermal route for administration of a ,B-adrenoceptor blocker is certainly one worth investigating. However, a transdermal route for the administration of 1B-adrenoceptor blockers has not been widely accepted in clinical practice. Transdermal application of ,-adrenoceptor blockers is reported to cause local irritation in human beings and animals (de Mey et al., 1989a,b; Spieker et
Correspondence and present address: Dr Kiyoshi Kubota, Department of Dermatology, University of California, San Francisco, School of Medicine, Box 0989, Surge 110, San Francisco, CA, 94143-0989, USA
471
472
K. Kubota, E. Koyama & K. Yasuda
was applied. This is because when this relationship is scrutinized, it may indicate if it is possible to reduce the skin irritation while a sufficient amount of timolol to produce systemic effect(s) is absorbed.
Methods
Subjects and drug The study held in the Tokyo Research Center of Clinical Pharmacology (Takata-no-baba, Tokyo, Japan), where phase I studies of various newly developed drugs are mainly conducted, was approved by the Ethics Committee of Tokyo (Chairman, Professor C. Ibukiyama, Tokyo College of Medicine). Six volunteers (subjects 1 to 6, aged 36 to 51 years with a mean of 46.5 years and weighing 49.5 to 66.9 kg with a mean of 59.0 kg) agreed to participate in the study after receiving full explanation and giving written consent. They were judged to be in good health on the basis of history, physical examination, routine laboratory data (complete haemogram, urinalysis, serum creatinine, transaminases, alkaline phosphatase, total bilirubin, total cholesterol, blood glucose, total serum protein, electrolyte) and electrocardiogram. No subject had a history of asthma. Each of four therapeutic timolol patches (2.5 x 6 cm2) containing 20% (w/v) timolol free base in acrylic co-polymer, 40 ,um in thickness (Kubota et al., 1990), was applied on the same site on the inner surface of the left forearm near the wrist. Each timolol patch contained 12 mg of timolol free base. Four placebo patches containing no timolol were also applied consecutively on the same site of the right forearm symmetrically to the timolol patch. An area, 4 cm proximal to the placebo patch, was used as a control. Each patch was applied for 2.5 h, then removed and erythema developed beneath the patch was evaluated. Next timolol and placebo patches were applied 0.5 h after the removal of the previous timolol and placebo patches, respectively. Evaluation of effects The study was conducted on the same day for all six volunteers in the room where the temperature and relative humidity were conditioned at 26 ± 1° C and 71 ± 2%, respectively. The evaluation of magnitude of erythema and measurements of blood pressure and heart rate were executed just before the application of active and placebo patches (at 0 h), after the removal of each patch (at 2.5, 5.5, 8.5 and 11.5 h) as well as at 14, 26 and 29 h. Blood pressure and heart rate were measured in duplicate after subjects had been supine for 5 min and after standing for 2 min with an automatic sphygmomanometer (Japan Colin BP 203-N, Komaki City, Japan). Erythema was evaluated by a visual score, laser doppler flowmeter and reflectance spectrophotometry at least 10 min after the removal of the patch. Erythema was graded by a dermatologist according to a visual score modified from that by Frosch & Kligman (1979) as: (0) no erythema; (0.5) slight spotty erythema; (1) weak diffuse erythema; (2) moderate, uniform erythema and (3) intense redness. A He-Ne laser doppler flowmeter (ALF 2100, Advance,
Tokyo, Japan) was employed to measure blood volume (mass) per unit volume of tissue expressed by an arbitrary unit as well as blood flow rate expressed by a unit, ml min-1 100 g-1 (Cao et al., 1989; Yamaguchi, 1989). The blood volume and blood flow were measured in triplicate for each of the active patches, placebo patches and control sites. An index of haemoglobin concentration (IHB) expressed by an arbitrary unit was recorded in duplicate for each of the three sites by a reflectance spectrophotometry (Tissue Spectrum Analyzer, TS-200, Sumitomo electric industries, Osaka, Japan). An IHB defined as the difference of absorbances between 569 nm (green light) and 650 nm (red light) (Leung et al., 1987) was employed to evaluate the extent of erythema, because an 'erythema index' (Diffey et al., 1984; Feather et al., 1982) has been defined as the difference between the absorbances at wave lengths corresponding to green and red lights. Sample collection Blood samples (10 ml) were collected from the left antecubital vein exactly 30 s after the application of a rubber tourniquet at 0 (pre), 3, 6, 9, 12, 14, 26 and 29 h. Venous blood samples (10 ml) were also collected from the right arm at 0 (pre), 12, 14, 26 and 29 h. Plasma was separated immediately and stored at -20° C until
analyzed. Drug analyses Plasma timolol concentrations were measured using the h.p.l.c. method by a Hitachi L-6000 pump (Tokyo, Japan) equipped with a Hitachi L-4000 u.v. detector. To 1-2 ml plasma, 100 ,u 4 M sodium hydroxide, 10 ,ug metoprolol-(+) tartrate, as internal standard, and 5 ml dichloromethane were added and vortexed for 180 s. After centrifugation for 5 min at 1500 g, the organic phase was evaporated to dryness. The residue was reconstituted with 100 ,ul of mobile phase and 50 to 75 RI of the solution was injected onto the chromatograph. The h.p.l.c. column, mobile phase and other chromatographic conditions were the same as previously described (Kubota et al., 1990). The absolute recoveries of timolol and internal standard in plasma were more than 97%. The detection limit was 0.3 ng ml-' when 2 ml of plasma sample was used and a 75 pI aliquot was injected onto the chromatograph. At 5 ng ml-' of plasma timolol concentration, the coefficient of variation (c.v.) in the within-day study was 1.7% (n = 9), while that in the dayto-day study was 7.5% (n = 7).
Statistical analysis All data were expressed as mean ± s.e. mean. Statistical significances were calculated by one-way analysis of variance (ANOVA) for blood pressure and heart rate measured at t = 0 to 29 h. The difference in the time courses among the three skin sites where active, placebo and no patches were applied was tested by two-way ANOVA for each of the changes in blood volume (mass), blood flow and IHB from the respective values at t = 0, which are defined as AMass, AFlow and AIHB, respectively. That for visual score was also tested by two-way
473
Skin irritation induced by topically applied timolol ANOVA. When there was a statistically significant difference in the time courses among three skin sites for either of AMass, AFlow, AIHB and visual score, the analysis of significance of differences of the time courses between two skin sites (i.e. timolol vs control sites, timolol vs placebo sites, and placebo vs control sites) were conducted by the Tukey Tmethod. The correlation between the plasma concentration collected from the left antecubital vein vs either of AMass, AFlow, AIHB and visual score was tested using Spearman's rank correlation coefficient, rs, as well as the simple linear correlation coefficient, r. P < 0.05 was considered statistically significant.
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Figure 2 Time courses of the mean ( s.e. mean, n 6) change in blood volume (mass) per unit volume of tissue (arbitrary unit) from that at t = 0, AMass, measured at the sites where the active (0), placebo (@) and no (C) patches were applied (a), change in blood flow, AFlow (b), change in the index of haemoglobin concentration (arbitrary unit), AIHB (c), and visual score (d).
The resting supine and upright blood pressure and heart rate did not change significantly throughout the study (ANOVA). No erythema developed in subjects 1 and 2 by a visual score. In subjects 3 to 6, erythema graded as weak (1) eventually developed on the left forearm where the active patch containing timolol was applied. In subjects 3 and 4, erythema assessed by a visual score where active, placebo and no patches were applied was developed also on the right forearm where the placebo statistically significant for all of AMass (P < 0.05), patch was applied. However, in subjects 5 and 6, no AFlow (P < 0.05), AIHB (P < 0.01) and visual score erythema was visually recognized except for the active (P < 0.01) (ANOVA). Using the Tukey T method, for patch site. No erythema was graded as moderate (2) or AMass, the difference between timolol and control sites (P < 0.01) and that between placebo and control sites more. Erythema was confined to the area where the active or placebo patch was applied and not accompanied (P < 0.01) were significant. For AFlow, that between by oedema or any other effect. No symptoms developed placebo and control sites (P < 0.05) was significant. For AIHB and visual score, that between timolol and placebo in any subject. After the removal of the patches, erythema eventually disappeared in 48 h without sites (P < 0.01) and that between timolol and control sites (P < 0.01) were significant. There was a significant sequelae. correlation between the plasma concentration collected timolol s.e. mean) plasma In Figure 1, the mean (± concentrations collected from the left and right arms are from the left antecubital vein and one of AMass, AFlow, AIHB and visual score (r, = 0.670, 0.760, 0.549 and shown. The mean plasma timolol concentrations from the left antecubital vein were 2.4 to 10.7 times higher 0.708, respectively; P < 0.001; r = 0.574, 0.708, 0.485 and 0.642, respectively; P < 0.01; Figure 3). The median than those from the right arm and had a great interindividual variation (coefficient of variation, c.v. 160%). The time courses of the mean (± s.e. mean)avf ;,,., 4^. ;b .4A t. .~ c fi values for AMass, AFlow, AIHB and visual score are shown in Figure 2, where those for the active patch site were greater than the placebo and control sites. The difference of those values among the three skin sites 0
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Time (h) Figure 1 Time courses of the mean (± s.e. mean, n = 6) plasma timolol concentration collected from the antecubital vein of the left arm (0) where the timolol patch was applied and the right arm (OJ) where the placebo patch was applied.
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Figure 3 Relation between the plasma timolol concentration collected from the left arm where the active patch containing timolol free base was applied and one of (a) change in blood volume (mass) per unit volume of tissue (arbitrary units) from that at t = O, i\Mass, (b) change in blood flow AvFlow, (c) change in index of haemoglobin concentration (arbitrary units), AIHB and (d) visual score. Symbols, 0, A, *, A, O and-l represent subjects 1 to 6, respectively.
474
K. Kubota, E. Koyama & K. Yasuda
plasma timolol concentration corresponding to erythema graded as slightly spotty (0.5) was 3.0 ng ml-', whereas that graded as weak diffuse (1.0) was 12.4 ng ml-' (Figure 3d). The correlation between visual score vs one of AMass, AFlow and AIHB was also significant (rs = 0.731, 0.772 and 0.731, respectively; P < 0.001; r = 0.692, 0.674 and 0.704, respectively; P < 0.001).
Discussion Blood flow measured by a laser doppler flowmeter (Nilsson et al., 1982) and 'erythema index' by a reflectance spectrophotometry (Diffey et al., 1984; Feather et al., 1982) have been considered to be the quantitative expres sions of the extent of erythema. Because the visual score was significantly correlated with AMass, AFlow and AIHB, those values could also be regarded as the quantitative expression of the intensity of erythema in this study. The logarithm of plasma concentration collected from the left antecubital vein and those changes seem to have the linear correlation in Figure 3. In other words, in Figure 3, there seems to be no specific 'threshold' plasma concentration which is critical for the development of erythema. It is of note that all the timolol concentrations in plasma drawn from the left antecubital vein of subjects 1 and 2 designated by open circles and triangles, respectively in Figure 3, were less than 5 ng ml-'. Thus, the reason why no erythema developed in these two subjects is explained merely by the fact that the plasma concentra tions from the left antecubital vein were very low in these subjects, rather than the fact that those concentrations did not reach some critical 'threshold' value, neither had they low reactivity to topically applied timolol. The great interindividual variation of timolol permeation was also noted in the previous in vivo study where 30 to 60 mg of timolol free base was given to six volunteers (Vlasses et al., 1985). According to Cargill et al. (1986), the timolol-induced erythema may be caused by the alkaline nature of timolol free base, its ,B-adrenoceptor blocking effect, or both. Though the data shown in Figure 3 do not offer any direct evidence, we may suggest that the former is a likely mechanism. This is because the plasma timolol concentration collected from the left arm in Figure 3 could be reflected by the extent of the derangement from the physiological pH in the local tissue. On the other hand, most 3-adrenoceptor blockers cause vasoconstric tion and blanching of skin rather than erythema (Hornqvist et al., 1984), even though a 3-adrenoceptor blocker is able to augment skin reactivity where the
chemical mediator(s) are released from dermal mast cells by the challenge of antigen (Shereff et al., 1973) or when histamine is given exogenously (Lamkin et al., 1976). It should be noted that erythema observed in this study occurs in a modest range of dose absorbed. The peak plasma concentrations in the systemic circulation (collected from the right arm) were lower than the detection limit of 6.9 ng ml-1 in six subjects in this study where 15 cm2 of skin was used. On the other hand, those were approximately 10 to 50 ng ml-' in the previous study (Vlasses et al., 1985) where 25 cm2 of skin was used. When the plasma concentrations from the left antecubital vein are considered to be roughly proportional to the drug flux from skin to local circulation, the results shown in Figure 3 indicate that timolol flux should be much smaller than its possible maximum (unit activity) flux (Flynn & Stewart, 1988; Kubota & Yamada, 1990) to inhibit the development of erythema completely. In this laboratory, in an in vitro study using excised human skin, this maximum flux or that from the saturated timolol aqueous solution was measured to be near 30 ,ug cm-2 h-1 (unpublished data) and that from acrylic copolymer was calculated to be near 45 ,ug cm-2 h-1 (Kubota & Yamada, 1990). If a daily dose of 10 mg timolol, the minimum oral daily dose (Frishman, 1982), is to be absorbed through skin using a flux, 4 ,ug cm-2 h-1, which is approximately one tenth of the possible attainable maximum flux, timolol should be applied over an area of 100 cm2. Because erythema developed in a wide range of the timolol concentrations collected from the left arm in this study (Figure 3), erythema at least graded as slight (0.5) will likely develop at the flux, 4 ,ug cm-2 h-1. On the other hand, the area, 100 cm2, may be considered near or more than the maximum acceptable area. Thus, it seems difficult to provide sufficient amounts of timolol to deliver the desired systemic effect(s) with no concurrent erythema. However, more extensive studies where timolol is applied on various sites other than the forearm (e.g. the abdomen, chest and back) are necessary to draw the final conclusion whether a ,-adrenoceptor blocker can be topically administered without skin irritation because it is well appreciated that there is regional variation in certain skin reactions including allergic and irritant ones (Maibach & Prystowsky, 1977; Lahti & Maibach, 1984; Van der Valk & Maibach, 1989). Similarly, more studies are necessary to know whether erythema graded as slight to weak in this study and graded as mild or minimal in the previous studies (Cargill etal., 1986; Vlasses et al., 1985) should exclude transdermal application as an alternate route of administration of ,B-adrenoceptor blockers.
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Maibach, H. I. & Prystowsky, S. D. (1977). Glutaraldehyde (pentanedial) allergic contact dermatitis. Arch. Dermatol., 113, 170-171. Nilsson, G. E., Otto, U. & Wahlberg, J. E. (1982). Assessment of skin irritancy in man by laser doppler flowmetry. Contact Dermatitis, 8, 401406. Riddell, J. G., Harron, D. W. G. & Shanks, R. G. (1987). Clinical pharmacokinetics of P-adrenoceptor antagonists: an update. Clin. Pharmacokin., 12, 305-320. Shereff, R. H., Harwell, W., Lieberman, P., Rosenberg, E. W. & Robinson, H. (1973). Effect of beta adrenergic stimulation and blockade on immediate hypersensitivity skin test reactions. J. Allergy clin. Immunol., 52, 328-333. Spieker, C., Vetter, H., Liedtke, R., Zidek, W. & Vetter, W. (1988). Transdermal ,B-blocker therapy in essential hypertension. Am. J. Hypertension, 1, 199S-200S. Spieker, C., Vetter, W., Zidek, W. & Vetter, H. (1989). Evaluation of the therapeutic effect of transdermal 1blocker therapy in patients with essential hypertension. Arzneim. Forsch., 39, 1512-1514. Van der Valk, P. G. M. & Maibach, H. I. (1989). Potential for irritation increased from the wrist to the cubital fossa. Br. J. Dermatol., 121, 709-712. Vlasses, P. H., Ribeiro, L. G. T., Rotmensch, H. H., Bondi, J. V., Loper, A. E., Hichens, M., Dunlay, M. C. & Ferguson, R. K. (1985). Initial evaluation of transdermal timolol: serum concentration and 13-blockade. J. cardiovasc. Pharmac., 7, 245-250. Wellstein, A., Kuppers, H., Pitschner, H. F. & Palm, D. (1986). Transdermal delivery of bupranolol: pharmacodynamics and beta-adrenoceptor occupancy. Eur. J. clin. Pharmac., 31, 419422. Yamaguchi, T. (1989). Continuous monitoring of gastroduodenal mucosal hemodynamics in rats by laser-doppler flowmetry and reflectance spectrophotometry. Gastro-
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(Received 28 June 1990, accepted 30 October 1990)
