Bronchodilator and antiallergic effects of thiazinamium chloride in guinea pigs, rats, cats and dogs

European Journal of Pharmacology, 80 (1982) 171-184

171

Elsevier Biomedical Press

BRONCHODILATOR AND ANTIALLERGIC EFFECTS OF THIAZINAMIUM CHLORIDE IN GUINEA PIGS, RATS, CATS AND DOGS * ALAN J. LEWIS, ALPHONSE DERVINIS and MARVIN E. ROSENTHALE **

Wyeth Laboratories Inc., P.O. Box 8299, Philadelphia, PA 19101, U.S.A. Received 7 August 1981, revised MS received 15 December 1981, accepted 1 February 1982

A.J. LEWIS, A. DERVINIS and M.E. ROSENTHALE, Bronchodilator and antiallergic effects of thiazinamium chloride in guinea pigs, rats, cats and dogs, European J. Pharmacol. 80 (1982) 171-184. This study characterized the in vivo pulmonary pharmacology of thiazinamium chloride administered largely by the aerosol route t in different animal species. The compound has greater anticholinergic but weaker antihistaminic activity than promethazine, the parent compound. It was less potent than atropine or ipratropium as an anticholinergic and had a shorter duration of action, but unlike these compounds it had long-lasting antihistaminic activity. It is effective in both IgG- and IgE-induced models of passive lung anaphylaxis in guinea pigs and rats, respectively. In Ascaris-induced allergic asthma in the conscious dog it produced a dose-related inhibition of the antigen-induced bronchospasm. No major side effects were observed in acute oral and inhalation toxicity studies in guinea pigs or rhesus monkeys. The results demonstrate that thiazinamium chloride is a safe, potent and efficacious bronchodilator after aerosol administration, with a rapid onset and moderate duration of action in animal models. Thiazinamium

Bronchodilator

Antiallergic

Antihistaminic

1. Introduction

Secretion of histamine and other mediators has been associated with extrinsic asthma, while vagally mediated reflex bronchoconstriction is likely to be predominant in intrinsic asthma (Lichtenstein, 1973; Gold, 1973; Widdicombe, 1975). Consequently, both antihistamines and anticholinerglcs ought to possess therapeutic activity in the treatment of asthma. Several Hi-receptor-blocking antihistamines, including promethazine and pyrilamine (Herxheimer, 1949), chlorpheniramine (Popa, 1977, 1980; Woenne et al., 1978) and clemastine (Nogrady et al., 1978), have been shown to cause bronchodilatation in asthmatics. However, anti-

* A preliminary report of some of these results was presented at the First International Conference on Immunopharmacology in July, 1980 (Int. J. Immunopharmacol. 2, 266,

1980). ** Present address: Ortho Pharmaceutical Corporation, Raritan, New Jersey 08869, U.S.A. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

Anticholinergic

histamines in general have not proved to be of great benefit in asthma after either oral or aerosol administration (Pearlman, 1976), suggesting that mediators other than histamine play a major role in the disease. This lack of success with antihistamines may also be a result of inadequate dose and consequently of insufficient drug at the site of histamine release (Popa, 1980). Furthermore, impaired central nervous function (Douglas, 1975) a n d / o r local irritant side effects of this class of compounds (Hawkins, 1955) have been implicated in their failure. Obstructive airways diseases have been successfully treated with anticholinergics such as atropine or ipratropium bromide by both aerosol and intravenous (i.v.) administration (Gandevia, 1975; Ulmer et al., 1975; Simonsson, 1977). However, the clinical use of such drugs has not been extensive, perhaps in view of their possibly hazardous side effects. Most antihistamines have some anticholinergic activity (Douglas, 1975). Thiazinamium methyl sulfate (Multergan®), a quaternary salt of pro-

172

methazine, has been shown to possess both potent antihistaminic and anticholinergic activity in animals (Ducrot and Decourt, 1950a,b), and moreover to be effective in obstructive airways diseases after intramuscular administration (Buuy-Noord et al., 1957; Simonsson, 1964; Van Bork et al., 1977). We have investigated the pulmonary pharmacology of the chloride salt of thiazinamium, primarily using the aerosol route, in attempts to improve bronchodilator efficacy and circumvent the systemic side effects of the parenterally administered compound. Furthermore, we have compared this profile with that obtained with promethazine and reference bronchodilator/antiallergic agents sfich as ipratropium and disodium cromoglycate (DSCG).

solution. Drugs (in 1 mi saline) were also administered via an indwelling catheter into the descending duodenum; stomach contents were kept from emptying into the abdomen by pyloric ligation. Activity was assessed 30 min after drug administration and thereafter at 30 min intervals for up to 2h. The maximal inhibition of bronchoconstriction was used for the assessment of drug effects calculated as follows: [% bronchoconstriction (pre-drug) - % bronchoconstriction (post-drug) ] / [% bronchoconstriction (pre-drug)] ×100

where % bronchoconstriction refers to % reduction in respitatory volume attributed to agonist.

2.2. Passive lung anaphylaxis (PLA) in the guinea pig 2. Materials and methods

2.1. Bronchoconstrictor agonists in the guinea pig Male Hartley guinea pigs (400-600g) were anesthetized with sodium pentobarbital (50 m g / k g i.p.). The jugular vein was cannulated for administration of bronchoconstrictor agonists and the trachea for artificial ventilation. Respiration was fully arrested with an additional 10 m g / k g i.v. of sodium pentobarbital and thereafter was maintained in a partially closed system of 64 strokes/rain by a miniature Starling pump. Pump stroke volume ranged from 6-8 ml. Lung volume changes were measured as overflow from the lung by a Konzett and R/Sssler (1940) apparatus as modified by Rosenthale and Dervinis (1968). Transient bronchoconstrictor responses were induced by i.v. administration of either acetylcholine (3.0-12.0 ~g/kg), histamine (1.0-8.0 # g / k g ) or 5-hydroxytryptamine (1.0-6.0 ffg/kg). In each case, alteration of the dose of agonist was made until bronchoconstrictor responses were constant and approximately at 50% of total overflow volume obtained by tracheal occlusion. Drugs were administered by aerosol using a Monaghan ultrasonic nebulizer. The duration of exposure and volume of the aerosol administered were kept constant at 10 inspiratory strokes of the pump. The aerosol dosage was varied by adjusting the concentration of the

Male Hartley guinea pigs (350-400g) were sensitized by the i.p. injection of 0.5 ml of a 0.9% saline solution containing 10 fig ovalbumin (Grade V, Sigma Co.), 20/~g lipopolysaccharide B (E. co# 055:B5, Difco Labs) and 0.485 ml Bordetella pertussis (20 O U / m l , Connaught Labs) on day 0. On day 14 the animals were given a booster dose (0.5 ml i.p.) of the same mixture. On day 21 they were killed and exsanguinated, and the serum was collected after leaving the blood overnight at - 4 ° C . Groups of 60-120 guinea pigs were used to provide a large bulk of antiserum. Serum was titered using a 4 h passive cutaneous anaphylaxis (PCA) to determine the dilution of serum that produced a wheal of 100 mm. The titer of the antiserum used was 1 : 506. Male Hartley guinea pigs (400-600 g) were passively sensitized by i.v. injection of 0.25 ml of antiserum into the cephalic vein. Two hours after sensitization, the animals were anesthetized with pentobarbital sodium (50 m g / k g i.p.). The jugular vein was cannulated for injection of drugs and antigen. Resistance to lung inflation was measured by the overflow technique as described in the preceding section. The respiratory volume of each guinea pig was determined by clamping off the trachea at the end of each experiment. Drugs or drug vehicle were administered i.v. or by aerosol (see preceding section for details) at different time

173

intervals prior to i.v. ovalbumin challenge (10 m g / k g dissolved in 0.9% saline). The inhibitory effect of drugs was determined at 1, 3 and 5 min after antigen challenge using the formula: [% bronchoconstr, in control group - % bronchoconstr, in test group] / [% bronchoconstr, in control group] × 100

These time intervals were chosen to reflect the pattern of bronchoconstriction and invariably included the maximal response. 2.3. Passive lung anaphylaxis in the rat

Male Sprague-Dawley~ rats (230-280g) were passively sensitized by i.v. injection of 0.15-0.6 ml of undiluted rat serum containing homocytotropic antibody to ovalbumin (prepared according to the method of Carlson et al., 1982). These amounts of serum produced a bronchoconstriction which was 50-70% of maximum. Twenty-four hours after sensitization, rats were anesthetized with 50 m g / k g sodium pentobarbital (i.p.) and spontaneous respiration was arrested with an i.v. infusion of 0.1 m l / m i n of a 3 m g / m l solution of succinylcholine chloride. The femoral vein was cannulated for i.v. injection of drugs and the jugular vein was cannulated for injection of antigen. Airways resistance was measured by the overflow technique described in a preceding section using the guinea pig. The respiratory volume of each rat was determined by clamping off the trachea at the end of each experiment. Drugs or drug vehicle were administered i.v. or by aerosol (as described for the guinea pig) at different times prior to ovalbumin challenge (25 m g / k g dissolved in 0.9% saline). The percentage bronchoconstriction was calculated by measuring the lung overflow at the time of maximal constriction and expressing it as a percentage of the respiratory volume. The inhibitory effect of drugs was determined using the formula described for the guinea pig PLA. 2.4. Cholinergic-induced tone in cats

The method used to produce bronchoconstriction in cats was similar to that described previously (Rosenthale et al., 1971). Cats of either sex,

weighing 2-5 kg, were anesthetized with i.p. pentobarbital-urethane (35 m g / k g pentobarbital, 240 m g / k g urethane). Respiration was arrested by the administration of an i.v. infusion of 1 m g / m l per min of succinylcholine chloride into the femoral vein. The animals were artificially respired with a Harvard pump (50-75 ml/breath, 28 breaths/ min). A solution of neostigmine methyl sulfate (0.1 mg/ml) was infused i.v. into the jugular vein at a rate of 1 m l / m i n until maximal or complete bronchoconstriction occurred. This initial severe bronchoconstriction was of brief duration and was followed by a period of partial recovery characterized by a period of stable bronchoconstriction. Excess mucus was aspirated from the trachea and lungs were overinflated to hasten stabilization; this procedure was also performed prior to the administration of drugs. The respiratory flow (V) was measured with a Fleisch pneumotachograph (type 00) and a Statham differential pressure transducer (PM ± 0.15-350 mm Hg). Transpulmonary pressure (Ptp) was measured with a Statham differential pressure transducer (PM 5 ± 0.7-350 mm Hg) bridged between an endotracheal tube and a Malecot catheter (size 12) placed in the pleural space. These measurements were monitored by an on-line analog pulmonary mechanics computer (Buxco Electronics, Inc.) which integrated V to obtain tidal volume (Vt). The computer determined dynamic lung compliance (Cdyn ) by dividing Vt by the change in Ptp and total lung resistance (R L) by dividing pressure differences at isovolumetric points by the changes in flow over the same time periods. The mean of five consecutive breaths was recorded and the output was displayed on a Beckman physiograph. Femoral arterial blood pressure and heart rate were also monitored. Drugs were administered as aerosols by means of a Monaghan ultrasonic nebulizer through a shunt in the afferent limb of the respirator, arranged so that inspired air passed through the nebulizer chamber before entering the animal's lungs. The duration and volume of the aerosol were kept constant at five inspiratory strokes of the pump. The dose was varied by altering the concentration of the solution. Bronchodilat0r activity was measured by the ability of the drug to

174 reverse the increased R r and decreased Cdyn characteristic of cholinergic stimulation induced by neostigmine. Baseline values of R L and Cdyn during succinylcholine infusion were first subtracted from those obtained during neostigmine-induced contraction and these differences were used to calculate percent changes of drug effect. The formula used to calculate the drug-induced changes was the same as that used for the guinea pig studies. 2.5. Ascaris-induced allergic asthma in the conscious dog A colony of female dogs of' mixed breed was used for these studies. Selection of dogs and the detailed experimental pro.cedure involved in measuring pulmonary mechanics have previously been described at length (Lewis et al., 1981). Briefly, '¢ and Pro were monitored in conscious dogs lying in the right lateral decubitus position and intubated with an endotracheal tube and esophageal balloon. ~' and Ptp signals were amplified and fed into an on-line analog pulmonary mechanics computer, as described for the studies in the cat, so that the mean R L and Cdyn were recorded for five consecutive breaths. Aerosols were generated by a Mark VII Bird respirator with an in-line nebulizer. Pressure, flow rate and sensitivity settings of the respirator were adjusted such that a maximal pleural pressure of 15 m m Hg was attained during delivery. The estimate of the total dose administered was made by weighing the nebulizer before and after aerosol dosing. Doses were altered by varying the concentration of the solution in the nebulizer. After a 3-min control period, each dog was exposed to five inhalations of a 0.05% histamine solution in physiological saline to establish histamine sensitivity and confirm that the system was functioning normally. When all parameters had returned to baseline values (approximately 15-25 min), drugs or saline vehicle were administered by aerosol 3 rain before the standard Ascaris antigen (SAA) was administered. Aerosol delivery of saline, drug and SAA was kept to 7 inhalations. Each dog was challenged with a different concentration of SAA which was predetermined by the respira-

tory sensitivity of each animal; individual concentrations varied from 1 : 100 to 1 : 160000. At least 3 weeks were allowed before rechallenge with antigen, and those animals that had been drugtreated were next subjected to saline vehicle. This allowed evaluation of any residual drug-induced changes in sensitivity to SAA. Initially, drug effects were calculated at 2-min intervals before and after the maximum changes in Cdyn and R L from control responses obtained just before SAA administration. It became apparent that the recording of maximum change in Cdyn and R L alone was indicative of any drug-induced effect, and consequently this procedure was routinely adopted. For each dog the mean of 2-3 responses produced after SAA alone was taken as the control, and the results from a single response to a dose of drug was used as the test value. The drug-induced change was calculated as a percentage of control using the formula: ( c o n t r o l - t e s t / c o n t r o l ) × 100. The mean of at least four values are reported for each dose. 2. 6. Guinea pig trachea in vitro Tracheal spirals (Constantine, 1965) from male Hartley guinea pigs (350-400 g) were equilibrated under an initial tension of 4 g in Krebs-Henseleit solution at 37°C (gassed with 5% CO 2 in 02). After the 1.5-h equilibration period, the resting tension was adjusted to 2 g. Tension was measured isometrically with Statham strain gauges (Model UC2) and was displayed on a Beckman RM dynograph. Initial studies were performed to examine the effect of drugs on the basal tone of the preparation using cumulative addition of the drug. The dose producing a significant and consistent (n = 7) relaxation over an untreated tracheal strip (usually 10% of resting tension) was recorded. Preparations were contracted to 80-90% of maximum tension with histamine (10 5 to 5 × 10 4M) or carbachol (5 × 10-7 to 10-6M). Log concentration/effect curves for relaxant drugs were obtained using the cumulative method described by Douglas et al. (1977) in which the concentration of a relaxant agonist in the bath was increased every 5 min without washing. This time interval was sufficient for each relaxation to reach a

175

plateau. The relaxation produced by drugs was expressed as a percentage of the tension developed with either histamine or carbachol. ECs0 values for relaxant drugs were determined from log concentration effect curves.

2.7. Drugs Thiazinamium chloride and promethazine hydrochloride were prepared at Wyeth Laboratories Inc. Acetylcholine chloride, histamine acid phosphate, carbachol, atropine sulfate, dl-isoproterenol hydrochloride and theophylline (all Sigma), chlorpheniramine maleate (Mann), and methysergide maleate (Sandoz) were obtained from the sources in parentheses. DSCG was a gift from Fisons Corporation and ipratropium bromide was a gift from C.H. Boehringer. All following data on drugs refer to the base salts.

2.8. Statistics Data are expressed as the means ± S.E. Statistical differences were calculated with the" Student's t-test. Regression line analysis (Finney, 1978) was utilized for the construction of all dose-response lines unless otherwise stated.

3. Results

3.1. Bronchoconstrictor agonists in the guinea pig Thiazinamium administered via the aerosol route possessed potent anticholinergic activity against acetylcholine-induced bronchoconstriction in the guinea pig (fig. 1). It was more potent than promethazine, but because the regression lines for thiazinamium (y = 33.82x + 44.34, r = 0.87) and promethazine (y = 25.0x + 13.21, r = 0.85) were not parallel, the potency was not constant throughout the dose range. Thiazinamium was 11 times less potent than atropine and 3 times less potent than ipratropium. In addition, the duration of effect for thiazinamium was greater than that for promethazine, but was less than that for either atropine or ipratropium (table 1). Aerosolized thiazinamium effectively inhibited histamine-induced bronchoconstriction; although it was less effective at low doses than promethazine (fig. IB), it possessed a similar duration of effect (table 1). Once again the regression line for thiazinamium (y = 30.09x + 10.66, r = 0.83) was not parallel with that constructed for promethazine (y = 16.75x + 67.91, r = 0.49). Atropine also possessed antihistaminic activity, but its effect was

TABLE 1 Duration of action of drugs administered via the aerosol route against acetylcholine- and histamine-induced bronchoconstriction in the guinea pig. Treatment

Duration of effect (min) a Acetylcholine

Thiazinamium Promethazine Atropine Ipratropium a b c d

Histamine

1.5 p,gb

15 p,gb

1.5 p,gb

15 p,gb

10.3 -*-2.0 5.3 -'- 2.0 >60 > 60

> 60 17.2 -+ 7.5 NT c NT

16.0 -+ 3.5 15.5 -+ 3.9 NA d NA

> 60 > 60 2.7 --+ 1.7 NA

Mean time (--+S.E.) for the bronchoconstriction to return to 75% of its control predrug response. Dose of antagonist. Not tested. Not active (i.e., less than 25% inhibition of control response).

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of short duration and it was 21 times less potent than thiazinamium. Ipratropium was virtually inactive in this model. Thiazinamium also exhibited inhibitory activity against serotonin (EDs0, 1.8 ~tg), and the duration of this effect (e.g., 4 6 . 2 +- 8.0 min at the 15 /zg dose) was slightly greater than that observed for the antihistaminic study. Administered intraduodenally promethazine was considerably less potent (EDso , 5.5 mg/kg) against acetylcholine-induced bronchoconstriction than it was against histamine (EDso, < 1 mg/kg) whereas thiazinamium was only slightly less effec-

tive against acetylcholine (EDso, 12.3 mg/kg) than against histamine (EDs0, 5.5 mg/kg). Ipratropium was inactive against histamine, but was the most potent agent tested against acetylcholine (EDs0, 2.2 mg/kg). 3.2. Guinea pig PLA

The bronchoconstriction produced in this model reaches a maximum within 30-45 s after antigen challenge. I.v. administration of thiazinamium and promethazine (both 10 min before antigen challenge) effectively suppressed the response at 1 min,

177

100

inhibited this response (fig. 2). Aerosolized thiazinamium (1 min before antigen) effectively inhibited bronchospasm in a doserelated fashion, and the inhibition was again most effective at 1 min (fig. 3). Isoproterenol but not promethazine, ipratropium, nor DSCG (all 1 min before antigen)' significantly affected antigen-induced bronchospasm.

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Thiazinamium inhibited the bronchoconstriction when administered by aerosol either at 1 rain (EDs0 70 /~g) or 10 min (EDs0 80 ttg) before antigen (fig. 4). Promethazine was equally effective using the 1-min aerosol pretreatment (EDs0 52 ~g), but was less effective at 10 min pretreatment (EDs0 > 150 /~g). Ipratropium was much more potent administered 10 min before antigen (EDs0 22/~g) than at 1 min. Conversely, DSCG was more potent at 1 min (EDs0 150 t~g) than at 10 min using the aerosol route although this was not statistically significant. I.v. thiazinamium (1 mg/kg) significantly inhibited the PLA response when administered at 1 (78%) to 10 min (31%) prior to antigen challenge. DSCG and atropine (both 1 mg/kg) significantly inhibited the response (75% and 52%, respectively) only when administered 1 min prior to antigen,

5

Fig. 2. Time course of the effectsof i.v. administration of drugs in the guinea pig PLA. Thiazinamium,promethazine,atropine and methysergidewere administered 10 min prior to antigen challenge, isoproterenol 3 min prior to challenge, and DSCG 1 min prior to challenge. Each point represents the mean of 5 animals. • Thiazinamium 1.0 mg/kg; O promethazine 1.0 mg/kg; • isoproterenol0.01 mg/kg; A atropine 1.0 mg/kg; × DSCG 10.0mg/kg; • methysergide10.0mg/kg. * P<0.05.

but their effectiveness (particularly that of promethazine) diminished thereafter (fig. 2). Isoproterenol (3 min before antigen) inhibited bronchospasm between 1 and 5 min, but neither DSCG (1 min prior to antigen), atropine, nor methysergide (both 10 min before antigen) significantly

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Fig. 4. Effect of aerosolized drugs administered at different times prior to antigen challenge in the rat PLA. Each point is the mean ±S.E. of 5 - 1 0 animals and represents the maximal inhibition of bronchoconstriction compared to vehicle-treated controls. • Thiazinamium; 0 promethazine; • ipratropium; A DSCG. * P<0.05.

whereas ipratropium (1 m g / k g ) was equiactive at 1 (43%) and 10 min (40%).

Atropine (EDso 0.15/~g) and ipratropium (EDs0 0.5 fig) also reduced R E and were both more potent than thiazinamium. Ipratropium possessed a much greater duration of action than either thiazinamium or atropine (fig. 5). None of the drugs produced major changes in mean arterial blood pressure or heart rate at any of the doses tested over the 60-120 rain period of assessment.

3.4. Cholinergic tone in cats Thiazinamium reduced the cholinergic-induced bronchoconstriction in the cat, as measured by a reduction in R L (EDs0 12 ~g; fig. 5). This effect was maximal within 5 min of drug administration. The duration of this effect increased with the dose, as seen from the time taken for R L to return to 20% of the constricted state shown in parentheses in fig. 5. C,~y,, reduced during bronchoconstriction, was elevated by thiazinamium, but this was only significant at 100 fig. d

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3.5. Ascaris-induced allergic asthma in the conscious dog Inhalation of SAA produces a bronchospasm, measured as a decrease in Coyn and an increase in

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179

RL, that reaches a maximum within 3-5 min and is prolonged for up to 30 rain (Lewis et al., 1981). Initial studies involved the measurement of drug effects on RE and Cdyn between 2 min prior to the peak response for each dog and at 2, 4 and 6 min after this maximum. However, the drug effects, beneficial or otherwise, were always reflected in changes at the peak bronchoconstrictor response and consequently only the drug effects at the peak response produced by each dog are recorded in this report. Thiazinamium produced a dose-related inhibition of the Ascaris control response (viz., it increased Coyn and reduced RE) , although the regression line was very shallow and the effect was maximal at approximately the 12 mg dose (fig. 6). Promethazine did not affect the Ascaris-induced bronchospasm in a dose-related fashion (fig. 6). There was a dose-related reduction in R E up to 2.5 mg, but thereafter higher doses potentiated the bronchoconstriction. The increase in Cdy~ after promethazine was inversely related to the dose of the drug. Isoproterenol was the most potent substance tested (EDs0 1/~g) and effectively reduced RE, but did not significantly change Cdyn after Ascaris (fig. 8). Ipratropium similarly only reduced R E•

3.6. Guinea pig trachea in vitro Initially, the cumulative effects of thiazinamium, promethazine, ipratropium, theophylline and isoproterenol on basal tone (i.e., approximately 2 g) were examined in this preparation, using log increments .beginning at 10 -8 M. Thiazinamium relaxed basal tone by 10% at 10-SM and isoproterenol and theophylline produced similar responses, but at 10 -8 M and 5 x 10 -5 M, respectively (n = 8). Neither promethazine, ipratropium, nor DSCG influenced basal tone at any dose examined (10-8-10 -4 M). Using histamine- or carbachol-contracted tracheal spirals, thiazinamium produced a cumulative dose-response relaxation, i.e., a staircase effect. It was more potent against histamine (ECso 5.1 × 10 -8 M) then carbachol (ECs0 1.1 × 10 -6 M). Isoproterenol produced a similar response and was also more potent against histamine (ECs0 2.2 ×

10 -9 M) than carbachol (ECs0 3.6 × 10 -7 M). Propranolol (10 -7 M), added 10 min prior to histamine, did not affect the cumulative dose-response to thiazinamium. Both atropine (10 -8 M) and promethazine (10 - 6 M) produced relaxation of histamine-induced contractions, but the initial relaxation was slow in onset and further addition of drug using log or semilog units did not produce the staircase effect seen with thiazinamium and isoproterenol. Similar responses were observed for a t r o p i n e (10 -9 M) and promethazine (5 X 10 _7 M) against carbachol-induced bronchoconstrictions. Furthermore, ipratropium (5X 10-9M) relaxed carbachol-induced tension, although no cumulative dose-response could be produced.

3. 7. Ancillary pharmacology and toxicology Thiazinamium (100 #g-3.2 mg), administered as an aerosol, caused negligible effects on mean arterial blood pressure, heart rate and myocardial contractile force in anesthetized dogs. Moreover, it did not significantly influence heart rate from 0-20 min after administration when given at 2-20 mg to conscious dogs. Atropine methyl bromide (2 mg) significantly elevated heart rate 15 min after its aerosol administration to the conscious dog. Intravenous administration (1-10 mg/kg) of thiazinamium tended to lower arterial blood pressure and increase heart rate in anesthetized dogs. Thiazinamium did not affect motor activity in mice when administered via the oral (10-100 mg/kg) or i.p. (10 mg/kg) routes over a 3 h evaluation period. Promethazine (p.o.) significantly reduced activity at doses > 25 mg/kg. Oral administration of thiazinamium (50-100 mg/kg) caused a dose-related increase in pupil diameter (mydriasis) in mice 1 h after administration but was 68 times less potent than atropine sulfate. It was equipotent with promethazine in producing mydriasis. The acute oral LDs0 of thiazinamium was 558 m g / k g for the male and 512 m g / k g for the female rat. An acute inhalation toxicity study in guinea pigs in which animals were exposed to the aerosolized drug at a metered concentration of 51.4 m g / l indicated that the LCs0 was greater than this con-

180

centration (estimated to be equivalent to 184.8 mg/kg). A subacute inhalation toxicity study in male and female rhesus monkeys in which animals were exposed to drug up to 19.95 mg/1 for 1 h per day, 5 days per week, for a 30-day experimental period indicated no major change in neurological or cardiovascular function. Dose-related mydriasis was observed and some nasal and ocular discharge was found at the highest dose. Some respiratory tract irritation was also observed at the higher doses.

4. Discussion

Thiazinamium has bronchodilator activity after aerosol administration in guinea pigs, rats, cats and dogs using a number of different procedures to induce bronchoconstriction. The compound has a rapid onset and an adequate duration of action in these animal models, two important features for bronchodilator activity. The bronchodilator effect 'is probably a result of its substantial anticholinergic, antihistaminic and antiserotoninergic properties. Thiazinamium is a less potent anticholinergic than either atropine or ipratropium in the guinea pig and cat in vivo and possesses a shorter duration of action than either of these drugs. It is more effective than promethazine as an anticholinergic only when compared in the guinea pig. Thiazinamium possesses antihistaminic properties, unlike ipratropium (this study and Offermeier, 1975), but it is less effective than promethazine. Atropine also possesses some antihistaminic activity in vivo, but only of a transient nature. In these guinea pig studies regression lines constructed for thiazinamium were parallel to those for atropine and ipratropium, but promethazine produced a more shallow response which was significantly different from that of the other compounds. It is likely that this is an indication of a different mechanism of action and may be the result of some nonspecific local action of promethazine, such as local anesthesia (Douglas, 1975). Administered intraduodenally, thiazinamium is effective at inhibiting both acetylcholine- and histamine-induced bronchoconstriction in the

guinea pig, but in both instances it is less potent than promethazine. This suggests that systemic absorption of thiazinamium is less than that of the parent tertiary structure, promethazine, and this further confirms the poor absorption of orally administered Multergan in humans, where a relative bioavailability of less than 5% of the drug (300 or 900 mg) was calculated from plasma concentrations (Jonkman et al., 1977). Studies using the guinea pig tracheal strip indicated a similarity between thiazinamium and isoproterenol, a fl-adrenergic stimulant, since both drugs caused spontaneous relaxation in tone and produced cumulative dose-response relaxations of histamine- and carbachol-induced bronchoconstriction. None of the other drugs produced either effect, although slow-onset relaxations of the contracted airways were observed. It seems highly unlikely that thiazinamium possesses fl-adrenoceptor stimulant activity, since propranolol did not modify the smooth muscle relaxant effect of thiazinamium while abolishing the response to isoproterenol. In addition, thiazinamium did not elevate cyclic AMP levels in rat peritoneal mast cells (D. Yu, personal communication). These in vitro responses to thiazinamium might be a result of rapid replacement of agonist and its attachment to receptor sites. The relaxation of basal tone of tracheal strips by thiazinamium is a property shared by isoproterenol and also theophylline. The mechanism of this effect for thiazinamium is unclear, but the effect cannot be attributed to inhibition of phosphodiesterase as in the case of theophylline, since thiazinamium (up to 10 -~M) does not alter cyclic AMP phosphodiesterase from guinea pig lung (D. Yu, personal communication). Further investigations into the mechanism of action of thiazinamium in airway smooth muscle and its comparison with reference antagonists are currently in progress in attempts to clarify some of these observations. In addition to its mediator antagonist properties, thiazinamium has good antiallergic activity in that it is capable of inhibiting allergic bronchospasm induced in rats, guinea pigs and dogs in vivo and is also capable of inhibiting histamine release from rat peritoneal mast cells in vitro (Rosenthale et al., 1980; Carlson et al., 1981).

181 These latter in vitro studies complement the work of others (Mota and Da Silva, 1960; Lichtenstein and Gillespie, 1975; Church and Gradidge, 1980), who demonstrated that several antihistamines, including promethazine, possess potent mast cellstabilizing properties in vitro. Furthermore, Church and Gradidge (1980) were tempted to speculate that prevention of mast cell degranulation contributed to any beneficial effects produced by antihistamines in asthma. In the rat PLA, thiazinamium has a rapid onset of action and also a longer duration of action than promethazine and DSCG. The activity of ipratropium and atropine in the rat PLA was somewhat surprising, since we and others (Farmer et al., 1975) have failed to establish a vagal reflex component in this model of IgE-induced bronchospasm. Indeed, mast cell serotonin release seems to play a major role in the response (Farmer et al., 1975). Thus the activity of thiazinamium in this allergic model may be attributable to the antiserotonergic activity of the compound a n d / o r its mast cell-stabilizing properties. In contrast, ipratropium does not possess antiserotonergic activity (Offermeier, 1975), so that its antiallergic activity may theoretically be explained by an inhibitory effect on mediator release from mast cells, as suggested for anticholinergic drugs (Kaliner et al., 1972). However, evidence for mast cell-stabilizing activity for ipratropium is not available; moreover, in antigen-induced histamine release from sensitized human lung in vitro, ipratropium was inactive, and the effectiveness of this drug in allergic asthma has not been consistent (Morr, 1979). In the IgG-induced allergic bronchospasm in the guinea pig, thiazinamium was also very effective. Although less potent than isoproterenol, it was more potent than promethazine; furthermore, neither aerosolized ipratropium nor DSCG significantly influenced this response. The latter observation confirms the failure of DSCG to affect IgGmediated passive cutaneous anaphylaxis (Cox, 1967) and IgG-mediated histamine release from chopped guinea pig lung (Taylor and Roitt, 1973). Anticholinergics do not affect this bronchospasm (atropine also failed to affect the response after i.v. administration); consequently, mediator release is likely to play the major role in eliciting broncho-

spasm. Histamine and slow reacting substance of anaphylaxis (SRS-A) released from lung mast cells have been implicated in this model of PLA (Blumenthal et al., 1981), as they have in similar active lung anaphylactic responses in this species (Collier and James, 1967). Since promethazine is a more effective antihistaminic in the guinea pig than thiazinamium, the greater and longer-lasting effect of thiazinamium in the guinea pig PLA is probably a consequence of its mast cell-stabilizing properties, in addition to its antihistaminic activity. Although Ascaris-induced bronchoconstriction in the conscious dog is also an allergic model, inhibition of the response can be attributed to suppression of vagal reflexes that are known to play an important role in this model of asthma (Gold et al., 1972; Dain and Gold, 1975; Krell et al., 1976; Zimmermann et al., 1976). However, the failure of thiazinamium and ipratropium to completely inhibit the response suggests that there is a substantial anticholinergic-resistant component. Ipratropium had previously been shown to inhibit Ascaris-induced bronchoconstriction in anesthetized dogs (Zimmermann et al., 1978) but it was less effective in our conscious dog model. The role of histamine in mediating Ascaris-induced bronchoconstriction is unclear; whereas Chiesa et al. (1975) demonstrated histamine release after SAA, Krell (1978) suggested that it does not play a major role. Indeed, neither i.v. diphenhydramine nor mepyramine influenced the resultant bronchospasm in the latter studies (Krell, 1978). The inhibition produced by promethazine in our study may be a result of the higher local concentration achieved using aerosol administration or, more likely, the anticholinergic component of this drug. The enhancement of the bronchospasm produced by a high dose of promethazine can be attributed to histamine release similar to that produced with high concentrations of promethazine in in vitro mast cell preparations (Church and Gradidge, 1980; Carlson et al., 1981). Since thiazinamium has potent anticholinergic and antihistaminic activity in the pulmonary studies, it is not surprising that the ancillary pharmacology studies indicate that oral and parenteral administration of the compound is indeed capable

182 of eliciting some effects associated with these activities. However, aerosol administration of the c o m p o u n d appears to minimize such 'side effects'. F o r example, no significant cardiovascular effects occurred in anesthetized or conscious dogs after aerosol administration of extremely high doses of thiazinamium chloride, whereas i.v. administration p r o d u c e d hypotension and tachycardia. The failure of thiazinamium to affect m o t o r activity even after extremely high oral and parenteral doses is most likely related to the fact that it is a quaternary a m m o n i u m c o m p o u n d and is consequently unable to penetrate the blood-brain barrier. Thiazinamium chloride contrasts with promethazine, which enters easily into the central nervous system (Schmiterlow, 1956), and with other c o m p o u n d s having antihistaminic activity, which all produce sedation after therapeutic doses (Douglas, 1975). This lack of CNS-depressant activity gives thiazinamium chloride a considerable advantage over promethazine in the treatment of airways obstruction. Thiazinamium does produce mydriasis, a comm o n side effect of anticholinergic therapy, but only at high oral doses. Indeed, atropine sulfate was a much more potent inducer of mydriasis in the mouse model used for these studies. Mydriasis was also one of the major effects noted in monkeys receiving aerosol exposure during a 4-week period. Multergan, a preparation of thiazinamium that has been administered largely intramuscularly, is effective in the symptomatic treatment of asthma ( B u u y - N o o r d et al., 1957; Van Bork et al., 1977) and in chronic airway obstruction in both the elderly (Orie, 1961; Simonsson, 1964) and in hemp workers (Bouhys and Ortega, 1976). In the latter study a comparison of the effect of Multergan (i.m.) with that of isoproterenol inhalation indicated that Multergan was superior both in reducing the s y m p t o m s and in improving lung function. Laros (1975) indicated that Multergan (i.m.) was effective in patients with chronic obstructive airways disease but that the same patients did not respond afterwards to ipratropium bromide inhalation. He concluded that these patients reacted to antihistamines only. However, it is established that Multergan (i.m.) possesses strong anticholinergic as well as antihistaminic activity (Ducrot and De-

court, 1950a,b; Bracq, 1952; Simonsson, 1964). In another comparative study using allergen inhalation, Multergan (i.m.) demonstrated a protective effect similar to that of disodium cromoglycate ( D S C G ) inhalation, although its duration of activity was less than that of D S C G (Buuy-Noord et al., 1970). Complete protection was achieved with Multergan (i.m.) against exercise-induced bronchoconstriction, yet atropine was later without effect in these same patients (Zielinski and Chodosowska, 1977). The protection was thus interpreted to be a consequence of the antihistaminic property of the drug. Oral activity for Multergan has also been reported in bronchial asthma and chronic obstructive lung disease, but only using very large doses of the drug (Guerrin et al., 1980), which confirms the previously mentioned poor oral absorption ( J o n k m a n et al., 1977).

Acknowledgments The authors would like to thank A. Blumenthal, T. Kirchner and M. Shefton for their excellent technical assistance, Dr. T. Baum for performing some of the cardiovascular studies, Dr. B. Baxter for CNS studies, and Dr. M. Green for toxicological data. In addition, we thank M. Ludovici for preparing the manuscript, P.H. Freimuth for graphic arts, Dr. T.W. Copenhaver for statistical advice, and Dr. R.J. Capetola for his encouragement throughout these studies.

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