Plasma levels and urinary excretion of disodium cromoglycate after inhalation by human volunteers

TOXICOLOGY

AND

APPLIED

Plasma

Levels

PHARMACOLOGY

20,147-156

(1971)

and Urinary Excretion of Disodium Cromoglycate After Inhalation by Human Volunteers

G. F. Moss, K. M. JONES,JEANT. RITCHIE, AND J. S. G. Cox Research and Development Laboratories, Fisons Limited, Pharmaceutical Division, Loughborough, England Received November 14,1969

PlasmaLevels and Urinary Excretion of Disodium CromoglycateAfter Inhalation by Human Volunteers. Moss, G. F., JONES, K. M., RITCHIE, JEAN T., and Cox, J. S. G. (1971). Toxicol. Appl. Pharmacol. 20, 147-156. The plasmalevels,rate of urinary excretion, total urinary excretion and the amount depositedin the mouth weremeasuredafter humanvolunteershad inhaled disodium cromoglycate (DSCG) (the disodium salt of 1,3-bis(2carboxychromon-5-yloxy)-2-hydroxypropane) from capsulesin the [email protected] after inhalation 30% to 50% of the dosewasdeposited in the mouth and wasrecoveredby meansof mouth washings;in clinical usethis proportion of the dosewould be swallowed.The powder which reachedthe lung was absorbedrapidly, the plasmalevelsreacheda peak concentration 20 min after inhalation, and the mean maximum rate of urinary excretion occurred during the first hour. Total urinary excretion of DSCG over 8 hr amountedto a meanof 4.2% of the dose(SD: 51.9). This indicateda total depositionin the lungsof about 7.5% (range5-10x) of the doseof DSCG. Disodium cromoglycate’ (DSCG) (the disodium salt of 1,3-bis(2-carboxychromon-5yloxy)-2-hydroxypropane) representsa new pharmacologic approach to the treatment of bronchial asthma. It is not a bronchodilator nor an antiinflammatory agent. It has no sympathomimetic, antihistaminic, or corticosteroid-like effects. In vitro, the drug inhibits the releaseof various mediators of anaphylaxis arising from the interaction of antigen with reagin-type antibodies; that is, “Type I” immediate allergic reactions. DSCG can also inhibit the releaseof histamine and slow-reacting substance of anaphylaxis (SRS-A) from portions of human lung which have been passively sensitized with human reaginic serum following exposure to specific antigens in vitro @heard and Blair, 1970). In clinical use DSCG is administered by a new inhalation device known as a [email protected] This device has been designed to deliver the drug as a fine powder aerosol cloud into the lungs when energized by the inspiratory effort of a patient. Using this apparatus the releaseof the drug is automatically coordinated with the inspiratory phase of respiration (J. H. Bell, personal communication). As might be expected with drugs administered by aerosol, the determination of the exact dosedeposited in the lung ’ Intal, Lomudal, registered trade mark of Fisons Limited, Pharmaceutical Division, Loughborough, L&s., England.

’ Registeredtrade mark of FisonsLimited, PharmaceuticalDivision, Loughborough,Leics., England. 147

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has proved difficult. This is due to the variable powder losses which occur during inhalation and the deposition of powder in the mouth, the latter contributing to the excretion products via oral absorption and metabolism of the drug. DSCG is the disodium salt of a strong acid (pKi = pK2 = 2.0) with a molecular weight of 512. The compound is freely soluble in water up to about 5 O0 and is insoluble in ethanol and higher alcohols. The absorption, distribution, and excretion characteristics have been studied in a number of laboratory animals (Moss et al., 1970; Moss and Ritchie, 1970). A very small proportion of the drug (approximately 1%) is absorbed after oral administration. Following inhalation, however, the compound is well absorbed and rapidly eliminated unchanged in the urine and bile; no accumulation has been detected. Its behavior is in fact entirely in keeping with that of a highly polar lipid-insoluble compound. To relate these studies to the clinical situation, our experiments were designed to measure the plasma levels and urinary excretion of DSCG following inhalation of the compound by humans. The usual clinical dose of DSCG is the content of 1 capsule (20 mg) inhaled 4 times a day although the dosage can be varied according to the needs of the patient. The plasma levels resulting from 1 capsule are too low for accurate estimation of DSCG; therefore, for the purpose of these experiments, the contents of 3 or 4 capsules were inhaled in rapid succession. In this study with human volunteers we describe methods for the estimation of DSCG in plasma and urine. Using these methods we have measured the plasma levels of DSCG after inhalation and the total DSCG excreted in the urine. The data has enabled us to calculate the rate of urinary excretion of DSCG after inhalation and to determine an approximate dose deposited in and absorbed from the lungs. These gave an indication of the rate of clearance of DSCG from the lungs.

METHODS DSCG DSCG is fluorescent in aqueous solutions with pH greater than 2. A characteristic spectrum can be obtained having an excitation maximum at 350 nm and fluorescent maximum at 450 nm (uncorrected). It was possible to utilize this property to measure DSCG in plasma, following a preliminary purification to remove endogenous fluorescent material. The fluorescent materials present in urine, however, caused a lack of specificity and prevented the use of this method for measurement of DSCG. Therefore, in order to measure DSCG in urine, use was made of the instability of the chromone ring to alkali. When DSCG is heated in alkaline solution the bis-o-hydroxyacetophenone is formed; this complexes with diazotized p-nitroaniline to form a compound, having an absorption spectrum with a peak at 490 nm. Plasma. Plasma (2 ml) was diluted with glass-distilled water (2 ml) and the proteins precipitated by the addition of 10% w/v sodium tungstate (3 ml), followed by 0.7 N sulfuric acid (3 ml). The pH of the solution was adjusted to 3.5-4.5 before the precipitate was centrifuged to produce a clear supernatant. A portion (5 ml) of supernatant was used for the DSCG assay. The supernatant was first extracted with ethyl acetate (2 x 5 ml). The organic extracts were discarded. The Measuremenf

of

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aqueous layer was acidified with 2 N hydrochloric acid (1.5 ml) and the DSCG extracted with ethyl acetate (3 x 5 ml). The organic extracts were combined and centrifuged to facilitate removal of water droplets. The DSCG was reextracted from the ethyl acetate solution with 1% ammonium hydroxide solution (1.5 ml) and the extract concentrated to a small volume (approximately 25 ~1) in vacua at 40°C. The concentrate, together with flask washings, was applied to a silica gel thin-layer chromatography (TLC) plate (5 x 20 cm, 0.3 mm layer). The plate was developed in isopropanol : ammonia: water (100: 10: 20) and subsequently dried in air at room temperature, and the DSCG was located by its fluorescence under a longwave UV light (366 nm). The area of silica gel containing the DSCG was removed from the plate and placed in a small test tube (capacity l-2 ml). The DSCG was eluted from the silica gel with 2% w/v aqueous potassium dihydrogen phosphate (1.0 ml) and the fluorescence of the centrifuged supernatant measured in a Baird Atomic Fluorispec Spectrofluorimeter at an activation wavelength of 350 nm and a fluorescent wavelength of 450 nm (uncorrected) using microcuvettes. A standard recovery sample and blank were run through the whole assay procedure along with the test samples; the recovery sample contained 1 pg DSCG in 2 ml of plasma. It was found that there was both an inter and intra individual “blank” fluorescence level variation: it was time dependent and was especially noticeable after meals. The blank sample was therefore taken immediately prior to dosing. It was considered that this sample would represent the plasma blank for up to 2 hr provided that no food was consumed during the period. The concentration of DSCG in the plasma at the time of sampling was calculated from the equation:

FT - FB x 0.5 = pg DSCG/ml FR- Fa

plasma

where FT is the fluorescence of the test plasma extract, FR is the fluorescence of the recovery plasma extract, and FB is the fluorescence of the blank plasma extract. Urine. Amberlite CG 4B resin, cleared from fines by repeated washing, was converted to the formate form by soaking in 98-100% AR formic acid for 2 hr with occasional mixing (100 g of resin is converted by 300 ml of acid). The resin was subsequently washed with 30 ‘A aqueous formic acid (2 x 300 ml) and finally prepared as a 50% v/v suspension in 30% v/v aqueous formic acid. Small ion exchange columns were made from 2-ml plastic disposable syringes. A disk of Whatman No. 1 filter paper was placed at the bottom of the syringe barrel and a Pharmaseal K 75 stopcock3 fitted to the nozzle to provide a means for controlling the flow. Resin slurry (1.6 ml) was added to the syringe to give a column with bed volume between 0.75 and 0.85 ml. A portion (10 ml) of the urine was acidified with 3 ml of formic acid AR (98-100x). The sample was applied to the column at a rate of approximately 0.5 ml/min. Faster rates gave lower recoveries, whereas slower rates gave higher blank readings. The column was washed at a rate of 1 ml/min with 30;/, v/v aqueous formic acid, the eluate was discarded. The DSCG was eluted from the column with 5 ml of formic acid AR (98-100’4). (The plastic stopcocks were removed prior to this stage as they were attacked by the concentrated formic acid solution.) The collected eluate was 3 Pharmaseal 3-way stopcocks supplied by Chas. F. Thackray Ltd., Park St., Leeds, England.

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evaporated to dryness in vacua at 50-60°C and the residue was dissolved in 2.5”, aqueous potassium carbonate (2.5 ml). Two l-ml portions of the extract were measured into 5-ml test tubes. One tube was heated in a boiling water bath for 1 hr and then cooled, the other was kept at room temperature. Three ml of diazotized p-nitroaniline was added to each of these tubes. Diazotized p-nitroaniline was prepared at approximately 4°C by mixing a solution of p-nitroaniline (5 ml of a 0.1 p/, w/v solution in 3 N HCI) with a solution of sodium nitrite (1 ml of a 0.5% w/v aqueous solution) and, after 2 min, diluting with 54 ml distilled water. The optical densities of the 2 solutions were measuredat 490 nm in a l-cm cuvette using a Unicam SP 600 spectrophotometer.4 Standard solutions were prepared by adding DSCG to 2.5 % potassium carbonate to give concentrations of lo-50 pug/mlDSCG. Thesesolutions were used to determine the percent recovery and standard curve for the method. The concentration of DSCG in the original urine sample was calculated from the equation :

ODt, - OQ, 4 _

’

Cont. of Standard = pg DSCG/ml urine OD of Standard

where f1 is the heated tube, tz is the unheated tube, and 2.5 ml of extract represented the original urine volume of 10 ml.

Inhalation

Studies

In all these experiments volunteers inhaled DSCG from capsules contained in a Spinhaler@, the clinical method for administering the drug. The volunteers were experienced in the use of the Spinhaler, which minimized errors in administering the drug. The compound as prepared for use by inhalation is contained in a capsule (No. 2 hard gelatin) together with lactose which servesto improve the flow properties of the drug. The DSCG powder has a particle size range in which more than 507; by weight is between 2 and 6 p. The lactose crystals are in the size range 30-60 p. The 2 compounds are present in the capsule (20 mg each of DSCG and lactose) as a mixture of drug agglomerates and lactose. Upon inhalation the turbulent air stream inside the Spinhaler @causesa proportion of theseagglomeratesof the drug to break up into fine particles which can consequently penetrate into the lung. Three or 4 deep steady inhalations are usually sufficient to empty the capsule. Twenty human volunteers (12 male and 8 female) took part in these experiments. The number of volunteers used for each experiment is given with the individual experimental details. The age and weight ranges of the males and females, respectively, were, 1g-35 yr, 56-80 kg; and 19-35 yr, SO-62kg. During the period of the experiments the volunteers kept to a normal diet and were not receiving any other medication. No attempt was made to regulate or measurethe frequency or depth of breathing. Plasma levels. Three human volunteers (male) each inhaled the contents of 3 capsules(3 x 20 mg DSCG) within a period of 5 min. Venous blood samples(10 ml) were taken prior to inhalation and at intervals after inhalation as shown in Fig. 1. All 4 Unicam S.P.600 supplied by Pye-Unicam Ltd., York St., Cambridge, England.

DSCG

CLEARANCE

AFTER

INHALATION

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BY HUMANS

the blood samples were heparinized and the plasma separated. The plasma concentration of DSCG was estimated. Rate ofurinary excretion of DSCG. A group of 10 volunteers (6 male, 4 female) each inhaled the contents of 4 capsules (4 x 20 mg DSCG) within a period of 10 min. To study in detail the initial rate of excretion of DSCG, urine was collected at 15-min intervals for the first hour after inhalation and at subsequent collection periods as shown in Table 1. The DSCG content of each urine sample was estimated. TABLE 1 TOTALS-HRURINARYEXCRETIONOF DSCG BYHUMANVOLUNTEERS,FOLLOWINGINHALATION OF 80 MGOFTHEPOWDER

No. of occasions tested

Volunteer K.J. J.W. S.B. J.A. A.D. J.G. S.W. G.M. H.C. P.M.

d d

J”k”* S-.6. A.C. P.L. Mean

z

: a” : 2

! ?

7 7 7 7 4 4 4 4 4 4 6 4 4 4

5

Mean urinary excretion DSCG (mg) 4.1 4.7 3.2 4.5

2.9 4.6

2.9 3.3 1.7 4.1 1.6 3.2 2.0 3.5 4.3

Range (mg) 2.0-5.9 2.9-7.0 1.8-5.4 2.2-6.1 2.4-4.2 3.2-6.0 2.2-3.2 2.3-4.6

1.5-1.9 2.3-6.0 1.0-2.1 1.8-3.8 1.6-2.2 2.5-6.0 3.7-4.8

3.4 f 1.48 (SD)

Total urinary excretion of DSCG. A group of 20 volunteers (12 male, 8 female) each inhaled the contents of 4 capsules (4 x 20 mg DSCG) over a lo-min period. Urine was collected for 6 hr after inhalation, and the DSCG concentration was estimated. The number of occasions each individual took the compound is shown in Table 1; in all a total of 75 determinations were made. Deposition of DSCG in the mouth after inhalation. After rinsing their mouths with water, 4 volunteers each inhaled the content of 1 DSCG capsule (1 x 20 mg). Immediately after the inhalation each volunteer washed his mouth out several times with water, using a total volume of 150 ml. The mouth washings from each volunteer were collected, combined, and made up to 200 ml for the estimation of DSCG content. Oral Administration Nine volunteers (male) were each given an oral dose of 500 mg DSCG in aqueous solution (2 mg/ml). Urine samples from each volunteer were collected for the subsequent 24-hr period. The DSCG content of the samples was measured.

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RESULTS

Methods of Estimation qf DSCG The recoveries pertaining to the 2 methods for estimation of DSCG were found to be 90 f 6% (SD) for the fluorimetric method and 97 h 1.5:); for the calorimetric method. Inhalation Studies Plasma levels. DSCG plasma levels after inhalation of the compound by the 3 volunteers are given in Fig. 1. In each case the highest plasma level (range 0.3 to 0.2 pg/ml)

I 20

10 MINUTES

60 AFTER

80

100

120

140

160

I a0

DOSE

FIG. 1. Plasma levels of DSCG after inhalation of the compound by 3 volunteers. Each inhaled the contents of 3 capsules (3 x 20 mg).

was found in the earliest blood sample taken (10-15 min after inhalation); these represented, however, only very small amounts of the drug. The lowest limit of accuracy for this method of estimation is 0.1 pg/ml; therefore, the later samples could only give an approximation to the amount of compound actually present. Rate of urinary excretion. The rate of excretion of DSCG during the 8 hr following inhalation of the compound is shown in Fig. 2. The rate was such that the highest percentage of the dose was excreted within 1 hr following inhalation. During this period, a mean of 1.3 mg or 1.6 % of the dose administered was excreted in the urine. The amount excreted per hour decreased during the subsequent periods, amounting to 0,6x, OS%, 0.3x, and 0.2% of the dose. Total urinary DSCG. Table 1 shows the results of the determination of total urinary excretion of DSCG following inhalation. The table shows mean excretion of DSCG

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for individual volunteers and the range of results when they were tested on several occasions. The amount excreted in the urine varied considerably between different volunteers and for the same volunteer tested on different occasions. The largest amount excreted was 7 mg while the smallest amount excreted was 1 mg. The overall mean urinary excretion for the whole group represents 4.3 % of the dose administered. Deposition of DSCG in the mouth. Following inhalation of the content of 1 DSCG capsule, the DSCG content of the mouth washings from the 4 volunteers was 30x, 32 %, 42 %, and 54 % of the dose.

FIG. 2. The rate of urinary excretion of DSCG by human volunteers following inhalation of the compound (80 mg). The results are expressed as a percentage of the total 8-hr excretion of DSCG and are the mean values from 10 different volunteers. The vertical bars represent the range of values recorded.

Oral Administration The mean urinary excretion of DSCG following oral administration (SD) % (range 0.2-0.8 %) of the dose administered.

was 0.5 f 0.2

DISCUSSION The metabolic fate of DSCG has been studied in several animal species (mouse, rat, rabbit, dog, silky marmoset, baboon, and in squirrel, cynomolus and macaque monkeys, (Cox et al., 1970; Moss et al., 1970). DSCG is handled by all these species in a similar manner; the compound is poorly absorbed from the gastrointestinal tract, but after iv injection, it is excreted unchanged in the urine and bile. There is no evidence of the compound being metabolized. In contrast to its poor absorption from the gastrointestinal tract, following deposition of DSCG in the lung of the rat, rabbit, and monkey, the compound was well absorbed and excreted in the urine and bile (Moss and Ritchie, 1970). In these studies with humans the rate of decrease in concentration of DSCG in the plasma (Fig. 1) and the subsequent early and relatively high concentrations of the compound in the urine indicate that DSCG was quickly cleared from the plasma to be excreted by the urine and bile. This is paralleled by results from our animal experiments which showed a rapid appearance of DSCG in the urine following both iv

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injection and inhalation. Accumulation of the compound in animal tissue was also noticeably absent. This similarity between the urinary excretion rates of DSCG in animals and man suggests that the lung clearance rate of DSCG in humans would follow the same pattern as that found in animals. Eight hours after inhalation urinary excretion of DSCG had fallen to negligible proportions, implying that no further absorption from the lungs was taking place, and also by analogy with the animal experiments, that negligible amounts of DSCG remained in the lung after this time. The urinary excretion rate of DSCG for humans has therefore been used in the derivation of a human lung clearance rate having an initial half-life of 1.0 hr. After iv injection of DSCG into human volunteers 40-50 yli of the dose was excreted in the urine and 55-60% in the feces (E. Forchielli and E. Segre, personal communication). By analogy with results from animal experiments and in view of the poor oral absorption of DSCG, all the compound found in the feces must have originated from the bile. Therefore it is possible to say that any DSCG absorbed systemically in humans is excreted in almost equal proportions in the urine and bile. There will of course be a small variation among individuals in the ratio of DSCG excreted in the urine to that excreted in the bile. For the purpose of this experiment, however, we have assumed that the compound is excreted in approximately equal proportions in the urine and bile and hence that quantity of DSCG excreted in the urine represents half the absorbed dose. No attempt has been made in this study to define the anatomical location in the airways from which drug absorption occurs. We have referred to the dose absorbed as that absorbed from the lung, i.e., bronchioles and alveoli, although we are aware that absorption of DSCG via the trachea and major bronchi may have also contributed to the value obtained. There are, however, other sites of deposition of DSCG; a significant proportion of the capsule content is deposited in the mouth and back of the throat. This amount varies according to the manner in which the Spinhaler @is used, and if used incorrectly, up to 50 % of the dose given is deposited in this way. After instruction and training, asthmatic patients become very proficient with the inhaler, and at flow rates above 80 I/min, considerably less powder is deposited in the mouth (J. H. Bell, personal communication). Therefore, in any consideration of the dose absorbed from the lung the contribution of orally ingested DSCG (i.e., via mouth, throat, and gastrointestinal tract) to the excretion pattern has to be taken into account. We know from studies on the oral absorption of DSCG, that approximately 1% of an oral dose is absorbed, of which 50% appears in the urine. Hence from an inhaled dose of 80 mg (4 capsules), approximately 0.4 mg appears in the urine; this has to be subtracted from the total urinary excretion value for DSCG following inhalation (3.4 mg) to give the dose absorbed from the lungs (3.0 mg). This means that an average of 6.0 mg or 7.5 % (range 5-10 %) of the original dose is absorbed from the lung. In these deductions we have assumed that there is no metabolic transformation -of DSCG. Previous studies have shown that no metabolites of DSCG are formed by any of the animal species studied (unpublished observations). Also, after iv injection of 14C-labeled DSCG to human volunteers, no metabolites were found (E. Forchielli, personal communication). Therefore, we can say that man behaves in an identical

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manner to all the other animal species studied in not forming any metabolites and that the DSCG measured in the urine represents the total urinary excretion of absorbed compound. The degree and depth of penetration of particulate matter, in the respiratory tract and its subsequent deposition, is an integrated effect of independent variables associated with aerosol cloud characteristics and the respiratory cycle. In order to achieve reproducible particle deposition, some degree of control must be achieved over such physiological events as rate and volume of inspiration. However, in clinical practice it is very difficult to train patients to inhale with a specific pattern, and simultaneously coordinate dose administration with a particular point in the inhalation cycle. Consequently the amount and sites of drug deposition in the airways and lungs may be variable, notwithstanding high precision of dose metering by the administration device. Even with devices such as the breath-actuated nebulizer and Spinhaler@, where there is a good degree of coordination of dose administration with inhalation into the inspired air stream, individual volunteers can vary considerably in the amount of drug they inhale. Nevertheless with practice and correct use of the inhaler these variations can be minimized and the range of results shown in Table 1 reflect this. Because individual volunteers studied on several occasions still showed a significant range in the total amount of DSCG excreted, the estimate of the total amount inhaled has been based upon the mean of a large number of determinations. The dose of DSCG we administered was 34 times that normally recommended. At first sight such a dose could appear to have little relevance to the clinical situation, where a smaller dose inhaled by asthmatic subjects with probably reduced inspiratory flow rates. However, separate experiments have shown that the figures obtained for a single capsule are of the same order of magnitude as those calculated from the multidose determinations. Furthermore, although asthmatics have increased airway resistance and reduced inspiratory flow volumes, their inspiratory flow rates are adequate to empty the drug from the capsule using the Spinhalerm, (Morrison-Smith and Devey, 1968). While it would be wrong not to recognize the limitations of these investigations, they provide a useful guide to the methodology and order of magnitude of the adsorbed dose of DSCG, and give an estimate of drug characteristics after inhalation in man. ACKNOWLEDGMENTS The authors are grateful to those members of the staff of the Research and Development Laboratories, who volunteered for the experiments. They are also indebted to Dr. R. E. C. Altounyan for assistance with clinical aspects of the work and Mr. J. H. Bell for helpful discussions. REFERENCES Cox,

J. S. G., and BEACH, J. E., BLAIR, A. M. J. N., CLARKE, A. J., KING, J., LEE, T. B., LOVEDAY, D. E. E., Moss, G. F., ORR, T. S. C., RITCHIE, J. T., and SHEARD, P. (1970). Disodium cromoglycate (Intal). Advun. Drug Res. 5, 115-196. MORRISON-SMITH, S., and DEVEY, G. F. (1968). A clinical trial of disodium cromoglycate

(“Intal”) in the treatment of asthmain children. Brir. Med. J. 2, 340-344.

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Moss, G. F., and RITCHIE, J. T. (1970). The absorption and clearance of disodium cromoglycate from the lung in rat, rabbit, and monkey. Toxicol. Appl. Pharmacol. 17,699-707. Moss, G. F., JONES, K. M., RITCHIE, J. T., and Cox, J. S. G. (1970).Distribution and metabolism of disodiumcromoglycatein rats. Toxicol. Appl. Pharmacol. 17, 691-698. SHEARD, P., and BLAIR, A. M. J. N. (1970).Disodium cromoglycateactivity in three in uitro modelsof the immediatehypersensitivityreaction in lung. Int. Arch. Allergy Appl. Immunol. 38, 217-224.