Assay bronchodilator

In the clinical area, the largest share of analytical methods development and publication has centered on the determination of theophylline in various body fluids, since theophylline is used as a bronchodilator in asthma. Monitoring serum theophylline levels is much more helpful than monitoring dosage levels.44 Interest in the assay of other methylxanthines and their metabolites has been on the increase, as evidenced by the citations in the literature with a focus on the analysis of various xanthines and methylxanthines. 

Though better tolerated, significantly less bronchodilating activity vs theophylline. Serious dosing errors possible if dyphylline monitored with theophylline serum assays... 

This relationship was of interest for several reasons. Firstly it indicated that the biological assay was sufficiently precise to enable the QSAR approach to be used Secondly the observation that only 2dkyl substituents R affected the activity whereas alkyl substituents in the R position apparently had little influence. This parallels the observations in the 6-thioxanthine series where a similar relationship was derived for the bronchodilating activity ( ) Thirdly, bulky substituents in the Rp position had a beneficial effect. Benzyl-substituted conqiounds e.g. (v) were more active than equation 1 indicated, possibly because the usual value of Es for benzyl did not reflect the buttressing effect of the adjacent triazole ring as revealed by a study of space-filling models ( ). 

None of the above noted 11-substituted analogs was more than minimally effective in our bronchodilator assays. However, lla-(2-hydroxyethylthio)PGE2 methyl ester (235) did prove to be a potent bronchodilator of sufficient interest to warrant clinical investigation (see Section VB). This compound was prepared as a separable mixture with its llp-epimer 236 by the addition of 2-mercaptoethanol to I-PGA2 methyl ester (2W) (69,107). 

These studies were carried out with ll-deoxy-13,14-dihydro-PGEi (2 ), since this compound was of high potency in our bronchodilator assays, and was readily available from I-PGA2 diester (21 ) by catalytic hydrogenation and saponification. Treatment with Ae requisite reagent under standard conditions afforded the 9-carbonyl derivatives 238-243 shown in Table III (108). 

For further evaluation, selected compounds are submitted to a dog assay (117,118) in which the prostaglandin is administered by aerosol to an anesthetized pilocarpine bronchoconstricted dog (n=3 to 6) and the decrease in airway resistance is recorded at the same time effects on the cardiovascular system (femoral pressure, pulmonary pressure, heart rate) are noted. This experiment is allowed to proceed for one hour, which also permits an assessment of the compound s ability to produce a prolonged bronchodilation. In this assay salbutamol maintains its effect for the entire hour, whereas isoproterenol and 1-PGEi lose theirs within the first twenty minutes. At the conclusion of the study a standard dose of isoproterenol is administered to determine the animal s maximum capacity to respond. 

The significance of the 11- and/or 15-hydroxy functions for bronchodilator activity is demonstrated in Table VI. Substantial activity clearly obtains in the 11-deoxy series, but the 15a-hy-droxy group apparently is an essential feature, although this requirement can be satisfied by a hydroxy group at Cje see section VIII F. Nevertheless it is noteworthy, that in the Konzett as well as other assays, even a primitive prostaglandin such as 11,15-bisdeoxy-PGEi produces a real PG-like effect, albeit with much diminished potency. 

Muchowski and co-workers have described the synthesis and bronchodilator activity of a series of ring halogenated prostaglandins. Several of these compounds showed excellent activity in the Konzett assay (ys. histamine) on intravenous or aerosol administration (Table X). Phase I evaluation of the most potent member of the series, 9-deoxy-9p-fluoro-PGE2 (IX), however, revealed a cough liability which precluded further clinical investigation (126,130). 

A large number of prostaglandin esters and amides have been reported in the patent literature. Although little is known concerning the biology of these derivatives, it generally is accepted that at least the simple methyl ester usually produces effects equivalent to that of the parent acid. From our own experience we are able to confirm this view as it applies to our bronchodilator assays. In a limited study we have found that a decyl ester and certain amides retain activity, but of considerably diminished potency. 

Many other a-chain modifications have been reported (Table XIV). Most of these changes appear to have been consistent with biological activity in one or another assay, but except for entry 6, no reports concerning bronchodilator activity have apeared. 

In our Konzett assay, ll-deoxy-16-methyl derivatives showed exceptionally high potency (48), an observation also made by a Wyeth group (113). However, Turther examination of 11-deoxy-16(R/S)-methyl-PGEi (XXIV) in the pilocarpine dog assay indicated this compound to be relatively ineffective and of no interest. Another member of this series, 16(S)-methyl-20-methoxy-PGE2 (XXV, YPG-209), has been reported to be 230 times as potent as PGE2 in the guinea pig vs. histamine-induced spasms. It also is claimed to be orally effective in this model without concommitant hypotension or diarrhea (169). This is the first claim, that we are aware of, for oral activity for any prostaglandin bronchodilator and we await the results of further studies with this compound. 

Since all eight failures apparently were selected, at least in part, on the basis of data obtained from a guinea pig Konzett-Rossler assay, the question arises as to the predictive capability of this widely used model for the selection of effective prostaglandin bronchodilators. However, in point of fact, four of the failures (entries 2-5) provided relatively modest Konzett responses, about 1-20% that of 1-PGEi, and therefore only one of these compounds (3) can be considered to have been tested at an appropriate multiple of the apparent 1-PGEi minimally effective dose [(50-100 jq)10.11.1641. Accordingly, the possibility remains that "weak" candidates may not have been studied at sufficiently high dose levels. 

The first example, depicted in Fig. 6, describes the synthesis and evaluation of a polymer imprinted with the bronchodilating drug theophylline, which is used in the treatment of asthma. Originally published in the journal Nature [2], this work drew considerable attention to the field of molecular imprinting because it was the first study to show that an MIP could be substituted for a natural antibody in a standard clinical assay. The MIP and antibody-based assays exhibit similar selectivities, and both can discriminate between theophylline and structurally related compounds. An equilibrium binding assay is described which uses radiolabeled theophylline as a marker. Data are presented for which nonradioactive theophylline, caffeine, and theobromine are used in competitive binding assays. These assays provide valuable information about the capacity and selectivity of the MIP. 

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