Getting new bronchodilator compounds from molecular topology

European Journal of Pharmaceutical Sciences 22 (2004) 271–277

Getting new bronchodilator compounds from molecular topology Inmaculada R´ıos-Santamarina a , Ramón Garc´ıa-Domenech a,∗ , Jorge Gálvez a , Jesús Morcillo Esteban b , Pedro Santamar´ıa b , Julio Cortijo b a

b

Unidad de Investigación de Diseño de Fármacos y Conectividad Molecular, Departamento de Quimica-F´ısica, Facultat de Farmàcia, Universitat de València, 46100 Burjasot, Valencia, Spain Departamento de Farmacolog´ıa, Facultad de Medicina y Odontolog´ıa, Universitat de València, Avda. Blasco Ibañez, 15, 46010-Valencia, Spain Received 5 November 2003; received in revised form 2 March 2004; accepted 22 March 2004 Available online 18 May 2004

Abstract Molecular topology has been used to select new lead bronchodilator compounds. The main advantage of this method, as compared to others frequently used, is that it does not require a previous explicit knowledge of the mechanism of action (MOA) of the compounds analyzed. A large database (12,000 chemicals) has been examined in this study to find less than 5% compounds with bronchodilator activity. After removing those compounds already described as bronchodilators, we present here the results for 20 among these compounds, some of them showing other pharmacological activities. Some of the compounds selected in this study showed higher relaxation and higher potency than theophylline, which is the reference drug used in the bronchodilator assay performed. For instance, tetrahydro-papaveroline showed significantly higher values than theophylline (93.9% versus 77.0% and pD2 = 7.30 versus pD2 = 4.69, respectively). Other compounds, although eliciting small or no relaxation at 0.1 mM, produced larger relaxation at higher concentrations (1 mM). In conclusion, the molecular topology based approach used in this work has demonstrated to be effective in the search of new bronchodilators. © 2004 Elsevier B.V. All rights reserved. Keywords: Molecular connectivity; Drug design; Bronchodilators; Topology; Drug research

1. Introduction A significant increase in the number of patients with asthma and other respiratory allergic diseases has been recently noticed. Thus, it is estimated that there is nowadays twice the number of asthmatics compared to 20 years ago. Moreover, the incidence of childhood asthma has also doubled along the last 15 years, thus becoming one of the most frequent diseases in pediatrics, and causing most of the hospital admissions as well. Recent epidemiological studies evaluate asthma incidence at a range between 10 and 17% of the children and adolescents in Western countries. The importance of an adequate pharmacological treatment during the childhood is critical, since about 80% of the children correctly treated will be free of disease at their adult age (Holgate, 1999; Nadel and Busse, 1998).

∗

Corresponding author. Tel.: +34-6-3544891; fax: +34-6-3544892. E-mail address: [email protected] (R. Garc´ıa-Domenech).

0928-0987/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2004.03.013

During the last decade, a new insight over the drug design methods has been developed, so that the High Throughput Screening methods as well as the virtual combinatorial techniques have become important tools in the rational drug design. The topological method (Kier and Hall, 1986) allows not only the prediction of molecular properties, but the design of new lead drugs as well (Gálvez et al., 1995; Estrada et al., 1998; de Julián-Ortiz et al., 1999; Gozalbes et al., 2000; Bruno-Blanch et al., 2003; Garc´ıa-Domenech et al., 2003). Furthermore, this can be achieved without an explicit knowledge of the mechanism of action (MOA). This is a very important difference with respect to other conventional methods based on quantum chemistry or molecular mechanics, which may be very efficient but as long as the MOA is known. Moreover, it is possible to speculate on the nature of MOA from the compound’s topological profile (Duart et al., 2001). The pharmacological distribution diagrams (Gálvez et al., 1996), introduced by our group, are particularly useful in this goal. Thus, regarding the bronchodilator activity, a topological pattern of activity was obtained in such a way

272

I. R´ıos-Santamarina et al. / European Journal of Pharmaceutical Sciences 22 (2004) 271–277

that those drugs following a common MOA are gathered into the same region of the diagram. The aim of this work was to apply a topological model to the search of new bronchodilators from a wide molecular database. The assessment of the pharmacological activity was carried out by performing the appropriate functional tests.

2. Materials and methods 2.1. Topological pattern of bronchodilator activity The search of a topological-mathematical model to select molecular candidates as bronchodilator drugs was carried out by linear discriminant analysis (LDA). The objective of LDA is to find linear functions capable to discriminate between two or more categories or objects (Johnson and Wichern, 1998; Gnanadesikan, 1988). In our case, two groups of compounds, the first including structures with a proven pharmacological bronchodilator activity and the second made up by presumably inactive compounds, were considered for analysis. Both, the active plus the inactive sets, constitute the training group. The way to select the pool of compounds in which to apply LDA is a key question since it conditions the success of the discrimination. There are different algorithms to assure that, but the basic input is the inclusion of balanced groups (the number of active and inactive compounds should be similar), a sufficient number of data (what is taken into account by the statistic results) and a significant degree of structural heterogeneity, so that the results are really evaluating bronchodilator activity rather than a particular mechanism of action or a particular category of molecules (for instance xanthines). LDA was performed with the BMDP 7M package (Dixon, 1990). The selection of the descriptors was based on the F–Snedecor parameter. The variables were introduced stepwise and the variable that adds the most to the separation of the groups is entered into (or the variable that adds the least is removed from) the discriminant function (DF). The classification criterion was the shortest Mahalanobis distance. The quality of the DF was evaluated by the Wilks’ parameter λ, as well as by the percentage of correct classifications into each group (discriminant capability). The independent variables in this study were the topological indices whereas bronchodilator activity was the discrimination property. The validation of the DF selected was performed by two tests: the first one is internal (the program chooses and pulls out, randomly, a number of compounds included as data in the LDA and rates them as test), and the other one external (involving the application of the DF to a number of compounds, active and inactive, not used in LDA, and their correct classification was verified). These series of compounds, constrained to internal and external test, constituted the “test group”.

The topological-mathematical model used to find out new compounds endowed with bronchodilator activity includes two discriminant functions: The first, namely DF1 , was obtained by inputing a huge set of more than 300 bronchodilator compounds including xanthines, beta-adrenergic agonists, anti-cholinergics, leukotriene antagonists as well as other structurally heterogeneous drugs showing some extent of bronchodilator activity. Just to improve the DF1 discriminant efficiency, a second discriminant function, DF2 , was also introduced. That function was obtained throughout a much shorter set of bronchodilator drugs, about 70, but including as many representative compounds as possible in order to consider drugs belonging to every family of bronchodilators. The discriminant functions achieved were (R´ıosSantamarina et al., 1998): DF1 = 3.071 χV − 3.58 G1 + 15.32J2 + 55.50J4 − 1.68 PR1 + 0.879 PR2 − 11.71 N = 739

U-statistics(Wilks λ) = 0.271

Fs = 286.5

DF2 = 17.40 (3 χP −3 χPV ) − 12.27 (4 χP −4 χPV ) − 6.61 N = 192

U-statistics (Wilks λ) = 0.315

Fs = 128.5

The topological descriptors used in these functions are the well known Kier and Hall’s connectivity indices (Kier and Hall, 1983), m χt , as well as the more recently introduced topological charge indices (Gálvez et al., 1994), Gi , Ji and some ad hoc indices (Gálvez et al., 1995), as for example PR1 and PR2 (number of pairs of ramifications i.e. vertices with valence three, separated by one and two edges, respectively). The topological charge indices evaluate the global charge transferred between pairs of atoms inside the molecule. Based on this model, a given compound was classified as a potential bronchodilator if either DF1 > −1 and DF1 < 10, or DF2 > 0 and DF2 < 17. Otherwise the compound is classified as inactive (R´ıos-Santamarina et al., 1998). Most of the active compounds were found within this range (a more detailed description is at the reader’s disposal on reference R´ıos-Santamarina et al., 1998 and the files containing the values of all the descriptors used in this work are also at reader’s disposal upon request). The topological model proposed here was used recently to search for natural products showing bronchodilator activity (R´ıos-Santamarina et al., 2002). Among the selected compounds, all of them derivatives of coumarine, flavonoids and antocianosides, stand out fisetin, a non-toxic vegetal pigment, and hesperetine (hesperidine’s aglycone) which at 0.1 mM elicited 88.9 and 87.4% relaxation of guinea-pig isolated trachea, respectively. As reference drug it was used theophylline which relaxed up to 77% at 0.1 mM.

I. R´ıos-Santamarina et al. / European Journal of Pharmaceutical Sciences 22 (2004) 271–277

were from Sigma–Aldrich. Most of compounds from the databases are organic compounds showing a huge structural heterogeneity (molecular weights ranging between 42.08 for cyclopropane and 1268.89 for ioxaglic acid). Once calculated the topological indices for every compound, the selected discriminant model was applied in the database, so that those compounds satisfying the required conditions were selected as candidates.

2.2. Application of the bronchodilator topological pattern to the selection of new active compounds using molecular databases The database used in this work was formed by approximately 12,000 compounds (50% of compounds were from the Merck Index database, which includes many different drugs or drug-like compounds. The rest of analyzed compounds, most of them just chemical reagents,

Table 1 Structures of some of the compounds selected as new potential bronchodilators

Compounds

Structure R1

R2

5-Bromo indol. 5-Bromoindoxyl acetate 3-(2-Bromoethyl)indol

I I I

H –O–COCH3 –Ethyl–Br

Br Br H

R3

R4

R5 R6

p-Piperidine-acetophenone II

–COCH3 H

H

H

H

o-Vanillin Vanillin

–CHO –CHO

–OCH3 H –OCH3 –OH

H H

H H

II II

–OH H

273

274

I. R´ıos-Santamarina et al. / European Journal of Pharmaceutical Sciences 22 (2004) 271–277

2.3. Pharmacological study of bronchodilator activity

3. Results and discussion

The viability of the topological–mathematical model used in this work to search and select new compounds with bronchodilator activity was confirmed by an appropriate experimental bronchodilator assay. The compounds selected were tested to assess their relaxation capacity at concentrations of 0.01, 0.1 or 1 mM, in trachea isolated from guinea-pigs (350–500 g) of either sex. This method is widely used for the in vitro testing of bronchodilators (Cortijo et al., 1994, 1997a). Tracheal rings were opened by cutting longitudinally through the cartilage rings diametrically opposite the trachealis muscle, and were suspended in jacketed 10 mL tissue chambers containing Krebs-Henseleit solution. The tension changes were recorded with isometric transducers coupled to a multi-channel polygraph. The preparations were subjected to an initial imposed tension of 2 g. A cumulative log concentration–relaxation curve for each compound was constructed, and results from measurement of graphs are expressed as percentage of the inhibition produced by theophylline (1 mM) added at the end of each experiment. The effective concentration 50% (EC50 ) was calculated by interpolation and expressed as pD2 (i.e. negative log of EC50 ). In order to avoid solubility problems which were detected in our previous work (R´ıos-Santamarina et al., 1998), Tween 80 (0.05 ml) was used as vehicle to prepare solutions of those compounds showing a low water solubility. No significant changes in the relaxant activity was found in controls carried out with drug vehicle.

After running the topological model on the selected database, we realized that no more than 5% of compounds were selected as potential bronchodilators (about 600 compounds). After filtering those candidates which had been previously reported as bronchodilators, the number of candidates shorted significantly. We present here the results for 20 among these compounds (see Tables 1 and 2). Some of them show other pharmacological activities, such as 5-flucytosine (antifungal), dapsone (antibacterial), camphtotecin (antileukemic and antitumor alkaloid, topoisomerase I inhibitor) or 5-bromoindoxyl acetate (substrate for stearase histochemical demonstration). Other candidates are used in the food industry, as for instance vanillin (flavoring agent) or active principles from plants, as for example, ellagic acid (isolated from the Eucalyptus maculata, hemostatic agent) and catechin (flavonoid found primarily in higher woody plants and used as antidiarrheal). Other compounds were metabolites coming from wide spectra drugs, such as 4-amino-antipyrine and 4-hydroxy-antipyrine (metabolites of aminopyrine, analgesic and antipyretic). Finally, other compounds were simple chemical reagents with unknown (or at least not reported) activity. The fact that some of the selected candidates were drugs with known pharmacological properties encouraged us to perform the bronchodilator activity assay since their known activities would favor a possible later development for

Table 2 Percent of relaxation, either at 0.1 mM or 1 mM, of compounds selected Compounds selected by molecular topology

Relaxation (%) 0.1 mM (1 mM)

pD2 (−log EC50 )

Theophylline (reference drug) p-Piperidine acetophenone (±)-Tetrahydro-papaveroline BrH

77.0 ± 0.0 72.1 ± 5.1 93.9 ± 2.1

4.69 4.65 7.30

Catechin. Thioxanthen-9-one.

0.0 ± 0.0, 87.8 ± 3.6 (1 mM) 69.7 ± 5.1

3.50 4.90

2-Chloro-thioxanthen-9-one. Ellagic acid o-Vanillin. Camptothecin 4-Amino-antipyrine 4-Hydroxy-antipyrine 4-4 -Diantipyrylmethane 5-Flucytosine Dapsone (4-aminophenyl-sulfone). 3-(2-Bromoethyl)indol 5-Bromo indol 1,5-Decalindiol.

48.1 31.1 0.0 14.2 66.0 47.8 27.2 42.3 47.3 55.9 74.1 28.5

± ± ± ± ± ± ± ± ± ± ± ±

4.6 4.0, 55.4 ± 4.2 (1 mM) 0.0, 53.1 ± 10.0 (1 mM) 1.2, 39.3 ± 0.2 (1 mM) 7.0 5.6 4.5 9.0 6.2 4.0 4.7 4.8

2-Bromoadamantane. 5-Bromoindoxyl acetate Vanillin Tetraethylene-glicol-bis-8-quinoline-ether

40.2 0.0 18.6 87.8

± ± ± ±

4.2 0.0, 78.1 ± 5.1 (1 mM) 2.8, 74.8 ± 3.5 (1 mM) 4.0

DF1 (DF2 )

Classification

Number assays

+ + +

6 8 14

−3.31 (7.19) 3.58

− (+) +

10 13

4.50 4.20 3.50 3.80 4.90. 4.90 5.10 4.80 4.80 4.70 4.50 5.60

4.01 0.50 0.04 5.58 −0.56 (2.58) −0.76 (3.46) 0.36 1.61 −0.19 (0.91) 6.23 3.33 −0.50

+ + + + − (+) − (+) + + − (+) + + +

11 8 7 4 11 13 9 6 8 7 11 6

4.70 3.50 3.70 4.60

0.82 0.65 −0.60 14.25

+ + + +

4.08 1.26 1.52

8 9 9 8

Values of pD2 i.e. −log EC50 , as well as the DF1 or DF2 values are also outlined. According to these values, the compounds are classified either as active (+) or inactive (−) as indicated in column five. The last column indicates the number of tests carried out for each compound.

I. R´ıos-Santamarina et al. / European Journal of Pharmaceutical Sciences 22 (2004) 271–277

Among the selected compounds, it is to be emphasized the high potency and almost complete relaxation produced by tetrahydro-papaveroline, a dopamine derivative without relevant acute toxicity, which is also a biosynthetic precursor of morphine, and shows in vivo and in vitro activity on pituitary function as well as on dopaminergic neurons. Other compounds also showed relaxations close to 50% at 0.1 mM, such as 2-chloro-thioxanthene-9-one, 4-hidroxyantipyrine (oxidated metabolite of 4-aminoantipyrine), 5flucytosine, dapsone (a sulphonamide-like drug used to treat leprosy), and 2-bromoadamantane. Furthermore, eight of the selected compounds were more potent than theophylline, with pD2 values between 4.8 and 7.3, other five compounds had potency values similar to that of theophylline (pD2 = 4.7), and the potency of other two was only slightly lower. One of the compounds tested, 1,5-decalindiol, exhibited a small relaxant effect but was relatively potent (pD2 = 5.6). Other compounds showed weak or no relaxant activity at 0.1 mM yet relaxation was much greater at 1 mM (vanillin, o-vanillin, ellagic acid, camphtotecin, catechin and 5-bromoindoxyl acetate). Of the 20 compounds tested, only three were inactive at 0.1 mM, but even these compounds showed relaxant activity at 1 mM.

-10

-10

10

10

30

30 Emax (%)

Emax (%)

clinical use as bronchodilators or, at least, it would make shorter the set of tests necessary to such aim. Table 1 shows the structures of each compound selected and Table 2 illustrates the values of DF1 and DF2 as well as the classification for each candidate. Table 2 shows the relaxation values (percentages) for the compounds selected, as well as their potency values (pD2 ). The relaxation of the reference drug theophylline (0.1 mM) was 77% (expressed as percentage of the maximal relaxation, i.e., 100%, obtained for 1 mM), and its pD2 was 4.69. The experimental protocol followed was the making of concentration–relaxation curves in guinea pig isolated trachea which is widely used for testing bronchodilators. As noted in Table 2, seven out of the 20 compounds selected showed a significant bronchodilator activity with more than 50% relaxation at 0.1 mM, and relaxation or potency values were higher than those of theophylline in some cases (p-piperidine-acetophenone, tetrahydro-papaveroline, thioxanthene-9-one, 4-amino-antipyrine, 3-(2-bromoethyl)indol, 5-bromo indol and tetraethylene-glicol-bis-8-quinoline-ether). Fig. 1 shows the values of percentage of relaxation obtained for four of the compounds tested as possible bronchodilators.

50

275

50

70

70

90

90

110

110 9

8

7

6

5

4

3

9

8

7

6

5

4

3

-log C

-log C

Tetrahydro-papaveroline

Tetraethylene-glycol-bis-8-quinoline-ether

-10 -10

10 10 30 Emax (%)

Emax (%)

30 50

50

70

70

90

90

110

110

9

8

7

6

5

-log C

Thioxanthen-9-one

4

3

9

8

7

6

5

4

3

-log C

4-amino-antipyrine

Fig. 1. Dose-response curves obtained for the selected compounds (triangle) and the reference drug theophylline (circle).

276

I. R´ıos-Santamarina et al. / European Journal of Pharmaceutical Sciences 22 (2004) 271–277

Table 3 Potency values (pD2 ) for the bronchodilatation produced by drugs representative of different mechanisms of action and clinical uses including theophylline as reference compound Drug

pD2

Mechanism of action; therapeutic use or potential clinical use

Reference

Theophylline

4.7

This study

Caffeine

3.3

Fenspiride

4.0

PF-904a

3.6

Rolipram Glaucine

6.2 4.5

SKF94120b

5.8

SCA40c

6.5

Sodium nitroprusside Levcromakalim

5.9 6.0

H-7d Dantrolene

5.0 4.5

Non-selective phosphodiesterase inhibitor; drug used in asthma and COPD Non-selective phosphodiesterase inhibitor; psychostimulant and enhancer of analgesia Non-selective phosphodiesterase inhibitor; drug used in COPD in some countries Non-selective phosphodiesterase inhibitor; pre-clinical development for asthma Archetypal PDE4 inhibitor; potential anti-depressant drug Selective PDE4 inhibitor and calcium channel blocker; antitussive (discontinued) Selective inhibitor of phosphodiesterase type 3; pre-clinical development as bronchodilator Phosphodiesterase types 3, 4 and 5inhibitor; preclinical development for asthma Donor of nitric oxide; anti-hypertensive Potassium channel opener; pre-clinical development for asthma and myocardial ischaemia Protein kinase C inhibitor; potential anti-cancer drug Blockade of Ca2+ release from sarcoplasmic reticulum; used in the treatment of spasticity disorders

a b c d

Sarria et al., 2000 Cortijo et al., 1998 Cortijo et al., 1997b Cortijo et al., 1996 Cortijo et al., 1999 Cortijo et al., 1996 Cortijo et al., 1997a Cortijo et al., 1998 Sarria et al., 2000 de Diego et al., 1995 Cortijo et al., 1991

4-Amino-1-ethyl-6-methylpyrazino[2,3-c][1,2,6]thiadiazine 2,2-dioxide. 5-(4-Acetamidophenyl)-pyrazin-2(H1)-one acetamidophenyl. 6-Bromo-8-methylaminoimidazol-[1,2-a] pyrazine-2-carbonitrile. 1-(5-Isoquinolinyl-sulfonyl)-2-methylpiperazine.

Considering these results, any pharmacologist could wonder: how unusual or not is the observation of bronchodilator activity in this assay at these modest levels of potency? This question is interesting and pertinent for the pharmacologist. When theophylline was selected as the reference bronchodilator in this work, it was done on the assumption that this xanthine is in clinical use for asthma and chronic obstructive pulmonary disease (COPD). In other words, its relatively low potency as bronchodilator has not precluded its therapeutic usefulness. Therefore, a low potency (i.e. in the high micromolar range) as bronchodilator may be acceptable and useful in the clinical setting. With respect to the commonness of such potency values, data from our own laboratory obtained in airway smooth muscle in vitro (same tissue organ bath technique used in the present work) stand as follows: (a) In two previous studies using molecular topology to select bronchodilator compounds we found that 16 compounds (89%) have potency values (expressed as pD2 ) within the range 4.5–5 (i.e. like theophylline) while only 2 (11%) were found with potency values >5 (R´ıos-Santamarina et al., 2002) and 12 compounds (60%) had potency values <5 while 8 compounds (40%) had potencies >5 (R´ıos-Santamarina et al., 1998). (b) In previous functional studies from this laboratory with different natural or synthetic compounds representative of different mechanisms of action, the relaxant potency

values found were close to that of theophylline, as shown in Table 3. In contrast, the finding of very potent bronchodilators is relatively unusual. A known example is isoprenaline (Sarria et al., 2000) with a pD2 of 7.5 and in general the beta-adrenoceptor agonists, and peptides like vasoactive intestinal peptide (VIP) which has a potency of 7 (Iriarte et al., 1993). Both are mimetics of neurotransmitters or neurotransmitters themselves. In summary, potency values such as those found here through molecular topology are relatively common and found also for compounds in clinical use or in pre-clinical development. Therefore, we believe that our method may provide useful compounds for further pharmacological studies as well as potentially useful in therapeutics.

4. Conclusions Molecular topology has demonstrated to be a useful methodology for identifying new compounds with bronchodilator activity. A pattern of topological similarity of bronchodilator activity has been obtained by using the LDA. This pattern has been applied successfully for the selection of drugs that in addition to other established pharmacological activities, also show bronchodilator activity. This study further confirms that one additional advantage of molecular topology is that it affords the screening of large databases with relatively low time consuming.

I. R´ıos-Santamarina et al. / European Journal of Pharmaceutical Sciences 22 (2004) 271–277

Acknowledgements This study has been supported by Generalitat Valenciana (GV2001-47), the Spanish Ministry of Science and Technology (SAF2000-0223-C03-02; SAF2002-04667 and SAF2003-07206-C02-01) and the Red de Investigación de Centros de Enfermedades Tropicales, RICET (C03/04).

References Bruno-Blanch, L., Gálvez, J., Garc´ıa-Domenech, R., 2003. Topological virtual screening: a way to find new anticonvulsant drugs from chemical diversity. Bioorg. Med. Chem. Lett. 13, 2749–2754. Cortijo, J., Sanz, C.M., Ortiz, J.L., Morcillo, E.J., 1991. Effects of dantrolene on the responses to methylxanthines in the isolated guinea-pig trachea. Eur. J. Pharmacol. 198 (2-3), 171–176. Cortijo, J., Sanz, C.M., Villagrasa, V., Morcillo, E.J., Small, R.C., 1994. The effects of phorbol 12,13-diacetate on responses of guinea-pig isolated trachea to methylxanthines, isoprenaline and ryanodine. Br. J. Pharmacol. 111, 769–776. Cortijo, J., Villagrasa, V., Navarrete, C., Sanz, C., Berto, L., Michel, A., Bonnet, P.A., Morcillo, E.J., 1996. Effects of SCA40 on human isolated bronchus and human polymorphonuclear leukocytes: comparison with rolipram, SKF94120 and levcromakalim. Br. J. Pharmacol. 119 (1), 99–106. Cortijo, J., Pons, R., Dasi, F., Marin, N., Martinez-Losa, M., Advenier, C., Morcillo, E.J., 1997a. Bronchodilator and anti-inflammatory activities of sca40: studies in human isolated bronchus, human eosinophils, and in the guinea-pig in vivo. Naunyn Schmiedebergs Arch. Pharmacol. 356, 806–814. Cortijo, J., Marti-Cabrera, M., Berto, L., Anton, F., Carrasco, E., Grau, M., Morcillo, E.J., 1997b. Pharmacological activity of PF-904 in guinea pig in vivo, and on human bronchus and neutrophils in vitro. Eur. J. Pharmacol. 333 (1), 69–78. Cortijo, J., Naline, E., Ortiz, J.L., Berto, L., Girard, V., Malbezin, M., Advenier, C., Morcillo, E.J., 1998. Effects of fenspiride on human bronchial cyclic nucleotide phosphodiesterase isoenzymes: functional and biochemical study. Eur. J. Pharmacol. 341 (1), 79–86. Cortijo, J., Villagrasa, V., Pons, R., Berto, L., Marti-Cabrera, M., MartinezLosa, M., Domenech, T., Beleta, J., Morcillo, E.J., 1999. Bronchodilator and anti-inflammatory activities of glaucine: In vitro studies in human airway smooth muscle and polymorphonuclear leukocytes. Br. J. Pharmacol. 127 (7), 1641–1651. de Diego, A., Cortijo, J., Villagrasa, V., Perpina, M., Morcillo, E.J., 1995. H-7, a protein kinase C inhibitor, inhibits spontaneous tone and spasmogenic responses in normal and sensitized guinea pig trachea. Gen. Pharmacol. 26 (8), 1747–1755. de Julián-Ortiz, J.V., Gálvez, J., Muñoz-Collado, C., Garc´ıa-Domenech, R., Gimeno-Cardona, C., 1999. Virtual combinatorial syntheses and computational screening of new potential anti-herpes compounds. J. Med. Chem. 42 (17), 3308–3314.

277

Dixon, W.J., 1990. BMDP Statistical Software University of California, Berkeley. Duart, M.J., Garc´ıa-Domenech, R., Antón-Fos, G.M., Gálvez, J., 2001. Optimization of a mathematical topological pattern for the prediction of antihistaminic activity. J. Comput. Aided. Mol. Des. 15, 561– 572. Estrada, E., Peña, A., Garc´ıa-Domenech, R., 1998. Designing sedative/hypnotic compounds from a novel substructural graph-theoretical approach. J. Comput. Aided. Mol. Des. 12, 583–595. Gálvez, J., Garc´ıa, R., Salabert, M.T., Soler, R., 1994. Charge indexes. New topological descriptors. J. Chem. Inf. Comput. Sci. 34, 520–525. Gálvez, J., Garc´ıa-Domenech, R., de Julián-Ortiz, J.V., Soler, R., 1995. Topological approach to drug design. J. Chem. Inf. Comput. Sci. 35, 272–284. Gálvez, J., Garc´ıa-Domenech, R., de Gregorio Alapont, C., de Julián-Ortiz, J.V., Popa, L., 1996. Pharmacological distribution diagrams: a tool for de novo drug design. J. Mol. Graph. 14, 272–276. Garc´ıa-Domenech, R., Rios-Santamarina, I., Catalá, A., Calabuig, C., del Castillo, L., Gálvez, J., 2003. Application of molecular topology to the prediction of antifungal activity for a set of dication-substituted carbazoles, furans and benzimidazoles. J. Mol. Struct. (Theochem). 624, 97–107. Gnanadesikan, R., 1988. Discriminant Analysis and Clustering. National Academy Press, Washington, D.C. Gozalbes, R., Brun-Pascaud, M., Garc´ıa-Domenech, R., Gálvez, J., Girard, P.M., Doucet, J.P., Derouin, F., 2000. Prediction of quinolone activity against mycobacterium avium by molecular topology and virtual computational screening. Antimicrob. Agents Chemother. 44 (10), 2764–2770. Holgate, S.T., 1999. The epidemic of allergy and asthma. Nature 402 (Suppl. 6760), B2–B4. Iriarte, M.F., Diaz-Juarez, J.L., Arilla, E., Pascual, R., Cortijo, J., Advenier, C., Prieto, J.C., Morcillo, E.J., 1993. Effects of sensitization on vasoactive intestinal polypeptide-induced relaxation and its concentration and binding in guinea-pig airways. Eur. J. Pharmacol. 250 (2), 295–302. Johnson, R.A., Wichern, D.W., 1998. Applied multivariate statistical analysis, fourth ed. Prentice Hall. Kier, L.B., Hall, L.H., 1983. General definition of valence delta-values for molecular connectivity. J. Pharm. Sci. 72 (10), 1170–1173. Kier, L.B., Hall, L.H., 1986. Molecular connectivity in structure-activity analysis, Research studies. Press, Letch Worth, England. Nadel, J.A., Busse, W.W., 1998. Asthma. Am. J. Respir. Crit. Care Med. 157, S130–S138. R´ıos-Santamarina, I., Garc´ıa-Domenech, R., Gálvez, J., Cortijo, J., Santamar´ıa, P., Morcillo, E.J., 1998. New bronchodilators selected by molecular topology. Bioorg. Med. Chem. Lett. 8, 477–482. R´ıos-Santamarina, I., Garc´ıa-Domenech, R., Cortijo, J., Santamaria, P., Morcillo, E.J., Gálvez, J., 2002. Natural compounds with bronchodilator activity selected by molecular topology. Internet Electron. J. Mol. Des. 1, 70–79. Sarria, B., Pedrós, C., Galán, G., Cortijo, J., Morcillo, E.J., 2000. Effects of phorbol 12,13-diacetate on human isolated bronchus. Eur. J. Pharmacol. 399 (1), 65–73.