WO2006030322A2

WO2006030322A2 - Use of fla vone/fla vanone derivatives fn the treatment or prevention of respiratory conditions

Info

Publication number: WO2006030322A2
Application number: PCT/IB2005/003591
Authority: WO
Inventors: Edwin A. Kroeger
Original assignee: University Of Manitoba
Priority date: 2004-09-17
Filing date: 2005-09-13
Publication date: 2006-03-23
Also published as: WO2006030322A3

Abstract

The present invention is directed to the use of compounds of the formula I below as bronchodilators and anti-inflammatory compounds for the treatment and/or prevention of diseases of the respiratory or nasal tract by administering a therapeutically effective amount of a compound of formula I to a patient in need of such treatment. The compounds of formula I are: where R is hydroxyl, methoxy or amino; R1 is hydroxyl, methoxy or amino; R2 is hydroxyl, methoxy or amino, m is an integer from 0 to 3; n is an integer from 0 to 3; and p is an integer from 0 to 3, and the pharmaceutically acceptable salts thereof.

Description

FLAVONE/FLAVANONE DERIVATIVES

Background of the Invention

Certain flavanone derivatives have been reported to possess anti-allergic effects (U.S. Patent No. 4,937,257) and to be useful for the treatment of diseases of the respiratory tract (published UK Patent Application GB 2145718A).

It is an object of the present invention to treat diseases of the respiratory tract using compounds and compositions having bronchodilation activity and anti-inflammatory activity.

It is a further object of the present invention to provide compounds and compositions useful for the treatment and/or the prevention of various respiratory conditions in humans and animals such as asthma, other chronic obstructive pulmonary diseases ("COPD") and rhinitis in humans, analogous conditions in cats and dogs and the asthma-like condition of "heaves" in horses.

Summary of the Invention

The present invention is directed to the use of compounds of the formula I below as bronchodilators and anti-inflammatory compounds for the treatment and/or prevention of diseases of the respiratory or nasal tract by administering a therapeutically effective amount of a compound of formula I to a patient in need of such treatment. The compounds of formula I are:

where R is hydroxyl, methoxy or amino;

R¹ is hydroxyl, methoxy or amino;

R² is hydroxyl, methoxy or amino; m is an integer from 0 to 3; n is an integer from 0 to 3; and p is an integer from 0 to 3; and the pharmaceutically acceptable salts thereof.

Detailed Description of the Invention There are two major aspects of biological activity which were considered to be essential to the choice of compound for therapy in asthmatic and COPD patients: bronchodilation and inhibition of synthesis of inflammatory mediators known to cause bronchoconstriction in asthma (primarily leukotrienes). With respect to bronchodilation, effectiveness was assessed on the basis of the rapidity (half-time) and completeness (% of maximal) of airway smooth muscle relaxation (equine tracheal smooth muscle pre-contracted with high-K solution). With respect to inflammatory mediators, these are arachidonic acid metabolites, whose role in asthma has been widely recognized (and prominent pharmaceutical approaches have targeted the development of leukotriene receptor antagonists for treatment of asthma). As there are a number of bronchoconstrictor leukotrienes produced by 5'- lipoxygenase, it was deemed desirable to inhibit this entire metabolic pathway, rather than attempt to block the individual metabolic products. Arachidonic acid metabolism can be divided broadly into cyclooxygenase and lipoxygenase pathways. The products of cyclooxygenase metabolism include both bronchoconstrictor and bronchodilator compounds and thus, depending on the individual, highly variable respiratory responses are produced. The products of 5 '-lipoxygenase metabolism, however, are uniformly bronchoconstrictors. The profile of the compound selected, therefore, identified anti-lipoxygenase activity as the preferred characteristic. Our selection, therefore involved measuring the anti-inflammatory profile of the most potent bronchodilator compounds and selecting the one with the optimal anti-inflammatory profile. This approach targets bronchodilation both directly and broadly (the bronchodilation is effective against a wide array of chemically unrelated bronchoconstrictors) as well as the upstream production of bronchodilator metabolites. Representative compounds useful in the present invention include:

3,3'-dimethoxyflavone

6,2',3 '-trihydroxyflavone

5,4'-dimethoxyflavone 3,7,4'-trihydroxyflavone

4'-hydroxy-3 -methoxyflavone

5,6,7,3',4',5'-hexamethoxyflavone

3,3'-dihydroxyflavone

3,6,3',4'-tetrahydroxyflavone 2'-hydroxyflavanone

P inocembrine (5 , 7-dihydroxyflavanone)

3AFW (5'-aminoflavone)

(4',5,7-trimethoxyisoflavone)

3,6,2',3'-tetramethoxyflavone 5,7,4'-trimethoxyflavone

3,7,3'-trihydroxyflavone

3,4'-dimethoxyflavone

5,7-dimethoxyflavanone

(3 ', 5 -dimethoxyflavone) 5 -methoxyflavone

3,7-dihydroxy-3',4'-dimethoxyflavone

5,7-dihydroxy-4'-methoxyisoflavone

(6-methoxyflavone)

Flavanone 3,6,3 -trihydroxyflavone

4'-hydroxyflavanone

5,2'-dimethoxyflavone

(Naringenin, or 4',5,7-trihydroxyflavanone)

3 ,6-dihydroxyflavone 3,3',4'-trihydroxyflavone

2',3 '-dihydroxyflavone Presently preferred compounds that are useful in the present invention are 5,7- dihydroxyflavone (pinocembrine); 3,3'-dihydroxyflavone; and 4'-hydroxy-3-methoxyflavone. The most preferred compound is 3,3'-dihydroxyflavone.

Actual dosage levels of the compounds useful in the methods of this invention can be varied to obtain an amount of the active compounds which is effective to achieve the desired therapeutic response for a particular patient, compositions and mode of administration. The selected dosage level will depend upon the activity of the particular compound, the route of administration, the severity of the condition being treated and the condition and prior medical history of the patient being treated. However, it is within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

The phrase "therapeutically effective amount" of the composition of the invention means a sufficient amount of the active compounds to treat disorders, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.

The total daily dose of the compounds useful in the methods of this invention administered to a human or lower animal may range from about 0.0001 to about 1000 mg/kg/day. For purposes of oral administration, more preferable doses can be in the range of from about 0.001 to about 5 mg/kg/day. If desired, the effective daily dose can be divided into multiple doses for purposes of administration; consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. A presently preferred concentration is less than 180 micromolars, more preferably from 10 micromolars to 180 micromolars and most preferably from 25 micromolars to 180 micromolars.

The pharmaceutical compositions useful in this invention can be administered to humans and other mammals, particularly horses, cats and dogs, orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneal Iy, bucally or as an oral or nasal spray or by inhalation. The term "parenterally," as used herein, refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion. A presently preferred method of administration is inhalation.

The present invention includes one or more compounds as described above formulated into compositions together with one or more non-toxic physiologically tolerable or acceptable diluents, carriers, adjuvants or vehicles that are collectively referred to herein as diluents, for parenteral injection, for intranasal delivery, for oral administration in solid or liquid form, for rectal or topical administration, among others. The compositions can also be delivered through a catheter for local delivery at a target site, via an intracoronary stent (a tubular device composed of a fine wire mesh), or via a biodegradable polymer. The compounds may also be complexed to ligands, such as antibodies, for targeted delivery.

Compositions suitable for parenteral injection may comprise physiologically acceptable, sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), vegetable oils (such as olive oil), injectable organic esters such as ethyl oleate, and suitable mixtures thereof.

These compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin. Suspensions, in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

In some cases, in order to prolong the effect of the drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound may be mixed with at least one inert, pharmaceutically acceptable excipient or carrier, such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid; b) binders such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings well-known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

The active compounds can also be in micro-encapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

In a presently preferred formulation for inhalation therapy, the active ingredient has a particle size of less than 5 microns and the carrier has a particle size of more than 5 microns. Presently preferred carriers are lactose, lactose monohyrdrate, anhydrous lactose, glucose, mannitol, maltodextrin and mixtures thereof.

Compounds useful in the present invention can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals which are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients and the like. The preferred lipids are natural and synthetic phospholipids and phosphatidyl cholines (lecithins) used separately or together.

Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq. When administered by inhalation the pharmaceutical composition useful in the present invention may be in a liquid or solid dosage form that is suitable for nebulization or dry powder delivery, as for example, by means of aerosolization in jet, ultrasonic, pressurized or vibrating porous plate nebulizers or by conversion of dry powder into aerosol particles in an appropriate particle size and at an appropriate salinity and pH for administration by a dry powder inhaler or other suitable device.

The term "pharmaceutically acceptable prodrugs" as used herein represents those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention. Prodrugs of the present invention may be rapidly transformed in vivo to the parent compound of the above formula, for example, by hydrolysis in blood. A thorough discussion is provided in T.

Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, V. 14 of the A.C.S.

Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press (1987), hereby incorporated by reference.

Compounds that are useful in the present invention that are formed by in vivo conversion of a different compound that was administered to a mammal are intended to be included within the scope of the present invention. Compounds that are useful in the compositions of the present invention may exist as stereoisomers wherein asymmetric or chiral centers are present. These stereoisomers are "R" or "S" depending on the configuration of substituents around the chiral carbon atom. The present invention contemplates various stereoisomers and mixtures thereof.

Stereoisomers include enantiomers and diastereomers, and mixtures of enantiomers or diastereomers. Individual stereoisomers of compounds may be prepared synthetically from commercially available starting materials which contain asymmetric or chiral centers or by preparation of racemic mixtures followed by resolution well-known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary or (2) direct separation of the mixture of optical enantiomers on chiral chromatographic columns.

Compounds useful in the present invention can exist in unsolvated as well as solvated forms, including hydrated forms, such as hemi-hydrates. In general, the solvated forms, with pharmaceutically acceptable solvents such as water and ethanol among others are equivalent to the unsolvated forms for the purposes of the invention.

While the mechanism(s) of flavonoid-induced dilation is not totally understood, pharmacological and physiological responses of equine airway smooth muscle have indicated both a Ca-antagonist action and a Ca-independent (intracellular) action relevant to the relaxation effected by our flavonoids. Fluorescence microscopy of a primary smooth muscle cell culture using the Ca indicator Fura-2 was employed to investigate these possible actions, specifically to evaluate an impact on the Ca influx and to determine whether an uncoupling of Ca from contraction occurred.

The experimental model system involved pre-incubating smooth muscle cells first with Fura-2 (which is trapped intracellular Iy) and then with the flavonoid (3,3'- dihydroxyflavone, at 18OuM, a concentration which produces complete relaxation) for 1 hour (experimental) or with a non-flavonoid-containing solution (control) (rat vascular smooth muscle, incubated for two weeks prior to the experiment in primary culture) and washing out the (fluorescent) flavonoid. The cells are then visualized with fluorescence microscopy and the Ca-related signal measured. The Perkin Elmer Imaging Suite (ver 4.0) was utilized to image and analyze the fluorescent signal for intracellular Ca. Ca influx was induced with ionomycin at 0.3 and 1.2 uM.

The Ca influx spike was decreased (but not abolished) in the presence of flavonoid, thus confirming the partial inhibition of Ca influx predicted by the physiological experiments. Insofar as there was still substantially elevated intracellular Ca under conditions known to produce complete relaxation in in vitro muscle experiments, a dissociation of the Ca signal from mechanical sequellae is suggested. These results are consistent with a direct effect of the flavonoid at the level of myosin light chain phosphorylation (either by inhibiting myosin light chain kinase or by activating the phosphorylase).

Bronchodilator (equine airway smooth muscle relaxant) activity may be evaluated by the following procedure in which equine trachea is obtained fresh from the abattoir and stored ice-cold until dissection. Transverse strips of the tracheal smooth muscle approximately lmm in width are dissected free of the mucosa and cartilage. These strips (up to eight in a typical experimental paradigm) are placed in organ baths containing aerated (95% 0₂/5%C0₂) physiological salt solution (PSS, a modified Krebs-Henseleit solution) at 37° C and connected isometrically, at a length approximately equal to their in vivo length, to force transducers and electronic recording equipment. After 90 minutes equilibration, muscles are caused to contract by a short exposure to a high-K (depolarizing) PSS (in which Na Salts have been replaced with K). When the contraction has peaked (approximately 5 min.), the high-K PSS is replaced with normal PSS to allow the muscle to relax. The contraction/relaxation cycle is repeated after 10 minutes rest time until muscle responses are stable. All muscles are exposed to high-K PSS for a period of 45 minutes, after which the contraction has become stable (tonic contraction). Muscles are then exposed to test compounds at equal concentrations (including muscles for solvent vehicle controls as appropriate) and the response monitored for the succeeding hour (see typical mechanograms attached). Analysis for bronchodilator activity involves both rapidity (half-time) and completeness (% relaxation) of action. Based upon experience with the most active compound identified in the natural compound mixture, the relaxation achieved after 50 minutes exposure to the compound is taken as the maximal relation. "% relaxation" is calculated from the measured basal tone of the muscle, the contraction produced before exposure to the experimental compound and the contraction remaining after 50 minutes exposure to the compound. The half-time of response is the time required for the relaxation to be 50% of its final value. The preferred bronchodilator will combine the optimum combination of rapidity and completeness of dilator action. Cyclooxygenase activity was estimated by measuring the production of prostaglandin E₂,(via its stable metabolite), a primary product of arachidonic acid metabolism via cyclooxygenase, by activated U-937 cells. The method of Yan, J. et al, Biochim Biophys Acta. 2003 JuI 4;1633(l):51-60 was used. The Prostaglandin E₂ EIA (Enzyme Immunoassay) Kit - Monoclonal supplied by the Cayman Chemical Co (Ann Arbor, MI) was used for these assays (limit of detection: 15 pg/ml). 5-lipoxygenase activity (LOX) was estimated by measuring the production of leukotriene C₄ (LTC₄), a primary product of arachidonic acid metabolism via 5-lipoxygenase activity, by activated RBL-2H3 cells. Note that, while metabolism Of LTC₄ to LTD₄ and LTE₄ is rapid in vivo, cells in vitro release LTC₄ into the medium where it accumulates and is stable. The Leukotriene C₄ EIA (Enzyme Immunoassay) Kit supplied by the Cayman Chemical Co (Ann Arbor, MI) was used for these assays (limit of detection: 10 pg/ml). Activated (ionomycin, 0.3 X 10^"6 M) RBL-2H3 cells were incubated (in Modified

Eagle's Medium + 15% fetal bovine serum) for defined periods of time (20 - 60 min), during which period LTC₄ produced by the cells accumulated in the medium. The incubation was terminated by removing the medium and centrifugation. The amount OfLTC₄ in the medium was measured using the Cayman Assay kit. The % inhibition of LOX activity was calculated by comparing the amount OfLTC₄ produced by cells in the presence and absence of test flavonoids of interest (with the amount produced in the absence of the flavonoid being taken as 100%). Internal controls included the use of MMK-886, a specific 5- lipoxygenase inhibitor.

Representative compounds, with their respective activities as bioassayed, are set forth in Table 1 below.

TABLE 1

Flavonoid Relaxant Effects² anti-COX¹ anti-LOX²

% Inhib % %

(min)

3,3 '-dimethoxyflavone 6.0 99.7 10.3

6,2',3 '-trihydroxyflavone 9.18 99.3 61.0

5,4' -dimethoxyflavone 9.2 99.2 0

3 ,7,4 ' -trihydroxyflavone 10.6 99.0 34.8

4'hydroxy-3'-methoxyflavone 5.9 98.8 93 37.1

5,6,7,3',4',5'-hexamethoxyflavone

3.5 98.5 0

3,3'-dihydroxyflavone 5.9 98.5 79 63.9

3,6,3 ',4'-tetrahydroxyflavone 10.9 98.2 95.7

2 ' -hydroxyflavanone 10.5 98.1 77.3

5,7-dihydroxyflavanone 9.4 97.0 35 38.7

5'-aminoflavone 4.3 96.4 39.0

4',5,7-trimethoxyisoflavone 10.0 96.2 37.2

3,6,2',3'-tetramethoxyflavone 3.3 94.6 0

5,7,4'-trimethoxyflavone 6.6 94.2 31.0

3,7,3 '-trihydroxyflavone 13.2 93.7 0

3,4'-dimethoxyflavone 7.6 93.1 0

5 ,7-dimethoxyflavanone 6.0 93.0 18.9

3 -hydroxyflavone N/A 0 97 53

MK-866³ (300 nM) N/A N/A N/A 76

¹ activity measured at 18 uM flavonoid

² activity measured at 180 uM flavonoid ³ MK-866 is a non-flavonoid 5'LOX inhibitor which binds to FLAP to prevent activation of LOX - a positive control

The bronchodilator activity set forth in Table 1 was confirmed in an animal (dog) model for one representative compound (3,3'-dihydroxyflavone) by the following procedure.

The animals were anesthetized with pentobarbital sodium (30 mg/kg intravenously). A large bore armored endotracheal tube (ID 10 mm, Willy Rusch AG, Karman- Germany) was advanced into the trachea, and the lungs were mechanically ventilated (20 ml/kg). The animals breathed room air mixed with oxygen to yield an inspiratory oxygen concentration of approximately 40%. The esophageal balloon technique was used to measure pleural surface pressure (PpI) as previously described (11,12), in which the distal end of the 70 cm length catheter attached to the esophageal balloon was connected to one port of a differential pressure transducer (Validyne Engineering Corp, Northridge, CA). Pressure at the airway opening (Pao) was determined by means of a catheter attached to a port placed into the endotracheal tube. By means of two 3-way stopcocks mounted on the respective ports of the differential pressure transducer, either PpI, Pao, or transpulmonary pressure (Ptp= Pao-Ppl) were measured.

Measurements were obtained with the animal positioned in the left lateral decubitus position in a pressure compensated volume displacement plethysmograph (10-12). Lung volume was measured by a Krogh spirometer attached to the rear of the plethysmograph, while flow was measured by a pneumotachygraph placed in series with the Krogh spirometer. Signals for flow, volume, and pressure were recorded on an oscilloscope and oscillograph (Astro-Med, West Warwick, RI).

For the airway resistance experiments, the animal was paralyzed with succinylcholine (40 mg intravenously). Airway resistance (R_L) at 4 Hz was measured at a flow rate of 1 L/S (12,14) by the subtraction technique of Mead and Whittenberger (14) in which the oscillatory component of ΔPtp in phase with flow divided by flow determined R_L.

The protocol was as follows. After a standard volume history to total lung capacity

(Ptp= 30 CmH₂O), baseline measurements of RL were performed. Four to five measurements were obtained at 1 minute intervals to ensure consistency of the response. Nebulization (by means of a Cardinal Health, Airline Brand Misty Max 10™ Nebulizer, McGaw Park, IL) of methacholine (2.5 ml;. 8 mg/ml) was then performed over an approximately 6 - 8 minute period. Repeated measurements of RL were obtained immediately after nebulization over a ten minute period. Assessments were made at approximately 1 minute intervals. Another intravenous injection of succinylcholine (49 mg) was given. Bronchodilator therapy (2.5 ml) or placebo (2.5 ml) was then administered over a 3 to 5 minute period. Measurements after bronchodilator therapy were performed over a fifteen minute interval in which assessments were obtained at approximately 1 minute intervals.

After the animal had recovered and was breathing spontaneously without the need for oxygen it was returned its cage. All animals had received antibiotics (clindamycin 4 mg/kg) and gentamicin (2 mg/kg) before nebulization to prevent bacterial infection. REFERENCES:

8. National Institutes of Health: Guide for the Care and Use of Laboratory Animals (NIH Publication No 85-23), Bethesda, MD. National Institutes of Health, 1996. 10. Mink SN, Expiratory flow limitation and the response to breathing a helium-oxygen gas mixture in a canine model of pulmonary emphysema. J Clin Invest 1984; 73: 1321-1334. 11. Mink S, Gomez A, Whitley L, et al. Hemodynamics in dogs with pulmonary hypertension due to emphysema. Lung 1986; 164: 41-54.

12. Mink S, Coalson JJ, Whitley L, et al. Pulmonary function tests in the detection of small airways obstruction in a canine model of bronchiolitis obliterans. Am Rev Respir Dis 1984; 130: 1125-1133. 14.Mead J, Whittenberger JC. Physical properties of human lung measured during spontaneous respiration. J Appl Physiol 1953; 5: 779-796. The results are set forth in Table 2.

TABLE 2

% Decrease in Airways Resistance (after methacholine challenge)

Dog # Control Bronchodilator

1 0 61

2 23 73

3 18 42

4 26 61

5 12 24

6 9 59

Mean 14.7 53.3

Standard deviation 9.6 17.5 p<0.001

Claims

1. A method of treating or preventing a respiratory condition in a mammal comprising administering to a mammal in need of such treatment a bronchodilating and anti-inflammatory effective amount of a compound of the formula

where R is hydroxyl, methoxy or amino; R¹ is hydroxyl, methoxy or amino; R² is hydroxyl, methoxy or amino; m is an integer from 0 to 3; n is an integer from 0 to 3; and p is an integer from 3; and the pharmaceutically acceptable salts thereof.

2. The method of claim 1 wherein the concentration of the compound is less than 180 micromolars.

3. The method of claim 1 wherein the compound is 3,3'-dihydroxyflavone.

4. The method of claim 1 wherein the compound is administered by inhalation.