WO1993007265A1

WO1993007265A1 - Treatment of cystic fibrosis

Info

Publication number: WO1993007265A1
Application number: PCT/US1992/008346
Authority: WO
Inventors: Francis S. Collins; Mitchell L. Drumm; David C. Dawson; Daniel W. Wilkinson
Original assignee: The Regents Of The University Of Michigan
Priority date: 1991-10-01
Filing date: 1992-09-30
Publication date: 1993-04-15

Abstract

Cystic fibrosis (CF), a lethal genetic disease associated with a defect in Cl transport, is caused by mutations in the gene coding for cystic fibrosis transmembrane conductance regulator (CFTR). Surprisingly, not only wild type CFTR, but several naturally-occurring CFTR mutants carrying a defect in the first nucleotide binding fold (NFB1) all expressed cAMP-activatable Cl currents. Treatment of the CFTR mutants with appropriate concentrations of methylxanthine phosphodiesterase inhibitor (which increases cAMP levels) activated Cl conductance to near wild type levels. The present invention thus provides a new avenue for treating cystic fibrosis by the administration of therapeutically effective amounts of compounds which elevate cAMP levels. Dosage and patient responsiveness to treatment, as well as relative efficacies of the compounds being or to be administered can also be determined in accordance with the methods of present invention.

Description

TREATMENT OF CYSTIC FIBROSIS SPONSORSHIP

Work on this invention was supported by the Cystic Fibrosis Foundation, the Howard Hughes Medical Institute and by the United States Government under Grant Nos. DK427185, DK39690 and DK29786 awarded by the National Institutes of Health. The Government has certain rights in the invention.

RELATED APPLICATIONS This is a continuation-in-part application of U.S. Application Serial No. 584,275, entitled "Gene Therapy for Cystic Fibrosis," filed September 18, 1990, and also filed September 16,1991 as International Application PCT/US91 /06660, which is a continuation- in-part of U.S. Application Serial No. 401,609, entitled "Cystic Fibrosis Gene," filed on August 31, 1989, which is a continuation-in-part of U.S. Application Serial No. 399, 945, entitled "Cystic Fibrosis Gene," filed on August 24, 1989, now abandoned, which is a continuation-in-part of U.S. Application Serial No.396,894, entitled "Cystic Fibrosis Gene," filed on August 22, 1989, now abandoned, all of which applications are specifically incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates generally to the treatment of chloride secretion insufficiencies caused by defects in cystic fibrosis transmembrane conductance regulator (CFTR) with compounds which increase or supplement cyclic AMP (cAMP) levels and, more specifically, to the treatment of cystic fibrosis (CF) with therapeutically effective amounts of phosphodiesterase inhibitors such as methyixanthines.

BACKGROUND OF THE INVENTION

Cystic fibrosis (CF) is the most common lethal autosomal recessive disease among Caucasians, affecting nearly 1 in 2500 newborns. Boat et al., Metabolic Basis of Inherited

Disease. (McGraw-Hill, NY 1989) 2649-2680. CF is caused by mutations in the gene coding for cystic fibrosis transmembrane conductance regulator (CFTR), a 1480 amino acid protein which has been associated with the expression of chloride conductance in a variety of eukaryotic cell types. See Rommens et al., Science 245: 1059 (1989); Riorden et al., Science 245:1066 (1989); Kerem et al., Science 245: 1073 (1989); Drumm et al., Cell

64:681 (1991); Kartner et al., Cell 64:681 (1991); Gregory et al., Nature 347: 382 (1990);

Rich et al., Nature 347:358 (1990); Rommens et al., PNAS (USA) 88:7500 (1991). Defects in CFTR destroy or reduce the ability of epithelial cells in the airways, sweat glands, pancreas and other tissues to secret Cl in response to cAMP-mediated agonists and impair activation of apical membrane channels by cAMP-dependent protein kinase A (PKA). See Frizell et a , Trends Nβurosci. 10:190 (1987); Welsh, FASEB J. 4:2718 (1990).

Although over 100 different mutations have been identified in the CFTR gene, a single NBF1 mutation, the deletion of phenyialanine 508 (ΔF508), accounts for almost 70% of the CF alleles in the population. Kerem et al., Science 245:1073 (1989); The Cystic Fibrosis Genetic Analysis Consortium, Am. J. Hum. Genet. (1990). Patients homozygous for the ΔF508 mutation present a similar clinical picture, including elevated sweat chloride levels, chronic pulmonary disease, and pancreatic insufficiency and are generally classified as severely affected. Kerem et al., N. Engl. J. Med. 323:1517 (1990). The clinical profiles of patients carrying other mutations, however, show a broad spectrum of severity. Kerem et al., N. Engl. J. Med. 323:1517 (1990); Cutting et al., Nature 346:366 (1990); Osborne et al., Am. J. Hum. Genet 48:608 (1990); White et al., Nature 344:665 (1990); lannuzi et al., Am. J. Hum. Genet. 48:226 (1991).

Given the high incidence and devastating nature of this disease, the development of effective CF treatments is imperative. For any therapeutic approach to have a great impact on the CF population, it must also be effective in treating the ΔF508 mutation, since it is estimated that as many as 92% of CF patients carry at least one ΔF508 aliele. Kerem et al., N. Engl. J. Med. 323:1517 (1990).

SUMMARY OF THE INVENTION The present invention provides a method of treatment of chloride secretion insufficiencies caused by CFTR defects generally comprising the administration to the patient of a therapeutically effective amount of a compound, or pharmaceutically acceptable form thereof, which increases or supplements cyclic AMP (cAMP) levels. The method of the invention thus further provides a method of treatment of CF though the administration of therapeutically effective amounts of such compounds, which include phosphodiesterase inhibitors, cAMP and its analogs and β adrenergic receptor agonists. The present invention also provides a method for in vitro dosage determination and screening for CF patient-responsiveness to treatment with the aforementioned compounds. Testing for relative efficacy of these compounds in treating specific CFTR defects and individual patients is also contemplated as falling within the scope of the present invention. Other features and advantages of the present invention will be become apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 A shows membrane currents at a holding potential of -60 mV from oocytes injected with either wild type or ΔF508 cRNA-injected and uninjected oocytes.

Figure 1B is a dose response curve comparing the effects of increasing IBMX concentrations on wild type and ΔF508 cRNA-injected oocytes.

Figure 2A is a bar graph comparing the maximal currents elicited in oocytes expressing each CFTR variant studied.

Figure 2B is a dose response curve showing activated currents (expressed as % activation) for five CFTR variants plotted against the concentration of IBMX in the bath. Figure 3A shows representative l-V plots obtained at maximal activation in oocytes injected with wild type cRNA.

Figure 3B shows representative i-V plots obtained at maximal activation in oocytes injected with ΔF508 cRNA.

Figure 4 is a Table comparing CF patient genotype with clinical phenotype. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Cystic fibrosis, a genetic disease associated with a defect in Cl transport, is caused by mutations in the gene coding for cystic fibrosis transmembrane conductance regulator (CFTR), which has been proposed to function as a Cl channel. The functional significance of naturally-occurring, single amino acid mutations in the first nucleotide binding fold (NBF1) of CFTR was investigated by assaying the expression of cAMP-activated Cl conductance in Xenopus oocytes injected with CFTR cRNA. Unexpectedly, oocytes injected with cRNAfrom several NBF1 mutants, including the most common ΔF508 defect, expressed readily discernable, albeit reduced compared to wild type, Cl currents. Sensitivity of the CFTR mutants to a phosphodiesterase inhibitor (IBMX) and forskolin inversely correlated with the severity of cystic fibrosis in patients carrying the mutations, i.e., mutations associated with severe disease being less sensitive than those associated with mild disease. Even the least sensitive mutants (ΔF508, G551 ), however, could be activated to a Cl conductance level approaching that of wild type by increasing the concentration of phosphodiesterase inhibitor (IBMX) and forskolin. These results are consistent wfth the theory that CFTRs bearing disease-causing mutations in NBF1 are inserted in the plasma membrane, but are defective in their ability to be activated by the appropriate stimulus.

The present invention thus comprises a method of treatment of cAMP-activatable chloride secretion insufficiencies associated wfth CFTR defects, such as cystic fibrosis, by the administration to the patient of therapeutically effective amounts of a compound, or pharmaceuticaily acceptable form thereof, which increases or supplements cAMP levels. In accordance with the present invention an increase or supplementation of cAMP levels can be achieved through an increase in cAMP production, inhibition of its breakdown or the administration of supplemental cAMP or analogs thereof. An increase in production, decrease in breakdown or supplementation of cAMP with cAMP or its analogs are hereinafter collectively referred to as "elevation of cAMP levels."

The present invention also provides a method for determining appropriate dosages and relative efficacy of compounds for specific CFTR defects and individual patients, and allows the design and monitoring of appropriate treatments in accordance with the invention. For example, as illustrated in the Specific .Examples below, the levels of response for different CFTR mutations may vary with a specific compound. CF patient tissues or cell populations carrying the defect of interest can thus be tested, for example in culture, to assist in determining which type and dosage of compound or combination of compounds are most beneficial for carriers of that mutation or the individual patient. Preferred compounds for administration in the practice of the method of the invention which elevate cAMP levels include phosphodiesterase inhibitors which increase cAMP levels by inhibiting cAMP breakdown. Supplemental cAMP and analogs thereof or β adrenergic receptor agonists, such as isoproterenol and albuterol, can also be employed in the practice of the invention. Preferred phosphodiesterase inhibitors which increase cAMP levels include nonspecific inhibitors such as alkylxanthines and cAMP-specific inhibitors such as Rolipram (Shearing AG). A review of phosphodiesterase inhibitors and their selection is presented by Nicholson et al., Trends Pharmacol. Sciences 12:19 (1991) and Beavo et al., Trends Pharmacol. Sciences 11:150 (1990), which are incorporated herein by reference. Preferred alkylxanthines include the methylxanthines, such as 3-isobutyl-1 -methylxanthine (IBMX) and 1 , 3-dimethylxanthine (theophyliine) and other xanthines such as papaverine, pentoxifiliine and caffeine. Methylxanthines such as IBMX are most preferred.

Although the aforementioned compounds may be administered alone, they are preferably administered as part of a pharmaceutical formulation. Such formulations can include pharmaceutically acceptable carriers known to those skilled in the art, as well as other therapeutic agents. It will also be appreciated that the compounds of the method of the present invention can be administered in various pharmaceutically acceptable forms, e.g., such as pharmaceutically acceptable salts thereof.

Appropriate dosages of the compounds and formulations administered in accordance with the invention will depend on the type of CFTR mutation and severity of the condition being treated and may also vary from patient to patient. Determining an acceptable or optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the dose and treatment of the present invention. For a dose to be "therapeutically effective," it must have the desired effect, i.e., an elevation in cAMP levels as defined herein, resulting in increased Cl secretion by the cell population being or to be treated with the dosage. An optimal dose will be one which, when administered to the patient carrying a CFTR defect, results in Cl secretion at or near wild type CFTR levels.

Administration may be by any suitable route including oral, nasal, topical (including buccal and sublingual), parenteral (including subcutaneous, intramuscular, intravenous and intradermal), vaginal or rectal, with oral and nasal administration being preferred. The formulations thus include those suitable for administration through such routes. It will be appreciated that the preferred route may vary with, for example, the condition and age of the patient. The formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and may be prepared and administered by any methods well known in the art of pharmacy, including liposomal delivery systems.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, as a powder or granules, or as a solution, suspension or emulsion. Formulations suitable for oral topical administration further include lozenges, pastilles, mouthwashes and inhalation mists administered in a suitable base or liquid carrier. Formulations suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising the compound to be administered and a pharmaceutically acceptable carrier or in a transdermal patch. Formulations suitable for nasal administration wherein the carrier is a solid include powders of a particle size, for example about 20 to 500 microns, which can be administered by rapid inhalation through the nasal passage. Suitable formulations wherein the carrier is a liquid can be administered, for example, as a nasal spray or drops.

Formulations suitable for parenteral administration include aqueous and non- aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic wfth the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit or mufti-dose containers, for example, sealed ampules and vials, and may be lyophilized, requiring only the addition of the sterile liquid carrier such as water for injections immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray; formulations for rectal administration may be presented as a suppository with a suitable base. it will be appreciated that in addition to the ingredients specifically mentioned above, the formulations used in the methods of this invention may include other agents conventional in the art having regard to the type of formulation in question.

SPECIFIC EXAMPLES EXAMPLE 1. Activation of Cl Current by Phosphodiesterase Inhibitor. A. Activation of Cl current by IBMX.

Ovarian lobes were removed from anesthetized frogs via a small abdominal incision. Stage V oocytes were separated from follicular membranes by exposure to collagenase (Gibco) and gentle dissection, and were injected with 10-15 ng of cRNA. Three to 12 days after injection, oocytes were assayed for activatable membrane currents by means of two electrode voltage clamps. Outward membrane current was defined as positive. During these experiments, the oocytes were continuously perfused with amphibian Ringers containing 100 mM Na; 106 mM Cl; 2.0 mM K; 5 mM HEPES; 1.8 mM Ca and 1.0 mM Mg 100 at pH 7.4. Where indicated, Cl was replaced by aspartate. Membrane currents (I_M) were recorded from Xenopus oocytes at a holding potential

(V_H) of -60 mV. Shown in Figure 1A are results from oocytes harvested from the same frog on the dame day and injected 3 days previously with cRNA transcribed from wild type or ΔF508 CFTR and an uninjected oocyte control. Shown in Figure 1A is the prior to stimulation, during exposure to a stimulatory cocktail containing 200μM 8-chlorophenylthio cAMP (8-cpt cAMP), 10μM forskolin and 1 mM IBMX, and during washout of the cocktail. As illustrated in Figure 1A, exposure of the wild type-injected oocyte to the stimulatory cocktail activated a large inward current which reached a maximum value in about 4 minutes, whereas the membrane current in the uninjected oocyte was unaffected. In the ΔF508 injected oocyte, the stimulatory cocktail also induced inward current, but the activation differed markedly from wild type. The rate of increase of l^ was slower and the final steady-state level was significantly lower than wild type. Washing out the forskolin and IBMX reversed the activation and returned both currents to the previous baseline. B. Effect of Concentration of IBMX on Cl Current.

Oocytes were harvested from the same frog as described in Example 1 A, but about 2 weeks later, and injected with wild type and ΔF508 cRNA. Three days after injection, the oocytes were exposed to a stimulation cocktail containing 10μM forskolin and, successively, 1 mM and 5 mM IBMX. 8-cpt cAMP was eliminated from the cocktail on the basis of other experiments which showed that it had little effect alone or in combination with forskolin and IBMX. The difference in the currents seen with 1 mM IBMX in Figure 1 A and 1 B is typical of the variability seen in expression in oocytes taken from the same frog at different times. As shown in Figure 1 B, raising the concentration of IBMX to 5 mM elevated the current in the ΔF508-injected oocyte to a level comparable to wild type, whereas current in the wild type-injected oocyte was little changed by the same maneuver.

In principle, the reduced current obtained with ΔF508 could be due to altered processing of the mutant protein (Cheng et al., Cell 63:827 (1990)) or simply altered sensitivity to the rise in cellular cAMP presumed to occur in response to forskolin and IBMX. The result depicted in Figure 1 B supports the latter explanation. .EXAMPLE 2. Activation of Cl Current in Five CFTR Variants. A. Activation by IBMX.

The striking difference in the sensitivity of oocytes injected with wild type and Δ F508 forms of CFTR cRNA to the stimulatory cocktail prompted us to examine three other naturally occurring CFTR variants for which information pertaining to the severity of disease was available. Table 1 , shown in Figure 4, compares CF patient genotype with clinical phenotype. Patient genotypes are listed in order of disease severity, and are designated by both alleles separated by a /. The ΔF508/F508C compound heterozygotes are clinically unaffected, G551S homozygotes are mildly affected, and the G551 D and ΔF508 homozygotes are severely affected.

To facilitate possible comparisons between expression of Cl conductance and disease severity, we chose, where possible, mutations found in homozygotes so that only a single mutation contributed to the phenotype. To synthesize CFTR cRNA, 1 μg of linearized plasmid was incubated with 50 U of T7 RNA polymerase (BRL), 40 U RNAsin (Promega), 2 mM each ATP, UTP, CTP, GTP, in buffer supplied by the enzyme manufacturer at 37°C for 60-90 minutes. RNA was phenol extracted and precipitated, resuspended in H₂O. RNA quality and quantity was estimated by comparing to standards on an agarose gel. Mutation constructs were generated by oligonucleotide-mediated site- specific mutagenesis of 1.7 kb cDNA fragment containing the first third of the CFTR coding sequence. This fragment was cloned into the pSelect Vector (Promega), and oligonudeotides corresponding to the desired mutation were synthesized and used for second strand priming as described by the supplier. After sequencing to verify the presence of the mutation, the fragment was shuttled into a full length cDNA construct in the vector pBluescript (Stratagene). Figure 2A compares the maximal currents activated by forskolin and IBMX for oocytes injected with cRNA representing five forms of CFTR: wild type, F508C, G551 S, ΔF508 and G551D. Maximal responses were defined as current elicited by 5 mM IBMX and 50 μM forskolin. Attempts to utilize higher concentrations of IBMX were compromised by the lack of solubility of the compound in the Ringers. Values are mean ± standard error. As shown in Figure 2A, if the level of the stimulus is raised sufficiently (10-50 μM forskolin, 5 mM IBMX) even mutants associated with the most severe clinical symptoms exhibit activity which is 50-60% that of wild type. B. Effects of concentration of IBMX.

Preliminary studies of the response of wild type-injected oocytes indicated that IBMX was the most potent component of the stimulatory cocktail. To circumvent variability between oocytes, each oocyte was exposed to 10μM forskolin and a series of IBMX concentrations. Figure 2B shows steady state activated currents for the five CFTR variants described above plotted against the concentration of IBMX in the perfusion solutions expressed as a percent of maximum activation. Values represent mean ± S.E. for 3-5 oocytes (see .Example 2A). In some cases the error bars are within the size of the symbol. In each case, the sensitivity of the CFTR variant to IBMX fell as the severity of disease expression in patients increased as shown in Table 1 of Figure 4.

As expected, oocytes expressing wild type CFTR exhibited the greatest sensitivity to activating conditions. Currents in oocytes expressing F508C, a cysteine for phenylalanine substitution at position 508, were only slightly reduced relative to those seen with wild type. This change has been found in combination with ΔF508 in an unaffected individual and is considered to be neutral. Kobayashi et al., Am. J. Hum. Genet. 47:611 (1990). Next in the rank order was G5518, a serine for glycine substitution at position 551 , which is associated with mild disease. Strong et al., manuscript submitted to N. Engl. J. Med. (1991 ). Accordingly, the expression of Cl conductance, although less than that seen with wild type, was nevertheless substantial at 1 mM IBMX. The greatest reduction in sensitivity was seen with ΔF508 and G551 D (ah aspartate for glycine substitution), both of which are associated with severe disease. See Strong et al., manuscript submitted to N. Engl. J. Med. (1991); Cutting et al., Nature 346:366 (1990); Kerem et al., N. Engl. J. Med. 323:1517 (1990). EXAMPLE 3. Cl Selectivity of Membrane.

The Cl selectivity of the oocyte membranes was examined by comparing current- voltage relations in the presence of normal extracellular Cl ([Cl]₀ = 106 mM) to those obtained after reducing [Cl]₀ to 4 mM. Representative l-V plots were obtained at maximal activation in oocytes injected with either wild type or ΔF508 cRNAs, by ramping the command potential from -100 mV to +80 mV at 100 mV/second. Records were corrected for the small capacitative component of the current. Shown in Figures 3A and 3B are records for wild type and ΔF508 cRNA-injected oocytes respectively, obtained prior to activation by forskolin and IBMX, after maximal activation and after reducing [Cl]₀ from 106 to 4 mM. The small outward current seen in Figure 3A prior to activation at depolarizing potential was also noted in uninjected oocytes and appears to represent an endogenous Cl conductance activated as the result of voltage-dependant Ca²⁺ entry. This current was eliminated in Figure 3B by perfusing the oocyte with a nominally calcium-free Ringers solution. Because this maneuver had no effect on the activation of l_M in injected oocytes, all l-V relations (except prestimulation in Figure 3A) were obtained in the Ca -free condition.

As shown in Figure 3, reducing [Cl]₀ shifted the reversal potential in the positive direction by about 50 mV and reduced the conductance for outward current by about 50%. Similar results were obtained with the other mutants (data not illustrated in Figures) indicating that all of these NBF1 mutants were associated with the expression of a Cl conductance. Discussion of Data Presented in -Examples.

The data presented in the .Examples strongly suggest that the naturally- occurring defects in NBF1 of CFTR examined here do not abolish function, but rather reduce the sensitivity to an activating stimulus. This result has several important implications. First, the similarity in Cl conducting properties associated with expression of the five forms of

CFTR suggests that, if CFTR functions as a channel as has been proposed (Anderson et al., Science 253:202 (1991)), then the conduction pathway perse is not compromised by these mutations. One theory which is consistent with the proposed domain structure of CFTR (Riordan et al. Science 245:1066 (1989)) and recent results of site-directed mutagenesis (Anderson et al., Science 253:202 (1991)) is that NBF1 is not a part of the anion selective pore. The behavior of naturally-occurring NBF1 mutant CFTRs also strongly suggests that they are defective in their ability to be activated by the appropriate stimulus. Although the normal sequence of events linking stimulus to activation is not completely clear, some of the elements of the transduction process have been identified. Phosphoryiation of the R domain by activated subun^'rt of protein kinase A (PKA) appears to be necessary for normal activation of Cl conductance in intact cells transfected with wild type CFTR constructs (Cheng et al., Cell 66:1027 (1991)); in excised membrane patches from wild type-transfected cells, the presence of PKA and ATP appears to be sufficient for activation of Cl channels. Tabcharani et al., Nature 352:628 (1991). All of the variants examined here result from single amino acid changes in NBF1 , suggesting that although this region does not contain consensus sequences for cAMP-dependent phosphorylation by PKA, it is intimately involved in the transduction process. A similar conclusion was reached regarding an interaction between the R domain and NBF2 (Rich et al., Science 253:205 (1991)) in response to forskolin. It has also been reported that Cl channels in CF and normal epithelial cells can be activated by application of ATP or UTP to the external apical membrane. Knowles et al., N. Engl. J. Med. 325:533 (1991).

In previous studies, introduction of the ΔF508 construct into eukaryotic cells was not associated with the expression of an activated conductance, a result which may reflect inadequate levels of stimulation. Rich et al., Nature 347:358 (1990) ; Rommens et al., PNAS (USA) 88:7500 (1991); Gregory et al., Mol, Cell. Biol. 11:3886 (1991); Cheng et al., Cell 63:827 (1990). In the present experiments, for example Figure 2A, ΔF508-injected oocytes exposed to 10 μM forskolin did not exhibit readily detectable activation unless the concentration of IBMX exceeded 200 μM. Based on the results presented herein, appropriate pharmacological intervention in accordance with the present invention can increase the activity of mutant CFTRs to ameliorate disease symptoms related to insufficient Cl secretion.

The foregoing discussion discloses and describes merely exemplary embodiments of the present invention. One skilled in the art will readily recognize from such discussion, and from the accompanying drawings and claims, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the invention as defined in the following claims. All publications cited herein are hereby incorporated by reference.

Claims

WE CLAIM:

1. A method of treating cAMP-activatable chloride secretion insufficiencies associated with a CFTR defect in a patient, generally comprising the step of: administering to the patient a therapeutically effective amount of a compound or a pharmaceutically acceptable form thereof which elevates cAMP levels.

2. The method of Claim 1 , wherein the compound is a phosphodiesterase inhibitor.

3. The method of Claim 1 , wherein the compound is cAMP or an analog thereof.

4. The method of Claim 1 , wherein the compound is a β adrenergic receptor agonist.

5. The method of Claim 2, wherein the phosphodiesterase inhibitor is a methylxanthine.

6. The method of Claim 6, wherein the methylxanthine is 3-isobutyl-1 - methylxanthine.

7. A method of treating chloride secretion insufficiencies associated with cystic fibrosis in a patient, generally comprising the step of: administering to the patient a therapeutically effective amount of a compound, or a pharmaceutically acceptable form thereof, which elevates cAMP levels.

8. The method of Claim 6, wherein the compound is a phosphodiesterase inhibitor.

9. The method of Claim 8, wherein the phosphodiesterase inhibitor is nonspecific.

10. The method of Claim 8, wherein the phosphodiesterase inhibitor is cAMP- specific.

11. The method of Claim 8, wherein the phosphodiesterase inhibitor is an alkylxanthine.

12. The method of Claim 11 , wherein the alkylxanthine is a methylxanthine.

13. The method of Claim 12, wherein the methylxanthine is 3-isobutyl-1 - methylxanthine.

14. The method of Claim 9, wherein the methylxanthine is 1 ,3-dimethylxanthine (theophyliine).

15. The method of Claim 10, wherein the phosphodiesterase inhibitor is Rolipram.

16. A method for testing the level of Cl secretion responsiveness of a cell population carrying a CFTR defect to a preselected cAMP level elevating compound, or pharmaceutically acceptable form thereof, generally comprising the steps of: administering a preselected amount of the cAMP level elevating compound to the cell population; and measuring the change in Cl secretion levels in at least a portion of the cell population after administration.

17. The method of Claim 16, wherein the compound is a phosphodiesterase inhibitor. 18. The method of Claim 17, wherein the phosphodiesterase inhibitor is an alkylxanthine.

19. The method of Claim 17, wherein the phosphodiesterase inhibitor is cAMP- specific.

20. The method of Claim 16, further comprising the step of dosage selection, the dosage selection step further comprising the steps of: administering a preselected dosage of the compound to the cell population; comparing the change in Cl secretion levels to the change in Cl secretion levels after of administration to the same or a comparable cell population of a different preselected dosage of the compound; and selecting the preferred dosage.

23. The method of Claim 18, wherein the cell population is an in vitro cell population from a human with cystic fibrosis.

AMENDED CLAIMS

[received by the International Bureau on 12 March 1993 (12.03.93) ; original claims 1-4, 6-8,14,15,20 and 21 amended; new claims 22 and 23 added; other claims unchanged (3 pages) ]

1. A method of treating cAMP-activatable chloride secretion insufficiencies associated with a CFTR defect in a patient, generally comprising the step of: administering to the patient a therapeutically effective amount of a composition which elevates cAMP levels, the composition comprising at least two compounds or pharmaceutically acceptable forms thereof, selected from the group consisting essentially of a compound which increases the production of cAMP, a compound which decreases the breakdown of cAMP, cAMP and a cAMP analog, wherein the therapeutically effective amount is sufficient to increase chloride secretion. 2. The method of Claim 1 , wherein one of the compounds selected is a phosphodiesterase inhibitor.

3. The method of Claim 1 , wherein one of the compounds selected is cAMP or an analog thereof.

4. The method of Claim 1 , wherein one of the compounds selected is a β adrenergic receptor agonist.

6. The method of Claim 5, wherein the methylxanthine is 3 - isobutyl - 1 - methylxanthine.

7. A method of treating chloride secretion insufficiencies associated with cystic fibrosis in a patient, generally comprising the step of: administering to the patient a therapeutically effective amount of a composition which elevates cAMP levels, the composition comprising at least two compounds or pharmaceutically acceptable forms thereof, selected from the group consisting essentially of a compound which increases the production of cAMP, a compound which decreases the breakdown of cAMP, cAMP and a cAMP analog, wherein the therapeutically effective amount is sufficient to increase chloride secretion.

8. The method of Claim 7, wherein one of the compounds. selected is a phosphodiesterase inhibitor.

10. The method of Claim 8, wherein the phosphodiesterase inhibitor is cAMP- specific. 11. The method of Claim 8, wherein the phosphodiesterase inhibitor is an alkylxanthine.

12. The method of Claim 11 , wherein the alkylxanthine is a methylxanthine.

13. The method of Claim 12, wherein the methylxanthine is 3-isobutyl-1 - methylxanthine. 14. The method of Claim 12, wherein the methylxanthine is 1 , 3 - dimethyl - xanthine.

15. The method of Claim 10, wherein the phosphodiesterase inhibitor is 4 - [3 - (cyclopentyloxy) - 4 - methoxyphenyl] - 2 - pyrrolidinone.

17. The method of Claim 16, wherein the compound is a phosphodiesterase inhibitor.

18. The method of Claim 17, wherein the phosphodiesterase inhibitor is an alkylxanthine.

19. The method of Claim 17, wherein the phosphodiesterase inhibitor is cAMP-specific.

20. The method of Claim 16, further comprising the step of dosage selection, the dosage selection step further comprising the steps of: administering a preselected dosage of the compound to the cell population; comparing the change in Cl secretion levels to the change in Cl secretion levels after administration to the same or a comparable cell population of a different preselected dosage of the compound; and selecting the preferred dosage.

21. The method of Claim 18, wherein the cell population is an in vitro cell population from a human with cystic fibrosis.

22. The method of Claim 1 , wherein the compounds selected include a compound which increases the production of cAMP and a compound which decreases the breakdown of cAMP.

23. The method of Claim 22, wherein the compound which increases the production of cAMP is a β adrenergic receptor agonist and the compound which decreases the breakdown of cAMP is a phosphodiesterase inhibitor.