WO1998019679A1

WO1998019679A1 - Cyclopentylxanthine derivatives for use in the treatment of cystic fibrosis

Info

Publication number: WO1998019679A1
Application number: PCT/GB1997/003036
Authority: WO
Inventors: Margaret Ann Mcpherson; Robert Leslie Dormer; Malcolm Martin Claude Pereira
Original assignee: University Of Wales College Of Medicine
Priority date: 1996-11-05
Filing date: 1997-11-05
Publication date: 1998-05-14
Also published as: GB9622981D0; AU4875197A; CA2270355A1; EP0973521A1

Abstract

The use of derivatives of theophylline for the preparation of medicaments suitable for the treatment of Cystic Fibrosis is described. In particular, the use of 8-cyclopentyl theophylline (CPT) is described for the preparation of medicaments for the treatment of cystic fibrosis. Such medicaments may be administered orally or directly to the lung, for example, in the form of an aerosol.

Description

CYCLOPENTYLXANTHINE DERIVATIVES FOR USE IN THE TREATMENT OF CYSTIC FIBROSIS

FIELD OF THE INVENTION

The present invention relates to the use of derivatives of theophylline for the preparation of medicaments suitable for the treatment of cystic fibrosis. In particular, it relates to the use of 8-cyclopentyl theophylline (CPT) for the preparation of medicaments for the treatment of cystic fibrosis. Such medicaments may be administered orally or directly to the lung, for example, in the form of an aerosol.

BACKGROUND TO THE INVENTION

Cystic fibrosis (CF) is one of the most common lethal inherited disorders occurring in Caucasian populations. It is characterized as an exocrinopathy involving disturbances in mucin secretion, i.e. resulting in an altered composition of epithelial cell mucous secretions, and in electrolyte transport, in particular chloride transport. These manifestations of the disorder are caused by mutations in the cystic fibrosis (CF) gene, which encodes the cystic fibrosis transmembrane conductance regulator protein (CFTR) .

Various hypotheses have been put forward to connect the mutations in the CFTR gene with the overt symptoms of the disease. Pollard et al in US Patent No. 5 366 977 claimed that mutations in the CFTR gene resulted in an abnormal potential difference across CF epithelia. The abnormality, they state, is due to a reduced cellular apical Cl^— conductance, with consequential abnormal chloride and sodium transport across mucous membranes . Pollard claimed to have found a method of treating cells having a reduced apical Cl^— conductance, such as cystic fibrosis cells, by contacting the cells with 8- cyclopentyl-1, 3-dipropylxanthine (CPX) or xanthine amino congener (XAC) .

In US Patent No. 5 366 977 it stated that other compounds that resemble theophylline in basic structure had been tested but had not been found to be useful in the treatment of cystic fibrosis. 3-Isobutyl-l-methylxanthine (IBMX) , a methyl xanthine structurally similar to theophylline, was non specific in activity and highly toxic and, therefore, lacked utility in the treatment of CF. Also ineffective in the activation of chloride efflux were 2- thio-CPX and 8-cyclopentyl -theophylline (CPT) . Similarly, substitution of either of the propyl groups of CPX with a one-carbon group generated a compound that was ineffective in activating chloride efflux from CF cells. Pollard concluded that, clearly, minor structural differences have a significant, if not substantial, impact on the effectiveness of the compound in the treatment of CF .

It has now, unexpectedly, been found that the CFTR mediated defect in mucin secretion can be corrected using CPT in the absence of an excessive rise in cyclic AMP (cAMP) levels, thus offering a specific drug treatment for cystic fibrosis which will correct the basic gene protein defect, whilst retaining cAMP at levels within the normal physiological range.

Using a CFTR antibody-inhibited submandibular acini model it has now been found that the defective mucin secretory response to the β-adrenergic agonist isoproterenol can be corrected by the Al receptor agonist CPT, but not by the A2 receptor agonist DMPX (3,7 - dimethyl -1-propargyl xanthine) . The results indicated that CPT was not acting via an Al receptor mechanism and that it did not potentiate isoproterenol induced increases in cyclic AMP.

According to one aspect of the invention, there is provided the use of a compound of formula (1)

wherein R¹ and R² are the same or different and each represents an alkyl group having from 1 to 5 carbon atoms, provided that R¹ and R² are not both propyl groups, for the manufacture of a medicament for the treatment of cystic fibrosis .

Preferably the compound of formula (1) used for the manufacture of a medicament is 8-cyclopentyl-theophylline, where R¹ and R² in formula (1) are both methyl groups.

According to a second aspect of this invention there is provided a pharmaceutical formulation comprising a compound of formula (1)

R^:

wherein R¹ and R² are the same or different and each represents an alkyl group having from 1 to 5 carbon atoms, provided R¹ and R² are not both propyl groups, together with a pharmaceutically acceptable carrier therefor.

According to yet another aspect of the invention there is provided a compound of formula (1)

wherein R¹ and R² are the same or different and each represents an alkyl group having from 1 to 5 carbon atoms, provided that R¹ and R² are not both methyl groups or propyl groups . DESCRIPTION OF THE DRAWINGS

Figure 1 shows the structure of IBMX, DMPX and CPT; Figure 2 shows the effects of different concentrations of CPT and DMPX on mucin secretion from normal rat submandibular acini .

Figure 3 shows the effect of CPT on CFTR antibody inhibited β-adrenergic stimulation of mucin secretion.

The medicament or pharmaceutical formulation according to the invention which contains a compound of formula (1) may be presented in a form suitable for oral administration in a pharmaceutical vehicle convenient for that administrative route. Thus, for example, the medicament may be presented as tablets, capsules, ingestible liquid or a powder preparation. Such formulations can include pharmaceutically acceptable carriers known to those skilled in the art. Formulations suitable for oral administration further include lozenges, pastilles, aerosols and mouthwashes .

Alternatively, the medicament or pharmaceutical formulation may be administered direct to the lung via the nasal passage. Formulations suitable for nasal administration, where the carrier is a solid, include powders. Where the carrier is a liquid the formulation can be administered as a nasal spray or aerosol, or as drops. In yet another alternative, the medicament may be presented in a formulation suitable for parenteral or intravenous administration, such as aqueous or non-aqueous sterile injectable solutions. Such solutions may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Alternatively, the formulation for parenteral administration may be presented as an aqueous or non aqueous sterile suspension which may include suspending agents and thickening agents.

Appropriate dosages of the compounds and formulations administered to the patient will depend on the severity of the condition and the size and age of the patient. Determining an acceptable or optimal dosage will involve the balancing of the level of therapeutic benefit against the risk of deleterious side effects. For oral or parenteral administration a therapeutically effective dose may typically comprise administering the active ingredient at the rate of 0.001 to 25 mg/kg body weight per day, and more preferably at a rate of from 0.01 to 0.5 mg/kg/day. For nasal administration the medicament may contain from 0.001 to 0.01% w/w of the active ingredient.

CPT (8-cyclopentyl theophylline) may be prepared by mixing 5, 6-diamino-l , 3 -dimethyl uracil with 1 - 1.25M equivalent of cyclopentanecarboxylic acid followed by heating at 120 - 200°C for 1 to 4 hours to give the solid amide. The solid amide is then powdered and heated with 5 -

10% sodium hydroxide until solution is complete. The solution is then saturated with carbon dioxide and the 8- cyclopentyl theoaphylline recrystallised from dry ethanol . To prepare compounds of formula (1) where R¹ and R² are other than methyl a similar process may be employed but using a 5, 6-diamino-uracil substituted with different appropriate 1,3-dialkyl groups. According to yet another aspect of the invention there is provided a method of treating cystic fibrosis comprising administering a compound of formula (1)

! R²

where R¹ and R² are the same or different and each represents an alkyl group having from 1 to 5 carbon atoms provided that R¹ and R² are not both propyl groups .

The invention will now be further described by way of reference to the following examples.

Example 1 Methods

Production of anti-peptide CFTR antibodies A peptide consisting of 14 amino acids (524-537) , residing in the first nucleotide binding domain (NBD) region, of CFTR, was synthesized and coupled to keyhole limpet haemocyanin (KLH) (lOmg peptide/8mg KLH) by Cambridge Research Biochemicals Ltd, Northwich, Cheshire. The peptide sequence was searched for in the Swissprot database (BLASTP and FASTA3_T, 1997) and only CFTR was found to contain a perfect match. Antisera were prepared as described in C.

Lloyd Mills et al Bioche . Biophys . Res. Comm, [88(3), (1992) , 1146-52] and affinity-purified using KLH or peptide coupled to CH-Sepharose 46 (Pharmacia) . The IgG content of antisera was estimated either following precipitation with 24% Na₂S0₄ for 2h at room temp. (KLH-Sepharose purified) or directly (peptide-Sepharose purified) , using the BioRad protein assay kit.

Isolation of rat submandibular acini, incorporation of antibodies into intact acini and measurement of mucin secretion.

Procedures were carried out by minor modification of those previously described in C. Lloyd Mills et al .

Briefly, acini were pulse-chase labelled with [³H] - glucosamine and suspended in TES-buffered saline (lOmM TES,

(N-tris [Hydroxymethyl] methyl-2-aminoethanesulfonic acid;

2 - ( [2 -Hydroxy- 1 , 1 -bis (hydroxymethyl ) -ethyl ] amino) ethanesulphonic acid)) . pH 7.4 containing 143mM NaCl, 4.7mM KC1, 1. ImM MgCl_2/ lmg/ml bovine serum albumin (BSA) ) . To 200/il of acini suspension, 800μl of either lOmM TES, pH 7.4 (swollen) or TES-buffered saline (unswollen) , each containing 5mM ATP and antibody (approx lmg IgG/ml) was added for 1.5 min at room temperature, followed by washing and resuspension in Krebs-Henseleit bicarbonate (KHB) buffer containing 20mg/ml BSA. Following a 15 min recovery incubation at 37°C in KHB buffer, acini were washed and incubated under experimental conditions at 37°C. Isoproterenol and 3-isobutylmethylxanthine (IBMX) were dissolved directly into KHB medium at the concentrations used; the adenosine antagonists were dissolved initially in dimethylsulphoxide (DMSO) and diluted to give a final concentration of 1% DMSO in the incubations. An equivalent amount of DMSO was added to control incubations. [³H] - labelled mucins, released -into the medium at zero time and after 30 min, were acid-precipitated and their radioactivity measured. Protein content of cell pellets was determined using the BioRad protein assay kit and mucin release over 30 min expressed as dpm/mg protein or as % basal secretion to take account of variation in unstimulated mucin release between experiments.

Measurement of cyclic AMP content Aliquots of acini suspensions (0.25ml) were added to an equal volume of ice cold trichloroacetic acid (20%) , extracted and assayed using a specific radioimmunoassay kit for cyclic AMP (Amersham) .

Results The actions of selective Al and A2 receptor antagonists on correction of CFTR-antibody-inhibited 6-adrenergic stimulation of mucin secretion has been investigated. Fig. 1 shows the structure of CPT which is more selective for the Al receptor, and DMPX (3 , 7-dimethyl-1-propargylxanthine) which is more selective for the A2 receptor compared to that of IBMX.

Fig.2 shows the effects of different concentrations of CPT and DMPX on mucin secretion from normal rat submandibular acini, isolated as described in the Methods. DMPX was the more effective stimulator, and at ImM increased mucin secretion to a similar degree to that obtained with isoproterenol or IBMX (Table 2) . CPT was much less potent and at ImM gave a small but significant increase above basal levels. Doses of CPT close to the K_± for Al adenosine receptor antagonism did not stimulate mucin secretion (lOnM CPT; 104% basal) . The data indicate that the effect of CPT in increasing mucin secretion is unlikely to be related to Al receptor antagonism.

IBMX was shown to correct defective CFTR function at a maximum concentration which did not significantly stimulate mucin secretion. Thus a concentration of CPT giving marginal stimulation (ImM) and a similar concentration of DMPX were tested for correction of defective CFTR function. Table 1 shows that the A2 adenosine receptor antagonist, DMPX did not correct defective β-adrenergic stimulation of mucin secretion in CFTR antibody containing cells. As shown previously, mucin secretion in response to isoproterenol from cells containing CFTR antibody, introduced by hypotonic swelling as described in the Methods, was significantly decreased compared to cells swollen in an equivalent amount of non-immune IgG. It can be seen (Table 1) that DMPX had no effect on the normal isoproterenol response in cells containing non-immune IgG (swollen + NI) or on the decreased isoproterenol response in CFTR antibody-containing cells (swollen + NBD) .

Fig. 3 shows that the Al receptor antagonist CPT corrected the defective isoproterenol -stimulated mucin secretion in submandibular cells containing CFTR antibody, at mM concentrations. The actions of CPT in increasing the CFTR antibody-inhibited response were almost as effective as that of mM cpt-cyclic AMP (8- (4-chlorophenylthio) -cyclic AMP) which has been previously shown to restore secretory responsiveness to approx: 75% of that seen in cells containing similar amount of non-immune IgG. CPT, at high concentration, corrected the CFTR antibody-inhibited mucin secretory response suggesting that a mechanism other than Al receptor antagonism was operating. Since correction was effected by IBMX, which massively potentiates the cyclic AMP rise induced by isoproterenol and by cpt-cyclic AMP, it was proposed that an excessive increase in cyclic AMP is the mechanism by which defective CFTR function is restored. It was therefore investigated whether high concentrations of adenosine receptor antagonists increase cyclic AMP in submandibular cells and potentiate the isoproterenol induced cyclic AMP rise. Our previous results have shown that the cyclic AMP response of non-swollen cells or cells swollen in the presence of either non-immune IgG or CFTR antibody are not different. Table 2. shows that CPT is a less effective stimulator of mucin secretion than IBMX; but it induces greater increases in cyclic AMP. DMPX, which did not correct CFTR function, increases mucin secretion to a greater degree than CPT (Fig. 2) and induces increases in cyclic AMP greater than those induced by isoproterenol alone

(129.2 ± 7.1% of isoproterenol stimulation; p<0.01). In the presence of isoproterenol, CPT does not increase the isoproterenol induced cyclic AMP rise (Table 2) , whereas

DMPX gave a 157.4 ± 12.4% (p<0.01) increase and IBMX at a concentration which corrected CFTR function gave an approx.

2 fold increase above levels induced by isoproterenol alone

(C. Lloyd Mills et al) . Neither of the adenosine receptor antagonists, nor IBMX can increase mucin secretion above the maximum in response to the 6-agonist. Table 2 also shows that unlike IBMX, CPT did not potentiate the cyclic AMP rise induced by isoproterenol. Thus, correction of CFTR function did not correlate with increase in mucin secretion or excessive increase in cyclic AMP.

In this experiment, a CFTR antibody containing cell model has been used for monitoring defective CFTR activity in mediating mucin secretion. The results show that the Al receptor antagonist CPT can mimic the action of IBMX in correcting defective 6- adrenergic stimulation of mucin secretion in CFTR antibody containing cells. However, the data suggest that the mechanism of action is not adenosine receptor antagonism: since high (mM) concentrations were required and a similar concentration of DMPX (Ki for Al receptor antagonism 45μM) would be predicted to act as an Al receptor antagonist and did not correct CFTR function (Table 1) .

IBMX also acts as a non-selective cyclic nucleotide phosphodiesterase inhibitor which increases cyclic AMP levels in submandibular acini and potentiates the cyclic AMP rise induced by isoproterenol. Furthermore, since the correction of CFTR-mediated mucin secretion was mimicked by cpt-cyclic AMP, it was postulated that excessive increase in cellular cyclic AMP could be a mechanism by which CFTR activity is restored. This hypothesis was tested by investigating the effects of the adenosine receptor antagonist on cyclic AMP levels in the presence or absence of isoproterenol. It is clear from the results in Table 3 that CFTR function could be corrected without potentiating the isoproterenol induced cyclic AMP rise although CPT increased cyclic AMP levels alone.

The present results have demonstrated a lack of correlation between correction of the CF secretory defect and excessive increase in cyclic AMP. It is possible that an inherent structural feature of these closely related compounds (Fig. 1) enables them to interact with CFTR itself, thus in the cell model neutralising the action of the CFTR antibody.

It is interesting that IBMX can apparently correct the function of the most common mutant form of CFTR (dF₅₀₈ CFTR) with loss of phenylalanine at position 508 in the protein in severely affected CF submandibular glands. There is evidence that at least some dF₅₀₈ CFTR reaches the apical membrane in CF airway epithelial cells. Whether this is able to be directly activated by IBMX and CPT to give significant effects on function requires investigation. Another possible mechanism is that a CFTR bypass exists and can be stimulated by IBMX and related compounds. This however seems to be unlikely at least in submandibular glands since cyclic AMP and Ca²⁺-dependent stimulators of mucin secretion converge via a common pathway.

Table 1. Effect of DMPX on isoproterenol -stimulated mucin secretion in rat submandibular acini containing IgG or CFTR NBD antibody.

Mucin Secretion (% basal) Swollen + NI Swollen + NBD

Isoproterenol (lOμM) 228.0 ± 35.0 155.0 ± 28.0 Isoproterenol + 245.1 ± 53.2 167.8 + 14.8 DMPX (ImM) Results are mean ± SEM for 4 experiments

Table 2. Actions of adenosine receptor antagonists on mucin secretion and cyclic AMP levels in rat submandibular acini.

Mucin Secretion Cyclic AMP (dpm/mg protein) (pmol/mg protein)

No addition 794 ± 78 8.8 ± 2.0 IBMX (ImM) *3092 ± 449 '27.8 ± 4.9 CPT (ImM) *1826 + 303 '113.5 ± 20.5 Isoproterenol (lOμM) *3311 ± 324 199.2 ± 37.8 Isoproterenol + 3240 ± 348 178.8 + 37.6 CPT ( IMm)

Mucin release was measured at 30 min and cyclic AMP at

5 min as described in the Methods, in the presence or absence of agonists. Results are means ± SEM for the number of experiments shown in parentheses. Significance of differences was assessed by Students t-test: *, p<0.0005 for difference from basal mucin secretion. All cyclic AMP values were significantly different (p_<0.0001) from basal; ^!, p<0.02 for difference from cyclic AMP in the presence of isoproterenol .

Example 2 Tablet Formulation

8-Cyclopentyl theophylline 200 mg

Nonpareil seeds Lactose

Hydroxypropyl methyl cellulose Magnesium stearate

with added trace amounts of Glyceryl monostearate White wax Cellulose acetate Diethyl phthalate Acetyl alcohol Myristoyl alcohol

Example 3 Nasal Drops

A solution suitable for use as nasal drops is prepared by dissolving CPT (0.2 mg/ml) in isotonic saline containing hypromellose, with the optional addition of a suitable buffer.

Example 4 Intravenous Formulation

A solution suitable for intravenous injection is prepared by dissolving CPT (0.2 mg/ml) in isotonic saline containing sodium EDTA, with the optional addition of a suitable buffer.

Example 5 Aerosol Formulation A solution suitable for aerosolisation into the lungs is prepared by mixing CPT (0.2 mg/ml) with oleic acid and priolene, which mixture can then be delivered to the lungs using the following propellents - 11 (trichlorofluoromethane) 12 (dichloro difluoromethane) .

Claims

1. The use of a compound of formula (1) ,

R

wherein R¹ and R² are the same or different and each represents an alkyl group having from 1 to 5 carbon atoms, provided that R¹ and R² are not both propyl groups , for the preparation of a medicament for the treatment of cystic fibrosis .

2. The use according to Claim 1 of a compound of formula (1) wherein R¹ and R² are both methyl groups.

3. The use according to Claim 1 or Claim 2 wherein the medicament is suitable for oral or parenteral administration.

4. The use according to Claim 6 wherein the medicament is administered in an amount of 0.001 to 25 mg/kg body weight per day.

5. The use according to Claim 1 or Claim 2 wherein the medicament is suitable for administering directly to the lung.

6. The use according to Claim 5 wherein the medicament is a pharmaceutically acceptable aerosol .

7. The use according to Claim 6 wherein the aerosol contains from 0.001 to 0.01% w/w of the compound of formula (1) •

8. A compound of formula (1)

R

wherein R¹ and R² are the same or different and each represents an alkyl group having from 1 to 5 carbon atoms, provided R¹ and R² are not both methyl groups or propyl groups .

9. A pharmaceutical formulation comprising a compound of formula (1)

R^:

10. The formulation according to Claim 9 which is suitable for oral administration.

11. The formulation according to Claim 9 which is suitable for administration direct to the lung.

12. The formulation according to Claim 11 which is in the form of a pharmaceutically acceptable aerosol.

13. The formulation according to Claim 9 which is suitable for parenteral administration.

14. A method of treating cystic fibrosis comprising administering a compound of formula (1)

R² where R¹ and R² are the same or different and each represents an alkyl group having from 1 to 5 carbon atoms provided that R¹ and R² are the same or different and each represents an alkyl group having from 1 to 5 carbon atoms provided that R¹ and R² are not both propyl groups .

15. The method of claim 14 wherein the compound of formula

(1) is administered orally.

16. The method of claim 14 wherein the compound of formula

(1) is administered intranasally .

17. The method of claim 14 wherein the compound of formula

(1) is administered parenterally .