Composition for the treatment of cystic fibrosis

Abstract

The present invention provides compositions comprising a prostacyclin or prostacyclin analog, or a pharmaceutically acceptable salt thereof for use in preventing or treating cystic fibrosis. The invention also provides the use of a kit comprising a prostacyclin or prostacyclin analog for treating or preventing a condition associated with cystic fibrosis in a subject.

Claims

1. A method of enhancing chloride channel function of cystic fibrosis transmembrane conductance regulator (CFTR) in lung epithelial cells of a subject with cystic fibrosis who expresses the F508 mutation of CFTR, comprising administering to the subject in need thereof an effective amount of treprostinil together with one or more pharmaceutically acceptable carriers and/or additives.

2. The method of claim 1, wherein the treprostinil is in a form suitable for intravenous administration.

3. The method of claim 1, wherein the treprostinil is in a form suitable for inhalation.

4. The method of claim 1, wherein the treprostinil is in a form suitable for oral administration.

5. The method of claim 4, wherein the treprostinil is in a form selected from the group consisting of tablets and capsules.

6. The method of claim 1, wherein the treprostinil is administered in an amount of at least 1.0 ng/kg of the subject's body weight/minute.

7. The method of claim 1, wherein the treprostinil is selected from the group consisting of a sustained release form of treprostinil, an inhaled form of treprostinil, and an oral form of treprostinil.

Description

FIGURES

(1) FIG. 1: Accumulation of cAMP in IB3-1 cells after incubation with Treprostinil.

(2) FIG. 2: Activation of a Cl-current by Treprostinil in the human bronchial epithelial IB3-1 cell line transiently expressing CFTR-wt.

DETAILED DESCRIPTION OF THE INVENTION

(3) It has been surprisingly found by the inventors that prostacyclin or analogues or derivatives or a pharmaceutically acceptable salt thereof can be used for treating cystic fibrosis. Synthetic prostacyclin analogues can be for example, but are not limited to Treprostinil, Iloprost, Cisaprost or Beraprost.

(4) Suitable prostacyclin derivatives include but are not limited to acid derivatives, prodrugs, sustained release forms, inhaled forms and oral forms of Treprostinil, Iloprost, Cisaprost or Beraprost.

(5) A pharmaceutically acceptable salt of a prostacyclin or prostacyclin analogue of this invention can be formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a carboxyl functional group. According to another embodiment, the compound is a pharmaceutically acceptable acid addition salt.

(6) Specifically, Treprostinil or its derivative is useful according to the invention. Treprostinil can successfully enhance the expression of F508 CFTR and/or the chloride channel function in epithelial cells of the lung of cystic fibrosis patients.

(7) Specifically, physiologically acceptable salts of Treprostinil include salts derived from bases. Base salts include ammonium salts (such as quaternary ammonium salts), alkali metal salts such as those of sodium and potassium, alkaline earth metal salts such as those of calcium and magnesium, salts with organic bases such as dicyclohexylamine and N-methyl-D-glucamine, and salts with amino acids such as arginine and lysine.

(8) It has been surprisingly shown that a prostacyclin or analogue or derivative thereof leads to stimulation of production of cAMP in bronchoepithelial cells. This mode of action might be via induced activation of the IP receptor. Interestingly the phosphodiesterase inhibitor (PDE3 inhibitor), Anagrelide, did not induce any accumulation cAMP in experiments.

(9) Given this ability to stimulate cAMP production through the IP receptor, and the limited presence of IP receptors to a small number of cell-types (such as epithelial lung cells), a prostacyclin or analogue thereof, for example Treprostinil or a derivative or salt thereof might induce expression and gating of CFTR and mutCFTR in a specific manner can be used for treatment of CF.

(10) The current invention therefore also relates to a composition comprising a prostacyclin or prostacyclin analogue, specifically Treprostinil or its derivative, or a pharmaceutically acceptable salt thereof for use in treating cystic fibrosis as well as therapies of cystic fibrosis using a prostacyclin or prostacyclin analogue, specifically Treprostinil or its derivative, or a pharmaceutically acceptable salt thereof.

(11) Treprostinil is a synthetic analogue of prostacyclin. Treprostinil is marketed as Remodulin. Treprostinil is a (1R,2R,3aS,9aS)-[[2,3,3a,4,9,9a-hexahydro-2-hydroxy-1-[(3S)-3-hydroxyoctyl]-1H-benz[f]inden-5-yl]oxy]acetic acid monosodium salt.

(12) According to the invention the derivatives of Treprostinil can be for example acid derivatives of Treprostinil, prodrugs of Treprostinil, sustained release forms of Treprostinil, inhaled forms of Treprostinil, oral forms of Treprostinil, polymorphs of Treprostinil or isomers of Treprostinil.

(13) The composition of the invention can be present in any form which can be used for administration.

(14) The composition of the invention can be administered as liquid or powder. It can be administered topically, intravenously, subcutaneously, by inhalation or by using a nebulizer or in orally available form like tablets or capsules. Due to the high metabolic stability of some prostacyclin analogues like Treprostinil, or if provided as lipid based or pegylated forms of the prostacyclins or prostacyclin analogues, the substances can also be administered as depot medicaments.

(15) Aerosolized delivery of the prostacyclin analogue may result in a more homogeneous distribution of the agent in a lung, so that deep lung delivery is obtained. Thereby the dosage of application might be reduced to the sustained presence of the agent at the site of action in the lung.

(16) The composition can be administered with any pharmaceutically acceptable substances or carriers or excipients as known in the art. These can be for example, but are not restricted to water, neutralizing agents like NaOH, KOH, stabilizers, DMSO, saline, betaine, taurine etc.

(17) The term pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S.

(18) The term carrier refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E. W. Martin. The formulation should be selected according to the mode of administration.

(19) Treprostinil is of high metabolic stability which specifically allows for administration by various routes.

(20) The amount of the inventive composition can be selected by any skilled person, preferably the amount of the prostacyclins or prostacyclin analogues or pharmaceutically acceptable salts thereof, specifically of Treprostinil is at least 1.0 ng/kg of body weight/min.

(21) The invention further provides a kit for treating or preventing a condition associated with cystic fibrosis in a subject, comprising (i) an effective amount of a prostacyclin or prostacyclin analogue or derivative or a pharmaceutically acceptable salt thereof, (ii) one or more pharmaceutically acceptable carriers and/or additives, and (iii) instructions for use in treating or preventing cystic fibrosis.

(22) According to the embodiment of the invention, the kit comprising (i) an effective amount of a prostacyclin or prostacyclin analogue or a pharmaceutically acceptable salt thereof, (ii) one or more pharmaceutically acceptable carriers and/or additives, and (iii) instructions for use in treating or preventing cystic fibrosis is provided for use in the treatment or prevention of a condition associated with cystic fibrosis in a subject, preferably a human.

(23) Said component (i) can be in a form suitable for intravenous administration, for inhalation or for oral administration. The component (i) can be in a form suitable for intravenous administration, for inhalation or for oral administration.

(24) More specifically, the present invention provides the use of a kit comprising i) an effective amount of Treprostinil or a pharmaceutically acceptable salt thereof, (ii) one or more pharmaceutically acceptable carriers and/or additives, and (iii) instructions for use in treating or preventing cystic fibrosis in the treatment or prevention of a condition associated with cystic fibrosis in a subject, preferably a human.

(25) Specifically, the component (i) is a pharmaceutically acceptable salt of Treprostinil. According to a specific embodiment for the use of the kit, component (i) is in a form suitable for intravenous administration or suitable for inhalation or suitable for oral administration.

(26) The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Different embodiments of the present invention have been described according to the present invention. Many modifications and variations may be made to the techniques described and illustrated herein without departing from the spirit and scope of the invention. Accordingly, it should be understood that the examples are illustrative only and are not limiting upon the scope of the invention.

EXAMPLES

Example 1

(27) IB3-1 cells were plated on 6 well-plates (0.2*106 cells/well) in complete growth medium (LHC-8+5% FCS). The following day, the adenine nucleotide pool was metabolically labelled by incubation with [3H]adenine (1 Ci/well) in Dulbecco's Modified Eagle Medium (DMEM) containing adenosine deaminase (1 unit/ml) for 4 h. Cyclc AMP formation was stimulated by 20 M forskolin or the PGI2-analogue treprostinil (Remodulin). The assay was performed in triplicates. The formation of [.sup.3H]cAMP was determined by sequential chromatography on Dowex 50WX-4 and neutral alumina columns followed by liquid scintillation counting of the eluate.

(28) Treprostinil caused a concentration-dependent accumulation of cAMP in IB3-1 cells (FIG. 1). Half-maximum stimulation was seen in the range of 0.3 to 1 M.

Example 2

(29) IB3-1 cells endogenously express only mutated CFTR-F508, which is retained within the cells. Using appropriated manipulations (e.g., pharmacochaperones or low temperature incubations), it is possible to translocate the mutant CFTR-F508 from the endoplasmic reticulum to the ER; when inserted at the cell surface, a Cl-conductance can be stimulated by elevating cAMP. The resulting Cl-conductance, however, is small. In order to unequivocally prove that the cAMP accumulation induced by Treprostinil translated into an activation of CFTR, we transiently expressed a GFP-tagged version of wild type CFTR (the GFP tag allowed for the identification of cells that expressed the protein at the cell surface). As can be seen from FIG. 2, Treprostinil caused a robust activation of the current induced by a depolarization from 40 mV holding potential to +60 mV. The maximum effect was delayed, i.e. it was only observed several s after wash-in of the compound. Likewise, there was also a hysteresis in the turn-off reaction; the current decayed to basal only 100 s after washout. These delayed responses reflect the (i) intervening signalling cascade (i.e., the receptor-dependent activation of G.sub.s, G.sub.s-dependent activation of cAMP formation and the ensuing protein kinase A-dependent phosphorylation of CFTR) and (ii) the delayed deactivation of increased cAMP by phosphodiesterases. Similar delays were also seen, if cells were stimulated with forskolin, a direct activator a adenylyl cyclase, which was used as a positive control. These observations prove that Treprostinil can activate CFTR in bronchial epithelial cells.

(30) Methods:

(31) Electrophysiology

(32) The whole cell patch clamp technique was used for current recordings performed at 221.5 C. using an Axoclamp 200B patch clamp amplifier (Axon Instruments). Pipettes had resistances between 1 and 2 M when filled with the recording pipette solution (composition: 110 mM CsCl, 5 mM EGTA, 2 mM MgCl.sub.2, 1 mM K.sub.2.ATP, 10 mM Hepes, pH adjusted to 7.2 with CsOH). Voltage-clamp protocols and data acquisition were performed with pclamp 6.0 software (Axon Instruments). Data were low-pass filtered at 2 kHz (3 dB) and digitized at 10-20 kHz. Cells were continuously superfused with external solution (composition: 145 mM NaCl, 4.5 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 5 mM glucose, 10 mM Hepes, pH adjusted to 7.4 with NaOH). When indicated, the external solution contained Treprostinil (10 M) or forskolin (5 M), switching between solutions was achieved by electronically controlled pressure valves.

(33) Cell Culture:

(34) I3B-1 cells were grown on dishes (Nunc, 3.5 cm diameter) covered with fibronectin (10 g/mL) rat collagen 1 (30 g/mL) and BSA 10 g/mL) in LHC-8 medium (Gibco) containing 5% fetal calf serum (FCS). Cells were transiently transfected with a plasmid driving the expression of human GFP-tagged wild type CFTR by using Lipofectamine Plus (Invitrogen) according to the instructions of the manufacturer.

(35) Representative current amplitudes recorded in the whole cell patch clamp configuration at +60 mV. A transiently transfected I3B-1 cell expressing GFP-tagged wild type CFTR was selected under fluorescent light and clamped to a holding potential at 40 mV. Depolarization was induced by a voltage step to +60 mV for 50 ms and the current amplitude was recorded. Wash-in of Treprostinil (10 M final concentration, TP) was initiated at the time point 50 s and terminated at 125 s. Forskolin was washed in at 275 s and was removed at 375 s. Results are shown in FIG. 2.