WO2016179279A1 - Materials and methods for the treatment of cystic fibrosis - Google Patents

Abstract

In preferred embodiments, the invention pertains to the treatment of Cystic Fibrosis (CF) using an agonistic of CaSR. In a specific embodiment, the subject invention provides a method of treating CF in a subject by administering to the subject a composition comprising a calcimimetic. The calcimimetic can be administered alone or in combination with a CaSR orthosteric agonist and/or an agent capable of stimulating colonic HCO₃- secretion in CFTR-independent manner. The composition can be administered to the subject via inhalation. Accordingly, the invention further pertains to compositions comprising a calcimimetic and optionally, further comprising a CaSR orthosteric agonist and/or an agent capable of stimulating colonic HCO₃- secretion in CFTR- independent manner, in the form suitable for administration to the subject via inhalation. Accordingly, devices for administering the compositions of the current invention via inhalation are also provided.

Description

DESCRIPTION

MATERIALS AND METHODS FOR THE TREATMENT OF CYSTIC FIBROSIS

CROSS-REFERENCE TO A RELATED APPLICATION

This application claims the benefit of U.S. Provisional Patent Application Serial No.

62/156,606, filed May 4, 2015, which is incorporated herein by reference in its entirety.

This invention was made with government support under HD079674 (SXC) awarded by National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Defective bicarbonate (HCO₃ ^') secretion by transport epithelia is a hallmark of pathophysiology in patients with cystic fibrosis (CF). In CF, defective bicarbonate secretion results in defective mucus secretion, leading to respiratory airway epithelial malfunction and infections, bowel obstructions, pancreatic insufficiency, liver disease and infertility. Despite worldwide efforts in searching for methods to correct this defect, so far an efficacious method is not found. Current CF therapies include CFTR gene therapy, CFTR chaperones therapy, CFTR stimulators and other approaches are designed to rescue CFTR function.

BRIEF SUMMARY

The subject invention provides methods for treating cystic fibrosis (CF) in a subject by administering to the subject a composition comprising a CaSR agonist. Specifically exemplified herein is the use of a CaSR type II agonist together with a type I agonist and/or a co-factor. In one embodiment the agonist is a calcimimetic in combination with a CaSR orthosteric (type I) agonist and/or an agent capable of stimulating colonic HCO₃ secretion in a CFTR-independent manner.

In addition to treating CF, the compositions and methods of the subject invention can be used to generally promote and/or improve mucosal health.

In one embodiment, the method comprises treating a subject by administering a composition of the invention to the subject via inhalation. In another embodiment, the composition is administered via enema. In one embodiment the patient has been diagnosed with CF.

Accordingly, certain embodiments of the invention provide compositions for treating CF in a form suitable for administration to the subject via inhalation. Devices for administration of the compositions of the invention to a subject via inhalation are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent application contains at least one drawing executed in color. Copies of this patent with color drawings(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

Figures 1A-1B. Anion transport defects in CF.

Figures 2A-2B. CaSR can uncouple HC0₃ secretion mediated by CFTR from non CFTR transporters and differentially regulate them.

Figure 3. Experimental approach 1 : The Ussing chamber pH-stat set up used to monitor and measure HC0₃ secretion by colonic mucosa.

Figure 4. Experimental Approach 2: Measurement of HC0₃ ^~ secretion by colonic mucosa in the presence of apical NPPB/Glibenclamide.

Figures 5A-5F. Activation of CaSR by R568 inhibits CFTR-mediated HC0₃ secretion

(JHCO3) and I_sc but stimulates non CFTR-mediated HC0₃ secretion (JHCO3) and I_sc. Physiological approach assayed with, versus without, lumen CI^".

Figures 6A-6F. Activation of CaSR by R568 inhibits CFTR-mediated HC0₃ secretion (JHCO3) and I_sc but stimulates CFTR-independent HC0₃ secretion (JHCXM) and I_sc. Pharmacological approach assayed with, and without, CFTR transporter inhibitors or AE inhibitor DIDS.

Figures 7A-7C. Effects of extracellular Ca²⁺ and CaSR agonist R568 on basal HC0₃ ^~ secretion. A-B: Representatives of 3 recordings of luminal pH responses of distal colonic mucosa to absence and presence of a serosa-to-lumen directed HCO^ gradient and carbachol (CCH, 100 μΜ, serosa) (A), and normal vs. low [Ca²⁺]₀. (B). The presence or absence of HC0₃ ^~ in the lumen or serosa was shown as indicated. C: Serosal-to-mucosal JHCO_S responses to R568 (10 uM, serosa). The basal tissue resistance (Ω cm²) and I_sc ^Eq/hr/cm²) were 69 ± 7 and 1.44 ± 0.32 at 0.5 mM [Ca²⁺]₀ vs. 72 ± 6 and 1.10 ± 0.10 at 1.2 mM [Ca²⁺]₀ (p > 0.05). Data are means ± SEM of 5 experiments. * p < 0.05 vs. control (with no R568).

Figures 8A-8B. Effect of CaSR agonist R568 on acid-induced HC0₃ ^~ secretion. A: Representatives of pH recovery responses in the presence vs. absence of R568 (10 μΜ, serosa). The addition of R568 is shown as indicated. B: Summary of ΔρΗ recovery rates (stimulated peak values above basal levels before additions of R568 or vehicle). Basal levels of acid-induced pH recovery were 0.015 ± 0.001 pH unit/min/cm². The tissue resistance (Ω cm²) and I_sc ^Eq/hr/cm²) at the end of 60 min experiments were 50 ± 12 and 1.74 ± 0.20 without challenge and 46 ± 10 and 1.97 ± 0.23 with acid challenges (p > 0.05). Data are means ± SEM of 5 experiments. * p < 0.05 vs. control (with no R568).

Figures 9A-9D. Effect of CaSR agonist R568 on secretagogue-induced HC0₃ ^~ secretion and HC0₃ ^~ current. Shown are J_Hco₃ (A) and I_sc (C) responses to forskolin (FSK, 500 nM, serosa) ± R568 (10 μΜ, serosa), assayed in lumen CP-containing Ringer solution. The changes induced by FSK in the absence vs. presence of R568 are summarized in B (AJ_Hco₃ ^FSK) and D (AI_SC ^FSK). The basal tissue resistance and I_sc under these conditions were: 68 ± 4 Ω cm² and 1.51 ± 0.22 μΕς/Ιντ/αη². Data are means ± SEM of 8 experiments. ** p < 0.01 vs. control (with no R568); ## p < 0.01 vs. FSK.

Figures 10A-10B. Activation of CaSR stimulates CI -dependent HC0₃ ^~ secretion. A:

J_HCO3 responses to the presence vs. absence of lumen CP. B: the AJ_Hco3^C1 0-e., CP-dependent HC⁽V secretion) responses to the presence vs. absence of R568 (10 μΜ, serosa). The basal tissue resistance (Ω cm²) and I_sc (uEq hr/cm²) were 68 ± 1 1 and 1.44 ± 0.32 in the presence vs. 95 ± 15 and 1.01 ± 0.08 in the absence of lumen Cl^~ (p > 0.05). Data are means ± SEM of 3-5 experiments. * p < 0.05 and ** p < 0.01 vs. control (1^st bar).

Figures 11A-11D. Activation of CaSR stimulates SCFA-dependent HC0₃ ^" secretion. Two equally longitudinally divided pieces of mucosa from same colon were mounted into 2 Ussing chambers. One piece was treated first in the presence then absence of lumen SCFA whereas the other piece was treated first in the presence then absence of serosa HC0₃ ^~ . Shown are representative recordings (A & B) and quantitative summary (C) of these J_Hco₃ responses. The AJ_Hco3^SCFA (i-e-, SCFA-dependent HC0₃ ^~ secretion) was calculated and its responses to the presence vs. absence of R568 (10 μΜ, serosa) are shown in D. The basal tissue resistance (Ω cm²) and I_sc ^Eq/hr/cm²) were 126 ± 26 and 0.71 ± 0.17 in the presence vs. 95 ± 5 and 1.01 ± 0.08 in the absence of lumen 25 mM isobytyrate (p > 0.05). Data are means ± SEM of 3-5 experiments. * p < 0.05 and ** p < 0.01 vs. control (1^st bar).

Figures 12A-12D. Activation of CaSR inhibits cAMP-dependent HC0₃ ^" secretion and HC0₃ ^_ current. Shown are JHCOS (A) and I_sc (C) responses to forskolin (FSK, 500 nM, serosa) ± R568 (10 μΜ, serosa), assayed in lumen CP-free Ringer solution. The changes induced by FSK in the absence vs. presence of R568 are summarized in B (AJ_Hco₃ ^FSK) and D (AI_SC ^FSK). The basal tissue resistance and I_sc under these conditions were 103 ± 3 Ω cm² and 0.87 ± 0.05 μEq/hr/cm². Data are means ± SEM of 4-7 experiments. ** p O.01 vs. control (1^st bar); ## p < 0.01 vs. FSK alone (2^nd bar).

Figures 13A-13F. R568 fails to stimulate CP- and SCFA- dependent and inhibit cAMP- dependent HC0₃ secretion in colons of CaSR null mice. Colonic mucosa from wild type (CaSR^+/+) (A-C) and knockout (CaSR ^_) mice (D-F) were isolated and treated, and activities of the three HCCV transporters assayed as in rats. Data are means ± SEM of 4-6 experiments. * p < 0.05 vs. control (1^st bar).

Figure 14. Cellular model of CaSR regulation of HCC secretion in colonocytes of rat distal colon. Upper panel: R568 acting via CaSR causes enhancement of HCCV secretion in surface epithelial cells by stimulation of luminal Cl -dependent HCCV secretion via apical C17HCCV exchange and stimulation of luminal SCFA-dependent HCCV secretion mediated by apical SCFA/HCCV exchange. Lower panel: CaSR reduces HCCV secretion in the crypt epithelial cells through inhibition of cAMP-dependent HCCV secretory process that may involve a NPPB/glibenclamide-sensitive apical anion channel such as CFTR and/or a basolateral HCCV entry mechanism(s). +, stimulation; -, inhibition.

Figure 15. Cation transport defect in CF.

Figure 16. CaSR inhibits ENaC and R568 corrects CF-associated defect in cation transport.

Figure 17. The inhibitory effect of R568 on ENaC was examined electrophysiologically by measuring amiloride-sensitive short-circuit current responses in the presence or absence of R568. Weanling infant distal colonic mucosa, which expresses highest activity of ENaC, is employed and mounted in Ussing chamber.

Figures 18A-18C. Representatives (A-B) and summary (C) of 10 μΜ amiloride effects on basal I_sc in 2-3 week old Sprague-Dawley rat proximal (A) and distal (B) colon.

Figures 19A-19C. Representative (A-B) and summarized (C) rate changes in I_sc by 10 μΜ

R568 in 2-3 week old Sprague-Dawley rat distal colon. Here, the activity of ENaC is measured as amiloride-sensitive bumetanide-insensitive I_sc. The results indicate that activation of CaSR by R568 inhibited the activity of ENaC.

Figures 20A-20C. Representative (A-B) and summarized (C) changes in I_sc in proximal vs. distal colons of 3 week old Sprague-Dawley rats fed with normal (1% calcium) vs. high calcium (2.5%) diet. The activity of ENaC was measured as amiloride-sensitive bumetanide-insensitive I_sc. Activation of CaSR by dietary calcium suppressed the activity of ENaC.

Figure 21. Activation of CaSR inhibits activity and function of ENaC. DETAILED DISCLOSURE

The invention is based, in part, on the identification of the role of CaSR in HCCV secretion, including the role of CaSR in basal, acid- and secretagogue-induced HCCV secretions. The role of CaSR in normal physiology as well as during pathophysiology, such as, diarrhea and CF has been identified. Further, the effects of CaSR agonists, for example, R-568, on C17HCCV and SCFA/HCCV exchanges (which are electroneutral and can be measured by pH stat) and electrogenic HCCV movement (which can be measured by I_sc and pH stat) are provided. In accordance with the subject invention CaSR agonists can be used to improve mucosal physiology through differentially regulating HCCV secretion.

CF is caused by mutations in CFTR that cause defects in transepithelial CI transport resulting in inability to maintain luminal hydration and defects in transepithelial HC0₃ ^~ transport resulting in inability to secrete alkaline fluid. This leads to mucus plugging and destruction of affected organs in CF. Accordingly, CF affects the function of multiple organs.

HCCV is biological buffer that maintains acid-base balance, thereby preventing metabolic and respiratory acidosis. HCCV also buffers the pH of mucosal layers that line all epithelia, protecting them from injury. Being a chaotropic ion, HC0₃ is essential for solubilization of ions and macromolecules such as mucins and digestive enzymes in secreted fluids. HCCV is of particular importance in CF because pH and HC0₃ ^~ affect mucin viscosity and binding of bacteria to mucins.

The CF phenotype varies from very severe with pancreatic insufficiency (PI) to very mild with pancreatic sufficiency (PS). CFTR mutations causing no defect in HC0₃ transport result in mild CF phenotype with PS; whereas, CFTR mutations causing defects in HCCV transport produce severe CF phenotype with PI. Accordingly, in accordance with the subject invention, stimulating HCCV transport in CF is used in the prevention and/or treatment of CF.

The mechanism of HCCV secretion by CFTR-expressing epithelia is not well understood.

HCO₃ is secreted by two pathways mediated by CFTR (CFTR dependent manner) and non CFTR transporters (CFTR-independent manner). These two pathways are coupled together and diseases that turn off one pathway typically turn off the other.

In accordance with the subject invention it has been found that CaSR "uncouples" HCCV secretion mediated by CFTR-dependent from CFTR-independent mechanisms and differentially regulates them. In the absence of a CaSR agonist, for example, R568, CFTR couples with electrogenic PAT1 and electroneutral AE1. As a consequence, stimulation of CFTR stimulates PAT1 but inhibits AE1 (Figure 2A). In the presence of a CaSR agonist, for example, R568, the CFTR interactions with PAT1 and AE1 are "uncoupled". As a consequence, CFTR and PAT1 are inhibited but AE1 is stimulated (Figure 2B).

In accordance with the subject invention, activation of CaSR by a CaSR agonist, for example, R568, inhibits CFTR-dependent HCCV secretion but stimulates CFTR-independent HCCV secretion. In the presence of lumen CI , R568 was found to induce a two-phase response (Figure 5A): an immediate phase I 7_iC-stimulatory response followed by a sustained phase II I_SC inhibitory response. Phase I (red column) and II (blue column) responses are quantified and shown in Figure 5C. Despite different effects on HC0₃ ^~ secretory currents, R568 induced a net stimulation of HC0₃ secretion, as revealed in pH stat measurements (Figure 5E).

In the absence of lumen CF, the phase I stimulation induced by R568 was abolished (Figures 5B and 5D) suggesting that the stimulation by R568 is mediated by C17HC0₃ ^~ anion exchange; however, the phase II inhibition remained unchanged or slightly reduced (Figures 5B and 5D), consistent with inhibition of a CF-independent mechanism (e.g., CFTR). Under these conditions, R568 induced a net inhibition of HCC>3^~ secretion (Figure 5F).

The results suggest that R568 inhibits CFTR-dependent HC0₃ secretion but stimulates CFTR-independent HC0₃ ^~ transport (e.g., CF-dependent HC0₃ ^~ secretion). The C17HCCV exchange stimulated by R568 is electrogenic with stoichiometry consistent with Solute carrier family 26 member 6 (Slc26A6), an AE that mediates lCi /2HC0₃ ^~ exchange and generates a lumen negative potential difference (PD) and positive I_SC. R568 stimulation of C17HC0₃ ^~ exchange is associated with more inhibition of CFTR activity; however, the net effect of R568 in the presence of lumen Cl^~ is stimulatory in HC0₃ secretion (Figure 5E).

Inhibition of CFTR by NPPB/Glibenclamide diminished, not only I_SC-CFTR (blue columns in Figure 6B vs. 6A) but surprisingly also I_SC._AE (red columns in Figure 6B vs. 6A) while the actual HC0₃ ^~ secretion remained unchanged or slightly stimulated (Figure 6E with 6D). These data suggest that the function of AE requires an active CFTR and that an additional HC0₃ ^~ secretory mechanism that is normally inactivated by CFTR might now be reactivated upon CaSR stimulation by R568.

Similarly, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) partially inhibited not only the AE-associated current (red columns of Figure 6C vs. 6A) but also inhibited the CFTR activity (blue columns of Figure 6C vs. 6A). These data suggest that the function of CFTR also requires an active AE. Again, under these conditions the HC0₃ ^~ secretion rate was stimulated or did not change (Figure 6F with 6D).

These results indicate that CFTR controls two independent HCCV secretory mechanisms in the colon: it stimulates the electrogenic AE (e.g., PATl) while it simultaneously inhibits the other mechanism (possibly not a channel but an electroneutral AE (e.g., AE1)). CaSR may function as an "uncoupler", separating the function of CFTR from non CFTR transporters and differentially regulating them. In accordance with the subject invention, agents and methods that uncouple and separately turn on/off CFTR-dependent and CFTR-independent HC0₃ ^~ secretion provide novel therapies for treating CF. Accordingly, an embodiment of the invention provides a method of treating CF in a subject. The method comprises administering to the subject, a CaSR agonist. The CaSR agonist can be an orthosteric agonist (type I agonist), which is capable of activating the CaSR on its own, or an allosteric agonist, which binds to allosteric sites on CaSR and requires the binding of an orthosteric agonist to the receptor to produce the agonistic effects.

In one embodment, the subject invention provides a CaSR agonist, for example, R568, as an agent for the treatment of CF and/or to promote mucosal health, including preventing and/or treating diarrheal illnesses.

Ca²⁺ is the primary orthosteric agonist of the CaSR. Other orthosteric agonists include divalent and trivalent cations, including Mg²⁺, Al³⁺, Sr²⁺, Mn²⁺, Ni²⁺, Gd³⁺, and Ba²⁺; aminoglycoside antibiotics (e.g. neomycin, gentamycin, tobramycin) and polyamines (e.g. spermine, spermidine, putrescine), all of which are positively charged. In general, CaSR agonists with a high positive charge density tend to have higher potency.

Allosteric agonists of CaSR are also called calcimimetics. Allosteric agonists of CaSR include, but are not limited to, aromatic L-amino acids (e.g., L-phenylalanine, L-tryptophan, L- tyrosine, L-histidine) and small molecule calcimimetics. Non-limiting examples of small molecule calcimimetics include R568 (2-Chloro-N-[(lR)-l-(3-methoxyphenyl)ethyl]-benzenepropanamine hydrochloride), R467 ((R)-N-(3-phenylpropyl)- -methyl-3-methoxybenzylamine hydrochloride), Cinacalcet ((R)-N-[l-(l-naphthyl)ethyl]-3-[3-(trifluoromethyl)phenyl]propan-l -amine), Calindol ((R)- 2-[[[l-(l-Naphthyl)ethyl]amino]methyl]-lH-indole Hydrochloride). Additional examples of small molecule calcimimetics are known to a person of ordinary skill in the art and such embodiments are within the purview of the claimed invention.

Examples of calcimimetics can be found in U.S. Patent Nos: 8,791, 147; 8,609,655;

8,486,381; 8,349,831; and 8,334,317, all of which are incorporated herein by reference, in their entiries.

In one embodiment of the subject invention, the method of treating CF in a subject comprises administering to the subject a composition comprising a calcimimetic. The calcimimetic can be selected from, for example, L-phenylalanine, L-tryptophan, L-tyrosine, L-histidine, R568, R467, Cinacalcet, and Calindol. Additional examples of a calcimimetic are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention. In a further embodiment of the subject invention, the method of treating CF in a subject comprises administering to the subject a composition comprising a calcimimetic and a CaSR orthosteric agonist. For example, CF can be treated by administering a composition comprising R568 and Ca²⁺. Any combination of a calcimimetic and a CaSR orthosteric agonist known to a person of ordinary skill in the art can be used to treat CF in a subject. For example, a person of ordinary skill in the art can combine any of the aforementioned calcimimetics or another calcimimetic known in the art with any of the aforementioned CaSR orthosteric agonists or another CaSR orthosteric agonist known in the art to produce a composition for treatment of CF.

In another embodiment, the method of treating CF in a subject comprises administering to the subject a composition comprising a calcimimetic and an agent capable of stimulating colonic HC0₃ secretion in a CFTR-independent manner. Non-limiting examples of agents capable of stimulating colonic HC0₃ ^" secretion in a CFTR-independent manner include CF and SCFA. Additional examples of agents capable of stimulating colonic HC0₃ ^~ secretion in a CFTR-independent manner are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention.

In one embodiment, the method of treating CF in a subject comprises administering to the subject a composition comprising a calcimimetic and an agent capable of stimulating colonic HCOf secretion in a CFTR-independent manner. For example, CF can be treated by administering a composition comprising R568 and SCFA. Any combination of a calcimimetic and an agent capable of stimulating colonic HC0₃ ^~ secretion in a CFTR-independent manner known to a person of ordinary skill in the art can be used according to the subject invention to treat CF in a subject. For example, a person of ordinary skill in the art can combine any of the aforementioned calcimimetics or another calcimimetic known in the art with any of the aforementioned agents capable of stimulating colonic HC0₃ ^~ secretion in a CFTR-independent manner or another agent capable of stimulating colonic HCO₃ secretion in a CFTR-independent manner known in the art to produce a composition for treatment of CF.

In a further embodiment of the invention, the composition comprising a calcimimetic and an agent capable of stimulating colonic HCCV secretion in a CFTR-independent manner further comprises a CaSR orthosteric agonist. Accordingly, in one embodiment, the method of treating CF comprises administering, to a subject in need thereof, a composition comprising a calcimimetic, an agent capable of stimulating colonic HC0₃ ^" secretion in a CFTR-independent manner and a CaSR orthosteric agonist. Accordingly, one specific embodiment of the invention provides treating CF in a subject by administering to the subject a composition comprising R568, SCFA and Ca²⁺. Table 1 provides examples of calcimimetics, agents capable of stimulating colonic HC0₃ ^~ secretion in a CFTR-independent manner, and CaSR orthosteric agonists. Any combination of a calcimimetic, with either or both of an agent capable of stimulating colonic HC0₃ secretion in a CFTR-independent manner and a CaSR orthosteric agonist is within the purview of the invention.

Additional examples of calcimimetics, agents capable of stimulating colonic HCO₃ ^" secretion in a CFTR-independent manner, and CaSR orthosteric agonists are well known to a person of ordinary skill in the art and combinations of such compounds are also within the purview of the claimed invention.

Pharmaceutical Compositions

Certain embodiments of the invention provide pharmaceutical compositions comprising one or more of the compounds described herein and a pharmaceutically acceptable carrier and/or excipient. Pharmaceutical compositions, as disclosed herein, can be formulated in accordance with standard pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a person skilled in the art.

Compositions for parenteral administration are generally physiologically compatible sterile solutions or suspensions which can optionally be prepared immediately before use from solid or lyophilized form. Adjuvants such as a local anesthetic, preservative and buffering agents can be dissolved in the vehicle and a surfactant or wetting agent can be included in the composition to facilitate uniform distribution of the active ingredient. For oral administration, the composition can be formulated into conventional oral dosage forms such as tablets, capsules, powders, granules and liquid preparations such as syrups, elixirs, and concentrated drops. Non toxic solid carriers or diluents may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. For compressed tablets, binders, which are agents which impart cohesive qualities to powdered materials are also necessary. For example, starch, gelatin, sugars such as lactose or dextrose, and natural or synthetic gums can be used as binders. Disintegrants are also necessary in the tablets to facilitate break-up of the tablet. Disintegrants include starches, clays, celluloses, algins, gums and crosslinked polymers. Moreover, lubricants and glidants are also included in the tablets to prevent adhesion to the tablet material to surfaces in the manufacturing process and to improve the flow characteristics of the powder material during manufacture. Colloidal silicon dioxide is most commonly used as a glidant and compounds such as talc or stearic acids are most commonly used as lubricants.

For transdermal administration, the composition can be formulated into ointment, cream or gel form and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and dimethylformamide.

For transmucosal administration, nasal sprays, rectal or vaginal suppositories can be used. The active compound can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols (carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate.

In a particular embodiment, the method of treating CF comprises administering to a subject in need thereof, a composition comprising a calcimimetic and optionally further comprising an agent capable of stimulating colonic HCCV secretion in a CFTR-independent manner and/or CaSR orthosteric agonist via inhalation.

Compositions of the invention appropriate for administration via inhalation can be a solution, suspension, or powder. These formulations are typically administered via an aerosol or a dry powder inhaler. Aerosol is a colloidal suspension of particles dispersed in air or gas. In aerosols, liquid or suspension droplets are the internal phase and a gas is the external phase.

In one embodiment, an aerosol delivery device is used to administer the compositions of the invention. An aerosol delivery device is used to produce aerosols, for example, for delivery to a subject via inhalation. Metered dose inhalers (MDI) are aerosol delivery devices that deliver a fixed dose in a spray with each actuation of the device. In certain embodiments, atomizers, nebulizers, or vaporizers are used as aerosol delivery devices. Additional examples of aerosol delivery devices are well known to a person of ordinary skill in the art and such embodiments are within the purview of the current invention.

Atomizers break up a liquid into an aerosol. Typically, an atomizer comprises a squeeze bulb which is used to blow air into the device causing the drug solution to rise in a small dip tube and vaporizing in the air stream. The air stream is directed into a baffle or bead which breaks the droplets in to even smaller droplets as they collide with the device. The mixture of air and liquid then exits the atomizer in the form of an aerosol.

A nebulizer contains an atomizing unit within a chamber. When the rubber bulb is depressed, the medication solutions is drawn up a dip tube and aerosolized by the passing air stream. Baffles or beads may also be present in the chamber. The fine droplets exit the nebulizer. The larger droplets collect on the chamber and fall back into the reservoir where they can be used again.

Vaporizers produce a fine mist of steam. Volatile medication is added to the water in the vaporizer or to a special medication cup present in some models. The medication volatilizes and is inhaled by a patient as he/she breathes.

In one embodiment, the composition is a dry powder and the inhalers contain the dry powders in cartridges or disks. When a patient administers a dose, the device is first activated by some mechanical motion and the dry powder becomes ready for inspiration. The patient then inhales through the device mouthpiece and the powder is drawn into the pulmonary tract along with the inspired air. These devices have overcome a major problem of inhalation therapy, synchronizing deep inspiration with the administration of the drug. Some of the commercially available devices are Diskhaler®, Turbuhaler®, Diskus®, and Rotahaler®.

In a further embodiment, a powdered composition is administered with insufflators or puffers. Squeezing the rubber bulb of an insufflator causes turbulence within the powder reservoir which forces some of the powder into the air stream and out of the device. A puffer is a plastic accordion- shaped container with a spout on one end. The powder is placed inside the container and the puffer is actuated by squeezing the device. A portion of the powder is ejected from the spout.

Additional devices appropriate for administration of the composition of the claimed invention via inhalation are well known to a person of ordinary skill in the art and such embodiments are within the purview of the invention. For example, Advanced Drug Delivery Reviews (2014), Volume 75, Pages 1-148 contains several articles directed to "limproving the efficacy of inhaled drugs for severe lung diseases: emerging pulmonary delivery strategies." The contents of these articles are herein incorporated by reference in their entirety, particularly, Angelo et al., "Improving the efficacy of inhaled drugs in CF: Challenges and emerging drug delivery strategies." Certain devices and methods for delivery of therapeutic substances via inhalation are also described "A Guide to Aerosol Delivery Devices for Respiratory Therapists, 3rd Edition (2013)" published by American Association for Respiratory Care, the contents of which are incorporated herein by reference in their entirety.

Definitions

Terms such as "treating," "treatment," "to treat," refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of CF, and 2) prophylactic or preventive measures that prevent or slow the development of CF. Thus, those in need of treatment include those already with CF; those prone to having CF; and those in whom the CF is to be prevented. A subject is successfully "treated" according to the methods of the present invention if the patient shows one or more of the following: mucus clearance, absence of infection, good lung function, slower progression of lung disease, reduced inflammation, improved respiratory function, reduced exacerbations, increased mucociliary clearance, loosening and removal of thick, sticky mucus from the lungs, avoidance of blockages in the intestines, prevention of dehydration and reduction in problems with digestive system. Complete absence of any CF symptoms is not required for successful "treatment."

The term "administering" is defined herein as a means of providing an agent or a composition containing the agent to a subject in a manner that results in the agent being inside the subject's body. Such an administration can be by any route including, without limitation, oral, subcutaneous, intradermal, intravenous, intra-arterial, intratumoral, intraperitoneal, intramuscular, or via inhalation.

The term "subject" or "patient" refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a bovine, equine, canine, ovine, murine or feline. In certain embodiments, the treatment of humans is contemplated by this invention.

The term "effective amount" means the amount of an agent required to treat CF and to produce some desired therapeutic effect. The effective amount of compound(s) used to practice the present invention for prevention or treatment of CF varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician will decide the appropriate amount and dosage regimen.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about. Materials and Methods

Animals and tissue preparations

Experiments were performed using non-fasting male Sprague-Dawley rats and C57BL/6 mice. Rats weighting 100-400 g were obtained from Charles River Laboratories. Mice lacking CaSR expression in intestinal epithelial cells (CaSR ⁷ ) and their CaSR^{+ +} littermates were bred and maintained. CaSR^"/_ mice were generated as previously described. Briefly, CaSR flox flox mice were bred with transgenic mice expressing Cre Recombinase under the control of the villin 1 promoter and genotyped prior to all experiments after an approximate 10-12 generations. Mice were used at 5-10 weeks of age. Animals were fed and maintained on regular chow (Harlan) with free access to water before sacrifice. Animals were sacrificed with standard C0₂ inhalation followed by cervical dislocation. The colons were isolated, cut open along the mesenteric border into a flat sheet, and flushed with ice-cold Ringer solutions. Mucosa from the distal colon were carefully hand-stripped off of serosal, muscular and submucosal layers as described and a pair of adjacent mucosal segments were incised and mounted into Ussing chambers. In some experiments, stripped mucosa was incised longitudinally into two equal pieces in order to facilitate comparisons between control and treatment. Short-circuit current and transepithelial resistance differences between these two adjacent tissues were less than 15%.

Ussing chamber setup

Stripped mucosal sheets were mounted into Ussing chambers (window area = 0.3-0.5cm², Physiologic Instruments, San Diego, CA). The mucosal side of tissue was bathed with an unbuffered HC0₃ ^~-free Cl^~ Ringer solution (see Table 2 for detailed composition) circulated by a gas lift with 100% 0₂ while the serosal side was bathed with buffered CI Ringer solution (pH 7.4) that contained 25 mM HC0₃ ^~ and gassed with 5% C0₂/95% 0₂. Each side contained 3-5 ml of solution and the temperature of the solution was adjusted to and maintained at 37°C by heated water-jacketed reservoirs. Experiments were performed under short-circuit conditions (Voltage-Current Clamp, VCC MC8; Physiologic Instruments, San Diego, CA) to maintain the transepithelial potentials at 0 mV, except for brief interruption at 20-second intervals for recording of open-circuit potential (V_r, mV).

Two types of approaches were used in the measurement of colonic HC0 secretion, namely, measurement of lumen pH or pH stat titration of net alkalinization by colonic mucosa. Except for limited experiments that require direct measurements of lumen pH (e.g., Experiment 1 and Experiment 3 below), luminal pH was maintained at 7.4 by the continuous infusion of 1 mM HCI or H₂S0₄ in case of lumen CP-free conditions under the automatic control of a pH-stat system (Bi- burette TIM 856 pH meter; Radiometer Analytical, Villeurbanne, France). The amount of the acid delivered per unit time per surface area was used to quantitate HC0₃ ^~ secretion by the mucosa. Measurements were recorded continuously and mean values for consecutive 5 or 10 min periods were averaged. The rate of HC0₃ ^~ secretion (Jncoi) is expressed as μEq/hr/cm². The short-circuit current (I_sc) was measured in microamperes (μΑ) and converted into μEq/hr/cm². Tissue resistance (R, Ω cm²) was calculated from Ohm's law.

Experimental designs - Seven experiments for HC03- secretion were conducted.

Lumen alkalinization response to [Ca ⁺]₀

Two equal pieces of mucosa from each colon that were obtained by longitudinal division along the antimessenteric border were mounted into two Ussing chambers. One piece was bathed with Cl^~ Ringer solution that contained 1.2 mM [Ca²⁺]₀ while the other piece was bathed with Cl^~ Ringer solution that contained 0.5 mM [Ca²⁺]₀. Initially, tissues were bathed, both luminally and serosally, in HC0₃ ^~-free solution. After 15 to 30 min, when stabilization was achieved and basal lumen pH recordings performed, HC0₃ ^~-free Ringer solution in the serosal side was replaced by 25 mM HC0₃ ^~-containing Ringer solution, and changes in lumen pH were monitored and recorded for 15 to 30 min. In some tissues, carbachol was added to the serosal side before the experiment was concluded.

Basal HCO3^" secretion

Two adjacent sheets of mucosa from each colon were mounted into two Ussing chambers and bathed luminally with HC V-free Cl^~ Ringer solution and serosally with HC0₃ -containing CI^" Ringer solution. After 15 to 30 min, when I_sc and JHCO₃ had stabilized, the CaSR agonist, R568 was added to the serosal or mucosal side of tissue, and changes in I_sc and J_Hco₃ during the 15-30 min period ensuing after the addition of the agonist were determined.

Acid-induced HCCV secretion

Colon mucosa from each animal was divided longitudinally into 2 equal pieces and mounted into two Ussing chambers. Acid (hydrochloride) was added to the lumen to lower pH by approximately 0.3-0.4 units and changes in lumen pH monitored. In response to luminal addition of acid, HCCV is secreted and pH in the lumen rises. When lumen pH rose above 7.4, another acid challenge was applied. After 15 to 30 min, when lumen pH responses had stabilized, one piece of mucosa was treated with R568 in the serosal bathing solution while the other piece of mucosa was treated with vehicle control, and changes in lumen pH responses during the 30-45 min period ensuing after the addition of the agonist were determined. Initial studies demonstrated that under these experimental conditions the tissue tolerated acid challenges for at least 60 minutes without significantly compromising tissue responses and integrity. Initial studies also established that acid- induced pH recovery reflects HC0₃ secretion from tissue as additions of acid to no-tissue controls or tissue controls without the presence of serosal HCCV did not generated significant pH recovery. A lowering of 0.3-0.4 pH units was used because this is the range of luminal pH that this part of the intestine normally varies. Initial rates of acid-induced pH recovery were used for comparison and were expressed as pH unit recovered/min/cm².

Secretagogue-induced HCCV secretion

To examine CaSR effect on stimulated HCCV secretion, a model of forskolin-induced HCCV secretion was employed to mimic cholera toxin. The secretagogue forskolin was used to increase tissue cAMP content rapidly as cholera toxin's effect will not be seen for 1-2 hours. Tissues were divided and treated as in the basal HCCV secretion experiments except that after 15 to 30 min, when I_sc and JHCO3 had stabilized, forskolin was added to the serosal side of tissue for 15 to 30 min until I_so and JHCO3 ^nad plateaued before R568 was then added to the serosal solution. Changes in I_sc and JHCO₃ during the 15-30 min period ensuing after the addition of the agonist were determined. Rodent distal colon displays Na⁺, K⁺ and CI^" currents, in addition to HC0₃ ^~ conductance. To reduce current interference from non HC0₃ ^~ conductance, prior to the recording of HC0₃ ^~ secretory responses, a cocktail containing 10 μΜ amiloride and 5 mM barium was added to the mucosal side to selectively inhibit Na⁺ and K⁺ conductance, and 100 μΜ bumetanide to the serosal side to inhibit CI secretory current (26, 41 ). Preliminary studies indicate that these transport inhibitors did not significantly affect ½co₃ response although I_sc was inhibited by amiloride and stimulated by barium (see Table 3).

Table 3: Effect of transport inhibitors on basal HC0₃ secretion in rat distal colon

C1 HCOf exchange

Mucosa from each colon was divided longitudinally into 2 equal pieces and mounted into 2 Ussing chambers. One piece of mucosa was bathed luminally with zero HC0₃ ^~ Ringer solution that contained C while the other piece of mucosa was bathed luminally with zero HC0₃ ^~ Ringer solution that did not contain Cl^_. Both pieces of mucosa were bathed serosally with Ringer solution that contained C\^~ and HC0₃ ^~. After 15 to 30 min, when JHCXM had stabilized, R568 or vehicle control was added to the serosal or mucosal side of tissue, and changes in ½χ» during the 15-30 min period ensuing after the addition of the agonist or vehicle were determined and averaged. When inhibitor was used, it was added at 30 min before the agonist. Ci7HC0₃ ^~ exchange activity was calculated as AJHCO3 (the difference between JHCXB i the presence minus absence of luminal Cl^~).

SCFA/HC0₃ ^" exchange

Unless specifically described elsewhere, colon segments were prepared and treated as in the C17HC0₃ ^~ exchange studies except that they were bathed luminally with zero HC0₃ ^~ Ringer solution that either contained or did not contain 25 mM isobutyrate. SCFA/HCO₃ ^" exchange activity was calculated as AJ_Hco₃ (the difference between JHCO₃ in the presence minus absence of luminal isobutyrate). Cyclic nucleotide-dependent HCCV secretion

Colon segments were prepared and treated as in the secretagogue-induced HC0₃ ^~ secretion experiments except that they were bathed luminally with HCCV-free Ringer solution that contained no isobutyrate and no Cl^~. Cyclic nucleotide-dependent HCCV secretion was calculated as AJ_HCo3 (the difference between JHCO3 after minus JHCO3 before the addition of forskolin). When inhibitor was used, it was added at 30 min before R568 or forskolin.

Chemicals and solutions

Forskolin, 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), glibenclamide, 5-nitro-2- (3-pheny]propylamino)benzoic acid (NPPB), amiloride, barium, and bumetanide were obtained from Sigma and GlyH-101 from Santa Cruz Biotechnology while R-568 from Tocris Bioscience (Ellisville, MI). All stock solutions were prepared in DMSO. The detailed composition of Ringer solutions used in these studies is listed in Table 1. 5% CO2 and 95% 0₂ were used to oxygenate Ringer solutions that contained HCO₃ , while 100% 0₂ was used to oxygenate those solutions that did not contain

Statistical Analysis

Values are expressed as means ± SEM. Δ¼χ», AI_SC and ΔρΗ recovery rates refer to stimulated peak responses minus basal control levels. Data were analyzed by one-way ANOVA followed by Holm-Sidak's post hoc test or, when appropriate, by the paired or unpaired two-tailed Student's ?-test using Microsoft Excel 2010 for Windows or GraphPad Prism version 6 for Windows (GraphPad Software, San Diego, CA). p < 0.05 was considered significant.

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 - BASAL HCCV SECRETION

Lowering extracellular Ca²⁺ concentration reduced and activation of CaSR by R568 enhanced basal HC0₃ secretion. The initial experiments performed examined the role of extracellular Ca²⁺ in modulation of HCC secretion under basal (non-stimulated) conditions. Mucosa was first bathed with HCCV free, CT Ringer solution before 25 mM HCCV was added to the serosa. As shown in Figure 7, there was no change in lumen pH; thus lumen alkalinization did not occur in the absence of a serosa-to-lumen directed HCCV gradient. Consistent with HCCV secretion, subsequent addition of HCO₃ ion to serosal side induced significant lumen alkalinization [Figure 7A; mean ± SEM (n) absence vs. presence of HCO3^" ion: -0.01 1 ± 0.002 (3) vs. 0.035 ± 0.002 (3) pH unit/min/cm², p < 0.01], Carbachol (CCH), a known secretagogue for HCCV secretion was used as a positive control. CCH induced an initial transient increase followed by a sustained increase in lumen pH (Figure 1A) characteristic of HCCV secretion associated with cholinergic receptor activation.

Experiments were then performed to examine whether CaSR regulates basal HCC secretion. For this, the concentration of Ca²⁺ in the buffer was lowered from 1.2 mM to 0.5 mM and HCCV secretory response compared. Extracellular Ca^2T is a physiological ligand of CaSR. Previous studies have shown that this maneuver reduces CaSR activity and that at 0.5 mM Ca²⁺ concentration CaSR is only minimally stimulated. Reduction of [Ca²⁺]₀ from 1.2 mM to 0.5 mM significantly reduced lumen alkalinization rates [Figure IB; mean ± SE (n) [Ca²⁺]₀ = 1.2 vs. 0.5 mM: 0.031 ± 0.002 (3) vs. 0.013 ± 0.002 (3) pH unit/min/cm², p<0.01]. As extracellular Ca²⁺ is also a known determinant of paracellular permeability of intestinal epithelium, additional experiments were performed to exclude the possibility that the reduced rate of lumen alkalinization noted at reduced [Ca²⁺] was not simply caused by HCCV back leak" secondary to altered paracellular integrity; thus, transepithelial electrical resistances (TEER) were measured. No significant differences in TEER were noted between normal and reduced Ca ⁺ treated tissues (see details in Figure 7, legend). Thus, extracellular Ca²⁺ (and CaSR) may regulate transcellular, and not paracellular HCCV movement.

To further assess the role of CaSR as a regulator of HCCV secretion, another set of experiments measured the rate of serosal-to-mucosal HCCV flux (JHCOS) and HCCV secretory J_SC and tested the effect of R568, a specific pharmacological CaSR agonist. A relatively low basal JHCO_S was observed in the absence of R568. Addition of R568 to serosa (Figure 7C) significantly stimulated JHCO3- Similar but slightly less pronounced effects were also noted when R568 was added to lumen solutions (not shown). Such stimulatory changes were not observed in I_5C [mean ± SEM (n) absence vs. presence of R568: 1.36 ± 0.40 (5) vs. 1.48 ± 0.44. (5) pEq/hr/cm², p > 0.05]. These data suggested that CaSR stimulates electroneutral HCCV secretion.

EXAMPLE 2 - ACID-INDUCED HCCV SECRETION

Activation of CaSR by R568 enhances acid-induced HCCV secretion - Acid-induced HCCV secretion is a known mechanism that intestinal mucosa utilizes as a defense against acid-induced damage. Colonic mucosa is exposed to luminal acidic environment, generated as a result of bacteria fermentation of undigested carbohydrates. To assess if CaSR affects acid-induced HCCV secretion, colonic tissues were challenged by additions of acid (HCl) to lower pH in the lumen, rates of pH recovery monitored and recorded, and effect of R568 examined. Figure 8A shows changes in the rates of pH recovery in the presence vs. absence of R568. The Arate changes at peak responses above basal levels were calculated and are summarized in Figure 8B. R568 induced a transient but significant stimulation of acid-induced HC0₃ ^~ secretion.

EXAMPLE 3 - SECRET AGOGUE-INDUCED HC0₃ ^~ SECRETION

Activation of CaSR by R568 inhibits secretagogue-induced HC0₃ ^~ secretion - HC0₃ secretion is markedly increased in cholera and other secretagogue-induced diarrheal diseases. To assess if CaSR stimulates HC0₃ ^~ secretion under these diseased conditions, the R568 effect was examined in a model of secretagogue-induced secretory diarrhea. Forskolin was used to stimulate HC0₃ ^~ secretion, and HC0₃ secretory response was monitored by measuring HC0₃ secretory rate (J COS) (Figure 9A) and by recording I_SC (Figure 9B). Forskolin stimulated both J CO_S and I_SC; subsequent addition of R568 did not increase but rather decreased forskolin-induced HC0₃ ^~ secretion. EXAMPLE 4 - CL^" DEPENDENT HCOf SECRETION

Activation of CaSR stimulates CI HCO₃ ^" exchange activity - Since the basal and acid- induced HC0₃ ^" secretion were assayed in CL-containing Ringer, it is likely that the CaSR effects reflect stimulation of a Cl -dependent HC0₃ ^~ secretory mechanism such as C17HC0₃ ^~ exchange. To address this possibility, CI7HC0₃ ^" exchange activity was measured and is shown in Figure 10. Figure 1 OA demonstrates HC0₃ secretory responses to the presence vs. absence of lumen CP. A luminal Cl^~ -dependent HC0₃ ^~ secretory mechanism (C17HC0₃ ^~ exchange) was observed. The latter was partially abolished by pretreatment with 100 μΜ DIDS added to lumen side (data not shown). Activation of CaSR by R568, added to serosal side, resulted in stimulation of the C17HC0₃ ^~ exchange activity (Figure 10B). This CL-dependent HC0₃ ^" secretion was significantly higher in the presence than in the absence of R568 (Figure 10B). A similar but less pronounced stimulatory effect of R568 was noted when this agonist was added to the mucosal solution (data not shown).

EXAMPLE 5 - SCFA-DEPENDENT HCO₃ SECRETION

Activation of CaSR stimulates SCFA/HC0₃ ^~ exchange activity - Short-chain fatty acids (SCFA) are present in the colon and induce HC0₃ ^" secretion via the SCFA/ HC0₃ ^~ exchange. To assess if CaSR stimulates HC0₃ secretion under these conditions, the next series of experiments examined isobutyrate-dependent HC0₃ secretion and its response to R568. Figure 1 1A & 11 B show representative tracings and Figure 11C presents a summary of HC0₃ ^~ secretory responses to the presence vs. absence of lumen SCFA (25 mM isobutyrate) with vs. without a serosa-to-mucosa directed HC0₃ ^~ gradient. SCFA-dependent HC0₃ ^~ secretion was present and required serosal HC0₃ , consistent with HC0₃ ^~ secretion mediated by SCFA/HC0₃ ^~ exchange. Similar to the R568 effect on the C17HC0₃ ^~ exchange, isobyturate-dependent HC0₃ ^~ secretion was significantly stimulated by activation of CaSR by R568 (Figure 1 ID).

EXAMPLE 6 - CAMP-DEPENDENT HC0₃ ^~ SECRETION

Activation of CaSR by R568 inhibits cAMP-dependent HC0₃ ^" secretion - In contrast to R568 stimulation of basal HC0₃ secretion, HC0₃ ^~ secretion was inhibited by R568 under forskolin- stimulated condition (Figure 9). It is uncertain how R568 produces this inhibition. Since R568 inhibited neither CF-dependent nor SCFA-dependent HC0₃ ^" secretion (Figures 10-1 1), it is unlikely that the effect of the CaSR agonist is via inhibition of either of these anion exchanges. Rather, a CF /SCFA-independent HC0₃ ^~ transport mechanism(s) might be responsible. One such mechanism is a cyclic nucleotide-dependent electrogenic channel (e.g., CFTR)-mediated HC0₃ secretion. Thus, to address this latter possibility, stimulation of HC0₃ ^~ secretion by forskolin and its inhibition following addition of R568 were re-assessed in lumen C17SCFA-free solutions. As shown in Figure 12A, a low rate of HC0₃ ^~ transport was noted under basal condition prior to the addition of forskolin. Addition of forskolin significantly stimulated HC0₃ ^~ secretion. The subsequent addition of R568 almost completely reversed FSK-induced HC0₃ secretion.

Similar changes were observed in experiments that determined changes in I_sc (Figure 12B).

Forskolin stimulated I_sc; subsequent addition of R568 inhibited HC0₃ ^~ secretion. Removal of the serosa-to-mucosa HC0₃ ^~ gradient significantly diminished both basal and forskolin-stimulated I_sc (data not shown); in the absence of serosal HC0₃ ^", forskolin and R568 failed to stimulate or to inhibit I_sc, respectively (data not shown). Pretreatment with luminal glibenclamide (100 μΜ) or GlyH-101 (10 μΜ), CFTR channel blockers, and NPPB (100 μΜ), an anion channel inhibitor, added either before the addition of forskolin or R568, abolished the forskolin stimulatory and R568 inhibitory effects on I_sc (data not shown). As a consequence, these results suggest that R568 inhibits cAMP- dependent, glibenclamide/GlyH-lOl/NPPB-sensitive electrogenic HC0₃ ^" secretion. EXAMPLE 7 - EFFECT OF CaSR KNOCK-OUT

R568 fails to stimulate CF- and SCFA- dependent and inhibit cAMP-dependent HC0₃ secretion in colon of CaSR null mouse - R568 is a specific pharmacological agonist that has been widely used to stimulate CaSR. To verify that the effects of R568 occurred via the CaSR, additional studies on the effect of R568 were performed in intestinal epithelium-specific CaSR knockout mice (Figure 13). Intestinal epithelium-specific CaSR knockout mice were used together with their wild type littermates. Activation of CaSR by R568 stimulated Cl^~- and SCFA- dependent HC(¾^~ secretion and inhibited cAMP-dependent HC0₃ ^~ secretion in colon mucosa of wild type mice (Figure 13A-C); such effects were abolished in CaSR null mice (Figure 13D-F). These results indicate that the R568 effects occur via activating the CaSR in the intestinal epithelium.

EXAMPLE 8 -REGULATION OF HC0₃ ^" SECRETION IN THE COLON

The current invention provides a new model for regulation of HC0₃ secretion in the mammalian colon where activation of CaSR by R568 stimulated basal and acid-induced HCO₃ secretion but, in contrast, R568 inhibited cyclic nucleotide-mediated HCO₃ ^* secretion. The invention also indicates that the enhancement of HC0₃ ^" secretion is mediated via stimulation of electroneutral C17HC0₃ ^~ and SCFA/HC0₃ ^" exchanges that are localized on the apical membrane of colonic surface epithelial cells; in contrast, the CaSR inhibitory action is a consequence of CaSR inhibition of a cAMP-dependent, lumen glibenclamide/GlyH-lOl/NPPB-sensitive electrogenic HC0₃ ^{^} secretory process primarily located in the crypt cells. A model for this differential regulation of colonic HC0₃ secretion by CaSR is depicted in Figure 14.

According to the present model of colonic ion function, absorptive processes are primarily localized to surface cells whereas secretory processes are primarily present in crypt cells. Because CL -dependent HCO₃ exchange and SCFA-dependent HC0₃ ^~ exchange are present only in surface cells and are absent in crypts, Cl -dependent HC0₃ secretion and SCFA-dependent HC0₃ secretion are most likely both surface cell functions. In contrast, CFTR-mediated HC0₃ ^~ secretion is generally considered to represent a crypt cell function. Thus, in addition to mediating colonic HC0₃ ^" secretion, the primary function of these two anion exchanges in these absorptive surface cells is to absorb solutes/electrolytes. SCFA absorption is mediated by SCFA/HC0 exchange, and NaCl absorption is the result of 0Γ/Η0Ο₃ ^~ exchange coupled to Na FT^" exchange, which is also localized in surface cells. The ability of CaSR agonists to stimulate both anion exchanges (Figures 10-13) as well as Na⁺/H⁺ exchange suggests that, in addition to stimulation of HC0₃ ^" secretion, CaSR may function as a mechanism to enhance electrolyte and fluid absorption. Activation of CaSR also stimulates colonic acid-induced HC0₃ ^~ secretion (Figure 8). This latter function may also neutralize FT from bacterial fermentation and/or NaVH ^" exchange, further increasing solute absorption.

Consistent with a recent in vivo study in rat perfused duodenum, the invention demonstrated that CaSR activation stimulated basal HC0₃ ^~ secretion in ex vivo colonic mucosa. These findings may have important physiological significance as HCCV secretion is an integral part of mucosal defense mechanisms. HC0₃ ^" secretion is required for mucin secretion by goblet cells to establish a layer of mucus overlying the epithelium, an initial defense barrier that limits pathogen invasion. Defects in HC0₃ secretion have been shown to impair the formation of the mucus layer and compromise the integrity of the intestinal barrier, leading to bacteria translocation and development of intestinal inflammation. Thus, the ability for CaSR to stimulate HC0₃ secretion under basal conditions suggests that, through modulating mucus secretion and barrier function, this well-conserved nutrient- sensing receptor may play a role in intestinal immune function. Indeed, mice deficient in CaSR with deficiently regulated HC0₃ secretion in the colon have altered barrier integrity, enhanced bacteria translocation and increased inflammation whereas enteral nutrients, including the CaSR-activating nutrients/minerals, calcium, spermine and tryptophan, have been shown to improve intestinal permeability and immunity and inflammation.

Importantly, both HCCV and Cl^~ secretion are markedly induced in cyclic nucleotide- mediated secretory diarrheas (e.g., cholera). Although these secretory responses may be helpful in enhancement of the defensing mucus layer so as to limit pathogen invasion and also to flush out toxins, over production and secretion of these anions by the intestine under these pathological conditions is harmful and may result in dehydration, alkali deficit and metabolic acidosis. Systemic volume depletion and metabolic acidosis are the two major causes of death associated with acute diarrheal illnesses, especially in infants and young children. The ability of CaSR agonists both to inhibit cyclic nucleotide-stimulated Cl^~ and HCC secretion (Figures 12 & 13) and to promote Cf and SCFA absorption (Figures 10-13) as well as Na⁺ absorption suggests that this class of drugs may provide a unique therapeutic approach to prevent and treat these potentially lethal diarrheal illnesses. Since CaSR agonists are naturally occurring nutrients, CaSR-based anti-diarrheal therapies would be of particular utility among actively growing infants and children.

The net increases in I_sc induced by forskolin (AI_SC ^FSK) were greater than those in net JHCO3

(AJ_Hco₃ ^FS ) [compare the grey-colored columns in Figures 9D and 12D (mean values: 3.6 and 1.5 μΕ /1ΐΓ/«η²) vs. Figures 9B and 12B (mean values: 1.7 and 0.2 μEq/hr/cm²)]. These differences cannot be explained by a non-steady-state flux period as all measurements were made after 15-30 minutes when both I_sc and JHCO₃ had stabilized and were in steady state. The most likely explanation is that a component of AI_SC ^FS represents forskolin-induced CI^" secretion even though serosal bumetanide was present in both experiments. Serosal bumetanide was employed to prevent (or at least to reduce) such a contribution from forskolin-induced CI^" secretion. It is known that in rat distal colon bumetanide does not completely suppress Cl^~ secretion induced by forskolin. Only ~ 70% of such CI^" secretion was inhibited by bumetanide; the remainder of the C secretion was mediated by a C17HC0₃ ^" exchange located in the basolateral membrane of colonocytes. Consistent with this, we found that the non-HC0₃ ^~ portion of AI_SC ^FSK was greater when a serosa-to-mucosa transepithelial CF gradient was present than when a CI gradient was absent (compare 88% in Figure 12 vs. 53% in Figure 9).

Although most experiments were performed with a reduced concentration of Ca²⁺ ₀ in order to minimize background activation of the receptor before R568 addition, under normal Ca²⁺ ₀ condition the effects of R568 were qualitatively similar, albeit with a slightly less pronounced effect. As such, the invention provides that the calcimimetic R568 has physiological relevance and clinical utility. Indeed, this same class of drug has been employed successfully to inhibit parathyroid hormone secretion in hyperparathyroid patients, where Ca²⁺ ₀ in the serum can be either < 1.0 mM (secondary hyperparathyroidism) or > 1.5 mM (primary hyperparathyroidism).

In summary, the present invention confirms the presence of at least three distinct mechanisms for HC0₃ secretion in rodent distal colon, i.e., lumen Cl^~-dependent HC0₃ ^~ secretion, SCFA- dependent HC0₃ secretion, and cAMP-activated HC0₃ ^~ secretion. Further, CaSR agonists differently regulate HC0₃ secretion, depending on the physiological state of the intestine and the specific transporter in question. During physiological conditions when electroneutral C17HC0₃ ^_ and SCFA/HC0₃ ^" exchanges dominate, CaSR enhances HC0₃ ^~ secretion; however, in experimental conditions that result in stimulation of fluid and HC0₃ ^~ secretion that also occurs in cholera in which electrogenic CFTR-mediated HC0₃ ^~ conductance is dominant, CaSR also inhibits HCCV secretion. Both of these regulatory processes induced by CaSR are potentially beneficial. While the stimulatory effect may help expand the mucus layer, the inhibition of channel-mediated HC0₃ ^~ secretion may be of particular clinical significance as it may reduce and minimize HC0₃ losses in diarrhea. EXAMPLE 9 - CORRECTION OF CATION TRANSPORT IN CF BY R568

In addition to defects in anion transport, patients with CF also exhibit a defect in cation transport. Adequate luminal hydration required for the maintenance of health in all transporting epithelia is achieved by a fine balance between the two interconnected transport processes; the apical CFTR, which secretes anions (CF and HC0₃ ^") and the Epithelian Sodium Channel (ENaC), which absorbs cation (Na⁺).

Normally, ENaC is inhibited by CFTR. However, in CF, CFTR loses this regulatory function. As a consequence, ENaC becomes overly activated and transepithelial Na⁺ and water is inappropriately absorbed and the lumen becomes inadequately hydrated and mucus plugging is formed (Figure 15).

Therefore, ihibiting ENaC-mediated Na^÷ absorption is critical in prevention and treatment of CF. This hypothesis, depicted in Figure 16, was examined by measuring changes in ENaC current with Ussing chamber-short circuit current (I_sc) recording (which measures the electrogenic Na⁺ absorption mediated by ENaC) in conjunction with the use of bumetanide (which specifically inhibits transepithelial CI^" secretion mediated by CFTR and NKCC1).

The experimental setup is illustrated in Figure 17. In the distal colon of 2-3 week old weanling rats, the short-circuit current (Isc) is primarily made up by the following two electrogenic ion transport processes: 1) secretory CI current, and 2) absorptive Na current. Secretory CI current is mediated by apical CFTR. This CI secretory Isc is sensitive to bumetanide, applied to the basolateral side of the epithelium. There, bumetanide inhibits CI entry from blood into the cell; thus, in the presence of bumetanide, the secretory CI current is eliminated. Absorptive Na current is mediated by apically located epithelial Na channel (ENaC). This latter current is particularly high in this weanling age and is sensitive to amiloride, when it is applied luminally.

The basal I_sc in distal colon of weanling infant was mostly due to amiloride-sensitive Na¹ absorption mediated by ENaC (Figure 18). Activation of CaSR by R568 inhibited the bumetanide- insensitive amiloride-inhibitable ENaC-mediated basal I_sc in the distal colon of weanling infant (Figure 19). Activation of CaSR by calcium (2.5% w/w, added to diet) suppressed the amiloride- sensitive ENaC-mediated basal I_s0 in distal colon of weanling infant (Figure 20). Therefore, activated CaSR inhibits ENaC activity. Hence, CaSR agonists can be used for correcting CF-associated defects in cation transport (Figure 21).

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application. REFERENCES

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Claims

CLAIMS We claim:

1. A method of treating cystic fibrosis (CF) in a subject, the method comprising administering, to a subject who has been diagnosed with CF, a composition comprising a CaSR agonist.

2. The method of claim 1 , wherein the composition comprises an extracellular calcium- sensing receptor (CaSR) orthosteric agonist.

3. The method of claim 1, wherein the composition comprises an agent capable of stimulating colonic bicarbonate (HC0₃ ) secretion in a cystic fibrosis transmembrane conductance regulator (CFTR)-independent manner.

4. The method of claim 3, wherein the composition comprises a CaSR orthosteric agonist and the agent capable of stimulating colonic HCCV secretion in a CFTR-independent manner.

5. The method of claim 4, wherein the method comprises administering R568, R467, Calindol, Cinacalcet, L-phenylalanine, L-tryptophan, L-tyrosine, or L-histidine.

6. The method of claim 2, wherein the CaSR orthosteric agonist is Ca²⁺, Mg²⁺, Al ⁺, Sr² Mn²⁴, Ni²⁺, Gd ⁺, Ba²⁺, neomycin, gentamycin, tobramycin, spermine, spermidine, or putrescine.

7. The method of claim 3, wherein the agent capable of stimulating colonic HCCV secretion in a CFTR-independent manner is CF or SCFA.

8. The method of claim 1, comprising administering the composition to the subject via inhalation or enema.

9. The method of claim 8, wherein administering the composition to the subject via inhalation is performed by a device suitable for administration of the composition to the subject via inhalation.

10. The method of claim 9, wherein the device is an aerosol delivery device.

11. The method of claim 10, wherein the device is an inhaler, atomizer, nebulizer, vaporizer, insufflator or puffer.

12. A composition for treatment of CF, the composition comprising a calcimimetic.

13. The composition of claim 12, further comprising a CaSR orthosteric agonist.

14. The composition of claim 12, further comprising an agent capable of stimulating colonic HCC>3^~ secretion in a CFTR-independent manner.

15. The composition of claim 12, further comprising an CaSR orthosteric agonist and an agent capable of stimulating colonic HCO₃ secretion in a CFTR-independent manner.

16. The composition of claim 12, wherein the calcimimetic is R568, R467, Calindol, Cinacalcet, L-phenylalanine, L-tryptophan, L-tyrosine, or L-histidine.

17. The composition of claim 13, wherein the CaSR orthosteric agonist is Ca²⁺, Mg²⁺, Al³⁺, Sr²⁺, Mn²⁺, Ni²⁺, Gd³⁺, Ba^2r, neomycin, gentamycin, tobramycin, spermine, spermidine, or putrescine.

18. The composition of claim 14, wherein the agent capable of stimulating colonic HCC secretion in a CFTR-independent manner is CI or SCFA.

19. The composition of claim 12, formulated for administration to a subject via inhalation.

20. A device comprising the composition of claim 12, wherein the device is suitable for administering the composition to a subject via inhalation.

21. The device of claim 20, wherein the device is an inhaler, an atomizer, a nebulizer, a vaporizer, an insufflator or a puffer.