Patent Application
20070078111
The invention relates to compositions and methods for treating secretory diarrhea. More particularly, the invention relates to compositions and methods for treating cystic fibrosis transmembrane conductance regulator (CFTR)-mediated diarrhea and for inhibiting activation of CFTR.
Secretory diarrhea is often caused by infectious organisms such as Vibrio cholerae, Clostridium difficile, and enterotoxic Escherichia coli. Infectious diarrheal disease is a major cause of morbidity and mortality worldwide, and is of particular concern for children, especially in developing countries.
Cholera, for example, is an acute diarrheal illness caused by the bacterium Vibrio cholerae. The infection can be asymptomatic, or may produce symptoms that range from mild to severe. About five percent of infected individuals have severe disease. A hallmark of cholera is a watery diarrhea that can lead to rapid loss of body fluids, dehydration and shock. Without treatment, an affected individual may be dead within hours. Patients are most often treated through rehydration, and individuals are often administered a prepackaged combination of sugar and electrolytes admixed with water. In some areas, however, sufficient quantities of potable water may not be available for treatment. Furthermore, in severe cases of secretory diarrhea rehydration alone may not be sufficient to save the patient's life.
Cholera-associated diarrhea, for example, is induced primarily by the interaction of cholera toxin with the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a plasma-membrane cyclic AMP-activated Cl− channel expressed in a variety of tissues. In epithelial cells, CFTR mediates the secretion of chloride ions. In the intestine, CFTR-mediated Cl− secretion causes the diarrhea associated with Vibrio cholerae, Clostridium difficile, and Escherichia coli infections, for example.
Inhibition of CFTR is an attractive target for prevention and treatment of secretory diarrhea. What are needed are safe and effective compositions that can quickly stop the loss of fluids through the intestine as a result of CFTR activation.
The present invention relates to a method of treating or preventing diarrhea by administering to an individual a therapeutically effective amount of lysophosphatidic acid (LPA), a food or supplement comprising a therapeutically effective amount of LPA, a therapeutically effective amount of an LPA2 receptor agonist, or combinations thereof.
The invention also relates to a composition for treating diarrhea comprising an aqueous carrier comprising one or more electrolytes for rehydration and for restoring or maintaining electrolyte balance, the aqueous carrier and electrolytes being admixed with one or more LPA2 receptor agonists.
The invention further relates to a method of inhibiting CFTR activity by administering to an individual a therapeutically effective amount of an LPA2 receptor agonist.
The inventors have discovered that lysophosphatidic acid (LPA) inhibits cholera toxin-induced secretory diarrhea through CFTR-dependent protein interactions. Furthermore, the inventors have discovered that the mechanism by which LPA inhibits CFTR involves the LPA2 receptor and that LPA2 receptor agonists synthesized by the inventors provide compositions for preventing and treating CFTR-mediated secretory diarrhea. The inventors have also discovered that LPA and LPA2 receptor agonists can inhibit CFTR-dependent Cl− currents in response to adenosine activation. Bacterial diarrhea is most often the result of interaction of bacterial toxins with CFTR, but diarrhea can also result from an increased immunoinflammatory response triggered by cytokines or mediators such as adenosine secreted from the intestinal mucosal inflammatory cells in response to luminal factors. The invention therefore provides compositions and methods for treating secretory diarrhea of bacterial origin, as well as diarrhea induced by stress and/or an immunoinflammatory response.
Compositions and methods as described by the invention may be utilized for therapy in animals, particularly mammals, such as humans, non-human primates, horses, cows, pigs, goats, sheep, dogs, and cats, for example. Compositions described herein are described as compositions comprising LPA2 receptor agonists, but it should be understood by those of skill in the art that such compositions may comprise LPA, a food or supplement comprising LPA, one or more LPA2 receptor agonists, or combinations thereof. LPA may be found, for example, in dietary supplements.
Treatment for diarrheal illness may be administered prior to onset of symptoms (e.g., following exposure to one or more organisms known to cause secretory diarrhea) or after onset of symptoms. Compositions of the invention may be distributed to groups of individuals in areas where bacterial or viral epidemics, for example, are occurring, to prevent fluid loss and associated electrolyte imbalance.
Compounds have been identified by the inventors which are specific for certain LPA receptors, may bind to more than one LPA receptor in a relatively non-specific manner, or may bind to more than one LPA receptor but preferentially bind to certain of the LPA receptors. Compounds of the present invention will therefore comprise compounds that bind to the LPA2 receptor, but even more preferably will comprise compounds that preferentially bind to the LPA2 receptor. Compounds of the invention comprise those that act as agonists of the LPA2 receptor. Therapeutically effective amounts are amounts that produce sufficient inhibition of CFTR to decrease symptoms (e.g., fluid accumulation in the intestine and fluid loss through diarrhea) associated with CFTR activation.
Compounds listed in Table 1 and Table 2 have been synthesized by the inventors and are examples of compounds that may be used for the methods and compositions for treatment described herein. Methods of synthesis of these compounds are described in U.S. Pat. No. 6,875,757 (Miller et al.) and U.S. patent application Ser. No. 10/963,085 (Publication Number 20060009507; Miller et al.).
Of these compounds in Tables 1 and 2, thiophosphoric acid O-dodecyl ester, thiophosphoric acid O-tetradecyl ester, thiophosphoric acid O-dodec-9-enyl ester, thiophosphoric acid O-tetradec-9-enyl ester, and thiophosphoric acid O-octadec-9-enyl ester have been demonstrated to be particularly effective for binding to the LPA2 receptor and thereby inhibiting CFTR activation.
Therapeutic formulations can be administered by a variety of routes including, for example, oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal, as well as administration via nasogastric tube.
Compositions of the present invention comprise at least one LPA2 receptor agonist provided in conjunction with an acceptable pharmaceutical carrier. In one embodiment, for example, such a composition may comprise at least one LPA2 receptor agonist admixed in an aqueous carrier comprising sugar and electrolytes suitable for rehydration and maintaining or restoring electrolyte balance in an individual who has experienced an episode of secretory diarrhea. In another embodiment, one or more LPA2 receptor agonists may be provided in a powder comprising one or more electrolytes, and, optionally, sugar. Compositions may also comprise antibiotics, anti-inflammatory agents, or other agents that may act to provide a benefit to an individual at risk for or experiencing secretory diarrhea. Compositions of the invention are described for oral administration, and it is well within the skill of those in the art to prepare oral rehydration salt (ORS) comprising one or more LPA2 receptor agonists admixed with, or provided in conjunction with, the ORS. Pharmaceutically acceptable compositions for IV administration may be required for treatment of more serious cases of secretory diarrhea. Such compositions may comprise, for example, saline solutions containing therapeutically effective amounts of LPA2 receptor agonists or Ringer's lactate solutions containing therapeutically effective amounts of LPA2 receptor agonists. The formulation may be in dry or liquid form, or as an oral or intravenous sugar-electrolyte solution or dry composition.
For oral administration, Rehydron™ (Orion Pharma International, Finland) or Pedialyte™ (Ross, USA) solution can be administrated according to the manufacturer's instructions, and may be admixed with one or more LPA2 receptor agonists prior to administration. In yet another embodiment, a parenteral rehydration solution may contain, for example, one or more LPA2 receptor agonists, glucose, sodium chloride and potassium chloride as the one or more electrolytes. Administration of an electrolyte for rehydration therapy may be performed as recommended by the World Health Organization. (See, for example, World Health Organization, Diarrheal Diseases Control Program: A Manual For The Treatment Of Acute Diarrhea For Use By Physicians And Other Senior Health Workers. Geneva: WHO, 1984: WHO/CDD/SER/80.2(rev.1); World Health Organization; The Treatment Of Diarrhea: A Manual For Physicians And Other Senior Health Workers, available online at http://www.who.int.) Oral rehydration therapy may additionally comprise alanine, arginine, vitamin A, and zinc, for example, to increase the rate of repair of the intestinal epithelia.
Compositions of the invention may also contain additional ingredients such as buffering agents, preservatives, compatible carriers, and optionally additional therapeutic agents such as antibiotics. Suitable buffering agents include: acetic acid, citric acid, boric acid, phosphoric acid and salts thereof. Compositions may additionally comprise pharmaceutically acceptable preservatives.
In making the formulations of the present invention, the LPA2 receptor agonist is usually admixed with an excipient, diluted in an excipient or enclosed within a carrier that can be in the form of a capsule, sachet, or paper, for example. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Compositions provided herein may be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing for example up to 10% by weight of the one or more LPA2 receptor agonists, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. Compositions can be administered in the form quiescently frozen confections, ice milks, ice creams, frozen yogurts, or other frozen treats, for example. Compositions may also be administered as flavored drinks such as fruit-flavored drinks, juices, or drinks containing at least a percentage of fruit juice.
LPA2 receptor agonists may be milled to a suitable particle size prior to combining or admixing with other ingredients. If the LPA2 receptor agonist is substantially insoluble, it may be is milled to a particle size of less than 200 mesh. If the LPA2 receptor agonist is substantially water soluble, the particle size may be adjusted by milling to provide a substantially uniform distribution in the formulation, for example about 40 mesh.
Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Compositions of the present invention can be formulated so as to provide relatively immediate, sustained, or delayed release of the active ingredient after administration to the patient by employing procedures known to those of skill in the art.
Compositions of the invention may be provided in a unit dosage form or provided in bulk quantity comprising sufficient volume for multiple doses, each dosage containing from about 2.0 mg to about 1000 mg, more usually about 20 mg to about 500 mg, of at least one LPA2 receptor agonist. The term “unit dosage form” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
For preparing solid compositions such as tablets the one or more LPA2 receptor agonists are mixed with a pharmaceutical excipient to form a solid formulation preferably containing a homogeneous mixture of the one or more LPA2 receptor agonists to form a composition of the present invention. When referring to the formulation as homogeneous, it is meant that the LPA2 receptor agonist is dispersed relatively evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms. In some embodiments, for example, tablets or caplets may be provided, the tablets or caplets being scored to facilitate use of one-fourth, one-half, etc. of a tablet or caplet (for use in children, for example).
Tablets or pills of the present invention may be coated or otherwise compounded to provide a dosage form providing the advantage of modified or extended release. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
LPA2 receptor agonists may also be provided in the form of a transdermal delivery device. Such transdermal “patches” may be used to provide continuous or discontinuous infusion of the LPA2 receptor agonist in controlled amounts. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art and is described in, for example, U.S. Pat. No. 5,023,252. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
Injectable drug formulations include, for example, solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (for example, ethanol, propylene glycol and sucrose) and polymers (for example, polycaprylactones and PLGA's).
Compositions suitable for parenteral administration may comprise a sterile aqueous preparation of the LPA2 receptor agonist, which is preferably isotonic with the blood of the recipient. This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
Timed-release, delayed release or sustained release delivery systems may increase ease of administration and efficacy of compositions as provided by the invention. Such delivery systems are commercially available and known to those of ordinary skill in the art. They include polymer based systems such as polylactic and polyglycolic acid, polyanhydrides and polycaprolactone; nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di- and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like. Specific examples include, but are not limited to: (a) erosional systems in which the polysaccharide is contained in a form within a matrix, found in U.S. Pat. No. 4,452,775 to Kent; U.S. Pat. No. 4,667,014 to Nestor et al.; and U.S. Pat. No. 4,748,034 and U.S. Pat. No. 5,239,660 to Leonard; and (b) diffusional systems in which an active component permeates at a controlled rate through a polymer, found in U.S. Pat. No. 3,832,253 to Higuchi et al. and U.S. Pat. No. 3,854,480 to Zaffaroni. In addition, a pump-based hardware delivery system can be used, some of which are adapted for implantation.
Use of a long-term sustained release implant may be particularly suitable for treatment of diarrhea in immunodeficient patients, who may need continuous administration of the compositions of the present invention. “Long-term” release, as used herein, means that the therapeutic agent is delivered at therapeutic levels of the agent for at least 30 days, and preferably 60 days. Long-term sustained release compositions and devices are well known to those of ordinary skill in the art.
Administration of the LPA2 receptor agonist, or formulations containing one or more LPA2 receptor agonists, can be provided as either a single dosage treatment, if suitable, or a plurality of dosages that are repeated, e.g., at regular intervals. Periodic administration can be two or more times per day, on two or more consecutive or non-consecutive days, etc.
The present invention also provides a method of inhibiting activity of a cAMP activated chloride channel (CFTR). This method involves providing to an individual a therapeutically effective amount of at least one LPA2 receptor agonist, the amount of the LPA2 receptor agonist being effective to inhibit formation of a macromolecular complex between the LPA2 receptor, the CFTR, and a Na+/H+ exchanger regulatory factor-2 (NHERF-2), thereby inhibiting activity of the CFTR.
The invention also provides research reagents and kits for studying CFTR-mediated cellular functions and pathways. Kits may comprise, for example, one or more aliquots of one or more LPA2 receptor agonists, cell culture media, buffers, or other reagents to facilitate culture of cells with at least one LPA2 receptor agonist or introduction of one or more LPA2 receptor agonists into the cellular environment.
The invention may be further described by means of the following non-limiting examples.
Tissue Culture and Transfection
HT29-CL19A (human colonic epithelial origin), Calu-3 (serous gland epithelial cells), and BHK (baby hamster kidney cells) were cultured as described (26). For Ussing chamber experiments, HT29-CL19A and Calu-3 cells were grown on permeable filters. EVOM Epithelial Voltohmmeter (World Precision Instruments; Sarasota, Fla.) was used to measure the transepithelial resistances on a daily basis. BHK cells were transiently transfected with Flag-LPA receptors using a vaccinia virus expression system (27).
Antibodies, Reagents, and Constructs
CFTR antibodies R1104 monoclonal mouse antibody and NBD-R polyclonal rabbit antibody have been described previously (28). We generated peptide-specific anti-LPA2 antibody (rabbit-2143) against the last 11 amino acids (a.a. 341-351) of LPA2 (Genemed Synthesis, CA) which was affinity-purified using Protein-A column. Anti-NHERF2 antibody was a kind gift from Dr. Emanuel Strehler (Mayo Clinic, Rochester, Minn.). We also generated our own NHERF2-specific antibody (rabbit-2346) against the full-length NHERF2 protein (Genemed Synthesis, CA), which was affinity purified using Protein-A column and the NHERF2 antigen itself This isoform-specific antibody for NHERF2 does not cross-react with NHERF1, which shares approximately 50% sequence identity with NHERF2 (
Iodide Efflux Measurements
Iodide efflux was performed as described (30). Calu-3 and HT29-CL19A cells were grown to confluency in 60-mm dishes and then pretreated with or without LPA 18:1, 18:2, 20:4 (complexed with BSA), or a structurally related lysophospholipid, sphingosine-1-phosphate (S1P), for 20 min before iodide efflux measurement. The first four aliquots were used to establish a stable base line in efflux buffer alone. Agonists, cpt-cAMP (200 μM) or adenosine (2 μM), were added to the efflux buffer with or without various LPA, and samples were collected every minute for 6 min in the continued presence of agonists and LPA (i.e., the efflux buffer used for subsequent replacements also contained agonists and LPA at the same concentration). The iodide concentration of each aliquot was determined using an iodide-sensitive electrode (Orion Research Inc., Boston, Mass.) and converted to iodide content (i.e., the amount of iodide released during the 1-min interval in nanomoles). For some studies, the peak efflux of iodide (5 min time point) was presented as the percentage of control. For the pertussis toxin (PTX) experiments, the cells were pretreated with 100 ng/ml, or 200 ng/ml of PTX for 24 h at 37° C. incubator before iodide efflux measurement.
Short-Circuit Current (Isc) Measurements
Calu-3 and HT29-CL19A polarized cell monolayers were grown to confluency on Costar Transwell permeable supports (filter area is 0.33 cm2). Filters were mounted in an Ussing chamber, and short-circuit currents (Isc) mediated through CFTR Cl− channel were carried out as described previously (31). Epithelia were bathed in Ringer's solution (mM) (serosal/basolateral: 140 NaCl−, 5 KCl, 0.36 K2HPO4, 0.44 KH2PO4, 1.3 CaCl2, 0.5 MgCk2, 4.2 NaHCO3, 10 Hepes, 10 glucose, pH 7.2, [Cl−]=149), and low Cl-Ringer's solution (mM) (luminal/apical: 133.3 Na-gluconate, 5 K-gluconate, 2.5 NaCl, 0.36 K2HPO4, 0.44 KH2PO4, 5.7 CaCl2, 0.5 MgCl2, 4.2 NaHCO3, 10 Hepes, 10 mannitol, pH 7.2, [Cl−]=14.8) at 37° C., gassed with 95% O2 and 5%) was stripped of serosal and smooth muscle layers (32) before being mounted in a Ussing chamber. LPA (20:4; 0-35 μM) was added into both the luminal (apical) side and serosal (basolateral) side of the isolated mouse intestine for 30 min. Increasing concentrations of cpt-cAMP (0-100 μM) or ADO (0-100 μM) were applied to both sides to elicit a CFTR-dependent Cl current response. DPC (500 μM, added into luminal side) was used to inhibit the Cl currents towards the end of the experiment. CO2. LPA (20:4) was added into both the luminal (apical) side and serosal (basolateral) side of the cell monolayers for 30 min before adenosine (ADO) was added into the luminal/apical side. In parallel, some filters were also pretreated for 30 min with phosphatidic acid (PA; 0-35 μM) and sphingosine-1-phosphate (S1P; 0-35 μM) before ADO addition. CFTR Cl− channel inhibitor, diphenylamine-2-carboxylate (DPC; 500 μM; added into luminal side), was used to inhibit the Cl− currents towards the end of the experiment. To demonstrate the effect of LPA on Cl− transport in the mucosa, a patch of mouse intestine (0.112 cm2).
Intestinalfluid Secretion (In Vivo) Experiments
The method of Verkman's group was followed with modifications (33) with Institutional Animal Care and Use Committee approval. CD1 mice were held off food 24 h prior to inducing anesthesia using pentobarbital (60 mg/kg). Body temperature of the mice was maintained at 36-38° C. during surgery using a circulating water heating pad. A small abdominal incision was made to expose the small intestine. Ileal loops (˜20 mm) proximal to the cecum were exteriorized and isolated (two loops per mouse: 1 loop PBS, 1 loop PBS+toxin). The loops were then injected with 100 μl of PBS alone or PBS containing cholera toxin (1-100 μg). Both the control and cholera toxin-injected loops were tested in the presence and absence of LPA (10-40 μM). The abdominal incision and skin incision were closed with wound clips, and the mice were allowed to recover. Intestinal loops were collected in a terminal procedure 6 h later. PA (10-100 μM) and S1P (10-100 μM) were also used in parallel experiments as described above.
Macromolecular Complex Assembly
This assay (34) was performed using maltose binding protein (MBP)-CFTR-C tail fusion protein (1 μM) immobilized on amylose beads (20 μl) and incubated with GST-NHERF2. This step, which is called pairwise binding, was done in 200 μl of lysis buffer (PBS-0.2% Triton-X 100+protease inhibitors) and mixed at 22° C. for 2 h. The complex was washed once with the same buffer and allowed to bind Flag-tagged LPA2 from lysates of BHK cells expressing the receptor. The binding was done at 4° C. for 3 h with constant mixing. The complex was washed with lysis buffer, eluted with sample buffer, and analyzed by immunoblotting using anti-Flag monoclonal antibody.
Delivery of LPA2 Receptor-Specific Peptide and Immunostaining
Delivery of LPA2-specific peptide was performed using the Chariot™ system according to the manufacturer's instructions (Active Motif; Carlsbad, Calif.) (35). Briefly, 1.2 μM peptide containing the PDZ motif of LPA2 (last 11 amino acids; a.a. 341 to 351, ENGHPLMDSTL) was mixed with Chariot solution (total volume: 400 μl) at room temperature for 30 min, then the Chariot-peptide complex was added to both luminal and serosal sides of polarized Calu-3 and HT29-CL19A cells grown on permeable supports and incubated for 1 h at 37° C. in a humidified atmosphere containing 5% CO2 before mounting in a Ussing chamber. At the end of the experiment, the epithelial cells were fixed and immunostained for the efficiency of the peptide delivery using anti-LPA2 antibody (raised against the last 11 amino acids of LPA2) that also recognizes this LPA2-specific peptide and subjected the fixed cells to immunofluorescence confocal microscopy. Other peptide (CFTR a.a. 107-117) was also delivered as described above as negative control.
Data Analysis
Results are presented as mean±SEM for the indicated number of experiments. The unpaired Student's t test was used for statistical comparison of mean values between two experimental groups. A value of P<0.05, P<0.01, or P<0.001 was considered statistically significant.
LPA1 and LPA2, but not LPA3 transcripts, were present in mouse intestinal tissue (
LPA2 was next colocalized in part to the apical surface of HT29-CL19A (
The C-terminal tails of LPA1 and LPA2, but not LPA3, contain a consensus for the PDZ (PSD95/Dlg/ZO-1) motif, the short COOH-terminal protein sequences that specifically interact with the PDZ-binding domains (usually ˜70-90 amino acid sequences) of PDZ proteins (16) (
Although all three LPA receptors (LPA1, LPA2, and LPA3) respond to LPA, LPA2 has the highest affinity to the lipid (17), leading to the activation of at least three distinct G-protein pathways: Gi, Gq, and G12/13 (6, 7). Activation of the receptors by LPA results in inhibition of the adenylate cyclase pathway, which in turn decreases cAMP levels (5-7). The Gi activation pathway of the receptor is summarized in
To test the hypothesis that the inhibition of CFTR function by LPA is receptor-mediated, Calu-3 cells were pretreated with pertussis toxin (PTX), which has been demonstrated to catalyze the ADP-ribosylation of the Gαi subunit, thus specifically disrupting the Gi pathway (21). PTX pretreatment reversed LPA-dependent inhibition of CFTR function in a dose-dependent fashion (
The effect of LPA on CFTR-dependent short-circuit currents (Isc) was monitored on polarized epithelial monolayers (18). LPA significantly inhibited CFTR-dependent Cl− currents in response to adenosine activation in HT29 cells (
To provide proof of concept that LPA could be used in a therapeutic setting, the effect of LPA was tested on cholera toxin (CTX)-induced diarrhea model in mice (
The inventors hypothesized that the interaction between CFTR and LPA2 was likely mediated by NHERF2. To test this, we assembled a macromolecular complex of LPA2, NHERF2, and CFTR in vitro (
We next determined if a physical association of LPA2 and CFTR via NHERF2 was essential for the LPA2-mediated inhibition of the Cl− channel. To disrupt the complex, we delivered an LPA2 isoform-specific peptide containing the PDZ motif using the Chariot system (24) into polarized epithelial cells before short-circuit measurements. Isc in response to adenosine (0-20 μM) in the presence of LPA was monitored. LPA2-specific peptide significantly reversed the LPA-elicited inhibition on CFTR-dependent Cl− currents (
Given that LPA2 receptors are expressed in the gut (
An open-loop mouse model was used in determining the effect of fatty alcohol thiophosphate 18:1d9 (FAP) on cholera toxin induced Cl-secretion. The animal protocol was reviewed and approved by the Animal Care and Use Committee at UTHSC. Male C57BL/6 mice aged around 8 weeks were purchased from Harlan (Indianapolis, Ind.) and maintained on a 12:12-h light-dark cycle and fed standard laboratory mouse chow and water ad libitum. After 2 weeks of acclimatization, mice were fasted with free access to water for 24 hours prior to the open-loop experiment. Mice were lavaged with 100 μl 7% NaHCO3 buffer containing 10 μg/ml CTX in the presence or absence of 100 μM LPA (18:1) or 100 μM FAP using an orogastric feeding needle, and sacrificed 6 hours later. The entire small intestine from pylorus to cecum was exteriorized and stripped off mesenteric and connective tissue, and then weighed. The small intestine was weighed again after flushing of its content. Intestine weight ratio and intestinal fluid accumulation were calculated as described by Werkman et al. (20). A ratio of intestinal weight before to intestinal weight after luminal fluid removal was used to determine the Cl-secretion into the intestinal lumem. As shown in
Various embodiments of the invention have been described herein, but it will be apparent to those skilled in the art that modifications, additions, and substitutions, may be made without departing from the scope of the invention.
This application claims the benefit of priority of earlier-filed U.S. Provisional Patent Application No. 60/700,489, filed 19 Jul. 2005.
The present invention was made at least in part with finding received from the National Institutes of Health under grant numbers CA92160 and HL-61469. The U.S. government may therefore claim certain rights to this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60700489 | Jul 2005 | US |
