MXPA00006326A - Novel pharmaceutical compositions of uridine triphosphate - Google Patents

Abstract

Novel pharmaceutical compositions of uridine 5'-triphosphate (UTP) for use in promoting increased mucociliary clearance of retained mucous secretions of the human airways, middle/inner ears or sinuses are disclosed. Novel Formulation (I) comprises UTP and aqueous solution having a therapeutic concentration between 5 and 45 mg/mL, a controlled tonicity within the range of 250 to 100 mOsM, a pH between 7.0 and 7.5, and is sterile. The pH-adjusted composition is capable of long-term storage in the refrigerated state with a shelf life of up to 30 months. Formulation (I) may be delivered therapeutically either in a nebulized form or in a liquid form. Novel Formulation (II) comprises UTP in an aqueous solution having a therapeutic concentration approaching the limit of UTP's solubility at ambient temperature or in a suspension which exceeds drug solubility. This high concentration allows delivery of a therapeutic amount of UTP in very small volumes (50 to 100 mL) suitable for administration via portable hand-held devices such as metered dose inhalers. Formulation (II) has extended shelf life without the need for refrigeration after being dispensed to the patient.

Description

NOVEDOSE PHARMACEUTICAL COMPOSITIONS OF URIDI TRIFOSPHATE NA This is a continuing part of the U.S. Application, Serial No. 08 / 996,740, filed on December 23, 1997, which is a continuing portion of the U.S. Application, No. 08 / 675,555 filed on October 3, 1997. July 1996.

TECHNICAL FIELD This invention relates to novel pharmaceutical compositions of uridine 5'-triphosphate (UTP). The compositions are effective to promote mucociliary clearance of retained mucous secretions in airways, middle / inner ears or mammals' breasts. The compositions are capable of both therapeutic and diagnostic applications.

BACKGROUND OF THE I NVENTION The mucociliary cloud is an important defense mechanism of the airways and middle / inner ear tract. The rhythmic movements of the cilia in the airways and ears brings the mucous layer to the pharynx, carrying with it microorganisms and other particles captured in the mucosa. The normal function of this system depends on the frequency and coordination of ciliary movement and the properties of the mucosa itself. It has been found that extracellular nucleoside triphosphates, especially UTP, modulate the mucociliary clearance. Specifically, UTP stimulates the frequency of ciliary movement (D. Drutz, et al., Drug Development Research, 377 (3), (1996)), increases hydration of the mucosal layer on the luminal surface of the airway (R. Bouocher, et al., Adenosine and Adenine Nucleotides: From Molecular Biology to Integrative Physiology, pp. 525-32"Mechanisms and Therapeutic Actions of Uridine Triphosphates in the Lung" (L. Belardinelli, et al., Ed., Alumwer Academic Publishers, Boston , 1995)), and increases the mucosal release of goblet cells and smbmucosal glands (M. I. Lethem, et al., Goblet cell nucleotide regulation in epithelial explants of the airways: exocytosis in cystic fibrosis Am. J. Respir. Cell Mol. Biol., 1993; 9: 315-322). In addition, UTP and other nucleotides have been shown to stimulate the release of surfactant phospholipids from alveolar type II cells (S. Rooney, et al., Progr. Respir Res., 27, 84-91 (1994); L. Gobran, and collaborators, Am. J. Physiol., 267, L625-33 (1994)). It has been postulated that UTP is effective in the treatment of cystic fibrosis and other airway diseases involving retained mucous secretions as described in U.S. Patent No. 5,292,498 and U.S. Patent No. 5,420,116. It has also been shown that UTP is safe and improves clearance by expectoration in patients with primary ciliary dyskinesia (PCD) (P. Noone, et al., Am. J. Resp. Crit. Care Med A530, (1996)). Studies by Inspire Pharmaceuticals, Inc. (Durham, NC) have shown that UTP helps induce a sputum sample by hydrating mucous secretions and stimulating the frequency of ciliary movement for diagnostic analysis, such as cytopathology for cancer lung or bacillus. of fast acid for tuberculosis. UTP is likely to decrease the risk of retained secretions in patients who are immobilized or receiving mechanical ventilation, thereby preventing pneumonia, including ventilator-associated pneumonia (VAP). A proprietary product that includes UTP in the formulation and is sold under the trade name Uteplex®, with a pH of 9.0, has been sold in France by Wyeth for the treatment of lower back pain. The French biotechnology company Synthelabo has developed a formulation of adenosine triphosphate (ATP) under the trade name rhinATP ™ for the treatment of nasal co-administration with mucosal fluid. Belgian Patent No. 597,360, issued on December 14, 1960, entitled "Novel Therapeutic Composition Usable as a Stimulant of Muscles and Nerves", describes a formulation of UTP used in various myopathies and nerve stimulation therapy. With the prospect of increasing the clinical uses of UTP on the horizon, the need for more compatible formulations and less sensitive to temperature has arisen. Formulation I of the present invention differs from the prior art nucleotide pharmaceutical formulations in that it is sterilized by filtration instead of caior, has a controlled tonicity within the osmolarity range of 250 to 1000 mOsM, more preferably about 300 mOsM, and the pH is adjusted to give values in the range of 6.5 to 8.5, preferably 6.5 to 8.0 and most preferred of 7.0 to 7.5. An unexpected property of the present invention is that Formulation I is capable of shelf life prolonged if kept properly refrigerated. This novel UTP Formulation I is suitable for delivery using a commercially available atomizer (v. G., Jet atomizers, electronic atomizers, etc.) which facilitates administration in the clinical facility. Formulation I of liquid UTP of the present invention can be delivered to the airways of a patient via inhalation of a nebulized form or can be delivered to the nose, eye, sinuses or middle / inner ears by means of nasal drops, for eyes or for ears. In any of the forms of administration, an effective amount of UTP contacts the directly affected site. Formulation II of the present invention differs from the prior art nucleotide formulations in that it is particularly well suited for the administration of small pre-measured volumes via portable, compact handheld devices that are capable of efficiently generating breathable aqueous-based fine aerosols or for the measurement of a small volume of a liquid reservoir before administration. Suitable devices for the administration of Formulation I include, but are not limited to, those currently under development by the Aradigm Corporation (Hayward, CA) as described in the U.S. Patent. , No. 5,544,646 by AeroGen, initially Fluid Propulsion Technology as described in U.S. Patent No. 5, 164,740, and by the Technology Partnership (Hertfordshire, UK) as described in U.S. Patent No. 5,518, 179 In addition, systems based on a small volume topical delivery metering pump, such as the patented Bespak pie, UK described in the U.S. Patent. , No. 5,547, 131, can be used to deliver Formulation I I of the present invention via the nose or ear.

BRIEF DESCRIPTION OF THE INVENTION UTP novel pharmaceutical compositions are described. The compositions comprise a UTP raw material, v. g. , dihydrate salt of trisodium uridine 5'-triphosphate, in a sterile aqueous solution or suspension. Formulation I is sterilized, of controlled tonicity, adjusted in pH and capable of shelf life when kept properly refrigerated. It is compatible for use in a variety of commercially available atomizers, such as jet or ultrasonic atomizers. Formulation II is highly concentrated and suitable for administration via portable, small handheld devices that are capable of efficiently generating breathable aqueous-based fine aerosols from small pre-measured volumes or measuring a small volume from a liquid reservoir before it is subsequently aerosolized . Additionally, Formulation I I is well suited for topical delivery of small volumes via pump-based systems that would deliver a therapeutically effective amount of UTP via the nose or ear. These novel UTP formulations are well suited to be administered therapeutically in order to promote mucociliary clearance in patients suffering from mucosal secretions retained in the lungs, sinuses or middle / inner ears; increase expectoration and sputum production to induce sputum for the purposes of diagnostic analysis, clear the lungs of various toxins from the air, clear the lungs before radiological imaging, and clear the lungs before vaccination or gene therapy. The formulations of the present invention also encompass the pharmaceutically acceptable salts of UTP, v. g. , an alkali metal salt such as sodium or potassium; an alkaline earth salt; or an ammonium or tetraalkylammonium salt, that is, NX + where X is C? -4. Because UTP is heat unstable, Formulations I and I I of the present invention are sterilized by filtration instead of using heat. A second aspect of the present invention is that the tonicity of Formulation I can be controlled, v. g. , for an isotonic formulation; thus UTPNa3 is dissolved in the appropriate amount of saline or other aqueous solution to control the tonicity within the osmolarity range of 250 to 1000 mOsM; the preferred osmolarity level is between 250 to 450 mOsM; the most preferred osmolarity is approximately 300 mOsM, i.e., an isotonic solution. A third aspect of the present invention is that Formulation I is pH adjusted to be in the range of 6.0 to 8.5; the preferred level of pH is between 7.0 and 7.5. A fourth aspect of the present invention is that Formulation I can be formulated in multiple concentrations of UTP between 0.1 and 100 mg / mL; the preferred therapeutic UTP concentration is between 5 and 45 mg / mL. A fifth aspect of the present invention is that Formulation I GO? a PH value of 7.0 to 7.5 will remain stable for up to 30 months when stored at a temperature between 0o to 10 ° C. The preferred storage temperature is about 5o C. In a sixth aspect of the present invention, the Formulation II is highly concentrated, either approaching the solubility limit of UTP over a temperature range of -20 ° to + 40 ° C, or as a suspension of microfine particles in concentrations above the solubility limit or in a non-solvent vehicle. In a seventh aspect of the present invention, a very small volume of Formulation I I can be administered while maintaining therapeutic efficacy. The volume range of this concentrated UTP Formulation is from 10 to 1000 μL, preferably 25 to 500 μL and most preferably 50 to 100 μL. In an eighth aspect of the present invention, since the Formulation is delivered to the target site and is rapidly diluted by the surface fluid of the airways, it is not necessary to maintain the osmolarity of this formulation in a physiologically isotonic value or maintain a physiological pH. In a second aspect of the present invention, Formulation I I does not need refrigeration once it has been dispensed to the patient. In a tenth aspect of the present invention, Formulation I I does not require the inclusion of a preservative for packing unit dose volumes.

BRIEF DESCRIPTION OF THE DIAMETERS Figure 1 is a general graphical representation of the time course for the effect of Formulation I versus placebo on the percentage of sputum samples positive for alveolar macrophages (AMs) in non-smokers. Figure 2 is a general graphical representation of the effect of Formulation I versus placebo on sputum weights (grams) on three consecutive days of treatment in a two-period crossover study in smokers (n = 15). Figure 3 is a general graphic representation of cytological results in sputum expectorated by smokers receiving Formulation I of UTP versus placebo (positive samples defined by the presence of alveolar macrophages, AM) (n = 15). Figure 4 is a general graphical representation of the effect of Formulation I versus placebo on sputum weights (grams) in patients with chronic bronchitis (baseline versus immediate at 5 minutes post dosing). Figure 5 is a general graphic representation of the effect of the Formulation I versus placebo in sputum weights (grams) in patients with chronic bronchitis (baseline versus 6-30 minutes postdose). Figure 6 is a general graphical representation of the effect of Formulation I versus placebo on sputum weights (grams) in patients with chronic bronchitis (baseline versus 31 minutes post dosing at discharge). Figure 7 is a general graphic representation of cytology results from sputum samples collected in patients with chronic bronchitis (positive samples defined by the presence of alveolar macrophages, AM) (baseline versus 6 minutes at 30 minutes post-dosing) ). Figure 8 is a general graphic representation of cytology results from sputum samples collected in patients with chronic bronchitis (positive samples defined by the presence of ciliated respiratory epithelial cells, CRE cells) (baseline versus 6 minutes at 30 minutes post-dosing). Figure 9 is a general graphic representation of radio-labeled global lung retention curves for baseline (no UTP or placebo inhalation), two doses of UTP Formulation I (5 mg / mL is labeled as treatment 3; mg / mL is labeled as treatment 2), and placebo (marked as treatment 1) in a period of 2.5 hours. A decline in retention demonstrates a clearing of secretions in the lungs that carry the radio-labeled particles.

DESCRITION OF SPECIFIC MODALITIES The pharmaceutical formulations of the present invention are novel formulations of UTP and sterile aqueous solution which are well suited for clinical therapeutic administration.

Formulation I is sterilized, has a controlled tonicity, is pH adjusted, and is compatible for administration in a variety of commercially available sprays, such as jet or ultrasonic sprays. An unexpected property of Formulation I is that it is capable of shelf life when stored in a refrigerated state. Formulation II is a highly concentrated solution that allows a very small volume to be delivered to the target and still be therapeutically effective. An unexpected property of Formulation I I is that there is no need to include a conservator. The therapeutic purpose of the compositions of the present invention, Formulation I and Formulation II, is to increase the mucociliary clearance of mucosal secretions retained from the airways, lungs, sinuses and middle / inner ears and eyes of patients suffering from cystic fibrosis, brochitis, sinusitis, otitis media, primary ciliary dyskinesia, ventilator-associated pneumonia and other diseases involving retained mucosal secretions or uneven ciliary movement, as well as bedridden individuals who do not suffer from any diseases, but who have a high risk of accumulating mucous secretions, v. g. , quadriplegics. Formulations I and II also improve the expectoration and induction of sputum samples for diagnostic analysis, v. g. , for diagnosis of cancer, tuberculosis or other lung diseases. Additionally, due to the well-demonstrated ability of UTP, the active ingredient of the compositions of the present invention, to improve mucociliary clearance of the lung in normal subjects, the formulations of the present invention can be used to accelerate the clearance of any type of Foreign material inhaled from the respiratory tract. This would prove beneficial in a number of situations, v. g., biological warfare, smoke inhalation, industrial exposure and in mining to inhaled toxins (resulting in silicosis, anthracosis and the range of so-called pneumoconiosis); inhaled radio-marking or other agents to make diagnostic images, and allergic reaction to inhaled particles such as pollen. The pharmaceutical compositions of the present invention are primarily intended for administration to humans, but may also be administered to other mammalian beings, such as dogs, cats and horses for veterinary purposes. Formulation I can be administered to the lungs or nose in an aerosol form which is inhaled by the patient, or can be administered in a liquid form, v. g., drops for nose, eye or ear. With either the aerosol or liquid form, an effective amount of UTP is deposited at the site of action directly. Formulation II can be administered to the lungs, nose or ears via portable, small handheld devices that are capable of efficiently generating breathable aqueous fine aerosols from small pre-measured volumes or measuring a small volume of a liquid reservoir which is subsequently converted in spray. Suitable handheld devices include, but are not limited to, those currently in development at Aradigm Corp., AeroGen Corporation, initially Fluid Propulsion Technology, and the Technology Partnership. In addition, systems based on small volume topical delivery metering pump (such as the one patented by Bespie) can be used to deliver Formulation I I. Whether or not the administration is via a portable handheld device or a topical delivery metering pump system, an effective amount of UTP is deposited at the site of action. A delivery rate of between 3 and 50 mL per second with an average mass aerodynamic diameter (MMAD) for aerosol droplets in the range of 1 to 10 microns, with a preferred range of 2 to 7 microns, and a range at most Preferred 3-6 is suitable for pulmonary drug delivery. The formulations of the present invention also encompass the pharmaceutically acceptable salts of UTP, v. g. , an alkali metal salt such as sodium or potassium; an alkaline earth salt; or an ammonium or tetralkylammonium salt, that is, NX4 + where X is C? -4. The pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesirable toxic effects. The raw material for the pharmaceutical compositions of the present invention, v. g. , UTPNa3 dihydrate, can be made according to known procedures or variations thereof which will be apparent to those skilled in the art. For example, nucleoside phosphorylation can be performed by standard methods such as those described in J. Am. Chem. Soc, 87, 1785-88 (1965) D. Hoard and D. Ott; M. Yoshikawa et al., Tetrahedron Let. 5065-68 (1967) and Bull. Chem. Soc. 83, 649-59 (1961); and B. Fischer et al., J. Med. Chem. 36, 3937-46 (1993) and references cited therein. UTP is also available commercially (Sigma, St. Louis, MO, USA). To increase the stability of the solid drug substance, the UTP in powder form can be kept frozen at a temperature between -20 ° C and -80 ° C and in such circumstances should be allowed to warm to room temperature before proceeding with the formulation. Additionally, the raw material of UTP must be brought to the operating temperature before opening to minimize the absorption of water and / or moisture condensation present in the ambient air. The tonicity of Formulation I of the present invention can be controlled by adding dry powder of UTPNa3 to a previously prepared aqueous solution of salt, v. g. , saline solution, by techniques known to those skilled in the art to bring the tonicity to any desired level of osmolarity within the range of 250 to 1000 mOsM; preferably the osmolarity of the solution is within the range of 250 to 450 mOsM; most preferred the solution is isotonic with biological fluids, ie the osmolarity is approximately 300 mOsM. Alternatively, the UTP and sodium chloride powder can be dissolved in a suitable vehicle before filtration. The solution is pH adjusted by techniques known to those skilled in the art so that the pH of said solution is between 6.0 and 8.5; preferably the pH is between 7.0 and 7.5. The solution is then sterilized by filtration, using an appropriate micrometer filter.

An unexpected result of the present invention is that Formulation I of isotonic, pH adjusted and sterilized UTP remained in stable form for up to 30 months when stored at a temperature between 0o and 10 ° C. Formulation I of the present invention confers A practical benefit because it does not require a valuable space in the refrigerator. Formulation I can be prepared in multiple concentrations of UTP between 0.1 and 100 mg / mL; the preferred therapeutic UTP concentration is between 5 and 45 mg / mL. The clinical administration of Formulation I is facilitated because it is suitable for administration using most commercially available sprays, v. g. , the Pari LC Plus jet atomizer. For Formulation II, the concentration of the pharmaceutical composition can be controlled by adding dry powder of UTPNa3 to an aqueous or ethanolic solution by techniques known to those skilled in the art to bring the concentration to about 1 mg / mL, the limit of its solubility under environmental conditions. The solution can be sterilized by filtration. Alternatively, a suspension in microfine particles can be formulated to deliver a therapeutic dose of UTP. In this case, a drug substance can be added to a previously sterilized aqueous vehicle. Formulation I I, due to its high concentration of UTP, delivers a therapeutic dose with a volume range of only 10 to 1000 μL, with a preferred range of 25 to 500 μL and most preferably a range of 50 to 100 μL. Since a very small volume of Formulation I solution is delivered to the target site, it is immediately diluted by the surface fluid of the airways, eliminating the need to maintain the osmolarity at a physiologically isotonic value. Similarly, a solution with more basic pH can be tolerated by the patient. The pH range of Formulation II can be between 6.5 and 12; preferably between 7.5 and 12; more preferably between 7.5 and 1 1; even more preferable between 7 and 10; most preferably between 8 and 10, and optimally between 7 and 9. An unexpected advantage of Formulation II is that its higher pH and UTP degradation not dependent on solution concentration results in a pharmaceutical composition that does not have to be refrigerated once the patient is delivered. The delivery of a dose of UTP from a pre-measured, sealed solution volume is preferred, because there is no need to include a preservative in the formulation. However, a deposit system is also acceptable, from which a small volume for drug delivery is measured and converted into aerosols. For a deposit system, a single preservative or a combination of preservatives may be required depending on the osmolarity and / or pH of the solution. Acceptable preservatives include, but are not limited to, chlorobutanol or benzalkonium chloride. The pharmaceutical compositions of the present invention are explained in more detail in the following Examples. These Examples are intended to be illustrative of the invention and should not be taken as limiting thereof.

EXPERIMENTAL EXAMPLES Example 1: Formulation I (6 Liter GMP Pilot Pack) Before formulating the UTPNa3 dihydrate was kept frozen at -20 ° C. The UTP powder was allowed to warm to operating temperature for at least one hour before opening; this is to minimize the absorption of water. The raw material of UTP was dissolved in sterile aqueous solution (saline). An appropriate concentration of saline was used to bring the osmolarity to approximately 300 mOsM, i.e., an isotonic solution. Alternatively, UTP powder can be dissolved in sterile water and an appropriate amount of NaCl added to bring the osmolarity to about 300 mOsM. In any case, aqueous solution was added in sufficient volume to reach an optimum therapeutic UTP concentration level of 5 to 45 mg / mL. The liquid solution was adjusted to pH to bring the pH level between 7.0 and 7.5. The resulting UTP solution was sterilized by filtration with a 0.2 μ filter. Quality control tests were carried out before packing: bacteriostasis / fungistasis; assay for endotoxins; high pressure liquid chromatography, osmolarity assay; pH level test; concentration test; and visual inspection for particulate matter. The rejected bulbs were destroyed.

Approximately 1250 ampoule containers per batch of 6 liters were then filled to a level of 4.2 mL each, with a fill of 4.4 mL (the label claims 4.0 mL). The ampules are shipped after two weeks of manufacture.

Example 2: Formulation I I (Lot of 50 mL of UTP Solutions of 250 v 300 ma / mL) The UTP was taken out of the freezer and allowed to return to near ambient temperature in a desiccator. Four batches of UTP solution were prepared at each concentration weighing exactly the equivalent of 12.5 g and 15 g of UTP (allowing the purity and water content of chromatography of the substance of the drug) in order to give solutions with concentrations of 250 and 300 mg / mL respectively. Each quantity of heavy UTP was accurately transferred to a clean 50 mL volumetric flask and dissolved in one portion to the final volume of 50 mL with distilled water. Each solution was adjusted to the target pH of 7.0, 8.0, 9.0 or 10.0 with sodium hydroxide solution and then adjusted to the final volume. Portions of each solution were transferred to any glass ampoules (1 1 mL Type 1, Borosilicate Class B, with screw-on top black phenolic lids having polyvinyl-faced pulp coatings) or low density polyethylene (LDPE) bottles (Nalgene No. 2003-9025 8 mL with narrow mouth). The samples thus packed were stored vertically at 5 ° C, 25 ° C / 60% relative humidity and 40 ° C / 75% relative humidity in order to determine the stability of the solution: 300 mg / mL solutions showed minimal degradation (between 92.8 and 98.0 of original power for storage in glass and between 92.5 and 94% of original power for storage in LDPE) following storage for 12 weeks at 25 ° C. No degradation was seen in the same range of solutions when stored at 5 ° C in 6 months. The stability studies described above were conducted in Metrics Inc., Greenville, NC 29836 under protocol I P / 033. During development studies, prototype electro-mechanical delivery devices showed flow regimes of up to 3.9 μL for UTP solutions formulated at 300 mg / mL. Similar prototypes have generated aerosols with average mass aerodynamic diameters (MMADs) in the range of 3 to 6 microns.

Clinical Studies A. Healthy Normal Volunteers (Non-Smokers) Study Design: This single-center Phase I Unit was a double-blind, randomized, randomized, single-dose aerosol UTP assessment of 48 healthy male volunteers. Four successive groups of 12 volunteers were enrolled in each dose level and were randomly drawn in a 2: 1 mode to receive UTP or placebo. The placebo was normal saline, and the four dose levels of UTP evaluated were: 0.5 mg / mL, 5 mg / mL, 25 mg / mL and 45 mg / mL. At the time of dosing, 4 mL of placebo or 4 mL of the appropriate UTP solutions were placed in an atomizer to convert into an aerosol. The purpose of this study was to determine the safety and tolerance of UTP at various dose levels and to determine if UTP could increase the ability of normal, healthy subjects to expectorate sputum containing alveolar macrophages, cells indicative of a deep lung sample, when compare with placebo.

Assignments and Administration of Study Drug Treatments: Each subject was randomly assigned to receive a single dose of either UTP or placebo. Each dose consisted of 4 mL of placebo or the appropriate UTP solution (0.5, 5, 25, or 45 mg / mL) and was administered using a jet pump (Pari LC PLUS ™) moved by portable compressor (De Vilbis Pulmo- Aide®) placed in a flow rate of 14 L / min. Inhalation of placebo or UTP took approximately 8 to 15 minutes. Research Products: The test product was UTP Solution for Inhalation at a concentration of 0.5, 5, 25 or 45 mg / mL. The UTP solutions were packed in single-use glass ampoules with rubber stoppers and tear-off aluminum seals. The product was stored refrigerated at 2 ° - 8 ° C. Each vial delivered 4 mL of solution upon emptying. The pH of each solution was 7.2 and the tonicity of each solution was approximately 300 MOsm / L. Each vial was individually labeled with the contents, strength and storage instructions. The placebo was supplied as a USP solution of Sodium Chloride Inhalation, 0.9%, sterile, (Dey Laboratories, Napa, CA, USA). The placebo was packed in single-use vials containing 5 mL of solution. For USP specifications, the pH of the solution was within the range of 4.5 to 7.0, 4 mL of placebo was used for administration. The product was stored at 2 ° -8 ° C. Each vial was individually labeled with contents, strength and storage instructions. Efficacy of Results: Sputum was collected for the purpose of cytological examination to determine if the sample contained alveolar macrophages. The presence of alveolar macrophages in a sputum sample indicates that the specimen is a quality specimen that rises from deep within the lungs (not just salivary secretions). For this study, sputum was collected for cytological examination at the following times: basic lines (pre-dose), immediately post-dosing, post-dosing at 4 hours, from 4 hours to 8 hours post-dosing, from 8 hours to 12 hours post-dosing and when getting up the next day. For data analysis purposes, all patients who received placebo were combined (n = 16) and patients who received UTO (n = 32, all four dose levels) were combined. Data from all 48 subjects were combined for the baseline sputum sample time point. Figure 1 illustrates the course of time for the effect of placebo and UTP on the percentage of sputum samples determined to be positive for alveolar macrophages (AM). Although most subjects (pre-dose period) could produce sputum sample at the baseline (44/48 subjects spontaneously and 47/48 subjects produced a sample that followed a simple maneuver involving 3 deep breaths, followed for a sputum); only about 30% of these samples were considered to be a quality sputum sample as evidenced by cytological examination (presence of AMs). The single-dose aerosol production of placebo (normal saline) in 16 subjects failed to improve the percentage of samples determined to contain alveolar macrophages at the time point immediately post-dosing compared to the baseline. However, in the combined group of UTP (n = 32), over 80% of the sputum samples were considered positive for alveolar macrophages, representing a 2.5-fold improvement over the baseline and placebo. The effect of UTP to improve the percentage of samples positive for alveolar macrophages was also evident at the second point of time (from the end of the dosage to 4 hours post-dosing); 37% of the samples were positive following the administration of placebo versus 57% following the UTP. The differences observed between UTP and placebo were no longer evident at the time point of 4 hours to 8 hours at which point both groups, placebo and UTP were essentially the same as the pre-dose values. This is consistent with the relatively short duration of action of UTP as well as findings in a previous study indicating that single, inhaled doses of UTP increase the overall mucociliary clearance of the lung for approximately 1.5 hours.

B. Study in Smokers Study Design: This second study in smokers was a cross-over, double-blind, randomized, single-center, multi-dose assessment of the highest level of UTP dose converted to aerosol evaluated in the previous study. Fifteen smokers were enrolled in this study. The subjects had to be smoking normally and have relatively normal lung function (forced expiration volume in 1 second of more than 80% predicted) at the entrance to the study. The subjects received both UTP (45 mg / mL) placebo (normal saline) three times a day for three consecutive days, with a wash of 1 week between periods. The purpose of this study was to determine if the highest dose of UTP could be consistently effective in increasing the amount of expectorated sputum on several consecutive days. This mimics sputum collection for the purpose of diagnosing certain lung infections and other conditions which is often done by asking patients to collect their sputum every morning for several consecutive days.

Assignments and Administration of Study Drug Treatments: Each subject was randomly assigned to receive multiple doses daily (three times a day for three consecutive days) of placebo and UTP with at least 1 week between the two dosing periods. Each dose consisted of 4 mL of the 45 mg / mL solution or placebo (normal saline) administered using a jet spray (Pari LC PLUS ™) moved by portable compressor (De Vilbis Pulmo-Aide®) placed at 14 L / min. . Inhalation of placebo or UTP took approximately 8 to 15 minutes. Efficacy of Results: The amount of expectorated sputum (weight in grams) was collected pre- and post-dosing of UTP and placebo at various time points through three days of dosing in each study period. Sputum was also evaluated by cytological examination to determine if the sputum samples contained alveolar macrophages; indicative of a deep lung sample. Figure 2 illustrates the sputum weights (g) in pre-dosing and post-dosing of placebo and UTP in the three days of treatment. As shown in Figure 2, the placebo inhalation did not increase the amount of sputum expectorated immediately post-dosing during any of the three days of dosing (comparison of sputum weights in pre-dosing with immediately post-dosing). In contrast, inhalation of UTP consistently increased the amount of sputum expectorated immediately post-dosing on all three days of dosing (comparison of sputum weights in pre-dosing with immediately post-dosing); the magnitude of the increase in the amount of expectorated sputum (pre- to post-dosing) was consistent in each of the three days for the UTP dosing period. Figure 3 illustrates cytology data for sputum expectorated by smokers who received UTP versus placebo. As shown in Figure 3, smokers who received UTP most likely produced a sputum sample containing alveolar macrophages (AM) on any of the three days that smokers received placebo (the difference between placebo and UTP was more pronounced on the day 2). There appeared to be a tendency for placebo to decrease the probability of producing a specimen containing alveolar mechophages (comparison of pre-dose with post-dose in the placebo group). This study showed that UTP consistently increases the amount of expectorated sputum (containing alveolar macrophages) over that produced by placebo. This effect of UTP was demonstrated in three consecutive days of treatment. Research Products: The test product was UTP Solution for Inhalation at a concentration of 45 mg / mL. The UTP solutions were packed in single-use glass ampoules with rubber stoppers and tear-off aluminum seals. The product was stored refrigerated at 2 ° - 8 ° C. Each vial delivered 4 mL of solution upon emptying. The pH of each solution was 7.2 and the tonicity of each solution was approximately 300 MOsm / L. Each ampoule was labeled individually with the contents, resistance and storage instructions. The placebo was supplied as a USP Solution of Sodium Chloride for Inhalation, sterile, 0.9% (Dey Laboratories, Napa, CA, USA). The placebo was packed in single-use vials containing 5 mL of solution. By USP specifications, the pH of the solution was within the range of 4.5 to 7.0. 4 mL of placebo was used for administration. The product was stored at 2 ° -8 ° C. Each vial was individually labeled with contents, strength and storage instructions.

C. Clinical Study in Patients with Chronic Bronguitis This single-center study was a double-blind, randomized, cross-evaluation of simple inhaled doses of scaling in patients with chronic bronchitis. The patients enrolled in this study had to fill the definition of chronic bronchitis of the American Thoracic Society (excessive production of mucosa in three months of the year, during at least 2 successive years). Patients who had mild to moderate airflow obstruction (forced expiratory volume over 1 second greater than 65% at the time of study entry) were included. A total of 26 patients (14 women and 12 men) were enrolled in this study and the majority were normal smokers. Five successive groups of five subjects (one additional patient was added in a cohort due to an absence) received a single dose of placebo and the appropriate dose of UTP (2.5, 5, 15, 25, and 45 mg / mL) in an order random. The dose of UTP and placebo were separated for at least 24 hours. The purpose of this study was to more carefully define the time course by which the UTO increases sputum expectoration in a population of patients known to be at high risk of developing lung cancer. In contrast to previous studies in smokers, cytological examination included identification and quantification of both alveolar macrophages as well as respiratory ciliated epithelial cells in sputum samples. Assignments and Administration of Study Drug Treatments: Each subject was randomly assigned to receive single inhaled doses of placebo (normal saline) or one of five doses of Formulation I of UTP on two separate days. For dosing, each dose consisted of four mL of 2.5, 5, 15, 25, 45 mg / mL of solution or placebo administered a jet atomizer (Pari LC PLUS ™) moved by portable compressor placed at 14 L / min. Inhalation of placebo or UTP took approximately 8 to 15 minutes.

Research Products: The test product was UTP Solution for Inhalation at a concentration of 2.5, 5, 15, 25, or 45 mg / mL. The UTP solutions were packed in single-use glass ampoules with rubber stoppers and tear-off aluminum seals. The product was stored refrigerated at 2 ° - 8 ° C. Each vial delivered 4 mL of solution upon emptying. The pH of each solution was 7.2 and the tonicity of each solution was approximately 300 MOsm / L. Each vial was individually labeled with the contents, strength and storage instructions. The placebo was supplied as a USP Solution of Sodium Chloride for Inhalation, sterile, 0.9% (Dey Laboratories, Napa, CA, USA). The placebo was packed in single-use vials containing 5 mL of solution. By USP specifications, the pH of the solution was within the range of 4.5 to 7.0. 4 mL of placebo was used for administration. The product was stored at 2 ° -8 ° C. Each vial was individually labeled with contents, strength and storage instructions. Efficacy of Results: The amount of expectorated sputum (weight in grams) was collected in the baseline and at several time points post-dosing of UTP and placebo. The time points were immediately 5 minutes post-dosing, 6 minutes to 30 minutes post-dosing, 31 minutes post-dosing to the discharge (within several hours of post-dosing). For the purposes of analysis, patients who received placebo across all dose groups were combined (n = 25); Patients receiving UTP (all doses) were combined; and patients who received the three highest dose levels (15, 25 and 45 mg / mL) (n = 15) were combined. Figure 4 illustrates the effect of placebo versus UTP on the amount of expectorated sputum (weight in grams) at two time points: baseline (expectorate expectoration) versus immediately at 5 minutes post-dosing . UTP (all combined doses) significantly increases the amount of expectorated sputum compared to baseline and placebo (all doses combined). The effect of UTP was even more pronounced when comparing the three highest dosed levels of UTP (n = 15) with the placebo group. The ability of UTP to increase the amount of expectorated sputum over the placebo and baseline was also evident at the last time point from 6 minutes to 30 minutes post-dosing as shown in Figure 5. Figure 6 shows that a at 31 of the discharge time point there was essentially no difference between the effect of UTP and placebo on the amount of expectorated sputum, indicating the effect of UTP is manifested over a short time frame consistent with previous studies. The cytology results of the sputum samples collected from 6 to 30 minutes post-dosing of the time point are shown in Figure 7 (sputum containing alveolar macrophages) and Figure 8 (sputum containing respiratory ciliated epithelial cells). UTP (all combined doses) and UTP (the three highest combined levels) significantly improved the percentage of patients producing a sputum ours containing alveolar macrophages (AM) compared to placebo (Figure 7). The UTP (all combined doses) and UTP (the three highest combined levels) also significantly improved the percentage of patients producing a sputum ours containing respiratory ciliated epithelial cells compared to placebo (Figure 8). It is worth noting in Figure 8 that only 8% of patients were able to produce a sample containing ciliated epithelial cells at the baseline or after the aerosol conversion of the placebo; while 73% of patients were able to produce such a sample in the UTP group (three higher levels). These effects of UTP were also observed in the immediately 5 minutes post-dosing time point for alveolar ciliated epithelial cells (baselines / placebo = 8% / 4%; UTP (all doses) = 32%; UTO (three highest doses) = 50% UTP effect returned close to baseline / placebo values within 31 minutes of discharge time point consistent with sputum weight findings This study clearly demonstrates that aerosol dose of UTP (particularly the three highest dose levels of 15, 25 and 45 mg / mL) are more effective than placebo (normal saline) in causing the production of deep sputum samples containing cells that could be evaluated for lesions or pre-cancerous changes, In addition, UTP can induce the production of a quality sample (containing both alveolar macrophages and ciliated epithelial cells) within a short time frame (within 30 minutes postdose). n) D. Treatment of a Human Subject with Soft Chronic Bronguitis It has been shown that two different doses of Formulation I of UTP given by inhalation (4 mL of a liquid solution containing either 5 mg / mL or 25 mg / mL of UTP) increases mucociliary clearance at the baseline (non-inhaled treatment) and also on placebo in a patient with mild chronic bronchitis. In this study, the patient inhaled the different doses of UTP and placebo on separate days. The procedure to measure mucociliary clearance was achieved by having the patient inhale, in the following order: iron oxide particles labeled with technetium and then either of the two doses of UTP or placebo. After inhalation of the UTP or placebo, the clearance of radio-labeled particles was measured by serial gamma-gamma-ray images for 2.5 hours. In order to establish mucociliary clearance of baseline on the first day of the study, the patient inhaled only the radiolabel and then made 2.5-hour gamma-ray images. Safety information was collected by monitoring heart rate, ECG, blood pressure, oxyhemoglobin saturation by pulse oximetry before, during and after inhalation for all dosing periods. During all phases of the study the patient was monitored for any adverse reactions during each dosing period. Figure 9 shows the total lung retention curves for the baseline (non-inhalation of UTP or placebo), both doses of UTP (5 mg / mL is labeled as "treatment 3", 25 mg / mL is labeled as " treatment 2") and placebo (labeled" treatment 1") over the 2.5 hour time period. A decrease in retention demonstrates a clearance of secretions in the lungs carrying the radiolabelled particles. In this case, both doses of UTP clearly improved the clearance of secretions (mucociliary clearance) on the placebo and baseline. Research Products: The test product was UTP Solution for Inhalation at a concentration of 5 and 25 mg / mL. The UTP solutions were packed in single-use glass ampoules with rubber stoppers and tear-off aluminum seals. The product was stored refrigerated at 2 ° - 8 ° C. Each vial delivered 4 mL of solution upon emptying. The pH of each solution was 7.2 and the tonicity of each solution was approximately 300 MOsm / L. Each vial was individually labeled with the contents, strength and storage instructions. The placebo was supplied as a USP Solution of Sodium Chloride for Inhalation, sterile, 0.9% (Dey Laboratories, Napa, CA, USA). The placebo was packed in single-use vials containing 5 mL of solution. By USP specifications, the pH of the solution was within the range of 4.5 to 7.0. 4 mL of placebo was used for administration. The product was stored at 2 ° -8 ° C. Each vial was individually labeled with contents, resistance and storage instructions. Clinical studies in patients with cystic fibrosis (ages 6-40) have shown that Formulation I of UTP had no serious adverse effects in dose ranges up to 45 mg / mL. The invention now fully described will be apparent to one of ordinary skill in the art that changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims (10)

1. REVIVAL NAMES 1. A sterile pharmaceutical composition comprising uridine 5'-triphosphate (UTP) and a sterile aqueous solution with a UTP concentration between 0.1 and 100 mg / mL; an osmolarity value between 250 and 450 mOsM; and a pH level between 6.0 and 8.5.
2. 2. A composition according to claim 1 wherein the UTP concentration is between 5 and 45 mg / mL.
3. 3. A composition according to claim 1 which is isotonic with an osmolarity of approximately 300 mOsM.
4. 4. A composition according to claim 1 wherein the pH is between 7.0 and 7.5.
5. 5. A method for increasing expectoration by administering an amount of a composition according to claims 1-4 effective to increase expectoration.
6. 6. A sterile pharmaceutical composition comprising uridine 5'-triphosphate (UTP) and a sterile suspension with a UTP concentration between 45 and 1000 mg / mL, a pH between 6.5 and 12; such that the composition is capable of delivering a therapeutic amount of UTP in a volume range of 10 to 100 μL.
7. 7. A composition according to claim 6 wherein the concentration of UTP is between 100 and 500 mg / mL.
8. A composition according to claim 6 wherein the pH is between 7 and 9.
9. 9. A composition according to claim 6 wherein the volume is between 50 to 100 μL per therapeutic amount.
10. 10. A composition according to claim 6 wherein a preservative is absent. 1 1. A method for increasing expectoration by administering an amount of a composition according to claims 6-10 effective to increase expectoration.