Method for therapy of pulmonary diseases

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine, particularly to treating bronchopulmonary dysplasia. A method involves administering an effective amount of citrulline into the patient. What is also presented is a pharmaceutical composition applicable for intravenous administration, containing a pharmaceutically acceptable carrier and an amount of citrulline effective for increasing blood plasma citrulline. The level is measured by comparing plasma citrulline in the patient under treatment to that in the patient having no bronchopulmonary dysplasia. The given composition is presented for producing a preparation for injections.

EFFECT: inventions provide the effective treatment of bronchopulmonary dysplasia by the polycomponent effect of citrulline on the disease process.

11 cl, 8 dwg, 2 tbl, 4 ex

This patent application claims priority on provisional patent application U.S. serial No. 61/025,157, registered 31 January 2008, the contents of which are incorporated herein in its entirety by reference.

The present invention relates to the treatment of lung diseases such as bronchopulmonary dysplasia (BPD) and is caused by chronic hypoxia pulmonary hypertension, for example, in newborns.

Bronchopulmonary dysplasia (BPD) usually occurs in infants, especially in premature infants, and is characterized by an acute form of pulmonary lesion or due to oxygen and / or mechanical ventilation, leading to harmful effects or delay the development of the alveoli or pulmonary vascular (Jobe et al. (2001) Am J Respir Crit Care Med 163: 1723-1729). In animal models of inhaled NO improves gas exchange, and structural development of the lungs, but the use of this therapy in infants at risk of BPD is controversial (Ballard et al. (2006) N Engl J Med 355: 343-353).

Neonates with chronic lung disease and heart disease blue type often suffer from hypoxia. Because of the impact on existing and developing pulmonary artery chronic hypoxia causes a progressive change and the function and structure of the pulmonary circulation. Shimoda L, et al., Physiol Res (2000) 49: 549-560; Subhedar, N. V., Acta Paediatr suppl (2004) 444: 29-32. In the end, and�ESO chronic hypoxia leads to severe pulmonary hypertension, culminating in failure of the heart muscle on the right side and death.

Accordingly, in this area has been long felt and still need to Ways to treat lung diseases such as BPD and caused by chronic hypoxia pulmonary hypertension, and further such as in newborns.

The present invention provides Methods and compositions for the treatment of lung diseases such as bronchopulmonary dysplasia (BPD) and is caused by chronic hypoxia pulmonary hypertension.

In some embodiments, an effective amount of a precursor of nitric oxide administered to a patient suffering from BPD and(or) related complications, and / or at risk of BPD and / or complications associated with BPD. In some embodiments, the precursor of nitric oxide contains at least one citrulline, a precursor that generates citrulline in vivo, its pharmaceutically acceptable salt, and combinations thereof. In some embodiments, the precursor of nitric oxide, such as citrulline, administered orally. In some embodiments, the precursor of nitric oxide, such as citrulline, administered intravenously.

In some embodiments, an effective amount of a precursor of nitric oxide administered to the patient, the suffering caused by chronic�Oh hypoxia pulmonary hypertension and(or) related complications and / or risk of disease caused by chronic hypoxia pulmonary hypertension and / or complications associated with caused by chronic hypoxia pulmonary hypertension. In some embodiments, the precursor of nitric oxide contains at least one citrulline, a precursor that generates citrulline in vivo, its pharmaceutically acceptable salt, and combinations thereof. In some embodiments, the precursor of nitric oxide, such as citrulline, administered orally. In some embodiments, the precursor of nitric oxide, such as citrulline, administered intravenously.

Therefore, the aim of the present invention is to provide for the treatment of lung disease in a patient.

In accordance with the above purpose of the present invention, other objectives will be apparent in the subsequent description in conjunction with the accompanying drawings and examples below.

Fig. 1 schematically shows the urea cycle. Fig. 2 shows a block diagram of the testing procedures in accordance with examples.

Fig. 3 shows a bar chart showing the average measurements of pulmonary artery pressure in piglets of control (n=6), chronic hypoxia (n=11) and chronic hypoxia in the treatment of L-citrulline (n-6). All values are given as mean ± standard error of the average, different from control;⁺different from chronic hypoxia; p<0,05, one�about-factorial analysis of variance with subsequent comparative test.

Fig. 4 shows a bar chart showing the calculated pulmonary vascular resistance in piglets of control (n=6), chronic hypoxia (n=11) and chronic hypoxia in the treatment of L-citrulline (n=6). All values are given as mean ± standard error of the average, different from control;⁺different from chronic hypoxia; p<0,05, one-factor analysis of variance with subsequent comparative test.

Fig. 5 shows a bar chart showing the exhaled nitric oxide in piglets of control (n=6), chronic hypoxia (n=11) and chronic hypoxia in the treatment of L-citrulline (n=5). All values are given as mean ± standard error of the average. * different from control;⁺different from chronic hypoxia; p<0,05, one-factor analysis of variance with subsequent comparative test.

Fig. 6 shows a bar chart showing the accumulation of nitrite/nitrate in the lung perfusate piglets from control (n=17), chronic hypoxia (n=9) and chronic hypoxia in the treatment of L-citrulline (n=5). All values are given as mean ± standard error of the average. * different from control;⁺different from chronic hypoxia; p<0,05, one-factor analysis of variance with subsequent comparative test.

Fig. 7A pok�shown a picture of a Western blot test for protein endothelial NO synthase in re probing for actin lung tissues from control piglets (n=3), in chronic hypoxia (n=3) and chronic hypoxia in the treatment of L-citrulline (n=3).

Fig. 7B shows a bar chart showing densitometry endothelial NO synthase, normalized against actin for lung tissues from control piglets (n=3), chronic hypoxia (n=3) and chronic hypoxia in the treatment of L-citrulline (n=3).

Born prematurely, children continue to be a major problem in obstetrics and neonatology, accounting for the bulk of perinatal mortality and long term neurological morbidity among newborns. BPD is one of many oslojenni that may be associated with preterm birth. BPD may be associated with long hospitalizatio prematurity, repeated hospitalization in the first few years of life and a delay in development. Fortunately, BPD currently rare in newborns weighing over 1200 g when prodenia or the age of the fetus, pervyshin 30 weeks (Jobe et al. (2001) Am J Respir Crit Care Med 163: 1723-1729). The incidence of BPD, defined as oxygen demand at the age of 36 weeks after the last menstrual period is about 30% for infants with birth weight <1000 g (Jobe et al. (2001) Am J Respir Crit Care Med 163:1723-1729). Some of these newborns with severe lung disease requiring ve�tilesii and(or) the filing of supplemental oxygen for several months or even years.

Many factors contribute to BPD, and are probably additionally or synergistically, contributing to the disease. Traditional views are that BPD is due mainly to the disease caused by oxidants and ventilation (Jobe et al. (2001) Am J Respir Crit Care Med 163: 1723-1729). Mechanical ventilation and oxygen can prevent the development of alveoli and blood vessels in premature infants and to contribute to the development of BPD (Jobe et al. (2001) Am J Respir Crit Care Med 163: 1723-1729). Reduced number of alveoli may lead to a significant reduction of the surface area of the body, which is associated with a decrease dysmorphic pulmonary microvascular system. These anatomical changes are associated with ever increasing levels of leukocytes and cytokines in respiratory tract samples (Jobe et al. (2001) Am J Respir Crit Care Med 163: 1723-1729).

Inflammation also may play a role in the development of BPD. Many anti-inflammatory and chemotactic factors are present in the airspace of premature babies with lung ventilation, and discovered a higher concentration of these factors in the airspace in infants who subsequently developed BPD (Jobe et al. (2001) Am J Respir Crit Care Med 163: 1723-1729). Other factors which are considered important to the development of BPD include: bambusoideae peptides, hyperoxia, hypoxia, malnutrition, treatment with glucocorticoids and SVER�cytokine production, factor-α, TGF-α, IL-6, or IL-11, tumor necrosis (Jobe et al. (2001) Am J Respir Crit Care Med 163:1723-1729).

Diagnosis of BPD mainly includes monitoring of respiration of the newborn during the first weeks of life for signs of delayed lung development and is continuing and(or) increased dependence on artificially increased respiration. Diagnostic tests that may be performed to assist the diagnosis of BPD may include: tests for oxygen content in the blood, x-ray studies of the chest ehokardio-grams. BPD is traditionally diagnosed when the fetus requires supplemental oxygen at the age of 36 weeks postmenstrual age. Modern methods definitions used to identify and detect BPD include specific criteria for "minor", "moderate" and "severe" BPD (Ryan, R. M. (2006) J Perinatology 26:207-209).

Treatment of BPD may include a multi-faceted approach to the treatment of symptoms and providing opportunities for the development of the lungs of the newborn. Currently available Methods of treatment may include: appointment of surfactants to improve lung ventilation, mechanical ventilation to compensate for the insufficiency of breathing, extra oxygen to ensure sufficient oxygen in the blood, drug therapy bronchodilators to improve air �Otok in the lungs, corticosteroids to reduce swelling and inflammation of the respiratory tract, the fluid control to avoid pulmonary edema, treatment of patent ductus arteriosus, and proper nutrition.

It is shown that the introduction of nitric oxide by inhalation improves lung development in newborn animals models (Ballard et al. (2006) N Engl J Med 355:343-353). However, the introduction of NO by inhalation is controversial for humans. Thus, in accordance with some embodiments of implementation of the present invention, the introduction of citrulline or another precursor of NO to a patient suffering from BPD, to thereby increase the synthesis of NO in vivo, may represent an alternative to the inhalation of NO in the treatment of BPD.

Because of its impact on existing, and developing the pulmonary artery, chronic hypoxia causes a progressive change and the function and structure of the pulmonary circulation. Shimoda L, et al., Physiol Res (2000);49:549-560; Subhedar, N. V., Acta Paediatr suppl (2004); 444: 29-32. Eventually chronic hypoxia leads to severe pulmonary hypertension, culminating in a right-sided heart failure and death. Currently therapy of pulmonary hypertension in infants with chronic cardiopulmonary disorders associated with permanent or episodic hypoxia is largely limited uluchshyeniye the basis of cardiopulmonary diseases and attempts to achieve appropriate oxygenation. Abman, S. N.; Arch Dis Child Fetal Neonatal Ed (2002) 87: F15-F18; Allen, J. and ATS subcommittee AoP, Am J Respir Crit Care Med (2003) 168: 356-396; Mupanemunda, R. H., Early Human Development (1997) 47: 247-262; Subhedar, N. V., Acta Paediatr suppl (2004) 444:29-32. Thus, according to some variants of implementation of the present invention proposes a new therapeutic approach, including the introduction of citrulline suffering from pulmonary hypertension due to chronic hypoxia.

Citrulline is a key intermediate in the cycle of formation of urea and the generation of nitric oxide (NO). In the cycle of formation of urea citrulline is a precursor neoplasms of arginine. Arginine can be detalizirovan by arginase to produce urea, which can then be allocated to free the body from waste nitrogen, particularly ammonia. In an alternative embodiment, the arginine can provide the production by NO synthase nitric oxide. In this case, the function of unchanged urea cycle is important not only for the separation of ammonia, but also to maintain appropriate levels in the tissue of the arginine precursor of NO.

Nitric oxide is synthesized by the synthase nitric oxide with arginine as a substrate. The factor limiting the intensity of NO synthesis is the availability of cellular arginine, and the preferred source of arginine for NO synthesis is argin�n newly formed from citrulline. The path of synthesis in a living organism to begins with arginine ornithine. Ornithine is connected to carbamyl phosphate to produce citrulline, which, in turn, connects with the aspartate, in the presence of ATP, to produce ariniraarinira. At the final stage fumarate is cleaved from adrenocortical for arginine production. Cascade reactions for arginine by the hydrolytic action of arginase to produce ornithine and urea. These reactions constitute the cycle of production of urea. Cm. also Fig. 1.

Alternatively, to mitigate the synthesis of urea arginine may be a substrate required for the synthesis of NO synthase by nitric oxide. In addition, exogenous citrulline can get into the cycle of production of urea and ensure the synthesis of arginine in a living organism, that can subsequently provide the synthesis of NO. Accordingly, the appointment of citrulline patients, including but not limited to those who have suspected or diagnosed BPD or caused by chronic hypoxia pulmonary hypertension may increase the synthesis of arginine and subsequently increase the production of NO, thereby, prevent and / or cure BPD or caused by chronic hypoxia pulmonary hypertension. Can also be provided with a precursor of citrulline, which generate citrol�n in a living organism. Alternatively, the citrulline may be provided other NO precursors. For example, as a precursor of NO can be provided arginine, or a precursor that generates the arginine in a living organism.

I. METHODS of TREATMENT

The present invention provides methods and compositions for increasing the synthesis of NO. In some embodiments, to increase the synthesis of NO administered to a patient an effective amount of citrulline or another precursor of NO. In some embodiments, implementation of the NO precursor selected from the group including, but not limited to this, citrulline, a precursor that generates citrulline in vivo, arginine, a precursor that generates the arginine in a living organism or combination thereof. In some embodiments, citrulline or another precursor NO. administered orally. In some embodiments, citrulline or another precursor NO. administered intravenously.

The present invention also provides methods and compositions for the treatment of BPD and(or) related complications in the patient. In some embodiments, an effective amount of citrulline or another NO precursor is administered to a patient suffering from BPD and(or) accompanying complications and / or risk of complications associated with BPD. In some embodiments, the exce�NO COP is selected from the group including but not limited to this, citrulline, a precursor that generates citrulline in vivo, arginine, a precursor that generates the arginine in a living organism or combination. In some embodiments, citrulline or another precursor NO. administered orally. In some embodiments, citrulline or another precursor NO. administered intravenously. In some embodiments, the patient who needs treatment, is the patient suffering from an acute condition associated with BPD. The above are representative examples of such conditions.

The present invention also provides methods and compositions for treating a patient caused by chronic hypoxia pulmonary hypertension and(or) related complications. In some embodiments, an effective amount of citrulline or another precursor of NO administered to the patient, the suffering caused by chronic hypoxia pulmonary hypertension and(or) related complications and / or risk of complications caused by chronic hypoxia pulmonary hypertension. In some embodiments, the precursor N is selected from the group including, but not limited to this, citrulline, a precursor that generates citrulline in vivo, arginine, a precursor that generates �Linin in a living organism or combination thereof. In some embodiments, citrulline or another precursor NO. administered orally. In some embodiments, citrulline or another precursor NO. administered intravenously. In some embodiments, the patient who needs treatment, is the patient suffering from an acute condition associated with caused by chronic hypoxia pulmonary hypertension. The above are representative examples of such conditions.

In some embodiments, the precursor of nitric oxide contains at least one citrulline, a precursor that generates citrulline in a living organism, its pharmaceutically acceptable salt, and combinations thereof. Cm. Fig. 1. In some embodiments, a precursor of an oxide of nitrogen selected from the group including, but not limited to this, citrulline, arginine or a combination thereof. In some embodiments, the precursor of nitric oxide, such as citrulline, administered orally. In some embodiments, the precursor of nitric oxide, such as citrulline, administered intravenously.

In some embodiments, the patient suffers from hipotiroidiei. In some embodiments, hypocellularity characterized by levels of citrulline in plasma ≤37 µmol/l, in some embodiments, ≤25 µmol/l, in some embodiments, the OS�span ≤20 μmol/l, in some embodiments, ≤10 μmol/l, in some embodiments, the implementation of ≤5 mmol/L.

In some embodiments, the patient suffering described in this document disease, experiencing a relative Hypo-citrullinemia. The term "relative hypocellularity" refers to the condition in which the patient is suffering from some disease, reduced levels of citrulline in plasma in comparison with a person who is not afflicted with this disease.

As used herein, the term "treatment" refers to interventional procedures, designed to improve the condition of the patient {e.g., after the occurrence of the disease or after injury) to mitigate complications related to the patient's condition, as well as interventional procedures that are designed to prevent the occurrence of a condition in a patient. In other words, the terms "treating" and its grammatical variants suggest a broad interpretation to encompass values that relate to reducing the severity and / or curing the condition, as well as to the values prevention. Regarding the latter, "treatment" can refer to "prevention" to any degree, for example, but not limited to, the patient at risk of an outbreak, or other pileni� the patient's ability to resist the disease process.

A patient treated according to the present invention in its many embodiments, preferably is a human, although it is obvious that the principles of the present invention indicate that the present invention is effective against all vertebrate species, including warm-blooded species such as mammals and birds, which, as implied, covered by the term "patient". In this context, it is clear that the mammal includes any mammalian species, for which the treatment is preferably, for example, but not limited to, mammalian species used in agriculture, and domestic animals.

Thus, the proposed treatment of mammals such as man, as well as those mammals that are important because of the threat of their disappearance (such as Siberian tigers), economically important (animal, farmed for human consumption) and / or important for a person socially (animals kept as Pets or in zoos), for example, carnivorous animals besides humans (such as cats and dogs), swine (pigs, domestic pigs and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses. Also provided is the treatment of birds, including the treatment of such species of birds, over which� is threatened with extinction, contained in zoos, as well as fowl, and more particularly, poultry, i.e., bred poultry, such as Turkey, chicken, ducks, geese, Guinea fowl, etc., because they are also important economically. Thus, the proposed treatment of livestock, including, but not limited to, domestic pigs (sows and boars), ruminants, horses, poultry, etc.

II. The PHARMACEUTICAL COMPOSITION

Needy patient is administered an effective dose of a composition of the present invention. "An effective amount" is an amount of composition sufficient to cause a noticeable response {e.g., a biologically or clinically justified response in patients for which the treatment is carried out). Actual dosage levels of active ingredients in the compositions of the present invention may vary, to try a number of active compound(s) that is effective to achieve the desired therapeutic response for a particular patient. The selected dose level depends on the activity of therapeutic composition, the route of administration, combination with other medicines or treatment, the severity of the disease, which must be treated, and the status and history of disease from the patient who is the treatment. By way of example, but not limitation, the dosage formulations can start with �rowney less than required, to achieve the desired therapeutic effect and gradually increase the dosage until you reach the desired effect.The effectiveness of the composition may vary, and therefore can change, "an effective amount".

According to the description of the present invention presented herein, a specialist in this area can pick up the dose for the individual patient taking into account the particular formulation, the route of administration used with this composition, and specific diseases that must be treated. Additional calculations of the dose can accommodate the growth and body weight of the patient, gender, severity and stage of symptoms, and the presence of additional adverse physical conditions.

Through additional examples, the amount of the active ingredient, which can be combined with a substance carrier to obtain a single metered-dose form is different for different patients, for whom being treated and the particular mode of appointment. For example, a composition intended for administration to man, may contain from 0.5 mg to 5 g of the active substance in a mixture with an appropriate and convenient amount of carrier substances, which may vary from about 5 to about 95 percent of the total composition. For example, for an adult patient doses per person in appointing mainly comprise from 1 mg to 500 mg �a number of times per day. Therefore, the standard dosage forms mostly contain from about 1 mg to about 500 mg of the active ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg or 1000 mg.

Precursor of nitric oxide is administered in some embodiments, the dose range is from about 0.01 mg to about 1000 mg, in some embodiments, the dose range is from about 0.5 mg to about 500 mg, and in some embodiments, the dose range is from about 1.0 mg to about 250 mg. is a Precursor of nitric oxide may also be assigned in some embodiments, the dose range is from about 100 mg to about 30,000 mg, in some embodiments, the dose range is from about 250 mg to about 1000 mg. of a Representative dose of 3.8 g/m²/day of arginine or citrulline (molar equivalent, molecular weight of L-citrulline 175,2, L-arginine 174,2).

Representative intravenous solutions citrulline may contain 100 mg/ml (10%) solution. Representative intravenous dosages of citrulline may contain 200 mg/kg 400 mg/kg 600 mg/kg and 800 mg/kg. In some embodiments, for example, but without limitation, the doses of 600 or 800 mg/kg, the dose can be reduced to the amount in the range of 50 mg/kg and 100 mg/kg to mitigate observed adverse effects on blood �itemno pressure. In some embodiments, the dose can be assigned once or repeatedly within a predetermined time period, e.g., per day.

In some embodiments, the pharmaceutical composition comprises a quantity of citrulline effective to raise the level of citrulline in plasma for the treatment of a condition described herein to a patient. In some embodiments, this level is determined by comparing the levels of citrulline in plasma from the patient who carried out the treatment, with the observed level in a patient who is not suffering from this disease. In some embodiments, the amount of citrulline effective to raise the level of citrulline in plasma of the patient of at least 5 μmol/l, in some cases at least up to 10 µmol/l, in some cases at least up to 20 µmol/l, in some cases at least 25 μmol/l, and in some cases up to about 37 mol/L.

In some embodiments, the present invention proposes a pharmaceutical composition containing a precursor of nitric oxide and a pharmaceutically acceptable carrier such as a pharmaceutically acceptable carrier for the person. In some embodiments, the present invention offers pharmaceutical compositions containing citrulli� or arginine dosage, as described above.

The compositions of the present invention is usually administered orally or parenterally in a standard dosage zostavax containing standard netoksicski pharmaceutically acceptable carriers, adjuvant and fillers, if necessary. The term "parenteral" as used herein includes methods, intravenous, intramuscular, intra-arterial injection or infusion.

Preparations for injection, for example, sterile aqueous or oily suspension for injection, are in accordance with the prior art, using suitable dispersing or wetting agents and suspendida substances. A sterile preparation for injection may also be a sterile injectable solution or suspension in a nontoxic acceptable diluent or solvent, for example, in the form of a solution in 1,3-butanediol.

Acceptable fillers and solvents that can be used are water, ringer's solution and isotonic sodium chloride solution. In addition, as a solvent or suspendida environment typically use a sterile non-volatile oil. For this purpose you can use any facilitate non-volatile oil using synthetic mono - or diglycerides. In addition, fatty oils, such as oleic acid, and�used for preparing drugs for injection. Examples of carriers include neutral saline solutions with buffer as phosphate, lactate, Tris, etc.

In a representative embodiment, the dose can be assigned to the patient several times during the relevant period of treatment, including, but not limited to, 1, 2, 3, 4, 5, 6 or more dosages.

However, it is understood that the specific dose level for an individual patient depends on a number of factors, including the age, body weight, General ostatnie health, sex, diet, time of appointment, method of appointment, rate of excretion, combination of drugs and the severity of the particular disease, which treatment is performed.

EXAMPLES

The following examples are included to illustrate representative modes of implementing the present invention. In light of the present description and the General level of technology professionals it is clear that the following examples are for demonstration only and that numerous changes, modifications and variations may be utilized without deviation from the essence and scope of the present invention.

EXAMPLES 1-4

In the following examples evaluated, prevent oral feeding of L-citrulline the development of pulmonary hypertension and concomitant reduction of NO production in newborn piglets, for 10 days were exposed to chronic hypoxia.

The METHODS USED IN EXAMPLES 1-4 EXPERIMENTS ON ANIMALS

All investigated 17 piglets during hypoxia and 17 piglets of control. Cm. Fig. 2. Animal control was studied in day of their receipt from the farm at the age of 12 days. Piglets with hypoxia (aged 2 days) were placed in an Isobaric hypoxic chamber for a period of 10 to 11 days. Isobaric hypoxia was provided by compressed air and nitrogen to create concentrations of inhaled oxygen 8-11% (PO₂60-72 Torr), and CO₂supported at the level of 3-6 Torr by absorption with soda lime. In animals monitorial body weight and physical condition twice a day. They were fed without the preparation of a sow milk replacer from drinking devices in the cell.

APPENDIX L-CITRULLINE

Six of the seventeen piglets with hypoxia received Supplement, L-citrulline oral, starting with the first day of exposure to hypoxia. Cm. Fig. 2. The Supplement L-citrulline was provided at a dose of 0.13 GM/kg of body weight twice a day with a syringe to supply the dose of oral. If the staff assumed that the pig swallowed the majority of the dose, her input was repeated. L-citrulline was mixed using the drug (Sigma Pharmaceuticals, St. Louis, Missouri, USA, 98% purity) at a concentration of 0.13 g per milliliter of distilled water, and when it is fully dissolved, pass the solution through a 0.20 micron filter.

HEMODYNAMICS IN vivo

MEASUREMENT of EXHALED nitric OXIDE

To measure exhaled NO in animals with anesthesia exhaled gas was sampled two or three times during the periods of 3 minutes and the prop�Cali via chemiluminescence analyzer (model 270 IN NOA; Sievers, Boulder, Colorado, USA), to measure the concentration of NO, as described above. Fike, C. D., et al., American Journal of Physiology (Lung Cellular and Molecular Physiology 18) (1998) 274: L517-L526. Exhaled NO (nmol/min) was calculated using the minute ventilation and the measured concentration of exhaled NO.

PERFUSION DISABLED LUNG

Light shut off and perform perfusion in vivo with the use of bicarbonate solution of Krebs-ringer (KRB) containing 5% dextran, molecular weight 70000, at 37°C under ventilation gas mixture with normal oxygen content (21% O₂and 5% CO₂), as described above. Fike, C. D. et al, J Appl Physiol (2000) 88: 1797-1803. Perfusion of the lungs was performed for 30-60 minutes until it reached a stable pressure of the pulmonary artery. Samples of perfusate (1 ml) was then removed from the cannula of the left atrium every 10 min for 60 minutes period. Samples of perfusate was centrifuged, and the supernatant was stored at -80°C for future analysis of the concentrations of nitrite/nitrate (NOx^-as described next. At the end of perfusion was measured, the volume of perfusate remaining in the circuit and capacity. In some cases, lung tissue was collected immediately after perfusion, frozen, and then stored at a temperature of -80 degrees for the subsequent measurement of the endothelial NO synthase, as described below.

MEASUREMENT NITRITE�/NITRATE NITROGEN

To determine the concentration of NOx^-in the perfusate (nmol/ml) at the time of each sampling was used chemiluminescent analysis. Fike, C. D. et al, JAppl Physiol (2000) 88:1797-1803; Turley, J. E. et al., Am J Physiol Lung Cell Mol Physiol (2003) 284:L489-L500. The perfusate (20 µl) was injected into the reaction chamber chemiluminescent NO analyzer (model NO. 170 IN A, Sievers). The reaction chamber contained a vanadium chloride (III) in 1 mol HC1, heated to a temperature of 90°C, to restore the nitrite and nitrate to NO gas. NO gas was transferred into the analyzer, using a constant gas flow of N₂through the trap gas bubbles containing 1 mol NaOH to remove HCl vapor. Received standard curve by adding a known amount of NaNO₃to distilled water and completing the study, as described DLY samples of perfusion.

The NOx concentration^-in the perfusate (nmol/ml) was calculated for each time of collection of samples by multiplying the NOx concentration^-in the perfusate at the time of selection of the sample volume on the system (circuit of perfusate+capacity) at the time of sampling plus the number of NOx^-removed all previous samples. The rate of production of NOx^-was determined by the slope of the linear regression line approximated to the amount of NOx^-in the perfusate, depending on time for the first 60 minutes of sample collection period.

MEASUREMENT of PLASMA AMINO acids

While GE�dinamicheskih measurements and(or) research perfusion of lungs from control and animals with chronic gioscia in the treatment of L-citrulline and no treatment, blood was taken before the beginning of the study, and the plasma was frozen at a temperature of -80 degrees to determine amino acid levels. For animals with hypoxia in the treatment of L-citrulline time of obtaining the blood sample was approximately 12 hours after the last dose of L-citrulline, so it was a minimum level. In some animals in the treatment of L-citrulline (n=3) after blood collection to the minimum level dose of L-citrulline was given via nasogastric tube. After this dose, blood samples were taken every 30 minutes for 90 minutes (the duration of studies in natural conditions). All samples were separated and collected plasma was frozen at a temperature of -80 degrees for the analysis of amino acids.

The concentration of citrulline and arginine in plasma were determined by analysis of amino acids in the extracts without proteins. Amino acids were separated by katio-noobman chromatography using an amino acid analyzer Hitachi L8800 (Hitachi USA, San Jose, CA, USA). The calibration of the analyzer was performed before testing samples of pigs.

WESTERN BLOTTING of ENDOTHELIAL NO SYNTHASE IN the LUNG TISSUE

Using the standard Method of immunoblotting, as described above, carried out the analysis of samples of whole lung homogenate from control (n=3), animals with hypoxia without treatment (n=3) and animals with hypoxia in the treatment of L-citrulline (n=3) for endothelial NO synthase. And�used 10 micrograms of total protein, dilution of primary antibodies to endothelial NO synthase 1:500 (BD transduction) and dilution of the secondary anti-mouse antibody, conjugated with horseradish peroxidase, 1:5000. Fike, C. D., et al., American Journal of Physiology (Lung Cellular and Molecular Physiology 18) (1998) 274:L517-L526.

CALCULATIONS AND STATISTICS

Pulmonary vascular resistance was calculated by hemodynamic measurements in vivo: (pulmonary artery Pressure - end-diastolic pressure LV) (Minute volume/body weight).

The data are presented as mean+standard error. For comparison the data contrle, animals with hypoxia without treatment and in the treatment of L-citrulline used univariate analysis of variance with a posteriori comparative test of protected least reliable difference on the Fischer PLSD). The p value less than 0.05 was considered significant. Meier, U., Pharm Stat (2006) 5: 253-263.

EXAMPLE 1

HEMODYNAMIC MEASUREMENTS IN vivo

In animals with chronic hypoxia and in the treatment of L-citrulline, and no treatment was lower minute volume and weight, and higher LVEDP measurements per day studies at the age of 12-13 days than piglets from the controls of comparable age (table. 1). Measurement of intra-aortic pressure and indicators of blood gases were similar in different groups (paO₂in drawing up 74±5 Torr in piglets of control, 74±8 Torr � piglets with hypoxia without treatment and 78±7 Torr in piglets with hypoxia in the treatment of L-citrulline; paCO₂was 39±2 piglets from control 41±4 in piglets with hypoxia without treatment and 30±1,0 in piglets with hypoxia in the treatment of L-citrulline). Notably, as shown in Fig. 3 that animals with epoxie treatment with L-citrulline had significantly lower pulmonary artery pressure than animals during hypoxia without treatment (p value of 0.01). The pulmonary artery pressure was not different to control at the normal content of oxygen and animals with hypoxia in the treatment of L-citrulline (p=0,08).

In addition, as shown in Fig. 4, the calculated pulmonary vascular resistance in animals with hypoxia in the treatment of L-citrulline (0,071±0,003) is significantly lower than in animals with hypoxia without treatment (p value of 0.001). Moreover, the pulmonary vascular resistance is similar in animals with hypoxia in the treatment of L-citrulline and contrle under normal oxygen content (p 0,07).

EXAMPLE 2

The OUTPUT of EXHALED NO AND NOX^-The PERFUSATE

As shown in Fig. 5, the output of exhaled NO in control and animals with hypoxia in the treatment of L-citrulline is higher than the output of exhaled NO in animals with hypoxia without treatment (p 0.001 and 0.032, respectively). However, the yield of exhaled NO did not differ between control animals and animals with hypoxia in the treatment of L-citrulline (p=0,124).

As shown in Fig.6, light and control (p=0.02) and in animals with hypoxia� in the treatment of L-citrulline p=0.04) had a significantly higher rate of accumulation of NOx ^-than the lungs in animals with hypoxia without treatment. Moreover, there was no difference the rate of accumulation of NOx^-between the lungs in animals with hypoxia in the treatment of L-citrulline and control under normal oxygen conditions.

EXAMPLE 3 PLASMA AMINO acids

As shown in table. 2, although not achieved statistical significance (p=0.05), the levels of L-citrulline in plasma of piglets with hypoxia without treatment were lower than the minimum levels of L-citrulline in piglets with hypoxia during treatment. Moreover, when selecting ninety minutes after the dose levels of L-citrulline in animals with hypoxia in the treatment were almost twice higher than in animals with chronic hypoxia without treatment (p=0.001). However, despite the time of selection of the sample, the levels of plasma arginine were higher in the animals with chronic hypoxia in the treatment of L-citrulline compared with animals with hypoxia without treatment.

EXAMPLE 4

WESTERN BLOTTING FOR PROTEIN ENDOTHELIAL NO SYNTHASE LUNG

As shown in Fig. 7A and 7B, the amount of protein endothelial NO synthase present in the lung tissue of animal control, and significantly higher than in the lungs of animals with hypoxia without treatment. Moreover, the amount of protein endothelial NO synthase present in the lung tissue of piglets with hypoxia in the treatment of L-citrulline, did not differ significantly from the amounts� in animals with hypoxia without treatment and was significantly lower than protein levels of endothelial NO synthase in animals of the control.

DISCUSSION of EXAMPLES

In examples 1-4 discovered that adding L-citrulline attenuates the development of pulmonary hypertension in newborn piglets subjected to 10 days of chronic hypoxia. Other results of this issledovaniya indicate that the production of exhaled NO, and the speed of the pulmonary vascular deposition of NOx^-higher in piglets with hypoxia in the treatment of L-citrulline than in piglets with hypoxia without treatment. Thus, these results show that the addition of L-citrulline significantly increases the production of pulmonary NO. The amount of protein endothelial NO synthase did not increase in animals with hypoxia in the treatment.

Although it is undesirable to tie themselves to any particular theory of operation, it is assumed that the mechanism by which L-citrulline helps increase the production of NO, provided increasing amounts of L-arginine available as a carrier for endothelial NO synthase. The levels of plasma arginine in animals in the treatment of L-citrulline in the examples slightly increased compared with animals with hypoxia without treatment. This discrepancy between intracellular arginine and NO production, called "steam�the ACS arginine", probably present due to the increase in NO production despite unchanged levels of arginine plasma observed in the addition of L-citrulline in the examples. L-citrulline is an intermediate in the production cycle of urea is metabolized to arginine by the metabolic cycle of the two enzymes, adrenocortical synthase (AS) and adrenocortical lyase (AL). Found that these two enzymes, AS and AL, combined with the endothelial NO sentati in the cells of the pulmonary endothelium. Boger, R. N., Curr Opin Clin Nutr and Met Care (2008) 11: 55-61. Considered together, these enzymes create a separate subcellular supply of arginine is used exclusively for the synthesis of NO. Arginine levels of tissue and plasma, does not accurately measure this subcellular stock.

L-citrulline may also be enhanced NO production and endothelial NO synthase through additional mechanisms.

In addition, although it is undesirable to tie themselves to any particular theory of operation, another potential effect of L-citrulline in the examples is to prevent uncoupled endothelial NO synthase by maintaining appropriate levels of carrying it arginine.

In addition, although it is undesirable to tie themselves to any particular theory of operation, L-citrulline may also act on the biological availability for �Chet compensation increased NO oxidation. When exposed to chronic hypoxia peroxide production may increase due to the enzymatic sources in addition to endothelial NO synthase, such as NADPH oxidase. Liu, et al., Am J Physiol Lung Cell Mol Physiol (2006) 290: L2-L10. This excessive production of hydrogen peroxide can be directly connected with NO, reducing its local production. In this case it is possible that the provision of L-citrulline provides for sufficient production of NO compensation for recovery, in which the peroxide is an intermediate.

In summary, the examples show that L-citrulline softens caused by chronic hypoxia pulmonary hypertension in newborn piglets. It is also clear that the effectiveness of citrulline due to increased production of NO. Thus, L-citrulline is a useful therapy in neonates at risk of developing pulmonary hypertension due to chronic or periodic permanent hypoxia.

LINKS

Listed below are the links, and all links mentioned in the description, which is incorporated herein by reference to the extent that they Supplement, explain, provide a background or describe the methodology, methods and / or compositions mentioned herein.

Jobe et al. (2001) Am J Respir Crit Care Med 163: 1723-1729.

Ballard et al. (2006) N Eng lJ Med 355: 343-353.

Ryan, R. M. (2006) J Perinatology 26: 207-209.

Publication of PCT international application No. WO 2005/082042, registered on September 9, 2005 U.S. Patent No. 6343382. U.S. patent No. 6743823.

It is clear that various details of the present invention may be modified without departure from the scope of the present invention. Moreover, the above-mentioned description is given for illustrative purposes only and does not imply any restrictions.

1. A method of treating bronchopulmonary dysplasia involving intravenous administration to a patient of an effective amount of citrulline.

2. A method according to claim 1, wherein the patient is a child.

3. A method according to claim 2, in which the child is a premature child.

4. A method according to claim 1, wherein the citrulline is administered in a dose from about 100 mg to about 30000 mg.

5. A method according to claim 4, wherein the citrulline is administered in a dose of from about 250 mg to about 1000 mg.

6. A method according to claim 1, wherein the patient is suffering from hipotiroidiei, characterized by the level of citrulline in blood plasma <37 mmol/L.

7. Pharmaceutical formulation acceptable for intravenous administration and is intended for the treatment bronchopulmonary dysplasia, containing a pharmaceutically acceptable carrier and an amount of citrulline effective to increase the level of citrulline in blood plasma, PR�than the level determined by comparing the level of citrulline in plasma of the patient, finding on treatment, with the level of the patient is not suffering bronchopulmonary dysplasia.

8. Pharmaceutical composition according to claim 7, in which the amount of citrulline is effective to increase the level of citrulline in plasma of the patient to at least 5 mmol/l, in some cases at least up to 10 mmol/l, in some cases at least 20 mcmol/l, in some cases at least 25 mcmol/l and in some cases at least to 37 mmol/L.

9. Use of pharmaceutical composition according to claim 7 to obtain a preparation for injection, is effective for the treatment bronchopulmonary dysplasia.

10. A method according to claim 1, wherein treating the patient with bronchopulmonary dysplasia includes the introduction of pharmaceutical compositions according to claim 7.

11. A method according to claim 10, wherein the pharmaceutical composition includes such a quantity of citrulline, which is effective to increase the level of citrulline in plasma of the patient to at least 5 mmol/l, in some cases at least up to 10 mmol/l, in some cases at least 20 mcmol/l, in some cases at least 25 mcmol/l and in some cases at least to 37 mmol/L.