| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | June 18, 1991 |
| PATENT TITLE |
Natural pulmonary surfactant, method of preparation and pharamceutical compositions |
| PATENT ABSTRACT | A pulmonary surfactant of animal origin made up of a high percentage of phospholipids (.perspectiveto.99%), by a protein fraction and characterized by the absence of carbohydrates and cholesterol. The surfactant of the invention, obtained through filtration, centrifugation and extraction and by chromatography in inverse phase, allows better therapeutic results in the treatment of infant and adult respiratory distress syndromes (IRDS and ARDS). |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | April 5, 1988 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT CLAIMS |
We claim: 1. An animal pulmonary surfactant which consists of polar lipids and proteins wherein the polar lipids are mainly phospholipids and the proteins are hydrophobic low molecular weight proteins of 3-14 KD (Kilodaltons), the polar lipid content is 98.5-99%, the protein content is less than 1.5%, and the phospholipid fraction contains at least 70-75% by weight of phosphatidylcholine, 40-45% of which consists of diplamitoylphosphatidylcholine, said surfactant is free of carbohydrates, cholesterol, triglycerides and cholesterol esters. 2. A process of preparation of a pulmonary surfactant which consists of an animal pulmonary surfactant consisting of polar lipids and proteins wherein the polar lipids are mainly phospholipids and the proteins are hydrophobic low molecular weight proteins of 3-14 KD (Kilodaltons), the polar lipid content is 98.5%-99%, the protein content is less than 1.5%, the phospholipid fraction contains at least 70-74% by weight of phosphatidylcholine, 40-45% of which consists of dipalmitoylphosphatidylcholine, and is free of carbohydrates, cholesterol, triglycerides and cholesterol esters, which comprises the steps of: (a) triturating animal lungs to obtain triturated lungs; (b) washing said triturated lungs in a salt solution and filtering off the filtrates to obtain a solid fraction; (c) centrifuging the solid fraction; (d) extracting with an organic solvent; (e) evaporating the solvent and (f) recovering the polar components by gel chromatography. 3. The process according to claim 2, wherein step (f) is carried out by chromatography in inverse phase on LIPIDEX -5000.RTM. column, with a 1,2-dichloroethane/methanol 1:4 (V/V). 4. A pharmaceutical composition for the cure of Infant Respiratory Distress Syndrome in premature infants and Adult Respiratory Distress Syndrome, in a suspension in vials for inhalation or endotracheal administration containing as active principle a pulmonary surfactant according to claim 1, the surfactant being suspended in physiological solution of concentration between 50 and 100 mg of phospholipids/ml. |
| PATENT DESCRIPTION |
Subject of the invention is a pulmonary surfactant (PS) preparation of animal origin, with a low toxicity and optimal surface characteristics, for use in the prevention and therapy of IRDS (Infant Respiratory Distress Syndrome) and ARDS (Adult Respiratory Distress Syndrome) related to HMD (Hyaline Membrane Disease). Normal pulmonary functionality depends on the presence of a particular material, the pulmonary surfactant, which is in charge of stabilizing the alveoli by reducing surface tension, in particular during the expiration phase. The presence of pulmonary surfactant is of particular importance at the moment of birth. Lack of PS is a key factor in the pathogenesis of IRDS, a disease which affects 10-15% of premature newborn babies. These subjects require artificial ventilation with high oxygen concentration and high insufflation pressure. IRDS death rate is around 25% and several of the survivors are left with chronic pulmonary complications mainly due to the prolonged artificial ventilation, and with secondary neurologic disfunctions due to cerebral hypoxia damage. Lack of pulmonary surfactant is an important factor also in ARDS. This pathology can develop in cases of multiple trauma, aspiration, pancreatitis, etc. with a 40-70% death rate. Administration of supporting doses of the lacking surfactant has proved useful in the treatment of these pathologies. The pulmonary surfactants known to the art belong to three fundamental groups: 1. ARTIFICIAL PULMONARY SURFACTANT Preparations of artificial surfactants with a base of dipalmitoyldyphosphocholine or of phospholipid mixtures in variable concentrations and ratios, optionally coupled with components such as sugars, aminoacids, alcohols or fat acids have been described in several patents: DE 2900300 (Klitzing LV); JP 58222022 (Teijin KK); EP 110498; U.S. Pat. No. 4312860 (University of California); DE 3229179 (Natterman A & Cie GmbH); JP 61065821 (Tokyo Tanabe KK). At clinical-pharmacological level, however, the artificial surfactant did not prove to be very effective. 2. HUMAN PULMONARY SURFACTANT Derived by extraction from amniotic liquid. Although effective, it has proved to be of little practical utility, both for its high protein contents (which can lead to sensitization of the treated subject) and for difficulties of preparation on a large scale, as to obtain a dose an amniotic-liquid-taking from three terminal pregnancies is necessary. It also presents a high risk of viral contamination with possible transmission of pathologies as serious as AIDS. 3. NATURAL PULMONARY SURFACTANT Extracted from mammal lung and with an effectivenes comparable to the human surfactant, it presents the great advantage of simplicity in preparation and a lower protein contents. Pulmonary surfactants of natural origin of animal extraction have been prepared before. For instance, DE 3021006, JP 58045299 and EP 119056 (Tokyo Tanabe KK), describe a rather complex method which, through a series of operations such as repeated centrifugations (850.times.g to 20,000.times.g), lyophilization and various extractions, leads to a natural PS containing, besides phospholipids (75-95.5%) and proteins (0.5-5%), also carbohydrates (0.1-2%), neutral lipids (0.3-14%) and total cholesterol (0-8%), components of no use to the pharmacological action. Also the surfactant prepared according to EP 145005 (Veb Arzneimittel Dresden) contains, in an even higher percentage (5-40%), an apolar lipid fraction, of no use to the biological activity and has instead low contents (40-70%) of phospholipids which represent on the contrary the most important physiological component. Finally EP 55041, JP 58183620, JP 58183621, JP 58164513 (Tejin KK) describe natural, artificial or semi-natural (i.e. added with synthetic phospholipids) surfactants totally de-proteinized. And the most recent studies have yet attributed to the presence of proteins a considerable functional meaning. After long and profound studies, started several years ago, applicant has prepared a new pulmonary surfactant, subject of the present invention, whose composition is optimum for a balanced pharmacological activity. A preparation process of this surfactant has also been worked out which, through the use of animal lung, allows to attain with few operations separation of the required fraction. A first aspect of the invention refers therefore to an animal pulmonary surfactant presenting the following characteristics: (a) the highest polar lipids concentration (99%), mainly phospholipids, as regards to other preparations; (b) total absence both of free carbohydrates, cholesterol, triglycerides and cholesterol esters, components of no use for surface activity (Suzuki Y., J. Lipid Res. 23, 62-69, 1982) and of other neutral lipids ineffective from the pharmacological point of view (Nohara K., Eur. J. Resp. Dis. 69, 321-335, 1986); (c) the presence of a protein component, characterized by a particularly high presence of hydrophobic amino acids, whose maximum concentration is lower than 1-1.5%. The proteic part is made up exclusively of hydropholic proteins of molecural weight ranging from 3 to 4 K (K=kilodaltons), which are important for absorption of the phospholipids at the air-liquid interface level. A second aspect of the invention refers to a preparation method which through a simple process, reproduceable and feasible on industrial scale, allows to obtain a surfactant of the indicated characteristic. Triturated animal lungs are washed in a physiological solution. They are filtered and centrifuged at speeds between 1,000 and 5,000.times.g for a time of one to three hours, according to the speed. Extraction of the surfactant is then carried out with an organic solvent, preferably made up of a 1:2 methyl alcohol/chloroform mixture. The organic phase is water-washed and evaporated, thus obtaining a raw lipid fraction which is recovered with organic solvent, preferably formed by a 1,2-dichloroethane/dichloromethane mixture in a 1:4 ratio. Subsequently, by gel chromatography, the polar lipid component, made of phospholipids, is separated from the apolar one, made up of triglycerides, cholesterol and cholesterol esters. The phospholipid fraction, the one for clinical use, is sterilized by ultrafiltration and stored at a temperature of at least -20.degree. C. In the alternative, it can be lyophilized and stored at -20.degree. C. |
| PATENT EXAMPLES | Available on request |
| PATENT PHOTOCOPY | Available on request |
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