Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1996-11-01
2001-01-09
Borin, Michael (Department: 1631)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C514S021800
Reexamination Certificate
active
06172203
ABSTRACT:
BACKGROUND OF THE INVENTION
The exchange of oxygen and carbon dioxide between the inspired air and the blood to be oxygenated is done through the pulmonary alveoli which are grouped in racemes surrounded by a network of capillaries where the blood to be ventilated is circulating.
The exterior faces of such walls are covered with a very thin film, monomolecular, of a viscous tensioactive substance which is synthesized by the type II alveoli, which controls the alveoli expansion, reducing the tendency to collapse (that is to say, the occlusion) of the alveoli during the expiration phase.
On the other hand such a film, due to is tensioactive properties, facilitates the displacement of macrophages (alveolaris) that capture foreign elements trapped in this film (inspired particles, bacteria, viruses), that travel along the alveoli surface and move to the bronchial mucociliar zone.
This substance having tensioactives properties that covers the alveoli surface, named pulmonary surfactant (PS) is of complex composition, not completely elucidated; in addition it varies with age, health and also with the extraction method. In general, the PS extract from healthy persons and other mammals contains 90% lipids, 80% of which are phospholipids and the remainder neutral lipids, mainly cholesterol. Phospholypic fraction in mammals in general.
In addition to the lipid fraction mentioned, the PS have a protein fraction from which was extracted and characterized several proteins that have tensioactive properties and complement the lipid tensioactive properties, and these include PS-A, PS-B, PS-C, PS-D, and the like.
When there are not sufficient physiological quantities of PS and/or when the PS film is altered, it presents respiratory problems that produce clinical charts with severe characteristics such as generically denoted respiratory distress syndrome (RDS) and adult respiratory distress syndrome (ARDS).
The patients that exhibit the above mentioned syndrome are treated by providing exogenous PS, a product that is obtained by washing the pulmonary tract of healthy animals (especially pork and in less abundance cows or sheep) with saline solutions and further fractionation, purification and lyophilization. Given that the exogenous PS is an expensive product, the treatment with natural exogenous PS, in general, is limited to the treatment of premature infants.
The high price of the natural exogenous PS has motivated the search for synthetic exogenous surfactants that are less expensive. There are several products with the tensioactive characteristics of natural PS, such as mixtures of dypalmitoilphodphatidylcoline (DPPC), hexadecanol and phyloxapol (Exosurf), phosphatidylglycocerol (ALEC), etc.
Exogenous PS is a product that results from the extraction, by aqueous saline solution from a pig's respiratory system or from a cow or sheep. Also from lung divided in sheets with later fractionation and purification, and in accordance with a sequence of operations of filtration, dialysis, ion exchange chromatography and exclusion chromatography in order to arrive at a lyophilized product or product in dilute solutions with a cost in the order of 4,000 to 5,000 dollars per gram.
SUMMARY OF THE INVENTION
We have developed a natural pulmonary surfactant by a new much simpler method that produces a product analogous to the one obtained by the conventional method of extraction and purification, which is much less costly, of the order of ⅛ to {fraction (1/10)} of natural PS extracted by prior art methods.
The object of this invention is the production of a product that contains liposoluble lipids and proteins, in a chloroform solution, useful for preparing exogenous PS. These liposoluble lipids and proteins obtained from washing the bronchial-alveoli of pork, cows or sheep with slightly hypertonic saline solutions by a method that includes:
I. Fractionating by means of differential centrifugation the liquid obtained by the washing operation. This centrifugation is done in at least two scaled operations, the first made at a low velocity, in the range of 500-10,000 g from which is obtained the liquid phase and the pellets are discarded. In the next stage the centrifugation is done at high velocity in the range of 8,000-10,000 g, salvaging the formatted pellet.
II. Fractionating by centrifugation at 8,000-10,000 g the suspension formed when the pellet of the previous stage is suspended in a saline solution buffered at pH: 7.2-7.4, saving the residue and repeating this operation until the pellets are white in color and have a creamy consistency.
III. Suspending the pellet in a water soluble polar solvent such as acetone, saving the residue after centrifugation at 8,000-10,000 g. This pellet is made up of crude exogenous pulmonary surfactant.
IV. Dissolving the crude exogenous pulmonary surfactant pellet from the previous stage in a medium insoluble in water such as chloroform or dichloromethane:methanol in a 2:1 ratio and extracting the solution formed with slightly hypertonic saline solution, recuperating the yellow liquid phase by filtration, from which the solvent is eliminated and the solid pellet is dissolved in chloroform to form the product of the present invention, finally it is filtered by a porous membrane of 0.80, 0.45 and 0.22 &mgr;m.
Another objective of this invention is a method for obtaining a product that contains the liposoluble lipids and proteins extracted from the liquid obtained from washing the bronchio-alveoli of pork, cows or sheep with slightly hypertonic saline solutions obtained with the procedure indicated: lyophilized and dosaged in units or kits for instant reconstitution that involves:
I. Fractionation by means of differential centrifugation of the liquid obtained by the washing operation. This centrifugation is done in at least two scale operations, the first made at low velocity, in the range of 500-1,000 g, from which is obtained the liquid phase and the pellets are discarded: the next stage the centrifugation is done at high velocity in the range of 8,000-10,000 g, salvaging the formatted pellets.
II. Fractionation by centrifugation at 8,000-10,000 g produces the suspension formed when the pellet of the previous stage is suspended in a saline solution buffered at pH: 7.2-7.4, the pellet is recovered and repeating this operation until the pellets are white in color and have a creamy consistency.
III. Suspending the pellet in a water soluble polar solvent such as acetone, saving the pellet after centrifugation at 8,000-10,000 g. This pellet is the crude exogenous surfactant pulmonary.
IV. Dissolving the crude exogenous pulmonary surfactant pellet from the previous stage in a medium insoluble in water such as chloroform or dichloromethane:methanol in a 2:1 ratio and extracting the solution formed with slightly hypertonic saline solution, recuperating the yellow liquid phase by filtration, from which is eliminated the solvent and solid residue, dissolving in chloroform to form the product of the present invention, finally it is filtered by porous membrane of 0.80, 0.45 and 0.22 &mgr;m dimension.
Another object of this invention it is to obtain a product administrable by inhalation, useful for the treatment of cases with deficient pulmonary ventilation characteristic of the RDS and ARDS syndromes that involves the product obtained from the lyoliphiliation of the exogenous PS in a pharmaceutical acceptable vehicle. Such as physiological solutions having effective therapeutic concentrations and the possible combination with other therapeutic agents such as antibiotics, bronchidilators, antimicoticos, etc.
Another object of the present invention is a product useful in obtaining images from pulmonary centellography that include: Resuspension in an acceptable vehicle such as a physiological solution, the material obtained from the lyophilization of the product mentioned above in combination with radionuclei.
In addition another object of this invention is a product useful in obtaining images from pulmonary centellography in which the radioisotope is 99 mTc in combination w
DePaoli Tomas
Hager Alfredo Adolfo
Borin Michael
Fulbright & Jaworski L.L.P.
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