the anf bearing acidic proteinoid microspheres of this invention are suitably made by any of the procedures described in ser . no . 98 , 027 issued as u . s . pat . no . 4 , 925 , 673 ; e . g ., by mixing anf with water , acidifying the mixture to a ph at which the acidic proteinoid is insoluble , contacting the ph adjusted mixture with that powdered proteinoid and recovering the spontaneously formed microspheres . as in ser . no . 98 , 027 issued as u . s . pat . no . 4 , 925 , 673 , the preferred acidic proteinoid materials for use in this invention are thermal condensation polymers derived from at least one acidic amino acid and at least one other acidic , basic or neutral amino acid . although microsphere forming polymers can be derived from as few as two such amino acids , a greater diversity generally results in higher yields of uniform size microspheres . proteinoids derived exclusively from naturally occurring amino acids ( i . e ., those found in animal or vegetable protein , including optical isomers thereof ) are especially preferred as it has been shown that when such proteinoids dissolve in human serum or in the bloodstreams of rats and guinea pigs , they consistently break into low molecular weight fragments which produce no toxic or immunological response . a flask equipped with an electric heating mantle and containing equimolar quantities of anhydrous aspartic acid , glutamic acid , valine and glycine is heated at approximately 175 ° c . under a stream of nitrogen until the contents are molten . the nitrogen swept mixture then is agitated gently with a glass encased magnetic stirrer and the temperature is raised to and held at 180 ° c . for six hours . after cooling , the dark amber product is extracted with saturated aqueous sodium bicarbonate and the extract dialyzed through a cellulose membrane against distilled water at room temperature for 24 hours , the water being changed every four hours . the entire content of the dialysis tubes then is dried under vacuum at 65 ° and the residual solids are ground to a fine powder with mortar and pestle . an aqueous solution of proteinoid is produced by mixing 35 mg of this powder per ml of water , adjusting the ph to 7 . 4 with concentrated aqueous sodium bicarbonate and removing any insoluble materials by filtration . sufficient distilled water is added to cloned bovine anf provided by california biotechnics corporation to produce a mixture containing 0 . 4 mg of anf per ml and the mixture is adjusted to ph 3 . 5 with concentrated acetic acid . a portion of this mixture is diluted with an equal volume of ph 3 . 5 aqueous acetic acid to reduce the anf concentration to 0 . 2 mg / ml and 1 ml samples are set aside for use in experiments 3 and 4 . one part by volume of the solids free proteinoid solution of example 1 is rapidly injected into an equal volume of the 0 . 4 mg / ml aqueous mixture of unprotected anf of example 2a . the resulting mixture , which has a ph of approximately 4 . 5 , is chilled in an ice bath for 15 minutes and filtered to recover the resulting anf filled microspheres . these are washed with ph 3 . 5 aqueous acetic acid and 0 . 4 mg of the microspheres are made up to one volume by the addition of ph 3 . 5 aqueous acetic acid . microscopic examination of the resulting suspension reveals that the microspheres are predominately from about 0 . 1 to about 5 . 0 microns in diameter . one ml samples of this suspension of microencapsulated anf , which contain slightly less than 0 . 2 mg / ml of anf , are set aside for use in examples 3 and 4 . four normal young male rats are selected arbitrarily for this experiment . each of rats a and b is administered by gavage one ml of the 0 . 2 mg / ml unprotected anf of example 2a and each of rats c and d is similarly given a one ml dosage of the suspension of microencapsulated anf of example 2b . blood samples are withdrawn from the tails of the animals 90 minutes after administration and the anf levels in the serum are determined by radioimmunoassay . this assay distinguishes between natural rat anf and the cloned bovine anf that was administered . the bovine anf levels in rats a and b are 28 and 62 fmol / ml ( fempto moles per milliliter ), while those of rats c and d are 360 and 365 fmol / ml . these results demonstrate that the quantity of orally administered anf that is delivered to the bloodstream is greatly increased by microencapsulation in acidic proteinoid microspheres which are stable to stomach acidity and dissolve at the near neutral ph of the blood and which have diameters of less than about 10 microns so as to readily penetrate the gastrointestinal mucosa . six hypertensive male rats weighing about 500 g each are arbitrarily divided into three groups of two . each of the animals of group 1 is administered by gavage 1 ml of the suspension of microencapsulated anf of example 2b . those of group 2 each similarly receive 1 ml of a similar suspension of proteinoid microspheres which contain no anf and which are made by omitting anf from the procedure of example 2b . those of group 3 each similarly receive 1 ml of ph 3 . 5 aqueous acetic acid . blood pressure measurements are made on the animals immediately before and at intervals after administration of their doses . table 1 shows the average systolic blood pressures for the groups . table 1______________________________________systolic blood pressure ( mm hg ) time after dosage ( minutes ) 0 15 90______________________________________group 1 - micro - 207 175 178encapsulated anfgroup 2 - micro - 198 195 197capsules onlygroup 3 - ph 3 . 5 185 189 188aqueous acetic acid______________________________________ these results demonstrate the unexpected prolongation of the blood pressure suppressing effect when anf is delivered to the bloodstream by oral administration of same encapsulated within less than 10 micron diameter acidic proteinoid microspheres . it will be apparent to those of ordinary skill in the art that numerous changes and modifications can be made in the illustrative embodiments of the invention described above without departing from the spirit and scope of the invention as set forth in the following claims .
atrial naturiuretic factor is encapsulated within hollow acidic proteinoid microspheres which are stable and protective in the acidic stomach of a mammal but which dissolve at the near neutral ph of the blood . microspheres having diameters of less than about 10 microns readily penetrate the gastrointestinal mucosa and release the anf in the bloodstream in physiologically active form . such gastric administration significantly extends the period of effectiveness of anf as a diuretic and blood pressure suppressant .