FI102456B - A method for microencapsulating a pharmacologically active substance in microspheres - Google Patents

Description

102456

Method for adsorption of a microcapsule of a pharmacologically active substance into microspheres Adjusting a microcapsule and a pharmacologically active medium in a microsphere 5

BACKGROUND OF THE INVENTION 1. Field of the Invention This invention relates to pharmacologically active agents encapsulated in protective proteinaceous microspheres and their administration to warm-blooded animals. It relates in particular to orally administrable 15 microspheres containing pharmacological agents whose activity is otherwise lost in the gastro intestinal tract.

2. Description of the Related Art 20 There are often very few available modes of release of pharmaceutical and therapeutic substances for delivery to the body due to chemical or physical barriers or both. Generally, for example, '• a; oral release of many such substances, if not. * substances would face so many obstacles as they travel this path. Inappropriate pH of gastric »· ♦ '* 25 range conditions, presence of strong digestive •' * enzymes, gastrointestinal and tissue permeability • · * ··· * properties and other factors are important factors ··· • · · *. * * When evaluating the possibility of oral dosing of active substances in subjects. Among the numerous pharmacological agents known to be impaired or ineffective when administered orally are biologically active polypeptides and proteins such as insulin. These substances are rapidly ♦ destroyed in the stomach by acid hydrolysis. The stomach, and • · · • · stomach intestine in the lower area they are destroyed by those enzymes have, • * 35 which have the ability to split peptiidisidoksia and in addition they pass slow; purely, if at all, gastric intestinal wall.

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studies of gastric intestinal area is very much focused on the isolation of modifying or damaging conditions so that a pharmacological agent which would otherwise be labile, could be absorbed through the stomach or intestinal wall unchanged, and the pharmacologically active 5-form. Research in this area has focused primarily on three directions: the co-administration of excipients such as resorcinols and nonionic surfactants such as polyoxyethylene polyethylene ether and n-hexadecyl polyethylene ether; for co-administration of enzymatic inhibitors such as pancreatic trypsin inhibitor, diisopropyl-10-fluorophosphate (DFP) and trasylol; and the use of liposomes, such as water-in-oil-in-water emulsions, which provide a protective lipid layer around the incorporated pharmacological agent and which is the most successful form today. For example, the use of heparin-containing liposomes is disclosed in U.S. Patent No. 4,239,754 and several studies have focused on the use of insulin-containing liposomes; e.g., Pat el et al., FEBS Letters, 62, 60 (1976) and Hashimoto et al., Endocrinol. Japan, 26, 337 (1979). Despite the limited utility of these, the use of liposomes is still in development and is associated with ongoing problems, e.g. poor Stability and insufficient fastener stability.

: In view of the above, there is a need for better ways of targeting the release of active pharmacological substances in the body, and in particular •. acceptable levels of ways administered pharmacological agents for oral T ··· through 25 which are unstable in the gastro intestinal area conditions.

• · * ·· • · · «I I • · · *. SUMMARY OF THE INVENTION It is an object of the present invention to provide improved methods for delivering pharmacologically active substances in a physiologically active form to a target organ or fluid in the body.

• · · • · · • · / ··. A further object of the present invention is to provide an improved release system, · 'for such administration by the intestinal pharmacological agents 35, which otherwise would pass the gastro intestinal wall slowly or not 3 102 456 at all, and / or are caused by susceptible acids and enzyme chemical splitting and gastric intestinal tract.

A particular object is to provide such a delivery system, within which the five active pharmacological agent encapsulated in a protective substance, which is itself pharmacologically harmless, which does not alter the physiological active substance and the biological characteristics that will protect the gastro intestinal active agent in the area from harmful conditions and which to discard or vavapauttaa blood of active agent stream or other 10 destinations. A particular further object of the present invention is to provide a combination of active agent and the protective agent that is sufficiently lipophilic, and having a small particle size, so that it can pass through the gastro intestinal mucosa quickly and is easy to prepare a composition.

It is a further object of this invention to provide methods for producing such delivery systems and administering them to animals. In particular, it is an object to provide effective ways of administering insulin orally to mammals with diabetes.

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It has been found that these objects and other advantages which will become apparent from this description are achieved by the invention described below.

·. The method according to the invention for microencapsulating a pharmacologically active substance into microspheres for release in a certain range in a selected pH range comprises forming a mixture of said substance. with a pharmaceutically acceptable liquid, wherein the pH of the mixture is outside said range, and contacting said mixture with protenoids formed of mixed amino acids by thermal condensation, which protenoids are soluble in the selected pH range and insoluble in said mixture to form microspheres having substantially less than about 10 microns in diameter containing the active agent, said microspheres encapsulating said active agent.

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Broadly speaking, one aspect of the present invention is a delivery system for an active pharmacological agent comprising said agent embedded or encapsulated in proteininoid microspheres.

Another broad aspect of the present invention is a method of encapsulating an active pharmacological agent, comprising admixing said active agent with a pharmaceutically acceptable liquid and contacting said mixture with a proteinoid that interacts with said mixture to form hollow microspheres.

A third broad aspect of the present invention is a method of targeting the release of a pharmacologically active agent in an animal, comprising administering to said animal an effective amount of said active agent encapsulated in proteininoid microspheres, said microspheres being stable under conditions encountered during administration. in said animal to the target release zone and unstable in said zone.

20 Description of the Preferred Embodiments

The proteinoids that make up the protective capsules of this invention have been described as artificial polypeptides because they are man-made condensate polymers produced by random techniques or directly by combining natural or synthetic amino acids and / or small peptide chains. Taking advantage of the observation that • · · *. it was made in the late 1950s that the linear condensate polyolers of mixed natural amino acids • · * *** · are able to interact with water and form microspheres, proteininoids have been the subject of large-scale studies of the origin of life.

··· • · · • · · «

An excellent summary of these studies and extensive biographies can be found in Fox, S.W. and Dose, K., Molecular ‘1’ Evolution and the Origin of Life. Marcel Dekker, Inc., New York (1977), • *. · 35 The contents of which are incorporated herein by reference.

As a result of these and other studies, there is a wealth of information on the fabrication and properties of proteinoids and proteinoid microspheres. For example, it is known that proteinoids derived from natural alpha amino acids (found in animal or plant protein), as well as those containing other naturally occurring substances (such as polynucleotides, phosphoric acid, iron and calcium), are non-toxic. It has also been found that the incorporation of a stoichiometric excess of an acidic di- or polycarboxylic amino acid into a polymer results in an acidic proteinoid that is insoluble under acidic conditions and soluble in a basic environment, whereas the incorporation of an excess of a basic diamino- or polyaminomonomer results in soluble in acidic medium and insoluble at high pH. These solubility properties can be affected very accurately. Similarly, the size of microspheres formed by contact between proteinoids and water or other fluid can be controlled in the range of less than about 0.5 microns to about 10 microns or more by manipulating various physical or chemical parameters such as fluid pH, osmolarity, or salt content.

It has also been found that proteininoids are much more resistant to cracking by digestive enzymes than proteins.

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The present invention was based on the finding that pharmacologically; the active substance can be encapsulated in the proteininoid microspheres in a simple manner. By dissolving or suspending such a substance in a pharmaceutically acceptable liquid such as water or dimethyl sulfoxide. which interacts with said proteinolide to form microspheres ♦ ♦ · * *.

* · ♦ · * *. ·. · It has also been found that such encapsulation does not alter the pharmacological properties of the active substance and that the diameter of the active substance carrying the microspheres is less than about 10 microns and is thus • * . ···. small enough to pass through the gastro intestinal mucosa, easily and mennäk- • · * 1 * to the blood stream. The preferred value for rapid diffusion is about 0.5 to n.

M. ’35 5.0 microns because smaller sizes result in somewhat less« · ·. * ·: Stability and contain relatively little active substance 6 102456 and larger sizes diffuse more difficult. However, particles of about 5.0 to about 10 microns are useful in admixture with those having a preferred size range because their slower diffusion results in a sustained release of the active agent.

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By adapting both the solubility properties and the particle sizes of the microspheres to the acidic proteinoid in a known manner, it has been found that it is possible to produce an active substance which carries microspheres stable in the mouth (normal pH about 4 to about 6.8). oral mucosa and enter the bloodstream and which release the active substance into the blood (normal pH about 7.35 - about 7.45). Such systems are suitable for sublingual administration of pharmacological agents such as human or bovine growth hormone, interferon or interleukin-II.

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It is also possible to produce easily diffusible microspheres from acidic proteinolides that are stable in a very acidic stomach (normal pH about 2 to about 6) but soluble in almost neutral blood. Such systems are suitable for the oral administration of pepin-20 hormones, such as insulin or heparin, which would otherwise be rapidly destroyed in the stomach. They are also suitable for protecting the stomach from gastric irritants such as aspirin.

When such aspirin, which contains microspheres, is administered orally. through, the microspheres penetrate the stomach wall and release aspirin «· · ••• # 25 into the bloodstream much faster than the coated aspirin that has entered the bloodstream in the usual way • ·” 1, which must first be passed through the stomach and • · · • · · then come the blood stream through the intestinal wall only after the • * · · · · bowel coating of the inputs is dissolved.

• ♦ ·. ·. · 30 It is also possible to produce systems from basic proteinoids which are stable in a weakly basic lower digestive tract (normal pH approx. 8) but which release the active substance • ·] ..> t the blood. Such systems are suitable for the administration of pharmacological agents such as dopamine or gamma-aminobuturic acid, such as calcium regulators and reduction-oxidation-carrier systems.

7 102456 In addition to these visceral delivery systems, it is also possible to produce a near-neutral proteinoid microsphere system that is stable in the bloodstream but releases its pharmacological content depending on the subject's environment, such as higher or lower pH. value or the presence of specific enzymes. Such systems are almost neutral give a blood vessel, if the microspheres were not small enough to be included in the major proteinoiidimikrosfääreihin, which have diffused through the gastric and intestinal mucosa are stable until they reach the blood stream 10.

Although any pharmacological agent can be incorporated into the proteinoid microspheres, the protection of such agents appears to be of particular value in cases where the agents would otherwise be destroyed or become less effective under animal body conditions before reaching the target zone.

Example 1 below describes the preparation of an acidic proteininoid capable of interacting with an aqueous solution of a pharmacologically active substance by incorporating and protecting this substance in hollow microspheres. These microspheres are stable in the presence of digestive enzyme and gastric acid in the presence of less than 5.0 microns in diameter, readily penetrate the gastric wall and go slightly to the beginning. into the bloodstream where they dissolve and release the pharmacological "... 25 substance.

• · • · • · · · · · Example · la • · • · · · · ·

Mixed solution containing 52.3 g of aspartic acid (0.4 moles), • · ·. ·. * 30 42 g of arginine hydrochloride (0.2 moles), 26 g of isoleucine (0.2 moles) ·· · · and 50 ml of glycerol is heated under nitrogen to 160 ° C, whereupon gas evolution occurs. The temperature is then maintained at 155 ° C for 23 • · * · ... hours, after which the mixture is cooled to room temperature, extracted with 200 ml of 10% strength by weight aqueous sodium bicarbonate: and the extract is dialyzed. through the collodion membrane against distilled water for 26: / ·· hours, changing the water every 6 hours. The contents of the dialysis tubes are then evaporated to dryness at 50 ° C under vacuum to give a glassy solid acidic proteinoid which is ground to a fine powder.

Example Ib 35 mg of this powdered proteininoid is added to a mixture of 50 mg of porcine insulin crystals in 2 ml of distilled water and the mixture is allowed to stand at room temperature until microspheres are formed.

The insulin-bearing microspheres are separated by filtration, washed at pH 5.4 with aqueous acetic acid and then resuspended in 2 ml of aqueous acetic acid at pH 5.4. Microscopic examination of this suspension shows that it has stable microspheres with a diameter mainly between 0.1 and 5.0 microns. When part of the suspension is neutralized to pH 7.4 with concentrated ammonium hydroxy, the dissolution of the microspheres can be detected immediately.

Example le 20 Three adult white rats with normal blood glucose values are administered a 0.35 L dose of the insulin-bearing microsphere suspension of Example 2b by inserting a syringe through the mouth into the stomach.

• i At this dose, each animal shows a significant decrease in blood glucose; ·, as measured by blood samples taken from the tail.

25 • · • · '** Although hollow microspheres suitable for the incorporation of pharmacological agents may be formed, protenoids derived from a single acidic or basic amino acid and as little as one other animoic acid, better yields are generally obtained and • · ·. ·. · 30 more cohesive microspheres throughout using a larger distribution of amino acid components to the desired diameter distribution within 0.5 microns. Example 2 illustrates the efficacy of an oral dose of insulin incorporated into a proteininoid derived from 18 different amino acids that causes hypoglycemia in mammals.

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Example 2a A 250 ml filter flask containing 10 g of anhydrous diglutamic acid and 10 g of anhydrous di-aspartic acid under nitrogen is heated in an oil bath to about 200 ° C until the contents melt. To this is added 5 g of an anhydrous mixture of 16 neutral and basic amino acids found in the animal protein; which are alanine, arginine, asparagine, cysteine, glycine, histadine, leucine, lysine, methionine, phenylanaline, proline, serine, threonine, tyrosine-10, tryptophan and valine. The resulting mixture is stirred with a glass rod and kept at 200 ° C under nitrogen for 3 hours. After cooling, the golden brown product is extracted with saturated aqueous sodium dicarbonate solution and the resulting solution is dialyzed through a collodion membrane against distilled water at room temperature for 24 hours, changing the water every 6 hours. The contents of the dialysis tubes are then acidified to pH 5.4 with concentrated acetic acid and centrifuged. After separation of the supernatant, the insoluble solids are washed with aqueous acetic acid at pH 5.4 and centrifuged again. This washings are also separated and the solid proteinoid-20 product is dried over silica gel overnight and then ground to a fine powder with a mortar and pestle.

* · * ». Example 2b, «·« * · '.. • k 25 A mixture containing 50 mg of porcine insulin crystals in 2 ml of distilled • ·

Hl of water, is added to 35 mg of the dry powdered proteinaceous compound of Example 2 and the mixture is allowed to stand at room temperature until • · · * · * · * microspheres are formed. The mixture is then centrifuged for 15 minutes.

After separation of the supernatant, the remaining microspheres are washed once with pH 5,4 aqueous aqueous acetic acid at room temperature and centrifuged for a further 15 minutes. The supernatant is separated again and the insulin-bearing proteininoid microspheres are suspended. is again added to 2 ml of aqueous acetic acid having a pH of • <5.4. Microscopic examination of the suspension shows that the diameter of the microspheres: 35 is mainly between 0.5 and 5.0 microns.

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Example 2c

Twelve white male rats, each weighing about 500 g and each having a normal blood glucose value, are arbitrarily divided into four groups of three to demonstrate the physiological efficacy of oral administration of an aqueous suspension of insulin carried by the proteinoid microspheres produced by the method of Example 2b above. 0.35 to 0.5 ml of this aqueous suspension of microspheres is administered by gavage to the stomach of each group 1 rat. Group 10 rats 10 were similarly given 1.5 to 1.7 ml of suspension. Group 3 rats received 1.0 ml of similarly dosed distilled water. Group 4 rats similarly received 25.0 mg of porcine insulin in 1.0 ml of distilled water. Both before and during the experiment, all animals were free to drink water and eat food normally. Blood glucose values are measured from samples taken from the tail at certain intervals after treatment and the group mean results are shown in Table 1 and expressed in milligrams of glucose / dl of blood (mg / dl).

TABLE 1 20

Avg. blood glucose (mg / dl)

Group Pre-dose 30 min lh 1.5 h 2 h 2.5 h 3 h a · · • · · 25 One 135 85 88 66 44 27 • ·

Two - - - - - 49 «· · ***’ Three 120 - ... 119 • ·

Four 113 120 120 124 120 - 116::: 30 • ·: T: Group 4 h 6 h 12 h 24 h 36 h 48 h • «a V One 38 - 135 119 122

Two 58 - 80 125 125 122: 35 Three 122 - - 119 124: / j Four 111 123 11 102456

It is clear from the data in Table 1 that insulin is released in a physiologically appropriate and active manner via the oral route by means of acidic proteininoid microspheres. In all animals receiving the insulin-containing microspheres, blood glucose-5 values returned to pre-dose levels without any observed adverse effect. It should be noted that the dosing of microspheres to animals in group two that contain higher amounts of insulin appears to increase the duration of the effect rather than the potency of the effect. It should also be noted that much higher doses per unit of body weight of oral unprotected porcine or bovine insulin do not cause a noticeable decrease in blood glucose levels in laboratory animals and humans.

Similarly active insulin-containing proteinoid microspheres can be produced by contacting a dry powdered acidic proteinoid, such as Example 1a or 2a, with insulin suspended or dissolved in various pharmaceutically acceptable liquids, including ethanol, isopropanol, terpenol, dimethyl sulfoxide, starch oxide, Aqueous solutions of Tween 80 and cyclodex-20 tran.

Example 3 illustrates a particularly preferred method for producing insulin-containing proteinoid microspheres that reliably produces high yields of microspheres that fit in the desired 0.5 to 5.0 micron ... 25 diameter range and are readily soluble in the target. *** at blood pH.

Example 3a • · ·. ·, · 30 A flask containing 2 parts by weight of anhydrous 1-glutamic acid in a stream of nitrogen • · · Ι ^ ί I is heated in an oil bath to a temperature of n. 175 ° C until the contents melt. To this is added 2 parts by weight of anhydrous 1-aspartic acid • · * 1 and 1 part by weight of an anhydrous mixture of 16 neutral and basic amino acids found in the animal protein. The resulting mixture: 35 is mixed with a glass rod and kept at 175 ° C under nitrogen for three: '.' for an hour.

After cooling, the dark yellow amber product is extracted with saturated aqueous sodium bicarbonate and the extract is dialyzed against the distillate through a collodion membrane at room temperature for 24 hours, changing the water every 4 hours. The entire contents of the dialysis tubes are then dried in vacuo at 25 ° C and the resulting solids are ground to a fine powder with a mortar and spray.

Example 3b 10

An aqueous solution of the proteininoid is made by mixing 35 mg of the powder of Example 3a per 1 ml of water, adjusting the pH to 7.4 with concentrated aqueous sodium bicarbonate solution and removing all insolubles by filtration. One volume of this solids free protein solution is then rapidly injected into a similar volume of a freshly prepared 25 mg / ml solution of pork insulin in aqueous acetic acid, pH 2.25. The mixture, which has a pH of about 3.5, is stirred in an ice bath for 15 minutes and filtered to separate the insulin-bearing microspheres from the filtrate, which is discarded. After two washes, the microspheres are resuspended in aqueous acetic acid at a pH of 3.5 for 10 volumes. : to an aqueous solution of acetic acid at pH 3,5. Microscopic examination of a portion of this suspension shows that a large proportion of microspheres having a diameter of mainly 0.5 to 5.0 microbes will dissolve rapidly when the suspension is neutralized to pH • · • · · 1 · 7.4 by adding concentrated aqueous sodium bicarbonate.

In the following Example 4, the doses of the suspension of insulin-containing microspheres according to Example 3b are referred to as "insulin-filled microspheres". Microspheres that do not contain:: encapsulated insulin are made by repeating the procedure of Example 3b, except that insulin is omitted during microsphere formation and the microspheres are formed in a porcine insulin solution containing 2.5 mg insulin / distilled water; dilute acetic acid.

: 35 Doses of the resulting suspension that do not contain insulin in the microspheres are referred to as "microspheres with extrinsic insulin." Doses containing 2.5 mg / ml of porcine insulin solution are referred to as "crude insulin" alone.

Example 4 5

Twelve white male rats, each weighing approximately 500 g and having a normal blood glucose value, are arbitrarily divided into two groups of three animals and a third group of six animals. Three animals in group A are given insulin-filled microspheres by tubular feeding, and three animals in group B are similarly given microspheres containing external insulin. Six animals in group C receive crude insulin. All doses are 1 ml / 500 g body weight and all animals are tested for blood glucose immediately before dosing and at regular intervals thereafter. The mean blood glucose of each group of animals is reported in Table 2.

TABLE 2

Rat blood glucose (mg / dl) 20 Time (hours) Group (Treatment)

ABC

• | Insulin-Filled Microspheres with Raw .. ·. microspheres is external insulin insulin j · 25 0 109.7 92 92.7, 5 54.7 89.3 95.5 • · · 1 59 84.3 98.2 • · 2 50 80.7 95.8 3 57 , 7 86.7 86.2 30 4 64.7 84 91: T: 6 76 83.7 89.8 8 65.7 88.7 91 «« · * ': 12 81.7 92.3 92 • · 24 94.3 95.7 92: 35. <• 4 ...................................... 1 '....... ...................... 1.111 I .1 1 ^^ - 14 102456 These tests do not show any significant effect on blood glucose values in the case of crude insulin or microspheres. with external insulin. In contrast, insulin-filled microspheres cause a decrease in peak value of about 50% and long-term potency. This indicates that acidic proteininoid microspheres have no effect on blood glucose values and only protect the incorporated insulin from the harmful abdominal environment, thus allowing the incorporated insulin to enter the bloodstream in a physiologically active form.

10

Example 5a

Diabetes is implanted in rats weighing approximately 300 g by administering to each vein 75 mg / kg body weight of streptozotoxin. Ten rats with persistently high blood glucose levels, excessive urine output, and water cravings and to be given piglet insulin by subcutaneous injection are selected for this experiment.

Example 5b; Three diabetic rats are administered by gavage approximately 1 ml of an aqueous suspension of porcine insulin in the acidic proteinaceous microspheres of Example 3b. The fourth diabetic rat receives ... 25 3 ml of suspension in 50 ml of tap water mixed in its water bottle • o *** and this rat dispenses its own dosage. All rats are kept • · · • without food for 12 hours before dosing. In all subjects, oral administration of micronised insulin results in a significant and prolonged reduction in blood glucose.

: V: 30 • · • · · i Ϊ t Example 5c «· · • · · •»

The three remaining six diabetic rats are arbitrarily divided into three groups of two animals. Animals of the first group: 35 are administered by gavage 1 ml of an aqueous suspension of porcine insulin in the acidic proteininoid microspheres of Example 3b. Animals in the second and 102456 third groups receive subcutaneous injection of 0.25 mg (6.5 I.U.) and 0.125 mg (3.25 I.U.) of porcine insulin. Blood glucose is measured from all animals just before dosing and at intervals thereafter. Groups of animals are divided twice a week so that 5 all animals are treated with both insulin treatments.

The mean percentage decrease in glucose from the initial glucose value for each treatment is shown in Table 3.

Table 3 10 Percentage reduction from baseline blood glucose in rats

Insulin treatment Time (hours) after dosing 15 _0.5 1 1.5 2 3 4 6 8 12 24 48

Oral microspheres 29 38 45 44 48 50 51 40 37 24 -27 0.25 mg SC 28 55 72 77 84 79 34 4 3 -7 -5 0.125 mg SC 5 17 27 45 47 29 8 -8 -8 -8 - 8 20 • · ·, ·. These results indicate that insulin-filled microspheres. . the peak effect of oral administration in diabetic rats is comparable to a subcutaneous injection of 0.125 mg insulin and that the effect is significantly longer than that obtained with either 0.125 mg insulin. or 0.25 mg by subcutaneous injection.

• · • · · • m · • ·

Example 6 30: *; : 1 ml of an aqueous suspension of porcine insulin containing the acidic proteinolides of Example 3b is administered by gavage to the stomach of three adult test rabbits weighing about 800 g. Blood samples are taken: * just before dosing.

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A blood sample from the first test rabbit is tested for blood glucose, which drops from a pre-dose value of 160 mg / dl to 42 mg / dl in half an hour and to 25 mg / dl in one and a half hours, the animal is weaned with oral glucose.

Blood samples from test rabbits 2 and 3 are tested for porcine insulin using a radioimmunoassay marketed by Cambridge Medical Diagnostics.

10 This method, which separates the insulin from the piglet and the test rabbit, shows that the pre-dose insulin concentration of both test rabbits 2 and 3 is 0. In test rabbit 2, the concentration peaks at 250 micrograms / ml one and a half hours after oral administration of microspheres and peak value of 240 micrograms-15 country / ml in four hours.

These experiments show that orally administered porcine insulin has an effective effect on blood glucose deficiency in guinea pigs in that it does indeed enter the bloodstream and that its administration does not accelerate the production of guinea pig insulin.

: Example 7a

The procedure of Example 3b is repeated except that microspheres filled with insulin <«· ... 25 are suspended in acidic aqueous acetic acid; a pH of 2.25 instead of 3.5. Closed vial with

«I I

’· 1 1 of this suspension, stored at room temperature for 23 days.

• · · ti · • ·

Example 7b: Y: 30 • · • · ·::: The efficacy of this older encapsulated insulin is tested by dosing. suspension by gavage into the stomach of adult mice that are not ’. fed for 8 hours and then by measuring blood glucose levels at regular intervals after dosing. The results are shown in Table 4.

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Table 4

Blood glucose (mg / dl) 5 Rat weight (g) dose (ml) Time after dosing (hours) _0 0.5 1.0 1.5 2.0 3.0 40 398 0.40 64 49 44 34 33 35 41 415 0.42 79 89 35 82 80 80 10 42 479 0.48 79 50 34 37 37 4

A normal insulin solution can be expected to break down in a few days even when frozen. The magnitude of the reduction in blood glucose in all these rats and the prolonged effect in rats 40 and 42 indicate that the stability of the insulin solution is improved by its incorporation into acidic proteinoid microspheres.

Example 7c An old suspension of encapsulated insulin is added to human serum and using a standardized laboratory red cell counting technique,. : the number of microspheres is counted immediately after mixing and at certain intervals thereafter. Table 5 shows the observed micro-. 1 ’as a function of the number of spheres.

25 · 2 Table 5 • · · «· · • · · • • • · · .1.1 Solubility of microspheres in human serum ί ^ ϊ 30 Time (in minutes) 0 3 30 60

Microspheres (x 1000) 78 50 19 9 e · · 2 • «*» These data indicate that insulin-containing acidic protein::: 35 nuclide microspheres have remained unchanged for 23 days at room temperature in acidic acetic acid, which The pH is 2.25, still rapidly dissolving in almost neutral human serum.

Example 8a 5

An aqueous solution of heparin containing 250 mg / ml heparin is adjusted to pH 4.5 by adding concentrated acetic acid. To this is added 35 mg / ml of a dry powdered acidic proteinoid of Example 3a and the mixture is allowed to stand at room temperature until microspheres are formed. One volume of the mixture is then centrifuged after discarding the supernatant, and the heparin-containing microspheres are washed at pH 4.5 with aqueous acetic acid, filtered and resuspended in pH 4.5 aqueous acetic acid, and the suspension is diluted to one volume. Microscopic examination shows that the microspheres are mainly in the range of about 0.1 to about 5 microns in diameter, with the major part being between 1-2 microns.

Example 8b 20 Seven white male rats, each weighing about 600 grams, are allowed to fast without food for 12 hours before the start of the experiment. Rat 1: does not receive any treatment. Rat 2 is injected intravenously by injecting 250 mg of heparin in 1 ml of distilled water. Each of rats 3-7 receives 1 ml of the microspheres of Example 8a containing 1 ml of an aqueous suspension of heparin directly by gavage into the stomach. The potency of heparin is determined using the activated partial thromboplastin time test • · · * · 1 1 (APTT). This test measures the time it takes for a • · • · · * .1.1 serum sample taken from a tail vein to form a fibrin clot. The results for each rat at different time points after dosing are shown in Table 6.

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Table 6

Coagulation time in APTT test (seconds) 5 Rat Heparin Dose lh 2 h 3 h 4 h 24 h treatment 1 none 28 26 27 - 2 IV 25 54> 300 10 3 microspheres 24 35> 300 - - - 4 '' 26 35 59 118> 300 21 5 "29 41 63 106 248 26 6" 25 37 66 121> 300 23 7 "24 31 62 111> 300 37 15

In all animals that received heparin-containing microspheres, the coagulation time rises to a level comparable to that obtained by intravenous injection of heparin. From these data, it becomes clear that heparin is released into the bloodstream in a physiologically appropriate and active form when encapsulated in: acidic proteinoid microspheres and administered orally. Is ·. note that much higher oral doses (per unit body weight of unprotected heparin) in laboratory animals ... 25 and humans do not result in a significant increase in shedding time.

♦ · ·· · * ·· • · · "· * * Example 9a • · • · ·« · · •

An aqueous solution of physostigmine containing 50 mg / ml physostigmine is adjusted to pH 5 by the addition of concentrated acetic acid. To one volume of this solution is added 100 mg per ml of dry powdered acid, the proteininoid of Example 3a, and the mixture is allowed to stand at room temperature until microspheres are formed. It is then filtered, washed three times at pH 5 with acidic acetic acid and the separated microspheres are resuspended in one volume of 20 102456 acetic acid at pH 5. Microscopic examination shows that the diameter of the suspended microspheres is mainly in the range of 0.5 to 5 microns.

5 Example 9b

Each of the two rats, which weighs about 360 g, is administered 3 ml of microspheres containing a suspension of physostigmine by gavage. Within 30 minutes of dosing, both animals have died 10 and both animals have enlarged liver and peritoneal bleeding. These lethal microencapsulated doses of physostigmine are calculated to be less than 1% of the oral LD50 dose of unprotected physostigmine in rats.

In addition to the specific pharmacological agents shown in the above examples, which are intended to be released into the bloodstream in physiologically active form when administered orally in protective acidic protenoid microspheres, microsphere release systems are equally effective in various other agents that are labile in the abdomen. including nitroglycerin, Saiki polio vaccine, rubella vaccine and hepatitis B vaccine.

; \ j vaccine. However, there are many other pharmacological agents that • · could be adversely damaged even under the weakly acidic conditions that prevail during incorporation into acidic proteininoid microspheres.

• · ·· · »·· • · · • # The following experiment demonstrates the ability to form an alkaline proteinoiidin microscope •« · * · * · * fäärejä, which encapsulate and protect one such extremely acid sensitive pharmacological agent, dopamine derivative, gastric intestinal VA- • · ·. ·. · 30 sensory areas, as well as to release the substance into the circulatory system ···! from where it penetrates the blood-brain shield of the brain and releases dopamine into the brain. The dopamine derivative used in this experiment is ^ PR-21, an exclusive acylated dopamine composition bound to a reduced dihydropyridine / pyridine salt type reduction-oxidation support developed by Pharmatek, Inc. and which is described in U.S. Patent 4 479 932. Unprotected PR-21 21 102 456 the composition is unstable in the gastro intestinal tract only, and is particularly sensitive to the hap-PAM conditions. When injected intravenously into rats, significant amounts of deacylated quaternary dopamine precursor can be detected in homogenized rat brain by the method described by Bodir and Farog in Journal of Medicinal Chemistry, 26, 528 (1983).

Example 10a 10 A nitrogen swept mixture containing an equimolar mixture of sixteen parts by weight of arginine, two parts by weight of lysine and 1 part by weight of an animal protein derived from 16 neutral and acidic amino acids is stirred and heated at 180 ° C for 3 hours. The cooled reaction mixture is extracted at pH 2.25 with acidic acetic acid and the extract is dialyzed through a collodion membrane against a large amount of distilled water at room temperature for 48 hours, changing the water every six hours. The contents of the dialysis tubes are then heated in vacuo at 65 ° C to give a dry powdery basic proteinoid. When suspended in a reasonably strong alkaline liquid medium, this powdery proteinoid spontaneously formed hollow microspheres which are stable in these, ·. : conditions but which are soluble in the blood at near neutral pH. '. you.

Example 10b One volume of PR-21 ethanol solution (360 mg / ml) is diluted with an equal volume of distilled water and the pH of the solution. is adjusted to 8 by adding saturated aqueous monobasic potassium phosphate buffers • t * t · 30 discipline. A portion of this buffered solution containing 180 mg / ml PR- • · e::: 21 is set aside and its doses are referred to below. melted "unprotected PR-21".

• «·

The remainder of the buffered solution is mixed with the 25 mg / ml dry powdered basic protein protein of Example-35a and frozen in an ice bath until microspheres have formed. Doses of the resulting suspension, in which the microspheres are mainly in the range of 0.5 to 5 microns in diameter, are hereinafter referred to as "microencapsulated PR-21".

5 Example 10c

Two rats weighing approximately 500 g (rats DA-1 and DA-2) are anesthetized, the jejunum is taken out, and the sphincter muscle is severed to prevent re-entry into the abdomen. Two milliliters of microencapsulated PR-21 10 is then injected into the jejunum of each rat. Two identical control rats (rats DA-5 and DA-6) are prepared in the same manner but 2 ml of unprotected PR-21 is injected into their jejunum. Finally, two similar control rats (DA-3 and DA-4) are injected intravenously with 2 ml of unprotected PR-21. Table 7 shows the amount of deacylated 15 quaternary precursors of dopamine that can be detected in the homogenized brain of these six target animals.

Table 7 20 Quaternary precursor of dopamine in rat brain. : Rat Treatment Precursor. ·. (in micrograms / g) ♦ «•« • · 25 DA-1 Injection into the intestine, 1 *** microcapsulation PR-21 ··· ♦ * · * · * * DA-2 Injection into the intestine, 10 »e microcapsulation PR-21 DA -3 IV injection, 3 • · 30 unprotected PR-21 DA-4 IV injection, 4.5 unprotected PR-21! DA-5 of injection into the gut, 0 unprotected PR-21 · 35 DA-6 injection into the gut, 0 \: unprotected PR-21 23 102 456 These results show the basic proteinoiidimikrosfäärien ability to encapsulate and protect dopamiinijohdannaista digestive and intestinal basic environment, as well as the fact that these microspheres are transported across the intestinal wall near neutral blood stream 5, wherein the encapsulated dopamine derivative is released. For such successful oral release of an encapsulated pharmacological agent, acid-sensitive basic proteinaceous microspheres must be protected as they pass through the mouth and stomach. Successfully this is achieved by conventional intestinal coating which does not dissolve until it reaches the intestine.

Example 12

A stirred mixture of 2 moles of anhydrous glutamic acid, 15 and 2 moles of lysine and 1 mole of an equimolar mixture of neutral amino acids (alanine, glycine, leucine, phenylalanine, proline, tyrosine and valine) is heated at 170 ° C under nitrogen for 1 hour. I drive. The cooled reaction product is extracted at pH 2.25 with aqueous acetic acid, and the extract is dialyzed through a collodion membrane 20 against distilled water for 24 hours, at which time the water is changed at 4-hour intervals. The contents of the dialysis tubes are evaporated to dryness by heat. : at 65 ° C and the solids in vacuo and residue are ground to a fine powder. When then aqueous pharmacological • · «.! substance in a solution or suspension with a pH of 7.4, this * ... 25 neutral powdery proteinoid spontaneously forms a large number of hollow microspheres encapsulating this solution or suspension. * pension.

• These microspheres are stable in human serum but dissolve rapidly in aqueous acid at pH 2.5 and release their contents. When their stability is removed by exposing them to a lower pH than when ingested in macrophages, these neutral proteininoid microspheres are suitable for the administration of a pharmacological agent such as • · * «azidothyrnide to a vessel which, in unprotected form, is rapidly absorbed by many. target body tissue and • ί cell as well as target macrophages.

24 102456

It will be apparent to those skilled in the art that numerous modifications and variations can be made to the embodiments of the invention described above without departing from the spirit of the invention as set forth in the following claims.

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Claims (7)

A method of microencapsulating a pharmacologically active agent in microspheres for target-directed release within a selected pH range, characterized in that a mixture of said agent with a pharmaceutically acceptable liquid is formed, said mixture having a pH outside said selected range, and said mixture is contacted with proteininoids formed by mixed amino acids by thermal condensation, said proteininoids being soluble within said selected pH ranges and insoluble in said mixture to form microspheres having diameters substantially less than about 10 microns which contain the active agent, said spheres encapsulating said active substance. Process according to claim 1, characterized in that said pharmaceutically acceptable liquid is water. '. 3. A method according to claim 1, characterized in that it V includes preliminary purification of said proteininoid by admixing with water having a pH within said selected range and separating the resulting aqueous solution of said proteininoid from any insoluble material. 4. A method according to any one of claims 1-3, characterized in that the proteinoids are formed from linear heat condensation polymers of mixed amino acids. • · · • t 5. A method according to any one of claims 1-3, characterized by: ***; thereof, that the proteininoids have been formed from directed polymers of mixed amino acids. · *. \ 30 • · e 6. A method according to any one of claims 1-3, characterized in that the proteinoids are formed from random polymers of mixed amino acids. 35 7. A method according to claim 1, characterized in that inulin is mixed with water and said mixture is contacted with a thermal polycondensate derived from about 2 parts of glutamic acid, about 2 parts of aspartic acid and about 1 part of neutral or basic alpha-amino acid. microencapsulating said insulin in a proteininoid microsphere; and an orally administrable composition for delivering insulin to the blood stream in physiologically active form. • «« • «· • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · · · «« ··· • · · · · · · · ♦ · ♦ ♦ • • m • · • · ♦ · · • # · • ·