HUT69785A - Soybean protein or hydrolylates in pharmaceutical compositions to protect bioaltive peptides from enzymatic inactivation - Google Patents

Abstract

Proteins or peptidic substances, which may be prepared from naturally occurring proteins, enhance the bioavailability of proteolytically-labile therapeutic agents which, in the absence of the protein or peptidic substance would suffer enzymatic inactivation upon administration.

Description

FIELD OF THE INVENTION The present invention relates to pharmaceutical compositions and to the enhancement of the bioavailability of pharmaceutical agents that are labile to proteolysis by administering the pharmaceutical agent in combination with a safener which is a protein, a purified natural protein, a molecular weight fractionated protein or a partially hydrolyzed protein. contain.

Peptide-like drugs and peptidease labile binding drugs are among the most promising therapeutic agents of modern times; however, their instability with respect to proteolytic enzymes in the gastrointestinal tract and other mucous membranes in the presence of proteolytic enzymes generally requires parenteral administration. Although patients are able to learn parenteral injection, there has long been a need for the administration of peptide-like agents by non-invasive methods.

Protease inhibitors and permeation enhancers are considered to bypass enzymatic and permeation barriers to peptide and protein absorption following mucosal administration. As a result of these barriers, peptide and proteinaceous agents are only biologically poorly marketed after administration to mucous membranes.

In particular, non-parenteral administration of peptide agents results in very low bioavailability because the peptides are hydrolyzed by proteolytic enzymes. For example, in the case of leuprolide, the oral bioavailability is 0.05%, but up to 38% when administered vaginally

-3 is added; for insulin, these two values are 0.05% and 18%, respectively (Lee, J. of Controlled Release 13, 213 (1990)).

Examples of proteolytic enzymes that inactivate pharmaceutical agents that are labile to proteolysis include pepsin, trypsin, chymotrypsin, elastase, and carboxypeptidase in the small intestine lumen; and aminopeptidases located on the surface of the gastrointestinal tract (GI channel, nasal and vaginal mucosa, in short).

Transport of intact oligopeptides has been demonstrated in jejunum of mature mammals, both in vitro and in vivo, in combination with peptidase inhibitors (Friedman and Amman, Pharmaceutical Research 8, 93 (1991)).

U.S. Patent No. 4,639,435 to Fujii et al., Issued July 1987, claims the use of l-isopropyl-4- [4- (1,2,3,4-tetrahydronaphthyloxy) -benzoyl] -piperazine methanesulfonate as an inhibitor of chymotrypsin. as a substance that is administered orally or rectally with a chymotrypsin labile drug (kallikrein or calcitonin). This disclosure further includes the use of benzoylpiperazine esters for the same purpose. The mechanism of action of these inhibitors is not described herein.

Cho and Flynn in WO-90/03164 (1990) disclose the use of protease inhibitors in oral dosage form, but do not detail the nature of such inhibition; among the examples of the application, aprotinin is the only protease inhibitor compound.

Kidron et al., U.S. Patent 4,579,730 (1986) disclose the use of protease inhibitors in the oral dosage form of insulin. Soybean meal is reported to be a source of trypsin inhibitor from soybean (Bowman-Birk's trypsin / chymotrypsin inhibitor, molecular weight 8000 Daltons).

Ziv et al., Biochem. Pharmacol. 36: 1035-1039 (1987) describe the use of aprotinin as a protease inhibitor to increase the oral absorption of proteins.

Losse et al., DD 252 539 A1 (1987), disclose the use of ε-aminocaproic acid and aprotinin protease inhibitors in the preparation of oral dosage forms of peptides.

J. Lee, (Controlled Release 13, 213-223, 1990) reviews the use of protease inhibitors in formulating peptides for oral, nasal, buccal, rectal, vaginal, pulmonary and other dosage forms.

Some low-number peptides have been shown to increase the bioavailability of peptide-like drugs up to four amino acids.

Hussain et al., Biochemical and Biophysical Research Communications 133: 923 (1985), suggest that the administration of peptides via the nasal mucosa may play an important role, provided that the peptidases present in the nasal mucosa are co-administered with pharmacologically ineffective peptidase substrates.

Faraj et al., J. Med. of Pharmaceutical Sciences 79.

-5698 (1990)] have shown that in the presence of small peptides, such as Ltirosyl L-tyrosine and tri-L-tyrosine methyl ester, the hydrolysis of leucine-enkephalin is reduced, probably because small peptides are present in the nasal mucosa. peptidases are competitively inhibited.

Friedman and Amtimen, in their above-cited publication, demonstrate that the perfusion of YGG tripeptide with encephalin (YGGFL) results in increased absorption of YGGFL in the perfused rat intestine.

Hori et al., J. Med. Pharm. Sci. 72: 435-439 (1983) reported that various peptides protected at their amino groups protect insulin from degradation after subcutaneous injection. Peptides include (benzyloxycarbonyl) -Gly-Pro-Leu-Gly, (benzyloxycarbonyl) -Gly-Pro-Leu, (dinitrophenyl) -prop-Leu-Gly and (benzyloxycarbonyl) ) -Gly-Pro protected peptides were used.

Numerous publications describe enzymatic treatment of plant proteins. John R. Turner discloses a pepsin modified blowing component in older U.S. Patent 2,489,208. Glycine is extracted with an alkaline material such as sodium sulfite, sodium carbonate or sodium hydroxide at a pH of 6.4 to 6.8, and the glycine is extracted from the extract to an isoelectric pH of e.g. is precipitated (sulfur dioxide can be used as an acid for adjustment). The glycine product thus precipitated is then modified by pepsin under temperature and pH conditions which lead to the hydrolysis of the protein. Glycine With Pepsin Until *

-6hydrolyze until water solubility is increased to 40-50%. U.S. Patent No. 2,502,482 to Sair et al. Also discloses enzymatic modification of glycine with pepsin; obtaining an isolate in which at least 60% by weight of the pepsin modified isolate is water soluble at pH 5.0.

Puski describes the enzymatic modification of soy isolates (pH 4.5 precipitated product) by Aspergillus oryzae [Modification of functional properties of soy proteins by proteolytic enzymatic treatment; Cereal Chem. 52, 655-665 (1975)].

Numerous reports have been reported on the use of saline solutions to extract soy proteins. A. K. Smith et al., J. Am. Am. Chem. Soc., 60, 1316-1320 (1938)] shows that extraction of soybean meal with water at pH 6.7 produces more protein than when extraction with water containing neutral salts.

U.S. Patent No. 4,131,607 to Petit discloses two-step alkaline extraction. The first extraction is carried out in the presence of sodium sulfite and magnesium salt at a pH of 7.0-8.5 and then raised to 10.0-10.5 for complete extraction. The white jeck extracts are then precipitated or coagulated by adjusting the pH of the extract to 4.5-5.5. Martinez et al., U.S. Patent 3,579,496, also disclose a multi-step solvent extraction process.

We have found that proteins, peptides, purified, space.

-7 naturally occurring proteins, as well as filtered, solvent-extracted, molecular weight fractionated or partially hydrolysed proteins, hereinafter referred to as "preservatives", increase the bioavailability of oral, nasal, rectal and vaginal administration of therapeutic agents that are labile to proteolysis, after oral, nasal, rectal or vaginal administration, they would become inactive by enzymatic action.

The present invention relates to a pharmaceutical composition comprising a safener and a therapeutically effective amount of a proteolytically labile drug (proteolytically sensitive), with the proviso that if the safener is soy meal, the therapeutically active agent is other than insulin.

The invention further provides a method of increasing the bioavailability of proteolytically sensitive pharmaceutical agents in mammalian animals or other animals that require the particular pharmaceutical agent; this method comprises administering a therapeutically active agent in combination with a bioavailability enhancing agent, with the proviso that if the protective agent is soy flour, the therapeutically active agent is other than insulin.

The proteolytically labile pharmaceutical agents present in the pharmaceutical composition of the invention contain peptide bonds in their structure or are degraded, denatured by various proteolytic enzymes present in the digestive tract or in the mucosa of the nose or vagina.

-8 - become ineffective as a result of the process or other process. As a result, when these therapeutic agents are administered orally, nasally, rectally or vaginally, they are not absorbed or therapeutically effective.

Examples of such pharmaceutical agents labile to proteolysis include peptides such as calcitonin, prolactin, adrenocorticotropin, thyrotropin, growth hormone, gonadotropic hormone, oxytocin, vasopressin, gastrin, tetragastrin, pentagastrin, secretin, pancreozimine, substance P, and gonadotropin. Examples of therapeutic agents that are labile to proteolysis include luteinising hormone releasing hormone, leuprolide, enkephalin, follicle stimulating hormone, cholecystokinin, thymopentin, endothelin, neurotensin, interferon, interleukins, insulin, and insulin. Other examples include therapeutic antibodies, such as those used to treat septic shock.

Purified extracts of natural origin and chemically modified variants thereof, as well as tissue culture derived products obtained by culturing microorganisms or cells produced by genetic engineering can also be used as therapeutic agents labile to proteolysis. The therapeutic agent that is labile to proteolysis may also be a synthetic peptide or derivative thereof, such as terlakiren (chemically N- [N- (4-morpholinocarbonyl) -L-phenylalanine-S-methylcysteine] -2 (R)). • · ·

9-hydroxy-3 (S) -amino-4-cyclohexylbutanoic acid isopropyl ester; see U.S. Patent No. 4,814,342).

The protective agents of the present invention may be chemically synthesized proteins and peptides, natural proteins, purified natural proteins, chemically modified natural proteins, partially hydrolysed natural proteins, or proteins which are fractionated by molecular weight, polarity or charge, or mixtures thereof. Natural food grade protein or partially hydrolyzed food grade protein are preferred. The protective agent must have a molecular weight greater than 1000.

The molecular weight fractionation of the protective agent can be varied to obtain any desired molecular weight fraction of the native protein. Solvent extraction can be used to separate proteins by molecular weight, polarity, or charge. Enzymatic or chemical hydrolysis of the protein may be followed by separation of the desired molecular weight fraction on ultrafiltration or dialysis membranes. Molecular weight fractionation can be accomplished by gel chromatography or otherwise.

The hydrolysis of the proteins or peptides may be accomplished by heat treatment, cold acid, bromocyanide or other chemical means.

Enzymatic treatment can be performed with a single proteolytic enzyme or with combinations of different proteolytic enzymes acting simultaneously or sequentially. A wide variety of proteolytes

Examples of enzymes which may be used include, but are not limited to, trypsin, chymotrypsin, elastase, carboxypeptidase, aminopeptidase, peptide and collagenase.

Fractionation and enzymatic treatments useful in the preparation of the inventive safeners are applicable to a wide variety of protein substances. Preferably, naturally occurring proteins or proteins of animal or vegetable origin are used. Examples of such protein-type starting materials include, but are not limited to, soybean meal, soy protein, wheat gluten, almond powder, hazelnut powder, casein, and fish protein.

In their non-limiting view, the protective agents of the present invention act as self-sacrificing protease inhibitors and thus increase the bioavailability of therapeutic agents that are labile to proteases that are capable of disrupting the therapeutic agents by oral, nasal, vaginal or rectal administration. As a result of coadministration of these protective agents with labile therapeutic agents, (1) the protective agents competitively capture degradable proteases; (2) inhibition of protease degradation of therapeutic agents enhances absorption and therapeutic efficacy; and (3) protective agents are metabolized and absorbed.

With respect to the mechanism of action suggested above, it is believed that it is desirable to balance the dissolution rate of the protective agent with the dissolution rate of the therapeutic agent sensitive to proteolysis. In general, we have found that short-acting soluble protective agents are small molecules »·« ·

They are more effective for pharmaceutical agents of 11 wt%; it is preferred to use peptides having a molecular weight of greater than 1000 but less than 100,000, and particularly preferred between 1000 and 30,000.

The effective preservative fraction should be consistent with the lability characteristics of the therapeutically active agent that is labile to proteolysis. Thus, for a pharmaceutical agent that is sensitive to aminopeptidase, a protective fraction which is rapidly soluble and effective against aminopeptidase is preferred. For the aminopeptidase-sensitive D-Ala-D-Leu-enkephalin (YdAGFdL) therapeutic agent, the pepsin-treated decanted soybean meal has a molecular weight of less than 30,000, as shown in Example 15. Generally, a preferred protective agent for one or more protease active in the lumen or mucosa with respect to proteolytic activity is one that improves the systemic delivery of the therapeutic agent when administered in substantially all doses (as described above). For a particular therapeutic agent susceptible to proteolysis, the preferred protective agent fractions are obtained by the following decantation, filtration, extraction, hydrolysis and molecular size fractionation processes. For a particular therapeutic agent that is labile to proteolysis, the preferred protective agent fractions are identified by the in vitro and in vivo assays described in the examples below.

Preferably, the pharmaceutical composition of the present invention is administered to a mammal or other animal in need of such treatment, ············· ·

-12 can be administered in any form that provides intestinal preservation of the protective agent and the pharmaceutical agent which is labile to proteolysis, such as in the form of tablets, granules or capsules, each with or without an enteric coating. The composition may also be administered rectally or vaginally by means of suppositories prepared by placing both components in a commonly used suppository base. Similarly, the protective agent and the pharmaceutical agent labile to proteolysis may be administered together in the form of a nasal spray. If desired, various pharmaceutically acceptable additives such as diluents and emulsifiers may be added to these dosage forms.

Preferably, the dosage of the therapeutically active agent labile to proteolysis is 0.0001-1 times that of the prior art orally. The amount of protective agent will depend on the route of administration and the lability and dosage of the pharmaceutical agent. For oral, rectal, and vaginal administration, the dosage of the protective agent will generally be about 10-1500 mg. In the case of oral solutions or suspensions, the protective agent is added in an amount of about 10 mg to about 15 g. For nasal administration, the dosage of the protective agent will generally be in the range of about 1 mg to about 100 mg.

The efficacy of several enteric formulations of the invention has been evaluated; the results of these tests are described below.

• · • · · · · · · · · · · · · · · · · ·

The present invention is further illustrated by the following non-limiting examples.

Example 1

Processing of commercial proteins and flours; fractionation by solubilization; examination of soybean meal

4.5 g of soy flour (product of Sigma Chem. Co.) was mixed with 135 ml of 0.01 M phosphate buffer, pH 7.5, with 15 ml of 0.05% thymerose for 15 minutes, followed by sonication for 10 minutes, and stirred for 25 hours at room temperature. The mixture is then allowed to settle and the supernatant is suctioned off, centrifuged and filtered. This results in a directly soluble fraction which is isolated and used or subjected to further processing.

Example 2

Processing of commercial proteins and flours; molecular weight fractionation by solubilization and dialysis; examination of soybean meal

Following the procedure described in Example 1, a decanted and filtered solution of soybean meal is prepared and processed as follows by molecular weight (MW) distribution. The solubilized fraction was evaporated to dryness in a vacuum oven at 55 ° C. The residue was dissolved in water and dialyzed against water for 24 hours in a dialysis tube with a lower molecular weight range of 1000, while the dialysis medium was periodically refreshed. The residue was evaporated to give 1.17 g of solubilized soybean meal with a molecular weight greater than 1000 ··· 444 · 4444

Fraction 14 is obtained.

Example 3

Processing of commercial proteins and flours; fractionating the molecular weight by solubilization and ultrafiltration (UF); the molecular weight fraction from 1 kD to 30 kD and 30 kD to 100 kD

288 g of commercial soybean flour (Sigma item S-9633) are added with stirring to 8640 ml of 0.01 M phosphate buffer solution, pH 7.5, and 1920 ml of 0.1% thimerosal solution. The suspension was stirred with a magnetic stirrer for another 15 minutes, then sonicated for 10 minutes and then stirred at room temperature for 24 hours. The solution is then centrifuged at 2500-3000 rpm for 1 hour. The supernatant was collected by ultrafiltration using a Pellicon membrane (Millipore Corp., Bedford, MA, USA) with a nominal molecular weight of 100 kD. The remainder (> 100 kD) is discarded and the remaining portion (<100 kD) is separated using a Pellicon membrane with a nominal molecular weight range of 30 kD. The second fraction (30 kD to 100 kD) was retained and the third passage (<1 kD) was discarded. The 1 kD to 30 kD fraction was lyophilized and the 30 kD to 100 kD fraction dried in a vacuum cabinet. The yield of the 1 kD-30 kD fraction was 7.33 g, 2.5% based on the soybean meal used as starting material. The yield of the 30 kD-100 kD fraction was 5.25 g, ie 1.8% relative to the starting soybean meal.

-154. example

Processing of commercial proteins and flours; hydrolysis with pepsin and molecular weight fractionation by dialysis; examination of soybean meal

The soybean meal is hydrolyzed to lower molecular weight fragments using pepsin, and the hydrolyzate is broken down into 1000-3500, 3500-6 / 8 kD and 6/8 kD - 12/14 kD fractions according to the following procedure.

5.4 g of soy flour (product S-99633 of Sigma Chemical Co.) and thimerosal (50 ppm final) are added to 180 ml of a solution having a pH of 1.9 and 0.2 for both potassium chloride and hydrochloric acid. N concentration. After stirring for 30 minutes, 18.0 mg of pepsin (Pma-6887 from Sigma) were added. Twenty ml of the above solution are added to nine Spectrum dialysis tubes with a molecular weight range of 12,000 to 14,000 and dialyzed against shaking with 55 ml of pH 1.9 potassium chloride buffer solution at 37 ° C. After 2 hours and 6 hours, the buffer solution is changed and dialysis is continued for 24 hours. The passage was collected from time to time and concentrated in a vacuum oven at 55 ° C.

Samples from the 2, 6, and 24 hour periods are placed in 1000 molecular weight tubes and dialyzed against water. The residue (1 kD - 12/14 kD) was evaporated in a vacuum oven at 55 ° C to give a total of 449.5 mg of product; 418 mg of this are dissolved in 30 ml of deionized water and placed in two 3500 molecular weight dialysis tubes and dialyzed against 55 ml of water at room temperature for 16 hours. Dialysis 2.

After 6 and 24 hours, the water is changed; the passages (1 kD to 3.5 kD) are combined and concentrated in a vacuum oven at 55 ° C.

The remainder of each tube is placed individually in a 6000/8000 molecular weight tube and treated as described above. Evaporation of the passages yields fragments having a molecular weight range of 3500-6000/8000. The residues, representing the 6000 / 8000-12,000 / / 14,000 molecular weight fraction, were also combined and evaporated. The fractionation process described above is illustrated in Scheme I.

Sketch I

Pepsin treated soybean meal

12/12 kD molecular weight dialysis (> 12/14 kD)

Residual Bypass (<12/14 kD) kD molecular weight dialysis

Leftover Passage (1 kD - 12 kD) (<1 kD)

3.5 kD molecular weight-

-scale dialysis

Leftover Passage

(6/8 kD - 12/14 kD) (3.5 kD - 6/8 kD)

-185. example

Processing of proteins and flours: treatment with immobilized pepsin

Soya flour (product S-9633 of Sigma Chemical Co.) was processed and dried as in Example 1. The material thus obtained is subjected to ultrafiltration on a 30 kD molecular weight membrane. The remainder (> 30 kD) was collected and dried. 13.8 g of this product are dissolved in 900 ml of water, the pH is adjusted to 2.0 with 0.1 N hydrochloric acid and 1.19 g of immobilized pepsin (for this purpose 4% of cross-linked pepsin immobilized on beaded agarose) used at 40 units / mg (pepsin, P-3286 of Sigma Chemical Co.). The resulting suspension is maintained at 37 ° C and ultrafiltrated on three 30 kD molecular weight membranes. The passage (> 30 kD) was collected at 15 minute intervals and lyophilized. The residue (> 30 kD) is recycled by ultrafiltration. The products of pepsin hydrolysis are present in the passage. Table I gives the volume of each passage fraction as well as the weight of the hydrolyzed peptide in each dried fraction.

-19I »Table

Yield of peptides obtained by pepsin hydrolysis as described in Example 5 | Volume Mass collected I (minutes) (ml) | (mg) |

15 600 361.1 30 300 385.0 45 300 341.5 60 300 361.3 75 275 403.2 90 350 390.1 105 500 557.8 120 600 410.5 135 550 261.6

Example 6

Processing of commercial proteins and flours: hydrolysis by sequential enzyme treatment; use of trypsin and elastase; examination of soybean meal

600 mg of soy flour were dispersed in 20 ml of a 0.01 M solution of potassium phosphate buffer (pH 7.5) containing 50 ppm of thimerosal and 2 mg of trypsin was added. The resulting mixture was placed in a 12/14 kD molecular weight dialysis tube and dialyzed against shaking water in a water bath at 37 ° C against 50 ml of buffer solution. The buffer solution was changed after 2 and 6 hours and dialysis was performed for 24 hours. After 6 hours, 50 ppm of the buffer solution used ·········· · · · · · · · · · · · · · · · ·

-20rose is given. The procedure was performed in triplicate.

The dialysis portion of each of the three probes was pooled over 2, 6, and 24 hours, the absorbance at 280 nm was determined, and the samples were concentrated in a vacuum oven at 55 ° C. The three evaporation residues were redissolved in 20 ml each of deionized water containing 50 ppm thimerosal.

To each sample was added 2.0 mg of elastase (0.182 ml of elastase solution, 11 mg of protein / ml) and the samples were dialyzed against the buffer by placing in a 12/14 kD molecular weight dialysis tube. The buffer solution is changed after 6 hours and dialysis is continued for 24 hours. At 280 nm, the absorbance was determined and the passages evaporated as above.

The product thus obtained is termed sequentially soya treated with trypsin followed by elastase and has a molecular weight of less than 12/14 kD.

Example 7

Increasing oral absorption of terlakiren in dogs with protective agents

The renin antagonist terlakirene tripeptide (200 mg as a solid, crystalline powder in the form of a hard gelatin capsule) was added to four starved game bags together with 1 g of an aqueous suspension of test substance in 150 ml of water. Serum concentration of tripeptide was administered at six time points after administration, i.e. after administration

-2115 and 30 minutes, then 1, 2, 3 and 4 hours. For each study, 4 fasted dogs were used, the animals representing their own controls the previous week. The serum was extracted with n-butyl chloride and incubated with aqueous chymotrypsin solution. After derivatization with fluorescamine, the degradation product was measured. The fluorescence detector was a Kratos Spectroflow 280 and a Waters Novapak C-18 column. The emission wavelength is 380 nm. The mobile phase was a 75:25 mixture of water and acetonitrile at a flow rate of 1.0 mL / min. The limit of detection was found to be 10 mg / ml. For each dog, the area under the trapezoidal rule was plotted over a 0- to 4-hour curve.

II. Table 1A shows that the 1-30 kD fraction of commercially available soy protein (Protein Technologies Inc. PP 620) and the processed soybean meal (prepared according to Example 3) increase the oral dose of terracyrene (a chymotrypsin-sensitive compound). biological active ingredient).

II. spreadsheet

Area under the curve (AUC) as a function of plasma concentration in dogs

AUC (Mg Hour / ml) (Preparation | Dog number | | 34112 | 34101 | 04132 | 04094 | Average |

PP620 (1-30kD) 0.863 .436 0,117 0.417 .458 Soy 0.420 0.320 0.078 0.326 0.286 control 0,043 0,026 0,022 0.106 0.049 PP620 / control ratio 20.1 16.8 5.3 3.9 11.5 Soya (1-30 kD) / / control ratio 9.8 12.3 3.5 3.1 7.2

Example 8

Protection of terlakirene against chymotrypsin-induced degradation: an in vitro test method

The following standard procedure was used to evaluate the in vitro inhibitory activity of various proteins and their processing products against the chymotrypsin-induced degradation of terlakirene.

Assay solutions were prepared from alpha-chymotrypsin (0.67 x ΙΟ ^-7 Μ), terlechirene (0.065 mM) and the inhibitor to be tested (approximately 0.1 mg / ml to 0.5 mg / ml) 6, 5 in a buffer solution containing 0.10 M citric acid and 0.20 M disodium hydrogen phosphate at pH 5 to a final concentration of 300 mOsm; these solutions at 37 ° C in • ·

-23kubáltuk. Samples were taken at zero time and then at 5 minute intervals and adjusted to pH 2.0 by addition of hydrochloric acid for HPLC analysis. HPLC analysis of terlakirene was performed on a Waters Resolve 5u C-18 column. The mobile phase was a 50:50 mixture of water and acetonitrile, to which 1 ml of phosphoric acid was added per liter. Data are expressed as percent inhibition of terlakiren degradation relative to zero time as a control value. The calculation was based on the following equation:% Inhibition = 100 x [l ^_ kinh / cl where k ^ £ _n if terlakiren without degradation of the initial rate in the presence of protective agents and _c k the initial degradation rate of terlakiren preservatives.

Percent inhibition values were determined on the 1 kD to 30 kD fraction of decanted ultrafiltered soybean meal. The results are shown in Table III. are shown in the table.

III. spreadsheet

Inhibition of chymotrypsin-catalyzed decomposition of terlakirene by a 1-30 kD fraction of decanted ultrafiltrated soybean meal | Concentration |

Inhibiting substance concentration * Inhibition%

(Mg / 1) (mg protein / ml) 0.5 0.04905 75.4 0.25 0.02453 79.8 0.1 0.00981 21.5 0.05 0.00491 29.3 0.01 0.00098 4.8

-24 * Value corrected for protein content

Example 9

Α-chymotrypsin-catalyzed hydrolysis of terlakirene: a method for the determination of Kp values for inhibitors

The K? Value, defined as the Michaelis-Menton constant for inhibition, is a conventional measure of the affinity of the inhibitor for the active site and thus the degree of enzyme inhibitory activity. Determination of Kp can be performed from initial degradation rate data obtained with multiple concentrations of the inhibitor at the same substrate and enzyme concentrations. Initial velocities are expressed in millimoles of minute decay of terlakirene, as shown in Figure IV. table.

ARC. spreadsheet

Inhibition of chymotrypsin-catalyzed decomposition of terlakirene by 1 kD to 30 kD fraction of decanted ultrafiltrated soybean meal | Inhibiting substance Concentration | concentration ** k *

(Mg / 1) (mg protein / ml) (Mmol / min) 0.5 0.04905 1.93 X 10 ^-4 0.25 0.02453 1.59 X 10 " ⁴ 0.1 0.00981 6.17 X 10 ^-4 0.05 0.00491 5.56 X 10 " ⁴ 0.01 0.00098 7.48 X 10 " ⁴ Cumulative control value 7.86 X 10 ~ ⁴

• ·

-25 * Decrease in initial decay rate of terlakirene ** Corrected for protein content

Alternatively, Kf can be determined for a single concentration of inhibitor (single point value) using the standard Michaelis-Menton equation for competitive inhibition. Its value for multiple concentrations (multiple point KjJ can be determined using the same relationship by introducing data from non-linear regression analysis into the equation.

Example 10

Dissolution time is an essential factor in the performance of the protective agents of the invention. For the purposes of this specification, dissolution time is defined as the time required for complete dissolution of a suspension of the test substance in a concentration of 0.5 mg / ml containing 0.1 molar citric acid, 0.2 molar disodium hydrogen phosphate at pH 7 in buffer solution at room temperature with a stirring speed of 8 rpm. The end point of complete dissolution was determined visually.

Example 11

The percentages of protein content of various substances as protective agents were determined. The concentration of carbon, hydrogen and nitrogen in the sample was determined with 2400 Perkin-Elmer C, Η, N elemental analyzers. About 2 mg of sample was accurately weighed and placed in the analyzer. The percentage of nitrogen in the sample is 6.25 • ·

Multiplied by -26 gave the estimated protein content (%).

Example 12

The inhibitory effect of several commercial and processed protein fractions on the chymotrypsin-induced degradation of terracirene was determined. Soybean meal was purchased from Sigma Chem. Co., almond and hazelnut meal from Pert Labs Institute, and wheat gluten from Total Foods Corp..

Soya flour obtained from Sigma Chemical Co. is unroasted and thus contains Bowman-Birk, an effective inhibitor of trypsin and chymotrypsin, having a molecular weight of 8000.

Soy Protein (PP620) from Protein Technologies, Inc. is a heat-treated formulation in which the Bowman-Birk trypsin / chymotrypsin inhibitor is inactivated. Percent inhibition was determined as described in Example 8. Table V shows that the protective fractions tested reduce the chymotrypsin-catalyzed degradation of terlakiren (a chymotrypsin-sensitive renin inhibitor).

β · · · · ♦ · · · · · ··· · ···

Table V

Inhibition of Terlakirene Degradation by Commercial and Processed Proteins (0.5 mg / ml) under In vitro Conditions

Source of material and description % Inhibition (as in Example 8) 1. Pepsin treated, dialyzed soy meal (> 1 kD) 87 2. Pepsin treated, fractionated soybean meal (Example 4) Molecular Weight: 1000-3500 24.0 3500-6 / 8kD 46.6 6 / 8K-12/14 kD 85.1 3. Soya flour 69.9

4th

Decanted ultrafiltered soybean meal kD - 30 kD 75.4 and 93.3

75.4 and 86.8 kD - 100 kD (two preparations) (two preparations)

5. Soybean meal obtained by dialysis, molecular weight> 100097.6

6. Wheat gluten, decanted, ultrafilter, molecular weight 1 kD - 30 kD95,8

7. Wheat gluten 76.8

8. Decanted, ultra-filtered hazelnut flour, molecular weight 1 kD - 30 kD28.6

9. Decanted ultrafiltered almond meal, molecular weight 1 kD - 30 kD37,6

10. Soya Protein (Protein Techn., Inc.) 86.0

Example 13

For various commercially available protein products and processed protein fractions, the percentage protein content, dissolution time and K i constant (as described in Examples 11, 10, and 9 above) were determined. These data are set out in Annex VI. are summarized in Table. The results show that degradation reducing fractions can be obtained which have a K i it has a low constant and a short dissolution time.

Soya flour from Sigma Chemical Co. is unroasted and thus contains 8,000 molecular weight active Bowman-Birk trypsin / chymotrypsin inhibitors.

VI. spreadsheet

Dissolution rate and inhibition constant of typical commercially available and processed proteins

The source and description of the material Protein% Dissolution time Who measured value THE. Commercial proteins and flours: First Casein (Sigma C-5890) 87.94 > 4 days 0,240 * 2. soy flour 51.69 > 4 days 0.18 * Third wheat gluten 78.81 > 4 days 0.091 * B Ultrafiltrated (1 kD - 30 kD) materials: First soy flour 9.81 0.1 min 0.00765 Second almond Flour 10.37 2.3 minutes 0.0618 * Third Casein 71.31 1.8 min -

-29VI. Continue Table

Source of the material Protein dissolution K £ is measured and its description % time is

4th peanut flour 13.19 3.1 minute 0,120 * 5th wheat gluten 37.81 3.8 minute 0.0060 * C. Ultrafiltrated (30 kD - 100 kD) materials: First soy flour 44.44 2.6 minute 0.0029 D. Dialysis,> 1 kD First soy flour 76.5 > 4 1 Sun 0,010 E. Ultrafiltrated pepsin-mixed (<30 kD) materials First Soya flour (Example 16) 80.1 0.1 minute 0.0094

* Single point K | (concentration of inhibitory substance 0.5 mg / ml)

Example 14

Brushed Border Membrane vesicles (BBMV): inhibition of enzymatic degradation of cholecystokinin-8 (CCK-8) by soy protein fractions

Borderline membrane vesicles (BBMVs) of rat and intestinal feathers were prepared by the method of Kessler et al., Biochem. Biophys. Acta 506, 136 (1978)]. BBMV corresponding to 25 micrograms of protein was incubated with 10 micromolar CCK-8 in the presence or absence of 0.15 mg / ml protective agents in a total volume of 1 ml at 37 ° C.

· * ·· · • · · · ·

-30mérsékleten. Samples were taken after 1 and 3 minutes and mixed with acetonitrile in an ice bath. Undegraded CCK-8 was determined by HPLC. VII. The data shown in Table II show that two different molecular weight fractions of processed soybean protein effectively inhibited the degradation of CCK-8 by BBMV proteases, presumably aminopeptidases.

VII. spreadsheet

Decomposition of CCK-8 by BBMV protease; effect of fractionated soy protein on degradation

Percentage of CCK-8 remaining intact Incubation time 1 kD - 30 kD molecular weight 30 kD - 100 kD mo1eku1atömegű Minute inhibitory inhibitory inhibitory without in the presence of in the presence of 1 minute 42% 59% 67% 3 minutes 8% 10% 16%

Example 15

Protection against aminopeptidase-induced degradation under in vivo conditions

D-Ala-D-Leu-enkephalin (YdAGFdL) analogous to enkephalin pentapepdide (YdAGFdL) (1.0 mg) was administered directly into the ileum of chronically swollen rats. Included with each protective agent 1.12 micrograms radio • 4 ············································ ·

-31actively labeled YdAFGdL was added. The four protective agents are: (1) decanted soybean meal having a molecular weight of less than 1 kD; (2) decanted and ultrafiltered soybean meal having a molecular weight of 1 kD to 30 kD; (3) pepsin treated (FMC ACTI-MOD) soybean meal having a molecular weight of less than 30 kD; (4) amastatin, a potent inhibitor of the aminopeptidase enzyme (positive control). At various times after dosing, blood was drawn through a cannula inserted into the cervical vein, and undissolved YdAGFdL was quantified by radioactive thin layer chromatography (TLC) using reverse phase KC-18 thin layer plates (Whatman Co.). The TLC plates were developed with a 30:70 mixture of 1-propanol and 0.1 M phosphate buffer pH 4.1.

VIII. Table 2 summarizes the absolute values of the bioavailability achieved by the various treatments. Pepsin-treated (FMC ACTI-MOD) soy meal with a molecular weight of less than 30 kD has been shown to be a particularly effective protective agent against enteric aminopeptidase enzymes; this is evidenced by the fact that the percentage absorbed increased almost 11-fold. An effective inhibitor of amastatin, the aminopeptidase, was also effective; this suggests that the bioavailability of YdAGFdL is partially limited by the degradation induced by intestinal aminopeptidases. Unroasted soybean meal had no protective effect against intestinal aminopeptidase-induced degradation.

• · ··· · · ··· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·–ward - place ·

-32VIII. spreadsheet

preservative n YdAGFdL Biological Sales% None (control) 16 1.78 + 0.46 Decanted soybean meal (<1 kD) (100 mg) 4 l, 74 ± 0.68 Decanted ultrafiltered soybean meal (FMC ACTI-MOD; 1 kD - 30 kD; 150 mg) 4 2.76 & 1.43 Pepsin-treated soybean meal (<30 kD; 100 mg) 4 19.54 13.75 + Aastatin (1 mg; Sigma Chemical Co .; Positive Control) 6 8.76 + 4.47 Unroasted Soya Flour (Sigma; 50 mg) 2 1.88 & 0.67

Example 16

Preparation of pepsin-treated soybean meal using immobilized pepsin g soybean meal (product of Sigma Chem. Co.) was suspended in 1080 ml of water and 120 ml of 0.1% (w / v) thimerosal solution and stirred for 24 hours at room temperature. It was centrifuged for 1 hour at 3600 rpm. The supernatant was ultrafiltered on a 30 kD molecular weight membrane. The fraction having a molecular weight of less than 30 kD was collected and evaporated, and the residue was adjusted to pH 2 with 0.5 N hydrochloric acid. This 30th

The fraction having a molecular weight of less than -33kD was fed into an ACTI-MOD spiral reactor unit previously loaded with 8 g of immobilized pepsin (product of FMC Corp., Pinebrook, NJ, USA). The liquid leaving the enzyme reactor was ultrafiltered on a 30 kD molecular weight membrane. The fraction of molecular weight less than 30 kD was discarded and the fraction of molecular weight greater than 30 kD was recycled through the enzyme reactor. After a total of 2 hours of enzyme treatment and ultrafiltration, all fractions with a molecular weight of less than 30 kD were retained and designated as ultrafiltered pepsin treated (<30 kD) soybean meal.

Example 17

In vitro inhibition of trypsin-catalyzed degradation of benzoyl-arginine (para-nitroanilide) To evaluate the in vitro efficacy of a protective agent of the invention in inhibiting trypsin-catalyzed degradation of benzoyl-arginine (para-nitroanilide) (briefly: ΒΑΡΝΑ). The following procedure was used. Assay solution was prepared from trypsin (1.25 Mg / mL, 103 BAEE units / mL), 0.5 mg / mL BAPNA, and 0.5 mg / mL test inhibitor at pH 8 in TRIS (0.048 M). and 0.019 M calcium chloride and 3.75 pg / ml bovine serum albumin and incubated at 37 ° C. Samples were taken for a total of 40 minutes after the first 5 minutes and at 5 minute intervals, and after 40 minutes, the solutions were mixed with an equal volume of 30% acetic acid to prepare the assay. Hydrolysis of • • ♦ «

The degradation product from -34, i.e. 4-nitroaniline, was determined on a Perkin-Elmer Lambda 38 UV / Vis spectrophotometer.

The absorbance of the digested samples was measured at 410 nm. Results were expressed as percent inhibition of ΒΑΡΝΑ decay relative to non-inhibitory control using the following equation:

Inhibition% = 100% X [1 - (Sinh / ^S o) S ^ l wherein the presence of the inhibitory rate of change of absorbance with time material; S _o and speed without change in absorbance as a function of time Time retardant material.

Percent inhibition was determined with a 100 kD molecular weight fraction of filtered soybean meal. This processed soybean meal fraction was prepared according to the solubilization and ultrafiltration method described in Example 3. Our results are shown in Section IX. shown in the table; the data demonstrate that the processed protein fraction examined decreased the trypsin-catalyzed degradation of ΒΑΡΝΑ.

IX. spreadsheet

Reduction of ΒΑΡΝΑ trypsin-catalyzed degradation by a 30 kD to 100 kD molecular weight fraction of processed soybean meal

Inhibitory Concentration (mg / ml)

Inhibitory protein concentration (mg protein / ml) Inhibition%

0.5

0.41 • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · »

Example 18

Other fractionation and enzyme treatment procedures

Proteins and peptides are characterized by molecular weight fractionation, 1-8. By varying the methods described in Examples 1 to 8, any desired molecular weight fraction was prepared by appropriate selection of the membranes used for ultrafiltration and dialysis. Molecular weight fractionation was also performed by gel chromatography.

4-7. The enzymatic treatment described in Examples 1 to 5 was also performed with a single proteolytic enzyme or with different combinations of proteolytic enzymes, with simultaneous or sequential action. Various proteolytic enzymes such as, but not limited to, trypsin, chymotrypsin, elastase, carboxypeptidase, aminopeptidase, pepsin and collagenase were used.

1-8. The fractionation and enzymatic treatments described in Examples 1 to 5 were applied to various proteinaceous materials of animal or vegetable origin. Examples of such proteinaceous starting materials include, but are not limited to, soybean meal, soy protein, wheat gluten, almond powder, hazelnut powder, casein, and fish protein.

Example 19

The ability of the protective agent to dissolve rapidly and to act immediately upon release in vivo is an important factor in the performance of the degradation agents of the present invention. To this end, in vitro * ·

We compared the effect of soybean meal and 30 kD - 100 kD, decanted and ultrafiltered soybean meal on the decomposition of terlakirene under dynamic conditions. The in vitro method described in Example 8 was followed except that the test solution contained only enzyme and terlakiren in buffer. The inhibitory substance to be tested (soybean meal on the one hand and decanted and ultrafiltered soybean meal with a molecular weight of 30 kD to 100 kD) was added at 0.01 mg / ml without further stirring while the solution was shaken at 37 ° C in a water bath (5). , model YB531). Samples were taken after 19 seconds and 1 minute, then at 2 minute intervals until 11 minutes, then at 5 minute intervals for a total of 46 minutes. Stopping the process, HPLC analysis, and data analysis were performed as in Example 8.

Table X.

The inhibitory substance tested 0.01 mg / ml k * (Mmol / min) Inhibition% soy flour 2.96 x 10 ⁴ 27.1 Soya flour decanted and ultrafilter, 30 kD - 100 kD molecular size fraction 0.86 x 10 " ⁴ 78.8

However, the present invention has been described with reference to preferred embodiments thereof, which are currently provided by the inventors

37, it is understood that various changes and modifications, which are obvious to those skilled in the art, may be made without departing from the scope of the invention.

Claims (20)

Claims A pharmaceutical composition comprising a protective agent and a biologically effective amount of a pharmaceutical agent labile to proteolysis, with the proviso that when the protective agent is soybean meal, the therapeutic agent is other than insulin. Pharmaceutical composition according to claim 1, characterized in that it contains as a protective substance a protein, a peptide, a purified protein of natural origin, a molecular weight fractionated protein, a solvent extracted protein or a partially hydrolysed protein. Pharmaceutical composition according to claim 1, characterized in that it contains a partially hydrolyzed protein which has been previously hydrolyzed enzymatically. Pharmaceutical composition according to claim 3, characterized in that the hydrolyzing enzyme comprises trypsin, chymotrypsin, elastase, carboxypeptidase, aminopeptidase, pepsin or collagenase. Pharmaceutical composition according to claim 1, characterized in that it contains partially hydrolyzed molecular weight fractionated protein having a molecular weight greater than 1000, as a protective agent. 6. A pharmaceutical composition according to claim 1, wherein the protective agent is partially hydro-, -, -, -, -, -, -, -, -, -, -. -39 lysed molecular weight fractionated protein having a molecular weight of 1000 to 30,000. 7. A pharmaceutical composition according to claim 1, wherein the protective agent is soy flour, soy protein, wheat gluten, almond flour, hazelnut flour, casein or fish protein. Pharmaceutical composition according to claim 1, characterized in that it contains a protein derived from a protein of natural origin or a partially hydrolysed protein by molecular weight. 9. The pharmaceutical composition according to claim 1, wherein the pharmaceutical agent labile to proteolysis is calcitonin, prolactin, adrenocorticotropin, chirotropin, growth hormone, gonadotropic hormone, oxytocin, vasopressin, gastrin, tetragastrin, pentagastrin, glucagon, gonadotropin, immunoglobulin, leuprolide, luteinising hormone releasing hormone, enkephalin, cholecystokinin, follicle stimulating hormone, interferon, interleukin, thymopentin, endothelin, neurotensin, insulin, insulinotropin or terlakir. 10. A method of increasing the bioavailability of a pharmaceutical agent labile to proteolysis, comprising administering to a mammal in need of such a pharmaceutical agent an amount of a safener sufficient to increase the bioavailability of the pharmaceutical agent, provided that the soy meal, then the cure * · ♦ · * · «· V • · · · • <» · * · · < ···· 4 active ingredients other than insulin. 11. The method of claim 10, wherein the protective agent is a protein, a peptide, a purified protein of natural origin, a fractional molecular weight protein, a solvent-extracted protein, or a partially hydrolyzed protein. 12. The method of claim 10, wherein the partially hydrolyzed protein which has previously been enzymatically hydrolyzed is added as a protective agent. 13. The method of claim 12, wherein the hydrolyzing enzyme is trypsin, chymotrypsin, elastase, carboxypeptidase, aminopeptidase, pepsin or collagenase. 14. The method of claim 10, wherein said hydrolyzed protein having a molecular weight greater than 1000 is added as a protecting agent. 15. The method of claim 10, wherein said hydrolyzed protein is a partially hydrolyzed protein having a molecular weight of between 1,000 and 30,000. 16. The method of claim 10, wherein the protective agent is protein or partially hydrolyzed protein derived from soybean meal, soy protein, wheat gluten, almond meal, hazelnut meal, casein or fish protein. 17. The method of claim 10, wherein the protective agent is a naturally occurring protein or a partially hydrolyzed protein. 18. The method of claim 10, wherein the therapeutically labile drug is calcitonin, prolactin, adrenocorticotropin, thyrotropin, growth hormone, gonadotropic hormone, oxytocin, vasopressin, gastrin, tetragastrin, pentagastrin, glucagon, secec , gonadotropin, immunoglobulin, fibrinogen, leuprolide, luteinising hormone releasing hormone, enkephalin, cholecystokinin, follicle stimulating hormone, interferon, interleukin, thymopentin, endothelin, neurotensin, insulin, terrine, insulin. 19. The method of claim 18, wherein the therapeutically active drug is terlakiren. 20. The pharmaceutical composition of claim 9, wherein the pharmaceutical agent is terlakiren as a labile drug for proteolysis.