Liposomal composition of human calcitonin gene peptide and its preparation

 

(57) Abstract:

The invention relates to medicine. The pharmaceutical composition CCGP contains liposomes from natural soy phospholipid in a weight ratio of CCGP with phospholipid 1-2 to 100-8000. The half-life of the composition over 72 h and it also has a longer resistance. The composition can be administered intravenously by spraying the mouth and nose in an amount of 0.1-10 PG CCGP per kilogram of body weight for the treatment of hypertension and congestive heart failure. Bioavailability is approximately 80%. The invention provides effective prevention and treatment of cardiovascular diseases. 4 C. and 4 h.p. f-crystals, 8 PL.

The invention relates to liposomal complex composition of human calcitonin gene peptide (CCGP) and the receipt thereof, in particular to the product obtained by combining phospholipid with CCHP.

Human calcitonin gene peptide-type (CCGP) is an endogenous neuromodulator to the present time known as the most effective vasodilator agent. His product is delivered to the world market. But, like other peptides, CCHP is the difference neustoichivost this peptide is difficult to use as a drug for clinical use.

To increase the possibility of using CCGP as clinical drugs, the basis of the invention is the creation of liposomal CCGP and how to obtain it, due to the connection of phospholipids with CCGP to get a very stable and effective product. Liposomal CCGP can allocate CCGP of liposomes gradually, to ensure a lasting effect with a half-life of 72 min in vivo, which makes it useful for the prevention and treatment of cardiovascular diseases.

The invention

To achieve the objectives of the proposed pharmaceutical composition CCGP containing liposomes obtained from natural soy phospholipids. It differs in that the weight ratio CCGP soya phospholipids is 1-2 to 100-8000, preferably 1.5 to 2 to 2500-6000.

Most preferred is a pharmaceutical composition liposomal CCGP containing 20-2000 PG CCGP 5 ml of the composition.

In the above composition liposomal CCGP you can add mannitol, sorbitol, isotonic saline and dextrose or other suitable for pharmaceutical purposes the materials.

Also proposed is a method of obtaining f is fillerbunny water for cleaning and drying of soybean phospholipids when the weight ratio of lipid to water more than 1 to 1000, then perform destruction with ultrasound to obtain small odnoobraznyh bubbles lipid bilayer;

(2) mix CCGP dissolved in H2O in the ratio of peptide to H2About 1: 1000-25000, with these soy phospholipids in a ratio of peptide to lipid 1-2:100-8000, preferably 1.5 to 2:2500-6000 perform destruction with ultrasound and incubated at 37oC for 30-60 min to obtain a stable liposomal composition CCGP.

Thus obtained composition liposomal CCGP can optionally be lyophilized, and then dissolved in H2About to obtain an aqueous solution containing 20-2000 PG CCGP 5 ml.

Also, a method for treating hypertension and congestive heart failure person by assigning the patient the above pharmaceutical composition liposomal CCGP. This method includes intravenous and spraying the mouth or nose. Intravenous liposomal CCGP its dose is 0.1-10 PG CCGP per kilogram of body weight. The bioavailability of liposomal CCGP is approximately 80%.

The invention is further described in more detail.


(1) 8 of 37 amino acids CCGP are polar amino acids with hydrophilic side chains, and 16 of 37 amino acids are non-polar substances with hydrophobic side chains;

(2) 4 of 8 polar amino acids are basic amino acids with positive charges in the H2Oh, and pI CCGP equal to 10. One molecule CCGP contains 2 argininemia, 2 lysine (Lys) and 1 aspartic (Asp) acid. While Arg and Lys have a positive charge, a Asp - negative at physiological pH. CCGP ratio ( Lys + Arg)/( Glu + Asp) = 4 is very strong basic peptide. At physiological pH CCGP positively charged three positive charges. CCGP contains 16 of hydrophobic amino acids and 6 hydrophilic amino acids and is typical ampelinus molecule.

By analysis of phospholipid composition, in particular of soybean phospholipid, were as follows:

(1) acidic lipids with negative charges in the head groups in the H2O make up more than 40% of the total phospholipid,

(2) unsaturated fatty acids in soybean phosphoglyceride approximately 70% and provide a protective effect against oxidation and hydrolysis,

(3) at extremely low concentrations of lipid (in the weight of the full-time bilayer vesicles of soybean phospholipids. The size of these bubbles ranges from 20 to 50 nm.

According to the invention, components of soybean phospholipids were analyzed by thin-layer chromatography and gas chromatography, corrected and quantitatively analyzed in comparison with standard phospholipids (Sigma). Soy phospholipids used for clinical injections contain 44.9% of acidic phospholipids containing phosphatidylserine (17,2;), phosphatidylglycerol (8,1%), pohsphatidylserine (15.2%) and cardiolipin (4,4%) rich in negative charges in the H2O, linoleic acid (58,31%), palmitic acid (24,36%), linolenic acid (7,32%), oleic acid (5,9) and stearic acid (3,88%), consisting of 71,53% unsaturated fatty acids and 28,47% saturated fatty acids (). As noted above, liposomal structures are formed spontaneously in the H2O phospholipid molecules from a variety of phospholipids, and the most commonly used composition is a natural phospholipid extracted from the cell membrane, such as soybean phospholipid (Imperial Chemical Industrial Ltd. and National Research Development Corporation, patent UK 1523965, 1977). Small odnomomentnye liposomes have a diameter of approximately from 200 to 500 and consist of one lipid is the missing:

(1) the absence of osmotic sensitivity

(2) about 70% of all lipids are located in the outer petal bubble

(3) at extremely low concentrations of lipids thermodynamically stable state is the variance odnoobraznyh bubbles double layer of lipids (Gruler H., Microstructure and transport properties of bubbles with one shell and monolayer lipid mixtures and lipid-protein compounds, Liposomal drugs and functions of immune cells, edited by Claude Nicola, 1981, 99 15-27);

(4) medium to large liposomes (MLV and LUV) is rapidly cleared in the circulation after/in reception, with small single-layer liposomes provide the potential for a gradual release of the drug in the bloodstream and enter the tissues, in addition to endothelial cells. Special attention was paid to the liposomes as model biomembranes, which provides the possibility of using in vivo in medicine and research (Yang, F. Y. Application of liposomes in the study of biomembranes and pharmacology, SHENWUHUAXUE YU SHENWUWULI JINZHAN (Biochemistry and Biophysics, 1977, 6:36). Soy phospholipids are new lipids contained in the biomembrane, which was used for artificial IU the-WULI JINXHAN (Biochemistry and Biophysics, 1978 4:1).

With the destruction of ultrasound and incubation can get the opportunity at the expense of sufficient interaction CCGP and lipid molecules to have their polar groups of the outer surface of the membrane as a result of ionic bonding with H2O. Negatively charged groups of the phospholipid head associated with the positively charged groups of amino acid residues CCGP. Apolar groups are located in the hydrophobic region of the membrane due to hydrophobic forces, including the tails of phospholipids and hydrophobic amino acid residues CCGP. When this is achieved thermodynamic stability of liposomal CCGP with increased half-life from 9-12 to 72 min and long-term storage in aqueous solution within two years (the initial retention time was 15 days). Significantly reduced effective dose liposomal CCGP (up to 10-5), which can be absorbed by introduction through the mucous membrane of the mouth, nose and rectum, with a bioavailability of about 80%. Clinical trials showed a marked therapeutic effect of liposomal CCGP on 200 patients with hypertension and congestive heart failure, without any side effects.

When is omashu rotary evaporators model XZ-6, Zhongkeyuan Kelong Corp. from a solution of chloroform/methyl (with a volume ratio of 2:1) to obtain a thin film plated on the walls of the flask with a round bottom with a capacity of 1000 ml.

After removal of the last visible traces of solvent were evaporated with rotation for 15 min, then dried for another 15 min in nitrogen atmosphere. Lipid suspended in 250 ml of distilled water by shaking with a few glass beads in a shaker (HZS-D, manufactured by Harbin Donglian Corp.), and then was treated with an ultrasonic disintegrator (DF-6P3 c, produced by Ningbo Xinyi Research Institute) for 30 minutes

Recovery CCGP in liposomal membrane

10 mg CCGP was dissolved in 250 ml of distilled water and stirred for 5 minutes the Solution CCGP was mixed with the above-described liposomal solution (250 ml) was stirred for 5 min and subjected to destruction by ultrasound in 2-3 min, three times with an interval of 3-5 min (ultrasonic disintegrator DF-6P3 c, produced by Ningbo Xinyi Research Institute). The mixture is then incubated at 37oC for 40 min (using sonic disintegrator at bath, manufactured by Harbin Donglian Corp.).

The deposition of liposomal CCGP

The recovered solution was besieged with POM is therouanne water.

Lyophilization and dissolution

Besieged liposomal CCGP was subjected to lyophilization using lyophilizate LGJ, Instrumental produced by the plant at the Academy of military medical Sciences (China) and dissolved in distilled water (passing through 6 sterilizing filters) (lipid:H2O = 1:1000), then the resulting solution was sterilized at 100oC for 30 min and was closed.

Example 2

Repeating the procedure described in example 1, except that used other weight ratios of peptide : lipid. Compared restore CCGP the membrane, the efficiency of the recovery and stability of the finished product, as shown in table 1 at the end of the description.

Methodology: liposomal CCGP was prepared according to the procedure described in example 1, with different weight ratios of peptide:lipid, after which it was centrifuged and determined the content of CCHP in the supernatant as a free CCGP without recovered in the membrane. After that, each group of the samples was divided into two groups: one was stored at -70oC in nitrogen after sterilization and sealing as a control sample, and the second was dissolved in water in a weight ratio of lipid:water = 1:1000 and is the overall effect of samples in % of the control sample. Each indicator is the average of UP to (standard deviation) for five independent data.

From table 1 it is seen that if the ratio of lipid:peptide above 1000, CCHP recovered in the membrane, leaving a small amount of free CCGP, and the vasodilator effect remains above 95% after storage for 2 years.

Example 3

The following test was repeated a procedure similar to example 1, except that used a different ionic strength for studying the effect of ionic strength in the solution on the efficiency of recovery CCGP in the lipid membrane. In table 2 at the end of the description shows the effect of ionic strength on the recovery CCGP.

Methods: in an aqueous NaCl solution of different concentrations was restored CCGP in the membrane of soybean phospholipid, after which it was centrifuged and determined the content of free CCGP in the supernatant, as % of total CCGP. The weight ratio of peptide:lipid was 1:1000, and each indicator is the average of UP to five independent data.

Table 2 shows that the efficiency of recovery decreased with increasing ionic strength in solution. The purified solution of H2O can provide time favorable the measures 1

Repeating the procedure of example 1, except for using two uncharged lipids instead of soy phospholipids.

By determining the content of free CCGP in the supernatant after centrifugation and chromatographic analysis, it was proved that the efficiency of recovery CCGP in the membrane of soybean phospholipids was 99,9, and in the membranes of PC and PE only 21,2 and 30.3, respectively, indicating thereby that the negatively charged phospholipids are very important for a successful recovery CCGP in the membrane (table.3, at the end of the description).

Comparative example 2

For the preparation of liposomal CCGP used three different phospholipid - soybean phospholipid (SP), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) and analyzed the structural integrity CCGP in three liposomal membranes during storage for 24 months using liquid chromatography high resolution (IHVR), as shown in table 4 at the end of the description.

Methodology: CCGP sample were extracted with acidic solution and analyzed using GHUR reverse phase, while during the chromatography was detected retention time and peak area CCGP. Each indicator is the average of UP to platinum, Switzerland).

The results in table 4 show that after storage for 24 months there has been a slight change of purity CCGP 1.4 %, testified that CCGP, recovered in the membrane of soybean phospholipid, is very stable during storage and membrane PE and PC unstable and integrity CCGP remained only at the level 64,4 and 65,1%.

Comparative example 3

In the following test for the preparation of liposomal CCGP and comparison with liposomal CCGP from soy phospholipids used phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Determined the vasodilator effect of three different liposomal CCGP and compared, as shown in table 5 at the end of the description.

Methods: experiments were performed on new Zealand white rabbits weighing 2.5-3.5 kg, which was anestesiologi the sodium pentobarbital (30 mg/kg, in/in).

Rabbits were placed in a fixture for fixing the head and measured the diameter of the ocular vessels (conjunctiva) using a microscope equipped with a camera connected to the video monitor. Images were recorded in the computer and subsequently the diameters of the vessels were measured using the software image analyzer. System for the analysis of the microcirculation of the eye vessels recorded in the computer.

The values of the ED50(x1018mg/ml) vasodilator effect of liposomal CCGP was measured during storage for 24 months. Small changes soybean phospholipid liposomal membranes CCGP decreased 100-1000 times 24 months, indicating that the negative charges in the lipid membrane is very important for a stable connection between the peptide and lipid.

Experiment 1

Analysis of complex CHKP with liposomes

Methodology: liposomal CCGP and free CCGP analyzed according to the method of Burke (Burke J and Marcinka K., Gel-chromatography in separation methods. Ed. Dales, 1984, 217). Samples CGK restored with soybean phospholipid or without (1.0 ml in 0.1 M Tris-HCl, pH 8,8) were placed in a thin column size 1.5 x 46 cm (Sephadex G-50) in 0.1 M Tris-HCl, pH 8,8. Blue dextran 2000 (Pharmacia) and32PO4(England) was mixed with a separate sample label for Vo and Vi, respectively.

Free CCGP in a thin column (Sephadex G-50) for the chromatographic had Kd = 0,52, and liposomal CCGP - Kd = 0,44, as well as blue dextran 2000, indicating that the formed complex liposomes-CCGP. Data obtained by gel filtration, shown in table 5.

Each indicator represents the result of a separate ptx2">

From table 6 it is seen that after recovery CCGP soybean phospholipid formed much more complex CCGP with lipid than CCGP, i.e. properties CGCP and soy phospholipid provide new opportunities for sustainable liposomal CCGP.

Experiment 2

Analysis of physical and chemical stability of liposomal CCGP

It is clear that any liposomal formula must have the appropriate stability during the time interval between preparation and the ultimate use to be a pharmaceutical carrier. The surface of the liposomal membrane, as mentioned above, has a large number of negative charges, which prevent resizing induced fusion of liposomes. Surrounded by a large quantity of water is negatively charged particles are thermodynamically stable state and a large number of unsaturated phospholipid tails can reduce the likelihood that the water molecules in the double layer of lipid, preventing degradation of the lipid molecules and the peptide as a result of autohydrolysis and autoxidation.

Phospholipids undergo hydrolysis in aqueous medium, which leads first to the formation of the corresponding is pholipid was defined as the criterion for chemical resistance using thin-layer chromatography (TLC), and the size of the liposomes was measured using gel filtration, watching the position of the peak elution as a criterion of physical stability (Szoka F., et al. Comparison of the properties and methods of making lipid vesicles (liposomes), Ann. Rev. Biophys. Bioeng. 1980 9:467.5).

The analysis of the content of LPC

Changes in the content of LPC in soybean phospholipid liposomes. Storage conditions: at 25oC, the samples in H2O, the weight ratio of lipid : H2O = 1:1000, sterilization at 100oC for 30 min and sealed.

Method: content analysis of LPC in the liposomes was performed using TLC, silica gel H, type 60) was obtained from company E, Mere, Germany. Standard LPC was supplied from Sigma as a control LPC samples, its Rf value was 0.04 in experimental conditions. After that, the samples were sterilized at 100oC for 30 min, the chemical stability of the liposomal membrane was determined using the analysis of the content of LPC with an interval of 3 months during storage. Each indicator is a weighted average with UP to 5 independent TLC (i.e., n=5). The mixture of sample and standard LPC used for remedial analysis using one-way TLC and bidirectional TLC to show that LPC in the mix was the only component on silicagel what's phospholipid vesicles and from 1,90,22% to 3,40,46% (p<0,01) for liposomal CCGP respectively. The percentage decomposition of phospholipid molecules was of 2.6 and 1.5% for liposomes and liposomal CCGP respectively, indicating that the recovered CCGP can improve the stability of the membrane due to their positively charged groups, which interact with negatively charged groups of the phospholipid.

The size of liposomes was determined using gel filtration (Kd) during storage. The values of Kd has not changed for soybean phospholipid liposomes or reconstituted liposomes with CCHP, testifying that the proposed liposomes thermodynamically stable in an environment with a weight ratio of 1000:1 (H2O : phospholipid) within 24 months of storage after sterilization.

Experiment 3

The stability analysis CCGP restored in the liposomal membrane

Sustainability CCGP restored in the liposomal membrane was observed with (a) a thin gel filtration on Sephadex G-50 for measuring the dissociation CCGP from liposomes; and (b) systems monitoring microcirculation to see the vasodilator effect, and also in comparison with the free CCGP during storage.

A. the Stability of the connection CCGP with the membrane (table 8, at the end of the description).

Methods: in the process Granny from liposomes were determined using gel filtration through every 3 months. Absorption CCGP in ultraviolet radiation 206 nm was 0.52 distribution coefficient (Kd) for the correction to the standard CCGP. When this value of Kd, the authors were able to study, dissociated whether CCGP recovered from liposomes during storage. Was held thin gel filtration on Sephadex G-50, each indicator is the average with UP to three independent measurements.

When stored for 24 months CCGP restored in the liposomal membrane, not dissociable in free CCGP while monitoring the absorption at 206 nm) lentago solution at Kd=0,52. This result showed that CCGP can form a very stable complex with a soy phospholipid vesicles using the proposed experimental procedure due to the characteristics of their molecular structure. All samples were stored at 25oC after sterilization and sealing. The ratio of lipid : H2O is 1:1000 in the restored soy liposome phospholipid.

C. Measurement vasodilator effect CCGP

CCHP is an endogenous neuromodulator and the most effective vasodilator agent that is known ABT the lipid, and the comparison with the free CCGP in H2O and plasma during storage (table 7,at the end of the description).

Methods: experiments were performed on new Zealand white rabbits weighing 2.5-3.5 kg, which was anestesiologi the sodium pentobarbital (30 mg/kg, in/in). Rabbits were placed in a fixture for fixing the head and measured the diameter of the ocular vessels (conjunctiva) using a microscope equipped with a camera connected to the video monitor. Images were recorded in the computer and subsequently the diameters of the vessels were measured using the software image analyzer. System for analysis of microcirculation were acquired in the company DAHENG Co. , China. 10 Ál of the diluted samples was buried in the eyes of the rabbit and the image of the ocular vessels recorded in the computer. The diameter of the vessels in the images were analyzed using terminal vascular program.

IN H2O vasodilator effect ( % of diameter) free CCGP and CCGP restored in the liposomal membrane of soybean phospholipid, was changed from +1878,9% to +8312,4% (p<0,001, n=5) and from +19816,4% to +19614,3%, respectively, after storage for 90 days. In plasma, their efficiency was decreased from +19516,49% to +256,8% (p<0,001, n= 5) and from +19819,2% to +1846,1% soo is me, the membrane is more stable in comparison with the free CCGP.

Therapeutic experiment 1

The role of liposomal CCGP in the treatment of patients with congestive heart failure (CHF)

Patients: the study was conducted on 16 patients with CHF - seven men and nine women, mean age 66.3 years (from 54 to 75 years). Six of them had stage IV according to the new York health Association (NYHA), seven - III stage and three - stage II (Bruce R. A. Mod. Concepts Cardiovasc. Dis. 1956, 25: 321-326). All patients received liposomal CCGP stopped taking other medicines, such as digoxin (digoxin) for three days.

Drug: liposomal CCGP prepared in accordance with example 1, was used for the treatment of patients containing 20 PG CCGP 5 ml. The contents of the medicine 2000 BU/5 ml

How to use:

Through the mucous through the mucous membrane of the mouth and nose 40-80 BU (1-3 drops) three times a day, through the rectum 2000 BU 3 times a day.

Intravenous: 2000-8000 BU (2-4 capsules) liposomal CCGP was added to 5% GS or 100-250 ml of 0.9% NaCl solution, 1 time per day.

Measurements: respiratory rate, vesicular tone, frequency and rhythm of cardiac contractions, the magnitude of the liver, the degree of swelling, weight, urine volume and cardiac activity using ECG was measured before and after drug administration. Eyed the nutrient effect on patients with CHF (table. 8). The majority of patients had symptomatic improvement the next morning. 9 patients were observed dominant improving cardiac activity, 6 - effective, and only one was not marked changes. None of the patients complained of side effects such as headache, hot flushes. The medicine did not cause hypotension and did not have an impact on liver function or renal function during treatment.

Discussion: congestive heart failure (CHF) is usually caused by reduced cardiac ejection in the narrowing of the myocardium, the improvement which is the main aim in the treatment of patients with CHF. Calcitonin gene peptide (CSB) is a neuropeptide that is able to provide a vasodilator, positive chronotropic and ionotropic effect on the heart, allowing it to be used for the treatment of CHF. Recent studies have shown that intravenous administration of CSE (to 8.0 ng/kg/min) for 8 h causes a decrease of pressure in the right artery, pulmonary artery, pulmonary arterial wedge also systolic pressure.

This increases cardiac output, stroke volcermt, renal blood flow and glomerular filtration. Treatment of 16 patients with CHF p is) CCGP gradually released from liposomes, providing long-lasting effect, the average duration of 10 h, 5 times higher than known from other sources the results of applying CCGP,

(2) easily absorbed by the mucous membrane of the mouth, nose and rectum,

(3) the bioavailability of liposomal CCGP 10 times higher than known from other sources.

Therapeutic experiment 2

The role of liposomal CCGP in the treatment of patients with hypertension

Materials and methods:

Patients: 21 hospitalized patients with hypertension, 10 men and 11 women, mean age 62.2 years (from 45 to 73) with hypertension, one was aldosterone. Duration of disease ranged from three to 37 years, clinical information was given a clear diagnosis. According to the diagnostic standard of hypertension WHO/ISN 1993 (Beijng Renmin Weigheng Chubanshe 1996, 227-228) 11 patients had stage (?), and 10 patients in the third stage.

Drugs and dimensions: 16 patients stopped taking other anti-hypertensive drugs for two weeks, five received bepridil (mepramidil) and carvedilol (carvedilol). Liposomal CCGP, prepared as in example 1, was administered to all patients intravenously or through the mucous membranes of the nose or the mouth.

A. Through SL is">

B. Intravenous: one vial of liposomal CCGP containing 20 PG CCGP in 5 ml of an aqueous solution, were injected with 100-500 ml of 0.9% NaCl solution per day for five days.

Century Measurement: blood pressure (BP) was measured through 15,30,60,120,180 min after administration of the first day. In the following days the AD was measured 6 times a day before and after administration.

The definition of hypotension:

Hypotension liposomal CCGP was determined in accordance with the diagnostic standard 1979 cardiovascular epidemiology (Henan, Zhen Zhou, China) (J. Cardivascular Diseases 1979, 7, (2),18).

The dominant result: reduced diastolic pressure >100 mm RT.article and to a normal level, or only >20 mm RT.article.

Effective result: reduced diastolic blood pressure by 10 mm RT.article and to a normal level or 10-19 mm RT.article.

No result: diastolic blood pressure dropped to normal levels or decreased to <10 mm RT.article.

Patients who had only increased systolic pressure, hypotension was defined by the above standard plus the decrease in systolic blood pressure by 20 mm RT.article.

Results: the proposed liposomal CCGP was administered to 21 patients: 4 - through the mucous, 4 - intravenous, 13 - what th - through the mucous membranes of the nose.

Result of treatment: the decrease in systolic blood pressure 20-105 mm RT.art., on average, 17 mm RT.article (p>0,001, n=21).

The decrease in diastolic pressure of 5-25 mm RT.art, on average, 17 mm RT.article ((p>0,001, n=21). Liposomal CCGP gave dominant outcome in 13 patients, effective in 7 patients without result in 1 patient. Hypotension occurred in 5 (?) after admission and continued for about 10 hours

Side effects:

2 patients with chronic runny nose felt a mild discomfort when receiving via the nasal mucosa after administration of intravenous this symptom disappeared. While receiving liposomal CCGP was not observed headache and tides of blood and pathological changes of the liver and kidneys.

Discussion

1. MSE is an endogenous neuropeptide. Proposed liposomal CCGP eliminates the rapid decomposition of the MSE and to ensure long-lasting effect, due to the gradual release CCGP in vivo, making it easily absorbed by the cells of the tissue. In the treatment of hypertension he works quickly, efficiently and reliably. Of the 21 patients dominant result was 61.9 per cent, effective result, 95.2% and only one patient was not from the intravenous or through the mucous membranes were observed.

2. In some sources it is the dependence of the hypotensive effect of CSE on the dose animals, and the efficiency increases with increasing dose. However, 21 patients in the described experiment, the optimal efficiency hypotension has been observed when taking 40-80 BU liposomal CCGP through the mucous membranes and further increasing the dose did not lead to improved results that can be explained by the increase in cardiac output, caused a positive ionotropic effect CCGP heart.

3. Shekgar et al. (Shekhar YC et al, the Influence of continuous infusion of human calcitonin gene alpha-peptide on hemodynamics, renal blood flow and hormone levels in congestive heart failure. Am J Cardiol 1991; 67:-733) reported that intravenous CCGP (8,0 ng/kg/min) for 8 h for a total dose of 3840 ng/kg causes hypotension and reduces blood pressure by 18% (p<0.05) after 30 min after administration. In this experiment, anti-hypertensive dose was 8000 BU in the day when the content CCGP only 780 PG, i.e. 0,8 PG CCGP per kilogram of body weight that is 2.0 x 10-7the dose CCGP, reported by other sources.

4. There are no significant side effects, only 1 pacido nose. Therefore, patients with diseases of the nose should appoint another way of receiving liposomal CCGP.

1. Pharmaceutical composition having vasodilating action, on the basis of human calcitonin gene peptide (CCGP), characterized in that it is made in the form of liposomes from natural soy phospholipid and CCGP, and the weight ratio CCGP soya phospholipid is 1 - 2 100 - 8000.

2. The pharmaceutical composition CCGP under item 1, characterized in that the weight ratio CCGP soya phospholipid is 1.5 - 2 to 2500 - 6000.

3. The pharmaceutical composition CCGP under item 1, characterized in that it contains 20 to 2000 PG CCGP in 5 ml of composition.

4. A method of obtaining a pharmaceutical composition having vasodilating action, based on CCGP, characterized in that in the first stage, add sterilized distilled water to the cleaned and dried soybean phospholipid in a weight ratio of lipid to water above 1: 1000, followed by the destruction of ultrasound to obtain small odnoobraznyh bubbles lipid bilayer, in the second stage mix CCGP dissolved in water in the ratio of peptide:water = 1:1000-2501,5-2:2500-6000, carry out the destruction of ultrasound and incubation at 37oC for 30 - 60 min to obtain a stable liposomal composition CCGP.

5. The method according to p. 4, characterized in that in the second stage, the weight ratio CCGP soya phospholipid is 1.5-2 to 2500-6000.

6. The method according to p. 4 or 5, characterized in that it further perform lyophilization and subsequent dissolution of liposomal CCGP in water to obtain an aqueous solution containing 20-2000 PG CCGP 5 ml.

7. A method of treating hypertension through the introduction of the patient one of the pharmaceutical compositions according to PP.1 - 3 intravenous, spraying the oral mucosa, nose in an amount of 0.1 - 10 PG CCGP per kilogram of body weight.

8. A method of treating congestive heart failure through the introduction of the patient one of the pharmaceutical compositions according to PP.1 - 3 intravenous, spraying the oral mucosa, nose in an amount of 0.1 - 10 PG CCGP per kilogram of body weight.

Priority points:

29.11.1996 - PP.1 - 6;

17.10.1997. - PP.7 and 8.

 

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The invention relates to compounds of the formula I

R1-Q1-X-Q2-R2(I)

in which Q1, Q2in each case, independently of one another are either absent or represent-NH-(CH2)n-CO-,

R1, R2in each case, independently of each other either absent or represent a cyclo-(Arg-Gly-Asp-Z), where Z is linked to the side chain of Q1or Q2or, if Q1and/or Q2missing (et), with X and where at least one of the radicals R1or R2must always be present,

X represents a-CO-R18-CO-, and if R1-Q1or R2-Q2- no, there is an R10, R13, R16, Het-CO -, or the residue of a fluorescent dye that is chemically linked through CONH-, -COO-, -NH-C(=S)-NH- -NH-C(=O)-NH-, - SO2NH -, or-NHCO-bonds

Z in each case independently represents an amino acid residue or di-, tri - or tetrapeptides balance, where amino acids independently selected from the group comprising Ala, Asn, Asp, Arg, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val or M

where these amino acids can be derived and amino acid residues connected to one another like xilinix groups, and where M is always present,

M represents NH(R8)-CH(R3)COOH,

R3- R5-R4, -R6-R4or-R7-R4< / BR>
R4is a HE, NH2, SH or COOH,

R5- alkylene containing 1-6 carbon atoms,

R6- alkaliphiles containing 7 to 14 carbon atoms,

R7- alkylenediamine containing 8-15 carbon atoms,

R8-H, a or alkylether containing 7-12 carbon atoms,

A is alkyl containing 1-6 carbon atoms,

R10- alkanoyl containing 1-18 carbon atoms, which is unsubstituted or contains one Deputy from among COOH, COOA, SR11or NR12R12,

R11- H or trityl, pyridyl-2-thio - or allylthiourea containing 1-6 carbon atoms,

R12, R12'each, independently of one another, represent H, alkyl containing 1-8 carbon atoms or a protective group of amino group,

R13- aroyl, which contains 7 to 11 carbon atoms and is unsubstituted or substituted and contains one or two substituent selected from the group comprising alkyl containing 1-6 carbon atoms, alkoxygroup containing 1-4 carbon atoms, alkanoyl containing 1-8 tomsche independently of one another H or A,

R16is arkanoid, which contains 7-19 carbon atoms and which is not substituted or substituted in the aryl fragment of one, two or three deputies, including Hal, alkoxygroup containing 1-6 carbon atoms, or HE, and in which the aryl fragment may also represent a group:

< / BR>
E - CH2or,

D is carbonyl or [C(R17R17')]m,

R17R17'each independently represents H or A,

R18is absent or is an R19, R20, R19-R20-R19or phenylene, which is not substituted or substituted and contains one or two substituent R5the length of the chain which is in each case independently of each other,

R19is alkylene containing 1-8 carbon atoms, where 1 or 2 methylene groups can be replaced with S, -CH=CH - or,

R20- cycloalkyl containing 3-7 carbon atoms,

Hal is F, Cl, Br or I,

Het is a monocyclic or bicyclic saturated, unsaturated or aromatic heterocycle which contains from 1 to 4 atoms of N, O and/or S, attached cherepy, including Hal, A, R3, NR4R4', CN, NO2and/or carbonyl oxygen,

n- 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 and

m is 1 or 2,

where, assuming that residues are residues of optically active amino acids and derivatives of amino acids, contain both D and L forms, and their salts

The invention relates to inhibitors of retroviral proteases, in particular to new compounds, compositions and method for inhibiting retroviral proteases such as protease of human immunodeficiency virus (HIV, HIV)

The invention relates to gonadotropins, consisting ofand-subunits, and these gonadotropins include negativnye disulfide bridges, preferably, negativnye misbehavin disulfide bridges
The invention relates to medicine, namely to physiotherapy

The invention relates to the field of biochemical pharmacology and medicinal chemistry

The invention relates to medicine and applies ointment to heal burns and infected wounds of various etiologies, as well as for the prevention and formation of keloid scars

The invention relates to medicine and relates to methods for obtaining protein hydrolysate suppressive regulation of allergic reactions, methods of prevention or treatment of allergies
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