JPH01500114A - Increased vascular perfusion brought about by lipid administration - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] name of invention Increased vascular perfusion brought about by administration of lipids. This is a partial continuation of the accepted U.S. Patent No. 782°724.

Field of the invention The present invention provides treatment with substances that are effective in increasing angiogenesis and vascular perfusion in mammals. The invention relates to a method of promoting angiogenesis and increasing blood flow in mammals by.

Background and prior art In the parent application filed by the applicant, No. 782,724, for the creation of blood vessels. Compositions have been described that exhibit efficacy. Test methods that show such effects include CAM application. There is an assay method that uses live chicken embryos. This application is shown here for reference. However, pending application No. 642, 62 was received on August 20, 1984. It is related to 4. This application is based on a chloroform-methanol image of a retinal tissue extract. The animal model used to demonstrate the in vivo effects of ). The "cat's limb" model described there is based on the actual model described below. It is noted in the detailed description of the examples.

Around 1903, the greater retina (omentum) was identified as an ischemic area of the body. It has been reported that this drug is effective in supplying blood to patients (careas). for example Brenley et al., Berl, 1in, voch, - vol. 40; pp. 773-775. (1903): Morrison, Brlt, Med, J, vol. 1, pp. 76-78 (1 906): Willkie, Br1t, Med, J., Volume 2, 1103-1. See p. 106, (1911) 6 For example, Osha Lanecy, Brlt, J. ur, vol. 23, pp. 665-670, (1936); Later studies such as Illusion, Vol. 9, pp. 230-232 (1958) It has been shown to provide collateral blood vessels to the viscera. That by Vainberg et al. Later work (Can, Med, As5oc, J, vol. 87; 1074-76 ( 1962), Can, Med, As5oc, J, Volume 90; 717-72 2 pages (1964).

Am, Heart J, Vol. 72; pp. 79-93 (1966), Dis, Ch. est, vol. 54; 315-322 (1968) and Surgery, G. nacol, obstetrics, vol. 137; 564-578 (1973) ), the internal mammary artery is directly transplanted into the myocardium along with a piece of tissue around the coronary artery. It has been reported to treat patients with arterial artery disease.

Current interest is in using the net for central system vascular regeneration. Dosmith et al., Juku” Niroku, Vol. 88, p. 32-736 (1980), 5tr oke, Volume 9; 224-229 (1978); Am, J, Sul, Volume 1 Vol. 30; pp. 317-320 (1975); "Kashil" Etoe, Vol. 129; 2 pp.63-265 (1975) Page (1973); Rev, Surg, Vol. 24; 379-380 (1967) ), the brain is attenuated and blood vessels are formed in the brain, but there is no ischemia in these organs. Although there is no Ru. Following this, Goldsmith et al. (JAMA, Vol. 252; 2034-203 (p. 6) discovered a lipid fraction of the omentum with potential angiogenic activity.

Forch et al. (J. Biol. Chem., Vol. 226; pp. 497-509 (1) [957] describes a method for separating and purifying lipids from animal tissues. "Fuorhi References regarding ``Fuorch's fraction'' and ``Fuorch's device'' are published in this document. It shows you how to do it. Redden et al. 9-139 (1982)] on the separation and analysis of general gangliosides. It is taught in part by Williams et al. (J. Neurochem, Vol. 35; 2). pp. 26-269 (1980)] used cartons of C1m during the separation of gangliosides. Indicates the use of ridges. Cantay et al. (J, Biol, Chem, No. 25 Volume 7; 14865-14874 (1982)] describes the clichosphere of human red blood cells. Ngolipid reacts with monoclonal antibodies against the developmental antigen 5SEA-1. It shows.

However, none of the previous technical knowledge uses topical lipids or other lipids to localized blood flow to traumatized tissue that has not been achieved to generate blood flow in the blood vessels It is hoped that the flow will flow. Places where such blood flow is reduced are sometimes easily detected Yes, but usually not detectable. Find areas where blood flow is further reduced However, it is difficult to locally administer drugs that increase blood circulation and perfusion. Substances that increase perfusion systemically are often available to those skilled in the art. It is clearly not desirable, as is readily apparent.

Here, traumatized tissue in mammals compositions used to increase local perfusion to It is an object of the present invention to provide an object.

It is another aspect of the present invention to provide a method of increasing blood flow using such compositions. It is one purpose.

Specification of traumatized tissues to increase blood flow locally using systemic administration It is another object of the present invention to provide a method of delivering the above composition to the site of It is.

See below how these objects of the invention are achieved. This can be easily confirmed by this.

Brief description of the drawing Figure 1: A diagram summarizing the experimental design. The second figure from the left is an anatomical diagram of a cat's hind limb. represents the junction of the femoral artery and the lumen fold and shows the surgical portion of the Hunter duct. There is.

Figure 2: An example of an image of a cat's hindlimb using a radionucleus. The X-axis scans both sides (left and right) Note that the radioactivity counts appear as a separate peak for that limb. Be mindful. acerta in the scanned area to compare the radioactivity density in the two limbs. It was quantified by integrating the peak. Next, move the X axis to another part (A, B .

(See C, D) Repeat this operation. Figure 3 below shows how the 10 locations are summarized. are doing. Note: This cat receives angiogenic lipids daily away from the wound site. Injected intramuscularly. Nuclear scanning was performed on the third day.

Figure 3 The 10 parts of the cat's limb obtained as shown in Figure 2 were synthesized. Radioactivity density profile. The area of the peaks corresponding to the right and left limbs and the starboard (surgical) The ratio of The following "results" were obtained by statistical analysis of all values at 10 locations. The average value R (± standard deviation) is calculated as shown in .

Figure 4. Animals before and after surgery on day 0 and treated for 7 days without lipid injection. Distribution of the ratio of radioactivity density to the operated animals that were shaken (R = average ratio of all animals; A = number of animals). Horizontal bars represent ±1 standard deviation.

Figure 5: CM of the cat's omentum. Release of animals administered Fr or saline (control). Radiation density data.

Figure 6. Brain ganglioside mixture (shown in Table 1) intramuscularly administered (wound Seven days after surgery, animals were treated (away from the mouth) and animals treated normally (injected Distribution of radioactivity density ratio in (not). Horizontal bars represent ±1 standard deviation Ru.

Figure 7: Neutral lipids in the pig web and neutral lipids in the pig web and fetal bovine brain ganglio Seventh day after surgery, Sid's 1000:1 (w/w) mixture was injected intramuscularly into the wound area. distribution of radioactivity density ratio (normal distribution).

pipe na-1 The need for local tissue revascularization requires externalization of the treated portion of the femoral artery in the cat's hindlimb. It was created through medical surgery (Figure 1). Lipid substance or control substance (saline) into or around the wound (for hind limbs) or at a distance (for forelimbs). ) was injected intramuscularly. When administered to distant areas, lipids increase blood flow to the wound. It is important to know whether or not it exerts its effect by being absorbed into the overall circulation. It was administered to To demonstrate the effect of locally increasing blood flow by systemic administration directed against traumatized tissue has been well studied. Ru.

This experiment included four main steps. That is, 1), preparation of lipid components, 2) creating a standard surgical wound resulting in the need to increase blood supply; 3) , lipid or saline administration, 4) vascular perfusion This is a standard method for showing the difference in radioactivity before and after surgery.

- key The method for preparing the chloroform-methanol fraction CMFr is described in the application under review. However, I will briefly outline them here. Ketami adult cats weighing 2.5 to 3.0 kg. The animals were anesthetized with intramuscular injection (7.0 mg/kg), and then the abdomen was opened through a midline incision. . At this point the omentum was surgically removed and placed in a plastic bag pre-chilled to 4°C. It was done. Weigh the net (it weighs 30 to 60 g), spread it on the plastic surface, and then Cut into pieces approximately 4 cm2 in size and add 3 ml of phosphate buffered saline (PBS). I put it into a Waring blender with 0m Q. Waring blender and solution is pre-cooled to 4°C. 5 minutes 20. Homogenized with OOOrpm . The obtained homogenate was heated at 1600 g for 20 minutes in a 250 m Q-volume plastic plate. The mixture was centrifuged in a centrifuge tube using a centrifuge cooled to 4°C. After centrifugation, the centrifuge tube contains Three halves can be seen. i.e. sediment, cloudy homogenate and floating cream. It is a lump of gray-colored lipid. Decant the homogenate to remove lipid chunks. After removing and weighing, mix chloroform methanol mixture (2 :1, v/v). 10 mfl of solvent for 1 g of lipid mass The percentage of Particulate material was removed by centrifugation at 200g for 10 minutes at 4°C. Obtained The resulting transparent supernatant was distilled under reduced pressure at 37°C using a rotary evaporator to remove the solvent.

The remaining oily substance was called the chloroform-methanol lipid fraction (CMFr). . This material was stored at -20°C.

Adjustment of sonoya fat CMFr from pig mesh was prepared as above and dissolved in hexane. (approximately 60 mQ of hexane per CMFrlOg) and 0.66 volumes of 95% ethanol. Added tanol. The whole was thoroughly mixed and then separated. The upper layer (hexane) is Re-extracted with 95% ethanol, and the resulting lower layer was mixed with the original ethanol fraction. did. The mixed ethanol fraction was re-extracted with hexane, and the resulting hexane layer was mixed with the initial hexane layer. The hexane solvent was distilled under reduced pressure and used in this experiment. porcine omentum neutral lipids (PONL).

Bovine brain monosialo (GMI), disialo N (GDla) and trisialo -N (GTI) ganglioside was purchased from Superco and mixed in the following ratio. Ta. Tridialoganglioside-GTlb contains 10-30% GDlb. Ru.

Fetal bovine brain (within 6 months of fetal life) was used to isolate gangliosides. cow womb The infant brain was washed with saline and the brain membranes were carefully removed. Then triple the amount of brain (3 homogenized in cold acetone (volumes). The homogenate is filtered , the residue was extracted again with 3 times the amount of cold acetone. After filtration, the residue is removed by the method of Kantei et al. (1982). The residue was dissolved in 3 volumes of isopropanol. Homogenized in xane-water (55:20:25) and then stirred overnight at room temperature. Ta. The homogenate was filtered and the residue was re-extracted with 3 volumes of the same mixture. this It was filtered again and the two filtrates were combined and dried on a rotary evaporator. lipid residue The residue was dissolved in chloroform-methanol (2:1) and the method of Forch et al. (195 7), i.e. by adding 115 volumes of water and mixing thoroughly. . The upper layer is removed and the lower layer is the theoretical upper layer i.e. chloroform-methanol-water (3: 48:47) was re-extracted twice.

The upper layer was brought to a final concentration of 0.1 MKCQ and then processed using the method of William and McClure (1 The mixture was applied to a reversed phase C column according to 980). Gangliosides are metabolized after desalting. The column was eluted with alcohol. After drying the sample and treating with 0.3M alkali, vinegar Neutralize with acid and 01.0 as described above. Desalting was performed again using a column. Neutral and acidic The methanol fraction containing lycosphingolipids was dried.

Ion-exchange chromatography for neutral glycosphingolipids and gangliosides It was separated using The mixture was dissolved in methanol and recorded by Ready and Nee. (1982) on a DEAE-Sephadex column. During ~ The glycolipids were eluted with 3 volumes of methanol. total gangliosides The mixture was extracted from the anion exchange resin with 0.5M ammonium acetate (in methanol). Served. Ganglioside fractions were dried and desalted as described above.

Both components were redissolved in methanol and a portion was transferred to a Merck IPTLC plate. chloroform-methanol-〇, 25% CaCQ, (55:45: 10). Gangliosides were colored with resorcinol reagent. child The main component of the mixture is GDla.

GTlb, GMI, GDlb, GD3. GM2. GM3. GO Ru.

Standard fucked mouth Bendoparbitol soda (33mg/k) was administered to male and female cats. g) was administered intraperitoneally for anesthesia. After anesthesia, a vertical skin incision is made on one hind leg. The procedure was performed from the groin to the lower part of the thigh above the large blood vessel. During this experiment , animals were operated on alternating right or left hind limbs.

Fragment of the femoral artery from the groin to the lower end of the Hunter's canal at the bifurcation of the femoral artery has been removed. The separated ends of the femoral artery were joined with 3-0 silk suture. then the wound The femur was washed with saline and then separated using a 3-0 chromium sheep intestine suture. The muscles lining the arteries were loosely closed. The femoral artery in the limb that did not undergo surgery has a It was used as a control for comparison of radionuclear images.

Seed I fifth step star Lipid fractions were administered intramuscularly with a t119 gauge needle. The injected substance is injected into the needle. The substance was warmed to 37°C immediately before administration to reduce the viscosity of the substance for better passage. . In some cats, intramuscular administration was performed at and around the incision, while in other animals Injected intramuscularly into the forelimb. 3 cc of lipid fraction dissolved in PBS or neat ( (See details in the results below) was first administered, followed by 1 cc injection per day for 6 to 7 days. I attached it. Control cats received intramuscular saline in the same manner as lipid administration. all In other cats that did not undergo surgery, the injected lipids caused damage to the blood vessels even if the wound was intact. The femoral muscle adjacent to the unoperated femoral artery to know whether to stimulate perfusion was administered.

In this experiment, regional vascular perfusion of the hindlimb was performed. rfusion) allows cats to view images with a gamma camera connected to a computer. displayed by labeling red blood cells with technetium (Tc-99). . Nuclear images of the animal's hindlimb were taken on day 08 (just before the removal of the femoral artery fragment) and on days 3 and 6 after surgery. It was held on the 7th and 9th. Some cats had nuclear scans on all these days, but the strategy or due to scheduling or technical computer difficulties scanning these days It was not possible to do this for all cats. However, on the aforementioned day Image observation was not necessary in this experiment. Because there is only one result This is because it is based on the average ratio (R) of blood flow in one limb compared to that of the other limb. The ratio would be valid at that time.

Cats were given anesthesia by intraperitoneal administration of bendoparbitol sodium (33 mg/kg). I got drunk. Administered as stannous pyrophosphate (pyrolyte) for nuclear imaging specimens. to expose a suitable blood vessel for intravenous injection with 500 mg of stannous ion. A small skin incision was made on the forelimb. After 20 minutes, 10 mC1 of technetium (Tc -99) was administered intravenously. Animals were placed on a computer system immediately after Tc-99 administration. Images were taken from the front and back using a parallel gamma camera. Scanned images of each limb ( 5×10′ integrated counts/image) were saved for later data analysis. re-drawer In each image, a computer program (second (Fig.) was analyzed using the ``area of interest'' technique. The X-axis runs through both hindlimbs. The counts (Y-axis) are plotted in a narrow (3 pixel) horizontal channel. This is illustrated in the form of a radioactivity density profile. Specific areas of right hindlimb and left hindlimb The area of each peak representing the total counts for each part of the intercept is integrated and The fractional ratio (r) was calculated between the limbs that underwent medical surgery and the limbs that did not undergo medical surgery. . Such radioactivity density profiles were obtained at 10 locations equidistant from each hindlimb. Rare.

The average R (standard deviation) of each scan was calculated from the values (r) of each section (Figure 3).

The average (R) value derived from each radioactivity density profile is This served as the basis for evaluating the effects of lipid factors on the network. The radioactivity density is the blood present in the hind legs. This directly reflected the pipe flow.

blue fruit The average ratio of the radioactivity density profile of the hindlimb (Rth standard deviation) is the local reflected the perfusion of target blood vessels. An average ratio (R) of 1.00 means that between the two hindlimbs shows that equal radial densities and therefore equal vascular perfusions. On the other hand, 1.0 An average ratio (R) higher or lower than 0 indicates increased vascular perfusion in the limb compared to the contralateral limb. It shows whether it is present or decreasing.

6 about the front baseline The average ratio (R) of the radial density profile of the hindlimb taken on day ○ (immediately before surgery) is It was 1.045±0.11. This value indicates that the radioactivity density profile is individual. The 16 animals showed that the vascular perfusion between their hindlimbs was very similar. (Figure 4).

baseline of The average ratio (R) taken on the 8th day immediately after surgery was 0.93 ± 0.12 (4th day). figure). Eleven cats were used in this study. Therefore, the femoral artery fragment is surgically removed. As expected, there appears to be a slight decrease in blood flow locally.

Mad Zoku Immunity Officer Twelve cats were operated on and healed for 7 days without any injections.

The results in Figure 4 show that the average ratio R for these animals was 1.22±0.17. Ta. This means that during the natural healing process, there is an increase in blood venous flow at the surgical site. It shows.

Cat CM Fr Y Twelve cats were involved in this group. Six cats received omentum lipids intramuscularly and six control cats. were given normal saline. Radioactivity density of animals in images on days 3, 6, and 9 The mean ratio R of 1.16 ± 0.18 in cats given saline intramuscularly. 1.R, compared to 1.R for those injected intramuscularly with cat omentum lipid CMFr. It was 81±0.30. This difference was determined using the statistical methods of one-sided analysis of deviations and t-tests. It was found to be highly statistically significant (Po, 001). Animals were further divided into the following groups for further evaluation of the data. Sunawa Animals that received intramuscular injection directly into the wound (uneven injection group) and animals that received injection into an area far away from the wound. Those who received injections (distant injection group). Figure 5 shows the results shown below. Ru.

a.’Inspection: When the CMFr fraction of omentum lipids is injected intramuscularly into the injured part of the femoral artery. Significant increase in blood flow as confirmed by a large increase in the proportion of radioactive density An increase has taken place. The mean ratio R for animals receiving feline CMFr was 1.99±0 ．． 27, whereas the control cat that received physiological saline in the partial region had a score of 1.23. It showed an average ratio R of ±0.19, an increase of 62%. This difference in ratio is statistically high. It was significantly significant (PO, 0O1).

As previously reported, the average ratio (1,23 ± 0.19) for animals injected with saline was Pre-surgery mean for normal (non-surgical) limb to be 1.05±0.14 It is higher than the ratio (R) and is about 17%. In this saline-administered cat, The increase in the mean ratio (R) of It was caused by a surgical wound.

(b, far-injected group: omental CMFr was injected into the forelimb far from the lower limb wound. Animals receiving physical injections show a significant increase in radioactivity density (increased localized blood flow). compared to a mean ratio (R) of 1.05 ± 0.05 in animals that received saline in the forelimbs. The average ratio (R) was 1.63±0.20 (PO,001). this thing represents an increase of 55%. After receiving an intramuscular injection in the forelimb, blood flow begins at a wound far away from the forelimb. The action of the lipid components of the omentum that causes an increase in This strongly suggests a direct reaction.

It was observed that the percentage increase was almost the same in both cases (a) and (b).

(C) Application to normal parts: Muscle parts around the femoral artery that were not operated on or Cats injected with omentum lipids around the site of radioactivity at the injection site There was only a slight increase (1.28±0.17). This number refers to the femoral artery. This is comparable to the radiodensity value when saline was administered to the area around the cut (1 ．． 23±0.19).

This finding suggests that the effects of omentum lipids are enhanced when administered to the site of a wound. It shows.

In both cases (b) and (c), systemic administration directs the effect to the traumatized tissue. It should be noted that Furthermore, there is no activity when injected into other traumatized tissues. was not seen.

Effect of ganglioside combination Cat net CM F r contains gangliosides. Therefore this type of compound It was of interest to investigate the effect on blood flow characteristics of the operated animals. Table 1 is Bovine fetal brain (prepared as described in the literature) and bovine brain (Spelco) The total amount administered over 6 days with a mixture of gangliosides (obtained from There is. Each substance listed in Table 1 was administered to test animals. With R(+s, D, ) Values given are the average of 70 scans (10 per animal) performed for each animal. It shows. Gangliosides were dissolved in PBS and removed from the wound every day for 6 days. I injected it intramuscularly. Animals were observed for radiographic imaging on the 7th day after surgery.

All ganglioside mixtures showed higher R values than normally cured animals. (See Table 1). Figure 6 shows a normally cured cat and a ganglioside mixture. Figure 2 illustrates the distribution of R values among treated cats. The P value for the two groups is It was 0.0072.

Table 1 Effects of fetal bovine brain and bovine brain ganglioside mixture on local blood flow in operated animals influence Bovine fetal brain ganglioside (Dinotri) 10 1.43±0.40 Bovine fetal brain Ganglioside (Ginotri) 30 1.58 ± 0.18 Ishijijin @Egg - Pig net fat and blood mixed with bovine brain gangliosides Fruit mixtures of non-polar lipids (neutral lipids) and their polar lipids (e.g. gangliosides) In order to evaluate the effect, the following experiment was performed on 82 groups of 5 animals each, and each Porcine net neutral lipids (PONL) extracted with approximately 10 g of hexane or 10 m The same amount of PONL containing g of fetal bovine whole gangliosides (FCBG) was administered for 6 days. It was administered daily (intramuscularly) to the wound area. Animals were radioactively scanned on day 7. Figure 7 shows the results. Animals treated with PONL and animals that healed normally (R=1 ．． 22 ± 0.17) showed a significantly higher R value (R = 1.79 ± 0.09 ). However, adding only 10 mg of FCBG to jog PONL The R value increased dramatically to 2.59±0.18, an unexpected effect. Rukoto has been shown.

The terms and expressions used herein are used for purposes of explanation and not limitation. Use such terms and expressions with any applicable features shown or described herein. It is not intended to be limited to all or part of the It is recognized that various modifications are possible.

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Claims (15)

[Claims] 1. angiogenically active lipids and a pharmaceutically acceptable carrier in a mammal. the local vascular perfus of a mammal, including administration to sion). 2. The composition includes an extract of mammalian omental tissue. A method according to claim 1 comprising: 3. 2. The method of claim 1, wherein the composition comprises a ganglioside. 4. The amount of the composition ranges from about 0 mg to about 500 mg per kg of body weight of the mammal. A method according to claim 1, wherein the method is administered. 5. 4. The method of claim 3, wherein the composition is administered intravenously. 6. 4. The method of claim 3, wherein the composition is administered subcutaneously. 7. 4. The method of claim 1 or 3, wherein the composition is administered intramuscularly. 8. The patent claims that the composition comprises a mixture of mono-, di-, and tri-sialogangliosides. The method described in item 3 of the scope of the request. 9. The composition has a weight ratio of mono-, di-, and tri-sialogangliosides of 1:1:1. The method according to claim 3, wherein: 10. Claims wherein the composition comprises a mixture of di-, tri-sialogangliosides The method described in box 3. 11. The claimed invention has di-, tri-sialogangliosides in a ratio of 1:2 by weight. The method according to scope item 11. 12. Claims wherein the composition comprises a mixture of mammalian omentum extract and gangliosides The method described in item 1. 13. The method according to claim 3, wherein the mixture further contains a net neutral lipid. Law. 14. The composition is administered to the mammal at a location remote from the location where improvement of blood flow is desired. The method according to claim 1. 15. The composition is administered to a mammal at a site of traumatized tissue. 15. The method of claim 14, wherein the method is administered.