TW516958B - Water-soluble fullerenol pharmaceutical composition - Google Patents

Abstract

A pharmaceutical composition for treating free radical-induced diseases or syndromes, comprising: an effective amount of compound: F(-X)m wherein, F is a fullerene nucleus; each X is independently -OH, -(CH2)n-SO3H, or the metal salt of -(CH2)n-SO3<SP>-</SP>, wherein, each n is independently an integer of from 2 to 50; and m is an integer of from 2 to 40; and a pharmaceutically acceptable vehicle.

Description

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516958 V. Description of the invention (/) Conjugated caged olefins, such as carbon 60 (Cm) or the like, are particularly sensitive to many chemical reagents, especially their effect on organic free radicals The multiple addition reactions all exhibit a high degree of reactivity, and it is speculated that this reactivity is related to the strong anions of the molecules themselves. At present, several functionalized hydrazone derivatives (fullerene derivatives) have been proposed in related biochemical or medical research, such as: bis (succinic acid-phenylethylamine) Cm can inhibit HIV-1 (acquired immune deficiency Virus) protein enzymes (see Friedman, et al., J. Am. Chem. Soc. 1993, 1 15, 6506), another example is: water-soluble monofunctional Cm after photoactivation has the activity to split DNA, And in vitro cytotoxicity to the HeLa S3 cell line; however, both of these activities occur in the presence of a light source. The object of the present invention is to provide a medicinal composition to treat the related symptoms caused by free radicals. Such a medicinal composition includes an effective amount of a polyhydroxylated candela compound, a poly (sulfoalkylated) candela compound or a metal salt thereof, or a candela compound containing a mixed functional group (that is, it also contains And sulfofluorenylalkyl) or metal salts thereof; and a pharmaceutically acceptable carrier 0 The above-mentioned fumarium compounds have the following general formula: F (-X) m where F is a monogamous nucleus (ie c6〇 Fu core; fuiierene core); each X is independently a gold salt of 0H, (CH2) n_s〇3H, or (CH2) n S0f, wherein each n is independently ^ different: and ⑺ 为 孓 (Please read the notes on the back before filling out this page) _ i-line · Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 3 Printed by the Employee Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 516958 A7 R7 _________________ ° 7 V. Invention Measure (2) ~ &quot; &quot; ------ 40 〇 When X is OH, m is preferably 4-30, and most preferably 1020. In addition, when X is a metal salt of (CH2) n-S03H or (CH2) n_s03, a claw is preferably 2-10, and η is preferably 2-10. When X is a metal salt of (CH2) n-S03, the metal may be a monovalent metal (such as sodium or potassium) or a divalent metal (such as calcium or magnesium). The so-called "fu core" is a cage-shaped molecule, mainly composed of carbon atoms. For example: c6. , C6〇Hx, c61, c62, QQ, ^ Nχ, C610x 'c62ox, c70ox, c710x, C72〇x, C70NX, c76, c76ox, c78' c780x 'c82' C82Ox 'c84' C84ox, c92, c92ox, Or other similar molecules, where X ranges from 1 to 20 (eg, i_8). C600x (〇H) Y is an example of an English derivative in which C600Ox is used as a nucleus and linked with a hydroxyl group. QHJNRR% is another example of c6 () Hx as the core. In addition, "c6 (), especially C6., C6Qhx, C61 ', C62', C60Ox, C60Nx, C6iox, or c62ox. Another object of the present invention is to provide a method for treating the symptoms caused by free radicals. A method comprising: supplying an effective amount of one or more of the above compounds to a subject in need thereof, wherein F is a nucleus; each X is independently OH, (CH2) n-S03H, or (CH2 ) a metal salt of n-S03 ·, in which each η is independently 2-50; and m is 2-40. The scope of the present invention includes the above-mentioned pharmaceutical composition as a radical scavenger, and also includes this composition It is made into medicine to treat the related symptoms caused by free radicals. This paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm) (Please read the precautions on the back before filling this page}

516958 V. Description of the invention (In order to make the above and other objects of the present invention obvious and easy to understand, the following features and advantages can be further exemplified by the preferred embodiments, which are described in detail below. The pharmaceutical composition of the present invention Relevant symptoms that can be caused by substances, for example, can be used to edit: I / σ treatment of free radicals, bow I, ancient j τ to inhibit airway free radicals caused by contraction, inhibit peroxide-induced brain cell oxidative damage, inhibit self The soil causes the oxidation of blood proteins, inhibits the growth of blood cancer cells and smooth muscle cells, inhibits cell damage caused by free radicals caused by gastric transplantation, or clears excess free radicals in the blood of cancer patients. In other words, the free radical-induced correlation Symptoms include, for example, bronchoconstriction due to ischemia, cell destruction during reperfusion surgery, cancer, arteriosclerosis, and neurological diseases such as epilepsy and Parkinson's disease. It can be used to treat these symptoms. Water-soluble polyhydroxylated puff derivatives, such as c6G (〇H) x or C60Ox (〇H) y, the preparation method is here For example, phoritol-1 can be reacted with pure base of Qg or (^ (84%) and C7G (i6%) at room temperature with bowl-based tetrakis boric acid in organic acid (RC02H). The obtained saccharin products are hydrolyzed. (Chiang, et al., US Patent 5,177,248; Chiang, et al. US · Patent 5,294,732; printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs and Chiang, et al. 5 J Am. Chem. Soc. 1992, 114, 10154; Chiang, et al ·, J. Am. Chem. Soc. 1993, 115, 5453 ·). The structure of pentanol-1 is C60Ox (OH) y, where the average x is less than 5, and y is equal to 18. Another example is that pentanol-2 can be reacted in a sulfuric acid solution containing sulfur trioxide (30%) via a mixture of pure C6G or C6G (84%) and C70 (16%). The obtained fufu product is hydrolyzed. (Chiang, et al ·, J · Org Chem · This paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) 516958 A7 V. Description of the invention (4) Ministry of Economic Affairs wisdom Printed by the Consumer Cooperatives of the Property Bureau 1994, 59, 3960 ·). The structure of pentanol-2 is C6〇 (〇H) y, where y is equal to 12. On average (continued on the basis of fluorination) Compounds or metal salts thereof can be synthesized by Example 1 or a similar method, and if necessary, can be followed by a hydroxylation reaction. For example, the following Example i, after the poly (butylsulfonium sodium) fu , C60 (CH2CH2CH2CH2SO3Na) x, where the size of x will change with the reaction conditions, ranging from 2_20 (average 4_6), can be hydroxylated using the methods described in the following two paragraphs to obtain a mixed B moonyl Husband C60 (CH2CH2CH2CH2SO3Na) x (OH) y, where the size of y will change with the reaction conditions, ranging from 1_2〇 (mostly 1-10). There are currently many analytical methods, such as the following examples 2-8, can be used to screen out water-soluble fusiform compounds that have curative effects on free radical-related symptoms. The effective amount herein means that after administration of such a dose to a patient ', the amount of free radicals can be significantly reduced, and the symptoms of the patient due to excessive free radicals can be alleviated. The effective amount given to a patient will vary depending on the patient's age, weight, the disease to be treated, and the extent of the condition, and the final decision will be made by the attending physician. This is usually provided to patients based on the patient's body surface area, patient weight, and patient condition. Freireich, E.J., et al., Cancer Chemother. Rep., 50 (4): 219, 1966, described the relative relationship between animal and human doses (in mg / m2 body surface area). The body surface area can be roughly estimated by the patient's height and weight, such as reference (please read the precautions on the back before filling this page)

516958 A7 B7 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 5. Description of the invention (5) Scientific Tables, Geigy Pharmaceuticals, Ardley, New York, pages 537-538, 1970. The effective amount of the compound in the present invention ranges from about 5 mg / kg to 500 mg / kg, preferably from about 5 mg / kg to 250 mg / kg ', and most preferably from about 5 mg / kg to 150 mg / kg. As known to those skilled in the art, the effective dose will also vary depending on the following conditions, including the route of administration, the dose of excipients, and the possibility of combining other treatments, such as other anticancer agents or radiation therapy. The formulation can be made into a convenient unit dosage form and can be made by any known pharmaceutical technique. All methods include combining the active ingredient with a carrier having one or more accessory ingredients. Usually, the formula of medicine tablets or powders is to mix the active ingredient with the solid carrier that has been completely crushed. If you want to become a medicine bond, you can shape the pushed product into a certain size and shape. Since this active almond compound is water-soluble, a water-soluble carrier may be used as the carrier. The above compounds can be administered to patients by any suitable route, preferably by blood injection, but those skilled in the art will know that the route used, such as intravenous, subcutaneous, intramuscular, intraperitoneal, nasal cavity, or Oral and the like will vary depending on the symptoms to be treated and the activity of the analogs used. Those skilled in the art should fully utilize the present invention under the description of the present invention. The following examples are only used to illustrate the present invention and not to limit any other applications. Water-soluble poly (butyl sodium sulfonate) fu derivatives "(CHzCHzCE ^ CH ^ SC ^ NaL or poly (propyl sodium sulfonate) fu derivatives) (Please read the precautions on the back before filling this page)

Order I f 0 I —ϋ-Β 7 Ming 58 A7 V. Printed invention description printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs (6) Synthesis of object C60 (CH2CH2CH2SO3Na) x A 100 ml circle equipped with a magnetic stirrer Bottom bottle A is sealed with a septum and filled with nitrogen. Into the flask, 500 mg of saccharum (this saccharum can be pure Cm or saccharin mixture), 40 ml of toluene and butanesultone or 丨, 3-propane sultone (i, 3_pr〇 叩 such as sultone; 5 to 20 equivalent ratio to sulcus). The mixture was dried with a molecular sieve (4A). In another flask b, dissolve Nai (Danli ratio of 5 to 20) with dimethoxyethane (DME) which has been dried by molecular | 帛 (4 persons), and Sodium (5 to 20 equivalents ratio to fumars) reacts with it to give a sodium solution of sodium nyl. The sodium nyl solution was added into flask A through an injection tube, and the β substance was stirred under nitrogen for 4 hours at the temperature. When the reaction was completed, 2 ml of water was added to quench all the reactive intermediates. The final solution was added to 60 ml of methanol to precipitate a brown solid, which was then separated by centrifugation. After rinsing twice with methanol (20 ml each time) and vacuum drying at 40 ° C, water-soluble poly (butylsulfosulfonyl) furan derivative C6〇 (CH2CH2CH2CH2S〇3Na) x or The brown solid of poly (propyl cerana) fu derivatives C60 (CH2CH2CH2SO3Na) x, where the value of X will change with the amount of sodium naphthyl used. The IR data of poly (calcined sodium glutamate) derivatives are: IRVmax (KBr): 1642, 1570, 1384, 1192, 1038 5 797? 750 5 603 5 534cm'1 ° fullerenoM-1 Antioxidant effect In the absence of oxygen, such as ischemia caused by bleeding or exsanguination, the oxygen-derived free radicals generated in the respiratory tissues increase rapidly with the onset of blood loss (Frank, J. Appl · Physiol · , 1982, 53, This paper size is in accordance with Chinese national standard (qsjs) A4 specification (210 x 297 mm (please read the note on the back? Matters before filling out this page) Order · | Line _ Staff Consumption of Central Bureau of Standards, Ministry of Economic Affairs Printed by the cooperative 516958 A7 B7 5. Description of the invention (7) &quot;475; Prasad, et al. Angiology, 1988, 12, 1005). Through the metabolism of the central nervous system in the body, peroxide anions and peroxidation will be produced at the same time Hydrogen (Halliwell, J. Neurochem., 1992, 59, 1609), and iron complexes will react with peroxide anions or hydrogen peroxide to form the most reactive hydroxyl radical. Oxygen derived radical radical), especially Hydroxyl free radicals activate afferent C-fibers' and then release tachykinins. The released tachykinins cause non-cholinergic airway contractions. In addition to bronchoconstriction, reactive oxygen free It also causes mucus secretion and edema due to microvascular rupture (Cees, et al. Free Radicals Biol. Med ·, 1990, 9, 381). The analysis method described below is used to test phoritol · 1 for blood loss Inhibitory effect of rapid contraction of the respiratory tract caused by the acute side effects of JL alcohol-1 on the lung function of pigs The pigs (Hartly strain guinea pigs) were randomly divided into six groups: intravenous saline, intratracheal saline, intravenous salivary -1 (1), tracheal-1 (1), intravenous salivary ( 2) Intratracheal -1 (2). Each animal was itched with sodium pentobarbital (Pentobarital; 30-50mg / kg). Animals in this group were intravenously saline Intravenous injection of 0.75 ml of saline and intratracheal salt In this group of animals, the same amount of saline was instilled through the trachea. In the two groups of pentanol-1 (1), 200 mg / kg (by 0.75ml of solution) laughol. (Please read the notes on the back before filling this page)

9 516958 A7 printed by the Consumer Cooperatives of the Central Standards Bureau of the Five Ministry of Economic Affairs ______B7 Invention Description (8) The two groups of pentanol-1 (2) are administered to each animal through the trachea and vein by 2mg / kg (In 0.75 ml of solution) phoritol]. After comparison with the two groups of intravenous saline and intratracheal saline, unless the high-dose (200 mg / kg) drip was administered through the trachea, the supply of pentanol] did not cause significant abnormalities in respiratory function, which indicates that This compound has no side effects on the bronchi. After 30 minutes of administering the salt solution or phorbol-1 in the manner described above, a lung function test was performed. Thirty-two young boars weighing approximately 229 to 5 kg were divided into four groups on average: the control group, the chronic hypoxia group, the pentanol-1 group, and the deferoxamine mesylate group. Animals in the control group were placed in an open low-pressure chamber without sub-atmospheric pressure, while animals in the chronic hypoxia group were placed in a closed low-pressure chamber at 380 Torr for 7 days. These animals were exposed to hypoxic conditions (intermittent exposure) from 5 noon to 8 am during the day, and the rest of the day were exposed to indoor air. Animals in the phorbol-1 group were injected with human 10 mg / kg / day fuzyme-1 via the peritoneum two days before the start of the functional study, and 2 mg were reinjected via the vein 30 minutes before the blood drive. / kg of phoritol · 1. Each animal was itchy with sodium pentobarbital (30-50 mg / kg) and inserted into a catheter. During artificial ventilation of a respirator (Harvard Rodent Respirator; Model 680), each pig was placed supine in a whole body volume change angiograph, and when the air pressure flow passed through the three-layer barbed wire (325-mesh) of the logger At that time, the flow is monitored with a differential pressure converter from Validyne DP 45. The change in vital capacity is obtained by integrating the flow. The operation of the maximum expiratory flow (MEFV) is based on the Buxco Pulmonary operating system (sharon, CT). The paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm). (Please read the precautions on the back before filling (This page)

Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 516958 A7 ---------B7 V. Description of Invention (^ ~) ~ &quot; &quot; '一 —'- OK, blow the lungs to the total vital capacity ( The airway opening air pressure is about 30emH20, the vital capacity) four times. When the fourth blow reaches the maximum volume, the screw valve for blow is closed, and the other screw valve for debulking is automatically opened immediately. This debulking valve is connected to a 20 liter container, which is maintained at a sub-atmospheric pressure of -40 CmH20. At the same time, the computer program selects a forced expiratory volume (FEV0i) of ο · m seconds in the volume-time diagram. A functional residual capacity (FRC) is determined before and after each mefv operation. From frc to f, blow the lungs to 50% lung activity with a standard 0.5% neon gas mixture, and then draw back the equilibrium gas mixture and analyze it. The total volume is subtracted from the dead space of the instrument to get the frc value. Then, the aorta of the animal's lower abdomen was cut off to drive blood, artificial ventilation was stopped, and MEFV was performed at 5, 10, 15, 20, 25, and 30 minutes after the start of blood drive. The FEV () 1 value is used as an indicator of bronchoconstriction (change in airway size). Released P was used to determine the inhibitory effect of phoritol-1 on rapid contraction of the respiratory tract. Immediately after the above test was completed, the method of Saria (Saria, et al Am Rev. Respir Dis, 137, 133, 1998) was used. The lungs of pigs were extracted with lung substance P (tachykinin). The amount of released P was measured with an enzyme immunoassay kit (Ann Arbor, MI) manufactured by Cayman Chemical Co. The principle of this analysis is based on the competition of a limited amount of rabbit antisera binding sites with a free-release P-tracer (linkage of p-linked to acetylcholinesterase). The concentration of the released P tracer remains constant, while the concentration of the freely released P changes over time. Therefore, the amount of release P chasing agent that can be combined with rabbit antiserum is exactly 11 paper sizes applicable to China National Standard (CNS) A4 specifications (210X297 mm) — ~ (Please read the precautions on the back before filling (This page)

516958 Α7 Β7 5. Description of the invention (i0) is inversely proportional to the concentration of freely released substance P in the groove. The resulting rabbit anti-serum clear-released P complex will stick to the individual antibodies previously attached to the grooves. Wash off the unattached reagents before adding Ellman ’s reagent (an enzyme containing acetylcholinesterase). The product of this fermentation reaction has a distinct yellow color and strong absorption at 412 nm, and its absorption is inversely proportional to the released substance P in the sample. The inhibitory effect of pentanol-1 on rapid airway contraction was measured by the activity of a neutral peptide enzyme. Printed by the Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs (please read the precautions on the back before filling this page). Neutrapeptide endoenzyme activity was measured in the trachea (Orlowski et al. Biochemistry, 20, 4942, 1981 and Haxhiu-Poskurica, et al. J. Appl. Physiol. 72, 1090, 1992). The frozen trachea was thawed and cut into tubes containing 50 mM Tris (pH 7.4). The obtained tissue was sonicated at 4 ° C for 30 seconds, and centrifuged at 17,500 g for 15 minutes, and then the surface float (tissue extract) was taken out for analysis. The reaction mixture (250 ml) includes 50 ml of tissue extract, 1.25 mM glutaryl_alanine-alanine_phenylalanine_4-methoxy-2-naphthylamine enzyme, 10 mg of leucine aminopeptidase, and 50 mM Tris buffer (pH 7.4). After the sample was incubated at 37 ° C for 1 hour, the product was stained with 2% Brij 35 garnet GBC (0.0025% solution 2ml) to obtain an absorption peak at a wavelength of 530 nm. The amount of 2-naphthylamine released was calculated using the disappearance coefficient, and each experiment was performed in two groups. A 50ml portion of each extract was pre-cultured at room temperature for 15 minutes with 8xl (T7M phosphoramidon's 50mM Tris buffer (pH 7.4)). The enzyme and leucine were applied to Chinese paper standards ( CNS) A4 (210 × 297 mm) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 516958 A7 B7 V. Description of the invention μ) ~ &quot; ~~~ Amino peptidase and the mixture at room temperature are added to this solution . The protein concentration in the trachea was determined based on bovine serum protein. The activity of a neutral peptide with phosphoramidon inhibitory activity is expressed as: the amount of 2-naphthylamine (nmole) released per milligram of protein per hour. Mystery Example 3: Inhibitory Effect of Alcohol-1 on Oxidative Damage to Brain Cells Induced by Hydrogen Peroxide 10 (M 40g Wistar male rats were anesthetized with ether, their heads were cut off, and hippocampi was quickly removed and placed in Oxidation of ice bundles (95% 〇2 and 5% COD artificial cerebrospinal fluid, which contains the following components (mM): Naci 124, KC1 4.0 ·, κ 2P04 1.0, MgCl2.6H20 1 · 0, CaCl2 2 · 5,

NaHC03 26, and glucose OO. A Sorval tissue cutter was used to cut a section about 400 mm thick from the middle area of the hippocampal temporal septum (in a direction perpendicular to the long axis of the structure), and then placed it in a recording room at 33-35 ° C. The water bath surrounding the recording chamber was mixed with 95% 02 and 5% C02 gas mixture 'to maintain the height of the fluid above the upper surface of the section, and the perfusion rate was maintained at 6.0 ml / min. These sections were incubated for 1 hour before use. Stimulation and recording of hippocampal slices were performed using a two-pole stimulation electrode, which included a 300 mm diameter stainless steel wire insulated with Teflon, and a silver-gasified silver wire. With the help of a microscope, the stimulating electrode was placed on the radiation layer for forward stimulation of Schaffer's side nerve bundles to CA1 cone cells. The stimulation conditions were a rectangular pulse of 5-15V at 0.1 ms, and a low-frequency stimulation at a frequency of 0 · 2Ηζ. The extracellular recording electrode was at the top layer of CA1. The paper size applies the Chinese National Standard (CNS) A4 specification (210X297 mm). (Please read the notes on the back before filling this page)

516958 Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs A7 B7 V. Description of Invention (12) to record the response of the hull. The amplitude of population spikes is a function of the intensity of the stimulus, so increasing the intensity of the stimulus will also increase the amplitude of the spikes. In the experiment, the stimulus intensity which can reach one-half of the maximum amplitude was used and the same stimulus intensity was maintained throughout the process. In a pair of stimulating pulses, the time interval is 50 ms. The percentage difference between the amplitude of the second spike group and the first spike group is taken as the promotion value of the pulse wave. The criterion for taking a slice and starting recording is: the largest stimulus can cause a single spike group with an amplitude of at least 5mV. Adjust the stimulus voltage to achieve a maximum amplitude of one-half of the baseline spike group of about 3_5mV. The amplitude of the CA1 spike group was recorded as 39 ± 0 2 mv (n = 13), and this amplitude was maintained at the same level after 200 minutes of culture. The amplitude of the CA1 spike group will decrease with the influence of the concentration of hydrogen peroxide. When the ratio of thorium peroxide / Chen exceeds 0 006%, the amplitude of the spike group will decrease. After the supply of hydrogen peroxide has passed for 10 minutes (004%, 0.006%, 0.008 / 〇, and 0.002 /.), The amplitude of the peak group of the CA1 bow will decrease to The prodrug control level is 98 · 0 ± 1β0% (η = 3), 32 3 ± 4 · 9% (η = 8), 15 · 2 ± 2.8% (η = 3), and 8 · 2 ± 2 · 9% (η = 3). No side effects of furfurol-l (100 mM) on hippocampal slices were detected. After 5 minutes, 10 minutes, and 15 minutes of feeding, the amplitude of the CA1 spike group displayed was 103.5 soil. 0.5% ("5", 105.5 ± 0.5%), should be .5 ± 1.5% (n = 3). After the supply of hydrogen peroxide has passed for 10 minutes (0 ._%), the amplitude of the peak group rapidly decays with time to 32.3 ± 4.9% (η = 8). And in another supply phoritol_1 (1〇〇 福 (please read the precautions on the back before filling this page)

14 Printed by the Employees 'Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs 516958 A7' &quot; ~ * ---- * ------ B7 V. Description of the invention (13) — 'In the experiment', the amplitude of its peak group is the former The drug control amplitude series was ι03.ο ± 0.5% (η = 5). After adding hydrogen peroxide for @ ⑺ minutes, the amplitude dropped to 74.6 ± 5.5% ㈣). The same experiment was performed with "hydrogen peroxide instead of pentanol" medium solution-hydrogen peroxide-which would have a similar inhibitory behavior on neural responses. Inhibitory effect of re-enchantment L1 · pentanol-1 on oxidative damage to brain cells caused by isopropylbenzyl hydrogen peroxide This example was performed in an experimental procedure similar to that of Example 3. The CA1 spike group of the hippocampal slice decreases its amplitude with the influence of the concentration of cumene hydroperoxide. After adding 0.5 mM and LOmM of cumene hydroperoxide for 15 minutes, the amplitude of the CA1 spike group decreased to 70.5 ± 2.4% (η = 3) and 34 · 5 of the control amplitude series, respectively. ± 5.4% (η = 5). If these hippocampal slices were cultured with pentanol-1 for 5 minutes before adding cumene hydroperoxide, the peak group amplitude was 102.8 ± 1.9% (η = 4) of the amplitude of the prodrug control amplitude. After adding cumene hydroperoxide for 15 minutes, the amplitude dropped to 75.6 ± 31% (η = 4), and the intensity was about 2.11 times that without gefanol-1. Re-administration Example 5: The effect of phorbol-1 on the cell destruction caused by reperfusion surgery. The autogenous transplantation of dogs was used to evaluate the effect of phorbol-1 on free radical scavenging during reperfusion surgery. Mongrel dogs (n = 8_i 0 in each group) were anesthetized and cut from the peritoneal midline for bilateral nephrectomy. The excised kidneys were immersed in a 4 ° C perfusate after a period of blood loss. After 24 hours of cryopreservation, a subcutaneous cervical depression was implanted in the same dog. This paper size applies to China National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page)

Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 516958 A7 ~ ^ _— B7_ V. Description of the invention ¢ 4) '-~~~-~ Blood was collected for 30 minutes before resection and 30 minutes after transplantation With urine sample. The experimental variables included the presence or absence of pentanol-1 (20 mM) in Rugao / Shanbi perfusion, and the duration of ischemia (0 min vs. 30 min). The evaluation of renal function is based on the comparison of biochemical coefficients before and after cutting, including blood urea nitrogen (BUN), serum creatinine, creatine liver clearing power (CCr), urine flow rate, urine nitrogen or potassium ions. Reabsorption or excretion. And before the cutting, and before and after the vascular junction, the adenosine concentration of the kidney biopsy was analyzed by HPLC, and the histopathological observation was performed on each representative sample. In each arm, post-transplantation data show deteriorating renal function. The data in each group were roughly the same at the time of cutting. The two groups of data were compared at 30 minutes and 30 minutes after transplantation (the perfusion solution contained 20 μM of pentanol-l). The CCr were 4.5 · ± 1 · 6 ml / min and 2 · 8 ± 0 · 5 ml / min; total sodium reabsorption amounts were 456 ± 213 mmol / min and 218 ± 56 mmol / min, respectively. The control group had significant progress.The CCr were 6.2 ± 2.3ml / min and 5.1 ± 1.5ml / min; the total sodium reabsorption was 663 ± 308mmol / min and 459 ± 183mmol / min. The histopathological part of the biological sample was observed under a light microscope with H &amp; e stain. Kidney tissue using perfusate in experiments showed less inflammatory cell penetration, less tube swelling and necrosis, and fewer bleeding spots. These differences were more pronounced in these two groups at 30 minutes of hemostasis. Analysis of the concentration of adenosine in kidney biopsy showed that the paper was sterilized. The paper size applies the Chinese National Standard (CNS) A4 (210X297 mm) (Please read the precautions on the back before filling this page)

516958 V. Description of the invention (/ sentence Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs printed the peroxidation by optical method, ♦ η 66 M ^ ^ ^, when the field θ protein reached a stable state of severe peroxidation , Cool it and add edta (3 secret) and then use 01 =) Γ reaction stopped. The degree of oxidation of lipoproteins depends on whether or not it is cold. [Poly (butylsulfosulfonate) fusi (using a concentration between 10-5 沢 to distinguish fusi effects, or depending on the reaction before, or before f should spread Add this fusiform derivative and observe the difference in effectiveness of the fusiform derivative in the period of addition. Τ The kinetics of conjugated diene formation is inferred by continuously monitoring the absorbance at 234nm. The calculation of the delay time is the absorbance curve The slope when the straight line rises rapidly is obtained by extrapolation, which corresponds to the stage of the propagation reaction. The degree of oxidation of the lipid is deduced by the thiobarbituric acid active substance thorium (TBARS), which is based on malondialdehyde ( malondialdehyde) is estimated to be different. The reaction proceeds in the manner that lipoprotein is treated with thiobarbituric acid at 95 ° C for 1 hour. After the mixture is cooled to room temperature, the resulting derivative is n-butyl It is extracted with alcohol, and the extract is used for fluorescence detection. The wavelength of the excitation light is 515nm and the wavelength of the emitted light is Naaman. Put fresh human lipoprotein in PBS and AAPH, or in PBS and In AMVN, it will react for 24 hours at 37 ° C. Oxidation of lipoproteins. At the same time, the addition of butylsulfazone will inhibit the oxidation of lipoproteins, and the effect of inhibition depends on its concentration. In all cases, when 500mM butylsulfazone was added It can almost completely inhibit the oxidation reaction (most of them exceed 90%), and the minimum amount of TBARS products. The divalent Cu + 2 ions will cause all lipoproteins to have significant oxidation reactions, and HDL caused by Cu + 2 In the oxidation reaction, please add (please read the precautions on the back before filling this page). Order --------- line! -Ϋ n-- -nn II n ϋ-18 516958 A7 V. Description of the invention (/ 7) The addition of 20 mM sulfosulfuric acid produces an inhibition effect of more than 80%. According to kinetic measurements, 17% of the butyl disulfide was added to the co-light dilute formation reaction caused by Cu + 2. Fu (10 mM) will also reduce the rate of the propagation reaction, and make the delay time and spike time significantly prolong the high inhibitory effect of butyl ~ Fu on oxidation of lipoproteins, not only in the initial stage of the reaction, The same is true in the propagation stage of the oxidation reaction. "Column 7 • Inhibition of proliferation of human T-lymphocytic cancer CEM cells by pentanol and pentanol_2 This example measures cholesterol_1 and pentanol_2 on TEM of C-cells caused by fetal bovine serum The inhibitory effect of cell proliferation was analyzed by analyzing the binding of [Η3] thymidine during the synthesis of DNA by proliferating cells to evaluate the inhibitory effect of phoritol and pentol-2 (Watabe, et al, j Natl.

Inst. 1984, 72, 1365). Human D-lymphocytic cancer CEM cells were cultured in KPMI-164O medium containing 10% fetal bovine serum, and the vitality of the cells was detected by the cyanine dye isolation method. The experiment was performed by adding pentanol-1 or pentanol-2 to suspended tumor cells (5 x 10 cells / well), while tumor cells of the control group did not add pentanol. In each set of experiments, the cells were cultured in a CO 2 incubator at 37 ° C for 0.5 hours, and then [H] thymocyte faint (1 μ.) Was added. After 4 hours of incubation, cells were collected and calculated [ H3] Amount of thymidine binding. The control value of combined [H3] thymidine caused by 10% fetal bovine serum in human T-lymphocytes was 7031 ± 322 cpm / well. When human T_lymphocytes and cancer cells are also exposed to pentanol (10-6 to ι〇-4μ), [η3] 19 This paper size applies to China National Standard (CNS) A4 (210 X 297 mm)- --- f Jing first read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs --- !! ----- line! 516958 A7 V. Description of the invention (The binding degree of the printed thymic nucleus printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs will be greatly reduced, and the proliferative response will decrease rapidly as the dose of pentanol increases. Pentanol]% The value is 6.4 ± 2.0 μM. The concentration of phoritol * Tian ± &amp; Qn 〇,. 〇0 / Fu 転 1 can be Xinglailang large inhibition rate f 9 coffee. 9 / ... after the experiment After comparison, it can be found that if anti-corrosive = vitamin C) is required to reach a 5G% inhibition rate, the dosage required is 100 times that of wuol-1. The results show that water-soluble phoritol can be applied to the proliferation inhibition of cancer cells, such as human D- and P-lymphocytes. The proliferative effect of smooth muscle cells on fat lines and fibrous plaques, as a connection The appearance and growth of smooth muscle cells with a tangled structure of tissue and lipids is a key factor in determining the extent of atheromatous lesions (Ross, Nature, 1993, 362, 801). This a yoke example measures the inhibitory effect of bovine alcohol 1 and bovine alcohol 2 on the proliferation of cultured rabbit aortic smooth muscle cells caused by bovine blood π (Huang, et al · Eur · J. Pharmacol., 1992, 221, 381). The blood vessels were removed from the aorta of the rabbit, and the endothelium was removed. The middle layer with 2/3 cells was cut into strips with a width of about 1 mm. These strips were cut into squares and placed in a dry culture dish. The petri dish was filled with DMEM medium containing 10% fetal bovine serum. When the cells were free, the aortic block was removed, and the cells between the 3rd and 8th passages were used, and the vitality of the cells was detected by the cyanine dye method. The proliferation of aortic smooth muscle cells is detected by the intake of [H] thymic nucleus during DNA synthesis. Before the experiment, smooth muscle cells (2.5 × 104 cells / well) were allowed to stand for 48 hours in 0.5% fetal bovine serum (please read the precautions on the back before filling this page). Order --------- line! -· Ϋ ϋ 20 paper-size paper towels _ quasi (CNS) A4 size (21〇X 297 public love) II — n _ 516958 A7 Description of invention (ί 1) hours, then add stimulant (5% fetal bovine serum ) And phoritol, this mixture was left for 24 hours, and then [H3] thymidine (0.2 μ ci / weu) was added. After 24 hours of incubation, the cells were harvested, and the bound [h3] thymidine could be calculated by a liquid flash counter. Each experiment was performed in groups. The inhibitory effects of pentanol-1 and pentanol-2 are expressed as a percentage of the control value produced without the addition of pentanol daily stimulant. The concentration that can cause the maximum inhibition of M% is ICs. [H3] thymidine intake control value due to 5% fetal bovine serum cultured rabbit aortic smooth muscle cells. The addition of 7266 ± 755 cpm / wel bufanol (10-6 to 10-4M) It can greatly reduce the binding degree of [H3] thymidine, and the proliferative response will decrease as the dose of pentanol increases. The value (n = 7) of pentanol is 0.12 ± 0.3 mM. Phenol at a concentration of 0.1 lm] exhibited the largest inhibitory effect at 97.7 to 0.8%. After experimental comparison, it can be found that if ascorbic acid (vitamin C) is used to achieve 50% inhibition, the required dosage is similar. Such results indicate that water-soluble phoritol has an inhibitory effect on the proliferation of vascular smooth muscle cells, and therefore can be used to treat disorders related to dysplasia of smooth muscle cells, such as atherosclerosis and angioplasty restenosis. sis). Although the present invention has been disclosed as above with a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can make various modifications and decorations without departing from the spirit and scope of the present invention. The scope of protection of the invention shall be determined by the scope of the attached patent application. 2 1 (Please read the notes on the back before filling this page) 11 --------- line! Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs

Claims (1)

516958 A8 Application No. 86116108, amended the date of this amendment Six, the scope of patent application 1 · A primer for the treatment of free radicals, including: | related symptoms from the medical composition of an effective amount of compounds: where F is-nucleus; each Each χ is independently a metal salt of ⑽, CH2) n-S03H, or (CH2) n-S03_, wherein each n is independently an integer of 2-10; and m is an integer of 2-20 ; And a pharmaceutically acceptable carrier. 2. The pharmaceutical composition according to item 1 of the scope of patent application, wherein X is OH. 3. The pharmaceutical composition according to item 2 of the scope of patent application, wherein m is an integer of 4-20. 4. The pharmaceutical composition according to item 3 of the scope of patent application, wherein m is an integer of 10-20. 5. The pharmaceutical composition as described in item 4 of the scope of patent application, wherein the husband's core is a C60 core. 6. The pharmaceutical composition as described in item 丨 of the patent application scope, wherein χ is a metal salt of (CH2) n_S03H, or (CH2) n-S0 /. 7. The pharmaceutical composition according to item 6 of the scope of patent application, wherein m is an integer from 2-10. 8. The pharmaceutical composition as described in item 7 of the scope of patent application, wherein the husband's core is a C60 husband's core. 22 This paper size applies the Chinese National Standard (CNS) A4 specification (210 X 297 mm) (Please read the precautions on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs a — — — — — — — — I — 1 — — — — — — — — — — — — — — —