Fluorocarbon blood substitutes

Abstract: A brominated perfluorcarbon emulsion non-toxic for internal and intravenous use in animals including humans for use as an oxygen transport medium and as tumour and other element contrast enhancement medium is stable with very small size characteristics for extended periods in excess of eighteen months and after sterilization, with a stabilizing component selected from steroid hormones, tocopherols, cholesterols and their combinations. An anti-oxidizing component enhances delivery in oxygen transport.

Description: The present invention relates to the art of non-toxic oxygen transport and contrast enhancement agents, and more particularly to stable emulsions capable of sterilization and suitable for internal and intravenous animal, including human, use where the emulsion is a brominated perfluorocarbon in the discontinuous phase in the presence of certain what are believed to be stabilizing agents. Mono-brominated cyclic and acyclic perfluorocarbons in aqueous emulsions with a minor amount of an emulsifying agent have been known for medical applications involving animals, including humans, for both radiopacity and oxygen delivery. Oxygen is highly soluble in, for example, perfluoroctylbromides. See Long, U. S. letters patent No. 3,818,229; No. 3,975,512; and, No. 4,073,879. The present invention is directed toward improvements in the use of such bromofluorocarbons wherein the oxygen transport characteristics, as well as the storage characteristics of the emulsions are enhanced, while the toxicity is further minimized or decreased altogether. In the past, efforts to use emulsified fluorocarbons as an oxygen transport or carrier, as in a blood substitute, have encountered certain difficulties. Purity, non-toxicity, chemical and biological inertness and excretability are necessary objectives. The emulsified fluorocarbon must be capable of sterilization, preferrably by heat, have long-term size and function stability in the fluid or non-frozen state, be industrially feasible, persist for sufficiently long times in the blood stream when used intravascularly and be eliminated rapidly from the body. It has been conventionally believed that those fluorocarbons which have fast elimination times from the body do not form stable emulsions, and that those fluorocarbons which form stable emulsions are retained too long in the body. Non-brominated perfluorocarbons show a direct relationship between emulsion stability and molecular weight and an inverse relationship between molecular weight and excretion rates from the animal body. Both types of fluorocarbons are inadequate, and attempts to combine amounts of both types have merely combined the problems of each. For intravenous use, it is considered important to have small particle size. However, long term storage for extended periods of time for a month and longer, of fluorocarbon blood substitutes, or "synthetic blood" has heretofore resulted in conglomeration of the fluorocarbon particles of the emulsion into larger particles, especially after heat sterilization. For a general discussion of the objectives and a review of the efforts and problems in achieving these objectives in fluorocarbon blood substitutes, see "Reassessment of Criteria for the Selection of Perfluoro Chemicals for Second-Generation Blood Substitutes: Analysis of Structure/Property Relationship" by Jean G. Riess, 8 Artificial Organs , 34-56 (1984). Larger particle sizes are dangerous in intravenous use in that they tend to collect in the lung, spleen and some other organs, enlarging them and endangering their functioning. On the other hand, it is desired to have sufficient particle size in the fluorocarbon particles for them to collect in tumors and other areas when the fluorocarbons are used as a contrast enhancement medium. Larger particle sizes, also, are unobjectionable when used in other, non-venous systems in the body, such as, for example, the cerebrospinal fluid ventricles and cavities. Japanese patent publication 60-166626(A) refers to aqueous bromoperfluororcarbon (BPFC) emulsions of 5-50 w/v % of BPFC. The present invention, as represented by the terms of the appended claims, discloses that stable emulsions can be obtained with BPFC content greater than 50% (w/v) DESCRIPTION OF THE PREFERRED EMBODIMENTS In brief, one aspect of the present invention comprises mono-brominated perfluorocarbon emulsions. The bromofluorocarbon emulsions found suitable for use as an oxygen transport medium comprise mono-brominated perfluorocarbons having a minor amount of an emulsifying agent and further comprising a compound believed to be useful in stabilising the membrane of the bromofluorocarbon particle. The compound could be steroid hormones, cholesterol, tocopherols and mixtures thereof. A nine-alpha fluorinated corticosteroid in combination with cholesterol emulsified along with a phosphatidylcholine to particles of a perfluoroctylbromide having the formula CF 3 (CF 2 ) 6 CF 2 Br or C 8 F 17 Br, or or related brominated perfluorocarbon such as a perfluorohexylbromide (C 6 F 13 Br) or a perfluoroseptobromide (C 7 F 15 Br), together with a tocopherol as an anti-oxidant is preferred. It has been found that particle size stability can be maintained with emulsions of from 20% weight per volume to 125% weight per volume of the bromofluorocarbon without undesirable viscosity. Herein in this specification, the expression "weight per volume" or "w/v" will be used and understood to mean the ratio of 100 grams per 100 cubic centimeters or milliliters, or equivalent expressions or mathematical identities thereof. Emulsions with concentrations of from 20% to 100% weight per volume have a thixotropic viscosity profile less than that of whole human blood. Perfluoroctylbromide is excreted rapidly from the animal body, because of the lipotrophic nature of the brominated perfluorocarbon, it is believed. In any event and notwithstanding its high molecular weight and stability, mono-brominated perfluorocarbon has a relatively high excretion rate from the animal body. In some applications where high bromide concentration, such as when the emulsion is to be used as a contrast enhancement medium, or where a high oxygen transport is needed in an intravascular system where large volume impact is to be minimized, the larger concentration emulsion is preferred. While it is not certain, it is considered that these suitable and stable high bromofluorocarbon concentration emulsions are possible because (1) of the relatively high molecular weight of the brominated perfluorocarbon, and (2) of the good bonding between the bromine and the phospholipid emulsifying agent discussed below. The preferred emulsifying agent is a phospholipid, an anionic surfactant or a fluorinated surfactant. Suitable phospholipids include lecithin, such as phosphatidylcholine. Phospholipids are common and biologically accepted elements in the blood, and are not so readily phagocytosed by macrophages or other organisms in the animal body's fluids. The resultant emulsion thus is resistant to macrophage and other animal body organism attack. Preferred anionic surfactants include polyoxyethylene-polyoxypropylene copolymers, such as Pluronic. Suitable fluorinated surfactants include XMO10 and XMO20. The phospholipid emulsifying agent should be included in the range of from 2 to 14 grams weight per volume, with the preferred amount being 6 grams weight per volume for concentrations of 75% w/v bromofluorocarbon and 7 grams to 10 grams weight per volume for concentrations of 100% bromofluorocarbon. The phospholipid lecithin contains both hydrophilic and hydrophobic or lipophilic characteristics and is thus a suitable emulsifying agent for the perfluorocarbon particle. According to one embodiment of the present invention, an additional compound is made part of the particle in emulsion. The additional component is believed to be effectual in making the discontinuous particle membrane more compatible and stronger with respect to the continuous, aqueous phase of the emulsion. The additional component could be a tocopherol, a steroid hormone, a cholestrol or, preferrably, a combination of these three components. Suitable steriod hormones include fluorinated corticosteriods, fluorinated androgens and non-fluorinated hormones, such as proges- terones and estrogens. The preferred steroid is one that is fluorinated in either the nine-alpha or the six-alpha positions, such as, for examples, nine-alpha-fluoro-16-alpha-methylprednisolone, nine-alpha-fluoro-16-betamethylprednisolone, nine-alpha-fluoro-16-alpha-hydroxyprednisolone and six-alpha-fluoro-16-alpha-methylprednisolone, or combinations of these corticosteroids. While the actual reaction or membrane structure that takes place is not known, it is believed that the affinity of the fluorine in the fluorinated corticosteriod with the fluorine in the bromofluorocarbon creates a more compatible and reliable bond between the steroid and the perfluorocarbon particle to form a more stable membrane for the perfluorocarbon particle in the discontinuous phase of the emulsion. Red blood cells have cholesterol on their cell membranes removed to be joined with the membrane of the fluorocarbon particles, which form close union with and have an affinity for the fluorocarbon particles, it is believed. Fluorocarbon particles having a significant coating of the cholesterols will deter the removal of cholesterol from the red blood cells, it is believed. Somewhat similarly, tocopherols and steroid horomes enhance the stability of the membrane of the perfluorocarbon particle. The steroids nine-alpha-fluoro-16-alpha-methylprednisolone and nine-alpha-fluoro-16-beta-methylprednisolone, and other additional components if any are combined with them, should be included in an amount from 0.5 mg. to 5 mg. (or 0.0001 to 0.005 percent) weight per volume (w/v) in the emulsion. Six times this quantity of the steroid nine-alpha-fluoro-16-alpha-hydroxyprednisolone and combined additional components may be used. Three times the range given may be used if the steroid six-alpha-fluoro-16-alpha-methylprednisolone and any additional component is used. The actual amount of the additional component or components is a function of the contemplated dose, and of the amount of bromofluorocarbon in the ultimate emulsion. In this specification, the term "biocompatible" is used to denote that amount or quantity which is compatible with, and above which toxicity results in the biological system into which the emulsion containing the biocompatible element is to be introduced. There are biocompatible limits for steroids and cholesterols. It may be that additional amounts or quantities of the steroids and cholesterols are biocompatible, but the range given has been found to be sufficient to achieve the particle size stability and efficacious compatibility with red blood cells and other components in the blood stream and other fluid systems of the animal body. Other nutrients may be added to the ultimate emulsion, such as, for example, glucose, amino acids, proteins and lipids. Oxygen is highly soluble in the perfluorocarbons and in particular the mono-brominated perfluorocarbons of the present invention. In using the present invention as an oxygen transport medium, it is important to retain the oxygen as part of the perfluorocarbon particle for a reasonable period of time in order to transport the oxygen throughout the vascular system or to increase intravascular dwell time. It is found that the tocopherols such as the alpha-tocopherol, and water soluble analogs of tocopherols are suitable anti-oxidants which will retard rapid oxidation. Other anti-oxidants that are useful are ascorbic acid and calcium ascorbate. Adding anti-oxidants to the emulsion in an amount of from 0.01% to 0.5% weight per volume has been found useful to retard oxidation of the lipid emulsifier which diminishes the stability of the emulsion. Anti-oxidants also quench free radicals such as superoxide or hydroxyl atoms which are harmful to biological systems. For contrast enhancement use and for oxygen transport use internally in an animal, including humans in other than the blood stream, such as in the cerebrospinal system, in the eye and in the tracheobronchial passages, for example, larger particle sizes can be tolerated, and indeed may even be preferred. Such larger particle sizes may provide for a more even distribution of the gas, such as oxygen. Particles sizes of less than 400 nanometers diameter for the substantial portion, on the order of 95% of the particles, with a median particle diameter of less than 150 nanometers is to be preferred, however, for use in the blood stream. Effective oxygen unloading or de-oxygenation occurs in the blood stream primarily in the capillaries, and the small bromofluorocarbon particle size is advantageous in getting the oxygen to these capillaries. For these sizes for use in the blood stream, and even for the emulsions to be used in non-vascular systems, it is highly important to maintain particle size characteristics stable over extended periods of time, at least more than one month and of the order of eighteen months and more. I have achieved particle sizes of perfluorocarbon emulsions in commercially usable quantities having very small sizes or diameters on the order of hundreds of nanometers using conventional particle fractionalization methods, such as the homogenization techniques using the Gaulin mixer. Bromo-perfluorocarbon emulsions made with such a technique appear to be suitably stable where the concentration of the bromo-perfluorocarbon is relatively small, on the order of less than 50% weight per volume. Attempts using the Gaulin mixer to prepare commercially usable quantities of bromo-perfluorocarbon emulsions having w/v concentrations of 50%, 75% and more and having a median particle diameter size of less than 200 nanometers were unsuccessful. These higher concentration bromo-perfluorocarbon emulsions were observed to have a median particle diameter size of more than 200 nanometers. Long term, extended period of time small particle size stability of higher concentrations of mono-brominated perfluorocarbon emulsion in an aqueous phase with a phospholipid emulsifying agent has been found when the emulsion is formed or generated using a plural flow impingement apparatus. The aqueous phase was buffered with sodium mono-phosphate and sodium di-phosphate in such an amount to give a resultant emulsion pH of between 6.8 and 7.2. The aqueous phase, further, was in a solution of glycerol to control the osmolarity of the resultant emulsion for use in the blood stream. This buffered, aqueous phase solution in glycerol is sometimes designated the vehicle. The bromofluorocarbon was metered in a predetermined, measured rate into the vehicle or aqueous phase having the emulsifying agent mixed therein. The resulting mixture was placed into a flow path which was divided into a plurality of flow paths. The flows were redirected to impinge upon each other at velocities in excess of 1500 feet per second in sheets of interaction in a cavity under 4,000 pounds per square inch or more of pressure. The resulting bromofluorocarbon particles had a size characteristic of more than 95% smaller than 350 nanometers in diameter, with the median size diameter of less than 150 nanometers and, significantly, these size characteristics were maintained stable for up to sixteen months, and even after sterilization, such as by heat or by filtration. The present invention can be further understood by reference to the following illustrative examples. EXAMPLE I Exchange transfusions were performed in female rats weighing 180 to 220 grams. The rats were anesthetized and polyethylene catheters were inserted into the left or right jugular vein and carotid artery. After recovery from the anesthesia, the rats were placed into an atmosphere enriched with 50% to 60% oxygen. Blood was removed through the carotid artery cather and a comparable amount of the brominated perfluorocarbon emulsion comprising 25% w/v of perfluoroctylbromide, 4% w/v of lecithin, 0.04% w/v of L-alpha-tocopherol, 2.21% w/v of glycerol, 0.012% w/v of sodium di-phosphate, 0.057% w/v of sodium mono-phosphate and the aqueous phase. The transfusion was continued until the red blood cell count of the rat was reduced to 50% of the baseline value. The rats were kept in the oxygen enriched atmosphere for twenty-four hours, after which they were removed to the ordinary atmosphere. All rats survived for more than one month. EXAMPLE II The experiment of Example I was repeated, except that the brominated perfluorocarbon emulsion comprised 50% w/v of perfluoroctybromide. All other perameters were the same. All rats survived for more than one month. EXAMPLE III Balb C mice were administered intravenously the brominated perfluorocarbon emulsion at doses of 45 grams per kilogram of body weight, and were administered intraperitoneally the brominated perfluorocarbon emulsion in doses of 100 grams per kilogram of body weight. The emulsion comprised 100% w/v of perfluoroctylbromide, 9.1% w/v of lecithin, 0.02% w/v of 6-alpha-fluoro-16-alpha-methylprednisolone, 0.2%% w/v of alpha-tocopherol, 1.0% w/v of glycerol, 0.012% w/v of soduim di-phosphate, 0.057% w/v of sodium mono-phosphate and the aqueous phase. After seven days, the liver and spleen were enlarged, but the peritoneal cavity showed no signs of inflammation, and the lungs were normal and filled with oxygen. There were no signs of hemorrhage or pulmonary congestion, or of inflammation of the tissues of the abdominal wall. EXAMPLE IV A mono-brominated perfluorocarbon emulsion comprising 100% w/v of perfluoroctylbromide, 9.1% w/v of lecithin, 0.02% w/v of 6-alpha-fluoro-16-alpha-methylprednisolone, 0.2%% w/v of alpha-tocopherol, 1.0% w/v of glycerol, 0.012% w/v of soduim di-phosphate, 0.057% w/v of sodium mono-phosphate and the aqueous phase was prepared by first preparing the vehicle of the continuous or aqueous phase by blending in the lecithin, the 6-alpha-fluoro-16-alpha-methylprednisolone, the alpha-tocopherol, the glycerol, the soduim di-phosphate, sodium mono-phosphate. The perfluoroctylbromide was added in a measured rate into the vehicle while mixing. The resulting emulsion at 10 degrees C. was passed through a microfluidizing apparatus in the method described herein where a plurality of flows of the emulsion were impinged upon each other at velocities in excess of 1500 feet per second, for fifteen passes. The particle size distribution was analyzed in a Nicomp sub-micron particle sizer manufactured by Pacific Scientific Co. of Anaheim, California. This analyzer determines relative quantities of various sized particles by a method of dynamic light scattering. Results according to the following Table I were given, where the first column represents the diameter of the particles in nanometers, and the second column represents a quantitative value of the relative quantity of the particles detected at the corresponding particle size: The emulsion was then sterilized at 90 degrees C. for fifteen minutes. After sterization, the Nicomp emulsion particle size characteristics were measured on the Nicomp particle sizer. The results, given in the Table II below where the columns represent the same characteristics as set forth for Table I, showed no significant particle size characteristic deterioration or change: EXAMPLE V An emulsion particle size stability over an extended period of time was studied by analyzing the particle size distribution in a Nicomp sub-micron particle sizer identified above for the Example IV. The brominated perfluorocarbon emulsion first was made by the methods described above and comprised 25% w/v of perfluoroctylbromide, 4% w/v of lecithin, 0.04% w/v of L-alpha-tocopherol, 2.21% w/v of glycerol, 0.012% w/v of sodium di-phosphate, 0.057% w/v of sodium mono-phosphate and the aqueous phase. The emulsion was analyzed shortly after formulation, and the relative quantities of the emulsion's particle sizes is given in the following Table III, where the scale and columns represent the same characteristics as set forth for the tables in Example IV: The emulsion was stored at 4 degrees C., although for various interpretations during the time of storage the temperature was changed, frequently being very substantially higher. A second and substantially identical analysis was made using the Nicomp particle sizer as described above some fifteen months and 22 days after the analysis given in Table III. The results of this second analysis is given in the following Table IV, where the scale and columns represent the same characteristics as set forth for the tables in Example IV:

Claims: Claims for the following Contracting States : BE, CH, DE, FR, GB, IT, LI, LU, NL, SE A brominated perfluorocarbon (BPFC) aqueous phase emulsion, having a BPFC Content of greater than 50W/V%, with a minor amount of an emulsifying agent and a biocompatible quantity of a compound selected from steroid hormone, cholesterol, tocopherol and combinations thereof, and having long term particle size stability and being acceptable for oxygen transport or contrast enhancement in the human or animal body. A monobrominated perfluorocarbon (BPFC) aqueous phase emulsion, having a BPFC content of greater than 50W/V%, with a minor amount of an emulsifying agent, and having long term particle size stability and being acceptable for oxygen transport or contrast enhancement in the human or animal body. An emulsion according to claim 2, having long term stability in the non-frozen state. An emulsion according to claim 2 or claim 3, having long term stability following heat sterilization. A brominated perfluorocarbon (BPFC) aqueous phase emulsion, having a BPFC content of greater than 50 W/V%, with a minor amount of an emulsifying agent, wherein in the non-frozen state after heat sterilization 95% of the emulsified perfluorocarbon exists in particle sizes of less than 400 nanometres with a mean diameter of less than 150 nanometres. An emulsion according to any one of claims 2 to 5 further comprising a biocompatible quantity of a compound selected from steroid hormone, cholesterol, tocopherol and combinations thereof. An emulsion according to claim 6 wherein the selected compound is a combination of steroid hormone and cholesterol. An emulsion according to claim 7 wherein said steroid hormone is a fluorinated steroid. An emulsion according to claim 8, wherein the steroid is fluorinated in the 9-alpha or the 6-alpha position. An emulsion according to any one of claims 7 to 9 wherein the steroid is selected from 9-alpha-fluoro-16-alpha methyl-prednisolone, 9-alpha-fluoro-16-beta-methyl-prednisolone, 9-alpha-fluoro-16-alpha-hydroxy-prednisolone, 6-alpha-fluoro-16-alpha-methyl-prednisolone and combinations thereof. An emulsion according to any one of the preceding claims which has said acceptable particle size stability over a period of more than one month. An emulsion according to any one of the preceding claims which has said acceptable particle size stability over a period of about 16 months or more. An emulsion according to any one of the preceding claims, comprising an emulsifying agent which is a phospholipid or an anionic surfactant. An emulsion according to claim 13 wherein the anionic surfactant is a polyoxyethylene-polyoxpropylene copolymer. An emulsion according to claim 13 wherein the emulsifying agent is a fluorinated surfactant. An emulsion according to any one of the preceding claims comprising an anti-oxidant. An emulsion according to claim 16 wherein said anti-oxidant is selected from tocopherol and water soluble analogs thereof or is selected from ascorbic acid and calcium ascorbate. An emulsion according to any one of the preceding claims wherein the BPFC content is in the range 75-125 W/V%. An emulsion according to claim 18 wherein the BPFC content is in the range 100-125 W/V%. An emulsion according to any one of the preceding claims wherein the BPFC comprises perfluorooctylbromide (PFOB). A process for preparing an emulsin of any one of the preceding claims, comprising the steps of: (a) mixing the BPFC with an emulsifier in an aqueous phase (b) passing the BPFC-containing mixture through emulsification apparatus in which the mixture is subjected to a flow rate over 457 m/second (1500 feet/second) and pressure of at least 280 kg/cm 2 (4000 pounds per square inch) sufficient to form the stable emulsion. A process according to claim 21 wherein in step (b) the mixture is forced into a plurality of flow paths which are redirected into each other. A process according to claim 21 or claim 22 wherein the mixture is passed through the emulsification apparatus a plurality of times until said stable emulsion is obtained. Claims for the following Contracting States : AT, ES A process which comprises forming a brominated perfluorocarbon (BPFC) aqueous phase emulsion, having a BPFC content of greater than 50W/V%, with a minor amount of an emulsifying agent and a biocompatible quantity of a compound selected from steroid hormone, cholesterol, tocopherol and combinations thereof, and having long term particle size stability and being acceptable for oxygen transport or contrast enhancement in the human or animal body. A process which comprises forming a monobrominated perfluorocarbon (BPFC) aqueous phase emulsion, having a BPFC content of greater than 50W/V%, with a minor amount of an emulsifying agent, and having long term particle size stability and being acceptable for oxygen transport or contrast enhancement in the human or animal body. A process according to claim 2, wherein the emulsion has long term particle size stability in the non-frozen state. A process according to claim 2 or claim 3, wherein the emulsion has long term particle size stability following heat sterilization. A process which comprises forming a brominated perfluorocarbon (BPFC) aqueous phase emulsion, having a BPFC content of greater than 50 W/V%, with a minor amount of an emulsifying agent, wherein in the non-frozen state after heat sterilization 95% of the emulsified perfluorocarbon exists in particle sizes of less than 400 nanometres with a mean diameter of less than 150 nanometres. A process according to any one of claims 2 to 5, wherein the emulsion further comprises a biocompatible quantity of a compound selected from steroid hormone, cholesterol, tocopherol and combinations thereof. A process according to claim 6, wherein the selected compound is a combination of steroid hormone and cholesterol. A process according to claim 7, wherein said steroid hormone is a fluorinated steroid. A process according to claim 8, wherein the steroid is fluorinated in the 9-alpha or the 6-alpha position. A process according to any one of claims 7 to 9, wherein the steroid is selected from 9-alpha-fluoro-16-alpha methyl-prednisolone, 9-alpha-fluoro-16-beta-methyl-prednisolone, 9-alpha-fluoro-16-alpha-hydroxy-prednisolone, 6-alpha-fluoro-16-alpha-methyl-prednisolone and combinations thereof. A process according to any one of the preceding claims wherein the emulsion has said acceptable particle size stability over a period of more than one month. A process according to any one of the preceding claims wherein the emulsion has said acceptable particle size stability over a period of about 16 months or more. A process according to any one of the preceding claims, wherein the emulsion comprises an emulsifying agent which is a phospholipid or an anionic surfactant. A process according to claim, 13 wherein the anionic surfactant is a polyoxyethylene-polyoxpropylene copolymer. A process according to claim 13 wherein the emulsifying agent is a fluorinated surfactant. A process according to any one of the preceding claims wherein the emulsion comprises an anti-oxidant. A process according to claim 16 wherein said anti-oxidant is selected from tocopherol and water soluble analogs thereof or is selected from ascorbic acid and calcium ascorbate. A process according to any one of the preceding claims wherein the BPFC content is in the range 75-125 W/V%. A process according to claim 18 wherein the BPFC content is in the range 100-125 W/V%. A process according to any one of the preceding claims wherein the BPFC comprises perfluorooctylbromide (PFOB). A process of any one of the preceding claims, comprising the steps of: (a) mixing the BPFC with an emulsifier in an aqueous phase (b) passing the BPFC-containing mixture through emulsification apparatus in which the mixture is subjected to a flow rate over 457 m/second (1500 feet/second) and pressure of at least 280 kg/cm 2 (4000 pounds per square inch) sufficient to form the stable emulsion. A process according to claim 21 wherein in step (b) the mixture is forced into a plurality of flow paths which are redirected into each other. A process according to claim 21 or claim 22 wherein the mixture is passed through the emulsification apparatus a plurality of times until said stable emulsion is obtained.

Inventors: LONG DAVID M JR, LONG, DAVID M. JR.
Applicants: ALLIANCE PHARMA, ALLIANCE PHARMACEUTICAL CORP.
Application year: 1987
Main classification: A61K9
IPC codes: A61K9, A61K49, A61K31