US 3629071 A
Description (OCR text may contain errors)
United States Patent Sekhar, N. C. J. Medicinal Chem. 13: 39- 44 (1970) Eflnventor Neelkant C. Selghar Kalamazoo, Mich. App]. No. 10,318 Filed Feb. 10, 1970 Patented Dec. 21, 1971 Assignee The Upjohn Company Kalamazoo, Mich.
STORAGE-STABLE HEMOSTATIC TRANSFUSION SUSPENSIONS OF BLOOD PLATELETS, GLUCOSE, MAGNESIUM CHLORIDE AND CERTAIN PROSTAGLANDINS l Claim, No Drawings US. Cl 195/].8,
feet of Eight Prostaglandins on Platelet Aggregation."
Elkeles, R. S. et al., Lancet 2: l 1 l (1969) Prostaglandin E1 and Human Platelets."
O Brien, .1. R. Lancet 1: 149 (1968) Prostaglandins and Platelets."
Crevelds, von et al., Nature 218: 361- 362 (I968) lnfluence of the Prostaglandins E1 and E2 on Aggregation of Blood Platelets.
Chandrasekhar, N. Blood 30: 554 (1967) Inhibition of Platelet Aggregation by Prostaglandins."
Emmons, P. R. et al., BR. Med. J. 2: 468-472 (1967) Effect of Prostaglandin E1 on Platelet Behavior in vitro and in vivo" Primary Examiner-Shep K. Rose Attorneys-Edward G. Jones and John Kekich I ABSTRACT: The invention provides storage-stable aqueous STORAGE-STABLE HEMOSTATIC TRANSFUSION SUSPENSIONS OF BLOOD PLATELETS, GLUCOSE, MAGNESIUM CHLORIDE AND CERTAIN PROSTAGLANDINS BACKGROUND OF THE INVENTION A crude mixture, called prostaglandin, was reported by von Euler, Arch. Exp. Path, Pharm Abs. 175-78 (1934); 181 (1936); J. Physiol 72,74 (1931); 81,102 (1934); 84,21 (1935); 88,213 (1936); and Klin, Wschr 14, 1182 (1935). More recently essentially pure crystalline PGF (PGF or PGF a) has been isolated, British Pat. No. 851,827 and Acta Chemica Scandinavica 14, 1693 (1960). Microbiological conversions of unsaturated fatty acids with mammalian glandular tissue are described a in U.S. Pat. Nos. 3,290,226 and 3,296,091. In the latter patent PGF (PGF or PGF,a) is designated as 7-[3a, 5a-dihydroxy-2-(3-hydroxy-1-ocetenyl)- cyclopentyl1-heptanoic acid.
The PGF-type prostaglandins are characterized by the presence of the hydroxyl group at the five-position in the cyclopentane ring. The designation PGF a shows the configuration of the hydroxyl at the five-position. Various other members of the PGF-type are known and are named either systematically or in terms of their relationship to PGF. Illustrative thereof are PGF a or 7-[3a-5a-dihydroxy-2-(3-hydroxy-1-octenyl)-cyclopentyll-5-heptenoic acid, PGF or 7-[3a,5adihydroxy-2-( 3-hydroxy-l ,5-octadienyl )-cyclopentyl]-5-heptenoic acid, and dihydro PGF,a or 7-[3a, 5a-dihydroxy-2-(3- hydroxyoctyl)-cyclopentyll heptanoic acid. Details of preparations from available materials are disclosed for dihydro PGF a, PGF a, and PGF a in Biochimica and Biophysica Acta, 84, 707 (1964), and for PGF a in U.S. Pat. No. 3,069,322. Bergstrom, Carlson and Weeks, Pharmacological Reviews, Vol. 20, No. 1, P. 1 (1968) review The Prostaglandins.
In U.S. Pat. No. 3,290,226 PGE compounds are described including PGE,, PGE and PGE The PGE Series is characterized by the presence of the keto group at the five-position in the cyclopentane ring. More recently, Ramwell et al., Prostaglandins" in Progress in the Chemistry of Fats and other Lipids, Vol. 9 edited by R. Holman, pp. 73-115, Pergamon Press, Oxford, 1967 refer to prostaglandin PGE as I la, l5(S)-dihydroxy-9-oxo-l 3-trans-prostenoic acid, PGE as 1 la, l5( S )-dihydroxy-9-oxo-5-cis, l 3-trans-prostadienoic acid and PGE as 1 111,15(S)-dihydroxy-9-oxo-5-cis,13-trans, 17- cis-prostatrienoic acid.
PGE l5-formate is prepared as follows: A solution of sodium carbonate (50 mg.) in 7.5 ml. of dry formic acid is added to 250 mg. of PGE,. This mixture is stirred under nitrogen at 25 C. for 2 hours. The reaction mixture is evaporated under reduced pressure. Benzene is added to the residue and the mixture is again evaporated under reduced pressure. The residue is then chromatographed on 50 g. of silica gel (acid washed to pH 4; 100-200 U.S. mesh; Mallinckrodt Silicar CC-4), eluting with 2.5 1. ofa gradient of 25 to 75 percent ethyl acetate-isomeric hexane mixture (Skellysolve B), and collecting l00-m1. fractions. Eluate fractions 9, 10, and 11 are combined and evaporated to dryness under reduced pressure to give 99 mg. of PGE 15-formate. 8-iso-PGE, is described by Daniels, et al., J. Am. Chem. Soc. 90, 5894 (I968); dl-8-Iso- PGE,, methylester is described by Schneider et al., .I. Am. Chem. Soc. 90, 5896 (1968); and ll-dehydro-PGE a is described by Lands et al., J. Biol. Chem. 243, 4104 (1968).
Excepting PGE these prostaglandins are to be construed as including optically active compounds of the natural configuration and racemic compounds. These compounds are known in the art or are available by methods known in the art. See U.S. Pat. No. 3,296,091; Rec. Trav. Chim. 85; 1233 (1966), Ibid. 87, 461 (1968); .I. Am. Chem. Soc. 90, 5895 (1968). For the racemic compounds, see, for example, Chemical Communications, pp. 303-305 (1969).
Pharmaceutically acceptable salts for example, those of a1- kali metals and alkaline earth bases, such as the sodium, potassium, calcium and magnesium salts; those of ammonia or a basic amine such as mono-, di-, and triethyl amines, benzylamine, heterocyclic amines such as piperidine and morpholine, and amines containing water-solubilizing or hydrophilic groups such as triethanolamine and phenylmonoethanolamine are disclosed in U.S. Pat. No. 3,296,091. Carboxylate esters such as methyl, ethyl, cyclohexyl and the like having no more than eight carbon atoms are formed by the usual methods, e.g., reaction with diazomethane or similar diazohydrocarbons as in U.S. Pat. No. 3,296,091.
Biological studies of the prostaglandins, for example, actions on smooth muscle, reproductive system, nervous system, cardiovascular system, and relationship to lipid and carbohydrate metabolism, and miscellaneous effects are summarized by Bergstrom et al.; The Prostaglandins: A Family of Biologically Active Lipids, Pharmacological Reviews, Vol. 20, No. l, P.1 et sequitur, 1968, The Williams and Wilkins Company.
Blood platelets when stored for a few hours, and then infused, lose most of their ability to perform the hemostatic function. Consequently, all thrombocytopenias (congenital as well as drug-induced) must be treated with freshly prepared platelet suspensions. The instability of the physiological properties of platelets results in total waste of unused platelet suspensions and also presents a major problem in the treatment of thrombocytopenias. Inhibition of platelet aggregation by some prostaglandins, including POE has been demonstrated in various investigations. Kloeze, .l., Nobel Symposium 2, Prostaglandins, Ed. 5. Bergstrom and B. Samuelson, pp. 24l252. Almquist and Wiksell, Stockholm (1967). Emmons, P. R., Hampton, J. R., Harrison, M. .l. R., Honour, A. J., and Mitchell, J. R. A., Brit. Med. J., 2, 468 (I967). Chandrasekhar, N., Blood, 30, 554 (1967). However, until the present invention, storage-stable suspensions of platelets both human and animal were not available.
SUMMARY OF THE INVENTION This invention relates to the preservation of the hemostatic function of blood platelets. More specifically, it relates to storage-stable aqueous suspensions of mammalian blood platelets, for example, human and animal such as rabbit, rat, and monkey. The aqueous suspensions consist essentially of said platelets and a prostaglandin selected from the group consisting of PGE.; POE 15-formate; 8-Iso-PGE,; dI-8-lso-PGE methylester; and l l-dehydro-PGF,a.
The aqueous suspensions of the platelets are preferably in the form of platelet-rich plasma or an aqueous suspension in 0.9 percent NaCl. The amount of prostaglandin for preservation of the hemostatic function of the platelets is within the nontoxic effective range from about 0.025 ug./ml. to about 1.0 mg./ml.
Additional ingredients complementing the preservative action of the prostaglandin component are glucose from about I to about 2 mg./ml. and from about 2.5 to about 10 milliequivalents of Mg in the form of magnesium chloride. In the suspensions the hemostatic activity of the platelets is preserved permitting their use in clinical conditions such as idiopathic thrombocytopenia and drug-related thrombocytopenia sometimes accompanying therapy with, for example, chloramphenicol, mustard-type cytotoxic agents, and cobalt treatments. Normally the human platelet count is from about 300,000 to about 500,000 per ml. In severe thrombocytopenia it may be depressed to about 50,000 per liter or lower necessitating infusion of 5 to 6 liters of preserved platelet suspension containing the effective nontoxic amount of prostaglandin.
DESCRIPTION OF THE PREFERRED EMBODIMENTS A model system involving a decrease in the number of platelets in rats is used to show the storage stability of blood platelets in combination with the prostaglandin component.
Platelet counts of Platelet-Rich Plasma obtained from Rats X- Irradiated at four different levels (three animals per group; Mean Counts expressed as Xl /mm.
No. of days following X- lrradiation X-lrradiation Level 100 R 200 R 300 R 400 R Day 5 1,964t80 1,487t33 1,151t162 1,2422161 Day 7 1,8091-73 1,372t69 1109-552 9851140 Day 9 1,1271-137 880x87 593:97 471x105 Day II 1,8161232 1,3Z1t91 1,17421269 927195 Three groups (12 rats/group) of mature male Sprague- Dawley rats are X-irradiated at 300 R. On the 6th day following X-irradiation, fresh platelet-rich plasma (PRP) is collected by centrifuging citrated blood (from another set of nonirradiated Sprague-Dawley rats) at 1200 r.p.m. for 10 minutes. One aliquot of the PRP is incubated with PGE (I00 ug./ml.) for 48 hr. at 5. Another aliquot of PRP is incubated with an equal volume of physiological saline (positive control). On the 8th day following X-irradiation, another sample of fresh PRP is obtained. One group of 12 rats (negative controls) is given tail vein injections of the freshly prepared PRP (four injections of 2 ml. each, equally spaced within 8 hr.). Another group (positive controls) is given similar injections of saline-incubated PRP and the third group (experimental) is given PGE -incubated PRP. All rats are sacrificed 24 hr. after infusion and the platelet aggregation (collagen-induced) of their PRP samples is determined using the Aggregometer. Results show that negative controls with fresh platelets give the maximum aggregation values and the positive control group with saline-incubated PRP shows the poorest aggregation values compared to the positive control group, indicating that PGE preserves the hemostatic function of stored platelets.
Platelet-Aggregation in Thromocytopenic Rats after PRP- Infusion Treatment Collagemlnduced Aggregation in 5 Min.
Negative Control 8. 1:0.7
Positive Control 3.0105
Experimental (PGE incubated PRP) 3.710.!
INHIBITION OF PLATELET AGGREGATION to yield a 10 mg./ml. solution. Acidic solutions are then diluted to l mg./ml. concentration, with a 0.2 mg./ml. sodium carbonate solution; esters are diluted to a concentration of l mg./ml. with 0.9 percent sodium chloride. Subsequent dilutions of all prostaglandins are accomplished with 0.9 percent sodium chloride; freshly prepared solutions only are used in the experiments. Platelet-rich plasma (PRP) is used as the source oflplatelets. Freshl drawn cit'rated blood samples from adult mae Sprague-Daw ey rats (Spartan strain) are centrifuged at 1200 r.p.m. for 10 min. to obtain rat PRP. Three aggregation inducers are used: (I) 0.25M calcium chloride solution; (2) 50 mg./ml. adenosine diphosphate solution (ADP) freshly prepared using Ringer's solution; (3) collagen suspension made up in Tyrode solution without calcium.
Two methods are employed to measure platelet aggregation parameters: a modification of Chandler's visual technique using a revolving plastic loop and a photo-optical method using an Aggregometer (Chronolog Corporation, Broomall, Pa.). For the Chandler's loop method, 0.8 ml. volume of PRP is taken up in a plastic loop (Transflex No. 8, 3M Company), 0.1 ml. of a 1 mg. per ml. solution of the prostaglandin is added to the plasma and the loop is rotated at 12 r.p.m. for 1 minute to assure proper mixing. An 0.] ml. volume of 0.25M calcium chloride is then injected onto the plasma layer at one end of the loop and three stop watches and the loop are started simultaneously. Three end points which occur in succession are recorded: l aggregation of platelets visible to the naked eye (VA); (2) appearance of snowstormlike platelet clumps (SS); and (3) a well-packed platelet disc or head at one end of the plasma surface (PH). Similar experiments are carried out using physiological saline in place of prostaglandin solution, to obtain control values. If VA obtained with prostaglandin is greater than 10 seconds from that of the controls, successive dilutions of prostaglandins are made and tested. The lowest concentration of prostaglandin that prolongs VA for more than 10 seconds is tested on three separate rat PRP samples and the mean and standard error of VA, SS and PH are recorded.
Platelet aggregation phenomenon induced by ADP and collagen is studied in the Aggregometer. Freshly obtained rat PRP (0.8 ml.) is mixed with 0.1 ml. of the prostaglandin solution in an Aggregometer cuvette for 1 minute. O.l ml. of the collagen suspension is pipeted into this cuvette to induce aggregation. Platelet aggregation/adhesion that takes place in the cuvette produces changes in optical density which are recorded on a Bausch and Lomb VOM 6 recorder. The height of the curve recorded is taken as the aggregation index. Identical experiments are done using PRP from control animals also. If the aggregation index for the experimental samples is less than percent of that of the controls, prostaglandin (at that particular concentration) is considered active.
All prostaglandins are initially tested in the Chandler's loop to determine the lowest concentration that is active. The compound is again tested in the Aggregometer at the lowest active concentration, against ADP and collagen.
1. A storage-stable aqueous suspension in isotonic saline with effective complementary hemostatic activity preserving concentrations of glucose and magnesium chloride for transfusion consisting essentially of mammalian blood platelets and a prostaglandin selected from the group consisting of P615 PGE,-l5 formate; 8-Iso-PGE,, dl-8-Iso-PGE methylester', and l l-dehydro-PGF,a, the amount of the prostaglandin being within the nontoxic effective range from about 0.025 ug./ml. to about 1 mg./ml. of the suspension.