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    Us 5173488

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    Pingol Pham
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    United States Patent 11») Haeger AQUA CAA AA (S4]_ STABLE INJECTABLE PHARMACEUTICAL FORMULATION FOR FOLIC ACID AND LEUCOVORIN SALTS AND METHOD 175] Inventor: Bruce E, Haeger, Highland Mills, NY. (73] Assignee: American Cyanamid Company, Stamford, Conn, [21] Appl. No.: 696,335, [22] Filed: May 1, 1991 Related US. Application Data [63] Continuation of Ser. No. 396,573, Aug. 21, 1989, aban- doned. [51] Int. cus .. A6IK 31/508; A6IK 47/10; ‘AGIK 47/20; AIK 47/18 [2] Us. c. snes 514/2495 514/973; 544/258 [58] Field of Search ..-.cuoue $14/249, 973; 548/258 {56 References Cited US. PATENT DOCUMENTS 1,926797 9/1933 Sutton . 514/706 2688018 8/1954 Cosulich vee 544/258 2,695,860 11/1984 Weidenheimer eta. su4/2s8 2741,608 8/1956 Shive 548/258 US00S173488A, {11} Patent Number: 5,173,488 (45] Date of Patent: Dec, 22, 1992 3,696,195 10/1972. Crivellaro etal. 514/973 4071620 1/1978 Sklar 44/175 4,500,711 2/1985 Wisowaty et al 544/258 4,931,441 6/1990 Lawrence 5147249 FOREIGN PATENT DOCUMENTS WO88/04927 1/1988 PCT Int'l Appl. - OTHER PUBLICATIONS Physicians Desk Reference (Oradell, N.J, Medical Economics Co, 1988) pp. 1148-1152. Durst, et al. Clinical Chemistry 18, (3) 1972, pp. 206-208, United States Pharmacopeia, XX, 1980, pp. 832-833, and 1237. Primary Examiner-—Robert T. Bond Attorney, Agent, or Firm—Thomas S. Szatkowski 157) ABSTRACT Injectable aqueous compositions comprising folic acid and leucovorin and their salts, optionally including benzyl alcohol, sodium chloride and agents for adjust- ing pH are stabilized and buffered in the range of 6to 10 by adding a combination of tromethamine and monoth glycerol. Such compositions remain stable for pro- longed periods even when exposed to sunlight, 20 Claims, No Drawings 5,173,488 1 STABLE INJECTABLE PHARMACEUTICAL FORMULATION FOR FOLIC ACID AND LEUCOVORIN SALTS AND METHOD. This is a continuation of copending application Ser. ‘No. 07/396,573 filed on Aug. 21, 1989, now abandoned. The present invention relates to new improved injec- table compositions comprising folic acid salts and leu- covorin salts. More particularly, it relates to composi- tions comprising folic acid salts and leucovorin salts that remain stable for prolonged periods under normal storage conditions because they include tromethamine and monothioglycerol as a buffering agent/antioxidant combination. BACKGROUND OF THE INVENTION Folic acid and its salts and leucovorin and its salts are known to be pharmaceutically effective. See, Reming- ton’s Pharmaceutical Sciences, Seventeenth Edition, Mack Publishing Co., Easton, PA 1985 (Remington's 17th Ed) p. 1023. Folic acid is used to treat vitamin deficiencies. Cosulich, U.S, Pat. No. 2,688,018 describes the preparation of such compounds and their clinical use for controlling the toxicity of aminopterin and other antifolic acid compounds and as hematopoietic drugs. Active derivatives of such compounds are described in Shive, U.S. Pat. No. 2,741,608. In U.S. Pat. No. 4,500,711, Wisowaty et al., describe the purification of Ieucovorin and its salts. Kerwar et al., U.S. Pat. No. 4,746,662 disclose that the antiarthritic efficacy of meth- otrexate can be potentiated by injection of an aqueous solution of leucovorin or its salts. EPO Patent Publica- tion No. 0,266,042, May 4, 1988, describes using pure leucovorin isomers to manufacture medicaments for methotrexate rescue, for treatment of colorectal cancer in combination with 5-fluorouracil, and for treating folate deficiency. Both folic acid and leucovorin are only sparingly soluble in water. Therefore they are administered in the form of salts such as alkaline metal and alkaline earth ‘metal salts, such as the sodium salt of folic acid and the calcium salt of leucovorin, the l-isomer of the latter being preferred. ‘The compound N-4((2-Amino -1,4-Dihydro-4-ox0- 6-pteridinyl)methyl) -amino)(ben2oyl)-L-glutamic acid, sodium salt (Sodium Folate) having the formula 1s 50 ® i TX s wd \-con ° | HooccHcHy is used principally to stimulate specifically the produc- tion of red blood cells, white blood cells and platelets in persons suffering from certain megaloblastic anemias. ‘The compound N-(4-((2-Amino-S-formyl -1,4,5,6,7,8- hexahydro-4-oxo-6-pteridinyl)methyl)amino)benzoyl)- L-glutamic acid, calcium salt (1:1), pentahydrate, (Leu- ‘covorin Calcium USP) having the formula: 6 sgooecticry~ d -cooca 7 h is used principally as an antidote for folic acid antago- nists such as methotrexate, which block the conversion of folic acid into folnic acid. Merck Index, Tenth Edi- tion, p. 603, Both folic acid and leucovorin salts are formulated in water for injection and may contain suit- able preservatives, as described under Folvite @ Injec- tion and Leucovorin Calcium Injection in the Phys: cian’s Desk Reference, Medical Economics Company, Oradell, NJ 1989 (PDR) pp. 1120 and 1124, respec: tively ‘Both compounds, but especially Leucovorin Cal- cium, require an alkaline pH of 7.7-82 for maximum stability, They are also light sensitive and prone to oxi- dative degradation in aqueous solution, thus requiring the use of amber glass for protection against photosensi- tivity and the use of nitrogen gas as a protectant throughout the bulk liquid manufacturing process and as a package headspace gas. In the past, methy] and propyl parabens (p-hydroxy benzoates) were used as a preservative for such salts especially Leucovorin Calcium Injection. However, the parabens were later found to have short term effective- ness atthe alkaline pH required for maximum folic a and/or leucovorin stability. Subsequently, benzyl aleo- hhol was approved and widely used as the new preserva- tive for the products: See “Folvite @ Folic Acid Solu- tion and *"Leucovorin Calcium Injection”, PDR, pp. 1120 and 1124 respectively. Although benzyl alcohol is effective as a preservative, it lacks the mild buffering activity afforded by the parabens. As a consequence the PH of the products tend to be somewhat unstable, Slowly drifting downward with time toward pH 6.5-1.0. This is below the above-mentioned optimum range and may require expiration dating to be very short term. Tt has now been found that the factors of pH drift, nitrogen headspace variability and short term expiry ating can be overcome in accordance with this inven- tion by using a buffer/antioxidant combination. The buffer comprises 2-Amino-2-(hydroxymethyl)-1, 3+ propanedial, also known as tromethamine. The antioxi- Gant comprises 3-Mercapto-1,2-propanediol, also known as monothioglycerol Both compounds, tromethamine and monothio- tlycerol, are toxicologically acceptable, and are de- scribed in U.S. Pharmacopeia XXI, U.S, Pharmacopeial Convention, Rockville, MD 1985 (U.S.P. XI) at pages 1102 and 1580, respectively. Surprisingly, the new buffer/antioxidant composi- tions of the present invention, for tromethamine and monothioglycerol, have been found to be superior to the presently used formulations, in terms of ability to control pH in the desired region of folic acid and leu- ‘covorin stability, and in their ability to retard oxidation, fand therefore degradation, in sealed dosage forms These new buffer/antioxidant compositions make it 5,173,488 3 possible to extend the expiry dating of the product and provide the potential for development of a broader dosage line for such products, e.g. single or multi-dose ials containing greater volumes and higher concentra- tions of either folic acid or leucovorin salts than the 5 resent single dose (3-5 mg/ml) ampuls, such as a 10 ‘mg/ml, 100 mg/vial dosage preparation. Unexpectedly also, benzyl alcohol becomes an optional ingredient and this is desirable expecially in cases where large doses are needed in emergencies and too much benzyl alcohol is not recommended. SUMMARY OF THE INVENTION According to the present invention there are pro- ided stable, injectable aqueous compositions compris- 4 (i) an effective amount of water-soluble pharmaceuti- cally-acceptable salt of folic acid or leucovorin; option- ally, (i) a small, effective preservative amount of benzyl alcohol; and i) an effective amount of a buffer/antioxidant com- bination comprising (a) tromethamine and (b) monothi- glycerol, said combination (ji) being present in an amount at least sufficient to maintain the pH of said ‘composition in a predetermined range of from about 6 to about 10 and to protect the composition against deg- radation induced by oxygen or light. In preferred embodiments, the compositions com- prise those wherein said salt comprises a salt of dl- leucovorin; those wherein the compositions also include Gv) sodium chloride in an amount sufficient to render said composition isotonic; those which also include (v) a pH adjustor comprising an acid of a base in amount sufficient to adjust the pH to any value within said range; those wherein said pH adjustor comprises hydro- chloric acid or sodium hydroxide. Especially preferred are compositions wherein said I-leucovorin salt com- prises calcium leucovorin. Special mention is made of a formulation comprising from 3 mg/ml to 25 mg/ml of calcium leucovorin in an isotonic solution at a pH of from 6.5 to 8.5, with or without benzyl alcohol at a concentration of from 0.0% w/v to 0.909% w/v, TRIS (tromethamine) at a concentration of from 0.10% w/v t0 0.30% w/v, monothioglycerol at a concentration of 48, from 0.10% w/v to 0.30% w/v; sodium chloride at a concentration from 0.45% to 0.65% w/v, with hydro- chioric acid at a concentration of from 5.0 v/v% to 10.0, v/v% and sodium hydroxide at a concentration of from 1.0 w/v% to 5.0 w/v% added as required to adjust the pH; and particularly those in which the concentration of calcium leucovorin on the one hand is 3.0 mg to 3.54 mg/ml and 10 mg to 11 mg/ml, on the other, and the concentrations of benzyl alcohol, TRIS (trometha- ‘mine), monothioglycerol and sodium chloride are from 0.768% w/v to 1.035% w/v, 0.150% w/v, 0.200% w/v ‘and 0.560% w/v, respectively. ‘The present invention also contemplates a method for stabilizing compositions as above defined “comprising adding thereto an effective amount of the buffer/antiox- ‘dant combination comprising tromethamine and mono- thioglycerol. DETAILED DESCRIPTION OF THE INVENTION The injectable composition of this invention can be prepared by techniques well known to those skille the art of pharmaceutical formulation. The substantially 10 25 38 o 0 3 o 6 4 pure silts may be prepared, mixed with the other com- ponents, filtered, filled into containers, and sealed under aseptic conditions. Folic acid and its salts can be prepared by any conve- nient method, for example 2,3-dibromopropionalde- hyde, dissolved in a water miscible organic solvent (alcohol, dioxane), is added to a solution of equal molec lar quanie of 245.ramino--byéroxypyrimidine and p-aminobenzylgluiamic acid, maintaining a pH of about 4 by the controlled addition of alkali as the reac- tion progresses. If sodium hydroxide is used as the al- kali sodium folate is obtained. To make calcium leucov- rin, folic acid is, for example simultaneously hydroge- nated and formulated in 90 to 100% formic acid under the influence of platinum oxide catalyst to yield leucov- rin. Conversion to the calcium salt may be accom- plished by dissolving the leucovorin in sodium hydro ide solution, treating the calcium chloride, and preci tating with ethanol Benzyl alcohol is an item of commerce and, if used, is widely available from a number of sources (Reming ton’s 17th Ed. p. 1057). 1,3-Propanediol, 2-amino-(2-hydroxymethyl) -(tromethamine) is available commercially (Reming- ton’s 17th Ed. p. 836). It can be made by additively reacting nitromethane with formaldehyde to yield tris (hydroxymethyl) nitromethane, and the nitro com- pound is then hydrogenated with Raney nickel in accor- dance with U.S. Pat. No. 2,174,242. 3-Mercapto-1,2-propanediol (monothioglycerol) is readily made, for example, by heating an ethanolic solu- tion of 3-chloro-1,2-propanediol with potassium bisul- fite (Remington's 17th Ed. p. 1279). ‘The amounts of the respective components can vary feirly broadly, within conventional limits well known to ‘those skilled in this art Preferred embodiments will be exemplified hereinafter. Typically the folic acid salt o Teucovorin salt will comprise from about 0:5 to 50 mg/ml, preferably from about 1 to about 35 mg/ml and especially preferably from about 3 to about 25 mg/m. With folic acid sodium salt, special mention is made of ‘5 mg/ml and for calcium leucovorin, 3 mg/ml. Benzyl alcohol can be omitted, but if present, can comprise up to about 2.5 percent w/v, preferably up to 1.5 percent w/v with sodium folate and up to about 0.908 percent w/v with calcium leucovorin. The amounts of tromethamine and monothioglycerol relative to each other can vary broadly, e.g, from about 1 to 99 parts by weight, preferably from about 20 to about 80 parts by weight of the former to from about 99, to 1 parts by weight, preferably from about 80 to about 20 parts ofthe latter. Preferably the tromethamine and monothioglycerol each will comprise from about 0.05 percent w/v to about 0.6 percent, preferably from about O.1 to about 0.3 percent w/v of the composition. If sodium chloride is present, it can range from 0.1 to about 1.0 percent, preferably from about 0.45 to about (065 percent w/v. The pH adjustors can vary widely type and amount. Typically hydrochloric acid, 5.0 per- cent v/v and sodium hydroxide 4.0% w/v will be con- veniently employed. The injectable solutions prepared as described above and more fully exemplified hereinafter are used in con- ventional dosages. A typical daily dose is generally up to about 150 mg, e.,, in the range of from about 25 to 150 mg which is conveniently administered in divided doses, for example 2, 3 or 4 doses in @ 24 hour period (methotrexate rescue with calcium leucovorin injecta- 5,173,488 5 ble). For treating folate deficiency lower doses of leu- covorin are generally administered. For example, a typical daily dose for an adult human is generally in the range of 2 to 25 mg which may be conveniently admin- istered as a single dose (leucovorin calcium) or up to 1.0 img daily (sodium folate). DESCRIPTION OF THE PREFERRED EMBODIMENTS The following examples illustrate the invention but are not intended to limit the claims in any manner what- soever. In the data tables, the following abbreviations have the following meanings: RT=room temperature pO M=month PABG=N-(p/10-aminobenzoyl)glutamic acid FFA=folic acid) Leucovorin Potency leucovorin calcium (as free acid) % LP=percent of label potency % L.=based on label potency of leucovorin ot more than, * C. and 75% relative humidity in closed con- exposure to sunlight jone detected ative humidity LCAB=light cabinet having 1,000 footcandle intensity EXAMPLE 1 Leucovorin calcium salt is used as the active ingredi- ent in a composition for injection having the formula set forth in Table 1 TABLE 1 ‘COMPOSITION FOR INECTION COMPRISING EUCOVORIN CALCIUM. Fonction Ingedient WIV Composition Lescovoris Calum USP 0310" Acive Ingreient Benzyl AlcoboLReagent Grade 0909 Antimicrobial, Preservative ‘Tromethanine Reagent Grade 0.150 er Monothioglycerol N 0200 Antioxidant Sodium ChlondeReagent Grade 0560 Toniety Adjoer Hydrochloric Acid Reagent pH 80-82 pH Adjuster Grade gs od Sodium Hydroxide USP gsad_ pH 80-82 pH Adjuster Water for Injection USP. gs ad 1000(/v) Vehicle Nitrogen NF, Prepurifed = — Provetan’ ‘ied 0 Leveovrin anyon Ft Ac 007 Anda hentpace he fl Seed poke ing mite ‘The formulation is prepared as follows: ‘A. Production of Bulk Solution Water for Injection, representing approximately 75% of final batch volume, is added toa stainless steel mixing tank. The Water for Injection is sparged with nitro- gen until the water temperature has reached 25°-30" C. (The product is continuously sparged or blanketed with nitrogen throughout the remaining process to protect against oxidation). The ingredients are se- uentially added, mixed and dissolved in the follow- ing order: benzyl alcohol; sodium chloride; trometha- mine; monothioglycerol; and calcium leucovorin. The pH is then adjusted to 8.10.1 with 5% hydro- chloric acid and/or 1% sodium hydroxide. The batch isbrought toa final volume of 20-30 liters with Water for Injection sparged with nitrogen. The pH is re- checked and re-adjusted to 8.1:0.1 with hydrochlo- ric acid or sodium hydroxide if necessary. 2 25 3 45 50 35 6s 6 B. Production of Sterile Filtrate Prior to the sterile filtration, a 0.2 micrometer filtration ‘cartridge is tested for integrity by Bubble Point test- ing ata pressure of 30 psig. The bulk solution is then passed through a first stage pre-filtration unit contain- ing an AWI9/0.45 micrometer cartridge to an in-ser- ies sterile 0.2 micrometer second stage cartridge. Pump and Nitrogen gas pressure feed the solution through the filtration units to a tared sterile stainless steel collection drum, At the completion of filtration the 0.2 micrometer filtration unit is again tested for integrity by Bubble Point testing at 35 psig. Elasto- ‘meric silicone tubing is used for all product transfer. €. Production of Filled Product The bulk sterile solution is taken to a class 100 filling ‘area where it is pumped from the collection drum through a sterile 5 micrometer filter to a filling line surge bottle. It is fed from the surge bottle to filling needles which eject measured doses to conveyor fed ampuls and vials. (Two filling lines, one for each package style, are used) 1 cc Amber glass ampuls are filled at 1.15005 mi (1.00.18:0.05 ml USP overage). 10 cc Type 1 amber glass vials are filled at 10.5 ml (10.0:0.5 ml USP over- age). All ampul and vial fills receive a nitrogen head- space blanket prior to sealing. Ampuls are heat sealed in 4 gas flame, and vials are sealed with butyl closures and aluminum crimp seals. EXAMPLES 2 AND 3 A second batch (Example 2) corresponding to Exam- ple 1is prepared by the method described in Example 1 asin Table I. A third batch (Example 3) is prepared, but this is 30 liters in size instead of 20 liters, as shown in Table 1. COMPARATIVE EXAMPLE 14 A formulation like Example 1 without tromethamine ‘and monothioglycerol having the formula set forth in Table 2 is included for comparison purposes: TABLE 2 ‘COMPOSITION FOR INJECTION COMPRISING (CALCIUMALEUCOVORIN. Function in WA Composition 10270-0330" Active In arent Levcovorin Calcium USP. retin Benzyl Alcohol Reagent Grade 090 Preservative Sodium Chloride-Resgent Grade 00-0560 Tonicty Ad- sister Sodium Hydroxide NF. gs ad pHT7 & pH Aduster & Hydrochloric Acid Reagent Grade pH 7.7: pH Adjuster 82 gaa Water for Injection USP. gs ad Nitrogen NF Prepried pdt procive unio ead asain fhe pode ring mamfctre snd ac haiace pt the fl wid phage 1000(v/s), Vehicle ‘The general procedure of Example 1 is used to make filed dosage forms comprising the following: Back tA 0A” Ingredient Sw Hw GIN WN Cacium 030 om 027 O33 Ceacovoria (As Free Acid) 5,173,488 7 8 continued The filled dosage forms from Examples 1-3 are placed on an accelerated stability test. The analytical ie methodolgy is as follows: TA 7A A_—aA Ingredient Sun WN we wh Benn alco 9 090 090 090 ‘Anavial Methodology wed for Bramples 3 peat = = Sodium Chloride -— = 0% 056 = met Sodium Hydride gk gk gk Dasrpion inal 1 Solution escovora potency a ese oa, Hysrochlorc| . * . . romatonraphy Acid 10% “es 4% 10 Levsovorin elated ‘iptey Soltion-adjust compounds potencies worl? Benzyl Alcohol Gas Chromatography Water for nC ) pe pit meter Injection gs ad Use Paniculste Mauer USP XXI Parcuate res Mater, Injection, ght 5 sbscuraion ‘Amber glass ampules are the containers selected for Monothogyceot Gu Chromoography filing, {entcation) Microbiological evaluation Antinirobiat Preservative COMPARATIVE EXAMPLES 2A, 3A and 4A of preserve sraem ——_Erisovenss et USP XXT pp 1151-1156, A second, third, and fourth batch of formulae with- 20 out tromethamine’and monothioglycerol are prepared for comparative testing using the general procedure for Example 1 The results of the test performed on he packaged formulations of Examples 1-3 are reported in Tables 3, 4-and 5, respectively: TABLE 3 “Three Month Accelerated and Six Month Room Temperate Stabilty Data on ‘Leucovorin Calcium Injection Solutions 3 mg/ml (Example 1) “Amber Ampol Packaging ‘Other Leucovorin Benayt Folic Relate Storage Potency Alcohol Acid © PABG FFA. Compounds Condition LP GLP EL EL LL pH_ Description Specie 100-1185 #S-115% NMT MT NMT NMT 65-85 Clear Ligh cation 10% 13%(1) 10% 25%@) Yellow Solution Ieiiat loo 902s "om? 038438292 Clear Ligh Yellow Soltion RTM wo 97ND 16ND 191792 Clear Light, ‘Yellow Solution RT. 6M WS 9738582328287 Clear Light, ‘Yellow Solution ms 9830s 774 Clear Light ‘Yellow Solution RHCM = — 1H 97S 228038240277 Clear Light Yellow Solstion RHCIM = 25H ND 238) ND 221790. Clear Light, ‘Yellow Solution SUNIM 103 96 0392023298178 Clea Lip ‘Yellow Solution Specify 100-1186 85-1185 NMT NMT NMT NMT 65-85 Clear Light cation 0% 136() 10% 2580) ‘Yellow Solution Initia to 9025038138198 Clear Light Yellow Solution RTM fot ND ta? ND 149290. Char Light, Yellow Solution RT, Ws 97ND nt 038208292 Clear Light, Yellow Solution RHCIM 10598036184 O30226 298 Char Light, Yellow Solution RHCM = 1039) ND 2 NDS, 286. Clear Light, Yellow Solution RHCIM = 0210 ND 289 ND SUNIM 1059903833828 297 Char Lig ‘Yellow Solution (0) Not more as 135 for nl elem a por ware tan 355 for Gn ets on fe ab ci of acorn Storage Condition Lescovorin Potency LP. 9 Benzyl Alcott! &LP. Folie Acid 5,173,488 TABLE 4 "Three Month Acceletaed and Sa Month Room Temperature Siabiiy Dal on Lexcovorn Calcium Injection Solutions 3 mg/l Example 2) ‘Amber Ampol Packaging PABG FFA Compounds ‘Otier Relates 10 Desripton Speci Tl RTM RTM RHC Rica RHCIM suN-M specif ini! RTM RT RHIcAM RHCIM RHCAM UNM Too-ti6% 104 103 101 100-1185 108 102 102 102 100 101 103 ® 155 o 9s 9% % % o ss.1186 ” ” 36 sek, NNT, 10% ND on 005 040 ND 005 Nur 105 020 ND 036 SL. NNT 13% 0) 090 136 as 208 170 Lo wr LEO as oss Mas 10s wh TMT 108% ous oz el. NMT 25% @) 19 20° Nat 25% 0) 10 191 200 i o5-85 790 191 736 291 190 1% 65.85 ‘Gar Light Yellow Solution lear Ligt Yellow Soltion Gear Light ‘Yelow Solution Cleat Light ‘Yellow Solution Clear Light Yellow Soltion Cleat Light Yellow Soltion Clear Light Yellow Solution Glese Light Yellow Solution Clear Light Yellow Solution (Gear Light Yellows Solution Clear Light Yellow Solution lear Light Yellow Solution lear Ligt ‘Yellow Solution lear Ligt Yellow Solution lest Ligh Yellow Solution leat Light Yellow Solution Storage Cancion Speci ‘nial RTM RToM RHCAM RHCAM RHCM suNIM specif ‘ial RTM RTM RHCAM RHCOM Lescovorin Potency LP. Tole 108 108 103 107 1s 100-1186 108 07 103 107 106 Benzyl Aloha LP. W115 100) 101 » 8 101 % 98 10 Folie acid se, or 108 as om ow oss 049 nw 10% 04s ost 092 04s os TABLE 5 "Three Month Accclersed and Sa Month Room Temperatore Siabiliy Dna 00 ‘LeacovorinCalelam Injection Solutions 3 mg/ml (Example) “Amber Amul Pachaging PABG aL, or 1380) ost 10 os 096 oss Nur 13) ost 116 141 096 101 FFA Compounds el, NM 108 027 08 ars 026 ons yur 10% 2 on 02 02s ‘Other Related el, TMT 25%0) 108 17 1a au 10 NM 23% 0) Tot 11 186 ut 194 oH e585 08 198 195 296 00 196 oses 209 196 Description lear Light Yellow Solstion lear Light Yellow Solution Gear Light ‘Yellow Solution Gear Lig ‘Yellow Solution leat Light ‘Yellow Solution Clear Light Yellow Soltion Gear Light Yellow Solution lear Light Yellow Sotsion Gear Light ‘Yellow Solution Gest Light Yellow Slution Clear Light Yellow Solution lear Light ‘Yellow Selution Gear Light Yellow Solution Gest Ligh ‘Yellow Solution 5,173,488 n TABLE 5-continued “Three Month Ac 12 jefated and Six Month Room Temperate Sabiliy Data on Leucoverin Calum Injection Solutions 3 mg/ml Example 3) ‘Amber Amp Packaging ‘Orker Leacovorin Benzyl Foie Relates Storage Potency” Alcohol Acid -PABG FFA Compounds Condiion LPO RLP LL&L Lp Description RCI 108 —~«OL_——aaT 410010150798 Clear Light Yellow Solution SUNIM 10599 ast ost 07796 Clear Light {]) Rov ween 135 for al iene nd nt move tn 15 fr se ed one ‘Yellow Solution (2) Not more than sm o 235 for rete a at moe an fr tf ess PAG usd ie nel li of The effectiveness of the preservative system of this invention for Leucovorin Calcium Injectable Solutions, Examples 1-3, is tested in accordance with the Antimi- ccrobial Preservatives Effectiveness Test outlined in TABLE &-continued Microbiological Evaluation of Preservative Sytem > USP. XXI Method Eximple 5 U.S. Phamacopeia XI, pages 1151-1156. This pro- 7° Tnceslation 1 a vides « measure of the capacity of the solutions to de- 2:8 See crease microbial growth when individually challenged Pusiomonar 105500 <10_— o@ = So wo> > ms mM DZ OE = > 5 eh a 7 = 8 i 2% mC rae oa = > > m@ mw 8 mom = ow 6 fy Sos 2 The electivenes of the preservative system of Leu HS > __covorin Calcium Injectable Solutions, for Comparative Fy Examples 1A, 2, 3A,and 4A i tested in accordance tor = ~ = with the Antimicrobial Preservatives Effectiveness Test IE, = olin measure of the capacity of the solutions to w= ™ crease ‘microbial growth’ when individually chal = im © lenged with Staphylococcus aureus, Escherchio coi = s, = 106, ‘Psuedomonas aeruginusa, Candid albicans and Aspergil ma MH = Jus niger at a dose level of 100,000 to 1,000,000 microor- wey ZZ a ganisms per ml. The contaminated solutions ae stored 2 Fay = Sind sampled ata series of time intervals in order to = = Stain meroorganism counts The results ofthese tests treet forth in Table 12 TABLE 12 Mion] Eoin of eset Sen USHA Meal Troan ‘Son O$ Hour 24 Hour 1 Week _2Wesks_4 West = Scpoiorcione 200 mem <1on <<

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