This material may be protected by Copyright law (Title 17 U.S. Code)
`
`FRESENIUS KABI 1004-0001
`
`

`
`nierung des freien Elektronenpaares am Stickstoff erschwert.
`Neben dem pH-Wert hat die Chloridionenkonzen[ration
`einen wesentlichen Einfluli auf die Lage des Hydro1ysegleich-
`gewichtes. Mit stcigcnder Chloridionenkonzentration sollte
`bevorzugt die Regeneration des Bendamustinhydrochlorids
`erfolgen, wodurch die Bildung des Hydroxyderivates zur1'ick-
`gedréingl wird, Ffir Melphalan ist die Abhéingigkeir des
`HydrolysegleichgewichLes von der Ch1oridionenk0nzentra—
`tion [5] u11d der Temperatur dokumentierl [4]. In Kenntnis
`dieser Zusammenhange fur das strukturverwandte Melphalan
`und untcr dem Aspekt der Anwendung am Patienten als
`Infusion erscheint isotonischc Natriumchloridlijsung als Tra-
`gerlosung fiir Bendamustinhydrochlorid geeignet. Ungiinstig
`ist allerdings die nur méifiige Loslichkeit der Substanz in
`diesem Medium, so daI3 die Zersetzung bereits wiihrend des
`zeitaufwendigen Losevorgangs beginnt. In reinem Wasser ist
`Bendamustinhydrochlorid gut loslich. Es empfiehlt sich daher
`das Losen in einer geringen Menge Wasser fiir Injektions—
`zwecke und das sofortige Verdfinncn mit isotonischer Na-
`t1'iumch1orid16sung.
`
`mustin, was fiir unsere Fragestellung ausrcichcnd ist.
`Vor Versuchsbeginn hatte die frisch hergestellte Untersu.
`chungslésung einen pH-Wert Von 4. Die Bestimmung der Zer-
`setzung von Bendamustinhydrochlorid-Infusionslbsungen er.
`folgte zunéichst bei 4 “C. Die Mefiergebnisse si11d in Tabelle 1
`msamlnengefafit. Die Geschwindigkeitskonstante (lk) und
`die Zeit bis zum Verlust von 10% wurden mittels exponen.
`tieller Regression berechnet. Fiir 1k ergab sich ein Wert V011
`—0,00093 h” 1. Der Korrelationskoeffizient wurde mit 0,9892
`ermittelt. Hieraus erreehnel sich fur die Stabilitéil
`(I90) Von
`Bendainustinhydrochlorid in Natriumchloridldsung 0,81%
`bei 4 “C ein Wert von 118,5 h. Weiterhin wurde die Stabilitéit
`Von Bendamustinhydrochlorid—Infusionslosung bei 23 “C be.
`stimmt. Die Mefiergebnissc sind in Tabelle 2 zusammengefaBt_
`Die Geschwindigkeits.konstante (lk) und die Zeit bis zum
`Verlust Von 10% (I90) wurden mittels exponentieller Regres-
`sion berechnet. Fur ‘k ergab sich ein Wert Von —0,01165 h '1
`(Korrelationskoeffizient 0,9985). Hieraus errechnet sich fiir
`die Stabilitéit von Bendamustinhydrochlorid (tgo) in Nalrium-
`chloridlosung 0,81% bei 23 “C ein Wert Von 9,2 h.
`
`Tabellel: Stabilitéitsdaten fiir Bendamustinhydrochlorid bei 4 °C
`Zeit
`.\«Iille|wcrt dcr
`Standard-
`Variations»
`Gehall an
`[I1]
`gemessenen
`abweichung
`koeffizient
`Bendumustin-
`Km17enlr2i-
`["/u]
`HCI [%]
`‘Lioncn an
`(to : 100"/u)
`Bendamustin—
`HCI [mg/ml]
`{fir n = 9
`
`0,00
`19,77
`42,57
`71,63
`92,55
`118,27
`
`0,2499
`0,2478
`0,2422
`0,2369
`0,2316
`0,2232
`
`0,00160
`0,00175
`0,00230
`0,00047
`0,00052
`0,00049
`
`0,64
`0,71
`0,95
`0,20
`0,22
`0,22
`
`100
`99,14
`96,22
`94,79
`92,68
`89,29
`
`Schema
`
`T
`/—Cl
`R —N : C1’ + R —N +
`\—C1
`¥CI
`LHEO
`,
`on
`H7 + R —N
`
`¥ CI
`
`
`
`011
`
`F0”
`H20
`c1-+ R —+Nf
`\> :> R—N\¥OH+H"
`
`Aufgrund entsprechender Vorversuche wurden folgende Be-
`dingungen fur die SLabilitéitsuntersuchungen gewéihlt: 25 mg
`Bendamustinhydrochlorid (=1 Ampulle Ribomustini“) Wur-
`den zunéichst in 10ml Wasser gelést und anschliefiend mit
`09% Natriumchloridlosung zu 100 ml Verdiinnt. Die damit
`hergestellte Konzentrarion Von 0,25 mg/ml entspricht thera-
`peutischen Erfordernissen. Die Stabilitiitsuntersuchung wurde
`bei Temperaturen Von 4°C und 23 °C durchgeffihrt. Die
`Bestiinmung Von Bendamustinhydrochlorid erfolgt mittels
`Reversed Phase HPLC. Der
`fiir Bendamustin eharakte-
`ristische Peak (Abb.) erscheint bei einer Retentionszeit (RT)
`Von 5,93 min. Das erstc Zersetzungsprodukt, vermutlich das
`Monohydrolyseprodukt, erscheint mit einer RT Von 4,14 min.
`Bei dem als ,,unknown“ bezeichneten Peak bei einer RT Von
`5,23 min handeit es sich um ein Nebenprodukt aus der
`Synthese. Mitllerweile ist es durch Optimierung des Syn-
`theseverfahrens gelungen, die Verunreinigung viillig zu elimi-
`nieren [6]. Die Fléiche dieses Peaks éindert sich wéihrend der
`gesamten Analysezeit nicht. Nach einer Aufbewahrungszeit
`Von 119 h ist
`im Chromatogramm neben der deutlichen
`Zunahme des Monohydroxyderivates ein vierter Peak bei
`einer RT Von 3,78 min zu erkennen, wobei es sich um das
`Dihydroxyderivat handelt. Mit einer Probe der kfirzlich als
`Dihydroxyderivat charakterisierten Verbindung [7] war es
`moglich, dieses Zersetzungsprodukt in unserem HPLC Assay
`zn identifizieren.
`Aufgrnnd der rasch fortsehreitcndcn Hydrolyse Von wéiflrigen
`Bendamustinhydrochloridlosungen diirfen bei allcn chroma-
`tographischen Bestiinmungen und entsprechenden Va1idie-
`rungen nur friseh hergestellte Losungen verwendet werden,
`die unmittelbar nach ihrer Zubereitung injiziert werden mus-
`sen. Séimtliche Standardproben zur Validierung der Melhode
`wurden mit Ribomustini durchgefuhrt. Die Mefiergebnisse
`erlauben somit cine rclativc Aussage 7.ur Stabilitéit Von Benda-
`
`776
`
`-5.5:um.mun
`
`fT;“__é*-
`
`300
`
`0n:lLs
`
`0:1
`
`.41|Nynnlylul
`.5.z3unknwn
` u ‘an
`FlT[10‘ min]
`
`J’
`
`/'
`
`Zcit
`[h]
`
`0,00
`19,77
`42,52
`71,63
`92,55
`118,27
`
`Mittelwert der
`gemessenen
`Kon7.entratinnen
`an Bendamustin-
`HC1 [mg/ml]
`fiir n : 9
`
`Variations- Gehalt
`Standard-
`abweichung kneffizient
`an Benda-
`[”/u]
`muslin-HCI
`[°/i]
`(ID = 100%)
`
`0,2499
`0,2478
`0,2422
`0.2369
`0,2316
`0,2232
`
`0,0016O
`000175
`0.00230
`0,00047
`0,00052
`0,0004‘)
`
`0,64
`0,71
`0,95
`0,20
`0,22
`0,22
`
`100
`99,14
`96,22
`94,79
`92,68
`89,29
`
`Abb.: HPLC-Chromatogramm von Bendamusrinhydrochlorid (0,25 mg/ml in
`NaC1 0.81% unmittelbar nach Verdiinnung
`
`7
`Pharmazie 49 (1994 H. 10
`FRESENIUS KAB|)1004-0002
`FRESENIUS KABI 1004-0002
`
`

`
`'[‘uhelle2: Stnbilitiitsdaten fiir Bendamustinhydrnchlorid bei 23 °C
`
`Stand1ird-
`;iliweie|ning
`
`V2iiiations-
`I”/ii]
`koeffizieiil
`
`Geliiili 2m
`HCI [“/ii]
`Bcndamustinv
`(tn = 100%)
`
`0,00107
`000212
`0,00130
`0,00211
`0,00404
`0.00139
`
`0,43
`0,85
`0,54
`0,89
`1,80
`0,71
`
`100
`99,31
`95,06
`94,04
`88,84
`77,87
`
`Zen
`[11]
`
`0,00
`0,67
`4,17
`5,33
`10725
`21,50
`
`Miltelwert der
`Konzenlrzi-
`geinessenen
`Lion an
`Eendamiistim
`HCl [irig/ml]
`fiir n = 3
`
`0,2525
`0,2 508
`0,2401
`0,2375
`0,2243
`0,1966
`
`3. Diskussion
`
`Die Messungen zeigeii, daB sowohl bei 4 °C als auch 23 “C
`die hydrolytische Zersetzung wéifiriger Bendai11ustiiihydro-
`cliloridlésungen in dem gemesseneii Bereicli linear Verléiuft.
`Ffir die Zersetzung VDI] Melplialan ist eiiie Kinctik pseudo-
`erster Ordiiung beschrieben [8], die aufgrund des identischen
`Hydrolyseinechanismus aiich fiir das
`strukturverwaiidte
`Beiidamustin angenommcn werden l(aI111. Die mittels expo-
`ueiitieller Regression berechneten Korrelationskoeflizieiiteii
`untcrstiitzen diese Annahme. Neueste Uiitersucliurigeii zur
`Hydrolysekinetik Von Bendainustin in Wasser bei Raum-
`teinperatur fiber den gesainten Zersetzungszeitraum ven'fi-
`zieren die Zersetzungskinetik pseudo-erster Ordnung [7]. Alle
`Uritersucliungslfjsungen wiesen deii stabilitéitsbegfiristigenden
`pH-Wert von 4 aiif. In Vorversuchen wurde die Hydrolyse
`von Bendaniustinhydrochlorid in Wasser
`fiir 1iijektions-
`zwecke iiberpriift, wobei gezeigt werden korinte, dals in dieseiii
`chloridfreieii Medium das Ausmalfi der Hydrolyse trotz
`gleicheri pH-Wertes wesentlicli gr6Ber ist. Fiir Lésuiigen der
`Ausgaiigskoiizeiitration 0,251ng/ml wurde hci Raumten:ipe-
`ratur exeinplarisch eine tgo Von 4,211 bestimmt. Dies belegi
`den stabilitétserhéhenden Einflulfi der Chloridionen. Als Tréi-
`germcdium ffir Bendamustinzubereitung ist ausschliefllich
`isotoiiische Natriumchloridlésung zu verwenden.
`Die Hydrolyse Von Bendamustinhydrochlorid ist erwartungs—
`geméifi Stark temperaturabhéingig. Die Stabilitét der Lésung
`mit eiiier Koiizentration Von 0,25 mg/ml
`ist bei 4 °C (190
`= 118,5 h) um mehr als das Zchnfache gréifier als bei 23 °C
`(190 2 9,2 h). Fiir die klinische Praxis bedeutet dies, daI3
`Bendamustiii-Infusionslésungen kiihl zu lagern sind. Mit
`physiologischer Kochsalzlésung als Triigermediuni und unter
`Aufbewahruiig irn Kfihlschrank ist ffir Bendaniustinlésiingeii
`der Konzentration 0.25 mg/ml eine Haltbarkeit vori vier d
`gegebeu, so dalfi cine Versorgurig mit applikatioiisfertigeii
`Bendamustin-lnfusionslésungen im Rahmen eiiier zentralen
`Zytostatikazubercitung dureh die Krankenhausapotheke
`sichcr mfiglich ist. F1"1r die empfohlene Applikatioii als Kurz—
`zeitinfusion fiber 30 min sind ebenfalls keine Stabi1itéits-
`probleme zu erwarten, da bei Raurnteinperatur fur diese
`Bendamustiiizubereituiigen cine Stabilitéit Von 9 h gegeben
`ist.
`
`4. Experimenteller Teil
`4.1. Substanzeii
`
`Die Bcstirniiiuiigeii wurden mit Ribornustin-Ampullen der Firma ribose-
`pharm (Charge 0190393) durcligefiilirt. Jede Durclistechllasclic mit 55mg
`Trockeiisubsiaiiz eiithiill 25 mg Bendamiistinhydmchlorid (Hillsstollz Man-
`riitol).
`
`4.2. Cliromatograpliiselie Bediiigurigen
`Die Messuiig crfolgte mit eiiier Millipore/Waters HPLC-Anlage.
`Pumpe: Waters 510 HPLC-Pump. Detektor: UV—Detek1or Waters 490 E,
`Pmbengeber: Waters 717-Autosampler, Séiule: Waters Novapak C 18, 60 A,
`r-liiiii, 3,9x150 mm. Flieljinittelz wéifirigc Na3SO..-Lésuiig 20mM (pH 3,
`
`Pharrnazie 49 (1994), H. 10
`
`H1504), Methanol 40/60 (V/V), FlL1B1":Il€I 0,3 ml/min, Wellenliingez 254 mm,
`Irijektioiisvolumeii: 10 1.11. Mefllemperatur: 4 EC bzw. 23 °C‘.
`
`4.3. Préizisiun Lles Geriires und der Metliode
`
`Sechs Eirizelinjektionen nus seclis jeweils friscli hergestellten Referenzlésiiiigen
`der Konzcntration 0,25 rrig/ml warden beziiglicli ermittelter Konzentratioii urid
`Reteiitionszclt ausgewertet. Die Ergebnisse sind iri Tzibelle 3 dargestellt.
`
`Tabel1c3: Prrizision des Geréites und der Methode
`
`Pmhe Nr
`
`1
`2
`3
`4
`5
`6
`Mittelwert
`Standardabweichiing
`Variatioiiskoeffiziem
`
`Menge
`lmgl
`
`0,2499
`0.2494
`0.2497
`0,2493
`0.2486
`0,2492
`0,2494
`0.000433
`0.2 %
`
`Reteritionszeil
`["131
`
`5,93
`592
`5,92
`5,94
`5,93
`5,93
`5,93
`0.0063
`0,1 %
`
`4.4.1iic1iurig des Systems
`Die Eichung des Systems erfolgt miltels Melirpuiikteieliiing mit externem
`Standard. Es werdeii je drei Injektionen von 10%, 100% und 200% der 7.1.1
`bestimmeriden Konzeiitration ausgefiihrt (Tabelle 4). Dazu wird eine Ampulle
`Riboniustin“ zunficlist in 10 ml H30 gelést und anschlieflend mit N2iCl 0,9‘!/u
`zu 0,025 mg/ml, 0.25 mg/ml und 0,50 mg/ml \’e1'dl.lIl1‘1I. Die Eichgerade ergibt
`sich aus den bcrcchneteii Peakfliichen. aufgetragen gegen die tlieoretisclie
`Knnzentration. Der Korrclationskoeffizient betréigt 0,9997.
`
`Tabelle 4: Eichung dcs Systems
`Theoretisch
`Mittelweit der
`Konzentriition
`gefundenen
`[mg/ml]
`Konzeiitrationen
`1111
`ll : 3
`[mg/ml]
`
`St:indard-
`abweicliiing
`
`Variations-
`koeffizient
`[%]
`
`0.025
`0,25
`0,50
`
`0.0266
`0,2499
`0,5080
`
`0.00036
`0,0016
`0,0021
`
`1,4
`0,6
`0,4
`
`4.5. Probeiivorbereiiiiiig
`Jeweils eirie Ainpulle Rilmmu:tin“‘ (4.1.) wird in 10 ml Wasser [fir 1nje1<tioi1s-
`zwecke gclbst und aiiscliliefleiid mit NaCl-Lésurig aus Viatlcx" Beuteln der
`Firmzt Baxter (pl-I-Wert: 5,0-7,0) All 100 ml \’E1‘(‘ll11‘1I1[. Daraus resultiert cine
`NaC1-Konzeiitratioii von 0,81%. Die Zubereimng C161’ Staiidardproben zur
`Erstelluiig der Eicligeraden erfolgt eiitsprechend.
`
`4.6. Aiiswertimg
`Die Auswertung erfolgt durch uiitomatische Peakfliicheiiiiitegratioii
`Eicligcradc (Software Waters Maxirna 820).
`
`initials
`
`Literatur
`
`
`
`\IO‘vi~t>uJM-—-
`
`Riboniiistiifl“ Stziridardinfnnnatioii fijr Kmiikeiihausapothcker April 1993
`Preiss, R.: Solir, R.: Matthias, M.: Brnckniziiiu, B.; I-liillcr, H.: 1’1iarmazie
`-'10, 782 (1985)
`Ki‘é"'imer, 1.: Krankeiiliauspliarnia/.ie 8, 325 (1990)
`Stout, S. A.; Riley, C. M.: Int. J. Pharm. 24. 193 (1985)
`Chang, S. Y.; EV€11'1S, T. S.; Alberts, D. S.; J. Pliai-in. Plizirinacol. 31, 853
`(1979)
`Persénliclic Mitteiluiig der Firina ribosepliiirin 1994
`Gust, R.: Unveriiffeiitlichte Ergebnisse (liistitut fiii‘ Pliarmazie, Umversitéit
`Regensburg)
`8 Flora, K. P; Smith. S. L.; Cradock, J. C.: J. Chroiuzltogr. 177, 91 (1979)
`
`Eiiigegaiigen am 8. Juni 1994
`Aiigeiiommeu am 4. Juli 1994
`
`Dr. Irene Krzimer
`Apotheke des Kliiiikuius
`Laiigenbcckstr. 1
`D-55131 Nlaiiiz
`
`777
`
`
`
`FRESENIUS KABI 1004-0003
`FRESENIUS KABI 1004-0003
`
`

`
`Pharmacy1, III. Medical Clinic and Polyclinic, Division of Hematology2, of the University Medical Center of the
`Johannes Gutenberg University Mainz
`
`Stability of Bendamustine Hydrochloride in Infusions
`
`BIRGIT MAAS1, C. HUBER2 AND IRENE KRAMER1
`
`Bendamustine hydrochloride was identified by means of
`reverse-phase HPLC. The chemical stability (t90) of the
`cytostatic (0.25 mg/mL) in 0.9% sodium chloride is 120 h
`at 4°C and 9 h at 23°C. The pH value, temperature, and
`chloride ion concentration are influencing factors for
`stability. 0.9% sodium chloride solution must be used as
`the carrier solution for bendamustine infusion solution.
`The stability times ensure unproblematic storage and
`application in clinical practice.
`
`Stability of bendamustine hydrochloride in infusions
`
`(0.25
`stability of bendamustine hydrochloride
`The
`mg/mL) in 0.9% sodium chloride was studied after storage
`4°C and
`23°C using
`reverse-phase HPLC.
`at
`Bendamustine hydrochloride is stable at 4°C for 120 h and
`at 23°C for 9 h. Temperature, pH, and chloride
`concentration are key influencing factors for stability.
`Isotonic sodium chloride must be used for the preparation
`of bendamustine hydrochloride infusions.
`
`1.
`
`Introduction
`
`4-[5-bis(2-chlorethyl)amino]-1-
`(Ribomustin®,
`Bendamustine
`methyl-2-benzimidazolyl
`butyric
`acid)
`is
`an
`effective
`chemotherapeutic drug in the treatment of malignant diseases.
`
`The stability of the lyophilized dry substance is already known
`[1]. In studies of the pharmacokinetics of bendamustine in
`humans, in vitro measurements of the dissolved substance were
`carried out at a temperature of 37°C in a buffered medium (pH =
`7.5; t50 = 6.2 min) and in heparinized plasma (t90 = 1.67 h) [2].
`However, data is still lacking on the stability of bendamustine
`infusions. Since patient-specific, ready-to-use cytostatic infusions
`or injections are increasingly prepared in a central cytostatic
`preparation facility in the hospital pharmacy and the infusion is
`longer
`prepared
`immediately
`before
`administration,
`no
`information on the stability of bendamustine hydrochloride in an
`aqueous solution is imperative [3].
`
`2. Tests and results
`
`Bendamustine is very unstable in an aqueous solution. In a
`weakly acid, neutral, and alkaline solution, hydrolysis of the bis-
`2-chlorethylamino function occurs. As in the hydrolysis of
`melphalan, monohydroxy bendamustine presumably develops
`first via an aziridium cation, and then the dihydroxy derivative
`develops (see diagram) [4]. In a highly acidic solution, hydrolysis
`is inhibited by protonation of the free electron pair on the
`In addition to the pH value,
`the chloride ion
`nitrogen.
`concentration has a significant
`impact on the hydrolysis
`equilibrium. It is preferred that the bendamustine hydrochloride
`be regenerated as the chloride ion concentration increases, which
`the formation of
`the hydroxyl derivative. The
`suppresses
`
`Pharmazie 49 (1994), H. 10
`
`775
`
`FRESENIUS KABI 1004-0004
`
`

`
`dependence of the hydrolysis equilibrium on the chloride ion
`concentration [5] and temperature [4]
`is documented for
`melphalan. On account of these relationships for the structurally
`related melphalan and the fact that it is administered to the patient
`as an infusion, isotonic sodium chloride solution seems to be
`suitable as a carrier solution for bendamustine hydrochloride. The
`merely moderate solubility of the substance in this medium is,
`however, unfavorable, because decomposition already begins
`during the time-consuming dissolution process. In pure water,
`bendamustine has good solubility. It is therefore recommended
`that it be dissolved in a small amount of water for injection and
`then immediately diluted with an isotonic sodium chloride
`solution.
`
`Diagram
`
`for the stability (t90) of bendamustine hydrochloride in a 0.81%
`sodium chloride solution at 4°C. The stability of bendamustine
`hydrochloride infusion solution was also determined at 23°C. The
`measurement results are shown in Table 2. The rate constant (1k)
`and time to 10% loss (t90) were calculated by means of
`exponential regression. A value of -0.01165 h-1 was obtained for
`1k (coefficient of correlation 0.9985). This calculated to a value of
`9.2 h for the stability of bendamustine hydrochloride (t90) in a
`0.81% sodium chloride solution at 23°C.
`
`Table 1: Stability data for bendamustine hydrochloride at 4°C
`
`Time [h]
`
`Mean
`Concentration
`of
`Bendamustine
`HCl [mg/mL]
`Measured for
`n = 9
`
`Standard
`Deviation
`
`Coefficient of
`Variation [%]
`
`Content of
`Bendamustine
`HCl [%]
`(t0 = 100%)
`
`0.00
`19.77
`42.57
`71.63
`92.55
`118.27
`
`0.2499
`0.2478
`0.2422
`0.2369
`0.2316
`0.2232
`
`0.00160
`0.00175
`0.00230
`0.00047
`0.00052
`0.00049
`
`0.64
`0.71
`0.95
`0.20
`0.22
`0.22
`
`100
`99.14
`96.22
`94.79
`92.68
`89.29
`
`following
`the
`Based on corresponding preliminary tests,
`conditions were selected for
`stability testing: 25 mg of
`bendamustine hydrochloride (= 1 ampoule of Ribomustin®) was
`dissolved first in 10 mL of water and then diluted with 0.9%
`sodium chloride solution to 100 mL. The produced concentration
`of 0.25 mg/mL meets therapeutic requirements. The stability test
`was performed at
`temperatures of 4°C and 23°C. The
`bendamustine hydrochloride was identified by reverse-phase
`HPLC. The characteristic peak of bendamustine (see figure)
`occurs at a retention time (RT) of 5.93 min. The first
`decomposition product, presumably the monohydrolysis product,
`appears at an RT of 4.14 min. The “unknown” peak at an RT of
`5.23 min is a by-product from synthesis. By optimizing the
`synthesis procedure in the meantime, we have been able to
`completely eliminate the impurity [6]. The area of this peak does
`not change during the entire time of analysis. After a storage time
`of 119 h, in addition to the clear increase in the monohydroxy
`derivative, a fourth peak can be seen at an RT of 3.78 min, which
`is the dihydroxy derivative. With a sample of the compound
`recently characterized as a dihydroxy derivative [7], we were able
`to identify this decomposition product in our HPLC assay.
`Due
`to
`the
`rapid
`hydrolysis of
`aqueous bendamustine
`hydrochloride solutions, only freshly prepared solutions which
`must be injected immediately following their preparation may be
`used in chromatographic determinations and corresponding
`validations. All standard samples for validation of the method
`were performed with Ribomustin®. The measurement results thus
`allow us to determine the relative stability of bendamustine,
`which is sufficient for our investigation.
`Before the start of testing, the freshly prepared test solution had a
`pH of 4. The decomposition of bendamustine hydrochloride
`infusions was determined first at 4°C. The measurement results
`are shown in Table 1. The rate constant (1k) and the time to 10%
`loss were calculated by means of exponential regression. A value
`of -0.00093 h-1 was obtained for 1k. The coefficient of correlation
`was determined as 0.9892. This calculated to a value of 118.5 h
`
`776
`
`Figure: HPLC chromatogram of bendamustine hydrochloride (0.25
`mg/mL in 0.81% NaCl immediately after dilution
`
`[Translator’s note: See Table 1 for translation of figure.]
`
`Pharmazie 49 (1994), H. 10
`
`FRESENIUS KABI 1004-0005
`
`

`
`Table 2: Stability data for bendamustine hydrochloride at 23°C
`
`4.3
`
`E q u i p m en t a n d m et h od p r ec i si on
`
`Standard
`Deviation
`
`Coefficient of
`Variation [%]
`
`Content of
`Bendamustine
`HCl [%]
`(t0 = 100%)
`
`Six different injections from six freshly prepared reference solutions with
`a concentration of 0.25 mg/mL were analyzed for concentration obtained
`and retention time. The results are shown in Table 3.
`
`Table 3: Precision of equipment and method
`
`Retention Time
`[mg]
`
`5.93
`5.92
`5.92
`5.94
`5.93
`5.93
`5.93
`0.0063
`0.1%
`
`Quantity
`[mg]
`
`0.2499
`0.2494
`0.2497
`0.2493
`0.2486
`0.2492
`0.2494
`0.000433
`0.2%
`
`Sample No.
`
`1 2 34 5 6M
`
`ean value
`Standard deviation
`Coefficient of variation
`
`4.4
`
`S ys t em c a li b ra t i on
`
`The system was calibrated by means of multiple-point calibration with an
`external standard. Three injections of 10%, 100%, and 200% of the
`concentration to be determined were performed (Table 4). For this
`purpose one ampoule of Ribomustin® was first dissolved in 10 mL of
`H2O and then diluted with 0.9% NaCl to 0.025 mg/mL, 0.25 mg/mL, and
`0.50 mg/mL. The calibration curve was derived from the calculated peak
`areas plotted against the theoretical concentration. The coefficient of
`correlation was 0.9997.
`
`Table 4: System calibration
`
`Theoretical
`Concentration
`[mg/mL]
`
`Mean Value of
`Concentrations
`Found for n = 3
`[mg/mL]
`
`Standard
`Deviation
`
`Coefficient of
`Variation [%]
`
`0.025
`0.25
`0.50
`
`0.0266
`0.2499
`0.5080
`
`0.00036
`0.0016
`0.0021
`
`1.4
`0.6
`0.4
`
`4.5
`
`S a mp l e p r ep a r a t i on
`
`Each ampoule of Ribomustin® (4.1) was dissolved in 10 mL of water for
`injection and then diluted to 100 mL with NaCl solution from ViaFlex
`bags from the company Baxter (pH 5.0–7.0). This resulted in an NaCl
`concentration of 0.81%. The standard samples for the calibration curves
`were prepared in the same way.
`
`4.6 An a l ysi s
`
`The analysis was performed with automatic peak area integration using
`the calibration curve (Waters Maxima 820 software).
`
`Literature
`
`1 Ribomustin® Standardinformation für Krankenhausapotheker, April
`1993
`2 Preiss, R.; Sohr, R.; Matthias, M.; Brockmann, B.; Hüller, H. Pharmazie
`40, 782 (1985)
`3 Krämer, I.: Krankenhauspharmazie 8, 325 (1990)
`4 Stout, S.A.; Riley, C.M.: Int. J Pharm 24,193 (1985)
`5 Chang, S.Y.; Evans, T.S.; Alberts, D.S.: J Pharm. Pharmacol. 31, 853
`(1979)
`6 Personal memo from the Ribosepharm company, 1994
`7 Gust, R.: Unpublished results (Institute of Pharmacy, University of
`Regensburg)
`8 Flora, K.P.; Smith, S.L.; Cradock, J.C.: J Chromatogr. 177, 91 (1979)
`
`Submitted on June 8, 1994
`Accepted on July 4, 1994
`
`Dr. Irene Krämer
`Apotheke des Klinikums
`Langenbeckstr. 1
`D-55131 Mainz
`
`777
`
`FRESENIUS KABI 1004-0006
`
`Time [h]
`
`Mean
`Concentration
`of
`Bendamustine
`HCl [mg/mL]
`Measured for
`n = 9
`
`0.00
`0.67
`4.17
`6.33
`10.25
`21.50
`
`0.2525
`0.2508
`0.2401
`0.2375
`0.2243
`0.1966
`
`0.00107
`0.00212
`0.00130
`0.00211
`0.00404
`0.00139
`
`0.43
`0.85
`0.54
`0.89
`1.80
`0.71
`
`100
`99.31
`95.06
`94.04
`88.84
`77.87
`
`3. Discussion
`
`The measurements show that the hydrolytic decomposition of
`aqueous bendamustine hydrochloride solutions is linear over the
`range measured at both 4°C and 23°C. A pseudo first-order
`kinetic reaction is described for melphalan [8], which can also be
`assumed for the structurally related bendamustine because of the
`identical hydrolysis mechanism. The coefficients of correlation
`calculated by exponential regression support this assumption. The
`latest studies of the hydrolysis kinetics of bendamustine in water
`at room temperature verify the pseudo-first-order decomposition
`kinetics [7]. All test solutions had the stability-promoting pH of 4.
`In
`preliminary
`tests,
`the
`hydrolysis
`of
`bendamustine
`hydrochloride was tested in water for injection and,
`in this
`chloride-free medium, the degree of hydrolysis was substantially
`higher despite having the same pH value. Typically for solutions
`with the initial concentration of 0.25 mg/mL, a t90 of 4.2 h was
`determined at room temperature. This is evidence of the stability-
`increasing effect of the chloride ions. Isotonic sodium chloride
`solution must be used exclusively as the carrier medium for
`bendamustine preparation.
`As expected, the hydrolysis of bendamustine hydrochloride is
`highly temperature-dependent. The stability of the solution with a
`concentration of 0.25 mg/mL is more than ten times greater at
`4°C (t90 = 118.5 h) than at 23°C (t90 = 9.2 h). For clinical practice,
`this means that bendamustine infusions must be kept cool. If a
`physiological salt solution is used as the carrier medium and the
`infusions are stored in a refrigerator, the storage time is four days
`for bendamustine solutions with a concentration of 0.25 mg/mL,
`so a supply with ready-to-use bendamustine infusions is certainly
`possible within the framework of central cytostatic preparation in
`the
`hospital
`pharmacy. Likewise
`for
`the
`recommended
`administration as a short
`infusion over 30 min, no stability
`problems are expected, since these bendamustine preparations
`have a stability of 9 h at room temperature.
`
`4. Experiment
`
`4.1
`
`Substanc es
`
`The determinations were performed using Ribomustin ampoules from the
`company Ribosepharm (batch 0190393). Each vial with 55 mg of dry
`substance contains 25 mg of bendamustine hydrochloride (excipient:
`mannitol).
`
`4.2
`
`C h ro ma t og ra p h i c c on d i t i on s
`
`The measurement was performed using a Millipore/Waters HPLC system.
`Pump: Waters 510 HPLC pump; detector: Waters 490E UV detector;
`proton donor: Waters 717 autosampler; column: Waters Novapak C18,
`60Å. 4µm, 3.9 x 150 mm; solvent: aqueous Na2SO4 solution 20 mM (pH
`3, H2SO4), methanol 40/60 (v/v); flow rate 0.3 mL/min; wavelength: 254
`mm; injection volume: 10 µL; measuring temperature: 4°C and 23°C.
`
`Pharmazie 49 (1994), H. 10
`
`

`
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`
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`
`‘—~c._*.
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`NOTARWS DECLARATION
`
`On this 30th day olfilune, 2015. in King County, Seattle. WA. USA. Arnanda Olson identified
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`FRESENIUS KABI 1004-0007
`FRESENIUS KABI 1004-0007