columns were kept identical. Eluent: A: 0.05 M KHZPO4. pH : 2.5 (H3PO4):
`B; CH3OH Gradient (linear): 0~35 min from 90% A to 55% A, 35—40 min to
`40% A; 40-60 min to 40% A: 60-70 min to 25% A. 70—80 min to 20% A.
`Flow rate: 0.7 ml/min. Temperature: If not otherwise steated the experiments
`were performed at room temperature. Other selected temperatures have been
`adjusted by a column thermostat. Sample volume: 20 [.11. Absorbance: 285 nm.
`HPLC system: Gradient system 600 E (Fa. Millipore Waters). diodenarray
`detector 990 (Fzt. Millipore Waters). HPLC-column thermostat 5—85 :C (Fa.
`Knauer).
`
`3.2.2. Pu://r izlcnlifimrimi
`Retention times and UV—spectra of reference substances have been used to
`identify the peaks in the chromatograms. The different reference substances
`have been checked by ‘H- and “C-NMR-spectroscopy.
`
`3.3. Solid phase NMR spectroscopy
`NMR-Unit: Bruker AMX 300 with a CP-MAS Unit.
`
`3.3.]. 1°Si-CP-MAS-NMR
`1\/Ieasuring frequency: 59.63 MHZ. Proton resonance: 300.14 MHZ. Optimal
`contact-time: 20000 usec for LiChrospher 60 RP-select B. 5000 usec for
`Nucleosil 100 AB.
`
`3.3.2. “C-CP—iUAS-NMR
`Measuring frequency: 75.48 MHZ. Proton resonance: 300.14 MHZ. Optimal
`contact-time: 2000 [.lS€C for Nucleosil 100 AB, 2000 usec for LiChrospher 60
`RP-select B. 2000 usec for Eurospher 100.
`
`to our colleague Mr. Lubda (Fa.
`Acknowledgement: We are most grateful
`Merck) for running the CP-MAS—NMR spectra.
`
`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`References
`
`
`
`
`
`O\UI4>-UJlJ>—-
`
`
`
`Gilpin. R. K.: Burke. M. F.: Anal. Chem. 45. 1383 (1973)
`Dorsey, J. G.: Dill. K. A.: Chem. Rev. 89. 331 (1989)
`Eisenbeifi. 1-7.: Ber. Bunsenges. Phys. Chem. 93. 1019 (1989)
`Morterrn. C.: Low. M. J. D.: J. Catul. 28.265 (1973)
`Waksmundzki. A.; Rudzinski. W.; Suprynowicz, Z.: Leboda. R.: J. Chroma-
`togr. 92. 9 (1974)
`(Ed.):
`in: Horvath. Cs.:
`Karger. B. L.: Le Page. J. N.: Tanaka. N.:
`High-Performance Liquid Chromatography. Advances and Perspectives.
`Vol. I, S. 159. Academic Press. New York 1980
`7 Nakum, A.: Horvath. C.: J. Chromatogr. 203. 53 (1981)
`oo
`Kohler. J.: Kirkland, J. J.: Chromatogr. 385. 125 (1987)
`9 Kohler. J.: Chase. D. B.: Farlee. R. D.: Vega. A. J.: Kirkland. J. J.: J.
`Chromatogr. 352. 275 (1986)
`10 Unger. K. K.: Porous Silica. Journal of Chromatography Library. Vol. 16.
`Elsevier. Amsterdam 1979
`Pfleiderer. B.: Albert. K.: Bayer. E.: J. Chromatogr. 506. 343 (1990)
`Sinsdorf, D. W.: Maciel. G. E.: J. Am. Chem. Soc. 105, 1487 (1983)
`Sindorf, D. W.; Maciel. G. E.: J. Am. Chem. Soc. 103.4263 (1981)
`Bayer. E.: Albert. K.: Reiners. J.; Nieder. M.: Miiller. D.: J. Chromatogr.
`264, 197 (1983)
`15 Szabo. K.: Le Ha. N.: Schneider. P.: Zeltner. P.: Kovats. E.: Helv. Chim.
`Acta 67. 2128 (1984)
`16 Hetem. M.; van de Ven. L.: de Haan. J.: Cramers, C.: Albert. K.: Bayer.
`E.: J. Chromatogr. 479. 269 (1989)
`17 Kinkel. J. N.: Unger. K. K.: J. Chromatogr. 316. 193 (1984)
`18 Sokolowski. A.: Wahlund. K.-G.: J. Chromatogr. 189. 299 (1980)
`19 Freytag. W. E.; Stapf. W.: Pharm. Ztg. Wiss. 6./138. 126 (1993)
`20 Fulde. G.: Dissertation Marburg 1993
`21 Jones. R. R.: Harkrader. R. J.:Southard. G. L.: J. Nat. Prod. 49, 1109 (1986)
`22 Berthold. A.: J. Chromatogr. 549.
`1 (1991)
`23 Unger. K. K.: Porous Silica, Journal of Chromatography. Library. Vol. 16,
`S. 102. Elsevier. Amsterdam 1979
`24 Sander. L. C.; Wise. S. A.: Anal. Chem. 56. 504 (1984)
`
`,_.._.,_.,_
`
`-J;uJt\z._.
`
`Received January 27. 1994
`Accepted March 20. 1994
`
`Dr. Hanns Haberlein
`Institut fiir Pharmazeutische Biologic
`der Philipps-Universitat Marburg
`Deutschhausstra|3e 17‘/1
`D-35037 Marburg
`
`l||. Medizinische Klinik und Poliklinik Abteilung Hématologiez, des Klinikums der Johannes Gutenberg-
`Apotheke‘,
`Universitét, Mainz
`
`Stabilitéit von Bendamustinhydrochlorid in lnfusionsliisungen
`
`BIRGIT IVIAAS‘, C. HUBER2 und IRENE KRAMER‘
`
`Bendamustinhydrochlorid wurde mittels Reversed Pha-
`se HPLC bestimmt. Die chemische Stabilitéit (tgo) des
`Zytostatikums (0.25 mg/ml) betragt in 0.9% Kochsalz-
`losung bei4 QC 120 h und bei 23 “C 9 h. Stabilitéitsbeein-
`tlussende Faktoren sind pH-Wert, Temperatur und
`Chloridionenkonzentration. Kochsalzlosung 0,9"/o ist
`als Tréigerlosung fiir Bendamustin-Infusionslosungen
`einzusetzen. Die Stabilitatszeitréiume gewéihrleisten eine
`unproblematische Aufbewahrung und Applikation in
`der klinischen Praxis.
`
`Stability of bendamustine hydrochloride in infusions
`
`The stability of bendamustine hydrochloride (0.25 mg/
`ml) in 0.9% sodium chloride was studied after storage
`at 4 ‘C and 23 °C using Reversed Phase HPLC. Bend-
`amustinhydrochloride is stable at 4 “C for 120 h and at
`23 °C for 9 11. Temperature, pH and chloride concentra-
`tion are important factors which influence stability.
`Isotonic sodium chloride must be used for the prepa-
`ration of bendamustine hydrochloride infusions.
`
`1. Einleitung
`
`(Ribomustin‘5‘, 4-[5-Bis(2-chlorethyl)amino]-
`Bendamustin
`1-methy1-2-benzimidazolylbutanséiure) ist ein wirksames Che-
`
`motherapeutikum in derBehand1ung maligner Erkrankungen.
`Die Stabilitéit der lyophilisierten Trockensubstanz ist bereits
`bekannt [1]. Im Rahmen von Untersuchungen zur Pharmako-
`kinetik Von Bendamustin am Menschen wurden auch in vitro-
`Messungen zur Stabilitéit der gelosten Substanz bei einer
`Temperatur Von 37 "C in gepuffertem Medium (pH : 7,5;
`tso = 6,2 min) und in heparinisiertem Plasma (t90 = 1.67 h)
`durchgefiihrt [2]. Es fehlen aber bislang Angaben zur Halt-
`barkeit Von Bendamustin-Infusionslosungen. Da die Her-
`stellung patientenbezogener. applikationsfertiger Zytostatika—
`Injektions- oder -Infusionslosungen immer héiufiger durch eine
`zentrale Zytostatikazubereitung in der Krankenhausapotheke
`erfolgt und die Infusion nicht mehr unrnittelbar vor ihrer
`Applikation zubereitet wird, sind Kenntnisse zur Stabilitéit
`Von Bendamustinhydrochlorid in wéiBriger Losung erf0rder—
`lich [3].
`
`2. Untersuchungen und Ergebnisse
`
`in wéiffariger Losung sehr in-
`Bendamustinhydrochlorid ist
`stabil. In schwach saurer. neutraler sowie alkalischer Losung
`erfolgt eine Hydrolyse der Bis-2-Chloretylaminofunktion.
`Analog der Hydrolyse Von Melphalan entsteht vermutlich
`iiber ein Aziridium-Kation zunachst das Monohydroxybend-
`amustin und nachfolgend das Dihydroxyderivat (Schema) [4].
`In stark saurer Losung wird die Hydrolyse durch Proto-
`
`Pharmazie 49 (1994), H. 10
`
`AGILA ET AL - EXHIBIT 1007 775
`
`0001
`
`AGILA ET AL - EXHIBIT 1007
`
`0001
`
`

`
`ist allerdings die nur méifiige Loslichkeit der Substanz in
`diesem Medium, so daB die Zersetzung bereits walirend des
`zeitaufwendigen Lésevorgangs beginnt. In reinem Wasser ist
`Bendaniustinhydrochlorid gut léslich. Es empfiehlt sich daher
`das Losen in einer geringen Menge Wasser fiir Injektions-
`zwecke und das sofortige Verdiinnen mit isotonischer Na-
`triunichloridlésung.
`
`stimmt. Die Mefiergebnisse sind in Tabelle 2 zusammengefafit.
`Die Geschwindigkeitskonstante (lk) und die Zeit bis zum
`Verlust Von 10% (I90) wurden mittels exponentieller Regres-
`sion berechnet. Fiir ‘k ergab sich ein Wert von —0,01165 h‘1
`(Korrelationskoeffizient 0,9985). Hieraus errechnet sich fiir
`die Stabilitéit von Bendamustinhydrochlorid (tgo) in Natrium-
`chloridlosung 0,81% bei 23 “C ein Wert Von 9,2 h.
`
`Schema
`
`T
`/—c1
`R—N 4: C1’+R —N +
`\—c1
`\—ci
`
`LHZO
`
`_
`HT+R ~N
`
`OH
`
`\-C1
`
`
`+ fOH
`—
`_
`Cl +R N
`
`H10
`-
`
`R_N
`
`r0H .
`+H,
`
`\—oH
`
`Aufgrund entsprechender Vorversuche wurden folgende Be-
`dingungen fur die Stabilitéitsuntersuchungen gewahlt: 25 mg
`Bendamustinhydrochlorid (=1 Ampulle Ribomustin"—‘) Wur-
`den zunéichst in 10 ml Wasser gelést und anschliefiend rnit
`0,9% Natriumchloridlosung zu 100 ml Verdiinnt. Die damit
`hergestellte Konzentration Von 0,25 mg/ml entspricht thera-
`peutischen Erfordernissen. Die Stabilitéitsuntersuchung wurde
`bei Temperaturen Von 4°C und 23 °C durchgefiihrt. Die
`Bestimmung Von Bendamustinhydrochlorid erfolgt mittels
`Reversed Phase HPLC. Der
`fiir Bendamustin charakte-
`ristische Peak (Abb.) erscheint bei einer Retentionszeit (RT)
`Von 5,93 min. Das erste 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 handelt es sich um ein Nebenprodukt aus der
`Synthese. Mittlerweile ist es durch Optimierung des Syn-
`theseverfahrens gelungen, die Verunreinigung véllig zu elimi-
`nieren [6]. Die Fléiche dieses Peaks andert 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 kiirzlich als
`Dihydroxyderivat charakterisierten Verbindung [7] war es
`moglich, dieses Zersetzungsprodukt in unserem HPLC Assay
`zu identifizieren.
`
`Aufgrund der rasch fortschreitenden Hydrolyse von wafirigen
`Bendamustinhydrochloridlésungen di'11'fen bei allen chroma-
`tographischen Bestimmungen und entsprechenden Validie-
`rungen nur frisch hergestellte Losungen verwendet werden,
`die unrnittelbar nach ihrer Zubereitung injiziert werden mus-
`sen. Samtliche Standardproben zur Validierung der Methode
`wurden mit Rib0mustin‘~‘ durchgefiihrt. Die Mefiergebnisse
`erlauben somit eine relative Aussage zur Stabilitéit von Benda-
`
`776
`
`Tabelle 1: Stabilitéitsdaten fiir Bendamustinhydrochlorid bei 4 °C
`Zeil
`Mittelwerl der
`Slandurd-
`Variz1lions-
`Gehall an
`[11]
`gemessenen
`abweichung
`koeffizient
`Bendumus1in-
`Kon7.en1ra-
`[D/n]
`HCI [%]
`lionen an
`(to : 100%)
`Bendamuslim
`I-{Cl [mg/‘m|]
`fiir 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
`0,00l75
`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
`
`
`
`
`
`A}.-5.9:Eiannlnunun
`
`3 00»
`
`2.00-
`
`1 00>
`
`~1011/u1L's
`
`0 00
`
`O 20
`
`0 =10
`
`,——44*’
`
`4uHyamym
`523unknnvn
`1 r
`
`0 ED
`
`0 B0
`
`FIT[10‘ min]
`
`Zeit
`[h]
`
`0,00
`19,77
`42,52
`71,63
`92,55
`118,27
`
`Mittelwert der
`gemessenen
`Konzentrationen
`an Bendamustin-
`HCI [mg/ml]
`fur n : 9
`
`Varia1ions- Gehalt
`Standard—
`abweichung koeffizient
`an Benda-
`["/n]
`mustin-HCI
`[%]
`(10 2 100%)
`
`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
`
`Abb.: HPLC—Chr0matogramm von Bendamustinhydrochlorid (0,25 mg/ml in
`NaCl 0.81% unmiltelbar nuch Verdiinnung
`
`Pharmazie 49 (19®® @210
`
`0002
`
`

`
`
`
`
`
`
`
`
`
`Tabelle 2: Stabilitéitsdaten fiir Bendamustinhydrochlorid bei 23 °C
`
`HZSO4), Methanol 40/60 (V/V). Fluliratez 0,3 ml/min. Wellenliinge: 254 mm,
`lnjektionsvolumenz 101.11. Me13temperz1lur: 4 ‘C bzw. 23 °C.
`
`Sl‘dl‘ld7.1l’Cl-
`2\lJ\\’€lLZl‘|\ll1g
`
`Vz1rizitions-
`["/o]
`koeflizient
`
`Gehtilt an
`HCI [%]
`Bendumuslinv
`(tn = 100%)
`
`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
`
`Z5“
`[11]
`
`0500
`0,57
`4,17
`6,33
`10,25
`21,50
`
`Miltelwert der
`K0|'1Z€nlI'£\-
`gemessenen
`tion an
`Benclzunustin-
`l—1Cl [mg/ml]
`fiir n : 3
`
`0,2525
`0,2508
`0,2401
`0,2375
`0,2243
`0,1966
`
`3. Diskussion
`
`Die Messungen zeigen, dalfi sowohl bei 4 “C als auch 23 °C
`die hydrolytische Zersetzung wéifiriger Bendamustinhydro-
`chloridlosungen in dem gemessenen Bereich linear Verléiuft.
`Fiir die Zersetzung von Melphalan ist eine Kinetik pseudo-
`erster Ordnung beschriebeii [8], die aufgrund des identischen
`Hyd1'olysemechanismus auch fiir das
`strukturverwandte
`Bendamustin angenommen werden kann. Die mittels expo-
`nentieller Regression berechneten Korrelationskoeffizienten
`unterstiitzen diese Annahme. Neueste Untersuchungen zur
`Hydrolysekinetik Von Bendamustin in Wasser bei RE1L11I1-
`temperatur iiber den gesamten Zersetzungszeitraum verifi-
`zieren die Zersetzungskinetik pseudo-erster Ordnung [7]. Alle
`Untersuchungslésungen wiesen den stabilitéitsbegiinstigenden
`pH—Wert von 4 auf. In Vorversuchen wurde die Hydrolyse
`Von Bendamustinhydrochlorid in Wasser
`fiir
`Injektions-
`zwec1<ei'1be1‘pri'1ft, wobei gezeigt werden konnte, daB in diesem
`chloridfreien Medium das Ausmafi der Hydrolyse trotz
`gleichen pH-Wertes wesentlich gr6Ber ist. Fiir Losungen der
`Ausgangskonzentration 0,25 mg/ml wurde bei Raumtempe-
`ratur exemplarisch eine tgo Von 4,211 bestimmt. Dies belegt
`den stabilitéitserhéhenden Einllufi der Chloridionen. Als Tr'2i-
`germedium fiir Bendamustinzubereitung ist ausschliefilich
`isotonische Natriumchloridlosung zu verwenden.
`Die Hydrolyse von Bendamustinhydrochlorid ist erwartungs—
`geméili stark temperaturabhéingig. Die Stabilitfit der Losung
`mit einer Konzentration Von 0,25 mg/ml
`ist bei 4°C (190
`= 118,5 h) urn mehr als das Zehnfache gr6Ber als bei 23 °C
`(tgo = 9,211). Fiir die klinische Praxis bedeutet dies, da13
`Bendamustin-Infusionslosungen kiihl zu lagern sind. Mit
`physiologischer Kochsalzlosung als Triigermedium und unter
`Aufbewahrung im Kiihlschrank ist fur Bendamustinlésungen
`der Konzentration 0,25 mg/ml eine Haltbarkeit Von Vier d
`gegeben, so da13 eine Versorgung mit applikationsfertigen
`Bendamustin-Infusionslosungen im Rahmen einer zentralen
`Zytostatikazubereitung durch die Krankenhausapotheke
`sicher moglich ist. Fiir die empfohlene Applikation als Kurz—
`zeitinfusion iiber 30 min sind ebenfalls keine Stabi1itéits-
`probleme zu erwarten, da bei Raumtemperatur fiir diese
`Bendamustinzubereitungen eine Stabilitéit Von 9 h gegeben
`1st.
`
`4. Experimenteller Teil
`4.1. Substanzen
`
`Die Bestimmungen wurden mit Ribornustin—Ampullen der Firma ribose—
`pharm (Charge 0190393) durchgefiihrt. Jede Durclistecliflasclie mit 55 mg
`Trockensubstanz enthiilt 25 mg Bendamustinhydrochlorid (Hilfsstoll: Man-
`nitol).
`
`4.2. Clnomatograpliiselie Bedingungen
`Die Messung erlolgte mil einer Millipore/Waters HPLC-Anlage.
`Pumpe: Waters 510 HPLC-Pump, Detektor: UV-Detektor Waters 490 E.
`Probengeber: Waters 717»Autosampler. Séiule: Waters Novapak C 18, 60 A,
`4iun, 3.9>< 150 mm. Flieflmittelz wéifirige Nz1lSO4—L6sung 20 mM (pH 3.
`
`Pharmazie 49 (1994), H. 10
`
`4.3. Priizision des Geriites und der Methode
`
`Sechs Einzelinjektionen zius sechs jeweils frisch hergestellteri Referenzlésungen
`der Konzentrzition 0,25 mg/ml werden beziiglicli erinittelter Konzentrzition Lind
`Retentionszeit ziusgewertet. Die Ergebnisse sind in Tabelle 3 dargestellt.
`
`Tabe11e3: Priizision des Geréites und der Methode
`
`Probe Nr.
`
`1
`2
`3
`4
`5
`6
`Mittelwert
`Standardabweicliung
`Variationskoeflizient
`
`1\/lenge
`[mg]
`
`0,2499
`0.2494
`0.2497
`0,2493
`0.2486
`0.2492
`0,2494
`0.000433
`0.2%
`
`Retentions’/.eit
`[mg]
`
`5.93
`5.92
`5,92
`5,94
`5.93
`5.93
`5,93
`0.0063
`0.1%
`
`4.4. Eichung cles Systems
`Die Eichung des Systems erfolgt mittels Mehrpunkteichung mit externeni
`Standard. Es werden je drei Injektionen Von 10%, 100% und 200% der zu
`bestimnienden Konzentration ausgefiihrt (Tabelle 4). Dazu wird eine Ampulle
`Riboniustin“ zunficlist in 10 ml H10 gelést Lind anschlielfiend mit NaCl 0.9%
`zu 0.025 mg/ml. 0.25 mg/ml und 0,50 mg/ml verdiinnt. Die Eichgerade ergibt
`S101] aus den bereclmeten Peaklliichen. aulgetragen gegen die theoretische
`Konzentration. Der Korrelationskoeflizient betréigt 0.9997.
`
`Tabelle 4: Eichung des Systems
`Theoretiscli
`Mittelwert der
`Konzentrulion
`gefundenen
`[mg/ml]
`Konzenlrzitionen
`Hit‘ :1 : 3
`[mg/ml]
`
`Standard»
`iibweichung
`
`Vz1riations—
`koeffizient
`["/u]
`
`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. Probenvorbereitung
`
`Jeweils eine Ampulle Ribomustin“ (4.1.) wird in 10ml Wasser liir Injektions—
`zwecke gelost und €lI‘lSCl‘1llCB€11Cl mit NaCl—L6sung aus Viatlex" Beuteln der
`Firma Baxter (pl~1»Wert: 5.0—7,0) zu 1001111 verdilnnt. Daraus resultiert eine
`NaCl-Konzentration V011 0,81%. Die Zubereitung der Standzirdproben zur
`Erstellung der Eieligeraden erfolgt entsprecliend.
`
`4.6. Auswertung
`
`Die Auswertung erfolgt dureh uutoinatische Peakfléiclienintegration mittels
`Eichgerade (Software Waters Maxima 820).
`
`Literatur
`
`1 Ribomustin" Standzirdinforination fiir Krankeiiliausapotheker April 1993
`ix)
`Preiss. R.: Sohr. R.: lVl21[ll1ldS, M.: Brockmzmn, B.: Hfiller. 1-1.: Pl1'dI'I11'dZl€
`40, 782 (1985)
`Kréimer, 1.: Krankenliausphzirmzizie 8, 325 (1990)
`Stout, S. A.: Riley. C. M.: Int. J. Plmrm. 24. 193 (1985)
`Cliang, S. Y.: Evans, T. S.: Alberts. D. S.: J. Pliarm. Pharinacol. 31, 853
`(1979)
`Personliclie Mitteilung der Firina ribosepluum 1994
`Gust. R.: Unveréffentlichte Ergebnisse (Institut fur Pliarinzizie, Universitéit
`Regensburg)
`8 Flora, K. P.; Smith. S. L.; Cradock, J. C.: J. Chromatogr. 177, 91 (1979)
`
`
`
`\lO\u-42.1»
`
`Eingegangen tun 8. Juni 1994
`Angenommen am 4. Juli 1994
`
`D1". Irene Kréimer
`Apotheke des Klinikums
`Lzingcnbeckstr. 1
`D-55131 Mainz
`
`777
`
`0003
`
`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
`The
`stability of bendamustine hydrochloride
`(0.25
`mg/mL) in 0.9% sodium chloride was studied after storage
`at
`4°C and
`23°C using
`reverse-phase HPLC.
`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.
`
`Introduction
`1.
`4-[5-bis(2-chlorethyl)amino]-1-
`Bendamustine
`(Ribomustin®,
`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
`no
`longer
`prepared
`immediately
`before
`administration,
`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
`nitrogen.
`In addition to the pH value,
`the chloride ion
`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
`suppresses
`the formation of
`the hydroxyl derivative. The
`
`Pharmazie 49 (1994), H. 10
`
`775
`
`0004
`
`

`
`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
`Standard
`Coefficient of
`Content of
`Deviation
`Variation [%]
`Concentration
`Bendamustine
`of
`HCl [%]
`(t0 = 100%)
`Bendamustine
`HCl [mg/mL]
`Measured for
`n = 9
`0.2499
`0.2478
`0.2422
`0.2369
`0.2316
`0.2232
`
`0.00
`19.77
`42.57
`71.63
`92.55
`118.27
`
`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
`
`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
`
`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
`
`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.]
`
`776
`
`Pharmazie 49 (1994), H. 10
`
`0005
`
`

`
`Table 2: Stability data for bendamustine hydrochloride at 23°C
`Time [h]
`Mean
`Standard
`Coefficient of
`Content of
`Deviation
`Variation [%]
`Concentration
`Bendamustine
`of
`HCl [%]
`(t0 = 100%)
`Bendamustine
`HCl [mg/mL]
`Measured for
`n = 9
`0.2525
`0.2508
`0.2401
`0.2375
`0.2243
`0.1966
`
`0.00
`0.67
`4.17
`6.33
`10.25
`21.50
`
`0.00107
`0.00212
`0.00130
`0.00211
`0.00404
`0.00139
`
`0.43
`0.85
`0.54
`0.89
`1.80
`0.71
`
`Equipment and method precision
`4.3
`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.
`
`Retention Time
`[mg]
`5.93
`5.92
`5.92
`5.94
`5.93
`5.93
`5.93
`0.0063
`0.1%
`
`Table 3: Precision of equipment and method
`Sample No.
`Quantity
`[mg]
`0.2499
`0.2494
`0.2497
`0.2493
`0.2486
`0.2492
`0.2494
`0.000433
`0.2%
`
`ean value
`Standard deviation
`Coefficient of variation
`
`123456M
`
`System calibration
`4.4
`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
`Mean Value of
`Concentration
`Concentrations
`[mg/mL]
`Found for n = 3
`[mg/mL]
`0.0266
`0.2499
`0.5080
`
`0.025
`0.25
`0.50
`
`Standard
`Deviation
`
`Coefficient of
`Variation [%]
`
`0.00036
`0.0016
`0.0021
`
`1.4
`0.6
`0.4
`
`Sample preparation
`4.5
`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 Analysis
`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
`
`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
`Substances
`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).
`
`Chromatographic conditions
`4.2
`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
`
`0006
`
`

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