p.782
`
`Institute for Clinical Pharmacology (Director: OMR Prof. Dr. sc. med. H. Hiiller)r
`and Tumor Clinic (Director: OIvIR Prof. Dr. sc. med. K.-H. Riessbeck)2
`of the Medical Division (Charite) of the Humboldt University Berlin
`
`Studies on the Pharmacokinetics of Bendamustine (Cytostasan@) in Humans3
`
`R. PREISS', R. SOI{RI, M. MATTHIAS2, B. BROCKMANNz, ANd H. HUTTNR'
`
`Pharmacokinetics of Benilamustine (Cytostasane@) in Pa-
`tibnts
`;
`The pharmacokinctics of bendamustine (Cytostasane$
`was determimed in plasma on seveo patients aftcr its intra.
`venously and oral application, respcctively. Cytostasane
`was givcn in a dosis of 4,2-5.5 mg . kg-' as an intravenous
`iirfusion over 3 min and as gelatine capsules in a 7-d inter-
`vall, Iu elimination from the plasma is fast, monoexpo-
`nentially and rwo-phasic after intravenous application
`(ttn" = 9.6 grin, trw * 36.1 mia). The AUC was
`71.17 pg. ml-' . h, the central distribution volume 11.15 I
`and,the'distributjon volume in steady statc 20.51 I. The
`mean total clcarancc was 528.9 ml . min-' . After oral appli-
`cation maximal plasma levels of Qrtostasane were detecta-
`bl9 before I h. The mean oral bioavailability was 0.57, ran-
`gcd from 0.25 to 0,94. qrtostasane undergoes metabolism.
`Its bydrolysis h plasma is slow (trn = 1.67 h). After Cytosta-
`saoe the depressiou of leucocytes was mild.
`
`1. Introduction
`
`Bendamustine, Cytostasan@, 5-[bis(2-chloroethyl)amino]-1-methylbenzimidazolyl-2-butanoic
`acid, synthesized in 1963 by Ozegowski and Krebs [12] as "Imet 3393", is regarded :ui an
`effective chemotherapeutic agent in the treatment of neoplastic diseases. The tumoristatic, which
`belongs to the group of alkylating agents, is successfully used in the treatment of chronic
`lymphadenosis and multiple myeloma [4, 5, 9]. It only has mild toxic side effects when
`administered in a typical dosage [4, 5, 9].
`Whereas the clinical use [3, 5, 9] and pharmacodynamic effectiveness [8, l0' 13] of the
`substance have repeatedly been the subject of studies, there has been no information on its
`pharmacokinetics in humans thus far. The goal of the present studies was to obtain initial data on
`the pharmacokinetics of Cytostasan after its intermittent i.v. and oral adminishation to humans.
`
`2. Studies, Results, and Discussion
`The studies were performed in 7 patients, 4 women and 3 men, from 56.4 t 3.7 years of age and
`an average weight of 68.6 + 5.1 kg (tJ,s). All patients had no functional impairments of the
`liver or kidney and had a comparable tumor load. Two patients each had metastatic breast cancer
`or stage IV non-Hodgkins lymphoma. An immunoblastic lymphoma, fibrous sarcoma, and an
`adenocarcinoma were diagnosed in one case each.
`The patients received Cyostasan at a dosage of 4.2 to 5.5 mg ' kg-l as a 3-min intravenous drip
`or orally in the form of gelatin capsules l-1.5 h after a light breakfast between 8 and 9 a.m.
`'Pharmazie 40 (1985), No. 1l
`
`AGILA ET AL - EXHIBIT 1010
`
`

`
`Cytostasan was administered without comedication at a 7-day interval. A 1.5-mL blood sample
`was taken before and 5, 10, 15, 20, 30, 45, 60, 90, 120, 150, 180, and 240 min after
`administration of Cytostasan. The heparinized blood samples were placed immediately in an ice
`water bath and erythrocytes were then separated from plasma by centrifugation. The plasma was
`stored for 7 days at -20 oC until analysis.
`Cytostasan was determined in the plasma samples by HPLC: 5-[bis(2-chloroethyl)amino]-t-
`methylbenzimidazolyl-2-pentanoic acid was used as the internal standard. After protein
`precipitation and extraction with ethyl acetate, the samples were chromatographed using RP8
`columns, type Lichrosorb 5 F, and the Cytostasan concentration in the samples was determined
`fluorometrically.
`
`.N
`
`R3-
`
`sEoo
`
`"
`
`Fig. L HPLC analysis of Cytostasan in plasma 90 or 180 min after its i.v. administration. RT
`e.ig na 6.70 min: metabolite; RT 8.77 and 9.02 min: Cytostasan; RT 10.22 and 10.52 min: 5-
`acid as the intemal standard.
`[bis(2-chloroethyl)amino]-t-methylbenzimidazolyl-2-pentanoic
`Figure 1 shows the typical chromatogram f<jr plasma samples 90 or 180 min after Cytostasan
`administration. Cytostasan appears as a well-defined peak with a retention time of 8.77 and 9.02
`min. The intemal standard follows with a retention time of 10.22 and 10.52 min after Cytostasan
`and well separated from it. The chromatogam shows in addition a metabolite which apparently
`has a higher polarity than Cytostasan and is eluted before it with a retention time of 6.79 and
`6.70. Its identity as a mono- or dihydroxy derivative of Cytostasan or as 5-[bis(2-
`chloroethyl)aminol-1,2-dimethylbenzimidazole was ruled out. Analogous to 4-[4-bis(2-
`chloroethyl)aminophenyl]acetic acid [11] forming by p-oxidation from chlorambucil, this could
`be the acetic acid derivative of 5-[bis(2-chloroethyl)amino]-l-methylbenzimidazole.
`
`n.783
`
`The precise qualitative and quantitative detection is associated with a synthesis of the metabolite.
`The elimination of Cytostasan from plasma occurs rapidly with a clearly evident 2-phase
`behavior. The mean half-lives (Table 1) we determined after i.v. Cytostasan administration of 9.6
`
`Pharmazie 40 (1985), No. l1
`
`

`
`and 36.1 min for the c phase and p phase, respectively, agree well with those described
`literature for the structurally relaied alkylating agents, chlorambucil and melphalan.
`
`in the
`
`Table 1. Pharmacokinetic Parameters of Cytostasan
`
`Range of Variation
`'st
`1.10 5.80-13.80
`9.60
`[min]
`7.00 17.10-72.80
`36.10
`[min]
`4.32 20.03-49.38
`[pg. rnI.'t] 31.27
`[pg.mt,'t .h] 11.17 1.91 3.62-17'16
`11.15 1.74 5.56-18'72
`tl,l
`20.51 5.69 9J8-5123
`tl-l
`[mL' min-rJ 528.9 87.9 291.4-818.6
`
`ttna
`hn1
`
`ce
`
`@
`
`AUCo
`vc
`Vd,,
`cl,o,
`
`of 4.2-5.5 mg'kg-'
`0
`C, =A*B
`A = fictitious starting concentration of the o phase at time t = 0
`B = fictitious starting concentration of the B phase at time t = 0
`
`s"g"
`
`These were determined for the o phase and for the p or terminal phase on average as 7.0 and 8G-
`109min[2, 1l],andinsingletestsintherangesof6.2-13.3and54-109min[1,6'J] 1:1:t9lH
`to the strirt trUf-tife of ttri p ptrase, a low AUC results and with 529 mL ' mir' a high total
`clearance of the alkylating cytostatic (Table l). The central distribution volume, determined on
`average as ll.2l in the 7 patients,.orrrrponis substantially to the blood volume. The merely
`slightiy elevated determined V65s of 20.5i indicates that binding of the native substance in the
`peripheral compartment can be virtually ruled out. Interindividual differences in the elimination
`rate and in the distribution behavior on the substance are considerable and amount up to a factor
`of 5.
`The oral absorption from gelatin capsules with a content of 25 mg of bendamustine
`hydrochloride proceeds rapidf Gig. 2).^Ma,rimum plasma concentrations were measured before
`the elapse of I h in all 6.iu-in.o fatients. The value of 0.57 was determined as the average oral
`uUrorpiion rate from the studieJ in these 6 patients (Table 2). It exhibits considerable
`interindividual differences, however. A similariy large interindividual range of variation is
`described by Alberts et al. [2] for the oral bioavailability of melphalan'
`
`Pharmazie 40 (1985), No. l l
`
`

`
`E
`
`60 l2o ttort'3io
`
`Fig.2. Course of the plasma concenhation of Cytostasan following oral administration of 275
`mg (patient 1).
`
`Table 2. Behavior of the Area under the Serum Level Curve (AUC) after Lv. and Oral
`Administration of Cytostasan
`o
`Patient Dose
`lmgl AUCo;.v. AUC, oral
`[pg.mL-' 'h] [pg'mL'' 'h]
`
`@
`
`275
`250
`
`300
`
`350
`
`275
`
`300
`
`t4.32
`9.07
`17.t6
`
`8.30
`
`3.62
`
`8.80
`
`4.66
`
`5.81
`
`16.04
`
`2.10
`
`1.96
`6.n
`
`x S
`
`F
`
`AUC, or.al
`
`----- €
`AUC, i.v.
`0.325
`
`0.641
`
`0.935
`
`0.253
`
`0.541
`0.694
`
`0.565
`
`0.102
`
`Pharmazie 40 (1985), No. I I
`
`

`
`rvaGli
`
`30 60 eo ti?,n,.lto
`
`a.
`
`E :
`
`. od
`
`to
`oo
`(J
`
`Fig. 3. Hydrolysis of Cytostasan in vitro. 1:phosphate buffer (0'01 mol'L't); pH = 7'5; 37 oC;
`10 pglml,. Z;hepainized plasma; 37 'C;2 p{mL.
`
`N-mustard derivatives undergo rapid hydrolysis in an alkaline environment. Figure 3 shows that
`the hydrolysis of Cytostasan lroceeds very rapidly in a buffered medium with pH: 7 .5 (\rz= 6'2
`min), Uut much mtre slowly (trn : 1.67 h) in heparinized plasma. Similar.pH relations are
`expected in thq upper intestinai area. The superimposition of the rapid absorption by a likewise
`,"piO ttyO.olysis oi the substance could result in passage differences in the stomach and upper
`iniestinal area. This could be a partial explanation for the considerable range of interindividual
`variations in the oral bioavailability of Cytostasan'
`Despite the high dosage, only rather mild side effects occurred. In addition to nausea and
`uo.iting, orynJss ormJuin and mild changes in taste were observed in less than half of the tests'
`After 2i individual. doses of Cytostasan,leukocye depression was found in only 40o/o of the
`tests, whereby the values did not drop below a lower limit of 1800 cells per mm3 of blood'
`
`3. Experimental Section
`
`3. I. Sample preparation
`A total of 0.5 mL of heparinized plasma is combined at 0 oC with 500 ng of 5-[bis(2-chloro-
`ethyl)amino]-1-methylbenzimidazolyl-2-pentanoic hydrochloride as the internal standard,
`dissolved in 50 pL of CHTOH, and with 200 pL of CH:OH. For protein precipitation, the
`,u*pf* are combined with 0.5 mL of HCIO4 diluted 1:10, shaken, and centrifuged for l5 min at
`0 .i and 5000 rpm. The amount of I mL of the supernatant is transferred with ice cooling into
`I mL of K2HpO; (l mol . L'r; and extracted with 4 mL of CHICOOCzHs. After centritugation,
`the CH3COOCzHs extract is concentrated in a Nz sheam at room temperature. The prepared
`samples are stored at -20 oC until measurement. Immediately before the analysis, the residue is
`taken up in 50 pL of CH:CNAIzO (1 :1). Of this,40 pL is chromatographed.
`
`Pharmazie 40 (1985), No. l1
`
`

`
`p. 784
`
`3.2. HPLC
`
`The HPLC measurements are made with the HPLC system 1084 B (Hewlett-Packard, USA) with
`use of the fluorescence detector FS 970 (Schoeffel, USA). LiChrosorb RP8, 5 pL (Merck, BRD)
`in a 250,mm long column with a 4 mm inside diameter was used as the stationary phase. The
`column temperature was 40 "C. An especially pure CH:CN (VEB PCK Schwedt, DDR) and
`KHzPOa (0.1 mol . L-t), pH = 3.0, was used as the mobile phase in a mixture ratio of 70:30 with
`isocratic operation at a flow rate of 1.2 mL ' min-'. The fluorometric detection occurred with
`excitation a1240 nm and emission measurement above 418 nm. The evaluation was performed
`after peak height measurement with use of a daily calibration curve in the patient's blank plasma.
`The detection limit of the method is below 10 nglml-.
`
`3.3. Calculation of the kinetic parameters
`
`The kinetic parameters of Cytostasan were calculated with a Hewlett-Packard 9825 A computer
`using a standard method. The AUC was calculated up to 4 h after administration of Cytostasan
`by the trapezoidal method, and then on the basis of the actual blood level concentration and the
`eiperimentally determined half-life of the monoexponential terminal phase.
`
`The authors are grateful to VEB Jenapharm, DDR, and the Central lnstitute for Microbiology
`and Experimental Therapy of the Academy of the Sciences of the German Democratic Republic,
`in particular Dr. sc. rer. nat. W. Wemer, for providing suitable substance samples.
`
`3 Dedicated to the 60th birthday of MRProf. Dr. sc. med. K. Feller
`
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`
`Received on l5 March 1985
`
`Doz. Dr. sc. med. R. Preiss
`DDR- 1080 Berlin
`Clara-Zetkin-Str. 94
`
`Pharmazie 40 (1985), No. I I