t,
`
`Journal of Radtoanalyttcal snd Nucleor Chemistry, Artlc,les YoI. 121, No. 2 (tggS) 489-497
`
`'
`
`ITADIOCHEMICAL ASSAY OF STABIL TY OF I4C-CYTOSTASANI
`SOLUTIONS DURTNG PREPARATION AND STORAGE
`v. SCASNAR,* S. nezerg* T. TRNorruC,+ R. GRrrpr.,**
`L LISSE**
`
`t I n s ti nt t e of E x p e r ime n tal m arm a col ogt, C e n fte of Ph y siol ogica I .Sc r'errces,
`Slovak Academy of Sclences, Brctislava (CVechoslovakta)
`**Institutg for Pharmacological Ree'arch ln the Pliarmaceutical Industry
`GERMED, Bqltn
`
`(Reccived November 19, 198?)
`Cytostasan, 5:[Bis(2-c]rtoroethyl)aminol.l.methylbenzimidazolyl-2-butyric acid, ir an
`antineoplastic agent wltich degrades spontaneously tn wstcl solutions yielding two
`hydrolysis products, monohydroxy- and dihydroxycytostason. lile devoloped a stsbility-
`indicating radiochemlcal assay based on ion-pair extraction to investiggate the stability of
`solutions of I
`'C-cytostasen under conditions ttrat might be expected when the drug is being
`prepared and stored for pharmacokinetic studles in urlmals. The possibility of using'the
`distribution coefticient of I a C-cytostasan as an indicator of stability was investigated in ttre
`extraction system benzonodicarbolide of cobalt-0.SN HCIO.. The mechanism of extraction
`is believed to be that of ion-pair fornring ptoc€ss between the hydrophobic inion and the
`protonized cytostasan. Slncc no €xtraction of hydroxy derivates was obsin'ed the value of
`in" OisttiUution coeffIcient of the parent drug appears to be a suitable lndicctor of the
`stability of t aC-cytostasan solutions.
`
`Introduction
`
`Over the past two years we have been dealing with pharmacokinetics of cytostasan
`an antineoplastic drug of the nitrogen mustard type, which is clinicaliy used in the
`treatment of chronic lymphadenosis and'multiple myeloma. r Chernically, cytostasan
`is 5-[bls(2-chloroethyl)aminoJ.l.methylberuimidazolyl'2-butyric acid and belongs to
`the lroup of allqylating agents (melphanal, clilorambucil). The analytical chemistry
`of cytostasan was described by HESS.2 He found that the drug degrades sPontane'
`ously in watei solutions yielding two hydrolysis Products, monohydroxy'cytostasan
`a11d clihydroxycytostasan. Neither of these degradation Products has cytotoxic acti-
`vity. Kinetic data for the individual hydrolytic steps of cytostasan were obtained by
`the I H:NMR rnethod.3 Upon hydrolysi.s two chlorine atoms are replaced by an OtI
`group.Becausb of the high instability.of cytostasan in water we focused our atten-
`tior.r to its stability under conditions that might be expected when the drug is being
`prepared ancl storecl prior to administration to anirnals. We used the drug double la'
`Elsevier Eequola S. A., Iausanne
`AkadCmlal Klad6' BudaPest
`
`AGILA ET AL - EXHIBIT 1013
`
`

`
`Irt"
`
`v. $CSNAR et at.: RADTocHEMtcAL AssAY oF STABILITY
`
`cl-cH,-cH,
`
`cr * cH2- c";) *re[
`
`)e,
`|
`CHr
`Fig. 1. Chernibal structure of I 'C-cytostBsan (+ position of label)
`
`t"'-' crrr-
`
`c H,- 6*"
`
`belled with l4C. The structural formula of raC.cytostasan is shown in Fig. 1. As
`known, radiochenrical purity, the fraction of radioactivity present in the specified
`chemical form, is a major factor deterrnining the reproducibility in pharmacokinetic
`studies. Impurities may arise during preparation and storage of radioactively labelled
`drugs and will nrodify organ distributton and specificity of the assay, possibly leading
`to incorrect data. The aim of this work was to propose a simple and specific radio-
`chemical assay for the indication of the stability of water solutions of raCJabelled
`cytostasan to be lierformed before they are used in pharmacokinetic experlrnents..
`
`Experimental
`
`I aC-cytostdsan with a specific radioactivity of 290 MBq/nrrnol was prepared in
`the Zentralinstitut frlr Kernforschung, Rqsendoif, GDR. The product was supplied
`in the form of a powdef withou! any traces of humidity in.a sealed vial. Its radio-
`chemiqal purity was 98%; Nonradioagtive cytostasan d.nd dihydroxycytostasan were
`gifts frorn the Zentralinstitut ftr Mikrobiologie und Experirnentelle Therapie, Jena,
`GDR. Stock solutions of raC-cytostasiin were prepared by dissolving the proper
`anount of label in distilled water or saline and aliquots r.rf l0 prl were taken for thin
`leyer chromatography anatysis or extrac.tion experirnents at various storage tirne in-
`tervals. The polyhedral complex H*[(n-(3)-l,TBsCzHr r )rCo-J, further referred to
`as dicarbolide of oobalt (DC-HI), was synthesized in the Institute of Inorganic
`(hemistry, Czechoslovak Acaderny of Sciences, Prague, and supplied in the form
`of an orange powder. For extrdction the agen! was dissolved in benzene. All rea-
`gents and organic solvents used wer.e of analytical grade. .
`Ascending thin layer clrromatography was conducted on Silufol W 254 nm
`chromatoplates (Kavatier, CSSR) coatecl with silica gel. The solvent system butanol-
`acetic acid-water (4:l:l) rvas use.d. A small amount of nonladioactive c),tostasan
`and d.ihydroxy-cytostasan were spotted together with the sample being analyzed
`in order to visualize the.spots under a UV lamp, Monohydroxycvtostasan was pre-
`pared by storing nonradioactive cytostasan in distilled water foi several days. The
`chrornatoplates were analyzed with a scanner equipped with a gas flow propor-
`tional detector (Tesla Vrdbte, CSSR). For quantitative determination of radloche-
`
`490
`
`.
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`

`
`v. SEASNAR et aI.: RADIocHFMcer AssAY oF sTABTLITY
`
`mical purity, the chromatoplates were cut into several sections and individual strips
`werc counted for radioactivity in 10 ml of Bray's scintillation cocktail using the
`Trip,arb model 300 CD (Downers Grovc, IL, USA) liquid scintillation counter,
`The extraclion experiments were carried out in glass tubes at ambient ternpera.
`ture by shaking for 5 minutes at the phase ratio org/aq = tlt. After extraction
`both phases were separated by centrifugation. The distribution coefficient D was
`calculated as the radioactivity ratio of aliquots of the organic and aqueous plase.
`ln studying the effect of ternperature on the valtre of the d.istribution coeffi-
`cie-nt of raC-cytostasan.the solutions were kept at 37 oC on a water bath, and at
`5 oC and -15 "C using a commercial refrigerator and freezer. Thawing of sarnples
`was achieved at ambient temperafitre, and immediately afterwards the samples
`oc for at least 30 mintrtes.
`were analyzed and then refrozen at -15
`
`Results and discussiorr
`
`A representative radiochrornatogram of frestrly dissolved r aC-cytostasan in dis-
`filled water is shown in Fig. 2 (5 min): Its radiochemical purity was found to be
`96% and the Rg value 0.66. The same sample analyzed 20 days later provided one
`radioactive peak'with an rf value of 0.36, as is seen in Fig.3a. In both cases the
`radioactive peaks corresponded to the spots of fresfily dissolved nonradioactive
`drug and to the drug stored for 3 months in distilled water and to the spot of syn-
`thesized dihydrorycytostasan, the final degradation product of cytostasan.
`Figure 2 depicts the radiochrornatogam of carrier-free r'C+ytostasan held in
`distilled water at ambient temperature for 5, 15,30 and TOminutes. As is seen, a
`rapid degradation of the label occurred. The radiirchemical purity test revealed
`the following anrounts of the parent drug at the giran time intervals: 96, 80, 70
`and 65Vo, respectlvely. The corresponding spots under UV lamp are shown below.
`hak I corresponds to I aC.cytostasan, peak 2 is r aC.monohydroxycytostasan and
`peak 3 is I aC-dihydroxycytostasan.
`The degradation rate of raC-cytostasan was reduced by addition of l0Opg/ml
`of nonradioactivE cytostasan to the l aC-cytostaszur stock solution. Even after 2
`hour storage, about 9oo/o of the parent dnrg was present in the solution. The in-
`hibition of degradation .to a minimum rate wgs observed [n water solutions saturb-
`ted with nonradioactive cytostasan. A typical radiochronratogram of slowly hydro-
`lyzed raC-cytostasan kept for I4 days in saturated water solutions is given in
`Fig..3b. It is obvious that the radioactive peaks 1,2 ond 3 correspond to the parent
`drug, m on ohy droxycytostasa n an d dlhydroxycytostasan, respectively-
`From the rezults presented it ls clear that TLC provides an excellent proof on
`
`491
`
`

`
`t"
`
`V. SCASNAR et aI.: RADIOCHAMICAL ASSAY OF STABILTTY
`
`€:{
`e^
`e2
`Fig. 2. TLC radiochromatograms showing tho effoct of storing tirho on Ulo stabiUty of r'C*yto-
`stasan kept in distilled wator. fiie coreqlonding nonradioactive spots are drown below.
`The qpplication point is indicated by. an arrow; (1) t tC-cytostassr\ (2) r 4C-monohydroxy-
`cytostasan, (3) I 4Cdihydroxycytostasan
`
`ttre high instability of t 'r Ccytostasan in water solutions, but on the other hand
`the whole procedure including spotting developing and measuring is rather long-
`Iasting and fails to meet the criteria fqr rapidly degrading cytostasan. Thus, we
`searched for a much faster method which could give us equivalent infonnation
`rvithin l0 minutes.
`Figure 4 depicts ttre pH dependence of distribution coe fficient of t'C-cyto-
`stasan upon extraction from the phosphate buffer into benzene. As is seen tho pH
`does not influence the D value which remains low, approxirnately 2. We harc tes-
`ted a number of organic solvents ranging from polar to nonpolar but none of them
`was found to be effective. These findings support the assumption that cytostasan
`is a higNy hydrophiiic drug. We found the distribution ratio to be 0,62 in the
`n-octanol/buffer pH 7.4 extraction rystern- However, the distribution coefficient
`was dramatically enhanced in the presence of DC-H+ in benzene at pH 5.2.
`Table I gives the values of the distribution coefficibnt of laC-cytostasan upon
`extraction from O.sM HCIO4 into various organic solvents. In case of benzene-DGH*
`the value of D is even much higlrer than that from the phosphate buffer, All other
`D values are too low or negligible and therefore unsuitable for analytical .purposes.
`Such an unexpectedly itigh O value as found in the extraction system benzerie.DC-H*
`
`492
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`\
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`

`
`v. SCISNAR et al.:RADrocHEMIcALAssAy oF STABILITv
`
`-
`
`or oz :3
`
`Fig 3. TLC kadiochromatogramsr strowing tlre effect of drug concentrati.gn or the stablllty of
`r {C.cytostasan in distilled water; (a) casier-free t'C-cytostasanr (b) I'C'cytostasan in
`distilled water saturatod.with nonredioactive cytostasan The cortesponding nonradioaotivc
`spots are shown below, The application point is indbated by an arrow; (1) r'C'cytostasan,
`(2) t' C-monoh ydroxy cy tostasan, (3) I' C-dihydroxycytostasan
`
`Table I
`Valuos of the distribution coefficicnt @)
`of ' tC-cytostosan uPon extraction
`from 0.5M HCIO. lnto varlou organic solvents
`
`Organic solvent
`
`Chloroform
`Catbon tetractrloride
`n-lleptane
`Diethytether
`Benzene
`Benzene/dicarbollde*
`Ethyl Ecetate
`n Octanol
`Toluene
`
`D
`1.5 r 0.08
`0.001
`0.055 * 0.01
`0.040 t 0.01
`r.3 r 0.05
`188
`15
`8.9 r 0.1
`2.18 r 0.08
`0.21 t 0.03
`
`r Inltlsl concentration
`is co = 5.s . Io-s M.
`
`of DC-H+ ln bentonc
`
`493
`
`

`
`.-l
`
`v. SCISNAR et at.: RADIocHEMIcAL AssAY oF STABILI'TY
`I
`60oof-
`ol
`4ooor
`
`,oo+-
`1o.ooF- -
`
`Fig 4. pH dependence of the distribufion coefficient of I aC-cytostasan upon extraction from a
`phosphate buffcr into benz ene and benzene-DC-H* system. The concentration of DC-If
`is5.5. 10-rM
`
`10 t2
`pH
`
`OBH
`ocH
`Fig 5. Chemical sttuctrre of dicarbolide anion
`
`is probably caused by the fornration of an ion-pair between the hydrophobic di-
`carbolide anion (-) and the protonized form of cytostasan (t) in acid nredia. Di-
`carbotide of cobalt is a potyheclral complex with the central Co atorn in the oxida-
`tion state +3 and strong hydrophobic properties. The chemical structure of dicar-
`
`494
`
`\..,
`
`

`
`v. sdlsNAR et al.: RADToCHEN{ICAL AssAy oF sTABrLrry
`
`bolide anion is shown in Fig. 5. This agent, which beharaes like a strong acid, h,as
`already been used in extraction of stroirgly hydrophilic inorganic cation*. rs?gr+
`and eoSr.2* from biolo$cal materials.a,s-Here the. mechanism of extraction was
`believed to be that of ion-pair forming process betwben the hydrophobic dicarbo
`lide anion and the counter ion. Similarly, we assurne the same principle to be in-
`volved in the extraction of protonated cytostasan.
`Figure 6 docurnents the selectivity of extraction of the parent drug from the
`mixture of its degradation products upon extraction using the benzene-DC.H+ sys-
`tem. The upper radiochromatograrn depicts the partly hydrolyzed I aC-cytostasari
`in distilled water (a) and the lower one is the radiochrornatogram of the benzene-
`
`Fig, 6. Selectivity of extraction 6f t 'C-cytostisan frcim a mixhrrb of its degradation proilucts;
`(a) TLC radiochromatogprrt o'f partty hydrolfi:d t tC+ytostasan, (b) TLC radioctrto'mato
`gram of the benzene-EC-Ff extrait from the sarne.sarnple
`
`DC-H+ extract from the same sample (b). As is seen, under these conditions neither
`hydroxy derivative is extracted. This fact prompted us to use the distribution co-
`efficient of the parent drug as an indicator of the stability of raC-cytostasan solu'
`tions in rarious media and at different t'ime inteivals.
`As known, tfue.value of tfie distribution coefficient for a given drug is a constant
`parametcr wfiich characterizes the physiio-chemical properties of the drtrg itself
`under specified extraction conditions. e.g. type of solvent, pH of aqtreous phase,
`ionic strengt5. temperature etc. Accorclingly, in our particular case the differe0ces
`in the clisiribution ol" the imrnecliately clissolved parent drug and of that stored for
`
`495
`
`\*
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`

`
`.
`V. SCASNAR et gI.: RADIOCHEMICAL ASSAY OF STABILITY
`
`T
`
`varlous time intervals will indicate the presence of interfering hydro:gyderivatives-
`Figure 7 depicts the effect of temperature on the value'of the distribution coeffi-
`cient of raC+ytostasan stored in satine. It is evident tlrai the higher the tempera-
`' hrre, the faiter the decrease of the distribution coefficient. Figure 8 shows the effect
`- of the container material on the value of the distribution coefficient of I aC.cyto-
`stasan stored in saline at ambient temperature (21 "C1. As is seen, the stabiliry of
`the drug is higfrer when stored in a polyethylene container in comparison with a
`glass container. Moreover, adsorption of ttre degradation products on glass occurred.
`No adsorption of the parent drug on glass was observed. Because of the high adsorp-
`tion of hydroxy derivatives on glass the value of the distribution coefficient cannot
`
`2.O
`
`r.5
`
`LO
`
`oI
`
`a -15 0c
`a 50c
`a21oc
`o 37sC
`
`o
`.
`
`a
`e,lrl,l,l,.
`2168
`
`trll
`1?3t.
`
`56?g
`
`.llr
`-l
`-
`27'
`515
`min
`h
`d
`Fig. 7. Effect of temperature on the stablllty of I aC*ytostasan stored in saline
`
`o Polyethytene
`o Gloss
`
`oo
`
`oo
`
`OO
`
`OO
`
`o
`
`o ooo
`ooo
`o
`
`oc
`
`n.o
`
`o.
`
`i-g g -
`rrrQ-
`l23t
`
`o'
`
`5 h
`
`l,l
`
`rl
`3A
`
`.l
`
`uJJ-
`515
`min
`Fig g. Effect of container material on tfte stability of t 'C*ytostasan stored in saline rt ambient
`ternperature (21 "C)
`
`496
`
`\
`
`

`
`v. sdasNAn et al.:RADIocHEMIcALAssAy oF srABrLITy
`be used for quantitative assessnent of degradation of raC.cytostasan solutioris, but
`orly as a semiquantitative stability indicator. Only in tliose csses where the adsorp-
`tion process is negligible, o.8. polyethylene material, is the degree of decomposition
`of the drug directly determined by the value of the distribution coefficient. For ex.
`ample, the half-life of degradation corresponds to the distribution coefficient l- ln
`other words, the value of the distribution coefficient I will indicate the presence of
`the same :urlount of l4C-cytostasan in the organic phase as of its nonextractable
`degradation products in the aqueous phase. Therofore, for qualtitative assessmept
`of degradation in a glass container, the radioactivity extracted at various tirne i'-
`tervals was comPared with that extract'ed at zero tirne, which was considered as the
`standard value.
`
`Conclusion
`
`The radiochemical impurities occurring in the course of preparation and storage
`of raC-cytostasan solutions iue produced by the chemical decomposition of the
`drug itseli and trot by radiatiou induced decomposition. Accordingly, I aC-cyto
`stasan solutions must be pr.epared at 5 "C in an ice bath and used in experinents
`within the sirorteSt possible time,.but solutions of the label can be stored at least
`for 3 and possibly up to 6 months at -15 "C without significant deterioration.
`The extraction method proposed seems to be suitable for stability-indicating purpo-
`ses of aqueous I aC-cytostasan solutions. The method is very simple and fast. No
`TLC or HPLC is necessary.
`
`The authors wislr to thenk Monika LUKACSOVA for her cxcillent technical assistance.
`
`rt
`
`References
`S. gEzK, M. DuRrSovA, v. FABERovA, v.
`LISSE, Final report: Disposition of r4C+ytostasan
`G. HESS, Zentralbl. Pharm. 110 (1971) No, 10.
`G. KLOSE,, G, AUCSTEIN, A. BARTII. Z. SAMEK, OTg.
`l, 15.
`V. SCASNAR, v, KoPRDA, J. Radioanol. Chem.,59 (f 980) 389.
`v. SCASNAR, Anal. Chem., 56 (1984) 605.
`
`SCnSNAN, 'I.. TRNOvEC, R. GRUPE, I.
`in Mice and Rats, Bratislava-Berlin, f 986.
`
`Magnctic Resonance, 19 (1982) No.
`
`1.
`
`2.
`3.
`
`4.
`5.
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`497