Journal ofRadlaamz1ytica'l and Nuclear Chemany. Articles Vol. I2}, ivo. 2 (1988) 489-"49 7
`
`RAD.-IOCHEMICAL ASSAY o1= STABILITY OF I “C-CYTOSTASAN
`SOLUTIONS DURING PREPARATION AND STORAGE
`
`V. .§CASNAR,* S. BEZEK,* T. TRNOVEC."' R. GRUPE,“
`V
`I. LISSE M
`
`‘Institute ofExp erimen ta! Pharmacology, Cenrne ofPhysz'olagz’ca! Sciences.
`Slovak A cademy of Sciences, Bmrisla ya‘ (Czechoslovakia)
`" ‘Institu re for Pharm acological R esearch In th e Phat-maceu rice! Industry
`' GERMED, Berlin
`
`(Received November 19, 1987)
`
`is an
`Cytostasan, 5llBis(2-chloroethyl)amino]-1-methylbenzimidazoiyl-2-butyric acid,
`antineoplastic agent which degrades spontaneously in water solutions yielding two
`hydrolysis products, rnonohydroxyo and dihydroxycytostasan. We developed a stability-
`indicating radiochemical assay based on ion-pair extraction to investigate the stability of
`solutions of ‘ ‘C-cytostasan under conditions that might be expected when the drug is being
`prepared and stored for pharmacokinetic studies in animals.‘ The possibility of usingthe
`distribution coefficient of ' “ C-cytostasan as an indicator of stability was investigated in the
`extraction system benzene-dicarbolide of cobalt-0.SN I-ICIO. . The mechanism of extraction
`is believed to be that of ion—pair forming process between the hydrophobic anion and the
`protonized cytostasan. Since no extraction ‘of hydroxy derivates was observed the value of
`the distribution coefficient of the parent drug appears to be a suitable indicator of the
`stability of ‘ ‘C-cytostasan solutions.
`
`Introduction
`
`Over the past two years we have been "dealing with pharmacokinetics of cytostasan»
`an antineoplastic drug of the nitrogen mustard type, whichis clinically used in the
`treatment of ‘chronic lymphadenosis and-’mu1tiple myeloma.‘ Chemically, cytostasan
`is 5-[bls(2-chloroethyl)amino]-1-methylbenzimidazolyl-2—butyric acid and belongs to
`the group of elkylating agents (melphanal, chlorambucil). The analytical chemistry '
`of cytostasan was described by HESS.’ He-found that the drug degrades spontane-
`ously in water solutions yielding two hydrolysis products, monohydroxy-cytostasan
`and dihydroxycytostasan. Neither of these degradation products has cytotoxic acti-
`vity. Kinetic data for the individual hydrolytic steps of cytostasan were obtained by
`the ‘H-NMR method.3 Upon hydrolysis twouchlorine atoms are replaced by an OH
`group. Because of the high instabilitycf cytostasan in water we focused‘ our atten-
`tiou to its stability under conditionsihat might be expected when the drug is being
`prepared and stored prior to administration to animals. We used the drug double la-
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`Elsevier Sequoia S. A’., Lausanne
`Akadémiat Kiadd, Budapest
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`Cl-CH,-CH, N
`cu—-cH,-cH,> ‘
`
`:fé’°”"cH{'c”"cooH
`c'H,
`
`Fig. 1.. Chemical structure of ' ‘(.2-cytostasan (“ position of label)
`
`belied with “C. The structural formula of “C-cytostasan is shown in Fig. 1. As
`known, radiochemical purity, the fraction of radioactivity present in the specified
`chemical form, is a major factor determining the reproducibility in pharmacokine tic
`studies. impurities may arise during preparation and storage of radioactively labelled
`drugs and will modify organ distribution 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 “C-labelled
`cytostasan to be performed before they are used in pharmacokinetic experiments,
`
`Experimental
`
`“C-cytostasan with a specific radioactivity of 290 MBq/mmol was prepared in
`the Zentralinstitut fiir Kernforschung, R-osendorf, GDR. The product was supplied
`in the form of a powder_without.any traces of humidity in a sealed vial. Its radio- -
`chemical purityfwas 98%.’ Nonradioactive cytostasan and dihydroxycytostasan were
`gifts from the Zentralinstitut ‘fiir ,M.i'k’rob'io1o'gie und Experirnentelle Therapie, Jena,
`GDR. Stock solutions of “C-cytostasan were prepared by dissolving the proper
`amount of label in distilled water or saline and aliquots of 10 pl were taken for thin
`layer chromatography analysis or extraction experiments at various storage time in-
`tervals. The polyhedral complex I‘-I"‘[(1r-('3)-1,2-B9C-2H, , )2Co']_. further referred to
`as dicarbolide of cobalt (DC-Hf’), wassynthesized in the Institute of Inorganic
`Chemistry, Czechoslovak Academy of Sciences, Prague, and supplied in the form
`of an orange powder. For extraction the agent was dissolved in benzene. All rea-
`gents and organic solvents usedhwere of analytical grade. .
`Ascending thin layer cliromatography was conducted on Silufol UV 254 nm
`chromatoplates (Kavalier, CSSR) coated with silica gel. The solvent system butano1—
`acetic acid—water (421 :1) was use_d.,A small amount of nonradioactive cytostasan
`and dihydroxy-cytostasan were spotted together with the sample being analyzed
`in order to visualize the spots under a _UV lamp. Monohydroxycytostasan was pre-
`pared by storing nonradioactive cytostasan in distilled water for several days. The
`chromatoplates were analyzed with a scanner equipped with a gas flow propor-
`tional detector (Tesla Vrable, CSSR). For quantitative determination of radioche-
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`mical purity, the chromatoplates were cut into several sections and individual strips
`were counted for radioactivity in 10 ml of Bray's scintillation cocktail using the
`Tricarb model 300 CD (Downers Grove, IL, USA") liquid scintillation counter.
`The extraction experiments were carried out in glass tubes at ambient tempera-
`ture by shaking for 5 minutes at the phase ratio org/aq = 1/ 1. 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 phase.
`in studying the effect of temperature on the value of the distribution coeffi-
`. cient -of “C-cytostasan the solutions were kept at 37 °C on a water bath, and at
`5 °C and -15 °C using a commercial refrigerator and freezer. Thawing of samples
`was achieved at ambient temperature, and immediately afterwards the samples
`were analyzed and then refrozen at -15 °C for at least 30 minutes.
`
`Results and discussion
`
`A representative radiochromatogram of freshly dissolved 1 ‘C-cytostasan in dis-
`tilled water is shown in Fig. 2 (5 min); Its radiochemical purity wasfound to be
`96% and the Rt? value 0.66. The same sample analyzed 20 days later provided one
`radioactive pealc-with -an If value of 0.36, as is seen in Fig. 3a. In both cases the
`‘ radioactive peaks corresponded to the spots of freshly dissolved nonradioactive
`drug and to the drug stored for 3 months in distilled water and to the spot of syn-
`thesized dihydroxycytostasan, the final degradation product of cytostasan.
`Figure 2 depicts the radiochromatogram of carrier-free “C-cytostasan held in
`distilled water at ambient temperature for 5, 15, 30 and 70' minutes. As is seen, a
`rapid degradation of the label occurred. The radiochemical purity test revealed
`the following amounts of the parent drug at the given time intervals: 96, 80, 70
`and 65%, respectively. The corresponding spots under _UV lamp are shown below.
`Peak 1 corresponds to “C—cytostasan, peak 2 is “C-monohydroxycytostasan and
`peak 3 is “C-dihydroxycytostasan.
`The degradation rate of “C-cytostasan was reduced by addition of 100 pg/ml
`of nonradioactive cytostasan to the 1 ‘C-cytostasan stock solution. Even after 2
`hour storage, about 90% of the parent drug was present in the solution. The in-
`hibition of degradation to a minimum rate was observed in water solutions satura-
`ted with nonradioactive cytostasan. A typical radiochromatogram of slowly hydro-
`lyzed “C-cytostasan kept for 14 days in saturated water solutions is given in
`Fig. .3b. It is obvious that the radioactive peaks 1,2 and 3 correspond to the parent
`drug, monohydroxycytostasan and dihydroxycytostasan, respectively.
`From the results presented it is clear that TLC provides an excellent proof on
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`Fig. 2. TLC radiochromatograms showing the effect of storing time on the stability of ‘ ‘C-cyto-
`stasan kept in distilled water. The conresponding nonradioactive spots are shown below.
`The application point is indicated by. an arrow; (1) ’ ‘C-cytostasan, (2) ’ ‘C-rnonohydroxy-
`cytostasan, (3) ‘ ‘Gdihydroxycytostasan
`
`the high instability of ’ ‘Ccytostasan in water solutions, but on the other hand
`the whole procedure including spotting, developing and measuring is rather long-
`lasting and fails to meet the criteria for rapidly degrading cytostasan. Thus, we
`searched for a much faster method which could give us equivalent information
`within 10 minutes.
`'
`
`Figure 4 depicts the pH dependence of distribution coefficient of ‘ "C-cyto-
`stasan upon extraction from the phosphate buffer into benzene. As is seen the pH
`does not influence the D value which remains low, approximately 2. We have 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 highly hydrophilic drug‘. We found the distribution ratio to be 0.62 in the
`n-octanol/buffer pH 7.4 extraction system. However, the distribution coefficient
`was dramatically enhanced in the presence of DC-H* in benzene at pH 5.2.
`Table 1 gives the values of the distribution coeificient of “C-cytostasan upon
`extraction from 0.5M HCIO4 into various organic solvents. In case of benzene-DC-H"
`the value of D is even much higher than that from the phosphate buffer. All other
`D values are too low or negligible and therefore unsuitable for analytical purposes’.
`Such an unexpectedly high D value as found in the extraction system benzene-DC-I-1*
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`Fig. 3. TLC hadiochromatograms: showing the effect of drug concentration on the stability of
`' ‘C-cytostasan in distilled water; (a) canier-free ‘ “ C-cytostasan, (b) ‘ ‘C-cytostasan in
`distilled water saturated with nonradioactive cytostasan. The corresponding nonradioactive
`spots are shown below. The application point is indicated by an arrow; (1) ‘ ‘C-cytostnsan,
`(2) ‘ “C~monohydroxycytostasan, (3) "C-dihycltoxycytostasan
`
`Table 1
`
`Values of the distribution coefficient (D)
`of ’ ‘C-cytostosanupon extraction
`from 0.5M I-ICIO. into various organic solvents
`
`
`
`
` Organic solvent ' D
`
`
`
`Chloroform
`Carbon tetrachloride
`
`n-I-Ieptane
`Diethyl ether
`Benzene
`
`t 0.08
`
`1.5
`0.001
`
`0.055 :t 0.01
`0.040 t 0.01
`1.3
`t 0.05
`
`1 5
`1 88
`Benzene/dicarbo1ide"'
`1: 0.1
`8.9
`Ethyl acetate
`1 0. 08
`2.7 8
`n-O ctanol
`0.21 t 0.03
`Toluene
`
`
`.
`
`" Initial concentration of DC—l-I’ in benzene
`is c.= 5.5 -10” M.
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`Fig. 4. pH dependence of the distribution coefficient of ‘ ‘C-cytostasan upon extraction from a
`phosphate bufier into benzene and benzene-DC-H‘ system. The concentration of DC-I-1*
`is 5.5 - 10‘ ’ M
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`
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`Fig. 5. Chemical structure of dicarbolide anion
`
`is probably caused by the formation of an ion-pair between the hydrophobic di-
`carbolide anion (——) and the protonized form of cytostasan (+) in acidlmedia. Di~
`carbolide of cobalt is a polyhedral complex with the central Co atom in the oxida-
`tion state +3 and strong hydrophobic properties. The chemical’ structure of dicar-
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`bolide anion is shown in Fig. 5. This agent, which behaves like a strong acid, has
`already been used in extraction of strongly hydrophilic inorganic cations" ‘“Cs’’
`and “ST.” {Wm bi°1°8iCfi1 materials-4'5 Here The. mechanism of extraction was
`believed to be that of ion-pair forming process between the hydrophobic dicarbo-
`lide anion and the counter ion. Similarly, we assume the same principle to be in.
`volved in the extraction of protonated cytostasan_
`Figure 6 documents 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 radiochromatogram depicts the partly hydrolyzed "C-cytostasan‘
`in distilled water (a) and the lower one is the radiochromatogram of the benzene-
`
`
`
`D-
`
`‘
`Fig. 6. Selectivlty of extraction of ‘ ‘VC-cytostasan Erorna mixture of its degradation products;
`(a) TLC radiochzomatogram of partly hydrolyzed ‘ ‘C.-cytostasan, (b) TLC radiochtomato-
`gram of the benzene-DC-H’ extract from thesame sample
`
`DC-H* extract from the same sample (b). As is seen, under these conditions neither
`hydroxy derivative is extracted. This fact prornpted us to use the distribution co-
`efficient of the parent drug as an indicator of the stability of “C-cytostasan solu-
`tions in various media and at different time intervals.
`'
`As known, the ‘value of the distribution coefficient for a given drug is a constant
`parameter which characterizes the physico-chemical properties of the drug itself
`under specified extraction conditions, e.g‘.
`type of solvent, pH of aqueous phase,
`ionic strength. temperature etc. Accordingly, in our particular case the differences
`in the distribution of the immediately dissolved parent drug and of that stored for
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`various time intervals will indicate the presence of interfering hydroxyderivatives-
`Figure 7 depicts the effect of temperature on the value-of the distribution coeffi-
`cient of “C-cytostasan stored in saline. It is evident that the higher the temps ra~
`' ture, the faster the decrease of the distribution coefficient. Figure 8 shows the effect
`of the container material on the value of the distribution coefficient of ’ ‘C-cyto-
`stasan stored in saline at ambient temperature (21 °C). As is seen, the stability of
`the drug is higher when stored in a polyethylene container in comparison with a
`glass container. Moreover, adsorption of the 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
`
`0 Z5
`
`8‘
`
`1.5
`
`1.0
`
`0.5
`
`0
`
`L’.
`
`'
`
`O 37 °C
`
`5 -15 °C
`0
`5 °C
`
`4 21 °C'
`
`°
`
`.
`
`i_L_l_J ¢.l.1_Li..Li_.L._._.L__,..
`5351
`2
`3
`lo
`5
`5
`7
`8»
`2463
`27
`min
`rs
`d
`
`Fig. 7. Effect of texnperature on the stability of ‘ ‘C-cytostasan stored in saline
`
`'3 3.0
`3.
`‘
`
`— O
`
`O
`‘3 D O O O o
`0
`
`.
`
`c1
`
`9
`
`0
`
`0 Polyethylene
`0 Gloss
`
`° 0
`
`° 0
`
`.3 D
`
`D D
`
`°
`
`0
`
`O
`
`2.5 »
`
`’
`
`20
`
`1.5
`
`1.0
`
`0.5‘
`
`0
`
`D - D“‘OU
`i._l_l_»L_i._..L__J_L4_.L..i_l
`L.J._L.§_.
`5151
`2
`3
`4
`'5
`I234
`
`' mm
`
`h
`
`d
`
`Fig. 8. Effect of container material on the stability of ‘ ‘C-cytostasan stored in saline at ambient
`temperature (21 °C)
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`be used for quantitative assessment of degradation ‘of “C-cytostasan soluticris, but
`. only as_a semiquantitative stability indicator. Only in those cases where the. adsorp-
`tion process is negligible, e.g. 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 1. In
`other words, the value of the‘ distribution coefficient 1 will indicate the presence of
`the same amount of “C-cytostasan in the organic phase as of its nonextractable
`degradation products in the aqueous phase. Therefore, for quantitative assessment
`of degradation in a glass container, the radioactivity extracted at various time in-
`tervals was compared with that extracted at zero time, which was considered as the
`standard value.
`
`Conclusion
`
`The radiochemical impurities occurring in the course of preparation and storage
`of “C-cytostasan solutions are produced by the chemical decomposition of the
`drug itself and not by radiation induced decomposition. Accordingly, "C-cyto-
`stasan solutions must be prepared at 5 °C in an ice bath and usedlin experiments
`within the shortest 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 1 ‘C-c-ytostasan solutions. The method is very simple and fast. No
`TLC or HPLC is necessary.
`‘
`"
`
`The authors wish to thank Monika LUKACSOVA for her excellent technical assistance.
`
`it
`
`References
`
`'1‘. TRNOVEC, R. GRUPE, 1.
`i5Um§ovA, v. FABEROVA, v. SCASNAR,
`1. S. BEZEK, M.
`LISSE, Final report; Disposition of "C~cytostasan in Mice and Rats, Bratislava—-Berlin, 1986.
`2. G. HESS, Zentralbl. Pharm. 110 (1971) No. 10.
`'
`3. G. KLOSE, G. AUGSTEIN, A. BARTI-l. Z. SAMEK, Org. Magnetic Resonance, 19 (1982) No.
`1, 15.
`.
`4. V. SCASNAR, V. KOPRDA, J. Rndioanul. Chem., 59 (1980) 389.
`5. v. SCASNAR, Anal. Chem., 56 (1 984) 605.
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