Possible Intermediates in the Action of Adriamycin a Pulse Radiolysis Study E.J

Br. J. Cancer (1985), 51, 515-523

Possible intermediates in the action of adriamycin A pulse radiolysis study E.J. Land', T. Mukherjeel*, A.J. Swallow' & J.M. Bruce2 'Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester, M20 9BX and the 2Department of Chemistry, The University, Manchester M13 9PL, UK.

Summary Over a wide range of pH, the semiquinone free radicals formed by reduction of adriamycin exist as a form which is strongly stabilised by internal hydrogen bonding and resonance. They protonate with pKa = 2.9. Below this pH they exhibit absorption maxima at 430nm (smax = 13,200dm3mol-'cm-1) and -720nm (Cmax =4,200dm3mol3-1 cm -). Above pH 2.9 they have maxima at 480 nm (smax= 14,600 dM3mol-1 cm-') and - 700 nm (Smax = 3,400 dm mol -cm -'). In acid and alkaline solution the radicals rapidly disappear by disproportionation, but within the approximate pH range 6 to 11 they appear to be relatively stable for at least 10-20ms, existing in transient equilibrium with parent adriamycin and the full reduced form. Some rate constants for the formation and reactions of the semiquinone are given, including the reaction with oxygen to give 0i-. Fully reduced adriamycin has absorption maxima at 410 nm (smax = 11,000dm3 mol-lcm-') at pH 5 and 430 nm (Vmax=19,000dm3mol- cm -) at pH 11. It undergoes decomposition within a few hundred ms. The intermediates from daunomycin would be expected to have properties similar to those from adriamycin.

Anthracycline antibiotics constitute a major class of or hydroxyl (OH'), which could react with the chemotherapeutic agents for the treatment of proximal DNA, producing the strand-scission different kinds of cancer. Among these, adriamycin reported for adriamycin (Neidle & Waring, 1983) (doxorubicin) has the widest spectrum of clinical As well as the semiquinone free radicals, other anti-tumour activity (Henry, 1976; Arcamone, unstable one- and two- electron reduced products 1981). Unfortunately chemotherapy with adria- may also play an important role in the biological mycin is hampered by dose-related cytotoxic and action of the drug (Sinha & Gregory, 1981). cardiotoxic effects (Rosen et al., 1974; Green et al., The ability of adriamycin free radicals to enter 1984). the nucleus and cause specific destruction of The mechanism of action of adriamycin does not nuclear DNA would depend on their reactivity with necessarily involve free radicals (Johnston et al., oxygen and other cell constituents and on their 1983). On the other hand, adriamycin, like other reaction with each other. Since the semiquinones anthracyclines, is known to augment the flow of are expected to be short-lived, the technique of electrons from NADPH to molecular oxygen. In pulse radiolysis is ideally suited to generate and this reaction, an intermediate semiquinone free study them (Swallow, 1982). The one-electron radical is formed by interaction with mammalian reduction potential of adriamycin has already been microsomes (Sato et al., 1977; Bachur et al., 1978). investigated by this method (Land et al., 1983a). From ESR studies (Bachur et al., 1977; The present study was undertaken to determine the Kalyanaraman et al., 1980) it has been shown that basic physicochemical properties of the semi- the semiquinone free radicals may serve to shuttle quinone with special emphasis on the optical the electrons to oxygen, a reaction catalysed by absorption, kinetic and stability parameters. Some NADPH-cytochrome P-450 reductase (Bachur et al., properties of the fully reduced form of adriamycin 1979). The radicals could be responsible for the are also reported. Since adriamycin is chemically toxic and/or active principle of adriamycin similar to daunomycin, our findings would also be chemotherapy. They could be site-specific, binding expected to apply to daunomycin. Our results help to DNA. They could either react directly, or they to provide a firm physico-chemical basis on which could generate other radicals like superoxide (02-) speculations about the action of the agents can rest.

Materials and methods *Present address: Chemistry Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India. Adriamycin (doxorubicin) hydrochloride was Received 3 October 1984; and in revised form 19 obtained from Sigma and was used as received. A December 1984. medicinal sample of the drug, containing lactose ©) The Macmillan Press Ltd., 1985 516 E.J. LAND et al. additive, was a product of Farmitalia research, with a combined electrode was used for the pH obtained from Montedison Pharmaceuticals Ltd. determinations. Pye Unicam SP 8000 (recording) Solutions were kept at room temperature for the and Cecil Instruments CE292 (digital) spectro- minimum period necessary. For experiments in acid photometers were used for the absorbance or alkaline solution, the acid (HC1O4) or alkali measurements of stable compounds. Curve fittings (NaOH) were added immediately before the and kinetic analyses were carried out using a experiments to minimise any hydrolysis of the Hewlett-Packard 9845A computer fitted with a molecule. Necessary stability checks were made Ladd Orthoplex Co-ordinate sensor type 3825-1. using absorption spectra as well as thin layer Programmes were written in this laboratory. chromatography. Where stock solutions of the drug were required, they were stored under refrigeration Results and discussion for the shortest possible time. It has been shown (Hoffman et al., 1979) that solutions remain stable Parent and dimerisation under such conditions for months. Water was spectra, pKa redistille,d from alkaline permanganate. All other Absorption spectra of adriamycin at pH 5.7, 11.5 chemicals were AnalaR grades from either BDH or and - 14.3 are shown in Figure 1. Care was taken Hopkin and Williams. Solutions were buffered to monitor the spectrum in the most alkaline wherever necessary by borate/OH-, H2PO /HPO2- solution in less than 5min after adding the alkali, or HCOOH/HCOO-. All solutions were purged for but it is possible that there may have been partial at least 30 min using argon, oxygen or oxygen diluted hydrolysis of the aminosugar group in the solution with nitrogen (all from Air Products Ltd.), or N20 (Abdeen et al., unpublished data). However this (British Oxygen Company). should have little effect on the spectrum. The For the pulse radiolysis studies, the purged spectrum at pH 5.7 is that of fully protonated solutions containing adriamycin, buffer and sodium adriamycin, represented as +HAdH2 where the first formate (10 -moldm 3) or formic acid (I mol H refers to that on the aminosugar and the other dm-3), were passed through a capillary flow system two to those on the hydroquinone: into a quartz capillary optical cell of - 3 mm 0 OH diameter and optical path length 1 mm, 1 cm or 0 2.5 cm. An electron beam from the Paterson OH Laboratories linear accelerator (Keene, 1972) was used to irradiate the solution. The changes produced were analysed within a very short time-scale (few microseconds to few seconds depending on the type of study) using an analysing light beam from a xenon or tungsten lamp. Doses (0.6 to 10Gy, where 1 Gy = J Kg- 1) were such that the concentration of radicals produced was always 5% of the parent adriamycin or less. Appropriate cut-off filters were used to avoid photolysis of the solution by the analysing light. The light coming through the solution was passed through a Bausch E and Lomb monochromator into an EMI 9558Q 0 photomultiplier (using band widths of 5 or 10nm) 0 for wavelengths up to 760 nm or a UDT pin 10 E photodiode (band widths 20 or 30nm) for wave- E lengths from -700 to 1000nm. Changes in optical transmission with time were recorded either with a -0 Polaroid camera fitted to a Tetronix oscilloscope or on paper print-out using a Commodore PET x 2001 computer fitted with a Tektronix 7912 AD digitiser. Absorbed doses were determined from the transient (SCN)2- formation from oxygen-saturated -3 10-2mol dm potassium thiocyanate (Adams et al., 15UU 4UU bUU louu 700 1965), using G (number of molecules of the species AJnm formed per 100 eV of absorbed dose) = 2.9 and I Figure 1 Absorption spectra of aqueous solutions of E500nm=7.1 X 103 dm3 mol- cm.-1 adriamycin (5.6x 10-5moldm-3), (A) pH5.7; (b) A radiometer pHM 62 digital pH meter fitted pH 11.5, (c) pH- 14.3. INTERMEDIATES FROM ADRIAMYCIN 517

Throughout the work we adopt pKa values as (9). The difference between the absorption spectra of follows (Sturgeon & Schulman, 1977) for the several the semiquinone and the quinone was obtained possible dissociations. No allowance has been made from measurements after the essential completion of for any effect of ionic strength. reactions (6) to (10) but before any significant radical-radical reaction had time to take place. +HAdH2=H+ +AdH2, pKa=8.22 (1) Difference spectra for pH 1.1 and 9.1 are shown in Figure 2. It can be seen that the absorption of the +HAdH2H+H++HAdH-, pKa9.01 (2) semiquinone is markedly different from that of the quinone in the region up to 600nm, and that new +HAdH-=H+ +AdH-, pKa=9.36 (3) bands appear at > 700nm where the quinone does not absorb at all. AdH2H + +AdH -, pKa= 10.1 (4) AdH--=H+Ad2, pKa 13.2 (5) On this view the spectrum at pH 11.5 is that of the form AdH- and the spectrum at pH- 14.3 is predominantly that of Ad2-. Adriamycin and daunomycin undergo associative dimerisation in aqueous solution (Eksborg, 1978; 0 et The Barthelemy-Clavey al., 1974). extent of x dimerisation would be expected to depend on pH a) and ionic strength, but if the constant Kd defined by 0 Q Kd = [Dimer]/[Monomer]2 is taken to be 570- .0Em 0 700dm3mol-1, as on the basis of measurements on 0 .0 daunomycin by circular dichroism and NMR n1 (Barthelemy-Clavey et al., 1974). then a typical solution of 6 x 10-5moldm-3 adriamycin consists of > 90% of the monomeric form so that di- I merisation should not play a major part in the results reported here.

Difference in absorption between semiquinone and quinone, and acid dissociation constants Figure 2 Difference between absorption spectra of semiquinone and parent adriamycin measured lOus When single pulses of electrons are given to argon- after the pulse, [adriamycin]=6.3 x 10-moldm-3 purged solutions of adriamycin containing high optical path length 1 cm, dose 5.5 Gy, (0) pH 1.1 concentrations of formate or formic acid, (I moldm-3 formic acid+H2S04); (0) pH9.1 semiquinone free radicals are formed by the (10 - Imol dm - formate+ borate buffer). sequence: H2O"-H-, OH, e- , H2, H202 (6) The variation with pH of the absorption change at 475 nm, AA, is shown in Figure 3. Since the OH,(H) + HCO2 (HCO2H)-4CO?2(CO2H ) parent does not change in this region, the variation must be due to a dissociation of the semiquinone. + H20(H2) (7) The points were fitted to the equation: eeq + adriamycin-+semiquinone (8) AA2 AA = + p_K_+l±lOpK -pH (11) CO'- + adriamycin-+semiquinone + CO2 (9) AA-iopH In N20-saturated solutions, the following reaction where AA1 and AA2 are changes in absorbance well predominates over reaction (8): beyond the pKa of the semiquinone. A good fit was obtained with pKa=2.9+0.05. The pKa is likely to H20 (10) correspond to dissociation of a proton from the eeq +N20 + N2+OH+OH- semiquinone moiety itself rather than the -NH' group, since the -NH+ group is remote from the Reaction (10) will be followed by reactions (7) and chromophore. Redox analysis shows that the net 518 E.J. LAND et al.

such as those in Figure 2 assuming the yield of 20 .- o- 0 semiquinone to be 6.5 molecules per 100eV. Spectra for pH 1.1 and 9.1 are given in Figure 4. The spectra at pH 5 and 12 were identical to that at 0 10- / pH 9.1. Below 600nm the spectrum of the basic form is similar to an independently determined x but an additional a1) 0 spectrum (Svingen & Powis, 1981) 0- broad band is now seen at -700nm. No significant .0Eco change in spectrum was observed at pH 5 when the 0 adriamycin concentration was varied from 2 x 10-5 .0 to 5.5 x 10 -4moldm-3 showing no effect of any -10- association between radicals or between radical and /0 parent. The presence of lactose (90% w/w), as in clinical samples of the drug, had no effect on the -20 - __ 0 ' spectrum.

1 2 3 4 5 6 pH 18 - Figure 3 Variation with pH of change in absorbance at 475 nm produced by pulse, [adriamycin] = 5.6 x 16 - 10-5moldm-3. The solid line is a computed best fit (Equation 11) with =2.9. pKa 14

E 12- charge on the semiquinone is zero at pH 7 (Land et u al., 1983a). This is further corroborated by 7 0 conductivity measurements (Cercek et al. E 10- we may write: unpublished data). Consequently E 8- + - = HAdH* =-H +++HAdHi+ PKa 2.9 (12) I0 The of is lower than found for most x 6- pKa 2.9 Q; semiquinones (Swallow, 1982) but is similar to the value of pKa=2.7 found for the corresponding pKa 4- of the model compound naphthazarin (Land et al., 1983b) presumably for the same reason, viz. 2- extensive delocalisation in the strongly internally hydrogen-bonded semiquinone structure. No pKa could be found spectroscopically in the 0o I 400 500 600 700 800 900 region 5-12. However by analogy with K/nm naphthazarin (Land et al., 1983b) and quinizarin (Land et al., in preparation) a further pKa of the Figure 4 Absolute absorption spectra of adriamycin hydroquinone part at pH 14 seems likely. The semiquinone obtained from data of Figure 2, (0) 9.1. -NH+ group of the semiquinone has pKa = 9.2 pH 1.1, (-) pH (Land et al., 1983a):

+ HAdH'- =H + + AdH, -, pKa = 9.2 (13) Formation of the semiquinone The rate of reaction between eaq and adriamycin The zwitterionic structure of the semiquinone at was obtained from the rate of biological pH values will influence transport (reaction 8) through membranes and affect orientation of the disappearance of e- in solutions containing five species when in the close vicinity of other molecules different concentrations of adriamycin (5 x 10-6 to of biological interest. 3xlO-5moldm-3) at pH6.0 (+HAdH2) and 11.5 (AdH-). Allowance was made for reaction of eaq in the Second Absolute absorption spectra of the semiquinone with other components solution. order rate constants were obtained from the The absolute absorption spectra of the semiquinone dependence (linear) of the observed first order rate free radicals were obtained from difference spectra constant on adriamycin concentration. The INTERMEDIATES FROM ADRIAMYCIN 519

corresponding rates with CO- (or C02H) were attainment of an equilibrium: studied within the concentration range 1 x 10-5 to 9 x 10- 5 mol dm- 3 by monitoring the growth of the 2 semiquinone=parent quinone+hydroquinone semiquinone absorbance at 380 nm, 475 nm and 720 nm using N2 0-saturated solutions. Rate con- (14) stants are included in Table I. The reaction of hydrogen atoms with adriamycin With adriamycin the equilibrium appeared to be was observed in acid solutions (5 x 10- 2mol much more to the left than with naphthazarin. For dm- 3H2SO4) containing 10- 'mol dm- 't-butyl example after 10- 6mol dm- radicals had been alcohol in place of formate. The product of the introduced into a solution of 5 x 10- 5mol dm- 3 reaction had a different spectrum from that of the adriamycin at pH 9, the shape and magnitude of the nm reaction of e- or CO'-, no doubt due to extra absorption changes in the range 530-730 after reaction possibilities available to hydrogen atoms, - 20 ms were indistinguishable from that due to the such as addition to the aromatic ring or abstraction production of the semiquinone, consistent with the of hydrogen. The rate of the reaction is included in equilibrium constant for reaction (14) being less than or to so that the radicals did not Table I. equal 1, decay significantly during the period of Radical stability measurement. Unfortunately attempts to explain the measured pH dependence of the absorption changes Observations were made at 720 nm, where fully in terms of reaction (14), although successful with reduced adriamycin would not be expected to have naphthazarin, have not yet succeeded with a strong absorption and the parent has zero or adriamycin so that an extra factor must be present. negligible absorption. In strongly acid and strongly Nevertheless it is clear that the adriamycin semi- alkaline solutions the absorption at 720 nm quinone is rather stable to disproportionation, so decreased in a second order manner attributed to that in biological systems there will be ample scope disproportionation of the semiquinone to yield for reaction of the semiquinone with components of parent and fully reduced adriamycin (the hydro- the cell remote from the site of origin. quinone). The rate constants for the reactions at pH 1.1 and 13 are included in Table I. Within the Reaction with oxygen approximate pH range 6 to 11 the absorption at 720 nm at first decreased, and then within one or Adriamycin semiquinone radicals were found to two milliseconds attained a value which remained react rapidly with oxygen at all pH values between constant for at least 10-20 ms. The residual 4.5 and 12. Rates for two pH values are given in absorption was at its highest at pH about 9. This Table I. It may be noted that the direction of the behaviour is analogous to that seen with naphtha- reaction, i.e. virtually complete reaction of the zarin (Land et al., 1983c). In the case of naphtha- adriamycin radical with oxygen to produce zarin the absorption changes were due to the superoxide, is different from that seen in non-

Table I Rate constants for formation and reactions of adriamycin semiquinone Rate Constant Reaction pH d3mol- 1s- I

Caq+ +HAdH2+ HAdH; + H2 6.5 (2.5 + 0.3) x 1010 eaq + AdH - -AdH- + OH- 11.5 (1.5+0.2) x 1010 CO2H+ +HAdH2-+CO2+ +HAdH; 1.1 (3.5+0.4) x 109 H-+ +HAdH2-+products 1.1 (2.9 +0.3) x 109 COj- + +HAdH2CO2+ +HAdHy- 6.5 (3.4+0.4) x 109 COj- +AdH +H2O- CO2+AdH;- +OH- 11.5 (1.8 +0.2) x 108 2+HAdH; -+ HAdH2+hydroquinone 1.1 2k=(1.3+0.2) x 109 2AdH'-AdH-(Ad2 )+ hydroquinone 13 2k = (5.6± 0.8) x 108 +HAdH- + 02 - O7 ++HAdH2 6.0 (3.5 + 0.4) x 108 AdH- +O2+OH-90j + AdH +H20 11.5 (1.7+0.2) x 108 520 E.J. LAND et al.

a b I

r .-I .... 1.114

If| f~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~$ CN

1 ms 1 ms c FT I IT TI. d I

c 0 ' .E_

cn CiL IIItT T

0 0l< I ,I* q*

1 ms 500 tLs

-!R $ q*

500 ,us 500 ,Ls

Figure 5 Oscilloscope/computer traces showing the formation of fully reduced adriamycin (hydroquinone) by mutual reaction of semiquinone free radicals. [adriamycin] =4.9 x O-5mol dm [formate]=10-lmoldm-, optical path length 2.5 cm, dose 7 Gy. (a) pH 5.0, A = 370 nm; (b) pH 5.0; A=400 nm; (c) pH 5.0 A=480 nm; (d) pH 11.5, A=430nm; (e) pH 11.5, A=480nm; (f) pH 11.5, A=520nm. INTERMEDIATES FROM ADRIAMYCIN 521 aqueous solution (Afanas'ev et al., 1980). At spectra obtained are shown in Figure 6. The biological pH values the reaction may be expressed: principal uncertainty is in ascribing the absorption at 720nm exclusively to the semiquinone. As the absorptions at 720nm a few ms after the pulse were +HAdHW +O2 + HAdH2+O2 (15) at their highest between pH 6 and - 11 it was not Equation (15) bears directly on a possible mode possible to make a reliable determination of the of action of adriamycin in which the drug is absorption spectrum of the hydroquinone at pH enzymically reduced (Arcamone, 1981; Bachur et al., values in this range. However the marked difference 1977; Crooke & Reich, 1980; Kalyanaraman et al., between the spectra at pH 5.0 and 11.5 shows that 1980; El Khadem, 1982; Lown et al., 1982b; Muggia the hydroquinone exists in different protonated et al., 1982; Neidle & Waring, 1983) to the semi- forms at these two pH values, so it has at least one quinone and then regenerated by reaction with PKa in this region in addition to that of the amino oxygen with concomitant formation of superoxide, group, which would not be expected to appreciably 0;-, from which hydrogen peroxide and hydroxyl influence the optical absorption. radicals could arise and cause strand scission of DNA via abstraction of hydrogen from deoxyribose residues (Arcamone, 1981; Bates & Winterbourn, 1982; Crooke & Reich, 1980; El Khadem, 1982; 20 Lown, 1979; Lown et al., 1982a,b; Muggia et al., 1982; Neidle & Waring, 1983; Winterbourn, 1981b). Whilst the properties of the quinone and the semiquinone may be affected by intercalation, and 16 therefore under these conditions not be precisely represented by the present work, evidence is ac- E 0 cumulating (Tritton & Yee, 1982) that intercalation 7 may not be essential for the anti-tumour action of 3 12 adriamycin-like quinones. However, although the E semiquinone itself has been implicated E (Winterbourn, 1981a) the present results still point 20 to a destructive mechanism based on superoxide 0 8 formation, and in this respect may be particularly x relevant to adriamycin cardiotoxicity (Green et al., 1984; Lown et al., 1982a). Absorption spectrum of the hydroquinone 4 Hydroquinone is present at relatively high concentration a few ms after delivery of pulses to solutions at pH values where reaction (14) is largely 0 to the right. The absorption spectrum of the 300 400 500 hydroquinone was determined at pH 5.0 and 11.5 X/nm using the known extinction coefficients of the parent Figure 6 Absorption spectra of the hydroquinone (sQ) and the semiquinone (i-R) assuming that the obtained from traces like those in Figure 5, (0) absorption at 720 nm is a measure of the pH 5.0, (0) pH 11.5. semiquinone concentration, ae. Typical traces are shown in Figure 5. At any wavelength, the observed increase in absorbance, AA, is given by: Slow elimination of daunosaminefrom the hydroquinone AA = [aeeR + (R°2 e)HQ-([Qo] - [Qe])EQ]d Changes in absorption were observed for periods extending to some seconds after delivery of pulses (16) ( - 8 Gy) to solutions containing 5 x 0-5moldm - adriamycin and 10- 'mol dm- formate. At pH 9 where Ro is the concentration of radicals initially the absorptions at 720 nm (largely semiquinone), produced, [Qo] and [Qe] are the initial 480 nm (parent and semiquinone) and 420 nm concentration of quinone and the concentration of (mainly fully reduced form) decreased over the quinone at the time of measurement, respectively, hundreds of milliseconds, yielding species possessing and d is the optical path length. The absorption strong absorptions at 380 and 608 nm. The data 522 E.J. LAND et al.

appeared to be consistent with loss of daunosamine the hydroquinone. At pH 5, the rate of conversion from the hydroquinone, yielding a tautomer of 7- was about a quarter of that seen in the more deoxyadriamycinone, analogous to the alkaline solutions. Further work will be required to corresponding reaction seen after reduction of establish the spectra of the tautomer and other daunomycin in methanol by free radical electron possible intermediates as a function of pH, to donors (Kleyer & Koch, 1983). However there establish a full kinetic scheme and to determine could be an intermediate step. Full spectroscopic accurate rate constants. and kinetic data were not obtained, but the results so far seem to be consistent with a rate constant of ' about 5s- for the main process at pH 9. A This work was supported by grants from the Cancer somewhat similar conversion, occurring over a Research Campaign and the Medical Research Council. similar period, could be seen at pH 11.5, except that The paper is dedicated to Professor Schulte-Frohlinde on there was little semiquinone to disappear along with the occasion of his 60th birthday.

References

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