Radiotoxicity of Platinum-195m-Labeled trans-Platinum (II) in Mammalian Cells

Abstract

The chemotoxicity and radiotoxicity of trans-dichlorodiammineplatinum (II) labeled with ^195mPt (trans-^195mPt) are investigated to ascertain the potential of radioplatinum coordination complexes as antineoplastic agents. Platinum-195m, with a half-life of about 4 days, is a prolific emitter of low-energy Auger electrons because of the high probability of internal conversion in its isomeric transitions. The kinetics of cellular uptake and retention after incubation and the radiotoxicity of this Auger electron emitter in the form of trans-^195mPt is investigated using cells of the Chinese hamster V79 cell line. The cellular uptake of ^195mPt reaches a plateau in about 3 to 5 h of incubation and varies nonlinearly with the extracellular concentration of radioactivity. The radioactivity is eliminated from the cells after incubation with an effective half-life of 24 h. Cell survival data, when corrected for the chemical toxicity of nonradiolabeled trans-platinum, give a cell survival curve typical for radiations with high linear energy transfer. At 37% survival, the mean lethal cellular uptake is about 1.0 mBq/cell. Dosimetric considerations, based on subcellular distribution of the radionuclide, yield a value of 4.8 for the relative biological effectiveness when compared with 250 kVp X rays. Theoretical Monte Carlo track-structure calculations indicate that the density of radical species produced in liquid water in the immediate vicinity of a ^195mPt decay site is substantially greater than the density of species along the track of a 5.3 MeV α particle. This explains qualitatively the efficacy of ^195mPt in causing high-LET radiation type biological effects. The extreme radiotoxicity of intranuclearly localized ^195mPt, in conjunction with the proclivity of platinum chemotherapy agents to bind to DNA in the cell nucleus, suggests that the combination of chemical effects and the effects of Auger electrons that can be obtained with radio-platinum coordination complexes may have potential in the treatment of cancer.

Introduction

When radionuclides decay by orbital electron capture or internal conversion, inner atomic shell vacancies are created in the residual atom. The highly excited atom attains a stable electronic configuration quickly in a time scale of about 10⁻¹⁵ s via radiative and nonradiative transitions. In general, the atomic vacancy cascades are dominated by the latter involving Auger, Coster-Kronig and super Coster-Kronig processes (1). As a result, numerous electrons are ejected from the atom. Most of these Auger electrons have very low kinetic energies (∼20–500 eV) with extremely short ranges (∼a few nanometers) in water (2–5). Even though the energy carried by these electrons is only a small fraction of the total energy released in the decay, their collective local energy deposition is very high (2, 4, 6, 7). Hence, when the decays occur in the immediate vicinity of critical biological molecules (e.g. DNA), the biological effects are expected to be severe (8–17).

Auger electron emitters are used widely in nuclear medicine, and consequently their biological effects are of considerable interest. The biophysical aspects of the prolific Auger electron emitter ¹²⁵I have been studied extensively, and an in-depth review has been provided recently by Sastry (3). When ¹²⁵I, as 5-[¹²⁵I]iodo-2′-deoxyuridine (¹²⁵IdU), a thymidine analog, is incorporated into the DNA of proliferating cells in culture, the efficacy for cell killing is very high with a relative biological effectiveness (RBE) of 7 to 9 (11, 13, 15, 18). The findings are very much the same with 5-[¹²⁵I]iodo-2′-deoxycytidine (15, 19) as well as with the Auger electron emitters ¹²³I (20) and ⁷⁷Br (9) incorporated similarly into the DNA. On the other hand, when ¹²⁵I is localized in the cytoplasm of cells (14, 19, 21, 22), the targets in the cell nucleus are irradiated only by the sparsely ionizing components of the radiations emitted. In these cases, an RBE of ∼1 is reported, typical of low-LET radiations. The position-dependent effects of ¹²⁵I, reported originally by Hofer et al. (23), have been established for a number of other Auger electron emitters both in vitro and in vivo (9, 16, 19, 20, 23–28). The effects of Auger electron emitters therefore depend on their subcellular location, which is governed, in turn, by the chemical form of the molecular agent to which the radionuclide is attached (11, 14, 21–23, 26, 29).

Now that the basic determinants of the radiobiology of internal Auger electron emitters are fairly well understood (i.e. effects depend on subcellular distribution), a systematic exploration of the potential of Auger electron emitters in cancer therapy is warranted. The central idea is that efficient cell killing by Auger electron cascades depends intimately on our ability to direct the radionuclide to the DNA in the cell nucleus via suitable molecular carriers. Bloomer and Adelstein (30) and Baranowska-Kortylewicz et al. (31) drew attention to ¹²⁵IdU as an effective agent in the ascites tumor model. Although dehalogenation of iodinated compounds introduced into the blood has been of some concern, novel therapeutic approaches with high-specific-activity ¹²³IdU and ¹²⁵IdU (31–35) and ¹²³I/¹²⁵I-labeled estrogens are being explored (10, 16).

In addition to ¹²⁵I, there are several other Auger electron emitters that have potential for use in therapy (e.g. ¹¹¹In, ¹²³I, ^193mPt). Inasmuch as platinum-coordination complexes have been used widely as chemotherapeutic agents (36), and since they bind to DNA, complexes in which the platinum-metal center is replaced by the prolific Auger electron emitter ^193mPt or ^195mPt are of particular interest (37). These isomers, with half-lives of about 4.3 and 4 days, yield approximately 26 and 33 low-energy Auger electrons per decay, respectively (5), compared with about 25 per decay for ¹²⁵I (5). Insofar as the chemotoxicity of platinum-coordination complexes is the limiting factor in their use in cancer therapy, the expected high-LET radiation type effects of DNA-incorporated radioplatinum may offer a unique approach to combined chemotherapy and radionuclide therapy. This “chemo-Auger” combination therapy may provide an opportunity to reduce the chemotoxicity in critical organs while improving overall therapeutic efficacy (37, 38). Accordingly, this paper reports our studies on the radiotoxic effects of ^195mPt incorporated into cultured mammalian cells via the platinum-coordination complex trans-dichlorodiammineplatinum-195m (II).

Methods and Materials

Radiochemistry

Platinum-195m was produced by neutron bombardment of enriched (95%) ¹⁹⁴Pt (cross section 0.09 barns, ref. 39) in the Oak Ridge National Laboratory high-flux reactor. Cis-dichlorodiammineplatinum (II) radiolabeled with ^195mPt (cis-^195mPt) was obtained from Oak Ridge National Laboratory at a specific activity of ∼15 MBq/mg. Platinum-197 (T_p = 18 h) was present as a small impurity (∼1.8 MBq/mg). The cis-isomer was converted to the trans-isomer using the procedure of F. F. Knapp, Oak Ridge National Laboratory (personal communication). Briefly, the cis-^195mPt, dissolved in 2 ml normal saline, was transferred to a quartz crucible and heated gently after addition of an equal volume of 15 M NH₄OH. Gentle heating was maintained until the resulting white powder reached near dryness, whereupon an additional 2 ml of 15 M NH₄OH was added. After heating to near dryness, the residue was dissolved in 6N HCl and transferred to a 10-ml pear-shaped flask with a spin bar. The flask was fitted with a condenser, the contents were refluxed for 3 to 4 h, and the solution was transferred to a 12-ml Pyrex^® centrifuge tube and centrifuged (2000 rpm) while hot to remove solids. The supernatant was transferred to a clean tube and cooled on ice for 20 min. After centrifugation and removal of the supernatant, the crystals were washed with 200 μl of ice-cold absolute ethanol, dissolved in 200 μl of dimethyl formamide and centrifuged, and the pellet was discarded. The supernatant was diluted with 0.75 ml of 0.1N HCl and chilled on ice for 30 min. The resulting trans-dichlorodiammineplatinum-195m II (trans-^195mPt) crystals were washed twice with 200 μl of phosphate-buffered saline (PBS) and dissolved in several milliliters of PBS using a sonicator bath, and the solution was filtered though a sterile 0.22-μm Millipore Millex-GV (Bedford, MA) filter. The activity concentrations in the cis- and trans-^195mPt solutions were determined by NaI γ-ray spectroscopy using the characteristic platinum K X rays with a yield of 0.77 per decay (5). Radioactive trace impurities were assayed with a liquid-nitrogen-cooled Si(Li) detector.

The chemical purity of the cis-^195mPt and trans-^195mPt was ascertained by thin-layer chromatography (TLC) on Silica Gel-60 aluminum-backed TLC sheets without fluorescence indicator (E. Merck) using an acetone:toluene:water (76:20:4) solvent system as described by De Spiegeleer (40). Approximately 0.1 μg each of cis- and trans-^195mPt, as well as authentic nonradiolabeled compounds (Aldrich, Milwaukee, WI), were chromatographed. After drying, the chromatographs were dipped in a saturated solution of dithio-oxamide (Sigma, St. Louis, MO) in methanol and dried on a hotplate at 100°C. The authentic and radiolabeled platinum compounds appeared as brown spots with R_f values of 0.30 and 0.59 for the cis- and trans-isomers, respectively. Radioanalysis of the chromatogram revealed a radiochemical purity >82% for trans-^195mPt. The specific activity was ∼15 MBq/mg.

Cell Culture

Chinese hamster V79 lung fibroblasts were maintained as monolayers in 75-cm² flasks at 37°C, in 5% C0₂–95% air, and subcultured twice weekly. The cells were grown in minimum essential medium (MEM) supplemented with 2 mM l-glutamine, 10 mM Hepes, 0.1 mM minimum nonessential amino acids, 25 U/ml penicillin, 25 μg/ml streptomycin, 50 μg/ml gentamycin sulfate and 15% fetal bovine serum, all from Gibco (Grand Island, NY). For experiments, exponentially growing cells were removed from the flask with trypsin (Gibco), resuspended in 10 ml calcium-free MEM and diluted to 400,000 cells/ml. The growth of V79 cells was not affected by the absence of calcium in the culture medium (25). Our experimental procedures described previously (11, 15, 24, 25, 41), described briefly below, were employed to obtain dose–response curves for the cells after exposure to trans-^195mPt.

Kinetics of Cellular Uptake and Retention of trans-^195mPt

Freshly suspended V79 cells (2.5 ml at 400,000 cells/ml) were transferred to a 12-ml round-bottom culture tube and incubated at 37°C in 5% C0₂–95% air for 4 h on a rocker-roller. An equal volume of calcium-free MEM containing trans-^195mPt was added (final concentration 148 kBq/ml), and the tubes were returned to the roller. At various times, aliquots of the cells were removed and assayed for cellular uptake of radioactivity as described by Kassis and Adelstein (41). Additional tubes of cells remained rolling for 18 h, whereupon the cells were washed three times with 10 ml fresh calcium-free MEM and returned to the roller. Aliquots of the cells were taken periodically and assayed for cellular radioactivity (41).

Clonogenic Survival

Freshly suspended V79 cells (400,000 cells/ml) were aliquoted (1 ml) into 12-ml round-bottom culture tubes and incubated for 4 h under standard conditions on a rocker-roller. Equal volumes of calcium-free MEM containing various concentrations of trans-^195mPt were added, and the tubes were returned to the roller and incubated for 18 h (T_I). Aliquots were removed and assayed for cellular uptake of radioactivity (41). The remaining cells were washed, serially diluted and seeded into 25-cm² flasks. Approximately 35% of the cellular activity was removed from the cells during the washing process. Flasks were incubated for colony formation under standard conditions for 1 week (T_CF), and the colonies were washed with normal saline, fixed with methanol, stained with crystal violet and scored (11). The remaining washed cells were used to determine the fraction of cellular activity that was precipitable with trichloroacetic acid (11, 25). Detailed subcellular distribution of the radioactivity was determined according to our well-established procedures (25). The chemotoxicity of trans-^195mPt was ascertained by performing colony-forming assays with radiochemical that had been allowed to decay over a period of 7 to 8 half-lives.

Results

Cellular Uptake and Retention of trans-^195mPt

The temporal pattern of cellular uptake of radioactivity is shown in Fig. 1. Saturation is achieved in 4 to 5 h. A least-squares fit to the data using an exponential function yields f(t) = 1 – e^−0.95t, where f(t) = A(t)/A(t = 18 h), and A(t) is the cellular activity at time t in hours. The residence time τ_I of the activity in the cell during the 18-h uptake period is then calculated to be 17 h. The dependence of cellular uptake of radioactivity on the activity concentration of trans-^195mPt in the culture medium is shown in Fig. 2. A least-squares fit using a two-term polynomial function yields A_I (mBq/cell) = 4.07 × 10⁻³ κ + 3.55 × 10⁻⁵ κ², where κ is in kBq/ml. Figure 3 shows the effective elimination of trans-^195mPt from the washed V79 cells. The radioactivity was eliminated exponentially with an effective half-life of 24 h. This corresponds to a residence time τ_CF of 34.3 h for the 1-week colony-forming period.

Fig. 1.

Effective uptake of trans-^195mPt by V79 cells as a function of incubation time in calcium-free culture medium containing 148 kBq/ml of the radiopharmaceutical. Data points represent the average of five experiments and probable errors of the mean are indicated. No dependence on extracellular concentration was observed over the range used in survival studies.

Fig. 2.

Dependence of cellular uptake of trans-^195mPt by V79 cells on the extracellular activity concentration of the radiochemical after an 18-h incubation. Data points are the average of four experiments with probable errors of mean denoted.

Fig. 3.

Effective elimination of trans-^195mPt from V79 cells after exposure to 148 kBq/ml of drug. Four experiments were averaged, error bars representing probable errors of the mean.

Clonogenic Survival

Figure 4 illustrates the dependence of cell survival on the drug concentration in the culture medium for both trans-^195mPt and nonradioactive trans-platinum. The higher toxicity of trans-^195mPt compared to nonradioactive trans-platinum is evident. The extracellular concentration of the nonradioactive trans-platinum that is required to achieve 37% survival is 14 μg/ml for the 18-h exposure. As would be expected, this value is somewhat lower than the value of 50 μg/ml obtained by Douple and Richmond (42) for a much shorter exposure time (2 h) using the same cell line.

Fig. 4.

Survival of V79 cells after 18 h incubation in culture medium containing trans-platinum. Squares and circles represent response of cells incubated in the presence of trans-^195mPt and nonradioactive trans-platinum, respectively. Each curve is the average of four experiments with probable errors of mean indicated.

Cell survival data for trans-^195mPt, corrected for chemical toxicity, are plotted in Fig. 5 as a function of cellular uptake of radioactivity. Corrections for chemical toxicity were performed assuming that the chemotoxicity and radiotoxicity of the radiochemical were not synergistic. The validity of this assumption is supported by the data of Douple and Richmond (42) which demonstrate that no synergistic effects between platinum-coordination complexes and ionizing radiation are expected when the cells are maintained under oxygenated conditions. The survival curve thus corrected is exponential with a mean lethal uptake of 1.0 ± 0.1 mBq/cell (Fig. 5).

Fig. 5.

Dependence of survival of V79 cells on cellular uptake of radioactivity after 18-h incubation in culture medium containing trans-^195mPt Survival data are corrected for chemotoxicity of the radiochemical. In addition, survival as a function of absorbed dose to the cell nucleus is indicated by the scale on the upper horizontal axis. Note that scales for cellular uptake (lower axis) and absorbed dose (upper axis) are different. Error bars represent probable error of the mean of four independent experiments.

Subcellular Distribution of Radioactivity

Subcellular distribution studies revealed that 25% of the cellular activity was localized in the cell nucleus and the remaining 75% in the cytoplasm. Of the activity localized intranuclearly, 97% was bound to nuclear proteins, DNA and RNA. The fraction of activity in the cell nucleus specifically bound to DNA was 42%. These results and additional data for subcellular distribution are summarized in Table I.

Table I. Subcellular Distribution of trans-^195mPt in V79 Cells.

	Percentage activity
Cellular compartment
Nucleus	25
Cytoplasm	75
Nuclear fraction
Protein, DNA, RNAa	97
DNA aloneb	42
Cytoplasmic fraction
Protein, DNA, RNAa	44
Mitochondria	2.5

^aTrichloroacetic acid (TCA)-precipitable activity.

^bGuanidine-HCl-precipitable activity.

Discussion

Cellular Dosimetry

The results presented in Fig. 5 show that the lethality of ^195mPt Auger electron cascades is of a high-LET radiation type (dose–response curve without a shoulder in contrast to that found with low-LET radiations). However, because of their very different radiation spectra and physical half-lives, meaningful comparisons with other Auger electron emitters such as ¹²⁵I cannot be made on the basis of cellular uptake of radioactivity but should be done on the basis of absorbed dose. Accordingly, the absorbed dose to the cell nucleus is calculated using procedures described in detail elsewhere (11, 15, 24, 25). The choice of the cell nucleus as the target volume is based primarily on our incomplete knowledge of the precise nature and location of the primary radiosensitive targets in the cell nucleus (15). The contributions to the absorbed dose to the cell nucleus from ^195mPt decays are: (1) nontarget-to-target dose from extracellular decays that occur in the culture medium during the incubation period T_I (2) self-dose from intracellular decays during the incubation period T_I, (3) self-dose from intracellular decays during the colony-forming period T_CF, and (4) cross-irradiation from decays occurring in neighboring cells of the colony. Cross-irradiation from decays in neighboring cells of the colony is small relative to the other contributions (15) and can be ignored.

Calculation of contributions (1) though (3) requires knowledge of the cumulated activity in the extracellular medium (Ã_Medium) and in the cells during the uptake (Ã_I) and colony-forming (Ã_CF) periods. The quantities Ã_Medium and Ã_I are easily calculated to be 1.21 × 10⁵ κ and 6.12 × 10⁴ A_I, where A_I is the cellular uptake at the end of the 18-h incubation period. Because 35% of the activity is removed from the cells during washes prior to seeding for colony formation, Ã_CF = 0.65 τ_CF A_I = 8.03 × 10⁴ A_I

The absorbed dose to the cell nucleus from intracellular decays is D_N←Cell = (Ã_I + Ã_CF) (f_NS_N←N + f_Cy S_N←Cy), where F_N and f_Cy are the fraction of cellular activity in the nucleus and cytoplasm, respectively. The quantities S_N←N and S_N←Cy are the S values for calculating the dose to the cell nucleus from activity localized in the nucleus and cytoplasm, respectively (43). Using the radiation spectrum for ^195mPt given by Howell (5) and the computer code of Goddu et al. (43), one obtains S_N←N = 1.63 × 10⁻² Gy/Bq s, and S_N←Cy = 3.84 × 10⁻³ Gy/Bq s for V79 cells [radius of cell = 5.15 μm, radius of nucleus = 4.0 μm (24)]; with f_N = 0.25 and f_Cy = 0.75, then D_N←Cell = 984 A_I Gy/Bq.

The absorbed dose to the cell nucleus from the activity present in the culture medium during the incubation period is obtained by calculating the electron and photon contributions separately. The photon contribution is calculated using the absorbed fractions of Powsner and Raeside (44) for the cylindrical culture-tube geometry and the methods of Kassis et al. (25). The electron contribution is calculated by considering a large sphere of activity around the cell using the code of Howell et al. (45). These calculations yield the S value S_N←Medium = 1.18 × 10⁻¹¹ Gy/Bq s. Hence the dose to the cell nucleus from the activity in the culture medium is given by D_N←Medium = Ã_Medium S_N←Medium = 1.43 × 10⁻³ κ Gy/(kBq/ml).

Using the relationships above for D_N←Medium and D_N←cell, and the relationship established between A_I and κ (see Results), one obtains the total absorbed dose to the cell nucleus D_N for a given cellular uptake of radioactivity A_I and corresponding extracellular activity concentration κ. Accordingly, the survival data in Fig. 5 are replotted as a function of the total dose to the cell nucleus. This dose–response curve (Fig. 5, upper X axis) is also of a high-LET radiation type with a mean lethal dose (D₃₇) of 1.2 ± 0.12 Gy.

Relative Biological Effectiveness

Using our previously reported D₃₇ value of 5.8 Gy for acute external 250 kVp X rays (24), an RBE of 4.8 is obtained for the radiochemical trans-^195mPt. This RBE is appreciably lower than the value of 7 to 9 compared to acute X rays that was obtained for Auger electron emitters (⁷⁷Br, ¹²³I, ¹²⁵I) incorporated into the DNA of V79 cells via thymidine analogs (9, 11, 15, 20), but is in agreement with the value of 4.2 obtained when ¹²⁵I was brought close to DNA via a DNA intercalator (12). The RBE for trans-^195mPt may appear relatively low in view of the large number of Auger electrons emitted by ^195mPt (33/decay) compared to the number emitted by ⁷⁷Br (8/decay), ¹²³I (15/decay) and ¹²⁵I (25/decay), as given in a recent report by the American Association of Physicists in Medicine (5). However, it is well known that the biological effects of Auger electron emitters are highly dependent on the subcellular distribution of the radiochemical (14, 15, 21–26, 29). In the case of trans-^195mPt, only 25% of the cellular activity is localized in the cell nucleus, of which 42% is bound to DNA (Table I). In contrast, the thymidine analogs are found almost exclusively in the DNA. Therefore, insofar as DNA incorporation is the primary determinant of the biological effectiveness of Auger electron emitters, the RBE value of 4.8 for trans-^195mPt is not surprising. In fact, if the ^195mPt decays in the cytoplasm are assigned an RBE of 1, a simple calculation yields an RBE of 8.8 for the decays occurring in the nucleus. This value is consistent with the RBE for the radiolabeled thymidine analogs.

The dose–response relationship observed for trans-^195mPt (Fig. 5) is reminiscent of those observed for high-LET α particles. In fact, we reported an RBE of 6 in our earlier studies with intracellularly localized ²¹⁰Po, an emitter of MeV α particles (15). Similarly, RBE values of 5.2 and were obtained for extracellularly localized ²¹¹At (46) and ²¹²Bi (+daughters) (47), respectively, which emit α particles with mean energies of 6.8 and 7.8 MeV. Some insights into the similarity of the biological effects of internal a-particle emitters and DNA-incorporated Auger electron emitters may be gained from Monte Carlo track-structure calculations which simulate Auger electron cascades (5) and follow the physical and chemical development of the Auger electron tracks in liquid water from their earliest stages up to 10⁻¹¹ s after passage of the particles (7). Details of Monte Carlo track-structure calculations for Auger electron emitters may be found in ref. (7). Briefly, each interaction between an electron and a water molecule is recorded (collision location, energy transferred and type of interaction) as are the reactive chemical species (OH^•, H₃O⁺, H^• and hydrated electrons, ${\mathrm{e}}_{\text{aq}}^{-}$) that are produced. Figure 6 shows the positions (at 10⁻¹¹ s) of these reactive chemical species produced around a ^195mPt decay site and along the track of a 5.3 MeV α particle, superimposed on a segment of a cylindrical DNA model. The decay site for ^195mPt is chosen to be on the surface of the cylindrical model at its midpoint. The 5.3 MeV α particle is directed perpendicularly though the middle of the DNA segment. It can be seen clearly that the density of the chemical species around the ^195mPt decay site is substantially greater than the density along the α-particle track. These calculations suggest that, when ^195mPt is in the vicinity of the DNA, the lethal effects should be at least as severe as those caused by ¹²⁵I or high-LET α particles.

Fig. 6.

Track structure for 5.3 MeV α particles of ²¹⁰Po (7) compared to ^195mPt Auger electron cascades. Initial positions of reactive chemical species (OH^•, H^• etc.) at 10⁻¹¹ s are indicated. Note that density of chemical species around the ^195mPt decay site exceeds density along the α-particle track.

Implications for Chemo-Auger Combination Therapy

Platinum coordination compounds are being used extensively to treat a variety of cancers (36). Their effectiveness is limited largely by chemotoxicity in normal organs, which restricts the amount of drug that can be administered (36). Since these platinated compounds are known to bind to DNA (36), one approach to overcoming this limitation is to synthesize the platinum compounds with the Auger electron emitters ^193mPt or ^195mPt (37). The experimental results in Fig. 4 show that the Auger electron cascades of ^195mPt augment the chemical lethality of trans-platinum. Alternatively, the same degree of cell killing can be obtained at a lower chemical concentration of trans-platinum when the drug is labeled with ^195mPt. An approach to cancer therapy that combines chemotherapy and Auger electron emitters (chemo-Auger combination therapy) may provide an opportunity to enhance the therapeutic efficacy while maintaining or reducing the adverse chemotoxity that is generally observed for these chemotherapy compounds. The success of this approach may depend on differential radiosensitivity of tumor and critical (e.g. kidney) tissues, as well as careful balancing of the chemotherapy and Auger electron therapy to maximize their overall efficacy. In this regard, the relative radiosensitivity of bone marrow compared to tumor tissue is of concern.

We acknowledge that trans-Pt may not be the ideal carrier for radioplatinum in that it is not among the select group of therapeutically effective platinum-coordination compounds. Our preliminary studies (37) with intraperitoneal administered B16 melanoma cells in C56BL/GJ mice suggest that cis-^195mPt may be effective as an agent for chemo-Auger combination therapy. However, the relatively low specific activity of ^195mPt precluded proper balancing of the chemotherapy and Auger electron therapy (37). This problem was more pronounced in our cell culture studies with cis-^195mPt, where no radiotoxicity was observed above and beyond its chemical toxicity (A. I. Kassis, unpublished data). These problems may be circumvented by the use of ^193mPt, which may be produced readily at substantially higher specific activities or even in carrier-free form (37). Effective chemotherapy agents that are less chemotoxic than cis-platinum, such as carbo-platinum (cis-diammine-1,1-cyclobutane dicarboxylate), may also be promising as agents for combined therapy when labeled with ^193mPt. The potential of such radiochemicals is being explored (48).