SE0000130
Studies of radioactive cisplatin ( Pt) for tumour imaging and therapy
Johan Areberg Department of Radiation Physics, Malmo Lund University
LUND UNIVERSITY
Malmo 2000
31-14 Studies of radioactive cisplatin (191Pt) for tumour imaging and therapy
Johan Areberg
LUND UNIVERSITY
Malmo 2000
Department of Radiation Physics Lund University Malmo University Hospital SE-205 02 Malmo SWEDEN Doctoral Dissertation Lund University Institute of Radiology and Physiology Department of Radiation Physics, Malmo Malmo University Hospital SE-205 02 Malmo, SWEDEN
Copyright© 2000 Johan Areberg (pp 1-60) ISBN 91-628-3989-6 Printed in Sweden by Elanders Skogs Grafiska, Malmo 2000 To Cecilia and Adam ABSTRACT
A radioactive variant of the cytostatic agent «x-dichlorodiammineplatinvim(ir), cisplatin, was synthesised from 191PtCU. The 191Pt-cisplatin was found to be a sterile product of high radionuclide, radiochemical and chemical purity. The pharmacokinetics of platinum in tumour tissue and organs at risk of fourteen patients undergoing treatment with cisplatin were studied by exchanging a small fraction of the prescribed amount of cisplatin with 191Pt-cisplatin. The uptake and retention of platinum were investigated by gamma camera measurements up to ten days after infusion of 191Pt-cisplatin. Highest concentration of platinum was found in the liver, on average 5.7 ± 0.5 ug/g normalised to a given amount of 180 mg cisplatin. Corresponding value for the kidneys was 1.9 + 0.3 ug/g. Uptake of platinum in tumours was visualised in five patients with an average maximum concentration of 4.9 ±1.0 ug/g normalised to a given amount of 180 mg cisplatin. The data from the pharmacokinetic study was used together with data from the literature to estimate the absorbed dose and effective dose to patients receiving radioactive cisplatin. The effective doses were calculated to be 0.10 ± 0.02 mSv/MBq, 191 0.17 ± 0.04 mSv/MBq and 0.23± 0.05 mSv/MBq for Pt-, i53mpt.; ^d i95mpt.ciSpiatin respectively. The combined effect of the radio- and chemotoxicity from 191Pt-cisplatin was investigated both in vitro and in vivo. A cervical cancer cell line was incubated with cisplatin or 191Pt-cisplatin with various concentrations and specific activities. It was shown that the surviving fraction was smaller for cells treated with 191Pt-cisplatin than for cells treated with the same concentration of non-radioactive cisplatin. The surviving fraction decreased with increasing specific activity. Isobologram technique showed that the radio- and chemotoxicity interacted in a supra-additive (synergistic) manner. In an in vivo model, nude mice with xenografted tumours were given physiological saline (controls), cisplatin or 191Pt-cisplatin. The growth was significantly more retarded for the tumours on the mice given 191Pt-cisplatin compared with the tumours on the other mice. No significant change in general toxic effect, manifested as weight change or mortality, could be detected between the groups of mice given cisplatin and 191Pt- cisplatin respectively. 191Pt-cisplatin was shown to be a radiopharmaceutical suitable for both tumour imaging and therapy. There is potential for an increased use of radioactive cisplatin in the future. LIST OF PAPERS
This thesis is based on the following papers, which will be referred to in the text by their Roman numerals:
I Gamma camera imaging of platinum in tumours and tissues of patients after administration of *™Pt-cisplatin. Areberg J, Bjorkman S, Einarsson L, Frankenberg B, Lundqvist H, Mattsson S, Norrgren K, Scheike O, Wallin R Ada Oncol 38:221-228,1999
II Absorbed doses to patients from l^Pt-, 193mpt_ an III In vitro toxicity of 191Pt-labelled cisplatin to a human cervical carcinoma cell line (ME-180). Areberg J, Johnsson A, Wennerberg J Accepted for publication in Int] Radiat Oncol Biol Pbys TV The antitumour effect of radioactive cisplatin (191Pt) on nude mice. Areberg J, Wennerberg J, Johnsson A, Norrgren K, Mattsson S Submitted to IntJRadzat Oncol Biol Phys 191 V Radiation dosimetry of Pt-, i»3mpt_ atl(j wsmpt.cigpiatin fa tumour- bearing mice. Areberg J, Hindorf C, Liu X, Norrgren K, Ljungberg M, Mattsson S, Strand S-E Internal report, MA-RADFYS 2000:01 Preliminary reports were given at: • The Annual Meeting of the Swedish Medical Society, Stockholm, Sweden, 29 November - 1 December, 1995 • Radioactive Isotopes in Clinical Medicine and Research, Badgastein, Austria, 9 — 12 January, 1996 • First Easter School in Radiopharmaceutics, Liverpool, United Kingdom, 24 - 29 March, 1996 • Second International Conference on Isotopes (2ICI), Sydney, Australia, 12-16 October, 1997 • The Annual Meeting of the Swedish Medical Society, Goteborg, Sweden, 24 - 26 November, 1998 ABBREVIATIONS AAS atomic absorption spectrometry Adv adsorptive voltammetry CT computed tomography DNA deoxyribonucleic acid EC electron capture Gy gray HPGe high-purity germanium HPLC high-performance liquid chromatography IC50 50% inhibition concentration ICP-AES inductively coupled plasma atomic emission spectrometry ICP-MS inductively coupled plasma mass spectrometry ICRP International Commission on Radiological Protection IT isomeric transition MIRD Medical Internal Radiation Dose Committee (USA) MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylformazan bromide PET positron emission tomography SD standard deviation SEM standard error of the mean SGD specific growth delay Sv sievert TD time for a tumour to double its volume TLC thin-layer chromatography XRF X-ray fluorescence CONTENTS INTRODUCTION 11 AIMS OF THE STUDY 12 BACKGROUND 13 An overview of the use of radioactive cytostatic agents 13 Cisplatin in clinical use 13 Chemistry of cisplatin and formation of DNA-adducts 14 Toxicity of cisplatin 16 Other platinum analogues for chemotherapy 16 Methods for measuring platinum in biological material 16 Pharmacokinetics of platinum after cisplatin treatment 17 Platinum isotopes and their production 18 Gamma camera imaging of radioactive platinum and other radionuclides 20 Combination of cisplatin and radiation for therapeutic purpose 21 Calculation of absorbed and effective doses 24 MATERIAL AND METHODS 26 Production of 19IPt 26 Synthesis of 191Pt-cisplatin 26 Pharmacokinetic study of platinum in patients 29 In vitro toxicity of 19IPt-cisplatin 29 In vivo toxicity of 191Pt-cisplatin 30 Absorbed dose and effective dose calculations 30 RESULTS AND DISCUSSION 33 Synthesis of 191Pt-cisplatin 33 Gamma camera imaging of 191Pt 34 Pharmacokinetics of platinum after treatment with cisplatin 34 In vitro toxicity of 191Pt-cisplatin 37 General toxicity and effect on tumours of 191Pt-cisplatin in vivo 38 Absorbed dose and effective dose 41 General comments 42 FUTURE ASPECTS 44 Chemoembolization 44 Intratumoural administration 45 Electrochemotherapy 45 CONCLUSIONS 46 ACKNOWLEDGEMENTS 47 REFERENCES 49 NEXT PAGE(S) left BLANK INTRODUCTION It has been estimated that in Sweden about 40% of the population will develop cancer at some time in their life. Despite recent developments and improvements in the treatment, 23% of all deaths in Sweden are related to cancer (Swedish National Board of Health and Welfare, 1998). The dominating treatment modalities for cancer are surgery, radiotherapy, chemotherapy and hormone therapy. Hyperthermia and photodynamic therapy give just a marginal contribution and new methods like immunotherapy and genetherapy are still at a stage of research and very limited clinical trials. In chemotherapy, cytostatic agents that inhibit the proliferation of tumour cells are used. The optimal cytostatic agent acts on the tumour cell, but is inactive in normal cells. Unfortunately no such drug exists, why side-effects of chemotherapy are common. It is therefore of great importance to develop methods that can give information about how effective a treatment with a cytostatic agent will be and/or how the organs at risk will react. The chemotherapy regimens given today are very general and the sensitivity of the tumour cells to the drug is normally not taken into consideration in the planning of the treatment. A more individualised treatment planning like that in external radiotherapy would be one way to improve the therapeutic gain. The different treatment modalities are often combined to optimise the outcome of the treatment. The combination of external radiotherapy and chemotherapy has markedly improved the therapeutic results for those tumours which are both chemosensrtive and radiosensitive. By using cytostatic agents labelled with radioactive compounds, chemo- and radiotherapy can be combined, but such agents have so far not become an alternative for therapy. The benefit of using radioactive cytostatic agents could be twofold: 1. The radiation emitted from the decay of the radionuclide can be detected, e.g. by a gamma camera, whereby the uptake and distribution of the drug in the patient can be monitored after the administration. 2. The effect of the radiation, i.e. the radiotoxicity, interacts with the chemotoxic effect of the drug and optimally an enhanced effect of the combined treatment is reached. If the enhancement is more pronounced for tumour cells than for normal cells, there is potential for a therapeutic gain. 11 AIMS OF THE STUDY The main purpose of this thesis was to explore the usefulness of radioactive cisplatin for imaging, for pharmacokinetic studies and for tumour therapy. The specific aims were to • develop a method to synthesise sterile radioactive cisplatin, 191Pt-cisplatin, with a high chemical and radionuclide purity. • use 191Pt-cisplatin for pharmacokinetic studies and imaging in patients (in tumours as well as in normal tissues). • calculate the absorbed dose and the effective dose to patients receiving the radioactive drugs 191Pt-, 193mPt- and 195mPt-cisplatin for tumour imaging or pharmacokinetic studies. • investigate, both in vitro and in vivo, the therapeutic possibility of 191Pt-cisplatin and analyse how the chemical toxicity of cisplatin interacts with the radiotoxicity of 191Pt in vitro. 12 BACKGROUND An overview of the use of radioactive cytostatic agents During the years, most studies using radioactive cytostatic agents have been performed with cisplatin or bleomycin. Radioactive cisplatdn was first used by Lange et al. (1972) and patient studies were carried out by the same group (Lange etal. (1973)) and by Smith and Taylor (1974), Thatcher etal. (1982), Owens etal. (1985), Iosilevsky etal. (1989), Shani et al. (1989) and lind etal. (1991). Besides patient studies, a number of animal studies using radioactive cisplatin have been reported in the literature. Nouel etal. (1972a, b) published the first results with bleomycin labelled with 57Co and during the following years a number of studies were reported on 57Co-bleomycin as a tumour-seeking agent in patients. Due to the long physical half-life of 57Co, 270 days, attempts were made to label bleomycin with other radionuclides of which mIn has been the most commonly used. Some studies with mIn-bleomyin have shown promising therapeutic results (Hou etal., 1985,1989; Kairemo etal., 1996; Jaaskela-Saari etal., 1998). The radionuclide 5ICr has also been labelled to bleomycin and results of patient studies have been reported, but a recent study (Areberg and Bjorkman, 1999) showed that bleomycin could not be labelled with 51Cr without extensive chemical degradation of bleomycin. Another cytostatic agent that has been radiolabelled is the antimetabolite 5-fluorouracil (5-FU), used for treatment of cancers in the digestive system and the breast. This agent has been labelled with 18F and used in PET-studies of patients (Shani etal, 1982; Young etal., 1982; Kissel etal, 1997; Moehler etal, 1998). The antibiotic drug doxorubicin, which is particularly useful in the treatment of sarcomas, breast cancer, lymphoma and acute leukaemia, has been shown to be a potential radiotracer when labelled with 57Ni (Zweit et al., 1994). The antiestrogen tamoxifen, used for treatment of breast cancer, has been radioiodinated with 124I and used in a PET-study (Carnochan et al., 1994). Cisplatin in clinical use Since the discovery of Rosenberg et al. (1969) that the inorganic compound ds- dichlorodiammineplatinum^I), cisplatin, exhibits cytotoxic properties, the drug has become one of the most frequently used cytostatic agents. It is used to treat a variety of different malignancies, such as germ cell tumours, ovarian, limited small-cell lung, metastatic bladder, head and neck, esophageal and bladder cancer (Comis, 1994). Research on cisplatin has increased continuously during the last decades, see for example Fig. 1 which shows the number of scientific articles published per year having the words cisplatin or cis-dichlorodiammineplatinum(ir) in their titles (from Medline®). 13 ouu 500- £ a 4oo- a a. 1'S 300 3 200- 100 - .l.lll•ll " •• «•: "I 8>-\ O> O>J. ft,! Jft-' OS-: i i Year Figure 1. The number of scientific articles published per year, during the period 1970 — 1998, having the words cisplatin or cis- dichlorodiamrnineplatinumfll) in their titles (from Medline®). Chemistry of cisplatin and formation of DNA-adducts The cisplatin molecule consists of a platinum atom located centrally, surrounded by two chloride and two ammonia ligands in ar-configuration (Fig. 2). Dissolved in aqueous solution, the chloride ions are displaced whereby reactive hydrated species are created. It has earlier been supposed that cisplatin reach the cell via passive diffusion, but later studies have shown that pharmacological agents can stimulate or inhibit the intracellular levels of cisplatin which indicates that there might be active transport systems involved (Gately and Howell, 1993). Although cisplatin and its hydrated complexes have the ability to form covalent adducts with many biological molecules, there are strong evidences to assume DNA to be the critical target. One of the most convincing pieces of evidence is the hypersensitivity to cisplatin of cells deficient in DNA repair (Chu, 1994). Cisplatin can form a variety of DNA-adducts of which the 1,2-intrastrand crosslink is the most common (>90%). The inactive isomer of cisplatin, /ra^-dicrilorodiplatinurn (transplatin), cannot form this crosslink, suggesting that such a lesion might be responsible for the biological activity of cisplatin (Zamble and Iippard, 1995). Of the cellular platinum, approximately 1% is bound to DNA (Andrews and Howell, 1990). Proteins in mammalian cells capable of binding to cisplatin DNA-adducts with high affinity and specificity have been found (Pil and lippard, 1992). These proteins remove the cisplatin DNA-adducts whereby the DNA is repaired. Deficiency in the genes coding 14 NH NH Figure 2. The molecule wi'-dichlorodiammineplatinum^I), cisplatin. for these repair proteins may to some extent explain the tumour-specific cytotoxicity of cisplatin. It has also been argued that these proteins are overexpressed in cisplatin- resistant cells (Chu, 1994). Although the intact cisplatin molecule and its hydrated complexes form DNA- adducts, which give rise to the cytotoxicity of the drug, most of the cisplatin molecules bind to plasma proteins. These protein-bound components cannot form cisplatin DNA- adducts, see Fig. 3, and are considered to be biologically inactive. At 24 hours after administration more than 95% of the cisplatin molecules are bound to proteins (Johnsson, 1996). Intact cisplatin It Protein-bound Hydrated components complexes Figure 3. An overview of the chemical changes of the cisplatin molecule. 15 Toxicity of cisplatin Side-effects of therapy with cisplatin are nephrotoxicity, neurotoxicity, ototoxicity, nausea and vomiting. Nephrotoxicity was a dose-limiting factor in the early clinical use but is now prevented by pre- and posthydration and forced diuresis of the patient. Symptoms of neurotoxicity (reduced sensation and paresthesia in the distal extremities) are related to the cumulative amount of cisplatin given (Andersson, 1995). The ototoxicity can give rise to irreversible hearing loss. Nausea and vomiting can be very severe and persist up to several days after infusion of cisplatin. Other platinum analogues for chemotherapy The side-effects of treatment with cisplatin led to search for other platinum analogues with the intention to decrease or alter the side-effects but maintain the antitumoural effect. Among the different platinum compounds tested in clinical trials, Carboplatin (ris- diammine(l,l-cyklobutanedicarboxulato)platinum(II)) has become the most commonly used and is today a clinical alternative to cisplatin in the treatment of ovarian- and lung cancer (Go and Adjei, 1999). Other platinum analogues that have gained attention lately are oxaliplatin (trans-L-dach(lR, 2R-diamminocyclohexane) oxalatoplatinum, L-OHP) (Raymond eta/., 1998) and the orally active JM-216 (fe-acetato-ammine-dichloro- cyclohexylamine-platinum(IV); satraplatin) (Judson eta/., 1997; Sessa eta/., 1998). These compounds are however still at the stage of research and development. Methods for measuring platinum in biological material Platinum-based agents have a unique property compared with other cytostatic agents that make them well suited for pharmacokinetic studies: they include a heavy metal atom in the molecule. An analysis of elemental platinum is possible down to very low concentrations whereby the pharmacokinetics and tissue concentrations can be determined for long periods after the administration of the platinum based agent. The analysis of elemental platinum in biological material after exposure to cisplatin can be divided into non-invasive and invasive methods. The non-invasive methods consist of tracer studies with radioactive cisplatin and of X-ray fluorescence (XRF) measurements of stable platinum in vivo (Jonson et a/., 1988,1991; Ali et a/., 1998), where the former have been used more and can detect lower concentrations, below 0.1 ppm (e.g. Owens et a/., 1985) compared to 6 ppm for XRF (Ali eta/., 1998). For the invasive methods, tissue samples have to be collected from the subject of interest. After tissue sampling, e.g. blood and tumour biopsies, the concentration of platinum can be determined by a number of different methods: atomic absorption spectrometry (AAS), inductively coupled plasma-mass spectrometry (ICP-MS), inductively coupled plasma atomic emission spectrometry (ICP-AES) (Minami eta/. 1995), adsorptive voltammetry (Adv) 16 (Nygren et al., 1990) and neutron activation analysis (Esposito etal., 1987; Rietz eta/., 1994). Pharmacokinetics of platinum after cisplatin treatment Concerning the published pharmacokinetic studies of cisplatin, it ought to be emphasised that usually it is the kinetics of total platinum that has been investigated. As well intact as hydrated and protein-bound cisplatin are included, which is why the behaviour of these single compounds cannot be separated. Separate studies of intact, hydrated and protein-bound cisplatin have been performed on plasma. In order to separate platinum bound to plasma proteins from platinum in intact or hydrated cisplatin molecules, plasma has been ultrafiltrated whereby the filtrate includes both the intact and the hydrated platinum. The intact cisplatin can then be separated from the ultrafiltrate by means of HPLC. The concentration of total platinum in plasma in patients, after 2-3 hours infusions, showed a biexponential behaviour with a fast elimination phase of T1/2 = 40 minutes and a slow phase of T1/2 = 5-7 days (Vermorken et al., 1986). The concentration of ultrafiltrated platinum declined also in a biexponential manner with half-times of 8 and 36 minutes respectively (Vermorken et al., 1986), whereas a monoexponential function of T1/2 = 30 minutes was best fitted to data for intact cisplatin (Reece et al., 1987). The platinum is mainly excreted via the urine. The cumulated urinary excretion of platinum, expressed as a percentage of activity given, was after 1 day, 2 days and 5 days 26.2 ± 2.1, 33.0 ± 2.0 and 37.3 ± 2.1 respectively (+ SEM) (Smith and Taylor, 1974; Thatcher et al., 1982; Vermorken etal, 1986; Bonetti etal, 1994, 1995). Platinum concentration in human organs and tissues Several studies have shown that the liver is the organ which receives the highest concentration of platinum after treatment with cisplatin, approximately 5 -10% of the given amount directly after injection (see Fig. 4). Other organs/tissues where medium or high platinum concentrations have been reported are kidneys, skin, spleen, gastrointestinal tract, heart, testicle, prostate, uterus, lung and subcutaneous fat while the uptake in the brain is low (Stewart etal., 1982; Tochigi etal., 1988; Ishigaki etal., 1989; Tothill etal., 1992; Gorodetsky etal., 1993; Kusumoto etal., 1993). 17 16 14 - • Uozumi et al. c n Stewart etal. 12 - A Kusumoot et al. ° Smith and Taylor i 50 100 ISO 200 250 300 Time after injection (d) Figur 4. Content of platinum in the liver at different times after injection of cisplatin, expressed as percentage of the total amount given (Smith and Taylor 1974; Stewart etal., 1985; Kusumoto etal., 1993; Uozumi etal., 1993b). A liver weight of 1800 g was assumed. To estimate the amount of platinum given, a body surface of 1.8 m2 was assumed. Platinum concentration in human tumours Lagrange et al. (1996) showed a linear relation between platinum concentration in tumours and the cumulated amount of cisplatin in patients with uterine cervix tumours treated with low-dose cisplatin. In a review of published data (Lagrange etal., 1996), platinum concentrations in tumours were found to vary between 1 and 4 pg/g when determined 48 hours after a drug administration of 100 mg/m2. An exception was the study of Pujol et al. (1990) where ten times higher concentrations were reported in non- small cell lung cancer patients. Platinum isotopes and their production There are three isotopes of platinum that have been used for pharmacokinetic, imaging 191 193m and/or therapeutic studies with radioactive cisplatin: Pt, Pt an(^ i95mpt- Among the other platinum isotopes, only 188Pt and 197Pt are of interest since the other isotopes have inappropriate half-lives. Physical properties of the five isotopes of platinum (Kinsey etal., 1997; Howell, 1992) are presented in Table 1. Generally, an artificial radionuclide can be produced in two ways: by a reactor or by an accelerator. In reactor production, the target most often consists of the same element as the material to be produced. It is irradiated with neutrons whereby a number of interactions can occur. When radionuclides are produced by accelerators, charged particles are irradiating the target whereby also a number of interactions can occur, 18 which in this case give radionuclides from other elements. In the case of accelerator production, carrier-free radionuclides can be produced which is not the case for reactor production. This means that a higher specific activity can be achieved for accelerator production compared with reactor production. Table 1. Types of decay, half-lives, electron energies as well as energies and number of photons emitted per decay for 188Pt (Kinsey etal., 1997),191 Pt (Kinsey etal., 1997), 193mPt (Howell, 1992; Kinsey etal., 1997), 195mPt (Howell, 1992; Kinsey etal., 1997) and 197Pt (Kinsey etal., 1997). Isotope Type of Tl/2 Electrons: energy/decay (keV) Photonsb: decay 19 Gamma camera imaging of radioactive platinum and other radionuclides Gamma camera imaging of radioactive platinum in patients after treatment with cisplatin has been performed with 195mPt-cisplatin by Lange etal. (1973), Smith and Taylor (1974), Iosilevsky etal, (1989) and Shard etal. (1989) and with 191Pt-cisplatin by Owens etal. (1985). In gamma camera imaging both the energy and the number of photons emitted per decay in the energy window are factors that affect the pulse rate in the image. Imaging of the radioactive platinum isotopes listed in Table 1. is optimal if an energy window centred at 70 keV with a width of 20% is used. This energy window is most optimal for detecting superficial radioactivity. The photons with higher energies will impair the image quality since scattered radiation can be falsely detected as "true" events. This problem is most pronounced for 188Pt and 191Pt. Nordberg etal. (1993) have studied the detectability of 191Pt for a single-headed gamma camera (Toshiba GCA-901 A, Tokyo, Japan) equipped with a high-energy collimator. For the signal to background ratios of 1.3, 3.4 and 7.2 they found that the depths at which a sphere with a diameter of 20 mm could be detected were 0.7 cm, 4.6 cm and 6.5 cm respectively. They also found the effective linear attenuation coefficient for the radiation from 191Pt to be 0.176 cm4. The corresponding value for 99mTc for the same camera but with a high-resolution collimator has been reported to be 0.126 cm4 (Leide etal., 1992). Starck and Carlsson (1997) suggested that the effective attenuation coefficient for 201Tl, with energy window settings at 75 keV ± 20% and 167 keV ± 10% (two energy windows), which ought to be used is 0.184 cm1, i.e. quite similar to the 0.176 cm-1 Nordberg etal. (1994) found for 191Pt. Cisplatin has also been synthesised using 13N (Haber etal., 1984; De Spiegeleer et al, 1986) and used for pharmacokinetic studies in rat and man (Ginos et al., 1987). The short physical half-life of 13N, 10 minutes, makes longer measurement series impossible. It is the initial uptake that can be studied. On the other hand, the physical characteristics of 13N give rise to smaller absorbed and effective doses to the patients compared to cisplatin with radioactive platinum isotopes. Also studies with 99mTc labelled platinum compounds based on cisplatin have been reported (Awaluddin etal., 1987; Jacobs etal., 1990; Bourne etal., 1992). These 99mTc labelled platinum chelates had different biodistribution than cisplatin but revealed a good tumour to blood ratio in excess 2.3 (Awaluddin etal., 1987), which might indicate them useful for tumour imaging purposes. 20 Combination of cisplatin and radiation for therapeutic purpose Theory of interaction — the isobologram method The analysis of how different treatment methods interact into a combined effect can be both complex and time consuming. Different approaches for this analysis have been suggested during the years and the most established and most used is the isobole method (Berenbaum 1989). In this method the proportion of experimental subjects responding in a particular way is plotted against the doses or concentration of each of the components. This diagram is called an isobologram. To be able to do any analysis from the isobologram, the level of zero-interaction has to be known. Let da and db be the concentrations/doses of the agents A and B respectively, E(da) and E(db) the effects of da and db respectively, E(da,db) the combined effect and Da and Db the concentrations/doses of A and B separately that are isoeffective with the combination. For example, let E(da,db) be the cell survival after treatment with two agents, A and B, and let E(da) and E(db) be exponential functions of da and db respectively, = exp(-kb-db) (Eq.2) where ka and kb are constants. If we assume for the zero-interaction that E(da,db) can be calculated by multiplying E(da) and E(db) E(da ,db) = Qxp(-ka -da-kb-db) (Eq. 3) we then get K as the expected zero-interaction when db is expressed as a function of da and E(da,db). Points in the isobologram below this "curve of additivity" are said to be in the synergistic, or supra-additive, region while points above it are said to be in the antagonistic, or sub-additive, region. Fig. 5 shows an example of an isobologram where Da and Db both are 10, the curve of additivity is a line between Da and Db and the locations of the experimental points indicate that the interaction between drug A and B are additive and sub-additive (antagonistic). 21 Curve of additivity, zero-interaction Figure 5. An example of an isobologram to demonstrate the analysis of the interaction between drugs A and B. On the axes are the concentrations, da and db, of the drugs to achieve a predefined effect plotted. The concentration of the drugs to achieve the effect alone is denoted Da and Db respectively. This analysis shows that an additive interaction is present at low or high values of da and db while at intermediate values there exists a sub-additive (antagonistic) interaction. The interaction between radiation and cisplatin The combined effect of external radiotherapy and cisplatin has been studied in both preclinical and clinical investigations. The interaction between the two treatments has been found to vary from supra-additive (synergistic) to clearly sub-additive (antagonistic). Concurrent therapy with cisplatin, fluorouracil and external radiation was superior to radiation alone in patients with localised carcinoma of the esophagus (Herskovic et al., 1992). Regimens of radiotherapy and chemotherapy that contain cisplatin improved the rates of survival and progression-free survival among women with locally advanced cervical cancer (Rose et al., 1999). The primary mechanisms of the interaction have not yet been defined (Vokes, 1996). Proposed mechanisms are increased intracellular formation of free radicals, increased binding of platinum complexes to DNA during radiation and inhibition of repair of radiation damage (Vokes, 1996). Based on the concept of repair of radiation damage, it has been argued that cisplatin might have most effect in combination with low dose-rate irradiation because sub-lethal repair occurs during these conditions. Wilkins et al. (1996) showed that cisplatin was an effective sensitdzer to low dose-rate irradiation in glioma cell lines. Raaphorst et al. (1996) investigated concomitant low dose-rate irradiation and cisplatin treatment in ovarian carcinoma cell lines and found a large difference in cisplatin radiosensitization during low dose-rate irradiation compared with 22 high dose-rate irradiation. They stated that cisplatin during low dose-rate irradiation was an effective approach for radiosensitization if cisplatin levels were maintained during irradiation. The same conclusion was reached by Fu etal. (1985), who on a murine squamous cell carcinoma observed that the optimal effect between continuous low dose- rate irradiation and cisplatin was achieved when cisplatin was infused continuously during the course of irradiation. Several investigators have studied the time schedule-dependency of the interaction between radiation and cisplatin. Gorodetsky etal. (1998) found supra-additive effect only when cells (human ovarian carcinoma and murine mammary adenocarcinoma) were exposed to cisplatin for a very short time interval (two hours) after irradiation. Longer time intervals gave additive or sub-additive responses. Nakamoto eta/. (1996) observed that cisplatin had a supra-additive effect on two rat yolk sac tumour cell lines with different radiosensitivities in combination with radiation within six hours. Clinically, daily administration of cisplatin for five days before irradiation has been found to be superior to weekly cisplatin administered with the same schedule of radiation (Vokes, 1996). Even though severe side-effects of the combined treatment have been reported (Herskovic etal., 1992), most studies have reported moderate toxicities (Choo etal., 1986; Slotman etal., 1992; Chougule etal., 1994; Wheeler and Spencer, 1995; Birkenhake etal., 1999). It has been proposed that the dose-limiting effects of cisplatin (nephrotoxicity, ototoxicity and peripheral neuorpathy) are not further enhanced when cisplatin is combined with radiation, because cisplatin and radiation have differing degrees of late vs. early normal tissue toxicities (Rubin, 1984). Radiochemotherapy with cisplatin and internal radiation The promising results from studies with low dose-rate external irradiation and cisplatin and the fact that the interaction between them can be optimal when they are given simultaneously, implies that the combination of cisplatin and radiopharmaceuticals might have a therapeutic potential. Only few studies have been performed in this area. Mastrangelo etal. (1995,1997) used cisplatin in combination with 131I- metaiodobenzylguanidine (MIBG) in advanced neuroblastoma with encouraging results, but with hematological toxicity as a major limiting factor. Mertens etal. (1992) investigated the effect of 89Sr and low-dose cisplatin for patients with hormone refractory carcinoma metastatic to bone. They found the treatment to be relatively safe and effective but the number of patients was to small to draw more general conclusions. It has also been shown that the addition of cisplatin improved the efficacy of the treatment with 131I-labelled monoclonal antibodies in mice (Kievit etal., 1997). In vitro studies have revealed synergistic interaction between cisplatin and 131I (Chenoufi et al., 1998) and 185Re-hydroxyethylidene diphosphonate (HEDP) (Geldof etal., 1999). If radioactive cisplatin is used, one can be aware that the chemo- and radiotoxicity will act on the same spot. The only scientific report published in the literature on radiochemotherapy with radioactive cisplatin is the one by Howell et al. (1986) who investigated tumour-bearing mice given cisplatin or 193mPt-cisplatin (14.8 MBq/mg) where they found extended life spans for the animals treated with 193mPt-cisplatin. 23 Calculation of absorbed and effective doses The Medical Internal Radiation Dose Committee (MIRD) has developed a method for calculating absorbed doses in nuclear medicine investigations, often referred as the MIRD system. The base for this calculation is the biological distribution data, the physical properties of the radionuclide and the method or scheme that combines the biological and physical data into the dose estimate (Loevinger et al, 1991). From the biological data the activity of the radionuclide as a function of time is determined within a specified organ/tissue. The area under the activity-time curve is denoted the cumulated activity, A, and is equal to the total number of transitions in the organ for that specified radionuclide. By dividing the cumulated activity with the injected activity the residence time (X) is obtained. The absorbed dose to the target organ from activity in the source organ is determined by multiplying the cumulated activity in the source organ with the appropriate S value D = A- S (Gy) (Eq. 5) Multiplying the S value with the residence time, instead of the cumulated activity, gives the mean absorbed dose to the target organ per unit administered activity %=T-S (Gy/MBq) (Eq. 6) The S value converts the energy released in the nuclear transition in the source organ to the mean absorbed dose in the target organ and includes information on the mean energy of each particle emitted during a transition and how much of that energy that is absorbed by the target organ: Er^i (Gy) (Eq.7) where n is the number of particles emitted per transition, E is the mean energy of the particle and O is the specific absorbed fraction, summed up for all types of radiation from the transition. The specific absorbed fraction O is defined as the quotient between the absorbed energy in the target organ and the emitted energy from the source organ per unit mass of the target organ. For nonpenetrating radiations, as beta particles and electrons as well as photons with energies below 10 to 20 keV (sic!), the specific absorbed fraction is the inverse of the mass of the organ/tissue when source and target organ/tissue are the same and zero when they are separated (Eq. 8) 24 if k t- h where ft and rh are the target and source organ/tissue respectively and mh is the mass of the source organ/tissue (Loevinger etal, 1991). For organs with walls and with the content as the source, i.e. the bladder and the gastrointestinal tract, the specific absorbed fractions for nonpenetrating radiations are calculated as (Snyder eta/., 1975) (Eq. 9) *,(*<-rft) = 0 To take into consideration the varying biological effects per unit absorbed dose of various types of radiation, ICRP has been defined the equivalent dose in tissue T, HT, as (Sv) (Eq.10) where WR is the radiation weighting factor which is selected for the type and energy of radiation R and DT.RIS the absorbed dose averaged over the tissue/organ T, due to radiation R (ICRP, 1991). To take the different radiosensitivities of the organs/tissues into consideration, ICRP has introduced the quantity effective dose, which is defined as the equivalent doses multiplied by a tissue weighting factor n>r- Hence it is given that the effective dose can be calculated from the absorbed dose by means of A simple and fast method to calculate the absorbed and effective doses in nuclear medicine investigations is to use the computer program MIRDOSE 3.1 (Stabin, 1998). The input data are the residence times for different organs/tissues whereupon the program calculates the mean absorbed and effective dose per unit administered activity using S values and weighting factors stored in the program code. The method for dose calculations using the MIRD system mentioned above concerns the mean absorbed dose to a whole organ/tissue. On the microscopic level, MIRD also has published cellular S values (Goddu et al., 1997). Cellular S values are tabulated for various radii of the cell and the cell nucleus. Different source-region to target-region combinations including cell to cell, cell surface to cell, nucleus to nucleus, cytoplasm to nucleus and cell surface to nucleus are considered. The absorbed dose to a specific region is calculated as the product of the cumulated activity in the source region and the S value of interest. 25 MATERIAL AND METHODS Production of 191Pt 191Pt was produced at the The Svedberg laboratory in Uppsala, Sweden. A target of gold foil, 2 g/cm2, was irradiated with protons, 75 - 65 MeV, whereby the main reaction that occurred was 197Au(p,2p5n)191Pt The integrated beam current was 50 —100 uAh. The targets were allowed to decay for two days before processing. After separation of 191Pt from the target, the final solution, in the form of carrier-free 191PtCU, was sent to Malmo (paper I). Synthesis of 191Pt-cisplatin The synthesis of radioactive cisplatin used in this work is based on the papers by Hoeschele etal. (1979,1982). They used "s^Pt as the labelling platinum isotope, but their methods are applicable to 191Pt as well. Two syntheses were developed in the present thesis, the original one described briefly in Paper I (A) and a slightly modified version (B) used in Paper IV and Paper V. Synthesis A took 5 — 6 hours to perform while synthesis B took 4-5 hours. The two methods described below refer to an initial reaction scale of 0.1 mmole of Pt. In the text, Pt (non-radioactive) + 191Pt is denoted Pt*. The synthetic work was performed under aseptic conditions. The "original" synthesis, A Formation ofNa^>1*(lV)Cl6 Pt*(TV)a4 + 2NaCl -H> Na2Pt*(TV)Cl6 The radionuclide 191Pt, in the form of 191Pt(TV)CLt, arrived in solution in aqua regia in a sealed ampoule and was transferred by a syringe to a tube containing 34 mg of Pt(rV)CU (non-radioactive) as a carrier. First the solution was evaporated to near dryness on a Bunsen burner. The ampule was then rinsed with 2 ml of 12 M HC1, which was transferred with the syringe to the tube. Then 200 ul of 2M NaCl was added and the solution was evaporated to near dryness to remove the nitric ions. The addition of HC1 and the evaporation process were repeated once. Two ml of water were then added and the solution was evaporated to near dryness. An additional 2 ml of water was added and the volume of solution was reduced to approximately 0.3 ml. 26 Reduction ofPt*(lV) to Pt*(II) 2 Na2Pt*(TV)Cl6 + N2H4 -2HC1-^ 2 Na2Pt*(H)Cl4 + 6 HC1 + N2 The Na2Pt*(TV)Cl6 solution was transferred with a Pasteur pipette to a centrifuge tube with a screw cap. The tube was cooled in ice and the air in the tube was replaced with argon to prevent air oxidation of the reaction product. A volume of 100 Jil of 0.5 M hydrazine hydrochloride, N2H4 -2HC1, was added to the N&Pt*(TV)Cl6 solution. The tube was then placed in a 40 °C water bath for 60 minutes. Small bubbles of nitrogen gas were visible in the tube. The colour of the solution changed from light yellow to light red. Formation of Na2Pt*(lI)Cl4 + 2 KC1 -> K2Pt*(ir)Cl4 + 2 NaCl Under continued argon protection 105 (i.1 of 2M KC1 was added. The tube was cooled in ice and 2-3 ml of ice-cooled ethanol (99.5%) was added using a Pasteur pipette, whereby a white precipitate was formed in an orange coloured solution. The tube was cooled in ice for 10 minutes and then centrifuged for 10 minutes at 4000 rpm. The supernatant was removed with a Pasteur pipette and the precipitate was washed with 1 ml of ice- cooled acetone. After centrifugation (5 min at 4000 rpm) the supernatant was pipetted off and a stream of argon was used to dry the precipitate in the tube. Formation of K2Pt*(lI)Cl4 + 4 KI -> K2Pt*(I])I4+4 KC1 The dry product from the previous step was dissolved in 2-3 ml of H2O. Then 150 Jll of 4M KI was added, whereby the solution turned dark red. The mixture was allowed to stand for 20 minutes at room temperature. Formation ofds-~Pt*(II)(NH3)2[2 K2Pt*(H)I4 + 2 NH3 -> OT-Pt*(H)(NH3)2l2 + 2 KI K2Pt*(H)l4 was converted to 27 Conversion ofch-Pt*(lI)(NH3)J.2 to cis-Pt*(II)(NH})2(H2O)2(NO3)2 + 2 AgNO3 + 2 H2O -^ «f-[Pt*(n)(NH3)2CH2O)2](NO3)2 + 2 Agl To the solid product from the previous step was added 500 |^ of 0.4 M AgNC>3 and a white precipitate was formed. The tube was shaken and placed in a 60 °C water bath for 15 minutes and shaken at least five times during these 15 minutes. After centrifugation (10 min at 4000 rpm) the supernatant was transferred with a Pasteur pipette to a new centrifuge tube. The precipitate was washed once with 500 ul of 0.01 M NaNC>3 and this supernatant was also transferred to the new centrifuge tube. Formation ofcis-Pt*(II)(NH3)2Cl2 ^-[Pt*(H)(NH3)2(H2O)2](NO3)2 + 2 HC1 -> ^-Pt*(H)(NH3)2Cl2 + 2 H2O + 2 HNO3 To ensure that all Ag+ ions had been removed 80 ul of 1 M HC1 was added to the solution for precipitation of these as AgCl. The tube was placed in a 60 °C water bath for 5 minutes. After centrifugation (10 min, 4000 rpm), the presence of residual Ag+ ions was tested for by dripping 0.1 M HC1 into the solution. If no precipitation occurred, the supernatant was free from Ag+ ions. If not, additional 50 ul of 1 M HC1 was added and the tube was centrifuged for 10 minutes. The supernatant was then transferred with a Pasteur pipette to a new centrifuge tube and 100 |ll of concentrated HC1 was added. The tube was placed in the 60 °C water bath for 5 minutes and then cooled in ice for 20 minutes. Cisplatin was formed as yellow crystals. Purification by recrystallisation After centrifugation (10 min, 4000 rpm), the supernatant was pipetted off and 800 ul physiological saline was added to the tube. The tube was placed in boiling water until all precipitate (cisplatin) had dissolved, then allowed to attain room temperature and placed for 30 minutes in ice. After 10 minutes of centrifugation (4000 rpm) the supernatant was pipetted off and the recrystallised cisplatin was dissolved in 10 ml of physiological saline. The solution was filtered through a 0.22 um sterile filter. Modified synthesis, B In the modified synthesis, the formation of K2Pt*(H)CLt was omitted. The Na2Pt*(H)Cl4 was converted directly to K2Pt*(U)U by addition of KI. The K2Pt*(II)l4 was converted to 28 Quality controls The chemical yield, purity and identity of the synthesised cisplatin were determined by high-performance liquid chromatography (HPLC) (Riley etal., 1981). A commercially available cisplatin solution (Platinol™, Bristol-Myers Squibb) was used as a reference. The activity yield of the syntheses was determined by measuring the activity of the starting material (*Pt(TV)Cl4) and of the final product (0>-*Pt(II)(NH3)2Cl2) with an activity meter (Capintec, CRC-10 RB). The radionuclide purity was controlled with a high-purity germanium (HPGe)-detector (Schlumberger Enertec) while the radiochemical purity was checked by thin-layer chromatography (TLC) on 0.25 mm silica gel plates (Merck), developed with acetone-0.1 M HC1 7:3 (Paper I). Pharmacokinetic study of platinum in patients The uptake, distribution and retention of platinum was investigated in fourteen patients undergoing treatment with cisplatin (Paper I). This was done by exchanging a small amount, maximum 10%, of the prescribed cisplatin with in-house synthesised 191Pt- cisplatin. The activity given was maximised to 50 MBq. Gamma camera imaging, both whole-body scans and static imaging, was performed directly after the infusions and then at different times up to ten days after the infusions. The gamma cameras used was a single-headed Toshiba GCA-901 A for the five first patients while a dual-headed Siemens MultiSPECT2 was used for the last nine patients. An energy window centred at 70 keV and width of 20% was used. The high-energy photons in the decay of I91Pt necessitated the use of a high-energy collimator. Urine was collected from the patients for up to 48 hours after the start of the infusion and the excretion of platinum via the urine was calculated by measuring the activity of the urine with a HPGe-detector. To be able to quantify the uptake of platinum from the gamma camera images, phantom measurements were performed for liver, kidneys and tumours. Suspected tumorous platinum uptake was compared with clinical diagnostic investigations of the tumour tissue (CT and/or bone seeking radiopharmaceuticals) performed close in time to the 191Pt-cisplatin study. The pharmacokinetic study was approved by the Human Research Ethics Committee of Lund University, the Swedish Medical Products Agency and the Isotope Committee of Malmo University Hospital. In vitro toxicity of 191Pt-cisplatin A human cervical carcinoma cell line (ME-180) was used to investigate the effect of 191Pt-cisplatin in vitro (Paper III). This cell line was originally derived from a highly invasive squamous cell carcinoma by Sykes et al. (1970). The cells were incubated for one hour with non-radioactive cisplatin or 191Pt-cisplatin, with specific activities of 48 - 167 29 MBq/mg, whereby the number of viable cells were evaluated with an MTT assay (Mosmann, 1983) after seven days of growth. An analysis of the interaction between the radio- and chemotoxicity was performed with the isobologram technique with the concentration of cisplatin on one axis and the absorbed dose to the cell nucleus on the other. The absorbed dose to the cell nucleus was calculated using cellular S-values (Goddu etal., 1997). The radiosensitivity of the ME-180 cells was investigated with a 60Co source. In vivo toxicity of 191Pt-cisplatin To investigate the toxic effect of 191Pt-cisplatin in vivo, nude mice (BALB/c) with a human squamous cell carcinoma of the head and neck (AB) xenografted on theirs backs, were used (Paper IV). The mice, weighing 18 — 25 g at start, were intraperitoneally given physiological saline (controls), cisplatin 5 mg/kg or 191Pt-cisplati'n 5 mg/kg. The effect of the treatments on tumour growdi was analysed with calculations of relative tumour sizes (RTS), area under the logarithms of the RTSs (AUC-log(RTS)), times for the tumour to double its size (TD) and specific growth delays (SGD) where the latter is defined as (TDCOntroi-TDtreated)/TDconttoi. SGD can be regarded as die number of TDs gained by the treatment (Johnsson and Wennerberg, 1999). The survival and weight changes among the animals were used as parameters for evaluating possible toxic effects of die treatment. The study was approved by the Swedish National Board for Laboratory Animals. Absorbed dose and effective dose calculations Absorbed dose to mice In Papet V the absorbed dose to the liver, kidneys and tumours for nude mice given 191 Pt-, i93mpt or i95mpt_cjSp].atm were estimated. The kinetic of platinum in die organs was determined during a seven day period after injection of 191Pt-cisplatin (2.5 mg/kg) whereby the cumulated activities were calculated. Since there are no S-values for mice published, these had to be estimated through Monte-Carlo calculations of a mathematical mouse model, constructed accordingly to the anatomy of die real mice used, Fig. 6. Absorbed dose and effective dose to patients In Paper II, die absorbed dose and effective dose to patients receiving 191Pt~, 193mPt or 195mPt-cisplatin were calculated. Own pharmacokinetic data for platinum (Paper I) was used togedier with data from the literature and a pharmacokinetic model (Farris et al.> 1988). Retention functions for platinum, Pt(t), for various organs and tissues were 30 Kifrujs •- Figure 6. The mathematically described mouse phantom used to simulate mouse- specific S-values. calculated by fitting exponential functions to the pharmacokinetic data. Since no published S values are available for 191Pt or i93™Pt, these were calculated from the specific absorbed fractions using Eq. 7. The effective doses were calculated using weighting factors from ICRP (1991). The method for calculating the absorbed and effective closes is summarised in Fig. 7. 31 Uptake and retention data Model simulations Retention function Physical decay Cumulated activity Specific absorbed fractions A for photons (MIRD) S-values Absorbed doses Specific absorbed D fractions for electrons (MIRD) Tissue and radiation weighting facto rs(ICRP) Effective doses E Figure 7. Method for calculating the absorbed and effective doses from 191Pt-, 193mPt-, and 195mPt-cisplatb to patients. 32 RESULTS AND DISCUSSION Synthesis of 191Pt-cisplatin Chemical yield, purity and identity HPLC gave a single peak corresponding to that of cisplatin from Platinol™. The chemical yield varied between 18 and 52% for the original synthesis (A) (Paper I) and between 20 and 65% for the modified synthesis (B). Activity yield The activity yield of the original synthesis (A) was in the range 10-36% (Paper I) while for the modified synthesis (B) it was 20 - 50%. The divergence between the chemical and activity yield can in part be explained by the fact that there were temporary problems with the separation of 191Pt from the target where 191Pt was also present as compounds other than 191PtCLt. Hence that activity was lost in the first step of the synthesis (Paper I). Another possible explanation is adhesion of 191Pt to the walls of the ampoule, the syringe and the tube with the carrier. Specific activity The specific activity of the synthesised 191Pt-cisplatin, defined as activity in MBq per mg of cisplatin, was in the range 15 — 200 MBq/mg directly after the end of the syntheses. To our knowledge, this is the highest specific activity reported in the literature for cisplatin based on radioactive platinum isotopes. This can be explained by the fact that our starting material, 191PtCLt, was carrier-free which is not the case when reactor produced radioactive platinum is used. The amount of 191PtCU varied from batch to batch, which explains the variation in specific activity. The possibility to get a high specific activity is of the greatest importance when considering therapy with radioactive cisplatin. It was found that the amount of carrier for synthesis B could be decreased to 25 mg of PtCL). Even lower amounts, down to 15 mg, were possible (only tested with non-radioactive platinum) but the syntheses failed more often and the yields were lower. The chance to get even higher specific activities is thus dependent on the available 191 activity of PtCl4. Radionuclide and radiochemical purity The radionuclide purity of the synthesised 191Pt-cisplatin was 99.5% directly after the synthesis had been performed, which was 3-7 days after the production of 191Pt. The only other radionuclide found in the synthesised product was 188Pt. Gamma spectroscopy of the 191PtCU solution also detected impurities such as 191Au and 188Ir (daughter to 188Pt), but these were not carried through the synthesis and hence not found in the final product (Paper I). Since 188Pt has a longer half-life than 191Pt, 10.2 days as compared to 2.8 days (Kinsey etal., 1997), the radionuclide purity of 191Pt-cisplatin will 33 1 -I I 1 1 1 1 1 1 1 0.98 - - 0.96 - - 0.94 - X - 0.92 - - RP(t) 0.9 - \ \ 0.88 - 0.86 - 0.84 - 0.82 - - 1 1 1 1 1 1 1 1 1 0.8 0 2 4 6 8 10 12 14 16 18 2 t Time after synthesis (days) Figure 8. The radionuclide purity of 191Pt-cisplatin, RP(t), as a function of time after the synthesis assuming a time interval of three days between the production of 191PtCU and the synthesis. The relative importance of the radionuclide impurity increases with time due to the longer physical half- life of 188Pt compared to 191Pt. decrease with time. The radionuclide purity as function of time after synthesis assuming a time interval of three days between the production of 191PtCU and the synthesis, is shown in Fig. 8. The synthesised 191Pt-cisplatin was found to be radiochemically pure (> 95 %) by means of TLC measurements. Gamma camera imaging of 191Pt The system resolutions at 70 keV were 12.1 mm and 12.5 mm (FWHM) for the Siemens and Toshiba gamma camera respectively, while the sensitivities were 190 cps/MBq for both cameras (Paper I). For the static acquisition images it was found that the best image quality was gained after smoothing with a Butterworth filter of fourth order and with the Nyqvist parameter = 0.35 sA. Pharmacoldnetics of platinum after treatment with cisplatin Uptake of platinum in known tumour tissues was seen directly after the infusion in five of the fourteen patients and remained observable in later days images. Localisations of the tumours were lung, head and neck and intestine. The depths to the visualised tumours were all 1-2 cm except for one case where it was 12 cm in lung tissue. The maximum depth for which tumours can be detected depends on the activity concentration in the tumour, the activity concentration in the surrounding tissue and the size of the tumour. An approximate value of the maximum detection depth for the 34 visualised tumours in this study would probably be 3-4 cm. The average maximum platinum concentration in the tumours was calculated to be 4.9 ± 1.0 ug/g normalised to a given amount of 180 mg cisplatin, well in accordance with other studies (Lagrange et a/., 1996). The uptake of platinum in tumours is summarised in Table 2. The highest uptake of platinum was seen in the liver, on average 8.8% of the administered platinum directly after infusion which corresponds to a concentration of 5.7 + 0.5 ug/g normalised to a given amount of 180 mg cisplatin. This is well in accordance with the results reported by other investigators, see Fig. 4. Platinum uptake in kidneys was lower and was only visible for up to one day after infusion. Maximum average concentration in the kidney was 1.9 ± 0.3 ug/g normalised to a given amount of 180 mg cisplatin. The lower concentration in the kidneys compared to the liver was expected from other Table 2. Visualisation of tumours in the 191Pt-cisplatin gamma camera images. The grade of visualisation, "-", "+/?", "+" and "++", refers to invisible, borderline case, visible and clearly visible respectively. For the cases where tumours were visualised, the depths and concentrations of platinum are given (from Paper I). Pat. Localisation of Visua- Depth for Maximum Verification of no tumour tissue lisation visualised platinum the presence of tumours concen- the tumour (cm) tration (ng/e) 1 Bladder and ureter Inguinal lymph + /? nodes 2 Head and neck + /? 3 Lung + 2 4.7 ± 0.8 CT 4 Pelvis region - 5 Lung - 6 Head and neck + + 2 4.1 ± 1.2 CT, bone scan 7 Pelvis region - 8 Lung - 9 Lung - Right leg 10 Lung + 12 3.3 ± 1.0 CT Head and neck + + 2 3.5 ± 1.0 CT 11 Head and neck + 2 3.0 ±1.3 CT, bone scan 12 Head and neck + + 1 1.8 ±0.9 CT Intestine + 1.0 ±0.4 CT 13 Rectum - 14 Lung - 35 studies in patients (Owens etal., 1985; Tothill etal., 1992; Kusumoto etal., 1993) while in animal studies, most often mice or rats, equally or higher concentrations of platinum in the kidneys have been reported (Harrison etal., 1983; Boven etal., 1985; Johnsson etal., 1995). It is interesting to note that in the study by Jonson etal. (1991), where the concentration of platinum in the kidneys was investigated with XKF technique, considerable higher concentrations were found. Platinum was also visible in the gallbladder and later on in the gastrointestinal tract, consistent with intestinal transit. No data is reported in the literature about platinum elimination via faeces even if platinum uptake in the gastrointestinal tract has been reported (Lange etal., 1973; Smith and Taylor, 1974; Owens etal., 1985). An uptake in the urogenital region, below the bladder, was visible in both men and women. This could be the uterus, testicles and/or prostate where high platinum concentrations have been found (Stewart etal., 1982; Tochigi etal., 1988; Tothill etal., 1992; Kusumoto etal., 1993; Tokuhashi et al., 1997). A diffuse uptake at the neck and mediastinum was seen in all patients and in all images, consistent with findings of Owens et al. (1985) who found uptake in superior mediastinum in 2 of their 3 patients. The bladder was clearly visualised during the first two days after infusion. Measurements of the urine showed that on average 28% (range 15 - 42%) of the administered platinum was excreted during the first day and 42% (range 28 — 62%) after two days. The grade of visualisation and concentration of platinum in gamma camera images of patients given 191Pt-cisplatin is shown in Table 3. Table 3. Grade of visualisation and concentration of platinum in gamma camera images of patients given 191Pt-cisplatin. Organ/tissue Visualisation of Maximum average platinum concentration of platinum1 (ng/g) Bladder +++ liver +++ 5.7 ± 0.5 Gastrointestinal tract +++ Kidneys ++ 1.9 ±0.3 Neck and mediastinum ++ Tumours ++ 4.9 ± 1.0 Urogenital region ++ Elbows, knees and ankle + joints Gall bladder + Heart and larger blood + vessels Spleen + ' Normalised to a given amount of 180 mg cisplatin 36 In vitro toxicity of 191Pt-cisplatin The addition of radioactivity to the cisplatin molecule clearly enhanced the cytotoxic effect of the drug. The concentration of cisplatin necessary to kill 50% of the ME-180 cells, ICso, decreased from 3.24 + 0.08 (u,g/ml) for non-radioactive cisplatin to 0.76 ± 0.13 (ug/ml) for 191Pt-cisplatin with the highest specific activity (167 MBq/mg). From the experiments with non-radioactive cisplatin the survival of the cells to cisplatin alone, Schemo, could be described as an exponential function of the concentration c, Scte™=exp(-0.22-c) (Eq.12) To get the cell survival after exposure to ionising radiation alone, Sradio, experiments with 60Co radiation were performed and a linear quadratic model was fitted to the data Sradio = exp(-0.088 • D - 0.0043 • D2) (Eq. 13) where D is the absorbed dose to the cell nucleus. Combining these two survival equations by multiplication, the expected survival after treatment with 191Pt-cisplatin should be a = exp(-0.22 • c - 0.088 • D - 0.0043 • D2) CEq. 14) Expressing c as function of D at a cell survival of 50% gives the curve of additivity (zero interaction) as In2-0.088 D-0.0043-D2 c = (Eq. 15) 0.22 ^ 4 ; The isobologram analysis showed a supra-additive (synergistic) interaction between the chemo- and radiotoxicity for 191Pt-cisplatin and ME-180 cells for specific activities over 100 MBq/mg (see Fig. 9). 37 D(Gy) Figure 9. Isobologram at 50% survival for ME-180 cells with the concentration of cisplatin and the absorbed dose to the cell nucleus on the axes. The curve of additivity is plotted in the diagram. Points below the curve fall into the supra-additive (synergistic) region while points above the curve indicate sub-additive (antagonistic) interactions (from Paper III). General toxicity and effect on tumours of 191Pt-cisplatin in vivo For the period from injection to 21 days post injection, the mortality of the mice was limited and did not differ significantly between the various groups (Paper TV), see Table 4. From day 21 to day 24 post injection, when all animals were killed, there were not more deaths in the groups treated with 191Pt-cisplatin than in the other groups. The weight changes of the mice during the treatment showed no significant difference between the mice treated with cisplatin or 191Pt-cisplatin (Fig. 10). Hence it seems that no marked general toxic effect was induced by the addition of radioactivity (191Pt) to the cisplatin molecule. 38 Table 4. Outcome of the treatment with physiological saline (controls), non- radioactive cisplatin or 191Pt-cisplatin for tumour-bearing nude mice. Treatment Number of Number of deaths AUC-log(RTS) SGD group mice (period) 0-14 d Controls 5 0 (0-21 d) 11.6 ±0.8 0±0.1 2 (21-24 d) Non-radioactive 7 1 (0-21 d) 7.3 ±1.2 2.1 ± 0.7 cisplatin 3 (21-24 d) 80 MBq/mg 8 0 (0-21 d) 3.7 ± 0.8 3.0 ± 0.4 cisplatin 1 (21-24 d) 160 MBq/mg 7 0 (0-21 d) 4.0 ± 0.7 3.9 ± 0.8 cisplatin 1 (21-24 d) 1.05 O 0.95 -°- Controls ~B~ Non-radioactive 0.85 -^80 MBq/mg -*-160 MBq/mg 0.8 8 10 12 14 16 18 20 Days after injection Figure 10. The relative weight at various days after the injection of physiological saline (controls), non-radioactive cisplatin or 191Pt-cisplatin (80 MBq/mg cisplatin and 160 MBq/mg cisplatin) for the tumour-bearing mice (from Paper IV). 39 Radioactive 191Pt-cisplatin was shown to be a more effective drug than non-radioactive cisplatin in retarding tumour growth in the mice, using the area under the logarithm of the relative tumour size curve (AUC-log(RTS)) and specific growth delay (SGD) as parameters. The growth curves for the tumours are shown in Fig. 11. Significant differences (p<0.05) in the areas under the growth curves (AUC-log(RTS)) and specific growth delays (SGD) were present between the tumours in the group of mice treated with cisplatin and the tumours in the group of mice treated with 191Pt-cisplatin, see Table 4. By comparing the concentration of 191Pt-cisplatin with the concentration of non-radioactive cisplatin to get an equal effect (measured as the log(RTS)-value at day 7), it was shown that 5 mg/kg of 191Pt-cisplatin, 80 MBq/mg and 160 MBq/mg, corresponded to approximately 9 mg/kg and 10 mg/kg respectively of non-radioactive cisplatin, i.e. to get the same effect the concentration of cisplatin could be reduced to approximately the half by using 191Pt-cisplatin. -*- Controls -B- Non-radioactive -«-80 MBq/mg -6-160 MBq/mg Days after injection Figure 11. The logarithm of the relative tumour sizes, log(RTS), at various times after injection of physiological saline (controls), non-radioactive cisplatin or 191Pt-cisplatin (80 MBq/mg cisplatin and 160 MBq/mg cisplatin) for the tumour-bearing nude mice (from Paper TV). 40 Absorbed dose and effective dose Absorbed dose to mice For the mice which were treated with 80 MBqm Pt per mg of cisplatin (Paper IV), the absorbed doses to the kidneys, liver and tumours were 1.3 + 0.1 Gy, 1.4 + 0.3 Gy and 0.55 ± 0.04 Gy respectively. Notable is that although a marked effect of the radiation on the tumours was observed and the absorbed dose to the tumours was considerable lower than the absorbed dose to the kidneys and liver, no toxic effect, manifested as weight decrease or change in mortality, was noted for the liver and kidneys. It appears that a therapeutic gain exists, but before more detailed conclusions can be drawn further analysis of the toxic profile has to be performed. Absorbed dose and effective dose to patients After administration of radioactive cisplatin to patients the highest absorbed dose was received by the liver, followed by uterus, testes, lower large intestine and kidneys (Table 5.). The effective doses after administration of 191Pt-, ^^Vt~ and 195mPt-cisplatin were calculated to be 0.10 ± 0.02,0.17 ± 0.04 and 0.23 ± 0.05 mSv/MBq respectively. For the same count-rate in the gamma camera images, 195mPt-cisplatin will give an effective dose 3.1 times higher than for 191Pt-cisplatin. The activity of 191Pt-cisplatin given to the patients in Paper I was as maximum 50 MBq, corresponding to an effective dose of 5.0 mSv. This is of the same order of magnitude as for other nuclear medicine investigations, for example 37 MBq57 Co-bleomycin or 74 MBqin In-bleomycin, other radiolabelled chemotherapeutdc drugs commonly used, which give 2 mSv and 12 mSv respectively (ICRP, 1987). Table 5. Absorbed doses (mGy/MBq) to patients per unit activity from 191Pt-, i93mpt. an(j issmpt.cispiatin (+1 SD) (from Paper II) Organ 19lpt 193mpt 195mpt (mGy/MBq) (mGy/MBq) (mGy/MBq) Kidneys 0.16 ± 0.02 0.27 ± 0.04 0.34 ± 0.05 liver 0.39 ± 0.06 0.64 ± 0.09 0.87 ± 0.12 LLPwall 0.22 ± 0.07 0.46 ±0.15 0.61 ± 0.19 Skin 0.07 ± 0.02 0.17 ± 0.05 0.22 ± 0.07 Small intestine 0.08 ± 0.03 0.05 ± 0.02 0.08 ± 0.03 Spleen 0.13 ± 0.04 0.24 ± 0.06 0.32 ± 0.09 Testes 0.19 ± 0.06 0.40 ± 0.13 0.53 ± 0.17 ULPwall 0.17 ± 0.05 0.23 ± 0.07 0.32 ± 0.10 Urinary bladder wall 0.12 ± 0.02 0.15 ± 0.02 0.22 ±0.03 Uterus 0.24 ± 0.08 0.45 ± 0.14 0.58 ±0.18 ' Lower large intestine 2 Upper large intestine 41 General comments Since it is the uptake of platinum and not cisplatdn that is visualised in the gamma camera images, one can question the clinical value in the information gained from the investigation. What is the reason to visualise platinum when it is not possible to separate active platinum compounds form inactive? The answer is that there probably is a correlation between platinum concentration in the tumour and outcome of the treatment. A low uptake of platinum in the tumour shows that only small amounts of cisplatin and/or its platinum containing reaction products reach the tumour. On the other hand, a high uptake of platinum into the tumour indicates that also cisplatin reaches the tumour. Welters etal. (1997,1999) found a correlation between total platinum levels and tumour response to cisplatin for head and neck squamous carcinoma cells, both in vitro and in vivo in nude mice. In autopsy samples from various tumour types, Stewart etal. (1988) showed that patients whose tumours responded to cisplatin- containing regimens had higher mean tumour platinum concentrations than the patients whose tumours did not respond to the treatment. In contrast to these results, Holding et al. (1991) found no correlation between concentration of platinum in biopsy samples and survival time for head- and neck cancer patients. Concerning the platinum uptake in organs at risk and side-effects of the cisplatin treatment, nephrotoxicity of cisplatin, expressed as altered serum creatinine concentrations, was found to correlate to renal cortical platinum concentration in autopsy material (Stewart et al. (1985)). On the other hand, the platinum content in the kidney did not correlate to the decrease in creatinine clearance (Uozumi et al., 1993a). It has also been shown that the change in uric acid level, correlated with the total amount of cisplatin but not with the platinum concentration in the renal cortex and medulla (Nanji et al, 1985). Neither could a correlation be found between the platinum concentration in die kidney and impairment of renal function in a histopathological study (Tanaka etal., 1986). Even if liver toxicity is a rare complication after cisplatin treatment, Nanji et al. (1986) found a correlation between the decrease in serum albumin concentration, which is a measure of the status of the liver, and liver platinum levels. It has to be emphasised that the absorbed dose estimates for both mice and patients were based on macrodosimetry, i.e. the radionuclide was assumed to be uniformly distributed throughout the whole organ/tissue. In contrast to external radiation, the absorbed dose distribution in die cell after internal radiation is often very heterogeneous as a consequence of the non-uniform intracellular distribution of the radionuclides and die limited range of the radiation. For those radionuclides which emit a lot of low-energy electrons, the distribution of the radionuclide on cellular and sub- cellular level has to be taken into consideration. The absorbed dose to DNA, which is considered to be the main target, can be very different depending on the distribution of the radionuclide throughout the cell. For radionuclides which decay by electron capture or internal conversion, inner atomic shell vacancies are created which initiate a complex de-excitation process whereby numerous low-energy electrons are emitted. These electrons, collectively known as Auger electrons, have in most cases energies ranging 42 from eV to tens of keV which correspond to very short ranges in tissue (0.001 —10 urn). As a consequence, a dense shower of Auger electrons deposits energy in the immediate vicinity of the decay site, resulting in local energy densities that can exceed those along 191 the tracks of densely ionising a-particles (Howell eta/., 1993). Both Pt, i93mpt ^d i95mpt emits Auger electrons and specially i'3mPt and i95mpt have been considered as radionuclides with the potential to be used in therapy (Howell eta/, 1986). Based on these considerations it can be stated that the absorbed dose to DNA can be different for 191Pt~, i93mpt. and i95mpt-cjSplatin even though the macroscopic absorbed doses to the organs/tissues are the same in all three cases, since the three platinum isotopes have different electron spectra. In Paper III it was shown that the radio- and chemotoxicity of 191Pt-cisplatin interacted in a synergistic manner. Synergistic interaction between external radiation and cisplatdn has been reported in the literature and Begg (1990) tried to reexamine the interaction between the two agents on a sub-cellular level. He assumed that DNA was the most important target for both cisplatin and radiation and considered single strand breaks (radiation) and platinum-DNA adducts as the lesions of interest. If the number of adducts (A) and single strand breaks (B) at a specific base pair in a cell is NA=A/k A6) NB=B/k ^ where k is the number of base pair in the cell, Begg assumed that the number of both lesions occurring at a specific base pair is the product of these, and the number occurring in the whole cell is this product multiplied by k, i.e. NAB=A-Blk (Eq. 17) This reasoning holds if the locations of the single strand breaks and the adducts are independent of each other, which is the case for external radiation. For radioactive cisplatin, the locations of the two lesions are not independent since part of the decays in the cell originates from radioactive platinum isotopes bound to DNA (platinum-DNA adduct). This discussion indicates the possibility of a more optimal interaction between radiation and cisplatin if radioactive cisplatin is used instead of external radiation and non-radioactive cisplatin. 43 FUTURE ASPECTS The pharmacokinetics and corresponding absorbed dose values for radioactive platinum described in Paper II are based on intravenous infusion of cisplatin. By changing the way of administration it is possible to alter the kinetics of platinum. A higher uptake and a longer residence time for the platinum in die tumour tissue and lower uptake and shorter residence times in the organs at risk is desirable. Below, three methods that alter the kinetics of platinum are described. Chemoembolization The blood supply to hepatic tumours comes almost exclusively from the hepatic artery while the blood supply to the liver to 80% comes from the portal vein and only to 20% from the hepatic artery (Ho et al., 1998). This condition has been utilised for designing intraarterial treatments for liver tumours, often in combination with the oily contrast medium Iipiodol (Laboratorie Guerbet, France). Iipiodol has been shown to remain selectively in the vascular canals in the tumour tissue of the liver for a long time after injection (Nakakuma etal., 1983). A low blood flow in the liver artery after injection ensures a high drug exposure. By also using an occlusive medium, for example gelatine sponge, embolization of the hepatic artery is attained. Iipiodol has been used together with a number of cytostatic drugs for intraarterial chemoembolization but has also been labelled with 131I for internal radiation therapy. Sasaki et al. (1987) reported very promising results with chemoembolization therapy for hepatoma using Iipiodol, cisplatin and gelatine sponge. As a consequence of the promising results, Feun et al. (1994) performed a phase I study for primary and metastatic liver cancer. Their results for hepatocellular carcinoma were not as impressive as the results from Sasaki et al., but they concluded that chemoembolization with Lipiodol and cisplatin is feasible and generally well tolerated for doses of at least 100 mg/m2 and that antitumour activity in metastatic ocular melanoma is encouraging but requires further study. In a pharmacokinetic study of arterial chemoembolization of liver tumours with cisplatin and starch microspheres, the filterable plasma platinum levels were significantly (p<0.05) lower and the tumour platinum concentrations significantly (p<0.05) higher than after an intraarterial infusion of cisplatin alone (Civalleri et al., 1991). Because of the intrinsic radioresistance of liver tumours and the relative sensitivity to radiation of non-tumorous liver parenchyma, conventional external beam therapy has not been extensively used in the treatment of hepatic cancer (Ho etal., 1998). Therefore, interest has been focused on internal radiation therapy. Beside treatment with 90Y microspheres, 131I-Iipiodol has been widely used intraarterially in the treatments of hepatocellular carcinoma and colorectal liver metastases (Ho etal., 1998). The use of internal radiation therapy for hepatic cancer using intraarterial 131I-Iipiodol was summarised in a review by Ho etal. (1998). Intraarterial chemoembolization with radioactive cisplatin could be an interesting opportunity for the treatment of liver 44 tumours. The absorbed dose to the liver will after such a treatment probably be tolerable for the patient. For example, assume that a treatment with 150 mg of 191Pt-cisplatin with the specific activity of 100 MBq/mg is given and assume a concentration of platinum in the liver 1.5 times higher compared to intravenous infusion (Civalleri et al, 1991). The absorbed dose to the liver can then be calculated to be 9 Gy, using the results in Table 5. Intratumoural administration The idea with intratumoural administration of cytostatic agents is to get a high concentration of the drug in the tumour, which hopefully responds in an increased antitumoural effect compared with intravenous administration. Intratumoural administration gives different pharmacokinetics than intravenous administration. The intratumoural administration is often given in a gel delivery system to ensure a localised long-term release of the drug. Platinum concentration in the tumours of mice was enhanced when cisplatin was given by intratumoural administration together with slow- release devices compared to intraperitoneal administration (Deurloo etal., 1991). It has been shown that combined treatments with cisplatin and radiation on mice were more effective with intratumoural drug injection than with intraperitoneally administration (Begg etal., 1994; Yapp etal. 1998). These results indicate that intratumoural administration of radioactive cisplatin might be a potential treatment modality. The use of radioactive cisplatin in intratumoural administration makes it possible to monitor, by a gamma camera, the uptake of the drug in the tumour, i.e. to examine if the drug is distributed throughout the whole tumour. Electrochemotherapy One method to increase the uptake of drugs into the cells and tissues is to apply short intense electric pulses over the cells, so called electropermeabilisation, whereby an increase in the plasma membrane permeability occurs. In electrochemotherapy this approach is used to increase the intracellular concentration of cytostatic drugs. In an in vivo study Cemazar etal. (1999) showed that cell kill was increased by a factor 20, as determined from tumour growth curves, after electrochemotherapy with cisplatin compared with cisplatin treatment only. The platinum concentration in tumours and the amount of platinum bound to DNA was approximately two times higher. Sersa etal. (1998) reported from a study with electrochemotherapy and cisplatin on patients with malignant melanoma that electrochemotherapy was effective in treating cutaneous tumour nodules and that the treatment was well tolerated by all patients. These results indicates that electrochemotherapy might be suited to be tested together with radioactive cisplatin. 45 CONCLUSIONS Synthesising cisplatin from the radioactive platinum isotope 191Pt was shown to give a sterile product of high radionuclide, radiochemical and chemical purity. With this product, it is possible to monitor uptake of platinum in tumour tissues and organs at risk of patients after treatment with cisplatin. The liver is the organ with the largest uptake of platinum which results in the largest absorbed dose among the organs/tissues. For diagnostic and pharmacokinetic purposes, there is a clear advantage in using 191Pt- cisplatin instead of 195mPt-cisplatin, which for the same count-rate in the images gives about three times higher effective dose than 191Pt-cisplatin. The addition of radioactivity (191Pt) into the cisplatin molecule enhances the therapeutic effect of the drug. The radio- and chemotoxicity of 191Pt-cisplatin was shown to interact in a synergistic manner in an in vitro model. The growth of xenografted tumours in nude mice was more delayed when 191Pt-cisplatin was used instead of non-radioactive cisplatin. Radioactive cisplatin has shown to be useful for both imaging, pharmacokinetic and therapeutic purposes. There is potential for an increased use of radioactive cisplatin in the future. 46 ACKNOWLEDGEMENTS The main part of the work for this thesis was performed at the Department of Radiation Physics, Malmo University Hospital, with significant contributions from several other departments: Clinical Physiology (Nuclear Medicine), Oncology and Hospital Pharmacy at Malmo University Hospital, Oncology and Radiation Physics at the University Hospital in Lund and The Svedberg Laboratory at Uppsala University. I would like to express my sincere thanks to: Sb'ren Mattsson, my tutor and chief, for enthusiastic supervision and for always believing in the future of the project. Kristina Norrgren, my co-tutor, for teaching me nuclear medicine physics, for interesting discussions and for always having time. Sven Bjb'rkman, for taking time to guide a physicist through elementary chemistry and pharmacology needed, for always given valuable and competent comments and for fruitful discussions during the whole process. Ok Scheike, for introducing me into oncology and for patient selection of suitable patients. Lars Einarsson and Hans Ljtndqvist, for providing us with 191Pt and for helpful discussions. Anders Johnsson andjohan Wennerberg for a fruitful collaboration and for interesting discussions. Margareta Olsson and Kristina Boll, for teaching me cell culture techniques and taking care of the nude mice and for many good laughs. Cecilia Hindorf, for the anatomic mouse model and Monte Carlo simulations. Ronnie Wallin, for HPLC-analyses and for teaching me how to work aseptic. BodilRoth, for time and patience with the HPLC-analyses. Roland Andersson and Thomas Sjb'berg, for giving me time for writing instead of watching football. All my friends and colleagues at the Department of Radiation Physics in Malmo. Finally, I would like to thank Cecilia for all her love and patience during the years. 47 This work was supported by grants from the Swedish Cancer Society, Malmo University Hospital, Malmo University Hospital Cancer Foundation, John and Augusta Persson Foundation for Medical Scientific Research, Gunnar, Elisabeth and Arvid Nilsson Cancer Foundation, Franke and Margareta Bergqvist Foundation for Cancer Research, the Swedish Society for Medical Research and Anna lisa and Sven-Eric Lundgren Foundation for Medical Research. 48 REFERENCES Ali PA, Al-Hussany AF, Bennett CA, Hancock DA, El-Sharkawi AM (1998) Plane polarized x-ray fluorescence system for the in vivo measurement of platinum in head and neck tumours. PhysMed Bio/43:2337-2345. Andersson A (1995) Analytical and pharmacokinetic aspects of the antineoplastic drug cisplatin and its monohydrated complex. 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