Effect of the Chemoprotective Agent WR-2721 on Disposition and Biotransformations of Ormaplatin in the Fischer 344 Rat Bearing a Fibrosarcoma1

[CANCERRESEARCH55, 2837â€”2846,July1. 1995] Effect of the Chemoprotective Agent WR-2721 on Disposition and Biotransformations of Ormaplatin in the Fischer 344 Rat Bearing a Fibrosarcoma1

D. CharlesThompson,StevenD. Wyrick, David J. Holbrook, and StephenG. Chane?

Curriculum in Toxicology (D. C. T.. D. J. H., S. G. C.] and Department of Biochemistry and Biophysics ID. J. H., S. G. CI, School of Medicine, and Division of Medicinal Chemistry and Natural Products [S. D. WI, School of Pharmacy, University ofNorth Carolina, Chapel Hill, North Carolina 27599-7260

ABSTRA4@T be selective for nontumor tissues (4, 5). The proposed mechanism for this selectivity is a relative lack of alkaline phosphatase activity in The effects of the phosphorothloate agent, WR-2721, have been Eaves tumor tissues as compared to normal tissues and the lesser conversion ligated with respect to the biotransformations oformaplatin in the Fischer of the inactive parent compound, WR-2721, to its postulated active 344 rat bearinga transplantedfibrosarcoma.A number of different paradigms ofdosing route and schedule for the administration of the two thiol metabolite, WR-1065, within tumor tissues. Because WR-1065 agents have been investigated. In the first group ofexperiments, WR.2721 is postulated to bind to reactive platinum species within the cell, this (200mg/kg, l.p.) wasadministered30 mis beforeormaplatin (12.5mg/kg, would provide a mechanistic basis for selective protection of normal i.p.), and then peritoneal fluid, plasma, and tissues were harvested at 30 tissues as compared to tumor tissues (3, 6, 7). This hypothesis has mm after the ormaplatin administration. Our results suggest that a sig never been tested directly. Ormaplatin (formerly tetraplatin) is a nificant interaction between WR-2721 and ormaplatin is occurring in the platinum(IV) analogue of cisplatin recently in Phase I clinical trials @tonea1cavity.The interaction was evident in terms of both effects on (8â€”10).This laboratory has previously reported investigations of the distribution and disposition of total platinum and in alterations of the biotransformations of ormaplatin under a variety of conditions in vitro profilesof biotransformation products formed in the various tissues and (1 1), in cell culture (12, 13), and in vivo (14â€”16).These studies have fluids. Plasma protein binding oformaplatin was decreased by 50% in the shown that ormaplatin is very rapidly (tÂ½â€˜@3s) reduced to the presence ofWR-2721. Total platinum in the spleen was decreased by 66% and In the liver by 50%. There were no trends among the findings that corresponding platinum(II) complex, Pt(dach)C12, in the presence of would indicate any selectivity between tumor and nontumor tissue with plasma protein sulfhydryl (1 1), and that subsequent biotransforma respect to the effects of WR-2721 on the parameters measured. Subse tions of this complex appear to be similar to those reported for quent investigations examined the effects of dosing the WR-2721 by the cisplatin (17, 18). The previous studies have also shown that the LV. rOute while continuing with the i.p. administration of the ormaplatin. intracellular biotransformations of ormaplatin in vivo are very similar WR-2721 was administered either 30 or 5 mm before the ormaplatin, and in all tissues examined to date (19). The present report discusses the plasma and tissues were harvested at 15, 30, or 60 mm after ormapla investigations into the effects of WR-2721 on ormaplatin biotransfor tin administration. The reverse-phase HPLC peak, which behaved chro mations in tumor-bearing male rats as a direct test of the proposed matographically as a Pt(dach)(WR-1065) standard, was less prominent mechanism of action of WR-2721. after the Lv. administration of WR-2721 than It was after i.p. administra lion under any of the paradigms tested. There was again no evidence for selectivity between tumor and nontumor tissue in the findings from any of MATERIALS AND METHODS the paradigms. It is concluded that If WR-2721 is capable of selectively protecting nontumor tissue from the toxicities of platinum-based chemo Materials. [4,5-3H2(n)]Ormaplatin (formerly tetraplatin) was prepared in therapy, It is doing so by some mechanism other than its selective uptake the Radiosynthesis Laboratory, Division of Medicinal Chemistry and Natural Into normal tissue and subsequent nonspecific inactivation of any reactive Products, School of Pharmacy, at the University of North Carolina at Chapel cytosolic platinum species formed. Other possible mechanisms are briefly Hill with a specific activity of 0.520 Ci/mmol. The synthesis and purity of this compound have been described elsewhere (20). Reductive tritiation of the cyclohexene ring yields a radiolabel that is not chemically exchangeable (20). INTRODUCTION Preparation of a solution of the drug for administration was as described previously (14). WR-2721 and its corresponding free thiol, WR-1065, were WR-27213 is a phosphorothioate compound currently in Phase III obtained from US Bioscience (West Conshohocken, PA). This is different clinical trials investigating its efficacy at reducing the adverse side from the clinical formulation available through the National Cancer Institute, effects of both radiotherapy and alkylating agent chemotherapy of which contains a 1:1 mix by weight of WR-2721 and mannitol. The drug was neoplastic disease (1â€”3).This chemoprotective effect is purported to dissolved either in H20 (20 mg/ml) for i.p. injection or in PBS (pH 7.5; 100 mg/mi) for iv. injection and filtered immediately before use. YMT ultrafil tration membranes (M, cutoff, 30,000) were purchased from Amicon (Danvers, Received 2/6/95; accepted 5/3/95. Thecostsof publicationofthisarticleweredefrayedinpartby thepaymentofpage MA). HPLC grade reagents were obtained from commercial sources, and charges. This article must therefore be hereby marked advertisement in accordance with solutions were filtered and degassed before use. A Barnstead Nanopure water 18 U.S.C. Section 1734 solely to indicate this fact. purification system with an organics filter provided the water used with I This work was supported by NIH Grants CA55326 and ES07126. solvents and for dissolution. All other general laboratory reagents were com 2 To whom requests for reprints should be addressed, at Department of Biochemistry and Biophysics, 502 FLOB CB# 7260, University of North Carolina, Chapel Hill, NC mercial reagent grade or better and were used without additional purification. 27599-7260. Animals. Male Fischer 344 adult rats (150â€”200 g) were obtained from 3 â€˜FIse abbreviations and trivial names used are: WR-2721, S-2-(3-aminopropyl Charles River Breeding Laboratories (Raleigh, NC) and housed in clear plastic amino)ethylphosphorothioicacid(Ethiofos,Amifostine,Ethyol);WR-1065,S-2-(3-ami cages on a 12-h light/12-h dark cycle with access to water and Purina rodent nopropylamino)ethanethiol, the thiol metabolite of WR-2721; cisplatin, cis [PtCl2(NH3)2J, cis-diamminedichloroplatinum(!I) (cDDP); ormaplatin (formerly called chow ad libitum. Room temperature was maintained at approximately 22Â°C. tetraplatin), (d@l)trans-1,2-diaminocyclohexanetetrachloroplatinum(!V);dach,(d,l)trans Animals were allowed at least a 1-week acclimatization period before use in 1,2-diaminocyclohexane; Pt(dach)Cl2, (cU)trans-1,2-diaminocyclohexanedichloroplati experiments. A transplantable fibrosarcoma (originally induced by methylchol num(ll); P2, reverse phase; SCX, strong cation exchange chromatography; AAS, flame anthrene) was obtained from Dr. M. W. Dewhirst (Duke University Medical less atomic absorption spectroscopy; LSC, liquid scintillation counting; GSH, reduced glutathione. Platinum(II) complexes with various amino acids are indicated as: Pt Center, Durham, NC) with permission from Dr. J. M. C. Bull (University of (dachXaminoacid)orPt(aminoacid)@;inmostcases,theexactstoichiometryorchargeof Texas Health Science Center, Houston, TX), who originally developed the the complex is not known. tumor (21). The tumor was maintained via regular passage involving s.c. hind 2837

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1995 American Association for Cancer Research. WR-2721 AND ORMAPLATIN BIOTRANSFORMATIONSIN F344 RATS flank implantation of solid donor tumor (â€œ100mm3) under ketamine-xylazine material, the reaction products formed were identical.4 Henceforth, only the anesthesia. Experiments were routinely performed at 10 days posttransplanta Pt(dach)(WR-1065) was used in column standardization, along with the other tion, at which time tumor volume was on average 2.5â€”3.0cm3. standards routinely tested: Pt(dach@cysteine),Pt(dachXGSH),Pt(dachXmethi Methods. Animals were administered WR-2721 or corresponding vehicle onine), Pt(dachXserine),and the free dach ligand. These were the standards the by either of two protocols. For most of the experiments, a dual i.p. paradigm chromatographic characteristics of which most closely approximated those of was used, in which case the WR-2721 (20 mg/mI and 200 mg/kg; @34-fold the RP HPLC peaks that appeared routinely in greatest abundance in the molar excess to platinum on a per kg body weight basis) or H20 vehicle was biological samples. administered i.p. under light ether anesthesia at time zero (4). At t 30 mm, Statistics. Independent (unpaired) t tests on control (ormaplatin only) ormaplatin was administered (12.5 mg/kg, i.p.), also under light ether anes and treated (WR-2721 plus ormaplatin) variables were performed with the thesia. These animals were all then sacrificed at t = 60 mm. The second SYSTAT statisticalpackage (SYSTAT, Inc., Evanston,IL). Assessment of the significance of any effect of WR-2721 treatment on ormaplatin disposition protocol used an i.v./i.p. paradigm in which the WR-2721 (100 mg/ml in PBS, or biotransformation was at the P = 0.05 level. pH 7.5 vehicle, 200 mg/kg) was administered iv. via the lateral vein under light ether anesthesia at time zero. For this protocol, ormaplatin (12.5 mg/kg i.p.) was administered at either t = 5 mm or t = 30 mm. These animals were RESULTS then sacrificed at either 15, 30, or 60 mm after ormaplatin administration. Many preclinical studies of WR-2721 prevention of platinum Sacrifice was by exsanguination via the inferior vena cava under light ether induced toxicity have used a dual i.p. paradigm in which both the anesthesia; blood was collected immediately into heparinized tubes on ice. WR-2721 and the platinum drug were injected into the peritoneal Peritoneal fluid (in the dual i.p. paradigm) was aspirated from the open cavity in a variety of temporal relationships (4, 27â€”32).Some of the abdominal cavity and placed on ice immediately before catheterization of the vein. After exsanguination, tumor and normal tissues of interest were harvested earliest studies were those of Yuhas et a!. (4, 29), and one animal into ice-cold 0.15 M NaC1. Plasma was prepared by centrifugation of whole model they used was the F344 rat. Thus, for these initial studies, a blood at approximately 1000 X g for 15 mm at 4Â°C(22). Plasma and peritoneal similar paradigm (4, 29) was used in which WR-2721 (200 mg/kg, fluid were then diluted 1:5 in ice-cold PBS (pH 7.5) and filtered over Amicon i.p.) was administered 30 mm before the ormaplatin (12.5 mg/kg, i.p.). YMT membranes by centrifugation at approximately 2000 X g for 45 mm at We chose to examine biotransformation product profiles at 30 mm 4Â°C(Sorvall RC2-B with SS-34 rotor; Norwalk, CT; Refs. 15 and 23). after ormaplatin administration based on our previous biotransforma Aliquots from the diluted fluids and from the ultrafiltrates were removed and tion studies at this same time point in nontumor-bearing animals (19). allowed to stand overnight in scintillation cocktail before LSC. The biotransformation product proffles of ultrafiltrates of plasma, Slices from several lobes of the liver, the cortex, and outer medulla of each liver, and tumor from control and treated animals shown in Fig. 1 give kidney, the entire spleen, and portions of viable, nonnecrotic tumor tissue were dramatic evidence from a qualitative standpoint that WR-2721 does isolated on ice, weighed, and homogenized according to Mistry et a!. (18). affect the biotransformations of ormaplatin in the whole animal. The Cytosol and cytosolic ultrafiltrate were separated essentially as described (18). peaks are labeled according to nomenclature established previously Aliquots from homogenate, cytosol, and ultrafiltrate were removed and pre (13, 15, 26). In Fig. 1, peak b contains the reduced Pt(dach)Cl2; the pared for LSC as described for plasma. The remainder of the ultrafiltrates from peak/shoulder eluting immediately before the peak corresponds to the peritoneal fluid, plasma, and each of the tissues was frozen at â€”20Â°Cuntil unchanged parent drug, ormaplatin. In Fig. 1, peak e is predominantly analysis by HPLC. the cysteine complex; peakfcould contain the OSH, serine, threonine, Radioactivity was assessed with an LKB model 1215 scintillation counter. glutamine, or asparagine complexes; peak g (unlabeled on Fig. 1) is The difference between counts in the cytosol and in the ultrafiltrate was predominantly the methionine complex and generally appeared as a considered to represent macromolecular-bound platinum (15, 22). Counts in shoulder to peak h in these experiments; and peak h is primarily the the ultrafiltrate, although frequently referred to as â€œfreeplatinum,â€•are more free dach ligand. In Fig. 1, peak i in treated animals behaves chro aptly termed â€œfilterable,â€•becauseit is considered likely that they represent matographically like a standard of the Pt(dach)(WR-1065) complexes. platinum bound to low molecular weight substances from the plasma and/or The shoulder or peak most closely corresponding to peak i in control cytosol (24). For these experiments quantitation of platinum biodistribution and biotransformations was based on conversion of radioactivity to amounts of animals is most likely the Pt(dach)(citrato) complex (11). platinum by the use of specific activity of the ormaplatin (0.520 Ci/mmol). Because the identification of peaks g, h, and i are crucial for This conversion is based on the assumption that the platinum-nitrogen bond of interpretation of these studies, the complete HPLC profiles for the the parent drug was still intact. Previous findings from this laboratory (19) and relevant standards are shown in Fig. 2. The retention times of Pt others (25) using LSC in parallel with AAS analysis appear to validate this (dachXmethionine) (Fig. 2D), free dach carrier ligand (Fig. 2, V), assumption for the early time point used in this study (see â€œResultsâ€•). Pt(dach)(citrato) (Fig. 2G), and Pt(dach)(WR-1065) (Fig. 2A) were The biotransformation products from the ultrafiltrates of peritoneal fluid, similar by RP HPLC, although clearly distinguishable. Moreover, plasma, and tissues were separated by a two-column HPLC system developed SCX HPLC produced a distinctive profile that was different for each by Mauldin et a!. (26). Briefly, an initial separation on a Partisil octadecyl of the standards. For example, RP HPLC (Fig. 2A) resolved the silica-3 RP column with heptane sulfonate as the ion-pairing reagent provided Pt(dach)(WR-1065) complexes into at least two distinct peaks (the resolution of species as neutral paired ions on the basis of polarity. Peak third peak as seen in Fig. 2A was not reproducibly formed). SCX fractions of interest from this separation were pooled and then injected onto a HPLC of the major RP HPLC peak gave two peaks at pH 4 (Fig. 2B). Partisil 10 SCX cation exchange column for additional resolution of the The major peak at pH 4 was clearly resolved from the free dach carrier positively charged species. Fractions from both separations were collected and ligand (V), Pt(dach)(methionine) (Fig. 2E) and Pt(dachXcitrato) (Fig. analyzed for radioactivity by LSC. The total radioactivity of any peak of 2H). There was a minor peak on SCX HPLC that had the same interest was expressed as a percentage of the total radioactivity recovered from retention time as the free dach carrier ligand at both pH 4 (Fig. 2B) the column. Individual biotransformation products were identified by compar and pH 2.3 (Fig. 2C). This most likely represents free dach carrier ison of their retention times on RP and SCX HPLC with those of standards ligand released by trans-labilization between the RP and SCX HPLC prepared in the laboratory (13, 26). Initially, standards for the reaction products steps.4 The major peak at pH 4 (Fig. 28) is resolved into two distinct of both WR-2721 and WR-1065 with ormaplatin were prepared, as were other standards, by displacement of the malonate ligand from Pt(dach)(malonato) at 37Â°Cin the dark overnight. Subsequently, it was determined that the reaction 4 D. C. Thompson, S. D. Wyrick, D. J. Holbrook, and S. G. Chancy. HPLC and 31P NMR characterization of the reaction between antitumor platinum agents and the phos of platinum complexes with WR-2721 resulted in displacement of the phos phorothioate chemoprotective agent, S-2(3-aminopropylamino)ethylphosphorothioic acid phate moiety. Thus, whether WR-2721 or WR-1065 was used as the starting (WR-2721), Biochem. Pharmacol., in press, 1995. 2838

Pt(dach)(citrato) standard at pH 4 (Fig. 2H). In the SCX HPLC profiles from the control animal (Figs. 3, B and C), the peak with a retention time of 34â€”35mm at pH 4.0 (Fig. 3B), which is resolved into two peaks at 37â€”38mm and 44â€”45mm at pH 2.3 (Fig. 3C), has been shown previously to contain primarily free dach carrier ligand (Fig. 3C; 37â€”38mm) and Pt(dach)(methionine) (Fig. 3C; 44â€”45mm; Ref. 19). The biotransformation products resolved from peak i of the treated animals by SCX HPLC (Fig. 3, E and F) appear to represent at least two distinct Pt(dach)(WR-1065) complexes that form both in vivo and in vitro. The Pt(dach)(WR-1065) complex formed in vitro with a retention time of 42â€”44mm by SCX HPLC at pH 2.3 (Fig. 2C) does not appear to form to a significant extent in vivo (Fig. 3F). These complexes are characterized in a related paper4 and will simply be referred to as the Pt(dach)(WR-1065) complexes throughout the rest of this paper. After having observed the qualitative effects of WR-2721 on or maplatin biotransformations evident in Figs. 1 and 3, these effects were then quantified by comparing individual peak quantitations among treated and control animals (for each n = 4). Table 1 summa rizes the effects of WR-2721 treatment on total platinum distribution at 30 mm after ormaplatin administration. The platinum was quanti tated by conversion of radioactivity into an amount of platinum from the specific activity ofthe labeled ormaplatin. Previous work from this laboratory in the absence of WR-2721 (19) has shown that biodistri bution and HPLC profiles are essentially identical whether platinum is measured directly by AAS or by conversion from LSC. Parallel analyses with AAS and LSC were also done on one control and one treated animal in the group of experiments reported here (data not shown). No major differences were observed in the quantitation of Fig.1. QualitativecomparisonofRP HPLCprofilesfromcontroland treatedrats. Ultrafiltrates of plasma (A and D), liver (B and E), and tumor (C and F) were prepared platinum by the two methods; thus, treatment with WR-2721 did not as describedinâ€œMaterialsandMethodsâ€•fromcontrolrats(A-C)givenvehiclei.p.and from treated rats (D-F) given WR-2721 (200 mg/kg, i.p.), 30 mm before ormaplatin (12.5 mg/kg, i.p.). Separation of ultrafiltrates on RP HPLC was as described in â€œMaterialsand MethOdS.â€•Notethat Y-axes for the plasma profiles are on a different scale than are those for the liver and tumor profiles. The nomenclature used for peak identification has been describedpreviously(13,15,26).Thesepeaksarediscussedinthetextandelsewhere(13, 15, 26) in terms of the most likely biotransformation products of ormaplatin that they represent > F- Pt(dach)(WR-1065) complexes at pH 2.3 (Fig. 2C), which also have 0 retention times that are distinct from those of the free dach carrier 0 ligand (V) and Pt(dachXmethionine) (Fig. 2F). Pt(dach)(citrato) was 0 not run at pH 2.3 because its SCX HPLC profile at pH 4 was sufficiently distinct from all known standards. Thus, RP and SCX HPLC allow resolution of at least three distinct Pt(dach)(WR-1065) 0 complexes that form in vitro (two peaks by RP HPLC, with one of bJ the RP peaks being resolved into two peaks that are distinct from F- :@ free dach carrier ligand by SCX HPLC at pH 2.3). Possible structures -J for those complexes have been suggested elsewhere.4 All three bJ Pt(dach)(WR-1065) complexes are clearly distinguishable by RP and sCxHPLCfromallotherknowndach-platinumstandards,ofwhich zFâ€” over 40 have been prepared and characterized to date. It should also LU be noted that the dach-platinum complexes formed with WR-2721 are 0 indistinguishable from those formed with WR-1065, most likely be LU cause the phosphate group is lost from WR-2721 in the reaction with platinum(II) complexes.4 Fig. 3 shows SCX HPLC analyses of the prominent, late-eluting RP HPLC peaks shown in Fig. 1. The profiles derived from additional 0 25 500 25 500 25 50 SCx HPLC resolution of peak i (Fig. 3D) from the treated kidney at RETENTIONTIME(MIN) pH 4.0 (Fig. 3E) and at pH 2.3 (Fig. 3F) are remarkably similar to Fig. 2. Standards for the in vivo studies. Pt(dach@malonate) (0.05 mM) and either profiles derived from a similar resolution of the Pt(dach)(WR-1065) WR-1065 (A-C), WR-2721 (A-C), methionine (D-F), or citrate (G-H) (1 mM) were standard (Fig. 2, B and C) and very different from the SCX HPLC incubated at 37Â°Covernight.A. D. and G, RP HPLC separation. The peak fractions from RP HPLC were analyzed by SCX HPLC at pH 4 (B, E, and H) and 2.3 (C and F). The profiles shown of peak h (Fig. 3A) from control kidney at pH 4.0 (Fig. profiles obtained with WR-1065 and WR-2721 were identical. V, the elution position of 3B) and at pH 2.3 (Fig. 3C) and from the SCX HPLC profile of the free dach carrier ligand. 2839

centage and absolute terms. The data suggest (Table 1) that WR-2721 treatment had a similar effect (i.e., of decreasing the percentage of platinum bound to protein in the peritoneal compartment), but this could not be confirmed due to an unexplained variability of this parameter among our control animals. > WR-2721 treatment was also found to have measurable but variable 0 effects on total platinum distribution and disposition among the tis 0 sues examined in this study (Table 1). These tissues, as noted in 0 previous work from this laboratory (19), represent a range of target and nontarget sites for ormaplatin toxicity, with kidney and spleen exhibiting signfficant sensitivity to platinum toxicity and liver exhib 0 Li iting relatively no sensitivity. Our tumor model has been shown previously to have limited but detectable sensitivity to platinum -J induced cytotoxicity (21). As demonstrated in Table 1, effects of Li WR-2721 treatment were most pronounced in the spleen. Distribution F- z of total platinum to the spleens of treated rats was decreased by 67%, Li and all other parameters were decreased as well. Total platinum 0 distributed to liver was decreased by 50%, whereas in the kidney and Li 0@ tumor, total platinum was relatively unaffected by WR-2721 treat ment. The amount of total platinum that remained associated with the cytosolic fraction of the whole tissue homogenates was decreased in all normal tissue of treated rats but not in tumor tissue. The percentage of this cytosolic platinum that, in turn, was bound to cytosolic proteins and/or macromolecules was decreased in the spleen and tumor tissue of treated animals but not in the kidney and liver. Fig. 3. Kidney ultrafiltrates from control and treated rats resolved by RP and SCX HPLC. Shown are RP HPLC profiles (A and D) from kidney ultrafiltrates, select fractions As described, HPLC was used to resolve the various biotransfor of which are then further resolved by SCX HPLC at pH 4.0 (B and E) and at pH 2.3 (C mation products of ormaplatin found in the filterable fraction (or and F), as described in â€œMaterialsandMethods.â€•Forthe control rat (as defined in Fig. ultrafiltrates) of Table 1. The quantitation of these separations, exam 1) ultrafiltrate (A), RP HPLC fractions 37â€”38(hpeak overlap) are shown following additional resolution by SCX HPLC (B and C). For the treated rat ultrafiltrate (D), RP ples of which are shown in Figs. 1 and 3, are summarized in Table 2. HPLC fractions numbers 39â€”41(i peak) are shown following additional SOC resolution Several effects of WR-2721 treatment were detectable: (a) the amount (E and F). Note that Y-axes for the RP profiles are on a different scale than are those for of unchanged parent drug, ormaplatin, was decreased as a percentage the SOC profiles. V, elution position of free dach carrier ligand. of the ifiterable platinum in the peritoneal fluid and plasma. The amount of the reduced product, Pt(dach)C12 (i.e., Fig. 1, peak b), was appear to have affected the stability of the platinum-nitrogen bond of apparently unaffected by the presence of WR-2721, except that it, too, ormaplatin at 30 mm. was decreased on a percentage basis in the peritoneal fluid. In Fig. 1, It is evident from Table 1 that 50% of the total plasma platinum in peaks e/fand g, both in terms of absolute amounts and as a percentage the control animals was bound to plasma proteins at 30 min. This is of the filterable platinum, were decreased almost uniformly across all in contrast to previous findings from this laboratory (15, 19) of tissues and fluids. In Fig. 1, peak i, as a percentage of filterable approximately 70% of plasma platinum bound at 30 mm after i.v. platinum, was increased in the peritoneal fluid, plasma, kidney, and administration in animals without tumors. WR-2721 treatment had no spleen of treated animals. It was also increased in terms of absolute effect on total plasma platinum levels but decreased by 50% the amount in the peritoneal fluid, plasma, and kidney. Apparent differ amount of plasma platinum that was bound to protein in both per ences in Fig. 1, peak i, between treated and control livers and tumors

Table 1Biodistribution ofplatinwnafter ormaplatini.p.f'Body (12.5 mg/kg, ip.) Â±WR-2721 (2(K)mg/kg. tissueFractionPentoneal fluid or

LiverSpleenTumorTotal fluidPlasmaKidney

@@ Control Â±0.77 Â±2.88 12.65 Â±1.95 Â±2.44 Â±0.25 0.08CytosolicTreated15045b198.71 Â±11.217.53 7.38 Â±0.6835.28 28.75 Â±3.05 6.27 Â±0.40(0.044)c11.45 3.79 Â±0.35 (0.050)1.42 1.87 Â±

Control Â±1.48 8.70 Â±0.74 Â±0.80 Â±1.48 0.06FilterableTreatedN/Aâ€• N/Aâ€•N/A N/A24.36 16.83 Â±2.14(0.032) 4.30 Â±0.21(0.007)5.68 2.06 Â±0.23(0.017)1.12 1.02 Â±

Control Â±7.94 Â±0.39 Â±1.24 2.68 Â±0.33 Â±0.24 Â±0.07 Treated94.59 198.53 Â±8.54(0.000)3.76 5.46 Â±0.39(0.022)18.01 13.02 Â±1.39(0.037) 1.72 Â±0.102.30 1.25 Â±0.17(0.015)0.55 0.65 Â±0.04 Bound Control 55.870.071.88 Â±30.833.78 Â±0.386.34 Â±0.296.02 Â±0.413.38 Â±0.580.57 Â± Treated Â±1.881.92 Â±0.33 (0.011)3.81 Â±0.892.58 Â±0.25(0.001)0.81 Â±0.07(0.020)0.37 Â±0.06 % bound Control 28.75Â±14.01 50.25Â±0.85 26.25Â±0.95 69.50Â±1.50 58.75Â±2.32 50.75Â±3.2536.25 Treated 1.00 Â±1.00 25.25 Â±2.87(0.002) 22.00 Â±3.03 59.50 Â±3.40 40.00 Â±2.61(0.002) Â±4.61(0.047) a Values are @g/ml(body fluids) or @zg/g(tissues) of platinum as derived from cpm radioactivity with the use of specific activity of tritiated ormaplatin.

M@ Â± SEM; n = 4 (except treated peritoneal fluid, where n = 3).

C Values in parentheses are the P value, control versus treated; where no P value indicated, P > 0.05. d N/A, not applicable. 2840

Table 2 Quantitation ofmajor biotransfonnation products afteri.p.rBody ormaplatin (12.5 mg/kg. i.p.) Â±R-2721 (200 mg/kg, tissueFractionPeritoneal fluid or LiverSpleenTumorPeak fluidPlasmaKidney b'Control5.29k' @ Â±0.00Treated2.27 1.230.13 Â±0.030.18 Â±0.06 0.04 Â±0.010.04 Â±0.010.01 0.00Peakb'%Control5.42Â±0.390.07 Â±0.020.08 Â±0.04 0.02 Â±0.010.01 Â±0.000.01 Â±

0.37Treated1.16 Â±0.973.18 Â±0.631.01 Â±0.36 1.34 Â±0.331.52 Â±0.39.77 Â± 0.17Peak Â±0.23 (0.019)c1.18 Â±0.25 (0.043)0.73 Â±0.35 1.27 Â±0.401.10 Â±0.100.98 Â± bControl32.49 0.01Treated28.36 Â±3.380.97 Â±0.220.90 Â±0.24 0.05 Â±0.020.23 Â±0.070.02 Â± 0.01Peak Â±3.070.77 Â±0.170.21 Â±0.08 0.05 Â±0.020.11 Â±0.030.02 Â± b%Control34.47 1.33Treated14.44 Â±2.5124.69 Â±4.235.17 Â±1.43 1.81 Â±0.589.65 Â±2.284.08 Â± 1.05Peak Â±2.00(0.002)13.70 Â±2.271.86 Â±0.81 2.56 Â±1.298.21 Â±1.762.77 Â± c/fControl19.04 0.02Treated10.60 Â±3.350.61 Â±0.045.49 Â±0.70 0.74 Â±0.140.43 Â±0.040.13 Â± 0.01Peak Â±0.740.28 Â±0.05(0.003)1.98 Â±0.23 (0.011) 0.28 Â±0.01 (0.041)0.22 Â±0.03 (0.009)0.07 Â± c/f%Control20.17 3.68Treated5.37 Â±3.1816.93 Â±2.8130.41 Â±2.98 28.02 Â±4.4119.50 Â±3.1224.81 Â± (0.033)Peak Â±0.50 (0.017)5.09 Â±0.71(0.021)15.20 Â±0.45 (0.014) 16.38 Â±1.4117.69 Â±0.6511.25 Â±0.61 gControl4.45 0.01Treated3.67 Â±1.000.35 Â±0.052.12 Â±0.18 0.26 Â±0.040.24 Â±0.060.06 Â± (0.026)Peak Â±0.510.17 Â±0.03(0.032)0.57 Â±0.12 (0.001) 0.09 Â±0.01 (0.024)0.07 Â±0.000.02 Â±0.01 g%Control4.63Â±0.819.37Â± 156Treated1.84 1.4211.76Â±037 10.17Â± 1.8410.08Â± 1.2611.77Â± (0.010)Peak Â±0.19(0.038)3.21 Â±0.71(0.015)431 Â±0.61 (0.000) 5.38 Â±0.565.70 Â±0.76(0.031)3.64 Â±1.50 hControl5.52 0.02Treated20.96Â±7.870.61Â±0.920.39 Â±0.073.88 Â±0.42 035 Â±0.060.31 Â±0.070.10 Â± 0.39Â±0.110.18Â±0.060.10Â±0.04Peak Â±0.222.22Â±0.70 h%Contact5.90 4.57Treated10.27 Â±1.0111.26 Â±3.2721.53 Â±1.61 13.38 Â±1.7413.47 Â±3.1120.20 Â± 6.95Peak Â±3.4112.10 Â±4.6718.88 Â±7.32 22.08Â±5.8214.08 Â±4.2415.11 Â± iControl3.73 0.050.18Â±0.06Treated70.62Â±1.110.22 Â±0.041.28 Â±0.12 0.23 Â±0.070.27 Â± 0.05Peak Â±6.40(0.008)2.36 Â±0.22(0.002)5.32 Â±1.24 (0.046) 0.38 Â±0.080.32 Â±0.050.24 Â± i%Control3.75 11.41Treated35.43 Â±0.925.68 Â±0.507.09 Â±0.28 8.11 Â±1.7111.70 Â±1.2233.35 Â± Â±1.64(0.000)43.57 Â±4.59 (0.003)39.41 Â±6.61(0.016) 22.53 Â±5.5726.47 Â±4.53(0.043)37.97Â±8.02 a Values are pg/mI (body fluids) or @g/g(tissues) of platinum as derived from cpm radioactivity using specific activity of tritiated ormaplatin, and based on RP HPLC separations as shownin Fig. 1. In conformitywithpreviouscharacterizationandnomenclaturefromthis laboratory(5,15, 18)andour owncontemporaneousstandardization,thefollowing represents the general pattern of RP HPLC fractions included for purposes of peak quantitation: peak b', #7â€”9;peakb, #9â€”11;peakc/f, #30â€”34;peakg, #34â€”36;peakh, #36â€”38; peaki, #38â€”41.Theoverlappingfractionsbetweenpeaks(fractions#9,34,36,and38)wereproportionedbetweentheadjacentpeaksbasedon thesizeof thepeaks. M@ Â± SEM; n = 4 (except treated peritoneal fluid, where n = 3).

C Values in parentheses are the P value, control versus treated; where no P value is indicated, P > 0.05.

did not prove to be statistically significant. It is, however, important tion when administered by the i.p. route (34, 35). There are also to emphasize the qualitative difference of this peak between WR clinical situations where the malignancy being treated has a peritoneal 2721-treated and control animals for all tissues (Figs. 1 and 3). In Fig. exposure, such as ovarian carcinoma, and in these situations, i.p. 1, peak i from our treated animals behaveschromatographically administration of a platinum agent has demonstrated some benefit almost identically with our Pt(dach)(WR-1065) standard (Fig. 2, clinically (36). Furthermore, systemic protection from platinum-in A-C). In Fig. 1, peak i from our control animals appears to be duced toxicity has been reported after concurrent i.p. administration of primarily due to tailing from peak h, although a shoulder in this region cisplatin and i.v. administration of another sulfhydryl-containing com is occasionally seen in the liver and tumor. We have previously pound, sodium thiosulfate (37, 38). Thus, we also investigated the observed occasional peaks in this region, due to formation of a effects of i.v. administration of WR-2721 on biotransformations of Pt(dachXcitrato) complex (11), but the SCX HPLC profiles of this i.p.-administered ormaplatin. For this paradigm, we administered complex (Fig. 2H) bear no resemblance to those observed for Fig. 1, WR-2721 i.v., either 5 or 30 min before ormaplatin, and characterized peak i, from animals pretreated with WR-2721. the ormaplatin biotransformation products at 15, 30, and 60 min after Because WR-2721 pretreatment affected ormaplatin biotransforma ormaplatin administration. It was of particular interest to know, given tions in the peritoneal fluid, plasma, and all tissues examined, we the rapid tissue uptake and clearance of WR-1065 after i.v. adminis concluded that inactivation of ormaplatin most likely was occurring in tration of WR-2721 in rodents (39), if the effects on ormaplatin the peritoneal cavity when both drugs were administered by the i.p. biotransformations were similar after the i.v. administration of WR route (see â€œDiscussionâ€•).Clinicalregimens, in contrast, have gener 2721 to those after its i.p. administration. These experiments were ally used an i.v. infusion of WR-2721 (1, 2, 33). We retained the i.p. designed to determine whether the Pt(dach)(WR-1065) complexes route of ormaplatin administration because our previous biotransfor were detectable at any time after WR-2721 (i.v.)/ormaplatin (i.p.) marion data with ormaplatin in tumor-bearing animals (Figs. 1 and 3; administration, rather than to provide a quantitative comparison of Table 2) had been obtained via that route, and previous studies have control and treated animals at a single time point. As is evident from shown rapid influx of platinum complexes into the systemic circula Fig. 4, there are distinct qualitative differences between the RP 2841

A Quantitation of the major peaks in plasma and all tissues of animals 20 pretreated with WR-2721 by the i.v. route are summarized in Table 3. When WR-2721 was administered 5 mm before ormaplatin, peak i represented 14.3â€”19.0%,9.1â€”13.2%,16.3â€”22.6%,and 13.5â€”16.2%of @10 total radioactivity recovered from the RP HPLC column for the > kidney, liver, spleen, and tumor, respectively, at times between 15 and F- 0 60 mm after ormaplatin administration. The percentage of recovered 0- radioactivity represented by this same peak i was slightly less when 0 the WR-2721 was administered 30 mm before the ormaplatin. As <20 +44'DC@&JShSSmentioned previously, a shoulder or small peak in the region of peak i is also observed in control animals not pretreated with WR-2721 0 Li (Fig. 1; Table 2). Because peak i represents a relatively small per F- 10 centage of filterable platinum in the treated animals, and some mate -J rial also migrates in the region of peak i in control animals, a Li quantitative comparison between control and treated animals F- z would likely prove inconclusive. Thus, SCX HPLC was used to 020Li determine whether any of the material in peak i might represent the Pt(dach)(WR-1065) complexes. Li As discussed previously, based on over 40 Pt(dach) standards 0@ 10 characterized in this laboratory (13, 26), this peak/shoulder could represent either an overlap ofthe free dach carrier ligand in peak h, the Pt(dach)(citrato) complex or Pt(dachXWRlO65) complexes. There fore, this peak was further characterized by SCX HPLC of RP fraction 0 :[email protected]@ @80 0 25 50 RETEN11ONliME(mm) no. 39 from the kidney and tumor samples at the 15-mm time point (Fig. 5). The peak with a retention time of 39 mm on SCX HPLC at Fig. 4. Variation with time of RP HPLC profiles in animals given wR-2721 iv.. Shown are RP HPLC profiles of ultrafiltrates from kidney (A-C) and tumor (D-F) of rats pH 4 could represent free dach carrier ligand resulting from trans treated with WR-272l iv. 5 mm before ormaplatin i.p. and then sacrificed at 15 mm (A labilization of the Pt(dach)(WR-1065) complexes4 or overlap with the and D), 30 mm (B and E), and 60 mm (C and F) after ormaplatin administration as described in â€œMaterialsandMethods.â€• RP peak h containing the free dach carrier ligand. However, the peak with a retention time of 43 mm is characteristic of the Pt(dach)(WR 1065) complexes (Fig. 2B) and different from the SCX peak of either Table 3 Quantirarion of major peaks following WR-2721(200 mg/kg iv) + ormaplatin the free dach carrier ligand (Fig. 2B, V) or the Pt(dach)(citrato) (12.5 mg/kg. i.p.) via four different paradigms complex (Fig. 2H). Accordingly, these data suggest that some Pt (dach)(WR-1065) complex does form in tissues when WR-2721 is iPlasma30TissueParadigmPeakâ€•Peak bPeak e/fPeak gPeak hPeak administered by the i.v. route. However, Pt(dach)(WR-1065) com mm > 30 mm1' 5 mm > 15 mm 28.2 10.4 3.0 7.9 13.1 5 mm > 30 mm 36.7 11.2 3.6 7.2 15.5 16.7Kidney305 mm > 60 mm32.2 10.013.1 17.34.1 4.65.6 11.87.2 mm > 30 mm 5 mm > 15 mm 2.5 23.5 7.5 24.0 19.0 5 mm > 30 mm 6.2 24.6 5.7 23.7 15.1 14.3Liver305 mm > 60 mm3.2 0.627.5 26.17.7 7.425.2 31.612.0 > Fâ€” mm > 30 mm 0 5 mm > 15 mm 1.3 21.8 6.4 15.8 12.7 5 mm > 30 mm 4.6 15.6 4.6 22.5 13.2 0 9.1Spleen30 5 mm > 60 mm2.6 0.724.7 23.46.2 5.016.5 21.77.7 0 mm > 30 mm 5 mm > 15 mm 10.4 13.3 5.9 13.4 18.2 5 mm > 30 mm 13.3 13.8 5.6 14.7 16.3 22.6Tumor305 mm > 60 mm11.7 1.417.6 15.16.7 6.315.8 18.910.1 0 Li mm > 30 mm F- 5 mm > 15 mm 2.4 17.8 3.5 12.7 16.2 :D 5 mm > 30 mm 5.9 13.3 3.1 17.1 13.5 -I 5 mm > 60 mm9.4 0.714.5 18.33.9 3.812.6 17.09.8 15.1 Li aEachpeakisrepresentedintermsofitspercentageofthetotalradioactivityrecovered F- from the RP HPLC column. z b The first time of the paradigm refers to the interval between WR-2721 administration Li and ormaplatin administration; the second time refers to the interval between ormaplatin 0 administration and sacrifice of the animal (n = 1 for each paradigm). a:: LU 0@ profiles of ormaplatin biotransformations after i.v. pretreatment with WR-2721 compared to those after pretreatment by the i.p. route. In 20 40 none of the paradigms tested in Fig. 4 was peak i as prominent as it RETENTION TIME (mm) was after i.p. administration of WR-2721 (Figs. 1, D-F, and 3D). Fig. 5. Additional peak resolution in animals given WR-2721 i.v. Shown are profiles However, a small peak or shoulder in the position of peak i was of SCXHPLCat pH4.0of RPHPLCfraction39fromkidney(A)andfromtumor(B) of an animal administered WR-2721 iv. 5 mm before ormaplatin and sacrificed at 15 mm observed in all tissues of the treated animals (Fig. 4 and data not after the ormaplatin (Fig. 4, A and D, respectively). All procedures were as described in shown). â€œMaterialsandMethods.â€•V,elution position of free dach carrier ligand. 2842

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1995 American Association for Cancer Research. WR-2721 AND ORMAPLATIN BIOTRANSFORMATIONSIN F344 RATS plexes appear to represent only a very small percentage of filterable tissues tested from animals treated with WR-2721 and ormaplatin platinum at any time point and are found in both normal and tumor by the i.p./i.p. route. In contrast, the same data demonstrate that tissue. Pt(dach)(WR-1065) complexes are only a minor, but detectable, bio transformation product in the plasma and all tissues of animals re DISCUSSION ceiving WR-2721 by the i.v. route. We think that these differences between i.p. and i.v. administration of WR-2721 are best explained by Previous investigations from this laboratory have been concerned models in which most of the ormaplatin and Pt(dach)Cl2 are converted with the relationship between biotransformations of platinum(dach) to Pt(dach)(WR-1065) complexes in the peritoneal cavity with sub compounds and the toxicity/cytotoxicity of these agents (9, 11, 13â€”16, sequent distribution of the Pt(dach)(WR-1065) complexes to plasma 19, 26, 40). Most recently (19) we have examined the organ-specific and all tissues when WR-2721 is administered by the i.p. route, biotransformations of ormaplatin in nontumor-bearing Fischer 344 whereas little of the Pt(dach)(WR-1065) complexes form in either rats. The present studies extend this line of investigation to determine plasma or tissues when WR-2721 is administered i.v. because both if another drug, WR-2721, which has purported chemoprotective WR-2721 and WR-1065 are cleared from those compartments before activity that is selective for nontumor tissue, would have selective ormaplatin and/or Pt(dach)Cl2 can reach them. These models are effects on in vivo biotransformations of ormaplatin. We have exam discussed in more detail below. ined a select group of target and nontarget tissues of platinum-induced It was, of course, difficult to predict the optimal time for detection toxicity similar to our previous studies (19) but have included a of Pt(dach)(WR-1065) complexes in vivo. The highest cytosolic transplanted fibrosarcoma in the present investigation. Unfortunately, WR-1065 concentrations are detected immediately after WR-2721 given the recent reports of neurotoxicity in clinical trials with or administration and then rapidly decrease to much lower steady-state maplatin (9, 10), it was not possible to study the effects of WR-2721 levels (39, 45, 46). This would seem to suggest that the optimal time on onnaplatin biotransformations in neural tissue. As was discussed in for detection of Pt(dach)(WR-1065) complexes would be shortly after the previous report (19), the target site of both ormaplatin- and administration of WR-2721. However, Treskes et a!. (47) have pre cisplatin-induced neurotoxicity appears to be peripheral nerve fibers dicted, based on in vitro reaction rates and likely tissue levels in vivo, (41â€”43),which offer too little material for these types of biotransfor that the reaction between platinum complexes and WR-1065 would be marion studies. We have chosen WR-2721 for this investigation relatively slow. Thus, we examined tissue cytosols for the presence of because it has shown promise in both preclinical (4, 29, 44) and Pt(dach)(WR-1065) complexes when WR-2721 was administered i.v., clinical (1, 2) studies. Carfagna et a!. (16) have previously shown that either 30 or 5 mm before ormaplatin, and at times ranging from 15â€”60 WR-2721pretreatmentprotectedkidneyandintestinaltissuefrom mm after i.p. ormaplatin administration. This makes it very unlikely ormaplatin-induced toxicity but has little effect on toxicity to lymph that we missed significant formation of Pt(dach)(WR-1065) corn oid tissue. The present investigations examine the relationship be plexes in vivo because of an incorrect choice of sampling times. For tween this chemoprotective effect of WR-2721 and its effects on the i.p./i.p. paradigm, we used only a single time point for analysis ormaplatin biotransformations. because our data indicated the likely inactivation of ormaplatin and/or The effects of WR-2721 on ormaplatin biodistribution were incon Pt(dach)Cl2 in the peritoneal cavity by that route (see below). sistent and not readily interpretable. For example, i.p. administration The most frequently described mechanism (2, 3) by which WR of WR-2721 caused a significant decrease in total platinum in the 2721 is proposed to selectively protect normal tissue from platinum spleen and liver but not in the kidney and tumor (Table 1). In contrast, induced toxicity has to do with the relative amounts and activities of the percentage of cytosolic platinum bound to macromolecules was alkaline phosphatase between tumor and nontumor tissue. WR-2721 is decreased in the spleen and tumor but not in the kidney and liver a prodrug that is converted to the more active WR-1065 by in vivo (Table 1). Moreover, because the Pt(dach)(WR-1065) complex ap dephosphorylation. It has been postulated that the undeveloped vas pears to form by direct reaction of Pt(dach)Cl2 and/or ormaplatin cularity frequently encountered in tumor tissue results in a lowered pH with WR-2721 in the peritoneal cavity (see below), the altered in the tumor microenvironment, which yields a net deficit of alkaline biodistribution pattern most likely reflects differences between the phosphatase activity in tumor tissue. This results in a relative lack of biodistibution of Pt(dach)Cl2, which is the major plasma biotrans conversion of WR-2721 to and uptake of WR-1065 in tumor tissue as formation product in control animals, and Pt(dach)(WR-1065), compared to normal tissue. Because previous pharmacokinetic studies which is the major plasma biotransformation product in WR-2721- had indicated that WR-2721 is rapidly cleared from the plasma (39, treated animals. Thus, we consider the biotransformation data as 48),thepredictionhasbeenthatonlytheWR-1065thataccumulates providing a better assessment of the effects of WR-2721. preferentially in normal tissue would be present in sufficient concen The biotransformation studies rely primarily on HPLC separation tration to react with and inactivate any reactive platinum species of platinum complexes to assess the effects of WR-2721 on ormapla encountered. These predictions are summarized in schematic form in tin biotransformation. In data reported elsewhere, we have character Model A of Fig. 6. The essential points of Model A are: (a) there is ized the RP and SCX HPLC profiles of over 40 platinum(II) standards predicted to be little or no direct reactivity between the parent drug, (11, 12, 13, 15, 26). More importantly, we have characterizedor WR-2721, and ormaplatin or its biotransformation products in the maplatin biotransformations in vitro (11), in cell culture (12, 13), and peritoneal cavity or plasma; and (b) little or no WR-1065 should be in the Fischer 344 rat (14â€”16, 19). The only complexes that have encountered by any platinum species except in the cytosols of normal somewhat similar retention times to Pt(dach)(WR-1065) on RP HPLC tissues. are the free dach carrier ligand and the Pt(dach)(citrato) complex, and As stated previously, the primary reason for our use of an i.p./i.p. both of these are clearly resolved from the Pt(dach)(WR-1065) corn paradigm in the first group of experiments was the inhibition reported plexes by SCX HPLC. Because of the difficulties inherent in quanti previously of cisplatin-induced nephrotoxicity with this same animal taring Pt(dach)(WR.1065) complexes by SCX HPLC,4 we have relied model and paradigm (4). Past decisions to use this i.p./i.p. paradigm on RP HPLC for quantitative comparisons (Tables 2 and 3). However, have also been influenced by pharmacokinetic analyses showing rapid the combination of our RP (Figs. 1 and 4) and SCX (Figs. 3 and 5) absorption and distribution of WR-2721-derived radioactivity into HPLC data clearly demonstrate that the Pt(dach)(WR-1065) corn (jnimarily normal) tissues after i.p. administration in this same rat plexes are a major biotransformation product in all body fluids and model (45). However, that study did not analyze the pharmacokinetics 2843

ModelA Moreover, our findings would particularly call into question previous I Cavity reports from other investigators in which WR-2721 chemoprotection Tumor I(dach)Pt-prot@n(dach)Pt-S-R(dach)Pt-proteln (dach)Pt-S-R (dach)Pt-protetn (dach)Pt-S-R against platinum-induced toxicity was tested in a dual i.p./i.p. para I @, digm, but the effect of WR-2721 on antitumor efficacy of the platinum I (dach)Pt-C@2â€” (dach)Pt-C12 â€”@ (dach)Pt-C12 â€œ- t\ agent was tested with a WR-2721 i.p./platinum agent i.v. paradigm oP@ [email protected] â€˜â€”(dath)Pt-C14-VascularSpacWPlasma-@(dach)Pt@CL4\ (27, 32). I \ Normal I \TlssuWintracellular(dach)Pt-protetn (@ch)Pt-S-R The change to i.v. administration of WR-2721 in the second group of experiments, as described above, was in part an attempt to more @@@(dach)Pt-Cl21â€” IPerftoneal closely mimic clinical protocols in which the WR-2721 is usually WR-27214@WR-2721â€”â€”-WR-2721WR-1O65â€”--@I(dach)Pt-WR-1065 given as a 15-mm infusion before infusion of the platinum agent (33). @â€”â€”(dach)Pt-WR-lO65@â€”-â€” We, however, chose to continue administering the ormaplatin by the ModelBI i_p. route because we already had the data from the first group of (dach)Pt-prot@n(dach)Pt-S-R (dach)Pt-S-R (dach)Pt-S-R experiments that were also based on i.p. dosing of ormaplatin. Fur thermore, as mentioned above, there does exist a precedent for such a oP@ @)â€”(dach)Pt-Ci2- -@(dath)Pt-C12 - -@(dach)Pt-C$2 @ I :WR-2721 ti paradigm of i.p. administration of platinum agents in conjunction with @ (dach)Pt-C14 -(dach)Pt-protetn..(dach)Pt-C14 @(dach)Pt-protein i.v. chemoprotection (37, 38). Such an i.v./i.p. paradigm obviously would not be subject to the same sort of difficulties as the dual i.p./i.p. WR-272114:1 â€” WR-2721@: â€” â€”@ pt.*,â€”â€”â€”@@-â€˜: I paradigm, mainly because the WR-2721 has been shown to be very V(dach)Pt-WR-1065â€”â€”â€”â€˜@dach)Pt-WR-lO65â€”â€”Ã¸@dach)Pt-WR-1O65,WR-1065â€” rapidly cleared from the circulation when administered i.v. (tÂ½ 5 mm; Refs. 3 and 39). On the basis of the in vitro reactivity of Fig. 6. Schematic of possible reaction pathways of WR-2721 and ormaplatin in the WR-2721 and WR-1065 with cisplatin and achievable in vivo con F344rat.ModelA,predictedreactionpathwaysbasedonpostulatedmechanismofaction of WR-2721 found in the literature (2, 3). Model B, reaction pathways, which would centrations of these compounds, Treskes et a!. (47) have predicted that appear to be operative based on the findings of the investigations reported here. not only is WR-2721 unlikely to directly inactivate platinum corn plexes in plasma, but that WR-1065 is also unlikely to cause signif icant inactivation of cytosolic platinum complexes. Our findings ap of WR-2721 and/or WR-1065 in the peritoneal cavity. Moreover, in pear to confirm the predictions of Treskes et a!. (47) because we comparing the plasma pharmacokinetics of WR-2721-derived radio detected at most 10â€”20%of recovered radioactivity by HPLC in the activity after administration of WR-2721 by the i.p. and i.v. routes in form of Pt(dach)(WR-1065) complexes after i.v. administration of that study, it was clear that a significant amount of WR-2721 and/or WR-2721 (Table 3). This should be considered a maximum estimate WR-1065 never reached the plasma compartment when WR-2721 because only a portion of peak i (Fig. 4, A and D) can be identified was administered i.p. (45). Our data Suggest that this missing pool of unambiguously as a Pt(dach)(WR-1065) complex (Fig. 5). This WR-2721 remains in the peritoneal cavity at 30 mm and is present at amount of inactivation of filterable platinum species would hardly be sufficiently high concentrations to rapidly convert the majority of sufficient for protection from toxicity, nor would it be predicted to add ormaplatin and/or Pt(dach)C12 to Pt(dach)(WR-1065) complexes be much to the protective effects of the methionine, cysteine, and GSH fore the parent compounds are able to react with other biological complexes detected in peaks e/f and g (Fig. 4). nucleophiles or to reach other compartments. For example, Model A Thus, these data suggest that the classic model for WR-2721 action (Fig. 6) predicts that WR-2721 would not react with ormaplatin or (Fig. 6, Model A) is also inadequate to explain the protection of Pt(dach)Cl2 and, therefore, would have little effect on the levels of normal tissues when WR-2721 is administered i.v. That is, our data platinum bound to protein and low molecular weight nucleophiles in strongly suggest that mechanisms other than a simple, nonspecific either the peritoneal cavity or the plasma. However, our data show a inactivation of reactive platinum species in the cytosols of normal statistically significant decrease in protein-bound platinum in the tissues must be considered to explain the protective effects of WR plasma and platinum bound to low molecular weight nucleophiles 2721. There have been several alternative mechanisms proposed, e.g., (peaks elf and g) in both the peritoneal cavity and plasma. Because reduction in the formation of reactive oxygen species by depletion of data reported elsewhere show that platinum complexes can react oxygen (49), the inactivation of radicals by a direct interaction (49), directly with WR-2721 (47) and that this reaction results in the proton donation to DNA damaged by radicals (49), and modification displacement of inorganic phosphate and formation of the Pt of enzymes involved in DNA metabolism and repair (46, 50). The (dach)(WR-1065)complexes,4ourdataaremoreconsistentwithan effects of WR-2721 on the formation and/or inactivation of free alternative pathway of biotransformation for ormaplatin in the pres radicals could be significant in light of several reports that platinum ence of WR-2721, which we have delineated schematically in Model complexes cause an increase in lipid peroxidation by reducing the B of Fig. 6. In this model, Pt(dach)(WR-1065) complexes are postu concentration of protective sulthydryl compounds (51, 52). One re lated to form directly in the peritoneal cavity (and possibly in the cent report has suggested that platinum complexes can generate free plasma as well) and to subsequently distribute nonspecifically to all radicals when they react with DNA (53). Several investigators (3, 46, tissues and/or be excreted. The direct reaction between WR-2721 and 54) have also proposed that WR-1065, which is positively charged at ormaplatin and/or Pt(dach)Cl2 in the peritoneal cavity would readily cellular pH, has a tendency to concentrate near the DNA by counter explain the decreased level of protein-bound platinum and platinum ion condensation. Thus, this would act to create pockets of high bound to low molecular weight nucleophiles in the peritoneal cavity WR-1065 concentration amid an apparently low total cytosolic con and plasma. It would also account for our second major finding with centration and increase the rate of reaction with any platinum species this paradigm, which was a lack of any apparent specificity between or free radicals appearing in the vicinity of the DNA. Such a model tumor and nontumor tissue in the effects of WR-2721 on ormaplatin could, in theory, be used to explain the protective effects of WR-2721 biodistribution and biotransformations. These findings, taken to on DNA in the nucleus, although inactivation of reactive platinum gether, would seem to indicate clearly that a dual i.p./i.p. paradigm is species in the cytosol was minimal. Whether this concentration around not a useful model by which to investigate the ability of WR-2721 to the DNA actually occurs in the in vivo situation becomes a moot selectively protect normal tissue from platinum-induced toxicity. question, of course, if DNA is not the target for platinum-induced 2844

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1995 American Association for Cancer Research. WR-2721 AND ORMAPLATIN BIOTRANSFORMATIONS IN F344 RATS toxicity. Particularly with respect to platinum-induced nephrotoxicity, 6. Calabro-Jones, P. M., Aguilera, J. A., ward, J. F., Smoluk, G. D., and Fahey, R. C. Uptake of WR-2721 derivatives by cells in culture: identification of the transported this very much remains a subject for debate (55). form of the drug. Cancer Res., 48: 3634â€”3640, 1988. In terms of a mechanistic basis for the tissue specificity of the 7. Smoluk, G. D., Fahey, R. C., Calabro-Jones, P. M., Aguilera, J. A., and Ward, J. F. protective effects of WR-2721, our data demonstrated an apparent Radioprotection of cells in culture by WR-2721 and derivatives: form of the drug responsible for protection. Cancer Res., 48: 3641â€”3647,1988. lack of any specificity between tumor and nontumor tissue when the 8. Schilder, R. J., LaCreta, F. P., Perez, R. P., Johnson, S. W., Brennan, J. M., Rogatko, WR-2721 is given by the i.v. route because we detected Pt(dachXWR A., Nash, S., McAleer, C., Hamilton, T. C., Roby, D., Young, R. C., Ozols, R. F., and 1065) complexes in tumor and nonturnor tissue alike. There are two O'Dwyer, P. J. Phase I and pharmacokinetic study of ormaplatin (tetraplatin, NSC 363812) administered on a day 1 and day 8 schedule. Cancer Res., 54: 709â€”717, possible explanations for these data. Either the fibrosarcoma used in 1994. these experiments did not exclude WR-1065, or the selective protec 9. Petros, W. P., Chancy, S. G., Smith, D. C., Fangmeier, J., Sakata, M., Brown, T. D., tion of normal tissues by WR-2721 involves mechanisms other than and Trump, D. L. Pharmacokinetic and biotransformation studies of ormaplatin in conjunction with a phase I clinical trial. Cancer Chemother. Pharmacol., 33: 347â€”354, the selective inactivation of platinum complexes in the cytosols of 1994. normal tissues. Unfortunately, ormaplatin at nontoxic doses did not 10. O'Rourke, T. J., Weiss, G. R., New, P., Bums, H. A., III, Rodriguez, G., ECkhardt, J., Hardy, J., Kuhn, J. 0., Fields, S., Clark, G. M., and Von Hoff, D. D. Phase Iclinical cause a statistically significant increase in tumor growth delay with trial of ormaplatin (tetraplatin, NSC 363812). Anticancer Drugs, 5: 520â€”526, 1994. this fibrosarcorna (data not shown). Thus, we were unable to deter 11. Chancy, S. 0., Wyrick, S. D., and Till, G. K. in vitro biotransformations of tetra mine whether WR-2721 had any effect on the antitumor efficacy of chloro(d,l-trans)-1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) in rat plasma. Cancer Res., 50: 4539â€”4545,1990. ormaplatin in this model system and could, therefore, not distinguish 12. Chancy, S. G., Gibbons, G. R., Wyrick, S. D., and Podhasky, P. An unexpected between the two possible explanations described above for the lack of biotransformation pathway for tetrachloro-(d,l-trans)-1,2-diaminocyclohexaneplati selective inactivation of ormaplatin biotransformation products in num(IV) (tetraplatin) in the L1210 cell line. Cancer Res., 5!: 969â€”973,1991. 13. Mauldin, S. K., Gibbons, 0., Wyrick, S. D., and Chancy, S. G. Intracellular biotrans normal tissues. However, it is important to remember that although formation of platinum compounds with the 1,2-diaminocyclohexanecarrier ligand in selective exclusion of WR-2721/WR-1065 has been demonstrated for the L1210 cell line. Cancer Res., 48: 5136â€”5144,1988. some tumors (4, 45, 56), there have been reports oftumors that do take 14. Carfagna, P. F., Wyrick, S. D., Holbrook, D. J., and Chancy, S. G. Effects of diethyldithiocarbamate (DDTC) on the plasma biotransformations of tetrachloro up and are protected by WR-2721 (29). (d,l-trans)-1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) in Fischer 344 rats. In summary, our data suggest that when both WR-2721 and the J. Biochem. Toxicol., 6: 71â€”80,1991. 15. Carfagna, P. F., Poma, A., Wyrick, S. D., Holbrook, D. J., and Chancy, S. 0. platinum complex are administered by the i.p. route, the protective Comparisons of tetrachloro(d,l-trans)l,2-diaminocyclohexaneplatinum(IV) biotrans effects observed are most likely due to direct reaction between the formations in the plasma of Fischer 344 rats at therapeutic and toxic doses. Cancer platinum complex and WR-2721 in the peritoneal cavity. This is Chemother. Pharmacol., 27: 335â€”341,1991. 16. Carfagna, P. F., Chancy, S. 0., Chang, J., and Holbrook, D. J. Reduction of particularly troubling for those preclinical studies in which protection tetrachloro(d,l-trans)1,2-diaminocyclohexaneplatinum(lV) (tetraplatin) toxicity by from platinum-induced toxicity by WR-2721 has been demonstrated the administration of diethyldithiocarbamate (DDTC), S-2(3-aminopropylamino)eth by using dual i.p./i.p. administration, whereas the lack of tumor ylphosphorothioic acid (WR-2721), or sodium selenite in the Fischer 344 rat. Fun dam. Appl. Toxicol., 14: 706â€”719,1990. protection has been demonstrated by using i.p./i.v. administration. Our 17. Daley-Yates, P. T., and McBrien, D. C. H. Cisplatin metabolites in plasma, a study data also suggest that when WR-2721 is administered i.v., direct of their pharmacokinetics and importance in the nephrotoxic and antitumour activity of cisplatin. Biochem. Pharmacol., 33: 3063â€”3070,1984. reaction between WR-1065 and reactive platinum species in the 18. Mistry, P., Lee, C., and McBrien, D. C. H. Intracellular metabolites of cisplatin in the cytosols of normal tissues is minimal. This confirms the predictions of rat kidney. Cancer Chemother. Pharmacol., 24: 73â€”79,1989. Treskes et al. (47) and suggests that new models for the mechanism 19. Thompson, D. C., Vaisman, A., Sakata, M. K., Wyrick, S. D., Holbrook, D. J., and Chancy, S. 0. Organ-specific biotransformation of ormaplatin in the Fischer 344 rat. of WR-2721 protection of normal tissues from platinum-induced Cancer Chemother. Pharmacol., in press, 1995. toxicity need to be explored. Finally, our data showed no selective 20. Wyrick, S. D., and Chancy, S. 0. Tritiated platinum antitumor agents containing the effect of WR-2721 on normal versus tumor tissue when WR-2721 trans-(d,l)-1,2-diaminocyclohexane carrier ligand. J. Labelled Compd. Radiopharm., 25: 349â€”357,1988. was administered by either the i.p. or the i.v. route. These data 21. Wondergem, J., Bulger, R. E., Strebel, F. R., Newman, R. A., Travis, E. 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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1995 American Association for Cancer Research. Effect of the Chemoprotective Agent WR-2721 on Disposition and Biotransformations of Ormaplatin in the Fischer 344 Rat Bearing a Fibrosarcoma

D. Charles Thompson, Steven D. Wyrick, David J. Holbrook, et al.

Cancer Res 1995;55:2837-2846.

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