Improved targeting of platinum chemotherapeutics. the antitumour activity of the HPMA copolymer platinum agent AP5280 in murine tumour models.

AP5280 is a novel N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound platinum (Pt) therapeutic designed to increase the therapeutic index relative to conventional, small-molecule platinum agents. The platinum-polymer construct accumulates in solid tumours on the basis of increased capillary permeability. The bound platinum moiety is present as an N,O-Pt chelate at the distal end of a tetrapeptide linker, glycine-phenylalanine-leucine-glycine, and the weight-average molecular weight (Mw) of the construct is 22 kDa. The antitumour activity and toxicity of AP5280 were assessed in the syngeneic murine B16F10 and Lewis lung tumour models, and in the human ovarian carcinoma 2008 and head and neck squamous carcinoma UMSCC10b xenograft models. The maximum tolerated dose (MTD) of AP5280 was 6-fold greater than that of carboplatin (CBDCA) in vivo. AP5280 was active in all four tumour models, and it displayed a higher therapeutic index than CBDCA in each of these tumour models. The antitumour effect of AP5280 given at 16% of its MTD was equivalent to that produced by a MTD of CBDCA. Thus, consistent with the design goal for this drug, and despite being less potent than CBDCA, AP5280 produced less systemic toxicity relative to its antitumour activity and thus has a greater therapeutic index. On the basis of the improved therapeutic index evidenced in these models, AP5280 has been advanced into clinical trials.

Pharmaceutical development of a parenteral lyophilized formulation of the investigational polymer-conjugated platinum anticancer agent AP 5280.

AP 5280 is a novel polymer-conjugated platinum anticancer agent showing promising in vitro and in vivo activity against solid tumors. The aim of this study was to develop a parenteral pharmaceutical dosage form for phase I clinical trials. AP 5280 drug substance was characterized by using a wide range of analytical techniques and showed excellent solubility in water. However, as aqueous solutions of AP 5280 proved to be labile upon sterilization by moist heat, it was decided to develop a lyophilized dosage form. Initially, glass vials were used as primary packaging, but this led to a high breakage rate, which could be completely prevented by the use of CZ resin vials. Stability studies to date show that the lyophilized product in glass vials is stable for at least 12 months when stored at 2-8 degrees C in the dark and the lyophilized product in CZ resin vials is stable for at least 6 months under these conditions. Photostability testing revealed photolability of AP 5280 drug substance and lyophilized product in both types of primary container, necessitating storage in the dark. The first clinical experiences indicate that the proposed formulation is fully applicable for use in the clinical setting.

Technetium(V) oxo complexes of substituted propylene diamine dioxime (PnAO) ligands: water-dependent interconversion between syn and anti isomers.

99mTc and (99)Tc complexes of PnAO (propylene diamine dioxime) ligands monosubstituted in the 6-position [PnAO-6-R] were prepared and studied. Ligands substituted with an alkyl group or with no substituent (R = H, CH(3), or CH(2)CH(CH(3))(2)), gave only one Tc complex. However, for several other nonalkyl substituents (R = COOCH(3), OH, OCH(3), OCH(2)CH(3), F, CN, NHCOCH(3), and NHCOCH(2)CH(3)), two Tc complexes A and B were formed. Products A and B were assigned to the anti and syn TcO(PnAO-6-R) species, respectively, based on (1)H NMR results. X-ray structure analyses supported these assignments. The A (anti) isomer of TcO(PnAO-6-OH) had the chemical formula TcC(13)H(25)N(4)O(4) and crystallized in an orthorhombic system with space group P2(1)2(1)2(1) and Z = 4; a = 12.744(2) A, b = 13.591(2) A, c = 9.976(2) A. The B (syn) isomer of TcO(PnAO-6-CN) had the chemical formula TcC(14)H(24)N(5)O(3) and was a 1:4 mixture of two monoclinic polymorphs: individual rectangular prisms (space group P2(1)/c, Z = 4) and clusters of intergrown twinned rectangular rods (space group Cc, Z = 8). For the prisms, a = 12.457(1) A, b = 13.932(1) A, c = 10.336(1) A, and for the rods, a = 31.344(5) A, b = 6.993(1) A, c = 21.657(2) A. The syn and anti isomers interconverted in the presence of water; nonequilibrium mixtures of epimers remained unchanged under dry conditions. The HPLC behavior under reversed phase conditions was consistent with on-column interconversion (poor resolution), whereas the two isomers were cleanly resolved under drier normal phase conditions. An oxo inversion mechanism involving trans water attack is proposed for the interconversion process. Water also influenced the position of equilibrium of the two isomers. The syn isomer was stabilized in water relative to the anti isomer.

Dopamine transporter loss and clinical changes in MPTP-lesioned primates.

Single photon emission computed tomography (SPECT) and the dopamine (DA) transporter tracer, 2 beta-carboxymethoxy-3 beta-(4-iodophenyl)tropane ([123I]beta-CIT), were used to determine DA transporter density in 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-lesioned monkeys with varying degrees of parkinsonism. The clinical stage of parkinsonism corresponded to SPECT measures of striatal DA transporter density suggesting that more severe parkinsonism was associated with a greater degree of dopaminergic terminal degeneration. These findings are similar to those reported earlier using positron emission tomography (PET) and the DA metabolism tracer, 6-[18F]fluoro-L-m-tyrosine (FMT), indicating that both are good methods for evaluating nigrostriatal degeneration in MPTP primate models.

Delineation of the border zone of ischemic rabbit myocardium by a technetium-labeled nitroimidazole.

Delineation of viable ischemic myocardium is an important problem in nuclear cardiology. To determine the feasibility of using a technetium-labeled nitroimidazole as an indicator of ischemic myocardium at risk of infarction, we characterized the distribution of a 2-nitroimidazole-derivatized PnAO ligand and its 99mTc complex, 99mTcO(PnAO)-1-CH2-(2NI) (BMS-181321) in the ischemic territory of the left anterior descending (LAD) coronary artery of the rabbit. In preliminary experiments, the performance of 14C-deoxyglucose (14C-2DG) and 14C-misonidazole was assessed relative to apparent regional relative myocardial blood flow (rMBF) indicated by 99mTc-teboroxime using double-label autoradiography in the rabbit LAD occlusion model. After demonstrating that 14C-2DG and 14C-misonidazole are selectively retained in the lateral border of the ischemic territory, BMS-181321 was co-injected intravenously, with either 14C-2DG or 14C-misonidazole, 20 min after LAD occlusion. In a separate experiment, 99mTcO(PnAO)-6-CH3, a complex with the same lipophilicity (log k' 0.26 vs. 0.31) as BMS-181321 but which lacks the 2NI moiety, was co-injected with 14C-2DG. After 30 min, the rabbits were sacrificed and 14C/99mTc autoradiograms were obtained from the same tissue sections. The autoradiograms revealed that BMS-181321 was retained with the same microregional distribution as both 14C-2DG and 14C-misonidazole in the border zone of the ischemic LAD territory. The selective retention of BMS-181321 depends on the presence of the nitroimidazole group, since 99mTcO(PnAO)-6-CH3 has a uniformly low myocardial distribution in contrast to the enhanced uptake of co-injected 14C-2DG. These data demonstrate that BMS-181321 is selectively retained in hypoxic myocardium and demarcates the ischemic border zone in a manner similar to 14C-2DG and 14C-misonidazole.

BATO complexes derived from dimethoxy dioximes: synthesis, characterization and biodistribution.

To prepare less lipophilic BATO complexes, two new methoxy-substituted dioximes were synthesized: cis-4,5-dimethoxycyclohexane-1,2-dione dioxime (DMCDO) and 1,4-dimethoxybutane-2,3-dione dioxime (DMDMG). 99mTcCl(DMCDO)3BMe (BMe = methylboronic acid) was prepared and characterized. Reversed-phase HPLC analyses of 99mTcCl(DMCDO)3BMe and 99mTcCl(DMCDO)3-p-TBA (p-TBA = p - tolylboronic acid) indicated that both of these complexes were mixtures of four enantiomeric pairs of diastereomers. Attempted preparation of a BATO complex from DMDMG gave a mixture of products. In rats, 99mTcCl(DMCDO)3BMe displayed more rapid liver and renal clearance than 99mTcCl(CDO)3BMe, but 99mTcCl(DMCDO)3BMe and 99mTcCl(DMCDO)3-p-TBA displayed low uptake in both heart and brain.

Boronic acid adducts of technetium dioxime (BATO) complexes derived from quinuclidine benzilate (QNB) boronic acid stereoisomers: syntheses and studies of their binding to the muscarinic acetylcholine receptor.

We have investigated the possibility of using BATO complexes derivatized with the muscarinic acetylcholine receptor (mAChR) antagonist, quinuclidinyl benzilate (QNB), for mAChR imaging. The BATO complexes, TcCl(DMG)3B-QNB, were prepared using QNB derivatives containing a 4'-boronic acid substituent on one of the benzilic benzene rings (QNB-boronic acid). The QNB-boronic acid molecule has two chiral centers, and all four QNB-BATO stereoisomers were made and evaluated. When studied using in vitro receptor binding assays based on tissue from rat brain caudate-putamen (which contains primarily M1 and M4 mAChR) and rat heart (M2 mAChR), the QNB-boronic acid stereoisomers had binding affinities (KA) in the range 2 x 10(5)-1 x 10(8), at least 10-fold lower than the KA for QNB (ca 2 x 10(9)). The stereochemistry of both centers had some influence on the affinity constant. When the TcCl(DMG)3B-QNB complexes were studied, none of the stereoisomeric complexes displayed measurable specific binding (KA < 10(6)), but all showed high non-specific binding. In vitro autoradiography with rat brain slices confirmed the absence of specific binding in these tracers. In vivo, the 99mTcCl(DMG)3B-QNB complexes displayed minimal brain uptake, and modest heart uptake; the latter was unlikely to be related to uptake by the mAChR. In light of these findings, we conclude that the interaction between the TcCl(DMG)3B-QNB complexes and biological membranes is dominated by the hydrophobicity of the BATO moiety. The TcCl(DMG)3B-QNB complexes, therefore, have little potential for mAChR imaging.

The synthesis and in vitro evaluation of a 99mtechnetium-nitroimidazole complex based on a bis(amine-phenol) ligand: comparison to BMS-181321.

We have developed a 99mTechnetium complex for imaging of hypoxic tissue (BMS-181321). Recently, another nitroimidazole derivative, based upon a bis(amine-phenol) ligand, was described in the patent literature. To compare this compound to BMS-181321, we have synthesized the ligand, prepared its 99mTc complex, and evaluated its performance in two in vitro assays of bioefficacy: membrane permeability and uptake in normoxic and anoxic cardiocytes. In attempting to reproduce the synthesis of the ligand described in the patent application, we found that one intermediate could not be made by the method described, and alternative routes were investigated. Complexation of the bis(amine-phenol) nitroimidazole with 99mTc gave an apparent single complex; this appeared as a broad peak on HPLC analysis. Purification by a solid-phase method gave a complex with 95% radiochemical purity. This complex was not permeable to cultured bovine brain endothelial cells nor did it show preferential uptake in anoxic myocytes.

TcO(PnA.O-1-(2-nitroimidazole)) [BMS-181321], a new technetium-containing nitroimidazole complex for imaging hypoxia: synthesis, characterization, and xanthine oxidase-catalyzed reduction.

A technetium(V)oxo nitroimidazole complex that shows promise for imaging regional hypoxia in vivo, [BMS-181321, TcO(PnAO-1-(2-nitroimidazole))] (1) was prepared from 3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazol-1-yl)-4,8-diazaundecane -2,10-dione dioxime, a 2-nitroimidazole-containing derivative of propyleneamine oxime (PnAO). The 99Tc complex [99Tc]Oxo[[3,3,9,9-tetramethyl-1-(2-nitro-1H-imidazol-1-yl)-4,8- diazaundecane-2,10-dione dioximato]-(3-)-N,N',N'',N''']technetium (V) was synthesized both from pertechnetate and [TcO(Eg)2]- (Eg = ethylene glycol). A new synthetic route to TcO(PnAO) (2) is also described. 99TcO(PnAO-1-(2-nitroimidazole)) was characterized by 1H NMR, IR, and UV/vis spectroscopy, HPLC, FAB mass spectrometry, and X-ray crystallography. Electrochemistry of 1 reveals that the nitro redox chemistry found in the ligand is maintained upon coordination to technetium but shifts to a slightly more positive potential. Using chiral HPLC (Chiracel OD), 99mTc (1) was resolved into its two enantiomers. However, the two isomers were found to racemize quickly (t1/2 < 2 min) in the presence of water. Localization of 1 is believed to be mediated by enzymatically catalyzed reduction of the nitroimidazole group, so the in vitro reaction of 99Tc(1) with the nitroreductase enzyme xanthine oxidase (XOD) was studied. XOD catalyzed the quantitative reduction of the nitroimidazole group on the molecule under anaerobic conditions in the presence of hypoxanthine. No reaction was noted using a non-nitro-containing complex (2). The rate of reduction of the Tc-nitroimidazole complex (1.5 +/- 0.16 nmol/min per unit XOD) was faster than that observed previously for the nitroimidazole BATOs (BATO = boronic acid adduct of technetium dioxime) and was about two-thirds that of fluoromisonidazole, a compound that has proven useful for imaging hypoxia in humans when labeled with 18F. These data suggest that BMS-181321 (1) has the potential to be recognized by nitroreductase enzymes in vivo, thus satisfying one of the criteria required for this potential hypoxia imaging agent.

Synthesis, characterization, and in vitro evaluation of nitroimidazole--BATO complexes: new technetium compounds designed for imaging hypoxic tissue.

Several technetium-99 BATO (boronic acid adduct of technetium dioximes) complexes TcX(dioxime)3BR (X = Cl) that contain a boron cap R which bears a 2- or 4-nitroimidazole moiety have been prepared from either TcCl(dioxime)3 or from Tc(dioxime)3(mu-OH)SnCl3 [dioxime = dimethyl glyoxime (DMG) or cyclohexanedione dioxime (CDO)]. Two hydroxy analogs (X = OH) were isolated by treatment of the corresponding chloro complexes with aqueous NaOH. The complexes have been characterized by elemental analysis, mass spectrometry, NMR, UV/vis spectroscopy, and high-performance liquid chromatography. These complexes have the potential for selective retention in hypoxic tissue, by a mechanism believed to be the result of nitro reduction. The electrochemistry and enzymatic reduction of these complexes was studied to assess the potential for reduction in vivo. The nitroreductase enzyme xanthine oxidase was shown to reduce the nitroimidazole group on the complexes 99TcOH(DMG)3BBNO2 and 99TcOH(DMG)3BprenNO2 under anaerobic conditions in the presence of hypoxanthine. However, the results indicated that the rate of reduction might be slow in vivo, limiting the suitability of these compounds for imaging of regions of hypoxia.

Direct analysis of whole blood by internal surface reversed-phase chromatography: an examination of the binding and metabolism of technetium dioxime complexes.

We have developed a method using internal surface reversed-phase (ISRP) packing for rapid on-line separation of small hydrophobic compounds from cellular whole blood components. This is achieved by the use of 75-microns ISRP chromatographic material packed into a small high-performance liquid chromatographic (HPLC) column, in conjunction with column switching. We have applied this analytical method to study the in vitro metabolism of 99mTc-BATO (boronic acid adducts of technetium dioxime) cerebral and myocardial perfusion tracers in whole blood. The results from the ISRP procedure were compared with a conventional centrifugation method of analysis. This novel HPLC methods provides a rapid, convenient and reliable method for the analysis of radioactive and non-radioactive lipophilic components in whole blood.

Technetium labeling of monoclonal antibodies with functionalized BATOs: 2. TcCl(DMG)3CPITC labeling of B72.3 and NP-4 whole antibodies and NP-4 F(ab')2.

BATO (boronic acid adduct of technetium dioximes) complexes, TcCl(dioxime)3BR, were prepared in which the boron substituent (R) was the protein-reactive 2-carboxy-4-phenyl isothiocyanate (CPITC). The 99Tc complexes, where the dioxime was either dimethylglyoxime (DMG) or cyclohexanedione dioxime (CDO), were prepared and characterized. The 99mTc complex TcCl(DMG)3CPITC was prepared from a freeze-dried kit and used to label B72.3 (anti-TAG.72) and NP-4 (anti-CEA) whole antibodies, and the NP-4 F(ab')2 fragment. SDS-PAGE electrophoresis indicated that the labeling reagent was strongly bound to antibody. The labeled antibodies displayed high binding to affinity columns and good tumor uptake in GW39 tumor-bearing mice.

Technetium labeling of monoclonal antibodies with functionalized BATOs. 1. TcCl(DMG)3PITC.

BATO (boronic acid adduct of technetium dioximes) complexes, TcCl(dioxime)3BR, were prepared in which the boron substituent (R) was the protein-reactive m-phenyl isothiocyanate (PITC). The 99TcCl(dioxime)3PITC complexes [dioxime = dimethylglyoxime (DMG) or cyclohexanedione dioxime (CDO)] were prepared from 99Tc(dioxime)3(mu-OH)SnCl3 and characterized. The X-ray crystal structure of 99TcCl(DMG)3PITC was determined. The 99mTc complexes were prepared from 99mTcO4- in a process using a freeze-dried kit, either in a one-step procedure or via 99mTcCl(dioxime)3. Initial labeling studies with 99mTcCl(dioxime)3PITC were performed on glycine and polylysine and, subsequently, on mouse IgG and the B72.3 monoclonal antibody. Covalent attachment of 99mTcCl(DMG)3PITC to B72.3 was demonstrated by SDS-PAGE electrophoresis. B72.3 labeled with 99mTcCl(DMG)3PITC displayed high binding to a TAG 72 affinity column and had a distribution in normal mice similar to that reported for iodine-labeled B72.3.

Chloro----hydroxy substitution on technetium BATO [TcCl(dioxime)3 BR] complexes.

The neutral, seven coordinate complexes of technetium known as the BATO (Boronic acid Adducts of Technetium diOximes) complexes have shown their utility as myocardial and cerebral perfusion agents. The axial chloride ligand of the BATO complexes [99mTcCl(dioxime)3 BR] is labile to substitution by a competitive anion; under physiological conditions, the axial chloride ligand can be replaced by a hydroxy group. The chloro and hydroxy analogs have different biodistributions and single-pass cerebral extraction efficiencies. The influence of structure on the rate of the in vitro chloro/hydroxy exchange process has been studied. The mechanism of axial ligand exchange was found to be SN1-CB, which proceeds by way of a transient, neutral six coordinate complex. Evidence is presented which indicates that chloro/hydroxy exchange is not the mechanism by which BATO complexes are retained in the brain.

Technetium-99m d,l-HM-PAO: a new radiopharmaceutical for SPECT imaging of regional cerebral blood perfusion.

Following investigation of a large number of new ligands based upon propylene amine oxime (PnAO) the d,l-diastereoisomer of hexamethyl propyleneamine oxime (HM-PAO) was selected as the preferred ligand for 99mTc as a tracer for cerebral perfusion imaging. The neutral, lipophilic 99mTc complex of d,l-HM-PAO was formed in high yield by stannous reduction of 99Mo/99mTc generator eluate using a kit formulation of the ligand. Two minutes following i.v. administration of this complex in rats, 2.25% of the injected dose appears in the brain. Little washout of the tracer is observed up to 24 hr postinjection. By qualitative autoradiographic comparison with iodoantipyrine this new radiopharmaceutical displays blood flow dependent brain uptake with little redistribution of the tracer over time. The lipophilic 99mTc complex converts slowly in vitro to a secondary complex. This conversion process may account for the ability of [99mTc]d,l-HM-PAO to be retained within the brain without redistribution.

Technetium-99m-d, 1-HM-PAO: a new radiopharmaceutical for imaging regional brain perfusion using SPECT--a comparison with iodine-123 HIPDM.

A new radiopharmaceutical, technetium-99m hexamethylpropyleneamine oxime (99mTc-d, 1-HM-PAO), has been reported to cross the blood-brain-barrier and to distribute in brain in proportion to regional blood flow. This study reports brain imaging obtained with 99mTc-d,1 HM-PAO in 20 subjects; seven without evidence of cerebral disease and 13 with cerebrovascular disorders. In 16 patients comparative data were available with N,N,N'-trimethyl-N'-(2-hydroxy-3-methyl-5-iodobenzyl)-1,3-propanediamine ([123I]HIPDM). Technetium-99m-d, 1-HM-PAO is retained sufficiently long to allow single photon emission computed tomography (SPECT) with widely available rotating gamma camera systems. The kinetics demonstrated a rapid brain uptake and prolonged retention of activity in cerebral structures. Good tomographic images are obtained with much higher uptake in gray than in white matter. Blood flow maps are comparable to those achieved with [123I]HIPDM and established strokes were clearly seen, with similar details as in HIPDM studies. Delayed studies showed that the distribution in the brain remained virtually unchanged. Technetium-99m-d, 1-HM-PAO imaging appears particularly promising in routine examination of patients with cerebrovascular disorders.

Development of a 99Tcm-labelled radiopharmaceutical for cerebral blood flow imaging.

A new radiopharmaceutical, 99Tcm-hexamethylpropyleneamine oxime (99Tcm-HM-PAO) has shown considerable promise for single photon emission tomographic (SPECT) imaging of the brain. In animal biodistribution studies this complex demonstrates good brain uptake with prolonged retention of activity in brain. Further improvement of these properties, resulting in higher brain uptake with very slow washout and fixed regional distribution has been achieved following the isolation of the d,1-diastereoisomer of HM-PAO.