Antitenascin antibody 81C6 armed with 177Lu: in vivo comparison of macrocyclic and acyclic ligands.

INTRODUCTION: When labeled with iodine-131, the antitenascin monoclonal antibody (mAb) 81C6 has shown promise as a targeted radiotherapeutic in patients with brain tumors. Because of its more favorable gamma-ray properties, lutetium-177 might be a better low-energy beta-emitter for this type of therapy. MATERIALS AND METHODS: Chimeric 81C6 (ch81C6) was labeled with (177)Lu using the acyclic 1B4M ligand and the macrocyclic ligands NHS-DOTA and MeO-DOTA and evaluated for binding to tenascin. Three paired-label tissue distribution experiments were performed in normal mice receiving one of the (177)Lu-labeled immunoconjugates plus (125)I-labeled ch81C6 labeled using Iodogen. Paired-label experiments in athymic mice bearing subcutaneous D54 MG human glioma xenografts were done to directly compare the biodistribution of ch81C6-1B4M-(177)Lu and (125)I-labeled ch81C6, and ch81C6-MeO-DOTA-(177)Lu and (125)I-labeled ch81C6. Similar comparisons were done using murine (mu) instead of ch81C6. The primary parameter utilized for evaluation was the (177)Lu/(125)I uptake ratio in each tissue. RESULTS: In the studies performed in normal mice, the NHS-DOTA ligand yielded the highest (177)Lu/(125)I uptake ratios in tissues indicative of loss of label from the chelate; for this reason, only 1B4M and MeO-DOTA were evaluated further. The (177)Lu/(125)I ratio in bone increased gradually with time for the chimeric conjugates; however, there were no significant differences between ch81C6-1B4M-DTPA-(177)Lu and ch81C6-MeO-DOTA-(177)Lu. In contrast, mu81C6-1B4M-DTPA-(177)Lu and mu81C6-MeO-DOTA-(177)Lu showed a more dramatic increase in the (177)Lu/(125)I ratio in bone - from 2.4+/-0.3 and 1.7+/-0.2 at Day 1 to 8.5+/-1.1 and 4.2+/-0.5 at Day 7, respectively. CONCLUSION: With these antitenascin constructs, the nature of the mAb had a profound influence on the relative degree of loss of (177)Lu from these immunoconjugates. MeO-DOTA shows promise as a bifunctional chelate for labeling 81C6 mAbs with (177)Lu.

Ventilation-synchronous magnetic resonance microscopy of pulmonary structure and ventilation in mice.

Increasing use of transgenic animal models for pulmonary disease has raised the need for methods to assess pulmonary structure and function in a physiologically stable mouse. We report here an integrated protocol using magnetic resonance microscopy with gadolinium (Gd)-labeled starburst dendrimer (G6-1B4M-Gd, MW = 192 +/- 1 kDa, R(h) = 5.50 +/- 0.04 nm) and hyperpolarized (3)helium ((3)He) gas to acquire images that demonstrate pulmonary vasculature and ventilated airways in live mice (n = 9). Registered three-dimensional images of (1)H and (3)He were acquired during breath-hold at 2.0 T using radial acquisition (total acquisition time of 38 and 25 min, respectively). The macromolecular Gd-labeled dendrimer (a half-life of approximately 80 min) increased the signal-to-noise by 81 +/- 30% in the left ventricle, 43 +/- 22% in the lung periphery, and -4 +/- 5% in the chest wall, thus increasing the contrast of these structures relative to the less vascular surrounding tissues. A constant-flow ventilator was developed for the mouse to deliver varied gas mixtures of O(2) and N(2) (or (3)He) during imaging. To avoid hypoxemia, instrumental dead space was minimized and corrections were made to tidal volume lost due to gas compression. The stability of the physiologic support was assessed by the lack of spontaneous breathing and maintenance of a constant heart rate. We were able to stabilize the mouse for >8 hr using ventilation of 105 breath/min and approximately 0.2 mL/breath. The feasibility of acquiring both pulmonary vasculature and ventilated airways was demonstrated in the mouse lung with in-plane spatial resolution of 70 x 70 microm(2) and slice thickness of 800 microm.

Preparation and in vivo evaluation of novel linkers for 211At labeling of proteins.

The syntheses, radiolabeling, antibody conjugation and in vivo evaluation of new linkers for (211)At labeling of monoclonal antibodies are described. Syntheses of the N-succinimidyl esters and labeling with (211)At to form succinimidyl 4-methoxymethyl-3-[(211)At]astatobenzoate (9) and succinimidyl 4-methylthiomethyl-3-[(211)At]astatobenzoate (11) from the corresponding bromo-aryl esters is reported. Previously reported succinimidyl N-{4-[(211)At]astatophenethyl}succinamate (SAPS) is employed as a standard of in vivo stability. Each agent is conjugated with Herceptin in parallel with their respective (125)I analogue, succinimidyl 4-methoxymethyl-3-[(125)I]iodobenzoate (10), succinimidyl 4-methylthiomethyl-3-[(125)I]iodobenzoate (12) and succinimidyl N-{4-[(125)I]iodophenethyl}succinamate (SIPS), respectively, for comparative assessment in LS-174T xenograft-bearing mice. With 9 and 11, inclusion of an electron pair donor in the ortho position does not appear to provide in vivo stability comparable to SAPS. Variables in radiolabeling chemistry of these three agents with (211)At are notable. Sequential elimination of acetic acid and oxidizing agent, N-chlorosuccinimide (NCS), from the (211)At radiolabeling protocol for forming SAPS improves yield, product purity and consistency. NCS appears to be critical for the radiolabeling of 6 with (211)At. Formation of 11, however, is found to require the absence of NCS. Elimination of acetic acid is found to have no effect on radiolabeling efficiency or yield for either of these reactions.

Application of a macromolecular contrast agent for detection of alterations of tumor vessel permeability induced by radiation.

Permeability of tumor vasculature can be a major barrier to successful drug delivery, particularly for high molecular weight agents such as monoclonal antibodies and their diagnostic or therapeutic conjugates. In this study, changes in permeability of SCCVII tumor vessels after radiation treatment were evaluated by dynamic magnetic resonance imaging as a function of time after irradiation using a generation-8 polyamidoamine dendrimer (G8-Gd-D)-based magnetic resonance imaging contrast agent shown previously to be confined to tumor blood vessels. Tumor irradiation consisted of either single doses (2-15 Gy) or various daily fractionated doses (5 days). A single radiation dose of 15 Gy resulted in significant transient image enhancement of the tumor tissue with a maximum occurring between 7 and 24 hours after radiation treatment. No observable enhancement was recorded for fractionated radiation doses. Use of dynamic magnetic resonance imaging coupled with G8-Gd-D provides an exquisite methodology capable of defining the timing of enhanced permeability of macromolecules in tumors after irradiation. Such information might be applied to optimize the efficacy of subsequent or concurrent therapies including radiolabeled antibodies or other anticancer agents in combination with external beam therapies.

Systemic radioimmunotherapy using a monoclonal antibody, anti-Tac directed toward the alpha subunit of the IL-2 receptor armed with the alpha-emitting radionuclides (212)Bi or (211)At.

To exploit the fact that IL-2 receptors are expressed by T-cells responding to foreign antigens but not by resting T-cells, humanized anti-Tac (HAT) armed with alpha-emitting radionuclides (212)Bi and (211)At was evaluated in a cynomolgus cardiac allograft model. Control graft survival was 8.2+/- 0.5 days compared with 14.0+/-1.3 days (p<0.01) survival for monkeys treated with (212)Bi labeled HAT and 26.7+/-2.4 days survival (p<0.001 versus controls) with (211)At labeled HAT. Thus, (211)At labeled HAT may have application in organ transplantation and in treatment of IL-2 receptor expressing T-cell leukemia.

Convective distribution of macromolecules in the primate brain demonstrated using computerized tomography and magnetic resonance imaging.

OBJECT: Convection-enhanced delivery (CED), the delivery and distribution of drugs by the slow bulk movement of fluid in the extracellular space, allows delivery of therapeutic agents to large volumes of the brain at relatively uniform concentrations. This mode of drug delivery offers great potential for the treatment of many neurological disorders, including brain tumors, neurodegenerative diseases, and seizure disorders. An analysis of the treatment efficacy and toxicity of this approach requires confirmation that the infusion is distributed to the targeted region and that the drug concentrations are in the therapeutic range. METHODS: To confirm accurate delivery of therapeutic agents during CED and to monitor the extent of infusion in real time, albumin-linked surrogate tracers that are visible on images obtained using noninvasive techniques (iopanoic acid [IPA] for computerized tomography [CT] and Gd-diethylenetriamine pentaacetic acid for magnetic resonance [MR] imaging) were developed and investigated for their usefulness as surrogate tracers during convective distribution of a macromolecule. The authors infused albumin-linked tracers into the cerebral hemispheres of monkeys and measured the volumes of distribution by using CT and MR imaging. The distribution volumes measured by imaging were compared with tissue volumes measured using quantitative autoradiography with [14C]bovine serum albumin coinfused with the surrogate tracer. For in vivo determination of tracer concentration, the authors examined the correlation between the concentration of the tracer in brain homogenate standards and CT Hounsfield units. They also investigated the long-term effects of the surrogate tracer for CT scanning, IPA-albumin, on animal behavior, the histological characteristics of the tissue, and parenchymal toxicity after cerebral infusion. CONCLUSIONS: Distribution of a macromolecule to clinically significant volumes in the brain is possible using convection. The spatial dimensions of the tissue distribution can be accurately defined in vivo during infusion by using surrogate tracers and conventional imaging techniques, and it is expected that it will be possible to determine local concentrations of surrogate tracers in voxels of tissue in vivo by using CT scanning. Use of imaging surrogate tracers is a practical, safe, and essential tool for establishing treatment volumes during high-flow interstitial microinfusion of the central nervous system.

Acyl-protected hydroxylamines as spin label generators for EPR brain imaging.

In a search for novel electron paramagnetic resonance (EPR) brain imaging agents, we have designed and synthesized the acyl-protected hydroxylamines 1-acetoxy-4-methoxycarbonyl-2,2,6,6-tetramethylpiperidine (AMCPe), 1-acetoxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (AMCPy), and 1-acetoxy-3-(acetoxymethoxy)carbonyl-2,2,5,5-tetramethylpyrrolidine (DACPy), in which both the ring size and the number of ester functions were varied. In all of them, the nitroxide was first reduced and the resultant hydroxylamine was then protected with an acetyl group. These compounds are lipophilic, which is a major prerequisite for blood-brain barrier penetration. Once in the brain, esterases and oxidants quickly convert these derivatives into ionic, water-soluble radicals and thus EPR detectable species that then reside in the central nervous system for periods of time sufficient for detection and imaging. The biological relevancy of these new compounds in mice has been assessed, and their biodistribution patterns have been compared. The five-membered ring derivative AMCPy emerged as a potent EPR brain imaging agent while the other two derivatives, AMCPe and DACPy, were quite ineffective.

Calixarenes Derivatized with Sulfur-Containing Functionalities as Selective Extractants for Heavy and Precious Metal Ions.

The calix[4]arenes 5,11,17,23-tert-butyl-25,26,27,28-(2-methylthioethoxy)calix[4]arene, 25,26,27,28-(2-methylthioethoxy)calix[4]arene, and 5,11,17,23-tert-butyl-25,26,27,28-(2-(2-thiophenecarboxy)ethoxy)calix[4]arene have been prepared. The structure of 25,26,27,28-(2-methylthioethoxy)calix[4]arene has been verified by X-ray crystallography. The crystals with the empirical formula C(40)H(48)O(4)S(4) are monoclinic C2/c with a = 20.428(2) Å, b = 10.581(1) Å, c = 20.445(2) Å, beta = 118.461(5) degrees, Z = 4. These calix[4]arenes are effective extractants for transferring heavy metal ions from aqueous solution into chloroform. The extraction of Sn(II), Hg(II), Ag(I), Pd(II), Au(III), MeHg(II), Pb(II) and Cd(II) into chloroform with these calix[4]arenes is compared with that performed with 5,11,17,23-tert-butyl-25,26,27,28-(2-N,N-dimethyldithiocarbamoylethoxy)calix[4]arene, 25,26,27,28-(2,N,N-dimethyldithiocarbamoylethoxy)calix[4]arene, and 5,11,17,23-tert-butyl-25,26,27,28-(2-mercaptoethoxy)calix[4]arene.