Saed Mirzadeh

Metallofullerene Drug Design

Abstract Endohedral lanthanide metallofullerenes are new molecules that may have useful medicinal properties. In particular, endohedral holmium metallofullerenes have been utilized in a model metallofullerene radiotracer study. The 165 Ho metallofullerenes were chemically functionalized to impart water solubility and then neutron activated to 166 Ho in order to determine their biodistribution and metabolism properties. The results have been evaluated for potential applications of lanthanide metallofullerenes as new diagnostic or therapeutic radiopharmaceuticals. Use of metallofullerenes in conventional diagnostic radiology (MRI contrast and X-ray imaging agents) has also been considered.

The continuing important role of radionuclide generator systems for nuclear medicine

In this review, the continuing importance and status of development of radionuclide generator systems for nuclear medicine are discussed. Radioisotope costs and availability are two important factors, and both nuclear reactors and accelerator facilities are required for production of the parent radioisotopes. Radionuclide generator research is currently focused on the development of generators which provide radioisotopes for positron emission tomography (PET) applications and daughter radioisotopes for various therapeutic applications which decay primarily by particle emission. Generator research continues to be influenced by developments and requirements of complementary technologies, such as the increasing availability of PET. In addition, the availability of a wide spectrum of tumor-specific antibodies, fragments, and peptides for radio-immunodiagnosis and radioimmunotherapy has stimulated the need for generator-derived radioisotopes. The advantages of treatment of arthritis of the synovial joints with radioactive particles (radiation synovectomy) may be expected to be of increasing importance as the elderly population increases, and many of these agents are prepared using generator-derived radioisotopes such as yttrium-90 and rhenium-188. Therapeutic use of the “in vivo generator” is a new approach, where the less radio-toxic parent radioisotope is used to prepare tissue-speciic therapeutic agents. Following in vivo site localization, decay of the parent provides the daughter for therapy at the target site. The principal foundation of most diagnostic agents will continue to require technetium-99m from the molybdenum-99/technetium-99m (“Moly”) generator. With the limited availability of nuclear reactors and facilities necessary for production and processing of fission 99mTc and the significant issues and problems associated with radioactive waste processing, however, the possibility of utilizing lower specific activity 99Mo produced from neutron activation of enriched 98Mo may become practical in the future.

Processing of reactor-produced 188W for fabrication of clinical scale alumina-based 188W/188Re generators

Abstract The traditional technique for processing of reactor-irradiated 186 W-enriched tungsten oxide (WO 3 ) targets involves formation of 188 W-sodium tungstate solutions by target dissolution in 0.1 M NaOH. Following long irradiations (> 21 days) in the ORNL High Flux Isotope Reactor (HFIR) the 186 WO 3 targets contain a NaOH-insoluble 188 W-labeled black solid (approx. 30–50% of total activity) which decreases the yield and specific activity of the processed 188 W (e.g. 5–6 mCi/mg 186 W for a 79-day irradiation). The black material is postulated to represent a “tungsten blue” insoluble polymeric form of tungsten oxide, which we have now found to dissolve in 0.1 M NaOH containing 5% sodium hypochlorite solution. Complete dissolution results in a significant increase in the yield and specific activity of sodium 188 W-tungstate. As an alternative approach, irradiated 186 W-enriched metal targets dissolve in sodium hydroxide solution by cautious addition of 188 W-tungstate solutions prepared from processing of such metal targets show no evidence of residual black insoluble material. Specific activity values for completely dissolved HFIR-irradiated 186 W targets have increased to 10 mCi/mg (43.5 days) and 12.9 mCi/mg (49.2 days). Large clinical scale (> 1 Ci) generators prepared from hypochlorite-processed 186 W oxide or peroxide-processed 186 W metal targets exhibit the expected 188 Re high yield and low 188 W breakthrough.

Vascular targeted radioimmunotherapy with 213Bi-An α-particle emitter

Abstract To destroy both tumor blood vessels and adjacent tumor cells, an α-particle emitter, 213 Bi, has been targeted with a monoclonal antibody (MAb) to vessels that feed lung tumors in mice. Animals, bearing approximately 100 EMT-6 carcinomas each of 50–400 cells in size in the lung, that were treated with 120 μCi of 213 Bi-MAb 201B were all cured of their disease. Animals treated when tumors were larger (10 3 –10 4 cells) had extended life spans, but a small number of residual tumors eventually killed the animals. Significant extension of life span was also induced with another tumor model—rat tracheal carcinoma growing in the lungs of SCID mice that were then treated with 136 μCi 213 Bi-MAb 201B. These studies indicate that attack of both blood vessels and tumor cells simultaneously is an effective mode of cancer treatment.

Reactor-produced radioisotopes from ORNL for bone pain palliation

The treatment of painful skeletal metastases is a common clinical problem, and the use of therapeutic radionuclides which localize at metastatic sites has been found to be an effective method for treatment of pain, especially for multiple sites for which the use of external beam irradiation is impractical. There are currently several metastatic-targeted agents radiolabeled with various therapeutic radionuclides which are in various stages of clinical investigation. Since neutron-rich radionuclides are produced in research reactors and often decay by emission of beta- particles, most radionuclides used for bone pain palliation are reactor-produced. Key examples of radionuclides produced by single neutron capture of enriched targets include rhenium-186 and samarium-153. In addition, generator systems are also of interest which provide therapeutic daughter radionuclides from the decay of reactor-produced parent radionuclides. One important example is rhenium-188, available from generators via decay of reactor-produced tungsten-188. Tin-117m is an example of a reactor-produced radionuclide which decays with the emission of low-energy conversion electrons rather than by beta- decay. Each of these agents and/or radionuclides has specific advantages and disadvantages, however, the ideal agent for bone pain palliation has not yet been identified. The goal of this paper is to briefly review the production and use of several reactor-produced radionuclides for bone pain palliation, and to discuss the role of the ORNL High Flux Isotope Reactor (HFIR) for the production of many of these radionuclides.

Gold coated lanthanide phosphate nanoparticles for targeted alpha generator radiotherapy.

Targeted radiotherapies maximize cytotoxicty to cancer cells. In vivo α-generator targeted radiotherapies can deliver multiple α particles to a receptor site dramatically amplifying the radiation dose delivered to the target. The major challenge with α-generator radiotherapies is that traditional chelating moieties are unable to sequester the radioactive daughters in the bioconjugate which is critical to minimize toxicity to healthy, non-target tissue. The recoil energy of the 225Ac daughters following α decay will sever any metal-ligand bond used to form the bioconjugate. This work demonstrates that an engineered multilayered nanoparticle-antibody conjugate can deliver multiple α radiations and contain the decay daughters of 225Ac while targeting biologically relevant receptors in a female BALB/c mouse model. These multi-shell nanoparticles combine the radiation resistance of lanthanide phosphate to contain 225Ac and its radioactive decay daughters, the magnetic properties of gadolinium phosphate for easy separation, and established gold chemistry for attachment of targeting moieties.

The fate of MAb-targeted Cd125mTe/ZnS nanoparticles in vivo

INTRODUCTION Nanoparticles (NP) have potential as carriers for drugs and radioisotopes. Quantitative measures of NP biodistribution in vivo are needed to determine the effectiveness of these carriers. We have used a model system of radiolabeled quantum dots to document the competition between efficient vascular targeting and interaction of the NP with the reticuloendothelial (RE) system. METHODS We have prepared (125m)Te-labeled CdTe NP that are capped with ZnS. Te-125m has a half-life and decay characteristics very similar to those for (125)I. The synthesized particles are stable in aqueous solution and are derivatized with mercaptoacetic acid and then conjugated with specific antibody. To evaluate specific targeting, we used the monoclonal antibody MAb 201B that binds to murine thrombomodulin expressed in the lumen of lung blood vessels. The MAb-targeted NP were tested for targeting performance in vivo using single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging, tissue autoradiography and standard organ biodistribution techniques. Biodistribution was also determined in mice that had been depleted of phagocytic cells by use of clodronate-loaded liposomes. RESULTS Cd(125m)Te/ZnS NP coupled with MAb 201B retained radioisotope and antibody activity and accumulated in lung (>400% injected dose [ID]/g) within 1 h of intravenous injection. Control antibody-coupled NP did not accumulate in lung (<10% ID/g) but accumulated in liver and spleen. Images from microSPECT/CT and autoradiography studies of the targeted NP document this specific uptake and demonstrate uniform distribution in lung with minor accumulation in liver and spleen. Within a few hours, a large fraction of lung-targeted NP redistributed to spleen and liver or was excreted. We hypothesized that NP attract phagocytic cells that engulfed and removed them from circulation. This was confirmed by comparing biodistribution of targeted NP in normal mice versus those depleted of phagocytic cells. In mice treated with clodronate liposomes, accumulation of NP in liver was reduced by fivefold, while accumulation in lung at 1 h was enhanced by approximately 50%. By 24 h, loss of the targeted NP from lung was inhibited by several-fold, while accumulation in liver and spleen remained constant. Thus, the treated mice had a much larger accumulation and retention of the NP at the target site and a decrease in dose to other organs except spleen. CONCLUSION Nanoparticles composed of CdTe, labeled with (125m)Te and capped with ZnS, can be targeted with MAb to sites in the lumen of lung vasculature. In clodronate-treated mice, which have a temporary depletion of phagocytic cells, accumulation in liver was reduced dramatically, whereas that in spleen was not. The targeting to lung was several-fold more efficient in clodronate-treated mice due to larger initial accumulation and better retention of the MAb-targeted NP at that site. This model system indicates that targeting of NP preparations is a competition between the effectiveness of the targeting agent and the natural tendency for RE uptake of the particles. Temporary inhibition of the RE system may enhance the usefulness of NP for drug and radioisotope delivery.

LaPO4 nanoparticles doped with actinium-225 that partially sequester daughter radionuclides.

Nanoscale materials have been envisioned as carriers for various therapeutic drugs, including radioisotopes. Inorganic nanoparticles (NPs) are particularly appealing vehicles for targeted radiotherapy because they can package several radioactive atoms into a single carrier and can potentially retain daughter radioisotopes produced by in vivo generators such as actinium-225 ((225)Ac, t(1/2) = 10 d). Decay of this radioisotope to stable bismuth-209 proceeds through a chain of short-lived daughters accompanied by the emission of four α-particles that release >27 MeV of energy. The challenge in realizing the enhanced cytotoxic potential of in vivo generators lies in retaining the daughter nuclei at the therapy site. When (225)Ac is attached to targeting agents via standard chelate conjugation methods, all of the daughter radionuclides are released after the initial α-decay occurs. In this work, (225)Ac was incorporated into lanthanum phosphate NPs to determine whether the radioisotope and its daughters would be retained within the dense mineral lattice. Further, the (225)Ac-doped NPs were conjugated to the monoclonal antibody mAb 201B, which targets mouse lung endothelium through the vasculature, to ascertain the targeting efficacy and in vivo retention of radioisotopes. Standard biodistribution techniques and microSPECT/CT imaging of (225)Ac as well as the daughter radioisotopes showed that the NPs accumulated rapidly in mouse lung after intravenous injection. By showing that excess, competing, uncoupled antibodies or NPs coupled to control mAbs are deposited primarily in the liver and spleen, specific targeting of NP-mAb 201B conjugates was demonstrated. Biodistribution analysis showed that ∼30% of the total injected dose of La((225)Ac)PO(4) NPs accumulated in mouse lungs 1 h postinjection, yielding a value of % ID/g >200. Furthermore, after 24 h, 80% of the (213)Bi daughter produced from (225)Ac decay was retained within the target organ and (213)Bi retention increased to ∼87% at 120 h. In vitro analyses, conducted over a 1 month interval, demonstrated that ∼50% of the daughters were retained within the La((225)Ac)PO(4) NPs at any point over that time frame. Although most of the γ-rays from radionuclides in the (225)Ac decay chain are too energetic to be captured efficiently by SPECT detectors, appropriate energy windows were found that provided dramatic microSPECT images of the NP distribution in vivo. We conclude that La((225)Ac)PO(4)-mAb 201B conjugates can be targeted efficiently to mouse lung while partially retaining daughter products and that targeting can be monitored by biodistribution techniques and microSPECT imaging.

Evaluation of 225Ac for Vascular Targeted Radioimmunotherapy of Lung Tumors

Several alpha particle emitting radioisotopes have been studied for use in radioimmunotherapy. Ac-225 has the potential advantages of a relatively long half life of 10 days, and a yield of 4 alpha emissions in its decay chain with a total energy release of approximately 28 MeV. A new, 12 coordination site chelating ligand, HEHA, has been chemically modified for coupling to targeting proteins without loss of chelating ability. HEHA was coupled with MAb 201B which binds to thrombomodulin and accumulates efficiently in murine lung. Ac-225 was bound to the HEHA-MAb 201B conjugate and injected into BALB/c mice bearing lung tumor colonies of EMT-6 mammary carcinoma. Biodistribution data at 1 and 4 h postinjection indicated that, as expected, 225Ac was delivered to lung efficiently (> 300% ID/g). The 225Ac was slowly released from the lung with an initial t1/2 = 49 h, and the released 225Ac accumulated in the liver. Injection of free HEHA was only partially successful in scavenging free 225Ac. In addition to the slow release of 225Ac from the chelate, data indicated that decay daughters of 225Ac were also released from the lung. Immediately after organ harvest, the level of 213Bi, the third alpha-decay daughter, was found to be deficient in the lungs and to be in excess in the kidney, relative to equilibrium values. Injected doses of 225Ac MAb 201B of 1.0 microCi, delivering a minimum calculated absorbed dose of about 6 Gy to the lungs, was effective in killing lung tumors, but also proved acutely radiotoxic. Animals treated with 1.0 microCi or more of the 225Ac radioconjugate died of a wasting syndrome within days with a dose dependent relationship. We conclude that the potential for 225Ac as a radioimmunotherapeutic agent is compromised not only by the slow release of 225Ac from the HEHA chelator, but most importantly by the radiotoxicity associated with decay daughter radioisotopes released from the target organ.

In Vivo Evaluation of Bismuth-Labeled Monoclonal Antibody Comparing DTPA-Derived Bifunctional Chelates

Among the radionuclides considered for radioimmunotherapy, alpha-emitters such as the bismuth isotopes, 212Bi and 213Bi, are of particular interest. The macrocyclic ligand, DOTA, has been shown to form stable complexes with bismuth isotopes. The kinetics of the complexation of bismuth with the DOTA chelate, however, are slow and impractical for use with 212Bi and 213Bi that have half-lives of 60.6 and 45.6 min. The study described herein compares six DTPA derived bifunctional chelates with the goal of identifying an alternative to the DOTA ligand for radiolabeling with bismuth. Radioimmunoconjugates comprised of MAb B72.3, each of the six DTPA chelates, and radiolabeled with 206Bi, which facilitated the evaluation due to its readily detectable gamma-emission. In vitro studies showed that each of the radioimmunoconjugates retained immunoreactivity that was comparable to its 125I-labeled counterpart. The 206Bi- and 125I-labeled immunoconjugates were then co-injected i.p. into normal athymic mice. Injection of Afree@ 206Bi demonstrated that the kidneys were the critical organ to evaluate for retention of bismuth in the chelate complex. Major differences were identified among the six preparations. The CHX-A and -B immunoconjugates were found to have 1) the lowest %ID/gm in the kidney; 2) a level of 206Bi in the kidney that was comparable to that of 125I-B72.3; and 3) no significant uptake of 206Bi evident in other organs such as bone, lung and spleen. The results described herein suggest that either of the cyclohexyl derivatives of DTPA may be suitable candidates for the labeling of immunoconjugates with alpha-emitting bismuth isotopes for radioimmunotherapeutic applications.