Evaluating the Combination of Radioimmunotherapy and Immunotherapy in a Melanoma Mouse Model.

Immunotherapy has changed the oncology landscape during the last decade and become standard of care for several cancers. The combinations of immunotherapy with other treatment modalities are also being investigated. One of the challenges to investigate such combinations is to identify suitable mouse models for the pre-clinical experiments. In the past, we and other researchers showed that murine B16-F10 melanoma in C57Bl6 mice is refractory to treatment with immune checkpoint inhibitors. In this work we studied the suitability of an alternative syngeneic model, Cloudman S91 murine melanoma in DBA/2 mouse (DBA/2NCrl), to study the combination of immunotherapy targeting PD-1 and radioimmunotherapy targeting melanin. DBA/2 male and female mice were injected subcutaneously with 3-6 million Cloudman S91 cells. When the tumors reached ~150 mm3 volume, the animals were treated intraperitoneally with PBS (sham), h8C3 unlabeled (cold) antibody to melanin, immunotherapy with anti-PD-1 antibody, radioimmunotherapy with 213Bismuth (213Bi)-labeled h8C3 antibody, or several combinations of immunotherapy and radioimmunotherapy. Treatments with immunotherapy alone produced very modest effect on the tumor size, while combination therapy resulted in significant slowing down of the tumor growth, increased animal survival, and no decrease in animal body weight. We conclude that Cloudman S91 murine melanoma in DBA/2 mouse is a suitable model to evaluate combination of immunotherapy of melanoma with tangentially targeted treatments.

Preclinical testing of radiopharmaceuticals for novel applications in HIV, bacterial and fungal infectious diseases.

Antibiotics, antifungal and antiviral medications have traditionally been used in the management of infections. Due to widespread emergence of resistance to antimicrobial medications, and their side effects, there is a growing need for alternative approaches for management of such conditions. Antibiotic resistant bacterial pathogens are on the rise. A cure has not been achieved for viral infections like AIDS, while fungal and parasitic infections are constant threats to the health of general public. The incidence of opportunistic infections in immunocompromised individuals like HIV patients, patients receiving high dose steroids, chemotherapy patients, and organ transplant recipients is on the rise. Radioimmunotherapy (RIT) has the potential to be a suitable and viable therapeutic modality in the arena of infection management. Provided the target-associated antigen is expressed by the target cells and minimally or not expressed by other tissues, selective targeting of radiation to target sites can be theoretically accomplished with relative sparing normal tissues from radiation exposure. In our laboratory we successfully demonstrated the effectiveness of RIT for treating infectious diseases. We targeted murine cryptococcosis with a mAb to the Cryptococcus neoformans capsular glucuronoxylomannan labeled with Bismuth-213 ((213)Bi) or Rhenium-188 ((188)Re). We subsequently extended the applicability of RIT for treating bacterial and viral infections. One of the advantages of using RIT to treat infections as opposed to cancer is that, in contrast to tumor cells, cells expressing microbial antigens are antigenically very different from host tissues and thus provide the potential for exquisite specificity and low cross-reactivity. Ever increasing incidence of infectious pathologies, exhaustion of antimicrobial possibilities and rising drug resistance calls for use of alternative and novel therapeutic options and we believe RIT is the need of the hour to combat these infections.

Combined treatment of the experimental human papilloma virus-16-positive cervical and head and neck cancers with cisplatin and radioimmunotherapy targeting viral E6 oncoprotein.

BACKGROUND: Human papilloma virus (HPV) is implicated in >99% of cervical cancers and ∼40% of head and neck squamous cell carcinoma (HNSCC). We previously targeted E6 oncogene with (188)Rhenium-labelled monoclonal antibody (mAb) C1P5 to HPV16 E6 in cervical cancer and HNSCC. Intranuclear E6 can be accessed by mAbs in non-viable cells with leaky membranes. As radioimmunotherapy (RIT) efficacy depends on the availability of target protein-we hypothesised that pretreatment with cisplatin will kill some tumour cells and increase E6 availability for RIT. METHODS: Mice with subcutaneous HPV16+ cervical (CasKi) and HNSCC (2A3) tumours were pretreated with 0-7.5 mg kg(-1) per day cisplatin for 3 days followed by (188)Re-C1P5 and biodistribution was performed 24 h later. For RIT, the animals were treated with: 5 mg kg(-1) per day cisplatin for 3 days; or 5 mg kg(-1) per day cisplatin for 3 days followed 200 or 400µCi (188)Re-C1P5 mAb; or 200 or 400µCi (188)Re-C1P5 mAb; or left untreated, and observed for tumour growth for 24 days. RESULTS: Pretreatment with cisplatin increased the uptake of (188)Re-C1P5 in the tumours 2.5 to 3.5-fold and caused significant retardation in tumour growth for CasKi and 2A3 tumours in both RIT alone and cisplatin, and RIT groups in comparison with the untreated control and cisplatin alone groups (P<0.05). The combined treatment was more effective than either modality alone (P<0.05). CONCLUSION: Our study demonstrates that preceding RIT targeting E6 oncogene with chemotherapy is effective in suppressing tumour growth in mouse models of HPV16+ cancers.

Toward developing a universal treatment for fungal disease using radioimmunotherapy targeting common fungal antigens.

BACKGROUND: Previously, we demonstrated the ability of radiolabeled antibodies recognizing the cryptococcal polysaccharide capsule to kill Cryptococcus neoformans both in vitro and in infected mice. This approach, known as radioimmunotherapy (RIT), uses the exquisite ability of antibodies to bind antigens to deliver microbicidal radiation. To create RIT reagents which would be efficacious against all major medically important fungi, we have selected monoclonal antibodies (mAbs) to common surface fungal antigens such as heat shock protein 60 (HSP60), which is found on the surface of diverse fungi; beta (1,3)-glucan, which is a major constituent of fungal cell walls; ceramide which is found at the cell surface, and melanin, a polymer present in the fungal cell wall. METHODS: MAbs 4E12, an IgG2a to fungal HSP60; 2G8, an IgG2b to beta-(1,3)-glucan; and 6D2, an IgM to melanin, were labeled with the alpha particle emitting radionuclide 213-Bismuth ((213)Bi) using the chelator CHXA". B11, an IgM antibody to glucosylceramide, was labeled with the beta emitter 188-Rhenium ((188)Re). Model organisms Cryptococcus neoformans and Candida albicans were used to assess the cytotoxicity of these compounds after exposure to either radiolabeled mAbs or controls. RESULTS: (213)Bi-mAbs to HSP60 and to the beta-(1,3)-glucan each reduced the viability of both fungi by 80-100%. The (213)Bi-6D2 mAb to melanin killed 22% of C. neoformans, but did not kill C. albicans. B11 mAb against fungal ceramide was effective against wild-type C. neoformans, but was unable to kill a mutant lacking the ceramide target. Unlabeled mAbs and radiolabeled irrelevant control mAbs caused no killing. CONCLUSION: Our results suggest that it is feasible to develop RIT against fungal pathogens by targeting common antigens and such an approach could be developed against fungal diseases for which existing therapy is unsatisfactory.

Treating infections with ionizing radiation: a historical perspective and emerging techniques.

BACKGROUND: Widespread use and misuse of antibiotics have led to a dramatic increase in the emergence of antibiotic resistant bacteria, while the discovery and development of new antibiotics is declining. This has made certain implant-associated infections such as periprosthetic joint infections, where a biofilm is formed, very difficult to treat. Alternative treatment modalities are needed to treat these types of infections in the future. One candidate that has been used extensively in the past, is the use of ionizing radiation. This review aims to provide a historical overview and future perspective of radiation therapy in infectious diseases with a focus on orthopedic infections. METHODS: A systematic search strategy was designed to select studies that used radiation as treatment for bacterial or fungal infections. A total of 216 potentially relevant full-text publications were independently reviewed, of which 182 focused on external radiation and 34 on internal radiation. Due to the large number of studies, several topics were chosen. The main advantages, disadvantages, limitations, and implications of radiation treatment for infections were discussed. RESULTS: In the pre-antibiotic era, high mortality rates were seen in different infections such as pneumonia, gas gangrene and otitis media. In some cases, external radiation therapy decreased the mortality significantly but long-term follow-up of the patients was often not performed so long term radiation effects, as well as potential increased risk of malignancies could not be investigated. Internal radiation using alpha and beta emitting radionuclides show great promise in treating fungal and bacterial infections when combined with selective targeting through antibodies, thus minimizing possible collateral damage to healthy tissue. CONCLUSION: The novel prospects of radiation treatment strategies against planktonic and biofilm-related microbial infections seem feasible and are worth investigating further. However, potential risks involving radiation treatment must be considered in each individual patient.

Radioimmunotherapy of methicillin-resistant Staphylococcus aureus in planktonic state and biofilms.

BACKGROUND: Implant associated infections such as periprosthetic joint infections are difficult to treat as the bacteria form a biofilm on the prosthetic material. This biofilm complicates surgical and antibiotic treatment. With rising antibiotic resistance, alternative treatment options are needed to treat these infections in the future. The aim of this article is to provide proof-of-principle data required for further development of radioimmunotherapy for non-invasive treatment of implant associated infections. METHODS: Planktonic cells and biofilms of Methicillin-resistant staphylococcus aureus are grown and treated with radioimmunotherapy. The monoclonal antibodies used, target wall teichoic acids that are cell and biofilm specific. Three different radionuclides in different doses were used. Viability and metabolic activity of the bacterial cells and biofilms were measured by CFU dilution and XTT reduction. RESULTS: Alpha-RIT with Bismuth-213 showed significant and dose dependent killing in both planktonic MRSA and biofilm. When planktonic bacteria were treated with 370 kBq of 213Bi-RIT 99% of the bacteria were killed. Complete killing of the bacteria in the biofilm was seen at 185 kBq. Beta-RIT with Lutetium-177 and Actinium-225 showed little to no significant killing. CONCLUSION: Our results demonstrate the ability of specific antibodies loaded with an alpha-emitter Bismuth-213 to selectively kill staphylococcus aureus cells in vitro in both planktonic and biofilm state. RIT could therefore be a potentially alternative treatment modality against planktonic and biofilm-related microbial infections.

Structure-function analysis and therapeutic efficacy of antibodies to fungal melanin for melanoma radioimmunotherapy.

Metastatic melanoma remains difficult to treat despite recent approvals of several new drugs. Recently we reported encouraging results of Phase I clinical trial of radiolabeled with 188Re murine monoclonal IgM 6D2 to melanin in patients with Stage III/IV melanoma. Subsequently we generated a novel murine IgG 8C3 to melanin. IgGs are more amenable to humanization and cGMP (current Good Manufacturing Practice) manufacturing than IgMs. We performed comparative structural analysis of melanin-binding IgM 6D2 and IgG 8C3. The therapeutic efficacy of 213Bi- and 188Re-labeled 8C3 and its comparison with anti-CTLA4 immunotherapy was performed in B16-F10 murine melanoma model. The primary structures of these antibodies revealed significant homology, with the CDRs containing a high percentage of positively charged amino acids. The 8C3 model has a negatively charged binding surface and significant number of aromatic residues in its H3 domain, suggesting that hydrophobic interactions contribute to the antibody-melanin interaction. Radiolabeled IgG 8C3 showed significant therapeutic efficacy in murine melanoma, safety towards healthy melanin-containing tissues and favorable comparison with the anti-CTLA4 antibody. We have demonstrated that antibody binding to melanin relies on both charge and hydrophobic interactions while the in vivo data supports further development of 8C3 IgG as radioimmunotherapy reagent for metastatic melanoma.

Immunoconjugates of geldanamycin and anti-HER2 monoclonal antibodies: antiproliferative activity on human breast carcinoma cell lines.

BACKGROUND: HER2 is a membrane receptor whose overexpression is strongly associated with poor prognosis in breast carcinomas. Inhibition of HER2 activity can reduce tumor growth, which led to the development of Herceptin, an anti-HER2 monoclonal antibody (MAb) that is already in clinical use. However, the objective response rate to Herceptin monotherapy is quite low. HER2 activity can also be inhibited by the highly cytotoxic antibiotic geldanamycin (GA). However, GA is not used clinically because of its adverse toxicity. Our purpose was to enhance the inhibitory activity of anti-HER2 MAb by coupling it to GA. METHODS: We synthesized 17-(3-aminopropylamino)GA (17-APA-GA) and conjugated it to the anti-HER2 MAb e21, to form e21 : GA. The noninternalizing anti-HER2 MAb AE1 was used as a control. Internalization assays and western blot analyses were used to determine whether the anti-HER2 MAbs and their immunoconjugates were internalized into HER2-expressing cells and reduced HER2 levels. All statistical tests were two-sided. RESULTS: The immunoconjugate e21 : GA inhibited the proliferation of HER2-overexpressing cell lines better than unconjugated e21 (concentration required for 50% inhibition = 40 versus 1650 microg/mL, respectively). At 15 microg/mL, e21 : GA reduced HER2 levels by 86% within 16 hours, whereas unconjugated e21, 17-APA-GA, or AE1 : GA reduced HER2 levels by only 20%. These effects were not caused by release of 17-APA-GA from the immunoconjugate because immunoconjugates containing [(3)H]GA were stable in serum at 37 degrees C. Furthermore, e21 : GA did not significantly inhibit proliferation of the adult T-cell leukemia cell line HuT102, which is HER2 negative yet highly sensitive to GA. CONCLUSIONS: Our findings suggest that conjugating GA to internalizing MAbs enhances the inhibitory effect of the MAbs. This approach might also be applied in cellular targeting via growth factors and may be of clinical interest.

Targeted radionuclide therapies for pancreatic cancer.

Pancreatic malignancies, the fourth leading cause of cancer deaths, have an aggressive behavior with poor prognosis, resulting in a 5-year survival rate of only 4%. It is typically a silent malignancy until patients develop metastatic disease. Targeted radionuclide therapies of cancer such as radiolabeled peptides, which bind to the receptors overexpressed by cancer cells and radiolabeled antibodies to tumor-specific antigens provide a viable alternative to chemotherapy and external beam radiation of metastatic cancers. Multiple clinical trials of targeted radionuclide therapy of pancreatic cancer have been performed in the last decade and demonstrated safety and potential efficacy of radionuclide therapy for treatment of this formidable disease. Although a lot of progress has been made in treatment of pancreatic neuroendocrine tumors with radiolabeled (90)Y and (177)Lu somatostatin peptide analogs, pancreatic adenocarcinomas remain a major challenge. Novel approaches such as peptides and antibodies radiolabeled with alpha emitters, pre-targeting, bispecific antibodies and biological therapy based on the radioactive tumorlytic bacteria might offer a potential breakthrough in treatment of pancreatic adenocarcinomas.

Naive and radiolabeled antibodies to E6 and E7 HPV-16 oncoproteins show pronounced antitumor activity in experimental cervical cancer.

BACKGROUND: In spite of profound reduction in incidence, cervical cancer claims >275,000 lives annually. Previously we demonstrated efficacy and safety of radioimmunotherapy directed at HPV16 E6 oncoprotein in experimental cervical cancer. MATERIALS & METHODS: We undertook a direct comparison of targeting E7 and E6 oncoproteins with specific (188)Rhenium-labeled monoclonal antibodies in CasKi subcutaneous xenografts of cervical cancer cells in mice. RESULTS: The most significant tumor inhibition was seen in radioimmunotherapy-treated mice, followed by the unlabeled monoclonal antibodies to E6 and E7. No hematological toxicity was observed. Immunohistochemistry suggests that the effect of unlabeled antibodies is C3 complement mediated. CONCLUSION: We have demonstrated for the first time that radioimmunotherapy directed toward E7 oncoprotein inhibits experimental tumors growth, decreases E7 expression and may offer a novel approach to cervical cancer therapy.

Comparative cellular catabolism and retention of astatine-, bismuth-, and lead-radiolabeled internalizing monoclonal antibody.

UNLABELLED: Monoclonal antibodies (mAbs) labeled with alpha-emitting radionuclides such as (211)At, (212)Bi, (213)Bi, and (212)Pb (which decays by beta-emission to its alpha-emitting daughter, (212)Bi) are being evaluated for their potential applications for cancer therapy. The fate of these radionuclides after cells are targeted with mAbs is important in terms of dosimetry and tumor detection. METHODS: In this study, we attached various radionuclides that result in alpha-emissions to T101, a rapidly internalizing anti-CD5 mAb. We then evaluated the catabolism and cellular retention and compared them with those of (125)I- and (111)In-labeled T101. T101 was labeled with (211)At, (125)I, (205,6)Bi, (111)In, and (203)Pb. CD5 antigen-positive cells, peripheral blood mononuclear cells (PBMNC), and MOLT-4 leukemia cells were used. The labeled T101 was incubated with the cells for 1 h at 4 degrees C for surface labeling. Unbound activity was removed and 1 mL medium added. The cells were then incubated at 37 degrees C for 0, 1, 2, 4, 8, and 24 h. The activity on the cell surface that internalized and the activity on the cell surface remaining in the supernatant were determined. The protein in the supernatant was further precipitated by methanol for determining protein-bound and non-protein-bound radioactivity. Sites of internal cellular localization of radioactivity were determined by Percoll gradient centrifugation. RESULTS: All radiolabeled antibodies bound to the cells were internalized rapidly. After internalization, (205,6)Bi, (203)Pb, and (111)In radiolabels were retained in the cell, with little decrease of cell-associated radioactivity. However, (211)At and (125)I were released from cells rapidly ((211)At < (125)I) and most of the radioactivity in the supernatant was in a non-protein-bound form. Intracellular distribution of radioactivity revealed a transit of the radiolabel from the cell surface to the lysosome. The catabolism patterns of MOLT-4 cells and PBMNC were similar. CONCLUSION: (211)At catabolism and release from cells were somewhat similar to that of (125)I, whereas (205,6)Bi and (203)Pb showed prolonged cell retention similar to that of (111)In. These catabolism differences may be important in the selection of alpha-radionuclides for radioimmunotherapy.

Recent advances in radionuclide therapy.

A variety of radionuclides continue to be investigated and/or clinically used for different therapeutic applications in nuclear medicine. The choice of a particular radionuclide with regard to appropriate emissions, linear energy transfer, and physical half-life is dictated to a large extent by the character of the disease (eg, solid tumor or metastatic disease) and by the carrier used to selectively transport the radionuclide to the desired site. An impressive body of information has appeared in the recent literature that addresses many of these considerations. This article summarizes and discusses the many recent advances and the progress in the clinical applications of therapeutic radionuclides in relatively new and developing areas, such as radioimmunotherapy, peptide therapy, intravascular therapy to prevent restenosis, radiation synovectomy, and bone malignancy therapy. Projections are made as to the future directions and progress in these areas. The crucial issue of a reliable, year-round supply of new and emerging therapeutic radionuclides in quantities sufficient initially for research, and then for routine clinical use, is a very worthy goal which, in the United States, remains to be achieved.

The role of tin in the direct labelling of proteins with Rhenium-188.

In the process of direct labelling of proteins with 188Re, the influence of Sn(II) in the concentration range of 5 x 10(-4)-l mg/mL of protein was studied using 117mSn radiolabel in the presence of two transchelation buffers-sodium gluconate and sodium citrate. It was shown that Sn(II) readily binds to the thiol groups on the protein, and the fraction of Sn bound to the protein was 5 to 10 times higher in citrate than in gluconate for all Sn(II) concentrations studied. At saturation point of approximately 1 microgram (10(-8) M) Sn/mg protein in gluconate, 16% of the protein thiol groups were bound to Sn, and at approximately 2.4 micrograms (2 x 10(-8) M) in citrate, 32% of thiols were bound to Sn. A mechanism was proposed for the involvement of Sn(II) in labelling of pre-reduced proteins with 188Re via formation of protein-tin-188Re(V) reaction intermediate. It was further shown that the amount of Sn(II) in reaction mixture must exceed a certain level in order to achieve high labelling yields, and this level of Sn(II) was found to be different for citrate and gluconate buffers.

Spectrophotometric method for determination of bifunctional macrocyclic ligands in macrocyclic ligand-protein conjugates.

A simple spectrophotometric assay for determination of bifunctional polyazacarboxylate-macrocyclic ligands of different sizes that are conjugated to proteins has been developed for: 12-membered macrocycle DOTA (2-[4-nitrobenzyl]-1, 4, 7, 10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) and analogs, the 15-membered PEPA macrocycle (2-[4-nitrobenzyl]-1,4,7,10,13-pentaazacyclopentadecane-N,N',N'',N ''',N''''-pentaacetic acid), and the large 18-membered macrocycle HEHA (1,4,7,10,13,16-hexaazacyclooctadecane-N,N',N'',N''',N'''',N'''''- hexaacetic acid). The method is based on titration of the blue-colored 1:1 Pb(II)-Arsenazo II (AAIII) complex with the polyazacarboxylate macrocyclic ligand in the concentration range of 0-2.5 microM, wherein color change occurring upon transchelation of the Pb(II) from the AAIII to the polyazamacrocyclic ligand is monitored at 656 nm. The assay is performed at ambient temperature within 20 min without any interfering interaction between the protein and Pb(II)-AA(III) complex. Thus, this method also provides a ligand-to-protein ratio (L/P ratio) that reflects the effective number of ligands per protein molecule available to radiolabeling. The method is not suitable for 14-membered TETA macrocycle (2-[4-nitrobenzyl]-1, 4, 8, 11-tetraazacyclotetradecane N,N',N'',N'''-tetraacetic acid) because of low stability constant of Pb(II)-TETA complex. The method is rapid, simple and may be customized for other polyazacarboxylate macrocyclic ligands.

Rhenium-188 as an alternative to Iodine-131 for treatment of breast tumors expressing the sodium/iodide symporter (NIS).

The sodium-iodide symporter (NIS), which transports iodine into the cell, is expressed in thyroid tissue and was recently found to be expressed in approximately 80% of human breast cancers but not in healthy breast tissue. These findings raised the possibility that therapeutics targeting uptake by NIS may be used for breast cancer treatment. To increase the efficacy of such therapy it would be ideal to identify a radioactive therapy with enhanced local emission. The feasibility of using the powerful beta-emitting radiometal (188)Re in the form of (188)Re-perrhenate was therefore compared with 131I for treatment of NIS-expressing mammary tumors. In the current studies, using a xenografted breast cancer model induced by the ErbB2 oncogene in nude mice, (188)Re-perrhenate exhibited NIS-dependent uptake into the mammary tumor. Dosimetry calculations in the mammary tumor demonstrate that (188)Re-perrhenate is able to deliver a dose 4.5 times higher than (131)I suggesting it may provide enhanced therapeutic efficacy.

Synthesis, characterization, and evaluation of a novel bifunctional chelating agent for the lead isotopes 203Pb and 212Pb.

Radioisotopes of Pb(II) have been of some interest in radioimmunotherapy and radioimmunoimaging (RII). However, the absence of a kinetically stable bifunctional chelating agent for Pb(II) has hampered its use for these applications. 203Pb (T(1/2) = 52.02 h) has application potential in RII, with a gamma-emission that is ideal for single photon emission computerized tomography, whereas 212Pb (T(1/2) = 10 h) is a source of highly cytotoxic alpha-particles via its decay to its 212Bi (T(1/2) = 60 min) daughter. The synthesis of the novel bifunctional chelating agent 2-(4-isothiocyanotobenzyl)-1,4,7,10-tetraaza-1,4,7,10-tetra- (2-carbamoyl methyl)-cyclododecane (4-NCS-Bz-TCMC) is reported herein. The Pb[TCMC]2+ complex was less labile to metal ion release than Pb[DOTA]2- at pH 3.5 and below in isotopic exchange experiments. In addition to increased stability to Pb2+ ion release at low pH, the bifunctional TCMC ligand was found to have many other advantages over the bifunctional 1,4,7,10-tetraazacyclodocane-1,4,7,10-tetraacetic acid (DOTA) ligand. These include a shorter and more straightforward synthetic route, a more efficient conjugation reaction to a monoclonal antibody (mAb), with a higher chelate to protein ratio, a higher percent immuroreactivity, and a more efficient radiolabeling reaction of the mAb-ligand conjugate with 203Pb.

In vitro and in vivo characterization of 67Ga(3+) complexes with cis,cis-1,3,5-triamino-cyclohexane-N,N',N"-triacetic acid derivatives.

The aim of this study was to investigate the in vitro and in vivo performance of a 67Ga complex with cis,cis-1,3,5-triaminocyclohexane-N,N',N"-triacetic acid (tachta) as a potential ligand for use as a Ga(III) radiopharmaceutical for PET imaging. The radiolabeling procedure, electrophoretic properties, lipophilicity, acid stability, human serum stability and biodistribution in mice of 67Ga(tachta) were investigated. The 67Ga(tachta) complex forms at 10(-3) M tachta concentration at 40 degrees C in 100% yield; it is neutral, non-lipophilic, 90% stable at pH = 4 and 5 and 100% stable at pH = 6, for at least 8 d. Serum stability experiments demonstrated that at 5 hr 67Ga(tachta) exists in serum as a free complex. At 24 hr, 30% of 67Ga(tachta) is reversibly bound to transferrin-albumin fraction of serum, and that this percentage remains unchanged for a period of 4 d. Biodistribution in mice showed that 67Ga(tachta) rapidly clears via the kidneys from the body with less than 10% of injected activity left in the body at 3 hours and only 6% remaining after 24 hr. The complex also cleared rapidly from all of the major organs, with bone showing some slightly increased (1.15% ID/g) 24 hr accumulation, in comparison with the 3 hr time point. Based upon these data, 67Ga(tachta) may be considered as a candidate for developing new Ga(III) radiopharmaceuticals for PET.

188Re(V)-DMSA revisited: preparation and biodistribution of a potential radiotherapeutic agent with low kidney uptake.

Methods of preparation and biodistribution in mice of tin-free 99Tcm(V)-DMSA and 188Re(V)-DMSA, a potential matching pair of radiopharmaceuticals for diagnosis and therapy of certain cancers, are described. Preparation of tin-free 188Re(V)-DMSA (I) is based on reduction with either SO2-releasing compounds like Na2S2O4 (30 mg Na2S2O4, 10 mg DMSA, 1 mg L-ascorbic acid, 37 degrees C, 60 min incubation), Na2S2O5 (as before, 70 degrees C, 15 min incubation), or HBr (0.2 ml 48% HBr, 0.2 ml 7 M HCl, 10 mg DMSA, 1 mg L-ascorbic acid, 70 degrees C, 60 min incubation). I exhibits significantly lower kidney uptake than tin-containing 188Re(V)-DMSA (II) (2-3% and 49% injected dose per gram organ, 1 h post-injection, respectively). HPLC profiles of I and II are similar. DMSA excess in tin-free 188Re(V)-DMSA is not responsible for the low kidney uptake of I. High kidney uptake of II is explained by formation of a mixed 188Re(V)-Sn-DMSA complex in vivo. Age-linked bone uptake in mice dependent on the maturation of the bone is demonstrated for both I and II.

Development of a titanium tungstate-based 188W/188Re gel generator using tungsten of natural isotopic abundance.

The feasibility of developing titanium tungstate-based 188W/188Re gel generator using tungsten of natural isotopic abundance irradiated in a moderate flux reactor has been investigated. Influence of temperature, pH and eluent concentration on generator performance was studied. It was found that "post-formed" approach allows to construct gel generators with elution performance and 188Re elution yields very close to those of conventional alumina 188W/188Re generator. Curie-level 185W radionuclidic impurity presents a challenge during the processing of target material and subsequent elution of the generator. In the future use of semi-enriched with 186W target material (50-60% enrichment) would be beneficial in the development of titanium tungstate-based 188W/188Re gel generators.

Radiolabeling antibodies with holmium-166.

We report the preliminary results from radiolabeling of a chelate-conjugated antibody with 166Ho produced from the beta(-)-decay of 166Dy. Ho-166 was separated from mg quantities of Dy target by reverse phase ion-exchange chromatography employing a cation exchange HPLC column and 0.085 M alpha-HIBA at pH = 4.3 as eluent. Evaporation to dryness of 166Ho fraction (up to 25 mL) and thermal decomposition of alpha-HIBA yielded 166Ho in a dry state which was then solubilized in 0.5 mL of 0.1 M HCl. Subsequent radiolabeling of CHX-B-DTPA conjugated 135-14 monoclonal antibodies with purified 166 Ho was readily achieved with approximately 80% efficiency and with a specific activity of 3-4 mCi of 166Ho per mg of protein. 166Ho-antibody conjugates are stable with regards to transferrin challenge for a period of 50 h. Further, it was shown that any Fe3+ ions present in alpha-HIBA as an impurity interfere with the labeling.