Preclinical evaluation of PAC1 targeting with radiolabeled Maxadilan.

There is an ongoing search for new tracers to optimize imaging of beta cell-derived tumors (insulinomas). The PAC1 receptor, expressed by insulinomas, can be used for targeting of these tumors. Here, we investigated whether radiolabeled maxadilan could be used for insulinoma imaging. Maxadilan was C- or N-terminally conjugated with DTPA (termed maxadilan-DPTA or DTPA-maxadilan respectively). BALB/c nude mice bearing subcutaneous INS-1 tumors were injected with either In-111-labeled maxadilan-DTPA or In-111-DTPA-maxadilan. Biodistribution studies were carried out at 1, 2 and 4 hours after injection and SPECT/CT imaging 1 and 4 hours after injection of maxadilan-DTPA-111In. Radiolabeling of maxadilan-DTPA (680 MBq/nmol) was more efficient than of DTPA-maxadilan (55 MBq/nmol). Conjugation with DTPA slightly reduced receptor binding affinity in vitro: IC50 values were 3.2, 21.0 and 21.0 nM for maxadilan, natIn-DTPA-maxadilan and maxadilan-DTPA-natIn respectively. Upon i.v. injection maxadilan-DTPA-111In accumulated specifically in INS-1 tumors (7.30 ± 1.87%ID/g) and in the pancreas (3.82 ± 0.22%ID/g). INS-1 tumors were clearly visualized by small animal SPECT/CT. In conclusion, this study showed that the high affinity of maxadilan to the PAC1 receptor was maintained after DTPA conjugation. Furthermore, radiolabeled maxadilan-DTPA accumulated specifically in INS-1 tumors and, therefore, may qualify as a useful tracer to image insulinomas.

Can 111In-RGD2 monitor response to therapy in head and neck tumor xenografts?

UNLABELLED: RGD (arginylglycylaspartic acid)-based imaging tracers allow specific imaging of integrin αvß3 expression, proteins overexpressed during angiogenesis; however, few studies have investigated the potential of these tracers to monitor responses of antiangiogenic or radiation therapy. In the studies presented here, (111)In-RGD2 was assessed for its potential as an imaging tool to monitor such responses to therapies. METHODS: DOTA-E-[c(RGDfK)]2 was radiolabeled with (111)In ((111)In-RGD2), and biodistribution studies were performed in mice with subcutaneous FaDu or SK-RC-52 xenografts after treatment with either antiangiogenic therapy (bevacizumab or sorafenib) or tumor irradiation (10 Gy). Micro-SPECT imaging studies and subsequent quantitative analysis were also performed. The effect of bevacizumab, sorafenib, or radiation therapy on tumor growth was determined. RESULTS: The uptake of (111)In-RGD2 in tumors, as determined from biodistribution studies, correlated well with that quantified from micro-SPECT images, and both showed that 15 d after irradiation (111)In-RGD2 uptake was enhanced. Specific or nonspecific uptake of (111)In-RGD2 in FaDu or SK-RC-52 xenografts was not affected after antiangiogenic therapy, except in head and neck squamous cell carcinoma 19 d after the start of sorafenib therapy (P < 0.05). The uptake of (111)In-RGD2 followed tumor volume in studies featuring antiangiogenic therapy. However, the uptake of (111)In-RGD2 in FaDu xenografts was decreased as early as 4 h after tumor irradiation, despite nonspecific uptake remaining unaltered. Tumor growth was inhibited after antiangiogenic or radiation therapy. CONCLUSION: Here, it is suggested that (111)In-RGD2 could allow in vivo monitoring of angiogenic responses after radiotherapy and may therefore prove a good clinical tool to monitor angiogenic responses early after the start of radiotherapy in patients with head and neck squamous cell carcinoma. Despite clear antitumor efficacy, antiangiogenic therapy did not alter tumor uptake of (111)In-RGD2, indicating that integrin expression was not altered.

Tyrosine kinase inhibitor sorafenib decreases 111In-girentuximab uptake in patients with clear cell renal cell carcinoma.

UNLABELLED: Tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of metastatic clear cell renal cell carcinoma (RCC). Although TKIs have demonstrated good clinical efficacy, the lack of complete responses, the chronic nature of the treatment, and the side effects are clear disadvantages. An interesting new approach in the treatment of clear cell RCC is antibody-mediated therapy with the chimeric anti-carbonic anhydrase IX (CAIX) antibody girentuximab (cG250). As the results of several girentuximab trials become available, the question arises of whether TKI treatment can be combined with girentuximab-based therapy. In this study, we assessed the effect of the widely used TKI sorafenib on the tumor-targeting potential of (111)In-labeled girentuximab. METHODS: (111)In-girentuximab imaging was performed on 15 patients suspected of having a renal malignancy, with surgery being part of their treatment plan. Of these, 10 patients were treated in a neoadjuvant setting with sorafenib (400 mg orally twice daily). Five patients received treatment during 1 wk, and 5 patients received treatment during 4 wk. In both sorafenib-treated groups, baseline and posttreatment tumor targeting of (111)In-girentuximab were compared. Surgery was performed 3 d after the last image acquisition. Five additional patients were included as a control group and had only a single (111)In-girentuximab injection and scintigraphy without any treatment. Distribution of (111)In-girentuximab was determined scintigraphically ex vivo in a 1-cm lamella of the resected tumorous kidney. Expression of CAIX and of the vascular marker CD31 was determined immunohistochemically on specimens of both tumor and normal kidney tissue. RESULTS: Treatment with sorafenib resulted in a marked decrease of (111)In-girentuximab uptake in the tumor in clear cell RCC patients, especially in the group treated for 4 wk (mean change in both sorafenib-treated groups, -38.4%; range, +9.1% to -79.4%). Immunohistochemical analysis showed markedly reduced CD31 expression and vessel density in the sorafenib-treated groups but no differences in CAIX expression between the sorafenib-treated groups and the nontreated patients. CONCLUSION: Treatment with sorafenib resulted in a treatment duration-dependent significantly decreased uptake of (111)In-girentumab in clear cell RCC lesions. These results indicate that the efficacy of antibody-mediated treatment or diagnosis modalities is hampered by TKI treatment.

111In-bevacizumab imaging of renal cell cancer and evaluation of neoadjuvant treatment with the vascular endothelial growth factor receptor inhibitor sorafenib.

UNLABELLED: Clear cell renal cell cancer (ccRCC) prominently expresses vascular endothelial growth factor-A (VEGF-A), and new treatment strategies for renal cell cancer (RCC) aim at the inhibition of VEGF-VEGF receptor signaling. This study explores the ability of (111)In-bevacizumab scintigraphy to depict RCC and to evaluate response to neoadjuvant treatment with sorafenib, a VEGF receptor inhibitor. METHODS: The ability to depict RCC with (111)In-bevacizumab scintigraphy was tested in 14 patients scheduled to undergo a tumor nephrectomy; of these, 9 RCC patients were treated in a neoadjuvant setting with sorafenib (400 mg orally twice a day). In the latter group, baseline and posttreatment (111)In-bevacizumab scans were compared. The intratumoral distribution of (111)In-bevacizumab was determined scintigraphically ex vivo in a 1-cm lamella of the resected tumorous kidney. Expression of VEGF-A, glucose transporter-1, carbonic anhydrase IX, α-smooth-muscle actin, and Ki67 was determined by immunohistochemistry and compared with the local concentration of (111)In-bevacizumab. Additionally, the VEGF-A content in tumor samples was determined quantitatively by enzyme-linked immunosorbent assay. RESULTS: In all 5 non-neoadjuvant-treated patients, preferential accumulation of (111)In-bevacizumab was observed in the tumors. All ccRCC lesions with enhanced (111)In-bevacizumab targeting expressed high levels of VEGF-A. Treatment with sorafenib resulted in a significant decrease of (111)In-bevacizumab uptake in the tumor in the patients with ccRCC (mean change, -60.5%; range, +1.5% to -90.1%). The decrease in uptake was due to destruction of the tumor neovasculature, whereas the VEGF-A expression remained intact. In the patient with papillary RCC, limited uptake without change after sorafenib was observed. CONCLUSION: RCC lesions were clearly delineated with (111)In-bevacizumab scintigraphy. Neoadjuvant treatment with sorafenib resulted in a significant decrease of (111)In-bevacizumab uptake in RCC. (111)In-bevacizumab scintigraphy can be an attractive biomarker for response and needs further study.

Hyperthermia and fibrinolytic therapy do not improve the beneficial effect of radioimmunotherapy following cytoreductive surgery in rats with peritoneal carcinomatosis of colorectal origin.

BACKGROUND AND OBJECTIVE: Cytoreductive surgery (CS) and heated intraperitoneal chemotherapy (HIPEC) are standard treatment for peritoneal carcinomatosis (PC) of colorectal cancer. Previously, we demonstrated that preclinical radioimmunotherapy (RIT) adjuvant to surgery in PC is a good alternative for HIPEC. Now we aimed to improve the effectiveness of RIT by combining it with whole-body hyperthermia (WBH) or fibrinolytic therapy. METHODS: Rats were inoculated intraperitoneally with colon carcinoma cells. Animals underwent CS, CS + WBH (40 degrees C, 3 hours), CS + RIT (74 MBq 177Lu-labeled MG1), or CS + WBH + RIT. In the second experiment, rats underwent CS, CS + RIT, CS + recombinant tissue plasminogen activator (rtPA, twice daily, 3 days), or CS + RIT + rtPA. RESULTS: Median survival after CS and CS + WBH was 34 and 37 days. Median survival after CS + RIT or CS + RIT + WBH was 63 and 86 days (p < 0.0003, p < 0.0006 compared to CS + WBH). Median survival after CS and CS + rtPA was 50 and 42 days (p = 0.1). Median survival was 106 days after CS + RIT and 103 days after CS + RIT + rtPA (p < 0.0001 compared to CS + rtPA). No difference was found between CS + RIT and CS + RIT + rtPA (p = 0.83). CONCLUSIONS: The application of WBH or rtPA in combination with adjuvant RIT after CS for the treatment of PC of colonic was feasible but did not significantly potentiate the efficacy of RIT.

The kinetic and biological activity of different loaded rhBMP-2 calcium phosphate cement implants in rats.

The healing of large bone defects can be improved by osteogenic bone graft substitutes, due to growth factor inclusion. A sustained release of these growth factors provides more efficient bioactivity when compared with burst release and might reduce the dose required for bone regeneration, which is desirable for socioeconomical and safety reasons. In this study, we compared different rhBMP-2 loadings in a sustained release system of CaP cement and PLGA-microparticles and were able to couple kinetic to biological activity data. Fifty-two rats received a critical-size cranial defect, which was left open or filled with the cement composites. The implants consisted of plain, high, and five-fold lower dose rhBMP-2 groups. Implantation time was 4 and 12 weeks. Longitudinal in vivo release was monitored by scintigraphic imaging of (131)I-labeled rhBMP-2. Quantitative analysis of the scintigraphic images revealed a sustained release of (131)I-rhBMP-2 for both doses, with different release profiles between the two loadings. However, around 70% of the initial dose was retained in both implant formulations. Although low amounts of rhBMP-2 were released (2.4 +/- 0.8 mug in 5 weeks), histology showed defect bridging in the high-dose implants. Release out of the low-dose implants was not sufficient to enhance bone formation. Implant degradation was limited in all formulations, but was mainly seen in the high-dose group. Low amounts of sustained released rhBMP-2 were sufficient to bridge critically sized defects. A substantial amount of rhBMP-2 was retained in the implants because of the slow release rate and the limited degradation.

Preclinical evaluation of technetium 99m-labeled P1827DS for infection imaging and comparison with technetium 99m IL-8.

BACKGROUND: The technetium 99 m (99mTc)-radiolabeled, leukocyte-avid peptide-glycoseaminoglycan complex, [99mTc]P1827DS, has been synthesized as an improved infection/inflammation imaging agent to [99mTc]P483H (LeukoTect, Diatide). In a phase I/II clinical trail, [99mTc]P483H images were equivalent to those obtained with 111In ex vivo labeled leukocytes. However, there was physiologic accumulation of radioactivity in the body that could hamper interpretation of the images. In this study, the potential of [99mTc]P1827DS for infection imaging was assessed in comparison with [99mTc]P483H and the well-described imaging agent [99mTc] hydrazinonicotinamide (HYNIC)-interleukin 8 (IL-8). METHODS: The binding of [99mTc]P1827DS to human blood cell was studied in vitro. A rabbit Escherichia coli infection model was used to perform the biodistribution and imaging studies with [99mTc]P1827DS, [99mTc]P483H and [99mTc]HYNIC-IL-8. RESULTS: [99mTc]P1827DS binds to leukocytes but not to erythrocytes. The leukocyte binding was not saturable up to an investigated concentration of 10 microM. The accumulation of [99mTc]P1827/DS at the infection site strongly depends on the P1827/DS ratio and was optimal at a molar ratio of 10:1. [99mTc]P1827DS shows improved biodistribution over [99mTc]P483H with similar uptake at the infection site. Abscess uptake of [99mTc]HYNIC-IL-8 was approximately three times higher than that of [99mTc]P1827DS. [99mTc]HYNIC-IL-8 showed high accumulation in the kidneys, whereas [99mTc]P1827DS showed high lung uptake and slightly higher accumulation in the liver and spleen. CONCLUSION: [99mTc]P1827DS is a potential new inflammation imaging agent, which clearly visualized the abscess in the rabbit E. coli infection model and showed improved biodistribution compared to [99mTc]P483H. However, the infection uptake and biodistribution of [99mTc]P1827DS is not superior to that of [99mTc]HYNIC-IL-8 in this animal model.

Enzymosomes with surface-exposed superoxide dismutase: in vivo behaviour and therapeutic activity in a model of adjuvant arthritis.

Acylated Superoxide Dismutase (Ac-SOD) enzymosomes, liposomal enzymatic systems expressing catalytic activity in the intact form, were previously characterized. The main scope of the present work was to investigate the biological behaviour of Ac-SOD inserted in the lipid bilayer of liposomes, in comparison with SOD located in the aqueous compartment of liposomes. Two types of liposomes were used: conventional liposomes presenting an unmodified external surface and long circulating liposomes coated with poly (ethylene glycol) (PEG). Liposomal formulations of Ac-SOD and SOD were prepared and labelled with indium-111 and their in vivo fate compared. Data obtained led us to the conclusion that, for liposomes coated with PEG the in vivo fate was not influenced by the insertion of Ac-SOD in the lipid bilayers. The potential therapeutic effect of Ac-SOD enzymosomes was compared with SOD liposomes in a rat model of adjuvant arthritis. A faster anti-inflammatory effect was observed for Ac-SOD enzymosomes by monitoring the volume of the inflamed paws. The present results allowed us to conclude that Ac-SOD enzymosomes are nano-carriers combining the advantages of expressing enzymatic activity in intact form and thus being able to exert therapeutic effect even before liposomes disruption, as well as acting as a sustained release of the enzyme.

Radioimmunotherapy is an effective adjuvant treatment after cytoreductive surgery of experimental colonic peritoneal carcinomatosis.

UNLABELLED: Because tumor targeting with radiolabeled monoclonal antibodies is more efficient in small lesions, radioimmunotherapy is considered most suitable for minimal or microscopic residual disease. The aim of the present studies was to assess the efficacy of adjuvant radiommunotherapy using radiolabeled monoclonal antibodies after cytoreductive surgery in rats with peritoneal carcinomatosis of colonic origin. METHODS: We used a tumor model, in which peritoneal carcinomatosis was induced by intraperitoneal inoculation of CC-531 colon carcinoma cells in Wag/Rij rats. The biodistribution of the (125)I-/(111)In-labeled anti-CC-531 MG1 monoclonal antibody after intraperitoneal administration was assessed. Subsequently, the efficacy of (177)Lu-MG1 (74 MBq per rat) was compared with that of unlabeled MG1 or phosphate-buffered saline-bovine serum albumin in this model. Finally, rats with resectable intraperitoneal CC-531 tumors were subjected to exploratory laparotomy only, cytoreductive surgery only, exploratory laparotomy + radiommunotherapy (56 MBq (177)Lu-MG1 per rat), or cytoreductive surgery + radiommunotherapy. Survival was the primary endpoint. RESULTS: Both (125)I- and (111)In-labeled MG1 preferentially accumulated in intraperitoneal CC-531 tumors. The uptake of (111)In-MG1 in tumor was higher than that of (125)I-MG1 (4.1 +/- 2.3 %ID/g vs. 1.1 +/- 0.5 %ID/g [%ID/g is percentage of injected dose per gram], 72 h after injection; P = 0.053). Radiommunotherapy with 74 MBq (177)Lu-MG1 almost completely eradicated tumor growth, whereas unlabeled MG1 had no effect. In the surgery study, both cytoreductive surgery and radiommunotherapy were well tolerated. The median survival of the control rats that underwent exploratory laparotomy only was 41 d. The median survival of the rats that were treated with cytoreductive surgery only, exploratory laparotomy + radiommunotherapy, or cytoreductive surgery + radiommunotherapy was 51 d (P = 0.05 compared with control rats), 61.5 d (P = 0.03), and 88 d (P = 0.0001), respectively, which suggests an additive effect of both treatment modalities. There was a highly significant trend toward improved survival of cytoreductive surgery + radiommunotherapy compared with both monotherapies (P = 0.0004). CONCLUSION: This study provides proof of principle that radiommunotherapy can be an effective treatment modality when applied as an adjuvant treatment after resection of tumors with a high risk of recurrence, such as after cytoreductive surgery of peritoneal carcinomatosis.

Peritoneal carcinomatosis of colorectal origin: incidence and current treatment strategies.

OBJECTIVE: To review the literature with regard to the incidence and prognostic significance of peritoneal seeding during surgery for primary colorectal cancer (CRC), the incidence of intraperitoneal recurrence of CRC, and the current treatment strategies of established PC of colorectal origin, with special focus on cytoreductive surgery and intraperitoneal chemotherapy (IPEC). SUMMARY BACKGROUND DATA: Although hematogenous dissemination forms the greatest threat to patients with CRC, peritoneal carcinomatosis (PC), presumably arising from intraperitoneal seeding of cancer cells, is a relatively frequent event in patients with recurrent CRC. METHODS: The PubMed and Medline literature databases were searched for pertinent publications regarding the incidence and prognostic significance of exfoliated tumor cells in the peritoneal cavity during curative surgery for primary CRC, the incidence of intraperitoneal recurrence of CRC, and the therapeutic results of systemic chemotherapy or cytoreductive surgery followed by IPEC. RESULTS: The incidence of peritoneal seeding during potentially curative surgery for primary CRC, as reported in 12 patient series, varied widely, from 3% to 28%, which may be explained by differences in methods to detect tumor cells. PC is encountered in approximately 7% of patients at primary surgery, in approximately 4% to 19% of patients during follow-up after curative surgery, in up to 44% of patients with recurrent CRC who require relaparotomy, and in 40% to 80% of patients who succumb to CRC. The reported median survival after systemic 5-fluorouracil-based chemotherapy for PC varies from 5.2 to 12.6 months. Median survival after aggressive cytoreductive surgery followed by (hyperthermic) IPEC in selected patients, as reported in 16 patient series, tends to be better and varies from 12 to 32 months at the cost of morbidity and mortality rates of 14% to 55% and 0% to 19%, respectively. One randomized controlled trial has been published confirming the superiority of aggressive surgical cytoreduction and intraperitoneal chemotherapy over strictly palliative treatment. CONCLUSIONS: Peritoneal seeding of cancer cells possibly leading to PC is a rather common phenomenon in patients with CRC. Cytoreductive surgery and adjuvant (hyperthermic) IPEC have been shown to be efficacious in selected patients and should therefore be considered in patients with resectable PC of colorectal origin.

Radioimmunotherapy with [131I]cG250 in patients with metastasized renal cell cancer: dosimetric analysis and immunologic response.

PURPOSE: A study was designed to define the therapeutic efficacy, safety, and toxicity of two sequential high-dose treatments of radioimmunotherapy with [131I]cG250 in patients with metastasized renal cell carcinoma. Here, we report the dosimetric analysis and the relationship between the development of a human antichimeric antibody response and altered pharmacokinetics. EXPERIMENTAL DESIGN: Patients (n = 29) with progressive metastatic renal cell carcinoma received a low dose (222 MBq) of [131I]cG250 for dosimetric analysis, followed by the first radioimmunotherapy with 2,220 MBq/m2 [131I]cG250 (n = 27) 1 week later. If no grade 4 hematologic toxicity was observed, a second low dose of [131I]cG250 (n = 20) was given 3 months later. Provided that no accelerated blood clearance was observed, a second radioimmunotherapy of [131I]cG250 was administered at an activity-dose level of 1,110 MBq/m2 (n = 3) or 1,665 MBq/m2 (n = 16). After each administration, whole-body images were obtained and the pharmacokinetics and the development of human antichimeric antibody responses were determined. Radiation-absorbed doses were calculated for whole body, red marrow, organs, and metastases. RESULTS: No correlation was found between hematologic toxicity and radiation-absorbed dose to the whole body or bone marrow, nor administered activity (MBq and MBq/kg). The tumor-absorbed doses varied largely. An inverse relation between tumor size and radiation-absorbed dose was found. Most tumor lesions received <10 Gy, whereas only lesions <5 g absorbed >50 Gy. A relatively high number of patients developed a human antichimeric antibody response (8 of 27) with altered pharmacokinetics, hampering additional radioimmunotherapies in four of these patients. CONCLUSIONS: Dosimetric analysis did not adequately predict the degree of bone marrow toxicity. When human antichimeric antibody developed, the rapid clearance of radioactivity from the blood and body prohibited further treatment. According to the calculated absorbed dose in metastatic lesions, future radioimmunotherapy studies with radiolabeled cG250 should aim at treatment of small-volume disease or treatment in an adjuvant setting.

Lack of efficacy of two consecutive treatments of radioimmunotherapy with 131I-cG250 in patients with metastasized clear cell renal cell carcinoma.

PURPOSE: A previous activity dose-escalation study using 131I-labeled chimeric monoclonal antibody cG250 in patients with progressive metastatic renal cell carcinoma (RCC) resulted in occasional therapeutic responses. The present study was designed to determine the safety and therapeutic efficacy of two sequential high-dose treatments with 131I-cG250. PATIENTS AND METHODS: Patients (n = 29) with progressive metastatic RCC received a low dose of (131)I-cG250 for assessment of preferential targeting of metastatic lesions, followed by the first radioimmunotherapy (RIT) with 2220 MBq/m2 131I-cG250 (n = 27) 1 week later. If no grade 4 hematologic toxicity was observed, a second low-dose 131I-cG250 (n = 20) was given 3 months later. When blood clearance was not accelerated, a second RIT of 131I-cG250 was administered at an activity-dose of 1110 MBq/m2 (n = 3) or 1665 MBq/m2 (n = 16). Patients were monitored weekly for toxicity, and tumor size was evaluated by computed tomography once every 3 months intervals. RESULTS: The maximum-tolerated dose (MTD) of the second RIT was 1,665 MBq/m2 because of dose-limiting hematological toxicity. Based on an intention-to-treat analysis, after two RIT treatments, the disease stabilized in five of 29 patients, whereas it remained progressive in 14 of 29 patients. Two patients received no RIT, and eight of 29 received only one 131I-cG250 RIT because of grade 4 hematologic toxicity, formation of human antichimeric antibodies, or disease progression. CONCLUSION: In patients with progressive end-stage RCC, the MTD of the second treatment was 75% of the MTD of the first RIT. In the majority of patients, two cycles of 131I-cG250 could be safely administered without severe toxicity. No objective responses were observed, but occasionally two RIT doses resulted in stabilization of previously progressive disease.