Helen Heyerdahl

DISTRIBUTION OF 5‐AMINOLEVULINIC ACID‐INDUCED PORPHYRINS IN NODULOULCERATIVE BASAL CELL CARCINOMA

Abstract— Microscopic fluorescence photometry incorporating a light‐sensitive thermo‐electrically cooled charge‐coupled device (CCD) camera was employed to investigate the fluorescence distribution of 5‐aminolevulinic acid (ALA)‐induced porphyrins in 22 patients with a total number of 52 noduloul‐cerative basal cell carcinomas (BCC) after topical ALA application with or without dimethylsulfoxide (DMSO)/ethylenediaminetetraacetic acid (EDTA) or after intravenous administration of ALA. Both localization patterns and amounts of ALA‐induced porphyrins in the BCC were studied. The ALA‐induced porphyrins were localized only in the superficial layers of the noduloulcerative BCC lesions after topical application of 20% ALA alone for 3 h. However, both the penetration of ALA into deep lesions and the production of the ALA‐induced porphyrin fluorescence were increased after topical administration of 20% ALA and 20% DMSO/4% EDTA for 3 h. Prior treatment with 99% DMSO for 15 min further enhanced the ALA penetration into the BCC lesions after topical application of the ALA/DMSO/EDTA mixture and produced more ALA‐induced porphyrins by a factor of about three compared with those treated with ALA alone. The penetration of ALA into the deep BCC lesions could also be increased by prolonging the time of topical application of 20% ALA/4% EDTA to 29–48 h (without DMSO). Intravenous injection of ALA led to a more homogeneous distribution of the ALA‐derived porphyrins in the whole noduloulcerative BCC lesions.

Photodynamic therapy with 5-aminoolevulinic acid-induced porphyrins and DMSO/EDTA for basal cell carcinoma

Seven hundred sixty three basal cell carcinomas (BCCs) in 122 patients were treated by photodynamic therapy by 5-aminolevulinic acid (ALA) in cream topically applied, either alone, in combination with dimethyl sulphoxide (DMSO) and ethylenediaminetetraacetic acid disodium salt (EDTA), or with DMSO as a pretreatment. After 3 hours cream exposure 40 - 200 Joules/cm2 of 630 nm laser light was given. Fluorescence imaging of biopsies showed highly improved ALA penetration depth and doubled ALA-induced porphyrin production using DMSO/EDTA. Treatment response was recorded after 3 months. After a single treatment 90% of 393 superficial lesions responded completely, independent of using DMSO/EDTA. In 363 nodulo-ulcerative lesions the complete response rate increased from 67% to above 90% with DMSO/EDTA for lesions less than 2 mm thickness and from 34% to about 50% for lesions thicker than 2 mm. Recurrence rate observed during a follow-up period longer than 12 months was 2 - 5%. PDT of superficial thin BCCs with ALA-induced porphyrins and DMSO/EDTA equals surgery and radiotherapy with respect to cure rate and recurrence. Cosmetic results of ALA-based PDT seemed to be better than those after other therapies. In patients with the nevoid BCC syndrome the complete response rate after PDT was far lower.

Experimental α-particle radioimmunotherapy of breast cancer using 227Th-labeled p-benzyl-DOTA-trastuzumab

BackgroundThe aim of the present study was to explore the biodistribution, normal tissue toxicity, and therapeutic efficacy of the internalizing low-dose rate alpha-particle-emitting radioimmunoconjugate 227Th-trastuzumab in mice with HER2-expressing breast cancer xenografts.MethodsBiodistribution of 227Th-trastuzumab and 227Th-rituximab in nude mice bearing SKBR-3 xenografts were determined at different time points after injection. Tumor growth was measured after administration of 227Th-trastuzumab, 227Th-rituximab, cold trastuzumab, and saline. The toxicity of 227Th-trastuzumab was evaluated by measurements of body weight, blood cell, and clinical chemistry parameters, as well as histological examination of tissue specimens.ResultsThe tumor uptake reached peak levels of 34% ID/g (4.6 kBq/g) 3 days after injection of 400 kBq/kg of 227Th-trastuzumab. The absorbed radiation dose to tumor was 2.9 Gy, while it was 2.4 Gy to femur due to uptake of the daughter nuclide 223Ra in bone; the latter already explored in clinical phases I and II trials without serious toxicity. A significant dose-dependent antitumor effect was observed for dosages of 200, 400, and 600 kBq/kg of 227Th-trastuzumab but no effect of 400 and 600 kBq/kg 227Th-rituximab (non-tumor binding). No serious delayed bone marrow or normal organ toxicity was observed, but there was a statistical significant reduction in blood cell parameters for the highest-dose group of 227Th-trastuzumab treatment.ConclusionInternalizing 227Th-trastuzumab therapy was well tolerated and resulted in a dose-dependent inhibition of breast cancer xenograft growth. These results warrant further preclinical studies aiming at a clinical trial in breast cancer patients with metastases to bone.

Fractionated therapy of HER2-expressing breast and ovarian cancer xenografts in mice with targeted alpha emitting 227Th-DOTA-p-benzyl-trastuzumab

Background The aim of this study was to investigate therapeutic efficacy and normal tissue toxicity of single dosage and fractionated targeted alpha therapy (TAT) in mice with HER2-expressing breast and ovarian cancer xenografts using the low dose rate radioimmunoconjugate 227Th-DOTA-p-benzyl-trastuzumab. Methodology/Principal Findings Nude mice carrying HER2-overexpressing subcutaneous SKOV-3 or SKBR-3 xenografts were treated with 1000 kBq/kg 227Th-trastuzumab as single injection or four injections of 250 kBq/kg with intervals of 4–5 days, 2 weeks, or 4 weeks. Control animals were treated with normal saline or unlabeled trastuzumab. In SKOV-3 xenografts tumor growth to 10-fold size was delayed (p<0.01) and survival with tumor diameter less than 16 mm was prolonged (p<0.05) in all TAT groups compared to the control groups. No statistically significant differences were seen among the treated groups. In SKBR-3 xenografts tumor growth to 10-fold size was delayed in the single injection and 4–5 days interval groups (p<0.001) and all except the 4 weeks interval TAT group showed improved survival to the control groups (p<0.05). Toxicity was assessed by blood cell counts, clinical chemistry measurements and body weight. Transient reduction in white blood cells was seen for the single injection and 4–5 days interval groups (p<0.05). No significant changes were seen in red blood cells, platelets or clinical chemistry parameters. Survival without life threatening loss of body weight was significantly prolonged in 4 weeks interval group compared to single injection group (p<0.05) for SKOV-3 animals and in 2 weeks interval group compared with the 4–5 days interval groups (p<0.05) for SKBR-3 animals. Conclusions/Significance The same concentration of radioactivity split into several fractions may improve toxicity of 227Th-radioimmunotherapy while the therapeutic effect is maintained. Thus, it might be possible to increase the cumulative absorbed radiation dose to tumor with acceptable toxicity by fractionation of the dosage.

Assessment of long term radiotoxicity after treatment with the low dose rate {alpha}-particle emitting radioimmunoconjugate 227Th-rituximab

PurposeThe anti-CD20 antibody rituximab labelled with the α-particle-emitting radionuclide 227Th is of interest as a radiotherapeutic agent for treatment of lymphoma. Complete regression of human lymphoma Raji xenografts in 60% of mice treated with 200xa0kBq/kg 227Th-rituximab has been observed. To evaluate possible late side effects of 227Th-rituximab, the long-term radiotoxicity of this potential radiopharmaceutical was investigated.MethodsBALB/c mice were injected with saline, cold rituximab or 50, 200 or 1,000xa0kBq/kg 227Th-rituximab and followed for up to 1xa0year. In addition, nude mice with Raji xenografts treated with various doses of 227Th-rituximab were also included in the study. Toxicity was evaluated by measurements of mouse body weight, white blood cell (WBC) and platelet counts, serum clinical chemistry parameters and histological examination of tissues.ResultsOnly the 1,000xa0kBq/kg dosage resulted in decreased body weight of the BALB/c mice. There was a significant but temporary decrease in WBC and platelet count in mice treated with 400 and 1,000xa0kBq/kg 227Th-rituximab. Therefore, the no-observed-adverse-effect level (NOAEL) was 200xa0kBq/kg. The maximum tolerated activity was between 600 and 1,000xa0kBq/kg. No significant signs of toxicity were observed in histological sections in any examined tissue. There were significantly (pu2009<u20090.05), but transiently, higher concentrations of serum bile acids and aspartate aminotransferase in mice treated with either 227Th-rituximab or non-labelled antibody when compared with control mice. The maximum tolerated dose to bone marrow was between 2.1 and 3.5xa0Gy.ConclusionTherapeutically relevant dose levels of 227Th-rituximab were well tolerated in mice. Bone marrow suppression, as indicated by decrease in WBC count, was the dose-limiting radiotoxicity. These toxicity data together with anti-tumour activity data in a CD20-positive xenograft mouse model indicate that therapeutic effects could be obtained with relatively safe dosage levels of the radioimmunoconjugate.

Treatment of HER2-Expressing Breast Cancer and Ovarian Cancer Cells With Alpha Particle-Emitting 227Th-Trastuzumab

PURPOSEnTo evaluate the cytotoxic effects of low-dose-rate alpha particle-emitting radioimmunoconjugate (227)Th-p-isothiocyanato-benzyl-DOTA-trastuzumab ((227)Th-trastuzumab [where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]) internalized by breast and ovarian cancer cell lines in order to assess the potential of (227)Th-trastuzumab as a therapeutic agent against metastatic cancers that overexpress the HER2 oncogene.nnnMETHODS AND MATERIALSnClonogenic survival and cell growth rates of breast cancer cells treated with (227)Th-trastuzumab were compared with rates of cells treated with nonbinding (227)Th-rituximab, cold trastuzumab, and X-radiation. Cell growth experiments were also performed with ovarian cancer cells. Cell-associated radioactivity was measured at several time points, and the mean radiation dose to cells was calculated.nnnRESULTSnSKBR-3 cells got 50% of the mean absorbed radiation dose from internalized activity and 50% from cell surface-bound activity, while BT-474 and SKOV-3 cells got 75% radiation dose from internalized activity and 25% from cell surface-bound activity. Incubation of breast cancer cells with 2.5 kBq/ml (227)Th-trastuzumab for 1 h at 4°C, followed by washing, resulted in mean absorbed radiation doses of 2 to 2.5 Gy. A dose-dependent inhibition of cell growth and an increase in apoptosis were induced in all cell lines.nnnCONCLUSIONSnClinically relevant activity concentrations of (227)Th-trastuzumab induced a specific cytotoxic effect in three HER2-expressing cell lines. The cytotoxic effect of (227)Th-trastuzumab was higher than that of single-dose X-radiation (relative biological effectiveness = 1.2). These results warrant further studies of treatment of breast cancer and ovarian cancer with (227)Th-trastuzumab.

Modifications in dynamic contrast-enhanced magnetic resonance imaging parameters after α-particle-emitting 227Th-trastuzumab therapy of HER2-expressing ovarian cancer xenografts

PURPOSEnThe purpose of this study was to investigate the effect of α-particle-emitting (227)Th-trastuzumab radioimmunotherapy on tumor vasculature to increase the knowledge about the mechanisms of action of (227)Th-trastuzumab.nnnMETHODS AND MATERIALSnHuman HER2-expressing SKOV-3 ovarian cancer xenografts were grown bilaterally in athymic nude mice. Mice with tumor volumes 253 ± 36 mm(3) (mean ± SEM) were treated with a single injection of either (227)Th-trastuzumab at a dose of 1000 kBq/kg body weight (treated group, n=14 tumors) or 0.9% NaCl (control group, n=10 tumors). Dynamic T1-weighted contrast-enhanced magnetic resonance imaging (DCEMRI) was used to study the effect of (227)Th-trastuzumab on tumor vasculature. DCEMRI was performed before treatment and 1, 2, and 3 weeks after therapy. Tumor contrast-enhancement curves were extracted voxel by voxel and fitted to the Brix pharmacokinetic model. Pharmacokinetic parameters for the tumors that underwent radioimmunotherapy were compared with the corresponding parameters of control tumors.nnnRESULTSnSignificant increases of kep, the rate constant of diffusion from the extravascular extracellular space to the plasma (P<.05), and kel, the rate of clearance of contrast agent from the plasma (P<.01), were seen in the radioimmunotherapy group 2 and 3 weeks after injection, compared with the control group. The product of kep and the amplitude parameter A, associated with increased vessel permeability and perfusion, was also significantly increased in the radioimmunotherapy group 2 and 3 weeks after injection (P<.01).nnnCONCLUSIONSnPharmacokinetic modeling of MRI contrast-enhancement curves evidenced significant alterations in parameters associated with increased tumor vessel permeability and tumor perfusion after (227)Th-trastuzumab treatment of HER2-expressing ovarian cancer xenografts.

Comparing high LET 227Th- and low LET 177Lu-trastuzumab in mice with HER-2 positive SKBR-3 xenografts.

UNLABELLEDnThe aim of the present study was to compare the biodistribution, normal tissue toxicity and therapeutic effect of the alpha-particle emitting 227Th-trastuzumab and the beta-particle emitting 177Lu-trastuzumab in mice with HER2- expressing SKBR-3 breast cancer xenografts.nnnMETHODSnBiodistributions of the two radioimmunoconjugates were determined at different time points after i.v. injection. Inhibition of tumor growth was measured after single injection of 227Th-trastuzumab (200, 400, 600 or 1000 kBq/kg), 177Lu-trastuzumab (40 or 200 MBq/kg) or saline. The toxicity profiles were compared by measurements of body weight,clinical chemistry and hematological parameters, as well as histological examination of tissue specimens.nnnRESULTSn400 kBq/kg of 227Th-trastuzumab and 40 MBq/kg of 177Lu-trastuzumab both resulted in an absorbed radiation dose to tumor of approximately 3 Gy. A significant anti-tumor effect and increased survival were observed at injected dosages of 400-1000 kBq/kg of 227Th-trastuzumab and 200 MBq/kg of 177Lu-trastuzumab as compared to the saline control. When compared at the same therapeutic effect level (100% prolonged growth delay as compared to control) the absorbed radiation dose of 227Th-trastuzumab was 3 times lower than with 177Lu-trastuzumab, indicating a relative biological effect (RBE) of 2.8 for 227Th-trastuzumab. In contrast, when compared at the same temporary decrease of WBC count (50% decrease in number of white blood cells as compared to control), the growth delay was 3 times longer with 177Lutrastuzumab than with 227Th-trastuzumab, which indicates that the therapeutic index was three times higher for 177Lutrastuzumab than for 227Th-trastuzumab.nnnCONCLUSIONnIn this xenograft model the RBE was higher for 227Th-trastuzumab than for 177Lu-trastuzumab, while the therapeutic index of 177Lu-trastuzumab was superior to that of 227Th-trastuzumab.

Dosimetry and light distribution systems for photodynamic therapy at the Norwegian Radium Hospital

Photodynamic therapy has been used for treatment of malignant cutaneous lesions. Photosensitizer has been topically applied, 5-aminolevulinic acid, with light delivered at 630 nm. A study of response rate to varying light doses was performed, it might seem that light doses of 40 - 50 J/cm2 is sufficient for superficial lesions. For nodular lesions higher light doses are needed. Some patients with tumors in the gastrointestinal tract have been treated with systemic administration of 5-aminolevulinic acid and re-treated with Photofrin in case of unsatisfactory results. From the preliminary material available 5-aminolevulinic acid seems less promising as a photosensitizer for internal lesions compared to dermal lesions.

Measurements of sensitizer fluorescence in patients by means of an ordinary fluorescence spectrometer

An ordinary fluorescence spectrometer equipped with a dichroic mirror and fiber optics can be used to measure sensitizer fluorescence in tumors and normal tissues. Our findings with such an instrument showed that after oral administration of 60 mg/kg 5-aminolevulinic acid (ALA) mucosas and rectal adenomas fluoresced stronger than normal skin. Also basal cell carcinomas (BCCs) showed a stronger porphyrin fluorescence than skin after topical application of ALA. The porphyrin fluorescence of BCCs decayed rapidly during standard laser irradiation at 630 nm. The degradation rate of porphyrins in the tumor was approximately constant down to at least 0.3 mm below the tumor surface, in agreement with an optical penetration depth of more than 1 mm.