Gerard W. M. Visser

Targeting of a hydrophilic photosensitizer by use of internalizing monoclonal antibodies: A new possibility for use in photodynamic therapy.

Coupling of photosensitizers to tumor‐selective monoclonal antibodies ( MAbs ) is an attractive option for improving the selectivity of photodynamic therapy (PDT). For this purpose, hydrophilic sensitizers would be most suitable because of their solubility in water. However, such sensitizers are known to be ineffective in PDT, probably because they cannot readily pass the cell membrane and reach the critical intracellular target. We used the model compound TrisMPyP‐ΦCO2H, a hydrophilic porphyrin derivative, to test the hypothesis that hydrophilic photosensitizers might become of therapeutic value when directed into the tumor cell by use of internalizing MAbs. TrisMPyP‐ΦCO2H was conjugated using a labile ester. Conjugates showed no impairment of integrity on SDS‐PAGE, full stability in serum in vitro, and optimal immunoreactivity when the sensitizer :MAb ratio was ≤3. At higher molar ratios, the solubility of the conjugates decreased. In vitro internalization experiments showed that TrisMPyP‐ΦCONH–125I‐cMAb U36 and TrisMPyPΦCONH–125I‐mMAb 425 conjugates were internalized by A431 cells, in contrast to TrisMPyP‐ΦCONH–125I‐mMAb E48 conjugates. Data on the in vitro efficacy of PDT with MAb‐conjugated TrisMPyP‐ΦCO2H showed that the internalizing cMAb U36 and mMAb 425 conjugates were phototoxic to A431 cells, while the non‐internalizing E48 conjugate and the unconjugated sensitizer were not. Biodistribution data of conjugates with sensitizer:125I‐cMAb U36 ratios varying from 1:1 to 3:1 in tumor‐bearing nude mice revealed selective accumulation in the tumor. Conjugates with higher molar ratios were cleared more rapidly from the blood than the unconjugated 125I‐cMAb U36, resulting in lower tumor uptake but similar tumor‐to‐blood ratios. Our data suggest that hydrophilic photosensitizers might have therapeutic value when targeted to tumors by internalizing MAbs. Int. J. Cancer 88:108–114, 2000.

Comparison of aluminium (III) phthalocyanine tetrasulfonate-and meta-tetrahydroxyphenylchlorin-monoclonal antibody conjugates for their efficacy in photodynamic therapy in vitro

A challenge in photodynamic therapy (PDT) is to improve the tumour selectivity of the photosensitizers by using monoclonal antibodies (MAbs). With this aim, we developed MAb‐conjugates with the hydrophobic photosensitizer meta‐tetrahydroxyphenylchlorin (mTHPC) and with the hydrophilic sensitizer aluminium (III) phthalocyanine tetrasulfonate (AlPcS4). The capacity of these photoimmunoconjugates for selective targeting of squamous cell carcinoma (SCC) in vivo was demonstrated previously in SCC‐bearing nude mice. Preliminary in vitro PDT studies with the vulvar SCC cell line A431 showed promising phototoxicity with both sensitizers when coupled to the internalizing MAb 425. To rank the photosensitizers for their potential in photoimmunotherapy, we herein describe an extensive in vitro evaluation of mTHPC‐MAb and AlPcS4‐MAb conjugates. Both classes of conjugates were directly compared using 5 different SCC cell lines as target and 3 different MAbs (BIWA 4, E48 and 425) for tumour cell targeting. In contrast to free AlPcS4 (IC50 ≥ 700 nM), MAb‐conjugated AlPcS4 was found to be highly phototoxic in PDT in all 5 cell lines. AlPcS4‐BIWA 4 was most consistently effective with IC50 values ranging from 0.06–5.4 nM. mTHPC‐MAb conjugates were in general hardly effective. Phototoxicity (log IC50) of the AlPcS4‐MAb conjugates was found to be strongly correlated with their total cell binding capacity (internalized and surface bound) and to be less correlated with their internalization capacity. In conclusion, these data show a high potential of AlPcS4‐MAb conjugates in comparison to mTHPC‐MAb conjugates for use in PDT.

On the stereoselectivity of the reaction of [18F]acetylhypofluorite with glucals☆

Abstract The reaction of acetylhypofluorite with tri-O-acetyl- D -glucal results in products, besides the proposed 2-deoxy-2-fluoro-tetra-O-acetyl- D -glucose, formed by a trans- or a reversed-addition, as analysed by 1H-NMR. Consequently, the resulting end-product FDG (2-deoxy-2-fluoro- D -glucose) is contaminated with 15–20% of the corresponding mannose derivative. Reaction of acetylhypofluorite with D -glucal gives 2-deoxy-2-fluoro- D -mannose (FDM) as the main product, especially when this reaction is performed in acetic acid. A TLC-system was developed to separate FDG and FDM on monosodium-phosphate impregnated silica plates.

High dose rhenium-186-labeling of monoclonal antibodies for clinical application†

Rhenium‐186 (186Re) has ideal properties for adjuvant radioimmunotherapy (RIT). However, the lack of suitable methods for high dose 186Re labeling of monoclonal antibodies (MAbs) has hampered the use of 186Re in clinical RIT. After development of a chemically identical multistep procedure for the production of 186Re‐MAG3‐MAb and 99mTc/99Tc‐MAG3‐MAb conjugates for use as a matched pair, the authors now report on further progress to make this labeling method broadly applicable for high dose 186Re labeling.

Tissue distribution of [18F]-5-fluorouracil in mice: effects of route of administration, strain, tumour and dose

SummaryIn a study investigating the usefulness of 5-fluorouracil labelled with fluorine 18 ([18F]-5-FU) in cancer chemotherapy, the tissue distribution of the radiolabel was determined in mice at 2, 4 and 6 h after administration by varying several parameters such as the mode of administration, the strain of mouse, the presence of a tumour and the total dose of 5-FU. The tissue distribution of fluorine 18 after i. p. injection pointed to an altered behaviour of the drug and/or its metabolites when compared with values obtained after i.v. injection, but no difference was found in the accumulation of radiolabel in the tumour. A comparison of non-tumour-bearing BALB/c and C57Bl/6 mice revealed that the latter showed a higher radiolabel accumulation of the drug and its metabolites in the liver, kidney, intestines and coecum (P <0.05 at 2 and 4 h). In tumour-bearing mice, especially at 2 h, the tissue accumulation of radiolabel was found to be significantly higher than in non-tumour-bearing controls (in BALB/c mice bearing colon 26 carcinoma,P <0.05 for all tissues; in C57Bl/6 mice bearing colon 38 carcinoma,P <0.05 for the blood, lung, liver, kidney, large intestines, coecum and muscle). Finally, a comparison of injections of a tracer dose of [18F]-5-FU (2.5 mg/kg) vs a therapeutic dose (100 mg/kg) revealed only small differences in the accumulation of fluorine 18 in the liver and kidney.

Complete ablation of small squamous cell carcinoma xenografts with186Re-labeled monoclonal antibody E48

In previous studies we have shown that in mice bearing head and neck squamous cell carcinoma (HNSCC) xenografts, radioimmunotherapy (RIT) with186Re-labeled MAb E48 resulted in complete regressions in one-third of the tumors (followup >150 d). MAb E48 was labeled with186Re following a novel labeling procedure developed at our institute. The injected dose was 600 μCi, which was the maximum tolerated dose (MTD; <15% wt loss) in these studies. The mean size of the tumors was 140±60 mm3. To investigate whether the therapeutic efficacy of RIT in our xenograft model would be improved when treating smaller xenografts, mice bearing 2 HNSCC xenografts with a vol of 75±17 mm3 (number of mice,n=6; number of tumors,t=12) were treated with 600 μCi of186Re-labeled MAb E48 IgG. All tumors completely regressed and did not regrow during followup (>150 d). In all mice, weight loss did not exceed 10%. to obtain biodistribution data, mice bearing two xenografts with a vol of 58±31 mm3 were injected with 100 μCi of186Re-labeled MAb E48 IgG. The maximum uptake in blood was 26.4% injected dose/g (%ID·g−1) at 2 h pi and was 53.1%ID·g−1 in the tumor at d 7 pi. In normal tissues, no nonspecific accumulation was observed. Based on these biodistribution data, the absorbed cumulative radiation dose was calculated. The accumulated dose in blood and tumor was 2004 cGy and 8580 cGy, respectively. In other tissues, the dose was less than 8.1% of the dose delivered to tumor. These data implicate that RIT with186Re-labeled MAb E48 may be especially suited to be used as adjuvant therapy for the treatment of head and neck cancer patients with minimal residual disease.

Synthesis and evaluation of 99mTc/99Tc-MAG3-biotin conjugates for antibody pretargeting strategies

Four 99mTc-MAG3-biotin conjugates were synthesized to determine their potential use in antibody pretargeting strategies for radioimmunoscintigraphy (RIS). To use these 99mTc-MAG3-biotin conjugates as model compounds for 186Re-MAG3-biotin conjugates for radioimmunotherapy (RIT), nanomolar amounts of 99Tc were added as carrier to 99mTc. The biotin derivatives used for the preparation of the conjugates-biocytin, biotin hydrazide, biotinyl-piperazine, and biotinyl-diaminosuccinic acid-differed at the site that is regarded to be susceptible to hydrolysis by biotinidase present in human plasma. All four conjugates were produced with high radiochemical purity, were stable in PBS, and demonstrated full binding capacity to streptavidin. The 99mTc/99Tc-MAG3-labeled biotinyl-piperazine and biotinyl-diaminosuccinic acid conjugates were stable in mouse as well as human plasma, whereas the corresponding biocytin and biotin hydrazide conjugates were rapidly degraded. The biodistribution in nude mice at 30 min after injection was similar for all conjugates, and a rapid blood clearance and high intestinal excretion were both observed. It is concluded that the metabolic routing of a conjugate containing biotin and MAG3 is dominated by these two moieties. For this reason, MAG3-biotin conjugates do not seem suited for pretargeted RIT, for which quantitative and fast renal excretion is a prerequisite to minimize radiation toxicity. However, in a pretargeted RIS approach the 99mTc-MAG3-biotin conjugates might have potential.

A simplified synthesis of 18F-labelled cytosine- and uracil-nucleosides

Abstract 18 F-labelled cytosine- and uracil- nucleosides are synthesized starting from their unprotected parent nucleosides by reaction with acetylhypofluorite in acetic acid in an overall radiochemical yield of 20 and 30% respectively.

An optimized synthesis of 18F-labelled 5-fluorouracil and a reevaluation of its use as a prognostic agent

An optimized synthesis of 18F-labelled 5-fluorouracil (5-FU) is described. The biodynamics of this radiopharmaceutical were studied in nude mice bearing a 5-FU sensitive (colon 38 carcinoma) or a 5-FU resistant (R1-rhabdomyosarcoma) tumour. It was found that not the initial tumour uptake, but the efflux of the 18F activity from the tumour was correlated with the 5-FU sensitivity of the tumour.

In vitro antiproliferative and metabolic activity of eight novel 5-fluorinated uracil nucleosides

The in vitro growth inhibitory activity of eight novel 5-fluorinated uracil nucleosides was assessed in four human tumour cell lines, one of colon and three of head and neck squamous cell origin. These compounds are ribose or deoxyribose sugars with an acetoxy or an hydroxyl-group at the 6-position in the uracil part of the molecule, and their respective diastereoisomers. Antiproliferative effects were tested in an automated microculture assay based on the reduction of a tetrazolium dye, the MTT assay. Using a continuous drug exposure for four days, all novel nucleosides were more potent inhibitors of cell growth than 5-fluorouracil (5-FU). Most drugs were very active, having an IC50 value at least 10 fold lower than that of 5-FU, and this was consistently found for all cell lines. The 6-acetoxy compounds were generally more active than the compounds with a hydroxyl-group at the 6-position, while diastereoisomerism did not seem to influence the antiproliferative effect. Their capacity to inhibit the incorporation of tritiated deoxyuridine into DNA, which reflects the inhibition of thymidylate synthase, was measured in a short term assay. When tested at a concentration of 10(-6) mol/l, most of the compounds were found to block this incorporation more efficiently than 5-FU.