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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 (AlPcS(4)). 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 AlPcS(4)-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 AlPcS(4) (IC(50) > or = 700 nM), MAb-conjugated AlPcS(4) was found to be highly phototoxic in PDT in all 5 cell lines. AlPcS(4)-BIWA 4 was most consistently effective with IC(50) values ranging from 0.06-5.4 nM. mTHPC-MAb conjugates were in general hardly effective. Phototoxicity (log IC(50)) of the AlPcS(4)-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 AlPcS(4)-MAb conjugates in comparison to mTHPC-MAb conjugates for use in PDT., (Copyright 2002 Wiley-Liss, Inc.)

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-PhiCO(2)H, 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-PhiCO(2)H 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

Distribution of the photosensitizer Foscan (meta-tetrahydroxyphenylchlorin, mTHPC), after i.v. or i.p. injection, was investigated in Wag/Rij rats bearing i.p. tumours. These results were compared with the efficacy of mTHPC-mediated photodynamic therapy for illumination intervals of 4 hr to 3 days. For the distribution experiments a single tumour (CC53I colon carcinoma) was implanted intra-abdominally in a fat pad, or a cell suspension (1 x 10(6) CC531 cells) was injected into the peritoneal cavity, which results in a dissemination of tumour nodules on the peritoneum. 14C-mTHPC was not selectively taken up in the single-tumour model after i.v. or i.p. injection, but higher concentrations were achieved for i.p. administration. For this tumour model the concentration ratios between tumour and normal tissue never exceeded a value of 3. In the disseminated-tumour model, an uptake of up to 40% of the injected dose was found per gram tumour at 4 hr after an i.p. injection and this resulted in very high (> 14) concentration ratios of tumour to normal tissues. Low uptake was found after the i.v. injection route (1% of the injected dose per gram tumour) with lower tumour/normal tissue ratios (<8). The efficacy of i.p. photodynamic therapy (IPPDT) was evaluated using the single-tumour model only. The lower abdomen was illuminated at 4 hr to 3 days after mTHPC, and tumour size was repeatedly measured via a small laparoscopy. Significant delay in tumour regrowth was achieved for 6 J x cm-2 at 1 day after i.v., or at 4 hr after i.p. mTHPC (p values 0.019 and 0.045 respectively). Response to PDT, of tumours implanted in the fat pad, was not greater for i.p. administration of the photosensitizer and there was a poor correlation between times of maximum drug uptake in tumours and optimal illumination times for PDT efficacy.

Preferential retention of photosensitizers in tumours has always been one of the major goals in the search for new photosensitizers and has determined the design of clinical trials with respect to the interval between drug administration and illumination. The purpose of this study was to investigate the importance of tumour and plasma concentrations of Foscan (mTHPC, meta-tetrahydroxyphenylchlorin) in relation to PDT effect. Both pharmacokinetic and tumour response studies were carried out in mice bearing s.c. RIF1 tumours. mTHPC was injected in 1 or 2 doses of 0.3 mg x kg-1. For distribution studies, 14C-labelled mTHPC was given 5 min to 48 hr before determination of plasma and tumour drug levels. Non-labelled sensitizer was used to determine the PDT efficacy for illumination at 5 min to 48 hr after drug administration. PDT efficacy was greatest for illumination at 1 to 3 hr, and for an interval of 48 hr there was no significant tumour-growth delay. In contrast, mTHPC tumour drug levels reached a maximum 6 hr after injection and remained high for 48 hr. A comparison of pharmacokinetics and response studies revealed no significant correlation between tumour mTHPC levels and tumour response. There was, however, a significant correlation between plasma drug levels and tumour response for time intervals of 1 to 48 hr. This association may imply that PDT protocols should use shorter drug-light intervals in combination with lower drug doses. This would increase safety and decrease the extent and duration of normal tissue photosensitization.

The new photosensitizer, meso-tetrahydroxyphenylchlorin (mTPHC) was compared with Photofrin in the murine RIF1 tumour and in normal mouse skin. A range of mTHPC or Photofrin doses were given at intervals of 1 hr to 7 days before illumination. mTHPC-PDT resulted in much higher tumour phototoxicity with longer regrowth delays and more cures. The RIF1 tumour could be effectively treated with 30 J cm-1 (interstitial illumination) at 1 day after mTHPC, whereas 4 to 13 times higher light doses were required with Photofrin for an equivalent anti-tumour effect. High doses of mTHPC also caused more skin phototoxicity (superficial illumination) than Photofrin for the 1-day illumination interval. Evaluating both tumour and normal skin photosensitization, the largest therapeutic gain factor (TGF) for mTHPC-PDT was achieved with a low drug dose (0.15 mg kg-1) at 1 day before illumination (TGF = 5.6, relative to Photofrin PDT). The duration of cutaneous photosensitivity for mTHPC was shorter than for Photofrin. The light dose required to produce a desquamation response in 50% of the animals increased more than 20-fold over the period 1 to 7 days after high doses of mTHPC, whereas this light dose only increased by a factor of 2 from 1 to 7 days after Photofrin. The large therapeutic gains seen for mTHPC-mediated PDT compared to Photofrin, plus the rapid fading of skin photosensitization, suggest that mTHPC is a potent photosensitizer suitable for clinical testing.

The tumoricidal effect of interstitial photodynamic therapy (IPDT) using Photofrin was found to increase when combined with the bioreductive alkylating agent mitomycin C (MMC) and, to a lesser extent, with the indoloquinone EO9. When MMC was given prior to IPDT or RIF1 tumours, the light dose required for a given regrowth time or for 50% cure was reduced by a factor of 2 compared with IPDT alone. MMC given immediately after illumination did not increase the effects of IPDT, although MMC plus illumination without photosensitizer produced a significant increase in regrowth time compared with MMC or light alone. Combination of IPDT with EO9, given directly before illumination, only marginally increased the tumour regrowth times at non-toxic doses. These results demonstrate that combining IPDT with MMC greatly improves the tumour response. Factors such as PDT-induced hypoxia, pH changes, temperature increases and production of toxic reactive oxygen species by both drugs may play a role in the enhanced MMC cytotoxicity.

The influence of photodynamic therapy (PDT) on vascular perfusion was investigated in 2 s.c. mouse tumours, a radiation-induced fibrosarcoma (RIF I) and a squamous-cell carcinoma (SCCVII). The 86Rb extraction technique was used to measure changes in perfusion relative to cardiac output at various intervals after interstitial PDT. Control groups showed that vascular perfusion in the RIF I tumours decreased with increasing tumour size. For both tumours, of constant size, vascular perfusion decreased to less than 10% of control values within 5 min after high PDT doses. Significant decreases in vascular perfusion were also seen after lower, sub-curative doses. Thereafter there was slow recovery towards control levels. Photofrin given at shorter intervals before illumination generally resulted in even larger decreases in tumour perfusion, and slower recovery. Comparison of tumour perfusion measurements after PDT with tumour response revealed an inverse correlation with tumour growth delay both for the RIF I and for the SCCVII tumours. PDT with sub-curative light doses appears to decrease vascular perfusion in the RIF I and SCCVII for a period of at least 24 hr. The most severe reductions in tumour blood flow were associated with the longest regrowth delays, indicating a major role of vascular damage in tumour response to PDT.