Michael F. Grahn

DISTRIBUTION OF TEMOPORFIN, A NEW PHOTOSENSITIZER FOR THE PHOTODYNAMIC THERAPY OF CANCER, IN A MURINE TUMOR MODEL

Abstract— The biodistribution of temoporfin (tetra[m‐hydroxyphenyl]chlorin, m‐THPC), a recently developed photosensitizer, was investigated in BALB/c mice. The drug was administered intravenously (0.35‐0.75 μmol/kg) to tumor‐free mice or to mice implanted with the Colo 26 colorectal carcinoma. Blood and tissue samples were collected for up to 96 h post‐dose. Drug concentrations were determined by HPLC coupled to photometric detection at 423 nm. Concentrations in blood and liver fell relatively rapidly such that blood concentrations at later time points were below the limit of detection. Tumor concentrations rose at first and then remained constant from 24 h. Temoporfin concentrations in some tissues, notably heart and skeletal muscle, declined only slowly when compared to blood. The tumor: tissue ratios for those organs that showed a more rapid decline in temoporfin concentrations were higher at later times, whereas in tissues such as muscle the ratio remained relatively constant. The organs with the highest tumor: tissue ratios were small intestine (8.6), liver (6.9) and skeletal muscle (5.0).

Biodistribution and bioactivity of tetra-pegylated meta-tetra(hydroxyphenyl)chlorin compared to native meta-tetra(hydroxyphenyl)chlorin in a rat liver tumor model.

It has been proposed that the construction of a photosensitizer–polymer conjugate would lead to an increased selective retention of the drug in tumor tissue resulting in an enhancement of selective tumor destruction by light in photodynamic therapy. In this study the kinetics of a tetra‐pegylated derivative of meta‐tetra(hydroxyphenyl)chlorin (mTHPC–PEG) were compared with those of native meta‐tetra(hydroxyphenyl)chlorin (mTHPC) in a rat liver tumor model. In addition, the time course of bioactivity of both drugs was studied in normal liver tissue. Pegylation of mTHPC resulted in a two‐fold increase in the plasma half‐life time, a five‐fold decrease in liver uptake and an increase in the tumor selectivity at early time intervals after drug administration. However, although mTHPC concentrations in liver decrease rapidly with time, mTHPC–PEG liver concentrations increased as a function of time. This led to a loss of tumor selectivity at all but the earliest time points, whereas with mTHPC tumor selectivity increased with time. For both drugs the time course of bioactivity in the liver parallels drug concentration levels with extensive necrosis after irradiation of mTHPC–PEG‐sensitized liver tissue up to drug–light intervals of 120 h. It is concluded that on balance mTHPC–PEG does not appear to show any benefits over native mTHPC for the treatment of liver tumors, as normal liver tissue accumulates the compound. However, pegylation is a potentially promising strategy with an increase in tumor selectivity and reduced liver uptake if accumulation in the liver can be prevented.

Distribution and excretion of radiolabeled temoporfin in a murine tumor model.

The biodistribution and excretion of temoporfin (tetra[m‐hydroxyphenyl]chlorin, m‐THPC), a recently developed photosensitizer, was investigated in BALB/c mice. [14C]temoporfin was administered intravenously (0.73 μmol/kg) to tumor‐free mice or to mice implanted with the Colo 26 colorectal carcinoma. Blood, tissue and fecal samples were collected for 35 days and 10 days postdose from tumor‐free mice and tumor‐bearing mice, respectively. Blood concentrations fell rapidly such that at later time points they were indistinguishable from background counts. Tumor concentrations rose to a peak of 0.34 μg temoporfin equivalents/mL at 2 days and then declined in parallel (log plot) with the blood concentrations. Tumor : tissue ratios at 2 days for skin, adipose tissue and skeletal muscle underlying the tumor were 1.5, 2.3 and 3.8, respectively. By 4 days the corresponding values were 1.6, 3.4 and 4.0. Nearly 40% of the administered radioactivity was excreted in the feces in the first 24 h and more than 80% had been excreted by 20 days. Less than 0.2 % of the dose was recovered from the urine. An elimination half‐life of 10–12 days was calculated from the excretion data.

Intracellular uptake, absorption spectrum and stability of the bacteriochlorin photosensitizer 5,10,15,20-tetrakis(m-hydroxyphenyl) bacteriochlorin (mTHPBC). Comparison with 5,10,15,20-tetrakis(m-hydroxyphenyl) chlorin (mTHPC)

The bacteriochlorin photosensitizer 5,10,15,20-tetrakis (m-hydroxyphenyl) bacteriochlorin (mTHPBC) is a member of a series of related compounds which includes the well-known compound 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin(mTHPC) (temoporfin). Although this bacteriochlorin has near-ideal spectral characteristics in pure solvents, little is known of its stability or other characteristics within tumour cells. This study compares mTHPBC with mTHPC in both solvents in vitro and monolayers of the mouse colon tumour cell line Colo26. In aqueous protein-containing solvents, mTHPBC shows signs of aggregation and is oxidized to mTHPC at a rate of 2% h(-1). Both drugs are taken up by the cells at similar rates and to the same extent, with plateau levels being reached between 9 and 30 h of incubation. Between 25% and 33% of the bacteriochlorin within the cells is oxidized to chlorin in 24 h, after which no further net oxidation is observed. The intracellular absorption spectra suggest that mTHPBC exists in more than one form within the cells. Measurements of photodynamic therapy (PDT) activity confirm that mTHPBC is active within these cells, but with between 0.6 and 0.7 of the potency of mTHPC. Although aggregation and oxidation of the bacteriochlorin will reduce its overall effectiveness, this must be balanced against the potential effect of the greater red light penetration in vivo and the presence of a green light peak which may be employed to treat thin lesions where there is a risk of perforation of a hollow organ

Significantly increased lesion size by using the near-infrared photosensitizer 5,10,15,20-tetrakis (m-hydroxyphenyl) bacteriochlorin in interstitial photodynamic therapy of normal rat liver tissue

Penetration of tissues by activating light ultimately limits the size of the lesions achievable in interstitial photodynamic therapy. Measurements of the wavelength‐dependence of tissue optical properties suggest that substantial improvements may be possible, particularly in pigmented organs such as the liver, by using drugs absorbing at near infrared wavelengths.

mTHPC Polymer Conjugates: The In Vivo Photodynamic Activity of Four Candidate Compounds*

Abstract. The in vivo photodynamic activities of four poly(ethylene glycol) (PEG) conjugates of the photosensitiser 5,10,15,20-tetrakis-(m-hydroxyphenyl)chlorin (mTHPC, temoporfin, Foscan®) were compared with that of mTHPC over a range of drug–light intervals using acute tumour necrosis and skeletal muscles swelling in a mouse model in order to ascertain the influence of linking group stability and PEG chain length on the photodynamic activity. The four compounds examined contained either PEG 2000 or PEG 5000 attached by carbonate or triazine linkages at the phenol hydroxyl groups of the mTHPC.All compounds tested caused tumour necrosis at drug–light intervals of between one and four days. mTHPC produced tumour necrosis of over 5 mm at drug–light intervals of 1 and 2 days with limited muscle damage at early drug–light intervals. The relatively labile carbonate-linked conjugates gave tumour necrosis similar to mTHPC but produced severe muscle and systemic phototoxicity on irradiation at 4–24 h after injection. The more stable triazine-linked conjugates produced no significant muscle damage at any of the drug–light intervals tested, but gave only limited tumour necrosis under the conditions tested. PEG chain length had relatively little effect on the patterns of bioactivity.It is concluded that both classes of mTHPC PEG conjugates may be suitable for photodynamic therapy if the problems of stability and early photosensitivity in the case of the carbonates and reduced potency in the case of the triazines can be overcome through improved formulations and PDT treatment regimens.

Adjuvant intraoperative photodynamic therapy for colorectal carcinoma : a clinical study

The local recurrence rate of colorectal carcinoma after surgery is unacceptable in most series, and adjuvant therapies have made only a small impact on this. There is experimental evidence that adjuvant intraoperative photodynamic therapy (AIOPDT) may be effective. AIOPDT involves systematically photosensitizing the patient preoperatively with a drug (HpD) which relatively localizes to tumour and is activated using visible light. At operation the resected tumour bed is illuminated with a predetermined uniform light energy density to eradicate microscopic tumour deposits left at the lateral resection margin. We have previously investigated technical and biological factors leading to this clinical trial. Seventeen patients have received AIOPDT in a potentially effective dose, and safety and technical matters have been investigated. Cutaneous phototoxicity occurred in 3 patients. Three patients had anastomotic breakdown, none considered attributable to PDT. The intraoperative technique was a practical option. AIOPDT carried a low patient morbidity and should be investigated in prospective clinical trials to determine if local recurrence rates can be decreased.

A comparison of novel light sources for photodynamic therapy.

A diode laser, light-emitting diode (LED) array bandwidth 25 nm, full width half maximum (FWHM) and filtered arc lamp (bandwidth 40 nm, FWHM), all with peak emission at about 650 nm, suitable for the photosensitizer tetra(meta-hydroxyphenyl)chlorin (mTHPC), were compared with a copper vapour laser pumped dye laser, using depth of necrosis in normal rat liver as a measure of photodynamic effect.A three-way comparison between a DL10K dye laser, the LED array and the filtered arc lamp resulted in mean depths of necrosis of 4.64, 4.29 and 4.04 mm, respectively, at 20 J cm-2, the values for the laser and arc lamp being significantly different at the 5% level. A further comparison of a narrower linewidth DL20K dye laser with the LED array, using a light dose of 20 J cm-2, showed a significant difference between the mean depths of necrosis of 4.97 and 4.05 mm, respectively (p=0.01).A final study, comparing the DL20K dye laser with the diode laser and a light dose of 10 J cm-2, demonstrated no significant difference in depths of necrosis (3.23 and 3.25 mm, respectively). The results obtained in the three studies are attributed to the relative bandwidths of light emission for the various sources. A simple mathematical model is presented explaining the results in terms of the relative activation of the photosensitizer and the consequent threshold fluence required for the induction of necrosis.It is concluded that, in order to achieve the same depth of effect as a laser when using the broad band sources, the incident fluence would have to be approximately doubled. However, when the low cost and ease of use of the non-laser sources are taken into consideration, these devices are likely to find widespread applications in clinical photodynamic therapy.

Multiple fibre interstitial photodynamic therapy of patients with colorectal liver metastases

Multiple fibre interstitial photodynamic therapy (IPDT) was performed in patients with colorectal hepatic metastases (CRHM) to determine treatment response and side-effects. Ten patients aged 48–75 years with 16 CRHM (seven solitary) were sensitized with (111 mg m−2) haematoporphyrin derivative intravenously. Forty-eight hours later a custom-designed light delivery system guided by ultrasound allowed 630 nm light from a copper vapour pumped-dye laser split into four 200 μm optical fibres to be geometrically positioned within the metastasis. This was performed percutaneously in four patients and at laparotomy in six. Metastases were imaged using computerized tomography and volumes measured by planimetry. Growth was expressed as a ratio relative to the initial volume (RVG). Following IPDT no complications occurred. Three metastases were not treated because of technical problems. No patient suffered skin photosensitivity reactions. Twelve weeks following IPDT, 12 metastases of less than 60 cc initial volume had a mean RVG of 0.99 (s.d. 0.27). One large solitary metastasis continued to grow after IPDT with a RVG of 1.9 after 12 weeks. Untreated hepatic metastases had a mean RVG of 2.13 after 12 weeks. These data suggest that IPDT can reduce the growth of small hepatic metastases and can be performed with low morbidity when applied at laparotomy and with minimally invasive techniques.

Comparison of meso-tetrahydroxyphenyl-chlorin and meso-tetrahydroxyphenyl-bacteriochlorin with respect to photobleaching and PCT efficiency in vivo

Using BALB/c nude mice bearing WiDr human colon adenocarcinoma, we investigated photobleaching and photochemotherapeutic (PCT) effects of meso- tetrahydroxyphenyl-chlorin (mTHPC) and meso- tetrahydroxyphenyl-bacteroiochlorin (mTHPBC). For both studies, mice were injected i.p. 1 mg/kg mTHPC and mTHPBC, respectively, 24 hr before light irradiation. Photobleaching of the sensitizers in mouse skin was carried out using a dye laser (for mTHPC) and 1 KW xenon source equipped with a monochromator (for mTHPBC). Fluorescence measurements were made by means of a fiberoptic system, exciting and collecting the fluorescence from the mouse skin. The system was coupled to a PE L550 fluorimeter. For the PCT study, the mTHPC or mTHPBC-sensitized tumors were exposed to the laser at a fluence of 10 J/cm2. The responses of the treated tumors were evaluated by measuring the tumor growth. We found that both mTHPC and mTHPBC are photolabile. Approximately 80% of the fluorescence of the two dyes in the mouse skin is bleached by a fluence of 10 J/cm2. When mTHPBC in mouse skin was photobleached by light of 740 nm, the bacteriochlorin (peak at 740 nm) was significantly bleached while the chlorin (peak at 652 nm) was unaffected. The growth of the tumors was delayed by 13 days after PCT with 1 mg/kg mTHPC Irradiation at 652 nm) and 5 days delay after PCT with 1 mg/kg mTHPBC (irradiation at 515 nm). The present data indicate that (1) by proper choice of a low dose of mTHPC or MTHPBC, it is possible to photobleach the sensitizers in normal tissues without eliminating their PCT effect on tumor tissues; (2) in order to increase the efficiency of PCT with mTHPBC, fractionated irradiation with different wavelengths (740 nm and 652 nm) should be considered to be used.