Kevin Brown

Preclinical assessment of hypocrellin B and hypocrellin B derivatives as sensitizers for photodynamic therapy of cancer: progress update.

Abstract— Hypocrellins are perylenequinone pigments with substantial absorption in the red spectral region and high singlet oxygen yield. They are available in pure monomelic form and may be derivatized to optimize properties of red light absorption, tissue biodistribution and toxicity. In vitro screening of synthetic derivatives of the naturally occurring compound, hypocrellin B (HB), for optimal properties of cyto‐(dark) toxicity and phototoxicity resulted in selection of three compounds for preclinical evaluation: HBEA‐R1 (ethanolaminated HB), HBBA‐R2 (butylaminated HB) and HBDP‐R1 [2‐(N,N‐dimethylami‐no)‐propylamine‐HB]. Extinction coefficients at 630 nm (φ630) are 6230, 6190 and 4800, respectively; and 1O2 quantum yields, φ, 0.60, 0.32 and 0.42. Intracellular uptake is essentially complete within 2 h (HBEA‐R1, HBBA‐R2) and 20 h (HBDP‐R1). Greatest uptake is associated with lysosomes and Golgi. The HBEA‐R1 and HBBA‐R2 elicit phototoxicity in vitro primarily via the type II mechanism, with some type I activity under stringently hypoxic conditions. Transcutaneous phototherapy with HBEA‐R1 permanently ablates EMToVEd tumors growing in the flanks of Balb/c mice, with minimal cutaneous effects. The HBBA‐R2 does not elicit mutagenic activity in strains TA98 and TA100 of Salmonella typhi‐murium. Further development of selected hypocrellin derivatives as photosensitizers for photodynamic therapy is warranted.

Quality assurance of the dose delivered by small radiation segments

The use of intensity modulation with multiple static fields has been suggested by many authors as a way to achieve highly conformal fields in radiotherapy. However, quality assurance of linear accelerators is generally done only for beam segments of 100 MU or higher, and by measuring beam profiles once the beam has stabilized. We propose a set of measurements to check the stability of dose delivery in small segments, and present measured data from three radiotherapy centres. The dose delivered per monitor unit, MU, was measured for various numbers of MU segments. The field flatness and symmetry were measured using either photographic films that are subsequently scanned by a densitometer, or by using a diode array. We performed the set of measurements at the three radiotherapy centres on a set of five different Philips SL accelerators with energies of 6 MV, 8 MV, 10 MV and 18 MV. The dose per monitor unit over the range of 1 to 100 MU was found to be accurate to within +/-5% of the nominal dose per monitor unit as defined for the delivery of 100 MU for all the energies. For four out of the five accelerators the dose per monitor unit over the same range was even found to be accurate to within +/-2%. The flatness and symmetry were in some cases found to be larger for small segments by a maximum of 9% of the flatness/symmetry for large segments. The result of this study provides the dosimetric evidence that the delivery of small segment doses as top-up fields for beam intensity modulation is feasible. However, it should be stressed that linear accelerators have different characteristics for the delivery of small segments, hence this type of measurement should be performed for each machine before the delivery of small dose segments is approved. In some cases it may be advisable to use a low pulse repetition frequency (PRF) to obtain more accurate dose delivery of small segments.

UPTAKE KINETICS AND INTRACELLULAR LOCALIZATION OF HYPOCRELLIN PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY: A CONFOCAL MICROSCOPY STUDY

Hypocrellins are naturally occurring compounds with photosensitizing properties in biological systems. We have prepared synthetic derivatives of hypocrellin B, which have promise as photosensitizers in the clinical application of photodynamic therapy. The intracellular localization and uptake kinetics of hypocrellin B and several selected hypocrellin congeners were determined semiquantitatively by fluorescence confocal microscopy in monolayer cultures of EMT6/Ed murine tumor cells. Each compound had unique uptake kinetics. Although no compound tested to date has demonstrated nuclear labeling, most could be detected in lysosomes, Golgi, endoplasmic reticulum and, to a minor extent, in cellular membranes. No two compounds gave identical labeling distributions. The differences are assumed to originate in physicochemical properties characteristic of each compound, which may ultimately impact upon the primary modality of phototoxicity.

Hypocrellins as photosensitizers for photodynamic therapy : a screening evaluation and pharmacokinetic study

Abstract Hypocrellin compounds were selected as potential photosensitizers for photodynamic therapy (PDT) owing to their high quantum yields of singlet oxygen (1O2), and facility for site-directed chemical modification to enhance phototoxicity, pharmacokinetics, solubility, and light absorption in the red spectral region, among other properties. Parent hypocrellins A and B share an absorption peak at 658 nm. These molecules may therefore be considered useful progenitors of derivatives which absorb more strongly in the red, considering that the ideal sensitizer should absorb in the 650–800 nm range, beyond the absorption range of hemoglobin and melanin, and where light penetration in tissues is maximized through reduced scattering. A series of pure, monomeric hypocrellin derivatives was tested for properties of dark cytotoxicity and photosensitizing potential by clonogenic assay in monolayer cultures of EMT6/Ed murine tumor cells. Their respective toxicities are reported on a molar basis. The in vitro screening assay has, to date, resulted in the selection of four hypocrellin derivatives for further development as photosensitizers for PDT. Cellular uptake for photosensitizing doses of selected compounds was determined by fluorimetry. Dose escalation studies in rodents indicate that potentially photosensitizing doses promote no demonstrable systemic toxicity.

Biodistribution of Photofrin II® and 5-Aminolevulinic Acid-Induced Protoporphyrin IX in Normal Rat Bladder and Bladder Tumor Models: Implications for Photodynamic Therapy

Photodynamic therapy (PDT) has been considered as a potential therapy for superficial bladder carcinomas. Cutaneous photosensitivity and reduction of bladder capacity are the two well‐known complications following systemic administration of the commonly used photosensitizer, Photofrin II® (PII). The objective of the present study was to evaluate whether intravesical. (i.b.) instillation of photosensitizers for PDT of bladder cancer might be a more suitable treatment method. Female Fischer rats were utilized to develop orthotopic and heterotopic bladder tumor models. Rats bearing orthotopic bladder tumors were treated either intravesically or intravenously with graded doses of 5‐aminolevulinic acid (ALA) or PII. Normal rats received the same doses of ALA or PII. As well, rats bearing heterotopic tumor were studied for comparison. The biodistribution times (times allowed for tissue uptake and bioconversion following drug administration) were 2, 4 or 6 h. Porphyrin fluorescence intensities within tumor, urothelium, submucosa, bladder muscularis and abdominal muscle were quantitated by confocal laser scanning microscopy. Following intravenous (i.v.) injection of ALA, tumor protoporphyrin IX (PpIX) levels peaked at 4 h and diminished by 6 h. The PpIX ratios of tumor‐to‐bladder mucosa, submucosa and muscle layers were 3:1, 5:1 and 8:1, respectively, 4 h following 1000 mg/kg ALA injection. After ALA instillation, the optimal biodistribution time appeared to be 4 h. Bladder instillation provided comparable tumor labeling with the i.v. route, but lost selectivity of PpIX accumulation between tumor and normal urothelium. The PpIX ratio of tumor‐to‐bladder muscularis was 5:1. After i.b. instillation of PII, porphyrin fluorescence was detected only within tumor and urothelium, while porphyrin fluorescence was mainly located in bladder submucosa following i.v. injection. Intravesical administration of ALA or PII might be feasible for PDT of superficial bladder cancers.

Fast kilovoltage/megavoltage (kVMV) breathhold cone-beam CT for image-guided radiotherapy of lung cancer

Long image acquisition times of 60-120 s for cone-beam CT (CBCT) limit the number of patients with lung cancer who can undergo volume image guidance under breathhold. We developed a low-dose dual-energy kilovoltage-megavoltage-cone-beam CT (kVMV-CBCT) based on a clinical treatment unit reducing imaging time to < or =15 s. Simultaneous kVMV-imaging was achieved by dedicated synchronization hardware controlling the output of the linear accelerator (linac) based on detector panel readout signals, preventing imaging artifacts from interference of the linacs MV-irradiation and panel readouts. Optimization was performed to minimize the imaging dose. Single MV-projections, reconstructed MV-CBCT images and images of simultaneous 90 degrees kV- and 90 degrees MV-CBCT (180 degrees kVMV-CBCT) were acquired with different parameters. Image quality and imaging dose were evaluated and compared to kV-imaging. Hardware-based kVMV synchronization resulted in artifact-free projections. A combined 180 degrees kVMV-CBCT scan with a total MV-dose of 5 monitor units was acquired in 15 s and with sufficient image quality. The resolution was 5-6 line pairs cm(-1) (Catphan phantom). The combined kVMV-scan dose was equivalent to a kV-radiation scan dose of approximately 33 mGy. kVMV-CBCT based on a standard linac is promising and can provide ultra-fast online volume image guidance with low imaging dose and sufficient image quality for fast and accurate patient positioning for patients with lung cancer under breathhold.

WHOLE BLADDER PHOTODYNAMIC THERAPY FOR ORTHOTOPIC SUPERFICIAL BLADDER CANCER IN RATS: A STUDY OF INTRAVENOUS AND INTRAVESICAL ADMINISTRATION OF PHOTOSENSITIZERS

PURPOSE Photodynamic therapy after intravenous injection of Photofrin (QLT Phototherapeutics, Vancouver, British Columbia, Canada) results in a contracted bladder and skin photosensitivity, which limits its clinical application. In an attempt to overcome these limitations photodynamic therapy after intravesical instillation of Photofrin or 5-aminolevulinic acid (ALA) in an orthotopic rat bladder tumor model was explored and compared with intravenous Photofrin for photodynamic therapy efficacy and phototoxicity. MATERIALS AND METHODS At 2 weeks after bladder implantation of 1.5 x 10(6) AY-27 tumor cells animals were randomly grouped. Photofrin was administered (5 mg./kg. intravenously and 2 mg./ml. intravesically). The ALA concentration for intravesical instillation was 300 mM. Whole bladder photodynamic therapy with graded doses of light (lambda = 630 nm.) was performed 4 hours after drug administration. Tumor control and complications were evaluated. RESULTS Photodynamic therapy with intravenous Photofrin plus 100 J./cm.(2) light resulted in severe bladder damage. Of 10 rats 6 died and 2 of the 10 that received 50 J./cm.(2) died. There were no photodynamic therapy related deaths in groups receiving intravesical instillation of Photofrin or ALA that also received 50 to 100 J./cm.(2) Median survival in rats treated with ALA intravesically plus 75 J./cm.(2) (77 days), Photofrin intravesically plus 50 (67) or 100 J./cm.(2) (76) and Photofrin intravenously plus 50 J./cm.(2) (60) were significantly different from that in controls (44). CONCLUSIONS Intravesical instillation of Photofrin or ALA can achieve the same photodynamic therapy efficacy as intravenous Photofrin in this orthotopic rat bladder tumor model with less phototoxicity to normal tissues.

Interstitial photodynamic therapy in subcutaneously implanted urologic tumors in rats after intravenous administration of 5-aminolevulinic acid

Photodynamic therapy (PDT) may be an attractive option for treatment of early stage prostate cancer. Aminolevulinic acid (ALA) acts as a prodrug leading to a selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), in epithelial cells. We investigated the efficacy of ALA-mediated PDT for rat R3327-H prostate cancer, compared with the AY-27 bladder tumor. Rats bearing either AY-27 or R3327-H tumors were randomized to different groups when their tumors reached approximately 1000 mm3. At the day of PDT, animals were administered 500 mg/kg ALA intravenously 4 hours prior to laser therapy. The argon-pumped dye laser light (630 nm) was coupled to multiple quartz fibers with cylindrical diffusing tips, which were inserted into the tumor in icosahedral pattern. Light exposure was varied to yield doses of 1000 to 3000 J/tumor. Animals bearing R3327-H tumors were imaged with 99mTc-HMPAO scintigraphy to evaluate tumor perfusion changes induced by PDT. There was a light-dose dependent tumor response in both tumor models. The mean time for R3327-H tumor to re-grow to 4 x treatment volume was 79.7 days in the control group (light only), 159 days in 1000 J group, and 169 days in 2000 J group (P < 0.05). Tumors treated with 3000 J were clinically cured (P < 0.01). Likewise, for AY-27 tumors, the average time to re-grow to 4 x treatment volume was 13.7 days in the control group, 179.3, 183.3, and 185.7 days in groups of 1000, 1500, and 2000 J (P < 0.05), respectively. Tumors treated with 3000 J were clinically cured (P < 0.01). 99mTc-HMPAO scintigraphy demonstrated a mild perfusion impairment following PDT. Interstitial PDT with ALA/PpIX is equally effective in treating prostate cancer and TCC in these heterotopic rat models.

Radiance modelling using the P3 approximation

Light dosimetry is an essential component of effective photodynamic therapy (PDT) of tumours. Present PDT light dosimetry techniques rely on fluence-based models and measurements. However, in a previous paper by Barajas et al, radiance-based light dosimetry was explored as an alternative approach. Although successful in demonstrating the use of Monte Carlo (MC) simulations of radiance in tissue optical characterization, the MC proved time consuming and impractical for clinical applications. It was proposed that an analytical solution to the transport equation for radiance would be desirable as this would facilitate and increase the speed of tissue characterization. It has been found that the P3 approximation is one such potential solution. Radiance and fluence expressions based on the P3 approximation were used to optically characterize an Intralipid-based tissue phantom of varying concentration of scatterer (Intralipid) and absorber (methylene blue) using a plane wave illuminated, semi-infinite medium geometry. The results obtained compare favourably with the Grosjean approximation of fluence (a modified diffusion theory) using the same optical parameters (mu(a), mu(s), g). The results illustrate that radiance-based light dosimetry is a viable alternative approach to tissue characterization and dosimetry. It is potentially useful for clinical applications because of the limited number of invasive measurements needed and the speed at which the tissue can be characterized.

Intracellular uptake kinetics of hypocrellin photosensitizers for photodynamic therapy

Hypocrellins are naturally occurring compounds with photosensitizing properties in biological systems. We have prepared synthetic derivatives of hypocrellin B, which have promise as photosensitizers in the clinical application of photodynamic therapy. The inherent fluorescence of four selected compounds has provided a means of determining their uptake and distribution in monolayer cultures of EMT6/Ed murine tumor cells, via fluorescence confocal microscopy.