New somatostatin-drug conjugates for effective targeting pancreatic cancer.

Pancreatic cancer poorly responds to available drugs, and finding novel approaches to target this cancer type is of high significance. Here, based on a common property of pancreatic cancer cells to express somatostatin receptors (SSTR), we designed drug conjugates with novel somatostatin-derived cyclic peptides (SSTp) with broad selectivity towards SSTR types to facilitate drug targeting of the pancreatic cancer cells specifically. Uptake of our newly designed SSTps was facilitated by SSTRs expressed in the pancreatic cancers, including SSTR2, SSTR3, SSTR4 and SSTR5. Three major drugs were conjugated to our best SSTps that served as delivery vehicles, including Camptothecin (CPT), Combretastatin-4A (COMB) and Azatoxin (AZA). All designed drug conjugates demonstrated penetration to pancreatic cancer cell lines, and significant toxicity towards them. Furthermore, the drug conjugates specifically accumulated in tumors in the animal xenograft model, though some accumulation was also seen in kidney. Overall these findings lay the basis for development of novel drug series that could target the fatal pancreatic cancer.

A novel histochemical method for the visualization of thrombin activity in the nervous system.

Although thrombin has an important role in both central and peripheral nerve diseases, characterization of the anatomical distribution of its proteolytic activity has been limited by available methods. This study presents the development, challenges, validation and implementation of a novel histochemical method for visualization of thrombin activity in the nervous system. The method is based on the cleavage of the substrate, Boc-Asp(OBzl)-Pro-Arg-4MßNA by thrombin to liberate free 4-methoxy-2-naphthylamine (4MßNA). In the presence of 5-nitrosalicylaldehyde, free 4MßNA is captured, yielding an insoluble yellow fluorescent precipitate which marks the site of thrombin activity. The sensitivity of the method was determined in vitro using known concentrations of thrombin while the specificity was verified using a highly specific thrombin inhibitor. Using this method we determined the spatial distribution of thrombin activity in mouse brain following transient middle cerebral artery occlusion (tMCAo) and in mouse sciatic nerve following crush injury. Fluorescence microscopy revealed well-defined thrombin activity localized to the right ischemic hemisphere in cortical areas and in the striatum compared to negligible thrombin activity contralaterally. The histochemical localization of thrombin activity following tMCAo was in good correlation with the infarct areas per triphenyltetrazolium chloride staining and to thrombin activity measured biochemically in tissue punches (85 ± 35 and 20 ± 3 mU/ml, in the cortical and striatum areas respectively, compared to 7 ± 2 and 13 ± 2 mU/ml, in the corresponding contralateral areas; mean ± SEM; p<0.05). In addition, 24 h following crush injury, focal areas of highly elevated thrombin activity were detected in teased sciatic fibers. This observation was supported by the biochemical assay and western blot technique. The histochemical method developed in this study can serve as an important tool for studying the role of thrombin in physiological and pathological conditions.

Initiation of apoptosis by photodynamic therapy using a novel positively charged and water-soluble near infra-red photosensitizer and white light irradiation.

Our aim was to investigate the photophysical and photodynamic properties of a new, water-soluble positively charged and chemically stable photosensitizer: tetrahydroporphyrin tetratosylat (THPTS). Absorption, fluorescence and (1)H NMR spectra and the intracellular distribution of THPTS were measured. The apoptosis in choroidal melanoma cells was measured using cell death detection ELISA and caspase-8 activity assay. THPTS-PDT efficiency was studied in Balb/c mice bearing C26 colon carcinoma. Subcutaneously located tumors were irradiated with a white light source at a fluence rate of 100 mW/cm(2). THPTS was administrated 3 h before illumination. The tumoricidal effect was examined 24 h after THPTS-PDT by vital staining with 0.4-ml 1% Evans blue solution, intraperitoneally injected to each mouse. THPTS showed a strong absorption band at 760 nm. Its purity, measured by (1)H NMR, is better than 99%. At 24-h incubation period, CLSM revealed THPTS fluorescence in mitochondria and cell nucleus. THPTS possesses no toxic effect in preincubated CM cells without irradiation, and THPTS-PDT causes efficient apoptosis. THPTS-PDT using white light irradiation at a dose of 480 J/cm(2) caused necrosis with a depth of 8 mm in subcutaneously located C26 colon carcinoma in Balb/c-mice. In accordance with the present results, the THPTS seems to be of interest for further in vivo investigations with broad-band white light sources.

Photothermic treatment of pigmented B16 melanoma using a broadband pulsed light delivery system.

Pulsed photothermic treatment (PTT) of pigmented B16 mice melanoma tumors was carried out using a Photodyne incoherent light delivery system. Tumor heating with average temperature of 41-44 degrees C was observed during broadband photoirradiation (600-800 nm) at light doses of 60-120 J/cm(2) delivered using 0.6 J/cm(2) pulses (2 ms) at 1 Hz repetition rate. Electron microscopy of tumor samples revealed pronounced structural changes in microvasculature and melanosomes. Pulsed PTT caused damage to endothelial cells and vascular walls, swelling of mitochondria and melanosomal disruption without nuclear alteration. Significant tumor response with necrosis formation followed by tumor regression was observed by a tumor growth study after PTT at 120 J/cm(2).

Photodynamic therapy of cutaneous lymphoma using 5-aminolevulinic acid topical application.

BACKGROUND: Photodynamic therapy (PDT) with topical application of 5-aminolevulinic acid (ALA) is a new and effective modality for treatment of superficial basal and squamous cell carcinomas. OBJECTIVE: We present the kinetics of ALA-induced protoporphyrin IX (PP) accumulation and the results of ALA PDT treatment on two patients with different stages (stage I and stage III) of mycosis fungoides (MF)-type cutaneous T-cell lymphoma (CTCL). METHODS: ALA-Decoderm cream was applied to the lesions for 16 hours. Spectrofluorescence measurements of PP accumulation were carried out before, during, and 1 hour after photoirradiation (580-720 nm) using the VersaLight system. RESULTS: Different patterns of PP fluorescence kinetics were observed in patients with early and advanced stages of the disease. During photoirradiation the intensity of fluorescence decreased depending on the lesion thickness. One hour after the photoirradiation procedure no PP fluorescence was observed in the stage I MF lesion, while in the thick stage III MF lesions, PP fluorescence reappeared; after an additional 10-15 minutes of irradiation PP fluorescence disappeared. Complete response with excellent cosmetic results was observed in the stage I lesion after a single irradiation with a light dose of 170 J/cm2; in five stage III lesions, complete response was achieved after fractionated irradiation with a total light dose of 380 J/cm2 (follow-up at 27 and 24 months, respectively). CONCLUSION: The results showed a high response of both stage I and stage III MF lesions to ALA PDT. This modality appears to be very effective and can be used successfully for MF treatment.

Enhancement of ALA-PDT damage by IR-induced hyperthermia on a colon carcinoma model.

The interaction of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA) and hyperthermia is not well understood. In the present study, significant enhancement of tumor damage was observed after simultaneous application of ALA-PDT and IR-induced hyperthermia using a broad-band incoherent light source. One hour after systemic administration of ALA at a dose of 200 mg/kg, subcutaneously transplanted C26 colon carcinoma tumors were irradiated with two bands of the VersaLight system, red (R, 580-720 nm) and red plus IR (R + IR, 580-720 nm and 1250-1600 nm). Photoirradiation using the R + IR band at different fluence rates and exposures caused mild heating of the tumor to 39-43 degrees C at a 3 mm depth. Electron microscopy after ALA + R, ALA + R + IR and R + IR treatments showed early mitochondrial swelling that was more pronounced in the ALA + R + IR group. Tumor necrosis assessment, using histology and vital staining, revealed an enhancement of tumor necrosis depth in the ALA + R + IR group compared to ALA + R and R + IR. The results showed that subhyperthermic heating to 39-39.5 degrees C in the ALA + R + IR group decreased the threshold light dose required for 100% tumor necrosis from 210 J/cm2 (observed in the ALA + R group) to 140 J/cm2. A tumor growth delay test, based on tumor volume measurement, also revealed significant enhancement of antitumor effect after application of ALA + R + IR compared to ALA + R.

In vivo photodynamic therapy with the new near-IR absorbing water soluble photosensitizer lutetium texaphyrin and a high intensity pulsed light delivery system.

An in vivo fluorescence monitoring and photodynamic therapy (PDT) study was performed using the new photosensitizer lutetium texaphyrin (Lu-Tex). This photosensitizer is water soluble and has the additional advantage of strong absorption near 730 nm. C26 colon carcinoma was transplanted in the foot of BALB/c mice. In vivo fluorescence spectroscopy was applied to study Lu-Tex tissue distribution kinetics. For this purpose, fluorescence intensity both in the foot with the tumor and in the normal foot was measured in vivo by the laser-induced fluorescence (LIF) system. For PDT, both feet of the mice were irradiated simultaneously with the use of a new high intensity pulsed light delivery system, the Photodyne. The results of the LIF measurements showed that the maximal fluorescence intensity ratio between the normal and tumor bearing foot (FIR) was observed 24-48 h after the agent injection. Photoirradiation with doses from 90 to 240 J cm-2 (0.6 J cm-2 per 2 ms pulse, 1 Hz) 24 h after injection of Lu-Tex at a dose of 10 mg kg-1 caused significant tumor necrosis and delay in the tumor growth rate. The antitumor effect was enhanced with increasing light doses. Normal tissue response to PDT with Lu-Tex was determined as the damage index of the normal foot, which was irradiated simultaneously with the tumor bearing foot. The normal tissue response after PDT with Lu-Tex was compared with 5-aminolevulinic acid (ALA) induced protoporphyrin IX (PP), chlorin e6 (Chl) and Photofrin (PII) at the same values of antitumor effect. The results showed that at 50, 80 and 100% inhibition of tumor growth the orders of the values of normal foot damage indexes were as follows: ALA > Lu-Tex > or = PII > Chl, PII > ALA > Lu-Tex > Chl and PII > Lu-Tex > ALA > Chl respectively.

The kinetics of protoporphyrin fluorescence during ALA-PDT in human malignant skin tumors.

Fluorescence monitoring during photodynamic therapy (PDT) with the use of topical 5-aminolevulinic acid (ALA) was carried out in patients bearing superficial and nodular basal cell carcinomas (BCC), squamous cell carcinomas (SCC) and Kaposi's sarcomas. A new diagnostic-therapeutic system based on an incoherent CW light source was used for fluorescence spectral measurements and imaging. The results showed that photoirradiation reduced ALA-induced protoporphyrin IX (PP) fluorescence in all tumors. The rate of PP photobleaching in superficial BCC and SCC tumors was significantly higher than in large nodular BCC tumors. The results showed that the differences in kinetics of fluorescence reduction could be attributed to the tumor thickness. One hour after photoirradiation with a light dose of 170 J/cm2 a phenomenon of re-appearance and recovery of PP fluorescence was observed in the large deeply penetrating BCC tumors and Kaposi's sarcoma lesions. In such cases an additional light treatment was performed. The results of the study demonstrated that fluorescence monitoring is very appropriate for the definition of an optimal ALA-PDT clinical protocol.

A comparative study of tissue distribution and photodynamic therapy selectivity of chlorin e6, Photofrin II and ALA-induced protoporphyrin IX in a colon carcinoma model.

An in vivo study of tissue distribution kinetics and photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA), chlorin e6 (Chl) and Photofrin (PII) was performed to evaluate the selectivity of porphyrin accumulation and tissue damage effects in a tumour model compared with normal tissue. C26 colon carcinoma of mice transplanted to the foot was used as a model for selectivity assessment. Fluorescence measurements of porphyrin accumulation in the foot bearing the tumour and in the normal foot were performed by the laser-induced fluorescence (LIF) system. A new high-intensity pulsed light delivery system (HIPLS) was used for simultaneous irradiation of both feet by light in the range of 600-800 nm, with light doses from 120 to 300 J cm-2 (0.6 J cm-2 per pulse, 1 Hz). Photoirradiation was carried out 1 h after injection of ALA, 3 h after injection of Chl and 24 h after injection of PII. A ratio of porphyrin accumulation in tumour vs normal tissue was used as an index of accumulation selectivity for each agent. PDT selectivity was determined from the regression analysis of normal and tumour tissue responses to PDT as a function of the applied light dose. A normal tissue damage index was defined at various values (50, 80 and 100%) of antitumour effect. The results of the LIF measurements revealed different patterns of fluorescence intensity in tumour and normal tissues for ALA-induced protoporphyrin IX (ALA-PpIX), Chl and PII. The results of PDT demonstrated the differences in both anti-tumour efficiency and normal tissue damage for the agents used. The selectivity of porphyrin accumulation in the tumour at the time of photoirradiation, as obtained by the LIF measurements, was in the order ALA-PpIX > Chl > PII. PDT selectivity at an equal value of anti-tumour effect was in the order Chl > ALA-PpIX > PII. Histological examination revealed certain differences in structural changes of normal skin after PDT with the agents tested. The results of PDT selectivity assessment with respect to differences in mechanisms of action for ALA, Chl and PII are discussed.

Temperature monitoring during photodynamic therapy of skin tumors with topical 5-aminolevulinic acid application.

Temperature monitoring during photodynamic therapy (PDT) with topical 5-aminolevulinic acid (ALA) cream application was performed on 22 patients with solar keratoses (SK) and basal cell carcinoma (BCC). The lesions were located on the forehead, nose, ear and cheek. Temperature measurements during photoirradiation, with a power density of 100 mW/cm2 from an incoherent light source (light delivery system for PDT), were carried out by noncontact (infrared thermal imaging radiometer) and contact (thermocouple) methods. Thermal imaging analysis revealed nonuniform temperature distribution in the irradiated areas. The temperature gradually increased from the peripheral to the central zone of the area. The results showed that photoirradiation induced heating of the skin tumors to 39.5-42.5 degrees C during the PDT procedure. The temperature of normal skin areas disposed symmetrically to the lesions on the contralateral side at the same conditions of irradiation (without prior ALA application) was about 42-43.5 degrees C. The surface temperature differences (delta T) between the normal and tumor tissues after 10 min of irradiation were 3.3 +/- 0.5 degrees C in the forehead areas, 2.5 +/- 0.4 degrees C in the nose areas and 0.8 +/- 0.3 degrees C in the ear areas.

Topical application of 5-aminolevulinic acid, DMSO and EDTA: protoporphyrin IX accumulation in skin and tumours of mice.

Topical 5-aminolevulinic acid (ALA) application in three different creams was carried out on mice bearing subcutaneously transplanted C26 colon carcinoma. The creams contained (a) 20% ALA alone, (b) ALA with 2% dimethylsulphoxide (DMSO) and (c) ALA, DMSO and 2% edetic acid disodium salt (EDTA). Protoporphyrin IX (PP) production in the tumour and in the skin overlying the tumour was studied by two methods: laser-induced fluorescence (LIF) and chemical extraction. The kinetics of PP production in the skin and in the tumour, as studied by the LIF method, was similar for all three cream preparations. The PP fluorescence intensity in the tissues reached its maximum 4-6 h after application of the creams. Quantitative analysis showed that the PP concentration after treatment was more pronounced in the skin than in the tumour. The efficiency of porphyrin production in the skin by the creams used was in the following order: ALA-DMSO-EDTA > ALA-DMSO > ALA. In the tumour the enhancing effect of DMSO and EDTA on PP accumulation induced by ALA was observed mainly in the upper 2 mm section. However, the concentration of PP in the tumour was found to be approximately the same for ALA-DMSO and ALA-DMSO-EDTA cream combinations. The possible mechanisms of the effect of DMSO and EDTA are discussed.

Experimental grounds for using chlorin e6 in the photodynamic therapy of malignant tumors.

The toxicity, pharmacokinetics and antitumour effect of chlorin e6 after light irradiation were studied. The LD50 value of chlorin e6 in C3H mice is 189 +/- 9 mg kg-1 and in Wistar white rats is 113 +/- 18 mg kg-1 14 days after intraperitoneal injection. The concentration of chlorin e6 in blood, liver, kidney, spleen and tumors (sarcoma M-1 and sarcoma 45) of the rats was determined by a fluorescence method, 3, 6, 12, 18, 24, 48 and 72 h after administration at a dose of 10 mg kg-1. For this purpose, chlorin e6 was extracted from tissues by the detergent Triton X-100. The depth of necrosis in sarcoma 45, the regression rate of sarcoma M-1 and the animal cure rate were evaluated after chlorin e6 administration at doses of 1-10 mg kg-1 and subsequent irradiation with krypton laser light. Depending on the dose and the time interval between chlorin e6 injection and irradiation, the depth of necrosis in sarcoma 45 varied from 5.0 to 15.0 mm. The cure rate of the animals with sarcoma M-1 varied from 10% to 60%. The antitumor effect was directly proportional to the chlorin e6 dose and light energy exposure and inversely proportional to the time interval between photosensitizer injection and irradiation.

Sensitivity of different rat tumour strains to photodynamic treatment with chlorin e6.

Transplantable rat tumours (sarcoma M-1, sarcoma 45, alveolar liver cancer PC-1 and Pliss' lymphosarcoma) were used to study chlorin e6 accumulation in tumours and its photodynamic effect. Tumours were irradiated by krypton ion laser light (647 and 676 nm; 90 J cm-2) 15 min and 24 h after chlorin e6 injection at doses of 5 and 10 mg kg-1. The relationship between some morphological peculiarities of these tumour strains, photosensitizer accumulation in tumours and their response to the photodynamic treatment is discussed.

Photodynamic therapy with chlorin e6. A morphologic study of tumor damage efficiency in experiment.

Morphological changes in rat sarcoma M-1 after photodynamic treatment with chlorin e6 were studied. The frequency of necrosis appearance and the depth of its spreading in tumor tissue were evaluated after intraperitoneal injection of chlorin e6 in doses of 1-10 mg kg-1 and subsequent irradiation by a krypton laser with light energy density 22.5-135 J cm-2, using the method of vital staining with Evans blue. It was found that the antitumoral effect of photodynamic treatment was strengthened by increasing the dose of the agent and light and reduced by increasing the time interval between chlorin e6 injection and light irradiation. The treatment being given in the parameters mentioned produced a depth of tumor necrosis which varied from 4.0 mm to 16.6 mm. The mechanisms of tumor tissue damage after photodynamic treatment in vivo are discussed.