Additive effects of earthworm extract with HpD-laser on the enhancement of reactive oxygen production and the inhibition of[~3H]—thymidine.uptake in mouse ascitic tumor cells

The effects of combined use of earthworm extract（912）and HpD-laser on the produc-tion of reactive oxygen and the biosynthesis of DNA in S180 tumor cells were studied throughchemiluminescence measurement and[3H]-TdR incorporation assay.The results showed that as com-pared with the control,the intensity of chemiluminescence emitted by tumor cells treatedsimultaneously with 912 and HpD-laser was enhanced more than ten-folds,while that treated with912 or HpD-laser alone was increased only 2～4 folds.The[3H]-TdR incorporation into tumorcells of the former group was inhibited upto 74.1%,and that of the latter groups decreased onlyby 42.2% and 40.0%,respectively.In accordance with these biochemical changes,the ultrastructuraldamage of tumor cells of the former,combinedly treated group appeared to be the most serious.This suggests an additive effect of 912 with HpD-laser on tumor cells.In addition,if free radicalscavengers,such as catalase and superoxide dismutase,were added to the reaction systembefore chemiluminescence assay,the luminescent enhancement effect mentioned above was dramaticallyalleviated,implying the presence of O2 and H2O2 in the system.Therefore,as to the toxic effecton tumor cells,912 and HpD-laser are not only additive in efficiency,but also similar in theunderlying mechanism of action.

Studies on Preclinical Toxicology of a New Tumor Photolocalizing and Photochemotherapeutic Agent, Photocarcinorin(PsD-007)

Photocarcinorin was prepared in our Lab and its composition was differentfrom that of any other hematoporphyrin photosensitizers by TLC and HPLC analyses.The 95% fiducial limits of iv LD in mice were 176-236 mg·kg-1.The iv MLD indogs was 171 mg·kg-1.The acute and subacutc toxic tests in 37 dogs showed that theintoxicated manifestations were characterized by a complex syndrome always seen inporphyrias.The biological,laboratory and histopathologic findings revealed that theliver,kidney and erythroeytic series were the target organs.The damages were dose-related and reversible within 2 wk.he phototoxicity was determined in mice with UV ra-diation and compared with that of HpD.The extent of its phototoxic reactions waslower than that of HpD’s.

A new photolocalizing and photochemotherapeutic agent and radiosensitizer photocarcinorin(PsD-007)

Here is presented a brief review of studies on the home-made photolocalizing andphotochemotherapeutic agent as well as radiosensitizer for human malignancies called photocarcinorin(PsD-007).Experimental data and principal research conclusions of chemistry,pharmacodynamics,preclinical pharmacology and toxicology as well as phase-Ⅰ-Ⅱ clinical trials are summarized.

Immune response induced by hematoporphyrin derivatives mediated photodynamic therapy:Immunogenic cell death and elevated costimulatory molecules

Photodynamic therapy(PDT)not only destroys tumor cells directly but also induced anti-tumor immune response through damage-associated molecular patterns(DAMPs).It is reported that anti-tumor response was associated with light dose and photosensitizer used in PDT.In this study,4T1 tumor cells were implanted on both the right and left flanks of mice.Only the right tumor was treated by HpD-PDT,while the left tumor was not irradiated.The anti-tumor immune response induced by HpD-PDT was investigated.The expression of DAMPs and costimulatory molecules induced by HpD-PDT were tested by immuno fluorescence and flow cytometry in vivo.Different light doses of PDT were designed to treat 4T1 cells.The killing effect was assessed by CCK-8 kit and apoptosis kit.The expression of DAMPs on 4T1 cells after HpDPDT were evaluated by flow cytometry,western blot and ATP kit.This study showed that CD4^(+)T,CD8^(+)T and the production of IFN-γwere increased significantly on day 10 in righttumor after PDT treatment compared with control group.HpD-PDT enhanced the expression of calreticulin(CRT)on tumor tissue.Importantly,co-stimulatory molecular OX-40 and 4-1BB were elevated on CD8^(+)T cells.In vitro,immunogenic death of 4T1 cells was induced after PDT.Besides,the expression of DAMPs increased with the increasing of energy density.This study indicates that anti-tumor immune effect was induced by HpD-PDT.The knowledge of the involvement of CRT,ATP and co-stimulatory molecules uncovers important mechanistic insight into the anti-tumor immunogenicity.It was the first time that co-stimulatory molecules were investigated and found to elevate after PDT.

THE SPECIFIC PHOTODYNAMIC EFFECTS OF MONOCLONAL ANTIBODIES CONJUGATED WITH HEMATOPORPHYRIN DERIVATIVE ON GASTRIC CANCER IN VITRO AND IN VIVO

Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated with a hematoporphyrin derivative (HPD) by using carbodiimide. The qualitative analysis of this conjugate showed that the amount of free HPD was negligible and there were no IgG aggregates among the conjugates. The conjugate retained both the antibody and photochemical activity of HPD.In vitro, the phototoxic effect of this HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The quality of selective photocytotoxicity was proven by the greater cytotoxi-city this conjugate showed than that of corresponding normal mouse IgG (NIgG) conjugated with HPD. It showed less cytotoxicity to colon cancer cell line B-80 (negative reaction to MoAb BB4.3) than to BGC825. Moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody, but not by another MoAb 3G9, which combines with BGC823 at different binding sites from MoAb BB4.3.Nude mice inoculated with 2 × 10- BGC823 cells were given HPD-BB4.3, HPD, HPD-NIgG, HPD plus BB4.3 and PBS, respectively then exposed to light. Four out of six animals treated with the HPD-BB4.3 conjugate remained tumor-free for a long period. Although two developed tumors, there was a significant difference between the HPD-BB4.3-treated group and all the control groups in tumor induction time, tumor growth rate, and survival time (p<0.001). The HPD-BB4.3 conjugate inhibited the growth of established tumors by more than 40% in comparison with control groups (p<0.05).

Potential of nano carriers as cancer cell-specific drug delivery systems in photodynamic therapy

Photodynamic therapy (PDT) in combination with chemotherapy has great potential for cancer treatment (1, 2)However,there have been very few attempts to develop cancer-targeted co-delivery systems of photosensitizers and anticancer drugs.We developed hematoporphyrin (HP)-modified doxorubicin(DOX)-loaded bovine serum albumin nanoparticles (HP-NPs) in an attempt to improve the therapeutic effect of PDT in treating liver cancer.

EFFECT OF HEMATOPORPHYRIN DERIVATIVE (HPD) PLUS LIGHT ON TRANSCRIPTION ACTIVITY IN THE NUCLEUS

To elucidate the mechanism of photodynamic damage of cells, the effect of HPD plus light on transcriptlonal ectlvlty In the mucleus Isolate from the normal rat liver was studied in vitor by 3H- UTP incorporation into RNA. Measurements of fluorescence spectrum showed that HPD was bound to the nucleus and its fluorescence Intensity Increased with the Increase of HPD concentration. The experimental results Indicated that no changes could be observed when either HPD or light was used alone. Whereas the nuclear transcription activity was found to be inhibited significantly by both HPD and light treatment, and the degree of Inhibition was dependent on the HPD concentration and the time of exposure to light. After treatment by 3 μg/ ml HPD, the inhibition rate of the nuclear transcription activity was 23% ,45% ,69% ,80% and 90%, respectively for light exposure of 2, 5, 10, 20 and 30 minutes. Our results suggested that dose-dependent decreases in the nuclear transcription activity, and marked inhibition of the activity was found in the range from 3 to 7 μg/ml HPD following exposure to light.

The Observation of the Photodynamic Effect of Hematoporphyrin Derivative (HpD) Combined with Aidiron on Ascitic Hepatoma Cells in Vitro

In this study, we found, through biochemical synthesis of DNA with the incorporation of ~3HTdR and morphological observation of the tumor cells, that aidiron (912), an extract from earthworm, had a stronger kilting effect (27%) on mouse ascitic hepatoma tumor cells (H_(22)) in vitro, and that laser-hematoporphyrin derivative (HpD) combined with 912 markedly enhanced the kilting effect (74%) on tumor cells. By means of the fluoresoence spectrophotography we also dis- covered that the killing effect of laser-HpD-912 on tumor calls was closely related with the produc- tion of free radicals and lipid peroxidation.

PURGE OF MALIGNANT CELLS FROM BONE MARROW BY HEMATOPORPHYRIN DERIVATIVES AND LIGHT EXPOSURE IN VITRO

During autologous bone marrow graft in treatment of malignant diseases, it is critical to purge malignant cells from the marrow. In the present study, the sensitivity to photodynamic inactivation of 3 leukemic cell lines was compared with their counterpart normal hematopoietic cells. After mouse leukemic L1210 cells were treated with a preparation of hematoporphyrin derivatives, YHpD, 10 μg/ml for 1 hr. and irradiated with blacklight (peak wavelength 395 nm, light intensity 0.6 mW/cm2) for 5 minutes, the survival rate of clonogenic cells decreased to <10%, while that of bone marrow granulocyte macrophage progenitor cells (CFU-GM) in DBA/2 mice remained at nearly normal level (>80%). Similar results were obtained when human leukemic HL-60 cells were compared with human CFU-GM and mouse leukemic L615 cells with CFU-GM in 615 strain mice. It is suggested that hematoporphyrin photoradiation may be useful for Iselectively killing leukemic cells in bone marrow.

Impact of the photosensitizers hematoporphyrin coated gold nanoparticles on biomphalaria alexandrina snails

The present study was done using two concentrations of the photosensitizer Hematoporphyrin coated Gold Nanoparticles (HpdGNP3) (5x10–6 , 5x10–5 mole/dicemeter–3), to evaluate their efficacy on survival rate, egg-laying capacity of Biomphalaria alexandrina snails and on histological deteriorations in their hermaphrodite gland. B. alexandrina snails were incubated for 12 hours at each tested concentration in the dark, thereafter they were exposed to direct sunlight (336.2 W/m2) for either 2 or 4 hours followed by 24 hours of recovery. Control snails were treated with these concentrations without exposure to light irradiance. Another experiment was carried out simultaneously and the snails were left for 4 weeks of recovery to evaluate their egg-laying capacity (Mx). The results indicate that 5x10–5 Mdm–3 (HpdGNPs) with 4 hours of exposure sunlight suppressed the survival rate of B. alexandrina snails by 50%. Meanwhile, control snails incubated with 5x10–5 Mdm–3 HpdGNPs were not affected and still alive (100%). For snail’s fecundity (Mx), treated snails laid low number of eggs throughout the recov-ery period (4 weeks), in comparison with that of control ones. The highest value of Mx for snails treated with 5x10–5 Mdm–3 Hpd coated GNPs was recorded at the 3rd week of recovery period, being 6.7 eggs/snail, compared to 37.6 eggs/control snail. This has a negative reflect on the reproductive rate (Ro) of treated snails as it was reduced under these conditions by 76.6% and 86.1%, respectively.Histological tests re-vealed injuries in spermatocytes, oocytes, several degenerations of B.alexandrina hermaphrodite gland then evacuations in many gonad’s cells which severely suppressed their capacity for egg-laying. It is concluded from the present work that exposing B. alexandrina snails to sublethal concentrations of the photosensiter Hpd coated GNPs (12 hours incubation, 4 hours exposure to 336.2 W/m2) significantly reduced their reproductive capacity that may have a negative reflect on schistosomiasis transmis-sion.

Efficacy of Photogem（Hematoporphyrin Derivative） as a Photoactivatable Larvicide against Aedes aegypti （Diptera： Culicidae） Larvae

Dengue vector is responsible for millions of deaths every year and has caused disastrous impacts on health systems. The continuous use of chemical insecticides, such as carbamates, pyrethroids and organophosphates generates resistant populations of the mosquito, therefore, new control methods must be investigated. The joint action of the population and guidelines for preventing the reproduction of the mosquito associated with the use of photoactivatable insecticides can be the alternative for the control of epidemiological outbreaks in affected regions. In this study, the photo-larvicidal activity of Photogem^（PG）, a derivative of hematoporphyrin, was investigated against 2nd-early 3rd instar of Aedes aegypti larvae （Diptera： Culicidae） under different lighting conditions （artificial lighting system and sunlight）. The dynamics of PG accumulation was characterized by CLSM （confocal laser scanning microscopy） and total time PG eliminationin solution was investigated by ultraviolet-visible spectrophotometry. The maximum photo-activity of PG was observed in 0.5 h under sunlight exposure which achieved 100% larval mortality. Fluorescence images showed a uniform distribution of PG along the digestive tract. PG remained stable in the sunlight for 48 h and in an artificial lighting system for longer periods, therefore, it can be used for the control ofAedes aegypti larvae as a new alternative to chemical insecticides. The method is considered environmentally friendly due to its rapid degradation in the presence of light. Further studies are required, so that the potential of the technique can be explored in real breeding places.

Gold nanorods with a hematoporphyrin-loaded silica shell for dual-modality photodynamic and photothermal treatment of tumors in vivo

Nanocomposites （NCs） consisting of a gold nanorod core and a mesoporous silica shell doped with hematoporphyrin （HP） have been fabricated in order to improve the efficiency of cancer treatment by combining photothermal and photodynamic therapies （PDT ＋ PTT） in vivo. In addition to the long-wavelength plasmon resonance near 810-830 nm, the fabricated NCs exhibited a 400-nm absorbance peak corresponding to bound HP, generated singlet oxygen under 633-nm excitation near the 632.5-nm Q-band, and produced heat under a 808-nm near-infrared （NIR） laser irradiation. These modalities were used for a combined PDT ＋ PTT treatment of large （about 3 cm3） solid tumors in vivo with a xenorafted tumor rat model. NCs were directly injected into tumors and irradiated simultaneously with 633-nm and 808-nm lasers to stimulate the combined photodynamic and photothermal activities of NCs. The efficiency of the combined therapy was evaluated by optical coherence tomography, histological analysis, and by measurements of the tumor volume growth during a 21-day period. The NC-mediated PDT led to weak changes in tissue histology and to a moderate 20% decrease in the tumor volume. In contrast, the combined PDT ＋ PTT treatment resulted in the large-area tumor necrosis and led to dramatic decrease in the tumor volume.

Synergistic effects of focus ultrasound with different frequency and hematoporphyrin on human tumor cells

Human hematopoietic cell K 562 , human melenoma cell LiBr and human stomach cancer cells were exposed to ultrasound (US, 1.75 W/cm\+2, 1.4, 2.16 and 2.4 MHz) in vitro in the presence or absence of hematoporphyrin (Hp, 100 μg/mL). The cell damaging effects of treatments were determined by means of the Trypan Blue dye exclusion test, MTT test and FDA test. The experimental results showed that the same cell line had different sensibilities to the US of different frequencies, and different cell line had different damage at the same acoustical radiation. The combined treatment with US and Hp enhanced greatly the cell damage, and no sensibility of insonation cells to US with Hp was observed. The cell damage tests showed that the results of MTT test corresponded well with that of Trypan Blue dye test.

INQUIRY OF PHOTOSENSITIZATION MECHANISM OF YANGZHOU HEMATOPORPHYRIN DERIVATIVE(YHPD)

By the use of the advanced ESR technique and through comparing with BHPD, the characteristic of YHPD photosensitization is discussed in this paper in the respect of the primary process of photosensitization. The experiment results showed: (ⅰ) not only ~1O_2, but also free radicals(O_2· OH and YHPD)can be formed by the aid of YHPD; and (ⅱ) as to the ability of producing ~1O_2, YHPD<BHPD, while for generating O_2 and-OH, YHPD>BHPD. Two points are indicated: first, the photosensitized damage of YHPD is interrelated to not only ~1O_2, but also free radicals (O_2. OH and YHPD); second, although the photosensitized damage of YHPD is stronger than that of BHPD, yet the photosensitized damage is negatively correlated to the yield of ~1O_2 but positively correlated to those of O_2 and OH. Based on these two points, it is suggested that activated oxygen free radicals(O_2 and. OH) instead of ~1O_2 play the main role as instantaneously activated material in the photosensitized damage of YHPD.

NMR STUDY ON THE MAJOR COMPONENTS IN HEMATOPORPHYRIN DERIVATIVE YHPD

The hematoporphyrin derivative YHPD, a China-made product, has been clinically used in photodynamic therapy of tumors as a good photosensitizing drug. The NMR study on the structure of its major components is reported here. In terms of high performance liquid chromatography (HPLC) four major components A, B, C and D were isolated. The NMR results showed that the component A is O-acetylhematoporphyrin, B and C are two isomers of vinyldeuteroporphyrin. The spectra of 2-dimensional homonuclear correlation NMR, 2-dimensional NOE (nuclear overhauser enhancement), ^(13)C-NMR and off-resonance as well as FAB (fast atom bombarding) mass spectrum of component D indicate that it is a protoporphyrin dimer linked by carbon-carbon bond. This finding may providea chemical basis for understanding the difference in biological activity between YHPD and other foreign commercial HPD, as well as the composition of clincally used alkali-treated HPD and its effective component.

STUDIES ON THE EXPOSITION OF HYDROXYL RADICAL FROM HEMATOPORPHYRIN DERIVATIVE SOLUTION TO LIGHT BY SPIN-TRAPPING METHOD

That the photosensitive action of hematoporphyrin can damage tumor cells, has attracted much attention recently. However, its mechanism of action is not very clear. Some reported that the damage is due to the production of singlet oxygen （1O2）, a cy-

THE ELECTRON SPIN RESONANCE STUDY ON PRIMARY PHOTOCHEMICAL REACTION OF HEMATOPORPHYRIN DERIVATIVE

The rate of oxygen consumption and the yield of free radical anion of hematoporphyrin derivative (HPD) in aqueous solutions of HPD and pyrocatechol were measured by the probe 2,2,6,6-tetramethyl4-piperidone-1-oxyl.It has been found that both singlet oxygen and free radical mechanisms exist simultaneously in primary photochemical reactions, and there is a competition between both mechanisms. When the oxygen concentration in solutions comes down to 12-14% of the stanting level, the predominant mechanism can be changed from the singlet oxygen to the free radical.Whether HPD exists in aggregation state is very important to photosensitization mechanisms.In the presence of the aggregation state of HPD, the predominant mechanism is the free radical,and photosensitization effects of HPD are all the better.