Long-term infusions of p-boronophenylalanine for boron neutron capture therapy: evaluation using rat brain tumor and spinal cord models

Rat 9L gliosarcoma cells infiltrating the normal brain have been shown previously to accumulate only approximately 30% as much boron as the intact tumor after administration of the boronated amino acid p-boronophenylalanine (BPA). Long-term i.v. infusions of BPA were shown previously to increase the boron content of these infiltrating tumor cells significantly. Experiments to determine whether this improved BPA distribution into infiltrating tumor cells after a long-term i.v. infusion improves tumor control after BNCT in this brain tumor model and whether it has any deleterious effects in the response of the rat spinal cord to BNCT are the subjects of the present report. BPA was administered in a fructose solution at a dose of 650 mg BPA/kg by single i.p. injection or by i.v. infusion for 2 h or 6 h, at 330 mg BPA/kg h(-1). At 1 h after the end of either the 2-h or the 6-h infusion, the CNS:blood (10)B partition ratio was 0.9:1. At 3 h after the single i.p. injection, the ratio was 0.6:1. After spinal cord irradiations, the ED(50) for myeloparesis was 14.7 +/- 0.4 Gy after i.p. administration of BPA and 12.9 +/- 0.3 Gy in rats irradiated after a 6-h i.v. infusion of BPA; these values were significantly different (P < 0.001). After irradiation with 100 kVp X rays, the ED(50) was 18.6 +/- 0.1 Gy. The boron compound biological effectiveness (CBE) factors calculated for the boron neutron capture dose component were 1.2 +/- 0.1 for the i.p. BPA administration protocol and 1.5 +/- 0.1 after irradiation using the 6-h i.v. BPA infusion protocol (P < 0.05). In the rat 9L gliosarcoma brain tumor model, the blood boron concentrations at 1 h after the end of the 2-h infusion (330 mg BPA/kg h(-1); n = 15) or after the 6-h infusion (190 mg BPA/kg h(-1); n = 13) were 18.9 +/- 2.2 microg 10B/g and 20.7 +/- 1.8 microg 10B/g, respectively. The irradiation times were adjusted individually, based on the preirradiation blood sample, to deliver a predicted 50% tumor control dose of 8.2 Gy ( approximately 30 photon-equivalent Gy) to all tumors. In the present study, the long-term survival was approximately 50% and was not significantly different between the 2-h and the 6-h infusion groups. The mode of BPA administration and the time between administration and irradiation influence the 10B partition ratio between the CNS and the blood, which in turn influences the measured CBE factor. These findings underline the need for clinical biodistribution studies to be carried out to establish 10B partition ratios as a key component in the evaluation of modified administration protocols involving BPA.     

Intratumor Administration of the Photosensitizer Pc 4 Affords Photodynamic Therapy Efficacy and Selectivity at Short Drug-Light Intervals

We evaluated intratumor (IT) versus intravenous (IV) administration of the photosensitizer Pc 4 with respect to tumor photosensitizer concentration, specificity, and responses to irradiation. BALB/c mice bearing intradermal EMT6 tumors were given 0.3 mg/kg Pc 4 injected IT or IV through the tail vein. Photosensitizer concentration was evaluated by chloroform extraction and localization assessed by fluorescence imaging and spectroscopy in vivo. Tumors were irradiated at 667 nm, 50 mW/cm², and 100 J/cm². Cures were defined as no palpable tumor 90 days after irradiation. Tumor Pc 4 concentrations 1 hour after IT administration were 35,000-fold higher than measured 24 hours after IV administration (0.112 vs 0.317 x 10^-5 µg Pc 4/mg tumor). Exquisite tumor selectivity was observed 1 hour after IT injection. Fluorescence imaging of freshly sectioned tumors revealed no regions devoid of sensitizer at this time point, with pixel intensities in a midline section within a factor of 3 of the peak intensity. For identical photosensitizer doses, IT administration significantly improved tumor responses to irradiation, with more than 70% of tumors cured with IT-Pc 4-PDT. In this model, IT-Pc 4 administration provides improved tumor control, greater selectivity, and opportunity for a short drug-light interval.     

Increase in tumor oxygenation and radiosensitivity caused by pentoxifylline.

The effects of pentoxifylline (PTX), a drug commonly used for vascular disorders in humans, on the pO2 in SCK tumors of A/J mice and FSa-II tumors of C3Heb/FeJ mice as well as on the radioresponse of SCK tumors were investigated. When the host mice were injected intraperitoneally (ip) with 5 mg/kg PTX, the tumor pO2 increased slowly, peaked 20-50 min postinjection, and returned to its original level in 70-90 min. The magnitude of the increase in tumor pO2 varied markedly depending on the site and tumors. The magnitude of the changes in tumor pO2 after an ip injection of 25 or 50 mg/kg PTX was similar to that caused by 5 mg/kg PTX, but the pO2 tended to remain elevated longer with the higher dose of PTX. When the A/J mice bearing SCK tumors in the legs were injected ip with 50 mg/kg PTX and the tumors were X-irradiated 20 min later, the radiation-induced growth delay of the tumors was greater than that caused by X irradiation alone. The present study demonstrated that PTX is potentially useful for increasing the pO2 and the radioresponse of human tumors.     

Hepatoprotective effect of aqueous extract of Phyllanthus niruri on nimesulide-induced oxidative stress in vivo

Nimesulide (NIM), an atypical non-steroidal anti-inflammatory drug (NSAID) is also used as analgesic. In the present study, we evaluated its effect on the prooxidant-antioxidant system of liver and the hepatoprotective potential of aqueous extract of the herb Phyllanthus niruri (PN) on NIM-induced oxidative stress in vivo using a murine model, by determining the activities of hepatic anti-oxidant enzymes superoxide dismutase (SOD) and catalase (CAT), levels of reduced glutathione (GSH) and lipid peroxidation (expressed as malonaldialdehyde, MDA). Aqueous extract of PN at a dose of 50 or 100 mg/kg body wt was administered either intraperitoneally or orally for 7 days, before NIM administration at a dose of 8 mg/kg body wt twice daily for 7 days in mice. Animals were sacrificed 24 h after administration of final dose of NIM. In another set of experiments, both aqueous extract of PN (at a dose of 50 or 100 mg/kg body wt) and NIM (8 mg/kg body wt) were administered simultaneously for 7 days. Animals were sacrificed 24 h after administration of final dose of the extract and NIM, liver tissues were collected, and the activities of SOD and CAT and levels of GSH and lipid peroxidation end-product (as MDA), were determined from the livers of all the experimental animals. Appropriate NIM control was maintained for all sets of experiments. NIM administration (8 mg/kg body wt) for 7 days caused significant depletion of the levels of SOD, CAT and reduced GSH, along with the increased levels of lipid peroxidation. Intraperitoneal administration of the extract at a dose of 50 mg/kg body wt for 7 days,. prior to NIM treatment, significantly restored most of the NIM-induced changes and the effect was comparable to that obtained by administering 100 mg/kg body wt of the extract orally. Thus, results suggested that intraperitoneal administration of the extract could protect liver from NIM-induced hepatic damage more effectively than oral administration. Antioxidant property of the aqueous extract of PN was also compared with that of a known potent antioxidant, vitamin E. The PN extract at a dose of 100 mg/kg body wt along with NIM was more effective in suppressing the oxidative damage than the PN extract at a dose of 50 mg/kg body wt. Results suggested that beneficial effect of the aqueous extract of PN, probably through its antioxidant property, might control the NIM-induced oxidative stress in the liver.     

Schistosoma japonicum: effect of artemether on glutathione S-transferase and superoxide dismutase

Glutathione S-transferase (GST) and superoxide dismutase (SOD) are major antioxidant enzymes of schistosomes that are involved in detoxification processes. To study the effect of artemether on these enzymes, mice infected with adult Schistosoma japonicum, were treated with artemether either at a subcurative (100 mg/kg) or a curative dose (300 mg/kg). Schistosomes were recovered 24-72 h post-treatment separated by sex and used for GST and SOD activity measurements. Female worms showed consistently higher GST inhibitions than males. For instance, 24 h after administration of 100 mg/kg artemether, GST activities of female worms were inhibited by 23.3%, as compared to 12.7% in males. Both activities were significantly lower when compared to worms recovered from untreated mice. Slightly higher inhibitions were observed at the higher dose of artemether, which gradually increased to levels of 52.5-55.1%, 72 h post-treatment. GST inhibitions could be reversed by application of 1,4-dithiothreitol at a concentration of 10 mmol/L. Adding L-cysteine also reduced GST inhibitions, but in female worms, GST activities remained significantly higher than in worms from untreated animals. Administration of 300 mg/kg artemether resulted in significant reductions of SOD activities in both sexes. In conclusion, these results suggest that the inhibition of GST and, to a lesser extent also SOD enzymes, could lead to increased schistosome susceptibility to oxidant attacks and might be linked with the antischistosomal action of artemether.     

Modification of reperfusion-induced ionic imbalance by free radical scavengers in spontaneously hypertensive rat retina

We studied the effects of free radical scavengers, superoxide dismutase (SOD), vitamin E, and EGB 761, on ion shifts (Na⁺, K⁺, and Ca²⁺) induced by ischemia reperfusion in rat retina obtained from spontaneously hypertensive rats. Eyes were subjected to 90 min of retinal ischemia followed by 24 h of reperfusion. Two basic protocols were used: (1) chronic application, in which rats received SOD (7500, 15,000, and 30,000 U/kg, i.v.), vitamin E (50, 100, and 200 mg/kg, i.v.), and EGB 671 (50, 100, and 200 mg/kg, orally) for 10 d, respectively; and (2) acute administration, in which 7500, 15,000, and 30,000 U/kg of SOD, 50, 100, and 200 mg/kg of vitamin E, and 50, 100, and 200 mg/kg of EGB 761 were administered after an ischemic episode, at the onset of reperfusion, respectively. In the drug-free control group, 90 min ischemia followed by 24 h of reperfusion resulted in an accumulation of retinal sodium and calcium from their nonischemic control values of 76 ± 4 and 3.2 ± 0.1 μmol/g dry weight to 112 ± 6 (p < .001) and 6.2 (p < .001) μmol/g dry weight, respectively. Tissue potassium loss was also observed in this model of retinal ischemia reperfusion, and after 90 min ischemia followed by 24 h of reperfusion potassium content was significantly reduced from its nonischemic control value of 266 ± 5 to 207 ± 6 (p < .001) μmol/g dry weight. The chronic administration of SOD, vitamin E, and EGB 761 dose dependently reduced the reperfusion-induced ionic imbalance and improved the recovery of retinal ion contents. When these drugs were administered at the onset of reperfusion (acute administration), SOD and EGB 761 still significantly improved the recovery of retinal ion contents, but vitamin E failed to protect the ischemic reperfused retina. Our results indicate that the elimination of oxygen free radicals by free radicals scavengers may reduce the reperfusion-induced ionic imbalance and improve the ionic homeostasis in the injured retinal cells obtained from spontaneously hypertensive rats.     

Effects of laser photodynamic therapy on tumor phosphate levels and pH assessed by 31P-NMR spectroscopy

The effects of photodynamic therapy using 632 nm photoradiation emitted from an ion pumped dye laser system on the phosphate metabolite levels of rat mammary tumors were monitored by 31P-NMR spectroscopy. A dramatic decline to almost undetectable levels, in the ratio of whole tumor beta-ATP (NTP) to Pi was observed after systemic administration of 5 mg/kg Photofrin II 24 h prior to exposure of R3230AC rat mammary tumor to laser irradiation at 180 and 360 J/cm2 total fluence. This decline in ATP was accompanied by a concomitant increase in the levels of Pi relative to the total observable phosphate signals. Whole tumor pH was calculated from the chemical shift in inorganic phosphate using the water proton signal as reference. Under the same treatment conditions used to monitor the phosphate metabolites following Photodynamic Therapy, the pH of the tumor as a whole decreased approximately 0.35 units at the time when the beta-ATP to Pi ratios were lowest. This maximal decrease in whole tumor ATP levels and pH, which occurred at 4-6 h post irradiation, was followed by a gradual return to pre-treatment levels over a 24 h period. These results demonstrate that Photodynamic Therapy employing porphyrin photosensitization and monochromatic laser irradiation is effective in reducing both tumor high energy phosphate levels and pH. Depending on sensitizer dose and light fluence, metabolic inhibition, represented by depleted nucleoside triphosphates and elevated Pi, may be reversible.     

A novel drug to reduce tumor perfusion: antitumor effect alone and with hyperthermia

We have investigated the feasibility of enhancing damage induced by hyperthermia in SCK murine tumors by reducing tumor blood perfusion using a new agent, KB-R8498. Within several minutes of an i.v. injection, the tumor perfusion was reduced to less than 20% of the control value, and it recovered to 40-70% of the control value by 1 h after injection. The perfusion in normal tissues decreased or increased soon after drug administration depending on the tissue type. However, by 1 h after drug treatment, perfusion in five of the seven tissues examined had returned to the control level. The tumor pH was also reduced after i.v. drug administration. Control tumors grew to four times the initial volume in 6 days. Tumors that were heated at 42.5 degrees C for 60 min were delayed in growth by 4 days compared to control tumors. There was a growth delay of 14 days when an i.v. injection of KB-R8498 was given and the tumors were heated at 42.5 degrees C either immediately or 1 h later. In drug-alone studies, the tumor growth was delayed by 4 days when the drug was infused continuously at a rate of 30-50 mg/kg day(-1) for 7 days or about 2 days when mice were treated with five daily injections of 30 mg/kg KB-R8498.     

PLD repair in rat rhabdomyosarcoma tumor cells irradiated in vivo and in vitro with high-LET and low-LET radiation

Results are reported of studies to measure the extent of recovery of potentially lethal damage (PLD) in rat rhabdomyosarcoma tumor cells after irradiation both in vivo and in vitro with either high-LET or low-LET radiation. Stationary-phase cultures were found to exhibit repair of PLD following irradiation in vitro either with low-LET X rays or with high-LET neon ions in the extended-peak ionization region. Following a 9-Gy dose of 225-kVp X rays or a 3.5-Gy dose of peak neon ions, both of which reduced the initial cell survival to 6-8%, the maximum PLD recovery factors were 3.4 and 1.6, respectively. In contrast, the standard tumor excision assay procedure failed to reveal any recovery from PLD in tumors irradiated in situ with either X rays or peak neon ions. PLD repair by the in vivo tumor cells could be observed, however, when the excision assay procedure was altered by the addition of a known PLD repair inhibitor beta-arabinofuranosyladenine (beta-ara-A). When a noncytotoxic 50 microM concentration of beta-ara-A was added to the excised tumor cells immediately following a 14.5-Gy in situ dose of X rays, cell survival in the inhibitor-treated cells was lower than in the untreated cells (0.018 compared to 0.056), resulting in a PLD repair inhibition factor of 3.1. Delaying the addition of beta-ara-A for 1, 2, or 3 h following tumor excision reduced the PLD repair inhibition factor to 1.6, 1.5, and 0.9, respectively. Following tumor irradiation in situ with neon ions in the extended-peak ionization region (median LET = 145 keV/micron), less PLD repair was observed than after X irradiation. For 5.8 Gy of peak neon ions, the PLD repair inhibition factors were 2.1, 1.5, 1.3, and 1.1 at 0, 1, 2, and 3 h, respectively. We interpret the absence of measurable PLD repair using the standard tumor excision assay procedure as resulting from undetectable repair occurring during the long interval (about 2 h) required for the cell dissociation and plating procedures. We conclude that at least for our tumor system, PLD repair does occur after irradiation of tumors in situ, even though it is not detectable using the standard tumor excision assay procedure. Thus a failure to measure such repair by this assay in a given tumor system does not necessarily mean the cells are incapable of PLD repair.     

Pharmacokinetic profile of 3beta-hydroxy-17-(1H-1,2,3-triazol-1-yl)androsta-5,16-diene (VN/87-1), a potent androgen synthesis inhibitor, in mice

The objective of this study was to assess the pharmacokinetics and bioavailability of 3beta-hydroxy-17-(1H-1,2,3-triazol-1-yl)androsta-5,16-diene (VN/87-1) in normal male mice and in SCID mice bearing human LNCaP tumor xenografts. VN/87-1 is a novel potent steroidal inhibitor of human testicular 17-alpha-hydroxylase/C(17,20)-lyase. The steroid also shows anti-androgenic activity and inhibits the growth of human prostate cancer cell lines (LNCaP) in vitro and in vivo. Male Balb/c mice were given a single oral, subcutaneous (s.c.) or intravenous (i.v.) bolus dose of VN/87-1 (25, 50 or 100 mg/kg). Male SCID mice bearing LNCaP tumor xenografts were injected with a single s.c. dose of VN/87-1 (50 mg/kg). The animals were sacrificed at various times up to 24 h after drug administration and blood was collected. The plasma samples were prepared and analyzed by a reversed phase HPLC system equipped with a diode array detector. A non-compartmental pharmacokinetic approach was used to evaluate the plasma level versus time data. Following i.v. administration of VN/87-1, the plasma levels declined exponentially with an elimination half-life of 1.2+/-0.03 h. The absolute bioavailability of the 50 mg/kg dose after oral or s.c. administration was 12.08+/-2 or 57.2+/-4.5%, respectively. VN/87-1 is a high clearance (5.0+/-1.3 l/h per kg) compound in mice and its volume of distribution was relatively large (6.5+/-1.2 l/kg). The pharmacokinetic parameters of VN/87-1 were not significantly altered in SCID mice bearing human LNCaP tumor xenografts. VN/87-1 is well absorbed from the subcutaneous site compared with absorption from the gastrointestinal tract and shows linear kinetics at doses up to 100 mg/kg.     

New non-steroidal aromatase inhibitors: focus on R76713

R76713 is a novel triazole derivative which selectively blocks the cytochrome P450-dependent aromatase. In human placental microsomes, in FSH-stimulated rat and human granulosa cells and in human adipose stromal cells, 50% inhibition of estradiol biosynthesis was obtained at drug concentrations of 2-10 nM. In PMSG-injected female rats, R76713 lowered plasma estradiol levels by 50 and 90% 2 h after single oral doses of 0.005 and 0.05 mg/kg respectively. After 1 mg/kg, estradiol levels were suppressed by 90% for 16 h. In male cynomolgus monkeys, R76713 dose-dependently (0.03-10 micrograms/kg) inhibited peripheral aromatization with an ED50 of 0.13 microgram/kg without altering metabolic clearance rates and conversion ratios. In vitro R76713 had no effect on other P450-dependent steroidogenic enzymes up to 1000 nM at least. In rats, LHRH-, ACTH- and sodium-deprived diet stimulated plasma testosterone, corticosterone and aldosterone levels were not modified 2 h after single oral administrations of R76713 (up to 20 mg/kg). Furthermore, R76713 did not show any in vitro or in vivo estrogenic or antiestrogenic property. R76713 also induced regression of DMBA-induced mammary tumors after daily oral administration of 1 mg/kg b.i.d. In male volunteers (n = 4), a single oral dose of 5 and 10 mg lowered median plasma estradiol levels from 70 pM to the detection limit of the assay (40 pM) 4, 8 and 24 h after intake whereas no changes were detected after placebo administration. In premenopausal women (n = 15), receiving a single oral dose of 20 mg, median plasma estradiol levels decreased from 389 pM (before) to 168, 133 and 147 pM, 4, 8 and 24 h after intake whereas they remained above 420 pM after placebo (n = 7).     

Time Modulation Effect of Diethyldithiocarbamate (DDC) on Radiosensitization by Superoxide Dismutase (SOD) Inhibition

Superoxide dismutase (SOD) is known to protect cells from the lethal effects of ionizing radiation by the dismutation of oxygen radicals. Diethyldithiocarbamate (DDC) is known inhibitor of SOD and may therefore be useful as a radiosensitizer. DDC however, is also a thinly radio protector due to its ability to scavenge radiation induced free radicals. We have shown that DDC. if administered to turnours 1 hour prior to x-irradiation exerts a protective effect, whereas if administered 4 hours prior to irradiation, it radiosensitizes. This time modulation effect is not apparent after neutron irradiation where DDC protects in both situations. We have also examined the effect of DPC on the LD_50/30 in mice after total body irradiation.     

Metrifonate and dichlorvos: Effects of a single oral administration on cholinesterase activity in rat brain and blood

Cholinesterase activities in rat forebrain, erythrocytes, and plasma were assessed after a single oral administration of metrifonate or dichlorvos. In 3-month-old rats, the dichlorvos (10 mg/kg p.o.)-induced inhibition of cholinesterase reached its peak in brain after 15–45 min and after 10–30 min in erythrocytes and plasma. Cholinesterase activity recovered rapidly after the peak of inhibition, but did not reach control values in brain and erythrocytes within 24 h after drug administration. The recovery of plasma cholinesterase activity, in contrast, was already complete 12 h after dichlorvos treatment. Metrifonate (100 mg/kg p.o.) had qualitatively similar inhibition kinetics as dichlorvos, albeit with a slightly delayed onset. Peak values were attained 45–60 min (brain) and 20–45 min (blood), after drug administration. Apparently complete recovery of cholinesterase activity was noted in both tissues 24 h after treatment. The dose-dependence of drug-induced inhibition of cholinesterase in rat blood and brain was determined at the time of maximal inhibition, i.e., 30 min after dichlorvos treatment and 45 min after metrifonate treatment. The oral ED₅₀ values obtained for dichlorvos were 8 mg/kg for brain and 6 mg/kg for both erythrocyte and plasma cholinesterase. The corresponding oral ED₅₀ values for metrifonate were 10 to 15 times higher, i.e., 90 mg/kg in brain and 80 mg/kg in erythrocytes and plasma. In rats deprived of food for 18 h before drug treatment, the corresponding ED₅₀ values for metrifonate were 60 and 45 mg/kg, respectively, indicating an about two-fold higher sensitivity of fasted rats to metrifonate-induced cholinesterase inhibition compared to non-fasted rats. Compared to 3-month-old rats, 19-month-old rats showed a higher sensitivity towards metrifonate and dichlorvos. At the time of maximal inhibition, there was a strong correlation between the degree of cholinesterase inhibition in brain and blood. These results demonstrate that single oral administration of metrifonate and dichlorvos induces an inhibition of blood and brain cholinesterase in the conscious rat in a dose-dependent and apparently fully reversible manner. While the efficiency of a given dose of inhibitor may vary with the satiety status or age of the animal, the extent of brain ChE inhibition can be estimated from the level of blood ChE activity.     

Antioxidants and antioxidant enzymes protect against pulmonary oxygen toxicity in the rabbit

Two major lines of defense exist against oxidant lung injury: tissue antioxidants and antioxidant enzymes. We studied pretreatment with the antioxidants, vitamin E and butylated hydroxyanisole (BHA), and the antioxidant enzymes, superoxide dismutase (SOD) and catalase, in rabbits exposed to 100% O2 for 48 h. BHA (200 mg/kg ip) or vitamin E (50-100 mg/kg po) were given for 2 or 3 days, respectively, before O2 exposure. Combined therapy with polyethylene glycol- (PEG) conjugated SOD (12 mg/kg) and catalase (200,000 U/kg) was given intraperitoneally 1 h before and 24 h after beginning 100% O2. Hyperoxia significantly increased the pulmonary content of malondialdehyde, indicating enhanced lipid peroxidation. One hundred percent O2 also increased lung weight gain and alveolar-capillary permeability to aerosolized 99mTc-labeled diethylenetriaminepentaacetate (99mTc-DTPA, 500 mol wt) and fluorescein isothiocyanate-labeled dextran (7,000 mol wt). Pretreatment with vitamin E, BHA, or the combination of PEG-SOD and PEG-catalase prevented the increase in malondialdehyde, lung weight gain, and alveolar-capillary permeability caused by hyperoxia. These results indicate that augmenting either tissue antioxidants or antioxidant enzymes can prevent the pulmonary injury caused by 48 h of 100% O2 in rabbits.     

Time Modulation Effect of Diethyldithiocarbamate (DDC) on Radiosensitization by Superoxide Dismutase (SOD) Inhibition

Superoxide dismutase (SOD) is known to protect cells from the lethal effects of ionizing radiation by the dismutation of oxygen radicals. Diethyldithiocarbamate (DDC) is known inhibitor of SOD and may therefore be useful as a radiosensitizer. DDC however, is also a thinly radio protector due to its ability to scavenge radiation induced free radicals. We have shown that DDC. if administered to turnours 1 hour prior to x-irradiation exerts a protective effect, whereas if administered 4 hours prior to irradiation, it radiosensitizes. This time modulation effect is not apparent after neutron irradiation where DDC protects in both situations. We have also examined the effect of DPC on the LD_50/30 in mice after total body irradiation.     

Matrix metalloproteinases inhibition provides neuroprotection against hypoxia-ischemia in the developing brain

The present study was designed to investigate the role of matrix metalloproteinases (MMPs) in the immature brain and the long term effects of early MMPs inhibition after hypoxic-ischemic (HI) injury. HI was induced by unilateral ligation of the right carotid artery followed by hypoxia (8% O₂ for 2 h) in P7 rat pups. GM6001, a broad spectrum MMPs inhibitor, was injected (50 mg/kg or 100 mg/kg) intraperitoneally at 2 h and 24 h after HI injury. Blood-brain barrier (BBB) integrity, brain edema, MMP-2/-9 activity, TIMP-1/-2 and tight junction protein (TJP) level were evaluated using IgG staining, Evan's blue extravasation, brain water content, zymography and western blot. Doxycycline, another MMPs inhibitor, was injected (10 mg/kg or 30 mg/kg) intraperitoneally at 2 h after HI, then BBB integrity and brain edema were measured at 48 h post-HI using brain water content measurement and IgG staining. The long-term effects of early MMPs inhibition (GM6001, 100 mg/kg) were evaluated by neurobehavioral tests, body weight, and brain atrophy measurement. GM6001 attenuated brain edema and BBB disruption at the dosage of 100 mg/kg. MMP-2 activity increased at 24 h and peaked at 48 h after HI, whereas MMP-9 activity peaked at 24 h and tapered by 48 h after HI. MMP-9/-2 activities were significantly attenuated by GM6001 at 24 h and 48 h after HI. The degradation of TJPs (ZO-1 and occludin) at 48 h after HI was reversed by GM6001 treatment. Early MMPs inhibition had long-term effects that attenuated ipsilateral brain tissue loss, and improved neurobehavioral outcomes after HI. These results suggest that early MMPs inhibition with a broad-spectrum inhibitor provides both acute and long-term neuroprotection in the developing brain by reducing TJPs degradation, preserving BBB integrity, and ameliorating brain edema after neonatal HI injury.     

Antitumor effect of photodynamic therapy with zincphyrin, zinc-coproporphyrin III, in mice

We studied the antitumor effects of photodynamic therapy (PDT) with Zincphyrin, coproporphyrin III with zinc, derived from Streptomyces sp. AC8007, in vitro and in vivo. The photokilling effect of Zincphyrin in the presence of 0.78-100 microg/ml with visible light of 27.2 mW x min/cm2 for 10 min was lower than the hematoporphyrin (Hp) used as a control with L5178Y or sarcoma-180 cells. On the other hand, Zincphyrin apparently reduced tumor growth after intraperitoneal injection at doses of 12.5-50 mg/kg with light irradiation of 75.48 mW x min/cm2 for 10 min in sarcoma-180-bearing mice. Although no mice treated with Zincphyrin died, Hp did cause the death of mice. In B-16 melanoma-bearing mice, both Zincphyrin and Hp had a similar phototherapic effect. Further improvement of the phototherapic effect was observed with the continuous administration of Zincphyrin at 12.5 mg/kg per day for 3 days. The concentration of Zincphyrin in the serum reached a maximum level of 16 microg/ml within 20 min, and the concentration remained at 4.2 microg/ml at 1 hour after the onset of treatment, indicating its rapid action in the body. No animals died after the intraperitoneal administration of Zincphyrin at 100 mg/kg plus exposure to light of 10 mW x min/cm2 for 2 hours, and the body weight of the mice did not decrease. In contrast, all animals receiving 100 mg/kg of Hp under the same conditions died. These results indicate that Zincphyrin would be a useful photosensitizer with low phototoxicity.     

The antiangiogenic agents SU5416 and SU6668 increase the antitumor effects of fractionated irradiation.

Angiogenesis is critical for tumor development, growth and metastasis. The vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF) and their tyrosine kinase receptors are major regulators of angiogenesis. Radiation induces the production of VEGF, FGF and PDGF in many tumor cells. We hypothesized that inhibition of the function of these growth factors could inhibit tumor angiogenesis and thereby enhance the efficacy of radiation therapy. To test this hypothesis, we used the small molecule inhibitors SU5416 (an inhibitor for Vegf receptor) and SU6668 (an inhibitor for Vegf, Fgf and Pdgf receptors) alone and in combination with fractionated irradiation to treat C3H mice bearing SCC VII carcinomas. The SCC VII tumors express Vegf, Fgf2 (also known as bFGF), Pdgf and their associated receptors. Animals were given either SU5416 or SU6668 daily before or after irradiation (2 Gy per fraction per day for 5 days). The results from these experiments demonstrate that administration of either SU5416 or SU6668 without radiation delayed tumor growth. Administration of SU5416 at a dose of 25 mg/kg per day (the maximum tolerated effective dose) inhibited tumor growth by 17.9% on day 7 (P < 0.05 compared to untreated control mice) and produced an average tumor growth delay time of 0.5-2.0 days. When combined with fractionated irradiation, administration of SU5416 increased the inhibition of tumor growth to 50-53% on day 7 and the tumor growth delay time to 5.7-6.5 days (P < 0.001 compared with SU5416 alone; P < or = 0.05 compared with radiation alone). SU6668 alone inhibited tumor growth in a dose-dependent manner. Administration of SU6668 at a dose of 75 mg/kg per day (a suboptimal dose) inhibited tumor growth by 36% on day 7 and produced an average tumor growth delay time of 3.3 +/- 1.4 days. The combination of SU6668 with fractionated radiation increased inhibition of tumor growth to 66-70% and the tumor growth delay time from 3.3 days to 11.9 days (P < or = 0.001 compared with either radiation alone or SU6668 alone). Administration of these agents before or after irradiation produced similar results (P = 0.40 for SU5416; P = 0.98 for SU6668). SU5416 or SU6668 alone or in combination with radiation was very well tolerated with little or no toxicity. These results suggest that inhibition of Vegf, Fgf and Pdgf receptor function by SU5416 and SU6668 can enhance the efficacy of irradiation. The targeting of multiple tyrosine kinase receptors by SU6668 is more effective than inhibition of the Vegf receptor alone by SU5416 for the enhancement of tumor cell killing by fractionated irradiation.     

Diethyldithiocarbamate injection induces transient oxidative stress in goldfish tissues

The effects of intraperitoneal injection of diethyldithiocarbamate (DDC) on free radical processes were examined in brain, liver and kidney of goldfish (Carassius auratus). Levels of oxidatively modified lipids and proteins as well as the activities of antioxidant and associated enzymes were measured. Intraperitoneal injection of DDC at a concentration of 0.01 mg/g wet mass decreased SOD activities by about 30-50% after 48 and 168 h compared to corresponding sham-injected values. This treatment resulted in transient oxidative stress. Lipid peroxide content increased after DDC injection at all time points in the kidney, after 48 h in the liver and was elevated in most experimental groups in the brain. Thiobarbituric-acid reactive substances (end products of lipid peroxidation) rose within the first 48 h after injection, but returned to initial levels after 168 h. Two other indices of oxidative stress were also transiently modified: protein carbonyl levels in the brain and kidney increased 24h post-injection, and the low-molecular mass thiol content was reduced over the same period in all tissues examined. Activities of catalase, glutathione peroxidase, glutathione-S-transferase, glutathione reductase, and glucose-6-phosphate dehydrogenase showed differential responses to DDC treatment that rebounded by 168 h post-injection. Glutathione peroxidase activities were reduced by 60, 45 and 65% in the brain, liver and kidney, respectively, after 24h but rebounded thereafter. After 48 h post-injection with DDC significant decreases were also seen in liver and kidney catalase, GST activities in all three tissues, and kidney GR and G6PDH activities. In some cases, catalase, GST, GR and G6PDH activities transiently increased after 24 h. It was concluded that DDC injection depleted SOD and simultaneously stimulated lipid peroxidation, but did not require compensatory enhancement of other enzymatic defenses. Different actions of the superoxide anion in cellular metabolism and possible consequences of the impairment of superoxide dismutase are discussed.