Effects of lecithin-based nanoemulsions on skin: Short-time cytotoxicity MTT and BrdU studies, skin penetration of surfactants and additives and the delivery of curcumin.

Surfactants are important ingredients in pharmaceutical and cosmetic formulations, as in creams, shampoos or shower gels. As conventional emulsifiers such as sodium dodecyl sulfate (SDS) have fallen into disrepute due to their skin irritation potential, the naturally occurring lecithins are being investigated as a potential alternative. Thus, lecithin-based nanoemulsions with and without the drug curcumin, known for its wound healing properties, were produced and characterised in terms of their particle size, polydispersity index (PDI) and zeta potential and compared to SDS-based formulations. In vitro toxicity of the produced blank nanoemulsions was assessed with primary human keratinocytes and fibroblasts using two different cell viability assays (BrdU and EZ4U). Further, we investigated the penetration profiles of the deployed surfactants and oil components using combined ATR-FTIR/tape stripping experiments and confirmed the ability of the lecithin-based nanoemulsions to deliver curcumin into the stratum corneum in tape stripping-UV/Vis experiments. All manufactured nanoemulsions showed droplet sizes under 250 nm with satisfying PDI and zeta potential values. Viability assays with human skin cells clearly indicated that lecithin-based nanoemulsions were superior to SDS-based formulations. ATR-FTIR tests showed that lecithin and oil components remained in the superficial layers of the stratum corneum, suggesting a low risk for skin irritation. Ex vivo tape stripping experiments revealed that the kind of oil used in the nanoemulsion seemed to influence the depth of curcumin penetration into the stratum corneum.

RP101 (brivudine) binds to heat shock protein HSP27 (HSPB1) and enhances survival in animals and pancreatic cancer patients.

BACKGROUND: Several reports describe the importance of the chaperone HSP27 (HSPB1) in cancer progression, and the demand for drugs that modulate HSPB1-activity is increasing rapidly. We reported earlier that RP101 (Bromovinyldeoxyuridine, BVDU, Brivudine) improves the efficacy of chemotherapy in pancreatic cancer. METHODS: Chemistry: Binding of RP101 and HSPB1 was discovered by affinity chromatography. Molecular and cell biology: HSPB1 in vitro transcription/translation (TNT), Pull down using RP101-coupled magnetic beads, Immuno Co-precipitations, Structural modeling of HSP27 (HSPB1), Introduction of point mutations into linear expression templates by PCR, Heat shock, Tumor Invasion. Animal experiments: Treatment of AH13r Sarcomas in SD-rats. Clinical Studies with late-stage pancreatic cancer patients: Pilot study, Dose finding study, Phase II study (NCT00550004). RESULTS: Here, we report that RP101 binds in vitro to the heat shock protein HSPB1 and inhibits interaction with its binding partners. As a result, more activated CASP9 was detected in RP101-treated cancer cells. We modeled HSPB1-structure and identified the RP101 binding site. When we tested RP101 as an anti-cancer drug in a rat model, we found that it improved chemotherapy. In clinical studies with late-stage pancreatic cancer patients, the dose of 500 mg/day was safe and efficient, but 760 mg/day turned out to be too high for lightweight patients. CONCLUSIONS: The development of RP101 as a cancer drug represents a truly novel approach for prevention of chemoresistance and enhancement of chemosensitivity.

Significance of manipulating tumour hypoxia and radiation dose rate in terms of local tumour response and lung metastatic potential, referring to the response of quiescent cell populations.

The purpose of this study was to evaluate the influence of manipulating intratumour oxygenation status and radiation dose rate on local tumour response and lung metastases following radiotherapy, referring to the response of quiescent cell populations within irradiated tumours. B16-BL6 melanoma tumour-bearing C57BL/6 mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. They received gamma-ray irradiation at high dose rate (HDR) or reduced dose rate (RDR) following treatment with the acute hypoxia-releasing agent nicotinamide or local hyperthermia at mild temperatures (MTH). Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the quiescent (Q) and total (proliferating + Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In other tumour-bearing mice, 17 days after irradiation, macroscopic lung metastases were enumerated. Following HDR irradiation, nicotinamide and MTH enhanced the sensitivity of the total and Q-cell populations, respectively. The decrease in sensitivity at RDR irradiation compared with HDR irradiation was slightly inhibited by MTH, especially in Q cells. Without gamma-ray irradiation, nicotinamide treatment tended to reduce the number of lung metastases. With gamma-rays, in combination with nicotinamide or MTH, especially the former, HDR irradiation decreased the number of metastases more remarkably than RDR irradiation. Manipulating both tumour hypoxia and irradiation dose rate have the potential to influence lung metastasis. The combination with the acute hypoxia-releasing agent nicotinamide may be more promising in HDR than RDR irradiation in terms of reducing the number of lung metastases.

Influence of manipulating hypoxia in solid tumors on the radiation dose-rate effect in vivo, with reference to that in the quiescent cell population.

PURPOSE: The aim of this study was to clarify the effect of manipulating intratumor hypoxia on radiosensitivity under reduced dose-rate (RDR) irradiation. MATERIALS AND METHODS: Tumor-bearing mice were continuously given 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating (P) cells. They received gamma-rays or accelerated carbon-ion beams at high dose-rate (HDR) or RDR with or without tumor clamping to induce hypoxia. Some mice without clamping received nicotinamide, an acute hypoxia-releasing agent or misonidazole, a hypoxic cell radio-sensitizer before irradiation. The responses of quiescent (Q) and total (= P + Q) cells were assessed by the micronucleus frequency using immunofluorescence staining for BrdU. RESULTS: The clearer decrease in radiosensitivity in Q than total cells after RDR gamma-ray irradiation was suppressed with carbon-ion beams, especially with a higher linear energy transfer value. Repressing the decrease in the radiosensitivity under RDR irradiation through keeping tumors hypoxic during irradiation and enhancing the decrease in the radiosensitivity by nicotinamide were clearer with gamma-rays and in total cells than with carbon-ion beams and in Q cells, respectively. Inhibiting the decrease in the radiosensitivity by misonidazole was clearer with gamma-rays and in Q cells than with carbon-ion beams and in total cells, respectively. CONCLUSION: Manipulating hypoxia during RDR as well as HDR irradiation influences tumor radiosensitivity, especially with gamma-rays.

Neurogenesis in the hypothalamus of adult mice: potential role in energy balance.

Ciliary neurotrophic factor (CNTF) induces weight loss in obese rodents and humans, and for reasons that are not understood, its effects persist after the cessation of treatment. Here we demonstrate that centrally administered CNTF induces cell proliferation in feeding centers of the murine hypothalamus. Many of the newborn cells express neuronal markers and show functional phenotypes relevant for energy-balance control, including a capacity for leptin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Coadministration of the mitotic blocker cytosine-beta-d-arabinofuranoside (Ara-C) eliminates the proliferation of neural cells and abrogates the long-term, but not the short-term, effect of CNTF on body weight. These findings link the sustained effect of CNTF on energy balance to hypothalamic neurogenesis and suggest that regulated hypothalamic neurogenesis in adult mice may play a previously unappreciated role in physiology and disease.

Hippocampal cell proliferation and epileptogenesis after audiogenic kindling are not accompanied by mossy fiber sprouting or Fluoro-Jade staining.

Repetitive sound-induced seizures, known as audiogenic kindling (AK), gradually induce the transference of epileptic activity from brainstem to forebrain structures along with behavioral changes. The aim of our work was to correlate the behavioral changes observed during the AK with possible alterations in neuronal proliferation, cell death, hippocampal mossy fiber sprouting and in the EEG pattern of Wistar audiogenic rats, a genetically susceptible strain from our laboratory. Susceptible and non-susceptible animals were submitted to repeated sound stimulations for 14-16 days and hippocampal mitotic activity was studied through the incorporation of bromodeoxyuridine (BrdU). Cell death and mossy fiber sprouting were assessed, respectively, by using Fluoro-Jade and Timm staining, 2 and 32 days after the last kindling stimulation. In addition, we used immunofluorescent double labeling for a glial and a mitotic marker to evaluate newly born cell identity. Some animals had hippocampus and amygdala electrodes for EEG recordings. Our results show that kindled animals with 6-11 generalized limbic seizures (class IV-V) had increased cell proliferation in the dentate gyrus when compared with animals with zero or one to three seizures. BrdU-positive cells labeled on day 2 and on day 32 were both GFAP negative. In the later group, rounded and well-defined BrdU-positive/GFAP-negative nuclei were seen in different portions of the granule cell layer. We did not observe any Fluoro-Jade or differential Timm staining in kindled animals at both killing times. However, EEG recordings showed intense epileptic activity in the hippocampus and amygdala of all animals with limbic seizures.Therefore, our data indicate that AK-induced limbic epileptogenicity is able to increase the hippocampal mitotic rate, even though it does not seem to promote neuronal death or mossy fiber sprouting in the supragranular layer of the dentate gyrus.

Antenatal dexamethasone administration inhibits smooth-muscle-cell DNA synthesis in pulmonary-arterial media in nitrofen-induced congenital diaphragmatic hernia in rats.

The aim of this study was to investigate the effect of antenatal glucocorticoid therapy on smooth-muscle-cell (SMC) DNA synthesis in the pulmonary arteries (PA) in a nitrofen-induced congenital diaphragmatic hernia (CDH) rat model following nitrofen administration on day 9.5 of gestation. Antenatal dexamethasone (DEX) was given intraperitoneally on days 18.5 and 19.5 of gestation. Bromodeoxyuridine (BrdU) was injected via a jugular vein into the dam 1 h before the fetuses were killed by cesarean section at term. The fetuses were divided into three groups: group I (n = 10): normal controls; group II (n = 10): nitrofen-induced CDH; group III (n = 10): nitrofen-induced CDH with antenatal DEX treatment. Immunostaining of the lungs with anti-BrdU antibody was obtained by a standard avidin-biotin complex method. The number of immunopositive cells in the PA media and adventitia were counted using an image analyzer and analyzed statistically. The number of BrdU-immunopositive cells in the media was significantly increased in group II (16.83 +/- 3.01) compared to groups I (9.16 +/- 2.20) and III (6.83 +/- 1.70) (P < 0.01). There was no significant difference between groups I and III. The number of BrdU-immunopositive cells in the adventitia was not significantly different between the three groups. Antenatal DEX treatment inhibits SMC DNA synthesis in PA media in CDH lungs. This may be a possible mechanism by which antenatal DEX prevents structural PA changes in nitrofen-induced CDH in rats.

Enhancement of cisplatin sensitivity of quiescent cells in solid tumors by combined treatment with tirapazamine and low-temperature hyperthermia.

We examined the enhanced chemosensitivity of quiescent (Q) cells in solid tumors to cis-diamminedichloroplatinum (II) (cisplatin) by combined treatment with tirapazamine (TPZ) and mild heating. C3H/He and Balb/c mice bearing SCC VII and EMT6/KU tumors, respectively, received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days using implanted mini-osmotic pumps to label all proliferating (P) cells. TPZ was administered intraperitoneally 2 h before cisplatin injection and/or tumors were locally heated at 40 degrees C for 60 min immediately after cisplatin injection. Sixty minutes after cisplatin injection, the tumors were excised, minced and trypsinized. The tumor cell suspensions were incubated with cytochalasin-B (a cytokinesis blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total (P+Q) tumor cells was determined from the tumors that were not pretreated with BrdU. The sensitivity to cisplatin was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency). Other groups of tumor-bearing C3H/He and Balb/c mice not given BrdU were injected with 195mPt-radiolabeled cisplatin. In both tumor systems, the MN frequency in Q cells was lower than that in the total cells. TPZ and mild heat treatment elevated the MN frequency in total and Q cells in both tumor systems, and to a higher extent in Q cells. The combination of TPZ and mild heat treatment increased the MN frequency more markedly than treatment with either TPZ or mild heating alone. In total tumor cells, TPZ and mild heat treatment increased the MN frequency in EMT6/KU tumor cells more markedly than in SCC VII tumor cells. 195mPt-labeled cisplatin uptake into total tumor cells was increased by mild heat treatment but not by TPZ. The cisplatin-sensitivity of Q cells was lower than that of total cells in both tumor systems. TPZ was thought to sensitize Q cells by killing the hypoxic cells without influencing tumor blood flow, and mild hyperthermia appeared to sensitize Q cells by distributing more cisplatin with an increase in blood flow in solid tumors.

Evaluation of a new lipophilic prodrug 3', 5'-dioctanoyl-5-bromodeoxyuridine (BrdU-C8) suspended in Lipiodol as a radiosensitizer for the treatment of AH136B tumor.

The effect of anti-cancer treatment on the AH136B tumor was studied using external beam irradiation in combination with a new oil-soluble agent, 3', 5'-dioctanoyl-5-bromodeoxyuridine (BrdU-C8), a lipophilic prodrug of BrdU. BrdU-C8 was dissolved in an oily lymphographic agent, Lipiodol (BrdU-C8/Lipiodol). BrdU-C8/Lipiodol is selectively accumulated in the neovasculature of the tumor and gradually releases BrdU. The AH136B tumor cell, a transplantable rat ascites hepatoma cell line, was implanted in the dorsal foot of rats. In vivo labeling index (L.I.) of the tumor cells after the intraarterial infusion of BrdU-C8/Lipiodol was significantly increased compared to the L.I. observed after intraarterial or intravenous infusion of BrdU. In addition, X-ray irradiation in combination with intraarterial infusion of BrdU-C8/Lipiodol significantly inhibited the tumor growth, indicating the increased radiosensitizing effect.

Blood supply and drug delivery to primary and secondary human liver cancers studied with in vivo bromodeoxyuridine labeling.

BACKGROUND: Because bromodeoxyuridine (BrdU) is incorporated into DNA synthesizing (S-phase) cells, the blood supply of liver tumors can be traced by injecting BrdU into either the hepatic artery or portal vein. It also is possible to study the delivery of anti-cancer drugs acting during S-phase when they are injected by these routes. The blood supply of and drug delivery to liver tumors were examined using BrdU in patients with 19 metastatic liver cancers and 8 hepatocellular carcinomas. METHODS: At the time of hepatic resection, 200 mg of BrdU was injected by the various routes or 200 mg of BrdU suspended in 2 ml of a lipid contrast medium was injected into the hepatic artery by a reported method 2 weeks before hepatectomy. The liver tumors resected were stained immunohistochemically with an avidin-biotin-peroxidase complex method using anti-BrdU monoclonal antibody. RESULTS: BrdU injected into the hepatic artery or portal vein was incorporated into the metastatic liver tumor. After intraarterial infusion BrdU suspension, the delivery of BrdU was enhanced. The nuclei of hepatocellular carcinomas that received BrdU from the hepatic artery or portal vein incorporated BrdU. CONCLUSIONS: Metastatic liver cancers had both arterial and portal blood supplies. Hepatocellular carcinomas also had, not only an arterial, but also a portal blood supply. In both primary and secondary hepatic cancers, the delivery of anti-cancer agents acting during S-phase using the lipid contrast medium administration method was excellent.

[Clinical study of the drug delivery in metastatic liver cancer using bromodeoxyuridine].

As the model of an anti-cancer agent acting at DNA synthesizing phase, BrdU was infused for metastatic liver cancers into the portal vein and/or the hepatic artery. After administration, the liver tumors were resected, and immunohistochemical staining was performed using anti-BrdU monoclonal antibody. Two of 6 portal group tumors were stained. All 4 arterial and 3 arterio-portal group tumors were stained. Therefore, it was proved that metastatic liver tumors had both arterial and portal blood supply. But areas to which BrdU was delivered were only peripheral, and the inmost area was 2 mm from the tumor surface. After the intra-arterial infusion of BrdU suspended in lipiodol, the delivery of BrdU was highly enhanced and 4 of 6 tumors were stained even to the deepest areas. However, one tumor of which the major part was necrotic and one tumor which produced mutin were not stained at all. It was interesting that after intra-arterial administration of BrdU suspended in lipiodol, BrdU was also found in the cytoplasm of normal liver cells.

Enhancing effect of bromovinyldeoxyuridine on antitumor activity of 5-fluorouracil and ftorafur against adenocarcinoma 755 in mice.

When combined with bromovinyldeoxyuridine (BVdUrd), 5-fluorouracil (FUra) brought about a significant reduction in the growth of adenocarcinoma 755 tumors in mice, at doses at which either drug used alone (BVdUrd: 100 mg/kg) did not effect an appreciable antitumor activity. BVdUrd also increased the toxicity of FUra for the hosts but not commensurately with its enhancing effect on the antitumor activity of FUra. BVdUrd also potentiated the antitumor activity of ftorafur, so that doses of ftorafur (50 or 100 mg/kg) which by themselves did not cause a significant reduction in tumor growth became markedly effective when combined with BVdUrd at a dose as low as 10 mg/kg. For some combinations of BVdUrd with FUra, the antitumor potency was further enhanced by the administration of L-cysteine (300 mg/kg).

Combined radiosensitizer infusion and irradiation of osteogenic sarcomas.

Three children with osteogenic sarcomas which were either unresectable or whose parents refused permission to amputate were treated with combined intra-arterial 5'bromodeoxyuridine (BUdR) infusion and high-dose-per-fraction megavoltage irradiation to the primary site. Pulsed, 48-hour BUdR infusions were performed prior to each 600-rad radiation therapy fraction, with a total radiation dose to the primary site of 4,200-4,800 rads in five weeks. Local control was obtained in all 3 children. One child is alive two years after treatment, another died with metastatic disease and the third patient who received radiotherapy to the lungs for pulmonary metastases is without evidence of disease one year later.