Mild Local Hyperthermia Research Articles

Ultrasound-induced mild hyperthermia improves the anticancer efficacy of both Taxol® and paclitaxel-loaded nanocapsules

We study the influence of ultrasound on paclitaxel-loaded nanocapsules in vitro and in vivo. These nanocapsules possess a shell of poly(dl-lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) and a liquid core of perfluorooctyl bromide (PFOB). In vitro experiments show that mechanical effects such as cavitation are negligible for nanocapsules due to their small size and thick and rigid shell. As the mechanical effects were unable to increase paclitaxel delivery, we focused on the thermal effects of ultrasound in the in vivo studies. A focused ultrasound sequence was therefore optimized in vivo under magnetic resonance imaging guidance to obtain localized mild hyperthermia with high acoustic pressure. Ultrasound-induced mild hyperthermia (41–43°C) was then tested in vivo in a subcutaneous CT-26 colon cancer murine model. As hyperthermia is applied, an inhibition of tumor growth for both paclitaxel-loaded nanocapsules and the commercial formulation of paclitaxel, namely Taxol® have been observed (p<0.05). Ultrasound-induced mild hyperthermia at high acoustic pressure appears as an interesting strategy to enhance cytotoxic efficacy locally.

Formulation and optimization of idarubicin thermosensitive liposomes provides ultrafast triggered release at mild hyperthermia and improves tumor response

Drug delivery through thermosensitive liposomes (TSL) in combination with hyperthermia (HT) has shown great potential. HT can be applied locally forcing TSL to release their content in the heated tumor resulting in high peak concentrations. To perform optimally the drug is ideally released fast (seconds) and taken up rapidly by tumor cells. The aim of this study was to develop a novel thermosensitive liposome formulation of the anthracycline idarubicin (IDA-TSL). The hydrophobicity of idarubicin may improve its release from liposomes and subsequently rapid cellular uptake when combined mild hyperthermia. Here, we investigated a series of parameters to optimize IDA-TSL formulation. The results show that the optimal formulation for IDA-TSL is DPPC/DSPC/DSPE-PEG (6/3.5/0.5mol%), with ammonium EDTA of 6.5 pH as loading buffer and a size of ~85nm. In vitro studies demonstrated minimal leakage of ~20% in FCS at 37°C for 1h, while an ultrafast and complete triggered release of IDA was observed at 42°C. On tumor cells IDA-TSL showed comparable cytotoxicity to free IDA at 42°C, but low cytotoxicity at 37°C. Intravital microscopy imaging demonstrated an efficient in vivo intravascular triggered drug release of IDA-TSL under mild hyperthermia, and a subsequent massive IDA uptake by tumor cells. In animal efficacy studies, IDA-TSL plus mild HT demonstrated prominent tumor growth inhibition and superior survival rate over free IDA with HT or a clinically used Doxil treatment. These results suggest beneficial potential of IDA-TSL combined with local mild HT.

Triggered doxorubicin release in solid tumors from thermosensitive liposome-peptide hybrids: Critical parameters and therapeutic efficacy.

Temperature-sensitive vesicles designed by inclusion of leucine zipper peptides within a lipid bilayer (Lp-Peptide hybrids) encapsulating Doxorubicin (DOX) have been reported. Intravenous administration of these constructs prolonged blood circulation kinetics and increased tumor accumulation in vivo with local mild hyperthermia. In this study, the biological activity of the DOX-loaded Lp-Peptide hybrid vesicles was further investigated at the cellular level and in vivo compared to lysolipid-containing temperature-sensitive liposomes (LTSL) and traditional temperature-sensitive liposomes. Lp-Peptide vesicles were not toxic to cell cultures at 37°C, while effective cancer cell toxicity was observed after 1 hr of heating at 42°C. The activity of Lp-Peptide vesicles in vivo was studied using two different heating protocols to obtain tumor intravascular or interstitial drug release. Lp-Peptide vesicle treatment allowing intravascular DOX release showed equally effective tumor growth retardation and survival to that of LTSL treatment. The Lp-Peptide vesicles also offered therapeutic responses using the alternative heating protocol to maximise drug release within the tumor interstitium. Matching the drug release kinetics of temperature-sensitive vesicles with the heating protocol applied is considered the most critical factor to determine therapeutic efficacy in the clinical translation of such modalities.

Two phase I dose-escalation/pharmacokinetics studies of low temperature liposomal doxorubicin (LTLD) and mild local hyperthermia in heavily pretreated patients with local regionally recurrent breast cancer

Purpose: Unresectable chest wall recurrences of breast cancer (CWR) in heavily pretreated patients are especially difficult to treat. We hypothesised that thermally enhanced drug delivery using low temperature liposomal doxorubicin (LTLD), given with mild local hyperthermia (MLHT), will be safe and effective in this population.Patients and methods: This paper combines the results of two similarly designed phase I trials. Eligible CWR patients had progressed on the chest wall after prior hormone therapy, chemotherapy, and radiotherapy. Patients were to get six cycles of LTLD every 21–35 days, followed immediately by chest wall MLHT for 1 hour at 40–42 °C. In the first trial 18 subjects received LTLD at 20, 30, or 40 mg/m2; in the second trial, 11 subjects received LTLD at 40 or 50 mg/m2.Results: The median age of all 29 patients enrolled was 57 years. Thirteen patients (45%) had distant metastases on enrolment. Patients had received a median dose of 256 mg/m2 of prior anthracyclines and a median dose of 61 Gy of prior radiation. The median number of study treatments that subjects completed was four. The maximum tolerated dose was 50 mg/m2, with seven subjects (24%) developing reversible grade 3–4 neutropenia and four (14%) reversible grade 3–4 leucopenia. The rate of overall local response was 48% (14/29, 95% CI: 30–66%), with. five patients (17%) achieving complete local responses and nine patients (31%) having partial local responses.Conclusion: LTLD at 50 mg/m2 and MLHT is safe. This combined therapy produces objective responses in heavily pretreated CWR patients. Future work should test thermally enhanced LTLD delivery in a less advanced patient population.

High-Intensity Focused Ultrasound-Induced, Localized Mild Hyperthermia to Enhance Anti-cancer Efficacy of Systemic Doxorubicin: An Experimental Study

The aim of this study was to evaluate the enhancement of the efficacy of systemic doxorubicin by pulsed high-intensity focused ultrasound (HIFU)-induced, localized mild hyperthermia. For the in vitro study, the intranuclear uptake of doxorubicin by squamous cell carcinoma (SCC)-7 cells incubated at different temperatures was compared. For the in vivo study, mice with SCC-7 tumors were assigned to either the control, conventional hyperthermia, HIFU hyperthermia, doxorubicin-alone, conventional hyperthermia + doxorubicin or HIFU hyperthermia + doxorubicin group. Conventional hyperthermia was induced by immersing the tumor in warm water (42.5°C), and HIFU hyperthermia was induced by HIFU after optimizing the parameters with direct temperature measurements (frequency = 1 MHz, pulse repetition frequency = 5 Hz, power = 12 W, duty cycle = 50%). In the in vitro study, fluorescence was more intense at 42°C than at 37°C and was time dependent. In the in vivo study, tumor growth in the HIFU hyperthermia + doxorubicin group was most prominently suppressed with the highest apoptotic index compared with all other groups (p < 0.05). Pulsed HIFU-induced localized mild hyperthermia enhanced the anti-cancer efficacy of systemic doxorubicin more than conventional mild hyperthermia.

Abstract P4-15-05: Novel targeted therapy for breast cancer chest wall recurrence: Low temperature liposomal doxorubicin and mild local hyperthermia

Abstract Background: Unresectable breast cancer chest wall recurrence (CWR) following radiation is very difficult to treat and often responds poorly to standard chemotherapy. Symptoms include pain, reduced range of motion, disfigurement, and skin erosions with bleeding and infection. We hypothesized that thermally enhanced drug delivery using low temperature liposomal doxorubicin (LTLD, ThermoDox®), given with mild local hyperthermia (MLHT) would be a safe and effective targeted therapy. LTLD is given by iv infusion; it then localizes in CWR tumors due to their leaky vasculature. When heated to ≥ 39.5°C, LTLD releases a high concentration of the heat-enhanced cytotoxic doxorubicin. Methods: The results of 2 similarly-designed independent phase I trials were combined for analysis. Eligible patients had CWR progressing after radiation, hormone therapy, and chemotherapy. Subjects were to get up to 6 cycles of LTLD every 21-35 days, followed immediately by chest wall MLHT for 1 hour at 40°- 42°C. In Trial A, 18 subjects received LTLD at 20, 30, or 40 mg/m2; in Trial B, 11 subjects received LTLD at 40 or 50 mg/m2. The primary endpoint of each trial was to determine the maximum tolerated dose (MTD); secondary endpoints were local objective response and the pharmacokinetic (PK) and safety profiles of LTLD. Local response was assessed by serial photography and measurements of CWR. PK samples for total plasma doxorubicin and doxorubicinol were collected at Cycle 1 and Cycle 2 for both trials. Results: Twenty-nine subjects were enrolled and received ≥ 1 cycle (median 4, range 1-6). Median age was 57; 16 (55%) had triple negative disease and 13 (45%) had distant metastases. The median prior exposure to anthracylines was 256 mg/m2 and the median prior dose of radiation was 6,100 cGy. Thirteen subjects were evaluable for MTD in Trial A and 9 in Trial B. Trial B established a phase II dose of 50 mg/m2 recommended by a Data Safety Monitor Board, based on 1 of 6 subjects at the 50 mg/m2 dose level having a DLT (grade 3 hypokalaemia unrelated to study treatment). In Trial A, 2 of 7 subjects at 40 mg/m2 had a DLT (grade 4 neutropenia lasting &amp;gt; 5 days; grade 3 dehydration lasting 27 days). The Cmax concentrations between 18,400 to 20,700 ng/mL were consistent at an equal dose level (40 mg/m2) between trials. Altogether, 7 (24%) subjects developed reversible grade 3-4 neutropenia and 4 (14%) reversible grade 3-4 leukopenia. No cardiac toxicity or hand-foot syndrome was seen. One case of CW thermal burn (grade 3) and one case of radiation recall (grade 2) were reported. Five (17%) complete local responses and 9 (31%) partial local responses were seen. The rate of local response was 48% (14/29; 95% CI: 30%-66%). Seven of 29 subjects (24%) progressed outside the study treatment field. Conclusion: LTLD plus MLHT is a novel therapy that is safe and produces objective responses in heavily pretreated CWR patients with limited therapeutic options. The primary toxicity is reversible bone marrow suppression. A phase II trial is ongoing at the MTD (50 mg/m2). Future work should test thermally enhanced LTLD delivery in a less advanced, less heavily pretreated patient population. *Author note-M.W.D. and K.L.B. equally contributed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-05.

Tumor vascular permeabilization using localized mild hyperthermia to improve macromolecule transport

The abnormal tumor vasculature presents a major challenge to the adequate delivery of chemotherapeutics, often limiting efficacy. We developed a nanoparticle-based technique to deliver localized mild hyperthermia (MHT) used to transiently alter tumor vascular transport properties and enhance transport of macromolecules into tumor interstitium. The strategy involved administering and localizing accumulation of stealth gold nanorods (GNRs, 103μg of GNRs/g of tumor), and irradiating tumor with a low-photon laser flux (1W/cm2) to generate MHT. The treatment increased vascular permeability within 24h after treatment, allowing enhanced transport of macromolecules up to 54nm in size. A mathematical model is used to describe changes in tumor mass transport properties where the rate of macromolecular exchange between interstitial and vascular region (R) and maximum dye enhancement (Ymax) of 23-nm dextran dye is analytically solved. During enhanced permeability, R increased by 200% while Ymax increased by 30% relative to untreated group in pancreatic CAPAN-1 tumors. MHT treatment also enhanced transport of larger dextran dye (54nm) as assessed by intravital microscopy, without causing occlusive cellular damage. Enhanced vascular transport was prolonged for up to 24h after treatment, but reversible with transport parameters returning to basal levels after 36h. This study indicates that localized mild hyperthermia treatment opens a transient time-window with which to enable and augment macromolecule transport and potentially improve therapeutic efficacy. From the Clinical EditorIn this study, local intra-tumor mild hyperthermia is induced using a nanoparticle-based approach utilizing stealth gold nanorods and irradiating the tumor with low-photon laser flux, resulting in locally increased vascular permeability enabling enhanced delivery of therapeutics, including macromolecules up to 54nm in size. Similar approaches would be very helpful in addressing treatment-resistant malignancies in clinical practice.

Clearance of genital warts in pregnant women by mild local hyperthermia: a pilot report

Genital warts acquired during pregnancy tend to grow fast, and management is challenging. We treated two cases of primipara with extensive genital warts by local hyperthermia at 44°C for 30 minutes a day for 3 consecutive days plus 2 additional days 1 week later, then once a week till there showed signs of clinical regression. The warty lesions in the patients resolved in 5 and 7 weeks, respectively. There was no sign of recurrence during a 6-month follow-up. This suggests that local hyperthermia seems to be a promising method for treating genital warts in pregnant women.

Mild hyperthermia triggered doxorubicin release from optimized stealth thermosensitive liposomes improves intratumoral drug delivery and efficacy

Liposome mediated anticancer drug delivery has the advantage of reducing cytotoxicity in healthy tissues. However, undesired slow drug release impedes the therapeutic efficacy of clinically applied PEG-liposomal doxorubicin (Dox). The aim of this study is to combine stealth thermosensitive liposomes (TSL) and local mild hyperthermia (HT) to increase bioavailable Dox levels in tumors. Dox was encapsulated in stealth TSL (~80nm) with optimized PEG concentration in the membrane, and compared with lysolipid-based Dox-LTSL for in vitro stability, release kinetics, and in vivo tumor growth control. In vitro cytotoxicity of Dox-TSL against murine BFS-1 sarcoma and, human BLM melanoma cell lines and Human Umbilical Vein Endothelial Cells (HUVEC) under normothermia (37°C) and HT (42°C) was compared with non-encapsulated Dox. In vitro Dox uptake in nuclei was imaged in BLM and HUVEC. In vivo intravascular Dox release from TSL in BFS-1 tumors under local mild HT in dorsal skin flap window chamber models was captured by intravital confocal microscopy. Intravascular Dox-TSL release kinetics, penetration depth and interstitial Dox density were subjected to quantitative image analysis. Systemic Dox-TSL administration in combination with local mild HT on subcutaneous tumor growth control was compared to Dox-LTSL plus local mild HT. Dox-TSL was stable at 37°C, while released over 95% Dox within 1min in 90% serum at 42°C. Dox-TSL demonstrated efficient in vivo intratumoral Dox release under local mild HT, followed by significant Dox uptake by tumor and tumor vascular endothelial cells. Dox-TSL plus mild HT showed improved tumor growth control over Dox-LTSL plus mild HT. Survival after a single treatment of Dox-TSL plus mild HT was 67%, while survival after Dox-LTSL plus mild HT was 22%. This combination of Dox-TSL and local mild HT offers promising clinical opportunities to improve liposomal Dox delivery to solid tumors.

Improved intratumoral nanoparticle extravasation and penetration by mild hyperthermia

Accumulation of nanoparticles in solid tumors depends on their extravasation. However, vascular permeability is very heterogeneous within a tumor and among different tumor types, hampering efficient delivery. Local hyperthermia at a tumor can improve nanoparticle delivery by increasing tumor vasculature permeability, perfusion and interstitial fluid flow. The aim of this study is to investigate hyperthermia conditions required to improve tumor vasculature permeability, subsequent liposome extravasation and interstitial penetration in 4 tumor models. Tumors are implanted in dorsal skin flap window chambers and observed for liposome (~85nm) accumulation by intravital confocal microscopy. Local hyperthermia at 41°C for 30min initiates liposome extravasation through permeable tumor vasculature in all 4 tumor models. A further increase in nanoparticle extravasation occurs while continuing heating to 1h, which is a clinically relevant duration. After hyperthermia, the tumor vasculature remains permeable for 8h. We visualize gaps in the endothelial lining of up to 10μm induced by HT. Liposomes extravasate through these gaps and penetrate into the interstitial space to at least 27.5μm in radius from the vessel walls. Whole body optical imaging confirms HT induced extravasation while liposome extravasation was absent at normothermia. In conclusion, a thermal dose of 41°C for 1h is effective to induce long-lasting permeable tumor vasculature for liposome extravasation and interstitial penetration. These findings hold promise for improved intratumoral drug delivery upon application of local mild hyperthermia prior to administration of nanoparticle-based drug delivery systems.

Adjuvant Properties of Thermal Component of Hyperthermia Enhanced Transdermal Immunization: Effect on Dendritic Cells

Hyperthermia enhanced transdermal (HET) immunization is a novel needle free immunization strategy employing application of antigen along with mild local hyperthermia (42°C) to intact skin resulting in detectable antigen specific Ig in serum. In the present study, we investigated the adjuvant effect of thermal component of HET immunization in terms of maturation of dendritic cells and its implication on the quality of the immune outcome in terms of antibody production upon HET immunization with tetanus toxoid (TT). We have shown that in vitro hyperthermia exposure at 42°C for 30 minutes up regulates the surface expression of maturation markers on bone marrow derived DCs. This observation correlated in vivo with an increased and accelerated expression of maturation markers on DCs in the draining lymph node upon HET immunization in mice. This effect was found to be independent of the antigen delivered and depends only on the thermal component of HET immunization. In vitro hyperthermia also led to enhanced capacity to stimulate CD4+ T cells in allo MLR and promotes the secretion of IL-10 by BMDCs, suggesting a potential for Th2 skewing of T cell response. HET immunization also induced a systemic T cell response to TT, as suggested by proliferation of splenocytes from immunized animal upon in vitro stimulation by TT. Exposure to heat during primary immunization led to generation of mainly IgG class of antibodies upon boosting, similar to the use of conventional alum adjuvant, thus highlighting the adjuvant potential of heat during HET immunization. Lastly, we have shown that mice immunized by tetanus toxoid using HET route exhibited protection against challenge with a lethal dose of tetanus toxin. Thus, in addition to being a painless, needle free delivery system it also has an immune modulatory potential.

Reducing intratumour acute hypoxia through bevacizumab treatment, referring to the response of quiescent tumour cells and metastatic potential

The aim was to evaluate the influence of bevacizumab on intratumour oxygenation status and lung metastasis following radiotherapy, with specific reference to the response of quiescent (Q) 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 γ-ray irradiation following treatment with the acute hypoxia-releasing agent nicotinamide or local mild temperature hyperthermia (MTH) with or without the administration of bevacizumab under aerobic conditions or totally hypoxic conditions, achieved by clamping the proximal end of the tumours. Immediately after the irradiation, cells from some tumours were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (P + Q) cell populations were assessed based on the frequency of micronuclei using immunofluorescence staining for BrdU. In the other tumour-bearing mice, macroscopic lung metastases were enumerated 17 days after irradiation. 3 days after bevacizumab administration, acute hypoxia-rich total cell population in the tumour showed a remarkably enhanced radiosensitivity to γ-rays, and the hypoxic fraction (HF) was reduced, even after MTH treatment. However, the hypoxic fraction was not reduced after nicotinamide treatment. With or without γ-ray irradiation, bevacizumab administration showed some potential to reduce the number of lung metastases as well as nicotinamide treatment. Bevacizumab has the potential to reduce perfusion-limited acute hypoxia and some potential to cause a decrease in the number of lung metastases as well as nicotinamide.

Abstract 2650: Triggered content release from optimized stealth thermosensitive liposomes using mild hyperthermia

Abstract Liposomes are potent nanocarriers to deliver chemotherapeutic drugs to tumors. However, the inefficient drug release hinders their application. Liposomes around 100 nm can extravasate through leaky microvasculature and accumulate in tumor tissue. Local hyperthermia (HT) augments the permeability of tumor microvasculature, resulting in enhanced intratumoral liposomal drug accumulation. Subsequently, thermosensitive liposomes (TSL) release their contents at elevated temperature. The combination of stealth TSL with phase transition temperature at 41 – 43 °C and mild local HT offers a promising clinical advance. This study identified thermal dose for liposome extravasation through tumor vessels and the optimum DSPE-PEG2000 concentration for triggered drug release from TSL. TSL were prepared with DSPE-PEG2000 concentration from 1 to 10 mol% and size of around 80 nm. Quenched carboxyfluorescein (CF) in the aqueous phase represented encapsulated drugs. In vitro temperature/time-dependent CF release and TSL stability in serum were quantified by fluorometry. In vivo CF release was observed by confocal microscopy in dorsal skin flap window chamber models implanted with human BLM melanoma. A circular resistive electric heating coil provided local HT. In vivo liposome extravasation was also studied in window chamber models of syngeneic B16 melanoma in mice with constitutive vascular endothelial cell expression of an eNOS-Tag-GFP fusion protein. The study of liposome extravasation through tumor microvasculature illustrated a thermal dose of 41 °C for 30 min tuned up the leakiness of tumor vessels towards liposomes of around 80 nm. A significant amount of liposomes escaped from circulation and intratumorally accumulated in the interstitial space. The in vitro temperature-dependent CF release indicated in vitro CF release increased with increasing DSPE-PEG2000 density. Six mol% and higher DSPE-PEG2000 caused content leakage at physiological temperature. Thus the optimum DSPE-PEG2000 concentration was 5 mol% (p &amp;lt; 0.05). These TSL released over 60 % of CF in one minute, and released almost 100 % of CF in one hour at 42 °C with encapsulation efficiency of 0.08 (mole/mole of CF/lipid). Content retention in 90 % serum at 37 °C for one hour was 96.6 %. In vivo optical intravital imaging showed immediate massive CF release above 41 °C. Incorporation of 5 mol% of DSPE-PEG2000 in the liposomal bilayer optimized stealth TSL content release triggered by HT (&amp;gt; 41 °C). In vivo tumor models confirmed in vitro TSL release characteristics. The superior application of TSL and mild HT will achieve both maximal intratumoral liposomal drug accumulation and rapid triggered drug release to aid in liposomal chemotherapy in clinical practice. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2650.

Enhanced transdermal-immunization with diptheria-toxoid using local hyperthermia

In this study we have investigated how mild local hyperthermia could be used for transdermal-immunization. Mild hyperthermia is found unique in many ways. It activates the immune system and it also results in higher mass transport of high molecular weight molecules and nanometer size quantum dots labeled antigens across the skin in experimental animals. Hyperthermia increases the expression of co-stimulatory molecules such as CD80 and CD86. Mice transdermally immunized with diphtheria toxoid, without any adjuvant or penetration enhancing reagent but under the conditions of local hyperthermia generate an antibody response. In the memory recall assay, the splenocytes of hyperthermia enhanced transdermal-immunized mice undergo proliferation, when exposed to diphtheria toxoid. Comparable response was generated when mice were immunized with diphtheria toxoid by hyperthermia enhanced transdermal route or by conventional inter-muscular injection. Hyperthermia enhanced transdermal-immunization procedure is likely to have higher compliance as it does not cause any pain or visible damage to the skin.

Radiosensitization of two murine tumours with mild temperature hyperthermia and carbogen breathing

Purpose: To test the individual and combined effects of local mild temperature hyperthermia (MTH) at 41.5 C for 60min and carbogen breathing on tumour radiosensitivity. Materials and methods: The FSall fibrosarcoma of C3H mice and the SCK mammary carcinoma of A/J mice were used. The effect of various treatments on tumour cell survival was determined using the in vivo/in vitro tumour excision assay, and the radiobiological hypoxic fraction was calculated. The tumour radiation sensitivity was tested with the tumour growth delay assay. Results: The radiobiological hypoxic cell fraction (HF) in control FSall and SCK tumours was 0.45 and 0.78, respectively, and these values decreased to 0.12 in FSall tumours and 0.22 in SCK tumours when determined immediately after the tumours were treated with MTH. The HF was 0.32 in FSall tumours and 0.33 in SCK tumours after carbogen breathing was applied. When tumours were treated with MTH and the animals breathed carbogen the HF decreased to 0.03-0.04 in both FSall and SCK tumours. MTH treatment alone had only a small effect on tumour growth, but MTH treatment applied before irradiating the tumours significantly increased the radiation-induced tumour growth delay. Carbogen breathing modestly improved the radiation-induced tumour growth delay while the combination of MTH treatment and carbogen breathing caused the largest increase in radiation-induced tumour growth delay. Conclusions: MTH treatment alone and combined with carbogen breathing substantially increased the tumour radiation response probably through an increase in the tumour oxygenation status.

Drug delivery to the brain using thermosensitive liposome and local hyperthermia.

We investigated the possibilities of drug delivery to the brain using thermosensitive liposomes and hyperthermia. Thermosensitive liposomes are small vesicles containing some drugs, which are designed to release the drugs in response to hyperthermia. The first experiment consisted of four groups: (1) received free Cisplatin: cis-diamminedichloroplatinum (CDDP); (2) received free CDDP and above 41 degrees C local brain heating for 30 min; (3) received liposomes containing CDDP (CDDP-liposome); and (4) received CDDP-liposome and above 41 degrees C local brain heating for 30 min. Brain CDDP levels were significantly higher in (4), while those on the other groups were undetectable. In the second experiment, we studied the distribution of Evans blue (Eb) in the artificially heated region of mongrel dogs' brain. One group received free Eb and the other group received liposomes containing Eb (Eb-liposome). While the extravasation of free Eb was localized in regions heated > 44 degrees C, that of Eb-liposome was extended up to the regions heated at 41 degrees C. We concluded that the use of thermosensitive liposomes and hyperthermia not only contributes to the brain tumour killing as direct thermal killing does but also helps to increase the concentration of chemotherapeutic drugs into the tumour invaded zones with mild local hyperthermia of 41 degrees C.

Ultraviolet B-induced apoptosis of keratinocytes in murine skin is reduced by mild local hyperthermia.

Two components of sunlight, ultraviolet (UV) B (290-320 nm) and infrared (greater than 700 nm), each cause damage to the skin. However, we recently identified a protective response in which heat reduces UVB-induced killing of cultured keratinocytes. Here, this investigation is extended to the living epidermis. The effects of hyperthermic preconditioning upon UVB-induced apoptosis were studied morphologically with hematoxylin and eosin staining, and biochemically with TUNEL (terminal deoxynucleotide transferase nick-end labeling) to measure endonucleolytic cleavage of DNA in situ. Anesthetized SKH-1 hairless mice were exposed to UVB light (0 to 120 mJ/cm2), after which their skin was biopsied at 24 h and paraffin sections were stained with hematoxylin and eosin or with TUNEL. Apoptotic keratinocytes were found to increase after UVB in a dose-related manner. In contrast, if one flank of the mouse was pretreated at 40 degrees C for 1 h and both flanks subsequently were UVB-irradiated at 6 h, the resulting formation of apoptotic cells was reduced twofold or more in the heated flank. Protection appeared by 3 h, reached a maximum at 6 h, and disappeared by 12 h. In summary, heat induces a transient protective effect that reduces UVB-mediated death of keratinocytes in skin at physiologically attainable doses of heat and UVB.

Enhancement of misonidazole chemosensitization effect by mild local hyperthermia

In an attempt to increase the chemosensitization effect of the alkylating agents 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU) and cyclophosphamide (CY), by misonidazole (MISO) at the tumor site, mild hyperthermic treatment (41.5°C, 1 hr) was applied at various administration sequences. C3Hf/Sed mice bearing subcutaneous FSa-II tumors in the foot were used for a tumor growth time assay. Local hyperthermic treatment increased the antitumor activities of BCNU and CY by 1.4 and 2.4 fold, respectively. MISO at 2.5 mmole/kg potentiated the antitumor activities of BCNU, but not CY, at normal body temperature. There were no significant improvement of MISO chemosensitization when heat was given before the administration of BCNU and CY. However, a significant enhancement of chemosensitization was observed when heat was given after the administration of MISO and the alkylating agents. Enhancement ratios of about 2.4 and 4.7 were observed with BCNU and CY, respectively. There may be two mechanisms responsible for this thermal enhancement. First, the MISO pre-incubation time that was required for the expression of chemosensitization effect decreased substantially at elevated temperatures. This hypothesis was supported by the pharmacokinetic studies that MISO was rapidly eliminated from tumors in the first 10 min during the local heat treatment and remained at a plateau with a concentration of about 5-fold less than the peak MISO concentration in the control tumors. This rapid elimination might result from the increase in the rate of hypoxic metabolism of MISO in heated tumors. Second, heat may increase the MISO-alkylating agent interactions, which are independent of pre-incubation time. This effect was especially pronounced in CY because pre-incubation-induced chemosensitization of CY in unheated tumor was insignificant in this study. The significant improvement of MISO chemosensitization at moderately elevated temperatures can be useful clinically in combined hyperthermia and chemotherapy treatment.

Schedule dependent tumour growth delay, DNA cross-linking and pharmacokinetic parameters in target tissues withcis-diamminedichloroplatinum(II) and etanidazole with or without hyperthermia or radiation

It has been reported previously that striking increases in tumour growth delay and cytotoxicity are seen when cis-diamminedichloroplatinum(II) (CDDP) is combined with mild local hyperthermia (43 degrees C, 30 min) and/or etanidazole (ETA). This paper reports a study of CDDP pharmacology and the in vivo tumour DNA cross-linking produced by these combinations. In C3H mice bearing the FSaIIC murine fibrosarcoma, Pt plasma pharmacokinetics were not significantly altered by any of the combination of treatments. Although ETA caused no significant change in CDDP tissue pharmacokinetics, treatment of the tumour-bearing limb with hyperthermia immediately following an i.p. injection of CDDP (10 mg/kg) resulted in an increased peak Pt concentration (3.5 versus 2.8 micrograms Pt/g tumour wet weight) and doubled the t1/2 elimination of Pt (15 to 30 h) from the tumour. Similar heat-induced changes were observed in the Pt pharmacokinetics in skin. There was about a three-fold increase in the Pt area under the curve (AUC) for the tumour, a 1.5-fold increase in the AUC for skin and little change in the AUC for muscle with hyperthermia. When the tumour DNA cross-linking factor (CLF) was determined, it was found that local hyperthermia treatment (43 degrees C, 30 min) increased the CLF of CDDP from 1.7 to 2.7 and hyperthermia (43 degrees C, 1 h) further increased the CLF to 6.1. Misonidazole (MISO) (1 g/kg) increased the CDDP CLF to 2.0, 6.3 and 15.1 in conjunction with 37, 43 (30 min) and 43 degrees C (1 h), respectively. ETA (1 g/kg) was more effective than MISO at increasing the CDDP CLF, producing CLFs of 2.8, 9.1 and 21.5 at 37, 43 (30 min) and 43 degrees C (1 h), respectively. These changes in CLF were reflected in an increased tumour growth delay in the FSaIIC murine fibrosarcoma with CDDP (5 mg/kg) alone from 4.4 to 5.9 days with 43 degrees C (30 min) and then to 11.9 days with ETA (1 g/kg) and 20.9 days with both ETA and local hyperthermia (43 degrees C, 30 min). When CDDP, ETA and hyperthermia were added to a radiation schedule of 300 cGy daily for five days, it was found that giving ETA (1 g/kg), CDDP (5 mg/kg) and hyperthermia (43 degrees C, 30 min) together on day 1 produced the largest tumour growth delay (43 days) and that other schedules which divided the dose of ETA over the other days of the radiation treatment (including one schedule with a second heat treatment on day 4) were significantly inferior.(ABSTRACT TRUNCATED AT 400 WORDS)

Design of Liposomes for Enhanced Local Release of Drugs by Hyperthermia

Liposomes can be designed to release an entrapped drug preferentially at temperatures attainable by mild local hyperthermia. In a test system in vitro, protein synthesis by Escherichia coli is inhibited and killing of the cells is enhanced by heating neomycin-containing liposomes to their phase transition temperature to maximize drug release. In the presence of serum the ratio of release at 44 degrees C to that at 37 degrees C can be made greater than 100:1, suggesting possible applications in the treatment of tumors or local infection.