Timothy S. Wiedmann

Inhalable magnetic nanoparticles for targeted hyperthermia in lung cancer therapy.

Lung cancer (specifically, non-small cell lung cancer; NSCLC) is the leading cause of cancer-related deaths in the United States. Poor response rates and survival with current treatments clearly indicate the urgent need for developing an effective means to treat NSCLC. Magnetic hyperthermia is a non-invasive approach for tumor ablation, and is based on heat generation by magnetic materials, such as superparamagnetic iron oxide (SPIO) nanoparticles, when subjected to an alternating magnetic field. However, inadequate delivery of magnetic nanoparticles to tumor cells can result in sub-lethal temperature change and induce resistance while non-targeted delivery of these particles to the healthy tissues can result in toxicity. In our studies, we evaluated the effectiveness of tumor-targeted SPIO nanoparticles for magnetic hyperthermia of lung cancer. EGFR-targeted, inhalable SPIO nanoparticles were synthesized and characterized for targeting lung tumor cells as well as for magnetic hyperthermia-mediated antitumor efficacy in a mouse orthotopic model of NSCLC. Our results show that EGFR targeting enhances tumor retention of SPIO nanoparticles. Further, magnetic hyperthermia treatment using targeted SPIO nanoparticles resulted in significant inhibition of in vivo lung tumor growth. Overall, this work demonstrates the potential for developing an effective anticancer treatment modality for the treatment of NSCLC based on targeted magnetic hyperthermia.

Drug solubilization in lung surfactant.

The relative affinity of glucocorticosteroids for lung surfactant was determined for the purpose of identifying chemopreventive agents with a high therapeutic index for lung cancer. The aqueous solubility and the extent of solubilization in Survanta, a native extract of bovine lung, of budesonide, triamcinolone acetonide, dexamethasone, and flunisolide were determined as a function of temperature by a dialysis technique. The aqueous solubilites at 37 degrees C were 19.6, 35.8, 104 and 120 microg/ml for the above listed compounds, respectively. The temperature dependence of the solubilities was modest consistent with the hydrophobic properties of the steroids. The amount of drug in solution was significantly enhanced in the presence of Survanta with solubilization ratios of 0. 019, 0.023, 0.014, and 0.02 microg drug dissolved per microg of Survanta phospholipid, respectively. In addition, the extent of solubilization also generally increased with temperature, although the phase transition of the surfactant lipid appeared to complicate the functional relation between temperature and solubilization. These results show that there is enhanced solubilization of glucocortosteroids by lung surfactant which is secreted by the cancer susceptible type II cells of the lung.

A combination antioxidant therapy prevents age-related hearing loss in C57BL/6 mice

OBJECTIVE: Age-related hearing loss (ARHL) is characterized by gradual, progressive sensorineural hearing loss, which impairs communication, lending to clinical depression and social withdrawal. There are currently no effective treatments for ARHL. The purpose of this study is to evaluate the potential of a combination antioxidant therapy in preventing ARHL. STUDY DESIGN: Randomized controlled trial. SETTING: Animal study. SUBJECTS AND METHODS: C57BL/6 mice, a recognized animal model of ARHL, were assigned to one of three groups: early treatment (n = 12), late treatment (n = 9), or control group (n = 9). Treatment groups of mice were fed with a combination agent comprising six antioxidant agents that target four sites within the oxidative pathway: L-cysteine-glutathione mixed disulfide, ribose- cysteine, NW-nitro-L-arginine methyl ester, vitamin B12, folate, and ascorbic acid. Auditory brainstem response (ABR) thresholds were recorded at baseline and every three months following initiation of treatment. RESULTS: Threshold shifts from baseline were decreased in the treatment groups when compared to the control group at all tested frequencies (P < 0.001). The ABR threshold shift at 12 months of age for the control group was 34.7 dB with a 95% confidence interval (CI) of ± 1.6. The mean threshold shifts for the early and late treatment groups were 7.5 dB (±0.87, 95% CI) and 9.2 dB (±1.6, 95% CI). CONCLUSION: Combination antioxidant therapy effectively decreased threshold shifts on ABR within an animal model of ARHL. Combination antioxidant therapy, with further research and investigation, may provide a safe and cost-effective method of preventing presbycusis in the growing elderly population.

Solubilization of drugs by physiological mixtures of bile salts

AbstractPurpose. The solubilization of a number of steroids was determined in bile salt simple micelles and a bile salt/phospholipid micellar system to provide a better basis to predict the extent of drug solubilization in vivo. Methods. Excess solid drug was dispersed in taurodeoxycholate or mixed micelle solutions prepared with fixed mole ratios of taurocholate, taurodeoxycholate, taurochenodeoxycholate, glycodeoxycholate, glycocholate, and glycochenodeoxycholate with egg phosphatidylcholine. Drug concentrations were determined from the absorbance following centrifugation. Using NMR spectroscopy, the diffusivities of the simple and mixed micelles were 2 × 10-6 and 8 × 10-7 cm2/s, respectively. Results. From the change in the concentration of drug in solution with a change in the lipid concentration, the solubilization ratio (SR) was calculated. The SR and aqueous solubility were used to calculate the micelle/aqueous partition coefficients (Km/w). Km/w was correlated with octanol/water partition (Po/w) for the TDC and mixed micelle data sets with correlation lines of logKm/w = 0.74logPo/w + 1.55 (r2 = 0.91) and logKm/w = 0.61 logPo/w + 2.44 (r2 = 0.95), respectively. Conclusions. With such data, a refined, predictive relationship between the in vitro and the in vivo solubilization with additional information concerning the bile salt/lipid concentration in the human intestine appears possible.

Quantitation of the Relative Amounts of Anhydrous Carbamazepine (C15H12N2O) and Carbamazepine Dihydrate (C15H12N2O · 2H2O) in a Mixture by Solid-State Nuclear Magnetic Resonance (NMR)

The application of solid state nuclear magnetic resonance (NMR) for the quantitation of the relative amounts of carbamazepine anhydrate (I) and Carbamazepine dihydrate (II) in a mixture is presented. The techniques of cross polarization, dipolar decoupling, and magic angle spinning have been used to obtain high-resolution NMR spectra of the samples in the solid state. Although the chemical shifts of I and II were similar, the proton spin lattice relaxation time of II was much shorter than that of I. A delay time of 10 sec between pulses resulted in saturation of the signal from I and in a spectrum arising solely from II. The dependence of the observed signal intensity on the contact time was evaluated for II and glycine, the internal standard, to allow theoretical estimation of the peak area ratios. Various molar ratios of I and II were then mixed with glycine, and the resulting peak area ratios of II to the area of the alpha and the carbonyl carbons of glycine was linearly related to the relative proportion of II in the mixture.

Effective Elimination of Cancer Stem Cells by Magnetic Hyperthermia

Cancer stem cells (CSCs) are a subpopulation of cancer cells that have stem cell-like properties and are thought to be responsible for tumor drug resistance and relapse. Therapies that can effectively eliminate CSCs will, therefore, likely inhibit tumor recurrence. The objective of our study was to determine the susceptibility of CSCs to magnetic hyperthermia, a treatment that utilizes superparamagnetic iron oxide nanoparticles placed in an alternating magnetic field to generate localized heat and achieve selective tumor cell kill. SPIO NPs having a magnetite core of 12 nm were used to induce magnetic hyperthermia in A549 and MDA-MB-231 tumor cells. Multiple assays for CSCs, including side population phenotype, aldehyde dehydrogenase expression, mammosphere formation, and in vivo xenotransplantation, indicated that magnetic hyperthermia reduced or, in some cases, eliminated the CSC subpopulation in treated cells. Interestingly, conventional hyperthermia, induced by subjecting cells to elevated temperature (46 °C) in a water bath, was not effective in eliminating CSCs. Our studies show that magnetic hyperthermia has pleiotropic effects, inducing acute necrosis in some cells while stimulating reactive oxygen species generation and slower cell kill in others. These results suggest the potential for lower rates of tumor recurrence after magnetic hyperthermia compared to conventional cancer therapies.

Phase behavior of mixtures of DPPC and POPG

The phase relation of dipalmitoylphosphatidylcholine (DPPC) and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG) has been determined by measurement of the endothermic transitions of mixtures of DPPC and POPG in 100 mM NaCl, 50 mM PIPES (pH 7.0). With the use of differential scanning calorimetry, the gel-liquid crystalline phase transitions of pure POPG and DPPC were estimated to be 274 K and 315.8 K, respectively. With mixtures, there was considerable broadening of the endotherms, but there was no evidence of immiscibility. At high and low mole fractions of DPPC, the observed transition regions are not different from that calculated assuming ideal behavior. However in the central region of the phase diagram, there were deviations from both the ideal liquidus and solidus curves. The chemical shift anisotropy of the 13C-labelled carbonyl carbon of pure DPPC was determined as a function of temperature. At 298 K, a broad peak characteristic of axially symmetric motional averaging of the shielding tensor was observed. At a temperature of 300 K, a narrow peak at 173 ppm was superimposed upon the broad peak. The magnitude of the narrow resonance increased with temperature over the range of 300 to 315 K with the spectrum obtained at the latter point almost completely devoid of any broad features. Spectra obtained with a 9:1 mole ratio of DPPC/POPG was very similar to that obtained with pure DPPC. However, with increasing amounts of POPG, both the temperature at which the narrow resonance appeared and the temperature at which only a narrow resonance was observed were reduced. Over the range of 0 to 50 mol % POPG, there was no major change in the width or shape of the spectra which contained only a broad or narrow resonance. Also for mol % of POPG of 20% and less, there was agreement between the temperature at which only the narrow component was observed and the completion of the main phase transition based on the DSC scans. However, at the two higher mol % of 33 and 50%, the temperature at which only the narrow component was observed was lower than the temperature established for the completion of the main phase transition.

Chemoprevention of Cancer of the Upper Respiratory Tract of the Syrian Golden Hamster by Aerosol Administration of Difluoromethylornithine and 5-Fluorouracil

Research aimed at identifying effective chemopreventive compounds active against carcinogenesis of the upper respiratory tract (URT) has been largely unsuccessful. We are addressing this problem by efforts at agent identification and by using aerosol delivery. Two compounds, difluoromethylornithine (DFMO) and 5-fluorouracil (5-FU) were investigated. DFMO is an irreversible inhibitor of ornithine decarboxylase, an enzyme important in cell proliferation. It has been used widely by oral administration for chemoprevention. 5-FU is a pyrimidine analog used extensively as a chemotherapeutic agent. It is generally administered i.v. and can cause considerable toxicity. However, aerosol administration for therapy of lung cancer in humans has been reported to be without adverse effects (Tatsumura et al., Br J Cancer 1993;68:1146–9). The experimental model used herein entailed six intratracheal administrations of methylnitrosourea (MNU) to hamsters. Each of the test agents was started about 1 week after MNU and was continued for 29 weeks with DFMO. Infiltrating squamous cell carcinomas of the URT occurred in 92% of the controls and were reduced by 50% in animals receiving DFMO (P = 0.0001). The experiment with 5-FU was of shorter duration being terminated 20 weeks after MNU. Thirty percent of the controls had infiltrating carcinomas and were reduced by 60% in animals receiving 5-FU (P = 0.0274). Both compounds resulted in a significant increase in the percent of cancer-free animals. These two agents may have selected use in subjects at high risk of cancer of the URT.

Solubilization of Retinoids by Bile Salt/ Phospholipid Aggregates

AbstractPurpose. The capacity and specificity of bile salt (BS)/phosphatidylcholine (PC) mixed lipid aggregated systems in solubilizing four structurally related retinoids, etretinate, motretinid, fenretinide and N-ethyl retinamide, were determined. Methods. Excess solid drug was dispersed into sodium taurocholate (NaTC)/egg PC systems at lipid ratios of 10:0, 10:2 and 10 mM:10 mM in isotonic HEPES buffer, pH 6.5. A sensitive HPLC method was used to quantify the amount solubilized. The melting point and associated enthalpy change as well as the aqueous solubilities were also measured. Results. The retinoids had aqueous solubilities of less than 25 nM. The predicted aqueous solubility was less than 0.01 nM. The amount of retinoid in 10 mM NaTC was increased from three to four orders of magnitude relative to the aqueous solubility. Further increases in the amount solubilized were observed in the 10:10 mixed micelle dispersion. Fenretinide and N-ethyl retinamide were particularly well solubilized by BS and BS/PC aggregated systems which may be related to the presence of a cyclohexenyl ring. Conclusions. The discrepancy between the observed and predicted aqueous solubility may be due to self-association of the retinoids. Micellar/aqueous distribution ratios appear to be dominated by the hydrophobic effect, although specific interactions also are important. In considering intestinal absorption, the large increase in solubilization with BS/PC micelles would be capable of dramatically increasing the bioavailability in spite of the smaller effective diffusivity of the solubilized retinoid.

Note: Dissolution of Aerosol Particles of Budesonide in Survanta™, a Model Lung Surfactant

The effect of a pulmonary surfactant extract from bovine lung, Survanta, on the dissolution rate of aerosol particles of budesonide was determined. Aerosol particles of budesonide were generated from an ethanol solution, dried, and collected by a cascade impactor for characterization or by a liquid impinger for dissolution experiments. Powder x-ray diffraction, differential scanning calorimetry, differential thermal analysis, and scanning electron microscopy were used to characterize the aerosol particles and starting material. No change in phase was detected, although the aerosol particles appeared to contain residual solvent. The dissolution rate of the aerosol particles in saline was low and variable. Survanta increased the extent of dissolution of budesonide in proportion to the added concentration, which was also verified by equilibrium solubilization studies. Survanta also increased rate of dissolution, in a manner similar to sodium dodecyl sulfate. Analysis of the concentration of budesonide following ultracentrifugation indicated that there is rapid equilibration of budesonide between the Survanta and aqueous phase. These results show that lung surfactant has the potential of enhancing the rate and extent of dissolution of drugs administered to the lung.