Thermodynamic Solubility Profile of Temozolomide in Different Commonly Used Pharmaceutical Solvents.

The solubility parameters, and solution thermodynamics of temozolomide (TMZ) in 10 frequently used solvents were examined at five different temperatures. The maximum mole fraction solubility of TMZ was ascertained in dimethyl sulfoxide (1.35 × 10-2), followed by that in polyethylene glycol-400 (3.32 × 10-3) > Transcutol® (2.89 × 10-3) > ethylene glycol (1.64 × 10-3) > propylene glycol (1.47 × 10-3) > H2O (7.70 × 10-4) > ethyl acetate (5.44 × 10-4) > ethanol (1.80 × 10-4) > isopropyl alcohol (1.32 × 10-4) > 1-butanol (1.07 × 10-4) at 323.2 K. An analogous pattern was also observed for the other investigated temperatures. The quantitated TMZ solubility values were regressed using Apelblat and Van't Hoff models and showed overall deviances of 0.96% and 1.33%, respectively. Apparent thermodynamic analysis indicated endothermic, spontaneous, and entropy-driven dissolution of TMZ in all solvents. TMZ solubility data may help to formulate dosage forms, recrystallize, purify, and extract/separate TMZ.

A rapid screening method to select microdialysis carriers for hydrophobic compounds.

Microdialysis is a minimally invasive sampling technique which is widely applied in many fields including clinical studies. This technique usually has limitation on sampling hydrophobic compounds as aqueous solutions are commonly used as the perfusates. The relative recovery of hydrophobic compounds is often low and irreproducible because of the non-specific binding to microdialysis membranes or catheter tubing. Carriers such as cyclodextrins have been used to improve the recovery and consistency, however the identification of an optimal carrier can only be achieved after time-consuming and costly microdialysis experiments. We therefore developed a rapid, convenient, and low-cost method to identify the optimal carriers for sampling hydrophobic compounds with the use of centrifugal ultrafiltration. Doxorubicin was used as the model compound and its relative recoveries obtained from centrifugal ultrafiltration and from microdialysis were compared. The results show that the relative recoveries are highly correlated (correlation coefficient ≥ 0.9) between centrifugal ultrafiltration and microdialysis when different types or different concentrations of cyclodextrins were used as the carriers. In addition to doxorubicin, this method was further confirmed on three other drugs with different hydrophobicity. This method may facilitate and broaden the use of microdialysis perfusion on sampling or delivering hydrophobic substances in various applications.

Developing a clinically relevant radiosensitizer for temozolomide-resistant gliomas.

The prognosis for patients with glioblastoma (GB) remains grim. Concurrent temozolomide (TMZ) radiation-the cornerstone of glioma control-extends the overall median survival of GB patients by only a few months over radiotherapy alone. While these survival gains could be partly attributed to radiosensitization, this benefit is greatly minimized in tumors expressing O6-methylguanine DNA methyltransferase (MGMT), which specifically reverses O6-methylguanine lesions. Theoretically, non-O6-methylguanine lesions (i.e., the N-methylpurine adducts), which represent up to 90% of TMZ-generated DNA adducts, could also contribute to radiosensitization. Unfortunately, at concentrations attainable in clinical practice, the alkylation capacity of TMZ cannot overwhelm the repair of N-methylpurine adducts to efficiently exploit these lesions. The current therapeutic application of TMZ therefore faces two main obstacles: (i) the stochastic presence of MGMT and (ii) a blunted radiosensitization potential at physiologic concentrations. To circumvent these limitations, we are developing a novel molecule called NEO212-a derivatization of TMZ generated by coupling TMZ to perillyl alcohol. Based on gas chromatography/mass spectrometry and high-performance liquid chromatography analyses, we determined that NEO212 had greater tumor cell uptake than TMZ. In mouse models, NEO212 was more efficient than TMZ at crossing the blood-brain barrier, preferentially accumulating in tumoral over normal brain tissue. Moreover, in vitro analyses with GB cell lines, including TMZ-resistant isogenic variants, revealed more potent cytotoxic and radiosensitizing activities for NEO212 at physiologic concentrations. Mechanistically, these advantages of NEO212 over TMZ could be attributed to its enhanced tumor uptake presumably leading to more extensive DNA alkylation at equivalent dosages which, ultimately, allows for N-methylpurine lesions to be better exploited for radiosensitization. This effect cannot be achieved with TMZ at clinically relevant concentrations and is independent of MGMT. Our findings establish NEO212 as a superior radiosensitizer and a potentially better alternative to TMZ for newly diagnosed GB patients, irrespective of their MGMT status.

Voltammetric/UV-Vis study of temozolomide inclusion complexes with cyclodextrin derivatives.

Temozolomide (TMZ) - a chemotherapeutic agent possessing cytotoxic activity is used in single or combined therapies of human glioma. Difficulties in these applications, connected with low solubility and stability of temozolomide lead us to study the inclusion complexes between TMZ, and three cyclodextrins: ß-cyclodextrin (ßCD), monodeoxy-6-monoamino-ß-cyclodextrin hydrochloride (ßCDamine), and ß-cyclodextrin containing galactosamine and triazole ring in the side group (ßCDgal). The voltammetric and spectroscopy studies showed the improvement of the drug solubility and formation of stable complexes. Higuchi and Connors method was used to determine the solubilities of the drug in the presence of the selected cyclodextrins. Phase solubility diagrams showed increase of TMZ solubility and 1:1 stoichiometry of the complexes formed. The stability constant of TMZ- ßCDgal complex was pH - dependent, larger at pH 7.4 (corresponding to the pH of the body fluids), than at pH 5.5, characteristic for the cancer cells environment. ßCDgal ligand was an effective complexing agent for TMZ due to additional strong proton-acceptor π-π interactions between the triazole ring of the cyclodextrin and the ring of TMZ. The increased solubility and sustainability of TMZ complexes with ßCDgal allow to propose this cyclodextrin as a promising TMZ carrier for further studies in the biological cell environment.

Targeting of temozolomide using magnetic nanobeads: an in vitro study

Temozolomide, a chemotherapeutic drug that is often administered for the treatment of brain cancer has severe side effects and a poor aqueous solubility. In order to decrease the detrimental effect of the drug over healthy cells, a novel drug delivery vehicle was developed where the therapeutic drug was encapsulated within the hydrophobic cavities of b-CD modified magnetite nanoparticles, which are embedded in chitosan nanobeads prepared by salt addition. In-vitro studies have shown that the magnetic properties of the novel delivery vehicle are adequate for targeted drug delivery applications under an external magnetic field. Additionally, an increase in the amount of chitosan was shown to exhibit a strong shielding effect over the magnetic properties of the delivery vehicle, which lead to deterioration of the amount of captured drug at the targeted area, suggesting a delicate balance between the amounts of constituents composing the drug delivery vehicle.

Novel Pyrene Excimer and Fluorogenic Probe for the Detection of Alkylating Agents.

A pyrene-containing salicylic acid derivative (4) was found to be low in fluorescence, but its derivative pyrene-containing methyl salicylate (3) was found to be highly fluorescent in aqueous solution. This derivative has been tested in solution and found to be superior in the fluorogenic assay of pharmaceutical compounds, detection of chemical warfare agents, a preliminary toxicology test, mutagenicity of medicinal compounds, and other chemical analyses, including trimethylsilyl diazomethane; alkyl bromides and iodides; a sulfur mustard mimic 2-chloroethyl ethyl sulfide; and anticancer drugs, busulfan and pipobroman. The salicylic acid derivative (4) was applied as a fluorogenic probe for the detection of alkylating agents by esterification and generating fluorescence at 475 nm in solutions at low concentrations.

HPLC-UV method for temozolomide determination in complex biological matrices: Application for in vitro, ex vivo and in vivo studies.

A high-performance liquid chromatography method for temozolomide (TMZ) determination in complex biological matrices was developed and validated for application in in vitro, ex vivo and in vivo studies of new nanotechnology-based systems for TMZ nasal delivery. The method was able to quantify TMZ in nanoemulsions, following cellular uptake, in the porcine nasal mucosa and in mouse plasma and brain. Analyses were performed on a C18 column at 35°C, under UV detection at 330 nm. The mobile phase was methanol-acetic acid 0.5% (30:70, v/v), eluted at an isocratic flow rate of 1.1 mL/min. The method was found to be specific, precise, accurate, robust and linear (0.05 to 5 µg/mL) for TMZ determination in all matrices. No interference of TMZ degradation products was found under various stress conditions such as acidic, alkaline, oxidative, light and thermal exposure, demonstrating stability. The method was applied for the quantification of TMZ in different matrices, i.e. the efficiency of nanoemulsions in vitro in increasing TMZ cellular uptake, ex vivo TMZ permeation and retention in the porcine nasal mucosa tissue, and for in vivo TMZ quantification in mouse brain following intranasal nanoemulsion administration compared with free TMZ.