A Highly Efficient Approach To Construct (epi)-Podophyllotoxin-4-O-glycosidic Linkages as well as Its Application in Concise Syntheses of Etoposide and Teniposide.

By taking full advantage of the mild promotion conditions of an ortho-alkynylbenzoate glycosylation protocol, a highly efficient approach to construct the challenging (epi)-podophyllotoxin 4-O-glycosidic linkages was devised under the activation of a catalytic amount of a Au(I) complex. The novel method enjoys a quite broad substrate scope in terms of both glycosyl donors and podophyllotoxin derivative acceptors, providing the desired glycosides in excellent yields. Based on the new approach, concise syntheses of clinically used anticancer reagents etoposide and teniposide were accomplished, and the overall yields counting from easily available starting materials could reach as high as 18% and 9%, respectively.

Preparation and in vitro-in vivo evaluation of teniposide nanosuspensions.

Teniposide (TEN) is a potent, broad spectrum antitumor agent, especially for cerebroma. But the application in clinic was limited because of its poor solubility. In this paper, teniposide nanosuspensions drug delivery system (TEN-NSDDS) for intravenous administration was developed for the first time. Specifically, TEN nanosuspensions were prepared by an anti-solvent sonication-precipitation method and evaluated in comparison with teniposide injection (VUMON) in vitro and in vivo. TEN nanosuspensions prepared showed rod-like morphology and the size was 151 ± 11 nm with a narrow poly dispersion index 0.138 determined by dynamic light scattering. The obtained TEN nanosuspensions were physically stable at least 10 days at 4°C. And the freeze-drying preparations were stable during 3 months. The cytotoxicity of TEN nanosuspensions were considerable to that of VUMON against U87MG and C6 cells in vitro. When tested in rats bearing C6 tumors, the TEN concentration in the tumors treated by the nanosuspensions was more than 20 times than that by the TEN solution at 2h. The TEN nanosuspensions exhibited significant tumor growth inhibition. Overall, the results suggested that nanosuspensions was an alternative formulation for teniposide to be administered intravenously, and it would be a promising formulation in clinic.

Production of rubusoside from stevioside by using a thermostable lactase from Thermus thermophilus and solubility enhancement of liquiritin and teniposide.

Solubility is an important factor for achieving the desired plasma level of drug for pharmacological response. About 40% of drugs are not soluble in water in practice and therefore are slowly absorbed, which results in insufficient and uneven bioavailability and GI toxicity. Rubusoside (Ru) is a sweetener component in herbal tea and was discovered to enhance the solubility of a number of pharmaceutically and medicinally important compounds, including anticancer compounds. In this study, thirty-one hydrolyzing enzymes were screened for the conversion of stevioside (Ste) to Ru. Recombinant lactase from Thermus thermophiles which was expressed in Escherichia coli converted stevioside to rubusoside as a main product. Immobilized lactase was prepared and used for the production of rubusoside; twelve reaction cycles were repeated with 95.4% of Ste hydrolysis and 49 g L(-1) of Ru was produced. The optimum rubusoside synthesis yield was 86% at 200 g L(-1), 1200 U lactase. The purified 10% rubusoside solution showed increased water solubility of liquiritin from 0.98 mg mL(-1) to 4.70±0.12 mg mL(-1) and 0 mg mL(-1) to 3.42±0.11 mg mL(-1) in the case of teniposide.

Preparation and characterization of teniposide PLGA nanoparticles and their uptake in human glioblastoma U87MG cells.

Many studies have demonstrated the uptake mechanisms of various nanoparticle delivery systems with different physicochemical properties in different cells. In this study, we report for the first time the preparation and characterization of teniposide (VM-26) poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) and their cellular uptake pathways in human glioblastoma U87MG cells. The nanoparticles prepared with oil-in-water (O/W) single-emulsion solvent evaporation method had a small particle size and spherical shape and provided effective protection against degradation of teniposide in PBS solution. Differential scanning calorimeter (DSC) thermograms concluded that VM-26 was dispersed as amorphous or disordered crystalline phase in the PLGA matrix. A cytotoxicity study revealed that, in a 24h period, blank PLGA NPs had no cytotoxicity, whereas teniposide-loaded PLGA NPs (VM-26-NPs) had U87MG cytotoxicity levels similar to free teniposide. Confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM) images showed the distribution and degradation processes of nanoparticles in cells. An endocytosis inhibition test indicated that clathrin-mediated endocytosis and macropinocytosis were the primary modes of engulfment involved in the internalization of VM-26-NPs. Our findings suggest that PLGA nanoparticles containing a sustained release formula of teniposide may multiplex the therapeutic effect and ultimately degrade in lysosomal within human glioblastoma U87MG cells.

Recent advances in semisynthesis, biosynthesis, biological activities, mode of action, and structure-activity relationship of podophyllotoxins: an update (2008-2010).

Podophyllotoxin, one of the well-known naturally occurring aryltetralin lignans, has been used as the lead-compound for the preparation of potent anticancer agents, such as etoposide, teniposide, and etopophos. In our previous review, we described the advances of podophyllotoxin derivatives from 2003 and 2007. In recent years, an increased number of interesting research work has been carried out on the podophyllotoxins. As a continuation, the present review summarizes and highlights the update advances of podophyllotoxin derivatives from 2008 and 2010 in regard to semisynthesis, biosynthesis, biological activities, mode of action and structure-biological activity relationship.

A review on hemisynthesis, biosynthesis, biological activities, mode of action, and structure-activity relationship of podophyllotoxins: 2003-2007.

Podophyllotoxin is an important and much sought after antimitotic natural lead compound, since it paved the way for three hemisynthetic derivatives of podophyllotoxin, e.g., etoposide, teniposide and etopophos, which are widely used as anticancer drugs and show good clinical effects against several types of neoplasms. Although the publication of the recent reviews by Gordaliza in 2004 and You in 2005, which covered the literatures concerning podophyllotoxin until the early part of 2003, there have been significant number of works carried out on podophyllotoxin recently. Therefore, this review presents up-to-date coverage of podophyllotoxin in regard to hemisynthesis, biosynthesis, biological activities, mode of action and structure-activity relationship.

Chemical stability of teniposide in aqueous and parenteral lipid emulsions.

The purpose of this study was to investigate the degradation kinetics of teniposide in lipid emulsion and aqueous solution. The chemical stability of teniposide in lipid emulsion and aqueous solution at various pH values and temperatures was monitored by high-performance liquid chromatography. In addition, the viscosities of emulsion at different temperatures were investigated. The degradation of teniposide both in emulsion and in aqueous solution was shown to follow pseudo-first-order degradation kinetics. The t (1/2) values of teniposide lipid emulsion (TLE) and the aqueous solution were 80 and 2.6 days at 10 degrees C, respectively. Under the most stable pH range of 6.0-6.5, stability of teniposide in the emulsion increased more than 30-fold compared with that in aqueous solution. Furthermore, there was a difference between the shelf life of TLE actually measured (29 days) at 10 degrees C and the one deduced (15 days) from the degradation data of high temperatures by Arrhenius equation. It could be hypothesized that the difference was due to a slower diffusion of teniposide from oil phase to aqueous phase at the lower temperatures, which would be a speed-limited process in the degradation of TLE. The results of viscosity test confirmed the presumption.

Podophyllotoxin: distribution, sources, applications and new cytotoxic derivatives.

Several podophyllotoxin derivatives modified in the A, B, C, D and E rings were prepared from podophyllotoxin and methyl isoxazopodophyllic acid and evaluated for their cytotoxicity on several neoplastic cell lines. Chemical transformations performed on these compounds have yielded derivatives more potent and more selective that the parent compound. Most of the compounds maintained their cytotoxicity at the microM level. Distribution, biosynthesis, production, biotechnology, applications and synthesis have also been reviewed.

Discovery of podophyllotoxins.

This review deals with the historical discovery of particularly important lignan derivatives used in cancer chemotherapy. From isolation of the naturally occurring podophyllotoxin, an inhibitor of microtubule assembly, to hemisynthesis of the clinically important anticancer drugs etoposide and teniposide, it will be demonstrated how the activities and the ability of this class of compounds to inhibit topoisomerase II were discovered by different research teams. By virtue of these discoveries, new hemisynthetic derivatives, with different mechanisms of action, are bringing improvements in the ability to treat cancer.

Stabilization of teniposide in aqueous mixtures of detergent-phospholipid.

Teniposide-containing mixed micelles and liposomes consisting of detergent and phospholipid were investigated and compared for their teniposide latency as functions of the mixed micellar preparation method, stabilizers, type of detergent,lipid composition and serum proteins after storage at 10 degrees C, and 23 degrees C, and 45 degrees C or/and freezing and freeze-drying. There was no significant difference in teniposide loss from liposomes obtained using different micellar preparation methods. Sugars, dextrose or sorbitol, had no effect on teniposide loss from liposome but stabilized teniposide micelles. Glutamic acid had no effect on teniposide loss from micelles but increased the loss from liposomes. The presence of cholesterol in bile salt-egg PC micelles had little effect on teniposide loss at 10 degrees C but generally increased it at 23 degrees C, and 45 degrees C, while bile salt-egg PC-cholesterol (9:9:1) liposomes were more stable than bile salt-egg PC liposomes. In contrast, teniposide loss from bile salt-egg PC-egg PE (2:1:1) liposomes or bile salt-egg PC-egg PA (16:15:1) micelles and liposomes increased remarkably, probably due to the surface charge and/or the destabilization of PC bilayer. However, bile salt-egg PC-soy PC (2:1:1) micelles and liposomes lost less amounts of teniposide under the same storage conditions. Further, the stability of teniposide was greatly increased by neutral detergents (e.g., CHAPS or octylglucoside). The loss of teniposide from CHAPS- or octylglucoside-egg PC micelles and liposomes after six months' storage at the ambient temperature were approximately 16% and 10%, respectively. Teniposide-micelles and liposomes, prepared in the presence of serum or serum protein, were more stable than CHAPS- or octylglucoside-egg PC liposomes. Teniposide was physically stable for at least 12 months when micelles were stored as the frozen or freeze-dried state. This result suggested that long-term storage for teniposide in neutral detergent-egg PC-soy PC micelles may be feasible in the presence of serum proteins.

Physical stability of teniposide in bile salt-egg phosphatidylcholine mixed micelles and liposomes.

Teniposide-containing bile salt-egg PC mixed micelles and their liposomes formed from dilution were investigated and compared for (i) their teniposide contents as a function of bile salt species, drug dose, the egg PC/BS molar ratio, total lipid concentration, ionic strength and storage states (freeze-thawed and freeze-dried state, and the presence of light and oxygen) and (ii) their mean particle size, stored under different conditions. The physical stability of teniposide in micelles and their liposomes increased with increasing hydrophobicity of bile salt the order being deoxycholate > cholate > conjugated cholate at all temperatures (10 degrees C, 23 degrees C, and 45 degrees C) studied. Teniposide-micelles were more stable at lower egg PC/BS molar ratios, while teniposide-liposomes were more stable at higher ratios. Teniposide stability slightly increased at higher total lipid concentration, indicating that bile salt-egg PC mixed micellar systems were in dynamic states. The presence of neither light nor oxygen significantly affected the stability of teniposide over the time and temperature range studied. However, the presence of salt in micellar and liposomal solutions greatly reduced teniposide loss by precipitation. Freezing or freeze-drying of teniposide-micelles induced neither micellar aggregation nor drug leakage. The stabilization may be due to the presence of detergent-like bile salts in the system.

Clinical pharmacology and schedule dependency of the podophyllotoxin derivatives.

Etoposide and teniposide are closely related derivatives of podophyllotoxin, and both have a phase-specific action in the late S and early G2 phases of the cell cycle. Etoposide has attracted more widespread use and study, although no evidence suggests a differing mode of action or spectrum of anticancer activity. The drugs have significant differences in their clinical pharmacology, however. Teniposide exhibits greater protein-binding affinity, has a longer plasma terminal elimination half-life, and has reduced plasma and renal clearances. Little is accurately known about the metabolism of either drug, but the fact that 40% to 60% of administered etoposide is accounted for by excretion or metabolism, whereas the range is only 10% to 25% for teniposide, reflects a further difference between the drugs. Renal dysfunction impairs etoposide excretion, but the effect of hepatic impairment on drug clearance is unclear. A specific oral formulation exists only for etoposide, although the unpalatable intravenous preparations of both drugs can be taken orally. The bioavailability of oral etoposide is about 50% at doses of 200 mg or less and decreases as drug doses increase. There is considerable intrapatient and interpatient variation in etoposide absorption, but the reasons for this are unknown. In vitro, the efficacy of etoposide is highly dependent on the schedule of administration. The superior efficacy without increased toxicity of more prolonged schedules of etoposide administration has been demonstrated recently in patients with small cell lung cancer (SCLC). Although the optimal schedule in any specific tumor is not known, current pharmacodynamic evidence suggests that the efficacy of etoposide, at least in SCLC, is related to the maintenance of prolonged low blood concentrations of drug.

Mechanisms of action of teniposide (VM-26) and comparison with etoposide (VP-16).

Teniposide is the result of extensive, long-term efforts to refine and improve on the cytotoxic activity of naturally occurring compounds extracted from podophyllin resins and purified. Isolation of an extremely potent though minor component of one of the early podophyllin derivatives led in turn to the synthesis and evaluation of several aldehyde condensation products. Two of these, teniposide and etoposide, were further investigated when their considerable antitumor activity in animals became apparent. Recognition of transient DNA breaks induced by teniposide, etoposide, and other podophyllotoxin analogues established not only that their site of activity was DNA but also that their cytotoxic effect was dose-dependent. Extensive investigation has further indicated that a primary mechanism of action of these agents involves inhibition of the catalytic activity of eukaryote topoisomerase II and, more important, the consequent stabilization of the normally transient covalent intermediate formed between the DNA substrate and the enzyme. As a result of elevated enzyme levels or enzyme activity, or both, in transformed cells, topoisomerase II inhibitors are highly selective for cancer cells versus normal cells. Although teniposide is not substantially more potent than etoposide in terms of catalytic inhibition or stabilization of the DNA-enzyme intermediate, it is more readily taken up by cells, which results in greater teniposide accumulation within the cells and, thus, a greater capacity for cytotoxicity.

Chemical and physical stability of etoposide and teniposide in commonly used infusion fluids.

The chemical and physical stabilities of the cytotoxic drugs etoposide and teniposide have been investigated in three different, commonly used, infusion fluids. Chemical stability has been measured by a stability indicating reversed-phase high-performance liquid chromatographic assay with ultraviolet detection. Physical stability was checked by visual inspection and the presence of microparticles was inspected by viewing the admixtures against a white and dark background of a light box equipped with an incandescent lamp and a polarizing filter. Samples were also tested for changes in pH. It is concluded that etoposide and teniposide in 5% dextrose and 0.9% sodium chloride infusion fluids (concentration: 0.4 mg/mL) are chemically stable for at least four days at room temperature. Stability of the drugs is not influenced by the presence of normal room fluorescent light nor by the type of container material used (glass bottles or polyvinyl chloride minibags). Occasional precipitation occurred in etoposide infusion fluids with a concentration higher than 0.4 mg/mL. Teniposide infusion solutions were physically stable at the tested concentrations up to 0.7 mg/mL.