EndoTAG1
In EndoTAG®-1 the active component is hydrophobic. The drug is inserted into the lipid bilayer, and in particular its hydrophobic compartment (Fig. 4), which acts as a two-dimensional solvent for the compound. EndoTAG®-1 comprises the diterpenoid paclitaxel (44), a potent antimitotic agent widely used in cancer therapy (45). Paclitaxel has a very low solubility in water (in the order of 1 mg/L) and therefore its solubility must be increased for IV application. In Taxol® (Brystol-Myers Squibb), which is approved for treatment of advanced ovarian, breast, and non-small cell lung cancer in the United States and in Europe, paclitaxel is solubilized by a mixture of Cremophor® EL (BASF) and ethanol. However, Cremophor causes serious side effects such as hypersensitivity reactions and peripheral neuropathy (46,47). Prophylactic steroids and histamine receptor antagonists have to be coadministered with Taxol to reduce these effects. In addition, the maximum dose of paclitaxel is limited by neutropenia and neurotoxicity.
Great efforts to develop alternative formulations for paclitaxel are ongoing (48-50). Common goals are to provide sufficient solubility in aqueous environment, to improve pharmacokinetic and pharmacodynamic parameters, to reduce side effects and, possibly, to improve delivery to the target tissue of the drug.
Even though EndoTAG®-1 has another target than conventional paclitaxel-based products for cancer therapy, it can also be regarded as a lipo-somal approach for paclitaxel formulation (50). A significant number of studies on the liposomal formulation of paclitaxel has been published, and fundamental aspects of paclitaxel-membrane interactions have been investigated to detail by various methods (51-54). For development of EndoTAG®-1, an excessive screening of drug/lipid mixtures and formulation techniques has been carried out. Physicochemical characterization of drug-loaded model membranes was performed in order to get insight into general aspects of paclitaxel insertion into (cationic) lipid membranes. Inter alia, differential scanning calorimetry measurements, spectroscopic techniques, X-ray scattering, and Langmuir monolayer measurements (55,56) have been applied as tools to study paclitaxel membrane interactions and to define the formulation parameters.
Figure 6 shows the understanding of the molecular organization of paclitaxel in liposomal preparations which can be derived from such experiments (50). In addition to the paclitaxel, which is inserted into the liposomes,
Liposomal preparation of paclitaxel
Figure 6 Options for the partition of paclitaxel in liposome preparations. The drug is supposed to be inserted in the liposomal lipid bilayer. In addition, a fraction that is dissolved in water has to be taken into account. If the maximum solubility of pacli-taxel is exceeded, formation of microcrystals as a colloidal dispersion or a precipitate can occur.
Liposomal preparation of paclitaxel
Figure 6 Options for the partition of paclitaxel in liposome preparations. The drug is supposed to be inserted in the liposomal lipid bilayer. In addition, a fraction that is dissolved in water has to be taken into account. If the maximum solubility of pacli-taxel is exceeded, formation of microcrystals as a colloidal dispersion or a precipitate can occur.
it may be present in the aqueous phase, or as precipitated or colloidally dispersed crystallites. In equilibrium, there is a constant ratio between the concentration of paclitaxel in the liposome and in the aqueous phase (53). If liposomes are loaded with an amount of paclitaxel, which is higher than the equilibrium value, paclitaxel release and subsequent crystallization of the drug may have to be taken into account. In a practical pharmaceutical preparation, the concentration of paclitaxel should be about two to three orders of magnitude higher than its maximum solubility in water.
EndoTAG®-1, which is currently tested in clinical studies, comprises about 3 mol% paclitaxel in a DOTAP/DOPC lipid matrix. For application to a patient it is present as a colloidal dispersion of particles of uniform size of about 200 nm, where the total lipid concentration is 10 mM. For storage, the formulations are lyophilized, and they are reconstituted with water for injection directly prior use. A robust industrial scale process for manufacturing and lyophilization of liposomal products was developed (57), and is summarized in Figure 7. Lipids and paclitaxel are dissolved in ethanol at the appropriate molar ratio, and this concentrated solution is injected into the aqueous phase under stirring. Thus, drug-loaded, polydisperse liposomes are formed spontaneously by a self-assembly process. The size distribution of the liposomes is adjusted by several consecutive extrusion cycles through membranes of defined pore size. After sterile filtration, the preparation is filled into moulded vials and freeze-dried. By lyophilization of the product, a shelf life of more than two years is provided. Regular cGMP production has been performed with a bulk size of about 70 L, resulting in a reproducible and consistent quality of the final product in more than 15 production batches.
In a broad preclinical program, the biological effects of EndoTAG®-1 were studied in comparison to different controls, including Taxol. EndoTAG®-1 showed superior antitumor activity in a variety of different species and
Figure 7 Production scheme of EndoTAG®-1. Multilamellar liposomes are formed by ethanol injection of the lipid and drug solution into the aqueous phase. By extrusion and sterile filtration, monolamellar, monodisperse, and sterile liposomes are formed. Subsequently, the preparation is freeze-dried for storage.
Figure 7 Production scheme of EndoTAG®-1. Multilamellar liposomes are formed by ethanol injection of the lipid and drug solution into the aqueous phase. By extrusion and sterile filtration, monolamellar, monodisperse, and sterile liposomes are formed. Subsequently, the preparation is freeze-dried for storage.
tumor models. It significantly inhibited tumor growth, delayed the onset of metastasis, inhibited infiltration of healthy tissue surrounding the tumor, and increased the survival of tumor-bearing animals (16,58,59). Importantly and indicative for a different mode of action, EndoTAG®-1 inhibited tumor growth also in Taxol-resistant animal tumor models, as for example, B16 melanoma and Sk-Mel 28 melanoma. EndoTAG®-1 demonstrated a strong antivascular effect on the preexisting tumor vasculature and affected several tumor microcirculatory parameters. It reduced the endothelial cell mitotic rate in the vicinity of the tumor (58), caused a dramatic lasting reduction of tumor perfusion (59) and tumor vessel damage (59,60). There is evidence that continuous EndoTAG®-1 treatment can lead to tumor vessel leakage and enhanced accessibility of the tumor tissue for low-molecular-weight substances (60,61). EndoTAG®-1 treatment of mice bearing an orthotopically grown human pancreatic carcinoma led to a total suppression of liver metastasis. Combination of EndoTAG®-1 with the conventional chemotherapeutic drugs cisplatin (60) and gemcitabine (62) further enhanced tumor growth inhibition.
EndoTAG®-1 has passed a phase I clinical program with more than 150 patients with advanced metastatic cancer for determination of the safety and tolerability, and for investigation of the pharmacokinetic parameters.
EndoTAG® -1 appears to be a safe drug with an overall response rate of 8% to 14%; a range which is typical for effective drugs in phase I cancer trials. A large phase II trial for EndoTAG®-1 in combination with gemcitabine in patients with advanced or metastatic adenocarcinoma of the pancreas has started in 2005.
EndoTAG®-2
Also EndoTAG®-2 is a preparation for tumor therapy, but the molecular target of the active compound and the mechanism of loading the drug to the liposome are essentially different to those in EndoTAG®-1. The active compound in EndoTAG®-2 is camptothecin (CPT), a quinoline-based alkaloid, which can be isolated from the Chinese tree Camptotheca acuminata. CPT is a topoisomerase inhibitor, i.e., binding to the topoisomerase I-DNA complex induces DNA breaks and cell death (64).
A fundamental molecular property of CPT is its pH-dependent equilibrium between the lactone and the carboxylate form (Fig. 8, left side). The lactone form is lipophilic, whereas the carboxylate, which predominates at physiological pH and above, is water-soluble. Both molecular forms are present as equilibrium and one form can be transformed into the other one, for example, by changing the pH. The carboxylate form is considered to be less active and responsible for severe side reactions such as neutropenia, thrombocytopenia, and hemorrhagic cystitis (63). Therefore, efforts in the development of CPT drugs concentrated on the stabilization of the lactone
Lactone
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