Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
2000-06-09
2002-03-19
Owens, Amelia (Department: 1625)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C549S510000, C549S511000
Reexamination Certificate
active
06358996
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to the synthesis and composition of isotopically labeled paclitaxel and related taxane compounds. The present invention is more particularly directed to paclitaxel and related taxane molecules having stable isotopes synthetically incorporated therein at common positions in the chemical structure thereof. The present invention is more specifically directed to a carbon-13 labeled paclitaxel useful as an internal standard for HPLC/MS analysis of biological tissues for quantitation of taxanes. More particularly, the present invention is directed to synthesis and composition of carbon-13 labeled paclitaxel for use as an internal standard for the HPLC/MS analysis of biological tissue samples of clinical and toxicology pharmacokinetic and pharmacodynamic studies.
BACKGROUND OF THE INVENTION
Various taxane compounds are known to exhibit anti-tumor activity. As a result of this activity, taxanes have received increasing attention in the scientific and medical community. Primary among these is a compound known as “paclitaxel”, which is also referred to in the literature as “taxol”. Paclitaxel has been approved for the chemotherapeutic treatment of several different varieties of tumors, and the clinical trials indicate that paclitaxel promises a broad range of potent anti-leukemic and tumor-inhibiting activity. Paclitaxel has the formula:
Paclitaxel is a microtubule poison characteristic of a new class of anticancer drugs. In contrast to other natural spindle poisons, such as vinca alkaloids, taxol increases both the assembly and stability of cellular microtubules, and blocks the cell cycle in late G2 and M phases. It has shown significant activity in clinical trials against a number of human tumors, including breast, lung, head, neck, and ovarian carcinomas. Several pharmacokinetic studies, based on high-performance liquid chromatography (HPLC) and ultraviolet (UV) detection, have been reported. In most of these early investigations, the disappearance of taxol from plasma was found to follow a bi-exponential elimination model. In clinical pharmacological studies, several investigators have demonstrated that total urinary excretion of unmetabolized taxol ranges from 1.5% to 9% with a substantial inter-patient variability. These data suggest that renal clearance contributes little to systemic clearance. Conversely, metabolism, biliary excretion and/or extensive tissue binding probably account for the bulk of taxol disposition. Cytochrome P450 enzymes involved in the biotransformation of taxol have been identified using human liver microsomes and various over-expressed human cytochrome P450 isozymes. Preliminary studies showed that 6∝-hydroxytaxol, the major metabolite of taxol in human bile, was also present in plasma. Other putative minor metabolites were also detected in the plasma of human patients. Despite these promising observations, the low concentration of taxol derivatives presents a major difficulty for analysis of paclitaxel and its derivatives and metabolites in biological samples.
Mass spectrometry is a powerful technique for taxoid identification. Electron impact (EI), chemical ionization (CI), desorption chemical ionization (DCI), fast atom bombardment (FAB), electrospray (ES) ionization and matrix-assisted laser desorption/ionization (MALDI) techniques have all been used for the mass spectrometric analysis of taxol, taxol-related diterpenoids, and taxol metabolites. Characterization and quantification of taxanes by tandem mass spectrometry (MS/MS) has also been described. All of these mass spectrometry procedures require the separation of the various taxane derivatives from biological fluids. This purification is labor-intensive and requires large volumes of fluids, due to the low concentration of these compounds in human plasma, urine and bile. The direct combination of mass spectrometry with chromatographic techniques offers a new and effective alternative for metabolic studies which involve the analysis of complex biological samples, such as cell culture medium, plasma, bile and urine. HPLC has been successfully interfaced with thermospray (TSP) ionization and atmospheric pressure ionization (API). Recent reports show the application of LC/MS to the analysis of taxanes using thermospray ionization, split-flow LC/ES-MS and LC/ES-MS/MS to screen plant or cell culture extracts.
Pharmacokinetic and pharmacodynamic studies of paclitaxel treatment strategies require extensive analyses of biological samples. Evaluation of oral formulations of paclitaxel for bioavailability following oral dosing in animals requires large numbers of analyses of plasma samples. The average sample preparation plus analysis of HPLC takes 30-60 minutes per sample. Accordingly, it would be desirable to significantly reduce the time for sample preparation and analysis in pharmacokinetic and pharmacodynamic studies of paclitaxel treatment strategies.
A widely used technique of quantitation of analyses by HPLC involves the addition of an internal standard to compensate for various analytical errors. With this approach a known compound at a fixed concentration is added to the unknown sample to give a peak in the chromatograph which can be measured separately. This known compound is used as an internal marker to compensate for the effect of minor variations in separation parameters on peak size, including sample-size fluctuations. However, because the delivery of sample volumes is quite precise with microsampling valves, the main utility of the internal standard technique is in assays that require sample pretreatment (and/or solute derivatization) where variable recoveries of compounds of interest may occur.
To compensate for losses of the compound of interest during sample workup, an internal standard that is structurally similar to the compound(s) of interest is added at a known concentration to the original unknown sample, the pretreatment is carried out, and the resulting sample is analyzed. In this approach any loss of the compound of interest will be accompanied by the loss of an equivalent fraction of internal standard. The accuracy of this approach is obviously dependent on the structural equivalence of the compound(s) of interest and the internal standard i.e., for best results the internal standard and the compound(s) of interest should, among other things, extract equally.
The selection of the internal standard is critical for measurements. An internal standard generally must have a completely resolved peak such that there are no interferences; must elute close to compound(s) of interest (similar k′ values); must behave equivalently to compound(s) of interest for analyses involving pretreatments derivative formation, etc.; must not be present in the original sample; and must be stable such that it is unreactive with sample components, column packing, or the mobile phase. The internal standard must be added at a concentration that will produce a measurement ratio of about unity with compound(s) of interest, and it is desirable for the internal standard to be commercially available in high purity. More than one internal standard may be required for multicomponent mixtures to achieve highest precision.
A practical problem with the internal standard technique is that the standard must be located in a “vacant” region in the sample analysis. For simple mixtures this is usually not difficult. However, for complex samples the selection of an internal standard can be tedious. A satisfactory internal standard often is a compound that is structurally related to the compound to be measured (e.g., an isomer or close homolog). The internal standard should have similar k′, solubility, and detection response but be adequately separated from other sample components.
Accordingly, there remains a need to provide a new and improved internal standard for use with pharmacokinetic and pharmacodynamic studies of paclitaxel treatment strategies, and in particular studies utilizing high performance liquid chromatography/mass spectrometry for ana
Alexander Michael S.
McChesney James D.
Zygmunt Jan
Henson Michael R.
Martin Timothy J.
NaPro BioTherapeutics, Inc.
Owens Amelia
Weygandt Mark H.
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