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(12) United States Patent (10) Patent No.: US 6,358,996 B1 Alexander Et Al

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(12) United States Patent (10) Patent No.: US 6,358,996 B1 Alexander Et Al USOO6358996B1 (12) United States Patent (10) Patent No.: US 6,358,996 B1 Alexander et al. (45) Date of Patent: Mar. 19, 2002 (54) STABLE ISOTOPE LABELING OF 5,688.977 A 11/1997 Sisti et al. PACLITAXEL 5,750,736 A 5/1998 Sisti 5,750,737 A 5/1998 Sisti et al. (75) Inventors: Michael S. Alexander, McMinnville, SE A go Silet al. 2- - -2 S C a Oulder,t SS), Jan Aygmunt, D. Mey. Longmont, t 5.948,9195,939,566 A 9/19998/1999 SistiSwindell et al. et al. both of CO (US) 5,973,170 A 10/1999 Sisti et al. 6,048,990 A 4/2000 Li t al. (73) ASSignee: NaPro BioTherapeutics, Inc., Boulder, 6,066,749 A 5/2000 CO (US) 6,072,060 A 6/2000 Swindell et al. 6,107,497 A 8/2000 Sisti et al. (*) Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 Primary Examiner Amelia Owens U.S.C. 154(b) by 0 days (74) Attorney, Agent, or Firm Mark H. Weygandt; a -- y yS. Timothy J. Martin; Michael R. Henson (21) Appl. No.: 09/591,159 (57) ABSTRACT (22) Filed: Jun. 9, 2000 A chemical compound comprising an isotopically labeled 51) Int. Cl." A61K 31/335 analog of a Standard taxane molecule, wherein Said isoto ( O - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - pically labeled analog is Synthetically formed tO have incor (52) U.S. Cl. ........................ 514,449; 549/510; 549/511 porated therein at a Selected position a Stable isotope of an (58) Field of Search ................................. 549/510, 511; element existing at Said Selected position in Said Standard 514/449 taxane molecule, Said isotope having amass different form a mass of a mass of a most abundantly occurring isotope of (56) References Cited Said element in nature, Such that Said isotopically labeled U.S. PATENT DOCUMENTS analog has a molecular weight different form a molecular weight of Said Standard taxane molecule. 5,675,025 A 10/1997 Sisti et al. 5,684,175 A 11/1997 Sisti et al. 35 Claims, 3 Drawing Sheets U.S. Patent Mar. 19, 2002 Sheet 1 of 3 US 6,358,996 B1 O O OHNZGO |10: HQ<—————————————.O eInuulo-I)(Zelnuuo-)LH(|eInuuoº)(£ HO HNZGO|OHNZGO -N- U.S. Patent Mar. 19, 2002 Sheet 2 of 3 US 6,358,996 B1 SOTOXVILTW/50N00||NILNESEHdT.EXWLITOV.d| I O O E0,0|| C O w Sdo "All Sueu "Aisuelu U.S. Patent Mar. 19, 2002 Sheet 3 of 3 US 6,358,996 B1 || | | | 90€?TW/50N06TEXVLITOVd ~~~~~E/-*—––--------------------~–1-------------0’0 07 08 92 Scdo "All987002 Sueu ||730'G. Scdo "WISuelu US 6,358,996 B1 1 2 STABLE ISOTOPE LABELING OF bile, was also present in plasma. Other putative minor PACLITAXEL metabolites were also detected in the plasma of human patients. Despite these promising observations, the low FIELD OF THE INVENTION concentration of taxol derivatives presents a major difficulty for analysis of paclitaxel and its derivatives and metabolites The present invention is directed to the Synthesis and in biological Samples. composition of isotopically labeled paclitaxel and related Mass spectrometry is a powerful technique for taxoid taxane compounds. The present invention is more particu identification. Electron impact (EI), chemical ionization larly directed to paclitaxel and related taxane molecules (CI), desorption chemical ionization (DCI), fast atom bom having Stable isotopes Synthetically incorporated therein at bardment (FAB), electrospray (ES) ionization and matrix common positions in the chemical Structure thereof. The assisted laser desorption/ionization (MALDI) techniques present invention is more Specifically directed to a carbon have all been used for the mass spectrometric analysis of 13 labeled paclitaxel useful as an internal Standard for taxol, taxol-related diterpenoids, and taxol metabolites. HPLC/MS analysis of biological tissues for quantitation of Characterization and quantification of taxanes by tandem taxanes. More particularly, the present invention is directed mass spectrometry (MS/MS) has also been described. All of to Synthesis and composition of carbon-13 labeled paclitaxel 15 these mass spectrometry procedures require the Separation for use as an internal standard for the HPLC/MS analysis of of the various taxane derivatives from biological fluids. This biological tissue Samples of clinical and toxicology phar purification is labor-intensive and requires large Volumes of macokinetic and pharmacodynamic Studies. fluids, due to the low concentration of these compounds in human plasma, urine and bile. The direct combination of BACKGROUND OF THE INVENTION mass spectrometry with chromatographic techniqueS offers a Various taxane compounds are known to exhibit anti new and effective alternative for metabolic studies which tumor activity. As a result of this activity, taxanes have involve the analysis of complex biological Samples, Such as received increasing attention in the Scientific and medical cell culture medium, plasma, bile and urine. HPLC has been community. Primary among these is a compound known as 25 successfully interfaced with thermospray (TSP) ionization “paclitaxel', which is also referred to in the literature as and atmospheric pressure ionization (API). Recent reports “taxol”. Paclitaxel has been approved for the chemothera show the application of LC/MS to the analysis of taxanes peutic treatment of Several different varieties of tumors, and using thermospray ionization, split-flow LC/ES-MS and the clinical trials indicate that paclitaxel promises a broad LC/ES-MS/MS to screen plant or cell culture extracts. range of potent anti-leukemic and tumor-inhibiting activity. Pharmacokinetic and pharmacodynamic Studies of pacli Paclitaxel has the formula: taxel treatment Strategies require extensive analyses of bio logical Samples. Evaluation of oral formulations of pacli taxel for bioavailability following oral dosing in animals O requires large numbers of analyses of plasma Samples. The 35 average Sample preparation plus analysis of HPLC takes 30-60 minutes per sample. Accordingly, it would be desir able to Significantly reduce the time for Sample preparation and analysis in pharmacokinetic and pharmacodynamic Studies of paclitaxel treatment Strategies. 40 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 Paclitaxel is a microtubule poison characteristic of a new unknown Sample to give a peak in the chromatograph which class of anticancer drugs. In contrast to other natural Spindle 45 can be measured Separately. This known compound is used poisons, Such as Vinca alkaloids, taxol increases both the as an internal marker to compensate for the effect of minor assembly and stability of cellular microtubules, and blocks variations in Separation parameters on peak size, including the cell cycle in late G2 and M phases. It has shown Sample-size fluctuations. However, because the delivery of Significant activity in clinical trials against a number of Sample Volumes is quite precise with microSampling valves, human tumors, including breast, lung, head, neck, and 50 the main utility of the internal Standard technique is in assays ovarian carcinomas. Several pharmacokinetic Studies, based that require sample pretreatment (and/or Solute on high-performance liquid chromatography (HPLC) and derivatization) where variable recoveries of compounds of ultraviolet (UV) detection, have been reported. In most of interest may occur. these early investigations, the disappearance of taxol from To compensate for losses of the compound of interest plasma was found to follow a bi-exponential elimination 55 during Sample workup, an internal Standard that is structur model. In clinical pharmacological Studies, Several investi ally similar to the compound(s) of interest is added at a gators have demonstrated that total urinary excretion of known concentration to the original unknown Sample, the unmetabolized taxol ranges from 1.5% to 9% with a sub pretreatment is carried out, and the resulting Sample is Stantial inter-patient variability. These data Suggest that renal analyzed. In this approach any loSS of the compound of clearance contributes little to Systemic clearance. 60 interest will be accompanied by the loSS of an equivalent Conversely, metabolism, biliary excretion and/or extensive fraction of internal Standard. The accuracy of this approach tissue binding probably account for the bulk of taxol dis is obviously dependent on the Structural equivalence of the position. Cytochrome P450 enzymes involved in the compound(s) of interest and the internal Standard i.e., for biotransformation of taxol have been identified using human best results the internal Standard and the compound(s) of liver microSomes and various over-expressed human cyto 65 interest should, among other things, extract equally. chrome P450 isozymes. Preliminary studies showed that The selection of the internal standard is critical for 6 -hydroxytaxol, the major metabolite of taxol in human measurements. An internal Standard generally must have a US 6,358,996 B1 3 4 completely resolved peak Such that there are no interfer in the A-ring Sidechain of the isotopically labeled analog, ences, must elute close to compound(s) of interest (similar Such as in an aromatic ring. A plurality of Stable isotopes, k' values); must behave equivalently to compound(s) of which may be different isotopes or identical isotopes and interest for analyses involving pretreatments derivative particularly carbon-13, may be incorporated in the isotopi formation, etc., must not be present in the original Sample; cally labeled analog at corresponding Selected positions. The and must be stable Such that it is unreactive with Sample molecular weight of the isotopically labeled analog may components, column packing, or the mobile phase. The differ Significantly from the molecular weight of the Stan internal Standard must be added at a concentration that will produce a measurement ratio of about unity with compound dard taxane molecule, for example by between about 4 and (S) of interest, and it is desirable for the internal Standard to about 10 amus, and preferably about 6 amus, such that the be commercially available in high purity.
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