QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.009944v1 34/11/1817    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jost, L. M. Articles by Laurent, D. Search for Related Content PubMed PubMed Citation Articles by Jost, L. M. Articles by Laurent, D.

Metabolism and Disposition of Vatalanib (PTK787/ZK-222584) in Cancer Patients

Kantonsspital Bruderholz, Oncology Department, Bruderholz, Switzerland (L.M.J.); Novartis Pharma AG, Exploratory Development, Basel, Switzerland (H.-P.G., C.S., F.W., G.G.); Schering Oy, Research & Development, Helsinki, Finland (T.J.); Schering AG, Berlin, Germany (C.G., A.R., K.D., D.L.); and Novartis Pharmaceuticals Corporation, Oncology Business Unit, Florham Park, New Jersey (E.M., Y.W.)

(Received February 23, 2006; Accepted July 25, 2006)

	Abstract

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Vatalanib (PTK787/ZK-222584) is a new oral antiangiogenic molecule that inhibits all known vascular endothelial growth factor receptors. Vatalanib is under investigation for the treatment of solid tumors. Disposition and biotransformation of vatalanib were studied in an open-label, single-center study in patients with advanced cancer. Seven patients were given a single oral ¹⁴C-radiolabeled dose of 1000 mg of vatalanib administered at steady state, obtained after 14 consecutive daily oral doses of 1000 mg of nonradiolabeled vatalanib. Plasma, urine, and feces were analyzed for radioactivity, vatalanib, and its metabolites. Metabolite patterns were determined by high-performance liquid chromatography coupled to radioactivity detection with off-line microplate solid scintillation counting and characterized by LC-MS. Vatalanib was well tolerated. The majority of adverse effects corresponded to common toxicity criteria grade 1 or 2. Two patients had stable disease for at least 7 months. Plasma C_max values of ¹⁴C radioactivity (38.3 ± 26.0 µM; mean ± S.D., n = 7) and vatalanib (15.8 ± 9.5 µM) were reached after 2 and 1.5 h (median), respectively, indicating rapid onset of absorption. Terminal elimination half-lives in plasma were 23.4 ± 5.5 h for ¹⁴C radioactivity and 4.6 ± 1.1 h for vatalanib. Vatalanib cleared mainly through oxidative metabolism. Two pharmacologically inactive metabolites, CGP-84368/ZK-260120 [(4-chlorophenyl)-[4-(1-oxy-pyridin-4-yl-methyl)-phthalazin-1-yl]-amine] and NVP-AAW378/ZK-261557 [rac-4-[(4-chloro-phenyl)amino]--(1-oxido-4-pyridyl)phthalazine-1-methanol], having systemic exposure comparable to that of vatalanib, contributed mainly to the total systemic exposure. Vatalanib and its metabolites were excreted rapidly and mainly via the biliary-fecal route. Excretion of radioactivity was largely complete, with a radiocarbon recovery between 67% and 96% of dose within 7 days (42–74% in feces, 13–29% in urine).

Vatalanib is a new oral, small antiangiogenic molecule belonging to the chemical class of aminophthalazines. It inhibits all known VEGFRs (VEGFR-1 [Flt-1], VEGFR-2 [KDR], and VEGFR-3 [Flt-4]) with greater potency against VEGFR-1 and VEGFR-2. In an enzyme-based assay, vatalanib inhibited all the VEGF receptors and three other kinases belonging to the same family of protein tyrosine kinases (class III): the platelet-derived growth factor receptor tyrosine kinase, receptor for stem cell factor (c-Kit) protein tyrosine kinase, and colony-simulating factor 1 receptor (c-Fms). Vatalanib does not have cytotoxic or antiproliferative effects on cells that do not express VEGFR at concentrations up to 10 µM (Bold et al., 2000; Wood et al., 2000).

Specific inhibition of tumor-induced angiogenesis should prevent both the continued growth of many solid tumors and their metastatic potential (Fan et al., 1995). Based on its biologic specificity for the VEGFR tyrosine kinases, vatalanib is under investigation for the treatment of patients with solid tumors, including carcinomas of the breast (Scott et al., 1998), lung (Takahama et al., 1998), gastrointestinal tract (Takahashi et al., 1995), prostate (Ferrer et al., 1998), liver (Mross et al., 2005), ovary (Nakanishi et al., 1997; Sowter et al., 1997; Yamamoto et al., 1997), brain, renal cell (Nakagawa et al., 1997), and bladder (Crew et al., 1997), as well as Kaposi's sarcoma and von Hippel-Lindau syndrome.

The pharmacokinetics of vatalanib has been studied in several phase 1 and 2 clinical trials after single and multiple administration. Pharmacokinetic plasma profiles of vatalanib have been collected on day 1, around day 15, and day 28 in most trials. Upon multiple dosing of 1000 mg of vatalanib, the area under the concentration-time curve (AUC) declined by approximately 50% between day 1 and day 15 of treatment and remained at steady state thereafter (Morgan et al., 2003; Mross et al., 2005). This decline in AUC between day 1 and day 15 is consistent with autoinduction mainly by CYP3A in clearance (Morgan et al., 2003). The purpose of this study was to assess the absorption, disposition, kinetics, and biotransformation of radiolabeled drug and metabolites after a single oral dose of 1000 mg of ¹⁴C-radiolabeled vatalanib (given as 1340 mg of succinate salt) to patients with advanced cancer who are under treatment with daily 1000 mg of nonradiolabeled vatalanib. The dose of 1000 mg/day has been evaluated in phase 1 trials in advanced cancer patients and has been established as being safe. Administration of a single radioactive dose of vatalanib during steady state was chosen, because it mirrors closely the pharmacokinetics and disposition of vatalanib under clinical circumstances.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Reference Compounds and Other Materials. Reference standards were provided by either Novartis Pharmaceutical Corporation (East Hanover, NJ) or Schering AG (Berlin, Germany). The reference compounds were named according to International Union of Pure and Applied Chemistry (IUPAC) rules. The prefixes "CGP" and "NVP" denote compound identifications given by Novartis. The prefix "ZK" denotes compound identifications given by Schering AG. The prefix "M" denotes the compound/metabolite identification number given by Schering AG.

The reference compounds used were as follows: NVP-AAW378/ZK-261557 (M14; methyl-carbinol-pyridine-N-oxide of vatalanib): rac-4-[(4-chloro-phenyl)amino]--(1-oxido-4-pyridyl)phthalazine-1-methanol; CGP-84368/ZK-260120 (M17; pyridine N-oxide metabolite): (4-chloro-phenyl)-[4-(1-oxy-pyridin-4-yl-methyl)-phthalazin-1-yl]-amine; ZK-276224 (M19; 3''-pyridone metabolite): 4-({4-[(4-chloro-phenyl)amino]phthalazin-1-yl}methyl)pyridin-2(1H)-one; vatalanib, PTK787/ZK-226343 (base) (M21; parent drug): (4-chloro-phenyl)-(4-pyridin-4-yl-methyl-phthalazin-1-yl)-amine; CGP-85587/ZK-228353 (M24; 4-yl-methyl carbinol metabolite): [4-([1E,3E]-4-chloro-1-vinyl-penta-1,3-dienylamino)-phthalazin-1-yl]-pyridin-4-yl-methanol; ZK-264851 (M38; 2'-hydroxy metabolite): 5-chloro-2-(4-pyridin-4-yl-methyl-phthalazin-1-yl-amino)-phenol; CGP-85903/ZK-228354 (M25; ketopynalin): [4-(4-chloro-phenylamino)phthalazin-1-yl] (4-pyridyl) ketone; ZK-260116 (M26; 3'-hydroxy metabolite): 2-chloro-5-(4-pyridin-4-yl-methyl-phthalazin-1-yl-amino)-phenol; CGP-79469/ZK-260013 (M27; 4'-hydroxyphenyl des-chloro metabolite): 4-(4-pyridin-4-yl-methyl-phthalazin-1-yl-amino)-phenol.

The purity of the reference compounds was separately examined by liquid chromatography coupled to mass spectrometry (LC-MS). Solvents and reagents, all of analytical grade, were purchased from commercial manufacturers.

Study Design and Subjects. This was an open-label, single-center absorption, distribution, metabolism, and excretion (ADME) study design with a single oral ¹⁴C-radiolabeled dose of 1000 mg of vatalanib. As an established standard for human ADME trials, a total of six subjects was planned to complete the ADME evaluation phase. A total of eight patients with advanced cancer (breast, n = 1; colorectal, n = 2; lung, n = 2; ovarian, n = 1; pancreatic, n = 1; and thyroid, n = 1), with a range of two to eight prior anticancer chemotherapeutic regimens, were enrolled into the study. Patient demographics are provided in Table 1. Patients received daily oral doses of 1000 mg of nonradiolabeled vatalanib for 14 consecutive days to obtain steady state (days 1–14; loading phase). Based on several previous trials, the 1000 mg/day dose has been determined to be safe. On day 15, patients received a single oral ¹⁴C-radiolabeled dose of 1000 mg of vatalanib. The day after the radioactive dose was given, treatment with 1000 mg of nonradiolabeled vatalanib daily was continued, and the patients were monitored for 7 days (ADME phase) (days 16–22). After the ADME phase, the patients received for 11 consecutive days (days 23–33) a daily oral dose of 1000 mg of nonradiolabeled vatalanib for monitoring of complete elimination/excretion of the radioactive study drug. After the ADME follow-up period, the patients who wanted to continue with vatalanib treatment received a daily oral dose of 1000 mg of nonradiolabeled vatalanib until disease progression or unacceptable toxicity occurred. All patients were followed every 2 weeks for safety until disease progression or unless they prematurely discontinued from the study without evidence of disease progression. Inclusion in this study was irrespective of stage of disease or extent of prior therapy. Other criteria for inclusion were as follows: histologically confirmed malignancy in which no conventional therapies were available that offered reasonable hope of cure or significant palliation, a body weight within –20 to +15% of normal for height and frame size according to the Metropolitan Life Insurance Table, an absolute neutrophil count 1.5 x 10⁹/l, hemoglobin 9 g/dl, platelets 100 x 10⁹/l, aspartate aminotransferase and alanine aminotransferase 3.0 times upper limit of normal (ULN), serum bilirubin 1.5 times ULN, serum creatinine 1.5 times ULN, 24-h creatinine clearance 50 ml/min, total urinary protein in 24-h urine collection 0.5 g, a life expectancy of at least 3 months, adequate organ function, and a World Health Organization performance status of 0 to 2.

Prior therapies were permitted if administered 12 months before study entry for radiolabeled compounds, 6 weeks for nitrosoureas or mitomycin C, 2 weeks for radiotherapy or surgery, or 4 weeks for any other cytotoxic, cytostatic, or investigational drug. Patients were excluded if they had a history of brain metastases; were pregnant or breastfeeding; had concurrent severe and/or uncontrolled medical disease, acute or chronic liver disease, or impairment of gastrointestinal function that would alter vatalanib absorption; or concurrent warfarin administration.

Drug Administration. Seven of eight patients were successfully dosed with four daily tablets of unlabeled vatalanib, each containing 335 mg of succinate salt (= 250 mg free base), for 14 days (day 1 to day 14; loading period). One patient discontinued after the loading phase with nonradiolabeled vatalanib because of tumor progression. On day 15, the patients received a single dose of ¹⁴C-radiolabeled vatalanib (Fig. 1) given as five gelatin capsules, each containing a nominal 268 mg of succinate salt (= 200 mg free base) and were monitored for 7 days (ADME phase). The radioactive dose was 1.005 MBq (27.16 µCi). The capsules were swallowed with approximately 250 ml of water between 7:30 and 09:00 AM, after at least a 10-h fast. The patients were dosed within, at maximum, a 1-h interval. All patients fasted for at least 10 h before the dose of ¹⁴C-radiolabeled vatalanib and continued to fast for at least 4 h thereafter. The day after administration of the radiolabeled dose, the patients received, for 7 consecutive days (days 16–22), a daily oral dose of 1000 mg of nonradiolabeled vatalanib (as four tablets of 335 mg of succinate salt, corresponding to 250 mg free base). After the ADME phase, the patients received for 11 consecutive days (days 23–33) a daily oral dose of 1000 mg of nonradiolabeled vatalanib (as four tablets of 335 mg of succinate salt, corresponding to 250 mg free base). After day 33, the patients who wanted to continue with drug treatment received a daily oral dose of 1000 mg of nonradiolabeled vatalanib (as four tablets of 335 mg of succinate salt, corresponding to 250 mg free base) until disease progression or unacceptable toxicity.

View larger version (12K): [in this window] [in a new window]   FIG. 1. Chemical structure of 14C-radiolabeled vatalanib. Chemical name: 4-chloro-phenyl)-(4-pyridin-4-yl-methyl phthalazin-1-yl) amine succinate. Molecular formula: C20H15ClN4 + C4H6O4 = C24H21ClN4O4. Molecular weight: 346.82 + 118.09 = 464.91 (referring to the nonradiolabeled compound). Positions of 14C label: uniformly labeled (* = 14C) at the 4-chloro-phenylamine ring moiety.

Safety and Tolerability Assessments. Safety assessments consisted of monitoring and recording all AEs and serious adverse events, the regular monitoring of hematology and blood chemistry, the regular measurement of vital signs, repeat electrocardiographic assessment (if clinically indicated), and the performance of physical examinations. Chest X-ray was repeated as clinically indicated. A physical examination was performed at screening, at baseline, on day 14, and at study completion. Upper gastrointestinal examination by double-contrast barium meal to exclude duodenal pathology was performed at screening and at the beginning of every third cycle (i.e., at days 3, 6, 9, etc., during the post-ADME follow-up period) and at study discontinuation if not performed within 1 month of discontinuation.

Patients who discontinued because of a study-related AE or study-related abnormal laboratory value were followed at least once a week for 4 weeks (and subsequently at 4-week intervals) or until resolution or stabilization of the event. All patients were followed for AEs for 28 days after their last dose of study drug. Tumor assessment was performed only as a part of the safety evaluation to detect progression or improvement of the underlying disease (criteria for discontinuation). The Southwest Oncology Group criteria were used to assess tumor response. Tumor assessments were performed at screening and on day 1 of each cycle, and repeated as clinically indicated.

Radiation Protection. Radiation dosimetry calculations and the study protocol were approved by the Swiss National Radiation Protection Authority. Calculations were performed according to the rules of the International Commission on Radiological Protection. Retrospective examination confirmed that the whole-body dose (effective dose) was clearly below 1 mSv. The written informed consent was obtained from the patients before enrollment. For ethical reasons, it was planned to involve six subjects to minimize human radiation exposure. With this small number of subjects, only a basic descriptive statistical evaluation is sensible.

¹⁴C-Radiolabeled Vatalanib. The drug ¹⁴C-radiolabeled vatalanib was synthesized by the Isotope Laboratory of Novartis Pharma AG (Basel, Switzerland). It was purified by chromatography, blended by recrystallization with nonradiolabeled drug substance using a nonradiolabeled Good Manufacturing Practice batch released for human use, and analyzed for release for human use according to predefined specifications (Pharmaceutical Development, Novartis). The chemical and radiochemical purity was 98% as verified by HPLC before dose administration (Isotope Laboratory, Novartis), and specific radioactivity was 749.5 Bq/mg succinate salt (= 1005 Bq/mg free base; 27.2 nCi/mg).

Nonradiolabeled Vatalanib. Film-coated tablets (335 mg of succinate salt, corresponding to 250 mg free base) were manufactured by Schering AG.

Sample Collection. In total, 17 blood samples were taken by either direct venipuncture or an indwelling cannula inserted in a forearm vein, collected at predose, 0 h, and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 48, 72, 120, 168, 312, and 432 h postdose into heparinized tubes. The blood samples collected at 312 and 432 h postdose (end of days 13 and 18) were for radioactivity counting only, and were used to prove complete elimination of drug-related radioactivity from the systemic circulation. Each patient collected a blank urine sample (in total, approximately 250 ml) the day before dose administration (day 14) and voided his bladder before drug administration (–30 to –1 min). During the whole course of the ADME part of the study, urine was collected in separate portions corresponding to 0- to 6-, 6- to 12-, and 12- to 24-h periods, and then at 24-h sampling periods up to 168 h. Each patient voided his bladder at the end of each urine sampling period. The urine pools were collected in polypropylene containers, which were stored in a refrigerator (4–8°C) during the sampling period. Upon completion of the collection period, the urine was thoroughly mixed, and the total weight of the collected urine sample was measured. One gram of urine was assumed to be 1 ml of urine. Each patient collected one feces sample in the 2 days before dose administration (this sample serves as the blank). Feces samples (including paper) were collected throughout the collection period of 168 h. Each feces sample was collected separately in a polypropylene container. The feces sample containers were prenumbered and used in consecutive order. The exact feces production date and time and the sample weight were recorded on the sample containers and on the pharmacokinetics feces log form. Individual sample containers were stored in a refrigerator (4–8°C) until transferred to the clinical laboratory. In the clinical laboratory, the samples were frozen as soon as possible and stored at < –18°C pending processing. For study patients who vomited within the time period from dose administration to 48 h postdose, the vomitus had to be, if possible, collected in a separate container (spare container; e.g., of the same type as the feces container). The container was labeled with patient number, patient initials, and date and time of vomiting. The collected vomitus from two patients was included in the radioactivity measurements and considered in the determination of the mass balance. The complete vomitus was stored at < –18°C until shipment.

Sample Storage and Shipment Conditions. Immediately after sample collection, blood, plasma, urine, and feces samples were kept refrigerated at 4–8°C. For subsequent analysis, the samples were stored frozen at –18°C until and after laboratory analysis. The stability of vatalanib and its metabolites was ascertained under these conditions. Samples were shipped to other laboratories under dry ice for analysis.

Analysis of Unchanged Vatalanib. Concentrations of vatalanib as free base in plasma were measured by Applied Analytical Industries Inc. (Neu-Ulm, Germany) by a validated HPLC/UV assay as described previously (Morgan et al., 2003). The measurements were performed under Good Laboratory Practice. The lower limit of quantification was 2.5 ng/ml (6.9 nM).

Radioactivity Analysis of Blood, Plasma, Urine, Feces, and Vomitus Samples. Radioactivity in blood, plasma, urine, feces, and vomitus samples was measured by liquid scintillation counting (LSC) with typical counting times of 10 and 30 min. Standard counting procedures and quench correction were performed by an external standard method (Botta et al., 1985). Plasma samples collected between 8 and 168 h were counted for 60 or 240 min. LSC was performed using liquid scintillation counter models Tri-Carb 2500 TR, Tri-Carb 2900 TR, or a super-low level counter model, Tri-Carb 2550 TR/LL (60- and 240-min counting times) (PerkinElmer Life and Analytical Sciences, Boston, MA). Blood and plasma samples (triplicates of approximately 300 µl each, weighed) were counted after solubilization and mixing with liquid scintillation cocktail. Feces and vomitus samples (quadruplicates of approximately 400 mg each, weighed) were combusted in a PerkinElmer A307 oxidizer (PerkinElmer Life and Analytical Sciences), and the ¹⁴CO₂ produced was trapped into the alkaline scintillation cocktail (Carbo-Sorb; Packard); radioactivity was measured by means of LSC. Urine samples (duplicates of 1 ml each) were mixed with liquid scintillation cocktail (Pico Aqua, Packard) and counted by LSC. Background values were measured in predose samples of blood and plasma, urine, vomitus, and feces. Postdose sample counts were corrected for background by subtraction. For each matrix and series of measurements, the limit of quantification (LOQ) for radioactivity was determined according to the definitions and formulae defined below. Calculations were performed using Microsoft Excel (Microsoft, Redmond, WA).

LOQ Calculation for Low Levels of Radioactivity. Radioactivity data near background were evaluated for their statistical quality and to determine the LOQ values for blood, plasma, urine, and feces. The LOQ is defined as the minimal number of sample disintegrations, N_s, where the relative statistical uncertainty, rU_s, is equal to or smaller than 20%.

Definition of variables. N_s is the number of radioactivity disintegrations counted in a sample vial, during sample counting time T corrected for background:

The counting time T for background and sample must be the same.

N₀ is the number of radioactivity disintegrations counted in a background vial, during background counting time T

N_tot is the number of radioactivity disintegrations counted in a sample vial, during sample counting time T. Total count including background:

I₀ is the blank count rate, i.e., number of radioactivity disintegrations per minute counted in blank vial (= background). I_s is the sample count rate, i.e., number of radioactivity disintegrations per minute counted in sample.

If a number of sample replicates, n, is measured, then the uncertainty, rU_s, decreases with increasing n by

U is statistical uncertainty of a sample count, N, due to the stochastic nature of radioactivity (random process):

is statistical error of N counts.

corresponds to the standard deviation of a Gauss or Poisson distribution (fundamental formula). The true value of N is within one sigma (k = 1), with a probability of 68%. Constant k specifies the significance level: k = 2 implies 2, corresponding to a probability of 95.5%.

For low N_s near background:

where 2 · represents a probability of 95.5%. The formula considers the propagation of errors from background and total count to the sample count.

The formula above considers the propagation of errors from background and total count to the sample count, as described in the literature (Plesch, 1962, 1965; Currie, 1968; Tse and Jaffe, 1991; Kessler, 1998).

With standard sample counting times of 10 and 30 min, the LOQs for vatalanib-related radioactivity concentrations were 9 and 7 dpm, corresponding to 1.4 and 2.7 µM in blood and plasma, respectively. For urine and feces samples with counting times of 10 min, the LOQs were 19 and 17 dpm, corresponding to 0.9 and 2 µM, respectively. Late plasma samples were counted for 60 and 240 min, resulting in LOQs of 5.7 and 2.4 dpm, corresponding to 0.9 and 0.4 µM, respectively.

Metabolite Analysis. Metabolite pattern analysis and characterization of metabolites in plasma, urine, and feces were obtained by applying a two-step procedure, as described previously (Boernsen et al., 2000; Gschwind et al., 2005). In the first step, metabolites of vatalanib were separated by reversedphase HPLC with fraction collection into 96-deep well LumaPlates (Packard BioScience B.V., Groningen, the Netherlands). Radioactivity of collected fractions was determined semiquantitatively using off-line detection by TopCount NXT (PerkinElmer Life and Analytical Sciences). In the second step, selected radioactive fractions representing metabolite peaks were analyzed by LC-MS on a LCQ Duo LC-MS system (Thermo Finnigan, San Juan, Puerto Rico) or a Q-TOF Global system (Micromass, Manchester, UK) equipped with an electrospray interface working in positive and negative ion mode to gain structurally informative fragments. Interpretation of spectra and assignments was corroborated by comparison with a set of reference compounds. This two-step separation procedure increased the ion intensity due to the reduced biological impurities, which were reduced by the first separation step, allowed the distinguishing between different metabolite isomers, and enabled the separation of coeluting metabolites.

Sample preparation of plasma, urine, and feces for metabolite investigation. From the plasma of six patients at time points 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 12 h, weighted plasma AUC pools 0.5 to 12 h were prepared (Hop et al., 1998). Before injection onto the radio-HPLC system, plasma and urine samples were processed by using an on-line clean-up and injection system (Prospekt 9200; Varian, Palo Alto, CA). The recovery of radioactivity after sample clean-up was 76% to 81% for plasma and urine. Feces material, obtained as aqueous suspension, was extracted with methanol/water before radio-HPLC. The recovery of radioactivity after extraction and sample reconstitution was 68% to 83%.

HPLC instrumentation for metabolite pattern analysis (radio-HPLC). The HPLC system consisted of an HPLC Xterra MS C18 column of 150 x 3.0 mm and 5-µm particle size (Waters Corp, Milford, MA), protected by a precolumn of 10 x 2.1 mm (5 µm) of the same material; a P580A HPG high-pressure gradient pump (Dionex Corp., Sunnyvale, CA); and an HTS PAL autosampler (CTC Analytics, Swingen, Switzerland). Plasma, urine, or feces extract samples were injected and chromatographed in the above HPLC system. The column temperature was 40°C. Chromatography was monitored by a UV detector at 254 nm (model 430; Kontron, Poway, CA). The chromatographic components were eluted with a gradient of 10 mM aqueous ammonium acetate (mobile phase A) versus acetonitrile (mobile phase B) at a flow rate of 0.5 ml/min. Radioactivity was detected on-line by a Flow Scintillation Analyzer 500 TR radioactivity detector (PerkinElmer Life and Analytical Sciences). Radioactivity data were evaluated by a Chromeleon system (Dionex Corp.). Alternatively, chromatographic fractions were collected by a model FC204 fraction collector (Gilson, Middleton, WI) into a 96-well LumaPlate-96 (Packard BioScience). The fraction volume was 58 µl (7 s per fraction). Vatalanib-related radioactivity was measured by a TopCount NXT 1.05 radioactivity detection system (PerkinElmer Life and Analytical Sciences). Counting times of 10 to 60 min were chosen depending on the amount of radioactivity counts in the collected fractions. Radio-HPLC-peak integration was performed by FLO-ONE evaluation software (PerkinElmer Life and Analytical Sciences). Metabolite and parent drug concentrations obtained from metabolite pattern have to be taken as semiquantitative data.

Structural characterization of metabolites by LC-MS. Metabolite structures were characterized by LC-MS and LC-MSⁿ analysis (Schering AG) and/or comparison through chromatographic retention time and fragment pattern with available reference compounds. LC-MS analyses were performed on an LCQ Duo LC-MSⁿ system (Thermo Finnigan) with an electrospray interface or Q-TOF Global system (Micromass) operating in positive and negative ionization mode.

Evaluation of HPLC data. The amounts of metabolites or parent drug in plasma or excreta were derived from the radiochromatograms (metabolite patterns) by dividing the radioactivity eluting from the HPLC column (equal to radioactivity in original sample minus losses during sample preparation and chromatography) in proportion to the relative peak areas. Parent drug or metabolites were expressed as concentrations (µmol/l) in plasma or as percentage of dose in excreta. These values are to be considered as semiquantitative only, as opposed to those determined by the validated quantitative LC-MS assay (Morgan et al., 2003).

Pharmacokinetic evaluation. Descriptive statistics of pharmacokinetic parameters include mean and standard deviation. Pharmacokinetic parameters were calculated using noncompartmental method(s) and WinNonLin v4.0 software in combination with Microsoft Excel.

A linear least-squares regression method was used to calculate the slope b and intercept a of the regression line: ln(C_t) = b · t + a. A weighting factor of 1 was chosen. The apparent terminal half-life t_1/2z was determined from the slope of the regression line of ln(C_t) versus time t. The AUC_0-tn was calculated up to concentration value C_n at the last time point t_n by the linear-logarithmic trapezoidal method. The AUC was extrapolated to infinite time using the relationship AUC_tn- = C_n · t_1/2z/ln(2). The apparent plasma clearance was calculated by: CL/f = dose/AUC. The apparent volume of distribution during terminal phase was calculated by: V_z/f = (dose · t_1/2z)/(AUC · ln(2)).

The binding of total radioactivity in whole blood to red blood cells was calculated according to the formula below.

F_E (%) is fraction of total radioactivity confined to red blood cells. F_P (%) is fraction of total radioactivity confined to plasma. C_P is concentration of radioactivity in plasma, measured on day 15 (day 1 of ADME phase) between 1 and 12 h. C_EP is concentration of radioactivity in whole blood, measured on day 15 (day 1 of ADME phase) between 1 and 12 h. H is hematocrit (a value of 0.45 was assumed).

	Results

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Patient Selection Eight advanced cancer patients were enrolled into the study and started the nonradiolabeled vatalanib medication for 14 days. One patient discontinued after the 14-day loading phase because of disease progression. Seven patients received the dose of ¹⁴C-radiolabeled study medication. One patient discontinued during the ADME period of the study because of tumor progression. Finally, six patients completed the 33-day ADME period according to the study plan. Five patients continued in the post-ADME period and received the study medication until disease progression. One patient did not enter into the post-ADME period, but discontinued the study because of elevated liver enzymes suspected to be study drug-related.

Adverse Events All patients had at least one gastrointestinal AE (Table 2). The most common AEs were nausea and vomiting, each occurring in 50% of the patients. The majority of the observed AEs were mild to moderate. No patient was discontinued from the study because of an AE. Serious adverse events were reported in four patients, none of which were suspected of being drug-related. No patient deaths occurred during the study. Two patients died because of disease progression within 28 days of the last study dose.

View larger version (18K): [in this window] [in a new window]   FIG. 2. Pharmacokinetics of vatalanib and 14C radioactivity in plasma after a single dose of 1000 mg of 14C-radiolabeled vatalanib administered at steady state, obtained after 14 consecutive daily oral doses of 1000 mg of nonradiolabeled vatalanib. Plasma concentrations of total 14C radioactivity and vatalanib are as indicated: A, linear scale; B, semilogarithmic scale. Error bars represent ±S.D., n = 7.

Pharmacokinetics of Vatalanib and Total Radioactivity In most patients, C_max of the vatalanib and ¹⁴C radioactivity concentrations were quickly reached after 1.5 and 2 h, respectively, indicating rapid onset of oral absorption (Fig. 2A). Thereafter, parent drug and ¹⁴C radioactivity were eliminated with mean apparent halflives of 4.6 and 23.4 h, respectively. The ¹⁴C radioactivity was eliminated virtually monoexponentially (Fig. 2B). Average pharmacokinetic parameters of vatalanib at steady state after a daily dose of 1000 mg are displayed in Table 3. The blood-to-plasma distribution ratio was approximately 0.7, corresponding to approximately 80% of the ¹⁴C radioactivity confined to the plasma compartment. This indicated no special affinity of vatalanib and/or its metabolites to blood cells.

Excretion and Mass Balance in Urine and Feces The mass balance of ¹⁴C radioactivity in urine and feces is given in Table 4. The ¹⁴C radioactivity was excreted mainly with feces (range, 42–74% of dose). A minor proportion was excreted renally (range, 13–29% of dose). In three patients the bulk of the dose was recovered within 2 days. By 7 days, the excretion of the radioactivity was close to complete in four of six patients (range, 83–96%). Two patients showed a balance of excretion of 67% and 76% of dose, respectively, which was probably due to incomplete sample collection, probably caused by the underlying disease.

Metabolism Metabolite Characterization by LC-MS. Metabolite structures were by LC-MS and LC-MSⁿ and, where possible, confirmed by comparison of fragment spectra and retention times with authentic reference compounds [NVP-AAW378/ZK-261557 (M14), CGP-84368/ZK-260120 (M17), ZK-276224 (M19), CGP-85587/ZK-228353 (M24), ZK-264851 (M38), CGP-85903/ZK-228354 (M25), ZK-260116 (M26), and CGP-79469/ZK-260013 (M27)]. The proposed partial or complete structures are given in Table 5.

From a number of minor metabolites detected in the radiochromatograms of plasma (M10, M15), urine (M1, M2, M3, M4, M5, M6, M7, M8, M9, M10, M11, M12, M13, M15, M16), or feces (M9, M28, M29, M22, M23, M38), only partial structures could be determined. Most of them were oxidation products, some N- or O-glucuronidated or O-conjugated with sulfuric acid. M16 appears to contain a degraded glutathione residue.

Metabolites in Plasma. Essentially five metabolite peaks were observed in the radiochromatograms in Fig. 3, A and B. CGP-84368/ZK-260120 (pyridine-N-oxide metabolite) was the most prominent metabolite plasma (26% of ¹⁴C AUC_0.5–12h; Table 5), followed by the 4-yl-methyl-carbinol-pyridine-N-oxide metabolite NVP-AAW378/ZK-261557 (21%), comigrating with traces of the N-glucuronide of vatalanib (M15). Their chemical structures are given in Table 5.

View larger version (24K): [in this window] [in a new window]   FIG. 3. Metabolite patterns in plasma at 1.5 h (A) and 6 h (B) after dosing of the 14C-radiolabeled vatalanib. The plasma samples from one patient were extracted before HPLC analysis. Chemical structures of metabolites are listed in Table 5. a, peak HP14 was only observed in one patient. It mainly contains vatalanib with an additional trace component of molecular mass 363 Da (vatalanib + oxygen). NVP-AAW378, NVP-AAW378/ZK-261557; CGP-84368, CGP-84368/ZK-260120.

Unchanged Vatalanib and Metabolites in Excreta. Urine. Between 13.1 and 29.2% of the radioactive dose was excreted in urine (Table 4). The metabolite pattern in 0- to 48-h urine essentially consisted of approximately 15 peaks (Fig. 4). Unchanged vatalanib was present in urine at very low amounts (0.4% of dose; Table 5), indicating extensive metabolism of the systemically available vatalanib and suggesting that renal clearance of the drug is insignificant. NVP-AAW378/ZK-261557 (M14) and CGP-84368/ZK-260120 (M17) were the main metabolites in urine and were identified as the methyl-carbinol-pyridine-N-oxide (4.8% of dose) and the pyridine-N- oxide metabolite (2.5% of dose), respectively. Most of the metabolites consisted of O-glucuronic or O-sulfuric acid conjugates of oxidized/hydroxylated metabolites or, to a minor extent, of C- or N-oxidized metabolites, or of vatalanib [M1, M2, M3, M4, M5, M6, M7, M8, M9, M10, M11, M12, M13, M15, CGP-85587/ZK-228353 (M24), CGP-85903/ZK-228354 (M25)]. Some of the metabolites coeluted or were present as trace components, detected by LC-MS in native urine of one patient [M5, M6; or M8, M9; or M11, M12; or CGP-85587/ZK-228353 (M24), CGP-85903/ZK-228354 (M25)]. M16 was tentatively characterized to consist of a mercapturic acid pathway-degraded glutathione derivative of a pyridine-N-oxide metabolite of vatalanib (0.6% of dose). Overall, 21.5% of the dose (=90% of total ¹⁴C in 0- to 168-h urine) could be recovered mostly as metabolites and was structurally characterized.

View larger version (23K): [in this window] [in a new window]   FIG. 4. Metabolite pattern in urine collected between 0 and 48 h after dosing of the 14C-radiolabeled vatalanib. Urine from one patient was pooled and centrifuged before HPLC analysis. Chemical structures of metabolites are listed in Table 5. t, present as a trace component detected by LC-MS, mainly in native sample of one patient. NVP-AAW378, NVP-AAW378/ZK-261557; CGP-84368, CGP-84368/ZK 260120; CGP-85587, CGP-85587/ZK-228353.

View larger version (21K): [in this window] [in a new window]   FIG. 5. Metabolite pattern in feces extract collected between 0 and 48 h after dosing of the 14C-radiolabeled vatalanib. Feces from one patient was pooled and centrifuged before HPLC analysis. Chemical structures of metabolites are listed in Table 5. t, present as a trace component detected by LC-MS, mainly in native sample of one patient. CGP-85587, CGP-85587/ZK-228353; CGP-85903, CGP-85903/ZK-228354.

CGP-79469/ZK-260013 (M27) has lost the chlorine atom at the 4'-chloro-phenylamine moiety, most likely through oxidative dechlorination (0.9% of dose). CGP-85903/ZK-228354 (M25) represented the ketopynalin metabolite of vatalanib and was detected at trace amounts. Overall, more than 56% of dose (93% of total ¹⁴C in 0- to 168-h feces) could be covered by vatalanib and structurally characterized metabolites.

	Discussion

Top Abstract Materials and Methods Results Discussion Conclusions References

 
Tolerability. This study demonstrates that vatalanib was well tolerated in patients with advanced cancers without any unexpected findings. Most patients had at least one AE, with the most common being nausea and vomiting. The majority of AEs were Common Toxicity Criteria grade 1 or 2 in severity.

Absorption. Vatalanib administered orally (1000 mg) was rapidly absorbed (Mross et al., 2005; Thomas et al., 2005). On average, approximately 46% of the dose in the feces pool consisted of unchanged vatalanib (range, 26–69%; Table 5), representing mainly nonabsorbed drug, or unchanged drug eliminated by biliary elimination or by direct intestinal excretion after absorption. Trace amounts of unchanged vatalanib were detected in urine. Assuming that the drug is stable against intestinal bacterial enzymes, the mean oral absorption of vatalanib can be estimated to be approximately 35% of dose (approximately 23% renally excreted radioactivity, plus approximately 12% fecally as metabolites; absorption range, 18–57%). However, the extent of oral absorption may be underestimated, since the recovery of the ¹⁴C radioactivity from feces extraction was incomplete (range, 68–83%). N-Oxide metabolites of vatalanib may be reduced to parent drug by intestinal bacterial enzymes (el-Mekkawy et al., 1993) after biliary transport into the intestine, and N-glucuronic acid conjugates of vatalanib may also be reconverted by intestinal glucuronidases (Chourasia and Jain, 2003) to parent drug, resulting in its potential reabsorption.

View larger version (15K): [in this window] [in a new window]   FIG. 6. Chemical structure of vatalanib and identified metabolic processes in cancer patients. The main metabolic pathways were oxidation at the 4-yl-methyl carbon and/or N-oxidation at the pyridine nitrogen to NVP-AAW378/ZK-261557 and CGP-84368/ZK-260120, respectively. For details of chemical structures, see Table 5.

Pharmacokinetics. The pharmacokinetics of unchanged vatalanib at steady state were comparable to data previously reported in cancer patients given the same dose of vatalanib at steady state (Morgan et al., 2003; Mross et al., 2005; Thomas et al., 2005).

Metabolism. The absorbed part of the oral dose was cleared mainly by biotransformation. Phase 1 biotransformation of ¹⁴C-radiolabeled vatalanib occurred mainly through oxidation, yielding N-oxides and/or oxidation of the 4-pyridine-4-yl-methyl group to a secondary alcohol (methyl-carbinol) and its corresponding ketone and/or oxidations/hydroxylations in the aromatic part of the molecule. The main biotransformation pathways of vatalanib are depicted in Fig. 6. In vitro experiments with human liver microsomes and hepatocytes indicated that vatalanib is a substrate of human CYP3A4 and CYP2D6, and partly of CYP1A2 (K. Denner, Apr. 6, 2004, unpublished data, Schering AG). Vatalanib is predominantly metabolized by CYP3A4, accounting for approximately 95% of cytochrome P450-dependent metabolism. Vatalanib itself and several of its oxidized/hydroxylated metabolites were subsequently conjugated with glucuronic acid or sulfuric acid. In addition, two minor pathways were observed: 1) vatalanib and its metabolites underwent oxidative dechlorination at the 4-chlorophenylamine moiety (M27 in Table 5), and 2) oxidized metabolites of vatalanib underwent conjugation with glutathione (M16). The pyridine N-oxide metabolite (CGP-84368/ZK-260120) displayed a comparable systemic exposure in plasma as parent drug (Table 5), but is considered to contribute very little (32-fold less active than vatalanib in the VEGF-R2 kinase domain receptor in vitro assay) to the overall pharmacological activity in patients (T. Meyer, S. Barbet, M. Centeleghe, J. Koeppler, J. Liebetanz, D. Manfrina, M. Mele, L. Muller, D. Fabbro, and L. Carre, Oct. 20, 2004, unpublished data, Novartis). The 4-yl-methyl-carbinol metabolite (CGP-85587/ZK-228353), which also showed substantial systemic exposure in plasma, does not contribute to the overall pharmacological activity, since it is 25-fold less active than vatalanib in the same assay above.

Elimination. Vatalanib was rapidly eliminated from the systemic circulation because of extensive metabolism. The predominant elimination route of vatalanib and its metabolites was biliary/fecal excretion. Accumulation of vatalanib and/or its metabolites is not anticipated after chronic oral daily administration.

Excretion and Mass Balance. Radiolabeled material was excreted largely through the feces in the range of 42 to 66% of dose (mean, 60.5%) and through urine at 13 to 29% of dose (mean, 23.4%). The recovered radioactivity in the excreta up to 7 days after dosing was between 67 and 96% of dose (mean, 84%). In four patients excretion was still ongoing at a low daily rate on days 6 and 7. A mean terminal excretion half-life of roughly 16 h could be estimated. In three patients, the total recovery, more than 90% of dose, was almost complete, which represented a reasonably complete excretion. In contrast, the recovery in two patients was low, at 67% and 76% of dose; however, complete collection of excreta in very ill patients over a substantial period of time is highly demanding, and complete collection and excretion are hard to achieve.

	Conclusions

Top Abstract Materials and Methods Results Discussion Conclusions References

 
The pharmacokinetics of the parent drug at steady state were comparable to data reported in other trials with vatalanib given at the same dose. Peak concentrations of radioactivity and unchanged vatalanib after oral dosing showed rapid onset of absorption and significant systemic availability of the parent drug.

The systemic exposure to metabolites was high. The two main plasma metabolites, the pyridine-N-oxide (CGP-84368/ZK-260120) and 4-yl-methyl-carbinol-pyridine-N-oxide (NVP-AAW378/ZK-261557), displayed systemic exposure comparable to that of the parent drug. Among the prominent systemically available drug-related components, parent drug is considered to contribute the majority to the overall pharmacological activity of vatalanib in humans.

The extent of oral absorption was estimated to be, on average, 35% of dose. The parent drug was rapidly eliminated from the systemic circulation mainly due to metabolism. The predominant route of elimination occurred via feces (on average, 61% of dose). Renal elimination was minor (on average, 23% of dose). Excretion was close to complete in four of six patients after 7 days. Accumulation of vatalanib and/or its metabolites in the body after chronic oral daily administration is not anticipated from these data.

	Acknowledgments

 
We thank Päivi Paldánius (M. Med. Sci.) from Schering AG (former Clinical Trial Leader), Dr. med. Michael Seiberling and Dr. med. Rolf Pokorny from Swiss Pharma Contract, Allschwil, Switzerland (Co-Investigators), Uwe Knirk (Medical Development Management & Operation) from Schering AG, Bernard Wirz (Graduate Chemist) from Novartis (Super Low Level Counting), Dr. Thomas Schmitt from Novartis (Radioisotope Laboratory), and Dr. Bertrand Sutter from Novartis (Pharmaceutical Development).

	Footnotes

 
This work was supported by Schering AG, Germany and Novartis Pharma AG, Switzerland.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.009944.

ABBREVIATIONS: VEGF, vascular endothelial growth factor; ADME, absorption, distribution, metabolism, and excretion; AE, adverse event; AUC, area under the concentration-time curve; HPLC, high-performance liquid chromatography; LC-MS, liquid chromatography coupled to mass spectrometry; LOQ, limit of quantification; LSC, liquid scintillation counting; PTK787/ZK-222584, vatalanib; radio-HPLC, HPLC coupled to radioactivity detection; ULN, upper limit of normal; VEGFR, vascular endothelial growth factor receptor; NVP-AAW378/ZK-261557, rac-4-[(4-chloro-phenyl)amino]--(1-oxido-4-pyridyl)phthalazine-1-methanol; CGP-84368/ZK-260120, (4-chloro-phenyl)-[4-(1-oxy-pyridin-4-yl-methyl)-phthalazin-1-yl]-amine; PTK787/ZK-226343, vatalanib (base), (4-chloro-phenyl)-(4-pyridin-4-yl-methyl-phthalazin-1-yl)-amine; CGP-85587/ZK-228353, [4-([1E,3E]-4-chloro-1-vinyl-penta-1,3-dienylamino)-phthalazin-1-yl]-pyridin-4-yl-methanol; CGP-85903/ZK-228354, ketopynalin, [4-(4-chloro-phenylamino)phthalazin-1-yl] (4-pyridyl) ketone; CGP-79469/ZK-260013, 4-(4-pyridin-4-yl-methyl-phthalazin-1-yl-amino)-phenol; ZK-260116, 2-chloro-5-(4-pyridin-4-ylmethyl-phthalazin-1-ylamino)-phenol.

Address correspondence to: Dr. Hans-Peter Gschwind, Exploratory Development/Drug Metabolism & Pharmacokinetics, Novartis Pharma AG, Building WKL-135.2.25, CH-4002 Basel, Switzerland. E-mail: hans-peter.gschwind{at}novartis.com

	References

Top Abstract Materials and Methods Results Discussion Conclusions References

 


Bold G, Altmann KH, Frei J, Lang M, Manley PW, Traxler P, Wietfeld B, Bruggen J, Buchdunger E, Cozens R, et al. (2000) New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinase useful as antagonists of tumor driven angiogenesis. J Med Chem 43: 2310–2323.[CrossRef][Medline]

Boernsen KO, Floeckher JM, and Bruin GJ (2000) Use of a microplate scintillation counter as a radioactivity detector for miniaturized separation techniques in drug metabolism. Anal Chem 72: 3956–3959.[Medline]

Botta L, Gerber HU, and Schmid K (1985) Measurement of radioactivity in biological experiments, in Drug Fate and Metabolism, Methods and Techniques, vol 5 (Garrett ER and Hirtz JL eds) pp 99–134, Dekker, New York.

Chourasia MK and Jain SK (2003) Pharmaceutical approaches to colon targeted drug delivery systems. J Pharm Sci 6: 33–66.

Crew JP, O'Brien T, Bradburn M, Fuggle S, Bicknell R, Cranston D, and Harris AL (1997) Vascular endothelial growth factor is a predictor of relapse and stage progression in superficial bladder cancer. Cancer Res 57: 5281–5285.[Abstract/Free Full Text]

Currie LA (1968) Limits for qualitative detection and quantitative determination. Application to radiochemistry. Anal Chem 40: 586–593.

el-Mekkawy S, Meselhy MR, Kawata Y, Kadota S, Hattori M, and Namba T (1993) Metabolism of strychnine N-oxide and brucine N-oxide by human intestinal bacteria. Planta Med 59: 347–350.[Medline]

Fan TP, Jaggar R, and Bicknell R (1995) Controlling the vasculature: angiogenesis, antiangiogenesis and vascular targeting of gene therapy. Trends Pharmacol Sci 16: 57–66.[CrossRef][Medline]

Ferrer FA, Miller LJ, Andrawis RI, Kurtzman SH, Albertsen PC, Laudone VP, and Kreutzer DL (1998) Angiogenesis and prostate cancer: in vivo and in vitro expression of angiogenesis factors by prostate cancer cells. Urology 51: 161–167.[Medline]

Gschwind H-P, Pfaar U, Waldmeier F, Zollinger F, Sayer C, Zbinden P, Hayes M, Pokorny R, Seiberling M, Ben-Am M, et al. (2005) Metabolism and disposition of imatinib mesylate in healthy volunteers. Drug Metab Dispos 33: 1503–1512.[Abstract/Free Full Text]

Hop CE, Wang Z, Chen Q, and Kwei G (1998) Plasma-pooling methods to increase throughput for in vivo pharmacokinetic screening. J Pharm Sci 87: 901–903.[CrossRef][Medline]

Kessler M (1998) Statistical computations in counting, in Handbook of Radioactivity Analysis (L'Annunziata MF ed), p 394, Academic Press, San Diego.

Morgan B, Thomas AL, Drevs J, Hennig J, Buchert M, Jivan A, Horsfield MA, Mross K, Ball HA, Lee L, et al. (2003) Dynamic contrast-enhanced magnetic resonance imaging as a biomarker for the pharmacological response of PTK787/ZK 222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases, in patients with advanced colorectal cancer and liver metastases: results from two Phase I studies. J Clin Oncol 21: 1–10.[Free Full Text]

Mross K, Drevs J, Muller M, Medinger M, Marme D, Hennig J, Morgan B, Lebwohl D, Masson E, Ho YY, et al. (2005) Phase I clinical and pharmacokinetic study of PTK/ZK, a multiple VEGF receptor inhibitor, in patients with liver metastases from solid tumours. Eur J Cancer 41: 1291–1299.[CrossRef][Medline]

Nakagawa M, Emoto A, Hanada T, Nasu N, and Nomura Y (1997) Tubulogenesis by microvascular endothelial cells is mediated by vascular endothelial growth factor (VEGF) in renal cell carcinoma. Br J Urol 79: 681–687.[Medline]

Nakanishi Y, Kodama J, Yoshinouchi M, Tokumo K, Kamimura S, Okuda H, and Kudo T (1997) The expression of vascular endothelial growth factor and transforming growth factor-beta associates with angiogenesis in epithelial ovarian cancer. Int J Gynecol Pathol 16: 256–262.[Medline]

Plate KH, Breier G, and Risau W (1994) Molecular mechanisms of development and tumor angiogenesis. Brain Pathol 4: 207–218.[Medline]

Plesch R (1962) Unterscheidung und Nachweis von Aktivitäten. Atomwirtschaft 7: 602–605.

Plesch R (1965) Statistik und Wahrscheinlichkeit in der Strahlungsmesstechnik. G-I-T Fachzeitschrift Labor 9: 430–437.

Scott PA, Smith K, Poulsom R, De Benedetti A, Bicknell R, and Harris AL (1998) Differential expression of vascular endothelial growth factor mRNA protein isoform expression in human breast cancer and relationship to eIF-4E. Br J Cancer 77: 2120–2128.[Medline]

Sowter HM, Corps AN, Evans AL, Clark DE, Charnock-Jones DS, and Smith SK (1997) Expression and localization of the vascular endothelial growth factor family in ovarian epithelial tumors. Lab Investig 77: 607–614.[Medline]

Takahama M, Tsutsumi M, Tsujiuchi T, Kido A, Okajima E, Nezu K, Tojo T, Kushibe K, Kitamura S, and Konishi Y (1998) Frequent expression of the vascular endothelial growth factor in human non-small-cell lung cancers. Jpn J Clin Oncol 28: 176–181.[Abstract/Free Full Text]

Takahashi Y, Kitadai Y, Bucana CD, Cleary KR, and Ellis LM (1995) Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res 55: 3964–3968.[Abstract/Free Full Text]

Thomas AL, Morgan B, Horsfield MA, Higginson A, Kay A, Lee L, Masson E, Puccio-Pick M, Laurent D, and Steward WP (2005) Phase I study of the safety, tolerability, pharmacokinetics, and pharmacodynamics of PTK787/ZK 222584 administered twice daily in patients with advanced cancer. J Clin Oncol 23: 4162–4171.[Abstract/Free Full Text]

Tse FLS and Jaffe JM (1991) Use of radioactivity in drug disposition studies, in Preclinical Drug Disposition, A Laboratory Handbook, pp 23–26, Dekker, New York.

Wood JM, Bold G, Buchdunger E, Cozens R, Ferrari S, Frei J, Hofmann F, Mestan J, Mett H, O'Reilly T, et al. (2000) PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res 60: 2178–2189.[Abstract/Free Full Text]

Yamamoto S, Konishi I, Mandai M, Kuroda H, Komatsu T, Nanbu K, Sakahara H, and Mori T (1997) Expression of vascular endothelial growth factor (VEGF) in epithelial ovarian neoplasms: correlation with clinicopathology and patient survival, and analysis of serum VEGF levels. Br J Cancer 76: 1221–1227.[Medline]

This article has been cited by other articles:

				F. Waldmeier, U. Glaenzel, B. Wirz, L. Oberer, D. Schmid, M. Seiberling, J. Valencia, G.-J. Riviere, P. End, and S. Vaidyanathan Absorption, Distribution, Metabolism, and Elimination of the Direct Renin Inhibitor Aliskiren in Healthy Volunteers Drug Metab. Dispos., August 1, 2007; 35(8): 1418 - 1428. [Abstract] [Full Text] [PDF]

				M. Los, J. M. L. Roodhart, and E. E. Voest Target Practice: Lessons from Phase III Trials with Bevacizumab and Vatalanib in the Treatment of Advanced Colorectal Cancer Oncologist, April 1, 2007; 12(4): 443 - 450. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.106.009944v1 34/11/1817    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jost, L. M. Articles by Laurent, D. Search for Related Content PubMed PubMed Citation Articles by Jost, L. M. Articles by Laurent, D.