Predicting Pharmacokinetics and Drug Interactions in Patients from in Vitro and in Vivo Models: the Experience with 5,6-Dimethylxanthenone-4-Acetic Acid (DMXAA), an Anti-Cancer Drug Eliminated Mainly by Conjugation

The novel anti-tumor agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) was developed in the Auckland Cancer Society Research Center. Its pharmacokinetic properties have been investigated using both in vitro and in vivo models, and the resulting data extrapolated to patients. The metabolism of DMXAA has been extensively studied mainly using hepatic microsomes, which indicated that UGT1A9 and UGT2B7-catalyzed glucuronidation on its acetic acid side chain and to a lesser extent CYP1A2-catalyzed hydroxylation of the 6-methyl group are the major metabolic pathways, resulting in DMXAA acyl glucuronide (DMXAA-G) and 6-hydroxymethyl-5-methylxanthenone-4-acetic acid. The predominant metabolite in human urine (up to 60% of total dose) was identified as DMXAA-G, which was chemically reactive, undergoing hydrolysis, intramolecular rearrangement, and covalent binding to plasma proteins. In vivo formation of DMXAA–protein adducts were also observed in cancer patients receiving DMXAA treatment. The comparison of the in vitro human hepatic microsomal metabolism and inhibition of DMXAA by UGT and/or CYP substrates with animal species indicated species differences. Renal microsomes from all animal species examined had glucuronidation activity for DMXAA, but lower than the liver. In vitro–in vivo extrapolations based on human microsomal data indicated a 7-fold underestimation of plasma clearance in patients. In contrast, allometric scaling using in vivo data from the mouse, rat, and rabbit predicted a plasma clearance of 3.5 mL/min/kg, similar to that observed in patients (3.7 mL/min/kg). Based on in vitro metabolic inhibition studies, it appears possible to predict the effects on the plasma kinetic profile of DMXAA of drugs such as diclofenac, which are mainly metabolized by UGT2B7. However, it did not appear possible to predict the effect of thalidomide on the pharmacokinetics of DMXAA in patients based on in vitro inhibition and animal studies. These data indicate that preclincial pharmacokinetic studies using both in vitro and in vivo models play an important but different role in predicting pharmacokinetics and drug interactions in patients

Preclinical Factors Influencing the Relative Contributions of Phase I and II Enzymes to the Metabolism of the Experimental Anti-Cancer Drug 5,6-Dimethylxanthenone-4-Acetic Acid

It is important to determine the relative contribution of each metabolic pathway (fp) and of enzymes to the net metabolism of a drug. The aim of this study was to investigate, using a human liver bank, the fp of the anti-cancer drug 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and the effects of various inhibitors and inducers on fp. The mean apparent Km and Vmax values (N=14) were 21±5 μM and 0.04±0.02 nmol/min/mg, respectively, for 6-methylhydroxylation, and 143±79 μM and 0.71±0.52 nmol/min/mg, respectively, for acyl glucuronidation in human liver microsomes. 6-Methylhydroxylation and acyl glucuronidation contributed 26 and 74%, respectively, to DMXAA metabolism at 5 μM; values were 7 and 93% at 350 μM DMXAA. There was a significant relationship between the ratio of metabolic activity by Phase II and I reactions (RII/I) and uridine diphosphate glucuronosyltransferase (UGT2B7) protein level (r=0.605, P=0.022), whereas a reverse correlation between RII/I and cytochrome P450 (CYP1A) protein level was observed (r=−0.540, P=0.046). Various compounds inhibited either DMXAA glucuronidation or 6-methylhydroxylation, or both pathways. Pretreatment of rats with β-naphthoflavone, but not phenobarbitone and cimetidine, increased the percentage of the contribution by 6-methylhydroxylation to 17% from 4% of control at 5 μM DMXAA. Our results indicate that the fp of DMXAA is subject to substrate concentration, inhibition, induction, and the protein levels of enzymes that biotransform DMXAA. However, clinical studies are important to verify the conclusions drawn from in vitro data

Determination of Thalidomide in Transport Buffer for Caco-2 Cell Monolayers by High-Performance Liquid Chromatography with Ultraviolet Detectionr Caco-2 Cell Monolayers by High-Performance Liquid Chromatography with Ultraviolet Detection

We report simple validated HPLC methods for the determination of thalidomide in the transport buffer for the human colonic cell line (Caco-2) cell monolayers. An aliquot of 50 μl of the mixture was injected onto a Spherex C18 column (150×4.6 mm; 5 μm) at a flow-rate of 0.5 ml/min of mobile phase consisting of acetonitrile–10 mM ammonium acetate buffer (24:76, v/v, pH 5.5), and thalidomide was detected by ultraviolet detector at a wavelength of 220 nm. Calibration curves for thalidomide were constructed at the concentration range of 0.025–1.0 and 1.0–50 μM in transport buffer. The validated methods were used to determine the transport of thalidomide by Caco-2 monolayers. The transport across the monolayers from the apical (A) to basolateral (B) side was similar to that from B to A side. The apparent permeability coefficient (Papp) values of thalidomide at 10–300 μM from the A to B and from B to A side was 2–6×10−5 cm/s, with a marked decrease in Papp values from A to B side at increased thalidomide concentration. The A to B transport appears to be dependent on temperature and sodium ion. Sodium azide, 2,4-dinitrophenol (both ATP inhibitors), 5-fluorouracil, cytidine and glutamic acid significantly inhibited the transport of thalidomide. These results indicate that the transport of thalidomide by Caco-2 monolayers was rapid, which might involve an energy-dependent mechanism

Identification of the Human Liver Cytochrome P450 Isoenzyme Responsible for the 6-Methylhydroxylation of the Novel Anticancer Drug 5,6-Dimethylxanthenone-4-Acetic Acid

In vitro studies were conducted to identify the hepatic cytochrome P450 (CYP) isoenzyme involved in the 6-methylhydroxylation of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) by using a human liver library (n = 14). The metabolite 6-hydroxymethyl-5-methylxanthenone-4-acetic acid (6-OH-MXAA) was determined by HPLC with fluorescence detection. The metabolite formed in human liver microsomes and by cDNA-expressed CYP isoform was identified by liquid chromatography mass spectrometry as 6-OH-MXAA. In human liver microsomes (n = 14), 6-methylhydroxylation of DMXAA followed monophasic Michaelis-Menten kinetics, with a mean apparent Km of 21 ± 5 μM and Vmax of 0.043 ± 0.019 nmol/min/mg. An approximate 10-fold interindividual variation in the intrinsic clearance (Vmax/Km) of DMXAA 6-methylhydroxylation in human liver microsomes was observed. The involvement of CYP1A2 in DMXAA metabolism by human livers was demonstrated by the following: 1) the potent inhibition of DMXAA metabolism by furafylline (kinact = 0.23 ± 0.04 min−1, K′app= 15.6 ± 6.7 μM) and α-naphthoflavone (Ki = 0.036 μM), but not by cimetidine, ketoconazole, tolbutamide, quinidine, chlorzoxazone, diethyldithiocarbamate, troleandomycin, and sulfaphenazole; 2) when incubated with human lymphoblastoid cell microsomes containing cDNA-expressed CYP isoenzymes, DMXAA was metabolized only by CYP1A2, with an apparent Km of 6.2 ± 1.5 μM and Vmax of 0.014 ± 0.001 nmol/min/mg, but not by CYP2A6, CYP2B6, CYP2C9 (Arg144), CYP2C19, CYP2D6 (Val374), CYP2E1, and CYP3A4; 3) a significant correlation (r = 0.90; P \u3c .001) between 6-methylhydroxylation of DMXAA and 7-ethoxyresorufinO-deethylation; and 4) a significant correlation (r = 0.75; P \u3c .01) between the CYP1A protein level determined by Western blots and DMXAA 6-methylhydroxylation

Thalidomide in Cancer Treatment

There is increased interest in the treatment of cancer with thalidomide because of its antiangiogenic, immunomodulating and sedative effects. In animal models, the antitumour activity of thalidomide is dependent on the species, route of administration and coadministration of other drugs. For example, thalidomide has shown antitumour effects as a single agent in rabbits, but not in mice. In addition, the antitumour effects of the conventional cytotoxic drug cyclophosphamide and the tumour necrosis factor inducer 5,6-dimethylxanthenone-4-acetic acid (DMXAA) were found to be potentiated by thalidomide in mice bearing colon 38 adenocarcinoma tumours. Further studies have revealed that thalidomide upregulates intratumoral production of tumour necrosis factor-α 10-fold over that induced by DMXAA alone. Coadministration of thalidomide also significantly reduced the plasma clearance of DMXAA and cyclophosphamide. All these effects of thalidomide may contribute to the enhanced antitumour activity. Recent clinical trials of thalidomide have indicated that it has minimal anticancer activity for most patients with solid tumours when used as a single agent, although it was well tolerated. However, improved responses have been reported in patients with multiple myeloma. Palliative effects of thalidomide on cancer-related symptoms have also been observed, especially for geriatric patients with prostate cancer. Thalidomide also eliminates the dose-limiting gastrointestinal toxic effects of irinotecan. There is preliminary evidence indicating that the clearance of thalidomide may be reduced in the elderly. The exact role of thalidomide in the treatment of cancer and cancer cachexia in the elderly remains to be elucidated. However, it may have some value as part of a multimodality anticancer therapy, rather than as a single agent. Recent clinical trials of thalidomide have indicated that it has minimal anticancer activity for most patients with solid tumours when used as a single agent, although it was well tolerated. However, improved responses have been reported in patients with multiple myeloma. Palliative effects of thalidomide on cancer-related symptoms have also been observed, especially for geriatric patients with prostate cancer. Thalidomide also eliminates the dose-limiting gastrointestinal toxic effects of irinotecan. There is preliminary evidence indicating that the clearance of thalidomide may be reduced in the elderly. The exact role of thalidomide in the treatment of cancer and cancer cachexia in the elderly remains to be elucidated. However, it may have some value as part of a multimodality anticancer therapy, rather than as a single agent

A Difference between the Rat and Mouse in the Pharmacokinetic Interaction of 5,6-Dimethylxanthenone-4-Acetic Acid with Thalidomide

PURPOSE: Coadministration of thalidomide, cyproheptadine or diclofenac has been shown to increase the area under the plasma concentration-time curve (AUC) of the novel antitumour agent 5,6-dimethylxanthenone-4-acetic acid (DMXAA) in mice. The aim of this study was to further investigate these pharmacokinetic DMXAA-drug interactions in the rat model. Methods: The effects of coadministration of L-thalidomide, cyproheptadine or diclofenac on the pharmacokinetics of DMXAA were investigated in male Wistar Kyoto rats. The effects of L-thalidomide, cyproheptadine and diclofenac on microsomal metabolism and plasma protein binding of DMXAA were also investigated. Results: No significant alteration in the plasma concentration profile for DMXAA was observed following L-thalidomide pretreatment in rats. In contrast, when combined with diclofenac or cyproheptadine, the plasma AUC of DMXAA was significantly (P \u3c 0.05) increased by 48% and 88% and the T1/2 by 36% and 107%, respectively, compared to controls. Both diclofenac and cyproheptadine at 500 µM caused a significant inhibition of DMXAA metabolism in rat liver microsomes. In contrast, L-thalidomide had no or little inhibitory effect on DMXAA metabolism in rat liver microsomes except for causing a 32% decrease in 6-methylhydroxylation at 500 µM. None of the drugs had a significant effect on the plasma protein binding of DMXAA in the rat. Conclusion: These studies showed that coadministration of L-thalidomide did not alter the plasma DMXAA AUC in rats, in contrast to previous studies in mice, whereas diclofenac and cyproheptadine significantly reduced the plasma clearance of DMXAA in rats in a similar manner to their effect in mice. The cause of the species difference in the pharmacokinetic response to thalidomide by DMXAA is unknown, and indicates difficulties in predicting the outcome of such a combination in patients

Rat Tumor Response to the Vascular-Disrupting Agent 5,6-Dimethylxanthenone-4-Acetic Acid as Measured by Dynamic Contrast-Enhanced Magnetic Resonance Imaging, Plasma 5-Hydroxyindoleacetic Acid Levels, and Tumor Necrosis

The dose-dependent effects of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on rat GH3 prolactinomas were investigated in vivo. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to assess tumor blood flow/permeability pretreatment and 24 hours posttreatment with 0, 100, 200, or 350 mg/kg DMXAA. DCE-MRI data were analyzed using K(trans) and the integrated area under the gadolinium time curve (IAUGC) as response biomarkers. Highperformance liquid chromatography (HPLC) was used to determine the plasma concentration of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) following treatment to provide an index of increased vessel permeability and vascular damage. Finally, tumor necrosis was assessed by grading hematoxylin and eosin-stained sections cut from the same tumors investigated by MRI. Both tumor K(trans) and IAUGC were significantly reduced 24 hours posttreatment with 350 mg/kg DMXAA only, with no evidence of dose response. HPLC demonstrated a significant increase in plasma 5-HIAA 24 hours posttreatment with 200 and 350 mg/kg DMXAA. Histologic analysis revealed some evidence of tumor necrosis following treatment with 100 or 200 mg/kg DMXAA, reaching significance with 350 mg/kg DMXAA. The absence of any reduction in K(trans) or IAUGC following treatment with 200 mg/kg, despite a significant increase in 5-HIAA, raises concerns about the utility of established DCE-MRI biomarkers to assess tumor response to DMXAA