Correlation of pharmacokinetics with the antitumor activity of Cetuximab in nude mice bearing the GEO human colon carcinoma xenograft.

PURPOSE: The epidermal growth factor receptor (EGFR), a protein tyrosine kinase expressed in many types of human cancers including colon and breast, has been strongly associated with tumor progression. Cetuximab, an IgG1 anti-EGFR chimeric mouse/human monoclonal antibody, has been proven to be effective in the treatment of advanced colon cancer. To date, there has not been a study to systematically evaluate the pharmacokinetics (PK) of Cetuximab in a preclinical model and to further explore any correlation of drug exposure between animal models and cancer patients. In the present study, we characterized the PK of Cetuximab in nude mice at efficacious dose levels and further compared the preclinical optimal dose and active plasma drug concentration with those determined in clinical studies. EXPERIMENTAL DESIGN: The antitumor activity of Cetuximab was evaluated using the GEO human colon carcinoma xenografts implanted subcutaneously in nude mice. The drug was administered ip every 3 days for five total injections (inj) (q3dx5) at dose levels ranging from 1 mg/inj to 0.04 mg/inj. The plasma PK of Cetuximab was determined at dose levels of 1.0, 0.25, and 0.04 mg/inj with a single bolus iv or ip administration in nude mice. The tumoral PK of Cetuximab was determined at dose levels of 0.25, and 0.04 mg/inj with a single bolus ip administration in nude mice bearing GEO tumor xenografts. The plasma and tumoral levels of Cetuximab were quantitated by an ELISA assay. RESULTS: Cetuximab demonstrated a dose-dependent antitumor activity at dose levels of 0.25, 0.1, and 0.04 mg/inj, with a statistically significant tumor growth delay (in reaching a tumor target size of 1 gm) of 18 days (P < 0.001), 12.3 days (P < 0.01), and 10 days (P < 0.01) for 0.25, 0.1, and 0.04 mg/inj, respectively. A separate study employing the same treatment schedule showed that Cetuximab was equally active at dose levels ranging from 0.25 mg/inj to 1 mg/inj. Therefore, dose levels of Cetuximab from 1 mg/inj to 0.04 mg/inj can be considered to be within the efficacious range, while dose levels of 0.25 mg/inj or higher appeared to be optimal for the antitumor activity of Cetuximab in the GEO tumor model. When Cetuximab was given iv to mice, the elimination half life (t(1/2)) was 39.6, 37.8, and 42.2 h for doses of 1.0, 0.25, and 0.04 mg/inj, respectively, suggesting a similar disposition kinetics of Cetuximab within this dose range. The volume of distribution (V(d)) ranged from 0.062 l/kg to 0.070 l/kg, suggesting that Cetuximab is primarily confined to the plasma compartment with limited peripheral tissue distribution. Clearance (CL) was similar and no apparent PK saturation was observed across the dose ranging from 0.04 mg/inj to 1.0 mg/inj. When mice were administered with a single bolus ip administration at doses of 1, 0.25, and 0.04 mg/inj, the maximum plasma concentration (C(max)) was 407.6, 66.4, and 16.5 microg/ml. The area under the curve of plasma drug concentration (AUC) was 19212.4, 3182.4, and 534.5 microg/ml h, for 1.0, 0.25, and 0.04 mg/inj, respectively. The average steady state plasma concentration (C(ss avg)) for the multiple dosing schedule was estimated to be 73.1 microg/ml at 0.25 mg/inj and was considered as an active plasma drug concentration. The maximum tumoral concentration of Cetuximab was 2.6 and 0.53 ng/mg-tumor while the tumoral drug exposure was 112.6 and 18.3 ng/mg h for 0.25 and 0.04 mg/inj, respectively. The EGFR was estimated to be nearly completely occupied by Cetuximab at the optimal dose of 0.25 mg/inj. CONCLUSION: In the present study, we compared the preclinical optimal dose and the corresponding active plasma concentration determined in mice with those being observed in cancer patients, i.e. 65-100 microg/ml. The preclinical optimal dose of 0.25 mg/inj was significantly lower than the current clinical dose. However, the active plasma concentration at 0.25 mg/inj is within the range of the active drug concentrations in cancer patients treated with Cetuximab under the current optimal dosing regimen. It appears that the active plasma drug concentration determined in preclinical model predicts better than the optimal preclinical dose for the clinical development of antibody drugs.

Pharmacokinetics and biotransformations of oxaliplatin in comparison with ormaplatin following a single bolus intravenous injection in rats.

PURPOSE: Traditionally ultrafilterable Pt has been used to estimate the body exposure to platinum drugs. However, previous studies have shown that ultrafilterable Pt consists of both cytotoxic and inert biotransformation products of platinum drugs. Therefore, it has been proposed that pharmacokinetic parameters of the parent drug and its cytotoxic biotransformation products are more likely to be correlated with the drug toxicity and efficacy than those of ultrafilterable Pt. Oxaliplatin and ormaplatin are likely to form very similar biotransformation products in vivo based on previous studies. However, ormaplatin causes severe and irreversible neurotoxicity while oxaliplatin causes moderate and reversible neurotoxicity. To evaluate the hypothesis that the neurotoxicity is associated with the pharmacokinetics of active biotransformation products, we investigated the biotransformations and pharmacokinetics of oxaliplatin and ormaplatin in rats at equimolar doses. METHODS: 3H-oxaliplatin and 3H-ormaplatin were administered to Wistar male rats through single bolus i.v. injections (20 micromol/kg). Blood was sampled from 3.5 min to 360 min and centrifuged at 2000 g to separate the plasma from red blood cells (RBCs). The RBCs were sonicated and centrifuged at 13000 g to separate the cytosol from the membrane fraction. Both plasma and RBC cytosol were filtered through YMT30 membranes (Mr = 30000 kDa), and the ultrafiltrates were analyzed using a single column HPLC technique to identify and quantitate the biotransformation products. The pharmacokinetics of oxaliplatin, ormaplatin, and their biotransformation products were characterized utilizing the curve stripping and nonlinear least-squares fitting program RSTRIP. RESULTS: The decays of total, plasma, plasma ultrafilterable (PUF), RBC-bound, and plasma protein-bound Pt-dach (only Pt species with an intact dach carrier ligand were quantitated in this study) were described by biphasic curves. No significant kinetic differences between oxaliplatin and ormaplatin were observed for total, plasma, and PUF Pt-dach in the initial alpha decay phase. However, Pt-dach bound to plasma proteins fourfold more quickly for ormaplatin than for oxaliplatin, and the AUC for Pt-dach bound to plasma proteins was twofold higher for ormaplatin than for oxaliplatin. The concentration of RBC-bound Pt-dach was highest at the initial time-point of 3.5 min for both drugs, which suggested a very rapid RBC uptake. The binding of Pt-dach to RBCs was slightly greater initially for ormaplatin than for oxaliplatin. However, the RBC-bound Pt-dach decayed more rapidly for ormaplatin (t(1/2alphaRBC) = 5.1 min) than for oxaliplatin (t(1,2alphaRBC) = 15.3 min). Thus the AUC(RBC) was slightly greater for oxaliplatin than for ormaplatin. The AUC was also slightly greater for oxaliplatin than for ormaplatin for the Pt-dach associated with the RBC membrane and RBC cytosolic proteins. However, there was no significant difference between oxaliplatin and ormaplatin for Pt-dach in the RBC cytosolic ultrafiltrate. There was also no significant difference in the AUCpuf between oxaliplatin and ormaplatin. Both oxaliplatin and ormaplatin produced the same types of major plasma biotransformation products including Pt(dach)Cl2, Pt(dach)(Cys)2, Pt(dach)(GSH)2, Pt(dach)(GSH), Pt(dach)(Met), and free dach. The decays of oxaliplatin, ormaplatin, and their biotransformation products were described by biphasic curves. (ABSTRACT TRUNCATED)

Comparative neurotoxicity of oxaliplatin, ormaplatin, and their biotransformation products utilizing a rat dorsal root ganglia in vitro explant culture model.

PURPOSE: Neurotoxicity is one of the major toxicities of platinum-based anticancer drugs, especially oxaliplatin and ormaplatin. It has been postulated that biotransformation products are likely to be responsible for the toxicity of platinum drugs. In our preceding pharmacokinetic study, both oxaliplatin and ormaplatin were observed to produce the same types of major plasma biotransformation products. However, while the plasma concentration of ormaplatin was much lower than that of oxaliplatin at an equimolar dose, one of their common biotransformation products, Pt(dach)Cl2, was present at 29-fold higher concentrations in the plasma following the i.v. injection of ormaplatin than of oxaliplatin. Because ormaplatin has severe neurotoxicity and Pt(dach)Cl2 is very cytotoxic, we have postulated that Pt(dach)Cl2 is likely to be responsible for the differences in neurotoxicity between ormaplatin and oxaliplatin. In order to test this hypothesis, we compared the neurotoxicity of oxaliplatin, ormaplatin, and their biotransformation products. Since the dorsal root ganglia (DRGs) have been suggested to be the likely targtet for platinum drugs and in vitro DRG explant cultures have been suggested to be a valid model for studying cisplatin-associated neurotoxicity, our comparative neurotoxicity study was conducted with DRG explant cultures in vitro. METHODS: Based on the previous studies of cisplatin neurotoxicity, we established our in vitro DRG explant culture utilizing DRGs dissected from E-19 embryonic rats. Rat DRGs were incubated for 30 min with different platinum compounds to mimic in vivo exposure conditions; this was by followed by a 48-h incubation in culture medium at 37 degrees C. At the end of the incubation, the neurites were fixed and stained with toluidine blue, and neurite outgrowth was quantitated by phase-contrast microscopy. The inhibition of neurite outgrowth by platinum compounds was used as an indicator of in vitro neurotoxicity. Since an in vivo study has indicated that the order of neurotoxicity is ormaplatin > cisplatin > oxaliplatin > carboplatin as measured by morphometric changes to rat DRGs, we initially validated our DRG explant culture model by comparing the in vitro neurotoxicity of ormaplatin, cisplatin, oxaliplatin, and carboplatin. After observing the same neurotoxicity rank between this study and a previous in vivo study, we further compared the neurotoxicity of oxaliplatin, ormaplatin, and their biotransformation products including Pt(dach)Cl2, Pt(dach)(H2O)Cl, Pt(dach)(H2O)2, Pt(dach)(Met), and Pt(dach)(GSH) utilizing the DRG explant culture model. RESULTS: Our study indicated that Pt(dach)Cl2 and its hydrolysis products were more potent at inhibiting neurite outgrowth than the parent drugs oxaliplatin and ormaplatin. In contrast, no detectable inhibition of neurite outgrowth was observed for DRGs dosed with Pt(dach)(Met) and Pt(dach)(GSH). CONCLUSION: This study suggests that biotransformation products such as Pt(dach)Cl2 and its hydrolysis products are more neurotoxic than the parent drugs oxaliplatin and ormaplatin. The different neurotoxicity profiles of oxaliplatin and ormaplatin are more likely due to the different plasma concentrations of their common biotransformation product Pt(dach)Cl2 than to differences in their intrinsic neurotoxicity.

High-performance liquid chromatographic separation of the biotransformation products of oxaliplatin.

A novel single reversed-phase HPLC system was developed for separating oxaliplatin and its biotransformation products formed in rat plasma. The major stable biotransformation products of oxaliplatin formed in rat plasma were identified as Pt(dach)(Cys)2, Pt(dach)(Met) and free dach. The minor biotransformation products Pt(dach)Cl2, Pt(dach)(GSH) and Pt(dach)(GSH)2 could also be resolved from other Pt-dach complexes. Among these biotransformation products, the identification of Pt(dach)(Met) was further confirmed by LC-ESI-MS, and the identification of Pt(dach)(Cys)2, Pt(dach)(GSH), Pt(dach)(GSH)2 and free dach was confirmed by atomic absorption and double isotope labeling. This HPLC technique should prove useful for separating and identifying the biotransformation products of Pt-dach drugs such as oxaliplatin, ormaplatin and Pt(dach)(mal) in biological fluids. This will allow a more complete characterization of the pharmacokinetics and biotransformations of these Pt-dach drugs, which should in turn lead to a better understanding of the mechanisms leading to their toxicity and efficacy.

Biotransformations of oxaliplatin in rat blood in vitro.

The partitioning and biotransformations of oxaliplatin [trans-l-1,2-diaminocyclohexaneoxalatoplatinum(II)] were investigated in the blood of Wistar male rats in vitro. [3-H]-Oxaliplatin was incubated with rat blood at 37 degrees C in 5% CO2 and the concentrations of all Pt complexes containing the [3-H]-dach carrier ligand were followed for up to 12 hours. Decay for both oxaliplatin and Pt-dach in the plasma ultrafiltrate (PUF) was rapid (t 1/2 oxaliplatin = 0.68 h and t 1/2 for Pt-dach in the PUF = 0.85 h). After 9 hours, the concentration of oxaliplatin fell below the detection limit. By 4 hours, the PUF-Pt-dach reached a plateau, which was 12% of total Pt-dach. The binding of Pt-dach to red blood cells (RBCs) and plasma proteins was also very rapid (t 1/2 RBCs = 0.58 h and t 1/2 plasma proteins = 0.78 h) and reached equilibrium by 4 hours. At equilibrium, 35% of total Pt-dach was bound to plasma proteins, 12% was in the plasma ultrafiltrate, and 53% was found associated with RBCs. Of the Pt-dach associated with RBCs, 23% was bound to the RBC membrane, 58% was bound to RBC cytosolic proteins, and 19% was in the RBC cytosol ultrafiltrate. Thus, these studies confirm previous observations of oxaliplatin accumulation by rat RBCs. To better characterize the determinants of this accumulation, oxaliplatin and other Pt-dach complexes were compared with respect to both their uptake by rat RBCs and their partition coefficients in octanol and water. The rank order for the rate of uptake was ormaplatin approximately Pt(dach)Cl2 > oxaliplatin > Pt(dach)(mal); while the rank order for hydrophobicity was ormaplatin > Pt(dach)Cl2 > Pt(dach)(mal) > oxaliplatin. Thus, in general, Pt-dach complexes appeared to be taken up better by RBCs than cisplatin or carboplatin, and the hydrophobicity of most of the Pt-dach complexes appeared to correlate with uptake. However, factors other than the dach carrier ligand and hydrophobicity clearly influence uptake. The biotransformations of oxaliplatin in rat blood were characterized utilizing reverse-phase high-pressure liquid chromatography (HPLC). In the RBC cytosol, both oxaliplatin and Pt(dach)Cl2 were observed at early times, while Pt(dach)(GSH)2, Pt(dach)(Cys)2, Pt(dach)(GSH), and free dach accumulated and reached steady-state levels by 4 hours. Thus, in the RBC cytosol, only chemically unreactive biotransformation products such as free dach and Pt-dach complexes with cysteine and glutathione accumulated in significant amounts. Furthermore, only Pt(dach)(Cys)2 and free dach appeared to efflux from RBCs. Thus, RBCs do not appear to serve as a reservoir for cytotoxic Pt-dach complexes. Finally, the biotransformation products of oxaliplatin in the plasma were identified as Pt(dach)Cl2, Pt(dach)(Cys)2, Pt(dach)(GSH), Pt(dach)(Met), Pt(dach)(GSH)2, and free dach. Among these compounds, Pt(dach)Cl2 formed transiently, while Pt(dach)(Cys)2, Pt(dach)(Met), and free dach accumulated and were the major biotransformation products by 4 hours. Thus, this study has identified the major inert and reactive biotransformation products of oxaliplatin in both plasma and RBCs and thus provides the information required for detailed pharmacokinetic and biotransformation studies of oxaliplatin. [figure in text]

Cytotoxicity, cellular uptake, and cellular biotransformations of oxaliplatin in human colon carcinoma cells.

Biotransformation products of platinum anticancer drugs have been suggested to be responsible for drug efficacy and toxicity. This study was designed to determine whether the efficacy of the closely related 1,2-diaminocyclohexane-Pt (dach-Pt) compounds oxaliplatin and ormaplatin were determined primarily by the parent drugs or by one of their biotransformation products. Based on consideration of both in vitro cytotoxicity in human colon carcinoma cells (HT-29) and concentrations following oxaliplatin administration in vivo, our data suggest that the efficacy of oxaliplatin is primarily determined by the plasma levels of the parent drug, with the biotransformation products Pt(dach)Cl2, Pt(dach)(H2O)Cl, and Pt(dach)(H2O)2 making only minor contributions. The stable biotransformation products containing amino acids did not have any significant cytotoxicity. In contrast, our data suggest that the efficacy of ormaplatin is primarily determined by plasma levels of Pt(dach)Cl2. The cytotoxicity of oxaliplatin, Pt(dach)Cl2, and Pt(dach)(H2O)Cl was approximately proportional to their cellular uptake, whereas the cytotoxicity of ormaplatin, Pt(dach)(H2O)2, and Pt(dach)(Met) was less than predicted from their uptake. Treatment of HT-29 cells with equimolar external concentrations of Pt(dach)Cl2 and oxaliplatin resulted in the formation of twofold more Pt-DNA adducts following Pt(dach)Cl2 treatment than following oxaliplatin treatment. However, intracellular Pt(dach)Cl2 levels were 30-fold higher for Pt(dach)Cl2-treated cells than for oxaliplatin-treated cells. These data suggest that intracellular conversion of oxaliplatin to Pt(dach)Cl2 makes only a minor contribution to Pt-DNA adduct formation and the resultant cytotoxicity of oxaliplatin.