A 9-Month Toxicity and Toxicokinetic Assessment of Subcutaneous Pegylated Human C-peptide (CBX129801) in Cynomolgus Monkeys.

C-peptide is formed in the biosynthesis of insulin and is therefore deficient in patients with type 1 diabetes mellitus. A pegylated form of human synthetic C-peptide (CBX129801) has been developed to extend the half-life of the native peptide and is undergoing clinical investigation as replacement therapy to treat diabetic peripheral neuropathy. This monkey study was conducted to evaluate the toxicity of CBX129801 with weekly subcutaneous dosing for 39 weeks at dose levels of 0 (vehicle), 0.4, 1.33, and 4.0 mg/kg/wk. No systemic adverse effects were observed at any dose with maximal CBX129801 plasma concentrations of 735 to 1050 nmol/L during the dosing period (physiological range is 1-3 nmol/L). CBX129801-related effects were limited to minimal macrophagic vacuolization at the injection sites and in the associated draining (axillary) lymph nodes; these local effects largely resolved by the end of a 7-week recovery period. No systemic macrophagic vacuolization was observed. Additionally, there was no histological evidence for plaque formation in the major arteries of these nondiabetic animals.

Phase I study of intravenously administered ATI-1123, a liposomal docetaxel formulation in patients with advanced solid tumors.

PURPOSE: ATI-1123 is a liposomal formulation of docetaxel and may be administered without the premedications and hypersensitivity reactions. This Phase I study examines the safety, tolerability, pharmacokinetics (PKs), and antitumor activity of ATI-1123. METHODS: Patients with advanced solid malignancies received escalating doses of ATI-1123 intravenously over 1-h every 3 weeks. The dosing commenced using an accelerated titration design and was followed by a modified 3 + 3 Fibonacci schema to determine maximally tolerated dose (MTD). Plasma was analyzed for encapsulated/non-encapsulated docetaxel; PK analyses were performed using model independent method. Response was assessed using RECIST criteria. RESULTS: In total, 29 patients received doses ranging from 15 to 110 mg/m(2). At 110 mg/m(2), two of six patients experienced dose-limiting toxicities including grade 3 stomatitis and febrile neutropenia. The 90 mg/m(2) cohort was expanded to ten patients and identified as the MTD. The most common adverse events were fatigue, nausea, neutropenia, anemia, anorexia, and diarrhea. ATI-1123 exhibited linear and dose proportional PKs. One patient with lung cancer had confirmed partial response, and stable disease was observed in 75 % patients. CONCLUSIONS: ATI-1123 demonstrated an acceptable tolerability and favorable PK profile in patients with solid tumors. Our results provide support for Phase II trials to determine the antitumor activity of this drug.

Development of prodrug 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl benzoate (TG100801): a topically administered therapeutic candidate in clinical trials for the treatment of age-related macular degeneration.

Age-related macular degeneration (AMD) is one of the leading causes of loss of vision in the industrialized world. Attenuating the VEGF signal in the eye to treat AMD has been validated clinically. A large body of evidence suggests that inhibitors targeting the VEGFr pathway may be effective for the treatment of AMD. Recent studies using Src/YES knockout mice suggest that along with VEGF, Src and YES play a crucial role in vascular leak and might be useful in treating edema associated with AMD. Therefore, we have developed several potent benzotriazine inhibitors designed to target VEGFr2, Src, and YES. One of the most potent compounds is 4-chloro-3-{5-methyl-3-[4-(2-pyrrolidin-1-yl-ethoxy)phenylamino]benzo[1,2,4]triazin-7-yl}phenol ( 5), a dual inhibitor of both VEGFr2 and the Src family (Src and YES) kinases. Several ester analogues of 5 were prepared as prodrugs to improve the concentration of 5 at the back of the eye after topical administration. The thermal stability of these esters was studied, and it was found that benzoyl and substituted benzoyl esters of 5 showed good thermal stability. The hydrolysis rates of these prodrugs were studied to analyze their ability to undergo conversion to 5 in vivo so that appropriate concentrations of 5 are available in the back-of-the-eye tissues. From these studies, we identified 4-chloro-3-(5-methyl-3-{[4-(2-pyrrolidin-1-ylethoxy)phenyl]amino}-1,2,4-benzotriazin-7-yl)phenyl benzoate ( 12), a topically administered prodrug delivered as an eye drop that is readily converted to the active compound 5 in the eye. This topically delivered compound exhibited excellent ocular pharmacokinetics and poor systemic circulation and showed good efficacy in the laser induced choroidal neovascularization model. On the basis of its superior profile, compound 12 was advanced. It is currently in a clinical trial as a first in class, VEGFr2 targeting, topically applied compound for the treatment of AMD.

Cyclic conversion of the novel Src kinase inhibitor [7-(2,6-dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (TG100435) and Its N-oxide metabolite by flavin-containing monoxygenases and cytochrome P450 reductase.

[7-(2,6-Dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine (TG100435) is a novel multi-targeted Src family kinase inhibitor with demonstrated anticancer activity in preclinical species. Potent kinase inhibition is associated with TG100435 and its major N-oxide metabolite [7-(2,6-dichlorophenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-{4-[2-(1-oxy-pyrrolidin-1-yl)-ethoxy]-phenyl}-amine (TG100855). The objectives of the current study were to identify the hepatic enzyme(s) responsible for 1) the total metabolic flux of TG100435, 2) the formation of TG100855, and 3) the subsequent metabolism of TG100855. Flavin-containing monooxygenases (FMO) and cytochrome P450 monooxygenases (P450s) contribute to TG100435 total metabolic flux. TG100435 metabolic flux was completely inhibited by methimazole and ketoconazole, suggesting only FMO- and CYP3A4-mediated metabolism. TG100855 formation was markedly inhibited (~90%) by methimazole or heat inactivation (>99%). FMO3 was the primary enzyme responsible for TG100855 formation. In addition, an enzyme mediated retroreduction of TG100855 back to TG100435 was observed. The N-oxidation reaction was approximately 15 times faster than the retroreduction reaction. Interestingly, the retroreduction of TG100855 to TG100435 in recombinant P450 or liver microsomes lacked inhibition by the P450 inhibitors. TG100435 formation in the human liver microsomes or recombinant P450 increased as a function of cytochrome P450 reductase activity, suggesting potential involvement of cytochrome P450 reductase. The results of this in vitro study demonstrate the potential of TG100435 and TG100855 to be interconverted metabolically. FMO seem to be the major N-oxidizing enzymes, whereas cytochrome P450 reductase seems to be responsible for the retroreduction reaction.

An age-dependent physiologically based pharmacokinetic/pharmacodynamic model for the organophosphorus insecticide chlorpyrifos in the preweanling rat.

Juvenile rats are more susceptible than adults to the acute toxicity of organophosphorus insecticides like chlorpyrifos (CPF). Age- and dose-dependent differences in metabolism may be responsible. Of importance are CYP450 activation and detoxification of CPF to chlorpyrifos-oxon (CPF-oxon) and trichloropyridinol (TCP), as well as B-esterase (B-est) and PON-1 (A-esterase) detoxification of CPF-oxon to TCP. In the current study, a physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) model incorporating age-dependent changes in CYP450, PON-1, and tissue B-est levels for rats was developed. In this model, age was used as a dependent function to estimate body weight which was then used to allometrically scale both metabolism and tissue cholinesterase (ChE) levels. In addition, age-dependent changes in brain, liver, and fat volumes and brain blood flow were obtained from the literature and used in the simulations. Model simulations suggest that preweanling rats are particularly sensitive to CPF toxicity, with levels of CPF-oxon in blood and brain disproportionately increasing, relative to the response in adult rats. This age-dependent nonlinear increase in CPF-oxon concentration may potentially result from both the depletion of nontarget B-est and a lower PON-1 metabolic capacity in younger animals. The PBPK/PD model behaves consistently with the general understanding of CPF toxicity, pharmacokinetics, and tissue ChE inhibition in neonatal and adult rats. Hence, this model represents an important starting point for developing a computational model to assess the neurotoxic potential of environmentally relevant organophosphate exposures in infants and children.

Metabolism and pharmacokinetics of a novel Src kinase inhibitor TG100435 ([7-(2,6-dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine) and its active N-oxide metabolite TG100855 ([7-(2,6-dichloro-phenyl)-5-methylbenzo[1,2,4]triazin-3-yl]-{4-[2-(1-oxy-pyrrolidin-1-yl)-ethoxy]-phenyl}-amine).

TG100435 ([7-(2,6-dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-amine) is a novel multitargeted, orally active protein tyrosine kinase inhibitor. The inhibition constants (K(i)) of TG100435 against Src, Lyn, Abl, Yes, Lck, and EphB4 range from 13 to 64 nM. TG100435 has systemic clearance values of 20.1, 12.7, and 14.5 ml/min/kg and oral bioavailability of 74%, 23%, and 11% in mouse, rat, and dog, respectively. Four oxidation metabolites of TG100435 have been found in human, dog, and rat in vitro and in vivo. The ethylpyrrolidine N-oxide of TG100435 is the predominant metabolite (TG100855; [7-(2,6-dichloro-phenyl)-5-methyl-benzo[1,2,4]triazin-3-yl]-{4-[2-(1-oxy-pyrrolidin-1-yl)-ethoxy]-phenyl}-amine) in human, dog, and rat. TG100855 is 2 to 9 times more potent than the parent compound. Flavin-containing monooxygenases are the primary enzymes mediating the biotransformation. Significant conversion of TG100435 to TG100855 has been observed in rat and dog after oral administration. Systemic exposure of TG100855 is 1.1- and 2.1-fold greater than that of TG100435 in rat and dog after oral dosing of TG100435. Since TG100435 is predominantly converted to the more potent N-oxide metabolite across species in vivo and in vitro, the overall tyrosine kinase inhibition in animal models may be substantially increased after oral administration of TG100435.

Age-related brain cholinesterase inhibition kinetics following in vitro incubation with chlorpyrifos-oxon and diazinon-oxon.

Chlorpyrifos and diazinon are two commonly used organophosphorus insecticides (OPs), and their primary mechanism of action involves the inhibition of acetylcholinesterase by their metabolites chlorpyrifos-oxon (CPO) and diazinon-oxon (DZO), respectively. The study objectives were to assess the in vitro age-related inhibition kinetics of neonatal rat brain cholinesterase (ChE) for CPO and DZO by estimating the bimolecular inhibitory rate constant (k(i)) values. Brain ChE inhibition and k(i) values following CPO and DZO incubation with neonatal Sprague-Dawley rat brain homogenates were determined at postnatal day (PND) 5, 12, and 17 and compared with the corresponding inhibition and k(i) values obtained in the adult rat. A modified Ellman method was utilized for measuring the ChE activity. CPO caused a greater ChE inhibition than DZO as evidenced from the estimated k(i) values of both compounds. Neonatal brain ChE inhibition kinetics exhibited a marked age-related sensitivity to CPO, with the order of ChE inhibition being PND 5 > PND 7 > PND 17 with k(i) values of 0.95, 0.50, and 0.22 nM(-1)hr(-1), respectively. In contrast, DZO ChE inhibition was not age related in the neonatal brain, and the estimated k(i) value at all PND ages was 0.02 nM(-1)hr(-1). These results demonstrated an age- and OP-selective inhibition of rat brain ChE, which may be critically important in understanding the potential sensitivity of juveniles to specific OPs exposures.

Development of a non-invasive biomonitoring approach to determine exposure to the organophosphorus insecticide chlorpyrifos in rat saliva.

Non-invasive biomonitoring approaches are being developed using reliable portable analytical systems to quantify dosimetry utilizing readily obtainable body fluids, such as saliva. In the current study, rats were given single oral gavage doses (1, 10, or 50 mg/kg) of the insecticide chlorpyrifos (CPF). Saliva and blood were then collected from groups of animals (4/time-point) at 3, 6, and 12 h post-dosing, and were analyzed for the CPF metabolite trichloropyridinol (TCP). Trichloropyridinol was detected in both blood and saliva at all doses and the TCP concentration in blood exceeded saliva, although the kinetics in blood and saliva were comparable. A physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model for CPF incorporated a compartment model to describe the time-course of TCP in blood and saliva. The model adequately simulated the experimental results over the dose ranges evaluated. A rapid and sensitive sequential injection (SI) electrochemical immunoassay was developed to monitor TCP, and the reported detection limit for TCP was 6 ng/L (in water). Computer model simulation in the range of the Allowable Daily Intake (ADI) or Reference Dose (RfD) for CPF (0.01-0.003 mg/kg/day) suggests that the electrochemical immunoassay has adequate sensitivity to detect and quantify TCP in saliva at these low exposure levels. However, to validate this approach, further studies are needed to more fully understand the pharmacokinetics of CPF and TCP excretion in saliva. These initial findings suggest that the utilization of saliva as a biomonitoring matrix, coupled to real-time quantitation and PBPK/PD modeling represents a novel approach with broad application for evaluating both occupational and environmental exposures to CPF.

Discovery of [7-(2,6-dichlorophenyl)-5-methylbenzo [1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-ylethoxy)phenyl]amine--a potent, orally active Src kinase inhibitor with anti-tumor activity in preclinical assays.

We describe the identification of [7-(2,6-dichlorophenyl)-5-methylbenzo [1,2,4]triazin-3-yl]-[4-(2-pyrrolidin-1-ylethoxy)phenyl]amine (3), a potent, orally active Src inhibitor with desirable PK properties, demonstrated activity in human tumor cell lines and in animal models of tumor growth.

Noninvasive biomonitoring approaches to determine dosimetry and risk following acute chemical exposure: analysis of lead or organophosphate insecticide in saliva.

There is a need to develop approaches for assessing risk associated with acute exposures to a broad range of metals and chemical agents and to rapidly determine the potential implications to human health. Noninvasive biomonitoring approaches are being developed using reliable portable analytical systems to quantitate dosimetry utilizing readily obtainable body fluids, such as saliva. Saliva has been used to evaluate a broad range of biomarkers, drugs, and environmental contaminants, including heavy metals and pesticides. To advance the application of noninvasive biomonitoring a microfluidic/electrochemical device has also been developed for the analysis of lead (Pb), using square-wave anodic stripping voltametry. The system demonstrates a linear response over a broad concentration range (1-2000 ppb) and is capable of quantitating saliva Pb in rats orally administered acute doses of Pb acetate. Appropriate pharmacokinetic analyses have been used to quantitate systemic dosimetry based on determination of saliva Pb concentrations. In addition, saliva has recently been used to quantitate dosimetry following exposure to the organophosphate insecticide chlorpyrifos in a rodent model system by measuring the major metabolite, trichloropyridinol, and saliva cholinesterase inhibition following acute exposures. These results suggest that technology developed for noninvasive biomonitoring can provide a sensitive and portable analytical tool capable of assessing exposure and risk in real-time. By coupling these noninvasive technologies with pharmacokinetic modeling it is feasible to rapidly quantitate acute exposure to a broad range of chemical agents. In summary, it is envisioned that once fully developed, these monitoring and modeling approaches will be useful for evaluating acute exposure and health risk.

Continuous system modeling of equilibrium dialysis for determinations of tissue partitioning of parathion and paraoxon.

A tissue/blood partition coefficient, defined as the ratio of tissue chemical concentration to that of the venous outflow of the tissue when at equilibrium, is an important parameter required for physiological based pharmacokinetic models. While many techniques have been developed to quantify tissue/blood partition coefficients for various chemicals, there is no single best approach for their determination. In the current study, equilibrium dialysis of the organophosphorus insecticide parathion and its active metabolite paraoxon was undertaken to assess their partitioning into rat liver. A mass balance analysis of the contents of the dialysis cells suggested that significant levels of parathion and paraoxon were bound to the dialysis membranes. There was no evidence of metabolism of either parathion or paraoxon by the very dilute liver homogenate utilized in the dialysis. In order to investigate the potential impact of binding of a chemical to dialysis membrane during determination of partition coefficients, a computer model of a dialysis system was constructed. The model assumed that all processes occurring within the dialysis cell were first or second order in nature, and that binding to the dialysis membrane occurred symmetrically on both sides of the membrane. Variations in the total number of simulated binding sites on dialysis membrane revealed that increasing the degree of membrane binding resulted in decreased compound on the homogenate and buffer sides of the dialysis cells. However, the final tissue/buffer partition coefficient was unaffected by these alterations in membrane binding, although increased membrane binding prolonged the incubation time required to achieve equilibrium. These simulations suggest that loss of a compound to membrane binding does not preclude the use of equilibrium dialysis for determination of tissue/buffer, and therefore tissue/blood, partition coefficients, provided the dialysis system is allowed to proceed to equilibrium.