Search

Supplementary Fig. S2 from Pharmacokinetic-pharmacodynamic correlation from mouse to human with pazopanib, a multikinase angiogenesis inhibitor with potent antitumor and antiangiogenic activity

Supplementary Table S4 from Pharmacokinetic-pharmacodynamic correlation from mouse to human with pazopanib, a multikinase angiogenesis inhibitor with potent antitumor and antiangiogenic activity

With the development of targeted therapeutics, especially for small-molecule inhibitors, it is important to understand whether the observed in vivo efficacy correlates with the modulation of desired/intended target in vivo. We have developed a small-molecule inhibitor of all three vascular endothelial growth factor (VEGF) receptors (VEGFR), platelet-derived growth factor receptor, and c-Kit tyrosine kinases, pazopanib (GW786034), which selectively inhibits VEGF-induced endothelial cell proliferation. It has good oral exposure and inhibits angiogenesis and tumor growth in mice. Because bolus administration of the compound results in large differences in Cmax and Ctrough, we investigated the effect of continuous infusion of a VEGFR inhibitor on tumor growth and angiogenesis. GW771806, which has similar enzyme and cellular profiles to GW786034, was used for these studies due to higher solubility requirements for infusion studies. Comparing the pharmacokinetics by two different routes of administration (bolus p.o. dosing and continuous infusion), we showed that the antitumor and antiangiogenic activity of VEGFR inhibitors is dependent on steady-state concentration of the compound above a threshold. The steady-state concentration required for these effects is consistent with the concentration required for the inhibition of VEGF-induced VEGFR2 phosphorylation in mouse lungs. Furthermore, the steady-state concentration of pazopanib determined from preclinical activity showed a strong correlation with the pharmacodynamic effects and antitumor activity in the phase I clinical trial. [Mol Cancer Ther 2007;6(7):2012–21]

A series of dianilinopyrimidineureas demonstrate potency as VEGFR2 kinase inhibitors.

Administration of exogenous proteins and peptides as therapeutics carries with it the potential for immune system recognition and the development of neutralizing antibodies to endogenous regulatory proteins. PEGylation of proteins typically reduces their immunogenicity in vivo. GW395058 is a PEGylated peptide thrombopoietin receptor (TPOr) agonist being evaluated for the treatment of chemotherapy-induced thrombocytopenia. Although GW395058 shares no homology with TPO, it does compete with TPO for binding to a common receptor, and a similarity in local structure could result in shared epitopes. Thus GW395058 could elicit TPO-neutralizing antibodies. In this study, we evaluated the immunogenicity of GW395058 in mice, the potential of rabbit antibodies elicited by immunizations with the non-PEGylated parent peptide AF15705 to cross-react with recombinant human (rHu) TPO, and the potential of mouse anti-rHuTPO antibodies elicited by repeated dosing with rHuTPO to cross-react with AF15705. GW395058-dosed mice failed to produce antibodies to AF15705 or rHuTPO. Mouse anti-rHuTPO did not cross-react with AF15705 and rabbit anti-AF15705 antibodies failed to cross-react with rHuTPO. GW395058 caused no immune-mediated lesions in mice, but rHuTPO suppressed megakaryocytopoiesis and caused B-lymphocyte hyperplasia in lymphoid tissues consistent with antigenic stimulation. These data suggest that the potential for an immune response to GW395058 in man would be low.

Novel 2,4-diamino-6-methyl-5-ethynylpyrimidines were prepared via palladium catalyzed coupling of 2,4-diamino-5-iodo-6-methyl-pyrimidine with terminal acetylenes. The compounds were inhibitors of dihydrofolate reductase and showed in vitro activity against several species of opportunistic fungi and the protozoan Toxoplasma gondii.

A series of novel 1,3-diamino-7,8,9,10-tetrahydropyrido[3,2-f]quinazolines were synthesized starting from 6-amino-5-cyanoquinoline (4). These compounds inhibited Candida albicans dihydrofolate reductase with Ki values of ≤0.60 aM. One analogue exhibited moderate in vivo efficacy in a C. albicans-infected mouse model.

A series of 7,8-dialkylpyrrolo[3,2-f]quinazolines were prepared as inhibitors of dihydrofolate reductase (DHFR). On the basis of an apparent inverse relationship between compound size and antifungal activity, the compounds were designed to be relatively small and compact. Inhibitor design was aided by GRID analysis of the three-dimensional structure of Candida albicans DHFR, which suggested that relatively small, branched alkyl groups at the 7- and 8-positions of the pyrroloquinazoline ring system would provide optimal interactions with a hydrophobic region of the protein. The compounds were potent inhibitors of fungal and human DHFR, with K(i) values as low as 7.1 and 0.1 pM, respectively, and were highly active against C. albicans and an array of tumor cell lines. In contrast to known lipophilic inhibitors of DHFR such as trimetrexate and piritrexim, members of this series of pyrroloquinazolines were not susceptible to P-glycoprotein-mediated multidrug resistance and also showed significant distribution into lung and brain tissue. The compounds were active in lung and brain tumor models and displayed in vivo activity against Pneumocystis carinii and C. albicans.

Several 5-(4-substituted benzyl)-2,4-diaminoquinazolines were prepared as potentially selective inhibitors of Candida albicans dihydrofolate reductase. These compounds were synthesized by a novel route, which included as a key step the displacement of a fluoro group in 2,6-difluorobenzonitrile by the anions of ethyl or methyl 4-substituted phenylacetates. The resultant diarylacetates were saponified and decarboxylated to the 2-fluoro-6-(4-substituted phenyl)benzonitriles. Ring closure of these benzonitriles with guanidine carbonate gave the 5-(4-substituted benzyl)-2,4-diaminoquinazolines.

The recent increase in fungal infections, especially among AIDS patients, has resulted in the need for more effective antifungal agents. In our search for such agents, we focused on developing compounds which inhibit fungal dihydrofolate reductase (DHFR). A series of 25 5-(arylthio)-2,4-diaminoquinazolines were synthesized as potentially selective inhibitors of Candida albicans DHFR. The majority of the compounds were potent inhibitors of C. albicans DHFR and much less active against human DHFR. High selectivity, as defined by the ratio of the I50 values for human and C. albicans DHFR, was achieved by compounds with bulky and rigid 4-substituents in the phenylthio moiety. For example, 5-[(4-morpholinophenyl)thio]-2,4-diaminoquinazoline displayed a selectivity ratio of 540 and was the most selective inhibitor synthesized to date. Substitution in the 2- or 3-position of the 5-phenylthio group provided only marginal selectivity. 6-Substituted-5-[(4-tert-butylphenyl)thio]-2,4-diaminoquinazolines showed potent activity against the C. albicans enzyme but were equally active against human DHFR. Most of the selective compounds were also good inhibitors of C. albicans cell growth, with minimum inhibitory concentration values as low as 0.05 microgram/ mL.

A series of 7,8-dialkylpyrrolo[3,2-f]quinazolines were prepared as inhibitors of dihydrofolate reductase (DHFR). On the basis of an apparent inverse relationship between compound size and antifungal activity, the compounds were designed to be relatively small and compact. Inhibitor design was aided by a GRID analysis of the three-dimensional structure of Candida albicans DHFR, which suggested that relatively small, branched alkyl groups at the 7- and 8-positions of the pyrroloquinazoline ring system would provide optimal interactions with a hydrophobic region of the protein. The compounds were potent inhibitors of fungal and human DHFR, with Ki values as low as 7.1 and 0.1 pM, respectively, and were highly active against C. albicans and an array of tumor cell lines. In contrast to known lipophilic inhibitors of DHFR such as trimetrexate and piritrexim, members of this series of pyrroloquinazolines were not susceptible to P-glycoprotein-mediated multidrug resistance and also showed significant distribution...

Inhibition of the vascular endothelial growth factor (VEGF) signaling pathway has emerged as one of the most promising new approaches for cancer therapy. We describe herein the key steps starting from an initial screening hit leading to the discovery of pazopanib, N(4)-(2,3-dimethyl-2H-indazol-6-yl)-N(4)-methyl-N(2)-(4-methyl-3-sulfonamidophenyl)-2,4-pyrimidinediamine, a potent pan-VEGF receptor (VEGFR) inhibitor under clinical development for renal-cell cancer and other solid tumors.

With the development of targeted therapeutics, especially for small-molecule inhibitors, it is important to understand whether the observed in vivo efficacy correlates with the modulation of desired/intended target in vivo. We have developed a small-molecule inhibitor of all three vascular endothelial growth factor (VEGF) receptors (VEGFR), platelet-derived growth factor receptor, and c-Kit tyrosine kinases, pazopanib (GW786034), which selectively inhibits VEGF-induced endothelial cell proliferation. It has good oral exposure and inhibits angiogenesis and tumor growth in mice. Because bolus administration of the compound results in large differences in Cmax and Ctrough, we investigated the effect of continuous infusion of a VEGFR inhibitor on tumor growth and angiogenesis. GW771806, which has similar enzyme and cellular profiles to GW786034, was used for these studies due to higher solubility requirements for infusion studies. Comparing the pharmacokinetics by two different routes of administration (bolus p.o. dosing and continuous infusion), we showed that the antitumor and antiangiogenic activity of VEGFR inhibitors is dependent on steady-state concentration of the compound above a threshold. The steady-state concentration required for these effects is consistent with the concentration required for the inhibition of VEGF-induced VEGFR2 phosphorylation in mouse lungs. Furthermore, the steady-state concentration of pazopanib determined from preclinical activity showed a strong correlation with the pharmacodynamic effects and antitumor activity in the phase I clinical trial. [Mol Cancer Ther 2007;6(7):2012–21]

Anilinoalkynylpyrimidines were prepared and evaluated as dual EGFR/ErbB2 kinase inhibitors. A preference was found for substituted phenyl and heteroaromatic rings attached to the alkyne. In addition, the presence of a potential hydrogen bond donor appended to this ring was favored. Selected molecules in the series demonstrated some activity against human tumor cell lines.

The recent increase in fungal infections, especially among AIDS patients, has resulted in the need for more effective antifungal agents. In our search for such agents, we focused on developing compounds which inhibit fungal dihydrofolate reductase (DHFR). A series of 25 5-(arylthio)-2,4-diaminoquinazolines were synthesized as potentially selective inhibitors of Candida albicans DHFR. The majority of the compounds were potent inhibitors of C. albicans DHFR and much less active against human DHFR. High selectivity, as defined by the ratio of the I50 values for human and C. albicans DHFR, was achieved by compounds with bulky and rigid 4-substituents in the phenylthio moiety. For example, 5-[(4-morpholinophenyl)thio]-2,4-diaminoquinazoline displayed a selectivity ratio of 540 and was the most selective inhibitor synthesized to date. Substitution in the 2- or 3-position of the 5-phenylthio group provided only marginal selectivity. 6-Substituted-5-[(4-tert-butylphenyl)thio]-2,4-diaminoquinazolines showed potent activity against the C. albicans enzyme but were equally active against human DHFR. Most of the selective compounds were also good inhibitors of C. albicans cell growth, with minimum inhibitory concentration values as low as 0.05 microgram/ mL.

Abstract 4726 The thrombopoietin receptor agonists (TPO-RA), romiplostim and eltrombopag, are presently indicated for the treatment of certain patient groups with immune thrombocytopenia purpura. In a clinical study with romiplostim in patients with low-/intermediate-1 risk myelodysplastic syndromes (MDS), cases of transient increases in blast cell counts were observed and cases of MDS disease progression to acute myeloid leukemia (AML) were reported. In the present study, we evaluated the impact of romiplostim, eltrombopag, and recombinant human thrombopoietin (TPO) on the proliferation of 5 human AML and 1 TPO-dependent megakaryoblastic cell line. The cell lines evaluated include the TPO-dependent cell line, N2C TPO; the TPO-R positive AML lines, HEL92.1.7 and OCI-AML-3; and the TPO-R negative AML cell lines, HL60, THP-1, and NOMO-1. All cells were exposed to 11-point dose response curves of the 3 agents at concentrations sufficient to generate a full stimulatory response in the N2C TPO cell line. Cells were exposed to concentrations of romiplostim and eltrombopag that met or exceeded the reported Cmax achieved for each agent in high-dose clinical trials and were 3- (eltrombopag) to 30-fold (romiplostim) above trough levels from the same clinical trials. Neither romiplostim nor TPO treatment resulted in detectable stimulation or inhibition of leukemia cell growth at concentrations up to 10 μg/mL. Treatment with eltrombopag up to 40 μg/mL caused inhibition of all AML cell lines with mean IC50 values ranging from 6.4 to 13.5 μg/mL. These IC50 values reflect concentrations that are 3- to 6-fold below the Cmax of a 300 qd dose of eltrombopag (40.5 μg/mL) and at concentrations as low as 2-fold below Ctau levels (12.4 μg/mL). Cmax exceeded the IC90 for these AML cell lines, which ranged from 18.5 to 27.9 μg/mL. No stimulation of AML growth was evident through the range of the eltrombopag dose response curve on any of the cell lines evaluated. The results of this study confirm earlier in vitro studies (Will 2009, Erickson-Miller 2010) showing inhibitory effects of eltrombopag on leukemic cell lines and support clinical studies to evaluate a potential anti-leukemic effect of higher doses of eltrombopag in patients with AML. Disclosures: Rusnak: GlaxoSmithKline: Consultancy, Equity Ownership, Patents & Royalties. Off Label Use: Eltrombopag is an oral TPO agonist indicated for chronic ITP being studied in MDS/AML. Rudolph:GlaxoSmithKline: Consultancy, Equity Ownership. Erickson-Miller:GlaxoSmithKline: Employment, Equity Ownership, Patents & Royalties, Research Funding.