Objective: We characterized and compared the CNS distribution of intrathecally (IT) administered ... more Objective: We characterized and compared the CNS distribution of intrathecally (IT) administered nusinersen in the spherical nucleic acid (SNA) or unformulated (linear) format in rats. Background: SNAs are densely packed, radial arrangements of oligonucleotides around a nanoparticle core. As a consequence of their structure, SNAs have increased cellular uptake, nuclease stability, and affinity to targets compared with linear oligonucleotides. Our previous studies showed that nusinersen-SNAs substantially increased the median survival of spinal muscular atrophy (SMA) mice versus nusinersen following intracerebroventricular injection. We hypothesized that the increased survival was due, at least in part, to improved retention of SNAs in the CNS tissues. Design/Methods: 125I-labeled nusinersen-SNA and nusinersen were IT injected into wild-type rats. The nusinersen-SNA and nusinersen distribution in the spinal cord, brain, and peripheral tissues were profiled by single-photon emission computed tomography combined with computed tomography (SPECT/CT) imaging over 7 days. Results: The SPECT/CT imaging analyses revealed the presence of nusinersen-SNA and nusinersen throughout the spinal cord and brain tissues as early as 0.25 to 0.5 hr after dosing, and accumulation in all CNS tissues by 6 hr. Integration of the signal revealed that by 24 hours after dosing, the levels of nusinersen-SNA were greater than nusinersen in the brain and spinal cord tissues. At 7 days after dosing, the concentration of nusinersen-SNA in the spinal cord and brain was 50% to 100% higher than nusinersen. Conclusions: These data demonstrate that nusinersen-SNA delivered by IT injection to rats widely distributes throughout the CNS and persists to a greater extent than nusinersen, and explain, at least in part, our previous observations of improved survival duration of SMA mice. These data also support the therapeutic potential of SNAs for treating CNS disorders. Disclosure: Dr. Kandimalla has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Exicure, Inc.. Dr. Daniel has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Exicure, Inc. Dr. Mix has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Exicure, Inc. Dr. Nallagatla has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Exicure, Inc. Dr. Kentala has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Charles River Laboratories. Dr. Zasadny has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Invicro. Dr. Schook has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Exicure, Inc.. Dr. Cedrone has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Exicure, Inc. Dr. Marks has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Exicure, Inc.
A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators ... more A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators (NAMs). Starting from a high-throughput screen (HTS) hit (<b>1</b>), a systematic structure–activity relationship (SAR) study was conducted with a specific focus on balancing pharmacological potency with physicochemical and pharmacokinetic (PK) properties. This effort led to the discovery of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1<i>H</i>-pyrazoloÂ[3,4-<i>b</i>]Âpyrazine (PF470, <b>14</b>) as a highly potent, selective, and orally bioavailable mGluR5 NAM. Compound <b>14</b> demonstrated robust efficacy in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-rendered Parkinsonian nonhuman primate model of l-DOPA-induced dyskinesia (PD-LID). However, the progression of <b>14</b> to the clinic was terminated because of a potentially mechanism-mediated finding consistent with a delayed-type immune-me...
A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators ... more A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators (NAMs). Starting from a high-throughput screen (HTS) hit (1), a systematic structure-activity relationship (SAR) study was conducted with a specific focus on balancing pharmacological potency with physicochemical and pharmacokinetic (PK) properties. This effort led to the discovery of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (PF470, 14) as a highly potent, selective, and orally bioavailable mGluR5 NAM. Compound 14 demonstrated robust efficacy in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-rendered Parkinsonian nonhuman primate model of l-DOPA-induced dyskinesia (PD-LID). However, the progression of 14 to the clinic was terminated because of a potentially mechanism-mediated finding consistent with a delayed-type immune-mediated type IV hypersensitivity in a 90-day NHP regulatory toxicology study.
PURPOSE We assessed the feasibility of noninvasive metabolic monitoring of cancer chemohormonothe... more PURPOSE We assessed the feasibility of noninvasive metabolic monitoring of cancer chemohormonotherapy using sequential quantitative positron emission tomographic (PET) scans of tumor glucose metabolism with the glucose analog 2-[18F]-fluoro-2-deoxy-D-glucose (FDG). PATIENTS AND METHODS Eleven women with newly diagnosed primary breast cancers larger than 3 cm in diameter beginning a chemohormonotherapy program underwent a baseline and four follow-up quantitative PET scans during the first three cycles of treatment (days 0 to 63). Tumor response was sequentially determined clinically, radiographically, and then pathologically after nine treatment cycles. RESULTS Eight patients had partial or complete pathologic responses. Their maximal tumor uptake of FDG assessed by PET decreased promptly with treatment to the following: day 8, 78 +/- 9.2% (P < .03); day 21, 68.1 +/- 7.5% (P < .025); day 42, 60 +/- 5.1% (P < .001); day 63, 52.4 +/- 4.4% (P < .0001) of the basal values. Tu...
To accelerate the discovery of novel small molecule central nervous system (CNS) positron emissio... more To accelerate the discovery of novel small molecule central nervous system (CNS) positron emission tomography (PET) ligands, we aimed to define a property space that would facilitate ligand design and prioritization, thereby providing a higher probability of success for novel PET ligand development. Toward this end, we built a database consisting of 62 PET ligands that have successfully reached the clinic and 15 radioligands that failed in late-stage development as negative controls. A systematic analysis of these ligands identified a set of preferred parameters for physicochemical properties, brain permeability, and nonspecific binding (NSB). These preferred parameters have subsequently been applied to several programs and have led to the successful development of novel PET ligands with reduced resources and timelines. This strategy is illustrated here by the discovery of the novel phosphodiesterase 2A (PDE2A) PET ligand 4-(3-[(18)F]fluoroazetidin-1-yl)-7-methyl-5-{1-methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazol-4-yl}imidazo[5,1-f][1,2,4]triazine, [(18)F]PF-05270430 (5).
[18F]FLT-PET imaging has been increasingly employed as a surrogate biomarker in early clinical tr... more [18F]FLT-PET imaging has been increasingly employed as a surrogate biomarker in early clinical trials to evaluate the mechanism of cancer drug candidates. In this report, we have assessed the preclinical application of [18F]FLT-PET imaging modality for several investigative agents that are currently under clinical development, including PF-03732010 (P-cadherin mAb), PD-0332991 (CDK4 inhibitor) and PF-03084014 (γ-secretase inhibitor). Docetaxel was also utilized as a standard comparator. Our goals included 1) building a preclinical understanding of the [18F]FLT tracer avidity in various xenograft models; 2) selecting models with appropriate tracer avidity and target molecular profiles for respective agents; 3) correlating suppression of [18F]FLT uptake with the changes of other pharmacodynamic endpoints and measures of antitumor efficacy; and 4) bridging preclinical and clinical [18F]FLT-PET imaging studies. Among the tested tumor models, [18F]FLT tracer avidity did not always correlate with the tumor growth rate. Affymetrix array and LC/MS analyses indicated that multiple factors including the expression levels of thymidine kinase 1 (TK1) and pyrimidine transporters, as well as the intrinsic thymidine level in tumor each contributed to the [18F]FLT tracer avidity. In the MDA-MB-435HAL-subrenal capsule model, administration of PF-03732010 resulted in a time- and dose-dependent inhibition of [18F]FLT tracer uptake, which corresponded with the modulation of β-catenin and Ki67 levels via IHC analysis. As β-catenin partners with P-cadherin for mediating the proliferation and invasiveness of tumor cells, this result suggests that the suppression in [18F]FLT uptake correspond with the target modulation. However, when we tested the [18F]FLT-PET imaging in the MDA-MB-231 model, treatment with an efficacious dose of PF-03084014 (GSI) exhibited an insignificant effect on the tracer uptake despite evidence in modulation of target genes. The GSI-induced impact on the tumor BrdU uptake was also minimal suggesting that the observed antitumor efficacy was not primarily mediated by modulation of cell cycle. In the Rb wild-type MDA-MB-231 and HCT116 tumor models, administration of an efficacious dose level of PD-0332991 (CDK4/6i) demonstrated a significant decline of [18F]FLT tracer uptake. Concurrent hypophosphorylation of RbSer780 and reduced BrdU uptake by IHC was also observed, indicating that [18F]FLT tracer uptake highly reflected the target modulation and predicted therapy. Therefore [18F]FLT-PET imaging modality presents a ideal proof-of-mechanism biomarker for PD-0332991. Collectively, these results indicate a potential applications for [18F]FLT-PET imaging in early clinical studies to demonstrate an impact on target endpoints depending on the mechanism of action of anticancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1758.
Journal of nuclear medicine : official publication, Society of Nuclear Medicine, Jan 19, 2016
The enzyme phosphodiesterase 2A (PDE2A) is a potential target for development of novel therapeuti... more The enzyme phosphodiesterase 2A (PDE2A) is a potential target for development of novel therapeutic agents for the treatment of cognitive impairments. The goal of the present study was to evaluate the PDE2A ligand (18)F-PF-0150270430, 4-(3-fluoroazetidin-1-yl)-7-methyl-5-(1-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)imidazo[1,5-f][1,2,4]triazine, in nonhuman primates. (18)F-PF-05270430 was radiolabeled by two methods via nucleophilic substitution of its tosylate precursor. Tissue metabolite analysis in rodents and positron emission tomography (PET) imaging in non-human primates under baseline and blocking conditions were performed to determine the pharmacokinetic and binding characteristics of the new radioligand. Various kinetic modeling approaches were assessed to select the optimal method for analysis of imaging data. (18)F-PF-05270430 was synthesized in >98% radiochemical purity and high specific activity. In the non-human primate brain, uptake of (18)F-PF-05270430 w...
Objective: We characterized and compared the CNS distribution of intrathecally (IT) administered ... more Objective: We characterized and compared the CNS distribution of intrathecally (IT) administered nusinersen in the spherical nucleic acid (SNA) or unformulated (linear) format in rats. Background: SNAs are densely packed, radial arrangements of oligonucleotides around a nanoparticle core. As a consequence of their structure, SNAs have increased cellular uptake, nuclease stability, and affinity to targets compared with linear oligonucleotides. Our previous studies showed that nusinersen-SNAs substantially increased the median survival of spinal muscular atrophy (SMA) mice versus nusinersen following intracerebroventricular injection. We hypothesized that the increased survival was due, at least in part, to improved retention of SNAs in the CNS tissues. Design/Methods: 125I-labeled nusinersen-SNA and nusinersen were IT injected into wild-type rats. The nusinersen-SNA and nusinersen distribution in the spinal cord, brain, and peripheral tissues were profiled by single-photon emission c...
Objective: We characterized and compared the CNS distribution of intrathecally (IT) administered ... more Objective: We characterized and compared the CNS distribution of intrathecally (IT) administered nusinersen in the spherical nucleic acid (SNA) or unformulated (linear) format in rats. Background: SNAs are densely packed, radial arrangements of oligonucleotides around a nanoparticle core. As a consequence of their structure, SNAs have increased cellular uptake, nuclease stability, and affinity to targets compared with linear oligonucleotides. Our previous studies showed that nusinersen-SNAs substantially increased the median survival of spinal muscular atrophy (SMA) mice versus nusinersen following intracerebroventricular injection. We hypothesized that the increased survival was due, at least in part, to improved retention of SNAs in the CNS tissues. Design/Methods: 125I-labeled nusinersen-SNA and nusinersen were IT injected into wild-type rats. The nusinersen-SNA and nusinersen distribution in the spinal cord, brain, and peripheral tissues were profiled by single-photon emission computed tomography combined with computed tomography (SPECT/CT) imaging over 7 days. Results: The SPECT/CT imaging analyses revealed the presence of nusinersen-SNA and nusinersen throughout the spinal cord and brain tissues as early as 0.25 to 0.5 hr after dosing, and accumulation in all CNS tissues by 6 hr. Integration of the signal revealed that by 24 hours after dosing, the levels of nusinersen-SNA were greater than nusinersen in the brain and spinal cord tissues. At 7 days after dosing, the concentration of nusinersen-SNA in the spinal cord and brain was 50% to 100% higher than nusinersen. Conclusions: These data demonstrate that nusinersen-SNA delivered by IT injection to rats widely distributes throughout the CNS and persists to a greater extent than nusinersen, and explain, at least in part, our previous observations of improved survival duration of SMA mice. These data also support the therapeutic potential of SNAs for treating CNS disorders. Disclosure: Dr. Kandimalla has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Exicure, Inc.. Dr. Daniel has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Exicure, Inc. Dr. Mix has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Exicure, Inc. Dr. Nallagatla has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Exicure, Inc. Dr. Kentala has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Charles River Laboratories. Dr. Zasadny has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Invicro. Dr. Schook has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Exicure, Inc.. Dr. Cedrone has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Exicure, Inc. Dr. Marks has received personal compensation for consulting, serving on a scientific advisory board, speaking, or other activities with Exicure, Inc.
A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators ... more A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators (NAMs). Starting from a high-throughput screen (HTS) hit (<b>1</b>), a systematic structure–activity relationship (SAR) study was conducted with a specific focus on balancing pharmacological potency with physicochemical and pharmacokinetic (PK) properties. This effort led to the discovery of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1<i>H</i>-pyrazoloÂ[3,4-<i>b</i>]Âpyrazine (PF470, <b>14</b>) as a highly potent, selective, and orally bioavailable mGluR5 NAM. Compound <b>14</b> demonstrated robust efficacy in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-rendered Parkinsonian nonhuman primate model of l-DOPA-induced dyskinesia (PD-LID). However, the progression of <b>14</b> to the clinic was terminated because of a potentially mechanism-mediated finding consistent with a delayed-type immune-me...
A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators ... more A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators (NAMs). Starting from a high-throughput screen (HTS) hit (1), a systematic structure-activity relationship (SAR) study was conducted with a specific focus on balancing pharmacological potency with physicochemical and pharmacokinetic (PK) properties. This effort led to the discovery of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (PF470, 14) as a highly potent, selective, and orally bioavailable mGluR5 NAM. Compound 14 demonstrated robust efficacy in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-rendered Parkinsonian nonhuman primate model of l-DOPA-induced dyskinesia (PD-LID). However, the progression of 14 to the clinic was terminated because of a potentially mechanism-mediated finding consistent with a delayed-type immune-mediated type IV hypersensitivity in a 90-day NHP regulatory toxicology study.
PURPOSE We assessed the feasibility of noninvasive metabolic monitoring of cancer chemohormonothe... more PURPOSE We assessed the feasibility of noninvasive metabolic monitoring of cancer chemohormonotherapy using sequential quantitative positron emission tomographic (PET) scans of tumor glucose metabolism with the glucose analog 2-[18F]-fluoro-2-deoxy-D-glucose (FDG). PATIENTS AND METHODS Eleven women with newly diagnosed primary breast cancers larger than 3 cm in diameter beginning a chemohormonotherapy program underwent a baseline and four follow-up quantitative PET scans during the first three cycles of treatment (days 0 to 63). Tumor response was sequentially determined clinically, radiographically, and then pathologically after nine treatment cycles. RESULTS Eight patients had partial or complete pathologic responses. Their maximal tumor uptake of FDG assessed by PET decreased promptly with treatment to the following: day 8, 78 +/- 9.2% (P < .03); day 21, 68.1 +/- 7.5% (P < .025); day 42, 60 +/- 5.1% (P < .001); day 63, 52.4 +/- 4.4% (P < .0001) of the basal values. Tu...
To accelerate the discovery of novel small molecule central nervous system (CNS) positron emissio... more To accelerate the discovery of novel small molecule central nervous system (CNS) positron emission tomography (PET) ligands, we aimed to define a property space that would facilitate ligand design and prioritization, thereby providing a higher probability of success for novel PET ligand development. Toward this end, we built a database consisting of 62 PET ligands that have successfully reached the clinic and 15 radioligands that failed in late-stage development as negative controls. A systematic analysis of these ligands identified a set of preferred parameters for physicochemical properties, brain permeability, and nonspecific binding (NSB). These preferred parameters have subsequently been applied to several programs and have led to the successful development of novel PET ligands with reduced resources and timelines. This strategy is illustrated here by the discovery of the novel phosphodiesterase 2A (PDE2A) PET ligand 4-(3-[(18)F]fluoroazetidin-1-yl)-7-methyl-5-{1-methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazol-4-yl}imidazo[5,1-f][1,2,4]triazine, [(18)F]PF-05270430 (5).
[18F]FLT-PET imaging has been increasingly employed as a surrogate biomarker in early clinical tr... more [18F]FLT-PET imaging has been increasingly employed as a surrogate biomarker in early clinical trials to evaluate the mechanism of cancer drug candidates. In this report, we have assessed the preclinical application of [18F]FLT-PET imaging modality for several investigative agents that are currently under clinical development, including PF-03732010 (P-cadherin mAb), PD-0332991 (CDK4 inhibitor) and PF-03084014 (γ-secretase inhibitor). Docetaxel was also utilized as a standard comparator. Our goals included 1) building a preclinical understanding of the [18F]FLT tracer avidity in various xenograft models; 2) selecting models with appropriate tracer avidity and target molecular profiles for respective agents; 3) correlating suppression of [18F]FLT uptake with the changes of other pharmacodynamic endpoints and measures of antitumor efficacy; and 4) bridging preclinical and clinical [18F]FLT-PET imaging studies. Among the tested tumor models, [18F]FLT tracer avidity did not always correlate with the tumor growth rate. Affymetrix array and LC/MS analyses indicated that multiple factors including the expression levels of thymidine kinase 1 (TK1) and pyrimidine transporters, as well as the intrinsic thymidine level in tumor each contributed to the [18F]FLT tracer avidity. In the MDA-MB-435HAL-subrenal capsule model, administration of PF-03732010 resulted in a time- and dose-dependent inhibition of [18F]FLT tracer uptake, which corresponded with the modulation of β-catenin and Ki67 levels via IHC analysis. As β-catenin partners with P-cadherin for mediating the proliferation and invasiveness of tumor cells, this result suggests that the suppression in [18F]FLT uptake correspond with the target modulation. However, when we tested the [18F]FLT-PET imaging in the MDA-MB-231 model, treatment with an efficacious dose of PF-03084014 (GSI) exhibited an insignificant effect on the tracer uptake despite evidence in modulation of target genes. The GSI-induced impact on the tumor BrdU uptake was also minimal suggesting that the observed antitumor efficacy was not primarily mediated by modulation of cell cycle. In the Rb wild-type MDA-MB-231 and HCT116 tumor models, administration of an efficacious dose level of PD-0332991 (CDK4/6i) demonstrated a significant decline of [18F]FLT tracer uptake. Concurrent hypophosphorylation of RbSer780 and reduced BrdU uptake by IHC was also observed, indicating that [18F]FLT tracer uptake highly reflected the target modulation and predicted therapy. Therefore [18F]FLT-PET imaging modality presents a ideal proof-of-mechanism biomarker for PD-0332991. Collectively, these results indicate a potential applications for [18F]FLT-PET imaging in early clinical studies to demonstrate an impact on target endpoints depending on the mechanism of action of anticancer agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1758.
Journal of nuclear medicine : official publication, Society of Nuclear Medicine, Jan 19, 2016
The enzyme phosphodiesterase 2A (PDE2A) is a potential target for development of novel therapeuti... more The enzyme phosphodiesterase 2A (PDE2A) is a potential target for development of novel therapeutic agents for the treatment of cognitive impairments. The goal of the present study was to evaluate the PDE2A ligand (18)F-PF-0150270430, 4-(3-fluoroazetidin-1-yl)-7-methyl-5-(1-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)imidazo[1,5-f][1,2,4]triazine, in nonhuman primates. (18)F-PF-05270430 was radiolabeled by two methods via nucleophilic substitution of its tosylate precursor. Tissue metabolite analysis in rodents and positron emission tomography (PET) imaging in non-human primates under baseline and blocking conditions were performed to determine the pharmacokinetic and binding characteristics of the new radioligand. Various kinetic modeling approaches were assessed to select the optimal method for analysis of imaging data. (18)F-PF-05270430 was synthesized in >98% radiochemical purity and high specific activity. In the non-human primate brain, uptake of (18)F-PF-05270430 w...
Objective: We characterized and compared the CNS distribution of intrathecally (IT) administered ... more Objective: We characterized and compared the CNS distribution of intrathecally (IT) administered nusinersen in the spherical nucleic acid (SNA) or unformulated (linear) format in rats. Background: SNAs are densely packed, radial arrangements of oligonucleotides around a nanoparticle core. As a consequence of their structure, SNAs have increased cellular uptake, nuclease stability, and affinity to targets compared with linear oligonucleotides. Our previous studies showed that nusinersen-SNAs substantially increased the median survival of spinal muscular atrophy (SMA) mice versus nusinersen following intracerebroventricular injection. We hypothesized that the increased survival was due, at least in part, to improved retention of SNAs in the CNS tissues. Design/Methods: 125I-labeled nusinersen-SNA and nusinersen were IT injected into wild-type rats. The nusinersen-SNA and nusinersen distribution in the spinal cord, brain, and peripheral tissues were profiled by single-photon emission c...
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