d035f48d75b586c47b3aa1bca57148cb pharmaceutics-15-02381.pdf c8316ecbe367beb8a073ba504c9dd42e44b7abe5 pharmaceutics-15-02381.pdf 0f41c2e514554533e74f3ea5901a9bc0977d7328dd19c1b92d48f898da4dd8c1 pharmaceutics-15-02381.pdf Title: Preclinical Evaluation of an Imidazole-Linked Heterocycle for Alzheimer’s Disease Subject: Humanity is facing a vast prevalence of neurodegenerative diseases, with Alzheimer’s disease (AD) being the most dominant, without efficacious drugs, and with only a few therapeutic targets identified. In this scenario, we aim to find molecular entities that modulate imidazoline I2 receptors (I2-IRs) that have been pointed out as relevant targets in AD. In this work, we explored structural modifications of well-established I2-IR ligands, giving access to derivatives with an imidazole-linked heterocycle as a common key feature. We report the synthesis, the affinity in human I2-IRs, the brain penetration capabilities, the in silico ADMET studies, and the three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of this new bunch of I2-IR ligands. Selected compounds showed neuroprotective properties and beneficial effects in an in vitro model of Parkinson’s disease, rescued the human dopaminergic cell line SH-SY5Y from death after treatment with 6-hydroxydopamine, and showed crucial anti-inflammatory effects in a cellular model of neuroinflammation. After a preliminary pharmacokinetic study, we explored the action of our representative 2-(benzo[b]thiophen-2-yl)-1H-imidazole LSL33 in a mouse model of AD (5xFAD). Oral administration of LSL33 at 2 mg/Kg for 4 weeks ameliorated 5XFAD cognitive impairment and synaptic plasticity, as well as reduced neuroinflammation markers. In summary, this new I2-IR ligand that promoted beneficial effects in a well-established AD mouse model should be considered a promising therapeutic strategy for neurodegeneration. 20pt Keywords: imidazoline I2 receptor ligand; Alzheimer’s disease; imidazoline-linked heterocycle; 2-(benzo[b]thiophen-2-yl)-1H-imidazole; 5XFAD; 3D-QSAR; neuroprotection Author: Andrea Bagán, Sergio Rodriguez-Arévalo, Teresa Taboada-Jara, Christian Griñán-Ferré, Mercè Pallàs, Iria Brocos-Mosquera, Luis F. Callado, José A. Morales-García, Belén Pérez, Caridad Diaz, Rosario Fernández-Godino, Olga Genilloud, Milan Beljkas, Slavica Oljacic, Katarina Nikolic and Carmen Escolano Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Sep 26 10:21:18 2023 CEST ModDate: Tue Sep 26 10:59:01 2023 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 26 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 4589154 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- IBTYZX+VnURWPalladioL Type 1 Custom yes yes no 10 0 SSJMSB+URWPalladioL-Bold Type 1 Custom yes yes no 15 0 NRWLWJ+URWPalladioL-Roma Type 1 Custom yes yes no 20 0 UXBBCH+URWPalladioL-Ital Type 1 Custom yes yes no 24 0 JFLFCJ+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 IAFAOG+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 47 0 ACZNUE+EURM10 Type 1 Builtin yes yes no 75 0 PCMMMJ+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 89 0 PCMMML+PalatinoLinotype TrueType WinAnsi yes yes no 92 0 PCMMNM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 95 0 PCMNMI+PalatinoLinotype TrueType MacRoman yes yes no 98 0 KVEXVN+CMSY10 Type 1 Builtin yes yes no 105 0 XXJPYL+URWPalladioL-BoldItal Type 1 Custom yes yes no 109 0 QGSPAQ+PazoMath Type 1 Builtin yes yes no 131 0 CRJHCU+PazoMath-Italic Type 1 Builtin yes yes no 135 0 PCMMMJ+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 149 0 PCMMMK+PalatinoLinotype CID TrueType Identity-H yes yes yes 152 0 PCMMML+PalatinoLinotype TrueType WinAnsi yes yes no 158 0 PCMMNM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 161 0 PCMMMI+PalatinoLinotype,Bold CID TrueType Identity-H yes yes yes 202 0 PCNBJD+Arial CID TrueType Identity-H yes yes yes 208 0 PCNBJE+Arial,Bold CID TrueType Identity-H yes yes yes 214 0 PCMMMJ+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 220 0 PCMMMK+PalatinoLinotype CID TrueType Identity-H yes yes yes 223 0 PCMMML+PalatinoLinotype TrueType WinAnsi yes yes no 229 0 PCMMNM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 232 0 PCMNMI+PalatinoLinotype TrueType MacRoman yes yes no 235 0 PCMPLO+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 238 0 CZOINJ+EURB10 Type 1 Builtin yes yes no 251 0 PCMMMJ+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 265 0 PCMMMK+PalatinoLinotype CID TrueType Identity-H yes yes yes 268 0 PCMMML+PalatinoLinotype TrueType WinAnsi yes yes no 274 0 PCMMNM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 277 0 PCMNMI+PalatinoLinotype TrueType MacRoman yes yes no 280 0 PCMMMJ+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 301 0 PCMMMK+PalatinoLinotype CID TrueType Identity-H yes yes yes 304 0 PCMMML+PalatinoLinotype TrueType WinAnsi yes yes no 310 0 PCMMNM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 313 0 PCMNMI+PalatinoLinotype TrueType MacRoman yes yes no 316 0 PCMPLO+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 319 0 PCMMMJ+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 338 0 PCMMMK+PalatinoLinotype CID TrueType Identity-H yes yes yes 341 0 PCMMML+PalatinoLinotype TrueType WinAnsi yes yes no 347 0 PCMMNM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 350 0 PCMNMI+PalatinoLinotype TrueType MacRoman yes yes no 353 0 PCMPLO+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 356 0 PCMMMJ+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 376 0 PCMMMK+PalatinoLinotype CID TrueType Identity-H yes yes yes 379 0 PCMMML+PalatinoLinotype TrueType WinAnsi yes yes no 385 0 PCMMNM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 388 0 PCMNMI+PalatinoLinotype TrueType MacRoman yes yes no 391 0 PCMPLO+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 394 0 MWRHMH+EURM5 Type 1 Builtin yes yes no 405 0 FZLVCN+EURM7 Type 1 Builtin yes yes no 409 0 PCMMMJ+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 423 0 PCMMMK+PalatinoLinotype CID TrueType Identity-H yes yes yes 426 0 PCMMML+PalatinoLinotype TrueType WinAnsi yes yes no 432 0 PCMMNM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 435 0 PCMNMI+PalatinoLinotype TrueType MacRoman yes yes no 438 0 PCMMMJ+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 451 0 PCMMMK+PalatinoLinotype CID TrueType Identity-H yes yes yes 454 0 PCMMML+PalatinoLinotype TrueType WinAnsi yes yes no 460 0 PCMMNM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 463 0 PCMNMI+PalatinoLinotype TrueType MacRoman yes yes no 466 0 PCMMMJ+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 482 0 PCMMMK+PalatinoLinotype CID TrueType Identity-H yes yes yes 485 0 PCMMML+PalatinoLinotype TrueType WinAnsi yes yes no 491 0 PCMMNM+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 494 0 PCMNMI+PalatinoLinotype TrueType MacRoman yes yes no 497 0 Jhove (Rel. 1.28.0, 2023-05-18) Date: 2024-02-02 02:39:15 CET RepresentationInformation: pharmaceutics-15-02381.pdf ReportingModule: PDF-hul, Rel. 1.12.4 (2023-03-16) LastModified: 2024-02-01 18:02:13 CET Size: 4589154 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 795 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: section.1 Item: Title: Materials and Methods Destination: section.2 Children: Item: Title: Chemistry Destination: subsection.2.1 Children: Item: Title: Synthesis of LSL42, LSL39 and LSL33 B42-pharmaceutics-2550485 Destination: subsubsection.2.1.1 Item: Title: Synthesis of LSL35, LSL34 and LSL29 Destination: subsubsection.2.1.2 Item: Title: Binding Studies Destination: subsection.2.2 Children: Item: Title: Preparation of Cellular Membranes Destination: subsubsection.2.2.1 Item: Title: Competition Binding Assays Destination: subsubsection.2.2.2 Item: Title: 3D-QSAR Study Data Set Preparation Destination: subsection.2.3 Item: Title: In Vitro Model of Alzheimer’s and Parkinsons’ Disease Destination: subsection.2.4 Children: Item: Title: Treatments Destination: subsubsection.2.4.1 Item: Title: Cell Viability Assay Destination: subsubsection.2.4.2 Item: Title: Nitrite Measurement Destination: subsubsection.2.4.3 Item: Title: Statistical Analysis Destination: subsubsection.2.4.4 Item: Title: In Vivo Pharmacokinetics of LSL33 Destination: subsection.2.5 Children: Item: Title: Animals for PK Studies Destination: subsubsection.2.5.1 Item: Title: Method Validation for Quantitation of LSL33 in Mouse Plasma and in Mouse Brain Destination: subsubsection.2.5.2 Item: Title: In Vivo Experiments in Mice Destination: subsection.2.6 Children: Item: Title: Cognitive Tests Destination: subsubsection.2.6.1 Item: Title: Molecular Experiments Destination: subsection.2.7 Children: Item: Title: Brain Processing Destination: subsubsection.2.7.1 Item: Title: Protein Levels Determination by Western Blotting (WB) Destination: subsubsection.2.7.2 Item: Title: RNA Extraction and Gene Expression Determination Destination: subsubsection.2.7.3 Item: Title: Results and Discussion Destination: section.3 Children: Item: Title: Chemistry Destination: subsection.3.1 Item: Title: In Silico ADMET Analysis of Physicochemical and Pharmacokinetic Parameters Destination: subsection.3.2 Item: Title: Pharmacological Evaluation Destination: subsection.3.3 Children: Item: Title: Blood-Brain Barrier Permeation Assay Destination: subsubsection.3.3.1 Item: Title: Radioligand I2-IRs Binding Assays Destination: subsubsection.3.3.2 Item: Title: 3D-QSAR Study Destination: subsection.3.4 Item: Title: In Vitro Models of Neurodegenerative Diseases Destination: subsection.3.5 Children: Item: Title: I2-IR Ligands Protects Neurons from Excitotoxic Damage Destination: subsubsection.3.5.1 Item: Title: I2-IR Ligands Decrease Neuroinflammatory Activity Destination: subsubsection.3.5.2 Item: Title: Metabolic Stability of LSL33 in Human Liver Microsomes Destination: subsection.3.6 Item: Title: Pharmacokinetics of LSL33 Destination: subsection.3.7 Item: Title: Cognitive Studies Destination: subsection.3.8 Children: Item: Title: LSL33 Reduces Cognitive Impairment Presented in 5XFAD Destination: subsubsection.3.8.1 Item: Title: LSL33 Ameliorates Synaptic Plasticity in 5XFAD Destination: subsubsection.3.8.2 Item: Title: LSL33 Attenuates Neuroinflammation Presented in 5XFAD Destination: subsubsection.3.8.3 Item: Title: Conclusions Destination: section.4 Item: Title: References Destination: section.5 Info: Title: Preclinical Evaluation of an Imidazole-Linked Heterocycle for Alzheimer’s Disease Author: Andrea Bagán, Sergio Rodriguez-Arévalo, Teresa Taboada-Jara, Christian Griñán-Ferré, Mercè Pallàs, Iria Brocos-Mosquera, Luis F. Callado, José A. Morales-García, Belén Pérez, Caridad Diaz, Rosario Fernández-Godino, Olga Genilloud, Milan Beljkas, Slavica Oljacic, Katarina Nikolic and Carmen Escolano Subject: Humanity is facing a vast prevalence of neurodegenerative diseases, with Alzheimer’s disease (AD) being the most dominant, without efficacious drugs, and with only a few therapeutic targets identified. In this scenario, we aim to find molecular entities that modulate imidazoline I2 receptors (I2-IRs) that have been pointed out as relevant targets in AD. In this work, we explored structural modifications of well-established I2-IR ligands, giving access to derivatives with an imidazole-linked heterocycle as a common key feature. We report the synthesis, the affinity in human I2-IRs, the brain penetration capabilities, the in silico ADMET studies, and the three-dimensional quantitative structure-activity relationship (3D-QSAR) studies of this new bunch of I2-IR ligands. Selected compounds showed neuroprotective properties and beneficial effects in an in vitro model of Parkinson’s disease, rescued the human dopaminergic cell line SH-SY5Y from death after treatment with 6-hydroxydopamine, and showed crucial anti-inflammatory effects in a cellular model of neuroinflammation. After a preliminary pharmacokinetic study, we explored the action of our representative 2-(benzo[b]thiophen-2-yl)-1H-imidazole LSL33 in a mouse model of AD (5xFAD). Oral administration of LSL33 at 2 mg/Kg for 4 weeks ameliorated 5XFAD cognitive impairment and synaptic plasticity, as well as reduced neuroinflammation markers. In summary, this new I2-IR ligand that promoted beneficial effects in a well-established AD mouse model should be considered a promising therapeutic strategy for neurodegeneration. 20pt Keywords: imidazoline I2 receptor ligand; Alzheimer’s disease; imidazoline-linked heterocycle; 2-(benzo[b]thiophen-2-yl)-1H-imidazole; 5XFAD; 3D-QSAR; neuroprotection Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Sep 26 10:21:18 CEST 2023 ModDate: Tue Sep 26 10:59:01 CEST 2023 ID: 0x4cc824356f2f15b038f766dabaca06e6, 0x4cc824356f2f15b038f766dabaca06e6 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: QGSPAQ+PazoMath FontSubset: true FirstChar: 68 LastChar: 165 FontDescriptor: FontName: QGSPAQ+PazoMath Flags: Symbolic FontBBox: -40, -283, 878, 946 FontFile: true Font: BaseFont: CRJHCU+PazoMath-Italic FontSubset: true FirstChar: 98 LastChar: 98 FontDescriptor: FontName: CRJHCU+PazoMath-Italic Flags: Symbolic FontBBox: -70, -277, 902, 733 FontFile: true Font: BaseFont: KVEXVN+CMSY10 FontSubset: true FirstChar: 0 LastChar: 20 FontDescriptor: FontName: KVEXVN+CMSY10 Flags: Symbolic FontBBox: -29, -960, 1116, 775 FontFile: true Font: BaseFont: IBTYZX+VnURWPalladioL FontSubset: true FirstChar: 160 LastChar: 226 FontDescriptor: FontName: IBTYZX+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ACZNUE+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: ACZNUE+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: XXJPYL+URWPalladioL-BoldItal FontSubset: true FirstChar: 51 LastChar: 83 FontDescriptor: FontName: XXJPYL+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: SSJMSB+URWPalladioL-Bold FontSubset: true FirstChar: 2 LastChar: 241 FontDescriptor: FontName: SSJMSB+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: NRWLWJ+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: NRWLWJ+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: MWRHMH+EURM5 FontSubset: true FirstChar: 12 LastChar: 12 FontDescriptor: FontName: MWRHMH+EURM5 Flags: Symbolic FontBBox: 59, -243, 1369, 720 FontFile: true Font: BaseFont: UXBBCH+URWPalladioL-Ital FontSubset: true FirstChar: 39 LastChar: 151 FontDescriptor: FontName: UXBBCH+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: FZLVCN+EURM7 FontSubset: true FirstChar: 12 LastChar: 22 FontDescriptor: FontName: FZLVCN+EURM7 Flags: Symbolic FontBBox: -5, -243, 1229, 720 FontFile: true Font: BaseFont: CZOINJ+EURB10 FontSubset: true FirstChar: 11 LastChar: 11 FontDescriptor: FontName: CZOINJ+EURB10 Flags: Symbolic FontBBox: -30, -247, 1178, 723 FontFile: true Font: BaseFont: JFLFCJ+VnURWPalladioL-Bold FontSubset: true FirstChar: 160 LastChar: 189 FontDescriptor: FontName: JFLFCJ+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 82, 281, 152, 290 Annotation: Subtype: Link Rect: 34, 269, 136, 278 Annotation: Subtype: Link Rect: 34, 164, 96, 186 Annotation: Subtype: Link Rect: 126, 90, 154, 99 Annotation: Subtype: Link Rect: 34, 78, 147, 87 Annotation: Subtype: Link Rect: 34, 756, 168, 792 Annotation: Subtype: Link Rect: 515, 756, 560, 792 Annotation: Subtype: Link Rect: 352, 662, 363, 676 Annotation: Subtype: Link Rect: 483, 662, 494, 676 Annotation: Subtype: Link Rect: 105, 650, 117, 663 Annotation: Subtype: Link Rect: 324, 650, 335, 663 Annotation: Subtype: Link Rect: 449, 650, 460, 663 Annotation: Subtype: Link Rect: 523, 650, 534, 663 Annotation: Subtype: Link Rect: 102, 637, 113, 651 Annotation: Subtype: Link Rect: 246, 637, 257, 651 Annotation: Subtype: Link Rect: 338, 637, 349, 651 Annotation: Subtype: Link Rect: 121, 627, 132, 638 Annotation: Subtype: Link Rect: 245, 627, 256, 638 Annotation: Subtype: Link Rect: 135, 17, 311, 28 Annotation: Subtype: Link Rect: 385, 17, 560, 28 Annotation: Subtype: Link Rect: 34, 390, 87, 409 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 541, 644, 548, 653 ActionDest: 500 Annotation: Subtype: Link Rect: 549, 644, 556, 653 ActionDest: 500 Annotation: Subtype: Link Rect: 527, 619, 534, 628 ActionDest: 500 Annotation: Subtype: Link Rect: 341, 582, 348, 591 ActionDest: 500 Annotation: Subtype: Link Rect: 195, 519, 202, 528 ActionDest: 500 Annotation: Subtype: Link Rect: 267, 506, 274, 515 ActionDest: 500 Annotation: Subtype: Link Rect: 275, 506, 282, 515 ActionDest: 500 Annotation: Subtype: Link Rect: 370, 418, 377, 427 ActionDest: 500 Annotation: Subtype: Link Rect: 293, 406, 300, 415 ActionDest: 500 Annotation: Subtype: Link Rect: 198, 368, 210, 377 ActionDest: 500 Annotation: Subtype: Link Rect: 210, 368, 222, 377 ActionDest: 500 Annotation: Subtype: Link Rect: 513, 368, 525, 377 ActionDest: 500 Annotation: Subtype: Link Rect: 434, 343, 446, 352 ActionDest: 500 Annotation: Subtype: Link Rect: 260, 331, 272, 339 ActionDest: 500 Annotation: Subtype: Link Rect: 473, 328, 480, 340 ActionDest: 79 Annotation: Subtype: Link Rect: 272, 318, 284, 327 ActionDest: 503 Annotation: Subtype: Link Rect: 518, 318, 530, 327 ActionDest: 503 Annotation: Subtype: Link Rect: 276, 305, 288, 314 ActionDest: 503 Annotation: Subtype: Link Rect: 392, 305, 404, 314 ActionDest: 503 Annotation: Subtype: Link Rect: 544, 305, 556, 314 ActionDest: 503 Annotation: Subtype: Link Rect: 354, 230, 366, 239 ActionDest: 503 Annotation: Subtype: Link Rect: 283, 202, 290, 214 ActionDest: 79 Annotation: Subtype: Link Rect: 305, 205, 317, 214 ActionDest: 503 Annotation: Subtype: Link Rect: 318, 167, 330, 176 ActionDest: 503 Annotation: Subtype: Link Rect: 497, 167, 509, 176 ActionDest: 503 Annotation: Subtype: Link Rect: 423, 130, 434, 139 ActionDest: 503 Annotation: Subtype: Link Rect: 437, 130, 449, 139 ActionDest: 503 Annotation: Subtype: Link Rect: 243, 89, 250, 101 ActionDest: 79 Annotation: Subtype: Link Rect: 264, 92, 276, 101 ActionDest: 503 Annotation: Subtype: Link Rect: 544, 79, 556, 88 ActionDest: 503 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 250, 333, 257, 345 ActionDest: 79 Annotation: Subtype: Link Rect: 343, 323, 355, 332 ActionDest: 503 Annotation: Subtype: Link Rect: 549, 308, 556, 320 ActionDest: 79 Annotation: Subtype: Link Rect: 434, 273, 446, 282 ActionDest: 503 Annotation: Subtype: Link Rect: 299, 185, 311, 194 ActionDest: 503 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 376, 660, 388, 669 ActionDest: 503 Annotation: Subtype: Link Rect: 439, 647, 451, 657 ActionDest: 503 Annotation: Subtype: Link Rect: 532, 635, 544, 644 ActionDest: 503 Annotation: Subtype: Link Rect: 545, 635, 557, 644 ActionDest: 503 Annotation: Subtype: Link Rect: 308, 622, 320, 631 ActionDest: 503 Annotation: Subtype: Link Rect: 321, 622, 333, 631 ActionDest: 503 Annotation: Subtype: Link Rect: 418, 572, 430, 581 ActionDest: 506 Annotation: Subtype: Link Rect: 422, 560, 434, 569 ActionDest: 506 Annotation: Subtype: Link Rect: 224, 547, 236, 556 ActionDest: 506 Annotation: Subtype: Link Rect: 363, 448, 375, 457 ActionDest: 506 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 423, 256, 434, 265 ActionDest: 506 Annotation: Subtype: Link Rect: 429, 72, 441, 80 ActionDest: 506 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 394, 591, 406, 600 ActionDest: 506 Annotation: Subtype: Link Rect: 454, 273, 466, 282 ActionDest: 503 Annotation: Subtype: Link Rect: 405, 84, 417, 94 ActionDest: 506 Annotation: Subtype: Link Rect: 531, 72, 543, 81 ActionDest: 506 Annotation: Subtype: Link Rect: 543, 72, 555, 81 ActionDest: 506 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 404, 735, 416, 744 ActionDest: 506 Annotation: Subtype: Link Rect: 221, 723, 233, 732 ActionDest: 506 Annotation: Subtype: Link Rect: 444, 723, 456, 732 ActionDest: 506 Annotation: Subtype: Link Rect: 210, 685, 222, 694 ActionDest: 506 Annotation: Subtype: Link Rect: 455, 597, 467, 606 ActionDest: 506 Annotation: Subtype: Link Rect: 200, 371, 212, 380 ActionDest: 506 Annotation: Subtype: Link Rect: 398, 371, 410, 380 ActionDest: 506 Annotation: Subtype: Link Rect: 419, 271, 431, 280 ActionDest: 506 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 352, 302, 366, 314 ActionDest: 255 Page: Sequence: 9 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 544, 282, 556, 291 ActionDest: 503 Annotation: Subtype: Link Rect: 363, 257, 375, 266 ActionDest: 503 Annotation: Subtype: Link Rect: 537, 242, 544, 254 ActionDest: 79 Annotation: Subtype: Link Rect: 522, 207, 534, 216 ActionDest: 503 Annotation: Subtype: Link Rect: 288, 116, 295, 128 ActionDest: 139 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 204, 721, 211, 732 ActionDest: 139 Annotation: Subtype: Link Rect: 342, 472, 349, 484 ActionDest: 79 Annotation: Subtype: Link Rect: 422, 436, 434, 446 ActionDest: 506 Annotation: Subtype: Link Rect: 403, 409, 410, 421 ActionDest: 139 Annotation: Subtype: Link Rect: 452, 113, 464, 122 ActionDest: 506 Annotation: Subtype: Link Rect: 435, 76, 447, 85 ActionDest: 506 Page: Sequence: 12 Annotations: Annotation: Subtype: Link Rect: 465, 723, 476, 732 ActionDest: 506 Annotation: Subtype: Link Rect: 199, 695, 206, 707 ActionDest: 102 Annotation: Subtype: Link Rect: 422, 647, 434, 656 ActionDest: 506 Annotation: Subtype: Link Rect: 499, 632, 506, 644 ActionDest: 242 Annotation: Subtype: Link Rect: 514, 635, 526, 644 ActionDest: 506 Annotation: Subtype: Link Rect: 509, 425, 519, 432 ActionDest: 506 Annotation: Subtype: Link Rect: 228, 396, 238, 404 ActionDest: 506 Annotation: Subtype: Link Rect: 544, 396, 554, 404 ActionDest: 506 Annotation: Subtype: Link Rect: 448, 348, 459, 357 ActionDest: 506 Annotation: Subtype: Link Rect: 340, 322, 352, 332 ActionDest: 503 Annotation: Subtype: Link Rect: 255, 233, 262, 244 ActionDest: 242 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 201, 644, 216, 656 ActionDest: 242 Annotation: Subtype: Link Rect: 245, 619, 252, 631 ActionDest: 245 Annotation: Subtype: Link Rect: 427, 360, 434, 372 ActionDest: 79 Annotation: Subtype: Link Rect: 276, 262, 288, 271 ActionDest: 506 Annotation: Subtype: Link Rect: 321, 259, 328, 271 ActionDest: 248 Annotation: Subtype: Link Rect: 339, 224, 351, 233 ActionDest: 506 Annotation: Subtype: Link Rect: 425, 224, 437, 233 ActionDest: 506 Annotation: Subtype: Link Rect: 544, 212, 556, 221 ActionDest: 503 Annotation: Subtype: Link Rect: 200, 197, 207, 209 ActionDest: 79 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 341, 557, 347, 569 ActionDest: 248 Annotation: Subtype: Link Rect: 249, 547, 261, 556 ActionDest: 506 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 545, 735, 556, 744 ActionDest: 503 Annotation: Subtype: Link Rect: 353, 657, 360, 669 ActionDest: 255 Annotation: Subtype: Link Rect: 334, 105, 346, 114 ActionDest: 506 Page: Sequence: 16 Annotations: Annotation: Subtype: Link Rect: 207, 735, 219, 744 ActionDest: 509 Annotation: Subtype: Link Rect: 274, 720, 280, 732 ActionDest: 285 Annotation: Subtype: Link Rect: 274, 264, 281, 276 ActionDest: 328 Annotation: Subtype: Link Rect: 465, 241, 477, 251 ActionDest: 509 Annotation: Subtype: Link Rect: 497, 151, 504, 163 ActionDest: 328 Page: Sequence: 17 Annotations: Annotation: Subtype: Link Rect: 218, 107, 225, 119 ActionDest: 360 Page: Sequence: 18 Annotations: Annotation: Subtype: Link Rect: 214, 735, 226, 744 ActionDest: 500 Annotation: Subtype: Link Rect: 202, 137, 209, 149 ActionDest: 402 Page: Sequence: 19 Annotations: Annotation: Subtype: Link Rect: 532, 404, 539, 416 ActionDest: 402 Page: Sequence: 20 Annotations: Annotation: Subtype: Link Rect: 500, 695, 507, 707 ActionDest: 245 Annotation: Subtype: Link Rect: 314, 518, 321, 530 ActionDest: 413 Page: Sequence: 21 Annotations: Annotation: Subtype: Link Rect: 224, 707, 236, 716 ActionDest: 509 Annotation: Subtype: Link Rect: 247, 695, 259, 704 ActionDest: 506 Annotation: Subtype: Link Rect: 298, 629, 305, 641 ActionDest: 441 Annotation: Subtype: Link Rect: 199, 620, 211, 629 ActionDest: 509 Annotation: Subtype: Link Rect: 199, 592, 206, 604 ActionDest: 441 Annotation: Subtype: Link Rect: 541, 579, 548, 591 ActionDest: 441 Page: Sequence: 22 Annotations: Annotation: Subtype: Link Rect: 515, 707, 527, 716 ActionDest: 509 Annotation: Subtype: Link Rect: 200, 654, 207, 666 ActionDest: 472 Annotation: Subtype: Link Rect: 332, 629, 339, 641 ActionDest: 472 Annotation: Subtype: Link Rect: 536, 151, 560, 162 Annotation: Subtype: Link Rect: 165, 138, 424, 151 Annotation: Subtype: Link Rect: 466, 105, 477, 113 ActionDest: 509 Annotation: Subtype: Link Rect: 479, 105, 490, 113 ActionDest: 509 Page: Sequence: 23 Annotations: Annotation: Subtype: Link Rect: 232, 392, 270, 402 Annotation: Subtype: Link Rect: 519, 379, 557, 391 Annotation: Subtype: Link Rect: 59, 369, 95, 379 Annotation: Subtype: Link Rect: 519, 345, 557, 356 Annotation: Subtype: Link Rect: 59, 334, 95, 344 Annotation: Subtype: Link Rect: 278, 311, 316, 321 Annotation: Subtype: Link Rect: 322, 311, 358, 321 Annotation: Subtype: Link Rect: 261, 287, 299, 298 Annotation: Subtype: Link Rect: 305, 287, 341, 298 Annotation: Subtype: Link Rect: 59, 265, 96, 275 Annotation: Subtype: Link Rect: 188, 242, 226, 252 Annotation: Subtype: Link Rect: 369, 218, 406, 229 Annotation: Subtype: Link Rect: 104, 196, 142, 206 Annotation: Subtype: Link Rect: 59, 173, 96, 183 Annotation: Subtype: Link Rect: 59, 139, 96, 149 Annotation: Subtype: Link Rect: 102, 139, 138, 149 Annotation: Subtype: Link Rect: 59, 104, 96, 114 Annotation: Subtype: Link Rect: 140, 81, 178, 91 Annotation: Subtype: Link Rect: 184, 81, 220, 91 Annotation: Subtype: Link Rect: 149, 58, 187, 68 Annotation: Subtype: Link Rect: 193, 58, 229, 68 Page: Sequence: 24 Annotations: Annotation: Subtype: Link Rect: 368, 733, 405, 744 Annotation: Subtype: Link Rect: 411, 733, 447, 744 Annotation: Subtype: Link Rect: 150, 711, 188, 721 Annotation: Subtype: Link Rect: 331, 687, 368, 698 Annotation: Subtype: Link Rect: 279, 664, 317, 675 Annotation: Subtype: Link Rect: 519, 641, 557, 652 Annotation: Subtype: Link Rect: 430, 606, 468, 617 Annotation: Subtype: Link Rect: 104, 573, 141, 583 Annotation: Subtype: Link Rect: 147, 573, 183, 583 Annotation: Subtype: Link Rect: 519, 549, 557, 560 Annotation: Subtype: Link Rect: 59, 538, 95, 548 Annotation: Subtype: Link Rect: 226, 504, 263, 514 Annotation: Subtype: Link Rect: 269, 504, 305, 514 Annotation: Subtype: Link Rect: 256, 468, 294, 479 Annotation: Subtype: Link Rect: 300, 468, 336, 479 Annotation: Subtype: Link Rect: 226, 434, 264, 445 Annotation: Subtype: Link Rect: 517, 411, 554, 422 Annotation: Subtype: Link Rect: 95, 377, 133, 387 Annotation: Subtype: Link Rect: 157, 343, 194, 353 Annotation: Subtype: Link Rect: 188, 308, 225, 318 Annotation: Subtype: Link Rect: 348, 284, 385, 295 Annotation: Subtype: Link Rect: 131, 262, 168, 272 Annotation: Subtype: Link Rect: 175, 262, 211, 272 Annotation: Subtype: Link Rect: 301, 238, 337, 249 Annotation: Subtype: Link Rect: 284, 215, 322, 226 Annotation: Subtype: Link Rect: 328, 215, 364, 226 Annotation: Subtype: Link Rect: 302, 192, 340, 203 Annotation: Subtype: Link Rect: 346, 192, 382, 203 Annotation: Subtype: Link Rect: 95, 159, 133, 169 Annotation: Subtype: Link Rect: 157, 136, 194, 146 Annotation: Subtype: Link Rect: 140, 101, 177, 111 Annotation: Subtype: Link Rect: 243, 66, 280, 77 Page: Sequence: 25 Annotations: Annotation: Subtype: Link Rect: 456, 710, 493, 721 Annotation: Subtype: Link Rect: 109, 676, 146, 687 Annotation: Subtype: Link Rect: 413, 652, 451, 664 Annotation: Subtype: Link Rect: 457, 652, 493, 664 Annotation: Subtype: Link Rect: 216, 607, 254, 617 Annotation: Subtype: Link Rect: 324, 572, 362, 583 Annotation: Subtype: Link Rect: 368, 572, 404, 583 Annotation: Subtype: Link Rect: 247, 549, 285, 560 Annotation: Subtype: Link Rect: 403, 537, 523, 548 Annotation: Subtype: Link Rect: 500, 514, 537, 525 Annotation: Subtype: Link Rect: 520, 503, 557, 514 Annotation: Subtype: Link Rect: 95, 423, 133, 433 Annotation: Subtype: Link Rect: 286, 377, 323, 387 Annotation: Subtype: Link Rect: 200, 354, 238, 364 Annotation: Subtype: Link Rect: 374, 330, 412, 341 Annotation: Subtype: Link Rect: 256, 307, 293, 318 Annotation: Subtype: Link Rect: 299, 307, 335, 318 Annotation: Subtype: Link Rect: 104, 285, 141, 295 Annotation: Subtype: Link Rect: 147, 285, 183, 295 Annotation: Subtype: Link Rect: 246, 262, 284, 272 Annotation: Subtype: Link Rect: 109, 239, 146, 249 Annotation: Subtype: Link Rect: 430, 227, 560, 238 Annotation: Subtype: Link Rect: 56, 215, 75, 226 Annotation: Subtype: Link Rect: 59, 182, 96, 192 Annotation: Subtype: Link Rect: 519, 112, 556, 123 Annotation: Subtype: Link Rect: 273, 100, 560, 111 Annotation: Subtype: Link Rect: 56, 89, 431, 100 Annotation: Subtype: Link Rect: 462, 66, 500, 77 Page: Sequence: 26 Annotations: Annotation: Subtype: Link Rect: 118, 711, 155, 721 Annotation: Subtype: Link Rect: 133, 676, 171, 687 Annotation: Subtype: Link Rect: 228, 653, 266, 664 Annotation: Subtype: Link Rect: 519, 629, 557, 640 Annotation: Subtype: Link Rect: 59, 619, 95, 629 Annotation: Subtype: Link Rect: 454, 596, 491, 606 Annotation: Subtype: Link Rect: 236, 573, 273, 583 Annotation: Subtype: Link Rect: 279, 573, 315, 583 Annotation: Subtype: Link Rect: 519, 560, 557, 571 Annotation: Subtype: Link Rect: 59, 550, 95, 560 Annotation: Subtype: Link Rect: 359, 537, 396, 548 Annotation: Subtype: Link Rect: 242, 514, 279, 525 Checksum: 71eee483 Type: CRC32 Checksum: d035f48d75b586c47b3aa1bca57148cb Type: MD5 Checksum: c8316ecbe367beb8a073ba504c9dd42e44b7abe5 Type: SHA-1 Checksum: 0f41c2e514554533e74f3ea5901a9bc0977d7328dd19c1b92d48f898da4dd8c1 Type: SHA-256