0548ab4becf4d13f2f6186abd2106741 pmc_34681257.pdf e243ab23063a3e1358adbbe1668e167a31b5c224 pmc_34681257.pdf 7f3eff18585491c6d9248314f82782be5152f1bcab71981877de30f5d35f9b8d pmc_34681257.pdf Title: Development of a Topical Amphotericin B and Bursera graveolens Essential Oil-Loaded Gel for the Treatment of Dermal Candidiasis Subject: The higher molecular weight and low solubility of amphotericin B (AmB) hinders its topical administration. The aim of this study was to incorporate Bursera graveolens essential oil into an AmB topical gel (AmB + BGEO gel) in order to promote the diffusion of the drug through the skin in the treatment of cutaneous candidiasis. AmB + BGEO gel formulation was determined using a factorial experiment. Physical and chemical parameters, stability, in vitro release profile and ex vivo permeation in human skin were evaluated. In vitro antimicrobial activity was studied using strains of C. albicans, C. glabrata and C. parapsilosis. The tolerability was evaluated using in vitro and in vivo models. AmB + BGEO gel presented appropriate characteristics for topical administration, including pH of 5.85, pseudoplastic behavior, optimal extensibility, as well as high stability and acceptable tolerability. In vitro release studies showed that the formulation releases the drug following a Boltzmann sigmoidal model. Finally, AmB + BGEO gel exhibited higher amount of drug retained inside the skin and lower Minimum Inhibitory Concentration than a formulation sans essential oil. Therefore, these results suggest that the incorporation of B. graveolens essential oil in the formulation could be used as strategy to promote a local effect in the treatment of cutaneous candidiasis. Keywords: amphotericin B; candidiasis; gel; essential oil; Bursera graveolens; topical treatment Author: Lupe Carolina Espinoza, Lilian Sosa, Paulo C. Granda, Nuria Bozal, Natalia Díaz-Garrido, Brenda Chulca-Torres and Ana Cristina Calpena Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Oct 12 07:37:33 2021 CEST ModDate: Tue Oct 12 07:39:22 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 15 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2556317 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- AZBFJT+VnURWPalladioL Type 1 Custom yes yes no 10 0 XLAFBF+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 JWFJLG+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 RWULST+URWPalladioL-BoldItal Type 1 Custom yes yes no 24 0 DBQQJJ+URWPalladioL-Roma Type 1 Custom yes yes no 28 0 WLENKM+VnURWPalladioL-Bold Type 1 Custom yes yes no 32 0 VWRBXB+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 40 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 62 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 66 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 70 0 KVEXVN+CMSY10 Type 1 Builtin yes yes no 82 0 ZXDKIP+EURM10 Type 1 Builtin yes yes no 86 0 ODFGMH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 96 0 ODFGMJ+PalatinoLinotype TrueType WinAnsi yes yes no 99 0 ODFGNK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 102 0 ODFGMH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 111 0 ODFGMI+PalatinoLinotype CID TrueType Identity-H yes yes yes 114 0 ODFGMJ+PalatinoLinotype TrueType WinAnsi yes yes no 120 0 ODFGNK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 123 0 ODFGMH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 131 0 ODFGMI+PalatinoLinotype CID TrueType Identity-H yes yes yes 134 0 ODFGMJ+PalatinoLinotype TrueType WinAnsi yes yes no 140 0 ODFGNK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 143 0 ZPGVCH+CMEX10 Type 1 Builtin yes yes no 151 0 GIGFZE+CMR10 Type 1 Builtin yes yes no 155 0 ODFGMH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 164 0 ODFGMJ+PalatinoLinotype TrueType WinAnsi yes yes no 167 0 ODFGNK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 170 0 ODFJDP+CambriaMath CID TrueType Identity-H yes yes yes 173 0 XIRVNA+EURB10 Type 1 Builtin yes yes no 183 0 ODFJKN+Arial CID TrueType Identity-H yes yes yes 190 0 ODFGMH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 196 0 ODFGMJ+PalatinoLinotype TrueType WinAnsi yes yes no 199 0 ODFGNK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 202 0 ODFHGJ+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 205 0 ODFJKN+Arial CID TrueType Identity-H yes yes yes 212 0 ODFGMH+PalatinoLinotype,Bold TrueType WinAnsi yes yes no 218 0 ODFGMJ+PalatinoLinotype TrueType WinAnsi yes yes no 221 0 ODFGNK+PalatinoLinotype,Italic TrueType WinAnsi yes yes no 224 0 ODFHGJ+PalatinoLinotype,BoldItalic TrueType WinAnsi yes yes no 227 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2022-03-22 02:39:41 CET RepresentationInformation: pmc_34681257.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-01-10 08:30:39 CET Size: 2556317 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 458 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Children: Item: Title: Solubility Studies Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Design and Preparation of AmB + BGEO Gel Formulation Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Characterization of AmB + BGEO Gel Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Stability Studies Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: In Vitro Release Study Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Ex Vivo Permeation Study Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Efficacy Study: Antimicrobial Activity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Tolerance Studies Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: Cytotoxicity Studies by MTT Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: In Vivo Tolerance Studies Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Children: Item: Title: Materials Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: High-Performance Liquid Chromatography (HPLC) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Solubility Studies Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: Formulation: Design and Analysis of 23 Factorial Experiment Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Characterization of AmB + BGEO Gel Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Stability Studies Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: In Vitro Release Study Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Ex Vivo Permeation Study Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Efficacy Study: Antimicrobial Activity Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Tolerance Studies Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Children: Item: Title: Cytotoxicity Studies by MTT Method Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: In Vivo Tolerance Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Info: Title: Development of a Topical Amphotericin B and Bursera graveolens Essential Oil-Loaded Gel for the Treatment of Dermal Candidiasis Author: Lupe Carolina Espinoza, Lilian Sosa, Paulo C. Granda, Nuria Bozal, Natalia Díaz-Garrido, Brenda Chulca-Torres and Ana Cristina Calpena Subject: The higher molecular weight and low solubility of amphotericin B (AmB) hinders its topical administration. The aim of this study was to incorporate Bursera graveolens essential oil into an AmB topical gel (AmB + BGEO gel) in order to promote the diffusion of the drug through the skin in the treatment of cutaneous candidiasis. AmB + BGEO gel formulation was determined using a factorial experiment. Physical and chemical parameters, stability, in vitro release profile and ex vivo permeation in human skin were evaluated. In vitro antimicrobial activity was studied using strains of C. albicans, C. glabrata and C. parapsilosis. The tolerability was evaluated using in vitro and in vivo models. AmB + BGEO gel presented appropriate characteristics for topical administration, including pH of 5.85, pseudoplastic behavior, optimal extensibility, as well as high stability and acceptable tolerability. In vitro release studies showed that the formulation releases the drug following a Boltzmann sigmoidal model. Finally, AmB + BGEO gel exhibited higher amount of drug retained inside the skin and lower Minimum Inhibitory Concentration than a formulation sans essential oil. Therefore, these results suggest that the incorporation of B. graveolens essential oil in the formulation could be used as strategy to promote a local effect in the treatment of cutaneous candidiasis. Keywords: amphotericin B; candidiasis; gel; essential oil; Bursera graveolens; topical treatment Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Tue Oct 12 07:37:33 CEST 2021 ModDate: Tue Oct 12 07:39:22 CEST 2021 ID: 0x13b3cccffe176ae3399421bfa5101105, 0x13b3cccffe176ae3399421bfa5101105 Filters: FilterPipeline: FlateDecode Fonts: Type1: Font: BaseFont: WLENKM+VnURWPalladioL-Bold FontSubset: true FirstChar: 189 LastChar: 189 FontDescriptor: FontName: WLENKM+VnURWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: 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: JWFJLG+URWPalladioL-Bold FontSubset: true FirstChar: 3 LastChar: 122 FontDescriptor: FontName: JWFJLG+URWPalladioL-Bold Flags: Symbolic FontBBox: -152, -301, 1000, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: ZXDKIP+EURM10 FontSubset: true FirstChar: 11 LastChar: 22 FontDescriptor: FontName: ZXDKIP+EURM10 Flags: Symbolic FontBBox: -32, -243, 1060, 720 FontFile: true Font: BaseFont: ZPGVCH+CMEX10 FontSubset: true FirstChar: 18 LastChar: 19 FontDescriptor: FontName: ZPGVCH+CMEX10 Flags: Symbolic FontBBox: -24, -2960, 1454, 772 FontFile: true Font: BaseFont: XIRVNA+EURB10 FontSubset: true FirstChar: 22 LastChar: 22 FontDescriptor: FontName: XIRVNA+EURB10 Flags: Symbolic FontBBox: -30, -247, 1178, 723 FontFile: true Font: BaseFont: RWULST+URWPalladioL-BoldItal FontSubset: true FirstChar: 66 LastChar: 118 FontDescriptor: FontName: RWULST+URWPalladioL-BoldItal Flags: Symbolic FontBBox: -170, -300, 1073, 935 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: AZBFJT+VnURWPalladioL FontSubset: true FirstChar: 161 LastChar: 243 FontDescriptor: FontName: AZBFJT+VnURWPalladioL Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: GIGFZE+CMR10 FontSubset: true FirstChar: 40 LastChar: 61 FontDescriptor: FontName: GIGFZE+CMR10 Flags: Symbolic FontBBox: -40, -250, 1009, 750 FontFile: true Font: BaseFont: DBQQJJ+URWPalladioL-Roma FontSubset: true FirstChar: 2 LastChar: 252 FontDescriptor: FontName: DBQQJJ+URWPalladioL-Roma Flags: Symbolic FontBBox: -166, -283, 1021, 943 FontFile: true EncodingDictionary: Differences: true Font: BaseFont: XLAFBF+URWPalladioL-Ital FontSubset: true FirstChar: 2 LastChar: 122 FontDescriptor: FontName: XLAFBF+URWPalladioL-Ital Flags: Symbolic FontBBox: -170, -305, 1010, 941 FontFile: true EncodingDictionary: Differences: true Pages: Page: Sequence: 1 Annotations: Annotation: Subtype: Link Rect: 35, 759, 167, 795 Annotation: Subtype: Link Rect: 515, 759, 560, 795 Annotation: Subtype: Link Rect: 157, 643, 168, 656 Annotation: Subtype: Link Rect: 322, 643, 333, 656 Annotation: Subtype: Link Rect: 397, 643, 408, 656 Annotation: Subtype: Link Rect: 521, 643, 532, 656 Annotation: Subtype: Link Rect: 280, 630, 291, 644 Annotation: Subtype: Link Rect: 54, 346, 154, 355 Annotation: Subtype: Link Rect: 35, 334, 72, 343 Annotation: Subtype: Link Rect: 35, 162, 97, 184 Annotation: Subtype: Link Rect: 153, 89, 154, 98 Annotation: Subtype: Link Rect: 35, 77, 147, 86 Annotation: Subtype: Link Rect: 387, 143, 394, 152 ActionDest: 250 Annotation: Subtype: Link Rect: 441, 130, 448, 139 ActionDest: 250 Annotation: Subtype: Link Rect: 448, 130, 455, 139 ActionDest: 250 Annotation: Subtype: Link Rect: 208, 105, 215, 114 ActionDest: 250 Annotation: Subtype: Link Rect: 539, 92, 546, 102 ActionDest: 250 Annotation: Subtype: Link Rect: 549, 93, 556, 102 ActionDest: 250 Annotation: Subtype: Link Rect: 142, 17, 275, 28 Annotation: Subtype: Link Rect: 376, 17, 558, 28 Annotation: Subtype: Link Rect: 35, 458, 87, 477 Page: Sequence: 2 Annotations: Annotation: Subtype: Link Rect: 314, 724, 321, 733 ActionDest: 250 Annotation: Subtype: Link Rect: 324, 724, 336, 733 ActionDest: 250 Annotation: Subtype: Link Rect: 220, 661, 232, 670 ActionDest: 250 Annotation: Subtype: Link Rect: 268, 599, 280, 608 ActionDest: 250 Annotation: Subtype: Link Rect: 280, 598, 292, 608 ActionDest: 250 Annotation: Subtype: Link Rect: 347, 586, 359, 595 ActionDest: 250 Annotation: Subtype: Link Rect: 360, 586, 371, 595 ActionDest: 250 Annotation: Subtype: Link Rect: 237, 548, 249, 557 ActionDest: 250 Annotation: Subtype: Link Rect: 359, 523, 371, 532 ActionDest: 250 Annotation: Subtype: Link Rect: 544, 511, 556, 520 ActionDest: 250 Annotation: Subtype: Link Rect: 404, 473, 416, 482 ActionDest: 253 Annotation: Subtype: Link Rect: 282, 435, 294, 444 ActionDest: 253 Annotation: Subtype: Link Rect: 206, 410, 218, 419 ActionDest: 253 Annotation: Subtype: Link Rect: 219, 410, 231, 419 ActionDest: 253 Annotation: Subtype: Link Rect: 223, 373, 235, 382 ActionDest: 253 Annotation: Subtype: Link Rect: 544, 347, 556, 356 ActionDest: 253 Annotation: Subtype: Link Rect: 185, 310, 197, 319 ActionDest: 253 Annotation: Subtype: Link Rect: 197, 310, 209, 319 ActionDest: 253 Annotation: Subtype: Link Rect: 458, 285, 470, 294 ActionDest: 253 Annotation: Subtype: Link Rect: 470, 285, 482, 294 ActionDest: 253 Annotation: Subtype: Link Rect: 196, 247, 208, 256 ActionDest: 253 Annotation: Subtype: Link Rect: 263, 235, 275, 244 ActionDest: 253 Annotation: Subtype: Link Rect: 530, 222, 542, 231 ActionDest: 253 Annotation: Subtype: Link Rect: 542, 222, 554, 231 ActionDest: 253 Annotation: Subtype: Link Rect: 471, 118, 478, 130 ActionDest: 90 Page: Sequence: 3 Annotations: Annotation: Subtype: Link Rect: 213, 534, 221, 546 ActionDest: 90 Annotation: Subtype: Link Rect: 222, 225, 229, 237 ActionDest: 90 Page: Sequence: 4 Annotations: Annotation: Subtype: Link Rect: 311, 666, 318, 678 ActionDest: 105 Annotation: Subtype: Link Rect: 515, 430, 522, 442 ActionDest: 105 Annotation: Subtype: Link Rect: 460, 187, 467, 199 ActionDest: 148 Page: Sequence: 5 Annotations: Annotation: Subtype: Link Rect: 219, 612, 226, 624 ActionDest: 148 Annotation: Subtype: Link Rect: 444, 161, 451, 173 ActionDest: 180 Page: Sequence: 6 Annotations: Annotation: Subtype: Link Rect: 388, 548, 395, 560 ActionDest: 180 Annotation: Subtype: Link Rect: 316, 287, 323, 299 ActionDest: 180 Page: Sequence: 7 Annotations: Annotation: Subtype: Link Rect: 544, 719, 556, 728 ActionDest: 253 Annotation: Subtype: Link Rect: 424, 694, 436, 703 ActionDest: 250 Annotation: Subtype: Link Rect: 441, 591, 448, 603 ActionDest: 90 Annotation: Subtype: Link Rect: 259, 569, 271, 578 ActionDest: 253 Annotation: Subtype: Link Rect: 415, 543, 427, 553 ActionDest: 253 Annotation: Subtype: Link Rect: 353, 518, 365, 527 ActionDest: 253 Annotation: Subtype: Link Rect: 365, 518, 377, 527 ActionDest: 253 Annotation: Subtype: Link Rect: 232, 493, 244, 502 ActionDest: 253 Annotation: Subtype: Link Rect: 284, 466, 291, 478 ActionDest: 105 Annotation: Subtype: Link Rect: 314, 466, 321, 478 ActionDest: 105 Annotation: Subtype: Link Rect: 421, 443, 433, 452 ActionDest: 253 Annotation: Subtype: Link Rect: 311, 405, 323, 414 ActionDest: 253 Annotation: Subtype: Link Rect: 195, 328, 202, 339 ActionDest: 148 Annotation: Subtype: Link Rect: 349, 305, 361, 314 ActionDest: 253 Annotation: Subtype: Link Rect: 458, 290, 464, 302 ActionDest: 148 Annotation: Subtype: Link Rect: 406, 242, 418, 251 ActionDest: 253 Annotation: Subtype: Link Rect: 312, 205, 324, 214 ActionDest: 253 Annotation: Subtype: Link Rect: 202, 179, 214, 189 ActionDest: 253 Annotation: Subtype: Link Rect: 215, 179, 227, 189 ActionDest: 253 Annotation: Subtype: Link Rect: 345, 79, 357, 88 ActionDest: 253 Page: Sequence: 8 Annotations: Annotation: Subtype: Link Rect: 526, 722, 538, 731 ActionDest: 253 Annotation: Subtype: Link Rect: 538, 722, 550, 731 ActionDest: 253 Annotation: Subtype: Link Rect: 321, 622, 333, 631 ActionDest: 253 Annotation: Subtype: Link Rect: 421, 572, 433, 581 ActionDest: 253 Annotation: Subtype: Link Rect: 244, 534, 256, 543 ActionDest: 253 Annotation: Subtype: Link Rect: 259, 534, 271, 543 ActionDest: 256 Annotation: Subtype: Link Rect: 381, 421, 393, 430 ActionDest: 256 Annotation: Subtype: Link Rect: 346, 358, 358, 367 ActionDest: 253 Annotation: Subtype: Link Rect: 358, 358, 370, 367 ActionDest: 256 Annotation: Subtype: Link Rect: 347, 244, 359, 253 ActionDest: 253 Page: Sequence: 9 Annotations: Annotation: Subtype: Link Rect: 473, 719, 485, 728 ActionDest: 256 Annotation: Subtype: Link Rect: 376, 450, 383, 462 ActionDest: 237 Annotation: Subtype: Link Rect: 387, 453, 399, 462 ActionDest: 256 Annotation: Subtype: Link Rect: 390, 300, 402, 310 ActionDest: 256 Annotation: Subtype: Link Rect: 402, 300, 414, 310 ActionDest: 256 Annotation: Subtype: Link Rect: 313, 260, 320, 272 ActionDest: 237 Page: Sequence: 10 Annotations: Annotation: Subtype: Link Rect: 253, 421, 265, 430 ActionDest: 256 Annotation: Subtype: Link Rect: 379, 318, 394, 330 ActionDest: 237 Annotation: Subtype: Link Rect: 336, 245, 348, 254 ActionDest: 256 Annotation: Subtype: Link Rect: 336, 217, 351, 229 ActionDest: 240 Annotation: Subtype: Link Rect: 419, 66, 434, 78 ActionDest: 237 Page: Sequence: 11 Annotations: Annotation: Subtype: Link Rect: 472, 431, 487, 443 ActionDest: 237 Annotation: Subtype: Link Rect: 398, 232, 410, 241 ActionDest: 256 Annotation: Subtype: Link Rect: 411, 232, 423, 241 ActionDest: 256 Page: Sequence: 12 Page: Sequence: 13 Annotations: Annotation: Subtype: Link Rect: 520, 449, 557, 460 Annotation: Subtype: Link Rect: 59, 416, 97, 426 Annotation: Subtype: Link Rect: 103, 416, 139, 426 Annotation: Subtype: Link Rect: 259, 380, 297, 391 Annotation: Subtype: Link Rect: 303, 380, 339, 391 Annotation: Subtype: Link Rect: 196, 357, 233, 368 Annotation: Subtype: Link Rect: 239, 357, 275, 368 Annotation: Subtype: Link Rect: 402, 334, 439, 345 Annotation: Subtype: Link Rect: 446, 334, 482, 345 Annotation: Subtype: Link Rect: 519, 323, 557, 334 Annotation: Subtype: Link Rect: 59, 312, 95, 322 Annotation: Subtype: Link Rect: 159, 288, 197, 299 Annotation: Subtype: Link Rect: 292, 265, 330, 276 Annotation: Subtype: Link Rect: 520, 254, 557, 265 Annotation: Subtype: Link Rect: 227, 231, 265, 242 Annotation: Subtype: Link Rect: 59, 197, 97, 207 Annotation: Subtype: Link Rect: 103, 197, 139, 207 Annotation: Subtype: Link Rect: 59, 151, 97, 161 Annotation: Subtype: Link Rect: 95, 128, 133, 138 Annotation: Subtype: Link Rect: 59, 105, 97, 115 Annotation: Subtype: Link Rect: 103, 105, 139, 115 Annotation: Subtype: Link Rect: 420, 81, 457, 92 Annotation: Subtype: Link Rect: 387, 58, 425, 69 Annotation: Subtype: Link Rect: 431, 58, 467, 69 Page: Sequence: 14 Annotations: Annotation: Subtype: Link Rect: 59, 710, 97, 720 Annotation: Subtype: Link Rect: 394, 686, 432, 697 Annotation: Subtype: Link Rect: 309, 663, 347, 674 Annotation: Subtype: Link Rect: 470, 640, 508, 651 Annotation: Subtype: Link Rect: 514, 640, 550, 651 Annotation: Subtype: Link Rect: 243, 618, 281, 628 Annotation: Subtype: Link Rect: 316, 594, 354, 605 Annotation: Subtype: Link Rect: 269, 560, 307, 571 Annotation: Subtype: Link Rect: 232, 526, 269, 536 Annotation: Subtype: Link Rect: 259, 503, 296, 513 Annotation: Subtype: Link Rect: 194, 469, 232, 479 Annotation: Subtype: Link Rect: 507, 445, 544, 456 Annotation: Subtype: Link Rect: 520, 422, 557, 433 Annotation: Subtype: Link Rect: 165, 400, 202, 410 Annotation: Subtype: Link Rect: 209, 400, 245, 410 Annotation: Subtype: Link Rect: 229, 365, 266, 375 Annotation: Subtype: Link Rect: 272, 365, 308, 375 Annotation: Subtype: Link Rect: 159, 342, 197, 352 Annotation: Subtype: Link Rect: 247, 307, 284, 318 Annotation: Subtype: Link Rect: 352, 272, 389, 283 Annotation: Subtype: Link Rect: 477, 249, 515, 260 Annotation: Subtype: Link Rect: 521, 249, 557, 260 Annotation: Subtype: Link Rect: 140, 227, 178, 237 Annotation: Subtype: Link Rect: 95, 204, 133, 214 Annotation: Subtype: Link Rect: 139, 204, 175, 214 Annotation: Subtype: Link Rect: 363, 180, 401, 191 Annotation: Subtype: Link Rect: 407, 180, 443, 191 Annotation: Subtype: Link Rect: 436, 157, 474, 168 Annotation: Subtype: Link Rect: 240, 134, 277, 145 Annotation: Subtype: Link Rect: 504, 111, 541, 122 Annotation: Subtype: Link Rect: 494, 88, 531, 99 Annotation: Subtype: Link Rect: 307, 65, 345, 76 Page: Sequence: 15 Annotations: Annotation: Subtype: Link Rect: 307, 721, 345, 732 Annotation: Subtype: Link Rect: 376, 698, 413, 709 Annotation: Subtype: Link Rect: 160, 676, 198, 686 Annotation: Subtype: Link Rect: 223, 641, 261, 651 Annotation: Subtype: Link Rect: 378, 617, 416, 628 Annotation: Subtype: Link Rect: 397, 594, 435, 605 Annotation: Subtype: Link Rect: 333, 537, 370, 548 Annotation: Subtype: Link Rect: 377, 537, 413, 548 Annotation: Subtype: Link Rect: 342, 479, 379, 490 Annotation: Subtype: Link Rect: 385, 479, 421, 490 Checksum: 5a4addb9 Type: CRC32 Checksum: 0548ab4becf4d13f2f6186abd2106741 Type: MD5 Checksum: e243ab23063a3e1358adbbe1668e167a31b5c224 Type: SHA-1