IL28B SNP rs12979860 Is a Critical Predictor for On-Treatment and Sustained Virologic Response in Patients with Hepatitis C Virus Genotype-1 Infection

Single nucleotide polymorphisms (SNPs) of interleukin-28B (IL28B) have received considerable interest for their association with sustained virological response (SVR) when treating patients of genotype-1 hepatitis C virus (GT1-HCV) chronic infection with pegylated interferon and ribavirin (PegIFN/RBV). This study was to investigate the predictive power of IL28B SNPs for on-treatment responses and SVR in treatment-naïve patients with GT1-HCV chronic infection.We analyzed ten SNPs of IL28B in 191 treatment-naïve patients with GT1-HCV chronic infection who received PegIFN/RBV. In these patients, rapid virological response (RVR), early virological response (EVR) and SVR were achieved in 69.6%, 95.8% and 68.6% of the patients, respectively. Multivariate analysis (odds ratio; 95% confidence interval; P value) indicated age (0.96; 0.93-0.99; 0.012), low baseline viral load (4.65; 2.23-9.66; <0.001) and CC genotype of rs12979860 (7.74; 2.55-23.53; <0.001) but no other SNPs were independent predictors for SVR. In addition, none of the ten SNPs examined were associated with baseline viral load and stages of liver fibrosis. Regarding RVR, low baseline viral load (2.83; 1.40-5.73; 0.004) and CC genotype of rs12979860 (10.52; 3.45-32.04; <0.001) were two critical predictors. As for EVR, only CC genotype of rs12979860 (36.21; 6.68-196.38; <0.001) was the predictor. Similarly, for end of treatment response (ETR), CC genotype of rs12979860 (15.42; 4.62-51.18; <0.001) was the only predictor. For patients with RVR, only low baseline viral load (3.90; 1.57-9.68; 0.003) could predict the SVR. For patients without RVR, only rs12979860 (4.60; 1.13-18.65; 0.033) was the predictor for SVR.rs12979860 is the critical predictor for RVR, EVR, ETR and SVR in treatment-naïve patients of GT1-HCV chronic infection. Furthermore, this SNP is the only predictor for SVR in patients without RVR. These results have provided evidence that rs12979860 is the ideal IL28B SNP for genetic testing in treating patients of GT1-HCV chronic infection

Bioisosteres of phosphate and pyrophosphate ligands as inhibitors of therapeutic targets

Part IHuman farnesyl pyrophosphate synthase (hFPPS) represents a critical enzyme which controls the first branch point of mevalonate pathway. It catalyzes the consecutive condensations of dimethylallyl pyrophosphate (DMAPP) with two molecules of isopentenyl pyrophosphate (IPP) to generate farnesyl pyrophosphate (FPP), a vital metabolite for protein prenylation. Nitrogen-containing bisphosphonates (N-BPs), a class of hFPPS inhibitors targeting the allylic sub-pocket, have been approved for the treatment of bone resorptive diseases. They also exhibit direct and indirect anti-cancer effects in vitro and are considered as potential anti-malignant agents. However, they suffer from poor pharmaceutical properties, such as low bioavailability and tissue distributions, due to their negatively charged nature. In this study, several approaches have been explored to identify hFPPS inhibitors with improved biopharmaceutical properties. Increasing the overall lipophility of the inhibitors by modifying them with hydrophobic moieties was first investigated. Some biological experiments were performed to demonstrate their inhibition activity, anti-proliferation effects, and potential mechanism of action. X-ray crystallography was employed to reveal the novel structural rearrangements of hFPPS upon binding with one of our potent inhibitors. Moreover, less charged hFPPS allosteric inhibitors, such as sulfonylphosphoryamidic and monophosphonate derivatives, were explored as well. Several analogs were shown to exhibit moderate activity toward inhibiting hFPPS and considered as potential novel hits for further investigations. Furthermore, employment of pro-drug strategies to improve the biopharmaceutical characters is also discussed. Part IIPyrophosphate (PPi) is the by-product generated during nucleotide polymerization. Foscarnet, a PPi bioisostere, has been shown to inhibit HIV reverse transcriptase (RT) by trapping the HIV RT/DNA complex in the pre-translocational state. The bisphoshphonate moiety is structurally similar to PPi and represents a potential pharmacophore for the design of HIV RT inhibitors. Hybrids of the bisphosphonate moiety and pyridopyrimidines, that are known purine mimics, were designed as potential HIV RT inhibitors. Several analogs were shown to inhibit DNA polymerization catalyzed by RT at low micromolar ranges and considered as potential new chemotypes for further investigation.Partie ILa synthase farnésyl-pyrophosphate humaine (FPPSh) représente une enzyme essentielle qui contrôle le premier point de dérivation de la voie du mévalonate. Elle catalyse les condensations consécutives du diméthylallyl-pyrophosphate (DMAPP) avec deux molécules d'isopentényl-pyrophosphate (IPP) afin de produire la farnésyl-pyrophosphate (FPP), un métabolite nécessaire pour la prénylation de plusieurs protéines. Les bisphosphonates contenants un groupe d'azote (N-BPs), une famille des inhibiteurs qui visent la sous-poche allylique de la FPPSh, ont été approuvés pour le traitement des maladies qui causent la résorption des os. Ils présentent aussi un effet direct ainsi qu'indirect anti-cancer in Vitro et sont considérés comme des agents anticancéreux potentiels. Cependant, ils ont un mauvais profile pharmaceutique, ainsi qu'une mauvaise biodisponibilité et exposition des tissus, en raison de leur nature anionique. Dans cette étude, plusieurs façons ont été explorées pour identifier les inhibiteurs de la FPPSh avec des propriétés biopharmaceutiques améliorées. Premièrement, une investigation pour rendre les inhibiteurs moins polairs en ajoutant des groupes hydrophobiques a été poursuivie. Puis, des expérimentes biologiques pour démontrer leur efficacité d'inhibition, effet anti-prolifération, et mécanisme d'action potentielle. La cristallographie à rayons X a été utilisée pour démontrer le changement en conformation de la FPPSh occupé par notre inhibiteur plus actif. En plus, des inhibiteurs de la FPPSh moins anioniques, comme des dérivatifs sulfonophosphoamidiques et monophosphonatiques, ont été explorés. Quelques analogues ont été démontrés d'avoir une inhibition modérée vers FPPSh et pouvant être considérés comme inhibiteurs potentiels avec plus d'investigation. En outre, l'emploi d'une approche prodrogue pour améliorer les propriétés biopharmaceutiques a été faite. Partie IILa pyrophosponate (PPi) est un sous-produit généré dans la polymérisation des nucléotides. Le foscarnet, une bioisostère de la PPi, a été prouvé de terminer la polymerisation des nucleotides par la transcriptase inverse (RT) VIH en trappant la complexe RT VIH/ADN dans l'état pretranslocational. La bisphosphonate ressemble à la structure de la PPi et représente une pharmacophore potentielle pour la conception des nouveaux inhibiteurs pour la RT. Les hybrides du groupe bisphosphonate avec les dérivatifs pyridopyrimidines, les mimiques purines, ont été conçus comme des inhibiteurs potentiels de la RT VIH. Quelques analogues ont été démontrés pour inhibir la polymérisation de l'ADN catalysé par RT VIH dans le genre faible micromole et sont considérés comme des échafaudages potentiels pour des futures investigations

Development of glycan-based Paclitaxel prodrugs for targeting delivery to cancer cells

紫杉醇是一種由太平洋紫杉的樹皮中萃取出來的抗癌藥物,已經被應用於臨床治療。然而,由於紫杉醇缺乏對癌細胞的專一性,在治療癌症的同時也會傷害到其他的正常組織。這項缺點限制了紫杉醇在癌症治療上的廣泛運用。具有專一傳輸性質的前驅藥物將能解決這項缺點。前驅藥物以低毒性的形態在體內傳送,在到達目的地後,它的毒性才會被釋放出來,而達到專一消滅癌細胞的效果。 在這篇論文中,我們設計合成了四種分別以葡萄糖和葡萄糖醛酸做為傳輸載體的前驅藥物。這四種藥物的設計概念是基於葡萄糖傳輸孔道與葡萄糖醛酸水解酶在癌細胞上的過度表現。在這四種前驅藥物中,以prodrug 1對人類癌症細胞株(NCI-H838, Hep-3B, A498, MES-SA, HCT-116, NPC-TW01,和MKN-45)具有最高的毒殺性質,僅較紫杉醇低二至十六倍。除此之外,我們也測試了這些藥物對正常細胞株(HUV-EC-C和CHO-K1)的細胞毒性。除了prodrug 4之外,我們的醣化前驅藥物對癌細胞都展現了高度的選擇性。 在本研究中,我們也證明了prodrug 1確實可以被癌細胞所攝取。為了追蹤藥物,我們在紫杉醇的C7上加上了一個螢光基團—間位胺基苯甲酸(m-aminobenzoyl group)。經過五小時的藥物處理後,該藥物出現於細胞質中。這表示癌細胞可以在短時間內將該藥物吸收。若將該藥物施予癌症病患,將可能降低藥物被正常細胞吸收的機率,以減緩化學治療所帶來的副作用。 當細胞處理藥物二十四小時後,我們觀察到微質管(microtubule)在細胞內累積聚集的現象。我們利用可移除式的連接鍊(linker)暫時抑止這些前驅藥物的毒性。我們假設在醣基被切除後,連接鍊尾端的官能基將攻擊連接紫杉醇活性區的羧酸基(carboxylic group),並釋放出紫杉醇以執行毒殺癌細胞的功能。由雷射掃描共軛焦顯微鏡的影像顯示,無論細胞被處理紫杉醇或醣化前驅藥物,都有微質管累積的情形。這項結果支持了我們先前所提出的藥物釋放假說。 我們所設計的前驅藥物也具有與紫杉醇相似的另一項生物活性—促使細胞凋亡(apoptosis)。相對於壞死(necrosis),細胞凋亡是一種較為溫和的細胞死亡方式。為了驗證醣化前驅藥物是否將引發細胞凋亡,我們利用核染色的方式觀察染色體的型態。當細胞凋亡時,它的染色體會開始濃縮、聚集。當細胞處理我們的前驅藥物後也出現這個現象。這顯示前驅藥物是藉由引發細胞凋亡的路徑造成細胞死亡。 總結而論,我們已設計合成出四種醣化紫杉醇前驅藥物並測試其生物活性。這些藥物造成細胞內的微質管累積聚集並引發細胞凋亡以達到殺死癌細胞的藥理效果。我們也利用螢光標定prodrug 1的方式證明了該藥物確實可以在短時間內被癌細胞所攝取。在這四種藥物中,以prodrug 1的毒性與癌細胞專一性最佳,未來將做更深入的研究與探討。Paclitaxel (Taxol), a natural diterpene compound, is a promising anticancer drug that was shown substantial clinical efficacy to solid tumor. However, like other cytotoxic agent, taxol lacks of selectivity and contributes to serious side effects. The conversion of taxol into prodrugs that can be specifically delivered to cancer cells may prevent this shortcoming. In this study, we designed and synthesized four glycan-based paclitaxel prodrugs. Compound 1 and 2 are glucose-based prodrugs; and 3 and 4 are glucuronic acid-based ones. Given the high level expressions of glucose transporters and -glucuronidase in carcinomas, there is rationale to develop glycan-targeting prodrugs. Within these four compounds, prodrug 1 has the best cytotoxicity against seven human tumor cell lines (NCI-H838, Hep-3B, A498, MES-SA, HCT-116, NPC-Two1, and MKN-45). The cytotoxicity of prodrug 1 is only 2 to 16-fold lower than taxol. In addition, we also tested the toxicity of these drugs to normal cell lines (HUV-EC-C and CHO-K1). Except prodrug 4, the IC50 of other compounds are more than 100 μM. Our prodrugs showed good cytotoxicity and selectivity to cancer cells. We also confirmed the internalization of prodrugs 1 into cancer cells. In order to trace this drug, we added a chromophore, m-aminobenzoyl group, in C7 position of taxol skeleton. After five hours treatment, the drug was seen in the plasma regions of NPC-TW01 cells. This result suggested that glucose-based prodrug 1 can be taken up by cancer cells within short time treatment. By means of rapid internalization by cancer cells, our prodrug may ease the side effects when administrating to patients. Microtubule aggregation was observed in NPC-TW01 cells after stimulation with these prodrugs for 24 hours. In our prodrugs, the biological properties of taxol are masked temporarily by blocking its active site with a self-immolative linker. We except that paclitaxel will be liberated after glycan removal and spacer autocleavage. In confocoal images, microtubules in cells treated with taxol or prodrugs were observed to lose silk structures. This result supported the hypothesis that our prodrugs can be converted into taxol and interference the dynamics of microtubule. Like taxol, our prodrugs can cause cell death through apoptosis. In contrast to necrosis, apoptosis is a milder cell death pathway. In order to avoid inflammation caused by necrosis, we must demonstrate that these drugs kill cells in apoptosis pathway. Chromosome condensation is a distinct character of programmed cell death; and we found this phenomenon in cells treated with these four compound. This result indicated that apoptosis is the major death mechanism triggered by our prodrugs. In summary, we have synthesized four glycan-based paclitaxel prodrugs and analyzed their biological characters. These prodrugs showed antitumor effect on promoting microtubule assembly and inducing apoptosis. Within these four drugs, prodrug 1 has the best cytotoxicity and selectivity to cancer cells according to the result of cytotoxicity assay. Furthermore, prodrug 1 was also proved to internalize into cells after five-hour treatment. This compound is a potential candidate for further investigation.Acknowledge i Abstract (Chinese) ii Abstract (English) iv 1. Introduction 1 2. Result 2.1 Chemistry 2.1.1. Synthesis of prodrug 1 and 2 5 2.1.2. Synthesis of prodrug 3 and 4 6 2.1.3. Synthesis of fluorescence-labeled taxol (21) 8 2.2 Biology 2.2.1. Cytotoxicity of prodrugs 1, 2, 3, and 4 to cancer and normal cell lines 9 2.2.2. Internalize of fluorescence-label drugs in NPC-TW01 cell lines 11 2.2.3. Confocoal microscopy analysis of NPC-TW01 cells which were treated with prodrugs 1-4 11 3. Experimental 3.1. Materials 13 3.2. Chemistry 3.2.1. Succinic acid mono-(8-benzyloxy-6-methoxy-2-phenyl-hexahydro-pyrano [3,2-d][1,3]dioxin-7-yl) ester(5) 14 3.2.2. C15 (6) 15 3.2.3. Prodrug 1 (1) 16 3.2.4. Benzoic acid 4-(8-benzyloxy-6-methoxy-2-phenyl-hexahydro-pyrano[3,2-d] [1,3]dioxin-7-yloxy)-butyl ester (7) 18 3.2.5. 4-(8-Benzyloxy-6-methoxy-2-phenyl-hexahydro-pyrano[3,2-d] [1,3]dioxin-7-yloxy)-butan-1-ol (8) 19 3.2.6. 4-(8-Benzyloxy-6-methoxy-2-phenyl-hexahydro-pyrano[3,2-d] [1,3]dioxin-7-yloxy)-butyric acid (9) 20 3.2.7. Taxol-ether-C14 (10) 21 3.2.8. Prodrug (2) 23 3.2.9. Benzoic acid 8-benzyloxy-6-methoxy-2-phenyl-hexahydro-pyrano[3,2-d] [1,3]dioxin-7-yl ester (11) 25 3.2.10. Benzoic acid 4,5-bis-benzyloxy-6-hydroxymethyl-2-methoxy-tetrahydro- pyran-3-yl ester (12) 26 3.2.11. 3,4-Bis-benzyloxy-5-hydroxy-6-methoxy-tetrahydro-pyran- 2-carboxylic acid benzyl ester (13) 27 3.2.12. Succinic acid mono-(4,5-bis-benzyloxy-6-benzyloxycarbonyl-2-methoxy- tetrahydro-pyran-3-yl) ester (14) 28 3.2.13. Glucronic-ester-taxol (15) 29 3.2.14. Prodrug 3 (3) 31 3.2.15. Benzoic acid 4-(4,5-bis-benzyloxy-6-hydroxymethyl-2-methoxy-tetrahydro- pyran-3-yloxy)-butyl ester (16) 33 3.2.16. 3,4-Bis-benzyloxy-5-(3-carboxy-propoxy)-6-methoxy- tetrahydro-pyran-2-carboxylic acid (17) 34 3.2.17. Glucronic-ether-taxol (18) 35 3.2.18. Prodrug 4 (4) 37 3.2.19. Taxol-2’TES (19) 39 3.2.20. Taxol-2’TES-7F (20) 40 3.2.21. Taxol-2’OH-7F synthesis (21) 41 3.2.22. Fluoresce-labeled prodrug 1 (22) 43 3.3 Biology 3.3.1. Cell proliferation assay 44 3.3.2. Fluorescence microscopy 45 3.3.3. Confocoal microscopy 46 4. Discussion 47 5. References 52 6. Schemes 55 7. Figures 62 8. Tables 70 9. Appendix 7

None of the six SNPs of IL28B could predict treatment responses in genotype 2 chronic HCV infected patients by propensity score matching analysis.

Background & aimsA combination of pegylated interferon-alpha and ribavirin (PR) is the standard therapy for patients with chronic hepatitis C. The impact of polymorphism of interleukin-28B (IL28B) on sustained virological response (SVR) to PR has been well documented in patients with CHC genotype-1 (GT1), but it is controversial in genotype-2 (GT2) CHC patients. This study investigated the predictability of six single nucleotide polymorphisms (SNP) of IL28B on the treatment responses of PR in patients with CHC GT2.Method197 CHC GT2 consecutive patients who received PR treatment in our prospective cohort were enrolled. Hepatitis C virus (HCV) genotyping, quantification of HCV-RNA and genotyping of the ten SNPs of IL28B were performed. Six SNPs of IL28B were chosen for analysis. The propensity score matching (PSM) analysis was applied using patients with CHC GT1 in another prospective cohort as a positive comparison to avoid covariate bias.ResultsThe distribution of the six SNPs was similar in GT1 and GT2 patients. Five of these SNPs had strong association with treatment responses in GT1 but not in GT2 patients. After PSM analysis, these five SNPs still showed strong association with rapid virological response (RVR), cEVR and SVR in GT1 and had no influence in GT2 patients. Furthermore, rs12979860 and baseline viral load were the predictors for both RVR and SVR in GT1 patients. However, only baseline viral load could predict RVR and SVR in GT2 patients. In addition, in patients without RVR, rs12979860 was the only predictor for SVR in GT1 but no predictor for SVR was found in GT2.ConclusionsThe genetic polymorphisms of IL28B had no impact on treatment responses in GT2 patients

Patients younger than forty years old with hepatitis C virus genotype–1 chronic infection had treatment responses similar to genotype–2 infection and not related to interleukin–28B polymorphism

Background and rationale. Age is one of the predictors for sustained virological response (SVR) when treating chronic hepatitis C (CHC) patients with pegylated-interferon/ribavirin (PegIFN/RBV). However, the treatment responses of the young patients had not been analyzed before. Therefore, we conducted this study to investigate the treatment responses of CHC patients younger than 40 years old (y/o).Material and methods. We retrospectively analyzed our prospective cohort of genotype 1 (GT1)- and genotype 2 (GT2)-CHC patients who received 24-week PegIFN/RBV treatment. We divided these patients into two groups according to their age younger or older than 40 y/o. Clinical parameters including viral responses and single nucleotide polymorphisms (SNPs) of interleukin–28B (IL28B) had been analyzed.Results. In GT1-CHC patients, the rapid, complete early viral response rates and the SVR rate were significantly higher in patients younger than 40 y/o. In GT–1 CHC patients younger than 40 y/o, the SVR rate was similar to the GT2-CHC patients, either with high or low baseline viral load. As for the SVR predictors, in CHC patients younger than 40 y/o, only BMI but not the genotype of HCV, not baseline viral load, and not IL28B SNP was the predictor.Conclusions. GT1-CHC patients younger than 40 y/o had SVR rate similar to GT2-CHC patients. The IL28B polymorphism had no impact on the SVR rate in these young GT1-CHC patients

Modular Assembly of Purine-like Bisphosphonates as Inhibitors of HIV‑1 Reverse Transcriptase

Bisphosphonates can mimic the pyrophosphate leaving group of the nucleotidyl transfer reaction and effectively inhibit RNA/DNA polymerases. In a search of HIV-1 reverse transcriptase (RT) inhibitors, a new chemotype of nonhydrolyzable purine diphosphate mimic was synthesized. A modular synthetic protocol was developed, utilizing 2-amino-6-(methylthio)-4-(trimethylsilyl)­nicotinonitrile as the key synthon in the preparation of highly substituted 2-aminonicotinonitriles. These building blocks were subsequently elaborated to the pyrido­[2,3-<i>d</i>]­pyrimidine bisphosphonates (PYPY-BPs). Biochemical screening identified analogs of PYPY-BPs that inhibit HIV-1 RT-catalyzed DNA synthesis