IC50 Ratio Research Articles

KARAKTERISTIK FISIK, KIMIA, MIKROBIOLOGI WHEY KEFIR DAN AKTIVITASNYA TERHADAP PENGHAMBATAN ANGIOTENSIN CONVERTING ENZYME (ACE)

This study was conducted to evaluate the characteristics of whey-based kefir products and their activity to inhibit the angiotensin converting enzyme (ACE). Kefir was produced by using many types of whey, namely SK: skim milk based kefir (control); WK: gouda cheese whey based kefir; and WKB: commercial whey powder based kefir. The experimental design was a completely randomized design. Each treatment was conducted in triplicates. Kefirs were evaluated for physical and chemical properties (pH, total titratable acidity, viscosity, protein, fat, lactose, and alcohol), microbiological (lactic acid bacteria and yeast) population, peptide concentration, ACE inhibition, IC50 and Inhibition Efficiency Ratio (IER). The results showed that the types of whey used for kefir productions significantly affected the physical and chemical characteristics of the products (p 0.05). The peptide concentration and ACE inhibitory activity of WK, 1.54±0.02 mg/mL and 73.07±0.91%, was significantly higher (p 0.05) from the control (47.19±0.09% per mg/mL) but was significantly higher (p<0.05) than that of WKB (45.75±0.18% per mg/mL). This research indicated that whey kefir is a potential source of bioactive peptide for antihypertention agent.

A Multifactorial Approach to Hepatobiliary Transporter Assessment Enables Improved Therapeutic Compound Development

The bile salt export pump (BSEP) is expressed at the canalicular domain of hepatocytes, where it serves as the primary route of elimination for monovalent bile acids (BAs) into the bile canaliculi. The most compelling evidence linking dysfunction in BA transport with liver injury in humans is found with carriers of mutations that render BSEP nonfunctional. Based on mounting evidence, there appears to be a strong association between drug-induced BSEP interference and liver injury in humans; however, causality has not been established. For this reason, drug-induced BSEP interference is best considered a susceptibility factor for liver injury as other host- or drug-related properties may contribute to the development of hepatotoxicity. To better understand the association between BSEP interference and liver injury in humans, over 600 marketed or withdrawn drugs were evaluated in BSEP expressing membrane vesicles. The example of a compound that failed during phase 1 human trials is also described, AMG 009. AMG 009 showed evidence of liver injury in humans that was not predicted by preclinical safety studies, and BSEP inhibition was implicated. For 109 of the drugs with some effect on in vitro BSEP function, clinical use, associations with hepatotoxicity, pharmacokinetic data, and other information were annotated. A steady state concentration (C(ss)) for each of these annotated drugs was estimated, and a ratio between this value and measured IC₅₀ potency values were calculated in an attempt to relate exposure to in vitro potencies. When factoring for exposure, 95% of the annotated compounds with a C(ss)/BSEP IC₅₀ ratio ≥ 0.1 were associated with some form of liver injury. We then investigated the relationship between clinical evidence of liver injury and effects to multidrug resistance-associated proteins (MRPs) believed to play a role in BA homeostasis. The effect of 600+ drugs on MRP2, MRP3, and MRP4 function was also evaluated in membrane vesicle assays. Drugs with a C(ss)/BSEP IC₅₀ ratio ≥ 0.1 and a C(ss)/MRP IC₅₀ ratio ≥ 0.1 had almost a 100% correlation with some evidence of liver injury in humans. These data suggest that integration of exposure data, and knowledge of an effect to not only BSEP but also one or more of the MRPs, is a useful tool for informing the potential for liver injury due to altered BA transport.

Effect of CYP3A5 Expression on the Inhibition of CYP3A-Catalyzed Drug Metabolism: Impact on Modeling CYP3A-Mediated Drug-Drug Interactions

The purpose of this study was to determine the impact of CYP3A5 expression on inhibitory potency (Ki or IC50 values) of CYP3A inhibitors, using recombinant CYP3A4 and CYP3A5 (rCYP3A4 and rCYP3A5) and CYP3A5 genotyped human liver microsomes (HLMs). IC50 ratios between rCYP3A4 and rCYP3A5 (rCYP3A5/rCYP3A4) of ketoconazole (KTZ) and itraconazole (ITZ) were 8.5 and 8.8 for midazolam (MDZ), 4.7 and 9.1 for testosterone (TST), 1.3 and 2.8 for terfenadine, and 0.6 and 1.7 for vincristine, respectively, suggesting substrate- and inhibitor-dependent selectivity of the two azoles. Due to the difference in the IC50 values for CYP3A4 and CYP3A5, nonconcordant expression of CYP3A4 and CYP3A5 protein can significantly affect the observed magnitude of CYP3A-mediated drug-drug interactions in humans. Indeed, the IC50 values of KTZ and ITZ for CYP3A-catalyzed MDZ and TST metabolism were significantly higher in HLMs with CYP3A5*1/*1 and CYP3A5*1/*3 genotypes than in HLMs with the CYP3A5*3/*3 genotype, showing CYP3A5 expression-dependent IC50 values. Moreover, when IC50 values of KTZ and ITZ for different HLMs were kinetically simulated based on CYP3A5 expression level and enzyme-specific IC50 values, a good correlation between the simulated and the experimentally measured IC50 values was observed. Further simulation analysis revealed that both the Ki ratio (for inhibitors) and Vmax/Km ratio (for substrates) between CYP3A4 and CYP3A5 were major factors for CYP3A5 expression-dependent IC50 values. In conclusion, the present study demonstrates that CYP3A5 genotype and expression level have a significant impact on inhibitory potency for CYP3A-catalyzed drug metabolism, but that the magnitude of its effect is inhibitor-substrate pair specific.

Abstract 990: Efficacy of various chemotherapeutic agents on 5-FU-resistant human gastric cancer cell lines and their mechanisms.

Abstract Purpose: Four 5-fluorouracil (5-FU)-resistant human gastric cancer cell lines were established by the long-term exposure of parent cell lines (MKN45, MKN-74, KATO-III, and NCI-N87) to 5-FU. The cytotoxicity and mechanisms of several chemotherapeutic agents were then investigated using 5-FU-resistant gastric cancer cell lines. Method: 5-FU-resistant cell lines were established by continuous exposure to escalating concentrations (1-5 μM) of 5-FU over a 1-year period. The sensitivities of the cell lines to chemotherapeutic agents (paclitaxel, docetaxel, cisplatin, oxaliplatin, epirubicin, and SN-38) were then evaluated using the WST-8 colorimetric assay. To elucidate the mechanisms of resistance, the gene expressions and copy numbers were measured using an Agilent Technologies whole human genome oligo DNA microarray and a CGH microarray. The protein and mRNA expressions of a target enzyme of 5-FU, thymidylate synthase (TS), were also investigated using western blotting and RT-PCR, respectively. Results: The IC50 ratios of the 5-FU-resistant cell lines were 2.6 to 15.6 times higher than those of the parent cell lines in vitro. MKN-45/5-FU showed cross-resistance to cisplatin and oxaliplatin and KATO-III/5-FU showed cross-resistance to SN-38, while MKN-74/5-FU showed collateral sensitivities to docetaxel, cisplatin, and oxaliplatin, and NCI-N87/5-FU showed collateral sensitivities to cisplatin, oxaliplatin, SN-38, and epirubicin. RT-PCR showed that TS mRNA expression was higher in the parent cell lines than in the 5-FU-resistant cell lines, and the TS protein expression level was also higher in all the resistant cell lines. For MKN-45/5-FU and KATO-III/5-FU, not only the mRNA expression level but also the TS gene copy number was increased. Interestingly, the expression of MAP3K15, which is an apoptosis-facilitating factor, increased in NCI-N87/5-FU, which showed collateral sensitivity to cisplatin, oxaliplatin, SN-38, and epirubicin. Conclusion: Analyzing the molecular mechanisms of 5-FU-resistant cell lines may be useful for the development of effective chemotherapy regimens for relapsed gastric cancer after 5-FU-based chemotherapy. Citation Format: Mamoru Nukatsuka, Takashi Kobunai, Ayako Nakamura, Kazuhiko Hayashi, Teiji Takechi. Efficacy of various chemotherapeutic agents on 5-FU-resistant human gastric cancer cell lines and their mechanisms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 990. doi:10.1158/1538-7445.AM2013-990

ASP6537, a novel highly selective cyclooxygenase-1 inhibitor, exerts potent antithrombotic effect without “aspirin dilemma”

IntroductionAspirin inhibits both the cyclooxygenase (COX)-1-dependent production of thromboxane A2 (TXA2) in platelets and COX-2-dependent production of anti-aggregatory prostaglandin I2 (PGI2) in vessel walls, resulting in “aspirin dilemma.” Our objective is to investigate whether ASP6537 can overcome aspirin dilemma and exert a potent antithrombotic effect without a concurrent ulcerogenic effect. MethodsWe evaluated the inhibitory effects of ASP6537 on recombinant human COX-1 (rhCOX-1) and rhCOX-2 activities using a COX-1/2 selectivity test. To determine whether ASP6537 induces aspirin dilemma, we examined the effects of ASP6537 on in vitro TXA2 and PGI2 metabolite production from platelets and isolated aorta of guinea pigs, and on plasma concentrations of TXA2 and PGI2 metabolites in aged rats. Finally, we evaluated the antithrombotic effects and ulcerogenic activity of ASP6537 using an electrically induced carotid arterial thrombosis model and a gastric ulcer model in guinea pigs. ResultsThe IC50 ratios of rhCOX-2 to rhCOX-1 for ASP6537 and aspirin were >142,000 and 1.63 fold, respectively. ASP6537 inhibited TXA2 production more selectively than aspirin in in vitro and in vivo TXA2/PGI2 production studies. ASP6537 exerted a significant antithrombotic effect at ≥3mg/kg, while aspirin tended to inhibit thrombosis at 300mg/kg but it was not statistically significant. Further, ASP6537 did not induce ulcer formation at 100mg/kg, whereas aspirin exhibited an ulcerogenic effect at doses of ≥100mg/kg. ConclusionsASP6537 functions as a highly selective COX-1 inhibitor with a superior ability to aspirin for normalizing TXA2/PGI2 balance, and exerts antithrombotic effect without ulcerogenic effect.

Antimalarial activities of medicinal plants and herbal formulations used in Thai traditional medicine

Malaria is one of the world's leading killer infectious diseases with high incidence and morbidity. The problem of multidrug-resistant Plasmodium falciparum has been aggravating particularly in Southeast Asia. Therefore, development of new potential antimalarial drugs is urgently required. The present study aimed to investigate antimalarial activities of a total of 27 medicinal plants and 5 herbal formulations used in Thai traditional medicine against chloroquine-resistant (K1) and chloroquine-sensitive (3D7) P. falciparum clones. Antimalarial activity of the ethanolic extracts of all plants/herbal formulations against K1 and 3D7 P. falciparum clones was assessed using SYBR Green I-based assay. All plants were initially screened at the concentration of 50 μg/ml to select the candidate plants that inhibited malaria growth by ≥50%. Each candidate plant was further assessed for the IC50 value (concentration that inhibits malaria growth by 50%) to select the potential plants. Selectivity index (SI) of each extract was determined from the IC50 ratio obtained from human renal epithelial cell and K1 or 3D7 P. falciparum clone. The ethanolic extracts from 19 medicinal plants/herbal formulation exhibited promising activity against both K1 and 3D7 clones of P. falciparum with survival of less than 50% at the concentration of 50 μg/ml. Among these, the extracts from the eight medicinal plants (Plumbago indica Linn., Garcinia mangostana Linn., Dracaena loureiri Gagnep., Dioscorea membranacea Pierre., Artemisia annua Linn., Piper chaba Hunt., Myristica fragrans Houtt., Kaempferia galanga Linn.) and two herbal formulations (Benjakul Formulation 1 and Pra-Sa-Prao-Yhai Formulation) showed potent antimalarial activity with median range IC50 values of less than 10 μg/ml against K1 or 3D7 P. falciparum clone or both. All except G. mangostana Linn. and A. annua Linn. showed high selective antimalarial activity against both clones with SI>10. Further studies on antimalarial activities in an animal model including molecular mechanisms of action of the isolated active moieties are required.

Synthesis and biological evaluation of 1-(2-hydroxy-3-phenyloxypropyl)piperazine derivatives as T-type calcium channel blockers

To obtain selective and potent inhibitor for T-type calcium channel by ligand based drug design, 2-hydroxy-3-phenoxypropyl piperazine derivatives were synthesized and evaluated for in vitro activities. Compound 6m and 6q showed high selectivity over hERG channel (IC50 ratio of hERG/α1G6m=8.5, 6q=18.38) and they were subjected to measure pharmacokinetics profiles. Among them compound 6m showed an excellent pharmacokinetic profile in rats.

Inhibitory Effects of Psychotropic Drugs on the Acetylcholine Receptor-Operated Potassium Current (I&lt;sub&gt;K.ACh&lt;/sub&gt;) in Guinea-Pig Atrial Myocytes

Influences of psychotropic drugs, six antipsychotics and three antidepressants, on acetylcholine receptor-operated potassium current (IK.ACh) were examined by a whole-cell patch clamp method in freshly isolated guinea-pig atrial myocyte. IK.ACh was induced by a superfusion of carbachol (CCh) or by an intracellular application of guanosine 5'-[thio] triphosphate (GTPγS). To elucidate mechanism for anticholinergic action, IC50 ratio, the ratio of IC50 for GTPγS-activated IK.ACh to CCh-induced IK.ACh, was calculated. Antipsychotics and antidepressants inhibited CCh-induced IK.ACh in a concentration-dependent manner. The IC50 values were as follows; chlorpromazine 0.53 μM, clozapine 0.06 μM, fluphenazine 2.69 μM, haloperidol 2.66 μM, sulpiride 42.3 μM, thioridazine 0.07 μM, amitriptyline 0.03 μM, imipramine 0.22 μM and maprotiline 1.81 μM. The drugs, except for sulpiride, inhibited GTPγS-activated IK.ACh with following IC50 values; chlorpromazine 1.71 μM, clozapine 14.9 μM, fluphenazine 3.55 μM, haloperidol 2.73 μM, thioridazine 1.90 μM, amitriptyline 7.55 μM, imipramine 7.09 μM and maprotiline 5.93 μM. The IC50 ratio for fluphenazine and haloperidol was close to unity. The IC50 ratio for chlorpromazine, clozapine, thioridazine, amitriptyline, imipramine and maprotiline was much higher than unity. The present findings suggest that the psychotropics studied suppress IK.ACh. Chlorpromazine, clozapine, thioridazine, amitriptyline, imipramine, maprotiline and sulpiride are preferentially acting on muscarinic receptor. Fluphenazine and haloperidol may act on G protein and/or potassium channel.

Mechanism-based Inhibition of CYP1A1 and CYP3A4 by the Furanocoumarin Chalepensin

The cytochrome P450 (P450, CYP) 2A6 inhibitor chalepensin was found to inhibit human CYP1A1, CYP1A2, CYP2A13, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 to different extents. CYP1A1 and CYP3A4 underwent pronounced mechanism-based inactivation by chalepensin and had the smallest IC50 ratios of inhibition with NADPH-fortified pre-incubation (IC50(+)) to that without pre-incubation (IC50(-)). CYP2E1 had the least susceptibility to mechanism-based inactivation. This inactivation of CYP1A1 and CYP3A4 exhibited time-dependence, led to a loss of spectrophotometrically detected P450, and could not be fully recovered by dialysis. Pre-incubation with chalepensin did not affect NADPH-P450 reductase activity. Cytosol-supported glutathione conjugation protected CYP3A4 but not CYP1A1 against the inactivation by chalepensin. Cytosolic decomposition of chalepensin may contribute partially to the protection. The high epoxidation activities of CYP1A1, CYP2A6, and CYP3A4 were in agreement with their pronounced susceptibilities to mechanism-based inactivation by chalepensin. Considering both the IC50 values and inactivation kinetic parameters, the threshold concentrations of chalepensin for potential drug interactions through inhibition of CYP2A6 and CYP3A4 were estimated to be consistently low. These results demonstrate that chalepensin inhibits multiple P450s and that epoxidation activity is crucial for the potential drug interaction through mechanism-based inhibition.

Glutathione-sensitive nanoplatform for monitored intracellular delivery and controlled release of Camptothecin

We report the design, synthesis, characterization and in vitro testing of a novel nanodrug based on a covalent linking model that allows intracellular controlled release of the pharmaceutical payload. A new synthetic strategy is implemented by direct coupling of as-synthesized (pyridin-2-yldisulfanyl)alkyl carbonate derivatives of camptothecin (CPT) with thiol groups of silica hybrid nanoparticles containing a non-porous core and a mesoporous shell. Upon reaction with thiols in physiological conditions, disulfide bridge cleavage occurs, releasing the naked drug after an intramolecular cyclization mechanism. Additional incorporation of a fluorophore into particles core facilitates imaging at the subcellular level for the monitoring of uptake and delivery. Confocal microscopy experiments in HeLa cervix cancer cells confirms that nanoparticles enter the cells by endocytosis but are able to escape from endo-lysosomes and enter the cytosolic compartment to release their cargo. The incorporation to cells of L-buthionine-sulfoximine, a glutathione inhibitor allows concluding that the intracellular releasing mechanism is mainly driven by the reducing activity of this tripeptide. This camptothecin nanoplatform shows the same cytotoxic activity than the free drug and is clearly superior to those release systems depending on enzymatic hydrolysis (as determined by calculation of the IC50 ratios).

A Small Molecule Inhibitor of Ubiquitin-Specific Protease-7 Induces Apoptosis in Multiple Myeloma Cells and Overcomes Bortezomib Resistance

Bortezomib therapy has proven successful for the treatment of relapsed/refractory, relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit its long-term utility. Here, we show that P5091 is an inhibitor of deubiquitylating enzyme USP7, which induces apoptosis in MM cells resistant to conventional and bortezomib therapies. Biochemical and genetic studies show that blockade of HDM2 and p21 abrogates P5091-induced cytotoxicity. In animal tumor model studies, P5091 is well tolerated, inhibits tumor growth, and prolongs survival. Combining P5091 with lenalidomide, HDAC inhibitor SAHA, or dexamethasone triggers synergistic anti-MM activity. Our preclinical study therefore supports clinical evaluation of USP7 inhibitor, alone or in combination, as a potential MM therapy.

Evaluation of Six Proton Pump Inhibitors As Inhibitors of Various Human Cytochromes P450: Focus on Cytochrome P450 2C19

Six proton pump inhibitors (PPIs), omeprazole, lansoprazole, esomeprazole, dexlansoprazole, pantoprazole, and rabeprazole, were shown to be weak inhibitors of cytochromes P450 (CYP3A4, -2B6, -2D6, -2C9, -2C8, and -1A2) in human liver microsomes. In most cases, IC₅₀ values were greater than 40 μM, except for dexlansoprazole and lansoprazole with CYP1A2 (IC₅₀ = ∼8 μM) and esomeprazole with CYP2C8 (IC₅₀ = 31 μM). With the exception of CYP2C19 inhibition by omeprazole and esomeprazole (IC₅₀ ratio, 2.5 to 5.9), there was no evidence for a marked time-dependent shift in IC₅₀ (IC₅₀ ratio, ≤ 2) after a 30-min preincubation with NADPH. In the absence of preincubation, lansoprazole (IC₅₀ = 0.73 μM) and esomeprazole (IC₅₀ = 3.7 μM) were the most potent CYP2C19 inhibitors, followed by dexlansoprazole and omeprazole (IC₅₀ = ∼7.0 μM). Rabeprazole and pantoprazole (IC₅₀ = ≥ 25 μM) were the weakest. A similar ranking was obtained with recombinant CYP2C19. Despite the IC₅₀ ranking, after consideration of plasma levels (static and dynamic), protein binding, and metabolism-dependent inhibition, it is concluded that omeprazole and esomeprazole are the most potent CYP2C19 inhibitors. This was confirmed after the incubation of the individual PPIs with human primary hepatocytes (in the presence of human serum) and by monitoring their impact on diazepam N-demethylase activity at a low concentration of diazepam (2 μM). Data described herein are consistent with reports that PPIs are mostly weak inhibitors of cytochromes P450 in vivo. However, two members of the PPI class (esomeprazole and omeprazole) are more likely to serve as clinically relevant inhibitors of CYP2C19.

Abstract 2799: BAY 1082439, a highly selective and balanced PI3Kα/β inhibitor demonstrated potent activity in tumors with activated PI3Kα and loss-of-function of PTEN

Abstract The PI3K pathway plays critical roles in cancer cell growth and survival, as well as in intrinsic and acquired resistance to both chemotherapy and targeted agents. These essential roles have led to the clinical development of PI3K pathway inhibitors. Due to the complexity derived from the existence of various PI3K isoforms (≥,α,α,α), and their differential roles in signal transduction, as well as cancer pathology, development of PI3K inhibitors with differential pharmacological profiles would allow exploration in different indications, combinations and dosing regimens. Having identified BAY 80-6946, an intravenously dosed, highly potent and selective PI3K inhibitor which is particularly effective in PIK3CAmut and/or Her2+ tumors, we sought to develop a novel oral PI3K inhibitor particularly effective in PTEN-loss tumors with coexisting mutation or amplification of PIK3CA and/or activation of PI3Kα (e.g., through RTKs). Herein we report the pharmacological profile of a highly selective PI3Kα/α-balanced inhibitor, BAY 1082439. BAY 1082439 has an IC50 ratio of 1:3 in biochemical assays of PI3Kα (4.9 nM) vs. PI3Kα (15.0 nM), and &amp;gt;1000-fold selectivity against mTOR kinase. The balanced PI3Kα and PI3Kα activity of BAY 1082439 is also reflected in cellular mechanistic (p-AKT473) and proliferation assays in PI3Kα- (KPL4, BT474) vs. PI3Kα-driven (PC3, LNCaP) tumor cells. In vivo, BAY 1082439 showed clear advantages over the strong PI3Kα inhibitor BAY 80-6946 in PTEN/PI3Kα-driven tumor models (e.g., PC3 and HEC-1B), when the two compounds were compared at their MTDs. Furthermore, BAY 1082439 has unique pharmacokinetic (PK) properties with very high plasma free fractions across all species tested (33-50%), large Vss, high clearance and intermediate T1/2. The relationship of PK vs. PD and the efficacy vs. dosing regimens were investigated. BAY 1082439 showed strong p-AKT inhibition at 2 and 5 hours post-treatment while p-AKT returned to levels comparable to the vehicle group at 24 hours in all 4 tumor models tested. Interestingly, with once daily dosing, BAY 1082439 could induce tumor regression in KPL4 (PIK3CAmut and HER2+), and tumor stasis in HEC-1B (PTENdel) and in HEC-1A (PIK3CAmut) tumor models, suggesting that continuous inhibition of p-AKT may not be required for anti-tumor efficacy. In addition, comparison of different dosing regimens (QD, Q2D, D1-2/W, D1-3/W, D1-4/W to QW) at MTD indicated that QD and QW dosing produced optimal anti-tumor efficacy. These results support the hypothesis that strong pathway inhibition for a certain time period, rather than maintaining constant inhibition might lead to optimal anti-tumor efficacy along with a maximal therapeutic window. In conclusion, BAY 1082439 represents a new type of PI3K inhibitor with unique pharmacological and pharmacodynamic properties to be further explored in clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2799. doi:1538-7445.AM2012-2799

The dynamics of Aβ distribution after γ-secretase inhibitor treatment, as determined by experimental and modelling approaches in a wild type rat

Inhibition of the enzyme(s) that produce the Amyloid beta (Aβ) peptide, namely BACE and γ-secretase, is considered an attractive target for Alzheimer's disease therapy. However, the optimal pharmacokinetic-pharmacodynamic modelling method to describe the changes in Aβ levels after drug treatment is unclear. In this study, turnover models were employed to describe Aβ levels following treatment with the γ-secretase inhibitor RO5036450, in the wild type rat. Initially, Aβ level changes in the brain, cerebral spinal fluid (CSF) and plasma were modeled as separate biological compartments, which allowed the estimation of a compound IC₅₀ and Aβ turnover. While the data were well described, the model did not take into consideration that the CSF pool of Aβ most likely originates from the brain via the CSF drainage pathway. Therefore, a separate model was carried out, with the assumption that CSF Aβ levels originated from the brain. The optimal model that described the data involved two brain Aβ 40 sub-compartments, one with a rapid turnover, from which CSF Aβ 40 is derived, and a second quasi-static pool of ~20%. Importantly, the estimated in vivo brain IC₅₀ was in a good range of the in vitro IC₅₀ (ratio, 1.4). In conclusion, the PK/PD models presented here are well suited for describing the temporal changes in Aβ levels that occur after treatment with an Aβ lowering drug, and identifying physiological parameters.

Novel hybrid molecules based on 15-membered azalide as potential antimalarial agents

Malaria remains the most prevalent tropical disease, and due to the spread of resistant parasites novel therapeutics are urgently needed. Azithromycin has shown potential in malaria treatment so we designed hybrid azalide molecules with the aim to improve activity against and selectivity for the malaria parasite. Novel hybrid molecules comprising 4-aminoquinoline moiety covalently liked to 15-membered azalide scaffold at position C-3′ were synthesized and biologically evaluated. Antimalarial testing against Plasmodium falciparum sensitive and resistant strains confirmed the improved in vitro activity over azithromycin and chloroquine. Selectivity of the compounds (HepG2 IC50/P. falciparum IC50 ratio) for the parasite was high (100–2700) and their antibacterial activity diminished. Even though oral bioavailability determined for compound 12 was low, novel quinoline C-3′-substituted 15-membered azalides represent an interesting subclass of antimalarial macrolides that need further research and evaluation.

Targeted delivery of a novel palmitylated D-peptide for antiglioblastoma molecular therapy

Effective glioblastoma treatment with low toxicity is one of the most difficult challenges in cancer therapy. The interaction between tumor suppressor protein p53 and its negative regulator murine double minute 2 (MDM2) provides a promising target for specific therapy because an important subtype of glioblastoma harbors wild-type p53 and overexpressed MDM2. Several D-peptides have been previously reported to effectively antagonize MDM2 for binding to p53 with high affinity and unsurpassed specificity. However, poor cell penetration and lack of efficient delivery method hampered the therapeutic applicability of the most potent D-peptide, D-PMIβ. In this study, a novel lipophilic derivate of D-PMIβ (pDP) was developed. Liposome was chosen as a carrier for pDP, and cyclic pentapeptide c(RGDyK) was used as a targeting moiety for the treatment of glioblastoma. D-PMIβ was N-terminally modified with palmitic acid and the resultant c(RGDyK) decorated liposomes (RGD-liposomal pDP) showed almost 100% encapsulation efficiency and 10% loading efficiency. The abilities of palmitylated D-peptide to antagonize MDM2 and reactivate p53 specifically were confirmed by the western blot assay. The IC50 ratio of RGD-liposomal pDP in treating human umbilical vascular endothelial normal cells vs. U87 tumor cells was 10 times higher than that of RGD-liposomal doxorubicin. After intravenous administration, the median survival time of intracranial U87 glioblastoma-bearing nude mice treated with RGD-liposomal pDP (29 days) was significant longer than that of mice treated with blank RGD-liposome (23 days) (p<0.001). These results indicated that palmitylated D-peptide inhibitor of p53-MDM2 combined with RGD modified liposomes provided a potential molecular therapy for glioblastoma.

Targeting Interleukin-4 Receptor α with Hybrid Peptide for Effective Cancer Therapy

Interleukin-4 receptor α (IL-4Rα) chain is highly expressed on the surface of various human solid tumors. We designed a novel hybrid peptide termed IL-4Rα-lytic peptide that targets the IL-4Rα chain. The IL-4Rα-lytic peptide contains a target moiety to bind to IL-4Rα and a cellular toxic lytic peptide that selectively kills cancer cells. The anticancer activity of the IL-4Rα-lytic peptide was evaluated in vitro and in vivo. It was found that the IL-4Rα-lytic peptide has cytotoxic activity in cancer cell lines expressing IL-4Rα, determined by quantitative real-time PCR. The IC(50) ratios of the lytic peptide to the IL-4Rα-lytic peptide correlated well with the expression levels of IL-4Rα on cancer cells (r = 0.80). In addition, IL-4Rα-lytic peptide administered either intratumoraly or intravenously significantly inhibited tumor growth in xenograft model of human pancreatic cancer (BXPC-3) in mice. These results indicate that the IL-4Rα-lytic peptide generated in this study has a potent and selective anticancer potential against IL-4Rα-positive solid cancers.

Development of QSAR model for prediction of fold selectivity of phenyl benzoxazole as estrogen receptor inhibitors

QSAR model development of 37 phenyl benzoxazole was carried out to predict the fold selectivity for estrogen receptor. The ratio of IC50 for estrogen receptor-α and estrogen receptor-β (fold selectivity) was taken as biological activity. Physicochemical parameters were calculated using Dragon software, version 1.11. Sequential multiple linear regression analysis was carried out to derive QSAR models, which were further evaluated for statistical significance and predictive power by internal and external validation. The best quantitative structure activity relationship model was selected having a correlation coefficient (R2) of 0.948, cross-validated correlation coefficient (Q2) of 0.921, and Rpred2 of 0.853. The predictive ability of the selected model was also confirmed by leave one-out cross-validation. The QSAR model indicates that the descriptors (H1p, R5u, RTe) play an important role in estrogen receptor-β binding. The information generated from this study may be useful in the design of more potent substituted phenyl benzoxazole derivatives as estrogen receptor ligands.

Biological Activities of Two Ultra-Low Molecular Weight Heparins, Semuloparin and Bemiparin, Reveal That a Common Potency Standard Is Inadequate

Abstract 4324 Introduction:Ultra-low molecular weight heparins (U-LMWHs) are being developed to improve the safety and efficacy of antithrombotic therapy. Two of these agents, bemiparin (Rovi, Madrid, Spain) and semuloparin (Sanofi-Aventis, Paris, France), are produced by distinct methods. Bemiparin is produced by esterification of porcine mucosal heparin followed by alkaline hydrolysis and fractionation. Semuloparin is prepared by a highly selective depolymerization reaction using a phosphazene base which preserves the AT binding sequences from destruction. While both bemiparin and semuloparin are effective at reducing the incidence of post-surgical VTE, some biological differences have been observed in humans. The objective of this investigation was to determine whether a common standard could be used to define their potency. Methods:Activities were compared using typical clinical coagulation assays and pharmacological assays required for potency assessment. Anticoagulant activity was assessed using aPTT and ACT assays. Anti-Xa and anti-IIa activities were determined using amidolytic assays. Constrained and unconstrained curves were determined for the anti-Xa and anti-IIa data to assess parallelism of the concentration-response curves. Thrombin generation was measured using a fluorometric substrate kinetic method (Technothrombin TGA assay; Technoclone GmbH, Vienna, Austria). Platelet function was assessed using platelet aggregometry and the serotonin release assay (SRA). Results:A significantly larger prolongation of the aPTT was observed with bemiparin at concentrations >1 μ g/ml. Differences in anticoagulant activity measured by the Heptest assay were not observed at concentrations below 2.5 μ g/ml. When supplemented to whole blood samples at higher concentrations, bemiparin and semuloparin are able to prolong the kaolin ACT. Anti-FXa activity for bemiparin and semuloparin was the same over the concentration range tested. Bemiparin produced more anti-FIIa activity at each concentration tested. Depletion of AT led to a complete loss of anti-FXa and anti-FIIa activities for both agents while depletion of heparin cofactor-II had minimal impact on anti-IIa activity. Parallelism assessment is used to identify similarity or difference of products in biological assays. The constrained and unconstrained models using anti-FXa concentration-response data were equivalent (p=0.422), indicating that bemiparin and semuloparin were equivalent (IC50 ratio = 0.97, confidence interval= 0.95–0.98, CV = 0.7%). In contrast, the constrained and unconstrained models using anti-FIIa concentration-response data were not equivalent (p<0.001), indicating that the in vitro anti-FIIa activities of the two agents is not equivalent (IC50 ratio = 1.25, confidence interval = 1.17–1.34, CV = 3.4%). At clinically relevant concentrations, bemiparin more effectively inhibited thrombin generation than semuloparin. Platelet aggregation in response to collagen, ADP and arachidonic acid was not affected by either agent at concentrations up to 10 μ g/ml. At a concentration of 5 μ g/ml, bemiparin was able to completely inhibit thrombin-induced aggregation (0.5 U/ml), whereas no effect was observed with semuloparin. Cross-reactivity of bemiparin and semuloparin with anti-PF4/heparin HIT antibodies was tested using platelet aggregation and the SRA. The level of maximal aggregation was comparable in the presence of bemiparin and semuloparin, however, the rate of aggregation was significantly slower in the presence of semuloparin compared to bemiparin. The typical bell-shaped concentration-response curve was observed with both drugs in the SRA. Although the peak release was comparable for the two drugs (65.7 ± 16.0% for semuloparin vs. 75.5 ± 6.6% for bemiparin), the peak serotonin release was observed at lower concentrations for bemiparin (1 μ g/ml) than for semuloparin (10 μ g/ml) suggesting a higher sensitivity of bemiparin to (stronger interaction with) the anti-PF4/heparin antibodies. Conclusions:These data demonstrate that the molecular profiling of heparin-derived agents is insufficient to determine drug class. Additional structural characterization and multiple biologic activities relevant to clinical safety and efficacy need to be considered as well. As such, the use of a common reference standard for potency determination of ULMWH is not valid. Disclosures:Rigal: Sanofi-Aventis: Employment. Bayol:Sanofi-Aventis: Employment. Viskov:Sanofi-Aventis: Employment.

Ex vivo susceptibility of Plasmodium falciparum isolates from Dakar, Senegal, to seven standard anti-malarial drugs

BackgroundAs a result of widespread chloroquine and sulphadoxine-pyrimethamine resistance, artemisinin-based combination therapy (ACT) (which includes artemether-lumefantrine and artesunate-amodiaquine) has been recommended as a first-line anti-malarial regimen in Senegal since 2006. Since then, there have been very few reports on the ex vivo susceptibility of Plasmodium falciparum to anti-malarial drugs. To examine whether parasite susceptibility has been affected by the widespread use of ACT, the ex vivo susceptibility of local isolates was assessed at the military hospital of Dakar.MethodsThe ex vivo susceptibility of 93 P. falciparum isolates from Dakar was successfully determined using the Plasmodium lactate dehydrogenase (pLDH) ELISA for the following drugs: chloroquine (CQ), quinine (QN), mefloquine (MQ), monodesethylamodiaquine (MDAQ), lumefantrine (LMF), dihydroartemisinin (DHA) and doxycycline (DOX).ResultsAfter transformation of the isolate IC50 in ratio of IC50 according to the susceptibility of the 3D7 reference strain (isolate IC50/3D7 IC50), the prevalence of the in vitro resistant isolates with reduced susceptibility was 50% for MQ, 22% for CQ, 12% for DOX, 6% for both QN and MDAQ and 1% for the drugs LMF and DHA. The highest significant positive correlations were shown between responses to CQ and MDAQ (r = 0.569; P < 0.0001), LMF and QN (r = 0.511; P < 0.0001), LMF and DHA (r = 0.428; P = 0.0001), LMF and MQ (r = 0.413; P = 0.0002), QN and DHA (r = 0.402; P = 0.0003) and QN and MQ (r = 0.421; P = 0.0001).ConclusionsThe introduction of ACT in 2002 has not induced a decrease in P. falciparum susceptibility to the drugs DHA, MDAQ and LMF, which are common ACT components. However, the prevalence of P. falciparum isolates with reduced susceptibility has increased for both MQ and DOX. Taken together, these data suggest that intensive surveillance of the P. falciparum in vitro susceptibility to anti-malarial drugs in Senegal is required.