Ahmad Ali Shaik

Preparation of penta-O-galloyl-β-D-glucose from tannic acid and plasma pharmacokinetic analyses by liquid-liquid extraction and reverse-phase HPLC.

The gallotannin penta-O-galloyl-beta-D-glucose (PGG) has many biological activities including in vivo anti-cancer efficacy. We present in this paper a scaled-up protocol for its preparation in high purity from tannic acid by acidic methanolysis with typical yield of 15%. We also describe a method for the analysis of PGG in mouse plasma by HPLC and its application in preliminary pharmacokinetic studies. A liquid-liquid extraction (LLE) protocol was optimized for the extraction of PGG from mouse plasma. The extraction efficiency for PGG at 1 μg/mL in mouse plasma was 70.0±1.3% (n=5). The limit of detection (LOD) for PGG was approximately 0.2 μg/mL. Preliminary pharmacokinetic parameters of PGG following a single i.p. injection with 5% ethanol/saline vehicle in mice were established. The peak plasma PGG concentrations (C(max)) were approximately 3-4 μM at a dose of 0.5 mg per mouse (∼20 mg/kg) at 2 h post-injection (T(max)).

Identification of methysticin as a potent and non-toxic NF-κB inhibitor from kava, potentially responsible for kava's chemopreventive activity

Nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays an essential role in cancer development. The results of our recent chemopreventive study demonstrate that kava, a beverage in the South Pacific Islands, suppresses NF-kappaB activation in lung adenoma tissues, potentially a mechanism responsible for kavas chemopreventive activity. Methysticin is identified as a potent NF-kappaB inhibitor in kava with minimum toxicity. Other kava constituents, including four kavalactones of similar structures to methysticin, demonstrate minimum activities in inhibiting NF-kappaB.

Mouse prostate proteome changes induced by oral pentagalloylglucose treatment suggest targets for cancer chemoprevention.

Recent in vitro and in vivo preclinical studies have suggested that the Oriental herbal compound penta-1, 2, 3, 4, 6-O-galloyl-beta-D-glucose (PGG) is a promising chemopreventive agent for prostate cancer. Little is known of its safety for chronic chemoprevention use and virtually nothing is known of its in vivo responsive proteins in the target organ. Here we treated male C57BL/6 mice with daily oral administration of PGG at two dosages (1 and 2 mg per mouse) from 7 to 14 weeks of age and profiled proteomic patterns in the prostate with iTRAQ labeling and 2D LC-MS/MS analyses. While neither dose affected feed intake and body weight gain, the 2 mg dose (∼80-100 mg per kg) led to a minor but statistically significant decrease of the weight of prostate and thymus. For proteomic profiling, five prostates were pooled from each group for protein extraction. Proteins were denatured, reduced, alkylated and digested to peptides. The peptides were labeled with iTRAQ reagents, mixed and subjected to 2D LC-MS/MS analyses. PGG consumption suppressed the abundance of oncoproteins (e.g., fatty acid synthase, clusterin) and up-regulated that of tumor suppressor proteins (e.g., glutathione S-transferase M), signifying changes that may contribute to prostate cancer risk reduction.

Quantitative Determination of Decursin, Decursinol Angelate, and Decursinol in Mouse Plasma and Tumor Tissue Using Liquid-Liquid Extraction and HPLC

The pyranocoumarin compound decursin and its isomer decursinol angelate (DA) are the major hydrophobic phytochemicals in the root of Angelica gigas Nakai (AGN, Korean Angelica), a major traditional medicinal herb. The ethanol extract of AGN and especially the purified decursin and DA have been shown to exhibit antitumor activities by our collaborative team and others. Although decursinol has been identified as a major hydrolysis metabolite of decursin and DA in vivo in previous pharmacokinetic studies with mouse and rat, other recently published results sharply disputed this conclusion. In this study, we set up a practical method for the concurrent analysis of decursin, DA, and decursinol in mouse plasma and tumor tissues by liquid-liquid extraction and HPLC-UV and applied the method to several animal experiments. Plasma or tumor homogenate was extracted directly with ethyl acetate. The extraction efficiency for decursin/DA (quantitated together) and decursinol was between 82-95 % in both mouse plasma and tumor homogenate. The lower limit of quantitation (LLOQ) was approximately 0.25 µg/mL for decursin/DA and 0.2 µg/mL for decursinol in mouse plasma. In a pilot pharmacokinetic study, male C57BL/6 mice were given a single dose of 4.8 mg decursin/DA mixture (~240 mg/kg) per mouse either by oral gavage or intraperitoneal injection. Maximum plasma concentrations for decursin/DA and decursinol were 11.2 and 79.7 µg/mL, respectively, when decursin/DA was administered via intraperitoneal injection, and 0.54 and 14.9 µg/mL via oral gavage. Decursin/DA and decursinol contents in the tumor tissues from nude mouse xenografts correlated very well with those in plasma. Overall, our results confirm the conclusion that the majority of decursin/DA hydrolyze to decursinol in rodent models with a tiny fraction remaining as the intact compounds administered.

Dihydromethysticin from kava blocks tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis and differentially reduces DNA damage in A/J mice

We have previously shown that kava and its flavokavain-free Fraction B completely blocked 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice with a preferential reduction in NNK-induced O (6)-methylguanine (O (6)-mG). In this study, we first identified natural (+)-dihydromethysticin (DHM) as a lead compound through evaluating the in vivo efficacy of five major compounds in Fraction B on reducing O (6)-mG in lung tissues. (+)-DHM demonstrated outstanding chemopreventive activity against NNK-induced lung tumorigenesis in A/J mice with 97% reduction of adenoma multiplicity at a dose of 0.05mg/g of diet (50 ppm). Synthetic (±)-DHM was equally effective as the natural (+)-DHM in these bioassays while a structurally similar analog, (+)-dihydrokavain (DHK), was completely inactive, revealing a sharp in vivo structure-activity relationship. Analyses of an expanded panel of NNK-induced DNA adducts revealed that DHM reduced a subset of DNA adducts in lung tissues derived from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the active metabolite of NNK). Preliminary 17-week safety studies of DHM in A/J mice at a dose of 0.5mg/g of diet (at least 10× its minimum effective dose) revealed no adverse effects, suggesting that DHM is likely free of kavas hepatotoxic risk. These results demonstrate the outstanding efficacy and promising safety margin of DHM in preventing NNK-induced lung tumorigenesis in A/J mice, with a unique mechanism of action and high target specificity.

Kava Blocks 4-(Methylnitrosamino)-1-(3-pyridyl)-1-Butanone–Induced Lung Tumorigenesis in Association with Reducing O6-methylguanine DNA Adduct in A/J Mice

We previously reported the chemopreventive potential of kava against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- and benzo(a)pyrene (BaP)–induced lung tumorigenesis in A/J mice during the initiation and postinitiation stages. In this study, we investigated the tumorigenesis-stage specificity of kava, the potential active compounds, and the underlying mechanisms in NNK-induced lung tumorigenesis in A/J mice. In the first experiment, NNK-treated mice were given diets containing kava at a dose of 5 mg/g of diet during different periods. Kava treatments covering the initiation stage reduced the multiplicity of lung adenomas by approximately 99%. A minimum effective dose is yet to be defined because kava at two lower dosages (2.5 and 1.25 mg/g of diet) were equally effective as 5 mg/g of diet in completely inhibiting lung adenoma formation. Daily gavage of kava (one before, during, and after NNK treatment) completely blocked lung adenoma formation as well. Kavalactone-enriched fraction B fully recapitulated kavas chemopreventive efficacy, whereas kavalactone-free fractions A and C were much less effective. Mechanistically, kava and fraction B reduced NNK-induced DNA damage in lung tissues with a unique and preferential reduction in O6-methylguanine (O6-mG), the highly tumorigenic DNA damage by NNK, correlating and predictive of efficacy on blocking lung adenoma formation. Taken together, these results demonstrate the outstanding efficacy of kava in preventing NNK-induced lung tumorigenesis in A/J mice with high selectivity for the initiation stage in association with the reduction of O6-mG adduct in DNA. They also establish the knowledge basis for the identification of the active compound(s) in kava. Cancer Prev Res; 7(1); 86–96. ©2013 AACR.

Reduction in colon cancer risk by consumption of kava or kava fractions in carcinogen-treated rats.

Epidemiological studies suggest that kava reduces colon cancer risk. However, no experimental studies of the chemopreventive properties of kava toward colon cancer have been reported. Further, there are concerns regarding hepatotoxicity of kava. The goal of this study was to determine whether kava consumption reduces markers of colon cancer in an animal model and to study the safety of kava. An ethanolic extract and polar and nonpolar fractions of the kava extract were fed to rats for 12 days prior to, during, and after administration of dimethylhydrazine, a colon-specific carcinogen. After 14 wk, rats fed the nonpolar extract had a significant reduction in precancerous lesions [aberrant crypt (AC) foci (ACF)] as well as large (≥4 AC/ACF) sialomucin-only expressing foci, an indicator of greater tumorigenic potential, compared to the control group. Groups fed the ethanolic extract and polar kava fraction trended toward reductions in ACF and large sialomucin-only expressing foci. The combined kava groups had significantly fewer total AC, ACF, large ACF, and large sialomucin-only expressing foci compared to the control group. Histological examination found no hepatic lesions in animals consuming the kava diets, suggesting that kava is safe to consume. Our results support that kava may reduce colon cancer risk.

Abstract 4622: Determination of pyranocoumarin compounds decursin, decursinol angelate and decursinol simultaneously in mouse plasma and tumor tissue by liquid-liquid extraction and HPLC

Pyranocoumarin compound decursin and its isomer decursinol angelate (DA) are the major chemical components of the root of Angelica gigas Nakai (AGN, Korean Angelica), a major traditional medicinal herb. Recently, AGN has been shown to exhibit anti-tumor activities by our collaborative team. Decursinol has been identified as a major metabolite of decursin and DA in vivo by us and others, but another group has published very controversial results. To better define the mechanisms of in vivo action for AGN, pharmacokinetic studies are essential. In this study, we set up a practical method for the concurrent analysis of decursin, DA and decursinol in mouse plasma and tumor tissues by liquid-liquid extraction and HPLC-UV. In brief, plasma or tumor homogenate (0.1-0.2 ml) spiked with 1μg chrysin as internal standard (IS) was extracted directly with ethyl acetate. After extraction, decursin, DA and decursinol were quantitated by HPLC with UV detection at 330 nm. The method was validated in terms of recovery, lower limit of quantitation and linear range. The extraction efficiency for spiked decursin/DA and decursinol at 1 µg/ml was 90.1±3.2 % and 90.3±1.8% in mouse plasma, respectively. The lower limit of quantitation (LOQ) was approximately 0.25 μg/mL for decursin/DA, and 0.2 μg/mL for decursinol in mouse plasma. The linear range of the method was 0.25 -25 µg/ml and 0.2-50 µg/ml in mouse plasma for decursin/DA and decursinol, respectively. In a pilot pharmacokinetic study, male C57BL/6 mice were given a single dose of 4.8 mg decursin/DA (∼240mg/kg) per mouse either by gavage or intraperitoneal injection in 0.2 mL vehicle of ethanol:PEG400:Tween80:5% glucose (3:6:1:20). Maximum plasma concentrations for decursin/DA and decursinol were found to be 34.2 and 324 µM respectively when D/DA was administered via intraperitoneal injection and to be 1.7 and 60.7 µM via gavage. These findings further confirmed that decursinol was one of the major metabolites of decursin/DA in vivo. In athymic nude mouse xenograft study, the mice were given intraperitoneal injection of decursin/DA at a dose of 1mg (∼40mg/kg) daily, 5 days per week, for 7 weeks. The decursin/DA and decursinol concentration was 1.5 and 10μg/g, respectively, in the tumor tissue. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4622. doi:10.1158/1538-7445.AM2011-4622

Abstract 1848: Decursin derivatives with increased in vivo stability suppress androgen receptor signaling and prostate cancer cell growth: Structure-bioactivity relationship

Androgen receptor (AR) signaling is crucial for the genesis and progression of prostate cancer (PCa). Targeting AR signaling remains an attractive approach for PCa chemoprevention and therapy. Our previous studies suggest that decursin, which is isolated from the Korean medicinal herb Angelica gigas Nakai, has novel anti-androgen attributes in comparison to that of AR antagonist drug bicalutamide (Casodex) and stronger anti-proliferative and pro-apoptotic activities in cell culture models. However, decursin quickly de-esterifies to decursinol in vivo, which lacks the anti-androgen and anti-proliferative activities. We report here that a novel prototype derivative (decursinol thiocarbamate, DTC) shows in vivo stability in single dose pharmacokinetic studies, with no detection of decursinol. Molecular docking with the wild type AR ligand binding domain (LBD) suggested that its AR-binding energy is inferior to DHT, but higher than decursin. Mechanistically DTC retains all the desirable properties of decursin, including inhibiting AR nuclear translocation, decreasing cellular AR protein abundance by promoting its 26S proteasomal-mediated degradation and lacking agonist activity. Dose-response comparisons showed that DTC was half as potent as decursin to inhibit AR signaling and androgen sensitive LNCaP PCa cell growth. In animal studies with mice, daily i.p. injection of DTC (2 mg per mouse) decreased AR transcriptional targets such as probasin and NKX3.1 in the mouse prostates. Chemical deposits on the surface of liver and other organs were observed suggesting poor solubility. Additional derivatives aimed to improved solubility had been synthesized and are being investigated for structure-activity relationship. Overall, the data suggest that DTC is a prototype stable decursin derivative retaining the novel AR antagonist activity for PCa chemoprevention or treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1848. doi:10.1158/1538-7445.AM2011-1848

Abstract 2882: Penta-O-galloyl-beta-D-glucose induces DNA replicative S arrest and G1 arrest independent of p21Cip1, p27Kip1 and p53 in human breast cancer cells and is orally active against breast cancer xenograft

Natural herbal compounds with novel actions different from existing breast cancer targeting modalities are attractive for improving treatment and prevention efficacy and safety. We have recently shown that penta-1,2,3,4,6-O-galloyl-beta-D-glucose (PGG), a naturally occurring gallotannin polyphenol compound, induced S-phase arrest in prostate cancer cells through inhibiting DNA replicative synthesis, in addition to the induction of G 1 arrest and cell deaths at higher levels of exposure. We and others have shown that PGG through i.p. injection exerts a strong in vivo growth suppression of human prostate cancer xenograft models in athymic nude mice. We hypothesize that the novel DNA replication targeting action and the cellular arrest and death effects of PGG are applicable to breast cancer cells and PGG could be a novel agent for treatment or prevention of breast cancer, especially those without proven drugable targets such as triple-negative breast cancer. In cell culture studies, exposure to serum achievable level of PGG induced rapid S arrest in p53-wild type ER-dependent MCF-7 breast cancer cells and in p53-mutant ER-/PR- and Her2-negative (triple-negative) MDA-MB-231 breast cancer cells as indicated by the lack of BrdU incorporation into S-phase cells. Higher levels of PGG induced more caspase-mediated apoptosis in MCF-7 than in MDA-MB-231 cells. In addition to S-arrest, PGG induced G 1 arrest in both cell lines. Contrary to a study by others with questionable design, we have found no induction of CDK inhibitory proteins p21Cip1 and p27Kip1 with G 1 arrest. PGG treatment led to decreased cyclin D1 in both cell lines and over-expressing cyclin D1 abolished G 1 arrest and hastened S arrest. In serum-starvation synchronized MCF-7 cells, we observed a close association of down-regulation of cyclin D1 and de-phosphorylation of Rb by PGG shortly before G 1 -S transition stimulated by serum. Though PGG inhibited estrogen-stimulated cell growth in MCF-7 cells, it had little effect on estrogen receptor-alpha level. To test the in vivo efficacy of PGG, we treated female athymic mice inoculated with MDA-MB-231 xenograft with 10 mg/kg body weight by daily gavage and compared with weekly paclitaxel treatment at the same dosage. Oral administration of PGG led to a better growth inhibitory efficacy than the taxane drug. Together, our in vitro and in vivo data support PGG as a potential chemopreventve or treatment agent for breast cancer with novel targeting actions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2882.