Preclinical Pharmacokinetics Study of R- and S-Enantiomers of the Histone Deacetylase Inhibitor, AR-42 (NSC 731438), in Rodents.

AR-42, a new orally bioavailable, potent, hydroxamate-tethered phenylbutyrate class I/IIB histone deacetylase inhibitor currently is under evaluation in phase 1 and 2 clinical trials and has demonstrated activity in both hematologic and solid tumor malignancies. This report focuses on the preclinical characterization of the pharmacokinetics of AR-42 in mice and rats. A high-performance liquid chromatography-tandem mass spectrometry assay has been developed and applied to the pharmacokinetic study of the more active stereoisomer, S-AR-42, when administered via intravenous and oral routes in rodents, including plasma, bone marrow, and spleen pharmacokinetics (PK) in CD2F1 mice and plasma PK in F344 rats. Oral bioavailability was estimated to be 26 and 100% in mice and rats, respectively. R-AR-42 was also evaluated intravenously in rats and was shown to display different pharmacokinetics with a much shorter terminal half-life compared to that of S-AR-42. Renal clearance was a minor elimination pathway for parental S-AR-42. Oral administration of S-AR-42 to tumor-bearing mice demonstrated high uptake and exposure of the parent drug in the lymphoid tissues, spleen, and bone marrow. This is the first report of the pharmacokinetics of this novel agent, which is now in early phase clinical trials.

Optical metabolic imaging of treatment response in human head and neck squamous cell carcinoma.

Optical metabolic imaging measures fluorescence intensity and lifetimes from metabolic cofactors nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). These molecular level measurements provide unique biomarkers for early cellular responses to cancer treatments. Head and neck squamous cell carcinoma (HNSCC) is an attractive target for optical imaging because of easy access to the site using fiber optic probes. Two HNSCC cell lines, SCC25 and SCC61, were treated with Cetuximab (anti-EGFR antibody), BGT226 (PI3K/mTOR inhibitor), or cisplatin (chemotherapy) for 24 hours. Results show increased redox ratio, NADH α1 (contribution from free NADH), and FAD α1 (contribution from protein-bound FAD) for malignant cells compared with the nonmalignant cell line OKF6 (p<0.05). In SCC25 and SCC61 cells, the redox ratio is unaffected by cetuximab treatment and decreases with BGT226 and cisplatin treatment (p<0.05), and these results agree with standard measurements of proliferation rates after treatment. For SCC25, NADH α1 is reduced with BGT226 and cisplatin treatment. For SCC61, NADH α1 is reduced with cetuximab, BGT226, and cisplatin treatment. Trends in NADH α1 are statistically similar to changes in standard measurements of glycolytic rates after treatment. FAD α1 is reduced with cisplatin treatment (p<0.05). These shifts in optical endpoints reflect early metabolic changes induced by drug treatment. Overall, these results indicate that optical metabolic imaging has potential to detect early response to cancer treatment in HNSCC, enabling optimal treatment regimens and improved patient outcomes.

Extraction of plant secondary metabolites.

This chapter presents an overview of the preparation of extracts from plants using organic solvents, with emphasis on common problems encountered and methods for their reduction or elimination. In addition to generally applicable extraction protocols, methods are suggested for selectively extracting specific classes of plant-derived compounds, and phytochemical procedures are presented for the detection of classes of compounds encountered commonly during extraction, including selected groups of secondary metabolites and interfering compounds. Successful extraction begins with careful selection and preparation of plant samples and thorough review of the appropriate literature for suitable protocols for a particular class of compounds or plant species. During the extraction of plant material, it is important to minimize interference from compounds that may co-extract with the target compounds, and to avoid contamination of the extract, as well as to prevent decomposition of important metabolites or artifact formation as a result of extraction conditions or solvent impurities.

BIOLOGICALLY ACTIVE NATURAL PRODUCTS OF THE GENUS CALLICARPA.

About 20 species from Callicarpa have reported ethnobotanical and ethnomedical uses, and several members of this genus are well known in the traditional medical systems of China and South Asia. Ethnomedical reports indicate their use in the treatment of hepatitis, rheumatism, fever, headache, indigestion, and other ailments. Several species of Callicarpa have been reported to be used against cancer (e.g., Callicarpa americana root to treat skin cancer and Callicarpa rubella bark to treat tumors of the large intestine). Extracts from about 14 species in this genus have been evaluated for biological activity, including antibacterial, antifungal, anti-insect growth, cytotoxic, and phytotoxic activities. In addition to amino acids, benzenoids, simple carbohydrates, and lipids, numerous diterpenes, flavonoids, phenylpropanoids, phytosterols, sesquiterpenes, and triterpenes have been detected in or isolated from the genus Callicarpa. The essential oils of Callicarpa americana have recently been reported to have antialgal and phytotoxic activities, and several isolates from this species (and C. japonica) were identified as contributing to the mosquito bite-deterrent activity that was first indicated by folkloric usage. Recent bioassay-guided investigations of C. americana extracts have resulted in the isolation of several active compounds, mainly of the clerodane diterpene structural type.

Cytotoxic constituents from the fruiting branches of Callicarpa americana collected in southern Florida.

Bioassay-guided fractionation of the combined fruits, leaves, and twigs (fruiting branches) of Callicarpa americana, collected from a plot in a forested area in southern Florida, led to the isolation of six new clerodane diterpenes (1-6) and eight known compounds. The structures of 1-6 [12(S),16xi-dihydroxycleroda-3,13-dien-15,16-olide (1), 12(S)-hydroxy-16xi-methoxycleroda-3,13-dien-15,16-olide (2), 12(S)-hydroxycleroda-3,13-dien-15,16-olide (3), 16xi-hydroxycleroda-3,11(E),13-trien-15,16-olide (4), 3beta,12(S)-dihydroxycleroda-4(18),13-dien-15,16-olide (5), and 12(S)-hydroxycleroda-3,13-dien-16,15-olide (6)] were elucidated by interpretation of spectroscopic data and chemical methods. The absolute configuration at C-12 in 1 and 3 was ascertained using the Mosher ester technique. The cytotoxicity of all isolates was tested against a panel of human cancer cell lines, and compounds 1, 4, and 6, and the known compounds genkwanin, 16xi-hydroxycleroda-3,13-dien-15,16-olide, and 2-formyl-16xi-hydroxy-3-A-norcleroda-2,13-dien-15,16-olide were active (ED50 <5 microg/mL). However, 1 was found to be inactive against human cancer cells implanted in mice using a hollow-fiber tumor model.

Relationships between inhibitory activity against a cancer cell line panel, profiles of plants collected, and compound classes isolated in an anticancer drug discovery project.

In an attempt to determine the relationships between the plant profiles (country of collection, taxonomy, plant part) and the compound classes isolated with cytotoxic activity against a panel of human tumor cell lines, the data compiled from a 15-year anticancer drug-discovery project were subjected to an analysis of variance (ANOVA). The results indicate significant trends in cytotoxic activity relative to collection location, taxonomy, plant part, and compound classes isolated. Plant collections were made in tropical forests in six countries, with collections from Ecuador resulting in higher activity than those from Indonesia and Peru. Interestingly, collections from Florida were not statistically different than those from the countries with higher biodiversity. One hundred and forty-five families were represented in the collections, with the Clusiaceae, Elaeocarpaceae, Meliaceae, and Rubiaceae having low ED50 (half maximal effective dose) values. Especially active genera included Aglaia, Casearia, Exostema, Mallotus, and Trichosanthes. Roots and below-ground plant materials were significantly more active than above-ground materials. Cucurbitacins, flavaglines, anthraquinones, fatty acids, tropane alkaloids, lignans, and sesquiterpenoids were significantly more active than xanthones and oligorhamnosides. The results from this study should serve as a guide for future plant collection endeavors for anticancer drug discovery.

Cytotoxic clerodane diterpenoids from the leaves of Premna tomentosa.

Three clerodane diterpenoids, premnones A-C (1-3), were isolated from a chloroform-soluble fraction of Premna tomentosa along with four known flavonoids and three known triterpenoids. Among these isolates, premnones A-C exhibited cytotoxic activity when evaluated against a small panel of tumor cell lines. However, premnone A was found to be inactive when evaluated in a follow-up in vivo hollow fiber assay at the highest dose tested (50mg/kg), using LNCaP, Lu1, and MCF-7 cells.

The role of pharmacognosy in modern medicine and pharmacy.

This review details the contribution to modern medicine and pharmacy made by natural products and drugs derived from natural products, with an emphasis on essential medicines and new introductions to the market. Areas covered include recent advances in the development of drugs derived from marine organisms, microbes, terrestrial animals, and vascular plants, and current issues regarding botanical medicines. The role of natural products in drug discovery and development is evaluated, particularly with regard to their value as sources of drug leads with "drug-like" properties. A rationale for the success of natural products research in providing new drugs and drug prototypes is presented, drawing on lines of evidence from chemical informatics and chemical ecology. Several innovative strategies for natural products drug discovery and evaluation of botanical medicines are also reviewed.

Cytotoxic constituents from the stem bark of Dichapetalum gelonioides collected in the Philippines.

Fractionation of an ethyl acetate-soluble extract of the stem bark of Dichapetalum gelonioides, collected in the Philippines, using the LNCaP (hormone-dependent human prostate) cell line as a monitor, led to the purification of three dichapetalin-type triterpenoids [dichapetalins A (1), I (2), and J (3)], along with two dolabrane norditerpenoids (6, 7), and the additional triterpenoids zeylanol (8), 28-hydroxyzeylanol (9), and betulinic acid. Since compounds 1-3 exhibited promising selectivity against the SW626 (human ovarian cancer) cell line, a re-collection of the plant material was carried out, to obtain more of these compounds for additional biological testing. Two further phenylpyranotriterpenoids [dichapetalins K (4) and L (5)] were isolated from the re-collected plant material. The structures of the new compounds 2-5 and 9 were determined on the basis of spectroscopic data interpretation, and the relative configuration of 6 was confirmed using X-ray crystallography. Compounds 4-6 and the methyl ester, 6a, exhibited broad cytotoxic activity when tested against a panel of human tumor cell lines. Dichapetalin A (1) was not active when evaluated in an in vivo hollow fiber assay in the dose range 1-6 mg/kg.

Tropane aromatic ester alkaloids from a large-scale re-collection of Erythroxylum pervillei stem bark obtained in Madagascar.

Fractionation by pH zone-refining countercurrent chromatography of an extract of the stem bark of Erythroxylum pervillei, obtained on a kilogram scale in southern Madagascar, led to the isolation and characterization of four tropane aromatic ester alkaloids as minor constituents, namely, pervilleines G (5) and H (6) and cis-pervilleines B (7) and F (8). Their structures were determined by spectroscopic data interpretation.

Cytotoxic lignans from the stems of Helicteres hirsuta collected in Indonesia.

Cytotoxicity-guided fractionation of the stems of Helicteres hirsuta, of Indonesian origin, led to the isolation and identification of six lignans, namely, (+/-)-pinoresinol, (+/-)-medioresinol, (+/-)-syringaresinol, (-)-boehmenan, (-)-boehmenan H and (+/-)-trans-dihydrodiconiferyl alcohol. Of these isolates, (+/-)-pinoresinol exhibited potent cytotoxic effects when evaluated against a small panel of cancer cell lines.

Antitumour activity of 3-chlorodeoxylapachol, a naphthoquinone from Avicennia germinans collected from an experimental plot in southern Florida.

As part of an ongoing collaborative effort to discover new anticancer agents from plants, an extract obtained from the leaves and twigs of Avicennia germinans, collected in a coastal area of southern Florida, was identified as possessing cytotoxic activity in a panel of human cancer cell lines. Fractionation of the petroleum ether partition, using cytotoxicity to guide the fractionation, led to the isolation of 3-chlorodeoxylapachol. The antitumour potential of 3-chlorodeoxylapachol was demonstrated with the in-vivo hollow fibre assay, a model of antitumour activity using human cancer cell-filled fibres implanted into mice. The possibility that this compound is an artefact of the isolation procedure was ruled out by liquid chromatography-mass spectrometry analysis of extracts prepared without the use of chlorinated solvent. In conclusion, 3-chlordeoxylapachol, a secondary metabolite obtained from the chloroform-soluble extract of a mangrove tree, was cytotoxic in a panel of human cancer cells, and active against KB human cancer cells in the murine hollow fibre antitumour model, with selectivity in KB cells for the intravenous site at lower doses, indicating possible metabolic activation.

Limnophilaspiroketone, a highly oxygenated phenolic derivative from Limnophila geoffrayi.

A highly oxygenated phenolic spiroketone, limnophilaspiroketone (1), was isolated from the aerial parts of Limnophila geoffrayi collected in Thailand. The structure of 1 was determined based on spectroscopic data interpretation. This novel isolate, obtained as a major secondary metabolite constituent, was verified as a racemate using the Mosher ester method.

Constituents of the stems of Macrococculus pomiferus and their inhibitory activities against cyclooxygenases-1 and -2.

As part of our program directed towards the discovery of new cancer chemopreventive agents from plants, the EtOAc-soluble extract of the stems of M. pomiferus was found to inhibit the enzyme cyclooxygenase-2 (COX-2). Bioassay-directed fractionation of this extract led to the isolation of two dibenzylbutyrolactone lignans, (8R,8'R)-3'-O-demethyl-5-hydroxymatairesinol (1) and (8R,8'R)-3'-O-demethyl-5-methoxymatairesinol (2), as well as seven known compounds, (-)-5'-methoxyyatein (3), blumenol A, (-)-deoxypodophyllotoxin (anthricin), (-)-deoxypodorhizone, 2,6-dimethoxyhydroquinone, 4-hydroxybenzaldehyde, and beta-sitosterol glucoside. The structures of compounds 1 and 2 were determined using spectroscopic data (1D and 2D NMR, and HREIMS), and the 8R and 8'R absolute stereochemistry was established for both 1 and 2 on the basis of their CD spectra. All isolates obtained in the present study were evaluated for their inhibitory effects with both COX-1 and -2. Of these, only 5'-methoxyyatein (3) showed weak activity against COX-2, while all other compounds isolated were inactive. The COX-2 inhibitory activity of the EtOAc extract was also traced to the presence of several common fatty acids by LC-MS.