George R. Pettit

Isolation and structure of the strong cell growth and tubulin inhibitor combretastatin A-4

The African treeCombretum caffrum (Combretaceae) has been found to contain a powerful inhibitor of tubulin polymerization (IC502–3 μM), the growth of murine lymphocytic leukemia (L 1210 and P 388 with ED50≈0.003 μM and human colon cancer cell lines [(e.g. LoVo (ED50=0.005 μg/ml), HT29 (ED50 0.02 μg/ml, Colo 205 (ED50=0.07 μg/ml), DLD-1 (ED50=0.005 μg/ml) and HCT-15 (ED50=0.0009 μg/ml)] designated combretastatin A-4 (1c). The structure assigned by spectral techniques was confirmed by synthesis.

Microplate Alamar Blue Assay for Staphylococcus epidermidis Biofilm Susceptibility Testing

ABSTRACT Biofilms are at the root of many infections largely because they are much more antibiotic resistant than their planktonic counterparts. Antibiotics that target the biofilm phenotype are desperately needed, but there is still no standard method to assess biofilm drug susceptibility. Staphylococcus epidermidis ATCC 35984 biofilms treated with eight different approved antibiotics and five different experimental compounds were exposed to the oxidation reduction indicator Alamar blue for 60 min, and reduction relative to untreated controls was determined visually and spectrophotometrically. The minimum biofilm inhibitory concentration was defined as ≤50% reduction and a purplish well 60 min after the addition of Alamar blue. All of the approved antibiotics had biofilm MICs (MBICs) of >512 μg/ml (most >4,096 μg/ml), and four of the experimental compounds had MBICs of ≤128 μg/ml. The experimental aaptamine derivative hystatin 3 was used to correlate Alamar blue reduction with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction and viable counts (CFU/ml) for S. epidermidis ATCC 35984, ATCC 12228, and two clinical isolates. For all four strains, Alamar blue results correlated well with XTT (r = 0.83 to 0.97) and with CFU/ml results (r = 0.85 to 0.94). Alamar blues stability and lack of toxicity allowed CFU/ml to be determined from the same wells as Alamar blue absorbances. If the described method of microplate Alamar blue biofilm susceptibility testing, which is simple, reproducible, cost-effective, nontoxic, and amenable to high throughput, is applicable to other important biofilm forming species, it should greatly facilitate the discovery of biofilm specific agents.

In vivo and in vitro evaluation of combretastatin A-4 and its sodium phosphate prodrug

SummaryThe anti-tumour effects and mechanism of action of combretastatin A-4 and its prodrug, combretastatin A-4 disodium phosphate, were examined in subcutaneous and orthotopically transplanted experimental colon tumour models. Additionally, the ability of these compounds to directly interfere with endothelial cell behaviour was also examined in HUVEC cultures. Combretastatin A-4 (150 mg kg–1, intraperitoneally (i.p.)) and its water-soluble prodrug (100 mg kg–1, i.p.) caused almost complete vascular shutdown (at 4 h), extensive haemorrhagic necrosis which started at 1 h after treatment and significant tumour growth delay in MAC 15A subcutaneous (s.c.) colon tumours. Similar vascular effects were obtained in MAC 15 orthotopic tumours and SW620 human colon tumour xenografts treated with the prodrug. More importantly, in the orthotopic models, necrosis was seen in vascularized metastatic deposits but not in avascular secondary deposits. The possible mechanism giving rise to these effects was examined in HUVEC cells. Here cellular networks formed in type I calf-skin collagen layers and these networks were completely disrupted when incubated with a non-cytotoxic concentration of combretastatin A-4 or its prodrug. This effect started at 4 h and was complete by 24 h. The same non-cytotoxic concentrations resulted in disorganization of F-actin and β-tubulin at 1 h after treatment. In conclusion, combretastatin A-4 and its prodrug caused extensive necrosis in MAC 15A s.c. and orthotopic colon cancer and metastases, resulting in anti-tumour effects. Necrosis was not seen in avascular tumour nodules, suggesting a vascular mechanism of action.

CONVOLUTAMYDINE A, A NOVEL BIOACTIVE HYDROXYOXINDOLE ALKALOID FROM MARINE BRYOZOAN AMATHIA CONVOLUTA

Abstract Convolutamydine A ( 1 ), a new alkaloid containing a dibromohydroxyoxindole moiety, was isolated from the Floridian bryozoan Amathia convoluta and the structure was elucidated on the basis of spectroscopic data. Compound 1 exhibited a potent activity in the differentiation of HL-60 cells.

Bryostatins: potent, new mitogens that mimic phorbol ester tumor promoters.

Bryostatins (2 ng/ml), when combined with insulin in serum-free culture medium, are strongly mitogenic for Swiss 3T3 cells that have been arrested in the G1/G0 phase of the cell cycle. The mitogenic effect of the bryostatins is similar to that of 12-O-decanoylphorbol-13-acetate (TPA). A prior treatment of the cultures with TPA eliminated the mitogenic response to bryostatin and to a second addition of TPA. Conversely, a prior treatment of the cultures with bryostatin eliminated the mitogenic response to TPA. Bryostatin potently inhibited the binding of [3H]phorbol dibutyrate to a high affinity receptor in the cells. The findings suggest that the bryostatins and TPA act via the same receptor, possibly protein kinase C.

Phase I study of bryostatin 1 in patients with relapsed non-Hodgkin's lymphoma and chronic lymphocytic leukemia.

PURPOSE To define, in a phase I study in relapsed non-Hodgkins lymphoma (NHL) and chronic lymphocytic leukemia (CLL), the maximum-tolerated dose (MTD), major toxicities, and possible antitumor activity of bryostatin 1, a macrocyclic lactone. PATIENTS AND METHODS Bryostatin 1 was delivered by 72-hour continuous infusion every 2 weeks to patients with relapsed NHL or CLL, at doses that ranged from 12 microg/m2 to 180 microg/m2 per course. Correlative investigations included evaluations of total protein kinase C (PKC) in peripheral blood and lymphoid differentiation in patient tumor tissue. RESULTS Twenty-nine patients were treated, including three patients with CLL and 26 with NHL. Generalized myalgia was the dose-limiting toxicity (DLT) and occurred in two of three patients treated with bryostatin 1 at 180 microg/m2 per course. Myalgias were dose-related and cumulative, and often started in the thighs and calves, improved with activity, were somewhat responsive to analgesics, and often took weeks to resolve once taken off study. Six patients were treated at the MTD of 120 microg/m2 per course. Myalgia, headache, and fatigue were common. Hematologic toxicity was uncommon. Total cumulative doses of bryostatin 1 up to 1,134 microg/m2 have been administered without untoward toxicity. Eleven patients achieved stable disease for 2 to 19 months. An in vitro assay for total PKC evaluation in patient peripheral-blood samples demonstrated activation within the first 2 hours with subsequent downregulation by 24 hours, which was maintained throughout the duration of the 72-hour infusion. CONCLUSION This phase I study defined the MTD and recommended phase II dose of bryostatin 1, when administered over 72 hours every 2 weeks, to be 120 microg/m2 (40 microg/m2/d for 3 days). Generalized myalgia was the DLT. Future studies will define the precise activity of bryostatin 1 in subsets of patients with lymphoproliferative malignancies and its efficacy in combination with other agents.

Isolation of dolastatins 10–15 from the marine mollusc dolabella auricularia

Abstract A twenty year pursuit of the cell growth inhibitory and antineoplastic constituents of the Western Indian Ocean (Mauritius) sea hare Dolabella auricularia has resulted in the discovery of fifteen structurally unique peptide, cyclopeptide, depsipeptide, and cyclodepsipeptide type-substances designated dolastatins 1–15. Solution of the difficult isolation problems leading to discovery of dolastatins 10–15 in 10−6 to 10−7% yields required 1,600 kg of Dolabella aurlcularia. To date, this represents the largest scale separation of sea hare components. Of these dolastatin 10 (4) has displayed unprecedented potency in experimental antineoplastic and tubulin assembly systems. Dolastatin 15 (9), and to a lesser extent dolastatin 14 (8) were also found to exhibit unusually strong antineoplastic activity. Both dolastatina 10 and 15 are in advanced preclinical development. Details of the isolation strategies and structural summaries for dolastatins 10–15 have been recorded. Intensive Study of the cell growth inhibition and antineoplastic constituents of the sea hare Dolabella auricularia provided the structurally unique peptides designated dolastatins 1-15.

Antineoplastic agents 338. The cancer cell growth inhibitory. Constituents of Terminalia arjuna (Combretaceae)

By means of bioassay-guided separation methods, the cancer cell growth inhibitory constituents residing in the bark, stem and leaves of the Mauritius medicinal plant Terminalia arjuna (Combretaceae) were examined. The cancer cell line active components were found to be gallic acid, ethyl gallate, and the flavone luteolin. Only gallic acid was previously known to occur in this plant. Luteolin has a well established record of inhibiting various cancer cell lines and may account for most of the rationale underlying the use of T. arjuna in traditional cancer treatments. Luteolin was also found to exhibit specific activity against the pathogenic bacterium Neisseria gonorrhoeae.

Structure-cytotoxic activity relationship for the toad poison bufadienolides.

The toad poison bufadienolides including natural and derivatized compounds were tested for their cytotoxic effects on primary liver carcinoma cells PLC/PRF/5 and their structure-cytotoxic activity relationships were studied. For this study, a ligand-binding model was developed by using a pharmacophore mapping program, Distance Comparisons (DISCO). The structural features that are common to the 3D structures of active bufadienolides were identified to provide approach to a 3D QSAR method by using Comparative Molecular Field Analysis (CoMFA) study and to correlate the steric and electrostatic fields of the molecules to their activities. A valuable model which enables prediction of their activities was obtained from the CoMFA analysis, which may be employed for the drug designs of new bufadienolide analogues.

Effects of bryostatin 1 and other pharmacological activators of protein kinase C on 1-[β-d-arabinofuranosyl]cytosine-induced apoptosis in HL-60 human promyelocytic leukemia cells

We have demonstrated previously that bryostatin 1, a macrocylic lactone with putative protein kinase C (PKC)-activating properties, synergistically augments the antileukemic actions of the deoxycytidine analog 1-[beta-D-arabinofuranosyl]cytosine (ara-C) in HL-60 human promyelocytic leukemia cells (Grant et al., Biochem Pharmacol 42: 853-867, 1991), and that this effect appears to be related to sensitization to ara-C-induced apoptosis (Grant et al., Cancer Res 52: 6270-6278, 1992). In the present studies, we have assessed the extent of this damage by quantitative spectrofluorophotometry of small molecular weight, double-stranded DNA fragments in order to provide: (a) a more complete characterization of the interaction between ara-C and bryostatin 1, and (b) a direct comparison of the relative effects of bryostatin 1 treatment with other pharmacological manipulations known to modulate protein kinase C activity. Exposure of cells to ara-C (10(-9) to 10(-4) M; 1-24 hr) induced time- and concentration-related increases in the extent of DNA fragmentation. Treatment with bryostatin 1 (10(-11) to 10(-7) M; 1-24 hr) alone failed to induce DNA damage, but promoted substantial time- and concentration-related increases in the extent of fragmentation induced by a subsequent 6-hr exposure to ara-C. Maximal potentiation of fragmentation (e.g. 2- to 3-fold greater than that obtained with ara-C alone) was observed following a 24-hr pretreatment with 10(-8) M or 10(-7) M bryostatin 1, and correlated closely with enhanced inhibition of HL-60 cell clonogenicity. The stage-1 tumor-promoter phorbol dibutyrate potentiated the effects of ara-C in a biphasic manner, maximally augmenting the response at 2.5 x 10(-8) M, but exerting no effect at 10(-7) M, whereas the stage-2 tumor-promoter mezerein failed to augment ara-C-related DNA fragmentation at low concentrations, and antagonized ara-C action at high concentrations. In contrast, ara-C-related DNA fragmentation was attenuated or abolished either by continual preexposure to synthetic diglyceride or by pretreatment with exogenous phospholipase C at all concentrations tested. Increased DNA fragmentation was not specifically related to recruitment of cells into S-phase or enhancement of ara-C-related cellular differentiation. Finally, concentrations of bryostatin 1 that maximally potentiated ara-C-related DNA fragmentation were associated with virtually complete down-regulation of total cellular PKC activity, whereas diglyceride and phospholipase C, which suppressed the response to ara-C, moderately increased total PKC activity.(ABSTRACT TRUNCATED AT 400 WORDS)