Bioactivity and cytotoxicity profiling of vincosamide and strictosamide, anthelmintic epimers from Sarcocephalus latifolius (Smith) Bruce leaf.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaf of Sarcocephalus latifolius is known to be used traditionally by the Fulanis in Nigeria to deworm animals. As helminthosis remains a major constraint to profitable livestock production worldwide, a precarious situation aggravated by the advent of resistant parasites, the discovery of new anthelmintics is a priority, necessitating exploration of medicinal plants for their anthelmintic principles. AIM OF THE STUDY: To identify and characterise compounds with anthelmintic activity from the leaf of Sarcocephalus latifolius. MATERIALS AND METHODS: Powdered S. latifolius leaves were extracted by successive maceration with n-hexane, chloroform and acetone. The dried extracts were evaluated for anthelmintic activity against Haemonchus placei adult worms, and the most active extract was subjected to bioassay-guided chromatographic separations. The isolated compounds were evaluated for cytotoxicity against the mammalian HeLa and MC3T3-E1 cell lines, using alamar blue and CellTitreGloTM to quantify cell viability. LC50 values were computed from the in vitro anthelmintic activity data by fitting to a non-linear regression equation (variable slope). Isolated compounds were characterized using spectroscopic and mass spectrometric analyses. RESULTS: Anthelmintic activity LC50 values for n-hexane, chloroform and acetone extracts were 47.85, 35.76 and 5.72 (mg/mL), respectively. Chromatographic separation of acetone extract afforded two bioactive epimers, identified as vincosamide (LC50 14.7 mg/mL) and strictosamide (LC50 12.8 mg/mL). Cytotoxicity evaluation showed that, below 200 µg/mL (400 µM), neither compound was toxic to the HeLa or MC3T3-E1 cells. CONCLUSION: Vincosamide and strictosamide could serve as novel scaffolds for the development of anthelmintic derivatives with improved potency and helminth selectivity.

[BIOLOGICALLY ACTIVE ALKALOIDS FROM RHIZOMES WITH ROOTS OF VINCA HERBACEA WALDST. ET KIT, GROWING IN GEORGIA].

Roots and rhizomes of Vinca herbacea Waldst. et Kit, were collected during early flowering and fruiting. Рhenophases biologically active substances I and II were obtained by liquid-liquid extraction. Dominant alkaloids: tabersonin, reserpine, maidine, norfluorocurarin and copsinin were obtained after the dispertion in citrare-phosfhate buffer and subsequent TLC. Accelerated restitution of granulocytopoiesis was observed in mice during both irradiation and myelotoxic drug-induced acute leucopenia. Increase in total WBC over 200% was observed after treatment by substance I in drug-induced leucopenia model (fivefold oral administration) and over 130% after treatment by substance I in irradiate mice (fivefold intraperitoneal administration). Morphological and anatomical structures of the underground organs of V. herbacea have been studied. The main microstructural characteristics are revealed - Rhizomes are characterized by coutinized epidermis, lamellar collenchyma, fibers and the texture of the vascular system of a monocyclic structure. The root system shows the whole cortex, the endoderm with Kaspar spots; the outer, radially continuous phloem tissue is located in the conducting system and distinguishes the cylindrical xylem tissue with annular and spiral-circular blood vessels.

The double-edged sword: Neurotoxicity of chemotherapy.

The number of available therapies for hematologic malignancies continues to grow at a rapid pace. Unfortunately, many of these treatments carry both central and peripheral nervous system toxicities, potentially limiting a patient's ability to tolerate a full course of treatment. Neurotoxicity with chemotherapy is common and second only to myelosuppression as a reason to limit dosing. This review addresses the neurotoxicity of newly available therapeutic agents including brentuximab vedotin and blinatumomab as well as classic ones such as methotrexate, vinca alkaloids and platinums. Although peripheral neuropathy is common with many drugs, other complications such as seizures and encephalopathy may require more immediate attention. Rapid recognition of adverse neurologic effects may lead to earlier treatment and appropriate adjustment of dosing regimens. In addition, knowledge of common toxicities may help differentiate chemotherapy-related symptoms from actual progression of cancer into the CNS.

Novel sugar esters proniosomes for transdermal delivery of vinpocetine: preclinical and clinical studies.

Vinpocetine (Vin) existing oral formulations suffer poor bioavailability (∼7%) since Vin undergoes a marked first-pass effect (∼75%) and its absorption is dissolution rate-limited. In this study, a novel sustained release proniosomal system was designed using sugar esters (SEs) as non-ionic surfactants in which proniosomes were converted to niosomes upon skin water hydration following topical application under occlusive conditions. Different in vitro aspects (encapsulation efficiency, vesicle size and shape, effect of occlusion, in vitro release, skin permeation and stability) were studied leading to an optimized formula that was assessed clinically for transdermal pharmacokinetics and skin irritation. All formulae exhibited high entrapment efficiencies, regardless of the surfactant HLB. Vesicle size analysis showed that all vesicles were in the range from 0.63 µm to 2.52 µm which favored efficient transdermal delivery. The extent of drug permeation through the skin from the optimized formula--containing laurate SE with shorter fatty acid chain length and high HLB--was quite high (91%) after 48 h under occlusive conditions. The extent of absorption of Vin from proniosomes was larger when compared to the oral tablet with a relative bioavailability (F(rel)) of 206%. Histopathological evaluation revealed only moderate skin irritation when using SEs compared to skin inflammation when using Tween 80. Sugar esters proniosomes may be a promising carrier for vinpocetine, especially due to their simple scaling up and their ability to control drug release.

Reducing undesirable hepatic clearance of a tumor-targeted vinca alkaloid via novel saccharopeptidic modifications.

During a phase I trial of EC145 (a folate-targeted vinca alkaloid conjugate), constipation was identified as the dose-limiting toxicity, probably from a nonfolate receptor-related liver clearance process capable of releasing unconjugated vinca alkaloid from EC145 and shuttling it to the bile. Here, we report on the selective placement of novel carbohydrate segments (1-amino-1-deoxy-glucitolyl-γ-glutamate) spaced in-between the folate and vinca alkaloid moieties of EC145, which yielded a new agent (EC0489) that is equipotent but less toxic than EC145. Whereas both compounds could cure tumor-bearing mice reproducibly, EC0489 differed from EC145 with i) a shorter elimination half-life, ii) approximately 70% decrease in bile clearance, iii) a 4-fold increase in urinary excretion, and iv) improved tolerability in rodents. This combination of improvements justified the clinical evaluation of EC0489 where currently administered dose levels have exceeded the maximal tolerated dose of EC145 by approximately 70%, thereby reflecting the translational benefits to this new approach.

Cytotoxicity of Vinca minor.

Vinca minor L. (Apocynaceae) is a medicinal plant that has long been used to treat cerebral and memory disorders in European folk medicine. Furthermore, it contains more than 50 alkaloids, some of them having bisindole structure such as the antineoplastic alkaloids present in Catharanthus roseus (L.) G. Don (Apocynaceae). In this study, the plant's alkaloid extract was divided into three fractions and the cytotoxic effects on cell proliferation of HT-29, Caco-2, T47D, and NIH/3T3 cell lines were examined. All alkaloid fractions showed a dose-dependent cytotoxic effect on the cell lines. IC(50) values confirmed that the growth and proliferation of NIH/3T3 cells were less affected in comparison to other cell lines.

Olesoxime prevents microtubule-targeting drug neurotoxicity: selective preservation of EB comets in differentiated neuronal cells.

Microtubule-targeting agents (MTAs), anticancer drugs widely used in the clinic, often induce peripheral neuropathy, a main dose-limiting side effect. The mechanism for this neurotoxicity remains poorly understood and there are still no approved therapies for neuropathies triggered by MTAs. Olesoxime (cholest-4-en-3-one, oxime; TRO19622) has shown marked neuroprotective properties in animals treated with paclitaxel and vincristine. The purpose of this study was to investigate its mechanism of neuroprotection against MTA neurotoxicity by using rat and human differentiated neuronal cells. We first showed that olesoxime prevented neurite shrinkage induced by MTAs in differentiated PC-12 and SK-N-SH neuroblastoma cell lines by up to 90%. This neuroprotective effect was correlated with enhanced EB1 accumulation at microtubule plus-ends, increased growth cone microtubule growing rate (20%) and decreased microtubule attenuation duration (54%). The effects of olesoxime on EB comets were specific for differentiated neuronal cells and were not seen either in proliferating neuroblastoma cells, glioblastoma cells or primary endothelial cells. Importantly, olesoxime did not alter MTA cytotoxic properties in a wide range of MTA-sensitive tumor cells, a prerequisite for future clinical application. Finally, olesoxime also counteracted MTA inhibition of microtubule-dependent mitochondria trafficking. These results provide additional insight into the neuroprotective properties of olesoxime, highlighting a role for microtubule dynamics in preservation of neurite architecture and axoplasmic transport, which are both disturbed by MTAs. The neuron-specific protective properties of olesoxime support its further development to treat MTA-induced neuropathy.

Carbohydrate-based synthetic approach to control toxicity profiles of folate-drug conjugates.

To better regulate the biodistribution of the vinblastine-folate conjugate, EC145, a new folate-spacer that incorporates 1-amino-1-deoxy-D-glucitol-gamma-glutamate subunits into a peptidic backbone, was synthesized. Synthesis of Fmoc-3,4;5,6-di-O-isopropylidene-1-amino-1-deoxy-D-glucitol-gamma-glutamate 20, suitable for Fmoc-strategy solid-phase peptide synthesis (SPPS), was achieved in four steps from delta-gluconolactone. Addition of alternating glutamic acid and 20 moieties onto a cysteine-loaded resin, followed by the addition of folate, deprotection, and cleavage, resulted in the isolation of the new folate-spacer: Pte-gammaGlu-(Glu(1-amino-1-deoxy-D-glucitol)-Glu)(2)-Glu(1-amino-1-deoxy-D-glucitol)-Cys-OH (21). The addition of 21 to an appropriately modified desacetylvinblastine hydrazide (DAVLBH) resulted in a conjugate (25) with an improved therapeutic index. Treatment of 25 with DTT in neutral buffer at room temperature demonstrated that free DAVLBH would be released under the reductive environment of the internalized endosome.

Effects of strictosamide on mouse brain and kidney Na+, K+-ATPase and Mg2+-ATPase activities.

Present study reports on the general bioactivity of strictosamide and on its effects on Na(+),K(+)-ATPase and Mg(2+)-ATPase activities of Charles River male mouse. Strictosamide is the main glycoalkaloid of Sarcocephalus latifolius (Rubiaceae) leaves and roots, used as medicinal plant in folk medicine. In this work, we studied the in vitro effects of various concentrations of strictosamide (0.25, 0.5, 1 or 2 mg/mL) and the in vivo effects of single doses (50, 100 or 200 mg/kg, i.p.) of this compound on kidney and brain Na(+),K(+)-ATPase and Mg(2+)-ATPase activities. Results of general study showed that strictosamide is slightly toxic to Charles River mouse (LD(50)=723.17 mg/kg), producing CNS depression and kidney toxicity, but the exact mechanism of these effects could not be defined. Strictosamide inhibited the in vitro and in vivo Mg(2+)-ATPase activity on kidney but had nonsignificant effect on brain. Furthermore, strictosamide had nonsignificant in vitro and in vivo effect on kidney Na(+),K(+)-ATPase activity but produced an in vivo increase of Na(+),K(+)-ATPase activity of brain, these findings suggesting that strictosamine may be related to the induction of alpha(2) isoform of Na(+),K(+)-ATPase and may account for the folk use of Sarcocephalus latifolius root infusion on hypertension.

Synthesis and biological evaluation of vinca alkaloids and phomopsin hybrids.

Ten hybrids of vinca alkaloids and phomopsin A have been synthesized by linking the octahydrophomopsin lateral chain to the tertiary amine of the cleavamine moiety of anhydrovinblastine (AVLB) and vinorelbine. These compounds have been elaborated in order to obtain original products that may interfere with both binding sites of vinblastine (VLB) and phomopsin in tubulin. Although NMR and molecular modeling studies have shown that the orientation of the added peptide chains of these hybrids is not the same as those of phomopsin A, most of them are very potent inhibitors of microtubules assembly and they present good cytotoxicity against KB cell line. These interesting biological activities may eventually be explained by the fact that their lateral chain resides in a pocket distinct from that of the phomopsin A peptide, at the interface of tubulins beta and alpha.

The thermodynamics of vinca alkaloid-induced tubulin spirals formation.

Vinca alkaloids are antimitotic, anticancer agents that induce tubulin to form spiral polymers at physiological protein concentrations. We used sedimentation velocity to investigate the effects of six vinca alkaloids on tubulin spiraling. Fitting to a Wyman linkage model reveals a drug dependent change of over two orders of magnitude in spiraling potential, K(1)K(2). Thermodynamic analysis of LnK(1)K(2) data demonstrates large and positive DeltaS values, indicating that tubulin spiral formation is entropically-driven. From the curvature in van't Hoff plots of vinblastine data, we estimate DeltaC(p) for GTP and GDP conditions to be -439 and -396 cal/mol K. Partitioning of DeltaS into the hydrophobic effect, DeltaS(HE), change in rotational/translational freedom, DeltaS(RT) and change in protein conformation, DeltaS(other), demonstrates that the major driving force for tubulin spiral formation is burial of hydrophobic surfaces and that protein conformational changes do not make a significant contribution. Spiraling potential is an indicator of antimitotic activity in vivo, although turbidity studies indicate that there is no correlation between spiraling potential and microtubule inhibition in vitro. Mechanisms that explain this discrepancy are discussed.

Vinca alkaloid-induced tubulin spiral formation correlates with cytotoxicity in the leukemic L1210 cell line.

The ability of a class of C-20' modified vinca alkaloid congeners to induce tubulin spiral formation was investigated relative to their ability to inhibit microtubule assembly, their cytotoxicity against a leukemic cell line, L1210, and their measured and calculated partition coefficients. These studies were prompted by the observation that the energetics of vinca alkaloid-induced tubulin spiral polymers, or spiraling potential, is inversely related to their clinical dosage and are aimed at the long-term goal of developing the ability to predict the cytotoxic and antineoplastic properties of antimitotic drugs. We demonstrate here that vinca-induced tubulin-spiraling potential is significantly correlated with cytotoxicity against L1210 cells. This is consistent with the size of spirals formed being proportional to the relaxation time for polymer redistribution, the lifetime of cell retention, and effects on microtubule ends and dynamics. Spiraling potential also correlates with calculated but not measured partition coefficients. Surprisingly, spiraling potential does not correlate with the ability to inhibit microtubule formation with purified tubulin or microtubule protein. For the set of C-20' modified compounds studied, the largest inhibitory effects on spiraling potential and cytotoxicity are caused by multiple sites of halogen (-F, -Cl) substitution with the introduction of increased rigidity in the ring. This suggests the C-20' position interacts with a hydrogen bond acceptor or an electrophilic region on the protein that electrostatically disfavors halogen substitutions. These studies are discussed in terms of the cellular mode of action of antimitotic drugs, particularly the importance of microtubule dynamics during mitosis and the factors that regulate those dynamics.

Vinflunine, a new vinca alkaloid: cytotoxicity, cellular accumulation and action on the interphasic and mitotic microtubule cytoskeleton of PtK2 cells.

Vinflunine, a newly synthesized derivative, possesses marked in vivo antitumor properties and, like other alkaloids, inhibits in vitro tubulin assembly at microM concentrations. However, in contrast to other vinca alkaloids, vinflunine exhibits relatively low in vitro cytotoxic potency. The aim of this report was to investigate whether the action(s) of vinflunine on the microtubule cytoskeleton could account for its cytotoxicity or if its cellular action requires another molecular target. Four vinca alkaloids used in cancer therapy and vinflunine were studied using PtK2 cells. Their activities on the most dynamic microtubules were investigated in mitosis and in interphase by evaluating the disturbance of the metaphase plate and the splitting of the diplosome, respectively. No correlation was observed between the cellular accumulation of these compounds and either their cytotoxicity or their action(s) on the microtubule cytoskeleton. In contrast, cytotoxicity, mitotic disturbance and diplosome splitting were observed in the nM range for vinblastine, vincristine, vindesine and vinorelbine, although these events occurred at 10 times higher concentrations in the case of vinflunine. Hence, dynamic modifications of both the mitotic and interphasic microtubule cytoskeleton are compatible with in vitro cytotoxicity of vinflunine, raising questions about the conventional biochemical screening of these vinca alkaloids.

Errors involving pediatric patients receiving chemotherapy: a literature review.

A review of mishaps involving pediatric patients receiving anticancer chemotherapy was undertaken in order to assist intervention. Although the case literature is too sparse to provide definite recommendations, suggestions for management are made in the event of an error with a high risk (based on the case literature) of life-threatening toxicities. It is recommended that all incidents be reported in the literature in order to provide a basis for devising standard treatment protocols. It is also suggested that studies using animal models continue to be done in order to provide more experimental data about toxicities and potentially beneficial rescue therapies.

Methods for the analysis of the new vinca alkaloid derivative, S 12363, in plasma by high-performance liquid chromatography with fluorescence detection.

Two sensitive analytical methods for the analysis of S 12363 in plasma are described. A highly sensitive procedure for human and dog plasma using cyanopropyl solid-phase extraction with ion pairing chromatography and fluorescence detection, has a limit of quantification of 0.1 ng ml-1. The technique has an overall precision and accuracy of 4.8 and 5.4% respectively over the concentration range 0.1-20 ng ml-1. A second, less sensitive, assay specifically adapted for rodent plasma, uses benzene sulphonyl cation-exchange solid-phase extraction followed by reversed-phase chromatography, with post-column fluorescence enhancement. This method has a limit of quantitation of 1.0 ng ml-1, with overall accuracy and precision of 7.2 and 11.6% respectively, over the concentration range 1.0-20.0 ng ml-1. Both assays have been successfully applied to dog and mouse toxicokinetic studies.

Image analysis of neuritic regeneration by adult rat dorsal root ganglion neurons in culture: quantification of the neurotoxicity of anticancer agents and of its prevention by nerve growth factor or basic fibroblast growth factor but not brain-derived neurotrophic factor or neurotrophin-3.

Peripheral neuropathies are a common side effect of chemotherapeutic agents, particularly antineoplastic drugs such as taxol, cisplatin, or vinca-alkaloids (vincristine, vinblastine, vindesine). Using dissociated cultures of adult rat dorsal root ganglion (DRG) neurons and video image analysis after neurofilament immunostaining, we have designed a system that allows: (i) rapid screening of potential neurotoxic agents, with the establishment of dose-response curves and the calculation of IC50; (ii) quantification of neurotrophic effects; and (iii) demonstration of neuroprotection by trophic factors. In particular, we show that nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) stimulate in vitro neuritic regeneration by adult rat DRG neurons, while brain-derived neurotrophic factor and neurotrophin-3 lack such effects. Furthermore, 24 h of pretreatment by NGF or bFGF drastically decreases the neurotoxic effect of vincristine and cisplatin.

Plasma pharmacokinetics of vinblastine and the investigational Vinca alkaloid N-(deacetyl-O-4-vinblastoyl-23)-L-ethyl isoleucinate in mice as determined by high-performance liquid chromatography.

The plasma pharmacokinetics of vinblastine and N-(deacetyl-O-4-vinblastoyl-23)-L-ethyl isoleucinate (VileE) in mice have been studied as part of the preclinical investigations of VileE, a new investigational semisynthetic Vinca alkaloid. Groups of animals received the test compounds through i.v. bolus injection at LD10, 0.5 x LD10, and 0.1 x LD10 doses. VileE has also been administered p.o. Drug plasma levels have been analyzed with a sensitive and selective method using liquid-liquid extraction for sample clean-up and high-performance liquid chromatography combined with fluorescence detection for quantification. Following i.v. injection, plasma kinetics of both vinblastine and VileE can be described adequately by a three-compartment open model. VileE demonstrates nonlinear pharmacokinetics with decreasing clearance and increasing terminal half-lives at increasing doses. Comparison of the plasma concentration versus time curves for vinblastine in humans and mice indicates that the toxicity of these compounds may not be directly related to the drug exposure expressed by the area under curve in plasma but by the terminal half-life and the time that a toxic threshold level is attained. Pharmacokinetically guided dose escalation in coming phase I trials of VileE is, therefore, discouraged.

Modulation of drug cytotoxicity in wild-type and multidrug-resistant tumor cells by stereoisomeric series of C-20'-vinblastine congeners that lack antimicrotubule activity.

Seven binary vinca alkaloid congeners were newly synthesized as the C14' or C16'(20') or C14'16'(20') stereoisomers of C20'-modified VBL. These congeners lacked detectable antimicrotubule activity in assays of polymerization of purified microtubule protein and of mitotic arrest induction. The compounds modulated the cytotoxicity of VBL, VCR, and DOX in sarcoma and colon-tumor cell lines. In wild-type cell lines, each congener elicited a concentration-dependent enhancement of cytotoxicity that was drug- and cell-type-selective. For example, C20'-deoxy C14'16'20'-epi VBL sensitized sarcoma S180 cells 19-fold to DOX and 11-fold to VCR but had no effect on VBL cytotoxicity. In the rat colon-cancer cell lines there was preferential enhancement of VCR cytotoxicity by most congeners. In two MDR cell strains of S180, the modulation potency of each congener was independent of specific drug or of resistance level. As a result, the amount of modulator (concentration) required for reversal was proportional to the drug-resistance level. Such properties were not displayed by the monomeric vinca alkaloid modulator vindoline. The potency of drug modulation in both wild-type and MDR cells strains was dependent on the stereoisomeric form of the congener and its C20'-substituents.

Relationship between the cellular accumulation and the cytotoxicity of S12363, a new Vinca alkaloid derivative.

S12363, a new vinca alkaloid derivative, was considerably more cytotoxic to murine L1210 cells and five human tumor cell lines (HL60, HT-29, COLO 320DM, NCI-H460, and PANC-1) than was vincristine (VCR) or vinblastine (VLB). S 12,363 bound to tubulin in crude extracts from brain or L1210 cells with an affinity similar to that of VLB and VCR (apparent Kd value: 1.1-1.6, 1.2-1.7, and 0.6-0.8 microM, respectively). After 1 h exposure, the accumulation of 20 nM [3H]-S 12,363 by L1210 cells was 4- to 18-fold that of [3H]-VLB and [3H]-VCR, respectively. After the cells had been preloaded for 1 h with the labeled drugs and then incubated for 3 h in drug-free medium, 37%-55% of the [3H]-S 12,363 was retained by the cells vs 36%-47% of the [3H]-VCR and less than 6% of the [3H]-VLB. Similar results were obtained for the five human cell lines tested. The accumulation factors (intracellular vs extracellular concentrations) found for [3H]-S 12,363 (54- to 167-fold) were significantly higher than those observed for [3H]-VCR (5- to 14-fold) or [3H]-VLB (19- to 41-fold). Greater than 90% of the radioactivity extracted from L1210 cells that had been treated with [3H]-S 12,363 was recovered as unmodified drug, demonstrating that [3H]-S 12,363 was not metabolized by these cells. S 12,362, which differs from S 12,363 only in the absolute configuration of the asymmetric carbon atom of its alpha-aminophosphonic side chain, was 300 times less cytotoxic, bound to tubulin with a lower affinity (apparent Kd value, 4.9-9.6 microM), and was neither accumulated nor retained by the cells. Taken together, these results demonstrate that the potency of S 12,363 is due at least in part to its cellular accumulation and retention.