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CATHARANTHUS ALKALOIDS

DEEKSHA PAHWA B.PHARM PUNJAB UNIVERSITY CHANDIGARH

CONTENTS  INTRODUCTION  CATHARANTHUS ALKALOIDS 1. Introduction 2. Extraction procedures of Vinca alkaloids 3. Biosynthesis of Catharanthus alkaloids 4. Cell and organ cultures
5. Semi-synthetic procedures of production of vinblastine

and vincristine 6. Mechanism of action 7. Clinical indications of the Vinca alkaloids 8. Toxicity
9. Effect of weather on the production of alkaloids in C.

roseus 10.Chemical Modifications of the Vinca alkaloids leading to the synthesis of novel alkaloids
11. Other

novel

cytotoxic

alkaloids

isolated

from

Catharanthus roseus.
12. Vinca alkaloids in clinical trials. 

TABLE : Vinca Alkaloids: Comparative Pharmacokinetic and Toxicologic Characteristics

 REFERENCES

INTRODUCTION
The genus Catharanthus (Family- Apocynaceae) is comprised of eight species of mostly perennial herbs. The single species Catharanthus pusillus is native to India. All other species, are native to Madagascar. The chromosome number for all Catharanthus species is 2n = 16. The different species are :
• • • • • • • •

Catharanthus coriaceus Markgr. Madagascar. Catharanthus lanceus (Bojer ex A.DC.) Pichon. Madagascar. Catharanthus longifolius (Pichon) Pichon. Madagascar. Catharanthus ovalis Markgr. Madagascar. Catharanthus pusillus (Murray) G.Don. Indian subcontinent. Catharanthus roseus (L.) G.Don. Madagascar. Catharanthus scitulus (Pichon) Pichon. Madagascar. Catharanthus trichophyllus (Baker) Pichon. Madagascar

Ornamental cultivars of Catharanthus are all derived from species native to Madagascar. Germplasm of C. roseus and C. trichophyllus has provided the primary material for development of ornamental cultivars. As an ornamental, Catharanthus, more commonly known as Madagascar Periwinkle or Vinca is valued for its drought and heat tolerance. In addition to its value as an herbaceous ornamental, alkaloid extracts of Catharanthus roseus have been used in folk medicine as an antidiabetic, diuretic, and antidysenteric, an anti haemorrhagic and for wound healing. In Europe, it was mainly used as an antidiabetic, for easing lung congestion and throat inflammation, in south and central Africa; as a poultice to stop bleeding in the US; in India in case of insect stings; against eye inflammation in the Caribbean and as an astringent, diuretic and cough remedy in China. Research on this plant was stimulated by its mention in folklore and investigations started in 1950 for antidiabetic activities. Antidiabetic activity could not be confirmed. However, the test animals became susceptible to bacterial infections on account of transient depression of the bone marrow leading to a net decrease in the number of circulating leukocytes. This led the researchers to undertake extensive examination for the possible immunosuppressive principles causing these effects. A number of dimeric indole alkaloids showing antileukaemic activity have subsequently been isolated and two of these, vincaleukoblastine (vinblastine) and leurocristine (vincristine), are now extracted commercially from

C.roseus and used, either alone, or in combination with other forms of therapy for cancer treatment. Catharanthus roseus G. Don., has been variously designated Vinca rosea L. and Lochnera rosea L. It is a rich source of alkaloids belonging to the category of terpenoid indole alkaloids which are isolated from the three varieties of the plant: (i) ‘roseus’ with violet or rose coloured flowers (ii) ‘albus’ with white flowers and (iii) ‘ocellatus’ with white flowers having red eye. Of these, the red purple variety contains the highest amount of vincristine and vinblastine.

Catharanthus roseus var. roseus

Catharanthus roseus var. ocellatus

Catharanthus roseus var. albus

CATHARANTHUS ALKALOIDS 1. Introduction
The aerial parts of the plant contain from 0.2 to 1% alkaloids. [1] About 150 alkaloids have been isolated from Catharanthus roseus.[2] Of particular interest is a group of 20 dimeric alkaloids which contains those with antineoplastic activity, including vincristine and vinblastine. These alkaloids are formed by the coupling of two moieties: an indole moiety and a dihydroindole moiety. Thus, this led to referring them as “dimer alkaloids” or “bisindole alkaloids. [1] Vinblastine is produced by coupling of two monomer alkaloids catharanthine (indole) and vindoline (dihydroindole), both of which occur free in the plant. Vincristine is structurally similar to vinblastine, but has a formyl group rather than a methyl on the indole nitrogen in the vindoline derived portion. Because these alkaloids are only minor constituents of the plant (vincristine is obtained in about 0.0002% yield from the crude drug), large quantities of raw materials are employed in the extraction procedures. Also there is a growing demand for vincristine rather than vinblastine, but the plant produces a much higher proportion of vinblastine. Fortunately, it is now possible to convert vinblastine into vincristine either chemically or via a microbiological N-demethylation using Streptomyces albogriseolus. [2] Other binary alkaloids which are active are leurosidine (20’-epivinblastine), leurosine (15’, 20’-epoxy vinblastine). [1] Some alkaloids, for example, ajmalicine, lochnerine, serpentine and tetrahydroalstonine, also occur in other genera of the family. [2]

2. Extraction procedures of Vinca alkaloids
Various extraction procedures employed are: a. b. c. d. Supercritical fluid extraction (SFE) Soxhlet extraction Solid-liquid extraction Hot water extraction

Supercritical fluid extraction Supercritical temperature of a substance is one above which the substance can no longer exist as a liquid, no matter how much pressure is applied. Similarly, supercritical pressure is a pressure above which a substance can no longer exist as a gas, no matter how high the temperature is raised. Under these conditions, the gas and liquid phases both possess the same density and no division exists between the two phases. This is the “critical state”. In practice, conditions somewhat above the critical temperature and pressure for a particular substance are usually used and these “supercritical fluids” exhibit properties intermediate between those of the liquid and gaseous phases. Most commonly used is supercritical fluid carbon dioxide (tc = 31oC and pc = 74atm). To render it more polar, a small amount of modifier, e.g. methanol, may be added to the carbon dioxide.[6] Other substances under development for use as supercritical fluids in SFE are: ethane, ethane, propane and nitrous oxide. In case of vinca alkaloids, vindoline is extracted by SFE. According to an optimization study conducted for the process, a remarkably high content of vindoline, 58 wt% was extracted from the leaves of Catharanthus roseus using SFE under conditions of temperature as 35oC and the pressure being 300 bars. The addition of 3 wt% ethanol as a cosolvent, only slightly improved the extraction yields.[3] Solid-liquid extraction The plant extract from C. roseus is a complex mixture of alkaloids with a wide range of polarities. The traditional solid-liquid extraction procedure for Catharanthus alkaloids from an aqueous acidic medium is based on their general basic properties. The alkaloids form salts in aqueous acidic media, showing improved solubility and enhanced stability at low pH values. In addition, protons in the aqueous acidic media assist in breaking the sample matrix to release the analytes more easily. As a further modification of the above process, water insoluble embonic acid complexes of catharanthine and vindoline were prepared by adding an aqueous alkaline (pH 10.5) solution of embonic acid (4,4’-methylene-bis-3hydroxynaphtalenecarboxylic acid) to the aqueous acidic solution (pH 1.5) of the plant extract containing the alkaloids as their soluble hydrochloride salts. The alkaloids were exhaustively precipitated when pH 5 was reached during this process. The precipitate mostly consisted of stable embonate complexes of catharanthine and vindoline, which were useful starting materials for vinblastine synthesis.[4] According to a recent study conducted, SFE method of extraction from dried leaves of C.roseus was optimized to give higher yields of the pharmacologically important indole alkaloids. Quantification of the alkaloid concentration was in the

range of 0.18 - 31 microg/ml. The yields obtained for catharanthine, vindoline, vinblastine and vincristine were 2.7, 2.0, 1.3and 1.1 microg/g.[5] Also different methods of extraction were compared for various indole alkaloids and best recoveries for catharanthine (100%) were obtained using SFE at 250 bar and 80oC, using 6.6vol% methanol as modifier for 40 minutes; for vindoline by Soxhlet extraction using dichloromethane in a reflux for16 hours; and for 3’,4’-anhydrovinblastine by solid-liquid extraction using a solution of 0.5M H2SO4 and methanol (3:1v/v) in an ultrasonic bath for 3hours.[5]

3. Biosynthesis of Catharanthus alkaloids
The various Catharanthus alkaloids belong to the class of terpenoid indole alkaloids, that is, they consist of two moieties derived from two separate metabolic pathways- The Mevalonate pathway which gives the non tryptophan moiety; and the tryptophan moiety is obtained from tryptophan. The complex structure of these alkaloids usually contains two nitrogen atoms; one is the indole nitrogen (in the tryptophan- derived moiety) and the second is generally two carbons removed from the Beta- position of the indole ring. The non- tryptophan moiety is derived from mevalonic acid and it is a C10-geraniol (monoterpenoid) contribution in the case of these alkaloids. This portion with suitable rearrangements leads to formation of three types of alkaloids(i) Coryanthe- type alkaloids (ii) Iboga- type alkaloids (iii) Aspidosperma- type alkaloids it is believed that the coryanthe- type monoterpenoid moiety is metabolically most primitive. The reactive form of terpene involves an aldehyde group. The loss of one carbon atom during biogenesis, to give C9 unit is largely common. Geraniol by a series of conversions forms loganin and then secologanin (a monoterpenoid glucoside). The molecular characterization of CYP72A1 from Catharanthus roseus was described nearly a decade ago, but the enzyme function remained unknown. However in a recent study conducted, it was shown that CYP72A1 converts loganin into secologanin.[56] A key intermediate in the biogenesis of the monoterpene indole alkaloids is 3alpha (S)- strictosidine, formed by the enzymatic condensation of tryptamine and secologanin. The enzyme responsible for this important reaction, strictosidine synthase, has been isolated and characterized from cell cultures of a number of species including Catharanthus roseus. Strictosidine then leads to formation of cathenamine (a coryanthe- type of alkaloid); enzyme involved is cathenamine synthase. Cathenamine further gives

rise to ajmalicine (enzyme- ajmalicine synthase) and serpentine. Both ajmalicine and serpentine are also coryanthe- type alkaloids. Cathenamine through a series of reactions also leads to formation of catharanthine (iboga- type) and vindoline (aspidosperma- type). Catharanthine and vindoline are monomeric indole alkaloids and occur free in the plant. 3’,4’-Anhydrovinblastine is a key intermediate from the coupling of catharanthine and vindoline and the enzymes involved are peroxidases. It is further converted to vinblastine.

4. Cell and organ cultures
In efforts to improve the production of alkaloids, cell cultures of C.roseus have received considerable attention. Earlier attempts at production in cell cultures failed because a part of the complex pathway was not active, i.e. from tabersonine to vindoline. The enzyme responsible for the conversion is tabersonine 16hydroxylase (T16H), a cytochrome P450-dependent enzyme. However, later it was found that T16H is induced in the suspension culture by light. [51] To date success has been achieved in obtaining total alkaloid yields corresponding to 0.1 – 1.5% (dry weight, cultivated cells). Alkaloids including catharanthine, vindoline and ajmalicine have been identified and isolated from these cell lines; however, the more useful dimeric alkaloids have only been isolated in traces from the cell cultures. [2] It has been observed that leaf organ cultures of C.roseus synthesize a variety of alkaloids. Also dimeric alkaloids have been detected in organ cultures of C.roseus, suggesting the possibility of an efficient production system for these valuable alkaloids. The dimers occurred only in those cultures which also contained vindoline and catharanthine. [9] However in a research conducted recently, the final dimerization step of the terpinoid indole pathway, leading to the synthesis of the dimeric alkaloid, vinblastine, was demonstrated to be catalyzed by a basic peroxidase and for the first time the cloning, characterization and localization of a novel basic peroxidase, CrPrx (Catharanthus roseus peroxidase), from C. roseus was done. The CrPrx nucleotide sequence encodes a translation product of 330 amino acids with a 21 amino acid signal peptide, suggesting that CrPrx is secretory in nature. The molecular mass of this unprocessed and unmodified protein is estimated to be 37.43 kDa. CrPrx was found to belong to a 'three intron' category of gene that encodes a class III basic secretory peroxidase. CrPrx protein and mRNA were found to be present in specific organs and were regulated by different stress treatments.

It is proposed that CrPrx is involved in cell wall synthesis, and also that the gene is induced under methyl jasmonate treatment indicating its potential involvement in the terpenoid indole alkaloid biosynthetic pathway. [55] In one of the experiments conducted, a two-liquid-phase bioreactor was designed to extract indole alkaloids from C.roseus ‘hairy root cultures’, using silicon oil. Partition studies between silicon oil and culture medium showed that the affinity of tabersonine and löchnericine for silicon oil is nine times higher than for the aqueous phase. Also, all measured alkaloids' specific yields were higher using silicon oil and elicitation, suggesting that the silicon oil, by acting as a metabolic sink for tabersonine and löchnericine, decreased their negative feedback effect on the production of the other useful alkaloids in the aqueous medium and so was efficient in increasing metabolic fluxes of the secondary metabolism pathways. [10] The transformed root culture seems to be the most promising for alkaloid production. The genetically transformed roots, obtained by the infection with Agrobacterium rhizogenes, produce higher levels of secondary metabolites than intact plants. Also, whole plants can be regenerated from hairy roots. The content of indole alkaloids in the transformed roots was similar or even higher when compared to the amounts measured in studies of natural roots. The predominant alkaloids in transformed roots are ajmalicine, serpentine, vindoline and catharanthine, found in higher amounts than in untransformed roots. Transformed hairy roots have been also used for encapsulation in calcium alginate to form artificial seeds.[57] • Role of the Non-Mevalonate Pathway in Indole Alkaloid Production by Catharanthus roseus Hairy Roots :

The 1-deoxy-D-xylulose-5-phosphate (DXP) pathway (non-mevalonate pathway) leading to terpenoids via isopentenyl diphosphate (IPP) has been shown to occur in most bacteria and in all higher plants. Treatment with the antibiotic fosmidomycin, a specific inhibitor of DXP reductoisomerase, considerably inhibited the accumulation of the alkaloids ajmalicine, tabersonine, and lochnericine by Catharanthus roseus hairy root cultures in the exponential growth phase. These results suggest that the DXP pathway is a major provider of carbon for the monoterpenoid pathway leading to the formation of indole alkaloids in C. roseus hairy roots in the exponential phase.[58]

5. Semi-synthetic procedures of production of vinblastine and vincristine

Vindoline and catharanthine are the major monomer alkaloids as well as biosynthetic precursors for the "dimeric" alkaloids, vinblastine and vincristine. Low "dimeric" alkaloid contents in the plant have encouraged intense research for alternative production methods involving cell cultures,[18,19] metabolic engineering,[20] semi-synthesis,[21,22] or even total chemical synthesis.[23] Total synthesis has proved difficult due to structural complexity of the molecules and complicated reaction steps involving stereochemical constraints. Various semisynthetic procedures have been developed for these alkaloids on the basis of chemical[21,22] or enzymatic[24] coupling of commercially available catharanthine and vindoline. The present semi-synthetic process is partially based on an earlier method involving photoactivation of catharanthine, as well as photolytic singlet oxygen production in an aqueous reaction solution.[9] In that method the high energy needed for singlet oxygen production from water, however, results in excessive catharanthine consumption by many side reactions. As an improvement to the above photochemical method, singlet oxygen (1O2) is here produced in situ from hydrogen peroxide and sodium hypochlorite [19]. An allylic hydrogen in the protonated catharanthine moiety is presumably attacked by singlet oxygen to yield a hydroperoxide of catharanthine which then possibly undergoes reduction in the presence of sodium borohydride. Then a final aromatic substitution reaction yields vinblastine through the crucial C18'-C15 bond linking the top indole half of the catharanthine moiety (C18') and the bottom dihydroindole half of the vindoline moiety (C15).

6. Mechanism of action
Vinblastine and vincristine are antimitotics. They bind to tubulin and prevent the formation of the microtubules which help in the formation of the mitotic spindle. Thus, these compounds block mitosis and cause an accumulation of cells in the metaphase (“metaphase arrest”).[11-16] This contributes to their major pharmacological action.[1] The microtubule assembly also plays a role at other levels, particularly in neurotransmission (axon microtubules). Hence blockade of this activity is responsible for the neurotoxicity caused as a side effect by these alkaloids. [1] They are generally in vitro inhibitors of the biosynthesis of protein and nucleic acid, elevate oxidized glutathione, alter lipid metabolism and membrane lipids, elevate cyclic AMP and inhibit calcium-calmodulin regulated cyclic AMP phosphodiesterase.[17]

The treatment of cell population with vincristine or vinblastine, leads to an accumulation of cells in the M and G2 phase and the effect is lethal in the S phase.[1]

7. Clinical indications of the Vinca alkaloids
Vincristine is used more commonly to treat pediatric malignancies, which likely reflects a combination of the higher level of sensitivity of pediatric malignancies to vincristine and to the better tolerance of higher vincristine doses in children. In both children and adults, however, vincristine is an essential component of the chemotherapy regimens used to treat acute lymphocytic leukemia, lymphoid blast crisis of chronic myeloid leukemia, and both Hodgkin and non-Hodgkin lymphomas. It also plays a role in some multimodality therapies of Wilms tumor, Ewing sarcoma, neuroblastoma, and rhabdomyosarcoma, as well as in the treatment of multiple myeloma and small-cell lung cancer in adults.[34]

Vincristine On the other hand, vinblastine has been a mainstay component of chemotherapy regimens for germ cell malignancies and some types of advanced lymphomas. On a historical note, vinblastine has also been used alone or in combination with other agents to treat Kaposi sarcoma and bladder, breast, and some types of brain malignancies.[34] Vincristine has a superior antitumour activity as compared with vinblastine, but the former is more neurotoxic.[2]

Vinblastine Desacetyl vinblastine (vindesine), initially identified as a metabolite of vinblastine, was introduced in the 1970s.[25-29] Vindesine is available only for investigational purposes in the United States, but is registered elsewhere. The agent was principally evaluated in combination with other agents, particularly cisplatin and/or mitomycin C, in treating non-small-cell lung cancer, but it has also demonstrated consistently favorable results in several hematologic and solid malignancies.[28,29]

V inde sine The newer, orally active,[2] vinblastine derivative semi-synthesized by Potier et al. Vinorelbine (5’-norhydro Vinblastine), has broader anticancer activity and lower neurotoxic side-effects than the other Catharanthus alkaloids.[8] It is structurally modified on its catharanthine nucleus, resulting in substantially greater lipophilicity as compared to the other Vinca alkaloids. The potent antitumor effect of Vinorelbine with minor neurotoxicity was explained by Vinorelbine having stronger activity on mitotic microtubules than axonal microtubules.[53] It is effective in combination with chemotherapeutic agents such as anthracycline, fluorouracil and Taxol. It is approved in the United States for treating non-smallcell lung cancer as either a single agent or in combination with cisplatin, and has been registered to treat patients with advanced breast cancer elsewhere. [30-33] Vinorelbine has also demonstrated anticancer activity in advanced ovarian carcinoma and lymphoma; however, a unique role in the therapy of these malignancies has not been defined.

Vinorelbine Tartrate

8. Toxicity
Although the Vinca alkaloids are quite similar from a structural standpoint, their toxicologic profiles differ significantly. All of the Vinca alkaloids induce a characteristic peripheral neurotoxicity, but VCR is most potent in this regard. The neurotoxicity is principally characterized by a peripheral, symmetric mixed sensory-motor, and autonomic polyneuropathy.[32,35,36,37-41] The primary pathologic effect is axonal degeneration and decreased axonal transport, most likely caused by a drug-induced perturbation of microtubule function. Toxic manifestations include constipation, abdominal cramps, paralytic ileus, urinary retention, orthostatic hypotension, and hypertension. severe neurotoxicity is observed less frequently with VBL, VDS, and VRL, as compared to VCR.[42,43] VRL has a lower affinity for axonal microtubules than either VCR or VBL, which seems to be confirmed by clinical observations.[30-32,43,44] VCR treatment in patients with hepatic dysfunction or obstructive liver disease is associated with an increased risk of developing neuropathy because of impaired drug metabolism and delayed biliary excretion. Neutropenia is the principal doselimiting toxicity of VBL, VDS, and VRL. Thrombocytopenia and anemia are usually less common and less severe.

Gastrointestinal toxicities, aside from those caused by autonomic dysfunction, may be caused by all the Vinca alkaloids. [26,28,37,38,41,46] Mucositis occurs more frequently with VBL than VRL or VDS, and is least common with VCR. Nausea, vomiting, and diarrhea may also occur to a lesser extent. Pancreatitis has also been reported with VRL. [47] The Vinca alkaloids are potent vesicants and may cause significant tissue damage if extravasation occurs. If extravasation occurs or is suspected, treatment should be discontinued immediately and aspiration of any residual drug remaining in the tissues should be attempted. [48] The application of local heat and injection of hyaluronidase, 150 mg subcutaneously, in a circumferential manner around the needle site are thought to minimize both discomfort and latent cellulitis, perhaps by facilitating drug dispersion. Because of their remarkable vesicant properties, the Vinca alkaloids should not be administered intramuscularly, subcutaneously, intravesically, or intraperitoneally. Direct intrathecal injection of VCR and other Vinca alkaloids, which has occurred as inadvertent clinical mishaps, induces a severe myeloencephalopathy characterized by ascending motor and sensory neuropathies, encephalopathy, and rapid death.[26,28,49,50] Mild and reversible alopecia occurs in approximately 10% and 20% of patients treated with VRL and VCR, respectively. The only known effective intervention for Vinca alkaloid neurotoxicity is discontinuing treatment or reduction of the dose or frequency of drug administration. Although a number of antidotes, including thiamine, vitamin B12, folinic acid, pyridoxine, and neuroactive agents (eg, sedatives, tranquilizers, anticonvulsants), have been used, these treatments have not been clearly shown to be effective.[26,28] Folinic acid protects mice against otherwise lethal doses of the Vinca alkaloids, and there are anecdotal reports of its successful use following VCR overdosage in man; however, prospective studies have never been performed.

9. Effect of weather on the production of alkaloids in C. roseus
In a study one set of plants were grown in rainy season from March-April to SeptOct. Another set was grown in winter season from Sept-Oct to March-Apr. [62]

Vincristine was totally absent from root material. It is also reproted that bisindole alkaloids and vindoline accumulate only in green tissue and are not found in root or cell suspension cultures. Also full sunshine is reported to give a higher sontent of alkaloids than shade. Table-1 Amount of different alkaloid (mg/gm) under difference season in leaf and root material of two varieties of c-roseus Type of Tissue Variation in Season Rainy Leaf Winter Rainy Root Winter Variety roseus alba roseus alba roseus alba roseus alba Ajmaline 1.09 N 3.38 N 4.52 N 3.0 0.85 Vincristine 1.39 3.83 8.75 N N N N N Ajmalicine 0.4 N 0.91 N 2.1 N N N

Table -2 Season Rainy alba roseus Winter alba LSD at 5%
a = on dry weight basis

Variety roseus

Total Alkaloid Leaves (mg/kg)a 45 40 48 39 3.5

Roots (mg/kg)a 24 28 26 20 2.8

Total alkaloid content was highest in ‘roseus’ variety during winter. Highest amount of root alkaloid was noted in ‘alba’ variety when grown in rainy season and in ‘roseus’ in winter season. Minimum alkaloid content was noted in ‘alba’ variety during the winter months. Present study showed that seasons have impact on the biomass and alkaloid production, both qualitatively and quantitatively on genotypes of C. roseus and variety ‘roseus’ was found to be superior to variety ‘alba’.

10. Chemical Modifications of the Vinca alkaloids leading to the synthesis of novel alkaloids
Several hundred derivatives have been synthesized and evaluated for their pharmacological activities, the majority being modified in the vindoline moiety, bearing several reactive centers. These efforts led to the identification of the amido derivative vindesine, registered in Europe in 1980 and now available in several countries. Then novel chemistry permitted the semisynthesis of derivatives modified in the velbenamine "upper" part of the molecule, creating a new potential in the Vinca alkaloids medicinal chemistry: as a result, vinorelbine, obtained by C' ring contraction of anhydrovinblastine, and is now marketed worldwide. Several strategies aimed at the total synthesis of vinblastine derivatives have been investigated, giving the opportunity to design rationally certain compounds. Modifications in the D' ring appeared to induce dramatic changes in the tubulin interactions. These observations have been confirmed recently by the identification of unprecedented pharmacological properties exerted by the novel fluorinated Vinca alkaloid, vinflunine.[52] The two second-generation Vinca alkaloids, vinorelbine and vinflunine, affect microtubule dynamics very differently from vinblastine, a first generation Vinca alkaloid. For example, vinblastine strongly suppresses the rate and extent of microtubule shortening in vitro, whereas vinorelbine and vinflunine suppress the rate and extent of microtubule growing events. There was doubt whether these differences result in differences in mitotic spindle organization that might be responsible for the superior antitumor activities of the two second-generation Vinca alkaloids. However,Despite differences in their actions on individual dynamic instability parameters, morphologically detectable differences in spindle effects among the

three drugs were minimal, indicating that overall suppression of dynamics may be more important in blocking mitosis than specific effects on growth or shortening. the peak intracellular drug concentration at the mitotic IC(50) value was highest for vinflunine (4.2 +/- 0.2 microM), intermediate for vinorelbine (1.3 +/- 0.1 microM), and more than 10-fold lower for vinblastine (130 +/- 7 nM), suggesting that intracellular binding reservoir(s) may be partially responsible for vinflunine's high efficacy and minimal side effects. [54]

11. Other novel cytotoxic alkaloids isolated from Catharanthus roseus
(i) According to a recent study, BM6, a new semi-synthetic vinca alkaloid, exhibits its potent in vivo anti-tumor activities via its high binding affinity for tubulin and improved pharmacokinetic profiles. The aim of this study was to evaluate the anti-tumor activities and to establish the mechanism of the action of 3-decarboxyl-acetyloxylmethyl-anhydrovinblastine (BM6). BM6 was characterized by its superior in vivo activity to vinorelbine in preclinical tumor models, though BM6 exerted in vitro cytotoxic activity against a wide spectrum of tumor cell lines with IC(50) values generally 10-fold higher than the classic Vinca alkaloids. Of note, BM6 displayed more potent cytotoxic activity against multidrug-resistant sublines.

BM6 also induced significant cell cycle arrested in mitosis and cytoskeleton disruption via interacting with the Vinca binding site on tubulin. Encouragingly, the features in term of its higher tubulin binding affinities and better pharmacokinetic profiles highlight BM6 distinct from other Vinca alkaloids. [59] (ii) Two New bisindole alkaloids, Vingramine 1 and Methylvingramine 2, were isolated from the Seeds of Catharanthus roseus. Their structures were determined by one- and two-dimensional NMR experiments. They possess a new bisindole skeleton involving an indole alkaloid part B with loss of 5',6'-ethylene, a C7'-C16'

linkage, a 14'-O-19'-tetrahydrofuran, and a N-4'-isobutyramide group. The 12methyl vincorine part A and part B are connected via an 11,10'-biphenyl linkage. These alkaloids display, in vitro, cytotoxic activity against nasopharynx carcinoma KB cells. [61]

(iii) 16-Epi-Z-isositsirikine, a monomeric indole alkaloid with antineoplastic activity from Catharanthus roseus. The compound displayed antineoplastic activity in the KB test system in vitro and the P-388 test system in vivo. [60]

12. Vinca alkaloids in clinical trials
Vinflunine : Vinflunine is a new Vinca alkaloid uniquely fluorinated, by the use of superacid chemistry, in a little exploited region of the catharanthine moiety. In vitro investigations have confirmed the mitotic-arresting and tubulin-interacting properties of vinflunine shared by other Vinca alkaloids. However, differences in terms of the inhibitory effects of vinflunine on microtubules dynamics and its tubulin binding affinities have been identified which appear to distinguish it from the other Vinca alkaloids. Studies investigating the in vitro cytotoxicity of vinflunine in combination therapy have revealed a high level of synergy when vinflunine was combined with either cisplatin, mitomycin C, doxorubicin or 5-fluorouracil. Furthermore, although

vinflunine appears to participate in P-glycoprotein-mediated drug resistance mechanisms, it has proved only a weak substrate for this protein and a far less potent inducer of resistance than vinorelbine. Vinflunine was identified in preclinical studies as having marked antitumour activity in vivo against a large panel of experimental tumour models, with tumour regressions being recorded in human renal and small cell lung cancer tumour xenografts. Overall its level of activity was superior to that of vinorelbine in many of the experimental models used. Interestingly, an in vivo study using a well vascularised adenocarcinoma of the colon has suggested that vinflunine mediates its antitumour activity at least in part via an antivascular mechanism, even at sub-cytotoxic doses. Therefore, these data provide a favourable preclinical profile for vinflunine, supporting its promising candidacy for clinical development. Phase I and Phase II evaluations of vinflunine have been completed in Europe and phase III clinical trials are now ongoing. [62-63]

vinflunine Table-3 Vinca Alkaloids: Characteristics Comparative Pharmacokinetic and Toxicologic

Formula Standard adult dose range (mg/m2/wk) Route Bioavailability

Vincristine C46H56N4O10 1–2 i.v. n.a.

Vinblastine Vindesine Vinorelbine C46H58N4O9 C43H55N5O7 C45H54N4O8 6–8 i.v. n.a. 3–4 i.v. n.a. 15–30 i.v., oral 79-91%

Protein Binding ~75% Pharmacokinetic Triphasic behavior Plasma half-lives α (min) <5 β (min) 50–155 γ (h) 23–85 Clearance (L/h/kg) Primary route 0.16

~75% Triphasic

65-75% Triphasic

43% (oral) Triphasic

<5 53–99 20–64 0.74 Hepatic metabolism and biliary elimination Neutropenia Alopecia, neurotoxicity, mucositis

<5 55–99 20–24 0.25 Hepatic metabolism and biliary elimination Neutropenia Alopecia, neurotoxicity

<5 49–168 18–49 0.4–1.29 Hepatic metabolism and biliary elimination Neutropenia

Hepatic metabolism and biliary elimination Principal toxicity Neurotoxicity Other toxicities Constipation, SIADH

Neurotoxicity, vomiting, constipation, mucositis SIADH = syndrome of inappropriate secretion of antidiuretic hormone.

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TABLES : Table-1. and Table-2. : Gupta K, Influence of seasons on biomass and alkaloid productivity in Catharanthus roseus, Journal of Medicinal and Aromatic Plant Sciences, 24(3), 2002 : 664-68. Table-3 : Edited by Kufe, Pollock, Wiechselbaum, Bast, Gansler, Holland, Frei, ‘Cancer Medicine’, 2003, Hamilton•London, UK: BC Decker Inc, Section 12 (53).