Antimitotic, Antitumor and Anticancer Constituents

Antimitotic, Antitumor and Anticancer Constituents!

Cancer is a general term applied to a series of malignant diseases which may affect many differ­ent parts of the body. These diseases are charac­terized by a rapid and uncontrolled formation of abnormal cells which may macs together to form a growth or tumors, or proliferate throughout the body, initiating abnormal growth at other sites.

If the process is not arrested, it may progress until it causes the death of the organism, hence the search for plant drugs amongst others, which should kill or incapacitate cancer cells without causing ex­cessive damage to normal host cells. In 2000, there were an estimated 170,000 new cases of prostate cancer and 31,000 deaths in the United States. Cancer is therefore the second cause of death worldwide.

Research focused on discovery of plant anticancer constituents become increasingly productive, and agents discovered include the amoorastatins, aphanastatin, bauhinia-statins, combretastatin (of which CA-4 and CA-1 are in clinical trials), the lychno-statins, the meliastatins, multigilin, multistatin, narcistatin, the nootkastatins, palstatin, pancratistatin (in advanced preclini­cal development), pedilstatin, thephyllantostatins (1 and 2 completed phase I clinical trials), radiatin, rolliniastatins, the sansevistatins, and the schleicherastatins.

Pettitetal., 1998,2000 isolated the first cancer cell growth inhibitor, (-)-combretastatin from African tree Combretum caffrum, a representative of the tropical and subtropical trees of the family Combretaceae (about 600 species in 20 genera) with a reserve of constituents with potentially useful biological properties, as up to 25 species are used in the traditional medicine of Africa and India.

This research subsequently led to discovery of 20 cancer cell growth inhibitory stilbenes, bibenzyls, dihydrophenanthrenes, and phenanthrenes which had resulted in the development of the first well established anti-angiogenic/vascular-disrupting anticancer drug, sodium combretastatin A-4 phosphate (CA4P), which is in broad human cancer clinical trials.

The combretastatins, despte their relative sim­ple chemical structures are characterized by re­markable biological activity as inhibitors of tubu­lin polymerization, while some exhibit potent in vitro inhibition against human cancer lines, in vivo efficacy as vascular disrupting agents, and act as anti-angiogenesis agents.

Tumors receive their nutrients through existing vasculature using new­ly formed neovascularization (angiogenesis), and the combretastatin A-4 (CA4) and A-1 (CA1) phos­phate pro-drugs (CA4P and CA1P) selectively dam­age tumor neo-vasculature with induction of ex­tensive blood flow shutdown in the metastatic tu­mor compared to normal tissues, thereby causing hypoxia and necrosis of the tumor.

This led to four Phase I human cancer clinical trials of CA4P while later clinical trials have incorporated com­bination with other anticancer drugs such as paclitaxel (Taxol), Carboplatin, Avastin (anti-angiogenic monoclonal), and/or fractionated external beam radiotherapy.

In July of 2003, the US FDA awarded orphan drug status to CA4P for treatment of advanced anaplastic thyroid cancer and in May of 2006 orphan drug status to CA4P for treatment of ovarian cancer in combination with Carbopl­atin.

Highlights of parallel studies of microorgan­ism anticancer constituents include the isolation and structural elucidations of carminomycin (in clinical use), kitastatin 1, the labradorins, Montana statin and the streptomyces antitumor antibiot­ic 593A (clinical trials by the NCI), as well as con­venient syntheses of the Streptomyces anticancer drugs, DON and azotomycin.

Doskotch et al., 1983 and El Naggar et al., 1980 had also worked independently, particular­ly on the constituents of local North American flo­ra, with cytotoxic (antitumor) activity and insect stimulants (for the elm bark beetle) and insect antifeedants (for gypsy moth larvae). Of importance are the work of Sun et al., 1998 and Xue et al., 2001 on anticancer and chemo preventive agents from plants.

The efforts in discovering antitumor agents are based on bioactivity-directed fractionation and isolation from medicinal plants, especially those used in traditional Chinese medicine, followed by rational drug design-based methods to produce the most promising lead for development.

Isolat­ed therapeutic agents from plants can also serve as leads in the development of better and more effective drugs.

From podophyllotoxin, a lignan isolated from Podophyllum peltatum (American mandrake, which was previously used as cathar­tics in folklore medicine) was developed etopside and teniposide as potent drugs used in the treat­ment of small-cell lung cancer, testicular cancer, lymphomas and leukem.

Podophyllotoxin and derivatives are from Podo­phyllum plants, including the American P. peltatum L. and Tibetan P. emodi, were long prized as medicines by the native populations of North America and the Himalayas.

The complex path from the plan (Podophyllum) to bioactive lead compound (podophyllotoxin) to clinically used drugs (etoposide and teniposide) and forward to new generation anticancer candidates illustrates the successful development of clinically useful drugs from natural (plant) sources.

In 1942, studies found that podophyllin, the alcoholic extract of Podophyllum rhizome, exhibited curative effects on the benign tumor Condylomata accuminata. Further research showed that its major constituent, podophyllotoxin, inhibits assembly of the mitotic spindle and was effective against tumors in mice.

Mechanistically, podophyllotoxin binds reversibly to cellular tubulin, inhibits mitotic spindle formation, disturbs the dynamic equilibrium between microtubule assembly/disassembly, and eventually causes mitotic arrest.

Studies on Podophyllum glycosides in the 1950s led to the opportune discovery of etoposide and teniposide in the late 1960s. These two compounds displayed significant antineoplastic effects. In 1983, the FDA approved etoposide for the treatment of testicular cancer and in 1992 teniposide was brought into the US market.

These drugs are currently used against many cancers, including small cell tung cancer, testicular cancer, lymphoma, leukemia, and Kaposi’s sarcoma.

There­fore, the synthetic etoposide analog, GL-331, de­rived from podophyllotoxin, was designed successfully to combat solubility, drug resistance, and other detrimental issues of the an­ticancer drug etoposide, which has been devel­oped to Phase II clinical trials.

In addition JC-9, discovered based on the curcumin model, has been licensed and developed by Androscience also against prostate cancer.

The work of Potier et al., 1989 has produced two major anticancer drugs, Navelbine (vinorelbine), an analog of vinblastine isolated from the Madagascan periwinkle and Taxotere (docetaxel), an analog of Taxol (paclitaxel) extracted from the bark of the yew tree. Pierre Fabre Laboratories and Sanofi-Aventis manufac­ture these two drugs, respectively.

The diterpenoid taxol has become one of the most promising lead compounds to emerge from the anti-tumour screening of natural products in recent years. Isolated from the stem bark of the Pacific yew Taxus brevifolia Nutt. (Taxaceae) and Taxus baccata L. Taxol possesses an original, highly functionalized diterpene steleton.

The mode of action of taxol is also unique. In contrast to other antimitotic drugs, it enhances both the rate and yield of microtubule assembly.

The major obstacle in the development of tax­ol to the clinical stage has been its very limited supply from natural sources. The compound oc­curs only in the stem bark at low concentrations. In order to supply 2.5 kg of taxol for clinical trial, the N atior.al Cancer Institute contracted for 2 7 tons of T.brevifolia bark, requiring the sacrifice of some 12000 C trees.

With the current demand for taxol, it has been estimated that yew population world­wide would soon become extinct if no alternative source of taxol could be developed. A diterpe­noid alkaloid?, a natural product with major anticancer activity against breast and cancers was developed.

However, an economically or technically re­alistic approach to the supply problem has been devised by French researchers with the partial syn­thesis of taxol from a congener found in the leaves in appreciable amounts.

The side-chain analog RP 56976 has reportedly greater chemotherapeutic potential than taxol itself and it has been devel­oped and registered as Paclitel. A side chain ana­log of taxol (Taxus baccata), known as taxotere, has also beer, developed by semi-synthesis from 10-deacetyIbaccatir III.

It has improved water solubility and better activity than taxol in some assays, and is also currently available for drug use against resistant breast cancer. Similarly, elucida­tion of the structure of Ellipticine, an alkaloid ex­tracted from Ochrosia sp., led to the synthesis of Celiptium, used for a few years in the treatment of breast cancer.

Acronycine, another alkaloid found in an Australian Acronychia led Francois Tillequin to develop very active synthetic analogs,  isolated the anti­cancer withanolides from Withania somnifera which was, among other uses, used as an antie-pressant.

Throughout the 1990s the NCI Laboratory of Drug Discovery Research and Development nat­ural products efforts focused primarily on extracts with selective cytotoxicity profiles in the 60-ceII anticancer screen or extracts with HIV inhibiting properties in the anti-HIV screen. The significant anticancer schweinfurthins  are notable terrestrial plant anticancer agents.

Another notable natural products work are those of Crews et al., 2004, and Ireland et al., 2003 which include four marketed agents, one of them is topotecan, a camptothecin analog with superi­or properties to the parent compound that was dropped from clinical trial due to unacceptable toxicity, and is marketed by SmithKline Beecham (now GlaxoSmithKline).

Twenty-one more agents have entered clinical trials including four derived from natural products.

Dictyostatin-1 was originally reported by Pettit et at., 1994 as a potent cytotoxic agent. Further studies showed that it is a potent microtubule-stabilizing agent with very significant activity against paclitaxel and multi-drug resistant cancer cell lines.

Neopeltolide also demonstrated potent cyto­toxicity, and cell cycle analysis of tumor cells showed arrest at the G1/S transition.

The discov­eries of camptothecin in 1966 and taxol in 1971 (Wani et al., 1971) have had a huge impact on cancer chemotherapy, and as just one indicator, in 2002, the global market place for antineoplastic agents was estimated at $9 bil­lion annually with the combined sales of taxol and the camptothecin analogs totalling at least a third of that market.

Camptothecin and derivatives from Camptotheca acuminata showed broad-spectrum activity with some limitations. The natural 10-hydroxycamptothecin is more active than camptothecin, and is used in China against cancers of the neck and head.

The synthetic analogs 9-aminocamptothecin and particularly the water-soluble derivatives topotecan and irinotecan showed good responses in a number of cancers, topotecan and irinotecan are available for the treatment of ovarian cancer and colorectal cancer respectively. Thanks to the relentless efforts of Drs. Cordell, Farnsworth, Kinghorn, Pezzuto, and Soejarto among others.

By 1970 the cephalotaxine esters harringtonine, iso-harringtonine and homoharringtonine were identified by Powell et al., 1970, 1972 as the anticancer principles of Cephalotaxus harringtonia, a small tree native to Japan and China.

Homoharringtonine has shown encouraging ac­tivity in preclinical and Phase l-ll trials in patients with hematologic and some solid tumors. Early studies in China reported high response rates in patients with leukemia, and studies in the United States and England have shown promising results in patients with chronic myeloid leukemia.

This has been granted orphan drug sta­tus by the US FDA, which is being promoted by ChemGenex Pharmaceuticals under the trademark Ceflatonin. These actions have encouraged ex­panded clinical trials and evaluation of the poten­tial of this drug for treatment of various malignant diseases.

Maytansinoids/Ansamitocins have been im­plicated in anticancer therapy. Maytansine, the parent compound of this group of potent antitu­mor agents was discovered in 1972 by Kupchan et al., 1972 by bioassay-guided fractionation of the extracts of the Ethiopian shrub Maytenus serrata.

Since this report, about 50 additional mem­bers of this group of compounds have been isolat­ed by several investigators from the same and closely related plants and from members of two unrelated plant families.

The mode of action of the maytansinoids has been traced to inhibition of tubulin-polymerization by binding to the (β-subunit at a site overlapping the vincristine binding site, and different from the colchicines binding site.

After biological testing showed that the ex­tract obtained from the Western (or Pacific) yew, Taxus brevifolia had activity against the 9KB cell line, led to the isolation of tax with in vivo activity against P388 lymphocytic leukemia, which was found to act by promoting the assembly of tubulin into microtubules.

Taxol entered Phase I clinical trials in 1983 while the first positive Phase II clinical results showed activity against ovarian cancer and the result turned taxol from an obscure lead compound into an instant celebrity, and also found against breast cancer in 1991.

Taxol is presently marketed as paclitaxel and together with its rela­tive docetaxel, the worldwide sales totaled approx­imately US $4 billion in 2005.

The research efforts on the Southeast Asian Catharanthus roseus led to the isolation of vinc­ristine and vinblastine with activity against the P- 1534 leukemia.They have been developed as commercial drugs. Vin­cristine was approved by FDA and marketed in 1963.

A major importance of the discovery of vin­cristine was its novel mechanism of action (at the time); blocking mitosis with metaphase arrest and specifically binding with tubulin and preventing its polymerization.

Because of this, vincristine became an important component of combination chemotherapy. The major use of vinblastine is the treatment of patients with Hodgkin’s disease, non-Hodgkin’s lymphomas, and renal, testicular, head and neck cancer.

Two additional semi-synthetic alkaloids relat­ed to vincristine and vinblastine, vinorelbine and vindesine were marketed.

The types of anticancer agents isolated from plants was also presented by the review of Hartwell in 1976 in which the active compounds isolated from twenty-five plants were tannins, sterols (in­cluding simple glycosides but not saponins), qui­nines, terpenes, lignans, flavonoids, quassinoids, steroid lactones and alkaloids. The most interest­ing were the diterpenes, lignans, quassinoids, and alkaloids.

The tumor systems used included the regular screen at different times and also the slow- growing tumors, B16 melanoma and Lewis lung carcinoma. The interesting goal here was the structure-antitumor activity relationships, which had brought out the desirability for further investiga­tion of certain types of compounds as possibilities for clinical studies.

Indicine-N-oxidefrom Heliotropium indicum was found to possess significant antitumor activi­ty in acute leukemia while monocrotaline has been used against skin cancer in China.

A large number of natural and semi-synthetic derivatives of colchicine’s have been tested and several in clinical trials, e.g. demecolcine are be­ing used in cancer treatment in some countries. Acronycine isolated from Achronychia baueri ex­hibited a very broad antitumor spectrum.

Pyridocarbazole alkaloids ellipticine and 9-methoxyellipticine from Ochrosia elliptica (Apo- cynaceae) and their derivatives, e.g. elliptinium acetate has produced a highly active material of value in some forms of breast cancer, and perhaps also in renal cell cancer.

Nitidine, a benzophenan- thridine alkaloid isolated from Zanthoxylum nitidum has been selected for development based on its exceptional anti-leukemic activity but was dropped owing to erratic toxicity while fagaronine, a closely related alkaloid, isolated from Z.

Xanthoxyloides, has been found to have greater anti­tumor activity than any of the natural benzophenanthridine structures, coupled with excellent stabil­ity, and has entered clinical trials in Japan.

The quassinoids or simaroubolides are a group of terpenoid-related compounds isolated from a variety of plants in the Simaroubaceae, e.g. bruceantin from Brucea antidysenterica showed high anti-leukemic activity at low dosages and over a wide dose range. It acts through inhibition of protein synthesis and has undergone clinical tri­als in man.

The diterpenes triptolide and tripdiolide iso­lated from Tripterygium wilfordi are potent anti-­leukemic agents that contain a reactive triepoxide system. The Central American tree Phyllanthus acuminates contains in its roots a complex mixture of glycosides, two of which, phyllanthostatin 1 and phyllanthoside have demonstrated marked anti­tumor properties. Phyllanthoside is in early clini­cal trials.