Recent progress in the development of docetaxel and paclitaxel analoguesJoëlle DuboisInstitut de Chimie des Substances Naturelles, CNRS, 1 avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France
Docetaxel (Taxotere®, Sanofi-Aventis) and paclitaxel (Taxol®, Bristol-MyersSquibb) are potent anticancer agents commonly used for the treatment ofbreast, ovarian and other cancers. Chemotherapy with these compounds givesundesirable side effects due to their low solubility and to the lack of selectivityfor cancer cells. Studies have been performed to solve these problems and tofind more potent taxoids on drug-resistant cancers. This review summarises theprogress in these areas as reported in recent patent applications on docetaxeland paclitaxel analogues that have been published between January 2003 andDecember 2005. The review specifically focuses on new methods for the pro-duction of taxoids, new potent analogues, including taxoid conjugates withimproved solubility, tumour targeting taxoids and prodrugs, combinationswith new anticancer agents and taxoid formulation and delivery.
Among the new antitumour substances that have emerged in the past decades, tax-oid anticancer agents such as docetaxel (1, Taxotere®, Sanofi-Aventis) [1] and paclit-axel (2, Taxol®, Bristol-Myers Squibb) [2], have shown remarkable potency againstvarious cancers. Their therapeutic effect is due, at least in part, to their interactionwith microtubules, cytoskeletal elements essential in all eukaryotic cells, with func-tions extending from cellular transport to cell motility and mitosis. Docetaxel andpaclitaxel act by stabilising microtubules, thereby blocking cell-cycle progressionprocess during mitosis [3].
Microtubules are made up of repeating αβ-tubulin heterodimers whose structurewas determined in 1998 by electron crystallography on zinc-induced sheets of tubu-lin stabilised by paclitaxel (2) [4]. The 3.7 Å resolution of tubulin, refined at 3.5 Å [5],has allowed localisation of the paclitaxel (2) binding site on the β-tubulin, and led tothe proposal of the T-shaped structure of paclitaxel as its bioactive conformation [6].This hypothesis has been supported by the recent synthesis of bioactive macrocyclictaxoids that adopt the T-shaped conformation [7,8].
Today, docetaxel and paclitaxel are very effective anticancer agents most com-monly used for the treatment of breast, ovarian, non-small cell lung and prostatecancers [9], but they may be used for many other types of cancer. Although signifi-cant progress has been made in curing some cancers, many others are resistant orbecome resistant to chemotherapy after administration of cytotoxic drugs. Antitu-mour taxoids have been shown to be substrates of P-gp, which is one of the ATPbinding cassette (ABC) transporters responsible for multi-drug resistance (MDR) incancer cells. In addition to the problem of resistance, they have poor water solubilityand have to be administered in alcoholic solutions with solubilising agents, such asCremophor-EL® (CrEL) for paclitaxel, and polysorbate 80 for docetaxel, agents thatincrease the side effects of the drugs. One other major drawback of these cytotoxic
1. Introduction
2. Improved production of
antitumour taxoids
3. New docetaxel and paclitaxel
analogues
4. Conjugates and prodrugs
5. Taxoids as part of a drug
combination
6. Drug formulation and delivery
7. Expert opinion: conclusion and
perspective
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1482 Expert Opin. Ther. Patents (2006) 16(11)
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agents is their poor selectivity towards tumour cells leading tonumerous undesirable side effects.
All of these problems have led to many further studies tofind more efficient production of these antitumour taxoids, tosynthesise more potent analogues with improved solubilityand activity on resistant cancer types and to improve formula-tion, delivery and combination with other anticancer agents.The aim of this article is to review the patent literature dealingwith these studies on docetaxel and paclitaxel analogues forthe period of 2003 – 2005, and to update the previous reviewsby Lin and Ojima (1997 – 1999) [10] and by Dubois, Guénardand Guéritte (2000 – 2002) [11].
2. Improved production of antitumour taxoids
Paclitaxel was first isolated and identified from the bark of thePacific yew tree, Taxus brevifolia, a non-renewable source [2].Fortunately, the continuity of supply was guaranteed by thediscovery by Potier et al. that a natural precursor, 10-deacetyl-baccatin III (3, DAB), is present in higher amounts in theleaves and twigs of the European yew, Taxus baccata, and canbe isolated without harm to the trees [12,13]. Paclitaxel is nowproduced either by extraction and purification of yew materi-als, by cell culture [14] or, as docetaxel, by a semisyntheticprocess. The natural compounds DAB or baccatin III (4) arethe starting materials for these semisyntheses that requiredmany chemical transformation and isolation steps [15-19]. Thischapter reports the patents dealing with the production ofpaclitaxel, DAB and baccatin III from plants, cell cultures orbioengineering, as well as with the improved syntheses ofpaclitaxel, docetaxel and of their C13-side chain.
2.1 Extraction from plantsTaxanes are generally isolated from species of Taxus [20], butrecently paclitaxel and other taxanes have been found inhazelnut trees (Corylus) [21]. In a patent describing their discovery
in other angiosperms of the genus Corylus, such as huckleberry(Vacinium parvifolium), scotchbroom (Cytisus scoparius) andred alder (Alnus rubra), it is also disclosed that taxoids are pro-duced by endophytic fungi of these angiosperms, preferably ofthe genus Alternaria, a fungus that had not, until now, beendiscovered to produce taxoids. A method is claimed to increasethe extraction yield by sterilising the surface of the plant andby omitting the grinding of the plant part before extracting thetaxoid [101].
A method for the purification of paclitaxel from paclit-axel-containing materials is claimed. The method comprisesseveral steps: extraction of a paclitaxel-containing material(plant or cell culture extracts), purification by normal phasechromatography, precipitation of the paclitaxel-containingeluate and then purification of the precipitate with high liquidperformance chromatography. It is claimed that paclitaxel of> 99.5% purity can be easily obtained from a Taxus genusplant with a high yield by this method [102].
In crude extracts of yew plants, the mixture of taxoids com-prises all or some of the following taxoids: paclitaxel, DAB, bac-catin III, 9-dihydro-13-acetylbaccatin III, cephalomannine,10-deacetyl taxol, 7-xylosyl taxol and 10-deacetyl-7-xylosyltaxol. A process is claimed for the preparation of a mixture ofDAB and baccatin III from an initial mixture of taxoidsobtained from crude extracts of various species of yew plants.After normal phase silicagel chromatography, the paclitaxel-richfractions are pooled for further purification. The other tax-oid-containing fractions are combined to form a waste taxoidsolution. Several chemical steps are applied to this solution:protection of the C7-hydroxyl group and cleavage of theC13-linkage, oxidation of the C9-OH and deprotection of theC7-OH to afford a mixture of DAB and baccatin III [103,104].
2.2 Production from cell culturesPlant cell cultures have been developed to produce secondarymetabolites such as paclitaxel [14]. The progress made in this
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Expert Opin. Ther. Patents (2006) 16(11) 1483
area has allowed Bristol-Myers Squibb to shift entirely to a cellculture process for paclitaxel production [22]. Modification ofthe culture medium has been an important way to increaseproduction. In this way, a medium treated with alkanoic acidor salt thereof is claimed to enhance paclitaxel productionfrom Taxus chinensis cell culture by up to sixfold. In this case,the additive is sodium butyrate 0.1 – 20 mM [105]. In the sameway, addition of indanoyl amino acids in the culture mediumof suspension cells of Taxus sp. increases the taxoid pro-duction. In particular, 6-ethyl-indanoyl-isoleucine (5) and1-oxo-indane-carboxy-(L)-isoleucine-methyl ester amide (6)have been found to increase paclitaxel production from Taxuschinensis cells < 1.6-fold and 2.4-fold, respectively, and bacca-tin III production < 4.6-fold and 7.6-fold, respectively [106].One problem in taxoid production by cell culture is that tax-oids are retained intracellularly, complicating downstreamprocessing and purification. Addition of a polynucleotideencoding a polypeptide corresponding to an ABC transporteror fragment should solve this problem. It is claimed that theuse of a polynucleotide corresponding to the NpABC1 geneinduces or enhances the production and/or the secretion of atleast one taxoid-type diterpene. It is also disclosed that thispolynucleotide can protect plants or plant cells from damagecaused by pathogens [107].
2.3 Production by genetic engineeringDetailed understanding of the paclitaxel biosynthetic pathwayshould facilitate the improvement of the production rate ofpaclitaxel, which is produced in Taxus sp. by a sequence of19 distinct enzymatic steps. Genes encoding enzymes formany steps of the paclitaxel biosynthetic pathway have beenisolated [23] and functionally expressed in Escherichia coli,yeast or Spodoptera cells [24]. Overexpression of these genesusing genetic engineering and in vitro synthesis wouldincrease the production of useful taxoids. Therefore, it isimportant to isolate and characterise new genes of the biosyn-thetic pathway. In this patent, the nucleic acid and proteinsequences of novel P450 oxygenases, of which taxoids are sub-strates, are claimed. The disclosed oxygenases, such as a taxoid5α-hydroxylase [25], hydroxylate the C5-position of a taxoid[108]. It is also claimed that plants that are deficient in geran-ylgeranyl diphosphate metabolism are useful in the biosynthe-sis of taxa-4,11-diene, the first intermediate in thebiosynthesis, and other paclitaxel intermediates. Transfectionof plants, such as tomato and other carotenoid synthesisingplants, with taxadiene synthase gene has generated four trans-genic lines that have produced taxa-4,11-diene as proven byGC-MS analysis [109].
2.4 Production by semisynthesisDocetaxel and paclitaxel are produced by semisynthesis fromDAB, after protection at C7, protection or acetylation at C10 fordocetaxel or paclitaxel, respectively, esterification at C13 and finaldeprotection [26]. A number of methods have been described forthe synthesis and introduction of the phenylisoserine side chain
at the C13-position [27]. Many patents describe modifications inthe different steps of the semisynthesis.
2.4.1 Side chain synthesisTwo patents describe chemoenzymatic processes to selectivelyprovide the (2R,3S) β-phenylisoserine side chain through opti-cally active trans-alkyl-phenylglycidates, precursors of the sidechain. The key steps are enzymatic resolutions during thehydrolysis of an ester by lipases [110] or during trans-esterifica-tion by a lipozyme [111]. An improved process for the prepara-tion of the paclitaxel side chain is also claimed. The noveltyconsists in the use of an heterogenous catalyst containing palla-dium and osmium (LHD-PdOsW) that performs the forma-tion of methyl cinnamate from bromobenzene, and methylacrylate followed by the Sharpless dihydroxylation in one potwithout any trace of osmium residue. Thus, methyl 2,3-dihy-droxy-3-phenylpropionate is obtained from bromobenzene in67% yield with > 99% ee [112]. Novel protected side chainsbearing an oxazolidine ring have been also described in twopatents. The difference is that the N-substituent is either analk-2-ynyloxycarbonyl [113] or a (2-trialkylsilyl)ethoxycarbonylgroup [114]. These side chains have been coupled to 7-protectedbaccatin III to afford paclitaxel after deprotection [114,115]. Noadvantage for the use of these chains is claimed.
2.4.2 Semisynthesis from 10-deacetylbaccatin III or baccatin IIIThe phenylisoserine side chains are generally coupled withC7-protected baccatin III. Several methods for preparing thiscompound from DAB are claimed. In the first process,acetylation of the C10-hydroxyl group of DAB in the pres-ence of a tertiary amine base give baccatin III in a basicallyquantitative yield; the reaction is faster and cleaner than whena Lewis acid is used [28]. The following protection of theC7-hydroxyl group with 2,2,2-trichloroethyl chloroformateaffords the C7-protected baccatin III in very high yield(97 – 100%). This process is claimed to be highly efficientand to suppress additional purification steps [116]. The secondprocess provides a new method for the conversion of DABcompounds to C7-protected baccatin III compounds in onestep without the need for purification of any intermediate.The C7-protecting group is added first and after completeprotection, the acetylating agent is added. The key feature ofthis invention is claimed to be the addition of a secondaryamine (preferably imidazole) to the reaction in the presence ofa nitrogen-containing compound (preferably pyridine) [117].The C7 hydroxyl group of DAB has also been protected by a2-haloacyl group before acetylation at the C10-position.Selective removal of this protecting group in the presence ofan acetyl group can be achieved under mild alkaline condi-tions [118]. A process is also provided for the semisynthesis ofprotected taxoid intermediates in a one pot reaction of pro-tecting the C7- and C10-positions with a Boc group andattaching a side chain at the C13-position [119]. C7-protectedbaccatin III is also the starting material in a semisynthetic
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process for the preparation of N-debenzoylpaclitaxel throughesterification with a carboxylic acid derivative of a phenyloxa-zolidine. The main difference from reported procedures is the2-nitrophenylthio N-protecting group, which is removed dur-ing the final elimination step of all protecting groups [120]. Anovel method for the coupling of the phenylisoserine sidechain is also claimed. This procedure involves the formationof triazine esters of the protected side chain, which are usedwithout further purification for the esterification of theC13-hydroxyl group of baccatin III. It is disclosed that thismethod gives a higher yield and allows an easier purificationof paclitaxel [121].
2.4.3 Semisynthesis from 9-dihydro-13-acetylbaccatin III 9-Dihydro-13-acetylbaccatin III (7, 9DHB) is a naturally-occurring taxoid present in abundant quantities in Taxuscanadensis. Four patents report its use in the synthesis of tax-oid intermediates. In the first patent, the conversion of9DHB into DAB in only three steps is described: protectionof the C7-OH, oxidation at C9 and final deprotection [122].Two processes consist in the protection of the C-7 hydroxylgroup, followed by the oxidation of the C9-OH. The addi-tion of a strong base to remove the C13-acetyl group followedby the addition of a β-lactam affords the desired taxoid inter-mediates [123,124]. Formation of the C9-ketone from C7-pro-tected 9DHB has also been performed via a C9-azido orC9-bromo derivative that is further oxidised. The formationof a C13-β-aminoester by direct reaction of C7-protected9DHB with an imine is also disclosed [125].
2.4.4 Modification at the N3′ positionTaxane amides can be converted to other taxoids by reductivedeoxygenation of the amide group by a zirconocene chloridehydride (Schwartz’s reagent) to form an imine that is furtherhydrolysed. The amine group is then acetylated either bydirect acylation [126] or by transacylation with the C2′-acyl
moiety [127]. The preparation of taxoids free of ring chlorina-tion using the above-mentioned method, with a benzoylatingagent essentially free of ring chlorination, has also beenclaimed [128]. Two patents describe the transformation ofcephalomannine to paclitaxel or docetaxel via aziridine ana-logues of cephalomannine [129,130]. A semisynthetic conversionof paclitaxel to docetaxel is also claimed [131].
2.4.5 Purification of taxoidsPurification of semisynthetic paclitaxel and docetaxel is a chal-lenging problem due to formation of a number of degradationproducts along the synthetic route. Two patents propose proc-esses of purification either by successive crystallisations [132] orby chromatography on polyethylene-bounded resins [133]. Tri-hydrate crystalline forms of paclitaxel and docetaxel are morestable under storage conditions. Processes for the productionof such forms are also disclosed in two patents [134,135].
2.4.6 Production of other taxoidsTwo new processes for the production of pentacyclic taxoidsare provided, including the production of the crystal form ofDJ-927 (8, Daiichi Pharmaceuticals Co. Ltd) [136,137].
3. New docetaxel and paclitaxel analogues
Docetaxel and paclitaxel are efficient antitumour drugs, butpossess undesirable side effects and interact with P-gp, one ofthe transporters involved in multi-drug resistance. Therefore,studies are still going on to find more potent taxoids, especiallyagainst resistant cancers, with fewer undesirable side effectsand with increased oral bioavailability. Some new compoundshave been patented these last 3 years, but fewer than inthe former period. Their biological activities are summarisedin Table 1.
C2′-methylated docetaxel or paclitaxel analogues havealready been described and have demonstrated significantenhancement in the inhibition of microtubule disassemblyand a better cytotoxicity toward KB cell line [29,30]. NewC2′-methylated taxoids of general formula 9, wherein R is tri-fluoromethyl, phenyl, 2-furyl or 2-thienyl, R1 is t-butoxycarb-onyl or benzoyl, R2 is hydroxy, R3 is hydrogen or, togetherwith R2, form a cyclic carbonate, have been claimed to haveantitumour activity. These compounds are as active as paclit-axel on A2780 cell line (an ovarian cancer cell line) and showa 100-fold enhancement in cytotoxicity toward paclitaxel- oradriamamycin-resistant A2780 cell lines [138]. Other noveltaxoids functionalised at the C14-position and a process fortheir preparation using novel C13-ketobaccatin III areclaimed. These novel taxoids of general formula 10 are ana-logues of ortataxel (11, IDN5109, Indena Spa) and are statedto be active against taxoid-resistant and MDR cell lines. Thenovelty resides in the substituent at the C14-position (namedX in general formula 10) can be azido, amine, amide, sulfona-mide, ether or a substituted double bond. No biological dataare presented for these novel taxoids 10 [139].
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Due to the poor oral bioavailability of paclitaxel anddocetaxel, there has been an increasing demand for develop-ing taxoids that can be orally administered. Bristol-MyersSquibb has recently described a new taxoid BMS-275183 (12)that showed good oral efficacy in two tumour models, goodoral bioavailability and potency against cell lines resistant topaclitaxel due to MDR and altered tubulin [31]. Metabolites ofthis compound have been identified through biotransforma-tion and incubation from mouse, rat, dog, monkey andhuman liver microsomes. They are claimed to have oral activ-ity and to be useful as therapeutic agents, but no biologicaldata are presented [140]. The development of drugs having
improved MDR reversal properties is still a critical issue. New9,10-α,α-OH taxoid analogues of general formula 13 areclaimed to meet this property and a method for productionthereof is disclosed. One of these compounds (14) was shownto display dose-dependent inhibition of the growth of theMDR MCF-7 breast cancer cell line with a higher activitythan paclitaxel. Compound 14 also inhibited the growth ofovarian carcinoma IA9PTX10, neuroblastoma SK-N-AS andsquamous cell carcinoma FADU cell lines [141]. In the devel-opment of original taxoids designed to be more active and lesstoxic at the Florida State University, C7-lactyloxy derivativeshave been prepared (15) that have shown increased activity
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Table 1. Biological activities of novel taxoids.
Compound Property* In vitro‡
Sensitive cell linesIn vitro‡
Resistant cell linesIn vivo‡
i.v. or p.o.administration
9 R A2780 (+) A2780 (Txl)§ (++) –
10 R – – –
Metabolites of 12 O – – –
14 M SK-N-AS (+)FADU (+)
MCF-7 (++)IA9PTX10§ (+)
–
15 N HCT116 (+) VM46 (+) –
16 N HCT116 (++) VM46 (++)DLD-1 (++)
Panc-1 (+)HT-29 (+)
17 N A2780 (++) – –
18 N A2780 (– –) – –
*M: Designed to be active on MDR expressing cell lines; N: No specific property; O: Designed to be orally administered; R: Designed to be active on resistant cell lines.‡(+) active; (++) very active; (– –) much less active; – no available data.§Paclitaxel-resistant cell line.E
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1486 Expert Opin. Ther. Patents (2006) 16(11)
against HCT116 and VM46 (a resistant variant of humancolon carcinoma HCT116) cell lines compared with paclit-axel and docetaxel [142]. In a second patent, C10-cyclopentylester substituted taxoids are disclosed. Compound 16 (simo-taxel) is claimed to be active against cancers in a manner supe-rior to paclitaxel or docetaxel with certain tumour types,including paclitaxel-sensitive and -resistant cell lines.Although the activity is the same on the paclitaxel-sensitivecell line HCT116, compound 16 is more active than paclit-axel and docetaxel on VM46 and DLD-1 (another resistanthuman colon carcinoma) cell lines. Compound 16 was shownto be active by intravenous or oral administration on mousepancreas (Panc-1) and colon (HT-29) tumour xenograft mod-els and to compare favourably to paclitaxel and docetaxel withrespect to toxicity. Therefore, compound 16 is claimed to havethe potential as a safe and effective antitumour agent for oraland intravenous administration [143]. This compound is nowunder Phase I clinical trials.
As reported in the introduction, macrocyclic taxoids havebeen designed to elucidate the bioactive conformation of tax-oids [7,8]. Some of these constrained paclitaxel derivatives,which bear a bridge extending between the ortho C3′-phenylgroup and the C4-position, have been patented [144]. Many ofthe compounds have superior properties to natural paclitaxel.The most active, compound 17, inhibited the A2780 cell linesin vitro with a potency that was ∼ 20-fold greater than paclit-axel. A challenge in taxoid development is to find structurallymuch simpler compounds, while also retaining the activity ofdocetaxel and paclitaxel. This patent also presents the design,synthesis and bioactivity of simplified paclitaxel analoguesbased on the T-shaped conformation (general formula 18wherein X = O, bond or OCH2, R = H and R′ = OBz orR = OBz and R′ = H, and the bridge is cis or trans). All thecompounds were cytotoxic, but were significantly less activethan paclitaxel (500- to 1000-fold less active) and tubulinactivity was only 10% that of paclitaxel [144]. This work onsimplified paclitaxel analogues has recently been published [32].
4. Conjugates and prodrugs
4.1 Taxoids with improved solubilityA major difficulty in the development of paclitaxel anddocetaxel for clinical trial was their insolubility in water.Present formulations use surfactant vehicles to render thesecompounds soluble, but these can cause allergic reactions andinduce side effects. Many studies have been devoted to thesynthesis of more water soluble taxoids. Conjugation of tax-oids to polymers has been an attractive approach to reducesystemic toxicity and improve the chemotherapeutic index. Inthis way, taxoids covalently bonded to hyaluronic acid or itsderivatives, optionally via a spacer, and processes for theirpreparation are claimed. The conjugates are disclosed to beinstantly soluble in the blood and to be instantly released bythe action of enzymes such as esterases. Conjugates were asactive as paclitaxel against OVCAR-3 implants in mice and
showed superior activity in vitro against MCF-7 andMDA/MB/468 breast cancer cell lines, being 150- and50-fold more active, respectively [145]. In the same way, carbo-hydrates, including D-glucosamine and chitosan, have beenincorporated at the C2′- and/or C7-positions of the paclitaxeland docetaxel molecules. The compounds are claimed to haveimproved water solubility compared with the parent com-pounds [146]. Taxoids have also been conjugated to a water sol-uble polymer, such as polyglutamic acid, polylysine orpolyaspartic acid [147-151]. Paclitaxel poliglumex (Xytotax,CT-2103, Cell Therapeutics, Inc.) is a polyglutamic acid pol-ymer that has shown improved outcomes in clinical pilotstudies and is now under Phase III trial [33]. Taxoids have alsobeen conjugated to a water soluble polyamino acid polymer(preferably polyglutamic acid [PG]) having a molecularweight of 5000 – 50000 Da, conjugated to multiple polyeth-ylene glycol (PEG) molecules . These conjugates are claimedto have a higher water solubility and higher ability to accumu-late in a tumour than the unconjugated anticancer drug.PEG-PG-paclitaxel is 70 – 90 times less toxic than paclitaxeland shows the same in vivo activity [152]. In another patent,water soluble prodrugs have been designed. 2′-O-acyl-3′-N-debenzoyl paclitaxel hydrochloride analogues are claimed to bepaclitaxel prodrugs with improved solubility and acceptablestability [153].
4.2 Taxoid with reduced liability toward multi-drug resistancePaclitaxel hybrid derivatives are claimed to have enhancedmetabolic stability and/or solubility and to display reducedliability toward MDR. The derivatives are paclitaxel ana-logues substituted with at least one or more polar append-ages at either the C7- or C10-positions. The appendages arepartially protected amino acids or completely deprotectedamino acids or adducts (such as small peptides) that areattached to the carboxylic moiety of the amino acid [154].Coupling of an omega-3 fatty acid, such as docosahexaenoicacid (DHA), with paclitaxel has already proven its efficacy incancer treatment studies. DHA-paclitaxel (Taxoprexin®,Protarga, Inc.) [34] exhibits improved pharmacokinetics andtoxicity profiles compared with paclitaxel and is now underPhase III clinical trials [35]. In another patent, omega-3 fattyacids have been added to taxoids via an ester linkage at theC2′-position. The improvement comprises the use of a sec-ond generation taxoid [36]. A typical conjugate, 2′-docosa-hexaenoyl-3′-dephenyl-3′-(2-methyl-prop-1-enyl)-10-cyclopentane-carbonyl-docetaxel, proved to be very efficient onDLD-1 (pgp+) tumour xenograft implanted in mice. It wasfound that all mice implanted were tumour-free on day 201after implantation [155].
4.3 Tumour-targeted taxoidsA major drawback in taxoid chemotherapy is the non-specificaction of the drugs leading to undesirable side effects. One wayto overcome this non-specific action is to design compounds
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that can be converted to active substances selectively intumours, but not in normal growing cells [37]. Therefore, amethod for identification of tumour targeting enzymes isclaimed, together with conjugated anticancer agents bearing asubstrate moiety of these enzymes. Microsomal dipeptidase,phospholipase C and DT-diaphorase are among the preferableenzymes for tumour targeting with cytotoxic agents.C2′-O-dipeptidoyltaxoid is exemplified as a tumour targetedcytotoxic. Microsomal dipeptidase cleaves the dipeptide allow-ing the free amine to deprotect the C2′-hydroxyl group. Thiscompound is as active as paclitaxel against the HCT116/S5cell line, a human colon cancer cell line transfected with thehuman microsomal dipeptidase cDNA connected to thecytomegalovirus promoter [156]. Novel polyethylene glycolatedtaxoid derivatives linked at the taxoid C7- or C10-positions aredisclosed in another patent. The PEG substituents bear a link-ing group, such as sulfide, disulfide, sulfhydryl, acid labile orphotolabile group, enabling the linkage to a cell binding agentand, therefore, these taxoids can be delivered to specific cellpopulations in a targeted tumour. The PEG-taxoids have
improved water solubility and are disclosed as being highlyeffective against MCF-7 and A-431 cell lines [38,157]. Polypep-tides encoded by cancer-related genes and antibodies to themcan also be conjugated to taxoids. Methods for treating and/orprotecting against cancer with such immunoconjugates areclaimed [158]. New conjugate derivatives comprising an activeagent attached to a group that is a substrate for a protein kinaseor lipid kinase are claimed. Preparation of paclitaxel–linker–thymidine conjugates with carbamate linker attach-ment at the C10-position of paclitaxel is disclosed. Some ofthese conjugates displayed an HFF/MCF-7 cytotoxicity selec-tivity index value of 11.4 compared with 1.4 for the parentcompound [39,159]. In another patent, novel drug conjugatederivatives of general formula Y-A-Z, wherein A is a 5-, 6-or 7-membered ring, Y is a substituent adjacent to A bear-ing a cytotoxic agent linked to different spacers, and Z isanother substituent adjacent to A bearing a specific bindingagent linked to different spacers, are claimed. Several conju-gate derivatives, particularly comprising taxoid derivativesas the active agent and a specific binding agent, such as
R1
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mAbs, are exemplified in this patent but no biological dataare presented [160].
4.4 Other taxoid conjugatesTaxoid conjugate derivatives able to be transported throughthe blood–brain barrier have been patented. These conjugatesare novel taxoid–peptide derivatives wherein the peptide is avector for the taxoid and is capable of increasing the solubilityof the active compound, enabling its transport across theblood–brain barrier [40]. The linear peptides conjugated totaxoid derivatives comprise a transduction domain enablingthe conjugate to penetrate inside the cell. In tests in mice, thespecified compound demonstrated a 12-fold improvement inpassage through the blood–brain barrier compared with pacli-taxel, and was approximately equipotent to paclitaxel againstMCF-7, MDA-MB-435 and N-SH cells [161].
Camptothecin is a DNA topoisomerase I inhibitor withantitumour activity. One patent investigated whether a conju-gate derivative between paclitaxel and camptothecin woulddisplay multiple tumour activity or improved activity againstdrug-resistant cells. Amino acids were introduced on theC7-hydroxyl group of paclitaxel and then coupled with7-formyl-camptothecin. The conjugate derivatives were gener-ally less active than the parent compounds and none inhibitedDNA relaxation [41,162].
4.5 SummaryCarbohydrate conjugates of taxoids [146] and acyl derivatives[153,154] show improved solubility or better MDR reliability,but no biological activity is described in the patents. Taxoidsconjugated with hyaluronic acid [145] are active in vitro, and onOVCAR-3 tumour xenograft by intraperitoneal injection.Conjugation with PEG, polyamino acid or both lead to moresoluble compounds with improved cytotoxic and pharmacoki-netic properties. PEG-taxoids [157] have only been testedin vitro but the other conjugates [147-152] also show promisingin vivo activity. In the series of DHA–paclitaxel analogues, asecond generation taxoid conjugated to DHA shows a goodin vivo activity on a pgp+ tumour xenograft [155]. Addition ofadequate peptides to taxoids allow them to go through theblood–brain barrier and these derivatives are active in vitro onsome cancer cell lines [161]. Finally, tumour targeted taxoidsconjugated with either thymine [159] or a dipeptide [156] exhibitpromising in vitro activity contrary to camptothecin–paclitaxelconjugate [162].
5. Taxoids as part of a drug combination
Drug combinations are often used in the treatment of cancer.They allow lowering the amount of each product and, conse-quently, reducing the associated toxicity and the risk of devel-opment of acquired resistance. Synergistic effects may existand can increase the therapeutic effect, especially if the targetof each drug is different. Paclitaxel and docetaxel are involvedin a number of studies for drug combination either with
well-known anticancer agents or with new ones. In thisreview, only the studies where taxoids are specifically claimedand where the combination gives better results than the singledrugs are reported and summarised in Table 2. The combina-tion where taxoids are mentioned among other anticancerdrugs is not presented here.
Adenosine A3 receptor antagonists [42] have been shown toenhance the activity of anticancer agents on cancer cell linesexpressing adenosine A3 receptors [43], including cancers withpgp-dependent MDR. In one example against the humanmelanoma A375 cell line, the IC50 value of docetaxel wasreduced from 0.038 nM to 0.006 nM by the addition of com-pound 19 [163]. Administration of ZD-6474 (20), an inhibitorof vascular endothelial growth factor receptor (VEGFR) tyro-sine kinase activity [44], in association with a taxoid enhancesthe antitumour effect of both drugs. In one example using theGEO human colon tumour xenograft model, the tumour vol-ume observed on day 28 after tumour cell injection was0.1 cm3 for mice treated with ZD6474, 0.95cm3 for micetreated with paclitaxel and 0.01cm3 for the combination [164].In the same way, the combination of AZD2171 (21), anotherinhibitor of VEGFR tyrosine kinase activity [45], withdocetaxel induced a significantly greater tumour growth inhi-bition than with individual drugs alone [165]. In another pat-ent, a protein kinase inhibitor, preferably a quinoxazolinecompound [46], is administered in combination with docetaxeland induced a higher survival rate for nude mice implantedwith L3.6pL pancreatic tumours [166]. Another antivascularagent, DMXAA (22) [47], has been combined with paclitaxeland docetaxel [48]. Co-administration of DMXAA and paclit-axel or docetaxel to mice implanted with MDAH-Mca-4tumour xenograft provides a therapeutic gain against estab-lished mouse mammary tumours [167]. The combination ofpurvalanol A (23), a cyclin-dependent kinase (CDK)-1 antag-onist, administered, after paclitaxel, to mice implanted withMCF-7 cancer cells, inhibited tumour growth and increasedsurvival [168]. Combination of another CDK inhibitor andpaclitaxel has been recently published and compared withpaclitaxel–purvalanol combination [49]. Another combinationcomprising docetaxel and roscovitine (24, Cyc202, Seliciclib®,Cyclacel Ltd), a CDK inhibitor [50], has been patented. Admin-istration of docetaxel in combination with roscovitine producesan enhanced effect as compared with individual drug adminis-tered alone [169]. An anthelminthic compound, particularlyniclosamide (25), is claimed to show antiproliferative activityagainst five human cancer cell lines. When niclosamide wasused in combination with paclitaxel, a seven- and sixfold reduc-tion in paclitaxel IC50 values was observed on HCT116 andDU145 cell lines, respectively [170]. In another patent, the use ofciclopirox (26), an antifungal agent, in combination with pacli-taxel is claimed. The combination enhanced the activity ofpaclitaxel against non-small cell lung carcinoma cells A549 andreduced its IC50 value by 10 – 20 times [171]. Novel methodsusing a combination of clofarabine, a purine nucleoside antime-tabolite (27, Clolar®, Genzyme) [51], and docetaxel for the
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NH
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N
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N
N
O
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inhibition of tumour cell growth and for the treatment of alarge variety of cancers are claimed. The use of the combina-tion in a human prostate tumour xenograft model in miceshowed a delay in tumour growth of 33 days, compared with19 and 21 days for clofarabine and docetaxel alone, respec-tively [172]. A positive effect has also been described when aVEGF antagonist and a taxoid are combined. Female athymicnude mice were inoculated with OVCAR-3 cells, followed2 weeks later by treatment with VEGF trap plus paclitaxel.Tumour burden was reduced by 97.7% with a 100% reduc-tion in ascites. This compares with a 55.9% and 85.4% reduc-tion in tumour burden and ascites, respectively, for animalsgiven paclitaxel alone [52,173]. An improved combination bacte-riolytic therapy has been described for the treatment oftumours by administering spores of a toxin-defective anaerobicbacterium [53] and a taxoid. This combination is claimed torevert, slow or arrest tumour growth efficiently. Treatment ofnude mice harbouring xenografts of HCT116 cells treated
with docetaxel and C. novyi-NT intravenously gave a higherrate of tumour regression when compared with control [54,174].The use of specific antibodies in combination with taxoidscan also demonstrate synergistic activity. The antibody can beeither conjugated with another anticancer compound oralone. In this manner, the administration of an antibody reac-tive to Lewis Y antigen conjugated to doxorubicin followed≥ 30 h later by docetaxel or paclitaxel, is claimed to be usefulfor treating lung, colorectal, oesophageal, gastric and ovariantumours. This combination, evaluated in vivo using a mousemodel of human non-small cell lung cancer, resulted in a highresponse rate of 89% [175]. Another patent claims the synergis-tic combination of an oral taxoid and an epidermal growthfactor receptor antibody such as cetuximab (Erbitux®,ImClone Systems, Inc.). The combination of compound 12and cetuximab demonstrated synergistic activity in mice bear-ing L2987 or GEO xenografts [55,176]. Inhibition of geneexpression can also be used in combination with taxoids. A
Table 2. Activity of patented combinations with taxoids.*
composition comprising a first oligonucleotide capable ofhybridising to the promoter region of an oncogene (such asbcl-2 gene) and a method of introducing the oligonucleotide incancer cells are claimed. Administration of the oligonucleotideand docetaxel resulted in a significantly greater decrease in thetumour size [177]. Finally, four other combinations undergoingclinical trial have been patented. The use of docetaxel, doxo-rubicin and cyclophosphamide (TAC) in adjuvant therapy ofbreast and ovarian cancers [56,57] is claimed to enhance theactivity of docetaxel without the need to enhance doses, andthereby to limit the known toxicities of this anticancer agent.The results suggested that therapy with this combination waswell tolerated and superior to a 5-FU, doxorubicin and cyclo-phosphamide combination in tumours overexpressing ER/PRand HER2 [178]. Two other patents described the use of adiphenyl compound to inhibit the binding of intracellularhistamine [58]. It is stated that the diphenyl compound inhib-its normal cell proliferation and at the same time promotesthe proliferation of malignant cells. This enhances the toxiceffect of chemotherapeutic agents and reduces their adverseeffects on normal cells. Premedication with N,N-diethyl-2[4-(phenylmethyl)phen-oxy]-ethanamine (DPPE, 28) beforetreatment with epirubicin or doxorubicin and paclitaxel ordocetaxel was evaluated in a Phase II clinical trial, and majorresponses were observed in 79% of patients and someimprovement in 97% of patients with metastatic breast can-cer [179]. The combination of DPPE with paclitaxel ordocetaxel is also claimed to be useful for treating breast orovarian cancer [180]. Finally, a combination of paclitaxel andtrabectedin (29, ecteinascidin-743 [ET-743]) [59] is claimedto be useful for treating many cancers. A Phase I trial hasbeen described showing positive responses and antitumouractivity in doxorubicin/ifosphamide-resistant liposarcomapatients [181].
6. Drug formulation and delivery
Another way to overcome the drawbacks of poorly water-solu-ble drugs such as taxoids is to find formulations capable ofenhancing the bioavailability of the drug and allowing oraladministration. The main solutions are the use of liposomes,micelles or emulsions, the use of more solubilising solventswith fewer side effects, the use of inclusion complexes and theuse of dispersions of nanoparticles [35]. In this chapter, a briefsummary of newly patented methods is presented.
Paclitaxel in a microsphere formulation of biocompatiblepolymers containing phosphorus linkages (named Paclimer®) isclaimed to be useful for the treatment of prostate cancer [60,182]
and head and neck cancers [183], but only the in vivo efficacyon tumour xenografts implanted in mice has been evaluated.Paclitaxel has also been encapsulated in neutral [184] or cati-onic liposomes [185]. A negatively-charged derivative of paclit-axel has also been introduced into amphiphilic liposomes [186].Sterically-stabilised mixed micelles composed of PEG-2000-DSPE plus egg-PC incubated with paclitaxel are claimed to be
an improved delivery system [187]. Novel block copolymershave also been described to form micelles with paclitaxel [188].All these preparations are stated to have high efficacy and fewside effects, but have only been tested either in vitro or in vivoon mice bearing tumour xenografts.
A novel 2-hydroxypropyl-β-cyclodextrin inclusion complexof paclitaxel has been disclosed [189] as well as novel cyclodex-trin dimer derivatives, which are claimed to be useful indocetaxel solubilisation [190]. No biological data are given forthese complexes. A formulation comprising an active ingredi-ent such as paclitaxel in a crosslinked matrix of a phospholipidand a chitosan derivative is also claimed. The demonstrationof in vitro and in vivo release of paclitaxel from a biodegrada-ble implant has been realised. The use of this formulation isstated to protect the drug from degradation [191]. Some pat-ents described new oily compositions to improve solubilityand lower toxicity, but are not presented here. In other stud-ies, CrEL or polysorbate 80 have been replaced by vitamin Eα-tocopheryl PEG succinate. The formulation includes eitherpropylene glycol, ascorbyl palmitate, di-α-tocopherol and eth-anol [192] or tyloxapol, propylene glycol or anhydrous ethanol[193]. Paclitaxel has also been solubilised in a 2,5-di-O-methyl1:3:4:6 dianhydroglucitol derivative to provide a clear solutionwith high bioavailability and stability [194].
Some patents are devoted to the formation of nanoparticu-lates of antimitotic compounds. In this way, nanoparticulatesof paclitaxel having a particle size of 0.1 – 5 µm and theirintraperitoneal delivery is claimed. The effects of differentcompositions of paclitaxel in an ovary tumour model in miceare exemplified [195]. Methods for the production of nanopar-ticulate bioactive agents either by mixing solvents [196] or byprecipitation with a non-solvent, are also disclosed [197]. How-ever, this latter preparation administered orally to mice exhib-its poor bioavailability relative to paclitaxel in CrEL. Amethod for preparing a highly uniform nano-scale paclitaxelsolid dispersion with an improved solubility by a supercriticalfluid process is claimed. The nano-scale paclitaxel solid dis-persion has been prepared by spraying a mixture of paclitaxeland a pharmaceutically acceptable additive dissolved in amixed organic solvent into supercritical carbon dioxide toform particles of paclitaxel and the pharmaceutically accepta-ble additive. The organic solvents were removed by washingthe particles with the supercritical fluid [198]. No biologicaldata are given.
7. Expert opinion: conclusion and perspective
Over the last 2 decades, paclitaxel and docetaxel have playeda significant role in the treatment of various malignancies.Nowadays, production of paclitaxel either by semisynthesisor by plant cell culture is no longer troublesome. Taxol isnow going generic after the expiration of a 10-year protec-tion period. This may explain the large amount of new pat-ents disclosing improved methods for its production andpurification. However, only slight progress has been made in
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the semisynthetic process. New methods have been devel-oped to obtain DAB or baccatin III from unwanted taxoidsfound in the biomass of Taxus extracts [103,104] or to realise thesemisynthesis from 9-DHB [122-125]. Although plant cell cul-tures are now able to produce large quantities of paclitaxel, abreakthrough in taxoid production will be made when it isproduced by genetic engineering. Genes encoding for manysteps of the biosynthetic pathway have been isolated and func-tionally expressed. The next step for taxoid production wouldbe the installation of these genes in a single host strain. Cro-teau et al. [61] tried to install five sequential steps in Saccaromy-ces cerevisiae leading from the precursors of isoprenoidbiosynthesis (isopentenyl diphosphate and dimethylallyldiphosphate) to taxadien-5α-acetoxy-10β-ol. However, theattempt to reconstruct the entire target pathway was partlyunsuccessful because only the first two steps were functionallycoupled leading from the precursors to taxa-4(5),11(12)-diene.Nevertheless, this first study should prompt further attemptsto engineer baccatin biosynthesis in yeast, and bodes well forthe ultimate production of more advanced taxoids in thismicrobial host.
Since the discovery of paclitaxel and docetaxel, many newanalogues have been synthesised. Several new taxoids beingtested in the clinic show improvements on activity againstresistant tumours (XRP9881A, XRP6258) or on water solu-bility (ortataxel, DJ-927, BMS-275183) [62]. Among thenewly patented taxoids, compounds 14 and 15 are underPhase I clinical trials. However, as previously noted, there arefew novel taxoid derivatives described in the literature in thelast 3 years. A large amount of structure–activity relationshipstudies have been performed in 25 years and almost all theparts of the paclitaxel molecule have been explored. It seemsdifficult now to find original active compounds in this seriesunless the taxane skeleton is modified. Therefore, a challeng-ing issue would be to design structurally simpler compoundswhilst retaining the activity of taxoids. Some analogues havealready been designed in that perspective [32,63-69,145], butnone retain the biological activity of taxoids and all are muchless cytotoxic. These studies deserve further investigation.
The poor solubility of paclitaxel and docetaxel necessi-tates the inclusion of surfactant vehicles in their commer-cial formulations. A number of strategies have beendeveloped to make available formulations of surfactant-freetaxoids. They include nanoparticles [195-198], liposomes[182-188], emulsions, new surfactant vehicles [192-194] and tax-oid conjugates [145-152,155] and prodrugs. Some of these for-mulations have certain advantages, such as shorteradministration time, oral administration and absence ofrecognition by the P-gp. Taxoid conjugates, such as paclit-axel polyglumex and DHA–paclitaxel, are undergoingPhase III clinical trials and nanoparticles have alreadyproved their interest. Paclitaxel albumin-bound particles(nab paclitaxel, ABI-007, Abraxane®, American Pharma-ceutical Partners, Inc. and American Bioscience, Inc.) [70]
was approved by the FDA in January 2005 for the treat-ment of metastatic breast cancer after failure of combina-tion chemotherapy or relapse within 6 months of adjuvanttherapy. Therefore, although it is not known now whetherthese agents would improve survival, the novel formulationsof taxoids do hold some promise in cancer therapy.
Tumour-targeting drug delivery system is the most attrac-tive way to overcome undesirable severe side effects. In thatprospect, the targeted-taxoid strategy is full of promise. Tax-oids conjugated with a substrate of tumour specific enzymes[156,157,159] or with a cancer-related antibody [158,160] have beendescribed to be highly effective and to show selectivity forcancer cells. Although these strategies seem very promising,these results should be confirmed by in vivo experiments andclinical trials.
Combination of taxoids with other anticancer agents oftengives synergistic effects, leading to reduced administeredquantities of drugs and, therefore, reduced side effects. Newcombinations have been described involving docetaxel orpaclitaxel with new anticancer agents, such as antivascularagents [164-167,173], CDK inhibitors [168,169] or specific anti-bodies [175,176]. Paclitaxel has also been administered withspores of toxin-defective anaerobic bacterium [174]. All ofthese combinations have shown synergistic effects on mousemodels of human cancer cell xenografts and should be expe-rienced in clinical trials to confirm their synergy in patients.The most advanced combination is undergoing a Phase IIItrial. The TAC combination adjuvant therapy has proven tobe superior to the 5-FU, doxorubicin and cyclophosphamidecombination on breast cancers [178]. Premedication withDPPE, an inhibitor of the binding of intracellular histamine,before treatment with paclitaxel or docetaxel, alone or incombination with an anthracyclin, have been evaluated inPhase II clinical trials and have shown encouraging results onmetastatic breast cancers [179,180].
Docetaxel and paclitaxel are very potent anticancer agentsand are commonly used for the treatment of breast, ovarian,non-small cell lung and prostate cancers. The developmentof new combinations, new analogues, new conjugates andnew delivery systems should give rise to the possibility ofusing these compounds for the treatment of many othertypes of cancer. This series of antimitotic agents is very toxicagainst cancer cells. To take full advantage of this property,the aim of the future pharmaceutical development is to findthe best way to transport taxoids selectively into the cancercells and to avoid their rapid exclusion by ABC transportersas well as their extensive metabolism. All the studiesdescribed in the patents over the last 3 years are devoted tothat goal and hopefully new clinically relevant drugs shouldappear in the near future.
Acknowledgements
Dedicated to the memory of Pierre Potier.
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