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Paclitaxel As an Anticancer Agent: Isolation, Activity, Synthesis and Stability

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Paclitaxel As an Anticancer Agent: Isolation, Activity, Synthesis and Stability Cent. Eur. J. Med. • 6(5) • 2011 • 527-536 DOI: 10.2478/s11536-011-0074-5 Central European Journal of Medicine Paclitaxel as an anticancer agent: isolation, activity, synthesis and stability Review Article Vesna D. Nikolic1*, Ivan M. Savic1, Ivana M. Savic1, Ljubisa B. Nikolic1, Mihajlo Z. Stankovic1, Valentina D. Marinkovic2 1 Faculty of Technology, University of Nis, Bulevar oslobodjenja 124, 16000 Leskovac, Serbia 2 Faculty of Pharmacy, University of Belgrade, Vojvode Stepe 450, 11000 Belgrade, Serbia Received 1 April 2011; Accepted 14 June 2011 Abstract: PaclitaxelisisolatedfromthePacificyew.ItcanbeobtainedfromtheEuropeanyew,butonlyafterchemicalmodificationoftheisolated compoundbyasemi-synthesisprocedure.Theprocedurefortotalsynthesisofpaclitaxelisverycomplicated,involvingmultiplesteps, andtheyieldsofpaclitaxelaremeagre.Thissubstanceisalsoametaboliteofcertainkindsoffungus.Themicrobiologicalpathway forproducingpaclitaxelcomparedwithisolationfromplantmaterialinvolvesshorterprocuctiontimesbutasmallyield.Cyclodextrins areusuallyusedforimprovingthesolubilityofpaclitaxelinaqueousmedia,withpolymericandothersubstancesadded.Paclitaxelhas anticanceractivityanduseforpreparingtheformulationsintravenouslyadministratedtopatientswithtumors.Thepaclitaxelconcen- trationintheseformulationsisdeterminedusingvalidatedHPLCmethods. Keywords: Paclitaxel • Isolation • Activity • Synthesis • Complexation ©VersitaSp.zo.o. 1. Nature sources of paclitaxel Stierle et al. [6] indentified the fungus of Taxomyces andreanae that produces paclitaxel. Although the yield of paclitaxel was tiny (24–50 ng/dm3), this discovery was The National Cancer Institute analyzed the content of of major interest to scientists [7]. Several procedures an extract from a bark of Pacific yew (Taxus brevifolia), have been suggested for isolation from the endophytic first isolated from a plant material in 1963 [1]. The Pa- fungus [8-10], showing that microorganism of Taxus cific yew is a rare, low-growing evergreen plant, locating species can produce paclitaxel. Therefore, the process in the old forests of the northwestern Pacific. Preclini- of fermentation using a paclitaxel-produced microorgan- cal studies of the extract showed that it had a cytostatic ism represents an alterative procedure for paclitaxel activity on many types of tumors [2]. In 1971, paclitaxel production. The major problems involved in fungus fer- was identified as an active constituent of this extract. mentation are the very small yield of paclitaxel and its Development of paclitaxel for medicinal use proceeded instability: the yields ranged from 24 ng to 70 μg per slowly despite its antitumor activity because collecting liter of the investigated fungus [6,8]. The microspore of sufficient amount required much time and plant mate- Pestalotiopsis CP-4 [9] produced paclitaxel from 50 to rial (for about 1 kg yield, it is necessary to isolate the 1487 ng/dm3. The goal of the present research is dis- extract from 3,000 of Pacific yew trees). Interest in this covering the optimal fermentation conditions for Taxus substance was aroused in 1979, when its mechanism cell cultures. of action as an antitumor drug was identified [3-5]. Pa- Although the amount of the produced paclitaxel from clitaxel is also contained in Taxus yannanensis, and has fungus was lower than those that were obtained from been obtained from Taxus baccata by semi-synthesis. * E-mail: [email protected] 527 Paclitaxel as an anticancer agent: isolation, activity, synthesis and stability yews, the shorter production time and high rate of fun- temperature or three months at 4°C (presumably due to gus growth have had an important role in further studies. the ability of the acetic acid to neutralize traces of alkali After almost two years of research, scientists have iso- in the methanol) [13]. Paclitaxel is also soluble in etha- lated several endophytic funguses from Taxus chinensis nol and acetonitrile [14]. var. mairei and Taxus yunnanensis. Species of Ozonium Paclitaxel has low solubility in water and is rapidly BT2 can also produce paclitaxel and taxane baccatin III destroyed in weakly alkaline aqueous solutions [13]. (intermediates in the production of paclitaxel). The lowest amount of degradation in aqueous paclitaxel Production of paclitaxel by a microbiological pathway solutions occurs in pH 3 to 5. Paclitaxel solutions at 0.1 is also possible using a pure culture of Actinomycetes and 1 mg/cm3 in 5% dextrose injection or 0.9% sodium [11]. Microorganisms from the Actinomycetes group chloride injection remained active for at least three days usually belong to the suprageneric group of Streptomy- at 4, 22, or 32°C [15]. cetes, Actinoplanetes, Maduromycetes, Thermomonos- poras or Nocardioforms, but more often to the Strep- tomyces, Actinoplanes, Nocardiopsis, Micromonospora, 3. Paclitaxel procedures of isolation Actinomadura or Kitasatosporia genus, and mostly to from natural sources the Kitasatosporia genus. Among them, the most signifi- cant is Kitasatosporia sp. CECT 4991. The first step involves washing raw plant material using deionized water [16]. The temperature of water is in the range of 20 to 25°C, and duration of the process is 3 h. 2. General characteristics of paclitaxel Then, the water is removed along with all water soluble impurities. The second step is extraction with an organic The IUPAC name for paclitaxel is (2α,4α,5β,7β,10β,13α)- solvent. The adequate solvents for this extraction are 4,10-bis(acetyloxy)-13-{[(2R,3S)-3-(benzoylamino)- alcohols (methanol), ketones (acetone) and their mix- 2-hydroxy-3-phenylpropanoyl]oxy}-1,7-dihydroxy-9- tures. In cases where a mixture is used, the volume ratio oxo-5,20-epoxytax-11-en-2-yl benzoate. Its structural in the mixture is about 1:1. The obtained extract is fil- formula is shown at Figure 1, and Table 1 lists its char- tered to remove deposits, and then transferred to a dou- acteristics. ble walled tank, where the water temperature is in the Paclitaxel is soluble in dimethyl sulfoxide (DMSO) range of 65–70°C. The organic solvent is then removed (50 mg/cm3) [6]. A 0.01 mol/dm3 solution of paclitaxel by distillation. The remaining solution is drained into an- in DMSO has been stored as aliquots until use and fur- other tank. Because there is residual water, that solution ther diluted to 10-10 mol/dm3 with medium [12]. Paclitaxel is a non-concentrated extract. The third step is isolation is also soluble in methanol (50 mg/cm3). It undergoes hydrolysis and transesterification to ~30% of the peak Table 1. General characteristics of paclitaxel signal at 227 nm by a high performance liquid chro- CAS number 33069-62-4 matography method (HPLC) after two weeks at room Molecular mass 853.9 temperature. Paclitaxel is rapidly destroyed in weakly Molecular formula C47H51NO14 alkaline, methanolic solutions and in strongly acidic DMSO, methanol, ethanol. Unstable in methanolic solutions (1:1 of methanol : concentrated Solubility information Methanol. HCl). A sample with 0.1% acetic acid added to methanol Consistency White crystalline solid. showed no signs of degradation for seven days at room max 227, 273 nm λ Melting point (decomposition) 200-220 °C Clear colorless solution at 10 mg·cm-3 of Solubility methanol. Solubility in water is ~ 0,4 μg·cm-3. Specific optical -49 ~ -55 rotation Storage -20 °C. Protect from light. Warning! May cause birth defects. Irritates Warnings eyes, skin, respiratory system cyclodecane Classification Antitumor agent Figure 1. Structural formula of paclitaxel 528 V. D. Nikolic et al. of biomass from the solution obtained in the previous A simple and efficient procedure of extraction was step. The extract is diluted in methanol and water and developed and optimized using enzymes to obtain of then salted out to precipitate the biomass. Sodium chlo- paclitaxel and the related compounds 7-xyl-10-deacetyl- ride is quickly added to the extract under heavy stirring. paclitaxel, 10-deacetylpaclitaxel, cephalomannine and The formed biomass is separated from the solution by 7-epi-10-deaceylpaclitaxel from needles of Taxus chi- filtration or by centrifugation. The separated biomass, nensis [19]. The optimal procedure of extraction begins which is wet, can be dried by ventilation or lyophilization. with treatment with a cellulose solution of 1 mg·dm-3, a The fourth step removes the resin and natural pigments liquid to solid ratio 1:10 (cm3/g), and incubation at pH by treating the dried biomass with mixture of acetone 4 and 40°C for 24 h. Under these optimal conditions, and hexane. An even better procedure is to put the dried the yields of paclitaxel, 7-xyl-10-deacetylpaclitaxel, biomass back into solution by first adding the mixture of 10-deacetylpaclitaxel, cephalomannine and 7-epi- acetone and hexane and adding 1.5 additional volumes 10-deaceylpaclitaxel were 1.78, 1.26, 2.46, 3.01 and of pure hexane. The final ratio of acetone and hexane 2.25-times higher than in beta-glucosidase, respec- is 1:4. Then, pure water is added to the obtained solu- tively. Additionally, the influence of enzymes on the tion to form a paclitaxel-enriched oily phase, which is structural changes of the plant cells was observed by then transferred into decanting desk. The oily phase scanning electron microscopy (SEM). The extraction us- at the bottom of the flask contains paclitaxel and oth- ing enzymes was improved for ecologic and economic er taxanes. The fifth and final step is chromatographic reasons for large-scale natural product extraction. purification of a solution of the paclitaxel-enriched oily
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