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Plant sterols have been long known to reduce plasma LDL-cholestero levels, by competing with dietary and biliary cholesterol absorption in the intestine. After a number of animal studies in the early 1950s, B-sitostero was introduced in 1957 by Eli Lilly as a drug for the treatment of hyper cholesterolemia [159]. Because of its poor solubility and bioavailability doses of up to 18 g/day had to be given. Phytosterols were soon aban doned, but renewed interest arose more recently with the possible use o plant sterols and sterol derivatives as nutraceutical agents. Phytosterol: and phytostanols are esterified with fatty acids, and these higly lipophilic esters can be readily incorporated into the lipid phase of foods such a: margarines.

Figure 34 Plant sterols have been long known to reduce plasma LDL-cholestero levels, by competing with dietary and biliary cholesterol absorption in the intestine. After a number of animal studies in the early 1950s, B-sitostero was introduced in 1957 by Eli Lilly as a drug for the treatment of hyper cholesterolemia [159]. Because of its poor solubility and bioavailability doses of up to 18 g/day had to be given. Phytosterols were soon aban doned, but renewed interest arose more recently with the possible use o plant sterols and sterol derivatives as nutraceutical agents. Phytosterol: and phytostanols are esterified with fatty acids, and these higly lipophilic esters can be readily incorporated into the lipid phase of foods such a: margarines.

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In order to give extra ‘medicinal life’ to B-lactams that were no longer resistant to the common f-lactamases, in the late 1960s and early 1970s, efforts were made, particularly by Beecham (now part of GlaxoSmithKline) and Pfizer to find molecules that would have similar pharmacokinetics to the 6-lactams but would inhibit the ‘regular’ p-lactamases that were part of the pathogenic microbe’s defense systems. Beecham discovered the clavulanate family with clavulanic acid (9) being incorporated into the combination known as Augmentin® a 1:1 mixture of amoxicillin and clavulanic acid (9) launched in 1981, thus extending the franchise of this particular B-lactam well beyond its original patent date.
In order to give extra ‘medicinal life’ to B-lactams that were no longer resistant to the common f-lactamases, in the late 1960s and early 1970s, efforts were made, particularly by Beecham (now part of GlaxoSmithKline) and Pfizer to find molecules that would have similar pharmacokinetics to the 6-lactams but would inhibit the ‘regular’ p-lactamases that were part of the pathogenic microbe’s defense systems. Beecham discovered the clavulanate family with clavulanic acid (9) being incorporated into the combination known as Augmentin® a 1:1 mixture of amoxicillin and clavulanic acid (9) launched in 1981, thus extending the franchise of this particular B-lactam well beyond its original patent date.
[104, 105]. Intestinal microsporidiosis, which is caused by the spore-form. ing unicellular parasite Enterocytozoon bieneusi, causes chronic diarrhez and is a major concern to immunocompromised patients [106]. The anti- microsporidiosis and angiogenic activities of fumagillin (66) and relatec compounds are thought to be due to its strong methionine aminopeptidase 2 (MetAP2) binding [107, 108]. It has been shown using zebra fish models that MetAP-2 inhibition blocks noncanonical Wnt signaling, which plays a critical role in development, cell differentiation, and tumorigenesis [109, 110].
[104, 105]. Intestinal microsporidiosis, which is caused by the spore-form. ing unicellular parasite Enterocytozoon bieneusi, causes chronic diarrhez and is a major concern to immunocompromised patients [106]. The anti- microsporidiosis and angiogenic activities of fumagillin (66) and relatec compounds are thought to be due to its strong methionine aminopeptidase 2 (MetAP2) binding [107, 108]. It has been shown using zebra fish models that MetAP-2 inhibition blocks noncanonical Wnt signaling, which plays a critical role in development, cell differentiation, and tumorigenesis [109, 110].
tion is essential for solid tumor to grow beyond a threshold size and is also critical for the spread of tumor cells to distant organs [48]. Com- bretastatin selectively disrupts the vascular system of tumors leading to extensive tumor necrosis at doses less than one tenth of the maximum tolerated dose in mice [49]. The mechanism for the selective vascular col- lapse observed in tumors is not yet completely elucidated, but it has been shown that combretastatin has dramatic effects on the three dimensional shape of immature endothelial cells. Combretastatin 4A causes disruption of the tubulin cytoskeleton and remodelling of the actin cytoskeleton [50]. Clinical trials are being performed with the water-soluble prodrug combretastatin 4A phosphate (38), which is now produced synthetically in a five step process. Despite extensive tumor cell killing, significant delay of tumor growth is rarely obtained when combretastatin 4A phosphate is used as a single therapy. This has been attributed to the rapid re-growth from the rim of cells at the tumor periphery. On the other hand, results in experimental tumor models with combretastatin 4A phosphate in com- bination with conventional cytotoxic chemotherapy or radiotherapy are promising. Such combinations are currently being tested in Phase IJ clini- cal trials against a range of tumor types. Combretastatin 4A phosphate is developed by OxiGene but is not the only compound under investigation. The straightforward synthesis has facilitated the preparation of hundreds of combrestatin derivatives [51]. Molecules in development include com- bretastatin-1 phosphate also in preclinical trial at OxyGene, and AVE8062 (39), a prodrug with a serine residue which is currently in Phase I clinical
tion is essential for solid tumor to grow beyond a threshold size and is also critical for the spread of tumor cells to distant organs [48]. Com- bretastatin selectively disrupts the vascular system of tumors leading to extensive tumor necrosis at doses less than one tenth of the maximum tolerated dose in mice [49]. The mechanism for the selective vascular col- lapse observed in tumors is not yet completely elucidated, but it has been shown that combretastatin has dramatic effects on the three dimensional shape of immature endothelial cells. Combretastatin 4A causes disruption of the tubulin cytoskeleton and remodelling of the actin cytoskeleton [50]. Clinical trials are being performed with the water-soluble prodrug combretastatin 4A phosphate (38), which is now produced synthetically in a five step process. Despite extensive tumor cell killing, significant delay of tumor growth is rarely obtained when combretastatin 4A phosphate is used as a single therapy. This has been attributed to the rapid re-growth from the rim of cells at the tumor periphery. On the other hand, results in experimental tumor models with combretastatin 4A phosphate in com- bination with conventional cytotoxic chemotherapy or radiotherapy are promising. Such combinations are currently being tested in Phase IJ clini- cal trials against a range of tumor types. Combretastatin 4A phosphate is developed by OxiGene but is not the only compound under investigation. The straightforward synthesis has facilitated the preparation of hundreds of combrestatin derivatives [51]. Molecules in development include com- bretastatin-1 phosphate also in preclinical trial at OxyGene, and AVE8062 (39), a prodrug with a serine residue which is currently in Phase I clinical
1igher and more sustained activity compared to artesuanate. A common lownside of the hemisynthetic derivatives is that they require artemisinin is starting material. Artemisinin is extracted from the plant in low yield 0.01-0.8%) [87]; this could become a supply and cost issue for a drug to ye used on a large scale, in particular in developing countries. Therefore, everal groups have attempted to produce fully synthetic peroxide analogs, ome of which show remarkable activity. They include trioxane, tetraox- ine and endoperoxide analogs such as arteflene (65) [87, 92]. In addition, asily synthesized 1,2,4-trioxolanes substituted with an adamentane ring vere recently shown to be active in the nanomolar range, chemically table and orally active in mice [87]. Ranbaxy Laboratories started, in 004, with Phase I clinical trials on OZ-277 (66) [23]. Finally, quinoline- yeroxide hybrids known as trioxaquines (e.g., compound 67) have also een prepared [87].
1igher and more sustained activity compared to artesuanate. A common lownside of the hemisynthetic derivatives is that they require artemisinin is starting material. Artemisinin is extracted from the plant in low yield 0.01-0.8%) [87]; this could become a supply and cost issue for a drug to ye used on a large scale, in particular in developing countries. Therefore, everal groups have attempted to produce fully synthetic peroxide analogs, ome of which show remarkable activity. They include trioxane, tetraox- ine and endoperoxide analogs such as arteflene (65) [87, 92]. In addition, asily synthesized 1,2,4-trioxolanes substituted with an adamentane ring vere recently shown to be active in the nanomolar range, chemically table and orally active in mice [87]. Ranbaxy Laboratories started, in 004, with Phase I clinical trials on OZ-277 (66) [23]. Finally, quinoline- yeroxide hybrids known as trioxaquines (e.g., compound 67) have also een prepared [87].
transcriptase inhibitors. The clinical development of calanolide A startec in 1997. The compound is now obtained by synthesis and is currently in Phase IJ trials focussed on evaluation of its long-term activity in combina. tion with other anti-HIV agents. Calanolide B (costatolide), dihydrocala- nolide B (73) and oxocalanolide (74) are in preclinical development [95] Calanolides A and B were also found to be active against Mycobacteriun tuberculosis [100]. This property is unique among antiviral agents anc could be of great value for the treatment of patients infected with both HIV and tuberculosis [23]. Tuberculosis has become the first opportunistic infection affecting HIV-infected people in developing countries.
transcriptase inhibitors. The clinical development of calanolide A startec in 1997. The compound is now obtained by synthesis and is currently in Phase IJ trials focussed on evaluation of its long-term activity in combina. tion with other anti-HIV agents. Calanolide B (costatolide), dihydrocala- nolide B (73) and oxocalanolide (74) are in preclinical development [95] Calanolides A and B were also found to be active against Mycobacteriun tuberculosis [100]. This property is unique among antiviral agents anc could be of great value for the treatment of patients infected with both HIV and tuberculosis [23]. Tuberculosis has become the first opportunistic infection affecting HIV-infected people in developing countries.
of vaccines and has been tested so far in over 60 trials with a total of more than 3,000 patients [148, 150]. Quillaja saponins are able to form so-called immune-stimulating com- plexes (ISCOMS). These are cage-like, stable complexes of cholesterol, phospholipid and saponin which can serve as carriers for amphipathic protein antigens such as viral envelope proteins [152]. ISCOMS with Quil- laja saponins have been shown to stimulate both humoral and cellular immune response, similar to the effects of the saponins. However, the adjuvant effect was greater than for the micellar form. An interesting field of possible application of ISCOMS is in the development of mucosal vac- cines [153]. In view of possible pharmaceutical application in humans, the stability of these bidesmosidic Quillaja saponins has been investigated. Ata pH of 5.5-6, stability was satisfactory over 2 years. Currently, economically feasible supply of highly purified QS-21 seems to be a major obstacle in the commercial development of this promising adjuvant.
of vaccines and has been tested so far in over 60 trials with a total of more than 3,000 patients [148, 150]. Quillaja saponins are able to form so-called immune-stimulating com- plexes (ISCOMS). These are cage-like, stable complexes of cholesterol, phospholipid and saponin which can serve as carriers for amphipathic protein antigens such as viral envelope proteins [152]. ISCOMS with Quil- laja saponins have been shown to stimulate both humoral and cellular immune response, similar to the effects of the saponins. However, the adjuvant effect was greater than for the micellar form. An interesting field of possible application of ISCOMS is in the development of mucosal vac- cines [153]. In view of possible pharmaceutical application in humans, the stability of these bidesmosidic Quillaja saponins has been investigated. Ata pH of 5.5-6, stability was satisfactory over 2 years. Currently, economically feasible supply of highly purified QS-21 seems to be a major obstacle in the commercial development of this promising adjuvant.
involved in the regulation of cholesterol and bile acids. Upon activation by bile acids, the natural ligands, FXR regulates the expression of genes involved in cholesterol/bile acid homeostasis and thus appears to play a key role in steroid homeostasis. Gugulsterones inhibited the activation of FXR target genes in response to bile acids and a synthetic agonist, and decreased hepatic cholesterol levels in wild-type mice fed with a high cho- lesterol diet, but not in FXR null mice. Despite the steroidal skeleton of the compounds, they are reportedly devoid of estrogenic, antiestrogenic and progesterone-like activity, and also do not activate or inhibit transac- tivation of other nuclear receptors involved in lipid metabolisms [158]). However, weak activation of the pregnane X receptor was found which possibly explains the induction of certain CYP isoenzymes. Although no comprehensive regulatorv toxicity data have been pub-
involved in the regulation of cholesterol and bile acids. Upon activation by bile acids, the natural ligands, FXR regulates the expression of genes involved in cholesterol/bile acid homeostasis and thus appears to play a key role in steroid homeostasis. Gugulsterones inhibited the activation of FXR target genes in response to bile acids and a synthetic agonist, and decreased hepatic cholesterol levels in wild-type mice fed with a high cho- lesterol diet, but not in FXR null mice. Despite the steroidal skeleton of the compounds, they are reportedly devoid of estrogenic, antiestrogenic and progesterone-like activity, and also do not activate or inhibit transac- tivation of other nuclear receptors involved in lipid metabolisms [158]). However, weak activation of the pregnane X receptor was found which possibly explains the induction of certain CYP isoenzymes. Although no comprehensive regulatorv toxicity data have been pub-
The first and prototypical AChE inhibitor was physostigmine (115), an alkaloid from the calabar bean (Physostigma venenosum, Fabaceae). Physo- stigmine was isolated in 1864 as the toxic principle of an African ordeal poison and was subsequently introduced as a glaucoma treatment. Recent clinical studies with the compound showed cognitive benefits in normal and AD patients, but a short half-life prevented further development. The carbamate moiety of the compound is essential for its activity. It interacts with a serine in AChE which is normally involved in the cleavage of ACh by transient formation of a covalent bond with the acetyl moiety. Via a nucleophilic attack of the serine, the carbamoyl group of physostigmine is covalently bound to the catalytic site of the enzyme. Subsequent hydro- lysis is slow, and the alkaloid thus acts as a quasi-irreversible inhibitor. Modification of the side chain and introduction of quaternary ammonium derivatives led to peripherally active AChE inhibitors such as neostigmine (116) which has been used for the treatment of myasthenia gravis. Devel- opment of lipophilic analogs, able to cross the blood-brain barrier, led to rivastigmine (117) which was introduced in Alzheimer therapy in 2000 [173].
The first and prototypical AChE inhibitor was physostigmine (115), an alkaloid from the calabar bean (Physostigma venenosum, Fabaceae). Physo- stigmine was isolated in 1864 as the toxic principle of an African ordeal poison and was subsequently introduced as a glaucoma treatment. Recent clinical studies with the compound showed cognitive benefits in normal and AD patients, but a short half-life prevented further development. The carbamate moiety of the compound is essential for its activity. It interacts with a serine in AChE which is normally involved in the cleavage of ACh by transient formation of a covalent bond with the acetyl moiety. Via a nucleophilic attack of the serine, the carbamoyl group of physostigmine is covalently bound to the catalytic site of the enzyme. Subsequent hydro- lysis is slow, and the alkaloid thus acts as a quasi-irreversible inhibitor. Modification of the side chain and introduction of quaternary ammonium derivatives led to peripherally active AChE inhibitors such as neostigmine (116) which has been used for the treatment of myasthenia gravis. Devel- opment of lipophilic analogs, able to cross the blood-brain barrier, led to rivastigmine (117) which was introduced in Alzheimer therapy in 2000 [173].
butylcholinesterase [174]. The compound has no affinity for cholinergic receptors [175]. Interestingly, the alkaloid possesses some neuroprotective properties which may be due to its ability to block excitatory action at NMDaA-type glutamate receptors. Bioavailability and penetration through the blood-brain barrier are high. Several clinical studies have been con- ducted with huperzine A, and significant improvement over placebo was reported [174]. The template of huperzine A has been the starting point for numerous synthetic efforts to find compounds with an improved activ- ity and safety profile. ZT-1 (119), a Schiff base prepared by condensation of huperzine A with 5-chloro-o-vanillin, is reportedly more selective as an AChE inhibitor and shows less toxicity than the parent compound by retaining potency, bioavailability, and duration of action. ZT-I has success- fully passed Phase I clinical trials and is reportedly in a large multicenter Phase II study [176].
butylcholinesterase [174]. The compound has no affinity for cholinergic receptors [175]. Interestingly, the alkaloid possesses some neuroprotective properties which may be due to its ability to block excitatory action at NMDaA-type glutamate receptors. Bioavailability and penetration through the blood-brain barrier are high. Several clinical studies have been con- ducted with huperzine A, and significant improvement over placebo was reported [174]. The template of huperzine A has been the starting point for numerous synthetic efforts to find compounds with an improved activ- ity and safety profile. ZT-1 (119), a Schiff base prepared by condensation of huperzine A with 5-chloro-o-vanillin, is reportedly more selective as an AChE inhibitor and shows less toxicity than the parent compound by retaining potency, bioavailability, and duration of action. ZT-I has success- fully passed Phase I clinical trials and is reportedly in a large multicenter Phase II study [176].
ee ci die Schematic representation of the phylogenetic relationships between the different types of FAS and PKS based on amino acid sequences of KS domains. KS domains of diverse types of PKS in bacteria and plants descend from FabF and FabH type KS that are part of type Il FAS systems in eubacteria. The tree combines the results from Jenke-Kodama et al. (2005) and Gross et al. (2006) and unpublished data. The branch lengths are arbitrary. Clade credibility values are given in the original publications. Schematic representations of PKS types are shown in Figure 1.
ee ci die Schematic representation of the phylogenetic relationships between the different types of FAS and PKS based on amino acid sequences of KS domains. KS domains of diverse types of PKS in bacteria and plants descend from FabF and FabH type KS that are part of type Il FAS systems in eubacteria. The tree combines the results from Jenke-Kodama et al. (2005) and Gross et al. (2006) and unpublished data. The branch lengths are arbitrary. Clade credibility values are given in the original publications. Schematic representations of PKS types are shown in Figure 1.
Examples of microbial natural products relevant for the pharmaceutical industry
Examples of microbial natural products relevant for the pharmaceutical industry
are not of interest. The second major hurdle in the process, de novo struc- ture determination of compounds that are new molecular entities (NME), is an area that has been revolutionized by many advances in spectroscopic techniques, particularly high resolution NMR technologies. While many approaches have been developed to lower the third hurdle, that being the isolation and purification of the active principles (often a minor compo- nent) from a complex matrix, it remains as a major bottleneck that con- tinues to impact natural product drug discovery [3]. In the HTS approach, it is essential that new technologies be leveraged in order to facilitate the rapid navigation of natural product hits through the detection, validation, rapid navigation of natural product hits through the detection, validation,
are not of interest. The second major hurdle in the process, de novo struc- ture determination of compounds that are new molecular entities (NME), is an area that has been revolutionized by many advances in spectroscopic techniques, particularly high resolution NMR technologies. While many approaches have been developed to lower the third hurdle, that being the isolation and purification of the active principles (often a minor compo- nent) from a complex matrix, it remains as a major bottleneck that con- tinues to impact natural product drug discovery [3]. In the HTS approach, it is essential that new technologies be leveraged in order to facilitate the rapid navigation of natural product hits through the detection, validation, rapid navigation of natural product hits through the detection, validation,
yut the issue is exacerbated with natural products since the presence c hese interfering compounds may not be fully characterized. It has nov een shown that in kinase, protease and phosphatase assays, fluorescen ‘ompound and light scattering interferences can be overcome by increas ng the fluorophore concentration in the assay, by using red-shifted wave ength dyes [9], or by the technique of lifetime discriminated polarizatio: 8]. Further, an effective strategy that helps to alleviate these kinds of inte! erences as well as shorten the time needed to isolate the active principle i he use of purified or pre-fractionated screening samples from the origin rude extract (Fig. 2). When developing an overall screening strategy for natural product
yut the issue is exacerbated with natural products since the presence c hese interfering compounds may not be fully characterized. It has nov een shown that in kinase, protease and phosphatase assays, fluorescen ‘ompound and light scattering interferences can be overcome by increas ng the fluorophore concentration in the assay, by using red-shifted wave ength dyes [9], or by the technique of lifetime discriminated polarizatio: 8]. Further, an effective strategy that helps to alleviate these kinds of inte! erences as well as shorten the time needed to isolate the active principle i he use of purified or pre-fractionated screening samples from the origin rude extract (Fig. 2). When developing an overall screening strategy for natural product
Comparison of drug-like properties of natural product and synthetic drug molecules
Comparison of drug-like properties of natural product and synthetic drug molecules
cation. Recent advances in HPLC-MS have greatly facilitated this process. Additional criteria are employed to prioritize the hits if the library is of significant size, because the number of initial active samples is likely too large for each hit to undergo bio-assay guided fractionation. Criteria must be employed to ascertain the likelihood of the observed activity being due to the presence of a natural product of interest. One of these criteria is the presence of so-called non-specific compounds or pan-inhibitors such as those shown in Figure 5. For example, the potent pan-kinase inhibitor staurosporine is produced in many actinomycete fermentations and is frequently identified as active in kinase assays [27]. Indeed, staurosporine itself has served as a lead for the development of several clinical candidates [28], however it might not be of interest if novel structural types were
cation. Recent advances in HPLC-MS have greatly facilitated this process. Additional criteria are employed to prioritize the hits if the library is of significant size, because the number of initial active samples is likely too large for each hit to undergo bio-assay guided fractionation. Criteria must be employed to ascertain the likelihood of the observed activity being due to the presence of a natural product of interest. One of these criteria is the presence of so-called non-specific compounds or pan-inhibitors such as those shown in Figure 5. For example, the potent pan-kinase inhibitor staurosporine is produced in many actinomycete fermentations and is frequently identified as active in kinase assays [27]. Indeed, staurosporine itself has served as a lead for the development of several clinical candidates [28], however it might not be of interest if novel structural types were
. ee Principles of saturation transfer difference (STD) NMR in the STD experiment, a large excess of scree- ning small molecule mixture composed of binders (triangle) and non-binders (ellipse) is exposed to target protein. The process is composed of the following steps: 1) Free ligand molecule (triangle) binds protein at rate constant K,,,, 2) Irradiation saturation is applied to protein resonance but away from the ligand resonances. Bound ligand molecule (triangle) is saturated via spin diffusion, 3) Saturated ligand dissociates at rate constant K,,, and free ligand population (binders only) becomes saturated, 4) Saturation of free ligand is built up in bulk population, 5) Saturation of free ligand is lost by relaxation. Re-association to protein occurs and saturation transfer cycle is repeated. 6) Difference spectrum is measured between on and off protein resonance, resulting in signals (in box) only from those protons on binding ligands.
. ee Principles of saturation transfer difference (STD) NMR in the STD experiment, a large excess of scree- ning small molecule mixture composed of binders (triangle) and non-binders (ellipse) is exposed to target protein. The process is composed of the following steps: 1) Free ligand molecule (triangle) binds protein at rate constant K,,,, 2) Irradiation saturation is applied to protein resonance but away from the ligand resonances. Bound ligand molecule (triangle) is saturated via spin diffusion, 3) Saturated ligand dissociates at rate constant K,,, and free ligand population (binders only) becomes saturated, 4) Saturation of free ligand is built up in bulk population, 5) Saturation of free ligand is lost by relaxation. Re-association to protein occurs and saturation transfer cycle is repeated. 6) Difference spectrum is measured between on and off protein resonance, resulting in signals (in box) only from those protons on binding ligands.
Figure 2. Schematic correlation between bioinformatics and chemoinformatics
Figure 2. Schematic correlation between bioinformatics and chemoinformatics
trategy B for the discovery of bioactive NPs using an integrated virtual screening approach
trategy B for the discovery of bioactive NPs using an integrated virtual screening approach
Reproduced from [45] with the permission from the copyright holder, John Wiley & Sons, Inc. 23 Factorial design including all interactions
Reproduced from [45] with the permission from the copyright holder, John Wiley & Sons, Inc. 23 Factorial design including all interactions
First row of the Plackett-Burman arrays — signs are used to denote high and low levels of the variables, respectively. From this first row the rest of the rows are generated by moving the previ- ous row to the right. For example to generate the second row the last entry in the first row is placed in the first column of the second row and all othe entries in the first column are moved one position to the right. The third row is generated from the second row in the same manner [49]. This design has been very useful in rapidly screening a large number of variables.
First row of the Plackett-Burman arrays — signs are used to denote high and low levels of the variables, respectively. From this first row the rest of the rows are generated by moving the previ- ous row to the right. For example to generate the second row the last entry in the first row is placed in the first column of the second row and all othe entries in the first column are moved one position to the right. The third row is generated from the second row in the same manner [49]. This design has been very useful in rapidly screening a large number of variables.
A balanced incomplete block design for four variables in six blocks
A balanced incomplete block design for four variables in six blocks
List of antifoam agents the surface, and increase in bubble coalescence leading to reduction of interfacial area a [86]. Lowering of mass transfer was also reported by Yagi and Yoshida [87]. However, the observed effect on the OTR was smaller, which could have been due to the lower concentration of antifoam used. While these data were obtained in bubble reactors, reduction in OTR was also noted in stirred tanks [88, 89]. Some commonly used antifoam agents are listed in Table 6. ‘iia, a yy w@Ze « ss» ¢« = «a
List of antifoam agents the surface, and increase in bubble coalescence leading to reduction of interfacial area a [86]. Lowering of mass transfer was also reported by Yagi and Yoshida [87]. However, the observed effect on the OTR was smaller, which could have been due to the lower concentration of antifoam used. While these data were obtained in bubble reactors, reduction in OTR was also noted in stirred tanks [88, 89]. Some commonly used antifoam agents are listed in Table 6. ‘iia, a yy w@Ze « ss» ¢« = «a
Plant cell cultures used in industry (Adapted from reference [16])
Plant cell cultures used in industry (Adapted from reference [16])
EE eee a a ee ie Paclitaxel is found in the bark of Taxus brevifolia, the Pacific yew tree, in small amounts: approximately 0.01% of the dry weight of the bark [44]. The bark is non-renewable — harvesting of the bark results in the death of the tree — and for many years it appeared that no sustainable supply of the drug existed. The structural complexity of the molecule prevented an eco- nomic total synthesis for industrial scale production. A number of related diterpene compounds referred to as taxanes are found in a variety of other Taxus species. One of these related taxanes baccatin III, a paclitaxel inter- mediate, is found in the needles of Taxus baccata, the European Yew tree, mediate, is found in the needles of Taxus baccata, the European Yew tree,
EE eee a a ee ie Paclitaxel is found in the bark of Taxus brevifolia, the Pacific yew tree, in small amounts: approximately 0.01% of the dry weight of the bark [44]. The bark is non-renewable — harvesting of the bark results in the death of the tree — and for many years it appeared that no sustainable supply of the drug existed. The structural complexity of the molecule prevented an eco- nomic total synthesis for industrial scale production. A number of related diterpene compounds referred to as taxanes are found in a variety of other Taxus species. One of these related taxanes baccatin III, a paclitaxel inter- mediate, is found in the needles of Taxus baccata, the European Yew tree, mediate, is found in the needles of Taxus baccata, the European Yew tree,
Plant cell suspension cultures producing paclitaxel (Adapted from Baldi A, Bisaria VS, Srivasrava AK (2007) Biotechnological approaches for the production of some promising plant based chemothera- pies. In: O Kayser, W Quax (eds.): Medicinal plant biotechnology. Wiley VCH, Weinheim, 128)
Plant cell suspension cultures producing paclitaxel (Adapted from Baldi A, Bisaria VS, Srivasrava AK (2007) Biotechnological approaches for the production of some promising plant based chemothera- pies. In: O Kayser, W Quax (eds.): Medicinal plant biotechnology. Wiley VCH, Weinheim, 128)
wage Vv Paclitaxel biosynthetic pathway. GGPS (geranyl geranyl pyrophosphate synthase); TASY (taxadiene synthase); T5aH (taxadiene 5a hydroxylase); TDAT (taxadieneSa-ol O-acetyltransferase); T10BH (taxane 106-hydroxylase); TBT (taxane 2a-O-benzoyltransferase); DBAT (10-deacetylbaccatin III-10- O-acetyltransferase); PPT (phenylpropanoyl transferase); NDBzT (3’-N-debenzoyl-2-deoxytaxol-N- benzoyltransferase)
wage Vv Paclitaxel biosynthetic pathway. GGPS (geranyl geranyl pyrophosphate synthase); TASY (taxadiene synthase); T5aH (taxadiene 5a hydroxylase); TDAT (taxadieneSa-ol O-acetyltransferase); T10BH (taxane 106-hydroxylase); TBT (taxane 2a-O-benzoyltransferase); DBAT (10-deacetylbaccatin III-10- O-acetyltransferase); PPT (phenylpropanoyl transferase); NDBzT (3’-N-debenzoyl-2-deoxytaxol-N- benzoyltransferase)
Biosynthetic scheme for camptothecin. Structure of camptothecin and derivatives The initial step in biosynthesis of camptothecin — along with all other monoterpene indole alkaloid natural products — is the condensation of tryptamine and secologanin to form the biosynthetic intermediate stric- tosidine (Fig. 7). At this stage, the biosynthesis of camptothecin diverges from other terpene indole alkaloids; typically, strictosidine is immediately deglycosylated, but in the camptothecin pathway strictosidine instead forms the strictosamide intermediate (Fig. 7). The biosynthetic conversion of strictosamide to camptothecin is not known on the enzymatic level. However, intermediates observed in camptothecin producing cultures [63, 64] has led to the proposal of a reasonable series of biosynthetic trans- formations (Fig. 7). The lack of genetic and enzymatic knowledge of the biosynthesis of this important class of alkaloids greatly hinders metabolic engineering efforts to improve the production of camptothecin and its derivatives.
Biosynthetic scheme for camptothecin. Structure of camptothecin and derivatives The initial step in biosynthesis of camptothecin — along with all other monoterpene indole alkaloid natural products — is the condensation of tryptamine and secologanin to form the biosynthetic intermediate stric- tosidine (Fig. 7). At this stage, the biosynthesis of camptothecin diverges from other terpene indole alkaloids; typically, strictosidine is immediately deglycosylated, but in the camptothecin pathway strictosidine instead forms the strictosamide intermediate (Fig. 7). The biosynthetic conversion of strictosamide to camptothecin is not known on the enzymatic level. However, intermediates observed in camptothecin producing cultures [63, 64] has led to the proposal of a reasonable series of biosynthetic trans- formations (Fig. 7). The lack of genetic and enzymatic knowledge of the biosynthesis of this important class of alkaloids greatly hinders metabolic engineering efforts to improve the production of camptothecin and its derivatives.
HnH6H is Hyoscyamus niger H6H, NtPMT is Nicotiana tabacum PMT solonaceae plants (Tab. 4). The overexpression of H6H under control of the SMV 35S promoter in transgenic hairy root cultures of Atropa baetica [84] Tyoscyamus niger [85], Hyoscyamus muticus [86], Scopolia parviflora [87] ind a Duboisia hybrid [88] cultures systematically resulted in an increas« n the production of scopolamine when compared to control cultures no’ -xpressing H6H (Tab. 4). Therefore, transgenic expression of H6H appear: o be a general method for increasing scopolamine production in hairy ‘oot cultures of tropane alkaloid producing plants. OTe kw wee men’ mux well ceeeuikwoanatwan cn dhe che mw RY seemtllecoGance thaw asitwewm beowr ae bi nw wee
HnH6H is Hyoscyamus niger H6H, NtPMT is Nicotiana tabacum PMT solonaceae plants (Tab. 4). The overexpression of H6H under control of the SMV 35S promoter in transgenic hairy root cultures of Atropa baetica [84] Tyoscyamus niger [85], Hyoscyamus muticus [86], Scopolia parviflora [87] ind a Duboisia hybrid [88] cultures systematically resulted in an increas« n the production of scopolamine when compared to control cultures no’ -xpressing H6H (Tab. 4). Therefore, transgenic expression of H6H appear: o be a general method for increasing scopolamine production in hairy ‘oot cultures of tropane alkaloid producing plants. OTe kw wee men’ mux well ceeeuikwoanatwan cn dhe che mw RY seemtllecoGance thaw asitwewm beowr ae bi nw wee
x i—_ 7 Tetrahydroisoquinoline biosynthesis. NCS (norcoclaurine synthase); 6-OMT (O-methyl transferase); NMT (N-methyltransferase); CPY80B3 (cytochrome P450); 40MT (O-methyl transferase) BBE (ber- berine bridge enzyme); STS (salutaridinol synthase); SAT (salutidarinol-7-O-acetyltransferase), COR (codeine reductase)
x i—_ 7 Tetrahydroisoquinoline biosynthesis. NCS (norcoclaurine synthase); 6-OMT (O-methyl transferase); NMT (N-methyltransferase); CPY80B3 (cytochrome P450); 40MT (O-methyl transferase) BBE (ber- berine bridge enzyme); STS (salutaridinol synthase); SAT (salutidarinol-7-O-acetyltransferase), COR (codeine reductase)
anti-inflammatory and antitumor activity. Mechanistic studies indicate that shikonin acts as a topoisomerase I inhibitor and that it also induces apoptosis in certain leukemia cell lines [126]. Shikonin appears to inhibit angiogenesis in in vitro studies. Shikonin is also used as a lipstick pigment in certain countries [127]. It was first demonstrated that Lithospermum erythrorhizon callus cultures yielded shikonin acy] esters in the 1970s [128]. High producing cell lines, producing up to 1.2 mg/g (fresh weight), could be selected for visually, since shikonin is red. Production in cell suspension cultures was subjected to extensive optimization both in terms of genera- tion of cell biomass and product production. It was found that sucrose, oligogalacturonide and methyl jasmonate upregulated the production of shikonin [127]. A 750 L bioreactor culture yielded 1.4—2.3 g/L of shikonin in 23 days [129]. Commercial production of shikonin was reported by Mitsui Petrochemical Industry in 1983 [127]. , i Se ee a | Ee ee ee ee eS ae ee a A AN
anti-inflammatory and antitumor activity. Mechanistic studies indicate that shikonin acts as a topoisomerase I inhibitor and that it also induces apoptosis in certain leukemia cell lines [126]. Shikonin appears to inhibit angiogenesis in in vitro studies. Shikonin is also used as a lipstick pigment in certain countries [127]. It was first demonstrated that Lithospermum erythrorhizon callus cultures yielded shikonin acy] esters in the 1970s [128]. High producing cell lines, producing up to 1.2 mg/g (fresh weight), could be selected for visually, since shikonin is red. Production in cell suspension cultures was subjected to extensive optimization both in terms of genera- tion of cell biomass and product production. It was found that sucrose, oligogalacturonide and methyl jasmonate upregulated the production of shikonin [127]. A 750 L bioreactor culture yielded 1.4—2.3 g/L of shikonin in 23 days [129]. Commercial production of shikonin was reported by Mitsui Petrochemical Industry in 1983 [127]. , i Se ee a | Ee ee ee ee eS ae ee a A AN
Related topics:
Oral Hypoglycemic AgentsNeuroprotective AgentsPharmaceutical preparations
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