The process of in vitro-in vivo extrapolation (IVIVE) can be used to predict pharmacokinetics of drugs in patients using data from in vitro systems. This process relies on the use of experimentally obtained scaling factors, such as abundances of different drug-metabolising enzymes and microsomal protein content (MPPGL). The use of simulators is dependent on abundances and activities of pharmacokinetically relevant enzymes. The incorporation of inter-individual variability in abundances of enzymes, correlations between enzyme expression patterns, and relationships between genetic, physiological, and environmental factors and enzyme expression and activity can make predictions using IVIVE and simulations of pharmacokinetic experiments in virtual populations more accurate and realistic. Incorporation of variability and correlations can also assist in predicting extreme cases where drug therapy may be ineffective or may cause adverse effects. A meta-analysis of 52 studies was carried out to assess the reported abundances of cytochrome P450 and uridine glucuronosyltransferase (UGT) enzymes in adult Caucasian subjects. Some heterogeneity was found between studies and the weighted means and overall coefficients of variation were calculated. Some strong enzyme expression correlations were identified; CYP3A4/CYP3A5*1/*3 (rs = 0.66, p < 0.0001, n = 37), CYP3A4/CYP2C8 (rs = 0.79, p < 0.0001, n = 107), and CYP2C8/CYP2C9 (rs = 0.71, p < 0.0001, n = 72). A quantitative protocol based on targeted proteomics was used to quantify cytochrome P450 and UGT enzymes in adult liver samples (n = 24). The QconCAT standard used for quantification was successfully expressed in-house after optimisation of the expression protocol, and the utility of two strategies in expressing recalcitrant QconCAT proteins was highlighted; the use of a fusion partner and reshuffling the order of peptides in the sequence. The enzymes quantified in this study were CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2D6, 2J2, 3A4, 3A5, 3A7, 3A43, and 4F2, and UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B4, 2B7, and 2B15. Correlations of expression identified in the meta-analysis were confirmed and new correlations were demonstrated between UGT enzymes and between enzymes from the two families. Correlations between UGT enzymes were particularly strong and statistically significant. Relationships between enzyme expression levels and genotype, age, sex, smoking, and alcohol consumption were investigated. A significant effect of genotype on expression was seen for CYP3A5 (p < 0.0001). An overall moderate decline of expression with age was observed for all the enzymes under study; however, this relationship was not statistically significant in most cases. Gender did not have a considerable effect on expression, although some differences in expression were observed between male and female donors. Smoking seemed to induce the expression of all enzymes; however, statistically significant induction was demonstrated only in the cases of CYP2A6, CYP3A4, CYP3A7, and UGT1A1 (p < 0.05). Alcohol consumption was not shown to have a considerable effect on enzyme expression. Two pig livers were used to optimise some aspects of the experimental protocol including solubilisation and digestion of proteins. Pig MPPGL was measured and relative hepatic contents of drug-metabolising cytochrome P450 enzymes in pig liver were established using label-free quantification.

Alteration in diet and knockdown of detoxification genes impacts the response of C. elegans to oxygen deprivation stress. I hypothesized that feeding worms a vitamin D3-supplementation diet would result in differential oxygen deprivation stress response. We used a combination of wet lab and transcriptomics approach to investigate the effect of a vitamin-D3 supplemented diet on the global gene expression changes and the anoxia response phenotype of C. elegans (Chapter 2). C. elegans genome consists of 143 detoxification genes (cyp and ugt). The presence of a significant number of genes in these detoxification families was a challenge with identifying and selecting specific cyp and ugt genes for detailed analysis. Our goal was to understand the evolution, phylogenetic, and expression of the detoxification enzymes CYPs and UGTs in C. elegans (Chapter 3). We undertook a phylogenetic and bioinformatics approach to analyze the C. elegans, detoxification family. Phylogenetic analysis provided insight into the association of the human and C. elegans xenobiotic/endobiotic detoxification system. Protein coding genes in C. elegans have been predicted to be human orthologs. The results of this work demonstrate the role of C. elegans in the identification and characterization of vitamin D3 induced alterations in gene expression profile and anoxia response phenotypes and the identification of human orthologs for the detoxification enzymes and provides insight into the gene expression pattern.

The purported chemopreventive and chemotherapeutic properties of the dietary phytochemical resveratrol continue to undergo active investigations. Systemic pharmacokinetics of this compound revealed that it was rapidly and extensively metabolized into its sulfate and glucuronide conjugates. This extensive metabolism leads to high plasma levels of resveratrol sulfates and glucuronides and very low levels of the parent compound (low bioavailability). These observations raised many questions, some of which this body of work examined and has helped to explain. Chapter 1 presents a detailed introduction to resveratrol and its role in colorectal cancer chemoprevention. It also lays the foundation for the hypotheses generated and the studies presented in succeeding chapters. In chapter 2, we explored the possibility that resveratrol metabolites possess intrinsic activity and thus contribute to the observed effects of the parent. The mono-sulfated and glucuronidated conjugates of trans-resveratrol were synthesized and tested for antiproliferative activity in a panel of mammalian cell lines. Their activity was then compared with the parent compound. Resveratrol was shown to be antiproliferative in all cell lines studied while no discernible antiproliferative activity was observed for the metabolites. Chapter 3 details the results of the glucuronidation kinetics of cis and trans-resveratrol isomers across a wide concentration range chosen to mimic blood levels following high dose consumption. Human tissue microsomes and recombinant supersomes over-expressing the enzymes (UGTs) of interest were used for these studies. Our results show the presence of atypical kinetics for the formation of resveratrol glucuronides across most of the protein sources used. Prior to this study, the full glucuronidation kinetics of total resveratrol had not been conducted. In chapter 4, we examined the association between genetic polymorphisms in the major enzymes (UGT1A1 and UGT1A6) and rates of glucuronidation of trans and cis-resveratrol. We set out to correlate functional genetic variations in these UGTs with their catalytic rates and a positive association was made for cis-resveratrol and UGT1A6 where the UGT1A6 variants mediated higher glucuronidation rates compared to the reference genotype. Chapter 5 explored the inherent ability of resveratrol to induce its own glucuronidation upon chronic dosing. Enzyme induction has been proposed as a mechanism that may contribute to the low bioavailability of resveratrol. Since dietary polyphenols like resveratrol are not consumed in isolation, we also studied the effects of combining resveratrol with two dietary polyphenols (curcumin and chrysin) on two chemoprevention endpoints - i) antiproliferation and ii) UGT enzyme induction. Our results indicate that resveratrol is capable of inducing UGT1A1 expression and activity in a non-concentration dependent manner and this induction as well as its antiproliferative effects are enhanced by both curcumin and chrysin. In summary, en route to probing the activity of resveratrol metabolites, we optimized two synthetic routes and generated measurable quantities of these compounds for future use. While the in vitro kinetics of resveratrol did not allow for any in vivo predictions, we were able to show alterations in resveratrol metabolism with respect to genotypic differences and enzyme induction that may contribute to the observed low bioavailability profile. / Pharmaceutical Sciences

This thesis shows advancements towards the development of engineered bacteria for sensing and responding to environmental pollutants by exploring the use of UDP-glycosyltransferases (UGTs) for their metabolism of toxins, along with the use of engineered tetracycline repressor protein (TetR) based transcriptional regulators as sensors for environmental toxins. The importance and applicability of UGTs as well as the adaptability of TetR systems for future developments are shown through a function-based review of UGTs, the development of high-throughput fluorescent UGT assay technique, and the creation of novel TetR transcription regulatory sequences. The assays effectively measured UGT71G1 activity based on the presence of reaction byproducts, leading to the identification of several new substrates including the toxin bisphenol A. Next, hybrid TetRs were assembled from complementary DNA-binding and ligand-binding domains of TetR homologs. The ability to interchange these domains while retaining their function multiplies the unique TetR systems available for use in cellular systems. In future, novel TetR and UGT71G1 systems may be developed to detect and respond to substrates like bisphenol A.

This dissertation aimed at developing an inhibitor strategy to improve the oral bioavailability (Foral) and systemic exposure (AUC∞) of buprenorphine (BUP) as well as reduce the variability associated with them. Twenty-seven generally recognized as safe (GRAS) compounds or dietary substances were evaluated for their potential to inhibit the oxidative and conjugative metabolism of BUP, using pooled human intestinal and liver microsomes. In both the organs, oxidation appeared to be the major metabolic pathway with a 6 fold (intestine) and 4 fold (liver) higher intrinsic clearance than glucuronidation. Buprenorphine was predicted to show low and variable Foral, AUC∞, and a large total clearance. The biorelevant solubilities of 5 preferred inhibitors were incorporated in the final model. An inhibitor dosing strategy was identified to increase Foral and reduce the variability in oral BUP AUC∞. These results demonstrate the feasibility of the approach of using GRAS or dietary compounds to inhibit the presystemic metabolism of buprenorphine and thus improve its oral bioavailability. This inhibitor strategy has promising applicability to a variety of drugs suffering from low and variable oral bioavailability due to extensive presystemic oxidative and conjugative metabolism.

Made available in DSpace on 2016-01-26T12:51:20Z (GMT). No. of bitstreams: 1 gustavonavarrobetonico_tese.pdf: 2515372 bytes, checksum: c873c3382e155afb310d06df86d4d2a2 (MD5) Previous issue date: 2008-02-15 / Pharmacogenetic studies have been performed in an attempt to demonstrate possible influences of the genetic pattern on the variable responses to the immunosuppressive medications. This finding could be another tool to tailoring individual immunosuppression. Objective: This work aimed to analyze the side effects presented by kidney transplant patients on MMF-based immunosuppression and verify their relation with the genetic polymorphism of uridine glucuronosyltransferases (UGT) enzymes, major responsible for bioavailability of mycophenolic acid (MPA), the active metabolite of MMF. Methods: In this study, we retrospectively analyzed 74 kidney transplant patients who had used MMF as part of their immunosuppression regimen. Genotyping of polymorphisms in UGT1A8 (-999C>T, codon 255A>G, codon 277G>A), UGT1A9 (-2152C>T, -275T>A, -118T9>10, codon 33T>C) and UGT2B7 (-79G>A, codon 268C>T) was performed using an automated sequencer and the chromatograms were analyzed on program StadenGap and PreGap4. The genotypes were then compared to the occurrence of eventual side effects, mainly diarrhea, blood disorders and infections. Statistical analyses used Pearson´s chi-square test. Results: Seventy-four kidney transplant patients with 56 ± 41 months post-transplant were enrolled, with mean age of 42 ± 12 years. The glomerular filtration rate was 46 ± 19 ml/min/1,73m2 and the other immunosuppressors were prednisone (98,6%), cyclosporine (39,2%), tacrolimus (35,1%) and sirolimus (28,4%). All polymorphism could be identified on the population, except the UGT2B7-79G/A. Data analysis showed that infection episodes were more frequently observed in individuals who carried the variant UGT1A8 codon 277A (p=0.03) and received 2g/day of MMF. Within individuals receiving this dosage of the medication, infection could be related to the presence of haplotype UGT1A8H5 (-999C/códon 255A/códon 277A) (p=0.02) or diplotype UGT1A8H2/H5 (-999CC/códon 255AA/códon 277GA) (p=0.02). Hematological disturbances (p<0.01) and MMF dose reduction (p<0.01) were more frequent in individuals carrying the haplotype UGT1A9H4 (-2152T/-275A/-118T9/codon 33T) and receiving 2g/day of MMF. Conclusions: The clinical and molecular data of this study suggest that UGT1A9 e UGT1A8 polymorphisms seem to be an additional factor influencing the occurrence of side effects, mainly infection and hematologic disturbances, in patients receiving 2g/day of MMF as drug transplant therapy. / Estudos na área de farmacogenética têm sido realizados na tentativa de se demonstrar possível influência do padrão genético na variabilidade da resposta aos imunossupressores, criando assim uma nova ferramenta de ajuste na dosagem destes medicamentos. Objetivo: Avaliar possível associação dos efeitos adversos, principalmente hematológicos, gastrintestinais, infecciosos e imunológicos apresentados por indivíduos transplantados renais que receberam terapia com micofenolato mofetil (MMF), com os polimorfismos nos genes que codificam enzimas da família das uridino-glucuronosiltransferases (UGTs), responsáveis pela biodisponibilidade do ácido micofenólico, metabólito ativo do MMF. Métodos: Foram estudados transplantados renais adultos que receberam, por no mínimo 30 dias, doses de 1 a 2g/dia de MMF como parte do esquema imunossupressor. A genotipagem dos polimorfismos de UGT1A8 (-999C/T, códon 255A/G, códon 277G/A), UGT1A9 (-2152C/T, -275T/A, -118T9/10, códon 33T/C) e UGT2B7 (-79G/A, códon 268C/T) foi realizada por meio de seqüenciamento automático e os cromatogramas gerados foram analisados no programa Staden Gap and PreGap4. Os genótipos obtidos foram comparados com os efeitos adversos eventualmente apresentados e com a necessidade de suspensão ou redução da dose do MMF. A análise estatística foi realizada utilizando-se o teste de qui-quadrado de Pearson. Resultados: Foram genotipados 74 indivíduos com 56 ± 41 meses pós-transplante em acompanhamento ambulatorial. Destes, 68,9% eram do sexo masculino, com média de idade de 42 ± 12 anos. A taxa de filtração glomerular média foi de 46 ± 19 ml/min/1,73m2 e os diversos esquemas imunossupressores associados ao MMF se basearam em prednisona (98,6%), ciclosporina (39,2%), tacrolimus (35,1%) e sirolimus (28,4%). Foram encontrados todos os polimorfismos citados, em homo ou heterozigose, com exceção do UGT2B7-79G/A que só foi encontrado na forma selvagem. Os episódios de infecção foram mais freqüentes em indivíduos que receberam 2g/dia de MMF e eram portadores da variante UGT1A8 codon 277A (p=0.03), bem como nos portadores do haplótipo UGT1A8H5 (-999C/códon 255A/códon 277A) (p=0.02) e do diplótipo UGT1A8H2/H5 (-999CC/códon 255AA/códon 277GA) (p=0.02). Também naqueles que receberam 2 g/dia de MMF, a presença do haplótipo UGT1A9H4 (-2152T/-275A/-118T9/códon 33T) associou-se com o desenvolvimento de distúrbios hematológicos, principalmente leucopenia (p<0.01) e necessidade de interrupção da medicação (p<0.01). Conclusão: A presença de distúrbios hematológicos e infecções em indivíduos transplantados renais que receberam 2g/dia de MMF está associada com variantes dos genes UGT1A9 e UGT1A8. Este estudo sugere que estes polimorfismos influenciam a ocorrência de alguns efeitos colaterais, principalmente infecção e leucopenia em indivíduos recebendo 2 g/dia de MMF como parte da terapia imunossupressora.

Protein engineering has been a useful tool in the fight against human diseases. Human insulin was the first recombinant DNA-derived therapeutic protein (Humulin®) approved by the US FDA in 1982. However, many of the early protein drugs were only recombinant versions of natural proteins with no modification of their primary amino acid sequence and most of them did not make optimal drug products mainly due to their short half-life or suboptimal affinity, leading to poor therapeutic efficacy. The difficulty in the large-scale production of some therapeutic proteins was another important issue. In the past three decades, different protein engineering platforms have been developed to overcome the obstacles seen in the first generations of these treatments. With the help of these new techniques, proteins have been purposefully modified to improve their clinical potential. The focus of my dissertation is the engineering of potential protein drugs to make them therapeutically useful and more valuable. Previously, our research group has developed cocaine hydrolases (CocHs) from human butyrylcholinesterase (BChE) for treatment of cocaine addiction and prevention of acute cocaine intoxication. In the first project, CocHs were further engineered to improve their performance, e.g., Fc-fused CocHs with an extended serum half-life. Then, I investigated the potential application of a long-lasting CocH for protection against the acute toxic and stimulant effects of cocaine. In the second project, I investigated the potential inhibition of CocH-mediated cocaine hydrolysis by heroin (3,6-diacetylmorphine) or its initial host metabolite, 6-monoacetylmorphine (6-MAM). The investigation of this possible inhibition was important to determine the in vivo efficacy of CocHs, as heroin is one of the most commonly co-abused drugs by cocaine-dependent individuals, as well as a possible metabolite of CocHs. In the third project, I expressed and characterized the recombinant human UDP-glucuronosyltransferase 1A10 (UGT1A10) enzyme, which can inactivate many therapeutically valuable substances. In the fourth and final project, prostate apoptosis response-4 (Par-4), a tumor suppressor protein, was engineered to have a prolonged duration of action so that it may be more therapeutically valuable for cancer treatment.

The bioavailability of a drug can be described as the fraction of an orally administered dose that reaches the systemic circulation and is often limited by first-pass metabolism in the gut and the liver. It is important to have knowledge about these processes since the systemic blood drug concentration is tightly connected to the effect of the drug. The general aim of this project was to quantitatively examine the role of the intestine in relation to the liver in first-pass metabolism of orally administered drugs. The first-pass metabolism of verapamil and raloxifene was investigated in detail with in vivo, in vitro and simulation studies, using the pig as an experimental model. The intestine contributed to the same extent as the liver to first-pass metabolism of R/S-verapamil in vivo in pigs. The S-isomer of verapamil was found in lower plasma concentrations compared to the R-isomer after oral dosing. The in vitro metabolism of verapamil in pig and human liver showed interspecies similarity and indicated equal intrinsic clearance for R- and S-verapamil. Through physiologically based pharmacokinetic modeling the stereoselectivity was explained by a combination of several processes, including enantioselective plasma protein binding, blood-to-plasma partition, and gut and liver tissue distribution. For raloxifene the intestine was the dominating organ in first-pass glucuronidation in vivo in pigs. Furthermore, the raloxifene concentration entering the intestine or the dose administered in the gut did not influence the plasma PK of raloxifene and indicated that the intestinal metabolism was not saturable with clinical relevant doses. For both verapamil and raloxifene, a time-dependent hepatic metabolism was noted with major consequences to the pharmacokinetic of the drugs. This project has pointed out the importance of intestinal metabolism in the overall first-pass extraction of drugs and indicates that intestinal metabolism should be considered and evaluated early in drug development.

CUNHA, Aline Gonzaga. Atividade de enzimas da via de síntese dos fenólicos durante o desenvolvimento de pedúnculos de clones de cajueiro (Anacardium occidentale L.). 2015. 60 f. Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-08-24T14:29:59Z No. of bitstreams: 1 2015_dis_agcunha.pdf: 1915595 bytes, checksum: ddc88d25dd660aa93b503d82263a5624 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-08-26T18:37:01Z (GMT) No. of bitstreams: 1 2015_dis_agcunha.pdf: 1915595 bytes, checksum: ddc88d25dd660aa93b503d82263a5624 (MD5) / Made available in DSpace on 2016-08-26T18:37:01Z (GMT). No. of bitstreams: 1 2015_dis_agcunha.pdf: 1915595 bytes, checksum: ddc88d25dd660aa93b503d82263a5624 (MD5) Previous issue date: 2015 / The dwarf cashew apple (Anacardium occidentale L.) is a tropical plant, originated in Brazil, which is fruit species with high potential for fresh consumption and for industrial processing. With the discovery of bioactive compounds in the cashew apple are necessary studies of the activity of enzymes of the synthesis of phenolic compounds, such as phenylalanine ammonia lyase (PAL), UDP-glucose: trans-cinnamate β-D-glycosyltransferase (UGT), flavonol synthase (FLS) and leucoanthocyanidin reductase (LAR). Soon, this study aimed to determine the activity of these enzymes in CCP 76 and BRS 189 dwarf cashew clones in three different stages of maturation, and to establish the phenolic profile in these stages. Reaction products were identified and quantified by LC-DAD. PAL activity decreases with maturation for dwarf cashew clones. UGT enzyme in the CCP 76 clone stage 4 showed the highest enzyme specific activity than in other stages of development for that clone. Whereas BRS 189 clone, the highest activity was in the mature cashew apple. Quercetin synthesis was evidenced in stage 4 for the two clones analyzed. The enzymes involved in the metabolism phenolic behave differently, showing the complexity of this biosynthetic pathway that varies between clones and during the development of cashew apple. Further studies, with mass spectrometer linked HPLC, will elucidate the profile of the compounds present in cashews apple lyophilized for chemical characterization of these in different stages of maturation. Therefore, the understanding of the enzymatic metabolism between the clones and during cashew apple development represent a real potential for the development of new products with functional properties and promotion of human health. / O cajueiro (Anacardium occidentale L.) é uma planta tropical, originária do Brasil, que se destaca como espécie frutífera com elevada potencialidade para o consumo in natura e para o processamento industrial. Com a descoberta de compostos bioativos presentes nos pedúnculos, se fazem necessários estudos da atividade de enzimas da síntese dos compostos fenólicos, tais como a fenilalanina amônia liase (PAL), UDP-glicose: trans-cinamato β-D- glicosiltransferase (UGT), flavonol sintase (FLS) e leucoantocianidina redutase (LAR). Logo, esse estudo objetivou determinar a atividade dessas enzimas, em pseudofrutos de cajueiro-anão-precoce dos clones CCP 76 e BRS 189 em três diferentes estádios de maturação, além de estabelecer o perfil de fenólicos nestes referidos estádios. Os produtos resultantes das reações enzimáticas foram identificados e quantificados por LC-DAD. A atividade da PAL decresce com o amadurecimento dos pseudofrutos. A enzima UGT no estádio 4 do clone CCP 76 apresentou a maior atividade enzimática específica que nos outros estádios de desenvolvimento para o referido clone. Enquanto no clone BRS 189, a maior atividade deteve-se no caju maduro. A maior síntese de quercetina no caju foi evidenciada no estádio 4 para os dois clones estudados. As enzimas envolvidas no metabolismo de fenólicos se comportam diferentemente, demonstrando a complexidade dessa rota biossintética que varia entre os clones e durante o desenvolvimento dos pedúnculos de cajueiro. Estudos posteriores, com CLAE acoplada a espectrômetro de massa, irão elucidar melhor o perfil dos compostos presentes em cajus liofilizados para caraterização química destes em diferentes estádios de maturação. Portanto, o entendimento do metabolismo enzimático entre os clones e durante o desenvolvimento do pedúnculo representam um potencial real para o desenvolvimento de novos produtos com propriedades funcionais e promoção da saúde humana.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / The dwarf cashew apple (Anacardium occidentale L.) is a tropical plant, originated in Brazil, which is fruit species with high potential for fresh consumption and for industrial processing. With the discovery of bioactive compounds in the cashew apple are necessary studies of the activity of enzymes of the synthesis of phenolic compounds, such as phenylalanine ammonia lyase (PAL), UDP-glucose: trans-cinnamate &#946;-D-glycosyltransferase (UGT), flavonol synthase (FLS) and leucoanthocyanidin reductase (LAR). Soon, this study aimed to determine the activity of these enzymes in CCP 76 and BRS 189 dwarf cashew clones in three different stages of maturation, and to establish the phenolic profile in these stages. Reaction products were identified and quantified by LC-DAD. PAL activity decreases with maturation for dwarf cashew clones. UGT enzyme in the CCP 76 clone stage 4 showed the highest enzyme specific activity than in other stages of development for that clone. Whereas BRS 189 clone, the highest activity was in the mature cashew apple. Quercetin synthesis was evidenced in stage 4 for the two clones analyzed. The enzymes involved in the metabolism phenolic behave differently, showing the complexity of this biosynthetic pathway that varies between clones and during the development of cashew apple. Further studies, with mass spectrometer linked HPLC, will elucidate the profile of the compounds present in cashews apple lyophilized for chemical characterization of these in different stages of maturation. Therefore, the understanding of the enzymatic metabolism between the clones and during cashew apple development represent a real potential for the development of new products with functional properties and promotion of human health. / O cajueiro (Anacardium occidentale L.) Ã uma planta tropical, originÃria do Brasil, que se destaca como espÃcie frutÃfera com elevada potencialidade para o consumo in natura e para o processamento industrial. Com a descoberta de compostos bioativos presentes nos pedÃnculos, se fazem necessÃrios estudos da atividade de enzimas da sÃntese dos compostos fenÃlicos, tais como a fenilalanina amÃnia liase (PAL), UDP-glicose: trans-cinamato &#946;-D- glicosiltransferase (UGT), flavonol sintase (FLS) e leucoantocianidina redutase (LAR). Logo, esse estudo objetivou determinar a atividade dessas enzimas, em pseudofrutos de cajueiro-anÃo-precoce dos clones CCP 76 e BRS 189 em trÃs diferentes estÃdios de maturaÃÃo, alÃm de estabelecer o perfil de fenÃlicos nestes referidos estÃdios. Os produtos resultantes das reaÃÃes enzimÃticas foram identificados e quantificados por LC-DAD. A atividade da PAL decresce com o amadurecimento dos pseudofrutos. A enzima UGT no estÃdio 4 do clone CCP 76 apresentou a maior atividade enzimÃtica especÃfica que nos outros estÃdios de desenvolvimento para o referido clone. Enquanto no clone BRS 189, a maior atividade deteve-se no caju maduro. A maior sÃntese de quercetina no caju foi evidenciada no estÃdio 4 para os dois clones estudados. As enzimas envolvidas no metabolismo de fenÃlicos se comportam diferentemente, demonstrando a complexidade dessa rota biossintÃtica que varia entre os clones e durante o desenvolvimento dos pedÃnculos de cajueiro. Estudos posteriores, com CLAE acoplada a espectrÃmetro de massa, irÃo elucidar melhor o perfil dos compostos presentes em cajus liofilizados para caraterizaÃÃo quÃmica destes em diferentes estÃdios de maturaÃÃo. Portanto, o entendimento do metabolismo enzimÃtico entre os clones e durante o desenvolvimento do pedÃnculo representam um potencial real para o desenvolvimento de novos produtos com propriedades funcionais e promoÃÃo da saÃde humana.