Biotransformation of B-lapachone using microbial cultures: an alternative to in vitro metabolism studies

Author(s)

Camila Raquel Paludo

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2013-03-05

Examining board members

Niege Araçari Jacometti Cardoso Furtado; Roberto Gomes de Souza Berlinck; Gisele Baraldi Messiano

• Advisor: Niege Araçari Jacometti Cardoso Furtado

Abstract

B-lapachone is considered an important ortho-naphthoquinone by their pharmacological activities, mainly antitumor, but there is no description of microbial biotransformation studies of ?-lapachone. These researches may furnish new derivatives and significant information on its metabolism. Many studies describe that microorganisms can catalyze reactions mimicking human enzymes. ?-lapachone was obtained by semisynthetic procedure from lapachol. Biotransformation processes were carried out using the filamentous fungi Mucor rouxii, Cunninghamella elegans, Cunninghamella echinulata, Penicillium crustosum and Papulaspora immersa and the gastrointestinal bacteria Escherichia coli grown aerobically and anaerobically, Lactobacillus acidophilus, Bifidobacterium sp. and mixed culture with Lactobacillus acidophilus, Bifidobacterium sp. and Streptococcus salivarius subsp. thermophilus. In order to establish a comparison between ?-lapachone microbial transformation and those of its isomer ?-lapachone, biotransformation studies of ?-lapachone were also carried out using M. rouxii. Seven derivatives of ?-lapachone were produced in the process performed by M. rouxii, including one unpublished, five already described in a study of metabolism by mammalian and human blood and one spirobenzolactone reported in a syntetic study. Other two unpublished derivatives of ?-lapachone, which are regioisomers conjugated with glucose, were produced after formation of hydroquinone in the process carried out by C. elegans. P. immersa provided two isomeric lactones also obtained by biotransformation with M. rouxii. Possible biotransformation products were detected by using HPLC-DAD in the processes carried out by the bacteria E. coli under aerobic condition and Bifidobacterium sp. However, these processes exhibited a low yield, and it was possible to identify only one derivative produced by E. coli, which was also obtained in the process performed by M. rouxii. A glycosylated derivative of ?-lapachone was produced by biotransformation with M. rouxii after 24 hours of incubation and subsequently was converted in hydroxylapachol, which in turn gave rise to ?-lapachone again and also to ?-lapachone, which was also metabolized. The major derivative produced in the process carried out by C. elegans was submitted to cytotoxic activity evaluation using human breast cancer cell line SKBR3 showing IC50 312.5 ?M, being the ?-lapachone IC50 5.6 ?M against the same cell line. The major derivative did not show cytotoxicity to normal human fibroblast GM07492-A cell line, while ?-lapachone was highly cytotoxic (IC50 7.25 ?M). The same major derivative was also produced in smaller yield in the process performed by C. echinulata. In the ?-lapachone microbial transformation studies occurred phase I and phase II reactions, mimicking the metabolism of mammals, including humans, as reported in literature. (AU)

• FAPESP's process: 11/01303-1 - Biotransformation of ²-lapachone using microbial cultures: an alternative to in vitro metabolism studies
• Grantee: Camila Raquel Paludo
• Support Opportunities: Scholarships in Brazil - Master