Publication: Nikotin ara metabolitlerinin ve nikotin analoglarının sentez, analiz ve metabolizmaları
Abstract
Nikotin, tütün ürünlerinde en yüksek miktarda bulunan ve farmakolojik olarak en güçlü bir alkaloiddir. Nikotin metabolizması çok geniş kapsamda çalışılmış olmasına rağmen nikotin metabolitleri ve ara ürünlerinin in vitro metabolizması hakkında çok az bilgi bulunmaktadır. Bu çalışmada ilk amaç nornikotin, kotinin ve norkotininin yani minör tütün alkaloidleri ya da nikotin metabolitleri olarak isimlendirilen bileşiklerin metabolik akibetlerini araştırmaktır. İlgili substratlar ve olası metabolitlerinin ayrımı, teşhisi ve miktar tayini için hassas ve spesifik yöntemler geliştirildi. Hazır ve sentezlenmiş bileşikler UV- visible spektroskopi (UV), infrared spektroskopi (IR), mass spektrometresi (MS), yüksek basınçlı sıvı kromatografisi (HPLC) ve yüksek basınçlı sıvı kromatografisine bağlanan UV spektroskopisi (HPLC-UV) gibi çeşitli yöntemlerle ayrıldı ve yapıları aydınlatıldı. Enzim kaynağı olarak sıçandan elde edilen beyin homojenatı ve karaciğer mikrozomları kullanıldı ve bulgular NADPH ile kuvvetlendirilmiş kontrol ve fenobarbitalle indüklenmiş olmak üzere iki ayrı grup olarak sunuldu. Substrat ve metabolitlerinin biyolojik ortamdan ayrımı sıvı-sıvı ekstraksiyonu ile yapıldı. Bileşiklerin miktar tayini HPLC ile gerçekleştirildi. Enzim kaynağı olarak sıçan beyin homojenatı ile nornikotin, kotinin ve norkotinin inkübe edildiğinde kendilerine karşılık gelen metabolitlerinin oluşmadığı görüldü. Bu inkübatlarda yalnızca nikotinamid tayin edildi. Sıçan karaciğer mikrozomları ile nornikotinin metabolizmasında karşılık gelen laktam metaboliti norkotinin ve nikotinamid tespit edildi. Norkotinin metabolitinin miktarı fenobarbitalle indüklenmiş sıçan karaciğeri mikrozomları ile 127 nmol/ mL, kontrol karaciğeri mikrozomları ile 80 nmol/ mL olarak tayin edildi. Kotininin metabolizması ile 5'-hidroksikotinin metaboliti ve nikotinamid tespit edildi. Metabolik deneylerde 3'-hidroksikotinin ve norkotinin metabolitleri gözlenmedi. Fenobarbitalle indüklenmiş ve indüklenmemiş sıçan karaciğeri mikrozomları ile 5'-hidroksikotinin miktarı sırasıyla 28 ve 16 nmol/ mL bulunmuştur. Bu durum metabolitin fenobarbitalle indüklenebilen sitokrom P450 izoenzimleri aracılığıyla oluşturulduğunu göstermektedir. Norkotinin inkübatlarında karşılık gelen ketoamid metaboliti olan 4-(3-piridil)-4-oksobutiramid tayin edilemedi. Mikrozomlarla birlikte kofaktör karışımı kullanıldığında mikrozomal inkübatlarda nikotinamidin oluşumu gözlendi. Bununla birlikte kofaktör karışımı yerine tampon çözelti içeren kontrol deneylerinde nikotinamid oluşumu görülmedi. N-1'-benzilnornikotin (I) ve N-1'-(p-klorobenzil) nornikotin (II)'in in vitro mikrozomal metabolizması substratların kendilerine karşılık gelen aldehit, amid ve laktam metabolitlerini oluşturup oluşturmayacağını tayin etmek için araştırıldı. Tersiyer aminlere ek olarak N-1'-benzoilnornikotin (Ia) ve N-1'-(p-klorobenzoil) nornikotin (IIa) olarak isimlendirilen amidlerin in vitro mikrozomal metabolizması çalışıldı. Tersiyer aminlerin karşılık gelen dealkilasyon ürünleri olan aldehit ve nornikotini oluşturduğu saptandı. N-1'-benzilnornikotin (I) substratı ile amid ve laktam metaboliti oluşumu gözlenmezken diğer tersiyer aminin (II) karşılık gelen amid ve laktama dönüştüğü görüldü. Amid substratlarının sıçan karaciğeri mikrozomları ile nornikotine hidroliz olduğu tespit edildi. Sonuçlar, metabolik yolların mekanizmaları ve metabolitlerin olası toksik etkileri kapsamında tartışıldı.
SYNTHESIS, ANALYSIS and METABOLISM OF NICOTINE METABOLİTES and NICOTINE ANALOGUES Nicotine is the most abundant and pharmacologically potent alkaloid present in tobacco products. Although nicotine metabolism has been studied in detail, less information on the in vitro metabolism of nicotine metabolites and its intermediates is available. In this study, the first aim is to investigate the metabolic fates of nornicotine, cotinine and norcotinine, minor tobacco alkaloids or nicotine metabolites. The sensitive and specific methods for the separation, identification and quantitation of the related substrates and their potential metabolites were developed. These compounds were separated and characterised by several methods, including UV-visible spectroscopy (UV), infrared spectroscopy (IR), mass spectrometry (MS), high performance liquid chromatography (HPLC) and high performance liquid chromatography coupled to UV-spectroscopy (HPLC-UV). Brain homogenate and hepatic microsomes obtained from rats were used as enzyme sources and the findings have been reported as two different groups by using control and induced with phenobarbitone fortified with NADPH. Separation of substrates and their metabolites from biological media was achieved by liquid-liquid extraction. Quantitation of these compounds was carried out by HPLC. Nornicotine, cotinine and norcotinine failed to produce the corresponding metabolites when they were incubated with rat brain homogenate as an enzyme source. Only nicotinamide was detected in these incubates. Following nornicotine metabolism, two compounds i.e. the corresponding lactam metabolite of norcotinine and nicotinamide were detected with rat hepatic microsomes. The amount of norcotinine metabolite was detected as 127 nmol/ mL using phenobarbitone induced rat microsomes and 80 nmol/ mL with controls. Following the metabolism of cotinine, 5'-hydroxcotinine metabolite and nicotinamide were detected. No 3'-hydroxycotinine and norcotinine metabolites were observed in the metabolic experiments. The amount of 5'hydroxycotinine was 28 and 16 nmol/ mL by phenobarbitone induced and noninduced rat microsomes, respectively. It indicates that the metabolite is formed via a phenobarbitone inducible isozyme of CYP450. In the norcotinine incubates, the corresponding ketoamide metabolite, 4-(3-pyridyl)-4-oxobutyramide was not detected. The formation of nicotinamide was observed in microsomal incubates when the cofactor mixture was used together with microsomes. However, there was no nicotinamide formation in the control experiments containing buffer solution instead of cofactor mixture. The in vitro microsomal metabolism of N-1'-benzylnornicotine (I) and N-1'-(p-chlorobenzyl) nornicotine (II) was carried out to establish if the substrates produce the corresponding aldehyde, amide and lactam as metabolites. In addition to tertiary amines, the in vitro microsomal metabolism of amides which was named as N-1'-benzoylnornicotine (Ia) and N-1'-(p-chlorobenzoyl) nornicotine (IIa) was studied. The tertiary amines produced corresponding dealkylation products ie. the corresponding aldehyde and nornicotine. No amide and lactam metabolite were observed with N-1'-benzylnornicotine (I) as a substrate. However, the other tertiary amine (II) produced the corresponding amide and lactam. The amide substrate was hydrolysed by rat hepatic microsomes to nornicotine. The results were discussed in relation to the possible mechanism of metabolic pathways and the possible toxic effects of metabolites.
SYNTHESIS, ANALYSIS and METABOLISM OF NICOTINE METABOLİTES and NICOTINE ANALOGUES Nicotine is the most abundant and pharmacologically potent alkaloid present in tobacco products. Although nicotine metabolism has been studied in detail, less information on the in vitro metabolism of nicotine metabolites and its intermediates is available. In this study, the first aim is to investigate the metabolic fates of nornicotine, cotinine and norcotinine, minor tobacco alkaloids or nicotine metabolites. The sensitive and specific methods for the separation, identification and quantitation of the related substrates and their potential metabolites were developed. These compounds were separated and characterised by several methods, including UV-visible spectroscopy (UV), infrared spectroscopy (IR), mass spectrometry (MS), high performance liquid chromatography (HPLC) and high performance liquid chromatography coupled to UV-spectroscopy (HPLC-UV). Brain homogenate and hepatic microsomes obtained from rats were used as enzyme sources and the findings have been reported as two different groups by using control and induced with phenobarbitone fortified with NADPH. Separation of substrates and their metabolites from biological media was achieved by liquid-liquid extraction. Quantitation of these compounds was carried out by HPLC. Nornicotine, cotinine and norcotinine failed to produce the corresponding metabolites when they were incubated with rat brain homogenate as an enzyme source. Only nicotinamide was detected in these incubates. Following nornicotine metabolism, two compounds i.e. the corresponding lactam metabolite of norcotinine and nicotinamide were detected with rat hepatic microsomes. The amount of norcotinine metabolite was detected as 127 nmol/ mL using phenobarbitone induced rat microsomes and 80 nmol/ mL with controls. Following the metabolism of cotinine, 5'-hydroxcotinine metabolite and nicotinamide were detected. No 3'-hydroxycotinine and norcotinine metabolites were observed in the metabolic experiments. The amount of 5'hydroxycotinine was 28 and 16 nmol/ mL by phenobarbitone induced and noninduced rat microsomes, respectively. It indicates that the metabolite is formed via a phenobarbitone inducible isozyme of CYP450. In the norcotinine incubates, the corresponding ketoamide metabolite, 4-(3-pyridyl)-4-oxobutyramide was not detected. The formation of nicotinamide was observed in microsomal incubates when the cofactor mixture was used together with microsomes. However, there was no nicotinamide formation in the control experiments containing buffer solution instead of cofactor mixture. The in vitro microsomal metabolism of N-1'-benzylnornicotine (I) and N-1'-(p-chlorobenzyl) nornicotine (II) was carried out to establish if the substrates produce the corresponding aldehyde, amide and lactam as metabolites. In addition to tertiary amines, the in vitro microsomal metabolism of amides which was named as N-1'-benzoylnornicotine (Ia) and N-1'-(p-chlorobenzoyl) nornicotine (IIa) was studied. The tertiary amines produced corresponding dealkylation products ie. the corresponding aldehyde and nornicotine. No amide and lactam metabolite were observed with N-1'-benzylnornicotine (I) as a substrate. However, the other tertiary amine (II) produced the corresponding amide and lactam. The amide substrate was hydrolysed by rat hepatic microsomes to nornicotine. The results were discussed in relation to the possible mechanism of metabolic pathways and the possible toxic effects of metabolites.