INTRODUCTION:

Metabolism is a process of biotransformation by enzymatic reactions to raise hydrophilicity, decrease activity and toxicity for most of the drugs. Metabolite identification is an important aspect of drug discovery and development. Metabolite identification at the preclinical stage helps in optimizing the structure of the compound^1,2. Food and Drug Administration³ and European Medicines Agency [ICH Topic M3 (R2)]⁴ guidelines encourage the identification of any difference in drug metabolism between animals and humans during in vitro and in vivo studies.

Cancer is one of the leading causes of mortality all over the globe. Therefore, researchers are trying to find innovative solutions against cancer. Actinomycetes are also attracting more attention owing to their ability to produce variety of natural bioactive metabolites^5,6. Erlotinib [6,7-Bis-(2-methoxy-ethoxy)-quinazolin-4-yl]-(3-ethynyl-phenyl)-amine, (trade name Tarceva), is an orally active epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that is primarily used in the treatment of non-small cell lung cancer (NSCLC), pancreatic cancer and several other types of cancer. Mechanism of action is to reversibly bind the ATP pocket of the intracellular tyrosine kinase and prevent phosphorylation, cellular differentiation. Nowadays, therapeutic drug monitoring has been suggested for the usage of erlotinib⁷. Erlotinib is extensively metabolized mainly by the CYP 3A4 and, to a lesser extent by CYP1A1 and CYP1A2; only around 2% is eliminated in an unchanged form^7,8. Several metabolites of erlotinib were identified in rats and dogs⁹. Metabolism and excretion of erlotinib were very well characterized in humans after a 100mg oral dose of ¹⁴C-erlotinib⁸. Several validated methods including UV^10,11_,PDA¹², tandem mass spectrometry^13,14, capillary electrophoresis¹⁵ have been published for quantification of erlotinib, its metabolites including OSI-420 (active metabolite which accounts for ~10% concentration of erlotinib) and other metabolites. Studying the in vitro metabolic profile using liver microsomes and hepatocytes is a standard approach followed by many scientists^16,17,18. Liquid/Gas chromatographies coupled with mass spectrometry were commonly used for the identification of metabolites/drug and considered as rapid and unambiguous tool^16–22. LC-MS/MS is valuable tool that provide information about the molecular weight, structure, identity and quantity of compounds²³. Due to high specificity, precision and accuracy LC-MS/MS is the first choice for identification/quantification of drug/metabolite from biological matrices^24–30and pharmaceutical formulation³¹.

To our knowledge, till date differences and similarities in the metabolism of erlotinib across six different species have not been studied. The primary objective of the present study was to study the metabolic pattern in six different species under in vitro conditions with differences and similarities and to identify the new phase I metabolites for erlotinib.

MATERIALS AND METHODS:

Chemicals and reagents:

Erlotinib (purity 99%) was received as a gift sample from Aarti Drugs Ltd. (Palghar, Maharashtra, India). Liver microsomes of rat, mice, dog, human, hamster, and S9-fraction of mice were procured from XenoTech, LLC (Kansas City, USA). Nicotinamide adenine dinucleotide phosphate hydrogen (NADPH), Potassium phosphate monobasic and Potassium phosphate dibasic were procured from Sigma-Aldrich (St Louis, MO, USA). Acetonitrile, Dimethyl sulfoxide (DMSO), Methanol and Formic acid were obtained from Merck (Mumbai, India). Ammonium formate obtained from the Subodh trading company (Pune, India).

Liquid chromatographic and mass spectrometric conditions:

The HPLC system (Shimadzu) interfaced with API 4000 MS/MS (AB Sciex, Foster City, CA, USA) was used for chromatographic and mass spectral analysis in unit resolution. The HPLC system (Shimadzu Corporation, Kyoto, Japan) consisted of a LC-20AD binary pump, a DGU20A degasser, SIL-HTC an autosampler, and a CTO-20A column oven maintained at 40° during analysis. Analytical separation of erlotinib and its metabolite were achieved on Eclipse XDB-C18 (4.6*150, 5µM) column from Agilent using 10 mM ammonium formate with 0.1% Formic acid (mobile phase A) and acetonitrile (mobile phase B) with a flow rate of 0.5ml/min by gradient elution (initially, %B was 10% from 0-1.1 min, then a gradual increase of %B up to 95% from 1.1-14 min; kept constant 95% B from 14-15.9 min then switched to 10% B at 16 min and again kept constant till end time of 18 min). Source parameters namely, Curtain gas, 23 psi; Collision gas, 6 psi; GAS 1, 50 psi; GAS 2, 70 psi; Temperature, 500°; Ion Spray voltage, 5500 V and Collision energies at 40 ± 5 V and 17±5 V were used during analysis. The Analyst software version 1.6 from AB Sciex was used for data acquisition and interpretation.

In vitro incubation using liver microsomes and S9-fraction:

Erlotinib (10µM) was incubated with six different species namely liver microsome of human, mice, rat, dog, hamster, and S9-fraction of mice (1mg/ml protein) in 0.05 M potassium phosphate buffer (pH 7.4). Final microbial incubations were conducted in a water bath at 37° with a pre-incubation time of 10 min. Incubation reactions were initiated by the addition of 2mM NADPH. Samples were withdrawn at 0 min, 60 min and quenched by ice-cold acetonitrile. Control incubation was performed in the absence of cofactor NADPH to confirm the enzymatic metabolism. Samples were vortexed for ~ 30 s, centrifuged at 5000rpm for 10 min at 10° and supernatant was injected onto LC-MS/MS analysis.

Metabolite identification strategy:

Metabolites were thoroughly searched and confirmed using a highly selective and sensitive LC-MS/MS method. Metabolites were searched with a targeted approach based on available literature (Ling et al., 2006) also with an untargeted approach by the use of a full scan approach in the mass range of 100–1000 m/z. Fragmentation pattern (MS2 spectra) of erlotinib was very well understood to facilitate the structural identification of metabolites. Turbo ion spray (ESI) source was used in both positive and negative polarity with scan types of Q1MS, Product ion (MS2), Precursor ion and Neutral loss (NL). A large number of components in the matrix were ionized and detected in the mass spectrum, therefore metabolite peaks were evaluated concerning the mass spectrum of control samples. Initially, positive and negative Q1MS scans were acquired for all 0 min and 60 min samples in a range of 100–1000m/z. Metabolites were searched using XIC (extracted ion chromatogram of interest) and confirmed further using MS2 product ion spectra. The fragmentation pattern of erlotinib and each metabolite were compared for further confirmation of particular metabolite. Data checked for characteristic neutral losses or characteristic fragment ions in the product ion spectrum. Characteristic fragment ions were used to set up the precursor scans. A characteristic neutral loss was used to set up neutral loss scans. An occurrence of individual metabolite in various species was searched to find the differences and similarities in metabolism. Chem Sketch version 12.01 (Advanced Chemistry Development Inc.) was used for the chemical structure elucidation of predicted metabolites and fragment ions obtained from mass spectrometry.

RESULTS AND DISCUSSION:

Fragmentation of erlotinib standard:

The structural identification of metabolites was based on the MS2 fragmentation pattern of parent erlotinib. Erlotinib showed protonated base ion peak [M+H]⁺ at m/z 394.1 and produced series of characteristic (structure specific) product ions at m/z 336.3, 304.3, 278.2, 250.0 (Fig. 1). The ion at m/z 336 was formed by the loss of a methoxy-ethyl group from the parent and subsequent neutral loss of methanol from the m/z 336 leads to the formation of m/z 304. The prominent ion at m/z 278 formed by the loss of both methoxy-ethyl groups from the parent and subsequent neutral loss of CO from the fragment ion m/z 278 leads to the formation of m/z 250.

Figure 1: Product ion spectra (MS2) of Erlotinib

LC-MS/MS analysis of erlotinib metabolites:

Erlotinib undergoes extensive phase I metabolism. Phase I metabolites including 19 in HLM (Human liver microsomes); 12 in each of MLM (Mice liver microsomes), RLM (Rat liver microsomes) and S9-fraction of mice; 10 in DLM (Dog liver microsomes) and 7 in Hamster LM were detected. Metabolites were further confirmed and characterized using product ion spectra (MS2) in positive ion mode. Erlotinib along with its metabolites were not ionized in negative polarity. Metabolites of erlotinib are polar and thus eluted prior to parent in chromatography. Metabolite structures were elucidated based on comparative analysis of fragment ions between parent and each metabolite, their accurate masses and retention time. Anna et al., 2015⁷ and Ling et al., 2006⁸reported 11- and 9- oxidative metabolites, respectively; whereas, in the present study, a total of 19 oxidative metabolites were detected in human liver microsomes. 1- Novel metabolite (M23) was identified with its putative structure. However, structures of 6 metabolites (out of 19) namely M22 and M25–M29 were difficult to identify due to complex fragmentation. All major metabolites (M14, M11, M16, M6, M2, M13, and M14) previously reported in healthy human volunteers⁸ were found in human liver microsomes. Experimental results showing the biotransformation pathway for each metabolite is shown in Table 1.

Table 1: Biotransformation pathway of metabolites

Metabolite	[M+H]⁺	Biotransformation pathway
M6	428.2	Oxidation of acetylene moiety to carboxylic acid
M7	430.2	Hydroxylation of M23
M12	366.2	O-demethylation, Didesmethyl Erlotinib
M11	394.2	O-demethylation of side chain followed by oxidation to acid
M13	380.2	O-demethylation, Desmethyl Erlotinib
M14	380.2	O-demethylation, Desmethyl Erlotinib
M16	410.2	Hydroxylation of aromatic ring
M17	396.2	Demethylation followed by Hydroxylation
M19	428.2	Addition of -OH and -COCH3 group
M2/M24	414.2	O-demethylation of M6
M21	396.2	Demethylation followed by Hydroxylation
M22	410.2	Unable to derive
M23^Novel	414.2	Oxidation of acetylene moiety to alcohol
M24/M2	414.2	Acetylene moiety oxidize to get -COOH from M13 and M14
M25	396.2	Unable to derive
M26	410.2	Unable to derive
M27	414.2	Unable to derive
M28	430.2	Unable to derive
M29	430.2	Unable to derive

Metabolite M12: M12 eluted at a retention time of 9.69 min, and showed a protonated molecular ion at m/z 366.2, which is 28 Da lower than the parent drug. MS2 spectrum showed a fragment of m/z 322 (loss of side chain -C2H3OH; -44 Da), a prominent fragment of m/z 278 (loss of both side chains -C2H3OH; -88 Da), and observed spectra are by previous reports⁸, based on these facts structure was confirmed as Didesmethyl erlotinib.

Metabolite M13 and M14 (OSI-420): M13 and M14 eluted at a retention time of 10.40 min and 10.56 min, respectively, and showed a protonated molecular ion at m/z 380.2, which is 14 Da lower than the parent drug. MS2 spectrum showed a fragment of m/z 322 (loss of methoxyethyl; -58Da), remaining fragments are like a parent, elution pattern, and observed spectra are by previous reports⁸, based on these facts structure was confirmed as Desmethyl erlotinib.

Metabolite M11: M11 eluted at a retention time of 10 min, and showed a protonated molecular ion at m/z 394.2, with a mass difference of 0 Da than the parent drug. Fragmentation pattern reported by Ling et al., 2006⁸ perfectly matches with observed spectra, therefore, it is confirmed that M11 is a product of O-demethylation of side chain followed by oxidation to acid. XIC and fragmentation pattern of M11 is shown in Fig. 2.

Figure 2: XIC and Product ion spectra (MS2) of metabolite M11

Metabolite M21: M21 eluted at a retention time of 8.81 min, and showed a protonated molecular ion at m/z 396.2, with a mass difference of positive 2 Da than the parent drug. MS2 spectrum showed a fragment of m/z 338 (loss of methoxyethyl, -58 Da), m/z 306 (NL of methanol from 338, -90 Da), a prominent fragment of m/z 294 (loss of both side chains, -102 Da) and m/z 266 (NL of CO from 294, -130 Da), also observed spectra are by previous reports⁷; the structure was confirmed on these observations. M21 is a hydroxylated form of M13.

Metabolite M17: M17 eluted at a retention time of 8.91 min, and showed a protonated molecular ion at m/z 396.2, with a mass difference of positive 2 Da than the parent drug. MS2 spectrum showed a fragment of m/z 352 (loss of side chain -C2H3OH; -44 Da), m/z 338 (loss of methoxyethyl), a prominent fragment of m/z 294 (loss of both side chains, -102 Da) and m/z 266 (NL of CO from 294, -130 Da), also observed spectra are by previous reports⁸, the structure was confirmed on these observations. M17 is a hydroxylated form of M14.

Metabolite M16: M16 eluted at a retention time of 9.85 min, and showed a protonated molecular ion at m/z 410.2, with a mass difference of positive 16 Da than the parent drug. MS2 spectrum showed characteristic fragments of m/z 352, 320, 294 and 266; this is simply an addition of m/z +16 to the parent fragments also observed spectra are by previous reports⁸, the structure was confirmed on these observations. M16 is a hydroxylated form of a parent. XIC and fragmentation pattern of M16 is shown in Fig. 3.

Differences and similarities in the metabolism across various species:

MLM, RLM and S9-fraction of mice produced 12 metabolites versus 19 in HLM, 7 in hamster LM and 10 in DLM (Table 2). Similar metabolic behavior had shown by MLM, RLM and S9-fraction of mice. DLM and Hamster LM produced few metabolites as compare to HLM. The fragmentation spectrum was same in all tested species for the individual metabolite. Metabolite M12 (Didesmethyl erlotinib) was most prominent in RLM considering uniform ionization in mass spectrometry throughout all species. All phase I metabolites previously reported in healthy human volunteers were identified in human liver microsomes. Novel metabolite (M23, oxidation of acetylene moiety to alcohol) was seen only in HLM. M6 (oxidation of acetylene moiety to carboxylic acid), M11 (O-demethylation of side chain followed by oxidation to acid) and M16 (hydroxylation of aromatic ring) are produced from major biotransformation pathways. M6 and M16 were found in all 6 species; however, M11 found only in DLM in addition to HLM. These differences and similarities in the metabolism of erlotinib confirmed the role of CYP 450 (CYP3A4) enzymes and their distinct activity across various species. A summary of metabolite m/z, retention time, their characteristic fragment ions and detection across various species is shown in Table 2.

Table 2: Summary of metabolite identification across various species

Metabolite	RT (min)	[M+H]⁺	(∆m)	HLM	MLM, RLM, S9-fraction mice	DLM	Hamster LM	Product Ions (m/z)
M6	9.55	428.2	34	+	+	+	+	370, 338.4, 312, 266.3
M7	8.70	430.2	36	+	+	-	-	372, 354, 340, 314, 295.7, 266, 282
M12	9.69	366.2	-28	+	+	+	-	322.2, 290.2, 277.9, 249.4
M11	10.00	394.2	0	+	-	+	-	336, 318, 304, 289.5, 276
M13	10.40	380.2	-14	+	+	+	+	336, 322, 304, 278, 250
M14	10.56	380.2	-14	+	+	+	+	336, 322, 304, 278, 250
M16	9.85	410.2	16	+	+	+	+	352, 320, 294, 266
M17	8.91	396.2	2	+	+	-	-	351.4, 338, 294, 266
M19	9.35	428.2	34	+	+	+	-	370, 338.3, 312, 283
M2/M24	8.63	414.2	20	+	-	-	-	370, 355.5, 312.4, 266
M21	8.81	396.2	2	+	-	-	-	338.4, 306, 294, 265.9
M22	11.25	410.2	16	+	+	+	+	334.3, 304, 290.3, 276
M23^Novel	9.53	414.2	20	+	-	-	-	355.7, 324.1, 297.5, 262.1
M24/M2	8.73	414.2	20	+	-	-	-	369.5, 355.8, 312.2, 266.4
M25	10.20	396.2	2	+	+	+	+	378.4, 320, 290, 276.4
M26	10.84	410.2	16	+	+	-	+	352, 334.8, 294, 277
M27	8.45	414.2	20	+	-	-	-	369.4, 312.3, 282, 253.1
M28	9.33	430.2	36	+	-	-	-	372, 340, 314, 284, 255
M29	9.52	430.2	36	+	+	+	-	371.5, 338.4, 313.3, 285, 267.3
Erlotinib	11.77	394.1	NA					336.3, 304.3, 278.2, 250.0

Where, +, Metabolite detected; -, Metabolite not detected/below limit of detection; (∆m), Mass difference compared to parent drug.

CONCLUSION:

Erlotinib is extensively metabolized in HLM showing a total of 19 phase I metabolites including those previously reported in humans and human liver microsomes. A novel metabolite M23 (oxidation of acetylene moiety to alcohol) was identified with its putative structure in HLM. Metabolites were confirmed depending on their retention time, accurate m/z and MS2 fragmentation spectra. However, based on the only mass spectrum it was not possible to deduce the structure of 6 metabolites. Also, the pharmacological significance of these newly formed metabolites along with enzyme involved in their formation is not presently known. MLM, RLM and S9-fraction of mice can be used interchangeably to study the metabolism of erlotinib and its analogs due to similar metabolic behavior. Human liver microsomes are more relevant to study the phase I metabolism. However, animal species can provide guidance on metabolic fate in early drug discovery and helps in selecting an appropriate animal model to study the safety and efficacy of the compound. MS/MS confirms the identity of individual metabolite.

In the current study, it was not possible to deduce the structure of 6 metabolites (M22 and M25 to M29) due to the complex fragmentation pattern. Therefore, those structures need to derive upon co-relation of IR, NMR and Mass data. Additionally, the pharmacological relevance (safety and efficacy) of these newly found metabolites including M23^Novel needs to assess as metabolite formed in small quantities can also exert significant pharmacological and toxicological effects. To synthesize and study the safety and efficacy of the proposed metabolite is not feasible to author under current experimental setup.

ACKNOWLEDGMENTS:

The authors are thankful to Mr. Prashant Survey for providing the guidance during the conduct of metabolic incubation experiments and structural elucidation of individual metabolite.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 05.10.2020 Modified on 08.11.2020

Research J. Pharm. and Tech 2021; 14(11):5683-5690.

DOI: 10.52711/0974-360X.2021.00988