Art S8

DRUDIS 103943 No.
of Pages 16, Model NS
Drug Discovery Today d Volume xxx, Number xx d xxxx 2024 REVIEWS
KEYNOTE (GREEN)
of
4
Small molecule drug metabolite synthesis
and identification: why, when and how?
o
5
Julia Shanu-Wilson ⇑, Samuel Coe,
Pr
7
8 Liam Evans, Jonathan Steele, Stephen Wrigley Julia Shanu-Wilson has been associated with
Hypha Discovery since 2013 and is part of the
management team; her main interests are in bio-
9
10 Hypha Discovery, 154B Brook Drive, Milton Park, Oxfordshire OX14 4SD, UK transformation and active metabolites. Following
postdoctoral research in New Zealand on the
11
12 biosynthesis and detection of mycotoxins, she
13 ed joined the pharmaceutical industry as a research
scientist, becoming a programme discovery leader
14 The drug discovery and development process encom- for Cubist Pharmaceuticals (since acquired by
15 passes the interrogation of metabolites arising from Merck). She was responsible for a team discovering
new natural-product-derived antibacterial drugs,
16 the biotransformation of drugs. Here we look at why, and was part of a cross-continent team responsible
for bringing the cyclic lipopeptide daptomycin to
17 when and how metabolites of small-molecule drugs the market. She earned her degree and PhD in
ct
18 are synthesised from the perspective of a specialist biochemistry from Imperial College London, UK.
19 contract research organisation, with particular atten-

Samuel Coe is a medicinal chemist specialising in
20 tion paid to projects for which regulatory oversight is central nervous system drug discovery. Following
his PhD studies at the University of Warwick, UK,
relevant during this journey. To illustrate important
re
21
investigating the total synthesis of a novel herbi-
22 aspects, we look at recent case studies, trends and cide, he was involved in medicinal chemistry pro-
jects at several organisations, including the Nav1.8
23 learnings from our experience of making and identi- program at Vertex Pharmaceuticals, compounds
from which are currently in clinical trials. At Hypha
24 fying metabolites over the past ten years, along with Discovery, his work was focused on initiating pro-
or
25 with selected examples from the literature. jects for clients in need of metabolite synthesis. He
is currently a senior scientist at Evotec, leading
synthetic chemistry efforts for clients.
26 Keywords: metabolites in drug development;
27 regulatory framework; disproportionate metabolites;
28 metabolite synthesis case studies; active metabolites Stephen Wrigley has worked in the field of
nc
industrial microbial biotechnology for nearly 40

29
years. He is currently chief technical officer at
30 Introduction Hypha Discovery, where his scientific interests are
focused on the identification of investigational
31 The significance of the metabolites of small-molecule drugs surfaces at drug metabolites by various routes, primarily
32 various points in the drug discovery and development process. Earlier on in involving biotransformation. He specialises in
structure elucidation using nuclear magnetic reso-
U
33 lead identification, metabolic stability assays and soft spot analysis support
nance spectroscopy. Prior to joining Hypha, he was
34 the medicinal chemistry design cycle to generate new iterations of scaffolds a natural products chemist with managerial roles at
35 with improved properties. Analytical workflow initiatives coupled with in RecombinoGen, Cubist Pharmaceuticals (UK), Ter-
raGen Discovery, Xenova and Glaxo Group
36 silico predictions can be used in a semi-automated way to identify major Research. He obtained his BSc and PhD degrees,
37 routes driving in vitro metabolic clearance at this early stage.(p1) Later in the both in chemistry, from Imperial College, University
of London, in 1982 and 1985, respectively.
38 discovery process and during preclinical and clinical development, more
39 definitive identification of metabolites becomes more pertinent as part of
40 understanding clearance and risk mitigation and to satisfy regulatory
41 requirements.
⇑ Corresponding author. Shanu-Wilson, J. (julia.shanuwilson@hyphadiscovery.com)
1359-6446/Crown Copyright Ó 2024 Published by Elsevier Ltd.

https://doi.org/10.1016/j.drudis.2024.103943This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).www.drugdiscoverytoday.com 1
Please cite this article in press as: Shanu-Wilson J. et al., Drug Discovery Today (2024), https://doi.org/10.1016/j.drudis.2024.103943
DRUDIS 103943 No. of Pages 16, Model NS
KEYNOTE (GREEN) Drug Discovery Today d Volume xxx, Number xx d xxxx 2024
42 Although safety is the number one reason why the metabolites of delays in clinical development, and the FDA strongly recom- 97
43 drugs are accessed and interrogated in the discovery and develop- mends that metabolic evaluation in humans is conducted as 98
44 ment process, there are other valuable benefits. Metabolites early as is feasible, rather than late in clinical trials.(p7) 99
45 might have improved potency, selectivity, solubility or improved By definition, metabolite studies conducted before human 100
46 drug metabolism and pharmacokinetics (DMPK) properties, and studies might not always accurately highlight what could 101
47 they can also provide valuable data for intellectual property become significant or disproportionate metabolites, and these 102
48 (IP) coverage.(p2),(p3),(p4),(p5) are consequently found later during clinical development. Such 103
49 In this article we focus on accessing metabolites of small- surprises usually require prompt action to definitively identify 104
50 molecule drugs as part of the typical drug discovery and develop- metabolite structures and provide sufficient material for further 105
51 ment life cycle, and discuss commonly used methods to synthe- testing. Such was the case during the development of ganax- 106
KEYNOTE (GREEN)
52 sise and characterise them. Insights are derived from experiences olone, in which a human disproportionate metabolite became 107
of
53 acquired by a contract research organisation (CRO) specialising apparent during the 14C radiolabel human mass balance study. 108
54 in metabolite synthesis for more than 300 client companies, Ganaxolone (Ztalmy oral suspension, CV) is a neuroactive 109
55 ranging from large pharmaceutical firms to small- and medium- steroid that was approved by the FDA for the treatment of sei- 110
56 sized enterprises focusing on the development of single products. zures associated with cyclin-dependent kinase-like 5 (CDKL5) 111
o
deficiency disorder (CDD) in patients 2 years of age and older. 112
It is subject to complex metabolism, with 59 metabolites identi- 113
57 The need
fied,(p9) and is a synthetic C-3 methyl analogue of allopreg-
Pr
114
58 Through our interactions with scientists across all types of phar-
nanolone that binds to allosteric sites of the GABAA receptor. 115
59 maceutical company, we see a wide variety of triggers for when a
The major routes of ganaxolone metabolism involve multiple 116
60 metabolite becomes of sufficient interest to warrant synthesis
steps, comprising hydroxylation at the 16a-hydroxy position, 117
61 and definitive structure elucidation. Scientists are not only keen
stereoselective reduction of the 20-ketone to give the correspond- 118
62 to meet regulatory guidelines, but are also driven to ensure that
ing 20a-hydroxysterol, and sulfation of the 3a-hydroxy group. 119
63 they are being thorough, yet practical, in ensuring their drug is ed The sulfation reaction yields an unstable tertiary sulphate, which 120
64 safe for a patient.
undergoes elimination to form a double bond in the A ring. A 121
65 The use of modern analytical methods such as high-resolution
combination of these pathways, together with oxidation of the 122
66 mass spectrometry (HRMS) and nuclear magnetic resonance
3b-methyl substituent to a carboxylic acid and sulfation at the 123
67 (NMR) spectroscopy generates a wealth of data from which bio-
20a position, give rise to two major circulating metabolites in 124
68 transformation pathways populated with a multitude of metabo-
plasma, M2 and its C20-sulfate, M17. 125
ct
69 lites can be assembled.(p6) Among these, what is classified as a
In vitro and animal in vivo studies led to the initial belief that 126
70 significant metabolite or xenobiotic pathway of concern? And
alternative biotransformations were responsible for clearance of 127
71 when should further action be taken to interrogate it?
the drug. The major human liver microsome (HLM) metabolite 128
72 The answer is not black-and-white, because the decision is
re
was identified as 16a-hydroxy-ganaxolone (M1), mainly through 129

73 based on various factors including systemic exposure, the signif-
the action of CYP3A4, whereas in human hepatocytes the corre- 130
74 icance of the chemical structure of the metabolite and whether
sponding glucuronide, M11, was also observed as a major 131
75 the metabolite is disproportionate. In order to build a framework
metabolite along with a minor metabolite, M5. A later key find- 132
76 that provides guidance and promotes consistency of strategy,
ing was the high conversion of this dehydrated metabolite to M2 133
or
77 regulatory direction has been issued by the FDA, European

in hepatocytes, but not in HLMs. 134
78 Medicines Agency (EMA) and other regulatory bodies.
Key intermediates of M2 and M17, M5, M6 and M53, were 135
observed at low levels in human hepatocyte and human S9 incu- 136
79 Why? The impact of regulatory framework bations of ganaxolone, but their levels were overlooked in the 137
nc
80 Metabolites in safety testing (MIST) guidance for small-molecule presence of the much more dominant M1 and M11. 138
81 drugs published in 2020 recommends that if a metabolite consti- None of the preclinical species yielded detectable amounts of 139
82 tutes >10% of the total drug-related material in human plasma, M2 or M17, except trace levels in the female rat. However, find- 140
83 and at significantly greater levels than the maximum exposure ings in the 14C radiolabel human mass balance study pointed to 141
84 seen in the preclinical toxicity studies, non-clinical characteriza- the presence of an alternative situation, with a very slow rate of 142
U
85 tion is warranted before large-scale clinical trials.(p7) excretion and with only 72.7% ± 9.6% of the administered 143
86 Although human-specific metabolites are not common, the radioactivity recovered by the end of the study at 720 h. 144
87 formation of a metabolite at higher levels in humans or with Subsequently, M2 and M17 were shown to be disproportion- 145
88 lower clearance than in the animal species used in safety testing ate human metabolites that persisted in circulation, highlighting 146
89 of the parent drug is frequently encountered.(p8) If a relevant ani- the limitations of traditional animal studies and human in vitro 147
90 mal species that forms the metabolite cannot be identified, such systems in predicting major circulating metabolites in humans. 148
91 disproportionate metabolites are synthesised to ensure clinical Unfortunately, there was an added complication in that minimal 149
92 safety. This necessitates the generation of material for dosing in structural information could be gleaned from tandem mass spec- 150
93 an appropriate animal model, along with the development of trometry (MS/MS), and NMR was needed to elucidate the struc- 151
94 standards for analytical methods to identify and measure the ture of M2 and other metabolites. 152
95 levels of the metabolite in the non-clinical toxicity studies. Early Multiple enzymes are involved in ganaxolone’s biotransfor- 153
96 identification of disproportionate drug metabolites can prevent mation, including cytochrome P450 3A4 (CYP3A4), aldo–keto 154
2 www.drugdiscoverytoday.com
Drug Discovery Today d Volume xxx, Number xx d xxxx 2024 KEYNOTE (GREEN)
155 reductase family 1 (AKR1), sulfotransferases (SULTs) and uridine including acyl glucuronides, O-glucuronides, N-glucuronides 198
156 50 -diphospho-glucuronosyltransferases (UGTs). Interestingly, it is and carbamoyl glucuronides, have been shown to be substrates 199
157 proposed that the species-specific nature of one sulfation step is or time-dependent inhibitors of CYP2C8.(p10),(p12) As well as 200
158 the reason that preclinical species fail to produce appreciable inhibiting key drug-metabolising enzymes, metabolites might 201
159 amounts of the M5 or M5-derived intermediate metabolites.(p9) also inhibit transporters in the intestine, liver and kidney. In a 202
160 The involvement of extrahepatic SULT or SULTs also points to recent study investigating 25 drug metabolites, seven were found 203
161 the disconnect between hepatic in vitro systems and the in vivo to be potent inhibitors of either or both of the organic anion 204
162 situation. transporters OAT1 and OAT3 in the kidney, and four were pre- 205
163 Although M2 demonstrated no functional activity at the sent at clinically relevant concentrations.(p13) 206
164 GABAA receptor, either as a modulator or as a direct activator, All major metabolites of parent drugs (or those that contribute 207
KEYNOTE (GREEN)
165 safety studies including toxicology and carcinogenicity studies significantly to pharmacological activity or contain structural 208
of
166 were needed. This necessitated the development of a scalable alerts for known DDI mechanisms) are now required by major 209
167 synthesis of M2 that could readily be progressed to a multiple- regulatory bodies [the FDA, EMA and Japanese Pharmaceuticals 210
168 kilogram scale (unpublished results). The additional evaluation and Medical Devices Agency (PMDA)] to be assessed for DDI 211
169 of M17 as a metabolite under the MIST guidelines was not seen potential. They all recommend a similar approach to DDI test- 212
o
170 as necessary, and no separate studies were conducted for M17. ing,(p14) with the In Vitro Drug Interaction Studies Guidance 213
171 The initial eight-step synthesis of M2 was unfortunately diffi- advising that in vitro DDI assessments be conducted before clin- 214
ical testing starts.(p15),(p16)
Pr
172 cult to scale up beyond low gram quantities. As an alternative, a 215
173 much shorter regio- and stereoselective synthesis was developed, The In Vitro Drug Interaction Studies Guidance states that 216
174 as shown in Figure 1, which required only three synthetic steps metabolites should be evaluated for in vitro CYP enzyme and 217
175 from the commercially available diketone shown on the right- transporter inhibition studies in cases in which circulating 218
176 hand side. These steps comprise a regioselective nitroalkane con- metabolites occur at 25% of parent exposure for metabolites that 219
177 densation, an oxidation of the resulting nitroalkane to the corre- are less polar than the parent drug, and at 100% of parent expo- 220
178
179
180
181
sponding carboxylic acid and a diastereoselective enzymatic
ketone reduction.
The MIST guidance is somewhat less concerned about phase II
metabolites such as glucuronides, because they ‘generally render
ed sure for metabolites that are more polar than the parent drug. In
addition, any metabolite with a structural alert should be evalu-
ated for potential mechanism-based inhibition. A study analys-
ing 120 new molecular entities approved between 2013 and
221
222
223
224
182 a compound more water soluble and are pharmacologically inac- 2018 revealed that 63% of metabolites quantified in vivo had 225
183 tive’.(p7) However, more caution is recommended with poten- >25% of parent exposure and that 75% of these metabolites were 226
ct
184 tially toxic conjugates, such as acyl glucuronides of carboxylic- tested for CYP inhibition in vitro, resulting in the identification of 227
185 acid-containing drugs. In addition, intermediate reactive 15 metabolites with potential DDI risk. Interestingly, analysis of 228
186 metabolites can form stable products such as glutathione conju- the data did not support the use of metabolite polarity as a pre- 229
re
187 gates, which can be more readily detected and characterised. This dictor of the risk of DDIs caused by metabolites, and it also 230
188 does not mean that glucuronides should be disregarded, owing to revealed several metabolites that inhibited CYPs in vitro where 231
189 the potential for drug–drug interactions (DDIs). the parent drug did not. The overall results supported the use 232
190 In addition to potential toxicity or other off-target concerns, of a 25% of parent exposure cutoff for in vitro CYP inhibition test- 233
191 there is the need to interrogate the potential of circulating ing to adequately identify metabolites with the potential to cause 234
or
192 metabolites for DDIs. This is not confined just to phase I metabo- in vivo DDIs.(p17) The EMA also recommends the assessment of 235
193 lites, because common metabolites such as glucuronides have metabolites that represent >10% of drug-related exposure 236
194 also been shown to interact with cytochrome P450 enzymes observed during the 14C human in vivo absorbed, metabolised, 237
195 (CYPs), in particular CYP2C8 as a result of its distinctive active excreted (AME) mass balance study.(p18) This includes structural 238
nc
196 site, which allows binding to anionic and bulky ligands such as characterisation, as well as establishing any potential inhibitory 239
197 glucuronides.(p10),(p11) Several classes of glucuronide conjugates,
U
Drug Discovery Today
FIGURE 1
Major human disproportionate metabolite M2 of ganaxolone, formed via M5 in humans, and the starting diketone used for the three-step synthetic route.
www.drugdiscoverytoday.com 3
240 effects on common drug-metabolising enzymes for phase I samples (blood, plasma, bile, etc.) can take place to enable fur- 271
241 metabolites that meet the criteria. ther understanding of the biological fate of molecules. The 272
242 The assignment of structural alerts has been debated widely as definitive human 14C ADME study usually takes place in phase 273
243 a way of flagging the potential for reactive metabolite formation II, after which metabolite synthesis might be needed for defini- 274
244 that could cause idiosyncratic drug toxicity,(p19) especially con- tive structure identification and pharmacological and safety eval- 275
245 sidering that numerous drugs containing structural alerts are uations. Indeed, it has been strongly recommended that the 276
246 devoid of any adverse drug reactions. Analysis of the top 200 study results and those of linked studies to characterise metabo- 277
247 drugs (sold and prescribed) in the United States in 2009 revealed lites in non-clinical species, as well as an understanding of any 278
248 a significant proportion containing structural alerts, with the possible interactions, should be available before phase III.(p18) 279
249 majority involving reactive metabolite formation. Significantly, The FDA’s MIST guidance encourages the identification of any 280
KEYNOTE (GREEN)
250 the majority of these drugs were administered at low daily dose; differences in drug metabolism between animals used in non- 281
of
251 this seems to be a key factor. There were also competing detoxi- clinical safety assessments and humans as early as possible to 282
252 cation pathways and/or alternative non-metabolic clearance avoid unwelcome surprises late in drug development, which 283
253 routes that mitigated the risk.(p20) However, the interrogation can cause costly delays: such as that experienced during the 284
254 of the metabolites of new drugs with structural alerts needs to approval of ozanimod (Zeposia). The two-year delay before the 285
o
255 be performed, and risk-mitigation strategies must be considered. drug could be approved and marketed stemmed from the FDA 286
issuing a ‘Refuse to File’ letter owing to insufficient preclinical 287
characterization of an active metabolite of ozanimod.(p21)
Pr
288
256 When? More recently, there has been an emphasis on the identifica- 289
257 We see the synthesis of drug metabolites performed at various tion and quantification of human metabolites from early clinical 290
258 stages in the drug discovery and development process, ranging studies,(p18),(p22) which could then lead to an earlier need for syn- 291
259 from late discovery or preclinical evaluation to almost all phases thesis of a metabolite. This shift in timing of the mass balance 292
260 of clinical development (Figure 2). study enables more efficient handling of any metabolites that 293
261 Prior to development, in vitro studies provide a key map of the ed might need safety testing.(p18) 294
262 biotransformations of a drug. Such studies can be run pre- It is advised that metabolites present at >10% of dose in exc- 295
263 emptively to identify any potential human disproportionate reta or drug-related material in plasma should always have their 296
264 metabolites through a cross-species comparison of metabolites structures definitively identified,(p23) which can be challenging if 297
265 observed in hepatocytes of human and preclinical animal spe- the metabolite profile is complex. Proof of the chemical structure 298
266 cies. The testing of drugs in traditional 14C absorption, distribu- might require additional investigations obtained from compar- 299
ct
267 tion, metabolism and excretion (ADME) rodent species and, isons with a synthesised authentic standard of the metabolite. 300
268 more rarely, non-rodent animal species has been a key milestone Metabolite reference standards can also be isolated directly from 301
269 as a drug progresses through preclinical development into phase a biological matrix (e.g., human urine from a clinical study), 302
270 I. Often at this stage a full ex vivo metabolite study of various
re
or
nc
U
FIGURE 2
Consideration of metabolites in the drug discovery and development process, selected information taken from ref.(p83)
303 although this is limited by the material availability relative to species-specific reactions of enzymes involved in N- 361
304 how much metabolite is needed for further studies. In terms of glucuronidation. 362
305 activity testing of metabolites, although structural similarity to Although the glucuronidation of drugs is a common clearance 363
306 the parent drug is not an indicator that a metabolite will be phar- pathway, it is unusual to encounter linked or di-glucuronides/ 364
307 macologically active, there is a greater likelihood that this might glycosides, not least those that are also major human circulating 365
308 be the case. It is thus recommended that circulating metabolites metabolites. GDC-0180 (Brilanestrant) is a non-steroidal, orally 366
309 formed from relatively simple biotransformations, such as bioavailable selective oestrogen receptor degrader (SERD) that 367
310 hydroxylation, heteroatom demethylation and dehydrogena- was developed for the treatment of breast cancer. Metabolite pro- 368
311 tion, should be evaluated for pharmacological activity.(p23) filing during the FTIH study led to the identification of a novel 369
312 A recent paper highlights that a good time to interrogate any discrete diglucuronide metabolite (M2) as a primary circulating 370
KEYNOTE (GREEN)
313 major human circulating metabolites missing from in vitro stud- metabolite that had not been previously detected. M2 is an 371
of
314 ies is through the analysis of plasma samples acquired during the example of a discrete diglucuronide formed by glucuronidation 372
315 phase I multiple ascending dose (MAD) study,(p24) because it is on two different functional groups to a major circulating 373
316 the metabolite profile and exposure of any circulating metabo- metabolite in humans, formed from sequential acyl glucuronida- 374
317 lites at steady-state that are key. tion in the intestines followed by N-glucuronidation in the 375
o
318 A potential move towards a human first/human only liver.(p28) 376
319 approach is gaining momentum.(p25) Technological advances There are a few interesting facets to this biotransformation. 377
from the use of 14C-microtracer dosing with accelerator mass
Pr
320 First, that N-glucuronidation is more common in humans owing 378
321 spectrometry (AMS) permit earlier human-relevant knowledge to the activity of UGT1A4 and UGT2B10, resulting in the 379
322 to be gained about the metabolism of a drug in phase I. As such, absence of M4 in animal species. Second, that acyl glucuronida- 380
323 the assessment of metabolites of human relevance from first time tion to M6 was generally compartmentalised in the intestine 381
324 in human (FTIH) studies is now considered standard practice in rather than the liver owing to the involvement of the UGTs 382
325 many pharmaceutical companies. Consequently, metabolites 1A8/1A7 and 1A1. These elements help to explain the unpre- 383
326
327
328
329
identified in early work can then be synthesised so they are avail-
able, for example, as chromatographic standards. At the same
time, this enables a reduction in the use of animals during drug
development, as resolved by the European Parliament in
ed dicted appearance of the discrete diglucuronide M2 as a major
circulating metabolite in humans (Figure 3).
384
385
How? Typical routes to metabolite synthesis used at 386

330 2021.(p26) In addition to the information gained on human
331 metabolite profiles and routes and rates of excretion, the use of Hypha 387
ct
332
14
C-microtracing in early clinical development quickly proved Both synthetic and biological approaches are used to generate 388
333 useful to Lundbeck in uncovering unknown issues related to metabolites of interest, with special attention paid to procedures 389
334 the metabolism of a drug. In this instance, the use of a microdose that are capable of yielding specific stereo- and regioisomers and 390
of 14C-labelled drug and analysis by AMS revealed that the unex- complex conjugates. Other reviews discuss the comprehensive 391
re
335
336 pectedly low exposure of a drug was almost exclusively attributa- techniques that can be applied(p29),(p30),(p31),(p32); however, here 392
337 ble to extensive metabolism by aldehyde oxidase (AO).(p27) we focus on techniques used internally to make the most fre- 393
338 Additional guidelines also affect the timelines for accessing quently requested metabolites needed to support drug develop- 394
339 metabolites. Various regulatory frameworks have previously ment programmes. Our experiences show that there is not one 395
or
340 addressed DDI studies. The latest International Council for Har- magic technique that can be relied upon to access the wide range 396
341 monisation of Technical Requirements for Pharmaceuticals for of metabolite structures needed. Where possible, it is more 397
342 Human Use (ICH) draft guidance for drug interaction studies animal-friendly and cost-effective to identify an alternative to 398
343 (M12) consolidates recommendations internationally for design- biotransformation methods that use mammalian tissues for 399
nc
344 ing, conducting and interpreting enzyme- or transporter- scaling-up metabolites, and surrogate methods can often readily 400
345 mediated in vitro and clinical DDI studies during drug develop- satisfy the regulatory requirements. 401
346 ment. The overall recommendation is that information on DDI

347 potential should be generated as early in the drug development Late-stage chemical synthesis 402
348 as possible, including the DDI potential of metabolites with sig- Classical organic synthesis is often considered first for accessing 403
U
349 nificant plasma exposure or pharmacological activity similar to metabolites. However, depending on the nature and complexity 404
350 that of the parent drug. Within projects, any major metabolites of the drug candidate and the structure of the target metabolite, 405
351 need to be available for DDI studies to be performed in parallel the generation of the metabolites is not always straightforward 406
352 with the parent drug. and might necessitate complex syntheses. This could demand 407
353 Much work is focussed on understanding the metabolism of valuable medicinal or process chemistry resources to deliver even 408
354 drugs in the liver; however, although less common, extrahepatic minor structural changes to the original molecule. Where timely 409
355 metabolism can also drive the formation of major human confirmation of metabolite structure or access to reference stan- 410
356 metabolites. It can be more difficult to pinpoint such metabolites dards is important, late-stage functionalisation methods that 411
357 at an earlier stage where the phase I or phase II enzymes involved are commonly used for improving drug-like properties are often 412
358 are specific to non-liver tissues, illustrated by the identification of successful and quick.(p33),(p34),(p35) 413
359 a human disproportionate and rather unusual diglucuronide in Chemical synthesis of phase I metabolites is often challenging 414
360 the FTIH study of GDC-0810. This was further complicated by in cases where the regiospecific oxidation of non-activated car- 415
KEYNOTE (GREEN)
o of
Pr
FIGURE 3
Metabolism of GDC-0810, adapted, with permission, from ref.(p28) Metabolites were identified in samples from the FTIH study, various in vitro studies and
ed
in vivo rat mass balance studies by comparison with synthesised standards made at Genentech.
416 bons in complex molecules is required or where metabolites have 5 g of AZD9574 resulted in the synthesis of 605 mg of the glu- 447
417 specific stereochemistry. However, the use of late-stage chemical curonide. However, due to high lability resulting in release of 448
418 oxidation, electrochemistry and photochemistry methods for some of the aglycone under mild conditions, a further purifica- 449
ct
419 some oxidations is possible. tion step was necessary. The solid material was also found to be 450
420 Late-stage chemical synthesis has been commonly used for temperature sensitive and started hydrolysing as soon as the final 451
421 making phase II conjugates of drugs, with glucuronides being lyophilisation stage was complete, thus necessitating careful 452
re
422 the most common phase II metabolite required. Although syn- handling. The additional purification step resulted in a total of 453
423 thetic methods for all classes of glucuronides have been pub- 481 mg of what was later determined to be an O-glucuronide, 454
424 lished,(p36) attention still needs to be given to minimizing any with minimised levels of the parent drug. 455
425 stability issues, including isomerization and hydrolysis, during
Microbial biotransformation 456
or
426 their synthesis and purification.

427 Late-stage methods are generally conducted as a two-step pro- Where the chemical synthesis of a human metabolite is challeng- 457
428 cess, starting with a screen to expose the parent compound to a ing or even impossible, carefully selected microbes can provide a 458
429 wide variety of chemical conditions appropriate to the glu- route to obtain these metabolites as a result of them expressing 459
430 curonide type (if known). Once a suitable method has been iden- phase I and II metabolising enzymes that are homologous to 460
nc
431 tified, the reaction will be scaled-up according to the target mammalian systems.(p38),(p39) These enzyme types include CYP 461
432 amount of glucuronide needed, and the material can then be monooxygenases as well as enzymes that form the products of 462
433 purified. Generally, most synthesised glucuronides are stable; non-CYP phase I mammalian metabolising enzymes, such as 463
434 however, some require careful handling and purification follow- AO and flavin-containing monooxygenases. The expression of 464
435 ing synthesis, as encountered with purification of the O- conjugative enzymes, including UGTs, provides the key advan- 465
U
436 glucuronide of AZD9574. tage over chemical synthesis in that multistep reactions in a ‘sin- 466
437 The main route of metabolism of the poly(ADP-ribose) poly- gle pot’ are possible: for example, sequential hydroxylation and 467
438 merase 1 (PARP1) inhibitor AZD9574 in human hepatocytes is glucuronidation. The scalability of microbial incubations is 468
439 through glucuronidation (Figure 4).(p37) Screening of AZD9574 mostly sufficient to cater for milligram- to gram-scale material 469
440 in eight different late-stage chemical glucuronidation conditions requirements, and in contrast to liver fraction incubations, the 470
441 resulted in the conversion to three different glucuronides across cost of materials is low due to the amenability of microbes for 471
442 three conditions. Two of these were shown to form the desired use in metabolite production in multi-litre-volume fermenta- 472
443 glucuronide by liquid chromatography with MS/MS (LC-MS/ tions, and also because microbes generate the necessary cofactors 473
444 MS) matching with the hepatocyte sample. It was noted at this for metabolic reactions. 474
445 initial stage that the glucuronide was hydrolytically unstable in Microbial biotransformation can also be applied to make 475
446 acidic conditions. Scale-up of the most promising reaction with stable labelled and radiolabelled metabolites of drugs in cases 476
in which chemical synthesis is more challenging. Here, a process 477
FIGURE 4
Glucuronide of AZD9574 O-linked through the lactim tautomer of the quinoxalinone, made by late-stage chemical synthesis.
KEYNOTE (GREEN)
of
478 for producing the metabolite is defined and optimised using approval of the drug by two years. Its biotransformation results 523
479 unlabelled material, and then repeated using labelled parent in more than seven active metabolites that are as potent and as 524
480 material. In this way, a 14C-radiolabelled major CYP metabolite selective as the parent drug. 525
o
481 of deflazacort (DFZ) was generated for transporter studies. In addition to the generation of active metabolites, the 526
482 DFZ is a glucocorticoid that exists as an inactive pro-drug that involvement of gut bacteria in ozanimod’s metabolism led to 527
483 is rapidly converted by esterases to the active metabolite, 21- the formation of a major inactive circulating metabolite, 528
Pr
484 desacetyl DFZ, after oral administration. The active metabolite RP101124, formed as a result of a scission of the oxadiazole ring 529
485 is further metabolised by CYP3A4, primarily to 6b-hydroxy-21- system (Figure S2). RP101124 was discovered during an in vivo 530
14
486 desacetyl DFZ (6b-OH-21-desDFZ) (Figure S1). The pharmacoki- C-ADME study in rats, in which a lag time was observed before 531
487 netic drug interaction potential of 6b-OH-21-desDFZ was not its appearance in circulation. Gut bacteria are also involved in 532
488 fully characterised and was subsequently required by the FDA the breakdown of RP101124, via RP112533, through anaerobic 533
489 after approval of the drug.(p40) Chemical synthesis of 6b-OH- ed decarboxylation, a step which might have contributed to the 534
490 21-desDFZ is challenging because multiple steps as well as stere- low recovery in the human mass balance study, where radiolabel 535
491 ospecific reactions are required, which are time-consuming and was lost as 14CO2, in addition to the long half-life of metabolites. 536
492 costly. Microbial biotransformation was therefore explored as a For drugs that act locally in the gut, prominent metabolites in 537
493 route to obtain 14C-radiolabelled 6b-OH-21-desDFZ.(p40) Unla- the gastrointestinal tract are of more significance than circulating 538
494 belled DFZ was first screened against 37 microbial strains to iden- metabolites. The pan-Janus kinase (JAK) inhibitor izencitinib is a 539
ct
495 tify a suitable route to the metabolite. The highest yielding strain gut-selective drug that is extensively metabolised, but no one 540
496 was scaled up using 200 mg of DFZ to provide 7.9 mg of a metabolite exceeded 10% of total drug-related material in 541
497 hydroxylated metabolite, the NMR spectra of which matched plasma. However, given that izencitinib is locally acting, it was 542
498 an authentic commercial standard of the desired b-isomer. The proposed that two major faecal metabolites (M9 and M18), 543
re
499 process was then repeated using [20 -14C]-DFZ, and after iterative which together represented more than 50% of the dose, were rel- 544
500 purification steps generated 62.1 lCi (equivalent to 448 lg) of evant for evaluating the pharmacological and toxicological prop- 545
501 the metabolite from 5 mCi of parent drug, with a radiochemical erties of the drug. Their structures are unusual, resulting from 546
502 purity of 99.2% and chemical purity of 98.7%, at an overall yield oxidation and rearrangement of izencitinib to M18 with a pro- 547
or
503 of 1.2%. posed one-carbon addition with formaldehyde forming a tetra- 548
504 The metabolite was used for CYP induction and inhibition cyclic product M9 (Figure S3). Both metabolites were tested for 549
505 studies, and the radiolabelled metabolite for further pharmacological activity and were inactive. The authors point 550
506 transporter-mediated substrate accumulation studies using bidi- out that toxicological testing of these ‘local metabolites’ should 551
nc
507 rectional permeability assays. The metabolite modestly inhibi be the focus rather than systemic exposure, the latter being irrel- 552
508 ted CYP2C19, CYP3A4, MDR1, OATP1B1 and OATP1B3 activi- evant to this locally acting drug.(p45) 553
509 ties (IC50 20 lM).(p40) However, the metabolite was not Where gut metabolites are suspected to be implicated in the 554
510 expected to drive any clinically relevant DDIs owing to the low fate of an experimental drug, human faecal incubations can be 555
511 plasma exposure at the therapeutic dose of DFZ (0.34 lM). conducted with parent drugs under anaerobic conditions to shed 556
U
light on whether a metabolite is formed by gut microbiota or in 557
512 Gut metabolism the faecal matrix itself. Sufficient material can usually be gener- 558
513 Metabolism by gut microbes has recently gained more atten- ated to purify enough for structure elucidation by NMR spec- 559
514 tion,(p41),(p42),(p43) although, in our experience to date, it is rela- troscopy and biological testing. 560
515 tively rare that these metabolites are prominent enough to

516 warrant investigation. They can, however, be significant, such Recombinant enzymes 561
517 as the unexpected involvement of gut microbiota in the reduc- Recombinant enzymes are used as a tool to make metabolites. 562
518 tive metabolism of ozanimod, where oxadiazole ring scission Various sources can be used, such as engineered human CYPs, 563
519 and reabsorption results in the formation of RP101124, a major ancestral CYPs and bacterial derived BM3 (CYP102A1) 564
520 circulating metabolite.(p44) mutants.(p30),(p46) All but a handful made by us have been CYP- 565
521 Ozanimod has a complex disposition, aspects that were dis- derived metabolites created using PolyCYPs enzymes, a panel 566
522 covered late in phase III clinical development and which delayed of bacterial CYPs mined from the genomes of Actinomycete 567
568 strains and expressed in Escherichia Coli together with appropri- ture makes provision of material by other routes more 625
569 ate redox partners.(p47),(p48),(p49) Scale-up of reactions of each iso- challenging. This is especially relevant where many possible 626
570 form of interest is achieved using cell homogenates or whole cell structures of a metabolite exist, thereby making synthetic 627
571 biotransformation of PolyCYPs expressed in E. coli or Streptomyces approaches unattractive until the structure can be elucidated 628
572 lividans. Where desired metabolites have not been produced by by techniques such as NMR spectroscopy. Only small amounts 629
573 PolyCYPs, human recombinant CYPs have been used to access of materials (typically tens of micrograms) are needed for struc- 630
574 metabolites. Of 22 CYP metabolites made by recombinant CYPs ture elucidation using cryoprobe NMR technology. Knowledge 631
575 and structurally identified, four have been made using human of the structure can then enable chemical synthesis of the correct 632
576 recombinant CYPs: one from CYP1A1, one from CYP1A2 and metabolite. These purifications can prove to be challenging, 633
577 two from CYP2J2. Other human recombinant enzymes have also especially if the concentration of the metabolite or metabolites 634
KEYNOTE (GREEN)
578 been useful in making non-CYP-derived phase I metabolites, of interest is low or detection by high-performance liquid chro- 635
of
579 specifically human recombinant AO and flavin-containing matography (HPLC) with ultraviolet detection mass spectrome- 636
580 monooxygenases (FMOs) expressed in E. coli, but these products try (UV/MS) is difficult. Selective extraction methods are 637
581 are typically scaled up using microbial biotransformation due to needed to minimise downstream problems caused by matrix 638
582 the better scalability and lower cost of this method. components. Because hydroxylated or conjugated metabolites 639
o
583 Similar to other commercially available CYP enzyme systems are usually polar molecules, purification by reversed phase HPLC 640
584 for making metabolites,(p30) PolyCYPs enzymes are used to make methods guided by LC-MS assays is usually appropriate. Two or 641
Pr
585 metabolites via screening and scale-up kits, as illustrated in the three purification stages using different column chemistries 642
586 synthesis of a prominent CYP3A4 metabolite of BI 894416, and/or mobile phase modifiers are usually needed to achieve suf- 643
587 where access to a monohydroxylated metabolite was required ficient purity. 644
588 as part of early development studies. During early development
589 of BI 894416, the appearance of M398(2) was observed, but its Liver S9 fractions and microsome incubations 645
590 structure could not be deduced by LC-MS/MS. Because there were Rarely, when other biotransformation or chemical synthesis 646
591
592
593
594
multiple sites in the lactam ring system where the biotransforma-
tion could occur, and because of the creation of a new chiral cen-
tre through hydroxylation, the use of biotransformation
methods to access the metabolite was preferred.(p50)
ed methods fail to make a target metabolite, biotransformations
using microsomes or liver S9 fractions can be used, along with
the addition of the appropriate cofactors. In our experience,
647
648
649
these are reserved as ‘back-up’ techniques owing to the cost of 650
595 BI 894416 is a spleen tyrosine kinase inhibitor that was under the materials and the lack of scalability arising from the typically 651
596 development for the treatment of asthma. Its metabolism results
ct
low concentrations of drug that can be dosed in such incuba- 652
597 in several oxidised metabolites, with M398(2) comprising 11.4% tions. Only 7% of metabolites identified in-house were made 653
598 of total drug-related exposure in the single ascending dose (SAD) using these methods. It is also desirable to minimise the use of 654
599 study AUC0–24h pool. Both HLM incubations and the use of Poly- systems that necessitate large quantities of animal-derived tissues 655
re
600 CYPs recombinant enzymes were evaluated as a route to make for scaling up in order to reduce the use of animals in drug devel- 656
601 M398(2). opment.(p52) These biotransformations, however, can be an 657
602 Incubation of BI 894416 in HLMs gave a 5% conversion to important technique for producing metabolites that are difficult 658
603 M398(2). Screening of BI 894416 against a panel of 18 recombi- to make by other means. 659
604 nant microbial PolyCYPs, human recombinant AO and FMO3
or
605 resulted in the highest conversion of 40% by PolyCYP 152 to

606 yield both M398(1) and M398(2). The fragmentation pattern of Learnings from the past 10 years of making 660
607 putative M398(2) produced by PolyCYP 152 matched the human metabolites 661
608 plasma sample from the SAD study. A small scale-up (1 mg) Analysis of the 100 most commonly prescribed drugs in 2022 662
nc
609 yielded 3.6% conversion in HLMs and 65% using PolyCYP 152. showed that, of the 89 subject to drug metabolism, 75% were 663
610 M398(2) was purified from both the HLM and PolyCYP 152 incu- cleared via phase I mechanisms, with CYP3A4 being the major 664
611 bations, and the position of the hydroxylation of the structure enzyme involved in metabolism of 48% of drugs.(p53) Similar 665
612 was confirmed by NMR spectroscopy (Figure 5). Scientists at statistics were observed for the small molecules that were 666
613 Boehringer used an in-house-developed diastereomeric in silico approved by the FDA in 2022,(p54) but perhaps this is not surpris- 667
U
614 chiral elucidation (DiCE2) prediction tool to identify the ing given that we are analysing molecules that emerged from lab- 668
615 metabolite from both sources as the RSR isomer.(p51) Further oratory benches many years ago. In 2008, 90% of all drugs 669
616 scale-up of the PolyCYP 152 reaction was performed to generate currently approved for clinical use were metabolised by one of 670
617 milligrams of M398(2) for additional investigations; however, only seven CYP isoforms: CYP1A2, CYP2C9, CYP2C18, 671
618 the programme was terminated before DDI and pharmacological CYP2C19, CYP2D6, CYP2E1 and/or CYP3A4.(p55) In 2013, an 672
619 activity studies were conducted. analysis of 248 clinically used drugs with known CYP involve- 673
ment pointed to the involvement of ten CYPs: CYP3A4 674
620 Purification (30.2%), CYP2D6 (20%), CYP2C9 (12.8%), CYP1A2 (8.9%), 675
621 Direct isolation of metabolites from biological matrices such as CYP2B6 (7.2%), CYP2C19 (6.8%), CYP2C8 (4.7%), CYP2A6 676
622 plasma or urine samples can be particularly useful for metabolites (3.4%), CYP2J2 (3%) and CYP2E1 (3%).(p56) A wider comparison 677
623 with unanticipated structures resulting from multi-step biotrans- of the main biotransformation routes of the top 200 most pre- 678
624 formations, and where the complexity of the metabolite struc- scribed drugs with drugs approved by the FDA between 2005 679
KEYNOTE (GREEN)
o of
Pr
FIGURE 5
Hydroxylated CYP3A4 human metabolites of BI 894416 formed by PolyCYP 152.
ed
ct
680 and 2016 revealed that the role of CYP1A2, CYP2C19 and The majority of biotransformations observed result from 709
681 CYP2D6 had decreased as major metabolizing enzymes among phase I mechanisms, including hydroxylations, multiple oxida- 710
682 the more recently approved drugs.(p57) The contribution of alco- tions, N-oxidation and ring opening/hydrolysis reactions. Not 711
683 hol dehydrogenases (ADHs), aldehyde dehydrogenases (ALDHs) surprisingly, hydroxylation is the most common biotransforma- 712
re
684 and SULTS had also decreased. Interestingly, the contribution tion observed, resulting mainly (where known) from CYP- 713
685 of CYP3A4 as the major biotransformation pathway had mediated oxidations of alkyl and aryl moieties in drugs. Where 714
686 increased from 40% in the established drug list to 64% in the drugs featured nitrogen-containing heterocycles, selected AO- 715
687 2005–16 FDA approved drug list. An even more recent analysis mediated hydroxylation was also observed, along with N- 716
or
688 of the main metabolic route of 245 small and macromolecule oxidations catalysed by CYPs or FMOs. Examples of known 717
689 drugs approved by the FDA from 2015 to June 2020 points to CYP-mediated hydroxylated metabolites made in this set where 718
690 the continued importance of CYP3A4/5, with 52% of drugs structures are in the public domain include eight hydroxylated 719
691 being metabolised by this enzyme. Overall, 80% of drugs in this (and corresponding keto) metabolites of ruxolitinib,(p2) a radiola- 720
belled hydroxylated metabolite of DFZ(p40) and, more recently,
nc
692 list were cleared by CYP enzymes, with 15% by UGTs and only 721
693 5% by other mechanisms [2% FMO, 2% monoamine oxidase three hydroxylated metabolites of rezafungin.(p59),(p60) 722
694 (MAO) and 1% AO].(p58) Rezafungin is an echinocandin that was approved by the FDA 723
695 We conducted an analysis of 241 metabolites (referred to as to treat candidaemia and invasive candidiasis in adults with lim- 724
696 the ‘Hypha set’) on which Hypha Discovery performed structure ited or no other treatment options. It has a quaternary ammo- 725
U
697 elucidation at the request of clients (Figure 6). The analysis nium side that confers high stability and a long half-life, 726
698 revealed that 60% of the requests derived from phase I oxidative thereby preventing the spontaneous degradative metabolism to 727
699 mechanisms and 37% from phase II mechanisms, particularly via a ring-opened peptide observed in previously approved 728
700 UGTs, with a smaller percentage arising from mixed phase I and echinocandins. Instead, metabolism is diverted to the alkyl por- 729
701 II pathways and gut metabolism. Metabolites made were promi- tion of the terphenyl, pentyl ether side chain. In the single-dose 730
702 nent in in vitro and/or in vivo studies, and for the metabolites that human mass balance study, although rezafungin was the most 731
703 clients had attempted to elucidate themselves before approach- abundant circulating component in plasma, calculated at 69% 732
704 ing us, these generally could not be made by chemical synthesis. of the total plasma radioactivity exposure (by AUC0-t), three 733
705 Where the metabolism pathway was known, metabolism via hydroxylated metabolites were also in circulation, comprising 734
706 CYP3A4 was the dominant route, mirroring the published data 9.8%, 5.5% and 6.9% of the total plasma radioactivity exposure. 735
707 on approved drugs. However, significantly more glucuronide The rate of formation of these metabolites is slow and is consis- 736
708 conjugates were observed as major metabolites in the Hypha set. tent with the slower elimination of rezafungin. Owing to the 737
KEYNOTE (GREEN)
o of
Pr
ed Drug Discovery Today
FIGURE 6
An analysis illustrating the broad routes of formation of 241 metabolites made for clients where Hypha Discovery was involved in the structure elucidation
step.
ct
738 structural complexity of this cyclic lipopeptide, microbial bio- matic nitrogen-containing heterocycles, for example, indicates 767
739 transformation was used to make the metabolites for identifica- that the more nitrogen atoms that are present in the ring system, 768
740 tion, biological testing and to act as reference standards.(p60) the less likely it is that it will be subject to phase I type oxidations 769
re
741 Following screening of rezafungin against a panel of microbes, of the ring itself. For example, pyrroles, azoles, imidazoles and 770
742 a fungal strain was identified that produced all three hydroxy- thiazoles are extensively metabolised by CYPs, often resulting 771
743 lated metabolites, and a minor des-pentyl metabolite formed in oxidative ring opening.(p61) By contrast, pyrazoles, triazoles 772
744 via O-dealkylation. Leaving the biotransformation longer and tetrazoles are more resistant to oxidative cleavage, but can 773
745 resulted in generation of the minor despentyl metabolite, but be subject to N-glucuronidation.(p62),(p63),(p64),(p65) N-containing 774
or
746 at the expense of the hydroxylated target metabolites. The struc- heterocycles can also act as substrates for AO metabolism.(p66) 775
747 ture of each metabolite was elucidated by NMR spectroscopy to As highlighted previously, interspecies variability in N- 776
748 reveal three isomers resulting from hydroxylation of the ter- glucuronidation can be a barrier for accurate prediction of 777
749 phenyl, pentyl ether side chain (Figure S4). Biological testing in vivo glucuronidation of drugs in humans, as was discovered 778
nc
750 revealed that the metabolites were inactive. during the development of LEO compound 1. LEO compound 779
751 There is perhaps a surprising number of metabolites that are 1 is an oral interleukin-17A (IL-17A) protein–protein interaction 780
752 made from or characterised by glucuronidation (34% of total modulator under development for the treatment of psoriasis and 781
753 metabolites) among the Hypha set, possibly reflecting the chal- other inflammatory disorders. It is metabolised through multiple 782
754 lenge in accessing these metabolites versus Those that are more phase I and phase II routes, including various CYP3A4-mediated 783
U
755 straightforward to make by facile chemical synthesis. The need hydroxylations and N-dealkylation, as well as N-sulfation and N- 784
756 for glucuronides might also represent a divergence from the glucuronidation (Figure 7). Conjugation reactions occur in the 785
757 CYP-based mechanisms that have previously dominated as the pyrazole moiety, with an N-glucuronide being a major metabo- 786
758 latest drugs move through the various stages of clinical develop- lite in humans; only small amounts of the N-glucuronide were 787
759 ment. O- and N-glucuronides constitute the majority of these, as observed in other species. 788
760 well as acyl glucuronides derived from some carboxylic-acid- The interspecies variability in N-glucuronidation is particu- 789
761 containing drugs. larly high, especially for aliphatic tertiary amines and, relevant 790
762 Nitrogen-containing aromatic heterocycles are commonly to LEO compound 1, aromatic N-heterocycles. In addition, N- 791
763 used in medicinal chemistry for multiple reasons, including their glucuronidation rates in humans are reported to be much higher 792
764 reduced lipophilicity when compared with phenyl groups, their than in animals, largely due to UGT1A4 and UGT2B10.(p67) This 793
765 increased metabolic stability and the ease of substitution. Look- species difference in UGT activities can therefore be a barrier for 794
766 ing at the metabolism of commonly used five-membered aro- accurate prediction of in vivo glucuronidation of drugs in 795
KEYNOTE (GREEN)
o of
Pr
ed
ct
re
FIGURE 7
Metabolism of LEO compound 1 in human hepatocytes to a major N-glucuronide.
or
796 humans. In fact, rodents lack a human UGT1A4 homologue metabolite. Profiling of the N-glucuronide in dogs suggested 816
797 gene.(p68) UGT1A4 was shown to be responsible for the major excretion in the bile, hydrolysis and reabsorption of the parent, 817
798 human metabolite of LEO compound 1. but this was not predicted to lead to significant enterohepatic 818
nc
799 Originally, the N-glucuronide was produced in human female recirculation.(p69) 819
800 liver S9 incubations to generate tens of milligrams as a reference Acyl glucuronides are commonly requested for synthesis, 820
801 standard. High conversions (79%) were obtained using this because this phase II metabolite is specifically mentioned in 821
802 route. However, owing to the amount of material subsequently the FDA’s MIST guidance as having the potential to cause 822
803 needed, a late-stage chemical synthesis method was developed idiosyncratic drug toxicity through the direct acylation of pro- 823
U
804 to make both unlabelled and deuterated metabolite. Initially, teins or, if subject to intramolecular rearrangement, through 824
805 chemical glucuronidation resulted in only very low conversion; the formation of aldehydes that lead to protein glycation.(p7) 825
806 however, various modifications in reaction temperature, solvent However, it is important to note that many acyl glucuronides 826
807 and reagent stoichiometry allowed the identification of condi- of carboxylic-acid-containing drugs are not a cause for con- 827
808 tions that provided a higher and cleaner conversion, increasing cern,(p70) exemplified by the acyl glucuronide of AZD5991. 828
809 isolated yield from 1.1% to approaching 10%. Microbial bio- AZD5991 is a potent and selective inhibitor of Mcl-1 that 829
810 transformation was also explored as a route to make the N- entered clinical development for the treatment of haematologi- 830
811 glucuronide, but this was only possible at a maximum of 10% cal malignancies.(p71) In human hepatocytes, AZD5991 is bio- 831
812 in extract yield prior to purification. The late-stage chemical transformed to several metabolites, with an S-oxide and an acyl 832
813 route was therefore used to scale up to multiple grams. An addi- glucuronide being the major human metabolites. The abundant 833
814 tional purification step, not used in the previous production glucuronide metabolite required further characterisation during 834
815 route via S9, was introduced to purify low gram amounts of the preclinical development, and chemical synthesis of it was not 835
836 possible at the time. Separately, an in vivo rat bile duct cannu- Requirements for metabolites 892
837 lated study revealed abundant excretion of the glucuronide in The quantities of metabolites requested vary widely (Figure 8), 893
838 bile, enabling milligram quantities to be isolated. The material with 10–50 mg or sometimes higher amounts typically requested 894
839 obtained from bile excreta was of sufficient quantity and purity for use, for example, as certified reference standards for quantita- 895
840 to obtain an NMR spectrum that revealed an acyl glucuronide tive bioanalysis. Frequently, metabolites are needed for on– and 896
841 structure. This material was also sufficient to test in an acyl off-target pharmacological testing and DDI studies. Larger 897
842 migration assay, which showed that it did not undergo acyl amounts are less commonly needed for in vivo toxicity studies 898
843 migration under physiological conditions. To support future for drugs further along the developmental pathway. 899
844 bioanalytical-method development and validation needs for Where multiple metabolites of a drug are needed, and where 900
845 clinical studies, significantly larger quantities were required than these can be produced in a ‘one pot’ approach such as through 901
KEYNOTE (GREEN)
846 those that could be obtained from direct purification of preclin- microbial biotransformation or use of a recombinant enzyme 902
of
847 ical materials. AZD5991 was therefore screened against a panel of system, the reaction is tailored to the metabolite produced at 903
848 microbes, which revealed extensive metabolism via phase I and lowest yield. Hence, significantly greater quantities of the major 904
849 phase II pathways.(p72) As observed in preclinical studies, a metabolite (or metabolites) can be produced and purified at the 905
850 prominent glucuronide was formed by several microbes. The glu- same time. 906
o
851 curonide produced was demonstrated by LC-MS/MS and NMR to
852 match that observed from human hepatocyte incubations and
Adding value from metabolite synthesis 907
Pr
853 excreta in rat bile. Experiments pointed to earlier dosing result-
Meeting regulatory requirements is the prime goal for many of 908
854 ing in greater stability, with over 90% conversion of AZD5991
the projects undertaken. However, additional benefits can be 909
855 to the acyl glucuronide (Figure S5).
reaped by evaluating metabolites, both major and minor, in tar- 910
856 There is also evidence to suggest that on-target pharmacolog-
get and off-target assays. In rare but notable cases, a metabolite 911
857 ical studies of acyl glucuronides of drugs could be warranted.(p71)
might prove superior to the parent drug, or provide important 912
858 This is particularly relevant where acyl glucuronidation consti-
ed structure–activity relationship (SAR) insights and support intel- 913
859 tutes the primary clearance mechanism, or where the pharmaco-
lectual property protection around a chemical series.(p78) 914
860 logical target is in the extracellular matrix and does not require
861 penetration by the acyl glucuronide conjugate.
Added SAR: an in vivo active aldehyde oxidase metabolite 915
862 Glucuronides can also be responsible for clinically relevant
Although metabolite surprises during clinical trials are never wel- 916
863 DDIs, such as those attributed to the acyl glucuronides of clopi- come, an interesting example of key learnings arose from a 917
864 dogrel and gemfibrozil, which selectively inhibit CYP2C8.(p74),(-
ct
p75)
metabolite of PF-5190457, a spiro-azetidino-piperidine drug, dur- 918
865 Because humans readily oxidise acidic drugs, there is also a ing late phase I clinical trials (Figure S7).(p79) The metabolite cir- 919
866 potential complication arising from the presence of acyl glu- culates at 25% of the parent drug in humans, and both the 920
867 curonides of oxidised metabolites of the drug, which might later
parent and metabolite significantly inhibit food uptake in rats 921
re
868 alter conjugate reactivity if oxidation occurs on a moiety nearby.

at similar doses. The largely AO-mediated biotransformation 922
869 Issues can also arise as a result of b-glucuronidase-mediated
revealed some intriguing differences about the metabolite com- 923
870 hydrolysis of O-glucuronides to the parent drug, the propensity
pared with the parent drug, and unearthed some previously 924
871 for which differs owing to marked species differences in expres- unknown SARs. Although the metabolite has lower binding 925
872 sion of b-glucuronidases.(p76)
or
affinity and potency for inhibiting GHSR1a-induced inositol 926

873 In addition to ‘simple’ hydroxylations and glucuronidations, phosphate accumulation versus The parent, it has increased 927
874 metabolites arising from multiple or more complex biotransfor- GHSR1a-induced b-arrestin recruitment potency. The authors 928
875 mations are often seen. These have generally already proven
demonstrated that although the metabolite had 25-fold lower 929
876 more difficult to make, and the substrate set analysed will thus
nc
affinity binding to GHSR1a than the parent, it was four- to seven- 930
877 be biased towards such metabolites. Phase I metabolites involv-
fold more potent in recruitment of b-arrestin. Follow-up work 931
878 ing multiple enzymes provide the most challenge, such as the
characterising the pharmacology of the parent compound and 932
879 M2 metabolite of ganaxolone previously discussed. Other nota- the metabolite PF-6870961 revealed that PF-6870961 had no 933
880 ble phase I biotransformations observed involve oxidation of off-target interactions in a full screen of binding and enzymatic 934
881 N-linked pyrrolidine ring moieties followed by ring openings or
U
targets. The studies revealed some new insights into SARs that 935
882 rearrangements. The example shown in Figure S6 was an out- might affect the future design of such GHSR1a inverse agonists. 936
883 come of a collaboration between Hypha Discovery and the
The metabolite is postulated to form hydrogen bonds in an 937
884 University of Dundee Drug Discovery Unit (DDU) on a series of
amino acid region that confers biased inverse agonism of b- 938
885 lead compounds discovered for the treatment of neglected trop-
arrestin recruitment, possibly through disrupted flexibility in 939
886 ical diseases; the drug molecule was originally identified during a
these regions. 940
887 previous collaboration between DDU and GlaxoSmithKline.(p77) The widening of IP coverage and patenting of active metabo- 941
888 Some oxidised metabolites prove to be susceptible to degradation lites is a desirable outcome from studies of metabolites, as hap- 942
889 on handling and pose significant purification challenges, such as pened with CD-14547, a novel mammalian target of rapamycin 943
890 hemiaminal metabolites that present purification issues to vary- (mTOR) inhibitor. The CD-14547 metabolites, which were origi- 944
891 ing degrees, depending on reactivity.
nally observed in plasma samples from an in vivo rat pharmacoki- 945
KEYNOTE (GREEN)
o of
Pr
ed
ct
FIGURE 8
The range of quantities made for 228 metabolites and their use.
re
946 netics study, were included in the CD-14547 patent as poten- development programmes. But when synthesis is performed 969
947 tially useful treatments for skin conditions such as acne, atopic in late lead-optimisation, beneficial properties associated with 970
948 dermatitis, actinic keratosis and psoriasis.(p80) To identify the polar modifications akin to oxidised metabolites can be cap- 971
or
949 metabolites, CD-14547 was screened against a panel of 20 Poly- tured and incorporated into the eventual development candi- 972
950 CYPs enzymes. Seven isoforms biotransformed the parent drug date. For instance, improving aqueous solubility by 973
951 to a maximum conversion of 91.5%, with PolyCYP 359 provid- hydroxylation can boost the oral bioavailability of poorly sol- 974
952 ing the best conversion to three hydroxylated metabolites in sim- uble compounds, and there are numerous precedents of 975
ilar proportions to those seen in the rat plasma samples. The hydroxylated metabolites possessing improved metabolic sta-
nc
953 976
954 PolyCYP 359 enzyme reaction was scaled up to generate material bility, target selectivity and sometimes potency profiles when 977
955 for structure elucidation and in vitro pharmacology assays. NMR compared with the parent drug.(p81),(p82) A common medicinal 978
956 spectroscopy of M1 revealed a hydroxylation at the penultimate chemistry challenge in the optimisation of drug leads is to 979
957 carbon in the alkyl side chain that generated a tertiary alcohol maintain control of lipophilicity while increasing potency, 980
group, with M2 and M3 being diastereoisomers formed by with ligand lipophilicity efficiency (LLE, or lipE, a function
U
958 981
959 hydroxylation of the non-equivalent terminal methyl groups of target IC50 and LogP) used as the metric to measure ‘drug- 982
960 (Figure 9). In the pharmacological assay, M1, M2 and M3 all likeness’. Although a hydroxylated metabolite with equivalent 983
961 inhibited AKT phosphorylation in human skin A-431 cancer cells potency to the parent drug will in itself improve LLE, more 984
962 with IC50 values of 13 nM, 7 nM and 3.7 nM, respectively, com- significant gains in LLE will result from the detection of a 985
963 pared with an IC50 of 2 nM for the parent drug. A further struc- more potent hydroxylated metabolite. This means that, in con- 986
964 turally uncharacterised metabolite (CD-14547-H2) was also trast to the synthesis of metabolites to support drug develop- 987
965 found to be active, with an estimated IC50 value of 3.6 nM. ment programmes, producing and testing metabolites at the 988
lead optimisation stage can provide information that is often 989
complementary and orthogonal to conventional medicinal 990
966 Metabolite synthesis to support lead optimisation chemistry drug design strategies, by empirically probing for 991
967 The majority of metabolite synthesis projects completed by hitherto unknown polar interactions with a target protein. 992
968 Hypha Discovery have been to support small-molecule drug

KEYNOTE (GREEN)
of
FIGURE 9
Novel mTOR inhibitor metabolites of CD14547. An enzyme reaction catalysed by PolyCYP 359 was scaled up using 82.5 mg of parent compound in 272 ml of
enzyme incubation to generate material for structure elucidation and in vitro pharmacology assays, yielding 25.8 mg of M1, 3.5 mg of M2 and 2.1 mg of M3,
at a total isolatable yield of 38%.(p80)
o
993 Concluding thoughts as proteolysis targeting chimeras (PROTACs), molecular glues 1032
Pr
994 Although the majority of the metabolite projects we have and covalent binding. It will be interesting to see the net result 1033
995 worked on were related to drug compounds in phase II and III of the interplay between this growing structural complexity 1034
996 clinical trials, it is clear from our interactions with many of our and the drive to reduce metabolic clearance in the design phase 1035
997 clients that metabolites pertinent to regulatory guidelines are on the biotransformation mechanisms of these molecules. This 1036
998 being targeted and identified earlier in development. We antici- will affect the type and number of metabolites that might need 1037
999 pate that this trend will increase with the greater focus on to be definitively identified and assessed during clinical develop- 1038
1000
1001
1002
human studies earlier in clinical development, particularly if
the human first/human only approach is embraced across the
industry. As expected, the vast majority of metabolites we are
ed ment and the challenges around accessing metabolites for these
studies.
1039
1040
1003 commissioned to make are deemed to be major according to Uncited reference 1041
(p73)
1004 the regulatory guidance, and among these, many are dispropor- . 1042
ct
1005 tionate human metabolites. However, there is also interest in
1006 the interrogation of metabolites for pharmacological activity Competing interest statement 1043
1007 and beneficial DMPK properties. This does not just apply to those All listed authors were employed by Hypha Discovery at the time 1044
1008 that would be classified as major metabolites from a regulatory any client project work described in the paper was conducted. 1045
re
1009 standpoint. Companies are interested in ensuring comprehen-

1010 sive IP coverage as well as not discounting structures that could
CRediT authorship contribution statement 1046
1011 deliver an improvement on the parent drug. Timing is key!
Julia Shanu-Wilson: Writing – original draft, Formal analy- 1047
1012 Even though some metabolites formed by simple single-step
sis, Data curation. Samuel Coe: Data curation. Liam Evans: 1048
or
1013 biotransformations, such as hydroxylated or glucuronidated

Writing – original draft. Jonathan Steele: Writing – original 1049
1014 metabolites, can be challenging to produce by facile chemical
draft. Stephen Wrigley: Writing – original draft, Formal anal- 1050
1015 synthetic approaches, these can generally be made in a late-
ysis, Data curation. 1051
1016 stage manner using either biocatalytic methods or late-stage
nc
1017 chemical functionalisation. Although initial metabolite-

1018 identification (MetID) studies are generally useful in pinpointing
Data availability 1052
1019 structural changes to specific moieties, they are not always accu- The data that has been used is confidential. 1053
1020 rate at predicting structures. After a metabolite structure has been

1021 definitively identified by NMR, some quite different and unex- Acknowledgements 1054
U
1022 pected biotransformations become evident. Although not an The authors thank all Hypha Discovery scientists both past 1055
1023 everyday occurrence, be open to expecting the unexpected when and present for their contribution to work described in this 1056
1024 it comes to the metabolism of drugs! As a result of unanticipated paper. We also acknowledge clients who have kindly allowed 1057
1025 structural changes, more complex multi-step biotransformations selected case studies to be featured in this paper. 1058
1026 can provide a challenge, and often these require more effort to We are grateful to Scott Obach for his review of the manu- 1059
1027 make and scale up once metabolite structures have been accu- script and helpful comments. 1060
1028 rately determined.

1029 There is a growing prevalence of more structurally complex Appendix A. Supplementary data 1061
1030 drug candidates: for example, beyond-rule-of-five molecules Supplementary data to this article can be found online at 1062
1031 and those that act through non-traditional mechanisms, such https://doi.org/10.1016/j.drudis.2024.103943. 1063
1064 References
1065 1. Weston DJ et al. A discovery biotransformation strategy: combining in silico 25. Young GC et al. Considerations for human ADME strategy and design paradigm 1133
1066 tools with high-resolution mass spectrometry and software-assisted data analysis shift(s) – an industry white paper. Clin Pharmacol Ther. 2023;113:775–781. 1134
1067 for high-throughput metabolism. Xenobiotica. 2022;52:928–942. 26. European Parliament. European Parliament resolution of 16 September 2021 on 1135
1068 2. Shanu-Wilson J et al. Biotransformation: impact and application of metabolism plans and actions to accelerate the transition to innovation without the use of animals 1136
1069 in drug discovery. ACS Med Chem Lett. 2020;11:2087–2107. in research, regulatory testing and education (2021/2784(RSP)). European 1137
1070 3. Jones C et al. Biocatalysis for lead discovery and optimization. Ref Module Chem Parliament; 2021 (published 16 September 2021; accessed 10 October 2023) 1138
1071 Mol Sci Chem Eng. 2022. https://doi.org/10.1016/B978-0-32-390644-9.00080-9. https://www.europarl.europa.eu/doceo/document/TA-9-2021-0387_EN.html. 1139
1072 4. Siirola E et al. Evolution of biocatalysis at Novartis over the last 40 Years. Chimia. 27. Jensen KG et al. Lack of exposure in a FIM study due to aldehyde oxidase. Drug 1140
1073 1141
KEYNOTE (GREEN)
2023;77:376–383. Metab Dispos. 2017;45:68–75.
1074 5. Cerny MA et al. The effective application of metabolite profiling in drug design 28. Zhang C et al. Diglucuronidation of GDC-0810 in humans. Drug Metab Dispos. 1142
of
1075 and discovery. J Med Chem. 2020;63:6387–6406. 2023;51:1284–1294. 1143
1076 6. Schadt S et al. A decade in the MIST. Drug Metab Dispos. 2018;46:865–878. 29. Evans L. Chapter 4 – Methods for metabolite generation and characterization by 1144
1077 7. US Department of Health and Human Services et al. Safety testing of drug NMR. Elsevier; 2020. 119–150. 1145
1078 metabolites, guidance for industry. US Food & Drug Administration; 2020 30. Gillam EMJ, Kramlinger VM. Opportunities for accelerating drug discovery and 1146
1079 (published March 2020; accessed 10 October 2023) https://www. development by using engineered drug-metabolizing enzymes. Drug Metab 1147
o
1080 fda.gov/media/72279/download. Dispos. 2023;51:392–402. 1148
1081 8. Luffer-Atlas D et al. A MIST conception: what has been learned from twenty 31. Wohlgemuth R. Synthesis of metabolites and metabolite-like compounds using 1149
1082 years of human metabolite safety assessment? Med Chem Res. biocatalytic systems. Metabolites. 2023;13:1097. 1150
Pr
1083 2023;32:1933–1949. 32. Cusack KP et al. Emerging technologies for metabolite generation and structural 1151
1084 9. Fitch WL et al. Complex metabolism of the novel neurosteroid, ganaxolone, in diversification. Bioorg Med Chem Lett. 2013;23:5471–5483. 1152
1085 humans. A unique challenge for MIST assessment. Drug Metab Dispos. 33. Börgel J, Ritter T. Late-stage functionalization. Chem. 2020;6:1877–1887. 1153
1086 2023;51:753–763. 34. Castellino NJ et al. Late-stage functionalization for improving drug-like 1154
1087 10. Ma Y et al. Glucuronides as potential anionic substrates of human cytochrome molecular properties. Chem Rev. 2023;123:8127–8153. 1155
1088 P450 2C8 (CYP2C8). Miniperspective J Med Chem. 2017;60:8691–8705. 35. Chambers RK et al. A preparative small-molecule mimic of liver CYP450 1156
1089 11. Katsube Y et al. Candesartan glucuronide serves as a CYP2C8 inhibitor. Drug enzymes in the aliphatic C-H oxidation of carbocyclic N-heterocycles. Proc Natl 1157
1090 Metab Dispos. 2021;49:289–297. ed Acad Sci USA. 2023;120. 1158
1091 12. Shah MB. Inhibition of CYP2C8 by acyl glucuronides of gemfibrozil and 36. Stachulski AV, Meng X. Glucuronides from metabolites to medicines: a survey of 1159
1092 clopidogrel: pharmacological significance, progress and challenges. Biomolecules. the in vivo generation, chemical synthesis and properties of glucuronides. Nat 1160
1093 2022;12:1218. Prod Rep. 2013;30:806–848. 1161
1094 13. Zou L et al. Drug metabolites potently inhibit renal organic anion transporters, 37. Kukushkina A et al. Impact of a fluorine on the CNS penetration and metabolic fate of 1162
1095 OAT1 and OAT3. J Pharm Sci. 2021;110:347–353. AZD9574 [Poster]. UK: European ISSX DMDG conference; Hertfordshire; 2023 1163
1096 14. A comparison of FDA, EMA & PMDA regulatory guidance for in vitro drug-drug (Abstract available at: https://cdn.ymaws.com/www.issx.org/resource/resmgr/ 1164
ct
1097 interaction (DDI) assessments. Labcorp https://ddblog.labcorp.com/2020/06/a- FinalABSTRACTBOOK6.15-ISSXDM.pdf). 1165
1098 comparison-of-fda-ema-pmda-regulatory-agency-guidance-for-in-vitro-drug- 38. Ghisalba O, Kittelmann M. Preparation of drug metabolites using fungal and 1166
1099 drug-interaction-ddi-assessments/ (published 7 July 2021; accessed 10 October bacterial strains. In: Schmid RD, Urlacher VB, eds. Modern Biooxidation. Wiley; 1167
1100 2023). 2007:211–232. 1168
1101 15. FDA. In vitro drug interaction studies — cytochrome P450 enzyme- and transporter- 39. Salter R et al. Microbial biotransformation – an important tool for the study of 1169
re
1102 mediated drug interactions guidance for industry. US Food & Drug Administration; drug metabolism. Xenobiotica. 2018;49:877–886. 1170
1103 2020 (published January 2020; accessed 10 October 2023) https://www.fda.gov/ 40. Ma J et al. In vitro cytochrome P450- and transporter-mediated drug interaction 1171
1104 regulatory-information/search-fda-guidance-documents/in-vitro-drug- potential of 6b-hydroxy-21-desacetyl deflazacort – a major human metabolite of 1172
1105 interaction-studies-cytochrome-p450-enzyme-and-transporter-mediated-drug- deflazacort. Pharmacol Res Perspect. 2021;9:e00748. 1173
1106 interactions. 41. Li H et al. The influence of gut microbiota on drug metabolism and toxicity. 1174
or
1107 16. ICH. Guideline M12 on drug interaction studies: Step 2b. European Medicines Expert Opin Drug Metab Toxicol. 2016;12:31–40. 1175
1108 Agency; 2022 (published July 2022; accessed 5 February 2024) https://www.ema. 42. Noh K et al. Impact of gut microbiota on drug metabolism: an update for safe 1176
1109 europa.eu/en/documents/scientific-guideline/draft-ich-guideline-m12-drug- and effective use of drugs. Arch Pharm Res. 2017;40:1345–1355. 1177
1110 interaction-studies-step-2b_en.pdf. 43. Wilson ID, Nicholson JK. Gut microbiome interactions with drug metabolism, 1178
1111 17. Steinbronn C et al. Do inhibitory metabolites impact DDI risk assessment? efficacy, and toxicity. Transl Res. 2017;179:204–222. 1179
1112 Analysis of in vitro and in vivo data from NDA reviews between 2013 and 2018. 44. Surapaneni S et al. Absorption, metabolism, and excretion, in vitro 1180
nc
1113 Clin Pharmacol Ther. 2021;110:452–463. pharmacology, and clinical pharmacokinetics of ozanimod, a novel 1181
1114 18. Coppola P et al. The importance of the human mass balance study in regulatory sphingosine 1-phosphate receptor modulator. Drug Metab Dispos. 1182
1115 submissions. CPT Pharmacomet Syst Pharmacol. 2019;8:792–804. 2021;49:405–419. 1183
1116 19. Kalgutkar AS. Designing around structural alerts in drug discovery. J Med Chem. 45. Yeola S et al. The metabolic fate of izencitinib, a gut-selective pan-JAK inhibitor, 1184
1117 2020;63:6276–6302. in humans. Identification of unusual fecal metabolites and implications for 1185
1118 20. Stepan AF et al. Structural alert/reactive metabolite concept as applied in MIST evaluation. Med Chem Res. 2023;32:2071–2088. 1186
U
1119 medicinal chemistry to mitigate the risk of idiosyncratic drug toxicity: a 46. Jurva U et al. Thermostable ancestral P450s as biocatalysts for pharma. Drug 1187
1120 perspective based on the critical examination of trends in the top 200 drugs Metab Dispos. 2024;52:242–251. 1188
1121 marketed in the United States. Chem Res Toxicol. 2011;24:1345–1410. 47. Steele, J.C.P. et al. (2022) Hydroxylation of branched aliphatic or aromatic 1189
1122 21. Shanu-Wilson J. Billion-dollar biotransformations: on the metabolism of substrates employing the Amycolatopsis lurida cytochrome P450, WO2018/ 1190
1123 ozanimod. Drug Hunter; 2022 (published 5 December 2022; accessed 10 091885, US Patent 11,384,371. 1191
1124 October 2023) https://drughunter.com/articles/billion-dollar- 48. Steele, J.C.P. et al. (2019) Hydroxylation techniques, WO2019/220093, US 1192
1125 biotransformations-on-the-metabolism-of-ozanimod/. Patent 11,618,906. 1193
1126 22. Beaumont C et al. Human ADME properties of drug molecules: a plethora of 49. Steele, J.C.P. et al. (2019) Biocatalytic techniques, PCT/GB2019/053337, US 1194
1127 approaches. Br J Clin Pharmacol. 2014;78:1185–1200. Patent 11,891,642. 1195
1128 23. Spracklin DK et al. Mini-review: comprehensive drug disposition knowledge 50. Auclair, A.A. et al. (2023) P–4 - Biosynthesis of BI 894416 metabolite M398(2) 1196
1129 generated in the modern human radiolabeled ADME study. CPT Pharmacometrics using microbial enzymes. 25th North American ISSX meeting; Boston, 1197
1130 Syst Pharmacol. 2020;9:428–434. Massachusetts; September 2023. Abstract available at: https://cdn.ymaws.com/ 1198
1131 24. Wang S et al. The importance of tracking “missing” metabolites: how and why? J www.issx.org/resource/resmgr/meetings_-_general/25th_na_issx/abstracts/ 1199
1132 Med Chem. 2023;66:15586–15612. 25NAISSX_AbstractBook.pdf 1200
1201 51. Xin D et al. Development of a 13C NMR chemical shift prediction procedure 67. Kaivosaari S et al. N-glucuronidation of drugs and other xenobiotics by human 1253
1202 using B3LYP/cc-pVDZ and empirically derived systematic error correction terms: and animal UDP-glucuronosyltransferases. Xenobiotica. 2011;41:652–669. 1254
1203 a computational small molecule structure elucidation method. J Org Chem. 68. Kutsuno Y et al. Glucuronidation of drugs in humanized UDP- 1255
1204 2017;82:5135–5145. glucuronosyltransferase 1 mice: similarity with glucuronidation in human 1256
1205 52. Marshall LJ et al. Phase-in to phase-out-targeted, inclusive strategies are needed liver microsomes. Pharmacol Res Perspect. 2013;1:e00002. 1257
1206 to enable full replacement of animal use in the European Union. Animals. 69. Andrews, M. (2023) Discovery of an oral, rule-of-5 compliant, IL-17A protein- 1258
1207 2022;12:863. protein interaction modulator (PPIm) for the treatment of psoriasis and other 1259
1208 53. Iversen DB et al. Drug metabolism and drug transport of the 100 most prescribed inflammatory diseases. 3rd RSC Anglo-Nordic Medicinal Chemistry Symposium; 1260
1209 oral drugs. Basic Clin Pharmacol Toxicol. 2022;131:311–324. Snekkersten, Denmark; 13–16 June 2023 1261
1210 54. Shanu-Wilson J. Metabolism of 2022 FDA approved small molecule drugs. Hypha 70. Walles M et al. New perspectives on drug-induced liver injury risk assessment of 1262
1211 Discovery. 2023 (published January 2023; Accessed 10 October 2023) https:// acyl glucuronides. Chem Res Toxicol. 2020;33:1551–1560. 1263
1212 www.hyphadiscovery.com/blog/metabolism-of-2022-fda-approved-small- 71. Tron AE et al. Discovery of Mcl-1-specific inhibitor AZD5991 and preclinical 1264
KEYNOTE (GREEN)
1213 molecule-drugs/. activity in multiple myeloma and acute myeloid leukemia. Nat Commun. 1265
1214 55. Maréchal JD et al. Insights into drug metabolism by cytochromes P450 from 2018;9:5341. 1266
of
1215 modelling studies of CYP2D6-drug interactions. Br J Pharmacol. 2008;153: 72. Evans, L. et al. (2023) Structural identification and synthesis of the acyl 1267
1216 S82–S89. glucuronide of the macrolide Mcl-1 inhibitor AZD5991 [Poster]. Drug Discovery 1268
1217 56. Zanger UM, Schwab M. Cytochrome P450 enzymes in drug metabolism: Chemistry 2023; San Diego, USA; April 2023. Available at: https://www. 1269
1218 regulation of gene expression, enzyme activities, and impact of genetic hyphadiscovery.com/poster/structural-identification-and-synthesis-of-the-acyl- 1270
1219 variation. Pharmacol Therapeut. 2013;138:103–141. glucuronide-of-the-macrolide-mcl-1-inhibitor-azd5991/ 1271
o
1220 57. Saravanakumar A et al. Physicochemical properties, biotransformation, and 73. Ryder TF et al. Acyl glucuronide metabolites of 6-Chloro-5-[4-(1- 1272
1221 transport pathways of established and newly approved medications: a hydroxycyclobutyl)phenyl]-1 H-indole-3-carboxylic Acid (PF-06409577) and 1273
1222 systematic review of the top 200 most prescribed drugs vs. the FDA-approved related indole-3-carboxylic acid derivatives are direct activators of adenosine 1274
Pr
1223 drugs between 2005 and 2016. Clin Pharmacokinet. 2019;58:1281–1294. monophosphate-activated protein kinase (AMPK). J Med Chem. 1275
1224 58. Bhutani P et al. U.S. FDA approved drugs from 2015–June 2020: a perspective. J 2018;61:7273–7288. 1276
1225 Med Chem. 2021;64:2339–2381. 74. Tornio A et al. Glucuronidation converts clopidogrel to a strong time-dependent 1277
1226 59. Ong V et al. Metabolism, excretion, and mass balance of [14C]-rezafungin in inhibitor of CYP2C8: a phase II metabolite as a perpetrator of drug-drug 1278
1227 animals and humans. Antimicrob Agents Chemother. 2022;66:e0139021. interactions. Clin Pharmacol Ther. 2014;96:498–507. 1279
1228 60. Manohar, R. et al. (2023) P1–1 - Synthesis and characterization of the human 75. Ogilvie BW et al. Glucuronidation converts gemfibrozil to a potent, metabolism- 1280
1229 metabolites of the echinocandin drug rezafungin by microbial dependent inhibitor of CYP2C8: implications for drug-drug interactions. Drug 1281
1230 biotransformation. 25th North American ISSX Meeting; Boston, Massachusetts;
ed Metab Dispos. 2006;34:191–197. 1282
1231 September 2023. Abstract available at: https://cdn.ymaws.com/www.issx.org/ 76. Smith DA et al. Safety assessment of acyl glucuronides – a simplified paradigm. 1283
1232 resource/resmgr/meetings_-_general/25th_na_issx/abstracts/25NAISSX_ Drug Metab Dispos. 2018;46:908–912. 1284
1233 AbstractBook.pdf 77. Bailey, C. et al. Rapid generation and testing of analogues of lead compounds 1285
1234 61. Dalvie DK et al. Biotransformation reactions of five-membered aromatic from neglected tropical disease drug development programmes using 1286
1235 heterocyclic rings. Chem Res Toxicol. 2002;15:269–299. commercially available P450 enzymes. Manuscript in preparation. 1287
1236 62. Huskey SE et al. N-glucuronidation reactions. I. Tetrazole N-glucuronidation of 78. Obach RS. Pharmacologically active drug metabolites: impact on drug discovery 1288
ct
1237 selected angiotensin II receptor antagonists in hepatic microsomes from rats, and pharmacotherapy. Pharmacol Rev. 2013;65:578–640. 1289
1238 dogs, monkeys, and humans. Drug Metab Dispos. 1993;21:792–799. 79. Deschaine SL et al. Initial pharmacological characterization of a major hydroxy 1290
1239 63. Huskey SE et al. N-glucuronidation reactions. II. Relative N-glucuronidation metabolite of PF-5190457: inverse agonist activity of PF-6870961 at the ghrelin 1291
1240 reactivity of methylbiphenyl tetrazole, methylbiphenyl triazole, and receptor. J Pharmacol Exp Ther. 2023;386:117–128. 1292
1241 methylbiphenyl imidazole in rat, monkey, and human hepatic microsomes. 80. Harris, C.S. et al. (2023) Galderma Holding. Novel MTOR inhibitor compounds, 1293
re
1242 Drug Metab Dispos. 1994;22:651–658. WO2023031738A1. 1294

1243 64. Huskey SE et al. N-glucuronidation reactions. III. Regioselectivity of N- 81. Cerny MA et al. Effective application of metabolite profiling in drug design and 1295
1244 glucuronidation of methylbiphenyl tetrazole, methylbiphenyl triazole, and discovery. J Med Chem. 2020;63:6387–6406. 1296
1245 methylbiphenyl imidazole using human and rat recombinant UDP- 82. Obach RS et al. Lead diversification at the nanomole scale using liver 1297
1246 glucuronosyltransferases stably expressed in V79 cells. Drug Metab Dispos. microsomes and quantitative nuclear magnetic resonance spectroscopy: 1298
or
1247 1994;22:659–662. application to phosphodiesterase 2 inhibitors. J Med Chem. 2018;61:3626–3640. 1299

1248 65. Sica DA et al. Clinical pharmacokinetics of losartan. Clin Pharmacokinet. 83. Schadt S et al. Chapter 14 – Metabolites in safety testing (MIST). In: Ma S, 1300
1249 2005;44:797–814. Chowdhury SK, eds. Identification and Quantification of Drugs, Metabolites, Drug 1301
1250 66. Manevski N et al. Metabolism by aldehyde oxidase: drug design and Metabolizing Enzymes, and Transporters. Elsevier; 2020:419–438. 1302
1251 complementary approaches to challenges in drug discovery. J Med Chem. 1303
1252
nc
2019;62:10955–10994.
U

Art S8

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Art S8

Uploaded by

Copyright:

Available Formats

DRUDIS 103943 No.

of Pages 16, Model NS

Drug Discovery Today d Volume xxx, Number xx d xxxx 2024 REVIEWS

19 contract research organisation, with particular atten-

industrial microbial biotechnology for nearly 40

⇑ Corresponding author. Shanu-Wilson, J. (julia.shanuwilson@hyphadiscovery.com)

1359-6446/Crown Copyright Ó 2024 Published by Elsevier Ltd.

was identiﬁed as 16a-hydroxy-ganaxolone (M1), mainly through 129

77 regulatory direction has been issued by the FDA, European

Drug Discovery Today

Drug Discovery Today

How? Typical routes to metabolite synthesis used at 386

346 ment. The overall recommendation is that information on DDI

426 their synthesis and puriﬁcation.

Drug Discovery Today

light on whether a metabolite is formed by gut microbiota or in 557

515 tively rare that these metabolites are prominent enough to

605 resulted in the highest conversion of 40% by PolyCYP 152 to

Drug Discovery Today

868 alter conjugate reactivity if oxidation occurs on a moiety nearby.

afﬁnity and potency for inhibiting GHSR1a-induced inositol 926

Drug Discovery Today

1009 standpoint. Companies are interested in ensuring comprehen-

1013 biotransformations, such as hydroxylated or glucuronidated

1017 chemical functionalisation. Although initial metabolite-

1020 rate at predicting structures. After a metabolite structure has been

1028 rately determined.

1242 Drug Metab Dispos. 1994;22:651–658. WO2023031738A1. 1294

1247 1994;22:659–662. application to phosphodiesterase 2 inhibitors. J Med Chem. 2018;61:3626–3640. 1299

You might also like