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ADME Profiling

The impact of early ADME profiling on drug discovery and development strategy

The increased costs in the discovery and development of new drugs, due in part to the high attrition rate of drug candidates in development, has led to a new strategy to introduce early, parallel evaluation of and biopharmaceutical properties of drug candidates. Investigation of terminated projects revealed that the primary cause for drug failure in the development phase was the poor pharmacokinetic and ADMET (Absorption, Distribution, , Discretion and ) properties rather than unsatisfactory efficacy. In addition, the applications of parallel synthesis and combinatory chemistry to expedite lead finding and lead optimisation processes has shifted the chemical libraries towards poorer biopharmaceutical properties. Establishments of high throughput and fast ADMET profiling assays allow for the prioritisation of leads or drug candidates by their biopharmaceutical properties in parallel with optimisation of their efficacy at early discovery phases.This is expected to not only improve the overall quality of drug candidates and therefore the probability of their success, but also shorten the drug discovery and development process. In this article, we review the early ADME profiling approach, their timing in relation to the entire drug discovery and development process and the latest technologies of the selected assays will be reviewed.

iscovery and development of a new drug is a lead finding/selection process, which in general takes By Dr Jianling Wang long, labour-demanding process. Recent a minimum of two more years. Typically, the whole and Dr Laszlo Urban Dstudies1 revealed that the average time to process is fragmented into ‘Discovery’, ‘Development’ discover, develop and approve a new drug in the and ‘Registration’ phases. United States has steadily increased from 8.1 years in The ‘Discovery’ phase, routinely three to four the 1960s to 14.2 years by the 1990s. The actual time years, involves identification of new therapeutic tar- may be even longer as the above calculation consid- gets, lead finding and prioritisation, lead optimisation ered the starting point from chemical synthesis, there- and nomination of new chemical entities (NCEs). In by not including the time for target identification and the ‘Development’ phase, the drug candidates are

Drug Discovery World Fall 2004 73 ADME Profiling

Figure 1 Attrition rate of drug candidates, as denoted by the 10000.0 number of compounds that Discovery would be required to generate 1500.0 a new drug at the different Phase I phases of discovery and 1000.0 development8,15 Phase II

Phase III 100.0 Registration

9.1 Launch 10.0 5.5

1.8 1.3 1.0 1.0 TTRITION RATE (# of compounds) TTRITION RATE A

0.1 Discovery Phase I Phase II Phase III Registration Launch

PHASE OF DISCOVERY AND DEVELOPMENT

subject to preclinical testing in animals (also known have the urgency to improve the efficiency and as ‘Phase I’) for about two years. In addition, the pre- effectiveness of the drug discovery and develop- liminary clinical trials in man and the subsequent full ment strategy in order to appeal to the investors clinical trials (known as Phase II and III) will take a and to secure continuing growth. In addition to the much longer time (~8.6 years)1. According to increasingly tight regulatory hurdles, big pharma- DiMasi, the ‘Registration’ phase averaged around 1.8 ceutical firms also suffer from their failing discov- years in the late 1990s. Each pharmaceutical firm has ery of NCEs. Commonly, it is attributed to: their own collection of drug candidates which are being allocated to different phases of the discovery ● The selection of improper targets in early phas- and development process, often referred to as the es that lack proof of concept in man. ‘pipeline’. Discovery scientists primarily focus on ● High attrition rate during development phases ‘hunting’ for drug candidates, whereas in due to poor . Development one is trying to ‘promote’ NCEs as ● Poor toxicological and safety-related pharmaco- novel and safer commercial . logical properties. Discovery and development of a new drug are ● Elongated discovery and development time course. also extremely costly. Despite the drastic upsurge in R&D expenditures (by a factor of 20-50), the Attempts to address these challenges embrace: output of pharmaceuticals (number of new drugs launched per year) remains virtually flat from 1963 ● Pursuit of physiologically viable targets using to 19992-3. Concurrently, the productivity of the genomics and proteomics strategies. , as measured by NCE out- ● Initiation of tech- put per dollar, declined continuously during the niques which escalate production of NCEs enter- past decades4. As a result, the average cost to dis- ing the pipeline. cover and develop a new drug, taking into account the drug candidates dropped along the way, soared However, the genomics approach was counter- to $800-$900 million in 20032-3,5, in comparison acted by a large number of novel but risky targets to that of $138 million in 1979 and $318 million which attributed to a 50% rise in the failure rate in 19912. A separate analysis estimated the cost at over the clinically validated approaches8. The $1.3-1.6 billion in 20056. Despite the recent strong downside of combinatorial chemistry is to shift the performance in earnings7, major pharma firms still discovery compound libraries towards large,

74 Drug Discovery World Fall 2004 ADME Profiling

‘greasy’ and biologically inactive molecules which candidates in development due to ADME can rarely survive in the development phase. (PK/, formulation), and Indeed, Lipinski’s recent drugability analysis of (safety) remains near 50%. NCE collections from and Merck led to the findings that new NCEs tend to have higher molec- Where did the traditional drug ular weight and higher LogP and in turn, poorer discovery and development process go solubility and permeability9. Therefore, eliminat- wrong? ing compounds with the worst ADMET properties In principle, most of the above issues might have as early as possible has become an attractive been foreseen in early discovery by using ADME, approach10-14. Alternatively, expert advice con- toxicological and pharmacological profiling tools. cerning ADME properties could guide chemists to However, it takes time and effort to reform the structure-activity relationship (SAR) based modifi- mindset of scientists and managers in drug discov- cations to optimise for ‘drug like properties’ (eg, ery and development. Traditionally, optimisation of good absorption, high bioavailability with meta- efficacy was strongly associated with discovery and bolic stability, required distribution). drugability with development, applied sequentially in their own ‘kingdoms’. Specifically, the majority What is behind the high attrition rate? of the project teams in discovery concentrated on Even when a candidate reaches the development the improvement of efficacy during lead ‘pipeline’, this will not guarantee the launch of a selection and optimisation. In the end, the champi- commercial drug. This milestone indicates nothing on NCEs, always those with most potent inhibition else but a probability for success, as many develop- or binding properties to the in vitro target in test ment candidates will not pass through the preclinical tubes/microplates, were ‘pushed over the wall’ to and clinical testing. Companies reserving a large the development phase, as if the world leading uni- number of candidates with a higher probability for versities picked their best candidates simply by the success in development phases are viewed as possess- criteria of nothing else but the academic record of ing ‘strong pipelines’. Attrition rate analyses of NCEs the applicants. From the discovery point of view, in the development phases of 10 big pharma compa- the goal was achieved with the identification of nies in the US and Europe over 1991-2000 led to active candidates relative to the therapeutic target. interesting findings8. Albeit distinct among therapeu- However, from the development point of view, tic areas, the average success rate for drug candidates excellent in vitro efficacy would not always translate entering development Phase I will only be around into in vivo . First, full in vitro characterisa- 11% (Figure 1). In other words, the vast majority of tions of the drug candidate including pre-formula- the compounds that are being worked on in the tion assessment, biopharmaceutics, toxicology and development phases will end up in the ‘waste bin’, safety pharmacology give a fair prognostic picture with no return on the expenditure for the investment. for in vivo performance. As a next step, preclinical As the development process advances, the potential experiments provide confirmation of pharmacody- for the surviving candidates to become a drug grad- namic (PD)/pharmacokinetic (PK) behaviour of the ually escalates, in proportion to the growing expen- compound in selected animal species. To complete diture for development per target. A couple of com- the evaluation, pharmaceutical development has to parable studies led to similar conclusions14-15. assess the developability of the candidates as poten- Drug candidates might fail during development tial commercial drugs (eg, optimisation of drug because of numerous reasons. Kennedy analysed delivery by various formulations, etc). Although the causes by which 198 NCEs failed in clinical these processes are scheduled way before the more development15 and found that the most prominent challenging and costly clinical trials, many NCEs cause of the failures was associated with poor display signs of poor ‘drugability’. For example, pharmacokinetic (PK) and ADME properties some NCEs could neither dissolve in aqueous media (Figure 2). Although lack of efficacy was still one nor permeate across the gastrointestinal membrane of the main reasons for terminations, the unsatis- to reach the concentration at the required therapeu- factory PK/ADME, toxicology and adverse effects tic level, often referred to as ‘brick dust’ by develop- accounted for up to two-thirds of the total failures. ment colleagues. Fast (metabolically unstable) or A separate analysis8 also led to a similar conclu- ultra-slow (potential accumulation) metabolism, sion, particularly true for the early 1990s. toxic and adverse effects are not necessarily obvious However, Kolo & Landis’s report revealed that from animal experiments executed at single dose even with the latest improvement in most of the time. As a consequence, these matters PK/Bioavailability aspects, the total loss of drug are frequently misinterpreted during the assessment

Drug Discovery World Fall 2004 75 ADME Profiling

Figure 2 Analysis of the reasons for 5% All NCEs (n = 198) failure of 198 drug candidates in clinical development15 5%

30% 10%

Lack of efficacy

Poor PK/ADME

11% Animal toxicity

Adverse effects in man

Commercial reasons

Miscellaneous

39%

of the in vivo efficacy, which (without mechanistic always take place. Some notorious GPCR antago- data) imposes additional challenges during the nists were classic examples for these efforts. development process. Frequently, the clinical dose format may have to be No wonder, that the late discovery of poor drug- approved by marketing teams and/or patients. like properties and adverse side-effects is heart- Eventually, such projects may not survive after breaking for both development teams, often struggling for years and end up with a big expen- referred to as ‘teams with licences to kill’, and drug diture and disappointed scientists. discovery teams. Under such a condition, the ques- tion is whether to continue the development of the How to improve our drug discovery NCE and try to optimise it with great hardship, or and development process? just kill the compound and start from scratch. A new trend in the modern drug discovery and However, the latter decision, (to throw the NCEs development process is emerging to evaluate thera- back over the wall) is always hard to make, as this peutic and drug-like features of NCEs together as means potential delays to the product launch, loss early as possible. This requires that two major ele- in exclusive patent protection and deterioration of ments are in place: competitive position against other pharmaceutical firms that are working on the same therapeutic tar- 1) A new mentality to break up the wall between get. The matter is made worse by the discovery drug discovery and development. Only interdiscipli- teams which beg their development colleagues to nary and translational teams can drive the projects rescue their ‘babies’. Unfortunately, there is very effectively toward the same goal, the clinical launch little that development teams can do to salvage of the . It is imperative to migrate from NCEs with inadequate drug-like properties and sequentially assessing efficacy and drugability to a adverse side-effect profiles. For instance, develop- parallel process in the new drug discovery and ment of proper formulations may help address development strategy (Figure 3). Promotion of drug issues of poorly soluble compounds. candidates at each phase of the long discovery and However, such improvement can also be very cost- development process will rely not only on a particu- ly and time-consuming and miracles may not lar parameter but their overall performance and the

76 Drug Discovery World Fall 2004 ADME Profiling

potential to become a commercial drug, similar to and therefore are impractical for direct application References the admission of students to the top universities to in early discovery. Adaptation of these assays is not 1 DiMasi, JA. New drug foster the winners of the future requires all-around always straight-forward. New approaches and development in the United States from 1963 to 1999. young talent as the more favourable choice over stu- methods have been continuously invented to assist Clinical Pharmacology dents with solely an outstanding academic record. early drug discovery. For example, special sand- Therapeutics 69, 286-296 wich-type devices consisting of donor and receiver (2001). 2) The new strategy for parallel profiling of efficacy chambers partitioned by either artificial membrane 2 DiMasi, JA, Hansen, RW, and drugability of NCEs in early discovery raised a or cellular monolayer in micro-plate format are Grabowski, HG.The price of : new estimates of new, important question as most of the tools to used to simulate the membrane permeation course costs. appraise drugability and side-effects such as absorp- of drug candidates. Most of the pharmaceutical Journal of Health Economics tion, metabolism, distribution, , toxicity labs have dedicated resources and time to develop 22, 151–185 (2003). and pharmacology were only available in the devel- and integrate automated assay workstations as it is 3 Service, RF. Surviving the opment phase. These assays were usually costly, not always easy to acquire an off-the-shelf system Blockbuster Syndrome. Science 303, 1796-1799 (2004). labour-intensive, low throughput and required a to work for a particular customised profiling assay. 4 Booth, B, Zemmel, R. great deal of materials, thereby not feasible for early Furthermore, it is important to work out a viable Prospects for productivity. drug discovery phases. Recently, major efforts have strategy with development colleagues to assure that Nature Rev. 3, 451-457 (2004). been made to develop and implement high-through- the new profiling assays are developed and imple- 5 Rawlins, MD. Cutting the put (HT), miniaturised, fast profiling assays with mented in discovery labs to create ‘neither gap nor ? Nature Rev. 3, 360-364 (2004). good predictivity for in vivo drugability, at reason- redundancy’ with the existing assays in develop- 6 Hodgson, J.ADMET – turning able cost for usage during early drug discovery. ment labs. It is commonly a good practice to utilise chemicals into drugs. Nature These assays can assist directly the selection of early the expertise in discovery for fast screening of dru- Biotech. 19, 722-726 (2001). hits or leads and can be implemented for the opti- gability of NCEs and leave the small fraction of 7 Marx,V. Drug earnings rise, misation cycle of chemical synthesis. problematic compounds to the development teams. albeit unevenly. C & EN News 82, 15-16 (2004). Finally, the profiling assays in support of early 8 Kola, I, Landis, J. Can the What is feasible to profile in early drug discovery should have a good balance between pharmaceutical industry discovery? quality (predictivity and reproducibility), quantity reduce attrition rates? Nature First, one needs to be realistic and to implement (turn-around time, throughput) and cost (capital, Rev. 3, 711-715 (2004). profiling assays with the balance of two major cri- operating, manpower and compound materials)16. 9 Lipinski, CA. Drug-like properties and the causes of teria: overall importance of the matter addressed Within each organisation, the assay strategy is pri- poor solubility and poor by the assay (the impact) and availability of high marily defined by specific requirements and the permeability. J. Pharmacol. & quality assays (the quality and throughput). impact on the drug discovery process. For instance, To xicol. Met., 44, 235-249 (2000). Therefore assays are selected based on their pre- maintenance of a cell-based permeability assay, 10 Li,AP.Screening for human dictive value towards major reasons for failures in such as a full Caco-2 assay, for examination of ADME/Tox drug properties in drug discovery. Drug Discovery later development phases. Most of these assays transport mechanism during late discovery and Today, 6, 357-366 (2001). address instant hurdles in early discovery such as early development, is very costly. To offer a fast and 11 Kerns, EH, Li, D. solubility and metabolism with clinical conse- cost-effective screening tool for the assessment of Pharmaceutical profiling in quences. Table 1 outlines the most important permeation of candidates during the early drug dis- drug discovery. Drug Disc. assays recommended for implementation in early covery phase, permeability could be estimated using Today, 8, 316-323 (2003). 12 Roberts, SA. Drug drug discovery to tackle the above issues. artificial membranes, simple cell monolayer, such metabolism and Furthermore, the recognition that a single assay MDCK or fast-cultured Caco-2 models (eg 3-7 pharmacokinetics in drug usually has very limited prognostic values, lead to day). The throughput may be further enhanced and discovery. Current Opinion in the implementation of the ‘assay suite’ which the cost will be greatly reduced in conjunction with Drug Discovery & addresses various elements of the complex clinical a proper assay configuration (reducing number of Development 6, 66-80 (2003). 13 Biller, SA, Custer, L, performance of drugs, collectively (eg, solubility replicates and time/data points) and analytical plat- Dickinson, KE, Durham, SK, and cell permeability of NCEs affect hERG inhibi- form (reducing analytical time, pooling analytical Gavai,A, Hamann, LG, Josephs, tion). These grant project teams with greater power samples and parallel LC/UV or LC/MS)17. JL, Moulin, F,Pearl, GM, Flint, to solve the puzzles from divergent approaches. OP,Sanders, M,Tymiak,AA,Vaz, Establishment of such a large number of profil- Adequate ADME profiling assay suite R.The Challenge of quality in candidate optimization. in ing assays in early discovery is not trivial. First, for early drug discovery Pharmaceutical Profiling in most of the ADME assays listed above were pri- Although each major pharmaceutical firm has its Drug Discovery for Lead marily adopted from assays that have been own favourite approach to implement the profiling Selection (Borchardt, RT, Kerns, employed in development laboratories. Such assay suite, the underlying principle shares common- EH, Lipinski, CA,Thakker, DR, assays, albeit predictive, commonly tend to be ality. As projects advance from lead finding (LF) to Wang, B. Eds.),AAPS Press, pp.413-429 (2004). time-consuming, labour-intensive, not cost-effec- lead optimisation (LO) and eventually to the devel- tive, require a large amount of sample material, opment phase, the required throughput (number of Continued on page 79

Drug Discovery World Fall 2004 77 ADME Profiling

DISCOVERY DEVELOPMENT LAUNCH

Target Lead Lead Lead Phase I Phase II Phase III selection finding prioritisation optimisation

Figure 3 candidates to be considered) drastically declines and Classification System (BCS)18, as absorption of New strategy for drug the predictivity of the profiling assay (the content of passively transported drugs across the gastroin- discovery and development the information) proportionally escalates (Figure 4). testinal (GI) tract is the combined product of both where the optimisation of efficacy and drugability will be During phases prior to or during HTS stages (eg tar- permeability and solubility according to Fick’s first performed in parallel.The new get identification, etc.) the potential drug-like fea- law19. Data from in vitro assays such as HTS activ- strategy will require profiling tures are commonly addressed by tools such ity or membrane permeability assays could be mis- of NCEs occurring in early as ‘rule of five’ and polar surface area (PSA), etc. Due interpreted without considering solubility. Dosing discovery, allowing for only all- to their simplicity, high throughput and low cost, the poorly soluble compounds in pharmacological ani- around optimised candidates to be promoted to the in silico tools are particularly appropriate for the mal testing is also very risky as it commonly fails subsequent development design of novel chemical scaffolds or new combina- in deriving a correlation between dose and in vivo phase torial libraries. For parameters which are dependent efficacy. Under these circumstances, it is problem- primarily on chemical structure of NCEs such as ion- atic to differentiate the issues between solubility isation constant (pKa) or PSA, the prediction results and efficacy. On the other hand, solubility data are generally reasonable. The downside of the in sil- may help one to understand other PK or PD mech- ico approach, however, appears as the inauspicious anisms. For instance, highly soluble, low molecular predictivity for assays involving complicated process- weight compounds (MW <250) can be transported es or mechanisms (than just molecular configuration) via the paracellular route across the GI membrane. such as solubility and permeability. The challenge to reliably estimate solubility of By the end of HTS or lead prioritisation (LP), in drug substances should not be underestimated. In vitro profiling assays should be available to reality it is very difficult to precisely quantify or address drugability. One approach can segregate predict the aqueous solubility due to the compli- the in vitro suite into a ‘primary’ suite and a ‘sec- cated solubilisation process and solid phase ondary’ suite, as shown in Table 2. In vivo assays chemistry of drug candidates. A variety of may be addressed in a separate suite. The most approaches are employed (eg in silico, kinetic, inventive part of this process involves the continu- equilibrium solubility) which should be utilised in ous optimisation of the ‘spider graph’ (Figure 4) tiers to deal with the solubility and dissolution along with the enhancement of efficacy and intro- issues occurring at the different phases of drug duction of discrete, obligatory profiling filters at discovery and development. the designated phase transition milestones. During the cycles of chemical synthesis in LO phase, one – Thermodynamic or equilibrium solubility has should always avoid gaining the efficacy and activ- been considered as the ‘gold standard’ for solubil- ity at the expense of deteriorating drugability. ity determination. In the conventional approach known as the ‘saturation shake-flask’ method, Solubility and solubilisation solid compounds are agitated for a long period of Solubility is a critical factor as drug substances time (24-72 hours) to ensure that an equilibrium is have to be dissolved before they can be absorbed. achieved between an aqueous solution and the Solubility and rate of dissolution are the crucial solid material under specific conditions (buffer, players in the famous Biopharmaceutical pH, temperature, etc.). The saturated solutions are

78 Drug Discovery World Fall 2004 ADME Profiling

then separated from the residual solids using the species at a pH) solubility data are determined Continued from page 77 filtration or centrifugation techniques. The super- using Noyes-Whitney titration over the range of natant or clear saturated solutions are then quan- pH 1-12. In principle, when a test compound pre- 14 Penzotti, JE, Landrum, GA, Putta, S. Building predictive tified using high performance chromatographic cipitates from aqueous solution due to changes of ADMET models for early column (HPLC) or LC-MS (), in pH, the titration curve typically deviates marked- decisions in drug discovery. comparison to the standard curves created from ly from its respective curve obtained in aqueous Current Opinion in Drug solutions of the test compounds with known con- titration. Careful data analysis using sophisticated Discovery & Development 7, centration (typically in methanol or other organic software will lead to a solubility pH profile in the 49-60 (2004). 15 Kennedy T. Managing the solvents). The thermodynamic solubility is viewed equilibrium mode and the data were found to cor- drug discovery/development as being accurate and reliable over a reasonably relate well with the published data from the interface. Drug Discovery large solubility range (eg 1-50,000mg/L) provided ‘shake-flask’ method21. This approach provides a Tech. 2, 436-441 (1997). that enough material is available. In addition, it comprehensive profiling of equilibrium solubility, 16 Yu,H,Adedoyin,A.ADME- also reflects the packing information of the com- along with the intrinsic solubility, in a broad pH To x in drug discovery: integration of experimental pound in solid phase (polymorph) and thus is range. In addition, the data collection is in the and computational well-accepted by pharmaceutical industry and automated mode, which becomes very useful for technologies. Drug Discovery FDA. The drawbacks, however, are that the late discovery and development where a complete Today, 8, 852-861 (2003). method is quite time-consuming, labour-intensive characterisation is necessary. The downside of the 17 Kassel, DB.Applications of and sometimes requires a lot of sample material. assay is the low throughput (1-2 samples per day). high-throughput ADME in drug discovery. Current Opinion in Therefore, it is generally not feasible for high In addition, the quality of the data relies highly on Chemical Biology, 8, 339–345 throughput screening of aqueous solubility in the accuracy of the pKa data determined in aque- (2004). early drug discovery. ous solution, the experience of operators, and 18 Amidon, GL, Lennernäs, H, Comparable to the ‘shake-flask’ method, a ther- sometimes the nature of the test compounds (num- Shah,VP,Crison, JR.A modynamic or equilibrium solubility pH profile ber of pKa’s and solubility, etc). Thus, whereas it theoretical basis for a biopharmaceutic drug can be derived using the potentiometric titration is an ideal tool for solubility characterisation for classification: the correlation 20 approach . Specifically, the intrinsic (for neutral late discovery, it is not practical for HT solubility between in vitro drug product species only) and equilibrium (solubility of all screening in early discovery. dissolution and in vivo bioavailability. Pharm. Res. 12, 413-420 (1995). 19 Avdeef,A.Absorption and ASSAY SELECTED ISSUES TO BE ADDRESSED drug development – solubility, permeability and charge state. ADME Assays Wiley-Interscience (Hoboken, Solubility (equilibrium, kinetic solubility) Oral absorption New Jersey) (2003). Rate of dissolution Oral absorption 20 Avdeef,A. Physicochemical Membrane permeability (PAMPA, cell models, BBB) Oral absorption and BBB penetration Profiling. Curr.Topics Med. Active transport Oral absorption and drug-drug interaction Chem., 1, 277-351 (2001). Ionisation Constant (pKa) Oral absorption and binding mechanism 21 Avdeef,A, Berger, CM, Lipophilicity (LogP,LogD) Oral absorption, cell membrane penetration, Brownell, C. pH-metric distribution solubility. 2: correlation Chemical stability Chemical integrity in body fluids, tissues and oral between the acid-base titration absorption and the saturation shake-flask Metabolic Bioavailability and clearance solubility-pH methods. Pharm. CYP450 inhibition Metabolism and drug-drug interaction Res., 17, 85-89 (2000). CYP450 induction Metabolism and drug-drug interaction 22 Lipinski, CA, Lombardo, L, /-binding Clearance, distribution and bioavailability Dominy BW,Feeney, PJ. Identification Metabolic mechanism Experimental and computational approaches to estimate To xicological Assays solubility and permeability in hERG, other cardiac ion channels QT liability, cardiotoxicity drug discovery and Genetic toxicology Teratogenicity, mutagenicity development settings.Adv. Drug Target organ/cytotoxicity Hepatotoxicity, neurotoxicity, nephrotoxicity, Delivery Rev. 23, 3-25 (1997). haematological side effects, etc 23 Kibbey, CE, Poole, SK, Robinson, B, Jackson, JD, Durham, D.An integrated Safety Pharmacological Assays process for measuring the Broad scale pharmacology profiling Adverse effects associated with receptor, channel, physicochemical properties of enzyme activation/inhibition/modulation drug candidates in a preclinical discovery environment. J. Pharm. Sci., 90, 1164-1175 (2001).

Ta b le 1: Profiling assays that are useful in early discovery Continued on page 80

Drug Discovery World Fall 2004 79 ADME Profiling

Continued from page 79 Ta b le 2: Comparison of ‘in vitro primary suite’ and ‘in vitro secondary suite’ in ADME profiling

24 Bevan, CD, Lloyd, RS.A high-throughput screening IN VITRO PRIMARY SUITE IN VITRO SECONDARY SUITE method for the determination of aqueous drug solubility Applicable to most of the LP/LO candidates Applicable to promising compounds in late discovery using laser nephelometry in microtiter plates.Anal. Chem., Common issues or interests to discovery Full characterisation for promotion to development 72, 1781-1787 (2000). 25 Kariv, I, Rourick, RA, Kassel, DB, Chung,TDY.Improvement of Rank the compounds and flag the potential issues Address the warning raised in primary assays “hit-to-lead” optimization by integration of in vitro HTS experimental models for early Reasonable throughput and short turn-around time Limited throughput and longer turnaround time determination of pharmacokinetic properties. Combinatorial Minimal sample required (1-2mg for the suite) More compound material required Chemistry & High Throughput Screening, 5, 459-472 (2002). 26 Stresser, DM, Broudy, MI, Ho,T, User-friendly streamlined compound logistics Individual request may be offered Cargill, CE, Blanchard,AP,Sharma, R, Dandeneau,AA, Goodwin, JJ, Good cross-site validation and data reporting on a Cross-site validation and global standards remain Tu rner, SD, Erve, JCL, Patten, CJ, global standard Dehal, SS, and Crespi, CL. Highly selective inhibition of human CYP3A in vitro by azamulin and Good quality and reasonable predictive values Improved quality over primary assays evidence that inhibition is irreversible. Drug Metab. Dispos. 32, 105-112 (2004). 27 Pan, L, Ho, Q,Tsutsui, K, 22 Takahashi, L. Comparison of — In silico prediction is an alternative approach — Kinetic solubility, first introduced by Lipinski , chromatographic and to estimate water solubility. It provides an oppor- imparts a fast and economic avenue to estimate the spectroscopic methods used tunity to project solubility in a cost-effective and aqueous solubility of early discovery compounds to rank compounds for fast fashion. However, the major challenge for the where a higher throughput (50-100 per day) and min- aqueous solubility. J. Pharm. Sci., 90, 521-529 (2001). in silico solubility prediction lies in its reliability imal sample consumption are prerequisites (10s 28 Zhou, L, Snyder, J, Mehta, M, and predictivity. This is because solvation and sol- pmol). Although a number of analytical approaches Wang, J. Determination of ubilisation of drug substances does not only rely are utilised such as turbidity22-23, nephelometry24-25, aqueous solubility using on chemical structure but also on a complex inter- laser cytometry26 and a direct-UV method27, kinetic nephelometric approach in early play between hydrogen-bond acceptor and donor solubility assays always start with compounds that drug discovery.To be published. 29 Stouch,TR, Kenyon, JR, properties, conformational effects and crystal are pre-dissolved in organic solvents such as DMSO. Johnson, SR, Chen, X-Q, packing energy. For these reasons standard devia- The introduction of compound stock solutions into Doweyko,A, Li,Y. In silico tions of predicted water solubility from the exper- designated media in microtiter plates (or vials) allows ADME/Tox: why models fail. J. imentally determined values (thermodynamic sol- for monitoring the concentration at which com- Computer-Aided Mol. Design ubility) are still relatively substantial. Many com- pounds crash out of the solution. Kinetic approaches 17, 83–92, (2003). 30 Kerns, EH. High-throughput mercial products claiming satisfactory agreement generally do not necessarily involve a long incubation physicochemical profiling for between the computational and experimental data period with thorough agitation and assays can be drug discovery. J. Pharm. Sci., were unable to deliver adequate predictions for accomplished quickly and efficiently. Its predictivity 90, 1838-1858 (2001). drug-like NCEs. This is not surprising as most of for thermodynamic solubility perks up in comparison 31 Balimane, PV,Chong, S, the in silico tools were trained using non-drug-like to in silico tools, in particular for the well-known Morrison, RA. Current - methodologies used for evaluation chemicals in the non-relevant solubility range (10 commercial drugs, most of which have relatively 12 3 of intestinal permeability and -10 g/L). The new trend will greatly help this decent physicochemical properties and a homoge- absorption. J. Pharmacol.Toxicol. problem by establishing collaborations between neous solid format28. The method tends to be prob- 44, 301-312 (2000). commercial vendors that develop the new in silico lematic for NCEs in the early discovery phase where 32 Sun, D,Yu, LX, Hussain, MA, tools and pharmaceutical labs that have large col- compounds might be impure, unstable, colourful, and Wall, DA, Smith, RA,Amidon, 28-29 GL. In vitro testing of drug lections of thermodynamic solubility data derived in mixed solid polymorphs . The errors of kinet- absorption for drug from drug-like molecules. In addition, some in sil- ic solubility may originate from: ‘developability’ assessment: ico predictions may require other physico-chemi- Forming an interface between in cal parameters such as partition co-efficient (logP) ● Insufficient incubation time (instant readings vs vitro preclinical data and clinical or melting points (MP) of the tested compounds 24-72 hour incubation). outcome. 7, 75-85 (2004). that may not necessarily be available in the early ● Presence of DMSO (0.5-5%) in kinetic approach Continued on page 81 stage of drug discovery. (over-estimates solubility).

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● Loss of polymorph information due to pre-dis- — Parallel artificial membrane permeability assay, Continued from page 80 solution of solids in DMSO. known as PAMPA33, offers a fast and robust tool ● Interference by impurities, degradation products for screening permeability of NCEs in early dis- 33 Kansy, M, Senner, F, 33-37 Gubernator, K. or the colour of the samples. covery phases . The method monitors the Physicochemical high ● Different analytical approaches used than the capability of drug candidates to permeate through throughput screening: parallel conventional HPLC approach. a chemical membrane immobilised on a 96-well fil- artificial membrane ● Deviation in robotic liquid handler when ter plate. The fraction of NCEs pervaded through permeability assay in the automation is involved. the chemical membrane is quantified simply using description of passive absorption process. J. Med. a UV plate reader (primary) and LC/MS (supple- Chem. 41, 1007-1010, (1998). — Development and implementation of HT- and mentary for those lacking UV chromophores). As a 34 Wohnsland, F, Faller, B. miniaturised equilibrium solubility has become a new simple chemical model, however, it can only esti- High-Throughput Permeability trend to derive reliable aqueous solubility of NCEs in mate permeability for compounds with a passive pH Profile and High- early drug discovery28. The approach applies novel transcellular diffusion mechanism and small Throughput Alkane/Water log P with Artificial Membranes. J. technologies in a number of key steps (sample han- molecular weights (eg <500). Preferably it may Med. Chem. 44, 923-930 dling, incubation, phase separation and quantifica- serve as a pre-screening tool for permeability rank- (2001). tion, etc) to assure that the assay is miniaturised and ing when multiple in vitro models are introduced 35 Sugano, K,Takata, N, the throughput is significantly enhanced without to address GI permeability. Machida, M, Saitoh, K,Terada, noticeably sacrificing quality30. The predictivity of the K. Optimized conditions of biomimetic artificial membrane new approach is validated against not only a few com- — The Madin-Darby canine kidney (MDCK) cell permeability assay Int. J. Pharm. mercial drugs but also a collection of NCEs in the model is one of the commonly used cell monolayer 228, 181-188 (2001). early discovery pipelines with divergent chemistry and systems to assess the human intestine barrier70. 36 Zhu, C, Jiang, L, Chen,TM, possibly unfavourable physicochemical properties. MDCK cell lines can reach full differentiation in Hwang, KK.A comparative It is worthwhile to mention that efforts are devot- three to seven days and are therefore relatively easy study of artificial membrane permeability assay for high ed to adopt the direct-UV approach that is com- for cell culturing and assay maintenance, in com- throughput profiling of drug monly used in the determination of kinetic solubili- parison to other cell lines such as human colon absorption potential. Eur. J. Med. ty to derive equilibrium solubility. Typical tactics adenocarcinoma cells (Caco-2). However, MDCK Chem. 37, 399-407 (2002). include minimising (eg 0.5%) the involvement of cell lines originate from dog kidney and the expres- 37 Ruell, JA,Tsinman, KL, organic solvents such as DMSO, extending incuba- sion of transporters is quite different from human Avdeef,A. PAMPA-a drug 31 absorption in vitro model 5. tion time and developing state-of-the-art analytical intestine . As a result, the MDCK monolayer is Unstirred water layer in iso- algorithms to flag the spectral interference by impu- commonly used for permeability evaluation of pH mapping assays and pKa rities and degradation products19. The method has NCEs transported by the passive transcellular dif- flux-optimized design (pOD- demonstrated appreciable enhancement in through- fusion mechanism38-39 (as does the PAMPA PAMPA). Eur. J. Pharm. Sci. 20, put in comparison to the conventional ‘shake-flask’ model), rather than for accurately predicting per- 393-402 (2003). 38 Ishihama,Y, Nakamura, M, method and also exhibited improvement in quality meability of compounds involving active uptake Miwa,T, Kajima,T,Asakawa N. over some kinetic solubility approaches. In compar- and efflux mechanisms. Efforts are devoted to A rapid method for pKa ison to the ‘shake-flask’ method, the direct UV pro- extend the permeability estimation using P-glyco- determination of drugs using tocol may have compromised the quality in several protein (Pgp)-transferred MDCK to account for pressure-assisted capillary key steps to accommodate the required throughput. the contributions of efflux transporters40. electrophoresis with photodiode array detection in drug discovery. J. Pharm. Sci. Permeability and active transporters — The Caco-2 cell permeability model, exhibiting 91, 933-942 (2002). Permeability, referred to as the capability of NCEs morphological as well as functional similarities to 39 Ungell,AL, Karlsson, J. Cell to penetrate across the human GI tract, is another human intestinal enterocytes, has therefore been culture in drug discovery: an key factor governing human oral absorption31-32. better received in drug discovery and development industrial perspective. In Drug 39,41 Bioavailability (H.van de Ideally, oral absorption of drug substances is meas- than other epithelial cell cultures . Caco-2 cells Waterbeemd, H. Lennernas ured by quantifying the fraction of the designated extensively express a variety of transport systems and P.Artursson eds),Wiley- drug absorbed through the human GI tract. including efflux of not only Pgp but also VCH, pp. 90-131 (2003). Although the data derived are considered very reli- the dipeptide transporters (PEPT1) normally found 40 Bohets, H,Annaert, P, able and serve as ‘gold standards’ in the assessment in small intestinal enterocytes. The expression of Mannens, G,Van Beijsterveldt, L,Anciaux, K,Verboven, P, of oral absorption of drug substances, the multiple transport systems in Caco-2 cells offers Meuldermans,W, Lavrijsen, K. approach is impractical in early discovery due to great advantages over simplified transport models Strategies for absorption the intricate and costly experimental procedures. as it can investigate the interplay among different screening in drug discovery Alternatively, high throughput in vitro permeabili- transport systems and differentiate the relative con- and development. Curr.Top. ty assays using either artificial membranes or cell- tributions from passive and active transport mech- Med. Chem. 1, 367-383 (2001). based models, are becoming the methods of choice anisms to the overall permeability across the human in early drug discovery11,31. GI tract. Being human in origin, the Caco-2 cells Continued on page 83

Drug Discovery World Fall 2004 81 ADME Profiling

Figure 4 Schematic presentation of an ideal way to establish and (100) utilise comprehensive profiling Efficacy tools in drug discovery and development.The vertical axis represents the enhancement of in vitro and in vivo efficacy (increasing from bottom to (1000-1) top).Those perpendicular to Full PK/PD the ‘efficacy’ axis signify the improvement of divergent drugable properties (inward). Discrete drugability filters may be exploited at key phase secondary transition milestones to assure comprehensive profiling data PK/PD Early Drugability filters are assessed along with In vitro efficacy results (102-3) To xicity Metabolism primary

In vitro In vitro Drug interaction Absorption

6 (10 ) Distribution

In silico Adverse effects Lead finding Lead optimisation Development Content of Number of information compounds

provide information for human transporters in Overall, the 96-well Caco-2 permeability model well-differentiated enterocytes, such as tight junc- demonstrates a good correlation to human oral tion and brush-border enzymes that are found in absorption and offers a comprehensive tool to the human GI tract. accurately project the permeability across the GI The latest development of the assay involves tract for drug candidates using different transport seeding and culturing Caco-2 cells over 96-well mechanisms. The method is reliable, shows good transwell filter inserts consisting of polycarbonate predictivity to human absorption and requires or polyethylene terephthalate (PET). Whereas cell minimal material for testing. However, its draw- culturing process could be reduced to 3-10 days, it backs are complicated in cell culturing, the high is believed that the full course of 18-21 day cell cul- cost for assay maintenance and potential problems turing ensures that all necessary transporters are for profiling across a broad pH range. fully differentiated for the accurate permeability assessment of the compounds with passive or Ionisation constant (or dissociation active transport mechanisms39. The permeability constant) assay is commonly carried out in the bi-directional The ionisation of drug candidates and the distribu- mode (apical to basolateral chambers and basolat- tion of the neutral and ionised species under phys- eral to apical compartments) to estimate the per- iological pH drastically govern the ADME proper- meability and to differentiate the contribution of ties of NCEs. Ionisation constant, pKa, defined as the active transporters in the permeation process of the concentration at which the neutral species and NCEs. The assay is generally quantified by using ionised form are equally distributed, is a useful LC/MS method. The throughput of the assay can thermodynamic parameter to monitor the charge be substantially enhanced by automating the cell state of drug candidates. First, pKa data can help culturing, permeability assay and by streamlining predict ADME properties of NCEs due to the pH the quantification42. gradient of 1.7-8.0 present in the human GI

82 Drug Discovery World Fall 2004 ADME Profiling

tract19. For instance, solubility and permeability ● Spectral Gradient Analyzer (SGA) presents the lat- Continued from page 81 are modulated severely by the pKa value(s) of a est development in the high-throughput determina- drug candidate as the former is favoured by the tion of pKa45. The method establishes a stable and 41 Artursson, P,Tavelin, S. Caco-2 and emerging ionised form whereas the latter is proportional to well-defined pH gradient by rapidly mixing acidic alternatives for prediction of the concentration of its neutral species in solution. and basic buffers, during which drug candidates, pre- intestinal drug transport: a In addition, pKa data can be used for better under- dissolved in organic solvent, are introduced at differ- general overview. In Drug standing the binding mechanisms of therapeutic ent pH conditions. The spectral changes associated Bioavailability (H.van de events and also for the optimisation of chemical with the variations in ionisation are monitored using Waterbeemd, H. Lennernas and P.Artursson eds),Wiley- reactions. A number of ADME properties such as an on-line photo-diode-array UV detector to derive VCH, pp. 72-89 (2003). lipophilicity and solubility pH profiles are derived pKa values. With usage of co-solvent in the media, 42 Wang, J, Eddy, P,Phillips J, in combination with aqueous pKa data. the assay works effectively with poorly soluble Tam, CY. Determination of A number of approaches are used to estimate NCEs. The pKa data measured on SGA correlate Drug Permeability in 96-Well pKa values for NCEs: very well with those from the potentiometric titra- Cell Plates, to be published 43 Cleveland Jr, JA, Benko, MH, tion method. The method exhibits a number of Gluck, SJ,Walbroehl,YM. ● Of many in silico tools for pKa prediction, the advantages in measuring pKa in early discovery such Automated pKa determination software developed by Advanced Chemistry as being high throughput, automatic, reproducible at low solute concentrations Development (ACD/Labs) yields promising pKa data and economic. However, its major drawback is that by capillary electrophoresis. J. for commercial drugs, in comparison to data collect- the success (eg ~70%) greatly relies on the existence Chromatogr.A, 652, 301-308 (1993). ed experimentally. The prediction deteriorates for of UV chromophores in NCEs and the ionisation- 44 Oumada, FZ, Ràfols, C, real NCEs in early discovery phase (LP and LO associated UV alterations. In other words, this Rosés, M, Bosch, E. stages), establishing the necessity for determination approach will not work for small molecules such as Chromatographic of NCEs using in vitro assays. This is not surprising peptides or other chromophore-containing NCEs determination of aqueous as most of the prediction tools are trained by the sim- where the ionisation occurs at the molecular moiety dissociation constants of some water-insoluble nonsteroidal ilar set of commercial drugs. However, it is a useful that is distant from the UV chromophore. The anti-inflammatory drugs J. tool for screening virtual molecules or in the cases method may require expertise and good understand- Pharm. Sci., 91, 991-999 where no experimental alternatives are available. ing of the underlying principle for data processing in (2002). ● Potentiometric titration records the pH changes, particular for compounds containing multiple pKa 45 Box, K, Bevan, C, Comer, J, with a glass electrode, caused by introducing a values that are close to each other in pH. Hill,A,Allen, R, Reynolds, D. High-throughput measurement known volume of titrants to the well-mixed solu- Nonetheless, this tactic can be nicely combined with of pKa values in a mixed-buffer tion of a drug candidate. The method is very reli- the potentiometric approach. linear pH gradient system. able to determine pKa and can analyse about 5-10 Anal. Chem., 75, 883-892 compounds per day. For low soluble compounds, Lipophilicity (2003). the method employs co-solvents thereby requiring LogP and LogD are the logarithms of partition 46 Lombardo, F, Shalaeva, M, Tupper, Gao, F,Abraham, M. J. some experience for data interpretation. In addi- co-efficient and apparent partition co-efficient ElogPoct: a tool for tion, the current size of the titration device still of drug candidates in a lipophilic phase such as lipophilicity determination in needs a few mg of sample material. Therefore it octanol and a hydrophilic phase like water. The drug discovery. Med. Chem. 43, has been widely accepted in late discovery and data are valuable to predict the ADME proper- 2922-2928 (2000). early development. ties ranging from solubility, permeability to the 47 Yurek, DA, Branch, DL, Kuo, ● M-S. Development of a system Capillary electrophoresis (CE) can technically understanding of transport mechanism. The to evaluate compound identity, handle a small amount of sample material. Samples conventional approach for LogP and LogD purity, and concentration in a are loaded on one end of CE and the migration, or determination, the saturation shake-flask single experiment and its mobility of the compounds is monitored under method, is to measure the equilibrium distribu- application in quality assessment electric potential. The mobility is highly dependent tions of NCEs in octanol and water, not feasible of combinatorial libraries and screening hits. J. Comb. Chem. on the ionisation process and thus pKa data of for early discovery. The dual-phase potentiomet- 4, 138-148 (2002). 23,38,43 NCEs can be assessed accordingly . This ric titration method involves extracting LogP 48 Metha,AC.Analytical issues tactic demonstrates potential in dealing with some and LogD by analysing distinctions in the titra- with chemical stability testing issues in early discovery such as interference of tion curves of pKa in aqueous and octanol of drugs in solution.Anal. Proc. impurities and where only minimal sample materi- media19. The method is reliable but only feasible Incl.Anal. Comm. 32, 67-70 (1995). al is available. However, the inability to handle for compounds with measurable pKa data. For 49 Obach, RS.The prediction poorly soluble compounds that are highly populat- those lacking an ionisation centre, HPLC logP of human clearance from ed in early phase becomes the major hurdle for its technique, also known as eLogP can be hepatic microsomal applications in early discovery. A new approach applied46. Other tactics available to assess LogP metabolism data. Curr. Opin. utilising an HPLC method appears to address the and LogD in early stages include liposome chro- Drug Discov. Devel. 4, 36-44 (2001). issues raised when measuring pKa for sparingly matography, immobilised artificial membrane soluble compounds44. (IAM) chromatography and CE approaches (a Continued on page 84

Drug Discovery World Fall 2004 83 ADME Profiling

Continued from page 83 good review given by Avdeef)19. The latest orally absorbed, might never reach the required development for HT-LogP determination is to therapeutic concentration. On the other hand, a 50 Masimirembva, CM, utilise technology similar to PAMPA where certain degree of instability might be desirable for Bredberg, U,Andersson,TB. Metabolic stability for drug octanol serves as the immobilised phase in a a prodrug where a metabolite is more active than 34 discovery and development, microtiter filter plate . its parent. Clin. Pharmacokinet., 42, 515- There are a number of tactics to determine 528 (2003). Chemical integrity and stability metabolic clearance. Whereas comprehensive in 51 Obach, RS. Prediction of Chemical integrity is valuable for the confirmation vivo PK studies in man serve as the ideal source human clearance of twenty-nine drugs from hepatic microsomal of chemical identity, in particular for validated for ADME data including metabolism, bioavail- intrinsic clearance data: an hits derived from high-throughput screening ability and clearance, such experiments are not examination of in vitro half-life where compounds have been stored in DMSO for available until a relatively late phase in drug dis- approach and non-specific a long period of time11. For freshly synthesised covery and during clinical trials. This means that binding to microsomes. Drug NCEs in the lead optimisation phase, it is also lead optimisation might go down a blind alley in Metab. Dispos. 27, 1350-9 (1999). 52 Lau,YY, Krishna, G,Yumibe, beneficial to verify that their chemical structure is terms of ADME features and produce expensive NP,Grotz, DE, Sapidou, E, as designed as well as confirming the purity. This drug candidates with severe liabilities and ulti- Norton, L, Chu, I, Chen, C, information will be helpful for the proper inter- mate threat of termination. Animal models, Soares,AD, Lin, CC.The use of pretation of the in vitro ADME and efficacy albeit useful in predicting various aspects of the in vitro metabolic stability for results and the in vivo PK/PD data. A typical metabolism and PK issues of NCEs in man, rapid selection of compounds in early discovery based on method to measure it uses LC/MS to verify the require considerable cost and lack the required their expected hepatic identity via the designated molecular weight and throughput necessary in early discovery. extraction ratios. Pharm. Res. the impurity can be roughly quantified using Furthermore, they do not always show satisfac- 19, 1606-1610 (2002). either of the LC/UV, chemical luminescence nitro- tory predictivity to human clearance due to the 53 Berry, L,Wang, J. gen detector (CLND) or evaporative light scatter- difficulties in deriving proper scaling factors at Determination of metabolic 47 stability in liver microsomes ing (ELSD) methods . early stages of drug discovery. using the in vitro half-life Chemical stability in general characterises the Currently, in vitro approaches that are exten- approach on a fully automated ability of an NCE to preserve its chemical and sively utilised to monitor the metabolic stability as platform.To be published. physical characteristics under specific conditions. well as to predict the human clearance of drug can- 54 Korfmacher,WA, Palmer, From the drugability point of view, stability for didates include the use of recombinant CYP450 CA, Nardo, C, Dunn-Meynell, K, Grotz, D, Cox, K, Lin, CC, solution and solid phases are both critical to fore- enzymes, liver microsomes, S9 fraction (the 9000g Elicone, C, Liu, C, Duchoslav, E. see the potential issues of a candidate to become a supernatant of a liver homogenate), isolated hepa- Development of an automated marketed drug. In early discovery, however, the tocytes and liver slices50. To meet the needs to mass spectrometry system for interest is concentrated in the solution phase as measure metabolic stability of NCEs in early dis- the quantitative analysis of drug candidates in early phase are commonly in covery, the common and reliable approach is to liver microsomal incubation samples: a tool for rapid amorphous or meta-stable states and will be trans- probe the depletion of the test compounds in the screening of new compounds ferred to the more stable polymorphous forms in presence of liver microsomes using LC/MS and for metabolic stability. Rapid the development phase. The stability in solution derive the half-life and intrinsic clearance values51- Commun. Mass Spectrom. 13, can be assessed after a long period of incubation 53. The in vitro data derived also demonstrate a 901–907 (1999). or under extreme conditions (eg temperature or decent correlation with the in vivo hepatic clear- 55 Watt,AP,Mortishire-Smith, 23 RJ, Gerhard, U,Thomas, SR. pH, etc) and quantified using HPLC , CE, thin- ance values, which are very useful in estimating Metabolite identification in layer chromatography (TLC) or LC/MS. It is bioavailability and systemic clearance. drug discovery. Current worthwhile to mention that choosing an adequate The in vitro method has the advantage of pro- Opinion in Drug Discovery & analytical approach is pivotal to the quality of the filing the metabolism of NCEs in the most relevant Development, 6, 57-65 (2003). chemical stability assay48. With the stability data, species and to predict the human hepatic clearance 56 Riley, RJ, Kenna, JG. Cellular models for ADMET predictions one can better understand the in vitro ADME and with relatively high throughput and low sample and evaluation of drug-drug efficacy data and in vivo PK results. For example, consumption at the earliest stages of drug discov- interactions. Current Opinion chemical stability in buffer allows for differentiat- ery. The high quality of the assay also allows for in Drug Discovery & ing the issues of chemical and metabolic stability the data to be widely utilised in discovery and Development, 7, 86-99 (2004). of NCEs. development stages, appreciably reducing the 57 Ito, K, Iwatsubo,T,Kanamitsu, S, Ueda, K, Suzuki, H, Sugiyama,Y. requirement of animals and offering both com- Prediction of pharmacokinetic Metabolic stability and clearance mercial and ethical advantages. However, quan- alterations caused by drug-drug Metabolic stability or clearance, particularly tification using LC/MS remains the bottleneck of interactions: metabolic interaction hepatic, is recognised as one of the main determi- the assay, in particular when dealing with such a in the liver. Pharmacol. Rev. 50, nants of drug concentration in blood and has been large number of samples and data points, as is also 387-411 (1998). used effectively to predict bioavailability and toxi- observed with most of the cell or protein-based Continued on page 85 cokinetics49. A metabolically unstable NCE, albeit ADME assays. An alternative approach is to

84 Drug Discovery World Fall 2004 ADME Profiling

reduce the time points of the depletion determina- by monitoring the impact of the test compounds on Continued from page 84 tion (eg a single time point at 20-30 minutes, CYP450 metabolic activity using a known sub- etc)11,54. This modification may be sufficient to strate. This is based on the assumption that when- 58 Wienkers, LC, Hutzler, JM. Screening for cytochrome offer the metabolic stability rank-ordering in early ever DDI occurs, regardless of the mechanism(s) P450 inhibitors. Current Drug discovery, provided that comprehensive metabolic involved (eg competitive, non-competitive or un- Discovery, 23-26 (2002).59. profiling will follow in late discovery. Another competitive), the compound will interfere with the , NJ, & Gibson, GG. potential issue of the method involves compounds performance of a co-administered drug that is Evaluation of the toxicological or their metabolised products that inhibit metabolised by the same enzyme60. relevance of CYP3A4 induction. Current Opinion in metabolising enzymes such as CYP450 enzymes. Conventionally, the obtained are Drug Discovery & In these cases, it is advisable to collectively inter- separated and analysed by LC/MS (occasionally by Development, 6, 50-56 (2003). pret the metabolic data along with drug-drug LC/UV)61, 65-66. The LC/MS method offers reli- 59 Plant, NJ, Gibson, GG. interaction profiling derived from CYP450 inhibi- able data but its application in early discovery is Evaluation of the toxicological tion assay. greatly restricted by the throughput and resources relevance of CYP3A4 induction. Current Opinion in For NCEs exhibiting high metabolic clearance, it given for method development. For example, in Drug Discovery & is worthwhile to follow up the metabolism by fully order to accurately quantify the metabolites, one Development, 6, 50-56 (2003). profiling the metabolites, or called metabolite iden- has to ensure that all DDI-induced changes in the 60 Madan,A, Usuki, E, Burton, tification. Under those circumstances, the metabo- metabolism of the designated substrate are com- LA, Ogilvie, BW, Parkinson,A. lites could be active or inactive to the therapeutic pletely captured. Analytically it is not trivial as all In vitro approaches for studying the inhibition of drug- target, which will affect the total efficacy meas- potential metabolites, instead of just the parental metabolizing enzymes and ured. It could get even worse when the metabolites compounds as done in most of ADME assays, will identifying the drug- are found toxic, or show adverse effects, which will need to be quantified. Therefore, the method is metabolizing enzymes raise other toxic and safety issues. Therefore the more practical in the late discovery and early devel- responsible for the metabolism identification of all metabolites will help under- opment phases. of drugs (Rodrigues,AD. Ed.), Marcel Dekker, New York. stand the metabolism mechanism and identify the Recent development of fluorescence-based pp.217-294 (2002). 61,63-64 chemical ‘soft spots’ of NCEs, which can be approaches demonstrates excellent sensitiv- 61 Cohen, L, Remley, M, utilised to direct the next round of syntheses. A ity and specificity without metabolite separation Raunig, D,Vaz,A. In vitro drug number of methodologies that are used to profile and quantification by LC/MS. This novel method- interactions of cytochrome the metabolite identification were reviewed by ology therefore substantially improves the P450: an evaluation of 55 fluorogenic to conventional Watt et al . throughput, turn-around time and cost-effective- substrates. Drug Metab. & ness by parallel monitoring via a 384-well plate- Dispos. 31, 1005-1015 (2003). CYP-450 inhibition: metabolism-related reader. The fully automated method 64 enables us 62 Clarke, SE, Jones, BC. Human and drug-drug interactions to reliably measure IC50 in duplicate over a broad Whereas the drug-drug interactions (DDI) in man concentration range (0.068-50µM) with a small their role in metabolism-based drug-drug interactions. In Drug- are a broad subject covering protein-binding, amount of material consumed (10s pmol). In addi- drug interactions (Rodrigues, transport carriers or other pharmaco-dynamic tion, the NCEs that exhibited DDI issues in the AD. Ed.), Marcel Dekker, New interactions56, many DDI-related pre-clinical/clini- end-point experiments can be automatically cher- York. pp.55-88 (2002). cal failures to date have been attributed to ry-picked for further kinetic studies to monitor the 63 Crespi, CL, Stresser, DM. CYP450-related hepatic metabolism57-59. For whole inhibitory course and derive insights into the Fluorometric screening for metabolism-based drug-drug instance, a drug candidate that is a potent CYP450 mechanisms. The major concern of the fluorogenic interactions. J. Pharmacol. inhibitor may greatly inhibit the metabolism of a approach is for compounds or their metabolites To xicol. Met., 44, 325-331 co-administered , potentially leading to that are fluorescent at the wavelengths used. (2000). adverse clinical events. Therefore, it is essential to However, fluorogenic control experiments are run 64 Ommert, S, Berry, L,Wang, screen compounds in early drug discovery to reveal for all NCEs to raise the flags for compounds or J. Fully automated screening of cytochrome P450 inhibition: a their major metabolic pathways and to monitor metabolites with potential fluorescent interference. probe for metabolism-related their in vitro inhibition of the major CYP450 iso- The suspicious compounds can thus be further test- drug-drug interactions in early forms in order to predict their in vivo effects60-61. ed using the LC/MS approach. drug discovery.To be Despite the many CYP450 isozymes present in published. man, five major isoforms (3A4, 2C9, 2C19, 2D6, Looking ahead 65 Yan, Z, Caldwell, GW, Metabolism profiling and 1A2) account for the metabolism-related DDI In the last decade, pharmaceutical profiling has cytochrome P450 inhibition issues for greater than 90% of marketed made a great leap forward, not only in building and induction in drug drugs58,61-62. Hence, the inhibition of the above up the foundation of comprehensive ADMET discovery. Curr.Top. Med. five major isoforms should be measured in a high diagnostic tools, but also gradually modernising Chem., 1, 403-425 (2001). throughput and fully automated fashion. the drug discovery mindset. Nonetheless, this is Commonly one tends to probe the metabolism- just the beginning and many unmet needs remain related drug-drug interactions of drug candidates and current assays need to be improved. First, Continued on page 86

Drug Discovery World Fall 2004 85 ADME Profiling

Continued from page 85 while the pharmaceutical profiling paradise is still correlation (ISIVC) and in vitro-in vivo correlation in the build-up phase, it is imperative to carefully (IVIVC) could be properly established and wisely 66 Ayrton, J, Plumb, R, Leavens, and constantly appraise the strategy and technical applied in our own drug discovery and develop- WJ, Mallett, D, Dickins, M, Dear, GJ,Application of a platforms. This feedback should be utilised to ment labs. DDW generic fast gradient liquid direct our construction of a full in silico/in chromatography tandem mass vitro/in vivo-PK profiling architecture in support spectrometry method for the of drug discovery and development. In addition, analysis of cytochrome P450 continuous commitments are essential to the probe substrates. Rapid Common. Mass Spetrom., 12, development of novel technologies that are cru- 217-224 (2001). cial to the implementation of early profiling. The 67 Theil, F-P,Guentert,TW, technology should be focused on the Haddad, S, Poulin, P.Utility of improvement of quality such as increasing predic- physiologically based tivity of the in silico and in vitro suites to the in pharmacokinetic models to drug development and rational vivo-PK results. Miniaturisation, automation and drug discovery candidate cost-effectiveness will become challenges for selection.Toxicol. Lett., 138, ADMET profiling. In spite of the considerable 29-49 (2003). expansions in the number of profiling assays in 68 Poggesi, I. Predicting human last decade, we are still required to work with the pharmacokinetics from preclinical data, Current same amount of sample material provided. In Opinion in Drug Discovery & addition, the difficulties of sample logistics in Development, 7, 100-111 handling so many high-throughput profiling (2004). assays should never be underestimated. Finally, 69 Preziosi, P.Science, one should concentrate on knowledge manage- pharmacoeconomics and ethics in drug R&D: a ment of the large collection of profiling data. It sustainable future scenario? includes the maintenance of a user-friendly data- Dr Jianling Wang obtained his PhD in biophysical Nature Rev. 3, 521-526 (2004). base, exploring comprehensible tools chemistry in the US. After his postdoctoral training 70 Irvine, JD,Takahashi, L, to effectively present the comprehensive ADMET at AT&T Bell laboratories, he joined Novartis in Lockhart, K, Cheong, J,Tolan, profiling data (such as Spotfire)71 and developing 1995. He has been devoted to the establishment of JW, Selick, HE, Grove, JR. MDCK (Madin-Darby canine or evaluating the evocative in silico tools (such as novel techniques in HT ADME profiling and has kidney) cells:A tool for GastroPlus of Simulations Plus, Inc, or other successfully built up the physico-chemical profiling membrane permeability models) for the projection of in vivo PK/PD prop- programme in Novartis, US. Recently he has taken screening J. Pharm. Sci. 88, 28- erties using collectively the in vitro ADMET additional responsibilities by heading the early 33 (1999). data67-69. This leads to two outcomes: ADME programme in Novartis at Cambridge. 71 Stoner, CL, Gifford, E, Stankovic, C, Lepsy, CS, (1) The co-operative exploitation of all ADMET Brodfuehrer, J.Vara Prasad, profiling data, instead of just one or two parame- Dr Laszlo Urban received his MD and PhD JVN, Surendran, N. ters, will exponentially enhance the power of pro- degrees in Hungary and worked as a postdoctoral Implementation of an ADME filing tools to resolve the puzzles in hunting for fellow at Iowa State University and University of enabling Selection and better and safer drugs. Kentucky. He was a visiting professor at Duke visualization tool for drug discovery. J. Pharm. Sci., (2) The drug discovery and development teams University. He joined the Sandoz Institute for 93,1131-1141 (2004). should be trained to make full and adequate uses Medical Research, London in 1990, where he led of the in silico, in vitro and in vivo-PK profiling the Pharmacology Department. Using his previous tiers to prioritise the candidates. Eventually the experience in drug discovery and development he is productivity of pharmaceutical industry will now leading an effort to implement broad scale, unlikely be improved unless the in silico-in vitro early profiling of NCEs.

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