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Metabolism Profiling: Changing the Game
with Biomimetic Oxidation Technology
Abstract:
This document reviews the issues surrounding metabolism profiling in drug discovery and identifies the
challenges of current-day in vitro and in vivo processes. Most notably, these traditional practices involve
significant animal sacrifice, and lack the speed, stability, scalability and predictability needed to design
new chemical entities (NCEs) with confidence. This paper further describes and recommends a new in
vitro paradigm for metabolism profiling – a “chemosynthetic liver” technology called Biomimiks™.
PART 1: Addressing In Vitro Challenges
Today’s pharmaceutical industry faces tremendous financial and competitive pressures to discover and
select promising drug candidates more quickly and cost-effectively. Metabolism profiling, which is
conducted relatively late in the drug discovery process, is a widely-used means of identifying toxicity and
potential side effects, and selecting the best drug candidates for further study. Since 90% of drug
metabolites are implicated in adverse drug reactions, metabolic processes of drugs are always the
subject of intense scrutiny in pharmaceutical companies. However, the present-day process of studying
metabolites involves animal studies, is labor-intensive and produces results that are chemically
inconclusive. Clearly, in vitro metabolism profiling is one area where breakthrough technology can be
used to overcome current shortcomings.
Focusing on Metabolism by Liver Enzymes. In humans and other animals, most small molecule drugs
are metabolized in the liver – necessitating the current in vivo practice of studying cytochrome P-450
(liver enzymes) mediated drug metabolism. For a number of reasons, animal studies are sub-optimal for
metabolism profiling: they entail animal sacrifice, they involve liver slice preparations as well as slow
reacting hepatocytes and microsomes which vary in potency, and the resulting metabolites are difficult
to predict, confirm and quantify. Although animal sacrifice cannot be completely eliminated from drug
testing altogether, it can be reduced significantly during drug research with the use of more advanced
chemical systems (e.g., chemosynthetic livers).
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Overcoming Short Turnover, Slow Reaction Rates and Insufficient Quantities. The past few decades
have seen advances in the development of synthetic metalloporphyrins to mimic numerous CYP
enzymes; biological systems such as hepatocytes, microsomes and liver slices have also been used for
the study of oxidative metabolism of drugs. However, these porphyrins have been limited by low
catalytic turnover (5-100), low stability and inability to generate sufficient quantities of metabolites for
thorough toxicology testing. Conversely, biological systems have slow reaction rates (days), generate
pitifully small quantities of metabolites (milligram amounts) and are constrained by a limited ability for
isolation and comprehensive structure elucidation. These traditional solutions have lacked the speed,
stability and scalability needed to support ongoing nonclinical iteration and design of new chemical
entities (NCEs).
Getting the Full Picture. The liver’s inherent function is to metabolize drugs by transforming them into
separate (polar), water-soluble metabolites that can be more easily excreted from the body.
Metabolites are not easily isolated from aqueous systems. Challenges arise because many metabolites
are water soluble and not amenable to isolation and separation by conventional routes (e.g.,
degradation by traditional HPLC solvents like phosphoric acid). Consequently, scientists obtain a limited
picture of metabolites, leading to unidentified side effects and unpredictable patient outcomes.
Earlier is Better. Today, few mechanisms exist to predict and quantify the accurate formation of
metabolites, leading to a costly process of trial and error. In its MIST (Metabolites in Safety Testing)
Guidelines from 2008, the FDA suggests that “early identification of disproportionate drug metabolites
can provide clear justification for nonclinical testing in animals, assist in interpreting and planning clinical
studies, and prevent delays in drug development.” Bottom Line: When it comes to metabolite profiling,
early (and more complete) detection and structured proof is better – enabling pharmaceutical
companies to quickly focus their time and money on the most promising drug candidates, validate
research results and reduce the attrition rate of NCEs.
PART 2: Examining the Realities of Metabolism Profiling
Traditional processes involved in drug discovery and development are clearly established in the
pharmaceutical industry (Figure 1 below).
Figure 1: Traditional Workflow for Drug Discovery and Development
[Source: US Food and Drug Administration]
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Focusing on Drug Discovery
Drug discovery is all about finding a new compound that binds to a chosen biological receptor or target
for a particular disease. The current process begins with high-throughput screening in the laboratory (in
vitro) where large libraries of chemicals are tested for their ability to modify the target and, just as
importantly, to detect their toxicity levels (Step 1 above). After toxic compounds have been identified
and eliminated, lead compounds are further synthesized and tested (Step 2 above) during in vitro and in
vivo metabolism studies, using animal-based liver slices and cell lines such as hepatocytes and
microsomes. Based on the results of these metabolism studies, final drug candidates are typically
proposed for Phase I of drug development.
Recapping Traditional Drawbacks
Present-day metabolism profiling is slow and inconclusive – making it nearly impossible to predict the
success or failure of a drug candidate. It’s important to consider that:

The current-day screening process typically takes days, and sometimes weeks.

Animal-based models fail to accurately mimic human reactions to drugs, leaving a significant
margin for error.

Animal derived samples (liver slices, hepatocytes and microsomes) vary in potency and produce
insufficient amounts of metabolites for ongoing pharmacological testing.

Conventional chromatography and spectroscopy on aqueous systems (used in current-day
metabolite profiling) do not detect water-soluble metabolites, providing an incomplete profile.

Undetected metabolites can lead to adverse side effects, prolonged trial and error during
patient treatment, and increased potential for late-stage drug withdrawals.
PART 3: Leveraging a New In Vitro Paradigm (Biomimiks™)
Empiriko’s proprietary Biomimiks™ technology mimics the in vivo metabolism of pharmaceutical
compounds, with catalysts serving as a “chemosynthetic liver” for predicting metabolism patterns,
pathways and profiles. Our proprietary catalysts (tetra-azamacrocycles) are sterically protected (with a
chemical sheath) and electronically activated (driving oxidative rate acceleration), providing the speed,
stability and scalability required for next-generation drug discovery.
Rapid Screening & Structure Validation. With Biomimiks™, scientists can quickly screen a series of
compounds by reacting them with permutations of our patented oxidative catalysts, co-oxidants and
non-aqueous solvents. This powerful combination serves as an “in vitro cocktail” that reduces the
elapsed time for drug screening from several days to hours. And since significant volumes of metabolites
survive during screening, scientists can quickly validate metabolite structures via mass spectroscopy –
and have plenty of metabolite solution left for toxicology and pharmacology testing.
Lead Development & Candidate Selection. Following initial screening, scientists continue to test
compounds that were found active against the selected target – a process referred to as Hit-to-Lead
Optimization. The objective here is to test compounds for other key characteristics such as interference
with liver enzymes (toxicologic, pathologic, histopathalogic, genotoxic), membrane permeability, water
solubility, chemical stability and issues relating to commercialization. With Biomimiks™, scientists can
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conduct more complete in vitro metabolism studies, confirm structure and generate quantitative
measures of toxicity, focusing their time and resources – more confidently – on the most promising drug
candidates.
The Biomimiks™ Difference. The diagram below (Figure 2) provides a side-by-side comparison of
traditional metabolism profiling methods (in vitro + in vivo) versus Biomimiks™ (in vitro only).
Figure 2: Traditional Methods vs. Biomimiks™
PART 4: Proof-of-Concept – Uncovering Lidocaine Metabolites
This proof-of-concept example focuses on the metabolism profiling for Lidocaine, a well-known local
anesthetic that is often applied topically to relieve itching, burning and painful skin inflammation. This
drug is also used as a local anesthetic for minor surgery and injected as a dental anesthetic. The
Lidocaine compound was first synthesized under the name xylocaine by Swedish chemist Nils Lofgren in
1943, and it was first marketed in 1949.
Metabolism Profiling: Using Traditional Methods
For 35 years after Lidocaine was introduced to the market, scientists tested the drug by administering it
to rats and dogs, and the generated metabolites were isolated from bodily fluids. Structure proof was
not possible during those early years since chromatographic and spectroscopic tools had not been
invented.
In 1989, clinicians incubated Lidocaine with CYP “liver” enzymes (in vivo) and were able to isolate the
generated metabolites using new chromatography equipment (Figure 3 below).
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Figure 3: Lidocaine Metabolite Profile Using Traditional Methods
Initial Results. At that time, the structures were confirmed by spectroscopy and reference standards
were isolated in very small quantities. However, the complete profile of Lidocaine was not obtained
because the metabolites were deemed unstable, and they could not be isolated from aqueous solutions
and bodily fluids.
Bottom Line. Pharmacologists had a partial picture of the metabolite structures of Lidocaine, and the
quantities of metabolites produced were insufficient for more comprehensive clinical evaluation.
Metabolism Profiling: Using Biomimiks™
In 1996, Chorghade and Dolphin used high-powered chemical catalysts in non-aqueous solutions to
reproduce Lidocaine’s metabolism profile (Figure 4 below). Reactions between the active pharmaceutical
ingredient of Lidocaine with these newfound catalysts were conducted in organic solvents at ambient
temperatures and pressures. The products of Lidocaine oxidation were further subjected to rigorous
scrutiny for structures of plausible metabolites.
Figure 4: Lidocaine Metabolite Profile Using Biomimiks™
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Updated Results. Products were separated by HPLC, and their identity was revealed with authentic
reference samples of the major metabolites of these drugs. More importantly, two new and previously
unobserved metabolites were detected and separated, and the structures were then elucidated. These
new metabolites (see green box in Figure 4 above) had escaped detection in all previous studies.
Bottom Line. Our scientists had successfully predicted the complete profile of Lidocaine metabolites and
confirmed those results via experimentation – more than 45 years after the drug was introduced to the
market. The side effects of Lidocaine were subsequently attributed to one of the newly isolated
metabolites. The rationale for the observed side effects had been scientifically deciphered using a
combination of innovative science and leading edge tools.
Biomimiks™ Changes the Game
The success of these early experiments spurred the eventual development of Empiriko’s innovative
Biomimiks™ solutions. These proprietary chemical catalysts precisely mimic the in vivo metabolism of
chemical entities used in small molecule drugs. With Biomimiks™ scientists can now reduce the attrition
rate of NCEs by conducting metabolism studies early, generating quantitative measures of toxicity, and
reducing the time and cost of unnecessary animal experimentation.
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About Empiriko
Empiriko is a Clinical Intelligence Technology (CIT) company that is taking a unique, holistic approach to
drug discovery, development and patient outcomes by integrating and leveraging knowledge from
scientific research, clinical experiences, observations and available patient data using sophisticated
algorithms, advanced analytics, predictive modeling and scientific and clinical interpretation of research
data. To achieve a higher level of predictive power, Empiriko has designed biomimetic systems that
emulate biological structure, function, mechanism and reactivity. Unlike traditional in silico or
computerized models, Empiriko works collaboratively with academic institutes and drug discovery
companies to iteratively test and refine computerized models in a laboratory environment. Our
proprietary Biomimiks™ solutions generate metabolite data that contributes to making Empiriko models
(disease, patient and drug) more robust and improving the predictive power of the CIT platform. These
models are used to connect drug discovery to patient outcomes, so the right therapies are developed
for the right subpopulation, and physicians are able to treat patients more effectively.
For more information about Biomimiks™ solutions for drug discovery, visit our website:
www.empiriko.com or email: [email protected]
Copyright © 2013 Empiriko Corporation. All right reserved. Empiriko and Biomimiks are trademarks of Empiriko Corporation.
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