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Deliveringtoxicitytests

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Deliveringtoxicitytests

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Drug discovery is all about finding a new compound that hits a chosen biological receptor or target for a particular disease. The current process begins with

high-throughput screening in the laboratory, where vast libraries of chemicals are tested for their ability to modify the target and, just as importantly, to detect their toxicity levels. After toxic compounds have been identified and eliminated, lead compounds are further synthesised and tested during in vitro and in vivo metabolism studies, using animal-based livers. Based on the results of these metabolism studies, drug candidates are typically advanced for Phase 1 clinical studies.

Since 90% of drug metabolites are implicated in adverse drug reactions, it is important to decipher these metabolic processes of drugs with rigour and precision.

A new approach to testing drug toxicity substitutes

liver enzymes for chemical catalysts and promises to spare thousands of animal lives, reports Mukund Chorghade

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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; degradation by traditional HPLC solvents like phosphoric acid is a frequent problem. The unstable metabolites such as epoxides, imines, N-formyls and others are frequently toxic and are rapidly trapped by DNA, glutathiones and other biological nucleophiles. Consequently, scientists obtain a limited picture of metabolites, leading to unidentified side effects and unpredictable patient outcomes. Our catalysts work in organic solvents and it is easier to make and isolate these metabolites without decomposition.

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 US Food and Drug Administration (FDA) suggested that ‘early identification of disproportionate drug metabolites can provide clear justification for non-clinical testing in animals, assist in interpreting and planning clinical studies, and prevent delays in drug development’. When it comes to metabolite profiling, early and more complete detection and structure proof is better – enabling pharmaceutical

However, the process of studying metabolites has traditionally been labour intensive and produces results that are often chemically inconclusive. Animal studies have always been sub-optimal for metabolism profiling, involving animal sacrifice, liver slice preparations as well as slow reacting liver cells and cell artifacts called microsomes that vary in potency. The resulting metabolites are difficult to predict, confirm and quantify.

Many years ago, I embarked on a quest to invent chemical catalysts that mimic the oxidative metabolites generated by liver enzymes and succeeded with the development of proprietary catalysts (tetra and diaza-azamacrocycles) that are sterically protected – with a chemical sheath – and electronically activated, providing the speed, stability and scalability required for next-generation drug discovery. Products were separated and their identity revealed with authentic reference samples of the major metabolites of drugs previously isolated from the urine of rats or characterised from rat liver microsomal incubations. In preliminary experiments, the catalytic turnover numbers exceeded 100,000 and the reaction rates were also markedly increased.

Finding chemical catalysts that can perform as well or better than liver enzymes was a herculean effort. Most drugs and other foreign substances digested by the body are degraded in the liver, by a series of oxidation processes involving 57-72 of the cytochrome P450 mono-oxygenase enzymes (CYP450).

CYPP450 enzymes belong to a group of versatile heam proteins, which serve diversified biological roles including oxygen and electron transport and storage (haemoglobin and myoglobin), electron transfer (cytochromes) and biocatalysis (catalase, peroxidase, lignin peroxidase, CYP450). Recently, the discovery of new heam proteins, together with their function and structure, has provided a wealth of information on these diverse and biologically important molecules. Catalytic activity of haem enzymes is known to be controlled by iron-porphyrin complexes, which are the active centres of these proteins.

Changes associated with the heam prosthetic group and modifications of the core protein structure provide a suitable environment for binding of a target drug to the metal centre.

Most biological oxidations involve

primary catalysis by CYP enzymes. All heam proteins that are activated by oxygen, including catalases, peroxidases and ligninases function via a two electron oxidation of the ferric resting state to an oxoferryl porphyrin cation radical (1), (Figure 1). Most of these reactions and those of the biomimetic catalysts that we have developed can be accounted for by oxygen transfer from radical (1) to a variety of substrates to give characteristic reactions such as hydroxylation, epoxidation and heteroatom oxidation. Such products cannot be readily prepared by classical modes of oxidation.

Researchers have been interested in finding chemical alternatives to liver enzymes since the 1980s. The tetra and diaza-azamacrocycles that we have developed are examples of metalloporphyrins and salens. These structures, which replicate the core structure of the haem group, function by a similar mechanism involving an oxo-ferryl radical cation and furnish the identical oxidative transformations as the liver enzymes.

However, the earliest synthetic metalloporphyrins were found to be relatively poor oxidation catalysts. Few catalytic turnovers were seen due to rapid destruction of the porphyrin macrocycle. For example, Jay Groves at University of Michigan, Ann Arbor, US, in the late 1970s-early 1980s first prepared tetramesityl porphyrins that were sterically protected but suffered from undesirable oxidation of the peripheral methyl groups.

In 1994, David Dolphin at the University of British Columbia, Vancouver, Canada, and I at Abbott Laboratories in Chicago, US, showed that introduction of halogens onto the aryl groups of meso-tetraarylporphyrins and on the β-pyrrolic positions of the porphyrins increases the turnover of catalytic reactions by decreasing the rate of porphyrin destruction. In addition, the combined electronegativities of the halogen substituents are transmitted to the metal atom, making the corresponding oxo-complexes more electron deficient and thus more effective oxidation catalysts.

One advantage of our new catalysts over traditional biological methods concerns the sensitivity and yields with which we are able to detect the reactive and unstable metabolites and prepare them on larger scale. The liver’s inherent function is to metabolise drugs

Further reading1 P. S. Traylor, D. Dolphin

and T. G. Traylor; J. Chem. Soc., Chem. Commun., 1984, 279

2 T. Wijesekera and D. Dolphin; Metalloporphyrins in catalytic oxidations, R.A. Sheldon (ed). Marcel Dekker Inc., New York, 1994, Chapter 7, page 193

3 L. Y. Xie and D. Dolphin; Metalloporphyrin catalyzed oxidations, F. Montanari and L. Casella, (eds), Kluwer Academic Publishers, The Netherlands, 1994, 269

4 (i) K. E. Andersen et al; Tetrahedron, 1994, 50(29), 8699; erratum in Tetrahedron, 1996, 52(10), 3375 (ii)) J. E. Celebuski, M. S. Chorghade and E. C. Lee; Tetrahedron Lett., 1994, 35(23), 3837; corrigendum in Tetrahedron Lett., 1995, 36(52), 9414

5 J. V. Andersen et al; Bioorganic and Medicinal Chemistry Letters, 1994, 4 (24), 2867

6 M. S. Chorghade et al; Pure and Appl. Chem., 1996, 68(3), 753

7 R. H. David et al; Tetrahedron Lett., 1996, 37(6), 787; M. S. Chorghade et al; Synthesis, 1996, 1320

8 M. S. Chorghade (ed) and E. C. Lee (assoc ed); Proceedings of the XXth IUPAC Symposium on the Chemistry of Natural Products. Chicago, Sep. 1996, preface page vi

9 M. S. Chorghade; Drug metabolism: databases and high throughput testing during drug design and development, International Union of Pure and Applied Chemistry: DMDB Working Party, Erhardt P. W. (ed), Blackwell, 1999, p152

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Researchers have been inter-ested in finding chemical alterna-tives to liver en-zymes since the 1980s

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companies to quickly focus their time and money on the most promising drug candidates, validate research results and reduce the attrition rate of new chemical entities (NCEs).

We carried out the initial proof of concept study using our new catalysts in the 1990s on the metabolism profiling for Lidocaine, a local anaesthetic that is applied topically to relieve itching, burning and painful skin inflammation. Lidocaine was first synthesised under the name xylocaine by Swedish chemist Nils Lofgren in 1943, and for 35 years after Lidocaine was introduced to the market in 1949, scientists tested the drug by administering it to rats and dogs, and isolated the resulting metabolites from body fluids. Structure proof was not possible during those early years since most 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 high performance liquid chromatography (HPLC). The structures were confirmed by spectroscopy and reference standards were isolated in minute quantities. However, the complete profile of Lidocaine was not obtained because the metabolites were deemed unstable, and could not be isolated from aqueous solutions and bodily fluids.

In 1996, Dolphin and I used our new metalloporphyrin catalysts in non-aqueous solutions to reproduce

Lidocaine’s metabolism profile. Reactions between the active pharmaceutical ingredients with these 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.

Two new, previously unobserved, metabolites were detected, separated and their structures elucidated (Figure 2). These metabolites had escaped detection in all previous studies. 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.

Established in 2010, Empiriko is a clinical intelligence company based

in Newton, Massachusetts, US, that leverages chemistry-based experimental research and in silico modeling to provide solutions in drug discovery, development and patient outcomes. Biomimiks catalysts were launched in January 2014 as an animal-free approach for the systematic preparation and identification of the entire spectrum of oxidative products. Reaction conditions that produce the maximum number of products can be experimentally defined

and scaled-up. The products can then be separated and subjected to toxicologic, pathologic, histopathalogic or genotoxic testing.

With the speed of scientific and technology innovation, it is possible to imagine chemosynthetic livers at the forefront of drug r&d and patient treatment. Over the next three to five years, I envisage the enhancement of the Biomimiks platform to include the development of a ‘chemosynthetic-liver-on-a-chip’ and other applications for CYP-specific reactions and liver dialysis. Meanwhile, Biomimiks could also be harnessed to convert achiral compounds into chiral compounds.

It continues to be critical to derive inspiration from Nature to prepare chiral products, elaborate them into chemical hybrid ’molecular Lego‘ and to screen

the many diverse compounds against targets. Ultimately, my vision is to apply Biomimiks to drug discovery and development involving chiral natural products.

Mukund Chorghade is chief scientific officer at Empiriko Corporation in Newton, Massachusetts, US

Fe(III)porphyrin Fe(IV)porphyrin

OH2O2 H2OFigure 1: Haem proteins activated by hydrogen peroxide

Figure 2: Lidocaine metabolite profile using chemical catalysts

90% of drug metabolites are implicated in adverse drug reactions

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