Advanced mass spectrometry techniques to support your drug discovery and development research

Inside this book are a host of practical tools and techniques that enable you to fully leverage the capabilities of mass spectrometry in order to get the most out of drug metabolism and pharmacokinetics studies. You’ll learn the latest applications for determining how a drug is absorbed, distributed, metabolized, and excreted by the body.

Logically organized, Mass Spectrometry in Drug Metabolism and Pharmacokinetics starts off with an overview of the role of mass spectrometry in drug discovery and development. It then shows you how to perform routine applications before introducing you to sophisticated tools and techniques for solving complex drug metabolism and pharmacokinetics problems. Throughout the book, the main focus is on the use of LC-MS and MALDI-MS.

Each chapter has been written by one or more leading experts who share their own first-hand laboratory experience. You’ll gain a wealth of tips for performing successful analyses as well as avoiding common pitfalls. Moreover, the book is packed with features that help you grasp new concepts quickly and put your knowledge into practice, including:

• Figures and illustrations to help you understand how to perform specific techniques and applications

• Charts and tables that serve as quick summaries and reference guides

• Coverage of the latest tested-and-proven techniques

• References leading to the primary literature in the field

Carefully edited to ensure thoroughness and a uniform standard of quality, this book is sure to help you advance your own important drug research efforts.

Ragu Ramanathan received a B.Sc. in Chemistry from the University of Southern Mississippi and a Ph.D. in Physical Chemistry/Mass Spectrometry from the University of Florida. His graduate research focused on coupling of electrospray ionization (ESI) to Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. After spending three years as a postdoctoral research fellow with Professor Michael L. Gross at the Washington University, St. Louis, Missouri, Dr. Ramanathan managed the Center for Advanced Mass Spectrometry at the Analytical Bio-Chemistry Laboratories, Columbia, Missouri. In 1998, Dr. Ramanathan joined Schering-Plough Research Institute’s (SPRI) Drug Metabolism and Pharmacokinetics (DMPK) Department and completed his tenure as a senior principal scientist in 2008. While at SPRI, Dr. Ramanathan was involved in the application of LC–MS for profiling and characterization of metabolites of drug candidates in the preclinical development and clinical stages. Dr. Ramanathan was with Pfizer Global Research and Development from 1999 to 2002 as a group leader of the Ann Arbor site biotransformation group. Dr. Ramanathan is currently an associate director at the Bristol-Myers Squibb, Co. and is responsible for elucidating biotransformation pathways of development drug candidates. Dr. Ramanathan’s accomplishments include 35 peer-reviewed papers, 10 book chapters, and over 60 oral/poster presentations. He also served as a chairperson for the North Jersey ACS Mass Spectrometry Discussion Group and as a chairman for DMPK sessions of the American Society for Mass Spectrometry and Eastern Analytical Symposium meetings.

Mass Spectrometry in Drug Metabolism and Pharmacokinetics

John Wiley & Sons

Copyright © 2009 John Wiley & Sons, Inc.
All right reserved.
ISBN: 978-0-471-75158-8

Chapter One

Evolving Role of Mass Spectrometry in Drug Discovery and Development

Dil M. Ramanathan Kean University, New Jersey Center for Science, Technology & Mathematics Education, Union, New Jersey

Richard M. LeLacheur Taylor Technology, Princeton, New Jersey

1.1 ROUTE TO MARKET: DISCOVERY AND DEVELOPMENT OF NEW DRUGS

1.1.1 Industry Research and Development

The members of the modern biopharmaceutical industry are engaged in an on-going struggle to balance the needs of medicine and patient care with the demands of running a growing, profitable business. Moreover, new drugs must be proven to possess some combination of improved efficacy and safety compared with existing treatments. Success in drug research and development (R&D) is critical for meeting all of these objectives, and R&D efforts within the biopharmaceutical industry, as measured by spending, continue to grow steadily (Fig. 1.1). In recent years, the rate of annual growth in R&D spending has been between 5 and 10% in the United States, with the most recent data indicating that R&D spending in 2006 exceeded $50 billion (PhRMA, 2006).

The many essential steps in the discovery and development of new drugs can be measured by two primary benchmarks. The first, the number of filed and approved investigational new drug (IND) applications, represents the threshold to human (clinical) testing. The second, the number of filed and approved new drug applications (NDAs), represents the threshold to marketing a drug. These numbers and their trends can represent the relative success of R&D efforts.

Given the typical 12-15 years required to discover, develop, and test a new drug (Fig. 1.2), the NDA submission and approval data will in part represent R&D progress from several years earlier. Since the late 1990s, the annual rate of NDA submissions and approvals has declined. A similar decline has been observed in the number of NMEs (GAO, 2006). Of the 93 NDA approvals for 2006, only 18 are considered to represent NMEs (The Pink Sheet, January 15, 2007). While both total NDAs and NMEs are important, the number of NMEs approved represents a particularly critical measure of overall R&D success.

The statistics of expenditure and NDA approvals can mask a major source of R&D cost and frustration in the industry: late-stage development and postmarketing failures. These types of failures attract significant unwanted publicity and only occur after hundreds of millions of dollars have been spent. Well-publicized examples have included the recent late-stage failure of torcetrapib (Tall et al., 2007) and the postmarketing withdrawals of fenfluramine-phentermine (Fen-Phen) and Vioxx (Embi et al., 2006).

Consideration of IND trends is more encouraging (Fig. 1.3). IND filings occur years before NDA filings and represent a more recent state of R&D success. The number of compounds in clinical testing has approximately doubled over the last decade to approximately 3000 compounds in 2005 in the United States alone. A recent tally of new treatments in clinical testing for various indications is summarized in Table 1.1 (PhRMA, 2006). It is encouraging to see this increase in clinical testing, but it is also important to remember that only about 8% of early-stage clinical testing drugs will produce an approved NDA (Caskey, 2007).

1.1.2 Drug Discovery and Development Process

The overall process of bringing a new drug to market is typically divided into two principal areas: drug discovery and drug development. Examples of summaries describing the entire process include the publication entitled “Drug Discovery and Development: Understanding the R&D Process” (PhRMA, February 2007) and a tutorial written by Jens Eckstein, recently available online at www.alzforurm.org/drg/tut/tutorial.asp. The following description very briefly summarizes some of the steps in drug discovery and development.

1.1.2.1 Drug Discovery The first step in discovering a new medicine is to identify a therapeutic target. Drugs in today’s market as well as those in recent clinical testing target less than 500 biomolecules, with more than 10 times that many potential therapeutic targets waiting to be discovered and developed (Drews, 2000). More than 50% of the newly approved drugs result from R&D involving previously clinically tested and validated targets. Once a target has been validated (proven to be related to the disease process), high-throughput screening methods may be used to determine initial structural leads. Compounds are assessed for target affinity and for their “drug-like” properties, including absorption, distribution, metabolism, and excretion (ADME) using a series of in vivo and in vitro tests. The results of these tests are used to improve the structure and therefore the properties of the next round of test compounds, until ultimately one or more acceptable compounds are advanced forward in the process. This stage of discovery, which can be lengthy and difficult to predict, is generally referred to as lead optimization. The lead selection and lead optimization studies that are used to sift out the problematic compounds are summarized in Fig. 1.4.

Mass spectrometry enters into all phases of drug discovery (Feng, 2004; Lee, 2005). Early in the discovery, target proteins are identified and characterized by MS following LC or two-dimensional gel electrophoresis separation (Kopec et al., 2005; Deng and Sanyal, 2006). The make-up of an isolated protein is determined by enzymatically digesting the protein and then analyzing the peptides by MS (Link, 1999; Kopec et al., 2005; Kpke, 2006). Once a target is validated, compounds generated from any one of the following strategies are evaluated against the target: total synthetic process (33%), derivative of natural products (23%), total synthetic product with natural product mimic (20%), biological (12%), natural product (5%), total synthetic product based on a natural product (4%), and vaccine (3%) (Newman et al., 2003; Newman and Cragg, 2007). In almost all pharmaceutical companies, open-access MS laboratories have been set up to allow medicinal chemists to confirm and assess the purity of their synthesis or isolated products (Chen et al., 2007). Once the compounds or compound series are confirmed, high-throughput screening (HTS) assays are used to weed out compounds that do not show any activity toward a host [protein, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), etc.] (Fligge and Schuler, 2006). Mass spectrometric approaches also have been used to study noncovalent complexes involving protein-drug, DNA-drug and RNA-drug to identify structural details of the drug-binding sites (Benkestock et al., 2005; Siegel, 2005; Hofstadler and Sannes-Lowery, 2006; Jiang et al., 2007).

Compounds or compound series selected using HTS are further filtered using invitro-based solubility, chemical stability (Wilson et al., 2001), permeability (Bu et al., 2000a,b; 2001a-d; Mensch et al., 2007), and metabolic stability (Lipper, 1999; Thompson, 2000, 2005) assays before the lead selection/optimization stage (Lipper, 1999; Thompson, 2000, 2005). Most of these in vitro assays are faster, more efficient, and more sensitive due to unsurpassed involvement of the LC-MS (Thompson, 2001; Mandagere et al., 2002; Pelkonen and Raunio, 2005; Thompson, 2005). Results from such high-throughput in vitro assays are used to select compounds for additional in vitro tests and finally for in vivo testing in preclinical species (mouse, rat, dog, monkey, etc.). Similar to the early discovery stage high-throughput assays, LC-MS and LC-MS/MS assays are the methods of choice for the late-stage discovery studies (lead optimization stage, levels II and III) because they are rapid, sensitive, easy to automate, and robust.

All the discovery stage quantitative and qualitative LC-MS assays (levels I, II, and III), which are used to select drug candidates for development, are not rigorously validated and are not required to satisfy any of the good laboratory practices (GLPs) guidelines set forth by the regulatory agencies (Shah et al., 2000; Hsieh and Korfmacher, 2006; Jemal and Xia, 2006).

1.1.2.2 Drug Development The preclinical testing represents the bridge between discovery and later clinical (human) testing. As shown (Fig. 1.2), if 10,000 compounds enter the screening stage, only about 250 will make it into the preclinical testing stage. During this stage, critical assessments of drug candidate safety are obtained in toxicology studies. Also essential understanding of the ADME, pharmacokinetic (PK), and pharmacodynamic (PD) properties of the drug is established.

1.1.2.2.1 The Drug Substance Before starting any long-term toxicological studies in rodent (rat or mouse) and nonrodent (dog or monkey) species, it is imperative to work out all the chemical, pharmaceutical, large-scale synthesis, purification, stability, and formulation issues associated with the drug substance (Smith et al., 1996; van De Waterbeemd et al., 2001).

For a drug substance to move further in the development pipeline, its physical and salt forms have to be optimized in pharmacokinetics studies often using quantitative LC-MS/MS assays. Pharmaceuticals can exist as either a crystalline form (which has long- and short-range order in three dimensions) or an amorphous form (which lacks the long-range order present in crystalline material). In the discovery stage, usually all ADME assays (levels I, II, and III) are conducted using laboratory-grade amorphous drug substance without optimizing for physical and pharmaceutical properties of the drug (Kerns, 2001). Although the stability of an amorphous drug substance is sufficient for short-term discovery studies and for making internal recommendations, a crystalline form is the preferred form for long-term toxicological and clinical studies due to its long-term stability. However, the ability of a drug (organic molecule) to exist in more than one crystalline form leads to polymorphism. Polymorphs (same chemical composition but different internal crystal structure) of a given drug can have widely different pharmacokinetic parameters (Chapter 2), especially bioavailability due to differences in physicochemical properties such as dissolution rate, density, and melting point (Kobayashi et al., 2000; Agrawal et al., 2004; Panchagnula and Agrawal, 2004).

Changes in the method of synthesis during the large-scale manufacturing phase of drug development can also lead to changes in the crystalline form (Perng et al., 2003; Huang and Tong, 2004). A well-documented example of crystalline form change was observed with ritonavir (Norvir), a protease inhibitor approved in 1996 for treatment of HIV infections. In mid-1998, sales of ritonavir were temporarily halted due to manufacturing difficulties associated with multiple polymorphs (Bauer et al., 2001; Van Arnum, 2007). Later, in 1999, reformulation and additional LC-MS/MS-based pharmacokinetic studies allowed Abbott Laboratories to bring ritonavir back to the market. Today, the Food and Drug Administration (FDA) requires application of techniques such as X-ray diffraction and/or vibrational spectroscopic analysis [Fourier transform infrared (FTIR), near infrared (NIR), Raman] to characterize polymorphic, hydrated, or amorphous forms of drug substances and for further evaluation of pharmacokinetic parameters using the final thermodynamically stable form of the drug.

Salt form selection/finalization is another crucial step in preclinical development (Engel et al., 2000; Furfine et al., 2004). Some of the common pharmaceutical salts include hydrochloride, sulfate, mesylate, succinate, tartrate, acetate, and phosphate. Similar to the changes that occur in the crystalline form, the changes that occur in the salt form also alter the oral bioavailability of a drug. When the salt form of a drug substance is changed, quantitative LC-MS/MSassays are used to reassess the key pharmacokinetic parameters as well as bridge the new parameters with the discovery stage data, if necessary. Along with physical and salt form optimization, the drug substance is also subjected to acid, base, and photostability tests, and when necessary, degradants are identified using LC-MS and nuclear magnetic resonance (NMR) techniques.

Once the salt and physical forms of a drug substance are finalized and large-scale manufacturing issues are addressed, the NCE/NMEs recommended for development and human testing is often referred to as the active pharmaceutical ingredient (API). Around this stage of the preclinical development, several kilograms of the API are manufactured under good manufacturing practices (GMP) guidelines established by the regulatory authorities (Webster et al., 2001). At this stage, LC-MS and MS/MS methods are used to fully characterize the API and to identify any major impurities and degradants present in the starting materials and/or formed during API processing (Kovaleski et al., 2007). Once all the API impurity issues are worked out, the certified API is used for toxicological studies conducted in support of first-in-human clinical studies. The International Conference on Harmonization (ICH) guidelines on the API suggest that impurities .0.15% and .0.05% respectively for [less than or equal to] 2 g and .2 g daily dose should be characterized and the impurity levels should be reduced if there are any known human risks.

Before the start of toxicological studies, an LC-MS/MS method to quantify the drug substance and/or its metabolites in plasma is developed using the certified API. This quantitative LC-MS/MS assay is developed under GLP guidance. Most often a stable isotope labeled form of the drug is used as the internal standard to correct for any experimental limitations. Upon completion of the rodent and nonrodent toxicological studies using the quantitative LC-MS/MS assays, safe human doses to be used in the first-in-human study come to light and the pharmaceutical company is ready to file for an IND. For perspective, the total testing regime up to this stage is estimated to consume about one-quarter of the total R&D expenditure in the industry (PhRMA, 2006). Of the 250 compounds that entered preclinical testing, only 5 on average will advance into human clinical testing.

1.1.2.2.2 Clinical Trials Once an IND is approved, clinical trials take place typically in three sequential phases, phases 1-3. However, based on the recent FDA guidelines, traditional phase 1 studies could be preceded by “phase 0” or “exploratory IND” studies. These studies involve the administration of a single subtherapeutic dose of a radiolabeled NME to healthy adult volunteers to assess the human pharmacokinetics and/or metabolism (Lappin and Garner, 2005; Hill, 2007). Subtherapeutic doses are defined as the smaller of either 1/100 of the expected pharmacologically effective dose, or 100 mg. The FDA guidelines also require animal toxicity studies to be completed using doses above the human subtherapeutic doses to show no risk of toxicity before starting phase 0 clinical studies. Phase 0 studies may allow identification of “less promising” compounds earlier and at lower cost. According to a recent presentation, phase 0 studies can shorten the drug development process by 6-12 months (Kummar et al., 2007). However, most of the phase 0 studies cannot be completed using conventional LC-MS techniques because administered doses are around 100 g and require the use of accelerator mass spectrometry (AMS), the only ultrasensitive technique capable of quantifying [14.sup.C]-labeled compounds with attomole ([10.sup.-18] M) sensitivity (Chapters 2 and 7). However, several laboratories are hard at work developing ultrasensitive LC-MS techniques capable of detecting drugs and/or metabolites from microdosing studies (Lebre et al., 2007; Seto et al., 2007; Yamane et al., 2007).

Phase 1 clinical trials are conducted on a small number (20-100) of healthy adult volunteers to determine the potential toxicity of a drug, whether severe side effects can occur, and safe dosage ranges. An assessment of pharmacokinetics and drug metabolism is also included. For obtaining all the PK parameters, quantitative LC-MS/MS assays developed under GLP guidance are used. However, metabolism studies are conducted using non-GLP-based qualitative LC-MS and LC-MS/MS methods to get a glimpse of the metabolites present in human plasma and urine (Chowdhury, 2007; Ramanathan et al., 2007c; Ramanathan et al., 2007d). In specialized cases, phase 1 trials may include subjects with the targeted disease (e.g., oncology drugs). Overall, the critical criteria for phase 1 are the safety profile of the drug and determination of a safe dosage.

(Continues…)

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