CHAPTER 15

Mass Spectrometry for the Studyof Peptide Drug Metabolism

PATRICK J. RUDEWICZ

15.1 INTRODUCTION

The drug discovery and development process is a time and labor-intensive endeavor.

The time from identification of a biological target to the registration of a new drug

at the conclusion of clinical trials may take on average 12 years and cost 800 to

900 million dollars [1]. Drug discovery is often described as a funnel where hundreds

of thousands of compounds are initially screened for potency against a biological

target by using high-throughput screening. At this early stage, chemical hits are

ligands having a large affinity for a particular biological target. The hits, however, do

not necessarily have the pharmaceutical properties desired in a drug. It is in the hit-to-

lead and lead optimization stages that pharmaceutical scientists optimize absorption,

distribution, metabolism, and excretion (ADME) qualities. At this juncture of drug

discovery, drug metabolism scientists have the largest impact through the judicious

use of screening and mechanistic studies so that the resulting compounds not only

have the required in vitro and cellular potency but also enter systemic circulation and

reach optimal concentrations at the therapeutic target.

When a drug is administered orally, it becomes necessary to overcome several

barriers to reach levels in blood required to have the desired pharmacological

effect. First, the drug undergoes dissolution within the stomach where the com-

poundmust possess adequate solubility and stability at low pH. As it passes through

the intestines, the drug needs to possess the necessary permeability to be absorbed.

Permeability may be described as either passive diffusion through the lipid bilayer

or active uptake by transporter proteins anchored in the lipid bilayer. Once

absorbed, the drug passes through the portal vein into the liver where it may be

Protein and Peptide Mass Spectrometry in Drug Discovery, Edited by Michael L. Gross, Guodong Chen,and Birendra N. Pramanik.� 2012 John Wiley & Sons, Inc. Published 2012 by John Wiley & Sons, Inc.

435

metabolized by enzymes located in hepatocytes including the cytochrome (CYP)

P450 enzymes which are mainly responsible for what is referred to as Phase I

oxidative metabolism.

Phase II metabolic reactions are conjugation reactions including glucuronidation,

sulfation, and amino acid conjugation. Conjugation of small endogenous molecules

may occur directly to the parent drug molecule or to a Phase I metabolic product such

as a hydroxylated drug molecule. These reactions increase the polarity of the drug

molecule so that it may more easily be excreted via the kidneys or in bile. Apart from

the liver, metabolizing enzymes are located in blood and other organs and peripheral

tissues, including the intestines. Similarly uptake and efflux transporter proteins are

located in many tissues, including hepatocytes, and there exists an interplay between

transporters and P450 enzymes. For example, a drug molecule may be effluxed out of

an enterocyte in the small intestine by a P-gp transporter and then metabolized by

CYP3A4 [2].

The goal in drug metabolism and pharmacokinetics (DMPK) groups is to eliminate

compounds that are poor candidates for pharmaceutical development. This is achieved

by implementing metabolism screening assays that have a relatively high throughput,

meaningful endpoints, and low compound requirements. Typical in vitro screens

include P450 inhibition, permeability, metabolic stability, transporter, including

P-lycoprotein interaction, and plasma protein binding. In vivo screens often include

IV/PO pharmacokinetic screening in mouse and rats.

15.2 PEPTIDE DRUG METABOLISM

The metabolism of peptide and protein drugs is governed by hydrolysis of peptide

bonds by peptidases found in blood and in various tissues [3–5]. These metabolizing

enzymes can restrict the half-life of peptides administered orally to range from a few

minutes to an hour; hence the drug may not reach the desired concentration at the

target tissue. Peptide bond hydrolysis is catalyzed by peptidases, which are ubiquitous

in body tissue. Metabolism of peptide drugs can be assessed in vitro using micro-

somes, hepatocytes andCaco-2 cells [6–8]. Some endopeptidase activitymight be lost

in cryopreserved compared to fresh hepatocytes. Rat brain synaptosomes can be used

for the evaluation of synapticmetabolism of investigational neuropeptides [9]. In vivo

identification of peptide drug metabolites is often performed using samples from

plasma, urine, bile, and feces after an investigational drug is dosed in animal species.

In addition to metabolism, due to the hydrophilic nature of most peptides, renal

excretion also contributes to the rapid clearance of peptide drugs.

As an example, the peptide drug metkephamid undergoes extensive hydrolysis by

peptidases at the surface of endothelial cells in the intestine and also at the surface of

hepatocytes, limiting its overall bioavailability to 30% [10]. Certain peptides can also

undergo phase I oxidative metabolism typical of other small molecule drugs. The

metabolism of cyclosporine A, a cyclic undecapeptide, occurs principally in the liver

and yields mono- and di-hydroxlylated metabolites as well as N-demethylated

metabolites [11,12].

436 MASS SPECTROMETRY FOR THE STUDY OF PEPTIDE DRUG METABOLISM

Absorption of peptide drugs across intestinal cell membranes is limited by their

hydrophilicity and molecular mass. Foger et al. [6] reported the correlation of in vitro

models for studying peptide drug absorption and showed that the molecular mass

of peptides correlates linearly with permeability in rat in vitro models. In addition

some peptide drugs may be substrates for P-glycoprotein efflux transporters that

further limit their absorption across intestinal and blood–brain membranes [6].

Several approaches can be employed to improve the stability of peptide drugs

toward proteolytic enzyme degradation as well as enhance membrane permeability.

These include the inclusion of D or unnatural amino acids, chemical modification of

N or C termini, creating cyclic analogues, the synthesis of peptidomimetics, and the

introduction of structural constraints. These approaches often lead to peptides that

possess improvedmetabolic properties and better bioavailability. The plasma half-life

of somatostatin was improved from a few minutes to 1.5 h by shortening the number

of amino acids and replacing selective L-amino acids with D-amino acids [5].

The addition of polyethylene glycol (PEG) or polymers of N-acetylneuramic acid

(polysialic acids) to peptide or protein drugs can also dramatically improve half-

life [13]. This is accomplished by providing a steric barrier to proteolytic enzymatic

cleavage and by increasingmolecular mass, thereby reducing renal excretion. Several

PEGylated protein drugs are on the market including PEGylated alpha-interferon for

the treatment of hepatitis C. PEGylated alpha-interferon has a half-life of 50 h,

whereas native alpha-interferon has a half-life of only 5 h [13]. Cyclic peptides (both

head-to-tail and side-chain to side-chain) are known to be more resistant to peptidase

hydrolysis and consequently may possess improved half-lives for metabolism when

compared to their linear analogues [14]. For example, cyclic growth regulating factor

(GRF) (1–29)-NH2 has a half-life of 2 h whereas the half-life of the linear analogue is

only 13min [5]. Cyclic peptides also display improved membrane permeability

compared to that of linear analogues [14–15]. This may be explained not only by

their increased resistance to proteolytic cleavage but also by facilitation of internal

hydrogen-bonded conformations and by the reduction of charged termini. Branched

peptideshave alsobeen shown to resist proteolytic cleavage byproteases andpeptidases

in vivo [16].

15.3 LC-MS/MS FOR METABOLITE IDENTIFICATION

Metabolite identification of drug candidates is done at various stages of the drug

discovery and development process. In the lead optimization stage, structural

elucidation of metabolites is often sought to identify metabolism “hot spots” that

may lead to poor pharmacokinetic properties, including high clearance and short half-

life clearance. This is usually accomplished by using samples from both in vitro

experiments (e.g., hepatocyte incubations) and invivo studies in rodents [17].Another

important reason for performing metabolite identification in drug discovery is to

identify and minimize the formation of any reactive metabolites that may covalently

bind to macromolecules and potentially form organ toxicity or idiosyncratic drug

reactions [18,19].

LC-MS/MS FOR METABOLITE IDENTIFICATION 437

In preclinical development, metabolite identification aids in the selection of the

proper rodent and non-rodent species for toxicology studies [20]. The purpose is to

ensure that the biotransformation pathways in the preclinical toxicology species

are similar to humans and that there are no human-specific metabolites. As part of the

preclinical development package, mass balance studies are performed [21]. These are

done in rat and also in a non-rodent species (e.g., dog or monkey). The goal is to gain

an in-depth understanding of the metabolic fate and clearance of a drug. The studies

are performed by administering radiolabeled drug, usually 14C, to animals and

collecting plasma, urine, feces, and sometimes bile at various time intervals after

dosing. Radiometric scintillation detection is used to determine the percent of the dose

eliminated by liver or kidneys as unchanged drug or metabolites. LC/MS/MS with

flow scintillation detection is used to gather a profile of the metabolic fate of the drug

and to identifymetabolite structures.Given that authentic standards ofmetabolites are

not always available, quantitation is accomplished by using radiolabel detection. At

this stage the contributions ofmetabolites to clearance, efficacy, and toxicitymay also

be evaluated.

In clinical development, metabolite identification is carried out as part of the

human absorption, metabolism, and excretion study in which a radiolabeled drug is

administered to humans. Evaluation and identification of all metabolites formed in

humans is essential to ensure that they are also formed by the preclinical toxicology

species at an adequate level.

Metabolite identification is done using LC/MS/MSwith electrospray ionization in

conjunction with various types of mass spectrometers. In the 1980s, tandem MS was

introduced for metabolite identification of Phase I metabolites [22]. This approach

was extended to on-line LC-MS/MS with neutral loss and precursor ion scans for

rapid identification and structural elucidation of secondary drug conjugates, including

glucuronides and aryl sulfate esters [23].

Triple quadrupoles mass spectrometers with electrospray ionization are still used

today for metabolite identification in biological matrices. Other mass analyzers,

however, now supplement the triple quadrupole. Ion trapmass spectrometers have the

advantages of increased sensitivity in the full-scan product ion mode relative to triple

quadrupole mass spectrometers [24]. Another advantage is the capability of MSn

production scanning to enhance structural information for unknown metabolites.

Disadvantages include in-trap space charging, which limits dynamic range in

quantitative analysis, and the lack of conventional neutral loss and precursor ion

scanning. Hybrid triple quadrupole-linear ion trap mass spectrometers, introduced

in 2002, are now used extensively for both quantitative analysis and metabolite

identification [25]. They maintain all the functions of a triple quadrupole mass

spectrometer and hence can be used not only for routine quantitation but also for

acquisition of enhanced sensitivity product-ion spectra of metabolites. Hybrid

quadrupole-linear ion traps can also be employed for simultaneous metabolite

identification and quantitation of drugs and their metabolites in plasma, thereby

improving throughput for the metabolite identification process [26].

High mass-resolving power mass spectrometry is an important tool for metabolite

identification; it aids in the assignment of structurally informative fragment ions and

438 MASS SPECTROMETRY FOR THE STUDY OF PEPTIDE DRUG METABOLISM

alsohelps todifferentiate between isobaricmolecular ions. The advent of high resolving

power hybrid orthogonal quadrupole time-of-flight (QqTOF) instruments makes

accurate mass measurements more accessible to pharmaceutical metabolism laborato-

ries [27]. These instruments are routinely used at mass resolving powers of 30,000

allowing accurate masses to be assigned within 3 ppm mass accuracy. A more recent

development that has also had a large impact in metabolite identification is the

introduction of the LTQ-Orbitrap [28]. This instrument combines a linear ion trap

with an Orbitrap mass analyzer with Fourier-transform detection. With the Orbitrap,

online LC/MS/MS experiments may be done with a mass-resolving power of 100,000

and concomitant improved accurate mass measurements.

15.4 QUANTITATIVE ANALYSIS

Quantitation in drug metabolism and pharmacokinetic studies is most often per-

formed by using triple quadrupole mass spectrometers in the selected reaction

monitoring (SRM) mode with electrospray ionization. In the SRM mode the

protonated molecule of the compound of interest is selected with the first quadrupole

and undergoes collisional-induced dissociation in the second quadrupole to yield

product ions. Usually one or two of themost abundant fragment ions aremonitored by

using the third quadrupole. This approach has been routinely applied in drug

metabolism laboratories since the early 1990s. Analytical standards are used to

generate a calibration standard curve, and the amount of drug is determined by back

calculation from a linear regression.

Quantitative determination of larger molecular weight peptides is also done using

enzyme-linked immunosorbent assays (ELISA). The technology is very sensitive

with lower limits of quantitation of 1 to 10 pg/mL, and sample analysis is fast and

amenable to automation. Method development for an ELISA assay, however, may

take months owing to the time required to generate and screen antibodies. Further-

more ELISA assays have limited dynamic range, and the lack of selectivity is also a

concern. Endogenous antibodies directed against the therapeutic agent can interfere

with ELISA assays. Because of these limitations, ELISA assays for peptide drugs are

often replaced by more specific LC/MS/MS assays using either conventional sample

preparation such as solid-phase extraction or immuno-capturing of the analyte [29].

Quantitation in drug metabolism studies is also done by using 14C or 3H radio-

isotope labeled drug [30]. The use of radiolabeled compound obviates the need to

synthesize analytical standards for each drug metabolite to be used for normal “cold”

LC/MS/MS assays. The radiolabeled atoms, however, need to be incorporated into the

molecule in such a fashion that the majority of the metabolites also contain the

radiolabel. This approach presents a limitation in themetabolism of peptide drugs that

easily hydrolyze to release the labeled part of the molecule; thus, major metabolic

pathways may be missed during metabolic profiling when using radio-chemical

detectors. Hence dual radiolabels are sometimes employed for drugs that contain

peptide bonds so that the metabolism may be tracked at different sides of the

molecule [31].

QUANTITATIVE ANALYSIS 439

15.5 CASE STUDY: IL-1b PROTEASE INHIBITORS

Interleukin-1b (IL-1b) converting enzyme (ICE) is a cytoplasmic cysteine protease

that is responsible for the conversion of inactive 31 kDa precursor IL-1b to the active

17.5 kDa IL-1b by cleavage of the peptide bond between Asp 116 and Ala 117.

The 17.5 kDa active form is secreted into extracellular fluid and has systemic effects.

Elevated levels of IL-1b are present in patients with infections, inflammation,

rheumatoid arthritis, and other diseases [32]. The goal of this particular drug

discovery program was to produce an orally available compound with the possibility

of reducing IL-1b processing by inhibiting ICE. The rationale was that inhibition of

IL-1b processing might allow modulation of its pathological effects while leaving its

immunological functions intact.

The compounds that were initially screened for activity as ICE inhibitors were

small di- and tri-peptides that also contained various leaving groups necessary for

in-vitro enzyme and cellular activity against ICE (see Figure 15.1 for the structures

of three such ICE inhibitors). One is a tripeptide, referred to as Z-Val-Ala-Asp-TPP,

and two others are dipeptides, Z-Val- Asp-TPP and Z-Val-Asp-DPP.

As part of the later stage selection of a lead compound, oral bioavailability studies

are run, as they were here in the dog. The assessment of bioavailability is a key step in

the design of an orally active therapeutic molecule. For non-rodent species, like dogs

and monkeys, often the drug candidate is dosed to an animal intravenously (IV) and

FIGURE 15.1 Structures of IL-1b protease inhibitors.

440 MASS SPECTROMETRY FOR THE STUDY OF PEPTIDE DRUG METABOLISM

then after a washout period, the compound is administered to the same animal orally.

The ratio of the area under the curve of the concentration time profile after an oral dose

(AUCoral) to the AUCiv normalized to any difference in dose is defined as bioavail-

ability (%F). In this study the IVand oral doseswere given 3 h apart in the same animal

as a means to reduce intra-animal variability. Any contributions of AUCiv to AUCoral

were deconvoluted by using Monte Carlo calculations [33].

In addition compounds of interest can be administered together as a “cassette”

rather than individually administered on separate occasions. The cassette approach

has been utilized to enhance throughput of in vivo PK assessments in drug discovery.

For these studies the three compounds were administered as a mixture both IV

(2mg/kg each) and then 2.5 h later, orally (5mg/kg each). Plasma samples were

collected using EDTA as the anticoagulant at various time points out to 24 h. Protein

precipitation with acetonitrile was used for sample processing. Sample introduction

was accomplished with a fast 5-min gradient on a C18 HPLC column. The analytes

were quantitated by using ionspray on a triple quadrupole mass spectrometer. The use

of a triple quadrupole mass spectrometer in the SRM mode added specificity to this

analysis and allowed successful calibration from 1 to 1000 ng/mL.

The resulting IV/PO PK profiles for each compound (Figure 15.2) show that

the tri-peptide Z-Val-Ala-Asp-TPP and the di-peptide Z-Val-Asp-DPP had poor

bioavailability of less than 5%. The dipeptide Z-Val-Asp-TPP, however, had a much

better bioavailability of 47%. As shown in Figure 15.3, the plasma versus concentra-

tion profiles are similar when administered either as a cassette or individually.

To investigate the mechanistic reasons for the large difference in %F for Z-Val-

Ala-Asp-TPP and Z-Val-Asp-TPP a discovery excretion/ mass balance study was

performed in dog using 14C radiolabeled compounds. The radiolabel was located on

themethylene carbon of the Z group. The labeled compoundwas administered to dogs

at 2mg/kg oral, and urine and feces were collected at various time intervals out to

FIGURE 15.2 Plasma concentration versus time profiles.

CASE STUDY: IL-1b PROTEASE INHIBITORS 441

120 h; plasma was collected at 1 and 4 h. Total radioactivity was measured in feces,

plasma, and urine by using a scintillation counter.Metabolite profiles and LC/MS/MS

spectra for metabolite identification were recorded by splitting the LC effluent and

directing 33% of the flow to the mass spectrometer and 67% to a flow scintillation

detector (Figure 15.4). The plasma radio chromatogram for Z-Val-Ala-Asp-TPP from

a 4-h post oral dose time point is shown in Figure 15.5.

To ascertain the structure of themetabolites, product-ion spectra were obtained for

all radiochromatogram metabolites as well as for the parent drug. The product ion

spectrum for the parent compound, Z-Val-Ala-Asp-TPP (Figure 15.6) shows the

protonated molecule at m/z 662. Major product ions are produced by the loss of the

FIGURE 15.3 Comparison of plasma concentration versus time profiles for discrete versus

cassette dosing.

HPLC

Mass Spectrometer

Radiometric Flow Detector

Radiocarbon Chromatogram

LC/MS Chromatogram

0 5 10 15 20 25 30 35 40 45

0 5 10 15 20 25 30 35 40 45

Time (min)

Time (min)

Injector

100

80

60

40

20

0

100

80

60

40

20

0

Extracted Samples

FIGURE 15.4 Schematic for radio-profiling/metabolite identification experiment.

442 MASS SPECTROMETRY FOR THE STUDY OF PEPTIDE DRUG METABOLISM

TPP (ion of m/z 434), the loss of the Z group and TPP to give an ion of m/z 344, and

loss of CO2 from both ions of m/z 434 and 344. From the product ion spectra, it was

determined that the largest circulating metabolite in plasma, with a retention time of

5min, is Z-Val, which is formed by peptide-bond hydrolysis. Similarly the smaller

radiolabeledmetabolite isZ-Val-Ala. Thedogurine radiochromatogram (Figure 15.7)

shows only one peak corresponding to Z-Val indicating complete peptide-bond

hydrolysis and excretion of Z-Val as the major metabolite. As expected, the major

10000

Z-Val

DOG

Compound B

Z-Val-Ala

TIME (min)

8000

6000

DP

M

4000

2000

00 5 10 15 20

FIGURE 15.5 Radiochromatogram of dog plasma for Z-Val-Ala-Asp-TPP.

FIGURE 15.6 Product ion spectrum for Z-Val-Ala-Asp-TPP.

CASE STUDY: IL-1b PROTEASE INHIBITORS 443

biotransformation pathway for Z-Val-Ala-Asp-TPP is peptide bond hydrolysis,

resulting in low oral bioavailability (5%) for this compound (Figure 15.8).

To summarize the conclusions for the dog excretion/mass balance study for Z-Val-

Ala-Asp-TPP, themajor route ofmetabolismwas hydrolysis of peptide bonds to yield,

Z-Val (major) and Z-Val-Ala (minor). Themajor plasmametabolite at both 1- and 4-h

post oral dose is Z-Val. The major metabolite in urine is also Z-Val.

The results of the mass/balance study for the di-peptide Z-Val-Asp-TPP showed

that Z-Val-Asp-TPPwas excreted intact in feces, indicating that biliary excretion was

O NH

O

COOH

CH3 CH3A16168

8084

0

0 (min)

Time (min)

DPM

5 10 15

Z-VAL

FIGURE 15.7 Radiochromatogram of dog urine for Z-Val-Ala-Asp-TPP.

FIGURE 15.8 Metabolic pathway for Z-Val-Ala-Asp-TPP in dog.

444 MASS SPECTROMETRY FOR THE STUDY OF PEPTIDE DRUG METABOLISM

a major mechanism of clearance (data not shown). Interestingly peptide bond

hydrolysis is not a major route of metabolism for this compound. Presumably

transporters assist in the uptake of this compound across the gut endothelial cells

and result in improved absorption into the portal vein and higher levels in systemic

circulation. The result is better oral bioavailability of approximately 45%.

15.6 FUTURE DIRECTIONS

One areawhereMSwill play a larger role in the future is in the quantitative analysis of

protein and antibody drugs. As was previouslymentioned, this is presentlymost often

done using ELISA assays that are easily automated and have excellent sensitivitywith

limits of detection in the low pg/mL range. MS is more specific, and method

development is faster because antibodies do not need to be prepared and character-

ized. Recent reports in the literature describe the analogous use of MS for protein

quantitation by using LC/SRM-MS for the detection of specific signature peptides

released by enzymatic digestion [34,35].

MS will become increasingly integrated into labs that now use ELISA, at the very

least to verify specificity during optimization of ELISA assays and also to evaluate

novel biomarkers. The sensitivity of MS detection with conventional (nonimmuno-

affinity sample preparation) has reached the low ng/mL range; hence at this time, MS

is most useful for preclinical toxicology study support. Micro-chromatography and

nano-ESI will be exploited to achieve better sensitivity for target protein quantitation.

Continuous advances in MS instrumentation will enhance sensitivity and selectivity

so that MS will begin to play a larger role not only in peptide quantitation and

metabolism but also for proteins.

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