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European Biopharmaceutical Review
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Rohan A Thakur and Michael Koleto at
Taylor Technology explore fast pharmacokinetic and semi quantitative
analysis of metabolites using high resolution mass spectrometry
To fail fast and fail often, it becomes essential to eliminate poor
candidates as early as possible in the drug development process.
Unfortunately, despite significant strides made in computerised
prediction modelling (in silico), there remains a lack of congruence between the data from in vitro experiments and the in vivomodels. Unfavourable pharmacokinetic (PK) properties (such as poor
absorption, low bioavailability and rapid clearance) have frequently
been the reasons for failure of a new chemical entity (NCE) in the
clinic. This realisation led to the application of high-throughput
principles to the in vivolead optimisation process, wherein the NCE is first given to rodents to
determine the PK profile in a rapid and limited study. This rapid
primary screen is known as fast PK and several critical decisions are
based on this initial study.
While triple quadruple systems have been dominant in pharmaceutical
bioanalysis, recent fundamental advances in mass spectrometry (MS)
technology have resulted in a new type of mass analyser known as the
Exactive Orbitrap (OT), which has the potential to revolutionise drug
discovery. Much like a triple quadruple, OT technology can enhance the
lead optimisation process by providing PK data for the NCE, but with
the added capability to provide simultaneous, semi-quantitative
information on metabolites present in the incurred sample. This allows
informed decisions to be made further upstream in the discovery
process, thereby reducing the cost of new drug development.
THE IMPACT OF MASS SPECTROMETRY ON FAST PK
Terms like cassette dosing PK (NCE cocktail dosed per animal),
cassette accelerated rapid rat PK (one NCE per animal, every 24 hours),
fast PK (one NCE per animal, every eight hours) and snapshot PK (one
NCE mouse model, every three hours) became the norm for improving in vivocycle time for lead optimisation studies within Big Pharma. These
approaches are important because they can either reduce the number of
animals involved, or the number of samples to be analysed and, in many
cases, reduce both. In addition, for a large majority of cases, rodent
fast PK studies correctly predict human PK, thus increasing the success
rate of the NCE as it moves down the pipeline.
A basic fast PK rodent assay includes formulation, dosing and
bioanalysis (plasma) of six time-points in six rats (three intravenous
and three oral) in six hours. The full PK rodent assay would extend the
time-points to eight and out to 24-hours postdose. There are several
advantages to this approach. Firstly, it provides information to the
synthetic chemists so that the chemical structure of the NCE may be
fine-tuned. Secondly, it allows for better design of in vivoefficacy studies, benefiting PK observations. Essentially, the fast PK
study validates oral bioavailability (solubility) and intestinal
absorption (permeability) characteristics – the two key parameters
indicative of future success.
Fast PK quantitative studies are traditionally performed using a
technique known as selected reaction monitoring (SRM). In the SRM mode,
the ions specific to the NCE are focused onto the detection system,
while all other non-specific ions are filtered away. As a result, only
the NCE is quantified and its PK estimates determined, but any
metabolite information (what and how much) contained in the sample is
lost. Along with the understanding of this limitation of the
SRM technique is the acknowledgement that potentially efficacious NCEs
may be eliminated prematurely due to poor PK characteristics, when in
fact more complete feedback from the same fast PK study could provide
the basis for the decision to make a simple tweak of the chemical
structure to improve bioavailability. This is where the OT technology
has the potential to revolutionise lead optimisation, providing
metabolite information during the fast PK analysis.
The fundamental driver for the use of HRMS for quantitative bioanalysis
is ease-of-use. Bioanalysis can be performed without any prior
knowledge of the compound, thus avoiding the need for compound specific
tuning (setting up the SRM transition, for example). This has
significant ramifications in fast PK applications, especially since
biotransformation information can now be provided. The Thermo
Scientific Exactive Orbitrap, for example, offers the ability to
quantify both the NCE and its major biotransformation products
simultaneously. It uses high resolution and accurate mass capability to
filter away chemical noise, and captures more information per scan.
Triple quadruple MS instruments usually have a resolution value in the
few hundreds. The Exactive Orbitrap operates at a nominal value of
10,000, which can be increased with a click of a button to 100,000, if
needed.
When using HRMS, compound-specific MS/MS tuning is no longer required
for specificity; just a very high resolution (more than 10,000) in the
full MS mode is sufficient. Since the fragmentation process
intrinsically reduces sensitivity, only signal-to-noise ratio is
gained; HRMS full MS mode applications theoretically do not have this
limitation. The SRM scan can be viewed as the sum of the ionisation
efficiency and fragmentation efficiency, whereas HRMS is simply
dependent on the ionisation efficiency. Tuning several SRM transitions
every day in a discovery setting is time consuming and, although
automatic tuning algorithms have been developed, human nature precludes
blind acceptance. The OT’s accurate mass functionality means it can
provide elemental composition information, allowing identification of
biotransformation products (metabolites), while it is performing fast
PK bioanalysis. The goal of having an analyst simply put the 96-well
plate into the LC-MS platform and hit the ‘start’ button to perform the
bioanalysis with a high degree of confidence is now becoming possible.
BIOANALYSIS & SEMI-QUANTITATIVE ANALYSIS OF METABOLITES USING HRMS
Figure 1 (page 55) shows the comparison between bioanalysis results
from a triple quadruple and the Exactive Orbitrap. A perfect
correlation would indicate a slope of 1 and the quantitative
performance is almost indistinguishable between the two instrument
platforms for discovery applications where the lower limit of
quantification (LLOQ) is usually between 1 and 5ng/ml (see Figures 2
and 3).
To further prove the quantitative performance using HRMS, 17
commercially available compounds were quantified in the range of 1 to
50ng/ml without tuning any parameters in rat plasma (see Figure 4).
This is important because one key application of the Exactive OT
platform in discovery applications is the semiquantitative analysis of
metabolites during the first in vivoPK study. To test this hypothesis, Chlorprazomine was incubated with
microsomes for 60 minutes, and the metabolites identified and
quantified without any a priori knowledge of the Exactive OT platform (see Figure 5).
The key to identifying metabolites ‘on-the-fly’ using HRMS is utilising
the advantage provided by ‘mass defect’, which is the difference
between the exact mass and the integer mass (the sum of the number of
protons and neutrons), as a result of mass deficiency. This mass
deficiency is due to the fact that 12C is the only isotope with an
exact mass integer of 12.00000 (ad infinitum) and used arbitrarily to
set the atomic mass scale for all other elements. Therefore, 16O
has an isotopic mass of 15.9949u, and is deficient by 5.085mmu.
Interestingly, 1H precedes 12C and therefore weighs 1.0078u, gaining
almost 7.825mmu per 1H. Since 1H is ubiquitous, a typical small molecule containing 40 hydrogen atoms can lead to a positive mass defect of about +0.3Da.
A positive mass defect is also referred to ‘mass sufficiency’ as
opposed to ‘mass deficiency’, typically exhibited by elements with mass
numbers higher than 12C on the periodic table (14N, 16O, 32S, 35Cl). Instruments such as the OT can also determine the mass difference between the 1H (1.007824Da) and a proton +H
(1.007276Da), which is deficient of 1 electron or 0.000548Da. This
becomes a factor for the analysis of peptides which have multiple
charges (z=n, where n>1), and the difference in mass can be used to
an advantage.
Since the common Phase I and Phase II metabolites have their own mass
defects, upon biotransformation they become additive to the precursor
ion. Thus, their masses can be simply added or subtracted from the
exact measured mass of the precursor ion, and metabolites can be
quickly identified, and then quantified. The quantitative analysis is
performed against the precursor, and since the response factors for the
metabolites are not available at this early stage, the quantitative
analysis is at best semiquantitative or relative to the parent. This is
a new and exciting application of HRMS for drug discovery applications,
where metabolism information can be provided to the synthetic chemists
as well as the pharmacologist to further improve the viability of the
NCE under study.
For example, the rapid formation of an active ‘hydroxy’ metabolite (+15.9949) during the first in vivoPK study where the NCE is dosed both orally and via IV, can provide
valuable information in terms of potential bioavailability, despite
poor oral PK profile. Based on its plasma concentration, the common
β-blocker, Alprenolol, for example, is more active given a single oral
dose than when given via IV, despite exhibiting poor oral
bioavailability in terms of PK. Alprenolol forms an active 4-
hydroxyalprenolol metabolite responsible for therapeutic action. Using
HRMS, this information can now be available in the very first in vivo study,
compared with the traditional SRM technique, which focused only on the
precuror ion (NCE only) and is blind to all other information
(metabolites) in the incurred sample.
CONCLUSION
Mass spectrometry (LC-MS) has played a major role in drug discovery
applications and provides the ADME research scientist with a powerful
analytical tool. Over the past two decades, bioanalysis has been
performed using SRM on a triple quadruple with a singular focus on
small molecules. With the emergence of biopharmaceuticals and the
advances in TOF and OT MS, it is quite likely that the ease-of-use
offered by these techniques is catching up with the sensitivity and
specificity requirements of non-regulated bioanalysis and perhaps
signalling a paradigm shift.
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