Predicting the ADME and Toxicity Characteristics

14 Pages·2014·0.66 MB·English

Challenges and Prospects in
Predicting the
ADME and Toxicity
Characteristics
of Drugs in Humans
Dr. Nico Scheer, Ph.D
Abstract
This white paper attempts to address one of the key
challenges of drug development: Making research in
discovery and preclinical development more predictive
of the results that will be found in patients later
in clinical development.
We will address this issue by examining some of the key obstacles that prevent the
compatibility of early stage in vivo studies with clinical trial data and the limitations
in the quality and availability of currently used in vitro tools. We will also offer
insights on how such challenges are being overcome through the application
of new translational science tools.
The topic of this paper is of interest to scientists involved in commercial
drug development at biopharmaceutical firms, contract research organizations,
academia, and other organizations working in the areas of drug metabolism,
safety assessment, infectious diseases and liver cancer.
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The challenge of predictability
in non-clinical and clinical
development studies
One of the major challenges of the pharmaceutical industry is the notoriously
high drug attrition rate in clinical trials, with only about 10% of compounds
entering first-in-man testing actually getting market approval. In fact, the
number of annual approvals for new chemical entities has stagnated for
more than a decade, though the money spent on drug development has
significantly increased1, 2.
The low clinical success rates imply a limited predictability and/or poor quality of
preclinical test systems. In this regard, it has been recognized that due to species
differences it is often difficult to extrapolate results obtained from wild type
preclinical animal models to the human situation. For example, the utility of the
traditional 2-year rodent carcinogenicity study with its protracted duration, large
cohort sizes and significant cost has been questioned for a long time because of
high numbers of false positive results and ambiguous relevance to human risk
assessment. Furthermore, the pathways defining drug metabolism, disposition and
toxicity can differ significantly between species, limiting the utility of traditional
animal models to predict the pharmacokinetic, drug-drug interaction and safety
profiles of compounds in humans. Finally, though human hepatocytes are routinely
used to predict drug responses in man, the limited availability, inconsistent quality
and high costs of these cells have prevented their more widespread application.
Approaches to address the
challenges in drug development
Significant efforts have been made by researchers to address the challenges in
drug development in general and to improve human predictions with regards to
drug metabolism, disposition and toxicity in particular. Important achievements in
this regard, for example, are the increasing availability of well-characterized in vitro
tools and assays, the improvement of mathematic models and in silico methods,
and the progress in the field of personalized medicine. Taconic contributes to
these developments with its tADMET™ portfolio. This toolbox provides a unique
collection of translational in vivo and in vitro products for predicting the Absorption,
Distribution, Metabolism, Excretion and Toxicity characteristics of new compounds
in humans. The portfolio has the following components:
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• rasH2 & TSG-p53®: Transgenic carcinogenicity testing models accepted by
the FDA & other regulators for replacement of the 2-year mouse
bioassay with a 6-months transgenic study
• tADMET™ GEMs and derived hepatocytes: A comprehensive selection of
single and multiple genetically humanized and knockout mouse models for
xenobiotic receptors, drug metabolizing enzymes and transporters as
well as cryopreserved hepatocytes derived from these mice
• FRG® KO: Highly repopulated liver humanized mice
• HepaCur™ and upcyte®: Human hepatocytes derived from multiple donors
that are available in large quantities and with consistent quality to
meet typical study needs
As part of an integrated strategy of various approaches used in drug development,
the tADMET™ tools can help to better predict drug responses in humans and thus
can contribute to an increase in clinical success rates.
The following paragraphs provide further details about the different tADMET™
tools and their utility.
Transgenic carcinogenicity testing
models: rasH2 & TSG-p53®
A prime example for the utility of genetically engineered models to improve
research in drug development is provided by the transgenic carcinogenicity
testing models. rasH2 and TSG-p53® are transgenic models with very low
incidence of spontaneous tumors until 6-month of age, but which are more
sensitive than wild type mice to rapidly develop tumors after exposure to
carcinogens. Accordingly, compared to the traditional 2-year mouse assay these
models allow for a faster carcinogenic risk assessment in a 6-month transgenic
study. Such transgenic models, in particular the rasH2, are now increasingly being
used by the pharmaceutical industry as cost effective, faster and more reliable
alternatives to the 2-year mouse assay (Figure 1)3. Research indicates that more
than 50% of all carcinogenicity protocols submitted to the FDA in 2013 were
carried out in rasH2. The trend towards increased use of rasH2 likely reflects
regulators’ views that the TSG-p53® model is suitable only for pharmaceuticals
that are positive in at least one genotoxicity assay, whereas rasH2 can also be
used for non-genotoxic compounds. Furthermore, Tg.AC, a transgenic model
evaluated for carcinogenicity testing using dermal applications is rarely
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recommended today. The current discussion of prospective changes to the
ICH S1 guideline, according to which the mandatory 2-year rat assay might be
waived for certain compounds in the future and either only the 2-year mouse
or a 6-months transgenic study would be required, has further increased
the market adoption of rasH24.
Figure 1: Carcinogenicity testing in weeks.
Carcinogen
administered
M Many Tumors }
HORT TER()26 Weeks rasH2 Mouse Cadamrciinnoisgt22ee66rned CCNaaour cTsiuenm oogof erCsnancer: EASILC3YA JNU DGE
S No Cancer
Wild Type Mouse
Carcinogen
administered
L
A }
N)s 26 52 76 104 Cause of Cancer:
Oek Spontaneous ?
TIWe Vehicle Carcinogen
ADI104 Non-transgenic administered Dietary EASCAILNY NJUODTG E
R( Mouse 26 52 76 104 Environmental
T
Cancer symptoms from none to terminal
tADMET™ GEMs & derived hepatocytes
and liver humanized FRG® KO mice to
predict drug metabolism, disposition
& toxicity in humans
Species differences in the pathways that define drug metabolism
& disposition limit the utility of wild type animal models in predicting the
pharmacokinetic, drug-drug interaction and toxicity profiles of compounds in
humans. Taconic’s tADMET™ portfolio includes two types of in vivo models to
improve such predictions, genetically engineered models for proteins involved
in drug metabolism and disposition (tADMET™ GEMs) and liver humanized
FRG® KO mice.
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a) Genetically engineered tADMET™
models and derived hepatocytes
The tADMET™ GEMs are a unique and comprehensive collection of single and
multiple genetically humanized and knockout mouse models for key proteins
involved in drug metabolism and disposition (Table 1). On the one hand, the
overall pathway of drug metabolism and disposition is highly conserved and it
involves the same protein families, namely xenobiotic receptors, phase 1 and
2 drug metabolizing enzymes and drug transporters. On the other hand, the
individual proteins have diverged significantly between species. In some cases
no direct orthologous genes can be identified in rodents and humans. Importantly,
though a large variety of such proteins do exist, only a handful of key players are
of major clinical relevance. The tADMET™ GEMs collection takes advantage of
the fact that mouse models humanized or knocked out for these key proteins
can provide more predictive data for drug responses in humans5, 6.
Table 1: The tADMET™ GEMs collection
Xenoreceptor Models Cytochrome P450 Models Transporter Models
Knockout Humanized Knockout Humanized Knockout Humanized
Ahr AHR Cyp1a1/1a2 CYP1A1/1A2 Bcrp BCRP (Q3 2014)
Car CAR Cyp2c CYP2C9 Mdr1a, Mdr1a/1b MDR1 (Q3 2014)
Pparα Cyp2d CYP2D6 Bcrp/Mdr1a/1b
Pxr PXR Cyp3a (BL/6) CYP3A4/3A7 Mrp1
Pxr/Car PXR/CAR Cyp3a (FVB) Liver or gut CYP3A4 Mrp2 MRP2
Pxr/Car/Ahr PXR/CAR/AHR Liver & Gut CYP3A4 Oatp1a/1b OATP1B1 or 1B3
Pxr/Car/Pparα PXR/CAR/PPARα Cyp2c/Cyp2d/Cyp3a OATP1B3 & 1B3
HRN™ (CYP Oct1/2
Reductase)
HRN™ nude Oat1/3 OAT1/3
Humanized Phase 2 Models Humanized Composite Models
UGT1A (Q3 2014) PXR/CAR/CYP3A4/7
PXR/CAR/CYP3A4/7/CYP2D6
PXR/CAR/CYP3A4/7/CYP2D6/CYP2C9
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Examples for the demonstrated utility of these models for improved predictions include:
• Drug-drug interactions, for example, resulting from PXR-mediated CYP3A4
induction7 or CYP3A48, CYP2D69 and CYP2C910 inhibition
• Generation of human-specific or predominant metabolites9, 11, which according
to the MIST guideline might require further safety assessment12
• Investigation of the relative contribution of a particular enzyme or transporter
to the metabolism or distribution of a compound6, 13
• Assessment of the relative contribution of gut vs liver CYP3A4 metabolism to
the oral bioavailability of a compound14, 15
• Prediction of xenobiotic receptor or drug metabolizing enzyme-mediated
toxicity of a compound5, 6
The tADMET™ GEMs are typically used subsequent to first in vitro studies, so that initial
information about the proteins which contribute to the metabolism and disposition of
the test compound is available. Therefore, the selection of the appropriate tADMET™
GEM for the in vivo study can be made with knowledge of this information and
depending on the primary purpose of the experiment. The tADMET™ GEMs can be used
before first-in-man testing for mechanistic studies, to support drug candidate selection,
for the safety assessment of a human specific metabolite or to adjust the clinical
dosing regimen, for example in a drug-drug interaction study. In addition, these models
have been used to retrospectively understand clinical results.
Taconic is extending its existing tADMET™ GEM collection by bringing to market
multiple humanized mice and genetically engineered transporter models and
providing cryopreserved hepatocytes from selected tADMET™ GEMs, including:
• Multiple humanized models that can be used to study complex pathways of drug
metabolism and disposition, which are often observed in vivo. In this regard, Taconic
recently generated a mouse model with a replacement of the corresponding murine
genes or gene clusters with human PXR, CAR, CYP3A4/7, CYP2D6 and CYP2C9. This
model covers the enzymes which contribute to >80% of human phase 1 metabolism
of drugs in clinical use and the receptors which regulate their expression.
• Genetic humanized and knockout drug transporter mice are powerful complements
to in vitro and in silico approaches to assess transporter-limited or transporter-
mediated drug absorption, distribution, and excretion and transporter mediated
drug-drug interactions. The clinical relevance of the contribution of transporters
to the pharmacokinetics, safety and efficacy profiles of drugs is increasingly
recognized16, which is reflected by corresponding changes in transporter-related
guidelines from the FDA17. In addition to the existing collection of models,
the tADMET™ GEMs toolbox is currently being extended to cover knockout and
humanized mice for all drug transporters regarded as clinically most relevant
by the International Transporter Consortium16.
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• Cryopreserved hepatocytes from selected tADMET GEMs are a valuable
complement to the in vivo models and can be used as cost-effective in vitro
pre-screening tools before entering into an tADMET™ GEMs in vivo study.
Taconic together with In Vitro ADMET Laboratories (Columbia, MD) to provide
cryopreserved hepatocytes from selected tADMET™ GEMs. In this regard, the
tADMET™ GEMs and the derived hepatocytes can help to establish in vitro-in
vivo extrapolations. In addition to their combined use with the in vivo models,
the hepatocytes can be used as stand-alone tools to assess the contribution
of individual human proteins to drug metabolism and disposition.
b) Liver humanized FRG® KO mice
FRG® KO mice are another tool being applied by scientists to make non-clinical data
more predictive of human response to drugs in clinical development. Taconic joins
with Yecuris Corporation (Portland, OR) to offer the chimeric FRG® KO mice with
humanized livers. These mice are generated by transplantation of human
hepatocytes which subsequently replace the mouse hepatocytes18.
The FRG® KO mouse on a C57BL/6 genetic background carries two types of
genetic alterations enabling highly efficient repopulation with human hepatocytes:
(1) Mutations in the rag2 and Il2rg genes resulting in immune deficiency and
preventing the rejection of human cells by these mice, and (2) a deficiency in the
fumarylacetoacetate hydrolase (Fah) gene coding for an enzyme in the tyrosine
catabolic pathway leading to the intracellular accumulation of a toxic metabolite
in hepatocytes and thereby causing mouse liver injury in this model. The onset
and severity of hepatocellular injury in FRG® KO mice is controllable through the
administration and withdrawal of the small molecule NTBC, which blocks an upstream
enzyme in tyrosine catabolism and thereby prevents the accumulation of the toxic
metabolite. Accordingly, liver injury is circumvented as long as the FRG® KO mice are
maintained on NTBC, but it is induced when the compound is withdrawn (Figure 2).
Figure 2: Human hepatocyte repopulation in FRG® KO mice
Maintain on NTBC Remove NTBC Transplant human hepatocytes
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The FRG® KO mouse model has several advantages compared to other
approaches: Because liver toxicity is caused by a mutation in an endogenous
gene rather than the overexpression of a transgene, a reversion of phenotype
cannot occur. Furthermore, efficient repopulation with human hepatocytes of
various donor ages has been achieved in this model. It has been used for serial
transplantations to expand primary human hepatocytes from a given donor
and very high (>95%) human repopulation of the mouse liver is possible in the
FRG® KO mouse. Finally, the mice are robust, relatively large and have good
survivability, enabling serial blood sampling, chronic studies and shipment
of animals. The FRG® KO mouse is a powerful tool for studying human drug
metabolism and hepatotoxicity19, lipid metabolism20, infectious diseases of
the liver, such as hepatitis21 and malaria22, liver cancer and gene therapy23.
An advancement of the FRG® KO mouse model was its recent combination with a
mutation in the Sirpα gene, which results in macrophage inactivation. This FRG®
KO/NOD mouse can be used for dual reconstitution of the liver and the immune
system. First dual reconstituted FRG® KO/NOD mice were successfully generated
and optimization is currently ongoing. A potential benefit of these mice for use
in ADME and toxicology studies is to improve human hepatotoxicity testing,
which often involves adverse reactions of the immune system. Another important
milestone was the development of an FRG® KO rat. Liver repopulation with human
hepatocytes is currently being established for this model. The FRG® KO rat will
be a valuable model for studies that require larger sample sizes, frequent blood
draws, or for other reasons the rat would be the preferred preclinical model.
c) tADMET GEMs vs FRG® KO mice
While there is overlap in the utility of the tADMET™ GEMs and the liver humanized
FRG® KO mice for studies in drug metabolism and toxicity, both of these models
have their specific benefits and limitations. Advantages of the FRG® KO approach
include 1) the complete humanization on the cellular (hepatocyte) and almost
complete humanization on the organ (liver) level, 2) the ease of generating
donor variability, 3) the possible consistency with in vitro studies using human
hepatocytes, and 4) its utility for studying human infectious diseases of the liver
and liver cancer. On the other hand, key advantages of the tADMET™ GEMs include
1) their full immune competence, 2) the significantly lower costs of production
and higher consistency given the tADMET™ GEMs don’t require continuous
recreation by transplantation, 3) the expression of a particular human gene
in all liver cells and, if applicable, in extra hepatic tissues contributing to drug
metabolism and disposition, and 4) the possibility to determine the role of a
particular human protein in drug responses related to metabolism and disposition
through the combined use of genetically humanized and knockout mice.
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A couple of factors need to be taken into consideration when making a
choice between tADMET™ GEMs and liver humanized FRG® KO mice for studies
in drug metabolism and toxicity. Such factors must be evaluated on a case-by-case
basis depending upon the purpose of the study and its particular protocol.
The tADMET™ GEMs have benefits where the study is directed to assess the role of
a particular protein or only a few selected proteins need to be considered, when
extra-hepatic organs significantly contribute to the metabolism and disposition
of the compound, and if the budget constraints are very tight. On the other hand,
the FRG® KO mice are often the first choice when complex hepatic pathways of
drug metabolism are involved and the in vivo effect of a compound on human
hepatocytes is being investigated.
HepaCur™ and upcyte® human hepatocytes
to overcome current bottlenecks in quantity,
quality and costs of hepatocytes from
preferred donors
Primary human hepatocytes are considered to be the gold standard for many
applications related to drug metabolism, drug-drug interaction and toxicity.
Their use is mandated for certain studies by regulatory authorities before testing
is conducted in humans. However, the supply of good quality primary human
hepatocytes from preferred donors is limited by the low and sporadic availability
of human liver tissue. Furthermore, the costs of human hepatocytes - particularly
if they are plateable - is significant, which has limited their more widespread
use. Both the HepaCur™ and upcyte® technologies overcome the aforementioned
bottlenecks by providing large batches and consistent qualities of plateable
human hepatocytes from preferred donors at competitive prices. Moreover, both
HepaCur™ and upcyte® allow for the custom expansion of hepatocytes from
specific donors (for example with disease phenotypes of interest).
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a) HepaCur™ hepatocytes
The primary human HepaCur™ hepatocytes are derived from the liver humanized
FRG® KO mice. In this case the FRG® KO mice are used as bioreactors to produce
large quantities of primary human hepatocytes from a single donor with superior
quality for in vitro drug testing. Due to the ability to do serial transplantation
in the FRG® KO model, the human cells can be geometrically expanded in each
round of transplantation, with an expansion factor of ~200-fold per mouse.
By utilizing this approach thousands of vials from a given donor can be produced.
Remaining mouse cells are typically ablated during the plating process due to
their sensitivity to tyrosine in the media and they can be entirely removed by
negative selection methods using magnetic beads, yielding >99.9% purified
human hepatocytes. A large inventory of cryopreserved human HepaCur™
hepatocytes from different donors for off-the-shelf sales is being established at
Taconic’s collaboration partner Yecuris Corporation (Portland, OR). The HepaCur™
hepatocytes were shown to maintain the expression of various drug metabolizing
enzymes and transporters as well as other typical primary hepatocyte
characteristics18. Therefore, these cells can be used for routine applications of
traditional primary hepatocytes, such as for clearance predictions, drug-drug
interaction studies, metabolite identification and toxicity testing. An additional
benefit of this technology is that the combined use of the HepaCur™ hepatocytes
and the liver humanized FRG® KO mice allows for (donor) consistency between
in vitro and in vivo studies.
b) upcyte® hepatocytes
Large batch numbers of plateable human hepatocytes with consistent quality
are also provided by the upcyte® technology, which was invented by Taconic’s
collaboration partner Medicyte GmbH (Heidelberg, Germany). The upcyte®
approach uses a lentiviral gene transfer system to bypass cell cycle control
mechanisms in a controlled manner, thereby driving primary human hepatocytes
into proliferation without inducing immortalization, uncontrolled cell growth or
major changes in the typical hepatocyte phenotype24. A unique capability of
these cells is to proliferate in vitro while maintaining sufficient expression
of drug metabolizing activities, which render the hepatocytes highly suitable
for in vitro micronucleus testing to assess the genotoxicity of a compound25.
Other demonstrated applications include the use of the upcyte® human
hepatocytes in studies related to CYP induction26, CYP inhibition27,
Metabolism28 and Cytotoxicity29.
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