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Dear colleages,
Within my company we are currently discussing whether or not human ADME
studies with radiolabeled material are needed for recombinant
protein-drugs. In this particular case we have a protein analog that is
composed of one human protein that is extended with a small part of
another human protein. Both protein parts contain exactly the same amino
acids as their natural counterparts. We would like to address the
following questions:
1) are human ADME studies feasible with recombinant proteins ? Which
radiolabel should be used and is there not a significant risk of
incorporation of the radiolabel into the natural proteins of volunteers
that are on the study.
2) are human ADME studies needed with recombinant proteins (and
especially protein analogs) to get registration? What are good
scientific reasons to justify that these are not usefull. (e.g. the mass
balance is not expected to be complete due to incorporation of
degradation products into natural proteins ?)
I would be very pleased if colleagues in the pharma-industry and
hopefully also from regulatory authorities could reflect upon the above
questions.
Best regards,
Geertje van Beerendonk
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Geertje:
Let me address only that part of your question dealing with the use of
radiolabeled compounds in human volunteers, and discuss it from a
technical/scientific and a regulatory viewpoint.
If you are going your ADME studies are going to be limited to
measurements of your drug in blood, you may also wish to consider the
use of stable nuclides (2H for 3H and 13C for 14C) by using amino acids
enriched in these stable nuclides. The improvements in sensitivity of
LC/MS makes the use of stable nuclides more feasible, and avoids the
human use of ionizing radiation. If your ADME studies include
noninvasive measurements at the drug target site(s), then you may need
to consider 11C as the radiolabel, although use of one of the iodine
radionuclides (131I or 123I) would be an alternative. In either case
you need to factor in the amount of radiation that would be deposited
at the target site(s) and at critical sites to determine how much is
allowable.
From a regulatory viewpoint, that is country specific. Look at the FDA
site at http://www.fda.gov/cder/regulatory/RDRC/default.htm, where you
will find information on the Radioactive Drug Research Committees
(RDRC's), which can authorize in the US the human use of radiolabeled
compounds. While the original intent of the RDRC's was to mainly to
authorize studies such as the PK of 14C-Glucose or other known
biochemicals or drugs, there is active discussion going on whether to
authorize expanding the statutatory authority of RDRC's.
Hope this information helps.
--
Professor Walter Wolf, Ph.D. President, Correlative Imaging Council,
Society of Nuclear Medicine
Distinguished Professor of Pharmaceutical Sciences
Director, Pharmacokinetic Imaging Program
Department of Pharmaceutical Sciences, School of Pharmacy
University of Southern California 1985 Zonal Ave., Los Angeles, CA
90089-9121
E-Mail: wwolfw.-at-.usc.edu
Telephone: 323-442-1405
Fax: 323-442-9804
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Do you have to look at any possibility of formation of new antigenic
site?
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Dear Geertje and Group
This is a very interesting area in my view and tissue distribution
studies
in man are to my mind amongst the most elegant in clinical pharmacology.
My comments would be firstly to consult with independent experts in
calculating radiation exposure who do it frequently such as we have in
the
departments of oncology and nuclear medicine in our hospital. We have
done
this recently for Indium-99 that we use as a marker for total gut
transit
studies. Interestingly they gave me equivalents in terms of distances
flown
at high altitude in commercial aircraft where we are all exposed to
cosmic
rays.(Our trial was about the equivalent of a flight from Heathrow to
Capetown)
They will ask which isotope and you will probably want C-14 since it is
a
stable low energy beta emitter that would keep the molecule stable. The
exposure from a beta emitter is very small due to its minimal tissue
penetration (a few mm) so it would have to be pretty much permanently
incorporated into tissue proteins to have a significant deleterious
effect.
I think this is unlikely because that implies slow turnover that would
greatly limit incorporation in the first place. I would hope that the
usual
distribution studies in rats might identify which tissues the label
resides
in. If you make sure to keep some rats back for a late measurement it
should be possible to show when all the label has gone. That is
probably the
key and in this case it might pay to use a later time than usual. It
would
produce some reassurance that the duration of exposure would be
acceptable
in man.
I would like to know from the forum if anyone has done this and
rejected a
study in man due to over long residence in rat tissue in
autoradiographic
studies.
Best wishes for 2005 to all in what I find to be the most worthy and
stimulating forum.
Andrew Sutton
Guildford Clinical Pharmacology Ltd.
Tel: +44 (0)1483 455375. Direct: 688303
URL: www.gcpl.co.uk
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The following message was posted to: PharmPK
Geertje,
The need for a human ADME study for recombinant proteins is
covered in an ICH guidance (ICH S6). This guidance (Preclinical safety
evaluation of Biotechnology-derived pharmaceuticals; dated July 1997)
states: "The expected consequence of metabolism of biotechnology-derived
pharmaceuticals is the degradation to small peptides and individual
amino acids. Therefore, the metabolic pathways are generally understood.
Classical biotransformation studies as performed for pharmaceuticals are
not needed."
However, the guidance also gives some caveats for conducting the studies
that it has stated aren't necessary: "When using radiolabeled proteins,
it is important to show that the radiolabeled test material maintains
activity and biological properties equivalent to that of the unlabeled
material. Tissue concentrations of radioactivity and/or autoradiography
data using radiolabeled proteins may be difficult to interpret due to
rapid in vivo metabolism or unstable radiolabeled linkage. Care should
be taken in the interpretation of studies using radioactive tracers
incorporated into specific amino acids because of recycling of amino
acids into nondrug related proteins/peptides."
I haven't come across a regulatory submission that contains a human ADME
study for a recombinant protein. If you were to conduct such a study it
would be fraught with difficulties and very difficult to interpret. You
will most likely get answers that you wish that you hadn't got! Some of
the challenges will be:
1) Low overall mass balance due to incorporation of amino acids
into nondrug related proteins/peptides.
2) The need to keep the subjects in the Clinical Pharmacology unit
for a long time due to the slow excretion of radioactivity.
3) A complicated metabolite profile that will most probably show
the presence of cleavage products of the administered protein. You will
need to determine the contribution of these metabolites to the overall
exposure to compound-related material and potentially testing them for
activity. These metabolites could also potentially "test positive" in
the ELISA assay that you have determined for parent compound. You may
end up in the situation that by conducting the human ADME study you end
up invalidating your ELISA assay due to a lack of specificity.
4) Difficulty in preparing and characterizing the radiolabeled
material for use in the human ADME study
5) The need to potentially conduct a dosimetry study in a primate
species due to the lack of relevance of the rat as a model for
addressing the PK of your recombinant protein
It is my guess that the above concerns and the fact that the ICH
guidance states that metabolism information is not required has lead to
the dearth of human ADME studies that have been conducted in support of
regulatory submissions for marketing approval for recombinant proteins.
However, the ICH guidance may be a bit outdated as it did not seem to
take conjugated proteins into account. In these cases, an assessment of
the stability of the conjugation seems to be a regulatory expectation.
I believe that such an assessment was performed for PegIntron. In your
case (a dimer of two different proteins) I would recommend establishing
ELISAs that are capable of detecting the intact molecule as well as the
individual proteins (i.e. assessing the cleavage of the parent molecule
into its constituent parts).
Regards
Mark
Mark N. Milton
Senior Director, Development DMPK
Millennium Pharmaceuticals, Inc.
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Prof. Wolf,
While it is true that in the past, carbon-14 studies required
significant quantities of radiation to overcome sensitivity limitations
of decay counting, Accelerator Mass Spectrometry is poised to
reinvigorate the use of carbon-14. AMS is a direct atom counting
method, that treats 14C as a stable isotope. Accordingly,
nanocurie-sized doses can be used to provide quantitative tracing of
any 14C molecule down to the high zeptomolar level. Moreover, the
measurment is free of matrix effects.
I have performed numerous human absorption and distribution studies in
Academia using as little as 20 nanoCuires of activity. We can easily
perform balance measurements of urine and stool, and trace minor
metabolites through the coupling of the AMS measurment to HPLC. A
typical measurement consumes about 25 ul of plasma, so finger-prick
sized samples can be used. I was able to take 60 blood measurements
over a 48 hr study and consumed less than 2 ml of total plasma volume
(but not using finger-pricks as we only have 10 fingers!).
As a result, radiation exposure calculations are almost meaningless.
Even for some of our longest retained molecules and using worst case
assumptions, our studies never exceeded 3 millirem in total dose
equivalents. At our institution anything below 10 millirem did not
even qualify as a radioactive study, so IRB approval was never an
issue. Moreover, since a single DPM is a 'large' signal, all the waste
products can be treated as ordinary lab waste (after biohazard
treatment). A typical 70 kg human carries with them 100 nanoCuries of
14C for their entire life from food sources. A 48 hours elevation in
14C levels from a 20 nanoCuire dose for 2 days in the context of a 75
year lifespan is pretty trivial.
AMS can be applied to Phase 1 clinics that want the power of a
radioisotope (selectivity and distinctiveness in the matrix), but the
removal of the headaches and ethical concerns associated with ionizing
radiation.
The principle obstacle towards greater use of AMS has been a lack of
awareness and a lack of facilities that offer the measurement. My
company, Vitalea Science, is the first US company to offer AMS
services to the Pharmaceutical Industry. We also work with Academia.
Please refer to our company website at www.vitaleascience.com for
information on AMS and its capabilities. AMS costs are somewhat higher
than a high resolution MS measurment, but not far off. I am glad to
send more information in the form of brochures if requested.
There is a great deal of activity at the FDA concerning adoption of
ultralow dose 14C studies using AMS detection. An important
application will be the Phase 0 concept, where sub-toxic doses of NCE
are taken directly to man with a very limited toxicology package. This
will require a modification of the CFR. Such a modification has been
adopted by the EMEA in Europe. It is probably important that the FDA
act on this sooner than later, as economic activity will be lost to
Europe as the Phase 0 format becomes common.
Regards,
Stephen Dueker, PhD
President
Vitalea Science, Inc
www.vitaleascience.com
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Dear Dr Dueker,
I would like to know the web directions for the guidance with respect
to a modification has been adopted by the EMEA in Europe, concerning to
adoption of ultralow dose 14C studies using AMS detection or other.
Regards,
Piedad Restrepo
Biopharmaceutics Department
CIDEIM
Cali - Colombia
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Dear Dr. Restrepo,
Concerning Accelerator Mass Spectrometry and microdoses.
The position paper (position paper on non-clinical safety studies to
support clinical trials with a single microdose) on
microdosing can be found at the EMEA website. There are 2 ways to find
it as it is buried in the site.
1. go to www.emea.eu.int for the emea home page.
2. go to the search tool and type in 2599/02. The position paper
should be the first listing.
2a. alternatively, type both "cpmp" and 'microdose' and this should
find the paper as well.
[http://www.emea.eu.int/pdfs/human/swp/259902en.pdf - looks like the
right document - db]
If you like, I can send it to you directly as an attachment. As I have
not seen attached files on the boomer site, I am guessing attachments
are not encouraged.
[correct for PharmPK listings, some original material is on the website
- db]
The definition of what constitutes a "microdose" seems rather
arbitrary, but given the amount of toxins we are
exposed to in a stick of broccoli, it is hard to imagine how
submicrogram doses can present any real hazard to a
human subject. Because AMS quantifies only on the 14C nuclide, the
actual mass of the drug can even be in the
nanogram range, and still provide quantitive pharmacokinetics (using
higher specfic activity radiolabels than 1 14C
per molecule).
I will link the position paper to our website, www.vitaleascience.com,
later tonight if people prefer to download it
there.
The main argument against micrdosing relates to non-linearities from
microdoses to pharmacologic-sized doses, a valid
concern. A recent paper addressing this concern appears in Drug
Metabolism and Disposition, Vol. 32, No. 11 pp.
1254-59 (Lead Author Sandhu).
The benefits of AMS will surely extend beyond microdosing.
Stephen R. Dueker
President
Vitalea Science, Inc
www.vitaleascience.com
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Dear Dr. Dueker:
This discussion on the potential - and the possible problems - of
microdosing is very important. As you indicate correctly below, two of
the serious potential problems with microdosing are:
1. That we do not know, when doing a first-in-man study, what
constitutes a toxic dose
2. That extrapolation from low dose to high dose may be a problem when
there is non-linearity
In studies we did many many years ago, (Distribution of
18F-5-Fluorouracil in Tumor Bearing Mice and Rats. J. Shani, W. Wolf,
T. Schlesinger, H.L. Atkins, P.R. Bradley-Moore, V. Casella, J.S.
Fowler, D. Greenberg, T. Ido, R.M. Lambrecht, R. MacGregor, C.
Mantescu, R. Neirinckx, P. Som, A.P. Wolf, I. Wodinsky and K. Meaney.
Intl. J. Nuc. Med. and Biol., 5 ,19-28, 1978) we had used varying doses
of 18F-5FU to determine, among other issues, whether biodistribution
might be different at high (pharmacological) dose or low (tracer) dose.
We found no difference. Because this work was done 25 years before the
term "microdosing" was used, it may have been overlooked.
However, there is one key difference in between the methodology I
believe is desirable and what is published in the paper by Sandhu et
al. you reference below: that in the Sandhu paper they only measure
blood PK, and as been pointed out in the recent discussions on this
listserver, there are many drugs, especially anticancer drugs, where
there is no direct relation between the blood PK of a drug and its
pharmacodynamic effect.
Thus, to ascertain that microdosing using AMS measurements provides a
valid set of data to develop or to monitor a drug, comparisons must be
made between the drug labeled with 14C and the same drug labeled with
11C. The latter would allow a direct measurement of the target
pharmacokinetics, which is what is really critical for evaluating
therapeutics effectiveness. It might also provide an insight into
deposition of the drug into other organ/tissue sites where toxicity
might occur, and hence, allow measurements of the toxicokinetics of
that drug. Blood measurements are excellent - if all you want to know
is what happens in blood. But they are often not adequate and
sufficient to evaluate how much of the drug goes to the target site,
which is what is desirable and counts, and how much of the drug goes to
undesirable sites, which might negate the clinical usefulness of such a
drug.
--
Professor Walter Wolf, Ph.D. President, Correlative Imaging Council,
Society of Nuclear Medicine
Distinguished Professor of Pharmaceutical Sciences
Director, Pharmacokinetic Imaging Program
Department of Pharmaceutical Sciences, School of Pharmacy
University of Southern California 1985 Zonal Ave., Los Angeles, CA
90089-9121
E-Mail: wwolfw.aaa.usc.edu
Telephone: 323-442-1405
Fax: 323-442-9804
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Dear Prof. Wolf,
Many thanks for alerting me to the earlier microdose studies in mice
with the labeled fluoruracil. I was pleased to find
that a scanned electronic copy was available from the journal's
archives.
I would agree that microdosing does not appear to be a useful tool in
the area of pharmacodynamics or toxicokinetics
without the ability to directly take biopsies of the target or
vulnerable tissues. If small biopsies were permissible, then
the picture would be different. My only experience with 'tissues' in
healthy subjects would be with the red and white
blood cells. Although a very 'accessible' tissue, we were able to
measure the incorporation of a 'microdose' (low
microgram) of a drug candidate into these cells in healthy humans.
Interestingly, and directly relevant to what you
stated lack of concordance between plasma and tissue levels, the blood
cells accumulated and retained the drug--the
elimination was multicomponent-- while the plasma levels decayed in a
fairly typical mono/bi-exponential fashion. In
my opinion, this would have to be viewed as positive behavior if the
white blood cells are the target tissue (which I
believe to be true in the described case). Your paper from 1978 showed
similar non-concordance in terms of
elimination rates from the plasma and tumor tissue with fluoruracil.
Microdosing is largely described as a tool to 'toss-out' candidates
that have poor intestinal absorption early in the
discovery process. In this regard, I think it has real value. It MAY
have even greater value at the efficacy stages
(Phase 2) to explain differential responses among clinical patients to
a drug. In this format, the labeled drug would
simply be a tracer of the pharmacological dose. With AMS, this could
be performed at 'ambient' levels of
radioactivity, but still provide quantitative data on absorption and
elimination in such efficacy trials.
Short of direct biopsies, I would agree that imaging tools such as PET
are powerful and of increasing importance in
the age of rational designed drugs and new knowledge of receptors. A
biopsy, however, small will not always be
reasonable to take for AMS evaluation. Moreover, the time resolution
for a biopsy would be limited to a single point
in most cases. If tissue biopsies in clinical patients could be
taken, however, a great deal of information could be
obtained that may serve to explain idiosyncratic responses to a drug.
Metabolite profiles at the target tissue are
conceivable with AMS sensitivity and perhaps that is an area that needs
to be explored. A milligram of tissue will
provide ample signal to establish covalent binding or metabolite
profiles in well designed experiments.
PET and AMS are two great tools that are truly complementary. The
co-administration of C11/C14 isotopomers in
man would be quite interesting. I am not sure if this has been done
before.
Many Thanks,
Stephen Dueker, Ph.D.
President
Vitalea Science, Inc
www.vitaleascience.com
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Copyright 1995-2010 David W. A. Bourne (david@boomer.org)