Note: Descriptions are shown in the official language in which they were submitted.
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LIPID CLEAVAGE ENZYME
The lipid cleavage enzyme complex (lipid cleavage
enzyme; LCE) and its analogues, the subject matter of
the present invention, are membranous enzymes that have
previously not been described which can for example be
isolated from the cell membrane fractions of human
peripheral blood leucocytes or human macrophages and
which cleave conjugates of pharmacologically active
substances that are bound to a lipid-like carrier
molecule to release the pharmacologically active
substance or a monophosphate thereof. The invention also
concerns the use of these conjugates which serve as
substrates of this enzyme complex for the production of
pharmaceutical agents that contain these conjugates as a
pharmaceutically active substance. The pharmaceutical
agents are suitable for the specific release and
accumulation of pharmacologically active substances in
appropriate target cells. In addition the invention
concerns in vitro test systems which contain this enzyme
complex to screen for further substrates of this enzyme
complex as well as test systems for finding LCE
analogues. LCE is understood as the enzyme complex,
isolated enzyme as well as possible isoenzymes.
Apart from the inadequate efficacy of the therapeutically
active substances used, the therapy of malignant
neoplasias (carcinomas, sarcomas, haemoblastoses,
haematological neoplasias), inflammatory diseases or
autoimmune diseases as well as ~;~e~ses caused by viruses
or retroviruses such as for example AIDS, ARC (AIDS
related complex), cytomegaly, herpes or hepatitis, is
often accompanied by their extreme side effects. This
effect is due to inadequate in vivo selectivity or the
CA 02204908 1997-0~-08
limited therapeutic range of the pharmacologically active
substances used. The advantageous pharmacological in
vitro properties of the pharmacologically active
substances can often not be transferred to the in vivo
conditions.
Therefore for years attempts have been made to modify
the chemical structure of pharmacologically active
substances to provide new substances which have improved
properties with regard to therapeutic range. Moreover
new pharmaceutical forms of administration are often
developed with the aim of transporting the active
substance specifically to its site of action where it is
intended to display its therapeutic action. It is
intended in particular to avoid undesired interactions
with healthy cells. In the case of tumour cells which
have corresponding surface antigens, antibodies have for
example been produced that recognize these special
surface antigens and thus selectively bind to the cancer
cell. The antibodies are modified with suitable toxins
in such a way that the toxin is released after binding
to the cancer cell and the cancer cell is killed.
Another alternative for improving the therapeutic range
is to change the physical properties of the underlying
active substance by slight modification of the
pharmacologically active substance for example by
producing acid or base addition salts or by preparing
simple esters [for example fatty acid esters; J. Pharm.
Sci. 79, 531 (1990)] in such a way that the solubility
or compatibility of the active substance is improved.
These slightly chemically modified compounds are often
referred to as so-salled "prodrugs" since on contact
with body fluids or in the liver (first pass metabolism)
they are almost immediately converted into the actual
therapeutically active agent.
CA 02204908 1997-0~-08
The technical problem that forms the basis of the
present invention was to find a new target which occurs
as specifically as possible on or in cells that are a
target for the administration of pharmacologically
active substances. The target should interact with
appropriate pharmaceutically active substances so that
the active substances are transported as specifically as
possible to these target cells, and can be recognized,
bound and taken up by these. In this case the
pharmaceutically active substance should be essentially
composed of two components the first component being
responsible for the recognition and interaction with the
target (ligand-specific part) and the second component
being the actual active substance (active substance-
specific part) which only develops its activity after
specific binding to the target molecule and the actual
active agent or its monophosphate has been cleaved o~f
intracellularly. This should avoid the undesired release
of the pharmacologically active substance in the body
fluids so that healthy cells are not adversely effected
by the pharmacologically active agent and undesired
side-e~fects are substantially avoided. In this
connection the pharmaceutically active substance used
for this purpose for the production of the
pharmaceutical form should on the one hand serve as a
ligand for the target and on the other hand contain the
actual active pharmacological agent whereby this can
also in particular be based on already known
pharmaceutically active structures for which it is
in~n~eA to significantly improve the therapeutic range
in this manner.
It was now surprisingly found that LCE is suitable as a
target, LCE being mainly located on or in malignant,
activated or virus-infected cells in particular on human
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-- 4
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peripheral blood leucocytes, macrophages, kidney,
adrenal or ovarian cells, cells of the lymphatic system
or the lymphoid organs or cells of the brain. In the
following this enzyme complex and its analogues are also
abbreviated as LCE. Surprisingly LCE does not exhibit a
uniform statistical distribution over all organs but is
observed primarily in the membranes of certain cells
which come into consideration as the target for the
administration of pharmacologically active substances. A
relatively very low enzyme activity was found in
cardiac, bone marrow and liver cells. The homogenate and
membrane fractions of cells, organs or tissues used to
isolate the LCE have different specific activities or
affinities of the LCE, the specific activity or affinity
being strongly increased in activated cells compared to
non-activated cells. This can be demonstrated for human
peripheral blood leucocytes, lymphocytes and
granulocytes as well as for human and non-human and
murine mononuclear cells such as e.g. monocytes/
macrophages.
Surprisingly a characterisitic of LCE is that it cleaves
lipid-like compounds as the substrate between the lipid
backbone and the linker structure of a physiologically
active substance covalently bound to this bridge. Such
substrates can be described by the general formula I
L - B - D (I)
in which L represents a lipid residue, B a bridge and D
represents a pharmacologically active substance or B-D
represents an active substance phosphonate. Surprisingly
the pharmaceutically active substances of formula I have
a larger therapeutic range compared to the
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pharmacologically active free or unmodified substances D
or -B-D. Moreover they often improve their retention
time in the body, the bioavailability or the membrane
permeability which is often known to be a critical
factor (e.g. blood-brain barrier, cell membranes etc.)
of the pharmacologically active substances. Substrates
of formula I therefore serve as a carrier system
(carrier) for the pharmacologically active substance.
The conjugates of formula I can be referred to as
intracellular drug storage, drug targeting and drug
delivery systems with regard to their function. Their
effect is that the pharmacologically active substance or
its prodrug form is released intracellularly after oral
administration, this release taking place advantageously
not unspecifically in all cells, organs or tissues of
the body but rather specifically in those cells that
contain the LCE in the cell membrane or also partially
intracellularly. However, it is particularly surprising
that cleavage does not already take place during the
transport of the substrate through the body fluids such
as blood, serum or lymph fluid or through the liver but
only on or in the respective target cells. In this
manner the undesired excretion of the cleavage product
by the kidney or cleavage of the substrate in the liver
is avoided so that the major proportion of the active
substance is transported to the respective target cells.
As already stated above, such cells are in particular
pathophysiologically or physiologically activated cells
which come into consideration as a target for the
administration of pharmacologically active substances
such as for example blood leucocytes, lymphocytes,
macrophages and other cell populations of the
immunological lymphatic system. These are in particular
activated cells (e.g. macrophages, granulocytes,
lymphocytes, leucocytes, thrombocytes, monocytes etc.)
CA 02204908 1997-0~-08
which play a pathological, physiological, patho-
physiological or symptomatic role in the respective
disease process.
LCE is an enzyme complex which has previously not been
described. It is characterized in particular in that it
cleaves the compound (3'-deoxy-3'-azidothymidine)-5'-
phosphoric acid-(3-dodecyl-mercapto-2-decyloxy)-propyl
ester (abbreviated as AZT-DMDOPE in the following) as a
substrate to form 3'-deoxy-3'-azidothymidine-
monophosphate and (3-dodecylmercapto-2-decyloxy)-
propanol (DMDOP). A further preferred substrate of LCE
is the compound (3'-deoxy-3'-fluorothymidine)-5'-
phosphoric acid-(3-dodecyl-mercapto-2-decyloxy)-propyl
ester (FLT-DMDOPE) which is cleaved into 3'-deoxy-3'-
fluorothymidine-5'-monophosphate and DMDOP.
Alternatively (5-fluorouridine)-5'-phosphoric acid-(3-
dodecylmercapto-2-decyloxy)-propylester (5-FU-DMDOPE) is
also cleaved into (5-fluorouridine)-5'-monophosphate
(5-FU-MP) and DMDOP.
The preparations containing LCE that have been produced
are free of phospholipase C. This has been demonstrated
by the different cation dependency of the LCE activity
as well as by phospholipase C-specific inhibitors which
do not inhibit LCE.
An enzyme assay was established (example 6) to determine
the turnover rate of AZT-DMDOPE by cell homogenates,
membrane and cytosol fractions, preferably of various
human cell types.
The test principle is based on a cleavage of the parent
substance by the LCE into AZT-MP and the corresponding
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thioether lipid part. [l4C]-AZT-DMDOPE and non-
radioactively labelled AZT-DMDOPE were used for this. The
thioether lipid metabolite DMDOP was extracted from the
mixtures (example 7) and the amount of radioactively
labelled substance was measured in a liquid scintillation
analyzer. Since a defined amount of [l4C]-AZT-DMDopE was
used in the enzyme assay it was possible to determine the
turnover rate of the enzymatic cleavage.
The enzyme is preferably isolated from human peripheral
blood leucocytes which have previously been activated by
PHA or other stimulants (e.g. cytokines etc.) or from
murine kidney cells. Prel;r;n~ry investigations have
yielded a molecular weight of about 120,000 - 160,000
determined by the SDS-PAGE method. Cells which contain
this enzyme can be identified in a simple manner by
admixing them under suitable test conditions with a
solution of AZT-DMDOPE or FLT-DMDOPE and detecting the
cleavage products AZT-monophosphate or FLT-monophosphate
or DMDOP for example by thin layer chromatographic
methods or, in the case of radioactively labelled
samples by scintillatographic methods (see examples 4 -
7). In contrast to the biochemically related
phospholipases C or D, the LCE is not inhibited by known
inhibitors of phospholipase C or D nor is it activated
by activators of phospholipase C or D. In contrast to
phospholipase C the enzyme is activated by the substance
D 609 (tricyclodecane-9-yl-xanthogenate; C11H15OS2K)
whereas D 609 inhibits phospholipase C.
In its non-phosphorylated form intracellular AZT does
not have an inhibitory effect on viral reverse
transcriptase (RT) (Nakashima et al., 1986, Antimicrob.
Agents Chemother. 30, 933-937; Mitsuya et al., 1985
Proc. Natl. Acad. Sci. USA 82, 7096-7100). The
CA 02204908 1997-0~-OX
structural analogue of thymidine is converted by
successive phosphorylation by the intracellular enzymes
thymidine kinase, thymidilate kinase and pyrimidine
nucleoside diphosphate kinase (Yarchoan et al., 1989, N.
Engl. J. Med. 321, 726-738; Toyoshima et al., 1991,
Anal. Biochem. 196, 302-307) via AZT-MP and AZT-DP into
the therapeutically active RT-inhibiting ATZ-TP. AZT-
DMDOPE as a thioether lipid AZT conjugate was converted
by intracellular enzymatic cleavage into AZT or directly
into the already phosphorylated form AZT-MP. In example
8 the intracellular concentrations of phosphorylated and
non-phosphorylated AZT are determined after incubation
with equipotent concentrations of AZT-DMDOPE and AZT.
In addition the enzyme complex and its analogues do not
exhibit a uniform statistical organ distribution in
various species (e.g. human, mouse, rat, dog, ape) but
are only found in membranes of certain cells, organs and
tissues which serve as target cells for cleavable
conjugates of formula I. The natural substrates of these
enzymes are still unknown. ~In cytosolic fractions the
LCE activity is always below or just at the limit of
detection. A very low activity is for example also found
in bone marrow cells which indicates a very low or even
a complete absence of bone marrow toxicity of the
pharmaceutically active substances of formula I used as
substrates of the LCEs.
The LCE activity exhibits a linear protein and time
dependency, a specific dependency on metal cations
(inhibition by Ca2+, Zn2+ and Mn2+) and a classical
Michaelis-Menten kinetics (substrate dependency)
(example 9). In addition to the higher activity of the
enzyme complex in activated cells there is also a higher
affinity of the LCE to its substrate under these
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conditions.
The isolated LCE or LCE strongly enriched from membrane
fractions can also be used to screen for new potentially
cleavable substrates or for substrates which occur
naturally. The substrates found in this manner can then
be investigated more extensively with regard to their
essential ~tructural features which are necessary for
the recognition of the substrate and binding to LCE.
Such identified structural properties can then be used
to produce chemically modified substrates which contain
these essential features as well as in addition suitable
functional groups that are suitable for coupling to
pharmacologically active substances.
This also enables a screening for inhibitors or
activators of the lipid cleavage enzyme.
The isolated or strongly enriched enzyme can be used
diagnostically when for example increased or reduced
lipid cleavage enzyme activities lead in vitro or in
vivo to pathological changes or when corresponding
diseases or disease symptoms are associated with these
pathological changes.
The LCE can also be used to produce diagnostic agents
which are used to check the cleavage of these substrates
when the pharmaceutically active substances are
administered to patients which results in a specific and
individual adaptation of the therapy modalities in these
patients (drug monitoring).
Recombinant LCE can be produced by well-established
methods by determining the amino acid sequence of LCE or
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-- 10 --
LCE fragments and screening a human or other mammalian
gene library with oligonucleotide probes that have been
constructed appropriately. The found gene is then
expressed by an appropriate vector in a prokaryotic or
eukaryotic cell system. The recombinant LCE can then be
purified by methods known in protein chemistry (see e.g.
Maniatis, Molecular Cloning).
A particular characteristic of the LCE is that it can
cleave compounds of the general type I:
L - B - D (I)
Cleavage by LCE
in which L represents a lipid moiety, B represents a
bridge and D represents a pharmacologically active
substance or B-D denotes an active substance
phosphonate. The very specific cleavage takes place
between the lipid moiety and phosphate residue. An
unspecific cleavage of compounds of formula I at other
functional groups in the molecule is not observed.
In this connection the lipid moiety L of the conjugate
of formula I represents the following residue of
formula II
Rl--X--CH2
R2-Y-CH
¦ (II)
(CH2)m~
in which
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R1 is a straight-chain or branched, saturated or
unsaturated alkyl chain with 1 - 30 carbon atoms
which cab be optionally substituted once or several
times by halogen, C5-C7 cycloalkyl, phenyl, Cl-C6
alkoxy, C1-C6 alkylmercapto, C1-C6 alkoxycarbonyl,
C1-C6 alkylsulfinyl or Cl-C6 alkylsulfonyl groups
R2 is hydrogen, a straight-chain or branched,
saturated or unsaturated alkyl chain with 1 - 20
carbon atoms which can be optionally substituted
once or several times by halogen, C5-C7 cycloalkyl,
phenyl, Cl-C6 alkoxy, C1-C6 alkylmercapto, C1-C6
alkoxycarbonyl or Cl-C6 alkylsulfonyl groups
X represents a valency dash, oxygen, sulphur,
aminocarbonyl, oxycarbonyl, carboxyamino,
carbonyloxy, carbonylamido, amidocarbonyl, a
sulfinyl or sulfonyl group
Y is a valency dash, aminocarbonyl, oxycarbonyl,
carboxyamino, carbonyloxy, carbonylamido,
amidocarbonyl, an oxygen or sulphur atom and
m represents an integer between 1 and 5.
R1 in the general formula II preferably denotes a
straight-chain or branched C8-C15 alkyl group which can
additionally be substituted by a C1-C6 alkoxy or a C1-C6
alkylmercapto group. Rl in particular represents a
nonyl, decyl, undecyl, dodecyl, tridecyl or tetradecyl
group. Methoxy, ethoxy, butoxy and hexyloxy groups come
preferably into consideration as C1-C6 alkoxy
substituents of R1. If R1 is substituted by a C1-C6
alkylmercapto residue, then this is in particular
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- 12 -
understood as a methylmercapto, ethylmercapto,
propylmercapto, butylmercapto and hexylmercapto residue.
R2 preferably denotes a straight-chain or branched C8-
Cl5 alkyl group which can additionally be substituted by
a Cl-C6 alkoxy group or a Cl-C6 alkylmercapto group. R2
in particular represents an octyl, nonyl, decyl,
undecyl, dodecyl, tridecyl or tetradecyl group. A
methoxy, ethoxy, propoxy, butoxy and hexyloxy group
preferably come into consideration as C1-C6 alkoxy
substituents of R2. If R2 is substituted by a Cl-C6
alkylmercapto residue then this is in particular
understood as a methylmercapto, ethylmercapto,
butylmercapto and hexylmercapto residue.
X is preferably sulphur, sulfinyl or sulfonyl and Y is
oxygen. The heteroatoms X and Y in the lipid moiety L
can only be replaced in special cases by the carboxylic
acid ester known from lecithin since otherwise a
hydrolytic cleavage to form the corresponding
lysolecithin derivatives or glycerol esters with a
corresponding more rapid elimination of the
pharmacologically active substance would already occur
in the serum or in the liver (first pass effect). The
thioether lipids and ether lipids (X, Y = O,S) of this
application do not exhibit this cleavage in the serum of
various species including humans.
Compounds are also preferred in which X and Y represent
a valency dash, R2 is hydrogen and R1 represents a Cl-
C30 alkyl chain which can optionally be substituted by
C1 C6 alkoxy or C1-C6 alkylmercapto.
m is preferably 1 or 2 and particularly preferably 1.
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- 13 -
The bridge B represents a valency dash or is expressed
by the formula III
~~~[(Pz)(oH)A]n- (III)
in which n can be 1, 2 or 3 but is preferably 1 or 2 and
especially 1, Z is either O or S, and A is either O, S
or a valency dash and preferably o.
The lipid moiety L and the phosphate bridge B have the
above-mentioned meaning in which L preferably represents
a residue of formula II and B is preferably a phosphate
bridge of formula III. A phosphate bridge of formula III
is particularly preferred in which n = 1 and a lipid
moiety of formula II is particularly preferred in which
R1 and R2 represent an alkyl residue with 8 - 15 C
atoms, X equals sulphur and Y is oxygen. Furthermore
compounds are preferred in which B as a phosphonate is a
constituent of the structure of the active substance in
which case n = 1 and A is a valency dash.
The term "pharmacologically active substance" (named D
or B-D in the case of phosphonates in formula I) in this
application represents an active substance within the
legal pharmaceutical sense. This active substance can be
an active substance of a pharmaceutical agent that has
already been introduced and licenced by the
pharmaceutical authorities or an active substance which
is currently being registered as a pharmaceutical agent.
The definition "pharmacologically active substance" also
encompasses such derivatives of active substances that
can be chemically modified by introducing one or several
functional groups (for example such groups which enable
D to be coupled to the lipid carrier moiety L such as
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e.g. hydroxy or amino groups). The definition also
encompasses prodrug forms that are formed from the
active substance D which are also physiologically
active. In particular pharmacologically active
substances D come into consideration whose clinical
development has been discontinued or not been started
due to undesired side effects or which only have a very
narrow dose-effect spectrum so that the administration
of the therapeutically required amount would be
associated with high risks or virtually impossible to
get under control.
Surprisingly it was found that the therapeutic range of
a pharmacologically active substance D or B-D in the
case of phosphonates of the active substance is
significantly improved when the substance is coupled to
a lipid-like carrier molecule. The conjugate prepared in
this manner serves as a new active substance for the
production of pharmaceutical forms of a~' ; n i~tration.
Overall the coupling results in an increased activity of
the pharmaceutically active substance D or B-D in vivo
since, due to the drug-delivery transport system that is
formed, the pharmacologically active substance is
localized in target cells and thus the efficiency of the
pharmacologically active substance is increased. This
means that on the one hand the amount of
pharmacologically active substance that has to be
administered can be reduced or on the other hand that an
increased pharmacological effect is achieved while
retaining the same effective amount.
The chemical structure of the pharmacologically active
substances D or B-D can in addition be modified in such
a way that the substances are changed with regard to
their physical or chemical properties and for example
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have a higher or lower lipophilicity but have
essentially the same properties as the unmodified
substance D or B-D with regard to their therapeutic
effect. In particular it is advantageous when the
substance D is chemically modified by the introduction
of functional groups in such a way that it can be
coupled via a suitable bridge to the lipid moiety L.
This is for example achieved by the introduction of
hydroxy groups which are coupled via the phosphate group
B to the lipid.
The pharmacologically active substance D or B-D is a
chemically or biologically based substance (antibody,
peptide, protein, hormone, toxin etc.; INDEX NOMINUM,
International Drug Directory, Medpharm) with a
biological effect as well as derivatives thereof
chemically modified by the introduction of a functional
group (e.g. a hydroxy group). A prerequisite is that the
pharmacologically active substance or its prodrug form
are activated by this endogeneous enzyme via the
cleavage of the liponucleotide by the lipid cleavage
enzyme. In this connection pharmacologically active
substances are preferred which after cleavage by the LCE
act in vivo as an intermediate product as the
pharmacologically active substance monophosphate and are
further phosphorylated by cellular enzymes such as e.g.
nucleoside monophosphate to nucleoside triphosphate, or
are cleaved to form a free pharmacologically active
substance (see example 8).
Within the sense of the invention all pharmacologically
active substances come into consideration which are
effective in vitro but are toxic in vivo in the
therapeutic range i.e. all substances with a narrow
therapeutic range which have a chemical functional group
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- 16 -
for a covalent linkage to phosphate. In addition those
substances can also be used which, although at first
containing no functional group in their
pharmacologically active form, can have one introduced
by chemical modification without a loss in the effect of
the substance.
Those pharmacologically active substances are preferably
used for conjugation with a lipid residue L which
normally reach their active form after phosphorylation
(such as in the case of nucleosides) or phosphonates of
the active substance. The pharmacologically active
substance phosphate is then released from the conjugate
by enzymatic hydrolysis of the conjugate. The release of
the phosphorylated substance is particularly important
since this process can also take place in cells which do
not normally have the necessary enzymes (kinases) to
phosphorylate the pure pharmacologically active
substance. The conjugated pharmacologically active
substance that is released intracellularly or in the
cell membrane by LCE can for example have a cytostatic,
cytotoxic, antitumoral, antiviral, antiretroviral,
immunosuppressive or immunostimulating effect.
Compounds that are suitable as pharmacologically active
substances D which can be optionally converted into a
derivative capable of coupling by introduction of a
functional group which does not significantly influence
its action which then for example slows tumour growth,
is a substance which intercalates into DNA and/or RNA,
inhibits topoisomerase I and II, is a tubulin inhibitor,
is an alkylating agent, is a ribosome inactivating
compound, is a tyrosine phosphokinase inhibitor, is a
differentiation inducer, a hormone, hormone agonist or
hormone antagonist, is a substance which changes
CA 02204908 l997-0~-08
- 17 -
pleiotropic resistance to cytostatic agents, is a
calmodulin inhibitor, is a protein kinase C inhibitor,
is a P-glycoprotein inhibitor, is a modulator of
mitochondrially bound hexokinase, is an inhibitor of
~-glutamylcysteine synthetase or glutathione-S
transferase, is an inhibitor of superoxide dismutase,
is an inhibitor of reverse transcriptase of HIV-1 and
HIV-2 or inhibitors of hepatitis viruses A-E.
The pharmacologically active substance D or B-D can have
an antiinflammatory, antirheumatic, antiphlogistic,
analgetic or antipyretic action. It can in addition be
an antiarrhythmic agent, a calcium antagonist,
antihistamine drug, an inhibitor of phosphodiesterase or
a sympathomimetic or parasympathomimetic.
All substances are suitable as the pharmacologically
active substances D or B-D which have a short half-life,
in particular also compounds with different organ,
tissue or cell half-lives, a poor bioavailability i.e. a
poor resorption, high liver cleavage or rapid
elimination, poor membrane penetration (e.g. cell
membrane, blood-brain barrier), bone marrow toxicity or
other limiting organ toxicities (e.g. cardiotoxicities,
liver toxicities, nephrotoxicities, neurotoxicities
etc), whose active concentration in vivo is too low. In
addition those substances are suitable which interact
specifically with the cell nucleus of the target cells
and interfere with the molecular process at the DNA or
RNA level such as e.g. antisense oligonucleotides, DNA
fragments and those which can be used for gene therapy.
Pharmacologically active substances D in formula I are
for example: AZT (azidothymidine), FLT (fluoro-
CA 02204908 l997-0~-08
- 18 -
thymidine), 5-fluorouracil, 5-fluorouridine, 6-MPR,
fludarabin, cladribin, pentostatin, ara-C, ara-A, ara-G,
ara-H, Acyclovir, Ganciclovir, doxorubicin, 4'-epi-
doxorubicin, 4'-deoxy-doxorubicin, etoposide,
daunomycin, idarubicin, epirubicin, mitoxantron,
vincristine, vinblastine, Taxol, colchicine, melphalan,
3'-deoxy-2-fluoro-adenosine, FdA, 5-ethinyluracil-9-~-D-
arabinofuranoside, 5-propinyluracil-9-~-D-arabino-
furanoside, d4T, ddU, ddI, ddA, d2T, 2'-deoxy-2',2'-
difluorocytidine, 5-trifluoromethyl-2'-deoxyuridine, 5-
chloro-2',3'-dideoxy-3'-fluorouridine, 3'-deoxy-3'-
fluoromyoinositol, neplanocin A, ribavirin, myoinositol,
fialuridine, 3TC (Lamivudine), doxifluridine, Tegafur,
hypericin, pseudohypericin, Usevir, Famciclovir,
Penciclovir, Foscarnet, Carvedilol, actinomycin A,
bleomycin, daunorubicin, floxuridine, mithramycin,
mitomycin C, mitoxanthrone, streptozotocin, vindesin,
netilmycin, amikacin, gentamycin, streptomycin,
kanamycin A, tobramycin, neomycin B, plicamycin,
papamycin, amphotericin B, vancomycin, idoxuridine,
trifluridine, vidarabin as well as morphines,
prostaglandines, leukotrienes or cyclosporins. Moreover
terfenadin, dexamethasone, terbutalin, prednisolone,
fenoterol, orciprenaline, salbutamol, isoprenaline,
muscarine, bupranolol, oxyphenbutazone, oestrogen,
salicylic acid, propranolol, ascorbic acid, spongiadiol,
diclofenac, isospongiadiol, flufenamic acid, digoxin,
4-methyl-aminophenazone, allopurinol, theophylline,
epoprostenol, nifedipine, quinine, reserpine,
methotrexate, chloroambucil, spergualine, ibuprofen,
indomethacin, sulfasalazine, penicillinamine,
chloroquine, azathioprine also come into consideration.
Pharmacologically active substances B-D in formula I are
for example PMEA and other acyclic nucleoside
CA 02204908 1997-0~-08
phosphonate analogues, quinoline-phosphonic acids, MP-
101, fostedil, HAB-439, 2-amino-7-phosphonoheptanoic
acid, fosfosal, fosphenytoin, atrinositol, cyplatate,
difficidin, fosquidone, alafosfalin, foteumstine,
etoposide, encyt/E, 4-oxo-S-phosphono-D-norvalin and
other NMDA-antagonist phosphonates.
Preferred pharmacologically active substances are for
example also peptides, proteins and oligonucleotides
such as e.g. corticotropin, calcitonin, desmopressin,
gonadotropin, goserelin, insulin, zypressin, beta-
melanotropin, alpha-melanotropin, muramyldipeptide,
oxytocin, vasopressin, FK-506, octreotide or enalkiren.
The above-mentioned pharmacologically active substances
and the conjugates to be prepared therefrom only
represent examples and do not limit the inventive idea.
Compounds of formula I and the production thereof are
for example described in the applications WO 92/03462,
W0 93/16092, W0 93/16091, W0 94/03465, PCT/EP94/02123,
DE 4402492, DE 4418690 as well as for example in
WO 91/19726; EP o 350 287; US 5,223,263; US 5,194,654;
US 4,921,951; US 4,622,392; US 4,291,024; US 4,283,394.
In the case of antivirally effective nucleosides lipid
derivatives (diacylglycerol nucleosides) and their use
in liposomal form are described in EP 0 350 287 and
US 5,223,263. An uptake of the substance preferably in
the form of liposomes by cells of the reticulo-
endothelial system (RES) e.g. macrophages and monocytes
should be possible.
It was possible to show by appropriate comparative
experiments that the therapeutic effects in vivo of the
CA 02204908 1997-0~-08
- 20 -
diacylglycerol conjugates known from EP 0 595 133 are
inferior to the thioether or etherlipid conjugates from
W0 92/03462. This is due to unspecific hydrolysis of the
fatty acid esters which does not only take place at the
site of action. In contrast the non-hydrolysable
thioether and ether residues exhibit significant
advantages since a substance with biological action or
appropriate intermediate products are only released in
the membranes of the target tissue or intracellularly by
the special enzyme (LCE).
The conjugates of formula I (L-B-D) exhibit significant
advantages in comparison with the unconjugated
pharmacologically active substance D or B-D . The
specific carrier (L-B- or L) covalently bound to the
pharmacologically active substance improves the
bioavailability of the poorly resorbed pharmacologically
active substances, the tolerance of potentially toxic
active molecules, the retention time of rapidly
eliminated or metabolized pharmaceutical agents and the
membrane penetration of compounds with poor membrane
permeability (e.g. blood-brain, cells etc.). The
enzymatic cleavage in vivo into carrier and
pharmacologically active substance D (or substance
derivative) or into carrier and pharmacologically active
substance phosphate (D-monophosphate) or phosphonate B-D
usually does not occur in the serum but only
intracellularly. In addition the carrier moiety with its
lecithin-like structure, which is essential for the
claimed effect, improves the penetration or membrane
permeability of the pharmacologically active substance
and exhibits a depot effect in many cases. Moreover the
gastrointestinal tolerance of the lipid conjugates L-B-D
is considerably better than that of the pure
pharmacologically active substances D. The lipid
CA 02204908 l997-0~-08
- 21 -
conjugate also exhibits a better penetration through
membrane structures during resorption and thus it is
more able to overcome the resorption barriers. The same
also applies to penetration e.g. of the blood-brain
barrier by facilitated diffusion or possibly active
transport. Conjugates of formula I with a lipid moiety
as limited by formula II are particularly preferred
which cleave off a substance with biological activity by
enzymatic hydrolysis with the lipid cleavage enzyme
which leads to less side effects in vivo than after
administration of the pharmacologically active substance
alone.
In addition the in vivo distribution is improved by a
better binding of the conjugate to plasma and tissue
proteins. The conjugate is primarily oxidized by normal
biotransformation from a thioether (X = S) to a
sulfoxide (X = SO) which, however, due to the equipotent
action of the sulfoxide in comparison to the thioether,
does not represent a disadvantage. The slow release of
the pharmacologically active substance from the
conjugate ensures a low level of active substance that
is, however, constant over a longer period of time and
thus improves the efficacy or avoids toxic side-effects.
The released pharmacologically active substance in the
form of a monophosphate no longer penetrates out of the
cell due to its high hydrophilicity.
The total body, cell and the organ half-lives of the
pharmacologically active substance are considerably
extended by the conjugation due to the longer retention
time of the conjugate in the organism. Due to the lack
of LCE activity for cleavage in serum and in some
organs, almost no or only slight bone marrow and organ
toxicity can be observed. It is particularly
CA 02204908 1997-0~-08
- 22 -
advantageous that the conjugates of formula I are
specifically accumulated in various target organs,
tissues or cells.
The compounds of formula I can be used as active
substances for the production of pharmaceutical agents
which can be used for all diseases in which a high level
of pharmacologically active substance in cells, organs
or tissues is required or is beneficial. An essential
requirement for this transport system denoted "drug-
storage-delivery-targeting" is that the cells which are
to respond in accordance with the intended therapy have
the lipid cleavage enzyme in the inner or outer cell
membrane so that the active substance binds to the LCE
in a first step and is subsequently transported through
the cell membrane into the interior of the cell in the
process of which the active substance is cleaved to form
the-physiologically active substance either essentially
simultaneously with transport through the cell membrane
or even later partially within the cell. Intracellular
cleavage takes place especially in those cases in which
the LCE is also located within the cell. Within the
sense of the invention the cleavage of the active
substance linked with the intracellular release of the
pharmacologically active substance can also occur such
that either the physiologically or pharmacologically
active substance is formed directly in this process or
an appropriate precursor of this substance (prodrug
form). Suitable target cells are for example blood
leucocytes (PBLs), monocytes, kidney cells or
macrophages and cells of the immunological lymphatic
system.
The effect of compounds of formula I is in particular
immunomodulating, para/sympatholytic, /sympathomimetic,
CA 02204908 1997-0~-08
centrally and/or peripherally muscle-relaxing;
antihypertensive, antihypotensive, antiobstructive,
analgetic, anti-phlogistic, antiemetic, anti-
inflammatory, antiallergic, antiasthmatic, antipyretic,
antiulcerative, antacidic, antianginal, antiarrhythmic,
antipsychotic, antidepressive, antiepileptic,
anticonvulsive, antiparkinsonoid, antihistaminergic,
antimuscarineric, antiserotoninergic, antigabaergic,
antiadrenergic, anticholinergic, glycosidic,
chronotropic, bathmotropic, dromotropic, inotropic,
diuretic, antidiuretic, uricosuric, uricostatic,
antihypolipidaemic, antifibrinogenic, antidiabetic,
hypoglycaemic, antioestrogenic, antiandrogenic,
antigestagenic, antiosteoporetic, thyreostatic, bone-
growth stimulating, narcotic, anaesthetic, antihypnotic,
antiinfectious, antibiotic, antituberculostatic or
haematopoetic or they represent a vitamin.
The advantageous effect of one of the compounds
according to the invention can be increased by
combination with suitable pharmaceutical agents or
combination of two different conjugates of the present
application.
The effect of a cytostatic or cytotoxic conjugate of
this application can for example be increased by
combination with other cytostatic or cytotoxic compounds
preferably when components with different m~chAn;~r~ of
action are used or a combination of a cytostatic or
cytotoxic conjugate with an antiviral conjugate
(synergism e.g. in the case of AIDS).
The conjugate combinations are especially suitable
according to the invention in which one component has
CA 02204908 1997-0~-08
- 24 -
cytostatic or cytotoxic potential and the other
component overcomes for example multi drug resistance or
a component inhibits reverse transcriptase of HIV and
the other component inhibits the protease for example or
both liponucleotides of the combination do not exhibit
cross-resistance. Compounds of formula I and their
pharmaceutical preparations can also be used for the
production of pharmaceutical agents which are suitable
for the treatment and prophylaxis of various diseases in
combination with other pharmaceutical agents.
Alkali, alkaline-earth and ammonium salts of the
phosphate group come above all into consideration as
possible salts of the compounds of the general formula
I. Lithium, sodium and potassium salts are preferred as
the alkali salts. Magnesium and calcium salts come in
particular into consideration as alkaline-earth salts.
Ammonium salts are understood according to the invention
as salts which contain the ammonium ion that can be
substituted up to four times by alkyl residues with 1-4
carbon atoms and/or by aralkyl residues preferably by
benzyl residues. In this case the substituents can be
the same or different.
The compounds of the general formula I can contain basic
groups in particular amino groups which can be converted
using suitable inorganic or organic acids into acid
addition salts. Acids which come for example into
consideration are: hydrochloric acid, hydrobromic acid,
sulphuric acid, phosphoric acid, fumaric acid, succinic
acid, tartaric acid, citric acid, lactic acid, maleic
acid or methanesulfonic acid.
In addition to the compounds of the general formula I
CA 02204908 1997-05-08
- 25 -
and the specifications according to formula II and III
this application also claims tautomers thereof and their
physiologically tolerated salts of inorganic and organic
acids or bases as well as processes for their production
and pharmaceutical agents containing these compounds.
Since the compounds of the general formula I contain
asymmetric carbon atoms all optically active forms and
racemic mixtures of these compounds are also a subject
m~tter of the present invention.
This application also concerns new liponucleotides. The
synthesis of these conjugates is exemplified in examples
14-16. The LCE also cleaves 2-chloro-2'-deoxy-adenosine
conjugates (cladribin conjugates) of formula V, 9-(~-D-
arabino-furanosyl)-2-fluoroadenine conjugates
~fludarabine conjugates) of formula VI, 3-(2-deoxy-B-D-
~rythro-pentofuranosyl)-3~ 6, 7, 8-tetrahydroimidazo
[4,5-d] [1,3] diazepin-8-ol conjugates (pentostatin
conjugates) of formula VII. The said compounds can be
used therapeutically better than the corresponding
nucleosides alone since they have a very good efficacy
and a large therapeutic range and have the same
advantages as the above-mentioned derivatives of
formula I:
NH2
L--B--O I o N N Cl
~ ',~ ~
OH
CA 02204908 1997-05-08
- 26 -
,NH2
L--B--o ~ o N N F (Vl)
OH
'~V
OH
HO
N , ',
' .! NH
L--B--O , o , N ~ ~VII)
''J
1H
Within the sense of the present invention the following
active substances are used in particular as compounds of
formula I for producing pharmaceutical agents:
1. (3'-deoxy-3'-azidothymidine)-5'-phosphoric acid-(3-
dodecyl-mercapto-2-decyloxy)-propyl ester
2. (3'-deoxy-3'-azidothymidine)-5'-phosphoric acid-(3-
undecyl-mercapto-2-undecyloxy)-propyl ester
3. (3'-deoxy-3'-fluorothymidine)-5'-phosphoric acid-
(3-dodecyl-mercapto-2-decyloxy)-propyl ester
4. (3'-deoxy-3'-fluorothymidine)-5'-phosphoric acid-
(3-undecyl-mercapto-2-undecyloxy)-propyl ester
CA 02204908 1997-0~-08
- 27 -
5. (2',3'-dideoxycytidine)-5'-phosphoric acid-(3-
dodecyl-mercapto-2-decyloxy)-propyl ester
6. (2',3'-dideoxyinosine)-5'-phosphoric acid-(3-
dodecyl-mercapto-2-decyloxy)-propyl ester
7. (3'-deoxythymidine)-5'-phosphoric acid-(3-dodecyl-
mercapto-2-decyloxy)-propyl ester
8. (5'-fluorouridine)-5'-phosphoric acid-(3-dodecyl-
mercapto-2-decyloxy)-propyl ester
9. (6-mercaptopurine-9-~-D-ribofuranoside)-5'-
phosphoric acid-(3-dodecylmercapto-2-decyloxy)-
propyl ester
10. (5-trifluoromethyluridine)-5'-phosphoric acid-(3-
dodecyl-mercapto-2-decyloxy)-propyl ester
11. (1-~-D-arabinofuranosyl-5-ethinyluracil)-5'-
phosphoric acid-(3-dodecyl-mercapto-2-decyloxy)-
propyl ester
12. (2'-deoxy-5-propinyluridine)-5'-phosphoric acid-(3-
dodecyl-mercapto-2-decyloxy)-propyl ester
13. 2'-(9-{[(1-hydroxymethyl)ethoxy]methyl}guanine)
phosphoric acid-(3-dodecyl-mercapto-2-decyloxy)-
propyl ester
14. 2'-[9-(ethoxymethyl)guanine])phosphoric acid-(3-
dodecyl-mercapto-2-decyloxy)propyl ester
CA 02204908 1997-05-08
- 28 -
Example 1:
Enzymatic cleavage of AZT-DMDOPE
10 mg (3'-deoxy-3'-azidothymidine)-5'-phosphoric acid-
(3-dodecyl-mercapto-2-decyloxy-propyl ester (AZT-DMDOPE)
is suspended in 0.5 ml 0.1 M Tris buffer solution and
incubated for 15 hours at 37~C after addition of the
enzyme (O.5 mg phosphodiesterase and 0.1 mg
phospholipase/nuclease). Subsequently the solution is
e~;ned by thin layer chromatography. After addition of
5 drops of saturated NaHCO3 solution it was incubated
for a further 6 hours at 37~C and subsequently the
solution was examined by thin layer chromatography.
The solution was ~ ;ned for the formation of possible
cleavage products (AZT, AZT-monophosphate, DMDOP and
lipid monophosphate) by thin layer chromatography using
various eluting agents.
The followinq enzymes were used in the above-mentioned
test:
a) Phospholipase C from Bacillus cereus, 2000 U/0.5 ml,
Boehringer M~n~heim GmbH
b) Phospholipase D, type I, from cabbage, 150 - 300 U/mg,
Sigma
c) Phosphodiesterase from calf spleen, 2 U/mg,
Boehringer Mannheim GmbH
d) Phosphodiesterase from snake venom, Boehringer
M~nnheim GmbH
e) Nuclease from Staphylococcus aureus, 15,000 U/mg,
Boehringer M~nnheim GmbH
f) LCE (lipid cleavage enzyme)
CA 02204908 1997-0~-08
- 29 -
Result:
The following Table shows that a weak fluorescence
quenching at the level of the lipid phosphate is only
found in the case of phospholipase D. In none of the
cases a) - e) used as reference enzymes was AZT or AZT
monophosphate formation detected. Apart from the
unchanged AZT-DMDOPE no additional spot was found in
these experiments within the detection limit.
Enzyme Fluorescence quenching
TLC
Phospholipase C negative
Phospholipase D weak
Phosphodiesterase from negative
calf spleen
Phosphodiesterase from negative
snake venom
Nuclease from negative
Styphylococcus aureus
LCE positive
Example 2:
Figure legends
Fig. 1: This figure shows the activity of the LCE which
was obtained from the membrane fraction of human
PHA-stimulated peripheral blood leucocytes
relative to the protein concentration. There is
a linear relationship between the protein
concentration and the amount of the cleavage
product DMDOP formed.
CA 02204908 1997-0~-08
- 30 -
Fig. 2: This figure shows the substrate kinetics
(Michaelis-Menten kinetics) of the LCE which was
obtained from the membrane fraction of human,
PHA-stimulated peripheral blood leucocytes.
Fig. 3: This figure shows the substrate kinetics
(Michaelis-Menten kinetics) of the LCE which was
obtained from the membrane fraction of human,
PHA-stimulated peripheral blood leucocytes [-]
and from quiescent (non stimulated) peripheral
blood leucocytes [~].
Fig. 4: This figure shows the dependence of the specific
enzyme activity of the LCE which was obtained
from the membrane ~raction of human, PHA-
stimulated peripheral blood leucocytes on the
calcium ion concentration.
Fig. 5: This figure shows the organ-specific and tissue-
specific distribution of the LCE of cell
membrane preparations in the untreated dog.
Fig. 6: Intracellular concentrations of AZT and AZT
nucleotides in stimulated human PBL after 1, 3, 6
and 24 h incubation with:
(A) 0.03 ~g AZT/ml (-) or 1 ~g AZT-DMDOPE/ml (~)
(B) 0.3 ~g AZT/ml (-) or 10 ~g AZT-DMDOPE/ml (~)
(means, n = 2 determinations)
Fig. 7: Intracellular concentrations on AZT and AZT
nucleotides in non-stimulated human PBL after 1,
3, 6 and 24 h incubation with:
(A) 0.03 ~g AZT/ml (-) or 1 ~g AZT-DMDOPE/ml (~)
(B) 0.3 ~g AZT/ml (-) or 10 ~g AZT-DMDOPE/ml (~)
CA 02204908 l997-0~-OX
- 31 -
(means, n = 2 determinations)
Fig. 8: Intracellular concentrations of AZT and AZT
nucleotides in stimulated human PBL after 6 and
24 h incubation with 1 ~g AZT-DMDOPE/ml (A) and
10 ,ug AZT--DMDOPE/ml (B):
~ treatment with alkaline phosphatase
no treatment with alkaline phosphatase
~means, n = determinations)
Fig. 9: Intracellular concentration of AZT and AZT
nucleotides in P3X63-Ag8.653 cells after 6, 24 and
48 h incubation with:
(A) 0.03 ,llg AZT/ml (--) or 1 ,ug AZT-DMDOPE/ml (~)
(B) 0.3 ,ug AZT/ml (--) or 10 ,llg AZT-DMDOPE/ml (~)
(means, n = 2 determinations)
Fig.10: Intracellular concentration of AZT and AZT
nucleotides in P3X63-Ag8.653 cells after 6 and
24 h incubation with 1 ~g BM 21.1290 Na/ml (A) and
10 ,ug AZT-DMDOPE/ml (B):
~ treatment with alkaline phosphatase
no treatment with alkaline phosphatase
(means, n = determinations)
Fig.11: Intracellular decay kinetics of AZT and AZT
nucleotides in stimulated human PBL after 24 h
incubation with 0.3 ~g AZT/ml(A) or 10 ~g AZT-
DMDOPE/ml(B). Intracellular concentration of AZT
and AZT nucleotides 1, 3, 6, 24 and 48 h after
removing the AZT or ATZ-DMDOPE incubation solution
(means, n = 2 determinations)
CA 02204908 1997-0~-08
- 32 -
Fig.12: Enzymatic cleavage of [14C]-AZT-DMDOPE by membrane
fractions (100 ~g protein/preparation) of
stimulated human PBL. Thin layer chromatogram of
the n-heptane phase after adsorption of the
substrate to silica gel 60H and separation in the
IBA system (2-propanol:n-butyl acetate:redistilled
water, 10:6:4, v/v/v) using silica gel 60 as a
stationary phase.
Fig.13: Specific activity [pmol mg~l min~1] of the LCE in
cell homogenates, cytosol and membrane fractions
of stimulated human PBL (mean + SD, n = 6
determinations)
Fig.14: Cleavage of AZT-DMDOPE by the membrane fractions
of stimulated human PBL in relation to the protein
concentration (mean + SD, n = 3 determinations).
Fig.15: Cleavage of AZT-DMDOPE by the membrane fraction of
human monocytes/macrophages (stimulated monocytes)
in relation to the protein concentration (mean,
n = 2 determinations).
Fig.16: Specific activity of the LCE in the membrane
fractions of stimulated and non-stimulated human
PBL and of human monocytes and
monocytes/macrophages (stimulated monocytes)
(human PBL: mean + SD, n = 6 determinations)
(human monocytes: mean + SD, n = 4 determinations.
Fig.17: Cleavage of AZT-DMDOPE by membrane fractions of
stimulated human PBL in relation to the incubation
time (mean + SD, n = 3 determinations)
CA 02204908 1997-0~-08
- 33
Fig.18: Thin layer chromatogram of [14C]-AZT-DMDOPE (A)
and of [14C]-AZT-DMDOPE after enzymatic cleavage
by cell homogenates of 5 x 107 CEM-SS cells (B)
after an incubation of 6 h at 37~C. Extraction of
the thioether lipids with diethyl ether : 2
propanol (9:1, v/v) and separation in the IBAE
system (2-propanol : n-butyl acetate: redistilled
water: glacial acetic acid, 3:5:1:1, v/v/v/v)
using silica gel 60 as a stationary phase
Example 3:
Influence of various enzYme inhibitors or activators on
the LCE activity
Inhibitor/ Site of attack of LCE Influence on
activator the inhibition/ membrane LCE activity
activation fraction
SQ 22536 adenylate-cyclase kidney (Balb/c) none
7-nitro- NO synthesis kidney (Balb/c) none
indazole
RHC-80267 DAG lipase kidney (Balb/c) none
neomycin PLC, (PLD) kidney (Balb/c) none
wort~nn;n PLD, (PLC) kidney (Balb/c) none
acetyl- PLC kidney (Balb/c) none
salicylic
acid
GTP-~-S Gp activator kidney (Balb/c) none
D 609 PLC kidney (Balb/c) stimulation
CEM-SS stimulation
CA 02204908 1997-0~-08
- 34 -
Example 4:
Subcellular fractionation
a. Preparation of cell homogenates, membrane and
cYtosol fractions
The cells cultured for the preparation of membrane
and cytosol fractions were transferred to
polypropylene tubes (50 ml) and sedimented for 10
min at 1600 rpm in a Minifuge T at room
temperature. In order to remove residues of the
culture medium the cell sediment was washed three
times with cold PsS and taken up in lysis buffer at
a cell density of 1 - 5 x 108 cells/ml. The cell
suspension was subsequently transferred to a glass
homogenizer cooled on ice. The cells were
m~chAnically disrupted by several-fold movement of
a teflon piston during which the cell disruption
was monitored optically in a reverse phase contrast
microscope. The disrupted cells were subsequently
centrifuged for 10 min at 1700 rpm and 40C in a
Minifuge T to remove the cell nuclei and the non-
disrupted cells. The supernatant was carefully
removed with a pipette and diluted to 10 ~ (w/v)
sucrose with 50 mM Tris buffer (pH 7.5). The cell
homogenate was divided into portions and stored at
-70~C or used to isolate membrane and cytosol
fractions. For this 3.2 ml of the cell homogenate
was transferred to a thick-walled polycarbonate
tube (3.2 ml) cooled on ice and centrifuged for 1 h
at 4~C and 75,000 rpm in a Beckmann table-top
ultracentrifuge with a TLA-100.4 rotor. The
supernatants were carefully removed with a Combitip
CA 02204908 1997-0~-08
pipette and the readily visible membrane se~;m~nts
were each admixed with 1 ml 50 mM Tris buffer (pH
7.5)/10 % sucrose. The membrane sediment was
coarsely homogenized with the aid of a syringe
(5 ml) with a cannula (0.9 x 40 mm). The coarse
homogenates were combined and finely homogenized
using cannulae of a smaller diameter (0.8 x 40 mm
and 0.45 x 25 mm). The membrane and cytosol
fractions were divided into portions and stored at
-70~C.
LYsis buffer 50 mM Tris pH 7.5
70 % (w/v) sucrose
50 ~g/ml APMSF
In order to dissolve the sucrose the
buffer has to be gently warmed while
stirring.
b. Isolation of the plasma membrane of stimulated
human peripheral blood lymPhocytes (PBL) (Record et
al. fl985), Biochem. Biophys. Acta 8/9 1-9)
Human PBL were isolated by centrifugation in an
isotonic density gradient and stimulated for 72 h
with PHA-M. The cells were washed three times with
cold PBS to remove residues of the culture medium
and, after determination of the cell number, were
frozen in liquid N2 and subsequently stored at
-70~C. The cells were lysed by freezing and thawing
three times in buffer 1 whereby the cell density
was adjusted to 2.5 x 108 cells/ml. A mixture of
11 ml Percoll and 2.13 ml buffer 2 was placed in
Ti60 centrifuge tubes (30 ml) which had previously
CA 02204908 1997-0~-08
been adjusted to pH 9 with 70 ~1 2 N NaOH. For the
isolation of the plasma membrane 4 ml of the
homogenate was applied to the mixture and
subsequently centrifuged for 10 min at 4~C and
39,000 rpm in a Ti60 rotor of a Beckmann
ultracentrifuge L 5 50 with the brake switched off.
1 ml fractions were taken from the upper phase of
the centrifuge tubes and diluted with 2 ml buffer
3. The solutions were subsequently transferred to
Ti50 tubes (10.4 ml) and centrifuged for 45 min at
4~C and 45,000 rpm in a Beckmann ultracentrifuge L
5 50 with a Ti50 rotor to remove residues of the
separation medium. The supernatants were removed
with a pipette, fractionated, frozen in liquid N2
and stored at -70~C. The fractions were
characterized by determining the protein content
and the enzyme activity of alkaline phosphatase.
Buffer 1 loO mM KCl
5 mM MgC12 x 6 H2O
1 mM ATP
2 mM (4~ inophenyl)-methane
sulfonylfluoride (APMSF)
25 mM Tris pH 9.6
Buffer 2 400 mM KCl
20 mM Mgcl2 x H2O
400 mM Tris pH 9.6
Buffer 3 100 mM KCl
5 mM MgC12 x 6 H2O
50 mM Tris pH 7.42
CA 02204908 1997-0~-08
- 37 -
ExamPle 5:
Production of substrate solutions for the LCE assav
A mixture of [14C]-AZT-DMDOPE and non-radioactively
labelled AZT-DMDOPE was used as a substrate in the ~CE
assay.
Starting with a [14C]-AZT-DMDOPE stock solution, a
solution with a specific activity of 83. 27 kBq/ml was
prepared by dilution with ethanol. The ethanolic
solution could be stored for 2 months at 4~C under inert
gas.
In a separate mixture the non-radioactively labelled
component of the substance mixture was prepared. For
this a solution of AZT-DMDOPE at a concentration of
1 ~mol/ml in ethanol was prepared and continuously
sonicated at 30 ~ energy release for 1 min in an
ice/water bath with an ultrasonic probe.
In order to prepare the substrate mixture of radioactive
and non-radioactive AZT-DMDOPE, 1. 67 kBq of the
radioactively labelled [14C]-AZT-DMDOPE and O. 7 -- 5 nmol
of the non-labelled compound were transferred per
mixture from these ethanolic solutions into a glass tube
(10 ml). The solvent was evaporated in a N2 s~ream and
AZT-DMDOPE was taken up in a suitable volume of
redistilled water and continuously sonicated at 30 %
energy release for 1 min while cooling in an ice/water
bath.
=
CA 02204908 1997-05-08
- 38 -
Example 6:
LiPid cleavaae enzvme (LCE) assay
In order to e~ ;ne the enzymatic cleavage relative to
time and the protein concentration 5 nmol AZT-DMDOPE and
0.98 nmol [14C]-AZT-DMDoPE (1.67 kBq) In a volume of 200
~l redistilled water were usually used in the LCE assay.
The pipetting scheme is given in the following table.
Table Pipetting scheme for the LCE assay to determine
the turnover of AZT-DI~r~
Reaction mixture Control
EGTA [20 mM] 50 50
Tris, pH: 8.0 [1 M] 50 50
[~l/mixture]
AZT-DMDOPE/[14C]-AZT-
DMDOPE [nmol/mixture]0.10 - 5.980.10 - 5.98
[~l/mixture] 5 - 200 5 - 200
Protein
[mg/mixture] 0.0125 - 0.2
[~l/mixture] x
redistilled water ad 500 ad 500
[~1]
In order to examine the enzymatic cleavage in relation
to the substrate concentration, a substrate stock
solution of 0.98 nmol [l4C]-AZT-DMDOPE and 0.7 nmol AZT-
DMDOPE/80 ~l redistilled water was used in the LCE
assay. The various substrate concentrations were
adjusted in the LCE assay by continuously doubling or
halving the volume of the substrate solution. The
CA 02204908 1997-0~-08
- 39 -
mixtures were placed in glass tubes (5 ml) and started
by adding the substrate solution. The incubation was
carried out at 370C in a water bath while gently
shaking.
Example 7:
Extraction of DMDOP from an aqueous matrix
The reaction mixtures of the LCE assays were taken from
the water bath after an appropriate incubation period and
the reaction was stopped by adding 750 ~l 2-propanol.
After thorough mixing 700 ~1 n-heptane heated to room
temperature was added to the mixtures. The mixtures were
again thoroughly mixed for 30 sec and centrifuged for 15
min in a Minifuge T at 3200 rpm at room temperature to
accelerate the phase separation. Subsequently 500 ~l of
the upper phase (n-heptane) was removed and transferred
to new glass tubes (5 ml) containing 10 mg silica gel 60
H and 200 ~l n-heptane. The mixtures were thoroughly
mixed for 30 sec and centrifuged for 10 min under the
conditions mentioned above to sediment the silica gel.
Subsequently 500 ~l was taken from the supernatant and
admixed with 3 ml scintillation liquid. The
radioactivity was measured for 4 min in a liquid
scintillation analyzer. In order to determine the
absolute value of radioactivity, 200 ~l of the substrate
solution was mixed with 3 ml scintillation liquid and
measured under the same conditions in the liquid
scintillation analyzer.
CA 02204908 1997-0~-08
- - 40 -
ExamPle 8:
In vitro pharmakokinetic studies with AZT-DMDOPE and AZT
a) Determination of the intracellular concentrations
of AZT and/or AZT nucleotides in stimulated and
non-stimulated human PBL after incubation with AZT-
DMDOPE and AZT
Human PBL were isolated from the buffy coat in
order to determine the intracellular concentrations
of AZT and/or AZT nucleotides after incubation with
AZT-DMDOPE and AZT.
For this the buffy coat of healthy donors is mixed
and fractionated by centrifugation in an isotonic
medium with a density of 1.077 g/ml. Since mono-
nuclear blood cells (monocytes and lymphocytes)
have a lower density than erythrocytes and
polymorphonuclear granulocytes they form a ring at
the border between the separation medium and the
sample and can be removed with the aid of a
pipette. Erythrocytes and polymorphonuclear
granulocytes se~;~~nt through the medium as a
result of their higher density and can thus be
separated from the PBLs.
In order to stimulate cell proliferation
phytohaemagglutinin A (mucoprotein) (PHA-M) the
mitogenic lectin extract from the red scarlet
runner (Phaseolus vulgaris) was used.
Phytohaemagglutinin is a family of 5 isolectins
which are present as tetramers. The subunits are
composed of a lymphocyte-reactive type L and an
CA 02204908 l997-0~-08
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erythrocyte-reactive type E. Type L has a high
affinity to lymphocyte surface receptors and is
thus responsible for the mitogenic properties. The
lymphocytes were stimulated for 72 h at 37~C and
5 % CO2 in KPMI 1640 complete medium III. After the
stimulation was complete the cells were cultured
for a further 24 h in RPMI 1640 complete medium IV
to increase the cell number.
For the determination of the intracellular
concentrations of AZT and AZT nucleotides the cells
were transferred into RPMI 1640 complete medium II
at a density of l x 106 cells/ml in tissue culture
flasks (50 ml/25 cm2) and incubated for 1 to 24 h
in the presence of 0.03 and 0.3 ~g AZT/ml (lot 15)
and 1 or 10 ~g AZT-DMDOPE/ml.
The concentrations of AZT-DMDOPE and AZT in this
case depend on the IC50 values for the
antiretroviral activity which was determined for
AZT and AZT-DMDOPE in HIV-1 infected human PBL.
The cultures were subsequently incubated for 1, 3,
6 and 24 h at 37~C and 5 % CO2. AZT and its
nucleotides were subsequently extracted with 60 %
methanol. The extracted nucleotides were
quantitatively dephosphorylated to AZT with
alkaline phosphatase since only AZT but not the
corresponding nucleotides can be detected with the
aid of a radioimmunoassay.
Accordingly the concentrations of intracellular AZT
that were determined with the aid of a radioimmuno-
assay were composed of that of the AZT and that of
CA 02204908 1997-0~-08
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the AZT nucleotides together. A separate
quantification of the concentrations of AZT-MP,
AZT-DP and AZT-TP was not carried out. Experimental
observations on stimulated PBL were able to
demonstrate a 40-fold higher concentration of AZT-
MP compared to AZT-DP and AZT-TP (Arnér et al.,
1992, J. Biol. Chem. 267, 10968-10975).
Consequently it can be assumed that the nucleotide
fraction is almost exclusively composed of AZT-MP.
In stimulated human PBL a ~;mll~ concentrations of
2.90 and 30.97 ng/106 cells was reached after an
incubation of 6 and 3 h respectively when incubated
with 0.03 and 0.3 ~g AZT/ml respectively (Fig. 6).
The concentrations subsequently decreased as the
incubation period increased to 2.32 and
19.88 ng/106 cells respectively. After incubation
with 1 and 10 ~g AZT-DMDOPE/ml there was a
continuous increase in the intracellular level of
AZT and its nucleotides over the entire incubation
period. After 24 h incubation with AZT-DMDOPE and
AZT the intracellular levels were identical. In
further experiments it was possible to show that
the intracellular concentrations of AZT and AZT
nucleotides after incubation with AZT-DMDOPE for a
period of 18 - 48 h was even higher than after
incubation with an equipotent concentration of AZT.
In the case of non-stimulated human PBL the
opposite relationships were found (Fig. 7). Thus
after incubation with AZT-DMDOPE the intracellular
concentrations of AZT and AZT nucleotides were
higher than after incubation with an equipotent
concentration of AZT over the entire incubation
period. After incubation with 1 and 10 ~g AZT-
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DMDOPE/ml maximum intracellular concentrations of
AZT and AZT nucleotides of 0.62 and 4.74 ng/106
cells respectively were reached whereas after
incubation with 0.03 and 0.3 ~g AZT/ml
concentrations of 0.10 and 0.47 ng/106 cells
respectively were detected.
In order to separately quantify AZT and AZT
nucleotides after incubation with BM 21.1290 Na all
intracellular concentrations of AZT and AZT
nucleotides were determined with and without
treatment with alkaline phosphatase. These
experiments were carried out on stimulated human
PBL.
For this cell suspensions with a density of 106
cells/ml containing 1 and 10 ~g AZT-DMDOPE/ml were
incubated for 6 and 24 h at 37~C and 5 % CO2. The
~ ;~um intracellular concentrations of free AZT
after incubation with 1 and 10 ~g AZT-DMDOPE/ml
were detected as 0.03 and 0.15 ng/106 cells
respectively after 6 and 24 h respectively (Fig.
8). If after treatment with alkaline phosphatase
the phosphorylated intracellular AZT nucleotides
were additionally measured then maximum
concentrations of 3.85 and 6.41 ng/106 cells
respectively were found after 24 h.
b. Determination of the intracellular concentrations
of AZT and/or AZT nucleotides in P3X63Aq8.653 cells
after incubation with AZT-DMDOPE and AZT
The determination of the intracellular
concentrations of AZT and AZT nucleotides in
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thymidine kinase deficient cells should yield more
information about the intracellular cleavage of the
test substance AZT-DMDOPE. Intracellular AZT cannot
be converted into AZT-MP and thus into the
therapeutically effective AZT-TP due to a lack of
thymidine kinase (TK) in these cells. The
determination of the intracellular concentrations
of AZT and AZT nucleotides after incubation with
AZT-DMDOPE could thus give a further information on
the cleavage of the test substance.
For this 1 x 107 cells in RPMI 1640 complete medium
I containing 0.03 or 0.3 ~g AZT/ml and 1 or 10 ~g
AZT-DMDOPE/ml were incubated for 6, 24 and 48 h in
tissue culture plates (60 x 15 mm). After
extracting the AZT and AZT nucleotides and
treatment with alkaline phosphatase the
concentration of AZT was determined with the aid of
the radioimmunoassay.
The graphical plot of the experimental results
shows that the intracellular concentrations of AZT
and AZT nucleotides were considerably higher with
AZT-DMDOPE than after incubation with equipotent
concentrations of AZT. Thus after 6 h incubation
with 1 and 10 ~g AZT-DMDOPE/ml maximum
concentrations of AZT and AZT nucleotides of 0.55
and 4.40 ng/106 cells respectively were achieved
(Fig. 9).
The maximum intracellular concentrations of AZT and
AZT nucleotides after incubation with 0.03 and
0.3 ~g AZT/ml were determined as 0.03 and
0.31 ng/106 cells respectively.
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In order to quantify the proportion of
phosphorylated AZT the intracellular concentrations
of AZT and AZT nucleotides were compared before and
after treatment of the cellular extracts with
alkaline phosphatase. The substance level of
phosphorylated AZT after incubation with 1 and
10 ~g AZT-DMDOPE/ml yielded values in this case of
0.48 and 2. 42 ng/106 cells respectively (Fig. 10).
If only AZT was measured then maximum
concentrations of 0.05 and 1.10 ng/ml respectively
were reached after 24 h. The differences in the
intracellular concentrations of AZT nucleotides
after incubation with AZT and AZT-DMDOPE can be
explained by an intracellular cleavage o~ the test
substance AZT-DMDOPE to AZT-MP.
c. Kinetics of the decrease of AZT and AZT nucleotides
in stimulated human PBL after incubation with AZT-
DMDOPE and AZT
Stimulated human PBL were incubated for 24 h with
0.3 ~g AZT/ml or 10 ~g AZT-DMDOPE/ml. Subsequently
1 x 107 cells were transferred into tissue culture
flasks (50 ml/25 cm2) cont~in;ng RPMI 1640 complete
medium I. After 1, 3, 6, 24 and 48 h AZT and AZT
nucleotides were extracted from the cellular
matrix. After treatment with alkaline phosphatase
the concentration of AZT was determined with the
aid of a radioimmunoassay.
The graphic representation of the experimental
results shows that after incubating the cells with
AZT the intracellular concentrations of AZT and AZT
nucleotides decrease rapidly and reach a constant
CA 02204908 1997-0~-08
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value of 0.40 ng/106 cells after 6 h. In contrast
in the incubation with an equipotent concentration
of AZT-DMDOPE the AZT and AZT nucleotides decrease
less rapidly and after 3 h reach a substantially
higher concentration of 1.80 ng/106 cells which
remains constant over 48 h (Fig. 11). Since non-
phosphorylated AZT was removed from the cell by
washing several times with PBS, the intracellular
concentrations are mainly attributable to
phosphorylated AZT nucleotides. Thus an incubation
with AZT-DMDOPE provides the cell with a 4.5-fold
higher concentration of phosphorylated AZT compared
to an equipotent concentration of AZT. These large
differences in the cellular concentrations of AZT
nucleotides after incubation with AZT-DMDOPE and
AZT can be explained by a direct intracellular
cleavage of the thioether-lipid-AZT conjugate AZT-
DMDOPE to AZT-MP and the corresponding thioether
lipid moeity DMDOP.
ExamPle 9:
Characterization of the AZT-DMDOPE cleavaqe enzyme/
enzYme sYstem (LCE)
Investigations on the enzymatic cleavage of AZT-DMDOPE
by cell homogenates of human PBL and CEM-SS cells showed
that AZT-DMDOPE is metabolized to DMDOP and AZT-MP. The
characterization of the LCE was the object of the
following investigations. Thus the dependency of the
enzymatic cleavage of AZT-DMDOPE on the protein
concentration, the incubation period and divalent metal
cations was examined. The dependency of the apparent
Michaelis-Menten parameters KM and vmaX on various
CA 02204908 1997-0~-08
substrate concentrations is determined in enzyme kinetic
experiments.
For these experiments an enzyme assay is established in
which AZT-DMDOPE and [14C]-AZT-DMDOPE were used as the
substrate (example 6). In protein and time dependent
measurements 5 nmol AZT-DMDOPE and 0.98 nmol (1.67 kBq)
[l4C]-AZT-DMDOPE were used per mixture. In the case of
substrate-dependent conversions a stock solution of 0.98
nmol [l4C]-AZT-DMDopE and 0.7 nmol AZT-DMDOPE/80 ~1 was
used. The various substrate concentrations were
subsequently adjusted by continuously doubling or
halving the volume of the substrate solution.
Cell homogenates, cytosol and membrane fractions of
known protein concentrations from various human cells
can for example be used as the enzyme source.
a. Isolation and quantification of DMDOP after
enzymatic cleavage of AZT-DMDOPE
The quantification of the enzymatic cleavage of
AZT-DMDOPE was carried out by two-step extraction
of the metabolite DMDOP with n-heptane and
subsequent adsorption of residues of the substrate
AZT-DMDOPE on silica gel 60 H.
For this the reaction mixtures were admixed with 2-
propanol and n-heptane in the process of which the
DMDOP which is less polar than the substrate
accumulates in the n-heptane phase. The n-heptane
phase was then admixed with silica gel 60H which
adsorbs residues of the substrate AZT-DMDOPE. The
n-heptane phase was afterwards separated from the
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silica gel by centrifugation, transferred to 3 ml
aqua luma and the amount of radioactively labelled
DMDOP contained therein was determined over a
period of 5 min in a li~uid scintillation analyzer.
From these results it was possible to calculate the
percentage conversion of the substrate AZT-DMDOPE
to DMDOP and the specific activity of the LCE. In
this case the specific activity is defined as the
amount of DMDOP which is formed per minute per 1 mg
protein of the cell preparations used.
In order to check the selective extraction of DMDOP
from the reaction mixture, optimization experiments
were carried out with membrane fractions of
stimulated human PBL. In this case the protein
concentration was 100 ~g/mixture. Mixtures without
protein were tested in parallel. After the
extraction the n-heptane phase was evaporated in an
N2 stream and the residues were taken up in a
mixture of methanol and ethyl acetate (1:1, v/v).
The analysis by thin layer chromatography was
carried out in the IBA system. The reaction value
in thîs case only allowed the parent substance
[14C~-AZT-DMDOPE to be detected. In the reaction
mixture a substance peak was detected at the end of
the mobile solvent front which could be identified
unequivocally as the metabolite [14C]-DMDOPE on the
basis of its Rf value (Fig. 12). The peak which
runs immediately in front of the DMDOP substance
peak cannot be allocated to either of the known
compounds. It is presumably a compound which is
formed by oxidation of the sulphur in the thioether
lipid moeity o~ DMDOP. Hence an extraction with n-
heptane enables the enzymatic cleavage product
CA 02204908 1997-0~-08
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DMDOP to be isolated from the enzyme assay. A
simple and rapid quantification of the metabolite
DMDOP released from AZT-DMDOPE was thus ensured.
b. Cleavage of AZT-DMDOPE bY cell homoqenates,
membrane and cytosol fractions of stimulated human
PBL
In a first series of experiments the enzymatic
cleavage of AZT-DMDOPE by cell homogenates and by
cytosol and membrane fractions of stimulated human
PBL was examined.
After determining the protein concentration in cell
homogenates, cytosol and membrane fractions with
the aid of the bicinchoninic acid (BCS) test 0.025
- 0.20 mg protein/mixture were used. The reaction
mixtures were incubated for 2 h at 37~C,
subsequently the product DMDOP was isolated from
the mixtures by extraction with n-heptane and the
specific activity of the LCE was determined (Fig.
13).
The AZT-DMDOPE-cleaving activity in cell
homogenates and in cytosol fractions of 6.48 + 0.38
(n = 6) and 1.65 + 0.40 pmol mg~l min~1 (n = 6) was
1.59-fold and 6.3-fold smaller than the specific
activity in the membrane fractions which was 14.23
+ 0.70 (n = 6) pmol mg~l min~1. Apparently there is
an enrichment of the LCE when the membrane
fractions are isolated. Based on these results
membrane fractions were used in the subsequent
experiments for determining the enzymatic
parameters of the LCE.
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The membrane fraction which was obtained by
mechanically disrupting the cells and subsequent
ultracentrifugation of the cell homogenate is a
mixture of fragments of the plasma membrane and
nuclear membrane as well as membranes of the cell
organelles.
It was possible to differentiate the various
membrane fragments by ultracentrifugation of the
cell homogenate in Percoll density gradients
followed by a subsequent characterization by the
plasma membrane marker alkaline phosphatase.
For the isolation of the plasma membranes 2 ml of a
cell suspension of stimulated human PBL with a cell
density of 2.5 x 1o8 cells/ml was disrupted by
freezing and thawing three times and fractionated
by centrifugation in a Percoll density gradient. 10
fractions (1 ml) were taken from the centrifugate,
residues of the separating medium were removed and
the activity of alkaline phosphatase in the
individual fractions was measured at 305 nm using
p-nitrophenylphosphate as the substrate.
The highest activity of alkaline phosphatase was
determined in fraction 8. The absorbance of the
remaining fractions was much less. This indicates
an enrichment of the plasma membranes in fraction
8. The specific LCE activity was determined in each
fraction after determining the protein
concentration. For this the protein concentration
was adjusted to 0.05 mg/mixture and the reaction
mixtures were incubated for 2 h at 37~C. The
highest specific LCE activity of 4.40 pmol mg~l
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min~l was determined in this case in fraction 8.
In all other fractions lower specific activities of
1.10 - 1.90 pmol mg~1 min~l were measured (Fig.
4.32). The highest specific activity of LCE was
thus determined in the fraction which at the same
time had the highest activity of alkaline
phosphatase. Hence this finding shows that LCE
occurs in the fraction with the highest amount of
fragments of the plasma membrane.
c. Dependency of the specific LCE activit~ on the
protein concentration
In further investigations it was intended to
determine the dependency of the turnover of AZT-
DMDOPE on increasing protein concentrations of the
cell preparations.
For this membrane fractions of stimulated and non-
stimulated human PBL and of human blood monocytes
were used. The conversion of AZT-DMDOPE to DMDOP by
the membrane fractions of stimulated human PBL
exhibited a linear behaviour when using 0.025 -
0.2 mg protein/mixture (Fig. 14). The specific LCE
activity was 14.23 + 0.7 pmol mg~l min~1 (n = 6)
(Fig. 16). When AZT-DMDOPE was converted by
proteins of the membrane fraction of non-stimulated
human PBL there was a linear relationship only in
the concentration range up to 0.05 mg
protein/mixture. At higher protein concentrations
there was no longer a linearity. The specific LCE
activity which was calculated from the turnovers in
the linear range was 1.45 + 0.32 pmol mg~1 min~1
.:
CA 02204908 1997-0~-08
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(n = 6).
In order to determine the specific LCB activity in
human blood monocytes these were isolated in
hypertonic density gradients. Monocytes have a
density of 1.068 g/ml on average which is somewhat
lower than that of lymphocytes with 1.070 g/ml.
This difference in the density is however, very
small so that it is not possible to separate these
blood cells in an isotonic gradient.
Under hypertonic conditions lymphocytes lose water
more rapidly than monocytes resulting in an
increase in their density. Hence it is possible in
a hypertonic separating medium to isolate monocytes
from whole blood or leucocyte rich plasma. It is
also possible to isolate human blood monocytes from
buffy coat. For this mononuclear cells are
separated under isotonic conditions in a density
gradient and monocytes are subsequently separated
in tissue culture flasks (175 cm2/800 ml) on the
basis of their adherencè.
With the aid of flow cytofluorometric analysis it
was possible to detect a stimulation of these cells
by adherence after determination of size and
granularity as well as after specific antibody
staining. As a result of this stimulation the
monocytes which were isolated by adherence to the
bottom of tissue culture bottles were denoted
monocytes/macrophages (stimulated monocytes) in the
following.
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In the enzymatic turnover of AZT-DMDOPE by the
membrane fraction of human monocytes which had been
stimulated during the course of their isolation by
adherence, a linear dependency was found up to a
concentration of 0.1 mg protein/mixture (Fig. 15).
The specific activity was 8.8 + 0.26 pmol mg~1
min~l (n = 4) (Fig. 16). When the monocytes were
isolated directly in a hypertonic density gradient
then there was a linear behaviour of the enzymatic
cleavage only up to a protein concentration of
0.025 mg protein/mixture. At higher protein
concentrations the substrate turnover was almost
constant. The specific activity was calculated from
the linear range analogously to the turnover with
the membrane fractions of non-stimulated human PBL
and was 3.2 + 0.60 pmol mg~l min~1 (n = 4).
In summary it was established that higher specific
LCE activities were observed when AZT-DMDOPE was
converted by enzymes of the membrane fractions of
stimulated human PBL and monocytes than when AZT-
DMDOPE was converted by membrane fractions of non-
stimulated cells (Fig. 16).
d. DependencY of the sPecific LCE activity on the
incubation Period
The enzymatic cleavage of the experimental
substance AZT-DMDOPE in relation to the incubation
period was carried out with membrane fractions of
stimulated human PBL as the enzyme source. The
reactions mixtures were incubated for 0.5, 1, 2, 3
and 6 h at 37~C and subsequently the amount of the
metabolite DMDOP was determined. After plotting the
CA 02204908 1997-0~-08
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experimental results on a graph it was possible to
show that the conversion of AZT-DMDOPE to DMDOP is
linear over the entire period of incubation of 0.5
- 6 h (Fig. 17).
e. DePendencY of the sPecific LCE activitY on divalent
metal cations
The dependency of the enzymatic cleavage of AZT-
DMDOPE by LCE on divalent metal cations was
~ ;ned with membrane fractions of stimulated
human PBL as the enzyme source.
The divalent metal cations were used at a
concentration of 2 mM in the LCE assay. The protein
concentration was fixed at 0.068 mg/mixture.
An experiment with EGTA was carried out in
parallel. EGTA is a specific complexing agent for
Ca2+ which is essential for the activity of
phospholipase C. After an incubation time of 2 h at
37~C the conversion of the parent substance was
measured and the specific activity of the LCE was
determined (following table).
CA 02204908 1997-05-08
Table: Dependence of the cleavage of AZT-DMDOPE by
membrane fractions of stimulated human PBL
on EGTA and divalent metal cations (mean f
SD, n = 3 determinationsJ
Effector DMDOP Specific activity Inhibition
[2 mM] tpmol](pmol mg-1 min-1] [%]
EGTA 84.96 + 11.18 10.41 + 1.37 - 0
CaCl2 50.50 + 2.13 6.19 + 0.26 41.0 + 2.5
MgCl2 84.36 + 17.02 10.34 + 2.08 0
ZnC12 0 0 100. 0
MnC12 4.75 + o.63 o.58 + o.08 94 + 1
The highest specific activity achieved when using
EGTA and MgCl2 was 10.41 + 1.37 (n = 3) and 10.34 +
2.08 pmol mg~1 min~1 (n = 3) respectively. Thus it
was not possible to detect any inhibition by MgCl2
within the limits of the measurement accuracy. When
CaCl2 was used in the enzyme assay a specific
activity of 6.19 + 0.26 pmol mg~1 min~1 (n = 3) was
determined. In comparison with the mixture
containing EGTA this means an inhibition of the LCE
activity of 41.0 + 0.25 ~ (n = 3). In the case of
ZnCl2 no DMDOP was detectable within the limits of
the measurement accuracy. ZnCl2 and MgCl2 led to a
total inhibition of the conversion of AZT-DMDOPE to
DMDOP and AZT-MP.
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f. Determination of the apparent Michaelis-Menten
~arameters ~I and vmaX
In order to determine the apparent Michaelis-Menten
parameters for the LCE the dependence of the
enzymatic cleavage of AZT-DMDOPE on increasing
substrate concentrations was ~x~rined.
Table: Apparent Michaelis-Menten parameters KM and vmaX
of the LCE in stimulated and non-stimulated human
PBL and in human monocytes and monocytes/
macrophages (stimulated monocytes)
Vm ~ KM
[pmol mg~ min~l] ~M]
non--stimulated PBL2.03 + O.l95.51 + 0.99
~timulated human PBL 15.29 + 0.37 2.26 + 0.16
human monocytes1.61 + O.37 12.08 + 4.13
human 0.09 + 0.44 4.63 + 0.45
monocytes/macrophages
~stimulated monocytes)
For this membrane fractions of stimulated and non-
stimulated human PBL as well as human monocytes and
monocytes/macrophages (stimulated monocytes) were
used. The reaction mixtures contained a constant
protein amount of 0.068 mg/mixture and were
incubated for 2 h at 37~C. Subsequently the amount
of the metabolite DMDOP was determined.
CA 02204908 1997-0~-08
Example 10:
Ex~erimental animals
In order to determine the pharmakokinetic parameters of
the experimental substance AZT-DMDOPE, in Vivo
experiments were carried out on female Balb/c mice
(Charles River Wiga, Sulzfeld; Bormholtgard, Ry
(Denmark); Iffa Credo, L'Abresle (France)). The animal
deliveries were ~r;ned before the experiment started
for virus antibodies (mice hepatitis virus, reo virus,
paro virus). In the experiments only animals were used
which had a negative antibody titre.
Keepinq the experimental ~n; ~1 s
The animals were kept in fully air-conditioned animal
cages at a room temperature of 22 - 24~C, a relative air
humidity of 50 - 70 % and a day-night rhythm of 12
hours. Laminar flow boxes ensured that the air in the
animal cage was exchanged 15 - 20 times per hour. The
animals were a~;n;~tered a standard diet (Ssniff,
Soest) and water ad libitum via a drinking bottle.
Example 11:
Cell culture methods
a. Cryopreservation of cell lines
For the cryopreservation, cells were adjusted to a
cell density of 5 x 1o6 cells/ml in RPMI 1640
medium and se~;r?nted for 10 min at 1600 rpm in a
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Minifuge T. Subsequently the cell sediment was
taken up in an equal volume of cryopreservation
medium. After resuspension of the cell sediment
1 ml of the cell suspension was transferred in each
case to cryo-tubes (1. 8 ml) and frozen for 24 h at
-70~C. The cryo tubes were transferred to a thermo-
container containing liquid N2 for the final
storage.
CrYoPreservation 60 % (v/v) RPMI 1640 medium
medium 0.05 mM 2-mercaptoethanol
loo U/ml penicillin
100 ~g/ml streptomycin
30 % (v/v) foetal calf serum
10 % (v/v) DMS0
b. Type culturing and growth conditions
Culturinq of CEM-SS cells
The suspension cell line CEM-SS was cultured in
RPMI 1640 complete medium I. For this 1 x 107 cells
were transferred to a tissue culture flask (83
cm2/260 ml) containing 50 ml medium. Subsequently
the culture was incubated for 3 days at 37~C and
5 % C02. For the cell passage the cells were
removed from the tissue culture flask with a
sterile pipette and sedimented for 10 min at 1600
rpm in a Minifuge T. After resuspending the cells
in culture medium and determining the cell number
by eosin staining in a Neubauer counting chamber, 1
x 107 cells were used for passage into 50 ml fresh
medium.
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Culture of P3X63Aq8.653 cells
P3X63Ag8.653 cells were cultured as a monolayer in
RPMI 1640 complete medium I. For this 5 X 106 cells
were transferred to a tissue culture flask (83
cm2/260 ml) containing 40 ml medium. The cell
culture was incubated for 5 days at 37~C and 5 %
C~2 until the bottom of the tissue culture flask
was covered with a confluent cell monolayer. For
the cell passage the adherent cells were
mechanically detached from the bottom of the flask
using a cell scraper and subsequently se~;~~nted by
centrifugation ~or 10 min at 1600 rpm in a Minifuge
T. After resuspending the cells in culture medium
the cell number was determined by eosin staining in
a Neubauer counting chamber and 5 x 106 cells were
used for passage into 40 ml fresh medium
c. Determining the cell count
Determination of the cell count by eosin staininq
in a Neubauer countinq chamber (Lindl and Bauer,
1989, "Zell und Gewebekultur", P. 75-90, Gustav
Fisher Verlag, Stuttgart, New York)
For the determination of the cell count in
suspension and monolayer cultures a cell
concentration of 0.1 - 1 x 106 cells/ml was
adjusted in PBS and a portion of this cell
suspension was admixed with 20 ~1 eosin.
Subsequently the cell suspension was carefully
admixed with a pipette, incubated for 2 min at room
temperature and transferred to a Neubauer counting
chamber. The number of cells was determined
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immediately after the incubation period in a
reverse phase contrast microscope whereby the cells
were counted within four large squares. In this
process vital cells were not stained in contrast to
avital cells. Weakly stained cells were considered
to be avital.
The number of vital cells/ml was derived from the
number of non-stained cells of the four large
squares multiplied by the chamber factor of 104 and
the dilution factor of the cell suspension in the
eosin solution.
Determination of the cell number bY electronic
countin~ in a coulter Counter (Lindl and Bauer,
1989)
The measurement of the cell number in a Coulter
counter is based on the cells flowing between two
platinum electrodes. If a cell passes through the
opening of the two electrodes then the resistance
of the current flowing through the electrodes
changes proportionally to the size of the cell.
This generates a voltage impulse which is recorded
and thus enables the cells to be quantified.
In order to determine the number of cells 25 ~l of
the cell suspension was transferred to 3 ml Iso-
Osmol a carrier liquid for the Coulter counter and
injected into the apparatus.
CA 02204908 l997-0~-08
- 61 -
In this type of cell counting the total cell number
of the cell suspension is determined. It is not
possible to differentiate between vital and avital
cells.
d. Detachment of adherent cells by trYpsin/EDTA
Adherent cells could be detached enzymatically from
the bottom of the respective culture vessel by
treatment with trypsin/EDTA. For this the culture
supernatant was carefully decanted and the cells
were washed twice with prewarmed (37OC) culture
medium. In order to detach the cells from the
bottom of the culture vessel these were incubated
for 5 min with a trypsin/EDTA solution (1 x) at
room temperature. The volume of trypsin/EDTA
solution was selected in this case such that the
bottom of the respective culture vessel was covered
with 2 - 3 mm of the enzymatic solution. At the end
of the incubation period the cells were detached
from the bottom by carefully ~h~k;ng the flask,
removed from the culture vessel and admixed with an
equal volume of RPMI 1640 complete medium I.
Afterwards the cells were sedimented for 10 min at
1600 rpm in a Minifuge T. The supernatant was
discarded, the cell sediment was resuspended in
cold PBS and again centrifuged under the same
conditions. The cells were then resuspended in
culture medium or buffer depending on their further
use.
CA 02204908 l997-0~-08
_ - 62 -
e. Isolation of mononuclear cells from human blood
Isolation of human PBL from buffv coat bY
centrifugation in isotonic density qradients
(Boyum, 1968, Scand. J. Clin. Lab. Invest. 21
(SUppl 97), 77-89; Boyum 1976, Scand. J. Clin. Lab.
Invest. 5 (SuPpl. 5), 5-15)
In order to isolate human PBL the buffy coat of
healthy donors was mixed and diluted with RPMI 1640
complete medium II in a volume ratio of 1:2. 20 ml
cold lymphocyte separation medium was placed in
polypropylene tubes (50 ml) and carefully covered
with a layer of 15 ml of the diluted buffy coats.
The fractionation was carried out by centrifugation
for 30 min at room temperature and 1600 rpm
(acceleration 4/brake 4) in a Minifuge T. The
readily visible band between the sample and
separation medium was isolated with the aid of a
Combitip pipette. The collected fractions were
sedimented for 10 min at room temperature and
1600 rpm to remove the separation medium. The
supernatant was decanted and the cells were washed
three times with RPMI 1640 complete medium II or
cold PBS depending on their use.
Stimulation and culture of human PBL
After isolation of human PBL by means of
centrifugation in isotonic density gradients it was
possible to set up peripheral lymphocyte cultures.
Normally blood cells die relatively rapidly in
culture. However, lymphocytes can be kept in
culture for several generations. In order to
CA 02204908 1997-0~-08
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achieve a proliferation of these cells they were
stimulated with the mitogen PHA-M, the lectin
extract from the red scarlet runner (Phaseol us
vul garis) .
For the stimulation the isolated cells were
adjusted to a cell density of 1 x 106 cells/ml in
RPMI 1640 complete medium II in tissue culture
flasks (175 cm2/800 ml) and incubated for 72 h at
370C and 5 % Co2. In order to avoid the cell
density becoming too high, the cell suspension was
diluted after 24 h with culture medium in a volume
ratio of 1:1. After completion of the stimulation
period the mitogen PHA-M was removed by washing
three times with RPMI 1640 complete medium II. For
the cell expansion the stimulated lymphocytes were
adjusted to a cell density of 5 - 7 x 106 cells/ml
in RPMI 1640 complete medium IV and cultured for 24
hours.
Preparation of leucocyte-rich plasma from whole
blood (BoYum~ 1968)
Venous fresh blood from healthy donors was admixed
with sterile 10 ~ (w/v) EDTA solution in a volume
ratio of 49:1 to prevent coagulation. In order to
achieve a rapid mixing of these components small
volumes of ~ - 9 ml blood were collected from the
donor in polystyrene tubes (14 ml). In order to
prepare leucocyte-rich plasma, EDTA-blood and
dextran 75 were mixed in a volume ratio of 10:1.
The two components were thoroughly mixed and the
erythrocytes were sedimented for a period of 60 min
at room temperature. Subsequently the almost clear
CA 02204908 l997-05-08
- 64 -
supernatant, the leucocyte-rich plasma, was
carefully removed and used to isolate monocytes.
Isolation of human monocytes from leucocyte-rich
~lasma by centrifugation in a hY~ertonic densitY
gradient (Boyum, 1983, Scan. J. Clin. Lab. Invest.
17, 429-436)
It was possible to isolate non-stimulated monocytes
from whole blood or leucocyte-rich plasma in a
hypertonic solution. For this 3 ml of the
hypertonic separation medium NycoPrep 1.068 was
placed in polystyrene tubes (14 ml) and carefully
covered with a layer of 6 ml leucocyte-rich plasma.
The tubes were closed and centrifuged for 15 min
and 600 x g (acceleration 4/brake 4) in a Minifuge
T. After centrifugation the clear plasma phase was
removed and discarded up to 5 mm above the broad
diffuse band. The remaining plasma phase and half
of the broad diffuse band was removed and the cells
were sedimented by centrifugation in a Minifuge T
for 7 min under the same conditions. The cell
sediment was subsequently resuspended and washed
twice in 6 ml wash solution to remove residues of
the separation medium.
Nycopre~ 1.068 13 % (w/v) Nycodenz
0.58 ~ (w/v) NaCl
5 (mM) Tricine/NaOH pH 7.4
density 1.068 + 0.001 g/ml
(20~C)
osmolarity 335 + 5 mOsm
-
CA 02204908 l997-0~-08
- 65 -
Wash solution 0.9 ~ (w/v) NaCl
0.13 % (w/v) EDTA
1 ~ (w/v) BSA (fraction V)
Isolation of human monocytes from buffy coat by
adherence (Andreesen et a., 1983, J. Immun. Methods
56, 295-304)
Mononuclear cells were isolated from the buffy coat
by centrifugation in isotonic density gradients and
washed three times with serum-free RPMI 1640
medium. In order to isolate the monocytes the cells
were adjusted to a cell density of 5 x 106 cells/ml
in tissue culture flasks (175 cm2/800 ml)
containing 20 ml RPMI 1650 complete medium II and
incubated for 45 min at 37~C and 5 ~ C02. Non-
adherent cells were removed by decanting the medium
after the incubation was completed. The cell layer
at the bottom of the tissue culture flask was
subsequently washed twice with pre-heated (37~C)
serum-free RPMI 1640 medium to remove residues of
suspension cells. The cell layer was admixed with
30 ml RPMI 1640 complete medium V and incubated for
a further 24 h under the same conditions. The
adherent monocytes were detached by treatment with
trypsin/EDTA.
CA 02204908 1997-0~-08
- 66 -
Example 12:
Immunolo~ical methods
a. Flow cytofluorometric analYsis of cell
subPoPulations from human blood by direct and
sequential antibody stA; n; n~
For the flow cytofluorometric analysis the cells to
be characterized were adjusted in FACS-PBS to a
cell density of 1 x 107 cells/ml. The antibody
stock solutions were diluted with FACS-PBS in a
volume ratio of 1:50. For the direct and sequential
antibody staining 50 ~l of the cell suspension (1 x
107 cells/ml) was placed in the wells of a
microtitre plate with a conical bottom.
Antibodies for the flow cYtofluorometric analysis of
mononuclear human blood cells
Antibodies SpecificityFluorescent Antibody
dye stA;n;ng
anti-Leu-12 Leu-12 (CD19) PE direct
(CD19) B lymphocytes
anti-Leu-5b Leu-5 (CD2) FITC direct
(CD2) T lymphocytes
anti-monocyte gp 55 - sequential
(CD14) monocytes
macrophages
anti-leu-2a cytotoxic T FITC direct
(CD8) lymphocytes
anti-leu-3a helper T PE direct
(CD4) lymphocytes
CA 02204908 1997-0~-08
- 67 -
Subsequently the cell suspension was admixed with
an equal volume of the antibody which in the case
of a direct antibody st~; n; ng is coupled to a
fluorescent dye. The mixtures were incubated for 30
min at 4~C and subsequently centrifuged at 1400 rpm
and room temperature for 2 min in a Minifuge T to
sediment the cells.
The supernatants were removed and the cell s~ nt
was resuspended in 200 ~l FACS-PBS and centrifuged
again as described above to remove residues of non-
bound antibody. The supernatant was removed and the
cell sediment was resuspended in 100 ~l FACS-PBS in
the case of a direct antibody staining. In the case
of the sequential antibody staining the cell
sediment was taken up in 100 ~l of a solution of
the goat-anti mouse Ig-R-PE (3 ~g/ml) antibody
labelled with phycoerythrin after completion of the
centrifugation of the cell sediment and incubated
for 30 min at 4~C. Residues of the non-bound
antibody were removed as described by washing with
200 ~l FACS-PBS and the cell s~; ~nt was
resuspended in 100 ~l FACS-PBS for the flow
cytofluorometric cell analysis.
b. Determination of the cell vitalitv by Propidium
iodide stainin~ in the flow cytofluorometric
anal~zer
The number of vital and avital cells can be
determined by flow cytofluorometric analysis after
st~; n; ng the cells with the fluorescent dye
propidium iodide. This dye is only taken up by
avital cells. For this the cell suspension is
CA 02204908 1997-0~-08
- 68 -
adjusted in PBS to a concentration of 0.5 - 1 x 106
cells/ml. 180 ~l is taken from this cell suspension
and admixed with 20 ~l of a propidium iodide
solution (5 ~g/ml). After an incubation period of
5 min 100 ~l of the solution was injected into the
cell analyzer. The percentage distribution of the
vital and avital cells could be determined by a
statistical analysis in the Lysis II program of the
cell analyzer.
c. Ouantitative determination of AZT in cell extracts
by means of a l25I-AZT radioimmunoassay
A commercial 125I-AZT RIA test kit was used for the
quantitative determination of AZT. Buffers and
solutions were prepared according to the
instructions of the manufacturer and used in the
test kit. The reaction mixtures were pipetted
according to the scheme (Tab.) shown below,
carefully mixed and incubated for 2 h at room
temperature. Subsequently 500 ~l of the goat anti-
rabbit antibody was added to precipitate the
complex of 125I-AZT and the AZT antibody (from
rabbits). The mixtures were mixed and incubated for
a further 30 min at room temperature.
CA 02204908 1997-0~-08
- 69 -
Pipetting scheme of the l25I-AZT radioimmunoassay
for the quantitative determination of AZT in cell
extracts
non-binding zero st~n~rd sample
standard st~n~rd
zero standard 300 200
[lll ]
standard [~1] - - 200
sample [~1] - - - 200
125I--AZT [~1] 100 100 100 100
AZT antibody - 100 100 100
[~1 ]
The mixtures were subsequently centrifuged at 1000
x g for 20 min to s~; ?nt the precipitation
complex. After decanting the supernatant the
radioactivity of the sediment was measured for 60
sec in a liquid scintillation analyzer. In order to
determine the absolute value, the radioactivity of
100 ~1 125I-AZT was determined under the same
conditions after an incubation of 2 h at room
temperature. The concentrations of AZT in the
biological matrix were determined with the aid of a
calibration curve which was established with
standards from the test kit with a defined
substance concentration.
CA 02204908 1997-0~-08
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Example 13:
Characterization of the intracellular enzYmatic cleavage
of BM 21.1290 Na
The results of the in vitro pharmakokinetic studies with
AZT-DMDOPE and AZT in stimulated and non-stimulated
human PBL as well as in thymidine kinase deficient
P3X63Ag8.653 cells indicate an intracellular enzymatic
cleavage of AZT-DMDOPE with a direct release of AZT-MP
and of the corresponding thioether lipid moeity DMDOP.
It is intended to verify this intracellular enzymatic
cleavage demonstrated in further experiments at a
~ subcellular level by a direct detection of the
corresponding metabolites. Hence for an unequivocal
characterization of the cleavage it was necessary to
identify the previously known metabolites which can be
formed from the parent substance AZT-DMDOPE. These
include the substances DMDOP, AZT and AZT-MP as well as
substances which are formed by oxidation of the sulphur
in the thioether lipid moiety of the parent substance
AZT-DMDOPE.
a. Development of methods for the determination of the
Rf values of AZT-DMDOPE and its ~otential
metabolites
It was possible to identify the potential
metabolites of AZT-DMDOPE with the aid of TLC. For
this the non-radioactively labelled pure substances
were analysed with the aid of various separation
systems and subsequently the Rf values were
determined.
CA 02204908 l997-0~-OX
- 71 -
For this the substances were dissolved at a
concentration of 4 mg/ml in a mixture o~ ethyl
acetate and methanol (1:1, v/v). 5 ~1 of these
solutions was transferred with the aid of a
microcapillary to the stationary phase and
analysed.
Thioether lipids such as AZT-DMDOPE, DMDOP as well
as those which are formed by oxidation of the
sulphur could be labelled with the aid of a reagent
containing iodine which stains the thioether lipid
moiety of these compounds. AZT and AZT-MP could
only be made visible on the TLC plates with a
fluorescence indicator on the basis of their
absorbance in the ultraviolet range at 254 nm. AZT-
DMDOPE, the substance with a thioether lipid moiety
and a chromophoric group could be detected ~ith
both types of detection.
Three separation systems were established for the
thin layer chromatographic analysis of the
substances which differed with regard to their
stationary and mobile phases.
In the first separation system, the IBA system,
using silica gel 60 as the stationary phase the
parent substance AZT-DMDOPE could be identified in
addition to DMDOP. A mixture of 2-propanol:n-butyl
acetate: redistilled water (10:6:4 v/v/v) was used
as the mobile phase.
This separation system was developed further by
using 2-propanol:n-butyl acetate redistilled water:
glacial acetic acid (3:5:1:1, v/v/v/v) as the
CA 02204908 1997-0~-08
- 72 -
mobile phase (IBAE system). The substances AZT-
DMDOPE and DMDOP could be separated in this case on
silica gel 60 TLC plates as described for the IBA
separation system. In addition AZT and AZT-MP could
be separated with a fluorescence indicator in W
light at 254 nm and using a stationary phase of
silica gel 60. Due to the AZT moiety it was also
possible to detect the parent substance AZT-DMDOPE
as well as its oxidation products.
After the substances were visualized on the TLC
plate the Rf values were determined.
A further separation system was available to
differentiate the substance AZT-DMDOPE from DMDOP.
For this a mixture of n-heptane and ethyl acetate
(4:1, v/v) was used as the mobile phase and silica
gel 60 as the stationary phase. The detection was
carried out with the reagent containing iodine
already described above by staining the thioether
lipid moiety of the substances.
In comparison to the IBA and IBAE system a
considerably lower Rf value was determined for the
substance DMDOP with this separation system. The
reason for this is a unpolar mobile phase compared
to the IBA and IBAE system consisting of n-heptane
and ethyl acetate. The detection limit of the
substances after thin layer chromatographic
analysis and detection with an iodine-cont~;n;ng
reagent was determined to be 0.1 ~g in all
separation systems.
CA 02204908 1997-05-08
- 73 -
In summary it can be stated that with the aid of
the described thin layer chromatographic separation
systems it is possible to unequivocally
characterize all substances via their Rf values
which come into consideration according to the
present state of knowledge as potential metabolites
of AZT-DMDOPE.
Table Rf values of AZT - DMDOPE and DMDOP after
separation in the IBA system ~2-propanol:n-
butyl acstate:redistilled water, 10:6:4,
v/v/v) with silica gel 60 as the stationary
phase (meansl~D~ n = 6 determinations)
Test subqtance Rf value
AZT-DMDOPE 0.68 1 5.2 x 10-3
DMDOP ~ 0.931 0.O0
CA 02204908 1997-05-08
- 74 -
Table Rf values of AZT-DMDOPE, DMDOP, AZT and AZT-
MP after separation in the IBAE sy~tem (2-
propa~ol:n-butyl acetate:redistilled
water:glacial acetic acid, 3:5:1:1, v/v/v/v)
with silica gel 60 and 60 F 254 as the
stationary phase (mean~ I SD, n = 6
dete~ ;n~tions)
Test substance Rf value
AZT-DMDOPE 0.57 1 1 x 10-3
DMDOP 0-99 1 0-00
AZT 0.85 1 o.oo
AZT-MP 0-14 1 0-00
b. Enzymatic cleavage of AZT-DMDOPE by cell
homoqenates of stimulated and non-stimulated human
PBL and CEM-SS cells
The cleavage of AZT-DMDOPE was examined with the
aid of an enzyme assay using [l4C]-AZT-DMDoPE and
AZT-DMDOPE as the substrate. Firstly cell
homogenates of CEM-SS cells and human PBL were used
as the enzyme source.
CA 02204908 1997-05-08
- 75 -
Table Rf values of AZT-DMDOPE and DMDOP after thin
layer chromatographic separation in the HE
system (n-heptane:ethyl acetate, 4:1, v/v)
with silica gel 60 as the Stationary phase
~means_SD, n = 6 determination~)
Test substance Rf value
AZT-D~DOPE 0.0 1 0-00
DMDOP 0.36 1 0.00
The latter were homogenized directly after their
isolation after stimulation with PHA-M as well as
in the non-stimulated state and used in the enzyme
assay.
In order to prepare the cell homogenates,.cell
suspensions were mechanically disrupted in a glass
homogenizer at a density of 5 x 107 cells/ml 50 mM
Tris (pH 7.4) The cell disruption in this case was
optically monitored in a reverse phase contrast
microscope.
- = :
CA 02204908 1997-05-08
- 76 -
Table Rf value~ of the subst~n~ss l -4 (Fig. 18)
after 1, 3, 6 and 24 h ;n~h~tion and
e~zymatic cleavage of ~l4C]-AZT-DMDoPE by
cell homogenates of 5 X 107 stimulated and
~o~-stimulated human PB1 and CEN-S8 cells.
8eparation in the IBA/IBAE sy5tem with 8ilica
gel 60 as the Stationary phase (meanslSD, n =
4 determinations)
Rf value - Rf value Rf value Rf valu~
Sub8tance 1 Substance 2 Substance 3 Substance 4
CEN-8S 0.410.56 0.86 0.96l0.0
+2 . 5 x 10-2 +2 . 5 x 10-2 1 5 . 8 x 10-3
stimulated - 0.65 0.86 0.94l0.0
human PBL l9.5 x 10-3 +5 . 8 x 10-3
~on stimulated - 0. 65 - 0. 92
human PBIJ +1 . 7 x 10-2 1 1 . 2 X 10-2
Firstly 1 ml of this cell homogenate was used in
the enzyme assay without prior determination of the
protein concentration. 0.98 nmol t14C]-AZT-DMDOPE
(1.67 kBq) was used as the substrate.
In order to ensure a substrate saturation a further
50 nmol of the non-radioactively labelled compound
was added. [14C]-AZT-DMDOPE carries the radioactive
label in the thioether lipid moiety and thus
enables an unequivocal identification of the
potential metabolite [14C]-DMDOP.
CA 02204908 1997-05-08
The mixtures were incubated for 1, 3, 6 and 24 h in
a water bath at 37~C. A mixture without addition of
cell homogenate was also carried out as a
reference. Subsequently the cleavage products were
extracted with diethyl ether:2-propanol (9:1, v/v)
and analysed by thin layer chromatography in the
IBA and IBAE system (Fig.18). After the substances
were separated the TLC plate was measured for 15
min in a radio TLC analyzer to detect the
radioactively labelled substances. It was finally
possible to unequivocally identify the metabolites
by determining the Rf values.
Table Rf values of the substances 1 and 2 (Fig. 18)
after 1, 3, 6 and 24 h ;n~ tion of [14C]-BM
21.1290 Na. 8eparation in the IBA/IBAE sy~tem
with silica gel 60 as the stationary phase
(meanslSD, n = 4 determinations)
Rf value Rf value
8ubstance 1 8ubstance 2
CEM-88 0.41l5.7 x 10-3 0.56l5.8 x 10-3
stimulated - 0.65l9.6 x 10-3
human PBL
non-stimulated - 0.65l1.9 x 10-3
human PB~
The thin layer chromatographic analysis of the
enzymatic cleavage products of AZT-DMDOPE by cell
homogenates of stimulated and non-stimulated human
CA 02204908 1997-05-08
- 78 -
PBL was carried out by using the IBA separation
system. In the subsequent experiments with the CEM-
SS cell homogenate the analysis of the cleavage
products was carried out in the IBAE separation
system. Since the Rf values of the pure substances
have been determined for both separation systems it
was possible to directly compare the results to
identify the substances after enzymatic cleavage of
AZT-DMDOPE.
After enzymatic cleavage of [l4C]-AZT-DMDOPE by
cell homogenates of stimulated and non-stimulated
human PBL three unknown substances were detected in
the TLC chromatogram. With Rf values of 0.65 + 9.5
x 10-2 (n = 4) and 0.65 + 1.7 x 1o-2 (n = 4) for
stimulated and non-stimulated human PB~ the
substance peak 2 could be unequivocally assigned to
the parent substance tl4C]-AZT-DMDOPE. The Rf
values of substance peak 4 of 0.94 + 0.00 (n = 4)
and 0.92 + 1.2 x 10-2 (n = 4) were identical to the
values of DMDOP which were determined by TLC
analysis of the pure substances. Substance peak 3
having an Rf value of 0.86 + 5.8 x 10-3 (n = 4)
could not be allocated to any of the known
substances. The results were confirmed by analysis
of the enzymatic cleavage of [14C]-AZT-DMDOPE by
CEM-SS cell homogenates. Substance peaks 2 and 4
having Rf values of 0.56 + 2.5 x 10-2 (n = 4) and
0.96 + 0.00 (n = 4) could be unequivocally
identified as AZT-DMDOPE and DMDOP respectively
whereas substance peak 1 having an Rf value of 0.41
+ 2.5 x 10-2 (n = 4) could be assigned to an
oxidation product. It was not possible to assign
substance peak 3 even in the IBAE system after
comparison of the Rf values (Fig. 18). Furthermore
CA 02204908 1997-0~-08
- 79 -
it was possible to show that after incubation of
tl4C]-AZT-DMDOPE only the parent substance was
detected when the cell homogenate was not added
(Fig. 18).
The thin layer chromatographic analysis of the
reaction mixtures therefore proves that the
substance DMDOP is released from the parent
substance.
Example 14:
9-(~-D-Arabinofuranosyl)-2-fluoroadenine-5-phosphoric
acid-(3-dodecylmercaPto-2-decyloxy)-~ropyl ester
(fludarabine coniuqate)
34.8 g (0.07 mol) phosphoric acid (3-dodecylmercapto-2-
decyloxy)-propyl ester was dissolved in 130 ml absolute
pyridine, admixed with 15 g methanesulfonic acid
chloride under nitrogen and stirred for 3 hours at room
temperature.
Then 20 g fludarabine was carefully added and the
solution was stirred for a further 48 hours at room
temperature. Fludarabine was synthesized analogously to
J. Heterocyclic Chem. 16, 157 (1979).
After hydrolysis of the reaction mixture by adding 30 ml
1 M triethylammonium bicarbonate solution and stirring
for 1 hour, the pyridine was removed in a vacuum and the
residue was partitioned between 200 ml t-butylmethyl
ether (MTB) and 150 ml water, the organic phase was
separated and evaporated in a rotary evaporator.
CA 02204908 l997-05-08
- 80 -
The residue was purified chromatographically on RP-18
with methanol/0.02 M acetate buffer pH 4 8/2 as the
eluant.
The fractions containing product were concentrated down
to the water portion, extracted with MTB and the MTB
phase was adjus~ed with sodium methylate solution
against Friscolyt to pH 7.
After the solvent was removed by evaporation the residue
was suspended in acetone, the amorphous precipitate was
suction filtered and dried.
Yield: 23.7 g (43 %). Amorph. Rf = 0.45 (TLC-mobile
solvent: isopropanol/butyl acetate/water/ammonia
50/30/15/5).
Example 15:
2-Chloro-2'-deoxyadenosine-5'-phosPhoric acid-t3-
dodecYlmercaPto-2-decYloxy)-propYl ester (cladribine
conjuqate)
The cladribine conjugate was prepared in a 35 % yield
analogously to example 14 using 4.2 g phosphoric acid-
(3-dodecylmercapto-2-decyloxy)-propyl ester, 3 g
methanesulfonic acid, 100 ml pyridine and 2 g
cladribine. R~ = 0.41 (mobile solvent as in example 14).
Cladribine was prepared analogously to J. Am. Chem. Soc.
106, 6379 (1984).
CA 02204908 1997-05-08
- 81 -
Exam~le 16:
r 3-(2-Deoxy-~-D-erythro-pentofuranosyl)-3,6,7,8
tetrahydroimidazo r 4,5-d~ r 1,3ldiazepin-8-ol~-5'-
phosphoric acid-(3-dodecylmercapto-2-decyloxY)-pr
ester (pentostatin coniuqate)
The pentostatin conjugate was prepared in a 27 ~ yield
analogously to example 14 using 4.2 g phosphoric acid-
(3-dodecylmercapto-2-decyloxy)-propyl ester, 3 g
methanesulfonic acid, lOo ml pyridine and 2.1 g
pentostatin. Rf = 0.52 (mobile solvent as in example
14). Pentostatin was prepared analogously to J. org.
Chem. 47, 3457 (1982) and J. Am. Chem. Soc. 101, 6127
(1979).
