Note: Descriptions are shown in the official language in which they were submitted.
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DESCRIPTION
~~ atment of Androgen-AssQciatPd
baldness Us~ng Antisense O1~gomeTs
.- .-; to nv ' o
Androgens are steroid hormones found circulating at
varying levels in both men and women. They are esser_tial
in sex differentiation, development, and reproductive
function. However, androgens car. also play a role in
undesirable physiological conditions, including different
types of baldness.
One of the most prevalent types of baldness is male
pattern baldness (MPH>. This condition is widespread,
of fecting two of every three men. MPH, which is inherited
as a autosomal dominant trait with partial penetrance, is
known to be androgen-dependent. This is evidenced in the
fact that castrated males do not develop baldness.
Hair follicles initially appear in utero. No new
follicles are created after birth, and it is believed that
none are lost in adult life. However, in MP3, hair
follicles do become progressively smaller (miniaturized).
Hair follicles exhibit cyc::ic activity. Each period of
active growth of hair (anagen) al ternates with a resting
period (telogen), separated by a relatively short transi-
tion phase (catagen). Hair growth on the human scalp is
a mosaic of follic;:lar activity with each fol licl a at a
stage independent of its neighbors. At any one _imz,
between 4-24% (average 13 % ) of foil icl es are in t~;oger.
ana, <1% in cat.ager.. Hairs reach a te=urinal or definitive
length, w~=c~: depends mair.l.y or_ ti-.2 duratior. of anaae~,
WO 94/18835 , . PCT/fJS94/01748
2
and partly on the rate of growth. In the human scalp,
anagen may occupy three years or more; however, the
percentage of follicles in telogen increases with age,
resulting 'in a gradual thinning. In MPB, the ratio of
telogen to anagen is increased still further. Also, in
MPB the hairs in affected areas become steadily shorter
and finer, and ultimately may be reduced to the short
(<2cm), fine, unpigmented hair known as vellus hair.
Although the endocrine system does not directly
initiate or curtail the activity of the hair follicle,
androgens do accelerate or retard the normal cyclic
activity of hair growth described above.
Testosterone (T) is the major circulating androgen.
Because circulating T is largely bound to sex hormone
binding globulin (SHBG), the availability of T depends not
only on its total concentration, but also on the level of
SHBG. While plasma T levels in MPB appear to be normal,
SHBG levels tend to be low. This implies that bald males
may have higher levels of free testosterone. This
implication is borne out by the demonstration that bald
males have high T concentrations in their saliva.
T itself has minimal activity in the hair follicle.
A much more active metabolite which is believed to be
responsible for MPB is 5-alpha-dihydrotestosterone (DHT).
DHT is formed in the cytoplasm of hair follicle cells
after reduction of T by the enzyme 5-alpha-reductase.
Because balding men have increased 5-alpha-reductase
activity in the hair follicles and skin of the frontal
scalp, it has been suggested that this enzyme may be
involved in development of MPB. Twp genes have been
reported, each of which codes for a distinct 5-alpha- '
reductase enzyme (Genbank locus:HUM5AR and HUMSRDA).
The effects of androgens in MPB are mediated by the '
binding of an androgen (primarily DHT) to the androgen
receptor (AR). Androgens bind specifically to the AR,
which is either situated in the nucleus or transferred to
it from the cytoplasm. The AR belongs to a subfamily of
~~ ~ST~TUTE SH~~?' ~~~J~.~ 2&~
WO 94/18835 w ~ PCT/US94/01748
3
steroid/thyroid hormone/retinoic acid receptors, whose
activity is controlled by the tight and specific binding
of the cognate ligand. Evidence for the involvement of
the AR in MPB includes the demonstration that androgenic
alopecia (a type of pattern baldness in women) can be
alleviated by treatment with antiandrogens. These
antiandrogens, such as spironolactone, cyproterone
acetate, flutamide and cimetidine, bind to the AR and
competitively inhibit DHT binding. In addition, sebaceous
glands of bald scalps were found to have greater binding
affinity and capacity for androgens than those in hairy
scalps.
In the past, baldness was treated only with surgical
procedures, such as hair transplants and scalp reduction.
Recently, however, there have been some advances in
medical treatment of baldness. The most publicized of
these is minoxidil (Rogaine~"). Minoxidil is a potent
vasodilator which has been used as a treatment for
hypertension. A noted side effect of this treatment was
the growth of hair on parts of the body. This led to the
testing of topical minoxidil on balding areas of the
scalp. The result in some cases was an apparent decrease
in vellus hairs with a concomitant increase in terminal
hairs. Many of the subjects studied reported that their
rate of hair loss decreased. However, not all subjects
responded to treatment with minoxidil. It was found that
younger men who only recently (within five years) had
begun to bald responded better than older men, and that
minoxidil worked best on small areas of vertex baldness.
Research indicates that minoxidil will not help the
' majority of balding men, although it does help a specific
population of minimally balding young men. The reason for
the effectiveness of minoxidil is not known. It might be
due to an increase in blood flow caused by the
vasodilating effect of the drug. The longterm effects of
minoxidil treatment are not known.
SUBSTITUTE SHEET (RULE 26)
WO 94/18835 - PCT/US94/01748
4
Other treatments are directed at reducing the
production of DHT from testosterone, thereby preventing
its cytosol-nuclear binding and/or translocation. Topical
or intralesional progesterone can also be used to reduce
Y
the production of DHT from T. Since progesterone is
similar in structure to testosterone, it competes with
testosterone for 5-alpha-reductase, 'the enzyme that
converts testosterone to DHT.
Summary of the Invention
The present invention is directed to methods of
treating androgen-associated hair loss, particularly hair
loss in men, more particularly to methods of decreasing
the progression of male pattern baldness and also to
pharmaceutical compositions useful for these methods.
These methods and pharmaceutical compositions are parti-
cularly suited to the treatment of hair loss associated
with increased levels of protein-bound DHT in scalp.
According to one aspect, Oligomers complementary to a
target sequence in genes which result in increased amounts
of androgen receptor bound-5-a-dihydrotestosterone in
scalp tissue are used to down-regulate genes or their
transcription products.
The topography of male pattern baldness has to do
with both the number of androgen receptor ("AR") molecules
of the follicular cells and the activity of 5-alpha
reductase ("5-a-RE") in different areas of the scalp.
Thus, targeting 5-alpha reductase or the AR would be
useful in developing a treatment for MPB. However, it is
essential that the treatment act only at the scalp and is
cleared quickly from the body, since systemic inhibition Y
of testosterone or DHT activity would be highly disadvan-
tageous in men, resulting in undesirable feminization.
The androgen receptor may be involved in other types
of hair loss aside from MPB. For example, androgenic
alopecia, a type of hair loss in women, has been shown to
respond to treatment with antiandrogens. Accordingly, the
SUBSTITUTE SHEET c~ULE 26)
WO 94/18835 ~ PCT/US94/01748
methods and pharmaceutical compositions of the present
invention may be useful in the treatment of other types of
androgen-associated hair loss. Also, these methods and
compositions may be useful in treating other conditions
5 where localized (as opposed to systemic) down-regulation
of the AR or 5-cx-RE is desirable. Accordingly, the
present invention is also directed to methods of
decreasing levels of protein-bound 5-alpha-dihydro-
testosterone in a localized and tissue-specific manner
without significantly interfering with testosterone
metabolism in other tissues or systemically by exposing
the cells of the tissue to be treated with an Oligomer or
Oligomers which inhibit or alter expression of the AR or
5-a-RE. Such Oligomers include those which interact with
a target sequence selected from a gene coding for the AR
or 5-a-RE or a sequence immediately upstream from the
transcription site of the gene or their transcription
products.
Thus, in one aspect, the present invention is
directed to a method of treating androgen-associated hair
loss by decreasing levels of 5-alpha-dihydrotestosterone
which are present in follicles and bound to protein, and
according to a preferred aspect decreasing levels of DHT
bound to the androgen receptor in scalp tissue without
significantly interfering with testosterone synthesis
and/or metabolism in other tissues. This method comprises
exposing scalp cells to an amount of an Oligomer or
Oligomers sufficient to provide a decrease in the rate of
hair loss, preferably by a cosmetically significant
amount. The Oligomer or Oligomers interact with a gene
coding for the AR or 5-a-RE or a sequence immediately
upstream from the transcription start site of the gene or
their transcription products and thereby inhibit or alter
expression of the AR or 5-a-RE.
Suitable Oligomers for use in the methods and pharma-
ceutical compositions of the present invention include (a)
an antisense Oligomer having a sequence complementary to
F'' ~ ~ ~ -,'''
WO 94/18835 PCT/US94/01748
6
a sequence of RNA transcribed from a target gene present
in the cells; (b) an antisense Oligomer having a nucleo-
side sequence complementary to a single stranded DNA
target sequence; (c) an antisense 0ligomer having a
nucleoside sequence complementary to a single RNA or DNA
strand contained within a duplex (d) a Third Strand
Oligomer having a sequence complementary to a selected
double stranded nucleic acid sequence of a target gene
present in the cells; and (e) a Triplex Oligomer Pair
which is complementary to a single-stranded nucleic acid
sequence of a target gene or its transcription product or
to a single-stranded sequence contained within a duplex.
The target gene is advantageously selected from the group
consisting of those genes encoding 5-alpha-reductase and
the androgen receptor. According to a preferred aspect,
the Oligomer is applied topically to the scalp tissue.
According to an alternate aspect, the present
invention is directed to a method of treating androgen
associated hair loss which comprises exposing scalp to an
amount of an Oligomer which decreases the rate of hair
loss wherein said Oligomer is selected from an antisense
Oligomer having a sequence complementary to that of RNA
transcribed from a gene for androgen receptor or an
antisense Oligomer having a sequence complementary to a
sequence of RNA transcribed from a gene for 5-alpha-
reductase.
According to a preferred aspect the Oligomer is a
neutral Oligomer. Neutral Oligomers such as methylphos-
phonate Oligomers are cleared rapidly through the kidneys.
Especially preferred are methylphosphonate Oligomers,
which are rapidly cleared from the plasma and are excreted '
in the urine.
The Oligomers used according to the present invention
preferably comprise Oligomers which have a neutral back
bone. Neutral Oligomers are preferred, in part, due to
their advantageous uptake through the skin when applied
topically. Preferably these Oligomers are substantially
~~~~TiTU~'~ SHEET (~U1.E 26)
WO 94/18835 ~ PCT/US94/01~48
7
neutral. More preferably, neutral Oligomers are used.
Particularly preferred are substantially neutral methyl
phosphonate Oligomers. According to an especially pre
ferred aspect, neutral methylphosphonate Oligomers are
employed.
Definitions
As used herein, the following terms have the fol-
lowing meanings unless expressly stated to the contrary.
The term "purine" or "purine base" includes not only
the naturally occurring adenine and guanine bases, but
also modifications of those bases such as bases sub-
stituted at the 8-position, or guanine analogs modified at
the 6-position or the analog of adenine, 2-amino purine,
as well as analogs of purines having carbon replacing
nitrogen at the 9-position such as the 9-deaza purine
derivatives and other purine analogs.
The term "nucleoside" includes a nucleosidyl unit and
is used interchangeably therewith, and refers to a subunit
of a nucleic acid which comprises a 5-carbon sugar and a
nitrogen-containing base. The term includes not only
those nucleosidyl units having A, G, C, T and U as their
bases, but also analogs and modified forms of the
naturally-occurring bases, including the pyrimidine-5-
donor/acceptor bases such as pseudoisocytosine and
pseudouracil and other modified bases (such as 8
substituted purines). In RNA, the 5-carbon sugar is
ribose; in DNA, it is 2'-deoxyribose. The term nucleoside
also includes other analogs of such subunits, including
those which have modified sugars such as 2'-O-alkyl
ribose.
~BSTITUTE SHEET (RULE 26~
WO 94/18835 , ., PCT/US94/01748
8
0
The term "phosphonate" refers to the group O=P-R
I
O ,
I
wherein R is hydrogen or.an alkyl or aryl group. Suitable
alkyl or aryl groups include those which do not sterically
hinder the phosphonate linkage or interact with each
other. The phosphonate group may exist in either an "R"
or an "S" configuration. Phosphonate groups may be used
as internucleosidyl phosphorus group linkages (or links)
to connect nucleosidyl units.
The term "phosphodiester" or "diester" refers to
I
O
O
the group O=P-O-
I
O
I
wherein phosphodiester groups may be used as inter
nucleosidyl phosphorus group linkages (or links) to
connect nucleosidyl units.
A "non-nucleoside monomeric unit" refers to a mono-
meric unit wherein the base, the sugar and/or the phos-
phorus backbone has been replaced by other chemical
moieties.
A "nucleoside/non-nucleoside polymer" refers to a
polymer comprised of nucleoside and non-nucleoside
monomeric units.
The term "oligonucleoside" or "Oligomer" refers to a
chain of nucleosides which are linked by internucleoside
linkages which is generally from about 4 to about 100 ,
nucleosides in length, but which may be greater than about
100 nucleosides in length. They are usually synthesized ,
from nucleoside monomers, but may also be obtained by
enzymatic means. Thus, the term "Oligomer" refers to a
chain of oligonucleosides which have internucleosidyl
linkages linking the nucleoside monomers and, thus,
SUBSTITUTE SHEET (RULE 2b~
WO 94/18835 '~ PCT/US94/01748
9
includes oligonucleotides, nonionic oligonucleoside alkyl-
and aryl-phosphonate analogs, alkyl- and aryl-phosphono-
thioates, phosphorothioate or phosphorodithioate analogs
of oligonucleotides, phosphoramidate analogs of oligo-
nucleotides, neutral phosphate ester oligonucleoside
analogs, such as phosphotriesters and other oligonucleo-
side analogs and modified oligonucleosides, and also
includes nucleoside/non-nucleoside polymers. The term
also includes nucleoside/non-nucleoside polymers wherein
one or more of the phosphorus group linkages between
monomeric units has been replaced by a non-phosphorous
linkage such as a formacetal linkage, a thioformacetal
linkage, a sulfamate linkage, or a carbamate linkage. It
also includes nucleoside/non-nucleoside polymers wherein
both the sugar and the phosphorous mcs~iety have been
replaced or modified such as morpholino base analogs, or
polyamide base analogs. It also includes nucleoside/non-
nucleoside polymers wherein the base, the sugar, and the
phosphate backbone of a nucleoside are either replaced by
a non-nucleoside moiety or wherein a non-nucleoside moiety
is inserted into the nucleoside/non-nucleoside polymer.
Optionally, said non-nucleoside moiety may serve to link
other small molecules which may interact with target
sequences or alter uptake into target cells.
The term "alkyl- or aryl-phosphonate Oligomer" refers
to Oligomers having at least one alkyl- or aryl-phos-
phonate internucleosidyl linkage. Suitable alkyl- or
aryl- phosphonate groups include alkyl- or aryl- groups
which do not sterically hinder the phosphonate linkage or
interact with each other. Preferred alkyl groups include
' lower alkyl groups having from about 1 to about 6 carbon
atoms. Suitable aryl groups have at least one ring having
a conjugated pi electron system and include carbocyclic
aryl and heterocyclic aryl groups, which may be optionally
substituted and preferably having up to about 10 carbon
atoms.
SUBSTITUTE SNEET {RULE 26)
WO 94/18835 - PCT/US94/01748
The term "methylphosphonate Oligomer" (or "MP-
Oligomer") refers to Oligomers having at least one
methylphosphonate internucleosidyl linkage.
The term "neutral Oligomer" refers to Oligomers which
5 have nonionic internucleosidyl linkages between nucleoside
monomers (i.e., linkages having no positive or negative
ionic charge) and include, for example, Oligomers having
internucleosidyl linkages such as alkyl- or aryl- phos
phonate linkages, alkyl- or aryl-phosphonothioates,
10 neutral phosphate ester linkages such as phosphotriester
linkages, especially neutral ethyltriester linkages; and
non-phosphorus-containing internucleosidyl linkages, such
as sulfamate, morpholino, formacetal, thioformacetal, and
carbamate linkages. Optionally, a neutral Oligomer may
comprise a conjugate between an oligonucleoside or
nucleoside/non-nucleoside polymer and a second molecule
which comprises a conjugation partner. Such conjugation
partners may comprise intercalators, alkylating agents,
binding substances for cell surface receptors, lipophilic
agents, nucleic acid modifying groups including photo-
cross-linking agents such as psoralen and groups capable
of cleaving a targeted portion of a nucleic acid, and the
like. Such conjugation partners may further enhance the
uptake of the Oligomer, modify the interaction of the
Oligomer with the target sequence, or alter the pharma
cokinetic distribution of the Oligomer. The essential
requirement is that the oligonucleoside or nucleoside/non
nucleoside polymer that the Oligomer conjugate comprises
be substantially neutral and capable of hybridizing to its
complementary target sequence.
The term "substantially neutral" in referring to an
Oligomer refers to those Oligomers in which at least about
80 percent of the internucleosidyl linkages between the '
nucleoside monomers are nonionic linkages.
The term "neutral alkyl- or aryl- phosphonate
Oligomer" refers to neutral Oligomers having neutral
SUBSTITUTE SHEET (RULE 26)
WO 94/18835 ~ PCT/US94/01748
11
internucleosidyl linkages which comprise at least one
alkyl- or aryl- phosphonate linkage.
The term "neutral methylphosphonate Oligomer" refers
to neutral Oligomers having internucleosidyl linkages
which comprise at least one methylphosphonate linkage.
The term "tandem oligonucleotide" or "tandem
Oligomer" refers to an oligonucleotide or Oligomer which
is complementary to a sequence located either on the 5'-
or 3'- side of a target nucleic acid sequence and which is
co-hybridized with a second Oligomer which is comple-
mentary to the target sequence. Tandem Oligomers may
improve hybridization of these Oligomers to the target by
helping to make the target sequence more accessible to
such Oligomers, such as by decreasing the secondary
structure of the target nucleic acid sequence. In
addition, one member of a pair of tandem Oligomers may
improve the hybrid stability of the second tandem Oligomer
to the target nucleic acid sequence by promoting a helical
structure at either the 5'- or 3'-end of said second
Oligomer and vice-versa.
The term "short chain aliphatic alcohol" refers to an
alcohol having from about 2 to about 20 carbon atoms in
which the aliphatic (alkyl) chain may be either straight
chained or branch chained and includes primary, secondary
and tertiary alcohols, glycols and polyols.
The term "flux enhancer" refers to a substance which
is used to increase transdermal flux of a compound. A
flux enhancer is typically applied to skin or mucous
membrane in combination with the compound to increase
transdermal flux of the compound. Enhancers are believed
~ to function by disrupting the skin or mucous membrane
barrier or by changing the partitioning behavior of the
. drug in the skin or mucous membrane.
The term "Triplex Oligomer Pair" refers to first and
second Oligomers which are optionally covalently linked at
one or more sites and which are complementary to and are
capable of hydrogen bonding to a segment of a single
'.~.rc:~ ~ ~~~~~ ~~~~ ~~c~ ~~ ~v~
WO 94118835 PCT/US94101748
12
stranded target nucleic acid, such as RNA or DNA, and,
thus, together with the single stranded target nucleic
acid, are capable of forming a triple helix structure
therewith.
The term "Third Strand Oligomer" refers to Oligomers
which are capable of hybridizing to a segment of a double
stranded nucleic acid, such as a DNA duplex, an RNA duplex
or a DNA-RNA duplex, and forming a triple helix structure
therewith.
The term "complementary," when referring to a Triplex
Oligomer Pair (or first and second Oligomers) or to a
Third Strand Oligomer, refers to Oligomers having base
sequences which are capable of forming or recognizing
hydrogen bonds (and base pairing or hybridizing) with the
base sequence of the nucleic acid to form a triple helix
structure.
The term "substantially complementary" refers to
Oligomers, including Triplex Oligomer Pairs or Third
Strand Oligomers which may lack a complement for each
nucleoside in the target sequence, have sufficient binding
affinity for the target sequence to form a stable duplex
or triple helix complex, as the case may be, and thereby
specifically recognize the target sequence and selectively
inhibit or down-regulate its expression.
The term "triplet" or "triad" refers to a hydrogen
bonded complex of the bases of three nucleosides between
a base (if single stranded) or bases (if double stranded)
of a target sequence, a base of a Second Strand and a
Third Strand (if a single stranded target sequence) or a
base of a Third Strand (if a double-stranded target).
Brief Descrit~tion of the Drawings
Figure 1 depicts thermal denaturation profiles for
double stranded and triple-stranded complexes formed
between Oligomer 2 and a target sequence.
Figure 2 depicts clearance from plasma of a tritium-
labelled tetramer in a mouse model.
SUBSTITUTE SHEET (RUIN 26~
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Figure 3 depicts clearance from plasma of a tritium
labelled dodecamer in a mouse model.
Detailed Description of the Invention
According to the present invention, methods of
arresting and/or diminishing the progression of conditions
characterized by androgen-associated hair loss, parti
cularly scalp hair loss in men, and more particularly that
condition known as male pattern baldness are provided.
These methods diminish and/or arrest the progression of
hair loss by decreasir_g amounts of 5-alpha-dihydro-
testosterone-androgen receptor complex present in scalp
tissue. This decrease may be obtained by either down-
regulation of synthesis of androgen receptor or of 5-
alpha-dehydrotestosterone ("DHT"). Synthesis of DHT may
be down-regulated by decreasing levels of 5-alpha
reductase present in scalp tissue. Such down-regulation
may be effected by use of an Oligomer which may bind to a
protein's active site to modulate its function or
Oligomers such as antisense Oligomers, Third Strand
Oligomers and Triplex Oligomer pairs. Suitable nucleoside
sequences for these Oligomers may be determined from the
sequences of target genes. Preferred sequences of the
target region are described herein.
A. Preferred Oligomers
The Oligomer selected may be any of a number of
types, including those having a charged or uncharged
backbone.
Preferred Oligomers include alkyl- and aryl
phosphonate Oligomers, especially preferred are
methylphosphonate Oligomers. Other preferred Oligomers
include phosphorothioate Oligomers, morpholino analogs,
formacetal analogs and peptide nucleic acid ("PNA")
analogs.
Preferably the Oligomers each comprise from about 4
to about 40 nucleosides, more preferably, from about 6 to
SUBSTITUTE SiiEET ~RUI.E 2F~
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69666-43
14
30 nucleosides. Especially preferred are Cligomers of
about 8 to about 20 nucleosides.
According to an alternately preferred aspect, tandem
Oligomers are employed. Preferred tandem Oligomers
include those which comprise a total of about 20 to about
40 nucleosides.
Oligomers having the selected internucleoside
linkages may be conveniently prepared according to
synthetic technic~es known to those skilled in the art.
For example, commercial machines, reagents and protocols
are available for the synthesis of Oligomers having
phosphodiester and ~=ertain other phosphorus-containing
internucleoside linkages. S.ee also Gait, M.J.,
OliQpnucleotide Syr~thesis: A Practical Ap roach (IRL
Press, 1984?; Cohere, Jack S., Oli4odegxynucleotides
Anti~;~ense Inhib:.tors gf Gene Expression, (CRC Press, Boca
Rators, FL, 1989 ) ; and Ol i aonucl eotides anc~ ~lnaloc~ues : A
P~act:ical Approach, (F. Eckstein, 1991] . Preparation of
Oligomers having certain non-phosphorus-containing
internucleoside linkages is described in United States
Patent No. 5,142,047.
According to an alternately preferred aspect,
chira.lly pure Oligomers are used according to the present
invention. Alternatively, Oligomers comprising at least
one ehirally pure internucleosidyl linkage may be used and
may be preferred. Such Oligamers may be prepared using
methods such as those described in Lesnikowski et al.,
Nucleic Acids Research 8 8 :2109-2115 (1990) and Stec et
al., Nucleic Acids Research 19(2i):5883-5888 (1991).
Synthetic methods for preparing methylphosphcnate
Oligomers are described in Lee B.L., et al., Biochemist~v
27:3197-3203 (1988), and Miller, P.S., et al., Biochem
istry 25:5092-5097 (1986), and commonly-assigned published
PCT a~oplications WO 92/07864 and WO 92%07882.
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69666-4.'.
i~
Also preferred are.Oligomers which are nucleoside/
non--nucleoside polymers. Suitable Oligomers also include
chimeric oligonucleotides which are composite RNA, DNA
analogues (Inoue et al., FEBS Lett. 2115:327 (1987)).
Other suitable Oligomers include Oligomers having chimeric
bacl~;bones. Such chimeric backbone Oligomers include
Oligomers having mixed phosphate backbones including
nucleoside sequences which are capable of activating
RNaseH and nucleoside sequences which do not activate
RNaseH, and thus allow site directed cleavage of an RNA
molecule. See U.S. Patent No. 5,149,797,
Chimeris backbone
Oligomers also include Oligomers having a mixture of
internucleosidyl linkages which may or may not include
IS phosphorus atoms, such as morpholinyl linkages, formacetal
linkages, peptide nucleic acid (PNA) linkages and the
like. Oligomers having a neutral backbone, for example,
methylphosphonate Oligomers with cleaving or cross-linking
moieties attached, may prove advantageous in certain
circumstances; such Oligomers may have a longer half-life
~rivo since the neutral structure reduces the rate of
nuclease digestion while the cleaving or cross-linking
moiety may promote inactivation of target polynucleotide
sequences.
According to one aspect of the present invention,
these antisense Oligomers have a sequence which is
complementary to a portion of the RNA transcribed from the
selected target gene. Although the exact molecular
mechanism of inhibition has not been conclusively
determined, it has been suggested to result from formation
of dL~plexes between the' antisense Oligomer and the RNA
transcribed from the target gene. The duplexes so formed
may inhibit translation, processing or transport of an
mRNA sequence or may lead to digestion by the enzyme
RNaseH.
Single stranded Oligomers may also bind to a duplsx
DNA target such t hat a duplex is feed witz one of t:~~e
CA 02156512 2002-O1-31
69666-43
1. 6
two DNA strands, and the second DNA of the target strand
is displaced from the duplex. Preferred is the formation
of a. duplex by the Oligomer with the coding strand of the
DNA duplex target ("invading duplex"). The invading
duplex sv formed may inhibit transcription.
According to an alternate aspect of the present
invention, down regulation of 5-alpha reductase or the
androgen receptor may be accomplished by triple helix
forrnuation using a Third Strand Oligomer or a Triplex
Oligomer Pair having sequences selected such that the
Olig~omer(s) are complementary to and form a triple helix
complex with a target sequence of double stranded or
single stranded nucleic acid and thereby interfere with or
prevent expression of the targeted nucleic acid sequence.
Triple strand formation can occur in one of several ways.
A single stranded Oligomer may form a triple strand with
duplex DNA or RNA; two separate or connected Oligomers may
form a triple strand with single stranded DNA or RNA; two
separate or connected Oligomers may bind to one of the
duplex DNA or RNA strands and displace the other such that
it 1.s not involved in triple strand formation. Further
descriptions of the use of Oligomers (including Third
Strand Oligomers and Triplex Oligomer Pairs) to prevent or
interfere with the expression of a target sequence of
double or single stranded nucleic acid by formation of
triple helix complexes is described in the copending U.S
Patent Applications Serial Nos. 07/348,027, 07/751,813,
07/772,081 and 07/987,746.
As a general matter, the Oligomer employed will have
a sequence that is complementary to the sequence of the
target nucleic acid. However, absolute complementarity
may not be required; in general, any Oligomer having
sufficient complementarity t« form a stable duplex (or
trip:Le helix complex as the case may be) with the target
nuclE=is acid is considered to be suitable. Since stable
dupl E~x formation depends cn t::e se~:er_ce and length of the
WO 94/18835 ~ ~ ~ PCTIUS94/01748
17
hybridizing Oligomer and the degree of complementarity
between the antisense Oligomer and the target sequence,
the system can tolerate less fidelity (complementarity)
when longer Oligomers are used. This is also true with
Oligomers which form triple helix complexes. However,
Oligomers of about 8 to about 40 nucleosidyl units in
length which have sufficient complementarity to form a
duplex or triple helix structure having a melting
temperature of greater than about 40°C under physiological
conditions are particularly suitable for use according to
the methods of the present invention.
The concentration of Oligomer used may vary,
depending upon a number of factors, including the extent
of hair loss condition to be treated, the type and the
specificity of the particular antisense Oligomer, Triplex
Oligomer Pair, or Third Strand Oligomer selected. It is
believed that significant inhibition as demonstrated by a
cosmetically significant decrease in progression of hair
loss may be obtained at concentrations in about the 10 ~,M
range; however, under other conditions, higher or lower
concentrations of Oligomer may be preferred.
Where Oligomers are to be administered transdermally,
preferred are neutral Oligomers.
According to one preferred aspect, these Oligomers
may comprise a conjugate between a polynucleoside or
nucleoside/non-nucleoside polymer and a conjugation
partner. Suitable conjugation partners include inter
calating agents such as acridine, alkylating agents,
binding substances for cell surface receptors, lipophilic
agents, photo-crosslinking agents such as psoralen, other
cross-linking agents, pro-chelates, or nucleic acid
modifying agents, including groups capable of cleaving a
. targeted portion of a nucleic acid such as hydrolytic or
nucleolytic agents like o-phenanthroline copper or EDTA
iron, all of which may be incorporated in the Oligomers.
Conjugation partners may also be introduced into the
Oligomer by the incorporation of modified nucleosides or
SU~~T~'~~'~~ SH~~;' ~~~~.~ ~G)
CA 02156512 2002-O1-31
69666-43
18
nucleoside analogs through the use of enzymes or by
chemical modification of the Oligomer, for example, by the
use of non-nuleotide linker groups.
When used to prevent function or expression of a
single or double stranded nucleic acid sequence, these
Oligomers may be advantageously derivatized or modified to
incorporate a nucleic acid modifying group which may be
caused to react with said target nucleic acid and
irreversibly modify its structure, thereby rendering it
l0 non-functional. Conjugates may be introduced to alter the
pha:rmacodynamics or toxicity of the oligonucleotides in
the body. For example, a cleavable moiety may be attached
according to patent 4,588,525, such cleavable moiety being
particularly useful with topical application of the
conjugate. Upon application of the conjugate, that
portion of conjugate that enters the blood stream instead
of the tissue at the site of topical application is
cleaved to a more highly charged species which only poorly
enters non-target tissues and is readily excreted.
Commonly assigned USSN 565,299 discloses
psoralen-derivatized Oligomers.
As discussed above, a wide variety of nucleic acid
modifying groups may be used as conjugation partners to
derivatize these Oligomers. Nucleic acid modifying groups
include groups which, after the derivatized Oligomer forms
a complex with a single stranded or double stranded
nucleic acid segment, may be caused to cross-link,
alkylate, cleave, degrade, or otherwise inactivate or
destroy the target nucleic acid segment or a target
3_0 sequence portion thereof, and thereby irreversibly inhibit
the function and/or expression of that nucleic acid
segment.
The location of the nucleic acid modifying groups in
the Oligomer may be varied ar_d may depend on the
parr_icular nucleic acid modifying group employed and the
tar:~eted nucleic acid segme::t . According'_y, the nuc';eic
WO 94/18835 ~ PCTIUS94/01748
19
acid modifying group may be positioned at the end of the
Oligomer or intermediate between the ends. A plurality of
nucleic acid modifying groups may be included.
In one preferred aspect, the nucleic acid modifying
group is photoreactable (e. g., activated by a particular
wavelength, or range of.wavelengths of light), so as to
cause reaction and, thus, cross-linking between the
Oligomer and the nucleic acid target.
Exemplary of nucleic acid modifying groups which may
cause cross-linking are the psoralens, such as an
aminomethyltrimethyl psoralen group (AMT). The AMT is
advantageously photoreactable, and thus must be activated
by exposure to particular wavelength light before cross
linking is effectuated. Other cross-linking groups which
may or may not be photoreactable may be used to derivatize
these Oligomers.
Alternatively, the nucleic acid modifying groups may
comprise an alkylating agent group which is covalently
bonded to the nucleic acid segment to render the target
inactive. Suitable alkylating agent groups are known in
the chemical arts and include groups derived from alkyl
halides, haloacetamides and the like. Polynucleotide
modifying groups which may be caused to cleave the
polynucleotide segment include moieties which generate
radicals, as well as moieties which promote cleavage
through nucleophilic attack. Transition metal chelating
complexes, such as ethylenediaminetetraacetate (EDTA) or
a neutral derivative thereof, can be used to generate
radicals. Other groups which may be used to effect
radical mediated cleavage include phenanthroline,
porphyrin and the like. When EDTA is used, iron may be
advantageously tethered to the Oligomer to help generate
- the cleaving radicals. Although iron-EDTA is a-preferred
polynucleotide cleaving group, other nitrogen containing
materials, such as azo compounds or nitrenes or other
transition metal chelating complexes, may be used. Yet
other cleavage agents include nucleophilic agents and
~~~v~~~~~~ ~~~~~~~ L
WO 94/18835 PCT/iJS94/01748
2 0 ' -'
hydrolytic agents that promote the addition of water at
the phosphorus internucleotide linkages. Such agents
include amines, substituted guanidinium groups, imidazole
groups and the like.
1. Preferred Neutral Oliaomer Formulations
Preferred neutral Oligomers include neutral alkyl-
and aryl-phosphonate Oligomers and neutral Oligomers
comprising morpholino or phosphoramidate linkages.
Especially preferred are neutral methylphosphonate
Oligomers. In view of their demonstrated ability to
penetrate skin, including tape stripped skin, (which has
had the stratum corneum removed and which has been
reported as a model for mucous membrane), particularly
preferred are neutral methylphosphonate Oligomers having
only methylphosphonate internucleosidyl linkages.
According to another aspect of the present invention,
preferred are Oligomers which may be neutral until they
enter cells and once inside are converted to charged
species through chemical or biological processes. Such
charged oligonucleotides may contain other moieties that
stabilize the oligonucleotides to nuclease degradation.
Substituents such as 2'-O-methylribose groups, various
base modifications, and analogs of the phosphorous
backbone, such as phosphorothioates, can increase
resistance to nucleases. Additionally, the presence of
methylphosphonate or other neutral internucleoside
linkages in the Oligomer give exonuclease resistance.
Preferred are neutral Oligomers having from about 6
to about 40 nucleosides, more preferably from about 12 to
about 20 nucleosides. Although neutral Oligomers which
comprise more than 20 nucleosides may be used, where
complementarity to a longer sequence is desired, it may be
advantageous instead to employ shorter neutral tandem
Oligomers which total more than 20 nucleosides in order to
maximize solubility and penetration through the skin while
competing for the development of a secondary structure of
SUBSTITUTE Sf'E~T {RUSE 26)
WO 94/18835 PCT/US94/01748
21
the target nucleic acid, such as an mRNA. These tandem
Oligomers may also increase specificity of binding to the
target sequence. Alternatively, it may be advantageous to
use a plurality of neutral Oligomers, each Oligomer com-
plementary to a distinct target sequence which may be part
of the same gene or a different gene.
Where the neutral Oligomers comprise alkyl- or aryl-
phosphonate Oligomers, it may be advantageous to
incorporate nucleoside monomeric units having modified
ribosyl moieties. The use of nucleoside units having 2'-
O-alkyl- or 2'-halo- and, in particular, 2'-O-fluoro-or
2'-O-methyl-ribosyl moieties in these neutral Oligomers
may advantageously improve hybridization of the Oligomer
to its complementary target sequence.
Suitable formulations comprise about 0.0001% to about
10% by weight of neutral Oligomer.
In one preferred aspect, there are provided neutral
Oligomer formulations which comprise about 2% to about
100% of a short chain aliphatic alcohol. Suitable
alcohols include ethanol, isopropyl alcohol, propylene
glycol and glycerol. In certain studies, formulations of
neutral Oligomers comprising ethanol have demonstrated
advantageous transdermal flux.
In an especially preferred aspect, these neutral
Oligomer formulations may additionally comprise a flux
enhancer. Suitable flux enhancers include those known to
those skilled in the art and include decylmethylsulfoxide,
dimethylsulfoxide as well as cyclic ketones, lactones,
anhydrides and esters such as those described in PCT
Application No. PCT/US86/02583 (Publication Number W087/
03473). Some of these flux enhancers also increase
retention of the Oligomer and, thus, act to increase the
concentration of Oligomer within the skin itself.
Thus, for Oligomer formulations for direct (local}
treatment, such as topical application to skin, it is
preferred to use a flux enhancer which not only maximizes
transdermal flux, but increases Oligomer retention in the
SU~STfTUTE S~fEET ~RtJLE 2~
CA 02156512 2002-O1-31
69666-43
skin. Certain c.rc'_=c cetone 3na lactone snhancers have
aeen repor ted to increase 1 : cal =etert~.en as well and,
thus, comprise a prefer=-ed class or enhancers for topical
administration ~~f Oligcmer formulations.
S Accordi_Zg to one aspect, the present invention
includes liposomal delivery ef the Oligomers. Various
methods and types of liposcmal vesicles for drug delivery
have been described. ~, e.Q., Remington's
Pharrnaceuticai Scienc~_s (1990?. The Oligomers may be
ZO encapsulated by a liwosome. Such liposome complexes
advantageously may ac- to er~:ancs 3elive~r o= O1'_gemer .
or practical ;ise, she ~ormulat~.ons may be out in.
=cmmercial packages. Such commercial packages usuallyr carr~r
~~rritten matters ~escribi.~.g instructions t~:at t'~ese
:.S formulations are to be used ~:~r decreasiac na_r loss.
'ro r~ ~ v..-~ '~ r~ ,- '"' r T r.S ~ o S
r~~-~ ro=n~~o ~- !~ o
Ac_o...,~__g to a p_ . d aspec.., he pr_sent
invention is 3ioect_d to ;net::ods of ~rsve_~_tizg or rsduc_zg
hai= lcss using Cligomers ~rhich inter=ere with the
20 exFressicn oL t he e_::zyme 5-alpi:a-reductase, or with the
er~ress:.on of the androgen receetor itsel f . Suitable
Oligomers include aatisense 01_gemers, Third Strand
Olig~omers and T_-iplex Oligomer Pairs.
According to one aspect of the present invention,
25 here are provided methods of dec=easing hair loss by
preventing or inter~er:ng with the expression of the human
androgen =eceator or the S-alpha-r~_ductase enzyme by
administration of an Oligomer wi-,ich is complementary to a
target sequenc°_ pa the DNA or an mRNA transcribed
30 therefrom which codes for the androgen receptor or
S-alpha-reductase or to a sequence immediately upstream
_rom the transc=ipticn stn= . site for t he mRNA. The
Oligomer administered ,nay be ei=her an antisense Oligomer ,
a Th:.rd Strand 0..~ ome_, o_. a .__
i~g r r '~r;Flex Oligomer Pai=. '~he
35 a_~.t:.sense O1 igcme= is compleme~ta~y to a sequence oL RNA
CA 02156512 2002-O1-31
69666-43
22a
..=anscr ibed ==om a Target gene, t~ a singl s-stranded DN1
car3et sequence, or to a singl' RNA or DNA strand
ccrtaized within a dspiex. The Third Strand Oligvmer aas
a case sequence selected so that it is caeabie of hydrogen
be~c:. :g wi;.:: a secue.~.c~ o~ a dou: ? a st=anded auc~~ic ac~~
WO 94/18835 ~ ~ PCT/US94/01748
23
and forming ,a triple helix complex therewith. The first
and second Oligomers of the Triplex Oligomer Pair have
sequences selected such that they are complementary to and
capable of hydrogen bonding with a targeted
single-stranded nucleic acid sequence of a target gene or
its transcription product or to a single strand of a
duplex and together with the single stranded nucleic acid
form a triple helix complex.
The target gene is selected from the group consisting
of those genes encoding the androgen receptor or the
enzyme 5-alpha-reductase and is considered to include a
target sequence immediately upstream from the
transcription start site of that:gene. Preferably the
target sequence would include sequences from -500 to +20
(relative to the transcription start site) of the androgen
receptor gene or 5-alpha reductase gene. More perferably
suitable sequences would include target sequences in the
area from -100 to +20 (relative to the transcription start
site) of the androgen receptor gene or 5-alpha reductase
gene.
Oligomers of appropriate length, preferably from
about 8 to 40 nucleosides, more preferably from about 12
to about 20 nucleosides, are selected so as to be adjacent
to or cover these sites when hybridized to the target, in
part or in whole. Preferred sites when the target
sequence is mRNA include, in both the androgen receptor
and 5-alpha-reductase genes, the 5' untranslated region,
the translation initiation region including regions
slightly downstream of the AUG start codon (preferably up
3,0 to about 20 nucleotides downstream from the AUG initiation
codon), splice acceptors, splice donors, and the 3'
untranslated region.
As examples, in the case of the androgen receptor,
the preferred target sites would include the sequence
ranging from 18-50, with reference to the nucleotide
positions of the human androgen receptor gene (Genbank
~~~~~~i i ~ ~ ' ~~~~ ~~~L~ ~~~
WO 94/18835 , PCT/US94/01748
24
locus:HUMARB). A preferred target of this sequence range
would include the sequence:
[1] TTC CCC CAC TCT CTC TC,
corresponding to the nucleotide positions 28-44 of the
androgen receptor gene (Genbank locus:HUMARB). A second
preferred target of this sequence rar_ging would include
the sequence:
[2] CTC TCT CTC ACC TC,
corresponding to the nucleotide positions 36-50 of the
androgen receptor gene. A preferred triplex target site
would include the sequence range from 109-126 of exon 4 of
the human androgen receptor gene (Genbank locus:HUMARC4).
A preferred target of this sequence range would include
the sequence:
[3] UCU CUC UUC CUU CCC,
corresponding to the nucleotide positions 109-123 of exon
4 of the human androgen receptor gene.
Thus, according to a preferred aspect of the present
invention, Oligomers of the appropriate length, preferably
from about 8 to 40 nucleosides and more preferably from
about 12 to about 25 nucleosides especially from about 12
to about 20 nucleosides, are selected so as to have
sequences which hybridize to sites immediately adj acent to
these sites or hybridize with and cover these sites, in
part or wholly, as defined by the nucleotide positions
included above for 5-alpha-reductase and the androgen
receptor.
When antisense Oligomers are used, the sequence of
the Oligomers is the reverse complement of the sequence of
the targeted region so as to be able to hybridize to the
targeted region.
When Third Strand Oligomers are used, the Oligomers
are selected to form sequence-specific hydrogen bonding
interactions with the double stranded nucleic acid target.
When Triplex Oligomer Pairs are used, the first and
second Oligomers are selected so as to form sequence
specific hydrogen bonding interactions with a single
SLt~STI~UTE SHEE7- (~Ul.~ 26~
WO 94/18835 ~ ~ PCT/US94101748
stranded nucleic acid, and together form a triple helix
structure.
To assist in understanding the present invention, the
following examples are included which describe the results
5 of a series of experiments. The following examples
relating to this invention should not, of course, be
construed in specifically limiting the invention and such
variations of the invention, now known or later developed,
which would be within the purview of one skilled in the
10 art are considered to fall within the scope of the present
invention as hereinafter claimed.
Example 1
Preparation of OliQOribonucleosides
Oligoribonucleotides may be synthesized using the
15 following procedures:
The oligoribonucleotides were synthesized using 5'-O-
dimethoxytrityl-2'-O-tert-butyldimethylsilyl-3'-O-N,N-
diisopropyl-~i-cyanoethylphosphoramidite nucleosides
(purchased from either Millipore or Pennisula Labora-
20 tories). The syntheses were done on a 1 /Cmole scale with
a Milligen 8750 automated DNA synthesizer using standard
Milligen phosphoramidite procedures with the exception
that the coupling times were extended to 12 minutes to
allow adequate time for the more sterically hindered 2' -O-
25 tert-butyldimethylsilyl RNA monomers to react. The
syntheses were begun on control-pore glass bound 2'-O
tert-butyldimethylsilyl ribonucleosides purchased from
Pennisula Laboratories. All other oligonucleotide
synthesis reagents were as described in Milligen's
standard protocols.
After synthesis, the oligonucleotides were handled
under sterile, RNase-free conditions. Water was
sterilized by overnight treatment with 0.5% diethyl
pyrocarbonate followed by autoclaving. All glassware was
baked for at least 4 hours at 300°C.
SUBSI'fTUTE SHEET (RULE 2~j
WO 94/18835 PCT/US94/01748
26
The oligonucleotides were deprotected and cleaved
from the support by first treating the support bound
oligomer with 3/1 ammonium hydroxide/ethanol for 15 hours
at 55°C. The supernatant, which contained the oligo-
nucleotide, was then decanted and evaporated to dryness.
The resultant residue was then treated with 0.6 mL of 1 M
tetrabutylammonium fluoride in tetrahydrofuran (which
contained 5~ or less water) for 24 hours at room tempera-
ture. The reaction was quenched by the addition of 0.6 mL
of aqueous 2 M triethylammonium acetate, pH 7. Desalting
of the reaction mixture was accomplished by passing the
solution through a Bio-Rad lODG column using sterile
water. The desalted oligonucleotide was then dried.
Purification of the oligoribonucleotides was carried
out by polyacrylamide gel electrophoresis (PAGE) con
taining 15~ 19/1 polyacrylamide/bis-acrylamide and 7 M
urea using standard procedures (See Maniatis, T. et al.,
Molecular Clonina~ A Laboratory Manual, pages 184-185
(Cold Spring Harbor 1982)). The gels were 20 cm wide by
40 cm long and 6 mm in width. The oligoribonucleotides
(60 OD Units) were dissolved in 200 ~,L of water containing
1.25% bromophenol blue and loaded onto the gel. The gels
were run overnight at 300 V. The product bands were
visualized by UV backshadowing and excised, and the
product eluted with 0.5 M sodium acetate overnight. The
product was desalted with a Waters C18 Sep-Pak cartridge
using the manufacturer supplied protocol. The product was
then 32P labelled by kinasing and analyzed by PAGE.
Example 2
Thermal denaturation profiles
The stabilities of triple stranded complexes formed
between two MP oligomers and a complementary RNA oligomer
were determined by thermal denaturation analysis. Solu-
tions were prepared for analysis as follows: 2.4 ~.M MP
oligomer, 1.2 ~,M RNA oligomer (2:1 mole ratio MP:RNA) in
10 mM potassium phosphate, 0.1 M sodium chloride, 0.03
SUBSTITUTE SHEET (RU~.E 2G~
WO 94/18835 ~ ~ PCT/US94/01748
27
potassium sarkosylate, 0.1 mM EDTA, pH 7.2, final volume
-'1 mL. Each solution was heated to 80°C and allowed to
cool to 4°C over a period of about 4 hours. The solutions
were then transferred to quartz cuvettes (1 cm pathlength)
' S and placed in a Varian Model 3E spectrophotometer equipped
with a temperature control module interfaced to an IBM
compatible PC computer. Temperature was varied from 5°C
to 80°C at a rate of 1.5 °C/minute and absorbance was
measured continuously at 260 nm. Plots of A26o versus
temperature revealed single monophasic transitions for
each of the oligomer sets described in this example.
The melting temperatures (Tm) at which half of each
complex had dissociated to single strands was 45.8°C and
42.3°C (2:1 mole ratio MP:RNA) for Oligomer 1 and Oligomer
2, respectively (see Table I). The entire melting curve
for MP Oligomer 2 and its target at 2:1 and 1:1 ratios is
shown in Figure 1. Thus, above 2.4 micromolar MP oligomer
concentration at physiological temperatures (below 37°C in
human skin) these Oligomers would be substantially
hybridized.
Figure 1 depicts a thermodenaturation profiles for
double-stranded and triple-stranded complexes formed
between Oligomer 2 and a target sequence.
Table I
( MP : RNA
mole
Oliaomer Tm (°C) ratio)
[4] Oligomer 1 (Androgen Rec. #1 44.1°C (1:1)
target: 5' gag-aga-gag-tgg-ggg-aa) 45.8°C (2:1)
[5] Oligomer 2 (Androgen Rec. #2 41.1°C (1:1)
target: 5' gag-gtg-gag-aga-gag) 42.3°C (i:2)
Example 3
Clearance of Methvlphost~honate Oliaomer From Serum
Clearance of methylphosphonate oligomers from mouse
serum was measured with two oligomers: 3H-tetramer (1689-3)
SUBSTITUTE SH~~i' (~uLf 26)
WO 94/18835 PCT/US94/01748
~~.~..'~
28
3H- (dT) ~ and a 12-mer (2054-2 ) 3H-C2- (TC) 6 (where C2
referred to a 2-carbon non-nucleotide linker with a
primary amine).
BALB/C female mice (Jackson Laboratory) 9-10 weeks
old were injected in the tail vein with 27 nmol (3 x 105
dpm) of oligomer in 200 ~.1 phosphate buffered saline.
Samples were collected at the indicated times by eye
bleed. 100 ~.1 samples were collected in 200 ~.1
heparinized eye bleed capillary tubes. The mice were
mildly anesthetized with metofane (methoxyflurane) during
the procedure, and each mouse was bled no more than 7 or
8 times. The blood was transferred to polypropylene
microcentrifuge tubes and spun to remove cells. A 20 ~.l
aliquot of the serum was removed and combined with 5 ml of
scintillation fluid (ScintiVerse BD). The amount of
radioactivity was determined in a liquid scintillation
counter.
The plasma half-lives of both oligomers in mice were
found to be approximately 8 to 10 minutes. Figures 2 and
3 depict plots of the clearance from plasma of the 4-mer
(Figure 2) and the 12-mer (Figure 3).
Example 4
Preparation of Skin Samples for Permeability and Tissue
Level Studies
A. Hairless Mouse Skin
Hairless mice (male, HRS/J strain, 8 to 10 weeks old,
20 to 25 g) were sacrificed in a COz chamber and approx-
imately 5 cm2 of full-thickness skin (dermis and epidermis)
was removed from the abdomen. After removal of the
subcutaneous fat, the skins were rinsed with physiological '
saline and used within one hour.
The stratum corneum was removed from hairless mice
for permeability experiments by using cellophane tape.
The tape was gently applied to the skin of a recently
sacrificed animal and then pulled away from the body.
SUBSTITUTE SHEET (RULE 26~
WO 94/18835 PCT/US94/01748
29
This was, repeated 12 to 15 times with fresh pieces of
tape.
B. Human Cadaver Skin
Human cadaver skin was obtained at autopsy through
the Stanford University Medical Center. The skin was
excised using a dermatome from the thigh area of a 74 year
old male within 24 hours post-mortem. The thickness, as
measured with a Van Keuren light wave micrometer, ranged
from 125 to 450 ~.m. The average thickness was 200 to 300
/Cm. The skin was rinsed with phosphate buffered saline
(pH 7.4), blotted dry and frozen for 6 months in triple-
sealed bags evacuated of air. Prior to use, the skin was
thawed and rinsed in PBS.
Exam 1p a S
Permeability Experiments
A diffusion console containing nine glass Franz dif-
fusion cells was used in the permeability experiments.
The Franz cells were maintained at 37°C by thermostati-
cally controlled water, which was circulated through a
jacket surrounding the cell body. Each skin was mounted
and clamped between the cell body and the cell cap so that
the epidermal side faced upward (vehicle side) . The skins
were then allowed to equilibrate for 1 hour in the diffu-
sion cells prior to addition of the vehicle. The exposed
surface was 2.0 cma. The receptor was 0.01 M phosphate-
buffered saline (pH 7.4) isotonic saline with 0.05 sodium
azide added to prevent growth of microorganisms.
The Franz cells were closed to maximize drug concen
tration in the receptor phase. The volume of the cells
was 6.2 mL. The cells were stirred using a teflon-coated
stir bar at 600 rpm.
The drug/vehicle mixtures were pipetted through the
cell cap onto the skin [0.2 mL total vehicle added to 2.0
cm2 (0.1 nK.cmz)]. At certain times following addition of
the vehicles, a syringe needle was inserted through the
SUBST~T~T~ SHEET {RULE 2~~
WO 94/18835 PCT/US94101748
side arm into the receptor solution and 300 ~,L was with-
drawn. The volume removed was replaced by an equal volume
of fresh saline. The solution effect was accounted for in
the drug flux calculations.
5 Permeability results are tabulated in Table II.
Example 6
Chromatoaraphic Analvsis of Oliaomer
The 14-mer (neutral methylphosphonate Oligomer of 14
nucleosides having only methylphosphonate internucleosidyl
10 linkages) and 14-mer-IA (methylphosphonate Oligomer of 14
nucleosides having an internal anionic internucleosidyl
linkage) were measured in the receptor solution by HPLC.
These analyses were performed on a Waters 840 system
consisting of two model 510 pumps, a model 481 W
15 detector, a model 710B WISP sample processor, and a
Digital computer model 350 microprocessor/programmer.
A. 14-Mer
The column used to separate the 14-mer was a 3.9 mm
x 15 cm 4 ~.m, Waters Nova-Pak C18. A gradient elution was
20 performed as follows for the 14-mer:
Time (min) gyp, ~B
o loo 0
4 70 30
10 55 45
25 11 55 45
12 5 95
15 5 95
17 100 0
22 ~ 100 0
30 Flow rate - 1.1 mL/min, wavelength - 260 nm, retention
time = 9.6 min.
A = 0.05 M TEAR, pH 7.6
B = acetonitrile/A (75:25)
SUBSTITUTE SHEET (RULE 26)
WO 94/18835 PCT/US94/01748
31
B. 14-Mer-IA
The HPLC conditions were altered somewhat for measure-
ment of the 14-mer-IA. Again, a gradient elution profile
was used as described below.
Time (min) ~A ~B
0 0 100
5 28 72
14 45 55
14.2 45 55
15.5 98 2
98 2
22 0 100
28 0 100
Flow rate - 1.1 mL/min, wavelength - 260 nm, retention
15 time = 10.2 min.
A = Acetonitrile/B (75:25)
B = 0.05 M ammonium acetate, pH 7.4
Example 7
Tissue Level Measurements of Oliaomer Retained in Skin
20 Preliminary work was performed to determine the
amount of 14-mer oligomer retained in the skin samples at
the conclusion of the permeability experiments. The skins
were rinsed with a small amount of water for several
seconds, followed by washing for about 10 seconds with a
small amount of acetonitrile to remove solid drug from the
surface of the skin. The skins were then rinsed for sev-
eral seconds with water. The skins were then frozen until
analysis (up to several weeks). The skins were thawed and
the region not exposed to the donor vehicle was cut away
and discarded. The hydrated skin samples were weighed and
then homogenized in 0.01 M sodium phosphate, pH 7.4, using
a Polytron Homogenizer for approximately 2 minutes. The
homogenate was then centrifuged at 8,000 g for 15 minutes
at room temperature. The supernatant was removed and
analyzed directly by HPLC analysis (see below for conditions) .
SUBSTITUTE SHEEN RULE 26)
WO 94/18835 PCT/US94/OI748
1
32
The chromatographic conditions were similar to those
described above for the 14-mer in permeability experiments
with some minor changes noted below.
Time (min) ~A ~B
0 100 0
5 70 30
14 55 45
15.5 2 98
32 2 98
35 100 0
42 100 0
Flow rate - 1.1 mL/min, wavelength - 260 nm, retention
time = 11.1 min.
A = 0.05 M ammonium acetate, pH 7.0
B = acetonitrile/A (75:25)
Presence of the oligomer in the tissue homogenates
was confirmed by spiking the samples with 40 ~.L of a 1.8
~.g/mL solution of 14-mer.
Resuspension of the pellet obtained after centrifuga
tion, followed by homogenization and recentrifugation, led
to release of between 1 to 3~ of the total 14-mer recov
ered from the original sample. These results indicate
that the 14-mer was efficiently isolated in the first
extraction step.
Amounts of Oligomer isolated from skin after
permeability experiments using different vehicles are
tabulated in Table III.
Example 8
Measurement of Flux and Retention of Oliaomers in Human
Skin
Human skin which had been dermatomed to a thickness
of about 5-200 ~,m was used. The skin was mounted in a
closed glass Franz diffusion cell (as described in Exam-
ple 5 ) .
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Vehicle containing oligomer and, in some instances,
erihancer (100 ~,L/cm2) was placed on the surface of the skin
(2 cm2 exposed surface) .
The amount of oligomer diffusing through and remain-
s ing in the skin was measured by HPLC. (See Example 5).
Results are summarized in Table IV. Ethanol alone
was found to be an effective penetration enhancer. Addi-
tion of DMS (decylmethylsulfoxide) to ethanol generally
increased the penetration rate (and cumulative amount,
i.e. amount penetrated over 24 hour period) of the 6-, 10
and 14-mers through human skin relative to that from etha
nol alone. Addition of water to the ethanol/DMS vehicle
increased the flux (and cumulative amount) still further
for the 6-mer; however, flux (and cumulative amount) for
the 10-mer and 14-mer was reduced.
Addition of DMS to propylene glycol increased the
flux (and cumulative amount) of the 6-mer through human
skin; however, the flux (and cumulative amount) was still
an order of magnitude lower compared with the ethanol/DMS
vehicle. Removing the stratum corneum from human skin led
to a large increase in flux (and cumulative amount) of the
6-mer, although the increase was not as dramatic as that
observed with hairless mouse skin.
In comparing the cumulative amount data from hairless
mouse skin with human skin for the 10-mer and the 14-mer,
the cumulative amount was greater in hairless mouse skin,
but was generally within an order of magnitude.
Overall, an inverse relationship of permeation rate
with molecular weight was observed (i.e., the higher the
molecular weight, the lower the cumulative amount).
Generally, the highest retention of oligomer both in
the viable tissues (dermal layer) and stratum corneum was
observed from the ethanol/water/DMS vehicle. The ratio of
retained oligomer in stratum corneum to dermis was about
10:30 (Note: Since there was considerably more viable
tissue than stratum corneum, the majority of oligomer
retained was in the dermis). Tape stripping (to remove
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34
stratum corneum) of skin did not lead to a larger amount
of 6-mer being retained in dermis as compared to retention
in dermis using whole skin.
Table V reports retention of 14-mer.in dermis versus
stratum corneum after treatment with 14-mer in various
vehicle/enhancer combinations. Stratum corneum and dermis
were separated before analysis by microwave treatment as
described by Kumar et al. (Pharm. Res. 6:740-741 (1989)).
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TABLE II
Permeabilitv of Oliaomers in Hairless Mouse (HM)
and Human Skin (HS)
Cumulative
Amount
at 24 h
Skin Oligomer Donor Vehicles (~.g/cm2)
5 HM 14-mer H20 0.75
EtOH 0.28
EtOH/DMS (95:5) 5.5
EtOH/DMS (97.5:2.5) 4.4
EtOH/OA (95:5)b 0.30
EtOH/OA (97.5:2.5) 0.24
EtAc~ 1. 2
EtAc/DMS (95:5) 1.1
EtAc4/OA (95:5) 0.60
EtOHd 18 7
EtOH/DMS (95:5)d 186
EtOH/H20/DMS ( 8 0 :15 : 5 ) 3 . 5
EtOH/Hz0/DMS (80:15:5) 2.7
EtOH/H20/DMS (80:15:5)f 2.1
EtOH/H20/DMS (80:15:05)9 0.23
HM 14-mer-IA HBO 0
EtOH 0
EtOH/DMS (95:5) 0.61
HS 14-mer EtOH 0.26
EtOH/DMS (95:5) 0.24
EtOH/OA (95:5) 0.30
EtOH/Hz0/DMS (80:15:5)h 0.23
aUnless stated in the table footnotes, all the donor
vehicles were saturated with oligomer
10 bOA = oleic acid
~EtAc = ethylacetate
dThese skins were free of stratum corne um, which was
removed by tape stripping.
e14-mer concentration in the vehicle was 1. 0 mg/mL (below
15 saturation)
f14-mer concentration in the vehicle was 0. 5 mg/mL (below
saturation)
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36
g14-mer concentration in the vehicle was 0.05 mg/mL (below
saturation)
h14-mer concentration in the vehicle was 1.0 mg/mL (below
saturation)
TABLE III
A. Amosnts of 14-mer Recovered from Skin Samples
Skin Donor Vehicle ~.g/gma ~,Mb
IBM EtOH/DMS ( 95 : 5 ) 3 0 . 2 7 .1
EtOH/DMS (95:5)° 112 26.3
EtOH/H20/DMS (80:15:5) 77 17.9
EtOH/Hz0/DMS (80:15:5)d 18.4 4.3
HS EtOH/DMS ( 95 : 5 ) 67 .1 15 . 7
0
aTotal ,ug of 14-mer recovered from the homogenized skin
sample corrected for loss of 14-mer during homogenization
and centrifugation (see Example 6); the gm is the wet
weight of the skin as measured prior to homogenization
b ~,M concentration of 14-mer in the skin were obtained from
the molecular weight of the 14-mer and the assumed density
of 1.0 for the skin sample (i.e., 1.0 gm of skin is equal
to 1.0 cc of skin)
°The HM skin used in this experiment was stripped to remove
the stratum corneum
dThe concentration of 14-mer in this vehicle was 0.5 mg/mL
compared to all the other experimental vehicles, which
were saturated with excess solid 14-mer
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37
B. Retention of 14-mer in Whole Skin and Viable Tissuesa
Skin Section Donor Vehicle ~,g/gmb ~,M~
HM Whole EtOH/H20/DMS (80:15:5) 63.2 14.8
Viabled EtOH/H20/DMS (80:15:5) 35.2 8.2
HS Whole EtOH/H20./DMS (80:15:5) 105.9 24.7
Viabled EtOH/H20/DMS (80:15:5) 7.0 1.6
$The weighed skin samples (hydrated) were either homoge-
nized whole or the stratum corneum was removed, and the
14-mer content of the remaining tissue (viable epidermis
and dermis) was determined. In each case,
n = 2.
bTotal ~.g of 14-mer recovered from the homogenized skin
sample corrected for loss of 14-mer durix~ homogenization
and centrifugation (see Example 6); the gm is the wet
weight of the skin as measured prior to homogenization
°~,M concentration of 14-mer in the skin were obtained from
the molecular weight of the 14-mer and the assumed density
of 1.0 for the skin sample (i.e., 1.0 gm of skin is equal
to 1.0 cc of skin)
dThe viable tissue is the tissue after the stratum corneum
has been removed by microwave treatment (Kumar, et al.,
Pharm. Res. 6:740-741 (1989)). It is a combination of the
viable epidermis and the dermis.
WO 94118835 PCT/US94/01748
38
TALLE IV
Penetration of Oligomers Through Skin
A. Human Skin
Vehicle/ Ratio of 24Hr Cumulative Values:
~nhancer Components
nmoles/cm2
Mean and
SD
6 Mer 10 Mer 14 Mer
EtOH/H20/DMS 13.8(5.7) 0.94(1.3) 0.18(0.17)
(80:15:5)
2.2 (2.0)
EtOH/DMS (95:5) 8.2(5.4) 6.0(4.2) 0.83(1.0)
EtOH (100:0) 3.4(2.8) 4.0(6.7) 0.37(0.48)
PG (100:0) 0.21(0.37) No Data No Data
PG/DMS (95:5) 0.57(0.50) No Data No Data
EtOH/DMS (95:5) 34.0(4.8) No Data No Data
(Tape Stripped)
The second value for 6Mer came from a time tudy using
the s
a different otherwise the data for the first
skin donor,
three enhancers m the same donor.
came fro
The data for
the last three
enhancers came
from the same
experiment but from a fferent donor.
di
B. Hairless Mouse
Vehicle/ Ratio of 24Hr Cumulative Values:
Enchancer Components nmoles/cm2 and SD
Mean
6 Mer 0 Mer 4 Mer
EtOH/Hz0/DMS (80:15:5) No Data 2.08(0.82) 0.82
EtOH/DMS (95:5) No Data 1.94(0.22) 1.28
EtOH (100%) No Data 0.35(0.10) 0.065
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WO 94/18835 PCT/US94/01748
39
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