CA2110040A1 - Compositions and delivery systems for transdermal administration of neutral oligomers - Google Patents

Abstract

Neutral Oligomers may be delivered transdermally and across mucous membranes for therapeutic purposes, such as to block expression of a specific target nucleic acid sequence. These neutral Oligomers are useful in treating a variety of pathological conditions, including those of the skin and mucous membranes, as well as conditions effecting other tissues.

Description

WO9Z/21353 2 1 ~ ~ O ~ O PCT/USg2/0437~

DESCRIPTIO~

~ompositions and DeliverY Systems for Transdermal Administration of Neutral Oliaomers Back~round of the Invention~
The use of antisense oligodeoxyribonucleotides for the treatmen~ of conditions caused by ~iruses such as Herpes viruses, has~been proposed. In order for Oligomers to be useful as therapeutic agents, they must be able to gain access ~o their sites of action. Since most of these sites of action~are~within the body of the animal to be treated,~is necessary~that the Oligom~rs be ~ble to enter the body tissues. Moreover, such Oligomers may need to be able to ~cross~cell~ meioranes and to enter indi~idual cells~. It~is possible that aocess to the body be achieved by~ini;ection,~but this is not a preferred route of access, especially in~cases~here~repeated administration or self-inistration~is;~involved. ~ore preferred routes are by 15~ eithe~ skin or mùcous~me~brane absorption. Mucous mem-brane~absorption can;~o~cur within~the nasal passages, oral cavity,~lung,;~upper~ or~ lower gastrointestinal tract or ~aginal~tract. Topical skin delivery is also a preferred route~o~; administration.~ Local delivery of these thera-

2~0~;~ peutic~ agents~has~the~additional benefit o~ potentially concentrating ~these ~agents ~near ~he~site of act~on for e~hsnced effecti~ene6s of the applied dose~ This latter mode~ of application can also great1y reduce the cost of treatment.
25~ What is then~ n~eded are O~igomer compositions that can~maxima1ly~penetrate~skin and mucous membranes. Typi-ca1 drugs administered~transdermally are sma~l molecules having molecular;weights less than 400 dalt~ns. Oligomers which are macr ~lecules having molecular ~eights far in 30~ excess o~ 400 daltons would not be considered likely can-didates for transdermal deliv~ry 3 E~-9~
~: , :
: ~ : ;;
:: :: :::; :: : : ::
SUBSTITUTE SHEET

3 PCT/US92/04370 2 ~1 ~ 0 d~, 2 Summary~of the Invention The present invention is directed to a method of delivery of an antisense Oliqomer for therapeutic purposes by administration across skin or mucous membranes. Said Oligomers may have therapeutic activity by preventing or interfering with expression of a specific nucleic acid target sequence or the protein product of that sequence.
In one aspect, this method comprises the application to skin or mucous membrane of a formulation which comprises a neutral Oligomer which is complementary to and which can .
` bind to or interact with said nucleic acid target sequence. These~Ol;igomers may hybridize to a single-stranded target nucleic acid seauence or alternatively may interact with a double-stranded target sequence to form a triple-stranded complex. ~(See, e.a., USSN 368,027 "Forma-~ ~ .
tion~of Triple Helix;Complexes of Double Stranded DNA
Using ~Nualeoside~;Oligomers," the disclosure of which is incorpora~ted herein by reference).
Among other factors, the present invention is based 20 :~ ~upon~ ~our surprising~ finding that formulations comprising these neutral Oligo~mers which have no ionic internucleo-sidyl~linkages~are~able to penetrate skin or mucous mem-brane~at a substantia~lly~higher rate than Oligomers which have~ ionic inte~nuc~leosidyl linkages. In fact, we have 2;5~ fo n=d~that Oligo~ers~having only one ionic internucleo~
sidyl~linkage and~the~remainder of the internucleosidyl linkages neutra~l internucleosidyl linkages penetrate skin at a substantially lower rate than the neutral Oligomers of the present invention.
The delivery~ of these neutral Oligomers across skin or mucous membranes~can~be used in the direct or systemic treatment of various~conditions by preventing expression of a nucleic acid~target sequence. In particular, deliv-` ery of these neutral Oligomers across mucous membranes of the nose, the lung or~gastrointestinal tract is suitable for systemic treatment, as well as direct treatment of SlJBSTlTUTE SHEET

~92/21353 PCT/US~2/04370 2l~n4~

that tissue. These neutral oligomers may be used in direct treatmen~ of skin or vaginal tissue.
The direct deIivery of these neutral Oligomers across skin or mucous membranes may be used to pre~ent expression of specific nucleic acid sequences which may comprise either foreign nuclaic acids from, for example, a virus, or an endogenous nucleic a~id se~uence. Thus, according to the present invention, topical administration of these neutral Oligomers may be used against viral, fungal and bacterial infections of the skin and mucous membranes such ` as genital warts caused by the human papilloma virus and : infections caused by Herpes viruses. Alternatively, topi-: cal administration of: neutral Oligomers may be used to block mediators of inflammation. In another aspect, transdermal delivery of these neutral Olig~mers may be `~ used to treat conditions in which improper immune or ~:~ ; inflammatory reBponses have been implicated such as ~; :psoriasis, atopic dérmatitis, eczema, rheu~atoid arth-ritis, allergic rhinitis and the like.
20~ In an additional aspect,: tran~dermal delivery of ; these neutral oligomers may be used to treat certain cancers of:the skin and mucous me~branes such as melanoma, myoosis fungvides;, and squamous cell carcinoma (~ncluding of the cervix) by~blocking of the expression of certain 25 :cellular *actorB which are involved in their proliferation.
The delive~y of these neutral Oligomers across mucous membranes may be~used in systsmic treatment of a number of conditions. For example, these neutral Oligomers may be .
administered by inhalation to nasal and lung mucous mem-:branes. The systemic delivery of these Oligomers may be used ~o prevent:expression of specified nucleic acid tar-get sequences which may comprise either foreign nucleic acid from, for exa~ple a virus, or an endogenous nucleic acid sequence. One proposed target is to regulate over-: expression of renin in the kidney. Since these Olîgomers are reported to be cleared by $he body through the kidney SUBSllTUTE SHEET

4~

(see Colvin, O.M., et al., Drua Metabolism and Disposi-tion, Vol. 18, pages;815-818 (1990)), administration of Oligomers across mucous membranes should result in a therapeutically effective concentration of Oligomer in the kidney.
In one aspect, the present invention is directed to compositions and delivery systems for the direct or sys-temic administration of neutral Oligomers across skin or mucous membranes. In one preferred aspect, the composi-tions of the present invention comprise a neutral Oligomerin a vehicle which comprises a short chain aliphatic alco-hol, preferably~ethanol. Optionally, the vehicle com-prises~ a~flux enhancer which increases transdermal ~or transmembrane) flux of said oligomer. Suitable enhancers include compounds~which ~disrupt the skin or mucous mem-brane barrier to the Oligomer and/or change the partition-ing~behavior of the~Oligomer to the skin or mucous mem-brane.`Suitable~en~àncers include decylmethylsufoxide, and oyclic~ketones, lactones, anhydrides or esters such as 20 ~those described in~PCT application PCT/US86/02583 (Publi-cation~No`. W0 87/03473), and other enhancers known to those ~killed in the~art.
According ~to~another aspect of the present invention, co~positions~for~transdermal delivery may optionally 25~include a retention~enhancer which increaæes retention of ~; ; the~Oligomer in the~skin, particulàrly in the viable tis-sue (rather~than the~;stratum corneum). Suitable retention enhancers include~cyclio ketones such as those described in PCT/U586/025~83 (Publication No. W087/03473).

Definitions As used herein~,~the following terms have the follow-ing meanings, unless expressIy stated to the contrary:
The term "nucleoside" includes a nucleosidyl unit and is used interchangeably therewith, and refers to a subunit of a nucleic aoid whioh comprises a 5 carbon sugar and a nitrogen-containing base. In RNA the 5 carbon sugar is SUBSTITVTE SHEET

W~92/21353 PCTJUS92/04370 211~ ~

ribose; in DN~, it is a 2'-deoxyribose. The term also includes analogs of such subunits.
A ~'non-nucleoside monomeric unit" refers to a mono-meric unit which does not significantly participate in hybridization of an Oligomer to a target se~uence. Such monomeric units must not, for example, participate in any significant hyhdrogen bonding with a nucleoside, and would exclude monomeric units having as a component, one of the

5 nucleotide bases or analogs thereof.
A "nucleoside/non-nucleoside polymer" re~ers to a polymer comprised of nucleoside and non-~ucleoside mono-meric units.
The term "oIigonucleoside" or "Oligumer" refers to a chain of nucleosides which are linked by internucleoside linkages.~which is generally from about 6 to about 100 :~ nucleosides in length, but which may be greater than about 100 nucleosides in length. They are usually s~nthesized : : from nucleoside ~monomers, but may also b~ obtained by ~nzymatic means. Thus, the term Oligom~r refers to a :~ 20 chain of oligonucleosides which have internucleosidyl linka~es linking :the nucleoside mono~ers and thus, includes oligonucleotîdes, nonionic oligonucleoside alkyl ::~ and aryl-phosphonate analogs, alkyl- and ~ryl-pho~phono-~ ~ thioates phosphoro~hioate analogs of o}igonucleotîdes, : 25 phosphoramidate analogs of olîgonucleotîdes, neutral phosphate ester oligonucleosîde analogs, such as phospho-triesters and other oligonucleoside analogs and modîfîed olîgonu~leosides, and also includes nucleoside/non-nucleoside polymers. The te~m also includes nucleoside/
non-nucleoside polymers where~n one or more of the phos-phorous group lînkages between monomeric units has been replaced by a non-phosphorous lînkage such as a, morpho--lino lînkage, a formacetal lînkage, a sulfamate lînkage or a carbamate lînkage.
The term "alkyl- or aryl-phosphonate ~ligomer" refers to Oligomers having at least one alkyl- or aryl-phospho-nate internucleosidyl linkage.

SUBStlTUTE SHEET

WO92/213~3 P~T/US92/~370 ~ 6 The term "methylphosphonate Oligomer" (or "MP-Oligo-mer") refers to Oligomers having at least one methylphos-phonate internucleosidyl linkage. ..
The term "neutral Oligomer" refers to Oligomers which 5 have nonionic internucleosidyl linkages between nucleoside ~-monomers (i.e. linkages having no net positive or negative ~ ionic charge) and include, for example, Oligomers having internucleosidyl li ~ ages such as alkyl- or aryl-phospho-nate ~inkages, alkyl- or aryl-phosphonothioates, neutral phosphate ester linkages such as phosphotriester linkages, especially neutral ethyItriester linkages; and non-phos~
phorus-containing:internucleosidyl linkages, such as sul-. famate, morpholino, formacetal and carbamate linkages.Optionally, a neutral Oligomer may comprise a conjugate between a oligonucleoside or nucleoside/non-nucleoside ~: polymer and a second molecule which comprises a conjuga-:~ tion partner. Such~ conjugation partners may comprise - interca~1~ators, alkylating agents, binding substances for cell surface receptors, ~lipophilic agents, photo-cross-20~ lin~ing agents such as psoralen, and the like. Such con-jugation partners~may~ further enhance the uptake of the Oligomer, modify:the interaction of the Oligo~er with the tar~et sequence;,~or:;alter the~phar~acokinetic distribution Qf the:Oligonucleosi~e.~: The essential reguirement is that ;25 the~oligonucleoside or~nucleQside/non-nu~leoside polymer that~th¢ conjugate~co~pri~ses be neutralO
The term "neutral alkyl- or aryl- phosphonate oligo-mer" refers to neutra1 oligomers having neutral in~er-nuc}eosidyl linkageB which comprise at least one alkyl- or 30 :aryl- phosphonate linkage.
: ~ ~
~ The term Uneutral methylphosphonate oligomer'~ refers :: to neutral olig~mers ~having internucleosidyl linkages which comprise at:least one methylphosphonate linkage.
: In some of the various 0ligomer sequenc~s listed herein "p" in, e.g., as in ApA represents a phosphate diester linkag~,:and "~"~in, e.g., as in C~G represents a ::
methylphosphate linkage.

SVBSTITVTE SHEET

WOg2/21353 PCT/US92/~370 21~i~0~

The term "tandem oligonucleotide" or "tandem Oligo-mer" 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 complemen-tary 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 oli-gomérs, such à~s~by decreasing the secondary structure of the target nuoleio~aoid sequence. In addition, one of a pair of tandem Oligomers may improve the hybrid stability of the second tandèm Oligomer by promoting a helical struc-ture at either the~5'~-~or '-end of said second Oligomer ~and vicé-versa.
~; 15 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 ~nd~tertiary alcohols, glycols and polyols.
20~ The term~nflux`~enhancer~ refers to a substance which is~ uséd ~to increase transdermal flux of a compound. A
flux~enhancer~is~typioally applied to skin or mucous mem-brane in combi ~ on~with the compound to increase trans-"
dermàl flux of ;the~compound. Enhancers are believed to 25~function ~by~disrupting the skin or mucous membrane barrier or~by changing-~the~ partitioning beha~ior of t~e drug in the-skin or mucous~membrane.

Brief Description of the Drawings FIG. 1 depicts~a~plot of cumulative amount of Oligo-mer penetrating~hairless mouse skin versus time ~or sev-eral concentrations ~formulations of 14-mer in vehicle (EtOH/H20/DMS,~80:15:~5).
FIG. 2 depicts~a plot of cumulative amount of a 14-mer having on~ internal anionic internu~leosidyl link-age (nl4-mer-lA")~penetrating hairless mouse skin versus time (vehicle was EtOH (DMSl95-5).

SVBSTlTUTE SHEET

WO9~/2~353 PCT/US92/0437 FIG~ 3 depicts a plot of percentage of applied dose of Oligomer absorbed versus time for formulations having varying concentrations of 14-mer.
FIG~ 4 depicts a plot of cumulative amounts of 14-mer penetrating human cadaver skin versus times for several formulations of 14-mer. :~

Detailed Descri~tion of the Invention :: Preferred Neutral Oliqomer Formulations ~::
Preferred neutral Oligomers include neutral alkyl~
and aryl- phosphonate Oligomer~ and neutral Oligomers com-prising triester or phosphoramidate internucleosidyl link-ages. Especially preferred are neutral methylphosphonate Oligomers. In view of their demonstrated ability to pene-trate skin, including tape stripped skin (which has had :~: 15 the stratum corneum removed and which has been reported as ~ a model f or mucous membrane), particularly preferred are : ~ neutral methylphosphonate Oligomers ha~ing only methyl-phosphonate internucleosidyl linkages.
Synthetic met~ods~ for pxeparing methylphosphonate 2~0: Ol~igomers are described in Example 1 herein and also in Lee:;B.L., et al.,::BiochemistrY 27:3197-3203 (1988), and Miller, P.S., et 31~, BiochemistrY 25:5092-5097 (1986), : : the disclosuFes of which are incorporated herein by reference.
~: 25 : : According to another aspect of th~ present invention, preferred are Oligomers:~which comprise one or more tri-~: eæ~er internucleoside linkages, especially neutral ethyl-triester internucleoside linkages. It is believed that once such Oligomers have entered the cell, tha ester grsup 30 i8 cleaved to give:a nega~ively charged diester linkage which is better retained inside the cell. According to :~
: one preferred embodiment, such Oligo~ers may incorporate either one or more methylphosphonate internucleoside link-ages or one or more nucleosides having a 2'-0-methyl ribo-syl moiety. The pre~ence of me~hylphosphonate inter-nucleoside linkages at the ends of the Oligomer gives SUBSrlTVTE SHEET
.:

WO92/21353 PCT/US92~04370 21~00l~
g exonuclease resistance and the inCorporatiQn of methyl-phosphonate internucleoside linkages or nucleosides which comprise a 2'-O-methyl ribosyl moiety in the middle of the Oligomer increases resistance of the Oligomer to endonucleases.
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 to employ~shorter tandem neutral Oligomers to . ~
maximize solubility~and~penetration through the skin or mucous membranes~while~competing for the development of a secondary structure of the target nucleic acid, such as a 15~mRNA.~ Alternatively,~it~may be advantageous to use more ; than one~neutral O~1igomer, each Oligomer complementary to a~distinct target~;~sequence~which may be part of the same gene or~a~different gene.
Where~the~neutra}~Oligomers comprise alkyl- or aryl-Z~0~ ~ sphonate Oligomers~ it ay be advantageous to incor-porate~nucleoside~monomeric units having modified ribosyl mo~ieties.~The: use~of~nucleoside units having 2'-O-alkyl-and~ in'~pa~rticular,~ 2-O-methyl-ribosyl moieties in these neutral Oligomers~may~a vantageously improve hybridization 25~ of~the~01igomer to~its~complem~entary target sequence.
According~ to;~one~preferred aspect, these neutral Oligomers~may~ao pr~ise~a conjugate between a polynucleo-sidé~or nucleoside/non-nucleoside polymer and a conjuga-tion partner. Suitable conjugation partners include intercalating agents, alkylating agents, binding sub-stances~for cell~surfaoe receptors, lipophilic agents, photo-crosslinking~agents such as psoralen, hydrolytic or ;nucleolytic agents,; pro-chelates or DNA modifying agents.
These conjugation partners may inclu~e groups such as 35~ photo-crosslinking groups like psoralen, cross-linking agènts, ihtercalating agents such as acridine, or groups capable of cleaving a targeted portion of a nucleic acid SUBSrlTUTE: SHEET `:

such as hydrolytic or nucleolytic agents like o-phen- :
anthrolinecopper or EDTA-iron may be incorporated in the Oligomers. I ~
These Oligomers may be labelled by any of several well known methods. Useful labels include radioisotopas.
ISotopic labels include 3H, 355, 3Zp 125I Cobalt a d 14C
Most methods of isotopic labelling involve the use of enzymes and include the known methods of nick translation, and labelling, second strand synthesis, and re~erse transcription.
Conjugation partners may also be introduced into the Oligomer by the incorporation of modified nucleosides or nucleoside analogs through the use of enzymes or by chem-ical modification of the Oligomar, for example, by the use of nonnucleotide linker groups.
When used to prevent function or expression of a single or double s.randed nucleic acid sequene, these :: Oligomers may be advantageously derivati2ed or modified to i~corporated a nucleic acid modifying group which may be 20 :caused to react with said nucleic acid and irrev~rsibly modify its structure, thereby rendering it non-functisnal.
Commonly assigned USSN 565,299, the disclosure of which is incorporated ;herein by reference, discloses psoralen-derivatized~Oligomers.
: 25 A uide variety of~DNA modifying groups may be us~d as :: conjugation partners to derivatize these Oligomers. DNA
~odifying groups include groups which, after the deriva-tlzed Oligomer forms a complex with a singl~ stranded or double stranded DNA s~gment, may be caused to form a covalent linkage, cross-link, alkylate; cleave, degrade, or othexwise inactivate or destroy the DNA segment or a target sequence portion thereof, and ther~by irreversibly inhibit the function and/or expression o~ that DNA
segment~ ~
The location of the DNA modifying groups in the Ol~- :
gomer may be varied and may depend on the particular DNA
modifying group employed and the targeted double stranded SUBSTITUTE SHEET

WO92/213~3 PCT/US92/04370 . . .
2 ~ 4 ~3 DNA segment. Accordingly, the DNA modifying group may be - positioned at the end of the Oligomer or intermediate the ends. A plurality of DNA modifying groups may be included.
In one preferred aspect, the DNA modifying group is photoreactable (e~.g., activated by a particular wave-length, or range~of waveléngths of light), so as to cause reaction and, thus, cross-linking between the Oligomer and the double stranded~DNA.
~;10 Exemplary of DNA~modifying groups which may cause cross-linking are~the psoralens, such as an aminomethyl-;trimethyl psoralen~group~(AMT). The AMT is advantageously ` photoreactable~ and~thus~must be activated by exposure to particular wavelength light before cross-linking is effec-15 ~tuated. ~Other cross-linking groups which may or may not be~photoreactable~ may be~ used to derivatize these Oligomèrs.
Alternatively,~ the~;~DNA~modifying groups may comprise ~n ~aIkylating agent group which is covalently bonded to 20~ the~DNA~segment~ to~render it inactive~. Suitable alkyl-ati~ng~ agent~ groups~are ~known in the chemical art sand clude~groups~deriYed~from~alkyl halides, haloacetamides, and the like.
DNA modifying~groups~which may be caused to cleave 25- the~DNA~segment~include~ transition metal chelating com-plexes~such~as ethylene diamine tetraacetate (2DTA) or a neutral derivative~thereof. Other groups which may be used to effect;cleaving ~include phenanthroline, porphyrin and the like. When EDTA is used, iron may be advantage-3~ ously tethered to the Oligomer to help generate the cleav-ing radicals. ~Although~EDTA is a preferred DNA cleaving group, other nitrogen containing materia}s, such as azo compounds or nitrenes or~other transition metal chelating ; -complexes may be~used ~-i 35 ~ Suitable formulations comprise about 0.0001% to about ~ .
~ 2% by weight of neutral Oligomer. '~
- .

, ;: SUBSTITUTE SHEET -~

W092/21353 PCT/US92/~370 In one preferred aspect, these neutral Oligomer for-mulations comprises about 2% to about lOO~ of a short chain aliphatic alcohol. Suitable alcohols include etha-nol, isopropyl alcohol, propylene glycol and glycerol. In certain studies, formulations of neutral Oligomers com-prising 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 disclosed in PCT
Application No. PCT/US86f02583 (Publication Number W087/
1~5 03473). Spme of these~flux enhancers also increase reten-tion of the OIigomer~a~nd, thus, act to increase the con-centration~of Oligomer~within the skin itself.
Thus~,~ for Oligomer~formulations for direct ~local) ; trea~tment, such~as~ topical application to skin, it is 20~ preferred to use a~flux enhancer which not only maximizes transdermal flux,~but increases Oligomer retention in the skin. Certain cyclic~ketone and lactone enhancers have beèn reported~ to~increase local retention as well and, thus,~comprise~a~prèferred class of enhancers for topical 25~ administration~of~01igomer formulations.
In- Oligomer formu~ations for systemic treatment, it is prefèrable to use a f1ux enhancer which maximizes flux with a~minimal~increase~of locaI retention of Oligomer.
; ~ ~
Preferred Taraets and Taraet Seouences 3~0 ~ According~to~one~;aspect, the present invention pro-vi~es methods of preventing or interfering with expression of a specific target~nucleic acid sequence by the trans-dermal administrati:on of a neutral Oligomer which is com-plementary to and~ which can bind to or interact with a specific target nùcIeic se~uence. These neutral Oligomers can penetrate skin or mucous membrane tissue. These neu-:: : :
SUBSrITUTE SHEET

W092/21353 PCT/US92/~370 2110~

tral Oligomers may be applied to skin or mucous membraneto treat a variety of conditions either lacally in the skin or mucous membrane ~such as lung, gastrointestinal tract or vaginal tissue) or systemically by blocking or interfering with the expression of a specific target nucleic acid sequence. The sequence to be blocked may ~comprise a "foreign" target nucleic acid se~uence (i.e., one from an outside source such as a pathogen) or one of endogenous origin~
Thus, in one aspect these neutral Oligomers may be used to directly treat viral, bacterial, fungal and other infections of the~skin or mucous membranes caused by patho-gens. Such conditions include those caused by a Herpes virus, a human papilloma virus ~such as genital warts), or specieæ~of Pseudomonas,~Staphylococcus or Helicobacter.
These neutral Oligomers may also be used to block mediators of inflammation. These mediators include cyto-;kin , growth~factors, cell adhesion molecules or their igands and receptors~thereof as well as key enzymes in 20~ pathways~;1eading~to~;infla~ation. These blocking actions include preventing the expression of cytokines (such as IL-l)~ ;growth~ factors (such as TGF-~ and ~EGF), or cell adhes~ion molecules ~(such as ELAM and ICAM); or the recep-tors~for c~ okines~ such as IL-l), growth factors, or cell 2S~ ~adhesion molec les.~ Key enzymes whose expression may be blocked~includ~e~protein kinase ~ and phospholipase A
or C. Neutral~Oligomers which block the expression of phospholipase~ (P~ or the~receptor for IL-l may be ; ~ useful to treat~inflammation of joints due to rheumatoid arthritis.
These neutral~O1igomers may be~ used therapeutically to treat immune~or~inflammatory-response related diseases or conditions.~ Neutral Oliqomers which block the expres-sion of IL-l, Transforming Growth Factor ~ ($GF~), amphi-35~ regulin, or IL-6 may be useful in treating psoriasis.
These neutral Oligomers may also be useful in the treat-ment of atopic dermatitis. Furthermore, these Oligomers SUBSTITVTE SHEET
, W092/213~3 PCT/US92/~370 9*~

may be used to block overexpression of IgE, said to be mediated by mast cells releasing histamine, and to be useful in the treatment;of atopic dermatitis, ecz~ma and asthma. Oligomers; which block IL-4 expression may be useful in the treatment of allergic rhinitis.
Additionally, these Oligomers may be used in therapy of certain skin-related cancers such as melanoma and squamous cell rarc~inoma, by blocking the expression of certain factors~which~promote cell growth and/or adhesion and are believed to~be involved in metastasis. These fac-tors include Epidermal Growth Factor (EGF), and certain cellular adhesion factors.
These neutral`Oligomers may be used to prevent expres-sion of mediators of cell adhesion or their receptors so 15~ there will be no focus for ce}l recruitment or cell adhe-sion at a~point of~in~ury. These mediators include ELAM, ICAM, and thrombospondin. These Oligomers may also be used to block expression 0f retinoic acid receptors. ~-These Oligomers~may~be usefu} to treat septic shock 20~;by~ ~;blocking the~ expression of the TL-l receptor antagonist.
In~ general~ preferred are target nucleic acid s ~ ences~;which~comprise ~a region of the gene to be blocXed~that is~required~for;its expression. These target 2~5~sequencés may comprise a portion of the gene, or a portion of~a~mRNA transcript~;thereof which is "available", i.e., is~in a~state where~the complementary neutral Oligomer is a~le~to hybridize~to~the ~target ~sequence. Thus, these target sequences are~ preferably single stranded and rela-30 tively free of secondary structure and bound protein. -~
Suitable target sequences include se~uences which are at or proximate to~a~5'-terminal translational start, a 3'-terminal polyadenylation signal, a mRNA cap site or a splice junction.
According to one preferred aspect of the present invention, expression~ of renin, especially in the juxta-glomerular apparatus (JGA), is blocked by administration ~:
: ~ :
: SUE~STITUTE SHEET
:~ ~

WOg2/213~3 PCT/US9~/~370 2 1 l 1) ~

of these neutral Oligomers to skin or mucous membranes.
Thus, a preferred target sequence for the neutral Oligo-mers of the present invention comprises a sequence within the renin gene or its~mRNA transcript. Renin is an enzyme which catalyzes the cleavage of angiotensinogen to produce angiotensin I. Angiotensin I is further cleaved by angio-tensin converting enzyme to angiotensin II, which is a potent extracellular messenger involved in vasoconstric-tion. ~ngiotension II also stimulates the adrenal cortex :
to secrete aldosterone. Neutral Oligomers which are com-plementary to targets in the renin gene or mRNA tran-scripts thereof~may;be~preferably administered by inhala-tion to the~mucous membrane of the lung, thereby passing into the bloodstream, and reaching the kidney where renin is produced. Heretofore~,~it has been difficult to inhibit renin~without~side~;effects.
These~ neutral~;Oligomers may also be used to treat chronic myelogenous~leùkemia by preventing expression of a~"~ (See~the~¢ommonly assigned, co-pending pa~ent 20~ application, USSN 565,299 nPsoralen-Conjugated Methylphos-phonate~oligonucleotides as;Therapeutic Agents for Chronic Myel ~ ous Léukemia",~ the disclosure cf which is incor-porated~herein~by~reference.~

Permeability of Oliaomer Formulations 25~ The ability~of~a~neutral methylphosphonate Oligomer having~14 nucleosides~and having only methylphosphonate internucleosidyl~linkaqes (referred to as "14~mer" herein) to penetrate hairless mouse skin was measured using sev~
eral concentrat~ions~of }4-mer and using several vehicle 3~0 ~for~ulations.~ (See Table I~) In comparison~, the ability of a second Oligomer (referred to as~ l4-mer-IA" herein~ which was a methyl-phosphonate Oligomer~which contained an internal anionic internucleosidyl~ l}nkage ~a phosphodiester linkage) to penetrate hairless mouse skin was measured using several vehicle formulations. ~See Table I.~ There was no mea-:: : :
~: ~ 8 UBSrITUTE SHEET
:

WO92/21353 , ~ ~ PCT/USg2/04370 surable amount of 14-mer-IA detected in the receptor solu-tion over the 24 hour test period with either the water or ethanol vehicle formulations, in contrast to the 14-mer in the same vehicle formulations. For the ethanol/decylmeth-ylsulfoxide (95:5) vehicle formulation, the permeabilityof hairless mouse skin for 14-mer-IA was about 10-times less than for 14-mer; the total amount of 14-mer which penetrated after 24 hours was about 5.5 ~g/cm2 in compar-ison to about Q.6 ;~g/cm2 of 14-mer-IA. Therefore, the introduction of just one~ionic (negative) internucleosidyl ; ~ ~ lin~age into the neutral Oligomer was demonstrated to sig-nificantly decrease the ability of the Oligomer to pene-trate skin when ;compared to the neutral 01igomer. See also Figures 1 and 2 which depict cumulative amount of 14-mer and 14-mer-IA,~respectively, which penetrates hair-; less mouse~skin over~time.
The stratum corneum~is the primary barrier to absorp-tion;through the~skin for~most drugs, including macromole-uies.~In order~ to~determine the degree to which the20 ~stratum oorne _ ~limits~;the absorption of methylphosphonate Oligomers,~àn experiment was~performed wherein the stratum corneum~ was removed ~from the~skin by tape stripping.
Also,~tape stripped s~in~has been reported to have simllar permeability~characteristics to D cou8 membrane an~, thus, 25~ has~been proposed;~as~a~model system for mucous membrane.
Penetration o~ the~14-mer tbrough normal (unstrippeq) hairless mouse ~skin and s~in following removal of the stratum corneum~was examined and the rates compared. (See Table I.) Table I gives~the~cumulative amount of neutral methyl-phosphonate 14-mer~penetrating stripped skins from two ; vehicles: Et ~tdecylmethyl sulfoxide (DNS) (95:5, vtv) and ethanol (EtOH)~.~ The amount of the neutral methyl-phosphonate 14~-mer~penetrating skin was increased drama-tically when the stratum corneum was removed by tape s~ripping. Removal of the strat~m corneum by tape strip-ping resulted in absorption of the entire dose of 14-mer :
SUBSl'ITUTE SHEET
:
.. .. .. . . . . . ..

WO92~21353 PCT/US92/~370 2 ~ 4~ ~?

absorbed through the stripped skin from both vehicles tested by 24 hours.
An experiment was performed to investigate the effect of changing the concentration of the neutral methylphos-phonate 14-mer in the vehicle. Because the EtOH-based vehicles, even with small amounts of a cosolvent such as DMS added, were poor solvents for the neutral methylphos-phonate 14-mer, water was added to the vehicle. The vehi-cle tested was~EtOH~/H20/DMS (80:15:05) in which neutral methylphosphonate 14-mer was added to saturation (3.8 ~` mg/mL), or in concentrations of 0.5 mg/mL, or 0.Q5 mg/mL.
The skin used in~these experiments was full-thickness hair~less mouse.~The cumulative amount of 14-~er pene-trating the skin over 24 hours is shown in Figure 1~ The concentration~of neutral~methylphosphonate 14-mer in the vehiclè ~was related~to~the amount of drug penetrating a unit area~of skln.~The~drivinq force is related to the concentrat~ion of~the~neutral methylphosphonate 14-mer in e~vehicle. ;
20~ Table~ depicts~ the penetration of 14-mer through hairle~s~mouse~skin~using several different vehicles and at~sevèral~different~concentrations of 14-mer~
T~e cumulative amount~of 14-mer which absorbed from a ~test ~ehic~le~over;~time is depicted in Figure 1. The 25 ~percent of the applied dose absorbed fro~ the test vehi-c~les for~ those Gonoentrations of 14-mer is shown in Fig-ure~3~ ~ The 0~05~ mgtmL and 0~5 mg1m~ formulations were both~solutions,~whi~le~the saturated (3~8 mg/mL) formula- ;
tion was a suspénsion. The sparing solubility of the 14-mer in the vehicle~may effect the rate at which it was absorbed.
The cumulative~ amount of neutral methylphosphonate ;14-mer which penetrated after 24 hours from the EtOHIH2O/
DMS~ (80:15:5) vehicle~saturated with drug was about 3.5 35 ~ ~g/cm2. This was~less than the cumulative amount which penetrated per cm2 after 24 hours from the EtOH/DMS (95:s) vehicle (saturated) (~wherein 5.5 ~gJcm2 was found to pene-S(IBSTITUTE SHEET

W~92~21353 PCT/US92~04370 ~ ~ 18 trate). (See Table I.) The flux using the EtOH/H2O/DMSvehicle may have been reduced because the solubility of 14-mer in that vehicle was higher than EtOH/DNS and, thus, there was less driving force to partition the 14-mer out of the vehicle into the skin.
The permeability of human skin to the 14-mer was - studied using the EtOH/H2O/DMS (80:15:5) vehicle saturated with neutral methylphosphonate 14-mer. The results of this experiment are;~ depicted in Figure 4 along with the data from thé same~experiment performed with the EtOH/DMS
(95:5) and EtON vehicles. While the permeability of the human ~skin toward the 14-mer from the EtOH/H20/DMS
~(80:15:5) vehicle~was lower than that from the water-free vehicles, the permeability profile was very similar. Com-I5~ pared to the hairless mouBe data, the differences in per-meability were minimal~. As~had been the case with all the vehicles test d with human skin, human skin was much less sensitive to changes~in~vehicle composition than was hair-ess~mouse skin~

20 ~etention of Oliaomer in the Skin r ~ c ~ ~ ~ lication to skin should maximize;the amount~of drug retained in the target area within the skin.~ In~the case of these neutral Oiigomers, for~many of~their;intended~targets, the basal cells of the 25~ derois a~re~the~regions;to which the drug should bè deli-véred. ~Perm-ability~expériments may indicate whether drug is being delivered; through~the skin and, therefore, that drug is being delivered~to the dermal layer. However, it is important to know how~much drug is retained in the skin at~various times during~the permeation experiment. There-fore~,~the skins used~in~the permeability experiments were routinely collected~to analyze the amount of drug retained after 24 hours of exposure to the drug-containing vehicle.
:: :
There are several ways that have been reported to assess drug retention. One method has been to homogenize a section of the whole skin used in the permeability ~ ' SUBSrlTUTE SHEET

WO92/21353 PCT/~92/0437~
21~00 ~

experiments and measure the amount of drug (i.e. oligomer) released from the sample. This technique detects the amount of drug in the entire skin sample (an average of the various strata~. A ~econd approach that can be used is the separation of skin into layers (~stratum corneum, viable epidermis, and dermis). A number of t~chniques have been used to separate skin into its component layers.
Most techniques suffer from one or more problems, such as leaching of the drug into water d-~ring the separation pro-cess. For instance, a heat treatment technique for sepa-ration of th¢ epidermis and the dermis requires submersion of the skin into hot water for several minutes prior to separation of the layers. To avoid this problem, a micro-wave technique to separate the dermis and epidermis has been described (Kumar, S., et al., Pharm. Res. 6:740-741 lg8gj)~
The first type of experiment~ used to determine drug Oligomer retention were~ those in which the entir~ skin sample was examined for~Qligomer. The experi~ental tech-~nique used to ~easure~retention is described in ~xample 5.In Table II, the~amounts of 14-mer recovered ~r~m skin samples following~the 24 hour permeability experiments are listed.
The stratum~ corne ~ was removed fr~m whole skin 25~ samples using microwave exposure for 5-8 seconds~ The amQunts of 14-me~r~recovered from the samples is shown in Table IIB. Both hairless mouse and human skin wer~ used;
two sa~ples o~ each were examined ~or total tissue reten-tion. Following removal of the strat~m corneum fro~ two additional skin samples, the 14-mer content wa~ ~easured in the remaining viable epidermis and dermis of the sam-ples. Differences~were observed between the hairless mouse and human~s~in d~ta.
To assist in understanding the present in~ention, the f~llowing examples are included which describe the results of a series of experiments. The following examples relat-ing to this invention should not, of ourse, be construed SlJBSrITUTE SHEE~T

W092/21353 ~ PCT/US92/~370 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 art are considered to fall within the scope of the present inven-tion as hereinafter claimed.

Examles Example 1 Pre~aration of MethvlDhosphonate Oliqomers ~, Neutral methylphosphonate oligomers are synthesized using lO methylphosphonamidite~monomers, according to the chemical ~
methods described~ by~P.S. Mi}ler ~et al. (Nucleic Acids ~`
Res.~ 6225-6242~ 1983))~, A. Jager and J. Engels (Tetra~
hedron Letters 25:1437-1440 (1984)) and M.A. Dorman et ,al.
~. : , Tetrahedron Letters`~40:'95-102 (1984)). Solid phase syn-thesis is performed on~a Biosearch Model 8750 DNA synthe-sizer~according~to the manufacturer's recommendations with ; the~following ~odifications~ GN and ~C" monomers are dis-olved"in 1:1 acetonitr~ile/dichloromethane at a concentra-tion~ of~;lOO~mM.~ A"~,;and "T" monomers are dissolved in 20 ~"acetonitrile,~at~a~ concentration of lO0 mM. DEBL~CK rea-gent~ 2~.5% ~dichloroacetic~ acid in dichloromethane.
OXIDI-~ER~ reagent~25~-g/L iodine~ in 2.5% water, 25% 2,6-lutidine,~72.5%;~;~tetrahydrofuran. CAP A = 10% acetic 'anhydride~'in~acetonitrile. CAP B = ).625% N,N-dimethyl-'2~5~; aminopyridine'in~pyridine. The~5!-dimethoxytrityi pro-tecting group~is~left~ on at the end of the synthesis to facilitate purification of the oligomers, as described below. , ; ;~
The crude,,protected methylphosphonate oligo~ers are 30 ~;~,re oved from~the solid~support~by mixing with concentrated ammonium~hydroxide~;for two hours at room temperature~ The solution is dra~ined~from the support using an Econo-Column~ (Bio-Rad, Richmond, CA) and the support i~ washed f`ive times with l:l acetonitrile/water. The eluted oligo-mer is evaporated to dryness under vacuum at room tempera-ture. Next, the protecting groups are removed from the SUBSrlTUTE SHEET
~: :

WO92~21353 PCT/US92/~370 bases with a solution of ethylenediamine/ethanol/aceto~
nitrile/water (50:23.5:23.5:2.5) for 6 hours at room tem-perature. The resulting solutions are then evaporated to dryness under a vacuum.
: 5The 5'-dimethoxytrityl (Ntrityl") containing oligo-mers are purified from non-tritylated failure sequences using a Sep-PakT~ Cl8 cartridge (Millipore/Waters Bedford, MA) as follows: The~cartridge is washed with acetoni-~::: trile, 50% acetonitrile::in lO0 mM, triethylammonium bicar-lO ~bonate (TEAB, pH 7.~5):~ and 25 mM TEAB. Next, the crude :methylphosphonate~oligomer is dissolved in a small volume of~ 1 acetonitrile/water and then diluted with 25 mM TEAB
to a~:final concentration of 5% acetonitrile. This solu-tion is then passed through:the cartridge. Next, the car-;lS~;:tridge is washed~with~;~lS-20% acetonitrile in 25 mM TEAB to eluate~ failure~ sequences~from the cartridge. The tri-;tyl-on:oligomer:remaining:bound to the cartridge is then de ~ lated by ~washing::with 2S mM TEAB, 2% trifluoro-acetic~acid,:and~2S.mM~TEAB, in that order. Finally, the 20~ trityl-selécted~oligo~er~is eluted.from the cartridge Jith 50%~acetonitrile/water;~;and evaporated to dryness under vacuum at~room`~temperature. :
The~methylphosphonàte oligomers are further purified by~ reverse-phase~ HPLC chromatography as follows: A
25~ Beckman~System ~Gold.~NPLC is used with a Hamilton PRP-l coIumn~(Reno, NV,~l0~ 7 mm i.d. x 305 mm long)~ Buffer ~50 mM triethylammoniùm acetate ~pH 7); Buffer B = 50%
~acetonitrile in ~50 ~mM~ triethylammonium acetate (pH 7).
: The sample, dissolved in a small volume of 10-50% acetoni-trile/water, is ~loaded onto the column while flowing at : 2~5-3:ml/minute~with~100-% Buffer A. Next, a linear gra-, dient of 0-70%:Buffer B~is run over about 30-50 minutes at a~flow rate of about~2.5~to 3.0 ml/minute. Fractions con-taining full length methylphosphonate oligomer are c~
, :~: 35 lected, evaporated under vacuum and resuspended in 50%
acetonitrile/water.
: ., ~
;
SUBSllTVTE Sr~EET
:

WOg2/213S3 PCT/US92/~370 ~ 2 Example 2 Pre~aration of Skin Samples for Permeability and Tissue Level Studies A. Hairless Mouse Skin S The hairless mice (male, HRS~J strain, 8 to lO weeks old, 20 to 25 g) were sacrificed in a C02 chamber and approximately 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 physio-logical 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 ~rom the body.
This was repeated 12~ to 15 times with fresh pieces of : ~ :
~ tape.
: ~ .
B. Human Cadaver~-Skin Hu an cad ver~skin~was obtained at autopsy through the Stanford~University~MedicaI Center. The skin was 20~ excised~using a~dérmatome from the thigh area of a 74 year o1d~ma1e~within 2~4~hours post-mortem. The thickness, as measured with~a Van~Keuren light wave micrometer, ranged from;~125 to 450 ~m.~ The average thicknesæ was 200 to 300 m~. ~The skin~was~'~'rinsed with phosphate buffered sa}ine 25~ pH 7.4)~ b1Otted~ dry and frozen for 6 months in triple-sea~led bags evacuated;of air. Prior to use, the skin was thawed and rinsed~in~PBS.

ExamD1e 3 ~ ~
Permeabilitv Exoe~iments A diffusion~console containing nine glass Franz dif-fusion cells was used in the permeability experiments. ~-The Franz cells~were~maintained at 37C by thermostati-cally controlled water, which was rirculated through a jacket surrounding~the cell body. Each skin was mounted and clamped between the cell body and the cell cap so that ;~
SUBSTITUTE SHEET
..

W092~1353 PCT/US92/04370 23 2~i~a~
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 cmZ. The receptor was 0.01 M phosphate-buffered saline ~pH 6.4) isotonic saline with 0.05% sodiumazide added to prevent qrowth 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 on to~the skin tO.2 mL total vehicle added to 2.0 cm2 (0.1 nK.cm2~)}~. At certain times following addition of the vehicles, a syringe needle was inserted through the 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 calcu1ations.

Exam~le~4 20~ ~ ~raDhic~AnalYsis of Oliaomer A. ~ 4~-Mer The 14-mer (neutral methylphosphonate Oligomer having only~methylphosphonate~internucleosidyl linkages) and 14-mér-IA ~methylphosphonate Oligomer having an internal ani-25~onic internucleosidyl~linkage) were measured in the recep-tor solution by HPLC.~These analyses were performed on a Waters~ 840 system` cons~isting~of two model 510 pumps, a model 481 W detector, a model 710B WISP ~sample proces-sor), and a Diqital computer model 350 microprocessor/ ~;~; 30 ~programmer. The~column used to separate the 14-mer was a 3.9 mm x 15 cm~4 ~m,~Waters Nova-Pak C18. A gradient elu-tion was performed as follows for the 14-mer:
~ , . , ~SUBSI-ITUTE SHEET
.

WO~2/21353 PCT/US92/04370 i ~ 24 ~;~
Time (min) %A %B -~:
0 100 0 ,.

~2 5 95 :
~:

22 100 0 :
Flow rate = 1.1 mLlmin, wavelength = 260 nm, retentio~
ti~e = 9.6 min.
A = O.05 M TEAA, pH:7.6 B = acetonitrile/A (75:25) ~ B. 1 : 15 The HPLC conditions w2re altered somewhat for mea-suremant of the 14~mer-IA. Again, a gradient elution profile was used ~described below.

Time (mi~l 100 ~.
20: :5 28 72 ~ :
: 14 ~ ~5 55 ~;
14.2~ 5 55 : 15.:5~ g8 2 : : 2~ 98 2 25 ~ :2~ 0 100 28~; 0 100 ;Flow rate = ~.1 mL/min, wavelength = 260 ~, retention time~= 10.2 min. ~:
A =~Acetonitrile/B:175:25) : 30 B - 0.05 ~ ~ammonium~acetate; pH 7.4 ::
:: ; : . :: .
: ~ A chromatogram~of the 14-mer-IA is shown in Figure 9 ; using the elution profile outlined above.

:
:~: '' ' : : : .~:
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..

: SUB9~7TVTE SHEET ~

WOg~/21353 PCT/US92/~370 21~0~
Exam~le 5 Tissue Level Measurements of Oliqomer Retained in Skin 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 surf ace of the skin. The skins were then rinsed for sev-eral seconds with water. The skins were then frozen untilready for 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 weîghed and then homogenized in 0.01 M sodium phosphate, pH 7.4 using a Polytron Homogenizer for approximately 2 minutes. The homogenate was then centri~uged at 8,000 g for l5~minutes at room~temperature. The supernatant was ~ removed and analyzed directly by HPLC analysis (see below : f~or conditions).
The chromatographic conditions were similar to those ~ described above for the 14-mer with some minor changes : noted~below.
Time~(minL %A
~ O 100 0 : ~ 2S 5 70 3~
: : 14 ~ 55 45 ~: 15.5 2 98 : 32 ~ 2 98 }00 o 42: 100 0 Flow rate = I.l mL/min, wavelength = 260 nm, retention time = 11.1 min.
A = 0.05 M ammonium acetate, pH 7.0 B = acetonitrile/A (75:25) ~ copy of a chromatogram obtained from a homogenized skin sample is found in Figure 10. Presence of the oligo-mer in the tissue homo~enates was confirmed by spiking ~he samples with 40 ~L o~ a 1.8 ~g/mL solution of 14-mer~

SUBS7-1Tl~TE SHEET
- .~, WO92~213~3 PCT/US~2/~370 ~ ~ 26 Resuspension of the pellet obtained after centrifuga- ~
tion, followed by homogenization and recentrifugation, led -to release of between l to 3% of the total 14-mer recov-ered from the original sample. These results indicate that the 14-mer was efficiently in the first extraction step.
, : Example 6 Me~surement of Flux and Retention of Oliq-omers in Human Skin :` I0 Human skin: which had been dermatomed to a thickness -of about 5-200;~m~wa9: used. The skin was mounted in a closed glas~ Franz ~:diffusion cell (as described in Exa~-ple 3).
Vehicle containing oligomer and optionally enhancer 15~ ~(lOO~L/cm2) was p~laced~on the surface of the skin (2 exposed surface cm2)~.:
The amount:of oligomer diffusing through and remain-Ing in the ski~:was~measur~d by HPLC. (See Example 3) Results are~summarized in Table ITI. Ethanol alone 20~ was` found tQ be~an::~effeotive penetration enhancer. Addi-tion;:of DMS (decylmethylsulfoxide) to ethanol generally increased the :penetration rate (and cumulative amount, i.:e.~amount:penetrate~ oYer 24 hour period) of the 6-, lO-and~14-mers through~h ~ an skin relative to that from etha-;25~ nol alone. Addition~of water to the ethanol/D~S vehicleincreased the flux (and cumulative amount) still further :
for the 6-mer; however, flux (and cumulative amount) for : the lO-mer and 14-mer was reduced.
Addition of DMS~ to propylene glycol increased the 30~ 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 hlaman skin led . .
to a large increase~in flux ~and cumulative amount) of the ~:35 6-mer, a~though~ the increase was not as dramatic as that observed with hairless mouse skin.
~ .
: ::
SUBSTITUTE SHEET
:: .

W092/2l353 PCT/US92/04370 21~0~'10 In comparing the cumulative amount data from hairless mouse skin with human skin for the lO-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 on 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 ma~ority of oligomer retained was in the dermis). Tape stripping (to remove 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 wholé skin.
;Table IV reports retention of 14-mer in dermis versus strateum corneum after treatment with 14-mer in various vehicle/enhancer combinations. St:ratum corneum and dermis were ~separated~ by ~micrcwave treatment as described by Kumar et~al. (Pharm. Res. 6:740-741 (19893~.

:

'~, SVBSTITUTF SHEET :`~

WO g2~21353 ~ PCr/US92/04370 TABLE I
PermeabilitY of Oliqomers in Hairless Mouse (HM) and Human_Skin ~S~

Skin Oligomer Donor Vehiclea Cumulative Amount at 24 h (~Lg/cmZ) HM 14-mer H20 O . 75 EtOH 0 . 2 8 EtOHlDMS (95: 5) 5 . 5 O EtOH/DMS ( 97 . 5: 2 . 5 ) 4 . 4 EtOH/OA (95:5)b 0.30 : ~EtO~/OA (97 . 5: 2 . 5) 0. 24 EtAcC l . 2 Et:ACJDMS (95:5) 1.1 ~ ~ EtAC4/OA (95:5) 0060 Et~Hd 187 EtOHlDMS ( 95: 5 ) d 18 6 . : EtOH/~2O/DNS (80::15:5) 3.5 EtOH/H2O!DMS t80: 15: S~ ' 2 . 7 20~ EtOHjH2o/D~s (80:15:5)f 2.1 EtOH/H2O/DMS ~80:15:05)9 0.23 14-mer-IA~HzO;:~ 0 EtOH 0 ::
EtOH/DMS ( 95: 5 ~ 0 . 61 25~ ~ HS : 14-mer ~ EtOH 0.26 EtOH/DMS (95:5) 0.24 ~;~
~: : Et~HjoA ~95:5) 0.3~ .:
EtOH/H2O/DMS (80:15:5)h 0.23 -30 aUnless stated i~ the table footnote~, all the donor vehicl s were saturated with: oligomer boA = oleic acid 'EtAc = ethylacetate ~:
dTh~se skins were free of stratum corneum , which was ~:
3 5 removed by tape stripping .
::

SUBSllTUTE SHEET :`
:,`

W092/2~353 PCT/US9~/04370 2 I ~

14-mer concentration in the vehicle was 1.0 mg/mL ~below saturation) fl4-mer concentration in the vehicle was 0.5 mg/mL (below saturation~
914-mer concentration in the vehicle was 0.05 mg~mL (below saturation) hl4-mer concentration in the vehicle was l.0 mg/m~ (below saturation) : ~

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WO92/213~3 ~ P~TJ~S92/04370 9~

TABLE II

A. Amounts of 14-mer Re~overed from Skin Samples Skin Donor Vehicle ~g/gm~ ~P

S HM EtOH/DMS ~95:5j~ 30.2 7.1 EtOH/DMS (95.5)C 112 26.3 EtOH/H20/DMS ~80:1S:5) 77 17.9 . :.
EtOH/H20/DMS (80 1S 5)d18.4 4.3 HS : EtOH/DMS (95:5~) 67.1 15~7 ::~

1 0 _ _ _ _ :
:
otal~ ~g of:14 mer recovered from the homogenized skin sample co~rect~d for~loss~of 14-mer during homogenizat;on ;and~centrifugat:ion~ (see~ Example 4~; the gm is the wet :.
wei~ t of the skin as~meisured prior to homogenization :;
~15~ b;~M~concentration of 14-mer in the skin were obtain~d from the~molecular;weight~of;the 14-msr and the assumed density :of;1.~0~for:the~skin~sample t e., 1.0 gm of skin is equal ``
to~1:.O~cc~:of~skin)~
'The~HM:skin used in~thi~experiment was stripped to remove 20~ thë~stratum:corneum:~
The~concentration~of~14-mer in this vehicle was 0.5 mg/mL
çoDpared to~all ~the~;other experimental vehicles, which . -we~e~s2turated with~excess solid 14-mer ~:

B. Retention of 14-mer in Whole Skin and Vîable Tissuesa : .
25~ Skin~ Section :Donor~Vehicle ~g/gmb HN : Whole EtOH/H20/DMS (80:15:5) 63.2 14.$
Viabled EtOH/H2O/DMS (80:15:5~ 35.2 8.2 :

SUBSTITUTE SHEET

W092/213~3 PCT/US92/04370 31 2 ~ :1 0 ~
HS Whole EtOH/H20tDMS (80:15:5) 105.9 24.7 Viabled EtOH/H20/DMS (80:15:5) 7.0 1.6 aThe 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 during homogenization and centrifugation (see Example 4); the gm is the wet weight of the s~in as measured prior to homogenization C~M concentration of 14-mer in the skin were obtained from the molecular weight of the 14-mer and the assumed density of~1.O for the skin sample ~ e.f 1.0 gm of skin is equal }5 ;to 1.0 cc of skin) `dThe viable tissue i5 the::tissue after the stratum corneum has::been removed by microwave tr~atment (Kumar, et al., Pharm. Res. 6:740-741 (1989)). It is a combination of the iable epidermis and the dermis. -`

2~0~ ;TA~E_III :~
Penetration of Oli~_mers_Throuqh Skin ; A.~ Human Skin Yehiole/ Ratio~c~f~ 24Hr cumulative Valùe~
Enhan~er Components nmole~/cm2_Mean_and SD
~ 6 Mer lQ_n~ 4 Mer EtOH/H20/DMS (80:15:5) 13.8(5.7) 0.94tl.3) 0.18~0.17 2.2(~.0) -~
EtOH/DMS~95:5j; 8.2(5~4)6.0(4.2) 0.83(1.0) : EtOH (100:0): 3.4(2.8)4-0(6D7) 9-37~0-48) ~ 30: 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 ., SUBSI ITUTE SHEET

W092/21353 ~t.9 PCI/US92/04370 3~
The second value for the 6Mer came from a time study using a different skin donor~ otherwise the data f or the f irst three enhancers came from the same donor.
The data for the last three enhancers came from the same experiment but ~rom a different donor.

,' .~

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' SUBSTITUTE SHEET

WO 92/213~3 PCl`/US92/04370 2~1DO~

B. Hairless Mouse Vehicle/ Ratio of24Hr Cumulative Values:
Enchancer Comonentsn3~01es!cm2 Mean and SD
6 Mer 10 Mer 14 Mer EtOH/H2O/DMS (80:15:5)No Data2.08(0.82~ 0.82 EtOH/DMS (95:5) No Data1.94(0.22) 1.28 EtON ~100%) No Data0.35~0.10) 0.065 , :.
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SUBSTITIJTE SHEET

WO92/21353 'l-~ PCI/U592/04370 34 _ Ul _~_ ~ O--O~ ~ CLU7 ~1 _ ____ ___ O ~S ~1 N _ _ __ _ O ~ ~ ~ --__ ___ .
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SUBSI ITUTE SHEET

Claims (42)

Claims

1. A method of preventing or interfering with expression of a specific nucleic acid target sequence which comprises administration across skin or mucous membrane tissue of a neutral Oligomer which is comple-mentary to and which can bind to or interact with said specific nucleic acid sequence or a modulating nucleic acid sequence which modulates expression of the target sequence.

2. A method according to claim 1 wherein said neutral Oligomer comprises an alkyl- or aryl-phosphonate Oligomer, a phosphotriester Oligomer, a phosphoramidate Oligomer, a carbamate Oligomer, a sulfamate Oligomer, a morpholino Oligomer, an alkyl or aryl- phosphonothioate Oligomer, or a formacetal Oligomer.

3. A method according to claim 2 wherein said neutral Oligomer comprises a neutral methylphosphonate Oligomer.

4. A method according to claim 3 wherein said neutral Oligomer is applied in a formulation which comprises a short chain aliphatic alcohol.

5. A method according to claim 1 wherein said nucleic acid target sequence codes for a mediator of inflammation.

6. A method according to claim 5 wherein said mediator is selected from a cytokine, a growth factor, a cell adhesion molecule or a receptor thereof.

7. A method according to claim 1 wherein said tar-get sequence comprises an RNA region which codes for an initiation codon region, a polyadenylation region, a mRNA
cap site, or a splice junction.

8. A method according to claim 1 wherein said tar-get sequence comprises a region of the gene which codes for renin or its mRNA transcript.

9. A method according to claim 8 wherein said target sequence comprises an initiator codon region, a polyadenylation region, a mRNA cap site or a splice junction.

10. A method according to any of claims 1 or 2 wherein said Oligomer comprises a conjugation partner.

11. A method according to claim 10 wherein said con-jugation partner is selected from intercalators, alkylat-ing agents, binding substances for cell surface receptors, lipophilic agents, cross-linking agents, hydrolytic or nucleolytic agents, prochelates, or DNA modifying groups.

12. A method of delivery of an antisense oligomer to skin or mucous membrane tissue which prevents or inter-feres with expression of a specific nucleic acid target sequence which comprises application of a formulation which comprises a neutral Oligomer which is complementary to and which can bind to or interact with said nucleic acid sequence or a modulating nucleic acid sequence which alter expression of the target sequence.

13. A method according to claim 12 wherein said neutral oligomer comprises an alkyl- or aryl-phosphonate Oligomer, a phosphotriester Oligomer, a phosphoramidate Oligomer, a carbamate Oligomer, a sulfamate Oligomer, a morpholino Oligomer, an alkyl or aryl-phosphonothioate Oligomer or a formacetal Oligomer.

14. A method according to claim 13 wherein said neutral Oligomer comprises a methylphosphonate oligomer.

15. A method according to claim 14 wherein said formulation comprises a short chain aliphatic alcohol.

16. A method according to claim 15 wherein said formulation further comprises a flux enhancer.

17. A method according to claim 12 or 13 wherein said Oligomer comprises a conjugation partner.

18. A method according to claim 17 wherein said conjugation partner is selected from intercalators, alkylating agents, binding substances for cell surface receptors, lipophilic agents, cross-linking agents, hydrolytic or nucleolytic agents, prochelates, or DNA
modifying groups.

19. A neutral Oligomer which is capable of penetrat-ing skin or mucous membranes and which is capable of selectively binding to or interacting with a specific nucleic acid target sequence.

20. An Oligomer according to claim 19 wherein said target sequence comprises an RNA region which codes for an initiator codon region, a polyadenylation region, a mRNA
cap site or a splice junction or a DNA region which results in alteration of gene expression.

21. An Oligomer according to claim 20 which com-prises an alkyl- or aryl-phosphonate Oligomer, a phos-photriester Oligomer, a phosphoramidate Oligomer, a carbamate Oligomer, a sulfamate Oligomer, a morpholino Oligomer, an alkyl- or aryl-phosphonothioate Oligomer or a formacetal Oligomer.

22. An Oligomer according to claim 21 which com-prises a methylphosphonate Oligomer.

23. An Oligomer according to claim 19 which com-prises an alkyl- or aryl-phosphonate Oligomer, a phos-photriester Oligomer, a phosphoramidate Oligomer, a carbamate Oligomer, a sulfamate Oligomer, a morpholino Oligomer, an alkyl- or aryl-phosphonothioate Oligomer, or a formacetal Oligomer.

24. An Oligomer according to claim 23 which com-prises a methylphosphonate Oligomer.

25. An Oligomer according to claim 19 wherein said nucleic acid target sequence comprises a region of the gene which codes for renin or its mRNA transcript or a region which results in alteration of renin expression.

26. An Oligomer according to claim 25 wherein said target sequence comprises an initiator codon region, a polyadenylation region, or a mRNA cap site or a splice junction.

27. An Oligomer according to any of claims 19, 20 or 25 which comprises a conjugation partner.

28. An Oligomer according to claim 27 wherein said conjugation partner is elected from intercalators, alky-lating agents, binding substances for cell surface recep-tors, lipophilic agents, cross-linking agents, hydrolytic or nucleolytic agents, pro-chelates, or DNA modifying groups.

29. A method of delivering an Oligomer to an animal for therapeutic purposes which comprises administration of a neutral Oligomer across skin or mucous membranes.

30. A method according to claim 29 wherein said Oligomer is administered transdermally.

31. A method according to claim 29 wherein said Oligomer is administered to mucous membranes.

32. A method according to claim 31 wherein said Oligomer is administered by inhalation.

33. A method according to claim 29 wherein said Oligomer prevents or interferes with expression or function of a mediator of inflammation.

34. A method according to claim 29 wherein said Oligomer comprises an alkyl- or aryl-phosphonate Oligomer, a phosphotriester Oligomer, a carbamate Oligomer, a sul-famate Oligomer, a morpholino Oligomer, an alkyl- or aryl phosphonothioate Oligomer or a formacetal Oligomer.

35. A method according to claim 34 wherein said Oligomer is a methylphosphonate Oligomer.

36. A method according to claim 35 wherein all internucleosidyl linkages of said Oligomer are methyl-phosphonate internucleosidyl linkages.

37. A method according to any of claims 29, 34, 35 or 36 wherein said Oligomer comprises a conjugation partner.

38. A method according to claim 37 wherein said con-jugation partner is selected from intercalators, alky-lating agents, binding substances for cell surface recep-tors, lipophilic agents, cross-linking agents, hydrolytic or nucleolytic agents, prochelates or DNA modifying groups.

39. A method according to claim 29 wherein said Oligomer prevents or interferes with expression or func-tion of a mediator of cell adhesion.

40. A method according to claim 29 wherein said Oli-gomer prevents or interferes with an agent which mediates cell proliferation.

41. A method according to claim 29 wherein said Oligomer prevents or interferes with cancer cell proliferation.

42. A method according to claim 29 wherein said target sequence comprises a region of the gene which codes for renin or its mRNA transcript or a region which results in alteration of renin expression.