WO2005033282A2 - Polyamide compositions and therapeutic methods for treatment of human papilloma virus - Google Patents

Abstract

Processes and compositions are provided for regulating replication in a human papilloma virus. The process includes selecting a polyamide composition comprising N-methyl pyrrole (Py) and either des-amino-N-methyl imidazole (dIm) or a fused, non-tautomerizing heteroaromatic bicyclic structure (L) that contains at least one hydrogen bond acceptor moiety, to provide specific binding to DNA at the E1 binding site in a human papilloma virus episome. The polyamide is then combined with the cell containing a human papilloma virus, wherein the polyamide binds to the human papilloma virus E1 binding site and regulates replication of the human papilloma virus.

Description

POLYAMIDE COMPOSITIONS AND THERAPEUTIC METHODS FOR TREATMENT OF HUMAN PAPILLOMA VIRUS

FIELD OF THE INVENTION [0001] The present invention relates to polyamide compositions and therapies for treatment of cells infected with human papilloma virus (HPV) . More particularly, the reduction in the replication of the HPV DNA present in infected cells using polyamides designed to bind to the El binding sites in the origin of replication of the HPV.

BACKGROUND OF THE INVENTION [0002] Human papilloma virus is a small double-stranded DNA virus that colonizes various stratified epithelia like skin, oral and genital mucosa, and induce the formation of self-limiting benign tumors known as papillomas (warts) or condylomas . Most of these benign tumors naturally regress due to the influence of host immunological defenses. Some HPVs, however, have oncogenic potential and have been associated with certain types of cancers. See, Lorincz et al . , Obstetrics & Gynecology, 79:328-337 (1992); Beaudenon et al . , Nature, 321:246-249 (1986); and Holloway et al., Gynecol . One, 41:123-128 (1991). For example, the majority of human cervical carcinomas (95%) contain and express HPV DNA and it is the expression of two viral oncoproteins, E6 and E7, which appears to be critical for cellular transformation and maintenance of the transformed state. Specifically, four HPV types (HPV-16, HPV-18, HPV-31, and HPV-45) have been connected to 75-93% of the cases of cervical cancer in the United States. It has been estimated that perhaps twenty percent (20%) of all cancer deaths in women worldwide are from cancers that are associated with HPV. [0003] Generally speaking, HPVs are grouped into types based on the similarity of their DNA sequence. Two HPVs are taxonomically classified as being of the same type if their DNA cross-hybridizes to greater than 50% as measured by hybridization in solution under moderately stringent hybridization conditions. HPVs can be further classified either high or low risk on the basis of the clinical lesions with which they are associated and the relative propensity for these lesions to progress to cancer. Low risk types, such as HPV types HPV-1, HPV-2, HPV-3, HPV-4, HPV-5, HPV-7, HPV-8, and HPV-9 cause common warts (verrucae vulgaris), plantar warts (verrucae plantaris), mosaic warts, flat warts (verrucae plana) , and butcher warts. Furthermore, HPV types HPV-6 and HPV-11 cause warts of the external genitalia, anus, and cervix. High-risk types, such as HPV-16, HPV-18, HPV-31, and HPV-33 are particularly common in intraepithelial neoplasias and cancers. In particular, the genomes of two HPV types, HPV-16 and HPV-18, have been found to be associated with about 70% of invasive carcinomas of the uterine cervix. [0004] Current treatment for HPV infection is extremely limited. Management normally involves physical destruction of the wart by surgical, cryosurgical, chemical, or laser removal of infected tissue. Some of these current treatments, like laser removal and surgery, are expensive and require the use of anesthesia to numb the area to be treated. Cryosurgical removal requires the use of special equipment. Furthermore, most patients experience moderate pain during and after procedure. [0005] Topical creams and solutions such as 5-fluorouracil, imiquimod, cidofovir, formaldehyde, glutaral, cimetidine, tricholoroacetic acid, bleomycin, podofilox and podophyllum preparations have also been used. Reichman in Harrison's Principles of Internal Medicine, 13th Ed. (Isselbacher et al . , eds.); McGraw-Hill, Inc., NY (1993) pp. 801-803). Reoccurrence after these treatments, however, is common (95%) , most likely due to the fact that the virus remains latent within the skin cells. Therefore, subsequent repetitive treatments must be used, which can destroy healthy tissue. [0006] Interferon has so far been the most effective treatment for HPV; however, its effectiveness is limited. Chang et al. (2002) Journal of Virology 76: 8864-8874 (finding some cells infected with HPV genomes became resistant to interferon treatment after only a few applications) . See also Cowsert (1994) Interirol. 37:226-230; Bornstein et al . (1993) Obstetrics Gynecol. Sur. 4504:252-260; Browder et al . (1992) Ann. Phannacother . 26:42-45. [0007] Recently, the understanding of the synthesis, the analysis, and the manipulation of DNA has led to an explosion of opportunities for the diagnosis and treatment of various illnesses and conditions, such as those caused by HPV. The specific interaction of proteins with DNA, such as transcription factors, affects the expression of genes, and hence, the regulation of cellular processes as well. Roeder, R.G., TIBBS . 9, 327-335 (1996) . A wide variety of human conditions ranging from cancer to viral infection arise from malfunctions in the biochemical machinery that regulates gene expression. (R. Tjian, Sci . Am. , 2, 54-61 (1995)). Therefore, researchers have focused on identifying specific sequences of DNA that, when expressed as a result of biochemical malfunction or otherwise, cause disease, defect, and discomfort. [0008] In recent years, researchers have learned that certain chemical compounds can be used to regulate the phenotypic effects of the genetic machinery. The expression of proteins, the end product of nucleic acid translation, can be affected by the application of certain natural and synthetic compounds. In research, these molecules can be used to modulate the activity of a particular gene in order to identify the function and cellular characteristics of that particular gene. In therapeutics, these molecules can be used to inhibit the proliferation of cells which may act as pathogens, where proliferation has an adverse effect on the host, or to combat life threatening diseases which result from misregulation in transcription. [0009] It has become known that certain oligomers of nitrogen heterocycles can be used to bind to particular regions of double stranded DNA. Particularly, N-methyl imidazole (Im) , des-amino-N-methyl imidazole (^dIm) , and N-methyl pyrrole (Py) have a specific affinity for particular bases. This specificity can be modified based upon the order in which these compounds are linked. It has been shown that there is specificity in that G/C is complemented by ^dIm/Py or Im/Py, C/G is complemented by Py/^dIm or Py/lm, and A/T and T/A are redundantly complemented by Py/Py. In effect, N-methyl imidazole and des-amino-N-methyl imidazole tend to be associated with guanine, while N-methyl pyrrole is associated with cytosine, adenine, and thymine . By providing for two chains of the heterocycles, as 1 or 2 molecules, a 2:1 complex with double stranded DNA is formed, with the two chains of the oligomer antiparallel, where G/C pairs have ^dIm/Py or Im/Py in juxtaposition, C/G pairs have Py/^dIm or Py/lm, and T/A pairs have Py/Py in juxtaposition. The heterocycle oligomers are joined by amide (carbamyl) groups, where the NH may participate in hydrogen bonding with nitrogen unpaired electrons, particularly of adenine. [0010] Polyamides may be synthesized to form hair-pin compounds by incorporating compounds, such as gamma-aminobutyric acid (y) or gamma-amino-beta-aminobutyric acid (Y^) _> to allow a single polyamide to form a complex with DNA. Such a structure has been found to significantly increase the binding affinity of the polyamide to a target sequence of DNA. [0011] Beta-alanine (β) may be substituted for a pair of N-methyl pyrrole groups when an AT or TA base pair is the target sequence. The added flexibility of the beta-alanine can help the entire polyamide stay "in register" with the target sequence of DNA. [0012] The polyamide molecule typically begins with des- amino-N-methyl imidazole which has a specific affinity for guanosine. The polyamide molecule typically ends with either 3- (Dimethylamino) propylamine (Da) or 3 , 3 ' -Diamino-N- methyldipropylamine (Ta) . Dye molecules can be incorporated at the amino groups of the γ-amino-β-amino-butyric acid, the Ta, or at both of these sites if both are available in the same molecule . [0013] More recently it has been discovered that the inclusion of a new aromatic amino acid, 3-hydroxy-N-methylpyrrole (Hp) , when incorporated into a polyamide and paired opposite Py, provides the means to discriminate A-T from T-A. White S., et al., Nature 391, 436-438 (1998). Unexpectedly, the replacement of a single hydrogen atom on the pyrrole with a hydroxy group in an Hp/Py pair regulates the affinity and the specificity of a polyamide by an order of magnitude. Utilizing Hp together with Py and ^dIm or Im in polyamides to form six aromatic amino acid pairs (^dIm/Py, Im/Py, Py/^dIm, Py/lm, Hp/Py, and Py/Hp) provides a code to distinguish all four Watson-Crick base pairs in the minor groove of DNA. The individual chemical units that comprise a polyamide molecule are illustrated in Fig. 1.

SUMMARY OF THE INVENTION [0014] Among the various aspects of the present invention, therefore, is the provision of polyamide compounds that bind to human papilloma virus DNA in the El binding site region, the provision of a process to regulate replication of a human papilloma virus using a polyamide compound, the provision of a process to suppress the regulation of a human papilloma virus using a polyamide compound, the provision of a process for reducing the concentration of human papilloma virus episo es within an infected cell, and the provision of a method for therapeutically treating a human papilloma virus infection. [0015] Briefly, therefore, the present invention is directed to a process for regulating gene expression of a human papilloma virus in an infected cell. The process comprises administering a polyamide in a pharmaceutically acceptable concentration to the infected cell, to tissue containing the infected cell, or to infected organisms comprising the human papilloma virus DNA. The polyamide comprising N-methyl pyrrole (Py) and either des-amino-N-methyl imidazole (^dIm) or a fused, non-tautomerizing heteroaromatic bicyclic structure that contains at least one hydrogen bond acceptor moiety. The polyamide being administered specifically binds to DNA at a El binding site in a human papilloma virus episome and regulates replication of the human papilloma virus. [0016] The present invention is further directed to a polyamide compound for regulating replication of a human papilloma virus. The polyamide comprises N-methyl pyrrole (Py) and either des-amino-N-methyl imidazole (^dIm) or a fused, non- tautomerizing heteroaromatic bicyclic structure that contains at least one hydrogen bond acceptor moiety wherein the polyamide specifically binds to the El binding site of DNA in human papilloma virus. [0017] Other objects and features of this invention will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS [0018] Fig. 1 is an illustration of polyamide component structures. [0019] Fig. 2 is an illustration of nucleotide regions containing El binding sites of various human papilloma viruses. [0020] Figs. 3A and 3B are illustrations of targeted binding locations in nucleotide regions containing El binding sites of various human papilloma viruses of polyamides wherein the targeted binding location is 5 ' -WWGWWWW-3' to 5' -WWGWWWWWWWWWW-3' (SEQ ID NO 15) or 3 ' -WWGWWWW-5' to 3' -WWGWWWWWWWWWW-5' (SEQ ID NO 16). [0021] Figs. 4A-4D are bar graphs illustrating the effect of polyamides on the replication of copies of HPV-31 episomes in 31NHFK-10 cells. [0022] Fig. 5 is an illustration of a polyamide binding to the El binding site of HPV-31. The illustrated polyamides include HPV2 where R = H and HPV4 where R = NH₂. The polyamides bind to DNA regions having a 10 base pair length. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0023] In accordance with the present invention, it has surprisingly been discovered that polyamides and analogs of polyamide polymers and oligomers (hereinafter collectively referred to as "polyamides") may be designed, synthesized, and utilized to regulate the replication of a human papilloma virus (HPV) by binding to HPV DNA in the El binding site region. More particularly, the present invention provides a process for suppressing or inhibiting the replication of a human papilloma virus by utilizing polyamides that bind in the El binding site region. The present invention also provides compositions and processes for promoting the clearance of HPV episomes from infected cells. Additionally, the present invention provides a process for the treatment of a human papilloma virus infection. [0024] Human papilloma viruses are a subgroup of a large family of double-stranded DNA viruses. Human papilloma viruses contain a highly conserved region called the long control region (LCR) which contains viral transcription and replication control genes . Within the long control region is the El binding site which is responsible for controlling replication of the viral DNA. Specifically, viral DNA replication is initiated when an El binding protein forms a replication complex with other components such as, inter alia, the E2 protein, and binds to the El binding site. In addition to being a part of the replication complex, the El protein is a helicase enzyme that removes the supercoiling from DNA (i.e., unwinds the DNA) so that replication can occur. Therefore, the helicase activity of the El protein and the binding of the replication complex at the El binding site function to initiate viral DNA replication. [0025] The nucleotide sequences containing the El binding site are highly conserved among the HPV genotypes. Due to variability of genome length, the positions of the El binding sites occur at different nucleotide positions within different strains of human papilloma viruses. In general, however, the El binding sites are located near nucleotide "1" of the HPV genome near the origin of replication (ORI) , although there may be exceptions. For example, the El binding site of HPV-31 is predicted to be contained within nucleotides 7902-7912/1-13 of the HPV-31 genome. Fig. 2 illustrates the nucleotide sequence regions of several human papilloma viruses which are believed to contain the El binding sites. [0026] The present invention relates to the design and use of polyamides to suppress or inhibit the replication of a human papilloma virus within affected cells of an infected individual. Polyamides with a particular binding specificity can be designed according to the present invention to bind to HPV DNA minor groove regions containing the El binding site near the origin of replication of a human papilloma virus. The bound polyamides disrupt the replication complex containing the El protein from binding at the El binding site. By binding in the region containing the El binding site, polyamides prevent the replication complex from binding at the El binding site and also interfere with the El protein helicase activity. This in turn suppresses or inhibits initiation of HPV episomal DNA replication. Thus, the use of polyamides suppresses or inhibits the replication of human papilloma virus episomal DNA and therefore affects the levels of human papilloma virus present in infected cells. [0027] The present invention also relates to the use of polyamides to provide clearance of HPV episomes from infected cells. After the cells of an individual become exposed and infected with a human papilloma virus, a number of HPV episome copies may become established within an infected cell . The HPV episomes further replicate as the cells divide, forming approximately the same number of HPV episome copies in each new cell (e.g., upon cell division, a cell containing 20-100 copies will form two new cells, each containing approximately 20-100 episome copies) . It has been surprisingly discovered that the use of polyamides designed to target the El binding site region of a HPV DNA not only suppresses or inhibits the replication of HPV episomes, but actually promotes the clearance of HPV episomes. Thus, for example, if polyamides binding to El binding sites completely inhibit HPV replication but the HPV episomes are not cleared from the cells, the infected cells will contain one- half the copies of episomes after the first cell division (e.g., if the infected cells contain an average of about 100 HPV episome copies of per cell, the cells will contain an average of about 50 copies per cell after one division) . However, experimental data has surprisingly indicated that at some doses of some polyamides ( e . g. , 0.1 μM to 10 μM) , the number of HPV episomes per cell dramatically decreases, and in some cases, the number of HPV episomes per cell decreases to almost zero. Thus, the process of the present invention can also be beneficially used as a therapeutic method of treatment for HPV. [0028] In general, polyamides are designed and synthesized to selectively bind in the region of the El binding site of a human papilloma virus episome. Research studies, outlined in the examples below, were conducted and the inhibitory characteristics of the tested polyamides were determined. By utilizing polyamides designed to target the El binding sites, the replication of the human papilloma virus can be suppressed or inhibited. Fig. 2 is an illustration of the nucleotide region of various strains of human papilloma virus that contain the El binding site. [0029] The polyamides of the present invention are designed to bind in desired areas of the El binding site wherein the target nucleotides begin with a W-^G at the "N-terminus," where W is the international DNA code for adenine (A) or thymine (T) , and G signifies guanine. The polyamides are designed to bind to an additional four to thirteen nucleotides. The polyamides are therefore designed to target a HPV DNA site having the sequence 5'- (W)₁.₂G(W)_x-3' (SEQ ID NO 17) or 3 ' - (W) _X.₂G (W)_x-5' (SEQ ID NO 18), where X = 4-13. The designed polyamides are provided in Table 1 of Example 1. Fig. 3 provides an illustration of target binding sites for polyamides that target 5 ' -WWGWWWW-3 ' to 5" -WWGWWWWWWWWWW-3 ' (SEQ ID NO 15) or 3 ' -WWGWWWW-5 ' to 3'-WWGWWWWWWWWWW-5' (SEQ ID NO 16) nucleotide sequences. [0030] In one embodiment of the present invention, the designed polyamides start with a des-amino-N-methylimidazole group followed by N-methylpyrrole and β-alanine building blocks. Hairpin turns are incorporated with either γ-aminobutyric acid or γ-amino-β-amino-butyric acid. Some degeneracy occurs in the polyamide-DNA recognition rules, allowing several polyamides of different compositions to be targeted to the same DNA sequence. Thus, a pair of β-alanine building blocks can be substituted for a pair of N-methylpyrrole groups when an AT or TA base pair is the target sequence. The added flexibility of the β-alanine building blocks can help the entire polyamide stay "in register" with the DNA. Furthermore, when appropriate sequences exist, some polyamides can simply be lengthened to bind and recognize a large DNA target sequence. The polyamides are typically capped at the "carboxy terminus" with either Da or Ta (see Fig. 1) . Additionally, dye molecules may be incorporated at the amino groups of the γ-amino-β-amino-butyric acid, the Ta, or at both of these sites if both sites were available in the same molecule. [0031] In another embodiment, the designed polyamide compounds comprise at least one heteroaromatic fused, bicyclic structure (i.e., wherein one of the rings thereof is heteroaromatic and the other is aromatic or heteroaromatic) , said structure having a heteroatom therein which acts as a hydrogen bond acceptor to bind guanine in the minor groove of dsDNA, and further wherein said structure cannot form a tautomer in which said heteroatom becomes a H-bond donor. [0032] In one embodiment, associated with, or bound directly or indirectly to, this fused, bicyclic structure are optionally other cyclic or heterocyclic compounds, which may or may not serve has H-bond donors or acceptors. Additionally, the compounds may comprise linking moieties (e.g., H-bond donors, such as amido (i.e., -C(O)NH-) or amido-containing linking moieties) . Accordingly, in one embodiment the compound may comprise a series of at least about 2, 4, 6, 8, 10 or more cyclic moieties (e.g., heterocyclic, including heteroaromatic, moieties and fused, bicyclic structures as described herein) which are bound with one or more linking moieties, in order to form a complementary pairing with target nucleotides of the dsDNA. [0033] In some instances, the compounds may be described as analogs of synthetic and/or non-naturally occurring polyamide oligomers or polymers, the binding affinity and/or selectivity potentially being improved, relative to conventional polyamides, by the inclusion of one or more moieties having said fused, bicyclic structure which serves as a H-bond acceptor. For example, the compounds may alternatively be described as oligomers in those instances wherein they comprise at least about 2, 4, 6, 8, 10 or more H-bond donor and/or H-bond acceptor moieties, while the present compounds may alternatively be described as polymers when two or more of said oligomers are linked (e.g., multiple hairpin oligomers may be linked to form a polyamide, as described and/or illustrated elsewhere herein) . [0034] In one embodiment of the compound of the present invention the fused, bicyclic structure is directly bound to another fused, bicyclic structure or a heterocyclic moiety (e.g., a pyrrole or imidazole ring) . Accordingly, the addition of each fused, bicyclic structure in the polyamide enables the elimination of a H-bond donor (e.g., an amido linker or amido- containing moiety) . Thus, the polyamides which are capable of altered, and preferably enhanced, interactions in the minor groove of HPV DNA (as compared to conventional polyamides) . An exemplary embodiment of such a fused, bicyclic structure is:

,^X1-~^,--^X3 < ■ > N' -X₄

[0035] wherein X¹, X³ and X⁴ (which may be the same or different) are as further described herein, and provided: X⁴ is a heteroatom as described herein (i.e., a H-bond acceptor heteroatom) ; each ring of the fused, bicyclic structure is unsaturated and has 5-members or 6-members (with the exception that both rings do not have 5-members) . The dotted lines in the above structure indicate the fact that the rings are unsaturated (aromatic or, in the case of a heteroatom-containing ring, heteroaromatic) . [0036] The fused, bicyclic structure which serves as a H- bond acceptor in the compound may be characterized, in one embodiment , as : X<ι -» - T - ^' - χ₃ / \ 1 1 * V' ^*-X4

[0037] wherein: [0038] X and X₂ are independently selected from 0, S, N, NR², CR³, CR⁴=CR⁴', CR⁴=N, N=CR⁴, N=N and CR⁴", provided that (i) when each one of X_α or X₂ is independently selected from O, S or NR², the other is independently selected from CR³ or N, and (ii) when each one of X_x or X₂ is independently selected from CR⁴=CR^4*, CR⁴=N, N=CR⁴ or N=N, the other is independently selected from CR⁴" or N; [0039] X₃ is independently selected from N, O, S, CR⁵, NR⁵, CR⁵=CR⁵', CR⁵=N, N=CR⁵ and N=N, and X₄ is independently selected from 0, S, N and CH, provided that (i) when each X₃ is independently selected from CR⁵ or N, X₄ is independently selected from O or S, and (ii) when each X₃ is independently selected from 0, S, NR⁵, CR⁵=CR⁵ ' , CR⁵=N, N=CR⁵ or N=N, X₄ is independently selected from CH or N; and, [0040] each R substituent (i.e., R², R³, R⁴, R⁴ • , R⁴", R⁵, R^5') generally represents a hydrogen or some other substituent, as defined herein, which does not detrimentally hinder binding of the oligomer to the dsDNA or, alternatively, acts to enhance such binding, provided that the structure cannot form a tautomer in which the heteroatom for binding guanine becomes a H-bond donor (e.g., when X¹ or X² is NR², R² is other than H, and when X³ is NR⁵, R⁵ is other than H) . [0041] The dotted lines in the above structure indicate the fact that the rings are unsaturated (aromatic or, in the case of a heteroatom-containing ring, heteroaromatic) . As detailed elsewhere herein, and in view of the exceptions noted above and elsewhere herein, acceptable substituents (e.g., R) may include, for example, those independently selected from H, hydroxy, N-acetyl, benzyl, substituted or unsubstituted C_1-e alkyl, substituted or unsubstituted C_x__e alkylamine, substituted or unsubstituted C_1-s alkyldiamine, substituted or unsubstituted Ci.g alkylcarboxylate, substituted or unsubstituted C₂_₆ alkenyl, substituted or unsubstituted C₂._s alkynyl, and the like, and when attached to a carbon optionally halo (e.g., chloro) . [0042] In instances or embodiments wherein the first ring of the fused, bicyclic structure is heteroaromatic and the structure occupies an initial terminal (i.e., "cap") position, X₂ may be, for example, C-H when X_x is NR² (e.g., N-CH₃) . Accordingly, when the fused, bicyclic structure serves as a cap the carbon present between X₃ and X₄ is typically the point of attachment of the second ring to the remaining portion of the compound. In such instances, the structure may be represented, for example, as:

[0043] wherein the bond extending from the noted sp² hybridized carbon serves to connect the structure to the remaining portion of the compound. [0044] In one embodiment, the first ring of the fused, bicyclic structure is:

[0045] wherein X = S or NMe. [0046] In another embodiment, the first ring of the fused, bicyclic structure is:

[0047] wherein X = S or NMe. [0048] The polyamides that may be used in accordance with the present invention are further described in Phillion et al . , U.S. patent Serial No. 60/482,292, pages 3, line 22 to page 69, line 29, the contents of which are incorporated herein in its entirety. [0049] Without being held to theory, it is believed that the polyamides of the present invention that target the El binding site of the HPV genome interfere with the helicase activity of the El protein, and alter the DNA around the origin of replication by stiffening the DNA structure. These alterations prevent local unwinding, force altered states of winding elsewhere along the HPV genome, and open the HPV genome up for nuclease degradation at one or more types of sites. These sites can include: 1) the under-coiled, stiffened DNA near the origin of replication (ORI) ; 2) the junctions between the polyamide-bound DNA and the adjacent sequences; and 3) the over- coiled DNA at some distance from the ORI where polyamides are bound. These structural disruptions are hypothesized to activate a noncytolytic mechanism of viral DNA clearance by imposing an altered structural state upon the viral DNA that is recognized by the cellular components as "foreign." This altered structural state could occur by bound polyamides interfering with topoisomerase action on the HPV genome or through other structural disruptions that block virus particle formation. These structural changes prevent the virus from maintaining the protection normally afforded by the viral protein coat and interfere with tethering of viral DNA to human chromosomes and segregation of viral DNA upon cell division. [0050] In one embodiment, a polyamide is used to suppress HPV replication wherein the polyamide is designed to target HPV DNA in the El binding site region. The targeted DNA binding site having a sequence of 5' - (W) _₂G (W) _x-3 ' (SEQ ID NO 17) or 3' - (W)₁_₂G(W)_x-5' (SEQ ID NO 18), where X = 4-13; preferably, X = 4-10; more preferably, X = 4-7; more preferably, X = 7. Still more preferably, the polyamide is selected from the group consisting of Im-Py-Py-β-Py-Py-Py-γ-Py-Py-Py-β-Py-Py-Py-β-Ta (HPV2- Ta) , ^dIm-Py-Py-β-Py-Py-Py-γ-Py-Py-Py-β-Py-Py-Py-β-Ta-FITC (HPV2-Ta- FITC) , ^m-Py-Py-β-Py-Py-Py-Y_u^-Py-Py-Py-β-Py-Py-Py-β-Ta (HPV4-Ta) , L-Py-Py-β-Py-Py-Py-γ-Py-Py-Py-β-Py-Py-Py-β-Ta, L-Py-Py-β-Py-Py-Py-γ- Py-Py-Py-β-Py-Py-Py-β-Ta-FITC, and L-Py-Py-β-Py-Py-Py-γ_NH2-Py-Py-Py-β- Py-Py-Py-β-Ta, wherein "L" is a fused, non-tautomerizing heteroaromatic bicyclic structure that contains at least one hydrogen bond acceptor moiety. [0051] In another embodiment, a polyamide is used to suppress HPV replication, wherein the polyamide is designed to target HPV DNA at two or more sites in the El binding site region. The targeted DNA binding sites having a sequence of 5^,-(W)₁._aG( )_x-3' (SEQ ID NO 17) or 3 ' - ( ) _X.₂G (W) _x-5' (SEQ ID NO 18), where X = 4-13; preferably, X = 4-10; more preferably, X = 4-7; still more preferably, X = 7. Still more preferably, a polyamide selected from the group consisting of ^dIm-Py-Py-β-Py-Py- Py-γ-Py-Py-Py-β-Py-Py-Py-β-Ta (HPV2-Ta) , ^dIm-Py-Py-β-Py-Py-Py-γ-Py-Py- Py-β-Py-Py-Py-β-Ta-FITC (HPV2-Ta-FITC) , ^dIm-Py-Py-β-Py-Py-Py-γ_NH2-Py-Py- Py-β-Py-Py-Py-β-Ta (HPV4-Ta) , L-Py-Py-β-Py-Py-Py-γ-Py-Py-Py-β-Py-Py- Py-β-Ta, L-Py-Py-β-Py-Py-Py-γ-Py-Py-Py-β-Py-Py-Py-β-Ta-FITC, and L-Py- Py-β-Py-Py-Py-γ_NH2-Py-Py-Py-β-Py-Py- y-β-Ta is used to suppress HPV replication. [0052] In another embodiment, a mixture of polyamides is used to suppress HPV replication wherein the polyamides are designed to target HPV DNA in the El binding site region. The targeted DNA binding site having a sequence of 5' - (W) _1-2G (W) _x-3 ' (SEQ ID NO 17) or 3 ' - (W) _₂G (W)_x-5' (SEQ ID NO 18), where X = 4-13; preferably, X = 4-10; more preferably, X = 4-7; still more preferably, X = 7. Preferably, the mixture of polyamides is selected from the group consisting of ^dIm-Py-Py-β-Py-Py-Py-γ-Py-Py- Py-β-Py-Py-Py-β-Ta (HPV2-Ta) , ^dIm-Py-Py-β-Py-Py-Py-γ-Py-Py-Py-β-Py-Py- Py-β-Ta-FITC (HPV2-Ta-FITC) , ^dIm-Py-Py-β-Py-Py-Py-γ_NH2-Py-Py-Py-β-Py-Py- Py-β-Ta (HPV4-Ta) , L-Py-Py-β-Py-Py-Py-γ-Py-Py-Py-β-Py-Py-Py-β-Ta, L-Py-Py-β-Py-Py-Py-γ-Py-Py-Py-β-Py-Py-Py-β-Ta-FITC, and L-Py-Py-β-Py- Py-Py-γ_NH2-Py-Py-Py-β-Py-Py-Py-β-Ta. More preferably, the mixture of polyamides is selected from the group consisting of ^dIm-Py-Py-β- Py-Py-Py-γ-Py-Py-Py-β-Py-Py-Py-β-Ta (HPV2-Ta) and ^dIm-Py-Py-β-Py-Py-Py-

Y_N∞-Py-Py-py-β-Py-Py-Py-β-Ta (HPV4-τa) . [0053] In another embodiment, a mixture of polyamides is used to suppress HPV replication wherein the polyamides are designed to target HPV DNA at two or more sites in the El binding site region. The targeted DNA binding site having a sequence of 5'-(W)₁_₂G(W)_x-3' (SEQ ID NO 17) or 3 ' - (W) ₁.₂G (W)_x-5' (SEQ ID NO 18), where X = 4-13; preferably, X = 4-10; more preferably, X = 4-7; still more preferably, X = 7. Preferably, the mixture of polyamides is selected from the group consisting of ^dlm-Py-Py- β-Py-Py-Py~γ-Py-Py-Py-β-Py-Py-Py-β-Ta (HPV2-Ta) , ^dIm-Py-Py-β-Py-Py-Py- γ-Py-Py-Py-β-Py-Py-Py-β-Ta-FITC (HPV2-Ta-FITC) , ^dIm-Py-Py-β-Py-Py-Py- Y_NH2-^py-Py- y-β-^py-^py-^py-β-^Ta (HPV4-Ta) , L-Py-Py-β-Py-Py-Py-γ-Py-Py-Py- β-Py-Py-Py-β-Ta, L-Py-Py-β-Py-Py-Py-γ-Py-Py-Py-β-Py-Py-Py-β-Ta-FITC, and L-Py-Py-β-Py-Py-Py-γ_NH2-Py-^py-^py-β-^py-^py-^py-β-^Ta- More preferably, the mixture of polyamides is selected from the group consisting of ^dIm-Py-Py-β-Py-Py-Py-γ-Py-Py-Py-β-Py-Py-Py-β-Ta (HPV2-Ta) and ^dIm-Py-Py-β-Py-Py-Py-γ_NH2-Py-Py-Py-β-Py-Py- y-β-Ta (HPV4-Ta) . [0054] In another embodiment, a polyamide is used to suppress HPV replication wherein the polyamide is designed to target HPV DNA in the El binding site region of HPV-6b, HPV-11, HPV-16, HPV-18, or HPV-31. The targeted DNA binding site having the sequence 5' - (W) _1-2G (W) _x-3 ' (SEQ ID NO 17) or 3 ' - (W) _1-2G (W) _x-5' (SEQ ID NO 18), where X = 4-13; preferably, X = 4-10; more preferably, X = 4-7; still more preferably, X = 7. [0055] In another embodiment, a polyamide is used to suppress HPV replication wherein the polyamide is designed to target two or more locations in a El binding site region. Preferably, the polyamide is designed to target two or more locations in the El binding site region of HPV-6b, HPV-11, HPV-16, HPV-18, and HPV-31. More preferably, the polyamide is designed to target two or more locations in the El binding site region of HPV-16, HPV-18, and HPV-31. Still more preferably, the polyamide is designed to target two or more locations in the El binding site region of HPV-31. [0056] In another embodiment, a polyamide is used to reduce the cellular concentration of HPV episomes in infected cells below the cellular concentration of HPV episomes attributable to inhibition of replication of viral DNA. The polyamide being designed to target the HPV El binding site region of a human papilloma virus. Preferably, the polyamide reduces the cellular concentration of HPV-6b, HPV-11, HPV-16, HPV-18, or HPV-31 episomes in infected cells below the cellular concentration of episomes solely attributable to inhibition of replication of viral DNA. More preferably, the polyamides reduces the cellular concentration of HPV DNA episomes through a non-cytolytic mechanism of viral DNA clearance. [0057] In some instances, administered polyamides may not enter the nucleus of a cell in sufficient quantity to significantly suppress or inhibit HPV replication. U.S. Patent Application Pub. No. US 2003/01-9448 Al describes methods in which nuclear uptake of polyamides may be enhanced by administering polyamides with a molecular trafficking compound. The molecular trafficking compound disclosures of U.S. Patent Application Pub. No. US 2003/01-9448 Al at paragraphs 25 to 67 are incorporated herein in their entirety. In one embodiment, the polyamides of the present invention are administered with a molecular trafficking compound selected from the group consisting of P-glycoprotein inhibitors, ATPase affecting chemicals, pH or proton gradient disrupters, calcium channel blockers, ATP depleting chemicals, sodium/potassium channel blockers, MRP inhibitors, protein kinase inhibitors, Multidrug Resistance Compounds and combinations thereof.

Dosage [0058] The aforementioned polyamide compounds may be administered in pharmaceutically acceptable concentrations to the infected cells possessing the target DNA according to methods known in the art . More than one polyamide compound may be administered, separately, simultaneously, or sequentially to the infected cells, to tissue containing the infected cells, or to infected organisms. The polyamides may be administered to the subject orally, intravenously, intraperitoneally, subcutaneously, transdermally, and the like. [0059] The polyamides may be administered generally to an organism through oral or parenteral routes ( e . g. , intravenously, subcutaneously, intraperitoneally, transdermally, etc.). The polyamide may also be administered by injection or catheter to localize the polyamides to specific organs or tissues containing the target cells to be treated by polyamide therapy. The polyamide may also be administered by topical or transdermal application. [0060] The dosing regimen of polyamide compounds in the present invention is selected in accordance with a variety of factors. These factors include the selected polyamide compound or compounds, the type, age, weight, sex, diet, and medical condition of the patient, the type and severity of the condition being treated with polyamide therapy, the target cell type being treated with polyamide therapy, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetics and toxicology profiles of the particular inhibitors employed, whether a drug delivery system is utilized, and whether the inhibitors are administered with other ingredients. The dosage can be determined routinely using standard methods known in the art. The dosage regimen actually employed may therefore vary widely based upon the treated subject and therefore deviate from the exemplary dosage regimen set forth below. [0061] Administration of the polyamide compounds may be with a regimen calling for a single daily dose, multiple, spaced doses throughout the day, a single dose every other day, a single dose every several days, or other appropriate regimens. The polyamides may be prepared in physiologically acceptable media in an appropriate form for the route of administration. Polyamide compositions may be prepared as powders, solutions, and dispersions in media for both oral and parenteral routes of administration. [0062] In one embodiment, the polyamides are administered at a dosage that provides a polyamide concentration in the intracellular or extracellular location of the target cells of about 1 nM to about 1 mM. Preferably, the polyamides are provided at a dosage that provides a polyamide concentration in the intracellular or extracellular location of the target cells of about 0.1 μM to about 100 μM, more preferably between about 1 μM to 10 μM. In order to attain a desired concentration of polyamides inside the cell, the concentration of polyamides outside the cell in the extracellular sera should be approximately 2 to 1,000 times greater in concentration than the intracellular concentration. [0063] The polyamides may also be administered in combination with one or more additional therapeutic agents. Depending on the condition being treated, the combination therapy may also include antibiotics, vaccines, cytokines, molecular trafficking compounds which facilitate cellular uptake and nuclear concentration of polyamides, and the like. [0064] The following example will further illustrate the invention.

EXAMPLE 1 - Polyamide Design [0065] Polyamides were designed to bind to DNA minor groove regions that either partially or completely overlap DNA sequences where El binding proteins bind to the human papilloma virus long control region (LCR) . The El binding site sequences in the LCR region of various HPV strains are outlined in Fig. 2. Polyamides were designed to bind at seven to ten nucleotide sequences wherein the third nucleotide in the sequence is a guanine and all other nucleotides are either adenine (A) or thy ine (T) . All polyamides were targeted to 5' - (W) ₁.₂G(W)_x-3 ' (SEQ ID NO 17) or 3'- (W)₁_₂G(W)_x-5' (SEQ ID NO 18) motifs, where X = 4-7, and W = A or T. The targeted nucleotide sequences in the El binding site region that are bound by polyamides of the present invention are illustrated in bold typeface in Fig. 3. [0066] Table 1 provides a listing of the polyamides that were synthesized to bind to the El binding site in the human papilloma virus. These polyamides were prepared by solid phase synthesis and purified by reverse phase chromatography. Abbreviations used in the table include W = A or T, ^dIm = des- amino-N-methyl imidazole, -Im = 4-amino-N-methylimidazole-2- carbonyl, -Py = 4-amino-N-methylpyrrole-2-carbonyl, -y = 4-aminobutyryl, -Y_NH2 = γ-amino-β-amino-butyric acid, -β = 3-aminopropionyl, -Dp = 3- (dimethylamino) propylamino, -Ta = 3,3 ' -Diamino-N-methyldipropylamine, -Da = 3- (Dimethylamino) propylamine, NHAc = acetamide. Fluorescent tags are abbreviated as FITC, which is fluorescein-5-isothiocyanate (fluorescein) , and BOFLX, which is Bodipy-FLX fluorescent dye. Structures for building units 1 (BUI) , 7 (BU7) , 8 (BU8) and 8-β (BU8-β) are illustrated in Fig. 1. Amide bonds (-CONH-) connect the polyamide subunits.

Table 1

EXAMPLE 2 [0067] The polyamides outlined above in Example 1 and Table 1 were designed to bind to DNA minor groove regions of the El binding site in the origin of replication of the HPV long control region (LCR) in the human papilloma virus. The binding of some of the polyamides at the El binding site was discovered to suppress or inhibit replication of the HPV episome and surprisingly, promote the clearance of HPV episomes from infected cells . Cell Culture [0068] To evaluate the replication of the human papilloma virus in cells infected with HPV episomes, quantitative PCR was used. A cell culture of 31NHFK-10 (31-normal human foreskin keratinocytes-10) cells were grown on mitomycin C-treated J2 3t3 cells in E media containing three parts Dulbecco modified Eagle medium (DMEM) and one part F12 media. Media was supplemented with 0.4 μg/ml hydrocortisone, 10 ng/ml cholera toxin, 5 ug/ml insulin, 24 ug/ml adenine, 5 μg/ml transferrin, 5 ug/ml 3 , 3 ' , 5-triiodo-thyronine (T₃) , 10 ng/ml epidermal growth factor (EGF) , 1% penicillin/streptomycin, and 5% fetal bovine serum (FBS) .

Creation of a Stable HPV Cell Line [0069] A stable HPV-31 cell line was created using cloned full length HPV-31 DNA (obtained from American Type Culture Collection, Manassas, VA) . The full length HPV-31 DNA was grown in its bacterial plasmid vector and isolated by standard procedures. Full length viral DNA was liberated from plasmid DNA following restriction endonuclease cleavage with EcoRI . HPV DNA was isolated by gel purification using QIAquick Gell Extraction kit (Qiagen, Valencia, CA) , and then recircularized with T4 ligase and purified by ethanol precipitation. Recircularized DNA was resuspended in sterile H₂0. Transfections were carried out essentially as described by Fratinni et al . (1996) . Normal human foreskin keratinocytes (NHFK) were isolated by standard procedures (Watt, 1994) from foreskins obtained from the Neonatal Care Unit of Bronson Hospital (Kalamazoo, MI) . Passage 2 keratinocytes were plated at 2 X 10⁶ cells in 60 mm tissue culture dishes. The following day the cells were rinsed once with Keratinocyte-Serum Free Medium (K-SFM; GIGBCO/BRL) and then transfected in the following manner. A total of 5μg DNA (4 μg recircularized HPV DNA plus 1 μg pSV-NEO) was mixed with 200 μl K-SFM. This DNA solution was added to 200 μl K-SFM containing 16 μl LIPOFECTAMINE^® Reagent. Following a 30 minute incubation at room temperature to allow formation of DNA-lipid complexes, the mixture was diluted with 1.6 ml K-SFM and immediately applied to the NHFKs . Following an overnight incubation, the cells were rinsed and cultured for 24-48 hours in complete media, and then cultured for one week in media containing 200 μg/ml G418 (GIBCO/BRL) . Following selection, the cells were expanded in complete media and examined for HPV DNA content . Subclones were selected by limiting dilution into a 96-well plate. Wells containing single colonies were selected, amplified, and examined for HPV DNA content.

Treatment and Harvesting of Cells [0070] The cells were then harvested and treated with polyamides. Polyamides were dissolved at 10 mM in 100% DMSO then diluted with H₂0 to 1 mM. Polyamides were added to 31NFHK-10 cells at 0.1-10 μM with final DMSO concentration of 0.1% DMSO as controls. After incubation, cells were harvested from the plates by either trypsinization or direct lysis with proteinase K digestion buffer (PK buffer-100 mM NaCl, 10 mM Tris pH 8, 25 mM EDTA, 0.5% SDS, 0.1 mg/ml proteinase K) . Trypsinized cells were counted on a hemocytometer, spun down, and episomal HPV was isolated by the Hirt method (Hirt, 1967) . Briefly, cell pellets were lysed in 0 . 6% SDS with 10 mM EDTA. NaCl was added to a final concentration of 1 M. Following an overnight incubation at 4o C, precipitates containing the chromosomal DNA were spun down and episomal DNA precipitated by the addition of isopropanol. Cells directly lysed in PK buffer were transferred to microfuge tubes and incubated at 50° C for two hours. Lysates were then extracted with phenol/chloroform/isoamyl alcohol and spun through a phase lock gel (heavy-Eppendorf, Hamburg, Germany) . Total DNA was precipitated with 0.3 M NaOAc and 2.5 vol . ethanol and resuspended in TE (i . e . , Tris-EDTA, (10 mM Tris-HCl, 1 mM EDTA)) .

Quantization of HPV DNA [0071] To examine HPV DNA content, quantitative PCR was performed using Real-Time PCR technology on the ABI PRISM 7700 Sequence Detector (Applied Biosystems, Foster City, CA) . HPV-31 primers and probe were designed using Primer Express 1.0 (Applied Biosystems, Foster City, CA) within the Ll gene: sense 5'-CCTGCTATTTTGGAAGATTGGAAT-3^* (SEQ ID NO 19), antisense 5^*-GGCCTGTGAGGTRGACAAACC-3 ' (SEQ ID NO 20), antisense 5^*-TTGGATTGACCACACCTCCCTCAGGTT-3' (SEQ ID NO 21). Human ApoE primers, sense 5 ' -TGAACTTCTGGGCTCAAGCG-3 ' (SEQ ID NO 22), antisense 5 ' -AGAGAGAAGAAGGAGCTAGGAGG-3 ' (SEQ ID NO 23), and probe 5'-TGGGATTAGAGGCATGAGCACCTTGC-3' (SEQ ID NO 24). All primers and probes were synthesized and HPLC purified by Applied Biosystems. The HPV probe was labeled with 5 ' reporter dye FAM (6-carboxyfluorescein) and the 3' quencher dye TAMRA (6-carboxytetramethylrhodamine) . The ApoE probe was labeled 5' with VIC A standard curve developed using HPV-3l/pBR322 and designed to include a range of 10⁸ to 10 copies/reaction using the following formula:

[0072] (1.82 X 10¹⁵) (μg/μl stock DNA) / (length in base pairs) (2) = copies/ul stock DNA.

[0073] PCR reactions contained final concentrations of IX Universal Master Mix (PE Applied Biosystems) , 300 nM each primer, and 200 nM probe (PE Applied Biosystems) in a reaction volume of 50 μl . Each Hirt-isolated DNA was analyzed in triplicate reactions for episomal HPV. Copies/reaction was determined from the standard curve and copies/cell was figured as : [0074] (copies/reaction) (DNA dilution) / (cells/culture) = copies/cell .

[0075] Alternatively, total DNA was used in multiplex reactions with HPV-31 and ApoE amplified together. The relative ration of HPV-31 to genomic DNA (ApoE) was determined by: (HPV Ct - ApoE Ct)

[0076] A summary of the effect of various polyamides on the replication of the human papilloma virus in cells infected with HPV is illustrated in Fig. 4A - Fig. 4D. [0077] An illustration of the binding of a polyamide to the El binding site of HPV-31 is provided in Fig. 5. The illustrated polyamides include HPV2, wherein R = H, and HPV4 wherein R = NH₂. The illustrated polyamides bind to DNA regions having a 10 base pair length. [0078] Collectively, these results indicate that polyamides can suppress replication of the human papilloma virus in infected cells .

EXAMPLE 3 [0079] A single experiment was conducted on a cell line infected with HPV-16 following the procedures set forth above in Example 2. The results of the experiment did not identify any polyamide described in Example 1 that suppressed or inhibited replication of the HPV-16 in infected cells in a statistically significant manner. [0080] The HPV-16 cell line used in this experiment, however, was not as well-behaved and less predictable than the HPV-31 cell line used in the experiments outlined in Example 2. For example, whereas the number of copies of episomes within the cells of the HPV-31 cell line would be fairly predictable, e. g. , remaining around 1000 copies/cell in the control cells, the copies/cell in the HPV-16 control cells would vary widely. It was believed that the unpredictability may be due to the HPV-16 virus possibly causing greater genomic instability in the cell line than was observed in the HPV-31 cell line. Another theory is that HPV-16, being highly oncogenic, may cause instability in the cell line due to cell proliferation resulting from the oncogenic properties . [0081] For the reasons given above, the results of the single experiment conducted are therefore not believed to be necessarily indicative of results that may be anticipated from in vivo testing. Thus, as HPV-16 has two binding regions in its El binding site similar to those of the HPV-31 binding site, the in vivo effects of polyamides HPV2-Ta and HPV4-Ta are anticipated to be similar to those observed in the HPV-31 cell line.

EXAMPLE 4 SKIN DISTRIBUTION OF THE ANTI-GENITAL WARTS AGENT PNU-692435E AFTER TOPICAL ADMINISTRATION IN DMSO TO HAMSTER EARS

[0082] According to the present invention, a polyamide compound that may be used to treat HPV infections, including genital warts is, e.g., PNU-692435E. For treatment of genital warts, it would be desirable for this compound to show local therapeutic activity after topical application. PNU-692435E ' s molecular weight is in excess of 1000; thus, it was unknown whether or not this compound would penetrate the skin at all. The following experiment was designed to evaluate the penetration and effectiveness of this compound when administered topically. The effectiveness was determined by observing the distribution of labeled PNU-692435E in various layers and fractions of skin after topical administration of the drug in solution form. [0083] The most straightforward model for this is the hamster ear, a model for human scalp tissue that has been used to screen minoxidil hair growth formulations for delivery to the hair follicles. While properties of the hamster ear may not be identical to those of the type of skin tissue affected by genital warts, particularly since the barrier properties of the latter may be compromised by the disease, hamster ear is still a convenient and quick model for determining local drug delivery in the skin. [0084] In the normal procedure/use of this model, the contents of the sebaceous glands, and by extrapolation, the hair follicles, are assayed. However, the dissection procedure connected with isolation of the sebaceous gland fraction lends itself easily to isolation of individual skin fractions (stratum corneum, dermis/viable epidermis, sebaceous glands, tissue below dermis and associated with ear cartilage (representing subdermal skin tissues) , and other tissues accessible only by systemic circulation, e.g., on the dorsal side of the ear) . The relative amount of labeled drug in each skin fraction can then easily be determined through scintillation counting. [0085] Preparation of stock solution (labeled PNU-692435E + unlabelled PNU-470580E) [0086] Radiolabeled PNU-692435E (3.5 mCi) was used in this experiment. To be properly used as a tracer, the radiolabeled material must be mixed with a significantly greater quantity of unlabelled material; otherwise, there is the possibility that the trace amount of labeled material would get bound up in the stratum corneum. The labeled PNU-692435E was therefore dissolved in a 10 g/ml PNU-470580E solution. This latter compound is a close analog of PNU-692435E with slightly higher molecular weight and served as a surrogate "diluent" :

[0087] PNU-692425E (radiolabelled material) ; molecular weight 1628.38

[0088] PNU-470580E (cold material) ; molecular weight 1743.37 [0089] A 10 mg/ml stock solution of the unlabelled material was prepared in reagent grade DMSO (Burdick & Jackson) . The solid material dissolved rapidly into solution after only a fraction of the DMSO had been added, suggesting that the solubility of PNU-470580E in DMSO is significantly greater than 10 mg/ml. 300 ml of this solution was transferred, using a Hamilton syringe, to the heavy-duty conical vial containing the 3.5 mCi of labeled PNU-692435E. The solution was carefully swirled with a vortex mixer on a low setting. After five minutes, we noted that the yellow-brown material seen initially in the bottom of the vial had disappeared, leaving only a pin point-sized black speck at the tip of the conical vial. Both stock solutions were kept in the freezer (-20°C) in between experiments . [0090] The final volume of solution (300 ml) was chosen so that 20 ml, the amount usually applied to each hamster ear in the distribution experiments, would contain about 0.23 mCi or 500,000 dpm. Therefore there would be enough solution to dose about 10-12 ears plus one 20 ml sample to get a measurement for "total activity applied."

The hamster ear dissection procedure [0091] At 30 minutes, 1 hour, or 2 hours post application to the ears, the following dissection procedure was used to isolate skin tissue fractions for determination of drug distribution. This procedure has been previously described in Randall Stehle and Andrea Cory, SR aO093604, "Assessment of follicular delivery of minoxidil from topical formulations using the hamster ear model II," In vivo procedure, 15 March 2001. [0092] 1. The hamster, lying within a Plexiglas chamber maintained at 37°C, is kept under light inhalation anesthesia using metered delivery (-500 cc/min at 1 atm. and 70°F) of a predetermined (ca. 1.5:98.5, by experience) isoflurane USP: oxygen mixture.

[0093] 2. With a positive displacement pipette, 20 ml of solution is applied to the ventral surface of each ear of the lightly anesthetized hamster. The rounded end of a glass capillary melting point tube is used to distribute the material evenly on the skin (one tube for each ear) . Used capillary tube is placed into a scintillation vial (to evaluate loss via this route) .

[0094] 3. When the specified time for the study (usually 1 or 2 hours) has elapsed, the hamster is sacrificed and one ear is removed (the second ear is removed and dissected after completing dissection of the first) .

[0095] 4. The ear is mounted on a board with pushpins. Both ends of a Q-tip are used to swab excess remaining dosed material from the ventral ear surface, following which both Q-tip ends are placed in a scintillation vial .

[0096] 5. The inside surface of the ear is stripped repeatedly with transparent tape until the surface glistens and the last tape strip looks clean (meaning that stratum corneum removal is complete) . The usual total number of strippings is about 25; successive groups of five pieces of tape are placed in separate scintillation vials.

[0097] 6. The ear is cut along the body end for a nice sharply defined edge to facilitate the separation of the dermis layer from the cartilage. The trimmed-off piece is placed in a scintillation vial.

[0098] 7. The upper skin layers (dermis + viable epidermis) are carefully peeled back as a unit and set them aside, dermis side up, on a clean microscope slide with a few drops of water to keep them moist.

[0099] 8 With the cartilage part ("lower skin layers") on a separate microscope slide, the residual- tissue surface is carefully scraped and scrapings are rinsed with water into 2-3 scintillation vials.

[0100] Dorsal ear tissue (from cartilage to dorsal ear surface) is placed into a scintillation vial .

[0101] 10 Step 8 is repeated with the dermis/sebaceous gland tissue fraction from step 7.

[0102] 11 After scraping and rinsing (step 10) , the remaining dermal tissue is placed into another scintillation vial.

[0103] 12 To each scintillation vial containing tissue fractions, approximately 10 ml of scintillation fluid is added, mixed well, followed by obtaining ¹⁴C counts for radioactive drug.

[0104] 13 Each sample's result is reported as a fraction of applied dose.

[0105] The data for the various times and skin fractions appear in Tables 2 and 3. The results are reported as percentages of applied dose. As can be seen, there was radioactivity in all skin fractions. Therefore, it can be noted that despite having a molecular weight greater than 1000, this molecule, at least when applied as a DMSO solution, is well absorbed and widely distributed among the various layers of the skin. The measured mass balance concentrations of the molecule were significantly less than the dose applied and about 24-65% of theory. The lower molecule concentration thus suggests that the molecule was absorbed into the peripheral bloodstream and systemically distributed in the body. This would explain the amount of activity appearing on the back of each ear; moreover, systemic distribution of drug means, to a certain degree, possible cross-distribution between ears.

PHA 3121.2 34

Table 2. Distribution of Pnu-692435e in Skin Tissues after Topical Application in DMSO

%dose %dose %dose Tissue (30 mins) (lhour) (2 hours Skin surface 7.56, 18.1, 15.3, 40.0 40.9, 16.1, 30.6, 22.7, 10.9, 23.2, 17.5 15.8, 34. Stratum corneum 6.51, 8.58, 9.74, 13.9 19.9, 17.1, 18.4, 16.8, 11.1, 8.39, 11.6 10.7, 21. (6 vials) Ear trimming 4.03, 1.97, 6.16, 4.99 5.37, 2.71, 7.64, 8.84, 7.03, 3.32, 3.31 2.23, 1.5

Sebaceous glands 1.23, 1.39, 2.07, 1.29 1.01, 1.23, 1.11, 1.79, 1.75, 1.31, 0.52 0.88, 1.3 (5 vials) Dermis/viable epidermis 0.50, 0.49, 0.62, 0.45 0.58, 0.41, 0.37, 0.62, 0.66, 0.43, 0.16 0.35, 0.4 Subdermal tissue; 2.43, 1.14, 6.22, 2.98 1.65, 4.26, 3.28, 4.97, 2.17, 5.16, 1.81 8.00, 1.9 cartilage (3 vials) Dorsal ear tissues 1.50, 0.52, 2.81, 1.80 1.79, 3.36, 1.62, 2.51, 3.20, 1.58, 0.41 0.28, 1.3

Total recovery 23.7, 32.2, 42.9, 65.4 64.0, 45.1, 63.1, 58.1, 36.8, 43.4, 35.3 33.3, 63.

PHA 3121.2 35

Table 3. Average Tissue Contents as Percent of Applied Dose (No. of Experiments.)

Tissue 30 minutes (4) 1 hour ( 7 ) 2 hours (2) Skin surface 20.2% ± 13.9% 23.1% ± 10.0% 25.3% ± 13.4%

Stratum corneum 9.68% + 3.11% 14.8% ± 4.34% 16.3% ± 7.85%

Ear trimming 4.29% + 1.77% 5.46% ± 2.43% 1.88% ± 0.50%

Sebaceous glands 1.50% ± 0.38% 1.25% ± 0.44% 1.11% ± 0.33%

Dermis/ viable epidermis 0.52% ± 0.07% 0.46% ± 0.17% 0.40% ± 0.07%

Subdermal tissue 3.19% ± 2.16% 3.33% ± 1.49% 4.95% ± 4.31%

Dorsal ear tissues 1.66% + 0.94% 2.07% ± 1.03% 0.84% ± 0.78%

[0106] It should also be noted that since the dissections were done sequentially (right ear first and then the left ear) , the second ear' s absolute distribution of radioactivity can differ from that of the first ear. Furthermore, since it is generally needed about 30-40 minutes to perform a careful dissection, further diffusion and distribution may occur within the second ear in that time. [0107] From these results, it is evident that when administered topically in DMSO, PNU-692435E is quickly and widely distributed throughout different layers of the skin, such as stratum corneum, viable epidermis, dermis, sebaceous glands, and subdermal tissue. The observed less-than-100% mass balance, along with the rapid change in distribution with time, suggests significant and rapid systemic distribution of this drug after topical administration, most likely due to the highly penetration-enhancing nature of DMSO. [0108] Accordingly, PNU-692435E should be tested in formulation with other less-penetrating solvents in order to achieve a more selective drug delivery. By way of example, the solvents that may be tested in formulation with PNU-692435E may also include petroleum jelly; alcohols such as methanol, ethanol, propanol, isopropanyl, butanol, t-butanol, and the like or other solvents known in the art for use in topical delivery of pharmaceuticals. The efficacy of PNU-692435E with such solvents can be evaluated as described above for PNU-692435E and DMSO. [0109] Furthermore, additional polyamide compounds may be tested as described above; however, it should be noted that all of the above-mentioned procedures can be modified for a particular study, depending on factors such as the polyamine compound used, solvent that is used, etc. Such modifications can be designed by a skilled artisan without undue experimentation. EXAMPLE 5 Higher Specificity Polyamides for the HPV31 El Binding Site

[0110] Polyamides designed to the HPV-31 El binding site were synthesized with a combination of solid and solution phase synthesis. BIAcore measurements can establish their binding behavior to the HPV-31 El binding site.

The following polyamide has two match sites within the El binding site. The polyamide was "capped" with an acetamide group.

β-PyPyPyMϊCOCH₃

[0111] The following polyamide is a variation of the polyamide provided above that further includes a β -alanine group in the tail .

[0112] The following polyamide is a variation of the polyamide provided above that further includes a β -alanine group in the tail . ^dI PyPyPy-ι Lβ-PyPyPyNHCOCH, Da-β-PyPyPyPy-"

[0113 ] The following polyamide links the two hairpins with a linker intended to also bind in the minor groove (there are many examples of tandem hairpins which show good binding, but a tandem hairpin with a linker of this length is unprecedented) .

[0114] The following polyamide links two hairpins that bind to one side of the El binding site.

[0115] The following polyamide uses an extended tail with one hairpin.

TmPyPyPy-. DaPyPyPyβPyPyPyPy-βPyPyPyβPyPyPyPy JNH₇

[0116] The following polyamides are linear polypyrrole polyamides with an 8-amino-3, 6-dioxaoctonoic acid linker (L) . These types of polyamides bind DNA, the specificity of binding to the HPV-31 El binding site can be established by BIAcore measurements. The polyamide given immediately below is merely one example, but there are several AT rich sites in the El binding site that could all be targeted using linear polypyrrole polyamides with an 8-amino-3 , 6-dioxaoctonoic acid linker (L) .

Da-βPyPyPyPyPy-L-PyPyPyPyNHCOCH₃

[0117] The following polyamide is an example of a single hairpin polyamide that could be designed to bind with potentially lower specificity than would be expected from the polyamides provided above. However, these polyamides could be used to study the ability of polyamides to disrupt El-protein/DNA interactions and their suitability for in vi tro assays. ^dImPyPyPyPy-ι Da-β-PyPyPyPyPy-l^NH2 [0118] The following two polyamides are much longer than any of the other polyamides described above . They are made up of seven and four hairpin polyamide building units, respectively that are illustrated in Fig. 1. These polyamides should have extremely high binding constants to the HPV DNA. They can be prepared by solution phase synthesis, like connecting the individual hairpin units designated the Building Unit 1 (BUI) , BU8, and BU7. The nature of the solution phase synthesis will provide a series of compounds, each differing in length by one building unit. The final products are shown below, but there will also be eight intermediate products generated from the synthesis which may be utilized. These molecules may also be modified to be dye-labeled which may affect their cellular uptake and nuclear localization.

BU7- β-BU8- β-BU8- β-BU8 -Da

BU8- β-BU8- β-BU8- β-BU8- β-BU8- β-BU8- β-BUI-Da

[0119] In view of the above, it will be seen that the several objects of the invention are achieved.

As various changes could be made in the above compositions and processes without departing from the scope of the invention, it is intended that all matter contained in the above description be interpreted as illustrative and not in a limiting sense.

Claims

WHAT IS CLAIMED IS:

1. A process for regulating replication of a human papilloma virus in an infected cell, the process comprising administering a polyamide in pharmaceutically acceptable concentrations to the infected cell, to tissue containing the infected cell, or to infected organisms comprising the human papilloma virus DNA, the polyamide comprising N-methyl pyrrole (Py) and either des-amino-N-methyl imidazole (^dIm) or a fused, non-tautomerizing heteroaromatic bicyclic structure (L) that contains at least one hydrogen bond acceptor moiety, wherein said polyamide binds to a El binding site in a human papilloma virus episome and regulates replication of the human papilloma virus.

2. The process of claim 1, wherein the polyamide targets the El binding site at a sequence 5' - (W) _X.₂G (W)_x-3 ' (SEQ ID NO 17) or 3' - (W)₁.₂G(W)_x-5 (SEQ ID NO 18), wherein W is adenine or thymine, G is guanine, and X is 4-13.

3. The process of claim 2 , wherein X is 4-7.

4. The process of claim 2, wherein the polyamide is selected from the group consisting of ^dIm-Py-Py-β-Py-Py-Py-γ-Py-Py- Py-β-Py-Py-Py-β-Ta, ^dIm-Py-Py-β-Py-Py-Py-γ-Py-Py-Py-β-Py-Py-Py-β-Ta- FITC, ^dIm-Py-Py-β-Py-Py-Py-γ_NH2-Py-Py-Py-β-Py-Py-Py-β-Ta, L-Py-Py-β-Py- Py-Py-γ-Py-Py-Py-β-Py-Py-Py-β-Ta, L-Py-Py-β-Py-Py-Py-γ-Py-Py-Py-β-Py- Py-Py-β-Ta-FITC, and L-Py-Py-β-Py-Py-Py-Y_røa-Py-Py-Py-β-Py-Py-Py-β-Ta, wherein β is 3-aminopropionyl, γ is 4-aminobutyryl, γ_BH2 is γ- amino-β-amino-butyric acid, Ta is 3 , 3 ' -Diamino-N- methyldipropylamine, and FITC is fluorescein-5-isothiocyanate.

5. The process of claim 1, wherein the polyamide targets human papilloma virus DNA at two or more sites in the El binding site region.

6. The process of claim 1, wherein a mixture of polyamides suppresses human papilloma virus replication wherein the polyamides target human papilloma virus DNA in the El binding site region.

7. The process of claim 6, wherein the polyamides targets human papilloma virus DNA at two or more sites in the El binding site region.

8. The process of claim 1 wherein the human papilloma virus is selected from the group consisting of HPV-6b, HPV-11, HPV-16, HPV-18, and HPV-31.

9. The process of claim 1 wherein the human papilloma virus is administered with a molecular trafficking compound selected from the group consisting of P-glycoprotein inhibitors, ATPase affecting chemicals, pH or proton gradient disrupters, calcium channel blockers, ATP depleting chemicals, sodium/potassium channel blockers, MRP inhibitors, protein kinase inhibitors, multidrug resistance compounds and combinations thereof .

10. A polyamide compound for regulating replication of human papilloma virus comprising N-methyl pyrrole (Py) and either des-amino-N-methyl imidazole (^dIm) or a fused, non-tautomerizing heteroaromatic bicyclic structure (L) that contains at least one hydrogen bond acceptor moiety, wherein the polyamide specifically binds to the El binding site of DNA in human papilloma virus.

11. The polyamide compound of claim 10 wherein the polyamide binds to sequences which partially or completely overlaps an El binding site for the human papilloma virus.

12. The polyamide compound of claim 10, wherein the human papilloma virus is selected from the group consisting of HPV-6b, HPV-11, HPV-16, HPV-18, and HPV-31.

13. The polyamide compound of claim 10, wherein the polyamide targets the El binding site at a sequence

5'- (W)_1-2G(W)_x-3' (SEQ ID NO 17) or 3 ' - (W) .₂G (W) _x-5 ' (SEQ ID

NO 18) , wherein W is adenine or thymine, G is guanine, and X is

4-13.

14. The polyamide compound of claim 10, wherein X is 4-7.

15. The polyamide compound of claim 10, wherein the polyamide compound is selected from the group consisting of ^dIm- Py_Py_β-Py_Py_Py-γ-Py_Py-.py-β-Py-Py_Py_β_Ta, ^dIm-Py-Py-β-Py- y-Py-γ-Py- Py-Py-β-Py-Py-Py-β-Ta-FITC, ^dIm-Py-Py-β-Py-Py-Py-γ_NH2-Py-Py-Py-β-Py-Py- Py-β-Ta, L-Py-Py-β-Py-Py-Py-γ-Py-Py-Py-β-Py-Py-Py-β-Ta, L-Py-Py-β-Py- Py-Py-γ-Py-Py-Py-β-Py-Py-Py-β-Ta-FITC, and L-Py-Py-β-Py-Py-Py-γ_NH2-Py- Py-Py-β-Py-Py-Py-β-Ta.