Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Ester doai
Reexamination Certificate
1999-10-15
2001-04-10
Reamer, James H. (Department: 1614)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Ester doai
C514S721000, C560S174000
Reexamination Certificate
active
06214874
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the use of nordihydroguiaretic acid derivatives, in particular tetramethyl nordihydroguiaretic acid and tetraglycinal nordihydroguiaretic acid, for the treatment of tumors induced by human papillomavirus.
2. Background Information
Human papillomavirus (HPV) infection causes unregulated cell growth in many types of squamous epithelial cells, resulting in afflictions ranging from benign pallilomae (warts) to cervical, penile and mouth cancer. The strong association of these cancers with HPV and the widespread occurrence of infection denotes the importance of developing an anti HPV therapy.
Most, if not all, viruses, including those replicatively active mutants, are host dependent. They require the participation of certain cellular factors for supporting viral growth. Host cellular factors, unlike viral proteins, are not under mutational pressure and are in general, structurally invariable. Thus, compounds that block the usage of these cellular factors at different stages of the viral life cycle are likely to be good candidates as mutation insensitive antiviral drugs. Several studies using cellular factors as alternative targets for the inhibition of HIV-1 have been reviewed (1).
Applicants reported earlier that 3′-0-methylated NDGA (i.e. Mal.4), isolated from Creosote bush (Larrea tridentata) can specifically block basal HIV transcription, Tat-regulated transactivation, and HIV replication in human cell culture (2,3,4). Mal.4 exerts its effects by interfering with the binding of transcription factor Sp1 to the promoter of the HIV proviral template. The target of Mal.4 is mapped to nucleotides −87 to −40, the Sp1 binding sites of the HIV long terminal repeat (LTR). The unmodified NDGA, in vitro, does not inhibit HIV transcription and has no effect on Sp1 binding (2).
Isolation and purification of plant lignans, however, is labor intensive and costly. In anticipation of the possible clinical use of plant lignans in controlling Sp1-regulated viral and tumor growth in humans, nine different methylated NDGA activities were synthesized chemically using unmethylated NDGA as the parent substrate in large quantities with low cost (5). At drug concentrations below 30 &mgr;M, tetramethyl NDGA was found to be most effective in the control of replication HIV via inhibition of Sp1 regulated proviral transcription and transactivation (5). This study has since been extended to the control of the growth of Herpes simplex virus (HSV-1 and HSV-2) (6). Herpes simplex immediate early (IE) ICP4 gene is essential for HSV replication (7). Its promoter region possesses eight Sp1 consensus binding sites (8), five of which are required for ICP4 gene expression. It thus makes the ICP4 gene a good candidate for such testing. Applicants have found that both 3-0-methyl NDGA (Mal. 4) and tetramethyl NDGA (M
4
N) are effective transcriptional inhibitors for HSV ICP4 gene expression in Vero cells via the blocking of Sp1 protein binding to the ICP4 promoter as shown by the electrophoretic mobility shift assay (6).
When the anti-HSV activities of M
4
N and Mal. 4 were tested and compared to that of acycloguanosine (acyclovir, ACV) in infected Vero cells, Applicants observed that the IC
50
for M
4
N varied between 11.7 &mgr;M to 4 &mgr;M for 10 passages of HSV-1 and 4 passages of HSV-2 without obvious uprising trend for requirement of higher drug concentration. However, the IC
50
for ACV increased from 7 &mgr;M for the first viral passage to 444 &mgr;M for the tenth passage of HSV-1 and to >88 &mgr;M for the fourth passage of HSV-2 indicating their rapid build-up of drug resistance against ACV in Vero cells. Consequently, while the selective index, S.I. (TC
50
/IC
50
) remained relatively stable for M
4
N, the S.I. for ACV dropped 60 fold following the viral passages in Vero cells (6). Thus M
4
N is a mutation insensitive drug. It can inhibit ACV resistant HSV effectively (6).
Due to the fact that Sp1 is an important cellular transcription factor (9), the possible inhibitory effect of this class of compounds on the expression of Sp1-regulated cellular genes should be addressed. Mal.4 cannot displace Sp1 once it is stably bound to its enhancer sites (2). It therefore seemed likely that NDGA derivatives would have a greater effect on Sp1-regulated genes in proliferating cells than on the expression of Sp1-regulated housekeeping genes in stationary cells. In the former case, the drug will be able to compete with Sp1 protein for the Sp1 sites in gene promoters during DNA synthesis, while in the latter case, the drug may have little effect on the transcribing chromatin of housekeeping genes with Sp1 protein already stably bound at their promoters. This in fact has been shown to be the case. As will be demonstrated below, by using gene array studies with 9600 expressed genes, Applicants found products of most Sp1 regulated genes remained at similar levels, are not affected by the drug treatment of cervical cancer cells C3 in culture (FIG.
5
). Even so, the relatively low selective index of M
4
N certainly limits its use to the lowest effective concentration if the drug must be used systemically. On the other hand, human papilloma virus induces solid cervical and oral tumors initially through the Sp1 regulated expression of HPV E
6
/E
7
genes (10). Applicants reasoned that if drug can be delivered in situ, and be kept only in the tumor area, the drugs of high concentration may be used to effectively destroy the tumor with little damage to the patients.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide a method for the treatment of tumors induced by human papillomavirus. The method comprises local application of nordihydroguiaretic acid derivatives, in particular tetramethyl nordihydroguiaretic acid (M
4
N) and tetraglycinal nordihydroguiaretic acid (G
4
N), to HPV-induced tumors.
By nordihydroguiaretic acid derivatives is meant compounds of the structure
wherein R
1
, R
2
, R
3
and R
4
independently represent —OH, —OCH
3
, —O(C═O)CH
3
, or an amino acid residue, but are not each —OH simultaneously. Particularly preferred compounds for use according to the invention are M
4
N and G
4
N, which are shown in FIG.
1
.
HPV-induced tumors include in particular, but are not limited to, cervical, oral, penile and head and neck cancers which are associated with HPV infection.
It is contemplated that M
4
N and other derivatives will be administered by local injection into the tumors, generally along with pharmaceutically acceptable diluents, excipients and carriers. In preferred embodiments, M
4
N is injected into tumors in the form of a DMSO solution, and G
4
N is administered in PBS solution. The use of G
4
N will complement the use of M
4
N, particularly in larger tumors (>2 cm
3
), due to its water solubility, which allows it to spread to a larger region of the tumor. Other water-soluble and water-insoluble nordihydroguiaretic acid derivatives can be similarly employed, according to the invention.
By pharmaceutically acceptable diluents, excipients and carriers is meant such compounds as will be known to persons of skill in the art as being compatible with M
4
N, G
4
N and other similar derivatives and suitable for local administration to a human or other mammal according to the invention. Although the examples hereinbelow describe administration by means of local injection, other means of local administration, such as topical application or targeted delivery to the tumor site, may also be used.
The amount of compound administered to obtain the desired treatment effect will vary but can be readily determined by persons of skill in the art. The amount of dosage, frequency of administration, and length of treatment are dependent on the circumstances, primarily on the size and type of tumor. However, dosages of from 10 mg to 20 mg of either M
4
N alone or with similar amounts of G
4
N per gram tumor weight at intervals from daily to weekly or less frequently may be mentioned for purposes of
Heller Jonathan D.
Huang Ru Chih C.
John Hopkins University
Pillsbury Madison & Sutro LLP
Reamer James H.
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