Oligonucleotide mediated reversal of drug resistance

Details Oligonucleotide mediated reversal of drug resistance Oligonucleotide mediated reversal of drug resistance

1996-11-12

2002-08-27

Wang, Andrew (Department: 1635)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S325000, C435S375000, C536S023100, C536S024500

Reexamination Certificate

active

06440660

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to drug resistance among infectious organisms or agents. More particularly, this invention relates to the use of antisense oligonucleotides to reverse such drug resistance, thereby resensitizing the infectious agents to therapeutic drugs.
BACKGROUND OF THE INVENTION
A number of diseases are caused by infectious organisms which have become resistant to various chemotherapeutic drugs commonly used to treat such organisms. One such disease is malaria. Malaria is estimated to afflict more than 200 million people annually (WHO (1992)
Bull. W.H.O.
70:801-804). While presently confined primarily to the tropics where it is endemic, malaria was formerly very wide spread, including the United States. However, in the late 1980's small foci of periodic transmission of malaria in San Diego County were reported (Anonymous (1990)
JAMA
263:1617), demonstrating that transmission capacity is still present in the United States, and that under the right circumstances, malaria could possibly become endemic once again.
Malaria is caused by infection with one or more species of Plasmodium. Three species (
P. vivax, P. ovale
, and
P. malariae
) produce relatively mild symptoms consisting of spiking periodic fever, anemia, and some jaundice. In contrast, infection with
P. falciparum
can lead to coma and death unless chemotherapy is initiated, and is responsible for about 800,000 deaths per year among African children under 5 years (WHO (1992)
Bull. W.H.O.
70:801-804; and WHO (1992)
WHO Weekly Epidemiological Record
22: p. 161-167).
Malaria infection is transmitted via the bite of an infected Anopheline mosquito during her blood meal. The sporozoite stage of the parasite is injected into the human host in mosquito saliva, and sporozoites then migrate to the liver where they invade hepatocytes, becoming intracellular parasites. Multiplication occurs within hepatocytes, which then rupture to release merozoite-stage parasites. These in turn invade circulating erythrocytes, beginning the asexual erythrocytic cycle of the parasite life cycle. It is the erythrocytic stages which are responsible for pathology to the human host. Within the erythrocyte, merozoites first develop into ring stage parasites, then trophozoites, then schizonts. Parasite DNA replication occurs during the trophozoite stage, giving rise to 16-20 merozoites at the end of schizogony. Mature schizonts cause the host erythrocyte to lyse, releasing merozoites which then reinvade erythrocytes to continue the cycle.
A small proportion of merozoites develop into male or female gametocytes. When drawn into a mosquito midgut during her blood meal, these erupt from the erythrocytes, fertilization occurs, and the zygotes penetrate the mosquito midgut wall to become oocysts. After asexual multiplication within oocysts, sporozoites are released, which migrate to the insect salivary glands to await the next mosquito blood meal. Injection of sporozoites in mosquito saliva during that next meal reinitiates the parasite life cycle.
Malaria has been treated with a variety of drugs, including anti-folate compounds such as pyrimethamine, trimethoprim, and proguanil (which inhibit the enzyme dihydrofolate reductase (Bzik et al. (1987)
Proc. Natl. Acad. Sci. USA
84:8360-8364)), sulfonamides, (which inhibit dihydropteroate synthetase (Brooks et al. (1994)
Eur. J. Biochem.
224:397-405), 4-aminoquinolines such as chloroquine (quinine analogs), sulfones, sulfanamides, and tetracyclines.
The antifolate drugs work by binding their target enzymes, thereby preventing normal enzyme function. While effective, resistance to these drugs can be mediated by selection for one or at most two point mutations which prevent binding of the drug to the active site (Brooks et al. (1994)
Eur. J. Biochem.
224:397-405; Basco et al. (1995)
Mol. Biochem. Parricidal.
69:135-138). Consequently, resistance to these drugs appeared fairly soon after these drugs were introduced (Peters,
Chemotherapy and Drug Resistance in Malaria
. (1987) London: Academic Press, pp. 15-20).
Until recently, chloroquine was by far the most commonly used antimalarial compound, owing to its low cost and lack of side effects compared with the antifolates (Peters,
Chemotherapy and Drug Resistance in Malaria
. (1987) London: Academic Press, pp. 5-14). However, after years of widespread chloroquine use, foci of resistant
P. falciparum
have been identified wherever malaria is endemic. Because of this widespread resistance, first antifolates, and now mefloquine have largely replaced chloroquine for treatment of
P. falciparum
and
P. vivax
as well (Peters,
Chemotherapy and Drug Resistance in Malaria
. (1987) London: Academic Press, pp. 659-670).
Mefloquine (a quinalone-methanol) has been shown to be effective against multi-drug resistant strains of
P. falciparum
(Harinasuta et al. (1983)
Bull. WHO
61:299-305), and also been used for prophylactic use by travellers (Anonymous (1990)
JAMA
263:2729-2737). While somewhat expensive and not without side effects, it remains the drug of choice for treating multi-drug resistant malaria (White (1988)
Eur. J. Clin. Pharmacol.
34:1-14; and Anonymous (1990)
JAMA
263:2729-2737). However, despite extensive measures to protect the efficacy of mefloquine, resistance has developed rapidly, and has even been found in areas where the drug has not been used clinically. In addition, wide spread cross resistance to other drugs has been demonstrated including structurally unrelated compounds such as halofantrine (Ringwald et al. (1990)
Lancet
335:421-422; Gay et al. (1990)
Lancet
336:1262; and Wilson et al. (1993)
Mol. Biochem. Parricidal.
57:151-160), and artemesinin (Wilson et al. (1993)
Mol. Biochem. Parricidal.
57:151-160), in addition to quinine (Brasseur et al. (1992)
Am. J. Trop. Med. Hyg.
46:1-7; Brasseur et al. (1992)
Am. J. Trop. Med. Hyg.
46:8-14; and Suebsaeng et al. (1986)
Bull. WHO
64:759-765). The apparent cross-resistance to quinine is particularly significant because intravenous quinine remains the treatment of last recourse in cases of severe or cerebral malaria (Warrell et al. (1990)
Trans. R. Soc. Trop. Med. Hyg.
84(suppl 2):1-65).
There is thus a need for improved chemotherapeutic drugs whose use inhibits or controls parasite infection without ultimately resulting in widespread resistance to such drugs and to those related thereto.
New chemotherapeutic agents have been developed which are capable of modulating cellular and foreign gene expression (see, Zamecnik et al. (1978)
Proc. Natl. Acad. Sci.
(USA) 75:280-284). These agents, called antisense oligonucleotides, bind to target single-stranded nucleic acid molecules according to the Watson-Crick rule or by other modes of hydrogen bonding, as well as base stacking, or to double stranded nucleic acids by the Hoogsteen rule of base pairing, or to and in doing so, disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal transcription, splicing, or translation; by triggering the enzymatic destruction of mRNA by RNase H, or by destroying the target via reactive groups attached directly to the antisense oligonucleotide.
Antisense oligonucleotides have been developed as antiparasitic agents, although none have been demonstrated to reverse drug resistant phenotype of a drug resistant parasite strain. PCT publication No. WO 93/13740 discloses the use of antisense oligonucleotides directed to nucleic acids encoding the dihydrofolate reductase-thymidylate synthase gene of
P. falciparum
to inhibit propagation of drug-resistant malarial parasites. Rapaport et al. (
Proc. Natl. Acad. Sci
. (
USA
) (1992) 89:8577-8580) teaches inhibition of the growth of chloroquine-resistant and chloroquine-sensitive
P. falciparum
in vitro using oligonucleotides directed to the dihydrofolate reductase-thymidylate synthase gene. PCT publication No. WO 94/12643 discloses antisense oligonucleotides directed to nucleic acids encoding a carbamoyl phosphate synthetase of
P. falc

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