Recent studies have shown that GRN163, which belongs to a new class of modified N3′→P5′ thio
-phosphoramidates, has potent human telomerase inhibitory activity in several biochemical assays with 50% inhibitory concentration values less than 1 nM (Gryaznov et al., 2001; Herbert et al., 2002
). This oligonucleotide was designed to bind by formation of a complementary duplex with part of the template region of hTR. Also, it appears that the thio
-phosphoramidate backbone of GRN163 is capable of forming—in addition to Watson-Crick base pairing—a stabilizing interaction with telomerase protein subunit hTERT upon the duplex formation with hTR (Gryazov et al., 2001). A recent study also revealed that GRN163 effectively inhibits telomerase activity in spontaneously immortalized human breast epithelial HME50-5E cells, with recorded 50% inhibitory concentration values of approximately 0.5 to 5 nM and 0.5 to 1 μM with and without lipid uptake enhancers, respectively (Herbert et al., 2002
). Moreover, inhibition of telomerase activity by GRN163 in HME50-5E cells produced a gradual reduction of telomere length, followed by the onset of cellular senescence and apoptosis (Herbert et al., 2002
In the present study we tested the telomerase inhibitor GRN163 as a potential therapeutic agent in experimental models of human malignant glioma. We hypothesized that the specific inhibition of telomerase activity in malignant gliomas might be a promising therapeutic approach because of the relatively high telomerase activity detected in these tumors, but not in normal brain tissue (Chong et al., 1998; Langford et al., 1995; Le et al., 1998; Mori et al., 1997; Sano et al., 1998
). Our initial in vitro studies showed that a 3-day exposure of U-251 MG cells to GRN163 inhibited telomerase activity by up to 68% as compared to levels in untreated cells.
The results of our in vivo studies show that treatment of s.c. tumors with GRN163 inhibited their growth. Tumor-growth inhibition occurred in all three independently conducted experiments regardless of frequency of injection (4 or 9 injections) or tumor size at the time of initiation of the treatment. Failure to observe differences in outcome between the two treatment regimens may indicate that both were equally effective, or it may simply mean that the animal numbers used were too small to reveal differences in outcome. Further experimentation would be needed to determine which of these possibilities is correct. Comparisons of tumor growth for the individual mice (–) suggest that animals with smaller tumors at the time of treatment might respond better to treatment with GRN163; however, there was too much variability in tumor growth patterns in each group of GRN163-treated animals to make a definitive statement.
Our results are in general agreement with the findings of Kondo et al. (1998)
, who also used the U251-MG cell line for their in vitro and in vivo studies of antitelomerase 2-5A- oligonucleotide conjugates directed to the site of hTR. Although this site is different from the template region for GRN163, administration of that oligonucleotide in vivo also inhibited flank-tumor growth.
Our studies show that direct i.c. administration by slow infusion of fluorescein-labeled GRN163 formulated in PBS without any cellular-uptake enhancers produces efficient uptake of the oligonucleotide into human brain-tumor cells. Moreover, a significant amount of this compound was retained by the tumor cells for up to 4 days after termination of the infusion, as shown by our distribution study. These data indicate there is good bio-availability and hydrolytic stability of GRN163 in the lipid-rich environment of the brain and suggest the possibility of intermittent dosing. Although GRN163 was found in normal brain cells that were adjacent to tumor cells, no abnormal behavior was detected in any animals, and there were no pathological indications of toxicity. This finding was expected because normal brain cells do not express telomerase activity.
When animals were infused with GRN163 shortly after implantation of tumor cells, tumor growth was prevented in the majority of animals (). This result appears to be specific to the antitelomerase activity of GRN163 because tumor formation and growth was not impeded by the administration of MM Control at the time of tumor implantation. Our finding suggests that GRN163 might have applicability as a chemopreventive agent, and while it is unlikely to be used for that purpose, it might be useful for treating patients with minimal residual tumors or micrometastatic disease.
The results of our i.c. tumor efficacy study, in which rats were treated with a 7-day infusion of either 150 or 500 nmol of GRN163, showed that both doses of GRN163 prolonged survival of the rats as compared to survival of rats treated with MM Control; however, there was minimal, if any, difference between the 150- and 500-nmol GRN163 groups. We have not yet studied doses between 500 and 1500 nmol to see whether a dose-response relationship occurs; however, a preliminary experiment revealed that 50 nmol of GRN163 did not prolong animal survival and 1500 nmol of GRN163 was toxic to animals.
The inhibition of growth observed in GRN163-treated flank tumors, as well as the prolonged survival of GRN163-treated animals with i.c. tumors, provides evidence that GRN163 was active in these tumor models. Although we cannot say that this activity was due strictly to its antitelomerase activity, because we did not measure it, GRN163 is not known to exhibit any other biological activity within the experimental time periods used in our experiments. Moreover, it did not exhibit any adverse effects when injected into normal brains, which for the most part lack telomerase activity. Finally, results from our in vitro studies clearly showed that GRN163 could inhibit telomerase activity. Thus, we think it is reasonable to presume that the biological effects produced by GRN163 are attributable to its antitelomerase activity.
The results of our in vivo studies confirm that agents that target telomerase show promise as potential antitumor drugs for malignant glioma. They also suggest that these agents may have potential for treating patients with minimal residual disease or micrometastatic disease. The findings support further investigations of GRN163 and other antitelomerase agents for their possible use in brain-tumor therapy.