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ANGIOZYME, a synthetic ribozyme targeting the mRNA of VEGFR-1: clinical updateANGIOZYME was designed to test whether inhibition of angiogenesis through blockade of VEGF signaling can impact disease. ANGIOZYME is a synthetic ribozyme that selectively cleaves the messenger RNA for VEGFR-1. It contains chemically modified ribonucleotides, a small number of phosphorothioate linkages and an inverted sugar moiety which render it relatively resistant to the nucleases in biological fluids. ANGIOZYME has shown antitumor activity in five model systems in rodents {LLC-HM (lung), KM12L4a (colon), MCF-7 (breast), 4T1 (breast), ACHN (renal)}. This activity is manifested at doses of 30-600 mg/m2 through inhibition of angiogenesis, inhibition of the growth of the primary tumor and/or inhibition of the formation of metastases. Efficacy has been seen using IV, SC, and IP routes of administration. ANGIOZYME has been evaluated for toxicity in both rodents and primates with exposures up to 13 weeks. There was no effect on clinical, hematologic or biochemical parameters with the principal clinical finding of injection site reactions. In a single-dose Phase I study, ANGIOZYME was shown to have a bioavailability by subcutaneous injection of 90% in the 100 mg/m2 cohort. In a Phase I/II trial, 31 patients with metastatic cancer who had failed standard treatment received daily subcutaneous doses (30-300 mg/m2) of ANGIOZYME for up to 16 months. The principal toxicity was injection site erythema that occurred in most patients; these reactions were usually Grade 1, did not require treatment and did not lead to early terminations. Antibodies to ANGIOZYME were seen in four of five patients dosed longer than 100 days; however these antibodies were not associated with adverse events. Two patients (squamous cell cancer of the head and neck, melanoma) had minor responses. The pharmacokinetics were linear and did not show accumulation of ANGIOZYME with multiple dosing. Eight patients had tumor biopsies at least three weeks after initiation of treatment. Immunohistochemistry demonstrated the presence of ANGIOZYME in endothelial cells adjacent to the tumor. Phase II studies of ANGIOZYME (100mg/m2 subcutaneous daily) have been initiated in metastatic breast and colorectal cancer.
Background: VNP20009, a highly attenuated
strain of Salmonella typhimurium, has been observed
to preferentially accumulate in tumors following systemic
administration, reaching tumor to normal tissue to ratios
of > 1000:1 in mouse models. From data collected in phase
I trials conducted in advanced cancer patients, VNP20009 is
rapidly cleared from the blood following intravenous (IV)
administration. One approach to optimizing the tissue colonization
in species with rapid clearance, is to increase the exposure
of tissues to VNP20009. Pharmacokinetic (PK) studies were
conducted in both dogs and monkeys to determine the effect
of various parameters: infusion time, steroids and dosing
schedule on the systemic clearance of VNP20009.
Methods: VNP20009 was administered by IV injection
as either a 30 minute, 2 hour or 4 hour infusion to cynomolgus
monkeys or beagle dogs. Blood samples were taken and quantitatively
cultured to determine real-time levels of VNP20009 measured
as colony-forming units per ml (cfu/mL) of blood. Results:
In both dogs and monkeys, VNP20009 reached high levels in
the blood during infusion and rapidly cleared after the end
of infusion. By extending the duration of the infusion to
2 or 4 hours, high levels of VNP20009 (104 to 105
cfu/mL) could be maintained over the duration of the infusion.
In an effort to increase the dose level but maintain safety,
the steroid dexamethasome was administered to counter the
cytokine induction that follows a systemic dose of bacteria.
Although steroids permit higher doses of VNP20009 to be given
safely, the results demonstrated that steroids lower the peak
VNP20009 blood levels. Administration of VNP20009 on d x 5
schedule in dogs demonstrated that daily doses at the MTD
(3x108 cfu/kg) were tolerated and although the
PK profile did not change, a larger overall dose can be safely
administered without sacrificing peak blood levels.
Conclusion: Through the use of animal models with similar
PK profiles to humans we evaluated various parameters in an
effort to improve the PK profile of VNP20009. Our results
indicate that multiple dosing and increased infusion time
offer certain advantages (increased blood PK values, longer
exposure) and should be further explored in the clinic.
Background: Tumor-specific variable regions
of the clonal immunoglobulin (Idiotype or Id) expressed by
B cell Non-Hodgkin’s Lymphoma (NHL) can be exploited
as a target for active immunotherapy. Studies immunizing follicular
NHL patients (pts) with Id protein derived from tumor-myeloma
hybridomas have shown induction of specific cellular and humoral
anti-Id immune responses (IR) that correlate with improved
disease-free and overall survival. Tumor regressions and molecular
complete remissions following immunization have also been
observed. These results provide rationale for further study,
however, isolation of Id by traditional hybridoma methods
is inefficient and wide application is limited.
Objective: A phase 2 study was designed to assess the ability
of recombinant Id produced by a novel expression technology,
termed HiGET™, to elicit anti-Id IR in follicular NHL
patients in 1st clinical
remission. Methods: Patient specific Id genes are PCR amplified
from small numbers of tumor B-cells, cloned into plasmid vectors,
and transfected into mammalian cells that yield stable sources
of recombinant Id protein. Conjugation to the carrier keyhole
limpet hemocyanin (KLH) yields the final product, Id-KLH.
Treatment consists of sub-cutaneous (s.c) immunizations of
Id-KLH on day 1, along with GM-CSF (s.c. days 1-4). Immunizations
are administered every 4 weeks for 4 doses, with a 5th
immunization at week 24. Results: Twenty-one of 22 pts immunized
are evaluable for humoral and 16 are evaluable for cellular
anti-Id IR. Overall, 13 of 21 pts developed specific IR: 7
humoral, 2 cellular, and 4 both. IR were seen in both CR/CRU
patients (9/13) and PR patients (4/8). The overall 62% IR
rate compares favorably with the 50% rate seen in the previous
studies using hybridoma-derived Id proteins. The most common
side effects are mild and moderate injection site reactions
and flu-like symptoms. This study demonstrates that recombinant
Id immunotherapy can induce specific humoral and cellular
IR. A phase 3 randomized trial utilizing recombinant Id in
follicular NHL pts following 1st
clinical remission is underway at 22 sites in North America
to determine the efficacy in improving disease-free and overall
survival.
Zicao, a commonly used traditional Chinese medicine containing shikonin, exhibits a variety of biological activities including inhibiting HIV-1 infection. Our previous data showed that shikonin selectively blocked chemokine ligands binding to CCR1 receptor. In this study, we further characterized the anti-chemokine function of shikonin. At non-cytotoxic concentration, shikonin inhibited monocyte chemotatic response to a variety of CC chemokines (MCP-1, MIP-1α, RANTES), CXC chemokine (SDF-1α ) and classic chemoattractants (fLMP and C5a) that use numerous receptors, in a dose-dependent fashion. The IC50 of RANTES-induced monocytes migration was under 10-7 M. This inhibition was not reversible. Monocyte calcium mobilization induced by RANTES, MCP-1 and C5a was attenuated by shikonin in a time-dependent fashion. Shikonin did not significantly influence CCR1 receptor cell surface expression on HEK/CCR1 cells as shown by FACS assay, but reduced CCR5 receptor expression on HEK/CCR5 cells in a dose-dependent manner with an IC50 of approximately 5´10-6 M. Furthermore, treatment by shikonin synergistically enhanced the reduction of CCR5 surface expression induced by RANTES. In a study of human PBMC infected with HIV-1 (ROJO), shikonin inhibited HIV reverse transcriptase acitivity in a dose-dependent fashion with an IC50 of 1.261´10-7 M. The activity was not based on a toxic effect as IC50 of human PBMC viability under the same conditions was 1.993´10-6 M. Furthermore, shikonin inhibits CCR5 antibody binding to the receptor. Interference with both HIV co-receptors and chemokine receptor signal transduction contribute to the anti-HIV activity of shikonin. Overall, these results suggest that shikonin is a pan-chemokine receptor inhibitor by blockade of chemokine receptor function and downstream signaling.
Background: We have previously shown that
the level of expression of death receptors for TRAIL, in particular
TRAIL-R2 appeared an important determinant of TRAIL-induced
apoptosis in melanoma cell lines. However, TRAIL induces low
levels of apoptosis in some melanoma cell lines despite relative
high levels of TRAIL-R2 expression on the cell surface. Objective:
To elucidate the intracellular mechanisms that regulate the
sensitivity of melanoma cells to TRAIL induced apoptosis.
Methods: We examined the multiple checkpoints in the TRAIL-initiated
apoptotic signalling pathway by flow cytometry, western blot,
immunofluorescence staining. c-DNA for XIAP, Bcl-2 and Smac/DIABLO
were transfected into TRAIL-sensitive or TRAIL-resistant melanoma
cells, respectively, to confirm their role in regulation of
TRAIL-induced apoptosis. Results: We identified four melanoma
lines with high TRAIL-R2 expression but low sensitivity to
TRAIL induced apoptosis. The lines were shown to have similar
levels of TRAIL-induced activated caspase-3 as the TRAIL sensitive
lines but substrates downstream of caspase-3 (ICAD and PARP)
were not degraded in the insensitive cell lines. This appeared
to be due to inhibition of caspase-3 by XIAP in that XIAP
was bound to activated caspase-3 and transfection of XIAP
results in inhibition of TRAIL induced apoptosis. Reduction
of XIAP levels by pre-treatment with Actinomycin D or by specific
inhibition of XIAP by overexpression of Smac/DIABLO in the
resistant melanoma cells was associated with the appearance
of catalytic activity by caspase-3, increased TRAIL-induced
apoptosis and reduced XIAP levels. After exposure to TRAIL,
XIAP levels were markedly reduced in the TRAIL sensitive compared
to resistant lines. This appeared to be associated with Smac/DIABLO
release from mitochondria as this release was greater in TRAIL
sensitive compared to resistant lines and had similar kinetics
to downregulation of XIAP levels. Furthermore, overexpression
of Bcl-2 inhibited Smac/DIABLO release and downregulation
of XIAP levels. These results indicate that inhibition of
caspase-3 by XIAP appears an important mechanism for protection
of melanoma cells from TRAIL-induced apoptosis and provide
evidence that TRAIL induced Smac/DIABLO release from mitochondria
is a key regulator of TRAIL induced apoptosis of melanoma.
TRAIL has received much interest as a promising agent for cancer therapy, since it promotes apoptosis in many tumor cells, yet has little effect on most normal cells. The proteasome inhibitor PS-341 also induces apoptosis in many tumor cells, and its inhibition of NFκ-B may underlay some of its pro-apoptotic activities. Since TRAIL also activates NFκ -B in many cells, we combined PS-341 with TRAIL in an attempt to amplify the apoptotic effects of TRAIL. In overnight (18h) assays combinations of PS-341 and TRAIL were much more effective than either agent alone in promoting apoptosis of a murine myeloid leukemia C1498, a murine renal cancer RENCA and a human breast cancer MDA-341. Optimal doses of PS-341 were in the range of 1-100nM and the optimal amounts of TRAIL also varied depending on the individual cell line. Apoptosis in all tumor cell lines was blocked by the general caspase inhibitor ZVAD-FMK. Western blotting analysis showed that doses of PS-341 which sensitized C1498 to TRAIL-mediated lysis also significantly decreased levels of the anti-apoptotic protein cFLIP, whereas levels of bcl-2 and bax were unaffected. In a bone marrow purging model , lethally irradiated B6 mice received bone marrow (3X106 cells) containing 10,000 C1498 cells, or bone marrow plus C1498 cells which had been treated overnight with PS-341 or TRAIL. Treatment of bone marrow cells (BMC) overnight with 10nM PS-341 did not result in significant myelosuppression, as assessed by CFU-c colony formation in soft agar in vitro or hematopoetic reconstitution experiments in vivo. Mice receiving untreated or TRAIL- treated bone marrow plus C1498 all succumbed within 25 days due to extensive tumor growth, whereas treatment with PS-341 alone significantly increased survival times. These data suggest that PS-341 exerts direct antitumor effects in vivo, and can possibly be used in combination with TRAIL to enhance TRAIL-mediated apoptosis effects on C1498 tumor cells without significant toxicity on normal bone marrow cells. Current studies are examining whether the combination of PS-341 with TRAIL may result in greater antitumor effects in vivo.
