Antitumor activity and pharmacokinetics of a mixed-backbone antisense oligonucleotide targeted to the RIα subunit of protein kinase A after oral administration

Overexpression of the RIα subunit of cAMP-dependent protein kinase (PKA) has been demonstrated in various human cancers. PKA has been suggested as a potential target for cancer therapy. The goal of the present study was to evaluate an anti-PKA antisense oligonucleotide (mixed-backbone oligonucleotide) as a therapeutic approach to human cancer treatment. The identified oligonucleotide inhibited the growth of cell lines of human colon cancer (LS174T, DLD-1), leukemia (HL-60), breast cancer (MCF-7, MDA-MB-468), and lung cancer (A549) in a time-, concentration-, and sequence-dependent manner. In a dose-dependent manner, the oligonucleotide displayed in vivo antitumor activity in severe combined immunodeficient and nude mice bearing xenografts of human cancers of the colon (LS174T), breast (MDA-MB-468), and lung (A549). The routes of drug administration were intraperitoneal and oral. Synergistic effects were found when the antisense oligonucleotide was used in combination with the cancer chemotherapeutic agent cisplatin. The pharmacokinetics of the oligonucleotide after oral administration of 35S-labeled oligonucleotide into tumor-bearing mice indicated an accumulation and retention of the oligonucleotide in tumor tissue. This study further provides a basis for clinical studies of the antisense oligonucleotide targeted to the RIα subunit of PKA (GEM 231) as a cancer therapeutic agent used alone or in combination with conventional chemotherapy.

Iontophoresis for modulation of cardiac drug delivery in dogs.

Cardiac arrhythmias are a frequent cause of death and morbidity. Conventional antiarrhythmia therapy involving oral or intravenous medication is often ineffective and complicated by drug-associated side effects. Previous studies from our laboratory have demonstrated the advantages of cardiac drug-polymer implants for enhanced efficacy for cardiac arrhythmia therapy compared with conventional administration. However, these studies were based on systems that deliver drugs at a fixed release rate. Modulation of the drug delivery rate has the advantage of regulating the amount of the drug delivered depending upon the disease state of the patient. We hypothesized that iontophoresis could be used to modulate cardiac drug delivery. In this study, we report our investigations of a cardiac drug implant in dogs that is capable of iontophoretic modulation of the administration of the antiarrhythmic agent sotalol. We used a heterogeneous cation-exchange membrane (HCM) as an electrically sensitive and highly efficient rate-limiting barrier on the cardiac-contacting surface of the implant. Thus, electric current is passed only through the HCM and not the myocardium. The iontophoretic cardiac implant demonstrated in vitro drug release rates that were responsive to current modulation. In vivo results in dogs have confirmed that iontophoresis resulted in regional coronary enhancement of sotalol levels with current-responsive increases in drug concentrations. We also observed acute current-dependent changes in ventricular effective refractory periods reflecting sotalol-induced refractoriness due to regional drug administration. In 30-day dog experiments, iontophoretic cardiac implants demonstrated robust sustained function and reproducible modulation of drug delivery kinetics.

A model-based approach for assessing in vivo combination therapy interactions

We present an approach for evaluating the efficacy of combination antitumor agent schedules that accounts for order and timing of drug administration. Our model-based approach compares in vivo tumor volume data over a time course and offers a quantitative definition for additivity of drug effects, relative to which synergism and antagonism are interpreted. We begin by fitting data from individual mice receiving at most one drug to a differential equation tumor growth/drug effect model and combine individual parameter estimates to obtain population statistics. Using two null hypotheses: (i) combination therapy is consistent with additivity or (ii) combination therapy is equivalent to treating with the more effective single agent alone, we compute predicted tumor growth trajectories and their distribution for combination treated animals. We illustrate this approach by comparing entire observed and expected tumor volume trajectories for a data set in which HER-2/neu-overexpressing MCF-7 human breast cancer xenografts are treated with a humanized, anti-HER-2 monoclonal antibody (rhuMAb HER-2), doxorubicin, or one of five proposed combination therapy schedules.

Approaches to enhancing the retroviral transduction of human synoviocytes

This report concerns a clinical trial for rheumatoid arthritis (RA), approved by the US National Institutes of Health and the Food and Drug Administration. An amphotropic retrovirus (MFG-IRAP) was used ex vivo to transfer a cDNA encoding human interleukin-1 receptor antagonist (IL-1Ra) to synovium. The protocol required the transduced cells to secrete at least 30 ng IL-1Ra/106 cells per 48 h before reimplantation. Here we have evaluated various protocols for their efficiency in transducing cultures of human rheumatoid synoviocytes. The most reliably efficient methods used high titer retrovirus (approximately 108 infectious particles/ml). Transduction efficiency was increased further by exposing the cells to virus under flow-through conditions. The use of dioctadecylamidoglycylspermine (DOGS) as a polycation instead of Polybrene (hexadimethrine bromide) provided an additional small increment in efficiency. Under normal conditions of static transduction, standard titer, clinical grade retrovirus (approximately 5 × 105 infectious particles/ml) failed to achieve the expression levels required by the clinical trial. However, the shortfall could be remedied by increasing the time of transduction under static conditions, transducing under flow-through conditions, or transducing during centrifugation.

Immunomodulation of experimental autoimmune encephalomyelitis by oral administration of copolymer 1

The activity of copolymer 1 (Cop 1, Copaxone, glatiramer acetate) in suppressing experimental autoimmune encephalomyelitis (EAE) and in the treatment of multiple sclerosis patients when injected parenterally has been extensively demonstrated. In the present study we addressed the question of whether Cop 1 can induce oral tolerance to EAE similar to myelin basic protein (MBP). We now have demonstrated that oral Cop 1 inhibited EAE induction in both rats and mice. Furthermore, oral Cop 1 was more effective than oral MBP in suppressing EAE in rats. The beneficial effect of oral Cop 1 was found to be associated with specific inhibition of the proliferative and Th1 cytokine secretion responses to MBP of spleen cells from Cop 1-fed mice and rats. In all of these assays, oral Cop 1 was more effective than oral MBP. The tolerance induced by Cop 1 could be adoptively transferred with spleen cells from Cop 1-fed animals. Furthermore, Cop 1-specific T cell lines, which inhibit EAE induction in vivo, could be isolated from the above spleen cells. These T cell lines secrete the anti-inflammatory cytokines IL-10 and transforming growth factor type β, but not IL-4, in response to both Cop 1 and MBP. In conclusion, oral Cop 1 has a beneficial effect on the development of EAE that is associated with down-regulation of T cell immune responses to MBP and is mediated by Th2/3 type regulatory cells. These results suggest that oral administration of Cop 1 may modulate multiple sclerosis as well.