Inotersen Treatment for Patients with Hereditary Transthyretin Amyloidosis.

BACKGROUND: Hereditary transthyretin amyloidosis is caused by pathogenic single-nucleotide variants in the gene encoding transthyretin ( TTR) that induce transthyretin misfolding and systemic deposition of amyloid. Progressive amyloid accumulation leads to multiorgan dysfunction and death. Inotersen, a 2'- O-methoxyethyl-modified antisense oligonucleotide, inhibits hepatic production of transthyretin. METHODS: We conducted an international, randomized, double-blind, placebo-controlled, 15-month, phase 3 trial of inotersen in adults with stage 1 (patient is ambulatory) or stage 2 (patient is ambulatory with assistance) hereditary transthyretin amyloidosis with polyneuropathy. Patients were randomly assigned, in a 2:1 ratio, to receive weekly subcutaneous injections of inotersen (300 mg) or placebo. The primary end points were the change in the modified Neuropathy Impairment Score+7 (mNIS+7; range, -22.3 to 346.3, with higher scores indicating poorer function; minimal clinically meaningful change, 2 points) and the change in the score on the patient-reported Norfolk Quality of Life-Diabetic Neuropathy (QOL-DN) questionnaire (range, -4 to 136, with higher scores indicating poorer quality of life). A decrease in scores indicated improvement. RESULTS: A total of 172 patients (112 in the inotersen group and 60 in the placebo group) received at least one dose of a trial regimen, and 139 (81%) completed the intervention period. Both primary efficacy assessments favored inotersen: the difference in the least-squares mean change from baseline to week 66 between the two groups (inotersen minus placebo) was -19.7 points (95% confidence interval [CI], -26.4 to -13.0; P<0.001) for the mNIS+7 and -11.7 points (95% CI, -18.3 to -5.1; P<0.001) for the Norfolk QOL-DN score. These improvements were independent of disease stage, mutation type, or the presence of cardiomyopathy. There were five deaths in the inotersen group and none in the placebo group. The most frequent serious adverse events in the inotersen group were glomerulonephritis (in 3 patients [3%]) and thrombocytopenia (in 3 patients [3%]), with one death associated with one of the cases of grade 4 thrombocytopenia. Thereafter, all patients received enhanced monitoring. CONCLUSIONS: Inotersen improved the course of neurologic disease and quality of life in patients with hereditary transthyretin amyloidosis. Thrombocytopenia and glomerulonephritis were managed with enhanced monitoring. (Funded by Ionis Pharmaceuticals; NEURO-TTR ClinicalTrials.gov number, NCT01737398 .).

Integrated Safety Assessment of 2'-O-Methoxyethyl Chimeric Antisense Oligonucleotides in NonHuman Primates and Healthy Human Volunteers.

The common chemical and biological properties of antisense oligonucleotides provide the opportunity to identify and characterize chemical class effects across species. The chemical class that has proven to be the most versatile and best characterized is the 2'-O-methoxyethyl chimeric antisense oligonucleotides. In this report we present an integrated safety assessment of data obtained from controlled dose-ranging studies in nonhuman primates (macaques) and healthy human volunteers for 12 unique 2'-O-methoxyethyl chimeric antisense oligonucleotides. Safety was assessed by the incidence of safety signals in standardized laboratory tests for kidney and liver function, hematology, and complement activation; as well as by the mean test results as a function of dose level over time. At high doses a number of toxicities were observed in nonhuman primates. However, no class safety effects were identified in healthy human volunteers from this integrated data analysis. Effects on complement in nonhuman primates were not observed in humans. Nonhuman primates predicted safe doses in humans, but over predicted risk of complement activation and effects on platelets. Although limited to a single chemical class, comparisons from this analysis are considered valid and accurate based on the carefully controlled setting for the specified study populations and within the total exposures studied.

Correlations between preclinical BJAB assay ranking of antisense drugs and clinical trial adverse events.

This analysis sought to assess the clinical predictivity of an in vitro assay which utilized the human B-lymphoma BJAB cell line, for identification of antisense oligonucleotides (ASOs) with the potential to elicit innate immune activation in humans. Adverse events (AEs) from clinical trial data were analyzed based on prior clinical knowledge and network analysis of the clinical data to identify correlations with the BJAB assay. Clinically evaluated ASOs were ranked by the BJAB assay's mean log-fold increase in TNF expression levels. Flu-like reactions (FLRs) and injection site reactions (ISRs), were chosen as AEs of interest, along with those Medical Dictionary for Regulatory Activities preferred terms identified using AE network analysis. Fifteen different 2'-O-methoxyethyl (2'MOE) modified ASOs were ranked by the incidence of each AE group in the integrated safety data from 35 clinical trials. ISRs are considered to be local to the injection site, whereas FLRs are reflected by systemic constitutional symptoms. The correlations identified in this analysis of integrated clinical data provide evidence that the ASO sequences selected by the BJAB assay have a lower likelihood of causing systemic inflammatory AEs associated with FLRs, but not ISRs.

Integrated Assessment of Phase 2 Data on GalNAc3-Conjugated 2'-O-Methoxyethyl-Modified Antisense Oligonucleotides.

Receptor-mediated delivery of an antisense oligonucleotide (ASO) using the ligand-conjugated antisense technology is establishing a new benchmark for antisense therapeutics. The triantennary N-acetylgalactosamine (GalNAc3) cluster is the first conjugated ligand to yield a marked increase in ASO potency for RNA targets expressed by hepatocytes, compared to the unconjugated form. In this study, we present an integrated safety assessment of data available from randomized, placebo-controlled, phase 2 studies for six GalNAc3-conjugated 2'-O-methoxyethyl (2'MOE)-modified ASOs. The total study population included 642 participants (130 placebo; 512 ASO) with up to 1 year of exposure. The primary measures were the incidence of signals from standardized laboratory tests and the mean test results over time. The GalNAc3-conjugated ASOs were well tolerated with no class effect identified across all doses tested compared to placebo. These results extend prior observations from phase 1 studies, now with treatment up to 1 year.

Early-Stage Identification and Avoidance of Antisense Oligonucleotides Causing Species-Specific Inflammatory Responses in Human Volunteer Peripheral Blood Mononuclear Cells.

A human peripheral blood mononuclear cell (PBMC)-based assay was developed to identify antisense oligonucleotide (ASO) with the potential to activate a cellular innate immune response outside of an acceptable level. The development of this assay was initiated when ISIS 353512 targeting the messenger ribonucleic acid for human C-reactive protein (CRP) was tested in a phase I clinical trial, in which healthy human volunteers unexpectedly experienced increases in interleukin-6 (IL-6) and CRP. This level of immune stimulation was not anticipated following rodent and nonhuman primate safety studies in which no evidence of exaggerated proinflammatory effects were observed. The IL-6 increase induced by ISIS 353512 was caused by activation of B cells. The IL-6 induction was inhibited by chloroquine pretreatment of PBMCs and the nature of ASOs suggested that the response is mediated by a Toll-like receptor (TLR), in all likelihood TLR9. While assessing the inter PBMC donor variability, two classes of human PBMC responders to ISIS 353512 were identified (discriminator and nondiscriminators). The discriminator donor PBMCs were shown to produce low level of IL-6 after 24 h in culture, in the absence of ASO treatment. The PBMC assay using discriminator donors was shown to be reproducible, allowing to assess reliably the immune potential of ASOs by comparison to known benchmark ASO controls that were previously shown to be either safe or inflammatory in clinical trials. Clinical Trial registration numbers: NCT00048321 NCT00330330 NCT00519727.

Preclinical and Phase 1 Assessment of Antisense Oligonucleotide Bepirovirsen in Hepatitis B Virus-Transgenic Mice and Healthy Human Volunteers: Support for Clinical Dose Selection and Evaluation of Safety, Tolerability, and Pharmacokinetics of Single and Multiple Doses.

Dose-dependent reductions in hepatitis B virus (HBV) RNA, DNA, and viral proteins following bepirovirsen administration were observed in HepG2.2.15 cells. In HBV-transgenic mice treated at 50 mg/kg/wk, hepatic HBV RNA and DNA were reduced by 90% and 99%, respectively. Subsequently, a phase 1 first-in-human study assessed pharmacokinetics and tolerability of single (75-450 mg) and multiple (150-450 mg on days 1, 4, 8, 11, 15, and 22) subcutaneous bepirovirsen doses in 96 healthy volunteers. Bepirovirsen at all dose levels was rapidly absorbed (maximum plasma concentration 3-8 hours after dosing), rapidly distributed to peripheral tissues, and slowly eliminated (median plasma terminal half-life: 22.5-24.6 days across cohorts). Plasma exposure (dose-proportional at 150-450 mg) and concentration-time profiles were similar following the first and sixth doses, suggesting little to no plasma accumulation (steady state achieved by day 22). Renal elimination of full-length bepirovirsen accounted for <2% of the total dose. Across the single and multiple dose cohorts, 197 treatment-emergent adverse events were reported, with 99% and 65% classified as mild in severity and local injection site reactions, respectively. In conclusion, bepirovirsen showed an acceptable safety profile in humans with observed pharmacokinetics consistent with the chemical class, warranting further evaluation of bepirovirsen in chronic HBV infection.

Improvements in the Tolerability Profile of 2'-O-Methoxyethyl Chimeric Antisense Oligonucleotides in Parallel with Advances in Design, Screening, and Other Methods.

The development process of antisense oligonucleotides (ASOs) as therapeutic agents in humans has advanced through the implementation of chemical compound modifications as well as increasingly sophisticated toxicological preclinical screening techniques. The Ionis Integrated Safety Database was utilized to determine if advances in ASO screening and clinical lead identification methods have improved the tolerability profiles of 2'-O-methoxyethyl (2'MOE)-modified ASOs as a class, relative to the first 2'MOE ASO approved for use in humans, mipomersen. Tolerability was assessed by the incidence and percentage of subcutaneous doses leading to adverse events at the injection site or flu-like reactions (FLRs), as well as by the incidence of dose discontinuations due to these events. In randomized placebo-controlled phase 1 and phase 2 trials, the incidence of each measure of tolerability was lower in the test group of 12 ASOs (713 ASO-treated subjects) compared with the reference, mipomersen (266 ASO-treated subjects); with the most marked reduction in the incidence of FLRs (0.6% vs. 9.4%). A similar reduction in the incidence of dose discontinuation due to FLRs was also observed (0.2% vs. 0.9%). When compared with mipomersen, 8 of 12 ASOs showed significant improvements in their respective mean percentage of doses leading to adverse events at the injection site, whereas 7 ASOs showed a significant improvement in mean percentage of doses leading to FLRs. These results support an overall improvement in the tolerability profile in 2'MOE ASOs that entered development after mipomersen, in parallel with advances in the drug discovery screening process as well as the gains in clinical experience during development of each ASO.

Safety, tolerability and antiviral activity of the antisense oligonucleotide bepirovirsen in patients with chronic hepatitis B: a phase 2 randomized controlled trial.

Chronic infection with hepatitis B virus (HBV) leads to an increased risk of death from cirrhosis and hepatocellular carcinoma. Functional cure rates are low with current treatment options (nucleos(t)ide analogs (NAs) and pegylated interferons). Bepirovirsen is an antisense oligonucleotide targeting all HBV messenger RNAs; in cell culture and animal models, bepirovirsen leads to reductions in HBV-derived RNAs, HBV DNA and viral proteins. This phase 2 double-blinded, randomized, placebo-controlled trial is the first evaluation of the safety and activity of an antisense oligonucleotide targeting HBV RNA in both treatment-naïve and virally suppressed individuals with chronic HBV infection. The primary objective was to assess the safety and tolerability of bepirovirsen in individuals with chronic hepatitis B (CHB) (NCT02981602). The secondary objective was to assess antiviral activity, including the change from baseline to day 29 in serum hepatitis B surface antigen (HBsAg) concentration. Participants with CHB infection ≥6 months and serum HBsAg ≥50 IU ml-1 were enrolled from seven centers across Hong Kong and the Republic of Korea and randomized (3:1 within each dose cohort) to receive bepirovirsen or placebo via subcutaneous injection twice weekly during weeks 1 and 2 (days 1, 4, 8 and 11) and once weekly during weeks 3 and 4 (days 15 and 22). Participants were then followed for 26 weeks. Twenty-four participants were treatment-naïve and seven were receiving stable NA therapy. Treatment-emergent adverse events were mostly mild/moderate (most commonly injection site reactions). Eleven (61.1%) and three (50.0%) treatment-naïve participants experienced one or more treatment-emergent adverse event in the bepirovirsen and placebo groups, respectively. In participants receiving NA therapy, the corresponding numbers were three (60.0%) and one (50.0%). Transient, self-resolving alanine aminotransferase flares (≥2× upper limit of normal) were observed in eight treatment-naïve participants and three participants on stable NA regimens in the bepirovirsen treatment arms. HBsAg reductions were observed and were significant versus placebo for treatment-naïve participants receiving bepirovirsen 300 mg (P = 0.001), but not for the bepirovirsen 150 mg group (P = 0.245) or participants receiving stable NA therapy (P = 0.762). Two participants in each of the 300 mg dose groups achieved HBsAg levels below the lower limit of quantitation by day 29 (n = 3) or day 36 (n = 1). Bepirovirsen had a favorable safety profile. These preliminary observations warrant further investigation of the safety and activity of bepirovirsen in a larger CHB patient population.

Effect of apolipoprotein-B synthesis inhibition on liver triglyceride content in patients with familial hypercholesterolemia.

To investigate the impact of mipomersen, an apolipoprotein B-100 (apoB) synthesis inhibitor, on intra-hepatic triglyceride content (IHTG content), we conducted a randomized, double-blind, placebo-controlled study in 21 patients with familial hypercholesterolemia (FH). Subjects received a weekly subcutaneous dose of 200 mg mipomersen or placebo for 13 weeks while continuing conventional lipid lowering therapy. The primary endpoint was change in IHTG content from week 0 to week 15 as measured by localized proton magnetic resonance spectroscopy (1H-MRS). Thirteen weeks of mipomersen administration reduced LDL-cholesterol by 22.0 (17.8) % and apoB by 19.9 (17.4) % (both P < 0.01). One of 10 patients (10%) in the mipomersen-treated group developed mild hepatic steatosis at week 15, which was reversible following mipomersen discontinuation. For the group, there was a trend toward an increase in IHTG content [placebo; baseline: 1.2% and week 15: 1.1%; change -0.1 (0.9). Mipomersen; baseline: 1.2% and week 15: 2.1%; change 0.8 (1.7) (P = 0.0513)]. Mipomersen administration for 13 weeks to subjects with FH is associated with a trend toward an increase in IHTG content. Future studies evaluating the effects of long-term use of mipomersen reaching more profound reductions in apoB are required prior to broader use of this compound.

Underlying Immune Disorder May Predispose Some Transthyretin Amyloidosis Subjects to Inotersen-Mediated Thrombocytopenia.

Inotersen, a 2'-O-methoxyethyl (2'-MOE) phosphorothioate antisense oligonucleotide, reduced disease progression and improved quality of life in patients with hereditary transthyretin amyloidosis with polyneuropathy (hATTR-PN) in the NEURO-TTR and NEURO-TTR open-label extension (OLE) trials. However, 300 mg/week inotersen treatment was associated with platelet count reductions in several patients. Mean platelet counts in patients in the NEURO-TTR-inotersen group remained ≥140 × 109/L in 50% and ≥100 × 109/L in 80% of the subjects. However, grade 4 thrombocytopenia (<25 × 109/L) occurred in three subjects in NEURO-TTR trial, and one of these suffered a fatal intracranial hemorrhage. The two others were treated successfully with corticosteroids and discontinuation of inotersen. Investigations in a subset of subjects in NEURO-TTR (n = 17 placebo; n = 31 inotersen) and OLE (n = 33) trials ruled out direct myelotoxicity, consumptive coagulopathy, and heparin-induced thrombocytopenia. Antiplatelet immunoglobulin G (IgG) antibodies were detected at baseline in 5 of 31 (16%) inotersen-treated subjects in NEURO-TTR, 4 of whom eventually developed grade 1 or 2 thrombocytopenia while on the drug. In addition, 24 subjects in the same group developed treatment-emergent antiplatelet IgG antibodies, of which 2 developed grade 2, and 3 developed grade 4 thrombocytopenia. Antiplatelet IgG antibodies in two of the three grade 4 thrombocytopenia subjects targeted GPIIb/IIIa. Plasma cytokines previously implicated in immune dysregulation, such as interleukin (IL)-23 and a proliferation-inducing ligand (APRIL) were often above the normal range at baseline. Collectively, these findings suggest an underlying immunologic dysregulation predisposing some individuals to immune-mediated thrombocytopenia during inotersen treatment.

The Effects of 2'-O-Methoxyethyl Oligonucleotides on Renal Function in Humans.

Systemically administered 2'-O-methoxyethyl (2'MOE) antisense oligonucleotides (ASOs) accumulate in the kidney and metabolites are cleared in urine. The effects of eleven 2'MOE ASOs on renal function were assessed in 2,435 patients from 32 phase 2 and phase 3 trials. The principle analysis was on data from 28 randomized placebo-controlled trials. Mean levels of renal parameters remained within normal ranges over time across dose groups. Patient-level meta-analyses demonstrated a significant difference between placebo-treated and 2'MOE ASO-treated patients at doses >175 mg/week in the percentage and absolute change from baseline for serum creatinine and estimated glomerular filtration rate. However, these changes were not clinically significant or progressive. No dose-related effects were observed in the incidence of abnormal renal test results in the total population of patients, or subpopulation of diabetic patients or patients with renal dysfunction at baseline. The incidence of acute kidney injury [serum creatinine ≥0.3 mg/dL (26.5 µM) increases from baseline or ≥1.5 × baseline] in 2'MOE ASO-treated patients (2.4%) was not statistically different from placebo (1.7%, P = 0.411). In conclusion, in this database, encompassing 32 clinical trials and 11 different 2'MOE ASOs, we found no evidence of clinically significant renal dysfunction up to 52 weeks of randomized-controlled treatment.

The Effects of 2'-O-Methoxyethyl Containing Antisense Oligonucleotides on Platelets in Human Clinical Trials.

A thorough analysis of clinical trial data in the Ionis integrated safety database (ISDB) was performed to determine if there is a class effect on platelet numbers and function in subjects treated with 2'-O-methoxyethyl (2'MOE)-modified antisense oligonucleotides (ASOs). The Ionis ISDB includes over 2,600 human subjects treated with 16 different 2'MOE ASOs in placebo-controlled and open-label clinical trials over a range of doses up to 624 mg/week and treatment durations as long as 4.6 years. This analysis showed that there is no class generic effect on platelet numbers and no incidence of confirmed platelet levels below 50 K/µL in subjects treated with 2'MOE ASOs. Only 7 of 2,638 (0.3%) subjects treated with a 2'MOE ASO experienced a confirmed postbaseline (BSLN) platelet count between 100 and 50 K/µL. Three of sixteen 2'MOE ASOs had >10% incidence of platelet decreases >30% from BSLN, suggesting that certain sequences may associate with clinically insignificant platelet declines. Further to these results, we found no evidence that 2'MOE ASOs alter platelet function, as measured by the lack of clinically relevant bleeding in the presence or absence of other drugs that alter platelet function and/or number and by the results from trials conducted with the factor XI (FXI) ASO.

Virological effects of ISIS 14803, an antisense oligonucleotide inhibitor of hepatitis C virus (HCV) internal ribosome entry site (IRES), on HCV IRES in chronic hepatitis C patients and examination of the potential role of primary and secondary HCV resistance in the outcome of treatment.

Antisense oligonucleotides represent a promising class of antiviral agents. ISIS 14803 is a 20-unit phosphorothioate oligodeoxynucleotide that inhibited hepatitis C virus (HCV) replication and protein expression in cell culture and mouse models. A Phase I dose-escalation clinical study of ISIS 14803 was performed in 24 patients with HCV genotype 1 chronic hepatitis C. The patients received 0.5, 1.0, 2.0 or 3.0 mg/kg of ISIS 14803 for 4 weeks. Two of them receiving 2.0 mg/kg, experienced a significant (>1.0 log10) viral load reduction and nine other patients experienced minor (<1.0 log10) viral load reductions that were difficult to definitively distinguish from assay or patient variations. The aims of this study were to examine the effect of ISIS 14803 on its target site and neighbouring region quasispecies evolution, and to determine whether primary and secondary HCV resistance contributed to the observed virological response rate. The HCV internal ribosome entry site (IRES), including the ISIS 14803 target site in virus specimens collected from patients at baseline and end-of-treatment, was sequenced. An extensive IRES quasispecies analysis was performed in 10 of the patients at various time points before, during and after ISIS 14803 treatment. A significant IRES genetic evolution was found in three out of 10 patients through quasispecies analysis suggesting that treatment with ISIS 14803, a drug designed to bind to HCV RNA, exerted a selective pressure on HCV IRES. However, no mutations in the ISIS 14803 target site, which would inhibit binding of the oligonucleotide to HCV RNA, were detected before (primary resistance) or after treatment (secondary resistance) with the oligonucleotide. Furthermore, no obvious nucleotide changes in the surrounding IRES region that might possibly affect oligonucleotide binding were detected.

Phase I study of the c-raf-1 antisense oligonucleotide ISIS 5132 in combination with carboplatin and paclitaxel in patients with previously untreated, advanced non-small cell lung cancer.

BACKGROUND: A phase I trial was performed to evaluate the administration of carboplatin/paclitaxel in combination with ISIS-5132, a phosphorothioate antisense oligodeoxynucleotide inhibitor of c-raf-1 kinase expression, in patients with advanced non-small cell lung cancer (NSCLC). PATIENTS AND METHODS: Previously untreated patients with stage IIIB/IV NSCLC received ISIS 5132 by continuous intravenous infusion at 2.0 mg/kg/d for 14 days. Starting doses were paclitaxel 175 mg/m(2) and carboplatin targeting an area under the free platinum plasma concentration-time curve (AUC(fp)) of 5 mg . min/ml (dose level 1). The carboplatin dose was then increased to AUC(fp) 6 mg . min/ml (dose level 2) after which the paclitaxel dose was increased to 200 mg/m(2) (dose level 3). The maximum tolerated dose was established by toxicity during the first two 21-day cycles of therapy. The pharmacokinetics of all three agents was determined before and during the ISIS 5132 infusion. RESULTS: Thirteen patients were treated with the carboplatin/paclitaxel/ISIS 5132 combination. Dose-limiting neutropenia occurred in two patients at dose level 3. Grade 3 and 4 nonhematologic toxicities were infrequent and limited to nausea and constipation. The maximum tolerated doses were carboplatin AUC(fp) 6 mg . min/ml, paclitaxel 175 mg/m(2), and ISIS 5132 2.0 mg/kg/d for 14 days. There were no objective responses and the concurrent infusion of ISIS 5132 did not alter the plasma pharmacokinetics of paclitaxel or total platinum. CONCLUSION: ISIS 5132 can be safely combined with standard doses of carboplatin and paclitaxel. Combining cytotoxic chemotherapeutic agents with inhibitors of aberrant signal transduction mediated by Raf proteins produced no objective responses in the dose and schedule administered in this study.

A phase I trial of an antisense inhibitor of hepatitis C virus (ISIS 14803), administered to chronic hepatitis C patients.

BACKGROUND/AIMS: ISIS 14803 is a 20-unit antisense phosphorothioate oligodeoxynucleotide that binds to hepatitis C virus (HCV) RNA at the translation initiation region of the internal ribosome entry site (IRES) and inhibits protein expression in cell culture and mouse models. This Phase I, open-label, dose-escalation trial of ISIS 14803 was performed in chronic HCV patients. METHODS: At least 7 days after receiving an initial single dose, twenty-eight patients received 0.5-3 mg/kg ISIS 14803 thrice weekly for 4 weeks by intravenous infusion or subcutaneous injection. RESULTS: In most patients, the 4-week treatment did not reduce plasma HCV RNA. However, 3 patients receiving > or =2 mg/kg had transient HCV reductions of 1.2-1.7 log(10) that persisted < or =32 days. These reductions were accompanied by asymptomatic, self-resolving elevations in serum alanine transaminase (ALT) levels to >10x the upper limit of normal. Two other patients had ALT flares without plasma HCV reduction. No clinical signs, symptoms of hepatic dysfunction, or laboratory changes in albumin or prothrombin time accompanied ALT elevations. CONCLUSIONS: ISIS 14803 treatment was associated with HCV reductions in only 3/28 patients. ALT flares in 5 patients also occurred. Further studies to evaluate ISIS 14803 treatment and the mechanisms of the ALT flares are now required.

A phase I trial of aprinocarsen (ISIS 3521/LY900003), an antisense inhibitor of protein kinase C-alpha administered as a 24-hour weekly infusion schedule in patients with advanced cancer.

PURPOSE: A phase I study was performed to determine the maximum tolerated dose (MTD), safety profile and pharmacology of aprinocarsen (ISIS 3521), an antisense oligonucleotide to protein kinase C-alpha, in patients with refractory solid tumors. EXPERIMENTAL DESIGN: Fourteen patients were treated in sequential cohorts of aprinocarsen by 24-hour continuous infusion (CIV), weekly, at doses of 6, 12, 18 and 24 mg/kg. RESULTS: One grade 4 toxicity was observed, transient grade 4 neutropenia at 18 mg/kg. Grade 3 toxicities included neutropenia at 12 mg/kg, fever and hemorrhage at 18 mg/kg, and neutropenia, nausea, and chills at 24 mg/kg. Grade 2 toxicities included thrombocytopenia myalgias, chills, headache, fatigue, fever and nausea/vomiting. Mean prothrombin times and activated partial thromboplastin times (aPTT) increased by 10% and 29% from baseline (p = 0.006 and 0.005). Mean complement split products (Bb and C3a) increased 1.6-fold and 3.6-fold (from p = 0.014 and 0.004, respectively). These changes correlated with dose and were transient with recovery to baseline by day 7. Steady state plasma concentrations (Css) of aprinocarsen were achieved within four hours. Css better described changes in aPTT than dose. Clinical evidence of complement activation was not observed. CONCLUSIONS: In contrast to 21-day protracted infusion schedules, delivery of aprinocarsen over a 24-hour infusion schedule showed concentration-dependent effects on coagulation and complement, which are consistent with nonclinical toxicology studies performed in the phosphorothioate DNA antisense drug class. These coagulation and complement changes resulted in a maximum tolerated dose 24 mg/kg.