Improvements in Survival and Clinical Benefit With Gemcitabine as First-Line Therapy for Patients With Advanced Pancreas Cancer: A Randomized Trial.

PURPOSE: Most patients with advanced pancreas cancer experience pain and must limit their daily activities because of tumor-related symptoms. To date, no treatment has had a significant impact on the disease. In early studies with gemcitabine, patients with pancreas cancer experienced an improvement in disease-related symptoms. Based on those findings, a definitive trial was performed to assess the effectiveness of gemcitabine in patients with newly diagnosed advanced pancreas cancer. PATIENTS AND METHODS: One hundred twenty-six patients with advanced symptomatic pancreas cancer completed a lead-in period to characterize and stabilize pain and were randomized to receive either gemcitabine 1,000 mg/m2 weekly x 7 followed by 1 week of rest, then weekly x 3 every 4 weeks thereafter (63 patients), or to fluorouracil (5-FU) 600 mg/m2 once weekly (63 patients). The primary efficacy measure was clinical benefit response, which was a composite of measurements of pain (analgesic consumption and pain intensity), Karnofsky performance status, and weight. Clinical benefit required a sustained (> or = 4 weeks) improvement in at least one parameter without worsening in any others. Other measures of efficacy included response rate, time to progressive disease, and survival. RESULTS: Clinical benefit response was experienced by 23.8% of gemcitabine-treated patients compared with 4.8% of 5-FU-treated patients (P = .0022). The median survival durations were 5.65 and 4.41 months for gemcitabine-treated and 5-FU-treated patients, respectively (P = .0025). The survival rate at 12 months was 18% for gemcitabine patients and 2% for 5-FU patients. Treatment was well tolerated. CONCLUSION: This study demonstrates that gemcitabine is more effective than 5-FU in alleviation of some disease-related symptoms in patients with advanced, symptomatic pancreas cancer. Gemcitabine also confers a modest survival advantage over treatment with 5-FU.

Forced degradation study and characterization of main impurities of ibuprofen soft gelatin capsules by LC-MS-QTOF.

Ibuprofen soft gelatin capsules were subjected to degradation under acidic, basic, oxidation, photolytic, thermal, humidity, and metal ions conditions. To analyse the degradation products, a reversed-phase liquid chromatography (RP-LC) indicative stability method was successfully developed. Chromatographic separation was achieved using a Poroshell HPH-C18 150 x 4.6 mm, 4 µm, column at 25 °C, with a mobile phase constituted by 0.1% phosphoric acid and acetonitrile in gradient at a flow rate of 1.0 mL• min -1 , using ultraviolet detection at 220 nm and injection volume of 20 µL. In total, eight unknown impurities were found. The peaks RRt 0.49, RRt 0.75, and RRt 0.95 were above 0.17%, corresponding to the identification threshold. Those were identified and characterized by LC-MS-QTOF, with the same chromatographic conditions, except for the exchange of 0.1% phosphoric acid for 0.1% formic acid. The impurities originated from the interaction of ibuprofen with excipients: esterification with PEG, with sorbitol/sorbitan, and with glycerol by-products, which has not yet been reported in the literature. The developed method can be used in several pharmaceutical areas as quality control of impurities, studies of forced degradation, and for the development of future formulations.

A metal-free blue chromophore derived from plant pigments.

Blue natural pigments are rare, especially among plants. However, flowering species that evolved to attract Hymenoptera pollinators are colored by blue anthocyanin-metal complexes. Plants lacking anthocyanins are pigmented by betalains but are unable to produce blue hues. By extending the π-system of betalains, we designed a photostable and metal-free blue dye named BeetBlue that did not show toxicity to human hepatic and retinal pigment epithelial cells and does not affect zebrafish embryonal development. This chiral dye can be conveniently synthesized from betalamic acid obtained from hydrolyzed red beetroot juice or by enzymatic oxidation of l-dopa. BeetBlue is blue in the solid form and in solution of acidified polar molecular solvents, including water. Its capacity to dye natural matrices makes BeetBlue the prototype of a new class of low-cost bioinspired chromophores suitable for a myriad of applications requiring a blue hue.

A metal-free blue chromophore derived from plant pigments

Blue natural pigments are rare, especially among plants. However, flowering species that evolved to attract Hymenoptera pollinators are colored by blue anthocyanin-metal complexes. Plants lacking anthocyanins are pigmented by betalains but are unable to produce blue hues. By extending the p-system of betalains, we designed a photostable and metal-free blue dye named BeetBlue that did not show toxicity to human hepatic and retinal pigment epithelial cells and does not affect zebrafish embryonal development. This chiral dye can be conveniently synthesized from betalamic acid obtained from hydrolyzed red beetroot juice or by enzymatic oxidation of L-dopa. BeetBlue is blue in the solid form and in solution of acidified polar molecular solvents, including water. Its capacity to dye natural matrices makes BeetBlue the prototype of a new class of low-cost bioinspired chromophores suitable for a myriad of applications requiring a blue hue.

A metal-free blue chromophore derived from plant pigments

Blue natural pigments are rare, especially among plants. However, flowering species that evolved to attract Hymenoptera pollinators are colored by blue anthocyanin-metal complexes. Plants lacking anthocyanins are pigmented by betalains but are unable to produce blue hues. By extending the p-system of betalains, we designed a photostable and metal-free blue dye named BeetBlue that did not show toxicity to human hepatic and retinal pigment epithelial cells and does not affect zebrafish embryonal development. This chiral dye can be conveniently synthesized from betalamic acid obtained from hydrolyzed red beetroot juice or by enzymatic oxidation of L-dopa. BeetBlue is blue in the solid form and in solution of acidified polar molecular solvents, including water. Its capacity to dye natural matrices makes BeetBlue the prototype of a new class of low-cost bioinspired chromophores suitable for a myriad of applications requiring a blue hue.

Potential antiproliferative activity of polyphenol metabolites against human breast cancer cells and their urine excretion pattern in healthy subjects following acute intake of a polyphenol-rich juice of grumixama (Eugenia brasiliensis Lam.).

The bioavailability and metabolism of anthocyanins and ellagitannins following acute intake of grumixama fruit, native Brazilian cherry, by humans, and its in vitro antiproliferative activity against breast cancer cells (MDA-MB-231) were investigated. A single dose of grumixama juice was administered to healthy women (n = 10) and polyphenol metabolites were analyzed in urine and plasma samples collected over 24 h. The majority of the metabolites circulating and excreted in urine were phenolic acids and urolithin conjugates, the gut microbiota catabolites of both classes of polyphenols, respectively. According to pharmacokinetic parameters, the subjects were divided into two distinct groups, high and low urinary metabolite excretors. The pool of polyphenol metabolites found in urine samples showed a significant inhibition of cell proliferation and G2/M cell cycle arrest in MDA-MB-231 cells. Our findings demonstrate the large interindividual variability concerning the polyphenol metabolism, which possibly could reflect in health promotion.

Habitat Suitability Model for the Distribution of Ixodes scapularis (Acari: Ixodidae) in Minnesota.

Ixodes scapularis Say, the black-legged tick, is the primary vector in the eastern United States of several pathogens causing human diseases including Lyme disease, anaplasmosis, and babesiosis. Over the past two decades, I. scapularis-borne diseases have increased in incidence as well as geographic distribution. Lyme disease exists in two major foci in the United States, one encompassing northeastern states and the other in the Upper Midwest. Minnesota represents a state with an appreciable increase in counties reporting I. scapularis-borne illnesses, suggesting geographic expansion of vector populations in recent years. Recent tick distribution records support this assumption. Here, we used those records to create a fine resolution, subcounty-level distribution model for I. scapularis using variable response curves in addition to tests of variable importance. The model identified 19% of Minnesota as potentially suitable for establishment of the tick and indicated with high accuracy (AUC = 0.863) that the distribution is driven by land cover type, summer precipitation, maximum summer temperatures, and annual temperature variation. We provide updated records of established populations near the northwestern species range limit and present a model that increases our understanding of the potential distribution of I. scapularis in Minnesota.

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.

Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae).

The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4 degrees C and the supernatants were used in enzymatic assays at 30 degrees C, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 +/- 0.15 absorbance variation min(-1) mg protein(-1), at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 +/- 0.02 mmol p-nitroaniline min(-1) mg protein(-1). The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50 degrees C, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae)

The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4°C and the supernatants were used in enzymatic assays at 30°C, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis) was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis) was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50°C, a temperature that reduces enzyme stability to 80 and 60 percent of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

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.

Efficacy and toxicity of the antisense oligonucleotide aprinocarsen directed against protein kinase C-alpha delivered as a 21-day continuous intravenous infusion in patients with recurrent high-grade astrocytomas.

Protein kinase C alpha (PKC-alpha) is a cytoplasmic serine threonine kinase involved in regulating cell differentiation and proliferation. Aprinocarsen is an antisense oligonucleotide against PKC-alpha that reduces PKC-alphain human cell lines and inhibits a human glioblastoma tumor cell line in athymic mice. In this phase 2 study, aprinocarsen was administered to patients with recurrent high-grade gliomas by continuous intravenous infusion (2.0 mg/kg/day for 21 days per month). Twenty-one patients entered this trial. Their median age was 46 years (range, 28-68 years), median Karnofsky performance status was 80 (range, 60-100), median tumor volume was 58 cm3 (range, 16-254 cm3), and histology included glioblastoma multiforme (n = 16), anaplastic oligodendroglioma (n = 4), and anaplastic astrocytoma (n = 1). The number of prior chemotherapy regimens included none (n = 3), one (n = 10), and two (n = 8). No tumor responses were observed. Patients on this therapy rapidly developed symptoms of increased intracranial pressure with increased edema, enhancement, and mass effect on neuroimaging. The median time to progression was 36 days, and median survival was 3.4 months. The observed toxicities were mild, reversible, and uncommon (grade 3 thrombocytopenia [n = 3] and grade 4 AST [n = 1]), and no coagulopathy or CNS bleeding resulted from this therapy. Plasma concentrations of aprinocarsen during the infusion exhibited significant interpatient variability (mean = 1.06 mug/ml; range, 0.34-6.08 mug/ml). This is the first study to use an antisense oligonucleotide or a specific PKC-alpha inhibitor in patients with high-grade gliomas. No clinical benefit was seen. The rapid deterioration seen in these patients could result from tumor growth or an effect of aprinocarsen on bloodbrain barrier integrity.

A phase II trial of ISIS 3521 in patients with metastatic colorectal cancer.

This phase II study was designed to characterize the clinical activity of ISIS 3521 in patients with metastatic colorectal cancer (CRC). Sixteen patients with pretreated or refractory CRC were treated with ISIS 3521. Eleven patients were given a dose of 2.0 mg/kg per day, and 5 patients received 3.0 mg/kg per day given over 21 days followed by a 7-day rest period. Patients continued with study until evidence of disease progression or unacceptable toxicity was detected. Patients underwent baseline tumor biopsies followed by a second biopsy during the last week of the first 21-day infusion. All 16 patients underwent baseline tumor biopsies, and 12 of the 16 patients underwent on-study tumor biopsies. No evidence of tumor response was observed. One patient had stable disease after 2 cycles and remained on for 1 additional cycle only to demonstrate progression of disease at that time. No dose-limiting or other significant toxicities were observed at both dosages, which could not be explained by progression of disease. Fatigue was common in all patients treated but was not dose limiting, and there was no evidence of coagulopathy. Analysis of the tumor biopsies obtained from the 11 evaluable samples showed marked uptake of ISIS 3521 in the normal liver parenchyma. However, there was minimal uptake within the tumor cells. In addition, no evidence of any alteration in protein kinase C-a within the tumors or any downstream effects leading to apoptosis were observed. ISIS 3521 demonstrated no clinical activity or target modulation in refractory metastatic CRC.

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.

A phase I trial of ISIS 2503, an antisense inhibitor of H-ras, in combination with gemcitabine in patients with advanced cancer.

PURPOSE: The purpose of this study was to define the toxicity, pharmacokinetics, and clinical activity of the combination of ISIS 2503, an oligodeoxynucleotide antisense inhibitor of H-ras, and gemcitabine in patients with advanced solid tumors. EXPERIMENTAL DESIGN: The target dose of ISIS 2503 on this study was 6 mg/kg/day. Twenty-seven patients (16 male, 11 female) received 97 treatment courses (median, 2; range, 1-13). Nineteen patients were treated with a fixed gemcitabine dose of 1000 mg/m(2) on days 1 and 8 and two escalating doses of ISIS 2503 (4 and 6 mg/kg/day) as a 14-day continuous infusion starting on day 1. In addition, 8 patients (5 male, 3 female) received a flat dose of ISIS 2503 based on ideal body weight. Cycles were repeated every 3 weeks. Toxicities, graded according to the National Cancer Institute Common Toxicity Criteria, were recorded as maximum grade/patient for all treatment cycles. Pharmacokinetic analyses were performed to evaluate any interaction between these two agents. RESULTS: The most common nondose-limiting toxicity was hematological, manifested as neutropenia (5 grade 2, 7 grade 3, and 1 grade 4) and thrombocytopenia (10 grade 1, 5 grade 2, 5 grade 3, and 1 grade 4). Nonhematological toxicities included anorexia (7 grade 1, 3 grade 2, and 1 grade 3), nausea (10 grade 1 and 1 grade 3), fatigue (6 grade 1, 5 grade 2, and 3 grade 3), fever (6 grade 1, 2 grade 2, 1 and grade 3), and thrombosis associated with central lines (5). The plasma concentration of gemcitabine at the end of infusion was altered in the presence of ISIS 2503, leading to alterations on other pharmacokinetic parameters, but the observed differences were not clinically relevant. The plasma disposition of ISIS 2503 was not altered by gemcitabine coadministration. One partial response was documented in a heavily pretreated patient with metastatic breast cancer. Disease stabilization for greater than six cycles of treatment was observed in 5 patients. CONCLUSIONS: The combination of gemcitabine and ISIS 2503 was well tolerated and clinically active in this group of heavily pretreated patients. The recommended Phase II dose of gemcitabine (1000 mg/m(2)) and ISIS 2503 (6 mg/kg/day) warrants additional evaluation.

Phase I trial of ISIS 104838, a 2'-methoxyethyl modified antisense oligonucleotide targeting tumor necrosis factor-alpha.

ISIS 104838 is a 20-mer phosphorothioate antisense oligonucleotide (ASO) that binds tumor necrosis factor-alpha (TNF-alpha) mRNA. It carries a 2'-methoxyethyl modification on the five 3' and 5' nucleotide sugars, with 10 central unmodified deoxynucleotides. ISIS 104838 was identified from a 264 ASO screen in phorbol myristate acetate-activated keratinocytes, and the dose response was assessed in lipopolysaccharide (LPS)-activated monocytes. Healthy males received multiple intravenous (i.v.) ISIS 104838 infusions in a placebo-controlled dose escalation trial (0.1-6 mg/kg). Additional volunteers received single or multiple subcutaneous (s.c.) injections. ISIS 104838 suppressed TNF-alpha protein by 85% in stimulated keratinocytes. The IC50 for TNF-alpha mRNA inhibition in stimulated monocytes was <1 microM. For i.v., C(max) occurred at the end of infusion. The effective plasma half-life was 15 to 45 min at 0.1 to 0.5 mg/kg and 1 to 1.8 h for higher doses. The apparent terminal plasma elimination half-life approximated 25 days. Obese subjects had higher plasma levels following equivalent mg/kg doses. For s.c. injections, C(max) occurred at 2 to 4 h and was lower than with equivalent i.v. dosing. Plasma bioavailability compared with i.v. was 82% following a 200 mg/ml s.c. injection. Transient activated partial thromboplastin time prolongation occurred after i.v. infusions and minimally after s.c. injections. Two subjects experienced rash, one a reversible platelet decrease, and mild injection site tenderness was noted. TNF-alpha production by peripheral blood leukocytes, induced ex vivo by LPS, was decreased by ISIS 104838 (p < 0.01). ISIS 104838, a second-generation antisense oligonucleotide, was generally well tolerated intravenously and subcutaneously. The pharmacokinetics support an infrequent dosing interval. Inhibition of TNF-alpha production ex vivo was demonstrated.

Dose ranging pharmacokinetic trial of high-dose alicaforsen (intercellular adhesion molecule-1 antisense oligodeoxynucleotide) (ISIS 2302) in active Crohn's disease.

BACKGROUND AND AIMS: To evaluate the safety, pharmacokinetics and clinical efficacy of the intercellular adhesion molecule-1 antisense phosphorothioate oligonucleotide alicaforsen (ISIS 2302) at 250-350 mg in Crohn's disease. METHODS: : Patients (> 50 kg) with active Crohn's disease (Crohn's disease activity index > or = 220) were assigned by gender, randomly, to two alicaforsen treatment groups: 300 or 350 mg, infused intravenously three times a week for 4 weeks. All patients weighing 36-50 kg received 250 mg of alicaforsen. Background aminosalicylates, antibiotics, immunosuppressives and corticosteroids were permitted, but tumour necrosis factor-alphainhibitors were prohibited. The primary end-point was clinical remission (Crohn's disease activity index < or = 150). RESULTS: Twenty-two patients were enrolled with a mean baseline Crohn's disease activity index of 304. Steroids were used by 27%, 5-aminosalicylic acid by 68% and immunosuppressives by 27%; 23% had previously received infliximab. Five subjects withdrew after one to three infusions for infusion-related symptoms. Nine patients (41%) experienced clinical remission. Fifty-three per cent of the evaluable subjects receiving more than three infusions experienced remission (18% at week 8; 29% at week 12). The overall response, using a minimum decrease of 70 in the Crohn's disease activity index, was 41-47% for the evaluable group, at weeks 8 and 12. The median duration of remission was 14 weeks. Plasma pharmacokinetic results showed overlapping levels (Cmax, AUC) for the three doses. The infusion-related reaction profile consisted of fever, chills, headache, nausea, emesis or arthralgias, typically occurring 2-4 h after completion of the first infusion. Reactions were less frequent in patients receiving background corticosteroids. The 2-4-h transient post-infusion partial thromboplastin time prolongation values, a class effect of phosphorothioate oligonucleotides, were 18, 21 and 23 s for 250, 300 and 350 mg, respectively. CONCLUSIONS: Alicaforsen (ISIS 2302), at fixed doses of 300 and 350 mg, achieved the desired drug exposure and may be an effective therapy for Crohn's disease. Infusion-related reactions were observed less frequently in patients on corticosteroids, and with decreasing frequency with continued treatment.

Phase I clinical and pharmacokinetic study of protein kinase C-alpha antisense oligonucleotide ISIS 3521 administered in combination with 5-fluorouracil and leucovorin in patients with advanced cancer.

The present study was designed to determine the maximum tolerated dose (MTD), toxicity profile, pharmacokinetics (PKs), and antitumor activity of the protein kinase C-alpha antisense oligonucleotide ISIS 3521 (ISIS Pharmaceuticals, Inc., Carlsbad, CA) when administered in combination with 5-fluorouracil (5-FU) and leucovorin (LV). Patients with refractory solid tumors received ISIS 3521 as a 21-day continuous infusion administered simultaneously with 5-FU and LV given daily for 5 days repeated every 4-5 weeks (one cycle). 5-FU and ISIS 3521 PK analysis were performed on samples taken during the first cycle in all patients. Fifteen patients received ISIS 3521 at one of three dose levels: (a) 1.0 (n = 3 patients); (b) 1.5 (n = 3 patients); and (c) 2.0 (n = 9 patients) mg/kg/day. All patients simultaneously received 5-FU (425 mg/m(2)/day) and LV (20 mg/m(2)/day) for 5 consecutive days. Grade 1-2 toxicities included alopecia, fatigue, mucositis, diarrhea, anorexia, nausea/vomiting, and tumor pain. One patient had grade 3 chest pain considered to be related to 5-FU therapy, another patient had dose-limiting grade 3 mucositis resolving in <7 days, and one patient with a history of gastritis had an acute upper gastrointestinal bleed thought to be 5-FU-induced toxicity. Five patients developed cycle 1 grade 4 neutropenia, which resolved without colony-stimulating factors before the next treatment cycle. There were no effects on prothrombin time and activated partial thromboplastin time. A clinically defined MTD was not reached. The character and severity of these toxicities do not seem to be dose related, and, as such, there was no classical dose-limiting toxicity defining the MTD. ISIS 3521 PKs in the presence of 5-FU was consistent with those reported previously. 5-FU PK parameters were also similar in the presence or absence of ISIS 3521. Six of 14 patients ( approximately 43%) across all dose cohorts had an improvement in measurable tumor response ranging from minor reduction in tumor size (4 patients) to objective partial response (>50% reduction in tumor size, 2 patients). ISIS 3521 is tolerable at its recommended single-agent dose when given with 5-FU and LV. There is no apparent PK interaction between ISIS 3521 and 5-FU and LV. Antitumor activity was observed with the combination; however, it is uncertain whether clinical activity is a result of enhanced drug interaction. Our study warrants further exploration of efficacy in a Phase II and/or Phase III clinical trial setting.

Association of c-Raf expression with survival and its targeting with antisense oligonucleotides in ovarian cancer.

c-Raf is an essential component of the extracellular related kinase (ERK) signal transduction pathway. Immunohistochemical staining indicated that c-Raf was present in 49/53 ovarian adenocarcinomas investigated and high c-Raf expression correlated significantly with poor survival (P = 0.002). c-Raf protein was detected in 15 ovarian cancer cell lines. Antisense oligodeoxynucleotides (ODNs) (ISIS 5132 and ISIS 13650) reduced c-Raf protein levels and inhibited cell proliferation in vitro. Selectivity was demonstrated by the lack of effect of ISIS 5132 on A-Raf or ERK, while a random ODN produced only minor effects on growth and did not influence c-Raf expression. ISIS 5132 produced enhanced apoptosis and cells accumulated in S and G(2)/M phases of the cell cycle. In vivo, ISIS 5132 inhibited growth of the s.c. SKOV-3 xenograft while a mismatch ODN had no effect. These data indicate that high levels of c-Raf expression may be important in ovarian cancer and use of antisense ODNs targeted to c-Raf could provide a strategy for the treatment of this disease.