Phase 12 Trial Experience of Alicaforsen ISIS 2302 Antisense to ICAM1 in Crohns Disease

In 1997, Glover et al. (7) reported on a phase I study of ISIS 2302 in healthy volunteers. These investigators showed that single iv doses of ISIS 2302 from 0.06 to 2.0 mg kg and four doses every other day ranging from 0.5 to 2.0 mg kg given by 2-h infusion were safe. The pharmacokinetic data realized by this study were the same as those found in primates. The only drug-related adverse event was a dose-related (approx 1.5-fold at a dose of 2.0 mg kg) and transient (for 2-4 h after dosing)...

PKCa LY900003 ISIS 3521 Affinitac

PKC-a belongs to a class of serine-threonine kinases whose involvement in oncogenesis is suggested by the fact that they are the major intracellular receptors for tumor-inducing phorbol esters. The results of a phase I study suggested that an AS-ODN directed against PKC-a (ISIS 3521) may be effective in the treatment of low-grade lymphoma (11). In a phase I trial, ISIS 3521 was delivered over a period of 21 d by continuous iv infusion followed by a 7-d rest...

In Vitro Transdermal Delivery Methods

The feasibility of transdermal delivery studies for a given chemical is first examined in an in vitro system. Skin is placed in either a static Franz diffusion chamber (40), a static side-by-side chamber (41), or a flow-through Bronaugh diffusion cell system (42). In all three systems, the skin is sandwiched between two pieces of glass or plastic with a hole cut out of the center. The unit is then clamped tightly shut to prevent fluid leakage. The drug of interest is placed on the epidermal...

Sequence Selection

A recent review describes computational ways to select the best candidate regions to target from known cloned genes (10). If known genes are being targeted in different cell types, a useful strategy is to search antisense oligo databases, such as that from the University of Utah (http antisense. genetics.utah.edu). For new mRNA targets, identification of the best sequence for antisense activity is not always predictable by computational methods. Some potentially useful sequences in an mRNA may...

Hras ISIS 2503

Abnormal expression of Ras proteins is frequently found with oncogenic transformation, making ras a promising therapeutic target. A 20-base phosphorothioate AS-ODN that binds to the translation initiation region of human H-ras mRNA (ISIS 2503) selectively reduced the expression of H-ras mRNA and protein in cell culture. Expression of other family members including N-ras, Ki-rasA, and Ki-rasB was not affected by ISIS 2503 in vitro. In a phase I trial, ISIS 2503 caused no dose-limiting toxicity...

Characteristics of the Brain Capillary Endothelia

The term barrier does not fully explain the functions and roles of the BBB. This monolayer of endothelial cells has features that allow selective entry of molecules into the CNS that are required for brain growth and function, while excluding or effluxing molecules and substances that may be harmful. Unlike the cells of the systemic capillaries, the endothelial cells of the brain capillaries are not fenestrated but, instead, are sealed together by intercellular tight junctions (see Fig. 1)....

Antisense Strategies in Animal Myocytes SERCA2a Overexpression Compared to Phospholamban Downregulation in Rat and

Adenoviral gene transfer into isolated myocytes has allowed the investigation of changes in phospholamban in species other than mouse, as well as direct comparison of phospholamban depletion with SERCA2a overexpression in the same preparation. Rat is similar to mouse in the predominance of SERCA2a (92 ) compared to other mechanisms of calcium removal, while in rabbit heart (like human) SERCA2a controls approx 70 , and there is a much larger role for the Na+ Ca2+ exchanger to remove excess...

Pancreatic Cancer

Cancer of the exocrine pancreas will attack an estimated 31,000 Americans in 2004, causing an estimated 30,000 deaths (1). Cancer of the exocrine pancreas (hereafter simply referred to as pancreatic cancer) occurs more frequently in African-Americans than in European-Americans. The disease progresses rapidly. Pancreatic cancer is usually refractory to treatment, with a median survival time of 12 mo. Overall, only 1 of patients live more than 5 yr after diagnosis. However, if the disease is...

Cytochrome P450 Enzymes

CYPs are a superfamily of heme-thiolate monooxygenases that were originally characterized by their absorption spectrum at 450 nm in the presence of carbon monoxide. These enzymes are located on the endoplasmic reticulum and present predominantly in the liver, but significant quantities are also found in the small intestine, kidney, and skin (15-17). This family of enzymes oxidizes a wide variety of endogenous and exogenous substrates, including xenobiotics. The term xenobiotic refers to...

Unmodified Antisense and the BBB Are Modifications Necessary 41 Antisense Therapy in Pathophysiology Involving

Although many studies have investigated various modification strategies designed to enhance penetration of antisense across the BBB, few studies have considered if unmodified antisense can cross the BBB. It is possible that enzy-matically resistant analogs of unmodified AS-ODNs enter the CNS by binding to transporters receptors present in the BBB for the import of endogenous substances. By analogy, viruses often use this method to enter cells. For example, human immunodeficiency virus-1 binds...

Metabolism of Paclitaxel

Most of the information about paclitaxel's metabolism is derived from incubation with human microsome fractions. Two significant metabolites are detectable (Fig. 2) 1. 6a-Hydroxytaxol (6HOT), resulting from CYP2C8-mediated hydrolysis, is the major metabolite detected after adult liver microsomal incubations (8,50) formed by a single stereospecific hydroxylation at the six position of the taxane ring. This metabolite is not observed in rats (50), demonstrating important interspecies differences...

Tc99mAnnexin V Detection of Apoptosis

Preparation of Chelator-Annexin-V Human annexin-V produced by expression in Escherichia coli is obtained commercially from Sigma. Annexin-V is derivatized as described previously 123 with hydrozinonicotinamide HYNIC by gently mixing for 3 h at 22 C in the dark a mixture of 5.6 mg mL of annexin-V in 20 mM HEPES pH 7.4 , 100 mM NaCl, and 22 g of 6-HYNIC Anor MED, Langley, British Columbia dissolved in 18.5 L of N,N-dimethyl formamide 42 mM . At the end of the incubation period, the...

The Use of an Enema Formulation of ISIS 2302 in Ulcerative Colitis

The research experience to date with iv ISIS 2302 in ulcerative colitis is limited to two patients, one of whom received placebo. The treated patient had a sustained remission 7 yr after treatment with 13 doses of iv ISIS 2302 0.5 mg kg . Van Deventer et al. reported the largest clinical experience with ISIS 2302 in ulcerative colitis at the United European Gastroenterology Week 2001 . Using a nightly ISIS 2302 enema for 1 mo, they studied patients age gt 18 with active ulcerative colitis...

Pka Gem 231

A number of advanced chemistry modifications have been employed to improve the specificity, pharmacokinetics, and safety profiles of phosphor-othioate oglionucleotide. These so-called mixed-backbone oglionucleotides permit oral and colorectal administration as a result of their increased in vivo metabolic stability. One of these compounds, GEM 231, is designed to interfere with the production of the RIa regulatory subunit of PKA, a cellular growth promoter that is increased within a wide...

Conclusion

Drug resistance during cancer therapy can occur during exposure to subtoxic concentrations of death-inducing drugs. On the other hand, excessive cytotoxicity is damaging for both tumor and normal cells 13 . In contrast to the permanent drug resistance through constitutive, ectopic overexpression of antiapoptotic genes such as bcl-2 and bcl-xL, which may not be relevant to cellular conditions in naturally occurring tumors, the duration of antisense downregulation by a typical AS-ODN is usually...

Phospholamban Downregulation and Heart Failure

Although antisense per se has not been used to downregulate phospholamban in animal models of heart failure, a similar strategy has been pursued with dominant-negative constructs, with some success. A pseudophos-phorylated mutant of phospholamban was introduced using viral transfer to a hamster model of cardiomyopathy, and progression of heart failure was slowed 35 . The same method has been used to transfect rats with myocardial infarction, and again the deterioration of ventricular structure...

Pharmacokinetic Analysis of Plasma Paclitaxel Levels

The data were evaluated with the assistance of the computer program PKCALC as published previously 66 . The iv plasma data were fit to the following equation 67 In which Cp t is the plasma concentration at time t, A, and B are intercept terms, a is a distribution rate constant, and p is an elimination rate constant. All data are reported as the mean SD, and key parameters are presented in Table 1. Paclixel's pharmacokinetics have been typically modeled with a two-compartment system the drug is...

V

Antisense DNA red base-paired to mRNA target. plasma and cellular proteins 139 . The known modes of phosphorothioate toxicity invite further modifications to reduce sulfur content in therapeutic oligonucleotides. Encouraging results have recently been obtained suggesting that greater potency and specificity might be possible with 2'-O-alkyl RNA DNA 2'-O-alkyl RNA phosphorothioate chimeras 140,141 , PNA-peptide conjugates 142 , a p a-anomeric DAN chimeras 143,144 , DNA boranophosphates...

PMO Synthesis and Sequences

PMOs were synthesized at AVI BioPharma Corvallis, OR as previously described 62 . Purity was greater than 90 full length, as determined by reverse-phase high-performance liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. Lyophilized PMO compounds were dissolved in sterile apyrogenic saline and filtered through 0.2- m Acrodisc filters Gelman . The antisense PMO AVI-4472 is complementary to the AUG translation initiation region of the rat...

ASODN Design

Unmodified DNA oligonucleotides are either poorly taken up or rapidly degraded by serum and cell-associated nucleases. To prevent this, phosphor- othioate oligonucleotides P-ODNs approx 16-18 bases long in which one of the nonbridging oxygen atoms in the nucleotide is replaced with sulfur provide protection from nuclease degradation without significantly interfering with RNase H activity. 2'-Methoxy-ethoxy modifications in the first and last four or five nucleotides flanking the central region,...

Cu64 Chimeras

Using the methods described above, we will prepare Cu-64-PNA-peptides with complementary sequences known to hybridize specifically to mRNAs for activated K-RAS mutated in the twelfth codon, CCND1, HER2, MYC, and mutant tumor suppressor p53, and capable of binding to the receptor for IGF-1. We will test the specificity of uptake and mRNA hybridization of the Cu-64-PNA-peptide probes in normal human epithelial cells vs K-RAS mutant AsPC1 human pancreatic cancer cells and K-RAS wild-type BT474...

Construction of Antisense Kras Expressing Plasmids

Dusty Miller Fred Hutchinson Cancer Research Center, Seattle, WA generously provided the retroviral vector plasmid LNSX. LNSX contains the selectable neomycin phosphotransferase gene, which is expressed from the retroviral long terminal repeat LTR , and an inserted DNA is placed in the downstream of an internal SV40 early promoter 21 . In this experiment, the LNSX is used as an expression plasmid vector see Note 1 . A K-ras cDNA fragment spanning from nucleotide nt 171 in the first exon to...

Antisense Fundamentals

AS-ODNs are short stretches of 16-18 chemically modified nucleotides designed to hybridize with specific complementary mRNA to block production of proteins encoded by the targeted mRNA transcripts. AS-ODNs use Watson-Crick base pairing to specifically bind to complementary mRNAs. For this binding to occur, the targeted mRNA region must be accessible to the oligonucleotide. In addition, the oligonucleotide must be taken up efficiently by the target cells and must arrive intact to the site of the...

Chemical Penetration Enhancers

A wide variety of chemicals can act as penetration enhancers. The mechanisms by which these chemicals alter delivery are dependent on their chemical class. First, they may act on the drug itself by altering its thermodynamic activity, leading to greater partitioning into the skin. Second, enhancers may interact with the stratum corneum lipids, causing a reduced barrier 32 . Effective enhancers include water, alcohols methanol and ethanol , alkylmethyl sulfoxides, pyrrolidones, laurocapram,...

Phosphorodiamidate Morpholino Oligomers

The studies presented in this chapter utilized phosphorodiamidate morpholino oligomer PMO antisense agents. The chemistry of PMO overcomes some of the major shortcomings of phosphorothioate oligonucleotides see Chapter 13 . The PMO backbone is nonionic at physiological pH and consists of a six-membered morpholine ring instead of a deoxyribose sugar, and the charged phosphodiester internucleoside linkage is replaced by an uncharged phosphorodiamidate linkage 52 . PMOs are highly resistant to...

Info

Baseline, and at 24 h, 48 h, 72 h, 1 wk, and 2 wk postdose. Adverse experiences were collected on an ongoing basis from the time of dosing to discharge from the study at follow-up wk 2. The most frequent adverse events reported included lower-extremity aches and headache. The majority of adverse events were graded mild in intensity and were generally self-limiting. Serum complement C3a was measured. Four subjects had elevated C3a greater than twice the upper limit of normal normal 0-400 ng mL ,...

M Ian Phillips PhD dsc

Imo Immunomodulatory Oligonucleotides

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