The Physical Compatibility of Clinically Used Concentrations of Diltiazem Hydrochloride With Heparin Sodium.

Background: Acute treatment of atrial fibrillation often requires concomitant intravenous (IV) continuous infusions of unfractionated heparin and diltiazem. Concomitantly infusing these medications through the same IV line minimizes multiple IV sites. Diltiazem and heparin visual compatibility have been previously investigated but with limited drug dwell times and differing drug concentrations leading to inconsistent published results. Objective: To investigate the physical compatibility of diltiazem hydrochloride at concentrations of 5 and 1 mg/mL combined with an equal volume of heparin sodium 100 units/mL. Methods: Using a 0.22-µm filter, 15 mL of heparin sodium were placed into a polyvinyl chloride infusion bag followed by 15 mL of either diltiazem hydrochloride 5 or 1 mg/mL. Admixtures were prepared in triplicate. Each admixture was investigated for visual precipitation, spectrophotometric absorbance, and pH change at baseline and 1, 5, 8, and 24 hours after mixing. Physical incompatibility was determined by visual observation, increased spectrophotometric absorbance, and demonstrative pH changes. Results: Each diltiazem 5 mg/mL admixture exhibited a slight haze and enhanced absorbance readings indicating turbidity while none revealed a demonstrative pH change. None of the diltiazem 1 mg/mL assessments revealed visual precipitation or suggested turbidity. Only one pH reading at 5 hours revealed a demonstrative change from baseline. Conclusions: Our findings indicate that infusing diltiazem hydrochloride 5 mg/mL with heparin sodium 100 units/mL in the same IV line cannot be advocated. In contrast, our findings suggest that heparin sodium 100 units/mL infused with diltiazem hydrochloride 1 mg/mL is physically compatible but chemical stability was not assessed.

Considerations in the Use of Codon Optimization for Recombinant Protein Expression.

Codon optimization is a gene engineering approach that is commonly used for enhancing recombinant protein expression. This approach is possible because (1) degeneracy of the genetic code enables most amino acids to be encoded by multiple codons and (2) different mRNAs encoding the same protein can vary dramatically in the amount of protein expressed. However, because codon optimization potentially disrupts overlapping information encoded in mRNA coding regions, protein structure and function may be altered. This chapter discusses the use of codon optimization for various applications in mammalian cells as well as potential consequences, so that informed decisions can be made on the appropriateness of using this approach in each case.

Codon Optimization in the Production of Recombinant Biotherapeutics: Potential Risks and Considerations.

Biotherapeutics are increasingly becoming the mainstay in the treatment of a variety of human conditions, particularly in oncology and hematology. The production of therapeutic antibodies, cytokines, and fusion proteins have markedly accelerated these fields over the past decade and are probably the major contributor to improved patient outcomes. Today, most protein therapeutics are expressed as recombinant proteins in mammalian cell lines. An expression technology commonly used to increase protein levels involves codon optimization. This approach is possible because degeneracy of the genetic code enables most amino acids to be encoded by more than one synonymous codon and because codon usage can have a pronounced influence on levels of protein expression. Indeed, codon optimization has been reported to increase protein expression by >  1000-fold. The primary tactic of codon optimization is to increase the rate of translation elongation by overcoming limitations associated with species-specific differences in codon usage and transfer RNA (tRNA) abundance. However, in mammalian cells, assumptions underlying codon optimization appear to be poorly supported or unfounded. Moreover, because not all synonymous codon mutations are neutral, codon optimization can lead to alterations in protein conformation and function. This review discusses codon optimization for therapeutic protein production in mammalian cells.

Role of the Funny Current Inhibitor Ivabradine in Cardiac Pharmacotherapy: A Systematic Review.

The pharmacology, pharmacokinetics, efficacy and safety of ivabradine are reviewed. Ivabradine is an oral medication that directly and selectively inhibits the hyperpolarization-activated cyclic-nucleotide gated funny (If) current in the sinoatrial node resulting in heart rate reduction. It has a plasma elimination half-life of 6 hours and is administered twice daily. Ivabradine is extensively metabolized by cytochrome P450 3A4, and its metabolism is affected by inducers and inhibitors of the 3A4 enzyme. Studies in patients with heart failure indicate that ivabradine improves surrogate markers such as exercise tolerance. The results of (1) phase III trial demonstrated ivabradine significantly reduced heart failure hospitalizations but had no effect on mortality. Ivabradine has been extensively evaluated for coronary artery disease wherein (2) large trials was shown to have no mortality benefit. Ivabradine has been associated with improved symptoms in stable chronic angina pectoris. Ivabradine has been evaluated for other cardiovascular conditions including tachycardias of various natures, arrhythmia prevention postcardiac surgery, in acute coronary syndrome, and for heart rate control during coronary computed tomography angiogram. The most common adverse events reported in clinical trials were bradycardia, new-onset atrial fibrillation, and phosphenes. Ivabradine, a novel cardiac medication, has been studied in numerous cardiac conditions. It is only currently approved in the United States to reduce hospitalizations for systolic heart failure. The role of this medication in other conditions has not been fully elucidated.

Translation regulation by ribosomes: Increased complexity and expanded scope.

The primary function of ribosomes is to decode mRNAs into polypeptide chains; however, this description is overly simplistic. Accumulating evidence shows that ribosomes themselves can affect the relative efficiency with which various mRNAs are translated and indicates that these effects can be modulated by ribosome heterogeneity. The notion that ribosomes have regulatory capabilities was elaborated more than a decade ago in the ribosome filter hypothesis. Various lines of evidence support this idea and have shown that the translation of some mRNAs is affected by discrete binding interactions with rRNA or ribosomal proteins. Recent work from our laboratory has demonstrated that base-pairing of the Hepatitis C Virus (HCV) internal ribosome entry site (IRES) to 18S rRNA is required for IRES function, but only in the context of more complex ribosomal interactions. The HCV IRES provides an example of the ribosome filter that involves multiple binding interactions between mRNAs and ribosomal subunits.

A cell-free expression and purification process for rapid production of protein biologics.

Cell-free protein synthesis has emerged as a powerful technology for rapid and efficient protein production. Cell-free methods are also amenable to automation and such systems have been extensively used for high-throughput protein production and screening; however, current fluidic systems are not adequate for manufacturing protein biopharmaceuticals. In this work, we report on the initial development of a fluidic process for rapid end-to-end production of recombinant protein biologics. This process incorporates a bioreactor module that can be used with eukaryotic or prokaryotic lysates that are programmed for combined transcription/translation of an engineered DNA template encoding for specific protein targets. Purification of the cell-free expressed product occurs through a series of protein separation modules that are configurable for process-specific isolation of different proteins. Using this approach, we demonstrate production of two bioactive human protein therapeutics, erythropoietin and granulocyte-macrophage colony-stimulating factor, in yeast and bacterial extracts, respectively, each within 24 hours. This process is flexible, scalable and amenable to automation for rapid production at the point-of-need of proteins with significant pharmaceutical, medical, or biotechnological value.

Base pairing between hepatitis C virus RNA and 18S rRNA is required for IRES-dependent translation initiation in vivo.

Degeneracy in eukaryotic translation initiation is evident in the initiation strategies of various viruses. Hepatitis C virus (HCV) provides an exceptional example--translation of the HCV RNA is facilitated by an internal ribosome entry site (IRES) that can autonomously bind a 40S ribosomal subunit and accurately position it at the initiation codon. This binding involves both ribosomal protein and 18S ribosomal RNA (rRNA) interactions. In this study, we evaluate the functional significance of the rRNA interaction and show that HCV IRES activity requires a 3-nt Watson-Crick base-pairing interaction between the apical loop of subdomain IIId in the IRES and helix 26 in 18S rRNA. Mutations of these nucleotides in either RNA dramatically disrupted IRES activity. The activities of the mutated HCV IRESs could be restored by compensatory mutations in the 18S rRNA. The effects of the 18S rRNA mutations appeared to be specific inasmuch as ribosomes containing these mutations did not support translation mediated by the wild-type HCV IRES, but did not block translation mediated by the cap structure or other viral IRESs. The present study provides, to our knowledge, the first functional demonstration of mRNA-rRNA base pairing in mammalian cells. By contrast with other rRNA-binding sites in mRNAs that can enhance translation as independent elements, e.g., the Shine-Dalgarno sequence in prokaryotes, the rRNA-binding site in the HCV IRES functions as an essential component of a more complex interaction.

A critical analysis of codon optimization in human therapeutics.

Codon optimization describes gene engineering approaches that use synonymous codon changes to increase protein production. Applications for codon optimization include recombinant protein drugs and nucleic acid therapies, including gene therapy, mRNA therapy, and DNA/RNA vaccines. However, recent reports indicate that codon optimization can affect protein conformation and function, increase immunogenicity, and reduce efficacy. We critically review this subject, identifying additional potential hazards including some unique to nucleic acid therapies. This analysis highlights the evolved complexity of codon usage and challenges the scientific bases for codon optimization. Consequently, codon optimization may not provide the optimal strategy for increasing protein production and may decrease the safety and efficacy of biotech therapeutics. We suggest that the use of this approach is reconsidered, particularly for in vivo applications.

Illicit narcotic injection masquerading as acute pulmonary embolism.

A 23-year-old male presented from a nursing home with hypotension, tachycardia, diaphoresis and electrocardiographic evidence of right ventricular strain that was confirmed by echocardiography. His differential diagnosis included sepsis and pulmonary embolism. A high-resolution computed tomography scan demonstrated no pulmonary emboli but did demonstrate multiple bilateral pulmonary nodules. Upon questioning he admitted to injecting a long-acting narcotic that had been manually macerated, dissolved in saline, and injected through an indwelling intravenous line. Lung biopsy findings were consistent with cellulose-induced perivascular granulomatosis. Cellulose granulomatosis can be seen in patients who inject medications designed for oral use and should be considered in patients who present with acute pulmonary hypertension.

Physical evidence supporting a ribosomal shunting mechanism of translation initiation for BACE1 mRNA.

In Alzheimer disease, elevated levels of the BACE1 enzyme are correlated with increased production of amyloid peptides and disease pathology. The increase in BACE1 levels is post-transcriptional and may involve altered translation efficiency. Earlier studies have indicated that translation of BACE1 mRNA is cap-dependent. As ribosomal subunits move from the cap-structure to the initiation codon, they fail to recognize several AUG codons in the 5' leader. In this study, we looked for physical evidence of the mechanism underlying ribosomal scanning or shunting along the BACE1 5' leader by investigating structural stability in the 5' leaders of endogenous mRNAs in vivo. To perform this analysis, we probed RNAs using lead(II) acetate, a cell-permeable chemical that induces cleavage of unpaired nucleotides having conformational flexibility. The data revealed that the ≈440-nt 5' leader was generally resistant to cleavage except for a region upstream of the initiation codon. Cleavage continued into the coding region, consistent with destabilization of secondary structures by translating ribosomes. Evidence that a large segment of the BACE1 5' leader was not cleaved indicates that this region is structurally stable and suggests that it is not scanned. The data support a mechanism of translation initiation in which ribosomal subunits bypass (shunt) part of the BACE1 5' leader to reach the initiation codon. We suggest that a nucleotide bias in the 5' leader may predispose the initiation codon to be more accessible than other AUG codons in the 5' leader, leading to an increase in its relative utilization.

Practical considerations for the pharmacotherapy of pulmonary arterial hypertension.

Pulmonary arterial hypertension is a devastating disease. Before the 1990s, when pharmacologic treatment was finally approved, only supportive therapy was available, consisting of anticoagulation, digoxin, diuretics, and supplemental oxygen. Calcium channel blocker therapy was also an option, but only a small percentage of patients respond to it. However, starting with epoprostenol in 1996, the number of drugs approved to treat pulmonary arterial hypertension increased. Three distinct classes of drugs were developed based on the pathophysiology of the disease: the prostanoids, endothelin-1 receptor antagonists, and phosphodiesterase type 5 inhibitors. The prostanoids are administered either parenterally or by inhalation to replace the lack of prostacyclin within the pulmonary arterial vasculature. The endothelin-1 receptor antagonists were the first class of oral drugs to be developed, but drug interactions and adverse effects are prominent with this class. The phosphodiesterase type 5 inhibitors increase the second messenger cyclic guanosine monophosphate (GMP) that is induced by nitric oxide stimulation. All of the drugs within these three classes are distinct in and of themselves, and their clinical use requires in-depth knowledge of pulmonary arterial hypertension and its pathophysiology. Because these drugs have different mechanisms of action, combination therapy has shown promise in patients with severe disease, although data are still lacking. This article should serve as a practical guide for clinicians who encounter patients with pulmonary arterial hypertension and the drugs used for the treatment of this devastating disease.

Analysis of rRNA processing and translation in mammalian cells using a synthetic 18S rRNA expression system.

Analysis of processing, assembly, and function of higher eukaryotic ribosomal RNA (rRNA) has been hindered by the lack of an expression system that enables rRNA to be modified and then examined functionally. Given the potential usefulness of such a system, we have developed one for mammalian 18S rRNA. We inserted a sequence tag into expansion segment 3 of mouse 18S rRNA to monitor expression and cleavage by hybridization. Mutations were identified that confer resistance to pactamycin, allowing functional analysis of 40S ribosomal subunits containing synthetic 18S rRNAs by selectively blocking translation from endogenous (pactamycin-sensitive) subunits. rRNA constructs were suitably expressed in transfected cells, shown to process correctly, incorporate into ≈ 15% of 40S subunits, and function normally based on various criteria. After rigorous analysis, the system was used to investigate the importance of sequences that flank 18S rRNA in precursor transcripts. Although deletion analysis supported the requirement of binding sites for the U3 snoRNA, it showed that a large segment of the 5' external transcribed spacer and the entire first internal transcribed spacer, both of which flank 18S rRNA, are not required. The success of this approach opens the possibility of functional analyses of ribosomes, with applications in basic research and synthetic biology.

Incidence of bleeding in renally impaired patients receiving incorrectly dosed eptifibatide or bivalirudin while undergoing percutaneous coronary intervention.

BACKGROUND: Limited data are available regarding adverse bleeding events associated with antithrombotic agents incorrectly dosed based on renal function in patients receiving percutaneous coronary intervention (PCI). OBJECTIVE: To compare the incidence of bleeding during their hospital stay in patients with reduced renal function receiving incorrect doses of bivalirudin or eptifibatide to the incidence of correct doses, based on manufacturer recommendations; secondary objectives were to determine the incidence of correct dosing based on manufacturer recommendations and the incidence of TIMI (Thrombolysis in Myocardial Infarction) major bleeding. METHODS: A chart review over a 32-month period showed that patients with reduced renal function who received either eptifibatide or bivalirudin during PCI were evaluated for correct dosing based on manufacturer recommendations, bleeding incidence according to the TIMI criteria, and extent of bleeding according to the TIMI and GUSTO (Global Use of Strategies to Open Occluded Coronary Arteries) criteria. RESULTS: One hundred ninety patients met inclusion criteria, 56 who received eptifibatide and 134 who received bivalirudin. Eptifibatide was dosed incorrectly in 64% of the patients. Patients receiving incorrectly dosed compared to correctly dosed eptifibatide experienced significantly more bleeding (64% vs 35%, respectively, p = 0.04), a greater extent of bleeding based on the TIMI and GUSTO criteria (p = 0.03 and p = 0.009, respectively), and had more TIMI major bleeding (19% vs 5%, respectively). Bivalirudin was dosed incorrectly in 28% of the patients. Patients receiving incorrectly dosed compared to correctly dosed bivalirudin experienced a significantly greater extent of bleeding based on the GUSTO criteria (p = 0.01). There was no significant difference between the incidence of bleeding (37% vs 21%, respectively; p = 0.06), extent of bleeding based on the TIMI criteria (p = 0.058), or incidence of TIMI major bleeding (5% vs 3%). CONCLUSIONS: Patients receiving incorrectly dosed eptifibatide and bivalirudin are susceptible to adverse bleeding events. The occurrence of incorrect dosing offers an opportunity for pharmacist-driven institutional improvement.

Determinants of initiation codon selection during translation in mammalian cells.

Factors affecting translation of mRNA contribute to the complexity of eukaryotic proteomes. In some cases, translation of a particular mRNA can generate multiple proteins. However, the factors that determine whether ribosomes initiate translation from the first AUG codon in the transcript, from a downstream codon, or from multiple sites are not completely understood. Various mRNA properties, including AUG codon-accessibility and 5' leader length have been proposed as potential determinants that affect where ribosomes initiate translation. To explore this issue, we performed studies using synthetic mRNAs with two in-frame AUG codons-both in excellent context. Open reading frames initiating at AUG1 and AUG2 encode large and small isoforms of a reporter protein, respectively. Translation of such an mRNA in COS-7 cells was shown to be 5' cap-dependent and to occur efficiently from both AUG codons. AUG codon-accessibility was modified by using two different elements: an antisense locked nucleic acid oligonucleotide and an exon-junction complex. When either element was used to mask AUG1, the ratio of the proteins synthesized changed, favoring the smaller (AUG2-initiated) protein. In addition, we observed that increased leader length by itself changed the ratio of the proteins and favored initiation at AUG1. These observations demonstrate that initiation codon selection is affected by various factors, including AUG codon-accessibility and 5' leader length, and is not necessarily determined by the order of AUG codons (5'→3'). The modulation of AUG codon accessibility may provide a powerful means of translation regulation in eukaryotic cells.

The 5' leader of the mRNA encoding the marek's disease virus serotype 1 pp14 protein contains an intronic internal ribosome entry site with allosteric properties.

We demonstrate the presence of a functional internal ribosome entry site (IRES) within the 5' leader (designated 5L) from a variant of bicistronic mRNAs that encode the pp14 and RLORF9 proteins from Marek's disease virus (MDV) serotype 1. Transcribed as a 1.8-kb family of immediate-early genes, the mature bicistronic mRNAs have variable 5' leader sequences due to alternative splicing or promoter usage. Consequently, the presence or absence of the 5L IRES in the mRNA dictates the mode of pp14 translation and leads to the production of two pp14 isoforms that differ in their N-terminal sequences. Real-time reverse transcription-quantitative PCR indicates that the mRNA variants with the 5L IRES is two to three times more abundant in MDV-infected and transformed cells than the mRNA variants lacking the 5L IRES. A common feature to all members of the 1.8-kb family of transcripts is the presence of an intercistronic IRES that we have previously shown to control the translation of the second open reading frame (i.e., RLORF9). Investigation of the two IRESs residing in the same bicistronic reporter mRNA revealed functional synergism for translation efficiency. In analogy with allosteric models in proteins, we propose IRES allostery to describe such a novel phenomenon. The functional implications of our findings are discussed in relation to host-virus interactions and translational control.