The N-Acetylgalactosamine-conjugated small interfering RNA inclisiran can be coadministered safely with atorvastatin in cynomolgus monkeys resulting in additive low-density lipoprotein cholesterol reductions.

Inclisiran, a small interfering RNA, selectively inhibits proprotein convertase subtilisin/kexin type 9 (PCSK9) synthesis in the liver and has been shown to reduce low-density lipoprotein cholesterol (LDL-C) by ≥50% in patients with hypercholesterolemia receiving maximally tolerated statins. The toxicokinetic, pharmacodynamic, and safety profiles of inclisiran when coadministered with a statin were characterized in cynomolgus monkeys. Six cohorts of monkeys were administered either atorvastatin (40 mg/kg, reduced to 25 mg/kg during the study, daily, oral gavage), inclisiran (300 mg/kg every 28 days, subcutaneous administration), atorvastatin (40/25 mg/kg) and inclisiran combinations (30, 100, or 300 mg/kg), or control vehicles over 85 days followed by 90 days' recovery.

Evaluation of the distribution and excretion of [C]-inclisiran following single subcutaneous administration in cynomolgus monkeys.

Inclisiran is a small interfering RNA molecule that was designed to reduce plasma low-density lipoprotein cholesterol (LDL-C) levels by inhibiting proprotein convertase subtilisin/kexin type 9 synthesis in the liver. This study aimed to characterize the tissue distribution and excretion of inclisiran after dosing in monkeys. A single 20 mg/kg subcutaneous injection of [C]-inclisiran was administered to 12 male cynomolgus monkeys.

In Vitro Drug-Drug Interaction Evaluation of GalNAc Conjugated siRNAs Against CYP450 Enzymes and Transporters.

Small interfering RNAs (siRNAs) represent a new class of medicines that are smaller (∼16,000 Da) than biologic therapeutics (>150,000 Da) but much larger than small molecules (<900 Da). Current regulatory guidance on drug-drug interactions (DDIs) from the European Medicines Agency, Food and Drug Administration, and Pharmaceutical and Medical Devices Agency provides no recommendations for oligonucleotide therapeutics including siRNAs; therefore, small molecule guidance documents have historically been applied. Over ∼10 years, in vitro DDI investigations with siRNAs conjugated to a triantennary -acetylgalactosamine [(GalNAc)-siRNA] ligand have been conducted during nonclinical drug development to elucidate the potential clinical DDI liability.

Toxicologic and Inhibitory Receptor Actions of the Etomidate Analog ABP-700 and Its Metabolite CPM-Acid.

Background: The etomidate analog ABP-700 produces involuntary muscle movements that could be manifestations of seizures. To define the relationship (if any) between involuntary muscle movements and seizures, electroencephalographic studies were performed in Beagle dogs receiving supra-therapeutic (~10× clinical) ABP-700 doses. γ-aminobutyric acid type A (GABAA) and glycine receptor studies were undertaken to test receptor inhibition as the potential mechanism for ABP-700 seizures.

Effect of repeated apoA-IMilano/POPC infusion on lipids, (apo)lipoproteins, and serum cholesterol efflux capacity in cynomolgus monkeys.

MDCO-216, a complex of dimeric recombinant apoA-IMilano (apoA-IM) and palmitoyl-oleoyl-phosphatidylcholine (POPC), was administered to cynomolgus monkeys at 30, 100, and 300 mg/kg every other day for a total of 21 infusions, and effects on lipids, (apo)lipoproteins, and ex-vivo cholesterol efflux capacity were monitored. After 7 or 20 infusions, free cholesterol (FC) and phospholipids (PL) were strongly increased, and HDL-cholesterol (HDL-C), apoA-I, and apoA-II were strongly decreased. We then measured short-term effects on apoA-IM, lipids, and (apo)lipoproteins after the first or the last infusion.

Metalloproteinase inhibitors, nonantimicrobial chemically modified tetracyclines, and ilomastat block Bacillus anthracis lethal factor activity in viable cells.

Lethal toxin, produced by the bacterium Bacillus anthracis, is a major contributor to morbidity and mortality in animals and humans who have contracted anthrax. One component of this toxin, lethal factor (LF), proteolytically inactivates members of the mitogen-activated protein kinase kinase (MAPKK or MEK) family. In this study we show that CMT-300, CMT-308, and Ilomastat, agents initially characterized as matrix metalloproteinase inhibitors which are in early stages of development as pharmaceuticals, effectively inhibit the zinc metalloproteinase activity of LF.

Quantification of 6-deoxy-6-demethyl-4-dedimethylaminotetracycline (COL-3) in human plasma using liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

An accurate and reliable liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method has been developed and validated for the determination of 6-deoxy-6-demethyl-4-dedimethylaminotetracycline (COL-3) in human plasma. The assay used chrysin as an internal standard (I.S.

Gene array analysis of osteoblast differentiation.

We have used gene array technology to chart changes in gene expression during differentiation of the mouse calvarial-derived MC3T3-E1 cell line to an osteoblast-like phenotype. Expression was analyzed on a mouse gene array panel of 588 cDNAs representing tightly regulated genes with key roles in various biological processes. When compared with NIH3T3 fibroblasts, MC3T3-E1 cells showed generally higher expression of cyclins and Bcl-2 family members, as well as specific expression of products such as the CD44 antigen, which is consistent with their calvarial origin.

A role for the anti-inflammatory properties of tetracyclines in the prevention of acute lung injury.

The etiology of inflammatory disorders involves many cellular, plasmatic and humoral pathways of signaling culminating in the production of enzymatic and free radical mediated tissue damage. Multiple redundant pathways of initiation and elusive temporal expression of initiators pose formidable barriers to effective treatment. Nowhere is this more evident than in the adult respiratory distress syndrome (ARDS), a systemic inflammatory disorder leading to pulmonary failure where, despite significant advances in intensive care management, mortality has improved only 10% over the last decade.

Phosphate is a specific signal for induction of osteopontin gene expression.

Osteopontin is a phosphorylated glycoprotein secreted to the mineralizing extracellular matrix by osteoblasts during bone development. It is believed to facilitate the attachment of osteoblasts and osteoclasts to the extracellular matrix, allowing them to perform their respective functions during osteogenesis. Several other functions have been suggested for this protein, and its up-regulation is associated with various disease states related to calcification, including arterial plaque formation and the formation of kidney stones.

Effects of tetracyclines on angiogenesis in vitro.

Most tumors kill their hosts by the process of metastasis rather than by local growth of the primary mass. A significant factor contributing to the distant invasion of cancer cells is the ability of tumors to produce large numbers of new blood vessels in their midst, known as angiogenesis. This both provides access to nourishment for the primary cancer and enables the cells to escape from the tumor and enter the bloodstream.

Relationship between alkaline phosphatase levels, osteopontin expression, and mineralization in differentiating MC3T3-E1 osteoblasts.

We are using viral oncogene probes to study the pathways by which osteoblast-specific gene expression is induced in ascorbic acid-treated MC3T3-E1 cells. The 12S product of the adenovirus E1A gene binds directly to key cellular regulators and, as a result, represses tissue specific gene expression and blocks differentiation in a wide variety of cell types. The main cellular targets of the E1A 12S product are the pRB family and p300/CBP family.

Transgenic mice and transhybridomas producing chimeric mouse/human anti-human interleukin-2 receptor recombinant antibodies.

Transgenic mice were developed that secreted chimeric mouse/human anti-human interleukin-2 receptor (IL-2R) antibodies (Ab) into their serum. In addition, hybridomas producing the chimeric Ab in tissue culture were generated from the transgenic mice. The presence of the mouse/human immunoglobulin (Ig) transgene did not appear to affect rearrangement of endogenous murine Ig in the hybridomas.

Identification of specific adenovirus E1A N-terminal residues critical to the binding of cellular proteins and to the control of cell growth.

Adenovirus early region 1A (E1A) oncogene-encoded sequences essential for transformation- and cell growth-regulating activities are localized at the N terminus and in regions of highly conserved amino acid sequence designated conserved regions 1 and 2. These regions interact to form the binding sites for two classes of cellular proteins: those, such as the retinoblastoma gene product, whose association with the E1A products is specifically dependent on region 2, and another class which so far is known to include only a large cellular DNA-binding protein, p300, whose association with the E1A products is specifically dependent on the N-terminal region. Association between the E1A products and either class of cellular proteins can be disrupted by mutations in conserved region 1.

Differential effects of a murine and chimeric mouse/human anti-interleukin-2 receptor antibody on human T-cell proliferation.

The preference for interleukin-2 receptor (IL-2R) expression on activated, compared with resting T lymphocytes makes the IL-2R a promising target for selective immunosuppressive therapy. To increase the potential therapeutic effectiveness of anti-IL-2R monoclonals, a chimeric mouse/human variant was constructed from Ig genes isolated from a murine anti-human IL-2R hybridoma cell line, designated AHT54. AHT54 binds to the same or spatially related epitope as IL-2 on the p55 protein that constitutes the low- and high-affinity forms of IL-2R.

A chimeric mouse/human anti-IL-2 receptor antibody with enhanced biological activities.

A chimeric mouse/human MAb against the human p55 IL-2R was constructed from Ig genes isolated from a mouse hybridoma cell line, designated AHT107. AHT107 binds to a different epitope on p55 than IL-2, and similar to observations made for other rodent anti-IL-2R antibodies that do not recognize the same or spatially related epitope as IL-2, murine AHT107 did not efficiently inhibit proliferation of T-lymphocytes in mitogen and MLR PBMC stimulation assays. In contrast, the chimeric AHT107 antibodies containing a human IgG-1 constant region had substantially more anti-proliferative activity than their murine IgG-I counterparts.

The soluble interleukin-2 receptor as a marker for human neoplasia and immune status.

Activation of T cells is associated with a dramatic increase in expression of the interleukin-2 receptor. In addition to the intact receptor found at the cell surface, activated T cells produce a truncated form of the receptor (sIL-2R) that is secreted as a soluble molecule. Patients with neoplastic disease or diseases involving immune activation exhibit markedly elevated serum levels of sIL-2R.

Interactions between cell growth-regulating domains in the products of the adenovirus E1A oncogene.

Among the various biological activities expressed by the products of the adenovirus E1A gene are the abilities to induce cellular DNA synthesis and proliferation in quiescent primary baby rat kidney cells. The functional sites for these activities lie principally within two regions of the E1A proteins: an N-terminal region and a small second region of approximately 20 amino acids further downstream. To study the biological functions of the first domain, we constructed an in-frame deletion of amino acid positions 23 through 107 of the E1A products.

Different functional domains of the adenovirus E1A gene are involved in regulation of host cell cycle products.

We have analyzed the cell cycle effects that different domains of the adenovirus E1A proteins have on quiescent primary BRK cells. Studies with deletion mutants that in combination removed all but the N-terminal 85 amino acids common to both the 12S and 13S proteins suggest that this region may be sufficient for the induction of synthesis of proliferating cell nuclear antigen and the stimulation of DNA synthesis. A second domain also common to the N-terminal exon of the 12S and 13S proteins was required for the induction of mitosis and stimulation of proliferation of primary BRK cells.

Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135.

Adenovirus E1A coding sequences that enable ras and pmt oncogenes to transform cultured primary cells.

Plasmids expressing partial adenovirus early region 1A (E1A) coding sequences were tested for activities which facilitate in vitro establishment (immortalization) of primary baby rat kidney cells and which enable the T24 Harvey ras-related oncogene and the polyomavirus middle T antigen (pmt) gene to transform primary baby rat kidney cells. E1A cDNAs expressing the 289- and 243-amino acid proteins expressed both E1A transforming functions. Mutant hrA, which encodes a 140-amino acid protein derived from the amino-terminal domain shared by the 289- and 243-amino acid proteins, enabled ras (but not pmt) to transform and facilitated in vitro establishment to a low, but detectable, extent.

Lytic and transforming functions of individual products of the adenovirus E1A gene.

To distinguish the individual roles of the 13S, 12S, and 9S adenovirus E1A gene products, we isolated the corresponding cDNA clones and recombined them into both plasmids and viruses. Only the expected E1A mRNA products were made from the corresponding 12S and 13S viruses. The 9S mRNA was detected when the 9S virus was coinfected with the 13S virus but not when either virus was infected alone.

Repair-defective mutants of Alteromonas espejiana, the host for bacteriophage PM2.

The in vivo repair processes of Alteromonas espejiana, the host for bacteriophage PM2, were characterized, and UV- and methyl methanesulfonate (MMS)-sensitive mutants were isolated. Wild-type A. espejiana cells were capable of photoreactivation, excision, recombination, and inducible repair.