Leveraging Integrated Continuous Manufacturing to Address Critical Issues in the U.S. Military.

There is a tremendous opportunity to modernize the pharmaceutical manufacturing industry-relinquishing outdated machines that have been used for decades, and replacing them with state-of-the-art equipment that reflect more contemporary advanced technologies. This article describes how the implementation of continuous manufacturing, replacing outdated batch systems, can positively impact our health care sector. Important benefits will include the creation of advanced pharmaceutical manufacturing jobs in the United States, the establishment of capabilities and capacity to quickly produce drugs critical to U.S. citizens, the reduction of health care costs through more efficient manufacturing, and access to better quality drugs through more sophisticated and reliable production processes. Furthermore, the application of continuous manufacturing will enable the U.S. Government, in partnership with pharmaceutical companies, to address current issues such as drug shortages, national emergencies (eg, natural disasters or chemical, biological, radiological, or nuclear threats), the Strategic National Stockpile (ie, improving response time and reducing maintenance costs), and the delivery of critical drugs to distant geographies (eg, forward military bases). The article also provides a detailed example of a critical aspect of continuous manufacturing: the ability to overcome technical challenges encountered by batch technologies.

An automated modular assembly line for drugs in a miniaturized plant.

We report here a fully automated, end-to-end, integrated continuous manufacturing process for a small-molecule generic medication with built-in quality assurance. The entire process fits into a box of 30.7 m2 modular footprint and a total residence time of less than 30 h, with a throughput up to 40.3 × 106 tablets per year.

Downregulation of survivin is associated with reductions in TNF receptors' mRNA and protein and alterations in nuclear factor kappa B signaling in urothelial cancer cells.

Because survivin is selectively expressed in and associated with an unfavorable prognosis in transitional cell carcinoma of the bladder (TCC), we treated T-24 cells with survivin siRNA. Survivin siRNA treatment caused a profound decrease of survivin protein that was associated with decreased cell growth, a specific G2/M arrest and increased cytochrome c release. Microarray analysis of apoptosis genes showed that levels of 14/114 gene products were decreased after 72 hours treatment with survivin siRNA, including survivin, three TNF receptors, Akt, c-Abl, caspases and their related genes and Bcl-2 and NF-kappaB signaling related genes. TNFR1, pro-caspase-2 and Akt protein levels were decreased after survivin siRNA treatment for 48 and 72 hours. Downregulation of survivin causes changes in mitosis and apoptosis, which may be related to changes in TNF receptors and NF-kappaB signaling.

Neonatal hypoxia suppresses oligodendrocyte Nogo-A and increases axonal sprouting in a rodent model for human prematurity.

Premature human infants frequently suffer from periventricular leukomalacia (PVL) characterized by the loss of central myelinated tracts in the brain [Neuropathology, 22 (2002) 193]. Rodent chronic sublethal hypoxia (CSH) from P3 to 33 (postnatal day 3-33) provides a model for PVL characterized by cerebral ventriculomegaly and reductions in cerebral white matter volume [Brain Res. Dev. Brain Res. 111 (1998) 197; Proc. Natl. Acad. Sci. USA 100 (2003) 11718]. Here, we demonstrate that mice exposed to CSH from P3 to P33 followed by normoxia from P33 to P75 continue to exhibit a locomotor hyperactivity that resembles behavioral changes observed in some human children with very low birth weights. Because periventricular white matter is specifically lost in PVL, we examined the expression of oligodendrocyte proteins. Hypoxic rearing dramatically decreases the level of the axon outgrowth inhibitor Nogo-A in oligodendrocytes of CNS white matter at P12. The Nogo-A decrease exceeds the moderate decrease in another myelin protein, myelin associated glycoprotein (MAG). Although myelin protein expression returns to normal by maturity (P75), persistent abnormalities in axonal trajectories are detectable. Anterograde axonal tracing from motor cortex demonstrates ectopic corticofugal fibers in the corticospinal tract (CST), corpus callosum, and caudate nucleus of adult animals reared in CSH. Thus, hypoxia-induced reduction in myelin-derived axon outgrowth inhibitors appears to contribute axonal misconnection to the pathology of very low birth weight infants.

Rho kinase inhibition enhances axonal regeneration in the injured CNS.

Myelin-associated inhibitors limit axonal regeneration in the injured brain and spinal cord. A common target of many neurite outgrowth inhibitors is the Rho family of small GTPases. Activation of Rho and a downstream effector of Rho, p160ROCK, inhibits neurite outgrowth. Here, we demonstrate that Rho is directly activated by the myelin-associated inhibitor Nogo-66. Using a binding assay to measure Rho activity, we detected increased levels of GTP Rho in PC12 and dorsal root ganglion (DRG) cell lysates after Nogo-66 stimulation. Rho activity levels were not affected by Amino-Nogo stimulation. Rho inactivation with C3 transferase promotes neurite outgrowth of chick DRG neurons in vitro, but with the delivery method used here, it fails to promote neurite outgrowth after corticospinal tract (CST) lesions in the adult rat. Inhibition of p160ROCK with Y-27632 also promotes neurite outgrowth on myelin-associated inhibitors in vitro. Furthermore, Y-27632 enhances sprouting of CST fibers in vivo and accelerates locomotor recovery after CST lesions in adult rats.