Solution Oligonucleotide APIs: Regulatory Considerations.

Manufacture of oligonucleotide active pharmaceutical ingredients (APIs) typically consists of solid-phase synthesis, deprotection and cleavage, purification and filtration, and isolation from aqueous solutions through lyophilization. In the first step of drug product manufacture, the API is dissolved in water again and excipients are added. While isolation of oligonucleotide APIs can be meaningful in many cases, there may be cases where keeping the API in solution provides benefit, and multiple technical aspects must be taken into account and balanced when determining the appropriate API form. A significant factor is whether an API in solution will contain additional components. While APIs in solution containing additional components (so-called formulated APIs) are well established for biological products, there are regulatory guidelines in place that represent hurdles for industry to using a formulated API approach for oligonucleotide drugs. The present communication outlines conditions where a formulated API approach can be chosen in compliance with existing guidelines. Relevant aspects pertaining to risk management, GMP standards, facility design, control strategies, and regulatory submission content are discussed. In addition, the authors propose that existing guidelines be modernized to enable the use of a formulated API approach for additional reasons than the ones described in the existing regulatory framework. The manuscript aims to promote a dialog with regulators in this field.

Tin-free radical alkoxyamine addition and isomerization reactions by using the persistent radical effect: variation of the alkoxyamine structure.

Various C-centered radicals can efficiently be generated through thermal C-O-bond homolysis of alkoxyamines. This method is used to perform environmentally benign radical cyclization and intermolecular addition reactions. These alkoxyamine isomerizations and intermolecular carboaminoxylations are mediated by the persistent radical effect (PRE). In the paper, the effect of the variation of the alkoxyamine structure--in particular steric effects in the nitroxide moiety--on the outcome of the PRE mediated radical reactions will be discussed. Fourteen different nitroxides were used in the studies. It will be shown that reaction times can be shortened about 100 times upon careful tuning of the alkoxyamine structure. Activation energies for the C-O-bond homolysis of the various alkoxyamines are provided. The kinetic data are used to explain the reaction outcome of the PRE-mediated processes.

Steric and electronic effects in cyclic alkoxyamines--synthesis and applications as regulators for controlled/living radical polymerization.

The synthesis of new six- and seven-membered cyclic alkoxyamines bearing ethyl groups at the alpha-N position of the alkoxyamines is described. The key step in the synthesis of the sterically hindered six-membered cyclic alkoxyamines is a Wadsworth-Horner-Emmons olefination with bisphosphonate 1. The seven-membered cyclic alkoxyamines were prepared from the corresponding six-membered keto alkoxyamines by ring-enlargement with trimethylsilyl(TMS)-diazomethane. The use of the new alkoxyamines as regulators/initiators for radical polymerization is discussed. Efficient controlled and living polymerization of styrene and n-butyl acrylate was obtained with the six-membered tetraethyl alkoxyamine 13. Controlled polymerizations can be conducted even at 90 degrees C. In addition, alkoxyamine 13 can be used for the preparation of AB diblock and ABA triblock copolymers with narrow polydispersities. The influence of the replacement of methyl groups in the alpha-position of the N atom in cyclic alkoxyamines by larger ethyl groups on the styrene polymerization (reaction time, PDI, kinetics of the C-O bond homolysis) is discussed. In addition, thermal decomposition of the new alkoxyamines was studied. Furthermore, the synthesis of N,N-bissilylated alkoxyamines is described. The silylated alkoxyamines are not suitable as regulators/initiators for the controlled/living radical polymerization. The C-O bonds in silylated alkoxyamines are stronger than the C-O bonds in analogous N,N-dialkylated alkoxyamines. The experimental results are verified by calculations with Gaussian 98 (A. 9).

Microwave-assisted free radical chemistry using the persistent radical effect.

Environmentally benign radical carboaminoxylations of various nonactivated olefins and difficult radical cyclization reactions are performed in good to excellent yields and with short reaction times under microwave irradiation.

Increased muscle activity during rapid eye movement sleep correlates with decrease of striatal presynaptic dopamine transporters. IPT and IBZM SPECT imaging in subclinical and clinically manifest idiopathic REM sleep behavior disorder, Parkinson's disease, and controls.

STUDY OBJECTIVES: Rapid eye movement (REM) sleep behavior disorder (RBD) is characterized by complex behavior during REM sleep. The etiology of this disorder is still unknown, but a recent study showed that RBD precedes symptoms of Parkinson disease (PD) by several years, and in a previous study, we found reduced striatal dopamine transporters in idiopathic clinically manifest RBD. DESIGN: Hypothesizing that subclinical RBD shows a less severe reduction of striatal dopamine transporters than clinically manifest RBD, we studied striatal postsynaptic dopamine D2-receptors with (S)-2hydroxy-3iodo-6-methoxy-([1-ethyl-2-pyrrolidinyl]methyl) benzamide labeled with iodine 123 (IBZM) and the striatal presynaptic dopamine transporters with (N)-(3-iodopropene-2-yl)-2beta-carbomethoxy-3beta-(4-chlorophenyl) tropane labeled with iodine 123 (IPT) using single-photon emission computed tomography (SPECT) in the following groups: 8 patients with idiopathic subclinical RBD, 8 patients with idiopathic clinically manifest RBD, 11 controls, and 8 patients with PD stage Hoehn & Yahr I. RESULTS: The IPT uptake was highest in controls. There was a significant decrease in IPT uptake from controls to patients with subclinical RBD, from patients with subclinical RBD to clinically manifest RBD, and from patients with clinically manifest RBD to patients with PD (controls: right = 4.07 +/- 0.29, left = 4.07 +/- 0.30; subclinical RBD: right = 3.56 +/- 0.21, left = 3.55 +/- 0.25; clinically manifest RBD: right = 3.18 +/- 0.43, left = 3.2 +/- 0.43; PD: ipsilateral to the clinically affected body side = 3.25 +/- 0.35, contralateral to the clinically affected body side = 2.51 +/- 0.28). Muscle activity during REM sleep lasting persistently longer than 0.5 seconds was independently associated with reduction of striatal dopamine transporters (P = 0.001). The IBZM uptake was not significantly different between the groups. CONCLUSIONS: This study suggests that there is a continuum of reduced striatal dopamine transporters involved in the pathophysiologic mechanisms causing increased muscle activity during REM sleep in patients with subclinical RBD.

Intermolecular radical addition and addition/cyclization reactions of alkoxyamines onto nonactivated alkenes.

[reaction: see text] Alkoxyamines A, which are readily prepared from commercially available starting materials, undergo efficient thermal radical carboaminoxylations onto various nonactivated alkenes to provide 1,4-functionalized malonates B in good to excellent yields. The experiments are very easy to conduct. The carboaminoxylations can be combined with radical cyclization and fragmentation processes.