Bivalent recombinant vaccine for botulinum neurotoxin types A and B based on a polypeptide comprising their effector and translocation domains that is protective against the predominant A and B subtypes.

The botulinum neurotoxins (BoNTs) are a large family of extremely potent, neuroparalytic, dichain proteins which act at the peripheral nervous system. The wide genetic diversity observed with this neurotoxin family poses a significant challenge for the development of an effective botulinum vaccine. The present study describes a vaccine development platform based on protein fragments representing the N-terminal two-thirds of each toxin molecule. These fragments, designated LH(N), comprise the light chain and translocation domains of each neurotoxin and are devoid of any neuron-binding activity. Using codon-optimized genes, LH(N) fragments derived from BoNT serotypes A and B were expressed in Escherichia coli in high yield with >1 g of purified, soluble fragment recoverable from 4.5 liter-scale fermentations. The protective efficacy of LH(N)/A was significantly enhanced by treatment with formaldehyde, which induced intramolecular cross-linking but virtually no aggregation of the fragment. A single immunization of the modified fragment protected mice from challenge with a 10(3) 50% lethal dose (LD(50)) of BoNT/A(1) with an 50% effective dose (ED(50)) of 50 ng of the vaccine. In similar experiments, the LH(N)/A vaccine was shown to protect mice against challenge with BoNT/A subtypes A(1), A(2), and A(3), which is the first demonstration of single-dose protection by a vaccine against the principal toxin subtypes of BoNT/A. The LH(N)/B vaccine was also highly efficacious, giving an ED(50) of approximately 140 ng to a challenge of 10(3) LD(50) of BoNT/B(1). In addition, LH(N)/B provided single-dose protection in mice against BoNT/B(4) (nonproteolytic toxin subtype).

Molecular properties and preclinical pharmacology of JNJ-1250132, a steroidal progesterone receptor modulator that inhibits binding of the receptor to DNA in vitro.

Progesterone receptor modulators have diverse potential therapeutic uses, including the treatment of endometriosis, uterine fibroids and breast cancer. Here we describe the molecular properties and preclinical pharmacology of a new steroidal progestin antagonist, JNJ-1250132. The compound is a high affinity ligand for the progesterone receptor, possessing cross-reactivity with other steroid receptors comparable to that of steroidal antagonists such as mifepristone. It inhibits progestin-inducible alkaline phosphatase gene expression in T47D human breast cancer cells, and also inhibits their in vitro proliferation. It inhibits gestation in rats and progesterone-dependent endometrial transformation in rabbits with efficacies comparable to mifepristone. Like mifepristone, it is a glucocorticoid antagonist in vivo. In cell-free DNA binding assays, the compound inhibits binding of the human progesterone receptor to a progesterone response element, and thus is similar to onapristone in this regard. In contrast, as judged by proteolytic analysis, JNJ-1250132 induces a receptor conformation more similar to that induced by mifepristone, which promotes receptor binding to DNA. Therefore, JNJ-1250132 has unique effects on the progesterone receptor that may translate into a novel clinical profile.

Pyrimidinylpyrroloquinolones as highly potent and selective PDE5 inhibitors for treatment of erectile dysfunction.

A series of N-pyrimidinylpyrroloquinolones were discovered as extremely potent and selective PDE5 inhibitors. Representative compounds demonstrated in vivo efficacy in dog erectile dysfunction models and are orally bioavailable.

Structure-activity relationships of N-acyl pyrroloquinolone PDE-5 inhibitors.

The discovery of the potent and selective PDE-5 inhibitory activity of a pyrroloquinolone scaffold prompted us to explore the SAR of its acyl derivatives. During the course of these studies, three structural series were found with K(i) values for PDE-5 in the subnanomolar range. Systematic modification of one of these leads produced a compound with excellent selectivity for PDE-5 over other phosphodiesterases and oral bioavailability of 15% in male rats. This compound also displayed in vivo efficacy in an anesthetized canine model of erection when dosed intravenously.

Furoyl and benzofuroyl pyrroloquinolones as potent and selective PDE5 inhibitors for treatment of erectile dysfunction.

Synthesis of furoyl and benzofuroyl pyrroloquinolones as potent and selective PDE5 inhibitors was reported. Their in vitro potencies in inhibiting PDE5 and selectivity in inhibiting other PDE isozymes (PDE1-4 and PDE6) were evaluated. Some of these compounds are more potent than sildenafil with better selectivity toward PDE1 and PDE6. Incorporation of solublizing groups resulted in bioavailable analogues. Selected compounds showed in vivo efficacy in anesthetized dog model for penile erection.

Characterization of a novel phosphodiesterase type 5 inhibitor: JNJ-10258859.

We have characterized a novel, potent, and selective phosphodiesterase type 5 inhibitor, JNJ-10258859 ((R)-(-)-3-(2,3-dihydro-benzofuran-5-yl)-2-[5-(4-methoxy-phenyl)-pyrimidin-2-yl]-1,2,3,4-tetrahydro-pyrrolo[3,4-b]quinolin-9-one). Its inhibitory effects on phosphodiesterase 1-6 were determined using enzymes partially purified from human tissues. The compound inhibited phosphodiesterase type 5 with a K(i) of 0.23 nM and displayed excellent selectivity versus phosphodiesterase types 1-4 (>/=22,000 fold compared to phosphodiesterase type 5). It had 27-fold selectivity over phosphodiesterase type 6 as well. In a cell-based assay, JNJ-10258859 was more potent than sildenafil in potentiating nitric oxide (NO) induced accumulation of intracellular cGMP. The in vivo effect of JNJ-10258859 was evaluated in an anesthetized dog model via intravenous administration. The compound had similar efficacy to sildenafil in enhancing both the amplitude and duration of intracavernosal pressure increase induced by electrical stimulation to the pelvic nerve. No significant effects on either mean aortic pressure or heart rate were observed during the course of the experiments. This data suggests that JNJ-10258859 could be a useful treatment for erectile dysfunction.

Comparison of endothelin-1-mediated tissue tension and calcium mobilization effects in isolated rabbit corpus cavernosum.

OBJECTIVES: To directly compare and contrast the effects of endothelin-1 (ET-1) and adrenoreceptor agonists norepinephrine and phenylephrine on eliciting calcium influx in primary rabbit corpus cavernosum cells and their ability to elicit tissue contractions. The potent vasoconstrictor peptide ET-1 and the alpha-adrenoreceptor agonists are important modulators of smooth muscle tone in the penile corpus cavernosum. However, the mechanisms involved in maintaining smooth muscle tone and contraction are not clearly understood. METHODS: Intracellular calcium mobilization was measured in cultured corpus cavernosum smooth muscle cells using calcium-sensing dyes in conjunction with a fluorometric imaging plate reader. Tissue tension studies on rabbit corpus cavernosum were conducted using organ chambers. RESULTS: ET-1 at concentrations as low as 10 nM was sufficient to induce a transient increase of intracellular calcium in these cells. In contrast, concentrations of 1 mM and greater of norepinephrine and phenylephrine were required to elicit comparable calcium fluxes in cavernosum cells. Tissue bath studies indicated that ET-1 is a potent stimulator of corpus cavernosum smooth muscle contraction, with concentrations as low as 10 nM sufficient to initiate contraction. CONCLUSIONS: The potency of ET-1 in producing contraction on tissue strips and inducing calcium flux suggests that ET-1 might be an important mediator for modulating and maintaining corpus cavernosum smooth muscle tone. Therefore, additional exploration of the role of endothelins and their receptors in the tumescence and detumescence states of the penis would be extremely valuable.

Synthesis and biological activities of novel beta-carbolines as PDE5 inhibitors.

A series of N(2)-furoyl and N(2)pyrimidinyl beta-carbolines was discovered to possess potent inhibitory activity against PDE5. During the synthesis we developed a tandem resin quenching protocol, which allowed us to synthesize large number of target compounds in a rapid fashion. Representative compounds exhibit superior selectivity to sildenafil versus other isozymes of PDEs, and demonstrated in vivo efficacy in increasing introcavernosal pressure in dogs.