Intramembranous bone matrix is osteoinductive.

All known bone-derived osteoinductive factors have been isolated from endochondral (EC) bones and all initiate bone induction via EC ossification. However, to date no attempt has been made to isolate comparable factors from bones which form initially and completely via intramembranous (IM) ossification. The purpose of this work was to isolate osteoinductive proteins from IM bones. To accomplish this, we extracted proteins from bovine frontal bone matrix (intramembranous origin) using methods previously described for endochondral (EC) bone matrix (i.e., femur). Bone powder (< 1 mm) was decalcified and proteins extracted with 4 M guanidine hydrochloride. Ultrafiltration was used to isolate and concentrate a 10-100 kilodalton (kDa) fraction, upon which heparin-Sepharose (HS) affinity chromatography was performed. HS-binding (HS-B) and non-binding proteins (HS-NB) were lyophilized with bovine type I collagen (Vitrogen) to form pellets which were implanted subcutaneously in rats. Radiology as well as brightfield, fluorescent, and polarizing microscopy were used to assess the formation of ectopic bone at the site of pellet implantation. In this report we demonstrate that a heparin-Sepharose binding, osteoinductive factor can be extracted and partially purified from bovine intramembranous bone matrix. This factor has a different sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) banding pattern than a comparable osteoinductive/chondroinductive factor isolated from EC bone.

The matrix of endochondral bone differs from the matrix of intramembranous bone.

Osseous tissue develops via two distinctly different processes: endochondral (EC) ossification and intramembranous (IM) ossification. The present study tests the hypothesis that each type of osseous tissue contains unique inducing factors for the promotion of cartilage and bone development. Previous work suggests that subcutaneous implants of demineralized EC and IM bone matrices both induce endochondral ossification. Thus, it concludes that the bone growth promotion properties of the respective matrices are very similar. As it was unclear to us why EC and IM bone powders should possess identical osteoinductive properties, we attempted to reproduce these results. We implanted EC (femoral) demineralized bone matrix (DBM), IM (frontal) DBM, or a mixture of the two into the ventral thoracic subcutaneous tissue of 12 to 15-week-old male Sprague Dawley rats. Morphological and radiolabeling techniques in this study demonstrated that implants of EC bone matrix induce bone formation via EC ossification in contrast to implants of IM bone matrix which do not induce EC ossification. Our findings suggest that the matrix of EC bone differs qualitatively from the matrix of IM bone due to their respective abilities to induce cartilage and/or bone formation. These observations differ from those previously reported possibly because our IM DBM preparations were not contaminated with tissues of endochondral origin. In current clinical practice, EC DBM allografts are often used to induce new bone formation in defects involving both IM and EC bone. We conclude that there may be clinical settings in which it would be more appropriate to replace bone originally formed via IM ossification with IM DBM rather than EC DBM.

Synthesis of (aryloxy)alkylamines. 1. Novel antisecretory agents with H+K+-ATPase inhibitory activity.

A series of heterocyclic (aryloxy)alkylamines of structures II and III were prepared and found to possess gastric antisecretory activity. Of the variety of substituted thiazoles, benzoxazoles, and benzothiazoles prepared, thiazole 18, benzoxazole 32, and benzothiazole 47 exhibited gastric antisecretory potency comparable to that of ranitidine in vivo in the pylorous ligated rat model. In an isolated rabbit parietal system, the series of thiazoles, benzoxazoles, and benzothiazoles also demonstrated similar potency to that of ranitidine toward the inhibition of both histamine-stimulated and dcAMP-stimulated uptake of amino[14C]pyrine. These compounds inhibited the H+K+-sensitive ATPase enzyme in isolated gastric microsomes. A direct correlation existed between inhibition of 14C uptake, in vivo antisecretory activity, and inhibition of the H+K+-ATPase enzyme. The more potent antisecretory compounds 18, 32, and 47 were also the more potent enzyme inhibitors. These data suggest that the mechanism responsible for the observed in vitro and in vivo gastric antisecretory activity, in these series of compounds, is a consequence of the inhibition of the H+K+-sensitive ATPase enzyme.

Antigastrolesive, gastric antisecretory, diarrheagenic and mucus-stimulating effects in rats following topically applied rioprostil, a synthetic prostaglandin E1 analog.

Prostaglandins may have many biological actions including hypotensive and antipeptic ulcer activity. The purpose of this investigation was to determine if the primary alcohol prostaglandin E1 analog rioprostil1 prevents ethanol-induced gastric lesions (antigastrolesive activity), inhibits gastric acid secretion (antisecretory activity), or causes diarrhea in rats when administered topically, and to compare these responses to the effect of rioprostil following enteral (oral or intraduodenal) administration. Rioprostil exhibited antigastrolesive activity in rats when administered either orally or when applied topically. The topical antigastrolesive potency of rioprostil against ethanol-induced lesions [ED50 = 3.7 (0.5-12) micrograms/kg] was similar to its oral potency [ED50 = 1.9 (1.7-2.2) micrograms/kg]. In 4 hr pylorus-ligated rats, topically administered rioprostil inhibited total gastric acid output with a potency [ED50 = 5.1 (2.6-24) mg/kg] similar to intraduodenal administration [ED50 = 3.7 (2.8-5.3) mg/kg]. In addition, in these rats rioprostil increased mucin levels and did not cause dermal irritation. Finally, the incidence of diarrhea was lower when rioprostil was applied topically than when given orally with a 16-fold difference in potency between these two routes of administration. These data show that when rioprostil is applied via the skin it has antigastrolesive, gastric antisecretory and mucus stimulatory effects in rats equal to enteral administration, and a diarrheagenic potency lower than following oral administration. This profile suggests that topical administration of rioprostil may be a useful means of delivery for clinical treatment of peptic ulcer disease.

Studies on the mechanism of action of the gastric microsomal (H+ + K+)-ATPase inhibitors SCH 32651 and SCH 28080.

The novel antisecretory agents SCH 32651 and SCH 28080 were evaluated for their antisecretory activities in vitro as well as for their abilities to inhibit the (H+ + K+)-ATPase enzyme activity in preparations of microsomal membranes from rabbit fundic mucosa. SCH 32651 and SCH 28080 inhibited both the histamine- and dibutyryl cAMP-stimulated uptake of [14C]-aminopyrine into isolated parietal cells with IC50 values of about 1.5 and 0.02 microM respectively. SCH 32651 and SCH 28080 competitively inhibited the K+-stimulated hydrolysis of ATP catalyzed by the (H+ + K+)-ATPase with Ki values of 16.3 and 0.12 microM respectively. The inhibition of the enzyme by both compounds was not affected by the addition of the sulfhydryl reducing agents dithiothreitol or beta-mercaptoethanol, was readily reversible by dilution or washing, and was dependent upon the concentration of KCl used to stimulate the enzyme. These data suggest that SCH 32651 and SCH 28080 are reversible, competitive inhibitors of the K+-stimulated hydrolysis of ATP.

Pharmacological comparison of ORF 17910, a potent, long-acting histamine H2-receptor antagonist, to cimetidine and ranitidine.

This paper describes the pharmacology of ORF 17910, a specific, long-acting histamine H2-receptor antagonist. ORF 17910 (ED50 = 0.26 mg/kg) is 26 and 2.7 times more potent p.o. than cimetidine and ranitidine, respectively, at inhibiting acid output in betazole-stimulated total gastric fistula dogs. When given i.v., ORF 17910 (ED50 = 0.06 mg/kg) is 3.6 times more potent than ranitidine. Qualitatively similar antisecretory potency differences are seen in rats (ED50 = 3.7 mg/kg intraduodenal). ORF 17910 retains 43 and 37% of its antisecretory potency 16 hr after dosing in dogs and rats, respectively, suggesting a long duration of action, whereas ranitidine is either inactive (rats) or loses 97% of its potency (dogs) at this time. When the parenteral and enteral (p.o. or intraduodenal) potencies of ORF 17910 and ranitidine are compared, ORF 17910 appears less bioavailable than ranitidine, although this difference is greater in the rat than in the dog and diminishes with time. In rabbit isolated parietal cell (pA2 = 7.96) and guinea pig isolated atria preparations (pA2 = 7.51), ORF 17910 is more potent than both cimetidine and ranitidine at inhibiting the effects of histamine. At high concentrations, the inhibitory effect of ORF 17910 in atria can not be overcome completely, a property which may contribute to its long duration of action in vivo. In several additional test systems, ORF 17910 does not exhibit any biologically significant pharmacology and appears to be specific for the histamine H2-receptor.

Pharmacology of ORF 17578, a new histamine H2-receptor antagonist: comparison with cimetidine and ranitidine.

This paper describes the pharmacology of ORF 17578, a new histamine H2-receptor antagonist, in comparison to the standard H2-blockers cimetidine and ranitidine. ORF 17578 inhibited betazole-stimulated gastric acid output in gastric fistula dogs and gastric acid secretion in pylorus-ligated rats. When administered enterally (p.o. or i.d.) ORF 17578 (ED50 = 0.21 mg/kg in dogs and 2.5 mg/kg in rats) was 10 to 11 times more potent than cimetidine and 1.8 to 2.1 time more potent than ranitidine in dogs and rats. In both species, duration of p.o. antisecretory activity was longer than with ranitidine. When administered parenterally ORF 17578 (ED50 = 0.15 mg/kg i.v. in dogs and 1.1 mg/kg i.p. in rats) was more potent than cimetidine and ranitidine in rats and similar in potency to ranitidine in dogs. Comparison of parenteral to enteral potencies suggests that ORF 17578 was well absorbed from the gastrointestinal tract of both species with a bioavailability profile similar to that of ranitidine. In rabbit isolated parietal cells (pA2 = 7.53) and guinea-pig isolated atria (pA2 = 7.26), ORF 17578 competitively antagonized the effects of histamine with a potency greater than cimetidine and similar to ranitidine. Results from several additional test systems indicated that ORF 17578 was specific for the histamine H2-receptor and did not exhibit the additional pharmacology often associated with cimetidine.

Pharmacological profile of etintidine, a new histamine H2-receptor antagonist.

Etintidine is a potent competitive antagonist of histamine H2-receptors. It is 4.6 and 2.2 times more potent than cimetidine in the isolated guinea pig atrium (pA2 = 7.18) and the isolated rabbit parietal cell (pA2 = 6.51), respectively. Etintidine is 2.0 (ED50 = 1.8 mg/kg, i.g.) times more potent than cimetidine at inhibiting betazole-stimulated total acid output in the chronic gastric fistula dog. In the 4 hr pylorus-ligated rat etintidine is a potent inhibitor of total acid output (ED50 = 22 mg/kg, i.d.), total pepsin output, and is well absorbed from the gastrointestinal tract. Additional pharmacological and biochemical studies indicate that etintidine displays minimal competition with appropriate ligands for the alpha 1, alpha 2, cholinergic and neuroleptic receptors in vitro, and has minimal effects on the immunological, autonomic and central nervous systems at doses much higher than antisecretory doses. While high doses of both cimetidine and etintidine increase serum prolactin levels in rats, the effect of etintidine is less than that of cimetidine. Similarly, the prolongation of hexobarbital-induced sleep time in rats is less than with cimetidine. These data indicate that etintidine may be potentially useful and safe in the treatment of peptic ulcer disease and may offer some advantages over cimetidine in terms of less potential for side effects.

Inhibition of H+K+ATPase by SCH 28080 and SCH 32651.

The novel antiulcer agents, SCH 28080 and SCH 32651 were examined for their ability to inhibit the H+K+ ATPase enzyme activity in a preparation of microsomal membranes from rabbit fundic mucosa. SCH 28080 inhibited the isolated enzyme activity with a potency similar to omeprazole, IC50s of 2.5 and 4.0 microM respectively. SCH 32651 was less potent exhibiting an IC50 of 200.0 microM. Both compounds may therefore exert their antisecretory activity via a direct inhibition of the parietal cell H+K+ ATPase.

Heterogeneity of biochemical actions among vasodilators.

Thirty-four vasodilators were screened in several in vitro biochemical assays related to smooth muscle excitation-contraction coupling, binding to beta 1-,beta 2-, and alpha-adrenergic receptors, inhibition of phosphodiesterase activity, and antagonism of calcium accumulation. Isoproterenol and perhexiline only exhibited binding to beta-adrenergic sites. Ergocryptine, tolazoline, and amotriphene only bound to alpha-adrenergic receptors. Leniquinsin, papaverine, proquazone, dioxyline, hoquizil, quazodine, and theophylline were active only as phosphodiesterase inhibitors. Isoxsuprine, nylidrin, and bencyclane bound to alpha- and beta-receptors. Pentoxifylline bound to beta 1-sites and inhibited phosphodiesterase. Cyclandelate bound to beta 2-sites and blocked calcium accumulation. Cinnarizine and flunarizine antagonized calcium accumulation and bound to alpha-sites. Prazosin bound to alpha-sites and inhibited phosphodiesterase. Ethaverine and dipyridamole were inhibitors of phosphodiesterase and calcium accumulation. Nafronyl bound to beta 2- and alpha-sites and antagonized calcium accumulation. Mebeverine bound to beta 2- and alpha-receptors and inhibited phosphodiesterase activity and calcium accumulation. Verapamil bound to alpha-sites, and blocked phosphodiesterase and calcium accumulation. Quinazosin bound to beta 2- and alpha-receptors and antagonized both phosphodiesterase activity and calcium accumulation. Vasodilators that were inactive in all assays included niacin, nicotinyl alcohol, inositol nicotinate, amyl nitrite, sodium nitroprusside, diazoxide, hydralazine, and protoveratrine. Vasodilators should not be considered as a single drug class since they act on various mechanisms related to coupling of neuronal excitation to muscular contractility.