The establishment and validation of efficient assays for anti-IIa and anti-Xa activities of heparin sodium and heparin calcium.

Heparin is used as an anticoagulant drug. The anticoagulation process is mainly caused by the interaction of heparin with antithrombin followed by inhibition of anticoagulant factor IIa and factor Xa. The anti-factor IIa and anti-factor Xa activities of heparin are critical for its anticoagulant effect; however, physicochemical methods that can reflect these activities have not been established. Thus, the measurements of anti-IIa and anti-Xa activities by biological assay are critical for the quality control of heparin products. Currently in the Japanese Pharmacopoeia (JP), the activities of heparin sodium and heparin calcium are measured by an anti-Xa activity assay (anti-Xa assay), but anti-IIa activity is not measured. Here, we established an anti-IIa activity assay (anti-IIa assay) and an anti-Xa assay having good accuracy and precision. When samples having a relative activity of 0.8, 1.0 and 1.2 were measured by the established anti-IIa and anti-Xa assays in nine laboratories, good accuracy (100.0-102.8% and 101.6-102.8%, respectively), good intermediate precision (1.9-2.1% and 2.4-4.2%, respectively) and good reproducibility (4.0-4.8% and 3.6-6.4%, respectively) were obtained. The established anti-IIa and anti-Xa assays have similar protocols, and could be performed by a single person without a special machine. The established assays would be useful for quality control of heparin.

Participation of prostaglandin E2 receptor in nasal congestion of Brown Norway rats.

The aim of the present study was to clarify the involvement of prostaglandin E(2) (PGE(2)) in nasal congestion in Brown Norway (BN) rats. For this purpose, we studied the effects of PGE(2) receptor (EP(1), EP(2), EP(3) and EP(4)) agonists on nasal congestion and sneezing induced by toluene 2,4-diisocyanate (TDI). Enhanced pause (Penh) was increased 1 h (early phase) and 4 h (late phase) after TDI challenge. Sulprostone (an EP(3) receptor agonist) inhibited the increase of Penh, an index of nasal congestion, in both early and late phase responses. On the other hand, PGE(1) alcohol (an EP(4) agonist) increased Penh in the early phase response. Moreover, sulprostone inhibited sneezing, an immediate response by TDI challenge. These results indicate that EP(3) receptor is responsible for the relief of nasal congestion in both early and late phase responses, and EP(4) receptor is correlated with the development of nasal congestion in the early phase response. In addition, EP(3) receptor also participates in sneezing in allergic rhinitis induced by TDI challenge in BN rats.

Role of histamine H(4) receptor in allergic conjunctivitis in mice.

We investigated the character of histamine H(1) receptor and H(4) receptor in allergic conjunctivitis. Histamine is the most important mediator in allergic conjunctivitis. We measured eye scratching behavior and allergic-like symptoms score, that is, hyperemia and edema in ICR mice, and examined which receptors intimately involved in allergic conjunctivitis. Histamine caused a dose-dependent eye scratching behavior and allergic-like symptoms. Histamine H(1) receptor antagonist (levocabastine) and H(4) receptor antagonist (JNJ7777120) inhibited eye scratching behavior and histamine H(1) receptor antagonist inhibited allergic-like symptoms induced by histamine. Additionally, combination of levocabastine and JNJ7777120 caused more potent inhibition in allergic conjunctivitis. On the other hand, both selective histamine H(1) receptor agonist (HTMT) and selective H(4) receptor agonist (4-methylhistamine) induced a dose-dependent eye scratching behavior and allergic-like symptoms. JNJ7777120 inhibited the effect of HTMT. However, levocabastine caused no inhibition on the response of 4-methylhistamine. H(4) receptor was closely related with allergic conjunctivitis. H(4) receptor antagonists may be effective in allergic conjunctivitis which showed no inhibition by histamine H(1) receptor antagonists.