Poloxamer micellar system for intra-articular injection of 15-deoxy-Δ12,14-prostaglandin J2 with improved bioavailability and anti-inflammatory properties in the temporomandibular joint of rats.

Painful conditions of the temporomandibular joint (TMJ) are challenging to manage and most attempts often result in unsatisfactory outcomes. In such context, nanocarrier systems, such as polymeric micelles, have been showing encouraging results in solving therapeutic limitations. Poloxamers are widely used, especially PL 407, because of their high biocompatibility and approval by the Food and Drug Administration (FDA) for clinical use. 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has shown important antinociceptive and anti-inflammatory activity. The present study evaluated the efficacy and viability of the micellar system of PL-15dPGJ2 in a formalin-induced acute pain model in the temporomandibular joint of rats. The PL-15dPGJ2 was prepared and characterized. The animals were pretreated with an intra-articular injection of PL-15dPGJ2 followed by the formalin challenge. The nociceptive response was evaluated at different time-periods and the periarticular tissue and articular wash were collected for analysis. We found that intra-articular injection of PL-15d-PGJ2 produced pain relief at lower concentrations and in a sustained manner compared with free 15d-PGJ2. Moreover, a strong anti-inflammatory effect was observed with decreased levels of key pro-inflammatory cytokines and modulation of the leukocyte migration process. Our findings suggest that 15d-PGJ2 combined with a poloxamer micellar system provided clinical relevance in terms of bioavailability, long-lasting effect, and safe dosage. The formulation investigated herein is a promising micellar carrier system for managing pain conditions of the TMJ.

Elimination of the biphasic pharmacodynamics of 15d-PGJ2 by controlling its release from a nanoemulsion.

15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has a dual action of stimulating anti-inflammation and anti-proliferation when exogenously administered at high doses. However, at lower doses, it can be toxic inducing opposite actions, ie, stimulation of both inflammation and cell proliferation. This biphasic phenomenon of 15d-PGJ2 is believed to be due to its multitarget behavior. In this study, we provide a strategy for controlling such biphasic pharmacodynamics by separating its dual actions while retaining the beneficial one by using a nanoemulsion (NE). The 15d-PGJ2 was encapsulated in the NE composed of triolein/distearoyl phosphatidylcholine/Tween 80 at a high encapsulation ratio (>83%). Furthermore, NE enhanced drug retention by slowing down its release rate, which was, unconventionally, inversely dependent on the total surface area of the NE system. Next, focusing on the biphasic effect on cell proliferation, we found that the 15d-PGJ2-loaded slow-release NE showed only a dose-dependent inhibition of the viability of a mouse macrophage cell line, RAW264.7, although a fast-release NE as well as free 15d-PGJ2 exerted a biphasic effect. The observed slow-release kinetics are believed to be responsible for elimination of the biphasic pharmacodynamics of 15d-PGJ2 mainly for two reasons: 1) a high proportion of 15d-PGJ2 that is retained in the NE was delivered to the cytosol, where proapoptotic targets are located and 2) 15d-PGJ2 was able to bypass cell membrane-associated targets that lead to the induction of cellular proliferation. Collectively, our strategy of eliminating the 15d-PGJ2-induced biphasic pharmacodynamics was based on the delivery of 15d-PGJ2 to its desired site of action, excluding undesired sites, on a subcellular level.

[Delta12-prostaglandinJ2-nano capsule up-regulates growth factor expression and enhances bone regeneration in rats].

OBJECTIVE: To investigate the effect of local delivery of delta12-prostaglandinJ2-loaded poly (lactic-co-glycolic acid) (Δ(12)-PGJ2-NC) on growth factors expression and bone formation. METHODS: Δ(12)-PGJ2-NC was prepared by the emulsion solvent diffusion method. The physical and chemical properties of the nanoparticles were evaluated by particle size analysis, transmission electron microscopy, drug-loading ratio and the in vitro release study. Then standardized transcortical defect (5.0 mm × 1.5 mm) was conducted in the femur of 48 male Wistar rats which were randomly divided into four groups (n = 12), S, K, F, and N. Thirty microliter of saline (S), unloaded nanoparticles (K), Δ(12)-PGJ2 (F) and Δ(12)-PGJ2-NC(N) in a collagen vehicle were delivered inside a titanium chamber fixed over the defect. Then, four subgroups were randomly divided in each group named as D3, D7, D14, and D28 (n = 3) according to the days 3, 7, 14, and 28 after the surgery. At days 3, 7, 14, and 28, the mRNA expression of the bone morphogenetic protein-6 (BMP-6), platelet-derived growth factor-B (PDGF-B) in defect aera was analyzed by real time quantitive-polymerase blotting. HE staining was employed to reveal new bone formation in weeks 2 and 4. RESULTS: Δ(12)-PGJ2-NC appeared opalescent white and remained relatively stable, with an average particle size of (135.2 ± 0.85) nm. The images from transmission electron microscopy showed that Δ(12)-PGJ2-NC was spherical in shape and homogeneously distributed. The encapsulation efficiency of Δ(12)-PGJ2 with the poly (lactic-co-glycolic acid) (PLGA) nanocapsules was about 92%. The in vitro release of Δ(12)-PGJ2-NC at 37 °C showed a sustained fashion and the average accumulated amount was 30%, 52%, 77%, 91%, and 98% respectively, at 0.5, 1, 2, 4 and 6 h. Compared with the animals treated with saline, after dose of 100 mg/L Δ(12)-PGJ2 and Δ(12)-PGJ2-NC apllication, the mRNA expression level of BMP-6, PDGF-B increased significantly (P < 0.05, P < 0.001). The protein expression of BMP-6, Ephrin-B2 also was up-regulated. Histomorphometry revealed that new bone formation increased at the same dose of 100 mg/L. But the unloaded nanoparticles did not have the same effect (P > 0.05). CONCLUSIONS: A stable Δ(12)-PGJ2 loaded nanoparticle was successfully prepared. Δ(12)-PGJ2-NC may upregulate the expression of BMP-6, PDGF-B and Ephrin-B2, and promote new bone formation in bone defect area.

A clearance system for prostaglandin D2, a sleep-promoting factor, in cerebrospinal fluid: role of the blood-cerebrospinal barrier transporters.

Although the level of prostaglandin (PG) D(2) in cerebrospinal fluid (CSF) affects the action of D-type prostanoid receptors that promote physiological sleep, the regulatory system of PGD(2) clearance from the CSF is not fully understood. The purpose of this study was to investigate PGD(2) elimination from the CSF via the blood-CSF barrier (BCSFB). The in vivo PGD(2) elimination clearance from the CSF was 16-fold greater than that of inulin, which is considered to reflect CSF bulk flow. This process was inhibited by the simultaneous injection of unlabeled PGD(2). The characteristics of PGD(2) uptake by isolated choroid plexus were, at least partially, consistent with those of PG transporter (PGT) and organic anion transporter 3 (OAT3). Studies using an oocyte expression system showed that PGT and OAT3 were able to mediate PGD(2) transport with a Michaelis-Menten constant of 1.07 and 7.32 µM, respectively. Reverse transcription-polymerase chain reaction and immunohistochemical analyses revealed that PGT was localized on the brush-border membrane of the choroid plexus epithelial cells. These findings indicate that the system regulating the PGD(2) level in the CSF involves PGT- and OAT3-mediated PGD(2) uptake by the choroid plexus epithelial cells, acting as a pathway for PGD(2) clearance from the CSF via the BCSFB.

Nose-to-brain delivery of TS-002, prostaglandin D2 analogue.

This study was conducted to investigate the possibility of performing nose-to-brain delivery of TS-002, which is an analog compound of prostaglandin D2 (PGD2) and thus would be a natural sleep inducer. The absolute bioavailability (BA) and sleep-inducing effect (SIE) following intranasal (IN) administration of TS-002 dry powder to cynomolgus monkeys were evaluated in comparison with intravenous (IV) administration. The SIE was evaluated as the accumulated time of sleeping-posture for 3 h. The brain distribution of TS-002 following IN administration of the dry powder was examined in rats. The absolute bioavailability (BA) in monkeys following IN administration of the dry powder (0.4-1.2 mg/body) was comparatively high (43.4-78.0%). The SIE following IN administration (0.05-0.4 mg/body) showed dose-dependency and its effect at 0.4 mg/body was twice as strong as that for IV administration (P < 0.05). The brain concentrations in rats following IN administration (0.1 mg/kg) were obviously higher than that for IV administration at the same dose. The highest content was observed in the olfactory bulb. These results demonstrated that TS-002 was directly transported from the olfactory region to brain, thereby showing that it may be possible to develop a novel sleep-inducing drug based on nose-to-brain delivery.

Targeting 15d-prostaglandin J2 to hepatic stellate cells: two options evaluated.

PURPOSE: Delivery of apoptosis-inducing compounds to hepatic stellate cells (HSC) may be an effective strategy to reverse liver fibrosis. The aim of this study was therefore to examine the selective targeting of the apoptosis-inducing drug 15-deoxy-delta12,14-prostaglandin J2 (15dPGJ2) with two different HSC-carriers: human serum albumin modified with the sugar mannose-6-phosphate (M6PHSA) or albumin modified with PDGF-receptor recognizing peptides (pPBHSA). METHODS AND RESULTS: After chemical conjugation of 15dPGJ2 to the carriers, the constructs displayed pharmacological activity and specific receptor-mediated binding to HSC in vitro. Unlike 15dPGJ2-pPBHSA, the cellular binding of 15dPGJ2-M6PHSA was reduced by a scavenger receptor antagonist. In vivo, both conjugates rapidly accumulated in fibrotic livers. Intrahepatic analysis revealed that 15dPGJ2-M6PHSA mainly accumulated in HSC, and to a lesser extent in Kupffer cells. 15dPGJ2-pPBHSA also predominantly accumulated in HSC with additional uptake in hepatocytes. Assessment of target receptors in human cirrhotic livers revealed that M6P/IGFII-receptor expression was present in fibrotic areas. PDGF-P receptor expression was abundantly expressed on human fibroblasts. CONCLUSIONS: These studies show that 15dPGJ2 coupled to either M6PHSA or pPBHSA is specifically taken up by HSC and is highly effective within these cells. Both carriers differ with respect to receptor specificity, leading to differences in intrahepatic distribution. Nevertheless, both carriers can be used to deliver the apoptosis-inducing drug 15dPGJ2 to HSC in vivo.

Prostaglandin D2 inhibits TGF-beta1-induced epithelial-to-mesenchymal transition in MDCK cells.

In a separate study, we identified PGE2 as a potent inhibitor of TGF-beta1induced epithelial-mesenchymal transition (EMT) in cultured Madin-Darby canine kidney (MDCK) cells (Zhang A, Wang M-H, Dong Z, and Yang T. Am J Physiol Renal Physiol 291: F1323-F1331, 2006). This finding prompted us to examine the roles of other prostanoids: PGD2, PGF(2alpha), PGI2, and thromboxane A2 (TXA2). Treatment with 10 ng/ml TGF-beta1 for 3 days induced EMT as reflected by conversion to the spindle-like morphology, loss of E-cadherin, and activation of alpha-smooth muscle actin (alpha-SMA). Treatment with PGD2 remarkably preserved the epithelial-like morphology, restored the expression of E-cadherin, and abolished the activation of alpha-SMA. In contrast, PGF(2alpha), carbocyclic thromboxane A2, PGI2 and its stable analog beraprost were without an effect. MDCK cells expressed DP1 and DP2 receptors; however, the effect of PGD2 was neither prevented by DP1 antagonist BW-A868C or DP2 antagonist BAY-u3405 nor was mimicked by DP1 agonist BW-245C. cAMP-elevating agents forskolin and 8-Br-cAMP blocked EMT. However, cAMP blockers H89 and Rp-cAMP failed to block the effect of PGD2. PGD2 did not seem to act via its metabolites as 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) levels in the medium following incubation with 3 microM PGD2 were well below the values predicted from the cross activity of the assay. Exposure to TGF-beta1 induced a threefold increase in reactive oxygen species production that was completely abolished by PGD2. We conclude that 1) PGD2, but not PGI2, PGF(2alpha), and TXA2 inhibit EMT, 2) PGD2 inhibits EMT independently of DP1 and DP2 receptors, and 3) PGD2 exhibits antioxidant property which may, in part, account for the antifibrotic action of this PG.

Preparation and evaluation of o/w type emulsions containing antitumor prostaglandin.

Antitumor prostaglandins(PGs) such as Delta12-PGJ2 and Delta7-PGA1 possess a cyclopentenone or cross-conjugated dienone structures. Antitumor PGs are actively incorporated through cell membrane and control gene expression. Very recent studies clarified that P53 independent expression of p21 and gadd 45, activation of PPARgamma are involved in antitumor mechanism of these PGs. At the low concentration, these PGs exhibit physiological or pathological activity such as osteoblast calcification, promotion of colon cancer cell proliferation. COMPARE PROGRAM using human 38 tumor cell lines suggested that antitumor mechanism of Delta7-PGA1 and 13, 14-dihydro-15-deoxy-Delta7-PGA1 methyl ester (TEI-9826) are quite different from other anticancer agents which are clinically used. Lipid microspheres and Lipiodol formulation were examined as dosage form of the PGs and lipid microspheres were selected for further study. At first lipid microspheres integrated TEI-9038 (Lipo TEI-9038) was chosen as a candidate for clinical trial. However Lipo TEI-9038 failed to exhibit substantial antitumor effect because of its enzymatic instability and toxicity in vivo. Lipo TEI-9826 was then selected as promising candidate for clinical trial because of its stability in serum. Lipo TEI-9826 exhibited marked antitumor effect in several animal models including CDDP resistant nude mice model. Pharmacokinetic and toxicological studies using rats suggested that continuous infusion is the most suitable administration method for Lipo TEI-9826. New type emulsifier, Controlled High Pressure Process Homogenizer (De-BEE 2000 and mini De-BEE) was developed during the preclinical studies on manufacturing process of Lipo TEI-9826. These results warrant the clinical trial for Lipo TEI-9826 in CDDP resistant cancer.

[Pharmaceutical and pharmacological development of antitumor prostaglandins].

Antitumor Prostaglandins such as delta 12PGJ2 and delta 7PGA1 possess a cross-conjugated dienone unit and exhibit unique antitumor effect. Lipid microshere (w/o type emulsion) was selected as pharmaceutical formulation because of physicochemical properties of prostaglandin. 13,14-Dihydro-15-deoxy-delta 7-PGA1 methyl ester (TEI-9826) were selected as a candidate for clinical trial. In a rat and mouse serum in vitro, TEI-9826 rapidly metabolized to 13,14-dihydro-15-deoxy-delta 7-PGA1 (TOK-4528), but TOK-4528 is stable as well as delta 12PGJ2. Lipid microshere containing TEI-9826 at the content of 5 mg/ml exhibited administration route and schedule dependent antitumor effect in vivo using Colon 26 bearing mouse model, which suggested that duration of serum concentration was important for antitumor effect. One of the antitumor mechanism of antitumor PG might be an induction of the cyclin-dependent kinase inhibitor p21. PPAR gamma also might be important. New type homogenizer, high pressure jet flow type homogenizer was developed in the study of antitumor prostaglandin.

Permeability of brain structures and other peripheral tissues to prostaglandins D2, E2 and F2 alpha in rats.

Parenchymal tissue-uptake (TU) and permeability-surface area (PS) product of [3H]prostaglandins (PG) D2, E2 and F2 alpha [1.85 MBq, 0.5 mg/kg (270 nmol)] were examined in 98 regions of the brain and in 19 other tissues of urethane-anesthetized male rats (180-200 g) 15 sec after i.v. administration with [14C]dextran [0.185 MBq, 0.6 mg/kg (2 nmol)] used as a blood spacer. Slight and insignificant change in blood volume was observed in most of the tissues and brain regions between vehicle- and PG-administered groups. TU for the three PG was markedly high in kidney and lung (2388-3952 ng/g), exceeding the blood concentration (2021-2320 ng/ml), but low (less than 10% of the blood concentration) in epididymis, epididymal fat, testis (59-163 ng/g), brain and spinal cord (33-67 ng/g). TU in brain were detected about 0.1% of the administered PG. Based on a two-compartment model, the PS product for the three PG ranged from 0.75 to 4.16 microliters/g/sec in the latter tissues. The value of brain was 1.22 +/- 0.18 microliters/g/sec for PGD2, 1.69 +/- 0.05 for PGE2 and 1.33 +/- 0.13 for PGF2 alpha, indicating that PGE2 enters the brain more readily than PGD2 and PGF2 alpha. In various brain structures, the ranges of the PS product were large and completely overlapped among the three PG (PGD2, 0.14-1.56 microliters/g/sec; PGE2, 0.05-1.78; PGF2 alpha, 0.05-1.82). The highest PS product for the three PG was found in olfactory bulb and cerebellum (0.96-1.82 microliters/g/sec) and the lowest was in septum (0.05-0.53). However, the level of the PS product was different among the PG in each brain region as follows: PGD2 greater than PGE2, PGF2 alpha in septum and anterior part of pyriform cortex; PGE2 greater than PGD2, PGF2 alpha in olfactory bulb, frontal cortex, basal forebrain, middle part of pyriform cortex, thalamus, hippocampus and lateral neocortex; and PGF2 alpha greater than PGD2, PGE2 in posterior part of pyriform cortex, hypothalamus, amygdala and entorhinal and retrosplenial cortices. Low correlation coefficients (0.708, 0.522 and 0.562 for PGD2, PGE2 and PGF2 alpha, respectively) between the PS product and cerebrovascular volume in various regions revealed heterogeneous cerebrovascular permeabilities of PG.

Contraluminal p-aminohippurate transport in the proximal tubule of the rat kidney. VII. Specificity: cyclic nucleotides, eicosanoids.

Using the stop-flow peritubular capillary microperfusion method the inhibitory potency (apparent Ki values) of cyclic nucleotides and prostanoids against contraluminal p-aminohippurate (PAH), dicarboxylate and sulphate transport was evaluated. Conversely the contraluminal transport rate of labelled cAMP, cGMP, prostaglandin E2, and prostaglandin D2 was measured and the inhibition by different substrates was tested. Cyclic AMP and its 8-bromo and dibutyryl analogues inhibited contraluminal PAH transport with an app. Ki,PAH of 3.4, 0.63 and 0.52 mmol/l. The respective app. Ki,PAH values of cGMP and its analogues are with 0.27, 0.04 and 0.05 mmol/l, considerably lower. None of the cyclic nucleotides tested interacted with contraluminal dicarboxylate, sulphate and N1-methylnicotinamide transport. ATP, ADP, AMP, adenosine and adenine as well as GTP, GDP, GMP, guanosine and guanine did not inhibit PAH transport while most of the phosphodiesterase inhibitors tested did. Time-dependent contraluminal uptake of [3H]cAMP and [3H]cGMP was measured at different starting concentrations and showed facilitated diffusion kinetics with the following parameters for cAMP: Km = 1.5 mmol/l, Jmax = 0.34 pmol S-1 cm-1, r (extracellular/intracellular amount at steady state) = 0.91; for cGMP: Km = 0.29 mmol/l, Jmax = 0.31 pmol S-1 cm-1, r = 0.55. Comparison of app. Ki,cGMP with app. Ki,PAH of ten substrates gave a linear relation with a ratio of 1.83 +/- 0.5. All prostanoids applied inhibited the contraluminal PAH transport; the prostaglandins E1, F1 alpha, A1, B1, E2, F2 alpha, D2, A2 and B2 with an app. Ki,PAH between 0.08 and 0.18 mmol/l. The app. Ki of the prostacyclins 6,15-diketo-13,14-dihydroxy-F1 alpha (0.22 mmol/l) and Iloprost (0.17 mmol/l) as well as that of leukotrienes B4 (0.2 mmol/l) was in the same range, while the app. Ki,PAH of the prostacyclins PGI2 (0.55 mmol/l), 6-keto-PGF1 alpha (0.77 mmol/l) and 2,3-dinor-6-keto-PGF1 alpha (0.57 mmol/l) as well as that of thromboxane B2 (0.36 mmol/l) was somewhat higher. None of these prostanoids inhibited contraluminal dicarboxylate transport and only PGB1, E2 and D2 inhibited contraluminal sulphate transport (app. Ki,SO4(2-) 5.4, 11.0, 17.9 mmol/l respectively). Contraluminal influx of labelled PGE2 showed complex transport kinetics with a mixed Km = 0.61 mmol/l and Jmax of 4.26 pmol S-1 cm-1. It was inhibited by probenecid, sulphate and indomethacin. Contraluminal influx of PGD2, however, was only inhibited by probenecid. The data indicate that cyclic nucleotides as well as prostanoids are transported by the contraluminal PAH transporter. For prostaglandin E2 a significant uptake through the sulphate transporter occurs in addition.(ABSTRACT TRUNCATED AT 400 WORDS)

Dexamethasone does not inhibit the release of mediators from human mast cells residing in airway, intestine, or skin.

Glucocorticoids are potent anti-inflammatory drugs that are widely used in the treatment of allergic disorders. Their actions are often species specific or cell-type specific. Previous studies have demonstrated that glucocorticoids inhibit mediator release from mast cells derived from the peritoneum of mouse or rat and from guinea pig lung, but not those residing in human lung parenchymal tissue. In the present study, we have analyzed the effect of overnight culture with dexamethasone (10(-6) to 10(-7)M) on the subsequent IgE-dependent release of mediators from human mast cells derived from airway tissue, intestine, and skin. Airway tissue was passively sensitized with antigen-specific, IgE-rich serum during the culture period and subsequently challenged with ragweed antigen E. Skin and intestinal mast cells were challenged with anti-IgE. Histamine and immunoreactive LTC4 and PGD2 release was monitored in all experiments. Prostaglandin E release was quantitated in the experiments using airway tissue. Dexamethasone treatment failed to inhibit the release of mast cell mediators from all three tissues, but it inhibited the antigen-induced release of immunoreactive PGE from other cells residing in airway tissue. These results confirm earlier studies of the effects of glucocorticoids on human lung parenchymal mast cells, but contrast with the inhibitory effects of steroids observed in murine mast cells and human basophils.

Transformation of prostaglandin D2 to isomeric prostaglandin F2 compounds by human eosinophils. A potential mast cell-eosinophil interaction.

PGD2 undergoes extensive isomerization in vivo followed by metabolism by 11-ketoreductase to yield a family of biologically active isomeric PGF2 compounds, including 9, alpha 11 beta-PGF2. Because immunologically activated human mast cells produce substantial quantities of PGD2 and eosinophils accumulate around mast cells at sites of immediate hypersensitivity reactions, the ability of eosinophils to metabolize PGD2 was investigated. Purified human circulating eosinophils from four different donors transformed PGD2 to 9, alpha 11 beta-PGF2 and 12-epi-9 alpha, 11 beta-PGF2 in a time- and concentration-dependent manner. The formation of these compounds increased rapidly during the first 30 min of incubation of eosinophils with PGD2 and tended to plateau at approximately 2 h. Detection and quantification of the formation of 9 beta,11 beta-PGF2 and its 12-epi isomer was accomplished by a negative ion chemical ionization gas chromatography/mass spectrometry assay. On one occasion, eosinophils from one donor also transformed PGD2 to two additional isomeric PGF2 compounds, the stereochemical structures of which were not identified. The ability of eosinophils to produce PGD2 was then investigated. After stimulation with 2 microM A23187, the major cyclooxygenase product formed was thromboxane B2 (2247 pg/10(6) eosinophils) whereas only small quantities of PGD2 were produced (50 pg/10(6) eosinophils). Inasmuch as PGF2 compounds can exert biologic actions that differ from those of PGD2, this ability of eosinophils to transform PGD2 to PGF2 compounds could alter the local biologic effects of PGD2 released from adjacent mast cells and thus may represent a physiologically relevant mast cell-eosinophil interaction.