An extended phase Ib study of epertinib, an orally active reversible dual EGFR/HER2 tyrosine kinase inhibitor, in patients with solid tumours.

BACKGROUND: Dose-escalation of epertinib (S-222611), a new potent oral EGFR/HER2 inhibitor, has established a recommended daily dose of 800 mg in patients with solid tumours. In this study, we have recruited a larger number of patients to assess further the safety, tolerability, pharmacokinetics (PKs) and antitumour activity. PATIENTS AND METHODS: Patients with solid tumours expressing EGFR or HER2 received a single dose of epertinib at 800 mg on Day 1 to assess PK over 7 days, followed by continuous once-daily dosing from Day 8. RESULTS: We treated 76 patients with breast (n = 27), upper gastrointestinal (GI; n = 30), head and neck (n = 12) or renal cancers (n = 7). Epertinib was well-tolerated with mostly grade I and II adverse events (AEs). The most frequent AE was diarrhoea, which was generally manageable with loperamide. The objective response rate (ORR) in patients with heavily pretreated breast and upper GI cancers was 16.0% (4 PRs) and 8.3% (1CR, 1PR), respectively. All six responding patients had HER2-positive tumours; the ORR for HER2-positive breast and upper GI cancer populations was 19.0% and 20.0%. Partial response in the brain disease of one breast cancer patient lasted 7.5 months. CONCLUSION: Once-daily dosing of epertinib at 800 mg was well-tolerated and demonstrated promising antitumour activity in patients with heavily pretreated HER2-positive breast and upper GI cancer, including those with brain metastases. EUDRACT NUMBER: 2009-017817-31.

A phase I/II study of cancer peptide vaccine S-288310 in patients with advanced urothelial carcinoma of the bladder.

Background: S-288310, a cancer peptide vaccine composed of two HLA-A*24:02-restricted peptides derived from two oncoantigens, DEP domain-containing 1 (DEPDC1) and M-phase phosphoprotein 1 (MPHOSPH1), was investigated in urothelial carcinoma (UC) of the bladder. Patients and methods: Thirty eight HLA-A*24:02-positive patients with progressive UC were enrolled in this study. In the phase I part of the study, three patients each were treated with S-288310 at 1 mg or 2 mg/peptide subcutaneously once a week to evaluate safety and tolerability. In the phase II, 32 patients were randomized to receive either 1 mg or 2 mg to evaluate the difference in cytotoxic T lymphocytes (CTL) induction and safety. Results: S-288310 was safe and well tolerated in the phase I. Of 27 patients evaluable for immune responses in the phase II, there was no difference in CTL induction rate between the 1 mg (100%) and 2 mg (80.0%) groups. Of 32 patients receiving S-288310 in the phase II, the most frequent drug-related AE was the injection site reaction that was observed in 29 patients (90.6%), but none of the patients discontinued administration due to these reactions and no dose relationship in the frequency and severity was observed. The objective response rate of the 32 patients was 6.3% and the disease control rate was 56.3%. The median overall survival (OS) rates for patients vaccinated with S-288310 after one regimen of chemotherapy, 2 regimens, or 3 or more were 14.4, 9.1 and 3.7 months, respectively, and 32.2% of patients post first-line treatment were alive at 2 years. OS of patients who showed CTL induction to both peptides was longer than that of those with CTL induction to no or one peptide. Conclusion: S-288310 was well-tolerated and effectively induced peptide-specific CTLs, which were correlated with longer survival for patients with UC of the bladder. Trial registration ID: JapicCTI-090980.

A prostaglandin D2 receptor antagonist modifies experimental asthma in sheep.

BACKGROUND: Prostaglandin (PG) D(2) is the major cylooxygenase metabolite released by mast cells upon allergen stimulation, and elicits responses through either the prostanoid DP1 receptor and/or the chemoattractant receptor homologous molecule expressed on T-helper type 2 (Th2) cells (CRTH2/DP2). Experimental evidence suggests that stimulation of one or both these receptors contributes to asthma pathophysiology. OBJECTIVE: The aim of this study was to test the hypothesis that the prostanoid DP1 receptor contributes to asthma pathophysiology by determining the efficacy of an orally active antagonist for this receptor, S-5751, on allergen-induced bronchoconstriction, airway hyperresponsiveness (AHR) and cellular inflammation in the sheep model of asthma. METHODS: PGD(2)-induced cyclic adenosine monophosphate (cAMP) production in platelet-rich plasma was used to establish the in vitro efficacy of S-5751. In vivo, sheep naturally allergic to Ascaris suum were challenged with an aerosolized antigen with and without S-5751 treatment (given 4 days before and for 6 days after the challenge). RESULTS: S-5751 inhibited PGD(2)-induced cAMP production in platelet-rich plasma with an IC(50) value of 0.12 microm. S-5751 at 30 mg/kg, but not at 3 mg/kg, reduced the early bronchoconstriction and inhibited the late bronchoconstriction. AHR and inflammatory cell infiltration in bronchoalveolar lavage fluid at days 1 and 7 were also inhibited with the 30 mg/kg dose. The responses observed with S-5751 at 30 mg/kg were comparable with those with montelukast treatment (0.15 mg/kg, twice a day, intravenous); however, S-5751 did not block inhaled leukotrieneD(4)-induced broncoconstriction. CONCLUSION: Prostanoid DP1 receptor inhibition may represent an alternative target for asthma therapy.

Association of T-cell receptor Vbeta haplotypes with dry skin in DS-Nh mice.

BACKGROUND: Although dry skin and T cell-dependent disease exacerbation are characteristic features of atopic dermatitis (AD), the involvement of T cells in the development of dry skin remains unclear. AIMS: We aimed to elucidate the role of T cells in the development of dry skin in DS-Nh mice as a model for AD, and to evaluate this skin condition pharmacologically. METHODS: We prepared DS-Nh mice harbouring a T-cell receptor (TCR)Vbeta(a) haplotype with a central deletion in the TCRBV gene segments, and mice harbouring a TCRVbeta(b) haplotype without any deletion. We analysed the TCRVbeta chain usage and cytokine response to antimouse CD3 monoclonal antibodies in the splenocytes from the two mouse substrains. Transepidermal water loss (TEWL) was measured, and histochemical examination of these mice was carried out. Finally, a pharmacological analysis using loratadine was also performed to evaluate the features of spontaneous dry skin in DS-Nh mice as a model of AD. RESULTS: Although the deletion of TCRBV gene segments in the TCRVbeta(a) haplotype yielded different representations of each TCRVbeta mRNA, this deletion did not evoke distinct cytokine profiles in the splenocytes compared with those of mice with the TCRVbeta(b) haplotype. Furthermore, our results indicated that the onset of dry skin occurred earlier in mice with TCRVbeta(b) than in those with TCRVbeta(a). Pharmacologically, AD-like dry skin in DS-Nh with TCRVbeta(b) mice is susceptible to an H1 blocker. CONCLUSIONS: A specific lymphocyte subpopulation bearing T-cell receptors may be responsible for loratadine-responsive dermatitis in DS-Nh mice.

Spontaneous scratching behaviour in DS-Nh mice as a possible model for pruritus in atopic dermatitis.

Itching is one of the major clinical symptoms in atopic dermatitis (AD) and complicates the management of this pathological condition. An animal model of AD-like pruritus would contribute to a better understanding of AD and could lead to the development of safe and effective antipruritic agents. DS non-hair (DS-Nh) mice raised under conventional conditions spontaneously develop pruritus, which is associated with a dermatitis similar to human AD. There is a significant positive correlation between disease severity and the period of scratching behaviour in DS-Nh mice. In the present study, we found that levels of histamine and nerve growth factor (NGF) in serum and/or skin tissue were higher in DS-Nh mice with AD-like dermatitis than in age-matched mice without dermatitis. The histopathological data indicated that nerve fibres extend into and mast cells infiltrate the surrounding area of the skin lesion. NGF production by XB-2 cells, which was derived from mouse keratinocytes, was enhanced by histamine via the H1 receptor. We also found that prolonged treatment with an H1-antagonist was effective against pruritus through depression of the production of NGF, which is thought to be generated by keratinocytes. We conclude that DS-Nh mice can serve as a suitable model for gaining a better understanding of pruritus in AD, and that prolonged treatment with an H1-antagonist may be beneficial in patients with AD-associated pruritus.

Effect of PACAP on LH release studied by cell immunoblot assay depends on the gender, on the time of day and in female rats on the day of the estrous cycle.

We have previously demonstrated that pituitary adenylate cyclase activating polypeptide (PACAP) can be released from cultured rat anterior pituitary cells and when added to the medium in physiological concentration it releases LH from individual gonadotropes. In the present work, we studied whether the release of PACAP and the responsiveness of LH cells to PACAP depend on the gender, on the time of day when the animals were sacrificed, and in females on the stage of the estrous cycle. Anterior pituitary cells were cultured on nitrocellulose membrane. We found that the number of PACAP releasing cells was higher in proestrous than in diestrous female or in male rats and their number was always higher in the evening than at the other times. The effect of PACAP on LH cells was stimulatory in the morning of proestrus and diestrus. In proestrous rats, PACAP did not influence LH release in the afternoon or the evening, but in diestrous rats it decreased it in the afternoon and the evening. In males, there was a decrease of LH due to PACAP treatment at 10 and 20 h; however, PACAP did not influence LH at 16 h. It was concluded that in vivo PACAP might be involved in the circadian and episodic release of LH at pituitary level.

DS-Nh as an experimental model of atopic dermatitis induced by Staphylococcus aureus producing staphylococcal enterotoxin C.

DS-Nh mice raised under conventional conditions spontaneously develop dermatitis similar to human atopic dermatitis (AD), which is associated with staphylococcal infection. In the present study, we show that Staphylococcus aureus producing staphylococcus exotoxin C (SEC) was recovered from the culture of the skin lesions of DS-Nh mice with AD-like dermatitis and that the serum levels of anti-SEC antibodies from these mice were elevated. We describe here how to promote experimental AD by epicutaneous injection with SEC-producing S. aureus to DS-Nh mice. In order to assess the role of SEC in the pathogenesis of AD, the mitogenic activity, TCRBV repertoire analysis and the production of IL-4 and IFN-gamma from spleen mononuclear cells (MNC) from DS-Nh stimulated by SEC were compared with those due to SEA, SEB and TSST. The weakest was the mitogenic activity of SEC, and higher IL-4 responses and lower IFN-gamma responses to SEC showed correlation with TCRBV8S2-positive T cells, which were selectively stimulated by SEC. We also demonstrate that SEC-producing S. aureus was able to survive in DS-Nh after intradermal injection. These results suggest a possible role for SEC in the pathogenesis of AD through host-S. aureus relationships.

Gastric atrial natriuretic peptide regulates endocrine secretion in antrum and fundus of human and rat stomach.

Atrial natriuretic peptide (ANP) is present in gastric mucosa and preferentially binds to two subtypes of natriuretic peptide receptors (NPR), NPR-A and NPR-C. The present study examines the role of endogenous ANP in regulating endocrine secretion in rat and human stomachs. NPR-A protein expression and transcripts were identified in rat antral and fundic mucosa by Western blot and RT-PCR. In superfused rat and human antral and fundic segments, ANP (0.1 pM to 0.1 microM) caused a concentration-dependent increase in somatostatin secretion. In antrum, this was accompanied by a decrease in gastrin, and in fundus, this was accompanied by a decrease in histamine secretion. Changes in gastrin and histamine secretion reflected changes in somatostatin secretion and were abolished by somatostatin antibody. The NPR-A receptor antagonist anantin 1) inhibited basal somatostatin secretion and 2) abolished the somatostatin, gastrin, and histamine responses to ANP. We conclude that endogenous ANP, acting via the NPR-A receptor, stimulates somatostatin secretion from both antrum and fundus of rat and human stomach. Stimulation of somatostatin secretion is coupled to inhibition of gastrin secretion in the antrum and inhibition of histamine secretion in the fundus.

Reciprocal paracrine pathways link atrial natriuretic peptide and somatostatin secretion in the antrum of the stomach.

Atrial natriuretic peptide (ANP) as well as its receptor, NPR-A, have been identified in gastric antral mucosa, suggesting that ANP may act in a paracrine fashion to regulate gastric secretion. In the present study, we have superfused antral mucosal segments obtained from rat stomach to examine the paracrine pathways linking ANP and somatostatin secretion in this region.ANP (0.1 pM to 0.1 microM) caused a concentration-dependent increase in somatostatin secretion (EC(50), 0.3 nM). The somatostatin response to ANP was unaffected by the axonal blocker tetrodotoxin but abolished by addition of the selective NPR-A antagonist, anantin. Anantin alone inhibited somatostatin secretion by 18+/-3% (P<0.005), implying that endogenous ANP, acting via the NPR-A receptor, stimulates somatostatin secretion. Somatostatin (1 pM to 1 microM) caused a concentration-dependent decrease in ANP secretion (EC(50), 0.7 nM) that was abolished by addition of the somatostatin subtype 2 receptor (sst2) antagonist, PRL2903. Neutralization of ambient somatostatin with somatostatin antibody (final dilution 1:200) increased basal ANP secretion by 70+/-8% (P<001), implying that endogenous somatostatin inhibits ANP secretion. We conclude that antral ANP and somatostatin secretion are linked by paracrine feedback pathways: endogenous ANP, acting via the NPR-A receptor, stimulates somatostatin secretion, and endogenous somatostatin, acting via the sst2 receptor, inhibits ANP secretion.

Neonatal PACAP administration in rats delays puberty through the influence of the LHRH neuronal system.

The onset of puberty is a concerted action of many factors which leads to cyclic LHRH release in rats. It has been demonstrated that; in common with vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase activating polypeptide (PACAP) is also involved in the differentiation of the central nervous system. In our previous work, it was shown that a single PACAP injection into neonatal female rats delayed puberty. In the present work, neonatal administration of PACAP delayed the vaginal opening and decreased the weight of anterior pituitaries, the number of expelled ova at the first ovulation and the intensity of LHRH immunostaining in the septo-preoptico-infundibular system. PACAP antiserum had a reverse effect on LHRH immunoreactivity. The other studied parameters in the latter group remained unchanged compared to control rats. It was concluded that neonatal PACAP administration delayed the onset of puberty through the influence of the LHRH neuronal system.

Cell immunoblot assay study demonstrating the release of PACAP from individual anterior pituitary cells of rats and the effect of PACAP on LH release.

The presence of pituitary adenylate cyclase activating polypeptide (PACAP) was previously demonstrated in the anterior pituitary by radioimmunoassay, immunohistochemistry, and reverse transcript-polymerase chain reaction (RT-PCR). With the use of cell immunoblot assay (CIBA), when the pituitary cells were cultured on nitrocellulose membrane, the release of PACAP by individual anterior pituitary cells was observed. The released peptide, trapped by the nitrocellulose membrane forming a blot around the cells, was demonstrated by immunocytochemistry. Double labeling revealed that a part of PACAP-immunoreactive cells can release LH as well. With the use of sandwich enzyme immunoassay (S-EIA), it was found that the concentration of PACAP in the anterior pituitaries is 10(-10) M. In cell culture in a similar concentration, PACAP stimulated the LH release from female gonadotropes, but did not influence it from male ones. The stimulated release of LH was indicated by the enhancement in the diameter of LH blots compared to the untreated control cultures. We concluded that PACAP may be released from the anterior pituitary cells in a concentration which would be able to influence LH release not only in vitro but under in vivo conditions as well. The effect of PACAP on LH release was different in female and male pituitary cultures.

Effects of pretreatment with PACAP on the infarct size and functional outcome in rat permanent focal cerebral ischemia.

PACAP exerts neuroprotective effects under various neurotoxic conditions in vitro. In vivo, it reduces brain damage after global and transient focal ischemia. The present study investigated whether PACAP has neuroprotective effects when applied before the onset of permanent ischemia. Rats were given bolus injections of PACAP38 intracerebroventricularly, and then underwent permanent middle cerebral artery occlusion. The results show that 2 microg of PACAP significantly reduced the infarct size measured 12 and 24h after the onset of ischemia. No further reduction was obtained by a 7-day pretreatment. PACAP also ameliorated certain sensorimotor deficits. Our present study provides further evidence for the neuroprotective effects of PACAP, and implies that it might be a promising preventive therapeutic agent in ameliorating ischemic brain damage.

Comparative study on the appearance of various bioactive peptides in foregut derivates during the ontogenesis.

Bioactive peptides have an important multifunctional role in the gastrointestinal tract. In the present study we have investigated the dynamism of the appearance of PACAP (pituitary adenylate cyclase activating polypeptide), VIP (vasoactive intestinal polypeptide), gastrin, and secretin immunoreactivities in human foregut derivates during the ontogenesis using an immunohistochemical approach. None of these peptides were observed in the foregut derivates of an 8-week-old embryo. VIP immunoreactive nerve fibers appeared by the 11th week in the smooth muscle layers of the stomach. No other peptide immunoreactivities were observed of this stage. In 18- and 20-week old fetuses PACAP, secretin, and gastrin immunoreactive cells appeared in the developing glands of the stomach. In the duodenum gastrin immunoreactivity was present in the Lieberkühn's glands and secretin immunoreactive cells were seen between the surface epithelial cells. In the pancreas secretin immunoreactivity was found in the Langerhans islets; however, PACAP immunreactivity was observed in the exocrine portion. The distribution of VIP fibers did not change during the fetal life and it was similar to the adult pattern. According to our results the appearance of PACAP, secretin, and gastrin in the developing glands suggests their role in the proliferation and differentiation of the epithelial derivates.

Inhibitory effect of a TP-receptor antagonist, S-1452, on antigen-induced nasal plasma exudation in guinea pig model for allergic rhinitis.

S-1452, a selective thromboxane (Tx) A(2) receptor (TP-receptor) antagonist, was evaluated in antigen- and U-46619 (a TxA(2) mimetic)-induced guinea pig nasal plasma exudation models. Exposure of the nasal cavity of actively sensitized guinea pigs to aerosolized ovalbumin (OA) caused marked exudation of dye into both the nasal mucosa and nasal airway lumen. These responses were significantly inhibited by S-1452 (30 mg/kg, p.o.) as well as an H(1)-antihistamine, diphenhydramine (5 mg/kg, i.v.). In addition, exposure of the nasal cavity of nonsensitized guinea pigs to aerosolized U-46619 or histamine also resulted in nasal plasma exudation, and S-1452 (1 mg/kg, p.o.) almost completely suppressed the U-46619-induced response but did not affect the histamine-induced one, even at a high dose of 30 mg/kg. These results indicate that TxA(2) as well as histamine may play an important role in antigen-induced nasal plasma exudation in guinea pigs, and S-1452 can be expected to be useful for the treatment of allergic rhinitis.

Distribution and daily variations of PACAP in the chicken brain.

Levels of PACAP38 were measured in different areas of the chicken brain under various lighting conditions by radioimmunoassay (RIA). Selected groups of animals were maintained under light for 14 h alternating with 10 h of darkness (LD), reversed lighting conditions (DL) and constant light (LL) or constant dark (DD). Daily variations of PACAP levels were observed in the brainstem, diencephalon, telencephalon and retina. In the brainstem and diencephalon, levels of PACAP increased during subjective nighttime, except in the DL group where levels were elevated between 15-21 h. In the telencephalon, the lowest level of PACAP was measured between 12-21 h except in the DL group where two peaks occurred at 18 and 03 h. In the retina, all 4 groups showed a similar level and pattern, with lowest levels during midday hours. No daily variation was observed in the pineal gland. According to the present observations, it is suggested that PACAP levels differ in several areas of the chicken brain under various lighting conditions and photic stimuli do not appear to be the main regulators of the circadian variations of PACAP.

Prevention of allergic inflammation by a novel prostaglandin receptor antagonist, S-5751.

Prostaglandin (PG) D2, the major cyclooxygenase metabolite generated from immunologically stimulated mast cells, is thought to contribute to the pathogenesis of allergic diseases due to its various inflammatory effects. However, since no DP receptor antagonist has been developed as an antiallergic drug, the role of PGD2 in the pathogenesis of allergic diseases remains uncertain. Here, we report the in vivo efficacy of our newly established DP receptor antagonist, S-5751 [((Z)-7-[(1R,2R,3S,5S)-2-(5-hydroxy benzo[b]thiophen-3-ylcarbonylamino)-10-norpinan-3-yl]hept-5- enoic acid)], using various allergic inflammation guinea pig models. In allergic rhinitis models, oral administration of S-5751 dramatically inhibited not only early nasal responses, as assessed by sneezing, mucosal plasma exudation, and nasal blockage, but also late responses such as mucosal plasma exudation and eosinophil infiltration. Even when S-5751 was administered after recovery from the early responses, these late phase responses were almost completely suppressed. In addition, S-5751 alleviated allergen-induced plasma exudation in the conjunctiva in an allergic conjunctivitis model and antigen-induced eosinophil infiltration into the lung in an asthma model. These findings provide evidence for the crucial role of PGD2 as a mediator of allergic inflammation in guinea pigs and suggest that DP receptor antagonists may be useful in the treatment of allergic diseases triggered by mast cell activation.

Pituitary adenylate cyclase-activating polypeptide and its receptors in amphibians.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide of the secretin/glucagon/vasoactive intestinal polypeptide superfamily, has been initially characterized in mammals in 1989 and, only 2 years later, its counterpart has been isolated in amphibians. A number of studies conducted in the frog Rana ridibunda have demonstrated that PACAP is widely distributed in the central nervous system (particularly in the hypothalamus and the median eminence) and in peripheral organs including the adrenal gland. The cDNAs encoding the PACAP precursor and 3 types of PACAP receptors have been cloned in amphibians and their distribution has been determined by in situ hybridization histochemistry. Ontogenetic studies have revealed that PACAP is expressed early in the brain of tadpoles, soon after hatching. In the frog Rana ridibunda, PACAP exerts a large array of biological effects in the brain, pituitary, adrenal gland, and ovary, suggesting that, in amphibians as in mammals, PACAP may act as neurotrophic factor, a neurotransmitter and a neurohormone.

Pituitary adenylate cyclase activating polypeptide is highly abundant in the nervous system of anoxia-tolerant turtle, Pseudemys scripta elegans.

The levels of the pituitary adenylate cyclase activating polypeptide (PACAP) were measured in the central nervous system and in peripheral organs of the anoxia-tolerant freshwater turtle, Pseudemys scripta elegans by radioimmunoassay. The concentration of PACAP38 was strikingly high in the central nervous system and lower but considerable immunoreactivity was detected in the peripheral organs. Levels of PACAP38 in the turtle brain exceed those measured in rat and human brain areas by 10-100-fold. Based on these exceptionally high levels of PACAP and the known neuroprotective role of the peptide, it can be suggested that PACAP38 plays a role in the extraordinary resistance of the turtle brain from anoxia-induced neuronal damage.

Effects of leukotriene B4 receptor antagonist, LY293111Na, on antigen-induced bronchial hyperresponsiveness and leukocyte infiltration in sensitized guinea pigs.

OBJECTIVE AND DESIGN: LY29311 Na, 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy] propoxy] -phenoxy]-benzoic acid sodium salt, is a novel leukotriene B4 (LTB4) receptor antagonist. Its effects on guinea pig models of asthma were compared with those of dexamethasone. METHODS: Effects of LY293111Na were tested in antigen (ovalbumin, OA)-induced bronchial hyperresponsiveness (BHR) and leukocyte accumulation in actively sensitized guinea pigs. Its effects on antigen-induced acute bronchoconstriction in passively sensitized guinea pigs were also studied. RESULTS: LY293111 Na (10 to 30 mg/kg p.o., 1 h before and 6 h after OA challenge) inhibited BHR to acetylcholine. LY293111 Na (3 mg/kg p. o.) significantly inhibited accumulation of neutrophils in bronchoalveolar lavage (BAL) fluid 24 h after antigen challenge but it did not inhibit accumulation of eosinophils and macrophages at any doses used. In contrast, dexamethasone (30 mg/kg p.o., 4 h before OA challenge) not only inhibited BHR but also reduced the infiltration of all three types of leukocytes. A significant increase of LTB4 levels in BAL fluid was noted at 3 and 15 min after the antigen challenge. LY293111Na did not inhibit antigen-induced acute bronchoconstriction in passively sensitized guinea pigs. CONCLUSION: These results indicate that LTB4 may participate in antigen-induced BHR but not in eosinophil infiltration and acute bronchoconstriction in guinea pigs.