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We describe the design of a novel PDE4 scaffold and the exploration of the dual-soft concept to reduce systemic side effects via rapid elimination: introducing ester functionalities that can be inactivated in blood as well as by the liver (dual-soft) while being stable in human skin. Compound 40 was selected as a clinical candidate as it was potent and rapidly degraded by blood and liver to inactive metabolites and because in preclinical studies it showed high exposure at the target organ: the skin. Preclinical and clinical data are presented confirming the value of the dual-soft concept in reducing systemic exposure.

Chronic inflammatory diseases of the respiratory tract, such as chronic obstructive pulmonary disease (COPD) and asthma, are severe lung diseases that require effective treatments. In search for new medicines for these diseases, there is an unmet need for predictive and translatable disease-relevant in vitro/ex vivo models to determine the safety and efficacy of novel drug candidates. Here, we report the use of precision cut lung slices (PCLS) as a potential ex vivo platform to study compound effects in a physiologically relevant environment. PCLS derived from an elastase-challenged mouse model display key characteristics of increased inflammation ex vivo, which is exacerbated further upon challenge with LPS, mimicking the immune insult of a pathogen triggering disease exacerbation. Such LPS-induced inflammatory conditions are significantly abrogated by immunomodulatory agents targeting specific inflammatory signaling pathways in the absence of cytotoxic effects in lung slices. Thus, an ex vivo model of PCLS with a simulated pathogenic insult can replicate proposed in vivo pharmacological effects and thus could potentially act as a valuable tool to investigate the underlying mechanisms associated with lung safety, therapeutic efficacy and exacerbations with infection.

We describe the design of a novel PDE4 scaffold and the exploration of the dual-soft concept to reduce systemic side effects via rapid elimination: introducing ester functionalities that can be inactivated in blood as well as by the liver (dual-soft) while being stable in human skin. Compound

Steroidal glucocorticoids (GR agonists) have been widely used for the topical treatment of skin disorders, including atopic dermatitis. They are a very effective therapy, but they are associated with both unwanted local effects in the skin (skin thinning/atrophy) and systemic side effects. These effects can limit the long-term utility of potent steroids. Here we report on a topically delivered non-steroidal GR agonist, that has the potential to deliver high efficacy in the skin, but due to rapid metabolism in the blood & liver ("dual-soft") it should have greater systemic safety than existing treatments. In addition, compared to less selective steroidal GR agonists, the new non-steroidal Selective Glucocorticoid Agonists (SEGRAs) have the potential to avoid the skin atrophy observed with existing topical steroids. Due to its potential for reduced skin atrophy and low systemic exposure, LEO 134310 (17) may be suitable for long term topical treatment of skin diseases such as atopic dermatitis and psoriasis.

Introduction: The aim of the present study was to compare sensitivity in detecting the drug-induced QT interval prolongation in three dog models: conscious telemetered at sinus rhythm and conscious and anesthetized dogs during atrial pacing. The test substances used represent different chemical classes with different pharmacological and pharmacokinetic profiles. Method: Dofetilide and moxifloxacin were tested in all models, whereas cisapride and terfenadine were tested in the conscious telemetered and paced models. All substances were given as two consecutive 1.5-h intravenous infusions (infusions 1 and 2). The individual concentration–time courses of dofetilide, moxifloxacin, and cisapride were linked to the drug-induced effects on the QT interval and described with a pharmacokinetic–pharmacodynamic model to obtain an estimate of the unbound plasma concentrations at steady state that give a 10- and 20-ms drug-induced QT interval prolongation (CE10ms and CE20ms). Results: In the conscious telemetered, conscious paced, and anesthetized dog models, the mean CE10ms values were 1.4, 4.0, and 2.5 nM for dofetilide and 1300, 1800, and 12,200 nM for moxifloxacin. For cisapride, the CE10ms values were 8.0 and 4.4 nM in the conscious telemetered and conscious paced dog models. The drug-induced QT interval prolongation during the last 30 min of infusions 1 and 2 was comparable in the conscious models, but smaller in the anesthetized dog model. Terfenadine displayed a marked delay in onset of response, which could only be detected by the extended ECG recording. Discussion: All dog models investigated detected QT interval prolongation after administration of the investigated test substances with similar sensitivity, except for a lower sensitivity in the anesthetized dogs following moxifloxacin administration. The conscious telemetered dog model was favorable, mainly due to the extended continuous ECG recording, which facilitated detection and quantification of delayed temporal differences between systemic exposure and drug-induced QT interval prolongation.

Introduction: Drug-induced QT interval prolongation may lead to ventricular arrhythmias. The aim of the study was to optimize QT interval data processing to quantify drug-induced QT interval prolongation in the telemetry instrumented conscious dog model. Methods: The test substances cisapride, dofetilide, haloperidol, and terfenadine and corresponding vehicles were given to male and female beagle dogs during two consecutive 90-min intravenous infusions. Cardiovascular parameters were recorded for 24 h and exposure to the drugs was measured. The delayed response in the QT interval after an abrupt change in heart rate was investigated. Eight mathematical models to describe the QT interval–heart rate relationship were compared and different sets of covariates were used to quantify the drug-induced effect on the QT interval. Results: After an abrupt decrease in heart rate, a 75% adaptation of the QT interval was reached after 54 ± 9 s. A linear model was preferred to correct the drug-induced effect on the QT interval for heart rate, vehicle effect, serial correlation, plasma concentration and time of day. All test substances significantly prolonged the QT interval. Discussion: To optimize the processing of QT interval data, the delay in QT interval response after an abrupt change in heart rate should be considered. The QT interval–heart rate relationship and vehicle response were individual-specific and corrections were therefore made individually. When estimating the drug-induced effect on the QT interval it is considered advantageous to use plasma concentration as a covariate, as well as adjusting for vehicle effect and serial correlation in measurements. The conscious dog model detected significant increases in the QT interval for all test substances investigated.

The second coding exon of the adenosine A, receptor gene was eliminated by homologous recombination. The phenotype of mice (mixed C57B6/129OlaHsd background) was studied, using siblings from matings of heterozygous mice. Among the offspring the ratio between+/+, +/-and -/-animals was 1:2:1. Over the first half-year-at least-growth and viability were the same in all genotypes. Binding of A(1) ligands was eliminated in-/-mice and halved in+/-mice. Blood pressure was increased in-/-mice and this was paralleled by an increase in plasma renin. Heart rate was unaffected, as was contractility. Furthermore, the response of the perfused heart to ischemia was similar in+/+and -/-hearts. However, remote preconditioning was eliminated in-/-mouse hearts. Tubuloglomerular feedback in the kidney was also lost in-/-mice. The analgesic response to a non-selective adenosing receptor agonist was lost in-/-mice, which also showed hyperalgesia in the tail-flick test. There was a slight hypoactivity in-/-mice, but responses to caffeine were essentially normal. The inhibition of excitatory neurotransmission in hippocampus by adenosine was lost in-/-mice and reduced in+/-mice. Responses to ATP were affected similarly. Hypoxic depression of synaptic transmission was essentially eliminated in hippocampus and hypoxic decrease in spinal respiratory neuron firing was markedly reduced. These results show that adenosine A, receptors play a physiologically important role in the kidney, spinal cord, and hippocampus and that they are critically important in the adaptive responses to hypoxia. (C) 2003 Wiley-Liss, Inc. (Less)

The neuronal isoform of nitric oxide synthase (nNOS) in the kidney is predominantly located in the macula densa (MD) cells. These cells are known to be the sensor in the tubuloglomerular feedback, which influences the tonus of the afferent arteriole. This study investigated the effect of angiotensin II (Ang II) after chronic inhibition of nNOS on renal blood flow (RBF) and cytosolic calcium concentration [Ca(2+)]i in smooth muscle cells from afferent arterioles. Measurements of RBF were made in two control groups and two groups treated with a nNOS inhibitor, 7-nitro indazole (7-NI), for 1 and 4 weeks. At the time of the experiment Ang II bolus was given in the renal artery before and during i.v. l-NNA. [Ca(2+)]i was measured in arterioles from control rats and from rats treated for 1 week with 7-NI. RBF decreased after bolus Ang II by 60 +/- 11% in the control vs. 23 +/- 8% in the 1 week 7-NI treated group. The decreased sensitivity to Ang II after 1 week of 7-NI treatment compared with control rats persisted after l-NNA infusion. There were no differences from control in the group treated for 4 weeks. Ang II gave a transient [Ca(2+)]i increase in vessels from control rats whereas this response was absent in 1 week 7-NI-treated rats. A possible explanation for these findings could be a down regulation of Ang II receptors. The renal vasculature of rats exhibits a diminished RBF and [Ca(2+)]i response to Ang II after 1 week blockade of nNOS.

The tubuloglomerular feedback (TGF) mechanism is an important regulator of the glomerular filtration rate (GFR) and urine excretion rate. It operates by sensing the distal delivery of fluid at the macula densa site and adjusting the tone of the glomerular arterioles to control GFR. We found evidence that nitric oxide is an important modulator of the setting of the sensitivity of the TGF mechanism. Studies on adenosine A1 receptor deficient mice have shown that these animals lack the TGF response and have an increased renin release. These findings show the important role of adenosine as a mediator of the signal for the TGF mechanism and as an inhibitor of renin release.

Nitric oxide (NO) produced by neuronal NO-synthase (nNOS) in macula densa cells may be involved in the control of renin release. 7-Nitro indazole (7-NI) inhibits nNOS, and we investigated the effect of short- (4 days) and long-term (4 weeks) 7-NI treatment on blood pressure (BP), plasma renin concentration (PRC) and glomerular filtration rate (GFR) in rats on different salt diets. Rats were divided into three groups and given low-salt (LS), normal (C) and high-salt (HS) diets. Each diet group was subdivided into two groups treated either with 7-NI or vehicle. Long-term 7-NI-treated rats (LS and C) showed increased BP compared with controls (LS: 149 +/- 4 vs. 133 +/- 3; C: 146 +/- 4 vs. 127 +/- 4 mmHg). Blood pressure in HS rats did not differ from that in controls. Plasma renin concentration was stimulated in LS-rats (251 +/- 64 mGU mL(-1)) compared with C and HS rats (42 +/- 8 and 39 +/- 5 mGU mL(-1), respectively) but was not significantly affected by chronic 7-NI treatment (350 +/- 103, 49 +/- 10 and 50 +/- 15 mGU mL(-1) in LS, C and HS, respectively). In rats treated with 7-NI for 4 days, no effect on BP was seen, but PRC was increased in 7-NI treated LS rats compared with vehicle treated LS rats (107 +/- 15 vs. 56 +/- 1 mGU mL(-1)). Stimulation of PRC in LS rats was further enhanced by 7-NI after 4 days of treatment, but not affected in rats treated for 4 weeks. This suggests that inhibition of nNOS stimulates renin release but that this stimulatory effect in the long run might be depressed by the increase in blood pressure.

The juxtaglomerular apparatus (JGA) has the very important functions of detecting the fluid flow rate to the distal tubule and thus controlling the glomerular filtration rate (GFR) (tubuloglomerular feedback mechanism [TGF]) and renin release from the afferent arteriole. In studies of the TGF it has been evident that the sensitivity of this mechanism can be reset. Volume expansion will reset it to a low sensitivity leading to a high GFR and urine excretion rate, while dehydration will sensitize the TGF mechanism, giving rise to a low GFR and low urine excretion rate. Furthermore, we have found that in animals that spontaneously develop hypertension there is initially a sensitization of the TGF, leading to a reduced GFR and urine excretion rate, with fluid volume retention in the body and a consequent rise in blood pressure. When the pressure is raised, the TGF characteristics are normalized. In the macula densa (MD) cells in the JGA, there is a large production of NO from neuronal NOS. This production continuously reduces TGF sensitivity and is apparently impaired in animals that spontaneously develop hypertension. When we added an nNOS inhibitor to the drinking water for several weeks while measuring blood pressure, we found an increase in blood pressure after 3-4 weeks of treatment This effect was abolished by a high salt diet From these investigations, it also appeared as if nNOS-derived NO inhibited renin release. Experiments have also indicated that NO may resensitize inhibited G-protein coupled purinergic receptors.

Introduction To assure drug safety, the investigation of the relationship between plasma concentration and drug-induced prolongation of the QT interval of the ECG is a challenge in drug discovery. For this purpose, dofetilide was utilized to demonstrate the benefits of characterizing the complete time course of concentrations and effect in conscious beagle dogs in the assessment of drug safety. Method On two separate occasions, four male and two female beagle dogs were given vehicle or the test substance, dofetilide (0.25 μmol/kg), over a 3-h intravenous infusion. Cardiovascular parameters, including QT intervals, were recorded for 24-h using radiotelemetry. The QT interval was corrected individually for heart rate, vehicle treatment, and serial correlation (QT c ). Exposure (plasma concentration) to dofetilide was measured and described by a two-compartment model. The individual concentration–time course of dofetilide was linked to the QT c interval via an effect compartment and a pharmacodynamic E max model, to account for the observed hysteresis. Results Dofetilide induced a concentration-dependent increase in the QT c interval, with an EC 50 of 9 nM (3–30 nM, 95% C.I.) and an E max of 59 ± 9 ms. A hysteresis loop was observed by plotting plasma concentrations vs. QT interval in time order, indicating a delay in onset of effect. It was found to have an equilibrium half-life of 11 ± 8 min. Based on the parameters potency and E max , a representation was made of the drug-induced changes to the QT interval. Discussion An effect compartment model was found to accurately mimic the QT interval prolongation following administration of the test substance, dofetilide. The assessment of the individual concentration–effect relationship and confounding factors such as hysteresis might provide a better prediction of the safety profiles of new drug candidates.

The juxtaglomerular apparatus in the kidney is important in controlling extracellular fluid volume and renin release. The fluid load to the distal tubule is first sensed at the macula densa site via the entry of NaCl, through a Na, K, 2Cl co-transport mechanism. The next step is unclear, but there is recent evidence of an increased macula densa cell calcium concentration with a reduction in fluid load to the macula densa. An increase in macula densa cell calcium could activate phospholipase A2 to release arachidonic acid, the rate-limiting step in the formation of prostaglandins. Recent evidence suggests that the prostaglandin formed is PGE2, a potent stimulator for renin release. Recent evidence has also shown that adenosine has an important function in the juxtaglomerular apparatus. It stimulates calcium release in afferent arteriolar smooth muscle cells, leading to contraction of the afferent arteriole as part of the tubuloglomerular feedback mechanism, and inhibits renin release. Thus, renin release from the afferent arteriole is mediated partly through formation of PGE2, and partly through the reduction of adenosine formation that inhibits renin production.

While macro- and microscopic kidney development appear to proceed normally in mice that lack Foxi1, electron microscopy reveals an altered ultrastructure of cells lining the distal nephron. Northern blot analyses, cRNA in situ hybridizations, and immunohistochemistry demonstrate a complete loss of expression of several anion transporters, proton pumps, and anion exchange proteins expressed by intercalated cells of the collecting ducts, many of which have been implicated in hereditary forms of distal renal tubular acidosis (dRTA). In Foxi1-null mutants the normal epithelium with its two major cell types - principal and intercalated cells - has been replaced by a single cell type positive for both principal and intercalated cell markers. To test the functional consequences of these alterations, Foxi1(-/-) mice were compared with WT littermates in their response to an acidic load. This revealed an inability to acidify the urine as well as a lowered systemic buffer capacity and overt acidosis in null mutants. Thus, Foxi1(-/-) mice seem to develop dRTA due to altered cellular composition of the distal nephron epithelium, thereby denying this epithelium the proper gene expression pattern needed for maintaining adequate acid-base homeostasis.

The effects of increased intra-abdominal pressure (IAP) and volume expansion on renal function in the rat were studied to gain more knowledge of the oliguria seen during laparoscopic procedures and to reduce the detrimental renal effects of IAP.IAP was elevated to 5 or 10 mmHg by insufflation of CO(2) and maintained for 2 h in anaesthetized and mechanically ventilated rats. Rats with normal IAP served as controls. An angiotensin II receptor I antagonist, candesartan, was given as a bolus injection and a 5% volume expansion was achieved by i.v. saline infusion. An angiotensin-converting enzyme (ACE) inhibitor was also given. Renal parameters were the glomerular filtration rate (GFR), urine production, the urinary concentrations of sodium and potassium and the osmolality in the urine. The arterial acid-base balance and blood pressure were also monitored.The GFR deteriorated by 70% during pneumoperitoneum (PP) of 10 mmHg. There was a dramatic drop in sodium excretion (88-97%). With candesartan and elevated IAP, there was a drop in mean arterial pressure (from 90 to 55 mmHg) and the negative renal effects were very pronounced. Renal function was better preserved during elevated IAP in combination with volume expansion.Capnoperitoneum suppresses renal function, especially in combination with angiotensin II receptor 1 blockade and ACE inhibition. Volume expansion reduces the deleterious effects of PP on renal function during elevated IAP. The results suggest that patients should not be given pharmaceuticals blocking the renin-angiotensin-aldosterone system prior to procedures that may increase IAP. It may be beneficial, however, to reduce angiotensin II tension by volume expansion.

In the juxtaglomerular apparatus of the kidney the distal tubule returns to its own glomerulus where specialised cells, the macula densa (MD) cells, are located so that they come in contact with the tubular fluid. It is well documented that MD cells sense the distal tubular flow-rate and that when the flow is too high they are activated, which leads to vasoconstriction of the afferent arteriolar, the tubuloglomerular feedback (TGF). Neuronal nitric oxide synthase (nNOS) is largely expressed in the MD cells. Acute inhibition of MD nNOS leads to an enhanced TGF response, indicating that nitric oxide (NO) is an important short-term modulator for volume regulation in the body. By contrast, the NO from the nNOS of the MD cells does not seem to play a role in the long-term adaptations of renin release in low- or high-salt diets.

The characteristics of the tubuloglomerular feedback (TGF) mechanism were examined in streptozotocin-diabetic rats. This model is known to induce damage in the distal tubular system and thus Tamm-Horsfall protein (THP) secretion. Three groups of male Sprague-Dawley rats were studied: (A) diabetic rats with blood glucose levels (BG)19 mmol/l, (B) with BG/=19 mmol/l, and (C) control rats. After 50 days, the diabetic rats had higher arterial blood pressure and increased TGF reactivity (delta P(SF)) than control rats. The proximal tubular free-flow pressure (P(T)) and stop-flow pressure (P(SF)) were reduced, while the glomerular filtration was normal. This indicates that the diabetic animals of this study were severely vasoconstricted. Inhibition of renal nitric oxide synthase (NOS) resulted in a greater increase of TGF reactivity in diabetic rats than in control rats. Diabetic rats also showed increased excretion rates of albumin and THP. The excretion rate of THP was associated with P(SF) (r=-0.88, p0.01). In conclusion, diabetes mellitus was associated with an increased blood pressure and an increased TGF reactivity, which indicates that the diabetic rats were vasoconstricted. NOS inhibition increased the reactivity of TGF to greater extent in diabetic animals than in controls, indicating that the renal vasoconstriction was compensated for by an increased NO production.

Hypertension and impaired renal angiotensin II response in rats after chronic neuronal nitric oxide synthase inhibition. Nitric oxide (NO) produced by the macula densa cells is important for the control of tubuloglomerular feedback (TGF). Reduced production of NO by these cells activates TGF and could result in hypertension, although the TGF activity is then normalized in the hypertensive state. The normalization of TGF in this form of hypertension might be explained by an impaired ability of angiotensin II to constrict renal vessels or by up-regulation of some other vasodilator not affected by NO synthase inhibitors.

Acute unilateral renal denervation (aDNX) is associated with reduced tubuloglomerular feedback (TGF) sensitivity. Six days after denervation (cDNX) TGF sensitivity is somewhat restored, but TGF reactivity increased. This study aimed to investigate if the increased TGF reactivity that was seen in cDNX kidneys was owing to reduced production of nitric oxide (NO). TGF characteristics were determined with micropuncture experiments in anaesthetized rats, using the stop-flow pressure (PSF) technique. Maximal drop in PSF (DeltaPSF) was used as an index of TGF reactivity and the loop of Henle perfusion rate that elicited half-maximal DeltaPSF, the turning point (TP) was used as a measure of TGF sensitivity. In cDNX kidneys, TP was higher than in control rats (25.4 +/- 1.5 nL min-1 vs. 19.1 +/- 1.1 nL min-1), but clearly lower than in aDNX rats (37. 3 +/- 3.1 nL min-1). TGF was more reactive in cDNX rats (DeltaPSF=14. 7 +/- 1.1 mmHg) than in aDNX (7.9 +/- 1.1 mmHg) and control rats (9. 6 +/- 0.9 mmHg). Intratubular inhibition of NO synthase N omega-nitro-L-arginine (L-NA) in sham-DNX animals, decreased TP to 13.9 +/- 2.2 nL min-1 and DeltaPSF was increased with 92%. In cDNX kidneys TP was not significantly reduced by L-NA, and TGF reactivity was only moderately increased by 31%. Intratubular infusion of L-arginine (L-Arg) reduced DeltaPSF from 10.2 +/- 0.7 to 6.5 +/- 0.6 mmHg in sham-DNX kidneys, but TP was unaffected. In cDNX kidneys, there was no effect on either DeltaPSF or TP by the addition of L-Arg. However, when NO was delivered via sodium nitroprusside in the tubular perfusate, a clear reduction of DeltaPSF was seen in both sham-DNX and cDNX kidneys (from 9.9 +/- 0.5 to 4.4 +/- 1.0 and from14.9 +/- 1.3 to 8.1 +/- 1.5 mmHg, respectively). This indicates that cDNX is a state of low renal NO production and that this low level of NO resets TGF to a higher sensitivity and more pronounced reactivity.