An assessment of implications of adaptive licensing for pharmaceutical intellectual property and regulatory exclusivity rights in the European Union.

One of the key advantages of adaptive licensing (AL) is to align the licensing of new medicines more closely with patient needs for earlier access to beneficial treatments. From an innovators perspective, "earlier" market access may seem an obvious incentive to gain earlier revenue generation. However, this is offset with an "earlier" start to patent and regulatory protection periods, which, depending on the technology, disease, population, and timing of subsequent asset protection periods, can present a conflict.

An evaluation of regulatory and commercial barriers to stratified medicine development and adoption.

Today, a range of products based on genomics, proteomics and metabolomics have facilitated the development of 'stratified' medicines and companion diagnostics. This investigation profiles a series of targeted medicines and corresponding diagnostics, and their role(s) in supporting evidence-based medicine. Despite their potential benefits we found that scientific, financial and regulatory barriers impede the development and adoption of companion diagnostics. Therefore, in order to realise improvements to the risk/benefit profiles of health-care interventions-notably reducing clinical uncertainty-conferred by the use of companion diagnostics, industry representatives, health-care providers and regulators will need a coordinated response to overcome these barriers.

Review: novel insights into the regulation of vascular tone by sphingosine 1-phosphate.

Endothelial dysfunction leading to increased vascular tone is implicated in the pathogenesis of cardiovascular disease, hypertension and pregnancy-related complications like preeclampsia and intrauterine growth restriction. Vascular tone is regulated by a balance between vasoconstrictor and vasodilator signals. Some vascular mediators circulate in blood, whereas others are produced by the endothelium and are delivered to the underlying vascular smooth muscle cells (VSMCs). It is proposed that increased permeability of resistance arteries in preeclampsia allows access of circulating vasoactive factors to VSMCs leading to increased vascular tone. This review focuses on the role of sphingosine 1-phosphate (S1P). This sphingolipid enhances the endothelial barrier, but it can also disrupt the barrier under certain conditions. These S1P-mediated effects on the endothelial barrier have been demonstrated in cultured endothelial cells and in isolated venules. They depend on S1P concentrations, the S1P receptors expressed and the vascular bed. However, no studies have examined if vascular tone is regulated by S1P in resistance arteries through changes in endothelial permeability and the leakage of circulating vasoconstrictors. Our recent studies using the pressure myograph system show that access of infused vasoconstrictors to VSMCs is blocked under low S1P concentrations. Pathophysiological levels of infused S1P disrupt the barrier and maximally increase vascular tone by facilitating access of itself and a co-infused vasoconstrictor to the VSMCs. Interestingly, infusion of an intermediate physiological concentration of S1P showed a small increase in endothelial permeability with controlled leakage of a co-infused vasoconstrictor that led to sub-maximal vascular tone development. These and other studies delineate the important role of S1P in the regulation of vascular tone and emphasize how dysfunction of this regulation can lead to pregnancy-related disorders.

Autotaxin protects MCF-7 breast cancer and MDA-MB-435 melanoma cells against Taxol-induced apoptosis.

Autotaxin (ATX) promotes cancer cell survival, growth, migration, invasion and metastasis. ATX converts extracellular lysophosphatidylcholine (LPC) into lysophosphatidate (LPA). As these lipids have been reported to affect cell signaling through their own G-protein-coupled receptors, ATX could modify the balance of this signaling. Also, ATX affects cell adhesion independently of its catalytic activity. We investigated the interactions of ATX, LPC and LPA on the apoptotic effects of Taxol, which is commonly used in breast cancer treatment. LPC had no significant effect on Taxol-induced apoptosis in MCF-7 breast cancer cells, which do not secrete significant ATX. Addition of incubation medium from MDA-MB-435 melanoma cells, which secrete ATX, or recombinat ATX enabled LPC to inhibit Taxol-induced apoptosis of MCF-7 cells. Inhibiting ATX activity blocked this protection against apoptosis. We conclude that LPC has no significant effect in protecting MCF-7 cells against Taxol treatment unless it is converted to LPA by ATX. LPA strongly antagonized Taxol-induced apoptosis through stimulating phosphatidylinositol 3-kinase and inhibiting ceramide formation. LPA also partially reversed the Taxol-induced arrest in the G2/M phase of the cell cycle. Our results support the hypothesis that therapeutic inhibition of ATX activity could improve the efficacy of Taxol as a chemotherapeutic agent for cancer treatment.

Cell-permeable ceramides increase basal glucose incorporation into triacylglycerols but decrease the stimulation by insulin in 3T3-L1 adipocytes.

OBJECTIVE: To investigate mechanisms for the regulation of glucose incorporation into triacylgycerols in adipocytes by ceramides, which mediate some actions of tumour necrosis factor-alpha (TNFalpha). DESIGN: The effects of C(2)- and C(6)-ceramides (N-acetyl- and N-hexanoyl-sphingosines, respectively) on glucose uptake and incorporation into triacylglycerols and pathways of signal tansduction were measured in 3T3-L1 adipocytes. RESULTS: C(6)-ceramide increased basal 2-deooxyglucose uptake but decreased insulin-stimulated uptake without changing the EC(50) for insulin. Incubating 3T3-L1 adipocytes from 2 to 24 h with C(2)-ceramide progressively increased glucose incorporation into the fatty acid and especially the glycerol moieties of triacylglycerol. These effects were accompanied by increased GLUT1 synthesis resulting from ceramide-induced activation phosphatidylinositol 3-kinase, ribosomal S6 kinase and mitogen-activated protein kinase. C(2)-ceramide also increased p21-activated kinase and protein kinase B activities. However, C(2)-ceramide decreased the insulin-stimulated component of these signalling pathways and also glucose incorporation into triacylglycerol after 2 h. CONCLUSIONS: Cell-permeable ceramides can mimic some effects of TNFalpha in producing insulin resistance. However, ceramides also mediate long-term effects that enable 3T3 L1 adipocytes to take up glucose and store triacylglycerols in the absence of insulin. These observations help to explain part of the nature and consequence of TNFalpha-induced insulin resistance and the control of fat accumulation in adipocytes in insulin resistance and obesity.

Developmental changes in adipose and muscle lipoprotein lipase activity in the atherosclerosis-prone JCR:LA-corpulent rat.

OBJECTIVE: To characterize the developmental changes in adipose and muscle lipoprotein lipase (LPL) activity in the atherosclerosis-prone JCR:LA-corpulent rat, and to test the hypothesis that tissue-specific abnormalities in LPL activity precede the establishment of obesity. DESIGN: Lean (+/?) and obese cp/cp male JCR:LA rats were studied at 4, 5 and 8 weeks of age, that is at the onset of obesity, and at a time when obesity is well established. Assessment was made of plasma variables related to glucose and lipid metabolism and of LPL activity in several adipose depots, skeletal muscles and the heart. RESULTS: At week 4, body weights were identical in both genotypes and began to diverge at week 5. Eight-week-old cp/cp rats weighed 35% more than their lean counterparts. Perirenal and epididymal adipose depot weights were also identical in both genotypes at week 4 and began to increase in cp/cp rats at week 5, whereas the subcutaneous depot of 4-week-old cp/cp rats was slightly enlarged. At week 4, the cp/cp rats were hyperinsulinemic (5-fold), hyperleptinemic (30-fold) and hypertriglyceridemic (3-fold) compared to their lean counterparts, and their liver contained twice as much triglyceride. The 4-week-old cp/cp rats displayed 2-7-fold higher LPL specific activity in the various adipose depots compared to lean rats, and enzyme activity remained higher in obese than in lean rats at all subsequent ages. In contrast, LPL activity in the vastus lateralis, gastrocnemius and heart muscles of 4-week-old obese rats was approximately half that observed in lean animals. CONCLUSION: Profound, persistent alterations in the tissue-specific modulation of LPL activity are established in the JCR:LA cp/cp rat prior to the development of frank obesity. The increase in adipose tissue LPL activity and its decrease in muscle tissues are likely to be related to the concomitant alterations in insulinemia and triglyceridemia, respectively. The pre-obesity, tissue-specific alterations in LPL activity may be considered as an integrated adaptation to increased lipid flux aimed at driving lipids toward storage sites and limiting their uptake by triglyceride-laden muscles.

Co-ordinate regulation of growth factor receptors and lipid phosphate phosphatase-1 controls cell activation by exogenous lysophosphatidate.

The serum-derived lipid growth factors, lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P), activate cells selectively through different members of a family of endothelial differentiation gene (EDG) receptors. Activation of EDG receptors by LPA and S1P provides a variety of signalling cascades depending upon the G-protein coupling of the different EDG receptors. This leads to chemotactic and mitogenic responses, which are important in wound healing. For example, LPA stimulates fibroblast division and S1P stimulates the chemotaxis and division of endothelial cells leading to angiogenesis. Counteracting these effects of LPA and S1P, are the actions of lipid phosphate phosphatases (LPP, or phosphatidate phosphohydrolases, Type 2). The isoform LPP-1 is expressed in the plasma membrane with its active site outside the cell. This enzyme is responsible for 'ecto-phosphatase' activity leading to the degradation of exogenous lipid phosphate mediators, particularly LPA. Expression of LPP-1 decreases cell activation by exogenous LPA. The mechanism for this is controversial and several mechanisms have been proposed. Evidence will be presented that the LPPs cross-talk with EDG and other growth factor receptors, thus, regulating the responses of the cells to lipid phosphate mediators of signal transduction.

Tumor necrosis factor-alpha induces stress fiber formation through ceramide production: role of sphingosine kinase.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF-alpha-induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-alpha, sphingomyelinase, or C(2)-ceramide induced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, and stress fiber formation. Ly 294002, a phosphatidylinositol 3-kinase (PI 3-K) inhibitor, or expression of dominant/negative Ras (N17) completely blocked C(2)-ceramide- and sphingomyelinase-induced tyrosine phosphorylation of FAK and paxillin and severely decreased stress fiber formation. The TNF-alpha effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-alpha and C(2)-ceramide. TNF-alpha, sphingomyelinase, and C(2)-ceramide translocated Cdc42, Rac, and RhoA to membranes, and stimulated p21-activated protein kinase downstream of Ras-GTP, PI 3-K, and SK. Transfection with inactive RhoA inhibited the TNF-alpha- and C(2)-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-alpha, which increases sphingomyelinase activity and ceramide formation, activates sphingosine kinase, Rho family GTPases, focal adhesion kinase, and paxillin. This novel pathway of ceramide signaling can account for approximately 70% of TNF-alpha-induced stress fiber formation and cytoskeletal reorganization.

Ceramide inhibition of mammalian phospholipase D1 and D2 activities is antagonized by phosphatidylinositol 4,5-bisphosphate.

Ceramides inhibit phospholipase D (PLD) activity in several mammalian cell types. These effects have been related to preventing activation by ARF1, RhoA, and protein kinase C-alpha and -beta and therefore indicate that PLD1 is inhibited. In the present work, we investigated the effects of ceramides in inhibiting both PLD1 and PLD2 and the interaction with another activator, phosphatidylinositol 4,5-bisphosphate (PIP2). PLD1 and PLD2 were overexpressed separately in Sf9 insect cells using baculovirus vectors. In our cell-free system, PLD1 activity was inhibited completely by C2-ceramide at sub-optimum concentrations of PIP2 (3 and 6 microM), whereas at supra-optimum PIP2 concentrations (18 and 24 microM) C2-ceramide did not inhibit PLD1 activity. Partially purified PLD2 exhibited an absolute requirement for PIP2 when the activity was measured using Triton X-100 micelles. Ceramides inhibited PLD2 activity, and this inhibition was decreased as PIP2 concentrations increased. However, C2-ceramide also reversibly inhibited the activity of PLD1 and PLD2 mutants in which binding of PIP2 was decreased, indicating that ceramides are interacting with the catalytic core of the mammalian PLDs. By contrast, C2-ceramide failed to produce a significant inhibition of PLDs from bacteria and plants. Our results provide a novel demonstration that ceramides reversibly inhibit mammalian PLD2 as well as PLD1 activities and that both of these actions are more pronounced when PIP2 concentrations are rate-limiting.

Lipid phosphate phosphatase-1 dephosphorylates exogenous lysophosphatidate and thereby attenuates its effects on cell signalling.

The serum-derived phospholipid growth factor, lysophosphatidate (LPA), activates cells through a family of G-protein-coupled EDG receptors. The present article examines the role of lipid phosphate phosphatase-1 (LPP-1, or phosphatidate phosphate 2A) in regulating cell activation by LPA. Overexpressing LPP-1 approximately doubled the rate of dephosphorylation of exogenous LPA by Rat2 fibroblasts. The amount of LPA dephosphorylation was restricted to less than 10% of the total exogenous LPA. Over-expression of LPP-1 attenuated cell activation as indicated by diminished responses including cAMP, Ca2+, activation of phospholipase D and ERK, DNA synthesis and cell division. LPP-1 therefore provides a novel level of regulation for controlling cell signalling by exogenous LPA.

Platelet-released phospholipids link haemostasis and angiogenesis.

Considerable attention has focused on identifying mediators of neovascularization at sites of growth and abnormal tissue development. By contrast, mediators of angiogenesis at sites of injury and wound repair are not well defined but factors generated during blood coagulation (haemostasis) are attractive candidates. In addition to proteins generated, activated and released during the activation of clotting cascades, platelet-derived lipid mediators are now known to play a key role in many aspects of the angiogenic response. The first indication of lipid mediator involvement in angiogenesis was the discovery that lysophosphatidate (LPA), phosphatidic acid (PA) and sphingosine 1-phosphate (SPP) are high affinity agonists for G-protein coupled EDG (endothelial differentiation gene) receptors. The prototype for this family, EDG-1, was cloned from genes expressed when endothelial cells were activated to assume an angiogenic phenotype in vitro. The subsequent finding that SPP is a high affinity ligand for EDG-1 led Spiegel, Hla and associates (Lee et al., Science 1998;279:1552-1555) to hypothesize that platelet-released phospholipids play an important role in angiogenesis. These investigators and others demonstrated that SPP, LPA and phosphatidate (PA) induce many important endothelial cell responses associated with angiogenesis, including liberation of endothelial cells from established monolayers, chemotactic migration, proliferation, adherens junction assembly and morphogenesis into capillary-like structures. Although these studies indicated the potential involvement of platelet-derived phospholipids in angiogenesis, their physiological importance was not established. However, recent work demonstrates that >80% of the potent endothelial cell chemoattractive activity generated in human serum during clotting--an activity necessary for optimal angiogenesis--results from platelet-derived SPP. Other factors released from platelets during clotting, including LPA and PA, exert profound effects on endothelial cells that contribute unique aspects to the angiogenic response. These combined studies establish that SPP and other platelet-derived lipid mediators provide a novel link between haemostasis and angiogenesis.

Beneficial insulin-sensitizing and vascular effects of S15261 in the insulin-resistant JCR:LA-cp rat.

S15261, a compound developed for the oral treatment of type II diabetes, is cleaved by esterases to the fragments Y415 and S15511. The aim was to define the insulin-sensitizing effects of S15261, the cleavage products, and troglitazone and metformin in the JCR:LA-cp rat, an animal model of the obesity/insulin resistance syndrome that exhibits an associated vasculopathy and cardiovascular disease. Treatment of the animals from 8 to 12 weeks of age with S15261 or S15511 resulted in reductions in food intake and body weights, whereas Y415 had no effect. Troglitazone caused a small increase in food intake (P <.05). Treatment with S15261 or S15511 decreased plasma insulin levels in fed rats and prevented the postprandial peak in insulin levels in a meal tolerance test. Y415 had no effect on insulin levels. Troglitazone halved the insulin response to the test meal, but metformin gave no improvement. S15261 decreased the expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase and stimulated the expression of acetyl-CoA carboxylase and acyl-CoA synthase. S15261 also reduced the expression of carnitine palmitoyltransferase I and hydroxymethyl-glutaryl-CoA synthase. S15261, but not troglitazone, reduced the exaggerated contractile response of mesenteric resistance vessels to norepinephrine, and increased the maximal nitric oxide-mediated relaxation. S15261, through S15511, increased insulin sensitivity, decreased insulin levels, and reduced the vasculopathy of the JCR:LA-cp rat. S15261 may thus offer effective treatment for the insulin resistance syndrome and its associated vascular complications.

Sphingosine 1-phosphate released from platelets during clotting accounts for the potent endothelial cell chemotactic activity of blood serum and provides a novel link between hemostasis and angiogenesis.

Recent studies have identified factors responsible for angiogenesis within developing tumors, but mediators of vessel formation at sites of trauma, injury, and wound healing are not clearly established. Here we show that sphingosine 1-phosphate (S1P) released by platelets during blood clotting is a potent, specific, and selective endothelial cell chemoattractant that accounts for most of the strong endothelial cell chemotactic activity of blood serum, an activity that is markedly diminished in plasma. Preincubation of endothelial cells with pertussis toxin inhibited this effect of S1P, demonstrating the involvement of a Galphai-coupled receptor. After S1P-induced migration, endothelial cells proliferated avidly and differentiated forming multicellular structures suggestive of early blood vessel formation. S1P was strikingly effective in enhancing the ability of fibroblast growth factor to induce angiogenesis in the avascular mouse cornea. Our results show that blood coagulation initiates endothelial cell angiogenic responses through the release of S1P, a potent endothelial cell chemoattractant that exerts its effects by activating a receptor-dependent process.

The characterization of phospholipase D in FRTL-5 thyroid cells.

We previously demonstrated that TSH activates phospholipase D (PLD) in Fischer rat thyroid line (FRTL)-5 cells. To date, two types of mammalian phosphatidylcholine-specific PLD cDNAs, designated as PLD-1 and PLD-2, have been cloned. The present study determined the PLD isoform composition in FRTL-5 thyroid cells and which isoform is regulated by TSH. PLD-1 is activated by small molecular weight G-proteins, such as ADP-ribosylation factor (ARF) and RhoA family members, while PLD-2 is relatively independent of such stimuli. We established the presence of PLD-1 and PLD-2 by Western blot analysis and compared PLD activity in cytosol, membranes and combined fractions in the presence and absence of GTPgammaS. The membrane fraction showed very little activity in the absence of GTPgammaS, but this activity increased approximately 5-fold (P<0.05, ANOVA) in the presence of GTPgammaS. Maximal PLD activity was seen with the combination of membrane plus cytosolic fractions (which contained ARF and RhoA) where the addition of GTPgammaS increased PLD activity approximately 8-fold (P<0.05, ANOVA). To determine the relative activities of PLD-1 and PLD-2 in FRTL-5 thyroid cells, cell-free PLD assays were performed in the presence of GTPgammaS or GDPbetaS with varying concentrations of phosphatidylinositol 4,5-bisphosphate (PIP(2)). PLD-2 contributed only approximately 19% of the total amount of PLD activity in the membranes and PLD-1 was the predominant PLD isoform. TSH stimulated PLD-1 activity by up to 2. 3-fold over control values (P<0.01, ANOVA). To establish the dependence of PLD-1 on small molecular weight G-proteins, the translocations of ARF and RhoA to the membrane fractions was determined after stimulation by TSH. Both ARF and RhoA were maximally translocated to the membrane fraction after 10 min incubation with 100 microU/ml TSH by approximately 1.7- and 2.3-fold over control values, respectively (P<0.02 and P<0.03, ANOVA). It is concluded that TSH stimulates PLD-1 activity in FRTL-5 thyroid cells and this is accompanied by the translocation of ARF and RhoA to the membrane fraction.

Lipid phosphate phosphatase-1 and Ca2+ control lysophosphatidate signaling through EDG-2 receptors.

The serum-derived phospholipid growth factor, lysophosphatidate (LPA), activates cells through the EDG family of G protein-coupled receptors. The present study investigated mechanisms by which dephosphorylation of exogenous LPA by lipid phosphate phosphatase-1 (LPP-1) controls cell signaling. Overexpressing LPP-1 decreased the net specific cell association of LPA with Rat2 fibroblasts by approximately 50% at 37 degrees C when less than 10% of LPA was dephosphorylated. This attenuated cell activation as indicated by diminished responses, including cAMP, Ca(2+), activation of phospholipase D and ERK, DNA synthesis, and cell division. Conversely, decreasing LPP-1 expression increased net LPA association, ERK stimulation, and DNA synthesis. Whereas changing LPP-1 expression did not alter the apparent K(d) and B(max) for LPA binding at 4 degrees C, increasing Ca(2+) from 0 to 50 micrometer increased the K(d) from 40 to 900 nm. Decreasing extracellular Ca(2+) from 1.8 mm to 10 micrometer increased LPA binding by 20-fold, shifting the threshold for ERK activation to the nanomolar range. Hence the Ca(2+) dependence of the apparent K(d) values explains the long-standing discrepancy of why micromolar LPA is often needed to activate cells at physiological Ca(2+) levels. In addition, the work demonstrates that LPP-1 can regulate specific LPA association with cells without significantly depleting bulk LPA concentrations in the extracellular medium. This identifies a novel mechanism for controlling EDG-2 receptor activation.

Lipid phosphate phosphatase-1 in the regulation of lysophosphatidate signaling.

Mammalian lipid phosphate phosphatases (LPPs, or Type 2 phosphatidate phosphohydrolases) constitute a family of enzymes that belongs to a phosphatase superfamily. The LPPs dephosphorylate a variety of bioactive lipid phosphates including phosphatidate, lysophosphatidate, sphingosine 1-phosphate, and ceramide 1-phosphate. Mouse LPP-1 was stably expressed in rat2 fibroblasts to determine its structural and functional properties. Transduced cells showed increased dephosphorylation of exogenous lysophosphatidate. This result is compatible with mutational studies that show the active site of LPP-1 to be located on the external surface of the plasma membrane. Elevated LPP-1 activity attenuated the ability of lysophosphatidate to stimulate mitogen-activated protein kinase (ERK1 and 2) activities and DNA synthesis. It is concluded that one function of LPP-1 is to dephosphorylate exogenous lysophosphatidate, thereby attenuating cell signaling through endothelial cell differentiation gene (EDG) receptors.

Identification of structurally important domains of lipid phosphate phosphatase-1: implications for its sites of action.

Lipid phosphate phosphatase-1 (LPP-1) dephosphorylates exogenous lysophosphatidate and thereby regulates the activation of lysophosphatidate receptors and cell division. Mutation of seven amino acids in three conserved domains of mouse LPP-1 abolished its activity. A glycosylation site was demonstrated between conserved Domains 1 and 2. LPP-1 is expressed in the plasma membrane, and the present results demonstrate the active site to be located on the outer surface.