Antimicrobial photodynamic active biomaterials for periodontal regeneration.

OBJECTIVE: Biomaterials for periodontal regeneration may have insufficient mechanical and antimicrobial properties or are difficult to apply under clinical conditions. The aim of the present study was to develop a polymeric bone grafting material of suitable physical appearance and antimicrobial photodynamic activity. METHODS: Two light curable biomaterials based on urethane dimethacrylate (BioM1) and a tri-armed oligoester-urethane methacrylate (BioM2) that additionally contained a mixture of ß-tricalcium phosphate microparticles and 20wt% photosensitizer mTHPC (PS) were fabricated and analyzed by their compressive strength, flexural strength and modulus of elasticity. Cytotoxicity was observed by incubating eluates and in direct-contact to MC3T3-E1 cells. Antimicrobial activity was ascertained on Porphyromonas gingivalis and Enterococcus faecalis upon illumination with laser light (652nm, 1×100J/cm2, 2×100J/cm2). RESULTS: The compressive strength, flexural strength and elastic modulus were, respectively, 311.73MPa, 22.81MPa and 318.85MPa for BioM1+PS and 742.37MPa, 7.58MPa and 406.23MPa for BioM2+PS. Both materials did not show any cytotoxic behavior. Single laser-illumination (652nm) caused total suppression of P. gingivalis (BioM2+PS), while repeated irradiation reduced E. faecalis by 3.7 (BioM1+PS) and 3.1 (BioM2+PS) log-counts. SIGNIFICANCE: Both materials show excellent mechanical and cytocompatible properties. In addition, irradiation with 652nm induced significant bacterial suppression. The manufactured biomaterials might enable a more efficient cure of periodontal bone lesions. Due to the mechanical properties functional stability might be increased. Further, the materials are antimicrobial upon illumination with light that enables a trans-mucosal eradication of residual pathogens.

Positively charged calcium phosphate/polymer nanoparticles for photodynamic therapy.

The charge of nanoparticles influences their ability to pass through the cellular membrane, and a positive charge should be beneficial. The negative charge of calcium phosphate nanoparticles with an inner shell of carboxymethyl cellulose (CMC) was reversed by adding an outer shell of poly(ethyleneimine) (PEI) into which the photoactive dye 5,10,15,20-tetrakis(3-hydroxyphenyl)-porphyrin (mTHPP) was loaded. The aqueous dispersion of the nanoparticles was used for photodynamic therapy with HT29 cells (human colon adenocarcinoma cells), HIG-82 cells (rabbit synoviocytes), and J774A.1 cells (murine macrophages). A high photodynamic activity (killing) together with a very low dark toxicity was observed for HIG-82 and for J774.1 cells at 2 microM dye concentration. The killing efficiency was equivalent to the pure photoactive dye that, however, needs to be administered in alcoholic solution.

Calcium phosphate nanoparticles as efficient carriers for photodynamic therapy against cells and bacteria.

Calcium phosphate nanoparticles were surface-functionalized with different polymers, and photosensitizers were incorporated into this layer. The charge was adjusted by choosing the appropriate polymer. Methylene blue and 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP) were used as photosensitizers. The particles showed a good performance with HIG-82 synoviocytes. For J774A.1 macrophages, they were toxic also in the dark, probably due to a lethal uptake of calcium. For HT29 epithelial cells, a moderate activity was observed. A good photoxicity was observed against the bacterial strain Staphylococcus aureus (Gram-positive), both with positively and negatively charged nanoparticles loaded with mTHPP. Against Pseudomonas aeruginosa (Gram-negative), good photoxicity was observed only with positively charged nanoparticles loaded with mTHPP. At higher concentrations, methylene blue-loaded nanoparticles were active against S. aureus. Thus, it is possible to prepare a water-dispersable system of dye-loaded calcium phosphate nanoparticles, but the efficiency depends on a number of parameters, e.g. particle charge, kind of polymer, and cell culture medium (e.g. the presence of proteins).

Improved photoinactivation of gram-negative and gram-positive methicillin-resistant bacterial strains using a new near-infrared absorbing meso-tetrahydroporphyrin: a comparative study with a chlorine e6 photosensitizer photolon.

The antimicrobial activity of new tetracationic and water-soluble meso-substituted tetrahydroporphyrin tetratosylat (BL1065) and of dicationic water-soluble chlorine e6 Photolon (BLC1013) is described. The dark toxicity and photosensitizing potentials of both photosensitizers were tested on Gram-positive (Staphylococcus aureus and MRSA) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria in phosphate-buffered saline (PBS, pH 7.4), PBS + horse serum (HS) and PBS + sheep blood (SB). The results show that BLC1065 and BLC1013 did not inhibit the growth of S. aureus in the dark, but efficiently inactivated this Gram-positive bacterium after illumination. Contrary to BLC1013, BLC1065 has a photodynamic activity toward Gram-negative bacteria as well, at least in PBS. Results suggest that tetracationic BLC1065 can bind better to both Gram-positive and Gram-negative bacterial cell envelope than the dianionic chlorine BLC1013 resulting in better efficiency of photoinactivation.

Induction of apoptosis in HaCaT cells by photodynamic therapy with chlorin e6 or pheophorbide a.

The two photosensitizers, chlorin e6 and pheophorbide a, were tested in an in vitro model of topical photodynamic therapy (PDT). Both dyes accumulate in HaCaT keratinocytes as verified by fluorescence measurement but pheophorbide a is enriched fivefold more strongly than chlorin e6 after 24 h. HaCaT cells are susceptible to PDT with both dyes. The phototoxicity measured by ATP bioluminescence is caused by necrosis and apoptosis depending on the photosensitizer used and the treatment modality. Chlorin e6 shows higher toxic potential because it elicits nearly 90% cell mortality 24 h after PDT comparable to pheophorbide a but with a fivefold lower rate of accumulation. These results implicate caution with topical PDT of oncologic diseases due to the risk of serious side effects on healthy skin in the course of topical photodynamic treatment. But the lack of dark toxicity and the time-dependent enrichment of both dyes in HaCaT cells are arguments for the application of these sensitizers in topical PDT of non-malign skin disorders. Further studies are necessary to discover appropriate lower doses and mechanisms of action of topical PDT with both compounds.

Expedited discovery of second generation NK-1 antagonists: identification of a nonbasic aryloxy substituent.

Solution-phase, parallel-synthesis techniques were used to optimize a series of nonbasic NK-1 antagonists, resulting in the identification of (R)-26, an orally bioavailable compound with subnanomolar potency.

Dose-response study of the analgesic effect of lanepitant in patients with painful diabetic neuropathy.

Lanepitant is effective in the formalin analgesic model suggesting efficacy in painful neuropathy. This study was designed to evaluate the dose-response effect of lanepitant in patients with daily moderate to severe, bilateral, distal neuropathic pain. After a 1-to 3-week lead-in period, patients were randomly allocated to double-blind, parallel treatment with lanepitant 50 mg daily (n = 27), 100 mg daily (n = 27), 200 mg twice daily (n = 13), or placebo (n = 26) over 8 weeks. Patients reported average daytime pain and average nighttime pain intensity. Plasma concentrations and amount of adjunctive analgesic medication were obtained at all visits after baseline. Patient global evaluation and clinician global impression were obtained at weeks 3 and 8. Safety was assessed by adverse events, vital signs, laboratory analytes, and electrocardiogram. No dosage of lanepitant differed significantly from placebo. Efficacy did not increase with lanepitant dosage, and higher plasma concentrations were no more effective than lower plasma concentrations. The adverse event diarrhea was more frequent for lanepitant-treated patients. Although well tolerated, lanepitant was ineffective in relieving pain of diabetic neuropathy.

Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain.

Converging lines of evidence implicate the beta-amyloid peptide (Ass) as causative in Alzheimer's disease. We describe a novel class of compounds that reduce A beta production by functionally inhibiting gamma-secretase, the activity responsible for the carboxy-terminal cleavage required for A beta production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon A beta production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, to mice transgenic for human APP(V717F) reduces brain levels of Ass in a dose-dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain A beta in vivo. Development of such novel functional gamma-secretase inhibitors will enable a clinical examination of the A beta hypothesis that Ass peptide drives the neuropathology observed in Alzheimer's disease.

Regulation of amyloid precursor protein processing by Abeta in human glioma cells.

Amyloid precursor protein (APP) is cleaved to neurotoxic/proinflammatory amyloid beta protein (Abeta) or to the neuroprotective secreted alpha-APPs. A balance in APP metabolism may influence the outcome between toxicity and protection to central nervous system (CNS) neurons in Alzheimer's disease. Treatment of U-373 MG astrocytoma cells with aggregated Abeta (1-40) decreases APP secretion into the medium to 10-30% of control values. This decreased secretion appears to be specific for APP since Abeta treatment causes an approximately 2-fold increase in interleukin-8 (IL-8) secretion. Abeta treatment also causes a 4- to 9-fold increase in total cell-associated APP. This increase is due to cellular retention of alpha secretase-cleaved APP and a 2-fold increase in mature full-length APP. These data suggest that deposition of aggregated Abeta may contribute to Alzheimer's-associated neurotoxicity by altering the metabolism of the APP protein. Abeta may exert harmful effects by decreasing the secretion of neuroprotective or neurotrophic APP and, in addition, by increasing intracellular full-length APP; thereby providing increased substrate for generation of amyloidogenic peptide within astrocytes.

Study of the analgesic effect of lanepitant in patients with osteoarthritis pain.

OBJECTIVE: Lanepitant selectively blocks substance P binding to the neurokinin-1 receptor, preventing neurogenic inflammation and pain transmission. Substance P is present in synovial fluid and in excess in cerebral spinal fluid. We investigated the effect of lanepitant on pain caused by osteoarthritis to evaluate the role of neurokinin-1 blockade. METHODS: Outpatients (n = 214) with moderate to severe lower-limb osteoarthritis pain were treated for 3 weeks in a parallel, randomized double-blind study with initial doses of 20, 60, 200, or 600 mg lanepitant, 375 mg naproxen, or placebo, followed by 10, 30, 100, or 300 mg lanepitant twice a day, 375 mg naproxen twice a day, or placebo twice a day in the multiple-dose period. Pain intensity, pain relief, patient global impression, and adjunctive analgesic use were compared across treatments. Safety was evaluated with adverse events, vital signs, and laboratory assessments. RESULTS: There was no statistically significant difference in efficacy or safety across treatments for the initial dose assessment. After 1 week of therapy, naproxen was statistically significantly (P < .05) better than placebo and lanepitant in reducing average pain. During the second and third weeks of therapy, patients receiving naproxen continued to have statistically significantly (P < .05) less pain than those receiving placebo or lanepitant despite using significantly less adjunctive analgesic medication. There were no statistically significant differences in rates of discontinuation across treatments. Lanepitant treatment was associated with diarrhea, whereas naproxen treatment was associated with gastric discomfort. There were no clinically relevant changes in vital signs or laboratory analytes for any of the treatments. CONCLUSION: Lanepitant was ineffective in relieving osteoarthritis pain, possibly because neurokinin-1 binding of substance P does not play a significant role in osteoarthritis pain or because lanepitant fails to adequately penetrate the blood-brain barrier to affect central pain perception.

Human amylin stimulates inflammatory cytokine secretion from human glioma cells.

Chronic neurodegeneration in the brains of Alzheimer's disease (AD) patients may be mediated, at least in part, by the ability of amyloid beta (Abeta) to exacerbate inflammatory pathways in a conformation-dependent manner. In this regard, we previously reported that the Abeta-peptide-mediated potentiation of inflammatory cytokine secretion from interleukin-1beta (IL-1beta)-stimulated human astrocytoma cells was conformation dependent. Other amyloidogenic peptides, such as human amylin, which display similar conformation-dependent neurotoxic effects, may also elicit inflammatory cytokine secretion from glial cells. To test this hypothesis, we compared human and rat amylin for the effects on cytokine production in U-373 MG human astrocytoma cells. Human amylin alone stimulated U-373 MG cells to secrete IL-6 and IL-8 in a concentration-dependent manner with maximum effects seen at 10-25 microM peptide. In addition, human amylin markedly potentiated IL-1beta-stimulated cytokine production with a similar concentration dependence. In contrast, nonamyloidogenic rat amylin modestly stimulated cytokine secretion, either alone or combined with IL-1beta. Aging human amylin resulted in diminished cytokine secretion, probably due to the formation of large, less active aggregates. In agreement with our previous studies using Abeta, extracellular Ca(2+) was necessary for human amylin stimulation of cytokine secretion. Our data suggest that amyloidogenic peptides promote cytokine secretion through similar beta-sheeted secondary-structure- and extracellular-Ca(2+)-dependent mechanisms.

Substance P regulates PTH secretion through the neurokinin-1 receptor.

The primary regulator of PTH secretion is serum ionized Ca(2+); however, neuropeptide-containing nerve fibers have been localized to the parathyroid gland. The purpose of this study was to determine whether or not substance P (SP) regulates PTH secretion. In dispersed porcine parathyroid cells, SP reversibly inhibited 0.5 mM CaCl(2)-induced PTH secretion (IC(50) = 0.29 nM) and had no effect at CaCl(2) concentrations of 1.5 mM and greater. At 0.5 mM CaCl(2), treatment with a NK-1 selective receptor agonist resulted in a concentration-dependent decrease in PTH secretion (IC(50) = 0.21 nM). In contrast, NK-2 and NK-3 receptor agonists were approximately 100-fold less active than SP or the NK-1 receptor selective agonist. An enantiospecific reversal of the effects of SP on PTH secretion was observed with LY306740, a potent selective NK-1 receptor antagonist (K(i) = 0.125 nM). In porcine parathyroid cells, expression of mRNA for the NK-1 receptor was observed using RT-PCR. In summary, a novel neuroendocrine pathway is described whereby the neuropeptide, SP, regulates PTH secretion through NK-1 receptors.

Regulation of cytokine secretion and amyloid precursor protein processing by proinflammatory amyloid beta (A beta).

Neurodegenerative processes in Alzheimer's disease (AD) are thought to be driven, in part, by the deposition of amyloid beta (A beta), a 39-43-aminoacid peptide product resulting from an alternative cleavage of amyloid precursor protein (APP). In addition to its neurotoxic properties, A beta may influence neuropathology by stimulating glial cell cytokine and acute phase protein secretion in affected areas of the brain (e.g., cortex, hippocampus). Using an in vitro human astrocyte model (U-373 MG astrocytoma cells), the effects of A beta treatment on acute phase protein (APP and alpha-1-antichymotrypsin [alpha 1-ACT]) and interleukin-8 (IL-8) were examined. U-373 MG cells secreted increased levels of alpha 1-ACT and neurotrophic/neuroprotective alpha-cleaved APP (alpha APP) after exposure to interleukin-1 beta (IL-1 beta) for 24 hours. A beta treatment resulted in a similar, but modest increase in alpha 1-ACT secretion, a two- to threefold stimulation of IL-8 production, and, conversely, a profound reduction in the levels of secreted alpha APPs. A beta inhibited alpha APP secretion by U-373 MG cells in a concentration- and conformation-dependent manner. Moreover, the reduction in alpha APP secretion was accompanied by an increase in cell-associated APP. Another proinflammatory amyloidogenic peptide, human amylin, similarly affected APP processing in U-373 astrocytoma cells. These data suggest that A beta may contribute to Alzheimer's-associated neuropathology by lowering the production of neuroprotective/neurotrophic alpha APPs. Moreover, the concomitant increase in cell-associated APP may provide increased substrate for the generation of amyloidogenic peptides within astrocytes.

Structure-activity relationships of a series of 1-substituted-4-methylbenzimidazole neuropeptide Y-1 receptor antagonists.

The characterization of a novel series of NPY-1 receptor antagonists derived from the 4-methylbenzimidazole 4 is described. Appropriate substitution on the piperidyl nitrogen of 4 led to systematic increases in Y-1 receptor affinity, to approximately 50-fold, and to the discovery of the importance of a second basic substituent.

Influence of phospholipid composition on excretion of beta-lactamase from a stringent/relaxed Escherichia coli K 12 strain pair.

In comparison to stringent (relA+) controlled Escherichia coli cells, relaxed (relA) controlled E. coli cells excreted more recombinant beta-lactamase into the culture medium. The analysis of the composition of phospholipid fractions of the cells yielded increased levels of phosphatidylserine in relaxed controlled cells. We added various phospholipid vesicles to growing cells in order to influence the excretion rate via their incorporation in the membranes. The addition of vesicles to phosphatidic acid, phosphatidylglycerol and phosphatidylserine reduced the excretion of beta-lactamase whereas vesicles of phosphatidylethanolamine and phosphatidylcholine decreased or increased the excretion of beta-lactamase in dependence on the individual fatty acid residues of the added phospholipids. The lower the degree of saturation of the added phospholipids the more permeable was the cell envelope for beta-lactamase.

Influence of relA locus on electrophoretic mobility of a stringent/relaxed Escherichia coli K 12 strain pair.

Stringent (relA+) and relaxed (rel A-) controlled Escherichia coli cells differ in their regulation of many biochemical pathways such as phospholipid and lipopolysaccharide metabolism (LPS) after amino acid limitation. Because such differences could result in various cell envelopes, cells of stringent controlled E. coli strain CP78 (relA+) and relaxed controlled E. coli strain CP79 (relA) were studied regarding their electrophoretic mobility. The graphs of the mobility distributions of both strains were different: cells of strain CP79 caused secondary peaks in addition to the main peaks whereas the mobility distributions of cells of strain CP78 showed only one maximum. In the pH range from 6.0 to 8.0 the location of the main peaks of cells of strain CP79 were changed to less negative values after induction of relaxed response. In contrast to this the stringent response in strain CP78 caused no change of the mobility distributions.

[3H]LY303870, a novel nonpeptide radioligand for the NK-1 receptor.

We synthesized a potent and selective antagonist radioligand for the neurokinin (NK)-1 receptor and characterized its binding to guinea pig striatal membranes. (R)-N-[2-[Acetyl[3H3][(2-methoxyphenyl)-methyl]amino]- 1-(1H-indol-3-ylmethyl) ethyl][1,4'-bipiperidine]- 1'-acetamide ([3H]LY303870) binds to a single class of sites with an equilibrium KD of 0.22 nM and a Bmax of 723 fmol/mg of protein. Unlabeled LY303870 potently inhibited the binding with an IC50 of 0.56 nM, whereas the less active (S)-enantiomer (LY306155) was substantially less potent. The nonpeptide NK-1 antagonists (+/-)-CP96,345 and (+/-)-RP 67580 had IC50 values of 0.74 and 49 nM, respectively. Substance P (SP) was also a potent inhibitor with with an IC50 of 3.1 nM. The inhibition by SP could be separated into two components: a high-affinity component with a Ki of 0.53 nM and a lower-affinity component with a Ki of 155 nM. Addition of 100 microM guanylyl 5'-imidodiphosphate [Gpp(NH)p] in the incubation increased the relative amount of the low-affinity agonist state of the receptor. Consistent with the antagonist properties of LY303870, the dissociation rate of [3H]-LY303870 was not changed by the presence of 100 microM Gpp(NH)p. The distribution of [3H]LY303870 binding sites in the guinea pig brain closely matched the distribution of NK-1 receptors labeled by [3H]SP. Therefore, [3H]LY303870 is a potent and selective antagonist radioligand for NK-1 receptors in guinea pig brain. In addition, regulation of NK-1 agonist affinity by guanine nucleotides is similar to that seen for monoaminergic receptors.

3-Aryl-1,2-diacetamidopropane derivatives as novel and potent NK-1 receptor antagonists.

Early structure-activity studies on racemic tryptophan ester and amide NK-1 antagonists 5-7 led to the discovery that the potency of the series could be markedly increased by moving the carbonyl function in these molecules to an off-chain position as in the 3-aryl-1,2-diacetamidopropane 9. Further medicinal chemistry incorporating this change resulted in the discovery of a novel series of highly potent aryl amino acid derived NK-1 antagonists of the R stereoisomeric series (IC50's = 100 pM to > 5 microM). Compounds in this series were shown to be competitive antagonists using an in vitro NK-1 smooth muscle assay, and this data correlated well with observed human NK-1 binding affinities. Two of these agents, (R)-25 and (R)-32, blocked intrathecal NK-1 agonist-driven [Ac-[Arg6,Sar9,Met(O2)11]- substance P 6-11 (Ac-Sar9)] nociceptive behavior in mice. Both compounds potently blocked the neurogenic dural inflammation following trigeminal ganglion stimulation in the guinea pig after intravenous administration. Further, upon oral administration in this model, (R)-32 was observed to be very potent (ID50 = 91 ng/kg) and have a long duration of action (> 8 h at 1 micrograms/kg). Compound (R)-32, designated LY303870, is currently under clinical development as an NK-1 antagonist with a long duration of action.

Differential tachykinin receptor subtype activation in capsaicin and KCl contractions of guinea pig trachealis.

We assessed the role of endogenously secreted tachykinins in mediating contraction caused by potassium chloride (KCl) in guinea pig tracheal smooth muscle (TSM) strips in vitro. Maximal isometric contraction was elicited with approximately 45 mM KCl and was 196 +/- 8% of the response to electrical field stimulation (% EFS) in the same tissues. Muscarinic receptor blockade with atropine modestly attenuated this contraction caused by KCl to 175 +/- 9 %EFS (P < 0.05), and treatment with a selective neurokinin subtype 1 (NK1) receptor antagonist, LY-297911, caused even greater inhibition of KCl-elicited contraction to 124 +/- 8 %EFS (P < 0.001). By contrast, SR-48968, a selective NK2 antagonist, had no effect on contraction caused by KCl (183 +/- 9 %EFS; P = NS vs. KCl alone). However, given together at the same concentration, SR-48968 augmented the inhibition of contraction caused by LY-297911 to 93 +/- 15 %EFS (P < 0.05 vs. LY-297911 alone). In contrast to the effect on KCl-induced contraction, LY-297911 caused only moderate inhibition of the contraction caused by capsaicin to 81 +/- 13 %EFS (P < 0.05 vs. control, 114 +/- 15 %EFS), whereas SR-48968 caused substantial attenuation of contraction caused by capsaicin to 23 +/- 5 %EFS (P < 0.005 vs. LY-297911). We demonstrate that a significant portion of the contraction caused by KCl, in addition to capsaicin, is elicited in guinea pig TSM through neurokinin secretion. However, NK1 receptors predominantly mediate contraction caused by KCl, and NK2 receptors predominantly mediate contraction elicited by capsaicin in guinea pig airway smooth muscle.