Efficacy and Safety of AFN-1252, the First Staphylococcus-Specific Antibacterial Agent, in the Treatment of Acute Bacterial Skin and Skin Structure Infections, Including Those in Patients with Significant Comorbidities.

This open-label noncontrolled, phase II multicenter trial was designed to evaluate the safety, tolerability, and efficacy of 200 mg of AFN-1252, a selective inhibitor of Staphylococcus aureus enoyl-acyl carrier protein reductase (FabI), given by mouth twice daily in the treatment of acute bacterial skin and skin structure infections (ABSSSI) due to staphylococci. Important aspects of the current study included a comparison of early response efficacy endpoints with end-of-treatment and follow-up endpoints. Many patients in the intent-to-treat population (n = 103) had significant comorbidities. The overall early response rate at day 3 was 97.3% (wound, 100%; abscess, 96.6%; cellulitis, 94.4%) in the microbiologically evaluable (ME) population. Within the ME population, 82.9% of patients had a ≥ 20% decrease in the area of erythema, and 77.9% of patients had a ≥ 20% decrease in the area of induration, on day 3. S. aureus was detected in 97.7% of patients (n = 37 patients with methicillin-resistant S. aureus [MRSA], and n = 39 with methicillin-sensitive S. aureus [MSSA]). No isolates had increased AFN-1252 MICs posttreatment. Microbiologic eradication rates for S. aureus were 93.2% at short-term follow-up (STFU) and 91.9% at long-term follow-up (LTFU) in the ME population. Eradication rates for MRSA and MSSA were 91.9% and 92.3%, respectively, at STFU and 91.9% and 89.7%, respectively, at LTFU. The most frequently reported drug-related adverse events, which were mostly mild or moderate, were headache (26.2%) and nausea (21.4%). These studies demonstrate that AFN-1252 is generally well tolerated and effective in the treatment of ABSSSI due to S. aureus, including MRSA. (This study has been registered at ClinicalTrials.gov under registration no. NCT01519492.).

Effects of an acetylcholinesterase inhibitor and an N-methyl-D-aspartate receptor antagonist on inflammation and degeneration of the nucleus pulposus.

OBJECTIVE: The study aimed to examine the effects of two drugs, an acetylcholinesterase inhibitor (AChEI) and an N-methyl-D-aspartate receptor (NMDAR) antagonist, on degenerated annulus fibrosus (AF) and nucleus pulposus (NP) cells and the extracellular matrix (ECM) structure in vitro. PATIENTS AND METHODS: Tissue samples were obtained from patients with intervertebral disc herniation (four males and four females; classified as Pfirmann stage IV) and used to prepare cell cultures. Untreated cell culture samples served as the control group. Study group samples were treated with donepezil, memantine or a combination of the two drugs. Cell viability, toxicity and proliferation were evaluated in all groups. Western blotting was used to examine changes in protein expression of signal transducer and activator of transcription 3 (STAT3), phospho-STAT3 (ser727), hypoxia-inducible factor (HIF)-1 alpha (HIF-1α) and nucleotide-binding oligomerisation domain (NOD) leucine-rich repeat (LRR)-containing proteins (NLR) family pyrin domain containing 3 (NLRP3) inflammasome. The alpha significance value was < 0.05. RESULTS: Analysis of the microscopy and commercial kit results revealed that cell proliferation was suppressed, and no cell death was observed. The protein expression levels of NLRP3, STAT3, ser727 and HIF-1α were lower in the samples treated with donepezil and memantine at 72 h (p < 0.05). The protein expression levels of NLRP3, STAT3, ser727 and HIF-1α were higher in the samples treated with the combination of donepezil and memantine (p < 0.05). CONCLUSIONS: The combined administration of memantine a NMDAR antagonist which can prevent neurodegeneration and donepezil an AChEI used for pain relief increased the protein expression levels in the anabolic pathway. However, it did not reduce the protein expression levels in the catabolic pathway. Therefore, further studies are needed to provide extensive insight into whether it may be among the potential targets for the therapy of intervertebral disc (IVD) diseases.

Can transcription factors in the intervertebral disc of lopinavir/ritonavir prevent degeneration in the nucleus pulposus by mediating the regulation of inflammation through signaling pathways?

OBJECTIVE: This study was conducted to examine whether lopinavir/ritonavir (Lop/r), an HIV protease inhibitor, can improve disc physiology and slow down intervertebral disc (IVD) degeneration through in vitro experimental methods, as well as whether it can suppress inflammation with interleukin-1 beta (IL-1ß) and sex-determining region Y (SRY) protein-related high-mobility group box genes-9 (SOX9) through hypoxia-inducible factor 1-alpha (HIF-1α) and the nuclear factor kappa B (NF-κB) signaling pathway. The aim was to investigate whether Lop/r application is toxic to IVD cells and the microenvironment simultaneously. PATIENTS AND METHODS: Human primary cell cultures were prepared using herniated IVD tissues obtained from patients with lumbar disc hernia who were unresponsive to conservative and medical treatment, and thereby, were operated on. The untreated culture samples served as control group, and the samples treated with Lop/r served as study group. Microscopic evaluations were performed simultaneously using fluorescent and supravital dyes in all groups. In addition to cell viability, toxicity, and proliferation analysis through a commercial kit, IL-1ß, SOX9, HIF-1α, and NF-κB protein expressions were evaluated using Western blotting. In the statistical comparison of the obtained data, an alpha value less than 0.05 was considered significant. RESULTS: Cell proliferation decreased in the Lop/r group, but no cell death was observed (p < 0.05). Moreover, at the end of 72 hours after Lop/r application, IL-1ß and NF-kB protein expressions decreased by 40% and 52%, respectively, while HIF-1α and SOX9 protein expressions increased by 4% and 59%, respectively (p< 0.05). CONCLUSIONS: Although these data were obtained from an in vitro experimental study, it is believed that these findings could make significant contributions to the pharmaco-regenerative treatment modalities of IVD degeneration. Lop/r suppresses the IL-1ß and NF-κB and induces SOX9 and HIF-1α, since these signaling pathways may be related to human IVD degeneration.

In-vitro activity of tigecycline against multidrug-resistant Gram negative bacteria: The experience of a university hospital.

The emergence of multidrug-resistant Gram negative bacteria has given rise to significant therapeutic challenges. These pathogens may have developed resistance to tigecycline, which is an alternative antibiotic used empirically in the treatment of serious infections. The objectives of this study were to identify the in-vitro activity of tigecycline against multidrug-resistant Gram negative strains isolated from clinical specimens and their related genes, at a university hospital. For this, 150 clinical isolates of multidrug-resistant Gram negative cultures from various clinical specimens were collected. Bacterial isolates were cultured, identified and their antibiotic susceptibilities were determined. Polymerase chain reaction was performed to amplify AcrB, AmpC, RamR, MexR, AdeB, TetA genes. Results revealed that all isolates were multidrug-resistant. The resistance of isolates was 91.4% to aztreonam, 94.6% to piperacillin, 34% to imipenem, 38.7% to meropenem, 71.3% to levofloxacin, 97.3% to ceftriaxone, 94.7% to cefepime, 9.3% to colistin, 78% to tetracycline, 21.4% to tigecycline and 68% to trimethoprim. AcrB, AmpC, RamR, MexR, AdeB, TetA genes were present in multidrug-resistant Gram negative bacteria. AcrB, RamR, TetA genes were related to tigecycline resistance. It is concluded that infections caused by multidrug-resistant Gram negative bacteria occur at a high rate. Most isolates were multi drug resistant, with 21.4% being resistant to tigecycline.

LIPSS-based functional surfaces produced by multi-beam nanostructuring with 2601 beams and real-time thermal processes measurement.

A unique combination of the ultrashort high-energy pulsed laser system with exceptional beam quality and a novel Diffractive Optical Element (DOE) enables simultaneous production of 2601 spots organized in the square-shaped 1 × 1 mm matrix in less than 0.01 ms. By adjusting the laser and processing parameters each spot can contain Laser Induced Periodic Surface Structures (LIPSS, ripples), including high-spatial frequency LIPSS (HFSL) and low-spatial frequency LIPSS (LSFL). DOE placed before galvanometric scanner allows easy integration and stitching of the pattern over larger areas. In addition, the LIPSS formation was monitored for the first time using fast infrared radiometry for verification of real-time quality control possibilities. During the LIPSS fabrication, solidification plateaus were observed after each laser pulse, which enables process control by monitoring heat accumulation or plateau length using a new signal derivation approach. Analysis of solidification plateaus after each laser pulse enabled dynamic calibration of the measurement. Heat accumulation temperatures from 200 to 1000 °C were observed from measurement and compared to the theoretical model. The temperature measurements revealed interesting changes in the physics of the laser ablation process. Moreover, the highest throughput on the area of 40 × 40 mm reached 1910 cm2/min, which is the highest demonstrated throughput of LIPSS nanostructuring, to the best of our knowledge. Thus, showing great potential for the efficient production of LIPSS-based functional surfaces which can be used to improve surface mechanical, biological or optical properties.

Physiological concentrations of lactate dehydrogenases and substrate inhibition.

Lactate dehydrogenases at physiological concentrations are inhibited by high concentrations of pyruvate when the enzyme and the pyruvate are incubated in the presence of oxidized nicotinamide-adenine dinucleotide before assay. The inhibition is much more pronounced with the H-type than with the M-type lactate dehydrogenase. These results suggest that substrate inhibition may be operative in vivo.

D-lactate specific pyridine nucleotide lactate dehydrogenase in animals.

A survey of representative invertebrates has revealed the presence of pyridine nucleotide-linked (D)-lactate dehydrogenase in a number of groups. All species studied contained either D(D)-or (L)L-lactate dehydrogenase, but no species contained both enzymes. The (D)-lactate dehydrogenase from Limulus polyphemus has been purified and has a molecular weight of 65,000.

3-acetylpyridine: effects in vitro related to teratogenic activity in chicken embryos.

Production of skeletal muscle hypoplasia by 3-acetylpyridine and its complete reversal by nicotinamide in developing chicken embryos have been confirmed. Cultures of developing embryonic chicken muscle show degenerative effects produced by 3-acetylpyridine; these effects are reversed by nicotinamide. Cartilage production in cultured chondrogenic cells is potentiated by 3-acetylpyridine; this potentiation is completely reversed by nicotinamide. It is suggested that nicotinamide-or pyridine-nucleotide-dependent reactions influence normal differentiation of limb mesoderm cells by inhibiting chondrogenic-cell and potentiating muscle-cell expression or proliferation.

Enzymatic identification of fish products.

Comparative enzymological techniques were used to distinguish between the muscle lactate dehydrogenases of 26 fish species. Intergeneric differences in enzymatic properties were frequently encountered. The techniques revealed, in addition, that some commercial samples of frozen fish fillets, labeled "haddock," contained cod lactate dehydrogenase.

Implication of nonlinear kinetics on risk estimation in carcinogenesis.

Efforts in estimating carcinogenic risk in humans from long-term exposure to chemical carcinogens have centered on the problem of low-dose extrapolation. For chemicals with metabolites that interact with DNA, it may be more meaningful to relate tumor response to the concentration of the DNA adducts in the target organ rather than to the applied dose. Many data suggest that the relation between tumor response and concentration of DNA adducts in the target organ may be linear. This implies that the nonlinearities of the dose-response curve for tumor induction may be due to the kinetic processes involved in the formation of carcinogen metabolite--DNA adducts. Of particular importance is the possibility that the kinetic processes may show a nonlinear "hockey-stick" like behavior which results from saturation of detoxification or DNA repair processes. The mathematical models typically used for low-dose extrapolation are shown potentially to overestimate risk by several orders of magnitude when nonlinear kinetics are present.

The steroid content of adrenal adenomas and measurements of aldosterone production in patients with essential hypertension and primary aldosteronism.

The content of aldosterone, corticosterone, and cortisol has been assayed in normal adrenal tissue obtained at autopsy and in adrenal adenomas from both normotensive and hypertensive patients obtained at autopsy and from patients with primary aldosteronism obtained at surgery. The content of aldosterone and corticosterone in the adenomas of patients with essential hypertension was similar to that of normal adrenal tissue and much less than that of adenomas from patients with primary aldosteronism. Since these results do not appear to reflect various extraneous factors such as assay technique, degradation of steroids, and patient selection, they suggest that most adrenal adenomas found in patients with essential hypertension are not associated with increased aldosterone production. The presence of such adenomas should not, in itself, be considered evidence for primary aldosteronism. The excretion or secretion of aldosterone in 43 hypertensive patients was found to be similar to that of 39 normotensive patients. These results suggest that primary aldosteronism is rarely the cause of "essential" hypertension.

Adrenocortical steroidogenesis: studies on the mechanism of action of angiotensin and electrolytes.

The effects of adrenocorticotropin (ACTH), angiotensins I and II, increased potassium, and decreased sodium concentrations upon steroid synthesis were examined by incubation of beef adrenal tissue slices. Angiotensin II shared with ACTH the need for calcium and an inhibition of its effect in the presence of puromycin but differed in not stimulating cyclic adenosine monophosphate (AMP). Angiotensin I was effective in steroidogenesis. The stimulation of aldosterone synthesis by increased potassium concentration was accompanied by an increased level of cyclic AMP and was inhibited in the presence of puromycin. Decreased sodium concentration stimulated aldosterone synthesis but, alone of these stimuli, simultaneously decreased corticosterone levels. It therefore appears that ACTH and potassium stimulate steroidogenesis at an early step in the biosynthetic pathway through the activation of cyclic AMP, whereas the effect of angiotensins I and II involve another mechanism and decreased sodium concentration affects a later step in steroidogenesis.

Adrenocortical steroidogenesis: the effects of prostaglandins.

Prostaglandins E(1) and E(2) significantly stimulated the synthesis of aldosterone, corticosterone, and to a lesser degree, cortisol in the outer slices of beef adrenal tissue. PGA, PGF(1a), and PGF(2a) were ineffective.PGE(1) was found to stimulate steroidogenesis in a manner similar to that of adrenocorticotropin (ACTH) in (a) needing calcium, (b) being inhibited by puromycin but not actinomycin D, (c) increasing the levels of cyclic AMP, and (d) not having an additive effect to exogenous cyclic AMP. PGE(1) did not produce an additive effect with either submaximal or maximal amounts of ACTH but did have an additive effect with angiotensin. These results are in keeping with the hypothesis that PGE(1) shares a receptor site on the plasma membrane with ACTH.

Diphtheria toxin effects on human cells in tissue culture.

HeLa cells exposed to a single sublethal concentration of diphtheria toxin were found to have diminished sensitivity when subsequently reexposed to the toxin. Three cells strains exhibiting toxin resistance were developed. In the cells that had previously been exposed to toxin at 0.015 mug/ml, 50% inhibition of protein synthesis required a toxin concentration of 0.3 mug/ml, which is more than 10 times that required in normal HeLa cells. There appears to be a threshold level of diphtheria toxin action. Concentrations of toxin greater than that required for 50% inhibition of protein synthesis (0.01 mug/ml) are associated with cytotoxicity, whereas those below this concentration may not be lethal. Several established human cell lines of both normal and neoplastic origin were tested for their sensitivity to the effects of the toxin. No special sensitivity was observed with the cells of tumor origin. Fifty % inhibition of protein synthesis of HeLa cells was achieved with diphtheria toxin (0.01 mug/ml) as compared to the normal human cell lines tested (0.03 and 0.5 mug/ml) and a cell line derived from a human pancreatic adenocarcinoma (0.2 mug/ml). A human breast carcinoma cell line showed a maximum of 45% inhibition of protein synthesis. This required a diphtheria toxin concentration of 5 mug/ml. These results suggest that different human cell lines show wide variation in their sensitivity to the toxin.

New doxorubicin analogs active against doxorubicin-resistant colon tumor xenografts in the nude mouse.

The antitumor activity of doxorubicin and three new derivatives modified on the position 4' of the amino sugar was tested against five human colon tumors and two human rectal tumors (originating from different patients) and xenografted into nude mice. The drugs tested were: 4'-epidoxorubicin; 4'-deoxydoxorubicin; and 4'-O-methyldoxorubicin. Mice were treated i.v. on a weekly basis for 3 to 4 weeks, starting when the tumors were well established (advanced stage of tumor treatment). No statistically significant effect was observed against the tumors tested with the drugs doxorubicin and 4'-epidoxorubicin. 4'-Deoxydoxorubicin was active against all the colon tumors tested (4 of 5 statistically significant), and 4'-O-methuldoxorubicin was active against 4 of 5 colon tumors tested (statistically significant). Overall, the activity of 5'-O-methyldoxorubicin was less than that of 4'-deoxydoxorubicin against the colon carcinomas tested. Neither analog was active against the two rectal carcinomas tested. The results of these studies indicate that: (a) the modifications in the chemical structure of doxorubicin can alter the biological properties and thus create new drugs varying in activity against different human tumors; (b) the two antracycline derivatives, 4'-deoxydoxorubicin and 4'-O-methyldoxorubicin, appear to be good candidates for clinical trial against colon carcinoma; and (c) the nude mice system can offer a great potential for identification of new anthracycline analogs and, in general, new anticancer agents of clinical interest.

Therapeutic response of human tumor xenografts in athymic mice to doxorubicin.

In order to establish the usefulness of the human tumor-nude mouse system as a predictive screen for anticancer agents, 17 tumors (3 breast, 3 colon, 3 lung, 3 melanoma, 2 ovary, 1 prostate, 1 sarcoma, and 1 larynx), serially transplantable in athymic mice, were used to study antitumor activity of doxorubicin (Adriamycin). BALB/c nude mice were treated i.v. on a weekly basis for 3 to 4 weeks, starting when the tumor volume became relatively large (advanced stage of tumor treatment). All the tumors except lung tumor T 293 showed a 90 to 100% take rate and stable growth. Doxorubicin, at dose levels of 6 and 10 mg/kg/injection i.v. every week for 3 weeks, showed significant activity against all of the three breast tumors studied. As was expected on the basis of clinical data, doxorubicin showed no antitumor activity against the three different colon tumors. In the case of lung tumors, statistically significant activities against oat cell carcinoma T 293 and epidermoid carcinoma T 222 were observed. In contradiction to clinical data, doxorubicin was found to have significant activity against various melanomas studied and slight but not statistically significant activity against ovarian tumor T 17. Experimental results obtained using doxorubicin against prostate, sarcoma, and larynx tumors also parallel the reported clinical data.

Isolation and characterization of plasma membranes from transplantable human astrocytoma, oat cell carcinoma, and melanomas.

Purified plasma membranes were obtained from five transplantable human tumors, a grade IV astrocytoma, an oat cell carcinoma, and three melanomas. Plasma membrane fractions were isolated from tumor homogenates by differential and discontinuous sucrose gradient centrifugation. Determination of enzyme activities indicated that the plasma membranes were enriched 10- to 20-fold with respect to 5'-nucleotidase, nicotinamide adenine dinucleotide glycohydrolase, Mg2+-activated nucleoside triphosphatase, and sialic acid. Specific activities of nearly all the enzymes varied with the individual tumors, even among tumors of the same type, i.e., the melanomas. Electron micrographs of the plasma membrane fractions showed smooth single-membrane vesicles with slight contamination by lysosomes. Therefore, these membranes are suitable for comparative biochemical studies and for the preparation of tumor-specific monoclonal antibodies. Plasma membranes from all five tumors contained very high Mg2+-adenosine triphosphatase (ATPase) activities. The Na+-K+-ATPase was a minor component of the total ATPase of these membranes (less than 30%). The major component was an ATPase exhibiting similar activity toward several nucleoside triphosphates. The activity of such a nucleoside triphosphatase has been correlated with tumorigenicity in cultured liver epithelial cells. The nucleoside triphosphatase of the plasma membranes of astrocytoma and oat cell carcinoma was stimulated from 50 to 1005 by concanavalin A, whereas ATPase of the melanoma plasma membranes was not or only slightly stimulated. The different response to concanavalin A could be due to differences in the ATPase molecules of the individual tumors or to the different environment of the ATPase.

Kinetics of protein synthesis inactivation in human T-lymphocytes by selective monoclonal antibody-ricin conjugates.

Immunotoxins synthesized with the pan-T-cell monoclonal antibody T101 and ricin, acetylricin, or ricin A-chain have been compared. Native ricin was acetylated with N-acetylimidazole to block the galactose-binding site of the toxin B (binding)-chain. In the presence of lactose, both whole-ricin-containing immunotoxins were selectively cytotoxic but the ricin A-chain conjugate was less effective in blocking cellular protein synthesis. Immunotoxin-treated cells cultured in fresh growth medium exhibited no growth, declining viabilities, and no protein synthesis activity. Lymphocytes treated with T101:ricin or ricin did not form clusters or colonies when plated in 0.3% Bacto-agar. Ammonium chloride markedly enhanced the efficacy of T101:ricin and T101:ricin A-chain. Our results suggest that: (a) all immunotoxins were selectively cytotoxic; (b) in the presence of ammonium chloride the effectiveness of the T101:ricin A-chain conjugate approached that of T101:ricin; and (c) the toxin B-chain may facilitate conjugate internalization and/or processing.