Treatment Of Gram-negative Bacterial Sepsis Research Articles

Drug repositioning of TANK-binding kinase 1 inhibitor CYT387 as an alternative for the treatment of Gram-negative bacterial sepsis

There is currently no specific drug for the treatment of sepsis and antibiotic administration is considered the best option, despite numerous issues. Therefore, the development of drugs to control the pathogen-induced inflammatory responses associated with sepsis is essential. To address this, our study examined the transcriptomes of lipopolysaccharide (LPS)-induced dendritic cells (DCs), identifying TANK-binding kinase1 (Tbk1) as a key factor involved in the inflammatory response. These data suggested drug repositioning of the Tbk1 inhibitor CYT387, currently used for the treatment of myelofibrosis and some cancers, as a candidate for regulating the LPS-induced inflammatory response. CYT387 also inhibited pro-inflammatory cytokine and surface molecule expression by mature DCs after LPS exposure. These effects correlated with both Akt phosphorylation and IκBα degradation. Finally, CYT387 demonstrated therapeutic effects in LPS-induced endotoxemia and Escherichia coli K1-induced mouse models of sepsis and decreased the expression of pro-inflammatory cytokines. In conclusion, our study suggests that drug repositioning of CYT387 may serve as a potential therapeutic for sepsis.

O-2 Population and developmental pharmacokinetic analysis to evaluate and optimise cefotaxime dosing regimen in neonates and young infants

Background Cefotaxime is one of the most frequently prescribed antibiotics for the treatment of Gram-negative bacterial sepsis in neonates. However, the dosing regi-mens routinely used in clinical practice vary considerably. The objective of the present study was to conduct a population pharmacokinetic study of cefotaxime in neonates and young infants in order to evaluate and optimise the dosing regimen. Methods An opportunistic sampling strategy com-bined with population pharmacokinetic analysis using NONMEM software was performed. Cefotaxime concentrations were measured by high-performance liquid chromatography tandem mass spectrometry. Developmental pharmacokinetics-pharmacodynamics, the microbiological pathogens, and safety aspects were taken into account to optimise the dose. Results The pharmacokinetic data from 100 neonates (gestational age [GA] range, 23 to 42 weeks) were modeled with an allometric two compartment model with first-order elimination. The median values for clearance and volume of distribution at steady state were 0.12 litre/h/kg of body weight and 0.64 litre/kg, respectively. The covariate analysis showed that current weight, GA, and postnatal age [PNA] had significant impacts on ce-fotaxime pharmacokinetics. Monte Carlo simulations demonstrated that the current dose recommendations underdosed the older newborns. A model-based dosing regimen of 50 mg/kg twice a day to four times a day, according to GA and PNA, was established. The associated risk of overdose for the proposed dosing regimen was 0.01%. Conclusion We determined the population pharma cokinetics of cefotaxime and established a model based dosing regimen to optimise treatment for neonates and young infants.

A Population and Developmental Pharmacokinetic Analysis To Evaluate and Optimize Cefotaxime Dosing Regimen in Neonates and Young Infants.

Cefotaxime is one of the most frequently prescribed antibiotics for the treatment of Gram-negative bacterial sepsis in neonates. However, the dosing regimens routinely used in clinical practice vary considerably. The objective of the present study was to conduct a population pharmacokinetic study of cefotaxime in neonates and young infants in order to evaluate and optimize the dosing regimen. An opportunistic sampling strategy combined with population pharmacokinetic analysis using NONMEM software was performed. Cefotaxime concentrations were measured by high-performance liquid chromatography-tandem mass spectrometry. Developmental pharmacokinetics-pharmacodynamics, the microbiological pathogens, and safety aspects were taken into account to optimize the dose. The pharmacokinetic data from 100 neonates (gestational age [GA] range, 23 to 42 weeks) were modeled with an allometric two-compartment model with first-order elimination. The median values for clearance and the volume of distribution at steady state were 0.12 liter/h/kg of body weight and 0.64 liter/kg, respectively. The covariate analysis showed that current weight, GA, and postnatal age (PNA) had significant impacts on cefotaxime pharmacokinetics. Monte Carlo simulations demonstrated that the current dose recommendations underdosed older newborns. A model-based dosing regimen of 50 mg/kg twice a day to four times a day, according to GA and PNA, was established. The associated risk of overdose for the proposed dosing regimen was 0.01%. We determined the population pharmacokinetics of cefotaxime and established a model-based dosing regimen to optimize treatment for neonates and young infants.

Determination of Optimal Amikacin Dosing Regimens for Pediatric Patients With Burn Wound Sepsis.

This study aimed to develop optimal amikacin dosing regimens for the empirical treatment of Gram-negative bacterial sepsis in pediatric patients with burn injuries. A pharmacodynamic (PD) target in which the peak concentration (Cmax) is ≥8 times the minimum inhibitory concentration (MIC) (Cmax/MIC ≥ 8) is reflective of optimal bactericidal activity and has been used to predict clinical outcomes. Population pharmacokinetic modeling was performed in NONMEM 7.2 for pediatric patients with and without burn injuries. Amikacin pharmacokinetic parameters were compared between the two groups and multiple dosing regimens were simulated using MATLAB to achieve the PD target in ≥90% of patients with burn injuries. The pharmacokinetic analysis included 282 amikacin concentrations from 70 pediatric patients with burn injuries and 99 concentrations from 32 pediatric patients without burns. A one-compartment model with first-order elimination described amikacin pharmacokinetics well for both groups. Clearance (CL) was significantly higher in patients with burn injuries than in patients without (7.22 vs 5.36 L/h, P < .001). The volume of distribution (V) was also significantly increased in patients with burn injuries (22.7 vs 18.7 L, P < .01). Weight significantly influenced amikacin CL (P < .001) and V (P < .001) for both groups. Model-based simulations showed that a higher amikacin dose (≥25 mg/kg) achieved a Cmax/MIC ≥8 in ≥90% of patients with assumed infections of organisms with an MIC = 8 mg/L. Amikacin pharmacokinetics are altered in patients with burn injuries, including a significant increase in CL and V. In simulations, increased doses (≥25 mg/kg) led to improved PD target attainment rates. Further clinical evaluation of this proposed dosing regimen is warranted to assess clinical and microbiological outcomes in pediatric patients with burn wound sepsis.

The search for molecular determinants of LPS inhibition by proteins and peptides.

Lipo-poly-saccharide (LPS) induced Gram-negative sepsis and septic shock remain lethal in up to 60 % of cases, and LPS antagonists that neutralize its endotoxic action are the subject of intensive research. The molecular motifs of specific binding of LPS by antiendotoxin proteins and peptides may lead to an understanding of LPS action at the atomic level and provide clues for the development of new immunomodulatory compounds for use as therapy in the treatment of Gram-negative bacterial sepsis. The interaction of LPS with its cognate binding proteins has been structurally elucidated in the single case of the X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from E. coli K-12 (Ferguson et al., Science 1999, 282, 2215). This structure and other known structures of LPS binding proteins have been used to propose a common binding motif of LPS to proteins. Another independent source of structural information are solution structures of peptides in complex with LPS that can be determined using the transferred NOE effect. The molecular mechanisms of biological activity of bacterial endotoxins can additionally be probed by theoretical means. The growing structural knowledge is opening pathways to the design of peptides or peptidomimetics with improved antiendotoxin properties.

Molecular biology of endotoxin antagonism.

The development of new therapies for the treatment of gram-negative bacterial sepsis has been the focus of extensive investigation. Molecular and cellular biologic techniques have led to important advances, including (1) identification of naturally occurring lipopolysaccharide (LPS)-binding proteins; (2) generation of novel LPS-binding antibodies, proteins, and peptides; and (3) characterization of the molecular determinants of LPS binding. In composite, these advances have facilitated comprehension of the manner in which gram-negative bacterial infection and concurrent endotoxemia exert detrimental effects on the mammalian host. The purpose of this review was to examine recent information regarding molecular determinants of LPS binding and the initial cellular signal transduction events, which lead to the local and systemic cytokine response and sepsis syndrome. Concurrently, the status of studies examining the effects of various endotoxin antagonists is reviewed. Data from these studies provide an indication of potential sites for therapeutic intervention as well as increasingly detailed understanding of the molecular mechanism of endotoxin antagonism. Taken together, these advances can be expected to further the development of the next generation of novel, adjuvant therapies for the treatment of sepsis syndrome caused by gram-negative bacterial infection and endotoxemia.

Identification of single amino acid residues essential for the binding of lipopolysaccharide (LPS) to LPS binding protein (LBP) residues 86-99 by using an Ala-scanning library.

Lipopolysaccharide binding protein (LBP) is a 60 kDa acute phase glycoprotein capable of binding to LPS of Gram-negative bacteria and facilitating its interaction with cellular receptors. This process is thought to be of great importance in systemic inflammatory reactions such as septic shock. A peptide corresponding to residues 86-99 of human LBP (LBP86-99) has been reported to bind specifically with high affinity the lipid A moiety of LPS and to inhibit the interaction of LPS with LBP. We identified essential amino acids in LBP86-99 for binding to LPS by using a peptide library corresponding to the Ala-scanning of human LBP residues 86-99. Amino acids Trp91 and Lys92 were indispensable for peptide-LPS interaction and inhibition of LBP-LPS binding. In addition, several alanine-substituted synthetic LBP-derived peptides inhibited LPS-LBP interaction. Substitution of amino acids Arg94, Lys95 and Phe98 by Ala increased the inhibitory effect. The mutant Lys95 was the most active in blocking LPS binding to LBP. These findings emphasize the importance of single amino acids in the LPS binding capacity of small peptides and may contribute to the development of new drugs for use in the treatment of Gram-negative bacterial sepsis.

Lipopolyamines as a therapeutic strategy in experimental Gram-negative bacterial sepsis

Lipopolyamines are a class of polycationic amphiphilic compounds that have been shown to bind with high affinity to polyanionic macromolecules, including both DNA and bacterial lipopoly-saccharide (LPS). One of these compounds, termed DOSPER (1,3-di-oleoyloxy-2-(6-carboxyl-spermyl)- propylamide), is non-cytotoxic and has been shown to inhibit LPS-mediated cytokine release and lethality in endotoxin challenge models. In the study reported here, the activity of DOSPER was tested in neutropenic rats with invasive Gram-negative bacteremia caused by Pseudomonas aeruginosa. DOSPER alone was ineffective (0/8) at influencing mortality, but provided a significant survival advantage if administered in combination with a bactericidal antibiotic, ceftazidime (10/12; P<0.05). Ceftazidime alone was partially protective (6/12) while the control group had no survivors (0/8). DOSPER administration markedly reduced circulating endotoxin levels (P<0.01) and interleukin-6 levels (P<0.05) but had no significant effect on bacteremia and bacterial concentrations of P. aeruginosa in liver or spleen tissue. Lipopolyamines may be potentially valuable as a therapeutic adjunct in treatment of Gram-negative bacterial sepsis.

Peptide derivatives of three distinct lipopolysaccharide binding proteins inhibit lipopolysaccharide-induced tumor necrosis factor-alpha secretion in vitro.

Bactericidal permeability increasing protein (BPI), Limulus anti-lipopolysaccharide factor (LALF), and lipopolysaccharide binding protein (LBP) are three distinct proteins that bind to lipopolysaccharide (LPS). Intriguingly, binding of BPI and LALF to LPS results in neutralization of LPS activity, whereas the binding of LBP to LPS creates a complex that results in augmentation of LPS activity. Despite their different effector functions, we hypothesized that peptides based on the sequences of the proposed LPS-binding motif from each protein would neutralize LPS in vitro. Three peptide sequences, each 27 amino acids in length, of the proposed LPS-binding motif of BPI (BG38), LALF (BG42), and LBP (BG43) were synthesized. These peptides were then tested for their: (1) ability to inhibit macrophage secretion of TNF-alpha after stimulation by LPS derived from Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Serratia marcescens; and (2) bactericidal activity against these same four gram-negative bacteria in vitro. Synthetic peptides BG38 (BPI-derived), BG42 (LALF-derived), and BG43 (LBP-derived) but not control peptide significantly inhibited LPS-induced tumor necrosis factor-alpha secretion by macrophages and mediated the lysis of gram-negative bacteria in vitro. In addition, preincubation of LPS with peptide BG38 mediated complete protection subsequent to lethal endotoxin challenge. These data demonstrate that small peptides derived from BPI, LALF, and LBP retained significant endotoxin-neutralizing and bactericidal activity against many different gram-negative bacteria in vitro. Identification of this conserved LPS-binding region within each protein may aid in the development of new immunomodulatory reagents for use as adjuvant therapy in the treatment of gram-negative bacterial sepsis.

Assessment of ability of murine and human anti-lipid A monoclonal antibodies to bind and neutralize lipopolysaccharide.

The use of monoclonal antibodies (mAbs) directed to lipid A for the therapy of gram-negative sepsis is controversial. In an attempt to understand their biologic basis of action, we used a fluid-phase radioimmunoassay to measure binding between bacterial lipopolysaccharide (LPS) and two IgM mAbs directed to lipid A that are being evaluated for the treatment of gram-negative bacterial sepsis. Both antibodies bound 3H-LPS prepared from multiple strains of gram-negative bacteria when large excesses of antibody were used, although binding was modest and only slightly greater than control preparations. We also studied the ability of each anti-lipid A antibody to neutralize some of the biological effects of LPS in vitro. Despite large molar excesses, neither antibody neutralized LPS as assessed by the limulus lysate test, by a mitogenic assay for murine splenocytes, or by the production of cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor from human monocytes in culture medium or in whole blood. Our experiments do not support the hypothesis that either of these anti-lipid A mAbs function by neutralizing the toxic effects of LPS.

Studies of the effect of a platelet-activating factor antagonist, CL 184,005, in animal models of gram-negative bacterial sepsis.

The effect of CL 184,005, a potent and specific platelet-activating factor antagonist, has been examined in a variety of animal models relevant to gram-negative bacterial sepsis. Pretreatment of mice with CL 184,005 protected them from the lethal effects of platelet-activating factor. When rats or primates rendered hypotensive with endotoxin were treated with CL 184,005, blood pressure was normalized. Pretreatment of rats with CL 184,005 protected them from the gastrointestinal lesions induced by endotoxin. Pretreatment of rats and mice with CL 184,005 protected them from the lethal effects of endotoxin. Plasma tumor necrosis factor levels in endotoxin-treated mice were lower when the mice were pretreated with CL 184,005. These observations suggest that CL 184,005 may be potentially useful in the treatment of gram-negative bacterial sepsis, and the agent is undergoing clinical evaluation.

Development and Potential Use of Antibody Directed Against Lipopolysaccharide for the Treatment of Gram-negative Bacterial Sepsis

Gram-negative bacterial sepsis remains a major cause of lethality in hospitalized patients, despite routine therapy consisting of antimicrobial agents, hemodynamic monitoring and fluid resuscitation, and metabolic support. Because a large body of evidence supports the concept that Gram-negative bacterial lipopolysaccharide (endotoxin, LPS) is responsible for many of the direct and host mediator-induced deleterious effects, recent work has been centered on the development and use of anti-LPS antibody preparations in order to ameliorate lethality. Both polyclonal and monoclonal antibody preparations directed against the common deep core/lipid A region of LPS are cross-reactive in vitro and cross-protective in vivo against a wide range of challenge organisms and LPS, and preliminary clinical trials indicate that a reduction in lethality may be possible. The precise endotoxin epitope against which antibody should be directed in order to maximize protection, however, has not been established. This modality most probably will become a standard form of adjunctive therapy within the next several years for the treatment of Gram-negative bacterial sepsis.

Immunotherapeutic advances in the treatment of gram-negative bacterial sepsis.

AbstractGram‐negative bacterial sepsis with the frequent sequelae of shock, multi‐system organ failure, and death represents one of the most severe infections that can occur in the surgical patient. Fatality in most series has paralleled the presence and severity of underlying host disease processes, polymicrobial bacteremia, shock, and lack of early appropriate antimicrobial therapy. Even patients with no underlying disease state have a significant mortality (10–20%). Therapy of gram‐negative bacterial sepsis and shock at present consists of antimicrobial agents, hemodynamic monitoring, aggressive fluid resuscitation, and metabolic support. The use of these treatment modalities in concert has reduced, but not eliminated, the severe consequences that may ensue. Administration of antibody directed against the comon core lipopolysaccharide antigen of gramnegative microorganisms to patients with gram‐negative sepsis represents a means by which the morbidity and mortality of this disease process may be further reduced. This is supported by a large body of experimental evidence, as well as several preliminary clinical studies. It is necessary to determine how the clinical efficacy of such antibody preparations can be maximized. This will entail rigorously controlled studies in which the timing of antibody administration and dose utilized can be correlated with clinical efficacy. Should such antibody preparations continue to prove efficacious as an additive form of therapy, it may be possible to identify high‐risk groups of patients who would benefit from antibody prophylaxis.