Preparation of aminated fish scale collagen and oxidized sodium alginate hybrid hydrogel for enhanced full-thickness wound healing.

Acute full-thickness wounds require a more extended healing period, thus increasing the risk of infection. Severe infection frequently resulted in wound ulceration, necrosis, and even life-threatening complications. Here, a hybrid hydrogel comprising aminated collagen (AC), oxidized sodium alginate (OSA), and antimicrobial peptides (polymyxin B sulfate and bacitracin) was developed to enhance full-thickness wound healing. The AC with low immunogenicity and high biocompatibility was made from marine fish scales, which are eco-friendly, low-cost, and sustainable. The cross-linked hydrogel was formed by a Schiff base reaction without any catalysts and additional procedures. As expected, the presented hybrid hydrogel can effectively against E. coli and S. aureus, as well as promote cell growth and angiogenesis in vitro. In addition, the hydrogel can promote full-thickness wound healing in a rat model through accelerating reepithelialization, collagen deposition, and angiogenesis. Our work demonstrated that the hybrid hydrogel has promising applications in the field of wound healing, which would prompt the utilization of marine fish resources during food processing.

Direct modifications of the cyclic peptide Polymyxin B leading to analogues with enhanced in vitro antibacterial activity.

Synthetic modifications have been made directly to the cyclic peptide core of polymyxin B, enabling the further understanding of structure activity relationships of this antimicrobial peptide. Such modified polymyxins are also substrates for enzymic hydrolysis, enabling the synthesis of a variety of semi-synthetic analogues, resulting in compounds with increased in vitro antibacterial activity.

Enhancing biocompatibility and neuronal anti-inflammatory activity of polymyxin B through conjugation with gellan gum.

Polymyxins, as strong antibiotics with high liposaccharide deactivation abilities, are rarely used as neuronal anti-inflammatory agent because of their high cytotoxicity. In this study, polymyxin B (PMB) was conjugated with deacylated gellan gum for the sustained release of PMB to reduce its cytotoxicity at high concentration without affecting the antibacterial and liposaccharide binding activities. For the conjugate of original PMB/GN ratio of 1.0 (GPC), the conjugating rate was 96.40%, and the releasing ratio of PMB was 30.12% within 60 h. The FT-IR spectra of GPC indicated that the amino groups of PMB were covalently bonded with the COOH groups of gellan and other PMB molecules. Most GPCs were micelle shaped regardless of whether they were under dry conditions or in an aqueous solution. The inhibition zones of PMB against Escherichia coli and Pseudomonas aeruginosa were small, but the half maximal inhibitory concentration value against BV-2 cells increased from 15.63 µg/mL to 2000.00 µg/mL after conjugation. GPC can also effectively depress the liposaccharide-stimulated overexpression of cytotoxic nitric oxide by BV-2 cells. This study revealed the possibility of using polymyxins for neuronal anti-inflammation and that this gellan/PMB conjugate can potentially be applied to wound healing and implants.

Tryptophan-containing lipopeptide antibiotics derived from polymyxin B with activity against Gram positive and Gram negative bacteria.

Resistance to all known antibiotics is a growing concern worldwide, and has renewed the interest in antimicrobial peptides, a structurally diverse class of amphipathic molecules that essentially act on the bacterial membrane. Propelled by the antimicrobial potential of this compound class, we have designed three new lipopeptides derived from polymyxin B, sp-34, sp-96 and sp-100, with potent antimicrobial activity against both Gram positive and Gram negative bacteria. The three peptides bind with high affinity to lipopolysaccharide as demonstrated by monolayer penetration and dansyl-displacement. The interaction with the cytoplasmic membrane has been elucidated by biophysical experiments with model membranes of POPG or POPE/POPG (6:4), mimicking the Gram positive and Gram negative bacterial membrane. Trp-based fluorescence experiments including steady-state, quenching, anisotropy and FRET, reveal selectivity for anionic phospholipids and deep insertion into the membrane. All three lipopeptides induce membrane fusion and leakage from anionic vesicles, a process that is favored by the presence of POPE. The molecules bind to zwitterionic POPC vesicles, a model of the eukaryotic membrane, but in a different way, with lower affinity, less penetration into the bilayer and no fusion or permeabilization of the membrane. Results in model membranes are consistent with flow cytometry experiments in Escherichia coli and Staphylococcus aureus using a membrane potential sensitive dye (bis-oxonol) and a nucleic acid dye (propidium iodide), suggesting that the mechanism of action is based on membrane binding and collapse of membrane integrity by depolarization and permeabilization.

Cellular Uptake and Localization of Polymyxins in Renal Tubular Cells Using Rationally Designed Fluorescent Probes.

Polymyxins are cyclic lipopeptide antibiotics that serve as a last line of defense against Gram-negative bacterial superbugs. However, the extensive accumulation of polymyxins in renal tubular cells can lead to nephrotoxicity, which is the major dose-limiting factor in clinical use. In order to gain further insights into the mechanism of polymyxin-induced nephrotoxicity, we have rationally designed novel fluorescent polymyxin probes to examine the localization of polymyxins in rat renal tubular (NRK-52E) cells. Our design strategy focused on incorporating a dansyl fluorophore at the hydrophobic centers of the polymyxin core structure. To this end, four novel regioselectively labeled monodansylated polymyxin B probes (MIPS-9541, MIPS-9542, MIPS-9543, and MIPS-9544) were designed, synthesized, and screened for their antimicrobial activities and apoptotic effects against rat kidney proximal tubular cells. On the basis of the assessment of antimicrobial activities, cellular uptake, and apoptotic effects on renal tubular cells, incorporation of a dansyl fluorophore at either position 6 or 7 (MIPS-9543 and MIPS-9544, respectively) of the polymyxin core structure appears to be an appropriate strategy for generating representative fluorescent polymyxin probes to be utilized in intracellular imaging and mechanistic studies. Furthermore, confocal imaging experiments utilizing these probes showed evidence of partial colocalization of the polymyxins with both the endoplasmic reticulum and mitochondria in rat renal tubular cells. Our results highlight the value of these new fluorescent polymyxin probes and provide further insights into the mechanism of polymyxin-induced nephrotoxicity.

Synthesis of a polymyxin derivative for photolabeling studies in the gram-negative bacterium Escherichia coli.

The antimicrobial activity of polymyxins against Gram-negative bacteria has been known for several decades, but the mechanism of action leading to cell death has not been fully explored. A key step after binding of the antibiotic to lipopolysaccharide (LPS) exposed at the cell surface is 'self-promoted uptake' across the outer membrane (OM), in which the antibiotic traverses the asymmetric LPS-phospholipid bilayer before reaching the periplasm and finally targeting and disrupting the bacterial phospholipid inner membrane. The work described here was prompted by the hypothesis that polymyxins might interact with proteins in the OM, as part of their self-promoted uptake and permeabilizing effects. One way to test this is through photolabeling experiments. We describe the design and synthesis of a photoprobe based upon polymyxin B, containing photoleucine and an N-acyl group with a terminal alkyne suitable for coupling to a biotin tag using click chemistry. The resulting photoprobe retains potent antimicrobial activity, and in initial photolabeling experiments with Escherichia coli ATCC25922 is shown to photolabel several OM proteins. This photoprobe might be a valuable tool in more detailed studies on the mechanism of action of this family of antibiotics.

Novel des-fatty acyl-polymyxin B derivatives with Pseudomonas aeruginosa-specific antimicrobial activity.

Polymyxin B (PMB) is a cationic cyclic decapeptide antibiotic with a fatty acyl (FA) modification at the α-amino group of Dab¹ (Dab: L-α,γ-diaminobutyric acid). In this study, which is part of a series of PMB structure-activity relationship investigations focused on identifying clinically useful peptide antibiotics, we synthesized ten des-FA PMB derivatives whose N-terminal moieties were changed to basic or hydrophilic amino acids. The antimicrobial and lipopolysaccharide (LPS) binding activities of these synthetic analogs were tested. The analogs showed more potent antimicrobial activity against Pseudomonas aeruginosa (P. aeruginosa) compared with the PMB nonapeptide. In particular, [Ser²-Dap³]-PMB(2-10), Guanyl-[Thr²-Dab³]-PMB(2-10), Guanyl-[Dab¹-Thr²-Dab³]-PMB(1-10), and N(α,γ)-diguanyl-[Dap³]-PMB(3-10) had antimicrobial activity equivalent to PMB. In LPS binding assays, the displacement curves shifted in a manner proportional to the number of positive charges available to bind to Escherichia coli (E. coli) and P. aeruginosa. Furthermore, peptides with basic side chains were comparable to PMB in binding activity assays against E. coli and P. aeruginosa. The acute toxicities of the peptides were evaluated by intravenously administering the peptides to mice through the tail vein. The toxicities of [Ser²-Dap³]-PMB(2-10), [Dap³]-PMB(3-10), and [Ser³]-PMB(3-10) were lower that of PMB (LD50, 4.8 µmol/kg).

Development of des-fatty acyl-polymyxin B decapeptide analogs with Pseudomonas aeruginosa-specific antimicrobial activity.

Twelve N-terminal analogs of des-fatty acyl-polymyxin B (Des-FA-[X(1)]-PMB, X=various amino acids or peptides) were synthesized and examined for their antimicrobial activity against Escherichia coli (E. coli), Salmonella Typhimurium (S. Typhimurium) and Pseudomonas aeruginosa (P. aeruginosa). It was found that Des-FA-[Dap(1)]-, Des-FA-[Ser(1)]-, Des-FA-[Dab-Dab-Dab(1)]- and Des-FA-[Arg-Arg-Arg(1)]-PMB had potent activity only against P. aeruginosa, with MIC values of 0.5-1 nmol/ml. Analogs in which X was Lys, Arg, Leu or Ala did not have increased antimicrobial activity against the three bacterial species tested compared with the lead compounds Des-FA-[Dab(1)]-PMB and polymyxin B (PMB). Des-FA-[Trp(1)]-PMB and Des-FA-[Phe(1)]-PMB had reduced activity against P. aeruginosa. The results indicate that compact hydrophilic amino acids (C3) or basic tripeptides at the N-terminal provide specificity for bactericidal activity towards P. aeruginosa. For LPS-binding activity, Des-FA-[Dab-Dab-Dab(1)]-PMB and Des-FA-[Arg-Arg-Arg(1)]-PMB showed activity comparable to PMB, while Des-FA-[Ala-Ala-Ala(1)]-PMB showed very low activity. Reduced acute toxicity of Des-FA-[Dap(1)]-PMB and Des-FA-[Trp(1)]-PMB was demonstrated by a mouse tail intravenous administration test, with LD(50) values of 23.5 and 19.0 micromol/kg, respectively, in contrast to PMB (LD(50), 4.8 micromol/kg).

Contribution of each amino acid residue in polymyxin B(3) to antimicrobial and lipopolysaccharide binding activity.

This study on the structure-activity relationship of polymyxin B, a cyclic peptide antibiotic, used sixteen synthetic polymyxin B(3) analogs including alanine scanning analogs to elucidate the contribution of the side chains to antimicrobial activity and lipopolysaccharide (LPS) binding. Of these analogs, [Ala(5)]-polymyxin B(3) showed greatly reduced antimicrobial activity against Escherichia coli (E. coli), Salmonella Typhimurium (S. Typhimurium) and Pseudomonas aeruginosa (P. aeruginosa) with MIC values of 4-16 nmol/ml, suggesting that the Dab (alpha,gamma-diaminobutyric acid) residue at position 5 is the most important residue contributing to bactericidal activity. The antibacterial contribution of Dab when located within the lactam ring (positions 5, 8 and 9) was greater than when located outside the ring (positions 1 and 3). [D-Ala(6)]-, [L-Phe(6)]-, [Ala(7)]-, and [Gly(7)]-polymyxin B(3) analogs retained potent antimicrobial activity, indicating that neither the reduction of hydrophobic character of the D-Phe(6)-Leu(7) region nor the D-configuration at position 6 is indispensable for antimicrobial activity. LPS binding studies showed that decreased hydrophobicity of the lactam ring had little effect, but the N(gamma)-amino function of the Dab residues at position 1, 3, 5, 8 and 9 greatly affected LPS binding, with the contribution of Dab(5) being the most significant.

Semi-synthesis of polymyxin B (2-10) and colistin (2-10) analogs employing the Trichloroethoxycarbonyl (Troc) group for side chain protection of alpha,gamma-diaminobutyric acid residues.

Improved strategies for the chemical conversion of natural polymyxin B and colistin to their N-terminal analogs are reported. First, the protection of the side chains of five L-alpha,gamma-diaminobutyric acid (Dab) residues in natural polymyxin B and colistin was achieved with trichloroethoxycarbonyl (Troc), then the resulting pentakis(N gamma-Troc)-polymyxin B and pentakis(N gamma)Troc)-colistin were treated with trifluoroacetic acid (TFA) : methanesulfonic acid (MSA) : dimethylformamide (DMF) : H2O (10 : 30 : 55 : 5) at 40 degrees C in order to remove N alpha-alkanoyl-Dab(Troc)-OH selectively. The new key compounds, tetrakis(N gamma-Troc)-polymyxin B (2-10) and tetrakis(N gamma-Troc)-colistin (2-10), were obtained in 19% and 15% yields, respectively, which is higher than previous reports using trifluoroacetyl (Tfa) for tetrakis(N gamma-Tfa)-polymyxin B (2-10) and tetrakis(N gamma-Tfa)-colistin (2-10), respectively. Acylation of tetrakis(N gamma-Troc)-polymyxin B (2-10) and tetrakis(N gamma-Troc)-colistin (2-10) with various hydrophobic acids bearing aliphatic or aromatic ring structures, followed by the deprotection of Troc by Zn in AcOH, produced polymyxin B (2-10) and colistin (2-10) analogs which were used for structure-activity relationship studies. It was found that cyclohexylbutanoyl-, 4-biphenylacetyl-, and 1-adamantaneacetyl-polymyxin B (2-10) showed potent antimicrobial activity equal to that of polymyxin B against three Gram-negative bacterial strains. The lipopolysacharide (LPS) binding activity of cyclohexylbutanoyl-, 4-biphenylacetyl-, and cyclododecanecarbonyl-polymyxin B (2-10) increased greatly in comparison with that of polymyxin B (2-10). The various N alpha-acylated polymyxin B (2-10) analogs showed slightly higher antimicrobial and LPS binding activities than the corresponding N alpha-acylated colistin (2-10) analogs.

Neopeptide antibiotics that function as opsonins and membrane-permeabilizing agents for gram-negative bacteria.

We suggest a novel approach to enhancing antimicrobial drug action by utilizing engineered peptide conjugates. Our most potent conjugates, [fMLF]PMBN and [fMLF]PMEN, are nonapeptides derived from polymyxin B's (PMB's) cyclic moiety (Thr-Dab-cyclo[Dab-Dab-d-Phe-Leu-Dab-Dab-Thr], where Dab is 2,4-diaminobutyric acid) and polymyxin E's (PME's) cyclic moiety (Thr-Dab-cyclo[Dab-Dab-d-Leu-Leu-Dab-Dab-Thr]), respectively, attached to a linear tail comprised of formyl-Met-Leu-Phe (fMLF). The cyclic part binds to gram-negative lipopolysaccharides, rendering the bacterial outer membrane permeable to hydrophobic antibiotics. The tail confers chemotactic and opsonic activities upon the conjugates. These two activities appear to be the basis for the conjugates' antibacterial activities. The conjugates are 8 to 10 times less toxic than the parent PMB or PME antibiotics. Fourteen of 18 mice lethally challenged with erythromycin-resistant Klebsiella pneumoniae survived following intraperitoneal administration of erythromycin and [fMLF]PMBN, whereas erythromycin or the peptide conjugate alone had no effect. Moreover, the clearance of Klebsiella from blood was markedly enhanced by intravenous injection of the [fMLF]PMEN peptide conjugate compared to the clearance of the organism from the mice treated with buffer alone as a control and was similar to that achieved by the PME antibiotic. Blood clearance was also significantly enhanced by administration of PMEN either alone or in a mixture with fMLF, although the effect was less than that produced by the peptide conjugate. Since resistance to polymyxins, the parent molecules of the synthetic cyclic peptides, is rare, the emergence of bacteria resistant to the antimicrobial properties of the peptide conjugates may be precluded as well.

Synthesis and membrane action of polymyxin B analogues.

We have designed synthetic peptides that mimic the primary and secondary structure of the cationic lipopeptide antibiotic polymyxin B (PxB) in order to determine the structural requirements for membrane action and to assess possible therapeutic potential. Two analogues with related sequences to that of PxB, but including synthetic simplifications (disulphide bridge between two cysteines in positions 4 and 10, N-terminal nonanoic acid), have been synthesized. Peptide-lipid interactions have been studied by fluorescence resonance energy transfer between pyrene and 4,4-difluoro-5-methyl-4-bora-3alpha,4alpha-diaza-s-indacene-3-dodecanoyl (BODIPY)probes covalently linked to phospholipids, and the possibility of membrane disruption or permeabilization has been assessed by light scattering and fluorescence quenching assays. The synthetic peptide sP-B, which closely mimics the primary and secondary structures of PxB, binds to vesicles of anionic 1-palmitoyl-2-oleoylglycero-sn-3-phosphoglycerol (POPG) or of lipids extracted from Escherichia coli membranes, and induces apposition of the vesicles and selective lipid exchange without permeabilization of the membrane. We conclude that sP-B forms functional vesicle-vesicle contacts that are selective, as previously described for PxB. The second analogue, sP-C, has a permutation of two amino acids that breaks the hydrophobic patch formed by D-Phe and Leu residues on the cyclic part of the sequence. sP-C lipopeptide is more effective than sP-B in inducing lipid mixing, but shows no selectivity for the lipids that exchange through the vesicle-vesicle contacts, and at high concentrations has a membrane-permeabilizing effect. The deacylated and non-antibiotic derivative PxB-nonapeptide (PxB-NP) does not induce the formation of functional intervesicle contacts in the range of concentrations studied.

Modulation of the hydrophobic domain of polymyxin B nonapeptide: effect on outer-membrane permeabilization and lipopolysaccharide neutralization.

Polymyxin B nonapeptide (PMBN), a cationic cyclic peptide derived from the antibacterial peptide polymyxin B, is capable of specifically increasing the permeability of the outer membrane (OM) of Gram-negative bacteria toward hydrophobic antibiotics. In this study, we evaluated the contribution of the hydrophobic segment of PMBN (i.e., D-Phe(5)-Leu(6)) to this activity. Accordingly, we synthesized four analogs of PMBN by replacing D-Phe(5) with either with D-Trp or D-Tyr and Leu(6) with Phe or Ala and evaluated their ability to bind cell-free lipopolysaccharide (LPS) and increase bacterial OM permeability. Compared with PMBN, [D-Tyr(5)]PMBN and [Ala(6)]PMBN possessed reduced LPS affinity (IC(50) = 2.5, 25, and 12 microM, respectively) and significantly reduced OM permeability and LPS neutralization activity. [Phe(6)]PMBN exhibited rather similar affinity to cell-free LPS (IC(50) = 5 microM) and the same OM permeability capacity as PMBN. However, [D-Trp(5)]PMBN, despite its similar affinity to cell-free LPS (IC(50) = 4 microM), had moderately reduced OM permeability capacity. These results demonstrate the significant role of the PMBN hydrophobic segment in promoting biological activity.

The functional association of polymyxin B with bacterial lipopolysaccharide is stereospecific: studies on polymyxin B nonapeptide.

The Gram-negative bacterial endotoxin lipopolysaccharide (LPS) is a major inducer of sepsis. The natural cyclic peptide polymyxin B (PMB) is a potent antimicrobial agent, albeit highly toxic, by virtue of its capacity to neutralize the devastating effects of LPS. However, the exact mode of association between PMB and LPS is not clear. In this study, we have synthesized polymyxin B nonapeptide, the LPS-binding cyclic domain of PMB, and its enantiomeric analogue and studied several parameters related to their interaction with LPS and their capacity to sensitize Gram-negative bacteria toward hydrophobic antibiotics. The results suggest that whereas the binding of the two enantiomeric peptides to E. coli and to E. coli LPS is rather similar, functional association with the bacterial cell is stereospecific. Thus, the L-enantiomer is capable of synergism with the hydrophobic antimicrobial drugs novobiocin and erythromycin, whereas the D-enantiomer is devoid of such activity. The potential of understanding and consequently utilizing the PMB-LPS association for novel, nontoxic PMB-derived drugs is discussed.

Structure-function studies of polymyxin B nonapeptide: implications to sensitization of gram-negative bacteria.

Polymyxin B nonapeptide (PMBN), a cationic cyclic peptide derived by enzymatic processing from the naturally occurring peptide polymyxin B, is able to increase the permeability of the outer membrane of Gram-negative bacteria toward hydrophobic antibiotics probably by binding to the bacterial lipopolysaccharide (LPS). We have synthesized 11 cyclic analogues of PMBN and evaluated their activities compared to that of PMBN. The synthetic peptides were much less potent than PMBN in their capacity to sensitize Escherichia coli and Klebsiella pneumoniae toward novobiocin and to displace dansyl-PMBN from Escherichia coli LPS. Moreover, unlike PMBN, none of the analogues were able to inhibit the growth of Pseudomonas aeruginosa. The structural-functional features of PMBN were characterized and identified with regard to the ring size, the distance between positive charges and peptide backbone, the chirality of the DPhe-Leu domain, and the nature of the charged groups. Apparently, the structure of PMBN is highly specific for efficient perturbation of the outer membrane of Gram-negative bacteria as well as for LPS binding. The present study further increases our understanding of the complex PMBN-LPS and may, potentially, enable the design of compounds having enhanced permeabilization potency of the Gram-negative outer membrane.

Unique molecular conformation of aureobasidin A, a highly amide N-methylated cyclic depsipeptide with potent antifungal activity: X-ray crystal structure and molecular modeling studies.

A structural feature of aureobasidins, cyclic depsipeptide antibiotics produced by Aureobasidium pullulans R106, is the N-methylation of four out of seven amide bonds. In order to investigate possible relationship between the molecular conformation and the amide N-methylation, aureobasidin A (AbA), which exhibits the potent antifungal activity, was subjected to X-ray crystal analysis. The crystal, recrystallized from ether (orthorhombic, space group P2(1)2(1)2(1), a = 21.643 (3) A, b = 49.865(10) A, c = 12.427 (1) A, z= 8), contained two independent conformers per asymmetric unit and they took on a similar arrowhead-like conformation. The conformation consisted of three secondary structures of antiparallel beta-sheet, and beta- and gamma-turns, and was stabilized by three intramolecular and transannular N-H O=C hydrogen bonds. The beta-hydroxy-N-methyl-l-valine residue, which is indispensable for its bioactivity, was located at the tip of the corner. Since a nearly identical conformation has been observed for aureobasidin E, a related cyclic depsipeptide, this arrowhead-like conformation may be energetically stable and important for biological activity. The contribution of the amide N-methylation to the conformation was investigated by model building and energy calculations. The energy-minimizations of AbA analogs, in which some (one to four) of four N-methylated amide bonds were replaced with usual amide bond, led to some conformers which are fairly different from the arrowhead form of AbA, although they are stabilized by three intramolecular N-H...O=C hydrogen bonds. This result explains the reason why four out of the seven amide bonds have to be methylated to manifest biological activity, i.e. the high N-methylation of aureobasidin is necessary to form only one well-defined conformation.

Solid-phase total synthesis of polymyxin B1.

The total solid-phase synthesis of polymyxin B1 (PMB1) has been achieved in 20% yield using the orthogonal protecting group N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl-(Dde). This report demonstrates that a complex peptide macrocycle can be synthesized in high yields using solid-phase synthesis. According to MS and HPLC, the synthetic peptide was identical to the naturally occurring antibiotic.

Conformation of polymyxin B analogs in DMSO from NMR spectra and molecular modeling.

The tertiary structures of two polymyxin analogues: [formula: see text] and [formula: see text] in DMSO, from solid-phase peptide synthesis and aerobic oxidation were determined from two-dimensional NMR spectra and distance geometry calculations followed by restrained molecular dynamics simulation. The backbone of peptide I had a rectangular shape stabilized by at least two hydrogen bonds and the hydrophilic side chains of five lysine residues, and the hydrophobic side chains of Phe and Leu resided at both sides to form an amphiphilic molecule. This amphiphilic structure of I is likely to interact with lipid A mainly via a hydrophobic interaction. Compared with I, peptide II, which lacks three N-terminal amino-acid residues, exhibits neither amphiphilic property nor binding ability with lipid A.

Inhibition of lipopolysaccharide-induced TNF-alpha production by semisynthetic polymyxin-B conjugated dextran.

During episode of severe endotoxemia, concentrations of both lipopolysaccharide and its lipid A-core subfraction are liberated from gram-negative bacteria and become elevated within the systemic circulation. Lipid-A core is the most homogeneous and physiologically toxic segment of the lipopolysaccharide molecule. Polymyxin-B has profound binding avidity for the lipid A-core subfraction of lipopolysaccharide. The mechanism of this binding avidity involves the development of attractive forces between the cationic groups of polymyxin-B and the anionic groups of the lipid A-core moiety of lipopolysaccharide. Complementary attractive forces include hydrophobic interactions which additionally become established between the octylheptanoyl group of polymyxin-B and the saturated carbon chains of the lipid A-core moiety. This paper describes a method for the semisynthetic production of polymyxin-B conjugated dextran in the form of polymyxin-B.ABH.dextran applying the photoreactive crosslinking reagent azidobenzoyl hydrazide (ABH). Molecular design and development of a semisynthetic technique for the conjugation of polymyxin-B to purified dextran fractions was motivated by the pronounced nephrotoxicity associated with this cationic polypeptide antibiotic. Conjugation of polymyxin-B to a relatively large molecular weight carrier compound would increase the overall size of the complex to a degree sufficient to theoretically reduce clearance through glomerular filtration mechanisms. Attributes of such a large molecular weight polymyxin-B conjugated biopharmaceutical would include diminished levels of nephrotoxicity due to a reduction of renal tubular concentrations and a simultaneous prolongation of its intravascular half-life (t (1/2)) and pharmacokinetic profile. Lipopolysaccharide (LPS) binding avidity of polymyxin-B.ABH.dextran was verified by Dot-Blot analysis in conjunction with the application of fluorescein isothiocyanate conjugated E. coli (0.55:B5) LPS (FITC-LPS). Capacity of polymyxin-B.ABH.dextran conjugates to inhibit in vitro LIP-induced synthesis of tumor necrosis factor-alpha (TNF-alpha) was assessed by the application of a tissue culture based biological assay system capable of detecting cytotoxicity mediated by this potent monokine. Semisynthetic conjugates of polymyxin-B.ABH.dextran conjugates (0.6 microns/mL), thereby providing cytoprotectivity (95%; p < or - 0.001. to WEHI 164 clone 13 cell populations relative to untreated reference controls. Since TNF-alpha is currently believed to be the principal endogenous mediator involved in the host's inflammatory response during episodes of endotoxemia, results from these investigations provide a scientific foundation for warranting the elevation of the in vivo efficacy of large molecular weight semisynthetic polymyxin-B conjugates. Results from these investigations may ultimately lead to the application of semisynthetic polymyxin-B.ABH.dextran as a model for the molecular design of semisynthetic production of alternative biopharmaceutical or pharmaceutical agents possessing prophylactic and/or therapeutic efficacy for the management of severe endotoxemia conditions.

Polymyxin B octapeptide and polymyxin B heptapeptide are potent outer membrane permeability-increasing agents.

Polymyxin B octapeptide (PBOP) and polymyxin B heptapeptide (PBHP) were found to be effective permeabilizers of the outer membrane of Escherichia coli and Salmonella typhimurium. PBOP was as effective as polymyxin B nonapeptide (PMBN), the known very potent permeabilizer. As low a PBOP concentration as 1 microgram/ml sensitized E. coli to rifampicin by a factor of 100. Three micrograms of PBOP per ml was sufficient to sensitize this target to all the other tested hydrophobic antibiotics (erythromycin, fusidic acid, clindamycin, and novobiocin) by a factor of 30. Only a slightly higher (3-fold) concentration of PBHP was required for a similar sensitizing effect.