Current treatments for septic shock caused by gram-negative bacteria include antibiotic therapy and intensive-care support to correct the resulting dysfunctions of cardiovascular, endocrine, and other organ systems. Clearly, there is a great interest in identifying novel strategies to treat not only infections, but also the underlying inflammatory responses; agents that can modulate inflammatory responses, in addition to having direct antimicrobial activity.
With a novel approache to investigating the applicability of sophorolipids as a treatment for sepsis, Biomedica has developed a libray of sophorolipid derivatives that act primarily through decreasing inflammatory cytokines and elicit other synergistic anti-inflammatory mechanisms. These compounds are biodegradable and have low toxicity profiles. The proposed studies challenge existing paradigms in that the proposed treatment appears treat sepsis without any adverse side effects at effective/therapeutic doses. Indeed the development of glycolipid structural analogues can have a very important therapeutic impact in the treatment of septic patients. A future opportunity would be to bio-engineer various compounds that inhibit signaling molecules (e.g. selectin blockers) by linking molecules to the sophorolipid carboxylic acid group.The company has assembled a unique team of bio-organic chemists, trauma specialists, and molecular immunologists to synthesize the sophrolipid components, conceive experiments, and analyze data
We have identified eight primary derivatives of sophrolipids in the natural mixture, and developed methods to prepare each in sufficient quantities and purity for animal experiments. These isoforms selectively modulate cytokine expression in vitro. In vitro experiments indicate that sophrolipids have immunomodulatory characteristics after exposure to lipopolysaccharide, specifically by decreasing production of pro-inflammatory cytokines. Furthermore, sophorolipids affect other important anti-inflammatory immune responses including regulation of cell surface adhesion molecule expression, and the ability to exert antimicrobial effects.
Sophorolipids are fermentatively produced by yeasts such as Candida bombicola, Yarrowia lipolytica, Candida apicola, and Candida bogoriensis. These compounds have widespread implications in many areas of clinical medicine, including pharmaceuticals, drug delivery and components of biomaterials. Studies in our laboratory indicate that sophorolipids possess anti-bacterial anti-viral and anti-inflammatory properties. The mechanisms involved in sepsis related anti-inflammatory effects includes reduction of nitric oxide, regulation of inflammatory cytokines and modulation of cell surface adhesion molecules. Other recent studies have demonstrated that sophorolipids are able to downregulate expression of pro-inflammatory cytokines including IL-1a, IL-1ß and IL-6 among others. Biomedica is investigating
Our researchers have bioenineered a range of pure single components from the natural mixture of sophorolipid or structural variants of them in high quantity and purity using biological or biological-coupled chemical synthesis. We have demonstrated that natural sophorolipid mixture and select isoforms can be safely given to rodents via IP and IV routes at doses of 5mg/kg. The lethal dose of 50% of animals (LD50) of naturally occurring sophorolipids in rodents is 6-7 gm/kg (38,39). Others have observed no measurable consequences with 12.5 g/kg SL in rats or 6 g/kg in mice (40). The oral LD of sophorolipid is estimated at 12.5 g/kg. However, the ideal derivative(s) , dose, route and for optimal anti-sepsis effect has not yet been defined.
We have also demonstrated that sophorolipid administration affects expression of cell surface adhesion, integrin and selectin molecules, including ICAM (CD54), L-selectin (CD62L) (40) which likely play a role in our observed decrease in sepsis related mortality.
Preliminary Data on the effects of natural sophorolipid mixture on mortality in intra-abdominal sepsis-
Control rats and experimental rats were divided into four groups (n=25/group) and induced with septic peritonitis via CLP and treated with saline or natural sophorolipid mixture (5mg/kg), either IP or IV. Doses were given at the end of the surgery, and animals were followed for 36 hours.
Intraperitoneal administration: Following CLP and IP placebo, the survival rate in the control group was 53%. The survival rate was increased to 67% in the experimental group treated with IP injection of sophorolipid (P=0.08).
Intravenous administration: Following CLP and IV placebo, the survival rate at 36 hours in the control group was 47.8%, and increased to 81.8% in those animals treated with IV injection of sophorolipid (P<0.05) (Fig. 1A).
This significant improvement in survival was achieved with a single dose of natural sophorolipid mixture given at the induction of sepsis with intravenous administration. The CLP model was chosen for its reproducible mortality rates and its ability to mimic fecal peritonitis. This dose is well below the LD50 (6 - 7 gm/kg) of naturally occurring sophorolipids in rodents. We have also demonstrates the effect of multiple sequential (q24 hr x 3 doses) IV dosing regimens of sophorolipid administration in septic rats (CLP). Sophorolipid treatment showed a trend toward improved survival of rats with CLP-induced septic shock by 28% at 24hr and 42% at 72hr for single dose and 39% at 24hr and 26% at 72hr for sequential doses when compared with vehicle control (p>0.05).
We have recently investigated the effect of sophorolipids on sepsis related mortality when administered as a natural mixture vs. individual isoforms compared with vehicle alone. We have found that there is a 25% overall decrease in mortality for select sophorolipid isoforms (i.e. ethyl ester) when compared with vehicle control (Fig 1B). Isoforms (lactonic sophorolipid) trended toward increased mortality, which although not statistically significant, implies that there may be differences in the activity of these isoforms with respect to pro- and anti-inflammatory effects (Fig 1B).
Decrease In Mortality Fig. 1A Fig.1B
In addition, we have studied the effects of delayed sophorolipid administration in a different model of endotoxic shock. This model employs galactosamine-sensitized LPS endotoxic shock induction in a mouse model of disease. This model has been shown to increase animal sensitivity to the lethal effects of lipopolysaccharide several thousand fold. Therefore, treatment after 1.5hr can be compared to treatment after 24hrs or later in conventional models. In these studies sophorolipids administered to septic animals 1.5 hr after insult decreased endotoxin related mortality by 53% (Table I) The fact that sophorolipids demonstrated such a robust response in an accelerated animal mortality model is remarkable and provides further support of therapeutic utility. The lack of protective effect with simultaneous administration (Table 1, b vs. rat data, Fig 2A) may be due to differences in animal (rat vs mouse), model (CLP vs, LPS), or route (IV vs IP). Our proposed studies will clarify these issues.
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Table I. Sophorolipid (SL) mediated protection of BALB/C mice against galactosamine-LPS induced endotoxic shock. Mice were induced with endotoxic shock by IP injection of Salmonella typhimurium lipopolysaccharide (LPS, 1mg/mouse) following sensitization by IP injection of D-galactosamine (18mg/mouse). SL (5mg/kg) were injected (a) 1.5 hours before LPS, (b) simultaneously, or (c) 1.5 hours after LPS injection. Key: 0=time 0 for injection |
Effect of natural sophorolipid mixture on cytokine production in CLP sepsis:
Using RNase Protection Assay, we demonstrated that mRNA derived from splenocytes, obtained from control CLP-septic rats (at 6hrs), expressed high levels of IL-1 ß (Fig. 3A). In contrast, mRNA derived from splenocytes, obtained from septic rats treated afterward with natural sophorolipid mixture (5mg/kg), had a 42.5% ± 4.7% (P < .05) reduction in IL-1 ß expression (Fig. 3A). Similarly, mRNA derived from splenocytes, obtained from CLP-septic rats treated with saline, expressed TGF-ß1 (Fig. 3B). In contrast, mRNA derived from splenocytes, obtained from septic rats treated with sophorolipid, showed an 11.7 ± 1.5% (P < .05) increase in expression TGF-ß1 (Fig. 3B). Additional data show changes in cell viability and clumping in macrophages treated with LPS +/- sophorolipid, in that LPS treatment alone demonstrated changes and clumping and cell viability whereas addition of sophorolipid reversed this effect. Furthermore, sophorolipid treatment alone had no effect on cell morphology or viability (trypan blue exclusion) (not shown).
Fig. 3A) IL-1ß and 3B) TGF-ß1 production in splenic lymphocytes of rats treated with natural sophorolipid mixtures. Data are expressed as percent control (CLP + vehicle) +/- SEM. Treatment groups were significant (p<0.05) compared with control using student's T test. CLP=cecal ligation and puncture; SL= sophorolipid
Microarray analysis of natural sophorolipid mixture mediated changes in gene expression in models of intra-abdominal sepsis and macrophages: We have previously reported that mononuclear cells (MC), obtained from blood (PBMC) or spleen have the ability to provide a "reporter function" which differs in pancreatitis when compared with other inflammatory states (septic shock) or healthy rats. We have also used microarray analysis of mouse macrophages cultured with LPS +/- natural sophorolipid mixture to show similar regulation of gene expression and data-mined for key sepsis related cytokines which are affected by sophorolipid treatment.
MS-23 Sophorolipids decrease inflammatory cytokine responses in vitro
a) Natural mixture: Since we have found decreases in the expression of proinflammatory cytokines through mining of microarray data, we set out to confirm these observations through multiplex PCR and determine if these decreases persisted over time. As shown in Fig. 4, low dose (200/ng/ml) sophorolipid treatment decreased (a) LPS induced inflammatory cytokine expression of IL-1a, IL-1ß, TGF-ß, and TNF-a by 37%, 26%, 44% and 37% respectively at 12-24 hours and (b) LPS induced cell death by 13% at 48 hours, compared with LPS alone in a macrophage cell line. Sophorolipid treatment alone had no effect on cytokine gene expression or viability (trypan blue exclusion) when compared with control The lack of significance at 36 hr may be due to a short half life for sophorolipid; additional dosing may be able prolong the observed anti-inflammatory effects.
Fig. 3. Cytokine expression profiles in response to LPS +/- sophorolipid (SL) confirmed by multiplex PCR.Y axis represents cytokine expression as Final Adjusted Volume in (INTxmm2). X axis represents hours post treatment.
Sophorolipid isoforms
We have also determined the cytokine responses of individual sophorolipid isoforms. As shown in Fig 5, select SL isoforms decreased IL-1 and IL-8 cytokine responses when compared with the natural mixture responses. For example cells cultured with the methyl ester isoform had an >85% decrease in IL-1 expression compared with natural mixture whereas culture with the lactonic isoform did not differ with regard to cytokine expression. Furthermore, the mono and di-acetate ethyl ester isoforms showed a 50% reduction in IL-1 production but approached a 75% reduction in IL-8 expression when compared with the natural mixture. These data suggest that select isoforms possess potent anti-inflammatory responses and may provide such in in vivo models of inflammatory disease.
Fig. 4. Pro-inflammatory cytokine induction (A) IL-1; (B) IL-8
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Effect of MS-23 sophorolipids on adhesion molecules. We have previously demonstrated that sophorolipids decreased sepsis related mortality in vivo in a rat model of peritonitis and in vitro by analysis of cytokine production (35). In order to better understand possible mechanisms of sophorolipid action, we investigated changes in cell surface expression profiles of helper/cytotoxic T cells (CD4, CD8), and adhesion molecules including ICAM (CD54), L-selectin (CD62L) and integrins (CD11a, CD11b/c) on blood leukocytes obtained from sophorolipid treated septic rats, compared with untreated and sham (laparotomy) controls. Intra-abdominal sepsis was induced in rats via cecal ligation and puncture (CLP). Sophorolipids (SL)(5mg/kg) or vehicle alone were injected intravenously (IV) via tail vein at the end of the operation.
Sophorolipid treated rats showed a 67% increase in lymphocyte CD11b/c expression when compared with untreated controls (15% vs. 9%, respectively, p<0.05) (Fig. 6A). Sophorolipid treatment also demonstrated a trend toward decreased lymphocyte CD54 and CD62L expression when compared with untreated controls (59% and 45%, respectively; 55% and 47%, respectively, p>0.05), and lymphocyte CD11a expression was similar in both groups (Fig. 6B). CD4+ and CD8+ cells were significantly reduced in both CLP groups (+ sophorolipid treatment) when compared with sham group (7%+1% and 13%, respectively; 8%+2% and 39%, respectively, p<0.05) (data not shown).
Antimicrobial effect of MS-23 Sophorolipids
Sophorolipids have been shown to be antifungal and are thought to elicit its effect by destabilizing the cell membrane (77).(53) We investigated the ability of sophorolipid mixtures and select isoforms to provide antimicrobial effects. As shown in Table 2 select sophorolipid isoforms (monoacetate ethyl ester and diacetate ethyl ester) demonstrated antimicrobial effects against a variety of microbial organisms. We will determine whether sophorolipids and select isoforms provide antimicrobial effects against relevant clinical pathogens in vitro and if it decreases bacterial load in vivo in experimental sepsis.
Table 2: Microbicidal activity of SLs Bacteriums (Bacillus subtilis, Escherichia coli, Moraxella sp., Ralstonia eutropha, Rhodococcus Rhodococcus, Rhodoccoccus erythropolis, Salmonella cholerasesuis, Streptococcus agolatiae)
Effect of M23 sophorolipids on Toll-like receptors (TLR)
TLR's are becoming increasingly important in the understanding of inflammatory diseases and appear to be increased in sepsis (48). Using microarray technology, we investigated the effect of sophorolipids on TLR expression. As shown in Fig. 7, LPS treatment increased expression levels of TLR6 at 24 and 48 hours. In contrast these increases were reduced in the presence of natural sophorolipids. Interestingly, sophorolipid treatment alone did not appear to increase TLR6 expression. We postulate that sophorolipids can decrease TLR's including TLR6 which are important in sepsis pathogenesis (49). (54) We will investigate the ability of sophorolipids to regulate TLR protein expression through flow cytometric analysis (as described in Materials and Methods)
................................................TLR 6
................ 24hr ......................................48hr
Fig. 6. Cytokine expression profiles of mined TLR6 in response to LPS +/- sophorolipid (SL). These data parallel those in Fig 7B. LPS induced TLR6 expression were mined from gene array experiments performed at 24 and 48 hrs
--------- LPS--------- SL----- LPS+SL----------- LPS--------- SL ------LPS+SL