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 infections as well as the underlying inflammatory response.
With a novel approach to investigating the use of glycolipids as a treatment for sepsis, Biomedica has developed and studied a libray of 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 the Glyci 23 family can have a very important therapeutic impact in the treatment of septic patients.
The company has assembled a unique team of bio-organic chemists, trauma specialists, and molecular immunologists to bring this agent to the market.
Our researchers have synthezided a range of pure single isoforms that 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 inhibit microbial effects.
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 Glyco 23 is able to downregulate expression of pro-inflammatory cytokines including IL-1a, IL-1ß and IL-6 among others.
We have demonstrated that select isoforms can be safely given to rodents via IP and IV routes at doses of 50mg/kg. The lethal dose of 50% of animals (LD50) in rodents is 6-7 gm/kg.
Preliminary Data on the effects of Glyco23 on mortality in intra-abdominal sepsis
We conducted a study on the preclinical application of selected isoforms and mixtures as clinically protective agents in sepsis.
Effect of Glyco 23 on mortality in intra-abdominal sepsis. Animals were randomized into two groups: control and experimental, (n=25/group); induced with septic peritonitis via CLP; and treated with saline or natural sophorolipid mixture (5mg/kg), IV.
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Doses were given at the end of the surgery, and animals were followed for
36 hours. Kaplan Meier statistics were performed on survival. Preliminary data
showed that we can obtain reproducible mortality rates of 60% to 70% with a 16 gauge needle. 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) . This significant improvement in survival was achieved with a single dose of Glyco23 mixture given at the induction of sepsis with intravenous administration. |
Reduced Mortality at 48Hs
Decrease in mortality compared with vehicle alone. Control rats and experimental rats were induced with septic peritonitis via CLP and treated with saline or natural sophorolipid mixture and individual isoforms (5mg/kg), IV.
Shows the effect of Glyco 23 on sepsis-related reduced mortality at 48 Hs. when administered as a natural mixture (SL) vs. individual isoforms compared with vehicle alone. Data shows a 28% overall decrease in mortality for select isoforms (e-SL) (i.e. ethyl ester) when compared with vehicle control (Fig 4). Isoforms (lactonic sophorolipid) (L-SL) trended to be less effective in reducing mortality, which although implies that there may be differences in the activity of these isoforms with respect to pro- and anti-inflammatory effects.
Survival at 3 hours and 24 hours post insult
Experimental rats were induced with septic peritonitis via CLP and treated with saline or natural sophorolipid mixture . Doses were given at the end of the surgery, and animals were followed for 24 hours (Table 1).
| 3 hours post insult | 24 Hs. post insult | ||||||
| Rat# | Survival% | WBC 16 cells/Ml |
Blood Culture | Survival% | WBC | Blood Culture | |
| Saline Controls | 6 | 84 | 6.3±2.5 | E.coli, P.mirabilis, Enterococcus sp, E.coli,Strep vir grp, Staph sp Co Neg Enterococcus sp |
32 | NA | NA |
| Sham Controls | 6 | 100 | 4±1.5 | Staph sp Co Neg Strep vir grp, Enterococcus sp, |
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| Glyco 23 | 6 | 100 | 4.±1.2 | 100 | 4.±1.6 | Strep vir grp Staphylococcus aureus Enterococcus sp, Staph sp Co Neg |
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1.5 Cytokine Response
Serum samples were obtained from rats (N=5 animals/group) 3 hrs. after (CLP) to establish polymicrobial sepsis, and intravenous injection of saline (control), sophorolipids - natural mixture (SL) - free acid (SLFA). Serum concentrations of Interleukin-6 (IL-6) were determined using commercial ELISA tests according to manufacturer's instructions. Results were evaluated for statistical significance using ANOVA.
It was observed that serum IL-6 increased dramatically (more than 40-fold) at 3 hrs after CLP procedure, and that administration of Glyco 23 was accompanied by a profound reduction (12-fold) of serum IL-6 concentrations; although they did not reach the low levels observed in sham-treated animals. No significant difference was observed between the inhibitory effects of natural mixture and free acid form of sophorolipids.
1.6. Effect of natural Glyco mixture on cytokine production in CLP sepsis Using RNase Protection Assay,
Cytokine Response
| Serum samples were obtained from rats (N=5 animals/group) 3 hrs. after (CLP) to establish polymicrobial sepsis, and intravenous injection of saline (control), natural mixture (SL) or free acid (SLFA). Serum concentrations of Interleukin-6 (IL-6) were determined using commercial ELISA tests according to manufacturer's instructions. Results were evaluated for statistical significance using ANOVA. |
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Histology
Tissue samples were taken from the lung, liver and kidney at 24 Hs. post insult. Tissue was fixed in formaline and embedded in paraffin and sections cut at 5 micron. Treated animals were injected with the sophorolipid mixture.
Lung - control (sham)
H+E x40 Interlacing pattern of evenly dispersed patent alveoli. Lung - CLP
H+E x 40 Irregular thickening of the interalveolar septa, in some places impinging on the alveolar spaces. There is also prominent congestion of branches of the pulmonary vein. Lung - CLP
H+E x 100 Marked thickening of the interalveolar septa and alveolar capillaries engorged with blood. Alveolar spaces contain finely granular pink precipitate characteristic of pulmonary edema. Marked passive congestion and edema from failure of the left ventricle of the heart. Lung - CLP+SL
H+E x 100 Normal pulmonary structure. The alveolar septa are uniform and no features of passive congestion or edema are evident.
Liver - control (sham)
H+E x 40 Plates of uniform hepatocytes arranged radially in a lobule. The sinusoids between the plates are uniform and are not distended. Liver CLP
H+E x 100 Prominent
vascular congestion of the centrolobular sinusoids, Liver - CLP
H+E x 400 Congestion of the sinusoids accompanied by attenuation and degenerative changes of hepatocytes. Changes of congestion result from right-sided cardiac decompensation. Liver - CLP+SL
H+E x 100
Intact hepatic architecture with no evidence of congestion of the sinusoids.
Kidney - control (sham)
H+E x 40 Two kidney glomeruli with proximal convoluted tubules which are lined by uniform intact cuboidal epithelial cells. Kidney - CLP
H+E x100 Kidney cortex tubules many of whose lining epithelial cells demonstrate swelling with numerous cytoplasmic vacuoles, changes which are consistent with tubular ischemic injury. 6734 - Kidney - CLP
H+E x 100 Pronounced congestion of the medullary vessels observed in shock.
Kidney - CLP+SL
H+E x100
Tubules lined by uniform intact epithelial cells.
Conclusion: Sections of the lung and liver of untreated animals following ligation and puncture showed pronounced changes related to cardiac failure resulting from sepsis. These sections of the kidney show both tubular ischemic damage, as well as changes related to shock. Sections of animals treated with glyco 23 following ligation and puncture appeared normal, very similar to shams, with no evidence of changes related to cardiac failure or shock.
In addition to histology data, mortality data from table 1 presents compelling evidence of the protective effects of sophorolipids. All treated animals survived 24 hours compared to controls (treated with saline solution) following CLP, which all died within 24 hours.
White Blood Cells (WBC) counts from blood samples obtained 24 hours post-insult in both groups are comparable to the number of WBC from 3 hour post-insult in the saline control group. (Table 1). As the rats in our saline control groups did not survive 24 hours, there is no comparative data at this time point. The WBC counts at 24 hours would have been expected to be much higher than the ones obtained after 3 hours post-insult. Our sham controls seem to show this trend as well. The low number of WBC in samples obtained 24 hour post-insult in animals treated with sophorolipids also indicate their protective effect.
Specific Aim 2. Determine most effective dose of glycolipid therapeutic lead compound(s) and the effect of delayed dosage survival
2.1. Dosage To determine the optimum dose that can be administered, 3 different doses 6 mg/kg, 12 mg/kg and 24 mg/kg of rat body weight (in 50 % ethanol-sucrose) were administered to three groups of five rats each using tail vein IV following the Cecal Ligation and Puncture (CLP). Control rats (n=5) were injected with 0.5 ml of (Ethanol-sucrose). Rats that received 24 mg/kg dose died within 2 hours indicating that this dose might be lethal, probably as a result of higher concentration and toxicity of the media components (Ethanol). Rats that received 6 mg/kg, and 12 mg/kg survived after 24 hours. Blood was obtained before they were sacrificed at 24 hours.
Mononuclear cell response to sophorolipids in rats: Peripheral blood mononuclear cells (PBMC) were obtained from rats 24 hrs after intravenous injection of sophorolipids. Blood (2 ml/animal) was collected with anticoagulant (EDTA, purple top tubes) and PBMC isolated by Ficoll-Hypaque discontinuous gradient centrifugation. PBMC from individual animals were placed in 24-well tissue culture wells in1 ml of minimal essential medium supplemented with 10% fetal calf serum, 2 mM glutamine and antibiotics (penicillin, 500 U/ml, streptomycin, 500 µg/ml, bacitracin, 25 µg/ml) (complete MEM). Cultures were incubated for 24 hrs at 37oC in a humidified atmosphere containing 5% CO2. Non-adherent cells were removed by pipetting; 1 ml of fresh complete MEM was added to each culture with the remaining adherent cells (monocytes), and these cultures were examined using an inverted microscope. A minimum of 4 microscopic fields were examined for each culture, and the number and appearance of adherent cells/field determined. The data are summarized in the table below (Table 2)
1. Control (Ethanol-sucrose)
2. Glyco (6 mg/kg, IV)
3. Glyco (12 mg/kg, IV)
Table 2. Mononuclear cell response to Glyco 23 in rats
Group Cells/field + SEM Estimated cell diameter (µm)
1 36+4 18
2 68+5 32
3 41+3 22
Monocyte appearance, numbers, and size suggest that at 6mg/kg, sophorolipids may cause monocyte activation. Culture supernatants will be collected at 5 days and the amounts of characteristic monocyte/macrophage activation products (nitric oxide, TNF-a) will be determined using the modified Griess reaction and ELISA, respectively. We determined that 12 mg/kg of rat body weight was the optimum dose and was used for the subsequent experiments.
2.2. Delayed dosage
We studied the effects of delayed sophorolipid administration in a different model of endotoxic shock. We used this model instead the previous cecal ligation model in order to trigger a very acute septic shock that reduces the time to endpoint. This model employs galactosamine-sensitized LPS endotoxic shock induction in a mouse model of disease (81). This model has been shown to increase animal sensitivity to the lethal effects of lipopolysaccharide several thousand fold. Therefore, treatment after 1.5 hr. can be compared to treatment after 24hrs or later in conventional models.
Table 3. Time of Injection (hours)
D-galactosamine CLP
CLP+ SL Mortality (%)
Sham Time 0 -- -- 0 (n=6)
Control non treated Time 0 Time 0 -- 83 (n=6)
Treated Time 0 Time 90 m. Time 0 80 (n=10)
treated Time 0 Time 0 Time 0 64 (n=9)
treated Time 0 Time 0 Time 90 30 (n=10)
In these studies sophorolipids administered to septic animals 1.5 hr after insult decreased endotoxin related mortality by 53% (Table 3) The fact that sophorolipids demonstrated such a robust response in an accelerated animal mortality model is remarkable and provides further support of therapeutic utility since it mimics the clinical situations in which therapy is applied at a time after sepsis occurs.
We have also demonstrates the effect of multiple sequential (q24 hr x 3 doses) IV dosing regimens of sophorolipid administration in septic rats (CLP) (79). 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) (79).
Doses were given at the end of the surgery, and animals were followed for 36 hours. Kaplan Meier statistics were performed on survival. Preliminary data in our lab (47) others (49, 50) showed that we can obtain reproducible mortality rates of 60% to 70% with a 16 gauge needle (Fig. 3).
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. 2). This significant improvement in survival was achieved with a single dose of natural 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. (38,39)
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 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 |
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.