Introduction Mouse Lethal Challenge Influenza Model ...

29 downloads 0 Views 681KB Size Report
The question as to which of the four isomers of beraprost sodium, GP1681,. GP1682, GP1683 or GP1684 would be a superior influenza therapy needed to be.
Mode of Action of GP1681 as a Therapeutic for Influenza Infections Daryl

1* Faulds ,

Hsiao-Lai

1 Liu ,

Jiing-Huey

1 Lin ,

Dale

2 Barnard

and William

1 Guilford

Gemmus Pharma Inc.1 San Francisco, California, United States, Utah State University2, Logan, Utah, United States * Corresponding author. Telephone: 415-978-2151; E-mail: [email protected]

Introduction The ‘flu-like-symptoms’ of influenza infection, high fever, headache, myalgia, fatigue and malaise, are independent of viral strain, but are typical of a proinflammatory response. Gemmus has identified a prostacyclin analog, beraprost sodium (GP1001) is an immunomodulator that has been shown to significantly decrease the level of pro-inflammatory cytokines in vitro and in other disease models. Since 2008, Gemmus-sponsored and NIH-funded studies have demonstrated that GP1001, Figure 1, increases survival, maintains lung function, and reduces the level of pro-inflammatory cytokines in mice infected with different strains of the Influenza A virus. The data supports the continued development of GP1001 as a treatment for influenza infections. The question as to which of the four isomers of beraprost sodium, GP1681, GP1682, GP1683 or GP1684 would be a superior influenza therapy needed to be addressed. Therefore, we sought to determine which isomer or mixture of isomers could ameliorate an Influenza A H5N1 virus infection in BALB/c mice without assistance of an antiviral agent. In addition we sought to determine if alternative endpoints such as cell infiltration into the lung and cytokine expression in the lung of infected mice significantly differs between placebo and treatment groups.

Mouse Lethal Challenge Influenza Model Virus: Influenza A/Duck/MN/1525/81 (H5N1) was obtained from Dr. Robert Webster of St. Jude Hospital, Memphis, TN. It was passaged through mice until adapted to the point of being capable of inducing pneumonia-associated death in the animals. When mice are exposed to lethal dose of the virus (1 X105 CCID50), infected animals die between days 4-8, with 90-100% mortality achieved by day 8 at this dose. The lungs are severely inflamed and exhibit extreme lung consolidation. Weight loss is excessive; animals that die lose greater than 30% of their total weight by day 7 after virus exposure.

**

****

****

Figure 2. Cytokine Inhibition Assay

****

****

****

On day 6 the lungs from 5 animals from each treatment group were harvested, weighed, scored for gross pathology (Figure 3), and then homogenized individually in 2ml DMEM. Homogenates were clarified by centrifugation and were assayed for cytokines (Meso Scale Diagnostics); (Figure 4). Homogenates were digested with Collagenase (Roche), red blood cells were removed and immune cells which make up the cellular infiltrate were isolated by centrifugation. Cell number per ml was determined by microscopic counting using Reichert Bright-Line hemacytometer.

PSS GP1681 GP1682 GP1683 GP1684 GP1001 Ribavirin

*

****

21 days after viral infection Influenza A/Duck/MN/1525/81 (H5N1) Female BALB/c Survival, lung score, lung weight 10 cytokines and chemokines 5 days for Ribavirin, 10 days for all others

**** **

**

****

**** **** **** **** **** **** ****

Phosphate Saline Solution (ip, bid) 0.8 mg/kg/day (ip, bid) 0.8 mg/kg/day (ip, bid) 0.8 mg/kg/day (ip, bid) 0.8 mg/kg/day (ip, bid) 1.6 mg/kg/day (ip, bid) 75 mg/kg/day (ip, bid)

* **

**

****

Mouse Survival after Viral Infection

Human PBMCs

Cmpd

EC50 (nM) 4 >10,000 22 >10,000 9

Death Parameter PSS GP1681 GP1682 GP1683 Total Deaths / # mice 19 / 20 6 / 10 9/9 10 / 10 Mean Day of Death ± SD 7.7 ± 1.3 11.0 ± 1.8** 7.1 ± 0.8 8.6 ± 3.5

Mouse RAW264 macrophage cell line EC50 GP1681: >100nM Method human PBMCs: Serial dilutions of test articles were added to cultured human peripheral blood mononuclear cells to yield 750 nM, 250 nM, 83 nM, 27.8 nM, 9 nM, 3 nM, 1 nM, and 0.34 nM. Poly r(I:C) (2 mg/ml) was added. Cultures were incubated 18 hr at 37oC and supernatants were harvested. ELISA were performed using commercial kits for human TNFa (R&D Systems). Cell assays were performed in triplicate. ELISA were performed in duplicate and the average of these values were plotted using the 4-parameter logistic non-linear model (GraphPad Prism). One representative donor, assay, and the ELISA plot is shown. The inhibition curves are sigmoidal over two log units. Method mouse RAW264 macrophage cell line: Same as above with the exception of cell type and mouse TNFa (R&D Systems) ELISA kit

****

GP1684

GP1001

10 / 10

7 / 10

7.2 ± 0.8

10.4 ± 2.1*

****

Figure 6. Correlation of Alternative Endpoints with Lung Weight

Pearson r = 0.80 p = 0.0001

Cell infiltrate level is associated with lung weight

Figure 5. Lung Cell Culture Cytokines

**

Treatment Groups

Results

Figure 4. Lung Cytokines

***

Groups of 15 BALB/c mice were administered GP1001 at 1.6 mg/kg/da i.p. and isomers at 0.8 mg/kg/da ip for 10 days beginning on day 0 the day of virus exposure to mice. Phosphate saline solution (PSS), representing the placebo group, was administered to 25 mice using the same dosing regimen as was used for GP1001. Similarly treated mice that were not exposed to virus were toxicity controls.

Duration Virus Mouse Endpoints Markers Dosing

Figure 1. Beraprost Sodium (GP1001)

Figure 3. Lung Parameters

Methods

Pearson r = 0.80 p = 0.0001

Cell infiltrate level is associated with chemokine CCL2 concentration

Methods: On day 6 the lungs from 5 animals from each treatment group were homogenized individually in 2ml DMEM. Lung Homogenates were digested with Collagenase (Roche) for 20 minutes at room temperature with gentle rocking. Each digested lung suspension was passed through a 40 mM cell strainer (Falcon) and the strainer was washed twice with phosphate buffered saline (PBS). Cells were collected by centrifugation at 400g. Red blood cells were removed by selective lysis using ammonium chloride solution. Immune cells were collected by centrifugation as above, and washed twice with PBS. Each cell culture was resuspended in 2ml RPMI plus 10% defined fetal bovine serum (HyClone), penicillin, streptomycin and beta mercaptoethanol. Cells were dispensed into 96 well cell culture plates in triplicate and incubated 18 hours at 37º 5% CO2 95% humidity. Culture supernatants were collected and frozen at -80º. Cytokine concentration was determined in duplicate by mouse multiplex ELISA (Quansys) , ANOVA statistical analysis by GraphPad Prism.

Summary

Pearson r = 0.80 p < 0.0001

Lung weight is associated with chemokine CCL2 concentration

Pearson r = 0.81 p < 0.0001

Lung weight is associated with cytokine IL-6 concentration

Methods: Data tables comprised of related individual mouse values from PSS, GP1681, GP1682 and Ribavirin treatment groups were created using GraphPad (Prism). One data point for each lung parameter and one ELISA value for lung homogenate made up the tables. Correlation coefficients were computed using Pearson correlation for two tailed P values. Discussion: The lung weight at necropsy was significantly correlated with the number of cells per mL determined for the cell suspension prepared from enyzymatically digested lung (p=0.0001). This correlation between a classic parameter of lung pathology is consistent with previous studies which showed an accumulation of mononuclear cells and neutrophils is associated with lung injury. The correlation between lung weight, lung cell infiltrate and chemokine CCL2 (MCP-1) levels is shown above. Our results are supported by a report that CCR2+ monocyte-derived cells are the predominant cause of lung edema during influenza infection and that such immune pathology is markedly abrogated in the absence of CCR2 (Lin et al. Monocyte-Derived Dendritic Cells and Exudate Macrophages Produce Influenza-Induced Pulmonary Immune Pathology and Mortality. The Journal of Immunology. v180: 2562-2572, 2008). CCR2 is the receptor for CCL2. The correlation between lung weight and cytokine IL-6 levels are shown above right. The significant correlation between the pulmonary pathology measure of lung weight, which is an indicator of lung edema, and the levels of cytokine and chemokine in the lung of infected mice is striking. Mice with the highest cytokine or chemokine levels and highest lung weight were from the placebo-treatment group, in which the survival rate was only 5%.

• Compound-treated mice had no adverse effects relative to placebo-treated mice • GP1681 is the most active isomer of GP1001 in human PBMC • In mouse lethal challenge influenza model • GP1681 is the most active isomer • GP1681 significantly decreased lung cytokine levels • GP1681 significantly decreased lung cellular infiltrate • GP1681 significantly increased MDD • GP1681 significantly decreased lung pathology