Diagnostic Method and Compounds for the Treatment of Infections Caused by a Pathogen

Abstract

The present invention provides a new family of compounds characterized as being long chain polyunsaturated fatty acids oxidized at certain positions, and which are useful for the treatment of infections caused by a pathogen in the respiratory system. Furthermore, the present invention shows a method of diagnosing and/or monitoring of this type of diseases to be treated with the compounds of the invention.

Claims

1-50. (canceled)

51. A method for diagnosing and/or monitoring the status of a patient suffering from a disease caused by the presence of a pathogen in the respiratory system based on the identification and quantification of the concentration of pro-resolutive lipid mediators in blood, plasma, and/or serum from said patient.

52. A method for diagnosing and/or monitoring the status of a patient according to claim 51, wherein the pro-resolutive lipid mediator is MaR1.

53. A method for diagnosing and/or monitoring the status of a patient according to claim 51, wherein the pro-resolutive lipid mediators are MaR1, and PDX.

54. A method for diagnosing and/or monitoring the status of a patient according to claim 51, wherein the pro-resolutive lipid mediators are RvD2, MaR1, and PDX.

55. A method for diagnosing and/or monitoring the status of a patient according to claim 51, wherein the pro-resolutive lipid mediators are RvD2, MaR1, and PDX, and the pro-inflammatory lipid mediator LTB4.

56. A method for diagnosing and/or monitoring the status of a patient according to claim 51, wherein the pro-resolutive lipid mediators are RvD2, MaR1, and PDX, and the pro-inflammatory lipid mediator LTB4, wherein the increase in the pro-inflammatory lipid mediator LTB4 is at least 100%, with respect to a healthy individual.

57. A method for diagnosing and/or monitoring the status of a patient according to claim 51, wherein the pro-resolutive lipid mediators are RvE1, RvD2, RvD4, MaR1, and PDX.

58. A method for diagnosing and/or monitoring the status of a patient according to claim 51, when a decrease of at least 30% is observed of the concentration in blood, plasma and/or serum of each of the following pro-resolutive lipid mediators RvE1, RvD2, RvD4, MaR1, and PDX, with respect to a healthy individual.

59. A method for diagnosing and/or monitoring the status of a patient according to claim 51, where the pro-resolutive lipid mediators are RvE1, RvD2, RvD4, MaR1 and PDX, and the pro-inflammatory lipid mediators are PGD2 and LTB4.

60. A method for diagnosing and/or monitoring the status of a patient according to claim 51, when a decrease of at least 40% is observed of the concentration in blood, plasma and/or serum of each of the following pro-resolutive lipid mediators RvE1, RvD2, RvD4, MaR1, and PDX, and an increase of at least 200% in each of the pro-inflammatory mediators PGD2 and LTB4, with respect to a healthy individual.

61. A method for diagnosing and/or monitoring the status of a patient according to claim 51, when a decrease of at least 50% is observed of the concentration in blood, plasma and/or serum of each of the following pro-resolutive lipid mediators RvE1, RvD2, RvD4, MaR1, and PDX, and an increase of at least 200%, and at least 100%, of the pro-inflammatory lipid mediators PGD2 and LTB4, respectively.

62. A method for diagnosing and/or monitoring the status of a patient according to claim 51, wherein the measurement of the concentration values of the pro-resolutive lipid mediators is carried out in plasma.

63. A method for diagnosing and/or monitoring the status of a patient according to claim 51, wherein the measurement of the concentration values of the pro-resolutive lipid mediators is carried out in serum.

64. A method for diagnosing and/or monitoring the status of a patient according to claim 51, wherein the blood sample is treated with an adenosine inhibitor selected from: adenosine deaminase (ADA), caffeine, 8-(3-chlorostyril)caffeine, 2-phenylaminoadenosine, 2-p-2-carboxyethylphenylamino-5′-N-ethylcarboxamide-adenosine, 5-N-ethylcarboxamide-adenosine, 5′-N-cyclopropyladenosine, 5′N-methylcarboxamide-adenosine, PD-1259444, 1,3-dipropyl-phenylxanthine, or 4-{2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a]-[1,3,5]-triazin-5-ilamine]ethyl}phenol (ZM 241385).

65. A method for diagnosing and/or monitoring the status of a patient according to claim 51, wherein the blood sample is treated with the adenosine inhibitor caffeine.

66. A method for diagnosing and/or monitoring the status of a patient according to claim 51, where the disease caused by the presence of a pathogen in the respiratory system is a disease that also occurs with cytokine release syndrome or cytokine storm syndrome in a mammal.

67. A method for diagnosing and/or monitoring of the status of a patient according to claim 51, wherein the disease caused by the presence of a pathogen in the respiratory system is selected from: pneumonia, bacterial pneumonia, viral pneumonia, or atypical pneumonia.

68. A method for diagnosing and/or monitoring the status of a patient according to claim 51, wherein the disease caused by the presence of a pathogen in the respiratory system is a viral pneumonia.

69. A method for diagnosing and/or monitoring the status of a patient according to claim 51, wherein the disease caused by the presence of a pathogen in the respiratory system is selected from COVID-19, SARS-CoV1, sepsis, Ebola, bird flu, smallpox, or common flu.

70. A method for diagnosing and/or monitoring the status of a patient according to claim 51, wherein the disease caused by the presence of a pathogen in the respiratory system is COVID-19.

Description

DESCRIPTION OF THE DRAWINGS

[0337] FIG. 1. Shows the weight variation of the group of animals infected with SARS-CoV-2, treated or not with a compound of formula (I), the methyl ester of 7R,14S-maresin 1 (MaR1 ME) and Lipinova® (a composition comprising the compound of formula (IV), 14S-hydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid, in the triglyceride form, or 14-HDHA), compared to the untreated group not inoculated (Control). Thus, it can be observed that body weight loss decreases significantly in animals exposed to SARS-CoV-2 treated with MaR1 ME and Lipinova®. In the case of groups treated with MaR1 ME, the improvement in weight loss is dose dependent, the highest dose of MaR1 ME leads to a greatly reduced body weight loss, of less than 5%.

[0338] FIG. 2. Shows the TNF-α cytokine profile obtained by the qRT-PCR assay in the lung tissues of SARS-CoV-2 infected hamsters treated and untreated at 4 dpi, 7 dpi and 14 dpi (n=6 per day). Treatments began on the second day after infection.

[0339] FIG. 3. Shows the effect produced by treatment with MaR1 ME and Lipinova® of infected animals, finding that the viral load in the lungs is significantly reduced after a few days of treatment.

EXAMPLES

Example 1. Determination of the Content of SPMs in Plasma

[0340] Obtaining plasma samples: For the present assay, blood was collected from (5) COVID-19 patients and (5) healthy subjects. For this, butterfly needles of 21 caliber were used to draw blood into a 10 ml syringe containing heparin (10 units/ml). The collected sample was then transferred to a 15 ml polypropylene tube and centrifuged for 20 minutes at 120G. The supernatant (plasma) was then collected with a Pasteur pipette and placed in another 15 ml polypropylene tube. The tube with the plasma was stored at −80° C.

[0341] Processing prior to LC-MS/MS analysis: the plasma sample was thawed at room temperature, centrifuged at 1000G for 30 minutes before solid phase extraction. Internal standards (500 pg each) d4-PGE2, d5-LXA4, d4-LTB4, d8-5S-HETE, and d5-RvD2, representing each region in chromatographic analysis, were added to the sample in 4V of methanol. To induce protein precipitation, the samples were kept at −20° C. for 45 min. The supernatant was taken and a solid phase extraction was carried out. The eicosanoids and SPMs bound to the matrix were dried and suspended in a methanol/H2O (1:1) mixture to be injected into a system of LC-MS/MS mass spectrometer of hybrid triple quadrupole SCIEX QTRAP 5500.

[0342] Concentration of SPMs (pg/ml) in human plasma samples from COVID-19 patients and healthy subjects (nd=not detected).

TABLE-US-00001 TABLE 1 Bioactive metabolome related to docosahexaenoic acid (DHA) RvD1 RvD2 RvD3 RvD4 RvD5 PDX MaRl Healthy 3.1 ± 0.5 3.1 ±0.5 0.4 ± 0.4 1.2 ± 0.4 1.8 ± 0.4 1.2 ± 0.7 0.6 ± 0.2 (n = 5) COVID-19 2.4 ± 0.6 2.1 ± 0.6 0.3 ± 0.3 0.3 ± 0.3 1.3 ± 0.4 0.5 ± 0.4 nd (n = 5)

TABLE-US-00002 TABLE 2 Bioactive metabolome related to eicosapentaenoic acid (EPA) RvE1 RvE2 RvE3 Healthy 4.0 ± 1.0 2.4 ± 1.1 1.3 ± 0.6 (n = 5) COVID-19 2.2 ± 1.0 2.0 ± 1.0 1.6 ± 0.7 (n = 5)

TABLE-US-00003 TABLE 3 Bioactive metabolome related to arachidonic acid (AA) LXA.sub.4 LXB.sub.4 LXB.sub.2 PGD.sub.2 PGE.sub.2 PGF.sub.2α LTB.sub.4 Healthy nd nd 3.1 ± 1.3 5.5 ± 2.2  3.6 ± 1.1 nd 1.4 ± 0.7 (n = 5) COVID-19 5.2 ± 0.6 2.7 ± 0.2 7.9 ± 2.3  24 ± 4.4 12.4 ± 2.5 2.7 ± 0.3 3.7 ± 0.6 (n = 5)

[0343] The analysis of the bioactive metabolome of Covid-19 patients shows a number of differences from the metabolome of healthy people. In the case of the compounds of the present invention, represented by the compound of formula (I), 7R,14S-dihydroxy-4Z, 8E,10E,12Z,16Z,19Z-docosahexaenoic acid (MaR1), it is surprisingly observed that unlike healthy individuals, this lipid mediator is not present in blood (plasma) or, if it is found, it does so at undetected concentrations, in Covid-19 patients.

[0344] In addition, marked decreases are observed in the concentration of RvE1 (−45%), RvD2 (−32%), RvD4 (−75%), and PDX (−58%) in plasma. These highly differentiated values between healthy people and Covid-19 patients indicate that these pro-resolution lipid mediators are relevant in this pathology. A decrease in the values of these lipid mediators in a subject are indicative of suffering from a disease caused by a pathogen in the respiratory system, while a patient suffering from one of the diseases described in the present invention whose levels of RvE1, RvD2, RvD4, PDX, and MaR1 are increased over time will be indicative of patient improvement.

[0345] On the other hand, a substantial increase is observed in two pro-inflammatory lipid mediators, PGD2 (+436%), and LTB4 (+264%). Therefore, these lipid mediators are relevant in this pathology. Its decrease in time will indicate that the patient tends towards an optimal or healthy state, while its increase is indicative of a worsening of the patient.

Example 2

Virus

[0346] SARS-CoV-2 virus was isolated from the nasopharyngeal aspirate specimen of a laboratory-confirmed COVID-19 patient. The plaque purified viral isolate was amplified by one additional passage in VeroE6 cells to make working stocks of the virus as described previously. (Chan J F, Yip C C, To K K, et al. Improved molecular diagnosis of COVID-19 by the novel, highly sensitive and specific COVID-19-RdRp/Hel real-time reverse transcription-polymerase chain reaction assay validated in vitro and with clinical specimens. J Clin Microbiol., 2020; March 4).

Animal Experiments

[0347] Male LVG Golden Syrian Hamster (Body Weight of 100-150 g, from Charles River, strain code 049) were kept in biosafety Level-2 housing and given access to standard pellet feed and water ad libitum for 6-10 days prior to the virus challenge, which was made in biosafety Level-3 animal facility. Phosphate-buffered saline (PBS) was used to dilute virus stocks to the desired concentration, and inocula were back-titrated to verify the dose given. Dulbecco's Modified Eagle Medium (DMEM) containing 105 plaque-forming units in 100 μl of SARS-CoV-2 was intranasally inoculated under intraperitoneal ketamine (200 mg/kg) and xylazine (10 mg/kg) anesthesia. Mock-infected animals were challenged with 100 ul of PBS. During all the experiment, animals were monitored twice daily for clinical signs of disease. Their body weight and survival were monitored for 14 days post-inoculation.

[0348] Seventy-six (76) hamsters were inoculated with SARS-CoV-2 virus titrated solution on Day 0, twelve (12) animals were mock inoculated the same day. Body weight and clinical signs were evaluated daily and twice daily respectively, starting three days before inoculation (Day −3). On day 2 post inoculation, four (4) inoculated animals and three (3) mock inoculated animals were sacrificed were euthanized by intra-peritoneal injection (i.p.) of pentobarbital at 200 mg/kg and appropriate tissues (nasal turbinate, trachea, lungs, selected portion of GI tract) were collected for viral load and histopathology. Blood profiles of selected cytokine/chemokine (TNF-α, IL-6, IL-1, IL-8, and MCP-1) and compounds of the present invention (Methyl 7R,14S-dihydroxy-4Z,8E,10E,12Z,16Z,19Z-docosahexaenoate or MaR1 ME which is a compound of formula (I), and Lipinova®, which is a composition comprising 14S-hydroxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid or 14-HDHA, which is a compound of formula (IV), in the triglyceride form) were also measured. The remaining inoculated animals (72) animals were separated into four (4) cohorts of eighty (18) animals. Two cohorts were treated twice daily (i.p.) with MaR1 ME (at dosages of 0.05 and 0.5 mg/kg), one cohort was treated twice daily i.p. with vehicle, and the remaining group was treated twice daily (by gavage) with Lipinova®.

[0349] On day 4, six (6) animals from each inoculated cohorts (treated and non-treated) and three (3) mock inoculated animals were sacrificed, appropriate tissues were collected for viral load and histopathology, blood cytokine/chemokine profiles were measured. On day 7, six (6) animals from each inoculated cohorts (treated and non-treated) and three (3) mock inoculated animals were sacrificed, appropriate tissues were collected for viral load and histopathology, blood cytokine/chemokine and SPM profiles were measured.

[0350] On day 14, six (6) animals from each inoculated cohorts (treated and non-treated) and three (3) mock inoculated animals were sacrificed, appropriate tissues were collected for viral load and histopathology, blood cytokine/chemokine and SPM profiles were measured. Blood and major organ tissues at necropsy were separated into two parts, one immediately fixed in 10% PBS-buffered formalin, the other immediately frozen at −80° C. until use.

TABLE-US-00004 TABLE 4 General description of the animal test. Group Group No - Inicio No - No - No - Code No description Inoculation Day 2 Tratamiento Treatment Day 4 Day 7 Day 14 1 12 Inoculados PBS 3* 2 day post Vehicle 3 3 3 simulados inoculation control 2 19 Inoculados COVID-19 1* Vehicle 6 6 6 control 3 19 Inoculados 1* Mar1 ME 6 6 6 tratados 0.05 mg/kg 4 19 Inoculados 1* Mar1 ME 6 6 6 tratados 0.5 mg/kg 5 19 Inoculados 1* Lipinova ® 6 6 6 tratados (1.20 mg/kg) *Basal injury

Variation in Body Weight

[0351] The variation of the body weight of the test animals of the group of animals infected with SARS-CoV-2, treated or not with a compound of formula (I), MaR1 ME and Lipinova® (composition comprising 14-HDHA, a compound of formula (IV), in the triglyceride form), and the results were compared with those of the uninoculated and untreated group (Control). Thus, animals inoculated with SARS-CoV-2 but not treated exhibited a progressive loss of body weight of up to ˜15% between days 1 and 5 post injection (dpi), and then gradually regained their weight until 14 dpi. These animals developed lethargy, curly skins, hunched posture, and rapid breathing from 2 dpi, and began to recover at 7 dpi. While in animals exposed to SARS-CoV-2 and treated with MaR1 ME and Lipinova®, it can be observed that the loss of body weight decreases significantly. In the case of the MaR1 ME treated groups, the improvement in weight loss is dose dependent, the highest dose of MaR1 ME leading to the lowest body weight loss, which was less than 5%. None of the SARS-CoV-2 infected or mock animals died.

Histopathology and Immunohistochemistry.

[0352] Tissue (nasal turbinate, trachea, lungs, selected portion of gastrointestinal tract) were fixed in 4% paraformaldehyde and were processed for paraffin embedding. The 4 μm sections were stained with hematoxylin and eosin for histopathological examinations.

[0353] For immunohistochemistry, SARS-CoV-2 N protein was detected using monoclonal antibody (4D11) (Nicholls, M. et al. “Time course and cellular localization of SARS-CoV nucleoprotein and RNA in lungs from fatal cases of SARS”. PLoS Med 3, e27, (2006)).

[0354] Cytokine/chemokine profile Chemokine/cytokine profiling was performed on the lung tissues and blood of the virus-challenged and mock-infected animals by qRT-PCR (Espitia C M, Zhao W, Saldarriaga O, et al. “Duplex real-time reverse transcriptase PCR to determine cytokine mRNA expression in a hamster model of New World cutaneous leishmaniasis”. BMC Immunol 2010; 11:31).

[0355] Both treatment with MaR1 ME and Lipinova® have both a significant impact on the chemokine and cytokine profile in infected animals. FIG. 2 specifically illustrates the increased profile of the cytokine TNF-α in the lungs of the group of animals infected with SARS-CoV-2 where the animals that have been treated in parallel with MaR1 ME and Lipinova® present levels of TNF-α significantly lower than untreated animals.

[0356] Viral load determination by quantitative real-time RT-PCR.

[0357] RNA was extracted from 140 μL tissue homogenate using QIAamp viral RNA mini kit (Qiagen) and eluted with 60 μL of water. The N gene of SARS-CoV-2 virus was detected and quantified using TaqMan™ Fast Virus 1-Step Master Mix as described (Chu, K. W. et al. “Molecular Diagnosis of a Novel Coronavirus (2019-nCoV) Causing an Outbreak of Pneumonia”. Clin Chem, doi:10.1093/clinchem/hvaa029 (2020).

[0358] MaR1 ME and Lipinova® treatments have a significant impact on viral load in tissues. FIG. 3 shows that in infected animals treated with MaR1 ME and Lipinova®, the viral load in the lungs is significantly reduced after a few days of treatment.