DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Priority
The instant application, filed on February 9, 2024, is a national stage entry of International Application No. PCT/EP2022/072963, filed August 17, 2022, which claims priority to European Patent Application No. EP21191639.0, filed on August 17, 2021. Receipt was acknowledged of certified copies of papers required by 37 CFR § 1.55.
Status of Claims
Applicant’s amendments to claims, received on June 22, 2026, are acknowledged and entered. Claims 1 and 6 are amended.
Claims 1-13 are pending.
Information Disclosure Statement
The Information Disclosure Statement received on June 22, 2026, is acknowledged and found to be in compliance with the provisions of 37 CFR § 1.97. Accordingly, the Information Disclosure Statement has been considered.
Claim Rejections - 35 USC § 112 (b) - Withdrawn
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Response to Arguments
Applicant’s arguments, see Remarks Received 06/22/2026, at 5-6 of 18, with respect to the rejections of claims 1 and 6 under 35 U.S.C. 112(b) have been fully considered and are persuasive. The rejections under 35 U.S.C. 112(b) have been withdrawn in view of the present amendments to the claims.
Claim Rejections - 35 USC § 103 – Maintained / Necessitated by Amendment
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Previous Grounds of Rejection are Maintained
Claims 1-5 and 7-13 are rejected under 35 U.S.C. 103 as being unpatentable over US’404,1 in view of Theriault 2012,2 the ML228 in vivo Studies,3 and Li 2021,4 and in further view of Wise 2021.5
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over US’404, in view of Theriault 2012, the ML228 in vivo Studies, Li 2021, and Lunn 2011,6 and in further view of Wise 2021.
The rejections are reiterated following the Response to Arguments.
Response to Arguments:
Applicant's arguments, see Remarks Received 06/22/2026, at 6-17 of 18, with respect to the rejections of claims 1-5 and 7-13 under 35 U.S.C. 103 over US’404, in view of Theriault 2012, the ML228 in vivo Studies, and Li 2021, and in further view of Wise 2021, and claim 6 over US’404, in view of Theriault 2012, the ML228 in vivo Studies, Li 2021, and Lunn 2011, in further view of Wise 2021, have been fully considered but they are not persuasive.
First, in Remarks Received at 6-7, Applicant distinguishes “activation” from “stabilization” within the context of the claimed invention.
As background to the following discussion, Applicant’s claims are drawn to the use of the research compound and active pharmaceutical ingredient (“API”) ML228 for the treatment or prophylaxis of certain “neurotropic” viral infections. ML228 has the following chemical structure:
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ML228 is an established, off the shelf research compound that was developed by the inventors of Theriault 2012. The ML228 in vivo Studies exemplified its use as an API in models of human disease. ML228 is known to “activate” hypoxia-inducible factor 1-alpha. See, e.g., Theriault 2012 at Title (“Discovery of a New Molecular Probe ML228: An Activator of the Hypoxia Inducible Factor (HIF) Pathway”). For clarity, hypoxia-inducible factor 1-alpha is abbreviated in different ways by different authors, e.g., HIF-1α. Further, when the art discusses activation of HIF-1α, it does so interchangeably with the term “stabilization” of HIF-1α.
However, as the examiner understands, Applicant utilizes a more nuanced definition of the term “activation” within the context of its invention. While Applicant’s position is acknowledged, the previous grounds of rejection do not turn on the particular definition of “activation”. The claims require administering ML228 to certain patient populations. The references teach the administration of ML228 to the claimed patient populations. Further, the references teach that ML228 activates HIF-1α.
Second, beginning in Remarks Received at 7, Applicant argues that US’404 is not enabled because “none of the working examples provided in US’404 show any anti-viral effect of HIF1-alpha activation.” Applicant’s position is not persuasive because it would not take undue experimentation to test a compound which stabilizes HIF-1α under normoxic conditions against viruses as expressly taught by US’404, claim 20. Further, US’404 explained the mechanism by which stabilized/activated HIF-1α triggers the immune system to engage in the killing of “microbial pathogens”. As the examiner pointed out, US’404 states that the killing of “microbial pathogens” within the context of US’404 means the killing of “bacterial and viral pathogens”. Further, as the examiner explained, had ML228 been invented before US’404 was filed, US’404 would have anticipated the instant claims.
Second, in Remarks Received at 8-9, Applicant argues that the reference Reyes A, et al. published 2020, hereinafter “Reyes 2020”, teaches in its “review of HIF-1 and its relevance to viral infections”, that results from others teach or suggest that upregulating HIF-1 would actually have a beneficial effect on certain viruses (e.g., causing them to replicate). Reyes 2020 was not cited in the rejections of the instant claims.
However, Applicant’s position is not persuasive because Reyes 2020 reviewed the art prior to the publication of Wise 2021. Wise 2021 advances the understanding of the role of HIF-1α in viral infections.
For example, while Reyes 2020 at 1487 states “that HCMV infection may be favored by HIF-1 activation”, Wise 2021, published after Reyes 2020, explains that the prior studies which proposed that HIF1-alpha was induced by HCMV to support virus replication and pathogenesis were incorrect. Indeed, Wise 2021 expressly states that its “observation suggests that HIF1α activity in infected cells alters metabolism as a protective strategy to limit viral infection”. Wise 2021 therefore is entirely consistent with the broad-spectrum anti-viral activity taught in US’404.
Further, because Wise 2021 corrected the past misunderstanding of the role of HIF-1α in viral infections, it calls into question the other prior studies reviewed in Reyes 2020 that contradict the observation from Wise 2021. E.g., Reyes 2020 at 1489 indicates that for H1N1, deficiency in HIF1-alpha facilitates viral replication. That result required reevaluation in view of Wise 2021. See also, HHV-1, see Reyes 2020 at 1487 (inconclusive effects of HIF1-alpha on HHV-1), also requiring reinvestigation.
Other results reviewed in Reyes 2020 were consistent with both US’404 and Wise 2021. E.g., regarding VSV, Reyes 2020 at 1491 teaches that inhibiting HIF-1a “enhanced the infection of cells with this virus”. It follows that activating HIF-1a may dampen infection with this virus. Such a result would be consistent with both US’404 and Wise 2021, and provide an even stronger reasonable expectation of success in making the claimed invention (see VSV recited in the instant claim 1).
Third, in Remarks Received at 9-10, Applicant argues that its submission of unpublished data that was not disclosed in the Specification pertaining to the APIs daprodustat and molidustat confirms that HIF activators “that act through PHD inhibition” do not show a consistent ability to reduce viral replication. Applicant’s position is not persuasive because 1) ML228 was not known to act through PHD inhibition, so the data from daprodustat and molidustat are of limited relevance, 2) this new evidence was submitted without an appropriate affidavit / declaration, and 3) a reasonable expectation of success does not require absolute certainty. This is shown even in Applicant’s data. While the graph is blurry, it appears that daprodustat suppressed viral replication in HSV-2.
Fourth, in Remarks Received at 10-12, Applicant argues that there is “no teaching or suggestion, pointers or links between US’404 and Theriault 2012”. Applicant’s position is not persuasive because US’404 teaches the administration of a compound which stabilizes HIF-1α under normoxic conditions. Theriault 2012 teaches the compound ML228 which stabilizes HIF-1α under normoxic conditions. The other references, e.g., the ML228 in vivo Studies, teach that ML228 was commercially available. Therefore, ML228 represented a convenient activator of HIF-1α that one could utilize in a drug repurposing strategy, as outlined in Li 2021. Moreover, given its ease of access, anyone seeking to confirm the findings of Wise 2021 would have been able to access it with ease.
Further, US’404 teaches the administration of such a compound as a broad-spectrum antiviral agent. As the examiner stated, had ML228 been invented before US’404 was filed, US’404 would have anticipated the instant claims. The inventors of US’404 listed many examples of compounds that were known in the art. ML228 represented a commercially available research compound and API that was optimized by its inventors for activating HIF-1α. The inventors of US’404 would have listed it in their Specification because of how well it performed both in vivo and in vitro.
Fifth, in Remarks Received at 11-12, Applicant argues that “the precise biological mechanism of action of ML228 remains unclear…”, and cites Farahani et al., which published in 2024, in support of its position. Applicant’s position is not persuasive because
Applicant does not explain how a publication that disclosed nuanced applications of ML228 after the filing of the claimed invention impacts the examiner’s conclusion that one of ordinary skill in the art at the time of filing would have a reasonable expectation of success in making the claimed invention, and
Even if this hypothetical person had the ability read Farahani et al., the paper does not refute the clear findings of Theriault 2012, which provide in Table 4 at 79 stellar EC50s for HIF-1α nuclear translocation and RT-PCR VEGF. Applicant’s focus on the “iron chelation” mechanism appears misplaced as it is not required for the mechanism of US’404 to occur.
Sixth, in in Remarks Received at 12-13, Applicant acknowledges that it has amended its claims to remove HHV-5 (HCMV) as virus treated in its methods. Applicant does not expressly admit that Wise 2021 shows that there was a reasonable expectation of success in treating HCMV in its original claims. Applicant instead states “the relevance of Wise 2021 is eliminated” (emphasis in the original). Applicant’s position is not persuasive because contrary to Applicant’s assertion, as explained above in the discussion of Reyes 2020, the disclosure of Wise 2021 called into question the accuracy of reports that indicated activating HIF-1α instead enhanced viral replication. Wise 2021 in fact corrected Reyes 2020 with respect to HCMV. Accordingly, Wise 2021 remains highly relevant to the claimed invention. Further, as stated, its observation at page 2 that “suggests that HIF1α activity in infected cells alters metabolism as a protective strategy to limit viral infection” was entirely consistent with US’404.
Seventh, in Remarks Received at 14-15, Applicant argues that the ML228 in vivo Studies (teaching that ML228 was a commercially available activator of HIF-1α that had been extensively investigated in vivo), and Li 2021 (teaching the drug repurposing strategy of taking well-characterized APIs and repurposing them as anti-viral agents) do “not appear to remedy the shortcomings [of the combination of US’404 and Theriault 2021].”
Applicant’s first position articulated in this section is against the cumulative number of references applied in the obviousness analysis.
Applicant’s first position against the cumulative number of references applied in the obviousness analysis of its claims is not persuasive. In response to Applicant's argument that the examiner has combined an excessive number of references, reliance on a large number of references in a rejection does not, without more, weigh against the obviousness of the claimed invention. See In re Gorman, 933 F.2d 982, 18 USPQ2d 1885 (Fed. Cir. 1991).
Further, Applicant claims broad-spectrum anti-viral activity in its invention. The combination of the references captures the claimed broad-spectrum anti-viral activity.
Applicant’s second position is in this section is that the ML228 in vivo Studies fail to disclose anti-viral use of ML228. Applicant’s second position in this section is not persuasive because the drug repurposing strategy of Li 2021 does not require selection of drugs previously utilized for their anti-viral activity.
Applicant’s third position in this section is consistent with the second, and is that Li 2021 exemplifies the drug repurposing strategy with known antivirals that have had a Phase I/Phase II trial. Applicant’s position is not persuasive because
Li 2021 at 2 expressly states that “Drug repurposing (also called drug repositioning) is a strategy for identifying new uses for approved or investigational drugs that [sic] beyond the original indicative scope to facilitate antiviral development”,
Li 2021 does not require an original antiviral indication for the repurposed drug,
Li 2021 does not require a public Phase I trial for the repurposed drug,
The examiner acknowledged in the Non-Final rejection that even though there was no formal designation of ML228 as an “investigational drug”, it was a distinction without a difference because independent new drug (IND) applications concerning new “investigational drug[s]” are held in confidence by regulatory authorities, and
The ML228 in vivo Studies show that ML228 was investigated in various models of human disease.
Eighth, in Remarks Received at 15-16, Applicant argues that in light of the arguments and amendments to the claims, that the references fail to establish obviousness of the instantly claimed invention.
Applicant’s first position in this section is that “the mechanism described in US’404 can never raise the amount of HIF1-alpha, as it can merely stabilize existing proteins, contrary to the mechanism found for ML-228 in the present application.” Applicant’s position is not persuasive because the mechanism of US’404 engages the immune system to engage in microbial killing. Applicant’s arguments regarding the mechanism of ML-228 activation appear misplaced because it regards an inherent property of ML-228 that is neither required for the mechanism of US’404 nor the instant claims.
Applicant’s second position in this section is that the rejections require hindsight. Applicant’s second position is not persuasive because it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Here, Applicant has offered no argument that the rejections contained knowledge gleaned only from the Applicant's disclosure. Instead, the prior art established that there was a reasonable expectation of success in making the claimed invention at the time of filing.
Applicant’s third position in this section restates its main positions, that 1) “US’404 explores anti-bacterial properties of activating the HIF1-alpha pathway”, 2) “there is no link between US’404 and Theriault 2012”, 3) “Wise 2021 specifically related to HCMV, which is no longer part of the claimed subject matter”, 4) “Li 2021 teaches that existing antivirals may be repurposed”, and 5) the “ML228 in vivo studies … are not relevant in combination with Li 2021”.
The examiner has addressed each of Applicant’s five positions in the above Response to Arguments, and finds each of them not persuasive.
Ninth, in Remarks Received at 16-17, Applicant argues that the reference Lunn 2011 teaching the link between CMV and GBS is no longer relevant because CMV and CBS were amended from the claims. Applicant’s position is not persuasive because Applicant did not remove “inflammatory neuropathies” from the list of diseases treated or prevented by administering ML228. Lunn 2011 explains that these neuropathies are prevalent in patients with CMV, and the instant claim 6 still reads on administering ML228 to treat or prevent “inflammatory neuropathies”, irrespective of the specific route of viral infection.
The examiner recommends that Applicant amend ML228 out of its claims. The drug-repurposing position does not apply to certain other embodiments of the claimed invention, such as select formula (II) compounds disclosed in the Specification at 11-12. Using ML228 as a commercially available research compound and a known API to confirm the studies of Wise 2021 in other viruses was obvious at the time of filing.
Reiterated Rejections:
Claim Rejections - 35 USC § 103 – Maintained / Necessitated by Amendment
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Rejection Statements
Claims 1-5 and 7-13 are rejected under 35 U.S.C. 103 as being unpatentable over US’404, in view of Theriault 2012, the ML228 in vivo Studies, and Li 2021, and in further view of Wise 2021.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over US’404, in view of Theriault 2012, the ML228 in vivo Studies, Li 2021, and Lunn 2011, and in further view of Wise 2021.
Initial Discussion of Claim 1
Claim 1 is generally directed to repurposing an off the shelf, commercially available research compound, ML228, which is a known activator of hypoxia-inducible factor 1-alpha,7 as a “broad-spectrum antiviral compound”.8
In particular, claim 1 recites a method of treatment or prophylaxis of a neurotropic viral infection caused by a neurotropic virus,9 wherein said treatment or said prophylaxis comprises administering to a subject a compound which is an activator of hypoxia-inducible factor 1-alpha (HIF1-alpha) of formula (I):
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The instant Specification discloses the structure, biological activity towards HIF1-alpha, and data from in vitro methods of using compounds of formula (I) for a single compound, ML228, reproduced below from Specification at 12.
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Specification at 12.
Claim 1 previously stated that the neurotropic virus is selected from the group consisting of human alphaherpesvirus 1 (HHV-1), human alphaherpesvirus 2 (HHV-2), human alphaherpesvirus 3 (HHV-3), human betaherpesvirus 5 (HHV-5), human betaherpesvirus 6A (HHV-6A), human betaherpesvirus 6B (HHV-6B), human betaherpesvirus 7 (HHV-7), human gammaherpesvirus 8 (HHV-8), human adenovirus (HAdV), Monkeypox virus Zaire-96-1-16, Human mastadenovirus, Vaccinia virus, horsepox virus HSPV0S0, cowpox virus, variola virus, human enterovirus, human rhinovirus, human papillomavirus, severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), encephalomyocarditis virus (EMCV), poliovirus, influenza A virus, influenza B virus, human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), vesicular stomatitis, Indiana virus (VSV or VSIV), and rabies lyssavirus.
Applicant amended human betaherpesvirus 5 (HHV-5) from its claimed indications following the Non-Final rejection mailed 03/27/2026.
The Specification at 6 explains that a neurotropic virus is one capable of infecting nerve cells:
In the present context, the term "neurotropic virus" refers to a virus that is capable of infecting nerve cells. A neurotropic virus may also possess other capabilities apart from being able to infect nerve cells. For example, a neurotropic virus may also be able to infecting other cell types, such as for example epithelial cells.
Specification at 6.
The Specification further explains that cytomegalovirus is an example of a neurotropic virus that may cause a neurotropic viral infection.
The neurotropic virus infection may be caused by genus specific neurotropic virus. An embodiment of the present invention relates to the compound for use as described herein, wherein the neurotropic virus is a member of a genus selected from the group consisting of simplexvirus, varicellovirus, lymphocryptovirus, cytomegalovirus, roseolovirus, rhadinovirus, alphacoronavirus, betacoronavirus, gammacoronavirus, deltacoronavirus, aphtovirus, avihepatovirus, cardiovirus, enteroviruses, erbovirus, hepatovirus, kobuvirus, parechovirus, tescovirus, tremovirus, sapelovirus, senecaviruses, alphainfluenzavirus, betainfluenzavirus, gammainfluenzavirus, deltainfluenzavirus, lentivirus, flavivirus, alphavirus, rubivirus, vesiculovirus, and lyssavirus.
Specification at 13 (emphasis added).
Human cytomegalovirus, is typically abbreviated as “HCMV”, and is also known as human betaherpesvirus 5, which is typically abbreviated as “HHV-5”. See NCBI entry for Human betaherpesvirus 5, attached hereto.
Claim Interpretation
The examiner finds that “stabilization” and “activation” of HIF1-alpha are synonymous.
First, the examiner further notes that hypoxia-inducible factor 1-alpha is abbreviated in different ways by different authors. While the examiner attempts to stay consistent with Applicants abbreviation, i.e., HIF1-alpha, the examiner notes that other authors refer to the same hypoxia-inducible factor 1-alpha as HIF-1α, or HIF1α, or similar variants thereof.
Claim 1 states that a compound of formula (I) is an “activator of hypoxia-inducible factor 1-alpha (HIF1-alpha)” (emphasis added). As explained above, a compound of formula (I) encompasses ML228. Accordingly, Applicants state that ML228 “activates” HIF1-alpha.
As discussed below in Theriault 2012, “ML228 was demonstrated to potently activate HIF in vitro….” Theriault 2012 at 81 (emphasis added). “HIF” is hypoxia-inducible factor, and one type of HIF is HIF1. Hypoxia-inducible factor 1-alpha (“HIF1-alpha”) is a subunit of hypoxia-inducible factor 1 (“HIF1” or “HIF-1”).10
Theriault 2012 explains that ML228 activates HIF through iron chelation. See Theriault 2012 at 79-80. “Activating HIF” through iron chelation is synonymous with “activating HIF1-alpha” and “stabilization of HIF-1α”, and other various variants thereof. Theriault 2012 at 76.11
Accordingly, by stating that ML228 “activates” HIF1-alpha, Applicants are also stating that ML228 “stabilizes” HIF1-alpha, which is consistent with Theriault 2012.
US’404
Generally, the idea of using activators of HIF1-alpha to treat viral infections is not new.
For example, US’404 teaches the use of activators of HIF1-alpha as broad-spectrum antiviral compounds for treating viral infections. US’404 discloses methods for the prophylaxis and/or treatment of and infection or virulence in a subject in need thereof, comprising administering to said subject a pharmaceutically effective amount of a HIF-1 modulating compound. See, e.g., US’404 at 35, claim 8. Claim 20 explains that the HIF-1 modulating compound is a compound which stabilizes HIF-1α under normoxic conditions. Id.12
US’404 explains that such methods are effective against a broad-spectrum of viruses:
In yet another embodiment, the microbes to be tested or treated are viruses. There are a number of viruses that are recognized and affected by the innate immune system, particularly by macrophage activity. Viruses that are contemplated include, but are not limited to, retroviruses proviruses, lentivriurses such as immunodeficiency viruses, togaviruses. Togaviruses, as used herein includes the Togaviridae family, including the Alphavirus and Rubivirus genera, as well as the flavivirus family (Flaviviridae) and the Flavivirus and Pestivirus genera. A review of virus taxonomy and the biology of these viruses may be found at, e.g., B. N. Fields, et al., editors, Fundamental Virology, 3rd edition, 1996, Lippencott-Raven Publishers, chapter I (pages 15-58) and chapter 17 (pages 523-540), which is incorporated herein by reference.
US’404 at 10, paragraph [0075] (emphases added).
US’404 at 7-8 discloses the following reasons why one of ordinary skill in the art at the time of filing would be motivated to administer an activator of HIF1-alpha to treat virulence in subjects.
[0064] Our studies have used conditional gene targeting in the myeloid cell lineage to demonstrate that HIF-1α transcriptional regulation plays an important role in innate immunity to infection. Activation of HIF-1α under hypoxia enhances microbicidal activity, and HIF-1α pathways are responsive to microbial stimulation even under normoxia. While certain myeloid cell functions including endothelial transmigration and respiratory burst activation appear independent of HIF-1α control, the present invention describes, without being limited to any particular mechanism, that the transcription factor HIF-1α is involved directly or indirectly in the regulation of specific immune functions including NO, granule proteases (cathepsin G, neutrophil elastase) and cathelicidin antimicrobial peptides. The marked reduction of granule protease and cathelicidin expression in HIF-1α-deficient neutrophils correlates to diminished microbicidal activity in vitro and failure to control infection in vivo, lending support to recent studies uncovering a key role for these neutrophil effectors in mammalian innate immunity (15, 24). The effectiveness of neutrophils and macrophages in innate antimicrobial defense reflects a diverse array of highly specialized cellular functions including phagocytic uptake of the microbe, production of reactive oxygen species, activation of iNOS, and release of antimicrobial peptides (e.g., cathelicidins, defensins) and granule proteases (e.g., elastase, cathepsin).
[0065] Successful control of infection in the peripheral tissues requires that host myeloid phagocytic cells function effectively in hypoxic environments. The challenge to immune defense is made more critical when the microbial toxins or local edema damage host cells and the vascular supply of oxygen to the tissues becomes further compromised. The placement of essential microbial killing functions of myeloid cells under regulation of HIF-1α therefore represents an elegant controlled-response system (FIG. 8). Microbicidal mechanisms can be maintained in an "off" state while the myeloid cells circulate in the oxygen-rich bloodstream, and then be activated in response to the declining oxygen gradient encountered upon diapedesis and entry into the infected tissues. Additional more potent stimulation of the HIF-1α transcriptional pathway is then provided by direct encounter with the microbe (FIG. 1A). A regulatory mechanism by which HIF-1α targets genes involved in microbial killing ensures that the corresponding inflammatory mediators are expressed preferentially in tissue foci of infection, but not in healthy tissues where inflammatory damage might otherwise harm host cells.
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[0066] Our experiments also reveal that NO production is a myeloid cell killing mechanism principally regulated by HIF-1α during microbial infection. Further, we suggest that NO is likely to play a key role in the amplification of the inflammatory response through stimulation of TNF-α. Although the effects of inflammatory cytokines on regulating NO production have been extensively studied (38-40), the reverse relationship, pertaining to the effect of NO on cytokines, remains controversial (41-44). A recent study demonstrated that suppression of NO could inhibit LPS-induced TNF-α and interleukin-1 release, and pinpointed such modulation to the pretranslational level (45). We find here that macrophage production of TNF-α is dependent on NO levels controlled in turn by HIF-1α-transcriptional regulation of iNOS.
[0067] Recent data has established that HIF-1α is subjected to stability regulation by soluble intracellular messengers, such as NO and TNF-α (33, 34). With such processes at play, one can envision that HIF-1α is situated at the center of an amplification loop mechanism for innate immune activation: stimulation of HIF-1α by oxygen depletion and microbial exposure induces the production of NO and TNF-α, which function not only to generate inflammation and control bacterial proliferation, but also as regulatory molecules to further stabilize HIF-L a in myeloid cells recruited to the infectious focus.
[0068] The relative contributions of HIF-1 and HIF-2 to the regulation of gene expression in hypoxic macrophages is still under debate. Detectable levels of HIF-2α, but not HIF-1α, have been found in a human promonocytic cell line following hypoxic induction in vitro and in tumor-associated macrophages (46, 47). In contrast, immunoreactive HIF-1α has been detected in human macrophages in the hypoxic synovia of arthritic human joints (10), and human macrophages accumulate higher levels of HIF-1 than of HIF-2 when exposed to tumor-specific levels of hypoxia in vitro (9). Our present results also clearly support a specific and independent action of HIF-1α. These findings suggest that HIF-1 may be the major hypoxia-inducible transcription factor in macrophages.
[0069] In summary, our results demonstrate that HIF-1α not only helps myeloid cells shift to glycolytic metabolism (11) but also functions in coordinating a proper innate immune response for microbial killing. The in vivo studies confirm that the HIF-1α pathway can play a critical role in controlling proliferation of a pathogen in compromised tissues. Recent commentaries based on our work have suggested that downregulation of HIF-1α could have a therapeutic effect in disease states characterized by chronic inflammation (48, 49). We now have shown that medically important microbial species such as GAS, methicillin-resistant S. aureus (MRSA), P. aeruginosa, and Salmonella species can trigger HIF-1α expression. Thus, the present studies suggest the design and use of pharmaceutical HIF-1α agonists (or vHL antagonists) to boost myeloid cell microbicidal activity for a novel approach for adjunctive therapy of complicated infections due to antibiotic-resistant pathogens or compromised host immunity.
US’404 at 7-8, and at Figure 8 (emphases added).
Had ML228 been invented at the time the inventors of US’404 conceptualized the use activators of HIF1-alpha as broad-spectrum antiviral compounds, it is reasonable that expect that the inventors of US’404 would have expressly taught the use of ML228 as a broad spectrum antiviral compound as well. Coincidentally, Applicants now seek patent protection for this use of ML228.
Nevertheless, one of ordinary skill in the art at the time of filing would have a clear motivation to select ML228 as the drug used to activate HIF1-alpha – ML228 is a commercially available research compound that “potently” activates HIF-1alpha (Theriault 2012). Theriault 2012 details the development of ML228, which discloses the various compounds the ML228 inventors developed in their path towards the discovery of ML228. Many of these compounds are also now encompassed by the instant compound of Formula (I).
Theriault 2012 and ML228
Theriault 2012 generally discloses that compounds based upon the following triazine chemical scaffold exhibit the ability to activate HIF1-alpha:
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Theriault 2012 Triazine Chemical Scaffold13
For example, Theriault 2012 at 78-79 discloses the following compounds that show the ability to activate HIF1-alpha with varying degrees of strength, including ML228 (see middle right figure below) under normoxic conditions:
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Theriault 2012 at 78-79.
Theriault 2012 explains that ML228 activates HIF through iron chelation, Theriault 2012 at 79-80. “Activating HIF” through iron chelation is synonymous with “activating HIF1-alpha” and “stabilization of HIF-1α”, and other various variants thereof. Theriault 2012 at 76.14
The remainder of Theriault 2012 is focused on characterizing ML228. Regarding ML228, Theriault 2012 specifically concludes that:
ML228 represents a novel chemotype available to the research community for the study of HIF activation and its therapeutic po-tential. Not only is the compound substantially different in struc-ture from known HIF activators, ML228 lacks the acidic functional group almost universally present in PHD inhibitors, which may be important for certain disease applications. ML228 was demonstrated to potently activate HIF in vitro as well as its downstream target VEGF. Further biological evaluation of ML228 is ongoing and will be reported in due course.
Theriault 2012 at 81 (emphasis added).
Wise 2021
Wise 2021 provides motivation for activating HIF1-alpha to treat viral infections, even in cases where inoculation with the virus is known to increase HIF1-alpha activity. In particular, Wise 2021 teaches that active HIF1-alpha suppresses HCMV replication through suppressing the IDO1 – KYN – AhR Pathway. Wise 2021 further teaches that this role of active HIF1-alpha appears to be a general mechanism to limit viral infection.
Wise 2021 explains that prior studies proposed that HIF1-alpha was induced by HCMV to support virus replication and pathogenesis:
Human cytomegalovirus (HCMV) is a herpesvirus that establishes lifelong asymp-tomatic infection in most people. HCMV infection in people with a compromised immune system causes disease that can lead to death. Additionally, HCMV infection is a leading cause of congenital disabilities (1). Replication of HCMV depends on evading cellular innate antiviral responses and hijacking host processes to support virus replication. Infection alters activity in many pathways in the host metabolic network, such as increasing glycolysis and the flow of carbons into lipid synthesis to support vi-rus replication (2–8).
Metabolic reprogramming following HCMV infection involves hijacking the activity of host metabolic regulators (2). Hypoxia-inducible factor 1α (HIF1α) is a metabolic reg-ulator that is altered by HCMV infection. Cell sensing of HCMV infection increases HIF1a protein levels under conditions with normal oxygen levels (i.e., normoxia) (9). Infected cells sustain HIF1a activity upon expression of HCMV early genes (10). These previous works proposed that HIF1α was induced by HCMV to support virus replication and pathogenesis (9, 10).
Wise 2021 at 1-2 (emphasis added).
Contrary to the prior assumptions, Wise 2021 discloses HCMV replication is enhanced in cells that lack HIF1-alpha, and that this appears to be a general mechanism to limit viral infection.
In contrast to the proposal in the previous work, we find that HCMV replication is enhanced in HIF1a KO cells. This observation suggests that HIF1α activity in infected cells alters metabolism as a protective strategy to limit viral infection.
Wise 2021 at 2 (emphases added).
HIF1-alpha is Active in HCMV Infected Cells
First, Wise 2021 explains that HIF1-alpha is active in HCMV infected cells
[W]e examined if HCMV infection alters HIF1α activity by measuring the expression of the gene encod-ing vascular endothelial growth factor (VEGF), which is transcriptionally regulated by HIF1a. VEGF transcripts were 20-fold higher in HCMV-infected cells than mock-infected cells, indicating that HIF1a is active in HCMV-infected cells maintained under normal oxygen conditions (see Fig. S1A in the supplemental material).
Wise 2021 at 2-3 (emphases added).
Active HIF1-alpha Suppresses HCMV Replication
Next, Wise 2021 explains that active HIF1-alpha suppresses HCMV replication at low multiplicity of infection:
We infected HIF1a KO and NT control cells at a low MOI of 0.05 infectious unit per cell and quantified the production of new virus progeny over 16 days. At 9 dpi, HIF1α KO cells produced ~60-fold more HCMV progeny than NT cells (Fig. 1E; Fig. S1B). At 12 and 16 dpi, ~30-fold more infectious virus was produced by HIF1a KO cells than NT cells.
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Wise 2021 at 3 (emphases added), and at 3, Fig. 1E (showing that active HIF1-alpha suppresses HCMV replication at low multiplicity of infection).
Wise 2021 explains that at high multiplicity of infection, active HIF1-alpha may contribute to an innate cellular antiviral response that is suppressed or not measured in a single-cycle high-MOI virus replication:
Since the 16-day virus growth curves shown in Fig. 1E measure multiple rounds of HCMV replication, we next tested virus replica-tion in a single replication cycle by infecting cells at an MOI of 3 and measuring infec-tious viral progeny at 4 dpi. Under these conditions, HCMV replication in HIF1a KO cells is similar to that in NT cells (Fig. 1F). Overall, our observations indicate that HIF1α sup-presses HCMV replication at a low MOI and may contribute to an innate cellular antivi-ral response that is suppressed or not measured in a single-cycle high-MOI virus repli-cation assay.
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Wise 2021 at 3-4 (emphasis added), and at 3, Fig. 1F (showing that at high multiplicity of infection, active HIF1-alpha suppression of HCMV is not observed).
Active HIF1-alpha Lowers KYN Levels Raised by HCMV Infection
Wise 2021 explains that HCMV infection raises KYN levels, and that KYN levels are lowered by HIF1-alpha:
KYN is a metabolite in tryptophan degradation that is made by the indoleamine 2,3-dioxygenase 1 (IDO1) pathway.
Wise 2021 at 2,
In uninfected cells grow-ing in normoxia, the loss of HIF1a has little or no effect on intracellular or extracellular KYN levels (Fig. 3A and B). In the NT cells that express HIF1a, HCMV-infected cells have a 3.5-fold increase in intracellular KYN relative to uninfected cells at 2 dpi (Fig. 3A). Similarly, KYN levels were greater in the extracellular fraction of HCMV-infected NT cells than uninfected NT cells (Fig. 3B). These data demonstrate that HCMV infection, in cells with HIF1α, enhances intracellular and extracellular KYN levels.
Relative to HCMV-infected NT cells, infected HIF1a KO cells had 3.5-fold- and 2.5-fold-higher intracellular and extracellular KYN levels (Fig. 3A and B).…
Wise 2021 at 6-7 (emphases added). Fig. 3A and 3B are reproduced below.
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Wise 2021 at 6, Fig. 3A and 3B (showing that active HIF1-alpha reduces KYN levels).
Restoring HIF1-alpha Activity Reduces KYN Levels
Wise 2021 further explains that restoring HIF1-alpha activity significantly reduced KYN levels:
For further confirmation that HIF1a suppresses KYN levels in HCMV-infected cells, we re-expressed HIF1a in our KO cells using a doxycycline-inducible system (Fig. 3C). In these cells, we engineered the re-expressed HIF1a to contain a silent mutation that removes the Cas9 protospacer-adjacent motif (PAM) recognition site while leaving the amino acid sequence of the protein unaffected. As a control, we expressed green fluorescent protein (GFP) in HIF1a KO cells. We infected these cells for 1 h, washed them, and treated them with doxycycline to induce HIF1a or GFP expression. KO cells reexpressing HIF1a had almost 2-fold-lower levels of KYN than HIF1a KO cells expressing GFP (Fig. 3D). These observations provide further evidence that HIF1a suppresses KYN levels in HCMV-infected cells.
Wise 2021 at 7 (emphases added). Fig. 3C and 3D are reproduced below.
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Wise 2021 at 6, Fig. 3C and 3D (showing that restoring HIF1-alpha reduces KYN levels).
Active HIF1-alpha Reduces IDO1 Expression
Wise 2021 explains that the expression of IDO1, the rate-limiting enzyme in KYN synthesis, is regulated by a HIF1-alpha dependent mechanism:
Since KYN is elevated in HCMV-infected HIF1a KO cells, we determined if the expression of the rate-limiting enzyme in KYN synthesis, indoleamine 2,3-dioxygenase 1 (IDO1), is regulated by a HIF1a-dependent mechanism. At 2 dpi, IDO1 transcripts were increased by >2-fold in HCMV-infected HIF1a KO cells relative to infected NT cells (Fig. 4A).
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Wise 2021 at 7 (emphases added) and at 7, Fig. 4A (showing that active HIF1-alpha reduces IDO1 activity).
Inhibiting IDO1 Activity Reduces HCMV Replication
Wise 2021 explains that inhibiting IDO1 activity with IDO1 inhibitor NLG919 reduces HCMV replication:
Our observations that IDO1 transcripts, KYN levels, and HCMV replication are enhanced in HIF1a KO cells led us to hypothesize that IDO1 activity promotes HCMV replication. We tested this hy-pothesis by inhibiting IDO1 activity using the IDO1 inhibitor NLG919. First, we exam-ined the effect of NLG919 treatment on HCMV replication in fibroblast cells that had not been genetically modified by CRISPR/Cas9 or any other means. Cells were infected at an MOI of 1 and then treated with NLG919 at concentrations ranging from 100 to 2,000 nM. Dimethyl sulfoxide (DMSO)-treated cells were used as a control. The medium was replaced at 2 dpi to renew the level of NLG919. At these conditions, NLG919 treat-ment had little or no effect on the survival of uninfected cells (Fig. S4A). At 5 dpi, the amount of infectious viral progeny produced was measured by determining the 50% tissue culture infective dose (TCID50). At 2,000 nM, NLG919 treatment reduced HCMV replication by 10-fold, relative to DMSO-treated control cells (Fig. 4B). NLG919 treat-ment at 100 to 1,000 nM reduced HCMV replication by 2- to 9-fold.
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Wise 2021 at 7-8 (emphases added), and at 7, Fig. 4B (showing the effect of inhibiting IDO1 activity with NLG919 on HCMV replication), and
We further tested if HCMV replication depends on IDO1 activity using HIF1a KO cells and a low MOI under the conditions used for the virus growth assays shown in Fig. 1E. In this case, the cells were treated at 1 h postinfection (hpi), and the medium was replaced every third day to renew the level of NLG919. At 9 dpi, the amount of in-fectious viral progeny produced was measured by TCID50. Since the cells were treated for 9 days, we focused on the lowest NLG919 concentration examined—100 nM—to limit any off-target effects treatment might have on the health of the cells. Under these conditions, 100 nM had little or no effect on cell survival (Fig. S4B). At 9 dpi, infectious HCMV progeny production was >2-fold lower in cells treated with 100 nM NLG919 than DMSO-treated cells (Fig. 4C). We conclude that IDO1 activity promotes HCMV rep-lication in non-genetically modified primary human fibroblasts and in HIF1a KO cells.
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Wise 2021 at 8 (emphasis added), and at 7, Fig. 4C (showing the effect of inhibiting IDO1 activity with NLG919 on HCMV replication)
The IDO1 – KYN – AhR Pathway
Wise 2021 explains that IDO1 controls the synthesis of KYN, that KYN is an important mediator in metabolite signaling through AhR, and that starving AhR from KYN suppresses HCMV replication:
IDO1 controls the synthesis of KYN, which is an important mediator in metabolite signaling through the aryl hydrocarbon receptor (AhR). We tested if AhR is required for HCMV replication using CH223191, an AhR inhibitor that blocks KYN binding (20, 21). As we did for NLG919 treatment, we first examined the effect of CH223191 treatment on HCMV replication in fibroblast cells that had not been genetically modified. Cells were infected at an MOI of 1 and then treated with DMSO or CH223191 at concentra-tions ranging from 3 to 24 mM. The medium was replaced at 2 dpi to renew the level of CH223191. Under these conditions, a reduction of less than 5% cell survival was observed when uninfected cells were treated at 24 mM (Fig. S4A). Lower concentrations of CH223191 had little to no effect on cell survival. At 5 dpi, CH223191 treatment at 24 mM reduced HCMV replication by 11-fold (Fig. 5A). Treatment at 3 to 12 mM reduced HCMV replication by 2- to 7-fold. We further tested if HCMV replication depends on AhR activity by infecting NT and HIF1a KO cells at a low MOI. Again, we used the low-est concentration of inhibitor tested, and the medium was replaced every third day to renew the level of CH223191. At 9 days posttreatment, DMSO-treated uninfected cells and 3 mM CH223191-treated uninfected cells had the same level of cell survival (Fig. S4). At 9 dpi, HIF1a KO and NT cells treated with CH223191 produced fewer infec-tious progeny than those treated with DMSO (Fig. 5B).
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Wise 2021 at 8 (emphases added), and at 9, Fig. 5A and 5B (showing the effect of inhibiting KYN binding to AhR with CH223191 on HCMV replication).
Wise 2021 further shows that activating AhR drives HCMV replication:
Conversely, activation of AhR with an exogenous dioxin ligand enhances HCMV rep-lication (22). We tested if AhR activation by an exogenous ligand would enhance HCMV replication by treating cells with the AhR activator 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Since TCDD is a liquid at room temperature, we diluted TCDD directly in the cell growth medium. As a control, we added an equal volume of water to the growth medium. Since TCDD is known to promote HCMV replication in primary human fibroblasts at 3.1 nM, we examined if TCDD treatment at this level would alter HCMV replication in NT and HIF1a KO cells. At 9 days posttreatment, cells survived equally in water and 3.1 nM TCDD treatment (Fig. S4). In NT cells, HCMV infectious-progeny pro-duction was 2.5-fold higher in TCDD-treated cells than water-treated cells (Fig. 5C). In HIF1a KO cells, virus progeny production was 1.5-fold higher in TCDD-treated cells than water-treated cells (Fig. 5C).
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Wise 2021 at 8 (emphasis added), and at 9, Fig. 5C (showing the effect of activating AhR with TCDD on HCMV replication).
KYN Promotes HCMV Replication
Wise 2021 further shows that feeding KYN to cells promotes HCMV replication:
Based on our observations, we hypothesize that KYN enhances HCMV replication. We tested this hypothesis by feeding KYN to cells fol-lowing a low-MOI infection and monitoring HCMV spread in cell culture.… In KYN-treated cells, the average plaque size was 15 to 23 cells (Fig. 6B). Since plaque size allows us to visualize HCMV replication and cell-to-cell spread, we next examined if KYN treatment would affect HCMV replication by meas-uring infectious-progeny production at 9 dpi. In this case, fibroblast cells were infected at an MOI of 0.05 infectious unit per cell, washed, and fed growth medium containing 0 or 1 mM KYN. At 9 dpi, the amount of infectious progeny was measured by TCID50. KYN treatment increased the level of HCMV produced by 3-fold (Fig. 6C). Since KYN treatment increased HCMV plaque size at 6 dpi and infectious-progeny production at 9 dpi, we con-clude that KYN promotes HCMV replication.
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Wise 2021 at 8,10 (emphasis added), and at 10, Fig. 6B and 6C (showing the effect of exogenous KYN on HCMV replication).
As discussed above, Wise 2021 generally discloses that contrary to prior assumptions that supported a pro-viral role of active HIF1-alpha in HCMV replication and pathogenesis, active HIF1-alpha instead suppresses viral replication through suppression of indoleamine 2,3-dioxygenase 1 (“IDO1”) expression. To summarize Wise 2021, it explains that
HIF1-alpha is active in HCMV infected cells,
the active HIF1-alpha lowers expression of IDO1,
IDO1 is the rate-limiting enzyme in kynurenine (“KYN”) synthesis,
by lowering IDO1 expression, KYN levels are reduced,
KYN is involved in metabolite-mediated activation of aryl hydrocarbon receptor (“AhR”), and
that starving AhR from KYN suppresses HCMV replication.
Accordingly, Wise 2021 teaches that active HIF1-alpha suppresses HCMV replication through suppressing the IDO1 – KYN – AhR Pathway.
Wise 2021 further teaches administering a compound to a subject in order to suppress the IDO1 – KYN – AhR Pathway and suppress HCMV replication. See supra discussion of NLG919 (inhibiting IDO1 activity suppresses HCMV replication); see supra discussion of CH223191 (inhibiting KYN binding to AhR suppresses HCMV replication).
The ML228 in vivo Studies
These references show that ML228 was a commercially available research compound and was used as an activator of HIF1-alpha to study various animal models of human disease.
Zhu, D., et al., "ORAI3 contributes to hypoxia-inducible factor 1/2α-sensitive colon cell migration" Physiology International, vol. 108, no. 2 pp. 221-237 (June 2021), hereinafter “Zhu 2021”.
Teaches administration of ML228 to rats and tumor samples from colon cancer patients.
Xing, Jihong, and Jian Lu, "HIF-1α activation attenuates IL-6 and TNF-α pathways in hippocampus of rats following transient global ischemia", Cellular Physiology and Biochemistry vol. 39, no. 2, pp. 511-520 (2016), hereinafter “Xing 2016”.
Teaches administration of ML228 to rats. “2 mg/kg of ML228 (i.p., twice) was injected after CA”. Xing 2016 at 513.
Liu, Xiao-Liang, Jian Lu, and Jihong Xing, "Stabilization of HIF-1α modulates VEGF and Caspase-3 in the hippocampus of rats following transient global ischemia induced by asphyxial cardiac arrest", Life sciences, vol. 151, pp. 243-249 (2016), hereinafter “Liu 2016”.
Teaches administration of ML228 to rats “2 mg/kg of ML228 (i.p., twice) was injected after CA)”. Liu 2016 at 244.
Chen, Hailong, et al., "Effect of hypoxia-inducible factor-1/vascular endothelial growth factor signaling pathway on spinal cord injury in rats", Experimental and therapeutic medicine, vol. 13, no. 3, pp. 861-866 (2017), hereinafter “Chen 2017”.
Teaches administration of ML228 to rats “treatment group (SCI rats that received 1 μg/kg ML228 treatment)”. Chen 2017 at 862.
Therefore, the results of the current study suggest that ML228 may effectively activate the HIF-1α/VEGF signaling pathway to promote the expression of HIF-1α and VEGF proteins within the injured segment of the spinal cord, which promotes neural functional recovery following SCI in rats. Therefore, treatment with ML228 may be developed as a novel therapeutic strategy to treat SCI.
Chen 2017 at Abstract (emphasis added).
ML228 represents a novel chemotype available to the research community for the study of HIF activation and its therapeutic potential.
Chen 2017 at 861.
Endo, Yori, et al., "Loss of ARNT in skeletal muscle limits muscle regeneration in aging", The FASEB Journal, vol. 34, no. 12, pp. 16086-16104 (2020), hereinafter “Endo 2020”.
“For ML228 treatment, ML228 (TOCRIS #1357171-62-0, Cayman Chemical, Ann Arbor, MI, USA) was dissolved in DMSO (0.03 mg ML228 in 1 mL DMSO) and was injected intraperitoneally (0.1 mL) once daily for a total of 5 days.” Endo 2020 at 16087.
Li 2021
Li 2021 provides motivation for repurposing drugs in the context of antiviral development, and stands for a finding that the reasonable expectation of success for translating candidate molecules to approved drugs is low. However, this reasonable expectation of success is increased when a candidate molecule is a “repurposed” investigational drug:
Drug repurposing (also called drug repositioning) is a strategy for identifying new uses for approved or investigational drugs that beyond the original indicative scope to facilitate antiviral development. Typically, antiviral discovery development is time and resource-consuming, which involves three major stages including drug discovery (3–6 years), preclinical studies in experimental animal models (about 3 years), clinical trials in humans from phase I to III (about 5 years). Finally, if a therapeutic succeeds to pass all the processes, it needs to get approved by the appropriate agency. It is estimated that only 5% of the candidate molecules are finally approved and up to 3 billion dollars are consumed. Given that the repurposed drugs have been proven to be safe in humans, drug repurposing likely can skip phase I and probably the phase II clinical trials. Thus, the attrition rate to be a novel antiviral is reduced, although the phase III trial is still needed. Remdesivir, an adenosine analog to inhibit EBOV RNA-dependent RNA polymerase (RdRp) (Tchesnokov et al., 2019), is the latest example. Although remdesvir did not show therapeutic activity against EBOV infection in a real-world phase III clinical trial (Nakkazi, 2018), remdesivir shows potent antiviral activity against SARS-CoV-2, SARS-CoV, and MERS-CoV in vitro or in vivo in preclinical animal models (de Wit et al., 2020; Wang et al., 2020a). Two randomized phase III clinical trials indicate that patients who received remdesivir had a shorter time to recover (Spinner et al., 2020; Wang et al., 2020c), based upon which the U.S. Food and Drug Administration (FDA) has approved remdesivir for use in COVID-19 patients, less than 1 year after the outbreak of the pandemic. From the above example, drug repurposing could significantly facilitate antiviral development for emergency use. Given the urgent need for therapeutics for emerging or re-emerging viruses and a great number of approved or developmental therapeutics, drug repurposing represents a better way for antiviral discovery. In this review, we discussed the strategies of drug repurposing for antiviral development, summarized the promising drug candidates that have the antiviral potency with broad-spectrum activity, and analyzed the possible caveats of this strategy of drug discovery.
Li 2021 at 2-3 (emphases added).
Claim 1 was Obvious at the Time of Filing
The examiner finds that “stabilization” and “activation” of HIF1-alpha are synonymous.15
US’404 discloses methods for the prophylaxis and/or treatment of and infection or virulence in a subject in need thereof, comprising administering to said subject a pharmaceutically effective amount of a compound which stabilizes HIF-1α under normoxic conditions. See, e.g., US’404 at 35, claims 8 and 20. As explained above, the methods that US’404 teaches are effective against a broad-spectrum of viruses.
For example, US’404 explains that HIF1-alpha activators
“boost myeloid cell microbicidal activity for a novel approach for adjunctive therapy of complicated infections due to antibiotic-resistant pathogens or compromised host immunity.”
US’404 at 8 (emphasis added).
US’404 explains that HIF1-alpha
not only helps myeloid cells shift to glycolytic metabolism (11) but also functions in coordinating a proper innate immune response for microbial killing. The in vivo studies confirm that the HIF-1α pathway can play a critical role in controlling proliferation of a pathogen in compromised tissues.
US’404 at 8 (emphasis added).
US’404 explains that microbial killing includes “killing microbial pathogens, such as bacterial and viral pathogens.” US’404 at 2 (emphases added).
US’404 explains that such methods are effective against a broad spectrum of viruses, including neurotropic viruses:
In yet another embodiment, the microbes to be tested or treated are viruses. There are a number of viruses that are recognized and affected by the innate immune system, particularly by macrophage activity. Viruses that are contemplated include, but are not limited to, retroviruses proviruses, lentivriurses such as immunodeficiency viruses, togaviruses. Togaviruses, as used herein includes the Togaviridae family, including the Alphavirus and Rubivirus genera, as well as the flavivirus family (Flaviviridae) and the Flavivirus and Pestivirus genera. A review of virus taxonomy and the biology of these viruses may be found at, e.g., B. N. Fields, et al., editors, Fundamental Virology, 3rd edition, 1996, Lippencott-Raven Publishers, chapter I (pages 15-58) and chapter 17 (pages 523-540), which is incorporated herein by reference.
US’404 at 10, paragraph [0075] (emphases added).
However, US’404 does not disclose using ML228 to accomplish these goals, because ML228 had not been invented yet. See US’404 at 14-18 (disclosing HIF1-alpha activators and modulating compounds that were precursors to ML228).16
Nevertheless, one having ordinary skill in the art at the time of a filing would have a reasonable expectation of success in selecting ML228 as the compound to administer to a subject in order to activate HIF1-alpha for the prophylaxis and/or treatment of a broad-spectrum of viral infections including neurotropic viruses as taught by US’404, because
ML228 was known to “potently activate HIF in vitro” by stabilization of HIF1-alpha under normoxic conditions, see supra discussion of Theriault 2012, and
the ML228 in vivo Studies show that ML228 was a commercially available research compound used in vivo to activate HIF1-alpha in animal models of human disease,
see, e.g., Chen 2017 at 861 (“ML228 represents a novel chemotype available to the research community for the study of HIF activation and its therapeutic potential.”) (emphasis added).
One having ordinary skill in the art at the time of filing would be strongly motivated to select ML228 as the compound to administer to a subject in order to activate HIF1-alpha, because ML228 could be repurposed to significantly facilitate antiviral development, as using the drug repurposing strategy explained by Li 2021.
Li 2021 discloses that drug repurposing “is a strategy for identifying new uses for approved or investigational drugs”. Li 2021 at 2 (emphases added).
Li 2021 at 3 explains:
Typically, antiviral discovery development is time and resource-consuming, which involves three major stages including drug discovery (3–6 years), preclinical studies in experimental animal models (about 3 years), clinical trials in humans from phase I to III (about 5 years).
Li 2021 at 3 (emphasis added), and that
[D]rug repurposing could significantly facilitate antiviral development for emergency use. Given the urgent need for therapeutics for emerging or re-emerging viruses and a great number of approved or developmental therapeutics, drug repurposing represents a better way for antiviral discovery.
Li 2021 at 3 (emphasis added).
As the ML228 in vivo Studies show, ML228 exhibited all the indications of an investigational drug that could be repurposed to significantly facilitate antiviral development, since it already had established in vitro and in vivo profiles for cellular and animal models of human disease. That there was no formal designation of ML228 as an “investigational drug” is a distinction without a difference, because investigational new drug (“IND”) applications are held in confidence by regulatory authorities, such as FDA.
Accordingly, one of ordinary skill in the art at the time of time would be strongly motivated to select ML228 as the compound to administer to a subject in order to activate HIF1-alpha, because repurposing the commercially available research compound ML228 used in vivo to activate HIF1-alpha in other animal models of human disease “could significantly facilitate antiviral development” and address “the urgent need for therapeutics for emerging or re-emerging viruses”.
For the above reasons, one having ordinary skill in the art at the time of filing would be motivated to select, and have a reasonable expectation of success in selecting, ML228 as the compound to administer to a subject in order to activate HIF1-alpha for the prophylaxis and/or treatment of a broad-spectrum of viral infections including neurotropic viruses.
Therefore, claim 1 was obvious at the time of filing over US’404, in view of Theriault 2012, the ML228 in vivo Studies, and Li 2021.
Furthermore, one of ordinary skill in the art at the time of filing would have a reasonable expectation of success in combining the above teachings of US’404, Theriault 2012, the ML228 in vivo Studies, and Li 2021 with Wise 2021 in selecting ML228 as the compound to administer to a subject in order to activate HIF1-alpha for the prophylaxis and/or treatment of a broad-spectrum of viral infections including neurotropic viruses, because Wise 2021 advances the understanding of the role of active HIF1-alpha in response to viral infections.
As discussed above, Wise 2021 teaches that active HIF1-alpha suppresses HCMV replication through suppressing the IDO1 – KYN – AhR Pathway. Wise 2021 explains that this appears to be a general mechanism of suppressing metabolism to limit viral infections. See, e.g., Wise 2021 at 2 (“This observation suggests that HIF1α activity in infected cells alters metabolism as a protective strategy to limit viral infection.”).
One of ordinary skill in the art would reasonably expect that by combining the teachings of Wise 2021 with US’404, one could treat an HMCV infection by administering to a subject in need a compound that activated HIF1-alpha, because the administration would cause the HIF1-alpha in the subject to activate and suppress HCMV replication, as taught by Wise 2021.
One having ordinary skill in the art at the time of filing would have a reasonable expectation of success in treating other neurotropic viruses in such a manner, because Wise 2021 explains that this role of active HIF1-alpha in infected cells alters metabolism as a protective strategy to limit viral infection. Given that some of the other neurotropic viruses are in the same virus family as HCMV, such as HSV-1 and HSV-2 that belong to the same herpesviridae family as HCMV, one having ordinary skill in the art at the time of filing would reasonably expect that this general protective strategy to limit viral infection would extend to HSV-1 and HSV-2 infections, since the herpesviridae family of viruses share similar characteristics. Moreover, given that neurotropic viruses as a class of viruses infect similar cellular targets, one having ordinary skill in the art at the time of filing would reasonably expect that altering cellular metabolism through suppressing the IDO1 – KYN – AhR Pathway would similarly achieve the goals of suppressing viral replication of other neurotropic viruses.
Moreover, such active HIF1-alpha would further boost myeloid cell microbicidal activity in coordinating a proper innate immune response for microbial killing of viral pathogens, as taught by US’404.
Accordingly, one having ordinary skill in the art at the time of filing would be motivated to select, and have a reasonable expectation of success in selecting, ML228 as the compound to administer to a subject in order to activate HIF1-alpha for the prophylaxis and/or treatment of a broad-spectrum of viral infections including neurotropic viruses.
Therefore, claim 1 was obvious at the time of filing over US’404, in view of Theriault 2012, the ML228 in vivo Studies, and Li 2021, and in further view of Wise 2021.
Claim 2 was Obvious at the Time of Filing
Claim 2 recites the method of claim 1 wherein the optionally substituted phenyl is substituted with several substituents, including phenyl.
The above rejections of claim 1 under 35 U.S.C. 103 are incorporated herein.
ML228 corresponds to a compound of formula (I) wherein R1 is –(CH2)n–R3, R2 is H, n is 1, and R3 is an optionally substituted phenyl, and the optional substitution of the phenyl is phenyl.
Accordingly, claim 2 was obvious at the time of filing for the same reasons claim 1 was obvious at the time of filing, and for the reasons stated above.
Claims 3 and 4 were Obvious at the Time of Filing
Claim 3 depends upon claim 1 and explains that these viruses are classified in the following families of viruses: herpesviridae, coronaviridae, picornaviridae, orthomyxoviridae, retroviridae, rhabdoviridae, flaviviridae, togaviridae, polyomaviridae, paramyxoviridae, peribunyaviridae, and matonaviridae.
Similarly, claim 4 depends from claim 1 recites that the neurotropic virus of is selected from the group consisting of: herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), encephalomyocarditis virus (EMCV), poliovirus, severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), influenza A virus, influenza B virus, human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), vesicular stomatitis Indiana virus (VSV or VSIV), and rabies lyssavirus.
The above rejections of claim 1 under 35 U.S.C. 103 are incorporated herein.
As discussed above for the rejection claim 1, Human cytomegalovirus, is typically abbreviated as “HCMV”, and is also known as human betaherpesvirus 5, which is typically abbreviated as “HHV-5”. It belongs to the family orthoherpesviridae. See NCBI entry for Human betaherpesvirus 5, attached hereto. The family orthoherpesviridae is also known as Herpesviridae. See NCBI entry for Orthoherpesviridae, attached hereto.
Further, US’404 explains that such methods of treating virulence comprising administering a compound which stabilizes HIF-1alpha under normoxic conditions are effective against a broad-spectrum of viruses, including, but not limited to, togaviridae, flaviviridae, lentivriurses such as immunodeficiency viruses (retroviridae, which includes HIV-1 and HIV-2) :
In yet another embodiment, the microbes to be tested or treated are viruses. There are a number of viruses that are recognized and affected by the innate immune system, particularly by macrophage activity. Viruses that are contemplated include, but are not limited to, retroviruses proviruses, lentivriurses such as immunodeficiency viruses, togaviruses. Togaviruses, as used herein includes the Togaviridae family, including the Alphavirus and Rubivirus genera, as well as the flavivirus family (Flaviviridae) and the Flavivirus and Pestivirus genera. A review of virus taxonomy and the biology of these viruses may be found at, e.g., B. N. Fields, et al., editors, Fundamental Virology, 3rd edition, 1996, Lippencott-Raven Publishers, chapter I (pages 15-58) and chapter 17 (pages 523-540), which is incorporated herein by reference.
US’404 at 10, paragraph [0075].
Accordingly, claims 3 and 4 were obvious at the time of filing for the same reasons claim 1 was obvious at the time of filing, and for the reasons stated above.17
Claims 5 and 6 were Obvious at the Time of Filing
Claim 5 depends upon claim 1 and recites that “a disease caused by the viral infection is also treated or prevented.” Claim 6 now depends upon claim 5 and recites that “the disease caused by the viral infection is selected from the group consisting of” a number of diseases caused by the viral infection.
The above rejections of claim 1 under 35 U.S.C. 103 are incorporated herein.
First, a portion of the claim recites that “a disease caused by the viral infection is also prevented”. This follows logically from preventing replication of the neurotropic virus causing the viral infection, and through boosting myeloid cell microbicidal activity to engage in killing microbial pathogens, such as viral pathogens.
Second, a portion of the claim recites that “a disease caused by the viral infection is also treated”. It is common knowledge that individuals suffering from viral infections develop secondary infections due to their compromised immune systems. Such a secondary infection is reasonably “a disease caused by the viral infection”. Killing the pathogens responsible for the secondary infection would logically treat the secondary infection.
US’404 explains that HIF1-alpha activators boost myeloid cell microbicidal activity in coordinating a proper innate immune response for microbial killing. US’404 further explains that microbial killing includes “killing microbial pathogens, such as bacterial and viral pathogens.” US’404 at 2 (emphases added).
Therefore, claim 5 was obvious at the time of filing for the same reasons claim 1 was obvious at the time of filing, and for the reasons stated above.
Regarding claim 6, Guillain-Barre syndrome, or GBS, is frequently observed following HCMV infection. See Lunn 201118 at 845:
The association between cytomegalovirus (CMV) and Guillain–Barre´ syndrome (GBS) was first noted in 1967 [1] and was soon endorsed by other reports and case series. The largest previous case series showed that CMV infection was associated with GBS in 8% of 229 patients from a European and North American trial [2]. In this issue of Clinical Infectious Diseases, Orlikowski et al [2] confirmed this finding in a larger population of people with GBS…
Lunn 2011 at 845.
While Applicant removed GBS from the claimed diseases recited in the instant claim 6, Applicant did not remove “inflammatory neuropathies” from the recited list of diseases treated or prevented.
As Lunn 2011 explains, patients with CMV-infections often have inflammatory neuropathies. See Lunn 2011 at 846 (“Seventy percent of the CMV-infected patients were classified neurophysiologically as having acute inflammatory demyelinating polyradiculoneuropathy (AIDP), and only 7% had axonal disease.”). See also id. (“One uncomfortable thought remains: whether the triggering infection directly invades the peripheral nervous system and contributes to the inflammation. Because GBS occurs after the symptoms of the acute infection have subsided, this possibility has been disregarded.”).
Preventing the CMV infection would still prevent the invasion of the peripheral nervous system and prevent the “inflammatory neuropathies”. Therefore, even though applicant amended out CMV and GBS from the claims, the claims still read on preventing diseases common in CMV patients, as well as other patients with invasion of the peripheral nervous system by a virus.
Therefore, one having ordinary skill in the art at the time of filing would have a reasonable expectation of success in preventing inflammatory neuropathies by treating an HCMV infection, or another viral infection that invades the peripheral nervous system, in a patient by administering ML228, because one having ordinary skill in the art at the time of filing would have a reasonable expectation of success in treating an HCMV infection in a patient, or another viral infection that invades the peripheral nervous system, by administering ML228, for the reasons stated in claim 1.
Therefore, claim 6 was obvious at the time of filing for the same reasons claim 1 was obvious at the time of filing, and for the reasons stated above.
Claim 7 was Obvious at the Time of Filing
Claim 7 recites the method of claim 1, wherein the compound is administered during a latency period, or during an incubation period, or during a disease period, of the viral infection.
The above rejections of claim 1 under 35 U.S.C. 103 are incorporated herein.
The examiner acknowledges that the Specification provides a special definition for each of these periods. See Specification at 7-8:
Latency period: In the present context the term "latency period" refers to a period in which a virus does not confer any disease conditions to the host and is unable to transmit from the host.
Incubation period: In the present context the term "incubation period" refers to a period in which a virus is replicating in a host, but without conferring any disease conditions to the host until the concentration of the virus reaches a high enough level.
Disease period: In the present context the term "disease period" refers to a period in which a virus is causing a disease in a host.
Specification at 7-8.
The examiner interprets the teachings of US’404 as covering the effects of active HIF1-alpha during latency, incubation, and disease periods, because at each of those periods, activating HIF1-alpha boost myeloid cell microbicidal activity in coordinating a proper innate immune response for microbial killing, which includes the killing of viral pathogens.
Furthermore, Wise 2021 discloses that active HIF1-alpha suppresses HCMV replication at low multiplicity of infection, see, e.g., supra discussion of Wise 2021 at 3, and at Fig. 1E.
Wise 2021 further discloses that active HIF1-alpha “may contribute to an innate cellular antiviral response” at high multiplicity of infection, but that response was “suppressed or not measured in single-cycle high-MOI replication assay.” Wise 2021 at 3-4. See also supra discussion of Wise 2021 at 3-4, and at 3, Fig. 1F.
The examiner interprets the teachings of Wise 2021 as covering the effects of active HIF1-alpha on viral replication during latency, incubation, and disease periods, because at each of those periods,
the viral infection may be at low multiplicity of infection, wherein active HIF1-alpha suppresses viral replication, or
the viral infection may be at high multiplicity of infection, wherein active HIF1-alpha “may contribute to an innate cellular antiviral response”, Wise 2021 at 4, and
Wise 2021 explains that this appears to be a general mechanism of suppressing metabolism to limit viral infections. See, e.g., Wise 2021 at 2 (“This observation suggests that HIF1α activity in infected cells alters metabolism as a protective strategy to limit viral infection.”).
Accordingly, claim 7 was obvious at the time of filing for the same reasons claim 1 was obvious at the time of filing, and for the reasons stated above.
Claim 8 was Obvious at the Time of Filing
Claim 8 recites the method of claim 1, wherein the compound is administered to a subject in need thereof.
The above rejections of claim 1 under 35 U.S.C. 103 are incorporated herein.
The examiner acknowledges that the “subject” recited in claim 1 is separate from the “subject” recited in claim 8, because the subject in claim 8 is now “in need” of the treatment method recited in claim 1.
To the extent that these subjects are different, the analysis is the same.
See, e.g., US’404 at claim 8 and 20 (the methods disclosed in US’404 pertain to a subject “in need”).
Accordingly, claim 8 was obvious at the time of filing for the same reasons claim 1 was obvious at the time of filing, and for the reasons stated above.
Claims 9 and 10 were Obvious at the Time of Filing
Claim 9 depends upon claim 8, and recites that the subject is a mammal. Claim 10 depends upon claim 9, and recites that the subject is selected from a list of mammals, preferably a human.
The above rejections of claim 8 under 35 U.S.C. 103 are incorporated herein.
As discussed above, US’404 generally discloses methods for the treatment of virulence in a subject in need thereof, comprising administering to said subject a pharmaceutically effective amount of a HIF-1 modulating compound. See, e.g., US’404 at 35, claim 8. Claim 20 explains that the HIF-1 modulating compound is a compound which stabilizes HIF-1alpha under normoxic conditions. Id.
US’404 further discloses that the methods may be used for plants and animals, including humans. See US’404 at 6, paragraph [0057].
Theriault 2012 discloses that “ML228 was demonstrated to potently activate HIF in vitro as well as its downstream target VEGF. Further biological evaluation of ML228 is ongoing and will be reported in due course.” Theriault 2012 at 81 (emphasis added).
Theriault 2012 explains that the in vitro studies were performed on human U20S osteosarcoma cell line:
The primary screening assay utilized a previously described cell-based gene reporter assay with a stably transfected human U2OS osteosarcoma cell line expressing luciferase under control of hypoxia response elements (HREs).13
Theriault 2012 at 77 (emphasis added).
The ML228 in vivo Studies disclose in vivo administration of ML228 in rat models of human disease:
See Xing 2016 at 513 (“ML228 group: CA and CPR were carried out and 2 mg/kg of ML228 (i.p., twice) was injected after CA [16].”).
See Chen 2017 at 862 (“treatment group (SCI rats that received 1 μg/kg ML228 treatment).”).
Li 2021 discloses that the reasonable expectation of success for translating candidate molecules to approved drugs is low:
Drug repurposing (also called drug repositioning) is a strategy for identifying new uses for approved or investigational drugs that beyond the original indicative scope to facilitate antiviral development. Typically, antiviral discovery development is time and resource-consuming, which involves three major stages including drug discovery (3–6 years), preclinical studies in experimental animal models (about 3 years), clinical trials in humans from phase I to III (about 5 years). Finally, if a therapeutic succeeds to pass all the processes, it needs to get approved by the appropriate agency. It is estimated that only 5% of the candidate molecules are finally approved and up to 3 billion dollars are consumed. Given that the repurposed drugs have been proven to be safe in humans, drug repurposing likely can skip phase I and probably the phase II clinical trials. Thus, the attrition rate to be a novel antiviral is reduced, although the phase III trial is still needed. Remdesivir, an adenosine analog to inhibit EBOV RNA-dependent RNA polymerase (RdRp) (Tchesnokov et al., 2019), is the latest example. Although remdesvir did not show therapeutic activity against EBOV infection in a real-world phase III clinical trial (Nakkazi, 2018), remdesivir shows potent antiviral activity against SARS-CoV-2, SARS-CoV, and MERS-CoV in vitro or in vivo in preclinical animal models (de Wit et al., 2020; Wang et al., 2020a). Two randomized phase III clinical trials indicate that patients who received remdesivir had a shorter time to recover (Spinner et al., 2020; Wang et al., 2020c), based upon which the U.S. Food and Drug Administration (FDA) has approved remdesivir for use in COVID-19 patients, less than 1 year after the outbreak of the pandemic. From the above example, drug repurposing could significantly facilitate antiviral development for emergency use. Given the urgent need for therapeutics for emerging or re-emerging viruses and a great number of approved or developmental therapeutics, drug repurposing represents a better way for antiviral discovery. In this review, we discussed the strategies of drug repurposing for antiviral development, summarized the promising drug candidates that have the antiviral potency with broad-spectrum activity, and analyzed the possible caveats of this strategy of drug discovery.
Li 2021 at 2-3.
One having ordinary skill in the art at the time of filing would have a reasonable expectation of success in treating neurotropic viral infections in humans by administering ML228, because
US’404 generally discloses that HIF-1alpha stabilizers/activators may be administered to humans in order to treat viral infections,
ML228 was known to “potently activate HIF in vitro”, Theriault 2012 at 81,
the ML228 in vivo Studies disclose in vivo administration of ML228 in rat models of human disease, and
Li 2021 discloses that there is an estimated 5% success rate in taking a “candidate molecules” from “drug discovery” through “preclinical studies in experimental animal models” to regulatory approval.
Accordingly, claims 9 and 10 were obvious at the time of filing for the same reasons claims 8 and 1 were obvious at the time of filing, and for the reasons stated above.
Claim 11 was Obvious at the Time of Filing
Claim 11 depends upon claim 1, and recites that the compound is administered to the subject by various methods, including intraperitoneal injection.
The above rejections of claim 1 under 35 U.S.C. 103 are incorporated herein.
The ML228 in vivo Studies disclose in vivo administration of ML228 by intraperitoneal injection in rat models of human disease. See, e.g., Xing 2016 at 513 (“ML228 group: CA and CPR were carried out and 2 mg/kg of ML228 (i.p., twice) was injected after CA [16].”).
US’404 discloses that the HIF-1 modulating compound may be administered in a pharmaceutical composition, see, e.g., US’404 at 5, paragraph [0047], and provides for the following routes of administration:
Examples of routes of administration include parenteral, e.g., intravenous or intra-arterial, intradermal, subcutaneous, oral (e.g., inhala-tion), transdermal (topical), transmucosal, nasal, pulmonary, ocular, gastrointestinal, and rectal administration.
US’404 at 23, paragraph [0148].
Accordingly, claim 11 was obvious at the time of filing for the same reasons claim 1 was obvious at the time of filing, and for the reasons stated above.
Claim 12 was Obvious at the Time of Filing
Claim 12 depends upon claim 1, and recites that the compound is administered in conjunction with at least one pharmaceutically acceptable excipient and/or pharmaceutically acceptable carrier.
The above rejections of claim 1 under 35 U.S.C. 103 are incorporated herein.
US’404 discloses that the HIF-1 modulating compound may be administered in a pharmaceutical composition, see, e.g., US’404 at 5, paragraph [0047].
US’404 discloses that the pharmaceutical composition may include excipients and/or carriers, see, e.g., US’404 at 5, paragraph [0047].
Accordingly, claim 12 was obvious at the time of filing for the same reasons claim 1 was obvious at the time of filing, and for the reasons stated above.
Claim 13 was Obvious at the Time of Filing
Claim 13 depends upon claim 1, and recites that the compound is administered in a lipid-based drug delivery system (LBDDS).
The above rejections of claim 1 under 35 U.S.C. 103 are incorporated herein.
The examiner acknowledges that the Specification provides a special definition for the term “Lipid-based drug delivery system”. See Specification at 9 (“In the present context the term "lipid-based drug delivery system" refers to a formulation comprising a lipid excipient.”).
US’404 explains that a number of different excipients may be added to a compound to improve or facilitate various factors, such as drug release profile:
One or multiple excipients, also referred to as inactive ingredients, can be added to a compound of the invention to improve or facilitate manufacturing, stability, administration, and safety of the drug, and can provide a means to achieve a desired drug release profile. Therefore, the type of excipient(s) to be added to the drug can depend on various factors. such as, for example, the physical and chemical properties of the drug, the route of administration, and the manufacturing procedure. Pharmaceutically acceptable excipients are available in the art, and include those listed in various pharmacopoeias. (See, e.g., USP. JP, EP, and BP, FDA web page …, Inactive Ingredient Guide 1996, and Handbook of Pharma-ceutical Additives, ed. Ash; Synapse Information Resources, Inc. 2002.)
US’404 at 23, paragraph [0149].
For example, US’404 directly teaches oil based excipients:
For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams, emulsion, a solution, a suspension, or a foam, as generally known in the art. The penetration of the drug into the skin and underlying tissues can be regulated, for example, using penetration enhancers; the appropriate choice and combination of lipophilic, hydrophilic, and amphiphilic excipients, including water, organic solvents, waxes, oils, synthetic and natural polymers, surfactants, emulsifiers; by pH adjustments; use of complexing agents and other techniques, such as iontophoresis, may be used to regulate skin penetration of the active ingredient.
US’404 at 24, paragraph [0155] (emphasis added).
See also, US’404 at 23-24, paragraph [0152] (teaching vegetable oil as an excipient for injections).
Accordingly, claim 13 was obvious at the time of filing for the same reasons claim 1 was obvious at the time of filing, and for the reasons stated above.
Prior Art Cited but not Applied
The prior art made of record and not relied upon is considered pertinent to Applicant’s disclosure.
B. N. Fields, et al., editors, Fundamental Virology, 3rd edition, 1996, chapter 2. Cited in a passage from US’404 to provide a background of the taxonomy of several viruses. See, e.g., page 24, Table 3, reproduced below.
PNG
media_image18.png
459
554
media_image18.png
Greyscale
Various NCBI entries for several viruses to show virus taxonomy :
NCBI entry for Human betaherpesvirus 5.
NCBI entry for Orthoherpesviridae.
NCBI entry for HHV-1
Conclusion
No claims allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/C.E.R./ Examiner, Art Unit 1629
/JEFFREY S LUNDGREN/ Supervisory Patent Examiner, Art Unit 1629
1 Johnson, Randall S., et al., "Hif Modulating Compounds and Methods of Use Thereof", U.S. Patent Application Publication No. US 2008/0213404 A1 (2008), hereinafter “US’404”.
2 Theriault, Jimmy R., et al., “Discovery of a New Molecular Probe ML228: An Activator of the Hypoxia Inducible Factor (HIF) Pathway”, Bioorg Med Chem Lett., vol. 22, no. 1, pp. 76-81 (2012), hereinafter “Theriault 2012”.
3 The “ML228 in vivo Studies” refer to the following in vivo studies of ML228 in rat models of human disease.
Xing, Jihong, and Jian Lu, "HIF-1α activation attenuates IL-6 and TNF-α pathways in hippocampus of rats following transient global ischemia", Cellular Physiology and Biochemistry, vol. 39, no. 2, pp. 511-520 (2016), hereinafter “Xing 2016”.
Chen, Hailong, et al., "Effect of hypoxia inducible factor 1/vascular endothelial growth factor signaling pathway on spinal cord injury in rats", Experimental and therapeutic medicine, vol. 13, no. 3, pp. 861-866 (2017), hereinafter “Chen 2017”.
Zhu, D., et al., "ORAI3 contributes to hypoxia-inducible factor 1/2α-sensitive colon cell migration" Physiology International, vol. 108, no. 2 pp. 221-237 (June 2021), hereinafter “Zhu 2021”.
Liu, Xiao-Liang, Jian Lu, and Jihong Xing, "Stabilization of HIF-1α modulates VEGF and Caspase-3 in the hippocampus of rats following transient global ischemia induced by asphyxial cardiac arrest", Life sciences, vol. 151, pp. 243-249 (2016), hereinafter “Liu 2016”.
Endo, Yori, et al., "Loss of ARNT in skeletal muscle limits muscle regeneration in aging", The FASEB Journal, vol. 34, no. 12, pp. 16086-16104 (2020), hereinafter “Endo 2020”.
4 Li, Xinlei, and Tao Peng. "Strategy, progress, and challenges of drug repurposing for efficient antiviral discovery." Frontiers in Pharmacology, vol. 12, pp. 1-29 (May 2021), hereinafter “Li 2021”.
5 Wise, Lisa M., et al., "Hypoxia-inducible factor 1α (HIF1α) suppresses virus replication in human cytomegalovirus infection by limiting kynurenine synthesis", MBio, vol. 12, no. 2, pp. 1-16 (March 23, 2021), hereinafter “Wise 2021”.
6 Lunn, Michael, and Richard Hughes, "The relationship between cytomegalovirus infection and Guillain–Barré syndrome", Clinical infectious diseases, vol. 52, no. 7, pp. 845-847 (2011), hereinafter “Lunn 2011”.
7 See Theriault, Jimmy R., et al., “Discovery of a New Molecular Probe ML228: An Activator of the Hypoxia Inducible Factor (HIF) Pathway”, Bioorg Med Chem Lett., vol. 22, no. 1, pp. 76-81 (2012), hereinafter “Theriault 2012”, (also cited in the IDS received on April 22, 2024, as NPL cite no. 3), at 81 (“ML228 was demonstrated to potently activate HIF in vitro as well as its downstream target VEGF.”).
8 See, e.g., Specification at 1 (“The present invention relates to compounds for treatment and prevention of virus infections. In particular, the present invention relates to broad-spectrum antiviral compounds.”) (emphasis added).
9 See Specification at 6 (“In the present context, the term "neurotropic virus" refers to a virus that is capable of infecting nerve cells.”).
10 See, e.g., Theriault 2012 at 76 (“Hypoxia and ischemia are linked to several serious public health problems that affect most major organ systems. Specific examples include diseases of the cardiovascular, pulmonary, renal, neuro-logic, and musculoskeletal systems. The hypoxia inducible factor (HIF) pathway is the major pathway required for intracellular adaptation initiated by lower oxygen availability in the blood-stream. The HIF pathway is known to take part in the angiogenesis processes enhancing blood supply required for tissue repair and regeneration. A unique gene transcription program involving the activation of multiple transcriptional factors known as the hypoxia inducible factors (HIFs) is triggered in response to hypoxia.1 HIFs exists as a heterodimeric complex containing one of three a sub-units (HIF-1a, HIF-2a, or HIF-3a) associated with the aryl hydro-carbon receptor nuclear translocator (ARNT), also known as HIF-1b. Although the hypoxic response requires multiple HIF subunits to be functional, hypoxia only leads to changes in both the accu-mulation and the activity of the HIF-1a subunit.”).
11 See, e.g., Theriault 2012 at 76 (“To date, the most common strategy for HIF activation is through inhibition of PHDs. General iron chelators such as desferrioxamine (DFO) and the inorganic salt cobalt chloride (CoCl2) have been explored clinically. DFO activates HIF-1α by chelating iron, whereas cobalt displaces iron from PHDs.”) (emphases added); id. (“In most cells the HIF pathway is primarily regulated by the inhibition of the HIF-1a subunit degradation during hypoxia.2 In nor-moxia, the intracellular level of HIF-1α protein is typically low due to its on-going ubiquitination and proteasomal degradation. The degradation of the HIF-1α subunit is initiated through hydroxylation on a conserved proline residue, which is a process mediated by three prolyl hydroxylase (PHD) isoforms known as PHD1, PHD2, and PHD3.3 The presence of iron, oxygen, and 2-oxo-glutarate (2-OG) are required for hydroxylation. Prolyl hydroxyl-ation is blocked during hypoxia, which leads to the stabilization of HIF-1α and the establishment of a negative feedback loop whereby hypoxia and HIF-1a both upregulate PHD2 expression.4 Once HIF-1α is stabilized, it accumulates in the cells, dimerizes with ARNT, and translocates into the nucleus. Inside the nucleus, the HIF-1a/ARNT complex through interaction with its binding partners initiates the transactivation of HIF-responsive genes, such as the glucose transporter Glut1 and the angiogenic factor vascular endothelial growth factor (VEGF).5 Several strategies have been explored for manipulation of the HIF pathway at various points to either promote or retard angiogenesis.6”) (emphases added).
12 As explained above, in the context of HIF1-alpha, the terms stabilizers and activators are used interchangeably.
13 See, e.g., Theriault 2012 at 78.
14 See supra claim interpretation section.
15 See supra discussion of claim interpretation.
16 As previously stated, ML228 is encompassed by the compound of formula (I) recited in the instant claim. Specifically, ML228 corresponds to a compound of formula (I) wherein R1 is –(CH2)n–R3, R2 is H, n is 1, and R3 is an optionally substituted phenyl.
17 Regarding claim 4, the examiner notes that HSV-1 and HSV-2 was explicitly discussed in the rejection of claim 1 in further view of Wise 2021.
18 Lunn 2011 was cited previously. For convenience, the full cite is restated. Lunn, Michael, and Richard Hughes, "The relationship between cytomegalovirus infection and Guillain–Barré syndrome", Clinical infectious diseases, vol. 52, no. 7, pp. 845-847 (2011), hereinafter “Lunn 2011”.
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