Abstract
A New HIV Drug Holds Promise for Cancer
WO/2022/000019,
Non-nucleoside reverse transcriptase inhibitors, such as rilpivirine, which were developed to treat HIV infection, have been found to be effective against many cancers. In some cases, the rationale is evident and direct, for example, in epithelial cancers that overexpress the pericentromeric human satellite II (HSATII) sequence, which is reverse transcribed by human endogenous retrovirus-K reverse transcriptase (see WO/2020/142629). In other cases, activation of the pregnane X nuclear receptor by rilpivirine might play a decisive role. 1,2 In mouse neuroblastoma xenograft models, strong synergy has been demonstrated with platinum drugs and vincristine (WO/2021/104487).
The present patent document attempts to expand the range further by showing that rilpivirine is a potent inhibitor of Aurora A kinase (K i = 0.13 μM), and at 10 μM, it also has inhibitory activity against Aurora B, Pim1, Jak1, and Flt3 kinases, all of which are found to be overexpressed in cancer. 3 At 1 μM, rilpivirine is a selective inhibitor of Aurora A and has significant levels of inhibition only against Pim1 and two other Src family tyrosine kinases, Lyn and Yes. At 5, 10, and 20 μM for 24 h, rilpivirine halted the cell cycle of HL-60, NB4, Jurkat, K-562, and U-937 leukemia cell lines and T47D breast cancer, PC3 prostate cancer, and MIA PaCa-2 pancreatic cancer cell lines at the G2/M phase, causing apoptosis.
When p53 wild-type cells were incubated with rilpivirine for 24, 48, and 72 h, cells were arrested in the G1 phase at all time points at 10 μM; they accumulated in the sub-G1 phase at higher rilpivirine concentrations. In breast and ovarian cancer cell lines, a region of synergy was identified between rilpivirine 1–5 μM and docetaxel 2–4 nM, while antagonism was observed with higher concentrations of either agent.
Malaria Drugs for Graves' Ophthalmopathy
WO/2022/000492,
Artemisinins—a family of sesquiterpene trioxane lactones—were the first effective and well-characterized antimalarial agents, but their bioavailability and pharmacokinetics are poor. While drug repurposing is considered a promising strategy to develop malaria drugs that address critical points in the Plasmodium life cycle and circumvent resistance, artemisinin and its derivatives have become interesting repurposing candidates for treatment of cancers and various inflammatory disorders and, most recently, for ocular conditions such as uveitis and retinal vascular diseases. 4,5 The present invention extends this to Graves' ophthalmopathy, a manifestation of the autoimmune thyroid disease where autoantibodies cause fibroblasts in the orbital muscles to differentiate into adipocytes, which expand and become inflamed.
Artemisinin (10, 50, and 200 μM) and its derivatives, artesunate (0.5, 2, and 10 μM) and dihydroartemisinin (1, 5, and 20 μM), were used on patients' primary orbital fibroblasts after three culture passages and were shown to inhibit their excessive proliferation, adipogenic differentiation, hyaluronic acid secretion, and fibrosis. This is in line with the recently reported observation that dihydroartemisinin inhibits TGF-β-induced fibrosis in tenon fibroblasts. 6 Although artemisinins are relatively expensive, their ocular formulations should be much cheaper than the anti-inflammatory biologicals that are currently approved for treatment of severe cases of Graves' ophthalmopathy.
The Oldest Gout Drug Reduces Hyperphosphatemia
WO/2022/007007,
The plant alkaloid, colchicine, has been in medical use to treat systemic inflammatory conditions, especially gout attacks and Behcet's disease, for a very long time. At the center of its pharmacological mechanism is its accumulation in leukocytes where it binds to tubulin, preventing microtubule assembly and thereby inflammasome activation and chemotaxis. 7,8 Its ability to cause cell cycle arrest at the G2/M phase as well as decreased mitochondrial membrane potential in cancer cells endows it with antiproliferative potential 9 and makes it a valuable tool to synchronize cell division and generate polyploid cells.
Building on the fact that the rapid adaptive increase in apical brush border membrane Na-P(i) cotransporter abundance on renal proximal tubules, which is seen as an acute response to a low-phosphorous diet, is due to microtubule-dependent translocation of the presynthesized transporter, 10 the inventors have reasoned that blocking microtubule assembly by colchicine should reduce the intestinal uptake of inorganic phosphate and reduce hyperphosphatemia in renal failure. Data from 15 individual patients show that colchicine (0.5 mg b.i.d.) typically reduced free serum phosphate by 10%–15%, without a low-phosphate diet or phosphate binders. The problem with colchicine is its narrow therapeutic window. Disruption of cytoplasmic microtubules in renal cortical cells by colchicine interferes with phosphate transport, which has already been reported in a 1976 article. 11
Empagliflozin Might Become Part of Cardiac Arrest Emergency Kits
WO/2022/011239,
Sodium–glucose cotransporter type 2 (Sglt2) inhibitors—the gliflozin class—have been developed as antidiabetic drugs that inhibit reabsorption of glucose in the kidney. It has been firmly established that they also have protective potential in chronic heart failure, independently of the patients' glycemic status. 12,13 A meta-analysis of 34 randomized controlled trials has found that gliflozin therapy was associated with a significant reduction in the risk of incident atrial arrhythmias and sudden cardiac death in diabetics, but no correlation with cardiac arrest or incidence of ventricular arrhythmias was seen. 14 In isolated cardiomyocytes, empagliflozin may exert its myocardial effects through inhibition of the sodium–hydrogen exchanger 1 (NHE-1) isoform by occupying its Na+-binding site. 15
However, gliflozins have not yet been investigated to assess whether they could elicit comparable beneficial effects on the myocardium during resuscitation from cardiac arrest, as NHE-1 inhibitors do in animal models. The inventors have now closed this gap by first testing empagliflozin in an open-chest swine model of ventricular fibrillation that enabled direct access to the heart during extracorporeal circulation to simulate the low-flow states of cardiopulmonary resuscitation (CPR). They then followed up with a closed-chest model that simulated the CPR technique currently used clinically for cardiac resuscitation.
During the postresuscitation phase, animals treated with empagliflozin had better myocardial and hemodynamic function at lower norepinephrine doses in the open-chest series, attributed to a higher left ventricular stroke work index and coronary blood flow accompanied by less wall thickening and faster relaxation. The closed-chest experiments also showed concomitant systemic vasodilation, which could be beneficial during resuscitation, enabling higher blood flow to be generated by chest compression.
Gliflozins for the Prevention and Treatment of Kidney Stones
WO/2022/015743,
Sglt-2 inhibitors, the new class of antidiabetics that reduce glycemia without inducing excessive insulin secretion by increasing urinary glucose excretion, have demonstrated cardiovascular benefits in normoglycemic patients with chronic as well as acute myocardial conditions (see discussion of WO/2022/011239, above). The present patent document claims (without presenting any data) that the induced glucosuria can have an unexpected side effect in patients with nephrolithiasis: urinary glucose may undergo nonenzymatic glycation with lithogenic compounds such as cystine and carboxylic acids to produce soluble adducts that can be excreted. The dose of gliflozin should provide about 20%–80% inhibition of Sglt2, which is roughly the range used to treat diabetes.
This application might be unrelated to the ongoing Boehringer Ingelheim-sponsored SWEETSTONE trial (NCT04911660), which assesses the impact of empagliflozin on urinary supersaturation of calcium oxalate, calcium phosphate, and uric acid in kidney stone formers 16 ; it is scheduled for completion at the end of 2022. The drawback of this treatment strategy is that gliflozins are contraindicated in diabetics with compromised renal function or urinary tract infections, both of which frequently accompany urolithiasis.
Big Data and Artificial Intelligence for Mitochondrial Diseases
WO/2022/025635,
A broad range of rare congenital disorders is either caused by compromised mitochondrial function or has it as a consequence. The inventors applied artificial intelligence-assisted in silico approaches to drug repurposing models based on the drug-perturbed transcriptional expression pattern of genes (obtained from the Broad Institute LINCS project 17 ) and also to structural and drug-like similarity in a total of 4 million compounds represented by SMILES strings with physical activities from public databases. In a third module, each given vector of the drug-perturbed gene expression data in the latent space under optimized hyperparameters was trained and clustered.
A final filtering step removed compounds that had been reported as therapeutics for the primary mitochondrial diseases attributed to mitochondrial respiratory chain dysfunction, were known to exert mitochondrial toxicity, or were reported to be toxic to patients with mitochondrial diseases. Into this multistage algorithm, the researchers fed a seed set of 27 compounds known or suspected to induce mitochondrial biogenesis (inter alia, bezafibrate, acipimox, pioglitazone, rosiglitazone, and varenicline).
Agents from the output hits were validated as mitochondria protective by intracellular ATP assays, membrane potential measurements, and resistance against MPP+-induced SH-SY5Y cell death: the macrolide antibiotic, josamycin; the calcium channel blockers, felodipine and cilnidipine; trapidil, a vasodilator and platelet derived growth factor antagonist; and metyrapone, a reversible inhibitor of 11β-hydroxylase that blocks cortisol steroidogenesis and is used for adrenocorticotropic hormone stimulation tests to diagnose adrenal insufficiency. Trapidil, metyrapone, josamycin, cilnidipine, and cyproterone acetate (a potent antiandrogen) were further validated in vivo in the Caenorhabditis elegans gas-1 (fc21) strain that harbors a mitochondrial complex I subunit homozygous missense mutation and in a Surf1(−/−) complex IV disease zebrafish model.
Osanetant Might Block Fertility in Both Genders
WO/2022/026901,
The NK3 receptor (which responds to neurokinin B) and the kisspeptin receptor are expressed in granulosa cells of healthy women and contribute to the control of fertility by acting on GnRH/luteinizing hormone secretion; their reduced expression is associated with infertility. 18 Additional evidence suggests that the same systems also have potential regulatory roles in spermatogenesis, spermatozoa function and motility, testicular steroidogenesis, and hence male fertility. 19 Animal data suggest that NK3 agonists could be used as infertility treatment for both sexes. 20,21
That the discontinued NK3 antagonist, osanetant (which had been investigated for treatment of schizophrenia and other central nervous system disorders), could be useful as a nonhormonal female contraceptive and as an agent for chemically castrating a male does not require a great leap of imagination; indeed, an observation that osanetant reduces the litter size of female rats has been reported almost 10 years ago. 22 This could offer a new possibility for individuals with a predisposition for developing hormone-sensitive cancers, where the use of hormonal therapeutic agents to prevent procreation is contraindicated. No data are presented. Also see Acer's companion application WO/2022/026900 claiming the treatment of vasomotor symptoms in cancer patients with NK antagonists.
An Antifungal Could Prevent Opportunistic Nonfungal Infections
WO/2022/027052,
Clotrimazole is an antifungal drug that disrupts membrane synthesis by targeting lipid metabolism; because of its modest systemic absorption, it is mostly used topically. Recently, there have been reports implicating it not only in immunomodulation in the context of fungal infection by blocking expression of c-Fos and proinflammatory cytokines 23 but also as an enhancer of dendritic cell-mediated antigen presentation in antitumor immunity. 24 Clotrimazole has also been investigated for a variety of other diseases, including sickle cell anemia, malaria, and Chagas disease.
In the present application, the inventors describe—apparently for the first time at the July 2020 priority date—that clotrimazole can activate the innate inflammatory response of macrophages in vitro, inducing elevated production of inflammatory cytokines, including IL-1β, TNFα, and IL-6, which are critical mediators for mounting an immunological defense against foreign pathogens.
It was already known that clotrimazole dissociates hexokinase 1 from the outer membrane of mitochondria in HIV-1-infected macrophages, which destabilizes them. 25 The present experiments, supported by an HK1-MBD (hexokinase 1 mitochondrial binding domain) mouse model constructed using CRISPR-Cas9, show that clotrimazole-driven liberation of hexokinase into the cytosol of macrophages induces a hyperinflammatory response, while GAPDH activity is inhibited, and activity of the pentose phosphate pathway is increased. This could offer a new perspective to prevent infections in patients who suffer from immunodeficiency or are preventively immunosuppressed.
A 70-Year-Old Anticonvulsant Revealed as a Kinase Inhibitor
WO/2022/027240,
Although the barbiturate drug, primidone, has been used to control seizures and essential tremor for seven decades, 26 it is still good for pharmacological surprises: the present document reveals that it is an inhibitor of the protein kinase, Ripk1, a promising therapeutic target for treatment of a wide range of human neurodegenerative, autoimmune, and inflammatory diseases. 27 In vitro data are provided concerning prevention of induced necrosis and Ripk1-dependent apoptosis in HT-29 human colon cancer cells, HT-22 hippocampal neuronal cells, and RCG5 rat retinal ganglion cells; even low nanomolar concentrations of primidone are highly effective. The priority date of this patent application might be in tight competition with a December 2020 article by German and U.K. researchers disclosing primidone as a potent inhibitor of Ripk1 activation in vitro and in a murine model of TNFα-induced shock. 28
Also see the companion patent applications: WO/2022/027239, claiming primidone as an inhibitor of proinflammatory cytokine release, and WO/2022/027237, claiming quizartinib as an Ripk1-dependent necroptosis inhibitor.
A Leukemia Drug Reactivates Silenced X-Chromosomes in Rett Syndrome
WO/2022/031759,
In Rett syndrome, loss-of-function mutations in the methyl-CpG-binding protein (MeCP2) cause random X chromosome inactivation, resulting in cells expressing either mutant or wild-type MeCP2 and leading to a mosaic pattern of mutant MeCP2 protein expression throughout the body. The invention comprises identifying a subject with an X-chromosome mutation outside of the MeCP2 exon 2 region and administering the DNA methyltransferase inhibitor, decitabine (a cytidine analog used to treat myelodysplastic syndromes and acute myeloid leukemia) to activate inactive X-chromosomes. This treatment builds on the fact that azacitidine and its 2′-deoxy derivative, decitabine, have shown substantial potency in reactivating epigenetically silenced tumor suppressor genes in vitro. 29
The diagnostic steps consist in obtaining X-chromosome-linked disease fibroblasts from the patient, reprogramming them into pluripotent stem cells, which are induced to differentiate into brain organoids, exposing their dissociated cells to decitabine (0.1–0.3 μM for 2 weeks), and checking for MeCP2 expression by immunofluorescence. Treatment should be possible with decitabine dosing regimens used for leukemia treatment; in animal models of Rett syndrome, even 5%–10% MeCP2 restoration improves neuromotor function and extends life span by five- to eight-fold. 30 The approach described here avoids the use of Xist antisense oligonucleotides, as reported in the inventors' earlier work. 31
A Breast Cancer Breakthrough Drug to Treat Neurodegeneration
WO/2022/039421,
Dysregulation of cyclin-dependent kinases, among them Cdk4 and Cdk6, which are important targets for cancer drugs, occurs in neurons in a wide diversity of chronically progressive as well as acute neurological disorders that are associated with microglial activation. G1/S cell cycle arrest through Cdk4/6 inhibition has been suggested as a potential strategy for Alzheimer's disease, 32 traumatic central nervous system injuries, 33 stroke, 34 and retinal degeneration. 35 Abemaciclib, approved for the treatment of hormone receptor-positive and HER2-negative breast cancer since 2017/18, is a unique representative of this agent class because of its combination of high oral bioavailability, relatively low myelosuppression allowing continuous dosing, and efficient brain penetrance through Abcb1a/1b and Abcg2 transporters. 36,37
While abemaciclib has been investigated for treatment of brain cancers, no claims for neurodegenerative diseases seem to have been made so far. The inventors support these with data from rodent models of lipopolysaccharide-induced cognitive decline, as measured in Y-maze and new object recognition paradigms, and extensive immunohistochemistry showing inhibition of inflammatory cytokine expression (IL-1β and IL-6) as well as Cox-2 and iNOS in neurons, along with inhibition of Tlr-4 signaling and levels of p-Stat3. Abemaciclib also reduced the density of activated astrocytes interacting with amyloid plaques to about the same degree as the cholinesterase inhibitor, donepezil.
The Long and Diverse Development History of a Synthetic Retinoid
WO/2022/040782,
Some compounds are repurposed by their original developer; others before they are approved, if at all; and in some cases, repurposing goes along with a change in compound ownership. Most of this is the case with fenretinide, a synthetic retinoid with a long development history. Originated at Ortho-McNeil Pharmaceutical and investigated for various solid tumors from the late 1980s onward, 38 reVision Therapeutics switched it to geographic atrophy in “dry” age-related macular degeneration. 39 Sytera, Inc., continued development under the research code SYT101. It became ST-602 when Sirion Therapeutics, Inc., acquired the company with its assets in 2006. Development for dry age-related macular degeneration ended in 2015 after a Phase II trial. Rheumatoid arthritis and even obesity had also been considered as potential treatment targets.
Fenretinide underwent clinical investigation for cystic fibrosis 40 by Laurent Pharmaceuticals, which meanwhile brought it into Phase II/III clinical trials for treatment of COVID-19. Laurent now attempts a further expansion of fenretinide's application range to interstitial lung diseases. Although antifibrotic effects of fenretinide were reported in the context of liver and skin fibrosis, these findings cannot be extrapolated (as is sometimes done in the literature) to pulmonary fibrosis, which has a different pathogenesis and pathophysiology.
The inventors demonstrate that fenretinide dose-dependently attenuates expression of alpha smooth muscle actin and procollagen 1A1 in normal human lung fibroblasts after exposure to TGF-β, as measured by ELISA and quantitative Reverse Transcription Polymerase Chain Reaction. Ashcroft scoring of mouse lung tissue following induction of lung fibrosis by intratracheal bleomycin instillation and 21-day oral administration of LAU-7b (the formulation of fenretinide for COVID-19 trials) was improved, as was collagen deposition, as assessed by whole-lobe high-resolution imaging of mouse lung tissue stained with Sirius red and 14-day oral administration.
Jak Inhibitors: For Myelofibrosis and Probably for Otoprotection
WO/2022/051029,
Hearing impairments are typically not amenable to pharmacological treatment, although limited progress has been made in repurposing of sodium channel blockers and excitatory receptor antagonists for some forms of tinnitus. A special problem is permanent sensorineural hearing loss caused by macrolide antibiotics 41 or platinum drugs, 42 which can destroy the inner ear hair cells lining the cochlea.
The inventors sought compounds that would protect against hair cell apoptosis by comparing datasets from drug screen connectivity maps and pathway enrichment analysis based on mouse strains that are resistant (CAST) or sensitive (129/SvJ) to sensorineural hearing loss. The identified compounds exhibited overlaps in gene expression transcriptomic profiles between at least one of a plurality of cell lines or mouse strains treated with ototoxic insults (cisplatin or antibiotic) and at least one of a plurality of cell lines or mouse strains treated with one of the compounds. All of the primary hits were kinase inhibitors: momelotinib, fedratinib, axitinib, sunitinib, ruxolitinib, oclacitinib maleate, deucravacitinib, itacitinib, solcitinib, ritlecitinib, filgotinib, decernotinib, and others with only research codes, which seem to indicate a correspondingly focused candidate compound pool.
Momelotinib and fedratinib (two Jak inhibitors under development or approved for myeloproliferative disorders) were tested in Zebrafish larvae (whose neuromast hair cells are considered homologous to those in the mammalian inner ear and are readily accessible to drugs) at nanomolar to micromolar concentrations. Dose dependency was seen only for fedratinib, with protection ∼100% at 13 μM and an EC50 of about 1 μM; momelotinib had only marginal efficacy. It has already been reported that inhibition of the Jak2/Stat3 pathway is protective against noise-induced cochlear tissue damage. 43
Machine Learning Identifies a New Activity in a 50-Year-Old Drug
WO/2022/061019,
Cholinesterase inhibitors were the first class of drugs to provide symptomatic benefit (and to a very limited degree, disease-modifying properties) in Alzheimer's disease. Many derivatives of donepezil, rivastigmine, and galantamine have been patented and clinically investigated, but never completed development. Entirely new cholinesterase inhibitors are not very intensely searched for by screening or rational design because the three widely approved ones seem to have already exhausted the potential of cholinergic dementia therapy. Drug repurposing is even less likely to offer new candidates: significant cholinesterase inhibition is a property drug developers select against because of the quite significant intestinal and cardiovascular side effect profile.
It is therefore highly remarkable to see that tilorone, a polynucleotide binder and interferon inducer that is effective against both DNA and RNA viruses, 44,45 inhibits human acetylcholinesterase with an IC50 of 56–73 nM (less than donepezil, but almost an order of magnitude better than galantamine), but not butyrylcholinesterase (IC50 >50 μM). The applicant's ASSAY CENTRAL® software and all ChEMBL compound datasets with IC50 data for human and eel acetylcholinesterase and human butyrylcholinesterase were used to create a training set; other machine learning models (random forest, k-nearest neighbors, support vector classification, naive Bayesian, AdaBoost decision trees, and deep learning) gave very similar results.
The best hit from the screening pool, tilorone, was docked in the recombinant human AChE crystal structure. CDOCKER, a simulated annealing-based docking algorithm, was used to generate a maximum of 10 poses of tilorone using rigid docking. In the Eurofins Discovery SafetyScreen44™ panel, which includes 38 binding assays and six functional assays of well-established targets and pathways, tilorone at 1 μM was found to only inhibit one target >50%: acetylcholinesterase. For the peer review companion article, see the study by Vignaux et al. 46 Note that tilorone has also been found to be active against Lewis lung carcinoma and B-16 melanoma cell lines. 47
Topical Antidepressants Ameliorate Radiation Dermatitis
WO/2022/066791,
Almost all patients receiving radiation therapy for cancer will experience an adverse epithelial reaction; depending on the cancer site, this will manifest as radiation dermatitis, severe oral mucositis, or radiation proctopathy. Steroid creams and emollients can slightly delay the onset of dermatitis, but hardly ameliorate its intensity, 48 and only palliative measures are available for mucositis and proctopathy. It has been proposed that damage to healthy tissue from radiation therapy may involve an endothelial response associated with signaling from the plasma membrane, through the acid sphingomyelinase/ceramide pathway 49 ; it follows that functional inhibitors of acid sphingomyelinase (FIASMAs) could provide an opportunity.
Conveniently, many of the widely used selective serotonin reuptake inhibitors as well as the old tricyclics are FIASMAs, which might well be major contributors to their antidepressant and anxiolytic activities. 50 What remained to be done by the inventors was to develop suitable formulations, with sertraline and amitriptyline as the most preferred agents. Besides the obvious topical formulations (0.5%–1% w/w), claims include soft, compressible, and porous rectal plugs to deliver agents for the treatment or prevention of proctopathy.
Radiation-induced dermatitis experiments with a 4% sertraline oil-in-water cream were conducted in female C57BL/6J mice. During the first 2 weeks after 30-Gy irradiation, all of the mice that received treatment with 4% sertraline cream, once daily, had a skin injury score of 4.5, while mice that received vehicle treatment had less severe scores of 3.0–3.5. On day 21, both groups scored 3.0–3.5, and at the end of the experiment (day 28), the sertraline-treated group had less severe skin injury scores ranging from 1.5 to 2.0 with an average score of 1.7, while vehicle-treated mice maintained higher skin injury scores ranging from 1.5 to 3.5 with an average score of 3.0.
Footnotes
DISCLOSURE STATEMENT
No competing financial interests exist.
FUNDING INFORMATION
No funding was received for this work.