Patent Publication Number: US-2023141664-A1

Title: Compositions and methods for the treatment and prevention of vascular malformations

Description:
CROSS REFERENCE TO RELATED APPLICATION 
     The present application is a U.S. National Phase Application under 35 U.S.C. § 371 of International Patent Application No. PCT/US2021/032374, filed May 14, 2021, which claims priority to U.S. Patent Application Ser. No. 63/024,670, filed May 14, 2020, each of which is hereby incorporated by reference as if expressly set forth in their respective entirety herein. 
    
    
     STATEMENT OF GOVERNMENTAL INTEREST 
     The present invention was made with government support under grants W81XWH1910266 and W81XWH191027 awarded by the Department of Defense and R03 HD092662 awarded by the National Institutes of Health. The government has certain rights in the present invention. 
    
    
     FIELD 
     This disclosure is in the field of preventing and treating vascular malformations, as well as in the field of screening for agents which prevent and treat these vascular malformations. 
     BACKGROUND 
     Vascular malformations are congenital anomalies of the blood and lymphatic vascular systems with a prevalence ranging from 1:3000 for lymphatic malformations (LMs) to 1% for venous malformations (VMs) and classified by the affected vessel type; capillary (CM), lymphatic (LM), venous (VM), and arteriovenous (AVM) malformations (Greene and Goss 2018; Behravesh et al. 2016). Vascular malformations are often apparent at birth and affected patients have significant morbidities and even mortality. Lymphatic dysfunction in LMs leads to defects in immune surveillance and lipid absorption leading to repeat infections and sepsis to malabsorption (Potente and Makinen 2017). VMs are associated with hematologic anomalies and painful intravascular coagulopathy (Dompmartin et al. 2008). In combined lymphatic and venous malformations (LVMs), deep vein thrombosis can result in life-threatening pulmonary emboli (Mazereeuw-Hautier et al. 2007). AVMs are associated with potentially life-threatening bleeding and heart failure (Uller et al. 2014). Another co-morbidity that affects all types of vascular malformation is hemorrhaging that can require repeated transfusions. In sensitive locations such as the brain, VMs and AVMs can have neurologically devastating consequences that can lead to death (Nikolaev et al. 2018). Despite these severe morbidities, there are no FDA-approved treatment for any vascular malformations. 
     Currently available treatments (such as surgery and interventional procedures) alleviate symptoms, but are not curative, and recurrence is common often requiring repeat interventions (Greene and Goss 2018; Behravesh et al. 2016). When the malformation is extensive, the effects of these treatments are limited at best. More recently, vascular malformation treatments have been guided by the genetics of the malformation. Studies have shown both germline and postzygotic genetic variants leading to hyperactivation of the PI3K/AKT/mTOR and/or the RAS/RAF/MAPK signaling pathways contribute to vascular malformation pathogenesis (Greene and Goss 2018). Based on this knowledge, pharmacotherapies—often cancer drugs—targeting these pathways have been used off-label clinically. Sirolimus, an mTOR inhibitor targeting the PI3K/AKT hyperactivation, has been used most extensively for VMs and LMs, but has generated only partial response (Hammill et al. 2011). 
     More recently, there have been multiple anecdotal reports using alpelisib, a Pik3ca inhibitor which targets PI3K/AKT/mTOR hyperactivation (Lopez Gutierrez et al. 20191), and trametinib, a MEK inhibitor targeting hyperactivated RAS/RAF/MAPK pathway hyperactivation (Foster et al. 2020). However, no pharmacotherapy, or class of pharmacotherapies, have shown complete response, despite this targeted approach. 
     Development of effective, biologically targeted therapies is sorely needed, but hampered by our limited understanding of how their genetics influences therapeutic responses. 
     SUMMARY 
     Using high throughput screening of endothelial cells from patients with vascular malformations, which had been identified as having specific genetic mutations, pharmacological agents were identified which inhibited the growth and viability of the cells. These pharmacological agents can be used for the treatment and prevention of vascular malformations, as well as to develop therapeutic and preventative agents for vascular malformations. 
     Thus, one embodiment of the present disclosure is a method of preventing and/or treating vascular malformations, comprising administering to a subject in need thereof a therapeutically effective amount of a proteasome inhibitor. 
     In some embodiments, the proteasome inhibitor is an inhibitor of the 20s subunit of proteasomes. In some embodiments, the proteasome inhibitor is an inhibitor of the chymotrypsin-like activity (CT-L), trypsin-like activity (T-L) and/or caspase-like (C-L) activity of the 20s subunit. In some embodiments, the proteasome inhibitor is an inhibitor of all three activities. In some embodiments, the proteasome inhibitor is an inhibitor of two of the three activities. In some embodiments, the proteasome inhibitor is an inhibitor of one of the three activities. In some embodiments, the proteasome inhibitor is an inhibitor of chymotrypsin-like activity of the 20s subunit. 
     In some embodiments, the proteasome inhibitor acts indirectly to inhibit proteolytic activities of proteasome complex. 
     In some embodiments, the proteasome inhibitor is listed in Tables 1 and 2. 
     The proteasome inhibitor for use in the method includes but is not limited to carfilzomib, delanzomib, ixazomib, bortezomib, oprozomib, and marizomib. See Table 1. 
     Additional proteasome inhibitors for use in the method include but are not limited to those listed in Table 2. 
     A further embodiment of the present disclosure is a method of preventing and/or treating vascular malformations, comprising administering to a subject in need thereof a therapeutically effective amount of disulfiram. 
     A further embodiment of the present disclosure is a method of preventing and/or treating vascular malformations, comprising administering to a subject in need thereof a therapeutically effective amount of an agent or compound which has proteasome inhibiting ability. 
     A further embodiment of the present disclosure is a method of preventing and/or treating vascular malformations, comprising administering to a subject in need thereof a therapeutically effective amount of a copper-binding agent or compound. 
     A further embodiment of the present disclosure is a method of preventing and/or treating vascular malformations, comprising administering to a subject in need thereof a therapeutically effective amount of omipalisib. 
     A further embodiment of the present disclosure is a method of preventing and/or treating vascular malformations, comprising administering to a subject in need thereof a therapeutically effective amount of an agent which targets a mutation in a gene chosen from the group consisting of Pik3ca, Pik3r3, Tsc2, Rasa1, Map2k2, Glmn, and combinations thereof. 
     A further embodiment of the present disclosure is a method of preventing and/or treating vascular malformations, comprising administering to a subject in need thereof a therapeutically effective amount of an agent which targets a mutation in a gene in the PI3K/AKT/mTOR or RAS/RAF/MAPK pathway. Such genes include but are not limited to Kras and Nras. 
     Vascular malformations that can be prevented and/or treated by the disclosed methods include but are not limited to lymphatic malformations (LMs), venous malformations (VMs), capillary malformations (CM), arteriovenous malformations (AVM), and combinations thereof. 
     In some embodiments, the agent further comprises a pharmaceutically acceptable carrier and is part of a composition. 
     In some embodiment, the subject is over 18 years old. In some embodiments, the subject is under 18 years old. In some embodiments, the subject is under 16 years old. In some embodiments, the subject is under 10 years old. In some embodiments, the subject is under 5 years old. In some embodiments, the subject is under 1 year old. 
     In an additional embodiment, the disclosure provides for methods of obtaining cells from the vascular malformation of the subject and testing the efficacy and/or dosage of any agent prior to administration of the agent. In some embodiments, these cells are endothelial cells. In some embodiments, the cells are subject to sequencing or other analysis to determine the mutations and/or defects within the cells. 
     A further embodiment of the present disclosure are kits comprising compositions and agents for practicing the disclosed methods. 
     A further embodiment of the present disclosure is a method and/or assay for screening and/or identifying an agent for the treatment and/or prevention of vascular malformations including lymphatic, venous, capillary, and arteriovenous and combinations thereof, comprising contacting or incubating a test agent with a cell derived from a patient with a vascular malformation, wherein if the viability and/or proliferation of the cells is decreased after contact or incubation with the test agent, the test agent is identified as a therapeutic and/or preventative agent for vascular malformations. 
     In some embodiments, the cells are endothelial cells. In some embodiments, the cells harbor mutations in a gene in the PI3K/AKT/mTOR or RAS/RAF/MAPK pathway. In some embodiments, the cells harbor mutations in a gene chosen from the group consisting of Pik3ca, Pik3r3, Tsc2, Rasa1, Map2k2, Glmn, and combinations thereof. In some embodiments, the cells are chosen from the group of cells listed in Table 3. In some embodiments, high throughput screening is performed. In some embodiments, the cells are subject to sequencing or other analysis to determine the mutations and/or defects within the cells. 
     The disclosure also includes any agents identified by the screening methods described herein and methods of using the same for the prevention and/or treatment of vascular malformations including lymphatic, venous, capillary, and arteriovenous and combinations thereof. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       For the purpose of illustrating the invention, there are depicted in drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings. 
         FIG.  1   —Inhibitors that target the PI3K/AKT and RAS/MAPK pathways display different efficacies against LMECs.  FIG.  1 A  shows the results of LMECs with different genetic variants (PIK3CA, PIK3CA/TSC2, and RASA1) and control cells (HdLECs) treated with increasing log doses of sirolimus.  FIG.  1 B  shows the results of the same cells treated with increasing log doses of alpelisib.  FIG.  1 C  shows results of the same cells treated with increasing log doses of the MAPK kinase inhibitor U0126. Proliferation rate was determined by WST-8 assay and expressed as a fraction of the number of cells in vehicle controls ±SD. *p&lt;0.05. The large arrow denotes the range of human doses ( FIGS.  1 A and  1 B ) and murine doses ( FIG.  1 C ) used in clinical setting. 
         FIG.  2   —PI3K/AKT pathway inhibitors differentially affect cell survival in a HTPS assay. Six endothelial cell populations isolated from venous malformation (VM) and lymphatic malformation (LM) patients (VMECs and LMECs), carrying mutations that hyperactivate PI3K signaling, were subjected HTPS with all inhibitors at 1 μM. Cell survival was determined after 48 hours and data presented as percent cell survival of cells treated with 1 μM compound versus vehicle-treated control ±SD. Small molecules presented belonged to 2 classes of inhibitors: PI3K inhibitors and proteasome inhibitors (right). Dark gray line: 50% cell from starting number. Omipalisib and proteasome inhibitors were significantly more efficacious than sirolimus at inhibiting growth/survival of the ECs with PI3K hyperactivation. Sirolimus to PI3K/mTOR inhibitors: ANOVA p=0.0017, post-hoc TTEST sirolimus vs. omipalisib p=0.0005. Sirolimus vs proteasome Inhibitors: ANOVA p=0.0013, post-hoc TTEST sirolimus vs. carfilzomib p&lt;0.0001, sirolimus vs. delanzomib p=0.0093. 
         FIG.  3   —Omipalisib demonstrated as a specific inhibitor with improved efficacy against LMEC28. Cell survival was determined after 48 hours and data presented as percent cell survival of cells treated versus vehicle treated cells. Omipalisib, a PI3K/mTOR inhibitor, at a lower dose (*) was more efficacious than another PI3K/mTOR dual inhibitor, bimiralisib, in inhibiting LMEC proliferation. At 10 nM; TTEST p&lt;0.03. 
         FIG.  4   —Proteasome Inhibitors efficaciously suppressed VMEC and LMEC growth/viability more than that observed for drugs used off label for vascular malformation patients.  FIG.  4    is a graph of cell viability of treated cells relative to vehicle treated cells. Using HTPS, VMEC/LMECs carrying variants in genes in the RAS/RAF/MAPK (LMEC10, VMEC13) or PI3K/AKT/mTOR (VMEC7/9, LMEC8/28/94/99) pathways (Table 3) were treated with 1 μM of 1344 compounds. Cell viability relative to vehicle treated cells was determined after 48 hours. Data presented for three PIs tested, and sirolimus, alpelisib, and trametinib, all of which have been used off label for vascular malformation patients. 
         FIG.  5   —Dose Response Curves of PIs, sirolimus, alpelisib and omipalisib using HMVEC (n=2) (control/normal/wild-type cells to be compared to those from vascular malformation patients), VMEC7, VMEC9, LMEC94 and LMEC99 (Table 3). The graphs show the percent viability of the cells at the various dosages in μM (dose range, 0.00125 μM to 20 μM). The large arrows mark the reported plasma concentration in humans.  FIG.  5 A  shows marizomib.  FIG.  5 B  shows bortezomib.  FIG.  5 C  shows carfilzomib.  FIG.  5 D  show delanzomib.  FIG.  5 E  shows ixazomib.  FIG.  5 F  shows oprozomib.  FIG.  5 G  shows sirolimus.  FIG.  5 H  shows alpelisib.  FIG.  5 I  shows omipalisib. 
         FIG.  6   —Proteasome inhibitors (PIs) are more effective at reducing viable cells than current therapies currently used off-label for vascular malformation patients.  FIG.  6    is a graph of the results of VMECs (n=2) and LMECs (n=2) carrying Pik3ca mutations subjected to increasing amounts of designated PI (left 6) or sirolimus or alpelisib, and cell viability determined after 48 hours at doses corresponding to clinically reported plasma concentration in μM. Data represent plasma concentration achieved in humans and presented as % viable cells treated with drug relative to vehicle treated cells. 
         FIG.  7   —PI effect not limited to VM/LM ECs with Pik3ca variants.  FIG.  7    is a graph of the results of LMEC28 carrying Pik3r3;Tsc2 mutations subjected to increasing amounts of oprozomib and cell viability determined after 48 hours. Yellow arrow marks clinically relevant dose and presented as % viable cells treated with drug relative to vehicle treated cells. 
         FIG.  8   —In vivo testing of PIs. In a xenograft model, oprozomib inhibited VM vessel growth.  FIG.  8 A  are images of the gross appearance of implants after treatment with vehicle or oprozomib. The dotted line marks the border of the Matrigel®.  FIG.  8 B  are representative H&amp;E images of vehicle and oprozomib treated implants. Boxed area is enlarged to the right. Scale bar—50 μm.  FIG.  8 C  is a graph of the quantification of vascular area normalized to area. *p&lt;0.05. vc—vascular channels. 
         FIG.  9   —Disulfiram efficaciously suppressed VMEC and LMEC growth/viability more than that observed for drugs used off label for vascular malformation patients. Using HTPS, cells were treated with 1 μM of compounds. Cell viability relative to vehicle treated cells was determined after 48 hours. Data presented for disulfiram against sirolimus, alpelisib, and trametinib, all of which have been used off label for vascular malformation patients. 
     
    
    
     DETAILED DESCRIPTION 
     Definitions 
     The terms used in this specification generally have their ordinary meanings in the art, within the context of this invention and the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the methods of the invention and how to use them. Moreover, it will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of the other synonyms. The use of examples anywhere in the specification, including examples of any terms discussed herein, is illustrative only, and in no way limits the scope and meaning of the invention or any exemplified term. Likewise, the invention is not limited to its preferred embodiments. 
     The term “subject” as used in this application means an animal with an immune system such as avians and mammals. Mammals include canines, felines, rodents, bovine, equines, porcines, ovines, and primates. Avians include, but are not limited to, fowls, songbirds, and raptors. Thus, the invention can be used in veterinary medicine, e.g., to treat companion animals, farm animals, laboratory animals in zoological parks, and animals in the wild. The invention is particularly desirable for human medical applications. 
     The term “patient” as used in this application means a human subject. In some embodiments of the present invention, the “patient” is suffering with from a vascular malformation, or a disease or condition characterized by a vascular malformation including capillary (CM), lymphatic (LM), venous (VM), and arteriovenous (AVM), and combinations thereof. 
     The terms “treat”, “treatment”, and the like refer to a means to slow down, relieve, ameliorate or alleviate at least one of the symptoms of the disease, or reverse the disease after its onset. 
     The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease onset, to prevent the disease from developing or minimize the extent of the disease or slow its course of development. 
     The term “in need thereof” would be a subject known or suspected of having or being at risk of a vascular malformation or a disease or condition characterized by a vascular malformation including capillary (CM), lymphatic (LM), venous (VM), and arteriovenous (AVM), and combinations thereof. 
     A subject in need of treatment would be one that has already developed the disease or condition. A subject in need of prevention would be one with risk factors of the disease or condition. 
     The term “agent” as used herein means a substance that produces or is capable of producing an effect and would include, but is not limited to, chemicals, pharmaceuticals, biologics, small organic molecules, antibodies, nucleic acids, peptides, and proteins. 
     The phrase “therapeutically effective amount” is used herein to mean an amount sufficient to cause an improvement in a clinically significant condition in the subject, or delays or minimizes or mitigates one or more symptoms associated with the disease, or results in a desired beneficial change of physiology in the subject. 
     The terms “screen” and “screening” and the like as used herein means to test an agent to determine if it has a particular action or efficacy. 
     The terms “identification”, “identify”, “identifying” and the like as used herein means to recognize an agent as being effective for a particular use. 
     The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, “about” can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” meaning within an acceptable error range for the particular value should be assumed. 
     ABBREVIATIONS 
     LM lymphatic malformation
 
VM venous malformation
 
CM capillary malformation
 
AVM arteriovenous malformation
 
EC s endothelial cells
 
LMECs lymphatic malformation endothelial cells
 
VMECs venous malformation endothelial cells
 
HMVECs human microvascular endothelial cells (controls)
 
WES whole exome sequencing
 
HTPS high throughput screening
 
PI proteasome inhibitor
 
     Identification of Therapeutic and Prophylactic Agents for Vascular Malformations 
     Germline mutations have been identified in some vascular malformation subtypes yet screening for germline variants has often failed to identify causative mutations. Recent studies have shown that post-zygotic mutations contribute to a significant portion of AVMs, VMs, and LMs. Somatic mutations are present only in affected tissues, specifically the malformed endothelial cells (ECs). 
     Endothelial cells from vascular malformation tissues carrying known disease associated pathogenic variants, including those that target the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways were collected. It was hypothesized that ECs isolated from patients that carried pathogenic somatic, as well as yet to be identified variants, could be used in a drug screen to identify novel therapies for the treatment of vascular malformations. Whole exome sequencing (WES) was used in the patient derived endothelial cells from venous and lymphatic malformation patients to identify mosaic pathogenic variants. These cells were then used in high throughput drug screening (HTPS) to identify new potential therapies for the venous and lymphatic malformation population (Table 3). 
     Using these endothelial cells carrying variants leading to activation of the PI3K pathway in HTPS, omipalisib, a combined PI3K/mTOR inhibitor, as well as proteasome inhibitors, as a class, and disulfiram, were identified to be efficacious in inhibiting cell survival. In the HTPS and proliferation assays, omipalisib and proteasome inhibitors, as well as disulfiram, had significantly improved efficacy when compared to sirolimus and alpelisib which are the standard of care for patients with vascular malformations. 
     Methods and Compositions for the Prevention and Treatment of Vascular Malformations 
     Based upon the findings set forth herein, the present disclosure provides for methods of preventing and/or treating vascular malformations including lymphatic, venous, capillary, arteriovenous, and combinations thereof, comprising administering to a subject in need thereof a therapeutically effective amount of an agent which targets a mutation in a gene in the PI3K/AKT/mTOR or RAS/RAF/MAPK pathway. 
     The current disclosure further provides for methods of preventing and/or treating vascular malformations including lymphatic, venous, capillary, arteriovenous, and combinations thereof, comprising administering to a subject in need thereof a therapeutically effective amount of an agent which targets a mutation in a gene chosen from the group consisting of Pik3ca, Pik3r3, Tsc2, Rasa1, Map2k2, Glmn, and combinations thereof. 
     The current disclosure further provides for methods of preventing and/or treating vascular malformations including lymphatic, venous, capillary, arteriovenous, and combinations thereof, comprising administering to a subject in need thereof a therapeutically effective amount of omipalisib. 
     HTPS determined that omipalisib was the strongest PI3K pathway inhibitor of proliferation/viablity of vascular malformation endothelial cells with PI3K hyperactivation, relative to the 14 tested inhibitors of the PI3K pathway. Omipalisib is a dual PI3K pathway inhibitor which targets the p110 activation domain of PI3K and MTOR, suggesting targeting two protein complexes in the PI3K pathway is more effective than a single target. When another dual PI3K and MTOR inhibitor, bimiralisib, or the clinically used sirolimus were assessed, omipalisib was significantly more efficacious at inhibiting proliferation of LMECs at a lower concentration then either of the other drugs. 
     Together these data suggest omipalisib is more effective at targeting the pathogenic hyperactivation of PI3K in vascular malformations than current therapies. 
     Omipalisib has completed Phase I clinical trials for both solid tumors (NCT00972686) and idiopathic pulmonary fibrosis (NCT01725139). It has never been investigated as a candidate for therapy for vascular malformations. 
     Without being bound by any theory, it is hypothesized that this drug uniquely inhibits the pathological hyperactivation of the PI3K signaling in two different subtypes of vascular malformations, venous and lymphatic malformations. Both vascular malformations have been shown to arise from somatic mutations endothelial cells in genes of the PI3K signaling pathway, including Pik3ca, Pik3r3, Tsc2, and Akt, which lead to increased activity of the PI3K p110 subunit and mTOR proteins targeted by omipalisib. 
     Omipalisib: 
     formula: C 25 H 17 F 2 N 5 O 3 S 
     chemical name: 2,4-difluoro-N-(2-methoxy-5-(4-(pyridazin-4-yl)quinolin-6-yl)pyridin yl)benzenesulfonamide. 
     Dose: 0.5 to 2 mg BID 
     Effective concentration—74.6 ng/mL (=0.15 μM) (Lukey et al. 2019) 
     The current disclosure further provides for methods of preventing and/or treating vascular malformations including lymphatic, venous, capillary, arteriovenous, and combinations thereof, comprising administering to a subject in need thereof a therapeutically effective amount of one or more proteasome inhibitors. The disclosure further provides for methods of preventing and/or treating vascular malformations, including lymphatic, venous, capillary, arteriovenous, and combinations thereof, comprising administering to a subject in need thereof a therapeutically effective amount of an agent or compound which has proteasome inhibiting ability. 
     Proteasome inhibitors that can used in the method include but are not limited to bortezomib, oprozomib, carfilzomib, ixazomib, delanzomib, and marizomib. 
     Several proteasome inhibitors are already FDA-approved, while several are in different phases of clinical trials. The FDA-approved indication is for the treatment of multiple myeloma, while clinical trials are also investigating their use in solid tumors and non-Hodgkins lymphoma. 
     Proteasome inhibitors as a class has not been investigated for use in patients with vascular malformations. See Tables 1 and 2. 
     Some of the proteasome inhibitors identified target the 20S catalytic subunit in the proteasome complex and in some instances, target all three proteolytic activities of the 20S subunit including the chymotrypsin-like activity (CT-L), trypsin-like activity (T-L) and caspase-like (C-L) activity. In some instance, the PIs targeted the chymotrypsin-like activity. In some instances, the proteasome inhibitors act indirectly to inhibit the proteasome complex. 
     Mutations in genes that make up the proteasome complex or their involvement in vascular pathologies have not been reported for vascular malformations. 
     Without being bound by any theory, proteasomes function in protein degradation via autophagy and ubiquitin-dependent, or ubiquitin-independent proteasome degradation pathway and has been implicated as a regulator of PI3K signaling. Thus, proteasome inhibitors may also inhibit proliferation of vascular malformation cells by targeting genetic activation of the PI3K pathway. 
     Proteasomes have also been shown to modulate angiogenic signaling via regulation of VEGF-A/VEGFR2 signaling, as well as other angiogenic and lymphangiogenic pathways. 
     Additionally, disulfiram was also found to inhibit cell proliferation and viability. Disulfiram has shown inhibition of proteasome activity. Thus, the current disclosure further provides for methods of preventing and treating vascular malformations including lymphatic, venous, capillary, arteriovenous, and combinations thereof, comprising administering to a subject in need thereof a therapeutically effective amount of disulfiram. 
     Copper-binding compounds, such as disulfiram, Clioquinol and diethyldithiocarbamate have also been documented to have proteasome inhibiting ability (Chen et al. 2006; Chen and Dou 2008). Thus, current disclosure further provides for methods of preventing and/or treating vascular malformations including lymphatic, venous, capillary, arteriovenous, and combinations thereof, comprising administering to a subject in need thereof a therapeutically effective amount of a copper-binding agent or compound. Copper-binding agents or compounds for use in the current methods include but are not limited to NSC-109268, pyrrolidine dithiocarbamate, disulfiram, Clioquinol and diethyldithiocarbamate. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 FDA Approved Currently Available Proteasome Inhibitors 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                   
                   
                 Plasma 
               
               
                   
                   
                 FDA  
                   
                 Concentration 
               
               
                 Inhibitor 
                 Mechanism 
                 Approval Status 
                 Dose 
                 (ng/mL) 
               
               
                   
               
               
                 Bortezomib 
                 Selective, 
                 2005 
                 0.4-1.38 mg/m 2   
                 100-200  
               
               
                   
                 reversible 
                   
                   
                 (260-520 nM) 
               
               
                   
                 inhibitor of 
                   
                   
                 (Shabaneh et al. 
               
               
                   
                 chymotrypsin- 
                   
                   
                 2013) 
               
               
                   
                 like activity of 
                   
                   
                   
               
               
                   
                 the 20S 
                   
                   
                   
               
               
                   
                 proteasome 
                   
                   
                   
               
               
                 Carfilzomib 
                 Irreversible 
                 2012 
                 27-56 mg/m 2   
                 932-2733  
               
               
                   
                 epoxyketone 
                   
                   
                 (1294-3796 nM)  
               
               
                   
                 inhibitor of the 
                   
                   
                 (Brown 
               
               
                   
                 chymotrypsin- 
                   
                   
                 et al. 2017) 
               
               
                   
                 like activity of 
                   
                   
                   
               
               
                   
                 20S 
                   
                   
                   
               
               
                 Marizomib 
                 Irreversible 
                 Orphan drug 
                 0.075-0.9 mg/m 2   
                 2.76-57.75  
               
               
                   
                 inhibitor of all 
                 designation 2015 
                   
                 (8.8-184 nM) 
               
               
                   
                 three proteasome 
                   
                   
                 (Harrison et al. 
               
               
                   
                 protease 
                   
                   
                 2016) 
               
               
                   
                 activities 
                   
                   
                   
               
               
                   
                 (chymotrypsin- 
                   
                   
                   
               
               
                   
                 like, trypsin-like 
                   
                   
                   
               
               
                   
                 and caspase-like 
                   
                   
                   
               
               
                   
                 activity of 20S) 
                   
                   
                   
               
               
                 Izazomib (oral) 
                 Boronic acid 
                 2015 
                 1.5-4 mg/d 
                 26.1-61 
               
               
                   
                 proteasome 
                   
                   
                 (72.3-169 nM)  
               
               
                   
                 inhibitor of 
                   
                   
                 (Gupta 
               
               
                   
                 chymotrypsin- 
                   
                   
                 et al. 2016) 
               
               
                   
                 like activity of 
                   
                   
                   
               
               
                   
                 20S 
                   
                   
                   
               
               
                 Oprozomib (oral) 
                 Irreversible 
                 Orphan drug 
                 240 mg/day  
                 218-870 
               
               
                   
                 tripeptide 
                 designation 2014 
                 (30-150 mg/d) 
                 (409-1633 nM) 
               
               
                   
                 epoxyketone 
                   
                   
                 (Infante et al. 
               
               
                   
                 inhibitor of 
                   
                   
                 2016) 
               
               
                   
                 chymotrypsin- 
                   
                   
                   
               
               
                   
                 like activity of 
                   
                   
                   
               
               
                   
                 20S (orally 
                   
                   
                   
               
               
                   
                 bioavailable 
                   
                   
                   
               
               
                   
                 analog of 
                   
                   
                   
               
               
                   
                 Carfilzomib) 
                   
                   
                   
               
               
                 Delanzomib (IV 
                 Inhibitor of the 
                 Clinical trials 
                 0.1-1.8 mg/m 2   
                 88.4-557.3  
               
               
                 or oral) 
                 proteasome 
                   
                   
                 (214-1348 nM) 
               
               
                   
                 chymotrypsin- 
                   
                   
                 (Gallerani et al. 
               
               
                   
                 like activity of 
                   
                   
                 2013) 
               
               
                   
                 20S that 
                   
                   
                   
               
               
                   
                 decreases NF-kB 
                   
                   
                   
               
               
                   
                 activity 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Additional Proteasome Inhibitors 
               
            
           
           
               
               
               
               
               
               
            
               
                 Generic 
                   
                   
                 Mechanism  
                 Delivery  
                   
               
               
                 Name 
                 Drug Name 
                 Drug Disease 
                 of Action 
                 Route 
                 Notes 
               
               
                   
               
               
                 4SC-206 
                 4SC-206; SC-68896 
                 Cancer solid, 
                 Proteasome 
                 Injectable 
                 Injectable 
               
               
                   
                   
                 unspecified; brain; 
                 inhibitor 
                   
                 proteasome 
               
               
                   
                   
                 myeloma 
                   
                   
                 inhibitor, 
               
               
                   
                   
                   
                   
                   
                 which was 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 solid tumors 
               
               
                   
                   
                   
                   
                   
                 and 
               
               
                   
                   
                   
                   
                   
                 inflammation.  
               
               
                   
                   
                   
                   
                   
                 Indications 
               
               
                   
                   
                   
                   
                   
                 included 
               
               
                   
                   
                   
                   
                   
                 multiple 
               
               
                   
                   
                   
                   
                   
                 myeloma, 
               
               
                   
                   
                   
                   
                   
                 small cell 
               
               
                   
                   
                   
                   
                   
                 lung cancer 
               
               
                   
                   
                   
                   
                   
                 and 
               
               
                   
                   
                   
                   
                   
                 neurological 
               
               
                   
                   
                   
                   
                   
                 tumors. 
               
               
                   
                   
                   
                   
                   
                 Preclinical 
               
               
                   
                   
                   
                   
                   
                 testing in 
               
               
                   
                   
                   
                   
                   
                 colon cancer 
               
               
                   
                   
                   
                   
                   
                 and 
               
               
                   
                   
                   
                   
                   
                 glioblastoma. 
               
               
                 ACU-D1 
                 ACCU D1; 
                 Acne; dry eye 
                 Proteasome 
                 Topical 
                 Nuclear 
               
               
                   
                 ACCUD1; ACCU- 
                 syndrome; 
                 inhibitor 
                   
                 factor kappa 
               
               
                   
                 D1; ACU D1; 
                 psoriasis; rosacea 
                 Transcription 
                   
                 beta (NFkB) 
               
               
                   
                 ACUD1; ACU-D1 
                   
                 factor 
                   
                 inhibitor, 
               
               
                   
                   
                   
                 NF-kappaB 
                   
                 which was 
               
               
                   
                   
                   
                 inhibitor 
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 skin and 
               
               
                   
                   
                   
                   
                   
                 ocular 
               
               
                   
                   
                   
                   
                   
                 diseases 
               
               
                   
                   
                   
                   
                   
                 including 
               
               
                   
                   
                   
                   
                   
                 acne rosacea, 
               
               
                   
                   
                   
                   
                   
                 psoriasis and 
               
               
                   
                   
                   
                   
                   
                 acne. Phase II 
               
               
                   
                   
                   
                   
                   
                 testing for 
               
               
                   
                   
                   
                   
                   
                 acne rosacea. 
               
               
                   
                   
                   
                   
                   
                 It was 
               
               
                   
                   
                   
                   
                   
                 previously 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for dry eye 
               
               
                 anticancer 
                 anticancer therapy, 
                 Cancer, unspecified 
                 Proteasome 
                   
                 Teva was 
               
               
                 therapy, 
                 Teva 
                   
                 inhibitor 
                   
                 developing a 
               
               
                 Teva 
                   
                   
                   
                   
                 series of (2S, 
               
               
                   
                   
                   
                   
                   
                 3R)-2-amino- 
               
               
                   
                   
                   
                   
                   
                 3-hydroxy- 
               
               
                   
                   
                   
                   
                   
                 butyric  
               
               
                   
                   
                   
                   
                   
                 acid derived 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitors, for 
               
               
                   
                   
                   
                   
                   
                 the treatment 
               
               
                   
                   
                   
                   
                   
                 of cancer 
               
               
                 AVR-147 
                 AVR-147; CTK- 
                 Cancer, unspecified 
                 Proteasome 
                   
                 A novel, 
               
               
                   
                 000147; CTK- 
                   
                 inhibitor 
                   
                 natural 
               
               
                   
                 100147 
                   
                   
                   
                 product 
               
               
                   
                   
                   
                   
                   
                 small- 
               
               
                   
                   
                   
                   
                   
                 molecule 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitor, 
               
               
                   
                   
                   
                   
                   
                 which was 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development) 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 cancer. It is 
               
               
                   
                   
                   
                   
                   
                 highly 
               
               
                   
                   
                   
                   
                   
                 selective for 
               
               
                   
                   
                   
                   
                   
                 the 
               
               
                   
                   
                   
                   
                   
                 chymotrypsin- 
               
               
                   
                   
                   
                   
                   
                 like activity 
               
               
                   
                   
                   
                   
                   
                 of the 
               
               
                   
                   
                   
                   
                   
                 proteasome. 
               
               
                 BSc-2118 
                 BSc-2118 
                 Cancer, lymphoma; 
                 Proteasome 
                   
                 A novel 
               
               
                   
                   
                 unspecified; 
                 inhibitor; 
                   
                 tripeptide 
               
               
                   
                   
                 myeloma 
                 Apoptosis 
                   
                 with 
               
               
                   
                   
                   
                 stimulant 
                   
                 inhibitory 
               
               
                   
                   
                   
                   
                   
                 activity 
               
               
                   
                   
                   
                   
                   
                 against the 3 
               
               
                   
                   
                   
                   
                   
                 proteolytic 
               
               
                   
                   
                   
                   
                   
                 activities of 
               
               
                   
                   
                   
                   
                   
                 the 20S 
               
               
                   
                   
                   
                   
                   
                 proteasome, 
               
               
                   
                   
                   
                   
                   
                 which was 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 multiple 
               
               
                   
                   
                   
                   
                   
                 myeloma 
               
               
                   
                   
                   
                   
                   
                 (MM) and 
               
               
                   
                   
                   
                   
                   
                 mantle cell 
               
               
                   
                   
                   
                   
                   
                 lymphoma 
               
               
                   
                   
                   
                   
                   
                 (MCL). 
               
               
                 CEP-28331 
                 CEP-28331 
                 Cancer, myeloma 
                 Proteasome 
                 Oral; 
                 Oral 
               
               
                   
                   
                   
                 inhibitor 
                 Oral, 
                 proteasome 
               
               
                   
                   
                   
                   
                 swallowed 
                 inhibitor, 
               
               
                   
                   
                   
                   
                   
                 which was 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 multiple 
               
               
                   
                   
                   
                   
                   
                 myeloma 
               
               
                 CVT-857 
                 CVT-710; CVT-857 
                 Inflammatory 
                 Proteasome 
                   
                 CVT-857 and 
               
               
                   
                   
                 disease, unspecified 
                 inhibitor 
                   
                 CVT-710 are 
               
               
                   
                   
                   
                   
                   
                 alpha- 
               
               
                   
                   
                   
                   
                   
                 ketoamide- 
               
               
                   
                   
                   
                   
                   
                 terminated 
               
               
                   
                   
                   
                   
                   
                 retropeptides, 
               
               
                   
                   
                   
                   
                   
                 which were 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 investigation 
               
               
                   
                   
                   
                   
                   
                 as potential 
               
               
                   
                   
                   
                   
                   
                 anti- 
               
               
                   
                   
                   
                   
                   
                 inflammatories.  
               
               
                   
                   
                   
                   
                   
                 They 
               
               
                   
                   
                   
                   
                   
                 selectively 
               
               
                   
                   
                   
                   
                   
                 inhibit the 
               
               
                   
                   
                   
                   
                   
                 chymotrypsin- 
               
               
                   
                   
                   
                   
                   
                 like site of 
               
               
                   
                   
                   
                   
                   
                 the 20S 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 complex 
               
               
                   
                   
                   
                   
                   
                 without 
               
               
                   
                   
                   
                   
                   
                 inhibiting 
               
               
                   
                   
                   
                   
                   
                 either the 
               
               
                   
                   
                   
                   
                   
                 peptidylglutamyl-  
               
               
                   
                   
                   
                   
                   
                 or 
               
               
                   
                   
                   
                   
                   
                 trypsin-like 
               
               
                   
                   
                   
                   
                   
                 proteolytic 
               
               
                   
                   
                   
                   
                   
                 activities of 
               
               
                   
                   
                   
                   
                   
                 the 20S 
               
               
                   
                   
                   
                   
                   
                 complex, 
               
               
                   
                   
                   
                   
                   
                 suggesting 
               
               
                   
                   
                   
                   
                   
                 that the 
               
               
                   
                   
                   
                   
                   
                 active site, 
               
               
                   
                   
                   
                   
                   
                 threonine, in 
               
               
                   
                   
                   
                   
                   
                 the 20S 
               
               
                   
                   
                   
                   
                   
                 proteasome is 
               
               
                   
                   
                   
                   
                   
                 not 
               
               
                   
                   
                   
                   
                   
                 undergoing 
               
               
                   
                   
                   
                   
                   
                 nucleophilic 
               
               
                   
                   
                   
                   
                   
                 addition to 
               
               
                   
                   
                   
                   
                   
                 the 
               
               
                   
                   
                   
                   
                   
                 electrophilic 
               
               
                   
                   
                   
                   
                   
                 carbonyl of 
               
               
                   
                   
                   
                   
                   
                 the alpha- 
               
               
                   
                   
                   
                   
                   
                 ketomide 
               
               
                   
                   
                   
                   
                   
                 group. 
               
               
                 CX13-608 
                 CX13 608; 
                 Cancer, myeloma; 
                 Proteasome 
                 Injectable, 
                 A proteasome 
               
               
                   
                 CX13608; CX13- 
                 lymphoma; 
                 inhibitor 
                 intravenous 
                 inhibitor, 
               
               
                   
                 608 
                 unspecified 
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 multiple 
               
               
                   
                   
                   
                   
                   
                 myeloma. 
               
               
                   
                   
                   
                   
                   
                 Phase I and 
               
               
                   
                   
                   
                   
                   
                 Phase II 
               
               
                   
                   
                   
                   
                   
                 testing. 
               
               
                 CYS-006 
                 anticancers, 
                 Cancer, unspecified 
                 Proteasome 
                   
                 CYS-006 was 
               
               
                   
                 Cytomics Systems; 
                   
                 inhibitor 
                   
                 a series of 
               
               
                   
                 CYS-006 
                   
                   
                   
                 compounds 
               
               
                   
                   
                   
                   
                   
                 targeting the 
               
               
                   
                   
                   
                   
                   
                 ubiquitin- 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 protein 
               
               
                   
                   
                   
                   
                   
                 degradation 
               
               
                   
                   
                   
                   
                   
                 pathway, 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 as 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitors to 
               
               
                   
                   
                   
                   
                   
                 stabilize the 
               
               
                   
                   
                   
                   
                   
                 degradation 
               
               
                   
                   
                   
                   
                   
                 of a protein 
               
               
                   
                   
                   
                   
                   
                 that has been 
               
               
                   
                   
                   
                   
                   
                 implicated in 
               
               
                   
                   
                   
                   
                   
                 the 
               
               
                   
                   
                   
                   
                   
                 pathogenesis 
               
               
                   
                   
                   
                   
                   
                 of 
               
               
                   
                   
                   
                   
                   
                 malignancies. 
               
               
                 disulfiram + 
                 CX 02; CX02; CX- 
                 Cancer, pancreatic; 
                 Chemokine 
                 Oral; 
                 Dicopp (CX- 
               
               
                 copper, 
                 02; Dicopp; 
                 myeloma; 
                 receptor 
                 Oral, 
                 02) is an oral, 
               
               
                 Cantex 
                 disulfiram + copper 
                 sarcoma; 
                 antagonist; 
                 swallowed 
                 single-dose 
               
               
                   
                 gluconate, Cantex; 
                 unspecified 
                 Proteasome 
                   
                 combination 
               
               
                   
                 disulfiram + copper, 
                 brain; breast; lung, 
                 inhibitor 
                   
                 of disulfiram +  
               
               
                   
                 Cantex 
                 non-small cell; 
                   
                   
                 copper, 
               
               
                   
                   
                 prostate, 
                   
                   
                 under 
               
               
                   
                   
                 Poisoning, 
                   
                   
                 development 
               
               
                   
                   
                 radiation 
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 pancreatic 
               
               
                   
                   
                   
                   
                   
                 cancer, 
               
               
                   
                   
                   
                   
                   
                 pediatric 
               
               
                   
                   
                   
                   
                   
                 sarcoma and 
               
               
                   
                   
                   
                   
                   
                 myeloma. It 
               
               
                   
                   
                   
                   
                   
                 was 
               
               
                   
                   
                   
                   
                   
                 previously 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for brain, 
               
               
                   
                   
                   
                   
                   
                 prostate, lung 
               
               
                   
                   
                   
                   
                   
                 and breast 
               
               
                   
                   
                   
                   
                   
                 cancers. 
               
               
                   
                   
                   
                   
                   
                 Phase I and 
               
               
                   
                   
                   
                   
                   
                 Phase II 
               
               
                   
                   
                   
                   
                   
                 testing of 
               
               
                   
                   
                   
                   
                   
                 some 
               
               
                   
                   
                   
                   
                   
                 cancers. 
               
               
                 ER-807446 
                 ER-804191; ER- 
                 Cancer, unspecified 
                 Proteasome 
                   
                 A 
               
               
                   
                 807446 
                   
                 inhibitor 
                   
                 representative 
               
               
                   
                   
                   
                   
                   
                 compound in 
               
               
                   
                   
                   
                   
                   
                 a series of 
               
               
                   
                   
                   
                   
                   
                 epoxyketone 
               
               
                   
                   
                   
                   
                   
                 derivatives 
               
               
                   
                   
                   
                   
                   
                 based on the 
               
               
                   
                   
                   
                   
                   
                 natural 
               
               
                   
                   
                   
                   
                   
                 product 
               
               
                   
                   
                   
                   
                   
                 eponemycin, 
               
               
                   
                   
                   
                   
                   
                 which was 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 investigation 
               
               
                   
                   
                   
                   
                   
                 as 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitors for 
               
               
                   
                   
                   
                   
                   
                 the treatment 
               
               
                   
                   
                   
                   
                   
                 of cancer. 
               
               
                 FV-162 
                 anticancer therapy, 
                 Cancer, lymphoma, 
                 Proteasome 
                 Oral; 
                 A potent, 
               
               
                   
                 Fluorinov Pharma; 
                 non-Hodgkin&#39;s 
                 inhibitor 
                 Oral, 
                 orally- 
               
               
                   
                 FV162; FV-162 
                 Cancer, myeloma 
                   
                 swallowed 
                 delivered 
               
               
                   
                   
                   
                   
                   
                 small 
               
               
                   
                   
                   
                   
                   
                 molecule 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitor, was 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 multiple 
               
               
                   
                   
                   
                   
                   
                 myeloma. It 
               
               
                   
                   
                   
                   
                   
                 is a novel 
               
               
                   
                   
                   
                   
                   
                 fluorine- 
               
               
                   
                   
                   
                   
                   
                 based 
               
               
                   
                   
                   
                   
                   
                 therapy. 
               
               
                   
                   
                   
                   
                   
                 Fluorine 
               
               
                   
                   
                   
                   
                   
                 atoms are 
               
               
                   
                   
                   
                   
                   
                 introduced in 
               
               
                   
                   
                   
                   
                   
                 to drug 
               
               
                   
                   
                   
                   
                   
                 candidates to 
               
               
                   
                   
                   
                   
                   
                 modify the 
               
               
                   
                   
                   
                   
                   
                 specific 
               
               
                   
                   
                   
                   
                   
                 physical and 
               
               
                   
                   
                   
                   
                   
                 chemical 
               
               
                   
                   
                   
                   
                   
                 properties of 
               
               
                   
                   
                   
                   
                   
                 the parent 
               
               
                   
                   
                   
                   
                   
                 drug 
               
               
                   
                   
                   
                   
                   
                 molecules. It 
               
               
                   
                   
                   
                   
                   
                 works by 
               
               
                   
                   
                   
                   
                   
                 selectively 
               
               
                   
                   
                   
                   
                   
                 inhibiting the 
               
               
                   
                   
                   
                   
                   
                 chymotrypsin- 
               
               
                   
                   
                   
                   
                   
                 like 
               
               
                   
                   
                   
                   
                   
                 enzymatic 
               
               
                   
                   
                   
                   
                   
                 activity of the 
               
               
                   
                   
                   
                   
                   
                 proteasome. 
               
               
                   
                   
                   
                   
                   
                 Phase I 
               
               
                   
                   
                   
                   
                   
                 testing in 
               
               
                   
                   
                   
                   
                   
                 myeloma. 
               
               
                 FV-214 
                 FV214; FV-214 
                 Cancer, myeloma 
                 Proteasome 
                 Injectable, 
                 A potent, 
               
               
                   
                   
                 Waldenstrom&#39;s 
                 inhibitor 
                 intravenous 
                 intravenously 
               
               
                   
                   
                 hypergamma- 
                   
                   
                 (iv) delivered 
               
               
                   
                   
                 globulinaemia 
                   
                   
                 small 
               
               
                   
                   
                   
                   
                   
                 molecule 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitor, 
               
               
                   
                   
                   
                   
                   
                 which was 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 multiple 
               
               
                   
                   
                   
                   
                   
                 myeloma, 
               
               
                   
                   
                   
                   
                   
                 Waldenstrom&#39;s 
               
               
                   
                   
                   
                   
                   
                 macroglobulin- 
               
               
                   
                   
                   
                   
                   
                 aemia. It 
               
               
                   
                   
                   
                   
                   
                 works by 
               
               
                   
                   
                   
                   
                   
                 selectively 
               
               
                   
                   
                   
                   
                   
                 inhibiting the 
               
               
                   
                   
                   
                   
                   
                 chymotrypsin- 
               
               
                   
                   
                   
                   
                   
                 like 
               
               
                   
                   
                   
                   
                   
                 enzymatic 
               
               
                   
                   
                   
                   
                   
                 activity of the 
               
               
                   
                   
                   
                   
                   
                 proteasome. 
               
               
                 G4-1 
                 G4 1; G4-1 
                 Cancer, solid; 
                 Proteasome 
                   
                 A novel 
               
               
                   
                   
                 unspecified 
                 inhibitor 
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitor, 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 solid tumors. 
               
               
                 GNF-6702 
                 GNF6702; GNF- 
                 Infection, 
                 Proteasome 
                   
                 A selective 
               
               
                   
                 6702 
                 leishmaniasis; 
                 inhibitor 
                   
                 inhibitor of 
               
               
                   
                   
                 trypanosomiasis 
                   
                   
                 the 
               
               
                   
                   
                   
                   
                   
                 kinetoplastid 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 which was 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 leishmaniasis, 
               
               
                   
                   
                   
                   
                   
                 Chagas 
               
               
                   
                   
                   
                   
                   
                 disease and 
               
               
                   
                   
                   
                   
                   
                 sleeping 
               
               
                   
                   
                   
                   
                   
                 sickness. 
               
               
                 GSK439 
                   
                   
                 Proteasome 
                   
                   
               
               
                   
                   
                   
                 inhibitor 
                   
                   
               
               
                 GSK- 
                 DDD 1305143; 
                 Infection, 
                 Proteasome 
                 Oral; 
                 Under 
               
               
                 3494245 
                 DDD1305143; 
                 leishmaniasis 
                 inhibitor 
                 Oral, 
                 development 
               
               
                   
                 DDD-1305143; 
                   
                   
                 swallowed 
                 for the 
               
               
                   
                 GSK 3494245; 
                   
                   
                   
                 treatment of 
               
               
                   
                 GSK3494245; GSK- 
                   
                   
                   
                 Leishmaniasis.  
               
               
                   
                 3494245; 
                   
                   
                   
                 Phase I 
               
               
                   
                 GSK3494245/DDD 
                   
                   
                   
                 testing. 
               
               
                   
                 1305143, DNDi; 
                   
                   
                   
                   
               
               
                   
                 GSK3494245/DDD 
                   
                   
                   
                   
               
               
                   
                 1305143, 
                   
                   
                   
                   
               
               
                   
                 GlaxoSmithKline 
                   
                   
                   
                   
               
               
                 immuno- 
                 immunoproteasome 
                 Autoimmune 
                 Proteasome 
                 Oral; 
                 Oral 
               
               
                 proteasome 
                 inhibitors, AbbVie; 
                 disease, 
                 inhibitor 
                 Oral, 
                 immuno- 
               
               
                 inhibitors, 
                 immunoproteasome 
                 Inflammatory 
                   
                 swallowed 
                 proteasome 
               
               
                 Principia 
                 inhibitors, Principia 
                 disease 
                   
                   
                 inhibitors, for 
               
               
                 BioPharma 
                 BioPharma 
                   
                   
                   
                 the treatment 
               
               
                   
                   
                   
                   
                   
                 of 
               
               
                   
                   
                   
                   
                   
                 inflammatory 
               
               
                   
                   
                   
                   
                   
                 disease and 
               
               
                   
                   
                   
                   
                   
                 autoimmune 
               
               
                   
                   
                   
                   
                   
                 disorders 
               
               
                   
                   
                   
                   
                   
                 such as 
               
               
                   
                   
                   
                   
                   
                 inflammatory 
               
               
                   
                   
                   
                   
                   
                 bowel 
               
               
                   
                   
                   
                   
                   
                 diseases 
               
               
                   
                   
                   
                   
                   
                 (IBD), lupus 
               
               
                   
                   
                   
                   
                   
                 and psoriasis. 
               
               
                   
                   
                   
                   
                   
                 It was 
               
               
                   
                   
                   
                   
                   
                 previously 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 immunological  
               
               
                   
                   
                   
                   
                   
                 disease. 
               
               
                   
                   
                   
                   
                   
                 Some are 
               
               
                   
                   
                   
                   
                   
                 LMP2 and 
               
               
                   
                   
                   
                   
                   
                 LMP7 
               
               
                   
                   
                   
                   
                   
                 inhibitors 
               
               
                   
                   
                   
                   
                   
                 (Basler et al. 
               
               
                   
                   
                   
                   
                   
                 2018). 
               
               
                 KZR-616 
                 KZR616; KZR-616; 
                 Autoimmune 
                 Proteasome 
                 Injectable; 
                 Selective 
               
               
                   
                 KZR616 (IV); KZR- 
                 disease, 
                 inhibitor 
                 Injectable, 
                 small 
               
               
                   
                 616 (IV); KZR616 
                 Inflammatory 
                   
                 intravenous; 
                 molecule 
               
               
                   
                 (SC); KZR-616 
                 disease 
                   
                 Injectable, 
                 inhibitor of 
               
               
                   
                 (SC); ONX 0914; 
                   
                   
                 subcutaneous 
                 the 
               
               
                   
                 ONX-0914; 
                   
                   
                   
                 immuno- 
               
               
                   
                 ONYX0914; 
                   
                   
                   
                 proteasome, under 
               
               
                   
                 ONYX-0914; 
                   
                   
                   
                 development 
               
               
                   
                 PR957; PR-957 
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 autoimmune 
               
               
                   
                   
                   
                   
                   
                 diseases and 
               
               
                   
                   
                   
                   
                   
                 inflammatory 
               
               
                   
                   
                   
                   
                   
                 diseases. 
               
               
                   
                   
                   
                   
                   
                 Phase I and 
               
               
                   
                   
                   
                   
                   
                 Phase II 
               
               
                   
                   
                   
                   
                   
                 testing. 
               
               
                 LGP-07154 
                 LGP07154; LGP- 
                 Cancer, unspecified 
                 Proteasome 
                 Oral; 
                   
               
               
                   
                 07154; VAL-789- 
                 Immunological 
                 inhibitor 
                 Oral, 
                   
               
               
                   
                 CHUM 
                 disease, unspecified 
                 unspecified 
                 swallowed 
                   
               
               
                 LMP7 
                 LMP7 inhibitor, 
                 Cancer, 
                 proteasome 
                 Oral; 
                   
               
               
                 inhibitor, 
                 Shouyao Holdings 
                 hematological; 
                 subunit 
                 Oral, 
                   
               
               
                 Shouyao 
                   
                 unspecified, 
                 beta 
                 swallowed 
                   
               
               
                 Holdings 
                   
                 Autoimmune 
                 type-8 
                   
                   
               
               
                   
                   
                 disease, 
                 inhibitor 
                   
                   
               
               
                   
                   
                 unspecified, 
                   
                   
                   
               
               
                   
                   
                 Inflammatory 
                   
                   
                   
               
               
                   
                   
                 disease 
                   
                   
                   
               
               
                 LODO-141 
                 TIR199, TIR 199, 
                 Solid Tumors 
                   
                   
                 LODO-141 
               
               
                   
                 LODO141, LODO 
                   
                   
                   
                 (TIR-199) is 
               
               
                   
                 141,TIR-199 
                   
                   
                   
                 an 
               
               
                   
                   
                   
                   
                   
                 irreversible 
               
               
                   
                   
                   
                   
                   
                 and potent 
               
               
                   
                   
                   
                   
                   
                 hybrid cyclic 
               
               
                   
                   
                   
                   
                   
                 peptide 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitor 
               
               
                   
                   
                   
                   
                   
                 from the 
               
               
                   
                   
                   
                   
                   
                 syrbactin 
               
               
                   
                   
                   
                   
                   
                 natural 
               
               
                   
                   
                   
                   
                   
                 product 
               
               
                   
                   
                   
                   
                   
                 family. 
               
               
                 LXE-408 
                 LXE 408; LXE408; 
                 Infection, 
                 Proteasome 
                 Oral; 
                 Proteasome 
               
               
                   
                 LXE-408 
                 leishmaniasistry- 
                 inhibitor 
                 Oral, 
                 inhibitor, 
               
               
                   
                   
                 panosomiasis, 
                   
                 swallowed 
                 under 
               
               
                   
                   
                 American 
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 visceral 
               
               
                   
                   
                   
                   
                   
                 leishmaniasis.  
               
               
                   
                   
                   
                   
                   
                 It was 
               
               
                   
                   
                   
                   
                   
                 previously 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for Chagas 
               
               
                   
                   
                   
                   
                   
                 disease. 
               
               
                   
                   
                   
                   
                   
                 Phase I and 
               
               
                   
                   
                   
                   
                   
                 Phase II 
               
               
                   
                   
                   
                   
                   
                 testing. 
               
               
                 M-3258 
                 M 3258; M3258;  
                 Cancer, myeloma 
                 Proteasome 
                 Oral; 
                 Immuno- 
               
               
                   
                 M-3258 
                   
                 subunit 
                 Oral, 
                 proteasome 
               
               
                   
                   
                   
                 beta 
                 swallowed 
                 subunit 
               
               
                   
                   
                   
                 type-8 
                   
                 LMP7 
               
               
                   
                   
                   
                 inhibitor 
                   
                 inhibitor 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 multiple 
               
               
                   
                   
                   
                   
                   
                 myeloma. 
               
               
                   
                   
                   
                   
                   
                 Phase 1 
               
               
                   
                   
                   
                   
                   
                 testing. 
               
               
                 MCIT-375 
                 Anti- 
                 Cancer, myeloma 
                 Proteasome 
                 Injectable 
                 An anti- 
               
               
                   
                 CD38/bortezomib/ 
                   
                 inhibitor; 
                   
                 CD38 
               
               
                   
                 lenalidomide Dual 
                   
                 Cereblon  
                   
                 antibody drug 
               
               
                   
                 Drug Pay load ADC; 
                   
                 E3 
                   
                 conjugate 
               
               
                   
                 MCIT375; MCIT- 
                   
                 ubiquitin  
                   
                 with dual 
               
               
                   
                 375 
                   
                 ligase 
                   
                 drug 
               
               
                   
                   
                   
                 stimulant; 
                   
                 payloads 
               
               
                   
                   
                   
                 Angiogenesis 
                   
                 bortezomib 
               
               
                   
                   
                   
                 inhibitor; 
                   
                 and 
               
               
                   
                   
                   
                 Immuno- 
                   
                 lenalidomide, 
               
               
                   
                   
                   
                 oncology 
                   
                 which was 
               
               
                   
                   
                   
                 therapy 
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 multiple 
               
               
                   
                   
                   
                   
                   
                 myeloma. 
               
               
                 MLN519 
                   
                 Inflammatory 
                 Ubiquitin 
                   
                 Phase I and 
               
               
                   
                   
                 Disease and Stroke 
                 Proteasome 
                   
                 Phase II 
               
               
                   
                   
                   
                 inhibitor 
                   
                 testing 
               
               
                 OSH-101 
                 OSH-101 
                 Alopecia 
                 Proteasome 
                 Topical; 
                 A 
               
               
                   
                   
                   
                 inhibitor 
                 Topical, 
                 tetrapeptide 
               
               
                   
                   
                   
                   
                 skin 
                 aldehyde 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitor, 
               
               
                   
                   
                   
                   
                   
                 which was 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 male and 
               
               
                   
                   
                   
                   
                   
                 female 
               
               
                   
                   
                   
                   
                   
                 pattern 
               
               
                   
                   
                   
                   
                   
                 baldness and 
               
               
                   
                   
                   
                   
                   
                 chemotherapy- 
               
               
                   
                   
                   
                   
                   
                 induced 
               
               
                   
                   
                   
                   
                   
                 alopecia. 
               
               
                   
                   
                   
                   
                   
                 Other 
               
               
                   
                   
                   
                   
                   
                 possible 
               
               
                   
                   
                   
                   
                   
                 indications 
               
               
                   
                   
                   
                   
                   
                 include 
               
               
                   
                   
                   
                   
                   
                 alopecia 
               
               
                   
                   
                   
                   
                   
                 areata, age- 
               
               
                   
                   
                   
                   
                   
                 related hair 
               
               
                   
                   
                   
                   
                   
                 thinning and 
               
               
                   
                   
                   
                   
                   
                 post- 
               
               
                   
                   
                   
                   
                   
                 transplant 
               
               
                   
                   
                   
                   
                   
                 hair 
               
               
                   
                   
                   
                   
                   
                 regrowth. 
               
               
                   
                   
                   
                   
                   
                 Phase I 
               
               
                   
                   
                   
                   
                   
                 testing 
               
               
                   
                   
                   
                   
                   
                 complete, 
               
               
                   
                   
                   
                   
                   
                 Phase II 
               
               
                   
                   
                   
                   
                   
                 planned. 
               
               
                 peptide 
                 peptide 
                 Inflammatory 
                 Proteasome 
                   
                 Subunit 
               
               
                 epoxyketones,  
                 epoxyketones, Onyx 
                 disease, unspecified 
                 inhibitor 
                   
                 selective 
               
               
                 Onyx 
                 Pharmaceuticals 
                   
                   
                   
                 peptide 
               
               
                 Pharmaceuticals 
                   
                   
                   
                   
                 epoxyketones 
               
               
                   
                   
                   
                   
                   
                 as 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitors, for 
               
               
                   
                   
                   
                   
                   
                 the treatment 
               
               
                   
                   
                   
                   
                   
                 of 
               
               
                   
                   
                   
                   
                   
                 inflammatory 
               
               
                   
                   
                   
                   
                   
                 disease. 
               
               
                 proteasome 
                 proteasome 
                 Cancer, unspecified 
                 Proteasome 
                 Oral; 
                 A next 
               
               
                 inhibitor, 
                 inhibitor, Flaveome 
                   
                 inhibitor 
                 Oral, 
                 generation 
               
               
                 Flaveome 
                   
                   
                   
                 swallowed 
                 orally- 
               
               
                   
                   
                   
                   
                   
                 bioavailable 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitor for 
               
               
                   
                   
                   
                   
                   
                 the treatment 
               
               
                   
                   
                   
                   
                   
                 of cancer. It 
               
               
                   
                   
                   
                   
                   
                 also inhibits 
               
               
                   
                   
                   
                   
                   
                 PI3K/Akt 
               
               
                   
                   
                   
                   
                   
                 signalling. 
               
               
                   
                   
                   
                   
                   
                 They are 
               
               
                   
                   
                   
                   
                   
                 small 
               
               
                   
                   
                   
                   
                   
                 molecules 
               
               
                   
                   
                   
                   
                   
                 derived from 
               
               
                   
                   
                   
                   
                   
                 flavonoid 
               
               
                   
                   
                   
                   
                   
                 structures. 
               
               
                 proteasome 
                 proteasome 
                 Cancer, unspecified 
                 Proteasome 
                   
                 Small- 
               
               
                 inhibitors, 
                 inhibitors, EntreMe 
                   
                 inhibitor 
                   
                 molecule 
               
               
                 EntreMe 
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitors, for 
               
               
                   
                   
                   
                   
                   
                 the treatment 
               
               
                   
                   
                   
                   
                   
                 of cancer. 
               
               
                 proteasome 
                 proteasome 
                 Cancer, unspecified 
                 Proteasome 
                   
                 Novel 26S 
               
               
                 inhibitors, 
                 inhibitors, Ergon 
                   
                 inhibitor 
                   
                 proteasome 
               
               
                 Ergon 
                 Pharmaceuticals 
                   
                   
                   
                 inhibitors, for 
               
               
                 Pharmaceuticals 
                   
                   
                   
                   
                 the treatment 
               
               
                   
                   
                   
                   
                   
                 of cancer. 
               
               
                 proteasome 
                 proteasome 
                 Cancer, unspecified 
                 Proteasome 
                   
                   
               
               
                 inhibitors, 
                 inhibitors, Jeil 
                   
                 inhibitor 
                   
                   
               
               
                 Jeil 
                   
                   
                   
                   
                   
               
               
                 proteasome 
                 proteasome 
                 Infection, malaria 
                 Proteasome 
                   
                 Inhibitors of 
               
               
                 inhibitors, 
                 inhibitors, NYU 
                   
                 inhibitor 
                   
                 enzymes of 
               
               
                 NYU 
                   
                   
                   
                   
                 the ubiquitin- 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 pathway, 
               
               
                   
                   
                   
                   
                   
                 different 
               
               
                   
                   
                   
                   
                   
                 from those of 
               
               
                   
                   
                   
                   
                   
                 the PS-341 
               
               
                   
                   
                   
                   
                   
                 series (qv), 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 malaria and 
               
               
                   
                   
                   
                   
                   
                 other 
               
               
                   
                   
                   
                   
                   
                 parasitic 
               
               
                   
                   
                   
                   
                   
                 diseases. The 
               
               
                   
                   
                   
                   
                   
                 pathway is 
               
               
                   
                   
                   
                   
                   
                 involved in 
               
               
                   
                   
                   
                   
                   
                 proteolysis 
               
               
                   
                   
                   
                   
                   
                 which occurs 
               
               
                   
                   
                   
                   
                   
                 during 
               
               
                   
                   
                   
                   
                   
                 Plasmodium 
               
               
                   
                   
                   
                   
                   
                 falciparum&#39;s 
               
               
                   
                   
                   
                   
                   
                 lifecycle 
               
               
                   
                   
                   
                   
                   
                 (Scrip, 1997, 
               
               
                   
                   
                   
                   
                   
                 2239, 19). 
               
               
                 proteasome 
                 proteasome 
                 Autoimmune 
                 Proteasome 
                   
                 Peptide 
               
               
                 inhibitors, 
                 inhibitors, QLi5 
                 disease, unspecified 
                 inhibitor 
                   
                 mimetic 
               
               
                 QLi5 
                 Therapeutics 
                 Cancer, unspecified 
                   
                   
                 proteasome 
               
               
                 Therapeutics 
                   
                 Inflammatory 
                   
                   
                 inhibitors for 
               
               
                   
                   
                 disease, unspecified 
                   
                   
                 the treatment 
               
               
                   
                   
                   
                   
                   
                 of cancer and 
               
               
                   
                   
                   
                   
                   
                 inflammation. 
               
               
                 proteasome 
                 peptide mimetics, 
                 Inflammatory 
                 Proteasome 
                   
                   
               
               
                 inhibitors, 
                 Signature; 
                 disease, unspecified 
                 inhibitor 
                   
                   
               
               
                 Signature 
                 proteasome 
                   
                   
                   
                   
               
               
                   
                 inhibitors, Signature 
                   
                   
                   
                   
               
               
                 proteasome 
                 proteasome 
                 Cancer, unspecified 
                 Proteasome 
                   
                 A series of 
               
               
                 inhibitors, 
                 inhibitors, 
                   
                 inhibitor 
                   
                 human 20S 
               
               
                 Takeda 
                 Millenium; 
                   
                   
                   
                 proteasome 
               
               
                   
                 proteasome 
                   
                   
                   
                 non-covalent 
               
               
                   
                 inhibitors, Takeda 
                   
                   
                   
                 inhibitors 
               
               
                   
                   
                   
                   
                   
                 derived from 
               
               
                   
                   
                   
                   
                   
                 N-β- 
               
               
                   
                   
                   
                   
                   
                 neopentyl 
               
               
                   
                   
                   
                   
                   
                 asparagine, 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 cancer. 
               
               
                 proteasome 
                 proteasome 
                 Cancer, unspecified 
                 Proteasome 
                   
                 Amall- 
               
               
                 inhibitors, 
                 inhibitors, Telik 
                   
                 inhibitor 
                   
                 molecule, 
               
               
                 Telik 
                   
                   
                   
                   
                 orally-active, 
               
               
                   
                   
                   
                   
                   
                 non-peptide 
               
               
                   
                   
                   
                   
                   
                 and non- 
               
               
                   
                   
                   
                   
                   
                 boron-based 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitors, for 
               
               
                   
                   
                   
                   
                   
                 the treatment 
               
               
                   
                   
                   
                   
                   
                 of cancer. 
               
               
                 proteasome 
                 proteasome 
                 Cancer, unspecified 
                 Proteasome 
                   
                   
               
               
                 inhibitors, 
                 inhibitors, Novartis 
                   
                 inhibitor; 
                   
                   
               
               
                 Novartis 
                   
                   
                 Apoptosis 
                   
                   
               
               
                   
                   
                   
                 stimulant 
                   
                   
               
               
                 proteasome 
                 proteasome 
                 Cancer, 
                 Proteasome 
                   
                   
               
               
                 inihibitors, 
                 inihibitors, 
                 hematological; 
                 Inhibitor 
                   
                   
               
               
                 IkerChem 
                 IkerChem 
                 unspecified; solid 
                   
                   
                   
               
               
                 syrbactins, 
                 syrbactins, Pono 
                 Cancer, myeloma; 
                 Proteasome 
                   
                   
               
               
                 Pono 
                 Pharma 
                 unspecified 
                 inhibitor 
                   
                   
               
               
                 Pharma 
                   
                   
                   
                   
                   
               
               
                 timosaponin 
                 AA-101; AA102; 
                 Cancer, breast 
                 Apoptosis 
                 Oral; 
                 Oral plant- 
               
               
                 AIII 
                 AA-102; BN-108; 
                   
                 stimulant; 
                 Oral, 
                 based 
               
               
                   
                 timosaponin AIII 
                   
                 Caspase 
                 swallowed 
                 aqueous 
               
               
                   
                   
                   
                 stimulant; 
                   
                 extract from 
               
               
                   
                   
                   
                 Proteasome 
                   
                 
                   Anemarrhena 
                 
               
               
                   
                   
                   
                 inhibitor 
                   
                 
                   asphodeloides  
                 
               
               
                   
                   
                   
                   
                   
                   bunge , 
               
               
                   
                   
                   
                   
                   
                 which 
               
               
                   
                   
                   
                   
                   
                 attenuates 
               
               
                   
                   
                   
                   
                   
                 mitochondrial 
               
               
                   
                   
                   
                   
                   
                 membrane 
               
               
                   
                   
                   
                   
                   
                 potential to 
               
               
                   
                   
                   
                   
                   
                 cause 
               
               
                   
                   
                   
                   
                   
                 cytochrome 
               
               
                   
                   
                   
                   
                   
                 C release and 
               
               
                   
                   
                   
                   
                   
                 caspase 
               
               
                   
                   
                   
                   
                   
                 activation, 
               
               
                   
                   
                   
                   
                   
                 inducing low 
               
               
                   
                   
                   
                   
                   
                 molecular 
               
               
                   
                   
                   
                   
                   
                 grade 
               
               
                   
                   
                   
                   
                   
                 apoptosis, 
               
               
                   
                   
                   
                   
                   
                 which was 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 breast cancer. 
               
               
                   
                   
                   
                   
                   
                 It induces 
               
               
                   
                   
                   
                   
                   
                 cancer cell 
               
               
                   
                   
                   
                   
                   
                 death via 
               
               
                   
                   
                   
                   
                   
                 activation of 
               
               
                   
                   
                   
                   
                   
                 the AKT and 
               
               
                   
                   
                   
                   
                   
                 mTOR 
               
               
                   
                   
                   
                   
                   
                 pathways in 
               
               
                   
                   
                   
                   
                   
                 breast cancer 
               
               
                   
                   
                   
                   
                   
                 cells, but not 
               
               
                   
                   
                   
                   
                   
                 normal cells. 
               
               
                   
                   
                   
                   
                   
                 Phase II 
               
               
                   
                   
                   
                   
                   
                 testing. 
               
               
                 TMC-86A 
                 proteasome inhibs, 
                 Immunological 
                 Proteasome 
                   
                 TMC-86A, 
               
               
                   
                 Tanabe; TMC-86A; 
                 disease, unspecified 
                 inhibitor 
                   
                 TMC-86B 
               
               
                   
                 TMC-86B; TMC-96 
                 Inflammatory 
                   
                   
                 and TMC-96 
               
               
                   
                   
                 disease, unspecified 
                   
                   
                 are 20S 
               
               
                   
                   
                   
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitors 
               
               
                   
                   
                   
                   
                   
                 with epoxy- 
               
               
                   
                   
                   
                   
                   
                 β-aminoketone 
               
               
                   
                   
                   
                   
                   
                 moieties, 
               
               
                   
                   
                   
                   
                   
                 which were 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 investigation 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 inflammatory 
               
               
                   
                   
                   
                   
                   
                 disease, 
               
               
                   
                   
                   
                   
                   
                 autoimmune 
               
               
                   
                   
                   
                   
                   
                 diseases and 
               
               
                   
                   
                   
                   
                   
                 muscle 
               
               
                   
                   
                   
                   
                   
                 wasting 
               
               
                   
                   
                   
                   
                   
                 associated 
               
               
                   
                   
                   
                   
                   
                 with cancer 
               
               
                   
                   
                   
                   
                   
                 cachexia, 
               
               
                   
                   
                   
                   
                   
                 diabetes and 
               
               
                   
                   
                   
                   
                   
                 sepsis. The 
               
               
                   
                   
                   
                   
                   
                 compounds 
               
               
                   
                   
                   
                   
                   
                 were isolated 
               
               
                   
                   
                   
                   
                   
                 from 
               
               
                   
                   
                   
                   
                   
                 
                   Streptomyces 
                 
               
               
                   
                   
                   
                   
                   
                 sp TC-1084 
               
               
                   
                   
                   
                   
                   
                 and 
               
               
                   
                   
                   
                   
                   
                 
                   Saccharothrix  
                 
               
               
                   
                   
                   
                   
                   
                 sp TC-1094 
               
               
                   
                   
                   
                   
                   
                 (J Antibiot, 
               
               
                   
                   
                   
                   
                   
                 1999, 52, 
               
               
                   
                   
                   
                   
                   
                 1069). 
               
               
                 TMC-95A 
                 TMC-95A; TMC- 
                 Immunological 
                 Proteasome 
                   
                 TMC-95A 
               
               
                   
                 95B; TMC-95C; 
                 disease, unspecified 
                 inhibitor 
                   
                 and its 
               
               
                   
                 TMC-95D 
                 Inflammatory 
                   
                   
                 diastereomers,  
               
               
                   
                   
                 disease, unspecified 
                   
                   
                 TMC-95B 
               
               
                   
                   
                 Musculoskeletal 
                   
                   
                 to D are 20S 
               
               
                   
                   
                 disease, unspecified 
                   
                   
                 proteasome 
               
               
                   
                   
                   
                   
                   
                 inhibitors, 
               
               
                   
                   
                   
                   
                   
                 which were 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 investigation 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 inflammatory 
               
               
                   
                   
                   
                   
                   
                 and 
               
               
                   
                   
                   
                   
                   
                 autoimmune 
               
               
                   
                   
                   
                   
                   
                 diseases, and 
               
               
                   
                   
                   
                   
                   
                 rapid muscle 
               
               
                   
                   
                   
                   
                   
                 wasting 
               
               
                   
                   
                   
                   
                   
                 associated 
               
               
                   
                   
                   
                   
                   
                 with cancer 
               
               
                   
                   
                   
                   
                   
                 cachexia, 
               
               
                   
                   
                   
                   
                   
                 diabetes and 
               
               
                   
                   
                   
                   
                   
                 sepsis. This 
               
               
                   
                   
                   
                   
                   
                 series of 
               
               
                   
                   
                   
                   
                   
                 cyclic 
               
               
                   
                   
                   
                   
                   
                 peptides was 
               
               
                   
                   
                   
                   
                   
                 isolated from 
               
               
                   
                   
                   
                   
                   
                 
                   Apiospora 
                 
               
               
                   
                   
                   
                   
                   
                 
                   montagnei 
                 
               
               
                   
                   
                   
                   
                   
                   Sacc , TC- 
               
               
                   
                   
                   
                   
                   
                 1093. 
               
               
                 TQB-3602 
                 TQB 3602; 
                 Cancer, myeloma 
                 Proteasome 
                   
                 TQB-3602 is 
               
               
                   
                 TQB3602; TQB- 
                   
                 inhibitor 
                   
                 a proteasome 
               
               
                   
                 3602 
                   
                   
                   
                 inhibitor, 
               
               
                   
                   
                   
                   
                   
                 under 
               
               
                   
                   
                   
                   
                   
                 development 
               
               
                   
                   
                   
                   
                   
                 for the 
               
               
                   
                   
                   
                   
                   
                 treatment of 
               
               
                   
                   
                   
                   
                   
                 multiple 
               
               
                   
                   
                   
                   
                   
                 myeloma. 
               
               
                   
                   
                   
                   
                   
                 Phase I 
               
               
                   
                   
                   
                   
                   
                 testing. 
               
               
                 Ubiquitin 
                 ubiquitin 
                 Cancer 
                 Proteasome 
                   
                   
               
               
                 Proteasome 
                 proteasome system 
                   
                 inhibitor 
                   
                   
               
               
                 Program 
                   
                   
                   
                   
                   
               
               
                 VL-01 
                 4SC-206, SC-68896, 
                 Hepatitis C (HCV); 
                 Proteasome 
                   
                   
               
               
                   
                 SC68896 
                 HIV (Antiviral) 
                 Inhibitor 
                   
                   
               
               
                   
                   
                   
                 that 
                   
                   
               
               
                   
                   
                   
                 induces 
                   
                   
               
               
                   
                   
                   
                 apopotosis 
                   
                   
               
               
                 VLX157 
                 VLX1570 
                 Cancer-bone, 
                 Proteasome 
                   
                   
               
               
                   
                   
                 mantle cell 
                 inhibitor 
                   
                   
               
               
                   
                   
                 lymphoma, 
                   
                   
                   
               
               
                   
                   
                 multiple myeloma 
               
               
                   
               
            
           
         
       
     
     All of the agents discussed herein can be in the form of pharmaceutical compositions. 
     Most preferred methods of administration of the agents and compositions for use in the disclosed methods are oral and parental including intravenous and injection. The pharmacological agent must be in the appropriate form for administration of choice. 
     Such pharmaceutical compositions comprising one or more pharmacological agents for administration may comprise a therapeutically effective amount of the pharmacological agent and a pharmaceutically acceptable carrier. 
     The phrase “pharmaceutically acceptable” as used herein refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human, and approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. 
     The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. 
     Pharmaceutical compositions adapted for oral administration may be capsules, tablets, powders, granules, solutions, syrups, suspensions (in non-aqueous or aqueous liquids), or emulsions. Tablets or hard gelatin capsules may comprise lactose, starch or derivatives thereof, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof. Soft gelatin capsules may comprise vegetable oils, waxes, fats, semi-solid, or liquid polyols. Solutions and syrups may comprise water, polyols, and sugars. An active agent intended for oral administration may be coated with or admixed with a material that delays disintegration and/or absorption of the active agent in the gastrointestinal tract. Thus, the sustained release may be achieved over many hours and if necessary, the active agent can be protected from degradation within the stomach. Pharmaceutical compositions for oral administration may be formulated to facilitate release of an active agent at a particular gastrointestinal location due to specific pH or enzymatic conditions. 
     A further preferred form of administration is parenteral including intravenous administration. Pharmaceutical compositions adapted for parenteral administration, including intravenous administration, include aqueous and non-aqueous sterile injectable solutions or suspensions, which may contain anti-oxidants, buffers, bacteriostats, and solutes that render the compositions substantially isotonic with the blood of the subject. Other components which may be present in such compositions include water, alcohols, polyols, glycerine, and vegetable oils. Compositions adapted for parental administration may be presented in unit-dose or multi-dose containers, such as sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile carrier, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to those skilled in the art. Examples include: Water for Injection USP; aqueous vehicles such as Sodium Chloride Injection, Ringer&#39;s Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer&#39;s Injection; water-miscible vehicles such as ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. 
     Pharmaceutical compositions adapted for nasal and pulmonary administration may comprise solid carriers such as powders, which can be administered by rapid inhalation through the nose. Compositions for nasal administration may comprise liquid carriers, such as sprays or drops. Alternatively, inhalation directly through into the lungs may be accomplished by inhalation deeply or installation through a mouthpiece. These compositions may comprise aqueous or oil solutions of the active ingredient. Compositions for inhalation may be supplied in specially adapted devices including, but not limited to, pressurized aerosols, nebulizers or insufflators, which can be constructed so as to provide predetermined dosages of the active ingredient. 
     Further methods of administration include sublingual, vaginal, buccal, rectal, or transdermal administration to a subject. 
     In some instances, the agent is in a next generation formulation such as nanoencapsulation, such as bortezomib. Such formulations are under investigation for cancer treatment. 
     Selection of a therapeutically effective dose will be determined by the skilled artisan considering several factors, which will be known to one of ordinary skill in the art. Such factors include the particular form of the pharmacological agent, and its pharmacokinetic parameters such as bioavailability, metabolism, and half-life, which will have been established during the usual development procedures typically employed in obtaining regulatory approval for a pharmaceutical compound. Further factors in considering the dose include the condition or disease to be treated or the benefit to be achieved in a normal individual, the body mass of the patient, the route of administration, whether the administration is acute or chronic, concomitant medications, and other factors well known to affect the efficacy of administered pharmaceutical agents. Thus, the precise dose should be decided according to the judgment of the person of skill in the art, and each patient&#39;s circumstances, and according to standard clinical techniques. 
     Doses can be adjusted to optimize the effects in the subject. For example, the agent can be administered at a low dose to start and then increased over time to depending upon the subject&#39;s response. A subject can be monitored for improvement of their condition prior to changing, i.e., increasing or decreasing, the dosage. A subject can also be monitored for adverse effects prior to changing the dosage, i.e., increasing or decreasing, the dosage. 
     The FDA approved PIs and dosages are found in Table 1. It will be appreciated by those of skill in the art that the subjects on which the methods of the present disclosure are being practiced are under 10 years of age and in some cases under one year of age. Thus, the therapeutically effective dosages for these subjects may be less than the FDA approved dosages. 
     The agents may be administered daily, weekly, biweekly, several times daily, semi-weekly, every other day, bi-weekly, quarterly, several times per week, semi-weekly, monthly, or more. The duration and frequency of treatment may depend upon the subject&#39;s response to treatment. 
     The agents described herein can be co-administered with other agents including those disclosed herein and additional ones for the prevention and treatment of vascular malformations. The co-administration of agents can be by any administration described herein. Moreover, it can be in one composition, or in more than one composition. The administration of the agents can be simultaneous, concurrently or sequentially. 
     Treatment using the present methods can continue as long as needed. 
     Kits 
     Also within the scope of the present disclosure are kits for practicing the disclosed methods. 
     In some embodiments, the kit can comprise instructions for use in any of the methods described herein. The included instructions can comprise a description of administration of the agents to a subject to achieve the intended activity in a subject. The kit may further comprise a description of selecting a subject suitable for treatment based on identifying whether the subject is in need of the treatment. 
     The instructions relating to the use of the agents described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical compositions are used for treating, delaying the onset, and/or alleviating a disease or disorder in a subject. 
     The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device, or an infusion device. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port. 
     Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above. 
     EXAMPLES 
     The present invention may be better understood by reference to the following non-limiting examples, which are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed to limit the broad scope of the invention. 
     Example 1—Direct Targeting of Affected Pathways with Known Drugs 
     LMECs and matching tissue with an activating mutation in PIK3CA or an inactivating mutation in RASA1 had increased AKT and ERK activation when compared to control tissue and cells, respectively. LMECs carry inactivating mutations in PIK3R3/TSC2 activation unexpectedly displayed increased ERK activation as well as AKT activation. Using these cells, it was earlier found that the LMECs with MAPK pathway activation were resistant to sirolimus, a mTOR inhibitor commonly used to treat LMs. See  FIG.  1 A . 
     It was then asked whether this finding was specific to sirolimus, or to inhibitors that target the PI3K/AKT/MTOR pathway. The effect of inhibiting the MAPK (RAS/MAPK) pathway on proliferation of these cells was also assessed. 
     Cells with the same mutations (Pik3ca, Pik3r3/Tsc2, Rasa1; LMEC8, LMEC28, and LMEC10 respectively), (Table 3) were treated with increasing log doses of alpelisib, a PI3K inhibitor, ranging from 1 μM to 100 μM. Proliferation rate was determined by WST-8 assay and expressed as a fraction of the number of cells in vehicle controls. 
     It was found that alpelisib significantly inhibited both control and the LMECs at concentrations flanking the calculated human sera level of 75 μM; it was more effective than sirolimus. However, at lower doses, LMEC responses were different. 
     Cells with only PIK3CA mutation (and only increased AKT signaling) were more sensitive to alpelisib, while the cells with increased ERK signaling were more resistant when compared to control HDLECs ( FIG.  1 B ). These results were similar to sirolimus ( FIG.  1 A ). 
     Cells were also treated with U0126, a MAPK inhibitor, in increasing log doses ranging from 1 μM to 100 μM. At the concentration used in animal models (10 μM), U0126 suppressed the growth of all cells by 50% ( FIG.  1 C ). 
     Interestingly, cells with the RASA1 mutations were not any more sensitive to MAPK inhibition than those with the PIK3CA mutation or control cells. These data demonstrated that drug responses were affected by genetic variants, and MAPK hyperactivation may contribute to pathogenic proliferation of LMECs. 
     Example 2—Unbiased High Throughput Screening (HTS) of ECs Identified Omipalisib and Proteasome Inhibitors as Effective 
     ECs from vascular malformation tissues carrying known disease associated pathogenic variants, including those that target the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways have been collected. It was hypothesized that ECs isolated from patients that carried pathogenic somatic, as well as yet to be identified variants, could be used in a drug screen to identify novel therapies for the treatment of vascular malformations. 
     High throughput screening of drugs was performed using ECs from either LMs (LMECs) or VM (VMECs) carrying pathogenic variants in genes of the PI3K/AKT/mTOR pathway or variants predicted to hyperactivate the RAS/RAF/MAPK pathway (Table 3). 
     Per optimization studies, about 500 cells were seeded into the wells of a 384-well plate and the VMECs or LMECs were treated with 1 μM of approximately 2500 drugs, which were either in clinical trials or FDA approved for another condition. The effects of the drugs on the proliferation and cell viability of the cells were assessed. Cell viability relative to vehicle treated cells was determined after 48 hours. 
     HTPS identified several candidate drugs that inhibited the PI3K/AKT signaling pathways ( FIG.  2   ). Interestingly, the inhibitory effects on of the same class of drugs on all VMEC and LMEC proliferation were variable across all cell populations. As an example, omipalisib, a dual PI3K/mTOR inhibitor, was more efficacious in inhibiting vascular malformation cells than voxtalisib, another PI3K/mTOR dual inhibitor. As omipalisib was found as the strongest PI3K/mTOR dual inhibitor of LMEC and VMEC proliferation in the HTPS screen, another dual PI3K/mTOR inhibitor, bimiralisib, was assessed as it was not in the HTPS panel ( FIG.  3   ). Omipalisib was significantly more efficacious than bimiralisib at every dose in inhibiting an LMEC population, suggesting omipalisib may be the optimal drug in its class to target the pathological growth in LMs and VMs. 
     A novel class of therapeutics, proteasome inhibitors (PIs), were identified in the HTPS that were more efficacious at suppressing cell growth/viability than drug therapies currently used off-label for VM and LM patients, sirolimus and alpelisib ( FIG.  4   ; Table 4). The 3 PIs tested in the HTPS suppressed LMEC (n=5) and VMEC (n=3) cell numbers 75%-90% relative to vehicle treated cells, while sirolimus and alpelisib reduced their growth and viability only 40-50%. The PIs had a much stronger effect on the LMECs and VMECs regardless of the mutation. As can be seen in Table 4, the 3 PIs tested in the HTPS were: number 1 in effectiveness for all cell variants (carfilzomib); number 2 for ⅝ cell variants and number 3 and 4 for the ⅜ (defanzomib); and in the top 9 for all cell variants (ixazomib). In contrast, sirolimus and alpelisib ranged from number 28-383 for effectiveness for all variants. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 LMECs and VMECs Used in HTPS Screening 
               
            
           
           
               
               
               
            
               
                   
                 Cell Population 
                 Pathogenic Variant 
               
               
                   
                   
               
               
                   
                 LMEC8 
                 Pik3ca c.1636 C &gt; A 
               
               
                   
                 LMEC10 
                 Rasa frameshift c.617_621delTAAGA 
               
               
                   
                 LMEC28 
                 Pik3r3 Sp-Donor  1:46527598 T/TA; Tsc2 5’utr   
               
               
                   
                   
                 16-2110805-G-A 
               
               
                   
                 LMEC94 
                 Pik3ca c.3140A &gt; G 
               
               
                   
                 LMEC99 
                 Pik3ca c.1258T &gt; C 
               
               
                   
                 VMEC7 
                 Pik3ca c.1035T &gt; A 
               
               
                   
                 VMEC9 
                 Pik3ca c.1624G &gt; A 
               
               
                   
                 VMEC13 
                 Map2k2c.1112g &gt; A; Glmn Sp-Donor c.1214+2T &gt; C 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Ranking of Proteasome Inhibitors in HTPS for Suppressing Cell Proliferation and 
               
               
                 Growth Compared to Drugs used for Off-Label Treatment 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 Cell 
                   
                   
                   
                   
                   
                   
                   
               
               
                 Pop. 
                 Carfitzomib 
                 Delanzomib 
                 Ixazomib 
                 Disulfiram 
                 Sirolimus 
                 Alpelisib 
                 Trematinib 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 VMEC 
                 1 
                 2 
                 8 
                 8 
                 118 
                 107 
                 79 
               
               
                 13 
                   
                   
                   
                   
                   
                   
                   
               
               
                 LMEC 
                 1 
                 3 
                 5 
                 3 
                 383 
                 50 
                 1225 
               
               
                 10 
                   
                   
                   
                   
                   
                   
                   
               
               
                 VMEC 
                 1 
                 4 
                 7 
                 10 
                 32 
                 44 
                 71 
               
               
                 7 
                   
                   
                   
                   
                   
                   
                   
               
               
                 VMEC 
                 1 
                 2 
                 9 
                 5 
                 52 
                 58 
                 71 
               
               
                 9 
                   
                   
                   
                   
                   
                   
                   
               
               
                 LMEC 
                 1 
                 3 
                 8 
                 7 
                 38 
                 42 
                 72 
               
               
                 8 
                   
                   
                   
                   
                   
                   
                   
               
               
                 LMEC 
                 1 
                 2 
                 9 
                 19 
                 63 
                 81 
                 241 
               
               
                 28 
                   
                   
                   
                   
                   
                   
                   
               
               
                 LMEC 
                 1 
                 2 
                 6 
                 11 
                 45 
                 28 
                 62 
               
               
                 94 
                   
                   
                   
                   
                   
                   
                   
               
               
                 LMEC 
                 1 
                 2 
                 4 
                 8 
                 40 
                 83 
                 82 
               
               
                 99 
               
               
                   
               
            
           
         
       
     
     Example 3—Dose Response Studies of Proteasome Inhibitors and Omipalisib 
     Using the plasma concentrations reported in the literature for six commercially available PIs (Table 1) and omipalisib (Lukey et al. 2019), a dose response study was performed using VMEC7, VMEC9, LMEC94 and LMEC99, all carrying Pik3ca variants (Table 3) as well as HMVECs. Cell growth and viability was measured as in Example 2 after 48 hours. The 6 PIs and omipalisib were all more effective at suppressing growth/viability of LMEC and VMEC carrying Pik3ca variants, than either sirolimus or alpelisib at clinically relevant doses (as shown by the arrows in  FIG.  5   ) ( FIGS.  5  and  6   ). 
     Example 4—Effectiveness of Proteasome Inhibitors is not Limited to VM/LM ECs with Pik3ca Variants 
     LMEC28 cells which carries the Pik3r3;Tsc2 mutations were subjected to increasing amounts of oprozomib from 1 nM to 1 μM and cell viability determined after 48 hours as set forth in Example 2. 
     As shown in  FIG.  7   , there was a dose dependent inhibition of the LMECs carrying the Pik3r3; Tsc2 showing that the PI was effective in suppressing cell proliferation in cells harboring mutations other than Pik3ca. 
     Example 5—In Vivo Effectiveness of Proteasome Inhibitors on Treating VMs 
     To assess the efficacy of PIs in an animal, the effect of oprozomib was assessed in a xenograft model. VMECs carrying Pik3ca variants (n=2 VMECs; VMEC7 and VMEC9—Table 3; 4 implants each for treated and vehicle) were resuspended in Matrigel® (Corning) and subcutaneously implanted in the flanks of immunocompromised mice. VM vessels were allowed to develop and mice randomized for one week. After 1 week, mice were given either 30 mg/kg oprozomib or vehicle orally two days a week for 4 weeks. Oprozomib, a second generation of carfilzomib, was chosen as it is reported to be more tolerable, and orally available (Teicher and Tomaszewski 2017). After 4 weeks of treatment, mice were sacrificed, and the implants surgically removed. The vehicle-treated implant was vascular and easily distinguished from surrounding soft tissues. The oprozomib-treated implant had no vascularity on gross inspection. The outline of the Matrigel® was visualized as a translucent, oval-shaped foreign body in the subcutaneous space. The implants were then fixed by formalin, paraffin embedded for histological analysis. Five (5) micron sections were stained with Hematoxylin and Eosin and images captured and used to assess implant morphology and vascularity. 
     Findings were dramatic. The implants in the vehicle treated group were highly vascularized and hemorrhagic. In contrast, the vessels appeared to be regressed or normalized in the implants from the treatment group. See  FIG.  8   . Additionally, no adverse effects were seen in the treated mice. 
     Taken together, these results demonstrate that PIs are efficacious against pathological VMECs and LMECs carrying genetic variants in the two most commonly mutated pathways in vascular malformations, PI3K/AKT/mTOR and RAS/RAF/MAPK pathways. 
     Example 6—Unbiased High Throughput Screening (HTS) of ECs Additionally Identified Disulfiram as Effective 
     Using the HTPS described in Example 2, disulfiram was also shown to efficaciously suppress VMEC and LMEC growth/viability more than that observed for drugs used off label for vascular malformation patients. See  FIG.  9    and Table 4. 
     As can be seen in Table 4, the disulfiram tested in the HTPS ranged from number 3 to number 19 for effectiveness in the variants tested. In contrast, sirolimus and alpelisib ranged from number 28-383 for effectiveness for all variants. 
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