US 20200206143A1 IN (19 ) United States ( 12) Patent Application Publication (10 ) Pub . No.: US 2020/0206143 A1 Moskowitz et al. (43 ) Pub . Date : Jul. 2 , 2020 ( 54 ) PLATELETS LOADED WITH ANTI - CANCER Publication Classification AGENTS (51 ) Int. Ci. A61K 9/50 (2006.01 ) ( 71 ) Applicant: Cellphire, Inc., Rockville , MD (US ) A61K 31/704 (2006.01 ) ( 72 ) Inventors : Keith Andrew Moskowitz , Westfield , A61K 31/502 ( 2006.01 ) IN (US ) ; Rafael Jorda, Bethesda, MD A61K 31/337 (2006.01 ) (US ) ; Ying Yi Zheng , Rockville , MD AOIN 1/02 ( 2006.01) (US ) ; Daniel Sheik , Rockville , MD A61K 9/127 (2006.01 ) (US ) (52 ) U.S. CI. CPC A61K 9/5068 ( 2013.01 ) ; A61K 31/704 (2013.01 ) ; A61K 31/502 ( 2013.01) ; AOIN ( 21 ) Appl. No .: 16 /698,645 1/021 ( 2013.01 ) ; AOIN 1/0226 (2013.01 ) ; A61K 9/1271 (2013.01 ) ; A61K 31/337 ( 22 ) Filed : Nov. 27 , 2019 (2013.01 ) ( 57 ) ABSTRACT Related U.S. Application Data In some embodiments provided herein is a method of (60 ) Provisional application No. 62 / 773,931, filed on Nov. preparing cargo - loaded platelets , comprising : treating plate 30 , 2018 , provisional application No. 62/ 775,141 , lets with a cargo and with a loading buffer comprising a salt , filed on Dec. 4 , 2018, provisional application No. a base , a loading agent , and optionally ethanol, to form the 62 /828,041 , filed on Apr. 2 , 2019 . cargo - loaded platelets . Patent Application Publication Jul. 2 , 2020 Sheet 1 of 20 US 2020/0206143 A1 Amount of Doxorubicin (DOX ) load per CD42b * Platelet DOX(mfi) FIG . 1 Amount of DOX in drug loaded platelets following ADP and /or TRAP stimulation 227 FIG . 2 Patent Application Publication Jul. 2 , 2020 Sheet 2 of 20 US 2020/0206143 A1 Loading platelets with DOX encapsulated liposome DOXmfi() 0 Cells 250ul 100ml Lipo - DOX FIG . 3 Patent Application Publication Jul. 2 , 2020 Sheet 3 of 20 US 2020/0206143 A1 2h Incubation Control 0.1mM DOX W20.3mM DOX 0 104 106 Control 95.1 % DOX loaded B Doxorubicin FIG . 4A - C Patent Application Publication Jul. 2 , 2020 Sheet 4 of 20 US 2020/0206143 A1 Control 0.5h incubation 92.7 % 2.OK DOX loaded 1.5K CellCount 102 106 Doxorubicin FIG . 5 Patent Application Publication Jul. 2 , 2020 Sheet 5 of 20 US 2020/0206143 A1 15 Doxosome ( umol) FIGS. 6A (left ) & 6B (right ) 25000 y 996.6x - 344.92 20000 R2 a 0.9978 Fluorescence 10000 0 0 Doxorubicin (UM ) FIG.7 Patent Application Publication Jul. 2 , 2020 Sheet 6 of 20 US 2020/0206143 A1 DOX Quantification 2 3 Membrane bound 108K 235K 454K 915K 1448K 1745K Cells / ul FIG . 8 DOX(mg)/1platelet 2 26-06 108K 235K 454K 915K 1448K 1745K CellsiuL FIG.9 Patent Application Publication Jul. 2 , 2020 Sheet 7 of 20 US 2020/0206143 A1 2 5 FIG . 10 Patent Application Publication Jul. 2 , 2020 Sheet 8 of 20 US 2020/0206143 A1 N FIG . 11 Patent Application Publication Jul. 2 , 2020 Sheet 9 of 20 US 2020/0206143 A1 Loading buffer allowed higher drug load per cell than compared to HMTbuffer 52A FIG . 12 DOX Induces Aggregation of Platelets 100 96 90 85 . 80 70 60 50 40 TransmittanceLight% 30 de 20 14 . 10 0 HMT + 1mM Thrombin 0.36mg/ ml 0.72mg/ ml Img/ ml DOX 3mg/ ml DOX Mg liliDOX DOX FIG . 13A Patent Application Publication Jul. 2 , 2020 Sheet 10 of 20 US 2020/0206143 A1 FIG . 13B FIG . 13C Patent Application Publication Jul. 2 , 2020 Sheet 11 of 20 US 2020/0206143 A1 Gate : Singlet platelets 3 PARP -FL No drug 4.72 % 10?M PARP?- FL , 0.5MM PARPI count(x103/UL)Cell loaded 50.43 % 30uM PARPL- FL , 1.5MM PARPI 1.81.2 platelets 97.11 % 60LM PARP - FL , 3mM PARPI 99.97 % 120 M PARPL- FL , 6MM PARPI 0.6 Method : flow cytometry - 3. pooled random donor platelets 0 Post 3 hour incubation at 37 ° C 101 102 103 104 105 106 107.2 Patent Application Publication Jul. 2 , 2020 Sheet 12 of 20 US 2020/0206143 A1 Total drug load / cell * Method Tecan fluorometer 2.0E - 09 Exem , 450 /518nm )(mg/call (PARP?-FL+PARPI)/platelet pooled random donor platelets Ttest, 2 tails * p < 0.05 p < 0.01 PARPI- FL (UM ) 0 30 120 PARPI (MM ) 0 0.5 3.0 6.0 FIG . 15 FIG . 16 Patent Application Publication Jul. 2 , 2020 Sheet 13 of 20 US 2020/0206143 A1 FIG . 17 FIG . 18 Patent Application Publication Jul. 2 , 2020 Sheet 14 of 20 US 2020/0206143 A1 FIG . 19 FIG . 20 Patent Application Publication Jul . 2 , 2020 Sheet 15 of 20 US 2020/0206143 A1 . FIG . 21 FG. 22 Patent Application Publication Jul. 2 , 2020 Sheet 16 of 20 US 2020/0206143 A1 Unloaded Tsomes DOX loaded Tsomes DOX loaded Tsomes Cephalin 4 ThrombinPeakHeight Octaplas 5 20 0 everythin 30.67 36.67 42.67 72.67 Time (min ) FIG . 23 Patent Application Publication Jul. 2 , 2020 Sheet 17 of 20 US 2020/0206143 A1 HSA medium (neg control) 1 % Dox loaded 1 % unloaded 5 % Dox loaded muy 5 % unloaded 10 % Dox loaded Dox (positive control ) Populationdoublings Time after adding fresh medium (hrs ) FIG . 24 Patent Application Publication Jul. 2 , 2020 Sheet 18 of 20 US 2020/0206143 A1 E 30003 thanMore 0 2 FIG . 25A 0 4 6 (hours ) FIG . 25B Patent Application Publication Jul. 2 , 2020 Sheet 19 of 20 US 2020/0206143 A1 1000KM 1 FIG . 26A 1 100 0 3 2 (hours ) FIG . 260 Patent Application Publication Jul. 2 , 2020 Sheet 20 of 20 US 2020/0206143 A1 Brightfield Fluorescence Overlay FIG . 27 US 2020/0206143 A1 Jul . 2 , 2020 1 PLATELETS LOADED WITH ANTI - CANCER used to control bleeding after injury or during acquired AGENTS platelet function defects or deficiencies, for example those occurring during surgery and those due to the presence of CROSS - REFERENCE TO RELATED platelet inhibitors . APPLICATIONS [0006 ] Loading platelets with pharmaceutical drugs may allow targeted delivery of the drugs to sites of interest . [0001 ] This application claimspriority to U.S. Provisional Further, drug - loaded platelets may be lyophilized or cryo Patent Application No. 62/ 773,931 , filed on Nov. 30 , 2018 , preserved to allow for long - term storage . In some embodi U.S. Provisional Patent Application No. 62/ 775,141, filed on ments the loading of a drug in the platelets mitigates Dec. 4 , 2018 , and U.S. Provisional Patent Application No. systemic side effects associated with the drug and lowers the 62/ 828,041 , filed on Apr. 2 , 2019. The contents of each of threshold of therapeutic dose necessary by facilitating tar these applications are incorporated herein by reference in geted treatment at site of interest . See , Xu P. , et. al. , their entireties. Doxorubicin - loaded platelets as a smart drug delivery sys tem : An improved therapy for lymphoma, Scientific Reports , TECHNICAL FIELD 7 , Article Number: 42632 , (2017 ) , which is incorporated by [ 0002 ] The present disclosure in some embodiments reference herein in its entirety . relates to the use of platelets , platelet derivatives , or throm bosomes ( e.g., freeze -dried platelet derivatives ) as biologi SUMMARY OF THE INVENTION cal carriers of cargo , such as pharmaceutical drugs, also [0007 ] In some embodiments provided herein is a method referred to herein as drug - loaded platelets , platelet deriva of preparing cargo - loaded platelets , cargo - loaded platelet tives , or thrombosomes . Also provided herein in some derivatives , or cargo -loaded thrombosomes ( e.g., freeze embodiments are methods of preparing platelets , platelet dried platelet derivatives ) , comprising : derivatives , or thrombosomes loaded with the drug of inter [0008 ] treating platelets , platelet derivatives , or throm est . bosomes with a cargo and with at least one loading [0003 ] The present disclosure relates to the field of blood agent and optionally one or more plasticizers such as and blood products . More specifically , it relates to platelets , organic solvents , such as organic solvents selected from cryopreserved platelets , and /or lyopreserved platelet com the group consisting of ethanol, acetic acid , acetone, positions, including those containing stabilized platelets or acetonitrile , dimethylformamide , dimethyl sulfoxide, compositions derived from platelets . The drug - loaded plate dioxane , methanol, n - propanol , isopropanol , tetrahy lets can be stored under typical ambient conditions, refrig drofuran ( THF ), N -methyl pyrrolidone , dimethylacet erated , cryopreserved , for example with dimethyl sulfoxide amide (DMAC ) , or combinations thereof, (DMSO ) , and / or lyophilized after stabilization ( e.g. , throm to form the cargo - loaded platelets , cargo - loaded platelet bosomes ) derivatives, or cargo - loaded thrombosomes. [0009 ] In some embodiments , the method of preparing BACKGROUND cargo - loaded platelets can include treating the platelets , the [0004 ] Blood is a complex mixture of numerous compo platelet derivatives, and/ or the thrombosomes with the cargo nents . In general , blood can be described as comprising four with one loading agent. In some embodiments , the method main parts : red blood cells , white blood cells , platelets , and of preparing cargo - loaded platelets , cargo - loaded platelet plasma. The first three are cellular or cell- like components , derivatives , or cargo - loaded thrombosomes can include whereas the fourth ( plasma ) is a liquid component compris treating the platelets , the platelet derivatives , or the throm ing a wide and variable mixture of salts , proteins , and other bosomes with the cargo with multiple loading agents . factors necessary for numerous bodily functions . The com [0010 ] In some embodiments , suitable organic solvents ponents of blood can be separated from each other by include , but are not limited to alcohols, esters, ketones , various methods . In general, differential centrifugation is ethers , halogenated solvents , hydrocarbons, nitriles , glycols , most commonly used currently to separate the different alkyl nitrates, water or mixtures thereof.