Nucleoside preparations

Abstract

The present disclosure relates to a nucleoside formulation comprising a nucleoside or its phosphate and an amphipathic cell membrane penetrating RALA peptide. In one embodiment, the nucleoside formulation comprises gemcitabine or its phosphate and the peptide WEARLARALARALARHLARALARALRACEA.

Description

[Technical field]

[0001] The present disclosure relates to nucleoside formulations comprising a nucleoside or its phosphate and an amphiphilic cell membrane penetrating peptide of the RALA family of peptides. The present disclosure further relates to methods for preparing the nucleoside formulations and their use in therapy. [Background technology]

[0002] Nucleoside analogues are an important class of antiviral agents that are now commonly used in the treatment of viral infections and cancer. Once in vivo, nucleoside analogues are phosphorylated and incorporated into growing DNA chains to act as chain terminators.

[0003] As illustrated by the problems seen with gemcitabine, three main challenges exist with nucleoside analogues: 1) because the drug is widely distributed in the body and enters other non-tumor cells, adverse side effects such as bone marrow suppression, liver and kidney problems, and nausea limit the dose that can be administered to patients; 2) the very short drug stability and half-life, measured in minutes, necessitates the infusion of high doses, further increasing the risk of toxicity; and 3) drug resistance mutations appear to emerge quickly, reducing the overall clinical response rate.

[0004] Gemcitabine, a nucleoside analogue, is a chemotherapy drug that was first approved for use in 1995 and is used to treat many different types of cancer. Gemcitabine is prescribed as a first-line treatment for pancreatic cancer and for the treatment of other cancers when a patient's previous treatments have been unsuccessful. Pancreatic cancer still has a 5-year survival rate of only about 9% (Office for National Statistics, Cancer survival by stage at diagnosis for England, 2019) and is responsible for almost 450,000 deaths per year worldwide, which accounts for 4.5% of all cancer deaths (GLOBOCAN, 2018 estimate). Once inside the body, gemcitabine is transported into cells via molecular transporters. It is then phosphorylated three times to the pharmacologically active form, gemcitabine triphosphate (dFdCTP). This is then incorporated into the DNA of cancerous cells upon each replication, causing uncorrectable errors, leading to the inhibition of further DNA synthesis and ultimately cell death.

[0005] Unfortunately, the overall clinical response rate to gemcitabine is low at 6% (Ioka T et al., Jpn J Clin Oncol. 2013 Feb; 43(2):139-45). Furthermore, multidrug resistance mechanisms have been identified that are related to mutations in the active transporters required for cellular uptake, upregulation of metabolic enzymes that inactivate gemcitabine, and downregulation of cellular enzymes that convert gemcitabine to its active triphosphate form (Kim HA et al., Breast. 2008 Feb; 17(1):19-26). Summary of the Invention [Problem to be solved by the invention]

[0006] There is a clear need for formulations of nucleoside analogs that improve cell entry, circumvent resistance mechanisms, increase accumulation, and improve half-life and drug stability. Furthermore, a drug delivery system that can deliver an active form, such as the triphosphate form, of a drug, such as gemcitabine, to cells may automatically circumvent some of the mechanisms that lead to drug resistance (Galmarini CM et al., Int J Pharm. 2010 Aug. 16, 395(1-2):281-9). [Means for solving the problem]

[0007] The RALA family of peptides are amphipathic peptides composed of repeating RALA units, which can overcome biological barriers to gene delivery both in vitro and in vivo. The term "RALA" is used inconsistently in the literature, but typically refers to an amphipathic peptide or group of peptides composed of repeating RALA units, usually less than about 50 amino acid residues. Cohen-Avrahami M et al. (J. Phys. Chem. B 2011, 115:10 189-1 097, and Colloids Surf B Biointerfaces. 2010 Jun. 1, 77(2):131-8) disclose an amphipathic 16-mer peptide called "RALA". Nouri FS et al. (Biomacromolecules 2013, 14, 2033-40) use the term "RALA" to describe a 30-mer RALA peptide, as does McCarthy HO et al. (J Control Release. 2014 Sep. 10, 189:141-9), but it is a different 30-mer peptide. WO2014 / 087023 and WO2015 / 189205 define the term "RALA" as a general term for the group of peptides within the scope of the inventions described in these documents.

[0008] Peptides of the RALA family have been incorporated into genetic material such as plasmid DNA (McCarthy HO et al., J Control Release. 2014 Sep 10, 189:141-9) and (Ali AA et al., Nanomedicine. 2017 Apr 13(3):921-932), mRNA (Udhayakumar et al., Adv Healthc Mater. 2017 Jul 6(13)), siRNA (Mulholland EJ et al., J Control Release. 2019 Dec 28, 316:53-65), as well as small molecules such as bisphosphonates (Jena LN et al., J. Nanobiotechnology. 2021 May 4, 19(1):127) and calcium phosphate (Sathy BN et al., J. Mater. Chem. B. 2017 May 7, 5(9):1753-1764).

[0009] The specific RALA peptide WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) is a non-toxic peptide of 30 amino acids with a charge of +6 at physiological pH, which converts to a helical, cell membrane-permeant conformation of +8 in the acidic conditions found inside the endosomes of cells. It has been shown that when complexed in water with certain payloads, such as DNA and mRNA, it can spontaneously self-assemble into nanoparticles (McCarthy HO et al., J Control Release. 2014 Sep 10, 189:141-9; and Udhayakumar et al., Adv Healthc Mater. 2017 Jul 6(13)). This pH-dependent change allows escape of the peptide and cargo inside the cell, resulting in highly efficient cellular entry and cargo delivery at a physiological pH of 7.4 outside the cell without any toxicity. The nanoparticles have been shown to be very stable over a wide range of temperatures and over time, and the RALA peptide does not provoke an immunological response by itself.

[0010] This application describes improved formulations of nucleosides and / or their phosphates, which are formulated with amphiphilic cell membrane penetrating peptides of the RALA family of peptides. The formulations can spontaneously form nanoparticles when mixed with water. The formulations can provide improved target-selective delivery with the ability to improve pharmacokinetics, enhance pharmacodynamics, and reduce off-target toxicity while avoiding drug resistance mechanisms, facilitating more potent therapeutic effects at lower doses. Such improved properties may open the possibility of using the nucleoside analog family of compounds and their phosphates for new indications. When the nucleoside or its phosphate (gemcitabine triphosphate) is gemcitabine triphosphate, the pharmacologically active form of gemcitabine, the nanoparticles have superior tumor-selective delivery compared to existing gemcitabine formulations with a longer circulating half-life, enhancing drug accumulation two-fold in solid tumors after intravenous administration compared to other sites in vivo.

[0011] The present specification describes, in part, a nucleoside formulation comprising a nucleoside or a phosphate thereof and an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1), or a sequence having at least 80% sequence identity or homology thereto.

[0012] The present specification also describes, in part, a nucleoside formulation comprising gemcitabine or a phosphate thereof and an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1), or a sequence having at least 80% sequence identity or homology thereto.

[0013] This specification also describes, in part, the nucleoside formulations described herein that are nanoparticulate formulations, and methods for preparing them.

[0014] This specification also describes, in part, the nucleoside formulations described herein for use in therapy, particularly for use in the treatment of viral infections and in the treatment of cancer.

[0015] In the following paragraphs, RALA refers to the peptide WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1). [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 shows particle size (Z-average) distribution plots of RALA / dFdCTP nanoparticles (molar ratio 2.0 (MR2.0)) produced by (i) manual formulation and (ii) automated formulation. [Diagram 2] Figure 1 shows the cell cycle analysis of BxPC-3 cells after 48 hours of treatment with various molar ratios of manually formulated RALA / dFdCTP nanoparticles. The percentage of cells not in G2 phase was much higher when treated with RALA / dFdCTP nanoparticles compared to untreated, dFdCTP-treated, and gemcitabine hydrochloride-treated cells. This indicates a high level of efficacy of RALA / dFdCTP nanoparticles. The molar ratio with the lowest percentage of cells in G2 phase (maximum % of G0 / G1+S) was found to be MR2.0. [Diagram 3]FIG. 1 shows reported data (heatmap) following colony formation assay of Example 6 performed after treatment of both gemcitabine-sensitive (BxPC-3) and gemcitabine-resistant (PANC-1) cell lines with various concentrations of RALA / dFdCTP nanoparticles, dFdCTP, and gemcitabine hydrochloride. RALA / dFdCTP nanoparticles reduce cell proliferation compared to gemcitabine hydrochloride at low / medium / high doses in gemcitabine-sensitive cells (BxPC-3) and at medium / high doses in gemcitabine-resistant cells (PANC-1), demonstrating increased efficacy over gemcitabine hydrochloride or dFdCTP alone, with RALA / dFdCTP nanoparticles showing the best average functionality compared to gemcitabine hydrochloride or dFdCTP alone in both cell lines and at relevant concentrations. [Figure 4] Figure 2 shows the functionality of RALA / dFdCTP nanoparticles (MR2.0) in gemcitabine-sensitive cells (BxPC-3) and gemcitabine-resistant cells (PANC-1) at the EC50 dose of RALA / dFdCTP. At the EC50 concentration of RALA / dFdCTP nanoparticles, treatment was found to result in significantly higher levels of γH2AX expression in both BxPC-3 and PANC-1 cells compared to treatment with dFdCTP or gemcitabine hydrochloride. These results indicate that the functionality of RALA / dFdCTP nanoparticles is greatly increased over dFdCTP or gemcitabine hydrochloride, as higher levels of DNA damage are possible at lower treatment doses. [Diagram 5]Figure 1 shows the functionality of RALA / dFdCTP nanoparticles (MR2.0) in gemcitabine-sensitive cells (BxPC-3) at the EC50 dose of RALA / dFdCTP before and after treatment with 10 μM dipyridamole to induce gemcitabine resistance via blocking nucleotide transport channels to test the ability of RALA / dFdCTP to overcome cellular gemcitabine resistance mechanisms. Dipyridamole treatment was found to reduce the functionality of gemcitabine hydrochloride and dFdCTP with respect to double-stranded DNA breaks (γH2AX) to a much greater extent than that seen with RALA / dFdCTP nanoparticles. This differentiation may be due to the method of entry of RALA / dFdCTP nanoparticles, which relies on endocytosis rather than entry into cells via the hENT / hCNT channels that dipyridamole blocks. Although the level of functionality of RALA / dFdCTP nanoparticles was slightly reduced after treatment with dipyridamole, double-strand breaks were still significantly more prevalent compared to the other two treatment groups, and the levels were higher than with gemcitabine prior to dipyridamole treatment. [Figure 6] Therapeutic efficacy of RALA / dFdCTP nanoparticles (MR2.0) in athymic nude mice (n=6) implanted subcutaneously with 2.5×106 gemcitabine-resistant PANC-1 cells. When tumor size reached a volume of approximately 150 mm3, mice were injected intravenously with a single dose of vehicle (10% w / v trehalose), or 1 mg / kg dFdCTP, gemcitabine, or RALA / dFdCTP (MR2.0). Tumors were measured three times a week until total tumor volume quadrupled (≧600 mm3). (A) Mean tumor volume growth curves for all treatment groups show delayed tumor formation after treatment with a single dose of RALA / dFdCTP (MR2.0) compared to other treatment groups. (B) Kaplan-Meier survival estimates for mice treated as part of this study. The increased survival of mice treated with RALA / dFdCTP(MR2.0) can be clearly seen. (C) Tumor doubling time calculated from tumor volume measurements shows a significant increase in doubling time in mice treated with RALA / dFdCTP(MR2.0). [Figure 7] Figure 7 shows the increased circulating half-life of RALA / dFdCTP nanoparticles (MR2.0). C57BL / 6 mice (n=6) were treated intravenously with 40 μg of RALA / dFdCTP, dFdCTP, or gemcitabine. At pre-determined time points (0.25 / 0.5 / 1 / 2 / 4 / 6 / 12 hours) after treatment, whole blood was sampled from the tail of the mice and serum was extracted via centrifugation. Serum samples were analyzed via mass spectroscopy and compared to standard curves of the molecule of interest at various concentrations. 7A: PK profiles after intravenous injection of a single 40 μg dose of RALA / dFdCTP (MR2.0), dFdCTP, or gemcitabine into C57BL / 6 mice. 7B: Area under the curve (circulating concentrations) of gemcitabine, dFdCTP, and RALA / dFdCTP (MR2.0) obtained from representative PK profiles. [Figure 8] FIG. 1 shows in vivo levels of safety biomarkers following intravenous delivery of 40 μg of RALA / dFdCTP nanoparticles (MR2.0), dFdCTP, or gemcitabine. Safety markers examined included: (A) nephrotoxicity (creatine), (B, C) liver toxicity (AST and ALT), (D, E, F) immunogenicity markers (TNF-α, IFN-γ, and MIP-2). [Figure 9] Figure 1 shows measurements of red blood cell (RBC) lysis induced by RALA / dFdCTP measured in sheep blood. RALA / dFdCTP nanoparticles or dFdCTP in isotonic trehalose solution were added to RBC suspensions, with the added dFdCTP corresponding to 10-30 μg, and the solution was incubated for 1 h at 37 °C. After incubation, the red blood cell suspension was centrifuged at 400 g for 90 s to form a cell pellet. The absorbance of the supernatant was measured via UV-Vis spectrophotometry (541 nm). 1% Triton X-100 was used as a positive control corresponding to 100% hemolysis. The data show that combining dFdCTP with RALA results in no detectable hemolytic activity in RBCs, whereas dFdCTP alone results in >20% hemolysis. [Figure 10]Figure 1 shows the charge titration curve of RALA / dFdCTP nanoparticles. A sample of RALA / dFdCTP nanoparticles containing 462.5 μg of RALA peptide was titrated with 0.01 M poly(acrylic acid) and the charge of the resulting solution was measured using a multi-parameter bench meter. A non-linear standard curve fit (R2=0.991) was used to calculate the equivalence point and interpolate to account for the volume of titrant added (V=46.33). Using the volume of titrant (V), the concentration of PAA (C), and the mass of RALA (W) in the equation, the average charge density of RALA / dFdCTP nanoparticles was calculated to be ≦2.0 μmol / mg at 20° C., indicating that the nanoparticles are not cytotoxic due to their charge. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Many embodiments of the invention are detailed throughout this specification and will be apparent to those of skill in the art, and the invention is not to be construed as limited to any of the embodiments listed.

[0018] "A" means "at least one." In any embodiment where "a" is used to describe a given material or element, "a" can mean one.

[0019] "Comprising" means that a given material or element may contain other materials or elements. In any embodiment where "comprising" is referenced, the given material or element may be formed from at least 10% w / w, at least 20% w / w, at least 30% w / w, or at least 40% w / w of that material or element. In any embodiment where "comprising" is referenced, "comprising" can also mean "consisting of" or "consisting essentially of" the given material or element.

[0020] "Consisting of" means that a given material or element is formed entirely from that material or element. In any embodiment where "consisting of" is mentioned, the given material or element may be formed from 100% w / w of that material or element.

[0021] "Consisting essentially of" means that a given material or element makes up substantially all of that material or element. In any embodiment where reference is made to "consisting essentially of," the given material or element may be formed from at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 80% w / w, at least 90% w / w, at least 95% w / w, or at least 99% w / w of that material or element.

[0022] In any embodiment where "is" or "may be" is used to define a material or element, "is" or "may be" can mean that the material or element "consists of" or "consists essentially of" that material or element.

[0023] The claims are embodiments.

[0024] Nucleosides A nucleoside contains a nitrogenous base and a five-carbon sugar.

[0025] In one embodiment, nucleosides are monomeric units and are not polymerized into longer chains.

[0026] In one embodiment, the nucleoside is not RNA or DNA.

[0027] In one embodiment, a nucleoside comprises a nitrogenous base selected from adenine, guanine, thymine, uracil, and cytosine, or modified forms thereof.

[0028] Modifications of the nitrogen base include, for example, one or more of a halo group, a nitro group, a cyano group, a hydroxy group, a trifluoromethoxy group, a trifluoromethyl group, an amino group, a carboxy group, a carbamoyl group, a mercapto group, a sulfamoyl group, a methyl group, an ethyl group, a methoxy group, an ethoxy group, an acetyl group, an acetoxy group, a methylamino group, an ethylamino group, a dimethylamino group, a diethylamino group, an N-methyl-N-ethylamino group, an acetylamino group, an N-methylcarbamoyl group, an N-ethylcarbamoyl group, an N,N-dimethylcarbamoyl group, an N,N-diethylcarbamoyl ...diethylcarbamoyl group, an N-methylcarbamoyl group, an N-ethylcarbamoyl group, an N,N-dimethylcarbamoyl group, an N,N-diethylcarbamoyl group, an N-methylcarbamoyl group, an In some embodiments, this may include the addition, substitution or removal of a heteroaryl group, N-methyl-N-ethylcarbamoyl group, methylthio group, ethylthio group, methylsulfinyl group, ethylsulfinyl group, mesyl group, ethylsulfonyl group, methoxycarbonyl group, ethoxycarbonyl group, N-methylsulfamoyl group, N-ethylsulfamoyl group, N,N-dimethylsulfamoyl group, N,N-diethylsulfamoyl group, N-methyl-N-ethylsulfamoyl group, or cyclopropyl group, or rearrangement and / or removal and / or addition of further heteroatoms to the nitrogen base.

[0029] In one embodiment, the nucleoside comprises a nitrogenous base selected from adenine or modified forms thereof.

[0030] In one embodiment, the nucleoside comprises a nitrogenous base selected from guanine or modified forms thereof.

[0031] In one embodiment, the nucleoside comprises a nitrogenous base selected from thymine or modified forms thereof.

[0032] In one embodiment, the nucleoside comprises a nitrogenous base selected from uracil or modified forms thereof.

[0033] In one embodiment, the nucleoside comprises a nitrogenous base selected from cytosine or modified forms thereof.

[0034] In one embodiment, a nucleoside comprises a 5-carbon sugar selected from ribose or 2'-deoxyribose or modified versions thereof.

[0035] Modifications of 5-carbon sugars include, for example, one or more of a halo group, a nitro group, a cyano group, a hydroxy group, a trifluoromethoxy group, a trifluoromethyl group, an amino group, a carboxy group, a carbamoyl group, a mercapto group, a sulfamoyl group, a methyl group, an ethyl group, a methoxy group, an ethoxy group, an acetyl group, an acetoxy group, a methylamino group, an ethylamino group, a dimethylamino group, a diethylamino group, an N-methyl-N-ethylamino group, an acetylamino group, an N-methylcarbamoyl group, an N-ethylcarbamoyl group, an N,N-dimethylcarbamoyl group, an N,N-diethylcarbamoyl ...diethylcarbamoyl group, an N-methylcarbamoyl group, an N-ethylcarbamoyl group, an N,N-dimethylcarbamoyl group, an N,N-diethylcarbamoyl group, an N-methylcarbamoyl group, an N-ethylcarbamoyl group, an N,N-diethylcarbamoyl group, an N-methylcarbamoyl group, an N-ethylcarbamoyl group, In some embodiments, the addition, substitution, or removal of an isopropyl group, N-methyl-N-ethylcarbamoyl group, methylthio group, ethylthio group, methylsulfinyl group, ethylsulfinyl group, mesyl group, ethylsulfonyl group, methoxycarbonyl group, ethoxycarbonyl group, N-methylsulfamoyl group, N-ethylsulfamoyl group, N,N-dimethylsulfamoyl group, N,N-diethylsulfamoyl group, N-methyl-N-ethylsulfamoyl group, or cyclopropyl group, or the replacement of one or more carbons in the 5-carbon sugar with one or more heteroatoms, such as sulfur.

[0036] In one embodiment, a nucleoside comprises a five carbon sugar selected from ribose or modified forms thereof.

[0037] In one embodiment, a nucleoside comprises a 5-carbon sugar selected from 2'-deoxyribose or modified forms thereof.

[0038] In one embodiment, the nucleoside is a "nucleoside analog" of the nucleoside analog family of pharma- ceutical active agents.

[0039] In one embodiment, a nucleoside or phosphate thereof refers to an analog of deoxyadenosine, adenosine, deoxycytidine, guanosine, deoxyguanosine, thymidine, deoxythymidine, or deoxyuridine, or a phosphate thereof.

[0040] In one embodiment, the nucleoside or phosphate thereof refers to didanosine, vidarabine, galidesivir, remdesivir, cytarabine, gemcitabine, emtricitabine, lamivudine, zalcitabine, abacavir, acyclovir, entecavir, stavudine, telbivudine, zidovudine, idoxuridine, or trifluridine, or a phosphate thereof.

[0041] In one embodiment, the nucleoside or phosphate thereof refers to didanosine or its phosphate, which is useful in the treatment and / or prevention of HIV and AIDS.

[0042] In one embodiment, the nucleoside or phosphate thereof refers to vidarabine or a phosphate thereof, which is useful in the treatment and / or prevention of herpes simplex and varicella zoster viruses.

[0043] In one embodiment, the nucleoside or phosphate thereof refers to galidesivir or phosphate thereof, which is useful in the treatment and / or prevention of Hepatitis C, Ebola virus, Marburg virus, Zika virus, and coronavirus.

[0044] In one embodiment, the nucleoside or phosphate thereof refers to remdesivir or its phosphate, which is useful in the treatment and / or prevention of Hepatitis C, Ebola virus, Marburg virus, and as a post-infection treatment for COVID-19.

[0045] In one embodiment, the nucleoside or phosphate thereof refers to cytarabine or phosphate thereof, which is useful in the treatment and / or prevention of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), and non-Hodgkin's lymphoma.

[0046] In one embodiment, nucleoside or its phosphate refers to gemcitabine or its phosphate.Gemcitabine or its phosphate is useful in the treatment and / or prevention of testicular cancer, breast cancer, ovarian cancer, non-small cell lung cancer, pancreatic cancer and bladder cancer, in particular metastatic breast cancer, or in the first-line treatment of metastatic breast cancer after unsuccessful adjuvant chemotherapy, or in the first-line treatment of patients with inoperable locally advanced (stage IIIA or IIIB) or metastatic (stage IV) non-small cell lung cancer, or in the treatment of locally advanced (unresectable stage II or stage III) or metastatic (stage IV) pancreatic adenocarcinoma, or in the treatment of locally advanced or metastatic bladder cancer, or in the treatment of locally advanced or metastatic epithelial ovarian cancer.

[0047] In one embodiment, the nucleoside or phosphate thereof refers to emtricitabine or a phosphate thereof, which is useful in the treatment and / or prevention of HIV infection.

[0048] In one embodiment, the nucleoside or phosphate thereof refers to lamivudine or a phosphate thereof, which is useful in the treatment and / or prevention of HIV / AIDS and chronic hepatitis B.

[0049] In one embodiment, the nucleoside or phosphate thereof refers to zalcitabine or a phosphate thereof, which is useful in the treatment and / or prevention of HIV / AIDS.

[0050] In one embodiment, the nucleoside or phosphate thereof refers to abacavir or a phosphate thereof, which is useful in the treatment and / or prevention of HIV / AIDS.

[0051] In one embodiment, the nucleoside or phosphate thereof refers to acyclovir or phosphate thereof, which is useful in treating and / or preventing severe complications of herpes simplex virus infection, varicella zoster virus infection, cytomegalovirus infection, and Epstein-Barr virus infection.

[0052] In one embodiment, the nucleoside or phosphate thereof refers to entecavir or phosphate thereof, which is useful in the treatment and / or prevention of Hepatitis B virus (HBV) infection.

[0053] In one embodiment, the nucleoside or phosphate thereof refers to stavudine or a phosphate thereof, which is useful in the treatment and / or prevention of HIV / AIDS.

[0054] In one embodiment, the nucleoside or phosphate thereof refers to telbivudine or a phosphate thereof, which is useful in the treatment and / or prevention of Hepatitis B infection.

[0055] In one embodiment, the nucleoside or phosphate thereof refers to zidovudine or a phosphate thereof, which is useful in the treatment and / or prevention of HIV / AIDS.

[0056] In one embodiment, the nucleoside or phosphate thereof refers to idoxuridine or phosphate thereof, which is useful in the treatment and / or prevention of herpes simplex keratitis.

[0057] In one embodiment, the nucleoside or phosphate thereof refers to trifluridine or a phosphate thereof, which is useful in the treatment and / or prevention of keratitis and keratoconjunctivitis caused by herpes simplex virus types 1 and 2, and in the treatment of ocular vaccinia virus infections.

[0058] In one embodiment, the nucleoside or phosphate thereof refers to gemcitabine.

[0059] Gemcitabine, or 2',2'-difluoro-2'-deoxycytidine (dFdC) (CAS ID: 95058-81-4), has the structure:

[0060] [ka] has.

[0061] In one embodiment, the nucleoside or phosphate thereof refers to the phosphate of gemcitabine.

[0062] In one embodiment, the nucleoside or phosphate thereof refers to gemcitabine monophosphate.

[0063] Gemcitabine monophosphate, or 2',2'-difluorodeoxycytidine 5'-phosphate (dFdCMP) (CAS ID: 116371-67-6), has the structure:

[0064] [ka] has.

[0065] In one embodiment, the nucleoside or phosphate thereof refers to gemcitabine diphosphate.

[0066] Gemcitabine diphosphate, or 2',2'-difluorodeoxycytidine 5'-diphosphate (dFdCDP) (CAS ID: 116371-66-5), has the structure:

[0067] [ka] has.

[0068] In one embodiment, the nucleoside or phosphate thereof refers to gemcitabine triphosphate.

[0069] Gemcitabine triphosphate, or 2',2'-difluorodeoxycytidine 5'-triphosphate (dFdCTP) (CAS ID: 110988-86-8), has the structure:

[0070] [ka]

[0071] or its phosphate

[0072] A phosphate is an anion, salt, functional group, or ester derived from phosphoric acid.

[0073] In one embodiment, the phosphate is a derivative of orthophosphoric acid, H3PO4.

[0074] In one embodiment, the phosphate is (PO4) 3- Contains a group.

[0075] In one embodiment, the phosphate comprises a P(O)(OH)2O- group.

[0076] In one embodiment, the phosphate comprises a diphosphate, for example a P(O)(OH)-O-P(O)(OH)-O- group.

[0077] In one embodiment, the phosphate comprises a triphosphate, for example a P(O)(OH)-OP(O)(OH)-OP(O)(OH)- group.

[0078] In one embodiment, the nucleoside or its phosphate is a monomeric unit and is not polymerized into a longer chain.

[0079] In one embodiment, the nucleoside or its phosphate is not RNA or DNA.

[0080] Amphiphilic cell membrane penetrating peptides According to convention, peptides described herein are written with the "N-terminus" first, i.e. to the left.

[0081] In one embodiment, the amphipathic cell membrane penetrating peptide comprises or consists of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology.

[0082] In one embodiment, the amphipathic cell membrane penetrating peptide comprises the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 80% sequence identity or homology.

[0083] In one embodiment, the amphipathic cell membrane penetrating peptide consists of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology.

[0084] In one embodiment, the amphipathic cell membrane penetrating peptide comprises or consists of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 85% sequence identity or homology.

[0085] In one embodiment, the amphipathic cell membrane penetrating peptide comprises the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 85% sequence identity or homology.

[0086] In one embodiment, the amphipathic cell membrane penetrating peptide consists of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 85% sequence identity or homology.

[0087] In one embodiment, the amphipathic cell membrane penetrating peptide comprises or consists of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 90% sequence identity or homology.

[0088] In one embodiment, the amphipathic cell membrane penetrating peptide comprises the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 90% sequence identity or homology.

[0089] In one embodiment, the amphipathic cell membrane penetrating peptide consists of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 90% sequence identity or homology.

[0090] In one embodiment, the amphipathic cell membrane penetrating peptide comprises or consists of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 95% sequence identity or homology.

[0091] In one embodiment, the amphipathic cell membrane penetrating peptide comprises the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) or a sequence having at least 95% sequence identity or homology.

[0092] In one embodiment, the amphipathic cell membrane penetrating peptide consists of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 95% sequence identity or homology.

[0093] In one embodiment, the amphipathic cell membrane penetrating peptide comprises or consists of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1).

[0094] In one embodiment, the amphipathic cell membrane penetrating peptide comprises the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1).

[0095] In one embodiment, the amphipathic cell membrane penetrating peptide consists of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1).

[0096] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) consists of 35 or fewer amino acid residues.

[0097] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) consists of 30 or fewer amino acid residues.

[0098] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) consists of 26 to 30 amino acid residues.

[0099] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises at least five arginine residues (R).

[0100] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises at least 6 arginine residues (R).

[0101] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises at least 10 alanine residues (A).

[0102] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises at least 12 alanine residues (A).

[0103] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises at least 5 leucine residues (L).

[0104] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises at least 6 leucine residues (L).

[0105] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises at least one cysteine ​​residue (C).

[0106] In one embodiment, a sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) contains at least two, but not more than three, glutamic acid (E) residues.

[0107] In one embodiment, a sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) contains at least six arginine residues (R), at least twelve alanine residues (A), at least six leucine residues (L), optionally at least one cysteine ​​residue (C), and at least two, but not more than three, glutamic acid residues (E).

[0108] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises the consensus sequence EARLARALARALAR and / or LARALARALRA.

[0109] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises the consensus sequence EARLARALARALAR.

[0110] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) comprises the consensus sequence LARALARALRA.

[0111] In one embodiment, sequences having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) include the consensus sequences EARLARALARALAR and LARALARALRA.

[0112] In one embodiment, the sequence having at least 80% sequence identity or homology to WEARLARALARALARHLARALARALRACEA (SEQ ID NO:1) does not contain a glycine (G).

[0113] Sequences with at least 80% sequence identity or homology Any of a variety of sequence alignment methods can be used to determine percent sequence identity, including, but not limited to, global, local, and hybrid methods, such as segment approach methods. Protocols for determining percent identity are routine procedures within the skill of the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up the scores of individual residue pairs and by imposing gap penalties. Traditional methods include Altschul et al., Bull. Math. Bio. 48: 603-16, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992, in which two amino acid sequences are aligned to optimize alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff.

[0114] The percent sequence identity between two or more amino acid sequences depends on the number of identical positions shared by the sequences. Thus, the percent identity can be calculated as the number of identical amino acids divided by the total number of amino acids, multiplied by 100. The calculation of percent sequence identity can also take into account the number of gaps and the length of each gap that needs to be introduced to optimize the alignment of two or more sequences. The sequence comparison and percent identity determination between two or more sequences can be performed using a specific mathematical algorithm, such as BLAST, which will be familiar to those skilled in the art.

[0115] Homologous sequences may be characterized as having one or more amino acid substitutions, deletions, or additions. These changes are of a minor nature that do not significantly affect the folding or activity of the peptide. These may be minor amino acid substitutions, minor deletions, and minor amino- or carboxy-terminal extensions, or other minor additions.

[0116] In one embodiment, the term "sequence identity or sequence homology" refers to sequence identity.

[0117] In one embodiment, the term "sequence identity or sequence homology" refers to sequence homology.

[0118] Nanoparticles In one embodiment, the nucleoside formulations described herein comprise or consist of nanoparticles, which can be formed by self-assembly by adding together the nucleoside or its phosphate and the amphiphilic cell membrane penetrating peptide in ultrapure water.

[0119] In one embodiment, (i) a nucleoside or a phosphate thereof, and (ii) Amino acid sequence: WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or an amphipathic cell membrane penetrating peptide comprising or consisting of a sequence having at least 80% sequence identity or sequence homology thereto; A nanoparticle formulation is provided comprising:

[0120] In one embodiment, (i) gemcitabine or its phosphate, and (ii) Amino acid sequence: WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or an amphipathic cell membrane penetrating peptide comprising or consisting of a sequence having at least 80% sequence identity or sequence homology thereto; A nanoparticle formulation is provided comprising:

[0121] In one embodiment, (i) gemcitabine triphosphate, and (ii) Amino acid sequence: WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) Amphiphilic cell membrane penetrating peptides comprising A nanoparticle formulation is provided comprising:

[0122] In one embodiment, there is provided a nucleoside formulation described herein that is a nanoparticle formulation.

[0123] In one embodiment, nanoparticles are provided that include a nucleoside formulation as described herein.

[0124] In one embodiment, a nanoparticle formulation is provided comprising the nucleoside formulation described herein.

[0125] In one embodiment, there is provided a nucleoside formulation as described herein comprising nanoparticles.

[0126] In one embodiment, there is provided a nucleoside formulation as described herein comprising nanoparticles having a Z-average of 30-150 nm, where Z-average is the average hydrodynamic size of a collection of particles as measured by intensity-weighted dynamic light scattering (DLS).

[0127] In one embodiment, there is provided a nucleoside formulation as described herein comprising nanoparticles having a Z-average of 60-100 nm.

[0128] In one embodiment, there is provided a nucleoside formulation as described herein comprising nanoparticles having a polydispersity index of ≦0.3. The polydispersity index (PI) is a size-based measure of the heterogeneity of a sample.

[0129] In one embodiment, there is provided a nucleoside formulation as described herein comprising nanoparticles having a polydispersity index of ≦0.5.

[0130] Molar ratio of nucleoside or its phosphate:amphiphilic cell membrane penetrating peptide In one embodiment, the molar ratio (mol:mol) of the nucleoside or phosphate thereof described herein to the amphipathic cell membrane penetrating peptide may be varied, which may have beneficial effects on physiochemical characteristics (e.g., Z-average, Zeta potential (particle charge), and / or polydispersity index), cellular uptake, and / or therapeutic efficacy.

[0131] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane penetrating peptide is about 0.1:10.

[0132] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 0.1:10.

[0133] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane penetrating peptide is about 1:5.

[0134] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:5.

[0135] In one embodiment, the molar ratio of the nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is about 1:1-3.

[0136] In one embodiment, the molar ratio of the nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:1-3.

[0137] In one embodiment, the molar ratio of the nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is about 1:1.2-2.8.

[0138] In one embodiment, the molar ratio of the nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.2-2.8.

[0139] In one embodiment, the molar ratio of the nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is about 1:1.4-2.6.

[0140] In one embodiment, the molar ratio of the nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.4 to 2.6.

[0141] In one embodiment, the molar ratio of the nucleoside or its phosphate:amphiphilic cell membrane-permeable peptide is about 1:1.6-2.4.

[0142] In one embodiment, the molar ratio of the nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.6-2.4.

[0143] In one embodiment, the molar ratio of the nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is about 1:1.8-2.2.

[0144] In one embodiment, the molar ratio of the nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.8-2.2.

[0145] In one embodiment, the molar ratio of the nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is about 1:1.9-2.5.

[0146] In one embodiment, the molar ratio of the nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.9-2.5.

[0147] In one embodiment, the molar ratio of the nucleoside or its phosphate:amphiphilic cell membrane-permeable peptide is about 1:2.0-2.4.

[0148] In one embodiment, the molar ratio of the nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:2.0 to 2.4.

[0149] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane penetrating peptide is about 1:1.

[0150] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.

[0151] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane penetrating peptide is about 1:1.2.

[0152] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.2.

[0153] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane penetrating peptide is about 1:1.4.

[0154] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.4.

[0155] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane penetrating peptide is about 1:1.6.

[0156] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.6.

[0157] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane penetrating peptide is about 1:1.8.

[0158] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.8.

[0159] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is about 1:2.

[0160] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:2.

[0161] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is about 1:2.2.

[0162] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:2.2.

[0163] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane penetrating peptide is about 1:2.4.

[0164] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:2.4.

[0165] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is about 1:2.6.

[0166] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:2.6.

[0167] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is about 1:2.8.

[0168] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:2.8.

[0169] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane penetrating peptide is about 1:3.

[0170] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:3.

[0171] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane penetrating peptide is about 0.1:3.

[0172] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 0.1:3.

[0173] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane penetrating peptide is about 1:4.

[0174] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:4.

[0175] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane penetrating peptide is about 1:5.

[0176] In one embodiment, the molar ratio of nucleoside or its phosphate:amphipathic cell membrane-permeable peptide is 1:5.

[0177] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is about 0.1:1-10.

[0178] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is 0.1:1-3.

[0179] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is about 1:1-5.

[0180] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is 1:1-5.

[0181] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is about 1:1-3.

[0182] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is 1:1-3.

[0183] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is about 1:1.2-2.8.

[0184] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.2-2.8.

[0185] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is about 1:1.4-2.6.

[0186] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.4-2.6.

[0187] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is about 1:1.6-2.4.

[0188] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.6-2.4.

[0189] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is about 1:1.8-2.2.

[0190] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.8-2.2.

[0191] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is about 1:2.0-2.4.

[0192] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is 1:2.0-2.4.

[0193] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is about 1:1.9-2.5.

[0194] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane-permeable peptide is 1:1.9-2.5.

[0195] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is about 1:1.

[0196] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is 1:1.

[0197] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is about 1:1.2.

[0198] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is 1:1.2.

[0199] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is about 1:1.4.

[0200] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is 1:1.4.

[0201] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is about 1:1.6.

[0202] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is 1:1.6.

[0203] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is about 1:1.8.

[0204] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is 1:1.8.

[0205] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is about 1:2.

[0206] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is 1:2.

[0207] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is about 1:2.2.

[0208] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is 1:2.2.

[0209] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is about 1:2.4.

[0210] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is 1:2.4.

[0211] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is about 1:2.6.

[0212] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is 1:2.6.

[0213] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is about 1:2.8.

[0214] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is 1:2.8.

[0215] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is about 1:3.

[0216] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is 1:3.

[0217] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is about 1:5.

[0218] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is 1:5.

[0219] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is about 0.1:10.

[0220] In one embodiment, the molar ratio of gemcitabine or its phosphate:amphipathic cell membrane penetrating peptide is 0.1:10.

[0221] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is about 0.1:1-10.

[0222] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is 0.1:1-10.

[0223] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is about 1:1-5.

[0224] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is 1:1-5.

[0225] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is about 1:1-3.

[0226] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is 1:1-3.

[0227] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is about 1:1.2-2.8.

[0228] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is 1:1.2-2.8.

[0229] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is about 1:1.4-2.6.

[0230] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is 1:1.4-2.6.

[0231] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is about 1:1.6-2.4.

[0232] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is 1:1.6-2.4.

[0233] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is about 1:1.8-2.2.

[0234] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is 1:1.8-2.2.

[0235] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is about 1:2.0-2.4.

[0236] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is 1:2.0-2.4.

[0237] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is about 1:1.9-2.5.

[0238] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane-permeable peptide is 1:1.9-2.5.

[0239] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is about 1:1.

[0240] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is 1:1.

[0241] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is about 1:1.2.

[0242] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is 1:1.2.

[0243] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is about 1:1.4.

[0244] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is 1:1.4.

[0245] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is about 1:1.6.

[0246] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is 1:1.6.

[0247] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is about 1:1.8.

[0248] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is 1:1.8.

[0249] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is about 1:2.

[0250] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is 1:2.

[0251] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is about 1:2.2.

[0252] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is 1:2.2.

[0253] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is about 1:2.4.

[0254] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is 1:2.4.

[0255] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is about 1:2.6.

[0256] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is 1:2.6.

[0257] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is about 1:2.8.

[0258] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is 1:2.8.

[0259] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is 1:3.

[0260] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is about 1:3.

[0261] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is 1:5.

[0262] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is about 1:5.

[0263] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is 0.1:10.

[0264] In one embodiment, the molar ratio of gemcitabine triphosphate:amphipathic cell membrane penetrating peptide is about 0.1:10.

[0265] Bulking agents, cryoprotectants, and agents that infer tonicity (osmotic pressure) In one embodiment, bulking agents may be added prior to lyophilization of the nanoparticles for transport and storage. Bulking agents are additives that increase the bulk volume of a product without affecting its properties.

[0266] In one embodiment, a cryoprotectant may be added prior to freeze-drying of the nanoparticles. Cryoprotectants are substances used to protect biological tissues from the damage of freezing.

[0267] In one embodiment, solutes may be added to estimate tonicity, for example, such that when water is added to the formulation, it results in an isotonic formulation, which has the same solute concentration as blood, i.e., 290-310 mOsmol / kg.

[0268] In one embodiment, the osmolality of a solution of the nucleoside formulation described herein is between 10 and 1000 mOsmol / kg.

[0269] In one embodiment, the osmolality of a solution of the nucleoside formulation described herein is between 100 and 500 mOsmol / kg.

[0270] In one embodiment, the osmolality of a solution of the nucleoside formulation described herein is between 200 and 400 mOsmol / kg.

[0271] In one embodiment, the osmolality of a solution of the nucleoside formulation described herein is between 290 and 310 mOsmol / kg.

[0272] In one embodiment, the osmolality of a solution of the nucleoside formulations described herein is about 300 mOsmol / kg.

[0273] In one embodiment, the osmolality of a solution of the nucleoside formulation described herein is 300 mOsmol / kg.

[0274] Suitable bulking agents include trehalose, sucrose, mannose, dextrose, or any mixture of such agents, which may also be utilized as cryoprotectants and / or agents for estimating tonicity.

[0275] In one embodiment, the nucleoside formulations described herein further comprise trehalose, sucrose, mannose, dextrose, or any mixture of such agents.

[0276] In one embodiment, the nucleoside formulations described herein further comprise >85% w / w trehalose, sucrose, mannose, dextrose, or any mixture of such agents.

[0277] In one embodiment, the nucleoside formulations described herein further comprise >90% w / w trehalose, sucrose, mannose, dextrose, or any mixture of such agents.

[0278] In one embodiment, the nucleoside formulations described herein further comprise >95% w / w trehalose, sucrose, mannose, dextrose, or any mixture of such agents.

[0279] In one embodiment, the bulking agent is trehalose.

[0280] In one embodiment, the nucleoside formulation described herein further comprises trehalose.

[0281] In one embodiment, the nucleoside formulation described herein further comprises >85% w / w trehalose.

[0282] In one embodiment, the nucleoside formulation described herein further comprises >90% w / w trehalose.

[0283] In one embodiment, the nucleoside formulation described herein further comprises >95% w / w trehalose.

[0284] In one embodiment, the filler is sucrose.

[0285] In one embodiment, the nucleoside formulations described herein further comprise sucrose.

[0286] In one embodiment, the nucleoside formulations described herein further comprise >85% w / w sucrose.

[0287] In one embodiment, the nucleoside formulations described herein further comprise >90% w / w sucrose.

[0288] In one embodiment, the nucleoside formulations described herein further comprise >95% w / w sucrose.

[0289] In one embodiment, the bulking agent is mannose.

[0290] In one embodiment, the nucleoside formulation described herein further comprises mannose.

[0291] In one embodiment, the nucleoside formulation described herein further comprises >85% w / w mannose.

[0292] In one embodiment, the nucleoside formulation described herein further comprises >90% w / w mannose.

[0293] In one embodiment, the nucleoside formulation described herein further comprises >95% w / w mannose.

[0294] In one embodiment, the bulking agent comprises trehalose.

[0295] In one embodiment, the filler comprises sucrose.

[0296] In one embodiment, the bulking agent comprises mannose.

[0297] In one embodiment, the filler comprises dextrose.

[0298] In one embodiment, the nucleoside formulations described herein comprise: (i) 0.1 to 1% w / w of gemcitabine or its phosphate; (ii) 1 to 10% w / w of an amphipathic cell membrane-permeable peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 89 to 99% w / w of trehalose, sucrose, mannose, and / or dextrose Includes.

[0299] In one embodiment, the nucleoside formulations described herein comprise: (i) 0.2 to 0.5% w / w of gemcitabine or its phosphate; (ii) 3.5 to 4.5% w / w of an amphipathic cell membrane-permeable peptide having the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 95 to 97% w / w of trehalose, sucrose, mannose, and / or dextrose Includes.

[0300] In one embodiment, the nucleoside formulations described herein comprise: (i) 0.1-1% w / w of gemcitabine triphosphate; (ii) 1 to 10% w / w of an amphipathic cell membrane-permeable peptide having the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); and (iii) 89-99% w / w trehalose Includes.

[0301] In one embodiment, the nucleoside formulations described herein comprise: (i) 0.2-0.5% w / w of gemcitabine triphosphate; (ii) 3.5 to 4.5% w / w of an amphipathic cell membrane-permeable peptide having the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); and (iii) 95-97% w / w trehalose Includes.

[0302] In one embodiment, the nucleoside formulations described herein comprise: (i) about 0.3% w / w of gemcitabine triphosphate; (ii) about 4% w / w of an amphipathic cell membrane penetrating peptide consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); and (iii) about 96% w / w trehalose Includes.

[0303] In one embodiment, the nucleoside formulations described herein comprise: (i) 0.3% w / w of gemcitabine triphosphate; (ii) 4.03% w / w of an amphipathic cell membrane penetrating peptide consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); and (iii) 95.67% w / w trehalose Includes.

[0304] Water-based preparations The nucleoside formulations described herein can be conveniently formulated in water, especially ultrapure water, to facilitate administration, especially via intravenous injection.

[0305] The nucleoside formulations described herein include (i) 0.1 to 1% w / w of gemcitabine or its phosphate; (ii) 1 to 10% w / w of an amphipathic cell membrane-permeable peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 89 to 99% w / w of trehalose, sucrose, mannose, and / or dextrose Including, It is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0306] The nucleoside formulations described herein include (i) 0.2 to 0.5% w / w of gemcitabine or its phosphate; (ii) 3.5 to 4.5% w / w of an amphipathic cell membrane-permeable peptide having the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 95 to 97% w / w of trehalose, sucrose, mannose, and / or dextrose Including, It is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0307] The nucleoside formulations described herein include (i) 0.1-1% w / w of gemcitabine triphosphate; (ii) 1 to 10% w / w of an amphipathic cell membrane-permeable peptide having the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); and (iii) 89-99% w / w trehalose Including, It is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0308] The nucleoside formulations described herein include (i) 0.2-0.5% w / w of gemcitabine triphosphate; (ii) 3.5 to 4.5% w / w of an amphipathic cell membrane-permeable peptide having the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); and (iii) 95-97% w / w trehalose Including, It is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0309] The nucleoside formulations described herein include (i) about 0.3% w / w of gemcitabine triphosphate; (ii) about 4% w / w of an amphipathic cell membrane penetrating peptide consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); and (iii) about 96% w / w trehalose Including, It is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0310] The nucleoside formulations described herein include (i) 0.3% w / w of gemcitabine triphosphate; (ii) 4.03% w / w of an amphipathic cell membrane penetrating peptide consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); and (iii) 95.67% w / w trehalose Including, It is formulated as an isotonic preparation in water with an osmolality of 290-310 mOsmol / kg.

[0311] Automated Formulation In one embodiment, nanoparticles are prepared through an automated controllable mixing system, such as an automated microfluidic system, such as Precision Nanosystems Ignite NanoAssemblr. This technology allows both mixing speed and mixing ratio control during nanoparticle formulation, resulting in a reduction in Z-average particle size, and therefore a further reduction in polydispersity index, compared to manual formulation methods.

[0312] Microfluidics refers to the behavior, precise control, and manipulation of fluids that are geometrically constrained to small scales (typically sub-millimeter) where surface forces are greater than volumetric forces.

[0313] In one embodiment, the nanoparticles described herein are prepared via an automatically controlled mixing system.

[0314] In one embodiment, a method for preparing a nanoparticle formulation is provided, comprising formulating (i) a solution of WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or sequence homology with (ii) a pharmacologically active agent in an automated controlled mixing system, in particular an automated microfluidic system.

[0315] In one embodiment, a method for preparing a nanoparticle formulation is provided, comprising formulating (i) a solution of WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1), or a sequence having at least 80% sequence identity or homology, with (ii) a nucleic acid or other agent, preferably a negatively charged or hydrophilic compound, in an automated controlled mixing system, in particular an automated microfluidic system.

[0316] In one embodiment, a method for preparing a nanoparticle formulation is provided, comprising formulating (i) a solution of WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology, and (ii) DNA, RNA, shRNA, and siRNA in an automated controlled mixing system, in particular an automated microfluidic system.

[0317] In one embodiment, there is provided a method for preparing a nanoparticle formulation comprising formulating (i) a solution of WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1), or a sequence having at least 80% sequence identity or homology, with (ii) a bisphosphonate drug, including alendronate, etidronate, zolendrate, or any other nitrogen-based or non-nitrogen-based bisphosphonate drug, in an automated controlled mixing system, in particular an automated microfluidic system.

[0318] In one embodiment, there is provided a method for preparing a nanoparticle formulation, comprising formulating (i) a solution of WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology, and (ii) gold in an automated controlled mixing system, in particular an automated microfluidic system.

[0319] In one embodiment, a method for preparing a nanoparticle formulation is provided, comprising formulating (i) a solution of WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or sequence homology with (ii) a nucleoside or a phosphate thereof in an automated controlled mixing system, in particular an automated microfluidic system.

[0320] In one embodiment, there is provided a method for preparing a nanoparticle formulation, comprising formulating (i) a solution of WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology, and (ii) gemcitabine or a phosphate thereof in an automated controlled mixing system, in particular an automated microfluidic system.

[0321] In one embodiment, a method for preparing a nanoparticle formulation is provided, comprising formulating (i) a solution of WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology, and (ii) gemcitabine triphosphate in an automated controlled mixing system, in particular an automated microfluidic system.

[0322] In one embodiment, the flow ratio of amphiphilic peptides described herein to pharmacologically active agents in the automated microfluidic system is 1:1.

[0323] In one embodiment, the flow ratio of amphipathic peptides described herein to pharmacologically active agents in the automated microfluidic system is about 1:1.

[0324] formulation The nanoparticles can be formed by self-assembly by adding the nucleoside or its phosphate and the amphiphilic cell membrane penetrating peptide together in ultrapure water to produce a formulation that can be freeze-dried for transport and storage and then rehydrated in water for use.

[0325] The nucleoside formulations described herein can be utilized in a variety of routes of administration, for example, oral, nasal, rectal, topical, transdermal, intravitreal, intravenous, or intramuscular, intradermal, and particularly intravenous. In one embodiment, the nucleoside formulations of the present disclosure can be utilized in an injectable formulation, for example, intravenous injection.

[0326] charge density The average surface charge density of nanoparticles may be a contributing factor to nanoparticle toxicity by promoting oxidative stress mechanisms, which in turn may promote mitochondrial dysfunction and reduced viability.

[0327] Charge density can be measured by polyelectrolyte titration using the methods described in Ritz et al., Biomacromolecules. 2015 Apr 13, 16(4):1311-21. doi: 10.1021 / acs.biomac.5b00108. Epub 2015 Apr 3, and Weiss et al., J Nanobiotechnology. 2021 Jan 6, 19(1):5. doi: 10.1186 / s12951-020-00747-7.

[0328] Polyelectrolyte titration can be performed using 0.01 M poly(acrylic acid) (PAA) at pH 7.4, and the addition of PAA to the nanoparticles and measurement of the charge will result in a sigmoidal curve, and the volume of the curve (V) can be that of the equivalence point. Together, the mass of peptide in the system (w) and the concentration of PAA used (0.01 M) can be used to calculate the average charge density (Q ek ) to calculate

[0329] In one embodiment, the nanoparticles comprising gemcitabine or its phosphate and an amphiphilic cell membrane penetrating peptide described herein have an average surface charge density of ≦2 μmol / mg at 20° C. The average surface charge density profile was measured using an Orion STAR® multiparameter bench meter.

[0330] In one embodiment, the nanoparticles comprising gemcitabine or its phosphate and an amphiphilic cell membrane penetrating peptide described herein have an average surface charge density of ≦1.5 μmol / mg at 20° C.

[0331] In one embodiment, the nanoparticles comprising gemcitabine or its phosphate and an amphiphilic cell membrane penetrating peptide described herein have an average surface charge density of about 1 μmol / mg at 20°C.

[0332] In one embodiment, the nanoparticles comprising gemcitabine or its phosphate and an amphiphilic cell membrane penetrating peptide described herein have an average surface charge density of 1 μmol / mg at 20°C.

[0333] use In one embodiment, there is provided a nucleoside formulation described herein for use as a pharmaceutical.

[0334] In one embodiment, there is provided the use of a nucleoside formulation described herein as a pharmaceutical agent.

[0335] In one embodiment, there is provided a nucleoside formulation as described herein for use in therapy.

[0336] In one embodiment, there is provided a nucleoside formulation described herein for use in intravenous injection.

[0337] In one embodiment, there is provided a nucleoside formulation as described herein for use in treating a viral infection.

[0338] In one embodiment, there is provided a nucleoside formulation as described herein for use in the treatment of HIV, Ebola, Marburg, coronavirus, Hepatitis B, Hepatitis C, Vaccinia, Epstein-Barr, Cytomegalovirus, Zika, Herpes simplex, and / or Varicella-Zoster virus infection.

[0339] In one embodiment, there is provided a nucleoside formulation as described herein for use in the treatment of cancer.

[0340] As used herein, the terms "treatment" and "treating" refer to reversing, alleviating, delaying the onset, or inhibiting the progression of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be administered after symptoms have disappeared, for example to prevent or delay their recurrence.

[0341] In the present specification, when the use in the treatment of cancer is described, this may be early stage cancer, actively progressing cancer, metastatic cancer, and / or drug resistant cancer. In some embodiments referring to cancer, the cancer is early stage cancer. In some embodiments referring to cancer, the cancer is locally advanced cancer. In some embodiments referring to cancer, the cancer is locally advanced and / or metastatic cancer. In some embodiments referring to cancer, the cancer is metastatic cancer. In some embodiments referring to cancer, the cancer is invasive cancer.

[0342] In one embodiment, there is provided a nucleoside formulation as described herein for use in the treatment of breast cancer, bladder cancer, testicular cancer, pancreatic cancer, ovarian cancer, or non-small cell lung cancer.

[0343] In one embodiment, there is provided a nucleoside formulation as described herein for use in the treatment of breast cancer.

[0344] In one embodiment, there is provided a nucleoside formulation as described herein for use in the treatment of testicular cancer.

[0345] In one embodiment, there is provided a nucleoside formulation as described herein for use in the treatment of bladder cancer.

[0346] In one embodiment, there is provided a nucleoside formulation as described herein for use in the treatment of pancreatic cancer.

[0347] In one embodiment, there is provided a nucleoside formulation as described herein for use in the treatment of ovarian cancer.

[0348] In one embodiment, there is provided a nucleoside formulation as described herein for use in the treatment of non-small cell lung cancer.

[0349] In one embodiment, there is provided a nucleoside formulation as described herein for use in the treatment of metastatic breast cancer.

[0350] In one embodiment, there is provided a nucleoside formulation as described herein for use in the first line treatment of metastatic breast cancer.

[0351] In one embodiment, there is provided a nucleoside formulation as described herein for use in the first line treatment of metastatic breast cancer after failed adjuvant chemotherapy.

[0352] In one embodiment, there is provided a nucleoside formulation as described herein for use in the first line treatment of patients with inoperable, locally advanced (Stage IIIA or IIIB) or metastatic (Stage IV) non-small cell lung cancer.

[0353] In one embodiment, there is provided a nucleoside formulation as described herein for use in the treatment of locally advanced (unresectable stage II or stage III) or metastatic (stage IV) pancreatic adenocarcinoma.

[0354] In one embodiment, there is provided a nucleoside formulation as described herein for use in the treatment of locally advanced or metastatic bladder cancer.

[0355] In one embodiment, there is provided a nucleoside formulation as described herein for use in the treatment of locally advanced or metastatic epithelial ovarian cancer.

[0356] Pharmaceutical Compositions In one embodiment, a pharmaceutical composition is provided comprising a nucleoside formulation as described herein for use in intravenous injection.

[0357] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation described herein for use in treating a viral infection.

[0358] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation described herein for use in the treatment of HIV, Ebola, Marburg, coronavirus, Hepatitis B, Hepatitis C, Vaccinia, Epstein-Barr, Cytomegalovirus, Zika, Herpes simplex, and / or Varicella-Zoster virus infection.

[0359] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation described herein for use in the treatment of cancer.

[0360] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation described herein for use in the treatment of breast cancer, bladder cancer, testicular cancer, pancreatic cancer, ovarian cancer, or non-small cell lung cancer.

[0361] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation described herein for use in the treatment of breast cancer.

[0362] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation described herein for use in the treatment of testicular cancer.

[0363] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation described herein for use in the treatment of bladder cancer.

[0364] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation described herein for use in the treatment of pancreatic cancer.

[0365] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation described herein for use in the treatment of ovarian cancer.

[0366] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation described herein for use in the treatment of non-small cell lung cancer.

[0367] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation described herein for use in the treatment of metastatic breast cancer.

[0368] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation described herein for use in the first line treatment of metastatic breast cancer.

[0369] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation as described herein for use in the first line treatment of metastatic breast cancer after failed adjuvant chemotherapy.

[0370] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation as described herein for use in the first line treatment of patients with inoperable, locally advanced (Stage IIIA or IIIB) or metastatic (Stage IV) non-small cell lung cancer.

[0371] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation as described herein for use in the treatment of locally advanced (unresectable stage II or stage III) or metastatic (stage IV) pancreatic adenocarcinoma.

[0372] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation as described herein for use in the treatment of locally advanced or metastatic bladder cancer.

[0373] In one embodiment, there is provided a pharmaceutical composition comprising a nucleoside formulation as described herein for use in the treatment of locally advanced or metastatic epithelial ovarian cancer.

[0374] Treatment method In one embodiment, a method of intravenous injection is provided, comprising administering a nucleoside formulation described herein.

[0375] In one embodiment, a method of treating a viral infection is provided comprising administering to said animal an effective amount of a nucleoside formulation described herein.

[0376] In one embodiment, a method of treating HIV, Ebola, Marburg, coronavirus, Hepatitis B, Hepatitis C, Vaccinia, Epstein-Barr, Cytomegalovirus, Zika, Herpes simplex, and / or Varicella-Zoster virus infection is provided comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0377] In one embodiment, a method of treating cancer in a warm-blooded animal, such as a human, is provided comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0378] In one embodiment, a method is provided for treating breast cancer, bladder cancer, testicular cancer, pancreatic cancer, ovarian cancer, or non-small cell lung cancer in a warm-blooded animal, such as a human, comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0379] In one embodiment, a method of treating breast cancer in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0380] In one embodiment, a method of treating testicular cancer in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0381] In one embodiment, a method of treating bladder cancer in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0382] In one embodiment, a method of treating pancreatic cancer in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0383] In one embodiment, a method of treating ovarian cancer in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0384] In one embodiment, a method of treating non-small cell lung cancer in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0385] In one embodiment, a method of treating metastatic breast cancer in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0386] In one embodiment, a method of first line treatment of metastatic breast cancer in a warm-blooded animal, e.g., a human, is provided, comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0387] In one embodiment, a method is provided for first-line treatment of metastatic breast cancer in a warm-blooded animal, e.g., a human, following unsuccessful adjuvant chemotherapy, comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0388] In one embodiment, a method is provided for first-line treatment of inoperable, locally advanced (stage IIIA or IIIB) or metastatic (stage IV) non-small cell lung cancer in a warm-blooded animal, such as a human, comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0389] In one embodiment, a method is provided for treating locally advanced (unresectable stage II or stage III) or metastatic (stage IV) pancreatic adenocarcinoma in a warm-blooded animal, such as a human, comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0390] In one embodiment, a method of treating locally advanced or metastatic bladder cancer in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0391] In one embodiment, a method of treating locally advanced or metastatic epithelial ovarian cancer in a warm-blooded animal, such as a human, is provided, comprising administering to the animal an effective amount of a nucleoside formulation described herein.

[0392] Use of nucleoside preparations In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for intravenous injection.

[0393] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the treatment of a viral infection.

[0394] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the treatment of HIV, Ebola, Marburg, coronavirus, Hepatitis B, Hepatitis C, Vaccinia, Epstein-Barr, Cytomegalovirus, Zika, Herpes simplex, and / or Varicella-Zoster virus infection.

[0395] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the treatment of cancer.

[0396] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the treatment of breast cancer, bladder cancer, testicular cancer, pancreatic cancer, ovarian cancer, or non-small cell lung cancer.

[0397] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the treatment of breast cancer.

[0398] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the treatment of testicular cancer.

[0399] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the treatment of bladder cancer.

[0400] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the treatment of pancreatic cancer.

[0401] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the treatment of ovarian cancer.

[0402] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the treatment of non-small cell lung cancer.

[0403] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the treatment of metastatic breast cancer.

[0404] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the first line treatment of metastatic breast cancer.

[0405] In one embodiment, there is provided a use of a nucleoside formulation as described herein for the manufacture of a medicament for the first line treatment of metastatic breast cancer after unsuccessful adjuvant chemotherapy.

[0406] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the first line treatment of patients with inoperable, locally advanced (Stage IIIA or IIIB) or metastatic (Stage IV) non-small cell lung cancer.

[0407] In one embodiment, there is provided a use of a nucleoside formulation as described herein for the manufacture of a medicament for the treatment of locally advanced (unresectable stage II or stage III) or metastatic (stage IV) pancreatic adenocarcinoma.

[0408] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the treatment of locally advanced or metastatic bladder cancer.

[0409] In one embodiment, there is provided a use of a nucleoside formulation described herein for the manufacture of a medicament for the treatment of locally advanced or metastatic epithelial ovarian cancer.

[0410] combination In one embodiment, the nucleoside formulations described herein as a first active ingredient can be used in combination with a second active ingredient, such as a second anti-cancer medicament. Particularly suitable second active ingredients can be platinum compounds, such as carboplatin or cisplatin.

[0411] As used herein, when the term "combination" is used, it is understood that this term refers to simultaneous, separate, or sequential administration. In one embodiment, "combination" refers to simultaneous administration. In one embodiment, "combination" refers to separate administration. In one embodiment, "combination" refers to sequential administration. When administration is sequential or separate administration, a delay in administration of the second component should not result in the beneficial effect of the combination being lost.

[0412] In one embodiment, there is provided a nucleoside formulation as described herein in combination with a second active ingredient.

[0413] In one embodiment, there is provided a nucleoside formulation as described herein in combination with a second active ingredient for use to obtain an anti-cancer effect.

[0414] In one embodiment, there is provided a nucleoside formulation as described herein in combination with a second active ingredient for use in the treatment of breast cancer, bladder cancer, testicular cancer, pancreatic cancer, ovarian cancer, or non-small cell lung cancer.

[0415] kit In one embodiment, a) a nucleoside formulation as described herein; b) container means for containing said nucleoside formulation; A kit is provided comprising:

[0416] In one embodiment, a) a nucleoside formulation as described herein; b) a container means for containing said nucleoside formulation, and optionally c) Instructions for use A kit is provided comprising:

[0417] In one embodiment, a kit is provided that includes a nucleoside formulation described herein in combination with a second active ingredient.

[0418] In one embodiment, a) a nucleoside formulation as described herein in a first unit dosage form; b) a second active ingredient in a second unit dosage form, and c) container means for containing said first and second dosage forms. A kit is provided comprising:

[0419] [Example] General Experiment In the Examples section the following applies: - RALA refers to the peptide WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1), obtained in lyophilized powder form from Biomatik, Canada. - dFdCTP refers to gemcitabine triphosphate obtained from Biorbyt®, UK - dCTP refers to deoxycytidine triphosphate obtained from Biorbyt®, UK. - Gemcitabine refers to gemcitabine hydrochloride obtained from Fluorochem®, UK. - BxPC-3 cells and PANC-1 cells were obtained from ATCC, USA. - MR means molar ratio. The "MR" numbers cited in the examples and figures refer to the number of moles of amphipathic cell membrane-permeable peptide:1 mole of nucleoside or its phosphate, for example, MR2.0 refers to 2 moles of amphipathic cell membrane-permeable peptide per mole of nucleoside or its phosphate.

[0420] [Example 1] Preparation of RALA peptide RALA was reconstituted with molecular grade water to the desired concentration, aliquoted, and stored at -20°C until further use. Aliquots were taken as needed and thawed on ice. Aliquots were not refrozen once thawed.

[0421] [Example 2] Nanoparticle Formulation 2.1: Manual preparation RALA / dFdCTP nanoparticles (final concentration 0.02 mg / mL) were formulated at various RALA:dFdCTP molar ratios by first adding the required volume of ultrapure water to 10 μg of dFdCTP at a concentration of 1 mg / mL in solution to a final formulation volume of 500 μL. The corresponding volume (Table 1) of peptide solution at a concentration of 10 mg / mL was added to the diluted dFdCTP solution. The mixture was pipetted up and down approximately 5-10 times to ensure homogenous mixing. Nanoparticles formed spontaneously in the solution.

[0422] [Table 1]

[0423] 2.2: Automated formulation RALA / dFdCTP nanoparticles (final concentration 0.02 mg / mL) were formulated at various RALA:dFdCTP molar ratios by using an automated microfluidic system (e.g., Precision Nanosystems Ignite NanoAssemblr). The two solutions (Table 2) with appropriate concentrations were loaded into syringes, which were then loaded into the microfluidic system. Nanoparticles were produced using a total flow rate (TFR) of 10 mL / min and a flow rate ratio (FRR) of 1:1. The resulting solution from the system contained nanoparticles at a concentration of 0.02 mg / mL of dFdCTP.

[0424] [Table 2]

[0425] [Example 3] Freeze-drying of RALA / dFdCTP nanoparticles 500 μL of RALA / dFdCTP nanoparticles were transferred to a 2 mL lyophilization vial. Trehalose (20% w / v) was added to the nanoparticle solution in the vial such that the volume added was equal to half of the final reconstitution volume, ensuring that the resulting solution contained 10% w / v trehalose once reconstituted in water after lyophilization. After adding the trehalose, a rubber lyophilization vial stopper was partially placed on the vial so that the airflow notch still promoted sublimation during the lyophilization process. The vial was then loaded into a programmable lyophilizer (e.g., SP Scientific AdVantage Pro) following the lyophilization procedure.

[0426] [Table 3]

[0427] [Example 4] Size and charge analysis of nanoparticles Z-average particle size measurements and polydispersity (PdI) of RALA / dFdCTP nanoparticles were obtained using Dynamic Light Scattering (DLS) to obtain particle size and charge distribution. Surface charge measurements of RALA nanoparticles were determined by Laser Doppler Velocimetry. Zeta potential of particles was measured using disposable collapsible zeta cuvettes. Zeta cuvettes for zeta potential measurements were first washed with 70% ethanol and then rinsed twice with double distilled H2O before loading the samples. 50 μL of undiluted sample was used for size measurements, then diluted to 1 mL with ultrapure water, and then 700-800 μL of diluted sample was used for zeta potential determination. Nanoparticles were made in a wide range of molar ratios (MR1.4, MR2.0-3.0) using at least 1 μg of dFdCTP in each sample. The nanoparticles were analyzed on a Zetasizer-Nano-ZS (Malvern Instruments) with DTS software (Malvern Instruments, UK) and the results are shown in FIG.

[0428] [Table 4]

[0429] Results show that a molar ratio of ≥2.0 promotes the formation of nanoparticles that meet critical quality attributes (Z-average <150 nm, Zeta potential >+10 mV, PdI <0.500). Similarly, the physiochemical characteristics of these particles can be further enhanced (Z-average <100 nm, Zeta potential >+10 mV, PdI <0.300) using an automated microfluidic mixer.

[0430] [Example 5] Cell cycle analysis 1.5×10 5BxPC-3 cells were seeded in 6-well plates (Nunc, UK) and left to attach for 24 hours. Cells were transferred to serum-free medium for 24 hours to ensure cell synchronization. Cells were then incubated with RALA / dFdCTP nanoparticles at a concentration of 2 μM (1 μg) for 5 hours, after which the treatment medium was removed and fresh complete medium was added. After 48 hours of treatment, cells were trypsinized and fixed in ice-cold 70% ethanol for 1 hour at 4°C. Cells were washed twice for 5 minutes in phosphate-buffered saline (PBS) before being resuspended in 500 μL PBS and 50 μL 100 μg / ml RNase (Invitrogen, UK) was added. 10 minutes before samples were subjected to fluorescence-activated cell sorting (FACS), 10 μl of 100 μg / ml propidium iodide (PI) stock was added. Controls included untreated, dCTP (negative control), RALA / dCTP (RALA control), dFdCTP (drug control), and gemcitabine (comparison control).

[0431] Analysis of cell cycle after RALA / dFdCTP treatment was performed. If dFdCTP is successfully delivered to cells, it will be incorporated during DNA polymerization and prevent DNA strand elongation. This ultimately prevents active proliferation of cells. This means that during the cell cycle, the cycle is arrested in the S phase. Therefore, if the percentage of cells in (G0 / G1+-S) phase is higher compared to G2 phase, this is an indication that dFdCTP has affected the cell cycle. The results are shown in Figure 2 and Table 5.

[0432] [Table 5]

[0433] [Example 6] Clonogenic assay 1×10 5(BxPC-3 and PANC-1) cells were treated with 8 / 16 / 32 nM gemcitabine, dFdCTP, or manually formulated nanoparticles of RALA / dFdCTP at various molar ratios. Five hours after transfection, cells were trypsinized, counted, and re-seeded at low density (300 and 600 cells / well for BxPC-3, 400 and 800 cells / well for PANC-1). Colonies were allowed to form for 14 days and then stained with crystal violet and counted. The results are shown in Figure 3.

[0434] [Example 7] Functionality of RALA / dFdCTP (γH2AX) To test the functionality of the RALA / dFdCTP nanoparticles, transfection of BxPC-3 (gemcitabine-sensitive) or PANC-1 (gemcitabine-resistant) cells was performed. First, 1.5 × 10 5 The cells were seeded in 6-well microplates (Nunc, UK) and left to attach for 24 hours. The cells were then cultured using EC 50Concentrations (pre-determined via clonogenic assay) of manually formulated RALA / dFdCTP (MR2.0), dFdCTP, or gemcitabine hydrochloride were incubated for 5 h, after which the transfection medium was removed and complete cell culture medium was added. Two and 24 h after transfection, cells were trypsinized and fixed in 4% formaldehyde (Sigma, UK), then permeabilized overnight in methanol at -20°C. Cells were blocked in PBS containing 10% fetal bovine serum (FBS). Samples were incubated in primary γH2AX (phosphS139) antibody (1:100 dilution in PBS containing 10% FBS) (Abcam, UK) for 2 h. Cells were then washed for 5 min in PBS and then incubated with a secondary γH2AX antibody tagged with fluorescein isothiocyanate (FITC) (Abcam, UK) (diluted 1:50 in PBS containing 10% FBS) for 1 h at room temperature. Washing was repeated. The percentage of γH2AX expressing cells was measured using a FACS Calibur (BD Biosciences, UK) and analyzed using FlowJo analysis software. The results are shown in Figure 4.

[0435] [Example 8] Overcoming gemcitabine resistance using RALA / dFdCTP 1.5×10 5 The gemcitabine-sensitive cell line BxPC-3 was seeded in 6-well microplates (Nunc, UK) and left to attach for 24 hours. A subset of cells was then treated with 10 μM dipyridamole (Sigma, UK), an inhibitor of nucleotide transport channels (hENT / hCNT), followed by EC 50 Concentrations (previously determined via clonogenic assays) of manually formulated RALA / dFdCTP (MR2.0), dFdCTP, or gemcitabine hydrochloride were transfected for 5 hours. Subsequent determination of functionality was performed by examining the levels of γH2AX after transfection via the procedure outlined in Example 7. The results are shown in FIG.

[0436] [Example 9] In vivo efficacy Twenty-four female athymic (nude) mice were weighed and, under anesthesia (isoflurane), implanted subcutaneously in the right flank with 2 × 106 PANC-1 cells in a volume of 100 μl PBS. At the start of treatment, tumors were established and had a size of 100–150 mm 3 The tumors had to reach a volume of 600 mm3. Once the tumors reached the required volume, mice were then treated with a single, 100 μl total volume, 1 mg / kg intravenous dose of either 10% trehalose (vehicle control), RALA / dFdCTP (MR2.0), dFdCTP, or gemcitabine (n=6 per treatment arm). Tumor volume and mouse weight were then monitored and recorded three times per week. Mice were sacrificed once the tumors reached the endpoint (specified as a volume of 600 mm3). The results of this study are shown in FIG. 6.

[0437] [Example 10] Pharmacokinetic and safety profile C57BL / 6 mice (n=7 per treatment arm) were administered a single iv injection (40 μg) of gemcitabine, dFdCTP, or RALA / dFdCTP (MR2.0). Mice were bled at various time points and plasma was extracted for analysis. dFdCTP or gemcitabine content in the collected plasma was measured via mass spectrometry. Terminal cardiac blood sampling was performed at endpoint and serum was extracted for characterization of safety profile via multiplex ELISA and standard ELISA for nephrotoxicity (creatinine), hepatotoxicity (AST, ALT), and immunogenicity markers (TNF-α, IFN-γ, and MIP-2). Results are shown in Figures 7 and 8.

[0438] [Example 11] Hemolysis assay Defibriminated whole sheep blood (5 ml, TCS Biosciences Ltd, UK) was centrifuged at 500 g for 20 min in citrate-phosphate buffer (15 ml, 0.1 M citric acid, C6H8O7, and 0.2 M disodium hydrogen phosphate Na2HPO4) to separate red blood cells. The red blood cells were then washed three times with 20 mL of citrate-phosphate buffer by centrifugation and resuspended in citrate-phosphate buffer solution at a concentration of 1 x 108 cells / mL. Red blood cells were counted using a Countess II automated cell counter (ThermoFisher, UK) by first diluting the red blood cell suspension 1:10000 in buffer. Then, an isotonic solution (osmolarity range 290-310 mOsmol / kg) of RALA / dFdCTP (MR2.0) nanoparticles mixed with trehalose or with dFdCTP and trehalose was added to the erythrocyte suspension, the added dFdCTP amounting to 10-30 μg, and the solution was incubated at 37 °C for 1 h. After incubation, the erythrocyte suspension was centrifuged at 400 g for 90 s to form a cell pellet. The absorbance of the supernatant was measured at 541 nm via a Nanodrop 2000c UV-Vis spectrophotometer (Thermo Scientific, UK). 1% Triton X-100 was used as a positive control corresponding to 100% hemolysis, and citrate-phosphate buffer at pH 7.4 was used as a negative control. The percentage of hemolytic activity was calculated using formula 1 and the results are shown in Figure 9.

[0439]

number

[0440] [Example 12] charge density The charge density of the RALA / dFdCTP(MR2.0) nanoparticles was measured by polyelectrolyte titration using the method described in Ritz et al., Biomacromolecules. 2015 Apr 13, 16(4):1311-21. doi: 10.1021 / acs.biomac.5b00108. Epub 2015 Apr 3, and Weiss et al., J Nanobiotechnology. 2021 Jan 6, 19(1):5. doi: 10.1186 / s12951-020-00747-7. The polyelectrolyte titration was performed using poly(acrylic acid) (PAA) 0.01 M, pH 7.4. While measuring the charge, PAA was added to the RALA / dFdCTP(MR2.0) nanoparticles to create a sigmoidal curve whose volume (V) could be that of the equivalence point. The mass of peptide in the system (w) and the concentration of PAA used (0.01 M) were used to determine the average charge density of the nanoparticles (Q ek The results are shown in Figure 10. Qek=V×c / w V = volume of titrant = 46.33 μl c = concentration of titrant - 0.01M w = mass of RALA = 0.4625 mg Qek = 46.33 × (0.01 / 0.4625) Qek=1.002

[0441] statement Statement 1. (i) a nucleoside or a phosphate thereof, and (ii) Amino acid sequence: WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology An amphipathic cell membrane penetrating peptide comprising or consisting of 13. A nucleoside formulation comprising:

[0442] Statement 2. 2. The nucleoside formulation of statement 1, wherein the nucleoside or phosphate thereof is selected from didanosine, vidarabine, galidesivir, remdesivir, cytarabine, gemcitabine, emtricitabine, lamivudine, zalcitabine, uyabacavir, acyclovir, entecavir, stavudine, telbivudine, zidovudine, idoxuridine, or trifluridine, or a phosphate thereof.

[0443] Statement 3. 3. The nucleoside formulation of statement 1 or 2, wherein the nucleoside or phosphate thereof is gemcitabine or a phosphate thereof.

[0444] Statement 4. The nucleoside formulation of any one of the preceding statements, wherein the nucleoside or phosphate thereof is gemcitabine triphosphate.

[0445] Statement 5. The nucleoside formulation of any one of the preceding statements, wherein the phosphate comprises a P(O)(OH)2O- group.

[0446] Statement 6. The amphipathic cell membrane penetrating peptide has the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) 4. The nucleoside formulation of any one of the preceding statements, comprising:

[0447] Statement 7. The amphipathic cell membrane penetrating peptide has the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) 20. The nucleoside formulation of any one of the preceding statements, consisting of:

[0448] Statement 8. A nucleoside formulation according to any one of the preceding statements, wherein the molar ratio of nucleoside or its phosphate:amphiphilic cell membrane-penetrating peptide is 1:1-3.

[0449] Statement 9. A nucleoside formulation according to any one of the preceding statements, wherein the molar ratio of nucleoside or its phosphate:amphiphilic cell membrane penetrating peptide is about 1:2.

[0450] Statement 10. The nucleoside formulation of any one of the preceding statements, further comprising a bulking agent.

[0451] Statement 11. 11. The nucleoside formulation of statement 10, wherein the filler comprises trehalose, sucrose, mannose, and / or dextrose.

[0452] Statement 12. The nucleoside formulation of any one of the preceding statements, which is a nanoparticle formulation.

[0453] Statement 13. A method for preparing a nanoparticle formulation according to statement 12, comprising the step of formulating (i) a solution of WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or sequence homology with (ii) a nucleoside or a phosphate thereof in an automated controlled mixing system, in particular an automated microfluidic system.

[0454] Statement 14. 13. A nucleoside formulation according to any one of statements 1 to 12 for use as a pharmaceutical.

[0455] Statement 15. 13. A nucleoside formulation according to any one of statements 1 to 12 for use in the treatment of a viral infection.

[0456] Statement 16. 13. A pharmaceutical composition comprising a nucleoside formulation according to any one of statements 1 to 12 for use in the treatment of cancer.

[0457] Statement 17 A method of treating breast cancer, bladder cancer, testicular cancer, pancreatic cancer, ovarian cancer, or non-small cell lung cancer in a warm-blooded animal, such as a human, comprising administering to the animal an effective amount of a nucleoside formulation described in any one of statements 1-12.

[0458] Statement 18. 13. Use of a nucleoside formulation according to any one of statements 1 to 12 for the manufacture of a medicament for the treatment of HIV, Ebola, Marburg, coronavirus, hepatitis B, hepatitis C, vaccinia, Epstein-Barr, cytomegalovirus, Zika, herpes simplex, and / or varicella zoster virus infection.

Claims

1. (i) gemcitabine or its phosphate, and (ii) Amino acid sequence: WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or an amphipathic cell membrane penetrating peptide comprising or consisting of a sequence having at least 80% sequence identity or sequence homology thereto.

13. A nucleoside formulation comprising:

2. Phosphate is P(O)(OH) 2 2. The nucleoside formulation of claim 1, comprising an O-group.

3. 3. The nucleoside formulation of claim 1 or 2, wherein the gemcitabine or phosphate thereof is gemcitabine triphosphate.

4. The amphipathic cell membrane penetrating peptide has the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) The nucleoside formulation of any one of claims 1 to 3, comprising:

5. The amphipathic cell membrane penetrating peptide has the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) The nucleoside formulation according to any one of claims 1 to 4, comprising:

6. The nucleoside formulation according to any one of claims 1 to 5, wherein the molar ratio of gemcitabine or its phosphate to amphipathic cell membrane-permeable peptide is 1:1-3.

7. The nucleoside formulation according to any one of claims 1 to 6, wherein the molar ratio of gemcitabine or its phosphate to the amphipathic cell membrane-permeable peptide is about 1:

2.

8. 8. The nucleoside formulation of claim 1, further comprising trehalose, sucrose, mannose, and / or dextrose.

9. The nucleoside formulation of any one of claims 1 to 8, further comprising trehalose.

10. (i) 0.1 to 1% w / w of gemcitabine or its phosphate; (ii) 1-10% w / w of an amphipathic cell membrane penetrating peptide comprising or consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology thereto; and (iii) 89-99% w / w of trehalose, sucrose, mannose, and / or dextrose The nucleoside formulation of any one of claims 1 to 9, comprising:

11. The nucleoside formulation of any one of claims 8 to 10, formulated as an isotonic formulation in water.

12. (i) 0.2 to 0.5% w / w of gemcitabine triphosphate; (ii) 3.5 to 4.5% w / w of an amphipathic cell membrane penetrating peptide consisting of the amino acid sequence WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1); (iii) 95-97% w / w trehalose and formulated as an isotonic preparation in water having an osmolality of 290-310 mOsmol / kg.

13. The nucleoside formulation of any one of claims 1 to 12, which is a nanoparticle formulation.

14. 14. The nucleoside formulation of claim 13, wherein the nanoparticles have an average surface charge density of ≦2 μmol / mg at 20° C.

15. 15. A method for preparing a nucleoside formulation according to any one of claims 1 to 14, comprising formulating a solution of (i) WEARLARALARALARHLARALARALRACEA (SEQ ID NO: 1) or a sequence having at least 80% sequence identity or homology with (ii) gemcitabine or a phosphate thereof in an automated controlled mixing system, in particular an automated microfluidic system.

16. A nucleoside formulation according to any one of claims 1 to 14 for use as a medicament.

17. A nucleoside formulation according to any one of claims 1 to 14 for use in intravenous injection.

18. A pharmaceutical composition comprising a nucleoside formulation according to any one of claims 1 to 14 for use in the treatment of cancer.

19. 15. A method of treating breast cancer, bladder cancer, testicular cancer, pancreatic cancer, ovarian cancer, or non-small cell lung cancer in a warm-blooded animal, such as a human, comprising administering to the animal an effective amount of a nucleoside formulation of any one of claims 1 to 14.

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