Copper Complexes Increase the Efficacy In vitro of Tecovirimat against Pox Viruses

Copper Complexes Increase the Efficacy In vitro of Tecovirimat against Pox Viruses | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Copper Complexes Increase the Efficacy In vitro of Tecovirimat against Pox Viruses Thomas Manning, Capri Persaud, Akshil Patel, Madelyn Adair, Morgan Wynn, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6196055/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The anti-viral medication Tecovirimat (TPOXX) has in vitro efficacy against poxviruses. In this study, there are several formulations tested in vitro that involve Cu(II) complexes as potential excipients. While the individual components have little or no efficacy, when some are combined with TPOXX, they improve the SI 50 values. The medicinal agents were tested against Vaccinia virus (Copenhagen, Resistant Isolate) and Cowpox virus (Brighton, CPXVR/Resistant Isolate). The poxviruses have become of great concern worldwide because of their potential to be bioterrorism agents (1). Nuclear Magnetic Resonance is utilized to determine structural components of complexes. Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry at 21 Tesla (ESI-21 T FT-ICR MS) identifies elemental compositions that correlate to proposed molecular structures in the aqueous phase. Other work is cited utlizing Cu(II) in which it improved the efficacy of antibiotics (for Tb) and cancers (9 types) in in vitro studies. Figures Figure 1 Figure 2 Figure 3 Introduction The variola virus, a member of the Poxviridae family, and the genus Orthopoxvirus , is believed to have impacted humanity for at least 3,000 years ( 2 , 3 ). In 1796, British physician Edward Jenner was the first to demonstrate a vaccine for smallpox by utilizing the immunity generated by the cowpox virus. The variola virus is responsible for the infectious disease, smallpox. The spread of the disease worldwide was correlated with increasing exploration and trade. The transmission, mortality rate (30%), and the disfigurement that marked the survivors made it a dreaded disease throughout human history. In the 1950s, smallpox was eradicated in Europe and the United States, followed by South America (1971), Asia (1975), and finally Africa (1977). The WHO declared the disease eradicated in May of 1980 ( 4 ). Smallpox has again attracted the attention of the scientific community and governments worldwide, with concerns centering on the use of the virus as a bioterrorism agent. Other front-line infectious diseases of concern include Anthrax, Botulism, the Plague, Tularemia, Filoviruses (Ebola, Marburg), and Arenaviruses (5.6,7). The smallpox vaccine is derived from the vaccinia virus. Once inside the patient, the virus begins to multiply, which triggers an immune response. With new genetic technology, it is a reasonable assumption that previous vaccines would not be effective against new strains of genetically engineered viruses ( 8 , 9 ). Cowpox played a significant role in medical history, as it was used to build up immunity to the more lethal infection caused by the vaccinia virus. The Cowpox Brighton strain virus is responsible for bovine vaccinia. Unlike Smallpox and Monkeypox (Mpox), Cowpox is not considered a bioterrorism threat ( 10 ). Orthopoxvirus is a genus of viruses that can infect humans, mammals, and arthropods. There are twelve species in this genus, but only four infect humans: variola, Mpox, vaccinia, and Cowpox. Orthopoxviruses are enveloped, large, brick shaped viruses that have a double stranded DNA genome, composed of 186,000 base pairs. They can infect vertebrate and invertebrate species and replicate in the cytoplasm. There are several variations of smallpox including Ordinary Smallpox (> 80% of cases), Modified-type Smallpox (strikes vaccinated people), Flat-type Smallpox (harsher symptoms than ordinary smallpox, occurs in children), and hemorrhagic Smallpox (strikes pregnant women, high fatality rate) ( 11 ). Three primary medications (Tecovirimat (TPOXX), Cidofovir, and Brincidofovir) have been approved for smallpox. TPOXX’s mechanism of action (MOA) involves inhibiting the VP37 envelope protein, preventing cellular transmission. Cidofovir was awarded FDA (Food and Drug Administration) approval in 1996 and has demonstrated activity in vitro against several orthopox infections, including MPox, Molluscipox, Camelpox, vaccinia, and variola ( 12 , 13 ). Brincidofovir is a pro-drug in which the C 16 acts like an endogenous lysophosphatidyl choline and can enter infected cells. Brincidofovir was approved for the treatment of the Ebola virus and was administered to the first patient infected during the 2014 U.S. Ebola breakout ( 14 ). Some fatty acids have anti-viral properties ( 15 – 19 ). Decanoic acid (CH₃(CH₂)₈COOH) has activity against African swine fever virus, vesicular stomatitis virus, herpes simplex virus (HSV), human immunodeficiency virus, respiratory syncytial virus, Visna virus (VV), Cytomegalovirus (CMV), Epstein-Barr virus, Herpes Simplex Virus, influenza virus, leukemia virus, pneumono virus, and Hepatitis C. Docosahexaenoic acid (DHA, C 22 H 32 O 2 ) is an omega-3 fatty acid and has activity against HIV and SARS-CoV-2. Sucrose is composed of glucose and fructose. Since the reducing groups of glucose and fructose are involved in glycosidic bond formation, sucrose is a non-reducing sugar ( 20 ). Copper, in the form of Cu(0), Cu(I) and Cu(II) can undergo oxidation-reduction reactions (Eq. 1–5). They can generate reactive oxidation species (ROS), such as the hydroxyl radical (HO • ), triplet oxygen (O 2 2• ), superoxide anion (O 2 •− ), peroxide ion (O 2 − 2 ), hydrogen peroxide (H 2 O 2 ), nitric oxide (NO • ), singlet oxygen ( 1 O 2 ), alpha-oxygen, peroxyl (RO 2 *), alkoxyl (RO*), hydroperoxyl (HO 2 *), and ozone (O 3 ) ( 21 ). Copper (II) oxide, which can form from Cu(II) in an aqueous environment, generates several ROS. ROS generated by copper can result in damage to cellular DNA, proteins, and lipids ( 22 – 23 ). The reactants, along with the copper species, can be ubiquitous species, such as H 2 O, H + , OH - , or in biological processes, such as respiration and photosynthesis. Cu 2 O(s) + H 2 O + 2 e − ⇌ 2Cu(s) + 2 OH − -0.36 V ( 1 ) Cu (NH 3 ) +2 + e − ⇌ Cu(s) + 2NH 3 (aq) -0.1 V ( 2 ) Cu 2+ + e − ⇌ Cu + 0.159 V ( 3 ) Cu 2+ + 2 e − ⇌ Cu(s) 0.337 V ( 4 ) Cu + + e− ⇌ Cu(s) 0.52 V ( 5 ) Excipients used in TPOXX include silica, colloidal anhydrous croscarmellose sodium, hypromellose, lactose monohydrate, magnesium stearate, cellulose, and sodium laurilsulfate ( 24 ). These components do not provide medicinal activity for TPOXX. Naturally occurring proteins in the body that contain copper include: 1. Ceruloplasmin is responsible for transporting up to 90% of the copper in the blood, 2. Metallothioneins are active in copper storage, 3. Copper chaperone proteins active in copper transportation, 4. Copper-transporting ATPases are active in transporting copper across cell membranes, and 5. Cytochrome c oxidase is an enzyme used in the respiration of mitochondria. When copper is bound to proteins, it can have a reduction potential between + 0.3 V to + 0.7 V ( 25 ). Wilson disease is the result of high copper levels, and Menkes disease is the result of an irregular (high and low levels) copper distribution throughout the body ( 26 ). The treatment of low copper levels is the administration of Cu(II). Copper tablets have dosages that range from one to four milligrams, which represents a starting point for the possible use of Cu(II) in medications. High dosages of copper can result in abdominal pain, diarrhea, nausea, vomiting and liver damage. The excretion route for copper is via biliary excretion. Copper levels in the body are; free serum copper: 10–15 µg/dL, total copper: 63.7–140.12 µg/dL, serum ceruloplasmin: 18–35 µg/dL, 24-hour urine copper: 20–50 µg/dL, and liver copper: 20–50 µg/g in tissue ( 27 – 29 ). The Irving–William's series describes the strong bond that Cu(II) forms with an amine group ( 30 ). The Cu(II)-amine bond is significantly stronger than Mn(II), Fe(II), Co(II), Ni(II), Zn(II) and Ca(II) bonds to the same functional group. The Cu(II) cation and Cu(II) in the Cu(II)-sucrose complex can bind the amines in TPOXX. Experimental Nuclear Magnetic Resonance is used to measure the interaction between the Cu(II) and sucrose and TPOXX. Cu(II) is paramagnetic and can broaden and shift spectral features. Zn(II), which has similarities in radii and charge to Cu(II) but is diamagnetic, is utilized to confirm an interaction with sucrose via small shifts in spectral line positions. FT-ICR provides high resolution mass/charge values for molecular species. For example, benzene (C 6 H 6 + ) is routinely assigned an m/z value of 78 Da, but FT-ICR could assign a value of 78.04640 Da for the charged species (composed of 12 C and 1 H isotopes). The various isotopic variations that utilize 13 C and 2 H would have a similar level of accuracy and precision. In vitro measurements were conducted at the University of Alabama Birmingham, under the authority of the National Institutes of Health (NAID). Results and Discussion ESI-21 T FT-ICR MS was applied to several samples, including TPOXX, Cu(II)-sucrose, Cu(II)-TPOXX, and Cu(II)-sucrose-TPOXX. The results in Table 1 should be considered representative of structures suggested from the data and not a complete list. FT-ICR allowed for over 4,700 unique masses to be identified for the Cu(II)-sucrose-TPOXX formulation. Because of the isotopes for elements (i.e. 12 C vs. 13 C; 1 H vs 2 H; 63 Cu vs. 65 Cu; 14 N vs. 15 N; and 16 O, 17 O, 18 O), as well as isotopes associated with the spectator ions (i.e. 35 Cl vs. 37 Cl, S in SO 4 − 2 32 S, 33 S, 34 S and 36 S), provides the reason for the high number of detectable species. Signal to Noise Ratios (SNR) are provided in Table 1 . The key point in the data analysis is that the Cu(II)-TPOXX, Cu(II)-sucrose, and the Cu(II)-sucrose-TPOXX complexes were detected in the formulations. Sucrose never appeared as an individual molecule (C 12 H 22 O 11 + ) but was detected linked to cations (i.e. Na + , Cu + 2 ), in D 2 O. In the case of the Cu(II)-TPOXX, Cu(II)-sucrose-TPOXX, and the Cu(II)-sucrose formulations, the paramagnetic effect of the Cu(II) cation distorted the spectra, resulting in broadening of the spectral features but little shift in the line positions. Figure 2 a is the pure sucrose 1 H NMR spectra. Figure 2 b is the Cu(II)-sucrose in the spectra, which is significantly distorted indicating the paramagnetic Cu(II) is 10–20 Å from the disaccharide's hydrogen nuclei. Figure 3 a, b provides the 13 C spectra for sucrose and the Cu(II)-sucrose formulation. Figure 4 is a plot of the 13 C spectral features of sucrose verses the 13 C line position for the Cu(II)-sucrose complex. It demonstrates there is no shift in the 13 C spectral positions when the disaccharide is bound to Cu(II). Considering the results for the FT-ICR data with the NMR solution data, it indicates Cu(II) interacts with sucrose in the aqueous phase but does not form a metal-ligand complex, characterized by covalent bonds, and that Cu(II) can interact with sucrose and TPOXX simultaneously. Additional NMR spectra and the FT-ICR data tables are provided in appendices. Table 1 The 21 T FT-ICR results for some complexes identified by exact mass/charge ratios. Molecular Species Empirical formula Calculated FT-ICR Error (ppm) Experiment (data file) 1 sucrose C 12 H 22 O 11 + 342.115662 - - a Cu(II)-Sucrose 2 Sucrose-Na C 12 H 22 O 11 Na 1 + 365.105432 365.105435 + 0.00759 Cu(II)-Sucrose 3 Sucrose-Cu (Cu-63) C 12 H 22 O 11 Cu 1 + 405.04525 405.045262 + 0.06556 Cu(II)-Sucrose 4 Tecovirimat (TPOXX) C 19 H 15 F 3 N 2 O 3 + 376.102928 376.102937 -0.088431 TPOXX 5 TPOXX-Na NaC 19 H 15 F 3 N 2 O 3 + 399.09269 399.092703 0.032574 TPOXX 1 6 TPOXX-Na NaC 19 H 15 F 3 N 2 O 3 + 399.09269 399.092706 0.040091 Cu-TPOXX 2 7 TPOXX-Na NaC 19 H 15 F 3 N 2 O 3 + 399.09269 399.092704 -0.00250 Cu(II)-Sucrose-TPOXX 3 8 TPOXX-Na − H NaC 19 H 15 F 3 N 2 O 3 H + 400.09606 400.096063 0.007498 Cu(II)-Sucrose-TPOXX 9 Cl-TPOXX-Na NaC 19 H 15 F 3 N 2 O 3 Cl + 399.09269 399.092706 0.04009 Cu(II)-TPOXX 10 TPOXX-Cu-Sucrose-H CuC 31 H 36 O 14 N 2 F 3 H + 780.1409118 780.140937 0.032302 Cu(II)-Sucrose-TPOXX 4 11 Cu(II)-TPOXX-Cl CuC 19 H 15 F 3 N 2 O 3 Cl + 474.00137 474.001389 0.040084 Cu(II)-TPOXX 12 Cu(II)-(TPOXX) 2 Cu 1 C 38 H 30 O 6 N 4 F 6 + 814.128177 814.128177 0.000491 Cu(II)-TPOXX-Sucrose 13 (TPOXX) 2 -Na C 38 H 30 F 6 N 4 O 6 Na + 775.19617 775.196201 0.038700 Cu(II)-Sucrose-TPOXX 5 Cu(II) was introduced as copper (II) sulphate salt (strong electrolyte) Signal-to-Noise ratio at: 127,433.631 Signal to Noise ratio at: 63,840.664 Signal-to-Noise ratio at: 99,522.220 Signal to Noise ratio at: 179.787 Signal to Noise ratio at: 28,083.329 Copper metal, Cu(II) and Cu(I) can have a toxicity above certain concentrations ( 31 ). The LD 50 for copper in humans depend on the form; copper flakes, 300–500 mg/kg; CuO, > 2500 mg/kg; copper (II) sulfate, 481 mg/kg; copper in human peripheral blood mononuclear cells, 115 µM; and copper dusts and mists, 100 mg Cu/m 3 . For humans, the naturally occurring copper concentration in serum is between 70 and140 micrograms per 100 mls of blood. In a clinical study that examined patients with MPOX and HIV treated with TPOXX, the blood concentration of TPOXX ranged from 1455 ng/mL (healthy patients) to 891 ng/mL (HIV patients). TPOXX was measured between 77.3% and 82.2% bound to human plasma proteins ( 32 ), which suggests that the Cu(II)-sucrose-TPOXX formulation could be delivered using a protein molecule as a carrier. In vitro evaluations utilized CPXVR(SF) (Cowpox Resistant Isolate) and CDVR-1A (Cidofovir Resistant Isolate) strains (Table 2 , 3 , 4 , 5 ) ( 33 , 34 ). With an SI 50 value of 10 or higher considered moderately active, only TPOXX (SI 50 = 12) and Cu(II)-sucrose-TPOXX (SI 50 = 26) formulations achieved this level. When Cu(II) is formulated with just a fatty acid or sucrose, it has little efficacy against viruses (Table 2 , 4 , 5 ). The Cu(II)-TPOXX formulation had no efficacy (SI 50 = 1; Table 2 ), which suggests the Cu(II) may have (i) interfered with the ability of TPOXX to bind and inhibit the activity of VP37, which prevents the virus from interacting with cellular proteins, or (ii) disrupted the TPOXX structure. The effective Cu (II)-sucrose-TPOXX formulation may (i) increase the delivery efficiency of TPOXX because sucrose provides an energy source when it enters the cells’ cytoplasm, or (ii) impact the envelope formation that is needed for the virus to leave the host and infect other cells. The in vitro data for two complexes against the Cowpox strain (CPXVR (SF)) followed the same trend as the Brighton strain (Table 4 , 5 ). The Cu(II)-Sucrose-TPOXX complex has a higher efficacy (SI 50 value) compared to TPOXX, and has higher values compared to the two controls: Cidofovir (SI 50 = 1) and N-Methanocarbamate (SI 50 = 9). Table 2 In vitro data for the Brighton Strain of Cowpox against thirteen formulations. TPOXX, two fatty acids, the Cu(II) cation, and Sucrose are tested independently and in formulations. The medicinal agent Cidofovir is used as a control (SI 50 26 2. TPOXX > 12 3. Decanoic acid < 4 4. Cu(II)-TPOXX-Decanoate < 4 5. Cu(II) < 3 6. Cu(II)-Sucrose < 3 7. DHA < 3 8. Cu(II)-DHA < 2 9. Cu(II)-TPOXX-DHA- Decanoate -Sucrose < 2 10. Cu(II)-TPOXX-DHA < 2 11. Cu(II)-Decanoate 1 12. Sucrose 1 13. Cu(II)-TPOXX 1 Table 3 In vitro data for two formulations against the Cowpox strain (CPXVR(SF)); Resistant Isolate. Compound Name SI 50 1. Cu(II)-Sucrose-TPOXX > 131 2. TPOXX > 79 Table 4 In vitro data for several complexes used against the Copenhagen strain of the vaccinia virus. The control drug is Cidofovir (SI 50 > 36). Compound Name SI 50 1. Cu(II)-sucrose-TPOXX > 2128 2. TPOXX-Cu(II)-sucrose-Cu(II)-DHA-Cu(II)-Decanoate > 1539 3. Cu(II)-TPOXX-DHA > 1483 4. Cu(II)-TPOXX-Decanoate 1043 5. TPOXX > 1031 6. Cu(II)-TPOXX > 1000 7. Cu(II) < 3 8. Cu(II)-sucrose < 3 9. DHA < 3 10. Cu (II)-DHA < 2 11. Cu(II)-Decanoate 1 12. Decanoic acid 1 13. sucrose 1 Table 5 In vitro data for several formulations used against the CDVR-1A (Resistant Isolate) strain of the Vaccinia virus. The control medications are Cidofovir (SI50 > 2) and N-methanocarbamate (SI50 > 128). Compound Name SI 50 1. Cu(II)-Sucrose-TPOXX > 248 1. TPOXX > 180 2. Cu(II)-TPOXX-Decanoate 162 3. Cu(II)-TPOXX > 150 4. Cu(II)-TPOXX-Decanoate-DHA-Sucrose 120 5. Cu(II)-TPOXX-DHA 100 The vaccinia virus (Tables 4 , 5 ) in vitro datum followed the same trend as the Cowpox datum. The Cu(II)-sucrose-TPOXX complex has the highest efficacy of the tested formulations, and formulating Cu(II) with TPOXX lowered its efficacy. The four TPOXX complexes that contain sucrose, decanoate, DHA, and both DHA and decanoate all have higher SI 50 values than pure TPOXX. When cellular replication takes place, the cell can consume sucrose, decanoate, and DHA as nutrients and building blocks, which could result in the TPOXX entering the cell more efficiently (Trojan Horse effect). The virulent vaccinia strain Rabbitpox virus has infected rabbits in research facilities in New York and Utrecht. Another vaccina strain, Buffalopox, had outbreaks in India where buffaloes are used for their milk ( 35 – 37 ). Cowpox virus replication occurs in the cytoplasm of a cell and is like the replication process of the vaccinia virus in some ways but is different in others. An example of their differences is that Cowpox has a significant amount of guanylate kinase homolog (CPXV-GRI A59) in its mature virion, while the vaccinia virus has significant quantities of the A-type inclusion protein (CPXV-GRI A27) in its fully formed and functioning virus particles. An example of their similarities is the cowpox virus and vaccinia viruses can leave the cell during its lyses as a non-enveloped particle or as an enveloped particle. Non-enveloped particles leave the cell when the cell membrane lyses, while enveloped particles leave when the virus fuses with the Golgi apparatus and outer cell membranes. Conclusion The diffusion rate of free Cu(II) is significantly lower than sucrose when penetrating a cell membrane. Most biological membranes are designed to allow a small, neutral molecule, such as sucrose, to easily penetrate it ( 38 ). A charged species, such as Cu(II), typically requires a specific transport protein to penetrate the membrane ( 39 ). Free Cu(II) can randomly bind proteins, specifically amines ( 40 ), due to the Irving-Williams effect and minimize its ability to penetrate a cell membrane. Cu(II) can undergo an electrostatic attraction to the oxygen atoms in sucrose, the carboxylates on the fatty acids, and the amines, oxygen atoms, and \(\:\pi\:\) bonds in TPOXX. At room temperature, the hydrolysis of sucrose has a ΔG rxn of -26 kJ/mol, a spontaneous reaction ( 41 ). Factors, such as reactant concentration, pH, and the presence of a catalysis, can dissociate sucrose into D-Glucose and L-Fructose. The bond enthalpy 163 kJ/mol for the N-N single bond in TPOXX is low and can easily dissociate (for reference; N-H, 386 kJ/mol; N = N 418 kJ/mol; N-Cl, 313 kJ/mol; N = O 607 kJ/mol). The Irving-Williams series indicates the Cu(II)-amine bond is strong, and this attraction can further weaken the N-N bond. Cu(I) and Cu(II) can form oxides (i.e. CuO − 1 , CuO, Cu 2 O) in the aqueous phase at physiological pH (7.38). These oxides have efficacy against cancer cells, bacterium, and viral infections ( 42 – 45 ). Declarations Author Contribution The data was obtained from the National High Field Magnet Lab (NMR, FT-ICR) and the National Institutes of Health (Infectious Disease, in vitro data).Capri Persaud, Morgan Wynn, Akshil Patel, Madelyn, Adair and Thomas Manning synthesized the compounds used in this work. They also interpreted the NMR and FT-ICR data. Amy McKenna and Joseph Fry obtained the FT-ICR data used in this paper. They also provided expertise in purifying and preparing samples for analysis for FT-ICR and NMR studies. All co-authors contributed to the interpreation of different data sets, writing and proof reading the paper. Acknowledgements: Valdosta State University has utilized the non-clinical and preclinical services program offered by the National Institute of Allergy and Infectious Diseases under Contract No. 75N93019D00016/75N93023F00001, PI (Principal Investigator) Scott James, MD. We would like to thank the National Institute of Health, NIAID, and Ms. Amanda Ulloa, for their support of the in vitro testing conducted. A portion of this work was performed at the Ion Cyclotron Resonance user facility at the National High Magnetic Field Laboratory at Florida State University, which is supported by the National Science Foundation Division of Chemistry and Division of Materials Research through NSF DMR 2128556, and the State of Florida. We would like to thank Dr. James Rocca of the MBI, Magnet Lab (University of Florida), AMRIS, NSF, State of Florida for working with obtaining the NMR spectra. 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Emerg Infect Dis 29(12):2426. 10.3201/eid2912.231146 Krankowska DC, Woźniak PA, Cybula A, Izdebska J, Suchacz M, Samelska K, Wiercińska-Drapało A, Szaflik JP (2021) Cowpox: How dangerous could it be for humans? Case report. Int J Infect Dis 104:239–241. 10.1016/j.ijid.2020.12.061 Bruneau C, Tazi L, Rothenburg S (2023) Cowpox Viruses: A Zoo Full of Viral Diversity and Lurking Threats. Biomolecules 13(2):325. 10.3390/biom13020325 Adams MM, Rice AD, Moyer RW (2007) Rabbitpox virus and vaccinia virus infection of rabbits as a model for human smallpox. J Virol 81(20):11084–11095 Roy CJ, Voss TG (2010) Use of the Aerosol Rabbitpox Virus Model for Evaluation of Anti-Poxvirus Agents. Viruses 2(9):2096–2107. 10.3390/v2092096 Roy P, Chandramohan A (2021) Buffalopox Disease in Livestock and Milkers, India. Emerg Infect Dis 27(7):1989–1991. 10.3201/eid2707.202111 Ribeiro A, Esteso M, Lobo V, Valente A, Simoes S, Sobral A, Burrows H (2007) Interactions of copper (II) chloride with sucrose, glucose, and fructose in aqueous solutions. J Mol Struct 826:113–119. 10.1016/j.molstruc.2006.04.035 Ohrvik H, Thiele DJ (2014) How copper traverses' cellular membranes through the mammalian copper transporter 1, Ctr1. Ann N Y Acad Sci 1314:32–41. 10.1111/nyas.12371 Posadas Y, Sánchez-López C, Quintanar L (2023) Copper binding and protein aggregation: a journey from the brain to the human lens. RSC Chem Biol 4(12):974–985. 10.1039/d3cb00145h Tombari E, Salvetti G, Ferrari C, Johari GP (2007) Kinetics and thermodynamics of sucrose hydrolysis from real-time enthalpy and heat capacity measurements. J Phys Chem B 111(3):496–501. 10.1021/jp067061p Govind V, Bharadwaj S, Sai Ganesh MR, Vishnu J, Shankar KV, Shankar B, Rajesh R (2021) Antiviral properties of copper and its alloys to inactivate covid-19 virus: a review. Biometals 34(6):1217–1235. 10.1007/s10534-021-00339-4 Tavakoli A, Hashemzadeh MS (2020) Inhibition of herpes simplex virus type 1 by copper oxide nanoparticles. J Virol Methods 275:113688 Fahmy HM, Ebrahim M, Gaber H (2020) In-vitro evaluation of copper/copper oxide nanoparticles cytotoxicity and genotoxicity in normal and cancer lung cell lines. J Trace Elem Med Biol 60:126481. 10.1016/j.jtemb.2020.126481 Yang D, Klebl D, Zeng S, Sobott F, Prévost M, Soumillion P, Vandenbussche G, Fontaine V (2020) Interplays between copper and Mycobacterium tuberculosis GroEL1. Metallomics 12(8):1267–1277. 10.1039/d0mt00101e Manning T, Slaton C, Myers N, Patel P, Arrington D, Patel Z, Phillips D, Wylie G, Goddard R (2018) A Copper 10 -Paclitaxel Crystal; a Medicinally Active Drug Delivery Platform. Bioorg Med Chem Lett 28(20):3409–3417. 10.1016/j.bmcl.2018.08.019 Manning T, Plummer S, Woods R, Wylie G, Phillips D, Krajewski L (2017) Cell line studies and analytical measurements of three paclitaxel complex variations. Bioorg Med Chem Lett 27(12):2793–2799. 10.1016/j.bmcl.2017.04.070 Manning T, Phillips D, Wylie G, Bythell B, Clark S, Ogburn R, Ledwitch K, Collis C, Patterson S, Lasseter L (2014) Copper ion as a delivery platform for taxanes and taxane complexes. Bioorg Med Chem Lett 24(1):371–377. 10.1016/j.bmcl.2013.10.073 Manning T, Mikula R, Lee H, Calvin A, Darrah J, Wylie G, Phillips D, Bythell BJ (2014) The copper (II) ion as a carrier for the antibiotic capreomycin against Mycobacterium tuberculosis . Bioorg Med Chem Lett 24(3):976–982 Manning T, Plummer S, Baker T, Wylie G, Clingenpeel AC, Phillips D (2015) Development of a three-component complex to increase isoniazid efficacy against isoniazid resistant and nonresistant Mycobacterium tuberculosis . Bioorg Med Chem Lett 25(20):4621–4627. 10.1016/j.bmcl.2013.12.053 Manning TJ, Wilkerson K, Holder T, Bartley AC, Jackson C, Plummer S, Phillips D, Krajewski L, Wylie G (2017) Pharmacokinetic studies of a three-component complex that repurposes the front-line antibiotic isoniazid against Mycobacterium tuberculosis . Tuberculosis (Edinb) 107:149–155. 10.1016/j.tube.2017.08.011 Technical notes The variables SI 50 and SI 90 are used in this paper (and in appendix B). EC 50 = Compound concentration that reduces viral replication by 50%. Provided in μM concentration units. EC 90 = Compound concentration that reduces viral replication by 90%; Provided in μM concentration units. CC 50 = Compound concentration that reduces cell viability by 50%; provided μM units. SI 50 = Selectivity index is calculated by (CC 50 / EC 50 ) . SI 90 = Selectivity index calculated by (CC 90 / EC 90 ) . Appendices included: NIH NSAID In vitro data (original data): CC 50 , EC 50 and SI 90 values for all data sets. (not available with this version) NMR data for TPOXX, Cu-TPOXX, sucrose, Cu-sucrose, sucrose-Cu-TPOXX (1 and 2 D); 1 H, 13 C, 15 N (images, processed by Topspin) (not available with this version) FT-ICR data for TPOXX, Cu(II)-TPOXX, sucrose, Cu(II)-sucrose, Cu(II)-sucrose-TPOXX formulations. (.txt files) Additional Declarations No competing interests reported. Supplementary Files NIHManningPOXdataCopy.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6196055","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":430464838,"identity":"2455c0e9-e940-4a1e-b659-333a074b0bba","order_by":0,"name":"Thomas Manning","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYNACAwYGfgYGxgM8UL4EPsU8MC2SDQwMIC0SRGoB6TpArBZ7scPPHhcU3LHbfCP5wYE3FXV1/A3MB2/z4NHCI51mbjzD4FnythtpBgfnnDksIXGALdkav5YEM2keg8PJZmcOGBzmbTsgAXQeUASvlvRvYC3GPcc/ALXUScgf4P9GQEsO2BY7A/YekC3MEgYHeNjwa7mdUwbSkiBxvKcA5BfJjYfZjC3n4NHCPjt9mzTPn8P2/M3sGx8AQ4xf7njzwxtv8GiBgcQGOJOZCOUgYE+kulEwCkbBKBiJAAAw1UocYgwsHwAAAABJRU5ErkJggg==","orcid":"","institution":"Valdosta State University","correspondingAuthor":true,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Manning","suffix":""},{"id":430464839,"identity":"27f919c8-7e07-4bfe-9f7f-5a2be8b036d8","order_by":1,"name":"Capri Persaud","email":"","orcid":"","institution":"Valdosta State University","correspondingAuthor":false,"prefix":"","firstName":"Capri","middleName":"","lastName":"Persaud","suffix":""},{"id":430464840,"identity":"7eb40b5d-db56-44a2-8d57-19470882ea33","order_by":2,"name":"Akshil Patel","email":"","orcid":"","institution":"Valdosta State University","correspondingAuthor":false,"prefix":"","firstName":"Akshil","middleName":"","lastName":"Patel","suffix":""},{"id":430464841,"identity":"bb9ed20d-032e-4258-8462-bd787aec169f","order_by":3,"name":"Madelyn Adair","email":"","orcid":"","institution":"Valdosta State University","correspondingAuthor":false,"prefix":"","firstName":"Madelyn","middleName":"","lastName":"Adair","suffix":""},{"id":430464842,"identity":"338e70e7-c0d7-455f-a056-ae834ea45d06","order_by":4,"name":"Morgan Wynn","email":"","orcid":"","institution":"Valdosta State University","correspondingAuthor":false,"prefix":"","firstName":"Morgan","middleName":"","lastName":"Wynn","suffix":""},{"id":430464843,"identity":"c0a65dcf-86bd-413f-8be8-04fc2ed2753c","order_by":5,"name":"Joseph Frye-Jones","email":"","orcid":"","institution":"National High Magnetic Field Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Joseph","middleName":"","lastName":"Frye-Jones","suffix":""},{"id":430464844,"identity":"459df5f9-0be6-438a-abf6-4eb35ec0e09f","order_by":6,"name":"Amy M. McKenna","email":"","orcid":"","institution":"National High Magnetic Field Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Amy","middleName":"M.","lastName":"McKenna","suffix":""}],"badges":[],"createdAt":"2025-03-10 13:38:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6196055/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6196055/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79067243,"identity":"e4feeeee-fca7-4ced-b15e-2ba8cbd7245c","added_by":"auto","created_at":"2025-03-24 04:49:01","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":294928,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 2. \u0026nbsp;(top) The \u003csup\u003e1\u003c/sup\u003eH NMR of sucrose, and (bottom) \u003csup\u003e1\u003c/sup\u003eH NMR of Cu(II)-sucrose formulation, both in D\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6196055/v1/1228583f3c793ea4330be2da.jpeg"},{"id":79067250,"identity":"32a50d53-c8fb-49eb-a930-ebabc843126b","added_by":"auto","created_at":"2025-03-24 04:49:02","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":408873,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 3. (top) The \u003csup\u003e13\u003c/sup\u003eC NMR of Sucrose and (bottom) \u003csup\u003e13\u003c/sup\u003eC NMR of Cu(II)-sucrose formulation, both in D\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6196055/v1/900def285a360b99b9cb8f36.jpeg"},{"id":79067246,"identity":"5286d670-e164-4856-b6df-14ca43ec616e","added_by":"auto","created_at":"2025-03-24 04:49:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":30808,"visible":true,"origin":"","legend":"\u003cp\u003eFigure 4. A plot of the \u003csup\u003e13\u003c/sup\u003eC line positions for the sucrose (x axis) and the Cu(II)-sucrose formulation (y axis).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6196055/v1/f27b7f8b1273c25516655f17.png"},{"id":80341624,"identity":"8056c053-bc79-4b36-898f-586b581fd91d","added_by":"auto","created_at":"2025-04-10 18:01:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1437819,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6196055/v1/9a6ac5ff-6ba9-4e13-ab29-2fa757d60465.pdf"},{"id":79067239,"identity":"e1cfbb54-be1b-4a81-9128-842ee75ed853","added_by":"auto","created_at":"2025-03-24 04:49:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":544342,"visible":true,"origin":"","legend":"","description":"","filename":"NIHManningPOXdataCopy.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6196055/v1/925285ce8cebff2c42a07efc.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eCopper Complexes Increase the Efficacy In vitro of Tecovirimat against Pox Viruses \u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe variola virus, a member of the \u003cem\u003ePoxviridae\u003c/em\u003e family, and the genus \u003cem\u003eOrthopoxvirus\u003c/em\u003e, is believed to have impacted humanity for at least 3,000 years (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). In 1796, British physician Edward Jenner was the first to demonstrate a vaccine for smallpox by utilizing the immunity generated by the cowpox virus. The variola virus is responsible for the infectious disease, smallpox. The spread of the disease worldwide was correlated with increasing exploration and trade. The transmission, mortality rate (30%), and the disfigurement that marked the survivors made it a dreaded disease throughout human history. In the 1950s, smallpox was eradicated in Europe and the United States, followed by South America (1971), Asia (1975), and finally Africa (1977). The WHO declared the disease eradicated in May of 1980 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Smallpox has again attracted the attention of the scientific community and governments worldwide, with concerns centering on the use of the virus as a bioterrorism agent. Other front-line infectious diseases of concern include Anthrax, Botulism, the Plague, Tularemia, Filoviruses (Ebola, Marburg), and Arenaviruses (5.6,7).\u003c/p\u003e \u003cp\u003eThe smallpox vaccine is derived from the vaccinia virus. Once inside the patient, the virus begins to multiply, which triggers an immune response. With new genetic technology, it is a reasonable assumption that previous vaccines would not be effective against new strains of genetically engineered viruses (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Cowpox played a significant role in medical history, as it was used to build up immunity to the more lethal infection caused by the vaccinia virus. The Cowpox Brighton strain virus is responsible for bovine vaccinia. Unlike Smallpox and Monkeypox (Mpox), Cowpox is not considered a bioterrorism threat (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOrthopoxvirus is a genus of viruses that can infect humans, mammals, and arthropods. There are twelve species in this genus, but only four infect humans: variola, Mpox, vaccinia, and Cowpox. Orthopoxviruses are enveloped, large, brick shaped viruses that have a double stranded DNA genome, composed of 186,000 base pairs. They can infect vertebrate and invertebrate species and replicate in the cytoplasm. There are several variations of smallpox including Ordinary Smallpox (\u0026gt;\u0026thinsp;80% of cases), Modified-type Smallpox (strikes vaccinated people), Flat-type Smallpox (harsher symptoms than ordinary smallpox, occurs in children), and hemorrhagic Smallpox (strikes pregnant women, high fatality rate) (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThree primary medications (Tecovirimat (TPOXX), Cidofovir, and Brincidofovir) have been approved for smallpox. TPOXX\u0026rsquo;s mechanism of action (MOA) involves inhibiting the VP37 envelope protein, preventing cellular transmission. Cidofovir was awarded FDA (Food and Drug Administration) approval in 1996 and has demonstrated activity \u003cem\u003ein vitro\u003c/em\u003e against several orthopox infections, including MPox, Molluscipox, Camelpox, vaccinia, and variola (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Brincidofovir is a pro-drug in which the C\u003csub\u003e16\u003c/sub\u003e acts like an endogenous lysophosphatidyl choline and can enter infected cells. Brincidofovir was approved for the treatment of the Ebola virus and was administered to the first patient infected during the 2014 U.S. Ebola breakout (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSome fatty acids have anti-viral properties (\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Decanoic acid (CH₃(CH₂)₈COOH) has activity against African swine fever virus, vesicular stomatitis virus, herpes simplex virus (HSV), human immunodeficiency virus, respiratory syncytial virus, Visna virus (VV), Cytomegalovirus (CMV), Epstein-Barr virus, Herpes Simplex Virus, influenza virus, leukemia virus, pneumono virus, and Hepatitis C. Docosahexaenoic acid (DHA, C\u003csub\u003e22\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) is an omega-3 fatty acid and has activity against HIV and SARS-CoV-2.\u003c/p\u003e \u003cp\u003eSucrose is composed of glucose and fructose. Since the reducing groups of glucose and fructose are involved in glycosidic bond formation, sucrose is a non-reducing sugar (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Copper, in the form of Cu(0), Cu(I) and Cu(II) can undergo oxidation-reduction reactions (Eq.\u0026nbsp;1\u0026ndash;5). They can generate reactive oxidation species (ROS), such as the hydroxyl radical (HO\u003csup\u003e\u0026bull;\u003c/sup\u003e), triplet oxygen (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e2\u0026bull;\u003c/sup\u003e), superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e), peroxide ion (O\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e), hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), nitric oxide (NO\u003csup\u003e\u0026bull;\u003c/sup\u003e), singlet oxygen (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e ), alpha-oxygen, peroxyl (RO\u003csub\u003e2\u003c/sub\u003e*), alkoxyl (RO*), hydroperoxyl (HO\u003csub\u003e2\u003c/sub\u003e*), and ozone (O\u003csub\u003e3\u003c/sub\u003e) (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Copper (II) oxide, which can form from Cu(II) in an aqueous environment, generates several ROS. ROS generated by copper can result in damage to cellular DNA, proteins, and lipids (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The reactants, along with the copper species, can be ubiquitous species, such as H\u003csub\u003e2\u003c/sub\u003eO, H\u003csup\u003e+\u003c/sup\u003e, OH\u003csup\u003e-\u003c/sup\u003e, or in biological processes, such as respiration and photosynthesis.\u003c/p\u003e \u003cp\u003eCu\u003csub\u003e2\u003c/sub\u003eO(s)\u0026thinsp;+\u0026thinsp;H\u003csub\u003e2\u003c/sub\u003eO\u0026thinsp;+\u0026thinsp;2 e\u003csup\u003e\u0026minus;\u003c/sup\u003e ⇌ 2Cu(s)\u0026thinsp;+\u0026thinsp;2 OH\u003csup\u003e\u0026minus;\u003c/sup\u003e -0.36 V (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCu (NH\u003csub\u003e3\u003c/sub\u003e)\u003csup\u003e+2\u003c/sup\u003e + e\u003csup\u003e\u0026minus;\u003c/sup\u003e ⇌ Cu(s)\u0026thinsp;+\u0026thinsp;2NH\u003csub\u003e3\u003c/sub\u003e(aq) -0.1 V (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e + e\u003csup\u003e\u0026minus;\u003c/sup\u003e ⇌ Cu\u003csup\u003e+\u003c/sup\u003e 0.159 V (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eCu\u003csup\u003e2+\u003c/sup\u003e + 2 e\u003csup\u003e\u0026minus;\u003c/sup\u003e ⇌ Cu(s) 0.337 V (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eCu\u003csup\u003e+\u003c/sup\u003e + e\u0026minus; ⇌ Cu(s) 0.52 V (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eExcipients used in TPOXX include silica, colloidal anhydrous croscarmellose sodium, hypromellose, lactose monohydrate, magnesium stearate, cellulose, and sodium laurilsulfate (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). These components do not provide medicinal activity for TPOXX. Naturally occurring proteins in the body that contain copper include: 1. Ceruloplasmin is responsible for transporting up to 90% of the copper in the blood, 2. Metallothioneins are active in copper storage, 3. Copper chaperone proteins active in copper transportation, 4. Copper-transporting ATPases are active in transporting copper across cell membranes, and 5. Cytochrome c oxidase is an enzyme used in the respiration of mitochondria. When copper is bound to proteins, it can have a reduction potential between +\u0026thinsp;0.3 V to +\u0026thinsp;0.7 V (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWilson disease is the result of high copper levels, and Menkes disease is the result of an irregular (high and low levels) copper distribution throughout the body (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). The treatment of low copper levels is the administration of Cu(II). Copper tablets have dosages that range from one to four milligrams, which represents a starting point for the possible use of Cu(II) in medications. High dosages of copper can result in abdominal pain, diarrhea, nausea, vomiting and liver damage. The excretion route for copper is via biliary excretion. Copper levels in the body are; free serum copper: 10\u0026ndash;15 \u0026micro;g/dL, total copper: 63.7\u0026ndash;140.12 \u0026micro;g/dL, serum ceruloplasmin: 18\u0026ndash;35 \u0026micro;g/dL, 24-hour urine copper: 20\u0026ndash;50 \u0026micro;g/dL, and liver copper: 20\u0026ndash;50 \u0026micro;g/g in tissue (\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Irving\u0026ndash;William's series describes the strong bond that Cu(II) forms with an amine group (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). The Cu(II)-amine bond is significantly stronger than Mn(II), Fe(II), Co(II), Ni(II), Zn(II) and Ca(II) bonds to the same functional group. The Cu(II) cation and Cu(II) in the Cu(II)-sucrose complex can bind the amines in TPOXX.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cp\u003eNuclear Magnetic Resonance is used to measure the interaction between the Cu(II) and sucrose and TPOXX. Cu(II) is paramagnetic and can broaden and shift spectral features. Zn(II), which has similarities in radii and charge to Cu(II) but is diamagnetic, is utilized to confirm an interaction with sucrose via small shifts in spectral line positions. FT-ICR provides high resolution mass/charge values for molecular species. For example, benzene (C\u003csub\u003e6\u003c/sub\u003eH\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) is routinely assigned an m/z value of 78 Da, but FT-ICR could assign a value of 78.04640 Da for the charged species (composed of \u003csup\u003e12\u003c/sup\u003eC and \u003csup\u003e1\u003c/sup\u003eH isotopes). The various isotopic variations that utilize \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e2\u003c/sup\u003eH would have a similar level of accuracy and precision. \u003cem\u003eIn vitro\u003c/em\u003e measurements were conducted at the University of Alabama Birmingham, under the authority of the National Institutes of Health (NAID).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eESI-21 T FT-ICR MS was applied to several samples, including TPOXX, Cu(II)-sucrose, Cu(II)-TPOXX, and Cu(II)-sucrose-TPOXX. The results in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e should be considered representative of structures suggested from the data and not a complete list. FT-ICR allowed for over 4,700 unique masses to be identified for the Cu(II)-sucrose-TPOXX formulation. Because of the isotopes for elements (i.e. \u003csup\u003e12\u003c/sup\u003eC vs. \u003csup\u003e13\u003c/sup\u003eC; \u003csup\u003e1\u003c/sup\u003eH vs \u003csup\u003e2\u003c/sup\u003eH; \u003csup\u003e63\u003c/sup\u003eCu vs. \u003csup\u003e65\u003c/sup\u003eCu; \u003csup\u003e14\u003c/sup\u003eN vs.\u003csup\u003e15\u003c/sup\u003eN; and \u003csup\u003e16\u003c/sup\u003eO, \u003csup\u003e17\u003c/sup\u003eO, \u003csup\u003e18\u003c/sup\u003eO), as well as isotopes associated with the spectator ions (i.e. \u003csup\u003e35\u003c/sup\u003eCl vs. \u003csup\u003e37\u003c/sup\u003eCl, S in SO\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;\u003csup\u003e\u0026minus;\u0026thinsp;2 32\u003c/sup\u003eS, \u003csup\u003e33\u003c/sup\u003eS, \u003csup\u003e34\u003c/sup\u003eS and \u003csup\u003e36\u003c/sup\u003eS), provides the reason for the high number of detectable species. Signal to Noise Ratios (SNR) are provided in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The key point in the data analysis is that the Cu(II)-TPOXX, Cu(II)-sucrose, and the Cu(II)-sucrose-TPOXX complexes were detected in the formulations. Sucrose never appeared as an individual molecule (C\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e) but was detected linked to cations (i.e. Na\u003csup\u003e+\u003c/sup\u003e, Cu\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e), in D\u003csub\u003e2\u003c/sub\u003eO.\u003c/p\u003e \u003cp\u003eIn the case of the Cu(II)-TPOXX, Cu(II)-sucrose-TPOXX, and the Cu(II)-sucrose formulations, the paramagnetic effect of the Cu(II) cation distorted the spectra, resulting in broadening of the spectral features but little shift in the line positions. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003ea is the pure sucrose \u003csup\u003e1\u003c/sup\u003eH NMR spectra. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb is the Cu(II)-sucrose in the spectra, which is significantly distorted indicating the paramagnetic Cu(II) is 10\u0026ndash;20 \u0026Aring; from the disaccharide's hydrogen nuclei. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b provides the \u003csup\u003e13\u003c/sup\u003eC spectra for sucrose and the Cu(II)-sucrose formulation. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e is a plot of the \u003csup\u003e13\u003c/sup\u003eC spectral features of sucrose verses the \u003csup\u003e13\u003c/sup\u003eC line position for the Cu(II)-sucrose complex. It demonstrates there is no shift in the \u003csup\u003e13\u003c/sup\u003eC spectral positions when the disaccharide is bound to Cu(II). Considering the results for the FT-ICR data with the NMR solution data, it indicates Cu(II) interacts with sucrose in the aqueous phase but does not form a metal-ligand complex, characterized by covalent bonds, and that Cu(II) can interact with sucrose and TPOXX simultaneously. Additional NMR spectra and the FT-ICR data tables are provided in appendices.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe 21 T FT-ICR results for some complexes identified by exact mass/charge ratios.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMolecular Species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEmpirical formula\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCalculated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFT-ICR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eError (ppm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eExperiment (data file)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003esucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e342.115662\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003csup\u003ea\u003c/sup\u003eCu(II)-Sucrose\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSucrose-Na\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003eNa\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e365.105432\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e365.105435\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u0026thinsp;0.00759\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCu(II)-Sucrose\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSucrose-Cu\u003c/p\u003e \u003cp\u003e(Cu-63)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e12\u003c/sub\u003eH\u003csub\u003e22\u003c/sub\u003eO\u003csub\u003e11\u003c/sub\u003eCu\u003csub\u003e1\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e405.04525\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e405.045262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e+\u0026thinsp;0.06556\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCu(II)-Sucrose\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTecovirimat (TPOXX)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e376.102928\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e376.102937\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.088431\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTPOXX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTPOXX-Na\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNaC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e399.09269\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e399.092703\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.032574\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eTPOXX\u003csup\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTPOXX-Na\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNaC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e399.09269\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e399.092706\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.040091\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCu-TPOXX\u003csup\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTPOXX-Na\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNaC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e399.09269\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e399.092704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-0.00250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCu(II)-Sucrose-TPOXX\u003csup\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTPOXX-Na\u003csup\u003e\u0026minus;\u003c/sup\u003eH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNaC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eH\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e400.09606\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e400.096063\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.007498\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCu(II)-Sucrose-TPOXX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCl-TPOXX-Na\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNaC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eCl\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e399.09269\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e399.092706\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.04009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCu(II)-TPOXX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTPOXX-Cu-Sucrose-H\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCuC\u003csub\u003e31\u003c/sub\u003eH\u003csub\u003e36\u003c/sub\u003eO\u003csub\u003e14\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003eH\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e780.1409118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e780.140937\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.032302\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCu(II)-Sucrose-TPOXX\u003csup\u003e\u003cb\u003e4\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCu(II)-TPOXX-Cl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCuC\u003csub\u003e19\u003c/sub\u003eH\u003csub\u003e15\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eCl\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e474.00137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e474.001389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.040084\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCu(II)-TPOXX\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCu(II)-(TPOXX)\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCu\u003csub\u003e1\u003c/sub\u003eC\u003csub\u003e38\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eF\u003csub\u003e6\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e814.128177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e814.128177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.000491\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCu(II)-TPOXX-Sucrose\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(TPOXX)\u003csub\u003e2\u003c/sub\u003e-Na\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eC\u003csub\u003e38\u003c/sub\u003eH\u003csub\u003e30\u003c/sub\u003eF\u003csub\u003e6\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e6\u003c/sub\u003eNa\u003csup\u003e+\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e775.19617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e775.196201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.038700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCu(II)-Sucrose-TPOXX\u003csup\u003e\u003cb\u003e5\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003col style=\"list-style-type:lower-alpha;\"\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eCu(II) was introduced as copper (II) sulphate salt (strong electrolyte)\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eSignal-to-Noise ratio at: 127,433.631\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eSignal to Noise ratio at: 63,840.664\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eSignal-to-Noise ratio at: 99,522.220\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eSignal to Noise ratio at: 179.787\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eSignal to Noise ratio at: 28,083.329\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003eCopper metal, Cu(II) and Cu(I) can have a toxicity above certain concentrations (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). The LD\u003csub\u003e50\u003c/sub\u003e for copper in humans depend on the form; copper flakes, 300\u0026ndash;500 mg/kg; CuO, \u0026gt;\u0026thinsp;2500 mg/kg; copper (II) sulfate, 481 mg/kg; copper in human peripheral blood mononuclear cells, 115 \u0026micro;M; and copper dusts and mists, 100 mg Cu/m\u003csup\u003e3\u003c/sup\u003e. For humans, the naturally occurring copper concentration in serum is between 70 and140 micrograms per 100 mls of blood. In a clinical study that examined patients with MPOX and HIV treated with TPOXX, the blood concentration of TPOXX ranged from 1455 ng/mL (healthy patients) to 891 ng/mL (HIV patients). TPOXX was measured between 77.3% and 82.2% bound to human plasma proteins (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), which suggests that the Cu(II)-sucrose-TPOXX formulation could be delivered using a protein molecule as a carrier.\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e evaluations utilized CPXVR(SF) (Cowpox Resistant Isolate) and CDVR-1A (Cidofovir Resistant Isolate) strains (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e,\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e,\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e,\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). With an SI\u003csub\u003e50\u003c/sub\u003e value of 10 or higher considered moderately active, only TPOXX (SI\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;12) and Cu(II)-sucrose-TPOXX (SI\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;26) formulations achieved this level. When Cu(II) is formulated with just a fatty acid or sucrose, it has little efficacy against viruses (Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e,\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e,\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The Cu(II)-TPOXX formulation had no efficacy (SI\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1; Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), which suggests the Cu(II) may have (i) interfered with the ability of TPOXX to bind and inhibit the activity of VP37, which prevents the virus from interacting with cellular proteins, or (ii) disrupted the TPOXX structure. The effective Cu (II)-sucrose-TPOXX formulation may (i) increase the delivery efficiency of TPOXX because sucrose provides an energy source when it enters the cells\u0026rsquo; cytoplasm, or (ii) impact the envelope formation that is needed for the virus to leave the host and infect other cells. The \u003cem\u003ein vitro\u003c/em\u003e data for two complexes against the Cowpox strain (CPXVR (SF)) followed the same trend as the Brighton strain (Table \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e,\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The Cu(II)-Sucrose-TPOXX complex has a higher efficacy (SI\u003csub\u003e50\u003c/sub\u003e value) compared to TPOXX, and has higher values compared to the two controls: Cidofovir (SI\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1) and N-Methanocarbamate (SI\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;9).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e data for the Brighton Strain of Cowpox against thirteen formulations. TPOXX, two fatty acids, the Cu(II) cation, and Sucrose are tested independently and in formulations. The medicinal agent Cidofovir is used as a control (SI\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;9).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSI\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1. Cu(II)-Sucrose-TPOXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;26\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2. TPOXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3. Decanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4. Cu(II)-TPOXX-Decanoate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5. Cu(II)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6. Cu(II)-Sucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7. DHA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8. Cu(II)-DHA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9. Cu(II)-TPOXX-DHA- Decanoate -Sucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10. Cu(II)-TPOXX-DHA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11. Cu(II)-Decanoate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12. Sucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13. Cu(II)-TPOXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e data for two formulations against the Cowpox strain (CPXVR(SF)); Resistant Isolate.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSI\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1. Cu(II)-Sucrose-TPOXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;131\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2. TPOXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e data for several complexes used against the Copenhagen strain of the vaccinia virus. The control drug is Cidofovir (SI\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;36).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSI\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1. Cu(II)-sucrose-TPOXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;2128\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2. TPOXX-Cu(II)-sucrose-Cu(II)-DHA-Cu(II)-Decanoate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;1539\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3. Cu(II)-TPOXX-DHA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;1483\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4. Cu(II)-TPOXX-Decanoate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1043\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5. TPOXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;1031\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6. Cu(II)-TPOXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;1000\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7. Cu(II)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e8. Cu(II)-sucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e9. DHA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10. Cu (II)-DHA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e11. Cu(II)-Decanoate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12. Decanoic acid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13. sucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eIn vitro data for several formulations used against the CDVR-1A (Resistant Isolate) strain of the Vaccinia virus. The control medications are Cidofovir (SI50\u0026thinsp;\u0026gt;\u0026thinsp;2) and N-methanocarbamate (SI50\u0026thinsp;\u0026gt;\u0026thinsp;128).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSI\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1. Cu(II)-Sucrose-TPOXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;248\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1. TPOXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;180\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2. Cu(II)-TPOXX-Decanoate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e162\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3. Cu(II)-TPOXX\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;150\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4. Cu(II)-TPOXX-Decanoate-DHA-Sucrose\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5. Cu(II)-TPOXX-DHA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe vaccinia virus (Tables \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e,\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) \u003cem\u003ein vitro\u003c/em\u003e datum followed the same trend as the Cowpox datum. The Cu(II)-sucrose-TPOXX complex has the highest efficacy of the tested formulations, and formulating Cu(II) with TPOXX lowered its efficacy. The four TPOXX complexes that contain sucrose, decanoate, DHA, and both DHA and decanoate all have higher SI\u003csub\u003e50\u003c/sub\u003e values than pure TPOXX. When cellular replication takes place, the cell can consume sucrose, decanoate, and DHA as nutrients and building blocks, which could result in the TPOXX entering the cell more efficiently (Trojan Horse effect). The virulent vaccinia strain Rabbitpox virus has infected rabbits in research facilities in New York and Utrecht. Another \u003cem\u003evaccina\u003c/em\u003e strain, Buffalopox, had outbreaks in India where buffaloes are used for their milk (\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCowpox virus replication occurs in the cytoplasm of a cell and is like the replication process of the vaccinia virus in some ways but is different in others. An example of their differences is that Cowpox has a significant amount of guanylate kinase homolog (CPXV-GRI A59) in its mature virion, while the vaccinia virus has significant quantities of the A-type inclusion protein (CPXV-GRI A27) in its fully formed and functioning virus particles. An example of their similarities is the cowpox virus and vaccinia viruses can leave the cell during its lyses as a non-enveloped particle or as an enveloped particle. Non-enveloped particles leave the cell when the cell membrane lyses, while enveloped particles leave when the virus fuses with the Golgi apparatus and outer cell membranes.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe diffusion rate of free Cu(II) is significantly lower than sucrose when penetrating a cell membrane. Most biological membranes are designed to allow a small, neutral molecule, such as sucrose, to easily penetrate it (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). A charged species, such as Cu(II), typically requires a specific transport protein to penetrate the membrane (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Free Cu(II) can randomly bind proteins, specifically amines (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), due to the Irving-Williams effect and minimize its ability to penetrate a cell membrane. Cu(II) can undergo an electrostatic attraction to the oxygen atoms in sucrose, the carboxylates on the fatty acids, and the amines, oxygen atoms, and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pi\\:\\)\u003c/span\u003e\u003c/span\u003e bonds in TPOXX.\u003c/p\u003e \u003cp\u003eAt room temperature, the hydrolysis of sucrose has a ΔG\u003csub\u003erxn\u003c/sub\u003e of -26 kJ/mol, a spontaneous reaction (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Factors, such as reactant concentration, pH, and the presence of a catalysis, can dissociate sucrose into D-Glucose and L-Fructose. The bond enthalpy 163 kJ/mol for the N-N single bond in TPOXX is low and can easily dissociate (for reference; N-H, 386 kJ/mol; N\u0026thinsp;=\u0026thinsp;N 418 kJ/mol; N-Cl, 313 kJ/mol; N\u0026thinsp;=\u0026thinsp;O 607 kJ/mol). The Irving-Williams series indicates the Cu(II)-amine bond is strong, and this attraction can further weaken the N-N bond. Cu(I) and Cu(II) can form oxides (i.e. CuO\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, CuO, Cu\u003csub\u003e2\u003c/sub\u003eO) in the aqueous phase at physiological pH (7.38). These oxides have efficacy against cancer cells, bacterium, and viral infections (\u003cspan additionalcitationids=\"CR43 CR44\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe data was obtained from the National High Field Magnet Lab (NMR, FT-ICR) and the National Institutes of Health (Infectious Disease, in vitro data).Capri Persaud, Morgan Wynn, Akshil Patel, Madelyn, Adair and Thomas Manning synthesized the compounds used in this work. They also interpreted the NMR and FT-ICR data. Amy McKenna and Joseph Fry obtained the FT-ICR data used in this paper. They also provided expertise in purifying and preparing samples for analysis for FT-ICR and NMR studies. All co-authors contributed to the interpreation of different data sets, writing and proof reading the paper.\u003c/p\u003e\u003cp\u003eAcknowledgements:\u0026nbsp; Valdosta State University has utilized the non-clinical and preclinical services program offered by the National Institute of Allergy and Infectious Diseases under Contract No. 75N93019D00016/75N93023F00001, PI (Principal Investigator) Scott James, MD. We would like to thank the National Institute of Health, NIAID, and Ms. Amanda Ulloa, for their support of the \u003cem\u003ein vitro\u003c/em\u003e testing conducted. A portion of this work was performed at the Ion Cyclotron Resonance user facility at the National High Magnetic Field Laboratory at Florida State University, which is supported by the National Science Foundation Division of Chemistry and Division of Materials Research through NSF DMR 2128556, and the State of Florida. We would like to thank Dr. James Rocca of the MBI, Magnet Lab (University of Florida), AMRIS, NSF, State of Florida for working with obtaining the NMR spectra. We would like to thank Valdosta State University and the Chemistry Department (Dr. Kurt Winkelmann) for their support in this work.\u0026nbsp;\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eWe attached the data for NMR spectra, FT-ICR spectra, and NIH in vitro data.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ehttps://\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003c/span\u003e\u003cspan address=\"http://www.cdc.gov/smallpox/bioterrorism/public/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (Checked September 21st, 2024)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTh\u0026egrave;ves C, Crub\u0026eacute;zy E, Biagini P (2016) History of Smallpox and Its Spread in Human Populations. Microbiol Spectr 4(4). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/microbiolspec\u003c/span\u003e\u003cspan address=\"10.1128/microbiolspec\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMartin DB, Military Medicine M (2002) 167, 7:546, The Cause of Death in Smallpox: An Examination of the Pathology Record\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShchelkunova GA, Shchelkunov SN (2017 Oct-Dec) 40 Years without Smallpox. Acta Naturae 9(4):4\u0026ndash;12\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGreen MS, LeDuc J, Cohen D, Franz DR (2018) Confronting the threat of bioterrorism: realities, challenges, and defensive strategies. Lancet Infect Dis. 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Tuberculosis (Edinb) 107:149\u0026ndash;155. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tube.2017.08.011\u003c/span\u003e\u003cspan address=\"10.1016/j.tube.2017.08.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Technical notes","content":"\u003cp\u003eThe variables SI\u003csub\u003e50\u003c/sub\u003e and SI\u003csub\u003e90\u003c/sub\u003e are used in this paper (and in appendix B).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e = Compound concentration that reduces viral replication by 50%. Provided in \u0026mu;M concentration units.\u003c/p\u003e\n\u003cp\u003eEC\u003csub\u003e90\u003c/sub\u003e = Compound concentration that reduces viral replication by 90%; Provided in \u0026mu;M concentration units.\u003c/p\u003e\n\u003cp\u003eCC\u003csub\u003e50\u003c/sub\u003e = Compound concentration that reduces cell viability by 50%; provided \u0026mu;M units.\u003c/p\u003e\n\u003cp\u003eSI\u003csub\u003e50\u003c/sub\u003e = Selectivity index is calculated by (CC\u003csub\u003e50\u003c/sub\u003e / EC\u003csub\u003e50\u003c/sub\u003e)\u003csub\u003e.\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eSI\u003csub\u003e90\u003c/sub\u003e = Selectivity index calculated by (CC\u003csub\u003e90\u003c/sub\u003e / EC\u003csub\u003e90\u003c/sub\u003e)\u003csub\u003e.\u0026nbsp;\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAppendices included:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003col style=\"list-style-type: lower-alpha;\"\u003e\n \u003cli\u003eNIH NSAID \u003cem\u003eIn vitro\u003c/em\u003e data (original data): \u0026nbsp;CC\u003csub\u003e50\u003c/sub\u003e, EC\u003csub\u003e50\u003c/sub\u003e and SI\u003csub\u003e90\u0026nbsp;\u003c/sub\u003evalues for all data sets.\u003c/li\u003e\n \u003cli\u003e\u003cspan data-teams=\"true\"\u003e(not available with this version)\u0026nbsp;\u003c/span\u003eNMR data for TPOXX, Cu-TPOXX, sucrose, Cu-sucrose, sucrose-Cu-TPOXX (1 and 2 D); \u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC, \u003csup\u003e15\u003c/sup\u003eN \u0026nbsp;(images, processed by Topspin)\u003c/li\u003e\n \u003cli\u003e\u003cspan data-teams=\"true\"\u003e(not available with this version)\u0026nbsp;\u003c/span\u003eFT-ICR data for TPOXX, Cu(II)-TPOXX, sucrose, Cu(II)-sucrose, Cu(II)-sucrose-TPOXX formulations. (.txt files)\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6196055/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6196055/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe anti-viral medication Tecovirimat (TPOXX) has \u003cem\u003ein vitro\u003c/em\u003e efficacy against poxviruses. In this study, there are several formulations tested \u003cem\u003ein vitro\u003c/em\u003e that involve Cu(II) complexes as potential excipients. While the individual components have little or no efficacy, when some are combined with TPOXX, they improve the SI\u003csub\u003e50\u003c/sub\u003e values. The medicinal agents were tested against \u003cem\u003eVaccinia virus\u003c/em\u003e (Copenhagen, Resistant Isolate) and Cowpox virus (Brighton, CPXVR/Resistant Isolate). The poxviruses have become of great concern worldwide because of their potential to be bioterrorism agents (1). Nuclear Magnetic Resonance is utilized to determine structural components of complexes. Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry at 21 Tesla (ESI-21 T FT-ICR MS) identifies elemental compositions that correlate to proposed molecular structures in the aqueous phase. Other work is cited utlizing Cu(II) in which it improved the efficacy of antibiotics (for Tb) and cancers (9 types) in \u003cem\u003ein vitro\u003c/em\u003e studies.\u003c/p\u003e","manuscriptTitle":"Copper Complexes Increase the Efficacy In vitro of Tecovirimat against Pox Viruses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-24 04:48:56","doi":"10.21203/rs.3.rs-6196055/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"53ce017f-690a-4a39-8466-7fb99ed76662","owner":[],"postedDate":"March 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-04-10T17:53:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-24 04:48:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6196055","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6196055","identity":"rs-6196055","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}