Formulations of brilincidofovir

Abstract

The present invention discloses a formulation comprising brilincidofovir. The formulation may be an intravenous formulation. The preparation can be used for solving the problem of gastrointestinal tract when a subject orally takes brilincidofovir. The formulation can also be used in situations where the drug cannot be orally taken due to potential conditions or insufficient oral absorption.

Description

[0001] This application is a divisional application of an invention patent application with an application date of June 28, 2017, application number 201780040478.9, and invention name “Preparation of Brincidofovir”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 355,844, filed on June 28, 2016; U.S. Provisional Patent Application No. 62 / 394,665, filed on September 14, 2016; U.S. Provisional Patent Application No. 62 / 446,213, filed on January 13, 2017; U.S. Provisional Patent Application No. 62 / 465,053, filed on February 28, 2017; U.S. Provisional Patent Application No. 62 / 507,397, filed on May 17, 2017, and U.S. Provisional Patent Application No. 62 / 512,825, filed on May 31, 2017, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0004] The present invention relates to pharmaceutical compositions (eg, lyophilized and / or aqueous compositions) comprising brincidofovir and methods of use thereof. Background Art

[0005] Brincidofovir (BCV, CMX001) is an orally bioavailable lipid acyclic nucleoside phosphonate that is converted intracellularly to the active antiviral cidofovir diphosphate (CDV-PP). Brincidofovir has broad-spectrum antiviral activity against double-stranded DNA viruses. The structure of Brincidofovir is shown below:

[0006] Summary of the invention

[0007] The present invention provides compositions comprising brincidofovir and methods of use thereof. The compositions can be lyophilized (e.g., as a powder) for long-term storage. The lyophilized formulation can be reconstituted (e.g., in a sugar aqueous solution) as a biocompatible formulation for intravenous (IV) administration (e.g., to a subject in need thereof).

[0008] In one aspect, the present invention provides a pharmaceutical composition comprising: brincidofovir; a filler; a buffer; and water; wherein the pH of the composition is about 8.0.

[0009] In some embodiments, the filler is mannitol or sucrose. In some embodiments, the filler is mannitol. In some embodiments, the buffer is sodium phosphate, L-arginine or tromethamine. In some embodiments, the buffer is L-arginine. In some embodiments, the brincidofovir is present at a concentration of about 10.0 mg / mL. In some embodiments, the filler is present at a concentration of about 2.5-9% (w / v). In some embodiments, the filler is present at a concentration of about 2.5% (w / v). In some embodiments, the filler is present at a concentration of about 5% (w / v). In some embodiments, the buffer is present at a concentration of about 100-200mM. In some embodiments, the buffer is present at a concentration of about 100mM. In some embodiments, the pH is adjusted with HCl and / or NaOH.

[0010] In some embodiments, the pharmaceutical composition comprises: brincidofovir at a concentration of about 10.0 mg / mL; mannitol at a concentration of about 25-50 mg / mL; L-arginine at a concentration of about 17.4 mg / mL; and water; wherein the pH of the composition is about 8.0.

[0011] In some embodiments, the pharmaceutical composition comprises: brincidofovir at a concentration of about 17.8 mM; mannitol at a concentration of about 137.5-275 mM; L-arginine at a concentration of about 100 mM; and water; wherein the pH of the composition is about 8.0.

[0012] In some embodiments, the liquid pharmaceutical composition is lyophilized, for example to remove water, to form a lyophilized powder.

[0013] In another aspect, the present invention provides a lyophilized powder comprising: about 13-23% by weight of brincidofovir; about 48%-65% by weight of mannitol; and about 22-40% by weight of arginine.

[0014] In another aspect, the present invention provides a lyophilized powder comprising: about 13-19% by weight of brincidofovir; about 48%-65% by weight of mannitol; and about 22-33% by weight of arginine.

[0015] In some embodiments, the lyophilized powder contains about 19% by weight brincidofovir, about 48% by weight mannitol, and about 33% by weight arginine. In some embodiments, the lyophilized powder contains about 13% by weight brincidofovir, about 65% by weight mannitol, and about 22% by weight arginine.

[0016] In some embodiments, the pH of the lyophilized powder is about 8.0.

[0017] In another aspect, the present invention provides an aqueous pharmaceutical composition comprising: brincidofovir; a filler; a buffer; and an aqueous solution of a sugar alcohol, an aqueous solution of a sugar, a Ringer's solution, or an aqueous solution of a salt (eg, sodium chloride).

[0018] In some embodiments, the aqueous pharmaceutical composition comprises: brincidofovir; mannitol; L-arginine; and glucose.

[0019] In some embodiments, the aqueous pharmaceutical composition comprises: brincidofovir; mannitol; L-arginine; and glucose; wherein the pH of the composition is about 8.0.

[0020] In some embodiments, the sugar aqueous solution is a solution containing about 5% by weight glucose. In some embodiments, the saline solution is a sodium chloride aqueous solution. In some embodiments, the concentration of the sodium chloride solution is 0.9% by weight. In some embodiments, the aqueous pharmaceutical composition further comprises additional water.

[0021] In some embodiments, additional water is added (eg, to adjust the tonicity, concentration, or pH of the formulation).

[0022] In some embodiments, the aqueous pharmaceutical composition comprises: brincidofovir at a concentration of about 1.0 mg / mL; mannitol at a concentration of about 2.5-5 mg / mL; and L-arginine at a concentration of about 1.74 mg / mL; and glucose at a concentration of about 50 mg / mL. In some embodiments, the pH of the composition is about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, or less than about 6.0.

[0023] In some embodiments, the aqueous pharmaceutical composition comprises: brincidofovir at a concentration of about 1.78 mM; mannitol at a concentration of about 13.75-27.5 mM; L-arginine at a concentration of about 10 mM; and glucose at a concentration of about 287 mM. In some embodiments, the pH of the composition is about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, or less than about 6.0.

[0024] In some embodiments, the aqueous pharmaceutical composition comprises: brincidofovir at a concentration of about 0.5 mg / mL; mannitol at a concentration of about 1.25-2.5 mg / mL; and L-arginine at a concentration of about 0.87 mg / mL; and glucose at a concentration of about 50 mg / mL. In some embodiments, the pH of the composition is about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, or less than about 6.0.

[0025] In some embodiments, the aqueous pharmaceutical composition comprises: brincidofovir at a concentration of about 0.89 mM; mannitol at a concentration of about 6.85-13.7 mM; L-arginine at a concentration of about 5 mM; and glucose at a concentration of about 287 mM. In some embodiments, the pH of the composition is about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, or less than about 6.0.

[0026] In some embodiments, the aqueous pharmaceutical composition comprises: brincidofovir at a concentration of about 1.0 mg / mL; mannitol at a concentration of about 2.5-5 mg / mL; and L-arginine at a concentration of about 1.74 mg / mL; glucose at a concentration of about 50 mg / mL; wherein the pH of the composition is about 8.0.

[0027] In some embodiments, the aqueous pharmaceutical composition comprises: brincidofovir at a concentration of about 1.78 mM; mannitol at a concentration of about 13.75-27.5 mM; L-arginine at a concentration of about 10 mM; and glucose at a concentration of about 287 mM; wherein the pH of the composition is about 8.0.

[0028] In some embodiments, the aqueous pharmaceutical composition comprises: brincidofovir at a concentration of about 0.5 mg / mL; mannitol at a concentration of about 1.25-2.5 mg / mL; and L-arginine at a concentration of about 0.87 mg / mL; and glucose at a concentration of about 50 mg / mL; wherein the pH of the composition is about 8.0.

[0029] In some embodiments, the aqueous pharmaceutical composition comprises: brincidofovir at a concentration of about 0.89 mM; mannitol at a concentration of about 6.85-13.7 mM; L-arginine at a concentration of about 5 mM; and glucose at a concentration of about 287 mM; wherein the pH of the composition is about 8.0.

[0030] In some embodiments, the volume of the sugar aqueous solution, sugar alcohol aqueous solution, Ringer's solution or saline solution used to dissolve the lyophilized preparation of brincidofovir is about 100 or 200mL. In other words, in some embodiments, the lyophilized powder containing brincidofovir is dissolved in about 100mL or about 200mL of sugar aqueous solution, sugar alcohol aqueous solution, Ringer's solution, saline solution or water. In some embodiments, the lyophilized powder is dissolved in about 100mL, about 110mL, about 120mL, about 130mL, about 140mL, about 150mL, about 160mL, about 170mL, about 180mL, about 190mL or about 200mL. For example, in some embodiments, the lyophilized powder of the present invention can be dissolved in about 100mL of 5% glucose aqueous solution. In some embodiments, the lyophilized powder of the present invention can be dissolved in about 200mL of 5% glucose aqueous solution.

[0031] In some embodiments, the aqueous pharmaceutical composition comprises: about 100 mg of brincidofovir; about 250-500 mg of mannitol; about 174 mg of arginine; about 5 g of glucose; and about 100 mL of water. In some embodiments, the pH of the composition is about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, or less than about 6.0.

[0032] In some embodiments, the aqueous pharmaceutical composition comprises: about 200 mg of brincidofovir; about 500-1000 mg of mannitol; about 348 mg of arginine; about 10 g of glucose; and about 200 mL of water. In some embodiments, the pH of the composition is about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, or less than about 6.0.

[0033] In some embodiments, the aqueous pharmaceutical composition comprises: about 50 mg of brincidofovir; about 125-250 mg of mannitol; about 87 mg of arginine; about 5 g of glucose; and about 100 mL of water. In some embodiments, the pH of the composition is about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, or less than about 6.0.

[0034] In some embodiments, the aqueous pharmaceutical composition comprises: about 100 mg of brincidofovir; about 250-500 mg of mannitol; about 174 mg of arginine; about 10 g of glucose; and about 200 mL of water. In some embodiments, the pH of the composition is about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, or less than about 6.0.

[0035] In some embodiments, the aqueous pharmaceutical composition comprises: about 100 mg of brincidofovir; about 250-500 mg of mannitol; about 174 mg of arginine; about 5 g of glucose; and about 100 mL of water; wherein the pH of the composition is about 8.0.

[0036] In some embodiments, the aqueous pharmaceutical composition comprises: about 200 mg of brincidofovir; about 500-1000 mg of mannitol; about 348 mg of arginine; about 10 g of glucose; and about 200 mL of water; wherein the pH of the composition is about 8.0.

[0037] In some embodiments, the aqueous pharmaceutical composition comprises: about 50 mg of brincidofovir; about 125-250 mg of mannitol; about 87 mg of arginine; about 5 g of glucose; and about 100 mL of water; wherein the pH of the composition is about 8.0.

[0038] In some embodiments, the aqueous pharmaceutical composition comprises: about 100 mg of brincidofovir; about 250-500 mg of mannitol; about 174 mg of arginine; about 10 g of glucose; and about 200 mL of water; wherein the pH of the composition is about 8.0.

[0039] In some embodiments, one or more of the brincidofovir, bulking agent, and buffering agent are lyophilized prior to incorporation into an aqueous pharmaceutical composition described herein.

[0040] In some embodiments, the aqueous pharmaceutical composition is suitable for intravenous administration. In some embodiments, the aqueous pharmaceutical composition is sterile.

[0041] In another aspect, the present invention provides a sterile aqueous pharmaceutical composition for intravenous administration comprising: brincidofovir; a filler; a buffer and glucose.

[0042] In another aspect, the present invention provides a sterile aqueous pharmaceutical composition for intravenous administration, comprising: brincidofovir; a bulking agent; a buffer and glucose; wherein the pH of the composition is about 8.0.

[0043] In another aspect, the present invention provides an aqueous pharmaceutical composition for intravenous administration, comprising: brincidofovir at a concentration of about 0.5 mg / mL to about 1.0 mg / mL; a filler at a concentration of about 2.5 mg / mL to about 5 mg / mL; a buffer at a concentration of about 0.87 mg / mL to about 1.74 mg / mL; and glucose at a concentration of about 50 mg / mL. In some embodiments, the pH of the composition is about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, or less than about 6.0.

[0044] In another aspect, the present invention provides an aqueous pharmaceutical composition for intravenous administration, comprising: brincidofovir at a concentration of about 0.5 mg / mL to about 1.0 mg / mL; a filler at a concentration of about 2.5 mg / mL to about 5 mg / mL; a buffer at a concentration of about 0.87 mg / mL to about 1.74 mg / mL; and glucose at a concentration of about 50 mg / mL; wherein the pH of the composition is about 8.0.

[0045] In another aspect, the present invention provides a method for treating a subject suffering from a viral infection, the method comprising: administering to the subject a pharmaceutical composition or pharmaceutical formulation according to the present invention.

[0046] In another aspect, the present invention provides a method of treating a subject having a viral infection, the method comprising: administering to the subject an intravenous pharmaceutical composition comprising brincidofovir; a bulking agent; a buffer; and glucose.

[0047] In another aspect, the invention provides a method of treating a subject having a viral infection, the method comprising: administering to the subject an intravenous pharmaceutical composition comprising brincidofovir; a bulking agent; a buffering agent; and glucose, wherein the pH of the composition is about 8.0.

[0048] In another aspect, the present invention provides a method of treating a subject with a viral infection, the method comprising: administering to the subject an intravenous pharmaceutical composition comprising brincidofovir at a concentration of 0.5 mg / mL to about 1.0 mg / mL; a filler at a concentration of about 2.5 mg / mL to about 5 mg / mL; a buffer at a concentration of about 0.87 mg / mL to about 1.74 mg / mL; and glucose at a concentration of about 50 mg / mL%. In some embodiments, the pH of the composition is about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, or less than about 6.0.

[0049] In another aspect, the present invention provides a method of treating a subject having a viral infection, the method comprising: administering to the subject an intravenous pharmaceutical composition comprising brincidofovir at a concentration of about 0.5 mg / mL to about 1.0 mg / mL; a bulking agent at a concentration of about 2.5 mg / mL to about 5 mg / mL; a buffer at a concentration of about 0.87 mg / mL to about 1.74 mg / mL; and glucose at a concentration of about 50 mg / mL%; wherein the pH of the composition is about 8.0.

[0050] On the other hand, the present invention provides an aqueous pharmaceutical preparation or composition for treating viral infections, which is prepared by a method comprising the following steps: dissolving a certain amount of brincidofovir, a filler and a buffer in water in any order to form a first solution; freeze-drying the first solution to form a lyophilized powder; and dissolving the lyophilized powder in a sugar alcohol aqueous solution, a sugar aqueous solution, a Ringer's solution or a sodium chloride solution to form an aqueous pharmaceutical preparation or composition.

[0051] In another aspect, the present invention provides use of an aqueous pharmaceutical composition comprising brincidofovir, a bulking agent, a buffer, and an aqueous sugar alcohol solution or an aqueous sugar solution in the preparation of a medicament for treating viral infection.

[0052] In another aspect, the present invention provides use of an aqueous pharmaceutical composition comprising brincidofovir, a bulking agent, a buffer and an aqueous sugar alcohol solution or an aqueous sugar solution in treating viral infection.

[0053] In some embodiments of any of the aforementioned aspects, the viral infection to be treated is polyomavirus, papillomavirus, herpes virus, adenovirus, Epstein-Barr virus, cytomegalovirus, hepatitis B virus, hepatitis C virus, varicella zoster virus, adenovirus, poxvirus, or a combination thereof.

[0054] In some embodiments, administration of a pharmaceutical composition as described herein to a subject does not result in hemolysis. In some embodiments, administration of a pharmaceutical composition as described herein to a subject does not result in gastrointestinal toxicity.

[0055] In some embodiments, the pH of any aqueous formulation or composition for intravenous administration may have a pH below about 8.0 (e.g., about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, about 5.5, about 5.0, about 4.5, about 4.0, or about 4.0 or less). In some embodiments, the pH may be above 8.0 (e.g., about 8.5, about 9.0, about 9.5, about 10.0, or above about 10.0).

[0056] Without wishing to be bound by theory, oral administration of brincidofovir may result in significantly higher exposure in the intestine compared to other organs; this higher exposure may result in gastrointestinal toxicity following oral administration of brincidofovir. As described herein, intravenous administration of brincidofovir may prevent excessive exposure of the intestinal tract (e.g., intestine) to brincidofovir (e.g., compared to oral BCV), improve gastrointestinal (GI) tolerance and reduce GI toxicity. In some embodiments, IV administration of brincidofovir may provide comparable (e.g., compared to oral administration) drug exposure to plasma and organs such as the liver, kidney, and small intestine, even at a lower dose than that required for oral administration.

[0057] In some embodiments, intravenous brincidofovir can deliver drugs to organs (e.g., brain) that are difficult to reach with other routes of administration (e.g., oral doses). In some embodiments, intravenous brincidofovir can treat viral infections in the brain (e.g., herpes encephalitis in neonates and adults; HHV-6 encephalitis; JC virus / PML in transplant recipients or patients with multiple sclerosis) with higher CNS exposure.

[0058] Intravenous brincidofovir can avoid the GI side effects observed with oral administration of BCV, and provides the opportunity to treat DNA viruses (e.g., in patients experiencing the GI side effects associated with oral administration of BCV). For example, like oral administration of brincidofovir, intravenous administration of brincidofovir can be used as a broad-spectrum antiviral agent with limited toxicity (e.g., substantially without heme toxicity, and without nephrotoxicity), and can effectively treat, for example, adenovirus and smallpox. In addition, intravenous administration of brincidofovir can be used to treat and prevent, for example, cytomegalovirus, adenovirus, and BK or JC virus. For example, these diseases can be treated in hematopoietic cell transplant patients.

[0059] Additional features and advantages will be apparent to those skilled in the art and are set forth in the following detailed description of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Those skilled in the art will appreciate that the drawings are primarily for illustrative purposes and are not intended to limit the scope of the inventive subject matter described herein. The drawings are not necessarily drawn to scale; in some cases, various aspects of the inventive subject matter disclosed herein may be shown exaggerated or enlarged in the drawings to facilitate understanding of different features. In the drawings, the same reference numerals generally represent the same features (e.g., functionally similar and / or structurally similar elements).

[0061] The above and other features will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings.

[0062] Figure 1 is a graph showing the product temperature profile recorded by the product probe as described in Example 4.

[0063] Figure 2 are a pair of graphs illustrating shelf life evaluation in an accelerated stability study.

[0064] Figure 3 Is displayed [ 14C] Time course of blood and plasma radioactivity concentrations in male Sprague Dawley (SD) and Long-Evans (LE) rats following a single 2-hour intravenous infusion (IV) of brincidofovir at a target dose of 15 mg / kg or 2 mg / kg, and following a single oral (PO) dose of 15 mg / kg as described in Example 9.

[0065] Figure 4 The intravenous and oral administration was as described in Example 9 [ 14 C] Small intestinal tissue concentration of brincidofovir versus time.

[0066] Figure 5 The intravenous and oral administration was as described in Example 9 [ 14 C] Renal cortical tissue concentration of brincidofovir versus time.

[0067] Figure 6 Figure 2 is a graph of brincidofovir plasma concentrations following 100 mg oral, 10 mg IV, and 25 mg IV doses.

[0068] Fig. 7A is a histogram of rat intestine after oral administration of brincidofovir as described in Example 10.

[0069] Figure 7B is a histogram of rat intestine following intravenous administration of brincidofovir as described in Example 10.

[0070] Figure 8 is a graph showing mean (+ / -SE) alanine aminotransferase (ALT) levels observed in the IV BCV single ascending dose (SAD) trial.

[0071] Fig. 9 is a graph showing the mean plasma brincidofovir concentration over time for subjects in Groups 1-4, and the mean plasma BCV concentration over time for subjects administered BCV orally.

[0072] Fig.10 is a graph showing the median plasma cidofovir concentrations over time following IV and oral BCV doses. DETAILED DESCRIPTION

[0073] The present invention relates to pharmaceutical compositions (e.g., lyophilized or aqueous compositions) comprising brincidofovir and methods of use thereof. The present invention also relates to pharmaceutical preparations comprising the reconstituted lyophilized pharmaceutical compositions described herein. The compositions and preparations can be used for intravenous administration. In some embodiments, intravenous administration of the compositions or preparations of the present invention does not result in hemolysis. The preparations can also be used in situations where oral administration is impossible or substantially impossible, for example due to underlying conditions or insufficient oral absorption.

[0074] The present invention relates to at least three types of brincidofovir compositions. First, the present invention relates to bulk preparations of brincidofovir that can be lyophilized before storage (e.g., liquid preparations of the present invention or pre-lyophilized preparations of the present invention). Lyophilized preparations (e.g., powders) can be stored for a long time. In addition, the lyophilized preparations can be reconstituted (e.g., by dissolving in a sugar solution) before (e.g., immediately before) administration to a patient (e.g., a patient in need of treatment). Therefore, the present invention teaches brincidofovir pre-lyophilized preparations, lyophilized powders, and reconstituted preparations (e.g., pharmaceutical preparations) for administration.

[0075] Public preparations

[0076] Pre-lyophilized preparations

[0077] In some embodiments, brincidofovir is dissolved in water before lyophilization (e.g., freeze drying). For example, brincidofovir can be co-dissolved with a filler and a buffer. The dissolution of the components can occur individually or simultaneously, and can occur in any order.

[0078] In some embodiments, the pre-freeze-dried composition of the present invention comprises a buffer. In some embodiments, the pre-freeze-dried composition of the present invention comprises a filler. In some embodiments, the pre-freeze-dried composition of the present invention comprises a buffer and a filler. In some embodiments, the concentration of brincidofovir is 10 mg / mL. In some embodiments, the pH of the solution is adjusted with HCl and / or NaOH.

[0079] In some embodiments, the buffer is selected from sodium phosphate, L-arginine, tromethamine and pH adjusted water. In some embodiments, the buffer is sodium phosphate, L-arginine or tromethamine. In some embodiments, the buffer is sodium phosphate. In some embodiments, the buffer is arginine. In some embodiments, the buffer is L-arginine. In some embodiments, the buffer is tromethamine. In some embodiments, the buffer is pH adjusted water. In some embodiments, the buffer is present at a concentration of about 100mM to about 200mM. In some embodiments, the buffer is present in an amount of about 25mM, about 50mM, about 75mM, about 100mM, about 125mM, about 150mM, about 175mM or about 200mM.

[0080] In some embodiments, the filler is mannitol or sucrose. In some embodiments, the buffer is sodium phosphate, L-arginine or tromethamine. In some embodiments, the filler is present in a concentration of about 5-9% (w / v). In some embodiments, the filler is present in a concentration of about 2.5-9% (w / v). In some embodiments, the filler is present in a concentration of about 2.5% (w / v), about 5% (w / v) or about 9% (w / v).

[0081] In some embodiments, the compositions of the invention comprise brincidofovir at a concentration of about 10.0 mg / mL, about 6.4 mg / mL, or about 3.2 mg / mL.

[0082] In some embodiments, the composition of the invention comprises brincidofovir at a concentration of about 10.0 mg / mL, mannitol at a concentration of about 25-50 mg / mL, L-arginine at a concentration of about 17.4 mg / mL and water, and has a pH=8.0.

[0083] In some embodiments, the composition of the invention comprises brincidofovir at a concentration of about 17.8 mM, mannitol at a concentration of about 137.5-275 mM, L-arginine at a concentration of about 100 mM and water, and has a pH=8.0.

[0084] In some embodiments, any pre-lyophilized formulation described herein can be lyophilized to remove water (eg, for storage).

[0085] In some embodiments, pre-lyophilized formulations can be used directly as pharmaceutical formulations (ie, can be directly administered to a patient). However, in some embodiments, these formulations are lyophilized for storage and subsequently reconstituted (eg, in 5% dextrose in water) for administration to a patient.

[0086] In some embodiments, liquid formulations of brincidofovir are used for intravenous administration after dilution in an infusion vehicle (eg, 5% dextrose).

[0087] In some embodiments, the liquid preparation of the present invention (i.e., the pre-lyophilized preparation of the present invention) is compatible with materials (e.g., containers) used in infusion carriers and clinical settings. For example, the liquid preparation of the present invention, when in contact with materials such as sterile filters, vials, stoppers, infusion bags, or IV systems, does not show significant changes in appearance, pH, or the recovery rate of brincidofovir, or the introduction of impurities. In some embodiments, the liquid preparation of the present invention, when filtered through a syringe filter, does not experience significant changes in pH, loss of brincidofovir, or the introduction of impurities.

[0088] Lyophilized preparations

[0089] The pre-lyophilized formulation can be lyophilized to produce a lyophilized formulation (e.g., powder) of BCV, filler, and buffer. The lyophilized formulation can be stable for a long time and can be reconstituted before administration to a patient. In some embodiments, the lyophilized formulation is sterile.

[0090] In some embodiments, lyophilization comprises freezing, annealing and drying the lyophilized composition. In some embodiments, drying comprises primary drying and secondary drying. In some embodiments, freezing and annealing comprise exposing the formulation to a temperature between about 5°C and -50°C. In some embodiments, primary drying is performed at a temperature of about 35°C. In some embodiments, secondary drying is performed at a temperature of about 20°C.

[0091] In some embodiments, freezing and / or annealing lasts for about 16 hours. In some embodiments, the primary drying stage lasts for about 20 hours. In further embodiments, secondary drying requires about 22 hours (e.g., 21.7 hours) or about 28 hours (e.g., 27.7 hours).

[0092] In some embodiments, the liquid formulations of the present invention (eg, liquid formulations comprising brincidofovir, arginine, and mannitol) do not foam during the lyophilization process, compared to other formulations that foam during the lyophilization process.

[0093] Reconstituted pharmaceutical compositions for IV administration

[0094] Before IV administration to a patient, the above-mentioned lyophilized powder can be reconstituted, for example, by dissolving in an aqueous solvent such as water. In some embodiments, the aqueous solvent is water containing a sugar alcohol or sugar (e.g., glucose). In some embodiments, the aqueous solvent is a 5% dextrose aqueous solution. The aqueous solvent can also be sterile (e.g., similar to a sterile lyophilized formulation) and can be suitable for administration to a patient in need. In some embodiments, the reconstruction of the lyophilized powder of the present invention produces an aqueous composition of the present invention.

[0095] As described below, the pharmaceutical compositions for IV administration described herein can provide therapeutically relevant plasma concentrations of brincidofovir using lower doses of brincidofovir than those required for oral administration of brincidofovir. For example, intravenous administration of brincidofovir using the formulations of the present invention provides plasma concentrations of brincidofovir in humans that have previously been demonstrated to have antiviral efficacy in cytomegalovirus prevention and adenovirus treatment. The amount of BCV used in the IV formulation (e.g., about 10 mg or about 25 mg) is about one-tenth the amount of BCV required to achieve similar plasma concentrations using oral administration.

[0096] Therefore, in some embodiments, the present disclosure teaches a treatment of a viral infection, which includes administering an IV preparation of brincidofovir as described herein to a subject in need thereof. In some embodiments, the IV dose of brincidofovir is less than the oral dose (e.g., about 50% of the oral dose, about 40% of the oral dose, about 30% of the oral dose, about 20% of the oral dose, or about 10% of the oral dose) to achieve similar results (e.g., plasma concentration, antiviral activity, etc.). Therefore, in some embodiments, a smaller amount of IV brincidofovir than that used for oral administration can be used to achieve similar (e.g., substantially the same) clinical results. In addition, because IV administration results in a smaller amount of brincidofovir in the intestine, in some embodiments, the present invention also provides treatment of viral infections that do not cause gastrointestinal toxicity. The present invention teaches the treatment of patients who cannot be administered orally (e.g., patients with sensitive intestines or patients who cannot swallow oral medications).

[0097] In some embodiments, the composition of the invention comprises brincidofovir at a concentration of about 1.0 mg / mL, mannitol at a concentration of about 2.5-5 mg / mL, L-arginine at a concentration of about 1.74 mg / mL, and glucose at a concentration of about 50 mg / mL, and has a pH = 8.0.

[0098] In some embodiments, the composition of the invention comprises brincidofovir at a concentration of about 17.8 mM, mannitol at a concentration of about 13.75-27.5 mM, L-arginine at a concentration of about 10 mM, and glucose at a concentration of about 287 mM, and has a pH=8.0.

[0099] In some embodiments, the composition of the invention comprises brincidofovir at a concentration of about 0.5 mg / mL, mannitol at a concentration of about 1.25-2.5 mg / mL, L-arginine at a concentration of about 0.87 mg / mL, and glucose at a concentration of about 50 mg / mL, and has a pH = 8.0.

[0100] In some embodiments, the composition of the invention comprises brincidofovir at a concentration of about 0.89 mM, mannitol at a concentration of about 6.85-13.7 mM, L-arginine at a concentration of about 5 mM, and glucose at a concentration of about 287 mM, and has a pH=8.0.

[0101] In some embodiments, the pharmaceutical composition of the present invention further comprises a sugar. In some embodiments, the sugar is in a sugar aqueous solution. In some embodiments, the sugar aqueous solution is a 5% glucose solution. In some embodiments, the volume of the sugar aqueous solution is about 100 or 200 mL.

[0102] In some embodiments, the composition of the present invention comprises brincidofovir in an amount of about 100 mg, mannitol in an amount of about 250 mg to about 500 mg, arginine in an amount of about 174 mg, glucose in an amount of about 5 g, and water in an amount of about 100 mL, and has a pH=8.0.

[0103] In some embodiments, the composition of the present invention comprises brincidofovir in an amount of about 200 mg, mannitol in an amount of about 500 mg to about 1000 mg, arginine in an amount of about 348 mg, glucose in an amount of about 10 g, and water in an amount of about 100 mL, and has a pH = 8.0.

[0104] In some embodiments, the composition of the present invention comprises brincidofovir in an amount of about 50 mg, mannitol in an amount of about 125 mg to about 250 mg, arginine in an amount of about 87 mg, glucose in an amount of about 5 g, and water in an amount of about 100 mL, and has a pH=8.0.

[0105] In some embodiments, the composition of the present invention comprises brincidofovir in an amount of about 100 mg, mannitol in an amount of about 250 mg to about 500 mg, arginine in an amount of about 174 mg, glucose in an amount of about 10 g, and water in an amount of about 200 mL, and has a pH=8.0.

[0106] In some embodiments, the composition of the invention comprises brincidofovir at a concentration of about 0.1-1.0 mg / mL in 100-200 mL of 5% glucose solution. In some embodiments, the composition comprises: 10 mg brincidofovir, 100 mM arginine, and 5% mannitol (w / v), and has a pH=8.0.

[0107] In some embodiments, the lyophilized compositions of the invention are intended for IV administration by infusion following dilution.

[0108] In some embodiments, the buffer is present at about 200 mM. In some embodiments, brincidofovir has been previously lyophilized. In some embodiments, the composition of the present invention is lyophilized to remove water. In some embodiments, brincidofovir, filler, and buffer have been previously lyophilized.

[0109] In some embodiments, the present invention provides an aqueous pharmaceutical composition for intravenous administration, comprising brincidofovir, mannitol and arginine. In some embodiments, the pH of the formulation is about 8.0. In some embodiments, the pH of the composition is adjusted with HCl and / or NaOH.

[0110] In some embodiments, the compositions of the present invention are suitable for reconstitution with deionized water. In some embodiments, the compositions of the present invention do not foam when reconstituted. In some embodiments, the compositions of the present invention foam when reconstituted and the foam dissipates in less than 20 minutes, less than 10 minutes, less than 5 minutes, or less than 1 minute. In some embodiments, compositions comprising 100 mM or more arginine show faster foam or bubble dissipation when reconstituted compared to compositions comprising 50 mM or less arginine.

[0111] In some embodiments, the lyophilized compositions of the invention do not undergo changes in appearance, pH, or recovery of brincidofovir when diluted with an infusion vehicle (eg, 5% dextrose), such as prior to IV administration.

[0112] In some embodiments, reconstitution of the lyophilized composition of the present invention does not result in the loss of brincidofovir.In some embodiments, the lyophilized formulation of the present invention is clear and colorless after reconstitution.

[0113] Excipients and pharmaceutically acceptable carriers

[0114] In addition to brincidofovir, buffer and filler, the pharmaceutical composition of the present invention may include additional pharmaceutically acceptable carriers. Such pharmaceutically acceptable carriers may include any and all solvents, diluents or other liquid carriers, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid adhesives, lubricants, etc., to be suitable for the desired specific dosage form. Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers for preparing pharmaceutical compositions and known techniques for their preparation. Unless any conventional carrier medium is incompatible with the active compound (i.e., brincidofovir), for example, by producing any undesirable biological effect or interacting with any other component of the pharmaceutical composition in a harmful manner, its use is considered within the scope of the present invention. Some examples of materials that can be used as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered yarrow; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil and cottonseed oil; safflower oil and sesame oil; olive oil; corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution, ethanol, and phosphate buffered solutions, as well as other nontoxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavoring agents and fragrances, preservatives and antioxidants may also be present in the composition according to the judgment of the formulator.

[0115] Methods used

[0116] In some embodiments, the present invention provides a method of treating a subject having a viral infection, comprising administering to the subject a composition of the present invention. In some embodiments, the composition of the present invention is administered intravenously to the subject.

[0117] In some embodiments, the formulations of the present invention may comprise a volume of concentrated sterile liquid solution (e.g., for dilution) and / or lyophilized powder (e.g., for reconstitution). In some embodiments, the formulation can be stored for at least 12 months (e.g., at about 25° C.). In some embodiments, the formulation can be stored upside down (e.g., for about 24 months, or about 36 months). In some embodiments, the formulation can be stored in a refrigerator (e.g., for about 12 months). In some embodiments, the lyophilized powder is easily dissolved in the liquid (e.g., for reconstitution). In some embodiments, the liquid volume can be very small (e.g., for parenteral administration). In some embodiments, the formulation exhibits acceptable filter and tubing compatibility. In some embodiments, the formulation is compatible with, including but not limited to, D5W, NS, and D5 1 / 2NS is compatible with normal resuscitation fluid.

[0118] Patient Population

[0119] In some embodiments, administration of the compositions of the invention does not result in hemolysis. In some embodiments, administration of the compositions of the invention does not result in gastrointestinal toxicity.

[0120] Therefore, in some embodiments, the present disclosure provides a dosage of brincidofovir for a patient who may experience undesirable side effects associated with oral brincidofovir (e.g., gastrointestinal side effects, such as diarrhea, pain, constipation, etc.). For example, in some embodiments, IV preparations can be used for patients with symptoms such as anemia, irritable bowel syndrome, constipation, diarrhea, intestinal pain, etc. In addition, in some embodiments, the present invention provides for continuous and / or repeated administration of brincidofovir to subjects in need thereof. For example, a patient who has undergone a stem cell transplant can be administered an IV preparation of the present invention to prevent gastrointestinal side effects associated with oral administration (e.g., repeated oral administration) of brincidofovir.

[0121] Virus Indications

[0122] In some embodiments, the viral infection to be treated is selected from polyomavirus (including BK, John Cunningham virus (JCV), Merkel cell virus (MCV), KI polyomavirus (KIV), WU polyomavirus (WUV), Simian virus 40 (SV 40)), papillomavirus (including human papillomavirus, cottontail rabbit papillomavirus, equine papillomavirus and bovine papillomavirus), herpes virus (such as herpes simplex virus), adenovirus, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), hepatitis B virus, hepatitis C virus, varicella-zoster virus (VZV) or a combination thereof.

[0123] In some embodiments, the IV formulations can be used to prevent clinically significant cytomegalovirus infection in at-risk (e.g., CMV seropositive) adult and pediatric allogeneic hematopoietic stem cell transplant recipients. In some embodiments, the formulations of the present invention can be used to treat adenovirus infection in adult and pediatric immunocompromised hosts. In some embodiments, the formulations can be used in hematopoietic stem cell transplant or solid organ transplant patients.

[0124] Dosage regimen

[0125] In some embodiments, the formulations of the invention (e.g., IV formulations) are administered at a dose of about 1 mg to 1000 mg BCV. For example, the formulations can be administered at a dose of about 10 mg to 200 mg BCV. In some embodiments, the IV formulation may be administered at about 5 mg; about 10 mg; about 15 mg; about 20 mg; about 25 mg; about 30 mg; about 35 mg; about 40 mg; about 45 mg; about 50 mg; about 55 mg; about 60 mg; about 65 mg; about 70 mg; about 75 mg; about 80 mg; about 85 mg; about 90 mg; about 95 mg; about 100 mg; about 105 mg; about 110 mg; about 115 mg; about 120 mg; about 125 mg; about 130 mg; about 135 mg; about 140 mg; about 145 mg; about 150 mg; about 155 mg; about 160 mg; about 165 mg; about 170 mg; about 175 mg; about 180 mg; about 185 mg; about 190 mg; about 195 mg; or about 200 mg BCV.

[0126] In some embodiments, the IV formulations of the invention can be administered to deliver about 5 to about 50 mg; about 10 to about 50 mg; about 10 to about 40 mg; about 10 to about 30 mg; about 5 to about 25 mg; about 10 to about 25 mg; about 15 to about 25 mg of BCV. The formulations can be administered to humans (e.g., adults).

[0127] In some embodiments, when administered intravenously at a dosage of about 10 mg to 100 mg (e.g., about 10 mg or about 25 mg), the formulation is safe and well tolerated. For example, the 10 mg and 25 mg doses of brincidofovir formulations disclosed herein can give a favorable tolerability profile without adverse events. In some embodiments, the dosage of about 10 mg and 25 mg of the formulations described herein does not produce gastrointestinal side effects.

[0128] In some embodiments, the IV preparations taught herein can be administered multiple times per day, or can be administered as a single dose. For example, the IV preparation can be administered once a day or can be administered twice a day. In some embodiments, the preparations of the present invention are administered once a week, or twice a week. In some embodiments, the preparations of the present invention are administered every other day. In some embodiments, the preparations of the present invention are administered every other week. In some embodiments, the preparations of the present invention are administered once a month, or twice a month. Those skilled in the art will be able to determine the appropriate dosage regimen for the patient.

[0129] The duration of administration of IV brincidofovir (i.e., for each dose) can vary according to the needs of individual subjects or patients in need thereof, and it is within the expertise of those skilled in the art (e.g., a clinician such as a nurse or doctor) to determine the appropriate time for a subject to take brincidofovir. For example, intravenous administration of brincidofovir can last for about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, or about 120 minutes.

[0130] In some embodiments, the preparation of the present invention can be administered in combination with other therapeutic agents or treatments. For example, other therapeutic agents can be cisplatin, doxorubicin, etoposide, irinotecan, topotecan, paclitaxel, docetaxel, epothilone, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, lonafarib, tipifarnib, 4-((5-((4-(3-chlorophenyl)-3-oxopiperazine-1-yl)methyl)-1H-imidazole-1-yl)methyl)benzonitrile hydrochloride, (R)-1-((1H-imidazole-5-yl)methyl)-3-benzyl-4-(thiophene-2-ylsulfonyl)-2 ,3,4,5-tetrahydro-1H-benzodiazepine-7-carbonitrile, cetuximab, imatinib, interferon α-2b, pegylated interferon α-2b, aromatase combination, gemcitabine, uracil mustard, mechlorethamine, ifosfamide, melphalan, chlorambucil, guanidine, triethylene melamine, triethylenethiophosphamide, busulfan, carmustine, lomustine, streptozotocin, dacarbazine, fluorouracil, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, leucovorin, oxaliplatin, pentothrin tadalafil, vinblastine, vincristine, vindesine, bleomycin, actinomycin, daunorubicin, epirubicin, idarubicin, mithramycin, deoxycostapyrin, mitomycin C, L-asparaginase, teniposide 17, α-ethinyl estradiol, diethylstilbestrol, testosterone, prednisone, fluoxymesterone, drostanolone propionate, testolactone, megestrol acetate, methylprednisolone, methyltestosterone, prednisolone, triamcinolone acetonide, chloranthone, 17α-hydroxyprogesterone, aminoacetimide, estramustine, medroxyprogesterone acetate, leuprolide acetate, flutamide Amine, toremifene citrate, goserelin acetate, carboplatin, hydroxyurea, amsacrine, procarbazine, mitotane, mitoxantrone, levamisole, vinorelbine, anastrozole, letrozole, capecitabine, raloxifene, droloxafine, hexamethylmelamine, bevacizumab, trastuzumab, tositumomab, bortezomib, ibritumomab tiuxetan, arsenic trioxide, porfimer sodium, cetuximab, thiotepa, hexamethylmelamine, fulvestrant, exemestane, rituximab, alemtuzumab, dexamethasone, bicalutamide, chlorambucil or valrubicin. The agents may be administered as part of a treatment regimen and are not necessarily administered at the same time or on the same schedule.

[0131] the term

[0132] For the purpose of promoting understanding of the embodiments described herein, references to preferred embodiments and specific language are used to describe them. The terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the scope of the invention. As used throughout the present invention, the singular forms "one", "one" and "this" include plural references unless the context clearly indicates otherwise. Therefore, for example, reference to "composition" includes a variety of such compositions, as well as a single composition, and reference to "therapeutic agent" or "active compound" refers to one or more therapeutic agents and / or medicaments (e.g., brincidofovir) and equivalents known to those skilled in the art. Unless otherwise indicated, all percentages and ratios used herein are by weight.

[0133] The term "about" as used herein means approximately, in the vicinity, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the above numerical values. Typically, the term "about" is used herein to modify the numerical values ​​above and below the stated value by 20% variance.

[0134] As used herein, the terms "comprising" and "including," whether in transitional phrases or in the body of the claims, are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases "having at least" or "including at least." When used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a molecule, compound, or composition, the term "comprising" means that the compound or composition includes at least the recited features or components, but may also include additional features or components.

[0135] A "pharmaceutical composition" is a formulation containing a compound of the invention (e.g., brincidofovir) in a form suitable for administration to a subject. The term "pharmaceutical composition" includes formulations suitable for administration to a mammal, such as a human. The pharmaceutical composition can be, for example, an intravenous (IV) formulation or an oral formulation.

[0136] The term "monotherapy" is understood to mean the use of a single drug to treat a particular condition or disease. Monotherapy differs from combination therapy in that combination therapy involves the combined use of at least two drugs to treat a particular condition or disease.

[0137] As used herein, "treat," "treatment," or "treatment" describes the management and care of a patient for combating a disease, condition, or disorder, and includes the administration of an active compound of the invention (i.e., brincidofovir) to alleviate the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term "treatment" may also include treating in vitro cells or animal models.

[0138] As used herein, "a compound of the invention" refers to active compounds (eg, brincidofovir) as well as inactive compounds (eg, fillers, buffers, sweeteners, etc.).

[0139] The active compounds of the invention (i.e., brincidofovir) can also be used to prevent related diseases, conditions or disorders, or to identify suitable candidates for such purposes. As used herein, "prevention," "prevention," or "preventing" describes reducing, ameliorating, or eliminating the onset of symptoms or complications of such diseases, conditions, or disorders.

[0140] Brincidofovir may also be used to prevent diseases, such as viral infections. Prevention is understood to mean actions taken to prevent a disease. In the context of the present invention, prevention may mean treatment with, for example, brincidofovir.

[0141] As used herein, "therapeutically effective amount" refers to the amount necessary to make clinically observed improvements in patients. In some embodiments, the compounds of the present invention are formulated so that they contain an amount that does not cause one or more undesirable side effects. For example, the term "therapeutically effective amount" can refer to the amount of any one or more agents (e.g., brincidofovir) used to treat, improve or prevent an identified disease or condition (e.g., viral infection), or to show a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject depends on the subject's weight, size, and health; the nature and extent of the condition; and the dosing regimen of the therapeutic agent selected for administration.

[0142] The therapeutically effective amount for a given situation can be determined by routine experiments within the skill and judgment of the clinician. In a preferred aspect, the disease or condition to be treated is a viral infection. In addition, the medicament of an effective amount is an medicament that provides an objectively identifiable improvement noted by a clinician or other qualified observer.

[0143] As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, carriers and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment, without excessive toxicity, irritation, allergic response or other problems or complications, and commensurate with a reasonable benefit / risk ratio.

[0144] "Pharmaceutically acceptable excipient or carrier" refers to an excipient or carrier that can be used to prepare a pharmaceutical composition that is generally safe, non-toxic, neither biologically nor undesirably, and includes acceptable excipients for veterinary use as well as human drugs. "Pharmaceutically acceptable excipient" as used in the specification and claims includes one and more than one such excipient. Pharmaceutically acceptable excipients and carriers are listed above.

[0145] As used herein, "subject" is interchangeable with "subject in need thereof", both of which refer to subjects (e.g., patients) with a condition caused by a viral infection or subjects with an increased risk of developing a viral infection-related disease or condition relative to the population as a whole. "Subject" includes mammals. Mammals can be, for example, humans or suitable non-human mammals, such as primates, mice, rats, dogs, cats, cows, horses, goats, camels, sheep, or pigs. The subject can also be a bird or poultry. In one embodiment, the mammal is a human.

[0146] Representative "pharmaceutically acceptable salts" of brincidofovir include, for example, water-soluble and water-insoluble salts, such as acetate, stilbenesulfonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium salt, edetate, camphorsulfonate, carbonate, chloride, citrate, clavulanate, dihydrochloride, edetate, acetate, etopoate, ethanesulfonate, fumarate, glucoheptonate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinol, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxy The pharmaceutically acceptable salts include, but are not limited to, iodide, dithionate, lactate, lactobionate, laurate, magnesium, malate, maleate, mandelate, methanesulfonate, methyl bromide, methyl nitrate, methyl sulfate, mucate, naphthoate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1-methylene-bis-2-hydroxy-3-naphthoate, einbonate), pantothenate, phosphate / diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, acetate, succinate, sulfate, sulfosalicylate, succinate, tannate, tartrate, teoclate, toluenesulfonate, triethiodide, and valerate. In some embodiments, the pharmaceutically acceptable salt is a sodium, potassium, or calcium salt.

[0147] Brincidofovir

[0148] Brincidofovir (CMX001, BCV) is a lipid conjugate of cidofovir (CDV) that can be used to prevent and treat infections and diseases caused by double-stranded DNA viruses with high unmet medical needs. The present disclosure teaches IV formulations of brincidofovir not only as a way to deliver BCV to patients who cannot take oral medications, but also as a way to potentially reduce or avoid adverse GI events observed with oral administration.

[0149] As used herein, the term "brincidofovir" is understood to include the following neutral compounds:

[0150]

[0151] and any pharmaceutically acceptable salts thereof. In some of the examples below, brincidofovir is referred to as the "test item".

[0152] Cidofovir

[0153] Cidofovir, also known as CDV, has the following structure:

[0154]

[0155] Brincidofovir deamination products

[0156] In some embodiments, brincidofovir can be deaminated under aqueous conditions (e.g., at acidic pH) to produce the corresponding uracil derivative. The deaminated product is shown below:

[0157]

[0158] The present invention relates to aqueous and lyophilized formulations containing brincidofovir and methods of use thereof. As used herein, a "lyophilized formulation" refers to a formulation containing a certain amount of solid lyophilized brincidofovir, which is dissolved in a solvent (e.g., water or a sugar solution). A "lyophilized formulation" can be a pharmaceutical composition of the present invention.

[0159] Characteristics of pre-lyophilized preparations

[0160] As described in Example 4, the pre-freeze-dried preparation comprising arginine as a buffer and mannitol as a filler produces a white uniform solid, with only a small amount of remelting during freeze-drying. In addition, compared with other test preparations, the lyophilized product obtained by the pre-freeze-dried preparation comprising arginine as a buffer and mannitol as a filler only shows slight bubbling when reconstructed. For example, the preparation comprising sodium phosphate or pH-regulated water as a buffer shows bubbling when reconstructed, and generally requires a few minutes (for example, more than 30 minutes) before the foam dissipates. Similarly, it is found that the preparation comprising sucrose as a filler shows bubbling when reconstructed, and generally requires a few minutes (for example, more than 30 minutes) before the foam dissipates.

[0161] In addition, as shown in Example 4 below, the mannitol-based aqueous formulations of the present invention were also found to produce a more compact, uniform lyophilized cake after lyophilization compared to other formulations. For example, sucrose-based formulations exhibited at least partial cake collapse (and in some cases significant cake collapse) and increased remelting (e.g., extensive remelting) upon lyophilization. However, mannitol-based aqueous formulations were found to exhibit less cake collapse and less remelting.

[0162] Additionally, in some embodiments, phosphate-based buffers exhibit a pH change upon freezing during the lyophilization process compared to formulations using arginine as a buffer. Thus, the compositions described herein were found to have desirable properties (e.g., higher stability and better ease of use) compared to other formulations tested.

[0163] Liquid formulation stability and material compatibility

[0164] As described in Example 1, when the brincidofovir sample stock solution (50 μg / mL) in 50 mM phosphate buffer at pH 6.0-8.0 containing 20% ​​methanol was stored at 2-8°C, precipitation was observed at pH 7.0 and below. However, samples prepared at higher pH (e.g., pH = 7.5 and pH = 8.0) showed no observable changes in appearance (e.g., no precipitation was observed) throughout the experiment. For samples stored at 40°C, no physical changes in appearance were observed for the samples at any pH, and minimal changes in brincidofovir concentration were observed under all pH conditions studied at 40°C.

[0165] In the studies presented in Example 1, 5X sodium phosphate buffer stock solutions (e.g., at pH 6.0, 6.5, 7.0, 7.5, or 8.0), various tonicity modifiers (e.g., 66.5 mM NaCl, 68.8 mM glucose, 49.8 mM MgCl2, or 35.7 mM Ca D-gluconate), and various excipients (e.g., , disodium ethylenediaminetetraacetate (EDTA), hydroxypropyl β-cyclodextrin (HPbCD), polyethylene glycol (PEG) 300, ethanol, glycerol, propylene glycol (PG), N,N-dimethylacetamide (DMA) / N-methyl-2-pyrrolidone (NMP), polysorbate 80, albumin or benzyl alcohol, or a mixture thereof), a carrier for preparing a brincidofovir formulation, wherein the brincidofovir concentration is 6.4 mg / mL. Without wishing to be bound by theory, glucose or NaCl is included as a tension regulator and The test formulations containing 1, 2, 4, 5, 6, 7, 8, 12, 14, 16, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 67, 68, 70, 71, 72, 73, and ethanol did not precipitate at pH 7 within 12 days, but precipitated at lower pH values. The formulations did not precipitate at pH 7. Without wishing to be bound by theory, the HPLC recovery data described in Example 1 indicate that under storage conditions (2-8° C.) and c(BCV)=6.4 mg / mL, some formulations are not suitable for continuous dissolution of brincidofovir in a stable manner.

[0166] As shown in Example 2, the equilibrium solubility of brincidofovir was found to increase at higher pH in an aqueous infusion vehicle containing 100 mM phosphate and 69 mM glucose. For example, the equilibrium solubility of brincidofovir was determined to be approximately 25 mg / mL at pH = 6.9 and approximately > 270 mg / mL at pH = 8.0 in an aqueous infusion vehicle containing 100 mM phosphate and 69 mM glucose.

[0167] As described in Example 5, the solubility of the liquid formulation of the present invention in a 5% glucose solution to produce a liquid formulation comprising 10 mg / mL brincidofovir, 100 mM arginine and 5% (w / v) mannitol was 100% for at least 24 hours after dilution of the liquid formulation. In addition, the resulting solution remained clear and colorless, and no significant changes in pH were observed.

[0168] As described in Example 5 and Example 3, the compatibility of liquid formulations of brincidofovir (e.g., formulations comprising 10 mg / mL brincidofovir, 100 mM arginine and 5% (w / v) mannitol; formulations comprising 10 mg / mL or 15 mg / mL brincidofovir and 200 nM sodium phosphate; or formulations comprising 2 mg / mL brincidofovir and 4 mM sodium phosphate) with infusion carriers and a range of materials from manufacturing and clinical stages (e.g., sterilizing filters, vials and stoppers under upright and inverted conditions, infusion bags and infusion systems). In some cases, the formulations were diluted with 5% glucose before testing. Without wishing to be bound by theory, no significant loss of precipitation, pH changes, or recovery of brincidofovir was observed at the end of the study for any formulation.

[0169] Hemolytic Potential of Brincidofovir

[0170] In some embodiments, it is advantageous for a drug (eg, an intravenous drug) not to cause hemolysis (ie, not to destroy red blood cells).

[0171] Example 7 describes the hemolytic potential of brincidofovir using whole human or whole rat blood collected immediately (within 4 hours) using the cyanomethemoglobin method to assess hemolysis. This study measured hemoglobin released from erythrocytes in the presence of brincidofovir to determine the extent to which brincidofovir lyses erythrocytes.

[0172] Without wishing to be bound by theory, lysis of red blood cells may cause local reactions such as cellulitis, phlebitis and may affect organ function.The results presented herein demonstrate that the IV formulation of brincidofovir does not cause a hemolytic reaction when administered intravenously.

[0173] Rat and human whole blood (stabilized with K2EDTA) were obtained from 4 rats and 1 human donor. Each concentration (0.2, 0.5 and 1.6 mg / mL) of brincidofovir for rats was mixed with rat whole blood in a ratio of 1:4, while each concentration (0.2, 0.5 and 1.6 mg / mL) of brincidofovir for humans was mixed with human whole blood in a ratio of 1:4. All samples were then incubated at 37±1°C for 20 minutes. After incubation, blood samples containing BCV, as well as similarly prepared isotonic saline (negative control), 1% saponin (positive control), 5% glucose (vehicle control) and untreated whole blood control samples, were centrifuged and analyzed for hemolysis (supernatant hemoglobin concentration) using the cyanomethemoglobin method. Representative BCV samples in isotonic saline were analyzed to ensure that BCV did not interfere with the analysis of supernatant hemoglobin concentration.

[0174] Without wishing to be bound by theory, brincidofovir showed a hemolytic potential in rat whole blood that appeared to be concentration dependent, with signs of hemolysis observed at 1.6 mg / mL. However, brincidofovir was found to have no hemolytic potential in human whole blood up to the highest concentration tested, corresponding to final concentrations of 0.2, 0.5, and 1.6 mg / mL.

[0175] Thus, in some embodiments, brincidofovir can be safely administered to patients, including patients with blood disorders (eg, anemia).

[0176] Maximum Tolerated Dose and 7-Day Dose-Range Study (DRF) in Rats

[0177] Example 8 gives the maximum tolerated dose of BCV in rats and a 7-day dose range determination (DRF) study. The results describe the acute range determination of rats in detail. In some embodiments, this data can be used to generate information to allow people to reasonably set the dosage used in further toxicology studies.

[0178] Phase 1

[0179] Phase 1 is designed to determine the maximum tolerated dose (MTD) of brincidofovir after intravenous infusion (2 hours) in rats. Phase 1 contains a single step-by-step increasing dose arm to assess any acute toxicity (e.g., "toxic syndrome") that may present general clinical signs, typically within a 48+ / -hour period of exposure to BCV. Without wishing to be bound by theory, the increasing dose arm may identify any obvious signs or symptoms of an overdose.

[0180] In the phase 1 of the study, when administered by a single 2-hour IV infusion, all doses were well tolerated. Therefore, the maximum tolerated dose of brincidofovir is not determined. However, based on the in vitro evidence of hemolysis under the brincidofovir concentration of more than 0.75mg / mL, the 15mg / kg delivered by the solution of 2-hour IV infusion of 0.75mg / mL brincidofovir represents the maximum feasible dose. In other words, even at the maximum feasible dose tested, no corresponding effects (for example, on average body weight or weight gain) are observed in rats. In addition, there is no effect in ophthalmology, hematology, clinical chemistry or urinalysis parameters. Even at a higher dosage level than oral administration, rats do not find obvious toxicity or death.

[0181] Phase 2

[0182] Phase 2, the repeated dose range determination (DRF) phase (Phase II), is designed to evaluate the toxicity of brincidofovir and the plasma toxicokinetics of brincidofovir and one of its metabolites, cidofovir. When administered intravenously to animals and humans, cidofovir is known to cause nephrotoxicity. Toxicokinetic parameters were evaluated after the first and last doses of two dose levels of brincidofovir (1 mg / kg and 15 mg / kg) were administered by intravenous (2 hours) infusion on days 1, 3, and 7.

[0183] In the Phase 2 of this study, animals received short-term treatment (3 doses over the course of 9 days) with high (15 mg / kg) or low (1 mg / kg) BCV IV infusion doses. Without wishing to be bound by theory, the Phase II of this study was used to determine whether any potential cumulative toxicity might occur with repeated dosing. Without wishing to be bound by theory, the Phase II also included a toxicokinetic arm to determine the IV toxicokinetics established after a single (first dose) and repeated (last dose) BCV IV infusion doses.

[0184] In the second phase of the study, peak brincidofovir concentrations were generally observed at the end of the 2-hour intravenous infusion and declined rapidly.max The increase in AUC was roughly proportional to the dose from 1 to 15 mg / kg, although AUC was determined at several concentrations after the 1 mg / kg dose. max and AUC last The values ​​show a trend that is lower on the 7th day compared to the 1st day (the 7th day / 1st day cumulative ratio (AR) is 0.44 to 0.54). In addition, no gender differences in the TK parameters of brincidofovir are observed. Without wishing to be bound by theory, the exposure increase of the metabolite cidofovir is less than the ratio of the increase in the brincidofovir dosage, and there is no obvious gender difference in the TK parameters, and the TK parameters do not change after repeated administration. Under the conditions of this study, a single dose of 1 or 15 mg / kg of brincidofovir was well tolerated by 2 hours IV infusion on the 1st, 4th and 7th days.

[0185] Brincidofovir: 28-day intravenous infusion toxicity study in rats with a 14-day recovery period

[0186] Example 10 details a repeated dose subchronic study with IV BCV in rats. Without wishing to be bound by theory, this study explores the cumulative biological effects (e.g., clinical, macroscopic and microscopic) of brincidofovir administered at a range of doses. The effect can be qualitative, such as target organs, the nature of the effect or quantitative, such as the plasma or tissue levels at which the effect is observed. Without wishing to be bound by theory, this study can determine the possibility of toxicity and recovery or progression.

[0187] As described in Example 10, and without wishing to be bound by theory, animals did not experience diarrhea in this study. During the study, all animals gained the expected weight. There were no GI manifestations in the intestine at the final necropsy. There were no clinical findings in life at the highest dose of IV brincidofovir (15 mg / kg). No elevation of transaminases was observed.

[0188] Without wishing to be bound by theory, a significant reduction in epithelial cells in the intestinal villi was observed after oral administration of brincidofovir. In contrast, minimal single cell effects were noted after IV administration of brincidofovir. For example, Fig. 7A Histogram showing the histogram of rat intestine after oral administration of brincidofovir, Figure 7B A histogram is shown of the rat intestine following IV administration of brincidofovir. As shown, IV administration of brincidofovir was better tolerated in the intestine.

[0189] As described in Example 10, Sprague-Dawley leukocytes were administered at 0 (2X sodium phosphate buffer solution), 1, 4, or 15 mg / kg / dose. Rats (15 / sex / group) were dosed with brincidofovir by intravenous infusion over 2 hours twice weekly for 28 days. Up to 5 animals / sex / group recovered within 14 days after dosing.

[0190] Administration of brincidofovir resulted in intermittent (usually post-infusion) clinical signs (primarily abnormal breathing and hunched posture) in some animals of both sexes at ≥4 mg / kg / dose and minimally reduced food consumption in males and females at ≥1 mg / kg / dose and 15 mg / kg / dose, respectively.

[0191] Brincidofovir-related findings were present in the male reproductive tract (testicular germ cell loss, luminal cell debris in the epididymis and increased epithelial cell apoptosis in the seminal vesicle) and intestine (single cell necrosis) at ≥4 mg / kg / dose, and sebaceous glands (atrophy) at 15 mg / kg / dose. All findings associated with Brincidofovir were fully recovered, except for male testicular changes at ≥4 mg / kg / dose, which, without wishing to be bound by theory, were not expected to fully recover within 2 weeks. In addition, some animals administered 15 mg / kg / dose had reduced bone marrow cellularity and unilateral or bilateral flexibility and / or small testicles and epididymis at the end of recovery.

[0192] Under the conditions of this study, the no observed adverse effect level (NOAEL) for brincidofovir was considered to be 1 mg / kg / dose in males and 4 mg / kg / dose in females based on testicular germ cell depletion in males at ≥4 mg / kg / dose and moderate intestinal single cell necrosis in 1 female at 15 mg / kg / dose.

[0193] Therefore, in some embodiments, the study can provide support for the start or continuation of human clinical studies. In some embodiments, the study can also be used to establish the dosage for long-term studies. Without wishing to be bound by theory, this study can also guide parameters, such as the number of animals evaluated when conducting long-term studies. The study also includes a toxicokinetic arm after the first and last dose administration.

[0194] Single oral or intravenous dose [ 14 Mass balance, pharmacokinetics, and tissue distribution of brincidofovir in rats by quantitative whole-body autoradiography

[0195] Studies were conducted to characterize, 14 Tissue distribution of total radioactivity in male Sprague-Dawley (SD) and Long-Evans (LE) rats following a single intravenous (IV, 2-hour infusion) or oral (PO, gavage) dose of [C]brincidofovir. 14Excretion rate (mass balance) and pharmacokinetics (PK) of total radioactivity in male Sprague-Dawley rats following administration of [C] brincidofovir.

[0196] Administer 15 mg / kg of [ 14 C] Brincidofovir was administered to pigmented and non-pigmented rats. Tissue distribution was determined by quantitative whole-body autoradiography at a time point of 35 days after administration.

[0197] Radioactivity was well distributed after IV or oral administration, and the qualitative distribution pattern of radioactivity was similar. Quantitatively, tissue radioactivity concentrations in small intestinal tissues after IV administration were approximately 1 / 10 of those in small intestinal tissues after oral administration. For most other tissues, tissue radioactivity exposure after IV administration was generally higher than after oral gavage administration. Peak radioactivity concentrations in most tissues occurred 4 to 8 hours after oral administration, or at the end of a 2-hour intravenous infusion. Regardless of the route of administration, tissues with the highest radioactivity concentrations were associated with clearance or elimination organs, such as the liver, kidney, and small intestine. The tissue-to-plasma ratio (T / P) in these organs was high (>30), and the renal cortex and liver were similar for both IV and oral routes of administration. The tissues with the lowest radioactivity concentrations were the brain, spinal cord, skeletal muscle, white adipose tissue, and bone. The association of radioactivity in the brain and spinal cord after IV administration was higher (approximately 20% of plasma concentrations and approximately 5% after oral administration). At 35 days after administration, radioactivity was below the limit of quantification in all tissues except bone marrow, lymph nodes, spleen, and adrenal glands after IV administration. This is different from the residual concentration tissues (renal cortex, liver, and small intestine) after oral administration. No evidence of a specific association with melanin-containing tissue (eye, uvea) was detected.

[0198] Compared with the oral route, [ 14 IV administration of brincidofovir resulted in lower small intestinal concentrations of BCV-related radioactivity, which is consistent with the lower incidence and severity of GI findings in toxicology studies in rats following repeated IV BCV administration.

[0199] In some embodiments, single cell necrosis in the small and large intestine is occasionally noted following IV BCV infusion. These mild and reversible GI findings can be managed with IV infusion. 14 C] brincidofovir hindgut tissue lower radioactive concentrations explained. In addition, GI found that no dose-limiting GI toxicity (i.e., gastropathy, enteropathy or enteritis) was caused. Therefore, the present invention provides an IV preparation of BCV and an IV BCV administration method, which can be used to alleviate GI interference associated with BCV.

[0200] Compared with oral BCV, IV 14Distribution of radioactivity after brincidofovir infusion was higher in all tissues except the small intestine, where concentrations were approximately 1 / 10 of those after oral administration.

[0201] Summary of Excretion and Distribution

[0202] Excretion after IV administration

[0203] In male SD rats, 15 mg / kg was intravenously infused with 14 C] After 2 hours of brincidofovir, the major route of radioactivity elimination was urine, which accounted for a mean of 51.2% of the administered dose during the 168-hour collection period. A mean of 42.2% of the administered dose was recovered in the feces. The total recovery of radioactivity in urine and feces of Group 1 male rats averaged 93.5% of the dose during the 168-hour collection period. Most (~86%) of the urinary and fecal excretion of radioactivity occurred within the first 24 hours after dosing.

[0204] Distribution after IV administration

[0205] Two hours after IV infusion of 15 mg / kg (Group 2) in male SD rats, the plasma levels of 14 C]Brincidofovir total radioactivity max It is 10.3 μg equivalent / mL, which occurs at T max The concentration decreased to 0.045 μg eq / mL by 72 hours after administration. 14 C] AUC of total radioactivity of brincidofovir last is 64.4 μg equivalent·h / mL, and t 1 / 2 It is 13.0h.

[0206] [ 14 C]Brincidofovir-derived radioactivity was well distributed to most tissues of albino and pigmented male rats after 2 h of intravenous infusion at 15 mg / kg, and concentrations in most tissues were slightly higher than plasma, especially in excretory and digestive tract tissues, which were much higher than plasma (tissue AUC all When the concentrations in most tissues were between 1.0 and 6.0 μg equivalent / g, at 2 hours post-dose (i.e., end of infusion) [ 14 C] brincidofovir-derived radioactive C max was discovered.

[0207] The blood to plasma ratio generally shows that, after IV infusion into rats, [ 14[C]Brincidofovir-derived radioactivity was similarly distributed, ranging from 0.89 to 1.46 between 1 and 24 hours after the start of infusion. At 72 hours, blood-to-plasma ratios of 1.42 and 3.06 were observed after dosing with 2 mg / kg and 15 mg / kg, respectively.

[0208] 15 mg / kg intravenous infusion for 2 hours, at the corresponding T max The highest concentrations (>20.0 μg eq / g) were observed in the liver, renal cortex, small intestine, renal medulla, and bladder of albino and pigmented male rats at the corresponding T max The tissues with the lowest concentrations (<1.0 μg equivalent / g) were: brain, spinal cord, white fat, bone, skeletal muscle and lens of the eye.

[0209] After a 2-hour intravenous infusion of 15 mg / kg, the highest total concentrations measured by QWBA (>400 μg equiv / g) were observed in bladder contents, bile, and digestive tract contents, reflecting the 14 C] Elimination pathways of brincidofovir-derived radioactivity.

[0210] Tissue concentrations declined steadily in most tissues, but elimination of drug-related material was incomplete in most tissues by 168 hours postdose; however, all tissues containing radioactivity had concentrations <1.0 μg equivalent / g. Radioactivity was still present in the bone marrow, lymph nodes, spleen, and adrenal glands at 840 hours postdose (the time point of the last sampling), but they were close to the LLOQ in these tissues, except for the spleen. 1 / 2 (with sufficient data points and acceptable r 2 ) ranged from 14.2 hours in the blood (heart) to 378 hours in the spleen. 1 / 2 The tissues with the longest time were spleen (378 h), lymph nodes (345 h), non-pigmented skin (310 h), and bone marrow (276 h). T of tissues with quantifiable concentrations at 840 h 1 / 2 The range was 254 hours (adrenal gland) to 378 hours (spleen).

[0211] A similar tissue distribution pattern was observed in rats given a 2-hour IV infusion at a lower dose of 2 mg / kg. Most tissue concentrations after 2 mg / kg IV administration increased roughly proportionally to the dose compared to the 15 mg / kg dose (i.e., a 4- to 15-fold change in concentration for a 7.5-fold increase in dose). However, in the small intestine, the increase in small intestinal concentrations was greater than proportional as the dose increased from 2 mg / kg to 15 mg / kg.

[0212] Distribution after oral administration

[0213] After PO administration of 15 mg / kg to male LE rats, plasma [ 14 C]Brincidofol total radioactivity C max In T max At 8 hours, the concentration was 1.5 μg equivalent / mL, and 24 hours after administration, the concentration decreased to 0.180 μg equivalent / mL. 14 C] AUC of total radioactivity of Brincidofol last The t is 21.0 μg equivalent·h / mL. Due to insufficient data points, t cannot be determined. 1 / 2 .

[0214] Blood to plasma ratios showed that male LE rats given a PO dose of [ 14 C]brincidofovir, [ 14 C]Brincidofovir-derived radioactivity was similarly distributed from 1 to 24 h (range 0.76-1.13).

[0215] After PO administration at 15 mg / kg, [ 14 C]Brincidofovir-derived radioactivity was well distributed to most tissues of both pigmented and non-pigmented male rats, and most tissues had slightly higher plasma concentrations, especially for excretory and digestive tract tissues, where the levels were much higher (tissue AUC all When the concentrations in most tissues were between 0.7 and 17.8 μg equiv / g, [ 14 C] brincidofovir-derived radioactive C max was discovered.

[0216] After PO administration at 15 mg / kg, max The highest concentrations (>3.3 μg equivalent / g) were observed in the small intestine, liver and kidney cortex, stomach (gastric mucosa), renal medulla, cecum, esophagus, and large intestine of pigmented male rats under 40 °C. max The tissues with the lowest concentrations (<1.0 μg eq / g) were: spinal cord, brain, bone, white fat, and lens of the eye.

[0217] Following PO administration of 15 mg / kg, the highest total concentrations measured by QWBA (>36.0 μg equiv / g) were observed in bladder contents, bile, and digestive tract contents, reflecting the 14 C] Elimination pathways of brincidofovir drug-derived radioactivity.

[0218] Tissue concentrations in most tissues of pigmented male rats declined steadily, but elimination of drug-related material was incomplete in most tissues by 168 hours post-dose; however, concentrations were <0.4 μg equiv / g in all tissues except the small intestine (5.9 μg equiv / g). Radioactivity was still present in the renal cortex, liver, and small intestine at 840 hours post-dose (the time point of the last sampling), but was close to the LLOQ. 1 / 2 About 5 hours. All tissues 1 / 2 (with sufficient data points and acceptable r 2 ) ranged from esophageal 37.2 hours to fat (brown) 234 hours. 1 / 2 The only tissues with reliable t 1 / 2 and 840 h in the tissue with quantifiable concentrations was the small intestine at 98.1 h.

[0219] Comparison of tissue distribution after PO administration and 2-hour IV infusion

[0220] Long-Evans and Sprague-Dawley rats were given a 2-hour intravenous infusion or oral gavage. 14 C] After brincidofovir, AUC all The blood to plasma ratio ranged between 1.1 and 1.4. For individual points, the blood to plasma ratio ranged from 0.64 to 1.5, with a median of 1.00. Without wishing to be bound by theory, the similar distribution of radioactivity between blood and plasma following the two routes of administration suggests that [ 14 C] Minimal restriction of distribution of brincidofovir from plasma to blood cells.

[0221] A single oral dose of 15 mg / kg 14 C] The maximum observed tissue concentration of brincidofovir in rats was reached at 4 or 8 hours, and the peak concentration was generally about 10-25% of the peak tissue concentration observed in rats given a 2-hour IV infusion. However, in the small intestine, a 6-fold higher Cmax was observed after oral administration compared with IV. The oral bioavailability of radioactivity (AUC all PO / AUC all Compared with IV, lower tissue AUC ratios (AUC PO / AUC IV<25%), particularly in the central nervous system, skin, skeletal muscle, and some endocrine and reproductive tissues. By both routes, tissues with the highest concentrations of radioactivity were associated with organs of excretion (i.e., liver, kidney, and intestine). For each of these tissues, tissue-to-plasma ratios were high (>30), and in the renal cortex and liver, tissue / plasma ratios were similar between the IV and oral routes of administration. The small intestine had the highest tissue-to-plasma ratio after oral administration (T / P = 428), as expected based on the route of administration, however, following IV infusion, significant radioactivity was associated with the small intestine (tissue-to-plasma ratio 32). Tissue elimination was incomplete at 840 hours after oral administration, with radioactivity present in the renal cortex, liver, and small intestine after PO administration, which was different from that observed 2 hours after IV infusion at the same time point. In the small intestine, 168-hour residual concentrations were significantly higher in rats dosed orally (5.9 μg equiv / g) than in rats dosed by IV infusion (tissue concentration BQL) [ 14 C] brincidofovir in rats. At 840 hours, residual concentrations in the vascular / lymphoid tissue system were much higher after 2 hours of IV infusion compared with concentrations observed after PO administration (BQL).

[0222] Distribution in pigmented tissues

[0223] Comparison of the tissue distribution of radioactivity between non-pigmented and pigmented tissues showed that concentrations in pigmented tissues (i.e., uvea of ​​the eye and pigmented skin) were similar to those observed in non-pigmented tissues, suggesting that [ 14 C]Brincidofovir drug-derived radioactivity was not specifically associated with melanin.

[0224] Without wishing to be bound by theory, in most tissues, higher concentrations of radioactivity were observed after IV administration, which may reflect higher blood concentrations. An exception was the gastrointestinal tissues. From highest to lowest, the tissues with the highest concentrations were the liver, renal cortex, small intestine, and renal medulla for intravenous administration, and the small intestine, liver, renal cortex, renal medulla, and cecum for oral administration. The tissues with residual drug after 840 hours were the adrenal glands, spleen, lymph nodes, and bone marrow for IV administration, and the small intestine, liver, and renal cortex for oral administration.

[0225] Thus, in some embodiments, lower doses of BCV administered intravenously can provide similar plasma concentrations as observed at higher doses administered orally. Thus, the present disclosure provides for treating viral infections in subjects in need thereof using lower doses of BCV than required for oral administration.

[0226] Pharmacokinetics of brincidofovir IV administration

[0227] As described in Example 11 below, brincidofovir was administered orally and intravenously to healthy subjects. It was found that intravenous administration of 10 mg brincidofovir provided similar exposure to 100 mg of brincidofovir administered orally. Therefore, the present invention teaches intravenous administration of brincidofovir. In some embodiments, no drug-related adverse events (e.g., no gastrointestinal events) were observed when brincidofovir was administered intravenously. In some embodiments, no graded laboratory abnormalities (e.g., no hemolytic toxicity and no renal toxicity) were observed.

[0228] Example

[0229] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope or spirit of the present invention to the specific procedures described herein. It should be understood that examples are provided to illustrate certain embodiments, and are therefore not intended to limit the scope of the present invention. It should be further understood that, without departing from the spirit of the present invention and / or the scope of the appended claims, various other embodiments, modifications and equivalents that can be proposed by those skilled in the art can be solved.

[0230] This section provides abbreviations and definitions of terms and concepts that may be commonly used throughout.

[0231] AUC Area under the time-concentration curve

[0232] AUC all The area under the time-concentration curve including all time points

[0233] AUC inf_obs The area under the time-concentration curve extrapolated to infinite observations

[0234] AUC last The area under the time-concentration curve containing the last time point with data above the lower limit of quantitation

[0235] BQL Below Detection Limit

[0236] ℃ Celsius

[0237] C(BCV) Concentration of brincidofovir in liquid or aqueous preparations

[0238] C(Bulking Agent) Concentration of bulking agent in liquid or aqueous preparations ca Circa (approximately)

[0239] C max Maximum observed concentration

[0240] cpm counts per minute

[0241] %CV Percentage of coefficient of variation DMA N,N-Dimethylacetamide dpm disintegration per minute

[0242] EDTA Disodium Ethylenediaminetetraacetate FVC Femoral vein cannula

[0243] g

[0244] h hour

[0245] HPLC High Performance Liquid Chromatography

[0246] HPbCD Hydroxypropyl β-cyclodextrin

[0247] IV

[0248] kg kilogram

[0249] L Liter

[0250] LE Long-Evans

[0251] LLOQ Lower limit of detection

[0252] LSC Liquid Scintillation Counting

[0253] MB Mass Balance

[0254] MBq Megabecquerel

[0255] mCi / mmol Curie per millimole MCID Microcomputer Image Device MDC Molecular Dynamics Counting mg milligram

[0256] mSv millisievert

[0257] μg equivalent / g Microgram equivalent per gram of tissue min

[0258] mL milliliters

[0259] mm Millimeters

[0260] n is the number of observations

[0261] NA Not applicable

[0262] NI Not Identified

[0263] NC Not calculated

[0264] NS Not sampled

[0265] NMP N-Methyl-2-pyrrolidone

[0266] PEG Polyethylene glycol

[0267] PG Propylene Glycol

[0268] PK Pharmacokinetics

[0269] PO Oral (oral dosage)

[0270] ROA route of administration

[0271] SD Sprague Dawley or standard deviation

[0272] t 1 / 2 Terminal elimination half-life

[0273] T max C max Time

[0274] ULOQ Upper limit of detection

[0275] QWBA quantitative whole body autoradiography

[0276] Example 1 - Liquid formulation development

[0277] Various common liquid formulations (ie, pre-lyophilized formulations or bulk formulations) were prepared and tested to develop an intravenous formulation of brocidofovir.

[0278] pH stability of brincidofovir: brincidofovir sample stock solution (50 μg / mL) was prepared in 50 mM phosphate buffer at pH 6.0-8.0 containing 20% ​​methanol. The sample was aliquoted and stored at two temperatures (T = 2-8 ° C and T = 40 ° C), and pH and brincidofovir recovery were monitored at multiple time points over a two-week period. Under both conditions, the sample maintained pH levels throughout the experiment.

[0279] Storage at 2-8°C: For samples stored at 2-8°C, precipitation was observed at t=2 days for pH 6.0 and 6.5. For pH 7.0, precipitation was observed at t=4 days. Samples prepared at higher target pH (pH=7.5 and pH=8.0) showed no observable changes in appearance throughout the experiment, while samples prepared at pH 6.0, 6.5, and 7.0 showed significantly reduced brincidofovir concentrations relative to the initial time point. This reduction may be related to the observed precipitation. Changes in pH 7.0 were observed at t>8 days. An impurity (RT=4.7 minutes) was detected that exceeded the set total peak area threshold for peak integration. For samples at pH 6 and 6.5, the percentage of the total peak area was less than 0.4%, and for samples at higher pH, the percentage of the total peak area was less than 0.1%.

[0280] Storage at 40°C: For samples stored at 40°C, no physical changes in the appearance of the samples were observed at any pH. Minimal changes in brincidofovir concentrations were observed at 40°C for all pH conditions studied. Although the concentrations of brincidofovir showed some fluctuations, no significant trends were observed. The summary display of the HPLC chromatograms reveals differences in impurity formation under different pH conditions during the experiment. For the lower pH samples, peaks associated with deamination products of brincidofovir were observed, including 1.91 area % at pH 6.0 and 0.58 area % at pH 6.5.

[0281] No degradation product peaks were observed at neutral pH and above.

[0282]

[0283] Preparation and characterization of brincidofovir formulations

[0284] Preparation of carriers and formulations: The carrier of the brincidofovir formulation is prepared from an organic excipient, a 5X sodium phosphate buffer stock solution (pH 6.0, 6.5, 7.0, 7.5 and 8.0), and a stock solution of a tonicity regulator and EDTA. Solid brincidofovir was dissolved in the carrier to obtain a concentration of c (BCV) = 6.4 mg / mL. The appearance (including precipitation when stored at 2-8 ° C) was evaluated and the pH was measured. The results are summarized in Table 1.

[0285] Table 1: Precipitation status and pH of vehicle and formulations. All formulations except formulation #30-32 were prepared with sodium phosphate buffer (100 nM).

[0286]

[0287]

[0288] 1 Formulations were prepared at ambient temperature and maintained at ambient conditions until the time point of Day 1. On Day 1, hemolysis assays were performed, after which samples were stored at 2-8°C. 2 N: No precipitation was observed. 3 Y: Precipitation was observed. 4 N / A: Not applicable or not measured.

[0289] The vehicle was a clear, colorless solution. Precipitation was observed for vehicles #11 and #12, while vehicle #29 was a clear, light yellow solution. Formulations #11 and #12 inherited the precipitation of their vehicle. Precipitation was observed for formulations #18, #30, #31, and #32 on day 1 after preparation. Formulations were stored at 2-8°C starting on day 2 and appearance was monitored over 12 days.

[0290] Brincidofovir Recovery: The brincidofovir formulations of Table 1 were analyzed for c(BCV) on days 2 and 7 after preparation. Formulations showing initial precipitation were not tested on day 2. Formulations showing precipitation until day 7 were analyzed by centrifugation and HPLC recovery. The results are summarized in Table 2.

[0291] Table 2: HPLC recovery of brincidofovir formulations

[0292]

[0293] It was found that carriers #11 and #12 formed precipitates during formulation preparation (i.e., addition of brincidofovir). Formulations with low pH in the presence of solubilizing excipients such as PEG 300, polysorbate 80 and ethanol (Formulations #1, #6, #18 and #22) precipitated, while formulations with the same composition but higher pH (Formulations #2, #7, #19 and #24) retained the ability to solubilize brincidofovir (Table 1). Purely organic formulations (Formulations #30, #31 and #32) showed limited solubilization ability for brincidofovir.

[0294] HPLC recovery data indicated that some formulations were not suitable for continuous dissolution of brincidofovir under storage conditions (2-8°C) and c(BCV) = 6.4 mg / mL. In addition, the data indicated that formulations with a pH greater than or equal to about 8.0 could dissolve brincidofovir.

[0295] Additional formulations (Formulations #33-40 and #49-56) were prepared and tested for brincidofovir equilibrium solubility and filterability.

[0296] Formulation vehicles #33-40 and #49-56 were saturated with brincidofovir and rotated under ambient conditions. The recovery of brincidofovir in the sample supernatant was determined at various time points to determine when solubility equilibrium was reached.

[0297] The saturated supernatant of equilibrium solubility study is subjected to filterability assessment.For this reason, about 900 μ L of saturated solution is filtered by syringe filter (25 mm, 0.2 μ m PES membrane).The ease of filtration is recorded, the pH in the filtrate is measured, and the recovery of brincidofovir is determined by HPLC analysis.In order to assess whether aseptic filtration introduces impurities, the HPLC chromatogram of saturated formulation carrier is qualitatively compared and displayed before and after filtration.The composition of each in preparation #33-40 and #49-56, and their pH, c (BCV) and the ease of filtration under solubility equilibrium are summarized in Table 3.

[0298] Table 3: Summary of brincidofovir solubility and filterability evaluations. The buffer in all formulations was 100 mM sodium phosphate.

[0299]

[0300]

[0301] The equilibrium solubility range of brincidofovir in the test formulation is 4 to 30 mg / mL, and the average solubility c(BCV)=25 mg / mL in the range of apparent pH=6.3-6.9 (ambient temperature). In some embodiments, the relatively high brincidofovir concentrations observed in the equilibrium solubility test result in significant changes in the carrier pH. Filtration of the saturated formulation of brincidofovir through the selected syringe filter did not find any problems with ease of filtration, loss of brincidofovir, or introduction of impurities.

[0302] Example 2 - Brincidofovir Formulation Testing

[0303] A formulation vehicle containing 100 mM phosphate and 69 mM glucose (Vehicle / Formulation #57) was prepared and further tested for a) equilibrium solubility and b) filterability.

[0304] a) Brincidofovir Solubility Assessment

[0305] Two samples of carrier #57 were saturated with brincidofovir. One sample carrier was saturated with brincidofovir without further pH adjustment (resulting in a formulation pH of 6.9), and the pH of the second sample was readjusted to pH=8.0 using sodium hydroxide solution (1N). After brincidofovir dissolves, the pH drifts to lower pH values, which in turn changes the solubility of brincidofovir until equilibrium is reached. Therefore, before adding more brincidofovir, the pH must be brought close to the target pH. For pH-adjusted samples, the brincidofovir concentration was not reached under equilibrium solubility conditions. After supplementing the sample with approximately 300 mg / mL additional brincidofovir solid, the addition of solid brincidofovir was stopped.

[0306] The recovery of brincidofovir in the two sample solutions / supernatants was determined at equilibrium and after 24 hours (HPLC determination). At ambient temperature, at an apparent pH of pH = 6.9, the equilibrium solubility of brincidofovir for formulation 57 was c(BCV) = 25 mg / mL, and 26 mg / mL after 24 hours. For the sample at pH = 8, the observed concentration of brincidofovir after 24 hours was 348 mg / mL, and equilibrium solubility was not reached when c(BCV)>270 mg / mL.

[0307] b) Filterability evaluation

[0308] The filterability of preparation #57 was tested at c(BCV)=20, 10 and 0.5 mg / mL. The test solution of c(BCV)=20 mg / mL was adjusted to pH=8.0 using NaOH solution (1N) and applied as a stock solution to prepare a test solution of lower concentration by diluting with D5W. The test solution was filtered through a syringe filter (25 mm, 0.2 μm PES membrane) (5 mL filter passed). The ease of filtration was observed, and the recovery (HPLC determination) of pH and brincidofovir was recorded before and after filtration. No significant changes in pH, loss of brincidofovir or introduction of impurities were observed (Table 4).

[0309] Table 4: Filterability Evaluation of Brincidofovir in Formulation 57 Vehicle

[0310]

[0311] Example 3 - Formulation Studies and Evaluation

[0312] A composite process for the preparation of brincidofovir formulations was developed and the formulation without a tonicity regulator (glucose) was detailed. For stability studies and testing, the following brincidofovir formulations were prepared at scales up to 0.5 L:

[0313] 10mg / mL brincidofovir, 200mM sodium phosphate, pH=8.0, 69mM glucose

[0314] 15mg / mL brincidofovir, 200mM sodium phosphate, pH=8.0, 69mM glucose

[0315] 10 mg / mL brincidofovir, 200 mM sodium phosphate, pH = 8.0 (no tonicity modifier)

[0316] Preparations containing tonicity modifiers (including glucose)

[0317] Brincidofovir formulations containing glucose as a tonicity regulator were used in stability study I and infusion vehicle compatibility assessment. An outline of the corresponding formulation formulation is as follows:

[0318] 1. Prepare 500 mM (2.5X) sodium dihydrogen phosphate solution and 500 mM (2.5X) sodium hydrogen phosphate solution by dissolving the corresponding sodium phosphate species in deionized water using a volumetric flask.

[0319] 2. Mix 500 mM (2.5X) sodium dihydrogen phosphate and disodium hydrogen phosphate solutions at pH to prepare 500 mM (2.5X) sodium phosphate buffer solution at pH = 8.0.

[0320] 3. Prepare a carrier stock solution of 400 mM (2X) sodium phosphate, 138 mM (2X) glucose at pH = 8.0 by dissolving solid glucose in 500 mM (2.5X) sodium phosphate solution at pH = 8.0 and perform quantitative analysis (QS) with water.

[0321] 4. Use a volumetric flask to dissolve brincidofovir in a carrier stock solution (400mM (2X) sodium phosphate, 138mM (2X) glucose solution, pH = 8.0) at 2 times the desired final concentration. Under pH control, sodium hydroxide solution is added to adjust the solution to pH = 8.0. Quantitative analysis (QS) is performed with deionized water to obtain a formulation containing 200mM (1X) sodium phosphate, 69mM (1X) glucose, pH = 8.0 at the desired brincidofovir concentration.

[0322] Tonicity-free formulations (without glucose)

[0323] Brincidofovir formulations lacking glucose were used in stability study II and in adsorption assessments, and most experiments were related to material compatibility assessments. Formulations without tonicity modifiers were prepared as follows:

[0324] 1. Prepare 400 mM (2X) sodium dihydrogen phosphate solution and 400 mM (2X) sodium hydrogen phosphate solution by dissolving the corresponding sodium phosphate species in water (volume flask).

[0325] 2. Mix 400 mM (2X) sodium dihydrogen phosphate and disodium hydrogen phosphate solutions under pH control to prepare a 400 mM (2X) sodium phosphate buffer solution (carrier solution) with a pH of 8.0.

[0326] 3. Using a 500 mL volumetric flask, dissolve brincidofovir in a carrier solution (400 mM (2X) sodium phosphate solution at pH 8.0) at twice the final concentration. NaOH 1N sodium hydroxide solution was added to a volume of [mL] = 1.65 x mBCV [g]. Quantitative analysis (QS) was performed with deionized water to obtain a formulation with the desired brincidofovir concentration in 200 mM sodium phosphate buffer at pH = 8.0.

[0327] 4. To prepare lower brincidofovir concentrations, the prepared formulations were diluted with the corresponding carrier to the desired strength. Stability Study I: Brincidofovir formulations containing a tonicity modifier (glucose)

[0328] Design and Setup: In this study, the chemical and physical stability of Formulation 57 was evaluated. Testing of chemical degradation was performed in an accelerated stability study setup, which applied heat as an accelerator / stressor. Arrhenius analysis was performed to provide API half-life and degradation rates at lower temperatures. Physical stability was evaluated at the desired storage temperature (2-8°C); baseline / reference conditions were also included in the study. The setup of the study was as follows:

[0329] Test solution:

[0330] 10 mg / mL brincidofovir, 200 mM sodium phosphate, 69 mM glucose, pH = 8.0,

[0331] 15 mg / mL brincidofovir, 200 mM sodium phosphate, 69 mM glucose, pH = 8.0,

[0332] Vehicle Control 1: 200 mM sodium phosphate, 69 mM glucose, pH = 8.0 (only at 75°C),

[0333] Vehicle Control 2: 200 mM sodium phosphate, pH = 8.0 (only at 75°C).

[0334] Stabilizing station temperature:

[0335] Accelerated stability study: 50℃, 60℃, 65℃, 70℃, 75℃,

[0336] Physical degradation and benchmark stability studies: 2-8°C, 25°C.

[0337] Time:

[0338] Accelerated stability study: initial, 2 days, 6 days, 9 days, 14 days,

[0339] Physical degradation and baseline stability studies: initial, 14-day and 28-day,

[0340] Vehicle control: Initial, 1, 5, 8 and 14 days.

[0341] test:

[0342] Brincidofovir formulations: appearance, pH, c(BCV) recovery (HPLC), purity (HPLC), and liquid particle count (physical degradation and benchmark stability studies),

[0343] Vehicle control: appearance and pH.

[0344] The stability test results (accelerated conditions) of the 10 mg / mL and 15 mg / mL formulations during the stability study, i.e., the change in pH and the trend of brincidofovir recovery, are summarized in Tables 8 and 9, respectively. The accelerated stability study with a strong biphasic rate trend does not allow Arrhenius analysis and calculation of brincidofovir half-life or extrapolation to degradation rate under typical storage conditions. The results of the vehicle control test in the accelerated stability study (75°C station only) are summarized in Table 5.

[0345] Table 5: Accelerated stability study results of 10 mg / mL brincidofovir formulations (Study I)

[0346]

[0347]

[0348] 1 The yellowness of the solution is indicated on a scale of 0 to 10: 0, colorless; 5, dark yellow; 10, amber. 2 Average of three (3) replicates. 3 BCV recovery = c(BCV) n天 / c(BCV) 0天 ×100%.

[0349] Table 6: Accelerated stability study results of 15 mg / mL brincidofovir formulations (Study I)

[0350]

[0351]

[0352] 1 The yellowness of the solution is indicated on a scale of 0 to 10: 0, colorless; 5, dark yellow; 10, amber. 2 Average of three (3) replicates. 3 BCV recovery = c(BCV)n day / c(BCV)0 day x 100%. It is defined as 100% at the initial time point.

[0353] Table 7: Accelerated Stability Study: Vehicle Control Results (Study I)

[0354]

[0355] 1 The yellowness of the solution is indicated on a scale of 0 to 10: 0, colorless; 5, dark yellow; 10, amber.

[0356] The study showed that over a two-week period, brincidofovir decreased by 16% (50°C) to 22% (75°C). Although some loss of brincidofovir recovery (assay loss) was observed, the purity of brincidofovir remained at 95% and 93% for temperatures up to 70°C, respectively. At the 75°C station, up to 16% impurities were observed at both starting concentrations. Degradation at higher temperatures was accompanied by a strong pH shift to lower pH values ​​and staining of the solution. The pH change and degradation rate were high at the initial time point, and after 2 days, the degradation and pH change slowed down significantly (the biphasic trend did not follow the first-order decomposition).

[0357] Physical degradation and benchmark stability studies

[0358] The results of the physical / benchmark stability of the two test formulations are summarized in Tables 11 and 12, respectively. The purity of the formulations at different temperatures was determined by comparing the chromatograms obtained at different temperatures and time points during the study. Degradation between 6% (2-8°C) and 8% (25°C) was observed within one month, while the purity of brincidofovir stagnated at 99%, indicating that brincidofovir was of insufficient quality.

[0359] Table 8: Physical degradation and baseline stability study results of 10 mg / mL brincidofovir formulations (Study I)

[0360]

[0361]

[0362] 1 Average of three (3) replicates. 2 Comprehensive area: 2.00-13.00 minutes. 3 BCV recovery = c(BCV)n day / c(BCV)0 day x 100%. It is defined as 100% at the initial time point.

[0363] Table 9: Physical degradation and baseline stability study results of 15 mg / mL brincidofovir formulations (Study I)

[0364]

[0365] 1 Mean values ​​of three replicates. 2 Comprehensive area: 2.00-13.00 minutes. 3 BCV recovery = c(BCV) n天 / c(BCV) 0天 × 100%. It is defined as 100% at the initial time point.

[0366] Light Obscuration Measurement: To prepare samples for liquid particle counting, formulation samples from six serum vials (1.0 mL fill) were carefully combined (avoiding foaming) into particle-free centrifuge tubes under optimal cleaning conditions. The tubes were then capped and sonicated for 1 minute before the samples were subjected to liquid particle counting. Four sample strokes of 1 mL were analyzed. The pull rate of the liquid particle counter was 10 mL / min. No significant increase in particulate matter was observed during the study. The test results of the test are summarized in Table 10.

[0367] Table 10: Liquid Particle Count Results for 10 and 15 mg / mL Brincidofovir Formulations (Study I)

[0368]

[0369]

[0370] Stability Study II: Brincidofovir Formulation without Tonicity Modifier (Glucose)

[0371] Design and Setup: Stability Study I designed to evaluate the chemical and physical degradation properties of the liquid brincidofovir formulation was repeated in the absence of glucose. Color changes (potential Maillard browning) and pH changes of the solution may be associated with the presence of glucose, which may therefore affect the degradation pathways and impair the observed brincidofovir degradation rate. The study setup for this repeated analysis was modified for the reagents tested as well as the stability station parameters. The exact parameters are as follows: Test solution:

[0372] 15 mg / mL brincidofovir, 200 mM sodium phosphate, pH = 8.0,

[0373] Vehicle Control 1: 200 mM sodium phosphate, pH = 8.0.

[0374] Stabilizing station temperature:

[0375] Accelerated stability study: 50°C, 60°C, 70°C and 75°C

[0376] Physical degradation and benchmark stability studies: 2-8°C and 25°C.

[0377] Time:

[0378] Accelerated stability study: initial, 3 days, 6 days, 10 days, 14 days,

[0379] Physical degradation and baseline stability studies: initial, 14 days and 28 days.

[0380] test:

[0381] Brincidofovir formulations: appearance, pH, c(BCV) recovery (HPLC), purity (HPLC), and liquid particle count (physical degradation and baseline stability study samples only),

[0382] Vehicle Control: Appearance, pH and Liquid Particle Count (Physical Degradation and Benchmark Stability Study Samples Only).

[0383] Results: The stability testing results for brincidofovir recovery and purity of the test formulations and vehicle control are summarized in Table 11 and Table 12, respectively.

[0384] Table 11: Accelerated stability study results of 15 mg / mL brincidofovir formulations (Study II)

[0385]

[0386]

[0387] a Average of three (3) replicates. b Comprehensive area: 0.00-13.00 minutes. c The data at this point in time was compromised due to an oven malfunction and is not included in the graphical representations or calculations below.

[0388] Table 12: Accelerated stability study results of vehicle control (Study II)

[0389]

[0390]

[0391] a not applicable. b The data at this point in time was compromised due to an oven malfunction and is therefore not included in the graphical representation below.

[0392] It should be noted that the values ​​of brincidofovir recovery and purity are inconsistent. This is at least partially due to the fact that the injected peak area (0.00-2.1 minutes) of the chromatographic trace is not integrated, which may overestimate the purity value because the non-integrated retention time area may accommodate known degradation products. In addition, the baseline separation between brincidofovir and its deamination product cannot be achieved using existing methods, which may affect the recovery value (degradation) and purity value.

[0393] The recovery data obtained were applied to determine the degradation rate at different temperatures by Arrhenius analysis, i.e., by logarithmically plotting the brincidofovir recovery as a function of time and determining the rate constant by linear fitting. The half-life of the 15 mg / mL brincidofovir formulation without glucose was determined to be 729 days and 295 days at station temperatures of 5°C and 25°C, respectively. Arrhenius analysis was performed as Figure 2 shown.

[0394] Physical degradation and benchmark stability studies

[0395] The results of physical / benchmark stability of the formulations studied and vehicle control are summarized in Tables 13 and 14, respectively.

[0396] Table 13: Physical degradation and baseline stability study results of 15 mg / mL brincidofovir formulations (Study II)

[0397]

[0398] 1 Mean values ​​of three replicates. 3 BCV recovery = c(BCV) n天 / c(BCV) 0天 × 100%. It is defined as 100% at the initial time point.

[0399] Table 14: Physical Degradation and Benchmark Stability Study Results for Vehicle Control (Study II)

[0400]

[0401]

[0402] 1 not applicable.

[0403] For analysis of particulate matter, a sample of the formulation from one serum vial (5.0 mL fill) was sonicated for 1 minute and the vial was deconvoluted. Liquid particle counts were performed on samples without tonicity agents as described above for formulations containing tonicity modifiers. The results of the analysis are summarized in Table 15.

[0404] Table 15: Liquid Particle Count Results for 15 mg / mL Brincidofovir Formulations without Tonicity and Vehicle Control (Study II)

[0405]

[0406] Under storage conditions (2-8°C and 25°C), degradation of brincidofovir in the tonicity modifier-free formulation was negligible over the course of one month. The nominal degradation of 1% was within the error range of brincidofovir quantification; no mass deficiency or particulate matter was observed.

[0407] Evaluation of brincidofovir adsorption to vials and stoppers

[0408] Design and setup: Adsorption of brincidofovir to vials and / or stoppers was tested to evaluate the adsorption of brincidofovir. To this end, different storage conditions that may have an impact (e.g., vial orientation) were evaluated. Solutions of brincidofovir were studied at formulation strength (10 mg / mL) and low concentration (0.2 mg / mL).

[0409] In order to test adsorption, it was treated with an organic solvent (methanol) suitable for redissolving the adsorbed material at different time points and compared with samples treated with water or untreated. Higher brincidofovir concentrations (after dilution standardization) were observed in samples containing methanol, indicating the presence of an adsorption effect. The difference in volume contraction applied between the solvents used was not considered. The study used 10mg / mL and 0.2mg / mL of brincidofovir in 200mM sodium phosphate (pH=8.0) solutions at 2-8°C and 25°C. First, the appearance, pH, brincidofovir recovery (HPLC) and purity (HPLC) of the solution were tested and tested after 1, 2 and 7 days. Some samples were further treated by adding methanol (1.0mL) or water (1.0mL). The results are summarized in Tables 16 and 17.

[0410] Table 16: Test results of 10 mg / mL brincidofovir formulations in adsorption evaluation

[0411]

[0412]

[0413]

[0414] 1 The average of three (3) replicates of the samples receiving the treatment. 2 BCV recovery = c(BCV) n天 / c(BCV) 0天 × 100%. It is defined as 100% at the initial time point. For both treated and untreated samples, BCV recovery at the initial time point is defined as 100.00%. 3 Not suitable for untreated samples. 4 Samples at the initial time point were not subjected to any conditions.

[0415] Table 17: Test results of 0.2 mg / mL brincidofovir formulations in adsorption evaluation

[0416]

[0417]

[0418] a The average of three (3) replicates of the samples receiving the treatment. b BCV recovery = c(BCV) n天 / c(BCV) 0天 × 100%. It is defined as 100% at the initial time point. For both treated and untreated samples, BCV recovery at the initial time point is defined as 100.00%. c Samples at the initial time point were not subjected to any conditions.

[0419] Comparison of the differently treated samples did not show any evidence of adsorption phenomena; no significant differences in the studied parameters were observed, i.e. no significant changes in appearance or purity, and no apparent loss of brincidofovir. The observed quality deficiencies were within the limits of sample preparation error, as indicated by the standard deviation of the replicate preparations.

[0420] Compatibility assessment of formulations

[0421] A range of materials were tested for compatibility with the formulation. This included test materials from the manufacturing phase, preclinical toxicology, and clinical testing. The applied compatibility testing evaluated 1) the possible loss of brincidofovir by adsorption or precipitation (appearance, recovery of brincidofovir by HPLC) and 2) changes in pH that could impair stability. The materials tested included infusion vehicles, sterilizing filters, product vials and stoppers, infusion systems and infusion bags for testing animals (rat) and IV systems for clinical applications.

[0422] The brincidofovir formulation containing 10 mg / mL brincidofovir, 200 mM sodium phosphate and 69 mM glucose (pH = 8.0) was diluted with a test infusion vehicle (5% glucose solution) to obtain c (BCV) = 0.5 and 1.0 mg / mL solutions. The diluted solution was stored under ambient conditions, and samples were collected at the beginning of the experiment and after 2 hours, 8 hours and 24 hours and the recovery of appearance, pH and c (BCV) was evaluated (by HPLC). Under ambient conditions, no significant changes were observed in the appearance, pH or c (BCV) of the test solution within 24 hours. The detailed results of this study are summarized as follows (Table 18).

[0423] Table 18: Results of infusion vehicle compatibility assessment

[0424]

[0425] a Average of three (3) replicates. b BCV recovery = c(BCV) n小时 / c(BCV) 0小时 × 100%. The sample at the initial time point was defined as 100%.

[0426] Sterile filter compatibility

[0427] The sterilizing filter compatibility of the formulations of the invention was evaluated. Aliquots of the tested brincidofovir formulations were filtered through the corresponding syringe filters as follows:

[0428] A: Filter: 0.2μm, 25mm, syringe filter, Membrane (PES), sterile; test solution: 10 mg / mL brincidofovir, 200 mM sodium phosphate, pH=8.0; test volume: 10 mL.

[0429] B: Filter: 0.2μm, 25mm, syringe filter, Membrane, sterile; test solution: 10 mg / mL brincidofovir, 200 mM sodium phosphate, pH = 8.0; test volume: 10 mL.

[0430] C: Filter: 0.22μm, 33mm, syringe filter, PVDF membrane, γ-irradiation; test solution: 2 mg / mL brincidofovir, 4 mM sodium phosphate, pH ~ 8, about 5% glucose (50X dilution of 10 mg / mL brincidofovir, 200 mM sodium phosphate, pH = 8.0, containing 5% glucose solution); test volume: 50 mL.

[0431] The first and last 10% of the filtrate were collected. Samples of the collected filtrate as well as samples of the pre-filtration formulation were evaluated for appearance, pH and c(BCV).

[0432] Table 19: Results of sterilizing filter compatibility assessment

[0433]

[0434]

[0435] a Average of three (3) replicates. b BCV recovery = c(BCV) 过滤后 / c(BCV) 预过滤 × 100%; pre-filtered sample was defined as 100%.

[0436] No significant changes in appearance, pH, or c(BCV) were observed when the filtrate solutions were compared to the corresponding pre-filtration samples and no impurities were introduced during filtration.

[0437] Drug Vial and Stopper Compatibility

[0438] The formulations of the present invention were evaluated for sterilizing filter compatibility. The test formulation (5 mL, 10 mg / mL brincidofovir, 200 mM sodium phosphate, pH = 8.0) was dispensed into a set of Afton sterilized Ready-To- In a vial. Stopper the vial with Afton Ready-To- The sterile seal was crimped and stored for 6 hours in upright and inverted orientations under ambient conditions. Samples of the filling solution were collected before filling and after 6 hours of storage under corresponding conditions. The appearance, pH and c (BCV) of the collected samples were evaluated. After 6 hours of contact with the vial and stopper tested, no significant changes in the appearance, pH or c (BCV) of the test formulation were observed. No additional impurities were introduced after contact.

[0439] Table 20: Results of Compatibility Assessment of Drug Product Vials and Stoppers

[0440]

[0441] a Average of three (3) replicates. b BCV recovery = c(BCV) n小时 / c(BCV) 预填充 × 100%. The prefilled sample is defined as 100%.

[0442] Preclinical Material Compatibility Assessment (Rat)

[0443] Infusion System Components (Rat)

[0444] The following brincidofovir formulations were tested for compatibility with the components of the infusion system used to administer the drug to rats (consisting of the syringe, tether, catheter, and syringe pump):

[0445] a.) 0.2 mg / mL brincidofovir, 4 mM sodium phosphate, pH ~8.0, about 4.90% glucose;

[0446] b.) 1.5 mg / mL of brincidofovir, 30 mM sodium phosphate, pH ~8.0, about 4.25% glucose.

[0447] Two solutions were prepared by diluting the developed brincidofovir formulation (10 mg / mL brincidofovir, 200 mM sodium phosphate, pH=8.0) with 5% glucose solution. The test procedure can be summarized as follows:

[0448] 1. Evaluate bulk test solutions for appearance, pH, and c(BCV) prior to contact (0.2 mg / mL BCV, 4 mM sodium phosphate, pH ~8.0, approximately 4.90% glucose and 1.5 mg / mL BCV, 30 mM sodium phosphate, pH 8.0, approximately 4.25% glucose).

[0449] 2. Draw approximately 5 mL of the test solution into the syringe and then connect the syringe to the assembled infusion system (vascular access harness (VAH) tether kit, VAH port for rat cuff, and catheter).

[0450] 3. Manually fill the infusion system with the test solution. Approximately 200 μL of the test solution was pushed out of the infusion system and collected as an end-of-line sample (t0) for evaluation of appearance and c(BCV).

[0451] 4. Install the syringe of the test infusion system onto the syringe pump and set the flow rate to a flow rate of 1.2 mL / h (20 μL / min) with a total dispense volume of 2.4 mL (120 min).

[0452] 5. The entire flow-through was collected during the experiment. 10min , t 105min ), collect the end-of-line sample (about 200 μL) for analysis.

[0453] 6. At the end of the experiment (t 120min ) measured the pH of the entire flow-through because the sample volume obtained from the previous time point (200 μL) was too small for a standard pH measurement.

[0454] The study results are summarized in Table 21.

[0455] Table 21: Results of preclinical infusion system compatibility assessment

[0456]

[0457] a Average of three (3) replicates. b BCV recovery = c(BCV) n分钟 / c(BCV) 接触前 × 100%, the sample before contact is defined as 100%. na: not applicable or unavailable.

[0458] For test #1 (0.2 mg / mL brincidofovir), an initial 10% loss of c(BCV) was observed, which can be attributed to absorption of the test infusion system. At low concentrations, initial adsorption effects (surface saturation) are often observed, and in the present case, full recovery of the observed losses was not observed at the time of the test; at t=105 min, a 6% loss of brincidofovir was observed. No significant changes in appearance or pH were observed. For test #2, no significant loss of c(BCV) or changes in appearance or pH were observed during the experiment.

[0459] Clinical material evaluation

[0460] Infusion bag compatibility

[0461] Material compatibility testing was performed on standard microinfusion bags (100 mL) using the following formulations:

[0462] 15 mg / mL brincidofovir, 200 mM sodium phosphate, pH = 8.0.

[0463] The strength of the formulation tested after dilution into the infusion bag is approximately:

[0464] 0.1 mg / mL brincidofovir, 5.3 mM sodium phosphate, pH ≈ 8.0 (in 5% glucose),

[0465] 1.0 mg / mL brincidofovir, 13.3 mM sodium phosphate, pH≈8.0 (in 5% glucose).

[0466] Testing clinical infusion bags is specified as follows:

[0467] 5% Dextrose for Injection, USP, 100mL VIAFLEX Infusion Bag

[0468] The developed brincidofovir formulation (15 mg / mL brincidofovir, 200 mM sodium phosphate, pH=8.0) was diluted to c(BCV)=0.1 and c(BCV)=1.0 mg / mL by injection into infusion bags. The infusion bags were then stored at ambient conditions and the infusion bags were filled with water at time points (t0, t 1h ,t 8h and t 24h ) Collect samples from the infusion bag for evaluation of appearance, pH and c(BCV). As a control, take out 5% glucose solution from a separate infusion bag and use the same dilution factor to perform an external dilution (standard laboratory glassware) of the test preparation with 5% glucose solution. Analyze the sample of the external dilution solution equally. The results are summarized in Table 22.

[0469] Table 22: Results of clinical infusion bag compatibility assessment

[0470]

[0471]

[0472] a Average of three (3) replicates. b BCV recovery = c(BCV) n小时 / c(BCV) 0小时 × 100%. The t0 sample is defined as 100%.

[0473] No significant changes in appearance, pH, or c(BCV) were observed for the dilution solutions in the c(BCV) test infusion bags. The higher brincidofovir recovery obtained from external dilutions is likely due to the fact that the infusion bags were typically overfilled (105-115 mL, average 110 mL, for 100 mL bags - information conveyed by Baxter). The concentrations determined for external dilutions were overestimated by applying the same dilution factor and using the t0 time point (diluted into the infusion bag) for standardization (defined as 100%). Internal studies conducted on a single infusion bag evaluated the volume of the infusion solution in a 100 mL infusion bag, ≈109 mL.

[0474] Clinical infusion system compatibility

[0475] Two infusion systems were tested (Baxter Non-DEHP CONTINUO-FLO solution set with 2 INTERLINK injection sites, male Luer-lok adapter; and Regulator IV set with 15 micron filter, 1 needle-free injection site) compatibility with the following formulations:

[0476] 15 mg / mL brincidofovir, 200 mM sodium phosphate, pH = 8.0.

[0477] The strength of the formulation tested after dilution into the infusion bag is approximately:

[0478] 0.1 mg / mL brincidofovir, 5.3 mM sodium phosphate, pH ≈ 8.0 (in 5% glucose),

[0479] 1.0 mg / mL brincidofovir, 13.3 mM sodium phosphate, pH≈8.0 (in 5% glucose).

[0480] The test procedure can be summarized as follows:

[0481] 1. Inject the developed brincidofovir formulation (15 mg / mL brincidofovir, 200 mM sodium phosphate, pH 8.0) into the above infusion bag (reservoir function) to obtain a test solution with c(BCV) = 0.1 or 1.0 mg / mL. Collect samples from the infusion bag for evaluation of c(BCV) (t 24h，袋 ).

[0482] 2. Incubate ("age") the infusion bag at ambient conditions for 24 hours, then collect another sample from the infusion bag (t 0，袋 ) for evaluation (appearance, pH, c(BCV)).

[0483] 3. Connect the infusion system under test to the infusion bag and manually fill it with the solution in the infusion bag. Collect approximately 1 mL (in triplicate) of the test solution as a terminal sample for analysis (t 0，eol ).

[0484] 4. Close the infusion system under test and store at ambient conditions until the next time point. Drain approximately 17 mL of the test solution (volume of the infusion system) and collect another terminal sample (1 mL, in triplicate) for analysis (t 10min，eol , t 3h，eol and t 6h，eol )).

[0485] Table 23: Results of clinical infusion system compatibility assessment

[0486]

[0487]

[0488] a Average of three (3) replicates. b BCV recovery = c(BCV) n小时 / c(BCV) 0小时 × 100%. t 0,袋 The sample is defined as 100%.

[0489] In the clinical infusion systems tested in both, for the test solutions with low c(BCV)=0.1 mg / mL, an initial loss of c(BCV) (up to 6%) was observed. This observation is likely explained by the initial saturation adsorption of the tube material. During the experiment (6 h), the concentration of brincidofovir recovered to about 100%. For the infusion systems tested in both, no significant overall changes in appearance, pH or c(BCV) were observed.

[0490] Example 4 - Stability and characteristics of lyophilized and reconstituted formulations

[0491] Pre-lyophilized formulations were prepared and subjected to the lyophilization process as shown in Table 24 below.

[0492] Table 24: Pre-lyophilized formulations.

[0493]

[0494] The glass transition temperature Tg' (amorphous), eutectic melting point Tg' (amorphous), and eutectic melting point Tg' (amorphous) of formulations 1-8 of Table 27 were determined by differential scanning calorimetry (DSC) analysis. Teu (Crystallization), Melting T melt,onset The starting temperature and freezing temperature T freeze To this end, 5 μL of each formulation was dispensed into an aluminum sample pan and sealed. DSC scans were performed by downward and upward scanning in the temperature range of +25°C to -65°C to +25°C at a heating rate of 5°C / min. The tested formulations showed a clear freezing point and a well-defined melting transition. The results of the DSC studies are summarized in Table 25.

[0495] Table 25: Results of DSC analysis

[0496]

[0497] Freeze drying cycle

[0498] A conservative lyophilization cycle was used to lyophilize the brincidofovir formulation, including parameters obtained by DSC analysis (see Table 28). The cycle consists of the following steps: freezing, annealing, primary drying, and secondary drying. Table 26 summarizes the set point temperature, heating rate, step time, and loading time for each step in an exemplary lyophilization cycle.

[0499] Table 26: Lyophilization Cycle Parameters (Cycle #1)

[0500]

[0501] Sample vials (5 mL vial size, 1 mL fill volume) were processed under optimal cleaning conditions in a biosafety cabinet. Nine samples were prepared for each formulation. For each formulation condition, one sample vial was equipped with a product probe to monitor product temperature (T 产物 ) and the shelf temperature of the freeze dryer (T 架子 The end of primary drying is determined when T 产物 ≈T 架子 At the end of the lyophilization cycle, the sample vials were backfilled with nitrogen (N2), stoppered, and removed from the freeze dryer for inspection and analysis.

[0502] The step time of the primary drying stage was set to 20 hours. Figure 1The product temperature profiles for Formulations 1-8 in this step are shown. In the formulations tested, the product temperature was lowered to T after the vacuum pump was activated and after a total cycle time of about 16 hours. (产物) <T (架子) After drying, a temperature jump was observed in different formulations within 15 hours. The temperature jump indicates that the sample was dried successfully.

[0503] The freeze-dried products were evaluated based on the structure and uniformity of the freeze-dried cake. Observation of "melt back" or "cake collapse" resulted in a decrease in ranking. Four vials from each formulation were randomly selected for appearance evaluation. The evaluation and ranking are summarized in Table 27.

[0504] Table 27: Evaluation and reconstitution properties of lyophilized products

[0505]

[0506] Among the formulations tested, the mannitol-based formulations (Formulations 1, 3, 5, and 7) produced more compact and uniform lyophilized cakes, and the sucrose-based formulations (Formulations 2, 4, 6, and 8) showed partial cake collapse and increased remelting.

[0507] Reconstitution evaluation of lyophilized formulations

[0508] The lyophilized product vial was weighed before and after lyophilization to determine the volume of the reconstitution solution. The lyophilized product was reconstituted with deionized water. After adding the solvent, the vial was gently rotated to record the reconstitution appearance and the reconstitution time. The pH and brincidofovir recovery (measured by HPLC) of the reconstructed product were further analyzed. For the reconstructed ranking, the parameters were supplemented by recording the foaming intensity and the foam dissipation time. The reconstructed evaluation results of preparations 1-8 are summarized in Table 30.

[0509] Formulation 3 was found to have no foaming upon reconstitution, and Formulation 7 was found to be able to be reconstituted with different buffers or infusion vehicles.

[0510] Example 5 - Freeze Drying Process Development

[0511] To develop the lyophilized drug product of brincidofovir, five different activities have been performed: i) lyophilization feasibility assessment, ii) lyophilization process development, iii) short-term stability studies, iv) preparation of drug product batches for stability testing, and v) material compatibility assessment.

[0512] A project overview capturing the purpose, outcome, and results of each activity is summarized in Table 28. Details of the lyophilization cycle parameters for certain lyophilization runs during process development can be found in Table 29.

[0513] Table 28: Project overview and decision rationale.

[0514]

[0515]

[0516] Table 29: Lyophilization cycle parameters (cycle #2, #3, #5, #6)

[0517]

[0518] i) Feasibility Assessment: Formulation and Reconstruction

[0519] To further evaluate the formulations based on reconstitution after lyophilization at different lyophilization cycles, liquid fill solutions were prepared and evaluated as follows. For lyophilization cycles #1, #2, and #5, liquid fill solutions were prepared, including:

[0520] a) Brincidofovir stock solution c (BCV) = 10 mg / mL (pH adjusted to pH = 8.0),

[0521] b) 10X buffer stock solution or deionized water (pH adjusted to pH = 8.0), and

[0522] c) 12.5% ​​(w / v) mannitol stock solution (pH adjusted to pH = 8.0)

[0523] d) Deionized water.

[0524] For cycles #6 and #7, prepare a liquid fill solution consisting of:

[0525] a) brincidofovir (solid),

[0526] b) mannitol (solid),

[0527] c) 10X arginine stock solution (pH adjusted to pH = 8.0),

[0528] d) NaOH solution (1 M), and

[0529] e) Deionized water.

[0530] For cycles #1, #2, and #5, the reconstitution volumes applied were determined by gravimetric analysis of product vials before and after lyophilization of each formulation. In the case of cycles #1 and #2, specific measured volumes were applied. For reconstitution of the lyophilized product of cycle #5, the average reconstitution volume was applied to all formulations. For the lyophilized product of lyophilization cycle #7, the liquid fill volume was scaled according to the difference between the previously determined liquid fill and reconstitution volumes.

[0531] After each freeze-drying cycle, the filter cake structure, uniformity and appearance defects of the freeze-dried product were evaluated. After reconstruction with deionized water, the appearance (e.g., foaming intensity, foam dissipation time), pH and recovery of brincidofovir of the solution during and after reconstruction were evaluated. The recovery of brincidofovir was determined by HPLC analysis. The fraction of volatiles was measured by thermogravimetric analysis (TGA). Table 30 summarizes the results of reconstruction evaluation.

[0532] Table 30: Drug product evaluation and reconstitution process.

[0533]

[0534]

[0535] * Average of three vials. For cycles #1-2, the approximate time to dissipate was recorded (continuous observation). For cycles #5-7, the foam intensity was evaluated at 10, 20, and 30 minutes after reconstitution. <20 means the foam dissipated time was between 10 and 20 minutes. <30 means the foam dissipated time was between 20 and 30 minutes. If foaming still persisted for 30 minutes, it means the dissipation time was >30 minutes.

[0536] ii) Process development: stability study of lyophilized preparations - 48h study

[0537] To evaluate the physical and chemical stability of the reconstituted solution, the samples were analyzed at the time of reconstitution (t0), 1 day (t 24h ) and 2 days later (t 48h ), the appearance and pH of each of Formulations 1, 3, 3a, 3b, 3c and 7, and the recovery of c(BCV) were evaluated at ambient temperature. The recovery of c(BCV) was determined by HPLC.

[0538] All formulations tested (Formulations #1, 3, 3a, 3b, 3c and 7) were clear and colorless in appearance and fully recovered brincidofovir within 48 hours after reconstitution (i.e., no significant loss of brincidofovir, and the variation in recovery was within the margin of error). Formulations 3 and 3a were found to show faster dissipation of foam / bubbles than formulations 3b and 3c (Table 33). iii) Short-term stability study: Stability study of lyophilized formulations - 2 week study

[0539] The appearance and pH of each formulation, as well as the recovery of c(BCV), were evaluated at the time of reconstitution (t0), 2 days (t 2天 ), 7 days (t 7天 ), 10 days (t 10天 )、14 days(t 14天), the stability of Formulations 1, 3 and 3a was evaluated at 25°C and 60°C. In addition, the foaming intensity was evaluated 10, 20 and 30 minutes after reconstitution.

[0540] The recovery of brincidofovir was determined by HPLC. The degradation products observed in the HPLC chromatogram were identified as deamination products, which may be cidofovir, as well as other degradation products. The movement or change of pH was not related to degradation. Among the formulations tested, formulation 3a showed the lowest degree of degradation under the conditions applied, with a purity of 99.2% after 14 days at 60°C. The experimental results are summarized in Tables 31, 32 and 36.

[0541] Table 31: Stability Study Results of Formulation 1 (200 mM Phosphate)

[0542]

[0543]

[0544] *Average of three vials. Foam intensity was assessed at 10, 20, and 30 minutes after reconstitution. <20 means foam dissipated between 10 and 20 minutes. <30 means foam dissipated between 20 and 30 minutes. If foaming still persists at 30 minutes, it means dissipation time is >30 minutes.

[0545] a The mean of three replicates of the treated samples was obtained.

[0546] b BCV recovery = c(BCV) n天 / c(BCV) 0天 ×100%.

[0547] ** Samples at t0 at T = 25°C and t0 at T = 60°C are the same. Note: HPLC sample variability accounts for sample-to-sample analytical variability. Vial-to-vial variability accounts for drug product vial-to-vial variability.

[0548] Table 32: Stability study results of Formulation 3 (200 mM Arginine)

[0549]

[0550]

[0551] *Average of three vials. Foam intensity was assessed at 10, 20, and 30 minutes after reconstitution. <20 means foam dissipated between 10 and 20 minutes. <30 means foam dissipated between 20 and 30 minutes. If foaming still persists at 30 minutes, it means dissipation time is >30 minutes. aThe mean of three replicates of the treated samples was obtained. b BCV recovery = c(BCV) n天 / c(BCV) 0天 × 100%. **Samples with t0 at T = 25°C and t0 at T = 60°C are the same. Note: HPLC sample variability accounts for sample-to-sample analytical variability. Vial-to-vial variability accounts for drug product vial-to-vial variability.

[0552] Table 33: Stability Study Results for Formulation #3a (100 mM Arginine)

[0553]

[0554]

[0555] a The mean of three replicates of the treated samples was obtained. b BCV recovery = c(BCV) n天 / c(BCV) 0天 × 100%. **Samples with t0 at T = 25°C and t0 at T = 60°C are the same. Note: HPLC sample variability accounts for sample-to-sample analytical variability. Vial-to-vial variability accounts for drug product vial-to-vial variability.

[0556] iv) Preparation of drug product batches for stability testing

[0557] Preparation of pre-lyophilized formulations: Formulation 3a was prepared according to the following procedure: Before QS (quantitative analysis), the pH of a 1 M buffer solution of arginine in deionized water was adjusted to 8 using hydrochloric acid (35-37%) under pH control. Mannitol was then added to the buffer solution, followed by the BCV. The pH of the resulting solution was adjusted to 8 using sodium hydroxide. In a separate volumetric flask, deionized water was used for the QS (quantitative analysis) solution. The two solutions were then mixed and sterile filtered to obtain the desired formulation 3a. This method resulted in formulation 3a having an observed c (BCV) of 9.9 mg / mL.

[0558] Preparation of lyophilized formulations: Under optimal cleaning conditions, approximately 200 vials of liquid formulation 3a prepared according to the above method were stoppered in the lyophilization position and lyophilized according to lyophilization cycle #7. The appearance, reconstitution appearance, pH and recovery of brincidofovir of the resulting lyophilized product were tested. The recovery of brincidofovir was determined by HPLC and thermogravimetric analysis. Table 34 summarizes the test results of appearance, reconstitution appearance and recovery of brincidofovir in the lyophilized formulations prepared according to the procedures outlined herein.

[0559] Table 34: Appearance, reconstitution and recovery of batch formulations before and after lyophilization.

[0560]

[0561] *Average of three vials. Foam intensity was assessed at 10, 20, and 30 minutes after reconstitution. 20 means foam dissipated between 10 and 20 minutes. <30 means foam dissipated between 20 and 30 minutes. If foaming still persisted at 30 minutes, it means dissipation time >30 minutes. a Scan range = 25°C to 150°C, slope = 10°C / min. b The average of three (3) replicates of the samples receiving the treatment.

[0562] v) Compatibility of lyophilized preparations

[0563] The liquid fill solution for lyophilized formulation 3a was tested for compatibility with the infusion vehicle and a range of materials from the manufacturing and clinical phases. The results of the infusion vehicle compatibility testing are summarized in Table 38. No precipitation, pH changes, or significant loss of recovery of brincidofovir was observed at the end of the study.

[0564] Infusion vehicle compatibility

[0565] Formulation 3a was diluted with an infusion vehicle (5% glucose) solution to obtain c(BCV) = 0.5 mg / mL and 1.0 mg / mL solutions. 2h ), 8 hours (t 8h ) and 24 hours (t 24h ) were followed by evaluation of appearance, pH, and brincidofovir recovery (as determined by HPLC).

[0566] Table 35: Results of infusion vehicle compatibility assessment.

[0567]

[0568] a Mean values ​​of three replicates. b BCV recovery = c(BCV) n小时 / c(BCV) 0小时 × 100%. The BCV recovery value at t0 is defined as 100%.

[0569] Sterile filter compatibility

[0570] The compatibility of liquid formulation 3a with two syringe filters used for aseptic processing was tested. 10 mL of liquid formulation 3a was passed through one of the filters a (0.2 μm, 25 mm, syringe filter, membrane) or b (0.2 μm, 25 mm, syringe filter, The first and last 10% filtrates were evaluated for appearance, pH, and brincidofovir recovery.

[0571] The results of the evaluation are summarized in Table 36. No precipitation, pH change, or significant loss of brincidofovir recovery was observed. The sample appeared clear and colorless. No degradation peaks were observed.

[0572] Table 36: Results of sterilizing filter compatibility assessment of Formulation 3a.

[0573]

[0574]

[0575] a Mean values ​​of three replicates. b BCV recovery = c(BCV) n小时 / c(BCV) 填充前 × 100%. The BCV recovery value at t0 is defined as 100%.

[0576] Drug Vial and Stopper Compatibility

[0577] Liquid Formulation 3a was tested for compatibility with vials and stoppers. Vials were filled with 5 mL of Liquid Formulation 3a, stoppered, crimped, and stored in both upright and inverted orientations at ambient conditions for 6 hours. The vials used for testing were clear, 20 mL, grade A borosilicate glass serum vials with stoppers of Stopper with Fluro coating.

[0578] Table 37 summarizes the results of this study. No precipitation, pH shift, or significant loss of brincidofovir recovery was observed throughout the study. No degradation peaks were observed in HPLC.

[0579] Table 37: Results of drug product vial and stopper compatibility assessment.

[0580]

[0581] a Mean values ​​of three replicates. b BCV recovery = c(BCV) n小时 / c(BCV) 填充前 × 100%. The BCV recovery value at t0 is defined as 100%.

[0582] Infusion bag compatibility

[0583] The compatibility of liquid Formulation 3a with 100 mL infusion bags was tested. Solutions of Formulation 3a in 5% glucose injection vehicle were prepared at two different formulation strengths (1.0 mg / mL and 0.1 mg / mL of brincidofovir) and stored in infusion bags under ambient conditions. 8h ) and 24 hours later (t 24h ), the appearance, pH and brincidofovir recovery (determined by HPLC) of samples from the infusion bags and from externally diluted standards were evaluated. The results of the infusion bag compatibility evaluation are summarized in Table 38.

[0584] Table 38: Results of infusion bag compatibility assessment

[0585]

[0586] a Mean values ​​of three replicates. b BCV recovery = c(BCV) n小时 / c(BCV) 0小时 × 100%. The BCV recovery value at t0 is defined as 100%.

[0587] The samples appeared clear and colorless. For both sample concentrations, no significant loss of brincidofovir recovery was observed within 24 hours. For one bag repeat of c(BCV)=0.1 mg / mL, significant brincidofovir loss (19%) was observed; it was further observed that dilution of the brincidofovir formulation into the infusion vehicle to produce a concentration of c(BCV)=0.1 mg / mL resulted in a significant pH change to pH=7. No degradation was observed in the experiment.

[0588] Infusion system compatibility

[0589] The compatibility of a brincidofovir formulation (10 mg / mL brincidofovir, 100 mM arginine, 5% mannitol, pH = 8.0) with two infusion systems (Baxter, Non-DEHP CONTINU-FLO; B Braun, IV regulator group). As previously described, the liquid formulation was diluted into an infusion bag (final concentration of the test: 1.0 mg / mL and 0.1 mg / mL), and aliquots were collected from the infusion bag for analysis of brincidofovir recovery (t0, bag) after 24 hours. The infusion system was then connected to the infusion bag and the solution from the infusion bag was manually filled. Aliquots were collected as end samples for analysis (t0, eol). The IV line was closed between time points, wherein the infusion was stationary in the infusion system and stored at ambient temperature. At the test time point, about 17 mL (approximately the volume of the IV system) of infusion solution was discharged, which resulted in the solution in the IV system being replaced from the bag reservoir. After the replacement, another end-of-line sample was collected for evaluation (t10 min, eol, t3 h, eol and t6 h, eol). The appearance, pH and brincidofovir recovery of the sample were evaluated. The results of the infusion system compatibility test are summarized in Table 42.

[0590] Initial adsorption / saturation effects were observed for both IV systems at low c(BCV), indicating a 7-11% loss from brincidofovir recovery. This loss was partially recovered over the next 6 hours. No degradants were observed during this study. As previously described, samples with c(BCV)=0.1 mg / mL showed a pH change of approximately 1 pH unit to pH=7 upon dilution with 5% glucose solution. Samples appeared clear and colorless during the study.

[0591] Table 39: Results of the infusion system compatibility assessment.

[0592]

[0593]

[0594] a Mean values ​​of three replicates. b BCV recovery = c(BCV) n小时 / c(BCV) 0小时 × 100%. The BCV recovery value at t0 is defined as 100%.

[0595] Example 6 - Stability Test

[0596] The stability of bulk liquid preparations and lyophilized preparations of the present invention was further tested. The liquid preparation containing arginine, mannitol and 10mg / mL of brincidofovir was stored in a 10mL glass bottle, plugged (rubber stopper or 20mm serum stopper) and sealed (aluminum seal), and stored for up to 12 months at about 5°C or about 25°C. The appearance, pH value and brincidofovir content of the stored preparations, as well as impurities and particulate matter were monitored at different time intervals (i.e., after 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months and 12 months). Impurities were evaluated by HPLC and confirmed here by their relative retention time. For a solution of a lyophilized powder of brincidofovir containing 10mg / mL, the same experiment was repeated. For lyophilized preparations, 20mL glass bottles and 20mm rubber stoppers were used. In addition, the appearance of lyophilized powder (i.e., lyophilized cake) was recorded.

[0597] The results of the stability studies of bulk liquid formulations at 5°C and 25°C are summarized in Tables 40A and 40B, respectively. The results of the stability studies of lyophilized formulations at 5°C and 25°C are summarized in Tables 41A, 41B, and 42, respectively.

[0598] Table 40A: Stability of liquid formulations at 5°C (±3°C)

[0599]

[0600]

[0601] Table 40B: Stability of liquid formulations at 25°C (±2°C) and 60% relative humidity (±5% relative humidity)

[0602]

[0603]

[0604]

[0605]

[0606]

[0607]

[0608] Example 7 - Compatibility determination of rat and human whole blood

[0609] introduction

[0610] For rats, each concentration of the test item was mixed with rat whole blood at a ratio of 1:4, while for humans, each concentration of the test item was mixed with human whole blood at a ratio of 1:4. These samples plus control samples [vehicle control (vehicle + whole blood, 1 rat and 1 human), positive control (1% saponin + whole blood), untreated whole blood and negative control (saline + whole blood) were subjected to hemolysis analysis (supernatant hemoglobin concentration).

[0611] Rat and human whole blood samples were incubated in the test or control item at 37 ± 1 °C for 20 minutes and then immediately (within 2 minutes) centrifuged at 3500 ± 100 rpm (2740 g) for 5-10 minutes at room temperature. The supernatant was extracted and analyzed for hemoglobin using the cyanomethemoglobin method on a Siemens Advia 120 hematology analyzer. If the test item has the potential to cause hemolysis, the hemoglobin released from the lysed erythrocytes into the plasma supernatant can be quantified by a reaction that oxidizes the heme iron in the solution from the ferrous to the ferric state in the presence of potassium cyanide. This reaction results in the formation of methemoglobin, which then binds to cyanide to form stable cyanomethemoglobin. When measured spectrophotometrically at 546 nm, the absorbance of cyanomethemoglobin follows Beer's law and is proportional to the concentration of hemoglobin released into the solution. In addition, the formulated test item at each evaluated concentration was mixed with normal saline at a ratio of 1:4 and then analyzed directly to ensure that the test item formulation would not interfere with the hemoglobin measurement of Siemens Advia.

[0612] Materials and methods

[0613] As described below, the experiment was conducted in 2 stages:

[0614] Table 43: Brincidofovir in blood of rats, Phase 1

[0615]

[0616]

[0617] Table 44: Brincidofovir in human blood, Phase 2

[0618]

[0619] Control item (vehicle)

[0620] The dosage formulation of the control item was 2 parts sodium phosphate buffer (400 mM, pH=8.0); 1.0 N sodium hydroxide solution; sterile water for injection; and 5% glucose solution.

[0621] Test Preparation and Analysis

[0622] The brincidofovir injection bulk preparation was formulated into 15 mg / mL brincidofovir, and the complex from the bulk solution included three (3) steps: (1) preparing 2X sodium phosphate buffer (400 mM, pH = 8.0); (2) purchasing 1.0N sodium hydroxide solution; preparing the brincidofovir injection bulk preparation (15 mg / mL brincidofovir, 200 mM sodium phosphate, pH = 8.0) from solid brincidofovir, 1.0N sodium hydroxide solution, 2X sodium phosphate buffer and WFI (water for injection).

[0623] Under refrigerated conditions (2 to 8° C.), the brincidofovir injection bulk preparation can be stable for 28 days. The brincidofovir injection bulk preparation is diluted with 5% glucose to obtain the desired dosage concentration.

[0624] Prepare fresh dose formulations on the day of the experiment. Refrigerate (2 to 8°C) dose formulations when not in use.

[0625] On the day of the experiment, saponin was diluted with saline to an appropriate concentration.

[0626] Test system

[0627] Whole blood (~20 mL) was collected from rats (samples were collected from 4 animals for the experiment) in the presence of K2EDTA anticoagulant and mixed by inversion.

[0628] Human whole blood (~20 mL) was collected from one human volunteer in the presence of K2EDTA anticoagulant for this study.

[0629] After blood samples were collected, they were stored at room temperature for no more than 4 hours for the hemolytic potential test in this study.

[0630] Test Method

[0631] For rats, each concentration of the test item was mixed with rat whole blood at a ratio of 1:4, and for humans, each concentration of the test item was mixed with human whole blood at a ratio of 1:4. These samples plus control samples [vehicle control (vehicle + whole blood, 1 rat and 1 human), positive control (1% saponin + whole blood), untreated whole blood and negative control (saline + whole blood) were subjected to hemolysis analysis (supernatant hemoglobin concentration).

[0632] Rat and human whole blood samples were incubated in the test or control items at 37±1°C for 20 minutes and then immediately (within 2 minutes) centrifuged at 3500±100 rpm (2740 g) for 5-10 minutes at room temperature.

[0633] The supernatant was extracted and analyzed for hemoglobin on a Siemens Advia 120 hematology analyzer using the cyanomethemoglobin method. Without wishing to be bound by theory, if the test item has the potential to cause hemolysis, then the hemoglobin released from the lysed erythrocytes into the plasma supernatant can be quantified by a reaction that oxidizes the heme iron in the solution from the ferrous to the ferric state in the presence of potassium cyanide. This reaction results in the formation of methemoglobin, which then combines with cyanide to form stable cyanomethemoglobin. When measured spectrophotometrically at 546 nm, the absorbance of cyanomethemoglobin follows Beer's law and is proportional to the concentration of hemoglobin released into the solution.

[0634] In addition, the formulated test item at each evaluated concentration was mixed with normal saline at a ratio of 1:4 and then analyzed directly to ensure that the test item formulation would not interfere with the hemoglobin measurement of Siemens Advia.

[0635] The test was conducted in 2 phases.

[0636] Two independent analytical runs were performed using whole blood from 4 normal rats and 1 human donor to make up 1 batch.

[0637] The R&D Systems Whole Blood Quality Control Unit was run pre- and post-batch to verify the instrument's operational performance, using established standards for this control group and, for the control material, set to the specified manufacturer's range. To verify the control hierarchical order for the hemoglobin assay in the solution phase, a separate set of (Dot Scientific) low, medium, and high hemoglobin-containing controls were run. Negative and positive controls, and test samples were run in singlet.

[0638] Hemolytic potential standard

[0639] Operation standard:

[0640] The 1% saponin positive control must show a hemoglobin value > (negative control + 0.5 g / dL) for the test to be considered acceptable.

[0641] Unprocessed whole blood must show a hemoglobin value < (negative control + 0.5 g / dL).

[0642] Saline + untreated whole blood (negative control) must show a hemoglobin value < 0.5 g / dL.

[0643] The R&D Systems whole blood control group should be within the manufacturer's specified target range.

[0644] The Dot Science Liquid Hemoglobin Control does not have a target range established for the ADVIA instrument but should show a rank order based on responses (based on concentration) of High>Mid>Low.

[0645] Sample analysis standards

[0646] The test item + saline sample must show a hemoglobin value < (negative control + 0.5 g / dL) to be considered a valid test run.

[0647] Vehicle control samples must show a hemoglobin value < (negative control + 0.5 g / dL) to be considered a valid test run.

[0648] Liquid QC controls containing hemolysate prepared from low, medium, and high concentrations of human red blood cells (Pointe Scientific) were used to validate the ability to measure liquid hemolysate with Advia using the Advia cyanomethemoglobin process. The low, medium, and high liquid controls returned values ​​of 7.9, 11.7, and 15.6 g / dL, respectively, for Phase 1. The low, medium, and high liquid controls returned values ​​of 8.1, 11.9, and 15.6 g / dL, respectively, for Phase 2, as measured on the Siemens Advia. The rank order of the controls met protocol criteria.

[0649] A (R&D Systems) whole blood quality control group was used to validate the operational performance of the Siemens Advia 120 hematology instrument. The QC values ​​recorded in the raw data were within the acceptable range specified by the manufacturer for the Advia instrument.

[0650] Positive hemolysis controls were performed using amphipathic glycoside saponin that binds to cholesterol to form pores in the cell membrane bilayer and cause lysis of erythrocytes. Positive controls for hemolysis were 1% saponin treated rat and human whole blood. Hemolysis produced mean hemoglobin concentrations in the supernatant of 3.9 and 11.0 g / dL, respectively (Tables 47, 48, 49, and 50). These values ​​met the protocol defined allowable criteria for experimental hemoglobin concentrations (negative control + 0.5 g / dL).

[0651] Vehicle controls (5% glucose) for rat and human whole blood in Phases 1 and 2 produced mean hemoglobin concentrations of 0.0 g / dL (Tables 47, 48, 49, and 50). Vehicle controls in both phases met protocol defined sample analysis criteria (negative control + 0.5 g / dL).

[0652] Plasma from both phases and the negative control exhibited a mean hemoglobin concentration of 0.0 g / dL (Tables 47, 48, 49, and 50). These values ​​met the protocol-defined experimental acceptance criteria of untreated whole blood hemoglobin concentration < (negative control + 0.5 g / dL) and saline and untreated whole blood hemoglobin concentration < 0.5 g / dL.

[0653] Vehicle controls were negative and process controls provided expected target values. All control tests indicated acceptable performance in Phases 1 and 2.

[0654] Brincidofovir hemolytic potential

[0655] Rats exhibited mean hemoglobin concentrations of 0.0, 0.25, and 1.35 g / dL at 0.2, 0.5, and 1.6 mg / mL of brincidofovir in whole blood, respectively (Tables 47, 48, 49, and 50), and are summarized as follows:

[0656] Table 45: Brincidofovir in rat whole blood

[0657]

[0658] *Calculated as: (HgB plasma) / (normal HgB whole blood x dilution factor)*100

[0659] Based on normal whole blood rat hemoglobin value of 13g / dl (according to our HCD)

[0660] Point Scientific controls are systematic tests to validate hemoglobin measurements in the solution phase. As described herein, brincidofovir exhibited a hemolytic potential effect in rat whole blood that appeared to be concentration-dependent. Presumptive evidence of this effect was observed at a concentration of 0.5 mg / mL, with more pronounced evidence of hemolysis observed at a concentration of 1.6 mg / mL.

[0661] The expected % hemolysis for detectable levels of hemoglobin in this study can be calculated using the mean normal hemoglobin concentration in rat whole blood and the appropriate dilution factor. For example, the normal hemoglobin value of rat whole blood diluted experimentally corrected for 13 g / dL results in a value of 10.4 g / dL (13 x 0.8). The calculated % hemolysis for hemoglobin values ​​of 0.25 and 1.35 g / dL is 2.4% and 13.0%, respectively. Hemolysis values ​​<10% for a formulation are considered non-hemolytic, while values ​​>25% are considered at risk for hemolysis (Amin and Dannenfelser, 2006). Without wishing to be bound by theory, a concentration of 0.2 mg / mL showed undetectable hemoglobin and was therefore considered non-hemolytic. Concentrations of 0.5 and 1.6 mg / mL resulted in detectable hemoglobin levels in rat whole blood, but these levels were not considered to be at risk for hemolysis.

[0662] Results from two independent analytical runs (n=2) from Phase 2 showed that brincidofovir had no effect on the hemocompatibility of human whole blood at concentrations of 0.2, 0.5, and 1.6 mg / mL and are summarized as follows:

[0663] Table 46: Brincidofovir in human whole blood

[0664]

[0665]

[0666] *Calculated as: (HgB plasma) / (normal HgB whole blood x dilution factor)*100

[0667] Based on a normal whole blood hemoglobin value of 14 g / dL for women (based on Mayo Clinic online data)

[0668] In addition, brincidofovir was not found to interfere with the determination of hemoglobin concentrations in both Phase 1 and 2 (Tables 47, 48, 49, and 50).The test item controls met the protocol-defined sample analysis criteria, which indicated acceptable performance of Phases 1 and 2.

[0669] Results from two (n=2) independent analytical runs in Phase 1 indicate that, without wishing to be bound by theory, brincidofovir exhibits a possible hemolytic effect in rat whole blood that appears to be concentration-dependent. Without wishing to be bound by theory, presumptive evidence of this effect was observed at a concentration of 0.5 mg / mL, and clear evidence of hemolysis was observed at a concentration of 1.6 mg / mL. Formulations with hemolysis values ​​<10% are considered non-hemolytic, while formulations with values ​​>25% are considered at risk for hemolysis. The 0.2 mg / mL concentration exhibited no detectable hemoglobin and was therefore considered non-hemolytic. Concentrations of 0.5 and 1.6 mg / mL resulted in detectable hemoglobin levels in rat whole blood, but these levels were not considered a risk for hemolysis.

[0670] Results from two (n=2) independent analytical runs in Phase 2 indicated that brincidofovir had no effect on the hemocompatibility of human whole blood at concentrations of 0.2, 0.5, or 1.6 mg / mL.

[0671] Without wishing to be bound by theory, not all species react identically to positive hemolytic reagents, which may represent differences in RBC fragility between species.

[0672] Table 47: Summary of Hemolytic Potential Results; Brincidofovir in Rat Blood in Phase 1

[0673]

[0674] *Calculated as: (HgB plasma) / (normal HgB whole blood x dilution factor)*100

[0675] Based on normal whole blood rat hemoglobin value of 13g / dl (according to our HCD)

[0676] Table 48: Summary of Hemolytic Potential Results; Brincidofovir in Human Blood Phase 2

[0677]

[0678] *Calculated as: (HgB plasma) / (normal HgB whole blood x dilution factor)*100

[0679] Based on a normal whole blood hemoglobin value of 14 g / dL for women (based on Mayo Clinic online data)

[0680] Table 49: Hemolytic Potential Results (Hemoglobin Concentration g / dL): Brincidofovir in Blood of Rats in Phase 1

[0681]

[0682] Table 50: Hemolytic Potential Results (Hemoglobin Concentration g / dL): Brincidofovir in Human Blood Phase 2

[0683]

[0684]

[0685] Hemolysis test of the test item when 1A=0.2mg / mL

[0686] Hemolysis test of the test item when 1B = 0.5 mg / mL

[0687] Hemolysis test of the test item when 1C = 1.6 mg / mL

[0688] 2A=0.2mg / mL Test item interference control

[0689] 2B=0.5mg / mL Test item interference control

[0690] 2C=0.5mg / mL Test item interference control

[0691] 3 = Hemolysis of vehicle control

[0692] 4 = hemolysis positive control; 1% saponin as a positive control

[0693] 5 = Hemolysis of untreated whole blood control

[0694] 6 = Hemolysis of negative control

[0695] SD = Standard Deviation

[0696] Example 8 - Maximum Tolerated Dose and 7-Day Dose-Range-Finding (DRF) Study in Rats

[0697] The objectives of the Phase 2 study were to determine the maximum tolerated dose (MTD) of brincidofovir when administered to rats by a single 2-hour intravenous infusion and to assess toxicity, as well as to characterize the single, repeated, and toxicokinetics of brincidofovir when administered to rats by 2-hour intravenous infusions on days 1, 4, and 7.

[0698] The results of this study may allow decisions to be made regarding dose levels for subsequent toxicity studies of brincidofovir when administered by intravenous infusion.

[0699] The study design incorporated elements of general regulatory guidelines for toxicity studies.

[0700] Summarize

[0701] This study consists of two phases: Phase 1 is a maximum tolerated dose (MTD) study, and Phase 2 is a repeated dose ranging (DRF) study.

[0702] Parameters evaluated during Phase 1 were: survival rate and clinical observations.

[0703] For phase 1, 2, 4, 10, or 15 mg / kg of Sprague-Dawley leukemia was administered by a single 2-hour IV infusion. Rats (2 / sex / dosing interval) were dosed with brincidofovir. The maximum feasible dose (MFD) was limited to 15 mg / kg due to increased hemolytic potential when the concentration of brincidofovir in the dosing solution exceeded 1 mg / mL. The dose volume was 10 mL / kg / hr for all dosing intervals. An observation period of 2-3 days was performed after each dose administration. At the end of the Phase 1 treatment period, all animals were euthanized and discarded without macroscopic examination.

[0704] Parameters assessed during Phase 2 were: survival, clinical observations, body weight, food consumption, hematology (end of dosing), clinical chemistry (end of dosing), and macroscopic observations.

[0705] For Phase 2, Sprague-Dawley 100 mg / kg was administered once via 2-hour IV infusion on days 1, 4, and 7 at 0 [2X sodium phosphate buffer (400 mM, pH 8.0 ± 0.04)], 1, or 15 mg / kg. Rats (5 / sex / group) were administered brincidofovir. The dose volume for all groups was 10 mL / kg / hr. At the end of the Phase 1 treatment period, all animals were euthanized and autopsied. Similar doses were administered to Phase 2 satellite animals (3 / sex / group 1 and 6 / sex / groups 2-3), and blood samples were collected on days 1 and 7 for toxicokinetic analysis of brincidofovir and its metabolite cidofovir.

[0706] List of abbreviations and definitions of terms

[0707]

[0708] Phase 1

[0709] All animals were dosed with a single dose of brincidofovir at 2, 4, 10 or 15 mg / kg by 2-hour IV infusion and allowed to survive until their scheduled euthanasia.

[0710] When brincidofovir was administered at 2, 4 or 15 mg / kg, intermittent clinical symptoms were caused in some male and female animals. No clinical symptoms were observed in animals administered with 10 mg / kg brincidofovir. Intermittent clinical symptoms included rapid breathing and prone posture, with or without erect hair, partially closed eyes, and decreased activity, or irregular breathing, or with or without irregular breathing, erect hair and decreased activity of bilateral partially closed eyes. The clinical symptoms were reversible and in the 1st and 2nd group animals that showed symptoms, the clinical symptoms subsided at the end of the infusion or within 45 minutes after the end of the infusion. The clinical symptoms were reversible and in group 4 that showed symptoms, the clinical symptoms subsided within 3 hours and 22 minutes after the end of the infusion.

[0711] All doses were well tolerated under the conditions of this study. Therefore, no maximum tolerated dose level was determined and 15 mg / kg was considered the maximum feasible dose (MFD).

[0712] Phase 2

[0713] All animals were dosed with three total brincidofovir doses of 1 and 15 mg / kg / day over a period of 7 days and were allowed to survive until their scheduled euthanasia.

[0714] Administration of brincidofovir at 1 mg / kg / day or 15 mg / kg / day did not result in any brincidofovir-related clinical signs in male or female animals.

[0715] Plasma samples (108) were collected 2 hours after the start of dosing on Days 1 and 7 (Group 1), or 1, 2, 8, and 24 hours after the start of dosing on Day 1 and before dosing and 2, 8, and 24 hours after the start of dosing on Day 7 (Groups 2 and 3) and analyzed by LC / MS / MS to determine the concentrations of brincidofovir and its metabolite (cidofovir).

[0716] Peak brincidofovir concentrations are usually observed at the end of a 2-hour IV infusion and decline rapidly. Typically, mean brincidofovir C maxThe increase in AUC and AUC was generally consistent with the dose increase from 1 to 15 mg / kg, however, the AUC after 1 mg / kg was based on limited concentration-time data. max and AUC last The values ​​showed a trend towards lower values ​​on day 7 [Day 7 / Day 1 cumulative ratio (AR) was 0.44 to 0.54]. No gender differences in brincidofovir TK parameters were observed. Overall, the increase in the metabolite cidofovir was less than the proportion of the increase in brincidofovir dose. No significant gender differences in cidofovir TK parameters occurred and TK parameters did not change after repeated dosing.

[0717] Hematologic findings associated with brincidofovir were limited to a mild decrease in reticulocytes in males and females, with dosing of 15 mg / kg brincidofovir (-33% and -30% control, respectively; statistically significant only in males), and in females (+2.1% control, statistically significant) in relation to mean cellular hemoglobin concentration (MCHC). At the end of the 7-day dosing period, the decrease in red blood cell mass (hemoglobin, hematocrit, red blood cells) was negligible (relative to -4.2% control).

[0718] There were minimal brincidofovir-associated increases in total calcium in males dosed with 15 mg / kg brincidofovir (+3.8%; not statistically significant) and females dosed with ≥1 mg / kg brincidofovir (+4.9%; statistically significant), and minimal increases in urea (BUN) in females dosed with 15 mg / kg (+29%; statistically significant).

[0719] Brincidofovir, 1 and 15 mg / kg / day, was given by 2-hour IV infusion on days 1, 4, and 7 and was well tolerated.

[0720] Route, duration, and frequency of administration

[0721] In Phase 1, each animal was given a single dose of brincidofovir by 2 hours of intravenous infusion. At each dosing interval, naive animals were given increasing doses to determine the maximum tolerated dose (MTD). MTD data were used to select the dose for Phase 2.

[0722] In Phase 2, selected doses were administered by 2-hour intravenous infusion on Days 1, 4, and 7 to assess the toxicity of repeated doses of brincidofovir and to aid in the selection of doses for subsequent toxicity studies. The seven-day study may be suitable for selecting doses for subsequent repeated-dose GLP toxicity studies.

[0723] Number of animals

[0724] The number of animals in this study was considered the minimum necessary for statistical, regulatory, and scientific reasons.

[0725] The number of animals (2 / sex / dose interval) in Phase 1 (MTD) is the minimum number required to determine the maximum tolerated dose in male and female rats. It is expected that four dose levels of brincidofovir will be sufficient to determine the maximum tolerated dose. Although there is a 2-3 day observation period, new animals are used at each dose level to eliminate the possible additive effects of repeated dosing, which could confound the determination of the maximum tolerated single dose level.

[0726] The number of primary study animals (5 / sex / group) in Phase 2 (DRF) was the minimum number to control for expected variability between animals.

[0727] A negative control group and two brincidofovir-treated groups receiving low and high multiples of the proposed human dose were considered the minimum number of groups required to establish a baseline and provide a range of effects and to allow extrapolation of results for additional repeated-dose studies.

[0728] The number of animals (6 / sex / treatment group) in the satellite toxicokinetic study was considered the minimum necessary to provide meaningful data in the Phase 2 (DRF) toxicokinetic assessment, given the inherent variability in the processes of distribution, metabolism, and excretion. A control group of 3 animals per sex demonstrated no exposure to brincidofovir.

[0729] Dose selection

[0730] The starting dose for phase 1 of this study was 2 mg / kg.

[0731] Based on the results of the Phase 2 study of the present invention, the low and high doses (1 and 15 mg / kg) for Phase 2 of this study were selected. In some embodiments, during Phase 1 of this study, in animals dosed with 15 mg / kg, the low and high doses were selected due to the lack of clinical symptoms during the post-dose observation period.

[0732] Experiment Outline

[0733] For Phase 1 (MTD), brincidofovir was administered in single ascending doses up to four dose levels in naive male and female rats by intravenous infusion (2 hours). Each dose was accompanied by a 2-3 day observation period. Based on the response to the previous dose, each subsequent dose level was increased and administered to naive animals until the maximum tolerated dose (MTD) was determined, or until the maximum feasible dose (MFD) of 15 mg / kg based on previous evidence of hemolysis was achieved.

[0734] Table 51: Phase 1 (MTD) study consisting of 4 dosing intervals

[0735]

[0736] For Phase 2 (DRF), the test and control groups were dosed on days 1, 4, and 7 by 2-hour IV infusion into rats.

[0737] Table 52: Phase 2 study: 1 control group and 2 treatment groups

[0738]

[0739] Prepare 2X sodium phosphate buffer (400 mM, pH 8.0 ± 0.04) vehicle solution by mixing appropriate amounts of sodium dihydrogen phosphate solution (400 mM) and sodium monohydrogen phosphate solution (400 mM). Adjust the pH of the solution with sodium dihydrogen phosphate solution (400 mM) when necessary. Filter the solution through a 0.22 μm -GP filter and enter the sterile container.

[0740] The vehicle (control group) was stored under refrigeration at 2-8° C. Fresh vehicle solution was prepared once before each session and used within one month of preparation.

[0741] A 15 mg / mL brincidofovir stock was prepared by mixing appropriate amounts of brincidofovir with 2X sodium phosphate buffer, sterile water for injection, USP, and 1N sodium hydroxide (NaOH). The solution was filtered through a 0.22 μm -GP filter and enter the sterile container.

[0742] In a sterile bottle under a laminar flow hood, dilute the appropriate amount of brincidofovir stock solution (15 mg / mL) or the appropriate amount of stock vehicle (2X sodium phosphate buffer solution) with the appropriate amount of 5% dextrose injection, USP and invert 10 times to mix to prepare the dose formulations for Phase 1 and Phase 2. In Phase 1 and Phase 2, prepare fresh dose formulations on each dosing day and store refrigerated at 2-8°C and protect from light when not in use.

[0743] Dosage

[0744] way

[0745] The intravenous infusion lasted for 2 hours. Treatment was performed at a constant dose of 20 mL / kg / 2 hours.

[0746] Infusion catheter implantation procedure

[0747] Infusion catheters were implanted approximately 1-2 weeks prior to dosing. All animals, including spare animals, were surgically catheterized.

[0748] The surgical site was prepared according to the standard operating procedure (SOP) of the test facility for sterile, restorative surgery. Animals received pre-analgesia (flunixin meglumine (USP) 2 mg / kg, subcutaneously).

[0749] All animals were anesthetized (isoflurane) and shaved in the groin and back. A small incision was made and the femoral vein was isolated. A small incision was made in the vessel and a sterile catheter was placed in the vessel. The catheter was inserted through the femoral vein and retrograde into the inferior vena cava caudal to the kidney. The catheter was secured with sutures and then passed under the skin to drain in the back of the neck. A small pocket was made in the external area, in which a loop of catheter was left. The inguinal area was closed with sutures.

[0750] The animals were placed in a cuff and the implanted catheters were secured to pins using capped septum connectors. The catheters were blocked with taurolidine citrate blocking solution. Each animal received an intramuscular injection of 5.0 mg / kg of enrofloxacin on the day of surgery.

[0751] Infusion Catheter Maintenance Procedure

[0752] Prior to using the animals for the study, the patency of the implanted femoral vein catheter was assessed according to the testing facility's SOP.

[0753] Volume dose (rate)

[0754] 20 mL / kg / 2 hours (10 mL / kg / hour).

[0755] Individual dose concentration

[0756] Individual animal concentrations were calculated based on the most recently recorded scheduled body weight.

[0757] Frequency and duration

[0758] In Phase 1, each animal received a single dose by intravenous infusion, with a 2-3 day observation period following each dosing interval. Following a single intravenous (2 hour) infusion, the animal's pocket and dosing set were removed and the animal was not returned to saline maintenance.

[0759] In Phase 2, brincidofovir was administered to each animal by intravenous infusion (2 hours) on days 1, 4, and 7. Between doses, the animals were placed in sterile saline at a rate of 0.5 mL / hour. After dosing was completed on Day 7, the catheter was tied (the knot was placed in the catheter and the catheter was withdrawn under the skin) and the pocket was removed.

[0760] Administration site

[0761] A surgically implanted cannula in the femoral vein / vena cava.

[0762] Dosage Procedure

[0763] One to two days before dosing begins, the lock solution (if possible) is removed from each animal's implanted catheter and the catheter is flushed with saline before connection to the tether and infusion dosing set. After connection to the animal's catheter, sterile saline (0.9% NaCl, USP) is injected into the animal at a rate of 0.5 mL / hr via a calibrated Medfusion syringe pump until dosing on Day 1.

[0764] A saline flush was injected into the infusion line to deliver brincidofovir and to ensure delivery of the full dose (at the same rate as brincidofovir, with an additional ~0.5 to 1 mL volume to flush the catheter line).

[0765] Dosing accuracy

[0766] Calculate and check the quantifiable amount of the daily infusion (confirm actual dose administration) to make daily adjustments. During each phase, check the accuracy of the pump before the start of dosing and after the end of dosing.

[0767] Collection times and number of animals

[0768] Blood samples were obtained for determination of plasma concentrations of brincidofovir during period 2.

[0769] Table 53: Collection times and number of animals

[0770]

[0771] On days 1 and 7, blood samples were obtained and used for toxicokinetic determinations at the above time points.

[0772] Collection Procedure

[0773] Approximately 0.4 mL of whole blood was obtained from each animal through the tail vein at each time point. Animals were not fasted before blood collection. Before processing, according to the equipment SOP, blood was collected into tubes containing K2EDTA anticoagulant and placed on wet ice in an upright state. Plasma was separated by centrifugation (10 minutes, at about 2000g, at about 2-8°C). About 0.10 mL of plasma was transferred to a freezing tube and appropriately labeled with the number of studies, number of animals, time point, sampling date and sample type. The remaining plasma was transferred to a second freezing tube and retained as a spare sample. Within about 2 hours after each blood sample was collected, plasma was frozen at about 80±10°C until analysis. Animals were euthanized (CO2 inhalation) after the last blood collection.

[0774] All plasma sample tubes were stored frozen (80 ± 10 °C) and shipped (frozen, on dry ice) for analysis to Pyxant Laboratories, Colorado Springs, CO, USA. Samples were shipped within 1 month of collection.

[0775] In general, TK parameters were estimated using Phoenix WinNonLin (V 6.3) using mean plasma concentrations of brincidofovir and cidofovir derivatives from a complex blood sampling design.

[0776] Cages were observed twice daily for mortality and signs of severe toxicity or pharmacological effects.

[0777] For dose observation, Phase 1: Record any sub-health or symptoms of toxicity or pharmacological effects observed during the infusion (e.g., abnormalities in general condition, appearance, activity, behavior, breathing, etc.).

[0778] The animal is removed from its cage and subjected to a pre-examination. The examination includes observation of the general condition, skin and coat, eyes, nose, mouth, abdomen, and external genitalia, as well as an assessment of respiration.

[0779] weight

[0780] Phase 1: Animals were removed from their cages and pre-weighed twice before each dosing.

[0781] Phase 2: Animals were removed from cages and pre-weighed twice before each dosing and after the last dose during the afternoon of Day 7. Finally, fasting body weights were obtained only before necropsy.

[0782] Beginning one week prior to Phase 2 dosing, food consumption was measured (weighed) weekly.

[0783] Blood obtained as a terminal procedure through the orbital sinus (posterior bulbar venous plexus) under mild isoflurane anesthesia was used to analyze the hematology and clinical chemistry parameters of all animals at the end of dosing. Before blood collection, animals were fasted overnight. After blood collection, animals were not allowed to recover from anesthesia.

[0784] hematology

[0785] Blood samples (approximately 0.25 mL) were collected into tubes containing K2EDTA anticoagulant and analyzed using a Siemens ADVIA120 hematology analyzer: hemoglobin concentration (HGB); hematocrit (HCT); red blood cell count (RBC); platelet count (PLT); mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); mean corpuscular hemoglobin concentration (MCHC); red blood cell distribution width (RDW); total white blood cell count (WBC); reticulocyte count (RETIC); differentiated white blood cell count (manual differentiated white blood cell count was performed for verification, and absolute values ​​were calculated when necessary); neutrophils (ANEU); lymphocytes (ALYM); eosinophils (AEOS); basophils (ABASO); monocytes (AMONO); large unstained cells (ALUC);

[0786] Blood smears from peripheral areas were prepared for each animal at each blood collection interval for confirmation of automated results and / or other evaluations deemed necessary by the clinical pathologist.

[0787] Clinical Chemistry

[0788] Blood samples (approximately 1 mL) were collected into tubes without anticoagulant, allowed to clot, centrifuged to obtain serum, and analyzed using a Siemens ADVIA 1800 chemistry analyzer for the following: aspartate aminotransferase (AST) (kinetic-modified Bergmeyer); alanine aminotransferase (ALT) (kinetic-modified Bergmeyer); alkaline phosphatase (ALKP) (kinetic-Tietz AMP buffer); blood urea nitrogen (BUN) (enzyme-Roch-Ramek and urease); creatinine (CREAT) (Jaffe Picric acid in alkaline medium); glucose (GLU) (glucose hexokinase II method); cholesterol (CHOL) (enzyme esterase / oxidase-Trand endpoint); triglycerides (TRIG) (Fossati three-step method enzyme-Trinder endpoint); total protein (TP) (biuret technique); albumin (ALB) (bromocresol green method); total bilirubin (TBILI) (vanadate oxidation); sodium (NA + )(ion selective electrode); potassium (K + ) (ion selective electrode); chloride (Cl - ) (ion selective electrode); calcium (Ca ++ )(Michaylova&Ilkova, arsenazo III); inorganic phosphorus (phosphomolybdate-UV method).

[0789] Autopsy

[0790] Phase 1: All animals were euthanized by exsanguination following carbon dioxide inhalation and discarded without macroscopic examination at the end of the observation period.

[0791] Phase 2: Main DRF study animals were euthanized by exsanguination after isoflurane inhalation, and TK satellite animals were euthanized by carbon dioxide inhalation.

[0792] Main study animals from Phase 2 were necropsied on Day 8. Animals were fasted overnight prior to necropsy.

[0793] A complete macroscopic examination was performed on the main Phase 2 study animals. The macroscopic examination included inspection of the external surface and all foramina; the external surface of the brain and spinal cord; the organs and tissues of the skull, thorax, abdomen, pelvis, and neck; and the remainder of the carcass for any gross morphological abnormalities. All animals were discarded after the examination; no tissues were collected or saved.

[0794] The following abbreviations are used for hematology: CS: coagulation specimen; LA: laboratory accident; NVIM: invalid because result is unreliable; CLSE: severe platelet aggregation noted; CLSL: mild platelet aggregation noted; NCLP: no aggregation.

[0795] Blood Chemistry: TBILI values ​​below the analytical limit (<0.2) were not calculated.

[0796] According to: Total protein - albumin to calculate globulin (GLOB)

[0797] Calculate albumin / globulin according to: albumin / globulin ratio (A / G)

[0798] All statistical analyses were performed separately for males and females, using the individual animal as the basic experimental unit.

[0799] For Phase 2, the following data types were analyzed separately at each time point: body weight; change in body weight from interval to interval; cumulative change in body weight from baseline; food consumption, hematology, and clinical chemistry. Parameters analyzed were identified as continuous, discrete, or binary. The brincidofovir-treated and control groups were then compared using the following procedure.

[0800] Life Research Behavior

[0801] Table 54 below summarizes details regarding the conduct of the study, including but not limited to experimental animals, study materials, study design, dosing, observations, and results.

[0802] Table 54: Experimental Design Study Summary (Phase 2)

[0803]

[0804] sampling

[0805] Use composite blood sampling design, make to sample twice on each sampling day to each rat.Collect blood sample according to the scheme shown in Table 54.Collect blood from unanesthetized animals through tail vein into tube containing anticoagulant, and place it on wet ice in an upright state.In blood collection animal, not fasted.By centrifugation (when about 2000g, be 10 minutes, about 2-8 ℃) separate plasma, and transfer to separately labeled cryovial.The cryovials containing all collected plasma samples are appropriately labeled with research quantity, animal number, time point, sampling date and sample type.

[0806] All plasma samples were obtained and frozen at approximately -80°C (±10°C) within approximately 2 hours of collection of each blood sample until analysis.

[0807] All plasma samples were stored frozen [approximately -80°C (±10°C)] and shipped (frozen, on dry ice) to Pyxant Laboratories for analysis.

[0808] Bioanalytical methods

[0809] Rat plasma bioanalysis was performed by Pyxant Laboratories. After LC-MS / MS analysis, the concentrations of brincidofovir and cidofovir in plasma samples were determined using a protein precipitation extraction-based method; for 50 μL aliquots in rat plasma, the calibration ranges for brincidofovir and cidofovir were 1.00-1500 ng / mL and 5.00-750 ng / mL, respectively (Pyxant Study No. 3025). As part of a non-GLP study, ISR sample analysis was not performed.

[0810] Data analysis

[0811] Toxicokinetic analysis

[0812] To generate the mean curve, all sample concentrations below the limit of quantitation (BLQ) were set to zero.

[0813] These values ​​are recorded in Tables 61 and 62, with the raw BLQ in the column labeled [Concentration] and the estimated values ​​from the mean calculation in the column labeled [Estimated Concentration].

[0814] The processing of the mean curve BLQ values ​​for NCA purposes is recorded in Tables 63 and 64, with the original mean curve BLQ in the column labeled [Mean Concentration] and the estimated values ​​for NCA analysis in the column labeled [Estimated Mean Concentration]. max Before) the BLQ value is set to 0, and the maxThe BLQ values ​​at subsequent time points are set to lost.

[0815] All pharmacokinetic parameter estimates were completed using a non-compartmental model (Model 200-202 for IV infusion dosing). AUC was calculated using the linear-upper-log-lower trapezoidal rule. last and AUC inf .

[0816] Statistical analysis was limited to descriptive statistics, including arithmetic mean, standard deviation, and %CV of the arithmetic mean.

[0817] The coefficient of variation (%CV) of mean plasma concentrations ranged from (9.98 to 173% CV) for brincidofovir and from (4.82 to 173% CV) for cidofovir.

[0818] Peak concentrations of brincidofovir are usually observed at the end of a 2-hour IV infusion and decline rapidly; 8 hours after the start of the 1 mg / kg dose and 24 hours after the 15 mg / kg dose, brincidofovir concentrations are at or near the lower limit of quantification (LLOQ, 1 ng / mL). The terminal half-life (t1 / 2), CL, and Vss could not be determined due to insufficient data points to characterize the elimination phase. In general, the mean brincidofovir C max The AUC and AUC increased roughly proportionally from 1 to 15 mg / kg, although AUC was determined based on a small number of concentrations after 1 mg / kg. max and AUC last The values ​​showed a trend towards lower values ​​on day 7 [Day 7 / Day 1 cumulative ratio (AR) 0.44 to 0.53]. Further, no gender differences in brincidofovir TK parameters were observed.

[0819] Overall, the increase in exposure to the metabolite cidofovir was less than proportional to the increase in brincidofovir dosing, there were no significant gender differences in TK parameters, and TK parameters did not change after repeated dosing.

[0820] Bioanalysis was performed on 108 plasma samples collected only during Period 2 to determine the concentrations of brincidofovir and the cidofovir metabolite, cidofovir. Plasma samples were collected 2 hours after the start of dosing on Days 1 and 7 (Group 1), or 1, 2, 8, and 24 hours after the start of dosing on Day 1, and before dosing and 2, 8, and 24 hours after the start of dosing on Day 7 (Groups 2 and 3), and analyzed by LC / MS / MS.

[0821] Toxicokinetic analysis of plasma concentrations of brincidofovir and cidofovir was performed for Phase 2 only.

[0822] On days 1 and 7, brincidofovir C max The increase was roughly proportional to the dose (for female rats) or less than proportional to the dose (for male rats). When the dose increased 15 times from 1 mg / kg to 15 mg / kg, for female C max Increased by 17.3 to 18.2 times for male C max On days 1 and 7, the AUC of brincidofovir was increased by 9.2-fold to 18.6-fold. last Roughly proportional increases (for female rats); when the dose was increased 15-fold, AUC last AUC could not be determined due to insufficient data points in male rats following 1 mg / kg dosing. last proportion.

[0823] In female and male animals, for 1 or 15 mg / kg dosing, brincidofovir C on days 1 and 7 was not significantly different. max The brincidofovir AUC could not be compared for the 1 mg / kg dose due to insufficient data points to calculate the AUC in males. last For the 15 mg / kg dose, the AUC of brincidofovir on days 1 and 7 last It was similar between female and male animals (within 16%).

[0824] Compared with day 1, the C of brincidofovir on day 7 was significantly higher after twice-weekly dosing of 1 mg / kg brincidofovir in females and 15 mg / kg in both females and males. max and AUC last The C values ​​showed a trend towards lower values ​​on the 7th day. max and AUC last (Day 7 / Day 1 cumulative ratio (AR) ranged from 0.44 to 0.53).

[0825] Table 55: Brincidofovir TK parameters following single and multiple 2-hour intravenous infusions of brincidofovir in rats on days 1, 3, and 7 (Phase 2)

[0826]

[0827] NA, not applicable; NR, not reportable due to insufficient data points

[0828] As shown in Table 55 and Table 56, on Day 1 and Day 7, Cidofovir C maxThe increase in cidofovir was generally less than dose proportional. For a 15-fold increase in brincidofovir dose from 1 mg / kg to 15 mg / kg, females max On days 1 and 7, for female animals, the AUC of cidofovir was 2.9-fold to 4.1-fold, and for male animals it was 5.6-fold to 16.8-fold. last The increase was less than dose proportional; for a 15-fold increase in the brincidofovir dose, the cidofovir AUC last Increased by 9.4-fold to 16.2-fold. Due to insufficient data points in male rats after 1 mg / kg dosing, AUC could not be determined. last proportion.

[0829] Cidofovir C on days 1 and 7 after a 15 mg / kg brincidofovir dose max and AUC last It was similar between female and male animals (within 33%).

[0830] For female animals, after twice weekly dosing with 1 mg / kg of brincidofovir, the C of brincidofovir on day 7 was max and AUC last The values ​​showed a trend towards higher C values ​​on day 7 compared to day 1. max and AUC last (Day 7 / Day 1 AR ranged from 1.64 to 1.9). However, after 15 mg / kg (3 doses) in both female and male animals, C max and AUC last were similar (Day 7 / Day 1 AR 1.0 to 1.1).

[0831] The ratio of metabolite to parent (M / P) was calculated on a molar basis. For females, the AUC on day 1 for 1 mg / kg and 15 mg / kg brincidofovir doses was last For female animals, the M / P ratios on day 7 were 0.193 and 0.444 for the 1 mg / kg and 15 mg / kg brincidofovir doses, respectively.

[0832] Following dosing at 1 mg / kg in male rats, the AUC could not be determined due to insufficient data points. last After administration of 15 mg / kg brincidofovir, the M / P ratios on day 1 and day 7 were 0.126 and 0.307, respectively.

[0833] Table 56: Cidofovir TK parameters following single and multiple 2-hour intravenous infusions of brincidofovir in rats on days 1, 3, and 7

[0834]

[0835] For both Phase 1 and Phase 2, all animals were allowed to survive until scheduled termination.

[0836] Clinical observation

[0837] Phase 1

[0838] Dose 1 (2 mg / kg): Clinical signs in two male animals consisted of rapid breathing and prone posture with or without piloerection, partial eye closure, and decreased activity.

[0839] Dose 2 (4 mg / kg); clinical signs in male and female animals consisted of irregular breathing.

[0840] Dose 3 (10 mg / kg): No clinical signs were observed in either male or female animals.

[0841] Dose 4 (15 mg / kg): Clinical signs in two males and one female included decreased activity with or without respiratory irregularities, piloerection, and bilateral partial eye closure.

[0842] All clinical symptoms were reversible and disappeared immediately after the end of the infusion.

[0843] Phase 2

[0844] Administration of brincidofovir at 1 mg / kg / day or 15 mg / kg / day did not result in any clinical signs associated with brincidofovir in males or females. There were no effects on body weight (assessed in Phase 2 only) or food consumption associated with brincidofovir treatment.

[0845] Clinical Pathology

[0846] Brincidofovir-associated hematological findings were limited to a slight decrease in reticulocytes in males and females at 15 mg / kg (-33% and -30% in the control group, respectively, statistically significant only in males), which was associated with an increase in mean cellular hemoglobin concentration (MCHC) in males (+2.1% in the control group, statistically significant). At the end of the 7-day dosing period, the decrease in erythrocyte count (hemoglobin, hematocrit, red blood cells) was negligible (-4.2% relative to the control group). The minimal effect of reticulocytopenia on erythrocyte count was attributed to the long lifespan of rat erythrocytes (~45-68 days) relative to reticulocytes (2-5 days). With continued dosing, the decrease in erythrocyte count is expected to be more pronounced. Reticulocytopenia indicates decreased erythropoiesis in hematopoietic tissues. In the absence of food consumption or weight loss or poor clinical condition, they may be due to the inhibition of erythropoiesis associated with brincidofovir, which is not unexpected with this type of drug (nucleotide analog).

[0847] Males at 15 mg / kg (control group + 3.8%; not statistically significant) and females at ≥ 1 mg / kg (control group + 4.9%; statistically significant) had minimal increases in total calcium associated with brincidofovir, and females at 15 mg / kg (control group + 29%; statistically significant) had minimal increases in urea (BUN). Both findings were observed to be associated with changes in renal function (decreased urinary excretion). In addition, without wishing to be bound by theory, increases in total calcium may reflect increased mobilization from bone or increased intestinal absorption.

[0848] The changes in Animal 3048 (mild increases in BUN, creatinine, and phosphorus) were not considered to be related to brincidofovir and were thought to be secondary to a decrease in glomerular filtration rate (noted macroscopically) due to the absence of the left kidney.

[0849] All other differences from the control group, statistically significant or otherwise, were not considered to be related to brincidofovir because of their direction, small magnitude, rare occurrence, irrelevance to dose, inconsistency with relevant endpoints, and / or because values ​​were comparable to the study control range.

[0850] pathology

[0851] Macroscopic findings were rare, occurred sporadically, and were not considered to be related to brincidofovir because they occurred at a similar frequency in control animals, lacked a dose relationship, or were considered to be congenital anomalies (animal 3048). Male animal 3048 did not have a left seminal vesicle or left kidney; the absence of the left kidney was associated with elevated blood urea nitrogen (BUN), creatinine, and phosphorus in this animal.

[0852] For Phase 1, all doses were well tolerated when administered by a single 2-hour intravenous infusion under the conditions of the study. Therefore, a maximum tolerated dose of brincidofovir was not determined. However, 15 mg / kg delivered by a 2-hour intravenous infusion of a 0.75 mg / mL brincidofovir solution represents the maximum feasible dose based on in vitro evidence of hemolysis at brincidofovir concentrations above 0.75 mg / mL.

[0853] For Phase 2, peak brincidofovir concentrations were generally observed at the end of the 2-hour IV infusion and declined rapidly.

[0854] Typically, the average brincidofovir C max The AUC and AUC increased approximately proportionally with the dose from 1 to 15 mg / kg, although after the 1 mg / kg dose, the AUC was determined based on a small number of concentrations. max and AUC last The values ​​showed a trend of lower values ​​on the 7th day (the 7th day / 1st day cumulative ratio (AR) was 0.44 to 0.53). Further, no gender differences were observed in the brincidofovir TK parameters. Overall, the increase in metabolite cidofovir exposure was less than the ratio of the brincidofovir dose increase, there was no obvious gender difference in the TK parameters, and the TK parameters did not change after repeated administration. Under the conditions of this study, single-dose administration by 2-hour IV infusion on days 1, 4, and 7 was well tolerated.

[0855] Brincidofovir concentration in plasma

[0856] The concentration-time data of brincidofovir following single and repeated dosing at 1 mg / kg and 15 mg / kg are summarized in Tables 59 and 60.

[0857] The concentrations of brincidofovir and cidofovir in all control plasma samples were at the BLQ for each analyte (<1.0 ng / mL and <5.0 ng / mL, respectively).

[0858] After a single IV infusion of brincidofovir over a 2-hour period, plasma brincidofovir concentrations at both dose levels (1 mg / kg and 15 mg / kg) were detected in the first sample (1 hour). After twice weekly dosing (3 doses on days 1, 3, and 7), brincidofovir in the pre-dose sample was BLQ, but brincidofovir could be detected in the first pre-dose sample on day 7 (2 hours, end of infusion). The median time of peak concentration of brincidofovir occurred 2 hours after single and twice weekly 1 mg / kg dosing, 1 hour after a single 15 mg / kg dosing, and 2 hours after twice weekly 15 mg / kg dosing. The concentration of brincidofovir reached BLQ in all males and some females 8 hours after 1 mg / kg dosing, and reached or approached LLOQ 24 hours after 15 mg / kg dosing. The coefficient of variation (%CV) of the mean plasma concentration of brincidofovir for female animals ranged from 13.0% to 173%, and the coefficient of variation (%CV) of the mean plasma concentration of brincidofovir for male animals ranged from 9.98% to 28.1%. In general, the highest %CV was observed at later time points when the concentration reached or approached the LLOQ.

[0859] Toxicokinetics of brincidofovir in plasma

[0860] Mean plasma brincidofovir concentration-time information from the input file for the Phoenix WinNonlin analysis is contained in Table 63. Brincidofovir TK parameters following single and twice weekly brincidofovir dosing as an intravenous infusion to rats are summarized in Table 56. The terminal half-life (t1 / 2), CL, and Vss could not be determined due to insufficient data points to characterize the elimination phase.

[0861] On days 1 and 7, brincidofovir C max The increase was roughly in proportion to the dose (for female rats), or less than in proportion to the dose (for male rats). For a 15-fold increase in dose from 1 mg / kg to 15 mg / kg, for female animals, the max The AUC of brincidofovir was increased by 17.3-fold to 18.2-fold in males and 9.2-fold to 18.6-fold in females on days 1 and 7. last Roughly proportional increases (for female rats); for a 15-fold dose increase, AUC last AUC could not be determined due to insufficient data points in male rats following dosing at 1 mg / kg. last proportion.

[0862] Between female and male animals, for the 1 or 15 mg / kg dose, brincidofovir C on days 1 and 7 max The brincidofovir AUC could not be compared for the 1 mg / kg dose due to insufficient data points to calculate the AUC in males. last For the 15 mg / kg dose, the AUC of brincidofovir on days 1 and 7 was last It was similar between female and male animals (within 16%).

[0863] Compared with day 1, brincidofovir C increased on day 7 after twice weekly dosing at 1 mg / kg for females and 15 mg / kg for females and males. max and AUC last The values ​​showed a trend towards lower C on day 7. max and AUC last [Day 7 / Day 1 cumulative ratio (AR) was 0.44 to 0.53].

[0864] Table 57: Brincidofovir TK parameters following single and multiple 2-hour intravenous infusions of brincidofovir in rats on days 1, 3, and 7

[0865]

[0866]

[0867] Plasma concentrations of cidofovir

[0868] The concentration-time data for cidofovir following single and repeated daily dosing at 1 mg / kg and 15 mg / kg are summarized in Tables 59-60.

[0869] In female animals, after a single IV infusion of brincidofovir, the plasma concentration of cidofovir was detected 2 hours after administration of 1 mg / kg (second time point) or 1 hour after administration of 15 mg / kg (first time point). After twice weekly administration of brincidofovir, the plasma concentration of cidofovir in the pre-dose sample was BLQ, but it could be detected in all 2-hour samples on the 7th day (first pre-dose time point). 2 hours after single and twice weekly 1 mg / kg brincidofovir administration, and 8 hours after single and twice weekly 15 mg / kg brincidofovir administration, the median T of cidofovir appeared max .

[0870] In male animals, after a single IV infusion of brincidofovir, detectable plasma concentrations of cidofovir were observed only at a single time point 8 hours after 1 mg / kg administration. However, cidofovir was detected in the first sample (1 hour) after 15 mg / kg administration. After twice weekly brincidofovir administration, the plasma concentration of cidofovir was BLQ in the predose sample and the first predose sample (2 hours), but it can be detected in the 8-hour sample on the 7th day after 1 mg / kg administration and the 2-hour sample on the 7th day after 15 mg / kg administration (the first predose sample). The median T of cidofovir appeared 8 hours after single or twice weekly 1 mg / kg or 15 mg / kg brincidofovir administration. max . In male and female animals, cidofovir concentrations reached BLQ 24 hours after single and twice weekly dosing with 1 mg / kg brincidofovir, and BLQ was not reached 24 hours (the last time point) after single or twice weekly dosing with 15 mg / kg brincidofovir. The %CV of the mean cidofovir concentrations ranged from 4.82 to 88.4% for female animals and from 5.49 to 173% for male animals. The highest %CV was observed at early or later time points when cidofovir concentrations were at or near the LLOQ. Toxicokinetics of Cidofovir in Plasma

[0871] The mean plasma cidofovir concentration-time information in the input file for the Phoenix Winolin analysis is contained in Table 64. The brincidofovir TK parameters following single and twice weekly brincidofovir dosing as an intravenous infusion to rats are summarized in Table 58.

[0872] On days 1 and 7, cidofovir C max The increase was generally less than dose proportional. For a 15-fold increase in the brincidofovir dose from 1 mg / kg to 15 mg / kg, the C of cidofovir in females max Increased by 2.9 to 4.1 times for male animals with cidofovir C max On days 1 and 7, for females, the AUC of cidofovir was 5.7 to 16.8 times higher. last The increase was less than dose proportional; for a 15-fold increase in the brincidofovir dose, the cidofovir AUC last AUC could not be determined due to insufficient data points in male rats following the 1 mg / kg dose. last proportion.

[0873] In both female and male animals, after a 15 mg / kg dose of brincidofovir, the C of cidofovir on days 1 and 7 was significantly higher than that of brincidofovir on days 1 and 7. max and AUC last are similar (within 33%).

[0874] In female animals, after twice-weekly administration of 1 mg / kg brincidofovir, the C of cidofovir on day 7 was significantly higher than that on day 1. max and AUC last The C max and AUC last However, the cidofovir C in females and males given 15 mg / kg (3 doses) was significantly higher than that in females (range 1.6 to 1.9 on day 7 / day 1). max and AUC last were similar (Day 7 / Day 1 AR ranged from 1.0 to 1.1).

[0875] For females, the AUC on day 1 for 1 mg / kg and 15 mg / kg doses of brincidofovir last The metabolite / parent (M / P) ratios were 0.190 and 0.661, respectively. For female animals, the M / P ratios on day 7 were 0.193 and 0.444 for the 1 mg / kg and 15 mg / kg doses of brincidofovir, respectively.

[0876] Due to insufficient data points in male rats following the 1 mg / kg dose, the AUC could not be determined. last The M / P ratio of 15 mg / kg brincidofovir was 0.126 and 0.307 on day 1 and day 7, respectively.

[0877] Table 58: Cidofovir TK parameters following single and multiple 2-hour intravenous infusions of brincidofovir in rats on days 1, 3, and 7

[0878]

[0879] Table 59: Plasma concentrations of brincidofovir and cidofovir in female and male rats following single and multiple 2-hour intravenous infusions at the labeled brincidofovir dose of 1 mg / kg

[0880]

[0881]

[0882]

[0883] a Samples with BLQ bioassay results were zeroed.

[0884] Table 60: Plasma concentrations of brincidofovir and cidofovir in female and male rats following single and multiple 2-hour intravenous infusions at the labeled brincidofovir dose of 15 mg / kg

[0885]

[0886]

[0887]

[0888] a Samples with BLQ bioassay results were zeroed.

[0889] Table 61: Measured and estimated average generated brincidofovir concentration data

[0890]

[0891]

[0892] Table 62: Measured and estimated average generated cidofovir concentration data

[0893]

[0894]

[0895] Table 63: Measured and estimated NCA brincidofovir concentration data

[0896]

[0897]

[0898] Table 64: Measured and estimated NCA cidofovir concentration data

[0899]

[0900] Example 9-[ 14 Mass balance, pharmacokinetics, and tissue distribution of brincidofovir by quantitative whole-body autoradiography in rats after a single oral or intravenous dose of C]

[0901] The purpose of this study was to characterize the efficacy of a single intravenous (IV, 2-hour infusion) or oral (PO, gavage) administration of [ 14 C] Total radioactivity tissue distribution in male Sprague Dawley (SD) and Long-Evans (LE) rats after brincidofovir. In addition, the rate and extent of excretion (mass balance) and pharmacokinetics (PK) of total radioactivity in male Sprague Dawley rats after administration of a single infusion (IV, 2-hour infusion) of brincidofovir were examined. The residual plasma and excreta collected in this study were stored at -70°C for metabolite distribution and identification experiments performed under different protocols.

[0902] Materials and Methods

[0903] Four groups of male SD rats (albino) and three groups of male LE rats (pigmented) were used in this study; a total of 39 rats. All animals were obtained from Hilltop Lab Animals, Inc. (Scottdale, PA). At the time of dosing, rats weighed between 214 and 265 g. A single 2-hour (h) IV infusion of [ 14 C] Brincidofovir was administered to rats in Groups 1, 2, 3, and 4. A single 2-hour IV infusion of [ 14 C] Brincidofovir was administered to rats in Group 5. A single oral gavage was administered at a target dose of 15 mg / kg. 14 C] Brincidofovir was administered to rats in groups 6 and 7. On the day of administration, 3 administration formulations (one high dose IV, one low dose IV, and one PO dose) were prepared at QPS. The IV formulations of groups 1, 2, 3, and 4 contained a vehicle of 10 mM sodium phosphate buffer in a 5% glucose solution at pH 8.0 (± 0.1). The IV formulation of group 5 contained a vehicle of 16 mM sodium phosphate buffer in a 5% glucose solution at pH 8.0 (± 0.1), and the PO administration formulation contained a vehicle of 12.5 mM sodium phosphate buffer at pH 8.0 (± 0.1). The IV formulation of group 5 and the PO administration formulations of groups 6 and 7 contained a higher concentration of buffer, so that the final buffer concentration in all dose formulations was approximately 10 mM.

[0904] Test animals

[0905] 16 adult male SD rats and 23 adult male LE rats were obtained from Hilltop Lab Animals Co., Ltd. (Scottdale, PA) and used for this study. The rats in Groups 1-5 were surgically modified to have a built-in femoral vein catheter for IV infusion. The weight range, source, supplier and receipt date of the animals are all recorded in the raw data. A permanent identification number was assigned to the animals used in this study using a permanent marker on the tail, and unused standby animals were returned to inventory after successful dosing of the animals used in this study. Randomization was carried out by catheter patency and QPS SOP.

[0906] Tables 65 and 66 give an overview of the dosing and sample collection schedule.

[0907] Table 65: Summary of dosing in living studies

[0908]

[0909] a Femoral vein catheterization; b 2 hours infusion time

[0910] Table 66: Summary of Living Sample Collection

[0911]

[0912] ROA = Route of Administration NA = Not Applicable TBD = To Be Determined a Collect samples on dry ice

[0913] Dosage

[0914] Animal weight was used to determine dosing volume, and animal weight was measured prior to dosing on the day of dosing. Dosimetry results, animal weight, and target dosing parameters were entered into Debra LIMS, and the target dose volume for each animal was determined by Debra LIMS. Animals were dosed with [ 14 C] a single 2-hour IV infusion of brincidofovir, or animals were given [ 14 C] Oral gavage administration of brincidofovir. Actual dosing for each rat was determined by Debra LIMS, which uses individual animal weight data by deducting the weight of the empty dose syringe / needle or needle / infusion line after administration from the weight of the full syringe / needle or needle / infusion line before administration. The average pre-dose radioactivity concentration (dpm / g dosing solution) was multiplied by the net weight of the dosing solution administered to calculate the amount of radioactivity administered to each animal. The actual doses are listed in Table 67.

[0915] Table 67: Body weight and [ 14 C] Brincidofovir Dosage

[0916]

[0917]

[0918]

[0919] a The specific activity of the oral (PO) preparation was 14.2689 μCi / mg [ 14 C] Brincidofovir. The dose preparation activity of the PO formulation was 42.2151 μCi / g.

[0920] b The specific activity of the intravenous (IV) infusion preparation was 13.5694 (at 15 mg / kg) and 101.5100 (at 2 mg / kg) μCi / mg [ 14 C] Brincidofovir. The IV formulation had a dose preparation activity of 10.1074 μCi / g at 15 mg / kg and 82.8740 μCi / g at 2 mg / kg.

[0921] c Sample collection from animals was terminated during the IV infusion period. These animals were omitted from the mean dose calculation.

[0922] d The mean dose for all animals in Group 4, 188.836 μCi / kg was used for dosimetry estimation.

[0923] Note: SD = standard deviation; MB = mass balance; PK = pharmacokinetic; QWBA = quantitative whole body autoradiography sample collection

[0924] Urine, feces, blood / plasma, cage residue samples, and carcasses were collected during this study as described below for each group Row Waste collection (Group 1)

[0925] Three male albino rats were assigned to Group 1 (IV infusion) for fecal collection.

[0926] Before dosing (overnight) and at intervals of 0-8h, 8-24h and every 24h interval thereafter until 168h after dosing, urine samples were collected from each animal and collected into pre-labeled urine collection tubes. All urine specimens were collected on dry ice. The total weight of each urine collection was recorded, and the samples were stored frozen at approximately -70°C until LSC analysis. After analysis, the samples were kept at approximately -70°C and stored for further analysis, which was performed under a separate protocol.

[0927] Fecal samples were collected from each animal before dosing (overnight) and at 24h intervals until 168h after dosing and were collected into pre-labeled fecal collection tubes. All fecal specimens were collected on dry ice and the total weight of each fecal sample was recorded. Samples were stored at approximately -70°C until homogenization and LSC analysis and stored for further analysis, performed under a separate protocol.

[0928] Collect cage residue samples. Rinse cages with approximately 30 mL of deionized water after each daily post-dose fecal collection starting 24 h after dosing. Spray the interior surfaces of the metabolism cages with approximately 90 mL of Windex solution (or equivalent detergent solution) and wipe with a gauze pad after the last fecal collection. Collect cage rinse and cage wash samples into tared and pre-labeled containers. Record the total weight of each cage rinse and wash. Store cage rinse, cage wash, and cage wipe collections at approximately -20°C. Keep animal carcasses in Groups 1 and 2 at approximately -20°C. Carcasses and cage residues were not analyzed because the recovery rate of radioactivity in the feces was >90%.

[0929] Plasma pharmacokinetic sample collection (Group 2)

[0930] Blood was collected from each animal in Group 2 (IV infusion) for plasma and blood analyses at the following times.

[0931] Peripheral blood samples were collected by cardiac puncture into pre-labeled blood collection tubes containing K2EDTA as an anticoagulant. Peripheral samples were collected at 0.5, 1, 2, 4, 8, 24 and 72 h after the start of the infusion (N = 1 per time point), and the volume was unlimited. Three weighed blood aliquots (~ 0.100 g) were taken out and analyzed for total radioactivity using combustion and subsequent LSC. Blood aliquots were kept at about 4 ° C until analysis. The remaining blood samples were kept on wet ice (about 4 ° C) and then centrifuged within 1 h of the blood collection time to obtain plasma. The centrifuge settings were recorded in the research notebook. The radioactivity of replicate plasma (0.050 mL) aliquots was analyzed by direct counting by LSC, and the remaining plasma was stored at about -70 ° C and stored for analysis under a possible separate research program in the future. The remaining red blood cells and the animal carcasses of Group 2 were discarded as radioactive waste.

[0932] QWBA (Groups 3-7)

[0933] At each animal's respective termination time point, euthanasia was performed immediately after blood collection for immediate QWBA analysis. One mouse was euthanized per group per time point, as listed below.

[0934] Group 3 (IV) - 2 (end of infusion), 24 and 168 h

[0935] Group 4 (IV) - 1, 2 (end of infusion), 4, 8, 24, 72, 96, 168 and 840 h

[0936] Group 5 (IV) - 2 (end of infusion), 8, 24 and 72 h

[0937] Group 6 (PO) - 2, 24 and 168 hours

[0938] Group 7 (PO) - 2 (end of infusion), 0.5, 1, 2, 4, 8, 24, 72, 96, 168, and 840 h

[0939] Each rat was deeply anesthetized by isoflurane anesthesia, and blood samples (about 2 mL) were collected by cardiac puncture into tubes containing K2EDTA as an anticoagulant, and the rats were frozen in a hexane / solid carbon dioxide bath for at least 15 min to euthanize. Blood samples were stored on wet ice (about 4° C.) and then centrifuged within 1 h of the blood collection time to obtain plasma. The centrifuge settings were recorded in the research notebook. The radioactivity of replicate plasma (0.050 mL) aliquots was analyzed by LSC using direct counting, and the remaining plasma was stored at about -70° C. and stored for analysis under a possible future separate research program. The remaining red blood cells and the animal carcasses of Group 2 were discarded as radioactive waste.

[0940] Sample storage conditions

[0941] Carcasses and cage residue samples were stored frozen at -20°C until sample analysis, and remaining plasma and feces were stored frozen at -70°C.

[0942] Sample analysis

[0943] LSC analysis of blood, plasma and feces

[0944] Feces were homogenized and analyzed for total radioactivity. A weighed fecal sample from each rat was homogenized in approximately 3 volumes of water (approximately 3 times the weight of the fecal sample). The total weight of each homogenized product was determined and three weighed aliquots (~0.5 g) were combusted in a Packard sample oxidizer prior to LSC analysis. The actual weight of the individual aliquots and the amount of solvent used in the homogenization were recorded. A pre-weighed portion of each fecal homogenate was placed in a chamber containing a combustion chamber- (Combusto- )'s combustion chamber - (Combusto- ), allowed to dry overnight in a fume hood, and completely combusted in a sample oxidizer.

[0945] Three weighed aliquots (-0.100 g) of the blood aliquots (Group 2 only) were combusted in a Packard sample oxidizer prior to LSC analysis.

[0946] Released during combustion 14 CO2 is captured in the Carbo- In the solution, add (scintillating fluid) and the radioactivity was measured by LSC. Oxidized samples containing a known amount of radioactivity (0.100 mL Spec- ) and compared with an unburned standard to determine the recovery of the combustion process. These controls were analyzed in triplicate and the recovery from these control samples was determined on each day of the combustion sample. 14 Quantitative recovery of C. Blank cones (no added radioactivity) were burned to assess radioactivity carryover between samples.

[0947] The homogenized product of excreta was kept at approximately -70°C after analysis and stored for further analysis under separate research protocols.

[0948] Urine (0.300 mL) and plasma (0.050 mL) samples were thawed, divided into two aliquots by volume if necessary, and analyzed by LSC. The volume or weight of the sample aliquots was recorded in the study record. Ultima gold scintillation fluid (5 mL, PerkinElmer) was added to each urine aliquot, as well as the plasma aliquot, and the aliquots were mixed well before analyzing the radioactivity by LSC.

[0949] The radioactivity (counts per minute) in each sample is converted to disintegrations per minute (dpm) by external standardization and quenching curve. The radioactivity content is quantified by a 2800TR or 2900TR liquid scintillation analyzer (PerkinElmer). All samples are counted for at least 5 minutes or at least 100,000 times per minute (cpm). If there is enough volume, the LSC results of duplicate samples that differ from the average by more than 10% are re-divided and re-analyzed. If the CV% of the LSC results of the three samples is greater than 10%, if there is enough volume, the sample is re-homogenized and re-analyzed. These specific requirements apply to all sample aliquots with an average radioactivity greater than 500 dpm, and values ​​below 500 dpm are accepted. The lower limit of quantification (LLOQ) is applied to data equal to 2 times the counts per minute (CPM) value of the background sample.

[0950] QWB

[0951] The pinnae, distal limbs, hair, and tail were removed from each frozen carcass, and each frozen carcass was embedded in an aqueous suspension of approximately 2% (w / v) carboxymethylcellulose and frozen into blocks. The blocks were stored at approximately 20°C before sectioning. Each block was mounted on the stage of a cryostat (Leica CM3600 Cryomacrocut, Nussloch, Germany and Vibratome 9800, St. Louis, MO) and maintained at approximately -20°C and internal standard (3), a concentration (approximately 0.05 μCi / mL) of [14 C]-glucose plasma is put into freezing block for slice thickness quality control.Use whole body cryostat set at -20 ℃, take out multiple whole body slices (about 40 μm thick) in sagittal plane with various levels of interest.Sagittal slices (20-50 μm thick) of frozen corpse are exposed to phosphor imaging plate.Use standard to convert image area in tissue into concentration.All major tissues, organs and biological fluids are represented.These slices are collected on tape (Scotch 8210, 3M Corp., USA, and before taking out for mounting and exposure, dehydrate in cryostat for at least 48 hours.

[0952] Tissue concentration data were determined for the following tissues and / or contents: adipose (brown and white), adrenal glands, bile (ducts), blood (heart), bone marrow, brain (cerebrum, cerebellum, medulla), cecum (and contents), large intestine (and contents), epididymis, esophagus, eye (uvea and lens), Harderian glands, heart, kidney (cortex and medulla), liver, lungs, lymph nodes, pancreas, pituitary gland, prostate, salivary glands, seminal vesicles, skeletal muscle, skin (pigmented and non-pigmented), small intestine (and contents), stomach (gastric mucosa and contents), spleen, spinal cord, testicles, thymus, thyroid gland, and bladder (and contents).

[0953] A set of whole body sections from each rat was mounted on a cardboard backing, covered with thin plastic wrap, and exposed to the 14 C-sensitive phosphor imaging plates (Fuji Biomedical, Stamford, CT) were mixed with 10 different concentrations (0.0009595 to 7.806 μCi / g) of blood. 14 C-glucose. The imaging plates and sections were placed in a light-tight exposure box, placed in a copper-lined lead safe, and exposed for 4 days at room temperature. The imaging plates were scanned using a Typhoon 9410 image acquisition system (GE / Molecular Dynamics, Sunnyvale, CA, USA), and the resulting images were stored on a dedicated QPS computer server. MCID image analysis software (v.7.0, Interfocus Imaging Ltd) and a standard curve consisting of integrated responses [ie, Molecular Dynamics Counts persquare millimeter (MDC / mm 2 )]and[ 14The nominal concentration of [14C] brincidofovir calibration standards was quantified by image densitometry. Radioactivity concentrations were expressed as μg equivalents of [14C] brincidofovir per gram of sample (μg equiv / g). A lower limit of quantification (LLOQ) was applied to the data (see Section 6.7.4).

[0954] Tissue concentrations below the LLOQ were defined as below the limit of quantitation (BQL). Tissue areas not shown on the autoradiograph were defined as no sample (NS) and reported as BQL. If no tissue was seen on the animal autoradiograph, no calibration curve was generated and all tissue concentrations for that animal were reported as BQL.

[0955] The results are based on original, electronic, digital images selected from a complete set of autoradiographic photographs.

[0956] Animal Observation

[0957] In the present study, no observable abnormalities were observed in rats.

[0958] Group 1 - Mass balance in rats after 2-hour intravenous infusion

[0959] Table 68 summarizes the excretion data for individual animals and the mean total radioactivity for Group 1 male Sprague Dawley rats. Table 68: Results of a 2-hour intravenous infusion of [ 14 C] Total excretion of radioactivity after brincidofovir

[0960]

[0961] SD = standard deviation;

[0962] Male rats were given 15 mg / kg by IV infusion over 2 hours. 14 C] The major route of elimination of radioactivity following brincidofovir was urine, which accounted for 51.2% (mean) of the dose administered within 168 h. 42.2% (mean) of the dose administered was obtained in the feces. The total recovery of combined radioactivity in urine and feces of Group 1 male rats was an average of 93.5% of the dose administered during the 168 h collection period. The majority of radioactivity excreted in urine (43.8% of the dose or 86% of the radioactivity excreted in urine) and fecal excretion (36.7% of the dose or 87% of the radioactivity excreted in feces) occurred in the first 24 h after dosing.

[0963] Group 2 - Toxicokinetics in rats after IV administration of 15 mg / kg

[0964] Table 69 reports the plasma total radioactivity concentrations versus time, PK parameters, and blood to plasma ratios for Group 2 male SD rats.

[0965] Table 69: Single 2-hour intravenous infusion of 15 mg / kg [ 14 C] Blood and plasma concentrations and pharmacokinetic parameters in male Sprague Dawley rats following brincidofovir (Group 2)

[0966]

[0967] After 2-hour IV infusion of 15 mg / kg in male rats, the plasma 14 C]Brincidofovir total radioactivity max At 2h T max (i.e., end of infusion) was 10.3 μg equiv / mL, and the concentration decreased to 0.045 μg equiv / mL 72 h after administration. 14 C] AUC of brincidofovir last was 64.4 μg equiv / mL, and t 1 / 2 It is 13.0h.

[0968] The blood-to-plasma ratio of group 2 SD rats showed that [ 14 C]Brincidofovir-derived radioactivity initially partitioned preferentially into plasma rather than the cellular fraction of rat blood between 0.5 and 8 h (range 0.62-0.8), but then showed greater partitioning into the cellular aspect at 24 and 72 h post-dose with a ratio of 1.26 and 1.27, respectively.

[0969] Groups 3-7 - 15 mg / kg (IV and PO) and 2 mg / kg (IV) IV and PO in albino and pigmented rats Comparison of blood and plasma concentration ratios

[0970] Table 70 reports the blood to plasma concentration ratios for Groups 3 to 7 of male SD and LE rats. Figure 3 Shown is a comparison of blood and plasma concentration versus time profiles obtained from Groups 3-7.

[0971] Table 70: Single 2-hour IV infusion or oral gavage administration [ 14 C] Blood to plasma ratio in male rats after brincidofovir (Groups 3-7)

[0972]

[0973]

[0974] ND = Not determined, blood and / or plasma BQLNS = No sample was collected at this time point

[0975] Similar to group 2, after administration of IV[ 14C] In the other animal groups treated with brincidofovir (SD rats, Group 3; LE rats, Groups 4 and 5), a trend toward higher blood:plasma ratios at later time points was observed, although the ratios were roughly consistent at the early (sparse) time points. 14 C] After brincidofovir, blood:plasma ratios ranged from 0.64-1.13, with no observed trend toward higher blood:plasma ratios at later time points (up to 24 h post-dose).

[0976] Groups 3 and 4 - Tissue distribution of QWBA in albino and pigmented rats following IV administration of 15 mg / kg

[0977] Table 71 and Table 72 list the [ 14 C] Summary of brincidofovir tissue distribution.

[0978] Table 71: Single intravenous infusion at a target dose of 15 mg / kg [ 14 C] Radioactivity concentration in tissues of male Sprague Dawley (albino, group 3) rats after brincidofovir administration

[0979]

[0980]

[0981]

[0982] BQL = value below LLOQ; Ni = not identified on the acceptable portion.

[0983] LLOQ = 0.00048400 μCi / g / 0.0135694 μCi / μg = 0.036 μg equivalent / g tissue ULOQ = 20.97300000 μCi / g / 0.0135694 μCi / μg = 1545.610 μg equivalent / g tissue

[0984]

[0985]

[0986]

[0987] Group 3 - QWBA tissue distribution in male albino rats (sparse sampling)

[0988] After a 2-hour IV infusion of 15 mg / kg, [ 14C]Brincidofovir-derived radioactivity was evenly distributed in most tissues of albino male rats (n=1 animal at 2, 24, and 168 h after the start of dosing), and concentrations in most tissues were slightly lower than those in blood / plasma at the end of dosing but higher than those in blood / plasma at later time points. Fecal and gastrointestinal tissue concentrations were significantly higher than blood / plasma at all time points. 14 The highest concentration of brincidofovir-derived radioactivity was 6.9 μg equiv / g, which was observed at 2 h (the end of the infusion period and the first sampling time point for the sparse sampling group). [C] was found in most tissues (35 of 37 tissues) at 2 h after dosing (the first sampling time point). 14 C] brincidofovir-derived radioactive C max , at which point the concentration in most tissues is between 1.0 and 6.0 μg equiv / g.

[0989] In their respective T max The tissues with the highest concentrations (>20.0 μg equiv / g) were: liver (229.4 μg equiv / g), renal cortex (106.5 μg equiv / g), small intestine (43.7 μg equiv / g), bladder (36.0 μg equiv / g), and renal medulla (33.4 μg equiv / g). max The tissues with the lowest concentrations (<1.0 μg equiv / g) were: brain, spinal cord, white fat, bone, skeletal muscle and lens of the eye.

[0990] The highest total concentration determined by QWBA was in the digestive tract contents (C max A range of 26.4 μg equiv / g in the stomach at 24 h to 95.9 μg equiv / g in the small intestine at 2 h) and bladder contents (47.0 μg equiv / g at 2 h) was observed, reflecting the presence of 14 C] Elimination pathways of brincidofovir-derived radioactivity.

[0991] Elimination of drug-related material from most tissues was incomplete at 168 h after dosing; however, those tissues that were determined to contain radioactivity had concentrations < 1.0 μg equiv / g.

[0992] Group 4 - QWBA tissue distribution in pigmented male rats

[0993] After a 2-hour IV infusion at 15 mg / kg, [ 14C]Brincidofovir-derived radioactivity was evenly distributed in most tissues of pigmented male rats (n=1 animal at 1, 2, 4, 8, 24, 72, 96, 168, and 840 h), with concentrations in most tissues slightly lower than those in blood / plasma at early time points (up to 4 h after infusion start) but higher than those in blood / plasma at later time points. Based on AUC (AUC tissue :AUC plasma ; Table 78) showed that most tissues had values ​​> 1, indicating that tissue concentrations were higher than plasma. Fecal and digestive tissue concentrations were significantly higher than blood / plasma at all time points. 14 The highest concentration of brincidofovir-derived radioactivity was 5.3 μg equiv / g, which was observed at 2 h (the end of the infusion period and the second collection time point for the sparse sampling group). [C] was found in most tissues (33 of 38 tissues) at 2 h after dosing. 14 C] brincidofovir-derived radioactive C max , at which point the concentration in most tissues is between 1.0 and 6.0 μg equiv / g.

[0994] In their respective T max The tissues with the highest concentrations (>20.0 μg equiv / g) at the respective Tmax were: liver (229.7 μg equiv / g), renal cortex (97.3 μg equiv / g), small intestine (71.0 μg equiv / g), renal medulla (27.0 μg equiv / g), and bladder (22.8 μg equiv / g). The tissues with the lowest concentrations (<0.5 μg equiv / g) at the respective Tmax were: brain (cerebrum and bone marrow), spinal cord, white fat, prostate, bone, skeletal muscle, and lens of the eye.

[0995] The highest total concentrations determined by QWBA were in bladder contents (814.4 μg equiv / g at 4 h), bile contents (508.4 μg equiv / g at 2 h), and digestive tract contents (C max A range of 11.0 μg equiv / g in the stomach at 8 h to 423.0 μg equiv / g in the small intestine at 2 h was observed, reflecting the presence of 14 C] Elimination pathways of brincidofovir-derived radioactivity. Without wishing to be bound by theory, bile may be the source of the high concentrations of radioactivity in the intestinal contents with high concentrations of radioactivity in the early time points. High concentrations in the stomach would support this observation because the stomach is close to the entrance of bile into the digestive system, although gastric secretion is also possible based on the observation of radioactivity in the gastric mucosa at 168h.

[0996] Tissue concentrations in most tissues declined steadily, but clearance of drug-related material from most tissues was incomplete at 168 h post-dose. In tissues that contained radioactivity, concentrations were present at <1.0 μg equiv / g. At 840 h post-dose (last sampling time point), radioactivity was still present in the bone marrow, lymph nodes, spleen, and adrenal glands, but these tissues were close to the LLOQ (0.036 μg equiv / g) except for the spleen (0.270 μg equiv / g).

[0997] When SD rats (Group 3) were examined at sparse time points, no significant differences in tissue concentrations were observed when compared to LE rats given the same dose (Group 4). Comparison of tissue distribution of radioactivity between non-pigmented (SD rats Group 3) and pigmented tissues (LE rats Group 4) revealed that pigmented tissues (i.e., uveal tracts and pigmented skin) were similar to concentrations observed in non-pigmented tissues. This suggests that [ 14 C]Brincidofovir-derived radioactivity was not specifically associated with melanin.

[0998] Group 5 - QWBA tissue distribution in pigmented male rats following IV dosing at 2 mg / kg (sparse sampling)

[0999] Table 73 summarizes the tissue distribution of QWBA in male pigmented rats following a single 2-hour IV infusion at 2 mg / kg (n=1 animal at 2, 8, 24, and 72 h after initial dosing).

[1000] Table 73: Single 2-hour intravenous infusion at a target dose of 2 mg / kg [ 14 C] Radioactivity concentration in tissues of male Long-Evans (pigmented, group 5) rats after brincidofovir administration

[1001]

[1002]

[1003]

[1004] BQL = value below LLOQ

[1005] LLOQ = 0.00048400 μCi / g / 0.10151 μCi / μg = 0.005 μg equivalent / g tissue

[1006] ULOQ = 20.97300000 μCi / g / 0.10151 μCi / μg = 206.610 μg equivalent / g tissue

[1007] After 2 hours of IV infusion at 2 mg / kg, [ 14C]Brincidofovir-derived radioactivity was evenly distributed in most tissues of pigmented male rats, and concentrations in most tissues were slightly lower than those in blood / plasma at the end of infusion but higher than those in blood / plasma at later time points. Excreta and gastrointestinal tissues were significantly higher than blood / plasma at all time points. 14 The highest concentration of brincidofovir-derived radioactivity was 0.771 μg equiv / g, which was observed at 2 h (end of infusion and first sampling time point in the sparse sampling group), which was 7-fold lower than that observed after the 15 mg / kg dose. [C] was found in most tissues (35 of 38 tissues) at 2 h after dosing (first sampling time point). 14 C] brincidofovir-derived radioactive C max , at which point most tissue concentrations were between 0.2 and 0.7 μgequiv / g, 10-fold lower than that observed after dosing rats with 15 mg / kg. The mean tissue concentration ratios after IV dosing of 2 mg / kg (Group 5) compared to 15 mg / kg (Group 4) indicated that increases in tissue concentrations were approximately dose proportional in vascular lymph nodes, excretory organs / metabolism, central nervous, endocrine, secretory, adipose, dermal, reproductive, bone / muscle, and respiratory (lung) (Table 74). The mean tissue concentration ratios in the small intestine were generally greater, indicating that small intestinal concentrations increased in greater proportions as the dose increased from 2 to 15 mg / kg.

[1008]

[1009]

[1010]

[1011] In their respective T max The tissues with the highest concentrations >1.0 μg equiv / g at each T were: liver (34.2 μg equiv / g), renal cortex (11.0 μg equiv / g), renal medulla (3.4 μg equiv / g), small intestine (2.5 μg equiv / g), and large intestine (1.0 μg equiv / g). max The tissues with the lowest concentrations (<0.20 μg equiv / g) were: brain, spinal cord, white fat, prostate, skeletal muscle and lens of the eye.

[1012] The highest total concentration determined by QWBA was in the digestive tract contents (C maxA range of 0.051 μg equiv / g in the stomach at 8 h to 63.3 μg equiv / g in the small intestine at 2 h was observed in bile (32.7 μg equiv / g at 2 h), and bladder contents (22.9 μg equiv / g at 2 h), reflecting the presence of a high level of leukemia after a 2-hour IV infusion. 14 C] Elimination pathways of brincidofovir-derived radioactivity.

[1013] Tissue concentrations declined steadily in most tissues, but at 72 h post-dose (the last time point analyzed for this group), drug-related material was detected in all tissues, but concentrations in most tissues not associated with clearance organs were <0.10 μg equiv / g.

[1014] Groups 6 and 7 - Tissue distribution of QWBA in leukemic and pigmented male rats following PO administration at 15 mg / kg

[1015] Table 75 and Table 76 list the male albinism (Group 6; n=3) and pigmentation (Group 7; n=10) respectively. 14 C] Summary of tissue distribution of brincidofovir. The pharmacokinetic parameters are given in Table 77.

[1016] Table 75: Single oral administration at a target dose of 15 mg / kg 14 C] Radioactivity concentration in tissues of male Sprague Dawley (albino, group 6) rats after brincidofovir administration

[1017]

[1018]

[1019]

[1020] BQL = value below LLOQ LLOQ = 0.00048400 μCi / g / 0.0147 μCi / μg = 0.034 μg equivalent / g tissue

[1021] ULOQ = 20.97300000 μCi / g / 0.0147 μCi / μg = 1469.727 μg equivalent / g tissue

[1022] Table 76: Single oral administration at a target dose of 15 mg / kg 14 C] Radioactivity concentration in tissues of male Long-Evans (pigmented, group 7) rats after brincidofovir administration

[1023]

[1024]

[1025]

[1026] BQL = value below LLOQ LLOQ = 0.00048400 μCi / g / 0.01427 μCi / μg = 0.034 μg equivalent / g tissue

[1027] ULOQ = 20.97300000 μCi / g / 0.01427 μCi / μg = 1469.727 μg equivalent / g tissue

[1028] Table 77: Tissue Pharmacokinetic Parameters in Rats Following a Single Oral Administration of [14C]Brincidofovir at a Target Dose of 15 mg / kg

[1029]

[1030]

[1031] ND = not determined; the value could not be determined from the data.

[1032] a These tissues / organs 1 / 2 Values ​​are obtained from insufficient data points, and / or the resulting r 2 The values ​​fell outside the acceptance criteria (≥0.85), therefore, these values ​​should be considered as estimates.

[1033] Group 6 - Tissue distribution of QWBA in male albino rats after drug administration (sparse sampling)

[1034] After a single oral dose of 15 mg / kg, [ 14 C]Brincidofovir-derived radioactivity was uniformly distributed in albino male rats (n=1 animal at 2, 24, and 168 h post-dose), and concentrations in most tissues were slightly lower than those in blood / plasma at early time points (2 h post-dose), but higher than those in blood / plasma at later time points. Fecal and gastrointestinal tissue concentrations were significantly higher than blood / plasma at all time points. 14 The highest concentration of brincidofovir-derived radioactivity was 0.200 μg equiv / g, which was observed at 2 h (first sampling time point in the sparse sampling group). [C] was found in most tissues (31 of 37 tissues) at 24 h post-dose (second sampling time point). 14 C] brincidofovir-derived radioactive C max , at which point the concentration in most tissues is between 0.10 and 0.50 μgequiv / g.

[1035] In their respective T maxThe tissues with the highest concentrations (>7.0 μg equiv / g) were: small intestine (236.3 μg equiv / g), liver (20.6 μg equiv / g), renal cortex (18.4 μg equiv / g), bladder (17.3 μg equiv / g), stomach (11.2 μg equiv / g), renal medulla (7.9 μg equiv / g), and cecum (7.5 μg equiv / g). max Tissues with the lowest concentrations (<0.10 μg equiv / g) were: brain, spinal cord, white fat, skeletal muscle and lens of the eye.

[1036] The highest total concentration determined by QWBA was in the digestive tract contents (C max A range from 28.6 μg equiv / g in the cecum at 24 h to 877.5 μg equiv / g in the stomach at 2 h was observed, in bladder contents (39.9 μg equiv / g at 24 h), and in bile (9.7 μg equiv / g at 2 h), reflecting the increase in serum creatinine levels following a single oral dose of 15 mg / kg. 14 C] Dosing routes and / or elimination routes of brincidofovir-derived radioactivity.

[1037] At 168 h after dosing, clearance of drug-related substances was incomplete, with detectable concentrations in approximately half of the tissues (<0.60 μg equiv / g). In the small intestine, higher residual concentrations (2.7 μg equiv / g) were observed at 168 h relative to other tissues.

[1038] Group 7 - Tissue distribution of QWBA in pigmented male rats after PO administration

[1039] After a single oral dose of 15 mg / kg, [ 14 C]Brincidofovir-derived radioactivity was evenly distributed in most tissues of pigmented male rats (n=1 animal at 0.5, 1, 2, 4, 8, 24, 72, 96, 168, and 840 h post-dose), with concentrations in most tissues slightly lower than those in blood / plasma at time points up to 24 h post-dose, but higher than those in blood / plasma at later time points. Excreta and digestive tract tissue concentrations were significantly higher than blood / plasma at all time points. Based on AUC (AUC tissue :AUC plasma ; Table 78) showed that the tissue: plasma (T / P) ratios of most tissues were >1, indicating that tissue concentrations were higher than plasma. 14The highest concentration of brincidofovir-derived radioactivity was 1.1 μg equiv / g, which was observed at 8 h. [C] was found in most tissues (20 of 38 tissues) at 8 h after dosing. 14 C] brincidofovir-derived radioactive C max , at which point the concentration in most tissues is between 0.20 and 0.40 μgequiv / g.

[1040]

[1041]

[1042]

[1043] Figure 4 and Figure 5 The concentrations of brincidofovir in different tissues are shown as a function of time and route of administration. Figure 4 The concentrations of brincidofovir in the small intestine were compared as a function of time for oral and IV dosing. Figure 5 The concentrations of brincidofovir in the renal cortex were compared as a function of time for oral and IV dosing.

[1044] In their respective T max The tissues with the highest concentrations (>2.0 μg equiv / g) were: small intestine (458.5 μg equiv / g), liver (36.1 μg equiv / g), renal cortex (17.8 μg equiv / g), stomach (12.0 μg equiv / g), renal medulla (9.1 μg equiv / g), cecum (7.6 μg equiv / g), urinary bladder (3.7 μg equiv / g), and large intestine (3.3 μg equiv / g). max Tissues with the lowest concentrations (<0.10 μg equiv / g) were: brain, spinal cord, white fat, skeletal muscle and lens of the eye.

[1045] The highest total concentration determined by QWBA was in the digestive tract contents (C max A range of 187.6 μg equiv / g in the small intestine at 4 h to 1004.6 μg equiv / g in the stomach at 1 h was observed, in bladder contents (110.9 μg equiv / g at 8 h), and in bile (36.3 μg equiv / g at 4 h), reflecting the high concentrations of 1 μg equiv / g in the bile after a single oral dose. 14 C] Dosing routes and / or elimination routes of brincidofovir-derived radioactivity.

[1046] Tissue concentrations in most tissues decreased steadily, but at 168 h post-dose, drug-related substances were detected in about half of the tissues, with most concentrations <0.40 μg equiv / g, except for the small intestine (5.9 μg equiv / g). At 840 h post-dose (the last sampling time point), radioactivity was still present in the small intestine, renal cortex, and liver, but concentrations in all tissues were close to the LLOQ (0.034 μg equiv / g).

[1047] Male SD rats were given 15 mg / kg IV infusion over 2 hours. 14 C] The major route of radioactivity elimination after brincidofovir was urine, which accounted for 51.2% (mean) of the administered dose over the 168-hour collection period. 42.2% (mean) of the administered dose was obtained in the feces. Most (~86%) of the radioactivity in urine and feces occurred in the first 24 hours after dosing.

[1048] After a 2 h IV infusion, increases in radioactivity were dose-proportional in most tissues (except the small intestine) at each time point. In small intestinal tissues, increases were greater than dose-proportional. Following oral administration, radioactivity was readily absorbed. Radioactivity was well distributed, with peak radioactivity concentrations occurring in most tissues (34 of 38) 4-8 h after oral administration and at the end of IV infusion (2 h). Qualitative distribution patterns of radioactivity were similar following IV or oral administration; quantitatively, tissue radioactivity exposures adjusted for radioactivity bioavailability (approximately 50%) were generally greater following IV administration than after oral gavage. Tissues with the highest radioactivity concentrations were associated with organs of excretion (i.e., liver, kidney, and intestine). Tissue-to-plasma ratios were high (>30) in each tissue and were similar between IV and oral routes of administration in the renal cortex and liver. As expected based on the route of administration, the small intestinal tissue-to-plasma ratio was highest following oral administration (T / P = 428). However, following IV infusion, significant radioactivity was associated with the small intestine (tissue-to-plasma ratio of 32). High concentrations (C max ) is related to bile in the bile duct, which is in IV (C max 508μg equiv / g) after oral administration (C max =36 μg equiv / g) was 14-fold higher after IV administration, suggesting a possible source of high intestinal tissue concentrations after IV administration. The tissues with the lowest radioactivity were brain, spinal cord, skeletal muscle, white adipose tissue, and bone. max ~20% of the plasma C max There was a higher association of radioactivity with brain and spinal cord after 4-6 weeks of dosing (-5% of the concentration). No evidence of a specific association with melanin tissue (eye, uvea) was detected.

[1049] Four different approaches, including single tissue and whole-body exposure estimates, were used to estimate predicted human exposure following oral administration of 100 μCi, all of which resulted in human exposures that were 4% or less than the limits permitted by the FDA and ICRP.

[1050] No deviations from the SOP and / or protocol set were observed during the study that could adversely affect the quality or integrity of this study. Single ascending-dose study of IV BCV in healthy subjects

[1051] Brincidofovir was administered to healthy subjects at 10 mg and 25 mg. It was found that 10 mg IV brincidofovir provided similar exposure to 100 mg oral administration of brincidofovir. The results showed that IV brincidofovir doses (10 mg and 25 mg) were generally safe and well tolerated. No drug-related adverse events were observed, no gastrointestinal adverse events, no graded laboratory abnormalities (e.g., no hematologic toxicity and no renal toxicity).

[1052] Single doses of oral BCV at 100 mg and 200 mg resulted in approximately 5% of cases of diarrhea, and the 350 mg dose resulted in approximately 20% of cases of diarrhea.

[1053] Figure 6 Plasma brincidofovir concentrations (AUC inf (ng*h / mL)). Figure 6 As shown, IV administration of 10 mg of brincidofovir provided essentially the same plasma concentrations as oral administration of 100 mg of brincidofovir, while IV administration of 25 mg of IV brincidofovir provided higher plasma concentrations than both IV 10 mg administration and oral 100 mg administration.

[1054] Example 10 - Brincidofovir: 28-day intravenous infusion toxicity study in rats with a 14-day recovery period

[1055] Summarize

[1056] Sprague-Dawley via 2-hour intravenous infusion Rats (15 / sex / group) were administered 0 (2X sodium phosphate buffer), 1, 4 or 15 mg / kg / dose brincidofovir twice a week for a total of 9 times over 29 days. For all dosing groups, the dosing rate was 10 mL / kg / hr. At the end of the treatment period, up to 10 animals / sex / group were euthanized and autopsied. The remaining animals (up to 5 animals / sex / group) were retained for a 14-day recovery phase to determine the progression or reversibility of any effects associated with brincidofovir. Satellite animals (up to 3 / sex / group) were similarly dosed, and serial blood samples were collected from each animal on days 1 and 29 for toxicokinetic analysis of brincidofovir and brincidofovir metabolites (cidofovir, CDV). The parameters evaluated during the study were: survival, clinical observations, ophthalmology, body weight, food consumption, clinical pathology (termination of dosing and end of recovery), organ weights, macroscopic observations, and microscopic pathology.

[1057] Brincidofovir exposure increased with increasing dose on days 1 and 29, with AUC greater than last In general, without wishing to be bound by theory, there was no accumulation of brincidofovir over the course of the 29-day study. The male-to-female ratio of brincidofovir indicated no sex differences in exposure (based on C max and AUC last ).

[1058] After intravenous infusion of brincidofovir on days 1 and 29, CDV C increased with increasing doses of brincidofovir (from 1 to 15 mg / kg). max and AUC last Overall, there was little to no accumulation of CDV throughout the study, and the male to female ratio generally indicated no sex differences in CDV exposure.

[1059] There were no brincidofovir-related mortalities. There were 3 unplanned deaths in this study, 2 deaths in toxic animals that were determined to be unrelated to brincidofovir, and 1 death in a TK animal whose cause could not be determined.

[1060] There were no brincidofovir-related effects in ophthalmology, body weight, hematology, coagulation, clinical chemistry, or urinalysis.

[1061] Administration of brincidofovir resulted in intermittent (usually post-infusion) clinical signs (rapid breathing and prone posture with and without piloerection, partially closed eyes, decreased activity irregular breathing, and / or flat posture) in some but not all animals of both sexes at ≥4 mg / kg / dose, and minimally decreased food consumption at ≥1 mg / kg / dose in males and 15 mg / kg / dose in females.

[1062] At the end of dosing, in the male reproductive tract (testis, epididymis and seminal vesicle) and intestinal tract (duodenum, jejunum and / or ileum) at 4≥mg / kg / dose, and in male and female skin (sebaceous glands) at 15mg / kg / dose, there were findings related to brincidofovir. In the male reproductive tract, the changes in the testicles were characterized by testicular weight loss, and a slight to significant reduction in germ cells at ≥4mg / kg, with minimal spermatocyte retention and Sertoli cell vacuolization at 15mg / kg / dose. In addition, males had minimal epididymal luminal cell fragments and minimally increased epithelial cell apoptosis in the seminal vesicle at 15mg / kg / dose. In the intestinal tract, in all sexes, single cell necrosis was observed in the small intestine (crypts) at ≥4mg / kg / dose, and in the large intestine (glands) at 15mg / kg / dose. At 15mg / kg, there was atrophy of the sebaceous glands in the skin in males and females.

[1063] Table 79: Comparison of toxicity of oral and intravenous brincidofovir

[1064]

[1065]

[1066] After a 14-day recovery period, all findings fully recovered in males at ≥4 mg / kg / dose, except for decreased testicular weight and germ cell depletion; however, without wishing to be bound by theory, it is expected that germ cell depletion will not fully recover within 2 weeks. In addition, some animals had reduced bone marrow cellularity and soft and / or small testes and epididymis (males) at 15 mg / kg / dose (3 / 5 males; 2 / 5 females). Without wishing to be bound by theory, germ cell depletion in the testes and reduced sperm in the caput epididymis region reflect the observed depletion of maturation due to loss of germ cell populations at the end of the dosing period.

[1067] Under the conditions of this study, the no-observable-adverse-effect-level (NOAEL) for brincidofovir was considered to be 1 mg / kg / dose in males and 4 mg / kg / dose in females based on testicular germ cell depletion in males at ≥4 mg / kg / dose and moderate intestinal wall single-cell crypt necrosis in 1 female at 15 mg / kg / dose.

[1068] introduction

[1069] The following abbreviations are used in this study:

[1070] M Male

[1071] F Female

[1072] ~ Approx.

[1073] mg / kg Milligrams of the test item per kilogram of body weight

[1074] mL / kg mL of dosage formulation per kilogram of body weight

[1075] mL / kg / hr mL of dosage formulation per kilogram of body weight per hour

[1076] mg / mL Milligrams of the test item per milliliter of dosage form

[1077] IV intravenous MTD maximum tolerated dose

[1078] DRF Dose Range Finding

[1079] The objectives of this study were to evaluate the toxicity and toxicokinetics of brincidofovir when administered by intravenous (2-hour) infusion twice weekly to rats for a total of 9 doses over the course of 29 days and to assess the progression or reversibility of brincidofovir effects during a 14-day drug-free recovery period.

[1080] The study design incorporated elements of general regulatory guidelines for toxicity studies.

[1081] This study was conducted in accordance with the following good laboratory practices: Good Laboratory Practice of the U.S. Food and Drug Administration (21 CFR Part 58); and the OECD Principles of Good Laboratory Practice (revised 1997), ENV / MC / CHEM (98)17

[1082] These principles are consistent with good laboratory practice regulations set by the European Community, the United States (EPA and FDA) and Japan (MHLW, MAFF and METI) and other countries that have signed the OECD mutual acceptance of data agreement.

[1083] This study complied with all appropriate sections of the animal welfare regulations: 9 CFR Parts 1 and 2 Final Rule, Federal Register, Volume 54, Number 168, August 31, 1989, Pages 36112-36163, effective October 30, 1989 and 9 CFR Part 3 Animal Welfare Standards; Final Rule, Federal Register, Volume 56, Number 32, February 15, 1991, Pages 6426-6505, effective March 18, 1991.

[1084] Regulatory agencies (e.g., FDA) recommend that preclinical studies be performed in two or more species (one rodent, one non-rodent) to support human dosing. The rat is a commonly used animal model in toxicity studies. In addition, historical databases are available for comparative evaluation.

[1085] The test items were administered intravenously up to 2 times per week.

[1086] The number of animals in this study was considered the minimum necessary for statistical, regulatory, and scientific reasons. The purpose of this study was to monitor toxicity and the progression or reversibility of the effects of the test item. Historical control data indicated that clinical laboratory data, organ weight data, and microscopic examinations of tissues varied among individual animals. The number of rats used to conduct the subchronic study (10 animals / sex / group) was recommended by regulatory guidance documents and was considered the minimum number to account for the expected variability between animals. To determine the progression or reversibility of potential effects of the test item, additional post-treatment (recovery) animals were included in the study (5 animals / sex / group). Three test item treatment groups receiving low, medium, and high multiples of the proposed human dose and one negative control group were considered the minimum number of groups necessary to provide a range of effects and allow extrapolation of the results to humans.

[1087] The number of animals selected for toxicokinetic evaluation (3 / sex / treatment group) was considered the minimum number necessary to provide meaningful data, taking into account the inherent variability of the absorption, distribution, metabolism, and excretion processes. A control group with 3 animals / sex was required to evaluate the absence of a test item.

[1088] In the maximum tolerated dose (MTD) phase, the doses of this study (0, 1, 4 and 15 mg / kg / dose) were 2, 4, 10 or 15 mg / kg / dose over the course of 2 hours, then, in the dose range finding (DRF) phase, a second group of animals were given 3 doses of 1 or 15 mg / kg / dose over the course of 2 hours over the course of 9 days. The high dose (15 mg / kg) administered in this 2-phase study was determined to be the maximum feasible dose (MFD) based on the hemolysis observed at the higher concentrations.

[1089] Because the infusion catheter was returned or unavailable on the scheduled date of dosing and there was insufficient time to reposition or repair the line, the following animals were dosed via tail vein injection on Day 29; Animals 1511 and 1519 were found to have returned catheters on Day 28, and Animal 4581 was found to have an ulcer near the exteriorization site on Day 29. During this procedure, these animals were restrained in individual plastic cones for no more than 2.25 hours (15 minutes for dosing site preparation and 2 hours for dosing).

[1090] Table 80: Experimental Outline

[1091]

[1092] The first day of dosing was defined as study day 1. Brincidofovir was provided as a white powder and stored at room temperature. The purity was determined to be 99.8%.

[1093] The diluent was 5% Dextrose Solution for Injection, USP, and was supplied as a clear liquid (96.8% purity). It was stored at room temperature.

[1094] 2X sodium phosphate buffer was used as a control. It was prepared from sodium dihydrogen phosphate (anhydrous), supplied as a white crystalline powder and stored at room temperature (100% purity). Phosphate buffer (control) was dissolved in sterile water and the pH was adjusted with sodium hydroxide.

[1095] Preparation of dosage forms

[1096] Prepare 2X sodium phosphate buffer (400 mM, pH 8.0 ± 0.04) vehicle solution by mixing appropriate amounts of sodium dihydrogen phosphate solution (400 mM) and sodium monohydrogen phosphate solution (400 mM). Adjust the pH of the solution with sodium dihydrogen phosphate solution (400 mM) when necessary. Filter the solution through a 0.22 μm -GP(PES) filter into a sterile container.

[1097] The vehicle (control item) was stored refrigerated at 2-8°C. Fresh vehicle solutions were prepared twice; once before dose 1 and again before dose 4, and each formulation was used within one month of preparation.

[1098] A 15 mg / mL brincidofovir stock was prepared by mixing appropriate amounts of brincidofovir with 2X sodium phosphate buffer, sterile water for injection, USP, and 1N sodium hydroxide (NaOH). The solution was filtered through a 0.22 μm -GP filter into a sterile container. The brincidofovir stock was prepared once before dose 1 and again before dose 4 and refrigerated at 2-8°C.

[1099] In a sterile bottle under a laminar flow hood, dilute the appropriate amount of brincidofovir stock solution (15 mg / mL) or the appropriate amount of stock vehicle (2X sodium phosphate buffer solution) with the appropriate amount of 5% glucose injection, USP, and invert 10 times to mix it to prepare the dosage formulation. Prepare fresh dosing formulations weekly and store refrigerated at 2-8°C when not in use.

[1100] Table 81: Administration

[1101]

[1102] Detailed records of brincidofovir use were maintained. The amount of brincidofovir required to prepare the formulation and the amount actually used were determined at each preparation.

[1103] stability

[1104] Stability analysis showed that the brincidofovir formulations at these concentrations used in this study (0.05 mg / mL and 2.0 mg / mL) were stable for 24 hours when stored at room temperature and for 8 days when refrigerated at 2 to 8°C. When stored at room temperature (labeled 20°C), the 15.0 mg / mL brincidofovir stock solution was stable for 24 hours and for 8, 14, and 28 days when stored at 2 to 8°C.

[1105] Confirmation analysis

[1106] Two samples (5 mL each) were taken from the mid-area of ​​each dosing formulation (including controls) prepared each week on the day of preparation. One sample was analyzed in duplicate and one sample was stored refrigerated (2 to 8°C) for dose confirmation analysis (the Group 2 retained samples were reanalyzed at 1 and 2 weeks because the initial results were outside the study acceptance plan criteria (±10% of nominal).

[1107] Storage conditions

[1108] All dose formulation samples were stored refrigerated (2 to 8°C) until analysis or final dispatch.

[1109] Brincidofovir administration

[1110] Brincidofovir was administered by intravenous infusion over 2 hours. Animals were treated at a constant dose of 20 mL / kg / 2 hours.

[1111] Infusion catheter implantation procedure

[1112] Infusion catheters were implanted approximately 1-3 weeks prior to dosing. All animals, including spare animals, were surgically catheterized.

[1113] The surgical site was prepared according to the test facility's SOP for sterile, restorative surgery. Animals received pre-analgesia (flunixin meglumine (USP) 2 mg / kg, subcutaneously).

[1114] All animals were anesthetized (isoflurane) and shaved in the groin and back. A small incision was made and the femoral vein was isolated. A small incision was made in the vessel and a sterile catheter was placed in the vessel. The catheter was inserted through the femoral vein and retrograde into the inferior vena cava caudal to the kidney. The catheter was secured with sutures and then passed under the skin to drain in the back of the neck. A small pocket was made in the external area, in which a loop of catheter was left. The inguinal area was closed with sutures.

[1115] The animals were placed in a cuff and the implanted catheters were secured to pins using capped septum connectors. The catheters were blocked with taloprolol citrate blocking solution. Each animal received an intramuscular injection of 5.0 mg / kg of enrofloxacin on the day of surgery.

[1116] When necessary, surgical repairs were performed to restore catheter patency during the pre-experimental period and at the discretion of the experiment director (limit 1 repair per animal). Analgesics were pre-administered (flunixin meglumine (USP) 2 mg / kg, subcutaneously). Animals undergoing surgical repair received appropriate doses of antibiotics on the day of surgery (according to the standard operating procedures of the testing facility).

[1117] The catheter was tied off (the knot was placed in the catheter and the catheter was withdrawn under the skin) and the pocket removed only after the pre-recovery dose was completed.

[1118] Infusion Catheter Maintenance Procedure

[1119] Prior to using the animals for the study, the patency of the implanted femoral vein catheter was assessed according to the testing facility's SOP.

[1120] Volume dose (rate)

[1121] 20 mL / kg / 2 hours (10 mL / kg / hour).

[1122] Individual dose concentration

[1123] Individual animal concentrations were calculated from the most recently recorded scheduled body weight.

[1124] Frequency and duration

[1125] The test / control items were administered by 2-hour intravenous infusion twice a week for a total of 9 individual doses for 29 days. Test / control item administration continued until the day before terminal necropsy. After the last dose, the recovery animals were retained for an observation period of 14 days.

[1126] Administration site

[1127] Through a surgically implanted cannula in the femoral vein / vena cava or through the tail vein in the distal area.

[1128] Dosage Procedure

[1129] One to two days before dosing, the lock solution was removed from each animal's implanted catheter (if possible) and the catheter was flushed with saline before being connected to the tether and infusion dosing device. After connection to the animal's catheter, sterile saline (0.9% NaCl, USP) was injected into the animal at a rate of 0.5 mL / hr via a calibrated Medfusion syringe pump until dosing on Day 1.

[1130] A saline flush was injected into the infusion line to deliver brincidofovir and to ensure delivery of the full dose (at the same rate as brincidofovir, with an additional ~0.5 to 1 mL volume to flush the catheter line).

[1131] Animals were maintained on saline during the dosing period. Saline syringes were changed at least every 4 days.

[1132] Animals with inoperable / non-functioning catheters were surgically repaired when possible. If repair was not possible, these animals were dosed via the tail vein in the distal region in a restrainer at the discretion of the experiment director. These animals were placed in individual plastic cones for no longer than 2.25 hours (15 minutes for dosing site preparation and 2 hours for dosing via tail vein injection).

[1133] On day 29, animals Nos. 1511, 1519 and 4581 were administered dose 9 via the tail vein in the distal region.

[1134] Dosing accuracy

[1135] The quantification of daily infusions (confirming actual dose administration) was based on the pre- and post-dose weights of each syringe and checked for daily adjustments; data were saved in the study files. The pump accuracy was checked with saline before dosing began using the same dosing rate length as this study, and post-dose quantification was used to monitor pump performance throughout the study.

[1136] animal

[1137] Species / Variety

[1138] Albino rats (outbreeding) (Sprague-Dawley derived) [Crl: (SD)BR]

[1139] Number of animals

[1140] A total of 170 were received (85 males and 85 females); 144 were placed in testing (72 males and 72 females).

[1141] During the pre-test

[1142] The pre-test period is approximately 4 weeks. All animals are examined during the pre-test period to confirm their suitability for the study. No pre-test procedures are performed until the animals have been allowed to stabilize for 5 days.

[1143] Age of the primary study animals at the start of treatment

[1144] About 10 weeks

[1145] Weight range of primary study animals at the start of treatment

[1146] Male: 290.6g to 422.4g. Female: 203.8g to 294.7g

[1147] distribute

[1148] More animals than were required for the study were purchased and stabilized. Animals deemed unsuitable for the study were excluded based on pretest physical examination and catheter patency prior to random selection for group assignment. Animals deemed suitable for the study were divided into 4 groups of 18 per sex by a computer random classification program so that the mean body weights of each group were comparable. Some toxic animals were exchanged with satellite animals due to ophthalmic findings prior to testing, but remained in the same group. Individual weights of animals placed in testing were within ±20% of the mean body weight of each sex.

[1149] Animal identification

[1150] A BMDSIMI-1000 implantable radiofrequency transponder (microchip) programmed with a unique number is implanted in each rat. This number is cross-referenced with the animal number assigned by the testing facility; this number plus the study number comprises a unique identification number for each animal. In addition, each cage is equipped with a color-coded cage card for dose level identification and contains the study number and facility-assigned animal number information.

[1151] Veterinary care

[1152] Animals were monitored by technicians for any conditions requiring possible veterinary care and treated as needed. The study director reviewed all non-test project related veterinary evaluations for individual animals and documented them in the study files.

[1153] Serial observations on animals from the satellite group were limited to clinical observations, body weight, ophthalmology, and food consumption; these data are not reported.

[1154] Blood samples were obtained for determination of plasma concentrations of brincidofovir and the brincidofovir metabolite cidofovir.

[1155] On Day 1 and Day 29, blood samples were obtained for toxicokinetic assays at the time points indicated in the table below:

[1156] Table 82: Collection time and number of animals

[1157]

[1158] Collection Procedure

[1159] Obtain about 0.4mL of whole blood from each animal through the tail vein at each time point. Animals were not fasted before blood collection. Blood was collected in a tube containing K2EDTA anticoagulant and placed on wet ice in an upright state. By centrifugation of plasma (10 minutes, at about 2000g, at about 2-8°C). About 0.150mL of plasma was transferred to a freezing tube and appropriately labeled with the number of studies, number of animals, time point, sampling date and sample type. Remaining plasma was transferred to a second freezing tube and retained as a standby sample. Obtain plasma and freeze at about 80 ± 10°C until analysis within about 2 hours after each blood sample collection. Animals were euthanized (CO2 inhalation) after the last blood collection.

[1160] Storage and arrangement of samples

[1161] All sample tubes were stored frozen (-80 ± 10 °C) and shipped (frozen, on dry ice) to Pyxant Laboratories, Colorado Springs, CO, USA for analysis.

[1162] Analysis / Reporting

[1163] Bioanalytical samples were analyzed at Pyxant Laboratories using a calibrated liquid chromatography-triple quadruple mass spectrometry (LC-MS / MS) assay.

[1164] When plasma concentration data are received, non-compartmental toxicokinetic analyses are performed by Nuventra, Inc. Durham, North Carolina.

[1165] Daily observation

[1166] Animals were observed twice daily in cages for mortality and signs of severe toxicity or pharmacological effects. Animals in extremely poor health or in danger of death were identified for further monitoring and possible euthanasia.

[1167] Drug administration observation

[1168] Any symptoms of sub-health or toxicity or pharmacological effects (e.g., abnormalities in general condition, appearance, activity, behavior, breathing, etc.) were observed immediately and for 2 hours after the completion of the drug administration.

[1169] symptom

[1170] The animals were removed from their cages and examined once pretest and weekly during the study. The examination included observation of general condition, skin and coat, eyes, nose, mouth, abdomen, and external genitalia, as well as assessment of respiration.

[1171] Ophthalmoscopy

[1172] All animals were pretested. All surviving toxicity animals were examined at the end of dosing and at the end of recovery. Only animals within normal ranges in the pretest examination were placed in the experiment.

[1173] Visually inspect the eyelids, tear glands, and conjunctiva. Indirect ophthalmoscopy is used to inspect the cornea, anterior chamber, lens, iris, vitreous humor, retina, and optic disc.

[1174] The pupil of each animal was dilated with tropicamide eye drops before examination.

[1175] weight

[1176] Animals were removed from their cages and weighed twice before pre-testing, the day before each dosing during treatment, and weekly during recovery. Terminal, fasting body weights were obtained prior to scheduled necropsy intervals. Animals euthanized for welfare reasons were weighed prior to necropsy.

[1177] Food consumption

[1178] Food consumption was measured weekly starting one week before treatment.

[1179] Clinical Pathology

[1180] Hematology, coagulation, and clinical chemistry parameters were analyzed using blood obtained via the orbital sinus (posterior bulbar venous plexus) under light isoflurane anesthesia for up to 10 toxicity animals / sex / group at the end of dosing and up to 5 recovery animals / sex / group at the end of recovery. Animals were fasted overnight prior to each blood collection interval.

[1181] Urine was obtained and analyzed from up to 10 toxicity animals / sex / group at the end of dosing and up to 5 recovery animals / sex / group at the end of recovery over a 16-hour overnight collection period. Animals were fasted but not deprived of water during the collection period.

[1182] hematology

[1183] Blood samples (approximately 0.25 mL) were collected into tubes containing K2EDTA anticoagulant and analyzed using a Siemens ADVIA120 hematology analyzer for the following: hemoglobin concentration (HGB); hematocrit (HCT); red blood cell count (RBC); platelet count (PLT); mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); mean corpuscular hemoglobin concentration (MCHC); red blood cell distribution width (RDW); total white blood cell count (WBC); reticulocyte count (RETIC); differentiated white blood cell count (manual differentiated white blood cell count was performed for verification and absolute values ​​were calculated when necessary); neutrophils (ANEU); lymphocytes (ALYM); eosinophils (AEOS); basophils (ABASO); monocytes (AMONO); large unstained cells (ALUC).

[1184] Blood smears from the peripheral area were prepared for each animal at each blood collection interval for confirmation of automated results and / or other evaluations deemed necessary.

[1185] Condensation

[1186] Blood samples (approximately 1.0 mL) were collected into tubes containing sodium citrate anticoagulant and analyzed using the Diagnostica Stago Products STA Compact. ) Mechanical coagulation detection system performs the following analyses: prothrombin time (PT): activated partial thromboplastin time (APTT).

[1187] Clinical Chemistry

[1188] Blood samples (approximately 1.0 mL) were collected into tubes without anticoagulant, allowed to clot, centrifuged to obtain serum, and analyzed using a Siemens ADVIA 1800 chemistry analyzer as follows:

[1189] Aspartate aminotransferase (AST) (kinetics-modified Bergmeyer); alanine aminotransferase (ALT) (kinetics-modified Bergmeyer); alkaline phosphatase (ALKP) (kinetics-Tietz AMP buffer); blood urea nitrogen (BUN) (enzyme-Roch-Ramek and urease); creatinine (CREAT) (Jaffe Picric acid in alkaline medium); glucose (GLU) (glucose hexokinase II method); cholesterol (CHOL) (enzyme esterase / oxidase-Trand endpoint); triglycerides (TRIG) (Fossati three-step method enzyme-Trinder endpoint); total protein (TP) (biuret technique); albumin (ALB) (bromocresol green method); total bilirubin (TBILI) (vanadate oxidation); sodium (NA + )(ion selective electrode); potassium (K + ) (ion selective electrode); chloride (Cl - ) (ion selective electrode); calcium (Ca ++ )(Michaylova & Ilkova, arsenazo III); inorganic phosphorus (PHOS) (phosphomolybdate-UV method)

[1190] Urinalysis

[1191] Urine was collected from toxicity study animals placed in metabolic cages into frozen containers overnight (approximately 16 hours).

[1192] Urine samples were analyzed using multi-use reagent strips and interpreted using a Siemens Clinitek Advantus for: pH; protein (PRO); glucose (GLU); ketones (KET); bilirubin (BIL); and occult blood (BLD).

[1193] The samples were also analyzed manually for the following:

[1194] Appearance (APP); specific gravity (Sp.G.) (clinical refractometer, Atago Urea-N); volume (VOL).

[1195] Autopsy

[1196] Methods of euthanasia

[1197] Animals sacrificed for welfare reasons or at predetermined sacrifice intervals were euthanized by exsanguination after isoflurane inhalation. Toxicokinetic animals were euthanized by CO2 inhalation.

[1198] Autopsy scheduled

[1199] After the animals were treated for 29 days, up to 10 toxicity study animals / sex / group were necropsied, and after a 14-day treatment-free recovery period, up to 5 toxicity study animals / sex / group were necropsied. Animals were fasted overnight before necropsy. A necropsy schedule was established to ensure that animals of both sexes from each group were examined at similar times of day throughout the necropsy period.

[1200] After the last blood collection on day 29, toxicokinetics animals were euthanized and discarded without examination.

[1201] Macroscopic inspection

[1202] All animals were subjected to a complete macroscopic examination. The macroscopic examination included inspection of the external surface and all foramina; the external surface of the brain and spinal cord; the organs and tissues of the skull, thorax, abdomen, pelvis, and neck; and the remainder of the carcass for any gross morphological abnormalities.

[1203] Animal TK 4107, which was sacrificed for welfare reasons, was examined macroscopically and the tissue was preserved but not examined microscopically.

[1204] Organ weight

[1205] The following organs were weighed at predetermined sacrifice intervals for all surviving toxicity and recovery study animals. Prior to weighing, the organs were carefully dissected and appropriately trimmed to remove fat and other adjacent tissues in a uniform manner. Organs were weighed as soon as possible after dissection to avoid drying. Paired organs were weighed together.

[1206] Tissue preservation and pathological examination

[1207] The following tissues were obtained and stored from all animals. In addition, sections of the indicated tissues were prepared and examined microscopically. Any abnormalities observed during histological processing that were not noted during macroscopic examination were recorded.

[1208] Table 83: Tissues weighed, stored, and examined under a microscope

[1209]

[1210]

[1211]

[1212] a Qualitative check (no difference counts).

[1213] b The rectum was not examined microscopically.

[1214] cMale breast and GALT were evaluated only if present on routine sections.

[1215] d Weigh after fixation.

[1216] e The prostate gland and seminal vesicles were weighed together.

[1217] preservative

[1218] All tissues were preserved in 10% neutral buffered formalin (NBF). Eyes and testes were first placed in modified Davidson's solution and then kept in 10% NBF. Lungs were perfused with 10% NBF before being immersed in a larger volume of the same fixative. Smear preparations of bone marrow from femurs were air-dried and fixed in anhydrous methanol.

[1219] deal with

[1220] After fixation, tissues and organs from all animals were routinely processed, embedded in paraffin, sectioned on a microtome set at 4-7 microns, mounted on glass slides, stained with hematoxylin and eosin, and examined by light microscopy. TM Decalcifies bones.

[1221] The data collection system used to collect survival and postmortem data divided the study into several phases. The start of each phase was day 1 / week 1. The phases presented in this report are:

[1222] Pre-treatment: Begins on the day the animal arrives. Phase days / weeks are labeled P1, P2, P3, etc. During this phase, animals are not given medication and they are not assigned their permanent identification number.

[1223] Randomization: Begins on the day the animals are divided into groups and assigned their permanent identification numbers. Phase days / weeks are labeled Ra1, Ra2, Ra3, etc. Animals are not dosed during this phase.

[1224] Treatment: Start when each animal receives its first dose of the test / control item specified in the study plan.

[1225] Recovery: Begins on the day after each assigned animal stops receiving its assigned dose and continues until study completion. Period days / weeks are labeled R1, R2, R3, etc.

[1226] The death codes used have the following meanings:

[1227] “FD”: found dead; “S1”: scheduled euthanasia 1; “WE”: euthanasia for welfare reasons; “Z1”: recovery euthanasia 1.

[1228] Clinical observation

[1229] A clinical observation was performed on each animal showing symptoms, providing details of the type of symptom, day or week of onset and, where applicable, duration of symptoms. "AM": morning observation; "PM": afternoon observation; "Unsched": unscheduled observation; "Post Dose": immediately after completion of the dose; "2Hr PD": 2 hours after completion of the dose.

[1230] Eye examination

[1231] Observations were performed on each animal that exhibited a finding. Observations were bilateral unless otherwise stated.

[1232] Food consumption

[1233] Food consumption (g / day) was calculated as follows:

[1234] Grams of food consumed ÷ # days

[1235] Food consumption (g / animal / day) was calculated as follows:

[1236] Grams of food consumed ÷ Number of animal days a

[1237] a The total number of animal days that each animal in the cage survives for a full day during each consumption period. If an animal dies, the day of death is not counted as an animal day. For example: 4 animals in a cage for 5 days equals 20 animal days; if an animal dies on the 3rd day, then the animal day equals 1.

[1238] hematology

[1239] The following abbreviations were used: “ACMO”: automatic count invalidated by manual observation; “CS”: coagulation specimen; “NSAD”: no specimen animal died.

[1240] The following abbreviations were used to describe sample annotations (SCM1): "CLSE": severe platelet aggregation was noted; "CLSL": mild platelet aggregation was noted; "NCLP": no aggregation.

[1241] Condensation

[1242] The following abbreviations were used: “CS”: coagulation specimen; “NS”: no specimen; “NSAD”: no specimen animal died; “NVIR”: invalid due to irreproducible results.

[1243] The following abbreviations were used to describe the sample annotation (SCM1): "SLH": mild hemolysis.

[1244] Blood Chemistry

[1245] BUN values ​​below the analytical limit (<10) were not included in the calculation. TBILI values ​​below the analytical limit (<0.2) were not included in the calculation.

[1246] The following abbreviations are used: “NS”: no sample; “NSAD”: no sample animal died; “1+H”: minimal hemolysis.

[1247] Globulin (GLOB) is calculated as follows:

[1248] Total Protein-Albumin

[1249] The albumin / globulin ratio (A / G) was calculated as follows:

[1250] Albumin / Globulin

[1251] Urinalysis

[1252] Group averages are presented only for volume, pH, and specific gravity.

[1253] The following additional abbreviations are used: “NS”: not specimen available.

[1254] The following abbreviations are used to describe appearance and color: "CLOU": cloudy; "CLR": clear; "SLCL": slightly turbid; "STR": straw; "YELL": yellow.

[1255] Table 84: Qualitative results of multi-standard diagnostics are reported according to the following conventions:

[1256]

[1257] Organ weight

[1258] Organ weights were absolute, relative to terminal body weight recorded on the day of autopsy, and relative to brain weight.

[1259] pathology

[1260] Unless otherwise stated in the histopathology description, the organs / tissues examined were (routine) sections as required.

[1261] The following abbreviations are used: “B-”: benign; “Cath”: duct; “GALT”: gut-associated lymphoid tissue; “Infuse”: infuse; “LS”: longitudinal; “M-”: malignant; “TS”: transverse.

[1262] Histopathological findings were graded on the following scale:

[1263] Minimal such changes are barely discernible and / or few / very small lesions or areas are affected.

[1265] Mild changes are more noticeable but only a few / small lesions or areas are affected.

[1267] Moderate changes clearly exist and are of appreciable size and / or number.

[1269] This change is significant and is abundant in many areas of the slice and / or is of significant magnitude.

[1271] Severe changes affect a large portion of the tissue and / or are of large size.

[1272] All statistical analyses were performed separately for males and females using the individual animal as the basic experimental unit.

[1273] The following data types were analyzed separately at each time point: body weight; change from interval to interval body weight; food consumption; hematology; coagulation; clinical chemistry; urinalysis; and organ weights.

[1274] The parameter to be analyzed is defined as continuous, discrete or binary. The test item treatment group is then compared to the control using the following procedure.

[1275] Study plan deviation

[1276] The following deviations from the study plan occurred during the study, but without wishing to be bound by theory, they are not believed to have compromised the validity or integrity of the study.

[1277] On Day 1, the processed 8-hour post-dose toxicokinetic samples of Groups 2 to 4 were placed on dry ice overnight. The samples were found frozen the next morning. However, the required temperature (-80°C (±10°C)) was not monitored during the overnight period. The samples were immediately transferred to the SL301 freezer at the indicated -80°C (±10°C).

[1278] On pre-test day 13, animal 1015 did not undergo surgery due to significant low body weight.

[1279] The remaining animals were not examined for viability on day 13. However, the animals were within normal range one day before and one day after day 13.

[1280] The surgical repairs of animals No. 1592 (day 21) and No. 4078 (day 10) were not approved by the experiment director until 7 days and 2 days after the repair, respectively.

[1281] On Day 29, low blood volume toxicokinetic samples were collected from animals No. 2036 (8 hour post-dose time point) and No. 3050 (2 hour post-dose time point) because of "difficult to bleed" and "slow to bleed" animals, respectively.

[1282] Pituitary tissue from animal 4074 was collected at recovery necropsy but was later found to be lost during post-fixation trimming; weight was not obtained.

[1283] On Day 29, no immediate post-dose observations were performed on Animal No. 4580.

[1284] On Day 1, the 24-hour toxicokinetic samples from Animals 1016 and 2036 were processed for 1 minute within the "2 hours" specified in the study protocol.

[1285] At the time of terminal necropsy, the location of clinical pathology sample collection for animal 1004 was not recorded.

[1286] On day 23, toxicokinetic animal No. 4107 was transferred for necropsy for welfare reasons, however, this animal was not recorded in the appropriate records but was examined.

[1287] According to the research plan, spare animal No. 1015 was euthanized instead of transferred to the internal colony.

[1288] Documentation presented in the surgical records did not document the animals that had individual catheters placed postoperatively.

[1289] On Day 19, the dosing infusion for female TK animal No. 1528 was interrupted for 14 minutes due to a change of vehicle syringe.

[1290] No changes in saline syringes were recorded in the saline and injection cap changes for the toxicokinetic animals between days 4 and 12.

[1291] On day 29, male animal 4107 was euthanized for welfare reasons on day 23, so toxicokinetic samples were collected from only 2 males instead of 3 males / group / time point in Group 4.

[1292] On Day 29, collect the following samples over ±3 minutes after the designated time points (time allowed per SOP):

[1293] Animal No. Days / interval Schedule Actual time Minute deviation 2029 29-1hr 10:59 11:04 2

[1294] Various tissue losses were noted during clipping or microscopic examination of various animals and at necropsy intervals.

[1295] Due to the timing of the validation study, it was anticipated that stability might not have been established before the first day of dosing, so the formulation preparation was changed to daily preparation rather than weekly preparation per the original study plan. The Day 1 dose formulation was prepared the day before dosing, thus deviating from the study plan at the time. Once stability was established, the formulation preparation was changed back to weekly preparation.

[1296] Some dose intervals had inconsistencies in dose reliability, resulting in deviations from the study plan because animals received more or less than the intended dose (Table 84A). These may or may not result in an out-of-range dose accountability criterion of ±10.0%.

[1297] Table 84A: Samples with inconsistent dose quantification.

[1298]

[1299]

[1300] *TK Animals

[1301] A = The amount of time it took to detect a catheter leak is indeterminate B = No obvious cause

[1302] C = Tail vein catheterization difficulty; drug administration was stopped for welfare reasons

[1303] On one occasion, dosing was interrupted for some animals or pump settings needed to be adjusted, affecting the reliability of the documentation. However, the variation was less than a 10% deviation from the expected dose. In addition, animal 1017 (TK) did not receive the final 2-minute saline catheter flush.

[1304] On the day of terminal necropsy, animals 1010, 3047, 3048, 3049, 3057, 2538, 2539, and 2594 were fed for welfare reasons after being fasted for nearly 26 hours.

[1305] Analytical Chemistry

[1306] Analysis of the preliminary mixtures confirmed that the test item was stable in the excipient at room temperature (20°C labeled) for at least 24 hours and at 2 to 8°C (storage conditions used in this study) for at least 8 days. Analysis performed during the treatment period confirmed that the appropriate concentrations of the dosing solutions were administered, except for the Day 1, 1 mg / mL (Group 2) formulation. The Day 1, 1 mg / mL dosing solution was 86.7% labeled. Without wishing to be bound by theory, it is concluded that the volume collected for the dosing formulation analysis may have reduced the analytical results due to interactions at the meniscus. The volume collected for analysis was increased in subsequent weekly preparations and confirmed to be of appropriate concentration.

[1307] Table 85: Average Analytical Concentrations on Day 1

[1308]

[1309] Table 86: Mean Analytical Concentrations at Week 2

[1310]

[1311]

[1312] Table 87: Mean Analytical Concentrations at Week 3

[1313]

[1314] Table 88: Mean Analytical Concentrations at Week 4

[1315]

[1316] Table 89: Mean Analytical Concentrations at Week 5

[1317]

[1318] Brincidofovir

[1319] For the dose range from 1 to 15 mg / kg, in animals treated with brincidofovir, the peak plasma concentration of brincidofovir on day 1 occurred 1 to 2 hours after the start of the infusion, with a mean concentration range of 215 ng / mL to 3670 ng / mL. For the 4 and 15 mg / kg dose groups, the plasma concentration of brincidofovir could still be quantified at the 8 hour time point, but for the 1 mg / kg dose group, the plasma concentration of brincidofovir could only be quantified at the 2 hour time point. On day 1, the plasma concentration of brincidofovir increased as the dose increased from 1 to 15 mg / kg.

[1320] Table 90: Summary of Plasma Brincidofovir Toxicokinetic Parameters in Male and Female Rats by Treatment Combination on Day 1 (TK Parameter Population)

[1321]

[1322]

[1323] NC = Not Calculated; for the 1 mg / kg dose group, only 2 quantifiable data points were used to calculate AUC last value.

[1324] For the dose range from 1 to 15 mg / kg, the peak plasma concentration of brincidofovir in animals treated with brincidofovir on Day 29 occurred 1 to 2 hours after the start of infusion, with a mean concentration range of 297 ng / mL to 4500 ng / mL. For the 4 and 15 mg / kg dose groups, the plasma concentration of brincidofovir could still be quantified at the 8-hour time point, and in 1 of 6 animals in the 1 mg / kg dose group. For the 1 mg / kg dose group, the plasma concentration could be quantified at the 2-hour time point for all animals. For most animals, there were no quantifiable concentrations at the 24-hour or 48-hour time points at any dose; excluding one animal in the 4 mg / kg dose group that showed a measurable concentration for more than 48 hours. On Day 29, the plasma concentration of brincidofovir increased as the dose increased from 1 to 15 mg / kg. Table 91: Summary of male and female plasma brincidofovir toxicokinetic parameters by treatment combination in rats on Day 29 (TK parameter population)

[1325]

[1326]

[1327] NC = Not Calculated; for the 1 mg / kg dose group, only 2 quantifiable data points were used to calculate AUC last value.

[1328] Brincidofovir metabolite Cidofovir Individual plasma concentrations of the metabolite CDV in brincidofovir-treated animals at the 1 mg / kg (0 of 6 rats) and 4 mg / kg (1 of 6 rats) dose groups were generally not quantifiable using the first sample collected after the start of dosing on Day 1. However, individual plasma concentrations of CDV were quantifiable in 5 of 6 rats at the 1 hour sample collection time point in the 15 mg / kg dose group.

[1329] On Day 29, individual plasma concentrations of the metabolite CDV could not usually be quantified in the first sample collected after the start of dosing (1 hour after the start of infusion) in the 1 mg / kg (1 of 6) dose group. However, individual plasma concentrations of CDV could be quantified in 4 of 6 rats in the 4 mg / kg dose group and 5 of 5 rats in the 15 mg / kg dose group at the 1 hour sampling time point.

[1330] Plasma concentrations of CDV were still quantifiable at the 24-hour time point for animals dosed with 4 mg / kg and at the 48-hour time point for animals dosed with 15 mg / kg, but were only quantifiable at the 8-hour time point for the 1 mg / kg dose group. Plasma concentrations of CDV generally increased as the dose of brincidofovir increased from 1 to 15 mg / kg.

[1331] Table 92: Summary of male and female plasma CDV toxicokinetic parameters by treatment combination in rats on Day 1 (TK parameter population)

[1332]

[1333] NC = Not Calculated; for the 1 mg / kg dose group, only 2 quantifiable data points were used to calculate AUC last value.

[1334] Table 93: Summary of male and female plasma CDV toxicokinetic parameters by treatment combination in rats on Day 29 (TK parameter population)

[1335]

[1336]

[1337] NC = Not Calculated; for the 1 mg / kg dose group, only 2 quantifiable data points were used to calculate AUC last value.

[1338] Because animal 4107 was euthanized prior to TK sample collection on Day 29, N=5 for the 15 mg / kg dose group.

[1339] mortality rate

[1340] Drug administration phase

[1341] There were three unplanned deaths in this study; none were considered to be related to brincidofovir. The cause of death in two animals (Toxic Animals 2595 and 1005) was determined to be unrelated to brincidofovir. The cause of death in the third animal (TK Animal 4107) could not be determined.

[1342] Animal 2595 (toxic female at 1 mg / kg / dose) was euthanized on day 23 for welfare reasons due to skull swelling possibly related to accidental trauma. Dark areas on the skull associated with multifocal hemorrhages were observed under the microscope. On day 23, this rat was abnormally cool to the touch and showed reduced activity and abnormal (labored and irregular) breathing.

[1343] On day 23, animal No. 4107 (toxicokinetic male at 15 mg / kg / dose) was euthanized for welfare reasons. On day 23, this rat was abnormally cool to the touch and showed decreased activity, partially closed eyelids and dull eyes, and irregular breathing. The liver and spleen were distended and correct catheter tip position was noted at gross necropsy. The cause of death could not be determined from the macroscopic data, and no microscopic evaluation was performed to further determine the cause of death.

[1344] Animal No. 1005 (toxic male control) was found dead on Day 27. The animal had decreased activity and red, watery nasal discharge on Day 27. Macroscopic and microscopic findings were consistent with a bacterial infection originating at the catheter site and disseminating to multiple other organs.

[1345] Recovery Phase

[1346] There was no mortality associated with brincidofovir.

[1347] Clinical observation

[1348] Drug administration phase

[1349] Administration of brincidofovir at 4 and 15 mg / kg / dose resulted in intermittent clinical signs in some animals (both sexes). Intermittent clinical signs included rapid breathing and prone posture with or without piloerection, partially closed eyes, decreased activity, irregular breathing, and / or flat posture. Manual differential leukocyte counts were performed for verification and absolute values ​​were calculated if necessary. These symptoms were usually observed between days 22 and 29 after infusion and the incidence was dose-related.

[1350] Recovery Phase

[1351] All clinical symptoms observed during the treatment phase fully resolved on day 1 of the recovery phase. During the recovery phase, there were no clinical symptoms related to brincidofovir.

[1352] Ophthalmology

[1353] At the end of the dosing recovery period, there were no ophthalmologically related effec...

Claims

1. A pharmaceutical composition comprising: brincidofovir at a concentration of 10.0 mg / mL; Mannitol at a concentration of 2.5% (w / v)-9% (w / v); L-arginine at a concentration of 100 mM-200 mM; and water; wherein the pH of the composition is 8.0, and wherein the composition shows less than 0.05% of drug-related impurities when stored at 5(±3)°C for 1 month.

2. The pharmaceutical composition according to claim 1, wherein the pH is adjusted with HCl and / or NaOH.

3. The pharmaceutical composition according to claim 1, wherein the mannitol is present at a concentration of 2.5% (w / v).

4. The pharmaceutical composition according to claim 1, wherein the mannitol is present at a concentration of 5% (w / v).

5. The pharmaceutical composition of claim 1, wherein the L-arginine is present at a concentration of 100 mM.

6. The pharmaceutical composition according to claim 1, comprising: brincidofovir at a concentration of 10.0 mg / mL; Mannitol at a concentration of 25 mg / mL-50 mg / mL; L-arginine at a concentration of 17.4 mg / mL; and water; The pH of the composition is 8.

0.

7. The pharmaceutical composition according to claim 1, comprising: brincidofovir at a concentration of 17.8 mM; Mannitol at a concentration of 137.5 mM-275 mM; L-arginine at a concentration of 100 mM; and water; The pH of the composition is 8.

0.

8. The pharmaceutical composition according to claim 1, wherein the composition contains no visible particles when stored at 5 (± 3) °C for 6 months.

9. The pharmaceutical composition according to claim 1, wherein the composition contains no visible particles when stored at 25 (± 2) °C for 6 months.

10. The pharmaceutical composition according to claim 1, wherein the composition contains no visible particles when stored at 25 (± 2) °C for 12 months.

11. The pharmaceutical composition of claim 1, wherein the composition is suitable for freeze-drying.

12. Use of an aqueous pharmaceutical composition in the preparation of a medicament for treating viral infection, wherein the pharmaceutical composition comprises: brincidofovir at a concentration of 10.0 mg / mL; Mannitol at a concentration of 2.5% (w / v)-9% (w / v); L-arginine at a concentration of 100 mM-200 mM; and water; The pH of the composition is 8.

0.

13. Use of the aqueous pharmaceutical composition according to claim 12, wherein the viral infection is selected from polyomavirus, papillomavirus, herpes simplex virus, adenovirus, Epstein-Barr virus, cytomegalovirus, hepatitis B virus, hepatitis C virus, varicella zoster virus, poxvirus or a combination thereof.

14. Use of the aqueous pharmaceutical composition according to claim 12, wherein said use does not cause hemolysis.

15. Use of the aqueous pharmaceutical composition according to claim 12, wherein said use does not cause gastrointestinal toxicity.

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