Formulations of tegafur and related compounds

文档序号：602353 发布日期：2021-05-04 浏览：94次中文

阅读说明：本技术 特加维因和相关化合物的制剂 (Formulations of tegafur and related compounds ) 是由史蒂文·大卫·戴克斯特拉亨利·哈维尔斯蒂芬·霍里根罗杰·哈里森杰弗里·拉森乔纳森于 2019-05-31 设计创作，主要内容包括：特加维因和相关化合物的制剂,制备这类制剂的方法和利用这类制剂治疗各种病况的方法。(Formulations of tegafur and related compounds, methods of making such formulations, and methods of treating various conditions using such formulations.)

1. A composition, comprising:

a) particles of a compound of formula I

Wherein R is_AIs hydrogen, R₇And R₈Independently selected from H and SO₂NR₃R₄Wherein R is₇And R₈Is hydrogen, and wherein NR is₁R₂And NR₃R₄Independently a 6 to 15 membered heterocyclic ring containing one nitrogen in the ringAlkyl, or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof; and

b) a surfactant;

wherein the particles have an effective D50 of less than or equal to 500nm and a D90 of less than or equal to 1.0 micrometer (μm) when measured using laser diffraction.

2. The composition of claim 1, wherein the compound of formula I is tegavirine (tegavirent) or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof.

3. The composition of claim 1, wherein the composition is a nanoparticle composition.

4. The composition of claim 1, wherein the surfactant is a poloxamer surfactant.

5. The composition of claim 1, wherein the poloxamer surfactant is poloxamer 188.

6. The composition of claim 1, wherein the composition further comprises a stabilizer.

7. The composition of claim 6, wherein the stabilizer is selected from the group consisting of: sugar, polyol, polysorbate surfactant, and polyvinylpyrrolidone (PVP).

8. The composition of claim 7, wherein the sugar is selected from the group consisting of sucrose and trehalose.

9. The composition of claim 7, wherein the polyol comprises sorbitol and mannitol.

10. The composition of claim 1, wherein the concentration of the compound is between about 10mg/ml and about 25 mg/ml.

11. The composition of claim 1, wherein the concentration of the compound is about 25 mg/ml.

12. A composition, comprising:

a.10-25mg/ml of tergaverine or a pharmaceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof;

b. poloxamer 188; and

c. sorbitol;

wherein the tegafur or said pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof is in the form of a nanosuspension of particles comprising tegafur or said pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof, and wherein said particles have an effective D50 of less than or equal to 500nm and a D90 of less than or equal to 1.0 micrometer (μm) when measured using laser diffraction.

13. The composition of claim 12, wherein the amount of tergaverine or said pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof is 25 mg/ml; poloxamer 188 in an amount of 0.625%; and sorbitol in an amount of 10%, wherein the percentages are by weight of the composition.

14. The composition of claim 1, wherein the composition is prepared by milling.

15. The composition of claim 1, wherein the composition is prepared by LyoCell technology.

16. A method of making a composition comprising:

a) particles of the compound of formula I

or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof; mixing with a surfactant and an acceptable carrier to produce a suspension;

b) roller milling or milling the suspension of step (a) using a high energy mill; and

c) adding a polyol to the particles of step (b).

17. The composition of claim 16, wherein the composition exhibits long-term stability.

18. The composition of claim 16, wherein the compound of formula I is tergaverine or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof.

19. The composition of claim 1, wherein the composition is formulated as: (a) a dosage form selected from the group consisting of tablets and capsules; (b) a dosage form selected from the group consisting of: controlled release, fast dissolving, delayed release, extended release, pulsed release and mixed immediate and controlled release formulations; (c) formulations suitable for inhalation or parenteral administration, including intramuscular, subcutaneous, intravenous and intradermal injection; or (d) any combination of (a), (b), and (c).

20. A method of preventing, treating, or reducing cancer or tumor metastasis in a mammal in need thereof, comprising administering to the mammal an effective amount of the composition of claim 1.

21. A method for treating cancer, comprising administering to a subject in need thereof a combination of: 1) a pharmaceutically effective amount of the nanoparticle composition of claim 1; and 2) a pharmaceutically effective amount of at least one additional anti-cancer agent.

22. The method of claim 22, wherein the additional anti-cancer agent is selected from the group consisting of: anti-mitotic agents, anti-metabolic agents, HDAC inhibitors, proteasome inhibitors, immunotherapeutic agents, FLT-3EGFR, MEK, PI3K, and other protein kinase inhibitors, LSD1 inhibitors, and WNT pathway inhibitors, alkylating agents, and DNA repair pathway inhibitors, anti-hormonal agents, anti-cancer antibodies, and other cytotoxic chemotherapeutic agents.

23. A method of treating and/or preventing a fibrotic disease in a mammal in need thereof, comprising administering to the mammal an effective amount of the composition of claim 1.

24. The method of claim 24, wherein the fibrotic disease is selected from the group consisting of: pulmonary fibrosis, Dupuytren's contracture, scleroderma, systemic sclerosis, scleroderma-like disorders, scleroderma without skin sclerosis, cirrhosis of the liver, interstitial pulmonary fibrosis, keloids, chronic kidney disease, chronic transplant rejection, and other scar/wound healing abnormalities, post-operative adhesions, reactive fibrosis.

Technical Field

The present invention relates generally to formulations of tegavirenz (tegavirint) and related compounds, methods of making such formulations, and methods of treating various conditions using such formulations.

Background

Cancer is the second leading cause of death in the united states. It presents a complex challenge to develop new therapies. Cancer is characterized by the abnormal growth of malignant cells that have undergone a series of genetic changes, resulting in tumor mass growth and metastatic properties.

Beta-catenin (Beta-catenin/Beta-catenin) is part of the protein complex that constitutes the Adhesive Junction (AJ). AJ is necessary for the generation and maintenance of epithelial cell layers by regulating cell growth and adhesion between cells. Beta-catenin may also anchor the cytoskeleton of actin and may be responsible for transmitting contact inhibitory signals that stop cells from dividing once the epithelial sheet is intact.

The Wnt/β -catenin pathway has been shown to play a role in cancer. Aberrant β -catenin signaling plays an important role in tumor formation. Specifically, colorectal cancer is estimated to have greater than 80% mutations in the β -catenin pathway, resulting in unregulated oncogenic signaling. Aberrant β -catenin signaling has been shown to be associated with various cancer types, including but not limited to melanoma, breast cancer, lung cancer, colon cancer, liver cancer, stomach cancer, myeloma, multiple myeloma, chronic myelogenous leukemia, chronic lymphocytic leukemia, T-cell non-Hodgkin lymphoma, colorectal cancer, and Acute Myeloid Leukemia (AML). In addition, aberrant Wnt/β -catenin signaling has been found in a number of other disorders, including osteoporosis, osteoarthritis, polycystic kidney disease, diabetes, schizophrenia, vascular diseases, cardiac diseases, hyperproliferative disorders, neurodegenerative diseases, and fibrotic diseases, including but not limited to Idiopathic Pulmonary Fibrosis (IPF), Dupuytren's contracture, nonalcoholic steatohepatitis (NASH), and the like. Myeloproliferative neoplasms (MPNs) are a group of closely related hematological malignancies in which the myeloid cells that give rise to human blood cells develop abnormally and function. The three major myeloproliferative neoplasms are Polycythemia Vera (PV), primary thrombocythemia (ET), and Primary Myelofibrosis (PMF). JAK2 gene mutations are present in most PV patients and 50% of ET and PMF patients. In many cases, the β -catenin pathway is activated in MPN and is required for the survival of these cells.

Tegravine and related compounds are described, for example, in U.S. patent No. 8,129,519. The Tegaviine has the following structural formula:

the molecular formula of the Tegaviine is C₂₈H₃₆N₄O₆S₂。

The molecular weight of Tegaviine is 588.20763 amu.

There is a need in the art to provide stable, readily bioavailable formulations of tegafur and related compounds, wherein the formulations allow administration by different routes of administration, including but not limited to parenteral administration and via inhalation, and are stable to be suitable for clinical study and treatment of various diseases treatable with tegafur.

Disclosure of Invention

The development of stable, non-toxic formulations of tegafur has been very challenging and difficult. A large number of formulations were developed and tested; however, they have poor bioavailability and/or demonstrate instability on storage, and/or become highly toxic. These formulations include microemulsions, solid suspensions, liposome-based formulations, various oral formulations, and IV formulations.

The present inventors have unexpectedly and unexpectedly found that nanosuspensions of tegafur function, wherein the nanosuspensions comprise a surfactant, and wherein the particles of tegafur have an effective D50 of less than or equal to 500nm, and a D90 of less than or equal to 1.0 micrometer (μm), when measured using laser diffraction. It has also been found that a particularly preferred concentration of Tegaviine is from 10 to 25mg/ml, most preferably 25 mg/ml; preferred surfactants are poloxamer (poloxamer) surfactants (preferably poloxamer 188), preferably at a concentration of 0.625%; and the nanosuspension should preferably comprise a polyol, and more preferably sorbitol.

Thus, the most preferred formulation is one containing 25mg/ml of Tegaviine; a composition of 0.625% poloxamer 188 and 10% sorbitol, wherein the tegafur is in the form of a nanosuspension comprising particles of tegafur, and wherein the effective D50 of the particles is less than or equal to 500nm and D90 is less than or equal to 1.0 micron (μm) when measured using laser diffraction.

Accordingly, in one embodiment, the present invention provides a composition comprising:

a) particles of a compound of formula I

Wherein R is_AIs hydrogen, R₇And R₈Independently selected from H and SO₂NR₃R₄Wherein R is₇And R₈Is hydrogen, and wherein NR is₁R₂And NR₃R₄Independently a 6 to 15 membered heterocycloalkyl containing one nitrogen in the ring, or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof; and

b) a surfactant;

wherein the particles have an effective D50 of less than or equal to 500nm and a D90 of less than or equal to 1.0 micrometer (μm) when measured using laser diffraction.

In some embodiments, the effective average particle size of the compound is about 4900nm, about 4800nm, about 4700nm, about 4600nm, about 4500nm, about 4400nm, about 4300mm, about 4200nm, about 4100nm, about 4 μm, about 3900nm, about 3800nm, about 3700nm, about 3600nm, about 3500nm, about 3400mm, about 3300nm, about 3200nm, about 3100nm, about 3 microns, about 2900mm, about 2800nm, about 2700nm, about 2600nm, about 2500nm, about 2400nm, about 2300nm, about 2200nm, about 2100nm, about 2000nm, about 1900nm, about 1800nm, about 1700nm, about 1600nm, about 1500nm, about 1400nm, about 1300nm, about 1200nm, about 1100nm, about 1000nm, about 900nm, about 800nm, about 700nm, about 600nm, about 500nm, about 400nm, or about 300 nm.

Additionally, in some embodiments, the effective average particle size of the compound is less than 900nm, more preferably less than 500nm, and even more preferably less than 300 nm.

In a preferred embodiment, the surfactant is a poloxamer surfactant.

In another preferred embodiment, the poloxamer surfactant is poloxamer 188.

In a preferred embodiment, the particulate composition further comprises a stabilizer.

In a preferred embodiment, the stabilizer is selected from the group consisting of: sugar, polyol, polysorbate surfactant, and polyvinylpyrrolidone (PVP).

In another preferred embodiment, the sugar is selected from the group consisting of sucrose and/or trehalose.

In a preferred embodiment, the polyol comprises sorbitol and/or mannitol.

In one embodiment, the concentration of the compound in the provided compositions is between about 1mg/ml and about 100mg/ml, more preferably between about 10mg/ml and about 50mg/ml, more preferably between about 10mg/ml and about 25mg/ml, and even more preferably about 25 mg/ml.

In one embodiment, the composition of the present invention is prepared by milling.

In another embodiment, the compositions of the invention are prepared by LyoCell technology. Us patent 7,713,440 describes LyoCell technology. The contents of U.S. patent 7,713,440 are incorporated herein by reference in their entirety.

In another embodiment, the compositions of the present invention may be prepared by a dry milling process, as described in U.S. patent 8,808,751. The contents of U.S. patent 8,808,751 are incorporated herein by reference in their entirety. By appropriate selection of the grinding media and the appropriate milling compound, it is possible to produce nanoparticle compositions from conventional drug substance particles and to prevent agglomeration of the small particles generated in the dry milling apparatus.

In another example, the compositions of the present invention can be prepared by a method that utilizes human serum albumin as a carrier, such as the method described in U.S. Pat. No. 6,537,579. The contents of U.S. Pat. No. 6,537,579 are incorporated by reference herein in their entirety. The method may be particularly suitable for preparing nanoparticle compositions of poorly water soluble compounds. The compositions produced by such methods may allow for the effective administration of poorly water soluble biologically active compounds.

In another embodiment, nanoparticle compositions containing polymers such as poly (DL-lactide-co-glycolide) are capable of delivering poorly soluble bioactive compounds. As shown in U.S. patent No. 5,543,158, these compositions can be designed as long acting vehicles. The contents of U.S. Pat. No. 5,543,158 are incorporated herein by reference in their entirety.

In another embodiment, the compositions of the present invention can be prepared as polymeric micelles, which have been successful in improving the solubility of biologically active compounds. The commercial product, Genexol-PM, using this technology incorporates the anticancer drug paclitaxel and was approved in korea in 2007.

In one embodiment, the present invention provides a method of preparing a composition comprising the following steps (a) to (c):

a) particles of the compound of formula I

or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof;

mixing with a surfactant and an acceptable carrier to produce a suspension;

b) milling the suspension of step (a) using a roller mill or high energy mill; and

c) adding a polyol to the particles of step (b).

In one embodiment, the acceptable carrier is a liquid carrier (e.g., water).

In one embodiment, the suspension is an aqueous suspension.

In another embodiment, a method of preparing a composition comprises the following steps (a) to (b):

a) particles of the compound of formula I

or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof;

mixing with a surfactant, a polyol, and an acceptable carrier to produce a suspension; and

b) milling the suspension of step (a) using a roller mill or a high energy mill.

In one embodiment, the acceptable carrier is a liquid carrier (e.g., water).

In one embodiment, the suspension is an aqueous suspension.

In a preferred embodiment, the compositions of the present invention exhibit long-term stability.

In a preferred embodiment, the composition of the present invention is a nanoparticle composition.

In a preferred embodiment, the compounds of formula I are in the compositions of the invention

Has the following structure:

or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof.

The compound having the above formula is also known as tegafur (BC 2059).

In one embodiment, the compositions of the present invention may be formulated as: (a) a dosage form selected from the group consisting of tablets and capsules; (b) a dosage form selected from the group consisting of: controlled release, fast dissolving, delayed release, extended release, pulsed release and mixed immediate and controlled release formulations; (c) dosage forms suitable for administration by inhalation or parenteral, including intramuscular, subcutaneous, intravenous and intradermal injection; (d) any combination of (a), (b), and (c).

The compositions of the present invention may further comprise one or more pharmaceutically acceptable excipients, carriers, or combinations thereof.

In another embodiment, the present invention provides a method of preventing, treating or ameliorating cancer or tumor metastasis in a mammal in need thereof, comprising administering to the mammal an effective amount of a composition of the present invention.

Methods of administration are not limited to any particular route of administration, and include, but are not limited to, intravenous, parenteral, oral, inhalation (including aerosolized delivery), oral, intranasal, rectal, intralesional, intraabdominal, intradermal, transdermal, subcutaneous, intraarterial, intracardiac, intraventricular, intracranial, intratracheal, intrathecal, intramuscular, intravitreal, and topical methods of application.

In another embodiment, a method of preventing, treating, or ameliorating cancer or tumor metastasis in a mammal in need thereof can comprise administering an additional anti-cancer agent and/or cancer therapy (e.g., cancer vaccine, anti-cancer adoptive cell therapy, and radiation therapy).

In one embodiment, the additional anti-cancer agent is selected from the group consisting of: anti-mitotic agents, anti-metabolic agents, HDAC inhibitors, proteasome inhibitors, immunotherapeutic agents, FLT-3EGFR, MEK, PI3K, and other protein kinase inhibitors, LSD1 inhibitors, and WNT pathway inhibitors, alkylating agents, and DNA repair pathway inhibitors, anti-hormonal agents, anti-cancer antibodies, and other cytotoxic chemotherapeutic agents.

In another embodiment, the present invention provides a method of treating and/or preventing a fibrotic disease in a mammal in need thereof, comprising administering to the mammal an effective amount of a composition of the present invention.

In a preferred embodiment, the fibrotic disease is selected from the group consisting of: pulmonary fibrosis, dupuytren's contracture, scleroderma, systemic sclerosis, scleroderma-like disorders, scleroderma without skin sclerosis, cirrhosis of the liver, interstitial pulmonary fibrosis, keloids, chronic kidney disease, chronic transplant rejection, and other scar/wound healing abnormalities, post-operative adhesions, and reactive fibrosis.

In one embodiment, the method of treating and/or preventing a fibrotic disease in a mammal in need thereof may comprise administering an additional anti-fibrotic agent.

Drawings

FIG. 1 is a plot of the Particle Size Distribution (PSD) of one of the formulations of the present invention.

FIG. 2 is a plot of the PSD of another of the formulations of the present invention.

Detailed Description

Definition of

The terms used in this specification generally have their ordinary meaning in the art, both within the context of the invention and in the specific context in which each term is used. Certain terms used to describe the invention are discussed below or elsewhere in the specification to provide additional guidance to the practitioner regarding the description of the invention. Synonyms for certain terms are provided. The recitation of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. The invention is not limited to the various embodiments presented in this description.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present document, including definitions, will control.

The term "tegafur" refers to a compound having the structure:

the term "BC 2059" is used interchangeably with "tegravine".

The term "long term storage" or "long term stability" is understood to mean that the pharmaceutical composition can be stored for three months or more, six months or more, preferably one year or more. Long term storage is also understood to mean storage of the pharmaceutical composition at 2-8 ℃ or at room temperature 15-25 ℃.

With respect to long term storage, the term "stable" is understood to mean that the active ingredient contained in the pharmaceutical composition does not lose its activity by more than 20%, or more preferably by 15%, or even more preferably by 10%, and most preferably by 5%, relative to the activity of the composition at the start of storage.

The term "mammal" includes, but is not limited to, humans.

The term "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid or excipient of any conventional type. Pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations employed, and are compatible with other ingredients of the formulation.

The term "treatment" refers to any administration or use of a disease in a mammal, including inhibiting the disease, arresting the development of the disease, alleviating the disease (e.g., by causing regression, restoration or repair of lost, lost or defective function), or stimulating an inefficient process. The term includes obtaining a desired pharmacological and/or physiological effect and encompasses any treatment of a pathological condition or disorder in a mammal. The effect may be prophylactic in terms of completely or partially preventing the disorder or a symptom thereof, and/or may be therapeutic in terms of a partial or complete cure of the disorder and/or a side effect attributable to the disorder. It includes (1) preventing the occurrence or recurrence of a disorder in a subject who is susceptible to the disorder but not yet symptomatic, (2) inhibiting the disorder, such as arresting its development, (3) halting or terminating the disorder or at least its associated symptoms such that the host no longer suffers from the disorder or its symptoms, such as by restoring or repairing a process of lost, missing or defective function or stimulation efficiency, causing the disorder or its symptoms to disappear, or (4) alleviating, alleviating or ameliorating the disorder or its associated symptoms, wherein ameliorating means in a broad sense at least reducing the magnitude of a parameter, such as inflammation, pain, and/or tumor size.

The term "therapeutically effective amount" refers to an amount that, when administered to a living body, achieves a desired effect on the living body. For example, an effective amount of a composition of the invention for administration to a living body is an amount that prevents and/or treats any of the diseases mediated via the Wnt/β -catenin pathway. The exact amount will depend on the purpose of the treatment and will be determined by one of ordinary skill in the art using known techniques. As is known in the art, modulation of systemic versus local delivery, age, body weight, general health, sex, diet, time of administration, drug interactions, and severity of the condition may be desired and can be determined by one of skill in the art with routine experimentation.

The term "composition" or "formulation" refers to a mixture typically containing a carrier, such as a pharmaceutically acceptable carrier or excipient conventional in the art and suitable for administration to a subject for therapeutic, diagnostic or prophylactic purposes. For example, compositions for oral administration may form solutions, suspensions, tablets, pills, capsules, sustained release formulations, oral rinses, or powders. The terms "composition", "pharmaceutical composition" and "formulation" are used interchangeably.

The term "nanoparticle composition" refers to a composition in which all, or almost all, of the particles are less than 1000 nM.

Compositions of the invention

In one embodiment, the present invention provides a composition comprising:

a) particles of a compound of formula I

Formula I

Wherein Ra is hydrogen, R₇And R₈Independently selected from H and SO₂NR₃R₄Wherein R is₇And R₈Is hydrogen, and wherein NR is₁R₂And NR₃R₄Independently a 6 to 15 membered heterocycloalkyl group containing one nitrogen in the ring,

or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof; and

b) a surfactant;

wherein the particles have an effective D50 of less than or equal to 500nm and a D90 of less than or equal to 1.0 micrometer (μm) when measured using laser diffraction.

D50 is also referred to as the median diameter of the particle size distribution. It refers to the 50% particle diameter value in the cumulative distribution. In other words, when the value of D50 is less than or equal to 500nm, this means that 50% of the particles are less than 500nm in diameter.

D90 refers to the percentage of particles at the reported particle size. In other words, when the value of D90 is less than or equal to 1.0. mu.m, this means that 90% of the particles have a diameter of less than 1.0. mu.m.

In some embodiments, the effective average particle size of the compound is about 4900nm, about 4800nm, about 4700nm, about 4600nm, about 4500nm, about 4400nm, about 4300mm, about 4200nm, about 4100nm, about 4 microns, about 3900nm, about 3800nm, about 3700nm, about 3600nm, about 3500nm, about 3400mm, about 3300nm, about 3200nm, about 3 microns, about 2900mm, about 2800nm, about 2700nm, about 2600nm, about 2500nm, about 2400nm, about 2300nm, about 2200nm, about 2100nm, about 2000nm, about 1900nm, about 1800nm, about 1700nm, about 1600nm, about 1500nm, about 1400nm, about 1300nm, about 1200nm, about 1100nm, about 1000nm, about 900nm, about 800nm, about 700nm, about 600nm, about 500nm, about 400nm, or about 300 nm.

Additionally, in some embodiments, the effective average particle size of the compound is less than 900nm, more preferably less than 500nm, and even more preferably less than 300 nm.

In a preferred embodiment, the surfactant is a poloxamer surfactant.

In another preferred embodiment, the poloxamer surfactant is poloxamer 188.

In a preferred embodiment, the composition further comprises a stabilizer.

In a preferred embodiment, the stabilizer is selected from the group consisting of: sugar, polyol, polysorbate surfactant, and polyvinylpyrrolidone (PVP).

In another preferred embodiment, the sugar is selected from the group consisting of sucrose and/or trehalose.

In a preferred embodiment, the polyol comprises sorbitol and mannitol.

A particularly preferred concentration of tegafur is 10-25mg/ml, most preferably 25 mg/ml; preferred surfactants are poloxamer surfactants (preferably poloxamer 188), preferably at a concentration of 0.625%; and the nanosuspension should preferably comprise a polyol, and more preferably sorbitol.

Thus, the most preferred formulation is one containing 25mg/ml of Tegaviine; 0.625% poloxamer 188 and 10% sorbitol nanosuspension.

In one embodiment, the composition of the invention is prepared by milling, preferably wet milling.

In one embodiment, the present invention provides a method of preparing a composition comprising the following steps (a) to (c):

a) particles of the compound of formula I

or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof;

mixing with a surfactant and an acceptable carrier to produce a suspension;

b) milling the suspension of step (a) using a roller mill or high energy mill; and

c) adding a polyol to the particles of step (b).

In one embodiment, the acceptable carrier is a liquid carrier (e.g., water).

In one embodiment, the suspension is an aqueous suspension.

In another embodiment, a method of preparing a composition comprises the following steps (a) to (b):

a) particles of the compound of formula I

or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof;

mixing with a surfactant, a polyol, and an acceptable carrier to produce a suspension; and

b) milling the suspension of step (a) using a roller mill or a high energy mill.

In one embodiment, the acceptable carrier is a liquid carrier (e.g., water).

In one embodiment, the suspension is an aqueous suspension.

In yet another embodiment, the compositions of the present invention can be prepared by a method that utilizes human serum albumin as a carrier, such as the method described in U.S. Pat. No. 6,537,579. The contents of U.S. Pat. No. 6,537,579 are incorporated by reference herein in their entirety. The method may be particularly suitable for preparing nanoparticle compositions of poorly water soluble compounds. The compositions produced by such methods may allow for the effective administration of poorly water soluble biologically active compounds.

In a preferred embodiment, the compositions of the present invention exhibit long-term stability.

In one embodiment, the composition of the present invention is a nanoparticle composition.

In a preferred embodiment, the compound of formula I in the composition of the invention has the following structure:

or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof.

This compound is also known as tegafur.

The invention encompasses formulations comprising tergaverine and pharmaceutically acceptable salts, esters, amides, stereoisomers or geometric isomers thereof.

The solubility of tegafur in water was measured in the pH range of 2 to 10 and found to be <0.25mcg/ml throughout the range.

The solubility of Tegaviine in organic solvents is shown below: DMSO (334. mu.g/mL), ethanol (260. mu.g/mL), methanol (299. mu.g/mL), acetone (1mcg/mL), dichloromethane ethanol (1:4) (1 mg/mL).

The compositions of the present invention may further comprise one or more pharmaceutically acceptable excipients, carriers, or combinations thereof.

The pharmaceutically acceptable excipients used in the formulations of the present invention may function in more than one way. The role of the dispersant is, for example, to keep the individual particles separated, i.e. to minimize agglomeration. However, this ingredient may also alter the surface tension of the formulation and may serve to reduce viscosity, for example.

The pharmaceutically acceptable excipient may be, for example, a dispersion medium, a dispersion emulsifier, a dispersion enhancer, or a combination thereof.

Examples of propellants include, but are not limited to, HFA-134a (1,1,1, 2-tetrafluoroethane), HFA-227(1,1,1,2,3,3, 3-heptafluoropropane), combinations thereof, and the like.

The dispersion medium can be, for example, ethanol, propylene glycol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, glycerol, combinations thereof, and the like.

The dispersion emulsifier (enhancer) can be, for example, H₂O, oleic acid, sodium lauryl sulfate, polyethylene glycol 1000, ammonium alginate, potassium alginate, calcium stearate, glyceryl monooleate, polyoxyethylene stearate, emulsifying wax, polysorbate 20, polysorbate 40, and polysorbateEsters 60, polysorbate 80, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate, poloxamers, combinations thereof and the like.

Examples of dispersion enhancers include, but are not limited to, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sodium carboxymethylcellulose, hypromellose, ethylene glycol stearate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate, glycerol monostearate, lecithin, meglumine, poloxamers, polyoxyethylene alkyl ethers, polyoxyl 35 castor oil, polyoxyethylene stearate, polyoxyl esters, pyrrolidones, sorbitan esters, stearic acid, vitamin E polyethylene glycol succinate, polyethylene glycol 1000, povidone, combinations thereof, and the like.

The compositions of the present invention may be suitable for all routes of administration, including but not limited to intravenous, parenteral, oral, inhalation (including aerosolized delivery), oral, intranasal, rectal, intralesional, intraabdominal, intradermal, transdermal, subcutaneous, intraarterial, intracardiac, intraventricular, intracranial, intratracheal, intrathecal administration, intramuscular injection, intravitreal injection, and topical application methods.

The pharmaceutical compositions according to the present invention may also comprise one or more binders, fillers, lubricants, suspending agents, sweetening agents, flavoring agents, preservatives, buffering agents, wetting agents, disintegrating agents, effervescent agents and other excipients. Such excipients are known in the art.

Examples of fillers are lactose monohydrate, lactose anhydrous and various starches; examples of binders are various celluloses and crosslinked polyvinylpyrrolidones, microcrystalline celluloses, e.g. cellulose acetatePH101 andPH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC)^TM)。

Suitable lubricants, including agents acting on the flowability of the powder to be compacted, are colloidal silicas, e.g. silica200. Talc, stearic acid, magnesium stearate, calcium stearate and silica gel.

Examples of sweeteners are any natural or artificial sweeteners such as sucrose, xylitol, saccharin sodium, cyclamate, aspartame (aspartame), and acesulfame (acesulfame). Examples of flavoring agents are(trade mark of MAFCO), bubble gum flavoring, fruit flavoring, and the like.

Examples of preservatives are potassium sorbate, methyl paraben, propyl paraben, benzoic acid and its salts, other esters of parahydroxybenzoic acid, such as butyl paraben, alcohols, such as ethanol or benzyl alcohol, phenolic compounds, such as phenol, or quaternary ammonium compounds, such as benzalkonium chloride.

Suitable diluents include pharmaceutically acceptable inert fillers such as microcrystalline cellulose, lactose, dibasic calcium phosphate, sugars and/or mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such asPH101 andPH 102; lactose, e.g. lactose monohydrate, lactose anhydrous andDCL 21; dibasic calcium phosphates, e.g.Mannitol; starch; sorbitol; sucrose; and glucose.

Suitable disintegrants include lightly crosslinked polyvinylpyrrolidone, corn starch, potato starch, maize starch and modified starches, croscarmellose sodium, crospovidone, sodium starch glycolate, and mixtures thereof.

Examples of effervescent agents are effervescent combinations, such as organic acids and carbonates or bicarbonates. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids as well as anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent combination may be present.

In a preferred embodiment, the compounds of formula I have the following structure:

or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof.

The present invention is more particularly described in the following examples, which are intended as illustrations only, since numerous modifications and variations therein will be apparent to those skilled in the art. In the following examples, it will be understood that the weight percentages of the various ingredients are expressed as w/v percentages.

Examples of the invention

It is very challenging and difficult to obtain a formulation of tegafur that works (i.e. is stable and non-toxic).

The formulation demonstrated to function is a nanosuspension of tegravine, wherein the nanosuspension comprises a surfactant, and wherein the particles of tegravine have an effective D50 of less than or equal to 500nm and a D90 of less than or equal to 1.0 micrometer (μm), when measured using laser diffraction. It has also been found that a particularly preferred concentration of Tegaviine is from 10 to 25mg/ml, most preferably 25 mg/ml; preferred surfactants are poloxamer surfactants (preferably poloxamer 188), preferably at a concentration of 0.625%; and the nanosuspension should preferably comprise a polyol, and more preferably sorbitol.

The examples section first describes a number of experiments to formulate tegafur that eventually fail for various reasons. This section then describes grinding feasibility experiments, which demonstrate that tegafur can be roll milled when suspended in aqueous solution with various dispersants. However, even when roll-milled, various formulations of tegravine have not been successful.

Finally, it describes a successful experiment involving nanosuspensions of the claimed tegravine.

Unsuccessful experiments

Example 1

The microemulsion preparation of Tegaweiyin has extremely high toxicity

A microemulsion formulation of tegafur was developed in which the formulation contained 20mg/ml BC2059, 10% Tween (Tween) (polysorbate 80), 30% ethanol, 50% Propylene Glycol (PG) and 10% D-alpha-tocopheryl polyethylene glycol 1000 succinate.

Although good stability of the formulation was observed, the formulation was extremely toxic to rodents and was therefore not further investigated.

Example 2

Liposome-based formulations are unstable

Based on preliminary studies, two BC2059 liposome formulations were selected as lead for 100ml scale-up and stability assessment.

The first was a 100% ePC formulation with a 15:1 lipid to drug ratio.

Another lead preparation included 80% lipid to drug ratio 10:1 to 20% ePC: LysoPC liposomes.

Both formulations were shown to be unstable when stored at 5 ℃ (precipitation was observed). In addition, the formulation is also unstable when frozen.

Example 3

Oral and IV formulations were unsuccessful

The oral formulation of tegafur, which contained soy lecithin, PEG200, PEG400, PG and TPGS, was initially selected as the lead oral formulation. However, this lead oral formulation showed poor bioavailability in dog studies and was therefore not studied.

Upon further screening, the IV-based formulation was selected as the next lead. This IV formulation contains an oil phase (vegetable oil and polysorbate 80(PS80) as solubilizer) and soy lecithin as emulsifier. The formulation had the following composition (all figures are in weight%):

BC 2059: 1 percent; PS 80: 10 percent; miglyol 812: 12 percent; soybean lecithin (LIPOID S-100): 12 percent; propylene Glycol (PG): 50 percent; deionized water: proper amount.

This formulation shows the possibility of filtration to 0.2 micron with minimal loss and good physical and chemical stability. However, due to the high toxicity in rodent studies, this formulation was not further investigated.

Experiments with nanosuspensions comprising Tegaviine

Example 4

Feasibility of grinding

First, it is determined whether grinding is possible in principle. Experiments have shown that it is feasible.

Milling feasibility starting with a laboratory-scale batch of tegravine suspended in 5% (50mg/mL) in aqueous solution by roller milling, the following dispersants, which are suitable for intravenous administration, were selected for milling:

polysorbate 20 (0.5%)

Poloxamer 188 (0.5%)

Polyvinylpyrrolidone, K17 (1%)

Polyvinylpyrrolidone, K17 (1%) and sodium deoxycholate (0.25%)

Lecithin (1%)

5mL of the test suspension was milled with about 10mL of yttria stabilized zirconia (YTZ) milling media having a diameter of 0.5mm, respectively, and periodically sampled for particle size distribution analysis by laser diffraction. After twelve hours of milling, only poloxamer and polyvinylpyrrolidone suspensions were shown to produce a homogeneous nanoparticle dispersion, with lecithin suspensions showing no significant size reduction, polysorbate suspensions showing caking of the BC2059 in the milling vessel, and polyvinylpyrrolidone/sodium deoxycholate suspensions showing high aspect ratio crystals. The finished test suspension was allowed to stand under uncontrolled environmental conditions for four days for informal particle size stability. All suspensions tested showed a degree of particle growth with particle elongation similar to that initially observed in the polyvinylpyrrolidone/sodium deoxycholate suspension.

To try to prevent crystal growth, a preparation was prepared using poloxamer and polyvinylpyrrolidone (PVP) as dispersants, and adding sucrose, sorbitol and trehalose (10% each) at 5% (50mg/mL) of tegravine. Milling and storage were performed under conditions similar to those of the initial feasibility experiment. All formulations ground down to the nanosuspension, but there appeared to be no significant effect of the additive on the inhibition of crystal growth. For further work, poloxamer 188 was chosen as the primary dispersant.

Additional material was ground at 5% (50mg/mL) in poloxamer 188. To facilitate scale-up, the poloxamer content was increased from 1% to 1.5% to ensure a homogeneous nanosuspension. The milled nanosuspension was diluted to yield a 2% (20mg/mL) BC 2059/0.6% poloxamer/0.9% sodium chloride formulation for initial pharmacokinetic work by the third party. The remaining ground concentrated material was retained to test the effectiveness of lyophilization to prevent apparent crystal growth.

This formulation was then tested in the experiment described in example 5.

Example 5

Feasibility of lyophilization

This experiment should determine whether lyophilization is possible in principle. It is shown that in principle tegafur can be lyophilized.

Diluting the 5% (50mg/mL) aqueous suspension of texaverine poloxamer with various possible cryoprotectant-containing diluents to a final concentration of 2% (20mg/mL) texaverine, 0.6% poloxamer and the following:

sucrose (10%)

Mannitol (5%)

Sucrose (5%) and mannitol (2.5%)

Sorbitol (10%)

Sorbitol (5%) and mannitol (2.5%)

Trehalose (10%)

Trehalose (5%) and mannitol (2.5%)

A 5mL serum vial was filled to 2mL with each preparation and lyophilized at-40 ℃ and 100 millitorr pressure. The dried vials were resuspended in purified water and analyzed for particle size distribution. In the tested system, only the 10% sorbitol and 10% trehalose resuspension returned a particle size distribution comparable to the suspension before lyophilization. The additional nanosuspensions were milled to increase the component concentrations to 10% (100mg/mL) BC2059 and 3% poloxamer to increase milling efficiency and facilitate larger batch manufacturing.

The following suspensions were prepared from the ground material for low temperature Differential Scanning Calorimetry (DSC) analysis:

2% (20mg/mL) BC2059, with 0.6% polysorbate and 10% sorbitol

2% (20mg/mL) BC2059 with 0.6% polysorbate and 10% trehalose.

DSC analysis was performed at a rate of 1 ℃ per minute from 25 ℃ to-40 ℃ and then back to 25 ℃, resulting in the following glass transition values for the suspension:

sorbitol suspension: -18 ℃ C

Trehalose suspension: -33 ℃ C

The suspension was lyophilized, a 5mL vial was filled with 2mL, primary dried at-30 deg.C/150 mTorr, and secondary dried at-16 deg.C/550 mTorr. The informal lyophilized samples were shown to be physically stable with a reproducible uniform size distribution for ambient laboratory conditions. For subsequent work, sorbitol was chosen instead of trehalose because of the higher glass transition temperature and higher historical toxicity data availability.

The test batches were milled and lyophilized, with primary drying at-24 ℃/250 mtorr and secondary drying at-16 ℃/500 mtorr to provide materials for animal studies. Milling was performed at 20% (200mg/mL) tegravine with a poloxamer content of 5% and attempts to facilitate larger batch sizes and increase milling efficiency. The dried formulation was measured by Karl Fischer (Karl Fischer) at about 1% water and showed sufficient particle size stability when reconstituted with purified water for 24 hours.

These formulations were then tested in the experiment described in example 6.

Example 6

Non-clinical toxicology/pharmacokinetic batch production

The purpose of this experiment was to test a lyophilized formulation of tegravine.

Four suspension sub-batches, each representing 15g of tegafur, were prepared in sequence, milled at increasing loads, and extracted from the milling media using a diluent of 11.43% sorbitol aqueous solution to improve product yield and produce a suspension of 2% (20mg/mL) tegafur/0.5% poloxamer/10% sorbitol.

The sub-batches were filled at 2mL into 5mL vials and lyophilized under previously optimized conditions. Although some of the sample bottles showed signs of meltback, possibly due to increased batch size, the dried material was easily resuspended into a uniform nanosuspension. The intermediate measured PSD and water results for each sub-batch of vials showed acceptable batch-to-batch consistency, so four groups of vials were combined and processed as a single batch for stability and animal study use. See table 1 below.

TABLE 1

Sub-batches	Measurement (% LC)	D90(um)	Water (% w/w)
				1	97.3	0.19	1.9
2	103.1	0.18	1.5
				3	111.3	0.18	1.1
4	105.9	0.19	1.1

During milling, one of the sub-batches failed due to the generation of stable foam, which prevented further milling and produced permanent particle agglomeration. It was discarded and another batch made and replaced. In the production of failed batches, a 250mL serum bottle was used instead of the media bottle to facilitate PSD sampling during milling. Foaming is due to the difference in bottle size, allowing air to be trapped on the surface, resulting in batch failure. During extraction of all batches, dark insoluble particles were separated from the milling media. This material was later analyzed by XRPD and fusion aggregates of tegravine were found.

At the one month stabilization point, the composite batches showed a significant particle size increase due to aggregation of the poloxamer, rather than maturation or crystal growth of the API (drug substance). An attempt was made to determine a laboratory-adapted route by which the use of test items could be saved. The samples were reconstituted, resealed and heated to 50 ℃ for up to 3 hours without reducing aggregates. The resuspended vials were autoclaved at 121 ℃ for 10 minutes using a slow release liquid cycle and allowed to cool to ambient conditions, returning an acceptable particle size distribution.

Since there is no suitable forward route for this process, it is decided to reformulate the product.

Example 7

Reformulation of the composition and eventual failure of lyophilization, but liquid suspensions containing poloxamers appear promising

In this experiment, an additional reconstituted composition of tegafur was tested. Finally, lyophilization did not work, but liquid suspensions (nanosuspensions) showed promising results.

A viable scale batch was made in some of the initially tested dispersants, but with an increased 20% (200mg/mL) concentration of soda, this change could have made a viable dispersant, which at 5% (5mg/mL) has not shown promise. The following dispersants were tested with a 5% poloxamer 188 control:

polysorbate 20 (2%)

Polyvinylpyrrolidone (2%)

Polyvinylpyrrolidone (2%) and sodium deoxycholate (1%)

Polyvinylpyrrolidone (2%) and polysorbate 20 (2%)

Polyvinyl alcohol, partially hydrolyzed (5%)

After 12 hours of milling, the polysorbate ester preparation showed good homogeneity much faster than the control. The polyvinylpyrrolidone preparation showed the presence of non-crystalline particles, possibly aggregates or residues of polyvinylpyrrolidone, which did not significantly affect the particle size distribution measurement, but were visible by light microscopy. The polyvinyl alcohol preparation did not produce a significant size reduction, possibly due to the viscosity of the dispersant. The two-component polyvinylpyrrolidone preparation showed significant aggregates, but the polyvinylpyrrolidone/sodium deoxycholate preparation was determined to prove suitable for additional development.

Polyvinylpyrrolidone and polysorbate 20 preparations, as well as modified polyvinylpyrrolidone (1%) and sodium deoxycholate (0.5%) suspensions were used in lyophilization development experiments involving the following cryoprotectants:

sorbitol (10%)

Sucrose (10%)

Trehalose (10%)

Mannitol (5%

Mannitol (5%)

Sorbitol (5%) and mannitol (2.5%)

Sucrose (5%) and mannitol (2.5%)

Lyophilization was performed at-36 ℃/100 mtorr and-15 ℃/500 mtorr and was subjected to a-15 ℃ annealing step. Upon resuspension, only 10% of the sucrose preparation gave suitable particle size recovery. Although the additional suspension was milled using a 1% polyvinylpyrrolidone/0.5% sodium deoxycholate preparation, a citrate buffer was included to maintain the pH at about 7.0. Although the milled suspension preparation gelled reversibly on standing, it was combined with the following cryoprotectants:

sucrose (15%)

Sucrose at 25mg/mL BC2059 (10%)

Sorbitol (10%)

Lactose (5%)

Sucrose (5%) and sorbitol (5%)

The higher concentration of sucrose relative to the API was shown to provide the best particle size protection and although the formulation appeared to be susceptible to melt back, accelerated stability studies performed at 25 ℃/60% RH and 40 ℃/75RH showed that the formulation had good physical stability over 4 weeks.

However, in the dilution test, the formulation was found to flocculate in the saline diluent used for administration and the pharmacokinetic release of the polyvinylpyrrolidone/sodium deoxycholate formulation was significantly lower than that of the poloxamer formulation originally tested.

Nanosuspension containing poloxamer 188

200mg/mL (20%) of BC2059 was milled in poloxamer 188 and provided to a third party for lyophilization optimization. The suspensions were combined with a series of cryoprotectants listed in table 2 below and used in lyophilization experiments. Initially, the 2.5% dextran/2.5% sorbitol preparation showed the most promising particle size retention upon reconstitution, however, after one month at 40C/75% RH, the only preparation that retained the nanosuspension was the undried control.

Thus, these tests indicate that lyophilization does not work under the conditions evaluated. This finding also indicates that, surprisingly, the liquid suspension is more stable than previously observed. Initial particle elongation was determined to be an immediate and limited phenomenon, probably as a result of a small amount of re-precipitation occurring after stopping milling due to initial supersaturation of the dispersant. See table 2 below.

TABLE 2

Example 8

Feasibility of irradiation

Both formulation dispersant systems (polyvinylpyrrolidone/sodium deoxycholate and poloxamer) were developed to determine the feasibility of terminal sterilization by irradiation. Both samples were prepared for irradiation feasibility. Samples of both formulations were used in a parallel Pharmacokinetic (PK) study in laboratory animals, for which a diluent containing poloxamer 188 was also provided, which was used with the polyvinylpyrrolidone/sodium deoxycholate formulation to determine whether the bioavailability of the drug was correlated with poloxamer. The results of the PK studies show that bioavailability is unexpectedly related to poloxamer content.

Frozen vials carrying both formulations were irradiated. Gamma and e-beam irradiation were tested at 15 and 25 kGy. The vials were to be handled under refrigerated conditions and were worst case at 5 ℃, simulating possible thawing during irradiation. Degradation is independent of temperature, but appears to be dose dependent, regardless of the type of radiation.

However, primary particle size testing, supported by subsequent stability data, showed extensive particle aggregation. Aggregation was attributed to irradiation based on previously successful freeze/thaw testing; later freeze/thaw cycles on another suspension, however, showed similar aggregation.

It was determined that frozen storage resulted in unpredictable aggregation rather than continuing the GLP batch selection. The vials were stored in a-20C refrigerator prior to irradiation and may exhibit different freezing rates between vials at different locations on the shelf.

Example 9

Preclinical production

The production of 25mg/mL of the tegafur nanosuspension is done under optimal clean conditions, i.e. various control measures and precautions are taken to minimize microbial contamination, but sterility is not guaranteed.

The preparation is made using sterile water for injection to minimize not only microorganisms but also pyrogen contamination. All excipients were USP/NF grade. All product contact items were sterilized by autoclaving or, if not suitable for heating, by sterilization with 70% isopropanol. All exposed preparations were performed in an ISO 5 quality laminar flow hood using sterile handling techniques. The API used for the manufacture of preclinical batches was gamma-irradiated at 30kGy prior to use.

Example 10

Rat test article preparation

A 1,600 gram (nominal) batch of a suspension of tegafur was prepared for administration in rat toxicology studies.

Production was started with a 200 gram batch of concentrated (200mg/mL) BC2059 nanosuspension. 10g of poloxamer 188 was dissolved in 150g of water in a 250mL serum bottle. Add 200 grams of YTZ milling media and plug and seal the bottle. The entire composition and poloxamer solution preparation can be autoclaved at 121C for 15 minutes to minimize bioburden, allowing for small batch quantities. 40 grams of irradiated API (drug substance) was added and the bottle was likewise stoppered and sealed. This preparation was rolled on a roller mill so that the angle of rupture of the cascading media was visually determined to be about 45 degrees.

Due to the tendency of the formulation to fail due to entrapped air, it was noted that the amount of grinding media used was about half that typically used to process 200 grams of batch suspension. The bottles used are also smaller than typical bottles to minimize headspace. Grinding was allowed to occur over the weekend and the suspension was sampled through the septum via a hypodermic needle.

The D90 of the particle size distribution was 0.23 microns and was determined to be sufficient for extraction using an autoclaved solution of 160 grams sorbitol in 1240 grams of water and a glass pressure funnel containing a 60 micron sintered frit. The extracted suspensions were mixed and filled into autoclaved 10mL glass vials using a positive displacement pipette set at 5.00 mL. 295 vials were filled, stoppered and sealed, representing 92% yield. The batch was stored at 5 ℃ until use.

The nanosuspension appeared ready for administration to rats.

Example 11

Preparation of pig test article

A 10,400 gram (nominal) batch of a suspension of tegafur was prepared for administration in porcine toxicology studies. Production was started with a concentrated (200mg/mL) BC2059 nanosuspension of a 1,300 gram batch. 65g of poloxamer 188 was dissolved in 975g of water in a 2000mL media bottle. 1000 grams of YTZ milling media were rinsed and bagged for sterilization. The media and solution were autoclaved separately and combined with 260 grams of sterilized API in a media bottle. This preparation was rolled on a roller mill so that the angle of rupture of the cascading media was visually determined to be about 45 degrees. Milling was allowed to proceed for a total of about three days until the particle size distribution D90 was 0.33 microns and was determined to be sufficient for extraction. Two aliquots of the sorbitol solution prepared by dissolving 520g of sorbitol in 4030g of water were autoclaved and used to extract the milled suspension, similar to the treatment performed on the rat study batch.

Extraction is difficult because it is clear that unground or larger particle size API has plugged the 60 micron filter frit and therefore the media must be removed and the frit rinsed. The extracted suspensions were mixed and filled into autoclaved 10mL glass vials using a positive displacement pipette set at 10.0 mL. Fill, plug and seal 970 vials, representing 93% yield. The batch was stored at 5 ℃ until use.

The nanosuspension appears ready for administration to pigs.

Example 12

Autoclaving did not significantly affect degradation

Along with the production of test articles for preclinical studies, two batches of tegafur suspensions were prepared: one batch was made with sorbitol and the other without sorbitol. The stability evaluation of these suspensions stored at 5 ℃, 25 ℃/60% RH and 40 ℃/75% RH indicated that the suspensions were fairly stable under all conditions.

A portion of the vials from the porcine study batch were autoclaved at 121 ℃ for 20 minutes using the liquid circulation, and the formulations were designated as batch 515-76 and FID 5910.

The stability data indicate that autoclaving had no significant effect on degradation, but did exhibit increased particle size.

Example 13

Engineering of nanosuspensions

In the prediction of clinical manufacturing, nanosuspensions of tegafur of several engineering batches were prepared, and some changes to the process were necessary to maintain compliance and minimize losses and contamination. Partly for safety reasons, partly for extraction purposes, a large surface area filter was incorporated, replacing the glass pressure funnel originally used for extraction with a stainless steel in-line filter housing fitted with a 55um stainless steel filter element (Pall).

The extraction equipment previously using nitrogen was pressurized using a peristaltic pump, as this pump would also be incorporated into the process, allowing not only sterile filtration of the diluent and dispersant, but also autoclaving thereof. The vials were filled using a metering peristaltic pump unit. Given the tendency of autoclaving to increase the particle size of the formulation, the first engineering batch (RD4050-5) was submitted to gamma irradiation.

The stability results showed minimal degradation and stability similar to previous batches.

TABLE 3

Engineering batch of Tegaweiyin 25mg/mL

Based on preliminary particle testing, the treated formulation contained some larger particles that were not present enough to significantly affect the laser diffraction particle size measurement, but enough to significantly affect the USP <788> test. To reduce particulates, a "polished" filter is proposed that has a porosity small enough to retain larger particulates, but not small enough to affect the Tegaviet determination. However, previous attempts to filter the suspension resulted in significant assay loss. Accordingly, Pall Corporation (Pall Corporation) signed up to evaluate the suitability of certain membranes for tyveine nanosuspension filtration.

A portion of the non-optimal cleaning suspension was filtered using various 47mm membranes available from pall and a pressure feedback pumping system was used to determine how much material could be treated before the filter clogged and failed. The following membrane types were used, and the filtrate was tested for PSD and determination:

TABLE 4

A HDCII film of 6 microns was chosen as the best candidate because it showed no significant effect on the nanosuspension measurements or particle size distribution. However, the relatively long lead times of peltier filters are a limiting factor for the timely production of clinical material. Therefore, an alternative approach was sought where Sartorius 8 micron polypropylene filters were used to make two engineering batches for determining bioburden for sterilization verification purposes.

Disadvantageously, the measurements for both batches were negatively affected by filtration (80.9% LC and 91.9% LC, respectively). Since they do not represent the final clinical batch properties, the batch was discarded and another engineered batch was processed using a peltier HDC membrane filter. This batch was determined to be within 90% to 110% LC and a peltier HDC filter was incorporated into the last step of the manufacturing process before filling the vial.

Example 14

Tegaverine for administration of female Schopper-Doley (Sprague-Dawley) rats by slow intravenous bolus injection Pharmacokinetic study

The objective of this study was to study the pharmacokinetics of tegravine following a single intravenous slow bolus administration of tegravine in female schlagger-dori rats.

The study was performed using a parallel design (n-4/set) and consecutive sampling as summarized in table 5:

TABLE 5

The Stepogo-Dolly rats used in the source study were obtained from the indoor animal resources agency of Advinus Therapeutics Ltd. of Bangalore, India. On the day of dosing, these animals were about 10-11 weeks old.

Identification each animal was identified with a unique identification number indicated on the caged card and a turmeric solution tag on the animal. The cage cards identify each cage by study number, identification number, type and strain, dose and gender.

Housing and environment rats were acclimated to the conditions of the study area 3 days prior to dosing. Animals were housed in polypropylene cages (one per cage) and maintained under controlled environmental conditions with 12-hour cycles of light and 12-hour darkness. The temperature and humidity of the room are maintained between 22 + -3 deg.C and 40% -70%, respectively. The room was subjected to 10-15 fresh air change cycles per hour.

Food and water standard granular food (Teklad certified (2014C) 14% protein global rodent diet-rodent chow food, produced by Harlan Laboratories, inc, maraseweg 87C PO Box 553,5800, the netherlands).

Dose preparation and administration stock preparations (25mg/mL) were provided. Accurately 600. mu.L of the dosage formulation (stock, 25mg/mL) was transferred to a labeled glass container. 2.4mL of a 5% glucose solution was added thereto, vortex mixed and sonicated to obtain a homogeneous suspension with an intensity of 5 mg/mL. The animals are dosed under feeding conditions. Rats were administered a single dose of 10mg/kg of tegafur by slow intravenous bolus injection (over 1.5 minutes) of jugular catheter at a dose volume of 2mL/kg under the guidance of a 23G blunt needle using a 1mL BD syringe. The syringe used for administration was weighed before and after administration in order to calculate the actual administered dose.

Sample collection and processing blood samples were collected at 0.083, 0.25, 0.5, 1,2, 4, 6, 8,12 and 24h post-dose. At each time point, approximately 0.25mL of blood was withdrawn from the jugular vein of the cannulated rat and transferred to a labeled microcentrifuge tube containing 200mM K2EDTA (20 μ L per mL of blood). After sampling, an equal volume of heparinized saline was replaced into the catheter. Blood samples were kept on wet ice all the time immediately after collection and plasma was separated by centrifugation at 5000g for 5 minutes at 4 ± 2 ℃. Plasma samples were separated for 1h over the planned time and stored at below-60 ℃ until bioanalysis.

Bioanalysis was performed using a LC-MS/MS method suitable for the purpose for quantitative determination of BC2059 in rat plasma samples. The Calibration Curve (CC) of the method consists of a blank comprising at least 6 non-zero calibration standards and a blank with an internal standard sample, wherein the lower limit of quantitation (LLOQ) is 0.050 μ g/mL. Study samples were analyzed as well as three sets of quality control samples (9QC samples; low, medium and high QC samples were replicated three times).

Pharmacokinetic data analysis Using validatedNon-compartmental analysis of software (version 6.3)The tool (extravascular) calculates the pharmacokinetic parameters of tegravine. The area under the concentration time curve (AUCIast and AUCinf) was calculated by the linear trapezoidal rule. After intravenous bolus dose administration, CO was estimated by back-extrapolating the first two concentration values (back-extrapolated concentration at zero time). Total plasma Clearance (CL) and volume of distribution at steady state (Vss) are estimates. The elimination rate constant (k) is calculated by linear regression of the logarithmically linear end of the concentration-time curve, using at least 3 decreasing concentrations in the end phase, correlation coefficients>0.8. The terminal half-life value (T1/2) was calculated using the equation 0.693/k. The beta half-life was calculated and reported.

Results of the experiment

After a single slow intravenous bolus administration of Tegavidin (dose: 10mg/kg) to rats, the mean plasma Clearance (CL) was estimated to be 9.92mL/min/kg, which is about 5.5 times lower than the normal rat liver blood flow of 55 mL/min/kg. The mean plasma distribution volume at steady state (Vss) was found to be almost 9.34 times higher than normal body water 0.7L/kg, which might indicate that it is widely distributed in the tissue compartment. The semilog plasma concentration-time plot indicates that BC2059 exhibits a bi-exponential elimination pattern with a fast distribution half-life (T1/2. alpha.) of 0.546h and a long terminal plasma half-life (T1/2. beta.) of 13.8 hours.

TABLE 6

PK characterization of BC2059

Regression points 0.5, 1, and 2h for the alpha phase and 6, 8,12, and 24h for the terminal beta phase were selected to calculate the elimination rate constant.

Example 15

Dose escalation intravenous infusion study of tegafur in male Beagle Dogs (Beagle Dogs)

Study animals: xenometrics released four male non-naive beagle dogs on 20/4/2017 for study use. Animals were fed Harlan ad libitum throughout the studyGlobal 25% protein certified dog diet 2025C (except for a brief period of time during its lifetime when it is in

Administration: animals were administered via Intravenous (IV) infusion (via jugular vein) for 4 hours (h) [ ± 5 minutes (min) ].

TABLE 7

Overview of study dosing

Pharmacokinetic (PK) blood collection:

blood samples were taken on the last day of administration (dose 6; 15mg/kg) before the start of infusion and at 4, 12, 24, 36, 48 and 72h after the start of infusion. All blood samples were collected within 10 minutes of the target time and processed according to the protocol. The bioassay results indicated that tegafur was present in all plasma samples.

TABLE 8

Plasma BC-2059 values (ng/mL) calculated in beagle dogs after 15mg/kg 4h IV infusion

The pharmacokinetic parameters of BC-2059 were determined within 4 hours after the final 15mg/kg drug infusion. The average values are shown in table 8 above. The data indicated a half-life of 53.0h, and a total AUC0-120h of 73480ng x h/mL.

TABLE 9

Average PK parameter for BC-2059

Male (4) ═ n

Example 16

Spray delivery of tegafur formulations

The purpose of this experiment was to test the spray delivery of nanosuspensions. This experiment shows that spray delivery was successful.

Tegaviine particles suspended in poloxamer 188/sorbitol were used at a concentration of 25 mg/mL.

These formulations were applied to mice in the form of an aerosol by systemic exposure. The mice were placed in plastic boxes. This box is sealed and is connected on one side to the outlet of the sprayer unit and on the other side to the closed water system. The whole process is carried out in a fume hood of an animal house.

For the first experiment, a nebulizer kit of SATER LABS was used. The apparatus uses an air jet system. For 5 mice per group, the device was perfused with 5ml of drug (i.e., 125mg of tegafur (BC2059)) and then the device was connected to a power source for spraying. Energy is provided by a DeVilbiss compressor model 646, allowing 5-7 pounds of pressure, and 6-8 liters per minute of flow. For the second experiment, the device used was an Altera ultrasonic nebulizer.

For both experiments, 10 male bcat-Ex3 mice were used per group. These mice were divided into 2 groups of 5 mice each. The first group received the drug daily for 5 consecutive days. The second group received the drug only once (day five). On day 5, all mice were sacrificed, lungs were harvested, and samples were stored at-30 degrees in two labeled nylon bags, each containing 5 samples from each group.

As a result:

watch 10

"Aerosol" refers to a standard aerosol jet nebulizer (Safer Labs);

"nebulizer" refers to a nebulizer ultrasonic eRapid machine (Altera)

"day 1" means a single dose on day 5;

"5 days" means 5 daily doses on days 1-5.

Example 17

Porcine study with liquid formulations of tegafur and nanosuspension of tegafur

Poor tolerance of pigs to liquid suspensions

In a series of pharmacokinetic studies, tegafur was injected intravenously into mini-pigs to determine formulations suitable for GLP toxicology studies in terms of systemic exposure and tolerance to pharmaceutical product formulations. These studies were conducted at Sinclair Research (Sinclair Research) (Auxvasse, MO).

In the first study, the drug was obtained in a formulation consisting of tween 80, ethanol, polyethylene glycol (PEG) and vitamin E TGPS (d-alpha tocopheryl polyethylene glycol 1000 succinate). The stock preparation is 20%(phospholipid stabilized soybean oil) to final dose concentration. Two pigs were administered 1.7mg/kg over 6 hours and two pigs were administered 2.2mg/kg over 24 hours. The formulation provides good systemic exposure.

Comparing the dose normalization values for the shorter 6h duration with respect to the 24h duration results in a higher peak concentration (Cmax/dose) at the end of the infusion, since the total dose is given over a shorter duration. However, the overall systemic exposure time (AUCs/dose) between the two infusion durations was similar, which means that at longer infusion times it was possible to obtain similar overall systemic exposure while avoiding higher peak plasma concentrations.

However, despite the good systemic exposure observed with this formulation, a clear infusion reaction was observed and the mini-pigs were intolerant to this formulation during the 6h or 24h infusion. It is hypothesized that tween/ethanol/PEG/vitamin E/intralipid solvent based excipients and possible drug precipitation are responsible for the infusion reaction, not tegafur itself. Indeed, Tegaverine does not cause hemolysis of red blood cells, whether in nanoparticle form or dissolved in DMSO.

Lyophilized nanosuspensions of Tegaviine are better tolerated but are eventually abandoned due to stability problems

In subsequent studies, tegravine was milled to nanoparticle size and a non-solvent formulation was used. In this study, study B01-109, Tegaviine was obtained in lyophilized form and was reconstituted to provide a stock formulation consisting of a suspension of Tegaviine 10mg/mL, 2.5mg/mL poloxamer 188 and 5mg/mL sorbitol. This stock solution was diluted with physiological saline to the final desired concentration for intravenous administration. Within 4 hours of infusion, two pigs were infused at 2.9mg/kg and 2 pigs were infused at 12.3 mg/kg. One pig in the 12.1mg/kg dose group had a very high systemic exposure. Despite this pig, the dose-normalized AUC and, to a lesser extent, Cmax were linear within the given 2.8 to 12.1mg/kg dose over the same duration. With the exception of one pig in the 12.1mg/kg dose group, this lyophilized form of nanomilled tegafur had less standardized exposure than the tween/ethanol/PEG/vitamin E/lipid inner solvent based formulation used in study B01-107.

Nevertheless, because this formulation was well tolerated by mini-pigs, there was no infusion reaction observed in study B01-107, thus scaling up the lyophilization process for future work. However, in the scale-up method, we cannot obtain a lyophilized product with sufficient stability, and therefore an alternative lyophilized formulation to tegravine is required.

In the study of TXPK-006-2059-24h, lyophilized formulations of milled Tegaviine were used. The lyophilized formulation used was reconstituted in water to a final concentration of 25mg/mL for BC2059, 0.125% polyvinylpyrrolidone (PVP), 0.0625% sodium deoxycholate (NaDOC), and 10% sucrose. This bulk solution was diluted to the desired concentration in physiological saline for dosing to two pigs per treatment group at 12.3 or 49.2mg/kg over a 24 hour infusion. Flocculation of the test article was observed in the syringe and the syringe was agitated throughout the 24h infusion period. Nevertheless, systemic exposure was extremely low compared to studies B01-107 and B01-109. Subsequent formulation work showed that saline with this lyophilized formulation resulted in aggregation of the test article and no ionic (salt) diluent could be used.

Frozen liquid nanosuspensions of Tegaviine function

At this point, lyophilization was abandoned and the frozen liquid formulation of milled tegafur was investigated. In the study of TXPK-001-2059-porcine 24h PK, three mini-pigs were assigned to one of three groups, one per group, where the formulation was administered within 24 h. Particle Sciences provided two suspensions that were cryoground. BC2059-1 was 25mg/mL BC2059, 0.125% PVP, 0.0625% NaDOC, 10% sucrose suspension (batch No. 515-10), and BC2059-2 was 25mg/mL BC2059, 0.625% Poloxamer 188, 10% sorbitol suspension (batch No. 515-13). The diluent for both formulations was D5W. The third group was BC2059 PVP/NaDOC/sucrose freezer formulation BC2059-1 with poloxamer 188/saline diluent to study the possible effect of poloxamer 188 in systemic exposure.

Of these 3 frozen test article formulations, 25mg/mL of tegafur diluted with D5W, 0.625% poloxamer 188, 10% sorbitol nanosuspension showed the highest systemic exposure. The dose-normalized AUC for this formulation was slightly less than the dose-normalized exposure observed in the 24h infusion of study B01-107, but not significant. The dose normalized exposure was significantly higher than that observed in 3 of 4 pigs in study B01-109, indicating that the saline diluent in the study may have an effect on systemic exposure of the BC2059 lyophilized poloxamer 188 formulation, although to a much lesser extent than that observed with the PVP/NaDOC formulation of tegravine.

TABLE 11

Single dose pharmacokinetics of BC2059 in mini-pigs

^a20mg/mL BC2059 in 30% ethanol, 50% PG, 10% Tween 80 and 10% D- α -tocopheryl polyethylene glycol 1000 succinate (batch P492-01); report the values of 2 piglets

^b10mg/mL BC2059, 2.5mg/mL poloxamer 188 and 5mg/mL sorbitol (batch No. BET 1213-001-29); report the values of 2 piglets

^c25mg/mL BC2059, 0.125% PVP, 0.0625% NaDOC and 10% sucrose (batch No. BET 1213-; report the values of 2 piglets

^d25mg/mL BC2059, 0.125% PVP, 0.0625% NaDOC, 10% sucrose nanosuspension (batch 515-10) diluted to a final concentration of 2mg/mL in glucose 5%; single miniature pig

^e25mg/mL BC2059, 0.625% poloxamer 188, 10% sorbitol nanosuspension (batches 515-13) diluted to a final concentration of 2mg/mL in glucose 5%; single miniature pig

^f25mg/mL BC2059, 0.125% PVP, 0.0625% NaDOC, 10% sucrose, nanosuspension (batches 515-10) diluted to a final concentration of 2mg/mL in poloxamer 188/saline, a final concentration of 0.05% poloxamer; single miniature pig

Based on the results of these single dose infusion studies in mini-pigs, the formulation selected for the 2-dose toxicology study was a 25mg/mL BC2059 frozen formulation with 0.625% poloxamer 188, 10% sorbitol (nanosuspension) and diluted with D5W.

During dose selection in a 2-dose non-GLP study to support the IND-authorized GLP toxicology study, we learned the scale-up method and production of Particle Sciences lot No. 515-33, whose frozen formulation resulted in the accumulation of tegravine in vials.

In view of this aggregation under refrigeration, we subsequently decided to resort to 25mg/mL nanomilled BC2059 in 0.625% poloxamer 188 and 10% sorbitol formulations, maintained at 2 ℃ -4 ℃. No aggregation was observed in the milled BC2059 suspension in multiple batches, provided the formulation was not frozen. The poloxamer/sorbitol formulation frozen at 2-4 ℃ was used for IND-authorized GLP toxicology studies and non-GLP migluus studies.

Example 18

Efficacy of nebulized tegafur in mouse models of idiopathic pulmonary fibrosis

The objective of this experiment was to study the tegafur nanosuspensions in a bleomycin-induced Idiopathic Pulmonary Fibrosis (IPF) mouse model. The test articles were as follows:

tegavidine (BC2059)25mg/mL in a nano-milled suspension in 0.625% Poloxamer 188 and 10% sorbitol. The test article was frozen at about 4 ℃.

The spraying device was an Altera ultrasonic eRapid machine sprayer (model 678G 1002).

The animals were 8-12 week old C57BL/6 male mice (Jackson laboratory, Jackson Lab, Bar Harbor, ME, Maine).

Experimental procedures

TABLE 12

Mouse pulmonary fibrosis models were induced by Intratracheal (IT) injection of bleomycin (Schaumburg, IL) APP Pharmaceuticals). A dose of 0.025U bleomycin dissolved in 50 μ l saline 0.9% on day 0 was administered to each animal, or PBS was used as a control.

The tegravine nanosuspension was applied as an aerosol to group 3 by systemic exposure. The mice were placed in plastic boxes. This box is sealed and is connected on one side to the outlet of the sprayer unit and on the other side to the closed water system. The whole process is carried out in a fume hood of an animal house. In each treatment session, 5ml of 25mg/ml Tegavidine (125mg) was sprayed into 4-5 mice per group in the room within 15 min. To increase the exposure of the mice to the aerosol, the precipitated tegafur in the aerosol chamber was collected with a syringe and re-sprayed a second and third time. Mice were sprayed twice daily between day 5 and day 21 after bleomycin administration. Groups 1 and 2 received 5ml of spray vehicle in the same manner.

Animal body weights were recorded on days 0, 5, 8,12, 16, 19 and 21.

As previously described (Morales-Nebreda L et al, J. USA respiratory cells and molecular biology (AJRCMB) 2015), pulmonary mechanics measurements were performed on day 21 using a FlexiVent mouse ventilator (Montreal, PQ, Canada) Scireq, Quebec, Canada, Canadeq) according to protocols established by Scireq. A standard ventilation history for each mouse was obtained by three total lung volume procedures prior to forced oscillation and quasi-static pressure-volume curve protocols, which were used to calculate airway resistance, dynamic and quasi-static tissue compliance, and elasticity.

On day 21, all animals were sacrificed and lungs were harvested. Total lung collagen content was assessed using the hydroxyproline assay as previously described (Morales-Nebreda L et al, J. USA. respiring cell & molecular biology 2015). Briefly, mouse lungs were harvested and suspended in 1ml of 0.5M acetic acid and then homogenized, first using a tissue homogenizer (60 s on ice) and then using 15 strokes in a Dounce homogenizer (on ice). The resulting homogenate was spun (12,000 × g) for 10 minutes and the supernatant was used for subsequent analysis. Collagen standards were prepared using rat tail collagen (Sigma Aldrich, st. louis, MO) in 0.5M acetic acid. Sirius red dye was prepared by: 0.2g of sirius red F3B (sigma-aldrich) was mixed with 200ml of water; 1ml of sirius red dye was added to 100 μ l of collagen standard or lung homogenate and then mixed continuously for 30 minutes at room temperature on an orbital shaker. The precipitated collagen was then granulated and washed once with 0.5M acetic acid (12,000 × g, 15 minutes each). The resulting pellet was resuspended in 1ml of 0.5M NaOH and quantified spectrophotometrically (540nm) for sirius red staining using a colorimetric microplate reader (Bio-Rad, Hercules, Calif.).

Results

Group 2 showed statistically significant weight loss after bleomycin treatment, which is one of the indicators of IPF induction. In contrast, group 3 inhaled tergavir reversed weight loss due to bleomycin-induced lung injury treatment.

Watch 13

In addition, bleomycin-induced reduction in lung compliance in group 2 indicates induction of fibrosis. Inhalation of Tegaverine treatment after bleomycin injury in group 3 reversed compliance values to those close to sham treated controls in group 1.

TABLE 14

In addition, total lung collagen content as measured by hydroxyproline assay showed significant increase in group 2, indicating activation of fibrosis following bleomycin injury; in contrast, inhalation of tegafur treatment after bleomycin injury reversed this change in group 3 and collagen levels were close to those of sham-treated controls in group 1.

Watch 15

Thus, this experiment shows that tegravine has great potential for treating IPF.

Example 19

Evaluation of aerosolized Tegaviin formulations

A series of BC-2059 (tegravine) formulations were nebulized using a vibrating screen and a compressed air nebulizer to determine the most effective aerosol generation method. Aerosol concentration and particle size distribution were characterized for the aerosol in only the rodent nasal exposure chamber. Each formulation has different regulatory variables to assess the effect on aerosol performance. These include particle size reduction of the API, excipient characteristics and the nebulizer utilized.

The objective of this study was to identify a method of aerosolizing a test article BC-2059 for rodent inhalation studies.

Test article BC-2059 was suspended at a concentration of 15mg/mL in purified water of 0.1% Tween 80. The suspension was sonicated using a Covaris S220x sonicator (Covaris of Boston MA, massachusetts) and then mixed for one minute on a vortex. Sonication and vortex mixing were repeated a total of 15 times.

The remaining bulk of the BC-2059 powder was milled in a Planetary Ball Mill (Retsch, Germany) at 150RPM for 10 minutes using a 12mL Ball Mill jar and three metal balls. The milled BC-2059 powder was suspended at a concentration of 15mg/mL in purified water of 0.1% Tween 80. The suspension was sonicated using the procedure outlined above.

The remaining bulk of the BC-2059 powder was milled in a Planetary Ball Mill (Leichi, Germany) at 300RPM for 60 minutes using a 12mL Ball Mill pot and three metal balls. The milled BC-2059 powder was suspended at a concentration of 15mg/mL in purified water of 0.1% Tween 80 and 10% PEG 400. The suspension was sonicated using the procedure outlined above. An additional 15mg/mL BC-2059 suspension was prepared using 10% ethanol in purified water. The suspension was sonicated for 10 minutes using a VWR sonicator (VWR of Radnor PA, pennsylvania) and mixed for 4 minutes using a vortex mixer.

The additional formulation (nano-milled suspension of 25mg/mL BC-2059 in 0.625% poloxamer 188 and 10% sorbitol) was used as is without additional modification.

Aerosols were produced from a series of surfactant preparations using 4 separate nebulizers (Aeroneb Solo (Aerogen, Ireland)), Pari LC Plus (Pari Respiratory Equipment Inc., Loxon, Va.), Hospitak Up Mis, Hospitak Inc. Farmdale, N.Y.) and Hudson Micro-Mist (Research Triangle Park, NC, Teleflex Inc.) and transitioning to a 2 laminar flow past rodent exposure system.

The total concentration of aerosol in the exposed environment was determined by analysis of filter samples (GF/A47-mm filters). Filter samples were collected at a nominal flow rate of 0.3L/min. Filter samples collected throughout the study were gravimetrically analyzed to determine total aerosol concentration and submitted for HPLC analysis.

The filter with the test article was extracted with 1:1 acetonitrile: methanol and analyzed by HPLC-UV determination.

The Particle Size Distribution (PSD) of the test article was measured at the breathing zone using an In Tox, mercer cascade impactor.

Results

The aerosol concentrations (gravity and chemistry) are shown in table 16 below.

TABLE 16

Summary of Process development

The particle size of the test environment was measured for suspensions prepared from 0.1% tween 80 In ultrapure water using an In-Tox cascade impactor, and the sponsor provided the poloxamer suspension using a compressed air nebulizer. The mass median aerodynamic diameter and geometric standard deviation for each formulation are listed in table 17 below. The particle size distribution is shown in fig. 1 and 2.

TABLE 17

Particle size distribution

Conclusion

The formulation of BC-2059 was sprayed and introduced into the nasal inhalation only exposure chamber. The aerosol concentration of the exposure environment was characterized using weight and HPLC measurements. The highest gravimetric aerosol concentration of the poloxamer formulation was measured to be 2.47mg/L, which corresponds to 0.48mg/L of the activity test article. The particle size distribution of this formulation was measured with a cascade impactor and had an MMAD of 2.46 μm with a geometric standard deviation of 1.45 μm.

On reviewing the results of the poloxamer formulation versus previous results, a BC2059 aerosol concentration of 0.484mg/L would result in a lung deposition dose (10% DF) of 1.5mg/kg for 30 grams of mice over 30 minutes. This will result in 0.2mg/g in lung tissue based on standard mouse lung weight. Previous tests resulted in-0.02 mg/g (measured concentration).

Thus, this sprayed BC2059 nanomilled suspension resulted in the optimal concentration of aerosol compared to other BC2059 formulations.

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