WO1998055106A1 - Method for preparing pharmaceutical formulation - Google Patents

Abstract

A process for making a homogeneous reconstituble pharmaceutical formulation comprising an anti-endotoxic agent, said anti-endotoxic agent having formula (1).

Description

METHOD FOR PREPARING PHARMACEUTICAL FORMULATION

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority pursuant to 35 U.S. C. §119 based upon Provisional Application Serial No. 60/047,628 filed May 23, 1997, the entire disclosure of which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to the preparation of pharmaceutical formulations, and in particular, lyophilized formulations that are reconstituted for injection.

BACKGROUND OF THE INVENTION

Christ et al., U.S. Patent No. 5,530,113, incorporated by reference herein, describes anti-endotoxic compounds that are useful for treatment of septic shock, and LPS-mediated activation of viral infection. For example, one such compound and has the following structure:

O

and includes pharmaceutically acceptable salts of the compound. This compound is referred to herein as Compound I.

In the commercial production of pharmaceutically acceptable formulations of agents for injection, the agents are often dispersed in an aqueous solution and, after addition of any further necessary components of the desired formulation, lyophilized. The lyophilized formulation is then reconstituted for use when it is needed for injection.

As can be seen in the structure shown, compounds of U.S. Patent No. 5,530,113 contain two sugar moieties attached to fatty acids. The compounds, when placed in alkaline aqueous solution, form into aggregates. Such aggregates cause difficulties in handling and processing, as they are prone to sticking to glassware and to each other. Also, aggregates present in the pharmaceutical formulations of the compounds may interfere with therapeutic effectiveness.

When subjected to the stirring that is conventionally used to form a dispersion of agents prior to lyophilization, the dispersion of the όompounds is not visibly turbid. Nevertheless, it has been determined by measurement of turbidity according to conventional means, that sufficient aggregates of the compounds are present following conventional dispersion that upon lyophilization, the product obtained is non- homogeneous, i.e. , aggregates of the compounds remain. The reconstituted injectable formulation that is made from the lyophilized product is also non-homogeneous, containing the same aggregates.

Extended stirring was found ineffective at eliminating aggregates. Similarly, raising the temperature did not effectively eliminate aggregates. Furthermore, each of these methods can cause substantial degradation of the compound without dispersing the aggregates. Sonication was also found to cause degradation of compounds when used for the period required to sufficiently eliminate aggregates, and in any event is not suitable for large scale production.

Thus, prior to the present invention, it was not known whether means existed for effectively breaking up aggregates of the compounds of U.S. Patent No. 5,530,113 without substantially degrading them. It was not known in particular whether this could be accomplished in a large scale industrial process for their manufacture.

It is therefore an object of the invention to provide a dispersion process for making an injectable formulation of the compounds described in U.S. Patent No. 5,530,113, and which substantially reduces turbidity resulting from aggregate formation.

It is also an object of the invention to provide a dispersion process that does not substantially degrade these compounds.

It is also an object of the invention to provide a process that is useful in the large scale manufacture of these compounds.

SUMMARY OF THE INVENTION

The present invention relates to a process for making homogeneous dispersions of an anti-endotoxic agent having the following formula:

wherein at least two of R¹, R², R³, and R⁴ are, independently:

wherein each L is O, N, or C; each M is O or N; each m, independently, is an integer between 0 and 14 inclusive; each n, independently, is an integer between 0 and 14 inclusive; each p, independently, is an integer between 0 and 10 inclusive; and each q, independently, is an integer between 0 and 10 inclusive; each x, independently, is an integer between 0 and 14; each y, independently, is an integer between 0 and 14 inclusive; and each z, independently, is an integer between 0 and 10 inclusive;

each of the remaining R¹, R², R³, and R⁴, independently, is:

wherein each L is O, N, or C; each M is O or N; each E, independently, is an integer between 0 and 14 inclusive; each m, independently, is an integer between 0 and 14 inclusive; each n, independently, is an integer between 0 and 14 inclusive, each p, independently, is an integer between 0 and 10 inclusive; and each q, independently, is an integer between 0 and 10 inclusive; each x, independently, is an integer between 0 and 14 inclusive; each y, independently, is an integer between 0 and 14 inclusive; each z, independently, is an integer between 0 and 10 inclusive; and each G, independently, is N, O, S, SO, or SO₂; A¹ and A² are

o

II

— O— P— OH, OH x is CH₂OCH₃, and Y is OH, or a pharmaceutically acceptable salt thereof. The dispersion is used to make a lyophilized product that is particularly suitable for reconstitution for injection. The process comprises

(a) mixing the anti-endotoxic agent in alkaline aqueous solution while heating to obtain a substantially homogeneous dispersion of the agent;

(b) combining the dispersion with any agents required to render the dispersion pharmaceutically acceptable for injection; and

(b) lyophilizing the formulation formed thereby to obtain a substantially homogeneous product suitable for reconstitution for injection.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph displaying results that show the turbidity of a Compound I dispersion in a 0.003 N NaOH solution, as a function of stirring time and heating temperature.

Figure 2 is an enlargement of part of the graph in Figure 1 showing the higher heating temperatures.

Figure 3 is a graph that displays results showing turbidity of a Compound I dispersion in alkaline solution at 45°C, as a function of stirring time and NaOH concentration.

Figure 4 is an enlargement of part of the graph shown in Figure 3 showing the change in turbidity for the NaOH concentration range between 0.001 and 0.01 N.

Figure 5 is a graph of results showing the turbidity of a Compound I dispersion in alkaline solution at 45°C as a function of pH and stirring time.

Figure 6 is an enlargement of the results in Figure 5 for the concentration range between 0.001 N and 0.01 N. Figure 7 is a graph of results showing turbidity of a Compound I dispersion in alkaline solution at 50° C as a function of stirring time and NaOH concentration.

Figure 8 is an enlargement of Figure 7 for the concentration range between 0.001 and 0.01 N.

Figure 9 is a graph demonstrating the decrease in turbidity during dispersion of two Compound I concentrations (2 mg/ml and 0.67 mg/ml) in alkaline solution.

Figure 10 is a graph showing the turbidity of Compound I dispersed in 0.003 N NaOH solution as a function of stirring time and temperature.

Figure 11 is a graph showing the turbidity of Compound I dispersed in alkaline solution at 50°C as a function of stirring time and NaOH concentration.

Figure 12 is a graph showing the turbidity of Compound I dispersed in alkaline solution at 50°C as a function of pH and stirring time (15 and 30 min).

Figure 13 is a flow chart diagramming the steps in one possible process for preparing a lyophilized product contaimng anti-endotoxic compounds while employing the process of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Any and all patents, patent applications, and publications listed in the present specification are hereby incorporated by reference.

The process of the invention is carried out with anti-endotoxic compounds included in the formula shown in the Summary of the Invention. The anti-endotoxic compounds used in the present process can be obtained following the synthetic steps described in U.S. Patent No. 5,530,113. The method of the invention has been found to avoid turbidity problems associated with conventional dispersion of these anti-endotoxic compounds, while at the same time avoiding degradation of the compounds. Specifically, it has been found that mixing and heating the compounds in alkaline solution at effective pH or normality, results in breaking up of aggregates without degrading the compounds.

The mixing step of the invention can be performed in any conventional mixing apparatus. Conventional batch mixers can be used. The preferred means of mixing is by stirring, which is carried out until a substantially homogeneous dispersion of the compound is attained, preferably, for more than about 5 minutes and less than about six hours, more preferably for more than about 30 minutes and less than about three hours, and most preferably for about one hour.

The dispersed product preferably exhibits a turbidity of less than about 1.0 NTU, more preferably less than about 0.5 NTU, and most preferably less than about 0.2 NTU. Measurement of turbidity is well known in this art.

Heating is performed at a temperature sufficient to cause substantial homogenization of the dispersion without substantial degradation of the product, preferably at a temperature greater than about 35 °C (e.g., between about 35 °C and 85 °C), more preferably between about 40 and 60 ^*C and most preferably between about 45 and 55° C, e.g. , about 50 °C.

Any suitable alkaline base can be used in preparation of the dispersion, including any pharmaceutically acceptable base capable of effecting the required pH. NaOH is preferred. The dispersion is preferably maintained at a pH greater than about 10, and more preferably also less than about 12. A pH of about 11 is most preferred. In an alternate embodiment, sufficient NaOH is added to attain a normality of between about 0.0005 and 0.1 N, preferably between about 0.001 and 0.01 N, more preferably between about 0.001 and 0.005 N, and most preferably about 0.003 N.

Lyophilization is well known in this art. Any conventional lyophilization apparatus can be used.

It is conventional, following the step of forming of a homogeneous dispersion of the pharmaceutical agent, and prior to lyophilization, to perform additional steps, and to add further ingredients to arrive at the lyophilizable pharmaceutical formulation. For example, following formation of the dispersion, it is preferred to mix the dispersion with one or more pharmaceutically acceptable carriers and pH adjusting agents. Such carriers and agents are well known in the art and are described, e.g. , in Gilman et al. (eds.), 1990, Goodman and Gilman 's: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press; Remington's Pharmaceutical Sciences, 17th ed., 1990, Mack Publishing Co., Easton, PA; Avis et al. (eds.), 1993; Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, New York; and Lieberman et al. (eds), 1990, Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York.

It is also understood that, following formation of the dispersion, and prior to lyophilization, it is conventional to perform a filtration step to remove any particles or impurities that are undesirable in the final product. Such filtration is well known in the art.

The present invention is illustrated by the following examples, which are intended merely to illustrate the invention and not to limit its scope. Examples

Procedures

Preparation of 0.3% NaOH solution

About 250 ml of water were placed in a 500 mL volumetric flask and 1.5 g of NaOH added. The NaOH was dissolved by shaking and the final volume adjusted to 500 mL by adding water.

Preparation of buffer solution

A stirrer bar was placed in a 1 L glass beaker and lOOg of lactose hydrous, 0.45 g of Na₂HPO₄-7H₂O and 0.35 g of NaH₂PO₄-H₂O were added to the beaker. 450 ml of water warmed to 40°C was added to the beaker and stirring started to dissolve the compounds. The beaker was placed into a circulating water bath warmed at 40°C and the dissolution of the compounds was visually confirmed. The beaker was then placed in a cooling bath and cooled to 20±5°C. The pH of the solution was checked and an appropriate amount of 0.3% NaOH solution added. The final volume was adjusted to 600 mL by adding water.

Preparation of Compound I dispersion

A stirring bar was placed into a 100 ml Erlenmeyer flask and 0.3% NaOH solution and 50 ml of water warmed at the temperature were added to the flask to obtain the pH and temperature values shown in the Figures. 60 mg of Compound I substance was added and the container was rinsed with water warmed at the temperatures specified in the Figures. The final weight was adjusted to 90 g. A nitrogen gas needle was placed in the mouth of the flask and the mouth covered with parafilm. The flask was placed in a circulating water bath at the temperatures indicated in the Figures. The temperature was maintained and the solution stirred under nitrogen gas purging. The disappearance of

Compound I gel was confirmed by visual inspection.

Samples were obtained at 5, 15, 30, 45, 60, 90, 120, 150 and 180 minutes after starting to stir and the turbidity and pH values determined, as shown in the Figures.

Preparation of formulated solution

7.5 ml of Compound I alkaline solution (Compound I: 0.67 mg/ml) were sampled. 30 ml of buffer solution were added and the final volume was adjusted to 50 ml by adding water. The solution was lyophilized.

If lyophilization was not conducted, 1 ml of alkaline solution was sampled and 4 ml of buffer solution and 1.67 ml of water added and then mixed with a touch mixer. The solution was filtered through a 0.22 μm filter (Millipore Millex GS).

Filtration. Filing and Lyophilization

The formulated solution was filtered though a 0.22 μm filter (Millipore

Millex GS). 53 ml of the filtered solution were placed into each vial and the vials are semi-plugged and lyophilized. The lyophilization conditions were as follows:

Freezing temperature: -40°C

Primary drying temperature: +20°C

Vacuum during primary drying: 0.075+0.025 mbar

Rising time of shelf temperature (-40°C to +20°C): 3 hours

Secondary drying temperature: +27°C

Vacuum during secondary drying: Best chamber vacuum

Rising time of shelf temperature (+20°C to +32°C): 1 hour

Secondary drying time: More than 18 hours

After lyophilization, the vial was reconstituted with 5 ml of water. Example 1 : Dispersion of a low Compound I concentration (0.67 mg/ml)

A dispersion of 0.67 mg/ml of Compound I was made according to the invention which had a turbidity of less than about 0.2 NTU.

Figure 1 is a graph showing the turbidity of a Compound I dispersion in a 0.003 N NaOH solution, as a function of stirring time and heating temperature. Turbidity decreased with heating time and the rate of decrease was faster when the heating temperature was higher. Figure 1 also shows that increasing base treatment and stirring by itself, i.e., without concomitant heating, was insufficient to minimize turbidity.

Figure 2 is an enlargement of part of the graph of Figure 1. As seen in Figure 2, the decrease in turbidity was especially drastic when the temperature was above 40°C. Turbidity decreased sharply during the initial period of stirring with heating before reaching approximately constant values (less than 0.2 NTU). When the temperature range was between 45 and 55°C, the rate of decrease in turbidity was approximately the same. Heating within a temperature range of 45 - 55°C, with the target temperature 50°C, in combination with stirring was extremely effective to disperse Compound I in NaOH solution.

Figure 3 is a graph of turbidity of a Compound I dispersion in alkaline solution at 45°C, and stirring time, as a function of NaOH concentration. Turbidity was

substantially reduced at 0.001-0.01 N NaOH. When NaOH concentration was 0.0001 N,

the turbidity increased.

Figure 4 shows the changes in turbidity for the NaOH concentration range 0.001 - 0.01 N. When the NaOH concentration was 0.01 N, the rate of decrease in

turbidity was lower as compared with the other three concentrations (0.001 N, 0.003N and 0.005 N). The rates of decrease in turbidity were approximately the same when the

NaOH concentration was between 0.001 and 0.005 N. This range of normality is especially effective.

Figure 5 is a graph showing the relationship between the turbidity of a Compound I dispersion in alkaline solution at 45°C and pH (i.e, NaOH concentration) as a function of stirring time. Figure 6 is an enlargement of the results for the concentration range between 0.001 N and 0.01 N. Figure 7 is a graph of results showing turbidity of a Compound I dispersion in alkaline solution at 50 °C, and stirring time, as a function of NaOH concentration. Figure 8 is an enlargement of Figure 7 for the concentration range 0.001 - 0.01 N. These figures indicate that a pH of about 11.3 (0.003 N NaOH) was most effective at decreasing the turbidity of the Compound I dispersion.

Figures 1 to 8 show that the method of the invention effectively decreases turbidity and that use of NaOH, particularly 0.003 N NaOH at 50+5°C, is surprisingly effective at dispersing Compound I.

The purities of Compound I formulations were checked by HPLC. HPLC purity did not substantially decrease during stirring in 0.003 N NaOH at 50±5°C for 3 hours and was above 99% at all times.

Example 2: Dispersion of 2 mg/ml Compound I

The Compound I concentration was increased 3 fold, from 0.67 mg/ml to 2 mg/ml. The steps required for dispersing Compound I were investigated using a protocol similar to that employed in Example 1.

Figure 9 is a graph demonstrating the decrease in turbidity during dispersion of two Compound I concentrations (2 mg/ml and 0.67 mg/ml) in alkaline solution. The dispersion of Compound I at a concentration of 0.67 mg/ml was conducted by diluting the 2 mg/ml solutions 3 fold with 0.003 N NaOH. Based on these results, it can be concluded that a higher concentration of Compound I (2 mg/ml) can be dispersed using the method of the invention. The higher concentration also allows easier observation of changes in turbidity.

Effective dispersing conditions for Compound I at a concentration of 2 mg/ml were investigated. Figure 10 shows the relationship between the turbidity of Compound I dispersed in 0.003 N NaOH solution and stirring time as a function of heating temperature. Turbidity decreased with heating time and the rate of decrease was faster when the heating temperature was high. When the temperature was between 45 and 55°C, the rate of decrease in turbidity was approximately the same. The turbidity reached a constant value of less than 0.2 NTU up until 60 minutes. When the stirring temperature was 40°C, the turbidity did not reach 0.2 NTU within 180 minutes. It is concluded that a temperature range between 45 and 55°C, with the target temperature being 50°C, is particularly effective for reducing turbidity. This is the same result as that obtained for the 0.67 mg/ml Compound I dispersion, described above.

Figure 11 shows the relationship between the turbidity of Compound I dispersed in alkaline solution at 50°C and stirring time as a function of NaOH concentration. When the NaOH concentration was 0.01 N, the rate of decrease in turbidity was lower than that for the other three concentrations (0.001, 0.003 and 0.005 N), When the NaOH concentration was between 0.001 N and 0.005 N, the turbidity values were less than 0.2 NTU after about 60 minutes; thus this concentration was particularly effective at reducing turbidity. Figure 12 illustrates the relationship between the turbidity of Compound I dispersed in alkaline solution at 50°C and pH (NaOH concentration) as a function of stirring time (15 and 30 min). When the NaOH concentration was 0.003 N (a pH of about 11.0), the rate of decrease in turbidity was the highest. This concentration was the most effective at decreasing turbidity.

The purities of the dispersed Compound I were checked using HPLC. No degradation of Compound I occurred after stirring of Compound I in 0.003 N NaOH at 50+5°C for 3 hours.

100 μg/ml and 300 μg/ml formulations were made of dispersed Compound I, lyophilized, and reconstituted. When the turbidity of the alkaline solution (Compound I: 2 mg/ml) was about 0.2 NTU, the turbidities of the 100 μg/ml and 300 μg/ml reconstituted formulated solutions were also approximately 0.2 NTU, i.e. , they were not significantly different before and after lyophilization. Example 3: Process of manufacture

An example of a procedure for manufacturing Compound I is schematically shown in Figure 13. It shows a process in which 2 mg/ml of Compound I is dispersed in 0.003 N NaOH at 50°C and mixed with phosphate buffer containing lactose, formulated to 100 μg/ml, filled into vials (530 μg/vial) and lyophilized. "WFI" indicates USP water for injection.

An example of a lyophilizable formulation containing 500 μg Compound I follows.

* μg as ompoun ree ac .

** Water is driven off during lyophilization.

Claims

WE CLAIM:

1. A process for making a homogeneous reconstitutable pharmaceutical formulation comprising an anti-endotoxic agent, said anti-endotoxic agent having the following formula

wherein at least two of R¹, R², R\ and R⁴ are, independently:

O (CHjOyCHs X.

(CH₂)J3l , (CH₂)_z4.╬╣ (CH₂) CH3

wherein each L is O, N, or C; each M is O or N; each m, independently, is an integer between 0 and 14 inclusive; each n, independently, is an integer between 0 and 14 inclusive; each p, independently, is an integer between 0 and 10 inclusive; and each q, independently, is an integer between 0 and 10 inclusive; each x, independently, is an integer between 0 and 14; each y, independently, is an integer between 0 and 14 inclusive; and each z, independently, is an integer between 0 and 10 inclusive;

each of the remaining R¹, R², R³, and R⁴, independently, is:

(CΑΪhPXk.

O

«

wherein each L is O, N, or C; each M is O or N; each E, independently, is an integer between 0 and 14 inclusive; each m, independently, is an integer between 0 and 14 inclusive; each n, independently, is an integer between 0 and 14 inclusive, each p, independently, is an integer between 0 and 10 inclusive; and each q, independently, is an integer between 0 and 10 inclusive; each x, independently, is an integer between 0 and 14 inclusive; each y, independently, is an integer between 0 and 14 inclusive; each z, independently, is an integer between 0 and 10 inclusive; and each G, independently, is N,

O, S, SO, or SO₂;

A¹ and A² are

o

II

OΓÇö PΓÇö OH, OH

x is CH₂OCH₃, and Y is OH, or a pharmaceutically acceptable salt thereof said process comprising:

(a) mixing said agent in alkaline aqueous solution while heating to obtain a substantially homogeneous dispersion;

(b) combining said dispersion with any agents required to render said dispersion pharmaceutically acceptable for injection; and

(c) lyophilizing to obtain a substantially homogeneous product suitable for reconstitution for injection.

2. The method of claim 1 wherein said alkaline solution comprises

NaOH in a normality of between about 0.0005 and 0.1 N.

3. The method of claim 1 wherein said alkaline solution comprises NaOH in a normality of between about 0.001 and 0.01 N.

4. The method of claim 1 wherein said alkaline solution comprises NaOH in a normality of between about 0.001 and 0.005 N.

5. A process for making a homogeneous reconstitutable pharmaceutical formulation comprising an anti-endotoxic agent, said anti-endotoxic agent having the following formula

and pharmaceutically acceptable salts thereof, comprising:

(a) stirring said agent in alkaline aqueous solution at a pH greater than about 10 and a temperature greater than about 35 ┬░C to obtain a dispersion; (b) combining said dispersion with any agents required to render said dispersion pharmaceutically acceptable for injection; and

(c) lyophilizing to obtain a substantially homogeneous lyophilized product.

6. The method of claim 5 further wherein, after step (a) and before step (c), said solution is filtered.

7. The method of claim 5 wherein, in step (b), said dispersion is mixed with a pharmaceutically acceptable carrier.

8. The method of claim 5 wherein, in step (b), said dispersion is combined with a pharmaceutically acceptable excipient, a pH adjuster, and water.

9. The method of claim 7 wherein, in step (b), said dispersion is mixed with phosphate pH adjuster, water, and lactose.

10. The method of claim 5 wherein, in step (a), said pH is less than about 12.

11. The method of claim 5 wherein said pH is about 11.

12. The method of claim 5 wherein said temperature is between about 40 and 60┬░C.

13. The method of claim 5 wherein said temperature is between about

45 and 55 ┬░C.

14. The method of claim 5 wherein said alkaline solution comprises NaOH.

15. The method of claim 5 wherein, following step (a), said dispersion exhibits a turbidity of less than about 1.0 NTU.

16. The method of claim 5 wherein, following step (a), said dispersion exhibits a turbidity of less than about 0.5 NTU.

17. The method of claim 5 wherein, following step (a), said dispersion exhibits a turbidity of less than about 0.2 NTU.

18. The method of claim 5 wherein said dispersion, in step (a), is stirred for between about 5 minutes and 6 hours.

19. The method of claim 5 wherein said dispersion, in step (a), is stirred for between about 30 minutes and 3 hours.

20. The method of claim 21 wherein said solution is stirred for about 1 hour.

21. A process for making a homogeneous reconstitutable pharmaceutical formulation comprising a pharmaceutically acceptable salt of an anti-endotoxic agent, said anti-endotoxic agent having the following formula

comprising:

(a) stirring said agent for more than about 1 hour in alkaline aqueous solution at between about 0.003 N and at a temperature of between about 50 ┬░C; (b) combining said dispersion with agents to render said dispersion pharmaceutically acceptable for injection; and

(c) lyophilizing to obtain a substantially homogeneous product suitable for reconstitution for injection.