Powdered Compositions Of Sensitive Active Materials In An At Least Partially Amorphous State

Abstract

The invention provides a powdered formulation which is a freeze-dried mixture of a sensitive active material and an excipient containing: 0.1-50% by wt of the sensitive active material, 50-99.99% by wt of the excipient, wherein at least 0.1% by wt of the mixture is an amorphous state; the formulation has a substantially reduced hygroscopicity.

Description

The invention is illustrated by way of example with reference to the Figure of the accompanying drawings which shows a graph showing the percentage increase in weight for freeze dried samples of mannitol and trehalose exposed to a 75% relative humidity environment.

The following Examples which illustrate the invention are not intended to limit the scope of the claims.

METHOD EXAMPLE 1

The following freeze-drying cycle has been used effectively for formulations having Tg′ or Tc at c. −16 to −18° C. This means that the product temperature should be maintained at c. -−23° C. (i.e. −18° C. plus 5° C. for operational safety=−23° C.).

Freeze to −445° C.

Cooling rate 0.25° C. per minute

Hold 120 minutes

Main Drying:

Shelf Temperature (step 1) −20° C.

Warming rate 1.0° C. per minute

Hold 1200 minutes

Chamber pressure 50 mTorr

Shelf Temperature (step 2) 0° C.

Warming rate 1.0° C. per minute

Hold 720 minutes

Chamber pressure 50 mTorr

Shelf Temperature (step 3) 5° C.

Warming rate 1.0° C. per minute

Hold 1000 minutes

Chamber pressure 50 mTorr

Final Drying

Shelf Temperature 15° C.

Warming rate 1.0° C. per minute

Hold 700 minutes

Chamber pressure 50 mTorr

EXAMPLES 2 AND 3

The activity of the enzyme lactic dehydrogenase after freeze drying was measured for formulations according to the invention and for a comparative formulation. Measurement of activity was carried out chemically by the reaction of the enzyme with its reactant, lactose.

The formulations set out below were prepared using the freeze drying cycle set out in Method Example 1:

Comparative Example 1

0.1 to 1.5 g enzyme

2.0 g Mannitol

Made up to 100 g water as start formulation before freeze-drying

Example 2

0.1 to 1.5 g enzyme

2.0 g Mannitol

1.0 g Lactose (amorphous after freeze drying)

Made up to 100 g water as start formulation before freeze-drying

Example 3

0.1 to 1.5 g enzyme

2.0 g Mannitol

1.0 g Glucose (amorphous after freeze drying) 2.0 g Dextran (mw 70,000 amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

It was found that the enzyme in Comparative Example 1 had 8% activity after the freeze drying process. In comparison the enzyme of Examples 2 and 3 according to the invention had an activity after lyophilisation of 60%.

EXAMPLES 4 TO 7

The activity of enzyme L-asparaginase (which is a known anticancer drug) after freeze drying was measured for formulations according to the invention and for a comparative formulation. Measurement of activity was carried out chemically by the reaction of the enzyme with its reactant, L-asparagine.

The formulations set out below were prepared using the freeze drying cycle set out in Method Example 1:

Comparative Example 2

0.1 to 1.5 g enzyme

2.0 g Mannitol

Made up to 100 g water as start formulation before freeze-drying

Example 4

0.1 to 1.5 g enzyme

2.0 g Mannitol

1.0 g Glucose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

Example 5

0.1 to 1.5 g enzyme

2.0 g Mannitol

1.0 g Lactose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

Example 6

0.1 to 1.5 g enzyme

2.0 g Mannitol

1.0 g Sucrose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

Example 7

0.1 to 1.5 g enzyme

2.0 g Mannitol

1.0 g Trehalose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

It was found that the enzyme in Comparative Example 2 had less than 1% activity after the freeze drying process. In comparison the enzyme of Examples 4 to 7 according to the invention had an activity after lyophilisation of 100%.

EXAMPLE 8

The activity of enzyme phenylalanine ammonia lyase (a known pharmaceutical agent) after freeze drying was measured for a formulation according to the invention and for a comparative formulation. Measurement of activity was carried out chemically by the reaction of the enzyme with its reactant, phenylalanine.

The formulations set out below were prepared using the freeze drying cycle set out in Method Example 1:

Comparative Example 3

0.1 to 1.0 g enzyme

1.0 g Mannitol

Made up to 100 g water as start formulation before freeze-drying

Example 8

0.1 to 1.5 g enzyme

2.0 g Mannitol

1.0 g Lactose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

It was found that the enzyme in Comparative Example 3 had less than 5% activity after the freeze drying process. In comparison the enzyme of Example 8 according to the invention had an activity after lyophilisation of 70%.

EXAMPLES 9 AND 10

The viability of Saccharomyces cerevissiae (live yeast for seed culture) after freeze drying was measured for formulations according to the invention and for a comparative formulation. Measurement of viability was by titres expressed as the number of colony forming units (cfu) per ml of fungal suspension as plated using solid agar plates.

The formulations set out below were prepared using the freeze drying cycle set out in Method Example 1:

Comparative Example 4

10⁸˜10¹² colony forming units Saccharomyces cerevissiae

Made up to 100 g water as start formulation before freeze-drying

Example 9

10⁸˜10¹² colony forming units Saccharomyces cerevissiae

10 g Mannitol

1.0 gm Trehalose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

Example 10

10⁸˜10¹² colony forming units Saccharomyces cerevissiae

10 g Mannitol

1.0 gm Glucose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

It was found that Saccharomyces cerevissiae in Comparative Example 4 had less than 1% viability after the freeze drying process. In comparison Saccharomyces cerevissiae of Examples 9 and 10 according to the invention had a viability after lyophilisation of 25%.

EXAMPLE 11 TO 15

The viability of Escherichia coli (live bacterium for seed culture) after freeze drying was measured for formulations according to the invention and for a comparative formulation. Measurement of viability was by titres expressed as the number of colony forming units (cfu) per ml of bacterial suspension as plated using solid agar plates.

The formulations set out below were prepared using the freeze drying cycle set out in Method Example 1:

Comparative Example 5

10⁶˜10¹² colony forming units Escherichia coli

1.0-5.0 g Mannitol

Made up to 100 g water as start formulation before freeze-drying

Example 11

10⁶˜10¹² colony forming units Escherichia coli

1.0-5.0 g Mannitol

1.0 g Trehalose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

Example 12

10⁶˜10¹² colony forming units Escherichia coli

1.0-5.0 g Mannitol

1.0 g Sucrose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

Example 13

10⁶˜10¹² colony forming units Escherichia coli

1.0-5.0 g Mannitol

1.0 g Glucose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

Example 14

10⁶˜10¹² colony forming units Escherichia coli

2.0 g Mannitol

1.0 g Maltose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

Example 15

10⁶˜10¹² colony forming units Escherichia coli

2.0 g Mannitol

1.0 g Lactose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

It was found that Escherichia coli in Comparative Example 5 had less than 1% viability after the freeze drying process. In comparison Escherichia coli of Examples 11 to 15 according to the invention had a viability after lyophilisation of 60%.

In Examples 11a, 12a, 13a, 14a and 15a, each formulation was prepared in a manner identical to Examples 11 to 15 except that 30 mM thiourea was included as a free radical scavenger. Shelf stability for these formulations after freeze drying was improved from 30 days (for Comparative Example 5) to 200 days (measured as the time to lose 1 log viability) as measured by titres expressed as the number of colony forming units (cfu) per ml of bacterial suspension as plated using solid agar plates.

EXAMPLE 16 TO 18

The viability of Salmonella typhimurium (live bacterium for diagnostic use) after freeze drying was measured for formulations according to the invention and for a comparative formulation. Measurement of viability was by titres expressed as the number of colony forming units (cfu) per ml of bacterial suspension as plated using solid agar plates.

The formulations set out below were prepared using the freeze drying cycle set out in Method Example 1:

Comparative Example 6

10⁶˜10¹¹ colony forming units Salmonella typhimuriumi

5.0 g Mannitol

Made up to 100 g water as start formulation before freeze-drying

Example 16

10⁶˜10¹¹ colony forming units Salmonella typhimuriumi

5.0 g Mannitol

1.0 g Sucrose (amorphous after freeze-drying

Made up to 100 g water as start formulation before freeze-drying

Example 17

10⁶˜10¹¹ colony forming units Salmonella typhimuriumi

5.0 g Mannitol

1.0 g Trehalose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

Example 18

10⁶˜10¹¹ colony forming units Salmonella typhimuriumi

5.0 g Mannitol

1.0 g Lactose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

It was found that Salmonella typhimurium in Comparative Example 6 had less than 1% viability after the freeze drying process. In comparison Salmonella typhimurium of Examples 16 to 18 according to the invention had a viability after lyophilisation of 40%.

EXAMPLE 19 TO 22

The viability of Lactobacillus acidophilus (live bacterium for silaging use) after freeze drying was measured for formulations according to the invention and for a comparative formulation. Measurement of viability was by titres expressed as the number of colony forming units (cfu) per ml of bacterial suspension as plated using solid agar plates.

The formulations set out below were prepared using the freeze drying cycle set out in Method Example 1:

Comparative Example 7

10⁶˜10¹² colony forming units Lactohacillus acidophilus

5.0 g Mannitol

Made up to 100 g water as start formulation before freeze-drying

Example 19

10⁶˜10¹² colony forming units Lactobacillus acidophilus

10.0 g Mannitol

10.0 g Foetal Calf Serum (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

Example 20

10⁶˜10¹² colony forming units Lactobacillus acidophilus

5.0 g Mannitol

1.0 g Trehalose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

Example 21

10⁶˜10¹² colony forming units Lactobacillus acidophilus

5.0 g Mannitol

1.0 g Lactose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

Example 22

10⁶˜10¹² colony forming units Lactobacillus acidophilus

5.0 g Mannitol

1.0 g Sucrose (amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

It was found that Lactohacillus acidophilus in Comparative Example 7 had less than 1% viability after the freeze drying process. In comparison Lactobacillus acidophilus of Example 19 according to the invention had a viability after lyophilisation of 60 and the Lactobacillus acidophilus of Examples 20 to 22 according to the invention had a viability after lyophilisation of 40%.

EXAMPLES 23 TO 26

The infectivity of influenza virus strain WSN (live, attenuated vaccine) after freeze drying was measured for formulations according to the invention and for a comparative formulation. Measurement of infectivity was expressed as plaque forming units (pfu per ml where 1 pfu=one lesion termed as ‘plaque’) in a chick embryo cell monolayer (defined as ‘sheet’) before and after freeze drying.

The formulations set out below were prepared using the freeze drying cycle set out in Method Example 1:

Comparative Example 8

10⁸˜10¹¹ plaque forming units Influenza Virus strain WSN

1.0 g Sodium Chloride

Made up to 100 g water as start formulation before freeze-drying

Example 23

10⁸˜10¹¹ plaque forming units Influenza Virus strain WSN

2.0 g Sodium Chloride

1.0 g Human Serum Albumin (amorphous after freeze-drying)

1.0 g Calcium Lactobionate

20 ml Chick Allantoic Fluid

Made up to 100 g water as start formulation before freeze-drying

Example 24

10⁸˜10¹¹ plaque forming units Influenza Virus strain WSN

1.0 g Sodium Chloride

1.0 g Lactose (amorphous after freeze-drying)

2.0 g Dextran (mw 110,000, amorphous after freeze-drying)

Made up to 100 g water as start formulation before freeze-drying

Example 25

10⁸˜10¹¹ plaque forming units Influenza Virus strain WSN

1.0 g Sodium Chloride

1.0 g Lactose (amorphous after freeze-drying)

1.0 g Sodium Monoglutamate (Maillard Reaction inhibitor)

Made up to 100 g water as start formulation before freeze-drying

Example 26

10⁸˜10¹¹ plaque forming units Influenza Virus strain WSN

1.0 g Sodium Chloride

1.0 g Lactose (amorphous after freeze-drying)

1.0 g Ascorbic Acid (Antioxidant)

Made up to 100 g water as start formulation before freeze-drying

It was found that the influenza virus strain WSN in Comparative Example 8 had less than 1% infectivity after freeze drying. In comparison influenza virus strain WSN of Example 23 according to the invention had an infectivity after lyophilisation of 70% infectivity and the influenza virus strain WSN of Example 24 according to the invention had an infectivity after lyophilisation of 40%. The shelf stability of Examples 24 and 25 was improved one from a log loss of infectivity in approximately 40 days to a one loss of infectivity in greater than 1000 days as measured as plaque forming units (pfu per ml where pfu=one lesion termed ‘plaque’) in a chick embryo cell monolayer (defined as ‘sheet’) before and after freeze-drying (1 log loss=arithmetic loss of 90%) compared to Comparative Example 8.

EXAMPLE 27

The activity of phage (p174 (phage for therapeutic or diagnostic use) after freeze drying was measured for formulations according to the invention and for a comparative formulation. Measurement of activity was expressed as plaque forming units (pfu per ml where 1 pfu=one lesion termed as ‘plaque’) in a Escherichia coli bacteria cell culture (defined as ‘sheet’) before and after freeze drying.

The formulations set out below were prepared using the freeze drying cycle set out in Method Example 1

Comparative Example 9

10¹⁰ plaque forming units phage (p174

0.9 g Sodium Chloride

Made up to 100 g water as start formulation before freeze drying

Example 27

10¹⁰ plaque forming units phage p174

0.9 g Sodium Chloride

0.1 g Serum Albumin

0.1 g Dextran Polymer

0.1 g Sucrose

Made up to 100 g water as start formulation before freeze drying

It was found that the phage p174 in Comparative Example 9 had less than 1% activity after freeze drying. In comparison phage p174 of example 27 according to the invention had an activity of 60%

EXAMPLE 28

Examples 1 to 27 above demonstrate that to maintain the biological activity/viability of a formulation containing a sensitive active material during lyophilisation, it is necessary to include a component that is either induced into or retains an amorphous state during freeze-drying. However, it is known that freeze-dried, amorphous materials will absorb moisture from its environment leading to deterioration in the physical properties and often leading to the amorphous matrix become ‘sticky’ and unsuitable for further processing (such as de-aggregation, mixing, milling, dispensing and/or packaging etc.). Absorption of moisture will also adversely affect product storage stability. It is for this reason that freeze dried formulations of sensitive active materials are usually freeze dried in vials or other containers that can be sealed in the freeze dryer prior to exposure to an ambient environment. In contrast, formulations retaining a crystalline nature during the freeze drying process, while being ineffective at maintaining properties such as biological activity, possess stable physical properties, do not absorb appreciable quantities of moisture and, therefore, are highly shelf stable.

This is demonstrated in the example below. The following four solutions containing trehalose (known to be amorphous in nature following freeze drying) and mannitol (known to retain its crystallinity during freeze drying) were prepared and freeze dried in vials.

COMPARATIVE EXAMPLE 10

Mannitol 200 mg
Water to 2 mL
Providing a 10% w/v mannitol solution

COMPARATIVE EXAMPLE 11

Mannitol 20 mg
Water to 2 mL
Providing a 1% w/v mannitol solution

COMPARATIVE EXAMPLE 12

Trehalose 200 mg
Water to  2 mL
Providing a 10% w/v trehalose solution

COMPARATIVE EXAMPLE 13

Trehalose 20 mg
Water to  2 mL
Providing a 1% w/v trehalose solution

Following lyophilisation the resultant freeze dried matrices were exposed to a 75% RH (Relative Humidity) atmosphere for a period of 8 hours. At intervals over this period, moisture uptake of each sample was measured gravimetrically and calculated as a percentage weight increase of the sample. It is clear from FIG. 1 that while there is significant moisture uptake in the amorphous (trehalose) samples, there is no appreciable moisture uptake in the crystalline (mannitol) samples. The moisture uptake in the amorphous samples was accompanied by a deterioration in the physical quality of these samples.

However, by utilisation of a formulation containing a sensitive active material that comprises excipients with a crystalline/amorphous character according to the invention, it is possible both to retain the biological activity/viability of the sensitive active material (as seen in previous examples) and achieve stable physical properties that do not appreciably take up moisture. It was surprisingly found that Examples 1 to 27 of the invention had a substantially reduced moisture take up such that the increase in weight was of each sample was less than 3% by weight after exposure to a high (75%) relative humidity environment for eight hours.

Claims

1. A powdered formulation which is a freeze-dried mixture of a sensitive active material and an excipient comprising: from 0.01 preferably from 0.1, more preferably from 0.5 to 50% by wt of the sensitive active material,from 50 to 99.99, preferably to 99.9, more preferably to 99.5% by wt of the excipient, wherein of at least 0.1% by wt of the mixture is in an amorphous state.

2. A formulation according to claim 1, of from 0.1, preferably from 0.5, more preferably from 1 to 50% by wt of the freeze-dried mixture in an amorphous state.

3. A formulation according to claim 1, comprising: from 0.01, preferably from 0.1, more preferably from 0.5 to 50% by wt of sensitive active material in an amorphous state,from 50 to 99.99, preferably to 99.9, more preferably to 99.5% by wt of excipient in crystalline state,0-5% by wt of excipient in an amorphous state.

4. A formulation according to claim 1, comprising: from 0.01, preferably from 0.1, more preferably from 0.5 to 50% by wt of sensitive active material in a crystalline state,from 50 to 99.89, preferably to 99.8, more preferably to 99.4% by wt of excipient in crystalline state, and0.1-5% by wt of excipient in an amorphous state.

5. A formulation according to claim 1, comprising: from 0.01, preferably from 0.1, more preferably from 0.5 to 25% by wt of an amorphous or a crystalline state of sensitive active material,from 75 to 99.49, preferably to 99.4, more preferably to 99% by wt of a crystalline state excipient, and0.5-5% by wt of excipient in an amorphous state.

6. A formulation according to claim 1 in which a saccharide is used to provide an excipient in an amorphous state.

7. A formulation according to claim 1 in which a sugar alcohol is used to provide an excipient in a crystalline state.

8. A formulation according to claim 1 wherein the formulation additionally comprises from 0.1 to 10% by wt (preferably from 1 to 10% by wt) of additive/stabilizer.

9. A formulation as defined in claim 8 wherein the additive/stabilizer is an antioxidant, a free radical scavenger and/or a Maillard reaction suppresser.

10. A formulation according to claim 1 wherein the sensitive active material is a labile organic and/or inorganic molecule, a biopolymer, a polypeptide, protein, enzyme, hormone, vitamin, antibiotic, polysaccharide, lipid, killed or live whole live cell.

11. A formulation according to claim 10 wherein the sensitive active material is a virus (including phage), bacterium, fungus and/or eukaryote.

12. A formulation according to claim 1 which has a stable crystalline/amorphous matrix.

13. A formulation according to claim 1 which has a substantially reduced hygroscopicity.

14. A formulation according to claim 1 which has a hygroscopicity of less than 5% by weight, preferably less than 3% by weight, more preferably less than 2% by weight, wherein the hygroscopicity is measured by the percentage increase in the weight of the formulation after 8 hours in a 75% relative humidity environment.

15. A dosage form comprising a formulation according to claim 1.

16. A dosage form according to claim 15 which is a container which comprises the formulation or an article which has been formed from the formulation.

17. A method of preparing a powdered formulation which comprises forming a mixed solution of sensitive active material and excipient(s) containing: from 0.01 preferably from 0.1, more preferably from 0.5 to 50% by wt of the sensitive active material,from 50 to 99.99, preferably to 99.9, more preferably to 99.5% by wt of the excipient, and freeze-drying the solution so that at least 0.1% by wt of the freeze-dried blend is in an amorphous state.

18. A method according to claim 17 in which the active material freeze dries to a crystalline state and the mixed solution contains: from 0.01, preferably from 0.1, more preferably from 0.5 to 50% by wt of sensitive active material in amorphous state,from 50 to 99.99, preferably to 99.9, more preferably to 99.5% by wt of excipient in crystalline state,0.1-5% by wt of excipient which freeze dries to an amorphous state.

19. A method according to claim 17 in which the active material freeze dries to an amorphous state and the mixed solution contains: from 0.01, preferably from 0.1, more preferably from 0.5 to 50% by wt of sensitive active material in crystalline state,from 50 to 99.89, preferably to 99.8, more preferably to 99.4% by wt of excipient in crystalline state,0-5% by wt of excipient which freeze dries to an amorphous state.

20. A method according to claim 17, in which the mixed solution contains: from 0.01, preferably from 0.1, more preferably from 0.5 to 25% by wt of amorphous or crystalline state of sensitive active material,from 75 to 99.49, preferably to 99.4, more preferably to 99% by wt of crystalline state excipient, 0.1-5% by wt of excipient which freeze dries to an amorphous state.

21. A method according to claim 17 in which a sugar is used to provide an excipient in amorphous state.

22. A method according to claim 17 in which a sugar alcohol is used to provide an excipient in crystalline state.

23. A method of medical treatment which method comprises supplying to a human or animal patient a therapeutically effective amount of a formulation according to claim 1.

24. A method of medical treatment which method comprises supplying to a human or animal patient a therapeutically effective amount of a dosage form according to claim 15.

25. A method of reducing the hygroscopicity of a freeze dried formulation which is a freeze-dried mixture of a sensitive active material and an excipient containing: from 0.01 preferably from 0.1, more preferably from 0.5 to 50% by wt of the sensitive active material, andfrom 50 to 99.99, preferably to 99.9, more preferably to 99.5% by wt of the excipient,wherein the method comprises the step of including in the formulation at least 0.1% by wt of a sensitive active material and/or an excipient in an amorphous state.