Stable lipase formulation and method thereof

Abstract

Lipase compositions and formulations, as well as methods of treatment and manufacture, are disclosed.

Description

[Technical field]

[0001] The present invention relates to stable lipase compositions and dosage forms, methods of treatment, and methods of preparation. [Background technology]

[0002] Lipase 2 (LIP2) from Yarrowia lipolytica (i.e., adlulipase) is an autologous yeast recombinant lipase. The targeted indication of adlulipase is the compensation of exocrine pancreatic insufficiency (EPI) resulting from cystic fibrosis, chronic pancreatitis, and other indications involving the exocrine pancreas. The symptomatology of EPI is essentially due to pancreatic lipase deficiency, an enzyme that hydrolyzes triglycerides into monoglycerides and free fatty acids.

[0003] Chronic pancreatitis (CP), the most common cause of EPI, is a prolonged inflammation of the pancreas that alters its normal structure and function, which is associated with EPI in approximately 60% of patients. Cystic fibrosis (CF), another frequent etiology of EPI, is a severe genetic disease associated with chronic morbidity and reduced life span in most affected individuals. Approximately 80-90% of patients with CF develop EPI. In addition, EPI is common after surgical resection of the pancreas, usually as a result of cancer or complications of CP. Other less common etiologies of EPI include gastric surgery, certain intestinal diseases (e.g., severe celiac disease, small bowel resection, and artificial enteral feeding), and pancreatic diseases (e.g., pancreatic trauma, severe acute pancreatitis with pancreatic necrosis, and pancreatic cancer).

[0004] The cost of EPI, classically dependent on porcine pancreatic extract (PPE, also referred to as porcine pancreatic replacement therapy (PERT)), has been of concern to the Food and Drug Administration (FDA) due to the animal components used in the preparation of PPE and the associated risk of transmission of conventional and non-conventional infectious agents. Furthermore, the dose of PPE that can be given may be limited, especially in CF, due to the risk of fibrotic colon disease possibly associated with the presence of proteases and / or gastroprotectants.

[0005] In general, lipases have many pharmaceutical and industrial applications.

[0006] Thus, there continues to be a need in the art for pharmaceutical and industrial compositions that include lipases, such as adole lipase, for the treatment of EPI. Summary of the Invention

[0007] In certain embodiments, the present invention is directed to temperature stable lipase dosage forms, methods of treatment, and methods of manufacture.

[0008] In certain embodiments, the present invention is directed to temperature stable lipase industrial compositions, methods of use, and methods of production.

[0009] In certain embodiments, the present invention is directed to a composition comprising: (a) a lipase; and (b) one or more excipients, wherein after exposure to an environment of about 37° C. for 15 minutes, the composition exhibits an activity (nmoles / min / mg) of at least 125.

[0010] In certain embodiments, the present invention is directed to processes for making the compositions and dosage forms disclosed herein.

[0011] In certain embodiments, the present invention is directed to a method of treating exocrine pancreatic insufficiency comprising administering a dosage form disclosed herein. In certain embodiments, the insufficiency may be caused by one or more of acute or chronic pancreatitis, cystic fibrosis, pancreatic resection (whether or not associated with cancer, such as pancreatic cancer), age-related, Shwachman-Diamond syndrome, type 1 diabetes, type 2 diabetes, HIV, celiac disease, or inflammatory bowel disease (such as ulcerative colitis or Crohn's disease).

[0012] The present invention is also directed, in certain embodiments, to methods of treating a condition (e.g., a colon disease or other condition treatable by targeted administration of an active agent to the colon) by administering to a subject any of the compositions disclosed herein. Delivery can be to treat a colon disease or condition, and can also be used to treat a systemic condition with a drug that is suitable for absorption in the colon.

[0013] The present invention is also directed, in certain embodiments, to a method of delivering an active agent to the colon of a patient by orally administering a formulation disclosed herein, in which at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the active agent is delivered to the colon of the patient. [Brief description of the drawings]

[0014] [Figure 1] 1 shows the effect of temperature on adole lipase activity. [Diagram 2] 1 shows the effect of temperature on adole lipase structure. [Diagram 3] 1 shows the effect of temperature on adul lipase activity in various formulations. [Figure 4] FIG. 1 shows the effect of temperature on pancrelipase (Creon®) activity. [Diagram 5] 1 shows the effect of temperature on porcine lipase activity. [Figure 6] FIG. 2 shows a schematic diagram of the process and particle size data of Example 1. [Figure 7] FIG. 2 shows a schematic diagram of the process and particle size data of Example 1. [Figure 8] The activity of the granulated and non-granulated formulations of Example 1 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention advances the state of the art by developing stable lipase compositions, pharmaceutical compositions, methods of treatment, methods of use, and methods of preparation.

[0016] It has been discovered by the present invention that lipase compositions (e.g., adorlipase, pancrelipase, porcine lipase, PERT) exhibit a loss of activity over time due to temperature. As described in the Examples, it was found that there is a loss of activity at 37°C (physiological temperature) with less significant loss or complete retention of activity at 25°C (room temperature). pH and buffers do not appear to significantly affect this loss. This is important in lipase pharmaceutical therapy because the active agent is rapidly degraded upon introduction to physiological temperatures after administration (e.g., into the gastrointestinal tract).

[0017] In certain embodiments, the present invention is directed to a pharmaceutical composition comprising (a) an adole lipase and (b) one or more stable pharma- ceutically acceptable excipients, such that, e.g., after exposure to an environment of about 37° C. for 15 minutes, the composition exhibits an activity (nmoles / min / mg) of at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, or at least 160, e.g., as measured by the assay of Example 1.

[0018] In certain embodiments, after exposure to an environment of about 37° C. for 30 minutes, the adul lipase composition exhibits an activity (nmoles / min / mg) of at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, or at least 140, as measured, for example, by the assay of Example 1.

[0019] In certain embodiments, after exposure to an environment of about 37° C. for 45 minutes, the adole lipase composition exhibits an activity (nmoles / min / mg) of at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, or at least 155, as measured, for example, by the assay of Example 1.

[0020] In certain embodiments, after exposure to an environment of about 37° C. for 90 minutes, the adole lipase composition exhibits an activity (nmoles / min / mg) of at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, or at least 130, as measured, for example, by the assay of Example 1.

[0021] In certain embodiments, the present invention is directed to pharmaceutical compositions comprising: (a) a lipase (e.g., an adole lipase); and (b) one or more stable pharma- ceutically acceptable excipients, such that, e.g., after exposure to an environment at about 37° C. for 90 minutes, the composition exhibits an activity (nmoles / min / mg) that is decreased by no more than 50%, no more than 40%, no more than 35%, no more than 25%, no more than 20%, no more than 10%, or no more than 5%, as measured, e.g., by the assay of Example 1.

[0022] In certain embodiments, the exposing is in vivo. In other embodiments, the exposing is in vitro.

[0023] In certain embodiments, the exposure is following oral administration to a human subject.

[0024] In certain embodiments, the lipase and excipients are spray dried.

[0025] In certain embodiments, the lipase and excipients are granulated and / or extruded. Granulation can be, for example, wet granulation or dry granulation. Dry granulation is the process of forming granules or granules from powdered or solid substances to produce a granular material. Typically, granulation involves the agglomeration of fine particles into larger granules, e.g., in the size range of 0.01-3.0 mm (D50). Agglomeration processes can include, for example, the use of a blend, application of pressure to compress the blend (roller or tablet / slug compression), shredding or grinding the solid materials into fine granules or pellets, and sieving. Wet granulation is very similar, but can include the incorporation of a solvent in the mixing process to produce the agglomerates, and a drying method to remove the solvent once the granules are formed.

[0026] In certain embodiments, the lipase and excipients are compressed.

[0027] In certain embodiments, the lipase and excipients are spray dried, granulated, and / or extruded.

[0028] In certain embodiments, the lipase and excipients are spray dried and compressed.

[0029] In certain embodiments, the lipase and excipients are granulated and / or extruded and compressed.

[0030] In certain embodiments, the lipase and excipients are spray dried, granulated, and / or extruded and compressed.

[0031] In certain embodiments, the lipase is a non-porcine lipase.

[0032] In certain embodiments, the lipase is porcine lipase.

[0033] In certain embodiments, the non-porcine lipase is a triacylglycerol hydrolase.

[0034] In certain embodiments, the non-porcine lipase has a molecular weight of about 30 kDa to about 45 kDa.

[0035] In certain embodiments, the non-porcine lipase has a molecular weight of about 36-40 kDa, or about 36 kDa, about 37 kDa, about 39 kDa or about 40 kDa.

[0036] In certain embodiments, the non-porcine lipase comprises from about 295 to about 310 amino acids.

[0037] In certain embodiments, the non-porcine lipase comprises about 301 amino acids.

[0038] In certain embodiments, the non-porcine lipase is produced from Yarrowia lipolytica.

[0039] In certain embodiments, the non-porcine lipase is encoded by the LIP2 gene.

[0040] In certain embodiments, the non-porcine lipase is an adole lipase.

[0041] In certain embodiments, the porcine lipase is pancrelipase.

[0042] In certain embodiments, the excipient is an oligosaccharide.

[0043] In certain embodiments, spray drying forms particles having a D50 of about 1 micron to about 200 microns, about 1 micron to about 50 microns, 50 microns to about 150 microns, about 60 microns to about 120 microns, about 65 microns to about 85 microns, or about 70 microns to about 82 microns.

[0044] In certain embodiments, the excipient comprises maltodextrin, xylan, mannan, fucoidan, galactomannan, chitosan, raffinose, stachyose, pectin, inulin, levan, graminan, and amylopectin, sucrose, lactulose, lactose, maltose, trehalose, cellobiose, nigerotriose, maltotriose, melezitose, maltotriulose, raffinose, kestose, or mixtures thereof.

[0045] In certain embodiments, the ratio of lipase to one or more excipients is about 1:5 to about 5:1, about 1:3 to about 3:1, about 1:2 to about 2:1, about 1:1, or about 1:2.

[0046] In certain embodiments, spray drying is carried out at a pH of about 3 to about 5, about 2 to about 7, or about 6.

[0047] In certain embodiments, spray drying is carried out at an inlet temperature above about 125°C, or from about 100°C to about 250°C, or from about 150°C to about 180°C, or from about 155°C to about 165°C, or about 162°C, and / or an outlet temperature below about 150°C, or from about 50°C to about 125°C, or from about 60°C to about 100°C.

[0048] In certain embodiments, spray drying produces lipase in a yield of greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 99%.

[0049] In certain embodiments, the composition includes an enteric material.

[0050] In certain embodiments, the present invention is directed to a method of treating exocrine pancreatic insufficiency comprising administering a pharmaceutical composition disclosed herein.

[0051] In certain embodiments, the present invention is directed to a method of treating acute or chronic pancreatitis comprising administering a pharmaceutical composition disclosed herein.

[0052] In certain embodiments, the present invention is directed to a method of treating cystic fibrosis comprising administering a pharmaceutical composition disclosed herein.

[0053] In certain embodiments, the present invention is directed to a method of increasing lipase effectiveness comprising administering a pharmaceutical composition disclosed herein.

[0054] In certain embodiments, the failure is caused by pancreatic resection, such as due to pancreatic cancer.

[0055] In certain embodiments, the deficiency is age-related or due to Shwachman-Diamond syndrome, type 1 diabetes, type 2 diabetes, HIV, celiac disease, or inflammatory bowel disease.

[0056] In certain embodiments, the deficiency results from ulcerative colitis or Crohn's disease.

[0057] In certain embodiments, at least a portion of the lipase is delivered to the duodenum of the patient.

[0058] Certain embodiments of the present invention are directed to a process for preparing a pharmaceutical composition comprising combining a lipase and a pharma- ceutically acceptable excipient to form a composition disclosed herein.

[0059] Other embodiments In certain embodiments, the formulations disclosed herein are delayed or immediate or sustained release oral dosage forms that include a lipase, such as a non-porcine lipase. In alternative embodiments, the dosage form includes an enteric material that includes or is dispersed with the lipase.

[0060] In certain embodiments, the present invention is directed to a spray-dried lipase as disclosed herein.

[0061] In certain embodiments, the formulations disclosed herein include a second agent, which may be an additional lipase or active agent. The second agent may be selected from fat-soluble vitamins, proteases, amylases, porcine pancreatic enzyme supplements, other non-porcine supplements, or combinations thereof. In certain embodiments, the vitamins are A, D, E, K, or combinations thereof. In other embodiments, the second active agent is pancrelipase, lipotamase, or combinations thereof.

[0062] In certain embodiments, the lipase and the second active agent are each independently immediate release, delayed release, sustained release, or a combination thereof.

[0063] In certain embodiments, the dosage forms disclosed herein are contained in a capsule, which optionally includes an enteric material, e.g., coated on or dispersed within the capsule, hi other embodiments, the enteric material is spray dried with the active agent, or the spray dried active agent is mixed with the enteric material.

[0064] In certain embodiments, the dosage form comprises adullipase in an amount of about 0.5 g per day to about 10 g per day, about 2 g per day to about 5 g per day, or about 2 g per day to about 4 g per day. In certain embodiments, the dosage is about 1.6 g per day, about 2.2 g per day, or about 4.4 g per day.

[0065] In certain embodiments, the dosage forms disclosed herein include tablets that optionally include an enteric material, for example, coated on or dispersed within the tablet.

[0066] In certain embodiments, the lipase (e.g., adullipase) may be in the form of a powder, optionally including an enteric coating material, for example, by dry blending, wet granulation, or co-spray drying or co-lyophilization.

[0067] In certain embodiments, the formulation is a powder or particles and is contained in a capsule, sachet, or powder paper.

[0068] In certain embodiments, the enteric material comprises a naturally occurring or non-naturally occurring material.

[0069] In certain embodiments, the enteric material comprises a cellulosic material, an acrylic polymer, or a combination thereof.

[0070] In certain embodiments, the enteric material comprises hydroxypropyl methylcellulose acetate succinate.

[0071] In certain embodiments, the enteric material comprises a methacrylic acid polymer, a cellulose acetate phthalate polymer, a hydroxypropyl methylcellulose acetate succinate polymer, a hydroxypropyl methylcellulose phthalate polymer, a polyvinyl acetate phthalate polymer, or a combination thereof.

[0072] In certain embodiments, the enteric material comprises methyl acrylate-methacrylic acid copolymer, cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymer, shellac, or a combination thereof.

[0073] In certain embodiments, the enteric material is hydroxypropyl methylcellulose acetate succinate, hydroxypropyl methylcellulose acetate succinate, hydroxypropyl cellulose acetate succinate, hydroxyethyl methylcellulose succinate, hydroxyethyl cellulose acetate succinate, hydroxypropyl methylcellulose phthalate, hydroxyethyl methylcellulose acetate succinate, hydroxyethyl methylcellulose acetate phthalate, carboxyethyl cellulose, carboxymethyl cellulose, cellulose acetate phthalate, methylcellulose acetate phthalate, ethyl Cellulose acetate phthalate, hydroxypropyl cellulose acetate phthalate, hydroxypropyl methylcellulose acetate phthalate, hydroxypropyl cellulose acetate phthalate succinate, hydroxypropyl methylcellulose acetate succinate phthalate, hydroxypropyl methylcellulose succinate phthalate, cellulose propionate phthalate, hydroxypropyl cellulose butyrate phthalate, cellulose acetate trimellitate, methylcellulose acetate trimellitate, ethyl cellulose acetate trimellitate, hydroxypropyl cellulose acetate trimellitate, hydroxypropyl methylcellulose acetate trimellitate, hydroxypropyl cellulose acetate trimellitate succinate, cellulose propionate trimellitate, cellulose butyrate trimellitate, cellulose acetate terephthalate, cellulose acetate isophthalate, cellulose acetate pyridine dicarboxylate, cellulose acetate salicylate, hydroxypropyl salicylate cellulose acetate, ethyl benzoate cellulose acetate, hydroxypropyl ethyl benzoate cellulose acetate, ethyl phthalate cellulose acetate, ethyl nicotinate cellulose acetate, ethyl picolinate cellulose acetate, or combinations thereof.

[0074] In certain embodiments, the enteric material is insoluble or substantially insoluble at a pH of less than about 4, less than about 3, or less than about 2.

[0075] In certain embodiments, the enteric material does not crack, break, or burst at a pH of less than about 4, less than about 3, or less than about 2.

[0076] In certain embodiments, the enteric material is soluble or substantially soluble at a pH greater than about 5, greater than about 5.5, greater than about 6, greater than about 7, or greater than about 8.

[0077] In certain embodiments, the enteric material does not crack, break, or burst at a pH greater than about 5, greater than about 5.5, greater than about 6, greater than about 7, or greater than about 8.

[0078] In certain embodiments, the dosage forms disclosed herein release less than about 20%, less than about 18%, less than about 15%, less than about 12%, less than about 10%, less than about 5%, less than about 3%, or less than about 1% lipase (e.g., adole lipase) in 30 minutes when tested in 900 mL of simulated gastric fluid at 37° C. at 50 rpm, 75 rpm, or 100 rpm with or without a sinker (at one or more points of buffer pH below 3.0, e.g., 3.0 and / or 1.2).

[0079] In certain embodiments, the dosage form releases less than about 10%, less than about 5%, less than about 3%, or less than about 1% lipase (e.g., adole lipase) in 60 minutes when tested in 900 mL of simulated gastric fluid at 37° C., at 100 rpm, with or without a sinker, in a USP Apparatus II (at one or more points of buffer pH below 3.0, e.g., 3.0 and / or 1.2).

[0080] In certain embodiments, the dosage form releases less than about 10%, less than about 5%, less than about 3%, or less than about 1% lipase (e.g., adole lipase) in 90 minutes when tested in 900 mL of simulated gastric fluid (at one or more points of buffer pH below 3.0, e.g., 3.0 and / or 1.2) in a USP Apparatus II with or without a sinker at 100 rpm at 37°C.

[0081] In certain embodiments, the dosage form releases less than about 10%, less than about 5%, less than about 3%, or less than about 1% lipase (e.g., adole lipase) in 120 minutes when tested in 900 mL of simulated gastric fluid at 37° C., at 100 rpm, with or without a sinker, in a USP Apparatus II (at one or more points of buffer pH below 3.0, e.g., 3.0 and / or 1.2).

[0082] In certain embodiments, the dosage form releases less than about 10%, less than about 5%, less than about 3%, or less than about 1% of one or more of the active agents in 30 minutes when tested in 900 mL of simulated gastric fluid (at one or more points of buffer pH below 3.0, e.g., 3.0 and / or 1.2) in a USP Apparatus II with or without a sinker at 100 rpm at 37° C.

[0083] In certain embodiments, the dosage form releases less than about 10%, less than about 5%, less than about 3%, or less than about 1% of one or both of the active agents in 60 minutes when tested in 900 mL of simulated gastric fluid (at one or more points of buffer pH below 3.0, e.g., 3.0 and / or 1.2) at 37° C., at 100 rpm, with or without a sinker, in a USP Apparatus II.

[0084] In certain embodiments, the dosage form releases less than about 10%, less than about 5%, less than about 3%, or less than about 1% of one or both of the active agents in 90 minutes when tested in 900 mL of simulated gastric fluid (at one or more points of buffer pH below 3.0, e.g., 3.0 and / or 1.2) in a USP Apparatus II with or without a sinker at 100 rpm at 37° C.

[0085] In certain embodiments, the dosage form releases less than about 10%, less than about 5%, less than about 3%, or less than about 1% of one or both of the active agents in 120 minutes when tested in 900 mL of simulated gastric fluid (at one or more points of buffer pH below 3.0, e.g., 3.0 and / or 1.2) in a USP Apparatus II with or without a sinker at 100 rpm at 37° C.

[0086] In certain embodiments, the dosage form releases at least about 75%, at least about 90%, at least about 95%, or at least about 99% of the lipase (e.g., adole lipase) in 15 minutes when tested in 900 mL of simulated gastric fluid (at one or more points of buffer pH 5.5 or greater, e.g., 5.5 or 6.0) in a USP Apparatus II with or without a sinker at 100 rpm at 37°C.

[0087] In certain embodiments, the dosage form releases at least about 75%, at least about 90%, at least about 95%, or at least about 99% of the lipase (e.g., adole lipase) in 30 minutes when tested in 900 mL of simulated gastric fluid (at one or more points of buffer pH 5.5 or greater, e.g., 5.5 or 6.0) in a USP Apparatus II with or without a sinker at 100 rpm at 37° C.

[0088] In certain embodiments, the dosage form releases at least about 75%, at least about 90%, at least about 95%, or at least about 99% of the lipase (e.g., adole lipase) in 45 minutes when tested in 900 mL of simulated gastric fluid (at one or more points of buffer pH of 5.5 or greater, e.g., 5.5 or 6.0 or 6.5 or 6.6) at 37° C. in a USP Apparatus II with or without a sinker at 50 rpm, 75 rpm, or 100 rpm.

[0089] In certain embodiments, the dosage form releases at least about 75%, at least about 90%, at least about 95%, or at least about 99% of the lipase (e.g., adole lipase) in 60 minutes when tested in 900 mL of simulated gastric fluid (at one or more points of buffer pH 5.5 or greater, e.g., 5.5 or 6.0) in a USP Apparatus II with or without a sinker at 100 rpm at 37° C.

[0090] In certain embodiments, the dosage forms disclosed herein target the release of lipase (eg, adullipase) in the duodenum of a patient in need thereof.

[0091] In certain embodiments, the lipase (eg, adlulipase) lipase is prepared by a process that includes drying, such as freeze drying or spray drying.

[0092] In certain embodiments, the spray drying utilizes a stabilizer such as an oligosaccharide, for example, maltodextrin.

[0093] In certain embodiments, the dried non-porcine lipase is in the form of a powder or particles. The particles can have a particle size having a D50 of, for example, about 1 micron to about 200 microns, about 1 micron to about 50 microns, 50 microns to about 150 microns, about 60 microns to about 120 microns, about 65 microns to about 85 microns, or about 70 microns to about 82 microns.

[0094] In certain embodiments, the stabilizer is maltodextrin, xylan, mannan, fucoidan, galactomannan, chitosan, raffinose, stachyose, pectin, inulin, levan, graminan, and amylopectin, sucrose, lactulose, lactose, maltose, trehalose, cellobiose, nigerotriose, maltotriose, melezitose, maltotriulose, raffinose, kestose, arginine, glycine, CaCl2, or mixtures thereof.

[0095] In certain embodiments, the ratio of active agent to stabilizer is about 1:5 to about 5:1, about 1:3 to about 3:1, about 1:2 to about 2:1, about 1:1, or about 1:2.

[0096] In certain embodiments, spray drying is carried out at a pH of about 3 to about 5, about 2 to about 7, about 4, or about 6.

[0097] In certain embodiments, spray drying is carried out at a temperature greater than about 125°C, greater than about 150°C, or from about 100°C to about 250°C, or from about 150°C to about 180°C, or from about 155°C to about 165°C.

[0098] In certain embodiments, spray drying produces a non-porcine lipase in a yield of greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 99%.

[0099] In certain embodiments, the method of treatment is solely the non-porcine lipase formulations disclosed herein without the co-administration of a second active agent such as fat soluble vitamins (e.g., vitamins A, D, E, K, and combinations thereof), proteases, amylases, porcine pancreatic enzyme supplements, other non-porcine supplements, pancrelipase, liprotamase, a combination of the three enzymes: lipase, protease, and amylase, or combinations thereof.

[0100] In certain embodiments, the dosage form is administered through a feeding tube in the form of a solution or suspension, or in the form of a powder sprinkled on food, or administered as an oral dosage form such as a capsule, powder, tablet, liquid or semisolid.

[0101] In certain methods disclosed herein, at least a portion of the lipase (e.g., adullipase) is delivered to the duodenum of the patient, which portion can be, for example, at least about 75%, at least about 85%, or at least about 95%.

[0102] In certain embodiments, the formulations and methods provide a CFA% in an individual patient or subject (i.e., after treatment) of about 70 to about 99, about 75 to about 98, about 80 to about 92, about 85 to about 92, about 86 to about 92, or about 90 to about 92. In certain embodiments, the patient being treated (i.e., before treatment) has a CFA% of less than about 80, less than about 60, less than about 40, about 15 to about 80, or about 30 to about 60 in the individual patient or subject.

[0103] In certain embodiments, the formulations and methods provide a CNA% of about 90 to about 99, about 92 to about 99, about 95 to about 99, or about 99 to about 99 in an individual patient or subject.

[0104] In certain embodiments, the formulations and methods provide a CNA% of about 90 to about 99, about 92 to about 99, about 95 to about 99, or about 99 to about 99 in a population of patients or subjects.

[0105] In certain embodiments, the formulations and methods provide an increase in CFA of about 3% to about 12%, about 4% to about 10%, about 2% to about 6%, about 3% to about 6%, about 4%, about 5%, or about 6% relative to the average.

[0106] In certain embodiments, the formulations and methods provide a maximum individual relative CFA increase of about 5% to about 50%, about 10% to about 45%, about 15% to about 40%, about 20% to about 50%, about 30% to about 40%, about 30%, about 35%, or about 40%.

[0107] In certain embodiments, the present invention is directed to preparing the compositions and formulations disclosed herein.

[0108] In other embodiments, the compositions disclosed herein are utilized for industrial processes and uses, such as biocatalysis; detergents; food; environmental industries; production of citric acid and fragrances from various carbon sources including sugars, alkanes, vegetable oils, starch hydrolysates, ethanol, and glycerol; degradation of hydrophobic substrates; degradation of organic compounds; bioremediation of environments contaminated with oil spills, aliphatic and aromatic compounds, organic pollutants, 2,4,6-trinitrotoluene, and metals; and synthesis processes for beta-hydroxybutyrate, L-dopa, and emulsifiers.

[0109] In certain embodiments, the non-porcine lipase is an acid-stable lipase (LIP2) secreted from the yeast Yarrowia lipolytica. It belongs to the family of triacylglycerol lipases. It shares the common fold of a / b hydrolases and its crystal structure has been solved.

[0110] LIP2 is a 301 amino acid protein that is secreted in the culture medium as a glycosylated mature form after cleavage of a 39 amino acid signal peptide. Alternative cleavage has been demonstrated on the lipase resulting in N-terminal sequence heterogeneity. The predominant N-terminal sequence was identified as STETSHIDQESYNFF in the spray-dried powder. This protein is also referred to as MS1819 or adole lipase.

[0111] Two N-glycosylation sites have been identified at residues N113 and N134, and analysis by mass spectrometry revealed that each site has the following sugar moiety GlcNAc2-Manx (x=8). Five major glycoforms have been demonstrated by isoelectric focusing gel (IEF). In certain embodiments, the drug substance is defined as a spray-dried active agent bulk solution after addition of maltodextrin in a ratio of 1:3 to 3:1 or 2:1 (based on bulk dry weight). In other embodiments, the drug substance is defined as an active agent prepared by spray drying maltodextrin in a ratio of 1:3 or 3:1 or 2:1 and an enteric polymer (e.g., HPMCAS) in a ratio of 1:10 to 10:1 or about 5:1. The active agent may also have 1 to 15% salt.

[0112] The sequence is as follows (SEQ ID NO:1): VYTSTETSHIDQESYNFFEKYARLANIGYCVGPGTKIFKPFNCGLQCAHFPNVELIEEFHDPRLIFDVSGYLAVDHASKQIYLVIRGTHSLEDVITDIRIMQAPLTNFDLAANISSTATCDDCLVHNGFIQSYNNTYNQIGPKLDSVIEQY PDYQIAVTGHSLGGAAALLFGINLKVNGHDPLVVTLGQPIVGNAGFANWVDKLFFGQENPDVSKVSKDRKLYRITHRGDIVPQVPFWDGYQHCSGEVFIDWPLIHPPLSNVVMCQGQSNKQCSAGNTLLQQVNVIGNHLQYFVTEGVCGI.

[0113] The following examples are provided to aid in the understanding of the present invention and, of course, should not be construed as specifically limiting the invention described and claimed herein. Such variations of the present invention, including the substitution of all equivalents now known or later developed that would be within the skill of a person skilled in the art, and changes in formulations or minor changes in experimental design, should be construed as being included within the scope of the invention incorporated herein.

[0114] Example 1 Adole lipase powder was prepared by spray drying with (w / w%): 54.4% adole lipase: 27% maltodextrin: 10% HPMCAS: 8.4% salt. Other embodiments may have 40%-75% adole lipase, 10-40% maltodextrin, 5-20% HPMCAS, and 1-15% salt.

[0115] Granulation was prepared using Adole Lipase spray dried powder (69%), microcrystalline cellulose (30%, 10% x 3) and 1% magnesium stearate.

[0116] Microcrystalline cellulose was added in three portions, followed by geometric mixing for 5 minutes, the mixture was sieved through a 425 micron mesh, then compressed into 100 mg tablets at 1000 psi, followed by granulation through an 850 micron mesh to provide a powder with a D50 of 111 micrograms. Another portion was compressed into 200 mg tablets at 2000 psi, followed by granulation through an 850 micron mesh to provide a powder with a D50 of 455 microns. A schematic of the process and particle size data is presented in Figures 7A and 7B.

[0117] The activity of the spray dried powder and granules was tested according to the following procedure. Activation method Details of the activity methods used to generate all formulation development data discussed: Colorimetric activity assay (96 wells) ● Scope: Adole lipase is a lipase that hydrolyzes DMPTB to release a free thiol group. This thiol group reacts with DTNB (Ellman's reagent) to produce TNB (a colored product). The production of TNB is quantified using a standard curve and the values ​​are used to calculate the enzymatic activity of Adole lipase. DMPTB 2,3-Dimercapto-1-propanol tributyrate ○DTNB 5,5'-Dithiobis(2-nitrobenzoic acid) ○TNB 2-nitro-5-thiobenzoic acid Sample preparation Prepare a 0.5 mg / mL lipase solution in 50 mM Tris. Mix 50 μg of lipase (from above) with 0.05% Triton X-100, 0.6 mM DMPTB, and 0.8 mM DTNB and start the measurement immediately. ●Analysis ○Wavelength: 412nm Analysis type / mode: Kinetics Total duration: 30 minutes, reading every 30 seconds (61 readings) Plot the linearity curve of TNB, obtain the slope and intercept values, and calculate the activity.

[0118] The results are shown in Figure 8.

[0119] Figure 2 shows the effect of temperature on adole lipase structure using size exclusion chromatography (SEC). Details of the SEC method are described below. ●High performance liquid chromatography (HPLC) method ●Mobile phase 20mM phosphate / 100mM NaCl, pH 6 Sample preparation ○ Prepare a 1.5mg / mL lipase solution in the mobile phase. ●Analysis ○Column: TSKgel G2000SW XL ○Flow rate: 0.5ml / min Column temperature: 20℃ ○Wavelength: 280nm Run Time: 40 minutes Calculate purity based on total peak area in chromatogram Calculate the assay by peak area comparison with a reference standard

[0120] For ease of explanation, embodiments of the methods of the present disclosure are depicted and described as a series of operations. However, operations in accordance with the present disclosure may occur in various orders and / or simultaneously, as well as with other operations not shown and described herein. Moreover, not all illustrated operations may be required to implement a methodology in accordance with the disclosed subject matter. In addition, those skilled in the art will understand and appreciate that a methodology may alternatively be represented as a series of interrelated states via a state diagram or events.

[0121] In the above description, many specific details are set forth, such as specific materials, dimensions, process parameters, etc., to provide a thorough understanding of the invention. Particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments. The word "example" or "exemplary" is used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as "example" or "exemplary" should not necessarily be construed as preferred or advantageous over other aspects or designs. Rather, use of the word "example" or "exemplary" is intended to present concepts in a concrete manner. As used in this application, "or" is intended to mean an inclusive "or" rather than an exclusive "or." That is, unless otherwise specified or clear from the context, "X includes A or B" is intended to mean all natural inclusive permutations. That is, if X includes A, if X includes B, or if X includes both A and B, then "X includes A or B" is satisfied under any of the above illustrative examples. Furthermore, the articles "a" and "an" used in this application and the appended claims should generally be construed to mean "one or more" unless otherwise specified or clear from the context directed to the singular. References throughout this specification to "an embodiment," "certain embodiment," or "one embodiment" mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the appearances of the phrases "one embodiment," "certain embodiment," or "one embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment.

[0122] References to numerical ranges throughout this specification should not be construed as limiting, but should be understood to include the outer limits of the ranges as well as each numerical and / or narrower range within the recited numerical ranges.

[0123] The invention has been described with reference to specific exemplary embodiments thereof. The specification and drawings are therefore to be regarded in an illustrative rather than a restrictive sense. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended claims.

Claims

1. 1. A pharmaceutical composition comprising: (a) a lipase; (b) one or more pharma- ceutically acceptable excipients; The pharmaceutical composition, wherein after exposure to an environment of about 37° C. for 15 minutes, the composition exhibits an average specific activity (nmoles / min / mg) of at least 125.

2. 10. The pharmaceutical composition of claim 1, wherein after exposure to an environment of about 37°C for 30 minutes, the composition exhibits an activity (nmoles / min / mg) of at least 110.

3. 10. The pharmaceutical composition of claim 1, wherein after exposure to an environment of about 37°C for 45 minutes, the composition exhibits an activity (nmoles / min / mg) of at least 100.

4. 10. The pharmaceutical composition of claim 1, wherein after exposure to an environment of about 37°C for 90 minutes, the composition exhibits an activity (nmoles / min / mg) of at least 75.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the exposure is in vivo.

6. 6. The pharmaceutical composition of claim 5, wherein the exposure is after oral administration to a human subject.

7. The pharmaceutical composition of claim 1 , wherein the lipase and the excipients are spray dried.

8. The pharmaceutical composition of claim 1 , wherein the lipase and the excipients are granulated.

9. The pharmaceutical composition of claim 1 , wherein the lipase and the excipient are compressed.

10. 10. The pharmaceutical composition of claim 1, wherein the lipase and the excipients are spray dried and granulated.

11. 10. The pharmaceutical composition of claim 1, wherein the lipase and the excipients are spray dried and compressed.

12. The pharmaceutical composition of claim 1 , wherein the lipase and the excipients are granulated and compressed.

13. 10. The pharmaceutical composition of claim 1, wherein the lipase and the excipients are spray dried, granulated and compressed.

14. 2. The pharmaceutical composition of claim 1, wherein the lipase is a non-porcine lipase.

15. 2. The pharmaceutical composition of claim 1, wherein the lipase is porcine lipase.

16. 15. The pharmaceutical composition of claim 14, wherein the non-porcine lipase is a triacylglycerol hydrolase.

17. 15. The pharmaceutical composition of claim 14, wherein the non-porcine lipase has a molecular weight of about 30 kDa to about 45 kDa.

18. 15. The pharmaceutical composition of claim 14, wherein the non-porcine lipase has a molecular weight of about 38 kDa.

19. 15. The pharmaceutical composition of claim 14, wherein the non-porcine lipase comprises from about 295 to about 310 amino acids.

20. 15. The pharmaceutical composition of claim 14, wherein the non-porcine lipase comprises about 301 amino acids.

21. 15. The pharmaceutical composition of claim 14, wherein the non-porcine lipase is produced from Yarrowia lipolytica.

22. 15. The pharmaceutical composition of claim 14, wherein the non-porcine lipase is encoded by the LIP2 gene.

23. 15. The pharmaceutical composition of claim 14, wherein the non-porcine lipase is an adole lipase.

24. 16. The pharmaceutical composition of claim 15, wherein the porcine lipase is pancrelipase.

25. The pharmaceutical composition of claim 1 , wherein the excipient is an oligosaccharide.

26. 8. The pharmaceutical composition of claim 7, wherein the spray drying forms particles having a D50 of about 1 micron to about 200 microns, about 1 micron to about 50 microns, 50 microns to about 150 microns, about 60 microns to about 120 microns, about 65 microns to about 85 microns, or about 70 microns to about 82 microns.

27. 2. The pharmaceutical composition of claim 1, wherein the excipient comprises maltodextrin, xylan, mannan, fucoidan, galactomannan, chitosan, raffinose, stachyose, pectin, inulin, levan, graminan, and amylopectin, sucrose, lactulose, lactose, maltose, trehalose, cellobiose, nigerotriose, maltotriose, melezitose, maltotriulose, raffinose, kestose, or mixtures thereof.

28. 2. The pharmaceutical composition of claim 1, wherein the ratio of lipase to the one or more excipients is from about 1:5 to about 5:1, from about 1:3 to about 3:1, from about 1:2 to about 2:1, about 1:1 or about 1:

2.

29. 8. The pharmaceutical composition of claim 7, wherein the spray drying is carried out at a pH of about 3 to about 5, about 2 to about 7, or about 6.

30. 8. The pharmaceutical composition of claim 7, wherein the spray drying is carried out at an inlet temperature of above about 125°C, or from about 100°C to about 250°C, or from about 150°C to about 180°C, or from about 155°C to about 165°C, or about 162°C, and / or an outlet temperature of below about 150°C, or from about 50°C to about 125°C, or from about 60°C to about 100°C.

31. 8. The pharmaceutical composition of claim 7, wherein the spray drying produces the lipase in a yield of greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 99%.

32. 10. A pharmaceutical composition according to any preceding claim, wherein the composition comprises an enteric material.

33. A method for treating exocrine pancreatic insufficiency, comprising administering a pharmaceutical composition according to any one of claims 1 to 32.

34. A method for treating acute or chronic pancreatitis, comprising administering a pharmaceutical composition according to any one of claims 1 to 32.

35. A method for treating cystic fibrosis comprising administering a pharmaceutical composition according to any one of claims 1 to 32.

36. A method for increasing the effectiveness of lipase comprising administering a pharmaceutical composition according to any one of claims 1 to 32.

37. 34. The method of claim 33, wherein the deficiency is caused by pancreatic resection, such as due to pancreatic cancer.

38. 34. The method of claim 33, wherein the deficiency is age-related or due to Shwachman-Diamond syndrome, type 1 diabetes, type 2 diabetes, HIV, celiac disease, or inflammatory bowel disease.

39. 34. The method of claim 33, wherein the deficiency results from ulcerative colitis or Crohn's disease.

40. 40. The method of any of claims 33-39, wherein at least a portion of the lipase is delivered to the duodenum of the patient.

41. A process for preparing a pharmaceutical composition comprising combining a lipase and a pharma- ceutically acceptable excipient to form a composition form according to any one of claims 1 to 32.

42. 1. A composition comprising: (a) a lipase; (b) one or more excipients, wherein after exposure to an environment of about 37° C. for 15 minutes, the composition exhibits an average specific activity (nmoles / min / mg) of at least 125.

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