Multilayer vesicle pharmaceutical formulations

Abstract

The present invention provides a multilayer vesicle active pharmaceutical ingredient (MLV-API) pharmaceutical formulation comprising MLV dissolved in an aqueous alkaline solution wherein a multilayer vesicle (MLV) coats an active pharmaceutical ingredient (API), and wherein the API comprises a weak base compound. The preparation of MLV-API does not require the use of an extrusion or dialysis process, and can achieve a high coating rate of 90% and a high drug / lipid ratio of greater than 15% by weight. The invention also provides a method and a kit for preparing the MLV-API pharmaceutical preparation.

Description

Technical Field

[0001] The present invention relates to a composition and method for preparing a multi-layer vesicle drug preparation with a high coverage rate. The multi-layer vesicle drug preparation can be prepared without the need for extrusion or dialysis processes. Background Art

[0002] Most medicines only come in solid dosage forms, such as tablets and capsules, which are difficult for children to swallow. 1,2 Pediatric medications require temporary reconstitution of their adult dosage forms. This typically involves dividing the adult dosage form into smaller doses, which are then ground into a powder and mixed with food or liquids such as water, milk, or juice. Unfortunately, this common practice is associated with poor medication adherence. 3,4 First, dosing is often inaccurate, which can lead to safety and efficacy issues for many drugs with a narrow therapeutic window. Second, many drugs have an unpleasant taste that cannot be effectively masked by food or flavored liquids. Often, children spit out the medication or refuse to take it, leading to treatment failure. 5 .

[0003] Tang et al. 6 For the first time, researchers have demonstrated that small unilamellar vesicles (SUVs) are an effective platform for preparing child-friendly drug formulations. They encapsulated the highly bitter drug mefloquine (Mef) within the aqueous core of SUV liposomes, dispersing the drug in a solution for easier swallowing while preventing direct contact between Mef molecules and oral taste buds, thereby completely masking the taste. Following oral administration, SUV liposomes effectively released their payload into acidic gastric and bile-salting intestinal fluids, resulting in a slight increase in oral absorption compared to standard suspension formulations. However, the process for producing SUV liposomes is cumbersome, time-consuming, expensive, and difficult to scale up. The method involves hydrating a lipid film composed of cholesterol and a phospholipid (DSPC) with aqueous ammonium sulfate to form liposomal multilamellar vesicles (MLVs). Film extrusion is then used to reduce particle size and lamellarity, yielding SUVs. The SUVs were then dialyzed against 10% sucrose to create a transmembrane gradient. Finally, the drug is mixed with SUVs and cultured to enable the drug to be actively loaded into the SUV core. 6 . Thin film extrusion and dialysis processes are particularly challenging to scale up. In addition, it has long been believed that drugs cannot be actively loaded into MLVs due to the limited aqueous space between the lipid bilayers. However, the present invention successfully demonstrated for the first time an active loading method for MLV liposomes with high encapsulation efficiency and D / L ratio. Therefore, there is a need for an alternative MLV-based liposome technology that does not require thin film extrusion and dialysis, making it substantially easier to prepare and scale up manufacturing. Summary of the Invention

[0004] The present invention provides a multilamellar vesicle active pharmaceutical ingredient (MLV-API) pharmaceutical preparation, which comprises a multilamellar vesicle (MLV) and an active pharmaceutical ingredient (API) dissolved in an alkaline aqueous solution, wherein the API comprises a weak base drug; wherein the MLV coats the API; wherein the drug / lipid ratio of the API-coated MLV is between about 15% and about 25% by weight; and wherein the pH inside the MLV is lower than the pH of the alkaline aqueous solution outside the MLV.

[0005] The present invention also provides a method for preparing an MLV-API pharmaceutical preparation, which comprises the following steps: a. forming MLVs by hydrating an organic component with an acidic solution; d. adding an alkaline aqueous solution to the MLVs formed in step a; e. dissolving the API in water to form an API solution; f. mixing the API solution of step e with the alkaline aqueous solution containing MLVs of step d to produce an active loading solution, wherein a pH gradient is established between the interior and exterior of the MLVs to promote active loading of the API; g. culturing the active loading solution of step f to actively load the API into the MLVs, wherein the API comprises a weak base; wherein the organic component comprises one or more lipids capable of forming the MLVs; and wherein the preparation method does not include the use of dialysis or extrusion processes.

[0006] The present invention also provides an MLV-API pharmaceutical preparation preparation kit, which comprises an API, an alkaline aqueous solution and freeze-dried MLV, wherein the API comprises a weak base drug in powder form or in any form that can be ground into a powder form; wherein the three components are stored separately; and wherein mixing the three components causes the API to be actively loaded into the MLV, without the need for any additional ingredients and without the need for extrusion and dialysis processes to form an MLV-API pharmaceutical preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 The encapsulation efficiency of chloroquine (CQ) in various embodiments of the MLV drug formulations of the present invention, each having a different drug / lipid ratio (D / L), is illustrated.

[0008] Figure 2A The encapsulation efficiency of Nile Blue (NB) at different drug / lipid ratios (D / L) in various embodiments of the MLV drug formulations of the present invention is illustrated. Figure 2B The encapsulation efficiency of HCQ at different drug / lipid ratios (D / L) in the MLV drug formulation of the present invention is illustrated.

[0009] Figure 3 Characterization of empty MLV and MLV-CQ pharmaceutical formulations of the present invention is described.

[0010] Figure 4 The characterization of the MLV-NB and MLV-HCQ pharmaceutical preparations of the present invention is described.

[0011] Figure 5A illustrates cryo-electron microscopy (cryo-TEM) images of empty MLVs of the MLV pharmaceutical formulation of the present invention, and Figure 5B Cryo-TEM images of MLV-CQ of the MLV drug formulation of the present invention at a drug / lipid ratio (D / L) of 20 wt % are illustrated.

[0012] Figure 6 This schematic diagram illustrates an embodiment of an MLV pharmaceutical preparation kit and an embodiment of a preparation preparation process according to the present invention. The kit includes a vial of lyophilized MLV (A) and a vial of aqueous Na2CO3 solution (B). Step 1: Reconstitute the MLV with a predetermined amount of water; Step 2: Adjust the pH to 8-10 using a predetermined volume of aqueous NaCO3 solution; Step 3: Powder the tablets / capsules, add them to the MLV, and mix thoroughly; Step 4: Incubate the mixture at room temperature for 1 hour before use.

[0013] Figure 7A The chloroquine (CQ) encapsulation efficiency of the lyophilized MLV pharmaceutical formulation of the present invention when stored at room temperature for up to 8 weeks is demonstrated. Figure 7B The CQ encapsulation efficiency of the MLV-CQ pharmaceutical formulation of the present invention when stored at 4°C for up to 4 weeks is demonstrated.

[0014] Figure 8 The invention describes the encapsulation rate of hydroxychloroquine (HCQ) in an MLV pharmaceutical preparation according to an embodiment of the invention by formulating adult hydroxychloroquine (HCQ) tablets with a certain D / L ratio range into a liquid preparation using an embodiment of the MLV pharmaceutical preparation kit according to the invention.

[0015] Figure 9 The in vitro drug release profiles of examples of the MLV-CQ (20%, w / w) pharmaceutical formulations of the present invention in simulated saliva (SS), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) supplemented with 3 mM sodium taurocholate are illustrated.

[0016] Figure 10 Examples of the MLV-CQ pharmaceutical formulation of the present invention and the pharmacokinetic profile of free CQ following oral administration of 10 mg CQ / kg to female CD-1 mice are described.

[0017] Figure 11Examples of the MLV-CQ pharmaceutical formulation of the present invention and the pharmacokinetic parameters of the CQ solution after oral administration of 10 mg CQ / kg to female CD-1 mice are described.

[0018] Figure 12 The use of dynamic light scattering (DLS) to perform size and PDI measurements on empty MLV pharmaceutical formulations of the present invention is demonstrated.

[0019] Figure 13 The use of dynamic light scattering to perform size and PDI measurements on examples of MLV-CQ pharmaceutical formulations of the present invention is demonstrated.

[0020] Figure 14 The use of dynamic light scattering to perform size and PDI measurements on examples of MLV-HCQ pharmaceutical formulations of the present invention is described.

[0021] Figure 15 The use of dynamic light scattering to perform size and PDI measurements on examples of MLV-NB pharmaceutical formulations of the present invention is described.

[0022] Figure 16 The retention rate (RR, %) of clonidine in the MLV-clonidine pharmaceutical formulation of the present invention during storage at 4°C is illustrated.

[0023] Figure 17 The change of clonidine encapsulation efficiency (EE%) of the MLV-clonidine pharmaceutical formulation of the present invention over time during storage at 4°C is illustrated. DETAILED DESCRIPTION

[0024] The compositions of the present invention may comprise, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well as any additional or optional ingredients, components, or limitations described herein.

[0025] As used in this specification and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a" cell includes a plurality of cells, including mixtures thereof.

[0026] "About" in a quantity value means that the average deviation from the indicated value is at most ±20%, preferably ±10% or more preferably ±5%. For example, an amount of about 30 mol% anionic lipid means 30 mol% ±6 mol%, preferably 30 mol% ±3 mol% or more preferably 30 mol% ±1.5 mol% anionic lipid relative to the total lipid / amphiphile molar concentration.

[0027] A "therapeutically effective amount" of a compound includes the amount required to achieve the desired result. The exact amount required varies from subject to subject, depending on the subject's species, age, general condition, severity of disease, mode of administration, desired outcome, etc. In certain embodiments of the present invention, a "therapeutically effective amount" of a compound or pharmaceutical composition means an amount that effectively alleviates, inhibits progression of, or reverses any disease symptom disclosed herein in a subject or biological sample (e.g., cells). In certain embodiments, disease symptoms are alleviated by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%. In certain embodiments, disease progression is inhibited by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 99%. In certain embodiments, the compound inhibits disease progression by at least about 25%, at least about 50%, at least about 75%, or at least about 90%. In certain embodiments, the disease is reversed by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or any number or range of numbers within these values.

[0028] "Subject," "individual," or "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, murines, apes, humans, farm animals, sport animals, and pets.

[0029] "Unilamellar vesicles" are spherical vesicles that contain a lipid bilayer membrane that defines a single, closed aqueous compartment. The bilayer membrane consists of two lipid layers: an inner layer and an outer layer (leaflet). The outer layer of lipid molecules has its hydrophilic heads facing the external aqueous environment and its hydrophobic tails pointing downward toward the liposome interior. The inner layer of lipids is directly beneath the outer layer, with the heads of the lipids facing the aqueous interior of the liposome and their tails facing the tails of the outer layer of lipids.

[0030] "Multilamellar vesicles" consist of more than one lipid bilayer membrane defining more than one closed aqueous compartment. The membranes are arranged concentrically so that different membranes are separated by aqueous compartments, like an onion.

[0031] The present invention provides a multilamellar vesicle active pharmaceutical ingredient (MLV-API) pharmaceutical formulation comprising multilamellar vesicles (MLVs) dissolved in an alkaline aqueous solution, wherein the MLVs encapsulate an active pharmaceutical ingredient (API), wherein the API comprises a weakly basic compound. In one embodiment, the API encapsulated within the MLVs comprises chloroquine (CQ), a CQ derivative (e.g., hydroxychloroquine (HCQ)), clonidine, a clonidine derivative, imatinib, propranolol, pilocarpine, meloxicam, aripiprazole, chlorpromazine, R848, imiquimod, mefloquine, and primaquine, or pharmaceutically acceptable salts thereof. In one embodiment, any of the MLV-API formulations disclosed herein are prepared by actively loading the API into the MLVs, without the use of dialysis and / or extrusion processes. In one embodiment, the MLV-API pharmaceutical formulations of the present invention are prepared by actively loading the API into the MLVs of the present invention without the use of dialysis or extrusion, wherein the coverage is at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, such that less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the API is present in the alkaline aqueous solution outside the MVL. In one embodiment, the coverage of the MLV-API pharmaceutical formulations of the present invention is greater than about 95% when the D / L weight ratio in the active loading solution is between about 5 and about 20 wt%. In one embodiment, the coverage of MLV-CQ is greater than about 95% when the D / L weight ratio in the active loading solution is between about 15 and about 20 wt%. In one embodiment, the coverage of MLV-NB is greater than about 95% when the D / L weight ratio in the active loading solution is about 5 wt%. In one embodiment, when the D / L weight ratio of the active loading solution is between about 5 and about 15 weight %, the coverage of MLV-HCQ is greater than about 95%.

[0032] In one embodiment, the drug-to-lipid (D / L) ratio of the API-loaded MLVs of the present invention is between 5-30 wt%, such as about 5, about 10, about 15, about 20, about 25, or about 30 wt%, including any numbers and ranges of numbers within these values. In one embodiment, the drug-to-lipid (D / L) ratio of the CQ-loaded MLVs (MLV-CQ) of the present invention may be between about 10 and about 30 wt%, such as about 10, about 15, about 20, about 25, or about 30 wt%, including any numbers and ranges of numbers within these values. In one embodiment, the drug-to-lipid (D / L) ratio of the NB-loaded MLVs (MLV-NB) of the present invention may be between about 10 and about 30 wt%, such as about 10, about 15, about 20, about 25, or about 30 wt%, including any numbers and ranges of numbers within these values. In one embodiment, the drug-to-lipid (D / L) ratio of the HCQ-loaded MLV (MLV-HCQ) of the present invention may be between about 5 and about 25 wt %, such as about 5, about 10, about 15, about 20, about 25 wt %, including any numbers and ranges of numbers within these values.

[0033] In one embodiment, when subjected to physiological conditions of the oral cavity of a subject for up to 30 minutes, the MLV-API pharmaceutical formulation of the present invention retains up to about 90% to completely retained, such as about 95%, about 96%, about 97%, about 98%, about 99% or completely retained API, as described in Example 4 in combination with Figure 9 In one embodiment, when subjected to physiological conditions of the oral cavity of a subject for up to 30 minutes, the MLV-CQ drug formulation retains up to about 90% to completely, such as up to about 95%, about 96%, about 97%, about 98%, about 99% or completely retains the API, as described in Example 4 in combination with Figure 9 The high coverage rate of the MLV of the present invention and the high retention rate of the API in the oral cavity of the subject effectively mask the unpleasant taste of the API.

[0034] In one embodiment, as in Example 4, Figure 9 As described above, under physiological conditions and a temperature range equivalent to that of the stomach of a subject, the API-loaded MLVs of the present invention release an API instantaneously or in an instant burst at a percentage of about 30% to about 40%, such as about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, or any number or range of numbers within these values. In one embodiment, the instant burst is within about 1 to about 120 seconds, such as about 1 second, about 2 seconds, about 5 seconds, about 15 seconds, about 30 seconds, about 45 seconds, about 60 seconds, about 80 seconds, about 100 seconds, about 120 seconds, or any number or range of numbers within these values. In another embodiment, as in Example 4, in combination with Figure 9As illustrated, after about 4 hours under physiological conditions and at a temperature equivalent to the stomach of a subject, the percentage of API released from the API-loaded MLVs of the present invention is between about 40% and about 55%, such as about 40%, about 42%, about 44%, about 46%, about 48%, about 50%, about 52%, about 54%, about 56%, about 58%, about 60%, or any number or range of numbers falling within these values. In yet another embodiment, as in Example 4 combined with Figure 9 As shown in FIG1 , after about 1 hour under physiological conditions and a temperature range equivalent to the intestinal tract of a subject, the percentage of API released from the API-loaded MLV of the present invention is between about 15% and about 30%, such as about 15%, about 17%, about 20%, about 22%, about 25%, about 27%, about 30%, or any number or range of numbers falling within these values. Figure 9 As illustrated, after about 4 hours under physiological conditions and at a temperature equivalent to the intestinal tract of a subject, the percentage of API released from the API-loaded MLVs of the present invention is between about 15% and about 40%, such as about 15%, about 17%, about 20%, about 22%, about 26%, about 28%, about 30%, about 32%, about 34%, about 36%, about 38%, about 40% or any number and range of numbers falling within these values.

[0035] In one embodiment, as in Example 4, Figure 9 As described above, under physiological conditions and at a temperature equivalent to that of a subject's stomach, the CQ-loaded MLVs of the present invention release CQ instantaneously or in an instantaneous burst of about 30% to about 40%, such as about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, or any number or range of numbers within these values. In one embodiment, the instantaneous burst is within about 1 to about 120 seconds, such as about 1 second, about 2 seconds, about 5 seconds, about 15 seconds, about 30 seconds, about 45 seconds, about 60 seconds, about 80 seconds, about 100 seconds, about 120 seconds, or any number or range of numbers within these values. In another embodiment, as in Example 4, the CQ-loaded MLVs of the present invention release CQ instantaneously or in an instantaneous burst of about 30% to about 40%, such as about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, ... the CQ-loaded MLVs of the present invention release CQ instantaneously or in an instantaneous burst of about 30% to about 40%, such as about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, or any number or range of numbers within these values. In another embodiment, the CQ-loaded MLVs of the present invention release CQ instantaneously or in an instantaneous burst of about 30% to about 40%, such as about 30 Figure 9 As shown in FIG. 1 , after about 4 hours under physiological conditions and at a temperature equivalent to the stomach of a subject, the percentage of CQ released from the CQ-loaded MLVs of the present invention is between about 40% and about 55%, such as about 40%, about 42%, about 44%, about 46%, about 48%, about 50%, about 52%, about 54%, about 56%, about 58%, about 60%, or any number or range of numbers within these values. In yet another embodiment, as in Example 4, in combination with Figure 9As shown in FIG. 1 , after about 1 hour under physiological conditions and a temperature range equivalent to the intestinal tract of a subject, the percentage of CQ released from the CQ-loaded MLVs of the present invention is between about 15% and about 30%, such as about 15%, about 17%, about 20%, about 22%, about 25%, about 27%, about 30%, or any number or range of numbers within these values. Figure 9 As illustrated, after about 4 hours under physiological conditions and at a temperature equivalent to the intestinal tract of a subject, the percentage of CQ released from the CQ-loaded MLVs of the present invention is between about 15% and about 40%, such as about 15%, about 17%, about 20%, about 22%, about 26%, about 28%, about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, or any number or range of numbers falling within these values.

[0036] In one embodiment, the MLV-API pharmaceutical formulation of the present invention comprises API-loaded MLVs having a diameter of about 50 to about 500 nm, such as about 50, about 100, about 200, about 300, about 400, about 500 nm, or any number or range of numbers within these values. In one embodiment, the MLV-CQ pharmaceutical formulation of the present invention comprises CQ-loaded MLVs having a diameter of about 200 to about 300 nm, such as about 200, about 220, about 240, about 260, about 280, about 300 nm, or any number or range of numbers within these values. In one embodiment, the MLV-NB pharmaceutical formulation of the present invention comprises NB-loaded MLVs having a diameter of about 150 to about 250 nm, such as about 150, about 170, about 200, about 220, about 250, or any number or range of numbers within these values. In one embodiment, the MLV-HCQ pharmaceutical formulation of the present invention comprises HCQ-loaded MLVs, wherein the diameter is about 170 to about 270 nm, such as about 170, about 190, about 210, about 230, about 250, about 270 nm, or any number or range of numbers within these values.

[0037] In another embodiment, the polydispersity index (PDI) of the API-loaded MLVs in the MLV-API pharmaceutical formulation of the present invention is from about 0.3 to about 0.7, such as about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, or any number and range of numbers within these values. In another embodiment, the polydispersity index of the CQ-loaded MLVs in the MLV-CQ pharmaceutical formulation of the present invention is from about 0.3 to about 0.4, such as about 0.3, about 0.32, about 0.34, about 0.36, about 0.38, about 0.4, or any number and range of numbers within these values. In another embodiment, the polydispersity index of the NB-loaded MLVs in the MLV-NB pharmaceutical formulations of the present invention is from about 0.4 to about 0.6, such as about 0.4, about 0.42, about 0.45, about 0.47, about 0.50, about 0.52, about 0.55, about 0.57, about 0.60, or any number or range of numbers within these values. In another embodiment, the polydispersity index of the HCQ-loaded MLVs in the MLV-HCQ pharmaceutical formulations of the present invention is from about 0.4 to about 0.6, such as about 0.4, about 0.42, about 0.45, about 0.47, about 0.50, about 0.52, about 0.55, about 0.57, about 0.60, or any number or range of numbers within these values.

[0038] In another embodiment, the total charge of the API-loaded MLVs in the MLV-API pharmaceutical formulation of the present invention is neutral or nearly neutral. In one embodiment, the zeta potential of the MLV pharmaceutical formulation of the present invention is about -15 to about 15 mV, such as about -15, about -10, about -7.5, about -5, about -2.5, about 0, about 2.5, about 5, about 7.5, about 10, about -12.5, about 15 mV, or any number or range of numbers within these values. In one embodiment, the total charge of the API-loaded MLVs in the MLV-CQ pharmaceutical formulation of the present invention is neutral or nearly neutral at about -5 mV. In one embodiment, the total charge of the NB-loaded MLVs in the MLV-NB pharmaceutical formulation of the present invention is neutral or nearly neutral at about -4 mV. In one embodiment, the total charge of the HCQ-loaded MLVs in the MLV-HCQ pharmaceutical formulation of the present invention is neutral or nearly neutral at about -3 mV.

[0039] In one embodiment, the MLV-API pharmaceutical formulation of the present invention comprises an MLV having an interior pH lower than the pH of the alkaline aqueous solution in which the MLV is located. In one embodiment, the interior pH of the MLV is from about 1.0 to about 5.0, such as about 1.0, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, about 3.2, about 3.4, about 3.6, about 3.8, about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5.0, or any number or range of numbers within these values. In one embodiment, the pH of the alkaline aqueous solution is from about 7.2 to about 12.0, such as about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, about 11.0, about 11.5, about 12.0, or any number or range of numbers within these values. In one embodiment, the pH inside the MLV is from about 1.0 to about 3.0, such as about 1.0, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, or any number or range of numbers within these values. In one embodiment, the pH of the alkaline aqueous solution is from about 8.0 to about 10.0, such as about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, or any number or range of numbers within these values.

[0040] In one embodiment, the MLV-CQ pharmaceutical formulation of the present invention comprises an MLV having an interior pH lower than the pH of the alkaline aqueous solution in which the MLV is located. In one embodiment, the interior pH of the MLV is from about 1.0 to about 5.0, such as about 1.0, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, about 3.2, about 3.4, about 3.6, about 3.8, about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5.0, or any number or range of numbers within these values. In one embodiment, the pH of the alkaline aqueous solution is from about 7.2 to about 12.0, such as about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, about 11.0, about 11.5, about 12.0, or any number or range of numbers within these values. In one embodiment, the pH of the MLV-CQ interior is from about 1.0 to about 3.0, such as about 1.0, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, or any number or range of numbers within these values. In one embodiment, the pH of the alkaline aqueous solution is from about 8.0 to about 10.0, such as about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, or any number or range of numbers within these values.

[0041] In one embodiment, the MLV-NB pharmaceutical formulation of the present invention comprises an MLV having an interior pH lower than the pH of the alkaline aqueous solution in which the MLV is contained. In one embodiment, the interior pH of the MLV is between about 1 and 5, such as about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, about 3.2, about 3.4, about 3.6, about 3.8, about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5.0, or any number or range of numbers within these values. In one embodiment, the pH of the alkaline aqueous solution is from about 7.2 to about 12.0, such as about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, about 11.0, about 11.5, about 12.0, or any number or range of numbers within these values. In one embodiment, the pH within the MLV-NB is from about 1.0 to about 3.0, such as about 1.0, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, or any number or range of numbers within these values. In one embodiment, the pH of the alkaline aqueous solution is from about 8.0 to about 10.0, such as about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, or any number or range of numbers within these values.

[0042] In one embodiment, the MLV-HCQ pharmaceutical formulation of the present invention comprises an MLV having an interior pH lower than the pH of the alkaline aqueous solution in which the MLV is contained. In one embodiment, the interior pH of the MLV is between about 1 and 5, such as about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, about 3.2, about 3.4, about 3.6, about 3.8, about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5.0, or any number or range of numbers within these values. In one embodiment, the pH of the alkaline aqueous solution is from about 7.2 to about 12.0, such as about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, about 11.0, about 11.5, about 12.0, or any number or range of numbers within these values. In one embodiment, the pH of the MLV-HCQ interior is from about 1.0 to about 3.0, such as about 1.0, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, or any number or range of numbers within these values. In one embodiment, the pH of the alkaline aqueous solution is from about 8.0 to about 10.0, such as about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, or any number or range of numbers within these values.

[0043] In one embodiment, the MLV-API pharmaceutical formulation of the present invention further comprises a cryoprotectant, which includes sucrose, dextrose, glucose, trehalose, ethylene glycol, propylene glycol, dextran, cyclodextrin, arginine, P188, polyvinyl pyrrolidone, glycerol, sorbitol, albumin, mannitol, or a combination thereof. In one embodiment, the MLV pharmaceutical formulation of the present invention comprises a cryoprotectant at a concentration of about 15%, 20%, 25%, 30%, 35%, 40%, or 45%, or any number or range thereof.

[0044] In one embodiment, the MLV-API pharmaceutical formulation of the present invention may further comprise two or more APIs, wherein the first API is encapsulated inside the MLV of the present invention and the second compound is located in an alkaline aqueous solution outside the MLV. In one embodiment, the API contained in the second compound is different from the first API, so that the oral pharmaceutical formulation of the present invention can provide a combination therapy of two or more drugs. In one embodiment, a weak base (such as primaquine, mefloquine, paracetamol) can be loaded into the MLV, while a weak acid can be retained in the external phase. In another embodiment, the MLV pharmaceutical formulation of the present invention further comprises a third compound, which includes an adjuvant present in the alkaline aqueous solution inside the MLV or outside the MLV.

[0045] The present invention also provides an MLV-API pharmaceutical preparation kit comprising: 1. an API in powder form or any form capable of being made into a powder form, such as a tablet that can be ground into a powder form, 2. an alkaline aqueous solution, and 3. lyophilized MLV without any API coating, wherein the three components are preserved and stored separately. In one embodiment, the lyophilized MLV can be stored at room temperature for at least 4, 5, 6, 7, or 8 weeks, and when the MLV-API pharmaceutical preparation is prepared from the kit of the present invention, the coating efficiency can reach about 90% or more, as shown in Example 2. Figure 7A In one embodiment, the Figure 6 The MLV-API pharmaceutical formulation of the present invention can be prepared by mixing the three components shown in FIG. 1 without the need for dialysis or extrusion. In one embodiment, when the API is prepared in powder form, it can be prepared by manually mixing as shown in FIG. Figure 6The three components shown are used to prepare the MLV-API pharmaceutical formulation of the present invention without dialysis or extrusion processes, wherein manual mixing comprises mixing all three components in a single container and gently shaking the container by hand for about 1 to about 10 minutes, such as about 1 minute, about 2 minutes, about 5 minutes, about 7 minutes, about 10 minutes, or any time period and time range falling within these values. In one embodiment, manual mixing comprises mixing all three components in a single container without shaking the container. In one embodiment, manual mixing comprises mixing all three components in a single container and vigorously shaking the container by hand for about 1 to about 10 minutes, such as about 1 minute, about 2 minutes, about 5 minutes, about 7 minutes, about 10 minutes, or any time period and time range falling within these values. In one embodiment, manual mixing comprises mixing all three components in a single container and shaking the container by hand until the solution becomes homogenous.

[0046] In one embodiment, bases that can be used in place of or in combination with sodium carbonate include, but are not limited to, potassium carbonate, calcium carbonate, magnesium carbonate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and ammonia. In one embodiment, the API comprises chloroquine (CQ), a CQ derivative (e.g., hydroxychloroquine (HCQ)), clonidine, a clonidine derivative, imatinib, propranolol, pilocarpine, meloxicam, aripiprazole, chlorpromazine, R848, imiquimod, mefloquine, and primaquine, or a pharmaceutically acceptable salt thereof.

[0047] In one embodiment, the coating efficiency of the MLV-API pharmaceutical preparation prepared using the kit of the present invention can be maintained at >80% after storage at about 4° C. for about 20 to about 34 days, such as about 20 days, about 22 days, about 24 days, about 26 days, about 28 days, about 30 days, about 32 days, about 34 days, or any number and range of numbers within these values. In one embodiment, the coating efficiency of the MLV-API pharmaceutical preparation prepared using the kit of the present invention can be maintained at >94% after storage at about 4° C. for about 12 to about 24 days, such as about 12 days, about 14 days, about 16 days, about 18 days, about 20 days, about 22 days, about 24 days, or any number and range of numbers within these values.

[0048] In one embodiment, the MLV-API pharmaceutical formulation of the present invention can enhance the absorption of the encapsulated drug. In one embodiment, the 24-hour AUC of the formulation of the present invention is more than two times higher than that of the free form API, and T max Remain unchanged, such as Figure 10 and 11 In one embodiment, the C of the MLV of the present invention is maxDrug absorption was approximately 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, and 3.0 times higher than that of the free form of the drug in the subjects, while T max Remain unchanged, such as Figure 10 and 11 shown.

[0049] In one embodiment, the molar ratio of cholesterol to lipid of any embodiment of the MLV of the present invention can be about 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.2, or any ratio therebetween. In one embodiment, the lipid comprises DSPC, DOPC, DOPS, DPPS, DSPS, DOPE, or a combination thereof.

[0050] In one embodiment, any embodiment of the MLV-API pharmaceutical formulation of the present invention can be prepared without the need for extrusion and / or dialysis processes.

[0051] In one embodiment, any embodiment of the MLV-API pharmaceutical formulation of the present invention may include, but is not limited to, one or more of the following components: phospholipids, lysolipids, lysolipids, neurolipids, PEGylated lipids, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol (not limited to specific sugars), fatty acids, sterols, caprylic acid, capric acid, triglycerides, medium chain triglycerides (MCT), lauric acid, myristic acid, palmitic acid, stearic acid, corn oil, peanut oil, oleic acid, Span 80, any other edible oil, Tween, Gelucire 44 / 14, Gelucire 50 / 13, Brij 35, Brij 58, polyoxyl 20 monostearate, polyoxyl 40 monostearate, lecithin, tocophersolan (TPGS), labrasol ALF, ethanol, glycerol, PEG 300-4000, PEG 8000, transcutol P ( P), 2-pyrrolidone (Soluphor P), prepared in any ratio deemed acceptable by those skilled in the art.

[0052] The present invention further comprises a method for preparing the MLV-API pharmaceutical formulation of the present invention, comprising the following steps:

[0053] i. Preparation of MLV and establishment of pH gradient:

[0054] a. Forming MLVs by hydrating an organic component with an acidic solution. In one embodiment, the acidic solution may comprise citric acid, tartaric acid, malic acid, acetic acid, oxalic acid, ascorbic acid, sodium acetate, or a combination thereof. In one embodiment, the organic component of the MLVs comprises DSPC, DOPC, DOPS, DPPS, DSPS, DOPE, cholesterol (Chol), phospholipids, lysolipids, lysolipids, neurolipids, PEGylated lipids, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol (not limited to a specific sugar), or a combination thereof.

[0055] d. Adding the MLVs prepared in step (a) to an alkaline aqueous solution to form an alkaline aqueous solution containing the MLVs, and establishing a pH gradient between the interior of the MLVs and the alkaline aqueous solution to facilitate active loading of the API in step (ii). In one embodiment, the alkaline aqueous solution may comprise sodium carbonate, potassium carbonate, calcium carbonate, magnesium carbonate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia, or a combination thereof.

[0056] ii. Active Load API:

[0057] a. Dissolve the powdered API in water to form an API solution. In one embodiment, the water comprises distilled water. In one embodiment, the API may be ground into a powder first.

[0058] b. The API solution of step ii a is mixed with the MLV alkaline aqueous solution prepared in step i to form an active loading solution.

[0059] c. Incubating the active loading solution formed in step iib to actively load the API into the MLVs. In one embodiment, the incubation temperature can be in the range of about 15°C to about 70°C, such as about 15°C, about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, or any temperature or temperature range within these values, and the incubation time can be in the range of about 5 minutes to about 2 hours, such as about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, about 120 minutes, or any time period or time range within these values. In one embodiment, the incubation temperature may range from about 50°C to about 70°C, such as about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, or any temperature or temperature range within these values, and the incubation time may range from about 40 minutes to about 80 minutes, such as about 40 minutes, about 50 minutes, about 60 minutes, about 70 minutes, about 80 minutes, or any time period or time range within these values.

[0060] In one embodiment, the method for preparing MLV-API of the present invention further comprises adding a cryoprotectant to the solution containing the MLVs from step ia, and freeze-drying the MLVs formed in step ia. In one embodiment, the freeze-drying conditions are between about -80°C and about -100°C, such as about -80°C, about -85°C, about -90°C, about -95°C, about -100°C, or any temperature or temperature range within these values, and at about 0.05 mBar to about 0.2 mBar, such as about 0.05 mBar, about 0.1 mBar, about 0.15 mBar, about 0.2 mBar, or any pressure or pressure range within these values, overnight. In one embodiment, the cryoprotectant of step ib comprises sucrose, dextrose, trehalose, or a combination thereof. In one embodiment, the cryoprotectant concentration in the solution of step ib is about 15% to 45%, such as about 15, about 20, about 25, about 30, about 35, about 40, about 45%, or any concentration or concentration range within these values. In one embodiment, the method for preparing an MLV-API pharmaceutical formulation of step i of the present invention further comprises storing the lyophilized MLV of step ib at 4° C. for up to 8 weeks in step ic, followed by performing the remaining steps id and steps ii-ac, and after storage, when performing the remaining preparation steps, the coverage still reaches greater than about 80%, about 90%, or about 95%, and the D / L ratio in the active loading solution is between about 5% and about 20% by weight, such as about 5%, about 10%, about 15%, about 20%, or any percentage or percentage range within these values ​​of the active loading solution.

[0061] Any embodiment of the method for preparing the MLV-API pharmaceutical formulation of the present invention does not include a thin film extrusion step and a dialysis step. Liposome technology that does not require thin film extrusion and dialysis steps will be substantially easier and more economical to prepare and manufacture on a large scale.

[0062] In one embodiment, in the hydration step of step i, the organic component is in the form of a film before hydration. In one embodiment, the organic component in step i comprises cholesterol and lipid in a molar ratio of about 1:0.2 to about 1:2.2, such as 1:0.2, 1:0.4, 1:0.6, 1:0.8, 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.2, or any number or range of numbers within these values. In one embodiment, the hydration step of step ia of the present invention comprises adding an acidic solution having a pH of about 1.0 to about 5.0, such as about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, about 3.2, about 3.4, about 3.6, about 3.8, about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, about 5.0, or any number and range of numbers within these values, and a concentration of about 50 to about 500 mM, such as about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550 mM, or any number and range of numbers within these values. In one embodiment, the MLV preparation step in step id of the present invention comprises adding an alkaline solution having a pH of 7.2 to about 12.0, such as about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5, about 11.0, about 11.5, about 12.0, or any pH or pH range within these values, and a concentration of about 50 to about 550 mg / mL, such as about 50 mg / mL, about 100 mg / mL, about 150 mg / mL, about 200 mg / mL, about 250 mg / mL, about 300 mg / mL, about 350 mg / mL, about 400 mg / mL, about 450 mg / mL, about 500 mg / mL, about 550 mg / mL, or any concentration or concentration range within these values. In one embodiment, the hydration step in step ia of the present invention comprises adding an acidic solution having a pH of about 1.0 to about 3.0, such as about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 2.0, about 2.2, about 2.4, about 2.6, about 2.8, about 3.0, or any number and range of numbers within these values, and a concentration of about 50 to about 500 mM, such as about 50, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550 mM, or any concentration and range of concentrations within these values.In one embodiment, the MLV preparation step in step ib of the present invention comprises adding an alkaline solution having a pH of 7.2 to about 10.0, such as about 7.2, about 7.4, about 7.6, about 7.8, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, or any pH or pH range within these values, and a concentration of about 50 to about 550 mg / mL, such as about 50 mg / mL, about 100 mg / mL, about 150 mg / mL, about 200 mg / mL, about 250 mg / mL, about 300 mg / mL, about 350 mg / mL, about 400 mg / mL, about 450 mg / mL, about 500 mg / mL, about 550 mg / mL, or any concentration and concentration range within these values.

[0063] In another embodiment, the MLV-API formulation of the present invention is prepared by combining the following alternative components: caprylic acid, capric acid, triglycerides, medium chain triglycerides (MCT), lauric acid, myristic acid, palmitic acid, stearic acid, corn oil, peanut oil, oleic acid, Span 80, any other edible oil, Tween, Gelucire 44 / 14, Gelucire 50 / 13, Brij 35, Brij 58, polyoxyl 20 monostearate, polyoxyl 40 monostearate, lecithin, tocophersolan (TPGS), labrasol ALF, ethanol, glycerol, PEG 300-4000, PEG 8000, transcutol P, 2-pyrrolidone (Soluphor P), or a combination thereof.

[0064] In one embodiment, the active loading step in step ii of the present invention comprises an API, wherein the API comprises a weak base drug. In one embodiment, the API in the active loading step in step ii of the present invention comprises CQ, a CQ derivative, clonidine, a clonidine derivative, imatinib, propranolol, pilocarpine, meloxicam, aripiprazole, chlorpromazine, R848, imiquimod, mefloquine, and primaquine, or a pharmaceutically acceptable salt thereof.

[0065] In one embodiment, the D / L ratio of the MLV pharmaceutical formulation prepared using the method of the present invention is up to about 5 wt % to about 30 wt %, such as about 5 wt %, about 10 wt %, about 15 wt %, about 20 wt %, about 25 wt %, about 30 wt %, or any wt % or wt % range falling within these values.

[0066] The present invention also provides a method of treating a disease using any embodiment of the MLV pharmaceutical formulation of the present invention. In one embodiment, the method of treatment using any embodiment of the MLV pharmaceutical formulation of the present invention comprises treating a subject who is sensitive to the unpleasant taste of the drug in free form. In another embodiment, the method of treatment using any embodiment of the MLV pharmaceutical formulation of the present invention comprises treating a subject who has difficulty swallowing. In one embodiment, the method of treatment using any embodiment of the MLV pharmaceutical formulation of the present invention comprises treating children. In one embodiment, the disease treated using any embodiment of the MLV pharmaceutical formulation of the present invention comprises rheumatoid arthritis, lupus erythematosus, and malaria. In another embodiment, the disease treated using any embodiment of the MLV pharmaceutical formulation of the present invention comprises hypertension, ADHD, drug withdrawal, menopausal hot flashes, diarrhea, cramps, and certain pain conditions.

[0067] The following examples are for illustration only and are not intended to limit the scope of the present invention. Other general and specific configurations will be apparent to those skilled in the art.

[0068] Example of MLV carrying CQ, NB, and HCQ:

[0069] Materials and methods

[0070] Material

[0071] 1,2-Distearoyl-sn-glycero-3-phosphatidylcholine (DSPC) was purchased from Avanti Polar Lipids (Alabaster, AL). Cholesterol, chloroquine diphosphate (CQ), hydroxychloroquine sulfate (HCQ), Nile blue (NB), citric acid monohydrate, sodium carbonate (Na2CO3), phosphoric acid, phosphate-buffered saline (PBS), concentrated hydrochloric acid, and sodium taurocholate hydrate were purchased from Sigma Aldrich (St. Louis, MO). Hydroxychloroquine tablets (200 mg / tablet) were purchased from Regency Medical Centre Pharmacy (Vancouver, BC, Canada). All chemical reagents and organic solvents were of analytical grade.

[0072] Simulating fluids

[0073] Saliva (SS, pH 6.5) was simulated by titrating 10 mM PBS (pH 7.4) with 1 M phosphoric acid. Simulated gastric fluid (SGF, pH 1.2) and simulated intestinal fluid (SIF, pH 6.8) were both purchased from RICCA Chemical Company (Arlington, TX). Sodium taurocholate hydrate (3 mM) was added to the simulated intestinal fluid to simulate bile salts in the intestine.

[0074] Preparation of multilamellar vesicles (MLVs)

[0075] MLVs were prepared using the thin film hydration method as described above. 14 A cholesterol / DSPC (45 / 55, molar ratio) mixture was dissolved in a round-bottom flask containing chloroform at a total lipid concentration of 30 mg / mL. The chloroform was removed using rotary evaporation at a temperature of 60°C and a rotation speed of 400 rpm to obtain a lipid film. The lipid film was dried under high vacuum for 30 minutes to remove the chloroform residues. Subsequently, the film was hydrated with 2 mL of 300 mM citric acid (pH 2). The MLV suspension was heated and ultrasonicated at 40°C for 30 minutes to obtain a uniform milky white liquid preparation. The particle size, polydispersity index (PDI) and zeta potential of MLV were measured by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern Instruments, Malvern, UK). For freeze drying, MLV prepared in citric acid (pH 2) with 30% sucrose were aliquoted into 200 μL portions and lyophilized using Triad TM The samples were freeze-dried overnight at -90°C and 0.12 mBar using a freeze-drying system (LABCONCO, Kansas City, MO). The samples were sealed and stored at room temperature for stability monitoring.

[0076] Active loading of drugs

[0077] Approximately 34-40 μL of sodium carbonate (300 mg / mL) was added to 200 μL of liposome MLVs to raise the external pH to 8-10. CQ and HCQ powders were dissolved in distilled water to a final concentration of 10 mg / mL. HCQ tablets (200 mg) were first ground into a fine powder and dissolved in distilled water at 10 mg / mL. Drug loading was performed with a drug-lipid (D / L) ratio ranging from 5% to 30% (w / w) by mixing the drug solution with MLVs containing a pH gradient, followed by incubation at room temperature for 1 hour and quenching on ice for 2 minutes to terminate the loading process. Unencapsulated drug was removed by size exclusion chromatography (SEC) on a Sephadex-G25 column (GE Healthcare, Milwaukee, WI). The drug and lipid concentrations in the samples were quantified by UPLC as described below. The drug encapsulation efficiency (EE) was calculated by the following formula, where D / L before and D / L after Represents the drug / lipid ratio before and after SEC, respectively.

[0078]

[0079] Alternatively, the MLVs were centrifuged at 10,000 g for 10 minutes, and the supernatant containing the unencapsulated drug was analyzed by UPLC. The results were then compared with the total drug concentration in the MLVs to obtain the EE.

[0080] Cryogenic transmission electron microscopy (CryoTEM)

[0081] Empty and CQ-loaded MLVs (30 mg lipid / mL) were deposited on glow-discharged copper grids at a 20% D / L ratio (w / w), vitrified using a FEI Mark IV Vitrobot (FEI, Hillsboro, OR, USA), and imaged using a 200 kV Glacios microscope equipped with a Falcon III camera at the UBC High-Resolution Macromolecular Cryo-Electron Microscopy Facility (Vancouver, BC, Canada).

[0082] Ultra-performance liquid chromatography (UPLC)

[0083] ACQUITY UPLC H-Class System (Waters, Milford, MA) 15 The concentrations of drugs and lipids were quantified simultaneously. The mobile phase consisted of solvent A: an aqueous solution containing 0.1% trifluoroacetic acid (TFA) and solvent B: methanol containing 0.1% TFA. CQ and HCQ were quantified by a photodiode array (PDA) detector at wavelengths of 342 nm and 342 nm, respectively. DSPC and Chol were quantified by an evaporative light scattering (ELS) detector. The concentrations of drugs and lipids were measured by integrating the peak area under the curve and comparing it with a standard curve. For plasma CQ quantification, a Waters QDa mass spectrometer was used. Ionization was performed at 250°C (source temperature) using a cone voltage of 65 V, and the single ion recognition (SIR) peak was integrated for the single charged molecular ion (m / z 319.8) obtained at a capillary voltage of 0.5 V to determine the CQ concentration.

[0084] Storage stability studies

[0085] MLVs prepared in citric acid with 30% sucrose were lyophilized and stored at room temperature. At selected time points (1, 2, 3, 4 and 8 weeks), the lyophilized MLVs were reconstituted using Milli-Q water. Subsequently, sodium carbonate was added to the MLVs to produce a transmembrane pH gradient as described above, followed by loading CQ at 20 wt% D / L. A centrifugation method was used to measure drug loading efficiency. The prepared CQ-loaded MLV drug formulations were stored in liquid form at 4°C. At different time points, a centrifugation method was used to determine the CQ coverage.

[0086] In vitro drug release

[0087] The MLV-CQ drug formulation of the present invention (MLVs loaded with CQ at 20% D / L (w / w)) was mixed with various simulated gastrointestinal fluids at a 1:1 (v / v) ratio and incubated at 37°C with shaking (150 rpm). At selected time points, 200 μL of the mixture was collected, and the released drug was removed by SEC. UPLC was used to measure drug and lipid concentrations in the MLVs before and after SEC. The drug release rate (%) was calculated using Equation (2):

[0088]

[0089] animal

[0090] Female CD1 mice (18-20 g, 6-7 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME). All in vivo studies were performed according to established protocols (A18-0177) approved by the Animal Care Committee of the University of British Columbia (Vancouver, BC, Canada).

[0091] Pharmacokinetics

[0092] The MLV-CQ pharmaceutical preparation of the present invention or free CQ (dissolved in saline) was administered to mice via oral gavage at 10 mg CQ / kg (50 mg lipid / kg). Blood was collected via saphenous vein or cardiac puncture at 0.5, 1, 3, 4, 6 and 24 hours and quickly transferred to EDTA-coated tubes. Plasma was separated by centrifugation at 10,000 g for 10 minutes at 4°C. 45 μL of plasma was mixed with 300 μL of ethanol, vortexed for 30 seconds, placed on ice for 30 minutes, and centrifuged twice at 12,500 rpm for 5 minutes each. The supernatant (280 μL) was collected, freeze-dried, and reconstituted in 45 μL of ethanol. Subsequently, 10 μL of sample was injected into UPLC to measure the CQ concentration. PK solver was used respectively. 16 Pharmacokinetic parameters and AUC were analyzed using GraphPad Prisim version 8.0 (GraphPad Software, San Diego, CA, USA).

[0093] Statistical analysis

[0094] All data are expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism version 8.0. Comparisons between two groups were performed using unpaired t-tests, and comparisons between three or more groups were performed using one-way analysis of variance.

[0095] Example 1. Preparation of drug-loaded MLVs

[0096] MLV liposomes were spontaneously formed by hydrating DSPC and cholesterol with 300 mM citric acid (pH 2), without the need for tedious film extrusion or dialysis. A transmembrane pH gradient was generated by adding aqueous Na2CO3 to the MLVs, resulting in an external pH of 8-10. To confirm whether this technique could provide active and complete loading of CQ, gradient MLVs with a range of D / L were mixed with CQ, incubated at room temperature for 1 hour, and then analyzed by EE. Figure 1 As shown in Figure 2, complete (>95%) drug coverage was achieved at D / L of 15-20% (w / w). When D / L increased to 25% (w / w), drug coverage decreased to approximately 80% ( Figure 1 ), indicating that the drug loading has reached saturation. It has long been believed that drugs cannot be actively loaded into MLVs due to the limited aqueous space between the lipid bilayers. In the preparation method of the present invention, the inventors disclosed a method for actively loading APIs into MLVs via a pH gradient. In addition, a simple and scalable technology is used to prepare the MLV drug formulation of the present invention and provide complete encapsulation for weak base drugs. In addition to CQ, the inventors also successfully encapsulated other weak base small molecules (such as HCQ, NB and clonidine) into the MLV of the present invention at the same D / L (Figures 2 and 8), confirming that this can be used as a platform technology.

[0097] Empty and CQ-loaded MLVs were characterized by DLS and showed comparable size (~250 nm), PDI (0.3-0.5), and zeta potential (±5 mV) ( Figure 3 The D / L ratio, size, PDI, coverage and zeta potential of MLV-NB and MLV-HCQ were also characterized. Figure 4 The higher PDI is expected because film extrusion was not used in the preparation to control the particle size. The relatively small size of these MLVs may be due to the gradient introduction method, which promotes lipid rearrangement. The multilamellar morphology and variable size distribution of MLVs were further confirmed by cryo-TEM imaging (Figure 5). Unlike the previously reported SUV-Mef structure 6 , no electron-dense drug aggregates were shown inside the MLVs.

[0098] Example 2. Development of a user kit for the simple preparation of stable liquid pharmaceutical formulations

[0099] The inventors envision that this MLV platform can be used as a universal taste-masking formulation for weak base drugs. Lyophilized, drug-free MLV will be provided at pharmacies along with an aqueous Na2CO3 solution or as a kit with complete dosing instructions for caregivers. First, the MLV is reconstituted with a fixed amount of water, followed by the addition of a fixed volume of aqueous Na2CO3 solution to raise the external pH to 8-10. A fixed amount of drug powder (15-20% by weight) is mixed with the MLV and then incubated at room temperature for approximately 1 hour before use. Figure 6 shown.

[0100] To examine the feasibility of this approach, the inventors lyophilized MLV prepared in 300 mM citric acid (pH 2) with 30% sucrose into a dry powder and then stored at room temperature for up to 8 weeks. At different time points, the lyophilized MLV was reconstituted in MilliQ water, the pH was adjusted to 8-10 using aqueous Na2CO3 solution, mixed with 20 wt% CQ, incubated at room temperature for 1 hour, and the drug encapsulation efficiency was measured using a centrifugation method. Figure 7A As shown in Figure 2, the lyophilized MLV can be stored at room temperature for at least 2 months and still provide >90% CQ coverage, which indicates that the lyophilized MLV product exhibits good storage stability at room temperature and can be used to prepare a child-friendly liquid formulation by the following simple process. In addition, the prepared MLV-CQ is stable after storage at 4°C for 2 weeks. Figure 7B As shown, CQ coverage remained at >94% after 2 weeks of storage at 4°C, but decreased to approximately 80% within 3 weeks. These data suggest that once a pediatric formulation is prepared using this MLV kit, it can be stored at 4°C for up to 2 weeks in patients.

[0101] Example 3. Using the MLV pharmaceutical preparation kit to prepare hydroxychloroquine tablets into liquid preparations

[0102] Next, the inventors examined whether the MLV drug formulation kit could be used to reconstitute commercially available HCQ tablets into a liquid formulation. Since CQ tablets are unavailable in Canada, the inventors purchased HCQ tablets as a research alternative. When a child-friendly formulation is unavailable commercially, pharmacists and caregivers often expedite the adult formulation into a liquid formulation. As previously mentioned, this common practice often leads to poor medication adherence due to poor taste masking.

[0103] Tablets (200 mg HCQ / tablet) were ground into a fine powder and dissolved in MilliQ water to a final concentration of 10 mg HCQ / mL. MLVs containing a pH gradient were prepared and mixed with the HCQ suspension at a range of D / L ratios (5-20 wt%), followed by incubation at room temperature for 1 hour, after which the HCQ coating efficiency was measured. Figure 8As shown, MLV-HCQ prepared at a D / L of 15 wt% exhibited nearly complete drug coverage, while when the D / L was increased to 20 wt%, the efficiency dropped to approximately 85%. These data demonstrate that the MLV drug formulation kit can be used to support the efficient formulation of adult tablets into child-friendly liquid formulations using a simplified process.

[0104] Example 4. In vitro drug release study

[0105] Subsequently, the inventors examined whether CQ could be released from the MLV drug formulation of the present invention into the gastrointestinal fluid after oral administration, and conducted drug release studies in simulated saliva (SS), simulated gastric fluid (SGF), and simulated intestinal fluid (SIF) supplemented with 3mM sodium taurocholate to simulate the conditions of the oral cavity, stomach, and intestine, respectively. MLV-CQ was incubated with various simulated fluids at 37°C while shaking at 150 rpm. Drug release was measured by comparing the CQ concentration before and after SEC at selected time points. Figure 9 As shown, when the MLV-CQ drug formulation of the present invention was incubated with SS, no drug was released within 30 minutes, indicating that CQ was retained inside the MLVs in the oral cavity. As previously determined by the inventors, the unpleasant taste of a compound depends on the concentration of the drug in free or released form. Therefore, the data indicate that this MLV delivery system that can retain 100% of CQ will completely mask the bitter taste. Due to the short transit time of the formulation through the oral cavity, the inventors only monitored drug release for 30 minutes. Subsequently, the inventors examined drug release in SGF (pH 1.2). Figure 9 As shown, the formulation exhibited an instantaneous burst release rate (approximately 38%) after incubation with SGF, with a cumulative CQ release rate of approximately 50% after 4 hours. This burst release rate can be explained by the extremely acidic pH in SGF, where the phosphate groups of DSPC (pKa approximately 3) are protonated, leading to an imbalance between the hydrophobicity and hydrophilicity of the phospholipids and ultimately causing lipid bilayer instability. 6 Finally, CQ release was assessed in SIF supplemented with 3 mM sodium taurocholate, a bile salt commonly found in the intestine. MLV-CQ showed 20% drug release after 1 hour of incubation in SIF, followed by a gradual increase to 30% release within 4 hours. Figure 9 Bile salts can act as surfactants to disrupt the liposome membrane to release drugs, as previously demonstrated and described. 6 The data show that when the MLV-CQ pharmaceutical formulation of the present invention is administered orally, MLV can retain CQ, preventing it from contacting taste buds and masking its bitter taste. After entering the stomach and intestines, CQ is effectively released in the gastrointestinal tract due to the acidic environment and the presence of bile salt surfactants.

[0106] Example 5. Pharmacokinetics

[0107] A pharmacokinetic study was conducted to examine and compare the oral administration of MLV-CQ and CQ aqueous solution in vivo. CD-1 mice were orally administered with 10 mg / kg MLV-CQ (liquid) or CQ solution, and plasma was collected at 0.5, 1, 3, 4, 6, and 24 hours after administration to measure CQ concentrations. After oral administration, plasma CQ concentrations gradually increased for both MLV-CQ and CQ solution, reaching CQ at 3 hours. max , and then completely eliminated from plasma within 24 hours. Figure 10 and Figure 11 As shown in Figure 3, the plasma profiles of MLV-CQ and CQ solution were comparable; however, the C of MLV-CQ was significantly higher than that of CQ solution. max The AUC and AUC were 1.75-fold and 2-fold higher, respectively, indicating that MLV can enhance drug absorption. This data is consistent with previous reports demonstrating that lipids are effective absorption enhancers. 17

[0108] in conclusion

[0109] The inventors have developed MLV platform technology for manufacturing child-friendly oral liquid formulations of weak-base drugs. This platform can be developed into kits for pharmacists and caregivers to support the formulation of adult solid dosage forms into pediatric formulations. There is a high demand for solutions to medication adherence issues that contribute to treatment failure in this specific population.

[0110] Example of Clonidine-Loaded MLVs:

[0111] Materials and methods

[0112] Material

[0113] DSPC (18:0PC) and cholesterol (≥99%) were purchased from Sigma-Aldrich (Oakville, NO, Canada). Clonidine hydrochloride (≥98%) and other general laboratory chemicals were purchased from VWR Scientific (Mississauga, NO, Canada).

[0114] Hydration buffer solution

[0115] Buffer A contains 0.3 M citric acid solution (pH 1.6). Buffer B contains 0.3 M sodium citrate buffer (pH 3.6). Buffer C contains 0.3 M sodium citrate buffer (pH 5.5). Buffer D contains 0.3 M ammonium sulfate buffer (pH 5.0).

[0116] Preparation and Characterization of Clonidine-Loaded DSPC / Cholesterol Multilamellar Vesicles

[0117] A thin film hydration method was used to prepare clonidine-loaded DSPC / cholesterol multilamellar vesicles (MLVs). Here, the molar ratio between cholesterol and DSPC (CHOL / DSPC molar ratio), hydration buffer (buffer A, B, C, and D), and drug-lipid weight ratio (D / L weight ratio) were optimized for preparation. First, lipid mixtures with various CHOL / DSPC molar ratios (ranging from 95 / 5 to 45 / 55) were dissolved in chloroform, with total lipid concentrations ranging from 10 to 30 mg / mL. Next, rotary evaporation was used to remove the solvent at 400-500 rpm at 25-57°C. The resulting well-formed lipid film was hydrated with an equal volume of buffer solution under ultrasonic treatment to obtain a uniform emulsion suspension. For active drug loading, the suspension was initially adjusted to pH 8-9 using sodium carbonate solution (300 mg / mL). Subsequently, various amounts of clonidine hydrochloride were added to each suspension so that the drug-lipid weight ratio was maintained within the range of 1:4 to 1:10. The resulting mixture was then incubated for 1 hour at 37° C. Active drug loading was terminated by quenching the mixture on ice for 2 minutes, ultimately obtaining MLVs without the use of extrusion or dialysis procedures.

[0118] The encapsulation efficiency (EE, %) of each MLV sample was analyzed by UPLC and calculated using the following formula (3):

[0119]

[0120] Among them, M free refers to the amount of unencapsulated drug in the MLV suspension, and M0 is the amount of drug initially added to the suspension. Each sample was centrifuged at 20,000 g for 8 minutes, and the supernatant containing unencapsulated drug was analyzed by UPLC.

[0121] For long-term stability studies, freshly prepared MLVs were sealed and stored at 4°C for further analysis. Changes in total drug content, i.e., clonidine retention rate (RR, %) and MLV encapsulation efficiency (EE, %), were assessed at each time interval.

[0122] The retention rate (RR, %) of clonidine during storage was calculated using the following formula (4):

[0123]

[0124] Among them, M t = refers to the amount of clonidine remaining in the MLV suspension at each time point, and M0 is the amount of clonidine initially added to the suspension. An aliquot of MLVs was first dissolved in methanol and then centrifuged. The supernatant was collected to determine the amount of clonidine retained in the MLVs during storage.

[0125] Analysis of clonidine content using ultra-performance liquid chromatography (UPLC)

[0126] The clonidine concentration of each MLV sample was analyzed using an ACQUITY UPLC H-Class System (Waters, Milford, MA) coupled with a photodiode array (PDA) detector (wavelength 208 nm) and an evaporative light scattering detector (ELSD). Samples were separated on a Waters Acquity BEH-C18 column (particle size: 1.7 μm, inner diameter: 2.1 mm, column length: 100 mm) at a flow rate of 0.5 mL / min. The mobile phase consisted of solvent A (0.1% v / v trifluoroacetic acid in water) and solvent B (0.1% v / v trifluoroacetic acid in acetonitrile). The following gradient was applied: 0 min: A / B (95 / 5), 3.5 min: A / B (50 / 50), 3.8 min: A / B (0 / 100), 4.8 min: A / B (0 / 100), 5.3 min: A / B (95 / 5), 6.5 min: A / B (95 / 5). The concentration of free clonidine in the aqueous phase was determined by integrating the PDA peak at a retention time of 1.6 min. The concentration of clonidine dissolved in methanol was determined by integrating the ELSD peak at a retention time of 1.6 min. Data were analyzed using Empower 3.0 software (Waters).

[0127] result

[0128] Example 6: Preparation and characterization of MLV

[0129] Active drug loading is a rapid and effective method for encapsulating drugs within the core of liposome particles, thereby preventing them from contact with the external environment. Clonidine hydrochloride is a water-soluble drug with a bitter taste. Effective loading into the lipid bilayer not only masks the unpleasant taste of clonidine but also improves its stability. Here, DSPC / cholesterol MLVs were used for clonidine encapsulation. The key factors that affect drug encapsulation efficiency (EE) during MLV preparation and storage are discussed below, namely the CHOL / DSPC molar ratio, the hydration buffer (especially pH), and the drug / lipid weight ratio.

[0130] Table 1: Encapsulation efficiency of MLVs prepared using different CHOL / DSPC molar ratios and hydration buffers

[0131]

[0132]

[0133] Table 1 shows the EE of MLVs prepared at the same drug-to-lipid weight ratio (1:10). As shown in Table 1, the hydration buffer and its pH have a greater impact on drug loading efficiency than the CHOL / DSPC molar ratio. Hydration buffers with lower pH values ​​promote higher EE. For example, using hydration buffer A (pH 1.6), lipid films with different CHOL / DSPC ratios all exhibited EEs >95%. Furthermore, the lipid composition exhibited a stronger encapsulation effect for active drug loading. When the pH was increased to 5.5 (buffer C), MLVs with higher CHOL / DSPC molar ratios exhibited higher EEs.

[0134] Table 2: Encapsulation efficiency of MLVs prepared using different drug / lipid weight ratios and CHOL / DSPC molar ratios

[0135]

[0136]

[0137] The effect of the drug / lipid ratio on the EE of MLVs was considered. As shown in Table 2, a lower drug / lipid weight ratio not only resulted in higher encapsulation efficiency but also reduced drug input costs. Notably, at a drug / lipid ratio of 1:10, the EE ranged from 56-83%, while at a drug / lipid ratio of 1:5, the EE appeared more consistent, ranging from 70-80%. However, at a drug / lipid ratio of 1:4, the drug appeared to be in excess, resulting in a significant decrease in EE. Furthermore, a higher CHOL / DSPC molar ratio had a positive impact on EE, with the highest EE occurring at a DSPC:Chol ratio of 2:8.

[0138] Table 3: Effects of incubation temperature and other preparation parameters on active drug loading

[0139]

[0140]

[0141] Other parameters during active drug loading, such as incubation temperature and drug / lipid weight ratio, were also analyzed. As shown in Table 3, higher incubation temperatures promoted molecular motion during active loading, resulting in more efficient drug loading. The EE of each sample varied due to the complex influence of all preparation parameters.

[0142] Example 7: Stability of MLV

[0143] The long-term storage stability of MLV was evaluated by monitoring the changes in total drug content and EE during storage at 4°C. Figure 16As shown in Figure 2, the clonidine content in each sample did not change much during the 7-week storage period, with drug retention maintained at greater than 80%. However, the EE of each MLV changed significantly. Figure 17 As shown, most samples showed a decreasing EE trend during storage. In particular, after 4 weeks, samples prepared using buffers A and B exhibited EEs exceeding 95%. However, MLVs prepared using buffer C (pH 5.5) initially showed a sharp drop in EE to <60% within 2 weeks, followed by a gradual return to near 80%. These results further suggest that MLVs with higher pH values ​​hinder drug encapsulation during active loading.

[0144] References

[0145] 1.British National Formulary for Children|MedicinesComplete.https: / / about.medicinescomplete.com / publication / british-national-formulary-for-children / .Accessed May 17,2022.

[0146] 2.Preventing Medication Errors in Pediatric and Neonatal PatientsDefining medication errors.

[0147] 3. World Health Organization. Development of pediatric medicines: points to consider in pharmaceutical development (Working Document QAS / 08.257 / Rev.3). 2012; (August).

[0148] 4. Nunn AJ.Making medicines that children can take.Arch DisChild.2003;88(5):369-371.Doi:10.1136 / adc.88.5.369

[0149] 5.Cram A,Breitkreutz J,Desset-Brèthes S,Nunn T,Tuleu C.Challenges ofdeveloping palatable oral paediatric formulations.Int J Pharm.2009;365(1-2):1-3.Doi:10.1016 / J.IJPHARM.2008.09.015

[0150] 6.Tang WL,Tang WH,Chen WC,Diako C,Ross CF,Li SD.Development of aRapidly Dissolvable Oral Pediatric Formulation for Mefloquine UsingLiposomes.Mol Pharm.2017;14(6):1969-1979.Doi:10.1021 / ACS.MOLPHARMACEUT.7B00077 / SUPPL_FILE / MP7B00077_SI_002.AVI

[0151] 7.Rynes RI.Hydroxychloroquine treatment of rheumatoid arthritis.Am JMed.1988;85(4 SUPPL.1):18-22.Doi:10.1016 / 0002-9343(88)90357-9

[0152] 8.Tsakonas E,Joseph L,Esdaile JM,et al.A long-term study ofhydroxychloroquine withdrawal on exacerbations in systemic lupuserythematosus.Lupus.1998;7(2):80-85.Doi:10.1191 / 096120398678919778

[0153] 9.Ruiz-Irastorza G,Egurbide M V.,Pijoan JI,et al.Effect ofantimalarials on thrombosis and survival in patients with systemic lupuserythematosus.Lupus.2006;15(9):577-583.Doi:10.1177 / 0961203306071872

[0154] 10.James JA,Kim-Howard XR,Bruner BF,et al.Hydroxychloroquine sulfatetreatment is associated with later onset of systemic lupuserythematosus.Lupus.2007;16(6):401-409.Doi:10.1177 / 0961203307078579

[0155] 11.SisóA,Ramos-Casals M,BovéA,et al.Previous antimalarial therapy inpatients diagnosed with lupus nephritis:influence on outcomes andsurvival.Lupus.2008;17(4):281-288.Doi:10.1177 / 0961203307086503

[0156] 12.Pauli E,Joshi H,Vasavada A,Brackett J,Towa L.Evaluation of anImmediate-Release Formulation of Hydroxychloroquine Sulfate With anInterwoven Pediatric Taste-Masking System.J Pharm Sci.2020;109(4):1493-1497.Doi:10.1016 / J.XPHS.2019.12.014

[0157] 13.CDC.Medicines for the Prevention of Malaria While TravelingChloroquine.2007:1-2.

[0158] 14.Yu J,Zhou S,Li J,et al.Simple weak-acid derivatives of paclitaxelfor remote loading into liposomes and improved therapeutic effects.RSCAdv.2020;10(46):27676-27687.Doi:10.1039 / D0RA03190A

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Claims

1. A multilamellar vesicle active pharmaceutical ingredient (MLV-API) pharmaceutical preparation comprising multilamellar vesicles (MLV) and an active pharmaceutical ingredient (API) dissolved in an alkaline aqueous solution, Wherein the API comprises a weak base drug; The MLV carries the API; and The pH inside the MLV is lower than the pH of the alkaline aqueous solution outside the MLV.

2. The formulation of claim 1, wherein the drug / lipid ratio of the API-loaded MLV is at least about 15 wt%.

3. The formulation of claim 1, wherein the coverage of the API-loaded MLV is about 80% or higher.

4. The formulation of claim 1, wherein the pH inside the MLV is from about 1.0 to about 3.0, and the pH of the alkaline aqueous solution is from about 8.0 to about 10.

0.

5. The formulation of claim 1, wherein the API comprises chloroquine (CQ), a CQ derivative, clonidine, a clonidine derivative, imatinib, propranolol, pilocarpine, meloxicam, aripiprazole, chlorpromazine, R848, imiquimod, mefloquine, primaquine, or a pharmaceutically acceptable salt thereof.

6. The formulation of claim 1, wherein the MLV comprises lipid and cholesterol.

7. The formulation of claim 6, wherein the molar ratio of the cholesterol to the lipid is from about 1:0.2 to about 1:2.

2.

8. The formulation of claim 7, wherein the lipid comprises DSPC, DOPC, DOPS, DPPS, DSPS, DOPE, phospholipids, lysolipids, lysophospholipids, sphingolipids, pegylated lipids, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol (not limited to a specific sugar), or a combination thereof.

9. The formulation of claim 1, further comprising a cryoprotectant.

10. The formulation of claim 9, wherein the concentration of the cryoprotectant is about 15% to about 45%.

11. The formulation of claim 1, further comprising one or more compounds other than the API that are present in the alkaline aqueous solution but not encapsulated in the MLV, so that the oral pharmaceutical formulation of the present invention can provide combination therapy of two or more drugs.

12. The formulation of claim 11, wherein the one or more compounds comprises an adjuvant.

13. The formulation of claim 1, wherein the API-loaded MLVs have a diameter size of about 50 to about 500 nm.

14. The formulation of claim 1, wherein the polydispersity index of the API-loaded MLVs is from about 0.1 to about 0.

7.

15. The formulation of claim 1, wherein the API-loaded MLV has a zeta potential of about -15 to about 15 mV.

16. The formulation of claim 1, wherein the coating efficiency of the MLV-API pharmaceutical formulation can be maintained at ≥ about 80% after about 20 days.

17. The formulation of claim 1, wherein the coating efficiency of the MLV-API pharmaceutical formulation can be maintained at ≥ about 94% after about 12 days.

18. The formulation of claim 1, wherein the API-loaded MLV retains up to about 95% to about 100% of the API within the MLV when the API-loaded MLV is subjected to physiological conditions equivalent to those of a subject's oral cavity for up to 30 minutes.

19. The formulation of claim 1, wherein the API encapsulated within the MLV of the MLV-API pharmaceutical formulation is released from the MLV by up to about 30% to about 40% in less than about 120 seconds under physiological conditions equivalent to those of a subject's stomach.

20. The formulation of claim 1, wherein the API encapsulated within the MLV of the MLV-API pharmaceutical formulation is released from the MLV by at most about 30% to about 55% within 4 hours under physiological conditions equivalent to those in the stomach of a subject.

21. The formulation of claim 1, wherein the API encapsulated within the MLV of the MLV-API pharmaceutical formulation can be released from the MLV by at most about 15% to about 40% within 4 hours under physiological conditions equivalent to those in the intestinal tract of a subject.

22. The formulation of claim 1, wherein the drug absorption rate of the API-loaded MLV in a subject is about 1.2 to about 3.0 times greater than the drug absorption rate of the free form of the drug in the subject.

23. A method for preparing an MLV-API pharmaceutical preparation, comprising the following steps: a. forming an MLV by hydrating the organic component with an acidic solution; d. adding an alkaline aqueous solution to the MLV formed in step a to prepare an alkaline aqueous solution containing the MLV; e. dissolving the API in water to prepare an API solution; f. The API solution of step e is mixed with the alkaline aqueous solution containing MLV of step d to prepare an active loading solution, wherein a pH gradient is established between the inside and outside of the MLV to promote active loading of the API; g. culturing the active loading solution of step f, so that the API is actively loaded into the MLV to form an MLV loaded with API, wherein the API comprises a weak base; wherein the organic component comprises one or more lipids; and The preparation method does not use dialysis or extrusion process.

24. The method of claim 23, wherein the drug / lipid ratio of the API-loaded MLV is at least about 15 wt%.

25. The preparation method of claim 23, wherein when the drug / lipid ratio in the active loading solution of step f is about 5 to about 20 wt%, the encapsulation efficiency reaches 90% or higher.

26. The preparation method of claim 23, wherein when the drug / lipid ratio in the active loading solution of step f is about 15 to about 20 wt% and when the API is chloroquine (CQ), the encapsulation efficiency reaches 90% or higher.

27. The preparation method of claim 23, wherein when the drug / lipid ratio in the active loading solution of step f is about 5 wt% and when the API is Nile Blue (NB), the encapsulation efficiency reaches 90% or higher.

28. The preparation method of claim 23, wherein when the drug / lipid ratio in the active loading solution of step f is about 5 to about 15 wt % and when the API is hydroxychloroquine (HCQ), the encapsulation efficiency reaches 90% or higher.

29. The preparation method of claim 23, wherein when the drug / lipid ratio in the active loading solution of step f is about 10 to about 20 wt% and when the API is clonidine hydrochloride, the encapsulation efficiency reaches 90% or higher.

30. The preparation method of claim 23, wherein the culturing temperature in step g is between about 50°C and about 70°C.

31. The preparation method according to claim 23, wherein the organic component of step a is in the form of a film.

32. The method of claim 23, wherein the organic component of step a comprises DSPC, DOPC, DOPS, DPPS, DSPS, DOPE, phospholipids, lysolipids, phospholipids, neurolipids, PEGylated lipids, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol (not limited to a specific sugar), cholesterol, or a combination thereof.

33. The preparation method of claim 23, wherein the organic component of step a comprises cholesterol and lipid in a molar ratio of about 1:0.2 to about 1:2.

2.

34. The preparation method of claim 23, further comprising step b of adding a cryoprotectant and freeze-drying the MLVs formed in step a.

35. The preparation method according to claim 34, wherein the cryoprotectant is selected from sucrose, dextrose and trehalose or a combination thereof.

36. The preparation method of claim 34, wherein the concentration of the cryoprotectant is about 15% to about 45%.

37. The preparation method of claim 34, further comprising step c of storing the API-loaded MLVs at room temperature for up to 8 weeks after the freeze-drying step b and before performing steps d, e, f, and g, wherein when steps d, e, f, and g are performed after step c, a coverage rate greater than 90% can be achieved.

38. The preparation method of claim 23, wherein the pH of the acidic solution is about 1.0 to about 3.

0.

39. The preparation method of claim 23, wherein the API comprises chloroquine (CQ), CQ derivatives, clonidine, clonidine derivatives, imatinib, propranolol, pilocarpine, meloxicam, aripiprazole, chlorpromazine, R848, imiquimod, mefloquine and primaquine or pharmaceutically acceptable salts thereof.

40. The preparation method of claim 23, wherein the alkaline aqueous solution comprises sodium carbonate, potassium carbonate, calcium carbonate, magnesium carbonate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia, or a combination thereof.

41. The preparation method of claim 23, wherein the MLV-API pharmaceutical preparation comprises the preparation of claim 1.

42. A method of treating rheumatoid arthritis, lupus erythematosus or malaria, hypertension, ADHD, drug withdrawal, menopausal hot flashes, diarrhea, cramps and pain conditions comprising orally administering to a subject the MLV pharmaceutical formulation of claim 1.

43. The method of claim 42, wherein the subject is a child.

44. A kit for preparing an MLV-API pharmaceutical preparation, comprising an API, an alkaline aqueous solution, and freeze-dried MLV. wherein the API comprises a weak base drug in any form that is in powder form or capable of being ground into a powder form; wherein the three components are stored separately; and Mixing the three components results in active loading of the API into the MLVs without any additional ingredients and without the need for extrusion and dialysis processes to form an MLV-API pharmaceutical formulation.

45. The kit of claim 44, wherein the MLV-API pharmaceutical preparation comprises the composition of claim 1.

46. ​​The kit of claim 44, wherein the MLV-API pharmaceutical formulation prepared using the kit of claim 44 maintains a coverage of >80% for up to about 34 days at a storage temperature of about 4°C.

47. The kit of claim 44, wherein the MLV-API pharmaceutical formulation prepared using the kit of claim 44 has an encapsulation efficiency that is >94% for up to about 24 days at a storage temperature of about 4°C.

48. The kit of claim 44, wherein the MLV-API formulation prepared using the kit of claim 44 has an encapsulation rate of at least 90% after the lyophilized MLV of the kit of claim 45 is stored at room temperature between about 15°C and about 30°C and at a relative humidity between about 20% and about 80% for at least 8 weeks.

49. The kit of claim 48, wherein the MLV-API formulation prepared using the kit of claim 44 maintains at least 90% coverage when stored at 4°C for about 2 weeks.

50. The kit of claim 44, wherein the MLV-API preparation prepared using the kit of claim 44 has an encapsulation rate of about 90%, about 95%, about 96%, about 97%, about 98% or about 99%.

51. The kit of claim 44, wherein the drug / lipid ratio of the API-loaded MLV is at least about 15 wt%.

52. The kit of claim 44, wherein the API comprises chloroquine (CQ), a CQ derivative, clonidine, a clonidine derivative, imatinib, propranolol, pilocarpine, meloxicam, aripiprazole, chlorpromazine, R848, imiquimod, mefloquine, and primaquine, or a pharmaceutically acceptable salt thereof.

53. The kit of claim 44, wherein the alkaline aqueous solution comprises sodium carbonate, potassium carbonate, calcium carbonate, magnesium carbonate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium bicarbonate, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonia, or a combination thereof.

54. The kit of claim 44, wherein the lyophilized MLV comprises DSPC, DOPC, DOPS, DPPS, DSPS, DOPE, phospholipids, lysolipids, phospholipids, neurolipids, PEGylated lipids, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol (not limited to a specific sugar), cholesterol, or a combination thereof.

55. The composition of claim 44, wherein the pH inside the MLV is from about 1.0 to about 3.0 and the pH of the alkaline aqueous solution is from about 8 to about 10.

56. The kit of claim 44, wherein the molar ratio of cholesterol to DSPC is about 1:0.2 to about 1:2.2.

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