Multi-drug lipid nanoparticle compositions and related methods for extending drug levels in blood and lymphatic tissues

Abstract

The present invention provides multi-drug lipid nanoparticles that stably incorporate a variety of small molecule drugs having different hydrophobic and water soluble characteristics, as well as related methods of making and using the same. The disclosed compositions and methods provide enhanced stability of lipid nanoparticle pharmaceutical formulations that can reliably provide drugs that cope with targets of different mechanisms, with prolonged presence in vivo, for more effective treatment and avoidance of single drug resistance.

Description

[0001] This application is a divisional application of the patent application for invention with the application number 201680034823.3 and the invention name "Multidrug Lipid Nanoparticle Compositions and Related Methods for Prolonging Drug Levels in Blood and Lymphoid Tissue", which entered the Chinese national phase on December 14, 2017, from the PCT international application PCT / US2016 / 037651 filed on June 15, 2016.

[0002] Cross - Reference to Related Applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 175,565, filed on June 15, 2015, the entire content of which is incorporated herein by reference.

[0004] Statement of Government Licensing Rights

[0005] This invention was made with government support under grants AI077390 (S1, S2, and S3), P51OD010425, RR00166, RR025014, UL1-TR000423, and UM1-AI120176, awarded by the National Institutes of Health. The government has certain rights in the invention. Background

[0006] Combination therapy with multiple drugs has become the standard-of-care for treating diseases such as those caused by human immunodeficiency virus (HIV) infection, and there is increasing evidence that it is superior to monotherapy in treating cancer. Combination antiretroviral therapy (cART) for HIV infection consists of a daily regimen of multiple orally administered antiretroviral drugs with different viral targets, and its benefits in reducing drug resistance and increasing treatment efficacy are well established. However, due to the challenges of non-compliance and related viral relapse in currently treated cART patients, there is an urgent need to develop long-acting anti-HIV drug technologies that can deliver combination therapy on a weekly or less frequent basis. Even when implementing a single oral tablet or capsule containing multiple drugs as standard cART to reduce pill burden, each drug in its free form naturally has a different pharmacokinetic profile, presenting difficulties in maintaining consistent effective blood drug concentrations of each drug to optimally inhibit HIV without promoting drug resistance. In addition, the penetration of oral combination drugs into lymph nodes and other lymphoid tissues is poor, resulting in low and inconsistent intracellular lymph drug concentrations. The drug is insufficient to reach the residual virus in patients receiving cART, even when the virus in the patient's blood is low or undetectable, which can lead to a resurgence of viral levels.

[0007] Even when seemingly similar lipids or lipid excipients are used in drug-nanoparticle formulations, the preparation methods and resulting drug-excipient interactions can yield different drugs with unique pharmacologies, toxicologies, and distributions in target or clearance tissues in vivo. The standard process of incorporating drugs into enclosed membranes, such as liposomes, is well known. However, while liposomes and other enclosed membranes have been proposed as general carriers for lipophilic drugs (incorporated into the lipid shell) and water-soluble drugs (encapsulated within the spherical interior), the incorporation and encapsulation of many drugs are difficult.

[0008] Even when drugs are successfully bound to lipid particles, the resulting particles are often not stable enough for product development. Liposomal encapsulation of hydrophilic compounds has proven particularly difficult, including nucleoside analog reverse transcriptase inhibitors (RTIs), such as tenofovir (TFV), lamivudine (3TC), and emtricitabine (FTC), which are key components of first-line cART. Due to the large external aqueous space relative to the entrapped internal aqueous compartment of small unilamellar vesicles, the encapsulation efficiency of small hydrophilic molecules in conventional liposomes with a neutral charge is typically very low, often less than a few percent. Attempts to increase TFV capture require altering the membrane contents with positively charged fatty acids. Not only are fatty acids readily removed from the liposome membrane by proteins in serum, rendering the liposome carrier unstable and ineffective, but the positively charged cationic particles also interact with red blood cells and other cells in vivo, resulting in particle instability and cytotoxicity. In fact, the toxicity issues associated with positively charged lipids have been a major obstacle to the clinical application of cationic non-viral vectors 17.

[0009] Even for hydrophobic HIV drugs that should be more easily introduced into the lipid membrane, optimization studies using different lipid compositions have yielded incomplete and uneven incorporation of two HIV drugs into liposomes. In addition, these liposomes, even those including polyethylene glycol modification to improve stability, readily release drugs when incubated in only 10% serum, with approximately 80% of the drug released within the first hour of incubation. The rapid destabilization of liposome-bound drugs renders these particles ineffective for transporting drugs from the injection site to the target tissue. While solid polymer particles can incorporate a variety of hydrophobic drugs with efficiencies up to 81%, these particles are even larger than liposomes and lack an aqueous compartment, limiting their utility for accommodating multiple drug combinations. In addition, these large polymer drug carriers and smaller quantum dots are typically trapped at the local injection site and released slowly, rather than being transported to lymphoid tissues and cells as individual drug-particle units.

[0010] Numerous variations and alternatives to the standard liposome assembly process have been reported. For example, in reverse evaporation vesicles (REV), multilamellar vesicles, unilamellar vesicles, the ethanol injection liposome preparation method is used. These formulations can provide a high incorporation of single drug molecules, especially for lipophilic drugs. However, the ability to trap water-soluble hydrophilic drugs in solution is variable and depends on the amount and charge of the drug molecules trapped within the liposome-encapsulating membrane environment.

[0011] Based on the ability of the liposome membrane to trap charged molecules such as (NH4)2SO4, and the membrane permeability of doxorubicin hydrochloride, the in-liposome precipitation of doxorubicin sulfate provides a method for efficient drug loading in liposomes. However, this method, called remote loading, is only applicable to a limited number of drugs that are permeable and have counterions showing low solubility. Unfortunately, not all drugs are suitable for remote loading into liposomes.

[0012] To improve the encapsulation efficiency of liposomes and lipid-drug nanoparticles on a pharmaceutical scale, microcapsule carrier methods using single or double emulsion methods have been used with little success. For example, the microemulsion method produces varying degrees of reproducible drug incorporation levels and requires the removal of residual organic solvents from water in the final step, which can be difficult and may pose a toxicity risk if the removal process is incomplete. In terms of single agent incorporation, the drug of interest must be added to the organic (oil) phase (for hydrophobic drugs) or the aqueous phase (for water-soluble hydrophilic drugs). This process is difficult to incorporate multiple drugs, especially drugs showing different physical characteristics - hydrophobic drugs and hydrophilic drugs. A variation of the microemulsion method is the double emulsion method, where the drug and a precipitant such as KCl are placed in two separate water-in-oil (o / w / o) emulsions with lipid excipients and surfactants. When the two o / w / o emulsions are mixed, drug-KCl particles form nano-drug precipitates. These nano-precipitates with a single-layer lipid coating are subjected to a second step of coating in an organic or w / o emulsion of lipids such as cholesterol and DOTAP. The resulting nanoparticle drug incorporation efficiency is very low. For example, for tenofovir (also known as PMPA), the final drug binding % is less than 3%. In addition, this method is designed for signal drug nanoparticle formation. Although the drug loading amount may be high based on the lipid-to-drug ratio, the percentage of drug loss based on the drug portion bound from start to end is low.

[0013] To improve the incorporation of water-soluble drugs carrying a charge, positively charged lipids such as 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP) have been included in the lipid membrane for electrostatic interaction. Unfortunately, the positive charge causes a toxicity risk in animals, and the resulting particle size (greater than 100 nm in diameter) is more likely to be rapidly cleared and eliminated from the body. Similarly, incorporation of a positively charged fatty acid, stearylamine (SA), into phosphatidylcholine lipids can increase the binding of hydrophilic drugs such as TFV to large particles up to ~2000 nm in diameter to 70%. However, the positively charged particles have a positive ζ potential of 53 - 93 mV and also show mitochondrial toxicity. Thus, compositions incorporating charged lipids to facilitate the incorporation of hydrophilic drugs are unlikely to be suitable for clinical development.

[0014] Despite progress in the art, there remains a need for methods to effectively and inexpensively incorporate a variety of structurally different drugs into a single drug delivery vehicle that provides stable delivery to the intended tissues and targets in the body. In the case of HIV treatment, there remains a need for a drug delivery vehicle that can maintain drug concentration in the plasma and deliver the drug to sites of persistent viral infection in lymphoid tissues. The present invention is directed to these and related needs.

[0015] Overview

[0016] This summary is provided to introduce a series of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify the key features of the claimed subject matter nor is it intended to be used to assist in determining the scope of the claimed subject matter.

[0017] In one aspect, the present invention provides multi-drug lipid nanoparticles. The multi-drug lipid nanoparticles comprise:

[0018] a first small molecule pharmaceutical agent having a log P greater than 1 at 25 °C;

[0019] a second small molecule pharmaceutical agent having a log P less than 0 at 25 °C;

[0020] a first amphiphilic excipient, wherein the first amphiphilic excipient is a lipid comprising a hydrophilic domain having a molecular weight less than 300 grams per mole; and

[0021] a second amphiphilic excipient comprising a hydrophilic domain having a molecular weight greater than 500 grams per mole.

[0022] In another aspect, the present invention provides a method for preparing multi-drug lipid nanoparticles. The method of this aspect comprises:

[0023] Dissolve a first small molecule agent having a log P greater than 1 at 25 °C, a first amphiphilic excipient, a first amphiphilic excipient, and a second amphiphilic excipient in an organic solvent to provide an organic solvent solution, wherein the first amphiphilic excipient is a lipid comprising a hydrophilic domain having a molecular weight less than 300 g / mol; the second amphiphilic excipient comprises a hydrophilic domain having a molecular weight greater than 500 g / mol, wherein the organic solvent comprises a co-component miscible with water;

[0024] Dissolve a second small molecule agent having a log P less than 0 at 25 °C in an aqueous solvent to provide an aqueous solvent solution;

[0025] Mix the organic solvent solution and the aqueous solvent solution to provide a mixed solvent solution;

[0026] Remove the mixed solvent from the mixed solvent solution to provide a dehydrated product comprising the first small molecule agent, the first amphiphilic excipient, the second amphiphilic excipient, and the second small molecule agent; and

[0027] Rehydrate the dehydrated product in an aqueous solution to provide a solution having multi-drug lipid nanoparticles.

[0028] On the other hand, the present invention provides a method for preparing multi-drug lipid nanoparticles. The method of this aspect includes:

[0029] Dissolve in a miscible solvent comprising an organic component and an aqueous component in a ratio of about 20:1 to about 40:1 (v / v):

[0030] A first small molecule agent having a log P greater than 1 at 25 °C,

[0031] A second small molecule agent having a log P less than 0 at 25 °C,

[0032] A first amphiphilic excipient, wherein the first amphiphilic excipient is a lipid comprising a hydrophilic domain having a molecular weight less than 300 g / mol, and

[0033] A second amphiphilic excipient, which comprises a hydrophilic domain having a molecular weight greater than 500 g / mol,

[0034] Remove the miscible solvent to provide a dehydrated product comprising the first small molecule agent, the second small molecule agent, the first amphiphilic excipient, and the second amphiphilic excipient;

[0035] Heat the dehydrated product to a first temperature at least 3 °C higher than the gel-to-liquid phase transition temperature of the amphiphilic excipient; and

[0036] Rehydrate the dehydrated product in an aqueous solution to provide a solution containing multi-drug lipid nanoparticles.

[0037] The present invention also includes any multi-drug lipid nanoparticles prepared by the methods disclosed herein.

[0038] On the other hand, the present invention provides a pharmaceutical formulation comprising the multi-drug lipid nanoparticles disclosed herein.

[0039] On the other hand, the present invention provides a method of treating a subject infected with HIV, which comprises administering an effective amount of the multi-drug lipid nanoparticles or formulation disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Since the above aspects of the present invention and many attendant advantages become better understood when considered in conjunction with the accompanying drawings, and thus become more readily understood, wherein:

[0041] Figure 1A and 1B is a schematic diagram of an exemplary method for preparing multi-drug combination nanoparticles stabilized with a lipid excipient. Figure 1A is a schematic diagram of an embodiment of a method herein referred to as the "mixed double solvent" method. Figure 1B is a schematic diagram of an embodiment of a method herein referred to as the "single miscible solvent" method.

[0042] Figure 2 Illustrates the effects of atazanavir (ATV) and darunavir (DRV) on lipid phase transition behavior. The effects of the anti-HIV drugs atazanavir (ATV: ○) or darunavir (DRV: □) on lipid phase transition behavior were monitored by fluorescence anisotropy using the membrane polarity probe DPH. The DPH anisotropy data were plotted against temperature (°C). The lipid mixture without drug was also evaluated as a control (△). The data were fitted with the non-linear regression model described in the Materials and Methods. The midpoints representing the phase transition temperature (Tc) of each lipid-drug composition and the control were estimated. The estimated Tc for the control, ATV, and DRV lipid mixtures were 54.9, 54.2, and 5, respectively.

[0043] Figure 3A and 3B Illustrates the concentration- and time-dependent release of atazanavir and darunavir from lipid-drug nanoparticles. The release of atazanavir ( Figure 3A ) and darunavir ( Figure 3B ) from lipid-drug nanoparticles was monitored at 25°C at 5 mM (●), 25 mM (■), and 200 mM (▲). The data are represented as the mean ± SD% of drug release for samples in triplicate.

[0044] Figure 4Illustrated is the pH-dependent release of atazanavir from atazanavir lipid nanoparticles (ATV-LNP). The percentage of total atazanavir in ATV-LNP released after exposure to the designated pH was measured at 25 °C (○) and 37 °C (●). Data for each pH and temperature are represented as the mean ± SD% of total atazanavir release from triplicate samples.

[0045] Figures 5A - 5F Illustrated is the time course of plasma drug concentrations in two primates administered anti-HIV lipid nanoparticles containing a combination of three drugs, atazanavir (ATV), ritonavir (RTV), and tenofovir (TFV). Two primates, M11016 ( Figure 5A 、 5C 、5E) and M10088 ( Figure 5B 、 5D 、5F), were administered subcutaneously with anti-HIV lipid nanoparticles containing ATV, RTV, and TFV (25, 12.8, and 15.3 mg / kg, respectively). Plasma drug concentrations of ATV ( Figure 5A 、 5B ), RTV ( Figure 5C 、[[ID=!19]] 5D ), and TFV ( Figure 5E 、 5F ) were determined at the designated time points.

[0046] Figures 6A - 6F Illustrated is the plasma concentration over time after subcutaneous administration of lopinavir (LPV), ritonavir (RTV), and tenofovir (PMPA) at standardized doses of 25 mg / kg LPV, 14.3 mg / kg RTV, and 17.1 mg / kg PMPA in free ( Figure 6A 、 6C 、6E) or lipid nanoparticle ( Figure 6B 、 Figure 6D 、 Figure 6F ) formulations. Data points represent individual animals (circle, M10066; triangle, M10068; square, R10142; diamond, Z11084; open, free drug; solid, LNP). Time points with mean values less than 10 ng / ml are annotated. *Outlier omitted: 3285.76 ng / ml for animal M10068 at 8 h.

[0047] Figures 7A - 7C Illustrated is lopinavir (LPV) ( Figure 7A ), ritonavir (RTV) ( Figure 7B ), and tenofovir (PMPA) ( Figure 7CMean intracellular concentrations over time in peripheral blood mononuclear cells (PBMC) after subcutaneous administration at the standardized doses of 25 mg / kg LPV, 14.3 mg / kg RTV, and 17.1 mg / kg PMPA in free (hollow symbols) or lipid nanoparticle (solid symbols) formulations. Concentrations are shown in ng / ml (left y-axis) and nmol / l (right y-axis). Error bars show the standard error of the mean (SEM). For free drug, n = 4, except n = 2 at 48 h and 120 h. For anti-HIV LNP, n = 3, except n = 1 at 48 h and 120 h. *SEM for anti-HIV LNP could not be calculated at the 48 h and 120 h time points due to n = 1.

[0048] Figures 8A through 8C Illustrated are the physical characteristics of multi-drug lipid nanoparticles prepared by the disclosed single miscible solvent method ( Figure 8A ), and the mixed double solvent method ( Figure 8B ), and typical liposomes ( Figure 8C ). The illustrated polyethylene glycolylated lipids retain the hydrophilic drugs present during assembly but exclude or inhibit the incorporation of additional agents into the unstirred corona formed. Figure 8A This high ratio of tight binding of hydrophilic drugs requires well-mixed agents and excipients and complete dehydration prior to rehydration to form drug lipid nanoparticles. The use of the mixed double solvent method ( Figure 8B ) appears to result in relatively loose binding of hydrophilic agents. Thus, upon shear force from sucrose gradient centrifugation, the drug binding % is substantially reduced to approximately 7 - 20% relative to 78 - 85%. The ability of typical liposomes to encapsulate hydrophilic drugs is much lower and is limited by the excluded or unstirred water (hydration) shell ( Figure 8C ). Thus, the binding of hydrophilic drugs such as tenofovir (TFV) is approximately 3 - 5%, a value similar to those reported by others for those with similar lipid compositions.

[0049] Figures 9A - 9C Illustrated is the time course of individual drug concentrations in four primate macaques (M. nemestrina) after administration of multi-drug lipid nanoparticles containing lopinavir ( Figure 9A ), ritonavir ( Figure 9B ), and tenofovir ( Figure 9C ) (molar ratio of 2:1:3). Further analysis showed that over 90% of the drug in plasma could be attributed to the lipid-drug particle-bound form. In contrast, as previously described, after administration of the unformulated free drug, each drug dropped below detectable levels in plasma by 24 h.

[0050] Figure 10Illustrated is the separation of three drugs, lopinavir (LPV), ritonavir (RTV), and tenofovir (TFV or PMPA), formulated in lipid drug particles. The lipid drug combination mixture was subjected to 5 - 20% sucrose gradient centrifugation to separate the bound fraction from the free fraction of the three drugs. Fractions 2 - 10 were used as the bound fraction, while fractions 11 - 14 (with low sucrose density at the top) were used as the free fraction. The results showed the following percentages of binding to lipid drug particles: lopinavir (LPV) = 81.8%; ritonavir (RTV) = 76%; tenofovir (TFV or PMPA) = 75.5%.

[0051] Detailed Description

[0052] The present invention provides a novel, simple, and clinically useful method for preparing multi-drug lipid-stabilized nanoparticles. This method produces novel lipid nanoparticles that incorporate drug compounds with different structural features into a single delivery vehicle, resulting in sustained and enhanced plasma and intracellular drug concentrations for all bound drugs in vivo. The use of the disclosed method and the resulting multi-drug lipid nanoparticle compositions offers the potential to overcome drug deficiencies by providing a sustained-release pharmacokinetic profile of multiple drugs simultaneously at a single target site. For antiviral applications, the disclosed method and compositions can overcome residual infections in tissues and avoid the possibility of developing drug resistance to single-drug regimens. Although our studies have thus far focused on anti-HIV drug combinations, the various methods and compositions disclosed herein can be readily applied to the preparation of multi-drug therapeutic agents for treating cancer and other diseases.

[0053] As described in more detail in the following examples, the present inventors have developed new methods for preparing new lipid nanoparticle carriers that can incorporate high levels of small molecule agents having generally different structural characteristics, i.e., hydrophilic and hydrophobic agents. The disclosed methods can be applied to many different small molecule drugs. Thus, the resulting multi-drug lipid nanoparticles facilitate acting as a single carrier that can surprisingly result in high and prolonged levels of all incorporated drugs in the plasma and tissues of a subject. In addition, the disclosed multi-drug lipid nanoparticles have the beneficial feature of avoiding the toxicity issues associated with positively charged lipid excipients (as described above) and exhibit significantly stable storage characteristics. Further, the method of assembling the multi-drug lipid nanoparticles is reproducible, efficient, easily scalable, and avoids the need for expensive removal of unincorporated drug compositions, which is typically required for formulation safety. In the case of anti-HIV formulations, the method provides new compositions that allow for the combination of relevant combination formulations that include high levels of hydrophilic agents that are critical for anti-HIV cART regimens. The sustained plasma and lymphoid tissue drug levels provided by the disclosed multi-drug lipid nanoparticles can reduce the frequency of dosing, e.g., on the scale of weeks, to overcome the non-compliance issues seen with current daily oral medications.

[0054] Indispensable to the disclosed method is that the inventors have successfully designed and implemented a unique solvent method that allows for the simultaneous combination and mixing of hydrophilic and hydrophobic components during lipid nanoparticle assembly. Many organic solvents used to form lipid nanoparticles are immiscible with water, and these organic solvents have presented the aforementioned challenges in incorporating hydrophilic agents into lipid nanoparticles to date. However, the inventors have found that combining such water-immiscible organic solvents with water-miscible organic solvents, such as alcohols, enables the additional incorporation of a limited amount of aqueous solution containing hydrophilic agents while maintaining a substantially single phase. The ability to incorporate the aqueous solution and its components is provided by, and ultimately limited by, the ability of the water-miscible organic solvent to be saturated with water. The resulting single-phase solution enables the complete mixing of all components of the multi-drug lipid nanoparticles, both hydrophilic and hydrophobic. Dehydration removes the solvent and forces the close binding of all the different components. This method reduces the physical repulsion between drugs derived from lipid excipients, whether hydrophobic or hydrophilic drugs. The close contact also overcomes any need to add positively charged lipid excipients to increase the binding of drugs to the particles and reduces the toxicity risk associated with positive charges. Subsequently, the homogeneous mixture of components in an aqueous buffer is rehydrated to produce nanoscale lipid particles that incorporate a higher total drug-to-lipid ratio than conventional liposomes, including hydrophobic and hydrophilic agents, without harmful residual detergents or solvents. The larger particles are sized down to a final uniform size, such as 20 nm - 100 nm, by sonication, extrusion through a membrane filter, or mechanical methods, such as an emulsifier or a microfluidizer, to optimize stability and avoid in vivo clearance.

[0055] An exemplary method is herein referred to as the "mixed dual-solvent" method, as described in more detail in Examples 1 - 4 below. See also Figure 1A which schematically illustrates the exemplary method. In this method, excipients (lipids or others) and hydrophobic agents are dissolved in an organic solvent having water-miscible and water-immiscible components. Hydrophilic agents (multiple hydrophilic agents) are additionally mixed in an aqueous solvent. Since the organic solution has the ability to be saturated with water due to the water-miscible organic component, the organic solution and the aqueous solution are mixed together to form a mixed dual-solvent phase. Although a low level of initial emulsion may be produced, the mixed dual-solvent ultimately forms a single phase that is completely mixed with the multi-drug lipid nanoparticles. After removing the mixed solvent by drying and rehydrating the dried phase, the actual multi-drug lipid nanoparticles are formed.

[0056] An alternative exemplary method, herein referred to as the "single miscible solvent" method, is described in Example 5. See also Figure 1B, which schematically illustrates an exemplary method. In this alternative, a single miscible solvent is provided, which comprises a water-immiscible organic component, a water-miscible organic component, and a minor aqueous component. As described above, the ability to maintain miscibility (i.e., to keep the water well mixed without phase separation) is limited by the amount of water in the solution and the ability of the water-miscible organic component to be saturated with water. Once formed, all components of the multi-drug lipid nanoparticles can be fully and thoroughly mixed in the same solution. The solvent is completely removed to provide a homogeneous and fully mixed dry product. The dry product is rehydrated in an aqueous buffer to form lipid nanoparticles loaded with both high levels of hydrophilic and hydrophobic agents.

[0057] Generally speaking, this new method requires fewer steps, is scalable, yields nanoparticles with a high multi-drug binding rate, even for nanoparticles showing extreme differences in hydrophobic or hydrophilic characteristics. In addition, the final product is an aqueous buffer, free of harmful solvents or positively charged excipients, suitable for most drug applications. Finally, it is not necessary to remove unbound portions of water-soluble drugs. Ultimately, the disclosed method significantly reduces the risk of contamination due to multiple filtrations or solvent removal and further reduces the costly losses associated with removing unbound drugs.

[0058] In view of the above, on the one hand, the present invention provides multi-drug lipid nanoparticles. The multi-drug lipid nanoparticles comprise: a first small molecule agent with a log P greater than 1 at 25 °C, a second small molecule agent with a log P less than 0 at 25 °C, a first amphiphilic excipient, and a second amphiphilic excipient, wherein the first amphiphilic excipient is a lipid comprising a hydrophilic domain with a molecular weight less than 300 grams per mole; the second amphiphilic excipient comprises a hydrophilic domain with a molecular weight greater than 500 grams per mole.

[0059] As described herein, the disclosed multi-drug lipid nanoparticles can advantageously incorporate both hydrophilic and hydrophobic therapeutic agents to provide an effective carrier for specific combinations. Hydrophobic / lipophilic or substantially water-insoluble pharmaceutically active agents can be any bioactive agent with limited solubility in an aqueous or hydrophilic environment. For example, the solubility of these agents in water at 20 - 25 °C can be less than about 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0.02, or 0.01 mg / mL. Hydrophilic or substantially water-soluble pharmaceutically active agents can be any bioactive agent with high solubility in an aqueous or hydrophilic environment. For example, the solubility of these agents in water at 20 - 25 °C can be greater than about 5, 10, 20, 30, 40, 50, 75, 100, or more mg / mL.

[0060] Characteristics of hydrophilic and hydrophobic therapeutic agents suitable as candidate therapeutic agents incorporated into the lipid nanoparticle formulations of the present invention include therapeutic agents defined by their octanol / water partition coefficient X log P (Wang et al., Chem. Inf. Comput. Sci., 1997, 37, 615 - 621, the entire content of which is incorporated herein by reference). In the practice of the present invention, therapeutic small molecule agents with a log P greater than 1.0 are excellent candidates for incorporation as hydrophobic (or lipophilic) agents into the nanoparticles of the present invention. Examples of such anti-HIV agents are listed in Table 18 below. Thus, as used herein, the terms "hydrophobic", "lipophilic", and "substantially insoluble in water" refer to therapeutic small molecule agents with an octanol / water partition coefficient log P greater than 1.0. In some embodiments, useful hydrophobic small molecule agents have a log P greater than about 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, and 6. In the practice of the present invention, therapeutic small molecule agents with a negative log P are excellent candidates for incorporation as hydrophobic (or lipophilic) agents into the nanoparticles of the present invention. Examples of such anti-HIV agents are listed in Table 19 below. Thus, as used herein, the terms "hydrophilic" and "substantially soluble in water" refer to therapeutic small molecule agents with an octanol / water partition coefficient log P less than zero. In some embodiments, useful hydrophobic small molecule agents have a log P less than about -0.25, -0.5, -0.75, -1.0, -1.25, -1.5, -1.75, -2.0, -2.5, -3.0, and -3.5.

[0061] In some embodiments, log P is determined at about 25 °C.

[0062] As used herein, the term "small molecule agent" refers to a therapeutic molecule having a molecular weight of about 1500 grams per mole or less. For example, any small molecule agent can have a molecular weight of about 1500, 1250, 1000, 900, 800, 700, 600, 500, 400, 300, 200 g / mol or less.

[0063] Exemplary anti-HIV small molecule agents are listed in Tables 18-21, but it should be understood that the disclosed nanoparticles include any small molecule for combating other diseases, disorders or infections. In some embodiments, the first small molecule agent is selected from rilpivirine (RPV) or its prodrug, efavirenz (EFV) or its prodrug, dolutegravir (DTG) or its prodrug, indinavir (IDV) or its prodrug, atazanavir (ATV) or its prodrug, ritonavir (RTV) or its prodrug, lopinavir (LPV) or its prodrug, etc. In some embodiments, the second small molecule agent is selected from tenofovir (TNF) or its prodrug, emtricitabine (FTC) or its prodrug, lamivudine (3TC) or its prodrug, raltegravir (RAL) or its prodrug, zidovudine or its prodrug, etc. Those of ordinary skill in the art can readily identify prodrugs of relevant therapeutic small molecule agents. Additionally, those of ordinary skill in the art will readily recognize that small molecule agents are not limited to a particular structure and eligible candidates for use in practicing the disclosed lipid nanoparticles and related methods can be readily identified.

[0064] In some embodiments, the molar ratio of the total small molecule agents to the excipient of the multi-drug lipid nanoparticles is at least 1:10, the total small molecule agents including the first and second small molecule agents, and the excipient including the first and second amphiphilic excipients. In some embodiments, the molar ratio of the total small molecule agents to the total amphiphilic excipients is 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3 or higher. In some embodiments, the molar ratio of the total small molecule agents to the total amphiphilic lipid excipients is 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3 or higher. In this regard, as described in more detail herein, multi-drug lipid nanoparticles prepared by the double mixed solvent method are characterized by a small molecule drug to lipid excipient ratio of about 1:8 to about 1:3. Multi-drug lipid nanoparticles prepared by the single miscible solvent method are characterized by a small molecule drug to lipid excipient ratio of about 1:3. Both of these ranges indicate significantly higher drug incorporation than typical liposomal formulations, the drug to lipid ratio of which does not exceed 1:10 and is typically much lower.

[0065] The disclosed multi-drug lipid nanoparticles can include any relevant range of relative concentrations of a first and a second small molecule agent determined by the intended use of the multi-drug lipid nanoparticles. By way of illustration, the molar ratio of the first small molecule agent to the second small molecule agent in the multi-drug lipid nanoparticles can be from about 1:20 to about 20:1, such as 1:9, 1:18, 1:17, 1:16, 1:15, 1:14, 1:13, 1:2, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, etc.

[0066] The disclosed multi-drug lipid nanoparticles will generally have at least two different types of amphiphilic excipients. The first amphiphilic excipient is a lipid molecule that contains a small hydrophilic domain. This type generally provides most of the structure of the lipid nanoparticles. When in an aqueous environment, the small hydrophilic domain helps various lipid excipients to orient into a generally spherical structure. The small hydrophilic domain generally has a molecular weight of less than about 300 grams per mole. In some embodiments, the molecular weight of the small hydrophilic domain is less than about 275, 250, 225, 200, 175, 150, 125, 100, and 75 grams per mole.

[0067] In some embodiments, the first amphiphilic excipient can be selected from the following types: phospholipids, sphingolipids, cholesterol and steroid derivatives, bile acids and derivatives, cardiolipin, acyl glycerol esters and derivatives, glycolipids, acyl peptides, and fatty acids. Phospholipids can be selected from the following types: distearoyl phosphatidylcholine (DSPC); dipalmitoyl phosphatidylcholine; dimyristoyl phosphatidylcholine; dioleoyl phosphatidylcholine; transesterified phospholipids derived from eggs, soybeans, flaxseeds, etc.; phosphatidylethanolamine; phosphatidylglycerol; phosphatidylserine; and phosphatidic acid.

[0068] In some embodiments, the first amphiphilic excipient is a fatty acid or a fatty acid derivative, as understood in the art. In some embodiments, the fatty acid has an ionized carboxylic acid at neutral pH.

[0069] In some embodiments, the phospholipids are selected from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), dipalmitoyl phosphatidylcholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), etc.

[0070] In some embodiments, the first amphiphilic excipient is stearic acid or oleic acid, or includes stearic acid or oleic acid.

[0071] In some embodiments, the first amphiphilic excipient comprises at least a first fatty acid tail domain having at least 14 carbons. In some embodiments, the first amphiphilic excipient further comprises a second fatty acid tail domain having at least 14 carbons. In some embodiments, at least the first fatty acid tail domain and optionally the second fatty acid tail domain comprise 14, 16, 18, 20, and 22 carbon atoms. The first fatty acid tail domain can be fully saturated (i.e., having the maximum number of hydrogens and no carbon-carbon double bonds) or unsaturated (i.e., having any number of carbon-carbon double bonds). In some embodiments, the first fatty acid tail domain has one carbon-carbon bond or no carbon-carbon bonds. In some embodiments, the first fatty acid tail domain has one, two, three, or more carbon-carbon double bonds. In some embodiments, the first amphiphilic excipient comprises at least a first fatty acid tail domain and a second fatty acid tail domain, wherein at least one of the first and second fatty acid tail domains has one carbon-carbon bond or no carbon-carbon bonds. In one embodiment, the first amphiphilic excipient comprises at least a first fatty acid tail domain and a second fatty acid tail domain, wherein both the first and second fatty acid tail domains have one carbon-carbon bond or no carbon-carbon bonds. In one embodiment, at least one of the first and second fatty acid tail domains has no carbon-carbon double bonds (i.e., is fully saturated). In one embodiment, both the first and second fatty acid tail domains have no carbon-carbon double bonds (i.e., are fully saturated).

[0072] The first amphiphilic excipient can be characterized by a gel-to-liquid phase transition temperature, which is the temperature at which the aggregates of the excipient (e.g., nanoparticles) begin to depolymerize and transform into a liquid solution. The disclosed multi-drug lipid nanoparticles typically incorporate a first amphiphilic excipient having a gel-to-liquid phase transition temperature of at least 37 °C. In some embodiments, the gel-to-liquid phase transition temperature exceeds 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, or 70 °C (or any intermediate temperature therebetween). In some embodiments, the gel-to-liquid phase transition temperature of the excipients of a combination of lipid nanoparticles can also be determined (i.e., also taking into account the properties and amounts of a second amphiphilic excipient).

[0073] The second amphiphilic excipient comprises a large hydrophilic domain relative to the first amphiphilic excipient. Due to the incorporation of the second amphiphilic excipient into the lipid nanoparticles, the large hydrophilic domain typically extends beyond the hydrophobic component (e.g., mainly the lipid region) into the extracellular space of the particles. The structure of the hydrophobic domain can be highly variable and thus does not limit the scope of the disclosed nanoparticles. These structures give rise to a corona region, as Figure 8A and 8BAs shown, it generates a structural protection (refuge) region to enable stable binding of hydrophilic small molecule agents to nanoparticles. The small hydrophilic domain of the second excipient typically has a molecular weight greater than about 500 grams per mole. In some embodiments, the molecular weight of the large hydrophilic domain is greater than about 500, 750, 1000, 1500, 2000, 2500, 3000, 5000, 7500, 10,000, 15,000, 20,000, 50,000, 100,000, 500,000, 1,000,000 or more grams per mole (or any intermediate molecular weight therebetween). The selection of an excipient having a specific hydrophilic domain can be informed by the amount of structural protection intended to be provided to the hydrophilic small molecule agent (which can also be affected by the relative amount of the second excipient).

[0074] In some embodiments, the first amphiphilic excipient can be selected from the following types: glycoproteins, glycolipids, polyalkylene oxide-containing polymers, and polyalkylene oxide-containing lipids. In some embodiments, the polyalkylene oxide-containing lipids are selected from the following types: polyethylene oxide-containing lipids and polypropylene oxide-containing lipids. In some embodiments, the polyethylene oxide-containing lipid is a polyethylene glycol-functionalized phospholipid, wherein the number average molecular weight of the polyethylene glycol is from about 500 to about 20,000 g / mol, such as 500, 750, 1000, 1500, 2000, 2500, 3000, 5000, 7500, 10,000, 15,000, 20,000 g / mol (or any intermediate molecular weight therebetween). In some additional embodiments, the polyethylene glycol-functionalized phospholipid is N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG-2000-DSPE).

[0075] In some embodiments, the polyalkylene oxide-containing lipid is a phospholipid comprising at least a first fatty acid tail domain having at least 14 carbons. The phospholipid can also comprise a second fatty acid tail domain that also has at least 14 carbons. With respect to embodiments having a first and second fatty acid tail of 14 or more carbons and / or saturated or containing a carbon-carbon double bond, the discussion of the above embodiments of the first amphiphilic excipient equivalently applies to the fatty acid tail(s) of the phospholipid of the second amphiphilic excipient, and is not repeated here to avoid further duplication.

[0076] The disclosed multi-drug lipid nanoparticles typically have a greater number of first defined lipid excipient molecules compared to the second excipient molecule. For example, the molar ratio of the first amphiphilic excipient to the second amphiphilic excipient in the multi-drug lipid nanoparticles is from about 2:1 to about 20:1 or higher, such as 2:1, 5:1, 7:1, 10:1, 13:1, 15:1, 18:1, or 20:1 or higher (or any intermediate ratio therebetween). For example, as described in the following examples, the stable multi-drug lipid nanoparticles are assembled with a high level of drug binding, and the molar ratio of the first amphiphilic excipient to the second amphiphilic excipient (i.e., the DSPC:DSPE-mPEG2000 ratio) is about 4:1 and 9:1.

[0077] The resulting multi-drug lipid nanoparticles are substantially spherical, meaning that the measurements of the diameter from different points on the surface through the center point to the opposite side will not vary by more than 50% or less, and preferably will not vary by more than 25% or less. In some embodiments, the diameter of the multi-drug lipid nanoparticles (or the average diameter if multiple measurements are taken) is from about 20 nm to about 200 nm. In some embodiments, the diameter or average diameter of the multi-drug lipid nanoparticles is from about 20 nm to about 150 nm, from about 20 nm to about 125 nm, from about 20 nm to about 100 nm, from about 30 nm to about 90 nm, from about 40 nm to about 60 nm, or any intermediate diameter or diameter range therebetween.

[0078] As described above, the multi-drug lipid nanoparticles have a corona surrounding the outer surface formed at least in part by the hydrophilic domain of the first amphiphilic excipient. The corona is formed by the hydrophilic domain of the second amphiphilic excipient, which provides a structure in which hydrophilic small molecule agents can be stably captured during the assembly and dehydration steps. The corona is hydrophilic because water and other hydrophilic molecules can reside in the corona. However, due to structural constraints, it is difficult to enter the hydrophilic corona region from the external aqueous environment once formed. Thus, the hydrophilic domain of the second amphiphilic excipient can prevent molecules from entering the hydrophilic corona. In some embodiments, when observed in an isotonic buffer (e.g., physiological tonicity) and at physiological pH and 25 ºC, the hydrophilic corona has a thickness of from about 2 nm to about 15 nm. In some embodiments, the corona has a thickness of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nm. This corona thickness can be obtained or designed by selecting the hydrophilic domain on the second lipid excipient.

[0079] The multi-drug lipid nanoparticles are not liposomes. Liposomes are defined by a lamellar bilayer of amphiphilic lipids.

[0080] In some embodiments, the multi-drug lipid nanoparticles do not have a solid core. Such a solid core can include, for example, metal microparticles, etc., which are coated with lipids, etc.

[0081] As described herein, the multi-drug lipid nanoparticles prepared according to and included in the present invention have enhanced properties, including stability and a surprisingly high binding rate of different small molecule agents. Thus, in some embodiments, the multi-drug lipid nanoparticles are characterized in that at least about 70%, such as 70%, 75%, 80%, 85% or more of the first small molecule agent(s) remain bound to the nanoparticles after 24 hours at pH 7.4 and 25 °C. In some embodiments, the multi-drug lipid nanoparticles are characterized in that at least 7%, such as about 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more of the second small molecule agent(s) remain bound to the nanoparticles after 24 hours at pH 7.4 and 25 °C. As described in more detail below, the method of lipid nanopore assembly can affect the structure of the resulting lipid nanoparticles, and thus affect the ability to retain bound hydrophilic (i.e., second) small molecule agents. In this regard, nanoparticles prepared by the single miscible solvent method can retain about 75% or more of the hydrophilic drugs used during the assembly process (see Table 21), and the disclosed lipid nanoparticles are quite stable during long-term storage. In some embodiments, at least about 70%, such as about 75%, 80%, 85% or more of the combined first and second small molecule agents remain bound to the nanoparticles after 8 months at pH 7.4 and 4 °C. In some embodiments, at least about 60%, such as 70%, 75%, 80% or more of the combined first and second small molecule agents remain bound to the multi-drug lipid nanoparticles after 8 months at pH 7.4 and 4 °C.

[0082] In some embodiments, when the nanoparticles are subjected to a sucrose gradient test, at least 70%, such as about 75%, 80%, 85% or more of the small molecule agents remain bound to the nanoparticles. In some embodiments, when the nanoparticles are subjected to a sucrose gradient test, at least 70%, such as about 75%, 80%, 85% or more of the second (i.e., hydrophilic) small molecule agents remain bound to the nanoparticles. The sucrose gradient test for determining this characteristic is described in Example 5. Briefly, the sucrose gradient test involves subjecting the multi-drug lipid nanoparticles to a continuous 5%-20% sucrose gradient and applying a centrifugal force of 200,000 g for 4 hours. Unbound small molecules separate and float in the less dense fraction, while the bound small molecule agents precipitate with the stable lipid nanoparticles in the denser gradient. The relative amounts of unbound and bound small molecule reagents can be quantified and thus compared.

[0083] As described below, the multi-drug lipid nanoparticles can exhibit pH responsiveness because, as the pH decreases, an increasing amount of the bound small molecule agent is released from the lipid nanoparticles. Thus, for example, it is advantageous to deliver the drug mainly when the lipid nanoparticles are taken up by endocytosis and exposed to the reduced pH within the cell lysosomal vesicles. This preserves a much higher proportion of the administered drug for delivery to the actual intended target, rather than just providing an initial systemic peak followed by immediate clearance.

[0084] In some embodiments, after administration to a mammalian subject, the disclosed multi-drug lipid nanoparticles confer extended blood drug concentrations for a first small molecule agent and a second small molecule agent as compared to an equal amount of the free small molecule agents administered to the mammalian subject. For example, when administered in an amount effective to affect the state of a disease or infection, the amounts of the first small molecule agent and the second small molecule agent remain detectable in plasma or lymphoid tissue for a period of time exceeding 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days. This is in contrast to the presence of the first small molecule agent and the second small molecule agent when administered in free form (i.e., not bound to the lipid nanoparticle formulation), which typically drops below detectable levels within one or two days.

[0085] As described herein, the extended presence in plasma and lymphoid tissue is beneficial for maintaining pathogen or other disease targets exposed to the small molecule therapeutic agent. In addition, combining multiple small molecule agents into a single stable carrier ensures that both (or all) therapeutic agents interact simultaneously with their intended targets and at relevant effective levels to facilitate effective multi-drug combination therapies, such as cART. Thus, the disclosed multi-drug lipid nanoparticles enhance or improve the efficacy and / or effectiveness of the small molecule agents incorporated therein as compared to the efficacy / effectiveness of the small molecule agents in free form or in forms where they are formulated separately in distinct delivery carriers.

[0086] It should be understood that the disclosed multi-drug lipid nanoparticles may also include any relevant components that enhance or direct function. For example, it is within the skill of those in the art to routinely incorporate detectable labels to provide imaging functionality. Additionally, the multi-drug lipid nanoparticles may also include targeting molecules such as receptors, lectins, antibodies, and functional fragments or derivatives thereof. For example, for antibodies, this can include single-chain antibodies (e.g., single-chain variable fragments (scFv), single-chain Fab fragments (scFab), VHH fragments, VNARs, or nanobodies), bispecific antibodies, Fab fragments, or F(ab)2 fragments). These can facilitate the directed delivery of small molecule therapeutics that bind to a target of interest having a unique antigen specifically recognized by the targeting molecule. Additionally, the multi-drug lipid nanoparticles may also include other binding partners (e.g., biotin, streptavidin) to assist in binding or linking to other functional structures. Such exemplary additional components can be integrated or incorporated into the disclosed lipid nanoparticles using techniques well known in the art.

[0087] On the other hand, the present invention provides a pharmaceutical composition comprising the multi-drug lipid nanoparticles disclosed herein. The pharmaceutical composition may include a pharmaceutically acceptable aqueous carrier as understood in the art.

[0088] On the other hand, the present invention provides a pharmaceutical composition comprising the multi-drug lipid nanoparticles disclosed herein. However, in this aspect, the multi-drug lipid nanoparticles are provided in a dry form, such as a powder, which can be reconstituted with sterile saline buffer prior to administration. Embodiments of the dry powder form include a first small molecule agent having a log P greater than 1 at 25 °C, a second small molecule agent having a log P less than 0 at 25 °C, a first amphiphilic excipient, and a second amphiphilic excipient, wherein the first amphiphilic excipient is a lipid comprising a hydrophilic domain having a molecular weight less than 300 grams per mole; the second amphiphilic excipient comprises a hydrophilic domain having a molecular weight greater than 500 grams per mole.

[0089] In yet another aspect, the present invention provides a kit comprising the formulations disclosed herein. In some embodiments, the kit further comprises a reconstitution buffer and written instructions that direct the reconstitution and administration processes.

[0090] On the other hand, the present invention provides a method for preparing multi-drug lipid nanoparticles. The method in this aspect is also referred to as the mixed double-solvent method herein. The method includes dissolving a first small molecule drug agent, a first amphiphilic excipient, and a second amphiphilic excipient having a log P greater than 1 at 25 °C in an organic solvent, wherein the first amphiphilic excipient is a lipid containing a hydrophilic domain with a molecular weight less than 300 g / mol; the second amphiphilic excipient contains a hydrophilic domain with a molecular weight greater than 500 g / mol to provide an organic solvent solution. The method further includes dissolving a second small molecule drug agent having a log P less than 0 at 25 °C in an aqueous solvent to provide an aqueous solvent solution. The organic solvent solution and the aqueous solvent solution are mixed to provide a mixed solvent solution. Next, the mixed solvent is removed from the mixed solvent solution to provide a dehydrated product containing the first small molecule drug agent, the first amphiphilic excipient, the second amphiphilic excipient, and the second small molecule drug agent. The dehydrated product is rehydrated in an aqueous solution to provide a solution containing multi-drug lipid nanoparticles.

[0091] The organic solvent contains a water-miscible co-component. In some embodiments, the organic solvent contains another co-component that is immiscible with water. Miscibility refers to the ability to mix with water or another aqueous solution without forming an emulsion (i.e., phase separation). When present, water miscibility is typically limited by the amount of water contained in the solution. Thus, one of ordinary skill in the art will readily understand that the relative amount of the aqueous solvent in the mixed solution must be limited.

[0092] In some embodiments, the organic solvent solution and the aqueous solvent solution are mixed in a ratio of at least about 20:1 (v / v). In some embodiments, the organic solvent solution and the aqueous solvent solution are mixed in a ratio of at least about 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 35:1, 40:1, 60:1 or higher (v / v) (or any intermediate ratio included therein).

[0093] Organic solvents that are generally known to be water-miscible are known and include acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, ethanol, methanol, propanol (and other alcohols), and tetrahydrofuran. In one embodiment, the water-miscible co-component of the organic solvent is an alcohol. In additional embodiments, the alcohol is selected from methanol, ethanol, propanol, hexanol, and decanol. In another embodiment, the water-miscible co-component of the organic solvent is acetonitrile.

[0094] In some embodiments, the organic solvent consists of water-miscible sub-components. However, in other embodiments, the organic solvent further comprises water-immiscible sub-components. In this sense, upon separation, the sub-components do not mix with water or an aqueous solution without forming a separate phase. However, the water-immiscible component can be combined with water or an aqueous solution in a single phase only when blended with the above-mentioned water-miscible components. Organic solvents that are generally water-immiscible are known and include benzene, butanol, carbon tetrachloride, chloroform, cyclohexane, cyclopentane, dichloroethane, dichloromethane, ethyl acetate, diethyl ether, heptane, hexane, methyl ethyl ketone, octane, pentane, dipropyl ether, tetrachloroethane, toluene, trichloroethane, and xylene. In some embodiments, the water-immiscible sub-components are selected from chloroform, hexane, decane, dichloromethane, and combinations thereof.

[0095] In some embodiments, the water-immiscible sub-component is chloroform, and the water-miscible sub-component is an alcohol, such as any one of methanol, ethanol, propanol, hexanol, and decanol.

[0096] In some embodiments, the ratio (v / v) of the water-immiscible sub-component to the water-miscible sub-component of the organic solvent is from about 1:1 to about 4:1. In some embodiments, the ratio is from about 7:5 (i.e., 1.4:1) to about 4:1. In some embodiments, the ratio is from about 2:1 to about 3:1. In one embodiment described in more detail below, for chloroform and alcohol, the ratio (v / v) of the water-immiscible sub-component to the water-miscible sub-component of the organic solvent is 65:35 (i.e., 13:7).

[0097] The aqueous solvent can be any suitable solvent in which a selected hydrophilic small molecule is dissolved. Examples include aqueous solvents containing NaHCO3, phosphate, borate, citrate, glycine, or combinations thereof, but are not necessarily so limited.

[0098] Evaporation can be used to remove the mixed organic and aqueous solvents from the mixed solvent solution, such as by applying a vacuum promoted by vacuum drying. Other techniques for drying or removing solvents are known and are encompassed in the disclosed methods. In some embodiments, the dehydrated product is substantially dry, with trace levels (e.g., less than 3% of the original volume) of solvent remaining. In some embodiments, the dehydrated product is completely or almost completely dry.

[0099] In some embodiments of the method, the dehydrated product is rehydrated in an aqueous solution at a temperature at least about 3 °C above the gel-to-liquid phase temperature of the amphiphilic excipient. As described above, the gel-to-liquid phase temperature is the temperature at which the aggregates of the excipient (e.g., nanoparticles) begin to depolymerize and transform into a liquid solution. The gel-to-liquid phase transition temperature can be determined by the structural properties of the first amphiphilic excipient or, alternatively, by the combined excipients of the lipid nanoparticles (i.e., taking into account at least the properties and amounts of the amphiphilic excipients). In some embodiments, the dehydrated product is rehydrated in an aqueous solution at a temperature at least about 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C or more (or any intermediate temperature included therein) above the gel-to-liquid phase temperature of the amphiphilic excipient(s). In some embodiments, depending on the excipient used, the method includes maintaining the temperature of the aqueous solution at at least 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, 70 °C or higher (or any intermediate temperature included therein) during the mixing process.

[0100] In some embodiments, the molar ratio of the small molecule pharmaceutical agent in the mixed solvent phase to the excipient is at least about 1:10. In some embodiments, the molar ratio of the small molecule pharmaceutical agent to the excipient in the resulting multi-drug lipid nanoparticles is at least about 1:10. The other features of the disclosed multi-drug lipid nanoparticles described above apply to the multi-drug lipid nanoparticles prepared by the disclosed method.

[0101] On the other hand, the present invention provides a method for preparing multi-drug lipid nanoparticles. The method of this aspect is also referred to herein as the single miscible solvent method. The method includes dissolving the following in a miscible solvent: a first small molecule pharmaceutical agent having a log P greater than 1 at 25 °C, a second small molecule pharmaceutical agent having a log P less than 0 at 25 °C, a first amphiphilic excipient, and a second amphiphilic excipient, wherein the first amphiphilic excipient is a lipid containing a hydrophilic domain having a molecular weight less than 300 g / mol, and the second amphiphilic excipient contains a hydrophilic domain having a molecular weight greater than 500 g / mol. The miscible solvent is removed to provide a dehydrated product comprising the first small molecule pharmaceutical agent, the second small molecule pharmaceutical agent, the first amphiphilic excipient, and the second amphiphilic excipient. The dehydrated product is heated to a first temperature at least 3 °C above the gel-to-liquid phase transition temperature of the amphiphilic excipient. The dehydrated product is rehydrated in an aqueous solution to provide a solution containing multi-drug lipid nanoparticles.

[0102] In some embodiments, the miscible solvent comprises an organic component and an aqueous component in a ratio of from about 20:1 to about 40:1 (v / v), such as about 20:1, 22:1, 25:1, 27:1, 30:1, 32:1, 35:1, 37:1, and 40:1 (or any intermediate ratio therebetween). In some embodiments, the organic component of the miscible solvent comprises a co-component that is miscible with water. In additional embodiments, the organic component of the miscible solvent further comprises a co-component that is immiscible with water. The above description of water-miscible and water-immiscible co-components, and their relative proportions in the organic solvent, applies to the organic co-components in this regard and their proportions in the organic component of the miscible solvent, and will not be repeated here.

[0103] In some embodiments, the water-immiscible co-component is chloroform, and the water-miscible co-component is an alcohol, such as any one of methanol, ethanol, propanol, hexanol, and decanol.

[0104] The above description of the aqueous solvent applies to the aqueous co-component of the miscible solvent, and will not be repeated here.

[0105] In some embodiments, removing the miscible solvent includes evaporation, vacuum drying, spray drying, or a combination thereof. Other techniques for drying or removing solvents are known and are encompassed by the disclosed methods. In some embodiments, the dehydrated product is substantially dry, with a trace level (e.g., less than 3% of the original volume) of solvent remaining. In some embodiments, the dehydrated product is completely or almost completely dry.

[0106] In some embodiments, the first temperature is at least about 3ºC, 4ºC, 5ºC, 6ºC, 7ºC, 8ºC, 9ºC, 10ºC, 15ºC, 20ºC, or higher (or any intermediate temperature therebetween) above the gel-to-liquid phase temperature of the amphiphilic excipient(s).

[0107] In some embodiments, the aqueous solution is maintained at at least the first temperature during the rehydration step. As shown, the first temperature is at least about 3ºC above the gel-to-liquid phase temperature of the amphiphilic excipient(s). In some embodiments, depending on the excipient used, the method includes maintaining the temperature of the dehydrated product and / or the aqueous solution at at least 40ºC, 45ºC, 50ºC, 55ºC, 60ºC, 65ºC, 70ºC, or higher (or any intermediate temperature therebetween) during the mixing process. In one embodiment, the rehydration step includes gradually combining the dehydrated product and the aqueous solution at about the first temperature and maintaining the fully combined dehydrated product and aqueous solution at a second temperature for at least about 1 hour, the second temperature being at least the first temperature.

[0108] The average molar ratio of the small molecule agent to the excipient in the resulting multi-drug lipid nanoparticles can be at least about 1:5, such as about 1:4, 1:3 or higher. In another embodiment, at least about 70% of the first small molecule agent and at least about 70% of the first small molecule agent are stably bound to the resulting multi-drug lipid nanoparticles at neutral pH. The other features of the disclosed multi-drug lipid nanoparticles described above apply to the multi-drug lipid nanoparticles prepared by the disclosed method.

[0109] In addition, the aspects of the method disclosed above may also include additional features or be characterized by additional features.

[0110] In some embodiments, the method further includes stirring an aqueous solution containing the multi-drug lipid nanoparticles to reduce the size of the multi-drug lipid nanoparticles. The stirring step may include applying sonication, extrusion through one or more filters, or mechanical or hydrodynamic shearing. The stirring step may produce multi-drug lipid nanoparticles in which at least 90% by number of the lipid nanoparticles have a diameter of about 20 nm to about 200 nm. Thus, the average diameter can be about 20 nm to about 150 nm, about 20 nm to about 125 nm, about 20 nm to about 100 nm, about 30 nm to about 90 nm, about 40 nm to about 60 nm, or any intermediate diameter or diameter range included therein.

[0111] The aspects regarding the structure and function of the multi-drug lipid nanoparticles described in other aspects or parts of the present invention, for example, regarding the corona, component first small molecule, component second small molecule, component first amphiphilic excipient, second amphiphilic excipient and their respective log P values and hydrophilic domains, the relative proportions of the component parts (such as small molecule agents and excipients), etc., apply to the multi-drug lipid nanoparticles prepared by this method and will not be elaborated herein.

[0112] On the other hand, the present invention includes any multi-drug lipid nanoparticles prepared by the disclosed method.

[0113] As described below, the inventors have demonstrated the utility and efficacy of the assembly method for preparing multi-drug lipid nanoparticles incorporating hydrophilic and hydrophobic small molecule agents approved for anti-HIV infection. Thus, on the other hand, the present invention provides a method for treating a subject infected with HIV, including administering an effective amount of the multi-drug lipid nanoparticles disclosed herein or administering an effective amount of the pharmaceutical composition disclosed herein. As used herein, the term "treating" refers to slowing down, reducing or preventing viral replication and / or improving symptoms associated with viral infection.

[0114] Administration can be to any mammal that may be HIV - positive. Administration can be by any suitable route that provides systemic delivery of the multi - drug lipid nanoparticles, such as subcutaneous (SC), intravenous (IV), or intramuscular (IM).

[0115] In some embodiments, the viral load is detectably reduced in the lymphoid tissues of the subject. In some embodiments, after administration to a mammalian subject, the disclosed multi - drug lipid nanoparticles confer an extended plasma drug concentration for the first small - molecule agent and the second small - molecule agent compared to administration of the same amount of the free small - molecule agents to a mammalian subject. For example, when administered in an amount effective to affect the state of a disease or infection, the amounts of the first small - molecule agent and the second small - molecule agent remain detectable in the plasma or lymphoid tissues for a period of time exceeding 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days.

[0116] As described herein, the extended presence in plasma and lymphoid tissues is beneficial for maintaining viral exposure to the small - molecule therapeutic agents. Further, the combination of multiple small - molecule agents into a single, stable carrier ensures that two (or all) therapeutic agents interact with their intended targets simultaneously and at relevant effective levels to facilitate effective combination therapy, such as cART. Thus, the disclosed multi - drug lipid nanoparticles enhance or improve the efficacy and / or effectiveness of the small - molecule agents incorporated therein relative to the efficacy / effectiveness of the small - molecule agents in free form or in forms formulated separately in distinct delivery vehicles.

[0117] The invention also includes methods of treating other diseases and infections by incorporating suitable multiple small - molecule agents that target different mechanisms affecting a specified disease or infectious pathogen (e.g., by combining hydrophilic and hydrophobic small - molecule agents as described).

[0118] Unless specifically defined herein, all terms used herein have the same meaning as they have to one of ordinary skill in the art of this invention. Publications cited herein and the subject matter cited therein are specifically incorporated herein by reference in their entirety.

[0119] The term “or” as used in the claims is used to mean “and / or” unless explicitly indicated to refer only to alternative or alternative are mutually exclusive, but the invention supports definitions that refer only to alternatives as well as “and / or”.

[0120] According to long - standing patent law, unless specifically noted, the words “a” and “an” when used in conjunction with the word “comprising” in the claims or specification denote one or more.

[0121] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprise", "comprising", and the like shall be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is, to mean "including but not limited to". The use of the singular or plural words also includes the plural and the singular respectively. Additionally, as used in this application, the words "herein", "above", "below", and words of similar import shall refer to the application as a whole and not to any particular part of the application.

[0122] The present invention discloses methods and compositions that can be used for disclosure, materials, compositions, and components that can be used in conjunction with the disclosed methods and compositions, can be used to prepare the disclosed methods and compositions, or are products of the disclosed methods and compositions. It should be understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed, various individual and collective combinations can be specifically considered even without an explicit specific mention of each of these compounds and each single combination and permutation. This concept applies to all aspects of the invention, including but not limited to the steps in the described methods. Thus, specific elements of any of the foregoing embodiments can be combined or replaced by elements in other embodiments. For example, if there are various additional steps that can be performed, it is understood that each of these additional steps can be performed using any specific method step or combination of method steps of the disclosed method, and each such combination or subset of combinations is specifically considered and should be regarded as disclosed. Furthermore, it is understood that any suitable materials can be used to implement the embodiments described herein, such as materials described elsewhere herein or known in the art. Examples

[0123] The following examples are intended to illustrate but not limit the disclosed invention.

[0124] Example 1

[0125] This example describes the evaluation of the interaction of atazanavir and darunavir with lipids to develop pH-responsive anti-HIV drug combination nanoparticles.

[0126] Abstract

[0127] We evaluated the pH-dependent solubility, lipid binding, and drug release from lipid nanoparticles (LNPs) of two human immunodeficiency virus (HIV) protease inhibitors, atazanavir (ATV) and darunavir (DRV). Both ATV and DRV were incorporated into LNPs composed of pegylated and non-pegylated phospholipids with nearly 100% efficiency, but only ATV LNPs formed stable lipid-drug particles and showed pH-dependent drug release. DRV LNPs were unstable and formed mixed micelles at low drug-lipid concentrations and thus were not suitable for lipid-drug particle development. When ATV LNPs were prepared with the metabolic and cell membrane efflux inhibitor ritonavir (RTV) and the HIV reverse transcriptase inhibitor tenofovir (TFV), stable, scalable, and reproducible anti-HIV drug combination LNPs were produced. Drug incorporation efficiencies of 85.5 ± 8.2, 85.1 ± 7.1, and 6.1 ± 0.8% were achieved for ATV, ritonavir, and tenofovir, respectively. Preliminary primate pharmacokinetic studies with these pH-responsive anti-HIV drug combination LNPs administered subcutaneously resulted in detectable plasma concentrations that persisted for 7 days for all three drugs. These anti-HIV LNPs could be developed as long-acting targeted antiretroviral therapy.

[0128] Introduction

[0129] Highly active antiretroviral therapy (HAART), introduced in the late 1990s and directed against multiple viral proteins, cleared human immunodeficiency virus (HIV) from the blood and resulted in a significant improvement in the quality of life and life expectancy of HIV-infected patients. Although oral HAART therapy is highly effective in clearing HIV from the blood, residual virus persists in lymph nodes and other lymphoid tissues even with high drug doses. We previously described that intracellular drug concentrations in monocytes in lymph nodes in HIV-infected patients on the oral anti-HIV drug indinavir were shown to be one-third of the levels found in blood monocytes. These data were recently confirmed in a prospective clinical study of 12 HIV-infected patients in which intracellular drug levels in lymph node cells for two HIV drugs, atazanavir (ATV) and darunavir (DRV), were found to be as much as 99% lower than intracellular drug levels in the blood. These lower intracellular drug levels in lymph nodes were associated with residual virus in the patients.

[0130] We previously developed pH-sensitive indinavir lipid nanoparticles systematically and demonstrated that when administered subcutaneously they preferentially localize in lymph nodes and lymphoid tissues. In HIV-infected primates, we reported that these lipid–indinavir complexes increased indinavir concentration in lymph nodes throughout the body, with drug levels reaching 22.7-fold higher than in plasma. These studies showed significant reduction of plasma viral load and reversal of CD4+ T cell decline. No enhancement in lymph node drug accumulation or clinical impact was observed in control primates treated with free drug.

[0131] However, for clinical translation, combinations of anti-HIV drugs—and not just indinavir monotherapy—are necessary to address potential drug resistance. Recent acquired immunodeficiency syndrome (AIDS) treatment guidelines recommend many drug combinations, most of which include at least two or three different anti-HIV drugs. Among the protease inhibitors used in HAART, many of the currently available newer anti-HIV drugs exhibit 10–100-fold higher antiviral potency and lower rates of drug resistance. ATV and DRV are new-generation protease inhibitors that are commonly used in combination with another protease inhibitor, ritonavir (RTV), and the reverse transcriptase inhibitor tenofovir (TFV).

[0132] Therefore, the aim of this study was to characterize the lipid–drug interactions of the new protease inhibitors ATV and DRV in terms of membrane binding, degree of incorporation, stability, and pH-dependent release of the drugs. These studies provide a basis for the development of pH-responsive anti-HIV drug combination lipid nanoparticles composed of a mixture of lipids and phospholipids modified with polyethylene glycol polymers, which are stable and can be scaled up for primate studies with high protease inhibitor incorporation efficiency. Our results show that both ATV and DRV bind to lipids and are mainly incorporated into the lipid membrane, but only ATV-lipid nanoparticles (ATV-LNP) are stable and show pH sensitivity. Thus, ATV-containing nanoparticles are suitable for further development of anti-HIV drug combination lipid nanoparticles containing ATV, RTV, and TFV.

[0133] Materials and Methods

[0134] Materials

[0135] 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2000] (DSPE-mPEG2000) (both GMP grade) were purchased from Genzyme Pharmaceuticals (purity > 99%; Cambridge, MA). Reference standards of atazanavir (C38H52N6O7, ATV), darunavir (C27H37N3O7S, DRV), ritonavir (C37H48N6O5S2, RTV), and tenofovir (C9H14N5O4P, TFV) were provided by the AIDS Research and Reference Reagent Program of the National Institutes of Health (NIH). Some of the subsequent samples were purchased from Waterstone tech LLC (Carmel, IN) and verified using reference compounds. Cycloheximide was purchased from Sigma-Aldrich (St. Louis, MO). 1,6-Diphenyl-1,3,5-hexatriene (DPH) was obtained from Invitrogen (Eugene, OR). Other reagents were of analytical grade or higher.

[0136] Determination of the partition coefficients of atazanavir and darunavir in octanol and buffer

[0137] The octanol-buffer drug partition coefficients at room temperature were determined by the small-scale shake flask method described by Dittert LW et al. Phase Solubility Technique in Studying the Formation of Complex Salts of Triamterene. Journal of pharmaceutical sciences. 1964; 53:1325–1328. Briefly, phosphate-buffered saline (PBS) at pH 3, 5, and 7.4 was used as the aqueous phase. ATV or DRV at 0.2 mg / mL was dissolved in octanol, added to an equal volume of PBS, and vortexed for 10 min. The mixture was centrifuged at 14,000 rpm (18,078 g) (Beckman Coulter™ Microfuge® 18 centrifuge, Beckman Coulter Inc., Brea, CA) to separate the octanol and aqueous phases. The drug concentrations in the two phases were determined by high-performance liquid chromatography tandem mass spectrometry (HPLC / MS / MS). The partition coefficient was calculated as the ratio of the drug concentration in the octanol phase to the drug concentration in the aqueous phase. Samples were used in triplicate at each pH.

[0138] Lipid-drug nanoparticle preparation

[0139] Lipid-drug nanoparticles were prepared as previously described in the following references: Kinman L et al. Optimization of lipid-indinavir complexes for localization in lymphoid tissues of HIV-infected macaques. J Acquir Immune Defic Syndr. 2006; 42(2):155–161; Choi SU et al. pH-dependent interactions of indinavir and lipids in nanoparticles and their ability to entrap a solute. Journal of pharmaceutical sciences. 2008; 97(2):931–943; and Endsley AN and Ho RJ. Design and characterization of novel peptide-coated lipid nanoparticles for targeting anti-HIV drug to CD4 expressing cells. The AAPS journal. 2012; 14(2):225–235, the entire content of each of which is incorporated herein by reference. Briefly, DSPC and DSPE-mPEG2000 lipids (8:2, mol / mol) and ATV, DRV, and RTV were dissolved in chloroform in a glass tube and then dried under nitrogen until a uniform lipid-drug film was formed. Residual solvent was removed by vacuum drying overnight. Subsequently, the dried lipid-drug film was rehydrated with 0.9% NaCl (pH 7.4) containing 20 mM sodium bicarbonate. The lipid-drug sample was hydrated at 60 °C for 2 h. The sample was then sonicated (laboratory scale) or homogenized (preclinical scale) to obtain a uniform suspension.

[0140] For small-scale preparation of lipid nanoparticles, 200 μL of the sample was sonicated using a bath sonicator (Avanti® Polar Lipids Inc., Alabaster, AL) until the sample became clear. For large-scale preparation, 45 mL of the hydrated lipid-drug mixture was homogenized for 15 cycles using an Avestin EmulsiFlex-C5 (Avestin Inc., Ottawa Ontario, Canada) operating at 5,000 - 6,000 psi. The weight molar osmotic concentration of the final formulation was measured using a VAPRO™ 5520 vapor pressure osmometer (Wescor Inc., Logan, UT). The lipid-drug nanoparticles and liposome control samples were stored at 4 - 8 °C.

[0141] Drug incorporation efficiency

[0142] The percentages of ATV, DRV, RTV, and TFV incorporated into the lipid-drug nanoparticles were determined using dialysis. Briefly, a 50 μL aliquot of the lipid-drug nanoparticle formulation was transferred into a dialysis membrane (MW cut-off value = 6,000 - 8,000; Spectra / Por® 6, Spectrum Laboratories Inc., Rancho Dominguez, CA) and dialyzed in 1,000 mL of pH 7.4 buffer to separate the free drug from the drug incorporated into the nanoparticles. The incorporation efficiency was calculated as the ratio of the amount of drug incorporated to the total amount of drug loaded, multiplied by 100%.

[0143] Particle size analysis

[0144] The average particle sizes of the lipid-drug nanoparticles and liposome control were determined by photon correlation spectroscopy (PCS) using a NICOMP™ 380ZLS instrument (NICOMP Particle Sizing Systems, Santa Barbara, CA). The lipid nanoparticle formulation was diluted to 0.25 mM in saline buffer for size measurement (0.4 mL final volume), and evaluated at a 90° angle using a 5 mW HeNe laser (λ = 632.8 nm). Samples were diluted to the same final volume using the same buffer to analyze the effect of concentration. The light scattering data was analyzed based on intensity-weighted NICOMP size distribution and presented as mean ± SD.

[0145] Fluorescence anisotropy studies for measuring membrane fluidity

[0146] To evaluate the changes in membrane fluidity caused by drug insertion into the lipid membrane, 0.1% 1,6-diphenyl-1,3,5-hexatriene (DPH) was used as an in-membrane probe 18,20. Briefly, to incorporate DPH into the lipid membrane, 2 μL of a 2 mM DPH solution in tetrahydrofuran was added to the lipid nanoparticle suspension with or without the drug. The final formulation was incubated at 60 °C (above 55 °C - the phase transition temperature of DSPC) for 30 min and then slowly cooled to room temperature within 15 min. The DPH-containing (drug-loaded and empty) lipid nanoparticles were added to a cuvette heated to 45 °C and then gradually increased to 65 °C in 1-2 °C increments. The temperature inside the cuvette was controlled using a water-jacketed cuvette holder connected to a circulating water bath (PolyScience, model 1162, Niles, IL), and this temperature was measured by a digital thermometer. The cuvette was equilibrated for 10 min before each temperature reading. The fluorescence intensities parallel and perpendicular to the emitted light were continuously measured using an F-4500 fluorescence spectrometer (Hitachi, Minato-ku, Tokyo, Japan) set at λex / em = 360 / 430 nm; both the excitation and emission slits were 5 nm. The fluorescence anisotropy response r was calculated by the following formula:

[0147]

[0148] where IVV and IVH are the fluorescence intensities recorded with polarizers oriented parallel and perpendicular to the plane of polarization of the excitation beam.

[0149] Using SigmaPlot software (version 11.0, Systat Software, Inc., San Jose, CA), a curve of the fluorescence anisotropy response versus temperature was generated by non-linear regression. The midpoint of the phase transition temperature (Tc) was estimated based on the following formula:

[0150]

[0151] where y is the fluorescence anisotropy, x represents the temperature (°C), min is the minimum response, max is the maximum response, Tc is the temperature value exactly midway between the minimum and maximum parameters, and Hillslope is the slope at Tc.

[0152] Determination of drug release from lipid-drug nanoparticles

[0153] The time- and pH-dependent release of the drug from the lipid-drug nanoparticles was determined using a dialysis method similar to that described above. Briefly, 50 μL aliquots of the lipid-drug nanoparticle formulation were transferred into dialysis membranes (MW cut-off value = 6,000 - 8,000; Spectra / Por® 6, Spectrum Laboratories Inc., Rancho Dominguez, CA) and dialyzed in 1,000 mL of buffer at pH 3, 4, 5, 6, or 7.4 to evaluate pH-dependent drug release and to separate free drug from drug incorporated into the nanoparticles. The initial and final drug concentrations were determined by HPLC / MS / MS (method described below) to estimate the percentage of drug released.

[0154] Plasma Profiles of Anti-HIV Drug-Combined Lipid Nanoparticles in Primates

[0155] According to the guidelines of the Washington National Primate Research Center, rhesus monkeys (pigtail macaques (Macaca nemestrina), male, 2.9 - 5.0 kg) were used for the pharmacokinetic study. All animal procedures were conducted under an approved protocol reviewed by the University of Washington Institutional Animal Care and Use Committee.

[0156] Two young adult male rhesus macaques were given a single 20 mL subcutaneous injection of lipid nanoparticles containing a combination of ATV, RTV, and TFV at a dose of 25 mg / kg ATV (35 μmol / kg), 12.8 mg / kg RTV (18 μmol / kg), and 15.3 mg / kg TFV (53 μmol / kg). Venous blood samples were collected from the femoral vein at 0, 0.5, 1, 3, 5, 8, 24, 48, 120, and 168 hours (7 days). 2 mL of plasma was immediately separated from the blood by centrifugation at 1,200 rpm (252 g) (Jouan CR 312 centrifuge, Jouan Inc., Winchester, VA) for 10 minutes. ATV, RTV, and TFV in plasma were extracted in duplicate and analyzed by liquid chromatography–mass spectrometry using a validated reverse-phase HPLC / MS / MS method similar to that for lopinavir (LPV), RTV, and TFV. Koehn J and Ho RJ. A Novel LC / MS / MS Method for Simultaneous Detection of anti-HIV Drugs Lopinavir, Ritonavir and Tenofovir in Plasma. Antimicrobial agents and chemotherapy. 2014.

[0157] The area under the plasma drug concentration–time curve (AUC) for ATV, RTV, and TFV was calculated using the linear trapezoidal method during the time course up to approximately 168 h post-dose. Based on the reported plasma half-lives of ATV, RTV, and TFV (product labels), as well as our preliminary data in primates given these drugs (data not shown), most of the ATV, RTV, and TFV was cleared from the blood after 8 h. Therefore, to delineate the effect of free drug compared to lipid-bound drug, we analyzed the AUC for three consecutive time periods: 0–8 h, 8–168 h, and 0–168 h.

[0158] Analysis of multiple anti-HIV drugs by HPLC / MS / MS

[0159] All drugs in buffer and plasma were analyzed using a validated HPLC / MS / MS analytical method capable of analyzing ATV, DRV, LPV, RTV, and TFV. The one-step analysis of LPV, RTV, and TFV has been previously published. Koehn J and Ho RJ. A Novel LC / MS / MS Method for Simultaneous Detection of anti-HIV Drugs Lopinavir, Ritonavir and Tenofovir in Plasma. Antimicrobial agents and chemotherapy. 2014. Briefly, the HPLC system was equipped with a Shimadzu 20AD HPLC (Shimadzu Scientific Instruments, Inc., Pleasanton, CA). ATV, RTV, and TFV were separated on a reversed-phase Synergi™ column (100 × 2.0 mm2; 4 μm POLAR-RP 80Å, Phenomenex Inc., Torrance, CA) and detected using a triple quadrupole mass spectrometer MS / MS (AB SCIEX 3200 QTRAP®, Framingham, MA). An aliquot of the internal standard, cycloheptamide, was added to plasma samples at a final concentration of 50 ng / mL. A 5 μL sample (containing the internal standard) was injected onto the column and eluted with a mobile phase containing acetonitrile and water with 0.1% acetic acid at a flow rate of 0.35 mL / min with a gradient of 3-100%. Transitions from specific precursor ions to product ions were detected using multiple reaction monitoring (MRM). The following m / z transitions were used to detect the analytes: 705.5 / 168.2 for ATV, 548.3 / 392.3 for DRV, 721.3 / 296.1 for RTV, 288.1 / 176 for TFV, and 238.1 / 193.2 for cycloheptamide.

[0160] Statistical analysis

[0161] When analyzing control and test samples, the student t-test was used. To determine differences among multiple groups with different lipid-drug concentrations, analysis of variance (ANOVA) was used. A p-value of less than 0.05 was considered statistically significant.

[0162] Results

[0163] Characterization of atazanavir and darunavir and their ability to form lipid nanoparticles

[0164] In addition to lipophilicity, the degree of ionization of a drug at different pH values can play an important role in the ability of the drug to associate or bind to lipids. Many prediction tools are available to estimate drug lipophilicity, which is expressed as the logarithm of the octanol-water partition coefficient (Log P) at neutral pH and is commonly denoted as XLog P. However, the XLog P values of ATV and DRV do not account for the effects of varying pH. Therefore, we evaluated these values for ATV and DRV at pH 3, 5, and 7.4 and expressed them as estimates of Log D (octanol-buffer partition coefficient). As shown in Table 1, although ATV and DRV had similar Log D values at pH 3—3.40 versus 2.98—only ATV, but not DRV, showed pH-dependent Log D values. The Log D of ATV increased with increasing pH, having a value of 4.66 at pH 5 and 5.77 at pH 7.4. For DRV, the Log D values at pH 5 and 7.4 were 2.80 and 2.84, respectively; no significant pH-dependent changes in Log D were detected. In contrast, the predicted XLogP3-AA values for ATV and DRV were 5.6 and 2.9, respectively, which were consistent with the observed Log D values at pH 7.4 described above. Overall, these data indicate that although both ATV and DRV are lipophilic (Log D ~3 or higher at neutral pH), only ATV shows a gradual increase in Log D with increasing pH.

[0165] Table 1. Molecular structures and pH-dependent hydrophobicity of atazanavir and darunavir

[0166]

[0167] a As described in Materials and Methods, the partition coefficient log D was measured at each pH value and expressed as the mean ± SD in triplicate.

[0168] b Theoretical estimates were obtained from the PubChem database (reference: websites pubchem.ncbi.nlm.nij.gov and sioc-ccbg.ac.cn / software / xlogp3).

[0169] c Drug bank data estimated by ChemAxon (website for ATV: drugbank.ca / drugs / DB01072; website for DRV: drugbank.ca / drugs / DB01264).

[0170] To determine whether there are differences in the extent and manner of incorporation of two hydrophobic drugs, ATV and DRV, into lipids, each compound was dissolved in an organic solvent consisting of chloroform together with lipids. After removal of the solvent and hydration in buffer, followed by particle size reduction, the drug incorporation efficiency, particle size, and particle size stability were evaluated at different concentrations. To keep the lipid composition constant, we used lipids consisting of DSPC and DSPE-mPEG2000 (8:2, mol / mol). This composition has been shown to be biocompatible and proven effective for anti-HIV drug incorporation and cellular uptake. We found that in the absence of lipids in the mixture, both ATV and DRV formed precipitates and were unable to form drug suspensions. When ATV and DRV were mixed with lipids, no drug precipitation was observed, which enabled the determination of drug incorporation into lipid nanoparticles. As shown in Table 2, we found that at all lipid-to-drug molar ratios tested (5:1, 8:1, and 20:1), ATV and DRV were incorporated almost completely into lipid-drug nanoparticles. Regardless of the lipid-to-drug molar ratio, the ATV incorporation efficiency was almost 100% (ranging from 96.2 - 97.4%), while the DRV incorporation efficiency showed a decline with increasing drug density. Compared to the incorporation efficiency at a lipid-to-drug molar ratio of 20:1, DRV showed a lower incorporation efficiency of 8.5 - 6.3% at lipid-to-drug molar ratios of 5:1 and 8:1 (96.6% at 20:1, 94.4% at 8:1, and 88.1% at 5:1).

[0171] Table 2: Effect of lipid-to-drug ratio on the extent of drug incorporation into lipid-drug nanoparticles and particle size

[0172]

[0173] a Lipid-drug nanoparticles containing ATV or DRV with different lipid-to-drug molar ratios were prepared as described in Materials and Methods.[[ID=IO]]

[0174] b Drug incorporation efficiency was determined based on the percentage of drug bound to the particles after removal of free, unincorporated drug; data are expressed as mean ± SD of 4 replicates.

[0175] c Lipid-drug particle diameter was determined using PCS, and data are expressed as mean ± SD of 4 replicates.

[0176] Next, we determined the effect of different lipid-to-drug molar ratios on the size (diameter) of these lipid-drug nanoparticles. As shown in Table 2, the size of darunavir-lipid nanoparticles (DRV-LNP) did not change significantly with increasing lipid-to-drug molar ratio. The size of all DRV-LNP was approximately 33.6 - 35.6 nm, and this value was very similar to that of drug-free control lipid nanoparticles (d ~35.4 ± 4.4 nm) (p > 0.05). In contrast, with increasing drug density (or decreasing lipid-to-drug ratio), the diameter of ATV-LNP increased from 34.6 ± 3.1 nm to 68.2 ± 7.1 nm (p < 0.01; Table 2).

[0177] In summary, these data indicate that ATV, but not DRV, exhibits pH-dependent hydrophobicity. Although both ATV and DRV can be almost completely incorporated into lipid nanoparticles composed of DSPC and DSPE-mPEG2000, the incorporation efficiency of only DRV, but not ATV, decreases with increasing drug density. Based on these data, we fixed the lipid-to-drug molar ratio of ATV and DRV at 8:1 to evaluate how these two drugs affect membrane fluidity and stability at different concentrations in subsequent studies.

[0178] Effects of atazanavir and darunavir on membrane fluidity: Measurement of lipid-drug interactions

[0179] To characterize the interaction between the drug and lipid and the effect of the drug on membrane fluidity, we used DPH as a fluorescence polarization probe. As a planar hydrophobic fluorescent molecule, DPH inserted into the hydrophobic domain of the phospholipid bilayer is sensitive to changes in the phase behavior represented by changes in the degree of DPH polarization and fluorescence intensity. DPH has been successfully applied in multiple reports to study the order of membrane bilayers with fluorescence anisotropy. If the drug is bound and incorporated into the lipid, changes in lipid membrane disorder caused by the drug should be detected. Therefore, we monitored the fluorescence anisotropy of DPH in DSPC and DSPE-mPEG2000 lipid membranes with and without the drug as a function of temperature. Figure 2 Shows the fluorescence anisotropy of control lipid nanoparticles and lipid nanoparticles with ATV or DRV (at a lipid-to-drug molar ratio of 8:1) as a function of increasing temperature. The midpoint of the phase transition temperature (Tc) is also depicted.

[0180] As Figure 2As shown, incorporation of DRV into the lipid bilayer decreased the polarization of DPH in lipid nanoparticles compared to the drug-free lipid control. In contrast, incorporation of ATV showed a smaller effect on membrane order, as the fluorescence anisotropy values were similar to those of the lipid control. However, both ATV and DRV decreased the phase transition temperature to varying degrees. At a fixed lipid-to-drug molar ratio of 8:1, DRV decreased Tc by 1.6 °C, while ATV decreased Tc by only 0.7 °C (for control LNP, ATV-LNP, and DRV-LNP, Tc was 54.9, 54.2, 53.3 °C, respectively; Figure 2 ). The 54.9 °C Tc value of the drug-free control LNP was similar to the reported 55 °C Tc of DSPC. Overall, these data indicate that both ATV and DRV can bind and incorporate into the lipid bilayer, altering membrane fluidity and decreasing the phase transition temperature.

[0181] Stability of atazanavir and darunavir binding to lipids and concentration-dependent effects on lipid-drug nanoparticles

[0182] Although ATV and DRV bind to lipids and affect lipid structural behavior, the stability of this binding interaction is important for the development of stable lipid-drug nanoparticle formulations. To address this issue, we first tested the particle size stability at different concentrations before determining the time-dependent drug release profiles.

[0183] Our initial data indicated that ATV could bind stably to the lipid bilayer, while the binding of DRV was somewhat unstable. If the drug molecules cannot bind stably to lipids, insoluble drug molecules may cause particle aggregation, separate from lipids when the drug precipitates, or reconstitute into smaller particles such as mixed micelles or micelles. After the formation of ATV-LNP and DRV-LNP, we did not detect any drug precipitation, which is usually not observed for free drugs of ATV and DRV in physiological buffer under the same conditions. Since the formation of mixed micelles or micelle structures is concentration-dependent and becomes evident at low lipid-drug concentrations, lipid nanoparticles containing ATV or DRV together with control nanoparticles (drug-free) were serially diluted, and the change in their diameters was monitored by PCS. The initial lipid concentration was the same for the formulations (200 mM), and they were diluted to a series of concentrations between 0.01 - 10 mM. As shown in Table 3, dilution of the lipid-drug nanoparticles had no significant effect on the hydrodynamic diameter of ATV-LNP. However, for DRV-LNP, as the lipid-drug concentration decreased, the particle diameter showed a gradual decrease (Table 3). When the concentration of DRV-LNP was below 1 mM, the particle diameter decreased from the initial 33.9 ± 3.8 nm to 8.9 - 14.3 nm (p < 0.01). The smaller diameters of these particles were in the range of mixed micelles and micelles. In contrast, the diameter of ATV-LNP did not change upon dilution. As expected, dilution had no effect on the diameter of the drug-free control lipid nanoparticles (liposomes) (Table 3).

[0184] Table 3: Effect of concentration on the apparent size of lipid-drug nanoparticlesa

[0185]

[0186] a Lipid-drug nanoparticles containing ATV or DRV or control (drug-free) with a fixed lipid to drug molar ratio (8:1) were prepared as described in Materials and Methods.

[0187] b The apparent size of the particles at the specified concentrations and dilutions was measured by PCS, and the particle diameter was expressed as the mean ± SD of 4 replicates.

[0188] The concentration-dependent effect on DRV-LNP was further confirmed by preparing DRV-LNP with different initial concentrations to determine the drug incorporation efficiency. We found that the drug incorporation efficiencies of DRV at 5 and 0.5 mM lipids were 95% and 58%, respectively, compared to 96% and 97% for ATV.

[0189] Next, as part of the stability study, we determined the time course of drug release from ATV-LNP and DRV-LNP. These drug release studies were performed on 5, 25, and 200 mM lipid-drug formulations, and the percentage of drug released after 24 hours at pH 7.4 was evaluated. During this time period, we did not observe any appreciable fraction of drug released from ATV-LNP ( Figure 3A ). In contrast, for DRV-LNP, nearly 100% of DRV was released at 5 and 25 mM dilutions, and 53% drug release was detected in the 200 mM formulation. The instability of DRV-LNP was also evident by 4 hours, at which time 65.21%, 42.14%, and 29.99% of DRV was released from 5, 25, and 200 mM DRV-LNP, respectively ( Figure 3B ).

[0190] In summary, these data indicate that although DRV and ATV are equally effective in their ability to incorporate into lipid nanoparticles, only ATV incorporation into the lipid bilayer results in a stable structure that is resistant to dilution or rapid drug release. DRV-LNP is less stable, and the drug release rate and decrease in particle size exhibit a concentration-dependent increase. Therefore, for subsequent experiments, we used ATV-LNP for pH-dependent drug release characterization and for the construction of combined drug particles for formulation scale-up and primary primate studies.

[0191] pH-Dependent Release of Atazanavir from Lipid Nanoparticles

[0192] Since ATV exhibits pH-dependent lipophilicity, we determined whether changes in pH could induce drug release from ATV-LNP. To this end, we exposed ATV-LNP to buffers of different pHs and measured the fraction of ATV released from the lipid-drug nanoparticles. As Figure 4 shown, at 37 °C, after exposing ATV-LNP to gradually decreasing pH values for 24 hours, under these conditions, approximately 21.3 - 26.2% of ATV molecules were released from ATV-LNP. When the pH was lowered to 3, almost all ATV molecules were released within 24 hours. This behavior was also very similar to the pH-dependent ATV release at 25 °C ( Figure 4 ). Overall, these data indicate that the release of ATV from ATV-LNP depends on pH and to some extent on temperature.

[0193] Development and Scale-Up Preparation of Anti-HIV Drug-Combined Lipid Nanoparticles

[0194] Since the above lipid-drug interaction studies showed that ATV (but not DRV)-lipid interactions result in stable ATV-LNPs, ATV was chosen to develop anti-HIV drug combination nanoparticles. In accordance with current best clinical guidelines for reducing the potential for viral resistance, in addition to ATV, this anti-HIV drug combination also includes the ATV metabolic inhibitor / booster RTV, plus the reverse transcriptase inhibitor TFV. Tenofovir (TFV) was chosen because of its proven potency as a reverse transcriptase inhibitor and because its phosphorylated active metabolite is retained intracellularly for sustained response. Accordingly, we prepared three different laboratory-scale (0.2 mL) lipid nanoparticle formulations containing ATV, ATV + RTV, or ATV + RTV + TFV and evaluated their incorporation efficiency and physical characteristics. As shown in Table 4, addition of RTV or RTV + TFV to ATV-lipid nanoparticles did not change the ~100% ATV incorporation efficiency. We also found that under those conditions, the RTV incorporation efficiency was nearly equally 100% (Table 4). As a water-soluble drug, the TFV incorporation efficiency was approximately 2.4% and was reproducible between batches. Inclusion of all three drugs in the lipid nanoparticles had no effect on the physical characteristics as no significant change in particle diameter (d = 56 - 62 nm) was detected (Table 4). In preliminary experiments, we compared the ability of ATV and ATV + RTV lipid nanoparticles to produce pH-dependent drug release. We found that the pH-dependent release of ATV and RTV was very similar to that of ATV-LNP. Accordingly, we prepared these anti-HIV drug combination lipid nanoparticles (ATV + RTV + TFV LNP) in a larger volume for preliminary primate pharmacokinetic studies (described below). As shown in Table 4, the scaled-up formulation (~45 mL) prepared under sterile conditions also provided comparable particle size, pH, and osmolality to the laboratory-scale formulation (~0.2 mL). This preclinical scale provided nearly complete ATV and RTV incorporation and approximately 6% consistent TFV incorporation in a reproducible manner. Accordingly, these anti-HIV LNPs containing a drug combination of ATV, RTV, and TFV (2:1:3 molar ratio) were used in primate studies without removal of unbound (approximately 93.9% free) TFV from the formulation.

[0195] Table 4: Characterization of lipid nanoparticles containing anti-HIV drugs prepared at pilot scale or preclinical scale for primate studies

[0196]

[0197] As described in Materials and Methods, drugs formulated in lipid nanoparticles were prepared. For both pilot (0.2 mL) and preclinical (45 mL) scales, a three-drug combination ATV + RTV + TFV (2:1:3 (mol / mol)) was used for comparison. At the pilot scale, lipid nanoparticles containing ATV or ATV + RTV were also prepared at a lipid-to-drug ratio of 8:1 (mol / mol).

[0198] b The incorporation efficiency was determined as described in Table 2.

[0199] c The particle diameter of the lipid-drug nanoparticles was measured by PCS and expressed as mean ± SD.

[0200] d The osmolality of the lipid-drug nanoparticles was monitored as described in Materials and Methods and expressed as mean ± SD.

[0201] Plasma Profiles of Anti-HIV Drug-Combination Lipid Nanoparticles in Primates

[0202] To evaluate the plasma profiles of ATV, RTV, and TFV, two primates were used in a preliminary study. They were given a single subcutaneous dose of an anti-HIV drug-combination lipid nanoparticle containing 25 mg / kg ATV, 12.8 mg / kg RTV, and 15.3 mg / kg TFV. Plasma drug concentrations were monitored for 7 days (see Figures 5A - 5F ). As Figures 5A - 5F shown, in the two macaques receiving anti-HIV LNP, plasma drug concentrations remained at detectable levels for 7 days (168 hours). Two main peaks were detected in the plasma TFV concentration profiles in both animals. The first peak of TFV decreased after 8 hours ( Figure 5E and 5F ), followed by a more prominent and sustained plasma TFV level. The sustained plasma TFV level was unexpected because this anti-HIV formulation provided only 6.1% lipid-bound TFV (93.9% was in the free soluble form). For RTV and ATV, this turning point appeared less prominent and more variable ( Figures 5A - 5D ). However, both ATV and RTV were clearly detectable in the plasma 7 days after a single subcutaneous administration of the anti-HIV LNP composed of the three-drug combination.

[0203] We further analyzed the plasma drug concentration time course by calculating the plasma drug concentration AUC for each drug to determine the change in plasma drug exposure over time. Our experience with subcutaneous administration of protease inhibitors such as indinavir, LPV, and RTV in solution or suspension to primates, along with the reported plasma half-lives in their respective product labels, has shown that plasma drug concentrations are expected to decrease below the limit of detection by 4 - 5 hours and TFV in solution is expected to decrease below the limit of detection by 8 hours. Therefore, we analyzed the AUC for the early (0 - 8 hours) and late (8 - 168 hours) time courses. Although there was some variation between the two animals, it was clear that after subcutaneous administration of anti-HIV lipid nanoparticles, for all three drugs, including TFV, the early (0 - 8h) AUC was less than 20% of the total AUC (0 - 168h). Since the anti-HIV LNP contains 93.9% (only 6.1% bound to the LNP) free TFV, the low early AUC0 - 8h fraction of as low as 2.6% of TFV was unexpected. In fact, most of the TFV exposure (AUC fraction) was found in the late AUC8 - 168 h. In any case, these data indicate that anti-HIV LNP provides sustained plasma drug levels for the three drugs ATV, RTV, and TFV in primates and that the early drug exposure in plasma is less than 20% of the total drug exposure that persists for more than 168 hours or 7 days after a single subcutaneous dose.

[0204] Discussion

[0205] Although the clinical use of many oral anti-HIV drug combinations, including protease inhibitors such as indinavir, LPV, RTV, ATV, and DRV, has successfully reduced plasma HIV below the limit of detection, most oral drug therapies require at least one or more daily administrations. Single or multiple daily administrations of various liquid and solid oral formulations often face compliance challenges, especially in substance-abusing and high-risk populations with high rates of HIV transmission. Therefore, there is an urgent need in the medical community to develop once-weekly anti-HIV combination drug regimens that overcome the daily dosing requirements of oral dosage forms, particularly for patients with compliance (e.g., drug-abusing populations) or practicality (inability to swallow or gastrointestinal discomfort) issues. Taking advantage of the hydrophobicity of ATV and DRV and their ability to bind to and interact with lipids, we developed lipid nanoparticles containing one of these two drugs (ATZ) and characterized the stability of the lipid-drug interaction. We found that ATV, rather than DRV, binds stably to lipids, enabling the development of anti-HIV drug combination lipid nanoparticles composed of ATV, RTV, and TFV (a hydrophilic reverse transcriptase inhibitor). This drug combination was selected considering current clinical HAART recommendations, conferring greater clinical potential. We prepared these combination nanoparticles using a simple scale-up process that yielded reproducible characteristics at preclinical scales suitable for study in primates.

[0206] Previous studies using the HIV protease inhibitor indinavir have shown that the compound binds completely to lipids. Additional optimization studies have shown that phosphatidylcholine lipids with C18 fatty acyl chains (DSPC and the pegylated lipid DSPE-mPEG2000) provide stable drug incorporation and drug accumulation in lymphoid tissues when administered subcutaneously in primates. These reports also show that complete drug binding in lipid-drug nanoparticles contributes to the ability of the drug to localize in lymph nodes throughout the body and to generate sustained drug levels in plasma. In this report, we used the same lipid composition to demonstrate that two potent HIV protease inhibitors, ATV and DRV, both of which exhibit high lipophilicity at neutral pH, can bind to lipids at high density (up to 1 drug molecule per 5 lipid molecules). We were able to prepare lipid nanoparticles with diameters of 33.6 - 68.2 nm that showed nearly 100% incorporation efficiency of ATV or DRV (Table 2). The lipid insertion of ATV and DRV was evident in the ability of each drug to lower the lipid phase transition temperature and to reduce, to varying degrees, the order of lipid molecule organization within the lipid-drug nanoparticle structure. The effect of the drug on lipid packing was detectable as a decrease in the anisotropic behavior of the membrane polarization probe DPH ( Figure 2). Although both ATV and DRV showed an effect on the ordered state of the lipid membrane, DRV showed a higher effect than ATV, as a stronger inhibition of the lipid phase transition temperature was detected ( Figure 2 ). Additionally, concentration-dependent studies showed that the apparent size (diameter) of only DRV-LNP, but not ATV-LNP, was affected by dilution. Only the DRV-LNP particle size significantly decreased to the size range of mixed micelles. These data suggest that DRV, but not ATV, induces LNP to adopt micelle-like behavior upon dilution (Table 3). In contrast, when the lipid concentration was decreased, the diameter of the lipid nanoparticles incorporating ATV did not change significantly, indicating that they formed stable lipid-drug nanoparticles. This concentration-dependent size behavior is consistent with the much lower drug release rate observed for ATV-LNP compared to DRV-LNP, and the release rate of ATV-LNP also showed to be concentration-independent ( Figure 3A and 3B ).

[0207] Although the exact mechanism or extent of insertion of DRV and ATV into the lipid membrane is unclear, it is evident from the pH-dependent partition coefficient data that only ATV showed a pH-dependent change in lipophilicity. When the pH was increased from 3 to 7.4, the measured value of the pH-specific lipophilicity of ATV, Log D, increased from 3.40 to 5.77, while for DRV, Log D remained relatively constant (2.80 - 2.98) over the same pH range (Table 1). The higher Log P value of ATV compared to DRV may partly contribute to the more stable binding of ATV-LNP (Table 2 - 3 and Figure 3A and 3B ). Additionally, the pH-dependent lipophilicity of ATV may be related to the ability of ATV to dissociate from the lipid-drug nanoparticles in a pH-dependent manner between pH 3 - 7.4 ( Figure 4). In addition, the pKa of the solute can affect the degree and extent of pH-dependent release of the drug from the lipid-drug nanoparticles. As a compound that is chemically stable under acidic conditions, when the pH is below the pKa (acidic), the compound will be protonated and its solubility will increase. The environment of pH 3 is lower than the pKa of ATV, which is 4.42 (acidic, Table 1), which is why approximately 98.1% of ATV is released from ATV-LNP at pH 3. For DRV, the pH from 3 to 7.4 is higher than its pKa of 2.39 (acidic, Table 1). Under these conditions, DRV will be deprotonated, making it more hydrophobic and unable to undergo pH-dependent release. Therefore, ATV incorporated into lipid nanoparticles can be internalized by cells in lymph nodes and lymphoid tissues and subsequently released in endosomes and lysosomes where the pH drops to 5.5 and 4, respectively. pH-sensitive drug binding and dissociation from nanoparticles will be useful for improving the intracellular delivery of free drug molecules to lymph nodes and lymphoid tissues. Inside these intracellular acidic organelles, the free or unbound drug can be made available to enhance anti-protease activity and anti-HIV effects. These and other possibilities require further investigation. However, this research is beyond the scope of this report.

[0208] The stability of ATV binding and insertion into the lipid membrane and the detailed understanding of lipid-drug interactions enabled us to develop formulations containing two other clinically used anti-HIV drugs, RTV and TFV. RTV is a metabolism and efflux inhibitor and is usually used in combination with other protease inhibitors such as ATV to reduce its clearance rate. With the almost complete and reproducible incorporation of ATV and RTV into lipid nanoparticles (Table 4), the scale-up process was greatly simplified and there was no need to remove free protease inhibitors. Therefore, wasteful and potentially cost-prohibitive purification processes could be avoided. For example, the requirement to remove free human growth hormone (hGH) from polymeric particles was shown to be cost-prohibitive and was considered a key reason for the discontinuation of the production of sustained-release hGH after obtaining regulatory approval for marketing in the United States. The almost complete incorporation of ATV and RTV into lipids described in this report can be considered an important feature of these lipid-drug nanoparticles, which enables sustained drug levels in primates to exceed those achieved by oral or subcutaneous administration of soluble and suspension formulations.

[0209] Current clinical guidelines recommend prescribing at least two combinations of drugs that inhibit viral resistance or drug combinations that inhibit two HIV targets, such as protease inhibitors and reverse transcriptase. Therefore, we selected the proven reverse transcriptase inhibitor TFV, which phosphorylates intracellularly and is subsequently retained for a long time, to incorporate into lipid-drug nanoparticles containing ATV and RTV. However, due to its high water solubility (low lipophilicity), we found a low but consistent and reproducible incorporation of approximately 6% of TFV. Therefore, we continued with primate studies using the three drugs ATV, RTV, and TFV in lipid-drug nanoparticles containing 93.9% of the "free" soluble form of TFV. Administration of soluble ATV, RTV, or TFV has been shown to generally reduce plasma drug levels in primates below the detection level within 8 hours, and the drug is undetectable in plasma at 24 hours. When the same three drugs were formulated together into lipid-drug nanoparticles, we found that all three drugs were still detectable in the plasma of two primates after 7 days. While it was expected that ATV and RTV would provide sustained but low levels, surprisingly, the water-soluble TFV, which was only 6.1% bound to the particles (93.9% free form), also exhibited a high degree of plasma drug exposure, which exceeded the plasma drug exposure obtainable during the free drug release period. One would expect that the AUC ratio of TFV at early time points (0 - 8 hours) should be higher than at later time points, since 93.9% of TFV exists as free drug. It would be speculated that the free drug is directly absorbed from the subcutaneous space into the blood, or easily penetrates through the lymph nodes and rapidly appears in the blood. Therefore, one would expect high plasma drug concentrations and high AUC ratios at early time points (0 - 8 hours). However, we found that only 2.6% of the drug exposure (AUC0 - 8h) of TFV was detectable in plasma at 0 - 8 hours, while 97.4% was detectable at 8 - 168 hours (Table 5). These data suggest that TFV (in combination with ATV and RTV in the formulation) can interact with anti-HIV LNP by a mechanism yet to be defined, resulting in prolonged TFV retention in plasma. While the exact mechanism of the observed interaction and the plasma TFV time course extension warrant further investigation, the current data point to the possibility of once-weekly administration of the triple ATV + RTV + TFV LNP to provide sufficient antiviral levels in plasma.

[0210] Table 5: Comparative analysis of early (0 - 8 h) versus late (8 - 168 h) plasma drug exposure in primates administered anti-HIV nanoparticles containing three drugs with different incorporation efficiencies subcutaneously

[0211]

[0212] The mean AUC of two primates (M11016 and M10088) within the specified time spans (0 - 8 h, 8 - 168 h, and 0 - 168 h), in ng•h / mL.

[0213] b Compare the AUC fractions for the early (0 - 8 h) and late (8 - 168 h) periods with the total plasma exposure over 0 - 168 h. Data are presented as AUC% for each drug.

[0214] If needed, plasma drug levels can be elevated with an increase in dose or the same dosing frequency. Additional detailed pharmacokinetic studies in more animals are planned and will be required to elucidate the predicted pharmacokinetic parameters for defining the dose and frequency necessary to provide optimal antiviral plasma concentrations. However, such studies are beyond the scope of this report.

[0215] In summary, we have demonstrated that two protease inhibitors, ATV and DRV, can be incorporated into lipid nanoparticles almost completely. However, only ATV, but not DRV, binds stably to the lipid and enables the development of anti - HIV drug - combination lipid nanoparticles. A triple anti - HIV LNP containing ATV, RTV, and TFV can be prepared aseptically and adjusted proportionally to produce consistent particle size, pH, and osmolality characteristics suitable for subcutaneous administration to primates. These anti - HIV drug - combination lipid nanoparticles provide sustained plasma drug levels of all three drugs for more than 7 days, even for the hydrophilic drug TFV. Based on preliminary primate plasma concentration - time data, once - weekly administration of the anti - HIV nanoparticles containing ATV, RTV, and TFV is feasible.

[0216] Example 2

[0217] This example describes the design and characterization of additional multi - drug lipid nanoparticles that provide long - acting triple - drug combination anti - HIV nanoparticles with enhanced drug exposure in primate plasma and lymph nodes and blood - borne cells.

[0218] Insufficient levels of HIV drugs in lymph nodes are associated with viral persistence. To overcome lymphatic drug insufficiency, we developed and evaluated lipid - drug nanoparticles containing lopinavir, ritonavir, and tenofovir in primates. Compared with free drugs, these nanoparticles produce intracellular concentrations of lopinavir, ritonavir, and tenofovir in lymph nodes that are more than 50 - fold higher. Plasma and intracellular drug levels in blood are enhanced and sustained for 7 days after a single subcutaneous dose, exceeding those achievable with current oral therapies.

[0219] Combined antiretroviral therapy (cART) can clear HIV from the blood; however, residual virus remains in lymph nodes. Oral cART produces lower drug concentrations in lymphoid tissues than in plasma, which is associated with the virus remaining in lymph nodes and viral rebound upon treatment cessation. Drug nanoparticles have the potential to overcome lymphatic drug insufficiency. We previously developed and demonstrated in primates that lipid nanoparticles (LNPs) containing indinavir (IDV) enhanced drug levels in all analyzed lymph nodes. In addition, IDV in the LNP enhanced intracellular drug concentrations in peripheral blood mononuclear cells (PBMCs), prolonged plasma residence time, reversed CD4+ T cell decline, and inhibited viral RNA in both plasma and lymph nodes. Based on the findings with IDV-LNP, we have developed an anti-HIV LNP containing two protease inhibitors, lopinavir (LPV) and ritonavir (RTV), and a reverse transcriptase inhibitor, tenofovir (TFV), for simultaneous delivery of the triple drug to HIV host cells in blood and lymph. LPV and RTV were selected because of their stability and strong hydrophobic interactions with the LNP. Ritonavir enhances the efficacy of LPV through metabolic and drug transporter interactions. Inclusion of TFV provides a second target for antiviral action that further inhibits the potential for drug resistance, and intracellular retention of phosphorylated TFV prolongs antiviral activity.

[0220] We characterized an optimized anti-HIV LNP for primate studies. The aseptically prepared anti-HIV LNP showed 94, 91, and 12% incorporation of LPV, RTV, and TFV, respectively, with an average diameter of 52 nm. A well-defined unbound drug fraction was included for in vivo studies. The antiviral potency against HIV-1, evaluated at a fixed LPV:RTV:TFV 1:1:0.5 molar ratio, showed that the anti-HIV LNP was at least three-fold more potent than the soluble form of the drugs (EC50 for LPV, RTV, and TFV in the LNP form was 30 ± 0.8, 30 ± 0.8, 15 ± 0.1 nmol / l, compared to 98 ± 0.3, 98 ± 0.3, 49 ± 0.2 nmol / l for the free form).

[0221] Primates administered LPV, RTV, and TFV (25.0, 14.3, and 17.1 mg / kg) in anti-HPV LNP formulations subcutaneously showed elevated plasma concentrations of LPV and RTV within 7 days (168 hours). In contrast, plasma drug levels after co-administration of free drugs decreased to near or below the limit of detection by 24 h. The total drug exposure [area under the curve (AUC)] of LPV, RTV, and TFV provided by the LNP formulations increased by 18, 14, and 7-fold, respectively (paired t-test P = <0.05, 0.07, and 0.173) (Table 6). The AUCs for early (0 - 8 h) versus late drug exposure (8 - 168 h) were also compared. Primates treated with free drugs showed 92% of the total TFV exposure within the first 8 hours, while those treated with anti-HIV LNP showed only 9.1% within the first 8 hours, with the remainder between 8 - 168 hours (Table 6).

[0222] Intracellular drug accumulation is key to antiviral action. Persistent LPV, RTV, and TFV were detected in PBMCs for more than 7 days in primates treated with anti-HIV LNP, while those of free drugs decreased to near or below the limit of detection by 48 h (Table 6). The anti-HIV LNP to free drug ratio (LNP / free ratio) was used to compare PBMC drug concentrations between the two test groups. This ratio was greater than 1 at all time points beyond 5 h and greater than 20 at later time points (Table 6). For TFV, only 12% was bound to LNP, and the LNP / free ratio in blood PBMCs was less than 1 at early time points; however, by 8 h, this value increased to more than 50. Anti-HIV LNP also increased the intracellular drug concentration in lymph node monocytes (LNMCs). LNMCs isolated from inguinal lymph nodes at 24 h showed no detectable LPV and only low levels of RTV in animals treated with the free drug combination, while those treated with anti-HIV LNP showed intracellular concentrations more than 50-fold higher (Table 6). For TFV, with limited LNP binding, the LNP / free ratio was low, recorded as 0.7; however, the difference between the two groups of macaques was not statistically significant (P = 1.0).

[0223] Table 6. Effects of anti-HIV lipid nanoparticlesa on intracellular levels of lopinavir, ritonavir, and tenofovir in peripheral blood monocytes and inguinal lymph nodes and total drug exposureb in plasma.

[0224]

[0225] LNP, lipid nanoparticle. NA, not available.

[0226] a An anti-HIV LNP composed of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), N-(carbonyloxy-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG-2000-DSPE), lopinavir, ritonavir, and tenofovir (TFV) was prepared by the thin-film hydration method as previously disclosed. All drugs were dried with the lipid film, rehydrated in bicarbonate buffered saline, and reduced in size by high-pressure homogenization under sterile conditions to produce drug-lipid nanoparticles with an average diameter of 52 nm. Free drug suspension formulations were prepared in bicarbonate buffered saline using biocompatible solvents and surfactants to suspend highly hydrophobic drugs.

[0227] b In a crossover study, four primates (pigtail macaques) were subcutaneously administered anti-HIV LNP and free drugs at standardized doses of 25 mg / kg LPV, 14.3 mg / kg RTV, and 17.1 mg / kg tenofovir (TFV). All experiments were conducted under an approved Institutional Animal Care and Use Committee (IACUC) protocol. Blood and lymph nodes were collected within 7 days at designated time points, and drug concentrations were determined using a validated analytical method published for liquid chromatography tandem mass spectrometry (LC-MS / MS). Data are presented as mean ± SD. Ratios used for comparative analysis are shown in bold.

[0228] c The LNP / free ratio is the mean anti-HIV LNP drug concentration divided by the mean free drug concentration. In cases where the drug level is below the limit of detection, this number is used as "0.01" to calculate the ratio.

[0229] d PBMCs were isolated from whole blood by density gradient method, and cell pellets of 2 million each were analyzed. (n = 4 / group; at 48 h and 120 h, n = 2 / group).

[0230] e Inguinal lymph nodes were collected at 24 h (n = 2 / group), and monocytes were isolated by pressing the tissue through a 200 μm cell strainer. Cell pellets of 2 million each were analyzed.

[0231] f The area under the curve (AUC) was calculated from plasma drug concentrations using the trapezoidal rule. The fractional percentages of the total AUC in the early (0 - 8 h) and late (8 - 168 h) periods were calculated from the mean of the total AUC and the mean AUC in the designated time range. Values are expressed as a percentage of the total AUC (n = 4 / group).

[0232] Although the toxicity of the anti-HIV LNPs requires further study, the complete blood count, serum chemistry panel, C-reactive protein, and complement levels did not show treatment effects. Animals without anti-HIV LNP treatment showed elevated C-reactive protein, white blood cell count, blood urea nitrogen (BUN), creatinine, or liver enzymes. Total complement levels varied highly but not significantly. Additionally, when administered with anti-HIV LNPs, no significant increase in cholesterol levels was noted (162 ± 16.4 vs. 191 ± 14.8 mg / dl). In physical examinations at the injection site, naive animals receiving free drug exhibited local reactions consisting of firm, non-erythematous swelling that resolved within subsequent weeks. Animals treated with anti-HIV LNPs did not show local reactions, and their platelet counts remained within the normal range.

[0233] In summary, taking advantage of the high LNP incorporation efficiency of the two lipophilic protease inhibitors, LPV and RTV, and the ability to encapsulate the hydrophilic TFV, we constructed combinatorial anti-HIV LNPs and analyzed intracellular drug concentrations and plasma kinetics in macaques (pigtail macaques). Primates administered subcutaneously with anti-HIV LNPs showed elevated and prolonged intracellular drug levels in monocytes in both blood and lymph nodes, indicating the utility of this approach to overcome lymph node drug insufficiency and associated viral persistence in patients on oral cART therapy. The ability of anti-HIV LNPs to extend plasma drug levels with a higher total drug exposure for more than 1 week supports consideration as a long-acting agent to improve patient compliance. Importantly, packaging the three drugs together in anti-HIV LNPs can reduce the potential for drug resistance by delivering all three drugs consistently and simultaneously above therapeutic levels in the same cells. This reduces the likelihood of intracellular concentration variations for each drug, such as those achieved when delivered in free form or separate particles. The therapeutic efficacy of anti-HIV LNPs can be further enhanced by organelle targeting with pH-responsive drug release and targeting of HIV host cells by expressing a CD4+-binding peptide. In conclusion, anti-HIV LNPs show promise in overcoming drug insufficiency in lymphoid tissues and improving patient compliance in the quest for a cure for AIDS.

[0234] Example 3

[0235] This example provides further description of anti-HIV drug combination nanoparticles that enhance the plasma drug exposure duration as well as the triple-drug combination levels in cells in the lymph nodes and blood of primates.

[0236] Abstract

[0237] HIV patients on combination oral therapy experience insufficient drug levels in lymph nodes, which is associated with viral persistence. After successfully enhancing lymph node drug levels and prolonging the plasma residence time of indinavir formulated in lipid nanoparticles, we developed multi-drug anti-HIV lipid nanoparticles (anti-HIV LNPs) containing lopinavir (LPV), ritonavir (RTV), and tenofovir (PMPA). These anti-HIV LNPs were prepared, characterized, scaled up, and evaluated in primates, with a focus on plasma time course and intracellular drug exposure in blood and lymph nodes. Four macaques were subcutaneously administered anti-HIV LNPs and free drug suspensions in a crossover study. The time course of plasma drug concentrations and intracellular drug concentrations in blood and inguinal lymph nodes were analyzed to compare the effects of the LNP formulations. Anti-HIV LNPs incorporated LPV and RTV with high efficiency and reproducibly captured a fraction of the hydrophilic PMPA. In primates, anti-HIV LNPs produced intracellular LPV and RTV concentrations in lymph nodes that were over 50-fold higher than those of the free drugs. Plasma and intracellular drug levels in blood increased and persisted for up to 7 days, exceeding those achievable with their free drug counterparts. Thus, multiple antiretroviral agents can be co-incorporated into anti-HIV lipid nanoparticles to enhance intracellular drug concentrations in blood and lymph nodes where viral replication persists. Since these anti-HIV lipid nanoparticles also prolong plasma drug exposure, they hold promise as long-acting formulations for HIV patients to address residual virus in cells and tissues.

[0238] Introduction

[0239] Highly active antiretroviral therapy (HAART) is a combination of antiretroviral drugs with different viral targets that can clear virus from the blood and maintain aviremia for several years. However, if daily oral therapy is interrupted, plasma viremia rapidly rebounds. Viruses persist in lymph nodes and lymphoid tissues, and viral DNA and RNA can still be detected in the lymph nodes and lymphoid tissues of HAART patients even in the presence of plasma aviremia. Drug concentrations in the lymphoid tissues of HIV+ patients on HAART are lower relative to concurrent plasma concentrations, which is associated with persistent lymphoid viral replication. Enhancing and prolonging drug exposure in lymphoid tissues is necessary to clear residual virus in the pursuit of a cure for HIV.

[0240] We previously found that lipid nanoparticles (LNPs) containing the protease inhibitor indinavir (IDV) produced elevated drug levels in lymph nodes throughout the body and prolonged plasma residence times. In HIV-infected primates, treatment with these IDV-LNPs reversed CD4+ T cell decline and reduced viral RNA in plasma and lymph nodes. Incorporation of other protease inhibitors as well as the hydrophilic drug tenofovir (PMPA), an active drug in Viread and a nucleotide analogue reverse transcriptase inhibitor (NRTI), into these LNPs has been documented. Since monotherapy regimens can promote drug resistance, while combination drug therapies targeting multiple HIV proteins reduce mortality, multiple drugs formulated in a single particle can enhance therapeutic efficacy and tissue viral clearance.

[0241] Accordingly, we developed and evaluated in primates lipid nanoparticles (anti-HIV LNPs) containing a three-drug combination of lopinavir (LPV), ritonavir (RTV), and PMPA. LPV and RTV were chosen because of their acid stability and hydrophobicity, which facilitate lipid binding. Ritonavir is a metabolic and transport inhibitor that is clinically used to enhance the efficacy of co-administered drugs. PMPA, as an NRTI, provides an additional antiviral site of action, and its phosphorylated form remains intracellular, thereby prolonging antiviral activity. This combination is clinically relevant and favors prolonged and enhanced intracellular drug exposure. We found that subcutaneous injection of anti-HIV LNPs in primates produced enhanced intracellular drug concentrations in lymph node mononuclear cells (LNMC) and peripheral blood mononuclear cells (PBMC) and prolonged residence times in PBMC and plasma compared to free drug in suspension. Long-acting anti-HIV lipid nanoparticles have the potential to overcome drug deficiencies and associated viral persistence in lymphoid tissues.

[0242] Materials and Methods

[0243] Materials and Animals

[0244] 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) and N-(carbonyloxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt (MPEG-2000-DSPE) were purchased from CordenPharma (Liestal, Switzerland). LPV, RTV, and PMPA ([(2R)-1-(6-aminopurin-9-yl)propan-2-yl]oxymethylphosphonic acid) were purchased from Waterstone Technology (Carmel, IN). Other reagents were of analytical grade or higher.

[0245] Four young adult male rhesus macaques (cynomolgus monkeys, 2.9 - 4.0 kg) were housed and cared for by the Washington National Primate Research Center (WaNPRC) according to an approved Institutional Animal Care and Use Committee protocol. One animal developed an unrelated illness, and thus the second round of PBMC data from that individual had to be discarded.

[0246] Lipid nanoparticle preparation and in vitro characterization. Lipid nanoparticles (LNPs) composed of DSPC:MPEG-2000-DSPE (8:2 or 9:1 molar ratio), LPV, RTV, and PMPA (115:10:5:15 lipid:LPV:RTV:PMPA molar ratio) were prepared aseptically according to a previously established method. Kinman L, Brodie SJ, Tsai CC, et al.: Lipid-drug association enhanced HIV-1 protease inhibitor indinavir localization in lymphoid tissues and viral load reduction: A proof of concept study in HIV-2287-infected macaques. J Acquir Immune Defic Syndr 2003;34(4):387–397, the entire content of which is incorporated herein by reference. Briefly, the lipids and protease inhibitor were dissolved in chloroform:ethanol (3:1 v / v), and PMPA from a stock solution of 150 mM NaHCO3 aqueous solution at 30 mg / ml was added. The solvent was removed by rotary evaporation and vacuum drying. The dried film was rehydrated to 200 mM lipid in 0.4% NaCl with 20 mM NaHCO3 buffer at 60°C. The particle size was reduced at 60°C by bath sonication (laboratory scale) or high-pressure homogenization using an Emulsiflex-C5 (Avestin, Ottawa, Canada) (clinical scale). The LNPs were maintained at 60°C to anneal for 30 minutes before cooling and stored at 4°C. The particle size was determined by photon correlation spectroscopy using a NICOMP 380 ZLS (Particle Sizing Systems, Santa Barbara, CA). The osmolality (Vapro 5520 osmometer; Wescor, Logan, UT) and pH (Hydrion paper) were evaluated.

[0247] The anti-HIV LNP formulations contain unbound drug and drug bound to lipid nanoparticles. To determine the drug incorporation / encapsulation efficiency, the anti-HIV LNP was dialyzed (MWCO 6,000 - 8,000) against bicarbonate-buffered saline at 1,000× volume for 4 hours at room temperature to remove unbound drug. The drug was quantified by LC-MS / MS using acetonitrile precipitation. The encapsulation efficiency (EE) was calculated as follows:

[0248]

[0249] As previously described, the antiviral activity of the triple-drug combination in soluble or LNP form was evaluated in HIV-infected CEM-174 cells. Kinman L, Brodie SJ, Tsai CC, et al.: Lipid-drug association enhanced HIV-1 protease inhibitor indinavir localization in lymphoid tissues and viral load reduction: A proof of concept study in HIV-2287-infected macaques. J Acquir Immune Defic Syndr 2003;34(4):387–397, the entire content of which is incorporated herein by reference. After 4 days of incubation with the drug, syncytia of the cells were observed and viral infection was confirmed by ELISA for HIV-2 p27. For antiviral activity evaluation, the ratio of LPV, RTV, and PMPA was fixed at 1:1:0.5 (m / m / m). The drug dose added to the cells represents the sum of the bound and unbound drug in these anti-HIV LNP formulations.

[0250] Preparation of free drug suspensions for primate studies

[0251] Two free drug suspensions were prepared at the same molar ratio as the anti-HIV LNP (LPV:RTV:PMPA 2:1:3, m / m / m). The carrier of the first suspension was bicarbonate-buffered saline containing 3% ethanol and 0.2% bovine serum albumin. The carrier of the second suspension was bicarbonate-buffered saline containing 8% DMSO and 0.1% Tween 20.

[0252] Sterility of injectable formulations

[0253] All injectable preparations are prepared using injectable aqueous solutions and aseptic techniques. The aqueous components that are contaminated with bacteria are sterilized by passing through a 0.22-μm cellulose acetate filter. Sterility is verified by 7-day blood agar culture tests at 37°C.

[0254] The combination of free and lipid-bound drugs was administered subcutaneously to rhesus monkeys

[0255] Four rhesus monkeys were divided into two treatment groups (two animals per group). Each animal received free drug and anti-HIV LNP (a mixture of bound and free drugs) in a crossover study, with a washout period of more than 12 weeks between experiments. Twenty milliliters of anti-HIV LNP was delivered subcutaneously in the back at doses of 25.0, 14.3, and 17.1 mg / kg of LPV, RTV, and PMPA, respectively. Due to limited solubility, the free drug suspension was administered at slightly lower volume doses (20.0, 11.5, and 13.7 mg / kg of LPV, RTV, and PMPA, respectively). Data were normalized to the anti-HIV LNP dose for comparative analysis.

[0256] Collection and processing of blood and tissue samples

[0257] Blood samples were collected in EDTA by femoral vein puncture at 0, 0.5, 1, 3, 5, 8, 24, and 168 h during the second round of the study, plus 48 h (both groups) and 120 h (anti-HIV LNP group). Plasma was removed, PBMCs were isolated by density gradient centrifugation, and the cells were aliquoted into cell pellets of approximately 2 million PBMCs each. Inguinal lymph nodes were surgically removed 24 h after dosing (n = 2 per treatment), LNMCs were isolated by passing through a 100-μm nylon cell strainer (Corning, Tewksbury, MA), and the cells were aliquoted into cell pellets of approximately 1–2 million LNMCs each. All samples were stored at -80°C prior to drug analysis.

[0258] Determination of drug concentrations in plasma and cells

[0259] Using the recently published method, the plasma concentrations of all three drugs were analyzed simultaneously by liquid chromatography-tandem mass spectrometry. Koehn J and Ho RJ, Novel liquid chromatography-tandem mass spectrometry method for simultaneous detection of anti-HIV drugs lopinavir, ritonavir, and tenofovir in plasma. Antimicrob Agents Chemother 2014;58(5):2675–2680, the entire content of which is incorporated herein by reference. PBMC and LNMC cell pellets were dissolved in 200 μl of water / methanol (1:1 v / v), sonicated for 10 minutes, and then extracted and analyzed using the same method as described above. Assuming a cell volume of 4 × 10-9 ml per cell, the intracellular concentration was converted to ng / ml. Alberts B, Johnson A, Lewis J et al. Molecular Biology of the Cell, 4 ed. Garland Science, New York, 2002, the entire content of which is incorporated herein by reference.

[0260] Determination of Plasma Drug Exposure

[0261] The trapezoidal rule was used to calculate the total evaluation time period (0 - 168 h) and the area under the curve (AUC) for 0 - 24 and 24 - 168 h. The data were analyzed by paired two-sided Student t-test, and p < 0.05 was considered statistically significant.

[0262] Assessment of Immune and Inflammatory Responses

[0263] Blood was collected at least 1 week before dosing and exactly 7 days after dosing for analysis of complete blood count, serum chemistry, C-reactive protein, and total complement. Reference values for pigtail macaques were provided by WaNPRC. Due to limited primate data, human reference values were used for C-reactive protein and total complement. The animals were observed daily for physical or behavioral changes.

[0264] Results

[0265] The physicochemical properties and antiviral activities of anti-HIV nanoparticles composed of lopinavir, ritonavir, and tenofovir. In the formulations for primate studies, anti-HIV LNPs containing LPV, RTV, and PMPA were optimized at the laboratory scale (0.1–0.4 ml) and scaled up to the clinical scale (14–40 ml) using equipment with a multiliter capacity. High protease inhibitor incorporation efficiency was reproducible, and clinical-scale batches showed more than 90% incorporation of LPV and RTV (Table 7). PMPA binding was consistent and reproducible (Table 7). High-pressure homogenization produced particles slightly smaller (diameter ~50 nm) than those prepared by sonication (diameter ~70 nm). The pH and osmolality of the anti-HIV LNP formulations were physiologically compatible, and culture tests verified sterility suitable for primate studies.

[0266] Table 7. Physicochemical characteristics of anti-HIV lipid nanoparticles prepared at the laboratory or clinical scale

[0267]

[0268] a Particle size was determined by Nicomp intensity-weighted analysis.

[0269] b Anti-HIV LNP was reproduced in 0.1 - 0.4 ml batches, and the values are expressed as the mean ± standard deviation of five batches.

[0270] c Anti-HIV LNP was reproduced in 15 - 40 ml batches, and the values are expressed as the mean ± standard deviation of three batches.

[0271] Anti-HIV LNPs were prepared with a lipid:LPV:RTV:PMPA molar ratio of 115:10:5:15 and a final lipid concentration of 200 mM. The drug incorporation of the three drugs LPV:RTV:PMPA (2:1:3 molar ratio) was determined as described for reproducibility and other characteristics in Materials and Methods. Anti-HIV LNPs contained free and bound drugs in defined fractions.

[0272] LNP, lipid nanoparticle; LPV, lopinavir; RTV, ritonavir; PMPA, tenofovir. <P

[0273] The antiviral potency of LNP-formulated LPV, RTV, and PMPA was evaluated in HIV-infected CEM-174 cells and compared to the soluble free drugs in equimolar ratios or alone. At a drug ratio of 1:1:0.5 (m / m / m), the anti-HIV LNPs showed a 30-fold increase in potency compared to the drugs formulated in soluble form (LPV, RTV, and PMPA EC50 values of 3.0 ± 0.8, 3.0 ± 0.8, and 1.5 ± 0.1 nM in the LNP form, compared to 98 ± 0.3, 98 ± 0.3, and 49 ± 0.2 nM in the free form). The EC50 values for the single agents of LPV, RTV, and PMPA were 600 ± 0.04, 530 ± 0.2, and 1150 ± 0.7 nM, respectively. Control lipid nanoparticles showed no antiviral effect.

[0274] Effect of LNP formulation on plasma pharmacokinetics and total drug exposure

[0275] Primates received a single subcutaneous dose of anti-HIV LNP or LPV, RTV, and PMPA in free suspension form. After administration of the free drugs, plasma concentrations peaked within 8 h and declined to low or undetectable levels by 24 h ( Figures 6A - 6F ). In contrast, macaques treated with anti-HIV LNP showed elevated plasma concentrations of LPV and RTV at all time points evaluated, and higher peak plasma concentrations of LPV and RTV (p < 0.05), and all three drugs remained detectable in plasma at levels within the effective antiviral concentration for 7 days (168 h) after administration (even without further optimization). LNP-formulated PMPA showed two peaks: the first peak was similar to that seen after administration of the free drug, while the second peak was not seen with the free drug ( Figure 6E and 6F ).

[0276] Total drug exposure was evaluated by calculating the area under the plasma drug concentration curve (AUC). As shown in Table 8, compared to the free drug suspensions (LPV, RTV, and PMPA ≤ 0.05, 0.07, and 0.173, respectively), the anti-HIV LNPs provided a 7-fold or greater increase in total drug exposure for all three drugs. In addition, for all three drugs, the percentage of total drug exposure occurring after the first 24 h was increased with the anti-HIV LNPs. This change was most significant for the hydrophilic PMPA (0.1% with the free drug and 51.2% with the anti-HIV LNP) (Table 8). These data indicate that the LNP formulation provides enhanced and prolonged plasma drug exposure for all three drugs, including the water-soluble PMPA with only 12% drug binding. It was noted that the initial peak plasma concentration of tenofovir (PMPA or TFV) that occurred within the first 24 h was significantly reduced. This reduction in the peak plasma concentration of tenofovir can reduce the adverse effects associated with the high peak plasma concentration of this drug. It was also of interest to note the total plasma drug exposure of the drug combination formulated with lipid nanoparticles during the extended period of 24 - 168 h of the seven-day study (right column in Table 8). Considering the 7 - 18-fold enhancement in total exposure and the free drug doses selected in this study being equivalent to the therapeutic SHIV treatment doses in primates (e.g., 20 mg PAMA and 50 mg / kg FTC SC daily), it is equivalent to being able to provide the therapeutic effect with only one dose of the multi-drug lipid nanoparticle formulation that provides seven daily free drug doses. In addition, the reduction in total dose and the expected lower plasma peak levels of tenofovir can be easily optimized to result in a safer and more effective product.

[0277] Table 8. Effect of anti-HIV lipid nanoparticles on plasma drug exposure in primates treated with a combination of lopinavir, ritonavir, and tenofovir

[0278]

[0279] a Four animals were treated with lopinavir (25 mg / kg), ritonavir (14.3 mg / kg), and tenofovir (17.1 mg / kg) administered subcutaneously in free or LNP form. Plasma drug concentrations were measured and the area under the curve (AUC) was calculated using the trapezoidal rule. Data are presented as the mean - standard deviation for four animals over a 168-hour period.

[0280] b AUC was analyzed from 0 to 24 h, representing the standard dosing period for current HAART, and from 24 to 168 h to demonstrate the prolonged plasma exposure provided by the anti-HIV LNPs. Values are presented as the percentage of total AUC, mean ± standard deviation.

[0281] Effect of LNP binding on intracellular LPV, RTV, and PMPA concentrations in blood and lymph node monocytes

[0282] Although plasma drug levels indicate continuous drug exposure, intracellular levels are necessary for inhibiting viral replication. Therefore, monocytes in blood (PBMC) were isolated and the concentrations of intracellular LPV, RTV, and PMPA were determined. As Figures 7A through 7C shown, macaques treated with anti-HIV LNP had detectable intracellular drug levels at 7 days after dosing, which exceeded the experimental EC50 reported above. In contrast, intracellular drug levels after free drug administration generally decreased below the limit of detection by 48 h ( Figures 7A - 7C ). Concentrations of LPV and RTV after anti-HIV LNP dosing were comparable to free drug at early time points and up to 20-fold higher than free drug at later time points. For the first five time points of anti-HIV LNP, water-soluble PMPA showed lower intracellular drug levels; however, by 8 h after dosing, intracellular PMPA concentrations in LNP-treated animals were more than 50-fold higher ( Figures 7A - 7C ).

[0283] To determine the effect of LNP binding on intracellular drug concentrations in LNMC, inguinal lymph nodes were collected 24 h after dosing. LNMC were isolated and intracellular drug concentrations were compared between test groups (ratio of LNP to free) and compared to concurrent plasma drug concentrations (Table 9). Concentrations of LPV and RTV were low or undetectable in LNMC of animals treated with the free drug combination and were comparable to plasma concentrations. In contrast, concentrations of LPV and RTV in LNMC after anti-HIV LNP administration were 2-fold and 10-fold higher, respectively, than concurrent plasma concentrations and more than 50-fold higher than LNMC concentrations after free drug administration (p < 0.05). Additionally, plasma concentrations of LPV and RTV for anti-HIV LNP were approximately 25-fold higher at 24 h compared to free drug (Table 9). Although PMPA was always detectable in LNMC, there was no significant difference in intracellular lymph node concentrations between the two test groups (p = 1.0). Intracellular drug levels in PBMC and LNMC were comparable at 24 h. In summary, anti-HIV LNP containing LPV, RTV, and PMPA enhances and prolongs intracellular levels of all three drugs in blood as well as lymph nodes and produces higher concentrations of LPV and RTV in LNMC than in plasma at 24 h after dosing.

[0284] Table 9. Effect of anti-HIV lipid nanoparticles on intracellular concentrations of lopinavir, ritonavir, and tenofovir in lymph nodes compared to plasma concentrations at 24 h

[0285]

[0286] Animals were subcutaneously administered anti-HIV LNPs or free drugs at standardized doses of 25 mg / kg LPV, 14.3 mg / kg RTV, and 17.1 mg / kg PMPA. Blood samples and inguinal lymph nodes were collected 24 h after dosing, and lymph node mononuclear cells were isolated for drug analysis. Intracellular drug concentrations were calculated as described in Materials and Methods. Drug concentrations were reported as mean ± standard deviation (n = 2).

[0287] b The mean anti-HIV LNP drug concentration was divided by the mean free drug concentration. In cases where the drug level was below the limit of detection, the value was taken as “1” for calculating the ratio.

[0288] c The mean intracellular drug concentration in lymph node cells was divided by the mean plasma drug concentration. In cases where the drug level was below the limit of detection, the value was taken as “1” for calculating the ratio.

[0289] Safety evaluation of immune and inflammatory responses

[0290] To evaluate safety, complete blood counts, serum chemistry panels, C-reactive protein, and total complement were analyzed in primate blood samples taken before and after drug administration. The relevant values are summarized in Table 10. Renal and liver function indices (blood urea nitrogen, creatinine, liver enzymes) remained within normal limits. The animals did not show an increase in C-reactive protein or white blood cell count to values outside the reference range. Total complement, although highly variable, did not show a significant change. In the case of anti-HIV LNP treatment, a slight change in cholesterol level was not statistically significant (p = 0.07). Untreated animals receiving the free drug showed local reactions consisting of a firm non-erythematous swelling that resolved within the following weeks and a slightly increased platelet count. Untreated animals receiving anti-HIV LNPs did not show local reactions, and their platelet counts remained within normal limits.

[0291] Table 10. Changes in selected inflammatory indices and cholesterol after subcutaneous administration of lopinavir, ritonavir, and tenofovir in free or anti-HIV lipid nanoparticle form a

[0292]

[0293] a Complete blood counts, serum chemistry, total complement, and C-reactive protein were analyzed in venous blood samples. Values are expressed as mean ± standard deviation. No parameters were excluded from the table that showed an increase to values outside the reference range after administration in any primate (n = 4).

[0294] b Blood was collected at least 7 days before dosing to establish a baseline.

[0295] Blood was collected 7 days after c administration.

[0296] d The p-value was determined by paired two-tailed Student t-test.

[0297] Discussion

[0298] Taking advantage of the ability of lipid nanoparticles to efficiently incorporate two lipophilic protease inhibitors, LPV and RTV, and simultaneously encapsulate a significant portion of PMPA, we constructed a drug combination anti-HIV LNP and analyzed the intracellular drug concentration and plasma kinetics in rhesus macaques (cynomolgus monkeys). Primates administered subcutaneously with anti-HIV LNP exhibited prolonged plasma drug levels ( Figures 6A - 6F ), with higher total drug exposure (Table 8), and sustained intracellular concentrations of LPV, RTV, and PMPA in the blood ( Figures 7A - 7C ). Importantly, the drugs in anti-HIV LNP provided much higher intracellular levels of LPV and RTV in lymph nodes, exceeding those achieved with free drugs (Table 9). These findings suggest that lipid nanoparticles may overcome the reported drug deficiencies in HIV-infected individuals on oral HAART.

[0299] Using a simple, reproducible, and scalable preparation method, these nanoparticles were stably incorporated with LPV and RTV with an efficiency > 90%, while encapsulating the hydrophilic reverse transcriptase inhibitor PMPA (Table 7). PMPA binding was consistent and well-defined, and unbound PMPA remained in solution as free drug, reducing the risk of drug waste and contamination and eliminating the need for expensive and time-consuming purification. Due to space limitations, other details regarding the optimization and in vitro characterization of anti-HIV LNP are beyond the scope of this report and will be reported separately.

[0300] As demonstrated by the increased AUC, the triple-drug combination of subcutaneously administered anti-HIV LNP increased peak plasma concentration, prolonged plasma residence time, and increased plasma drug exposure ( Figures 7A - 7C and Table 8). Considering the 12% encapsulation efficiency of PMPA, these findings suggest that the limited encapsulation of hydrophilic PMPA still has a significant impact on drug distribution and / or metabolism. The enhanced stability provided by LNP and reduced renal and hepatic drug clearance may contribute to these findings, but the in vivo behavior and form of anti-HIV LNP remain to be explored. <L

[0301] Intracellular drug exposure is an important consideration if the drug maximally inhibits viral replication in cells. For all three drugs, lipid-drug binding increased the intracellular drug concentration in PBMC and LNMC, indicating that LNP was internalized and retained in monocytes ( Figures 7A - 7Cand Table 9). Although this non-terminating study was able to analyze only a single peripheral lymph node, previous work with indinavir-containing LNPs showed elevated indinavir concentrations in lymph nodes throughout the body (including mesenteric, tonsillar, bronchial, etc.). Thus, broad lymphatic distribution of drug-containing LNPs is achievable. Accordingly, anti-HIV LNPs hold promise as a vehicle for the simultaneous broad lymphatic distribution of multiple anti-HIV drugs, overcoming lymphatic drug insufficiency at viral reservoirs.

[0302] Providing multiple drugs with different viral targets within a single particle is important for improving antiviral efficacy and reducing the risk of viral resistance. Although many efforts have been made to develop single-drug nanoparticles that combine upon administration, this approach can lead to heterogeneity in intracellular drug concentrations and may shelter drug-resistant viruses. Solid polymer particles with diameters of 100 - 600 nm containing multiple crystalline drugs have been synthesized, but these are much larger than anti-HIV LNPs and may not be suitable for lymphatic uptake. Although the mechanism of action of the drugs in anti-HIV LNPs remains to be elucidated, cellular uptake of the intact nanoparticles, followed by pH- or phospholipase-dependent release of the active drugs, may result in elevated intracellular levels. This mechanism using a combination of acid-stable drugs provides a synergistic antiviral effect compared to free-form drugs or separate nanoparticles. Further studies are planned to evaluate the therapeutic efficacy of multi-drug anti-HIV LNPs in a rhesus macaque model infected with HIV.

[0303] In the context of daily oral HAART therapy, treatment interruption is usually associated with adverse side effects or drug abuse. Gastrointestinal side effects cause patients to avoid taking their medications, and patients with a history of drug abuse are three times more likely to discontinue treatment, leading to rapid viral rebound. The elevated and sustained plasma and intracellular drug exposures obtained with a single dose of anti-HIV LNP suggest the feasibility of less frequent (e.g., once-weekly) dosing, reducing the likelihood of treatment interruption. Although dose-range and multi-dose studies are needed to optimize the dose and dosing frequency, our predictive models suggest that once-weekly administration of anti-HIV LNP is feasible. Studies are planned to evaluate intracellular drug levels in lymph nodes throughout the body and lipid particle distribution using a gadolinium tracer, but these are beyond the scope of this report.

[0304] In addition to providing multiple antiviral compounds, the anti-HIV LNP platform also offers the possibility of pH-dependent drug release, expression of CD4-binding peptides, or other surface modifications for cell and organelle targeting. These anti-HIV lipid nanoparticles rarely or do not elicit local or systemic inflammatory responses and hold promise for delivering HIV drugs such as LPV, RTV, and PMPA to sites of viral persistence in blood and lymphoid tissues.

[0305] Example 4

[0306] This example describes additional studies of the characterization and long-term stability of extended multi-drug lipid nanoparticle formulations.

[0307] As described in Example 3, we developed multi-drug anti-HIV lipid nanoparticles containing the hydrophobic protease inhibitors lopinavir (LPV) and ritonavir (RTV) (sold together as Kaletra®) and the hydrophilic TFV (the active drug in Viread®). At bindings of greater than 90% of LPV and RTV and approximately 12% of TFV, these lipid-stabilized drug particles enhanced and prolonged the plasma and intracellular drug exposures of all three drugs in primates compared to the free drugs. The in vivo pharmacokinetic data designed and obtained by this LNP approach indicate that this new approach is scalable and clinically relevant in the preparation of multi-drug therapeutics for the treatment of HIV, cancer, and other diseases.

[0308] The described lipid-stabilized drug-combination nanoparticle compositions and related preparation methods are widely applicable and capable of incorporating hydrophobic and hydrophilic drugs simultaneously at levels not achievable with conventional liposomes. To further demonstrate the applicability of the described method and the resulting nanoparticle formulations, we evaluated the method with additional different drug combinations. Each combination was tested for high and reproducible binding of two hydrophobic drugs and stable binding of an acceptable and reproducible fraction of the hydrophilic drug (Table 11).

[0309] Table 11: Drug combinationsa that have been simultaneously incorporated into lipid-drug nanoparticles and prepared on a large scale (15 - 60 mL volume). The lipid-drug binding efficienciesb (mean ± standard deviation) are shown in parentheses.

[0310]

[0311] aAll compositions were formulated with DSPC:DSPE-mPEG2000 at an optimized molar ratio of 9:1. The lipid:drug molar ratios were as follows: for combination 1, lipid:LPV:RTV:TFV was 115:10:5:15; for combination 2, lipid:ATZ:RTV:TFV was 115:10:5:15; for combination 3, lipid:LPV:EFV:TFV was 200:26.6:20:40.

[0312] bThe lipid-drug binding efficiency was determined by dialyzing the drug-lipid particle product against at least 1000x volume of isotonic bicarbonate-buffered saline at physiological pH for 4 hours at room temperature. The drug concentration after dialysis (determined by LC-MS / MS method) was compared with the drug concentration before dialysis to determine the percentage of drug stably bound to the lipid.

[0313] To test the long-term stability, the multi-drug nanoparticles were stored refrigerated at 4 °C and their stability was tested after a long period of time. The lipid-drug combination particles were stable, as determined by particle size and degree of drug binding (Table 12). This is a key factor when evaluating the clinical potential of a drug, which must show an acceptable shelf life to be clinically feasible. Even hydrophilic compounds that easily diffuse out of most lipid particles within a few hours, lose only a very low fraction of the bound drug after 8, 12, and 17 months of storage. This excellent stability provides a substantial advantage for translation into clinical applications.

[0314] Table 12: Storage stability of the drug fraction bound to the drug combination nanoparticle mixturea

[0315]

[0316] a Storage stability of LPV / RTV / PMPA. Three to four formulations of the lipid storage of lopinavir / ritonavir / PMPA at 150 mg / mL in the multi-drug lipid nanoparticle mixture were stored at 4 °C under sterile conditions. At the designated storage time points (0 - 17 months), after removing the free drug from the formulation by dialysis, the fraction of drug bound to the multi-drug nanoparticles was determined. The data presented are the mean and standard deviation. At 17 months, only one batch of formulation was available for stability analysis. C = degree Celsius, LPV = lopinavir, mg = milligram, mL = milliliter, PMPA / TNF = tenofovir, RTV = ritonavir, SD = standard deviation.

[0317] As described above, we established the pH-dependent binding of various drugs to lipids at neutral pH and the concomitant drug release upon decreasing pH. Also as described above, this stable incorporation and pH-dependent release of drugs from multi-drug lipid nanoparticles is consistent for both lipophilic and water-soluble compounds. To confirm the pH-dependent release of individual drugs from multi-drug combination lipid nanoparticles, the LPV / RTV / PMPA (TFV) lipid nanoparticles assembled as described above were subjected to different pH environments, and the free drug fraction in the supernatant was analyzed to determine the amount released. Consistent with the results reported in the above examples, different drugs showed pH-dependent release from the multi-drug combination lipid nanoparticles. See Table 13. This confirmed pH-responsive property shown in Table 13 illustrates an additional benefit of the disclosed multi-drug combination lipid nanoparticles. When the lipid nanoparticles are in tissues and are taken up by HIV-infected cells, the incorporated antiretroviral drugs are subsequently released inside organelles (such as endosomes and lysosomes) upon decreasing pH. Due to the chemical stability of the proposed drugs under acidic conditions (most drugs are extracted to measure plasma drug levels for LC-MS analysis), this property can enhance antiviral potency by providing a more stable, prolonged, and effective drug delivery mode.

[0318] Table 13: pH-dependent release of drugs from lipid nanoparticles containing lipids conjugated with lopinavir (LPV), ritonavir (RTV), and tenofovir (PMPA / TFV). Drug release fractions over 2 hours at 37 ºC are expressed as mean ± SD%.

[0319]

[0320] The antiviral potency enhancement of the HIV drug combinations provided in the disclosed multi-drug - lipid nanoparticle compositions was evaluated, particularly the combination incorporating LPV, RTV, and PMPA (TFV). The efficacy of the multi-drug - lipid nanoparticle compositions was evaluated in HIV-infected CEM-174 cells and compared to the soluble free drugs combined in equivalent ratios or the soluble free drugs used alone. At a drug ratio of 1:1:0.5 (m / m / m), LPV, RTV, and PMPA in the multi-drug - lipid nanoparticle formulation enhanced the single-drug potency as well as the equivalent fixed combination in soluble form (Table 14). Control lipid particles did not show antiviral effects. The drugs formulated in multi-drug - lipid nanoparticles showed approximately 30-fold enhancement in the in vitro 50% inhibitory concentration (EC50). The exact enhancement will vary depending on the viral strain, but this data empirically shows that the antiviral potency caused by the multi-drug - lipid nanoparticle formulation was increased 3 to 30-fold.

[0321] Table 14: Role of multi-drug lipid nanoparticle formulations containing lopinavir, ritonavir, and PMPA in HIV-2 (287) replication (data are presented as 50% inhibitory drug concentration (nM) of antiviral replication). For combination therapy, the ratios of lopinavir, ritonavir, and PMPA in the formulation were fixed at 1:1:0.5 (m / m / m).

[0322]

[0323] All three lipid-drug combinations of Table 11 have been evaluated in vivo, and initial pharmacokinetic data from subsets of these combinations are described in Examples 1-3 above. Compared to the same combinations in free form, lipid-drug particle mixtures prepared using the disclosed large-scale preparation methods consistently demonstrated plasma drug levels lasting more than 7 days, enhanced plasma drug exposure, and higher intracellular drug concentrations in lymph nodes and blood. The total plasma drug exposure after administration of lipid-stabilized drugs, evaluated as the area under the plasma time-course curve (AUC), was generally significantly higher than the total plasma drug exposure achieved by free drugs and consistently shifted to later drug exposures (Table 15). This was most notable for TFV, which consistently showed a 10- to 20-fold increase in plasma exposure with only 8-10% of stable lipid binding, and a sharp shift of the AUC to later time points. Free drug-treated primates showed ~90% of the total TFV exposure within the first 8 hours, while lipid-stabilized drug particle-treated primates showed ~95% of the total TFV exposure after 8 hours, regardless of the drug combination (Table 15). Lipid-drug particles also enhanced and prolonged intracellular drug exposure in peripheral blood mononuclear cells (PBMCs), providing detectable intracellular drug levels for more than 7 days, while free drug levels decreased to near or below the limit of detection by 24 hours (Table 16). Blood tests and physical examinations did not show significant adverse reactions associated with subcutaneous administration of lipid-drug combination particles, indicating an acceptable level of safety.

[0324] Table 15: Effect of lipid stabilization on plasma drug exposure. Three different optimized multi-drug lipid formulations were studied to determine the change in area under the plasma curve (AUC) compared to the same drugs in free forma. Drugs used included lopinavir (LPV), ritonavir (RTV), tenofovir (TFV), atazanavir (ATZ), and efavirenz (EFV).

[0325]

[0326] a In the crossover study, lipid-drug particles and free drug (except in the case of LPV / EFV / PMPA) were administered to primates. Plasma concentrations were determined by LC-MS / MS method, and AUC was calculated using the trapezoidal rule. Data are presented as mean ± standard deviation (n = 2 or 4 per group).

[0327] b Fractional AUC was calculated by dividing the AUC of the specified time range by the total AUC.

[0328] c The lipid / free ratio is the mean total AUC generated by the lipid-drug mixture divided by the mean total AUC generated by the free drug.

[0329] Table 16: Effect of lipid binding on intracellular levels of lopinavir, ritonavir, and tenofovir in peripheral blood mononuclear cells (PBMCs) after subcutaneous administration a

[0330]

[0331] a In a crossover study, four primates (pigtail macaques) were administered lopinavir (LPV), ritonavir (RTV), and tenofovir (TFV) subcutaneously in the form of lipid-drug particles and free drug in combination, at standardized doses of 25 mg / kg LPV, 14.3 mg / kg RTV, and 17.1 mg / kg TFV. PBMCs were isolated from whole blood by density gradient method, and cell pellets of 2 million PBMCs each were analyzed. Data are presented as mean ± standard deviation.

[0332] b The lipid / free ratio is the mean drug concentration after administration of the lipid-drug particle mixture divided by the mean drug concentration after administration of the free drug. In cases where the drug level is below the limit of detection, the value "0.01" is taken to calculate the ratio.

[0333] These results further demonstrate that the binding of multiple drugs to the lipid excipient in the nanoparticle aggregates significantly improves the delivery performance and the sustained release / availability of the drugs. Specifically, the binding of the drug combination to the lipid excipient in the disclosed nanoparticles results in a significant increase in plasma and intracellular drug concentrations over a longer period of time. This can more effectively treat diseases such as HIV, where administration of multiple drugs that target different targets is necessary for virus clearance. This approach offers an exciting opportunity to overcome the deficiencies of existing strategies, in which multiple drugs may be administered separately and cannot provide high levels of all administered drugs in the same target tissues and cells, and thus cannot achieve complete virus clearance. While the present invention focuses on anti-HIV drugs and related therapies, it is obvious that the disclosed method can be applied to the preparation and administration of therapeutic agents for other diseases and / or infections, which involve small molecule agents that can similarly bind to the lipid excipient forming the particles.

[0334] Example 5

[0335] Examples 1-4 above describe multi-drug lipid nanoparticles assembled during the process of allowing hydrophilic and hydrophobic agents to be co-incorporated into stable lipid nanoparticles using a mixed dual-solvent method. As described, the agents are separately dissolved in an organic solvent or an aqueous solvent, and then the solvents are combined in a ratio that allows mixing without forming multiple phases (referred to herein as the "mixed dual-solvent" method). This enables the provision of a stable formulation that provides significantly enhanced and prolonged delivery of multiple drugs, each having different physical characteristics, such as different hydrophilicity / hydrophobicity, and potentially targeting different disease targets. Having a single formulation that can flexibly address multiple disease targets allows the formulation to be used to address diseases such as HIV / AIDS while avoiding potential drug resistance to single-target therapies. To achieve this specific goal, Table 17 provides a list of FDA-approved exemplary anti-HIV drugs and their respective targets / modes of action.

[0336] This example describes the successful assembly of multi-drug lipid nanoparticles incorporating selected anti-HIV drugs using an illustrative alternative assembly method. The alternative assembly method is designed to ensure or enhance the complete dissolution and mixing of hydrophilic and hydrophobic components (i.e., small molecule agents and excipient components) together in the final homogeneous composition. The alternative method involves completely dissolving all components, i.e., hydrophobic small molecule agents, hydrophilic small molecule agents, and excipient components, in a single miscible solvent. This is followed by controlled removal of the solvent to form a dry product, which is rehydrated to form multi-drug lipid nanoparticles. In some aspects, this illustrative alternative method produces multi-drug lipid nanoparticles with enhanced or optimized characteristics, including a higher degree of stability and hydrophilic drug binding.

[0337] Table 17. List of FDA-approved HIV drugs and their viral target proteins

[0338]

[0339] a Non-nucleoside reverse transcriptase inhibitor (NNRTI)

[0340] b Nucleoside analogue derivative (NRTI) that inhibits HIV reverse transcriptase

[0341] As described above, some organic solvents such as alcohols (e.g., methanol, ethanol, propanol, hexanol, etc.) can be mixed with a small portion of water to form a water-saturated single-phase miscible solvent without forming an emulsion. This can even be used in combination with other organic solvents such as chloroform (CHCl3), which is immiscible with water alone, as long as the entire mixture does not contain more water than the saturation capacity of the water-miscible organic solvent, a single phase can be maintained. Here, a single-phase completely miscible solvent capable of solvating all components, i.e., hydrophilic and hydrophobic pharmaceuticals and excipient components, is formed to allow the pharmaceuticals and excipients to dissolve and mix completely to achieve maximum interaction. In some embodiments, the organic component of the miscible solvent can be a combination of different organic molecules and aqueous solutions or buffers. For example, chloroform can be mixed with an alcohol. In this example, chloroform is mixed with ethanol in a ratio of 65:35 (volume / volume). In some embodiments, ethanol can be replaced with methanol, propanol, hexanol, etc. The presence of some water-insoluble organic components such as chloroform is believed to contribute to the drying of the mixture.

[0342] Selected lipophilic (hydrophobic / water-insoluble) small molecule pharmaceuticals (multiple small molecule pharmaceuticals), hydrophilic (water-soluble / lipid-insoluble) small molecule pharmaceuticals (multiple small molecule pharmaceuticals), lipid excipients, and excipients containing large hydrophilic domains are completely dissolved in the miscible solvent. For the purposes of this illustrative example, lipophilic small molecule pharmaceuticals (also referred to as "class 1") are selected from Table 18, and hydrophilic small molecule pharmaceuticals (also referred to as "class 2") are selected from Table 19, which each provide the structure and physical properties of each illustrative small molecule pharmaceutical.

[0343] Table 18: Illustrative lipophilic (hydrophobic, also referred to as "class 1") small molecule pharmaceuticals suitable for incorporation into the disclosed lipid nanoparticles for HIV treatment.

[0344] * Net formal charge at neutral pH

[0345] Table 19: Illustrative hydrophilic (also referred to as "class 2") small molecule pharmaceuticals suitable for incorporation into the disclosed lipid nanoparticles for HIV treatment.

[0346]

[0347] * Net formal charge at neutral pH

[0348] This example describes additional studies characterizing the multi-drug lipid nanoparticles assembled in the single miscible solvent method described above. Illustrative multi-drug lipid nanoparticles incorporate lopinavir (LPN), ritonavir (RTV), and tenofovir (TFV), and in vivo pharmacokinetic studies were conducted in primates to establish an extended presence in plasma. Additionally, to study the surprisingly high and sustained levels of the hydrophilic drug tenofovir upon administration of the lipid nanoparticles, sucrose gradient tests were performed, and high levels of drug binding were established in the nanoparticles even in the presence of shear forces applied during the gradient test.

[0349] Multi-drug lipid nanoparticles incorporating lopinavir (LPN), ritonavir (RTV), and tenofovir (TFV) were assembled according to the following general method:

[0350] 1. Select one or two hydrophobic and water-insoluble (Class 1) drugs from Table 18 (e.g., lopinavir and ritonavir).

[0351] 2. Select one or two hydrophilic and water-soluble (Class 2) drugs listed in Table 19 (e.g., tenofovir and / or emtricitabine and / or lamivudine).

[0352] 3. Select an excipient combination (e.g., 9 parts DSPC and 1 part PEG-DSPE).

[0353] 4. Prepare a single-phase miscible solvent that can fully dissolve the selected water-insoluble Class 1 drugs and water-soluble Class 2 drugs plus the excipient from step 3. Examples include CHCl3:methanol:water (65:35:4); CHCl3:ethanol:water (65:35:4); CHCl3:propanol:water (65:35:4); and CHCl3:hexanol:water (65:35:4).

[0354] 5. Dissolve the components from steps 1, 2, and 3 in the miscible solvent from step 4.

[0355] 6. Dry the drugs (steps 1 and 2), excipient (step 3), and miscible solvent (step 4) under reduced pressure using a combination of heat, reduced pressure, and / or atomization to provide uniformity and drying efficiency.

[0356] 7. Heat the dehydrated composition above the gel-to-liquid phase transition temperature of the excipient mixture (e.g., 50ºC).

[0357] 8. Prepare a buffered salt solution and equilibrate the temperature to the same temperature as the dehydrated composition.

[0358] 9. Gradually combine the buffered salt solution and the dehydrated composition and maintain at a specified temperature (e.g., 50o C) 3 hours.

[0359] 10. While maintaining at a defined temperature (in the liquid phase of the lipid excipient), the resulting hydrated multi-drug lipid nanoparticles are size-reduced by shearing or forcing through small holes or a pressure filter device. Typically, about 10 - 15 passes provide a defined size of 60 - 120 nm in diameter.

[0360] The molar ratio of drug to excipient is usually high, and for every 3 excipient molecules (i.e., as described in step 3), up to 1 drug molecule (i.e., the drug in steps 1 and 2 above) can be incorporated in the combination. The final product mixture contains more than 70 - 90% of the drugs bound to the multi-drug lipid nanoparticles listed in Table 18 (lipophilic; class 1) and Table 19 (water-soluble; class 2). Thus, a low fraction of free drug is allowed for therapeutic applications as a loading dose to saturate non-specific distribution and adsorb to tissues.

[0361] In the case of using miscible solvents and a rehydration control process called the single miscible solvent method, another part of the class 2 water-soluble pharmaceutical agents binds to the resulting lipid nanoparticles. The improvement in the binding of class 2 pharmaceutical agents can be noted in Table 21 below. Data on the binding of these hydrophobic (class 1) and hydrophilic (class 2) pharmaceutical agents are provided in Tables 20 and 21 below. These tables also provide comparative data of the multi-drug lipid nanoparticles prepared by the double solvent method and using conventional liposomal drug formulations.

[0362] Table 20: Percentage of binding of hydrophobic HIV drugs in the formed lipid nanoparticles.

[0363]

[0364] a Reported values

[0365] b Non-nucleoside reverse transcriptase inhibitor (NNRTI) targeting reverse transcriptase

[0366] c HIV target: protease

[0367] Table 21: Percentage of binding of hydrophilic HIV drugs in the formed lipid nanoparticles.

[0368]

[0369] a Reported values

[0370] b Nucleoside reverse transcriptase inhibitor (NRTI) targeting reverse transcriptase

[0371] c HIV target: integrase

[0372] To evaluate the extent of binding of the class 2 drugs stabilized in reported Table 21, we performed sucrose gradient tests on lipid - drug nanoparticles (LNPs) composed of lopinavir (LPV), ritonavir (RTV), and tenofovir (TFV). The sucrose gradient test centrifugation test sedimented the lipid - drug particles through a sucrose gradient of increasing density to remove unbound drugs, thereby separating any unbound drugs from the multi - drug lipid nanoparticles. Under these conditions, all free drugs in solution remained at (or floated to) the top of the gradient with low sucrose density. Specifically, a continuous gradient of 5 - 20% (with 65% buffer) sucrose was used, and the lipid - drug particle mixture was subjected to a centrifugal force of 200,000 g for 4 hours. By this method, we were able to separate the bound and unbound drug fractions. As Figure 10 shown, it was found that most of the water - soluble drug TFV co - precipitated with the lipophilic LPV and RTV (i.e., with the stable lipid nanoparticles). The data indicate that these particles are stable under shear forces when traversing the sucrose gradient. We estimated that the binding fraction of the hydrophilic drug TFV found in the particles was approximately 75.5%, far exceeding the 7 - 30% binding of TFV to LNPs prepared by the mixed - solvent method, or even lower values (~5%) achieved using liposomes (Table 21). The data indicate that the multi - drug lipid nanoparticles are very stable under shear forces when traversing the sucrose gradient, and the bound drugs, including the hydrophilic drug, remain strongly bound to the nanoparticles. Note that the percentages of multi - drug lipid nanoparticle binding for the other two drugs (LPV and RTV) observed are consistent with the data in Table 11.

[0373] Taking into account Figure 10 and the data in Table 21, there is a clear difference between the drug loading and drug binding between lipid - drug particles and liposomes. Drug loading is a measure of the amount of lipid fraction used to stabilize drugs that cannot remain alone in solution and suspension. Drug binding is a measure of the fraction of the total drug found to be absorbed or bound to the particles in the mixture. In both of these assessments, liposomes provide a much lower drug - loading capacity and fraction of bound hydrophilic drug in the final lipid - drug particle formulation.

[0374] The multi - drug lipid nanoparticles prepared by this single - miscible - solvent method are thus significantly more stable and provide a higher degree of hydrophilic drug binding. Without being bound by any particular theory, this effect may be due to the removal of bound water on the amphiphilic lipid corona of the nanoparticles. This enables hydrophilic drugs to be trapped within this unstirred layer on the surface of the lipid nanoparticle structure, which is mainly formed by hydrophobic drug and excipient components. Such trapping is less likely to occur after the formation of lipid nanoparticles. Thus, both hydrophilic and hydrophobic drugs remain highly bound to the stable lipid nanoparticles.

[0375] These results indicate that two assembly methods produce lipid nanoparticles that highly bind to hydrophobic agents. However, the single miscible solvent method described in this example results in a much higher binding rate of hydrophilic drugs. Finally, the lipid nanoparticles assembled by the single miscible solvent method show a lipid to total drug molar ratio of approximately 3:1, with a high percentage of the initially dissolved drug (hydrophobic or hydrophilic) bound to the formed lipid nanoparticles. The lipid nanoparticles assembled by the above-described mixed double solvent method exhibit a lipid to total drug molar ratio of approximately 3:1 to approximately 8:1, indicating a tendency to retain less total drug, most likely due to a lower degree of hydrophilic drug binding compared to the lipid nanoparticles developed by the single miscible solvent method.

[0376] In any case, compared to liposomes that can only encapsulate a limited amount of water-soluble drugs initially combined with the component lipids, the lipid nanoparticles assembled by the single miscible solvent method or the mixed double solvent method show a much higher binding, especially of hydrophilic agents. In this regard, liposomes generally only have a lipid to total drug ratio of more than 10:1, indicating that the final liposome particles are mainly lipids, which is necessary for forming a stable bilayer structure with limited encapsulation. Since an absolute number of lipid molecules is required to form a large and stable bilayer membrane, liposomes cannot be formed at the low lipid to drug ratio (high drug to lipid ratio) obtained with the disclosed lipid nanoparticles. In addition, as explained in more detail below, liposomes are particularly poor at stably retaining significant levels of hydrophilic (class 2) drug molecules. Therefore, there is a clear difference between drug loading and drug binding between lipid-drug particles and liposomes. "Drug loading" is a measure of the amount of lipid fraction used to stabilize drug particles that cannot remain alone in solution and suspension. "Drug binding" is a measure of the total drug fraction found absorbed or bound to the particles in the mixture. In both of these assessments, liposomes provide a much lower drug-loading capacity and fraction of bound hydrophilic drugs in the final lipid-drug particle formulation. Finally, the combination of different types (e.g., hydrophilic and hydrophobic) of agents in the same liposome formulation has not been achieved and does not provide the distinct advantages of the disclosed lipid nanoparticles.

[0377] The physical characteristics, stability, and pharmacokinetic parameters in primates (pigtail macaques) of these multi-drug lipid nanoparticles were also characterized according to the methods described in more detail in the above examples. Briefly, 25 mg / kg of multi-drug nanoparticles composed of lopinavir, ritonavir, and tenofovir in a 2:1:3 molar ratio using the single miscible solvent method were administered subcutaneously to four primate macaques (pigtail macaques). The concentration of each single drug was monitored over time. As previously described, for all three drugs, the free (unformulated) drug in solution dropped below the detectable level within 24 hours. However, as Figure 9A and 9BAs shown, we found that, as expected, two hydrophobic drugs, such as lopinavir and ritonavir, both provided sustained and extended plasma profiles. In fact, as observed in the initial studies of the mixed dual-solvent LNPs described above in Examples 1-4, the duration of the sustained plasma drug levels from nanoparticles assembled in a single miscible solvent technology was further extended to at least two weeks rather than one week. Additionally, as Figure 9C shown, we found that hydrophilic tenofovir (TFV) unexpectedly exhibited sustained plasma drug concentrations of more than 90% for two weeks. This was surprising considering that equilibrium dialysis analysis of the multi-drug lipid nanoparticles (prepared by the mixed dual-solvent method described above in Examples 1-4) showed a binding fraction of approximately 9-10%.

[0378] To illustrate the enhanced in vivo performance and stability, the results of primate studies of these lipid nanoparticles (prepared by the single miscible solvent method) were compared with those of similar multi-drug lipid nanoparticles assembled in the combined dual-solvent and miscible solvent methods described in the above examples. See Table 22. Notably, the single miscible solvent method resulted in additional presence of the drugs in the plasma, with plasma drug levels lasting from 1 to 2 weeks, reflecting an extended plasma half-life for all three drugs, TFV, LPV, and RTV. For both methods, the differences in drug exposure and time extension, which were particularly evident in pharmacokinetics, were parallel to the increased fraction of TFV bound to the LNP.

[0379] Table 22. Comparison of the physical characteristics and primate pharmacokinetic profiles of the anti-HIV (3) drug combination of lopinavir (LPV), ritonavir (RTV), and tenofovir (TFV) prepared using a single miscible solvent or a dual-solvent method.

[0380]

[0381] Multidrug-lipid nanoparticles prepared according to the single miscible solvent method are significantly more stable and provide a higher degree of hydrophilic drug binding, even when incorporating additional different (hydrophobic) agents. Without being bound to any particular theory, this is thought to be the result of removing bound water from the hydrophilic corona region surrounding the formed lipid nanoparticles. The hydrophilic corona is established by a large number of hydrophilic domains provided by some nanoparticle excipients located on the outer surface. During the assembly of the lipid nanoparticles, these hydrophilic domains provide protection for the water-soluble drugs present in the solution at this time. It is believed that hydrophilic drugs are largely captured within this corona region on the surface of the lipid-drug particles, while the structure of the nanoparticles is mainly provided by hydrophobic drugs and lipid excipients. Thus, both hydrophilic and hydrophobic drugs remain highly bound to the multiple drugs loaded into the lipid-stabilized drug-combination nanoparticles. Typical nanoparticle assembly methods cannot achieve this result because similar corona regions, while hydrophilic, are generally "unstirred", indicating that due to structural limitations, penetration of preformed corona regions from the external environment rarely occurs, even for small hydrophilic molecules. Thus, once assembled, lipid nanoparticles carrying hydrophobic small molecule agents cannot further load hydrophobic agents into the corona region.

[0382] To illustrate the structural differences between the disclosed multidrug nanoparticles and typical liposomal formulations, Figures 8A - 8C multidrug nanoparticles and liposomes assembled by both the single miscible solvent method ( Figure 8A ) and the mixed double solvent method ( Figure 8B ) are schematically depicted, Figure 8CThe structure of ( ). In the shown formulation, nanoparticles and liposomes are constructed using phosphatidylcholine (PC) lipids and polyethylene glycolylated (PEG)-lipids as lipid excipients and an excipient with a significantly hydrophilic domain. The particles are not drawn to scale and the liposomes are not depicted with a lamellar bilayer, but this is understood to be part of their structure. The excipient containing polyethylene glycol (PEG-lipid) on the particle surface retains hydrophilic drugs in the unperturbed corona while preventing additional agents (hydrophilic or otherwise) from entering after assembly. As described herein, the ability to retain hydrophilic drugs in the corona region requires that hydrophilic and hydrophobic drugs be mixed with the lipid and dehydrated, and then the nanoparticles be assembled during rehydration. As described in Examples 1-4, the use of a mixed dual-solvent enables the assembly of nanoparticles that retain a large amount of hydrophilic agents, which represents a significant improvement over liposomes that do not similarly retain hydrophilic agents. In contrast, liposomes can encapsulate a small amount of hydrophilic molecules within the core. However, this generally does not provide a large amount of stable retention. Additionally, liposomes have known stability issues when administered in vivo. Lipid nanoparticles produced by the single miscible solvent method have a higher degree of hydrophilic agent binding within the unperturbed corona, such that the drug retention rate approaches that observed for hydrophobic agents. Additionally, as shown by sucrose gradient (see Example 6 below), these particles appear to be endowed with enhanced stability, where the nanoparticles are able to retain up to ~85% of the hydrophilic agent under the shear forces of sucrose gradient centrifugation. Lipid nanoparticles assembled by the mixed dual-solvent method retain approximately 7-20% of the hydrophilic drug under similar conditions, and liposomes only retain approximately 3-5% of the binding. Ultimately, due to the relatively limited ability of liposomes to encapsulate hydrophilic drugs and the exclusion of the unperturbed corona, these two types of lipid nanoparticles are significantly superior to standard liposomes in terms of the combination of hydrophilic agent integration and retention.

[0383] Overall, these data at least demonstrate that (1) lipid-drug particle formulations are capable of not only high drug loading but also high levels of stable binding of the drug to the particle; (2) the high level of binding eliminates the requirement to remove unincorporated drug from the formulation prior to administration, thus simplifying the preparation process; (3) the unique formulation provides the ability to co-formulate drugs with extreme physical characteristics, i.e., lipophilic and hydrophilic molecules, such that both can be reliably and effectively delivered together to the target site; (4) the flexible and physically stable lipid-drug binding provides long-term behavior that is important for medical use; (5) the platform with high drug loading in the lipid-drug mixture is novel and significantly superior to any liposome of the lipid vesicle formulations or micelle formulations reported in the literature.

[0384] Although the illustrative embodiments have been illustrated and described, it should be understood that various changes can be made without departing from the spirit and scope of the invention.

Claims

1. A multi-drug lipid nanoparticle, comprising: ritonavir (RTV), and lopinavir (LPV); tenofovir (TNF) or a prodrug thereof; a first amphiphilic excipient, wherein the first amphiphilic excipient is a phospholipid comprising a hydrophilic domain with a molecular weight less than 300 g / mol, and wherein the phospholipid is distearoyl phosphatidylcholine (DSPC); and a second amphiphilic excipient, which is a polyalkylene oxide-containing lipid comprising a hydrophilic domain with a molecular weight greater than 500 g / mol, and wherein the polyalkylene oxide-containing lipid is polyethylene glycol-functionalized 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE), wherein the molar ratio of the first amphiphilic excipient to the second amphiphilic excipient of the nanoparticle is about 2:1 to about 20:1, and wherein the nanoparticle is assembled according to a single miscible solvent method, and wherein when the nanoparticle is subjected to a continuous 5%-20% sucrose gradient and a centrifugal force of 200,000 g is applied for 4 hours, at least 75% of tenofovir (TNF) or its prodrug, at least 90% of ritonavir (RTV) and lopinavir (LPV) remain bound to the nanoparticle; wherein the single miscible solvent method comprises the following steps: dissolving ritonavir (RTV), lopinavir (LPV), tenofovir (TNF) or its prodrug, the first amphiphilic excipient and the second amphiphilic excipient in a miscible solvent comprising CHCl3, alcohol and water in a ratio of 65:35:4 (v / v / v), wherein the alcohol is selected from methanol, ethanol, propanol or hexanol; removing the miscible solvent to provide a dehydrated product comprising ritonavir (RTV), lopinavir (LPV), tenofovir (TNF) or its prodrug, the first amphiphilic excipient and the second amphiphilic excipient; heating the dehydrated product to a first temperature at least 3 ºC higher than the gel-to-liquid phase transition temperature of the amphiphilic excipient; and rehydrating the dehydrated product in an aqueous solution to provide a solution containing multi-drug lipid nanoparticles.

2. The multi-drug lipid nanoparticle according to claim 1, wherein the molar ratio of tenofovir (TNF) or its prodrug, ritonavir (RTV) and lopinavir (LPV) of the nanoparticle to the first and second amphiphilic excipients is at least 1:

10.

3. The multi-drug lipid nanoparticle according to claim 1, wherein the molar ratio of ritonavir (RTV) and lopinavir (LPV) of the nanoparticle to tenofovir (TNF) or its prodrug is about 1:20 to about 20:

1.

4. The multi-drug lipid nanoparticle according to claim 1, wherein the first amphiphilic excipient has a gel-to-liquid phase transition temperature of at least 37 ºC.

5. The multi-drug lipid nanoparticle according to claim 1, wherein the polyethylene glycol has a number average molecular weight of about 500 to about 20,000 g / mol.

6. The multi-drug lipid nanoparticle according to claim 5, wherein the polyethylene glycol-functionalized phospholipid is N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG-2000-DSPE).

7. The multi-drug lipid nanoparticle according to claim 1, wherein the molar ratio of the first amphiphilic excipient to the second amphiphilic excipient of the nanoparticle is from about 4:1 to about 9:

1.

8. The multi-drug lipid nanoparticle according to claim 1, wherein the diameter of the lipid nanoparticle is from about 20 nm to about 200 nm.

9. The multi-drug lipid nanoparticle according to claim 1, wherein when in an isotonic buffer at physiological pH and 25 °C, the lipid nanoparticle comprises a hydrophilic corona having a thickness of from about 2 nm to about 15 nm.

10. The multi-drug lipid nanoparticle according to claim 1, wherein the lipid nanoparticle is not a liposome.

11. The multi-drug lipid nanoparticle according to claim 1, wherein the lipid nanoparticle has no solid core.

12. The multi-drug lipid nanoparticle according to claim 1, wherein at least 75% of ritonavir (RTV) and lopinavir (LPV) remain bound to the nanoparticle after 24 hours at pH 7.4 and 25 °C.

13. The multi-drug lipid nanoparticle according to claim 1, wherein at least 70% of ritonavir (RTV), lopinavir (LPV) and tenofovir (TNF) or its prodrug remain bound to the nanoparticle after 8 months at pH 7.4 and 4 °C.

14. A pharmaceutical composition comprising: A plurality of multi-drug lipid nanoparticles according to any one of claims 1-13, and a pharmaceutically acceptable aqueous carrier.

15. A method for preparing a multi-drug lipid nanoparticle, comprising: Dissolving in a miscible solvent comprising CHCl3, alcohol and water in a ratio of 65:35:4 (v / v / v): Ritonavir (RTV), and lopinavir (LPV), Tenofovir (TNF) or its prodrug, A first amphiphilic excipient, wherein the first amphiphilic excipient is a phospholipid comprising a hydrophilic domain having a molecular weight of less than 300 g / mol, wherein the phospholipid is distearoylphosphatidylcholine (DSPC) and A second amphiphilic excipient, wherein the second amphiphilic excipient is a polyalkylene oxide-containing lipid comprising a hydrophilic domain having a molecular weight of greater than 500 g / mol, wherein the molar ratio of the first amphiphilic excipient to the second amphiphilic excipient of the nanoparticle is from about 2:1 to about 20:1, wherein the polyalkylene oxide-containing lipid is polyethylene glycol-functionalized 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (PEG-DSPE), wherein the alcohol is selected from methanol, ethanol, propanol and hexanol; Removing the miscible solvent to provide a dehydrated product comprising ritonavir (RTV) and lopinavir (LPV), tenofovir (TNF) or its prodrug, the first amphiphilic excipient and the second amphiphilic excipient; Heat the dehydration product to a first temperature that is at least 3 °C higher than the gel-to-liquid phase transition temperature of the amphiphilic excipient; and Rehydrate the dehydration product in an aqueous solution to provide a solution containing multi-drug lipid nanoparticles.

16. The method of claim 15, wherein removing the miscible solvent comprises evaporation, drying under reduced pressure, spray drying, or a combination thereof.

17. The method of claim 15, wherein the aqueous solution is maintained at least at the first temperature during the rehydration step.

18. The method of claim 15, wherein prior to the rehydration step, the aqueous solution is preheated to about the first temperature.

19. The method of claim 15, wherein the rehydration step comprises gradually combining the dehydration product and the aqueous solution at about the first temperature and maintaining the fully combined dehydration product and aqueous solution at a second temperature that is at least the first temperature for at least about 1 hour.

20. The method of claim 15, wherein the average molar ratio of the small molecule drug agent to the excipient of the resulting multi-drug lipid nanoparticles is at least about 1:

5.

21. The method of claim 15, wherein at least about 70% of ritonavir (RTV) and lopinavir (LPV) and at least about 70% of tenofovir (TNF) or its prodrug are stably bound to the resulting multi-drug lipid nanoparticles at neutral pH.

22. The method according to any one of claims 15-21, further comprising stirring the aqueous solution containing the multi-drug lipid nanoparticles to reduce the size of the multi-drug lipid nanoparticles.

23. The method of claim 22, wherein the stirring step comprises applying sonication, extrusion through one or more filters, or mechanical or hydrodynamic shear disruption.

24. The method of claim 22, wherein the stirring step produces multi-drug lipid nanoparticles, wherein at least 90% by number of the lipid nanoparticles have a diameter of about 20 nm to about 200 nm.

25. The method according to any one of claims 15-24, wherein when in an isotonic buffer at physiological pH and 25 °C, the resulting multi-drug lipid nanoparticles comprise a hydrophilic corona having an average thickness of about 2 nm to about 15 nm.

26. The method according to any one of claims 15-25, wherein the average molar ratio of ritonavir (RTV) or its prodrug and lopinavir (LPV) or its prodrug to tenofovir (TNF) or its prodrug of the resulting multi-drug lipid nanoparticles is about 1:20 to about 20:

1.

27. The method of claim 15, wherein the polyethylene glycol has a number average molecular weight of about 500 to about 20,000 g / mol.

28. The method of claim 27, wherein the polyethylene glycol-functionalized phospholipid is N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (mPEG-2000-DSPE).

29. The method according to any one of claims 15-28, wherein the molar ratio of the first amphiphilic excipient to the second amphiphilic excipient of the nanoparticles is about 4:1 to about 9:

1.

30. The method according to any one of claims 15 - 28, wherein the molar ratio of the first amphiphilic excipient dissolved in the solvent to the second amphiphilic excipient is from about 4:1 to about 9:

1.

31. The method according to any one of claims 15 - 30, wherein the aqueous solution is a buffered salt solution.

32. A multi-drug lipid nanoparticle prepared by the method according to any one of claims 15 - 31.

33. The multi-drug lipid nanoparticle according to claim 32, wherein the multi-drug lipid nanoparticle is pH-responsive.

34. The multi-drug lipid nanoparticle according to claim 33, wherein the pH responsiveness comprises the characteristic of releasing an increased amount of the bound small molecule agent at a lower ambient pH.

35. The multi-drug lipid nanoparticle according to any one of claims 32 - 34, wherein the multi-drug lipid nanoparticle confers an extended plasma concentration of ritonavir (RTV), lopinavir (LPV), and tenofovir (TNF) or their prodrugs compared to administering the same amount of the free small molecule agent to a mammalian subject upon administration to the mammalian subject.

36. Use of the nanoparticle according to any one of claims 32 - 35 in the preparation of a formulation for treating a subject infected with HIV.

37. The use according to claim 36, wherein the nanoparticle is administered subcutaneously (SC), intravenously (IV), or intramuscularly (IM).

38. The use according to claim 36, wherein the viral load is detectably reduced in the lymphoid tissue of the subject.

39. The use according to claim 36, wherein ritonavir (RTV), lopinavir (LPV), and tenofovir (TNF) or their prodrugs remain detectable in plasma or lymphoid tissue for more than 7 days after administration.

40. The use according to claim 36, wherein ritonavir (RTV), lopinavir (LPV), and tenofovir (TNF) or their prodrugs remain detectable in plasma or lymphoid tissue for more than 14 days after administration.

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