WO1998047492A1

WO1998047492A1 - Nanosized aspartyl protease inhibitors

Info

Publication number: WO1998047492A1
Application number: PCT/US1998/007845
Authority: WO
Inventors: Pravin Ramsewak Chaturvedi; Roger Dennis Tung; Joshua S. Boger
Original assignee: Vertex Pharmaceuticals Incorporated
Priority date: 1997-04-18
Filing date: 1998-04-14
Publication date: 1998-10-29
Also published as: AU7133898A

Abstract

The present invention relates to particles of the free base form of aspartyl protease inhibitors and pharmaceutical dosage forms containing those particles. The invention also relates to methods of treating mammals with those pharmaceutical dosage forms.

Description

NANOSTZED ASPARTYL PROTEASE INHIBITORS

FIELD OF THE INVENTION

The present invention relates to submicron particles of the free base form of aspartyl protease inhibitors and pharmaceutical dosage forms containing those particles. The invention also relates to methods of treating mammals with those pharmaceutical dosage forms .

BACKGROUND OF THE INVENTION

Aspartyl protease inhibitors are considered the most effective current drug in the fight against HIV infection. These inhibitors, however, require certain physicochemical properties in order to achieve good potency against the enzyme. One of these properties is high hydrophobicity. Unfortunately, this property goes hand in hand with poor aqueous solubility resulting in poor oral bioavailability.

VX-478 (4-Amino-N-( (2 syn, 3S) -2-Hydroxy-4- phenyl-3- ( (S) -tetrahydrofuran-3-yl-oxycarbonylamino) - butyl) -N-isobutyl-benzenesulfonamide) is an HIV protease inhibitor that has been described in United States Patent 5,585,397. It is a classic example of the dilemma of formulating a hydrophobic aspartyl protease inhibitor into a pharmaceutical composition. In its mesylate salt form, VX-478 is slightly soluble. While the oral bioavailability of this inhibitor in a "solution" formulation is excellent, the dosage of VX-478 in this form is severely limited by the amount of liquid present in the particular liquid dosage from, e.g., encapsulated into a soft gelatin capsule.

Currently, this formulation approach produces an upper limit of containing 150 mg of VX-478 in each capsule. Given a therapeutic dose of 2400 mg/day of VX- 478, this formulation limitation would require a patient to consume 16 capsules per day. Such a high pill burden would likely result in poor patient compliance, thus producing sub-optimal therapeutic benefit of the drug.

Furthermore, these "solution" formulations are at a saturation solubility of VX-478. This creates the real potential of having the drug crystallize out of solution under various storage and/or shipping conditions. This, in turn, would likely result in a loss of some of the oral bioavailability achieved with VX-478.

Yet another problem with this "solution" formulation of VX-478 is poor taste-masking. This is probably due to the fact that the drug being in solution accelerates or facilitates the sensation of the bitterness. This particular feature presents a serious roadblock to the development of a suitable pediatric formulation of the drug.

It is assumed that other aspartyl protease inhibitors, particularly HIV protease inhibitors, also exhibit these same problems.

One way of overcoming these problems is to develop a standard solid dosage form, such as a capsule or a tablet, and/or a suspension form. Unfortunately, such solid dosage forms have much lower oral bioavailability of the drug.

United States patent 5,145,684 describes solid particle forms of drugs that are smaller than 400 nm as a potential solution to the low bioavailability of insoluble drugs. Although this "nanoparticle" technology is theoretically useful in increasing the bioavailability of any insoluble or poorly soluble drug, in practice many insoluble drugs do not exhibit increased bioavailability when milled into particles smaller than 400 nm. Thus, there is still a need to find a way to improve the drug load per unit dosage form for aspartyl protease inhibitors. Similarly, there is also a need to develop a suspension formulation of aspartyl protease inhibitors, which would minimize the bitterness of the drug and improve taste-masking, resulting in a more "patient-friendly" pediatric dosage form for treatment of HIV infection in children.

SUMMARY OF THE INVENTION

The present invention solves the problems set forth above by providing particles of the free base form of an aspartyl protease inhibitor having a surface modifier adsorbed on the particle surface, said particles having a size of less than about 400 nm. Applicants have discovered that when the free base form of an aspartyl protease inhibitor is wet milled to a size of less than 400 nm in the presence of a surface modifier ("nanosized particles") , the resulting composition is stable, dispersible and has a bioavailability equal to or greater than a "solution" form of the corresponding salt form of the same inhibitor. The nanosized particles also have significantly greater bioavailability than "solution" forms of the free base inhibitors. Moreover, applicants have discovered that, surprisingly, nanosized particles of the salt of the aspartyl protease inhibitor do not exhibit increased bioavailability. In fact, that salt form exhibits significantly lower bioavailability than either the "solution" form of the salt or the nanosized particles of the free base.

The invention also provides a stable dispersion consisting essentially of nanosized particles of the free base aspartyl protease inhibitor in a liquid dispersion medium.

According to another embodiment, the invention provides solid dosage and suspension dosage form pharmaceutical compositions comprising the nanosized particles of this invention in a pharmaceutically acceptable vehicle. Such compositions are able to provide equal or greater bioavailability as the corresponding solution forms and can advantageously be formulated to contain significantly more inhibitor per unit dosage form.

Furthermore, with the selection of proper excipients, these nanosized particles can be used to make suspension formulations, which can be converted into a free flowing powder. The free-flowing powder can then be used in capsule or tablet formulation. These high dose capsules or tablets allow a reduction in the number of pills that need to be taken in a day in order to reach the required daily intake of inhibitor. Additionally, when the nanosized particles of the aspartyl protease inhibitor are formulated in a suspension, the intense bitterness from the chemical is not felt immediately, as it is in the solution forms. This allows for better taste masking and therefore a better dosing alternative than a solution for the pediatric population.

DETAILED DESCRIPTION OF THE INVENTION

According to one embodiment, the present invention provides particles consisting essentially of 99.9-10% by weight of a crystalline, free base form of an aspartyl protease inhibitor having a solubility in water of less than 10 mg/ml, wherein the inhibitor has a non- crosslinked surface modifier adsorbed on its surface in an amount of 0.1-90% by weight and sufficient to maintain an effective average particle size of less than about 400 nm.

Even more preferably, the aspartyl protease inhibitor in the particles of this invention is the free base form of an HIV protease. Examples of HIV protease inhibitors include, but are not limited to VX-478 (Vertex, also known as 141W94 (Glaxo-Wellcome) and KVX-478 (Kissei)), saquinavir (Ro 31-8959, Roche), indinavir (L-735,524, Merck)), ritonavir (ABT 538,

Abbott) , nelfinavir (AG 1343, Agouron) , palinavir (Bila 2011 BS), U-103017 (Upjohn), XM 412 (DuPont Merck), XM 450 (DuPont Merck), BMS 186318 (Bristol-Meyers Squibb), CPG 53,437 (Ciba Geigy) , CPG 61,755 (Ciba Geigy) , CPG 70,726 (Ciba Geigy), ABT 378 (Abbott), GS 3333 (Gilead Sciences), GS 3403 (Gilead Sciences), GS 4023 (Gilead Sciences), GS 4035 (Gilead Sciences), GS 4145 (Gilead Sciences), GS 4234 (Gilead Sciences), and GS 4263 (Gilead Sciences). Most preferably, the inhibitor is VX-478. The invention is designed to increase the bioavailability of poorly soluble free base forms of aspartyl protease inhibitors. At a minimum, the free base form of the proteases inhibitor employed in the particles of this invention should have a solubility of less than about 10 mg/ml . More preferably, the solubility should be less than about 1 mg/ml. Most preferred are free base forms of aspartyl protease inhibitors that have a solubility of less than about 0.2 mg/ml . The particles of this invention additionally contain a surface modifier which adheres to, but does not form a chemical bond with, the aspartyl protease inhibitor.

Suitable surface modifiers are set forth in United States patent 5,145,684, the disclosure of which is herein incorporated by reference. Such surface modifiers are preferably be selected from known organic and inorganic pharmaceutical excipients. Such excipients include various polymers, low molecular weight oligomers, natural products and surfactants. Preferred surface modifiers include nonionic and anionic surfactants. Representative examples of excipients include gelatin, casein, lecithin (phosphatides) , gum acacia, cholesterol, tragacanth, stearic acid, benzalkonium chloride, calcium stearate, glyceryl onostearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, polyoxyethylene alkyl ethers, e.g., macrogol ethers such as cetomacrogol 1000, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, e.g., the commercially available Tweens, polyethylene glycols, polyoxyethylene stearates, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, carboxymethylcellulose calcium, carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethycellulose phthalate, noncrystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol, and polyvinylpyrrolidone (PVP) . Most of these excipients are described in detail in the Handbook of Pharmaceutical Excipients, published jointly by the American

Pharmaceutical Association and The Pharmaceutical Society of Great Britain, the Pharmaceutical Press, 1986, the disclosure of which is hereby incorporated by reference in its entirety. The surface modifiers are commercially available and/or can be prepared by techniques known in the art.

Particularly preferred surface modifiers include polyvinyl pyrrolidone, Pluronic F68 and F108, which are block copolymers of ethylene oxide and propylene oxide, Tetronic 908, which is a tetrafunctional block copolymer derived from sequential addition of ethylene oxide and propylene oxide to ethylenediamine, dextran, lecithin, Aerosol OT, which is a dioctyl ester of sodium sulfosuccinic acid, available from American Cyanamid, Duponol P, which is a sodium lauryl sulfate, available from DuPont, Triton X-200, which is an alkyl aryl polyether sulfonate, available from Rohm and Haas, Tween 80, which is a polyoxyethylene sorbitan fatty acid ester, available from ICI Specialty Chemicals, and Carbowax 3350 and 934, which are polyethylene glycols available from Union Carbide.

Surface modifiers which have found to be particularly useful include polyvinylpyrrolidone, Pluronic F-68, and lecithin. The surface modifier is adsorbed on the surface of the free base form of the protease inhibitor in an amount sufficient to maintain an effective average particle size of less than about 400 nm. As used herein, particle size refers to a number average particle size as measured by conventional particle size measuring techniques well known to those skilled in the art, such as sedimentation field flow fractionation, photon correlation spectroscopy, or disk centrifugation. By "an effective average particle size of less than about 400 nm" it is meant that at least 90% of the particles have a weight average particle size of less than about 400 nm when measured by the above-noted techniques. In preferred embodiments of the invention, the effective average particle size is less than about 250 nm. With reference to the effective average particle size, it is preferred that at least 95% and, more preferably, at least 99% of the particles have a particle size less than the effective average, e.g., 400 nm. In particularly preferred embodiments, essentially all of the particles have a size less than 400 nm. A general procedure for preparing the particles of this invention is set forth below. The free base form of the aspartyl protease inhibitor is obtained commercially and/or prepared by techniques known in the art in a conventional coarse form. If the aspartyl protease inhibitor is obtained as a salt, it may be converted to a free base form and recrystallized by methods well known in the art. It is preferred, but not essential, that the particle size of the coarse protease inhibitor be less than about 100 μm as determined by sieve analysis. If the coarse particle size of the inhibitor is greater than about 100 μm, then it is preferred that the particles of the drug substance be reduced in size to less than 100 μm using a conventional milling method such as airjet or fragmentation milling.

The coarse aspartyl protease can then be added to a liquid medium in which it is essentially insoluble to form a premix. The concentration of the inhibitor in the liquid medium can vary from about 0.1-60%, and preferably is from 5-30% (w/w) . It is preferred, but not essential, that the surface modifier be present in the premix. The concentration of the surface modifier can vary from about 0.1 to about 90%, and preferably is 1- 75%, more preferably 20-60%, by weight based on the total combined weight of the aspartyl protease inhibitor and surface modifier. The apparent viscosity of the premix suspension is preferably less than about 1000 centipoise.

The premix can be used directly by subjecting it to mechanical means to reduce the average particle size in the dispersion to less than 400 nm. It is preferred that the premix be used directly when a ball mill is used for attrition. Alternatively, the aspartyl protease inhibitor and, optionally, the surface modifier, can be dispersed in the liquid medium using suitable agitation, e.g., a roller mill or a Cowles type mixer, until a homogeneous dispersion is observed in which there are no large agglomerates visible to the naked eye. It is preferred that the premix be subjected to such a premilling dispersion step when a recirculating media mill is used for attrition.

The mechanical means applied to reduce the particle size of the drug substance conveniently can take the form of a dispersion mill. Suitable dispersion mills include a ball mill, an attritor mill, a vibratory mill, and media mills such as a sand mill and a bead mill. A media mill is preferred due to the relatively shorter milling time required to provide the intended result, i.e., the desired reduction in particle size. For media milling, the apparent viscosity of the premix preferably is from about 100 to about 1000 centipoise. For ball milling, the apparent viscosity of the premix preferably is from about 1 up to about 100 centipoise. Such ranges tend to afford an optimal balance between efficient particle fragmentation and media erosion. The grinding media for the particle size reduction step can be selected from rigid media preferably spherical or particulate in form having an average size less than about 3 mm and, more preferably, less than about 1 mm. Such media desirably can provide the particles of the invention with shorter processing times and impart less wear to the milling equipment. The selection of material for the grinding media is not believed to be critical. Suitable grinding media includes, but is not limited to, zirconium oxide, such as 95% ZrO stabilized with magnesia, zirconium silicate, glass grinding media, stainless steel, titania, alumina, and 95% ZrO stabilized with yttrium. Preferred media have a density greater than about 3 g/cm³.

The attrition time can vary widely and depends primarily upon the particular mechanical means and processing conditions selected. For ball mills, processing times of up to five days or longer may be required. On the other hand, processing times of less than 1 day (residence times of one minute up to several hours) have provided the desired results using a high shear media mill.

The particles must be reduced in size at a temperature which does not significantly degrade the protease inhibitor. Processing temperatures of less than about 30°-40° C. are ordinarily preferred. If desired, the processing equipment can be cooled with conventional cooling equipment. The method is conveniently carried out under conditions of ambient temperature and at processing pressures which are safe and effective for the milling process. For example, ambient processing pressures are typical of ball mills, attritor mills and vibratory mills. Processing pressures up to about 20 psi (1.4 kg/cm²) are typical of media milling.

The surface modifier, if it was not present in the premix, must be added to the dispersion after attrition in an amount as described for the premix above. Thereafter, the dispersion can be mixed, e.g., by shaking vigorously. Optionally, the dispersion can be subjected to a sonication step, e.g., using an ultrasonic power supply. For example, the dispersion can be subjected to ultrasonic energy having a frequency of 20-80 kHz for a time of about 1 to 120 seconds.

The relative amount of aspartyl protease inhibitor and surface modifier can vary widely and the optimal amount of the surface modifier can depend, for example, upon the particular inhibitor and surface modifier selected, the critical micelle concentration of the surface modifier if it forms micelles, etc. The surface modifier preferably is present in an amount of about 0.1-10 mg per square meter surface area of inhibitor. The surface modifier can be present in an amount of 0.1-90%, preferably 20-60% by weight based on the total weight of the dry particle.

According to another embodiment, the invention provides a stable dispersion of the particles described above. The dispersion consists of a liquid dispersion medium and the above-described particles. The dispersion of surface modified free base aspartyl protease nanoparticles can be spray coated onto sugar spheres or onto a pharmaceutical excipient in a fluid-bed spray coater by techniques well known in the art.

According to another embodiment, the present invention provides pharmaceutical compositions comprising the particles described above in an amount effective to inhibit an aspartyl protease in a patient and a pharmaceutically acceptable carrier therefor. An amount effective to inhibit an aspartyl protease in a patient is between about 5 - 500 mg/kg/day. Preferably, an effective amount is between about 10 - lOOmg/kg/day. Most preferably, an effective amount is between about 20 - 60 mg/kg/day.

Preferably, the particles in a unit dosage form of the composition will contain greater than 150 mg of the free base aspartyl protease inhibitor. The term "unit dosage form", as used herein with respect to a solid dosage form, refers to a single tablet, capsule or pill. With respect to a suspension formulation, that term refers to less than 2.5 ml of the suspension. More preferably, a single dosage form will contain greater than 400 mg of the aspartyl protease inhibitor.

The term "patient" as used herein, refers to a mammal. Preferably a "patient" is a human being.

Suitable pharmaceutically acceptable carriers for use in the pharmaceutical compositions of this invention are well known to those skilled in the art. These include non-toxic physiologically acceptable carriers, adjuvants or vehicles for parenteral injection, for oral administration in solid or liquid form, for rectal administration, and the like. In a preferred embodiment, the pharmaceutical compositions of this invention are in a tablet or capsule form. In this embodiment, the pharmaceutically acceptable carrier is a standard excipient. Standard excipients useful to manufacture nanoparticles of free base aspartyl proteases into a tablet or capsule form include fillers, such as sugars (e.g., lactose or sucrose) or celluloses (e.g., macrocrystalline or starch); disintegrants (e.g., Cab-O-Sil); lubricants (e.g, talc or magnesium stearate) ; and other necessary surfactants, such as sucrose esters, crodesta or sorbitanisters (e.g., Span/Tween) .

According to another preferred embodiment, the pharmaceutical compositions of this invention are in a suspension formulation. As stated above, nanoparticles of the free base form of aspartyl protease inhibitors are more easily taste masked than solutions of the corresponding salts (or solutions of the free base form to the extent they can be solubilized) . Thus, suspension formulations of the particles of this invention are ideally suited for pediatric formulations.

In these preferred formulations the pharmaceutically acceptable carriers include suspending agents, such as sodium methyl cellulose, methyl cellulose, gum acacia or tragacanth; sweeteners, such as sorbitol, sucrose, aspartame or saccharin; flavors, such as bitter orange, strawberry citrus, or mocha; preservatives, such as methyl and propyl parabenz, sodium benzoate, or parabenzoic acid; antioxidants, such as sodium sulfite, tocopherol, butyl hydroxy toluene, or butyl hydroxy anisole) ; necessary surfactants, such as Tween, Span, or crodestas; anticaking agents, such as sodium citrate or citric acid; cosolvents/vehicle systems, such as glycerin, ethanol, propylene glycol, polyethylene glycols, water, medium chain triglycerides, etc.

The particles of this invention and the compositions which comprise them may be employed in a conventional manner for the treatment of viruses, such as HIV and HTLV, which depend on aspartyl proteases for obligatory events in their life cycle. Such methods of treatment, their dosage levels and requirements may be selected by those of ordinary skill in the art from available methods and techniques. For example, the particles of this invention may be combined with a pharmaceutically acceptable adjuvant for administration to a virally-infected patient in a pharmaceutically acceptable manner and in an amount effective to lessen the severity of the viral infection or to alleviate pathological effects associated with HIV infection. Alternatively, the particles of this invention may be used in prophylactics and methods for protecting individuals against viral infection during a specific event, such as childbirth, or over an extended period of time. The particles may be employed in such prophylactics either alone or together with other antiretroviral agents to enhance the efficacy of each agent. As such, particles of this invention can be administered as agents for treating or preventing HIV infection in a mammal. The particles of this invention may be administered to a healthy or HIV-infected patient either alone or in combination with other anti-viral agents which interfere with the replication cycle of HIV. The additional agent may be part of the same composition which comprises the particles of this invention or it may be part of a separate composition which is administered to the patient sequentially or concurrently with the particle-containing composition. Thus, the terms "in combination with" and "coadministered with", as used herein, refer to both single and multiple dosage forms.

By administering the particles of this invention with other anti-viral agents which target different events in the viral life cycle, the therapeutic effect of these particles is potentiated. For instance, the co-administered anti-viral agent can be one which targets early events in the life cycle of the virus, such as cell entry, reverse transcription and viral DNA integration into cellular DNA. Anti-HIV agents targeting such early life cycle events include, didanosine (ddl) , dideoxycytidine (ddC) , d4T, zidovudine (AZT) , 3TC,

935U83, 1592U89, 524W91, polysulfated polysaccharides, sT4 (soluble CD4), ganiclovir, trisodium phosphonoformate, eflornithine, ribavirin, acyclovir, alpha interferon and trimenotrexate . Additionally, non- nucleoside inhibitors of reverse transcriptase, such as TIBO, delavirdine (U90) or nevirapine, may be used to potentiate the effect of the particles of this invention, as may viral uncoating inhibitors, inhibitors of trans- activating proteins such as tat or rev, or inhibitors of the viral integrase.

Combination therapies according to this invention exert an additive or synergistic effect in inhibiting HIV replication because each component agent of the combination acts on a different site of HIV replication. The use of such combination therapies also advantageously reduces the dosage of a given conventional anti-retroviral agent which would be required for a desired therapeutic or prophylactic effect, as compared to when that agent is administered as a monotherapy. Such combinations may reduce or eliminate the side effects of conventional single anti-retroviral agent therapies, while not interfering with the anti-retroviral activity of those agents. These combinations reduce potential of resistance to single agent therapies, while minimizing any associated toxicity. These combinations may also increase the efficacy of the conventional agent without increasing the associated toxicity. Preferred combination therapies include the administration of the particles of this invention with AZT, ddl, ddC, d4T, 3TC, 935U83, 1592U89, 524W91 or a combination thereof.

Alternatively, the particles of this invention may also be co-administered with other HIV protease inhibitors such as saquinavir (Ro 31-8959, Roche) , L- 735,524 (Merck), ABT 538 (A-80538, Abbott), AG 1341 (Agouron) , XM 412 (DuPont Merck), XM 450 (DuPont Merck), BMS 186318 (Bristol-Meyers Squibb) and CPG 53,437 (Ciba Geigy) or prodrugs of these or related particles to increase the effect of therapy or prophylaxis against various viral mutants or members of HIV quasi species. Preferably, the particles of this invention are administered as a single agent or in combination with retroviral reverse transcriptase inhibitors, such as derivatives of AZT, or other HIV aspartyl protease inhibitors, including multiple combinations comprising from 3-5 agents. We believe that the co-administration of the particles of this invention with retroviral reverse transcriptase inhibitors or HIV aspartyl protease inhibitors may exert a substantial additive or synergistic effect, thereby preventing, substantially reducing, or completely eliminating viral replication or infection or both, and symptoms associated therewith. The particles of this invention can also be administered in combination with immunomodulators and immunostimulators (e.g., bropirimine, anti-human alpha interferon antibody, IL-2, GM-CSF, interferon alpha, diethyldithiocarbamate, tumor necrosis factor, naltrexone, tuscarasol, and rEPO) ; and antibiotics (e.g., pentamidine isethiorate) to prevent or combat infection and disease associated with HIV infections, such as AIDS and ARC.

According to yet another embodiment, the invention provides methods for inhibiting aspartyl proteases, in particular inhibiting aspartyl proteases in a human. These include viral aspartyl proteases that essential for the life cycle of certain viruses, such as HIV and other AIDS-like diseases caused by retroviruses, such as simian immunodeficiency viruses, HTLV-I and HTLV- II; renin; and aspartyl proteases that process endothelin precursors. Preferably, the methods of this invention are used to treat or prevent HIV infections in humans.

In order that this invention be more fully understood, the following examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.

EXAMPLE 1 Synthesis of Free Base VX-478 The synthesis of the aspartyl protease inhibitor VX-478 is disclosed in United States patent 5,585,397. It is referred to as compound #168 in that document. The essentials of that synthesis are set forth below.

A solution of 102 mg of N- ( (2 syn, 3S)-2- hydroxy-4-phenyl-3- ( (S) -tetrahydrofuran 3-yl- oxycarbonylaminobutylamine in 4 : 1 CH₂Cl₂/saturated aqueous NaHC0₃ was treated sequentially, at ambient temperature under an atmosphere of nitrogen, with 65 mg of p- nitrobenzenesulfonyl chloride and 51 mg of sodium bicarbonate. The mixture was stirred for 14 h, diluted with CH₂C1₂, washed with saturated NaCl, then dried over MgS0₄, filtered, and concentrated in vacuo . The residue was purified by low pressure silica gel chromatography using 20% diethyl ether/CH₂Cl₂ as eluent to provide 124 mg of product as a white solid. TLC : Rf = 0.36, 20% diethyl ether/CH₂Cl₂. HPLC: Rt = 15.15 min. (ΙH)-NMR (CDC13) consistent with structure.

A solution of 124 mg of the resultant compound in ethyl acetate was treated, at ambient temperature, with 13 mg of 10% palladium on carbon. The mixture was stirred for 14 h under an atmosphere of hydrogen, filtered through a pad of Celite filter agent, and concentrated in vacuo. The residue was subjected to preparative HPLC to yield 82 mg of 4-Amino-N- ( (2 syn,3S)- 2-Hydroxy-4-phenyl-3- ( (S) -tetrahydrofuran-3-yl- oxycarbonylamino) -butyl) -N-isobutyl-benzenesulfonzmide (VX-478) as a white solid. TLC: Rf = 0.10, 20% ether/CH₂Cl₂. HPLC: Rt = 13.16 min. (ΙH)-NMR (CDC1₃) consistent with structure.

EXAMPLE 2

Preparation of VX-478 Mesylate Salt The mesylate salt of VX-478 is prepared by reacting the free base with 1 equivalent of methane sulfonic acid.

EXAMPLE 3

Preparation of Nanoparticles of Free Base VX-478 and VX-478 Mesylate Salt

The solid form of both the free base and the salt of VX-478 (2.0% w/v) were subjected to milling in the presence of the surface modifiers hydroxypropyl cellulose (HPC-L for the free base; HPC-SL for the salt)

(1.0% w/v) and SDS (0.01% w/v).

The resulting particles had a mean particle size of 157 nm for the free base and 122 nm for the salt.

A second preparation of VX-478 mesylate salt nanoparticles was prepared using 2.0% w/v of the salt,

1.0% w/v of PVP-30 and 0.01% SDS. This preparation produced particles having a mean size of 696 nm, but 90% of those particles were less than 288 nm in size, thus meeting the criteria of the invention.

EXAMPLE 4 Comparison of Solution Versus Nanoparticle Suspension Formulations of Free Base VX-478 Free base VX-478 was dissolved in a pharmaceutically acceptable aqueous buffer and administered orally to Sprague-Dawley rats daily at various dosages as a single dose (100 mg/kg) or for a period of either one (50 mg/kg) or three months (125 mg/kg and 500 mg/kg) . The nanoparticle form of VX-478 was suspended in a pharmaceutically acceptable aqueous buffer and administered orally to a separate population of Sprague-Dawley rats as a single dose. The pharmokinetic properties of each of these formulations was calculated and is presented in the table below. Table 1: Statistical summary of selected pharmacokinetic parameters of VX-478 (free base) when administered orally as a solution or a nanosized suspension in Sprague-Dawley rats

* Dose in mg/kg/day; AUC(0-24) calculated from a 3 month rat toxicology study with VX-478

** Dose in mg/kg; AUC(0-12) calculated from a 1 month rat toxicology study with VX-478 *** Harmonic mean

Based upon these results it is apparent that at higher dosages a higher bioavailability (represented by AUC m the table above) can be achieved with the nanoparticle formulation of free base VX-478 as compared to the solution formulation. Administering higher dosages of the solution form is impractical due to its limited solubility. This limitation is not a barrier when formulating the nanoparticle form in a suspension.

Moreover it should be noted that the C_max of the nanosized preparation was equal to or superior to that of the solution formulation at all dosages.

Thus, the nanoparticle form of free base VX-478 represents a better formulation of the drug than the solution form.

EXAMPLE 5

Comparison of Solution Versus Nanoparticle Suspension Formulations of Mesylate Salt VX-478

The mesylate salt of VX-478 was dissolved in a pharmaceutically acceptable aqueous buffer and administered orally to Sprague-Dawley as a single dose (96 mg/kg) . The nanoparticle form of the mesylate salt of VX-478 was suspended in a pharmaceutically acceptable aqueous buffer and administered orally to a separate population of Sprague-Dawley rats as a single dose (40.8 mg/kg) . The pharmokinetic properties of each of these formulations was calculated and is presented in the table below.

Table 2: Statistical summary of selected pharmacokinetic parameters for VX-478 mesylate salt when administered orally as a solution or a nanosized suspension in Sprague-Dawley rats

* p < 0.01 when compared to the solution dosage form

The results of this study demonstrate that the bioavailability (AUC) of the nanoparticle mesylate salt suspension is significantly reduced as compared to the corresponding salt solution. In addition, the C_max of the nanoparticle salt formulation is also significantly decreased. This demonstrates that, unlike the situation for the free base form of VX-478, nanoparticles of VX-478 mesylate salt do not provide superiority (and, in fact appear to be inferior) over the corresponding solution form.

A comparison of the results for the VX-478 nanoparticulate mesylate salt in the table above with the nanoparticulate free base form in Table 1 further confirms the surprising advantage of nanoparticle technology when applied to the free base form of aspartyl protease inhibitors. A single dose of the VX-478 free base nanoparticle formulation (43 mg/kg) produced almost 10-fold higher bioavailability and greater than 10-fold higher C_max than a corresponding single dose of then VX- 478 mesylate salt nanoparticle formulation (41 mg/kg) . Moreover, the VX-478 free base nanoparticle formulation produced slightly higher bioavailability and almost 3- fold higher C_max than a solution formulation of the mesylate salt form.

EXAMPLE 6

Comparison of Solution Versus Nanoparticle Solid Formulations of Free Base VX-478

In another experiment, a nanoparticle free base

VX-478 preparation was film dried to prepare a powder formulation. A single dose (802.5 mg/kg or 988.3 mg/kg) was orally administered to rats and the pharmokinetic properties of that preparation were measured. The table below shows the results of those measurements as compared to the pharmokinetic properties of a VX-478 solution preparation (as set forth in Example 4) .

Table 3: Statistical summary of selected pharmacokinetic parameters for VX-478 free base when administered orally as a solution formulation or as a film dried powder formulation in Sprague-Dawley rats

^** Harmonic mean

The results of this experiment confirm that solid formulations of VX-478 free base nanoparticles demonstrate increased bioavailability (due to the ability to provide higher dosages) than the corresponding solution formulation, while maintaining a similar C_max. The powder formulation described above can be used to formulate solid tablets and capsules containing greater amounts of drug than can be obtained in a solution formulation. This is demonstrated in the next example .

EXAMPLE 7

Bioavailability of Nanoparticle Solid Formulations in Dogs

In another experiment, a nanoparticle free base VX-478 preparation was film dried to prepare a powder formulation. The powder was then formulated into tablets containing 300 mg of VX-478 using either HPC-SL or PVP- K90 as an adjuvant. A single tablet was orally administered to dogs and the pharmacokinetic properties of that preparation were measured by taking plasma samples immediately after dosing. The table below shows the results of those measurements.

Table 4: Statistical summary of selected pharmacokinetic parameters for VX-478 free base when administered as a film dried powder tablet formulation in dogs

Compared to the above results, solid formulations of micronized VX-478 free base in tablet form (300 mg) showed no measurable bioavailability.

Thus, nanoparticles of VX-478 can be formulated into solid tablets and capsules containing greater amounts of drug than can be obtained in a solution formulation (either liquid, suspension or soft gel capsules) while still maintaining acceptable bioavailability. This provides the advantage of reducing pill load (and therefore increasing patient compliance) in patients taking the drug.

Qualitatively similar results as those set forth in Examples 1 through 6 will be obtained for nanoparticulate forms of other aspartyl protease inhibitors . While we have hereinbefore described a number of embodiments of this invention, it is apparent that our basic constructions can be altered to provide other embodiments of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claims appended hereto rather than by the specific embodiments which have been presented hereinbefore by way of example.

Claims

CLAIMSWe claim:

1. Particles consisting essentially of 99.9- 10% by weight of a crystalline, free base form of an aspartyl protease inhibitor having a solubility in water of less than 10 mg/ml, said inhibitor having a non- crosslinked surface modifier adsorbed on the surface thereof in an amount of 0.1-90% by weight and sufficient to maintain an effective average particle size of less than about 400 nm.

2. The particles according to claim 1, wherein said aspartyl protease inhibitor is an inhibitor of HIV protease.

3. The particles according to claim 2, wherein said inhibitor is selected from VX-478, saquinavir, indinavir, ritonavir, nelfinavir, palinavir, U-103017, XM 412, XM 450, BMS 186318, CPG 53,437, CPG 61,755, CPG 70,726, ABT 378, GS 3333, GS 3403, GS 4023, GS 4035, GS 4145, GS 4234, or GS 4263.

4. The particles according to claim 3, wherein said inhibitor is VX-478.

5. A stable dispersion consisting essentially of a liquid dispersion medium and the particles according to any one of claims 1 to 4.

6. A pharmaceutical composition comprising an amount of the particles according to any one of claims 1 to 4 effective to inhibit aspartyl protease activity and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, comprising greater than 150 mg per unit dosage form.

8. The pharmaceutical composition according to claim 7, wherein said unit dosage form is a single capsule or a tablet.

9. The pharmaceutical composition according to claim 6, wherein said unit dosage form is a suspension.

10. A method of treating a human suffering from an HIV infection comprising the step of administering to said animal a pharmaceutical composition according to any one of claims 6 to 9.

11. The method according to claim 10, wherein said human is a child and wherein said method comprises administering to said child a pharmaceutical composition according to claim 9.