Method and composition for pharmaceutical product

Abstract

This invention is directed to a composition comprising dry granulated tenofovir DF and emtricitabine, and a method for making same. Dry granulation was unexpectedly found to be important in preparing a tenofovir DF containing composition suitable for inclusion in a combination dosage form containing emtricitabine, efavirenz and tenofovir DF.

Description

BACKGROUND OF THE INVENTION

This application relates to products for the treatment of viral infections, in particular HIV infections, using the known antiviral compounds efavirenz (tradename Sustiva, also known as EFV), emtricitabine (tradename Emtriva, also known as FTC) and tenofovir DF (disoproxil fumarate, also known as TDF) (tradename Viread, sold in combination with emtricitabine under the tradename Truvada).

The Truvada product is produced by wet granulation of emtricitabine and tenofovir DF (WO 04/64845), which under the circumstances produces a chemically stable dosage form. This product does not contain efavirenz.

HIV therapy using efavirenz as well as emtricitabine and tenofovir DF has been considered desirable. (hereafter “triple combination”; see WO 04/64845). Manufacturing a commercially viable triple combination product, however, would require that the final product meet stringent FDA requirements for bioequivalence to the commercial products, Viread (tenofovir disoproxil fumarate), Emtriva (emtricitabine), and Sustiva (efavirenz), and that the tablet be of suitable size for patients to easily swallow.

Initial efforts to simply combine the three drugs (active pharmaceutical intermediates, or APIs) into a unitary, essentially homogeneous composition manufactured by wet granulation failed to produce a chemically stable tablet. The tenofovir DF in this combination tablet was highly unstable and rapidly degraded in stability studies. The efavirenz formulation was unexpectedly incompatible with tenofovir DF, a result now attributed to the surfactant (sodium lauryl sulfate) found in the efavirenz portion of the formulation.

Another attempt was made to produce the triple combination, this time using a dry granulation of the three part combination and omitting the surfactant. This resulted in a tablet that failed to achieve bioequivalence with respect to efavirenz in human clinical trials. The peak efavirenz concentration in the blood stream and total drug exposure (Cmax and AUC) were both below the parameters determined for the commercial comparator, Sustiva (efavirenz) tablets. The inventors concluded that at least the surfactant in the triple combination (efavirenz/emtricitabine/tenofovir disbproxil fumarate) tablets was necessary to achieve bioequivalence to Sustiva.

Next, combination tablets were manufactured by wet granulating the efavirenz component with the surfactant and other excipients, separately manufacturing the Truvada component using dry granulation, mixing the granulates together, compressing the mixture into tablets, and then film-coating the tablets. Unexpectedly, this approach also failed to produce the desired bioequivalence in between the commercial product, Sustiva (efavirenz), and clinical trial material (i.e., proposed commercial triple combination product). A novel and inventive step was needed to overcome the shortcomings of more straight-forward approaches to a triple combination dosage form.

As described further in copending U.S. Ser. No. 60/771,279 (filed of even date and expressly incorporated herein by reference) the stability and bioequivalence objectives for the triple combination tablet ultimately were achieved in an exemplary embodiment by dry granulating the emtricitabine/tenofovir disoproxil fumarate component, wet granulating the efavirenz component and, rather than using the straight-forward process of simply combining the granulates, instead organizing the granulates to produce a multilaminate dosage form, one component containing the emtricitabine/tenofovir disoproxil fumarate element, the other containing the efavirenz element. This minimized the contact of the tenfovir DF with surfactant, yet maintained the efavirenz excipients and process features that contributed to achieving bioequivalence.

An additional obstacle to the triple combination dosage form was presented, and it is this problem that the present application is directed to solving. As noted above, simply combining the excipients present in the known commercial products, Truvada and Sustiva tablets, was undesirable because the resulting tablet would contain the entire excipient load of the known tablets and thus would be large for a single tablet and present a dosage form that was difficult to swallow and therefore inconvenient for patient use. It thus was an objective to prepare a highly concentrated preparation of emtricitabine and tenofovir DF, which by reducing the amount of excipients in the preparation, would contribute to an overall reduction in the size of the triple combination tablet. However, simply reducing the proportion of excipient to API and wet granulating in accord with the known process was not effective in producing a stable composition.

While the prior art reports the successful manufacture of chemically stable Truvada preparations (WO04/64845) by wet granulation, these preparations typically contain relatively low proportions of excipient to API, on the order of to 1:1. Wet granulation of a preparation in which the proportion of excipient had been reduced to manageable amounts for a triple combination tablet unexpectedly resulted in a chemically unstable preparation. Without being held to any particular theory of operation, the inventors believe that so much water is required in the wet granulation of efavirenz (which has relatively low solubility in comparison to emtricitabine and tenofovir DF) that the latter two APIs dissolve into a eutectic mixture. These dissolved APIs, when dried during granulation, form a glassy or amorphous product, which is chemically unstable in comparison to the crystalline API. In the prior process enough excipient is present to ameliorate the effect of the excess water is, but this was not feasible when the ratio of excipient to API is reduced to a level required for a manageable triple combination oral dosage form.

SUMMARY OF THE INVENTION

In accordance with this invention, a stable preparation of emtricitabine/tenofovir DF is provided by dry granulating a composition comprising a pharmaceutically acceptable excipient, tenofovir DF and emtricitabine. The omission of destabilizing amounts of water from the granulation process eliminates the disadvantageous formation of an emtricitabine/tenofovir DF eutectic mixture and enhances the stability of the resulting pharmaceutical product. The practice of the method of this invention produces a composition comprising dry granulated emtricitabine and tenofovir DF.

DETAILED DESCRIPTION OF THE INVENTION

Dry granulation is a well-known pharmaceutical manufacturing process per se. In general, API is combined with excipients and lubricant excipient and then compressed to form a mass. This mass typically is then comminuted or milled, then sieved to obtain the desired size of particle. The granular product is compressed into tablets, filled into capsules or otherwise formed into a unitary dosage form in conventional fashion. This invention at least in part is directed to the products produced by this process.

Compression into a mass is accomplished by conventional equipment. Typically, the API and excipients are passed through a roller compactor or chilsonator apparatus for compaction. However, other means for compacting, e.g., compaction into slugs (or “slugging”), the API/excipient mixture optionally are used. This in turn is comminuted or milled, and then optionally sieved to produce the desired size granules.

A dry granulated composition comprising emtricitabine and tenofovir DF is defined as the product of a dry granulation process. This composition essentially retains the crystalline APIs and is substantially free of dried eutectic emtricitabine/tenofovir DF. It typically will contain less than about 15% by weight dried eutectic mixture, ordinarily less than about 10% and generally less than about 5%. Dry granulated compositions include the direct product of dry granulation, i.e., dry granules per se, as well as products made from such granules including tablets, capsules, suppositories and other pharmaceutical dosage forms. Forming the dry granules into such physical forms substantially retains the character of the dry granular starting material and does not result in a substantial change in the properties of the granular component of the physical form presented.

Dry granulation is conducted in the absence of a destabilizing amount of water, “destabilizing” being that amount of liquid water that is capable causing degradation (defined infra) of tenofovir DF and/or emtricitabine. Ordinarily, no water at all is added during the dry granulation process.

Bound, entrained or absorbed water are commonly present in excipients. This water will not significantly adversely affect the stability of tenofovir DF and thus is not excluded from the invention. In general, liquid water (added or generated in situ) from any source, e.g., chemical reactions, condensation, entrained ice, or the like is to be excluded from the granulation. However, minor amounts of liquid water optionally are added during granulation. These typically would be less than about 5% by weight, ordinarily less than about 1% by weight, however the water is generated or supplied. Water is present in the final granulation product up to about 10% by weight (Karl Fischer), but preferably is less, as low as 0.1% by weight. However, permitted quantities of water may vary depending upon other factors in the granulation, e.g., excipient type, temperature and so forth. For example, if a hygroscopic excipient is included this will convert added water into a bound form. All that is necessary is that the water not result in degradation of tenofovir DF in the final product. In general, water is excluded both from the pregranulation stage (preparation of the composition to be used directly in the granulation) as well as during the granulation process itself.

Absence of water or “dry” does not mean the absence of liquid. Granulations with organic solvents are optionally conducted in accordance with this invention provided that destabilizing amounts of water are excluded.

Dry granulation results in a product that contains minimal amounts of water. The amount of water in the product granulate or dosage forms made there from are measured by loss on drying (LOD) or by the Karl Fischer method. The LOD of compositions of this invention are about 15%, about 10%, about 5% or typically less than about 3% by weight. The Karl Fischer water is about from 0.1 to 10% by weight, usually less than about 5% by weight, or less than about 2%. The amount of water in the final preparations, as opposed to the granulates, is a function of granulate water as well as minor amounts of water used during subsequent process steps such as coating. These amounts of water added in later steps than granulation generally will not affect the stability of the emtricitabine/tenofovir DF APIs, and therefore are subject to considerable permitted variation.

“Degradation” of tenofovir DF is the generation—in pharmaceutically unacceptable amounts—of at least one of the degradation products mono-POC PMPA, dimer or mixed dimer. “Degradation” of FTC is defined as the generation—in pharmaceutically unacceptable amounts—of FTU. These degradation products are shown below.
Mono-POC PMPA
Dimeric Degradation Products

A “pharmaceutically unacceptable amount” is defined as the following amounts of each degradation product. Degradation products optionally are assayed in either an absolute or incremental amount. The absolute or total amount of degradation product is simply the amount found in the test article. The incremental amount is the additional amount of degradation product appearing in the product over that which was present (if any) in the API starting material. Moreover, the amount of degradation product(s) optionally are measured at either or both of two points in time. One is the time of release into the marketplace. The other is after exposure to storage conditions under the conditions described below, i.e., the shelf life as set forth below.

Total Amounts at Release (First Commercial Sale)

• No more than about 3%, ordinarily about 1.5%, of mono-POC PMPA,
• No more than about 1%, ordinarily about 0.5% of Dimer,
• No more than about 0.5%, ordinarily about 0.25% of Mixed Dimer.
• Less than about 0.5%, ordinarily about 0.2% of FTU
  Total Amounts at Shelf Life (Storage Under Desiccant at 25° C./60% RH for 24 Mo.)
• No more than about 10%, ordinarily about 5% of mono-POC PMPA,
• No more than about 2%, ordinarily about 1% of Dimer,
• No more than about 2%, ordinarily about 1% of Mixed Dimer.
• No more than about 4%, ordinarily about 2% of FTU
  Incremental Amounts at Release (First Commercial Sale)
• No more than about 2%, ordinarily about 0.5%, of mono-POC PMPA,
• No more than about 0.6%, ordinarily about 0.1% of Dimer,
• No more than about 0.3%, ordinarily about 0.05% of Mixed Dimer.
• Less than about 0.4%, ordinarily about 0.1% of FTU
  Incremental Amounts at Shelf Life (Storage Under Desiccant at 25° C./60% RH for 24 Mo.)
• No more than about 9%, ordinarily about 4% of mono-POC PMPA,
• No more than about 1.6%, ordinarily about 0.6% of Dimer,
• No more than about 1.8%, ordinarily about 0.8% of Mixed Dimer.
• No more than about 3.9%, ordinarily about 1.9% of FTU.

The percentage of degradation products is the amount of degradation product as measured by HPLC retention time comparison. In the HPLC retention time comparison, the retention time of the main peaks observed in the tablets is required to be within 2% of the retention time of the main peaks in the a reference standard preparation containing efavirenz, emtricitabine, and tenofovir DF in an assay which has been shown to be specific for efavirenz, emtricitabine, and tenofovir DF. The percentage is determined by dividing the total amount of tenofovir DF plus the three degradation products into the amount of individual degradation product as determined by the HPLC assay.

Thus, for example, it is conceivable that a small amount of water might be desirably present during a dry granulation. This water might be added in the liquid form as an incidental solubilizing agent for an excipient included in the composition to be compressed. It also might be added bound to a hygroscopic excipient containing an unusually large amount of absorbed water. If the resulting product upon release did not contain more than the specified approximate limits of any one or more of the 4 contaminants listed under any of the 4 assay paradigms above, then the process concerned would still be considered a dry granulation process. Of course, the artisan may adopt more stringent standards (i.e., the amounts of some contaminants may be less than set forth above), but this will be a matter of choice and shall not limit the scope of this invention.

The manufacturing process described below is directed to one embodiment of the invention. Other embodiments will be well within the skill of the artisan. This embodiment entails the preparation of a triple combination tablet containing efavirenz, emtricitabine, and tenofovir DF. In this particular embodiment the last two drugs/excipients are segregated in a portion of the tablet, which is separate from, but in contact with, the portion of the tablet containing efavirenz/excipients. It will be understood, however, that the emtricitabine and tenofovir DF component of the tablet, which is an embodiment of this invention, optionally is manufactured for example as a stand-alone product and not necessarily in assembly with an efavirenz component. In this case, the emtricitabine/tenofovir DF dry granulation intermediate described below is optionally combined with other APIs or excipients, and compressed into tablets or conventionally processed into other conventional unitary dosage forms such as capsules, cachets, suppositories, or the like.

The manufacturing method for the triple combination tablet employs two separate granulation steps. The efavirenz final blend (efavirenz and excipients) was produced by a wet granulation process whereas emtricitabine, tenofovir DF, and suitable excipients were blended and dry granulated by a roller compaction process. The final blends were compressed into a bilayer tablet which in turn was film-coated with an immediate release coating.

Materials

The quantitative compositions of the efavirenz powder blend, FTC/TDF powder blend, and film-coated bilayer EFV/FTC/TDF tablets are listed in Table 1, Table 2, and Table 3, respectively. The quantities of efavirenz, emtricitabine, and tenofovir DF were adjusted for drug content factors (DCF) if the value was less than 0.99 with a concomitant reduction to the quantity of microcrystalline cellulose in each granulation.

TABLE 1
Quantitative composition of efavirenz powder blend
			Unit
		% w/w	Formula
	Ingredient	of Total	(mg/tablet)
	Efavirenz	38.71	600.0
	Microcrystalline Cellulose, NF/EP	11.52	178.6
	Hydroxypropyl cellulose, NF/EP	2.48	38.4
	Sodium Lauryl Sulfate, USP/EP	0.77	12.0
	Croscarmellose Sodium, NF/EP	3.87	48.0
	Magnesium Stearate, NF/EP	0.58	9.6
	Total for Tablet Core	57.94	898.0

TABLE 2
Quantitative composition of FTC/TDF powder blend
			Unit
		% w/w	Formula
	Ingredient	of Total	(mg/tablet)
	Emtricitabine	12.90	200.0
	Tenofovir Disoproxil Fumarate	19.35	300.0
	Microcrystalline Cellulose, NF/EP	5.77	89.5
	Croscarmellose Sodium, NF/EPa	3.10	48.0
	Magnesium Stearate, NF/EPa	0.94	14.5
	Total for Tablet Core	42.06	652.0
	aTo be incorporated into both the intragranular and extragranular portions of the formulation during the manufacturing process.

TABLE 3
Quantitative composition of film-coated
bi-layer EFV/FTC/TDF Tablets
		% w/w	Unit Formula
	Ingredient	of Total	(mg/tablet)
	Efavirenz Powder Blend	57.94	898.0
	FTC/TDF Powder Blend	42.06	652.0
	Total for Tablet Cores	100.00	1550.0
	Opadry II Pink	3.00	46.5
	Purified Water, USP/EPa
	Total for Film-Coated Tablets		1596.5
	aWater removed during film-coating process.

The excipients were all compendial grade materials:
Efavirenz Wet Granulation

Efavirenz was wet granulated using a Niro-Fielder PMA-400 equipment train. Efavirenz, microcrystalline cellulose and sodium lauryl sulfate (Table 1) were added to the PMA-400 and blended for 3 minutes. Croscarmellose sodium and hydroxypropyl cellulose (Table 1) were added to the pre-mix and blended for an additional 2 minutes. Purified water was added to form a suitable granulation followed by additional wet massing after water addition. Table 4 lists the summary of granulation parameters used for two representative lots and sub parts. All sub parts used a water to efavirenz ratio of 1.30 except for AB509 Mix C which used a 1.25 ratio of water to efavirenz.

TABLE 4
Efavirenz wet granulation process parameter summary
	AB507	AB509
Process	Parameter	Mix A	Mix B	Mix C	Mix A	Mix B	Mix C
Granulation	Total Water Added (kg)	33.57	33.56	33.56	33.56	33.56	32.18
	Ratio of Water:EFV	1.30	1.30	1.30	1.30	1.30	1.25
	Total Addition Time (Min:Sec)	9:36	9:29	9:24	9:17	9:32	9:02
	Final Impeller Power (% Load)	10.4	9.8	8.5	11.3	11.3	9.9
Wet	Total Time (Min:Sec)	4:00	3:00	3:00	2:00	1:15	2:00
Massing	Final Impeller Power (% Load)	11.6	12.0	11.7	18.0	17.7	10.5
Dryinga	Inlet Temperature (° C.)	70	70
	Time (Hr:Min)	1:45	1:51
	Final Outlet Temp. (° C.)	50	50
	Final LOD (%)	0.3	0.8
aMixes A, B, and C for each lot were combined before drying.

In general, the wet granules were milled, then dried to an LOD less than or equal to 1.5%. The dried granules were milled and blended with magnesium stearate (Table 1).

The bulk density, particle size, and moisture content by LOD of the efavirenz granulations are listed in the first three lines of Table 5 (the B lot numbers are efavirenz products, the C lot numbers are emtricitabine/tenofovir DF). Particle size was determined by sifting 10-gram samples through 3-inch diameter screens using a sonic sifter (Model L3P, ATM Corporation, Milwaukee, Wis., USA). The following US Standard Mesh sizes (openings) were used: #20 (850 μm), #30 (600 μm), #40 (425 μm), #60 (250 μm), #80 (180 μm), and #250 (63 μm). The agitation and pulse were set at 7 and the sifting time was 5 minutes. The amount of powder retained on the sieves and the fines collector was determined by calculating the difference in weight before and after sifting. The geometric mean particle size was calculated by logarithmic weighting of the sieved distribution.

Bulk density was determined by filling a 100-mL graduated cylinder with sample and calculating the difference in weight between the empty and full graduated cylinder per unit volume. In typical embodiments the bulk density of the granules is about from 0.25 to 0.75 g/mL.

Moisture content measurements by loss on drying (LOD) were performed by heating a 2.5 g sample at 85° C. for 15 minutes using a heat lamp/balance system (Model LP16/PM400, Mettler-Toledo, Columbus, Ohio, USA).

The granulations had similar bulk densities (0.54 to 0.56 g/mL) and similar geometric mean particle size distributions (215 to 268 μm). The LOD values of the final blend were consistent from 0.98 to 1.80%. The individual sieve distributions for the efavirenz granulations are listed in Table 6.

TABLE 5
Summary of efavirenz powder blend and emtricitabine/tenofovir
DF powder blend physical properties
		Geometric Mean
		Diameter	Bulk
	Gilead	Particle Size	Density	LOD
	Lot Number	(μm)	(g/mL)	(%)
	AB507	247	0.56	1.80
	AB508	215	0.55	1.08
	AB509	268	0.54	0.98
	AC507	330	0.60	0.91
	AC508	344	0.60	1.02
	AC509	343	0.59	0.99

TABLE 6
Particle size distribution for efavirenz and FTC/TDF powder blends
	% Weight Retained on Screena
	US Standard Screen Size (mesh opening)
Gilead	20	30	40	60	80	230	pan
Lot No.	(>850 μm)	(600 (μm)	(425 μm)	(250 μm)	(180 μm)	(63 μm)	(<63 μm)
AB507	5.9	10.9	16.2	22.2	11.4	22.6	10.9
AB508	6.1	10.4	15.8	20.0	9.0	20.8	17.9
AB509	9.6	13.3	17.4	20.1	8.9	17.2	13.3
AC507	22.0	19.8	15.2	11.2	4.6	10.5	16.6
AC508	22.1	20.1	15.4	11.6	5.1	10.6	14.9
AC509	22.4	19.7	15.3	11.7	4.8	11.1	14.8

Emtricitabine/Tenofovir DF Dry Granulation

Emtricitabine, microcrystalline cellulose, tenofovir DF, and croscarmellose (Table 2) were blended in a 650 L tote bin using a Gallay blender for 10 minutes. Magnesium stearate (Table 2) was added and blended for an additional 5 minutes. This pre-blend was then transferred to a 320-L Matcon bin fitted with a cone valve discharging station to assist with material transfer into the roller compactor hopper.

The pre-blend was roller compacted using a Gerteis Macro-Pactor model 250/25/3 with 250 mm diameter by 50 mm wide smooth rolls. The roll gap thickness (2 mm), roll speed (10 rpm), compaction force (4 kN/cm), oscillating mill speed (75 rpm clockwise and counterclockwise), and oscillating mill screen opening (1.25 mm) were kept constant for all batches. The oscillating mill angle of rotation was also the same for all lots at 150° clockwise and 140° counterclockwise.

There was no material handling issues among all three batches while feeding into the roller compactor. The entire roller compaction process proceeded without any apparent sign of heat accumulation on the equipment, product build-up, or melting. The granulations then were blended with extragranular croscarmellose sodium (34% of total amount) and magnesium stearate (47% of total amount).

The particle size, bulk density, and LOD of the emtricitabine/tenofovir DF dry granulations were all similar for the three batches and are listed in Table 5 (bottom 3 compartments). The geometric particle sizes were very similar at from 330 to 344 μm. Bulk densities ranged from 0.59 to 0.60 g/mL. The final blend LOD values were consistent from 0.91 to 1.02%. The final powder blends have remarkably consistent physical properties.

The efavirenz and tenofovir DF granulations each have geometric mean particle sizes that optionally range about from 100 to 600 μm, bulk densities optionally ranging about from 0.1 to 1 g/mL and LOD values optionally ranging about from 0.1 to 5% by weight.

Final Blends

The mass of efavirenz granulation and extragranular magnesium stearate were adjusted appropriately based on the yield of emtricitabine/tenofovir DF dry granulation. Efavirenz granulation and emtricitabine/tenofovir DF dry granulation were blended in a 3 cubic foot V-blender for 10 minutes. Magnesium stearate was added and blended an additional 5 minutes. Samples of the final powder blend were taken from 10 different locations after blending and analyzed for blend uniformity. The efavirenz and emtricitabine/tenofovir DF final powder blends showed acceptable blend uniformity and homogeneity for all three active ingredients indicating the robustness of the formulation regardless of the particle size or bulk density of emtricitabine/tenofovir DF dry granulations and efavirenz granulations. The granulations and blending procedure would be satisfactory for the formulation on a larger scale.

Tablet Core Compression

Efavirenz/emtricitabine/tenofovir DF final powder blend was compressed into tablet cores using a Stokes Genesis Model 757, 41 station bilayer tablet press equipped plain-faced upper/ embossed “123” lower, capsule-shaped (20.0 mm×10.4 mm) punches. The target mass of the tablet cores was 1550 mg. Samples of the core tablets were taken from a minimum of 20 equally spaced locations during the compression run and analyzed for content uniformity. In general, all powder blends compressed satisfactory on the rotary tablet press with respect to tablet hardness, friability, tablet thickness, tablet appearance, and tablet weight variation. The compression operation was performed at a rate of approximately 500 tablets/minute (12 rpm press speed) or approximately 0.8 kg/minute to deliver satisfactory tablet weight uniformity.

Tablet Film-Coating

Suitable film coatings are selected by routine screening of commercially available preparations. This activity is well within the skill of the ordinary artisan. Each lot of tablet cores was divided into two coating sub-lots that were film coated in a 48-inch Thomas Engineering COMPU-LAB coating pan using a dual-nozzle spraying system. All the tablet cores were film-coated using a 15% w/w aqueous coating suspension Opadry II pink, which was used within 24 hours of preparation. All tablet cores were coated to a target weight gain of 3.0% using a target spray rate of 180 g/min, which corresponds to a normalized spray rate of 1.5 to 2.3 g/min/kg tablets.

HPLC Assay for Degradation Products

Efavirenz/emtricitabine/tenofovir DF tablets (EFV/FTC/TDF tablets) are assayed by HPLC for EFV, FTC, and TDF using external reference standards. The degradation products of EFV, FTC, and TDF are determined by area normalization with the application of relative response factors, as appropriate. The identity of EFV, FTC, and TDF are confirmed by comparison of their retention times with those of the reference standards.

Standard and Sample Solution Preparation

Standard and Sample Solvent

25 mM Phosphate Buffer, pH 3

Weigh and transfer 3.4 g of potassium phosphate monobasic, anhydrous into a 1 L volumetric flask. Add about 800 mL of water and mix until dissolved. Adjust the pH to 3.0±0.1 with phosphoric acid, then dilute to volume with water.

Sample Solvent (40:30:30 25 mM Phosphate Buffer, pH 3:Acetonitrile:Methanol)

Combine 400 mL of 25 mM Phosphate Buffer, pH 3, 300 mL of acetonitrile, and 300 mL of methanol and mix. Allow to equilibrate to ambient temperature.

50:50 Acetonitrile:Methanol

Combine 500 mL of acetonitrile and 500 mL of methanol and mix. Allow to equilibrate to ambient temperature.

Standard Solution

Accurately weigh approximately 60 mg of EFV reference standard, 20 mg of FTC reference standard, and 30 mg of TDF reference standard and transfer into a 100 mL volumetric flask. Add approximately 80 mL of sample solvent (40:30:30) to the flask and mix or sonicate until dissolved. Dilute to volume with sample solvent (40:30:30) and mix well. The final concentration of each component is approximately 0.6 mg/mL of EFV, 0.2 mg/mL of FTC, and 0.3 mg/mL of TDF.

System Suitability Test Solutions

Sensitivity Check Standard

Prepare a 10 μg/mL FTU stock solution by accurately weighing out approximately 10 mg of the FTU authentic substance into a 100 mL volumetric flask. Add sample solvent (40:30:30) to approximately 80% of volume and mix or sonicate until dissolved. Dilute to volume with sample solvent (40:30:30) and mix well. Pipet 10 mL of this solution into a 100 mL volumetric flask. Dilute to volume with sample solvent (40:30:30) and mix well.

Prepare the sensitivity check standard containing 0.2 mg/mL of FTC and 0.2 μg/mL of FTU (0.10% relative to FTC). Accurately weigh out 20 mg FTC into a 100 mL volumetric flask. Using a Class A pipet, transfer 2.0 mL of the FTU stock solution into the same flask. Add additional sample solvent (40:30:30) to the flask and mix or sonicate until dissolved. Dilute to volume with sample solvent (40:30:30) and mix well. Alternately, 2.0 mL of the 10 μg/mL FTU stock solution may be added to the standard solution prior to diluting to volume.

Sample Preparation for EFV/FTC/TDF Tablets

The strength and degradation product content of EFV/FTC/TDF tablets is determined by the analysis of a composite solution prepared from ten tablets.

The final concentration of each component in the sample solution is approximately 0.6 mg/m L of EFV, 0.2 mg/mL of FTC, and 0.3 mg/mL of TDF.

• • a) Place ten tablets into a 1 L volumetric flask and add 400 mL 25 mM phosphate buffer, pH 3 to the volumetric flask.
  • b) Mix by stirring vigorously for about 75 minutes.
  • c) Add 50:50 acetonitrile:methanol to the flask to approximately 2 cm below the volume mark.
  • d) Equilibrate the solution to ambient temperature by mixing for an hour. Dilute to volume with 50:50 acetonitrile:methanol. Mix well by inverting the flask or stirring with a magnetic stir bar.
  • e) Using a 0.45 μm syringe filter with a syringe, filter approximately 10 mL of step (d) for the next dilution. Discard the first 2 mL of filtrate.
  • f) Using a Class A pipet, transfer 5.0 mL of the filtrate from step (e) into a 50 mL volumetric flask and dilute to volume with sample solvent (40:30:30). Mix well.
    Chromotography

1. An HPLC equipped with a UV detector and an electronic data acquisition system is used.

2. An HPLC column, 4.6 mm i.d. by 250 mm long, packed with C12 reversed phase, 4 μm particle size, 80 Å pore size material is used.

3. Mobile phase buffer: Prepare a 20 mM ammonium acetate buffer, pH 4.6; adjust pH with acetic acid as needed.

4. Mobile phase gradient: Elute with Mobile Phase Buffer:acetonitrile from 99:1 to 1:99 over 67 minutes.

5. Peak detection: UV at 262 nm

6. Injection volume: 10 μL.

Under the stated chromatographic conditions, the retention times of the FTC, TDF and EFV peaks are typically 11, 33, and 50 minutes, respectively

Injection Sequence

Inject the sample solvent at least twice as a blank to ensure that the column is equilibrated and to identify any potential artifact peaks.

Inject the sensitivity check standard or standard solution containing approximately 0.10% FTU to measure the sensitivity of detection.

Inject five replicates of standard solution 1 (R1), followed by a single injection of standard solution 2 (R2). Calculate the theoretical plates and tailing factors from the standard solution injections.

For identity, strength, and degradation product determination, perform duplicate injections of the sample solution.

All sample solutions must be bracketed by standard solution injections. Generally, not more than ten sample solution injections between bracketing standard injections is recommended.

System Suitability

Theoretical Plates and Tailing Factor

Calculate the number of theoretical plates (N) and the tailing factors (T) for the EFV, FTC, and TDF peaks from the Standard Solution chromatogram. The formulas for N and T detrmination are defined in the current United States Pharmacopeia. The values of these parameters must conform to the criteria: N≦40,000 and 0.8≦T≧2.0.

Sensitivity Check

The sensitivity check will utilize the FTU peak in the sensitivity check standard present at approximately 0.10%. Calculate the area percent of the FTU peak with the appropriate RRF (listed in Table 2) applied for the sensitivity check standard using the calculation for percent individual degradation product. Compare this result to the theoretical percent of FTU for the sensitivity check standard as follows: $\mathrm{Sensitivity}=\frac{{\mathrm{FTU}}_{\mathrm{Determined}}}{{\mathrm{FTU}}_{\mathrm{Theoretical}}}$

Where: FTU_Determined=area percent of FTU determined for the sensitivity check standard or standard solution

• • FTU_Theoretical=theoretical area percent of FTU for the sensitivity check standard or standard solution

The sensitivity must be between 0.70-1.30.

Evaluation and Calculations

Identification of Degradation Products

Employ the appropriate detection parameters (such as peak threshold, minimum peak area, etc.) to allow detection of peaks present at 0.05% or less. Identify the impurities and degradation products of EFV, FTC, and TDF present in the chromatograms of the sample solution injections by noting the relative retention times (RRT) of the observed secondary peaks, discounting any peaks not related to the sample. Only degradation products are quantified. Calculate the average of the results from all sample solution injections to the nearest 0.01%. In cases where the degradation product was not detected or was below the threshold of integration in one injection and/or sample, use only the quantified results in the calculation (i.e., do not treat as a zero value). $\mathrm{RRT}=\frac{\mathrm{retention}\mathrm{time}\mathrm{of}\mathrm{the}\mathrm{secondary}\mathrm{peak}}{\mathrm{retention}\mathrm{time}\mathrm{of}\mathrm{the}\mathrm{tenofovir}\mathrm{disoproxil}\mathrm{peak}}$

The RRTs and the relative response factor (RRF) values of the potential impurities and degradation products for EFV are shown in Table 1, and the degradation products are shown in bold-face. The impurities and degradation products for FTC are shown in Table 2, and the degradation products are in bold-face. The impurities and degradation products for TDF are shown in Table 3, and the degradation products are in bold face.

As the RRT may vary, the identity of impurities and degradation products may be confirmed by comparison to authentic substances (or to impurity and degradation product peaks in the reference standard), if required.

Degradation Product Content Determination

Quantification of FTC Degradation Products

Determine the level of each degradation product of FTC observed in the chromatograms of the sample solution injections using the following formula: $\mathrm{Degradation}\mathrm{Product}\left(\%\right)=\frac{I}{\mathrm{TPA}}\times \mathrm{RRF}\times 100$

Where: I=Area of the degradation product peak

• • TPA=Total peak area (area of FTC and all related degradation products, excluding impurities and artifacts), corrected by RRF
  • RRF=Relative response factor with respect to FTC

Quantification of TDF Degradation Products

Determine the level of each degradation product of TDF observed in the chromatograms of the sample solution injections using the following formula: $\mathrm{Degradation}\mathrm{Product}\left(\%\right)=\frac{I}{\mathrm{TPA}}\times \mathrm{RRF}\times 100$

Where: I=Area of the degradation product peak or unassigned peak

• • TPA=Total peak area (area of the TDF main peak, all related degradation products, and all unassigned peaks, excluding impurities and artifacts), corrected by RRF
  • RRF=Relative response factor with respect to TDF
    Results and Reporting
    Degradation Product Content

Report individually the average of the results for each degradation product observed to the nearest 0.01%. Report the total degradation product content of EFV, FTC, and TDF respectively to the nearest 0.1%, as the sum of the average levels of all degradation product peaks observed. For degradation products found at levels less than 0.05%, report their levels as trace and do not include their levels in the calculation of total degradation product content.

REFERENCES

United States Pharmacopeia <621>

Pharmacopeial Forum 26(4) 2000

TABLE 1
EFV related impurities and degradation products
		Approximate
	EFV Related Compound	RRTa	RRFb
	SD-573c	1.46	0.5
	SR-695d	1.50
	EFV	1.50
	SP-234	1.57
	SW-965	1.60
	SE-563	1.73
	SM-097c	1.83	0.5
	aApproximate RRTs, and the values are relative to the TDF peak
	bRRFs for EFV related degradation products are relative to EFV
	cEFV related degradation products
	dSR-695 elutes before EFV (approximately 0.1 min separation)
	Degradation products are marked in bold face

TABLE 2
FTC related degradation product
FTC Related Compound	Approximate RRTa	RRFb
FTC	0.33
FTUc	0.38	0.7
aApproximate RRTs, and the values are relative to the TDF peak
bRRFs for FTC related degradation products are relative to FTC
cFTC related degradation products

TABLE 3
Tenofovir DF related degradation products
		Impurity	Approximate
	TDF Related Compound	Number	RRTa	RRFb
	mono-POC PMPAc	3	0.47	0.6
	Mixed Dimerc	13	0.98	1.0
	TDF		1.00
	Dimerc	14	1.34	0.9
	aApproximate RRTs, and the values are relative to the TDF peak
	bRRFs for TDF related degradation products are relative to TDF
	cTDF related degradation products

Claims

1. A composition comprising dry granulated emtricitabine and tenofovir DF.

2. The composition of claim 1 wherein the water content (Karl Fischer) is about from 0.1 to 10% by weight.

3. The composition of claim 1 wherein the bulk density of the granules is about from 0.1 to 1 g/mL.

4. The composition of claim 1 wherein the geometric mean diameter of the granules is about from 50 to 800 micrometers.

5. The composition of claim 1 further comprising a pharmaceutically acceptable disintegrant.

6. The composition of claim 5 wherein the disintegrant is croscarmellose sodium or crospovidone.

7. The composition of claim 1 further comprising a pharmaceutically acceptable filler.

8. The composition of claim 1 further comprising a pharmaceutically acceptable binder.

9. The composition of claim 1 further comprising a pharmaceutically acceptable lubricant.

10. The composition of claim 1 as a unitary dosage form.

11. The composition of claim 10 which is a tablet.

12. The composition of claim 1 wherein the amount of emtricitabine and tenofovir DF is greater than about 70% by weight of the granules.

13. The composition of claim 12 wherein the amount of emtricitabine and tenofovir DF is about 77% by weight of the granules.

14. The composition of claim 1 which further comprises at least one pharmaceutically acceptable excipient.

15. The composition of claim 1 comprising (by approximate weight percent) emtricitabine 30.6, tenofovir DF 46.0, microcrystalline cellulose 13.7, croscarmelose sodium 7.3 and magnesium stearate 2.2.

16. The composition of claim 1 wherein the LOD is about 10%.

17. A method comprising granulating a composition comprising emtricitabine and tenofovir DF without contacting the composition with a destabilizing amount of liquid water.

18. The method of claim 17 wherein liquid water is not combined with the composition prior to or during granulation.

19. The method of claim 17 wherein the composition further comprises at least one pharmaceutically acceptable excipient.

20. The method of claim 17 wherein granulation comprises aggregating the composition and comminuting it to desired dimensions.

21. The method of claim 20 wherein the aggregation is accomplished by slugging or roller compaction.

22. The method of claim 20 wherein the composition is sieved to recover granules of the desired dimensions.

23. The method of claim 22 wherein the granules are retained by a 1.25 mm mesh.

24. The method of claim 19 wherein the excipient is a lubricant.

25. The method of claim 24 wherein the lubricant is an alkali metal salt of a C8-C18 fatty acid.

26. A unitary dosage form made by a process comprising dry granulation of a composition comprising emtricitabine and tenofovir DF.

27. A composition comprising greater than about 75% by weight emtricitabine and tenofovir DF.

28. A composition comprising granules comprising tenofovir DF, emtricitabine and croscarmellose sodium in an extragranular matrix also comprising croscarmellose sodium.

29. A method for antiviral therapy comprising administering an antivirally effective amount of the composition of claim 1 to a patient in need of antiviral therapy.

30. The method of claim 29 wherein the antiviral therapy is anti-HIV therapy.