Micronized amoxicillin

Abstract

The present invention relates to amoxicillin trihydrate compositions having a surface area of from 1.0 to 2.5 m.sup.2.Math.g.sup.−1 that are free of organic contaminants such as dichloromethane, isopropanol, pivalic acid and triethyl amine and that have a purity of from 97.0% to 99.99%. Furthermore, the present invention relates to a method for the preparation of said amoxicillin trihydrate compositions and to the use of said compositions for the treatment of a bacterial infection.

Claims

1. A composition comprising: (i) from 97.0% to 99.99% (w/w) of amoxicillin trihydrate having a surface area of from 1.0 to 2.5 m.sup.2.Math.g.sup.−1 having an average particle size distribution of D.sub.10 from 1 μm to 3 μm, D.sub.50 from 5 μm to 15 μm, D.sub.90 from 15 μm to 30 μm, and D.sub.100 from 30 μm to 100 μm, and less than 500 ppm of at least one compound selected from the group consisting of dichloromethane, isopropanol, pivalic acid and triethyl amine.

2. The composition according to claim 1, wherein the surface area of the amoxicillin trihydrate is from 1.3 to 2.2 m.sup.2.Math.g.sup.−1.

3. The composition according to claim 2, wherein the composition has an average particle size distribution of D.sub.10 from 1.5 μm to 2.5 μm, D.sub.50 from 8 μm to 12 μm, D.sub.90 from 20 μm to 25 μm and D.sub.100 from 35 μm to 50 μm.

4. The composition according to claim 1, wherein the composition comprises from 2 to 35 ppm of protein.

5. The composition according to claim 1, wherein the amoxicillin trihydrate has a purity of 99.4±0.5%.

6. A method for the preparation of a composition according to claim 1, wherein the method comprises the steps of: (a) contacting 6-aminopenicillanic acid and an ester or an amide of D-4-hydroxyphenylglycine or a salt thereof together with an enzyme in water; (b) isolating, and drying solid amoxicillin trihydrate formed in step (a); and (c) reducing the particle size of the solid amoxicillin trihydrate obtained after step (b).

7. The method according to claim 6, wherein step (c) comprises reducing the particle size of the solid amoxicillin trihydrate by micronization.

8. The method according to claim 6, wherein the enzyme is a penicillin acylase or an α-amino acid ester hydrolase.

9. A method for the treatment of a bacterial infection comprising administering to a subject in need thereof an effective amount of the composition according to claim 1.

10. The method according to claim 9, wherein the composition is a veterinary composition.

11. A pharmaceutical composition comprising the amoxicillin trihydrate of claim 1.

12. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition further comprises a β-lactamase inhibitor.

13. The pharmaceutical composition of claim 1, wherein the β-lactamase inhibitor comprises one or more of clavulanic acid, sulbactam and tazobactam.

14. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is prepared by wet granulation.

15. The pharmaceutical composition of claim 11, wherein the pharmaceutical composition comprises a tablet.

16. The composition according to claim 1, wherein the composition comprises less than 500 ppm of protein.

17. The composition according to claim 1, wherein the composition comprises less than 50 ppm of protein.

18. The composition according to claim 1, Wherein the amoxicillin trihydrate has been synthesized in an enzymatic process.

19. The composition according to claim 1, wherein the amoxicillin trihydrate has a contact angle from 80-130.

20. A composition comprising: (iii) from 97.0% to 99.99% (w/w) of amoxicillin trihydrate having a surface area of from 1.0 to 2.5 m.sup.2.Math.g.sup.−1 having an average particle size distribution of D.sub.10 from 1 μm to 3 μm, D.sub.50 from 5 μm to 15 μm, D.sub.90 from 15 μm to 30 μm and D.sub.100 from 30 μm to 100 μm, and (iv) less than 500 ppm of at least one compound selected from the group consisting of dichloromethane, isopropanol, pivalic acid and triethyl amine, wherein the composition has a purity of 99.4±0.5%, a contact angle from 80-130, and less than 500 ppm of protein.

Description

LEGEND TO THE FIGURES

(1) FIG. 1 displays the dissolution profiles of amoxicillin trihydrate samples, determined according to the dissolution analysis protocol given in the General section of the Examples. X-axis: time in minutes; Y-axis: percentage amoxicillin released in solution. The insert depicts a zoomed portion of the percent amoxicillin released from 80 to 100%. Error bars indicate the standard deviation (SD).

(2) The following symbols are used (see also Table 1 in Example 1): ◯ Non-micronized amoxicillin powder sample 1 □ Non-micronized amoxicillin powder sample 2 ⋄ Non-micronized amoxicillin powder sample 3 .circle-solid. Micronized amoxicillin powder sample 4 (obtained from non-micronized amoxicillin powder sample 1) .square-solid. Micronized amoxicillin powder sample 5 (obtained from non-micronized amoxicillin powder sample 2) .diamond-solid. Micronized amoxicillin powder sample 6 (obtained from non-micronized amoxicillin powder sample 3)

EXAMPLES

General

(3) Amoxicillin trihydrate was prepared enzymatically according to known procedures such as described in WO 2004/082661 or WO 2010/072765.

(4) Residual solvents dichloromethane, isopropanol and triethyl amine were determined by head space gas chromatography using a Perkin Elmer Model Autosystem consisting of a Flame Ionisation detector (FID), a headspace autosampler model HS 40 and TOWS v 6.2.0 software. Sample vials for the autosampler where 50 mL injection vials with a crimp cap with PTFE-coated butyl rubber septum.

(5) Gas Chromatograph: Column: Varian CP-SIL 5CB 30 m×0.25 mm ID, df=0.25 μm Oven temperature: 70° C. Detector temperature: 220° C. Detector sensitivity (range): 1 Attenuation: 16 Injector temperature: 220° C. Carrier gas: Nitrogen Carrier flow rate: 9.0 psi Hydrogen flow rate: 40-45 mL.Math.min.sup.−1 Zero air: 400-446 mL.Math.min.sup.−1 Split flow: 20 mL.Math.min.sup.−1

(6) Autosampler: Sample temperature: 70° C. Needle temperature: 70° C. Transfer temperature: 90° C. Inject time: 0.06 min Thermostating time: 20 min Withdrawal time: 0.1 min Pressurization time: 0.5 min GC cycle time: 15 min
Residual pivalic acid was determined by gas chromatography using a Perkin Elmer Model Autosystem ‘XL’ consisting of a Flame Ionisation detector (FID) and TOWS v 6.2.0 software. Sample vials for the autosampler where 50 mL injection vials with a crimp cap with PTFE-coated butyl rubber septum.

(7) Gas Chromatograph:

(8) TABLE-US-00001 Column: Varian CP-SIL 5CB 50 m × 0.53 mm ID, df = 5.0 μm Oven temperature: 150° C.: 3 min 150° C. .fwdarw. 200° C.: ramp rate 15° C. .Math. min.sup.−1 200° C.: 10 min Equilibrium time: 0.2 min Detector temperature: 240° C. Detector sensitivity (range): 1 Attenuation: 16 Injector temperature: 225° C. Carrier gas: Nitrogen Carrier flow rate: 6.5 psi Hydrogen flow rate: 40-45 mL .Math. min.sup.−1 Zero air: 404-446 mL .Math. min.sup.−1 Inject volume: 5.0 μL Split flow: 65 ± 5 mL .Math. min.sup.−1

(9) Particle size distribution values D.sub.10, D.sub.50, D.sub.90 and D.sub.100 were determined using laser diffraction applying Malvern equipment. A suitable apparatus for determining D.sub.10, D.sub.50, D.sub.90 and D.sub.100 is a Malvern particle sizer 2600 C or a Malvern Zeta Sizer.

(10) Specific powder surface area was determined using nitrogen gas sorption (Smartsorb, Smart Instruments, Mumbai, India). Prior to measurements, samples were regenerated by degassing to remove moisture and contamination. The regenerated sample was dipped in liquid nitrogen and the quantity of the adsorbed gas was measured using a thermal conductivity detector and subsequently integrated using an electronic circuit in terms of counts. The instrument was calibrated by injecting a known quantity of nitrogen. The measured parameters were subsequently used to calculate the surface area of the sample by employing the adsorption theories of Brunauer, Emmett and Teller (BET).

(11) Dissolution analysis of amoxicillin trihydrate powder samples was carried out using the USP/NF paddle method at a rotational speed of 75 rpm. Ultrapure ELGA laboratory water (900 mL) was used as dissolution medium. Media were equilibrated to 37.0±0.5° C. prior to dissolution. Amoxicillin trihydrate sample powder (500 mg) was directly added to the dissolution apparatus (Electrolab USP-24, India). Samples were collected at 0, 1, 3, 5, 10, 15, 30, 60 and 90 min, filtered through 0.2 μm filters and analyzed by HPLC. Dissolution profiles were constructed by plotting the percentage amoxicillin released against time.

(12) The HPLC system (Shimadzu Corporation, Kyoto, Japan) comprised of an SCL-10A VP system controller, LC-10AT VP liquid chromatograph, FCV-10AL VP flow control valve, DGU-14A degasser, SIL-10AD VP auto-injector, CTO-10AS VP column oven, SPD-M20A prominence diode array (PDA) detector and data acquisition Class-VP 6.10 software. The HPLC method was adopted from the United States Pharmacopoeia (USP). The method was validated for linearity, precision, accuracy and intra- and inter-day variability. The mobile phase was 50 mM monobasic potassium phosphate:methanol (96:4, v/v, pH 5.0). All analyses were carried out using Lichrospher® 100 RP-18e analytical column (5 μm, Merck KGaA, Darmstadt, Germany) under isocratic conditions at a flow rate of 1.0 mL.Math.min.sup.−1 at 25° C. with 20 μL injection volume. Effluent was monitored at a wavelength of 230 nm. Samples for calibration curve generation were prepared in 50 mM monobasic potassium phosphate:methanol 1:1 (v/v) proportion at a concentration range of 1 to 600 μg.Math.mL.sup.−1.

(13) Contact angles of amoxicillin trihydrate powder samples were measured by the sessile drop method using an Drop Shape Analyzer instrument (FTA 1000, First Ten Angstrom, Virginia, USA). Powder samples were mounted on double sided adhesive tape adhered to a glass slide and excess powder was removed by tapping the slide. A drop of probe liquid (water) was dispersed onto the sample surface and video images were captured by the FTA image analyzer. The contact angle was calculated by the instrument by fitting a mathematical expression to the shape of the drop followed by calculating the slope of the tangent to the drop at the liquid-solid-vapor interface line. The surface tension of ultrapure ELGA laboratory water was measured to be 73±0.5 mN.Math.m.sup.−1 at 25° C. All measurements were performed in air under ambient conditions and an average of four measurements was reported in the below Examples.

Example 1

Micronization of Amoxicillin Trihydrate

(14) Three different batches of enzymatically prepared amoxicillin trihydrate were micronized in a jet mill micronizer with a capacity of 50-150 kg.Math.h.sup.−1. Micronization involves high speed particle collision thus creating increasingly smaller fines through particle-on-particle impact. Centrifugal force holds larger particles in grinding area while centripetal force drives fines towards the center for discharge. On stream product was fed through a feeding chamber for intended micronization. A suitable recipe based on Augar speed, primary and secondary air was put on auto mode to initiate the micronization process. Qualified compressed air was used as primary and secondary air. Multiple recipes were a result of experimental trials taken during the Performance Qualification (PQ) phase to generate a design space.

(15) TABLE-US-00002 TABLE 1 Characteristics of micronized and non-micronized amoxicillin Surface Area Contact (m.sup.2 .Math. g.sup.−1) Average Particle Size Angle Mean ± SD (μm) Mean ± SD (n = 3) D.sub.10 D.sub.50 D.sub.90 D.sub.100 (n = 4) 1 Non- 0.58 ± 0.09 12.3 39.8 93.3 275.9 75.50 ± 2.95 micron- ized 2 Non- 0.69 ± 0.03 13.5 40.6 94.3 237.5 67.70 ± 1.39 micron- ized 3 Non- 0.72 ± 0.16 13.1 39.3 91.7 230.1 71.54 ± 1.08 micron- ized 4 Micron- 1.78 ± 0.03 1.9 10.1 23.1 44.6 110.68 ± 0.23  ized 1 5 Micron- 2.03 ± 0.15 1.9 10.0 22.5 44.3 97.89 ± 1.22 ized 2 6 Micron- 1.69 ± 0.07 1.9 10.0 22.4 38.9 107.84 ± 1.72  ized 3
Parameters operated were: Augar Speed: 5-90 rpm Primary Air Pressure: 2-8 kg.Math.cm.sup.−2 Secondary Air Pressure: 4-10 kg.Math.cm.sup.−2
The surface area (in triplicate), average particle size and contact angle (in quadruplicate) were determined as outlined in the General section. The purity of the amoxicillin trihydrate samples was 99.4±0.3% and were void of dichloromethane, isopropanol, pivalic acid and triethyl amine. Other results are summarized in Table 1.

Example 2

Powder Dissolution of Micronized and Non-Micronized Amoxicillin Trihydrate (Dissolution Apparatus Electrolab USP-24)

(16) The three non-micronized (i.e. entries 1-3 of Table 1) and micronized (i.e. entries 4-6 of Table 1) amoxicillin trihydrate samples of Example 1 were subjected to a powder dissolution test (in triplicate) as outlined in the General section. The results are graphically displayed in FIG. 1 and Tables 2 to 7.

(17) TABLE-US-00003 TABLE 2 Dissolution data for non-micronized amoxicillin (entry 1 of Table 1) Amoxicillin released Time in solution (%) (min) #1 #2 #3 Mean SD 0 0 0 0 0 0 1 84.65 86.65 86.65 85.98 1.16 3 99.74 99.01 99.01 99.25 0.42 5 99.44 99.54 99.54 99.51 0.06 10 98.65 99.04 99.73 99.14 0.55 15 99.28 98.69 99.38 99.12 0.37 30 99.50 99.56 98.87 99.31 0.38 60 99.56 99.10 99.10 99.25 0.27 90 99.93 99.61 99.74 99.76 0.16

(18) TABLE-US-00004 TABLE 3 Dissolution data for non-micronized amoxicillin (entry 2 of Table 1) Amoxicillin released Time in solution (%) (min) #1 #2 #3 Mean SD 0 0 0 0 0 0 1 61.68 61.21 62.23 61.71 0.51 3 99.58 99.88 99.60 99.69 0.17 5 99.89 99.79 97.29 98.99 1.47 10 99.66 98.95 100.36 99.66 0.71 15 99.51 98.98 99.80 99.43 0.42 30 99.65 99.00 99.34 99.33 0.32 60 99.56 99.17 99.60 99.45 0.24 90 99.24 99.24 98.96 99.15 0.17

(19) TABLE-US-00005 TABLE 4 Dissolution data for non-micronized amoxicillin (entry 3 of Table 1) Amoxicillin released Time in solution (%) (min) #1 #2 #3 Mean SD 0 0 0 0 0 0 1 57.32 60.63 61.23 59.73 2.10 3 96.94 97.25 98.77 97.65 0.98 5 99.68 100.87 98.80 99.78 1.03 10 99.06 99.42 99.27 99.25 0.18 15 100.69 100.64 99.55 100.29 0.64 30 99.21 99.18 99.14 99.18 0.04 60 99.14 100.41 100.92 100.16 0.92 90 99.58 99.28 99.46 99.44 0.15

(20) TABLE-US-00006 TABLE 5 Dissolution data for micronized amoxicillin (entry 4 of Table 1) Amoxicillin released Time in solution (%) (min) #1 #2 #3 Mean SD 0 0 0 0 0 0 1 54.49 57.74 57.67 56.64 1.85 3 76.32 76.89 78.12 77.11 0.92 5 85.25 82.78 84.47 84.17 1.26 10 88.15 86.80 87.34 87.43 0.68 15 90.61 89.05 89.49 89.71 0.81 30 93.27 95.75 91.85 93.77 1.95 60 97.15 99.70 98.65 98.50 1.28 90 97.76 99.52 100.66 99.31 1.46

(21) TABLE-US-00007 TABLE 6 Dissolution data for micronized amoxicillin (entry 5 of Table 1) Amoxicillin released Time in solution (%) (min) #1 #2 #3 Mean SD 0 0 0 0 0 0 1 58.56 62.91 58.32 59.93 2.58 3 78.61 80.12 75.20 77.98 2.52 5 83.91 82.77 79.73 82.14 2.16 10 89.18 88.59 82.76 86.84 3.55 15 90.98 92.05 84.94 89.32 3.84 30 94.78 99.20 96.17 96.72 2.26 60 97.29 99.86 95.65 97.60 2.12 90 99.33 99.64 98.47 99.15 0.61

(22) TABLE-US-00008 TABLE 7 Dissolution data for micronized amoxicillin (entry 6 of Table 1) Amoxicillin released Time in solution (%) (min) #1 #2 #3 Mean SD 0 0 0 0 0 0 1 70.98 72.37 68.67 70.68 1.87 3 82.73 76.25 79.68 79.56 3.24 5 84.40 78.46 84.94 82.60 3.59 10 85.63 82.85 88.53 85.67 2.84 15 87.94 85.45 90.95 88.11 2.76 30 96.08 96.66 97.81 96.85 0.88 60 99.56 98.85 99.69 99.37 0.45 90 97.01 99.48 99.78 98.76 1.52

Example 3

Powder Dissolution of Micronized and Non-Micronized Amoxicillin Trihydrate (Visual Determination of Clarity)

(23) Non-micronized amoxicillin trihydrate (3.0 g) was added to a 2 L beaker and drinking water (1000 mL) was added. The mixture was stirred for 2 min at 300±10 rpm at 30±1° C. After two minutes the stirrer was stopped. The same procedure was applied to a sample of micronized amoxicillin trihydrate. Both mixtures were visually observed. The non-micronized sample appeared cloudy whereas the micronized sample appeared clear.