Compositions comprising buprenorphine

[0001] This disclosure relates to a sustained release buprenorphine delivery system for the treatment of conditions alleviated by buprenorphine compounds. The sustained release delivery system includes a free-flowing composition comprising a buprenorphine suspension, its metabolite or a prodrug.

BACKGROUND OF THE INVENTION [0002] Buprenorphine (also known as (2S) -2 - [(-) - (5R, 6R, 7R, 14S) -9a-cyclopropylmethyl-4,5-epoxy-6,14-ethane-3 hydroxy-6-methoxymorphinan-7-yl] -3, 3-di-methyl-butan-2-ol and sold under the tradenames SUBUTEX (TM) and SUBOXONE (TM) to relieve the symptoms of opioid addiction.

[0003] The chemical structure of buprenorphine is shown in formula (1).

HO

H bcH₃

Formula (1) [0004] Buprenorphine is most commonly used to treat the symptoms of opioid addiction for long-term pain relief. Currently, commercial opioid addiction products are SUBUTEX (TM) and SUBOXONE (TM) sold by RB Pharma Inc. These products are in the tablet formulation and are intended to deliver therapeutic levels of buprenorphine over short periods of up to several hours and are usually bucally or sublingually assayed. However, it is necessary for the patient to supplement this dose in

At regular intervals, the problems of distraction in patients with the problem of opioid addiction are frequent. There is therefore a need for a long-term, non-reversible method of administering buprenorphine which provides a stable and effective dose of the active agent to the patient for up to 30 days and which does not result in undesirable accumulation of the residual active agent in the patient's metabolism.

[0005] Various methods of sustained release are used in the pharmaceutical industry, for example, systems such as solid biodegradable sticks or non-degradable tanks. However, they usually require surgical implantation and further, for non-degradable delivery systems, a second surgical operation is required to remove the reservoir.

[0006] WO2007 / 103185 A2 presents dosage forms including buprenorphine drug-releasing suspensions within six days.

[0007] There is a continuous need to develop products that provide increased bioavailability of buprenorphine. In particular, there is a need to develop a sustained release buprenorphine formulation that does not have low bioavailability, poor release kinetics, toxicity at the injection site, relatively large injection volume, and inconveniently short release times.

SUMMARY OF THE INVENTION [0008] The present invention relates to a sustained release buprenorphine delivery system capable of delivering buprenorphine, its metabolite or prodrug over a period of about 7 days to about 1 month. The sustained release buprenorphine delivery system includes a free-flowing composition for sustained release of buprenorphine as defined in the claims. The sustained release buprenorphine delivery system provides release profiles of at least 7 days and up to 30 days, characterized by extremely high bioavailability and minimal risk of permanent tissue damage and usually no risk of muscle necrosis.

[0009] Surprisingly, it was found that such an extended delivery system

This release is achieved by a composition comprising a buprenorphine suspension in water in which buprenorphine is in the form of particles.

[0010] Accordingly, provided according to the first embodiment of the present invention, a composition comprising:

• a suspension of 5-20% by weight buprenorphine in water; and • a polyethylene glycol (PEG) polymer and a nonionic surfactant selected from the group consisting of Tween 20, Tween 80, poloxamers and phospholipids, where:

• buprenorphine is present as a free base (non-protonated), buprenorphine is in the form of particles with an average particle size of less than 50 micrometers; and - the composition does not contain a polylactide polymer or polyglycolide or a mixture thereof.

[0011] Preferably, the average particle size of buprenorphine in the composition as described herein before is less than 40 Pm.

[0012] Particularly preferably, the average particle size of buprenorphine in the composition as described herein before is less than 20 μm, more particularly, preferably less than 10 μm.

[0013] The composition as described hereinbefore includes buprenorphine present as a free base (non-protonated).

[0014] In yet another embodiment, the PEG has a MW of between 1000 and 10,000.

[0015] In a still further embodiment, a composition is provided wherein the surfactant is preferably one or both of Tween 20 or Tween 80.

[0016] In yet another embodiment, a composition is provided according to any one of the preceding claims, further comprising pharmaceutically acceptable salts or sugars for adjusting the tonicity of the composition and / or preservatives, preferably selected from the group consisting of methyl paraben, polyparaben and benzyl alcohol.

[0017] In a second embodiment of the present invention, it is shown

The use of the composition according to the first embodiment in a method of treating a patient from opioid addiction or pain relief, comprising administering said composition parenterally or non-vascular.

[0018] Preferably, the method according to the second embodiment provides treatment of opioid addiction or pain relief for a period of at least 7 days.

[0019] Preferably, the method according to the second embodiment provides treatment of opioid addiction or pain relief for a period of no more than 30 days.

[0020] Particularly preferably, the method of treating a patient according to the second embodiment provides treatment of opioid dependence or pain relief for a period of at least 7 days and no more than 28 days. [0021] Further preferred, the treatment method according to the second embodiment provides treatment of opioid addiction or pain relief for a period of at least 10 days and no more than 17 days.

[0022] Further preferred according to the second embodiment of the invention, there is provided a method of treating a patient comprising an initial dose regimen plus a follow-up dosing at regular intervals of between 7 and 30 days.

[0023] Still preferably according to the second embodiment of the invention, a method is described substantially as described hereinbefore which provides a therapeutically effective dose of buprenorphine, its metabolite or prodrug from about 0.1 to about 10 milligrams (mg) or from about 1 to about about 8 milligrams (mg) per day.

[0024] Even more preferably, according to a second embodiment of the invention, a method is described substantially as described hereinbefore, wherein the dosage achieves a therapeutically effective level of buprenorphine, its metabolite or prodrug, within about one day after administration of the composition; and wherein the therapeutically effective dose of buprenorphine, its metabolite or prodrug is provided for at least about 7 days after administration of the composition or for at least about 30 days after administration of the composition.

[0025] In a third embodiment of the present invention, a method of forming a composition according to the first embodiment of the invention, comprising the steps of:

(a) mixing water with any further optional ingredient (b) adding an opioid agonist together with the disintegrating agent (c) crushing the slurry until the required particle size is achieved [0026] In a preferred method according to the third embodiment of the present invention, the method further comprises a method of final sterilizing the composition, which method comprises the steps of:

(a) filling pharmaceutically acceptable vials or ampoules with the composition and tightly sealing the vials or ampoules;

(b) final sterilization of the vials or ampoules by autoclaving or irradiation (gamma or electron beam).

Examples [0027] Although the ranges and numerical parameters determining the broad scope of the invention are approximated, numerical values ​​set forth in specific examples are set forth as precisely as possible. However, any numerical values ​​inherently contain some errors resulting inevitably from the standard deviation of their individual test measurements.

STUDY ON RATS [0028] Experimental procedures. All preclinical studies were performed on Sprague-Dawley rats. Five rats per test product per time point were injected either intramuscularly or subcutaneously in full anesthesia into the dorsal-pectoral region (DT) with approximately 200 mg of the Test Product described above.

[0029] During the course of the study, the animals were observed for toxicity and any existing local abnormalities were observed, including redness, bleeding, swelling, leakage, bruising, pushing out of the Test Device at the injection site. In addition, the weight of the injection was recorded upon administration, and the body weight was weighed and recorded at the time of delivery and termination. In selected ones

At the time points, fist rats on the Test Product were anesthetized and exsanguinated (approximately 5 ml) by cardiac puncture. The blood was collected into labeled tubes of potassium ethylenediaminetetraacetic acid. Blood was centrifuged for 10 min at 3000 rpm. The plasma fraction was transferred to labeled 5 ml plastic culture tubes and stored at -86 ° C. The rat plasma samples were analyzed for buprenorphine concentration using the procedure described below. After collecting the blood, the rat was sacrificed in a chamber with carbon dioxide. The injection site was dissected and the residual drug and surrounding tissues were carefully removed and placed in a scintillation vial. The vials were stored at -20 ° C for analysis. The collected drug residue / tissue was analyzed for buprenorphine content using the implant analysis method described below.

Buprenorphine analysis in rat plasma samples [0030] This procedure is based on that described by Li-Heng Pao et al., Journal of Chromatography B, 746 (2000), 241-247.

[0031] High performance liquid chromatography had the following conditions: Mobile phase: 80/20 acetonitrile / 5 mM sodium acetate buffer (pH 3.75); flow rate: 1.2 ml / min; temperature of the autosampler: room temperature; column temperature: 25 ° C; detection: fluorescence (excitation at 215 nm and emission at 355 nm); total run time: 14 min; injection volume: 50 μΙ; column: Phenomenex Luna Silica (2) 250 x 4.6mm, 5μm; column storage: 100% acetonitrile; approximate retention time for buprenorphine and internal standard: 7.9 min and 8.7 min.

Implant extraction / analysis procedure [0032] To the vials containing the drug residue / tissue collected, exactly 10 ml of the dissolution solution formulation [90/5/5 acetonitrile / glacial acetic acid / water] was added exactly. The vials were then shaken at about 200 rpm at room temperature on an orbital shaker for at least 2 hours. The vials were then centrifuged at 2500 rpm for 10 minutes. After centrifugation, the vials were carefully removed from the centrifuge. A portion of the supernatant from the vials was transferred to the HPLC vial and, if necessary, the transferred solution in the vial was further diluted using the solution formulation to dissolve to the appropriate concentration for

HPLC analysis.

[0033] High performance liquid chromatography had the following conditions: Mobile phase A: 0.065% sodium octanesulfonic acid and 0.1% trifluoroacetic acid in water, mobile phase B: 90/10 acetonitrile / 0.065% sodium octanesulfonic acid and 0.1% trifluoroacetic acid in the water; flow rate: 1.0 ml / min; temperature of the autosampler: room temperature; column temperature: 30 ° C; detection: 285 waveform (UV); total run time: 21 min; injection volume: 20 μΙ; column: Phenomenex Luna C18 250 x 4.6 mm, 5 μm; column storage: 70/30 acetonitrile / water; each sample was passed according to the following gradient program:

<td>Time</td><td>Mobile phase A</td><td>Mobile phase B</td>

<td>0</td><td>100%</td><td>0%</td>

<td>2</td><td>100%</td><td>0%</td>

<td>16</td><td>20%</td><td>80%</td>

<td>18</td><td>0%</td><td>100%</td>

<td>20</td><td>100%</td><td>0%</td>

<td>21</td><td>100%</td><td>0,00%</td>

approximate retention time of buprenorphine: 15.4 minutes.

Preparation of a standard solution is as follows: a standard stock solution was made by dissolving approximately 10 mg of buprenorphine in 10 ml of a 1: 1 formulation of a solution of dissolution [90/5/5 acetonitrile / glacial acetic acid / water] / H2O. Standard series ranging from 40 ppm to 500 ppm were diluted with water from the original standard solution.

TESTS ON DOGS [0035] Experimental procedures. All preclinical studies in dogs were conducted on beagles with a body weight in the range of 8-12 kg. Six dogs per group were injected subcutaneously in the dorso-thoracic region or intramuscularly in the hind legs with a dose of buprenorphine equivalent to 60 mg per dog. The exact injection dose was obtained by weighing the injection syringe before and after each

Injection. After the injection, the dogs were bled periodically through the jugular vein into EDTA tubes. Plasma samples were then obtained and stored in the freezer at -80 ° C for analysis. Dogs were weighed once a day at blood sampling times. Test sites were evaluated for any abnormalities including redness, bleeding, swelling, leakage, bruising and pushing out of the Test Product on blood collection days. Dogs were also observed after administration in terms of signs of significant toxicity throughout the study period.

Analysis of buprenorphine in plasma samples of dogs [0036] Plasma samples from dog studies were analyzed for buprenorphine and norbuprenorphine levels using the LC-MS-MS method by analytical laboratory service on request. The method was developed and confirmed in the laboratory service on request. In the authorized method, a liquid-liquid extraction step was followed, followed by LC-MSMS analysis.

1. Preparation of an aqueous suspension of the buprenorphine free base [0037] Exactly 3.0 g of PEG3350, 0.2 g of Tween 80 and 0.9 g of sodium chloride were weighed in 100 ml volumetric flasks. Water was added to dissolve and made up to 100 ml. 17.6 g of this aqueous solution was transferred to a 60 ml glass jar, then the free base of buprenorphine (2.4 g) was weighed in this jar. The jar was then placed in about 20 inches of Burundum crushing balls. The jar was closed with a lid and then placed on a spinning mill with a jar at 60 rpm at room temperature for 24 hours. The buprenorphine suspension was then filled with 1 ml glass ampoules. The filled ampoules were sealed and autoclaved at 121 ° C for 15 minutes. The average particle size of the buprenorphine free base [d (0.5)] was measured at 8,

2. Subcutaneous injection of a micronized aqueous suspension of buprenorphine base in rats.

[0038] Formulation: 10% suspension of buprenorphine free base in 3% PEG3350 and 0.2% aqueous solution Tween 80, micronized, subcutaneous injection

EP 2 579 875 B1

0.2 ml of a rat formulation (20 mg bup on rat) (particle size 3.7 μ)

Results:

[0039]

Table 1. Buprenorphine release based on the analysis of the collected implant

<td>Time (day)</td><td>% of the released bup.</td><td>SD</td>

<td>0.083333</td><td>6.0</td><td>4.7</td>

<td>1</td><td>3.2</td><td>3.7</td>

<td>3</td><td>9.6</td><td>4.7</td>

<td>7</td><td>15.4</td><td>3.1</td>

<td>14</td><td>57.6</td><td>15.2</td>

<td>21</td><td>79.0</td><td>20.2</td>

<td>28</td><td>86.8</td><td>10.6</td>

<td>42</td><td>96.4</td><td>5.8</td>

<td>56</td><td>99.4</td><td>1.3</td>

Table 2. Buprenorphine plasma levels

<td>Time (day)</td><td>Bup level (ng / ml)</td><td>SD</td>

<td>0.083333</td><td>23.7</td><td>6.2</td>

<td>1</td><td>22.1</td><td>1.1</td>

<td>3</td><td>18.2</td><td>5.7</td>

<td>7</td><td>32.3</td><td>8.5</td>

<td>14</td><td>61.2</td><td>15.0</td>

<td>21</td><td>40.9</td><td>11.7</td>

<td>28</td><td>20.1</td><td>6.9</td>

<td>42</td><td>3.7</td><td>4.7</td>

<td>56</td><td>0.4</td><td>0.8</td>

Figure 1. Buprenorphine release and plasma levels after SC injection of an aqueous suspension of buprenorphine base in rats

EP 2 579 875 B1

<img file="PL2579875T3_D0001.tif" />

3. Intramuscular injection of a micronized aqueous suspension of the free buprenorphine base in rats.

[0040] Formulation: 13.3% suspension of buprenorphine free base in 3% PEG3350, 0.2% Tween 80 and 0.9% aqueous sodium chloride solution, micronized, intramuscular injection 0.15 ml rat formulation (20 mg bup on rat) (particle size 10.5 μ)

Results:

[0041]

Table 3. Buprenorphine release based on the analysis of the collected implant

<td>Time (day)</td><td>% of the released bup.</td><td>SD</td>

<td>0.083333</td><td>12.3</td><td>9.9</td>

<td>1</td><td>5.9</td><td>3.1</td>

<td>4</td><td>8.0</td><td>1.3</td>

<td>7</td><td>22.7</td><td>3.4</td>

<td>14</td><td>41.9</td><td>18.0</td>

<td>21</td><td>61.0</td><td>16.7</td>

<td>28</td><td>72.8</td><td>12.2</td>

<td>42</td><td>92.0</td><td>13.0</td>

EP 2 579 875 B1

<td>Time (day)</td><td>% of the released bup.</td><td>SD</td>

<td>56</td><td>94.8</td><td>10.5</td>

<td>69</td><td>98.0</td><td>4.0</td>

Table 4. Buprenorphine plasma levels

<td>Time (day)</td><td>Bup level (ng / ml)</td><td>SD</td>

<td>0.083333</td><td>26.3</td><td>17.2</td>

<td>1</td><td>25.6</td><td>6.3</td>

<td>4</td><td>39.7</td><td>15.1</td>

<td>7</td><td>45.5</td><td>13.2</td>

<td>14</td><td>22.6</td><td>10.0</td>

<td>21</td><td>19.5</td><td>8.5</td>

<td>28</td><td>27.4</td><td>8.2</td>

<td>42</td><td>15.3</td><td>8.3</td>

<td>56</td><td>1.8</td><td>1.7</td>

<td>69</td><td>1.3</td><td>1.2</td>

Figure 2. Buprenorphine release and plasma levels after IM injection of an aqueous suspension of buprenorphine free base in rats% released bup / plasma level (ng / ml)

<img file="PL2579875T3_D0002.tif" />

Days

EP 2 579 875 B1

4. Subcutaneous injection of aqueous suspensions of the free micronized buprenorphine / larger particle size, as well as suspensions of buprenorphine hydrochloride in rats (particle size Group I = 4.7μ, Group II = 40.3μ)

formulation:

[0042]

Group I: 10% suspension of free buprenorphine base in 0.2% Tween 80 and 0.9% sodium chloride aqueous solution, micronized, subcutaneous injection 0.20 ml rat formulation (20 mg bup on rat)

Group II: 10% suspension of free buprenorphine base in 0.2% Tween 80 and 0.9% sodium chloride aqueous solution, subcutaneous injection 0.20 ml rat formulation (20 mg bup on rat)

Group III: 10% suspension of buprenorphine hydrochloride in 0.2% Tween 80 and 0.9% aqueous sodium chloride solution, micronized, subcutaneous injection 0.20 ml rat formulation (20 mg bup on rat)

Results:

[0043]

Table 5. Buprenorphine release based on the analysis of the collected implant

<td>Time (day)</td><td>Group I:</td><td>SD</td><td>Group II:</td><td>SD</td><td>Group III:</td><td>SD</td>

<td>0.083333</td><td>0.1</td><td>4.5</td><td>19.6</td><td>7.8</td><td>13.9</td><td>8.5</td>

<td>1</td><td>3.9</td><td>2.1</td><td>1.4</td><td>18.6</td><td>41.4</td><td>3.7</td>

<td>3</td><td>5.1</td><td>1.2</td><td>15.4</td><td>31.4</td><td>20.1</td><td>10.3</td>

<td>7</td><td>20.5</td><td>4.4</td><td>2.6</td><td>10.4</td><td>26.3</td><td>5.4</td>

<td>14</td><td>49.5</td><td>18.0</td><td>37.7</td><td>33.5</td><td>36.7</td><td>8.7</td>

<td>21</td><td>75.0</td><td>17.1</td><td>55.9</td><td>31.2</td><td>49.6</td><td>15.5</td>

<td>28</td><td>93.8</td><td>6.6</td><td>78.5</td><td>22.3</td><td>70.7</td><td>16.6</td>

<td>42</td><td>99.9</td><td>0.1</td><td>96.6</td><td>2.1</td><td>77.0</td><td>21.8</td>

Table 6. Buprenorphine plasma levels

<td>Time (day)</td><td>Group I:</td><td>SD</td><td>Group II:</td><td>SD</td><td>Group III:</td><td>SD</td>

<td>0.083333</td><td>22.7</td><td>5.1</td><td>28.2</td><td>9.2</td><td>55.9</td><td>6.6</td>

<td>1</td><td>27.9</td><td>6.1</td><td>18.9</td><td>8.3</td><td>15.4</td><td>4.3</td>

<td>3</td><td>26.1</td><td>3.9</td><td>17.5</td><td>3.7</td><td>8.9</td><td>1.4</td>

EP 2 579 875 B1

<td>Time (day)</td><td>Group I:</td><td>SD</td><td>Group II:</td><td>SD</td><td>Group III:</td><td>SD</td>

<td>7</td><td>48.3</td><td>11.9</td><td>25.6</td><td>7.5</td><td>9.9</td><td>2.3</td>

<td>14</td><td>31.1</td><td>23.9</td><td>26.1</td><td>4.4</td><td>23.0</td><td>14.4</td>

<td>21</td><td>23.7</td><td>4.2</td><td>17.9</td><td>5.7</td><td>20.3</td><td>8.7</td>

<td>28</td><td>7.6</td><td>4.3</td><td>14.3</td><td>10.4</td><td>16.4</td><td>3.4</td>

<td>42</td><td>1.2</td><td>2.7</td><td>1.0</td><td>1.3</td><td>4.3</td><td>1.4</td>

<img file="PL2579875T3_D0003.tif" />

5. Subcutaneous and intramuscular injection of a micronized aqueous suspension of the free buprenorphine base in dogs) [0044] Formulation: 12% buprenorphine base free suspension in 3% PEG3350, 0.2% Tween 80 and 0.9% aqueous sodium chloride solution, micronized, injection subcutaneous and intramuscular 0.5 ml dog formula (60 mg bup per dog) (particle size 8.3 μ).

Results:

[0045]

Table 7. Buprenorphine plasma levels after sc injection of bup

EP 2 579 875 B1 (Group III)

<td>Day</td><td>BDT8</td><td>TWL5</td><td>SPT8</td><td>TAT8</td><td>WHV8</td><td>XPT8</td><td>Average</td><td>SD</td>

<td>-4</td><td>_*</td><td>0.156</td><td>_*</td><td>_*</td><td>_*</td><td>_*</td><td></td><td></td>

<td>1 hour</td><td>3.44</td><td>5.58</td><td>13.3</td><td>7.66</td><td>10.9</td><td>5.99</td><td>7.81</td><td>3.66</td>

<td>2 hours.</td><td>5.45</td><td>8.06</td><td>19.6</td><td>16.1</td><td>16.1</td><td>8.4</td><td>12.29</td><td>5.70</td>

<td>4 hours</td><td>6.04</td><td>10.1</td><td>18.5</td><td>19.5</td><td>21</td><td>9.28</td><td>14.07</td><td>6.33</td>

<td>8 hours</td><td>3.51</td><td>11.9</td><td>10.6</td><td>12.6</td><td>16.5</td><td>4.25</td><td>9.89</td><td>5.06</td>

<td>1</td><td>6.64</td><td>9.2</td><td>8.3</td><td>11.5</td><td>21.9</td><td>13.1</td><td>11.77</td><td>5.47</td>

<td>3</td><td>2.84</td><td>5.8</td><td>10.9</td><td>16.1</td><td>13.4</td><td>9.4</td><td>9.74</td><td>4.87</td>

<td>7</td><td>2.79</td><td>4.1</td><td>8.62</td><td>11</td><td>8.93</td><td>10.5</td><td>7.66</td><td>3.41</td>

<td>10</td><td>7.07</td><td>2.45</td><td>6.68</td><td>11.5</td><td>15.7</td><td>12.3</td><td>9.28</td><td>4.77</td>

<td>14</td><td>5.08</td><td>1.43</td><td>1.82</td><td>0.723</td><td>0</td><td>1.78</td><td>1.81</td><td>1.75</td>

<td>17</td><td>5.2</td><td>1.41</td><td>1.44</td><td>0.362</td><td>0.104</td><td>0754</td><td>1.55</td><td>1.87</td>

<td>20</td><td>5.64</td><td>1.37</td><td>1.16</td><td>0.295</td><td>_*</td><td>0.456</td><td>1.49</td><td>2.10</td>

<td>24</td><td>4.4</td><td>1.33</td><td>0.75</td><td>0.272</td><td>_*</td><td>_*</td><td>1.13</td><td>1.68</td>

<td>27</td><td>1.63</td><td>1.14</td><td>0.651</td><td>0.176</td><td>_*</td><td>_*</td><td>0.60</td><td>0.67</td>

<td>31</td><td>0.13</td><td>1.06</td><td>0.489</td><td>_*</td><td>_*</td><td>_*</td><td>0.28</td><td>0.43</td>

<td>38</td><td>_*</td><td>0.968</td><td>0322</td><td>_*</td><td>_*</td><td>_*</td><td>0.22</td><td>0.39</td>

<td>45</td><td>0.389</td><td>0.894</td><td>0.354</td><td></td><td></td><td></td><td>0.27</td><td>0.35</td>

<td>52</td><td>_*</td><td>0.767</td><td>0.232</td><td></td><td></td><td></td><td>0.17</td><td>0.31</td>

<td>66</td><td>_*</td><td>0.618</td><td>_*</td><td></td><td></td><td></td><td>0.10</td><td>0.25</td>

Table 8. Buprenorphine plasma levels after IM injection of bupp suspension (Group IV)

<td>Day</td><td>FLV8</td><td>ITV8</td><td>TEV8</td><td>TQT8</td><td>WOT8</td><td>YHT8</td><td>Average</td><td>SD</td>

<td>-4</td><td>0.267</td><td>_*</td><td>_*</td><td>_*</td><td>2.42</td><td>_*</td><td></td><td></td>

<td>1 hour</td><td>2.72</td><td>4.1</td><td>1.99</td><td>4.37</td><td>2.84</td><td>3.17</td><td>3.20</td><td>0.89</td>

<td>2 hours.</td><td>3.73</td><td>5.38</td><td>3.18</td><td>4.15</td><td>2.99</td><td>3.87</td><td>3.88</td><td>0.85</td>

<td>4 hours</td><td>4.23</td><td>6.36</td><td>2.91</td><td>5.62</td><td>3.7</td><td>5.57</td><td>4.73</td><td>1.32</td>

<td>8 hours</td><td>4.54</td><td>4.41</td><td>3.96</td><td>6.13</td><td>3.91</td><td>4.9</td><td>4.64</td><td>0.82</td>

<td>1</td><td>7.14</td><td>8.7</td><td>5.61</td><td>8.22</td><td>5.71</td><td>7.18</td><td>7.09</td><td>1.26</td>

<td>3</td><td>8.07</td><td>8.63</td><td>4.31</td><td>7.3</td><td>4.85</td><td>6.4</td><td>6.59</td><td>1.74</td>

<td>7</td><td>7.54</td><td>7.63</td><td>3.53</td><td>6.81</td><td>4.98</td><td>6.8</td><td>6.22</td><td>1.62</td>

<td>10</td><td>5.93</td><td>4.75</td><td>4.67</td><td>12.7</td><td>8.42</td><td>5.58</td><td>7.01</td><td>3.10</td>

<td>14</td><td>10.2</td><td>4.37</td><td>4.7</td><td>13.2</td><td>9.17</td><td>6.56</td><td>8.03</td><td>3.45</td>

<td>17</td><td>4.11</td><td>3.74</td><td>4.14</td><td>1.34</td><td>4.81</td><td>5.26</td><td>3.90</td><td>1.37</td>

<td>20</td><td>1.53</td><td>2.81</td><td>3.51</td><td>0227</td><td>2.4</td><td>0.506</td><td>1.83</td><td>1.31</td>

EP 2 579 875 B1

<td>Day</td><td>FLV8</td><td>ITV8</td><td>TEV8</td><td>TQT8</td><td>WOT8</td><td>YHT8</td><td>Average</td><td>SD</td>

<td>24</td><td>*</td><td>1.92</td><td>2.32</td><td>*</td><td>*</td><td>*</td><td>0.71</td><td>1.10</td>

<td>27</td><td>*</td><td>1.26</td><td>1.52</td><td>*</td><td>*</td><td>*</td><td>0.46</td><td>0.72</td>

<td>31</td><td>*</td><td>1.2</td><td>0.882</td><td>*</td><td>*</td><td>*</td><td>0.35</td><td>0.55</td>

<td>38</td><td>*</td><td>0797</td><td>0.511</td><td>*</td><td>*</td><td>*</td><td>0.22</td><td>0.35</td>

<td>45</td><td>*</td><td>1.03</td><td>0.159</td><td>*</td><td>*</td><td>*</td><td>0.20</td><td>0.41</td>

<td>52</td><td></td><td>0598</td><td>*</td><td></td><td></td><td></td><td>0.10</td><td>0.24</td>

<td>66</td><td></td><td>0.355</td><td>*</td><td></td><td></td><td></td><td>0.06</td><td>0.14</td>

<td colspan="9">{* = BLQ &lt;0.01}</td>

<img file="PL2579875T3_D0004.tif" />

EP 2 579 875 B1