Patent Publication Number: US-2005129768-A1

Title: Pharmaceutical formulation containing an LTB4-antagonist, as well as processes for the preparation thereof and the use thereof

Description:
The invention relates to a new pharmaceutical formulation containing an LTB 4 -antagonist which comprises a benzamidine group, processes for the preparation thereof and the use thereof as a medicament.  
     BACKGROUND OF THE INVENTION  
      LTB 4 -antagonists which comprise a benzamidine group are compounds with pharmacologically valuable properties. LTB 4 -antagonists may have considerable therapeutic benefit for example in the treatment of arthritis, asthma, chronic obstructive pulmonary disease, psoriasis, ulcerative colitis, Alzheimer&#39;s disease, shock, reperfusion damage/ischaemia, cystic fibrosis, arteriosclerosis and multiple sclerosis.  
      Compounds of this kind are known e.g. from International Patent Applications WO 93/16036, WO 94/11341, WO 96/02497, WO 97/21670, WO 98/11062, WO 98/11119, WO 01/25186, PCT/EP01/00262 and WO 03/07922.  
      These compounds have the chemical structure of formula I:  
                 
 
 wherein 
          A denotes a group of formula 
 
—O—C m H 2m —O-(PHE) n -  (II) 
    wherein 
            m is an integer from 2 to 6, preferably 2 to 5,     n is 0 or 1,     PHE denotes a 1,4-phenylene group optionally substituted by one or two C 1 -C 6 -alkyl groups, preferably a 1,4-phenylene group substituted by a C 2 -C 4 -alkyl group linked to the oxygen in the ortho-position;    
            or     A denotes a group of formula  
                 
            preferably of formula  
                 
 
 wherein 
   
                R 1  denotes H, OH, CN, COR 10 , COOR 10 , or CHO, preferably H or COOR 10 ;     R 2  denotes H, Br, Cl, F, CF 3 , CHF 2 , OH, HSO 3 —O, C 1 -C 6 -alkyl, C 1 -C 6 -alkoxy, C 5 -C 7 -cycloalkyl, CONR 8 R 9 , aryl, O-aryl, CH 2 -aryl, CR 5 R 6 -aryl, or C(CH 3 ) 2 —R 7 , preferably OH, HSO 3 —O, CONR 8 R 9 , or CR 5 R 6 -aryl,     R 3  denotes H, C 1 -C 6 -alkyl, C 1 -C 6 -alkoxy, OH, Cl, or F, preferably H or C 1 -C 3 -alkoxy,     R 4  denotes H or C 1 -C 6 -alkyl, preferably H;     R 5  denotes C 1 -C 4 -alkyl, CF 3 , CH 2 OH, COOH, or COO(C 1 -C 4 -alkyl), preferably C 1 -C 4 -alkyl, particularly methyl;     R 6  denotes H, C 1 -C 4 -alkyl, or CF 3 , preferably C 1 -C 4 -alkyl, particularly methyl;     R 7  denotes CH 2 OH, COOH, COO(C 1 -C 4 -alkyl), CONR 8 R 9 , or CH 2 NR 8 R 9 ;     R 8  denotes H, C 1 -C 6 -alkyl, phenyl, phenyl-(C 1 -C 6 -alkyl), COR 10 , COOR 10 , CHO, CONH 2 , CONHR 10 , SO 2 —(C 1 -C 6 -alkyl), SO 2 -phenyl, while the phenyl group may be mono- or di-substituted by Cl, F, CF 3 , C 1 -C 4 -alkyl, OH and/or C 1 -C 4 -alkoxy, and preferably denotes C 1 -C 4 -alkyl, particularly isopropyl;     R 9  denotes H or C 1 -C 6 -alkyl, preferably H or C 1 -C 4 -alkyl, particularly isopropyl; or     R 8  and R 9  taken together represent a C 4 -C 6 -alkylene group;     R 10  denotes C 1 -C 6 -alkyl, C 5 -C 7 -cycloalkyl, aryl, heteroaryl, aralkyl, or heteroaryl-(C 1 -C 6 -alkyl), preferably C 1 -C 4 -alkyl, 
 
 while the aryl groups mentioned under groups R 2  and R 10  denote phenyl or naphthyl, the heteroaryl groups denote pyrrole, pyrazole, imidazole, furanyl, thienyl, pyridine, or pyrimidine and in each case may be mono- or polysubstituted by Cl, F, CF 3 , C 1 -C 4 -alkyl, OH, HSO 3 —O, or C 1 -C 4 -alkoxy, preferably by OH or HSO 3 —O—. 
   

      The compounds according to formula I have extremely low solubility in water and solubility in the physiological pH range (approx. &lt;0.5 μg/ml) combined with poor wettability. In view of the importance of the LTB 4 -antagonists mentioned above there is therefore a constant need to discover ways of improving the bioavailability and hence efficacy of these compounds. Thus, WO 03/007922 describes how the bioavailability of the active substance may be increased if the active substance is formulated together with a wetting agent.  
      Consequently, an underlying aim of the present invention is to provide a formulation with improved bioavailability for LTB 4 -antagonists, i.e. to develop a formulation which releases an active substance of formula I relatively rapidly and completely and thus leads to an increased bioavailability of this active substance. In addition, an orally administered pharmaceutical formulation is to be provided. A further aim of the present invention is to prepare a formulation which is easily handled during the manufacturing process and thereby allows industrial production in reproducible manner while maintaining a consistently high quality. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       FIG. 1  shows the average plasma concentration of a glucuronide metabolite, on a linear scale, as recorded over 24 hours, after a single dose of the LTB 4  antagonist of formula IA in the form of the base, either as an embedding melt or in the form of a tablet under fasting conditions (parallel groups), according to Example 2;  
       FIG. 2  shows the average plasma concentration of the glucuronide metabolite, on a logarithmic scale, as recorded over 24 hours, after a single dose of the LTB 4  antagonist of formula IA in the form of the base, either as an embedding melt or in the form of a tablet under fasting conditions (parallel groups), according to Example 2;  
       FIG. 3  shows the average plasma concentration of the glucuronide metabolite, on a linear scale, as recorded over 72 hours, after a single dose of the LTB 4  antagonist of formula IA in the form of the base, either as an embedding melt or in the form of a tablet under fasting conditions (parallel groups), according to Example 2;  
       FIG. 4  shows the average plasma concentration of the glucuronide metabolite, on a logarithmic scale, as recorded over 72 hours, after a single dose of the LTB 4  antagonist of formula IA in the form of the base, either as an embedding melt or in the form of a tablet under fasting conditions (parallel groups), according to Example 2;  
       FIG. 5  shows the results of a release test carried out with a suspension containing a ground powder of a pharmaceutical formulation according to Example 3a containing 10% of an LTB 4 -antagonist.  
       FIG. 6  shows the results of a release test carried out with a suspension containing a ground powder of a pharmaceutical formulation according to Example 3b containing 5% of an LTB 4 -antagonist.  
       FIG. 7  shows the results of a release test carried out with a suspension containing a granulated pharmaceutical formulation according to Example 3c containing 10% of an LTB 4 -antagonist.  
       FIG. 8  shows the results of Example 3d, which is a release test carried out with a suspension containing a granulated pharmaceutical formulation according to the present invention containing 15% of an LTB 4 -antagonist.  
       FIG. 9  shows the plasma concentrations of the glucuronide metabolite zwitterion, standardized to a dose of 1 mg/kg, after oral administration to minipigs of various formulations of the LTB 4  antagonist of formula IA in the form of the base according ot Example 5; and  
       FIG. 10  shows the dose-standardized C max  and AUC 0-24 h values of the glucuronide metabolite zwitterion after oral administration to minipigs of various pharmaceutical formulations of the LTB 4  antagonist of formula IA in the form of the base according to Example 5. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
      The problem set out above is solved by the features of claim  1 . This provides a pharmaceutical formulation containing an LTB 4 -antagonist of formula 1, the pharmacologically acceptable acid addition salt, glycoside, O-sulphate, or glucuronide thereof as active substance as well as optionally at least one pharmacologically acceptable excipient and/or carrier, while the active substance is in the form of a solid solution or solid dispersion in a polymer matrix.  
      The active substance of formula I may be present in the pharmaceutical formulation according to the invention in the form of a physiologically acceptable acid addition salt, for example. By physiologically acceptable acid addition salts are meant, according to the invention, pharmacologically acceptable salts which are selected from the salts of hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, acetic acid, fumaric acid, succinic acid, lactic acid, citric acid, tartaric acid and maleic acid. If desired, mixtures of the above-mentioned acids may also be used to prepare the salts. According to the invention, the salts of formula I selected from the group consisting of hydrochloride, hydrobromide, sulphate, phosphate, fumarate, and methanesulphonate are preferred. The salts are particularly preferably selected from the hydrochloride, hydrobromide, and fumarate. The active substance may optionally be present in the form of a hydrate. However, according to the invention, the compound of formula I is preferably in the form of the free base.  
      A particularly preferred compound of formula I is the compound amelubant, i.e. [4-((3-((4-(1-(4-hydroxyphenyl)-1-methylethyl)phenoxy)methyl)benzyl)oxy)benzene-carboximidamide-N-ethylcarboxylate], shown below in formula IA:  
                 
 
      The compounds of formula I wherein R 1  is other than hydrogen are generally prodrugs which are converted in-vivo into the corresponding compounds of formula I wherein R 1  is hydrogen. For example, from the compound IA is formed, in-vivo, the compound of formula IA1:  
                 
 
 wherein X denotes OH, HSO 3 —O, a carbohydrate group of formula C 6 H 11 O 5 —O or a glycosyl group and are metabolites of the above compound. 
 
      The pharmaceutical formulation according to the invention is a solid solution or dispersion of the active substance in the form of an LTB4-antagonist of formula I, particularly of formula IA, in a matrix of one or more polymers. Polymers or polymer mixtures which are particularly suitable within the scope of the invention are hydrophilic or water-soluble polymers, which help to enable the active substance to be formulated as a solid solution/dispersion. By “water-soluble” in this context are meant not only true solutions but also colloidal solutions of the polymer or polymers in water.  
      The polymers used may be, for example, polyethyleneglycols, polypropyleneglycols, cellulose ethers, polyvinylpyrrolidones, polyvinyl acetates, copolymers, and mixtures thereof. Particularly preferred polymers are poloxamers, i.e. known copolymers of polyethyleneglycols and polypropyleneglycols, methylcellulose, ethylcellulose, propylcellulose, carboxymethylcellulose, ethylhydroxyethylcellulose, hydroxypropyl-cellulose, N-vinylpyrrolidone (NVP) homopolymers, such as those sold by BASF under the registered trademark Kollidon®, as well as mixed polymers of polyvinylpyrrolidone and polyvinyl acetate or polyethyleneglycols with various chain lengths. Most particularly preferred are poloxamers, such as for example Poloxamer 188.  
      The pharmaceutical formulation according to the invention optionally contains in addition to the active substance/s and the polymer matrix one or more excipients and/or carriers, such as fillers, binders, disintegrants, break-down agents, flow agents or flow regulators, lubricants, separators, pH correctors, particularly buffers, antioxidants and dyes.  
      Suitable fillers which have proved particularly advantageous for use within the scope of the present invention are carbohydrates such as lactose or mannose, particularly finely divided lactose, or sugar alcohols such as mannitol, sorbitol, or xylitol, particularly mannitol.  
      Binders which are preferred according to the invention are selected from the group consisting of: powdered cellulose, microcrystalline cellulose, sorbitol, starch, polyvinylpyrrolidone (povidone), copolymers of vinylpyrrolidone with other vinyl derivatives (copovidone), cellulose derivatives, particularly methylhydroxypropyl cellulose, e.g. Methocel® A15LV from Dow Chemical Company, and mixtures of these compounds. Preferably, powdered cellulose, particularly microcrystalline cellulose and/or copovidone are present as binders.  
      The pharmaceutical formulation according to the invention may also contain, in addition to the above-mentioned constituents, breakdown agents, which are sometimes also referred to as disintegrants. These are preferably selected according to the invention from the group consisting of sodium starch glycolate, cross-linked polyvinylpyrrolidones (crospovidone), croscarmellose-sodium salt (cellulose-carboxymethylether sodium salt, cross-linked), sodium carboxymethylcellulose, dried maize starch, and mixtures thereof. It is particularly preferred within the scope of the present invention to use sodium starch glycolate, crospovidone and preferably crospovidone or croscarmellose sodium salt.  
      The pharmaceutical formulation according to the invention may contain as further constituents flow agents or flow regulators as well as lubricants. These include within the scope of the present invention for example silicon dioxide, talc, stearic acid, sodium stearylfumarate, magnesium stearate, and glyceroltribehenate. Preferably, magnesium stearate is used according to the invention.  
      In addition, the pharmaceutical formulation according to the invention may contain one or more synthetic or natural pharmaceutically acceptable colorings, preferably indigo carmine.  
      Naturally, other excipients and carriers known to the skilled man may be contained in the pharmaceutical formulation according to the invention. The proportion of excipients and/or carriers, based on the total mass of the formulation, is preferably in the range from about 50 to about 99.5 wt.-%, particularly about 90 to about 99 wt.-%.  
      Based on the total mass of the pharmaceutical formulation according to the invention the compound of formula I, for example of formula IA, according to the invention is preferably present in an amount of up to about 0.5 to about 50 wt.-%, particularly preferably about 0.5 to about 25 wt.-%, particularly about 1 to about 10 wt.-%. Preferably the proportion of free base, based on the total mass of the formulation, is between about 0.5 and about 25 wt.-%, particularly preferably between about 1 and about 10 wt.-%.  
      Preferably, the active substance is used in crystalline, unground form or in ground form, particularly in jet-ground form.  
      Without being tied to a particular theory, it is assumed that the pharmaceutical formulation according to the invention may act as follows:  
      In the new galenic formulation in the form of solid solutions/dispersions provided according to the invention, the polymer matrix initially dissolves after administration when using water-soluble polymers, where “water-soluble” is defined as above. This takes place in the aqueous medium, i.e. in the gastrointestinal tract, leaving behind the active substance which is dissolved or finely divided in the polymer matrix. This dissolves partly in parallel with the dissolution of the polymer matrix and partly afterwards, producing a supersaturated solution. The dissolved concentration of the active substance obtained is higher than is theoretically possible and even achievable in a thermodynamically stable form. Nevertheless, it is surprisingly possible to produce supersaturation. This can be assisted by a suitable choice of excipients and polymers used. Thus, with the invention, the supersaturated solutions/dispersions are stabilized for different lengths of time to the desired extent. Thus, the high concentration of dissolved active substance leads to increased absorption of the active substance in vivo, i.e. the active substance is more available to an organism and can thus develop its activity to a significantly greater extent.  
      This supersaturation of the active substance concentration achieved according to the invention by the specially developed galenic formulation has been demonstrated by release tests in vitro and by bioavailability studies in vivo, as is shown in detail in the Examples.  
      The invention also relates to a process for preparing the pharmaceutical formulation described above comprising the steps of: 
      (1) melting a polymer or a mixture of polymers; 
        (2) dissolving or dispersing an active substance selected from the LTB 4  antagonists of formula I as defined above in the melt;    
        (3a) pouring the melt into suitable moulds and leaving the melt to harden while cooling or     (3b) leaving the solid solution or solid dispersion obtained to harden while cooling and then comminuting the solid solution or solid dispersion obtained into suitable shapes.    

      First, in step (1) according to the invention a melt of one or more polymers is produced. These are the polymers described above in detail. This is followed in step (2) by the dissolving or dispersing of the LTB 4  antagonist active substance of formula I as defined above, e.g. of formula IA, in the molten polymer or polymers. It is particular preferred to use the active substance in step (2) in crystalline, unground form or in ground form particularly in jet-ground form. In addition, it has been found particularly useful to screen the active substance after grinding. Preferably the active substance used then has a mean particle size (D 50) of about 1 μm to about 7 μm, particularly about 1.5 μm to about 3 μm. This was determined by the laser diffractometry method (e.g. Sympatec apparatus using HELOS software, RODOS dry disperser).  
      As soon as the active substance or substances are dissolved or dispersed in the melt, one of two alternative procedures can be followed. Either the melt is poured into suitable moulds and left to harden as they cool (step 3a) or the solid solution or solid dispersion obtained is left to go cold and then cut up into the required shapes (step 3b). Preferably, this is done by grinding but any known technique may be used. Then another screening may be carried out.  
      After cooling, so-called solid solutions are formed, in which the active substance is present in the hardened polymer matrix in molecular dispersion. If the active substance re-crystallizes during cooling or does not totally dissolve in the melt, so-called solid dispersions are formed. The products obtained in step (3a) or (3b) may expediently be processed into tablets, film-coated tablets, sugar-coated tablets, powders, or sachets of powder, or may be packed directly into capsules, such as hard gelatin capsules, for example.  
      The invention also relates to a solid solution or solid dispersion containing an LTB 4  antagonist of formula I as hereinbefore defined, in a polymer matrix.  
      The invention also relates to a solid solution or solid dispersion containing an LTB 4  antagonist of formula IA as hereinbefore defined, in a polymer matrix.  
      Another embodiment of the present invention for preparing the pharmaceutical formulation or the solid solutions or dispersions is preparation using a melt extrusion process, which is also known per se in the art and requires no further explanation.  
      The invention also relates to the use of the pharmaceutical formulation for preparing a pharmaceutical preparation with increased bioavailability for treating or preventing illnesses in which LTB 4  antagonists may be used therapeutically or preventively.  
      In particular, the invention also relates to the use of the pharmaceutical formulation according to the invention for preparing a pharmaceutical preparation for the treatment or prevention of arthritis, asthma, chronic obstructive pulmonary diseases, psoriasis, ulcerative colitis, Alzheimer&#39;s disease, shock, reperfusion injury/ischaemia, cystic fibrosis, arteriosclerosis, and multiple sclerosis.  
      The advantages associated with the present invention are numerous: The invention provides a new galenic formulation which is a solid solution or solid dispersion of an LTB 4  antagonist as active substance in a polymer matrix. This results in an extraordinary improvement in the dissolving characteristics and hence the bioavailability of the active substance, which yields an active substance concentration which is inherently thermodynamically unstable and therefore supersaturated. Nevertheless, the teaching according to the invention makes it possible to prepare a physically and chemically stable formulation. Surprisingly, therefore, it is possible to stabilize supersaturated solutions/dispersions with a high content of active substance, as a result of which the active substance, when released, is more readily available to the body and can develop its activity to a significantly higher degree.  
      This is demonstrated not only by in vitro release tests; it has also been found in tests carried out in vivo as shown, for example, by the bioavailability study and the release tests on mini-pigs in the Examples.  
      The exceedingly favorable stability of the pharmaceutical formulation according to the invention and the excellent bioavailability in humans and animals was unexpected and also unforeseeable.  
      The Examples that follow serve to illustrate the formulations according to the invention. They are intended solely as possible methods described by way of example without restricting the invention to their contents.  
     EXAMPLES  
      In the Examples that follow, the pharmaceutical formulation according to the invention is referred to simply as the “embedding melt” or “Embedding Polymer Melt” and abbreviated to “EPM.” 
     Example 1  
      Preparation of a embedding melt according to the invention containing a 1% charge of active substance  
      Active substance: LTB 4  antagonist of formula IA in the form of the base, amount: 75 mg polymer matrix amount: 7.5 g (1% embedding melt)  
      I. Composition  
      Ingredients  
                                      (01) LTB 4  antagonist of formula IA, base form, jet-ground    75 mg*       (02) Poloxamer 188 Pharm   7425 mg           Total   7500 mg                 *The content of active substance must be determined before weighing             
 
 II. Product Description 
 
      Granules/Powder  
                                                      Appearance:   fine, white, waxy powder           Particle size:   ≦500 μm           Color:   white           Fill volume:   7.5 g                      
 
 III. Manufacturing Process 
 
 1 Batch=606 g 
 
      1. Melting the Poloxamer 188 Pharm  
      650,000 g of Poloxamer 188 Pharm (02) are melted at 80° C. in a crystallizing dish of Ø190 mm in a vacuum drying cupboard at an absolute pressure of 100 to 200 mbar over a period of 4 hours.  
      2. Screening the Active Substance  
      6.0606 g of the LTB 4  antagonist of formula IA in the form of the base (01) are jet-ground and screened in an analytical screen with a mesh size of 800 μm.  
      3. Preparing the Embedding Melt  
      The laboratory reactor is preheated for approx. 30 min at a water bath temperature of 90° C. The laboratory reactor is filled with 600,0000 g of Poloxamer 188 Pharm (02) (liquid). The anchor stirrer is set to 20 rpm, the direction of rotation is set to the right and an absolute pressure of 100 to 200 mbar is applied. After 5 min., the reactor is opened and, within 5 min., all the LTB 4  antagonist of formula IA in the form of the base (01) in jet-ground form is added to the laboratory reactor in one go (6.0606 g) and the reactor is closed. The anchor stirrer is set to 20 rpm and again the direction of rotation is set to the right. 3 min after the addition of active substance the absolute pressure is set at 100 to 200 mbar. 5 min after the addition of active substance the anchor stirrer speed is increased to 100 rpm. 10 min after the addition of active substance the anchor stirrer is set to rotate to the left. From the 15th to the 20th minute the laboratory reactor is opened and any residues of active substance on the anchor stirrer, temperature sensor and glass wall are wiped off and returned to the melt. The laboratory reactor is closed again, an absolute pressure of 100 to 200 mbar is applied, the speed is left at 100 rpm, and the anchor stirrer is set to rotate to the right. 20 min after the addition of active substance the temperature setting of the water bath is returned to 86° C. 25 min after the addition of active substance the anchor stirrer is set to rotate to the left. Then, 35 min after the addition of active substance, the anchor stirrer is set to rotate to the right again. 40 min after the addition of active substance the anchor stirrer speed is set to 20 rpm. 60 min after the addition of active substance the laboratory reactor is opened and the embedding melt is poured out in a thin layer on a sheet of glass or stainless steel.  
      Process/Equipment Data:  
                                      Laboratory reactor:   IKA Laboratory reactor system LR-A 1000       Stirrer:   IKA stirrer       drive:   RE 162 A       control:   RE 162 P Analog       Circulating thermostat:   mgw Lauda C3       type:   T 1                  
 
      4. Hardening  
      The embedding melt is poured out in a thin layer on a sheet of glass or stainless steel (layer thickness approx. 1.5 to 2.5 mm) and left to harden. The hardening time is approx. 2 to 3 h. The hardened embedding melt is scraped off from the sheet of glass or stainless steel using a dough scraper and stored in a brown wide-necked flask.  
      5. Grinding and Screening  
      The individual flakes are ground up using a water-cooled IKA Universal Mill and the ground material is screened using a 500 μm Kressner screen. The grinding and screening process is repeated until all the embedding melt has been ground to ≦500 μm.  
      Process/Equipment Data:  
                                                      Mill:   IKA-cross-beater/universal mill           Type:   M 20           Grinding time:   3 × approx. 5 sec/filling                      
 
      6. Filling  
      Under GMP conditions (Good Manufacturing Practice) the granulated material is packed into the sterile-packaged glass flasks and the pilfer-proof closure is sealed using a PfP flanging machine.  
                                                      Contents:   7.500 g           Tolerance during filling   7.470 g to 7.530 g                      
 
 IV. In-Process Controls 
 
      1. Liquid Embedding Melt  
      About 30 to 35 min after the addition of active substance it is advantageous if the active substance is dissolved. From this time onwards, the embedding melt is clear. No undissolved flakes should be visible in the embedding melt shortly before the emptying of the laboratory reactor.  
      2. Solid Embedding Melt  
      As the embedding melt hardens its appearance changes from a clear liquid to a waxy white substance.  
      3. Granules/Powder  
      Fine particles, white, particle size ≦500 μm.  
      The correct particle size is achieved when everything passes through the 500 μm screen.  
      4. Fill quantity  
      The masses packed are between 7.470 g and 7.530 g.  
     Example 2  
      The embedding melt prepared in Example 1 containing a 1% charge of the active substance (75 mg of the LTB 4  antagonist of formula IA in the form of the base) was tested on humans. The control used was a tablet which also contained 75 mg of the LTB 4  antagonist of formula IA in the form of the base. The plasma concentration of the glucuronide metabolite was determined. The glucuronide metabolite is the LTB 4  antagonist of formula IA in the form of the base, whose structure was explained in Example 1, except that the ethylcarboxyl group has been cleaved (═N—CO 2 C 2 H 5 →═NH) and at the same time the hydroxy group has been glycosylated at the “left-hand” phenyl ring (LTB 4  antagonist of formula IA1). The glucuronide metabolite is present as a zwitterion. The LTB 4  antagonist of formula IA in the form of the base is converted into the glucuronide metabolite in the human body, as explained, and constitutes an active metabolite thereof.  
      The results are shown in FIGS.  1  to  4 . FIGS.  1  to  4  show the average plasma concentration of the glucuronide metabolite, recorded against time, after a single dose of 75 mg of the LTB 4  antagonist of formula IA in the form of the base, either as a embedding melt according to the invention (EPM) or in the form of a WIF tablet (wettability improved formulation, a formulation corresponding to the prior art according to WO 03/007922, containing a wetting agent)—under fasting conditions (parallel groups), at different times in each case.  
      The tests on humans clearly show that the plasma concentration of the formulation according to the invention exceeds that of a conventional tablet with the same active substance in the same dosage, i.e. the quantity of active substance available is many times greater with the formulation according to the invention than with a conventional tablet.  
     Example 3  
      Release results of embedding melts containing different loads of active substance Examples 3a) and 3b) that follow relate to a suspension of the EPM powder in water. Release tests were then carried out with this suspension. The results are shown in  FIGS. 5 and 6 . On the one hand, the amount of the LTB 4  antagonist of formula IA in the form of the base released (“unfiltered”) and, on the other hand, the colloidally dissolved fraction with particle sizes of less than 220 nm, which are marked as “filtered” in  FIGS. 5 and 6  were evaluated.  
      On the one hand, the total amount of active substance released by the pharmaceutical preparation was shown. The release curves normally used for characterizing solid oral preparations were used for this.  
      On the other hand, the pharmaceutical fractions which cannot be filtered off using a 0.22 μm filter are also shown. Their particle size is therefore less than 220 nm. Thus, the active substance is dissolved or at least colloidally dissolved, i.e. very finely divided, even if these concentrations are far higher than the saturation concentrations of the active substance. This supersaturation is maintained for a certain length of time before the active substance crystallizes and can therefore be removed by filtration, i.e. has a particle size &gt;220 nm. The other Examples 3c) and 3d) that follow relate to release tests on EPM powder packed into hard gelatin capsules. Here again, both the total quantity of the LTB 4  antagonist of formula IA in the form of the base released and also the colloidally dissolved fraction were investigated.  
      3a) EPM Ground—10% Active Substance Charge  
     
         
         
           
              (suspended) 
 
 Composition EPM 10%: 
 
 67.41 g of the LTB 4  antagonist of formula IA in the form of the base 
 
 600 g Poloxamer  188  Pharm (polymer matrix) 
 
 Preparation: In the beaker 
 
 Release of 499.8 mg of EPM, particle size &lt;0.5 mm, suspended in 5 mL of de-ionized water, added after 5 minutes to the release medium 
 
 Release medium: 500 mL of 0.1 N HCl with 50 mg of Methocel A 15 LV 
 
           
         
       
    
      The results are shown in  FIG. 5 .  
      3b) EPM Ground—5% Active Substance Charge  
     
         
         
           
              (suspended) 
 
 Composition EPM 5%: 
 
 31.9369 g the LTB 4  antagonist of formula IA in the form of the base 600.09 g Poloxamer 188 Pharm 
 
 Preparation: In the laboratory reactor 
 
 Release of 1000.2 mg of EPM, particle size &lt;0.5 mm, suspended in 5 mL of de-ionized water, added after 5 minutes to the release medium 
 
 Release medium: 500 mL of 0.1 N HCl with 50 mg of Methocel® A 15 LV 
 
           
         
       
    
      The results are shown in  FIG. 6 .  
      3c) Capsule Containing Granulated EPM—10% Active Substance Charge  
     
         
          Composition EPM 10%:  
          1002.4 mg the LTB 4  antagonist of formula IA in the form of the base 9021.8 mg Poloxamer  188  Pharm  
          Capsule filled with 300 mg EPM, particle size 0.5-0.8 mm  
          Release medium: 400 mL of 0.1 N HCl with 20 mg Methocel® A15LV  
       
    
      The results are shown in  FIG. 7 .  
      3d) Capsule with EPM Granules—15% Active Substance Charge  
     
         
          Composition EPM 15%:  
          4.5037 g the LTB 4  antagonist of formula IA in the form of the base 25.5067 g Poloxamer 188 Pharm  
          Capsule filled with 332.7 mg of EPM, particle size &lt;0.5 mm  
          Release medium: 500 mL of 0.1 N HCl with 50 mg Methocel® A15LV  
       
    
      The results are shown in  FIG. 8 .  
     Example 4  
      Preparation of an embedding melt according to the invention with a 10% active substance charge:  
      active substance: the LTB 4  antagonist of formula IA in the form of the base, amount: 75 mg amount of polymer matrix: 0.750 g (10% embedding melt)  
      I. Composition  
      Ingredients  
                                                      (01) The LTB 4  antagonist of formula IA    75 mg*               base form, jet-ground 98.9%           (02) Poloxamer 188 Pharm   675 mg               TOTAL   750 mg                         *The active substance content must be determined before weighing out             
 
 II. Product Description 
 
      Granules/Powder  
                                                      Appearance:   fine, white, slightly waxy powder           Particle size:   ≦500 μm           Color:   White           Fill volume:   750 mg                      
 
 III. Production Method 
 
 1 Batch=667 g=89 Bottles 
 
      1. Melt the Poloxamer 188 Pharm  
      As the melting poloxamer in the laboratory reactor would overload the anchor stirrer, the poloxamer is melted separately. In a crystallizing dish of Ø 190 mm 650,000 g Poloxamer 188 Pharm (02) are melted at 80° C. in a vacuum drying cupboard at an absolute pressure of 100 to 200 mbar over a period of 4 hours.  
      2. Screen the Active Substance  
      66.6667 g of the LTB 4  antagonist of formula IA in the form of the base (01) are jet-ground and screened in an analytical screen with a mesh size of 800 μm.  
      3. Prepare the Embedding Melt  
      The laboratory reactor is preheated for approx. 30 min at a water bath temperature of 90° C. The laboratory reactor is filled with 600,0000 g of Poloxamer 188 Pharm (02) (liquid). The anchor stirrer is set to 20 rpm, the direction of rotation is set to the right, and an absolute pressure of 100 to 200 mbar is applied. After 5 min., the reactor is opened and, within 5 min., all the LTB 4  antagonist of formula IA in the form of the base in jet-ground form is added to the laboratory reactor in one go (66.6667 g), and the reactor is closed. The anchor stirrer is set to 20 rpm and again the direction of rotation is set to the right. 3 min after the addition of active substance the absolute pressure is set at 100 to 200 mbar. 5 min after the addition of active substance the anchor stirrer speed is increased to 100 rpm. 10 min after the addition of active substance the anchor stirrer is set to rotate to the left. From the 15th to the 20th minute the laboratory reactor is opened and any residues of active substance on the anchor stirrer, temperature sensor and glass wall are wiped off and returned to the melt. The laboratory reactor is closed, an absolute pressure of 100 to 200 mbar is applied, the speed is left at 100 rpm, and the anchor stirrer is set to rotate to the right. 25 min after the addition of active substance the anchor stirrer is set to rotate to the left. 35 min after the addition of active substance, the water bath temperature setting is returned to 86° C. and the direction of rotation of the anchor stirrer is set to the right again. 40 min after the addition of active substance the anchor stirrer speed is set to 20 rpm. 60 min after the addition of active substance the laboratory reactor is opened and the embedding melt is poured out in a thin layer on a sheet of glass or stainless steel.  
      Process/Equipment Data (as in Example 1)  
      4. Hardening  
      The embedding melt is poured out in a thin layer on a sheet of glass or stainless steel (layer thickness approx. 1.5 to 2.5 mm) and left to harden. The hardening time is approx. 2 to 3 h. The hardened embedding melt is scraped off from the sheet of glass or stainless steel using a dough scraper and stored in a brown wide-necked flask.  
      5. Grinding and screening  
      The individual flakes are ground up using a water-cooled IKA Universal Mill and the ground material is screened using a 500 μm Kressner screen. The grinding and screening process is repeated until all the embedding melt has been ground to ≦500 μm.  
      Process/Equipment Data (as in Example 1)  
      6. Filling  
      Under GMP conditions, the granulated material is packed into the sterile-packaged glass flasks and the pilfer-proof closure is sealed using a PfP flanging machine. 
          Contents: 750 mg     Tolerance during filling 745 mg to 755 mg 
 
 IV. In-Process Controls (see Example 1) 
       

     Example 5  
      The embedding melts according to the invention containing 5 and 10% active substance charge, as prepared in Example 4, were tested on minipigs. A bioavailability study was carried out, the results of which are shown in  FIGS. 9 and 10 .  
      a) Summary  
      A relative bioavailability study with different formulations of the LTB 4  antagonist of formula IA in the form of the base (5% EPM, 10% EPM, and tablet) was carried out after oral administration to minipigs. Two EPM formulations, one with a 5% active substance charge, and one with a 10% active substance charge of the LTB 4  antagonist of formula IA in the form of the base, were examined.  
      b) Objective  
      The aim is to study the absorption of various pharmaceutical formulations of the LTB 4  antagonist of formula IA in the form of the base on minipigs.  
      c) Method  
      c1) Animal Tests  
      Two EPM formulations were used, one containing 5% of the LTB 4  antagonist of formula IA in the form of the base and one containing 10% of the LTB 4  antagonist of formula IA in the form of the base. The EPMs were stored in glass containers and suspended in 50 ml of tap water immediately before use. After the dose had been administered, the flasks were washed once with another 50 ml of tap water which was also given to the animals.  
      6 tablets containing 75 mg of the LTB 4  antagonist of formula IA in the form of the base were each placed in a capsule (size 000). After the capsules had been administered, the container was rinsed with 50 ml of tap water. The food was provided 3 to 4 hours after administration, except in the case of group 2, where the food was given 15 minutes after administration.  
                                   TABLE 1                       group   formulation   dose [mg]   sex   subject   weight [kg]                                                        1   EPM 10%   300   M   53918   19.3               300   M   53879   31.5               300   F   53825   36.0               300   F   53837   24.0       2   Tablet in capsule/food   1350   M   51853   27.3           15 min after taking   900   M   51726   28.4               1350   F   51826   29.9               900   F   51830   34.1       3   EPM 5%   300   M   53918   20.1               300   M   53879   31.6               300   F   53825   35.4               300   F   53837   24.9       4   Tablet/food   1350   M   51853   30.1           4 h after taking   1350   M   51726   31.9               1350   F   51826   30.0               1350   F   51830   33.5                 M . . . male            F . . . female             
 
 c2) Bioanalytical Parameters 
 
      The plasma concentrations of glucuronide metabolite in zwitterion form were quantified by HPLC-MS/MS.  
      d) Results  
      The dose-standardized concentrations of the glucuronide metabolite zwitterion, the individual and average dose-standardized AUC O-24 h and C max  values and the t max  values are shown in section f). In addition, the pharmacokinetic parameters are shown in Table 2.  
      The results are also shown in  FIGS. 9 and 10 .  FIG. 9  shows the plasma concentrations of the glucuronide metabolite zwitterion, standardized to a dose of 1 mg/kg, after oral administration to minipigs of various formulations of the LTB 4  antagonist of formula IA in the form of the base.  FIG. 10  shows the dose-standardized C max  and AUC 0-24h  values of the glucuronide metabolite zwitterion after oral administration to minipigs of various pharmaceutical formulations of the LTB 4  antagonist of formula IA in the form of the base.  
               TABLE 2                          Summary of the pharmacokinetic parameters                                 AUC 0-24 h     C max     t max                                                                   g mean   gCV       g mean   gCV   mean   range       group   formulation   N   [ng · h/ml]/dose   [%]   N   [ng/ml]/dose   [%]   [h]   [h]                                                             1   EPM (10%)   4   320   78   4   31   98   1.5   1-2       3   EPM (5%)   4   630   57   4   56   84   3   1-4       2   tablet + food   2   390   57   4   16   190   14    2-24       4   tablet   4   807   63   4   66   75   8   4-8                 N . . . number of experimental animals            AUC 0-24 h  . . . Area under the curve from 0 to 24 hours            C max  . . . maximum plasma concentration            t max  . . . time to reach maximum plasma level            g mean . . . geometric mean value            g CV . . . geometric standard deviation            mean . . . mean value             
 
      Variability: The inter-individual variability of the plasma concentrations was high. Differences in release were observed between male and female minipigs. However, the differences between the formulation groups were not uniform.  
      EPM: The dose-standardized AUC 0-24 h and C max  values of the EPM formulations which contained 5% the LTB 4  antagonist of formula IA in the form of the base were about twice as great as the corresponding values of the EPM formulations which contained 10% the LTB 4  antagonist of formula IA in the form of the base.  
      Tablet: Oral administration of the tablets also led to a substantial release in the animals. It should be pointed out that this formulation was tested on different animals from the EPM. A direct comparison of the results might possibly be misleading on account of the high inter-individual variability. However, the AUC O-24 h and C max  values were comparable with the 5% EPM values. T max  was significantly delayed compared with the EPM formulations.  
      a) Conclusions  
      The differences in release between male and female minipigs were not consistent between the different formulation groups and are therefore due to inter-individual variability rather than actual gender differences.  
      It was shown that the ratio of the LTB 4  antagonist of formula IA in the form of the base to Pluronics (=Poloxamer, polymer matrix) affects the release of the glucuronide metabolite zwitterion in the animals. A lower charging of the EPM with the active substance the LTB 4  antagonist of formula IA in the form of the base, i.e. a higher amount of Pluronics (polymer matrix), led to higher the glucuronide metabolite zwitterion plasma concentrations.  
      With a more conventional formulation, i.e., a tablet of the LTB 4  antagonist of formula IA in the form of the base, systemic release was also achieved in the animals, which was comparable with the release using EPM with a 5% charge of active substance.  
      f) Summary of the Values Measured  
               TABLE 3                       Dose-standardized plasma concentration of the glucuronide metabolite       zwitterion, after oral administration of 10 mg/kg or 1350 mg (tablet)/minipig, LTB4       antagonist of formula IA in the form of the base                  group 1, dose-standardized                                                     time   53918   53879   53825   53837       mean       g-mean           [h]   M   M   F   F   N   [ng/mL]   CV (%)   [ng/mL]   gCV (%)               1   11.23   25.35   91.25   10.24   4   34.52   111.4   22.71   133.7       2   12.69   18.17   68.17   30.40   4   32.36   77.3   26.29   83.8       4   8.000   15.25   71.530   29.34   4   31.03   91.6   22.49   118.4       8   7.626   11.63   44.920   14.44   4   19.65   86.9   15.49   88.1       24    4.285   5.068   7.002   4.899   4   5.314   22.1   5.224   21.1                         group 2, dose-standardized                                                     time   51853   51726   51828   51830       mean       g-mean           [h]   M   M   F   F   N   [ng/mL]   CV (%)   [ng/mL]   gCV (%)               1   6.822   13.45   2.500   2.500   4   6.318   81.9   4.894   98.5       2   23.96   65.46   0.4588   2.779   4   23.16   130.0   6.687   1170.6       4   20.530   74.07   2.324   5.155   4   25.52   130.6   11.62   305.5       8   7.945   21.17   4.317   3.456   4   9.222   88.9   7.078   96.4       24    11.150   7.599   5.049   6.822   4   7.655   33.5   7.350   33.6                         group 3, dose-standardized                                                     time   53918   53879   53825   53837       mean       g-mean           [h]   M   M   F   F   N   [ng/mL]   CV (%)   [ng/mL]   gCV (%)               1   13.81   21.75   132.1   58.30   4   56.49   95.6   39.00   133.5       2   22.44   34.53   117.6   70.53   4   61.28   69.8   50.35   84.9       4   23.32   46.73   78.79   47.63   4   49.12   46.3   44.97   53.3       8   18.85   42.54   73.33   26.90   4   40.41   59.5   35.46   64.2       24    7.309   4.525   14.58   8.933   4   8.837   48.0   8.101   51.5                         group 4, dose-standardized                                                     time   51853   51726   51828   51830       mean       g-mean           [h]   M   M   F   F   N   [ng/mL]   CV (%)   [ng/mL]   gCV (%)               1   11.48   3.603   0.7784   2.397   4   4.565   104.1   2.964   156.1       2   49.76   25.41   4.153   8.504   4   21.96   94.2   14.54   155.4       4   87.68   94.21   25.40   13.69   4   55.25   75.3   41.17   120.9       8   95.59   82.88   86.38   24.13   4   72.25   45.0   63.75   72.6       24    13.20   19.71   10.50   9.372   4   13.20   35.1   12.65   33.7                 N . . . number of experimental animals            CV . . . standard deviation (coefficient of variation)            gCV . . . geometric standard deviation            g mean . . . geometric mean value            mean . . . mean value             
 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                   
               
               
                 Individual and average dose-standardized AUC 0-24 h  of the glucuronide 
               
               
                 metabolite zwitterion, after oral administration to minipigs of different formulations of 
               
               
                 the LTB4 antagonist of formula IA in the form of the base. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 dose 
                   
                   
                 AUC (0-24 h) /dose 
                   
                 mean 
                 CV 
                 g-mean 
                 gCV 
               
               
                 group 
                 [mg/kg] 
                 sex 
                 subject 
                 [ng * h/mL] 
                 N 
                 [ng * h/mL] 
                 [%] 
                 [ng * h/mL] 
                 [%] 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 15.5 
                 M 
                 53918 
                 161.9 
                   
                   
                   
                   
                   
               
               
                 1 
                 9.5 
                 M 
                 53879 
                 247.4 
                 2 
                 204.6 
                 29.6 
                 200.1 
                 30.7 
               
               
                 1 
                 8.3 
                 F 
                 53825 
                 819.6 
               
               
                 1 
                 12.5 
                 F 
                 53837 
                 310.5 
                 2 
                 565.1 
                 63.7 
                 504.4 
                 77.6 
               
               
                   
                   
                 M&amp;F 
                   
                   
                 4 
                 384.8 
                 77.0 
                 317.7 
                 77.7 
               
               
                 2 
                 49.5 
                 M 
                 51853 
                 267.5 
               
               
                 2 
                 31.7 
                 M 
                 51726 
                 566.6 
                 2 
                 417.1 
                 50.7 
                 389.3 
                 57.0 
               
               
                 2 
                 45.1 
                 F 
                 51828 
                 NA 
               
               
                 2 
                 26.4 
                 F 
                 51830 
                 NA 
               
               
                   
                   
                 M&amp;F 
                   
                   
                 2 
                 417.1 
                 50.7 
                 389.3 
                 57.0 
               
               
                 3 
                 14.9 
                 M 
                 53918 
                 349.7 
               
               
                 3 
                 9.7 
                 M 
                 53879 
                 570.1 
                 2 
                 459.9 
                 33.9 
                 446.5 
                 35.6 
               
               
                 3 
                 8.5 
                 F 
                 53825 
                 1271 
               
               
                 3 
                 12 
                 F 
                 53837 
                 616.1 
                 2 
                 943.6 
                 49.1 
                 884.8 
                 54.7 
               
               
                   
                   
                 M&amp;F 
                   
                   
                 4 
                 701.7 
                 56.6 
                 628.6 
                 57.2 
               
               
                 4 
                 44.9 
                 M 
                 51853 
                 1206 
               
               
                 4 
                 42.3 
                 M 
                 51726 
                 1193 
                 2 
                 1199.5 
                 0.8 
                 1199 
                 0.8 
               
               
                 4 
                 45 
                 F 
                 51828 
                 832.1 
               
               
                 4 
                 40.3 
                 F 
                 51830 
                 354.2 
                 2 
                 593.1 
                 57.0 
                 542.9 
                 66.3 
               
               
                   
                   
                 M&amp;F 
                   
                   
                 4 
                 896.3 
                 44.7 
                 807.0 
                 62.7 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                   
               
               
                 Individual and average dose-standardized C max  of of the glucuronide 
               
               
                 metabolite zwitterion, after oral administration to minipigs of different formulations of 
               
               
                 the LTB4 antagonist of formula IA in the form of the base. 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 dose 
                   
                   
                 C(max)/dose 
                   
                 mean 
                 CV 
                 g-mean 
                 gCV 
               
               
                 group 
                 [mg/kg] 
                 sex 
                 subject 
                 [ng/mL] 
                 N 
                 [ng * h/mL] 
                 [%] 
                 [ng * h/mL] 
                 [%] 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 15.5 
                 M 
                 53918 
                 12.69 
                   
                   
                   
                   
                   
               
               
                 1 
                 9.5 
                 M 
                 53879 
                 25.35 
                 2 
                 19.02 
                 47.1 
                 17.94 
                 52.0 
               
               
                 1 
                 8.3 
                 F 
                 53825 
                 91.25 
               
               
                 1 
                 12.5 
                 F 
                 53837 
                 30.40 
                 2 
                 60.83 
                 70.7 
                 52.67 
                 91.1 
               
               
                   
                   
                 M&amp;F 
                   
                   
                 4 
                 39.92 
                 87.7 
                 30.74 
                 97.5 
               
               
                 2 
                 49.5 
                 M 
                 51853 
                 23.96 
               
               
                 2 
                 31.7 
                 M 
                 51726 
                 74.07 
                 2 
                 49.02 
                 72.3 
                 42.13 
                 94.4 
               
               
                 2 
                 45.1 
                 F 
                 51828 
                 5.049 
               
               
                 2 
                 26.4 
                 F 
                 51830 
                 6.822 
                 2 
                 5.936 
                 21.1 
                 5.869 
                 21.5 
               
               
                   
                   
                 M&amp;F 
                   
                   
                 4 
                 27.48 
                 117.2 
                 15.72 
                 189.3 
               
               
                 3 
                 14.9 
                 M 
                 53918 
                 23.32 
               
               
                 3 
                 9.7 
                 M 
                 53879 
                 46.73 
                 2 
                 35.03 
                 47.3 
                 33.01 
                 52.3 
               
               
                 3 
                 8.5 
                 F 
                 53825 
                 132.1 
               
               
                 3 
                 12 
                 F 
                 53837 
                 70.53 
                 2 
                 101.3 
                 43.0 
                 96.52 
                 46.6 
               
               
                   
                   
                 M&amp;F 
                   
                   
                 4 
                 68.17 
                 68.6 
                 56.45 
                 83.6 
               
               
                 4 
                 44.9 
                 M 
                 51853 
                 95.59 
               
               
                 4 
                 42.3 
                 M 
                 51726 
                 94.21 
                 2 
                 94.90 
                 1.0 
                 94.90 
                 1.0 
               
               
                 4 
                 45 
                 F 
                 51828 
                 86.38 
               
               
                 4 
                 40.3 
                 F 
                 51830 
                 24.13 
                 2 
                 55.26 
                 79.7 
                 45.65 
                 112.0 
               
               
                   
                   
                 M&amp;F 
                   
                   
                 4 
                 75.08 
                 45.6 
                 65.82 
                 75.3 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                   
               
               
                 Individual and average dose-standardized t max  of of the 
               
               
                 glucuronide metabolite zwitterion, after oral administration to 
               
               
                 minipigs of different formulations of the LTB4 antagonist 
               
               
                 of formula IA in the form of the base. 
               
               
                 t max   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 dose 
                   
                   
                 mean 
                 range 
               
               
                   
                 group 
                 [mg/kg] 
                 sex 
                 N 
                 [h] 
                 [h] 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                 1 
                  9.5-15.5 
                 M 
                 2 
                 1.5 
                 1-2 
               
               
                   
                 1 
                  8.3-12.5 
                 F 
                 2 
                 1.5 
                 1-2 
               
               
                   
                 1 
                  8.3-15.5 
                 M&amp;F 
                 4 
                 1.5 
                 1-2 
               
               
                   
                 2 
                 31.7-49.5 
                 M 
                 2 
                 3 
                 2-4 
               
               
                   
                 2 
                 26.4-45.1 
                 F 
                 2 
                 24 
                 24  
               
               
                   
                 2 
                 26.4-49.5 
                 M&amp;F 
                 4 
                 14 
                  2-24 
               
               
                   
                 3 
                  9.7-14.9 
                 M 
                 2 
                 4 
                 4 
               
               
                   
                 3 
                  8.5-12.0 
                 F 
                 2 
                 1.5 
                 1-2 
               
               
                   
                 3 
                  8.5-14.9 
                 M&amp;F 
                 4 
                 3 
                 1-4 
               
               
                   
                 4 
                 42.3-44.9 
                 M 
                 2 
                 6 
                 4-8 
               
               
                   
                 4 
                 40.3-45.0 
                 F 
                 2 
                 8 
                 8 
               
               
                   
                 4 
                 40.3-44.9 
                 M&amp;F 
                 4 
                 8 
                 4-8 
               
               
                   
                   
               
               
                   
                   M . . . male    
               
               
                   
                   F . . . female