HK1091743B - Semi-solid formulations for the oral administration of taxoids - Google Patents

Description

Semisolid formulations for oral administration of taxanes

The present invention relates to oral formulations of taxanes (taxoids).

The taxanes used in the formulations of the invention are preferably of formula (I),

wherein

R₁Is hydrogen, (C)₂-C₄) Acyl, (C)₁-C₃) An alkyl group, a carboxyl group,

R₂is OH, alkoxy, or R₂And R₃Is a methylene group, and is a linear or branched chain,

R₃is CH₃Or R is₂And R₃Is a methylene group, and is a linear or branched chain,

R₄is OCOCH₃Or OCOOCH₃，

R is phenyl or (C)₃-C₄) Alkoxy or (C)₃-C₄) Alkenyloxy, preferably phenyl or tert-butoxy, R' is aryl, preferably phenyl, optionally substituted, or (C)₂-C₄) Alkyl or (C)₂-C₄)alkylen。

The taxanes used in the formulations of the present invention are, for example, the taxanes of the following formulae (Ia) to (If).

Formula Ia: (Docetaxel/Docetaxel)

Formula Ib:

formula Ic:

formula Id: (Paclitaxel/Paclitaxel)

Formula Ie:

formula If:

taxanes of the general formulae (Ia) to (If) and their use are known, these taxanes being particularly advantageous for their use as chemotherapeutic agents.

Unfortunately, taxanes are poorly water soluble compounds. These molecules are weakly lipophilic and have relatively high molecular weights. To date, taxanes have been administered intravenously, especially using formulations consisting of high levels of PS80 or cremophor. The present invention aims to develop taxane preparations for oral administration.

Oral administration of PS80 or cremophor formulations of taxanes results in very low bioavailability in animals such as dogs, which is probably responsible for high metabolic rates. Furthermore, formulations consisting of high levels of PS80 (e.g. less than 40mg taxanes per gram of PS80) are not suitable for oral administration due to the potential toxicity of PS80 in contact with the intestinal mucosa. Moreover, dose escalation studies cannot use the expected dose because of the solubility limit, with the result that PS80 has limited ability to solubilize taxanes in gastrointestinal fluids. Finally, the development of pharmaceutical dosage forms is a major problem: indeed, immediate dilution of the PS80 solution with an aqueous medium is not conceivable for oral administration of cytotoxic agents.

A large number of documents describe systems suitable for dissolving and/or improving the bioavailability of hydrophobic active ingredients. However, the tested systems have so far proven to be ineffective for the preparation of pharmaceutical compositions containing stable and bioavailable taxanes, wherein the taxanes can be administered orally in effective concentrations.

WO95/24893 describes a delivery system for hydrophobic drugs. This application describes compositions comprising digestible oils, lipophilic surfactants and hydrophilic surfactants for formulating hydrophobic active ingredients and enhancing their bioavailability.

WO99/49848 describes pharmaceutical dosage forms for anticancer drugs such as paclitaxel in which the active drug is formulated as a stable self-emulsifying preconcentrate. WO99/49848 describes a composition comprising an anticancer drug in a carrier system comprising at least one hydrophobic component selected from a tri-, di-or monoglyceride, a free fatty acid, a fatty acid ester or a derivative thereof, and a hydrophilic component selected from a hydroxyalkane, a dihydroxyalkane or a polyethylene glycol (PEG), and comprising at least one surfactant.

EP 0152945B1 describes a transparent multicomponent system for pharmaceutical applications, containing one or several active ingredients in a system consisting of an oily ingredient, a surfactant, a co-surfactant and optionally water.

EP 0670715B1 describes pharmaceutical compositions for ingestion capable of forming microemulsions comprising at least one active ingredient, a lipophilic phase, a surfactant, a co-surfactant and a hydrophilic phase of specific composition.

EP 0334777B1 describes pharmaceutical microemulsions comprising a water-soluble phase and a lipid-soluble phase, containing at least one polyethylene glycol-based surfactant and at least one polyglycerol-based co-surfactant.

It has been found and forms the subject of the present invention that it is possible to prepare chemically and physically stable formulations of taxanes for oral administration. The present invention relates to a semisolid formulation for the oral administration of taxanes comprising at least one taxane and at least one agent selected from Vitamin EAnd G lu cire44Of (4) a polymeric material.

Preferably, the taxanes have the general formula (I):

wherein:

R₁is hydrogen, (C)₂-C₄) Acyl, (C)₁-C₃) An alkyl group, a carboxyl group,

R₂is OH, alkoxy, or R₂And R₃Is a methylene group, and is a linear or branched chain,

R₃is CH₃Or R is₂And R₃Is a methylene group, and is a linear or branched chain,

R₄is OCOCH₃Or OCOOCH₃，

R is phenyl or (C)₃-C₄) Alkoxy or (C)₃-C₄) Alkenyloxy, preferably phenyl or tert-butoxy, R' is aryl, preferably phenyl, optionally substituted, or (C)₂-C₄) Alkyl or (C)₂-C₄) An alkenyl group.

More preferred taxanes are selected from the compounds of formulae (Ia) to (If),

the semisolid formulations of the present invention are particularly suitable for the taxanes of formula (Ib) and formula (Ic).

One suitable semi-solid formulation of the invention contains no more than 200mg of taxane per gram of polymeric material, more preferably 50 to 200mg of taxane per gram of polymeric material. Suitable taxane contents may be appropriate for patient needs, e.g., taxane concentrations in the polymeric material of 5mg/g, 10mg/g, 20mg/g, 30mg/g, 40mg/g, 50mg/g, 60mg/g, 70mg/g, 80mg/g, 90mg/g, 100mg/g, 150mg/g, or 200 mg/g.

The semi-solid formulation of the invention may optionally further comprise at least one additional additive selected from a stabilizer, a preservative, an agent that may adjust the viscosity or an agent that may adjust the organoleptic properties.

In another aspect, the present invention relates to a process for preparing the above formulation, wherein a mixture of the main excipient is prepared after melting the semi-solid excipient by heating as appropriate, and then, if necessary, mixed with additional additives, and then the taxane is added, with stirring, to obtain a homogeneous mixture.

The strategy is to obtain formulations capable of enhancing the dissolution of taxanes in aqueous media by using amphiphilic and lipid based formulations capable of forming colloidal (miniemulsion or micellar solution) systems in vivo.

In amphiphilic and lipid based formulations, there are 3 classes identified:

amphiphilic polymers (micelle and emulsion formation)

Phospholipids (lipid vesicle formation)

SMES (self-microemulsifying system): oil + surfactant + co-surfactant (microemulsion formation)

The solubility of the taxane in the vehicle after first selecting the appropriate vehicle (for safety and development feasibility) is the first screening step in selecting the vehicle and selecting the prototype. Prototypes (solid or semi-solid) were then prepared and characterized for simulating in vitro behavior and chemical stability in GI medium. Finally, the physical properties and stability of the semi-solid prototype were investigated.

The solubility of taxanes was tested as described in the literature for different classes of excipients based on amphiphilicity and lipid formulation components:

1. oil (medium chain triglycerides, fatty acids.)

2. Amphiphilic surfactants with hydrophilic character (HLB > 10) (PEO sorbitan fatty acids, castor oil ethyl oxide, fatty acid ethyl oxide)

3. Amphiphilic surfactants with lipophilic character (HLB < 10) (fatty acid glycerides: glyceryl oleate/linoleate, oleoyl macrogolglycerides; propylene glycol derivatives: PG octanoate/linoleate)

4. Phospholipids (lecithin)

5. Hydrophilic solvent (PEG 400.)

All selected excipients are described as safe for oral administration and they can be developed (alone or in admixture) into pharmaceutical dosage forms (soft or hard capsules).

The chemical composition of the selected excipients in liquid form at room temperature and the solubility of the taxane of formula (Ib) are reported in table 1 below.

TABLE 1 solubility data for taxanes of formula (Ib) in liquid vehicles

Table 2 below reports the chemical composition of selected excipients in semi-solid form at room temperature and the solubility of the taxane of formula (Ib). The excipient is pre-dissolved at a temperature not higher than 70 deg.C for drug dissolution.

Table 2 solubility data in semisolid excipients (in the molten state) and solid excipients

The solubility of the taxane of formula (Ib) at room temperature is determined by X-ray diffraction.

In view of the solubility of the taxane of formula (Ib), for the class 3 drug delivery system the following excipients were retained:

vitamin E TPGS for micelle formation

Phosal 75SA and Phospholipon 90H for lipid vesicle formation

Labrasol and Gelucire 44/14 for emulsion formation

Forming a micro-emulsion: as the surfactant: myrj 45, PS80, Cremophor EL, Labrasol; as cosurfactant: maisine, Capryol 90, Peceol, Lauroglycol90, Imwitor 988; as the oil: miglyol 812N, Edenor.

For the first 3 classes, the excipients were formulated with the drugs in a 2-membered system at the following concentrations:

● Vitamin E TPGS (semi-solid matrix): 50. 100mg/g preparation

● Phosal 75SA (solution): 100mg/g preparation

● Phospholipon 90H (solid powder): 50. 100mg/g preparation

● Gelucire 44/14 (semi-solid matrix): 50. 100mg/g preparation

● Labrasol (solution): 50. 100, 200mg/g preparation

For the SMES class (three-component system), it is necessary to first screen the excipients which are oils, surfactants (HLB > 10) and cosurfactants (HLB < 10) mixed in different proportions in the absence of active ingredient to identify formulations which form microemulsions (droplet size < 30nm) after infinite dilution with water. By this screening, the following SMES were determined:

●Cremophor EL/Maisine/Miglyol 812N，50mg/g

●Cremophor EL/Lauroglycol 90/Miglyol 812N，50mg/g

●Cremophor EL/Capryol 90/Miglyol 812N，50mg/g

●Cremophor EL/Peceol/Miglyol 812N，50mg/g

●Cremophor EL/Imwitor 988/Miglyol 812N，50mg/g

the proportions of excipients in the retained formulation were as follows: the ratio of surfactant to co-surfactant was 3: 1 and the oil concentration was 20%.

It will be appreciated that the dosage may vary depending on the degree and nature of the condition being treated. Thus, the amount of active product in the composition of the invention determined will give a suitable dosage. The amount of taxane varies with its solubility in the mixture and also with the appropriate dosage for treating the patient. Preferably, care should be taken not to add more than 10% w/w taxane drug to avoid microemulsion destabilization.

For humans, it will be understood that to select the most appropriate daily dosage, the patient's weight, his general health, his age and all factors affecting the therapeutic effect should be considered. Preferably, the compositions are formulated in unit doses containing 0.1 to 50mg of active product.

Alternatively, the composition may contain 0.2 to 50mg when the second active ingredient is introduced. However, the amount may optionally be lower or may vary from 0.2 to 10 mg.

When the composition also contains other additives, the latter may be stabilizers, preservatives, agents possibly regulating the viscosity, or agents for example regulating the organoleptic properties.

The stabilizer may be, for example, an antioxidant, in particular selected from alpha-tocopherol, ascorbyl palmitate, BHT (butylhydroxytoluene), BHA (butylhydroxyanisole), propyl gallate or malic acid.

The preservative may be selected from, for example, sodium metabisulphite, propylene glycol, ethanol or glycerol.

Among the agents capable of regulating the viscosity, mention may be made, for example, of lecithin, phospholipids, propylene glycol alginate, sodium alginate or glycerol.

Agents capable of modulating the organoleptic properties of the composition are for example malic acid, fumaric acid, glycerol, vanillin or menthol.

When these additives are used, the latter may constitute from 0.001% to 5% by weight of the total composition.

According to the invention, the pharmaceutical composition can be obtained as follows: the main excipients (after melting the semi-solid excipients by heating) are mixed as appropriate, then, if necessary, with additional additives, followed by the addition of the taxane, and kept under stirring to obtain a homogeneous mixture.

The composition of the invention may be provided in a semi-paste form.

They are particularly suitable for presentation in the form of hard or soft gelatin capsules or in the form of oral solutions.

The compositions of the present invention are particularly advantageous because of their good physical and chemical stability and the increased bioavailability provided by oral administration of taxanes.

The following non-limiting examples illustrate the formulations of the present invention.

Drawings

FIG. 1: release profiles of the taxane of formula (Ib) in simulated gastric media at 100mg/g of different formulations.

FIG. 2: the release profiles of the taxane of formula (Ib) in simulated gastric media in 50 and 100mg/g semisolid formulations.

FIG. 3: particle size of taxane formulation of formula (Ib) in simulated gastric media.

FIG. 4: particle size of taxane formulation of formula (Ib) in simulated gastric media producing < 50nm droplets.

Examples

Example 1: preparation of the prototype

1.1 materials

● taxane of formula (Ib)

● Miglyol 812N (Condea Vista Co., Cranford, NJ, USA)

●Labrasol(Gattefossé，Saint Priest，F)

●Gelucire 44/14(Gattefossé，Saint Priest，F)

●Vitamin E TPGS(Eastman Chemical，Anglesey，UK)

●Cremophor EL(BASF AG，Ludwigshafen，DE)

●Capryol 90(Gattefossé，Saint Priest，F)

●Lauroglycol 90(Gattefossé，Saint Priest，F)

●Peceol(Gattefossé，Saint Priest，F)

●Maisine 35-1(Gattefossé，Saint Priest，F)

● Imwitor 988(Condea Vista, Cranford, NJ, USA)

●Phosal 75SA(Nattermann，Cologne，DE)

●Phospholipon 90H(Nattermann，Cologne，DE)

● PS80 VG DF (Seppic, Paris, France)

1.2 preparation of semi-solid scaffolds

Weighed amounts of drug are dispersed in molten vehicle and then kept at 50-60 ℃ until dissolved under mechanical agitation. The material was poured into a hard gelatin capsule (No. 0) and refrigerated overnight. The gelatin shell is then removed to avoid compatibility problems during this step.

1.3 chemical stability

Chemical stability of different formulations is a key parameter. The prototype whole (glass bottle) was stored for no more than 3 months at +5 ℃ (± 3 ℃), 25 ℃ (± 2 ℃) and 30 ℃ (± 2 ℃), with a Relative Humidity (RH) of 60% (± 5%) and at 40 ℃ (± 2 ℃) and a relative humidity of 75% (± 5%).

Stability was assessed by potency as measured by HPLC and evaluation of related substances. Prototypes used for the analysis of drug dose and stability studies are listed in the table below.

Table 3: prototype taxane formulations of formula (Ib) for stability studies

Prototype	Pharmaceutical preparation with a concentration of mg/g
		PS 80	100
Capryol 90	250
		Labrasol	100
Labrasol	200
		Phosal 75SA	100
Gélucire 44/14	80
		Gélucire 44/14	100
Vitamin E TPGS	60
		Vitamin E TPGS	100
Cremophor EL-/Miglyol 812N-Peceol	50
		Cremophor EL-/Miglyol 812N-Maisine	50
Cremophor EL-/Miglyol 812N-Lauroglycol 90	50
		Cremophor EL-/Miglyol 812N-Capryol 90	50
Cremophor EL-/Miglyol 812N-Imwitor 988	50
		Phospholipon 90H	50
Phospholipon 90H	100

All formulations, except the SMES formulation, were stable at 75% RH at 40 ℃ for 3 months. In fact, SMES is stable at 25 ℃ for 1 month, whereas at 40 ℃ impurities (hydrolysis) (t) of the taxane of formula (Ib) occur_{1 Month of year}1.15-3.88% depending on the nature of the co-surfactant), 3 month analysis of the samples allowed evaluation of whether an increase in impurities was critical: after 3 months, an increase in the impurity level of the taxane of formula (Ib) was noted.

SMES was stable at 5 ℃ for 7 months.

Example 2:in vitro behavior in simulated gastrointestinal media (gi ═ gastrointestinal tract)

Release profiles after incubation in simulated gastrointestinal media

2.1 composition of the simulated liquid

The following simulated liquids were selected for this experiment:

● stomach Medium USP, pH1.2

● fasted intestinal medium, pH6.8 (see Dressman et al, pharm. Res., 1998)

● intestinal medium, pH5 (see Dressman et al, pharm. Res., 1998)

Table 4: simulating the composition of the gastrointestinal medium

Stomach medium (G)

Sodium chloride 2g

About 100ml of 1N hydrogen chloride

Demineralized water qsp 1000ml

Fasted intestinal medium (Fassif) for 500ml

Potassium hydrogen phosphate 0.029M 1.97g

Sodium hydroxide in a suitable amount to pH6.8

Sodium taurocholate 5mM 1.34g

Lecithin (Phospholipon 90G) 1.5mM 0.58G

Potassium chloride 0.22M 8.2g

Demineralized water qsp1 l qsp 500ml

Fed intestinal medium (Fessif) for 500ml

Acetic acid 0.144M 4.33g

Sodium hydroxide to pH5 to 5

Sodium taurocholate 15mM 4.03g

Lecithin (Phospholipon 90G) 4mM 1.55G

Potassium chloride 0.19M 7.08g

Demineralized water qsp1 l qsp 500ml

2.2 Experimental conditions

In the first step of the experiment, the formulation (100mg drug/g formulation, 500mg formulation in hard gelatin capsule) was diluted 1: 500 (1 capsule/250 ml) in gastric medium and then incubated for 2 hours at 37 ℃ in a USP standard dissolution apparatus under stirring conditions (50 rpm).

To study the effect of drug/excipient and excipient ratio on the release profile, the same experiment was performed in gastric media using 2 capsules containing a lower concentration formulation (50mg drug/g formulation).

In the second step of the experiment, the gastric media was first incubated for 1 hour and then either fasted or fed intestinal media for 2 hours to simulate the gastric emptying process.

Samples were taken at 5-15-30-60 minutes and 2 hours. After centrifugation (6000rpm, 10min) the drug concentration was determined by HPLC. The homogeneity of the medium was evaluated by sampling at the bottom, middle and top of the vessel. 2.3 results

The drug release profile of the 100mg/g formulation in gastric media is shown in figure 1.

Only the formulation consisting of Vitamin ETPGS allowed an improvement in the in vitro solubility of the taxane of formula (Ib) by 2 hours (80% drug dissolution) compared to the PS80 formulation (evaluated as a reference).

The curves obtained with the data of the other Phosal and glucire formulations are less representative, since these formulations lead to very heterogeneous mixture formation after incubation. For Glucire 44/14, disintegration of the semisolid matrix only partially occurs and is not sufficiently dispersible in simulated gastric media. The Labrasol formulation resulted in a very homogeneous emulsion with the vehicle, although a small amount of drug was recovered after centrifugation (see release profile), suggesting that for a coarse emulsion, centrifugation (determining emulsion breaking of the emulsion) may underestimate its in vitro performance. The Phospholipon 90H experiment was stopped (no data acquisition) because the powder floated and could not form a uniform suspension.

Comparison of the drug release profiles of the 50 and 100mg/g semisolid formulations (Gelucire, Vitamin E TPGS and PEG 400) (FIG. 2; the profiles relating to 100mg drug/g Vitamin E TPGS and Gelucire are the same as reported in FIG. 1) shows that: the Vitamin E TPGS showed the highest dissolution profile, 80% for the 100mg/g dose and 100% for the 50mg/g dose. The 50mg/g Gelucire formulation allows about 80% drug dissolution as opposed to a 100mg/g dose, as previously described. Finally, as expected, hydrophilic PEG 4000 was demonstrated to be unable to dissolve hydrophobic drugs in aqueous media.

Example 3:particle size analysis (USP) after incubation in gastric media

The objective of this part of the study was to evaluate the self-emulsifying properties and colloidal stability of the emulsion/microemulsion/micellar solution of taxane of formula (Ib) after incubation in gastric medium by particle size determination.

3.1 conditions of the experiment

The formulation (concentration 100mg drug/g formulation, 100mg formulation) was diluted 1: 500 in gastric medium (50ml) and incubated at 37 ℃ for 2 hours with mechanical stirring (300 rpm).

The samples were immediately diluted with water for particle size determination or, if necessary, 2 μm filtration. Filtration can retain oil droplets > 2 μm and drug crystals > 2 μm to allow particle size determination by QELS (quasi-elastic light scattering) (Nanosizer N4+, Beckman-Coulter).

3.2 results

As shown in FIGS. 3 and 4, particle sizes of < 50nm were only obtained in formulations with an active concentration of 50 mg/g: 5 microemulsions (regardless of their composition), Gelucire (after 2 μm filtration) and Viamin ETPGS.

The results suggest that: formulations that form small and monodisperse droplets in the gastric medium are used to achieve better in vivo efficacy. To evaluate the effect of bile salts on formulation size and colloidal stability, further experiments should be conducted in simulated intestinal media.

3.3 preliminary conclusions for the evaluation of taxane formulations of formula (Ib)

All results regarding the in vitro behavior of the taxane oral formulation of formula (Ib) in simulated GI fluids and the chemical stability under accelerated conditions are summarized in the following table.

Table 5: summary of in vitro behavior of 50mg/g formulation

Preparation	Chemical stability	In vitro droplet size (in gastric medium 37 ℃, 2 hours)	In vitro homogeneity (stomach medium at 37 ℃ C., 2 hours)	Drug released in vitro after 2 hours (in stomach medium)	Drug released in vitro after 1 hour in gastric media and 2 hours Fassif%
						Vitamin ETPGS	More than 3 months at 40 ℃/75% RH	16nm	Good effect	100％	Is not made
Gélucire44/14	More than 3 months at 40 ℃/75% RH	45nm (after filtration)	Good effect	79％	＞60％

Table 6: summary of in vitro behavior of 100mg/g formulation

Preparation	Chemical stability	In vitro droplet size (in gastric medium 37 ℃, 2 hours)	In vitro homogeneity (stomach medium at 37 ℃ C., 2 hours)	Drug released in vitro after 2 hours (in stomach medium)	Drug released in vitro after 1 hour in gastric media and 2 hours Fassif%
						Vitamin ETPGS	More than 3 months at 40 ℃/75% RH	335nm	Good effect	82-84％	60％
Gélucire44/14	More than 3 months at 40 ℃/75% RH	118nm (after filtration)	Difference (D)	Not determined	Not determined

Gelucire forms a heterogeneous emulsion with GI media, so the concentration is discarded at this point. Thus, only the Vitamin E TPGS formulation showed valuable behavior (in terms of release profile and particle size) at 100 mg/g.

Example 4: physical characterization and stability of semi-solid scaffolds

4.1 Experimental methods

X-ray powder diffraction (XRPD)

Analysis was performed on a Siemens-Bruker D5000Matic diffractometer using a secondary focusing Bragg-Brento (theta-2 theta) -type geometry. If sufficient product was available, the powder was placed on a concave aluminum sample holder. Otherwise, a thin layer of the product is deposited on the single crystal silicon wafer, cut according to a crystallographic orientation that (510) impedes any Bragg reflection (ensuring extinction of the corresponding diffraction band system). A cobalt anticathode tube (40kV/30mA) gave an iron filtered incident beam. Two types of radiation are emitted: CoK alpha₁(λ＝1.7890) And CoK alpha₂(λ＝1.7929). A50M multicanal Braun linear detector performed the operation. It has a 10 deg. wide detection window at 2 theta angle. Maps were recorded under the following conditions: 2 theta angle 1.5 to 50.0 degree sweep, 2 theta angle per degree recording time 10-30 seconds, pressure, temperature and relative humidity% depending on the amount of powder analyzed, ambient conditions.

4.1.2 physical characterization

The taxane semisolid formulations of formula (Ib) studied in this section were prepared and characterized by t.borovac (DEA report, conference of convention et factitious details of the compositions semi-solid formulations of compounds alone-soluble excipients and a. atropriciple act and product of convention, 2003-6-19).

In the semi-solid formulation, XRPD is used to characterize the physical state (dissolved or dispersed) and physical form (if dispersed) of the drug substance a series of physical mixtures (Vitamin E TPGS or Gelucire and drug) were used to evaluate the detection limits of this technique: this limit is 2.5% or 25mg/g for both excipients.

4.2 physical stability

Storage conditions (temperature, pressure, time) may alter or induce recrystallization of the drug in a dissolved semi-solid formulation or polymorphism of the drug in a dispersed formulation.

Two semi-solid formulations (a 60mg/g Vitamin E TPGS or an 80mg/g Gelucire) were evaluated after 1 month at 30 ℃/60% RH or 40 ℃/75% RH. Both formulations were mostly dissolved after preparation and no recrystallization was observed after 1 month. We can expect that: both 50mg/g formulations were physically stable for at least one month.

4.3 conclusion and further study

Table 7: comparative Properties of recommended formulations according to selection criteria

Claims (2)

1. Semisolid preparation for oral administration of taxanes consisting essentially of a binary system containing a taxane

And one selected from Vitamin EAnd G elucire 44/Wherein the formulation contains 5 to 100mg taxane per gram of polymeric material.

2. A process for the preparation of a formulation as claimed in claim 1, wherein the semisolid excipient is formulated after melting by heating, if appropriate, and then the taxane is added, with stirring, to obtain a homogeneous mixture.