A collagen preparation for the controlled release of active substances is characterized in that it has mixtures of acid-insoluble collagens with different molecular weight distributions.

A number of polymers are used in the production of administration forms for pharmaceutical or cosmetic active substances. Depending on the site of application, these are used to impart to the respective administration form the desired properties. Agents for the treatment of wounds and implantable materials should be adapted to the surface of the respective application site, and they should not interfere with the function and activity of body cells, such as keratinocytes, fibroblasts, or endothelial cells. In these cases the spectrum of suitable polymers is therefore limited to those having an excellent compatibility on contact with connective tissue and which preferably are biodegradable.
For quite a long time now, collagen, the main protein of the connective tissue, has gained a special place among the polymers suitable for this purpose. This is due to the biological compatibility and degradability of the administration forms produced therefrom. Such collagen preparations are for the most part used as wound dressings without any additional additives. However, there have also been attempts to charge collagen matrices of most different kinds with biologically active substances and to influence the release of these substances, which in general follows the dissolution and/or enzymatic degradation of the collagen carrier matrix, by the kind, structure and composition of the matrix.
To produce collagen matricesxe2x80x94such as those described in, for example, U.S. Pat. No. 5,024,841, U.S. Pat. No. 4,789,663, U.S. Pat. No. 4,774,091, U.S. Pat. No. 4,563,350 U.S. Pat. No. 5,246,457, or EP 429 438xe2x80x94solutions of telopeptide-free, acid-soluble collagen are used in general. These are used to obtain reconstituted fibrils by means of dialysis, shift of the pH, or other processes; and these fibrils are then processed by means of different methods into preferably porous matrices. These preparations can exhibit an excellent biocompatibility since they consist of pure collagen, but they have the disadvantage that a retardation of the active substance release is limited owing to the use of soluble collagen.
Such a release retardation can be achieved by decreasing the solubility of the collagen matrix by using cross-linking agents or binders, as is described, for example in U.S. Pat. No. 4,409,332, DE 2 843 963, WO 93/00890, or WO 85/04413, or by using coating agents, e.g., described in DE 38 41 397, or by combining water-soluble collagen with other polymers, preferably natural anionic polymers or their derivatives. However, owing to the additional components, additional toxicological risks which, in particular when the known cross-linking agents for collagen are used, should not be underestimated must be accepted in each of these cases.
Another possibility of delaying the active substance release is described in EP 518 697. Here, laminates are produced from water-soluble and/or water-insoluble collagen, which consist of one or several active substance-containing reservoir layers and one layer retarding the active substance supply. As compared to the above-mentioned preparations, a retardation of the release while minimizing the toxicological risks could be achieved with such laminates. However, they have the disadvantage that their production is extremely expensive and that the adherence of the layers can only be achieved with xe2x80x9cmoistxe2x80x9d films. Dry films which are absolutely necessary in case of active substances susceptible to decomposition cannot be joined to form such laminates, for example.
However, a retardation of the active substance release is not always desired. EP 224 453 describes a collagen matrix which mainly consists of water-insoluble, reticulated collagen and additionally comprises water-soluble, non-reticulated collagen. In cosmetic preparations, e.g., face packs, said soluble collagen serves as active substance which, after application, is dissolved out by means of the natural cutaneous moisture or by extremely moistening the preparation which then takes effect on the skin. For pharmaceutical purposes, active substances can be incorporated into the preparation, these are then dissolved out and released from the collagen matrix together with the soluble collagen very quickly after application. Such a collagen preparation can be useful in cases where a rapid release is desired and appropriate.
Mechanisms to delay or accelerate the active substance release from collagen preparations have been described in many ways. However, due to the given composition, each of these prior art collagen preparations offers only one possibility of influencing the release, and therefore they have a given release profile each tailor-made to one specific problem, e.g., a certain active substance or certain active substance group, a certain therapeutic principle, or a certain disease. None of the prior art collagen preparations can achieve a selective and at the same time manifold control of the active substance release, i.e., permit a variable and individual adaptation of the release kinetics of active substances to differing problems, wherein factors, such as different active substance properties and differences in the onset and duration of action, can be considered adequately for each case.
Accordingly, it was the object of the present invention of find a collagen preparation which is not only suitable for a certain active substance, a certain active substance combination, or a certain release profile, but also permitsxe2x80x94for a great variety of usesxe2x80x94a wide-ranging, reliable control of the active substance release adapted to the respective problem.
Most surprisingly, the solution of this object has been found in a collagen preparation for the controlled release of active substances, comprising mixtures of acid-insoluble collagens having different molecular weight distributions.
According to one embodiment, the collagen preparation comprises different active substances. It may additionally comprise adjuvants, such as viscosity regulators, binders, humectants, softening agents, penetration enhancers, preservatives, disinfectants, pH-regulators, antioxidants, active substance stabilizers, oils, fats, waxes, emulsion stabilizers, odorous substances, dyes, and/or inert fillers.
According to a preferred embodiment, the insoluble collagen is telopeptide-free, native, uncross-linked Type-1-collagen. This may be an insoluble collagen which is a product obtained from calfskin by means of alkaline decomposition.
Furthermore, the collagen preparation may be present in the form of powders, dusts, microparticles, fibers, flakes, foams, sponges, needles, small rods, tablets, gels, creams, single-layer films, or laminates.
Advantageously, the collagen preparation may include combinations of different administration forms to achieve the desired release kinetics.
It is preferred that the collagen preparation be bioadhesive.
A process for the production of the collagen preparation may comprise spray drying, freeze-drying, coating or casting with subsequent drying, phase separation and coacervation processes, compression, or filling into containers.
According to the process, the active substance release can additionally be influenced and/or controlled by the mixing ratio of acid-insoluble collagens with different molecular weight distributions. According to the process, the active substance release can additionally be controlled by dissolution, swelling, or erosion of the collagen preparation. Another possibility of controlling the active substance release is by biological degradation of the collagen preparation.
The use of the collagen preparation according to the present invention consists in the controlled release of the active substance to wounds. However, the use may also be directed to the controlled release of active substances to intact skin. Finally, the use of the collagen preparation may serve to implant or inject active substances into a living organism.
In general, prior art collagen preparations for the active substance release are produced from acid-soluble collagen, that is collagen which is dissolved clearly in dilute acids at a pH of 2. This acid-soluble collagen can be isolated from several animal and vegetable tissues by means of a great variety of processes.
In contrast to this, the collagen used according to the present invention is an acid-insoluble collagen whichxe2x80x94when it is present in aqueous dispersionxe2x80x94cannot be brought into solution even when concentrated acetic acid is added.
It is preferred that this insoluble collagen be native collagen the greater part of which Is present as Type-I-collagen and the smaller part as Type-III-collagen. The term native collagen refers to a collagen molecule having an unchanged triple-helical tertiary structure.
The insoluble collagen used according to the present invention can be obtained from biomaterial of various origins by alkaline decomposition; for this purpose processes, such as those described, for example, in DE-OS 3034273, U.S. Pat. No. 4,021,522, or DE-OS 27 16 602 are slightly modified. It is preferred that the starting material be calfskin, aged for six months. In contrast to the normally used tissues and body parts of cattle, pigs, or horses, this ensures a starting material of a defined, constant quality, this in turn ensuring the reliable reproducibility of the process steps described in the following and illustrated in greater detail in Example 1 hereunder.
First, the calfskin is mechanically dehaired and degreased. Then, soluble collagens and non-collagenous soluble components of the connective tissue are extracted and rejected. Next, the connective tissue is first treated with an aqueous solution of an alkali hydroxide, preferably sodium hydroxide, and an alkali sulfate, preferably sodium sulfate, and then with an aqueous alkali sulfate solution to saponify and dissolve out sebaceous matter and to swell the collagen fibers in a controlled manner. When this is done, the terminal, non-helical portions of the collagen molecule, the so-called telopeptides which are mainly responsible for the antigenic properties of xenogeneic collagen, are also split off. In addition, the treatment with alkali hydroxide/alkali sulfate solution and with pure alkali sulfate solution splits a defined portion of the intermolecular collagen bonds in the fiber composite of the swollen connective tissue, said portion depending on the concentration and the duration of action of the solutions. The intramolecular bonds of the collagen are not attacked so that the helical structure of the molecule remains undamaged. The connective tissue so decomposed is washed out with water and dilute acids in several stages, purified and neutralized, and then mechanically comminuted and dispersed in water.
The decisive step for the isolation of the acid-insoluble collagen having different molecular weight distributions used according to the present invention is the selective alkaline cleavage of the intermolecular collagen bonds. If, for example, an aqueous solution of 9.75% sodium hydroxide and 9.2% sodium sulfate is allowed to react on the swollen connective tissue for 48 hours, the HPLC-analysis (column system Biosil TSK-400+Biosil TSK-125; eluent 0.5 m ammonium acetate buffer pH 6.7; detector UV 275 nm; sensitivity range 0.02 AUFS) with assessment against standard-protein chromatograms shows an average molecular weight of about 420,000 for the insoluble collagen after dispersion in water. In contrast to this, if the swollen connective tissue is treated with an aqueous solution of only 5% sodium hydroxide and 9.2% sodium sulfate for only 12 hours, far fewer intermolecular bonds are split, and the analysis of the insoluble molecular aggregates obtained after dispersion in water shows a mean molecular weight of about 2,500,000. These differences first of all affect the flow behavior and with that the processibility of the dispersions of insoluble collagen. Whereas a dispersion with 5% of low-molecular, insoluble collagen is a viscous but still free flowing mass, the flow limit of a dispersion of higher-molecular, insoluble collagen is at about 2.5%.
Thus, the variable factors of the decomposition process, by variation of which different molecular weight distributions can be achieved each time, are concentration and reaction time of sodium hydroxide. The higher the concentration of sodium hydroxide and/or the longer its duration of action, the lower the mean molecular weight of the isolated, acid-insoluble collagen, and vice versa. To represent the connection between the molecular weight that can be achieved and the described decomposition conditions, e.g., in the form of a curve, one of the influencing factors, i.e., concentration or duration of action, would have to be kept constant while the other one is changed. This is possible in principle, but from the production and operational point of view this is rather useless, since, for example, a free variation of the duration of action alone is not possible in general. For instance, very long reaction times with a correspondingly low sodium hydroxide concentration would increase both the machine running times and the requirement of personal; in view of the operating expenses this is not acceptable. On the other hand, owing to the required higher sodium hydroxide concentration, very short durations of action result in increased wear of the manufacturing facilities, e.g., of the conduits and filtering installation, and this cannot be realized either because of increasing working expenses. Therefore, the required duration of action and concentration of sodium hydroxide must individually be ascertained empirically for each problem in such as manner that they represent an optimum both with respect to the demands on the administration form, in particular regarding the release kinetics of the active substance, and to economic efficiency.
The differences in the molecular weight distribution of insoluble collagen distinctly influence the properties of the collagen preparations that are produced therefrom by means of drying. Example 2 shows that, depending on the mixing ratio, foams of insoluble collagen with different molecular weight distributions produced by means of freeze-drying have very different disintegration properties. While foams consisting of 100% of low-molecular, insoluble collagen completely decompose in artificial wound exudation of pH 6.4 after only 45 minutes, foams made of 100% of higher-molecular, insoluble collagen have not disintegrated even after 10 days under the same conditions.
The example shows that the use of high-molecular collagen aggregates has a clearly stabilizing effect on the foam; this is expressed in a clearly slowed down absorption of secretion and swelling, and in a very delayed disintegration. Form stabilization by means of maintaining a high natural cross-linking degree of the collagen has the important advantage, in particular from the toxicological point of view, that no additional manipulations to consolidate the structure, e.g., by tanning or cross-linking, are required. The product is rendered dimensionally stable by merely maintaining the collagen""s original quaternary structure, such as that present in the skin, to a substantial degree.
Thus, the collagen foams, each manufactured from only one single basic type of insoluble collagen, show clear differences, for example, with respect to interaction with the secretion of the wound. However, as mentioned above, the profile which is required for a collagen product and is based on a specific purpose can only in special cases be satisfied by such mono-preparations. Compared with that, the present invention offers two possibilities of exactly tailoring the properties of a collagen preparation to given demands on a product. On the one hand, the molecular weight distribution of the insoluble collagen can continuously be varied over a very wide range by selectively controlling the cleavage of the intermolecular bonds. On the other hand, the required product properties can be adjusted by mixing said variations of insoluble collagen each having different molecular weight distributions. If the already mentioned Example 2 is continued, this becomes clear, for instance, by the disintegration of freeze-dried foams, wherexe2x80x94prior to dryingxe2x80x94insoluble collagen having a mean molecular weight of about 420,000 and insoluble collagen having a mean molecular weight of about 2,500,000 were mixed at always different ratios. With different pH-values, the decay periods always show a gradual decrease when the percentage of insoluble collagen having a low average molecular weight is gradually increased.
The exact adjustment of the product properties to a demand profile representing the optimum for the respective therapy is of importance primarily in the therapy using pharmaceutical active substances.
Since the collagen preparation according to the present invention is preferably used on the skin, on external and internal wounds, and in internal body tissues and body cavities after implantation or injection, the active substances suitable for charging the collagen preparation preferably are active substances for the dermal and transdermal application, active substances for the treatment of wounds and for the promotion of wound-healing, as well as active substances usually administered by means of preparations for implantation or injection.
For the dermal treatment of local skin diseases the following substances are used: local anaesthetics, local antibiotics, antiseptics, antimycotics, antihistaminics, and antipruritic drugs; keratolytics and caustic drugs; virustatics, antiscabietic agents, steroids, as well as different substances for the treatment of acne, psoriasis, or photodermatoses. Active substances applied intradermally include, for example, steroid and non-steroid antirheumatics, substances stimulating the blood flow, or vasoprotectors and vaso constrictors for the treatment of vascular diseases. The active substances applied transdermally include, for example, neuroleptics, antidepressants, tranquilizers, hypnotics, psychostimulants, analgesics, muscle relaxants, antiparkinson drugs, ganglionic blockers, sympathomimetics, alpha-sympatholytics, beta-sympatholytics, antisympathotonics, antidiabetics, coronary therapeutic agents, antihypertensives, anti-asthmatics, or diuretics. The collagen preparation according to the present invention can also be used on the skin in cosmetic preparations, e.g., in the form of lather masks or films for the treatment of, for example, aged skin, wrinkles, or impure skin; for body care, depilation, reduction of perspiration, or for light protection.
Active substances which are used in the collagen preparations according to the present invention on external and internal wounds, preferably are styptic active substances, among which collagen itself has a special place; wound-cleansing substances, such as enzymes, antiseptics, disinfectants, and antibiotics; as well as active substances promoting wound healing which stimulate granulation, induce vascularization, or promote epithelization. Among the active substances promoting the wound healing, biologically active peptides and proteinsxe2x80x94which develop high activities at only very low concentrations and are mainly manufactured by recombinant technologiesxe2x80x94increasingly gain importance. The collagen preparation according to the present invention represents a particularly suitable carrier and release system for these substances which include the so-called growth factors, such as Platelet derived growth factor (PDGF), Epidermal growth factor (EGF), Platelet derived endothelial cell growth factor (PD-ECGF), acidic Fibroblast growth factor (aFGF), basic Fibroblast growth factor (bFGF), Transforming growth factor xcex1 (TGF xcex1), Transforming growth factor xcex2 (TGF xcex2), Keratinocyte growth factor (KGF), Insulin-like growth factors 1 and 2 (IGF1, IGF2), and Tumor necrosis factor (TNF).
The active substances administered parenterally by means of the collagen preparation according to the present invention include, for example, antibiotics, antiseptics, anaesthetics, analgesics of varying strengths; cytostatics, hormones, steroids, cytokinins, such as interleukins, interferons, and colony-stimulating factors, Hormone releasing and release inhibiting factors, prostaglandins, enzymes, as well as growth factors, in particular osteoinductively effective bony growth factors.
One of the greatest challenges for the development of active substance-containing preparations is to find formulations releasing the active substance in such a manner that the optimum action and best therapy is achieved. The half-life period of many active substances, in particular of the above-mentioned biologically active peptides and proteins, in the body is relatively short, and for this reason they must frequently be administered several times a day.
Therefore, active substance vehicles releasing the active substances in a delayed or even controlled manner gain increasing importance. Collagen preparations according to the present invention offer the possibility of developing carrier systems using relatively few formulation base components. These systems are very flexible both with regard to application and design, and can exactly be directed to the required solution of a problem. The mechanisms, which impart to the release kinetics of an active substance its characteristic features, can selectively be controlled by mixing of insoluble collagen having different molecular weights and by shaping of the collagen preparation. These factors, in addition to structure and density of the polymeric collagen skeleton, also influence the number and distribution of hydrophilic, hydrophobic and ionic bonds along the polymer skeleton. Since active substance can not only be enclosed in cavities of a collagen preparation according to the present invention, but can also be adsorbed to the surface of the collagen skeleton, the bonding force between collagen and active substancexe2x80x94and thus its release behaviorxe2x80x94is substantially-determined by ionic relations as well as by hydrophilic and hydrophobic interactions.
In the dermal, intradermal and transdermal application of an active substance using a collagen preparation according to the present invention, the solubility of the active substance in the preparation, the degree of charge and saturation, and the diffusion rate of active substance within the preparationxe2x80x94in addition to structure, density and bonding activity of the collagen preparationxe2x80x94have an influence on the release behavior.
In case of collagen preparations for the release of active substances to external or internal wounds and collagen preparations for implantation or injection another factor appears.
Since the collagen preparations according to the present invention come into contact with body fluid in the applications mentioned above, the rate and amount of liquid absorbed by the collagen preparation and consequently the swelling capacity and disintegration properties of the collagen preparation can be used to control the release, as is shown in Example 2 mentioned hereinbefore. In addition to the decay of the collagen preparation used as controlling mechanism, other suitable mechanisms with regard to the release control on contact with body fluid primarily include the dissolution of active substance out of the collagen preparation by means of body fluid and the fluid-induced diffusion of active substance from the center of the collagen preparation to its interface. Furthermore, the release is influenced by the biological degradation of the collagen preparation by means of hydrolysis and enzymatic reaction on contact with body fluid. The wide range of interaction between the liquid and preparations of mixtures of insoluble collagen having different molecular weight distributions has already been shown in Example 2. The way said interactions in combination with one or several of the above-mentioned influencing factors take effect on the release kinetics of an active substance is illustrated Examples 3 and 4. It is made clear that the mixing ratio of insoluble collagen having different molecular weight distributions significantly influences the kinetics of the active substance release. In the case of a given active substance and a given desired release per time unit, it is thus possible to adapt the collagen preparation selectively to the required demands; in this way an optimum active substance dosage for the respective therapy is achieved after application over the pre-determined application period. If active substance is to be released over an application period of 24 to 48 hours from the administration form chosen in Examples 3 and 4xe2x80x94manufactured of the preparations of insoluble collagen having different molecular weight distributions as described in Example 1xe2x80x94the mixing ratio of low-molecular and high-molecular insoluble collagen should not be lower than 3 to 1. If, on the other hand, an even, delaying active substance release from a comparable administration form is to take place over an application period of 7 to 14 days, the mixing ratio of low-molecular and high-molecular insoluble collagen should not be greater than 1 to 3.
In order to prove the controlling possibilities of preparations consisting of mixtures of insoluble collagen having different molecular weight distributions, free from any influence of other auxiliary agents, pure collagen preparations plus active substance were exclusively used in the Examples on purpose. In practice, however, it will not be possible to do without additional water-soluble or water-dispersible additives, since, in addition to the requirements regarding the active substance release, the intended purpose will usually also result in demands with respect to handling properties and stability of an active substance-collagen-preparation.
Such adjuvants may be additional polymers, e.g., serving as viscosity regulators when liquid preparations are used or as binders when solid forms are used, for example, cellulose derivatives, starch derivatives, galactomannan derivatives, dextrans; vegetable polysaccharides, such as alginates, pectins, carrageenan, or xanthan, chitosan, proteins, glycoproteins, proteoglycans, glucosaminoglycans, polyvinyl alcohol, polyvinylpyrrolidone, vinyl pyrrolidone-vinyl acetate copolymers, polyethylene glycol, polyacrylates and polymethacrylates, polylactides and polyglycolides, as well as polyamino acids.
The collagen preparation may comprise as additional adjuvants:
humectants, such as glycerol, sorbitol, polyethylene glycol, polypropylene glycol,
softening agents, such as citric acid ester, tartaric acid ester, or glycerol ester,
penetration enhancers, such as alkyl sulfates, alkyl sulfonates, alkali soaps, fatty acid salts of multivalent metals, betaines, amine oxides, fatty acid esters, mono-, di- or triglycerides, long-chain alcohols, sulfoxides, nicotinic acid ester, salicylic acid, N-methyl pyrrolidone, 2-pyrrolidone, or urea,
preservatives, such as p-Cl-m-cresol, phenylethyl alcohol, phenoxyethyl alcohol, chlorobutanol, 4-hydroxybenzoic acid methylester, 4-hydroxybenzoic acid propylester, benzalkonium chloride, cetylpyridinium chloride, chlorohexidine diacetate or digluconate, ethanol, or propylene glycol.
Disinfectants, for example, halogens, such as polyvidoneiodine; halogen compounds, such as sodium hypochloride or tosylchloride sodium; oxidants, such as hydrogen peroxide or potassium permanganate; aryl mercury compounds, such as phenylmercury borate or merbromin; alkyl mercury compounds, such as thiomersal; organotin compounds, such as tri-n-butyltin benzoate; silverwhite compounds, such as silverwhite acetyltannate; alcohols, such as ethanol, n-propanol, or isopropanol; phenols, such as thymol, o-phenylphenol, 2-benzyl-4 -chlorophenol, hexachlorophene, or hexylresorcinol; or organic nitrogen compounds, such as 8-hydroxyquinoline, chloroquinaldol, clioquinol, ethacridine, hexetidine, chlorohexidine, or ambazone.
pH-regulators, such as glycerol buffers, citrate buffers, borate buffers, phosphate buffers, or citric acid phosphate buffers.
Antioxidants, such as ascorbic acid, ascorbyl palmitate, tocopherol acetate, propyl gallate, butylhydroxyanisole, or butylated hydroxytoluene.
Active substance stabilizers, such as mannitol, glucose, lactose, fructose, saccharose,
emulsifyable inactive ingredients, such as oils, fats, and waxes,
odorous substances, dyes, cleaning agents, substances for personal hygiene,
emulsion stabilizers, such as non-ionogenic emulsifiers, amphoteric emulsifiers, cation-active emulsifiers, and anion-active emulsifiers,
fillers, such as micro-crystalline cellulose, aluminum oxide, zinc oxide, titanium oxide, talcum, silicon dioxide, magnesium silicate, magnesium aluminum silicate, kaolin, hydrophobic starch, calcium stearate, or calcium phosphate.
For example, adjuvants may be dissolved, dispersed, or emulsified in dispersions of insoluble collagen prior to shaping the administration form. However, they may also be introduced in a manner usual and known for the respective shaping processes after termination of a primary forming process (described hereinafter) of the mixtures of insoluble collagen during later stages of the drug shaping.
The embodiments wherein admixtures of insoluble collagen of different molecular weight distributions may be used for the controlled release of active substances are so numerous and manifold that they cannot be represented herein extensively.
These embodiments of the collagen preparation according to the present invention may be manufactured from mixtures of dispersions of insoluble collagen each having different molecular weight distributions according to various methods known to the skilled artisan; for example by spray drying, freeze-drying, coating or casting with subsequent drying, phase separation and coacervation processes for particles or emulsified droplets, drying and compression, as well as by simple filling into containers, e.g., tubes. They result in, for example, powders, dusts, microparticles, fibers, flakes, foams, sponges, needles, small rods, tablets, gels, creams, single-layer films, or laminates. Preferred embodiments are spraydried microparticles, freeze-dried foams, and gel-like films manufactured by coating.
The introduction of active substance into the collagen preparation according to the present invention may be effected such that the active substance is dissolved or dispersed in the finished mixture of dispersions of insoluble collagen having different molecular weight distributions, prior to forming the preparation. If more than one active substance is to be incorporated, these may also be dissolved or dispersed separately from one other in the individual fractions of insoluble collagen, prior to the mixing process. Active substance may also be introduced on or into the preparation after the shaping process of the preparation by means of coating, spraying, impregnating, dipping, or other adsorption methods.
The possible embodiments, the respective manufacturing processes, and the methods of incorporating the active substance may also be combined with one another in order to achieve certain properties.
For instance, the collagen preparation according to the present invention may comprise a shell of insoluble collagen with a low mean molecular weight, which is soluble or at least swellable in body fluid, in the form of a sponge or film containing a micro-particulate preparation of insoluble collagen having a high mean molecular weight dispersed therein. If only one active substance is incorporated, the outer sponge or film phase serves the quick release of active substance to achieve the required minimum active substance concentration very quickly, followed by a slower, more even release of active substance from the inner, particulate phase to maintain the required active substance concentration over the application period. Such release profiles may also be achieved with other preparation forms, such as fibers in hydrogels, spongy oil-in-water-emulsions, compressed mixtures for implantable tablets, multi-layer films, combinations of films and foams, and the like.
Such multi-phase preparations can also be used if a release of different active substances at different points of time with different release rates is desired, or if one or several active substances are to be released phase-like, i.e., with release-free intervals. In this case active substance A may, for example, be contained in a readily soluble, quick-releasing outer phase, whereas active substance B is contained in an inner phase which is slightly soluble and releases the active substances in a retarded and controlled sustained manner. If a phase-like release profile is desired for only one active substance, external and internal phase may, for example, release active substance at an even kinetics. However, the inner phase will then be surrounded by an active substance-free layer of insoluble collagen, which, after dissolution of the outer phase or active-substance-exhaustion thereof, must swell or dissolve itself first so that the active substance release from the inner phase can take place. By means of this arrangement, an interval without any active substance release can be obtained.
If the collagen preparation according to the present invention is used for the dermal, intradermal or transdermal application of pharmaceutical active substances or cosmetic active principles, plane embodiments, such as films, membranes, or thin sponges are preferred. These flat embodiments may consist of laminates which, for example, also include barrier layers which are free from active substances, permeable separating layers, controlling membranes, and adhesive layers. Substructures, such as laminae, powders, microparticles, or oil droplets, may then be introduced in or between the individual layers, in order to achieve the suitable release kinetics for one or several active substances. It is preferred that such single- or multi-layer collagen preparations for the mentioned applications, e.g., for protection against dehydration or growing in of germs, be provided with a backing layer and a removable protective layer located on the opposite side according to known processes, the backing layer and the protective layer consisting of materials known to the skilled artisan, for example, those used in the formulation of patches and adhesive tapes.
In a preferred embodiment of the collagen preparation according to the present invention for the use on external wounds and in the interior of the body, the preparation is paste-like, e.g., foamy or spongy. The size of the pores and the structure of the preparation are designed such that immigration of cells, e.g., fibroblasts or osteoblasts, into the preparation is possible and that the cells are given a structural orientation; this can particularly be attributed to the degree of orientation of the collagen in the preparation according to the present invention, which is similar to that of natural connective tissue. Immigration of the cells may be necessary, for example, for the degradation of the preparation or for the release or deposition of substances which are required, for example, for neo-formation of tissue or vascularization of a tissue which is to take the place of the collagen preparation according to the present invention.
In another preferred embodiment for the use on wounds or in the interior of the body, the collagen preparation according to the present invention is adjusted by admixing adjuvants, such as carboxymethylcellulose, polyacrylic acid, tragacanth, sodium alginate, or hydroxypropylcellulose in such a manner that it is bioadhesive, i.e., that it adheres to the surface tissue of the application site for a certain time by means of interfacial forces, in order to increase the retention time at the site of application or absorption.

Collagen preparation for the controlled release of active substances


A low level scaffold with ball screw drive is a lightweight, mobile, compact, electrically activated, self-erecting, scissor-type lifting platform. The electronic system utilizes an electric motor in direct drive arrangement with a ball screw assembly to vary the platform height from a retracted position to extended position. Electric switches limit reverse direction between the retracted and extended positions to vary platform height anywhere between the minimum and maximum heights above ground level. The design allows for transportation through household doorways and provides anti-tilt arms with threaded level mounts for use on non-level surfaces. The scaffold provides homeowners and small commercial contractors with an apparatus to perform small interior and exterior projects at elevated heights.

This invention relates to a scaffold having an electric drive for lifting a user, equipment, and materials from a ground level to an elevated working level within a contained area of height and width and to stabilize the scaffold on non-level ground, and mobility through interior passageways.
The homeowner and small commercial contractor struggle with small interior and exterior projects that are at elevated heights. They primarily rely are stepladders, stools, and extension ladders for these projects. The user must constantly travel up and down these structures to retrieve tools and materials. The work area is limited to the reach to the user. This places the user in an unsafe situation that may result in a fall from an elevated height. As the user leans to extend his reach, the ladder or stool may become unstable and tilt or the user may loose his grip or balance and fall to the ground. Also, the user needs to constantly reposition the structure as the work progress. This is very inefficient wasting precise time. Stability of the structure is also an issue when working on unleveled surfaces. This is a particular problem when outdoors.
Conventional aerial lifts are primarily designed for commercial and industrial users. The lifts initial purchase costs are high with significant reoccurring maintenance costs. The lifts employ hydraulics to elevate working platforms. The hydraulics requires significant maintenance and are constantly leaking making these machines unsuited for internal use for home or light industrial or small commercial operations. The relative size of the available machines also limits their use to external areas or internal spaces with wide doorways and high ceiling. Their use within a home or office is prohibited due to maneuverability through passageways and the leaking hydraulic fluid. Most conventional aerial lifts are powered by diesel engines, propane motors or large battery packs that also limits their usage within structures due to environment issues.
In order to work safety at elevated heights within a house or office for the purpose of installing or fixing electric fixtures, painting walls or ceiling, or changing light bulbs, it is necessary to have an apparatus that is lightweight, low cost, portable and mobile, anti-tilt, sufficient load lifting capabilities, compact design in full retracted position, transportable through doorways, platform work area sufficient to reduce up and down trips by operator, level mount features allow placement on non-level surfaces, and a motorized direct drive elevating means to eliminate the hydraulics.
Accordingly, several objects and advantages of my invention are to provide the consumer with a low cost, portable, mobile, multi-purpose scaffold for household use and light duty construction and maintenance.
It is a further object of this invention to provide a scaffold that is of lightweight construction with anti-tilt features.
It is a further object of this invention to provide a scaffold that has load-lifting capabilities up to 600 pounds.
It is another object of this invention to provide a scaffold that is compact in full-retracted position.
It is another object of this invention to provide a scaffold that is transportable through building doorways.
It is yet another object of this invention to provide a scaffold with a platform work area that limits up and down trips by operator.
It is a further object of this invention to a scaffold with level mount features that allow a scaffold to be placed on non-planar surfaces.
It is a further object of this invention to provide a scaffold with a motor direct drive system that eliminates oil leakages associated with hydraulic systems.
Still further objects and advantages will become apparent from a consideration of the ensuing description and accompanying drawings.
The objects set forth above as well as further and other objects and advantages of the present invention are achieved by the embodiments of the invention described hereinbelow.
Briefly stated, the preferred embodiment introduces an improvement in scissor advancement mechanism that enables significant reduction in the size of a lifting scaffold for multipurpose use. The present invention provides a means for lifting construction material, tools and workman to working heights within a confined working area. For example, one embodiment of the present invention provides a lifting platform of conventional design wherein the platform is lifted from ground level and maintained in a substantially level height by a known scissor arrangement. The invention is sized to allow transportation through household doorways and other limiting structures. The scissor arrangement raises a load by mechanical lifting means driven by at least on electric motor means powered by AC or DC power. Preferably the mechanical lifting means includes at least one ball screw. The ball screw is connected to the scissor arrangement such that as the electric motor rotates the screw, the scissors open and the platform raises. The present invention employs the advantages of using a mechanical drive rather than hydraulic fluid to elevate the platform.
An alternative embodiment includes electric micro switches provided in the assembly design to limit the extension and retraction position of the upper movable platform anywhere within a desired height range, preferably between 1 and 6.5 feet above surface level. These micro switches shut down electric power to the drive motor when mechanically activated.
Another alternative embodiment includes casters for maneuverability and allows the operator to transport the apparatus from location to location in similar manner to a dolly.
Yet another alternative embodiment includes stabilizing arms located on the stationary base to preclude the assembly from tipping over when the upper movable platform is fully extended. Leveling mounts located on the stabilizing arms allow the assembly to be mounted on non-level surfaces.
For a better understanding of the present invention, together with other and further objects thereof, reference is made to the accompanying drawings and detailed description and its scope will be pointed out in the appended claims.


A pesticidal composition comprising 4-phenoxyphenyl 2-(2-pyridyloxy)propyl ether and (E)-1-(2-chlorothiazol-5-ylmethyl)-3-methyl-2-nitroguanidine as active ingredients, especially, the composition having the weight ratio of 4-phenoxyphenyl 2-(2-pyridyloxy)propyl ether and (E)-1-(2-chlorothiazol-5-ylmethyl)-3-methyl-2-nitroguanidine in the composition is in the range of from 100:1 to 1:30. Further, a method for controlling pests applying an effective amount of 4-phenoxyphenyl 2-(2-pyridyloxy)propyl ether and (E)-1-(2-chlorothiazol-5-ylmethyl)-3-methyl-2-nitroguanidine to pest or place where pest inhabits.

The present invention relates to pesticidal compositions and pesticidal methods.
Hitherto, pesticides for controlling various pests such as agricultural and forestry pests, ectoparasites of animals and hygienically unfavorable pests have been developed. However, it cannot be said that old pesticides exhibit satisfactory effects in the practical use wherein pests in various growth stages exist.
The present invention provides pesticidal compositions and pesticidal methods which are very effective for controlling the pests in agricultural and forestry fields, such as whiteflies, and further effective for controlling ectoparasites of animals, hygienically unfavorable pests and the other various pests.
According to the present invention, a pesticidal composition comprising 4-phenoxyphenyl 2-(2-pyridyloxy)propyl ether and (E)-1-(2-chlorothiazol-5-ylmethyl)-3-methyl-2-nitroguanidine as active ingredients and a pesticidal method comprising utilizing 4-phenoxyphenyl 2-(2-pyridyloxy)propyl ether and (E)-1-(2-chlorothiazol-5-ylmethyl)-3-methyl-2-nitroguanidine together give satisfactory effect in practical use.
Namely, the present invention provides a pesticidal composition (hereinafter, referred to as the present pesticidal composition) comprising 4-phenoxyphenyl 2-(2-pyridyloxy)propyl ether (hereinafter, referred to as pyriproxyfen) and (E)-1-(2-chlorothiazol-5-ylmethyl)-3-methyl-2-nitroguanidine (hereinafter, referred to as chlothianidine) as active ingredients and a pesticidal method (hereinafter, referred to as the present pesticidal method) comprising utilizing pyriproxyfen and chlothianidine together.
Pyriproxyfen used in the present pesticidal composition and the present pesticidal method is a compound described in U.S. Pat. No. 4,751,225 specification and chlothianidine is a compound described in U.S. Pat. No. 5,034,404 specification. These compounds can be produced according to the descriptions in these specifications.
The combination of pyriproxyfen and chlothianidine produces a synergistic effect that is clearly larger than a sum of the sole effect of each compound as shown in the test examples described later.
In the present pesticidal composition and the present pesticidal method, pyriproxyfen and chlothianidine are combined and utilized in a ratio that can produce a synergistic effect. It is usually 0.01 to 30 parts by weight of chlothianidine, preferably 0.5 to 10 parts by weight based on 1 part by weight of pyriproxyfen.
The present pesticidal composition and the present pesticidal method can be applied for controlling various pests such as agricultural and forestry pests, ectoparasites of animals and hygienically unfavorable pests. Examples of the pest include arthropods such as insects and acarina and nematoda, namely Hemipteran pests such as Delphacidae (planthoppers) [e.g. Laodelphax striatellus (small brown planthopper), Nilaparvata lugens (brown planthopper) and Sogatella furcifera (white-backed rice planthopper)], Deltocephalidae (leafhoppers) [e.g. Nephotettix cincticeps and Nephotettix virescens], Aphididae (aphids) [e.g. Aphis gossypii (cotton aphids), Myzus persicae (green peach aphid), Aphis citricola, Lipaphis pserudobrassicae (turnip aphid), Nippolachnus piri, Toxoptera aurantii (black citrus apid) and Toxoptera ciidius (brown citrus apid)], stink bugs [e.g. Nezara antennata (green stink bug), Cletus punctiger, Riptortus clavetus (bean bug) and Plautia stali (oriental stink bug)], Aleyrodidae (whiteflies) [e.g. Trialeurodes vaporariorum (greenhouse whitefly), Bemisia tabaci (sweetpotato whitefly) and Bemisia argentifolli (silverleaf whitefly)], scales [e.g. Aonidiella aurantii (California red scale), Comstockaspis perniciosa (San Jose scale), Unaspis citri (citrus snow scale), Pseudaulacaspis pentagona (white peach scale), Saissetia oleae (brown olive scale), Lepidosaphes beckii (purple scale), Ceroplastes rubens (red wax scale) and Icerya purchasi (cottonycushion scale)], Tingidae (lace bugs) and Psyllidae (suckers); Lepidopteran pests such as Pyralidae [e.g. Chilo suppressalis (rice stem borer), Cnaphalocrocis medinalis (rice leafroller), Ostrinia nubilalis (European cornborer), Parapediasia teterrella (bluegrass webworm), Notarcha derogata (cotton leafroller) and Plodia interpunctella (Indian meal moth)], Noctuidae [e.g. Spodoptera litura (tobacco cutworm), Pseudaletia separata (rice armyworm), Mamestra brassicae (cabbage armyworm), Agrotis ipsilon (black cutworm), Trichoplusia spp., Heliothis spp. and Helicoverpa spp.], Pieridae [e.g. Pieris rapae], Tortricidae [e.g. Adoxophyes spp., Grapholita molesta (oriental fruit moth) and Cydia pomonella], Carposinidae [e.g. Carposina niponensis (peach fruit moth)], Lyonetiidae [e.g. Lyonetia spp.], Lymantriidae [e.g. Lymantria spp. and Euproctis spp.], Yponameutidae [e.g. Plutella xylostella], Gelechiidae [e.g. Pectinophora gossypiella (pink bollworm)], Arctiidae (tiger moths) [e.g. Hyphantria cunea (fall webworm)] and Tineidae [e.g. Tinea translucens (casemaking clothes moth) and Tineola bisselliella (webbing clothes moth)]; Dipteran pests such as Culex spp. [e.g. Culex pipiens pallens and Culex tritaeniorhynchus], Aedes spp. [e.g. Aedes albopictus], Anopheles spp. [e.g. Anopheles sinensis], Chironomidae (midges), Ceratopogonidae (biting midges) [e.g. Culicoides oxystoma], Muscidae [e.g. Musca domestica (housefly) and Muscina stabulans (false housefly)], Calliphoridae, Sarcophagidae, Fannia spp. (little house flies), Anthomyiidae [e.g. Delia platura (seedcorn maggot) and Delia antiqua (onion maggot)], Tephritidae (fruit flies), Drosophilidae (vinegar flies), Psychodidae (sand flies), Simuliidae (black flies), Tabanidae, Stomoxyidae (stable flies) [e.g. Haematobia irritans] and Agromyzidae (leafminer flies); Coleopteran pests such as corn rootworms [e.g. Diabrotica virgifera virgifera (western corn rootworm) and Diabrotica undecimpunctata howardi (southern corn rootworm)], Scarabaeidae [e.g. Anomala cuprea and Anomala rufocuprea], Curculionidae (weevils) [e.g. Sitophilus zeamais (maize weevil), Lissorhoptrus oryzophilus (ricewater weevil), Hypera pastica (alfalfa weevil) and Callosobruchuys chienensis (adzuki bean weevil)], Tenebrionidae (darkling beetles) [e.g. Tenebrio molitor (yellow mealworm) and Tribolium castaneum (red flour beetle)], Chrysomelidae (leaf beetles) [e.g. Aulacophora femoralis (cucurbit leaf beetle), Phyllotreta striolata (striped flea beetle) and Leptinotarsa decemlineata (Colorado beetle)], Anobiidae, Epilachna spp. [e.g. Epilachna vigintioctopunctata], Lyctidae (powderpost beetles), Bostrychidae, Cerambycidae and Paederus fuscipes; Dictyopteran pests such as Blattella germanica (German cockroach), Periplaneta fuliginosa (smokybrown cockroach), Periplaneta americana (American cockroach), Periplaneta brunnea (brown cockroach) and Blatta orientalis; Thysanopteran pests such as Thrips palmi, Thrips tabaci, Thrips hawaiiensis (flower thrips), Scirtothrips dorsalis (yellow tea thrips), Frankliniella intonsa (flower thrips), Frankliniella occidentalis (western flower thrips) and Ponticulothrips diospyrosi; Hymenopteran pests such as Formicidae (ants), Vespidae (hornets), Bethylidae and Tenthredinidae (sawflies) [e.g. Athalia japonica (cabbage sawfly)]; Orthopteran pests such as Gryllotalpidae (mole crickets) and Acrididae (grasshoppers); Siphonapteran pests such as Pulex irritans (human flea), Ctenocephalides felis (cat flea) and Ctenocephalides canis (dog flea); Anopluran pests such as Pediculus humanus corporis and Phthirus pubis (crab louse); Isopteran pests such as Reticulitermes speratus and Coptotermes formosanus; Acarina such as Tetranychidae (spider mites) [e.g. Tetranychus urticae (two-spotted spider mite), Tetranychus kanzawai (Kanzawa spider mite), Panonychus citri (citrus red mite), Panonychus ulmi (European red mite) and Oligonychus spp.], Eriophyidae [e.g. Aculops pelekassi (pink citrus rust mite) and Calacarus carinatus (purple tea mite)], Tarsonemidae [e.g. Polyphagotarsonemus latus], Tenuipalpidae (false spider mites), Tuckerellidae, Ixodidae [e.g. Haemaphysalis flava (Japanese tick), Haemaphysalis flava (yellow tick), Ixodes ovatus and Ixodes persulcatus], Acaridae [e.g. Tyrophagus putrescentiae (copra mite)], Dermanyssidae [e.g. Dermatophagoides farinae (American house dust mite), Dermatophagoides ptrenyssnus], Cheyletidae [e.g. Cheyletus eruditus, Cheyletus fortis, Cheyletus malaccensi and Cheyletus moorei] and chicken mites]; and Nematoda such as Pratylenchus coffeae (coffee root-lesion nematode), Pratylenchus fallax, Pratylenchus loosi, Pratylenchus vulnus (walnut root-lesion nematode), Heterodera glycines (soybean cyst nematode), Glohodera rostochiensis (potato cyst nematode), Meloidogyne hapla (northern root-knot nematode) and Meloidogyne incognita (southern root-knot nematode).
In the present pesticidal method, each of pyriproxyfen or its formulation and chlothianidine or its formulation can be used simultaneously, but it is convenient to use the present pesticidal composition in which pyriproxyfen and chlothianidine are mixed with each other in advance.
The present pesticidal composition comprises pyriproxyfen and chlothianidine as active ingredients and usually further comprises an inert carrier. The inert carrier can be solid carriers, liquid carriers and the like which are utilized for usual pesticidal formulations. The present pesticidal composition can optionally comprise formulation auxiliaries such as surfactant, dispersant, adhesive agent, stabilizer and propellant to be formulated to oil solution, emulsifiable concentrates, wettable powders, flowables such as aqueous suspension and aqueous emulsion, microcapsule formulation, spot-on formulation, pour-on formulation, shampoo formulation, granules, dusts, aerosol, ULV formulation, poison bait, sheet formulation and resin formulation.
The total amount of pyriproxyfen and chlothianidine in the present pesticidal composition is usually 0.01 to 90% by weight, preferably 0.1 to 80% by weight.
Examples of the solid carrier include fine powders and granules of clays such as kaolin clay, diatomaceous earth, bentonite, Fubasami clay and terra alba; synthetic hydrated silicon oxide; talc and the like; ceramics; the other inorganic minerals such as sericite, quartz, sulfur, activated carbon, calcium carbonate and hydrated silica; and chemical fertilizers such as ammonium sulfate, ammonium phosphate, ammonium nitrate, urea and ammonium chloride. Examples of the liquid carriers include water; alcohols such as methanol and ethanol; ketones such as acetone and methyl ethyl ketone; aromatic hydrocarbons such as benzene, toluene, xylene, ethylbenzene and methylnaphthalene; aliphatic hydrocarbons such as hexane, cyclohexane, kerosene and gas oil; esters such as ethyl acetate and butyl acetate; nitriles such as acetonitrile and isobutyronitrile; ethers such as diisopropyl ether and dioxane; acid amides such as N,N-dimethylformamide and N,N-dimethylacetamide; halogenated hydrocarbons such as dichloroethane, trichloroethane and carbon tetrachloride; dimethyl sulfoxide; and vegetable oils such as soybean oil and cottonseed oil.
Further, the present composition may be formulated by added a propellant such as flon gas, butane gas, liquefied petroleum gas, dimethyl ether and carbon dioxide thereto.
Examples of the surfactant include alkyl sulfate ester salts, alkylsulfonate salts, alkylarylsulfonate salts, alkylaryl ethers and polyoxyethylenated derivatives thereof, polyethyleneglycol ethers, polyvalent alcohol esters and sugar alcohol derivatives.
Examples of the other auxiliaries such as adhesive agents and dispersants include casein; gelatin; saccharides such as starch, gum arabic, cellulose derivatives and alginic acid; lignin derivatives; bentonite; and synthetic water-soluble polymers such as polyvinyl alcohol, polyvinylpyrrolidone and polyacrylic acids. Further, stabilizers including PAP (isopropyl acid phosphate), BHT (2,6-di-tert-butyl-4-methylphenol), BHA (2-tert-butyl-4-methoxyphenol and 3-tert-butyl-4-methoxyphenol), vegetable oils, mineral oils, fatty acids and fatty acid esters can be utilized as formulation auxiliaries.
Examples of the base materials for the poison bait include bait ingredients such as grain powder, vegetable oils, sugars and crystalline cellulose; antioxidants such as BHT and nordihydroguaiaretic acid; substances for preventing erroneous eating from children and pets such as red pepper powder; and attractants such as cheese flavor, onion flavor, leek flavor and peanut oil.
Examples of the base materials for resin formulations include olefin polymers, vinyl chloride polymers and polyurethanes. To these base materials, there can be added, if necessary, plasticizers such as phthalates and stearic acid; pest attractants such as pheromones; coloring agents with pest-attractive colors; and so on.
The resin formulations can be obtained by kneading the active ingredients into the base materials with a conventional kneader and then forming by injection, extrusion or compression molding. Further, they can be also obtained by supporting the active ingredients on the base materials which do not contain the active ingredients by impregnation, coating, printing or the like. In the operation, the active ingredients may be used in the form of oil solutions, emulsifiable concentrates, flowables or their dilutions. The resin formulations thus obtained can be formed through further steps, if necessary, such as shaping and cutting, into plates, films, tapes, nets, strings or the like, and can also be formed into sheets for mulching, attractive strings, supports for horticultural use, sheet formulations, wrapping films, animal collars and ear tags for animals.
The flowable formulations of the present pesticidal composition usually contain 1-75% by weight of the active ingredients, 0.5-15% by weight of dispersants, 0.1-10% by weight of suspension aids (e.g., protective colloids, thixotropy-conferring compounds), 0-10% by weight of auxiliaries (e.g., antifoaming agents, rust preventives, stabilizers, spreading agents, penetration aids, antifreezing agents, antibacterial agents, antifungal agents). The replacement of water with oils in which the active ingredients can hardly be dissolved makes it possible to produce oily suspensions. As the protective colloids, there can be used, for example, gelatin, casein, gums, cellulose ethers, polyvinyl alcohol and the like. The thixotropy-conferring compounds may include, for example, bentonite, alminum magnesium silicate, xanthan gum, polyacrylic acid and so on.
In the present pesticidal composition and the present pesticidal method, the other insecticide, nematocide, acaricide, repellent, fungicide, herbicide, plant growth regulator, synergist, fertilizer, soil improving agent or animal food may be used simultaneously.
Examples of the insecticide, nematocide and acaricide include pyrethroid compounds such as permethrin, cypermethrin, fenvarelate, esfenvarelate, fenpropathrin, biphenthrin, deltamethrin, fluvalinate, flucythrinate, allethrin, d-allethrin, prallethrin, cyphenothrin, phenothrin, resmethrin, tefluthrin, empenthrin, acrinathrin, cyhalothrin, cyfluthrin, etofenprox, halfenprox, silafluofen, tralomethrin, cycloprothrin, esbiothrin, transfluthrin, terallethrin, imiprothrin and 1-ethynyl-2-fluoro-2-pentenyl 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate; organophosphorus compounds such as cyanophos, fenthion, fenitrothion, parathion, methylparathion, pirimiphos-methyl, diazinon, isoxathion, pyridaphenthion, chlorpyrifos, chlorpyrifos-methyl, oxydeprofos, vamidothion, malathion, phenthoate, dimethoate, thiometon, disulfoton, phosalone, phosmet, methidathion, prothiofos, sulprofos, profenofos, azinphosmethyl, pyraclofos, calvinphos, salithion, tetrachlorvinphos, dichlorvos, monocrotophos, naled, dimethylvinphos, propaphos, acephate, metamidofos and ethion; carbamate compounds such as carbaryl, metolcarb, isoprocarb, fenobcarb, propoxur, XMC, ethiofencarb, bendiocarb, pyrimicarb, carbosulfan, carbofuran, benfuracarb, furathiocarb, methomyl, thiodicarb, oxamyl, alanycarb, fenothiocarb, metoxadiazone and fenothiocarb; nitroiminoimidazolidine derivatives; nereistoxin derivatives such as cartap, bensultap and thiocyclam; formamidine derivatives such as amitraz and chlordimeform; phenylpyrazole derivatives such as ethiprole; benzoylphenylurea compounds such as diflubenzuron, teflubenzuron, chlorfluazuron, flufenoxuron, triflumuron, hexaflumuron, lufenuron and novaluron; triazine derivatives such as cyromazine; thiadiazine derivatives such as buprofezine; juvenoid compounds such as methoprene, hydroprene, fenoxycarb and diofenolan; tebufenozide; methoxyfenozide; halofenozide; chromafenozide; chlorofenapir; phenisobromolate; quinomethionate; propargit; fenbutatin oxide; hexythiazox; clofentezine; pyridaben; fenpyroximate; tebufenpyrad; pyrimidifen; polynactin complex; milbemectin; avermectin; ivermectin and azadirachtin.
The present pesticidal method is an application of an effective amount of pyriproxyfen and chlothianidine to pest or a place where pests inhabit. Typical methods are explained as below.
In case of controlling agricultural or forestry pests by the present pesticidal method, the application rate of pyriproxyfen and chlothianidine is usually 10 g to 10000 g, preferably 100 g to 1000 g per 1 hectare at the total amount of the active ingredients. In the present pesticidal method, the concentration is usually 10 ppm to 1000 ppm at the total amount of the active ingredients, when the present pesticidal composition such as emulsifiable concentrates, wettable powders and flowables is diluted with water and applied. Granules and dusts of the present pesticidal composition are applied as they are.
In the present pesticidal method, pyriproxyfen and chlothianidine may be supplied to foliar application to plants those are crops to be protected from agricultural and forestry pests, or soil treatment to the surroundings of the plants for making the active ingredients absorbed from their roots. Soil treatment can also control agricultural and forestry pests those inhabit in soil. Further, the resin formulations formed to sheet or string can be applied by winding around the plants, setting or covering on the soil surface of the surroundings of the plants.
In case of controlling hygienically unfavorable pests by the present pesticidal method, pyriproxyfen and chlothianidine are usually applied indoors and the application rate is 0.1 mg to 1000 mg, preferably 0.5 mg to 200 mg per 1 m2 of the floor at the total amount of the ingredients. The application rate is also 0.01 mg to 300 mg, preferably 0.1 mg to 100 mg per 1 m3 of the space when spraying to the air. The concentration of the applied dilution is usually 0.1 ppm to 10000 ppm at the total amount of the active ingredients, when the present pesticidal composition such as emulsifiable concentrates, wettable powders and flowables is diluted with water and applied. Oil solutions, aerosols, smokings, ULV formulations and poison baits are applied as they are. When the present pesticidal compositions are directly applied to small animals for controlling ectoparasites of the animals such as dogs and cats, they are applied by conventional veterinary methods: for example, as tablets, by feed incorporation, as suppositories or by injection (e.g., intramuscular, subcutaneous, intravenous, intraperitoneal) for systemic control; or by spraying oily or aqueous liquid, by pour-on or spot-on treatment, or as shampoo formulations or resin formulations such as ear tags for non-systemic control. For such direct application to animal bodies, the amounts for application are usually 0.1 mg to 500 mg per 1 kg of the animal weight at the total amounts of the active ingredients.
The amounts or concentrations for application may vary depending upon type of formulations, times, places and methods of application, kinds of pests, degree of damage, and other factors; they can therefore be increased or decreased without limitation to the above ranges.

Insecticide compositions and method for destroying insects


The combination of erythropoietin and calcium, with or without phosphates, is useful for treating haemochromatoses. A method for treating primary haemochromatoses in a patient involves administering to the patient a combination therapy including an erythropoietin preparation and a calcium preparation, where the erythropoietin preparation and the calcium preparation are each administered from 1 to 5 times weekly. Beneficially, this combination therapy further administers a phosphorus preparation.

The invention relates to use of erythropoietin (Epo) in low doses for production of pharmaceutical preparations for treatment of haemochromatoses and pharmaceutical combination preparations containing erythropoietin (Epo) and calcium compounds and/or phosphate compounds. These combination preparations are used particularly for treatment of primary haemochromatoses (inherited disturbances through excess of iron).
More than ⅔ (about 70%) of newly-formed bone-marrow parent cells in the organism participate in metabolism of iron and bone into erythrocytes and osteoblasts or osteoclasts. These processes (erythropoiesis and bone formation) are controlled by the two differentiating hormones, i.e. erythropoietin (Epo) and parathyroid hormone (PTH), the normal PTH concentration in the serum (between 10 and 60 ng/l ) resulting in normal formation of osteoblasts and osteoclasts whereas normal concentrations of Epo, between 6 and 25 U/l, result in normal formation of erythrocytes.
Erythopoiesis in particular is vitally important, since 45% of newly-formed bone-marrow parent cells develop into erythrocytes. For this reason all parameters (iron resorption, iron incorporation, Epo, folate, vitamin B12) of the iron metabolism are extremely closely regulated.
As is known, Epo is a glycoprotein which is formed in the kidneys and can also be synthesised and stimulates blood formation (erythropoiesis) xe2x80x9chumorallyxe2x80x9d, i.e. by way of the bloodstream.
It is known to treat anaemia, particularly anaemia of haemodialysis patients caused by transfusion (renal anaemia) with recombinant human erythropoietin (rhEpo). Anaemia in chronic illness is the second most frequent form of anaemia throughout the world.
Reduced production of new erythrocytes is an important factor in anaemia when caused by reduced erythropoiesis in the bone or disturbances in iron re-utilisation. The daily iron requirement for normal erythropoiesis is 25 mg. Iron deficiency anaemia, which is the most frequent form of hypochromic anaemia, is regulated by addition of iron. Administration in combination with rhEpo is used in therapy in order to obtain a significant increase in the number of erythrocytes.
In clinical chemistry, disturbances in iron metabolism are diagnosed by determining the concentration of serum ferritin. The total body iron is about 3.5 g in men and 2.5 g in women. Iron occurs in active metabolism and in storage compartments. The active pool in a man on average contains 2100 mg in the haemoglobin, 200 mg in the myoglobin, 150 mg in enzymes in the tissue (haem and non-haem) and 3 mg in the iron transport compartment. Iron in the form of ferritin (700 mg) and haemosiderin (300 mg) is stored in tissue between the cells.
The total resorption mechanism for iron has not yet been fully explained (Gunshin et al., Nature 388, 482-488, 1997), but probably occurs via the protein divalent cation transporter (xe2x80x9cDCT1xe2x80x9d). Regulation is critically influenced by the small-intestine mucosa cells. In the case of the mucosa, the critical signal seems to be the total iron content of the body. It has been shown that the serum ferritin concentration is inversely related to the quantity of absorbed iron.
The iron is delivered by the intestinal mucosa cells to the transferrin. This iron transport protein has two iron-bonding sites. It is synthesised in the liver. There is therefore a mechanism whereby iron can be taken from cells (e.g. small-intestine mucosa or macrophages) and delivered to specific membrane receptors of erythroblasts, placenta cells or liver cells. Through endocytosis, the transferrin-iron-receptor complex arrives at the erythrocyte precursor cells, where the iron is delivered to the mitochondria, where haem is formed from iron and protoporphyrin.
Iron not needed for erythropoiesis is conveyed by transferrin to two kinds of storage pools. The most important store is ferritin. This is a heterogeneous family of proteins surrounding an iron nucleus. It is soluble and is the active storage form in the liver (hepacytes), bone marrow, spleen (macrophages), erythrocytes and in the serum (about 100 ng/ml). The tissue ferritin pool is very labile and quickly available when iron is needed. The circulating serum ferritin comes from the reticulo-endothelial system (RES) and the concentration in circulation varies in parallel with the total body iron (each ng/ml corresponds to 8 mg of iron supply).
To sum up, in iron metabolism
all cells of the RES are stores of ferritin and haemosiderin,
Hb-synthesising, iron-consuming parent cells are converted by Epo into erythrocytes and
iron is transported by transferrin from the store to the erythrocytes in process of formation and back from dead erythrocytes into the store.
In bone metabolism, on analogy with iron metabolism
the bone stores hydroxyl apatite crystals (and collagen) and the osteoclast xe2x80x9clabilisesxe2x80x9d the hydroxyl apatite by breaking it down to a basic skeleton.
If no disturbance occurs, the basic skeleton is used for synthesis.
parent cells which synthesise hydroxyl apatite (and collagen) and consume calcium and phosphate are converted by PTH (+IGF 1+2) into osteoblasts and
calcium and phosphate are transported from the store to the osteoblasts in process of formation and back to the store after synthesis of hydroxyl apatite.
As early as 1983, S. G. Massry (Kidney International 24, (16) 204-207) and also F. N. Hutchison and J. Jones (AJKD Vol. 29, No. 5, May 1997) described how in cases of hyperparathyroidism, i.e. at very high PTH concentrations, erythropoiesis is suppressed. In other words a high concentration of PTH ( greater than  greater than 60 ng/l) slows down the formation of Epo in the organism, thus inevitably reducing the formation of erythrocytes.
PTH is therefore an important regulator in the human organism, since both iron metabolism and bone metabolism are regulated by PTH. PTH of course regulates resorption of calcium, phosphate, magnesium and iron.
However, the following weak points may occur in iron and bone metabolism:
insufficient differentiation of the parent cells by Epo or PTH,
xe2x80x9clabilisationxe2x80x9d of the form of storage (iron nucleus in ferritin or hydroxyl apatite in collagen by osteoclast) or
insufficient transport of iron to erythrocytes or hydroxyl apatite (calcium and phosphate) to the osteoblasts.
Treatment is possible for disturbances of erythropoiesis in the case of renal anaemia or iron deficiency anaemia but no successful treatment has been found for haemochromatoses. Bloodletting is the only method at present.
Haemochromatoses are iron storage diseases resulting from an excess of iron in the organism, particularly in the parenchymatous organs, associated with increased iron absorption and massive deposition of iron in the form of haemosiderin in numerous organisms and in the monocyte-macrophage system. A distinction is made between primary (idiopathic) haemochromatoses and secondary (erythropoietic) haemochromatoses. Secondary haemochromatoses are disturbances in blood formation resulting in haemosiderosis (inherited excess of iron in the organism). Primary (idiopathic) haemochromatoses appear in the form of liver cirrhosis (pigment cirrhosis), bronze discoloration of the skin and failure of endocrine and exocrine glands (hypogonadism, insulin-dependent diabetes mellitus or xe2x80x9cbronze diabetesxe2x80x9d), cardiac insufficiency, and hair loss.
The object of the invention therefore is to provide pharmaceutical preparations suitable for treatment of haemochromatoses and containing optimum proportions of active substances.
It has unexpectedly been found that erythropoietin preparations in low dosage are suitable for treatment of haemochromatoses.
The invention therefore relates to use of Epo for producing pharmaceutical preparations for treatment of haemochromatoses.
According to the invention the preparations contain 500 to 5000 U of Epo and are preferably used for treatment of patients with secondary haemochromatoses.
The erythropoietin preparations according to the invention can be active substances comparable in physiological effect with human erythropoietin. According to the invention, these active substances will for short be called Epo or Epo-preparations. Recombinant Epo (rhEpo; see European Patent Specifications EP-B1 0 205 564 and EP-B1 0 411 678) or appropriate modifications of these proteins are examples of suitable Epo preparations. The modifications can e.g. be proteins with a molecular weight higher (see EP 0 148 605) or lower than 34000 Da (the SDS-PAGE molecular weight of urinary Epo) or isoforms of the enzyme or protein with varying glycosylation. More particularly, use can also be made of proteins chemically modified by (polyethylene glycol) polyethylene glycol (PEG). In principle, use can also be made of proteins derived by deletion, substitution or prolongation of one or more amino acids in the amino acid sequence of natural Epo having a length of 166 amino acids. The physiological properties of these proteins are substantially comparable with rhEpo. More particularly these proteins have biological properties causing bone marrow cells to increase production of reticulocytes and red blood corpuscles and/or to increase haemoglobin synthesis. Instead of these proteins, use can be made of low-molecular substances called Epo-mimetics, which bond to the same biological receptor. These mimetics can also preferably be administered orally. The preferred amount of these proteins or mimetics is obtained by comparing the biological activity of these active substances with that of Epo.
For use in therapy, the combination preparation according to the invention contains e.g. 500 to 5000 U of an Epo preparation (the abbreviation xe2x80x9cUxe2x80x9d is an alternative to xe2x80x9cIExe2x80x9d,=international units). The preferred dosages are 500 U, 1000 U, 2000 U or 5000 U per individual dose.
It has also been found, however, that iron resorption is quantitatively much less important than disturbance in calcium (phosphate) resorption (the concentration ratio is 1:1000) and that a disturbance in calcium (phosphate) resorption is the main factor, apart from disturbance in iron resorption, in primary haemochromatoses.
It has thus been found that on the one hand erythropoiesis is assisted only if transferrin can be fully loaded transferrin (xe2x80x9cTFxe2x80x9dxc3x972), i.e. if the iron store is full. Otherwise the store is filled with half-loaded transferrin (Tfxc3x971).
On the other hand osteoblasts are supplied with sufficient calcium and phosphate only when the store (bone) is full of hydroxyl apatite. If not, the consequence in the case of iron will be fatal. Too much iron is resorbed, because too much calcium (phosphate) is resorbed. Since Epo is brought to a stop by PTH, erythropoiesis is not assisted.
According to the invention this can be avoided by controlled administration of Epo in low dosage in combination with calcium and/or phosphate compounds.
The invention therefore also relates to use of Epo in low dosage and of calcium and/or phosphate compounds for producing a pharmaceutical preparation for treatment of primary haemochromatoses and a combination preparation consisting of an erythropoietin preparation together with calcium and/or phosphate compounds and suitable for treatment of primary haemochromatosis. The active substances in the combination preparation can be in separate forms of administration or in a single form.
The combination preparation contains optimum proportions of Epo and calcium and/or phosphate for treatment of primary haemochromatosis. According to the invention, in addition to Epo it preferably contains a calcium phosphate complex compound. Particularly preferably it contains an apatite compound having the formula Ca2+[Ca3(PO4)2]32, preferably in the form of a gluconate. Alternatively the combination preparation can contain a calcium compound in the form of calcium gluconate and/or a phosphate compound in the form of glucose-1-phosphate or potassium hydrogen phosphate.
The preferred ratio of calcium to phosphate (or phosphorus) in the combination preparation is from 11:5 to 9:7, preferably 10:6.
Alternatively the preparation can contain Epo in combination with a calcium compound only or in combination with a phosphate compound only.
In a preferred variant the combination preparation contains 500-5000 U of recombinant human erythropoietin (xe2x80x9crhEPOxe2x80x9d), 20-500 mg calcium and/or 10-250 mg phosphorus (or 30-750 mg phosphate). These values mean that a suitable calcium or phosphate compound must be applied in a quantity such that 20-500 mg calcium or 10 to 250 mg phosphorus (or 30-750 mg phosphate) are supplied.
The combination preparations according to the invention can contain Epo and the calcium and/or phosphate compounds in a unit package ready for sale (known as a combination package). The medicament packs can also contain either a suitable amount of Epo or a suitable amount of a calcium phosphate compound in the form of an individual preparation, the proportion of constituents of the individual preparation in the package being such that they can be administered according to the invention in combination with the respective other preparation. In such cases the manufacturer or importer of the medicament will label the medicament as legally prescribed in many countries, giving instructions or information about the combined administration of the individual preparations. This also applies to medicament packs which contain a suitable amount of Epo and a suitable amount of a calcium compound and/or a phosphate compound.
Calcium or phosphate preparations for oral or parenteral administration are of use according to the invention. In principle the substances can be individual preparations in which the active substance is a physiologically compatible calcium salt and/or a phosphate compound or a calcium phosphate complex compound, or combination preparations which contain the physiologically compatible preparation together with other active substances such as vitamins, folic acid, thiamine chloride, riboflavin, pyridoxine, ascorbic acid or nicotine amide.
Alternatively according to the invention, Epo and calcium and/or phosphate compounds in the form of separate pharmaceutical formulations can be applied simultaneously or successively (free combination). This free combination, which can be provided in a unit pack, has the advantage of great flexibility.
Usually the free combination is supplied in the form of a single unit pack containing at least two containers, the first being an Epo preparation in a suitable form for application (freeze-dried, injection or infusion solution or for oral application if required) whereas the second is the calcium phosphate or calcium and/or phosphate preparation in a suitable form for application. By this means, each patient can individually be provided with the prescribed quantity of Epo and calcium or phosphate compound. These combination preparations have the additional advantage of more successful treatment, since in each case the optimum adjusted amount of the individual preparations is fixed and it is practically impossible to confuse them with other commercial individual preparations on offer in different dosages.
The combination preparations according to the invention also reduce the risk of an accidental excess of calcium and/or phosphate, which might happen if conventional calcium or phosphate preparations from separate packages were applied together with Epo. The combination preparations according to the invention ensure reliable treatment and are easy to handle. Another possibility in the present case is to use one active substance as an injection solution and the other active substance in a form for oral administration.
In the case when the Epo preparation is available in freeze-dried form, the medicament packs (combination packs) contain the appropriate quantity of the Epo preparation in glass ampoules or in capsules. The calcium/phosphate preparation can be in solid form (tablets, powder, granulate, freeze-dried, etc) or in liquid form in separate containers. The combination pack also contains a reconstitution solution for dissolving the freeze-dried Epo, either alone or together with the solid calcium/phosphate preparation. If the calcium/phosphate preparation is in the form of a solution ready for use, the solution can be mixed with the Epo solution if they are to be applied together. Alternatively in principle the calcium/ phosphate preparation can be supplied in concentrated form for addition to conventional infusion solutions, so that application is slower and over a number of hours.
Another possibility according to the invention is to provide individual Epo preparations and calcium and/or phosphate compounds ready for sale in the form of independent medicaments suitable for application, the individual preparations being packaged so as to contain the amounts of individual substances required for the Epo preparation combined with calcium and/or phosphate according to the invention.
Another alternative when using the combination preparations is to administer Epo and calcium and/or phosphate compounds in a xe2x80x9cfixed combinationxe2x80x9d, i.e. in a single pharmaceutical formulation containing both compounds. The formulation can e.g. be an injection solution or infusion solution or freeze-dried, e.g. packed into ampoules. A fixed combination of active substances in freeze-dried form has the advantage of simple and safe handling. The freeze-dried substance is dissolved in the ampoule by adding conventional pharmaceutical injection media and is applied intravenously.
The pharmaceutical forms for administration are produced by conventional galenic methods using conventional pharmaceutical adjuvants.
In therapy using the combination preparation according to the invention
(a) the ferritin concentration must be determined, since it is a measure of the level of the iron store,
(b) the transferrin saturation and transferrin receptor must be determined, indicating the loading capacity of the transport protein, and
(c) the level of apatite and collagen in the bone must be determined, using various diagnostic parameters.
The target values, which usually indicate a good adjustment of the iron and bone metabolism, are given in Tables 1 and 2.
Accordingly the following are the main parameters to be checked during therapy:
a) For iron metabolism:
Ferritin (ng/ml)
Transferrin saturation (%) transferrin receptor (mg/l)
The synthesising capacity of the parent cells, based on haemotological control parameters such as haemoglobin (Hb), haematocrit (proportion of red blood corpuscles in the total volume), erythrocytes, hypochromic erythrocytes, reticulocytes, hypochromic reticulocytes.
Ferritin is an iron-storing glycoprotein. Isoferritins typical of tissue exist and can be determined immunologically in the serum. The ferritin value is a measure of the iron stored in the tissue. In most laboratories the normal range is between 30 and 300 ng/ml and the geometrical mean is 88 for men and 49 for women. The serum ferritin values are closely related to the total iron supply in the body. The levels are therefore higher when there is an excess of iron.
In order to determine the ferritin level, erythrocytes are separated from glucocytes and thrombocytes (which also contain ferritin) by centrifuging in blood treated with heparin (or EDTA or citrate). The erythrocytes are then lysed and the stored ferritin is determined immunologically. The erythrocyte ferritin indicates the level of the iron store during the preceding three months (i.e. during the life of an erythrocyte). The normal values are generally between 5 and 48 attograms (ag) per erythrocyte. Values below 5 occur in iron deficiency anaemia whereas elevated values (often  greater than 100) occur when there is an excess of iron.
Determination of the serum ferritin is another frequently-used method, e.g. using the Boehringer Mannheim ferritin test, which can be automated and is based on the principle of the immunological agglutination test with amplification of the reaction by latex.
Tranferrin saturation is defined as the ratio of the concentration of iron in the serum/plasma to the concentration of transferrin in the serum plasma (multiplied by a correction factor of 1.41). This number is dimensionless, irrespective of the state of the patient. It is calculated from the formula:
Transferrin saturation (%)=(iron[mg/dl]xc3x97100)/(transferrin[m/g/dl]xc3x971.41)
The serum transferrin receptor can also be determined by the enzyme-intensified immune absorption test (enzyme-linked immunosorbent assay =ELISA) or the latex test, using a monoclonal antibody against the soluble receptor. The reference range is between 0.5 and 3 mg/l.
b) The diagnostic parameters for bone metabolism are as follows:
Caxc3x97PO4 in the serum (in mg/dl)2, and calcium and phosphate in the serum and in the urine (in mmol/l ),
the level of apatite in the collagen, by determination of bone, apatite (xe2x80x9cAPxe2x80x9d), pyridinolines (xe2x80x9cCrosslinksxe2x80x9d and xe2x80x9cCrosslabsxe2x80x9d), 3-hydroxyproline and N-terminal crosslinked peptide (xe2x80x9cNPxxe2x80x9d),
the concentration of osteocalcine, which can also store and transport apatite. It occurs in the serum in a concentration comparable to that of ferritin (about 10-100 xcexcg/l).
The normal values for calcium and phosphate in the serum are 2.2-2.6 mmol/l corresponding to 8.6-10.2 mg/dl for calcium and 1.0-1.5 mmol/l corresponding to 2.7-4.5 mg/dl for phosphate. This results in a product for Caxc3x97PO4 which can be regarded as acceptable at values between 23 (mg/dl)2(8.6xc3x972.7) and 46 (mg/dl)2(10.2xc3x974.5).
With regard to the composition of the hydroxyl apatite (Ca10(PO4)6(OH)2) in the bone, optimum loading capacity occurs at a product Caxc3x97PO4 of about 40 (mg/dl)2 (see Table 3).
Collagen, the most important protein for storing apatite, is determined indirectly via markers of the bone construction in the serum such as bone ALP and terminal propeptides which split off during formation of collagen fibrils, and via markers of bone disintegration such as pyrodinolines (crosslinks), 3-hydroxy-proline or NTx (N-terminal crosslinked peptides) in the urine.
The serum also contains a concentration of about 10-100 xcexcg/l (normal value) of the matrix protein osteocalcine, which has glutaminic acid radicals enabling it to bond to crystals of the calcium mineral hydroxy apatite incorporated in the bone.
The product calciumxc3x97phosphate is the most important quantity in diagnostic investigations and is determined in the serum. It has been found that the bone is sufficiently filled with Ca and PO4 if the product Caxc3x97PO4 is 30-50 (mg/dl)2, the target value being 40 (mg/dl)2. A value above 60 (mg/dl)2 Caxc3x97PO4 indicates calcification of an organ. At values below 25 mg/dl there is a risk of hypocalcaemia or hypophosphataemia, which may result in increased extraction of PTH and cutting-off of Epo.
Accordingly the invention relates to use of Epo in low dosage for producing pharmaceutical preparations for treatment of haemochromatoses. For treatment of secondary haemochromatoses, the preparations contain 500-5000 U of Epo. Treatment is given once to five times, preferably up to four times, a week, without exceeding a total amount of 5000 U Epo per patient per week.
The invention also relates to use of Epo in combination with calcium and/or phosphate compounds for producing combination preparations for treatment of primary haemochromatoses and pharmaceutical combination preparations comprising erythropoietin (Epo) and calcium and/or phosphate compounds, which can be in separate forms for administration or in a uniform form for administration. The following embodiments are particularly important:
Combination preparations containing Epo and a calcium phosphate complex compound. Combination preparations in which the calcium phosphate complex compound is an apatite compound having the formula Ca2+[Ca3(PO4)2]32, preferably in the form of a gluconate. Combination preparations containing Epo, a calcium and a phosphate compound. Combination preparations in which the phosphate compound is glucose-1-phosphate or potassium hydrogen phosphate. Combination preparations where the ratio of calcium to phosphate is between 11:5 and 9:7, preferably 10:6. Combination preparations containing 500-5000 U Epo, 20-500 mg calcium and/or 10-250 mg phosphorus (or 30-750 mg phosphate) in separate forms for administration as injection or infusion solutions or freeze-dried or in a uniform form for administration.
In therapy using the combination preparation according to the invention, the maximum weekly dosage can be decided in simple manner. Treatment is given one to five times, preferably up to four times a week, the total amount of Epo not exceeding 5000 U and of calcium not exceeding 4xc3x97500 mg per patient per week.
To avoid over production of erythrocytes, the treatment may advantageously be accompanied by bloodletting.
The invention also relates to methods of producing pharmaceutical Epo preparations and combination preparations as described hereinbefore, wherein Epo, calcium and/or phosphate compounds are formulated with conventional pharmaceutical excipients or adjuvants and are provided in a uniform or separate forms for administration.
The invention also relates to use of Epo for producing a pharmaceutical preparation for treatment of secondary haemochromatoses and use of Epo and calcium and/or phosphate compounds for producing combination preparations for treatment of primary haemochromatoses.
The invention will now be explained in further detail with reference to examples.

Use of erythropoietin for the treatment of haemochromatoses