Botulinum toxin pharmaceutical compositions

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DESCRIPTION

Botulinum toxin pharmaceutical compositions

BACKGROUND

The present invention relates to pharmaceutical compositions as defined in the claims. In particular, the present invention relates to pharmaceutical compositions containing a botulinum toxin and a recombinantly manufactured human serum albumin.

A pharmaceutical composition is a formulation containing one or more active ingredients as well as one or more excipients, stabilizers or bulking agents, which is suitable for administration to a human patient to achieve a desired therapeutic effect or diagnostic result.

For its stability in storage and convenience in handling, a pharmaceutical composition can be formulated as a lyophilized (ie freeze dried) or vacuum dried powder that can be reconstituted with saline or water prior to administration to a patient. Alternatively, the pharmaceutical composition can be formulated in the form of an aqueous solution. A pharmaceutical composition can contain a protein active ingredient. Unfortunately, proteins can be very difficult to stabilize, resulting in loss of protein and / or loss of protein activity during formulation, reconstitution (if required), and during storage prior to use of a pharmaceutical composition that contains protein. Stability problems can occur due to denaturation, degradation, dimerization, and / or polymerization of the protein. Various excipients, such as albumin and gelatin have been used with varying degrees of success in trying to stabilize a protein active ingredient present in a pharmaceutical composition. Additionally, cryoprotectants such as alcohols have been used to reduce protein denaturation under the freeze-dried conditions of lyophilization.

Albumin

Albumins are small and abundant plasma proteins. Human serum albumin has a molecular weight of about 69 kilodaltons (kD) and has been used as a non-active ingredient in a pharmaceutical composition where it can serve as a filler and stabilizer for certain protein active ingredients present in a pharmaceutical composition.

The stabilizing function of albumin in a pharmaceutical composition can be present both during the multistage formulation of the pharmaceutical composition and in the subsequent reconstitution of the formulated pharmaceutical composition. Thus, stability by means of albumin can be imparted to a protein active ingredient in a pharmaceutical composition, for example, (1) by reducing the adhesion (commonly referred to as adhesiveness) of the protein active ingredient to surfaces, such as the surfaces of the laboratory glassware, containers, to the vial in which the pharmaceutical composition is reconstituted and to the inner surface of a syringe used to inject the pharmaceutical composition. Adhesion of a protein active ingredient to surfaces can lead to loss of active ingredient and denaturation of the remaining retained protein active ingredient, both of which reduce the total activity of the active ingredient present in the pharmaceutical composition, and; (2) reducing the denaturation of the active ingredient, which can occur when preparing a low dilution solution of the active ingredient.

In addition to being able to stabilize a protein active ingredient in a pharmaceutical composition, albumin also has the advantage of generally negligible immunogenicity when injected into a human patient. A compound with appreciable immunogenicity can cause the production of antibodies against it, which can lead to an anaphylactic reaction and / or the development of drug resistance, making the disease or disorder to be treated potentially refractory to the pharmaceutical composition that it has an immunogenic component.

Unfortunately, despite its known stabilizing effect, there are significant drawbacks to the use of albumin in a pharmaceutical composition. For example, albumins are expensive and increasingly difficult to obtain. Furthermore, blood products such as albumin, when administered to a patient may subject the patient to a potential risk of receiving blood-borne pathogens or infectious agents. Thus, it is known that the possibility exists that the presence of albumin in a pharmaceutical composition may result in the inadvertent incorporation of infectious elements into the pharmaceutical composition. For example, it has been reported that the use of albumin can transmit prions to a pharmaceutical composition. A prion is an infectious protein particle that is hypothesized to arise as an abnormal conformational form of the same nucleic acid sequence that makes normal protein. It has been further hypothesized that infectivity resides in a recruitment reaction of the normal protein soform to the prion protein protein at a posttranslational level. Apparently normal endogenous cellular protein is induced to erroneously fold into a pathogenic prion conformation. Significantly, several lots of human serum albumin have been withdrawn from distribution following a determination that a blood donor to a blood bank from which albumin was prepared was diagnosed with Creutzfeldt-Jacob disease.

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Creutzfeldt-Jacob disease (sometimes characterized as rapidly advancing Alzheimer's disease) is a rare neurodegenerative disorder of transmissible human spongiform encephalopathy, in which the transmissible agent is apparently an abnormal isoform of a prion protein. An individual with Creutzfeldt-Jacob disease can deteriorate from apparent perfect health to akinetic mutism within six months. The possible iatrogenic transmission of Creutzfeldt-Jacob disease by albumin transfusion has been published and it has been speculated that insufficient protection is provided against the transmission of Creutzfeldt-Jacob disease by the usual methods of preparing albumin, methods that They include the removal of cellular elements from the blood and heating to 60 degrees Celsius for 10 hours. Thus, there may be a potential risk of acquiring a prion-mediated disease, such as Creutzfeldt-Jacob disease, from the administration of a pharmaceutical composition containing human plasma protein concentrates, such as serum albumin.

Gelatin has been used in some protein active ingredient pharmaceutical compositions as a substitute for albumin. Notably, gelatin is an animal derived protein and therefore poses the same risk of potential infectivity that albumin may possess. Accordingly, it is desirable to find a substitute for albumin that is not a blood fraction, and preferably the substitute for albumin is not gelatin and is not derived from any animal source.

Botulinum toxin

The anaerobic gram positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, tobulin toxin, which causes a neuroparalytic disease in humans and animals called botulism. Clostridium botulinum and its spores are commonly found in soil, and the bacteria can grow in improperly sterilized and sealed food containers from household containers, which are the cause of many cases of botulism. The effects of botulism typically appear 18 to 36 hours after eating food infected with a Clostridium botulinum culture or spores. Botulinum toxin can apparently pass through the lining of the intestine and attack peripheral motor neurons. Symptoms of botulinum toxin poisoning can progress from difficulty walking, swallowing, and speaking to paralysis of the respiratory muscles and death.

Botulinum toxin type A is the deadliest natural biological agent known to man. About 50 picograms of botulinum toxin (purified neurotoxin complex) type A is an LD₅₀ in mice. Interestingly, on a molar basis, botulinum toxin type A is 1.8 billion times more lethal than diphtheria, 600 million times more lethal than sodium cyanide, 30 million times more lethal than cobrotoxin, and 12 million times times more lethal than cholera. Singh; Critical Aspects of Bacterial Protein Toxins, pages 63-84 (chapter 4) of Natural toxins II, edited by BR Singh et al., Plenum Press, New York (1976) (in which the said LD50 of botulinum toxin type A of 0.3 ng which is equal to 1U is corrected for the fact that around 0.05 ng of BOTOX® is equal to 1 unit). One unit (U) of botulinum toxin is defined as the LD50 per intraperitoneal injection in female Swiss Webster mice weighing 18-20 grams each. Seven immunologically distinct botulinum neurotoxins have been characterized, these being respectively botulinum toxin serotypes A, B, C1, D, E, F and G, each of which is distinguished by neutralization with a specific type of antibody. The different botulinum toxin serotypes vary in the animal species they affect and in the severity and duration of the paralysis they evoke. For example, botulinum toxin type A has been determined to be 500 times more potent, as measured by the rate of paralysis produced in the rat, than botulinum toxin type B. Additionally, botulinum toxin type Type B is non-toxic in primates at a dose of 480 U / kg which is about 12 times the primate LD50 for botulinum toxin type A. Botulinum toxins appear to bind with high affinity to cholinergic motor neurons, translocate within the neuron, and block presynaptic acetylcholine release.

Botulinum toxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by overactive skeletal muscles. Botulinum toxin type A was approved by the US Food and Drug Administration in 1989 for the treatment of essential blepharospasm, strabismus, and hemifacial spasm in patients around 12 years of age. The clinical effects of botulinum toxin type A are usually seen within a week of injection. The typical duration of symptomatic relief (ie, paralysis of flabby muscles) from a single intramuscular injection of botulinum toxin type A can be around three months.

Although all botulinum toxin serotypes apparently inhibit the shedding of the neurotransmitter acetylcholine at the neuromuscular junction, they do so by affecting different neurosecretory proteins and / or cleaving these proteins at different sites. Botulinum toxin A is a zinc endopeptidase that can specifically hydrolyze a peptide linkage of the intracellular vesicle-associated protein SNAP-25. Botulinum type E also cleaves the 25 kiloDalton (kD) synaptosomal associated protein (SNAP-25), but targets different amino acid sequences within this protein compared to botulinum toxin type A. types B, D, F and G act on the vesicle-associated protein (VAMP, also called synaptobrevin), each serotype cleaving the protein at a different site. Finally, botulinum toxin type C1 has been shown to cleave both syntaxin and SNAP-25. These differences in mechanism of action can affect the relative potency and / or duration of action of various botulinum toxin serotypes.

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The molecular weight of the botulinum toxin molecule, for all seven known botulinum toxin serotypes, is around 150 kD. Interestingly, botulinum toxins are shed by Clostridium bacteria in the form of complexes comprising the 150 kD botulinum toxin protein molecule along with associated notoxin proteins. Thus, the botulinum toxin type A complex can be produced by Clostridium bacteria in 900 kD, 500 kD and 300 kD forms. Botulinum toxin types B and Ci are apparently produced only as a 500 kD complex. Botulinum toxin type D is produced as both 300 kD and 500 kD complexes. Finally, botulinum toxins types E and F are produced only as roughly 300 kD complexes. The complexes (ie, molecular weight greater than about 150 kD) are believed to contain a non-toxin hemagglutinin protein and a non-toxin and non-toxic non-hemagglutinin protein. These two notoxin proteins (which together with the botulinum toxin molecule may comprise the relevant neurotoxin complex) can act to provide stability against denaturation of the botulinum toxin molecule and protection against digestive acids when the toxin is ingested. Additionally, it is possible that larger (greater than about 150 kD molecular weight) botulinum toxin complexes may result in a slower rate of diffusion of botulinum toxin away from the site of intramuscular injection of a botulinum toxin complex. . The toxin complexes can be dissociated into toxin protein and hemagglutinin proteins by treating the complex with red blood cells at pH 7.3. The toxin protein has notable instability when removing the hemagglutinin protein.

All botulinum toxin serotypes made by the bacterium Clostridium botulinum are inactive single chain proteins that must be cleaved or cleaved by proteases to become neuroactive. The bacterial strains that make botulinum toxins of serotypes A and G possess endogenous proteases and serotypes A and G can therefore be recovered from bacterial cultures predominantly in their active form. In contrast, botulinum toxins of serotypes Oi, D, and E are synthesized by non-proteolytic strains and are therefore typically inactivated when recovered from culture. Serotypes B and F are produced by both proteolytic and non-proteolytic strains and therefore can be removed in the active or inactive form. However, even proteolytic strains that produce, for example, botulinum toxin type B serotype only cleave a portion of the toxin produced. The exact ratio of cut to uncut molecules depends on the length of incubation and the temperature of the culture. Therefore, a certain percentage of any preparation of, for example, botulinum toxin type B is likely to be inactive, accounting for the known significantly lower potency of botulinum toxin type B compared to botulinum toxin type A. The presence of inactive botulinum toxin molecules in a clinical preparation will contribute to the total protein load of the preparation, which has been associated with increased antigenicity, without contributing to its clinical efficacy. Additionally, botulinum toxin type B is known to have, when injected intramuscularly, a shorter duration of activity and is also less potent than botulinum toxin type A at the same dose level.

High quality crystalline botulinum toxin type A can be produced from the Hall A strain of Clostridium botulinum with characteristics of> 3 χ 10⁷ U / mg, an A26o / A278 of less than 0.60, and a distinct band pattern on gel electrophoresis. The well-known Shantz procedure can be used to obtain crystalline botulinum toxin type A, as described in Shantz, EJ et al., Properties and use of Botulinum toxin and Other Microbial Neurotoxins in Medicine, Microbiol Rev. 56: 80:99 ( 1922). Generally, the botulinum toxin type A complex can be isolated and purified from anaerobic fermentation by culturing Clostridium botulinum type A in an appropriate medium. Crude toxin can be collected by precipitation with sulfuric acid and concentrated by ultramicrofiltration. Purification can be carried out by dissolving the acidic precipitate in calcium chloride. The toxin can then be precipitated with cold ethanol. The precipitate can be dissolved in sodium phosphate buffer and centrifuged. Upon drying, approximately 900 kD crystalline botulinum toxin type A complex with a specific potency of 3 × 10 can then be obtained.⁷ LD50 U / mg or higher. This known procedure can also be used, when separating non-toxin proteins, to obtain pure botulinum toxins, such as for example: purified botulinum toxin type A with a molecular weight of about 150 kD with a specific potency of 1-2 x 10⁸ LD50 U / mg or higher; Purified botulinum toxin type B with a molecular weight of approximately 156 kD with a specific potency of 1-2 χ 10⁸ LD50 U / mg or higher, and; Purified botulinum toxin type F with a molecular weight of approximately 155 kD with a specific potency of 1-2 x 10⁷ LD50 U / mg or higher.

Prepared and purified botulinum toxins and appropriate toxin complexes for preparing pharmaceutical formulations are available from List Biological Laboratoires, Inc. Campbell, California; the Center for Applied Microbiology and Research, Porton Down, UK; Wako (Osaka, Japan), as well as Sigma Chemicals of St Louis, Missouri.

Pure botulinum toxin is so labile that it has limited practical utility in preparing a pharmaceutical composition. Furthermore, botulinum toxin complexes, such as toxin type A complex are also extremely susceptible to denaturation due to surface denaturation, heat, and alkaline conditions. The inactivated toxin forms toxoid proteins that can be immunogenic. The resulting antibodies can make a patient refractory to toxin injection.

As with enzymes generally, the biological activities of botulinum toxins (which are intracellular peptidases) depend, at least in part, on their three-dimensional conformation. Thus, botulinum toxin type A is detoxified by heat, swelling of the surface with various chemicals, and drying of the surface. Additionally, it is known that dilution of the toxin complex obtained from culture, fermentation and

Known purification at the much lower concentrations used for formulation of the pharmaceutical composition results in rapid detoxification of the toxin unless an appropriate stabilizing agent is present. Dilution of the toxin from milligram quantities to a solution containing nanograms per milliliter presents significant difficulties due to the rapid loss of specific toxicity with such a large dilution. Since the toxin can be used months or years after the pharmaceutical composition containing the toxin is formulated, the toxin must be stabilized with a stabilizing agent. The only successful stabilizing agent for this purpose has been the animal derived proteins albumin and gelatin. And as noted, the presence of animal-derived proteins in the final formulation presents potential problems because certain stable viruses, prions, and other pathogenic or infectious compounds carried by donors can contaminate the toxin.

In addition, any of the severe conditions of pH, temperature, and concentration ranges required to lyophilize (freeze-dry) or vacuum dry a pharmaceutical composition containing a botulinum toxin in a toxin transport and storage format (ready-to-use or reconstitution by a physician) can detoxify the toxin. Thus, proteins derived from animals or from a donor bank such as gelatin and serum albumin have been used with some success to stabilize botulinum toxin.

A widely available pharmaceutical composition containing botulinum toxin is sold under the trade name BOTOX® (available from Allergan, Inc. of Irvine, California). BOTOX® consists of a complex of botulinum toxin type A, albumin and sodium chloride packaged in sterile vacuum-dried form. Botulinum toxin type A is made from a culture of the Hall strain of Clostridium botulinum grown in a medium containing NZ-amine and yeast extract. The botulinum toxin type A complex is purified from the culture solution by a series of acid precipitations to a crystalline complex consisting of the active high molecular weight toxin protein and an associated hemagglutinin protein. The crystalline complex is redissolved in a solution containing saline and albumin and sterile filtered (0.2 microns) prior to vacuum drying. BOTOX® can be reconstituted with sterile, non-preserved saline prior to intramuscular injection. Each vial of BOTOX® contains approximately 100 units (U) of Clostridium botulinum toxin type A complex, 0.5 milligrams of human serum albumin, and 0.9 milligrams of sodium chloride in a sterile, vacuum-dried form without a preservative. .

To reconstitute BOTOX® Sterile Normal Saline without a preservative; 0.9% sodium chloride injection is used by withdrawing the appropriate amount of diluent into the appropriate size syringe. Since BOTOX® is denatured by bubbling or similar violent shaking, the diluent is gently injected into the vial. BOTOX® must be administered within four hours after reconstitution. During this period of time, the reconstituted BOTOX® is stored in a refrigerator (2^S to 8^SC). Reconstituted BOTOX® is clear, colorless and free of particulate matter. Vacuum dried product is stored in a refrigerator at or below 5^SC. BOTOX® is administered within four hours after it is removed to the vial from the refrigerator and reconstituted. During these four hours, the reconstituted BOTOX® can be stored in a refrigerator (2^S to 8^SC).

It has been reported that a suitable alternative to albumin as a stabilizer for botulinum toxin may be another protein or alternatively a low molecular weight (non-protein) compound. Carpender et al., Interactions of Stabilizing Additives with Proteins During Freeze-Thawing and Freeze-Drying, International Symposium on Biological Product freeze-Drying and Formulation, 24-26 October 1990; Karger (1992), 225-239.

Many substances commonly used as carriers and bulking agents in pharmaceutical compositions have been shown to be unsuitable as substitutes for albumin in the pharmaceutical composition containing the neurotoxin. For example, cellobiose disaccharide has been found to be inappropriate as a toxin stabilizer. Thus, the use of cellobiose as an excipient together with albumin and sodium chloride is known to result in a much lower level of toxicity (10% recovery) after lyophilization of crystalline botulinum toxin type A with these excipients. , compared to toxicity after lyophilization with albumin alone (> 75% to> 90% recovery). Goodnough et al., Stabilization of Botulinum Toxin Type A During Lyophilization, App & Envir. Micro. 58 (10) 3426-3428 (1992).

Furthermore, saccharides, including polysaccharides, are generally poor candidates to serve as protein stabilizers. Thus, a pharmaceutical composition containing a protein active ingredient is known to be inherently unstable if the protein formulation comprises a saccharide (such as glucose or a glucose polymer) or carbohydrates because proteins and glucose are known to interact together. and they undergo the well-known Maillard reaction, due, ie, to the reducing nature of glucose and glucose polymers. Much work has been devoted to largely unsuccessful attempts to prevent this protein-saccharide reaction, for example by reducing moisture or using non-reducing sugars. Significantly, the degrading pathway of the Maillard reaction can result in a therapeutic insufficiency of the protein active ingredient. A pharmaceutical formulation comprising protein and a saccharide, carbohydrate, or reducing sugar, such as a glucose polymer, is therefore inherently unstable and cannot be stored for a long period of time without significant loss of the protein's desired biological activity. active ingredient.

Notably, one of the reasons that human serum albumin can function effectively as a stabilizer for a protein active ingredient in a pharmaceutical composition is because albumin, being a protein, does not undergo the Maillard reaction with the protein active ingredient in a pharmaceutical composition. Therefore, one would expect to search and find a substitute for albumin among other proteins.

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Finding an appropriate substitute for albumin as a stabilizer for botulinum toxin present in a pharmaceutical composition is difficult and problematic because it is believed that albumin functions in a pharmaceutical composition as more than a mere bulking agent. Thus, albumin appears to be able to interact with botulinum toxin to increase the potency of the neurotoxin. For example, it is known that bovine serum albumin can act as more than a mere stabilizing excipient for botulinum toxin type A, since bovine serum albumin apparently also accelerates the rate of catalysis of synthetic peptide substrates, substrates that resemble substrate intraneuronal SNAP-25 for botulinum toxin type A, Schmidt et al., Endoproteinase Activity of Type A Botulinum Neurotoxin Substrate Requirements and Activation by Serum Albumin, J. of Protein Chemistry, 16 (1), 19-26 (1997). Thus, albumin may have a potentiating effect, apparently affecting rate kinetics, on the intracellular proteolytic action of a botulinum toxin on the toxin substrate. This potentiating effect may be due to the albumin that has accompanied botulinum toxin in the endocytosis of the toxin in a target neuron or the potentiating effect may be due to the pre-existing presence of cytoplasmic albumin within the previously neuron protein. to the endocytosis of botulinum toxin.

The discovery of the presence of a kinetic rate-stimulating effect of bovine serum albumin on the proteolytic activity of botulinum toxin type A makes the search for an appropriate substitute for albumin in a pharmaceutical formulation containing toxin especially problematic. botulinum. Thus, an albumin substitute with desirable toxin stabilizing characteristics may have an unknown and possibly detrimental effect on the rate of toxin catalysis of the substrate, since at least relative to bovine serum albumin both Characteristics (stabilization of the toxin and enhancement of the catalysis of the toxin substrate) are apparently inherent in the albumin carrier itself. This enhancing effect of albumin shows that albumin does not act as a mere excipient in the formulation and therefore makes the search for an appropriate substitute for albumin more difficult.

Additionally there are many unique characteristics of botulinum toxin and its formulation in an appropriate pharmaceutical composition that restrict and prevent and make the search for a substitute for the albumin used in current botulinum toxin-containing pharmaceutical formulations very problematic. Examples of four of these unique characteristics follow. First, botulinum toxin is a relatively large protein for incorporation into a pharmaceutical formulation (the molecular weight of a botulinum toxin type A complex is 900 kD) and is therefore inherently brittle and labile. The size of the toxin complex makes it much more fragile and labile than smaller, less complex proteins, hence the difficulties of formulation and handling of the formulation if the stability of the toxin is to be maintained. Accordingly, a substitute for albumin must be capable of interacting with the toxin in a way that does not denature, fragment, or otherwise detoxify the toxin molecule or cause dissociation of the non-toxin proteins present in the toxin complex. . Second, as the most lethal known biological product, exceptional safety, precision, and accuracy are required for all stages of the formulation of a pharmaceutical composition containing botulinum toxin. Thus, a potential preferred substitute for albumin must not itself be toxic or difficult to handle so as not to exacerbate the already extremely demanding formulation requirements of the botulinum toxin-containing pharmaceutical composition.

Third, since botulinum toxin was the first microbial toxin to be approved for injection for the treatment of human disease, specific protocols had to be developed and approved for cultivation, mass production, formulation into a pharmaceutical compound, and use of the toxin. botulinum. The purity of the toxin and the dose for injection are important considerations. Production by culture and purification must be carried out in such a way that the toxin is not exposed to any substance that could contaminate the final product even in trace amounts and cause undue reactions in the patient. These restrictions require culture in a simplified medium without the use of animal meat products and purification by procedures that do not involve solvents or synthetic resins. Preparation of the toxin using enzymes, various exchangers, such as those present in chromatography columns, and synthetic solvents can introduce contaminants and are therefore excluded from the preferred formulation steps. Furthermore, botulinum toxin type A denatures already at temperatures above 40^sC, loses toxicity when bubbles form at the air / liquid interface, and denatures in the presence of nitrogen or carbon dioxide.

Fourth, there are particular difficulties in stabilizing botulinum toxin type A, because type A consists of a toxin molecule of around 150 kD in non-covalent association with non-toxin proteins weighing around 750 kD. Non-toxin proteins are believed to preserve or help stabilize the secondary and tertiary structures on which toxicity depends. Procedures or protocols applicable to the stabilization of non-proteins or relatively smaller proteins are not applicable to the problems inherent in the stabilization of botulinum toxin complexes, such as the 900 kD botulinum toxin type A complex. Thus, although from pH 3.5 to 6.8 toxin type A and non-toxin proteins are non-covalently bound together, under slightly alkaline conditions (pH> 7.1) the highly labile toxin is released from the complex. of toxin. Due to its lability, the pure toxin has no or limited utility for medical administration.

In view of the unique nature of botulinum toxin and the requirements described above, it should be seen that the probability of finding an appropriate albumin substitute for the albumin used in current botulinum toxin pharmaceutical compositions actually approaches zero. . Prior to the present invention, only animal derived proteins albumin and gelatin have been known to have utility as stabilizers.

ES 2 275 992 T5 of the botulinum toxin present in a pharmaceutical formulation. Thus, it is known that albumin, by itself or with one or more additional substances such as sodium phosphate or sodium citrate, allows high recovery from botulinum toxin type A toxicity after lyophilization. Unfortunately, as already described, albumin, as a mixed blood product, can, at least potentially, carry infectious or disease-causing elements when present in a pharmaceutical composition. Certainly, any animal product or protein such as gelatin can also potentially contain pyrogens or other substances that can cause adverse reactions when injected into a patient.

Chinese patent application CN 1215084A discusses a botulinum toxin type A formulated with gelatin, a protein derived from an animal. This formulation therefore does not eliminate the risk of transmitting an animal protein derivative or an accompanying infectious element.

Hydroxyethyl starch

A polysaccharide can be made up of hundreds or even thousands of saccharide units linked together by glycoside (ether) linkages. Two important polysaccharides are cellulose and starch. Cellulose is the main structural material in plants, giving plants their rigidity and shape. Starch is the reserve food supply of plants and is found primarily in various seeds and tubers.

Starch is presented in the form of granules whose size and shape are characteristic of the plant from which the starch is obtained. In general about 80% of starch is a water insoluble fraction called amylopectin. Amylopectin is composed of chains of D-glucose units (such as glucopyranose), each unit being linked by an alpha-glycoside linkage to the C-4 of the next glucose unit. Like starch, cellulose is also made up of chains of D-glucose units, in which each unit is linked by a glucoside linkage to the C-4 of the next unit. However, unlike in starch, the glycoside linkages in cellulose are beta linkages. Treatment of cellulose with sulfuric acid and acetic anhydride gives the disaccharide cellobiose. As previously described, attempts to stabilize botulinum toxin using cellobiose have been unsuccessful.

A particular starch derivative obtainable by treating starch with pinedine and ethylenechlorohydrin, 2-hydroxyethyl starch, also referred to as hetastarch. US Patent: No. 4,457,916 describes a combination of a nonionic surfactant and hydroxyethyl starch to stabilize aqueous solutions of tumor necrosis factor (TNF). Additionally, a 6% aqueous solution of 2-hydroxyethyl starch (hetastarch) (available from Du Pont Pharma, Wilmington, Delaware under the trade name HESPAN®, 6% hetastarch in 0.9% sodium chloride injection) is known. . Albumin is known to act as a plasma volume expander when administered intravenously to a patient. HESPAN® has also been administered to patients to achieve a plasma volume expansion effect and in this sense HESPAN® can be considered a substitute for intravenous albumin.

Hetastarch is an artificial colloid derived from a waxy starch composed almost entirely of amylopectin. Hetastarch can be obtained by introducing hydroxyethyl ether groups into glucose units of the starch, and the resulting material can then be hydrolyzed to give a product with an appropriate molecular weight for use as a plasma volume expander. Hetastarch is characterized by its molar substitution and also by its molecular weight. The molar substitution may be about 0.75, meaning that the hetastarch is etherified to the point that for every 100 glucose units of hetastarch there are, on average, about 75 substituent hydroxyethyl groups. The weight average molecular weight of hetastarch is approximately 670 kD with a range of 450 kD to 800 kD and at least 80% of the polymer units being within the range of 20 kD to 2,500 kD. Hydroxyethyl groups are linked by ether linkages mainly at C-2 of the glucose unit and to a lesser extent at O-3 and C-6. The polymer resembles glycogen, and the polymeginated D-glucose units are linked primarily by α-1,4 linkages with occasional α-1,6 branch linkages. The degree of branching is about 1:20, meaning that there is an average of about one α-1.6 branching per 20 glucose monomer units. Hetastarch comprises more than 90% amylopectin.

The plasma volume expansion produced by HESPAN® can be approximated to that obtained by albumin. Hetastarch molecules below 50 kD molecular weight are rapidly eliminated by renal excretion and a single dose of approximately 500 ml of HESPAN® (approximately 30 g) results in the elimination in the urine of approximately 33% of HESPAN® administered in about 24 hours. The hydroxyethyl group of hydroxyethyl starch is not cleaved in vivo, but remains intact and attached to glucose units when excreted. No significant amounts of glucose are produced as hydroxyethylation prevents complete metabolism of the smaller hydroxyethyl starch polymers.

Cellulose can be similarly converted to hydroxyethyl cellulose. The average molecular weight of 2-hydroxyethyl cellulose (a 2-hydroxyethyl cellulose ether) is around 90 kD. Unfortunately, hydroxyethyl cellulose, unlike hydroxyethyl starch, is highly reactive and therefore unsuitable for use as a stabilizer for an active protein ingredient in a pharmaceutical formulation.

What is needed therefore is an appropriate substitute for animal derived protein or donor bank albumin stabilizer used in neurotoxin-containing pharmaceutical compositions.

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Patent applications WO 96/11699 and WO 97/35604, as well as MC Goodnough et al., Appl. Environ. Microbiol., 58, 1992, 3426-3428 and JD Rollnik et al., Eur. Neurol. 2000, 43: 9-12 describe pharmaceutical compositions comprising botulinum neurotoxin and native albumin. However, none of these documents describe or suggest the use of a pharmaceutical composition comprising a botulinum toxin and a recombinantly manufactured albumin. Furthermore, patent application WO 00/15245 which was published after the priority date of the present invention, describes liquid formulations of botulinum toxin that additionally include a protein as an excipient, such as serum albumin, which does not confer additional significant biological activity. to the preparation.

SUMMARY

The present invention fulfills this need and provides for the replacement of the albumin present in a pharmaceutical composition with a compound that can stabilize the botulinum toxin present in the pharmaceutical composition. The albumin replacement compound has the characteristic of low and preferably negligible immunogenicity when injected into a human patient. Additionally, the preferred albumin substitute has a rapid clearance rate from the body after injection of the pharmaceutical composition.

Definitions

As used herein, the words or terms described below have the following definitions.

The word around means that the object, parameter, or term thus qualified includes a range of plus or minus ten percent above and below the value of the quoted object, parameter, or term.

The word amino acid includes polyamino acids.

The word polysaccharide means a polymer of more than two saccharide molecule monomers, which monomers can be identical or different.

The phrase "pharmaceutical composition" means a formulation in which an active ingredient is a neurotoxin, such as a Clostridial neurotoxin. The word formulation means that there is at least one additional ingredient in the pharmaceutical composition in addition to the neurotoxin active ingredient. A pharmaceutical composition is therefore a formulation that is suitable for diagnostic or therapeutic administration (ie, by intramuscular or subcutaneous injection) to a human patient. The pharmaceutical composition can be: in a lyophilized or vacuum dried state; a solution formed after reconstitution of the lyophilized or vacuum dried pharmaceutical composition with saline or water, or; as a solution that does not require reconstitution. The neurotoxin active ingredient can be one of the botulinum toxin serotypes A, B, Ci, D, E, F, or G or a tetanus toxin, all of which are manufactured by Clostridial bacteria.

The phrase "therapeutic formulation" means a formulation that can be used to treat and thereby alleviate a disorder or disease, such as a disorder or disease characterized by hyperactivity (ie, spasticity) of a peripheral muscle.

The words stabilizing, stabilizing, or stabilizing mean that when reconstituting with saline or water a pharmaceutical composition containing lyophilized, or vacuum-dried botulinum toxin that has been stored below about -2 degrees Celsius for between six months and four years, or for an aqueous solution of pharmaceutical composition containing botulinum toxin that has been stored at between about 2 degrees and about 8 degrees centigrade for six months to four years, the botulinum toxin present in the reconstituted pharmaceutical composition or in aqueous solution has more than about 20% and up to about 100% of the toxicity that the biologically active botulinum toxin had prior to being incorporated into the pharmaceutical composition.

The aspect of the present invention is a pharmaceutical composition comprising a botulinum toxin and a recombinantly manufactured human serum albumin under vacuum drying conditions or lyophilized for reconstitution. This composition also preferably includes an acetyltyptophanate or its salts or derivatives.

This invention also includes the addition of an ion zinc source to a pharmaceutical formulation containing botulinum toxin. Metals, especially divalent cations, are said to greatly influence the success of a lyophilized formulation due to various properties including the lattice structures of the ice formed. Foreign metals such as copper and iron species themselves lend themselves to radical oxidation and should generally be avoided. More specifically, botulinum toxin type A depends on bound zinc for its activity. Many of the proposed excipients or reaction products with the excipients will chelate metals. This could lead to the formation of unstable ice and / or inactivated toxin. By supplying abundant zinc in the formulation, the presence of desirable divalent cations is ensured, the probability of zinc depletion by the toxin is reduced, and stability is improved. This can be achieved by adding ZnSO₄ to a pharmaceutical composition of botulinum toxin.

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It is known to produce recombinant human serum albumin (rHSA) using a high productivity methylotrophic yeast expression strain Pichia pastoris. Pharmaceutical formulations containing botulinum toxin can be prepared containing stabilizing rHSA. RHSA expressed by genetically altered yeast host cells, while having the same primary amino acid sequence, differs in other respects from plasma-derived HSA. Thus, although eukaryotic, yeast lacks many intracellular processes found in mammals. Additionally, pHSA (plasma derived human serum albumin) is obtained in non-glycosylated form and undergoes extracellular, non-enzymatic addition of glucose. Thus, the carbohydrate moieties of pHSA and rHSA are different. Furthermore, it is known that the amounts of palmitic acid and stearic acid present in rHSA are much lower than those contained in pHSA. These differences are expected to result in differences in, among others, ligand binding, conformational stability, and molecular charge between rHSA and pHSA. It was therefore surprising to discover that rHSA can be used to stabilize botulinum toxin, particularly in view of the known kinetic rate effect of pHSA against botulinum toxin. An advantage of rHSA is that it is free of blood-borne pathogens. Thus, another aspect of the invention encompasses the replacement of blood-derived human serum albumin in a pharmaceutical composition with rHSA. Preferably, the rHSA is present in the pharmaceutical formulation containing botulinum toxin with acetyltryptophanate, as indicated above.

Commercially available human serum albumin is heated to 60^sC for ten hours as a requirement to eliminate potentially infectious agents derived from the pool of human blood. To prevent serious denaturation during this procedure, two stabilizers are added: sodium acetyltryptophanate and sodium caprylate. With an rHA it is not necessary to add these ingredients as there is no risk of disease and no heating stage is required. The addition of sodium acetyltryptophanate to rHA has been found to increase thermal stability more than shown by the use of sodium caprylate alone, even when the sodium caprylate concentration is doubled. Without wishing to be bound by theory, it is believed that it may be due to caprylate binding to only one site, whereas sodium acetyltryptophanate binds to two sites. It appears that binding to this second site increases resistance to thermal disturbance. It can also be postulated that the addition of sodium acetyltryptophanate can, in some way, increase the stability of botulinum toxin formulations, possibly maintaining a thermodynamically favorable conformation in the molecule, binding to the toxin or avoiding denaturation of serum albumin itself human.

This invention also encompasses the addition of a preservative, either in the diluent or in the formulation itself, to allow prolonged storage. A preferred preservative is preserved saline solution containing benzyl alcohol.

A liquid formulation can be advantageous. A single-stage presentation (eg, pre-filled syringe) or a product configuration that the user perceives as a single-stage presentation (eg, a two-compartment syringe) would be convenient, eliminating the reconstitution stage. Freeze drying is a complicated, expensive and difficult process. It is often easier and cheaper to produce liquid formulations. On the other hand, liquid formulations are dynamic systems and therefore are more susceptible to the interaction of excipients, rapid reactions, bacterial growth and oxidation, than freeze-dried formulations. A compatible preservative may be needed. Antioxidants such as methionine can also be useful as scavengers, especially if surfactants are used, to reduce adsorption as many of these compounds contain or produce peroxides. Any of the stabilizing excipients that can be used in a freeze-dried formulation (eg, hydroxyethyl starch or an amino acid such as lysine) can be adapted for use in a liquid formulation to help reduce adsorption and stabilize the toxin. Suspensions similar to those developed for insulin are also good candidates. Additionally, the stabilization of botulinum toxin in a liquid vehicle may require a low pH vehicle, since it is recognized that the toxin is labile above pH 7. This acidity could cause burns and burning after injection. A binary syringe could be used. The inclusion of a co-dispensing buffer, sufficient to raise the pH to physiological, would alleviate the discomfort of low pH injection while maintaining the toxin at a low pH during storage. Another two-compartment syringe option would include diluent and lyophilized material segregated into a separate compartment, mixing only after use. This option provides the benefits of a liquid formulation without the additional time and resources.

Thus, botulinum toxin can be prepared at low pH to be dispensed in conjunction with a buffer that raises the pH to or near physiological pH at the time of administration. The binary or two-compartment syringe may have in the first compartment (close to the plunger) a liquid formulation of a botulinum toxin with a pH between 3 to 6 (ie, at pH 4.0). The second compartment (near the tip of the needle) may contain a suitable buffer, such as phosphate buffered saline at a higher pH (ie, pH 7.0). Alternatively, the first compartment may contain a saline diluent and the second compartment may contain a freeze-dried or lyophilized neurotoxin formulation. The two compartments can be joined in such a way, and the buffer components selected in such a way that the solutions mix at or near the needle, thus bringing the final solution to a physiological pH. Two-compartment syringes suitable for use as pre-filled syringes for the purposes set forth herein are available from Vetter Pharma-Fertigung of Yardley, Pennsylvania.

There are several advantages to formulating botulinum toxin at a low pH. The toxin has a low isolectric point (pl) and formulating proteins near its pl is a known way to stabilize a protein. Additionally, the toxin is used

ES 2 275 992 T5 at a very low concentration, making surface adsorption a problem. The use of a low pH solution can suppress ionization of toxin sites that can interact with surfaces. The syringe and plunger materials are materials that reduce surface adsorption by toxin. Such suitable materials are polypropylene.

EXAMPLES

Example 1

Botulinum toxin pharmaceutical composition

As previously described, the botulinum toxin type A complex can be obtained from a culture of the Hall strain of Clostridium botulinum grown in a medium containing NZ-amine and yeast extract. The botulinum toxin type A complex is purified from the culture solution by a series of acid precipitations to a crystalline complex consisting of the active high molecular weight toxin protein and an associated hemagglutinin protein. The crystalline complex is then redissolved in a solution containing saline and albumin and sterile filtered (0.2 micron) prior to vacuum drying. BOTOX® is then reconstituted with sterile, unpreserved saline prior to intramuscular injection. Each vial of BOTOX® contained about 100 (U) Clostridium botulinum toxin type A complex, 0.5 milligrams of human serum albumin, and 0.9 milligrams of sodium chloride in sterile, vacuum-dried form without a preservative. According to the present invention, human serum albumin is replaced by a recombinantly manufactured albumin.

Reference example 2

Botulinum toxin pharmaceutical composition containing 2-hydroxyethyl-starch

Pharmaceutical formulations of purified botulinum toxin type A neurotoxin complex were prepared in the same manner as described in Example 1 above, except that the 0.5 milligrams of albumin was replaced by 500 pg or 600 pg of hetastarch. It was determined that full potency was maintained with the preparation of the hetastarch-containing formulations. Thus, with both hetastarch-containing formulations, the potency of the albumin-free, hetastarch-containing composition, as measured at reconstitution of the 100 U (± 20 U) butulinum toxin type A complex lyophilized, it was 96 to 128 units. Three different pharmaceutical compositions of botulinum toxin type A complex with hetastarch had potency measurements, at reconstitution, of 105, 111 and 128 units respectively. Potency was measured using the standard mouse administration toxin potency assay.

Reference example 3

Botulinum toxin pharmaceutical composition containing glycine

Pharmaceutical formulations of purified botulinum toxin type A neurotoxin complex were prepared in the same manner as described in Example 1 above, except that the 0.5 milligrams of albumin was replaced by 500 pg or 600 pg of hetastarch. Furthermore, 1 mg of glycine was added to the formulation. A lyophilized pharmaceutical composition of 100 U botulinum toxin type A complex, albumin-free, with hetastarch plus glycine was then stored for seven months at -5^SC. At the end of this seven-month period, the potency of this hetastarch plus glycine toxin formulation, using the mouse administration assay, was determined to be essentially unchanged (i.e., potency differed by less than 5% from the original power).

Reference example 4

Botulinum toxin pharmaceutical composition containing lysine

Pharmaceutical formulations of 100 U purified botulinum toxin type A neurotoxin complex were prepared in the same manner as described in Example 1 above, except that the 0.5 milligram of albumin was replaced by 600 pg of hetastarch. In addition, 1 mg of lysine was added to the formulation. A lyophilized pharmaceutical composition of 100 U botulinum toxin type A complex, albumin-free, with hetastarch plus lysine was then stored for one year at -5^SC At the end of this one year period the potency of this hetastarch plus glycine toxin formulation, using the mouse administration assay, was determined to be essentially unchanged (i.e. potency differed by less than 5% from original power).

Reference example 5

Botulinum toxin pharmaceutical composition containing histidine

Pharmaceutical formulations of 100 U botulinum toxin type A purified neurotoxin complex are prepared in the same manner as described in Example 1 above, except that the 0.5 milligrams of albumin was replaced by 600 pg of hetastarch. In addition, 1 mg of histidine was added to the formulation. A lyophilized pharmaceutical composition of 100 U botulinum toxin type A complex, albumin-free, with hetastarch plus histidine was stored at

ES 2 275 992 T5 continued for one year at -5^SC. At the end of this one year period, the potency of this hetastarch plus histidine toxin formulation, using the mouse administration assay, was determined to be essentially unchanged (i.e. potency differed by less than 5% from the original power).

Reference example 6

Botulinum toxin pharmaceutical composition containing arqinine

Pharmaceutical formulations of 100 U botulinum toxin type A purified neurotoxin complex were prepared in the same manner as described in Example 1 above, except that the 0.5 milligrams of albumin was replaced by 600 pg of hetastarch. In addition 1 mg of arginine was added to the formulation. A lyophilized pharmaceutical composition of 100 U botulinum toxin type A complex, albumin-free, with hetastarch plus arginine was then stored for one year at -5^SC. At the end of this one year period, the potency of this hetastarch plus glycine toxin formulation, using the mouse administration assay, was determined to be essentially unchanged (i.e., potency differed by less than 5% from the original power).

Reference example 7

Botulinum toxin pharmaceutical composition containing an amino acid

Pharmaceutical formulations of botulinum toxin type A purified neurotoxin complex can be prepared in the same manner as described in Example 1 above, except that the 0.5 milligrams of albumin can be replaced by approximately 1 mg of an amino acid such as lysine, glycine, histidine, or arginine. A lyophilized pharmaceutical composition of 100 U botulinum toxin type A complex, polysaccharide-free, albumin-free, plus glycine can be stored for at least one year at -5^SC, and at the end of this period you can have essentially unchanged power (that is, power may differ by less than 5% from original power).

Reference Example 8

Use of a pharmaceutical composition of botulinum toxin

A 48-year-old man is diagnosed with a spastic muscle condition, such as cervical dystonia. The patient is injected intramuscularly between about 1 x 10 '³ U / kg and 35 U / kg of a botulinum toxin type A pharmaceutical composition containing 600 pg of hetastarch and 1 mg of an amino acid such as lysine. In 1-7 days the symptoms of the spastic muscle state are relieved and the relief of symptoms persists for at least about 2 months to about 6 months.

A pharmaceutical composition according to the invention described herein has many advantages, including the following:

1. The pharmaceutical composition can be prepared free of any other blood product, such as albumin and therefore free of any infectious blood product such as a prion.

2. The pharmaceutical composition has stability and a high% recovery of the potency of the toxin, comparable to or superior to that achieved with currently available pharmaceutical compositions.