The present application relates to the field of pharmacology and medicinal chemistry, and provides improved pharmaceuticals, and methods for effective administration thereof.
Allergies often are chronic in nature. Medication that controllably releases over a long period of time would be most effective for the control of allergies. However, allergies typically are treated with injections, pills, or capsules, which do not provide controlled release of the allergy medication.
Motion sickness occurs in humans when they are exposed to unfamiliar movement or visual stimulus. The characteristic symptoms are nausea and vomiting that disrupt normal function until these symptoms ameliorate. Astronauts frequently experience space motion sickness and disorientation as a result of changes in gravitational level. This results in a loss of work time and a disruption of planned activities until symptoms are relieved, often resulting in a loss of expensive flight programs and experiments.
Effective pharmaceutical preparations are needed to treat motion sickness, allergies, and a wide variety of ailments, which can be easily and safely used over days to weeks with minimal side effects.
The present application provides a pharmaceutical preparation adapted for mucosal delivery of a pharmacologically effective dose of a pharmacologically active agent to a mammal. The pharmaceutical preparation comprises microcapsules adapted to provide controlled release of the pharmacologically effective dose. The microcapsules comprise a core and a shell, the shell comprising a release retardant, the core comprising the pharmacologically active agent and an excipient. The pharmacologically active agent is selected from the group consisting of antihistamines and anticholinergics.
In another aspect, the application provides a pharmaceutical preparation adapted for mucosal delivery of a pharmacologically effective dose of a pharmacologically active agent to a mammal. The pharmaceutical preparation comprises microcapsules adapted to provide controlled release of the pharmacologically effective dose. The microcapsules comprise a shell and a core, the core comprising a quantity of a single enantiomer of the pharmacologically active agent. The pharmacologically active agent is selected from the group consisting of antihistamines and anticholinergics.
In another aspect, the application provides a pharmaceutical preparation adapted for mucosal delivery of a pharmacologically effective dose of a pharmacologically active agent to a mammal. The pharmaceutical preparation comprises one or more absorption enhancers and microcapsules adapted to provide controlled release of the pharmacologically effective dose of the pharmacologically active agent. The pharmacologically active agent is selected from the group consisting of antihistamines and anticholinergics.
The application also provides a method for mucosal delivery of a pharmacologically effective dose of a pharmacologically active agent to a mammal. The method comprises:
In yet another aspect, the application provides a method for mucosal delivery of a pharmacologically effective dose of a pharmacologically active agent to a mammal. The method comprises:
In another aspect, the application provides a method for mucosal delivery of a pharmacologically effective dose of a pharmacologically active agent to a mammal. The method comprises:
In yet another aspect, the application provides a pharmaceutical preparation for mucosal delivery of a pharmacologically active agent to a mammal without cytotoxicity to mucosal epithelial cells. The pharmaceutical preparation comprises:
In another embodiment, the application provides a method for mucosal delivery of a pharmacologically active agent to a mammal. The method comprises:
In another aspect, the application provides a method for alleviating a condition in a mammal selected from the group consisting of motion sickness, allergy, and a combination thereof. The method comprises administering to the mammal a pharmacologically effective amount of a highest pharmacological activity enantiomer of a phenothiazine.
In another aspect, the application provides for resolving (+) enantiomer and (−) enantiomer of ethopropazine, said method comprising:
The present application provides pharmaceutical preparations adapted for mucosal delivery which can be easily and safely used over days to weeks with minimal side effects. A preferred type of mucosal delivery is nasal delivery.
The pharmaceutical preparations comprise microcapsules comprising at least one pharmacologically active agent selected from the group consisting of antihistamines and anticholinergics. The microcapsules provide controlled release of the pharmacologically active agent. Cytotoxicity is avoided for cytotoxic pharmacologically active agents and/or for cytotoxic release rates of the pharmacologically active agent by one or more of the following: (a) manipulating the mucosal transport rate of the pharmacologically active agent through the mucosal epithelial cells to achieve a mucosal transport rate which is substantially the same as the controlled release rate, and/or (b) selecting only a most active enantiomer, to allow less to be used, and/or a less cytotoxic enantiomer of the pharmacologically active agent for use in the pharmaceutical preparation.
Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, R and S, or (+)- or (−)-, are used to denote the absolute configuration of the molecule about its chiral center(s). The enantiomers of a racemic drug generally differ in biological activity as a consequence of stereoselective interaction with optically active biological macromolecules. For drugs having a specific action at receptors, one enantiomer may have all of the activity, whereas the other enantiomer appears to be inactive. Such a molecule may be marketed by the pharmaceutical industry as a racemate, assuming that the non-active enantiomer is insignificant from a therapeutic and a toxicological point of view. However, the non-active enantiomer may actually be deleterious rather than simply inert and it is likely that the side-effects encountered may be due to the non-active enantiomer.
Many biological receptors are chirally sensitive, including the histamine receptors. Waelbroeck M, Camus J, Tastenoy M, et al. Stereoselective interaction of procyliine, hexahydrodifenidol, hexabutinol and oxyphencyclimine and of related antagonists, with four muscarinic receptors. Eur. J. Pharmacol. 227:3342, (1992), incorporated herein by reference. Different enantiomers of various chiral antagonists also show differing levels of inhibition. Hence, phenothiazine enantiomers, such as PMZ enantiomers, may have different affinities for the histamine receptors, resulting in different efficacies in vivo.
Where the enantiomers of the particular pharmacologically active agent have different affinities for the relevant receptors, or demonstrate different cytotoxicity levels, a preferred embodiment comprises the use of only the (+)- or the (−)-enantiomer of the pharmacologically active agent. Preferably, the enantiomer exhibiting increased affinity for the receptor and/or lower cytotoxicity, preferably both, is chosen as the pharmacologically active agent in formulating the pharmaceutical preparation and in performing the method described herein.
Controlled delivery may be desirable for many pharmacologically active agents. Hence, mucosal delivery of pharmaceutical preparations comprising microcapsules comprising the pharmacologically active agent(s) may be used for a number of pharmacologically active agents, including but not necessarily limited to those selected from the group consisting of antihistamines and anticholinergics.
Controlled delivery of the pharmacologically active agent involves encapsulating the pharmacologically active agent in microcapsules. The microcapsules preferably comprise a core comprising one or more pharmacologically active agents. In a preferred embodiment, the core comprises an excipient. The core also preferably comprises one or more mono-, di-, and/or triglycerides, more preferably stearine, even more preferably partially hydrogenated palm oil. A preferred partially hydrogenated palm oil is CAS 68514-74-9. The core of the microcapsules is coated by a shell material comprising a release retardant, more preferably ethylcellulose, most preferably ethylcellulose of premium grade from about 4 to about 10, preferably comprising an ethoxyl content of from about 45 wt. % to about 47 wt. %. In a preferred embodiment, a 5% solution of ethylcellulose in 80% toluene and 20% ethanol has a viscosity of from about 9 centipoise (cP) to about 11 cP at 25° C. In a most preferred embodiment, the pharmaceutical formulation comprises absorption enhancers effective to increase the rate of mucosal transport of the pharmacologically active agent across the mucosal epithelium, preferably to a mucosal transport rate that is substantially the same as the controlled release rate.
The pharmaceutical preparations and methods will be described with reference to agents which are pharmacologically active to treat motion sickness and/or allergy. However, the pharmaceutical preparations and methods of the present application are not limited to pharmaceutical preparations and methods for treating motion sickness and/or allergy. Rather, the pharmaceutical preparations are useful to treat a variety of ailments using a pharmacologically active agent selected from the group consisting of antihistamines and anticholinergics.
Referring to agents for treating motion sickness, the source of the motion sickness response is complex. Although the semicircular canals and otolith organs are essential for the genesis of motion sickness, subsequent events leading to motion sickness take place in the CNS. Emesis, the final event in motion sickness, is a reflex controlled by the brain stem. A variety of pharmacological agents are effective in minimizing motion-induced emesis therapeutically. These agents include, but are not necessarily limited to antihistamines and anticholinergics.
Promethazine (PMZ) is a member of a class of compounds called phenothiazines. PMZ acts as a histamine receptor 1 (H1) antagonist. PMZ also is effective against allergy symptoms. PMZ commonly is used clinically to prevent the symptoms of motion sickness during space flight and sea voyaging because PMZ is capable of halting the nausea and disorientation after onset. The H1 receptor antagonism activity of PMZ is the apparent mechanism of action for the reduction of the symptoms of motion sickness. Interestingly, PMZ is a chiral compound that is used clinically as the racemate.
Promethazine (PMZ) has been isolated, resolved, and tested for cytotoxicity. Neither enantiomer of PMZ demonstrated a significant increase in cytotoxicity compared to the racemate. However, both of the enantiomers and the racemate of PMZ showed a significant level (10 −4 molar) of inherent cytotoxicity. The (+)-enantiomer (as measured in water) of promethazine (PMZ) has been found to be the highest activity enantiomer of the racemic PMZ mixture. The (−) enantiomer (as measured in water) of ethopropazine has been found to be the highest activity enantiomer of the racemic ethopropazine mixture. As hereinafter used, the terms (+) and (−)-enantiomer refer to optical rotation as measured in water.
Useful compounds for administration to a patient include pharmaceutically acceptable acid addition salts of the pharmacologically active agent, preferably the phenothiazines defined by the above formula. Acids commonly employed to form such salts are inorganic acids, such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids, such as p-toluenesulfonic, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, and the like.
Examples of such pharmaceutically acceptable salts thus are the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycollate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like. Preferred pharmaceutically acceptable acid addition salts are those formed with mineral acids such as hydrochloric acid, hydrobromic acid and organic acids such as acetic acid, oxalic acid, maleic acid or fumaric acid.
It is important to note that the pharmacologically active agent preferably is not obtained from a commercially available tablet that may contain a variety of non-active ingredients, including binders, which may exert a detrimental effect on the efficacy of the composition. If the source of a pharmacologically active agent is a commercial tablet, then the mixture obtained from the tablet preferably is treated to provide the active ingredient relatively free, preferably substantially free of the non-active components. Methods of purification are well known to those of ordinary skill and may include dissolution of the mixture in a solvent and recrystallization, for example.
The pharmaceutical preparation may be used prophylactically, or may be administered to a patient already suffering from an ailment or symptoms associated therewith, such as allergy or motion sickness. Once relief has been provided, the composition can be administered under a regimen to maintain a substantially symptom-free state. Generally, the dosage or frequency of administration of the pharmacologically active agent required to keep the patient essentially free of allergy or motion sickness symptoms (the “maintenance dosage”) is less than the dosage or frequency used in the initial phase of treatment (the “initial dosage”) and lower than the dosages used with the racemate. After administration of the initial dosage, the dosage or frequency can be cut back until the symptoms begin to manifest themselves once again. The dosage or frequency is then adjusted to just suppress the symptoms.
As used herein, the term “phenothiazine” refers to compounds having the following general structure:
wherein
Phenothiazines primarily differ by substitution of various alkylamino groups on the nitrogen atoms at the 10 position of the basic phenothiazine nucleus. The chemical group bound at the 10 position of the phenothiazine nucleus appears to determine histaminic response.
The method may use a racemic mixture, or only the (+)- or the (−)-enantiomer of a given pharmacologically active agent, such as a phenothiazine, to treat motion sickness, allergy, or other ailment. The following are the structures of certain preferred phenothiazines for use in the method:
Promethazine, ethopropazine, and trimeprazine are available commercially as racemic mixtures, for example, from Aldrich Chemical Co., or by prescription.
Promethazine hydrochloride is currently administered during space flight after onset of motion sickness by a painful and unwieldy intramuscular route. A less invasive, more selective delivery route is preferred for safer, more effective remedies. Mucosal delivery, preferably nasal delivery, is noninvasive and should be amenable to space flight use. Importantly, nasal delivery also enables high plasma loadings without first pass metabolism in the liver after administration. This route is ideal for drugs, such as promethazine, that are rapidly metabolized to their inactive sulfoxide by liver oxidases.
In previous research, racemic promethazine hydrochloride was encapsulated in a variety of shell materials and administered to beagles; however, severe nasal irritation was observed. R. Ramanathan, R. S. Geary, L. Putcha, “Bioavailability of Intranasal Promethazine Dosage Forms in Dogs”, Pharmacol. Res. 38 (1), 1998, pg. 36-39, incorporated herein by reference.
Nasal delivery can be done by powder insufflation, aerosol delivery of droplets, liquid dosing or by application of a cream or ointment. Insufflation, aerosol, and liquid all have disadvantages such as microbiological instability, short residence time of dose, variable site of deposition, and variable dose. Supporting work has shown the importance of nasal ciliary beat frequency and site of deposition on the absorption of insulin. S. Gizurarson, E. Bechgaard, “Intranasal Administration of Insulin to Humans”, Diab. Res. Clin. Pract. 12, 1991, pg. 71-84, incorporated herein by reference. Site of administration of nasally delivered drugs also is important.
Recent developments in nasal administration of creams or gels by addition of absorption enhancers, such as polyethylene glycol 300 or 400 and dimethylcyclodextrin, have made this delivery mode highly desirable since problems of variable site deposition, dose and residence time are more manageable. E. Martin, N. G. M. Schipper, F. W. H. M. Merkus, “Nasal Mucociliary Clearance as a Factor in Nasal Drug Delivery”, Adv. Drug Deliv. Rev., 29, 1998, pg. 13-38; R. Ramanathan, R. S. Geary, L. Putcha, “Bioavailability of Intranasal Promethazine Dosage Forms in Dogs”, Pharmacol. Res. 38 (1), 1998, pg. 36-39, incorporated herein by reference.
Microencapsulation of the pharmacologically active agent, such as phenothiazine, achieves “controlled release” of the agent. In the case of phenothiazine, the release rate is effective to enable the composition to act as an “H1 receptor antagonist.” By “H1 receptor antagonist” is understood to mean that the phenothiazine is capable of partially or completely inhibiting the biological effect of histamine on the H1 receptor. An H1 receptor antagonist induces a coherent pharmacological response (including or not including its binding to the H1 receptor), specifically a reduced production of IL-6 in comparison to a control, in the assay described in Delneste Y., Lassalle P. et al Histamine induces IL-6 production by human endothelial cells. Clin. Exp. Immunol. 98:344-349, (1994), incorporated herein by reference. A preferred microcapsule composition comprises about 0.1 to 50% by weight of the phenothiazine, preferably about 20% by weight of the phenothiazine. Preferably, the release rate into isotonic saline at 37° C. takes 20-360 minutes.
Cytotoxicity has been avoided even when the pharmacologically active agent is inherently cytotoxic, or when the release rate is sufficient to cause cytotoxicity, by combining microencapsulation effective to achieve controlled release of the pharmacologically active agent with the use of absorption enhancers which transport the pharmacologically active agent through the cells at the site of administration, typically mucosal bodies, at a mucosal transport rate which is substantially the same as the controlled release rate. This combination of controlled release and rapid absorption caused by the absorption enhancers maintains the effective concentration in the cells at the site of administration below the cytotoxic limit. The absence of cytotoxicity symptoms using the foregoing combination has been demonstrated in the case of cytotoxic phenothiazines and nasal administration (see examples). It is believed that use of the same technique will avoid cytotoxicity using other cytotoxic agents selected from the group consisting of antihistamines and anticholinergics.
Enantiomer Resolution
Where one enantiomer of the pharmacologically active agent is more active and/or less cytotoxic, preferably both, it is preferred to use the more active, less cytotoxic enantiomer only in the pharmaceutical preparation. Methods of resolving enantiomers are known. For example, in order to resolve a phenothiazine racemate into its two enantiomers, 0.5-25 grams of optically pure phenothiazine enantiomers are isolated using column chromatography. Nilsson, J. Lars G.; Hermansson, Joeergen; Hacksell, Uli; Sundell, Staffan “Promethazine-resolution, absolute configuration and direct chromatographic separation of the enantiomers” Accta Pharm. Suec. (1984), 21 (5), 309-16, incorporated herein by reference. Generally, the racemate is allowed to react with an optically active compound. The two products of the reaction are diastereomers, which are separated by virtue of differences in their physical properties, such as solubility. The diastereomers are decomposed, and the optically active components of the original racemate are recovered. If the racemate is a base, an optically active acid or derivative thereof such as tartaric acid, or mandelic acid, is used to split the enantiomeric pair. In the case of phenothiazines, a preferred optically active acid is dibenzoyl tartaric acid. The racemate is mixed with the acid, and diastereomerically related and optically active salts crystallize. Since the diastereomeric salts have different solubility properties, they are separated by fractional crystallization to give homogeneous substances.
Alternately, the racemate may be separated using chromatographic separation, such as gas chromatography (GC), high performance liquid chromatography (HPLC) [Ponder, Garratt W.; Butram Sandra L.; Adams, Amanda G.; Ramanathan Chandra S.; Stewart, James T. “Resolution of promethazine, ethopropazine, trimeprazine and trimipramine enantiomers on selected chiral stationary phases using high-performance liquid chromatography,” Journal of Chromatography A, (1995), 692, 173-182, incorporated herein by reference], and recently capillary electrophoresis (CE) [Wang, Rongying; Lu Xiaoning; Wu, Mingjia “Chiral separation of promethazine by capillary electrophoresis with end-column amperometric detection” J. Sep. Sci. (2001), 24, 658-62, incorporated herein by reference]. In these chromatographic separations, a variety of chiral selectors have been employed, including proteins, modified crown ethers, and cyclodextrins.
Enantiomer Characterization
The enantiomers of ethopropazine (EPZ), trimeprazine (TPZ), and promethazine (PMZ) have been isolated and resolved, and the enantiomers of PMZ and ethopropazine have been tested for efficacy (as discussed above). Each enantiomer lot of phenothiazine is characterized to provide consistency within the test articles and to protect against varied polymorphism surprises. Once isolated, each drug class is characterized to determine its polymorph fingerprint vs. the racemate by powder diffraction x-ray (XRD). The XRD characterization is important because polymorphisim often occurs in chiral compounds. J. Breu, H. Domel, N. Per-Ola, Eur. J. Inorg. Chem. 11, 2000, pg. 2409-2419. H. H Paradies, S. F. Clancy, Rigaku J. 17 (2), 2000, pg. 20-35, incorporated herein by reference. A change in polymorph of a compound can result in a significant difference in the solubility and bioavailability of that compound. Chiral High Performance Liquid Chromatograpy (“Chiral HPLC”) was used along with optical rotation measured in water to determine the optical purity of the samples prepared. G. W. Ponder, S. L. Butram, A. G. Adams, J. T. Stewart, Resolution of Promethazine, Ethopropazine, Trimeprazine and Trimipramine Enantiomers on Chiral Stationary Phases Using HPLC, Jrnl. Chrom. A, 692, 1995, pg. 173-182, incorporated herein by reference. Nuclear Magnetic Resonance (NMR) and Infrared (IR) spectra and melting point information were gathered on each enantiomer. After complete characterization of each enantiomer, samples were set aside and retained as standards. Each subsequent lot of phenothiazine enantiomer prepared was analyzed against these primary standards prior to formulation and dosing.
The optical purity of the phenothiazine enantiomers was determined using Chiral HPLC. Preferably, a chiral α1-acid glycoprotein column (α1-AGP column), containing 183 mg α1-AGP/g solid phase. The enantiomers were resolved using a mobile phase composition of phosphate buffer pH7.0 with addition of 2% v/v of ethanol (95% v/v) and 1.95 mM N,N-dimethyloctylamine.
Cytotoxicity and Efficacy
Cytotoxicity is evaluated by measuring cell survival after exposure to the relevant pharmacologically active agent. Cytotoxicity for purposes of mucosal delivery typically is determined by the level of tetrazolium salt reduction accomplished by surviving cells, preferably over four orders of magnitude. If the level of tetrazolium salt reduction is decreased, then cytotoxicity exists. One assay for measuring tetrazolium salt reduction is the WST-1 assay (Boehringer Mannheim) using L929 lung fibroblast cells. Other known assays include, but are not necessarily limited to assays which measure lactose dehydrogenase (“LDH”), which is released by cells upon death, and/or assays which measure the rate of DNA synthesis.
Various assays also exist for identifying a highest pharmacological activity enantiomer of a given pharmacologically active agent. Where the pharmaceutical activity is as a histamine antagonist, IL-6 production by HUVEC cells is a cell biomarker of histamine activity and is used to assess the relative antagonistic activity of prospective H1 blockers. IL-6 production in human endothelial cells is known to be induced by histamine due to H1 and H2 receptor binding with H1 the dominant effect. Delneste, et al. As H1 antagonism is directly linked to reduced emesis during motion sickness treatment, this assay serves as an in vitro methodology for the selection of potential motion sickness and antihistamine candidates. Realtime RT-PCR analysis of IL-6 mRNA synthesis in HUVEC cells stimulated with histamine is employed as an in vitro assay for the analysis of the relative efficacy of potential antihistaminic agents.
A preferred assay for measuring activity of phenothiazine and other histamine antagonists comprises: providing at least a first viable culture and a second viable culture comprising Huvec cells; exposing the first viable culture to a first combination comprising histamine and the (+)-enantiomer of the phenothiazine under conditions effective to inhibit IL-6 mRNA expression; exposing the second viable culture to a combination comprising histamine and the (−)-enantiomer of the phenothiazine under conditions effective to inhibit IL-6 mRNA expression; measuring inhibition of IL-6 mRNA expression induced by the first combination and the second combination after at least four hours to identify a (+)-enantiomer inhibition value and a (−)-enantiomer inhibition value; and selecting as the highest pharmacological activity enantiomer the enantiomer having the greater inhibition value selected from the group consisting of the (+)-enantiomer inhibition value and the (−)-enantiomer inhibition value.
In a preferred embodiment, the method further comprises providing a third viable culture comprising Huvec cells as a control; exposing the third viable culture to a third combination comprising histamine in the absence of the phenothiazine under conditions effective to induce IL-6 mRNA expression; and, measuring IL-6 mRNA expression induced by the third combination after at least four hours to identify a control expression value.
In a preferred embodiment, the method further comprises providing a fourth viable culture comprising Huvec cells; exposing the fourth viable culture to a fourth combination comprising histamine and a racemate mixture of the phenothiazine under conditions effective to inhibit IL-6 mRNA expression; measuring inhibition of IL-6 mRNA expression induced by the fourth combination after at least four hours to identify a racemate inhibition value. Depending on the results, this embodiment may comprise identifying the racemate mixture of the phenothiazine as the highest activity candidate.
The data for each enantiomer is compared to that of the racemate and the other enantiomers of the experimental group and a ‘highest efficacy’ (HE) candidate is selected. This data is a significant indicator for efficacy against motion sickness and/or allergy.
Formulation of Pharmaceutical Preparation
Pharmaceutical preparations comprising microcapsules, as described herein, are useful to deliver substantially any pharmacologically active agent selected from the group consisting of antihistamines and anticholinergics across the blood-brain barrier.
If one enantiomer of the particular pharmacologically active agent is superior, then the most potent and/or less cytotoxic enantiomer is used alone. In a preferred embodiment, the pharmacologically active agent is a phenothiazine, most preferably a single, most active enantiomer of the phenothiazine. In a preferred embodiment, the phenothiazine is selected from the group consisting of the (+)-enantiomer of promethazine and the (−)-enantiomer of ethopropazine.
The microcapsules are fabricated by the disk process. D. C. Johnson et al. J. Gas Chrom, 3, 345-347, (1965), incorporated herein by reference. The microcapsules comprise a core and a shell.
The core of the microcapsule preferably comprises an excipient. Suitable excipients include, but are not necessarily limited to mono-, di-, and triglycerides. Suitable mono- and/or di-glycerides are selected from the group consisting of MYVEROL™ and MYVOCET™ which are commercially available from Gillco Ingredients. Suitable triglycerides are selected from the group consisting of stearate, hydrogenated palm oil, cottonseed oil, soybean oil, and combinations thereof. The hydrogenated palm oil preferably is partially hydrogenated palm oil, most preferably STEARINE-27, a partially hydrogenated palm oil with a melting point of −135° F. STEARINE-27 is commercially available from Loders-Croklaan. In one embodiment, the triglyceride is mixed with the pharmacologically active agent. The core of the microcapsule also may comprise one or more absorption enhancer(s).
The microcapsules preferably are over coated with a release retardant. Suitable release retardants or shell materials include, but are not necessarily limited to shellac and ethylcellulose, most preferably ethylcellulose of premium grade from about 4 to about 10, preferably comprising an ethoxyl content of from about 45 wt. % to about 47 wt. %. In a preferred embodiment, a 5% solution of ethylcellulose in 80% toluene and 20% ethanol has a viscosity of from about 9 cP to about 11 cP at 25° C. The release retardant is effective to slow the release of the pharmacologically active agent and to reduce, and preferably to prevent mucosal tissue irritation, preferably nasal tissue irritation. The shell of the microcapsules also may comprise one or more absorption enhancer(s).
In a preferred embodiment, the pharmaceutical preparation comprises microcapsules in combination with one or more absorption enhancer(s). The one or more absorption enhancer(s) may be incorporated into the microcapsules themselves, or the absorption enhancer(s) may be incorporated into a carrier gel or cream. In a preferred embodiment, the absorption enhancer(s) are incorporated into the carrier gel or cream. The absorption enhancer(s) preferably are effective to transport the pharmacologically active agent through mucosal epithelial cells at a mucosal transport rate that is substantially the same as the controlled release rate from the microcapsules. Suitable absorption enhancers include, but are not necessarily limited to those selected from the group consisting of glycodeoxycholate (GDC), dimethyl-cyclodextrin, L-α-lysophosphatidylcholine (LPC), polyethylene glycol (PEG), glycofurol, and mixtures thereof. I. Gill, A. N. Fisher, M. Hinchcliffe, J. Whetstone, R. DePonte, L. Illum, “Cyclodextrins as Protection Agents Against Enhancer Damage in Nasal Delivery Systems”, Eur. J. Pharm. Sci., 1 (5), 1994, pg. 235-248, incorporated herein by reference. A preferred absorption enhancer PEG/glycofurol, more preferably 30/70 wt./wt. PEG/glycofurol, most preferably 30/70 wt./wt. PEG 400/glycofurol.
In a preferred embodiment, the pharmaceutical preparation comprises a microcapsule- gel or cream formulation comprising a suitable carrier. Suitable carriers include, but are not necessarily limited to polyethylene glycol (PEG), glycofurol, laureth-5, 6 or 9, aquaphor, plurfect, poloaxamer, and mixtures thereof, and the like. A preferred PEG is PEG 400. In a preferred embodiment, suitable for nasal delivery, a carrier gel or cream that will not irritate the nasal tissue or inhibit the ciliary beat frequency of the nostril is used.
Preferred pharmacologically effective formulations comprise microcapsules comprising the pharmacologically active agent and an absorption enhancer selected from the group consisting of glycodeoxycholate (GDC), L-α-lysophosphatidylcholine (LPC), and mixtures thereof. In a most preferred embodiment, the pharmacologically effective formulation further comprises a carrier comprising a gel or cream that does not irritate the nasal tissue or inhibit the ciliary beat frequency of the nostril. Preferred carriers are selected from the group consisting of polyethylene glycol, glycofurol, laureth-5, 6 or 9, aquaphor, plurfect, poloaxamer, and mixtures thereof.
Method of Delivery
The pharmaceutical preparation may be delivered in a variety of ways. In a preferred method, the pharmaceutical formulation comprising a pharmacologically active agent is mucosally delivered. In a preferred embodiment, the mucosal delivery is nasal delivery.
The method is effective to enable delivery of the pharmacologically active agent across the blood brain barrier. In a most preferred embodiment, in which the pharmacologically active agent is nasally administered, the microcapsules also can deliver the pharmacologically active agent through the axonal nerve found in the ostium, bypassing the blood brain barrier.
The following examples will better illustrate the application:
Enantiomers of PMZ were prepared, purified, and characterized. A chiral-high performance liquid chromatographic (HPLC) method was developed to enable analysis of the optical purity of the enantiomers prepared.
The methods developed for making the PMZ enantiomers are described below:
Promethazine Base Conversion:
1. To promethazine hydrochloride (12.5 g, 0.039 mol), obtained from Sigma (lot #128H1474) added 100 mL ether and 25 mL 2M sodium hydroxide (0.045 mol). The resulting suspension was shaken and the ether layer was collected. The aqueous layer was extracted twice with ether. The combined ether layers were dried over magnesium sulfate. Rotary evaporation gave 10 g (0.035 mol) promethazine. Yield 90%.
Promethazine-D-tartrate:
2. Promethazine (10 g, 0.035 mol) dissolved in 80 mL acetone was heated in a 60° C. bath while dibenzoyl-D-tartaric acid (12.789 g, 0.036 mol) was added. The resulting clear yellow solution was left at ambient temperature for 3 days.
3. A heavy precipitate formed which was filtered off and recrystallized from ethanol four times to give 4.0 g promethazine dibenzoyl-D-tartrate white crystals.
4. Promethazine-D-tartrate was converted to promethazine by reaction with sodium hydroxide aqueous solution in ether. Ether layer was separated. The aqueous layer was extracted with ether and the combined ether layer was dried over magnesium sulfate. Rotary evaporation gave 1.6 g promethazine.
5. (−)-Promethazine hydrochloride was obtained by precipitation of promethazine with 2M HCl/ether. After vacuum drying 1.34 g off-white powder was obtained.
Promethazine-L-tartrate:
6. From the acetone mother liquor (Step 2) 11.3 g of brownish liquid was obtained after rotary evaporation. This liquid was converted to promethazine 3.6 g (similar to Step 4).
7. To 3.6 g promethazine obtained from the previous step, 36 mL acetone was added, heated in a 60° C. bath and 4.6040 g dibenzoyl-L-tartaric acid was added. The resulting clear solution was left at ambient for 3 days.
8. A heavy precipitate formed which was filtered off and recrystallized (from ethanol 3 times, once from acetone, once more from ethanol) to give 1.2 g promethazine dibenzoyl-L-tartrate white crystals.
9. Similar to Step 4, promethazine-L-tartrate was converted to promethazine.
10. (+)-Promethazine hydrochloride was obtained by precipitation of promethazine with 2M HCl/ether. After vacuum drying, 0.48 g of off-white powder was obtained (purity 99.87% by HPLC).
Repeating Steps 1-5 with 5.7703 g promethazine gave about 0.95 g (−)-promethazine hydrochloride as an off-white powder (purity 99.82% by HPLC). X-ray of the promethazine racemate and enantiomers has also been completed and shows that the pure enantiomers are different crystal forms than the racemate (
Enantiomer cytotoxicity was evaluated by measuring cell survival using the WST-1 assay (Boehringer Mannheim). Cells, L929 lung fibroblast, were grown in culture until confluent. The cells were then treated with the enantiomer dissolved in DMSO (dimethyl sulfoxide, 1 g %) for 1 and 18 hours. Enantiomer cytotoxicity was tested over a four-fold range of concentration.
Following enantiomer incubation, the conversion of WST1 reagent by cells was measured spectrophotometrically as an indicator of cell number and, hence, cell survival. A total of 8 replicate wells of each test concentration were used per assay. One factor analysis of variance (ANOVA), using Fisher's LSD test for post-hoc analysis, was used to determine if the effects of the test substances were significant at the p<0.05 level for each concentration tested versus non-treated controls and DMSO-only treated controls. The data indicates that Ethopropazine, Trimeprazine and Promethazine are all cytotoxic at concentrations greater than 10−5 M.
Huvec cells were plated and grown to confluence in 6-well plates. At confluence, the cells were treated with either Histamine (10−4 M, H), Promethazine racemate (10−5 M) and Histamine (10−4 M, R), Promethazine (+) enantiomer (10−5 M) and Histamine (10−4 M), Promethazine (−) enantiomer (10−5 M) and Histamine (10−4 M) or left untreated (U/T) for 5 hours. Total RNA was isolated using Tri-reagent and subjected to reverse transcription polymerase chain reaction (RT-PCR) analysis of IL-6 production using semiquantitative analysis against HPRT expression (control gene).
As shown in
Ethopropazine (EPZ), obtained from Sigma as the racemate ethopropazine hydrochloride, was resolved using the procedures in Example 1 and subjected to the assays described in Examples 2 and 3. The results are given in
Trimeprazine (TPZ), obtained from Sigma as the racemate, was resolved by preparative column chromatography using CHIRALCEL® OJ-H® preparative column eluting with 99.9% methanol/0.1% diethylamine at room temperature. The isolated enantiomers were subjected to the assays described in Examples 2 and 3. The results are given in
Racemic ethopropazine hydrochloride salt was mixed with methylene chloride and 2M sodium hydroxide. The resulting suspension was agitated and organic layer collected. After drying the solvent was removed by rotary evaporation to give racemic ethopropazine base (4.0 g, 0.013 mol) that reacted with dibenzoyl-D-tartaric acid (4.4 g, 0.012 mol) in acetone with agitation. A white precipitate was collected after a few hours. After two recrystallization steps from absolute ethanol, a 99+% crystal was obtained which was converted to ethopropazine hydrochloride salt. Yield: 20%. From the mother liquor, another diastereomeric salt was obtained as white precipitate, which was also recrystallized twice from absolute ethanol before converting to hydrochloride salt. Yield: 20%.
The following were the peak results from chiral HPLC chromatograms of the ethopropazine HCl racemate:
The following were the peak results from chiral HPLC chromatograms of one of the recrystallized salts, which was determined by HNMR to be the (−)-enantiomer of ethopropazine HCl:
The following were the peak results from chiral HPLC chromatograms of the other recrystallized salt, which was determined by HNMR to be the (+)-enantiomer of ethopropazine HCl:
Optical rotations were measured in water at 27° C.
A hot melt of STEARINE-27 (Loders-Croklaan) with PMZ (Sigma) loading of 40% was used to make the core microcapsules by running off the disk at 6000 RPM at 50-55° C. Ethocel (10%) solutions in ethylacetate:acetone (60:40 wt/wt) were used to coat the PMZ or the PMZ stearine microcapsules. A picture of the PMZ microcapsules is displayed in
Enantiomer cytotoxicity was evaluated by measuring cell survival using the WST-1 assay (Boehringer Mannheim). Cells, L929 lung fibroblast, were grown in culture until confluent. The cells were then treated with the enantiomer dissolved in DMSO (dimethyl sulfoxide, 1 g %) for 1 and 18 hours. Enantiomer cytotoxicity was tested over a fourfold range of concentration. Following enantiomer incubation, the conversion of WST1 reagent by cells was measured spectrophotometrically as an indicator of cell number and, hence, cell survival. Eight replicate wells of each test concentration were used per assay. One factor analysis of variance (ANOVA) using Fisher's LSD test for post-hoc analysis, was used to determine if the effects of the test substances were significant at the p<0.05 level for each concentration tested versus nontreated controls and DMSO-only treated controls. The data is plotted in
The data indicates that the PMZ and enantiomers are cytotoxic at concentrations of 10−4 M and greater. EPZ and TPZ were also found to be cytotoxic at 10 −4 M and greater.
This study was undertaken to evaluate the effect of various formulations of promethazine (PMZ) on the rat nasal mucosa when given via a topical nasal mechanism. Six formulations were evaluated:
At 24 hours each animal was again given an aliquot of anesthesia cocktail as above. The abdominal segment of the aorta was then exposed and 1 ml of blood was drawn into heparinized tubes. The animal was decapitated, the anterior integument and lower jaw removed and a 10-mL volume of Millongs solution (5% Formalin in PBS) gently injected into the nasal cavity. The upper head was then fixed in 50 mL of Millongs for 48 hours with one fixative change. The head was decalcified in buffered formic acid for 14 days with changes of solution every 2 days until no evidence of calcium was found. At that time, the specimen was dissected into four segments stretching from the anterior to posterior nasal cavity (numbered C1 through C4) and paraffin embedded using standard protocols. Sections (5 microns) were cut from each tissue specimen and stained with H&E. The histology was documented with an Olympus microscope using Image Pro software. Blood samples were centrifuged at 1000×g for 5 minutes to pellet the cells and the plasma removed to pre-labeled vials which were stored at −80° C.
The results of the experiment are shown in Table 1, below, and the histology is shown in the
Blood PMZ concentration was measured in the 30 minute post-treatment plasma samples (Table 2). PMZ was detected in all animals tested. Microencapsulated PMZ delivery was not statistically different from that of the PMZ in freebase.
The procedures of Example 3 were repeated using the (+)-enantiomer (#2) and the (−)-enantiomer (#1) of EPZ at 10−5 molar and 10−6 molar, and TPZ at 10−5 molar. The results are given in
Persons of ordinary skill in the art will recognize that many modifications may be made to the present invention without departing from the spirit and scope of the present invention. The embodiment described herein is meant to be illustrative only and should not be taken as limiting the invention, which is defined in the claims.
The present application claims the benefit of U.S. Provisional Application Ser. No. 60/353,766 and U.S. Provisional Application Ser. No. 60/353,633, both filed Jan. 31, 2002.
The U.S. government has certain rights in this invention pursuant to grant number NAG 9-1300 from the National Aeronautics and Space Administration.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US03/02797 | 1/31/2003 | WO | 6/22/2004 |
Number | Date | Country | |
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60353633 | Jan 2002 | US | |
60353766 | Jan 2002 | US |