Patent application title: NOVEL TOPIRAMATE COMPOSITIONS AND AN ESCALATING DOSING STRATEGY FOR TREATING OBESITY AND RELATED DISORDERS
Inventors:
Thomas Najarian (Los Osos, CA, US)
Thomas Najarian (Los Osos, CA, US)
Peter Y. Tam (Redwood City, CA, US)
Peter Y. Tam (Redwood City, CA, US)
Leland F. Wilson (Menlo Park, CA, US)
Leland F. Wilson (Menlo Park, CA, US)
IPC8 Class: AA61K31357FI
USPC Class:
424457
Class name: Preparations characterized by special physical form capsules (e.g., of gelatin, of chocolate, etc.) sustained or differential release
Publication date: 2009-12-10
Patent application number: 20090304789
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Patent application title: NOVEL TOPIRAMATE COMPOSITIONS AND AN ESCALATING DOSING STRATEGY FOR TREATING OBESITY AND RELATED DISORDERS
Inventors:
Leland F. Wilson
Peter Y. Tam
Thomas Najarian
Agents:
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY AND POPEO, P.C
Assignees:
Origin: PALO ALTO, CA US
IPC8 Class: AA61K31357FI
USPC Class:
424457
Patent application number: 20090304789
Abstract:
The present invention is drawn to novel topiramate compositions as well as
methods for treating obesity and related conditions, including conditions
associated with and/or caused by obesity per se. The present invention
also features a pharmaceutical composition that includes, e.g.,
topiramate alone or in combination with a sympathomimetic agent and a
novel escalating dosing strategy for administering such compositions.Claims:
1. A controlled release composition for treating obesity, diabetes or a
related condition in a subject comprising:an effective amount of
topiramate;microcrystalline cellulose; andmethocellulose.
2. The composition of claim 1, wherein the topiramate, the microcrystalline cellulose, and the methocellulose are present in a matrix core of a bead.
3. The composition of claim 2, wherein the matrix core is coated with ethyl cellulose.
4. The composition of claim 3, wherein the matrix core is further coated with polyvinyl pyrrolidone.
5. The composition of claim 4, wherein the bead is encapsulated into a capsule.
6. The composition of claim 5, wherein the capsule further comprises a sympathomimetic agent.
7. The composition of claim 6, wherein the sympathomimetic agent is phentermine.
8. The composition of claim 7, wherein the phentermine is coated onto a sugar sphere.
9. The composition of claim 7, wherein the phentermine is coated onto the controlled release topiramate beads.
10. The composition of claim 7, wherein the phentermine is an immediate release form.
11. The composition of claim 7, wherein the phentermine is a controlled release form.
12. The composition of claim 6, wherein the sympathomimetic agent is bupropion.
13. The composition of claim 12, wherein the bupropion is coated onto a sugar sphere.
14. The composition of claim 12, wherein the bupropion is coated onto the controlled release topiramate beads.
15. The composition of claim 12, wherein the bupropion is an immediate release form.
16. The composition of claim 12, wherein the bupropion is a controlled release form.
17. The composition of claim 7, wherein the topiramate reduces phentermine exposure and reduces side effects associated with phentermine.
18. The composition of claim 12, wherein the topiramate reduces bupropion exposure and reduces side effects associated with bupropion.
19. A method for treating an individual for obesity or a related condition, the method comprising administering an effective amount of topiramate according to an escalating dosage strategy, wherein the topiramate is in a controlled-release form.
20. The method according to claim 19, wherein the dosing strategy comprises:administering an initial daily dosage of topiramate to the individual for a specific period of time; andincrementally increasing the dosage at various designated time points.
21. The method according to claim 20, wherein the topiramate is administered at a dosage ranging from 20-100 mg/day.
22. The method according to claim 21, wherein the initial dosage of topiramate is 23 mg/day.
23. The method according to claim 20, further comprising administering an effective amount of a sympathomimetic agent.
24. The method according to claim 23, wherein the sympathomimetic agent is phentermine.
25. The method according to claim 23, wherein the sympathomimetic agent is bupropion.
26. The method according to claim 23, wherein said sympathomimetic agent is in immediate release form.
27. A method for treating obesity or a related condition in an individual by administering a non-toxic, effective amount of topiramate according to an escalating dosage strategy, the method comprising:administering an initial dose of topiramate for a first week at a dosage of 23 mg/day;administering 46 mg/day of topiramate for a second one week administration after the first one week administration; andadministering 69 mg/day of topiramate for a third one week administration after the second one week administration; andadministering 92 mg/day of topiramate for a fourth one week administration after the third one week administration.
28. The method according to claim 27, further comprising administering 3.75 mg/day of phentermine in the first one week administration.
29. The method according to claim 28, further comprising administering 7.5 mg/day of phentermine in the second one week administration.
30. The method according to claim 29, further comprising administering 11.25 mg/day of phentermine in the third one week administration.
31. The method according to claim 30, further comprising administering 15.0 mg/day of phentermine in the fourth one week administration.
32. A kit comprising topiramate and instructions providing a dosing strategy for administering topiramate to an individual for treating obesity or a related condition.
33. The kit according to claim 32, wherein the dosing strategy provide a dosing strategy which includes administering a lower daily dosage of topiramate for a specific period of time and then incrementally increasing the dosage at various designated time points.
34. The kit according to claim 32, further comprising:a sympathomimetic agent;instructions providing a dosing strategy for administering the sympathomimetic agent in combination with the topiramate.
35. The kit according to claim 34, wherein the topiramate and sympathomimetic agent are provided in a titration card, wherein the card provides dosages for four weeks.
36. The kit according to claim 35, wherein the dosages increase each week.
Description:
BACKGROUND
[0001]The prevalence of obesity in both children and adults is on the rise in first world countries, especially in the United States, as well as in many developing countries such as China and India. Many aspects of a person's life are affected by obesity, from physical problems such as knee and ankle joint deterioration, to emotional problems resulting from self-esteem issues and society's attitude towards heavy people. The medical problems caused by obesity can be serious and often life-threatening and include diabetes, shortness of breath and other respiratory problems, gallbladder disease, hypertension, dyslipidemia (for example, high cholesterol or high levels of triglycerides), cancer, osteoarthritis, other orthopedic problems, reflux esophagitis (heartburn), snoring, sleep apnea, menstrual irregularities, infertility, gout, problems associated with pregnancy, heart trouble, muscular dystrophy and metabolic disorders, including hypoalphalipoproteinemia, familial combined hyperlipidemia, insulin resistant syndrome X or multiple metabolic disorder, coronary artery disease, and dyslipidemic hypertension. In addition, obesity has been associated with an increased incidence of certain cancers, notably cancers of the colon, rectum, prostate, breast, uterus, and cervix.
[0002]Moreover, obesity substantially increases the risk of morbidity from hypertension, dyslipidemia, type 2 diabetes, coronary heart disease, stroke, gallbladder disease, osteoarthritis and endometrial, breast, prostate, and colon cancers. Higher body weights are also associated with increases in all-cause mortality. Most or all of these problems are relieved or improved by permanent significant weight loss as well as a significant increase in longevity.
[0003]The currently available strategies for treating obesity and/or related disorders include dietary restriction, increased physical activity, pharmacological and surgical approaches which vary depending, at least in part, on the degree of weight loss one is attempting to achieve as well as on the severity of obesity exhibited by the subject. For example, treatments such as low-fat diet and/or regular exercise are often adequate in cases where a subject is only mildly overweight. The difficulty in maintaining long-term weight loss through diet and behavior modification has led to an increasing interest in other avenues for treatment, particularly pharmacotherapy.
[0004]Present pharmacological interventions typically induce a weight loss of between five and fifteen kilograms; if the medication is discontinued, renewed weight gain ensues. Surgical treatments are comparatively successful and are reserved for patients with extreme obesity and/or with serious medical complications.
[0005]The above treatments, i.e., diet, exercise, surgery, and pharmaceutical treatments, can be enhanced by controlled use of over-the-counter appetite suppressants including caffeine, ephedrine and phenylpropanolamine (Acutrim®, Dexatrim®). Moreover, prescription medications including amphetamine, diethylpropion (Tenuate®), mazindol (Mazanor®, Sanorex®), phentermine (Fastin®, Ionamin®), phenmetrazine (Preludin®), phendimetrazine (Bontrol®, Plegine®, Adipost®, Dital®, Dyrexan®, Melfiat®, Prelu-2®, Rexigen Forte®), benzphetamine (Didrexg®) and fluoxetine (Prozac®) are often used in the treatment of seriously overweight and/or obese subjects or patients.
[0006]Accordingly, while society has seen tremendous advances in the field of pharmaceuticals, there are, of course, drawbacks to the administration of any given pharmaceutical agent. Sometimes, the disadvantages, characterized as "side effects," are so severe as to preclude administration of a particular agent at a therapeutically effective dose. For example, these drugs may be effective only in a subset of patients and their long term use is limited by side effects, some of which are severe. In such a case, drug therapy is discontinued, and other pharmaceutical agents may be tried. Many agents in the same therapeutic class, however, display similar side effect profiles, meaning that patients either have to forego therapy or suffer from unpleasant side effects associated with a particular medication.
[0007]The present invention is directed to an escalating dosing strategy for administering topiramate alone or in combination with a second therapeutic agent which directly or indirectly reduces the side effects associated with one or both of the agents administered. By "indirectly" reducing side effects is meant that a first pharmaceutical agent allows the second agent to be administered at a lower dose without compromising therapeutic efficacy, thus resulting dose-dependent unwanted effects.
[0008]Topiramate (2,3,4,5-Bis-O-(1-methylethylidene)-β-D-fructopyranose sulfamate) is a broad-spectrum neurotherapeutic agent approved by the FDA and more than 75 countries around the world for selected seizure disorders (E. Faught et al. (1996) Neurology 46:1684-90.; Karim et al. (1995) Epilepsia 36 (S4):33; S. K. Sachdeo et al. (1995) Epilepsia 36(S4):33; T. A. Glauser (1999) Epilepsia 40 (S5):S71-80; R. C. Sachdeo (1998) Clin. Pharmacokinet. 34:335-346). There has also been evidence that topiramate is effective in the treatment of diabetes (U.S. Pat. Nos. 7,109,174 and 6,362,220), neurological disorders (U.S. Pat. No. 6,908,902), depression (U.S. Pat. No. 6,627,653), psychosis (U.S. Pat. No. 6,620,819), headaches (U.S. Pat. No.6,319,903) and hypertension (U.S. Pat. No.6,201,010). However there have been adverse effects associated with the use of topiramate in humans, such as diarrhea and anal leakage, which can discourage many obese patients from taking this drug.
[0009]As such, there is interest in the development of additional methods and compositions for treating obesity and related conditions in which the therapeutic efficacy of known compositions are improved. In addition, combination treatment may be employed to decrease the doses of the individual components in the resulting combinations while still preventing unwanted or harmful side effects of the individual components. Thus, there is an urgent need to discover suitable methods for the treatment of obesity and/or a related condition, including combination treatments that result in reduction of toxicity, decreased side effects and effective treatment.
SUMMARY OF THE INVENTION
[0010]The present invention provides novel topiramate compositions and methods for treating obesity and related conditions, including conditions associated with and/or caused by obesity per se.
[0011]The present invention also features a pharmaceutical composition that includes, e.g., topiramate alone or in combination with a sympathomimetic agent. The term "sympathomimetic agent" is a term of art and refers to agents or compounds which mimic or alter stimulation of the sympathetic nervous system. In certain aspects, the combination compositions include topiramate with two or more sympathomimetic agents.
[0012]The present invention also features a pharmaceutical composition that includes topiramate in combination with phentermine for treating obesity or a related condition.
[0013]The present invention also features a pharmaceutical composition that includes topiramate in combination with bupropion for treating obesity or a related condition.
[0014]The present invention also features a novel escalating dosing strategy for administering topiramate alone or in combination with a sympathomimetic agent.
[0015]In certain embodiments, the present invention features a controlled release composition for treating obesity or a related condition.
[0016]In certain embodiments, the present invention is used for treating diabetes.
[0017]In certain aspects, the present invention comprises an effective amount of topiramate, microcrystalline cellulose and methocellulose.
[0018]In certain aspects of the invention, the topiramate, the microcrystalline cellulose, and the methocellulose are present in a matrix core of a bead.
[0019]In certain aspects of the invention, the matrix core is coated with ethyl cellulose.
[0020]In certain aspects of the invention, the matrix core is further coated with polyvinyl pyrrolidone.
[0021]In certain aspects of the invention, the bead is encapsulated into a capsule.
[0022]In certain aspects of the invention, the capsule further comprises a sympathomimetic agent.
[0023]In certain aspects of the invention, the sympathomimetic agent is phentermine.
[0024]In certain aspects of the invention, the phentermine is coated onto a sugar sphere.
[0025]In certain aspects of the invention, the phentermine is coated onto the topiramate controlled release beads.
[0026]In certain aspects of the invention, the phentermine is an immediate release form.
[0027]In certain aspects of the invention, the phentermine is a controlled release form.
[0028]In certain aspects of the invention, the topiramate reduces phentermine exposure and reduces side effects associated with phentermine.
[0029]In certain aspects of the invention, the sympathomimetic agent is bupropion.
[0030]In certain aspects of the invention, the bupropion is coated onto a sugar sphere.
[0031]In certain aspects of the invention, the bupropion is coated onto the topiramate controlled released beads.
[0032]In certain aspects of the invention, the bupropion is an immediate release form.
[0033]In certain aspects of the invention, the bupropion is a controlled release form.
[0034]In certain aspects of the invention, the topiramate reduces bupropion exposure and reduces side effects associated with bupropion.
[0035]In certain embodiments, the present invention features a method for treating an individual for obesity or a related condition, the method comprising administering an effective amount of topiramate according to an escalating dosage strategy, wherein the topiramate is in a controlled-release form.
[0036]In certain aspects of the invention, the dosing strategy includes administering an initial daily dosage of topiramate to the individual for a specific period of time and incrementally increasing the dosage at various designated time points.
[0037]In certain aspects of the invention, the method includes administering the topiramate at a dosage ranging from 20-150 mg/day.
[0038]In certain aspects of the invention, the method includes administering an initial dosage of 23 mg/day of topiramate.
[0039]In certain aspects of the invention, the method further includes administering an effective amount of a sympathomimetic agent.
[0040]In certain aspects of the invention, the sympathomimetic agent is phentermine.
[0041]In certain aspects of the invention, the sympathomimetic agent is bupropion.
[0042]In certain aspects of the invention, the sympathomimetic agent is in immediate release form.
[0043]In certain embodiments, the present invention features a method for treating obesity or a related condition in an individual by administering a non-toxic, effective amount of topiramate according to an escalating dosage strategy.
[0044]In certain aspects of the invention, the method includes administering an initial dose of 23 mg/day of topiramate in a first one-week administration.
[0045]In certain aspects of the invention, the method further includes administering a dosage of 46 mg/day of topiramate for a second one-week administration.
[0046]In certain aspects of the invention, the method further includes administering a dosage of 69 mg/day of topiramate for a third one week administration.
[0047]In certain aspects of the invention, the method further includes administering a dosage of 92 mg/day of topiramate for a fourth one week administration.
[0048]In certain aspects of the invention, the method further includes administering 3.75 mg/day of phentermine in the first one week administration.
[0049]In certain aspects of the invention, the method further includes administering a 7.5 mg/day of phentermine in the second one week administration.
[0050]In certain aspects of the invention, the method further includes administering a dosage of 11.25 mg/day of phentermine in the third one week administration.
[0051]In certain aspects of the invention, the method further includes administering a 15.0 mg/day of phentermine in the fourth one week administration.
[0052]In certain embodiments, the present invention features a kit comprising topiramate and instructions providing a dosing strategy for administering topiramate to an individual for treating obesity or a related condition.
[0053]In certain aspects of the invention, the kit includes a dosing strategy which includes administering a lower daily dosage of topiramate for a specific period of time and then incrementally increasing the dosage at various designated time points.
[0054]In certain aspects of the invention, the kit includes a sympathomimetic agent and instructions providing a dosing strategy for administering the sympathomimetic agent in combination with the topiramate.
[0055]In certain aspects of the invention, the kit also includes the topiramate and sympathomimetic agent in a titration card, wherein the card provides dosages for four weeks.
[0056]In certain aspects of the invention, the dosages in the titration card increase each week.
BRIEF DESCRIPTION OF THE DRAWINGS
[0057]FIG. 1 provides a summary of the plasma concentration of controlled release topiramate according to the present invention versus topiramate (Topomax®) in normal obese subjects.
[0058]FIG. 2 depicts the mean plasma phentermine concentrations versus time for subjects administered phentermine in combination with controlled release topiramate and phentermine in combination with immediate release topiramate (Topomax®).
DEFINITIONS AND NOMENCLATURE
[0059]It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, "an active agent" refers not only to a single active agent but also to a combination of two or more different active agents, "a dosage form" refers to a combination of dosage forms as well as to a single dosage form, and the like. Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein may be useful in the practice or testing of the present invention, preferred methods and materials are described below. Specific terminology of particular importance to the description of the present invention is defined below.
[0060]When referring to an active agent, applicants intend the term "active agent" to encompass not only the specified molecular entity but also its pharmaceutically acceptable, pharmacologically active analogs, including, but not limited to, salts, esters, amides, prodrugs, conjugates, active metabolites, and other such derivatives, analogs, and related compounds.
[0061]The terms "treating" and "treatment" as used herein refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, and improvement or remediation of damage. In certain aspects, the term "treating" and "treatment" as used herein refer to the prevention of the occurrence of symptoms. In other aspects, the term "treating" and "treatment" as used herein refer to the prevention of the underlying cause of symptoms associated with obesity and/or a related condition.
[0062]By the terms "effective amount" and "therapeutically effective amount" of an agent, compound, drug, composition or combination of the invention which is nontoxic and effective for producing some desired therapeutic effect upon administration to a subject or patient (e.g., a human subject or patient).
[0063]The term "dosage form" denotes any form of a pharmaceutical composition that contains an amount of active agent sufficient to achieve a therapeutic effect with a single administration. When the formulation is a tablet or capsule, the dosage form is usually one such tablet or capsule. The frequency of administration that will provide the most effective results in an efficient manner without overdosing will vary with the characteristics of the particular active agent, including both its pharmacological characteristics and its physical characteristics, such as hydrophilicity.
[0064]The term "controlled release" refers to a drug-containing formulation or fraction thereof in which release of the drug is not immediate, i.e., with a "controlled release" formulation, administration does not result in immediate release of the drug into an absorption pool. The term is used interchangeably with "nonimmediate release" as defined in Remington: The Science and Practice of Pharmacy, Nineteenth Ed. (Easton, Pa.: Mack Publishing Company, 1995). In general, the term "controlled release" as used herein includes sustained release, modified release and delayed release formulations.
[0065]The term "sustained release" (synonymous with "extended release") is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period. The term "delayed release" is also used in its conventional sense, to refer to a drug formulation which, following administration to a patient provides a measurable time delay before drug is released from the formulation into the patient's body.
[0066]By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. When the term "pharmaceutically acceptable" is used to refer to a pharmaceutical carrier or excipient, it is implied that the carrier or excipient has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration. "Pharmacologically active" (or simply "active") as in a "pharmacologically active" derivative or analog, refers to a derivative or analog having the same type of pharmacological activity as the parent compound and approximately equivalent in degree. The term "pharmaceutically acceptable salts" include acid addition salts which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
[0067]As used herein, "subject" or "individual" or "patient" refers to any subject for whom or which therapy is desired, and generally refers to the recipient of the therapy to be practiced according to the invention. The subject can be any vertebrate, but will typically be a mammal. If a mammal, the subject will in many embodiments be a human, but may also be a domestic livestock, laboratory subject or pet animal.
[0068]Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[0069]Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[0070]Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
[0071]The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
DETAILED DESCRIPTION
[0072]The present invention provides novel topiramate compositions and methods of treating obesity and conditions associated with and/or caused by obesity per se. The present invention also features a pharmaceutical composition that includes, e.g., topiramate alone or in combination with a sympathomimetic agent. In certain aspects, the sympathomimetic agent is phentermine or bupropion. The present invention, in certain aspects, is directed to an escalating dosing strategy for administering topiramate alone or in combination with one or more sympathomimetic agents.
Topiramate Mono Therapy
[0073]Topiramate is an anticonvulsant sulfamate compound (Topamax®), which is also referred to in the art as 2,3:4,5-bis-O-(1-methylethylidene)-β-D-fructopyranose sulfamate.
[0074]In certain aspects, the pharmaceutical compositions of the subject invention include an effective amount of topiramate as the active agent. In some embodiments, an "effective amount" of topiramate is an amount that results in a reduction of at least one pathological parameter associated with obesity and/or a related disorder. Thus, e.g., in some embodiments, an effective amount of topiramate is an amount that is effective to achieve a reduction of at least about 10%, at least about 15%, at least about 20%, or at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, compared to the expected reduction in the parameter in an individual suffering from obesity and/or a related disorder and not treated with the topiramate compositions.
[0075]A suitable dose range for topiramate is one which ranges from 10-1500 mg in a single daily dosage which is administered to a patient over a specific period of time. For example, 10 mg, 20 mg, 30 mg, 60 mg, 90 mg, 120 mg, 150 mg, 180 mg, 210 mg, 240 mg, 270 mg, 300 mg, 330 mg, 360 mg, 390 mg, 420 mg, 450 mg, 480 mg, 500 mg, 600 mg, 800 mg, 1000 mg, 1200 mg, 1500 mg or the like is administered to a patient as a single daily dosage. In another example, 23 mg, 46 mg, 69 mg and 92 mg or the like is administered to a patient as a single daily dosage. In some embodiments, the single dosing range of the present invention is from 10-150 mg of topiramate. In certain embodiments, the single dosing range of the present invention is from 10-100 mg of topiramate.
[0076]Further, the patient may receive a specific dosage of topiramate over a period of weeks, months, or years. For example, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years and the like.
Topiramate Formulations
[0077]Aspects of the invention provide a topiramate monotherapy or combination therapy in which the subject topiramate formulation is effective when administered at a initial dose as low as 10-23 mg. In certain aspects, the topiramate formulation is effective at a dose of approximately 20 mg. The novel topiramate formulations of the present invention have a lower maximum concentration (Cmax) without decreasing total drug exposure defined by the area under the concentration-time curve (AUC). Further, the novel topiramate formulations of the present invention have a delay in time after administration of a drug when the maximum plasma concentration is reached (Tmax) by six to eight hours. As depicted in FIG. 1, drug exposure as measured by AUC for the control release (CR) formulation capsule is the same as the 100 mg of immediate release topiramate (Topamax®) tablet despite a 20% reduction in the Cmax. Therefore, this formulation is capable of reducing the Cmax which would reduce side effects without compromising the efficacy of the treatment, since the AUC is the same. This reduction in Cmax is preferred as topiramate can be sedating and a delay in the time to reach maximum plasma concentration to the late afternoon or evening time would improve the tolerability of the drug.
[0078]As such, the effective amount of topiramate is decreased, thereby further reducing any toxicity or harmful side effects in the patient. The amount of topiramate administered to the patient is less than an amount that would cause toxicity in the patient. In certain embodiments, the amount of the compound that is administered to the patient is less than the amount that causes a concentration of the compound in the patient's plasma to equal or exceed the toxic level of the compound. The optimal amount of the compound that should be administered to the patient in the practice of the present invention will depend on the individual as well as the severity of the individual's symptoms.
[0079]Depending on the intended mode of administration, the pharmaceutical formulation may be a solid, semi-solid or liquid, such as, for example, a tablet, a capsule, a caplet, a liquid, a suspension, an emulsion, a suppository, granules, pellets, beads, a powder, or the like, preferably in unit dosage form suitable for single administration of a precise dosage. Suitable pharmaceutical compositions and dosage forms may be prepared using conventional methods known to those in the field of pharmaceutical formulation and described in the pertinent texts and literature, e.g., in Remington. The Science and Practice of Pharmacy (Easton, Pa.: Mack Publishing Co., 1995). For those compounds that are orally active, oral dosage forms are generally preferred, and include tablets, capsules, caplets, solutions, suspensions and syrups, and may also comprise a plurality of granules, beads, powders, or pellets that may or may not be encapsulated. Preferred oral dosage forms are tablets and capsules.
[0080]As noted above, it is especially advantageous to formulate compositions of the invention in unit dosage form for ease of administration and uniformity of dosage. The term "unit dosage forms" as used herein refers to physically discrete units suited as unitary dosages for the individuals to be treated. That is, the compositions are formulated into discrete dosage units each containing a predetermined, "unit dosage" quantity of an active agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of unit dosage forms of the invention are dependent on the unique characteristics of the active agent to be delivered. Dosages can further be determined by reference to the usual dose and manner of administration of the ingredients. It should be noted that, in some cases, two or more individual dosage units in combination provide a therapeutically effective amount of the active agent, e.g., two tablets or capsules taken together may provide a therapeutically effective dosage of topiramate, such that the unit dosage in each tablet or capsule is approximately 50% of the therapeutically effective amount.
[0081]Tablets may be manufactured using standard tablet processing procedures and equipment. Direct compression and granulation techniques are preferred. In addition to the active agent, tablets will generally contain inactive, pharmaceutically acceptable carrier materials such as binders, lubricants, disintegrants, fillers, stabilizers, surfactants, coloring agents, and the like.
[0082]Capsules are also preferred oral dosage forms for those pharmaceutical active agents that are orally active, in which case the active agent-containing composition may be encapsulated in the form of a liquid or solid (including particulates such as granules, beads, powders or pellets). Suitable capsules may be either hard or soft, and are generally made of gelatin, starch, or a cellulosic material, with gelatin capsules preferred. Two-piece hard gelatin capsules are preferably sealed, such as with gelatin bands or the like. See, for example, Remington: The Science and Practice of Pharmacy, cited earlier herein, which describes materials and methods for preparing encapsulated pharmaceuticals.
[0083]Oral dosage forms, whether tablets, capsules, caplets, or particulates, may, if desired, be formulated so as to provide for controlled release of topiramate, and in a preferred embodiment, the present formulations are controlled release oral dosage forms. Generally, the dosage forms provide for sustained release, i.e., gradual, release of topiramate, from the dosage form to the patient's body over an extended time period, typically providing for a substantially constant blood level of the agent over a time period in the range of about 4 to about 12 hours, typically in the range of about 6 to about 10 hours or 6 to about 8 hours.
[0084]Generally, as will be appreciated by those of ordinary skill in the art, sustained release dosage forms are formulated by dispersing the active agent within a matrix of a gradually hydrolyzable material such as a hydrophilic polymer, or by coating a solid, drug-containing dosage form with such a material. Hydrophilic polymers useful for providing a sustained release coating or matrix include, by way of example: cellulosic polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose e.g., Methocel® A15LV, ethyl cellulose, cellulose acetate, and carboxymethylcellulose sodium; acrylic acid polymers and copolymers, preferably formed from acrylic acid, methacrylic acid, acrylic acid alkyl esters, methacrylic acid alkyl esters, and the like, e.g. copolymers of acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, methyl methacrylate and/or ethyl methacrylate; and vinyl polymers and copolymers such as polyvinyl pyrrolidone e.g., Povidone K30, polyvinyl acetate, and ethylene-vinyl acetate copolymer.
[0085]When sustained release preparations are prepared, tablets, granules, powder, capsules, and the like can be produced according to a conventional method after adding excipient, and as necessary, binder, disintegrating agent, lubricant, coloring agent, taste-modifying agent, flavoring agent, and the like. These additives may be ones generally used in the field, and for example, lactose, sodium chloride, glucose, starch, microcrystalline cellulose, e.g., Avicel®, and silicic acid as the excipient, water, ethanol, propanol, simple syrup, gelatin solution, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, and polyvinylpyrrolidone as the binder, agar powder, sodium hydrogencarbonate, sodium lauryl sulfate, and stearic acid monoglyceride as the disintegrating agent, purified talc, stearic acid salt, borax, and polyethylene glycol as the lubricant, β-carotene, yellow iron sesquioxide, and caramel as the coloring agent, and saccharose and orange peel as the taste-modifying agent can be listed as examples.
[0086]Additional sustained release dosage forms of the present invention may be composed of the acrylate and methacrylate copolymers available under the trade name "Eudragit" from Rohm Pharma (Germany). The Eudragit series E, L, S, RL, RS, and NE copolymers are available as solubilized in organic solvent, in an aqueous dispersion, or as a dry powder. Preferred acrylate polymers are copolymers of methacrylic acid and methyl methacrylate, such as the Eudragit L and Eudragit S series polymers. Particularly preferred such copolymers are Eudragit L-30D-55 and Eudragit L-100-55 (the latter copolymer is a spray-dried form of Eudragit L-30D-55 that can be reconstituted with water). The molecular weight of the Eudragit L-30D-55 and Eudragit L-100-55 copolymer is approximately 135,000 Da, with a ratio of free carboxyl groups to ester groups of approximately 1:1. The copolymer is generally insoluble in aqueous fluids having a pH below 5.5. Another particularly suitable methacrylic acid-methyl methacrylate copolymer is Eudragit S-100, which differs from Eudragit L-30D-55 in that the ratio of free carboxyl groups to ester groups is approximately 1:2. Eudragit S-100 is insoluble at pH below 5.5, but unlike Eudragit L-30D-55, is poorly soluble in aqueous fluids having a pH in the range of 5.5 to 7.0. This copolymer is soluble at pH 7.0 and above. Eudragit L-100 may also be used, which has a pH-dependent solubility profile between that of Eudragit L-30D-55 and Eudragit S-100, insofar as it is insoluble at a pH below 6.0. It will be appreciated by those skilled in the art that Eudragit L-30D-55, L-100-55, L-100, and S-100 can be replaced with other acceptable polymers having similar pH-dependent solubility characteristics. Other preferred Eudragit polymers are cationic, such as the Eudragit E, RS, and RL series polymers. Eudragit E100 and E PO are cationic copolymers of dimethylaminoethyl methacrylate and neutral methacrylates (e.g., methyl methacrylate), while Eudragit RS and Eudragit RL polymers are analogous polymers, composed of neutral methacrylic acid esters and a small proportion of trimethylammonioethyl methacrylate.
[0087]In a specific embodiment, controlled release topiramate beads are made using an extrusion spheronization process to produce a matrix core comprised of topiramate: 40.0% w/w; microcrystalline cellulose, Avicel® PH102: 56.5% w/w; and Methocel® A15 LV: 3.5% w/w. The topiramate cores are then coated with ethyl cellulose: 5.47% w/w and Povidone K30: 2.39% w/w.
[0088]Preparations according to this invention for parenteral administration include sterile aqueous and nonaqueous solutions, suspensions, and emulsions. Injectable aqueous solutions contain the active agent in water-soluble form. Examples of nonaqueous solvents or vehicles include fatty oils, such as olive oil and corn oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, low molecular weight alcohols such as propylene glycol, synthetic hydrophilic polymers such as polyethylene glycol, liposomes, and the like. Parenteral formulations may also contain adjuvants such as solubilizers, preservatives, wetting agents, emulsifiers, dispersants, and stabilizers, and aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, and dextran. Injectable formulations are rendered sterile by incorporation of a sterilizing agent, filtration through a bacteria-retaining filter, irradiation, or heat. They can also be manufactured using a sterile injectable medium. The active agent may also be in dried, e.g., lyophilized, form that may be rehydrated with a suitable vehicle immediately prior to administration via injection.
[0089]The active agent may also be administered through the skin using conventional transdermal drug delivery systems, wherein the active agent is contained within a laminated structure that serves as a drug delivery device to be affixed to the skin. In such a structure, the drug composition is contained in a layer, or "reservoir," underlying an upper backing layer. The laminated structure may contain a single reservoir, or it may contain multiple reservoirs. In one embodiment, the reservoir comprises a polymeric matrix of a pharmaceutically acceptable contact adhesive material that serves to affix the system to the skin during drug delivery. Alternatively, the drug-containing reservoir and skin contact adhesive are present as separate and distinct layers, with the adhesive underlying the reservoir which, in this case, may be either a polymeric matrix as described above, or it may be a liquid or hydrogel reservoir, or may take some other form. Transdermal drug delivery systems may in addition contain a skin permeation enhancer.
[0090]In addition to the formulations described previously, the active agent may be formulated as a depot preparation for controlled release of the active agent, preferably sustained release over an extended time period. These sustained release dosage forms are generally administered by implantation (e.g., subcutaneously or intramuscularly or by intramuscular injection).
[0091]Although the present compositions will generally be administered orally, parenterally, transdermally, or via an implanted depot, other modes of administration are suitable as well. For example, administration may be transmucosal, e.g., rectal or vaginal, preferably using a suppository that contains, in addition to the active agent, excipients such as a suppository wax. Transmucosal administration also encompasses transurethral administration, as described, for example, in U.S. Pat. Nos. 5,242,391, 5,474,535, and 5,773,020 to Place et al. Formulations for nasal or sublingual administration are also prepared with standard excipients well known in the art. The pharmaceutical compositions of the invention may also be formulated for inhalation, e.g., as a solution in saline, as a dry powder, or as an aerosol.
Combination Therapy
[0092]In some embodiments, a subject method of treating obesity and/or a related disorder comprises administering topiramate in an individual and a sympathomimetic agent. In certain aspects, the subject methods may comprise administering topiramate with two or more sympathomimetic agents.
[0093]Sympathomimetic agents for use in the present invention and their general clinical uses or effects are set forth in Table I.
TABLE-US-00001 TABLE I Sympathomimetic Agents and Clinical Uses Thereof ##STR00001## Ring α Receptor β Receptor CNS, Agent name substituent(s) R.sup.α R.sup.β R.sup.γ A N P V B C 0 Bupropion 3-Cl ═O CH3 C(CH3)3 Phenylethylamine H H H Epinephrine 3-OH, 4-OH OH H CH3 A, P, V B, C Norepinephrine 3-OH, 4-OH OH H H P Epinine 3-OH, 4-OH H H CH3 Dopamine 3-OH, 4-OH H H H P Dobutamine 3-OH, 4-OH H H 1* C Nordefrin 3-OH, 4-OH OH CH3 H V Ethylnorepinephrine 3-OH, 4-OH OH CH2CH3 H B Isoproterenol 3-OH, 4-OH OH H CH(CH3)3 B, C Protokylol 3-OH, 4-OH OH H 2* B Isoetharine 3-OH, 4-OH OH CH2CH3 CH(CH3)2 B Metaproterenol 3-OH, 5-OH OH H CH(CH3)2 B Terbutaline 3-OH, 5-OH OH H C(CH3)3 B Metaraminol 3-OH OH CH3 H P Phenylephrine 3-OH OH H CH3 N, P Tyramine 4-OH H H H Hydroxyamphetamine 4-OH H CH3 H N, P C Methoxyphenamine 2-OCH3 H CH3 CH3 B Methoxamine 2-OCH3, 5- OH CH3 H P OCH3 Albuterol 3-CH2OH, 4- OH H C(CH3)3 B OH Amphethamine H CH3 H CNS, 0 Methamphetamine H CH3 CH3 P CNS, 0 Benzphetamine H CH3 --NHR.sup.γ is 0 replaced with 3* Ephedrine OH CH3 CH3 N, P B, C Phenylpropanolamine OH CH3 H N Mephentermine H --CHR.sup.β-- is CH3 N, P replaced with 4* Phentermine H --CHR.sup.β-- is H 0 replaced with 4* Chlorphentermine 4-Cl H --CHR.sup.β-- is H 0 replaced with 4* Fenfluramine 3-CF3 H CH3 C2H5 0 Propylhexedrine 5*: phenyl ring H CH3 CH3 N is replaced with cyclohexyl Diethylpropion 6*: The substituent at the 1- 0 position is replaced with 6, below. Phenmetrazine 7*: The substituent at the 1- 0 position is replaced with 7, below. Phendimetrazine 8*: The substituent at the 1- 0 position is replaced with 8, below. *1: ##STR00002## *2: ##STR00003## *3: ##STR00004## *4: ##STR00005## *5: ##STR00006## *6: ##STR00007## *7: ##STR00008## α Activity A = Allergic reactions (includes β action) N = Nasal decongestion P = Pressor (may include β action) V = Other local vasoconstriction (e.g. in local anesthesia) β Activity B = Bronchodilator C = Cardiac CNS = Central nervous system 0 = Anorectic *Numbers bearing an asterisk refer to the substituents numbered in the bottom rows of the table; substituent 5 replaces the phenyl rings, and 6, 7 and 8 are attached directly to the phenyl ring, replacing the ethylamine side chain. .sup.†The α and β in the prototype formula refer to positions of the C atoms in the ethylamine side chain.
[0094]In certain embodiments, the sympathomimetic agent is phentermine or a phentermine-like compound. As defined herein, a phentermine-like compound is a compound structurally related to phentermine (e.g., an analog or derivative) which maintains an anorectic activity similar to that of phentermine. One phentermine-like compound is chlorphentermine. In yet another embodiment, the sympathomimetic agent is amphetamine or an amphetamine-like compound. As used herein, an "amphetamine-like compound" is a compound structurally related to amphetamine (e.g., an analog or derivative) which maintains an anorectic effect of amphetamine. In yet another embodiment, the sympathomimetic agent is phenmetrazine or a phenmetrazine-like compound. As defined herein, a "phenmetrazine-like compound" is a compound structurally related to phenmetrazine (e.g., an analog or derivative) which maintains an anorectic effect of phenmetrazine. One phenmetrazine-like compound is phendimetrazine. Analogs and/or derivatives of the compounds of the present invention can be tested for their ability to suppress appetite (e.g., suppress food intake) in a subject (e.g., a mammalian subject).
[0095]In other embodiments, the sympathomimetic agent is bupropion or a bupropion-like compound. As defined herein, a "bupropion-like compound" is a compound structurally related to bupropion (e.g., an analog or derivative) which maintains an anti-depressive activity similar to that of bupropion.
[0096]In an exemplary embodiment, the sympathomimetic agent is selected from bupropion, amphetamine, methamphetamine, benzphetamine, phenylpropanolamine, phentermine, chlorphentermine, diethylpropion, phenmetrazine, and phendimetrazine (as set forth in Table I).
[0097]In one embodiment, the sympathomimetic agent is phentermine. It is also within the scope of the present invention to utilize other sympathomimetic agents including pseudo ephedrine (a stereoisomer of ephedrine, SUDAFED®), methylphenidate (RITALIN®), tuaminoheptane, other CNS stimulants including, for example, caffeine and bupropion.
[0098]The choice of appropriate dosages for the drugs used in combination therapy according to the present invention can be determined and optimized by the skilled artisan, e.g., by observation of the patient, including the patient's overall health, the response to the combination therapy, and the like. Optimization, for example, may be necessary if it is determined that a patient is not exhibiting the desired therapeutic effect or conversely, if the patient is experiencing undesirable or adverse side effects that are too many in number or are of a troublesome severity.
[0099]Although the dosage used will vary depending on the clinical goals to be achieved, a suitable dose range is one which ranges from 2-1500 mg of a sympathomimetic agent in a single daily dosage which is administered to a patient over a specific period of time. For example, about 2 mg, 4 mg, 10 mg, 20 mg, 30 mg, 60 mg, 90 mg, 120 mg, 150 mg, 180 mg, 210 mg, 240 mg, 270 mg, 300 mg, 330 mg, 360 mg, 390 mg, 420 mg, 450 mg, 480 mg, 500 mg, 600 mg, 800 mg, 1000 mg, 1200 mg, 1500 mg or the like is administered to a patient as a single daily dosage.
[0100]In another example, 3.75 mg, 7.5 mg, 11.75 mg, 15 mg or the like is administered to a patient as a single daily dosage.
[0101]In one embodiment, each component of the combination (e.g., (i) topiramate, and (ii) a sympathomimetic drug,) is prescribed at a dose that is below the typically described dose for each component as a monotherapy. The components may be prescribed separately or as a combination dosage. In one embodiment, each component of the combination (e.g., (i) topiramate, and (ii) a sympathomimetic drug,) is prescribed at a dose that is above the typically described dose for each component as a monotherapy. The components may be prescribed separately or as a combination dosage.
[0102]In another embodiment, the prescribed dosage of the sympathomimetic drug is above the typically described dose for monotherapy, and topiramate is prescribed at a dosage that is at or below the typically described dose for monotherapy. In another embodiment, the prescribed dosage of the sympathomimetic drug is at or below the typically described dose for monotherapy, and topiramate is prescribed at a dosage that is above the typically described dose for monotherapy.
[0103]In certain embodiments, when phentermine is the sympathomimetic agent, phentermine may be, for example, administered at a dosage ranging from 2-60 mg per day. In one aspect, the phentermine is administered at a dosage ranging from 2-30 mg per day. In another aspect, the phentermine is administered at a dosage ranging from 2-15 mg per day.
[0104]In certain embodiments, when bupropion is the sympathomimetic agent, bupropion may be, for example, administered at a dosage ranging from 50-400 mg per day. In one aspect, the bupropion is administered at a dosage ranging from 50-200 mg per day.
[0105]The method of administration of compositions or combinations of the invention will depend, in particular, on the type of sympathomimetic agent used. Topiramate and the sympathomimetic agent may be administered together in the same composition or simultaneously or sequentially in two separate compositions. Also, one or more sympathomimetic agents may be administered to a subject or patient either in the form of a therapeutic composition or in combination, e.g., in the form of one or more separate compositions administered simultaneously or sequentially. The schedule of administration will be dependent on the type of sympathomimetic agent(s) chosen. For example, a sympathomimetic agent can have a stimulant effect and the degree of such stimulant effect may vary depending on the sympathomimetic agent chosen. Accordingly, a sympathomimetic agent having a significant stimulant effect might be administered earlier in the day than administration of a sympathomimetic agent having a lesser stimulant effect. Accordingly, topiramate has a significant sedative effect and may be administered later in the day than administration of a compound having a lesser sedative effect.
[0106]In a specific embodiment, topiramate is administered as a controlled release form and phentermine is administered as an immediate release form. As such, the phentermine may be taken in the morning because the drug is a stimulant as well as an appetite suppressant. In this embodiment, topiramate may be taken later in the day than the phentermine. Preferably, the patient takes the topiramate just before dinner or later in the evening because the drug is sedating.
[0107]In yet another embodiment, topiramate is administered as a controlled release form and bupropion is administered as an immediate release form. As such, the bupropion may be taken in the morning because the drug is a stimulant as well as an appetite suppressant. In this embodiment, topiramate may be taken later in the day than the bupropion. Preferably, the patient takes the topiramate just before dinner or later in the evening because the drug is sedating.
[0108]In one embodiment, the controlled release topiramate beads are made using an extrusion spheronization process to produce a matrix core comprised of topiramate: 40.0% w/w; microcrystalline cellulose, Avicel® PH102: 56.5% w/w; and Methocel® A15 LV: 3.5% w/w. The topiramate cores are then coated with ethyl cellulose: 5.48% w/w and Povidone K30: 2.39% w/w. The phentermine beads are straight immediate release drug coated onto sugar spheres. Both sets of beads are then encapsulated into one capsule.
[0109]In certain embodiments, the phentermine beads may be controlled release drugs coated onto the sugar spheres. In other aspects, the phentermine beads may be coated onto the controlled release topiramate beads.
[0110]In another embodiment, the controlled release topiramate beads are made using an extrusion spheronization process to produce a matrix core comprised of topiramate: 40.0% w/w; microcrystalline cellulose, Avicel® PH102: 56.5% w/w; and Methocel® A15 LV: 3.5% w/w. The topiramate cores are then coated with ethyl cellulose: 5.48% w/w and Povidone K30: 2.39% w/w. The bupropion beads are straight immediate release drug coated onto sugar spheres. Both sets of beads are then encapsulated into one capsule.
[0111]In certain embodiments, the bupropion beads may be controlled release drugs coated onto the sugar spheres. In other aspects, the bupropion beads may be coated onto the controlled release topiramate beads.
Escalating Dosage Strategy
[0112]The subject methods include a dosing strategy for the administration of topiramate alone or in combination with a sympathomimetic agent. In certain aspects, the present invention provides a dosing strategy for the administration of a pharmaceutical composition that includes, e.g., topiramate in combination with bupropion or phentermine.
[0113]In the subject methods, the dosing strategy includes administering a lower daily dosage of topiramate alone or in combination with a sympathomimetic agent to a patient for a specific period of time and then incrementally increasing the dosage at various designated time points.
[0114]For example, when treating a patient for obesity and/or a related condition, the patient receives a dosage of 23 mg/day of topiramate for 1 week. Next, the patient receives a dosage of 46 mg/day of topiramate for a second week. Thereafter, the patient receives a dosage of 69 mg/day of topiramate for a third week which is followed by a final dosage of 92 mg/day of topiramate for a fourth week.
[0115]In another example, when treating a patient for obesity and/or a related condition, the patient receives a dosage of 23 mg/day of topiramate in combination with a dosage of 3.75 mg/day of phentermine for 1 week. The patient next receives a dosage of 46 mg/day of topiramate in combination with a dosage of 7.5 mg/day of phentermine for a second week. Thereafter, the patient receives a dosage of 69 mg/day of topiramate in combination with a dosage of 11.25 mg/day of phentermine for a third week which is followed by a final dosage of 92 mg/day of topiramate in combination with a dosage of 15 mg/day of phentermine for a fourth week.
[0116]Aspects of the invention include after the fourth week of administration, the further administration of topiramate alone or topiramate in combination with phentermine indefinitely or until a sufficient reduction of symptoms has been achieved. In certain aspects, the final dose of 92 mg/day of topiramate alone or in combination with a dosage of 15 mg/day of phentermine indefinitely or until a sufficient reduction of symptoms has been achieved. In other aspects, the final dose of topiramate alone or topiramate in combination with phentermine is decreased to the initial starting dose of the regimen and maintained indefinitely or until a sufficient reduction of symptoms has been achieved.
Subjects Suitable for Treatment
[0117]Subjects suitable for treatment with a subject monotherapy or combination therapy treatment strategy include individuals suffering from obesity and/or a related disorder. As such, the subject methods at least provide for improvement in at least one of the indications provided below.
Indications
[0118]While the invention is useful in conjunction with numerous pharmaceutical agents and therapeutic regimens, conditions of particular interest include obesity and related conditions, such as those often associated with and/or caused by obesity and/or a related condition. The topiramate monotherapy or combination therapy with one or more sympathomimetic agents such as phentermine and/or bupropion provide increased therapeutic effects, and reduced adverse effects, making these pharmaceutical combinations extremely effective therapeutics, especially in the treatment of obesity or related conditions, including conditions associated with and/or caused by obesity per se. Subjects suitable for treatment with the subject combination therapy treatment regimen include individuals suffering from the following conditions associated with obesity, including, hypertension, diabetes or glucose intolerance and insulin resistance, hyperlipidemia, and often tiredness and sleepiness associated with sleep apnea. Patients are often treated with combinations of antihypertensives, lipid lowering agents, insulin or oral diabetic drugs, and various mechanical and surgical methods for treating sleep apnea. However, such treatments are often costly and do not treat the underlying problem of obesity. Moreover, some of the treatments for diabetes including insulin and oral diabetic agents actually aggravate the conditions associated with obesity by increasing insulin levels, increasing appetite, and increasing weight. This can lead to higher blood pressure and even higher cholesterol.
Overweight and Obesity:
[0119]"Overweight and Obesity" are defined by the Centers for Disease Control and Prevention (the CDC) with respect to body mass index, or BMI, of a patient. Factors such as weight and height are used to determine whether a person is underweight, normal, overweight or obese. The CDC also notes other methods for analysis of amount and location of fat in a person by using measurements of skinfold thickness and waist circumference, waist-to-hip circumference ratios, and techniques such as ultrasound, computed tomography, and magnetic resonance imaging (MRI).
[0120]Symptoms of overweight and obesity are usually quite obvious, as excess fat is often easy to see on a person. individuals. The medical problems caused by overweight and obesity can be serious and often life-threatening, and include diabetes, shortness of breath and other respiratory problems, gallbladder disease, hypertension, dyslipidemia (for example, high cholesterol or high levels of triglycerides), cancer, osteoarthritis, other orthopedic problems, reflux esophagitis (heartburn), snoring, sleep apnea, menstrual irregularities, infertility and heart trouble. Moreover, obesity and overweight substantially increase the risk of morbidity from hypertension, dyslipidemia, type 2 diabetes, coronary heart disease, stroke, gallbladder disease, osteoarthritis and endometrial, breast, prostate, and colon cancers. Higher body weights are also associated with increases in all-cause mortality. Most or all of these problems are relieved or improved by permanent significant weight loss. Longevity is likewise significantly increased by permanent significant weight loss.
Diabetes:
[0121]Diabetes mellitus is associated with continuous and pathologically elevated blood glucose concentration. It is one of the leading causes of death in the United States and is responsible for about 5% of all mortality. Diabetes is divided into two major sub-classes: Type I, also known as juvenile diabetes, or Insulin-Dependent Diabetes Mellitus (IDDM); and Type II, also known as adult onset diabetes, or Non-Insulin-Dependent Diabetes Mellitus (NIDDM).
[0122]According to the American Diabetes Association, there are over one million juvenile diabetics in the United States. Type I Diabetes is a form of autoimmune disease. Autoantibodies produced by the patients completely or partially destroy the insulin producing cells of the pancreas. Juvenile diabetics must, therefore, receive exogenous insulin during their lifetime. Without treatment, excessive acidosis, dehydration, kidney damage, and death may result. Even with treatment, complications such as blindness, atherosclerosis, and impotence can occur.
[0123]There are more than five million Type II (adult onset) diabetics diagnosed in the United States. Type II disease usually begins during middle age; the exact cause is unknown. In Type II diabetics, rising blood glucose levels after meals do not properly stimulate insulin production by the pancreas. Additionally, peripheral tissues are generally resistant to the effects of insulin. The resulting high blood glucose levels (hyperglycemia) can cause extensive tissue damage. Type II diabetics are often referred to as insulin resistant. They often have higher than normal plasma insulin levels (hyperinsulinemia) as the body attempts to overcome its insulin resistance. Some researchers now believe that hyperinsulinemia may be a causative factor in the development of high blood pressure, high levels of circulating low density lipo-proteins (LDLs), and lower than normal levels of the beneficial high density lipo-proteins (HDLs). While moderate insulin resistance can be compensated for in the early stages of Type II diabetes by increased insulin secretion, in advanced disease states insulin secretion is also impaired.
[0124]Insulin resistance and hyperinsulinemia have also been linked with two other metabolic disorders that pose considerable health risks: impaired glucose tolerance and metabolic obesity. Impaired glucose tolerance is characterized by normal glucose levels before eating, with a tendency toward elevated levels (hyperglycemia) following a meal. According to the World Health Organization, approximately 11% of the U.S. population between the ages of 20 and 74 are estimated to have impaired glucose tolerance. These individuals are considered to be at higher risk for diabetes and coronary artery disease.
[0125]Obesity may also be associated with insulin resistance. A causal linkage among obesity, impaired glucose tolerance, and Type II diabetes has been proposed, but a physiological basis has not yet been established. Some researchers believe that impaired glucose tolerance and diabetes are clinically observed and diagnosed only later in the disease process after a person has developed insulin resistance and hyperinsulinemia.
[0126]Insulin resistance is frequently associated with hypertension, coronary artery disease (arteriosclerosis), and lactic acidosis, as well as related disease states. The fundamental relationship between these disease states, and a method of treatment, has not been established.
Hypertension:
[0127]Hypertension is a condition that occurs when the blood pressure inside the large arteries is too high. Hypertension is very common, affecting about 50 million people in the United States alone. It is more common as people grow older and is both more common and more serious in African Americans. Most cases of hypertension are of unknown etiology. It is known that the tendency to develop hypertension can be inherited. Environment also plays a very important role in hypertension. For example, hypertension may be avoided by keeping body weight under control, keeping physically fit, eating a healthy diet, limiting alcohol intake, and avoiding medications that might increase blood pressure. Other less common causes of hypertension include disorders of the kidneys or endocrine glands. Hypertension has been called "the silent killer" because it has no specific symptoms and yet can lead to death. People with untreated hypertension are much more likely to die from or be disabled by cardiovascular complications such as strokes, heart attacks, heart failure, heart rhythm irregularities, and kidney failure, than people who have normal blood pressure.
[0128]Current treatments for hypertension include lifestyle changes (diet, exercise, nonsmoking, etc.) as well as drug therapy. The major classes of medications currently used to treat hypertension include adrenergic neuron antagonists (which are peripherally acting), alpha adrenergic agonists (which are centrally acting), alpha adrenergic blockers, alpha and beta blockers, angiotensin II receptor blockers, angiotensin converting enzyme (ACE) inhibitors, beta adrenergic blockers, calcium channel blockers, thiazides (benzothiadiazine derivatives) and related diuretics, and vasodilators (which act by direct relaxation of vascular smooth muscles).
[0129]A particularly serious hypertensive disorder is primary pulmonary hypertension, also known as idiopathic pulmonary hypertension. This is a condition in which the blood pressure in the pulmonary arteries is abnormally high in the absence of other diseases of the heart or lungs. The cause of primary pulmonary hypertension is unknown. Pulmonary hypertension develops in response to increased resistance to blood flow. Narrowing of the pulmonary arterioles occurs and the right side of the heart becomes enlarged due to the increased work of pumping blood against the resistance. Eventually, progressive heart failure develops. Currently, there is no known cure for primary pulmonary hypertension. Treatment is primarily directed towards controlling the symptoms, although some success has occurred with the use of vasodilators. Other medications used to treat the symptoms of primary pulmonary hypertension include diuretics and calcium channel blockers. Typically, as the disease progresses, oxygen is often required. In certain cases, a heart-lung transplant may be indicated for certain suitable candidates, although the availability of donor organs continues to be extremely limited. Unfortunately, primary pulmonary hypertension is a progressive disease, usually leading to congestive heart failure and respiratory failure.
[0130]Secondary pulmonary hypertension is a serious disorder that arises as a complication of other conditions such as, for example, scleroderma. Treatments are similar as those for primary pulmonary hypertension and, unfortunately, the prognosis is the same as well.
Sleep Apnea:
[0131]Sleep apnea occurs in two main types: obstructive sleep apnea, the more common form that occurs when throat muscles relax; and central sleep apnea, which occurs when the brain doesn't send proper signals to the muscles that control breathing. Additionally, some people have mixed sleep apnea, which is a combination of both obstructive and central sleep apneas. Sleep apnea literally means "cessation of breath." It is characterized by repetitive episodes of upper airway obstruction that occur during sleep, usually associated with a reduction in blood oxygen saturation. In other words, the airway becomes obstructed at several possible sites. The upper airway can be obstructed by excess tissue in the airway, large tonsils, and a large tongue and usually includes the airway muscles relaxing and collapsing when asleep. Another site of obstruction can be the nasal passages. Sometimes the structure of the jaw and airway can be a factor in sleep apnea.
[0132]The signs and symptoms of obstructive and central sleep apneas overlap, sometimes making the type of sleep apnea more difficult to determine. The most common signs and symptoms of obstructive and central sleep apneas include: excessive daytime sleepiness (hypersomnia); loud snoring; observed episodes of breathing cessation during sleep; abrupt awakenings accompanied by shortness of breath; awakening with a dry mouth or sore throat; morning headache; and/or difficulty staying asleep (insomnia). Disruptive snoring may be a more prominent characteristic of obstructive sleep apnea, while awakening with shortness of breath may be more common with central sleep apnea.
[0133]Sleep apnea is a progressive condition and should not be taken lightly. It is a potentially life-threatening condition that requires immediate medical attention. The risks of undiagnosed obstructive sleep apnea include heart attacks, strokes, impotence, irregular heartbeat, high blood pressure and heart disease. In addition, obstructive sleep apnea causes daytime sleepiness that can result in accidents, lost productivity and interpersonal relationship problems. The severity of the symptoms may be mild, moderate or severe.
[0134]Sleep apnea is diagnosed utilizing a sleep test, called polysomnography but treatment methodologies differ depending on the severity of the disorder. Mild Sleep Apnea is usually treated by some behavioral changes. Losing weight, sleeping on your side are often recommended. There are oral mouth devices (that help keep the airway open) that may help to reduce snoring in three different ways. Some devices (1) bring the jaw forward or (2) elevate the soft palate or (3) retain the tongue (from falling back in the airway and blocking breathing).
[0135]Moderate to severe sleep apnea is usually treated with a continuous positive airway pressure (C-PAP). C-PAP is a machine that blows air into your nose via a nose mask, keeping the airway open and unobstructed. For more severe apnea, there is a Bi-level (Bi-PAP) machine. The Bi-level machine is different in that it blows air at two different pressures. When a person inhales, the pressure is higher and in exhaling, the pressure is lower.
[0136]Some people have facial deformities that may cause the sleep apnea. It simply may be that their jaw is smaller than it should be or they could have a smaller opening at the back of the throat. Some people have enlarged tonsils, a large tongue or some other tissues partially blocking the airway. Fixing a deviated septum may help to open the nasal passages. Removing the tonsils and adenoids or polyps may help also. Children are much more likely to have their tonsils and adenoids removed. Surgical procedures, such as tracheostomy, uvulopalatopharyngoplasty (UPPP), laser assisted uvuloplasty (LAUP), somnoplasty, or mandibular myotomy, are often required to effectively treat sleep apnea.
Epilepsy:
[0137]"Epilepsy" is defined as a brain disorder with recurrent, unprovoked seizures. Epilepsy includes seizures of focal onset and generalized seizures. The types of focal onset seizures are partial seizures of temporal lobe origin, frontal lobe origin or others. Focal epilepsies with genetic components include benign childhood epilepsy with centrotemporal spikes, childhood epilepsy with occipital paroxysms or primary reading epilepsy. The generalized genetic epilepsies include benign neonatal familial convulsions, benign neonatal convulsions, benign myoclonic epilepsy in infancy, childhood absence epilepsy, juvenile absence epilepsy, juvenile myoclonic epilepsy, and epilepsy with grand mal seizures on awakening. The cryptogenic childhood epilepsies with generalized seizures include infantile spasms without tuberous sclerosis, West syndrome, Lennox-Gastaut syndrome, epilepsy with myoclonic-astatic seizures, epilepsy with myoclonic absences, and symptomatic epilepsy such as infantile spasms associated with tuberous sclerosis, epilepsy with continuous spike and wave EEG during slow-wave sleep, and acquired epileptic aphasia (Landau-Kleffner syndrome).
[0138]The treatment of any convulsive disorders is also contemplated using the methods and compositions of the invention. These convulsive disorders include all forms of epilepsies, for example, temporal lobe epilepsy, focal epilepsies, including idiopathic epilepsies such as benign childhood epilepsy with centrotemporal spikes, childhood epilepsy with occipital paroxysms or primary reading epilepsy, symptomatic epilepsies with simple partial seizures, complex partial seizures, secondarily generalized seizures, generalized epilepsies and syndromes; generalized epilepsies including idiopathic epilepsies such as benign neonatal familial convulsions, benign neonatal convulsions, benign myoclonic epilepsy in infancy, childhood absence epilepsy, juvenile absence epilepsy, juvenile myoclonic epilepsy, and epilepsy with grand mal seizures on awakening; cryptogenic or symptomatic epilepsies including West syndrome, Lennox-Gastaut syndrome, epilepsy with myoclonic-astatic seizures, epilepsy with myoclonic absences, and symptomatic epilepsy such as early myoclonic encephalopathy or specific syndromes; epilepsies with undetermined origin including neonatal seizures, severe myoclonic epilepsy in infancy, epilepsy with continuous spike and wave EEG during slow-wave sleep, and acquired epileptic aphasia (Landau-Kleffner syndrome). It also includes seizures caused by metabolic derangements, acute brain injury, acute head trauma, drug withdrawal, alcohol withdrawal, and toxins.
[0139]Symptoms associated with, or arising from epilepsy, include convulsions, grand mal seizures, absence seizures, petit mal seizures, focal seizures, temporal lobe seizures, psychomotor seizures, muscle spasms, loss of consciousness, strange sensations, strange emotions and strange behavior.
Migraines:
[0140]"Migraines" are generally headaches that are typically associated with various psychological (e.g., irritability, depression, fatigue, drowsiness, and restlessness), neurological (e.g., photophobia, and phonophobia), and gastrointestinal symptoms. The headache starts with mild pain, which increases in intensity over a short period of time. There are two major types of migraines. The common migraine affects 80-85% of migraine sufferers and classical migraine with aura affects 15% of migraine sufferers. Symptoms associated with migraines include headaches, psychological symptomatology such as irritability, depression, fatigue, drowsiness, restlessness; neurological symptoms such as photophobia, phonophobia or gastrointestinal symptoms such as change in bowel habit, change of food intake or urinary symptoms such as urinary frequency, auras which are neurological deficits and can be a variety of deficits for the migraine population but in the individual is usually stereotyped. These deficits may be visual scotoma or visual designs, hemiplegia, migrating paraesthesia, dysarthria, dysphasia, or deja-vu. The headache is usually accompanied by light or sound sensitivity, photophobia or phonophobia, irritability and impaired concentration.
Depression:
[0141]"Depression" is manifested by a combination of symptoms that interfere with the ability to work, study, sleep, eat, and enjoy once pleasurable activities. Depression includes major depression, especially refractory depression, bipolar depression, and the degeneration associated with depression. Symptoms of depression include persistent sad, anxious, or "empty" mood, feelings of hopelessness, pessimism, feelings of guilt, worthlessness, helplessness, loss of interest or pleasure in hobbies and activities that were once enjoyed, including sex, decreased energy, fatigue, being "slowed down", difficulty concentrating, remembering, making decisions, insomnia, early-morning awakening, or oversleeping, appetite and/or weight loss or overeating and weight gain, thoughts of death or suicide; suicide attempts, restlessness, irritability, persistent physical symptoms that do not respond to treatment, such as headaches, digestive disorders, and chronic pain.
Others:
[0142]Other psychiatric disorders may also be treated using the compositions and methods of the invention. These disorders include panic syndrome, general anxiety disorder, phobic syndromes of all types, mania, manic depressive illness, hypomania, unipolar depression, stress disorders, PTSD, somatoform disorders, personality disorders, psychosis, and schizophrenia.
[0143]"Impulse Control Disorders" are characterized by harmful behaviors performed in response to irresistible impulses. The essential feature of an impulse control disorder is the failure to resist an impulse, drive, or temptation to perform an act that is harmful to the person or to others. Symptoms include an increasing sense of tension or arousal before committing an act, and then experiences pleasure, gratification, or release at the time of committing the act. After the act is performed, there may or may not be regret or guilt. Numerous disorders can be characterized as impulse control disorders including intermittent explosive disorder, kleptomania, pathological gambling, pyromania, trichotillomania, compulsive buying or shopping, repetitive self-mutilation, nonparaphilic sexual addictions, severe nail biting, compulsive skin picking, personality disorders with impulsive features, attention deficit/hyperactivity disorder, eating disorders characterized by binge eating, and substance use disorders.
[0144]"Alcohol addiction" is characterized in a subject by the presence of one or more of the following symptoms. The subject has a tolerance for alcohol. The subject has withdrawal symptoms after stopping drinking alcohol. The subject takes alcohol in larger amounts than was intended. The subject lacks the ability to decrease the amount of alcohol consumed. The subject spends a great deal of time attempting to acquire alcohol. Lastly, the subject continues to use alcohol even though the subject should know that there are reoccurring physical or psychological problems being caused by the alcohol.
[0145]Symptoms associated with alcohol addiction include death from alcohol toxemia, cirrhosis of the liver, pancreatitis, heart disease, polyneuropathy, alcoholic dementia, increased incidence of many types of cancer: breast cancer, head and neck cancer, esophageal cancer and colorectal cancer, nutritional deficiency involving deficiencies in folic acid, thiamine (vitamin B1), sexual dysfunction, osteoporosis and osteonecrosis.
Kits:
[0146]Also provided are kits for practicing the subject methods. The subject kits may vary greatly in regards to the components included. The subject kits at least include a dosage form of topiramate according to the present invention. In other embodiments, the subject kits include topiramate and at least one sympathomimetic agent but in certain embodiments, the kit may include two or more symapthomimetic agents. In certain aspects, the topiramate alone or in combination with the sympathomimetic agent may be provided in a titration card to enhance patient compliance. In some embodiments, the titration card may include the different escalating dosages according to a specific dosing strategy. For example, the titration card may include four weeks of dose titration medication in which the dose level increases each week as specified in a dosing strategy according to the subject invention.
[0147]In certain embodiments, the subject kits include instructions for a patient to carry out drug administration to treat obesity and/or those conditions associated with obesity. The instructions may further include the dosing strategy for the administration of topiramate alone or in combination with a sympathomimetic agent. In certain aspects, the subject kits include a dosing strategy for the administration of a pharmaceutical composition that includes, e.g., topiramate in combination with bupropion or phentermine. In one embodiment, the subject kits provide a dosing strategy which includes administering a lower daily dosage of topiramate alone or in combination with a sympathomimetic agent to a patient for a specific period of time and then incrementally increasing the dosage at various designated time points. The instructions may be recorded on a suitable recording medium or substrate. For example, the instructions may be printed on a substrate, such as paper or plastic, etc. As such, the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging) etc. In other embodiments, the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc. In yet other embodiments, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate
[0148]Some or all components of the subject kits may be packaged in suitable packaging to maintain sterility. In many embodiments of the subject kits, the components of the kit are packaged in a kit containment element to make a single, easily handled unit, where the kit containment element, e.g., box or analogous structure, may or may not be an airtight container, e.g., to further preserve the sterility of some or all of the components of the kit. In certain aspects, the subject kit comprises a sealed package of controlled release dosage forms wherein the dosage forms provide for immediate release of the second pharmaceutical agent and delayed release of the first pharmaceutical agent.
[0149]This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference.
EXAMPLES
[0150]The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric.
Example 1
[0151]The controlled release topiramate beads were made using an extrusion spheronization process to produce a matrix core comprised of topiramate: 40.0% w/w; microcrystalline cellulose, Avicel® PH102: 56.5% w/w; and Methocel® A15 LV: 3.5% w/w. The topiramate cores were then coated with ethyl cellulose: 5.47% w/w and Povidone K30: 2.39% w/w.
[0152]The final coated beads % w/w were as follows:
TABLE-US-00002 Ingredient % w/w topiramate 36.85 microcrystalline cellulose, 52.05 Avicel ® PH102 Methocel ® A15 LV 3.22 ethyl cellulose 5.47 Povidone K30 2.39
[0153]The phentermine beads were straight immediate release drug coated onto sugar spheres. Both sets of beads were then encapsulated into one capsule.
Example 2
[0154]In a study comparing controlled-release formulation of topiramate according to the present invention versus immediate release topiramate (Topamax®) in combination with phentermine, the controlled release formulation of the instant invention of topiramate had a 10-15% lower effect on phentermine exposure (FIG. 2).
[0155]The mean and statistical comparisons for plasma phentermine PK parameters at steady state in multiple dose administrations are summarized in Table 2.
TABLE-US-00003 TABLE 2 Arithmetic Mean (SD) and Statistical Comparison of Pharmacokinetic Parameters for Plasma Phentermine Mean ± SD Treatment 2 Versus Treatment 4 Pharmacokinetic Treatment 2 Treatment 4 90% Confidence Parameters (N = 13) (N = 12) Intervals % Mean Ratio AUC0-tau (ng * hr/mL) 2250 ± 563 2530 ± 644 (75.6, 105.3) 89.2 AUC0-96 (ng * hr/mL) 4640 ± 1570 5550 ± 1960 (67.1, 105.0) 84.0 AUC0-t (ng * hr/mL) 4640 ± 1570 5550 ± 1960 (67.1, 105.0) 84.0 Cmax,96 (ng/mL) 114 ± 23.6 127 ± 27.6 (78.8, 104.5) 90.7 C.sub.min,68 (ng/mL) 9.84 ± 7.24 14.6 ± 11.3 (42.5, 109.0) 68.1 tmax (hr) 4.01 (1.04, 7.00) 4.54 (1.00, 10.0) t1/2 (hr) 23.3 ± 6.17 26.3 ± 7.43 CLss/F (L/hr) 7.10 ± 1.89 6.38 ± 2.00 V2/F (L) 229 ± 45.3 232 ± 58.5 tmax is presented as median (minimum, maximum) Parameters were dose-normalized and in-transformed prior to analysis. % Mean Ratio = 100 * exp(Treatment 2 - Treatment 4) for in-transformed parameters Treatment 1 (Test): 7.5 mg phentermine/50 mg topiramate (Formulation A) Treatment 2 (Test): 15 mg phentermine/100 mg topiramate (Formulation A) Treatment 4 (Reference): 15 mg phentermine/100 mg topiramate Source: Tables 14.2.1.8., 14.2.1.10, 14.2.1.12. and 14.2.1.17
[0156]These data indicate a lower maximum and extent of phentermine exposure between test versus reference treatments after multiple-dose administration.
[0157]As such, the controlled release formulation of topiramate reduced drug interaction with phentermine which in turn will reduce further side effects associated with phentermine.
Sequence CWU
1
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Thr Lys Glu Asp Asn Gln Ile Ser Ala Thr1 5
10 15Ser Glu Gln Thr Glu Asp Ser Ala Cys Leu Ser Ala
Met Val Leu Thr 20 25 30Thr
Asn Leu Val Tyr Pro Ala Val Leu Asn Ala Ala Ile Asp Leu Asn 35
40 45Leu Phe Glu Ile Ile Ala Lys Ala Thr
Pro Pro Gly Ala Phe Met Ser 50 55
60Pro Ser Glu Ile Ala Ser Lys Leu Pro Ala Ser Thr Gln His Ser Asp65
70 75 80Leu Pro Asn Arg Leu
Asp Arg Met Leu Arg Leu Leu Ala Ser Tyr Ser 85
90 95Val Leu Thr Ser Thr Thr Arg Thr Ile Glu Asp
Gly Gly Ala Glu Arg 100 105
110Val Tyr Gly Leu Ser Met Val Gly Lys Tyr Leu Val Pro Asp Glu Ser
115 120 125Arg Gly Tyr Leu Ala Ser Phe
Thr Thr Phe Leu Cys Tyr Pro Ala Leu 130 135
140Leu Gln Val Trp Met Asn Phe Lys Glu Ala Val Val Asp Glu Asp
Ile145 150 155 160Asp Leu
Phe Lys Asn Val His Gly Val Thr Lys Tyr Glu Phe Met Gly
165 170 175Lys Asp Lys Lys Met Asn Gln
Ile Phe Asn Lys Ser Met Val Asp Val 180 185
190Cys Ala Thr Glu Met Lys Arg Met Leu Glu Ile Tyr Thr Gly
Phe Glu 195 200 205Gly Ile Ser Thr
Leu Val Asp Val Gly Gly Gly Ser Gly Arg Asn Leu 210
215 220Glu Leu Ile Ile Ser Lys Tyr Pro Leu Ile Lys Gly
Ile Asn Phe Asp225 230 235
240Leu Pro Gln Val Ile Glu Asn Ala Pro Pro Leu Ser Gly Ile Glu His
245 250 255Val Gly Gly Asp Met
Phe Ala Ser Val Pro Gln Gly Asp Ala Met Ile 260
265 270Leu Lys Ala Val Cys His Asn Trp Ser Asp Glu Lys
Cys Ile Glu Phe 275 280 285Leu Ser
Asn Cys His Lys Ala Leu Ser Pro Asn Gly Lys Val Ile Ile 290
295 300Val Glu Phe Ile Leu Pro Glu Glu Pro Asn Thr
Ser Glu Glu Ser Lys305 310 315
320Leu Val Ser Thr Leu Asp Asn Leu Met Phe Ile Thr Val Gly Gly Arg
325 330 335Glu Arg Thr Glu
Lys Gln Tyr Glu Lys Leu Ser Lys Leu Ser Gly Phe 340
345 350Ser Lys Phe Gln Val Ala Cys Arg Ala Phe Asn
Ser Leu Gly Val Met 355 360 365Glu
Phe Tyr Lys 37031227DNAMedicago sativa 3caaaaattca tttgcaaaaa
aaaatggcgt catcaattaa tggcagaaaa ccaagtgaaa 60ttttcaaagc acaagcttta
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ggaacaactg ctaagattat ttgtgagact tttcctaagt 660tgaaatgtat tgtgtttgat
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aaaaaaa 12274352PRTMedicago sativa
4Met Ala Ser Ser Ile Asn Gly Arg Lys Pro Ser Glu Ile Phe Lys Ala1
5 10 15Gln Ala Leu Leu Tyr Lys
His Ile Tyr Ala Phe Ile Asp Ser Met Ser 20 25
30Leu Lys Trp Ala Val Gly Met Asn Ile Pro Asn Ile Ile
His Asn His 35 40 45Gly Lys Pro
Ile Ser Leu Ser Asn Leu Val Ser Ile Leu Gln Val Pro 50
55 60Ser Ser Lys Ile Gly Asn Val Arg Arg Leu Met Arg
Tyr Leu Ala His65 70 75
80Asn Gly Phe Phe Glu Ile Ile Thr Lys Glu Glu Glu Ser Tyr Ala Leu
85 90 95Thr Val Ala Ser Glu Leu
Leu Val Arg Gly Ser Asp Leu Cys Leu Ala 100
105 110Pro Met Val Glu Cys Val Leu Asp Pro Thr Leu Ser
Gly Ser Tyr His 115 120 125Glu Leu
Lys Lys Trp Ile Tyr Glu Glu Asp Leu Thr Leu Phe Gly Val 130
135 140Thr Leu Gly Ser Gly Phe Trp Asp Phe Leu Asp
Lys Asn Pro Glu Tyr145 150 155
160Asn Thr Ser Phe Asn Asp Ala Met Ala Ser Asp Ser Lys Leu Ile Asn
165 170 175Leu Ala Leu Arg
Asp Cys Asp Phe Val Phe Asp Gly Leu Glu Ser Ile 180
185 190Val Asp Val Gly Gly Gly Thr Gly Thr Thr Ala
Lys Ile Ile Cys Glu 195 200 205Thr
Phe Pro Lys Leu Lys Cys Ile Val Phe Asp Arg Pro Gln Val Val 210
215 220Glu Asn Leu Ser Gly Ser Asn Asn Leu Thr
Tyr Val Gly Gly Asp Met225 230 235
240Phe Thr Ser Ile Pro Asn Ala Asp Ala Val Leu Leu Lys Tyr Ile
Leu 245 250 255His Asn Trp
Thr Asp Lys Asp Cys Leu Arg Ile Leu Lys Lys Cys Lys 260
265 270Glu Ala Val Thr Asn Asp Gly Lys Arg Gly
Lys Val Thr Ile Ile Asp 275 280
285Met Val Ile Asn Glu Lys Lys Asp Glu Asn Gln Val Thr Gln Ile Lys 290
295 300Leu Leu Met Asp Val Asn Met Ala
Cys Leu Asn Gly Lys Glu Arg Asn305 310
315 320Glu Glu Glu Trp Lys Lys Leu Phe Ile Glu Ala Gly
Phe Gln His Tyr 325 330
335Lys Ile Ser Pro Leu Thr Gly Phe Leu Ser Leu Ile Glu Ile Tyr Pro
340 345 3505365PRTMedicago sativa
5Met Gly Ser Thr Gly Glu Thr Gln Ile Thr Pro Thr His Ile Ser Asp1
5 10 15Glu Glu Ala Asn Leu Phe
Ala Met Gln Leu Ala Ser Ala Ser Val Leu 20 25
30Pro Met Ile Leu Lys Ser Ala Leu Glu Leu Asp Leu Leu
Glu Ile Ile 35 40 45Ala Lys Ala
Gly Pro Gly Ala Gln Ile Ser Pro Ile Glu Ile Ala Ser 50
55 60Gln Leu Pro Thr Thr Asn Pro Asp Ala Pro Val Met
Leu Asp Arg Met65 70 75
80Leu Arg Leu Leu Ala Cys Tyr Ile Ile Leu Thr Cys Ser Val Arg Thr
85 90 95Gln Gln Asp Gly Lys Val
Gln Arg Leu Tyr Gly Leu Ala Thr Val Ala 100
105 110Lys Tyr Leu Val Lys Asn Glu Asp Gly Val Ser Ile
Ser Ala Leu Asn 115 120 125Leu Met
Asn Gln Asp Lys Val Leu Met Glu Ser Trp Tyr His Leu Lys 130
135 140Asp Ala Val Leu Asp Gly Gly Ile Pro Asp Asn
Lys Ala Tyr Gly Met145 150 155
160Thr Ala Phe Glu Tyr His Gly Thr Asp Pro Arg Phe Asn Lys Val Phe
165 170 175Asn Lys Gly Met
Ser Asp His Ser Thr Ile Thr Met Lys Lys Ile Leu 180
185 190Glu Thr Tyr Thr Gly Phe Glu Gly Leu Lys Ser
Leu Val Asp Val Gly 195 200 205Gly
Gly Thr Gly Ala Val Ile Asn Thr Ile Val Ser Lys Tyr Pro Thr 210
215 220Ile Lys Gly Ile Asn Phe Asp Leu Pro His
Val Ile Glu Asp Ala Pro225 230 235
240Ser Tyr Pro Gly Val Glu His Val Gly Gly Asp Met Phe Val Ser
Ile 245 250 255Pro Lys Ala
Asp Ala Val Phe Met Lys Trp Ile Cys His Asp Trp Ser 260
265 270Asp Glu His Cys Leu Lys Phe Leu Lys Asn
Cys Tyr Glu Ala Leu Pro 275 280
285Asp Asn Gly Lys Val Ile Val Ala Glu Cys Ile Leu Pro Val Ala Pro 290
295 300Asp Ser Ser Leu Ala Thr Lys Gly
Val Val His Ile Asp Val Ile Met305 310
315 320Leu Ala His Asn Pro Gly Gly Lys Glu Arg Thr Gln
Lys Glu Phe Glu 325 330
335Asp Leu Ala Lys Gly Ala Gly Phe Gln Gly Phe Lys Val His Cys Asn
340 345 350Ala Phe Asn Thr Tyr Ile
Met Glu Phe Leu Lys Lys Val 355 360
3656381PRTCoptis trifolia 6Met Cys Thr Ser Leu Ser Glu Leu Lys Cys Pro
Val Phe Ser Thr Lys1 5 10
15Arg Lys Leu Leu Leu Glu Phe Ala Leu Arg Thr Ser Val Asp Met Ala
20 25 30Ala Gln Glu Gly Val Asn Tyr
Leu Ser Gly Leu Gly Leu Ser Arg Leu 35 40
45Ile Cys Leu Pro Met Ala Leu Arg Ala Ala Ile Glu Leu Asn Val
Phe 50 55 60Glu Ile Ile Ser Gln Ala
Gly Pro Asp Ala Gln Leu Ser Pro Ser Asp65 70
75 80Ile Val Ala Lys Ile Pro Thr Lys Asn Pro Ser
Ala Ala Ile Ser Leu 85 90
95Asp Arg Ile Leu Arg Met Leu Gly Ala Ser Ser Ile Leu Ser Val Ser
100 105 110Thr Thr Lys Ser Gly Arg
Val Tyr Gly Leu Asn Glu Glu Ser Arg Cys 115 120
125Leu Val Ala Ser Glu Asp Lys Val Ser Val Val Pro Met Leu
Leu Phe 130 135 140Thr Ser Asp Lys Ala
Val Val Glu Ser Phe Tyr Asn Ile Lys Asp Val145 150
155 160Val Leu Glu Glu Gly Val Ile Pro Phe Asp
Arg Thr His Gly Met Asp 165 170
175Phe Phe Gln Tyr Ala Gly Lys Glu Glu Arg Val Asn Lys Ser Phe Asn
180 185 190Gln Ala Met Gly Ala
Gly Ser Thr Ile Ala Phe Asp Glu Val Phe Lys 195
200 205Val Tyr Lys Gly Phe Asp Asn Leu Lys Glu Leu Val
Asp Val Gly Gly 210 215 220Gly Ile Gly
Thr Ser Leu Ser Asn Ile Val Ala Lys His Pro His Ile225
230 235 240Arg Gly Ile Asn Phe Glu Leu
Pro His Val Ile Gly Asp Ala Pro Asp 245
250 255Tyr Pro Gly Val Glu His Val Pro Gly Asp Met Phe
Glu Gly Val Pro 260 265 270Asn
Ala Gln Asn Ile Leu Leu Lys Trp Val Leu His Asp Trp Asp Asp 275
280 285Asp Arg Ser Ile Lys Ile Leu Lys Asn
Cys Trp Lys Ala Leu Pro Glu 290 295
300Asn Gly Thr Val Ile Val Ile Glu Phe Val Leu Pro Gln Val Leu Gly305
310 315 320Asn Asn Ala Glu
Ser Phe Asn Ala Leu Thr Pro Asp Leu Leu Met Met 325
330 335Ala Leu Asn Pro Gly Gly Lys Glu Arg Thr
Thr Ile Glu Phe Asp Gly 340 345
350Leu Ala Lys Ala Ala Gly Phe Ala Glu Thr Lys Phe Phe Pro Ile Ser
355 360 365Gln Gly Leu His Val Met Glu
Phe His Lys Ile Asn Cys 370 375
3807368PRTScaevola aemula 7Met Gly Ser Thr Gly Asn Ala Glu Ile Gln Ile
Ile Pro Thr His Ser1 5 10
15Ser Asp Glu Glu Ala Asn Leu Phe Ala Met Gln Leu Ala Ser Ala Ala
20 25 30Val Leu Pro Met Ala Leu Lys
Ala Ala Ile Glu Leu Asp Val Leu Glu 35 40
45Ile Met Ala Lys Ser Val Pro Pro Ser Gly Tyr Ile Ser Pro Ala
Glu 50 55 60Ile Ala Ala Gln Leu Pro
Thr Thr Asn Pro Glu Ala Pro Val Met Leu65 70
75 80Asp Arg Val Leu Arg Leu Leu Ala Ser Tyr Ser
Val Val Thr Tyr Thr 85 90
95Leu Arg Glu Leu Pro Ser Gly Lys Val Glu Arg Leu Tyr Gly Leu Ala
100 105 110Pro Val Cys Lys Phe Leu
Thr Lys Asn Glu Asp Gly Val Ser Leu Ala 115 120
125Pro Phe Leu Leu Thr Ala Thr Asp Lys Val Leu Leu Glu Pro
Trp Phe 130 135 140Tyr Leu Lys Asp Ala
Ile Leu Glu Gly Gly Ile Pro Phe Asn Lys Ala145 150
155 160Tyr Gly Met Asn Glu Phe Asp Tyr His Gly
Thr Asp His Arg Phe Asn 165 170
175Lys Val Phe Asn Lys Gly Met Ser Ser Asn Ser Thr Ile Thr Met Lys
180 185 190Lys Ile Leu Glu Met
Tyr Asn Gly Phe Glu Gly Leu Thr Thr Ile Val 195
200 205Asp Val Gly Gly Gly Thr Gly Ala Val Ala Ser Met
Ile Val Ala Lys 210 215 220Tyr Pro Ser
Ile Asn Ala Ile Asn Phe Asp Leu Pro His Val Ile Gln225
230 235 240Asp Ala Pro Ala Phe Ser Gly
Val Glu His Leu Gly Gly Asp Met Phe 245
250 255Asp Gly Val Pro Lys Gly Asp Ala Ile Phe Ile Lys
Trp Ile Cys His 260 265 270Asp
Trp Ser Asp Glu His Cys Leu Lys Leu Leu Lys Asn Cys Tyr Ala 275
280 285Ala Leu Pro Asp His Gly Lys Val Ile
Val Ala Glu Tyr Ile Leu Pro 290 295
300Pro Ser Pro Asp Pro Ser Ile Ala Thr Lys Val Val Ile His Thr Asp305
310 315 320Ala Leu Met Leu
Ala Tyr Asn Pro Gly Gly Lys Glu Arg Thr Glu Lys 325
330 335Glu Phe Gln Ala Leu Ala Met Ala Ser Gly
Phe Arg Gly Phe Lys Val 340 345
350Ala Ser Cys Ala Phe Asn Thr Tyr Val Met Glu Phe Leu Lys Thr Ala
355 360 3658365PRTPopulus tremula 8Met
Gly Ser Thr Gly Glu Thr Gln Met Thr Pro Thr Gln Val Ser Asp1
5 10 15Glu Glu Ala His Leu Phe Ala
Met Gln Leu Ala Ser Ala Ser Val Leu 20 25
30Pro Met Ile Leu Lys Thr Ala Ile Glu Leu Asp Leu Leu Glu
Ile Met 35 40 45Ala Lys Ala Gly
Pro Gly Ala Phe Leu Ser Thr Ser Glu Ile Ala Ser 50 55
60His Leu Pro Thr Lys Asn Pro Asp Ala Pro Val Met Leu
Asp Arg Ile65 70 75
80Leu Arg Leu Leu Ala Ser Tyr Ser Ile Leu Thr Cys Ser Leu Lys Asp
85 90 95Leu Pro Asp Gly Lys Val
Glu Arg Leu Tyr Gly Leu Ala Pro Val Cys 100
105 110Lys Phe Leu Thr Lys Asn Glu Asp Gly Val Ser Val
Ser Pro Leu Cys 115 120 125Leu Met
Asn Gln Asp Lys Val Leu Met Glu Ser Trp Tyr Tyr Leu Lys 130
135 140Asp Ala Ile Leu Asp Gly Gly Ile Pro Phe Asn
Phe Ala Tyr Gly Met145 150 155
160Thr Ala Phe Glu Tyr His Gly Thr Asp Pro Arg Phe Asn Lys Val Phe
165 170 175Asn Lys Gly Met
Ser Asp His Ser Thr Ile Thr Met Lys Lys Ile Leu 180
185 190Glu Thr Tyr Lys Gly Phe Glu Gly Leu Thr Ser
Leu Val Asp Val Gly 195 200 205Gly
Gly Thr Gly Ala Val Val Asn Thr Ile Val Ser Lys Tyr Pro Ser 210
215 220Ile Lys Gly Ile Asn Phe Asp Leu Pro His
Val Ile Glu Asp Ala Pro225 230 235
240Ser Tyr Pro Gly Val Glu His Val Gly Gly Asp Met Phe Val Ser
Val 245 250 255Pro Lys Ala
Asp Ala Val Phe Met Lys Trp Ile Cys His Asp Trp Ser 260
265 270Asp Ala His Cys Leu Lys Phe Leu Lys Asn
Cys Tyr Asp Ala Leu Pro 275 280
285Glu Asn Gly Lys Val Ile Leu Val Glu Cys Ile Leu Pro Val Ala Pro 290
295 300Asp Thr Ser Leu Ala Thr Lys Gly
Val Val His Val Asp Val Ile Met305 310
315 320Leu Ala His Asn Pro Gly Gly Lys Glu Arg Thr Glu
Lys Glu Phe Glu 325 330
335Gly Leu Ala Lys Gly Ala Gly Phe Gln Gly Phe Glu Val Met Cys Cys
340 345 350Ala Phe Asn Thr His Val
Ile Glu Leu Arg Lys Lys Ala 355 360
3659343PRTSaxifraga sp. 9Met Leu Phe Ala Met Gln Leu Ala Ser Ala Ser Val
Leu Pro Met Val1 5 10
15Leu Lys Ser Ala Ile Glu Leu Asp Leu Leu Glu Ile Ile Ala Ser Gln
20 25 30Asp Thr Cys Met Ser Pro Thr
Glu Ile Ala Ser His Leu Pro Thr Thr 35 40
45Asn Pro His Ala Pro Thr Met Ile Asp Arg Ile Leu Arg Leu Leu
Ser 50 55 60Ser Tyr Ser Ile Val Thr
Cys Ser Val Arg Ser Val Asp Asp Gln Arg65 70
75 80Val Tyr Ser Pro Ala Pro Val Cys Lys Tyr Leu
Thr Lys Asn Gln Asp 85 90
95Gly Val Ser Ile Ala Ala Leu Cys Val Ala Ala Gln Asp Lys Val Leu
100 105 110Met Glu Cys Trp Tyr His
Met Lys Asp Ala Val Leu Asp Gly Gly Ile 115 120
125Pro Phe Asn Lys Ala Tyr Gly Met Pro Ile Phe Asp Tyr Phe
Ala Lys 130 135 140Asp Leu Gly Ser Asn
Lys Val Phe Asn Lys Gly Met Ser Asp Phe Ser145 150
155 160Ser Met Ile Ile Lys Lys Ile Leu Glu Thr
Tyr Lys Gly Phe Gln Gly 165 170
175Leu Thr Ser Leu Val Asp Val Gly Gly Gly Thr Gly Ala Thr Leu Thr
180 185 190Lys Ile Leu Ser Lys
Tyr Pro Thr Ile Arg Gly Ile Asn Phe Asp Leu 195
200 205Pro His Val Ile Gln Asp Ala Pro Glu Tyr Pro Gly
Ile Glu His Val 210 215 220Gly Gly Asp
Met Phe Val Ser Val Pro Lys Gly Asp Ala Ile Phe Met225
230 235 240Lys Trp Ile Cys His Asp Trp
Asn Glu Glu Gln Cys Leu Lys Leu Leu 245
250 255Lys Asn Cys Tyr Asp Ala Leu Pro Asn Asn Gly Lys
Val Ile Val Ala 260 265 270Glu
Tyr Ile Leu Pro Val Val Pro Asp Ser Ser Leu Ala Ser Lys Leu 275
280 285Ser Val Thr Ala Asp Val Met Ile Val
Thr Gln Asn Ser Gly Gly Lys 290 295
300Glu Arg Thr Glu Lys Glu Phe Glu Ala Leu Ala Lys Ala Ala Gly Phe305
310 315 320Gln Gly Phe Gln
Val Phe Cys Asn Ala Phe Thr Ile Tyr Ile Ile Glu 325
330 335Phe Ser Lys Asn Ile Ser Asn
34010360PRTPisum sativum 10Met Asp Phe Ser Thr Asn Gly Ser Glu Glu Ser
Glu Leu Tyr His Ala1 5 10
15Gln Ile His Leu Tyr Lys His Val Tyr Asn Phe Val Ser Ser Met Ala
20 25 30Leu Lys Ser Ala Met Glu Leu
Gly Ile Ala Asp Ala Ile His Asn His 35 40
45Gly Lys Pro Met Thr Leu Pro Glu Leu Ser Ser Ser Leu Lys Leu
His 50 55 60Pro Ser Lys Val Asn Ile
Leu Tyr Arg Phe Leu Arg Leu Leu Thr His65 70
75 80Asn Gly Phe Phe Ala Lys Thr Thr Val Lys Ser
Asn Glu Gly Glu Glu 85 90
95Glu Thr Ala Tyr Val Leu Thr Pro Ser Ser Lys Leu Leu Val Ser Gly
100 105 110Lys Ser Thr Cys Leu Ser
Ser Leu Val Lys Gly Ala Leu His Pro Ser 115 120
125Ser Leu Asp Met Trp Gly Val Ser Lys Lys Trp Phe His Glu
Asp Lys 130 135 140Glu Gln Thr Leu Phe
Glu Cys Ala Thr Gly Glu Asn Tyr Trp Asp Phe145 150
155 160Leu Asn Lys Asp Ser Asp Ser Leu Ser Met
Phe Gln Asp Ala Met Ala 165 170
175Ala Asp Ser Arg Leu Phe Lys Leu Ala Ile Gln Glu Asn Lys His Val
180 185 190Phe Glu Gly Leu Glu
Ser Leu Val Asp Val Ala Gly Gly Thr Gly Gly 195
200 205Val Ala Lys Leu Ile His Glu Ala Phe Pro His Ile
Lys Cys Thr Val 210 215 220Phe Asp Gln
Pro Gln Val Val Gly Asn Leu Thr Gly Asn Glu Asn Leu225
230 235 240Asn Phe Val Gly Gly Asp Met
Phe Lys Ser Val Pro Ser Ala Asp Ala 245
250 255Val Leu Leu Lys Trp Val Leu His Asp Trp Asn Asp
Glu Leu Ser Leu 260 265 270Lys
Ile Leu Lys Asn Ser Lys Glu Ala Ile Ser His Lys Gly Lys Asp 275
280 285Gly Lys Val Ile Ile Ile Asp Ile Ser
Ile Asp Glu Asn Ser Asp Asp 290 295
300Arg Gly Leu Thr Glu Leu Gln Leu Glu Tyr Asp Val Val Met Leu Thr305
310 315 320Met Phe Leu Gly
Lys Glu Arg Thr Lys Lys Glu Trp Glu Lys Leu Ile 325
330 335Tyr Asp Ala Gly Phe Ser Arg Tyr Lys Ile
Thr Pro Ile Cys Gly Phe 340 345
350Lys Ser Leu Ile Glu Val Tyr Pro 355
36011359PRTCapsicum sp. 11Met Asp Ser Thr Asn Gln Asn Leu Thr Gln Thr Glu
Asp Glu Ala Phe1 5 10
15Leu Phe Ala Met Gln Leu Ala Ser Ala Ser Val Leu Pro Met Val Leu
20 25 30Lys Ser Ala Leu Glu Leu Asp
Leu Leu Glu Ile Met Ala Lys Ala Gly 35 40
45Pro Gly Ala Ala Ile Ser Pro Ser Glu Leu Ala Ala Gln Leu Pro
Thr 50 55 60Lys Asn Pro Glu Ala Pro
Val Met Leu Asp Arg Met Leu Arg Leu Leu65 70
75 80Ala Thr Tyr Ser Val Leu Asn Cys Thr Leu Arg
Thr Leu Pro Asp Gly 85 90
95Arg Val Glu Arg Leu Tyr Ser Leu Ala Pro Val Cys Lys Leu Leu Thr
100 105 110Lys Asn Ala Asp Gly Val
Ser Val Ala Pro Leu Leu Leu Met Asn Gln 115 120
125Asp Lys Val Leu Met Glu Ser Trp Tyr His Leu Thr Asp Ala
Val Leu 130 135 140Asp Gly Gly Val Pro
Phe Asn Lys Ala Tyr Gly Met Thr Ala Phe Glu145 150
155 160Tyr His Gly Thr Asp Pro Arg Phe Asn Lys
Val Phe Asn Arg Gly Met 165 170
175Ser Asp His Ser Thr Met Thr Met Lys Lys Ile Leu Glu Asp Tyr Lys
180 185 190Gly Phe Glu Gly Leu
Asn Ser Ile Val Asp Val Gly Gly Gly Thr Gly 195
200 205Ala Thr Val Asn Met Ile Val Ser Lys Tyr Pro Ser
Ile Lys Gly Ile 210 215 220Asn Phe Asp
Leu Ser His Val Ile Glu Asp Ala Pro Ala Tyr Pro Gly225
230 235 240Val Glu His Val Gly Arg Asp
Met Phe Val Ser Val Pro Lys Ala Asp 245
250 255Ala Ile Phe Met Lys Trp Ile Cys His Asp Trp Ser
Asp Glu His Cys 260 265 270Leu
Lys Phe Leu Lys Asn Cys Tyr Glu Ala Leu Pro Ala Asn Gly Lys 275
280 285Val Leu Val Ala Glu Cys Ile Leu Pro
Glu Thr Pro Asp Thr Ser Ala 290 295
300Ala Thr Lys Asn Ala Val His Val Asp Ile Val Met Leu Ala His Asn305
310 315 320Pro Gly Gly Lys
Glu Arg Thr Glu Lys Glu Phe Glu Ala Leu Ala Lys 325
330 335Gly Ala Gly Phe Thr Gly Phe Arg Arg Ala
Cys Cys Ala Tyr Gln Thr 340 345
350Trp Val Met Glu Phe His Lys 35512364PRTNicotiana tabacum 12Met
Gly Ser Thr Ser Glu Ser Gln Ser Asn Ser Leu Thr His Thr Glu1
5 10 15 Asp Glu Ala Phe Leu Phe Ala
Met Gln Leu Cys Ser Ala Ser Val Leu 20 25
30Pro Met Val Leu Lys Ser Ala Val Glu Leu Asp Leu Leu Glu
Leu Met 35 40 45Ala Lys Ala Gly
Pro Gly Ala Ala Ile Ser Pro Ser Glu Leu Ala Ala 50 55
60Gln Leu Ser Thr Gln Asn Pro Glu Ala Pro Val Met Leu
Asp Arg Met65 70 75
80Leu Arg Leu Leu Ala Ser Tyr Ser Val Leu Asn Cys Thr Leu Arg Thr
85 90 95Leu Pro Asp Ser Ser Val
Glu Arg Leu Tyr Ser Leu Ala Pro Val Cys 100
105 110Lys Tyr Leu Thr Lys Asn Ala Asp Gly Val Ser Val
Ala Pro Leu Leu 115 120 125Leu Met
Asn Gln Asp Lys Val Leu Met Glu Ser Trp Tyr His Leu Lys 130
135 140Asp Ala Val Leu Asp Gly Gly Ile Pro Phe Asn
Lys Ala Tyr Gly Met145 150 155
160Thr Ala Phe Glu Tyr His Gly Thr Asp Pro Arg Phe Asn Lys Val Phe
165 170 175Asn Arg Gly Met
Ser Asp His Ser Thr Met Ser Met Lys Lys Ile Leu 180
185 190Glu Asp Tyr Lys Gly Phe Glu Gly Leu Asn Ser
Ile Val Asp Val Gly 195 200 205Gly
Gly Thr Gly Ala Thr Val Asn Met Ile Val Ser Lys Tyr Pro Ser 210
215 220Ile Lys Gly Ile Asn Phe Asp Leu Pro His
Val Ile Gly Asp Ala Pro225 230 235
240Thr Tyr Pro Gly Val Glu His Val Gly Gly Asp Met Phe Ala Ser
Val 245 250 255Pro Lys Ala
Asp Ala Ile Phe Met Lys Trp Ile Cys His Asp Trp Ser 260
265 270Asp Glu His Cys Leu Lys Phe Leu Lys Asn
Cys Tyr Glu Ala Leu Pro 275 280
285Ala Asn Gly Lys Val Ile Ile Ala Glu Cys Ile Leu Pro Glu Ala Pro 290
295 300Asp Thr Ser Leu Ala Thr Lys Asn
Thr Val His Val Asp Ile Val Met305 310
315 320Leu Ala His Asn Pro Gly Gly Lys Glu Arg Thr Glu
Lys Glu Phe Glu 325 330
335Ala Leu Ala Lys Gly Ala Gly Phe Thr Gly Phe Ala Arg Leu Val Ala
340 345 350Leu Thr Thr Leu Gly Ser
Trp Asn Ser Thr Ser Asn 355 36013390PRTHordeum sp.
13Met Gln Asp Thr Ser Ser Thr Gln His Lys Ser Leu Pro Asn Asn Ile1
5 10 15Ile Glu Met Asp Met Val
Thr Ser Met Pro Leu Glu Ala Asn Ser Asn 20 25
30Gly Gln Ile Leu Gln Ala Glu Ala Glu Leu Phe Cys His
Ser Phe Gly 35 40 45Tyr Leu Lys
Ser Met Ala Leu Gln Ser Val Val Lys Leu Arg Ile Pro 50
55 60Asp Val Leu His Arg Tyr Gly Gly Ala Ala Ser Leu
Pro Glu Leu Leu65 70 75
80Ser Thr Val Pro Ile His Pro Asn Lys Leu Pro Tyr Leu Pro Arg Leu
85 90 95Met Lys Met Leu Ala Ala
Ala Gly Ile Phe Thr Ala Glu Asp Val Pro 100
105 110Ala Thr Val Gly Asp Gly Glu Pro Thr Thr Leu Tyr
His Leu Asn Ala 115 120 125Val Ser
Arg Leu Leu Val Asp Asp Ala Ser Val Asn Gly Gly Ala Ser 130
135 140Met Ser Pro Cys Val Leu Leu Gly Thr Val Pro
Leu Phe Leu Gly Ala145 150 155
160Ser Leu Lys Leu His Glu Trp Leu Gln Ser Glu Glu Gln Ala Thr Thr
165 170 175Glu Thr Pro Phe
Met Leu Ala His Gly Gly Thr Leu Tyr Gly Ile Gly 180
185 190Gly Arg Asp Ser Glu Phe Asn Thr Val Phe Asn
Lys Ala Met Gly Ala 195 200 205Ser
Ser Glu Phe Val Ala Ala Leu Ala Val Arg Glu Cys Arg Asp Val 210
215 220Phe Ala Gly Ile Lys Ser Leu Val Asp Val
Ala Gly Gly Asn Gly Thr225 230 235
240Thr Ala Arg Thr Ile Ala Glu Ala Phe Pro Tyr Val Lys Cys Ser
Val 245 250 255Leu Asp Leu
Pro Gln Val Ile Gln Gly Ile Ser Ser His Gly Thr Val 260
265 270Glu Phe Val Ala Gly Asp Met Met Glu Phe
Val Pro Pro Ala Glu Ala 275 280
285Val Leu Leu Lys Tyr Val Leu His Asn Trp Ser Asp Gln Asp Cys Val 290
295 300Lys Ile Leu Thr Arg Cys Arg Glu
Ala Ile Ser His Gly Glu Lys Ala305 310
315 320Gly Lys Val Ile Ile Ile Asp Thr Val Val Gly Ser
Pro Ser Gln Gln 325 330
335Ile Leu Glu Ser Gln Val Thr Met Asp Leu Ser Met Met Met Leu Phe
340 345 350Asn Gly Lys Val Arg Glu
Glu Gln Asn Trp His Lys Ile Phe Leu Glu 355 360
365Ala Gly Phe Ser His Tyr Lys Ile His Asn Val Leu Gly Met
Arg Ser 370 375 380Leu Ile Glu Val Gln
Pro385 39014364PRTHordeum sp. 14Met Gly Ser Ala Asn Pro
Asp Asn Lys Asn Ser Met Thr Lys Glu Glu1 5
10 15Glu Glu Ala Cys Leu Ser Ala Met Ala Leu Ala Ser
Ala Ser Val Leu 20 25 30Pro
Met Val Leu Lys Ser Ala Ile Glu Asp Leu Asp Leu Leu Glu Leu 35
40 45Ile Lys Lys Ser Gly Pro Gly Ala Tyr
Val Ser Pro Ser Glu Leu Ala 50 55
60Ala Gln Leu Pro Thr Gln Asn Pro Asp Ala Phe Val Met Leu Asp Arg65
70 75 80Ile Leu Arg Leu Leu
Ala Ser Tyr Ser Val Leu Asn Cys Thr Leu Lys 85
90 95Asp Leu Pro Asp Gly Gly Ile Glu Arg Leu Tyr
Ser Leu Ala Pro Val 100 105
110Cys Lys Phe Leu Thr Lys Asn Glu Asp Gly Val Ser Met Ala Ala Leu
115 120 125Leu Leu Met Asn Gln Asp Lys
Val Leu Met Glu Ser Trp Tyr His Leu 130 135
140Lys Asp Ala Val Leu Glu Gly Gly Ile Pro Phe Asn Lys Ala Tyr
Gly145 150 155 160Met Thr
Ala Phe Glu Tyr His Gly Lys Asp Pro Arg Phe Asn Lys Val
165 170 175Phe Asn Gln Gly Met Ser Asn
His Ser Thr Ile Ile Met Lys Lys Ile 180 185
190Leu Glu Ile Tyr Gln Phe Gly Gln Gly Leu Lys Thr Val Val
Asp Val 195 200 205Gly Gly Gly Thr
Gly Ala Thr Leu Asn Met Ile Val Ser Lys Tyr Pro 210
215 220Ser Ile Lys Gly Ile Asn Phe Gly Leu Pro His Val
Ile Glu Asp Ala225 230 235
240Pro Ser Tyr Pro Gly Val Asp His Val Gly Gly Asp Met Phe Val Ser
245 250 255Val Pro Lys Gly Asp
Ala Ile Phe Met Lys Trp Ile Cys His Asp Trp 260
265 270Ser Asp Ala His Cys Leu Lys Phe Leu Lys Asn Cys
His Glu Ala Leu 275 280 285Pro Glu
Asn Gly Lys Val Ile Leu Ala Glu Cys Leu Leu Pro Glu Ala 290
295 300Pro Asp Ser Thr Leu Ser Thr Gln Asn Thr Val
His Val Asp Val Ile305 310 315
320Met Leu Ala His Asn Pro Gly Gly Lys Glu Arg Thr Glu Lys Glu Phe
325 330 335Glu Ala Leu Ala
Lys Gly Ala Gly Phe Arg Gly Phe Ile Lys Val Cys 340
345 350Cys Ala Tyr Asn Ser Trp Ile Met Glu Leu Leu
Lys 355 36015367PRTGlycyrrhiza glabra 15Met Ser
Asp Ser Tyr Ser Thr Lys Asp Asn Asn Leu Phe Ser Thr Ser1 5
10 15Ser Glu Gln Thr Glu Asp Gly Ala
Cys Leu Ser Ala Met Arg Leu Val 20 25
30Thr Asn Leu Val Tyr Pro Ala Val Leu Asn Ala Ala Ile Asp Leu
Asn 35 40 45Leu Phe Glu Ile Ile
Ala Lys Ala Thr Pro Pro Gly Ala Phe Met Ser 50 55
60Ala Ser Glu Ile Ala Ser Lys Leu Pro Leu Pro Thr Gln His
Ser Asp65 70 75 80Leu
Pro Asn Arg Leu Asp Arg Met Leu Arg Leu Leu Ala Ser Tyr Ser
85 90 95Val Leu Thr Cys Ala Thr Arg
Ser Thr Glu Arg Val Tyr Gly Leu Ser 100 105
110Gln Val Gly Lys Tyr Leu Val Pro Asp Gly Ser Arg Gly Tyr
Leu Ala 115 120 125Ser Phe Thr Thr
Phe Leu Cys Tyr Pro Ala Leu Met Asn Val Trp Leu 130
135 140Asn Phe Lys Glu Ala Val Val Asp Glu Asp Ile Asp
Leu Phe Lys Lys145 150 155
160Leu His Gly Val Ser Lys Tyr Glu Tyr Met Glu Thr Asp Pro Lys Met
165 170 175Asn His Ile Phe Asn
Lys Ser Met Ala Asp Val Cys Ala Thr Glu Met 180
185 190Lys Arg Ile Leu Gln Ile Tyr Lys Gly Phe Glu Gly
Ile Ser Thr Leu 195 200 205Val Asp
Val Gly Gly Gly Asn Gly Gln Asn Leu Lys Met Ile Ile Ser 210
215 220Lys Tyr Pro Leu Ile Lys Gly Ile Asn Phe Asp
Leu Pro Gln Val Ile225 230 235
240Glu Asn Ala Pro Pro Ile Pro Gly Ile Glu Leu Val Gly Gly Asp Met
245 250 255Phe Ala Ser Val
Pro Gln Gly Asp Ala Met Ile Leu Lys Ala Val Cys 260
265 270His Asn Trp Ser Asp Glu Lys Cys Leu Glu Phe
Leu Ser Asn Cys His 275 280 285Lys
Ala Leu Ser Pro Asn Gly Lys Val Ile Val Val Glu Phe Ile Leu 290
295 300Pro Glu Glu Pro Glu Pro Thr Glu Glu Ser
Gln Leu Ala Ser Thr Leu305 310 315
320Asp Asn Ile Met Phe Ile Thr Val Gly Gly Arg Glu Arg Thr Gln
Lys 325 330 335Gln Tyr Glu
Asn Met Cys Lys Leu Ala Gly Phe Ser Lys Phe Gln Val 340
345 350Ala Cys Arg Ala Phe Ser Ser Leu Gly Val
Met Glu Phe Tyr Lys 355 360
36516372PRTMedicago sativaMOD_RES(1)Met or Mse 16Xaa Gly Asn Ser Tyr Ile
Thr Lys Glu Asp Asn Gln Ile Ser Ala Thr1 5
10 15Ser Glu Gln Thr Glu Asp Ser Ala Cys Leu Ser Ala
Xaa Val Leu Thr 20 25 30Thr
Asn Leu Val Tyr Pro Ala Val Leu Asn Ala Ala Ile Asp Leu Asn 35
40 45Leu Phe Glu Ile Ile Ala Lys Ala Thr
Pro Pro Gly Ala Phe Xaa Ser 50 55
60Pro Ser Glu Ile Ala Ser Lys Leu Pro Ala Ser Thr Gln His Ser Asp65
70 75 80Leu Pro Asn Arg Leu
Asp Arg Xaa Leu Arg Leu Leu Ala Ser Tyr Ser 85
90 95Val Leu Thr Ser Thr Thr Arg Thr Ile Glu Asp
Gly Gly Ala Glu Arg 100 105
110Val Tyr Gly Leu Ser Xaa Val Gly Lys Tyr Leu Val Pro Asp Glu Ser
115 120 125Arg Gly Tyr Leu Ala Ser Phe
Thr Thr Phe Leu Cys Tyr Pro Ala Leu 130 135
140Leu Gln Val Trp Xaa Asn Phe Lys Glu Ala Val Val Asp Glu Asp
Ile145 150 155 160Asp Leu
Phe Lys Asn Val His Gly Val Thr Lys Tyr Glu Phe Xaa Gly
165 170 175Lys Asp Lys Lys Xaa Asn Gln
Ile Phe Asn Lys Ser Xaa Val Asp Val 180 185
190Cys Ala Thr Glu Xaa Lys Arg Xaa Leu Glu Ile Tyr Thr Gly
Phe Glu 195 200 205Gly Ile Ser Thr
Leu Val Asp Val Gly Gly Gly Ser Gly Arg Asn Leu 210
215 220Glu Leu Ile Ile Ser Lys Tyr Pro Leu Ile Lys Gly
Ile Asn Phe Asp225 230 235
240Leu Pro Gln Val Ile Glu Asn Ala Pro Pro Leu Ser Gly Ile Glu His
245 250 255Val Gly Gly Asp Xaa
Phe Ala Ser Val Pro Gln Gly Asp Ala Xaa Ile 260
265 270Leu Lys Ala Val Cys His Asn Trp Ser Asp Glu Lys
Cys Ile Glu Phe 275 280 285Leu Ser
Asn Cys His Lys Ala Leu Ser Pro Asn Gly Lys Val Ile Ile 290
295 300Val Glu Phe Ile Leu Pro Glu Glu Pro Asn Thr
Ser Glu Glu Ser Lys305 310 315
320Leu Val Ser Thr Leu Asp Asn Leu Xaa Phe Ile Thr Val Gly Gly Arg
325 330 335Glu Arg Thr Glu
Lys Gln Tyr Glu Lys Leu Ser Lys Leu Ser Gly Phe 340
345 350Ser Lys Phe Gln Val Ala Cys Arg Ala Phe Asn
Ser Leu Gly Val Xaa 355 360 365Glu
Phe Tyr Lys 37017352PRTMedicago sativaMOD_RES(1)Met or Mse 17Xaa Ala
Ser Ser Ile Asn Gly Arg Lys Pro Ser Glu Ile Phe Lys Ala1 5
10 15Gln Ala Leu Leu Tyr Lys His Ile
Tyr Ala Phe Ile Asp Ser Xaa Ser 20 25
30Leu Lys Trp Ala Val Glu Xaa Asn Ile Pro Asn Ile Ile Gln Asn
His 35 40 45Gly Lys Pro Ile Ser
Leu Ser Asn Leu Val Ser Ile Leu Gln Val Pro 50 55
60Ser Ser Lys Ile Gly Asn Val Arg Arg Leu Xaa Arg Tyr Leu
Ala His65 70 75 80Asn
Gly Phe Phe Glu Ile Ile Thr Lys Glu Glu Glu Ser Tyr Ala Leu
85 90 95Thr Val Ala Ser Glu Leu Leu
Val Arg Gly Ser Asp Leu Cys Leu Ala 100 105
110Pro Xaa Val Glu Cys Val Leu Asp Pro Thr Leu Ser Gly Ser
Tyr His 115 120 125Glu Leu Lys Lys
Trp Ile Tyr Glu Glu Asp Leu Thr Leu Phe Gly Val 130
135 140Thr Leu Gly Ser Gly Phe Trp Asp Phe Leu Asp Lys
Asn Pro Glu Tyr145 150 155
160Asn Thr Ser Phe Asn Asp Ala Xaa Ala Ser Asp Ser Lys Leu Ile Asn
165 170 175Leu Ala Leu Arg Asp
Cys Asp Phe Val Phe Asp Gly Leu Glu Ser Ile 180
185 190Val Asp Val Gly Gly Gly Thr Gly Thr Thr Ala Lys
Ile Ile Cys Glu 195 200 205Thr Phe
Pro Lys Leu Lys Cys Ile Val Phe Asp Arg Pro Gln Val Val 210
215 220Glu Asn Leu Ser Gly Ser Asn Asn Leu Thr Tyr
Val Gly Gly Asp Xaa225 230 235
240Phe Thr Ser Ile Pro Asn Ala Asp Ala Val Leu Leu Lys Tyr Ile Leu
245 250 255His Asn Trp Thr
Asp Lys Asp Cys Leu Arg Ile Leu Lys Lys Cys Lys 260
265 270Glu Ala Val Thr Asn Asp Gly Lys Arg Gly Lys
Val Thr Ile Ile Asp 275 280 285Xaa
Val Ile Asp Lys Lys Lys Asp Glu Asn Gln Val Thr Gln Ile Lys 290
295 300Leu Leu Xaa Asp Val Asn Xaa Ala Cys Leu
Asn Gly Lys Glu Arg Asn305 310 315
320Glu Glu Glu Trp Lys Lys Leu Phe Ile Glu Ala Gly Phe Gln His
Tyr 325 330 335Lys Ile Ser
Pro Leu Thr Gly Phe Leu Ser Leu Ile Glu Ile Tyr Pro 340
345 35018140PRTMedicago sativaMOD_RES(10)Mse
18Thr Glu Asp Ser Ala Cys Leu Ser Ala Xaa Val Leu Thr Thr Asn Leu1
5 10 15Val Tyr Pro Ala Val Leu
Asn Ala Ala Ile Asp Leu Asn Leu Phe Glu 20 25
30Ile Ile Ala Lys Ala Thr Pro Pro Gly Ala Phe Xaa Ser
Pro Ser Glu 35 40 45Ile Ala Ser
Lys Leu Pro Ala Ser Thr Gln His Ser Asp Leu Pro Asn 50
55 60Arg Leu Asp Arg Xaa Leu Arg Leu Leu Ala Ser Tyr
Ser Val Leu Thr65 70 75
80Ser Thr Thr Arg Thr Ile Glu Asp Gly Gly Ala Glu Arg Val Tyr Gly
85 90 95Leu Ser Xaa Val Gly Lys
Tyr Leu Val Pro Asp Glu Ser Arg Gly Tyr 100
105 110Leu Ala Ser Phe Thr Thr Phe Leu Cys Tyr Pro Ala
Leu Leu Gln Val 115 120 125Trp Xaa
Asn Phe Lys Glu Ala Val Val Asp Glu Asp 130 135
14019193PRTMedicago sativaMOD_RES(2)Mse 19Lys Xaa Asn Gln Ile
Phe Asn Lys Ser Xaa Val Asp Val Cys Ala Thr1 5
10 15Glu Xaa Lys Arg Xaa Leu Glu Ile Tyr Thr Gly
Phe Glu Gly Ile Ser 20 25
30Thr Leu Val Asp Val Gly Gly Gly Ser Gly Arg Asn Leu Glu Leu Ile
35 40 45Ile Ser Lys Tyr Pro Leu Ile Lys
Gly Ile Asn Phe Asp Leu Pro Gln 50 55
60Val Ile Glu Asn Ala Pro Pro Leu Ser Gly Ile Glu His Val Gly Gly65
70 75 80Asp Xaa Phe Ala Ser
Val Pro Gln Gly Asp Ala Xaa Ile Leu Lys Ala 85
90 95Val Cys His Asn Trp Ser Asp Glu Lys Cys Ile
Glu Phe Leu Ser Asn 100 105
110Cys His Lys Ala Leu Ser Pro Asn Gly Lys Val Ile Ile Val Glu Phe
115 120 125Ile Leu Pro Glu Glu Pro Asn
Thr Ser Glu Glu Ser Lys Leu Val Ser 130 135
140Thr Leu Asp Asn Leu Xaa Phe Ile Thr Val Gly Gly Arg Glu Arg
Thr145 150 155 160Glu Lys
Gln Tyr Glu Lys Leu Ser Lys Leu Ser Gly Phe Ser Lys Phe
165 170 175Gln Val Ala Cys Arg Ala Phe
Asn Ser Leu Gly Val Xaa Glu Phe Tyr 180 185
190Lys 20345PRTMedicago sativaMOD_RES(24)Met or Mse 20Arg
Lys Pro Ser Glu Ile Phe Lys Ala Gln Ala Leu Leu Tyr Lys His1
5 10 15Ile Tyr Ala Phe Ile Asp Ser
Xaa Ser Leu Lys Trp Ala Val Glu Xaa 20 25
30Asn Ile Pro Asn Ile Ile Gln Asn His Gly Lys Pro Ile Ser
Leu Ser 35 40 45Asn Leu Val Ser
Ile Leu Gln Val Pro Ser Ser Lys Ile Gly Asn Val 50 55
60Arg Arg Leu Xaa Arg Tyr Leu Ala His Asn Gly Phe Phe
Glu Ile Ile65 70 75
80Thr Lys Glu Glu Glu Ser Tyr Ala Leu Thr Val Ala Ser Glu Leu Leu
85 90 95Val Arg Gly Ser Asp Leu
Cys Leu Ala Pro Xaa Val Glu Cys Val Leu 100
105 110Asp Pro Thr Leu Ser Gly Ser Tyr His Glu Leu Lys
Lys Trp Ile Tyr 115 120 125Glu Glu
Asp Leu Thr Leu Phe Gly Val Thr Leu Gly Ser Gly Phe Trp 130
135 140Asp Phe Leu Asp Lys Asn Pro Glu Tyr Asn Thr
Ser Phe Asn Asp Ala145 150 155
160Xaa Ala Ser Asp Ser Lys Leu Ile Asn Leu Ala Leu Arg Asp Cys Asp
165 170 175Phe Val Phe Asp
Gly Leu Glu Ser Ile Val Asp Val Gly Gly Gly Thr 180
185 190Gly Thr Thr Ala Lys Ile Ile Cys Glu Thr Phe
Pro Lys Leu Lys Cys 195 200 205Ile
Val Phe Asp Arg Pro Gln Val Val Glu Asn Leu Ser Gly Ser Asn 210
215 220Asn Leu Thr Tyr Val Gly Gly Asp Xaa Phe
Thr Ser Ile Pro Asn Ala225 230 235
240Asp Ala Val Leu Leu Lys Tyr Ile Leu His Asn Trp Thr Asp Lys
Asp 245 250 255Cys Leu Arg
Ile Leu Lys Lys Cys Lys Glu Ala Val Thr Asn Asp Gly 260
265 270Lys Arg Gly Lys Val Thr Ile Ile Asp Xaa
Val Ile Asp Lys Lys Lys 275 280
285Asp Glu Asn Gln Val Thr Gln Ile Lys Leu Leu Xaa Asp Val Asn Xaa 290
295 300Ala Cys Leu Asn Gly Lys Glu Arg
Asn Glu Glu Glu Trp Lys Lys Leu305 310
315 320Phe Ile Glu Ala Gly Phe Gln His Tyr Lys Ile Ser
Pro Leu Thr Gly 325 330
335Phe Leu Ser Leu Ile Glu Ile Tyr Pro 340
34521141PRTMedicago sativa 21Gln Thr Glu Asp Ser Ala Cys Leu Ser Ala Met
Val Leu Thr Thr Asn1 5 10
15Leu Val Tyr Pro Ala Val Leu Asn Ala Ala Ile Asp Leu Asn Leu Phe
20 25 30Glu Ile Ile Ala Lys Ala Thr
Pro Pro Gly Ala Phe Met Ser Pro Ser 35 40
45Glu Ile Ala Ser Lys Leu Pro Ala Ser Thr Gln His Ser Asp Leu
Pro 50 55 60Asn Arg Leu Asp Arg Met
Leu Arg Leu Leu Ala Ser Tyr Ser Val Leu65 70
75 80Thr Ser Thr Thr Arg Thr Ile Glu Asp Gly Gly
Ala Glu Arg Val Tyr 85 90
95Gly Leu Ser Met Val Gly Lys Tyr Leu Val Pro Asp Glu Ser Arg Gly
100 105 110Tyr Leu Ala Ser Phe Thr
Thr Phe Leu Cys Tyr Pro Ala Leu Leu Gln 115 120
125Val Trp Met Asn Phe Lys Glu Ala Val Val Asp Glu Asp
130 135 14022199PRTMedicago sativa 22Phe
Met Gly Lys Asp Lys Lys Met Asn Gln Ile Phe Asn Lys Ser Met1
5 10 15Val Asp Val Cys Ala Thr Glu
Met Lys Arg Met Leu Glu Ile Tyr Thr 20 25
30Gly Phe Glu Gly Ile Ser Thr Leu Val Asp Val Gly Gly Gly
Ser Gly 35 40 45Arg Asn Leu Glu
Leu Ile Ile Ser Lys Tyr Pro Leu Ile Lys Gly Ile 50 55
60Asn Phe Asp Leu Pro Gln Val Ile Glu Asn Ala Pro Pro
Leu Ser Gly65 70 75
80Ile Glu His Val Gly Gly Asp Met Phe Ala Ser Val Pro Gln Gly Asp
85 90 95Ala Met Ile Leu Lys Ala
Val Cys His Asn Trp Ser Asp Glu Lys Cys 100
105 110Ile Glu Phe Leu Ser Asn Cys His Lys Ala Leu Ser
Pro Asn Gly Lys 115 120 125Val Ile
Ile Val Glu Phe Ile Leu Pro Glu Glu Pro Asn Thr Ser Glu 130
135 140Glu Ser Lys Leu Val Ser Thr Leu Asp Asn Leu
Met Phe Ile Thr Val145 150 155
160Gly Gly Arg Glu Arg Thr Glu Lys Gln Tyr Glu Lys Leu Ser Lys Leu
165 170 175Ser Gly Phe Ser
Lys Phe Gln Val Ala Cys Arg Ala Phe Asn Ser Leu 180
185 190Gly Val Met Glu Phe Tyr Lys 195
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