Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
  • C07K5/10—Tetrapeptides
  • C07K5/1002—Tetrapeptides with the first amino acid being neutral
  • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
  • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
  • A61P39/06—Free radical scavengers or antioxidants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the invention is referred to the field of medicine and may be employed as a geroprotective substance for the prevention of premature ageing of the organism.
• Gerovital a Novocain-containing drug, is used as a geroprotective substance.
• the detriments of this preparation consist in its possible negative influence on the functions of the cardiovascular system, its allergic impact, sometimes sleep impairment, the feeling of anxiety, muscular and articular pains.
• the claimed invention is aimed at obtaining a new biologically active compound of peptide origin capable of geroprotective activity.
• the claimed peptide compound - tetrapeptide - has no structural analogues.
• the tetrapeptide L-alanyl-L-gluthamyl-L-asparagyl-glycine with the following amino acid sequence: L-Ala-L-Glu-L-Asp-Gly reveals biological activity, namely, geroprotective activity due to the stimulation of indices of the anti-oxidation defence system and due to the process of melatonin synthesis in structures of the diffusive neuroendocrine system.
• the tetrapeptide is obtained by a classical method of peptide synthesis in a solution (19).
• the geroprotective activity of L-Ala-L-Glu-L-Asp-GIy tetrapeptide was stated in experimental trials.
• the study of geroprotective activity was conducted in Drosophila melanogaster by analysing the indices of anti-oxidation defence, life span, and the length of reproduction period, as well as in rats by examining the synthesis of extra-pineal melatonin.
• the pharmacological substance capable of geroprotective activity includes as its active base an effective amount of tetrapeptide of the formula L-alanyl-L-gluthamyl-L-asparagyl-glycine (L-Ala-L-Glu-L-Asp-Gly) or its salts.
• the pharmacological substance capable of geroprotective activity may contain salts of the amino group (acetate, hydrochloride, and oxalate) or of carboxyl groups (the salts of metals - Sodium, Potassium, Calcium, Lithium, Zinc, Magnesium, and other organic and inorganic cations - ammonium, triethylammonium).
• the pharmacological substance is meant for parenteral, intranasal, oral administration, or local application.
• the claimed pharmacological substance revealing geroprotective activity is capable of stimulating the indices of the anti-oxidation defence system and the processes of melatonin synthesis in structures of the diffusive neuroendocrine system, which, in their turn, inhibit ageing processes and promote a life span increase.
• geroprotective substance used in this application, implies the substance, which inhibits ageing and prolongs life by means of preventing premature ageing, which needs stimulation of the anti-oxidation defence system and the regulatory influence on metabolic processes in structures of the diffusive neuroendocrine system.
• the proposed tetrapeptide or its pharmaceutically applicable derivatives are stirred as an active ingredient and a pharmaceutical carrier in accordance with the methods of compounding accepted in pharmaceutics.
• the carrier may have various forms, which depend on the drug form of the preparation desirable for administration, for example: parenteral, intranasal, or oral. All known pharmacological components may be used for the preparation of compositions in doses preferable for oral application.
• the carrier usually includes sterile water, although there may be included other ingredients instrumental for stability or maintaining sterility.
• the method embraces prophylactic or therapeutic exposure of patients to the claimed pharmacological substance in doses 0.01 to 100 mg/kg of the body weight, at least once a day during a period necessary for achieving a therapeutic effect - 10 to 40 days with respect to the character and severity of a pathologic process.
• the tetrapeptide is active in case it is administered in doses
• the invention is illustrated by an example of synthesis of the tetrapeptide, whose formula is L- alanyl-L-gluthamyl-L-asparagyl-glycine (L-Ala-L-Glu-L-Asp-Gly) (Example 1), by the examples of the tests for toxicity and biological activity of the tetrapeptide (Examples 2-8), and also by the examples, which show the results of clinical application of the tetrapeptide, thus demonstrating its pharmacological properties and attesting the possibility of achieving prophylactic and/or therapeutic effect (Examples 9, 10).
• Example 1 Synthesis of L-Ala-L-Glu-L-Asp-Gly tetrapeptide 1.
• Product name L-ala ⁇ yl-L-gluthamyl-L-asparagyl-glycine.
• Structural formula H-Ala-GIu-Asp-Gly-OH
• T RULE 91 Z - benzyloxycarbonyl group; BOC - tertbutyloxycarbonyl group; OSu - N-oxysuccinimide ester; OBzl - benzyl ester;
• N, N ' -dimethylformamide was used as a solvent.
• aspartic acid there was applied the defence of the ⁇ -COOH group by the salification method with the use of triethylamine.
• the removal of the BOC-protecting group was performed with the solution of trifluoracetic acid (TFA); the removal of the Z- protecting group was performed with catalytic hydrogenation.
• the extraction and purification of the product were conducted by the method of preparative high-performance liquid chromatography (HPLC) on the column with a reversed phase.
• N-carbobenzoxyalanyl-( ⁇ -benzyl)gluthamyl( ⁇ -benzyl)aspartylglycine benzyl ester (III) are hydrogenated in the methanol-water-acetic acid system (3:1:1) over Pd/C. Progress of the reaction is monitored by TLC method in the benzol-aceton (2:1) and acetonitryl-acetic acid-water (5:1:3) systems. After the end of the reaction the catalyst is filtered, the filtrate is removed in vacuo, and the residue is recrystallised in the water- methanol system. The product is dried in vacuo over KOH. The yield is 20mg (95%).
• R f 0.73 (acetonitryl-acetic acid-water, 5:1:3).
• 390mg of the product is dissolved in 4ml of 0.01%-triflouracetic acid and subject to HPLC on the column with a reversed phase 50x250mm Diasorb-130-C16T, 7 ⁇ m.
• the chromatograph used is Beckman System Gold, 126 Solvent Module, 168 Diode Array Detector Module.
• the obtained peptide in the form of acetate is transferred to a free form by processing it with IRA anionite or with an analogous substance in the (OH " )-form. Afterwards, salts of the amino group are obtained with the subsequent addition of an equivalent of a corresponding acid (hydrochloride or oxalic). The obtained water solution is lyophilised and analysed as a ready product.
• the free tetrapeptide is added a calculated amount of the water solution of hydroxide of a corresponding metal (NaOH, KOH, Zn(OH) 2 , LiOH, Ca(OH) 2 , Mg(OH) 2 , N ⁇ OH).
• a corresponding metal NaOH, KOH, Zn(OH) 2 , LiOH, Ca(OH) 2 , Mg(OH) 2 , N ⁇ OH.
• triethylammonium salt the processing is carried out similarly, with the use of triethylamine as a base.
• Peptide content is defined by the HPLC method on the Surelco LC-18-DB column, 4.6x250mm, grad. LC-18-DB.
• the flow speed constitutes lml/min. Detection by 220nm, scanning - by 190-600nm, the sample volume is 20 ⁇ l. Peptide content - 98.45%.
• TLC individual, (acetonitryl-acetic acid-water, 5: 1 :3). Sorbfil plates, 8-12 ⁇ m Silicagel, developing in chlorine/benzydine.
• Example 2 Study of L-Ala-L-Glu-L-Asp-Gly tetrapeptide for toxicity.
• the purpose of study consisted in defining the tolerable toxic doses of the preparation, in estimating the stage and character of pathologic alterations in various organs and systems of the organism, and in identifying the correlation between the toxic dose-related effect and the duration of drug application.
• L-Ala-L-Glu-L-Asp-Gly tetrapeptide in doses several thousand times exceeding the therapeutic one, recommended for clinical trials, does not induce acute toxic reactions, which confirms a wide therapeutic applicability of the preparation.
• RECTIFIED SHEET (RULE 91) ISA/EP single administration of the drug intramuscularly for 90 days in doses l ⁇ g/kg, 0.3mg/kg, 3mg/kg in 0.5ml of sodium chloride solution. Animals of the control group were administered the same amount of sodium chloride.
• the animals were exposed daily to a single administration of the drug intramuscularly in doses I ⁇ g kg, 0.1 mg/kg, 1 mg/kg in 0.5ml of sodium chloride solution during 6 months. Behaviour of the animals, their rations and water consumption, the state of hairy integument and mucous membranes were registered. The animals were weighed daily during the first 3 months of the experiment and then once a month. Haematological and biochemical studies were carried out in 3 months after the onset of the drug administration and on the experiment completion. Functions of the cardiovascular system, liver, pancreas, kidney, and adrenal gland were estimated.
• Example 3 Impact of L-Ala-L-Glu-L-Asp-Gly tetrapeptide on the ageing rate and free radical processes in Drosophila melanogaster of HEM strain selected for high embryonic mortality rate.
• HEM strain L-Ala-L-Glu-L-Asp-Gly tetrapeptide was applied in Drosophila melanogaster larvae of the 2 nd age of HEM strain selected for a high ageing rate (20).
• HEM strain is characterised by the unique dynamics of embryonic mortality, which consists in an increased frequency of early
• RECTIFIED SHEET (RULE 91) ISA/EP embryonic lethal outcomes from 65% on the 1 st day of egg-laying up to 95% on the 4 th day, in the reduced average life span (29 days), and in the raised intensity of lipid peroxide oxidation.
• L-Ala-L-Glu-L-Asp-Gly tetrapeptide was added in proportion- 0.00001% of the culture medium weight, which is an extremely low dose.
• the doses stated in literature on the influence of various substances on Drosophila melanogaster usually ranged from 0.001 to 0.01%. Lower doses do not normally produce any effect.
• Biochemical parameters were studied on 14-day old flies. Among them the activity of catalase and the intensity of tissue chemoluminescence were analysed (21, 22, 23). Catalase is the basic enzyme of anti-oxidation defence. The degree of its activity suggests the ability of the organism to withstand oxidative stress. Luminol-dependent chemoluminescence, induced by hydrogen peroxide, reflects the stage of active oxygen forms in tissues. Thus, a decrease in the chemoluminescence intensity exhibits an increase in the organism ability to resist oxidative stress.
• Table 2 displays the results of the life span analysis in different experimental groups.
• L-Ala-L-Glu-L-Asp-GIy tetrapeptide produces a pronounced geroprotective effect.
• P ⁇ 0.001 the average life span
• R the ageing rate
• P ⁇ 0.05 the ageing rate
• L-Ala-L-Glu-L-Asp-GIy tetrapeptide increases the average life span by 26% and reduces the population real ageing rate, estimated by the quantity of Gomperz parameter, by 40%.
• Example 4 Influence of L-Ala-L-Glu-L-Asp-Gly tetrapeptide on the reproductive functions in Drosophila melanogaster of HEM strain, selected for high embryonic mortality rate.
• RECTIFIED SHEET (RULE 91) medium in dose 0.00001% of the medium weight, which approximately made 0.015mg/l 00ml.
• the coefficients of lineal regression are presented in Table 4. As it is shown in the Table, the coefficients of the control regression line differ significantly from the experimental one. The "a"-coeff ⁇ cient (reflecting the slope of the straight line) in the control is 1.8 times higher than the regression coefficient of the experimental variant. Thus, it is demonstrated that the share of fertile cultures in the control variant reduces twice faster in comparison with the L-Ala-L-Glu-L-Asp-Gly tetrapeptide variant. Duration of the reproductive period amounted to 30 days for the cultures, not exposed to the tetrapeptide, and to 47 days for the cultures processed with the preparation. It is stated that L-Ala-L-Glu-L-Asp-Gly tetrapeptide 1.5-fold extends the reproduction period. The data obtained on the life span were analysed correspondingly. The data on the regression analysis are displayed in Table 5.
• Example 5 Influence of L-Ala-L-Glu-L-Asp-Gly tetrapeptide on the ageing rate and free radical processes in Drosophila melanogaster of low activity (LA) strain selected for a low sexual activity of males.
• LA low activity
• L-Ala-L-Glu-L-Asp-Gly tetrapeptide The impact produced by L-Ala-L-Glu-L-Asp-Gly tetrapeptide on catalase activity, the content of the products of lipid peroxide oxidation (LPO), the life span, and the ageing rate were studied on Drosophila melanogaster of LA strain.
• ISA/EP L-Ala-L-Glu-L-Asp-Gly tetrapeptide was applied in the larvae of the 3 rd age in dose 0.00001% of the culture medium weight, which constituted approximately 0.015mg/l 00ml.
• Catalase activity was estimated by generally accepted methods in the homogenates of adult 14 days old flies. The experiment was carried out with a subsequent repetition. Regression and dispersal analyses served as the basic technique of statistic processing. The authenticity of the differences was confirmed by the method of the minimal relevant diversity.
• L-Ala-L-Glu-L-Asp-Gly tetrapeptide in females did not induce any alterations in the average life span, but did increase the maximal life span (LS ⁇ x ).
• the analysis of the survival rate curves and Gomperz charts showed that L-Ala-L-Glu-L-Asp-Gly tetrapeptide in females served as a geroprotector of the 1 st type (ALS and LS max were increased, though the ageing rate did not change in comparison with the control).
• Drosophila melanogaster of EA strain served as a model for studying the influence of L-Ala-L-Glu-L-Asp-Gly tetrapeptide on catalase activity, tissue chemoluminescence, and life span.
• One-day old flies were placed in test tubes, 10 individuals of the same sex in each. Every 5 to 7 days they were transferred to a fresh medium, taking into account the number of deceased individuals. The average and maximal life spans were defined. The ageing rate was calculated by the parameters of Gomperz equation.
• L-Ala-L-GIu-L-Asp-Gly tetrapeptide was applied in the larvae of the 3 rd age in dose
• L-Ala-L-Glu-L-Asp-Gly tetrapeptide produces an effect on luminol- dependent chemoluminescence (Table 14).
• the reduction of this index evidences the activation of the anti-oxidation defence system in flies.
• the raise of intensity in the tissue anti-oxidation defence in this case is presumably associated with the activation of low-molecular endogenous anti-oxidants, such as glutathione, tocopherol, and the like.
• Table 1 Influence of L-Ala-L-Glu-L-Asp-Gly tetrapeptide on the life span parameters in male Drosophila melanogaster of EA strain (Experiment 1).
• Example 7 Influence of L-Ala-L-Glu-L-Asp-Gly tetrapeptide on the life span of Drosophila melanogaster of "wild " Canton-S strain.
• 4-day old female flies underwent a randomly selected mating with males for 72 hours, a couple per each test tube, after which parental couples were removed from the test tubes.
• Table 15 displays the aftermath of the life span distribution characteristics (average values and the standard errors of these average values) for all the experimental and control groups.
• concentrations of L-AIa-L-Glu-L-Asp-Gly tetrapeptide were selected: O.Olx, O.lx, lx, 5x, 7.5x, and 12.5x10 " % of the culture medium weight.
• L-Ala-L-Glu-L-Asp-Gly tetrapeptide in Drosophila melanogaster did not alter the duration of their stage development, which, in general, reflected the lack of genotoxic effect produced by the studied substance.
• Example 8 Influence of L-Ala-L-Glu-L-Asp-Gly tetrapeptide on the extra-pineal synthesis of melatonin in rats after epiphysectomy.
• the research was carried out on male Wistar rats weighing 130-140g. The total number of the animals (23) was divided into 5 groups. The I s " group (control) consisted of intact animals.
• the animals of groups 2-5 underwent epiphysectomy (EE). In 3 weeks after the surgery (on the 21 s * day) the animals of the 2 nd and 3 rd groups were subcutaneously injected with sodium chloride in dose 0.5ml for the next 10 days. The animals of groups 4 and 5 were administered to L-Ala-L-
• Glu-L-Asp-GIy tetrapeptide in dose 0.5 ⁇ g according to the same scheme.
• DNES diffusive neuroendocrine system
• Fragments of the extracted organs were fixed for 24 hours with Buen sour liquid for optic microscopy examination and according to Karnovsky method - for electron microscopy. Material dehydration and filling it in with paraffin for optic microscopy, as well as epone mixture for ultra-structural examination were prepared according to generally accepted techniques. Paraffin sections (7 ⁇ m) were placed on a microscope slide covered with a poly-L-lysine film (Sigma). Ultra-thin sections (lOOnm) prepared on LKB-7A microtome (L B) were contrasted with uranyl acetate and plumbum citrate.
• EC-cells enterochromophine-cells
• uniclonic antibodies were identified according to avidin-biotin-peroxidase (ABP) method (Vectastain kit) in order to reveal immune serum globulins of mice.
• ABSP avidin-biotin-peroxidase
• the received data confirm that the main point of L-AIa-L-Glu-L-Asp-Gly tetrapeptide application is the gastric EC-cells, in which L-Ala-L-Glu-L-Asp-Gly tetrapeptide promotes the extra-pineal synthesis of serotonin and melatonin (Table 16), thus, in essence, providing a complete compensation for the extracted epiphysis.
• N EN rJlmm the density of entero-endocrine cells
• N s ⁇ lmm 2 the density of serotonin- positive cells.
• L-Ala-L-GIu-L-Asp-Gly tetrapeptide properties revealed in the pre-clinical experiment provide its manifested prophylactic and/or therapeutic application as a geroprotective substance.
• Example 9 Efficacy of the application of the pharmacological substance containing L-Ala-L-Glu-L-Asp-Gly tetrapeptide in patients with disturbances of the anti- oxidation defence system for premature ageing prevention.
• the studied pharmacological substance was applied in 14 patients (34 to 69 years old) with age-related pathology (hypertension disease, ischemic heart disease, chronic gastritis, non-insulin-dependent diabetes mellitus) and reduction of the anti-oxidation defence indices in blood.
• age-related pathology hypertension disease, ischemic heart disease, chronic gastritis, non-insulin-dependent diabetes mellitus
• the substance containing L-Ala-L-Glu-L-Asp-Gly tetrapeptide was administered intramuscularly in the form of an injection solution in a single daily dose lO ⁇ g per an injection for 10 days.
• Results of the therapy prove the restorative effect of the pharmacological substance containing L-Ala-L-Glu-L-Asp-Gly tetrapeptide on the anti-oxidation defence system of the organism.
• N-acetyl-5-methoxykynurenamine N-AMK
• N-AMK an analogue of acetylsalicylic acid in its chemical structure
• the substance was administered intramuscularly in the form of an injection solution daily in the morning in dose lO ⁇ g for 10 days.
• the patients were treated in the phase of fading exacerbation or disease remission accompanied by permanent anti-asthma therapy, which included inhalation and oral glucocorticoids.
• Clinical effect produced by the preparation was estimated immediately after the treatment and within the next 7 months on the basis of analyses reported by the patients, which included the data on their health state dynamics, capacity for work, psychological condition, sleep impairments, and daily doses of the anti-asthmatic drugs taken.
• VBC vital breathing capacity
• TV tidal volume
• TLC total lung capacity
• FVBC forced vital breathing capacity
• VexhR maximum volumetric exhalation rate
• VBC 50 and VBC 75 forced exhalation volume per second
• 6-sulfatoxymelatonin the basic melatonin metabolite
• Harman D. Free radical theory of ageing effect of free radical reaction inhibitors on the mortality rate of male LAF, mice // J. Gerontol. - 1968. - Vol. 23. - P. 476.

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