WO2001010422A9 - Use of hepoxilins or hepoxilin analogs as antidiabetics, antiinframmatory agents - Google Patents
Use of hepoxilins or hepoxilin analogs as antidiabetics, antiinframmatory agentsInfo
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- WO2001010422A9 WO2001010422A9 PCT/CA2000/000896 CA0000896W WO0110422A9 WO 2001010422 A9 WO2001010422 A9 WO 2001010422A9 CA 0000896 W CA0000896 W CA 0000896W WO 0110422 A9 WO0110422 A9 WO 0110422A9
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- alkene
- lower alkyl
- eicosa
- mammal
- hydroxy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
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- 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
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- 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
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
Definitions
- the present invention relates to therapeutic methods and pharmaceutical compositions employing native hepoxilins and hepoxilin analogs.
- Hepoxilins are biologically active hydroxy epoxide derivatives of arachidonic acid formed through the 12-lipoxygenase pathway (Pace-Asciak et al. (1983;, J. Biol. Chem., v. 258, pp. 6835-6840; Pace-Asciak et al. (1983), Prostaglandins, v. 25, pp. 79-84; Pace-Asciak et al. (1984), Biochem. Biophys. Ada, v. 793, pp. 485-488). They are formed from 12-HPETE, an unstable hydroperoxide derivative of arachidonic acid (Pace-Asciak et al. (1983), Adv.
- hepoxilin A 3 [8(S,R)-hydroxy- 11 (S),12(S)-epoxy- eicosa-5Z, 9E, 14Z-trienoic acid] and hepoxilin B 3 [IO(S,R)-hydroxy- 1 1 (S),12(S)-epoxy-eicosa-5Z, 8Z, 14Z-trienoic acid].
- Hepoxilins have been demonstrated to raise intracellular calcium in human neutrophils in vitro. Hepoxilins have also been demonstrated to be involved in the in vitro release of insulin by pancreatic islet cells (Pace-Asciak et al. (1986), Progr. Lipid Res., v. 25, pp. 625-628), although nothing has been published on the action of hepoxilin analogs on such cells.
- U.S. Patent No. 5,616,607 discloses hepoxilin analogs found to antagonize hepoxilin activity and stated to be useful in the modulation of hepoxilin-mediated processes such as inflammation and in the modulation of other processes mediated by cellular calcium levels. It is suggested that hepoxilin analogs may have utility in diabetes. However, the in vivo activity of hepoxilin analogs has never been demonstrated nor has an increased in vivo efficacy of any hepoxilin analogs been demonstrated with respect to either insulin secretion or the inflammatory process.
- hepoxilin analogs for use in the regulation of insulin secretion, for the treatment and prevention of both acute and chronic inflammation in a mammal and for decreasing/preventing lung fibrosis in vivo.
- HX Native hepoxilin
- a method for regulating insulin secretion in a mammal comprising administering an effective amount of a native hepoxilin or hepoxilin analog to said mammal.
- a method for changing the plasma insulin levels in a mammal comprising administering an effective amount of a native hepoxilin or hepoxilin analog to said mammal.
- a method for altering plasma glucose levels in a mammal the method comprising administering an effective amount of a native hepoxilin or hepoxilin analog to said mammal.
- the hepoxilin analogs of the present invention also have use in various types of binding assays to identify hepoxilin binding proteins and to identify other forms of such binding proteins as exist for example in diabetes and as such provide a means for diagnosing diabetes in the prediabetic stage.
- the hepoxilin analogs may also be used in various in vitro assays to study second messenger downstream effects after receptor binding.
- hepoxilin analog photoligands for use in binding studies, most preferably for use in binding studies identifying the hepoxilin binding protein. It is understood by one skilled in the art, that the hepoxilin analogs of the present invention can be chemically modified, for example radioactively labelled as required, for various uses in different assay systems.
- hepoxilin binding proteins have been demonstrated to be G protein coupled as the binding of hepoxilin as well as calcium release is inhibited by non-hydrolyzable analogs of GTP. Additionally, hepoxilin binding causes a rise in cyclic AMP formation indicating that binding is coupled to a Gs-protein.
- hepoxilin analogs to increase cAMP formation. Hepoxilin and its analogs can be used to elicit second messenger systems via binding to the hepoxilin binding protein.
- a further embodiment of the invention is a method for treating an inflammatory disorder in a animal comprising administering to the mammal in need of such treatment a therapeutically effective amount of a hepoxilin analog.
- compositions for treating an inflammatory disorder in a mammal comprising a therapeutically effective amount of a hepoxilin analog and a pharmaceutically acceptable carrier.
- a method for treating in a mammal a disorder which involves the development of lung fibrosis comprising administering to the mammal in need of such treatment a therapeutically effective amount of a hepoxilin analog.
- compositions for treating a disorder in a mammal which involves the development of lung fibrosis comprising a therapeutically effective amount of a hepoxilin analog and a pharmaceutically acceptable carrier.
- a further embodiment of the invention is a method for treating conditions where an anti-coagulative effect is desired, in a mammal, the method comprising administering to the mammal in need of such treatment a therapeutically effective amount of native hepoxilin or a hepoxilin analog.
- a method for modulating vascular tone in a mammal the method comprising administering to the mammal in need of such treatment a therapeutically effective amount of native hepoxilin or a hepoxilin analog.
- According to another embodiment of the present invention is a method for decreasing vascular permeability in a mammal, the method comprising administering to the mammal in need of such treatment a therapeutically effective amount of native hepoxilin or a hepoxilin analog.
- a native hepoxilin or a hepoxilin analog for decreasing the vascular side effects of chemotherapeutic agents.
- According to another embodiment of the invention is a method for reducing or preventing side effects of treatment for malignancy in a mammal wherein the mammal is concurrently receiving a chemotherapeutic agent, the method comprising administering to the mammal in need of such treatment a therapeutically effective amount of a hepoxilin analog and a pharmaceutically acceptable carrier.
- hepoxilin or hepoxilin analog for decreasing collagen synthesis associated with tissue fibrosis.
- Figure 1 shows changes in plasma insulin and plasma glucose concentrations after bolus i.a. administration of glucose (50mg/100 ⁇ l) or ethanol (10 ⁇ l), the vehicle for hepoxilin (HX) analogs, in the fed rat.
- the injection of the test compounds was carried out in a Y -cannula attached to the carotid artery through which the test compound in 10 ⁇ l ethanol was injected while 300 ⁇ l saline was being injected. Hence the test compound was 'carried' into the circulation by the saline. The total time of injection was 5 sec.
- Figure 2 shows changes in plasma insulin concentration produced by each of the four HX analogs, H1-H4, at 100 ⁇ g /10 ⁇ l ethanol (i.a. details as in Figure 1 ).
- Figure 3 shows changes in plasma glucose concentrations after i.a. administration of each of the four HX analogs as in Figure 2.
- Figure 4 shows changes in plasma insulin concentration produced by each of the four native HXs, at 100 ⁇ g /10 ⁇ l ethanol (i.a. details as in Figure 1 ).
- Figure 5 shows changes in plasma glucose concentrations after i.a. administration of each of the four native HXs as in Figure 4.
- Figure 6 shows a comparison of Kd and Bmax for the binding of tritiated HX to neutrophil membranes from human normals, Type 1 and Type 2 diabetic subjects.
- Figure 7 shows 1 D-PAGE analysis of the neutrophil HX binding protein from normal and Type 1 diabetic subjects after u.v. cross-linking with radioiodinated hepoxilin photoligand.
- Figure 8 shows 2D-PAGE analysis of the neutrophil binding protein from normal subjects showing the effect of addition of unlabeled HX. Note the single discreet radioactive 'spot'.
- Figure 9 shows 2D-PAGE analysis comparing the normal binding protein pattern with the protein pattern (isoforms) in Type 1 diabetic subjects.
- Figure 10 shows a comparison between the normal and Type 1 diabetic neutrophils in their response to a rise in free calcium caused by different amounts of native HX.
- Figure 11 shows diabetes onset (glucosuria) in NOD mice treated with cyclophosphamide i.p. and the effect of HX analogs (H1-H4) and Indomethacin administered before and every second day after cyclophosphamide injection.
- HX analogs H1-H4
- Indomethacin administered before and every second day after cyclophosphamide injection.
- Each group was subdivided into subgroups of 5 and 6 mice and each subgroup was injected with test compounds on alternate days. All test compound-treated animals from both subgroups behaved in the same way, and the data are combined as shown.
- Figure 12 shows the insulitis scores of the experiment in Figure 11.
- the pancreas from the experiment in Figure 11 was fixed and stained (H&E).
- H&E histoneum
- Four sections were cut from each specimen and over 100 islets were observed for each group and scored for the degree of insulitis. Only islets from the non-diabetic animals were scored, as pancreata from diabetic animals would be devoid of islets.
- the graph shows the % insulitis for each group.
- Figure 13 shows bleomycin-induced lung injury mouse model. Left panel: saline control (no bleomycin). Right panel: bleomycin and saline.
- Figure 15 shows the protective actions of HX analogs in a bleomycin- induced lung injury mouse model.
- Upper left panel saline control (no bleomycin); upper right panel: (bleomycin + saline);middle left panel: (bleomycin + H1 in saline); middle right panel: (bleomycin + H2 in saline); lower left panel: (bleomycin + H3 in saline); lower right panel: (bleomycin + H4 in saline). All compounds (except bleomycin which was injected at the start only) were injected i.p. daily for 8 days. At the end of the study, lungs were inflated, perfused and fixed for H&E staining.
- Figure 16 shows collagen staining in a bleomycin-induced lung injury mouse model.
- Left panel is a saline control and right panel is bleomycin.
- Figure 17 shows collagen staining in a bleomycin-induced lung injury mouse model at various concentrations of H1 (HXA-1 ).
- Upper left panel saline control (no bleomycin); upper right panel: (bleomycin + saline); middle left panel: (bleomycin + 400 ⁇ g/kg H1 in saline); middle right panel: (bleomycin + 2mg/kg H1 in saline); and, lower left panel: (bleomycin + 4mg/kg H1 in saline).
- Figure 18 shows the actions of HX analogs H1 to H4 (HXA-1 to HXA-4) on collagen synthesis by collagen staining in a bleomycin-induced lung injury mouse model.
- Upper left panel saline control (no bleomycin); upper right panel: (bleomycin + saline); middle left panel: (bleomycin + H1 in saline); middle right panel: (bleomycin + H2 in saline); lower left panel: (bleomycin + H3 in saline); and lower right panel: (bleomycin + H4 in saline).
- Figure 21 shows the inhibition of the hepoxilin A 3 -induced rise in intracellular calcium and binding of tritiated native hepoxilin A 3 ([ 3 H]-HxA 3 (8S) in neutrophils into which cytosolic GTP- ⁇ -S was incorporated through brief hypertonic/hypotonic shock (see Methods for details).
- Neutrophils (2 x 10 6 cells/determination) were used in 1 mL calcium-free buffer at 37°C with continuous stirring. Control experiments (not shown) with Lucifer yellow indicated that while no dye was incorporated in normal cells, the brief hypertonic/hypotonic shock procedure led to the internalization of dye equivalent to an approximate cytosolic concentration of GTP- ⁇ -S (or GTP) of 21 ⁇ M.
- Figure 22 shows the changes in the cellular content of cAMP (left panel) and cGMP (right panel) evoked by different concentrations of HxA 3 (8S). The effect of 10 ⁇ M Forskolin is shown (middle panel).
- the present invention provides methods and pharmaceutical compositions for treating a number of disorders. These methods and compositions employ native hepoxilins or hepoxilin analogs.
- Hepoxilin analogs which may be used in the methods of the invention comprise compounds of the formula
- alkyl means a branched or unbranched alkyl radical.
- Lower alkyl or alkene means C1 to C22 alkyl or alkene.
- Native hepoxilins and the hepoxilin analogs described herein have been shown to stimulate insulin release and to increase blood insulin levels m vivo in mammals. These compounds therefore provide a new method and new pharmaceutical compositions for regulating insulin secretion and glycemia in diabetes especially in Type II diabetes.
- the hepoxilin analogs described herein have also been shown to delay the onset of diabetes in NOD mice injected with cyclophosphamide and provide a means of delaying or preventing the development of diabetes in susceptible subjects.
- a specific hepoxilin-binding protein has also been demonstrated in normal human neutrophils and has been resolved on 2D-PAGE to show one main protein band using an HX photoaffinity radioligand.
- the hepoxilin-binding protein from Type 1 and Type II diabetic subjects was resolved into at least three isoforms on 2D-PAGE.
- the present study demonstrates that intact human neutrophils from diabetic subjects are more responsive to a dose regimen of native HX than those from normal subjects in terms of calcium release in vitro. It appears that the hepoxilin analogs herein disclosed can be used in various assay systems to identify and characterize the hepoxilin receptor in both normal and diabetic patients.
- a diagnostic test for pre-diabetes may therefore by carried out by examining the neutrophil HX-binding protein by 2D-PAGE, with detection using an HX photo affinity radioligand, as described in the examples herein.
- the lung reacts to a number of insults by undergoing fibrosis, which impairs lung function and leads to morbidity and eventually, in severe cases, death.
- Lung fibrosis can occur, for example, as a result of bacterial infections or shock or as a result of hyperoxygenation when patients breathe pure oxygen or oxygen-enriched air. Lung fibrosis is also a frequent and serious side-effect of cancer chemotherapy, for example, with the drug bleomycin.
- Bleomycin-induced lung injury provides an accepted animal model for lung fibrosis. It has been shown that treatment with the hepoxilin analogs described herein provided protection against lung fibrosis in this model, with reduced pulmonary fibrosis and edema, reduced collagen synthesis, reduced vascular permeability and reduced inflammatory response.
- one or more hepoxilin analogs may be administered to a mammal in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art.
- the compositions of the invention may be administered orally or parentally, the latter route including intravenous and subcutaneous administration. Parenteral administration may be by continuous infusion over a selected period of time.
- Forms for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.
- the hepoxilin analog may be orally administered with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, compressed into tablets or incorporated directly with the food of the diet.
- a hepoxilin analog may be incorporated with excipient and used in the form in ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers and the like.
- Compositions containing one or more hepoxilin analogs of the present invention can also be administered in a solution or emulsion contained within phospholipid vesicles called liposomes.
- the liposomes may be unilamellar or multilamellar and are formed of constituents selected from phosphatidylcholine, dipalnmitoylphosphatidylcholine, cholesterol, phosphatidylethanolamtine, phosphatidylserine. dimyristoylphosphatidylcholine and combinations thereof.
- the multilamellar liposomes comprise multilamellar vesicles of similar composition to unilamellar vesicles, but are prepared so as to result in a plurality of compartments in which the analogs containing solution or emulsion is entrapped. Additionally, other adjuvants and modifiers may be included in the liposomal formulation such as polyethyleneglycol, or other materials.
- the liposomes containing the hepoxilin analog compositions may also have modifications such as having antibodies immobilized on the surface of the liposome in order to target their delivery.
- the composition may be administered to any living organism in need of such treatment including humans and animals as the composition has efficacy in vivo.
- safe and effective as used herein, is meant providing sufficient potency in order to decrease, prevent, ameliorate or treat the disease affecting the subject while avoiding serious side effects.
- a safe and effective amount will vary depending on the age of the subject, the physical condition of the subject being treated, the severity of the disorder, the duration of treatment and the nature of any concurrent therapy, and its determination is within the skill of the ordinary physician.
- a therapeutically active amount of a pharmaceutical composition of the present invention means an amount effective, at dosages and for periods of time necessary to achieve the desired result. This may also vary according to factors such as the disease state, age, sex, and weight of the subject and the ability of the hepoxilin analog to elicit a desired response in the subject. Dosage procedures may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
- pharmaceutically acceptable carrier as used herein is meant one or more compatible solid or liquid delivery systems.
- Some examples include but are not limited to starches, sugars, cellulose and its derivatives, powdered tragacanth, malt, gelatin, collagen, talc, stearic acids, magnesium stearate, calcium sulfate, vegetable oils, polyols, agar, alginic acids, pyrogen free water, isotonic saline, phosphate buffer, and other suitable non-toxic substances used in pharmaceutical formulations.
- Other excipients such as wetting agents and lubricants, tableting agents, stabilizers, anti-oxidants and preservatives are also contemplated.
- compositions described herein can be prepared by known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the hepoxilin analog or analogs is combined in a mixture with a pharmaceutically acceptable carrier.
- Suitable carriers are described for example in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton, PA, USA, 1985).
- the compositions include, albeit not exclusively, solutions of the hepoxilin analog(s) in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
- Hepoxilin analogs may be prepared as described, for example, in U.S. P. 5,616,607.
- hepoxilin analogs may have use in the general activation of G proteins and increasing cAMP levels. This may have a general significance where such activation is shown to have a desired physiological effect such as in platelet aggregation.
- native hepoxilin and hepoxilin analogs may have clinical relevance with respect to anti-coagulation and therefore in various disease states associated with the cardiovascular system for example where it would be beneficial to prevent/treat blood clots in vessels.
- Both native hepoxilin and hepoxilin analog action on second messenger systems may also be significant with respect to use for modulating vascular tone.
- native hepoxilin and hepoxilin analogs also have clinical relevance with respect to therapeutic treatment in cancer and treatment-associated lung disease, preventing the serious vascular side effects of chemotherapeutic agents such as bleomycin for example.
- Example 1 Hepoxilin analogs cause a rise in plasma insulin in the Inactin- anaesthetised rat after i.a. administration in the fed but not the fasted rat Rats were divided into groups of 5, and after Inactin anaesthesia, tracheotomy was performed. The animals were heparinized.
- the carotid artery was cannulated for sample injection (glucose at 50 mg/100 ⁇ l saline and test compounds (100 ⁇ g) as a bolus in 10 ⁇ l ethanol injected during flushing of the cannula with 300 ⁇ l saline) and blood sampling.
- sample injection glucose at 50 mg/100 ⁇ l saline and test compounds (100 ⁇ g) as a bolus in 10 ⁇ l ethanol injected during flushing of the cannula with 300 ⁇ l saline
- Each animal was subjected to blood sampling at the following time points, i.e. -10 and -5 minutes, 30 sec after injection of test compound and 2, 5, 10, 30 and 60 min. Blood samples at the end of the experiment were centrifuged and the serum was transferred to plastic tubes and analyzed for both insulin and glucose. Insulin was measured by RIA.
- hepoxilin analogs H1 , H2, H3, H4; natural hepoxilins: HxA 3 -8S, HxA 3 -8R, HxB 3 -10S, HxB 3 -10R.
- Glucose was tested at a concentration of 50 mg/1 OO ⁇ l injection which yields a blood level of approx. 13 mM, and ethanol vehicle (10 ⁇ l) was also tested in a set of animals. All hepoxilins were tested at 100 ⁇ g/200g rat body in weight in 10 ⁇ l ethanol and added through a Y injector into a 300 ⁇ l stream of heparinized saline. The sample injection took 5 sec.
- Figures 4 and 5 compare the effect of the 4 native hepoxilins (A3 (8S and 8R) and B 3 (I0S and 10R)) on plasma insulin and glucose respectively at the same doses. All scales were maintained the same for easy comparison with the exception of the experiments where glucose was administered. Again changes in plasma insulin levels but not in glucose levels, were seen with the four native hepoxilins.
- Example 2 A specific Hepoxilin-binding protein in human neutrophils exists which has been resolved on 2D-PAGE into one main protein band using an HX photoaffinitv radioligand.
- FIG. 7 shows gel patterns comparing diabetic and normal hepoxilin binding in human neutrophils. A protein band around 60 - 70 kD is seen which is competitively antagonized by unlabeled hepoxilin showing specificity of binding. This band is diffuse in the Type 1 diabetic subject while that from the normal is quite distinct. This confirms earlier findings that the diabetic binding protein is possibly made up of isoforms or that the protein is modified. These studies have diagnostic potential in the pre-diabetic stage.
- Figure 8 is shown a 2D PAGE for the normal protein; four panels show ⁇ HxA 3 for both Radioactivity (top panels) and Coomassie Blue (lower panels) staining. It is clear that the ⁇ 60 kD protein observed in the 1 D PAGE is a distinct band with a pi around pH6. The resolution of the proteins is good enough to permit initial testing for 'purity' and to prepare tryptic digests to determine composition of the peptides as well as to investigate the localisation of the radioligand to specific peptides providing information on the binding sites.
- Example 3 The Hepoxilin-binding protein from Type 1 diabetic subjects is resolved into at least three bands on 2D-PAGE'
- Example 4 Human neutrophils from diabetic subjects are more responsive to a dose regimen of native Hepoxilin than those from normal subjects in terms of calcium release in vitro. It was previously reported that the native HX cause a rise in intracellular calcium in suspensions of human neutrophils. This rise is unaffected by extracellular medium devoid of calcium; hence HX causes a release of calcium from intracellular stores. Additionally it was shown that if HX is added a second time (5 min later) to the neutrophils, the second calcium response is inversely related to the rise elicited by the first challenge, suggesting depletion of calcium stores. Earlier studies also demonstrated that the calcium-release step was dependent on hepoxilin binding to its intracellular receptor.
- mice (20g body weight 6-8 wks old) were divided at random into 6 groups (11 /group); all were administered cyclophosphamide (250mg/kg) i.p. in a single time. Each group was further subdivided into 5 and 6 mice which were injected on separate days. Saline was given i.p. to group 1; indomethacin to group 2; the HX analogs to groups 3-6. HX analogs and the indomethacin were administered i.p one day before cyclophosphamide and subsequently every second day for 2 weeks at the dose of 50 ⁇ g/mouse (2500 ⁇ g/kg). After the first week, urine was tested for glucosuria with test strips.
- the animal was rendered diabetic if it tested positively for glucose in urine two days in a row. Diabetes onset is shown in Figure 11. These results clearly show a difference in the rate of onset in the HX analog-treated groups relative to the saline-treated (cyclophosphamide-treated) groups. Histological examination of the pancreatic and splenic tissue in the animals confirmed that the severity of the disease (insulitis) was lessened in the HX analog-treated groups (attached 2 necropsy reports- pancreas and spleen).
- Cyclophosphamide administration causes splenic white pulp atrophy; this is observed in the saline-cyclophosphamide group while it appears to be 'milder' in the HX-analog-cyclophosphamide treated groups.
- Figure 12 shows the results of these scores.
- mice were divided into 7 groups, each containing 5 animals. All groups were administered i.p. saline +7% ethanol as vehicle. Group 1 received only vehicle. Groups 2-7 received intratracheal bleomycin. Bleomycin is an antibiotic having antineoplastic activity. It is used clinically to treat malignancies such as lymphoma and testicular cancer. However, it has several side effects such as causing lung fibrosis characterized by diffuse alveolar hemorrhage and marked accumulation of inflammatory cells and an increase in collagen synthesis ( Figure 13).
- Group 3 received Trolox antioxidant
- Group 4 received H1
- Group 5 received H2
- Group 6 received H3
- Group 7 received H4.
- All hepoxilins were dissolved in 7 ⁇ l ethanol and diluted to 100 ⁇ l saline for i.p. administration. All compounds were administered daily at the dose of 100 ⁇ g/20g (5000 ⁇ g/kg) mouse for 8 days. Lungs from all animals were perfused and fixed for histology.
- H1 Figure 14
- H2 hepoxilin analogs H1 ( Figure 14) and H2 (data not shown) were administered in various concentrations. These studies indicated that hepoxilin analogs inhibited with different potencies the bleomycin-evoked lung fibrosis with H1 and 2 being most potent. H1 activity was dose dependent with a threshold activity at 400 ⁇ g/kg, i.p. H1 was more potent than Amifostine (200 mg/kg) or Trolox (30 mg/kg).
- H1 and 2 blocked the bleomycin (12 ⁇ g) - evoked vascular permeability at 100 ng intradermally.
- Figures 19, 20 and 21 vascular permeability was measured using Evans Blue I.V. Hepoxilin analogs were intradermally applied and within 30 minutes, a formamide extraction of punched skin was done. Extraction and absorbance was read at 620 nm.
- the cells were hypotonically shocked during 2 min at 37°C by the addition of 1 mL of hypotonic medium made up of 1 part normal medium and 0.66 parts of water [Hallett & Campbell. 1983, Immunology, 50, 487-495]. The cells were then centrifuged at 900 rpm x 5 min and resuspended in 2 mL of calcium-free assay medium for the binding studies or in RPMI 1640 for the calcium measurements. The extent of permeabilization was approximated in 4 x 10 6 cells using Lucifer yellow [Jaconi et al 1993, J. Biol. Chem. 268, 26075-26078] at a concentration of 1 mg/mL in the permeabilizing medium.
- Binding studies The binding assay was conducted in a clear medium (composition in mM: NaC1 140, KCI 5, MgC1 2 1 , CaCI 2 l, HEPES sodium-free 10, and glucose 10, pH 7.3) containing the neutrophil suspension (2 x 10 6 cells/tube) and [ 3 H 6 ]- HXA 3 (8S) (50,000 cpm) in the presence or absence of 1 ⁇ g of unlabeled HxA 3 (8S) (in 2 ⁇ l DMS0) to determine the total and non-specific binding. All binding experiments were carried out in 1 mL at 37°C for 60 min and performed in duplicates.
- the reaction was started by the addition of the cells in the medium containing the radioactive ligand (and the unlabeled ligand where specified).
- the binding reactions were terminated after the appropriate incubation times by isolation of the ligand-receptor complexes through vacuum filtration through Whatman GF/B glass fiber filters (Maidstone, England) prewashed with the clear medium.
- the tubes and the filters were washed with 3 x 3 mL of ice-cold medium.
- the radioactivity retained on the filters was counted in 10 mL of Ecolite scintillation fluid (ICN, St Laurent, Quebec) in a Beckman (Model LS 3800) liquid scintillation counter.
- Neutrophil membranes equivalent to 2 x 106 cells / tube were prepared by sonication (Model XL 2020, Sonicator Ultrasonic Processor, Heat Systems Inc., Farmingdale, NY) of the cells and centrifugation.
- Intracellular free calcium concentrations were monitored continuously in a Perkin-Elmer fluorescence spectrophotometer (Model 65040) using Indo-I AM-loaded neutrophils. Excitation wavelength was set at 331 nm, emission wavelength at 410 nm., with slits of excitation and emission set at 3 and 15 nm respectively.
- Neutrophil suspensions (10 7 cells) (normal or shocked cells) were loaded in RPMI 1640 medium with 3 ⁇ L of 1 mM (final concentration 3 ⁇ M) of the acetoxymethyl ester precursor of Indo-I for 30 min at 37°C. Unloaded dye was removed by centrifugafion and the cells were resuspended in fresh RPM1 1640(1 mL).
- Dye-loaded cells were kept at room temperature on a rotator (RotoTorque, Cole-Palmer model 7637, USA) turning at about 10- 15 rotations/min.
- Typical measurements involved 2 x 10 6 cells in 1 mL of calcium-free assay medium (composition in mM: NaC1 140, KCI 5, MgCI 2 1 , Hcpcs sodium-free 10, glucose 10, and EGTA 1, pH 7.3) in a temperature controlled plastic cuvette (Diamed Labs., Canada) at 37°C with constant stirring.
- Human neutrophils (1 x 10 7 cells) in 1 ⁇ L of assay buffer were preincubated with/without Forskolin (10 ⁇ M) during 10 min at 37°.
- DMSO (1 ⁇ L) alone (control) or containing various concentrations of HxA 3 (10 "9 to 10 "6 M final concentration) was added and the cells were incubated for a further 5 min.
- the reaction was stopped by the addition of 12% TCA and the samples were sonicated (4 x 3 sec). The samples were left on ice for 60 min to extract cAMP and cGMP. After centrifugation at 2500g for 15 min, the supernatants were transferred and extracted with 5 x 5 mL water-saturated diethyl ether to remove the TCA.
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP00952812A EP1207887A2 (en) | 1999-08-04 | 2000-08-03 | Use of hepoxilins or hepoxilin analogs as antidiabetic, antiinflammatory agents |
US10/048,863 US6673785B1 (en) | 1999-08-04 | 2000-08-03 | Use of hepoxilins or hepoxilin analogs as antidiabetics, antiinflammatory agents |
AU65508/00A AU6550800A (en) | 1999-08-04 | 2000-08-03 | Hepoxilin analogs |
CA2393904A CA2393904C (en) | 1999-08-04 | 2000-08-03 | Hepoxilin analogs |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14714399P | 1999-08-04 | 1999-08-04 | |
US60/147,143 | 1999-08-04 | ||
US20830400P | 2000-06-01 | 2000-06-01 | |
US60/208,304 | 2000-06-01 |
Publications (3)
Publication Number | Publication Date |
---|---|
WO2001010422A2 WO2001010422A2 (en) | 2001-02-15 |
WO2001010422A3 WO2001010422A3 (en) | 2001-08-23 |
WO2001010422A9 true WO2001010422A9 (en) | 2002-08-29 |
Family
ID=26844634
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CA2000/000896 WO2001010422A2 (en) | 1999-08-04 | 2000-08-03 | Use of hepoxilins or hepoxilin analogs as antidiabetics, antiinframmatory agents |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1207887A2 (en) |
AU (1) | AU6550800A (en) |
CA (1) | CA2393904C (en) |
WO (1) | WO2001010422A2 (en) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2002218084A1 (en) * | 2000-11-09 | 2002-05-21 | The Hospital For Sick Children | Inhibitors of thromboxane formation and action |
AU2003229202A1 (en) | 2002-05-28 | 2003-12-12 | The Hospital For Sick Children | Compositions comprising hepoxilin analogs and their use in the treatment of cancer |
WO2004104022A2 (en) * | 2003-05-16 | 2004-12-02 | The General Hospital Corporation | Compositions comprising pathogen elicited epithelial chemoattractant (eicosanoid hepoxilin a3), inhibitors thereof and methods of use thereof |
WO2006106438A2 (en) * | 2005-01-11 | 2006-10-12 | Evolva Sa | Modulators of ppar activity |
WO2007118335A1 (en) | 2006-04-19 | 2007-10-25 | Evolva S.A. | Hepoxilin analog enantiomers |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE168372T1 (en) * | 1993-03-29 | 1998-08-15 | Hsc Res Dev Lp | HEPOXILIN ANALOGUE USABLE AS ANTI-INFLAMMATORY AGENTS |
KR100325581B1 (en) * | 1998-08-07 | 2002-08-24 | 오우택 | Analgesic compositions containing substances that result from lipoxygenase metabolism of arachidonic acid |
-
2000
- 2000-08-03 AU AU65508/00A patent/AU6550800A/en not_active Abandoned
- 2000-08-03 CA CA2393904A patent/CA2393904C/en not_active Expired - Lifetime
- 2000-08-03 WO PCT/CA2000/000896 patent/WO2001010422A2/en not_active Application Discontinuation
- 2000-08-03 EP EP00952812A patent/EP1207887A2/en not_active Withdrawn
Also Published As
Publication number | Publication date |
---|---|
EP1207887A2 (en) | 2002-05-29 |
CA2393904A1 (en) | 2001-02-15 |
AU6550800A (en) | 2001-03-05 |
WO2001010422A2 (en) | 2001-02-15 |
CA2393904C (en) | 2012-10-16 |
WO2001010422A3 (en) | 2001-08-23 |
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