Patent Application: US-201314433734-A

Abstract:
the invention involves the preparation of cholesterol lecithin nanosomes , without propylene glycol , from the complex formed by quercetin and 2 - hydroxypropyl - β - cyclodextrin , by means of a process that allows the safe , effective intravenous use thereof in the treatment of cerebral pathological conditions in adults and newborns . the preparation is safe , stabilizes the altered haemodynamic parameters in severe neonatal hypoxia in newborn pigs and is effective in protecting cerebral function in experimental parkinson &# 39 ; s disease models and in newborn pigs subject to hypoxia .

Description:
the set of results obtained by the group of innovators in experimental studies in vitro confirmed the antioxidant and neuroprotective role of quercetin . [ 20 - 23 ]. however , in aqueous preparations , quercetin is not detected in the brain due to its low bioavailability . for the purpose of providing a vehicle to quercetin to facilitate its access to the brain , the inventors formulated in first instance liposomes containing only a mixture of lecithin with quercetin . cerebral protection was then demonstrated experimentally in a model of ischemia in the rat , when the liposomes were injected intraperitoneally ( 22 ). liposomes have been widely used in the pharmaceutical industry in a variety of clinical applications to transport and deliver a wide range of active ingredients [ 50 , 51 ]. it should be noted that , for use in conditions such as stroke or asphyxia of the newborn , the preparation of quercetin should be administered acutely by the intravenous route . in research conducted by the inventors , they were able to show that the preparation of liposomes of lecithin and quercetin that had been beneficial effects administered intraperitoneally in rats ( 27 ), produced significant adverse effects when administered intravenously in pigs . this is evident when the effect on the pressure of the pulmonary arterial is observed in fig1 , where an increase during the intravenous administration of a preparation of liposomes with quercetin under baseline conditions is analyzed ( arrow ). since there are no formulations for intravenous use , a formulation of a nanosomal carrier was first obtained with the addition of cholesterol to improve solubility . quercetin crystals decreased as it can be observed in the in the microphotographs of phase contrast microscope as shown in a and b in fig2 , where the crystals present in a , disappear in b . to improve bioavailability 2 - hydroxypropyl - cyclodextrin ( hpbcd ) was added to the nanosome preparation . in our case the hpbcd is used since it improves solubility and is non - toxic in several animal models and in humans , including children [ 32 ]. it has been reported that quercetin complexes with hpbcd resulting in a molecular encapsulation that has even been postulated for cerebral use in previous studies ( chinese patent documents cn101301477b and cn101301477a ). this innovation differs from these previous ones in the fact that beyond obtaining the quercetin / hpbcd complex it was encapsulated in a lecithin / cholesterol nanosome . the novelty regarding the effects on cerebral protection is that the complex quercetin / hpbcd achieves synergistic neuroprotective action , an aspect shown by us in the protection model in experimental parkinson and not mentioned in the chinese patents listed above . this innovation differs from the state of the art in the field in that it does not use polyethylene glycol ( peg ) in its formulation . quercetin and peg - liposomes given orally have been studied in mice with solid tumors [ 28 , 29 ] and anxiety in adult rats [ 1 ]. in the patent application cn102058536a of may 2011 the combination of phospholipid / cholesterol with cyclodextrins ( cds ) and peg was used in order to obtain greater stability and solubility of liposomes . studies conducted by the inventors including peg in the nanosomes showed lower size stability of nanosomes as assessed with or without peg in the preparation , when the size is determined by polydispersity index up to two months after its preparation ( fig3 ). furthermore it has been shown that the general use of glycols in solubilizing drugs is associated with adverse effects such as phlebitis , pain and hemolysis . particularly in the pediatric and neonatal age ( under 4 years ) and in patients with impaired alcohol deshldrogenase enzyme or low glomerular filtration or liver disease or pregnancy , they are prone to accumulation of glycols increasing therefore the risk of toxicity . in the neonatal period , no risk assessment of peg has been done and its use is not recommended , for example by esnee ( european study for neonatal exposure excipients ). because of these reasons peg is not included in the formulation proposed in this patent , making a distinct difference with available nanosomal preparations , and making also a difference in reference to obviousness . against this background it can be concluded that though liposomal preparations of quercetin have already been used for neuron recovery , the present preparation differs from the state of art in the size (& lt ; 200 nm ) of the particles , in which it does not include peg in the formulation and in that the active molecule is not only the quercetin molecule , but the quercetin / hpbcd complex . besides , the resulting preparation has adequate hemodynamic tolerance for intravenous administration . although it may seem obvious to include hpbcd in a liposome , from the data available it should be noted that the inventors obtained hpbcd experimental evidence showing that it is necessary for the safety of intravenous administration of the formulation ( fig4 ). the present invention also relates to a method of preparing an injectable formulation for intravenous administration comprising nanosomes of lecithin / cholesterol complex with quercetin or other flavone or flavonol or its alkyl and / or sulfur derivatives with 2 - hydroxypropyl - - cyclodextrin , prepared according to the following steps : i — preparation of the complex mixing 2 - hydroxypropyl - β - cyclodextrin and quercetin or other flavonol or flavone on a ratio of quercetin or other flavonol or flavone to 2 - hydroxypropyl - β - cyclodextrin of 1 : 20 to 1 : 50 times by a period of time of 12 to 90 hours , in a medium containing ethanol with continuous stirring in sterile conditions . i — inclusion of complex obtained in step i into a nanosome of lecithin / cholesterol in a ratio of 5 to 10 respectively , with sonication for a period of 5 min to 2 hours . iii — the preparation obtained in step ii is mixed with the complex obtained in step i in a ratio of flavonoid / cholesterol of 0 . 5 to 2 times . iv — the formulation obtained in iii is injected in a saline solution at a temperature between 60 and 100 degrees celsius at a rate of 5 to 30 ml / h to result in the formation of nanosomes . for the preparation of lipid nanosomes cover cholesterol plus lecithin are used . for incorporation of quercetin / hpbcd in the bilayer of phosphatidylcholine and cholesterol , 0 . 5 to 5 ml of ethanol solution are mixed with 30 - 95 % quercetin and hp - β - cd ( ratio 1 - 20 to 50 times ). this solution is maintained in a laminar flow chamber with magnetic stirring over 48 h to form complexes between the components . subsequently cholesterol and phosphatidylcholine are added to this mixture and saline , ph between 6 . 2 and 7 . 8 is injected . the injection is made between 40 and 80 ° c . in a reactor , with magnetic stirring and with a variable flow . throughout the procedure aseptic conditions are kept to allow the production of a preparation that can be used intravenously . in fig4 , an electron microscopy image of the nanosomes can be observed . to evaluate the safety and effectiveness of the nanosomal preparation the following tests were performed . the preparation obtained according to the previously described formulation was evaluated in its hemodynamic tolerance in rats and newborn pigs . the latter were anesthetized with electrocardiographic monitoring , and monitoring of systemic blood pressure ( sbp ), pulmonary pressure ( pap ), heart rate ( hr ), core temperature ( tc ), and oxygen saturation ( sat o 2 ). acid - base status , electrolytes , hemoglobin , hematocrit , ph and blood gases were also evaluated . the nanosomal preparation of quercetin / hpbcd was injected to the animal intravenously at quercetin concentrations of 10 mg / kg . as shown in fig5 , no changes were observed in hr , sat o 2 , sbp , and pap after administration of nanosomes , demonstrating the hemodynamic safety as the basis for the systemic administration of the preparation . administered intravenously to rats , via the femoral vein with continuous flow for 30 minutes at the same quercetin concentration of 10 mg / kg , the nanosomal preparation of quercetin / hpbcd showed no clinical changes . b — neuroprotective effectiveness . stabilization of hemodynamic parameters altered by severe hypoxia in newborn pigs in newborn pigs anesthetized and monitored as described previously , hypoxia was induced decreasing inspired oxygen by the endotracheal tube to 8 %. during hypoxia , the o 2 - saturation level decreases to below 20 %. after completion of the hypoxia the fraction of inspired oxygen ( fio 2 ) is set to 1 for 30 min , to start resuscitation . after that time it is maintained for 8 h at adequate levels ( above 90 %) applying an increased fraction of inspired o 2 if it drops below the minimum required . to get adequate oxygen saturations animals that had been subjected to hypoxia required more oxygen ( fio 2 0 . 34 ± 0 . 25 ), while those receiving hypoxia and the nanosomal preparation required an fio 2 of 0 . 22 ± 0 . 04 , significantly reduced as demonstrated in table 1 , which shows a chi square analysis of the proportion of animals requiring only air or oxygen . these results indicate a lower requirement for oxygen in the group treated with the nanosomal preparation to maintain an adequate exchange and to deliver to different organs an appropriate oxygen supply . in particular they indicate a better physiological condition , with protection of lung injury induced by the hypoxic event and subsequent re oxygenation . a progressive fall in systemic blood pressure , which often fails to maintain the required levels for life is observed throughout the experiment . after completion of hypoxia several animals showed a tendency to hypotension , making necessary to add inotropic medication to maintain stability . in this case continuous infusion of adrenaline was used according to the requirement of each animal . this hemodynamic response is more stable in the animals that received the preparation in doses of 10 mg / kg quercetin . those animals maintained systemic blood pressure levels next to the controls without the need for inotropic addition . in fig6 , the monitoring of sbp is observed . the sbp is stable after injury in animals that received the nanosomal preparation ( bottom records ), while it was instable subsequent to resuscitation in only hypoxic newborn piglets ( up records in figure ), with repeated episodes of hypotension , which were only reversed with the addition of adrenaline . with respect to acid - base balance , electrolytes and glucose metabolism , no significant differences between the hypoxia group and the group that also received single - dose of 10 mg / kg quercetin were detected . c . neuroprotective effectiveness ii : improvement of brain electrical activity by treatment with nanosomes in newborn piglets subjected to hypoxia . in newborn pigs subjected to severe hypoxia by restricting the fraction of oxygen received in the same model as previously described , electrical brain activity was recorded with an electroencephalogram amplitude monitor ( cfm ). during the experimental situation ( hypoxia ), the amplitude of cerebral activity is maintained below a value of 7 μν for a minimum time of 17 min . during hypoxia amplitude markedly decreased in all animals and remained lowered after resuscitation with 100 % oxygen at the time of peripheral collapse ( as hemodynamic blood pressure and heart rate markers decrease ). in animals that received treatment with quercetin nanosomes with 10 mg / kg of quercetin there was a significant recovery of brain electrical activity that was maintained up to 8 hours after the end of hypoxia ( fig7 ) in the model of severe hypoxia in newborn pigs mentioned in the preceding paragraphs , following the experimental period of 8 hours , the animals were kept under intensive care for 72 hours , ensuring its survival . at 72 hours the electroencephalographic improvement seen at the end of experimental hypoxia persisted in animals treated with quercetin nanosomes . these animals also could suck a bottle and feed ( 80 %) while only 20 % of the animals subjected to hypoxia without treatment could . the latter required a greater oxygen supply assistance and inotropic and did not walk . treated animals were able to walk , albeit with some difficulties . e . neuroprotective effectiveness iv : recovery of dopamine levels in the striatum of rats in experimental model of parkinson &# 39 ; s disease . in an experimental model of parkinson &# 39 ; s disease , the toxin 6 - hydroxydopamine ( 6 - ohda ) was injected into the substance nigra ( sn ) of adult male rats in a similar lesion to that observed in patients with parkinson &# 39 ; s disease . the neurotransmitter dopamine decreased in in the sn and striatum ( es ), which is the area of the terminals of neurons of the sn . the administration of the nanosomal preparation in doses of 10 mg of quercetin 1 hour and 24 hours after the injury by 6 - ohda , significantly recovered the levels of dopamine in the es , an index generally accepted as functional neurological recovery in the model ( fig5 ). 1 . reitsma , j b , et al , epidemiology of stroke in the netherlands from 1972 to 1994 . the end of the decline in stroke mortality . neuroepidemiology , 1998 17 ( 3 ):. p . 121 - 31 . 2 . roger , v i . , et al , executive summary :. heart disease and stroke statistics 2012 update ˜: a report from the american heart association . circulation , 2012 125 ( 1 ): p .. 188 - 97 . 7 . evans , d j , levene m i , and m . tsakmakis , anticonvulsants for mortality and morbidity preventative in full term newborns with perinatal asphyxia . cochrane database syst rev , 2007 ( 3 ): p . cd001240 . 9 . shankaran , s ., et al ., acute neonatal morbidity and long - 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