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US010675269B2

(12 ) United States Patent ( 10 ) Patent No.: US 10,675,269 B2 Sela et al. (45 ) Date of Patent : Jun . 9 , 2020

( 54 ) COMPOSITIONS COMPRISING (58 ) Field of Classification Search None See application file for complete search history . (71 ) Applicant: TRINUTRA LTD ., Nes Ziona ( IL ) (56 ) References Cited (72 ) Inventors : Yoram Sela , Ra'anana ( IL ) ; Mor Zeilkha , Ramat Gan ( IL ) ; Itschak U.S. PATENT DOCUMENTS Lamensdorf, Modi'in ( IL ) 6,277,396 B1 8/2001 Dente 6,383,526 B1 5/2002 Andrews et al. ( 73 ) Assignee : TRINUTRA LTD . , Nes Ziona ( IL ) 2002/0182196 Al 12/2002 McCleary 2004/0001817 Al 1/2004 Giampapa 2006/0264497 Al 11/2006 Zeligs ( * ) Notice : Subject to any disclaimer, the term of this 2009/0068255 A1 * 3/2009 Yu A61K 8/0212 patent is extended or adjusted under 35 424/450 U.S.C. 154( b ) by 0 days. 2012/0294952 A1 11/2012 Zarbock et al. 2013/0004599 A1 1/2013 Schwartz ( 21 ) Appl. No.: 16 / 070,925 2013/0012824 Al 1/2013 Vanney et al . 2013/0315983 Al 11/2013 Einbond et al . ( 22 ) PCT Filed : Jan. 17 , 2017 2015/0071993 Al 3/2015 Patel et al . ( 86 ) PCT No .: PCT / IL2017 /050060 FOREIGN PATENT DOCUMENTS $ 371 (c )( 1 ), WO 01/26646 4/2001 (2 ) Date : Jul. 18 , 2018 WO 2008/036979 3/2008 ( 87 ) PCT Pub . No .: W02017 / 125919 OTHER PUBLICATIONS PCT Pub . Date : Jul. 27 , 2017 International Search Report for PCT/ IL2017 /050060 , dated Jun . 19 , 2017 , 7 pages . (65 ) Prior Publication Data Written Opinion of the ISA for PCT/ IL 2017/050060 , dated Jun . 19 , 2017 , 9 pages. US 2019/0029999 A1 Jan. 31 , 2019 Kirubakaran Shanmugam et al: “ Plant- derived polyphenols attenu ate lipopolysaccharide- induced and tumour necrosis factor production in murine microglia and macrophages ” , Mol . Related U.S. Application Data Nutr. Food Res, vol . 52 , Jan. 1, 2008 ( Jan. 1, 2008 ), pp . 427-438 . Roberta Foresti et al : " Small molecule activators of the Nrf2 -HO - 1 (60 ) Provisional application No. 62 / 279,936 , filed on Jan. antioxidant axis modulate heme metabolism and inflammation in 18 , 2016 . BV2 microglia cells ” , Pharmacological Research ., vol. 76 , dated Oct. 1 , 2013 ( Oct. 1 , 2013 ) , GB , pp . 917-919 , XP055230780 . Jacobo -Herrera et al : Nf- [kappal ] B Modulators , Jan. 1 , 2006 ( Jan. (51 ) Int. Ci. 1, 2006 ), pp . 917-919 . A61K 36/00 ( 2006.01 ) Clark , I. A. and Vissel, B. Inflammation - sleep interface in brain A61K 31/4045 ( 2006.01) disease : TNF, insulin , orexin (2014 ) . A61K 31/185 ( 2006.01 ) A61K 36/185 ( 2006.01 ) * cited by examiner A61K 31/191 ( 2006.01 ) A61P 25/20 (2006.01 ) Primary Examiner Qiuwen Mi A61P 25/22 ( 2006.01) (74 ) Attorney , Agent, or Firm — Nixon & Vanderhye P.C. A61P 29/00 (2006.01 ) A61K 9/00 ( 2006.01) (57 ) ABSTRACT A61K 31/192 ( 2006.01 ) The present invention provides a composition comprising a A61K 36/84 ( 2006.01 ) synergistic combination of melatonin , valerenic acid and (52 ) U.S. Cl. carnosic acid . The composition may be used in the treatment CPC A61K 31/4045 (2013.01 ) ; A61K 9/006 and prevention of several different disorders including (2013.01 ) ; A61K 31/185 (2013.01 ); A61K inflammatory conditions, sleep disorders and anxiety . The 31/191 (2013.01 ) ; A61K 36/185 ( 2013.01) ; invention also relates to several dosage forms which are A61P 25/20 ( 2018.01 ) ; A61P 25/22 ( 2018.01) ; suitable for the delivery of the claimed composition to A61P 29/00 ( 2018.01) ; A61K 31/192 subjects in need of treatment therewith . (2013.01 ) ; A61K 36/84 (2013.01 ) ; A61K 2300/00 (2013.01 ) 13 Claims, 3 Drawing Sheets U.S. Patent Jun . 9 , 2020 Sheet 1 of 3 US 10,675,269 B2

Fig . 1

NO (UM ) 25 lililli

Fig . 2 NO (UM )

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Fig . 3 ill U.S. Patent Jun . 9. 2020 Sheet 3 of 3 US 10,675,269 B2

Fig . 4 JU Ist bar of each pair : No caffeine 2nd bar of each pair : With caffeine US 10,675,269 B2 1 2 COMPOSITIONS COMPRISING gastro - intestinal diseases , inflammatory conditions of the MELATONIN central nervous system and certain forms of asthma. Con sequently , it has been proposed that inhibition of NO pro This application is the U.S. national phase of International duction could provide a useful therapeutic mechanism for Application No. PCT/ IL2017 /050060 filed 17 Jan. 2017 , 5 the treatment and /or management of these inflammatory which designated the U.S. and claims the benefit of U.S. disorders. Provisional Application No. 62/ 279,936 filed 18 Jan. 2016 , Melatonin may also play a role in several other physi the entire contents of each of which are hereby incorporated ological and pathological processes, and it has been sug by reference . gested that adequate levels of this hormone may play a role 10 in protecting against cardiovascular disease, various differ FIELD OF THE INVENTION ent cancers and several different degenerative conditions affecting the central nervous system . The present invention relates to novel compositions that Several plant- derived preparations have also been used to comprise melatonin , and which may further comprise one or treat and other sleep disorders, with various more additional active ingredients that synergistically 15 degrees of success. These plant -derived treatments include enhance the biological activity of said compositions . extracts , tinctures, teas, oils or whole -plant material from inter alia the following species : ( Valeriana offici BACKGROUND OF THE INVENTION nalis) , ( Passiflora incarnata ), chamomile ( Anthe mis nobilis ) , Hops (Humulus lupulus) , lavender (Lavandula Melatonin (N -acetyl -5 -methoxytryptamine ) is a naturally 20 officinalis ), wild lettuce (Lactuca virosa ), california poppy occurring substance that is found in animals , plants , fungi (Eschsholzia californica ), kava (Piper methysticum ) and bacteria . In humans and other mammals , melatonin is and St. John's wort (Hypericum perforatum ). Although produced by the pineal gland and acts as a neurohormone , various degrees of success have been reported with regard to playing a key role in the maintenance of circadian rhythms. the treatment of insomnia using these various herbal treat As such , it has a significant influence on many physiological 25 ments , there is no consensus regarding their use , recom functions including sleep . In this regard, it has been found mended dosage, standardization of the preparation used or that melatonin may be used in order to regularize sleep and their effectiveness in relation to other, more standard stand to treat certain sleep disorders , notably insomnia . alone preparations. Furthermore , many plant -derived mate Various clinical studies of the use of melatonin in the rials ( including inter alia some of those listed above ) have treatment of insomnia have been performed , and the results 30 been shown to possess anti - inflammatory activity . of some of these studies indicate that prolonged - release Several different formulations containing melatonin for melatonin may indeed be effective in improving sleep use in the treatment of insomnia are known in the art . These latency, sleep quality and daytime alertness in adults . Other formulations include inter alia oral dosage forms with either studies have also shown that melatonin administration may immediate - release or prolonged - release characteristics ( liq provide an effective treatment sleep -wake cycle disorders 35 uids, capsules or tablets ) , sublingual dosage forms and associated with neurodevelopmental disorders in children . transdermal patches. Administration of Immediate -release In addition to its effects on sleep , a number of additional dosage forms generally lead to peak blood levels being and different health benefits have been associated with obtained within about one hour. In the case of currently melatonin . For example , melatonin has been found , in a available prolonged - release formulations, peak blood levels number of well documented studies , to have a variety of 40 of melatonin are reached gradually over period of 8-10 effects on inflammatory and immune processes (both as an hours , thereby mimicking the body's internal secretion activator and an inhibitor of these processes ). In this regard , profile. The sublingual route for the delivery of pharmaceu it has been found that melatonin controls the production of tical and nutraceutical agents , whereby the active ingredi many different inflammatory mediators , including but not ents are allowed to diffuse into the bloodstream through limited to leukotrienes and cytokines . In addition to specific 45 tissues under the tongue , is well known in the art . Many actions on various mediator systems, melatonin may also different classes of pharmaceutical agents have been formu modulate ongoing inflammatory processes by virtue of its lated for sublingual administration , including : cardiovascu potent antioxidant effects . lar drugs, steroids, , enzymes , and increasingly , Several studies have indicated that sleep patterns are vitamins and minerals . Various different dosage forms may altered during inflammatory disease , and to some degree 50 be used for sublingual delivery , including sublingual tablets may be considered as markers of the presence and severity ( regular or fast disintegrating) , lipid matrix sublingual tab of the inflammatory process . lets , thin films and sublingual sprays . Furthermore, several different inflammatory mediator There are several advantages associated with drug deliv systems have been implicated in causing changes in sleep ery by the sublingual route , in comparison with oral admin patterns. It may therefore be concluded that there is a type 55 istration . Firstly , the direct entry of the medicament into the of inflammation - sleep interface (Clark , I. A. and Vissel , B. blood stream ( i.e. via the capillaries located in the sub (2014 ) J. Neuroinflammation 11: 51 Inflammation - sleep epithelial connective tissue in the floor of the mouth ), often interface in brain disease : TNF, insulin , orexin ), and thus leads to a shorter onset time. In addition , the more efficient agents which modulate various aspects of the inflammatory uptake of the medicament may, in some cases , permit process may also be capable of influencing sleep patterns 60 smaller dosages to be used . However , there is a further and behaviour. advantage of the sublingual route that is often even more A very large number of different chemicalmediators have significant. This advantage relates to the fact that since been shown to be involved in the development and control sublingually administered medicaments are not absorbed of the inflammatory process . Recent studies by a number of through the intestinal wall into the portal circulation , they different laboratories have implicated nitric oxide (NO ) as 65 are not subject to first -pass metabolism in the liver prior to an important modulator of a variety of acute and chronic entering the general circulation . Similarly , the drugs admin inflammatory disorders , including various types of arthritis , istered by this route are protected from the hostile gastro US 10,675,269 B2 3 4 intestinal environment and thus are not degraded by stomach The present invention is further directed to compositions acid , bile and /or enzymes such as monoamine oxidase disclosedherein for the treatment of inflammatory disorders . (MAO ) . This is a significant advantage, in particular for In another embodiment, the invention is directed to compo active ingredients that are metabolized by MAO , such as sitions of the present invention for the treatment of anxiety melatonin . In summary, it may be appreciated that admin- 5 and /or sleep disorders . istration via the sublingual route may often improve the bioavailability of the medicament. Many of the advantages BRIEF DESCRIPTION OF THE DRAWINGS described hereinabove are also features of other formula tions from which the active ingredient is absorbed within the FIG . 1 presents results showing the synergistic interaction oral cavity , such as buccal formulations and chewable tab- 10 between melatonin, carnosic acid and valerenic acid in a murine cell model of nitric oxide release, wherein these lets . components are present at a molar ratio of 1 : 1.4 : 2 (mela Despite the many advantages discussed above ,many of tonin :carnosic acid :valerenic acid ) . the known sublingual delivery systems suffer from the FIG . 2 presents results showing the absence of a syner disadvantage that the delivery performance and the bioavail 15 gistic interaction between melatonin , carnosic acid and ability of the active ingredient are affected by its physical valerenic acid in a murine cell model of nitric oxide release , properties, including solubility , crystal morphology , particle when used at a molar ratio of 1 : 1.4 : 20 (melatonin :carnosic size , hygroscopicity , compressibility and polarity . acid :valerenic acid ) . One of the aims of the present invention is to provide new FIG . 3 presents results obtained from a laboratory rat and improved therapeutic compositions comprising mela- 20 spontaneous activity model, in which a synergistic anxi tonin , particularly suitable for use in the treatment of insom olytic interaction between melatonin and valerian is nia and / or inflammatory conditions. observed . Another aim is to provide melatonin - containing compo FIG . 4 presents results indicating a synergistic interaction sitions which provide better onset/ offset times, greater effi between melatonin and valerian as measured by changes in cacy and reduced undesired effects ( such as excessive morn- 25 the ‘ center time' observed in an open - field rat anxiety model. ing sleepiness ), when compared with prior art treatments . A still further aim of the present invention is to provide DETAILED DESCRIPTION OF PREFERRED compositions comprising synergistic combinations of mela EMBODIMENTS tonin and certain plant extracts . Further aims and objectives will become apparent as the 30 As disclosed hereinabove, in one aspect the present inven description proceeds. tion is directed to compositions comprising melatonin as an active ingredient. Such compositions may be provided as SUMMARY OF THE PRESENT INVENTION pharmaceutical compositions, nutraceutical compositions, compositions for use in addition to food and beverage The present invention provides novel compositions com- 35 products , and so on . prising melatonin . These compositions are suitable for use In one particularly preferred embodiment of this aspect of inter alia in the treatment of a number of different conditions the invention , the composition comprises a synergistic com including, but not limited to , inflammatory conditions , anxi bination ofmelatonin , valerenic acid and carnosic acid . The ety and sleep disorders . latter two components may be present in synthetic form , as In some preferred embodiments the compositions of the 40 purified compounds obtained from natural plant material present invention further comprise additional active agents ( Valeriana species and Rosmarinus officinalis, respectively ) , including, but not limited to , natural material derived from partially -purified extracts of said natural plant material, a number of plant species including Valeriana species , crude extracts of said plant material, and so on . rosmarinus species , passiflora species , and others , as well be The term “ synergistic combination ” , as used herein , refers described in detail hereinbelow . 45 to the fact that when combined together , the total biological In another aspect , the present invention is directed to activity of said combination is greater than the sum of the novel dosage forms that are suitable for the administration of biological activities of each individual component of said the presently -disclosed compositions. These dosage forms combination when assayed individually , at the same con include ingestible oral formulations as well as sub -lingual centrations as when combined . In particular, the present dosage forms. 50 inventors have unexpectedly found that when used in com In a further aspect , the present invention encompasses bination ,melatonin , valerenic acid and carnosic acid interact methods of treating inflammatory disorders by means of synergistically such that said combination possesses signifi administering a composition comprising a synergistic com cantly greater anti - inflammatory activity than would be bination of melatonin , valerenic acid and carnosic acid . predicted from the anti- inflammatory activity of each of In other embodiments , the present invention provides 55 these agents when tested alone at the same concentration . methods of treating anxiety and / or sleep disorders by means The synergistic anti- inflammatory activity of this ternary of administering a composition comprising melatonin , and ( i.e. three -component ) composition is demonstrated and optionally further comprising additional substances includ compared with the activities of the binary combinations and ing , but not limited to , materials derived from Valeriana individual agents (melatonin , valerenic acid and carnosic species , rosmarinus species, passiflora species, and others . 60 acid ) in Working Examples 1-5 , hereinbelow . In a still further aspect, the present invention is directed In one preferred embodiment of this aspect of the inven to the use of the compositions disclosed hereinabove and tion , the three obligatory active components of the compo described in detail hereinbelow in the preparation of medi sition are present in the following molar ratio ranges: caments . In some embodiments , these medicaments are Melatonin : carnosic acid : valerenic acid suitable for the treatment of inflammatory disorders . In other 65 1 : 0.1-10 : 0.5-10 embodiments , the medicaments are suitable for the treat In another preferred embodiment, the three components ment of anxiety and /or sleep disorders. are present in the following molar ratio ranges: US 10,675,269 B2 5 6 Melatonin : carnosic acid : valerenic acid valerenic acid having amounts of each of the three main 1 : 0.5-5 : 1-5 active components within the following preferred ranges: In one preferred embodiment, the three components are Melatonin 0.2-5 mg present in a molar ratio of: Carnosic Acid 2-5 mg Melatonin : carnosic acid :valerenic acid 5 Valerenic Acid 1-5 mg 1 : 0.7 : 1.3 In one particularly preferred embodiment, the amount of In another preferred embodiment, the three components valerenic acid in each dosage unit is not less than 2 mg. In another aspect , the present invention is directed to a are present in a molar ratio of: method of treating and / or preventing inflammatory disorders Melatonin : carnosic acid : valerenic acid 10 in a mammalian (preferably human ) subject, by means of 1 : 2.1 : 4 administering a composition comprising a synergistic com In a further preferred embodiment, the three components bination ofmelatonin , valerenic acid and carnosic acid ( i.e. are present in a molar ratio of: the composition disclosed in the previous section and Melatonin :carnosic acid : valerenic acid described in more detail hereinbelow ) . 1 : 4 : 1 15 In one preferred embodiment of this method , the inflam In another preferred embodiment, the three components matory condition to be prevented or treated may be one are present in a molar ratio of: selected from the group consisting of acute inflammatory Melatonin : carnosic acid : valerenic acid conditions, chronic inflammatory conditions , osteoarthritis 1 : 1.5 : 2 rheumatoid arthritis , inflammatory conditions of the upper The composition of this aspect of the present invention 20 and lower respiratory tracts, peritonitis , cardiovascular may optionally comprise further active ingredients , includ inflammation , sepsis , trauma, inflammation of the skin ( such ing, but not limited to plant- derived material , 5 -hy as in psoriasis , acne , dermatitis and so on ) and various types droxytryptophan (5 -HTP ) , and other amino of gastrointestinal inflammation . This list is , of course , not acids, vitamins and minerals . exhaustive and is provided for exemplary purposes only . In some preferred embodiments of this aspect of the 25 In one preferred embodiment of this method of treatment invention , the additional active agents are plant- derived and /or prevention of inflammatory conditions, the amounts substances obtained from one or more of the following of each of the three abovementioned active ingredients species: (melatonin , carnosic acid and valerenic acid ) administered Passiflora (Passiflora incarnata ), chamomile (Anthemis each day are : nobilis ), Hops (Humulus lupulus ), lavender (Lavandula 30 Melatonin 0.3-4 mg/ day officinalis ), wild lettuce (Lactuca virosa ), california Carnosic acid 2-4 mg/ day poppy (Eschsholzia californica ), Kava kava (Piper Valerenic acid 3-5 mg/ day methysticum ) , St. John's wort (Hypericum perforatum ), In another aspect , the present invention is directed to a rosemary ( Rosmarinus officinalis ) and the 5 HTP method of treating and /or preventing sleep disorders ( such containing plant Griffonia simplicifolia . 35 as insomnia ) and / or anxiety in a mammalian ( preferably It is to be noted that the species listed above are given for human ) subject, by means of administering a composition the sake of illustration only , and other related species may comprising a synergistic combination of melatonin , valer also be used without deviating from the scope of the present enic acid and carnosic acid ( i.e. the composition disclosed in invention . the previous section and described in more detail hereinbe In addition , the compositions of this aspect of the present 40 low ). invention may further comprise biologically inactive ingre In a further aspect, the present invention is directed to the dients , including, but not limited to , one or more pharma use of a composition comprising a synergistic combination ceutical excipients , such as binding agents , bulking agents , of melatonin , valerenic acid and carnosic acid in the prepa fillers , diluents, additional release -control polymers , poly ration of a medicament. In one preferred embodiment , the meric matrices, disintegrants , flavorings, coating agents , 45 medicament is intended for use in the treatment and / or penetration enhancers , and so on . prevention of inflammatory disorders, as described herein The compositions of this aspect of the present invention above. In another preferred embodiment, the medicament is may be formulated in several different dosage forms for intended for use in the treatment and / or prevention of administration to mammalian ( particularly human ) subjects . anxiety and /or sleep disorders ( such as insomnia of different Suitable dosage forms include (but are not limited to ) oral 50 types ). Suitable dosage forms, routes of administration and dosage forms, sub - lingual dosage forms, injectable formu details of the synergistic combination are provided herein lations, suppositories , patches for use on skin or mucous above. membranes, inhalable formulations, topical formulations In a still further aspect, the present invention is directed and so on . Further details of the preparation of such formu to a composition comprising a synergistic combination of lations and dosage forms can be obtained from any standard 55 melatonin , valerenic acid and carnosic acid for the treatment reference on the subject, such as Remington's Pharmaceu and /or prevention of inflammatory disorders ( as described tical Sciences , Mack Publishing Co , Easton , Pa. , USA , 21st hereinabove ) . edition ( 2006 ) . In another aspect , the present invention is directed to a The details of a novel sublingual formulation that is composition comprising a synergistic combination of mela suitable for the delivery of the synergistic composition of 60 tonin , valerenic acid and carnosic acid for the treatment this aspect of the present invention are provided hereinbe and/ or prevention of anxiety and / or sleep disorders ( such as low . insomnia ) . In some preferred embodiments of this aspect of the In another aspect, the present invention provides a novel invention , the composition is formulated ( e.g. as a sublin dosage form , which contains melatonin in a combination of gual tablet or swallowable tablet, caplet or capsule ) , such 65 both immediate release ( IR ) and controlled release (CR ; also that each dosage unit (i.e. a single table , caplet or capsule ) sometimes referred to as “ extended release ( ER )) forms. may comprise a combination ofmelatonin , carnosic acid and This combination when given sublingually (or in the form of US 10,675,269 B2 7 8 buccal or chewable dosage forms) enables fast onset of the encapsulation is used , whereby an inert core is coated with melatonin activity , together with prolonged activity that the active ingredient and then surrounded by one or more ensures a long -lasting therapeutic effect due to the controlled layers of insoluble envelope materials , the number, type and release component, which is capable of increasing themela thickness of which may be selected in order to obtain the tonin short half- life. In this way, the subject taking the 5 desired release characteristics. Many different types of con dosage form is able to enjoy continuous undisturbed sleep . trolled release polymer may be used to manufacture these In addition , the combined IR /CR composition of the present various types of CR formulations , including (but not limited invention also displays improved bioavailability and effi to ): hydroxypropylmethyl cellulose , microcrystalline cellu cacy in relation to melatonin compositions of the prior art. lose , methyl cellulose , hydroxypropyl cellulose, hydroxy A further advantageous feature of the presently -claimed 10 ethyl cellulose , sodium carboxymethyl cellulose, carboxym composition is that when administered to a subject suffering ethyl ethyl cellulose, ethyl cellulose, and other cellulose from insomnia , it leads to both a rapid initial onset of sleep derivatives, polymethacrylic copolymers , poloxamers, poly as well as a quick return to sleep after waking up mid -sleep . oxyethylene stearate , polyvinylpyrrolidone, polyvinylpyr It has also been unexpectedly found that when the above rolidone -polyvinylacetate copolymers , polyvinyl , disclosed melatonin dosage form further comprises certain 15 polyethylene oxide, and gums (e.g. , Xanthan gum or guar plant -derived material from species such as Valeriana offi gum ). cinalis , Rosmarinus officinalis , and others , said plant- de In one preferred embodiment of the present invention , the rived material interacts synergistically with the melatonin in controlled release components of the dosage form are matrix enhancing its anti -insomnia and anxiolytic activities . It has minitabs , coated minitabs or coated microcapsules prepared also been found that these activities are further enhanced by 20 by multistage layering or extrusion and spheronization fol adding additional substances such as passiflora extracts and lowed by functional coating . whole plant material , other plant- derived material , green tea In addition to the two different forms of the active extracts , antioxidants and so on . ingredient, melatonin , the dosage form of the present inven While these advantages of the present invention are seen tion may further comprise one or more pharmaceutical when the melatonin IR /CR combination is formulated as a 25 excipients , including (but not limited to ) binding agents , sublingual, buccal or chewable dosage form , other dosage bulking agents , fillers , diluents , additional release -control forms, particularly oral dosage forms, containing this com polymers, polymeric matrices, disintegrants , flavors , coating bination are also included within the scope of the present agents , penetration enhancers , and so on . invention . In one preferred embodiment , the dosage form further The present invention is thus primarily directed to a 30 comprises one or more penetration enhancers . Examples of sublingual , buccal or chewable dosage form comprising a two suitable penetration enhancers are and vitamin combination of melatonin IR and melatonin CR compo E TPGS . nents , wherein said IR and CR components work synergis In another aspect, the present invention also encompasses tically to improve sleep quality . In addition , these combi a sublingual dosage form comprising a combination of nations contain antioxidants that may have benefits in 35 melatonin IR and melatonin CR components . As disclosed improving sleep quality and in some cases , such as with hereinabove, said dosage form may further comprise one or Vitamin E TPGS ( d -alpha tocopheryl polyethylene glycol more additional active insomnia - treating agents , wherein 1000 succinate ), can serve as penetration enhancers as well. said agents are selected from the group consisting of plant Generally , each dosage form unit will contain about 1-10 derived material , 5 -hydroxytryptophan ( 5 -HTP ), tryptophan mg of melatonin in the IR component and 1-10 mg of 40 and other amino acids, vitamins and minerals . melatonin in the CR component . In some preferred embodiments of this aspect of the In a highly -preferred embodiment, the dosage form fur invention , the additional active agents are plant- derived ther comprises vitamin E TPGS, which acts both as an substances obtained from one or more of the following anti -oxidant and as a penetration enhancer . species: Preferably — but not exclusively — the dosage form of the 45 valerian (Valeriana officinalis ) , passiflora (Passiflora present invention is formulated as a sublingual tablet . In one incarnata ) , chamomile ( Anthemis nobilis ), Hops (Hu preferred embodiment , the sublingual tablet can be prepared mulus lupulus) , lavender (Lavandula officinalis ), wild as a double layer tablet where one part of the tablet dissolves lettuce (Lactuca virosa ), california poppy (Eschsholzia and absorbed sublingually , while the ER components are californica ) ,Kava kava (Piper methysticum ), St. John's swallowed and released continuously in the GI tract during 50 wort (Hypericum perforatum ), rosemary (Rosmarinus the subsequent 1-12 hours . officinalis ) and the 5 HTP -containing plant Griffonia In some preferred embodiments , the sublingual tablet can simplicifolia . be prepared as a presscoat tablet, wherein the external layer It is to be noted that the species listed above are given for contains melatonin in IR form , while the internal core the sake of illustration only , and other related species may contains melatonin in CR form . 55 also be used without deviating from the scope of the present In other preferred embodiments , the dosage form is for invention . mulated as a buccal tablet or chewable tablet . In one preferred embodiment, the present invention pro The controlled release components of the dosage form vides a sublingual dosage form comprising a combination of may be prepared in accordance with any of the methods melatonin IR and melatonin CR components and material known to the skilled artisan in this field . Generally , in CR 60 derived from Valeriana officinalis and /or other related Val dosage forms the active pharmaceutical (or nutraceutical ) eriana species . Preferably , the Valeriana officinalis material ingredient is embedded within an insoluble matrix . The is present in the dosage form in an amount that contains in matrix is generally porous, and the active ingredient exits the the range of about 1-10 mg valerenic acid . matrix via the pores. In other types of CR formulation , the In a further preferred embodiment, the present invention active ingredient is dissolved within a polymeric matrix 65 provides a sublingual dosage form comprising a combina which swells , thereby forming a gel through which said tion of melatonin IR and melatonin CR components and active ingredient needs to exit . In another approach , micro material derived from Valeriana officinalis and /or other US 10,675,269 B2 9 10 related Valeriana species , and material derived from Ros Furthermore , the present invention is also directed to a marinus officialis . Preferably , the Valeriana officinalis mate method for reducing anxiety in mammalian subjects (par rial is present in the dosage form in an amount that contains ticularly human subjects ), wherein said method comprises in the range of about 1-10 mg valerenic acid . the administration of a dosage form containing a combina In another preferred embodiment, the present invention 5 tion ofmelatonin IR and melatonin CR components . Pref provides a sublingual dosage form comprising a combina erably , said dosage form is a sublingual tablet as disclosed tion of melatonin IR and melatonin CR components and and described hereinabove , and preferably , the administra material derived from Passiflora incarnata and /or other tion of the dosage form is performed sublingually . related Passiflora species. Preferably , the Passiflora incar In any of the methods of treatment defined above , the nata material is present in the dosage form in an amount in 10 dosage form used may further comprise one or more further the range of about 10-300 mg. active ingredients such as plant- derived material, vitamins, In yet another preferred embodiment, the sublingual dos minerals , 5 -HTP , tryptophan and other amino acids, as age form comprises a combination of melatonin IR and described hereinabove . melatonin CR components , an extract of Valeriana offici nalis containing 1-10 mg of valerenic acid , 10-100 mg of an 15 EXAMPLES extract of Passiflora incarnata and 10-100 mg of 5HTP. In the following non -limiting examples , unless otherwise It is to be noted that, as mentioned hereinabove , the stated , the melatonin used was a synthetic melatonin plant -derived components of these dosage forms interact obtained from Luotian Xinpusheng Pharmaceutica . Co. Ltd. , synergistically with melatonin , thereby enhancing its anti- 20 Hubei, China . The valerian extracts were purchased from insomnia activity . Indo World Trading Corp., Badarpur , India , while the pas It is to be noted that the term “ plant- derived material” and siflora extract was obtained from Bara Herbs , Yokneam , similar expressions refer to material that is obtained in any Israel. The rosemary extract was obtained from Vitiva , of the various forms well known to the skilled artisan in this Markovci, Slovenia (product name Inolens 25 ) . Other stan field , including : dried plant material obtained from roots , 25 dard reagents and materials were purchased from several leaves, shoots , flowers and whole plants ; aqueous and non different laboratory and pharmaceutical suppliers . aqueous extracts ; teas or tisanes ; decoctions and tinctures; oils . WORKING EXAMPLES : As explained hereinabove , sublingual tablets are the pre ANTI- INFLAMMATORY EFFECTS OF TESTED ferred type of formulation for the present invention . How- 30 COMBINATIONS ever , even though said tablets are intended for sublingual delivery ( thereby providing rapid onset of activity ), they Objectives may also be swallowed in the same way as standard oral The objective of the studies reported in working examples dosage forms, when quick onset is not required . 1-5 hereinbelow was to evaluate the anti - inflammatory prop In addition to the advantages of the dosage form of the 35 erties of Melatonin , carnosic acid (Rosemary extract) , Val present invention in relation to prior art melatonin prepara erenic acid and combinations thereof on a cell line derived tions, it is to be noted that said dosage form also possesses from murine macrophages (RAW 264.7 cells ) activated by improved efficacy with minimal side effects and residual lipopolysaccharide (LPS ). tiredness , when compared with other anti- insomnia treat General Methods : ments of the prior art such as . 40 10 % RAW 264.7 cells were seeded in 96 - well plates in a It is to be noted that the above - disclosed dosage forms and medium containing 1 % FBS , 1 % and 1 % Pen / compositions have also been found by the inventors to strep in DMEM . possess significant anxiolytic activity . Similarly , the addition For dose response assays , cells were treated with 2- fold of certain plant- derived (and other) agents , materials and concentrations of Melatonin ( starting from 15.6 um up to substances to these compositions has now been found to 45 1000 UM LOT # XPS150311, Yes Pharma ), Rosemary cause synergistic enhancement of said anxiolytic activity . extract (0.1 , 0.2 , 0.4 , 1 , 2 , 4 , or 10 uM , Product # 302659, Without wishing to be bound by theory, it is believed that Batch # LAB.16-659001, Vitiva ) or 10 % Valerenic acid anxiety is often a significant factor in the development of ( starting from 1.56 mg/ ml up to 100 mg/ ml , Batch # IWTC / insomnia and other sleep disorders. It is thus believed that 5949/15001 , Indo World Trading Corporation ) for 6 hours . the anxiolytic effect of the compositions of the present 50 Then , the test solutions were removed and the cells were invention contribute (directly or indirectly ) to the anti induced with lipopolysaccharide (LPS ) ( 5 ng/ ml ) . In addi insomnia effects of said compositions . tion , positive control cells were treated with 10 uM dexam In another aspect , the present invention is directed to a ethasone . Nitric oxide (NO ) release was measured after method for treating insomnia in a human subject, wherein incubation at 37 ° C., 5 % CO2 using a Griess Reagent said method comprises the administration of a dosage form 55 System . The method is based on the chemical diazotization containing a combination ofmelatonin IR and melatonin CR reaction that was originally described by Griess in 1879 , components . Preferably , said dosage form is a sublingual which uses sulfanilamide and N - 1 - napthylethylenediamine tablet as disclosed and described hereinabove , and prefer dihydrochloride (NED ) under acidic (phosphoric acid ) con ably , the administration of the dosage form is performed ditions. This system detects NO , in a variety of biological sublingually . 60 and experimental liquid matrices such as tissue culture Similarly , the present invention also provides a method medium that was tested in this study . for improving sleep quality in a human subject, said method For combination assays, cells were treated with various comprises the administration of a dosage form containing a concentrations ofMelatonin , Rosemary extract ( containing combination of melatonin IR and melatonin CR compo 25 % carnosic acid ) and 10 % Valerenic acid , at the final nents . Preferably , said dosage form is a sublingual tablet as 65 concentration indicated in each experiment , hereinbelow , for disclosed and described hereinabove , and preferably , the 22 hours together with LPS induction (5 ng /ml ) . The LPS administration of the dosage form is performed sublingually . concentration was chosen based on previous studies which US 10,675,269 B2 11 12 showed no anti- inflammatory effect with the tested Items. In stimulated baseline cells (column 1) : 0.05 um Melatonin addition , positive control cells were treated with 10 UM ( column 3 ) , 0.07 UM carnosic acid ( column 4 ) and 1.3 UM dexamethasone. Nitric oxide (NO ) release was tested after valerenic acid (column 5 ) . incubation at 37 ° C., 5 % CO2 using the Griess Reagent It may thus be concluded that the synergistic interaction System . 5 between the three components tested is dependent on the relative amounts of each of said components when used in Example 1 combination . In particular , a significantly elevated amount of valerenic acid appears to abolish the synergistic anti Anti - Inflammatory Effects of a Synergistic inflammatory effect seen with the near -equimolar combina Combination of Melatonin , Carnosic Acid and 10 tion (as shown in Example 1) . Valerenic Acid WORKING EXAMPLES : ANXIOLYTIC All anti - inflammatory studies were performed using the EFFECTS OF TESTED COMBINATIONS techniques described in the 'General Methods' section , hereinabove . 15 Example 3 To assess the anti - inflammatory effects of tested combi nations in a preliminary study it was found that tested item Synergistic Interaction Between Melatonin and should be incubated for 22 hours together with 5 ng /ml LPS . Valerian : Anxiolytic Effect The results from various treatments are summarized in 20 Open field tests were used as a measure of the spontane FIG . 1 as follows: when tested separately none of the ous activity of laboratory rats , in order to measure the following individual components produced any significant anxiolytic effects of melatonin , valerian and a combination inhibition of NO production when compared with the LPS of these wo substances . stimulated baseline cells ( column 1 ) : 0.5 uM Melatonin Method : ( column 3 ) , 0.7 um carnosic acid ( column 4 ) and 1.3 UM 25 Rats were placed in a behavioral room one hour before valerenic acid (column 5 ). Similarly , the following binary testing procedures, for purpose of habituation . combinations were also without effect on the level of NO Activity was tested in open boxes ( open - fields: 80 cm production : 0.5 uM Melatonin and 0.7 um carnosic acid wide, 80 cm large , 60 cm high ) made of transparent Plexi ( column 6 ) ; 0.5 UM Melatonin and 1.3 uM valerenic acid glas. Animals were individually placed in the open - fields for ( column 7 ); 0.7 ?M carnosic acid and 1.3 ° M valerenic acid 30 15 minute sessions. The following parameters were (column 8 ). However, the ternary combination of 0.5 UM recorded : Indices ofmotor exploratory activity : the distance Melatonin , 0.7 um carnosic acid and 1.3 uM valerenic acid traveled , mobility time, Open Field spontaneous activity is (column 9 ) caused marked inhibition of NO production by performed on the testing day, 30 minutes after the admin istration of a test substance (melatonin , valerian , a combi the cultured cells : 15 uM of NO instead of 40 uM in the 35 nation of the two, or vehicle ) . LPS -stimulated , untreated cells ( column 1 ) . It is of interest Indices of habituation : distance traveled . to note that this inhibition is far greater than that caused by During the last 5 minute period in percent of the first as the dexamethasone positive control ( 30 uM ; column 2 ) . last 5 minute periods. The fact that none of the three components tested dem Indices of anxiety : onstrated any effect on this model when tested individually 40 Percentage of distance traveled in the central zone of or even in binary combinations, indicates that there is a the open - field . unexpected synergistic interaction between all three com Percentage of time spent in the central zone of the ponents when presenttogeth at the indicated relative molar open - field . ratio (0.5 uM Melatonin , 0.7 uM carnosic acid and 1.3 UM Results : valerenic acid ). Of particular interest is the fact that, at 45 The effect of the test substances are shown in the bar concentrations of the individual components which do not graph presented in FIG . 3. It may be seen from this graph display an anti- inflammatory effect, the combination thereof that both melatonin and valerian (when administered sepa at this ratio results in a significant inhibition of NO produc rately ) increased the mobility time of the rats (in comparison tion . to the vehicle control) , indicating they both possess anxi 50 olytic effects . However , the difference in mobility time Example 2 between test substance and vehicle control only reached statistical significance when melatonin was administered in Absence of Anti - Inflammatory Effects of a combination with valerian ( last bar in FIG . 3 ) . Combination of Melatonin , Carnosic and Valerenic These results are highly suggestive of a synergistic inter Acid when Valerenic Acid is Present at a High 55 action between melatonin and valerian with respect to their Concentration anxiolytic activity . All anti - inflammatory studies were performed using the Example 4 techniques described in the 'General Methods' section , hereinabove . This study was performed in the same general 60 Synergistic Interaction Between Melatonin and manner as described in Example 1 , hereinabove . However, Valerian : Base -Line and Caffeine - Induced in this study, the relative amount of valerenic acid used was Anxiolytic Effect ten -times that used in Example 1 . The results from the various treatments are summarized in As in Example 3 , above , open field tests were used to FIG . 1 as follows: when tested separately none of the 65 measure the anxiolytic effects ofmelatonin , valerian and a following individual components produced any significant combination of these two substances. However, in some of inhibition of NO production when compared with the LPS the rats in the present study, the effect of the test substances US 10,675,269 B2 13 14 wasmeasured in rats which had also received caffeine . Thus , 6. Add to the sieved mixture : 24 g of the controlled release while the study reported in Example 3 provided insights into minitabs prepared in Example 5 , 19 g stevia sweetener , 24 the effect ofmelatonin and valerian on base - line anxiety , the g starch 1500 ( pregelatinized ) , 5.6 g menthol crystals, 3.8 g results obtained in this study relate to the effect of these aerosol 200. Mix 10 minutes . substances on both base - line and caffeine - induced ( “ caf- 5 7. Add 1.9 g magnesium stearate and mix for 3 minutes . feine -relaxed " ) subjects . 8. Compress the mixture into sublingual tablets , each Method : containing about 3 mg melatonin immediate release and 3 The same open - field method was used as in Example 3 . mg controlled release melatonin . However, in this study, the parameter that was measured was Example 7 the " center time” that is , the time that the rats spend in the 10 center of tested arena . The center time was used as an index Preparation of Sublingual Tablets Containing for the anxiolytic effect of the test substances in both Immediate and Slow Release Melatonin and “ base - line ” and “ caffeine - relaxed ” rats . Valerian Extract Results : The effect of the test substances is shown in FIG . 4. Itmay 15 1. Dissolve in 100 g ethanol: 2.9 g melatonin , 3.8 g be seen from this graph that in the “ base - line ” animals ( i.e. valerenic acid ( 38 g valerian extract, 10 % valerenic acid ), those that had not received caffeine ; left- hand bar of each 9.6 g Vitamin E TPGS and 19.2 g PVP K25 . pair of bars ) , both melatonin and valerian ( when adminis 2. Mix together: 108 g lactose and 200 g mannitol. tered separately ) increased the mobility time of the rats (in 3. Add the granulation solution from stage 1 and granulate comparison to the vehicle control ), indicating they both 20 for 1 minute in a high shear granulator , Diosna 1 type. possess anxiolytic effects . However, in the case of the 4. Dry the wet granulate in a fluid bed dryer for 45 caffeine - relaxed rats ( right- hand bar of each pair of bars ) , minutes with an inlet temperature of 60 degrees C. only valerian (both when used separately and when com 5. Screen through a 0.8 mm sieve . bined with melatonin ) affected caffeine induced center 6. Add to the sieved mixture : 24 g of the controlled release 25 minitabs prepared in Example 5 , 19 g stevia sweetener, 24 motility . g starch 1500 (pregelatinized ) , 5.6 g menthol crystals and These results suggest that melatonin may operate as an 3.8 g aerosol 200. Mix for 10 minutes . anxiolytic and agent via different receptors and 7. Add 1.9 g magnesium stearate and mix for 3 minutes . pharmacological mechanisms than those used by valerian , 8. Compress the mixture into sublingual tablets each and that these two substances interact synergistically when containing about 3 mg melatonin immediate release, 4 mg used in combination . 30 valerenic acid and 3 mg controlled release melatonin . 9. The tablets are further coated with Sepifilm LP 770 PREPARATIVE EXAMPLES humidity protecting coating . Example 5 Example 8 35 Preparation of Controlled Release Melatonin Preparation of Sublingual Tablets Containing Minitabs Immediate and Slow Release Melatonin , Rosemary Extract and Valerian Extract 1. Dissolve 12 g melatonin together with 12 g Vitamin E TPGS, 8 g polyvinylpyrrolidone (PVP K25 ) in 150 g abso- 40 1. Dissolve in 100 g ethanol : 2.9 g melatonin , 3.8 g lute ethanol. valerenic acid ( 38 g valerian extract, 10 % valerenic acid ) , 9.6 g Vitamin E TPGS and 19.2 g PVP K25 . 2. Mix together 40 g Hydroxypropylmethyl cellulose 2. Mix together : 108 g lactose , 12 gr rosemary extract (HPMC K15M ), 24 g HPMC E50 , 20 g Kucel® LF, 40 g powder (25 % carnosic acid ) and 200 g mannitol . Avicel® pH 101. 3. Add the granulation solution from stage 1 and granulate 3. Add the melatonin solution from stage 1 and granulate 45 for 1 minute in a high shear granulator, Diosna 1 type . in high shear granulator for 1 minute . 4. Dry the wet granulate in a fluid bed dryer for 45 4. Dry the wet granulate in Glatt fluid bed dryer, inlet minutes with an inlet temperature of 60 degrees C. temperature 60 degrees C. for 45 minutes . 5. Screen through a 0.8 mm sieve . 5. Sieve the dry granules through a 0.8 mm sieve . 6. Add to the sieved mixture : 24 g of the controlled release 6. Add 2 g aerosol and mix for 5 minutes. Add 1 g 50 minitabs prepared in Example 5 , 19 g stevia sweetener , 24 magnesium stearate and mix for 1 minute . g starch 1500 (pregelatinized ), 5.6 g menthol crystals and 7. Compress the granules into 2 mm controlled release 3.8 g aerosol 200. Mix for 10 minutes. minitabs . 7. Add 1.9 g magnesium stearate and mix for 3 minutes. 8. Compress the mixture into sublingual tablets each Example 6 55 containing about 3 mg melatonin immediate release , 4 mg valerenic acid and 3 mg controlled release melatonin . Preparation of Sublingual Tablets Containing 9. The tablets are further coated with Sepifilm LP 770 Immediate and Slow Release Melatonin humidity protecting coating . 1. Dissolve in 100 g ethanol : 2.9 g melatonin , 9.6 g 60 Example 9 Vitamin E TPGS, 19.2 g PVP K25 . 2. Mix together: 108 g lactose , 204 g mannitol. Preparation of Sublingual Tablets Containing 3. Add the granulation solution from stage 1 and granulate Immediate and Slow Release Melatonin and for 1 minute in high shear granulator, Diosna 1 type . Passiflora Extract 4. Dry the wet granulate in a fluid bed dryer for 45 65 minutes with an inlet temperature of 60 degrees C. 1. Dissolve in 100 g ethanol : 2.9 g melatonin , 9.6 g 5. Screen through a 0.8 mm sieve . Vitamin E TPGS and 19.2 g PVP K25 . US 10,675,269 B2 15 16 2. Mix together: 108 g lactose , 104 g mannitol and 100 g Melatonin : carnosic acid : valerenic acid Passiflora incarnate extract. 1 : 1.5 : 2 . 3. Add the granulation solution from stage 1 and granulate 5. The composition according to claim 1 , wherein the for 1 minute in a high shear granulator, Diosna 1 type . melatonin , valerenic and carnosic acid are each provided in 4. Dry the wet granulate in a fluid bed dryer for 45 5 a form selected from the group consisting of synthetic minutes with an inlet temperature of 60 degrees C. compounds , purified compounds obtained from plant mate 5. Screen through a 0.8 mm sieve . rial , partially purified extracts of plant material and crude 6.Add to the sieved mixture: 24 g of the controlled release extracts of plant material . minitabs prepared in Example 5 , 19 g stevia sweetener , 24 6. A method for treating and /or preventing inflammatory g starch 1500 (pregelatinized ) , 5.6 g menthol crystals and 10 disorders in a mammalian subject, comprising administering 3.8 g aerosol 200. Mix for 10 minutes. a composition according to claim 1 . 7. Add 1.9 g magnesium stearate and mix for 3 minutes. 7. The methods according to claim 6 , wherein the mam 8. Compress the mixture into sublingual tablets each malian subject is a human subject. containing about 3 mg melatonin immediate release , 100 mg 8. A method for treating and /or preventing sleep disorders Passiflora incarnate and 3 mg controlled release melatonin . 15 in a mammalian subject, comprising administering a com The invention claimed is : position according to claim 1 . 1. A composition comprising a synergistic combination of 9. A method for treating and /or preventing anxiety in a melatonin , valerenic acid and carnosic acid . mammalian subject, comprising administering a composi 2. The composition according to claim 1 , wherein the tion according to claim 1 . melatonin , valerenic acid and carnosic acid are present in a 20 10. A method comprising preparing a medicament includ molar ratio of: ing a composition according to claim 1 . Melatonin :Carnosic acid : Valerenic acid 11. The method according to claim 10 , further comprising 1 : 0.1-10 : 0.5-10 . administering the medicament for treatment and /or preven 3. The composition according to claim 1 , wherein the tion of inflammatory disorders. melatonin , valerenic acid and carnosic acid are present in a 25 12. The method according to claim 10, further comprising molar ratio of: administering the medicament for treatment and /or preven Melatonin : Carnosic acid : Valerenic acid tion of sleep disorders . 1 : 0.5-5 : 1-5 . 13. The method according to claim 10 , further comprising 4. The composition according to claim 1, where the administering the medicament for treatment and / or preven melatonin , valerenic acid and carnosic acid are present in a 30 tion of anxiety . molar ratio of: