WO 2008/027395
`
`PCT/U82007/018942
`
`Further, formulations without PEG also appeared to be preferred in order to improve
`
`clonazepam stability.
`
`[00205]
`
`Addition of an anti-oxidant to the formulations of the present invention
`
`used for intranasal delivery of benzodiazepines may provide desirable protective benefits
`
`to such formulations. Examples of a suitable anti-oxidants include, but are not limited to,
`
`tocopherol and derivatives thereof, ascorbic acid and derivatives thereof,
`
`butylhydroxyanisole, butylhydroxytoluene, fumaric acid, malic acid, propyl gallate,
`sodium sulfite, metabisulfites (including sodium metabisulfite) and derivatives thereof, as
`
`well as EDTA disodium, trisodium and the tetrasodium salts. Soluble, organic anti-
`
`oxidants are preferred, for example, butylhydroxytoluene.
`
`[00206]
`
`Further, the data indicate protective effects resulting from the inclusion of
`
`pH modifiers when an aqueous solvent was used. Microbial challenge with 5 organisms
`
`(staphylococcus aureas, pseudomonas aeruginosa, escherichia coli, candida albicans and
`
`aspergillus niger) showed a log plate count of less than 1/mL observed after a period of
`28 days, indicating that the liquid formulation itself is microcidal and therefore a non-
`
`sterile product is likely acceptable.
`
`Example 4
`
`Screening Formulations for Nasal Irritation Potential Using a Rat Model
`
`[00207]
`
`A number of formulations were tested in a rat irritation model. The first
`
`objective was to establish the irritation threshold of Transcutol®. Two formulations were
`
`tested containing 20% and 50% Transcutol® in PEG 200. The blood pressure signals
`
`integrated as a fimction of time were as shown in Figure 3.
`
`[00208]
`
`In Figure 3, the designations were as follows: CLZ2080 -- 10 mg/mL
`
`clonazepam, 20% Transcutol® (TC), 80% Polyethylene Glycol (PEG); CLZSOSO -- 10
`
`mg/mL clonazepam, 50% TC, 50% PEG; CLZ7OG30T -- 10 mg/mL clonazepam, 70%
`
`GF, 30% TA; CL220T8OP02T, 10 mg/mL clonazepam, 10% TC, 90% PEG 200 and
`
`0.2% TWEEN 20; Saline (negative control); Acetic Acid (HOAc) 0.3% (positive
`
`irritation control); Acetic Acid (HOAc) 1.5% (positive irritation control); Setron (positive
`
`irritation control).
`
`[00209]
`
`The data shown in Figure 3 demonstrated slight, transient irritation
`
`apparent in the test animals. After instillation of compositions, irritation typically lasted
`
`51
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2451
`
`page 2451
`
`

`

`WO 2008/027395
`
`PCT/U82007/018942
`
`less than 1.5 minutes in rats (range 0.7 to 2.2 minutes). Irritation was generally greater
`
`than saline and similar to irritation from 1.5% acetic acid. Veterinary evaluation of the
`
`data resulted in the conclusion that nasal irritation from these formulations was not
`
`significant.
`
`[00210]
`
`Two other clonazepam formulations were tested (70% PEG and 30% GF;
`
`and 10% TC, 90% PEG 200 and 0.2% Tween 20) and similar results were obtained.
`
`Tween 20 (polyethylene glycol sorbitan monolaurate) was used as a possible irritation
`
`reducer.
`
`[00211]
`
`One formulation, CLZSOSO appeared to produce more intense irritation
`
`than the other formulations as instillation was associated with a blood pressure drop. The
`
`drop biased the drawing of a base line and therefore the integration of the signal.
`
`[00212]
`
`In a second rat nasal irritation experiment, eight clonazepam formulations
`
`and one formulation matrix without clonazepam (K: 30% TA, 70% GF) were tested in the
`
`irritation model and compared with irritation results obtained using 0.9% acetic acid. The
`
`formulations used in and results from the rat nasal irritation study are presented in Table
`
`11. In the table, Iden. -— is the identifier associated with the formulation; MBP —-
`
`integrated mean blood pressure over the duration of the irritation response; T -- duration
`
`of irritation response (minutes); TC — Transcutol®; PEG -- polyethylene glyco; TA --
`
`triacetin; GF —— glycofurol; PG -- propylene glycol; H20 -- water; Tw -— TWEEN 20; w/o
`
`clz = without clonazepam. Fifty uL of each formulation containing 20 mg/mL
`
`clonazepam was administered to each animal.
`
`52
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2452
`
`page 2452
`
`

`

`WO 2008/027395
`
`PCT/U82007/018942
`
`Table 11
`
`Rat Nasal Irritation Study
`E
`i
`h; 6‘
`Formulation
`><.
`HO.OB
`0an
`.
`TC
`
`TA GF
`
`PG H20
`
`PEG
`
`9993999NNm;_NNN NtoNANNNNMaxNMNNNHmm
`.0.O‘-lNN€
`.HHHHIHHII
`v—-N—-—-—-o'0‘i»is.)mmuxi
`\l(Ab)b.)b.)\l
`K w/o-- L»)b.)OO
`
`
`
`r—‘
`
`O
`
`_
`
`_O
`
`nu
`
`- a
`
`n---
`
`--
`_-
`
`_-
`m-
`m-
`
`A _o W
`a = blood pressure drop must be due to intense irritation. The drop biases the drawing of
`a base line and therefore the integration of the signal.
`b = the third animal in the group reacted very strongly to administration, received cardiac
`resuscitation. The results from this animal were not included in the data processing.
`0 = numbers in parentheses are used when n < 3.
`d = includes citrate buffer pH4, and sodium metabisulphite.
`
`Sodium metabisulfite and citric acid were each present at less than 1% (w/w) basis in the
`above formulations.
`
`[00213]
`
`Acetic acid 0.9% has been found to be tolerated by volunteers in a human
`
`trial. The objective of this experiment was to provide a preliminary test a variety of
`
`formulations and compare them with respect to irritation. Because a slightly irritating
`
`profile would be tolerated for an intranasal formulation against seizure clusters and other
`
`acute indications such as panic attacks,
`
`the major concern was that volunteers
`
`participating in a clinical Phase I trial would not suffer unnecessary pain. The irritation
`
`scores based on measurement of blood pressure are presented in Figure 4 and Table 11.
`
`In Figure 4, the columns for saline, acetic acid solutions and a setron formulation (i.e., the
`
`right-most four columns) represented data from previous experiments and were inserted
`
`for comparison. Table 11 also present the duration of the irritation response in minutes.
`
`The results showed that all formulations tested gave a relatively short—lived irritation
`
`response, in the range from 0.7 to 2.2 minutes.
`
`[00214]
`
`To test the irritation of Transcutol®, two formulations were tested
`
`containing 20% and 50% Transcutol® with PEG 200 as the cosolvent. Transcutol® at
`53
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2453
`
`page 2453
`
`

`

`WO 2008/027395
`
`PCT/US2007/018942
`
`20% (I) or 50% (H) demonstrated similar irritation scores suggesting that Transcutol® is
`
`no more irritating than PEG. A third formulation containing Transcutol® was the same
`
`as I but with 0.2% Tween. Comparison of I and] with Tween-20 suggested that Tween
`
`did not provide a substantial reduction of irritation in this formulation.
`
`[00215]
`
`Formulation K (30% TA, 70% GF), which showed a good
`
`pharmacokinetic profile (see Example 5), had the lowest irritation score of the
`
`formulations tested in this experiment.
`
`[00216]
`
`Formulation K was also tested without clonazepam to obtain information
`
`on the effects of clonazepam on imitation. Comparison of the irritation scores of K and K
`
`without clonazepam showed that elonazepam appeared to have an irritation reducing
`
`effect.
`
`[00217]
`
`Formulations M and T contain the same amount of glycofurol but M
`
`contains 70% PEG while T contains propylene glycol as the cosolvent. The irritation
`
`score of these two does not differ significantly indicating a similar degree of irritation by
`
`PG and PEG.
`
`[00218]
`
`Formulation R with 10% buffer (citrate/1‘ween/bisulphite) or with 10%
`
`water was the only water containing formulation tested. The formulation with water only
`
`appeared to be significantly more irritating than the formulation that contained
`
`buffer/Tween/bisulphite. Formulation R with buffer demonstrated similar irritation
`
`profile as did the non-aqueous formulations I, H, M and T.
`
`[00219]
`
`’
`
`Acetic acid 0.9% has been tested for irritation in a human trial and was
`
`found to be irritating but tolerable. All formulations tested except R with water
`
`demonstrate irritation equal or lower than this reference formulation (Figure 4) suggesting
`
`that they had minor irritation but were tolerable.
`
`[00220]
`
`These nasal irritation data suggested that the clonazepam formulations of
`
`the present invention were suitable for intranasal delivery.
`
`Example 5
`
`Pharmacokinetics and Tolerability
`
`[00221]
`
`Different clonazepam formulations were delivered intranasally and
`
`intravenously to rabbits. Many of the administrated formulations demonstrated intranasal
`
`54
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2454
`
`page 2454
`
`

`

`WO 2008/027395
`
`PCT/U82007/018942
`
`bioavailability higher than 70% that of the intravenous formulations. Those that
`
`contained Transcutol® at concentrations 20-100% demonstrating that Transcutol® was a
`
`useful absorption enhancer and solvent for clonazepam
`
`[00222]
`
`Additionally, the intranasal clonazepam formulations containing
`
`Transcutol® in the concentration range 20-lOO% yielded pharmacokinetic (PK) profiles
`
`with tmax lower than 4 minutes. The absorption enhancing effects of Transcutol® were
`
`also demonstrated by this performance index.
`
`[00223]
`
`Some exemplary phannacokinetic data for clonazepam formulations is
`
`presented in Table 12A (N=1 for each formulation). In the table, Tw or tween is
`
`TWEENZO, EtOH is ethanol, Triac or TA is Triacetin, phosph is phosphate buffer,
`metabisulph is sodium metabisulphite, citr is citrate buffer, AUC is area under the curve
`
`me is minutes, Cmax is in ng/ml, and F% is bioavailability of the intranasal formulation
`
`as compared to the intravenous formulation-- other abbreviations are as used herein
`
`above.
`
`55
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2455
`
`page 2455
`
`

`

`WO 2008/027395
`
`PCT/U82007/018942
`
`LL
`
`D°O
`
`
`.:I%°‘
`
`\°
`
`v <2-
`0: Ox
`
`"‘1
`Q m a co 0l\
`
`Egg
`
`:5:a:aE:5:Na:!anE:a:I$2$2I“£23v'5
`gaglmllll
`.xNSN23iSS
`<323gaNa;
`EE'H-EEI'IIagaaglfilll
`
`EII:ala22
`$8anlEa;
`$393!Ea;
`:3Elm-EH
`IEEIi—‘i
`{NewNNEEagllla
` 5&0mo3HUmm0OH(Pamum
`NEEEII'
`NEEIIII
`EEIIII
`Mglolllgll
`III-
`III
`
`.ollllillfi:3+=quxNo+,5:2
`
`
`
`
`
`5.5ozouiooafihaamE;gown—55.8%Savage—UBan—3m
`
`<23an
`
`saga
`
`2°!N
`
`O
`l‘
`
`m m
`
`m
`
`O
`l‘
`
`m
`
`00
`0°06
`
`OO O
`NN I11
`
`2:
`
`GO
`WN
`
`O
`In
`
`m
`
`M
`
`m
`
`m
`
`M
`
`ONI$S+OH$8+STF$°M
`
`Buic$2+0h$oo+<b$om
`
`Uh$oe+0m$ov
`
`3m£0$3+oh$oo+<r~$om
`
`$2.+UH$$+«F$cm
`
`v2.28:0$2+523
`
`$3+EH83?$3
`
`ON:$2+OH$3
`
`@8385;
`
`$3+552:;$3
`
`@6385;$2:
`
`®_8=om=8._.
`
`®_2=om§¢$2:
`
`UH$om+m0$3
`
`UH$om+"a$3
`
`W
`
`UH$ow+ocNOmE$8
`
`O~:$c_+0H$om+de~$9m
`
`:.mo:.$o#+0H$oo+<rr$om
`
`Uk$oo+0m$ov
`
`8-503%$m+“—0$3
`
`ON:$2+Up.$3
`
`UH$3+m0$om
`
`m0$3+<H$om
`
`_o._£oa5$2:
`
`56
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2456
`
`page 2456
`
`
`
`
`
`
`
`

`

`WO 2008/027395
`
`PCT/U82007/018942
`
`
`
`
`
`I'llIII-Ell!
`
`EEEEIIIIIEIE
`IllEEIEIEII
`
`I'll-IIIIII'I8”Q:
`EEEEEIIIIEIII"6$2:
`
`EggaaEIE'mo:2+Ex:
`EEAEE'IIIEIII!
`3““a:a283HE8IEEéssuésoataoxs
`N:
`
`II"
`
`goo+:85gmd+no$2
`
`comum:
`
`x8+5233.2+8x2
`
`8Nom:
`
`+zoflm§m+u§c~+o§a
`
`a.smsfiésoatgoxe
`
`E9m§N+o§8+o§a
`
`EEHEEEIIIIEE.
`
`I'llIIIIIEIE
`Egggglllllllggm2:x2:
`
`
`EEEEEIII'IIIE8“om:5:
`
`88m§81§2EE'HEEIIIIIHIIIE28m:sale:2Eggg:gIIIIIEIIIE
`
`
`.g:2”ES208oa$5925an
`
`
`
`$3+:uoEXRd+OH$0—vI.3920o\oo#+:00?“
`
`
`
`o\°m.o+Proaoe+«a.foam
`
`852.88me
`
`com0mm
`
`EEEEEII
`
` 8.c1“.ocmd--E-E
`EEQEHIIIEEIII3
`
`Ecstguxoiofixslo
`
`$2
`
`8“cm::3+no0%
`
`EonoxoM
`
`4.E552.
`
`:§2+88m§8¢o§mE”QE38gom5.3553592:
`
`g§2+88m§8£o§m
`
`57
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2457
`
`page 2457
`
`
`
`
`
`
`

`

`WO 2008/027395
`
`PCT/USZOO7/018942
`
`
`
`
`
`.maocflspzom“Sons05E2mgA55.x;55mm“:a£085:08Eu?3%0E62805—3532:838m
`
`
`
`
`
`83
`
`8mm
`
`96
`
`SN!
`
`x
`VIn
`
`82‘In
`
`.—
`
`00
`ON
`
`°\"01
`
`N °
`
`N
`
`04".
`
`O¢“
`
`5O
`
`fig
`
` EEIHIE
`x:8»!2m3x:5%2a8a.afiassosaaabeoxa
`:2amIRE85%R»l«.2s
`
`
`u--I-£:..smBSoEES9EIIIgH—IIom:5:8530:2acom.o
`
`
`
`
`HIHIEl
`1NNHE "a
`:Sil
`\:13V.
`gl-""
`
`°OVl\
`
`mN
`
`'
`
`“201V.
`noun»
`
`0<
`
`:>
`3
`
`N:
`
`icmxaémmxoniwxe
`
`r—.EmBMwDEEOOBbbmugo
`
`Exchioxa
`
`o§2¢o§m
`
`$2
`
`Esztasxeémmxaio
`
`esmssoe
`
`Iomxaémmxomioxs
`
`\cooBtEuXoiOmmgowlo
`
`$2
`
`58
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2458
`
`page 2458
`
`
`
`
`
`
`
`

`

`WO 2008/027395
`
`PCT/U52007/018942
`
`[00225]
`
`Some clonazepam formulations without Transcutol® also provided a rapid
`
`rise in blood levels post-intranasal closing including, for example, 95% GF, 5% Tween-20,
`
`100% GF, 10%GF, 90% PEG, 100% PEG and 30% TA, 70% GF.
`
`[00226]
`
`The pharmacokinetic data presented above illustrated that clonazepam
`
`compositions formulated for intranasal administration are pharmaceutically efficacious to
`
`deliver clinically relevant amounts of clonazepam into the bloodstream in a short time period
`
`-- making such intranasal formulations clinically useful, for example, for the treatment of
`
`seizure clusters. Such clonazepam compositions comprise, for example, one or more
`
`solvents selected from the group including, but not limited to, Transcutol®(diethylene glycol
`
`monoethylether) and similar alkylethers, propylene glycol, triacetin, Glycofurol (ethoxylated
`
`furanyl alcohol or tetrahydrofurfuryl alcohol polyethyleneglycol ether) and similar
`
`ethoxylated tetrahydrofurfuryl alcohols, as well as polyethylene glycol (e. g., PEG 200, PEG
`
`300, etc.). However, as noted above, free PEG polymers lead to reduced stability of
`
`clonazepam formulations.
`
`[00227]
`
`The data shown in Table 12 above were reanalyzed. The rabbit pilot PK
`
`experiments had been performed in two groups of ten animals, JCOl (Group 1) and JC02
`
`(Group 2). The JC01 experiments were performed in a group of rabbits which were older
`
`and heavier than the J C02 group of rabbits. Each group of rabbits had their own set of
`
`intravenous clonazepam PK data for the calculations of bioavailability.
`
`[00228]
`
`The intranasal formulations were 4 mg/mL. The animals were administered 25
`
`[LL of formulation to each nostril, 50 uL in all, with an Eppendorf dosing pipette. The animal
`
`was held in a supine position while being dosed and for about 10 seconds after. The
`
`intravenous formulation, Rivotril® injectable, was administered as SOOuL injected over 30
`
`seconds into the marginal ear vein on opposite site to the blood sampling ear. All rabbits
`
`received 0.2 mg clonazepam.
`
`[00229]
`
`Five formulations were tested on each study day, where each of the
`
`formulations was administered to two rabbits. The data were analyzed before the
`
`composition of the formulations administered to the next group of animals was decided.
`
`59
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2459
`
`page 2459
`
`

`

`WO 2008/027395
`
`PCT/USZOO7/018942
`
`[00230]
`
`Due to the different body weights of the two rabbit groups, the Cmax and the
`
`60 minute AUC results from the two groups were not directly comparable. The relative
`
`bioavailability was corrected for weight differences between the two groups, based on results
`
`of the IV administrations to each group. The me was not directly comparable between the
`
`two groups, but was included in the table as a relative indication peak levels within each
`
`group.
`
`Table 12B: Example Clonazepam Formulations and Pharmacokinetic Data
`
`Group 1 Rabbits
`
`Fo’rm'ulation
`ID
`
`V
`V
`‘
`'
`IV m i
`_———_~~—_—_*_
`832 'Um0 u8
`
`V
`
`n
`
`‘
`
`
`Rabbit no.
`'21-i5
`21
`
`
`
`
`100% ,
`
`62%
`
`44%
`
`
`
` NNNNEim‘mu
`
`
`100% PEG 200
`
`100% Glycofurol
`
`27
`
`23
`28
`
`
`
`
`
`N ‘1
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2460
`
`page 2460
`
`

`

`WO 2008/027395
`
`PCT/USZOO7/018942
`
`
`
`Group 2 Rabbits
`
`
`
`
`"In. -
`—._ m
`%=m
`
`Relative
`BA
`
`100%
`
`53%
`
`73%
`
`.
`
`39.7
`
`0.212
`0.216
`
`
`
`IV-
`
`80% GF + 20% TC
`
`
`
`F G
`
` J—
`
`73%
`69%
`
`29.6
`17.0
`
`78%
`79%
`37
`I“ ,
`50% en 50% TC _
`u ”‘3
`IE- 25%
`.
`51%
`
`70% GF+20% TC+10°/o H20
`
`20%
`
`‘i-
`“Ii-IE“ 105"!
`90% TC + 10% H20
`76%:I
`
`
`
`84%
`30%Triac.+60%TC+10%H20
`
`103 °/n
`
`47.2
`94%
`
`50% PEG200+50% TC
`
`
`80%PEGZOO+20%TC
`
`53%
`
`
`95% GF + 5% Tween-20
`
`
`30% Triacetin + 70% thcofurol
`
`
`40%PG + 60%TC
`
`
`30°loGF+70%PEG200
`
`
`[ [
`
` L
`
`24.9
`
`84%
`
`-‘—m%
`
`m 85%
`
`J48%
`In
`105%
`33. 7
`
`-so 9
`30°loTA+60°lnTC+1 0%citrate
`94%
`
`19 a
`76% _
`72%
`
`30%TA+60%TC+10°/ophosph.
`
`
`61
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2461
`
`page 2461
`
`

`

`WO 2008/027395
`
`PCT/U82007/018942
`
`10% GF 4- 80% PEGZDO + 10% cilrltweenlmetab
`
`10% GF + 50% Peczoo + 30% PG + 10%
`citrltweenlmetab
`
`30% GF + 60% PG + 10% citr/tweenlmetab
`
`
`
`
`
`30% GF 4- 70% PG
`
`50% PG + 20% EtOH + 20% TC + 10% citrltween/metab
`
`Note: Tw or tween is TWEENZO, EtOH is ethanol, Triac or TA is Triacetin, phosph is phosphate buffer,
`
`metabisulph is sodium metabisulphite, citr is citrate buffer.
`
`[00231]
`
`As exemplified in Tables 12A and 123 above, the composition may comprise
`
`a solvent matrix of two solvents, for example, a first solvent that provides high solublization
`
`of clonazepam (for example, TC or GF) that, after application to nasal mucosa, is absorbed
`
`by the nasal mucosa leading to clonazepam super saturation, and a second solvent (for
`
`example, TA or PG) in which clonazepam has lower solubility relative to the first solvent.
`
`In
`
`preferred embodiments, the compositions are substantially non-aqueous or anhydrous;
`
`however, the compositions may further comprise an aqueous component (for example, of less
`
`than about 10% aqueous content, preferably of less than about 5% aqueous content, more
`
`preferably of less than about 2% aqueous content, wherein the aqueous content is preferably
`
`buffered with a physiologically acceptable buffer to obtain a pH range of about pH 4 to about
`
`pH 7, preferably between about pH 4 to about pH 6.5). The benzodiazepine compositions of
`
`the present invention may comprise further components as well, for example, anti-oxidants
`
`(for example, sodium metabisulfite or butylhydroxytoluene (BHT). Preferred embodiments
`
`typically do not include polyethylene glycol polymers as a solvent but may include solvents
`
`like tetrahydrofurfiu’yl alcohol polyethyleneglycol ether (Glycofurol) wherein the solvent
`
`molecules contain polyethylene glycol polymers as an intrinsic part of their molecular
`
`structure, that is, polyethylene glycol polymers as substituent groups of a larger chemical
`
`62
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2462
`
`page 2462
`
`

`

`WO 2008/027395
`
`PCT/US2007/018942
`
`structure (also, see, for example, published P.C.T. International Patent Application Nos. W0
`
`03/070273 and W0 03/070280).
`
`[00232]
`
`The pharmacokinetics and tolerability of four clonazepam compositions
`
`comprising binary solvent systems were further evaluated. The four formulations were as
`
`follows in Table 13.
`
`[00233]
`
`Table 13
`
`Compositions of binary solvent systems (10 mg/mL clonazepam)
`
`Solvent S stem
`
`-_
`50% dieth lene 1 col monoeth lether + 50% triacetin
`_]- 50% dieth lenegl col monoeth lether+ 50%001‘0 lene _1 col
`‘1— 50% _1 cofuml + 50% triacetin
`50% l cofurol + 50% ro- lene g1 col
`
`
`
`
`
`
`
`[00234]
`
`The pharmacokinetics of the formulations in Table 13 were evaluated by nasal
`
`administration to rabbits and compared to intravenous (i.v.) administration of clonazepam in
`
`rabbits. Sample size for each formulation was N=1O with instillation of 10 mg/mL
`
`clonazepam dose adjusted to body weight. A summary of the data is presented in Figure 5.
`
`[00235]
`
`The data is further summarized in Table 14.
`
`Table 14
`
`PK Data for Selected Formulations
`
`Dose (mg)
`
`
`
`
`
`
`
`
`
`—_—----
`mm—
`“mm
`
`[00236]
`
`The intranasal PK profiles of the formulations presented above demonstrated a
`
`rapid absorption of clonazepam such that clinically relevant amounts of clonazepam reach
`
`the bloodstream in a short period of time. Short-term bioavailability does not necessarily
`63
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2463
`
`page 2463
`
`

`

`WO 2008/027395
`
`PCT/U82007/018942
`
`need to be high; it is of higher importance that the blood levels become high in as short a
`
`time as possible. Lower bioavailability can be balanced out, for example, with higher dose.
`
`An advantage of a higher dose and low short term bioavailability may be passage of the drug
`
`that is not absorbed intranasally into the gastro-intestinal tract resulting in the remainder of
`
`the drug undergoing classical GI absoprtion leading to a sustained release profile.
`[00237]
`I
`As can be seen from the PK data in rabbits, benzodiazepine compositions of
`
`the present invention formulated for intranasal delivery may be characterized, for example,
`
`by a Tm“ of benzodiazepine, after a single intranasal administration (in one or both nostrils),
`
`of 2 hours, ofien less than 1 hour likely less than 30 minutes or less than 15 minutes. Further,
`
`pharmaceutical compositions of benzodiazepines for intranasal delivery, as described herein,
`
`may be characterized, for example, by providing at least one of a mean maximum plasma
`
`concentration (me) of benzodiazepine of at least about 3.0 ng/mL or at least about 15% of
`
`the concentration of an intravenously delivered dose often 30% of an intravenously delivered
`
`dose or 50% or an intravenously delivered dose, and a mean plasma Area Under the Curve
`
`over 60 minutes (AUC) value ofclonazepam of at least about 400 ng-hr/mL, when a single
`
`dose of the composition is administered intranasally to deliver a dose of at least about 0.2 mg
`
`of clonazepam. Further, the bioavailability of benzodiazepine compositions of the present
`
`invention, after intranasal administration, is typically greater than 30% often greater than
`
`40% and frequently greater than 50% of that of intravenous administration.
`
`[00238]
`
`In addition to the PK parameters discussed above, the experiments performed
`
`in support of the present invention evaluated the local tolerance in the upper and lower
`
`respiratory tract of formulations I-IV containing clonazepam as active drug. This tolerance
`
`was assessed in the rabbit as model. Treatments were performed during seven consecutive
`
`days before histopathological evaluation of selected tissues.
`
`[00239]
`
`The rabbits used in these experiments were as follows: Breed, New Zealand
`
`White; Sex, 30 males and 30 females; Weight, Mean body weight 2.466 d: 0.093 (SD) kg for
`
`the male rabbits, 2.465 :I: 0.114 (SD) kg for the female rabbits. Animals showing any
`
`concurrent disease at the time of the treatment were not included. Rabbits were obtained
`
`from Charles River Laboratories, L’Arbresle Cedex, France.
`
`64
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2464
`
`page 2464
`
`

`

`WO 2008/027395
`
`PCT/U82007/018942
`
`[00240]
`
`Animals were weighed during the acclimatisation period for allocation, within
`
`the 3 days prior to treatment and just before slaughter. The dose-level of 10 mg/mL (1 mg
`
`Clonazepam in 100 uL solution) was selected to be comparable to an anticipated dose to be
`
`administered in humans.
`
`[00241]
`
`The treatment groups are detailed in Table 15. Formulation 5 is a vehicle
`
`control -- 50 % glycofurol; 50 % propylene glycol (with no Clonazepam). Formulation 6 is a
`
`saline control (0.9% NaCl in water).
`
`Table 15
`
`Allocation of treatments into groups
`
`Group
`
`Treatment
`
`Nmber Of
`animals
`
`1
`
`2
`
`3
`
`4
`
`Formulation l
`TC/TA+
`
`Formulation II
`TC/PG+
`
`5 males
`5 females
`
`5 males
`5 females
`
`Formulation III
`GF/TA+
`
`5 males
`5 females
`
`Formulation IV
`GF/PG+
`
`5 males
`5 females
`
`5
`Formulation 5
`5 males
`
`GF/PG-
`5 females
`Formulation 6
`5 males
`S-
`5 females
`
`6
`
`Concentration of
`Clonazepam
`[ngng
`10
`
`10
`
`10
`
`10
`
`0
`
`0
`
`Number Of
`treatments
`
`7
`
`7
`
`7
`
`7
`
`7
`
`7
`
`[00242]
`
`The selected route of administration was the route of administration of the
`
`final product.
`
`[00243]
`
`Whatever the formulation, 0.] mL of the formulation was daily administered
`
`to all animals by nasal instillation during seven consecutive days.
`
`[00244]
`
`All administrations were performed in the right nostril using a 1 mL pipette
`
`(Bl3, Adjustable pipettes Pipetman P200 from Gilson) fitted with a plastic cone. The
`
`required volume of item was measured with the pipette and placed just inside the nostril of
`
`the animal.
`
`65
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2465
`
`page 2465
`
`

`

`WO 2008/027395
`
`PCT/U82007/018942
`
`[00245]
`
`Treatment details were recorded in the raw data including dose administered,
`
`formulation identification, date and time of administration.
`[00246]
`Six animals per group, three males and three females at Day 8, and the
`
`remaining animals at Day 15, after a seven-day recovery period, were sacrificed by ex-
`
`sanguination from abdominal aorta under isoflurane anaesthesia-
`
`[00247]
`
`Following euthanasia, macroscopical examination of larynx, trachea, bronchi,
`
`lungs and oesophagus were performed.
`
`,
`
`[00248]
`
`The head of the animal, with the larynx and specimens of trachea, bronchi,
`
`lungs and oesophagus were taken at necropsy and fixed in formalin for histopathology.
`
`[00249]
`
`From head, nasal mucosa, turbinates, in addition to larynx and trachea were
`
`sampled afier specific preparation and examined. Any observed macroscopic abnormalities
`
`or lesions were also sampled and fixed, with a border of surrounding tissue, for
`
`histopathology.
`
`[00250]
`
`Nasal mucosa and turbinates were examined in the nasal cavities on three
`
`head sections corresponding to nasal cavities proximal, nasal cavities turbinates and nasal
`
`cavities olfactory.
`
`[00251]
`
`Histopathological examinations were performed and the results evaluated by a
`
`pathologist. All results were tabulated per group, means and standard deviations were
`
`calculated on each organ. Statistical comparisons were performed between group using
`
`ANOVA. There were no obvious differences in. growth between groups.
`
`[00252]
`
`Severity of the eventual modifications observed in the histological
`
`preparations were scored by the pathologist as follows: 0, no lesions; 1, slight; 2, moderate;
`
`and 3, severe.
`
`[00253]
`
`Figure 6 summarizes the histopathology results for the nasal cavities of the
`
`animals. Severity scores in group 3 was statistically higher than scores of groups 4, 5 and 6
`
`(p=0.003). Irritative modifications like erosion and fibrino-leucocytic material in turbinates
`
`lumen were observed mainly in group 3 (2/3 females and 1/3 males), also for group 1 (1/3
`
`females) and group 5 (2/3 males) but not for other treated or control groups. These
`
`modifications were not observed in necropsy on day 15. Mild epithelial atrophy on
`
`turbinates was noted in necropsy on day 8 and also in necropsy on day 15 mainly for treated
`
`66
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2466
`
`page 2466
`
`

`

`WO 2008/027395
`
`PCT/USZOO7/018942
`
`group 1 and slighter for other treated groups. Control group 6 showed no epithelial atrophy.
`
`Blood was sometimes observed in aerian lumen for larynx and also nasal cavities both in
`
`treated and control groups and are probably of traumatic origin. The best local tolerance was
`
`observed for treated group 2 and 4. These results indicate generally good nasal tolerance for
`
`the tested formulations.
`
`[00254]
`
`Blood or petechia were found in larynx on 15 animals (2 from group 1, 4 from
`
`group 2, 3 from group 3, 2 from group 4, 4 from group 5) during necropsy and on 6 animals
`
`(1 from group 2, 2 from group 4, 3 from group 5) at histopathology examination. Table 16
`
`presents mean and SD severity scores in each group.
`
`Table 16
`
`Mean and SD severity score on larynx in each group
`
`Group 4 Group 5 Grouv 6
`l
`—mmm 0 2
`0-3
`mi-m—m 0-5
`
`Slight epithelial desquamation were observed on oesophagus from two animals from group 4.
`
`Table 17 presents mean and SD severity scores in each group.
`
`Table 17
`
`Mean and SD severity score on oesophagus in each group
`
`l
`
`—mmm—mm
`“mm-lumina-
`
`[00255]
`
`Petechia or blood were observed during necropsy on 17 animals (4 from
`
`group 1, 3 from group 2, 5 from group 3, 1 from group 4, 4 from group 5). No
`
`histopathological lesions were observed in bronchi and trachea.
`
`[00256]
`
`Lung modifications were observed during necropsy on 17 animals (1 from
`
`group 1, 3 from group 2, 3 from group 3, 1 from group 6). Congestive foci were
`
`histologically recorded on two animals from group 2 at Day 15. Table 18 presents mean and
`
`SD severity scores in each group.
`
`67
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2467
`
`page 2467
`
`

`

`WO 2008/027395
`
`PCT/U82007/018942
`
`Table 18
`
`Mean and SD severity score on lungs in each group
`
`Group ]
`
`Mean scorelllEllillat:
`
`Group 2 Grou 3
`0.2 mum-n
`mun-m
`
`[00257]
`
`Considering the whole respiratory tract, microscopical lesions were mainly
`
`observed in the very upper part, the nasal cavities. As no lesions were recorded in the control
`
`group, it is likely that all the lesions were related to the treatments. Petechia recorded at
`
`necropsy and presence of blood observed during histopathological examination can be due to
`
`a trauma induced by the treatment. In the majority of animals (20 out of the 26 presenting
`
`petechia or blood at necropsy), lesions recorded at necropsy were associated with
`
`histopathological findings. Irritative lesions were observed just after treatment and were not
`
`present afier a one week recovery. Mild epithelial atrophy was observed after a one week
`
`recovery. Considering severity scores, formulation 3 induced significantly the most severe
`
`lesions. Local tolerances of the other formulation were nearly similar.
`
`[00258]
`
`In conclusion, the results of necropsy and histopathological examination,
`
`including comparison of severity scores, suggested that the clonazepam compositions of the
`
`present invention comprising formulations for intranasal delivery have acceptable tolerability
`
`for pharmaceutical use.
`
`Example 6
`
`Sprayability and Viscosig of Solvent Matrices
`
`[00259]
`
`Fourteen representative solvent matrices used for clonazepam formulations
`
`were tested for spray pattern and compared with water. The solvent mixtures were made up,
`
`spiked with minute amounts of Coomassie Brilliant Blue Dye and 100 uL were subsequently
`
`filled into Pfeiffer unit-dose devices (Pfeiffer of America, Princeton, NJ). To measure the
`
`spray pattern, the devices were actuated below a sheet of paper that was located 3 cm above
`
`the spray nozzle. All measurements were made at ambient room temperature (20-25°C).
`
`The smallest (Dmin) and the largest (Dmax) diameter of the blue pattern formed on the sheet of
`
`paper were measured and the results used to calculate the Dmx/Dmin ratio, the area of the
`68
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2468
`
`page 2468
`
`

`

`WO 2008/027395
`
`PCT/U82007/018942
`
`pattern and the average spray angle. The plume area at 3 cm was calculated using the
`
`equation for the area of an ellipse using the half of the two diameters as the ellipse radii.
`
`Viscosity of all formulations was measured using Brookfield DV-I viscometer (Brookfield
`
`Engineering Laboratories, Inc., Middleboro, Massachusetts). The results from the
`
`measurements are shown in Table 19. Table 19 presents data related to sprayability and
`
`viscosity of solvent mixtures used in clonazeparn formulations. Viscosity, plume area at 3
`
`cm, spray angle and Dmx/Dmil1 ratio reflecting the symmetry of the spray plume are
`
`presented.
`
`Table 19
`
`Sprayability and Viscosity
`
`Avg.
`viscosity
`cP ’
`
`Plume area
`
`at 3 cm cm2
`
`Com - osition
`
`80% Glycofurol + 20%
`Transcutol®
`
`50% Glycofurol + 50%
`Transcutol®
`
`95% Glycofurol + 5%
`H20
`
`70% Glycofurol + 30%
`Triacetin
`60% Transcutol® + 30%
`
`TriaCetin + 10% H20
`60% Transcutol® + 40%
`
`Pro lene 1 col
`70% PEGZOO + 30%
`
`H20
`80% PEGZOO + 20%
`
`H20
`90% PEGZOO + 10% .
`
`
`
`H20
`
`_80% PEG200+10%
`GF+10% H20
`50% PEGZOO+30%
`
`PG+10% GF+10% H20
`60% PG+30%GF+10%
`
`H20
`70% PG + 30% GF
`Water
`
`AQUESTIVE EXHIBIT 1004
`
`AQUESTIVE EXHIBIT 1004 page 2469
`
`page 2469
`
`

`

`WO 2008/027395
`
`PCT/U82007/018942
`
`[00260]
`
`While water had a viscosity of 1 .0 CP the solvent mixtures tested range from
`
`5.8 (60% Transcutol® + 30% Triacetin + 10% H20) to 42.8 CF (80% PEG200+10%
`
`GF+10% H20). The viscosity of the solvent mixtures had a negative correlation with plume
`
`area and spray angle and plume asymmetry as shown in Figure 7, Figure 8 and Figure 9.
`
`[00261]
`
`From the data shown in Table 19, Figure 7, and Figure 8 it is evident that the
`
`spray angle became smaller with increasing viscosity of solvent matrix in the standard
`
`Pfeiffer Unit-dose devices. Figure 9 shows that the plume asymmetry remained within the
`
`range 1.0 to 1.2 up to solution viscosity about 20 cP above which irregularity in the plume
`
`shape increased. Visual inspection of the appearance of the spray plume of three solutions in
`
`the viscosity range from 5.8 to 41.7 revealed that a plume was formed and none of them
`
`“squirted.”
`
`[00262]
`
`These results demonstrated that at 20-25°C all solvent matrices tested spray
`
`well from Pfeiffer unit dose devices. The results also suggested that viscosity is a good
`
`predictor of sprayability for the formulations of the present invention. As weather may
`
`dictate substantially different conditions of use, the effect of temperature on viscosity was
`
`determined. A Gilmont falling ball viscometer was filled with diethylene glycol monoethyl
`
`ether and calibrated fo