WO 2008/027395
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`PCT/US2007/018942
`
`Further, formulations without PEGalso 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 mayprovide 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, pseudomonasaeruginosa, 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 productis likely acceptable.
`
`Screening Formulations for Nasal Irritation Potential Using a Rat Model
`
`Example 4
`
`
`
`[00207] A numberof formulations were tested inarat irritation model. Thefirst
`
`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 function 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); CLZ5050 -- 10
`
`mg/mL clonazepam, 50% TC, 50% PEG; CLZ70G30T -- 10 mg/mL clonazepam, 70%
`
`GF, 30% TA; CLZ20T80P02T, 10 mg/mL clonazepam, 10% TC, 90% PEG 200 and
`
`0.2% TWEEN20; 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
`
`apparentin the test animals. After instillation of compositions, irritation typically lasted
`
`51
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`less than 1.5 minutes in rats (range 0.7 to 2.2 minutes). Irritation was generally greater
`than saline and similarto irritation from 1.5% acetic acid. Veterinary evaluation of the
`
`data resulted in the conclusionthat nasalirritation from these formulations was not
`
`significant.
`
`[00210]
`
`Twoother clonazepam formulations were tested (70% PEG and 30% GF;
`
`and 10% TC, 90% PEG 200 and 0.2% Tween 20) andsimilar results were obtained.
`
`Tween 20 (polyethylene glycol sorbitan monolaurate) was used as a possibleirritation
`
`reducer.
`
`[00211]
`
`One formulation, CLZ5050 appeared to produce moreintenseirritation
`
`than the other formulationsasinstillation was associated with a blood pressure drop. The
`
`drop biased the drawing ofa baseline and therefore the integration of the signal.
`
`[00212]
`
`In a secondrat nasalirritation experiment, eight clonazepam formulations
`
`and one formulation matrix without clonazepam (K: 30% TA, 70% GF) weretested in the
`
`irritation model and compared withirritation results obtained using 0.9% acetic acid. The
`
`formulations used in and results from the rat nasalirritation 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 ofthe irritation response; T -- duration
`ofirritation response (minutes); TC — Transcutol®; PEG-- polyethylene glyco; TA --
`triacetin; GF -- glycofurol; PG -- propylene glycol; H2O -- water; Tw -- TWEEN 20; w/o
`
`clz = without clonazepam. Fifty pL of each formulation containing 20 mg/mL
`
`clonazepam was administered to each animal.
`
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`
`Table 11
`
`Rat NasalIrritation Study
`
`
`
`MBP
`T (min
`Formulation
`Q.Q
`} —¢
`1 TC|PEG|TA |GF|PG/|H2
`
`|B
`
`INIA, g=
`aver.|stdev’|Avg
`ojo|gNM[NAL]
`
` So|9/SS/2NoNA}]tNHS)woMN]|—ralon
`_Npoet|poem|ome|>¢ioNNiwa
`
`—_ |S
`
`
`
`
`
`ooNS)iu
`
`PL| 16|(0.32 |26 |11
`
`
`
`
`
`
`2a
`a= blood pressure drop must be dueto intenseirritation. 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.
`c = numbers in parentheses are used when n < 3,
`d = includescitrate buffer pH4, and sodium metabisulphite.
`
`_—
`
`
`
`Sodium metabisulfite and citric acid were each presentat 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 respectto irritation. Because a slightly irritating
`
`profile would be tolerated for an intranasal formulation against seizure clusters and other
`
`the major concern wasthat volunteers
`acute indications such as panic attacks,
`participating in a Clinical Phase I trial would not suffer unnecessary pain. Theirritation
`scores based on measurement of blood pressure are presented in Figure 4 and Table 11.
`
`In Figure 4, the columnsforsaline, acetic acid solutions and a setron formulation(i.e., the
`
`right-most four columns) represented data from previous experiments and wereinserted
`
`for comparison. Table 11 also present the duration ofthe irritation response in minutes.
`
`The results showedthat all formulations tested gavea relatively short-livedirritation
`
`response, in the range from 0.7 to 2.2 minutes.
`
`[00214]
`
`Totest the irritation of Transcutol®, two formulations were tested
`
`containing 20% and 50% Transcutol® with PEG 200 as the cosolvent. Transcutol® at
`53
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`20% (I) or 50% (H) demonstrated similar irritation scores suggesting that Transcutol® is
`
`no moreirritating than PEG. A third formulation containing Transcutol® was the same
`
`as I but with 0.2% Tween. Comparison of I and I with Tween-20 suggested that Tween
`
`did not provide a substantial reduction ofirritation in this formulation.
`
`{00215}
`
`Formulation K (30% TA, 70% GF), which showed a good
`
`pharmacokinetic profile (see Example 5), had the lowestirritation score of the
`
`formulations tested in this experiment.
`
`[00216]
`
`Formulation K wasalso tested without clonazepam to obtain information
`
`on the effects of clonazepam onirritation. Comparisonoftheirritation scores of K and K
`
`without clonazepam showed that clonazepam appearedto haveanirritation reducing
`
`effect.
`
`[00217]
`Formulations M and T contain the same amountof glycofurol but M
`contains 70% PEG while T contains propylene glycol as the cosolvent. Theirritation
`
`score of these two doesnot differ significantly indicating a similar degree ofirritation by
`
`PG and PEG.
`
`[00218]
`
`Formulation R with 10% buffer (citrate/Tween/bisulphite) or with 10%
`
`water was the only water containing formulation tested. The formulation with water only
`
`appeared to be significantly moreirritating 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.
`
`Acetic acid 0.9% has beentested for irritation in a human trial and was
`—
`[00219]
`foundto 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 minorirritation but weretolerable.
`
`[00220]
`
`Thesenasal 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
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`bioavailability higher than 70% that of the intravenous formulations. Those that
`contained Transcuto!l® at concentrations 20-100% demonstrating that Transcutol® wasa
`useful absorption enhancerand solvent for clonazepam
`
`[00222]
`
`Additionally, the intranasal clonazepam formulations containing
`
`Transcutol® in the concentration range 20-100% 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 pharmacokinetic data for clonazepam formulationsis
`
`presented in Table 12A (N=1 for each formulation). In the table, Tw or tweenis
`
`TWEEN20, EtOHis ethanol, Triac or TA is Triacetin, phosph is phosphate buffer,
`metabisulph is sodium metabisulphite,citr is citrate buffer, AUCis area under the curve
`Tmax is minutes, Cmax is in ng/ml, and F% is bioavailability of the intranasal formulation
`
`as comparedto the intravenous formulation-- other abbreviations are as used herein
`
`above.
`
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`
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`AQUESTIVE EXHIBIT 1004=page 2456
`
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`
`56
`
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`

`

`WO 2008/027395
`
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`
`
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`AQUESTIVE EXHIBIT 1004=page 2457
`
`AQUESTIVE EXHIBIT 1004 page 2457
`
`57
`
`
`€
`
`

`

`WO 2008/027395
`
`PCT/US2007/018942
`
`
`
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`38
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`

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`
`{00225}
`
`Some clonazepam formulations without Transcutol® also provided a rapid
`
`rise in blood levels post-intranasal dosing 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 aboveillustrated 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 formulationsclinically 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 alcoholor 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 reducedstability of
`
`clonazepam formulations.
`
`[90227]
`
`The data shown in Table 12 above were reanalyzed. The rabbit pilot PK
`
`experiments had been performedin two groups of ten animals, JCO1 (Group 1) and JCQ2
`
`(Group 2). The JCO1 experiments were performedin a group of rabbits which were older
`
`and heavier than the JCO2 group of rabbits. Each group of rabbits had their own set of
`
`intravenous clonazepam PK datafor the calculations of bioavailability.
`
`{00228]
`
`Theintranasal formulations were 4 mg/mL. The animals were administered 25
`
`pL of formulation to each nostril, 50 wL in all, with an Eppendorf dosing pipette. The animal
`
`washeld in a supine position while being dosed and for about 10 secondsafter. The
`intravenous formulation, Rivotril® injectable, was administered as 500pL injected over 30
`
`secondsinto 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
`
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`

`WO 2008/027395
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`
`[00230]
`
`Dueto the different body weights of the two rabbit groups, the Cmax and the
`
`60 minute AUCresults 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 Cmax was not directly comparable between the
`
`two groups, but wasincludedin the table as a relative indication peak levels within each
`
`group.
`
`Table 12B: Example Clonazepam Formulations and Pharmacokinetic Data
`
`Group 1 Rabbits
`
`Relative BA
`
`
`RL
`
`a32
`
`1.3
`
`14.6
`
`100%Transcutot
`
`Formulation
`Intravenous
`
`
`100% PEG 300
`
`100% PEG 200
`
`ID
`Iv
`
`r=
`
` ee
`
`100% Glycofurol
`
`
`
` 8 9
`
`30%TA+GO%TC + 0.2% tween + 10%
`citrate pH 4
`
`10
`
`60
`
`AQUESTIVE EXHIBIT 1004=page 2460
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`

`WO 2008/027395
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`
`
`Group 2 Rabbits
`
`
`Relative
`BA
`
`100%
`53%
`
`73%
`
`78%
`79%
`‘oO
`
`39.7
`
`N;=]§NI@ a#
`
`
`
`78%se 111%ov
`
`
`Le
`
`F
`
`G
`
`J
`
`bf
`
`—
`
`100% GF
`
`Pt
`
`80% GF + 20% TC
`
`30%GF+70%PEG200
`
`30%TA+60%TC+10%phosph.
`
`
`0.212
`:
`
`.
`
`51%
`,
`
`70% GF+20% TC+10% H20
`
`.
`20%
`
`105%
`| ee
`90% TC + 10% H20
`
`76%
`
`
`84%
`30%Triac.+60%TC+10%H20
`
`103%
`
`94%
`47.2
`50% PEG200+50% TC
`
`$ a
`-
`
`80%PEG200+20%TC
`
`53%
`
`
`47%
`95% GF + 5% Tween-20
`eeeennneUNOnnEnES
`
`29.6
`73%
`
`
`
`.
`17.0
`69%
`
`30% Triacetin + 70% Gtycofurol
`.
`24.9
`84%
`
`51.3
`
`
`30%TAtE0%TC+10%citrate
`
`
`
`
`309|94%
`
`
`
`48%
`
`|
`
`20
`
`77%
`|
`
`AQUESTIVE EXHIBIT 1004
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`

`WO 2008/027395
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`
`10% GF + 50% PEG200 + 30% PG + 10%
`citr/tween/metab
`
`30% GF + 60% PG + 10% citr/tween/metab
`
`25%
`I.
`0,200
`36
`
`0.200
`“31
`37%
`0.200
`R
`49%
`39%
`
`
`
`
`
`Note: Tw or tween is TWEEN20, EtOH is ethanol, Triac or TA is Triacetin, Shosphis phosphate buffer,
`metabisulph is sodium metabisulphite, citr is citrate buffer.
`
`[00231]
`
`As exemplified in Tables 12A and 12B 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 supersaturation, and a second solvent (for
`
`example, TA or PG) in which clonazepam has lowersolubility 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% aqucous content, wherein the aqueouscontentis 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 tetrahydrofurfuryl alcohol polyethyleneglycol ether (Glycofurol) wherein the solvent
`
`molecules contain polyethylene glycol polymersas an intrinsic part of their molecular
`
`structure,that is, polyethylene glycol polymers as substituent groupsofa larger chemical
`
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`
`structure (also, see, for example, published P.C.T. International Patent Application Nos. WO
`
`03/070273 and WO 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)
`
`
`FU 50% diethyleneglycol monoethylether + 50% triacetin
`
`
`
`
`[50%diethyleneglycolmonoethylether+50%propyleneglycol_|
`
`PMT50%glycofurol+50%triacetin,
`
`50% glycofurol + 50% propyleneglycol
`
`
`
`[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=10 with instillation of 10 mg/mL
`clonazepam dose adjusted to body weight. A summary ofthe data is presented in Figure 5.
`
`[00235]
`
`The data is further summarized in Table 14.
`
`
`
`
`Table 14
`
`PK Data for Selected Formulations
`
`=_
`
`
`
`
`[sonerersorera[oni[203|902[ea|
`[nfsovercssoneo[oni[ast|ast[708|eee
`[itsoncresonra[one[328[Tora[a4[a
`
`[00236]
`
`The intranasal PK profiles of the formulations presented above demonstrated a
`
`rapid absorption of clonazepam suchthatclinically relevant amounts of clonazepam reach
`
`the bloodstream in a short period of time. Short-term bioavailability does not necessarily
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`need to be high; it is of higher importance that the blood levels become highin as short a
`
`time as possible. Lowerbioavailability 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 undergoingclassical GI absoprtion leading to a sustained release profile.
`[00237]
`|
`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 Tax Of benzodiazepine, after a single intranasal administration (in one or both nostrils),
`
`of 2 hours, often 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 (Cmax) of benzodiazepine ofat least about 3.0 ng/mL orat 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 Underthe Curve
`
`over 60 minutes (AUC)value of clonazepam ofat least about 400 ng-hr/mL, when a single
`
`dose of the composition is administered intranasally to deliver a dose ofat 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% ofthat 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 asactive 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 + 0.093 (SD) kg for
`
`the male rabbits, 2.465 + 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.
`
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`[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 pL solution) was selected to be comparable to an anticipated dose to be
`
`administered in humans.
`
`{00241}
`
`The treatment groupsare detailed in Table 15. Formulation 5 is a vehicle
`
`control -- 50 % glycofurol; 50 % propylene glycol (with no clonazepam). Formulation6 is a
`
`saline control (0.9% NaCl in water).
`
`Table 15
`
`Allocation of treatments into groups
`
`Group
`
`Treatment
`
`Number of
`
`animals
`
`Concentration of
`
`Clonazepam
`(mg/mL)
`10
`
`.
`
`10
`
`10
`
`10
`
`0
`
`0
`
`Number of
`
`treatments
`
`7
`
`7
`
`7
`
`7
`
`7
`
`7
`
`1
`
`2
`
`3
`
`5 males
`Formulation |
`5 females
`TC/TA+
`5 males
`Formulation II
`5 females
`TC/PG+
`5 males
`Formulation III
`5 females
`GF/TA+
`5 males
`Formulation IV
`5 females
`GF/PG+
`5 males
`Formulation 5
`5
`
`GF/PG-
`5 females
`Formulation 6
`5 males
`S-
`5 females
`
`4
`
`6
`
`[00242]
`
`The selected route of administration was the route of administration of the
`
`final product.
`
`[00243]
`
`Whatever the formulation, 0.1 mL of the formulation was daily administered
`
`to all animals by nasalinstillation during seven consecutive days.
`
`[00244]
`
`All administrations were performed in the right nostril using a 1 mL pipette
`
`(B13, 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.
`
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`[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 underisoflurane anaesthesia.
`
`[00247]
`
`Following euthanasia, macroscopical examination of larynx, trachea, bronchi,
`
`lungs and oesophaguswere performed.
`
`The head of the animal, with the larynx and specimensof trachea, bronchi,
`[00248]
`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 after 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 examinedin the nasal cavities on three
`
`head sections correspondingto nasal cavities proximal, nasal cavities turbinates and nasal
`
`cavities olfactory.
`
`[00251]
`
`Histopathological examinations were performedandthe 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 wasstatistically 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) butnotfor 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 day8 andalso in necropsy on day 15 mainly for treated
`
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`group 1 andslighter for other treated groups. Control group 6 showed noepithelial 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. Theseresults 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
`
`|
`|Meanscore|0.0|0.1|0.0|02|03|0.0|
`SSee2
`
`MeanandSDseverity score on larynx in each group
`
`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
`
`MeanandSDseverity score on oesophagusin each group
`[| Group1[Group2|Group3|Group4Group5|Group 6
`[Meanscore|0.0|0.0|00|02|0.0|
`rsp|00[|00|00|04|00|
`
`[00255]
`
`Petechia or blood were observed during necropsy on 17 animals (4 from
`
`group 1, 3 from group 2, 5S 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.
`
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`Table 18
`
`Mean and SDseverity score on lungs in each group
`
`|
`[Meanscore|0.0{|02|00|0.0|0.0|00|
`|spfoo|04fToofToofToo|00|
`
`{00257}
`
`Considering the whole respiratory tract, microscopical lesions were mainly
`
`observedin the very upperpart, the nasal cavities. As no lesions were recordedin 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 inducedbythe 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 after 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 Viscosity 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 pL 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 andthe results used to calculate the Dmax/Dmin ratio, the area of the
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`pattern and the average spray angle. The plumearea 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 clonazepam formulations. Viscosity, plume area at 3
`
`cm,spray angle and Dimax/Dmin ratio reflecting the symmetry of the spray plume are
`
`presented.
`
`Table 19
`
`Sprayability and Viscosity
`
`Composition
`
`Plume area
`at 3 cm (cm
`
`Spray
`angle (°
`
`Ratio
`Dynax/Dmin
`
`Avg.
`viscosity
`,
`cP
`80% Glycofurol + 20% bag
`
`11.5
`
`
`
`
`
`
`
`
`
`
`
`
`|tas
`
`
`
`
`
`
`Transcutol®
`50% Glycofuro] + 50%
`Transcutol®
`95% Glycofurol + 5%
`H20
`70% Glycofurol + 30%
`Triacetin
`60% Transcutol® + 30%
`Triacetin + 10% H2O
`
`60% Transcuto]l® + 40% P|
`
`Propylene glycol
`70% PEG200 + 30%
`
`8.8
`
`
`
`
`
`
`
`
`
`23.3
`a
`4.92
`
`
`+>|-io N
`31.
`15.52
`|WaterCTCCiCOT
`
`80% PEG200 + 20%
`H20
`90% PEG200 + 10%
`H2O0
`80% PEG200+10%
`GF+10% H,O0
`50% PEG200+30%
`PG+10% GF+10% H,O
`60% PG+30%GF+10%
`
`46.3
`
`1.79
`
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`

`WO 2008/027395
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`PCT/US2007/018942
`
`[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 cP (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 whichirregularity in the plume
`
`shape increased. Visual inspection of the appearance of the spray plumeofthree solutions in
`
`the viscosity range from 5.8 to 41.7 revealed