WO2009046444A2

WO2009046444A2 - Formulation for intranasal administration of diazepam

Info

Publication number: WO2009046444A2
Application number: PCT/US2008/078983
Authority: WO
Inventors: Henry R. Costantino; Ching-Yuan Li; Gopi Krishna Kasi
Original assignee: Mdrna, Inc.
Priority date: 2007-10-05
Filing date: 2008-10-06
Publication date: 2009-04-09
Also published as: WO2009046444A3

Abstract

The present disclosure provides intranasal mucosal delivery formulations containing diazepam.

Description

FORMULATION FOR INTRANASAL ADMINISTRATION OF DIAZEPAM

BACKGROUND

Diazepam ^-chloro-l-methyl-S-phenyl-l, 3-dihydro-2H-l, 4-benzodiazepin-2- one) is a central nervous system depressant with a molecular weight of 284.7 g/mol. Diazepam was first marketed as Valium; it is a benzodiazepine derivative drug. Diazepam possesses anxiolytic, sedative, anticonvulsant, skeletal muscle relaxant and amnestic properties. It is commonly used for treating anxiety, seizures, alcohol withdrawal, and muscle spasms.

Diazepam plays a crucial role in decreasing neurological activity. Diazepam acts as a positive allosteric modulator of gamma-aminobutyric acid (GABA); it binds to a specific subunit on the GABAA receptor at a site distinct from the endogenous GABA molecule. The GABA_A receptor is an inhibitory channel that decreases neurological activity once activated. Diazepam appears to act on areas of the limbic system, thalamus and hypothalamus, inducing anxiolytic effects. The anticonvulsant properties of diazepam and other benzodiazepines may be in part or may be entirely due to binding to voltage- dependent sodium channels rather than benzodiazepine receptors. Sustained repetitive firing seems to be limited by benzodiazepines' effect of slowing recovery of sodium channels from inactivation.

Diazepam was the second benzodiazepine to be characterized and was approved for human use in 1963. It is five times more potent than its predecessor, chlordiazepoxide. Diazepam is a Schedule IV controlled drug substance. Diazepam is soluble in 1 in 16 of ethyl alcohol, 1 in 2 of chloroform, 1 in 39 of ether, and is not very soluble in water, which has, in part, limited its use via non-invasive (e.g., nasal) routes of administration. The pH of diazepam is neutral (pH 7).

Diazepam has been administered orally, intravenously, intramuscularly, or as a suppository. When diazepam is administered orally, it is rapidly absorbed and has a fast onset of action. For example, the onset of action is 1-5 minutes for intravenous administration and 15-30 minutes for intramuscular administration. The duration of diazepam's main pharmacological effects is 15 minutes to 1 hour for both routes of administration. When diazepam is administered as an intramuscular injection, absorption is slow, erratic and incomplete. Peak plasma levels are achieved 30 minutes to 2 hours after oral administration. Diazepam is known to be an effective anticonvulsant when administered via intravenous or rectal routes at a dose in the range of 2.5-20 mg. However, intravenous administration requires a trained health care professional and is typically performed in a hospital setting. Rectal administration (e.g., Diastat^® AcuDial™) is another route of administration, but the major drawback to this method is that it presents a socially awkward situation, especially for adults and teens. Additionally, this method of administration also shows a delayed absorption and biological activity profile.

Diazepam is highly lipid-soluble, is widely distributed throughout the body after administration, and easily crosses both the blood-brain barrier and placenta. Diazepam is known to be metabolized in the liver via the cytochrome P450 enzyme system. It has a biphasic half -life of 1-2 and 2-5 days, and has several pharmacologically active metabolites. The main active metabolite of diazepam is desmethyldiazepam. Other active metabolites include temazepam and oxazepam, which become conjugated with glucorinide and are excreted primarily in the urine. Because of these pharmacologically active metabolites, the serum values of diazepam alone are not useful in predicting the biological effects of the drug.

The pharmacokinetics (PK) of diazepam and the resulting pharmacodynamic (PD) effect of this drug has been well-studied in humans for injectable and inhaled diazepam. However, there are few reports of the pharmacokinetic-pharmacodynamic relationship when diazepam is administered intranasally. Consequently, there is a need to develop pharmaceutical formulations for intranasal administration of a formulation comprising diazepam at higher concentrations than currently available and which are able to provide peak plasma concentrations in patient's between 10-20 minutes after dosing.

BREF SUMMARY

The present disclosure relates generally to the identification of pharmaceutical formulations capable of solubilizing diazepam at sufficiently high concentration to allow intranasal administration of therapeutically effective dosage forms to a patient in need thereof. DETAILED DESCRIPTION OF THE DISCLOSURE

In one aspect, this disclosure relates to the pharmacodynamic effects of diazepam by way of a nasal spray delivery method. The pharmaceutical formulation delivered may be a multi-component formulation. In this regard, a delivered dose of about 5 to about 10 or about 20 mg is expected to be therapeutically effective. This dose may be administered by a single spray in each nostril of a patient in need thereof, or following multiple administrations over a relatively short period of time (e.g., 5-30 minutes). For example, if the volume delivered by each actuation of a nasal spray delivery device is from about 150 μl to about 200 μl, a therapeutically effective dose of diazepam of about 10 mg would be manufactured (e.g., formulated) as a pharmaceutical formulation and delivered in a volume of about 300-400 μl, and thereby requires a formulation capable of providing a concentration (i.e., solubility) of diazepam of at least about 25 mg/ml, including from about 50 mg/ml to about 60 mg/ml, and to about 100 mg/ml, and higher. In order to provide for the therapeutically effective intranasal administration of diazepam and since diazepam shows relatively low solubility (-25 μg/ml) in water, several formulations are disclosed herein, with the expected capability of attaining the pharmacodynamic response of rectally administered diazepam with a pharmacokinetic profile of intranasally administered diazepam. In this regard, the pharmacokinetic profile and consequent pharmacodynamic response of diazepam delivered by nasal administration is expected to be faster than diazepam delivered by rectal administration.

As used herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, "about" or "consisting essentially of" mean ± 20% of the indicated range, value, or structure, unless otherwise indicated. As used herein, the terms "include" and "comprise" are used synonymously. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components. The use of the alternative (e.g., "or") should be understood to mean either one, both or any combination thereof of the alternatives. In addition, it should be understood that the individual compounds, or groups of compounds, derived from the various combinations of the structures and substituents described herein, are disclosed by the present application to the same extent as if each compound or group of compounds was set forth individually. Thus, selection of particular structures or particular substituents is within the scope of the present disclosure.

"Analog" or "analogue" as used herein refers to a chemical compound that is structurally similar to a parent compound (e.g., a peptide, protein, small molecule, or a mucosal delivery enhancing agent), but differs slightly in composition (e.g., one atom or functional group is different, added, or removed). The analog may or may not have different chemical or physical properties than the original compound and may or may not have improved biological or chemical activity. For example, the analog may be more hydrophilic or it may have altered activity as compared to a parent compound. The analog may mimic the chemical or biological activity of the parent compound (i.e., it may have similar or identical activity), or, in some cases, may have increased or decreased activity. The analog may be a naturally or non-naturally occurring (e.g., chemically-modified, synthetic or recombinant) variant of the original compound. An example of an analog is a mutein (i.e., a protein analog in which at least one amino acid is deleted, added, or substituted with another amino acid). Other types of analogs include isomers (enantiomers, diastereomers, and the like) and other types of chiral variants of a compound, as well as structural isomers.

"Derivative" as used herein refers to a chemically or biologically modified version of a chemical compound (including an analog) that is structurally similar to a parent compound and (actually or theoretically) derivable from that parent compound. Generally, a "derivative" differs from an "analog" in that a parent compound may be the starting material to generate a "derivative," whereas the parent compound may not necessarily be used as the starting material to generate an "analog." In certain aspects of this disclosure, a formulation for nasal administration may be used to deliver more than one diazepam (e.g., diazepam and a diazepam analog or derivative).

In the case of diazepam, although oral, rectal and injection modes of delivery are possible, nasal administration is equally viable as it may provide certain advantages such as increasing the rate at which the drug becomes bioavailable due, at least in part, as a consequence of avoiding hepatic first-pass metabolism and degradation in the gastrointestinal tract, convenience and speed of delivery, as well as reducing or eliminating concerns regarding patient compliance, and side effects that may be associated with oral, rectal and/or injection modes of delivery. However, mucosal delivery of biologically active agents is limited by barrier functions of a mucosal surface and other factors. As used herein, a mucosa includes the nasal, oral, intestinal, buccal, rectal, vaginal, and bronchopulmonary mucosal surfaces, and further encompasses all mucus-secreting membranes lining all body cavities or passages that mediate communication between the external environment and internal organs.

Epithelial cells make up the mucosal barrier and provide a crucial interface between the external environment and mucosal and submucosal tissue compartments. One of the most important functions of mucosal epithelial cells is to determine and regulate mucosal permeability. In this context, epithelial cells create selective permeability barriers between different physiological compartments. Selective permeability is the result of regulated transport of molecules through the cytoplasm (the transcellular pathway) and the regulated permeability which occurs through spaces between the cells (the paracellular pathway). Accordingly, in certain aspects of this disclosure, a pharmaceutical formulation comprising diazepam and containing one or more mucosal delivery enhancing agent is capable of reducing the transepithelial electrical resistance (TEER) between or otherwise across epithelial cells making up a epithelial cell barrier of a mucosal surface by modulating epithelial cell-epithelial cell interactions (e.g., tight junctions), as maybe measured using an in vitro model system. Such in vitro model systems may also include the evaluation of cell viability, cytotoxicity and diazepam permeation. A reduction in TEER is thought to be a necessary prerequisite for effective paracellular transport of an active pharmaceutical ingredient (e.g., diazepam).

Intercellular junctions such as tight junctions (TJ) of epithelial and endothelial cells are particularly important in the regulation of permeability via the paracellular pathway, and also divide the cell surface into apical and basolateral compartments. Tight junctions form continuous circumferential intercellular contacts between epithelial cells and create a regulated barrier to the paracellular movement of water, solutes, and immune cells. They also provide a second type of barrier that contributes to cell polarity by limiting exchange of membrane lipids between the apical and basolateral membrane domains.

The mucosal delivery may proceed transcellularly or paracellularly. Paracellular transport involves only passive diffusion, whereas transcellular transport can occur by passive, facilitated or active processes. Generally, hydrophilic, passively transported, polar solutes diffuse through the paracellular route, while more lipophilic solutes use the transcellular route. Absorption and bioavailability (e.g., as reflected by a permeability coefficient or physiological assay), for diverse, passively and actively absorbed solutes, can be readily evaluated, in terms of both paracellular and transcellular delivery components, for any selected diazepam, an analog, or derivative thereof, within this disclosure. For passively absorbed drugs, the relative contribution of paracellular and transcellular pathways to drug transport depends upon the pKa, partition coefficient, molecular radius and charge of the drug, the pH of the luminal environment in which the drug is delivered, and the area of the absorbing surface. The paracellular route represents a relatively small fraction of accessible surface area of the nasal mucosal epithelium. In general terms, it has been reported that cell membranes occupy a mucosal surface area that is a thousand times greater than the area occupied by the paracellular spaces. Thus, the smaller accessible area, and the size- and charge-based discrimination against macromolecular permeation would suggest that the paracellular route would be a generally less favorable route than transcellular delivery for drug transport. Surprisingly, the methods and compositions of this disclosure provide for significantly improved transport of biotherapeutic drugs into and across mucosal epithelia via the paracellular route.

As disclosed herein, a pharmaceutical formulation for intranasal delivery of diazepam, an analog, or derivative thereof, may comprise a formulation delivery vehicle that is an aqueous or non-aqueous solution, or a multi-phase formulation such as a suspension, or emulsion, and which further comprise one or more mucosal delivery enhancing agent. In certain aspects, a pharmaceutical formulation for intranasal delivery of diazepam may be a dry powder or solid surface dispersion, and which further comprise one or more mucosal delivery enhancing agent. In certain aspects, a pharmaceutical formulation for intranasal delivery of diazepam may comprise a delivery vehicle composed of micelles or liposomes, and which further comprise one or more mucosal delivery enhancing agent.

As used herein, "mucosal delivery-enhancing agents" are defined as chemicals, excipients and/or polypeptides, or combinations thereof, that, when added to a formulation comprising water, salts and/or common buffers and diazepam produce a pharmaceutical formulation that results in a significant increase in transport of diazepam, an analog, or derivative thereof across a mucosa (relative to a formulation without such enhancing agent(s)), as measured by maximum blood, serum, or cerebral spinal fluid concentration (C_max) or by the area under the curve (AUC) in a plot of concentration versus time. The one or more mucosal delivery-enhancing agent contained in a pharmaceutical formulation for intranasal delivery of diazepam may also provide for improved solubility and/or stability of diazepam (e.g., stability as sold, or in use stability) relative to a formulation without such mucosal delivery-enhancing agent(s). As used herein, mucosal delivery-enhancing agents may be referred to as or include carriers, solvents or co- solvents, excipients, additives, enhancing agents, enhancers or a thickening agent.

Mucosal delivery-enhancing agents include chemicals, polypeptides (e.g., the polypeptide PN159) and other excipients that, when added to an aqueous or non-aqueous pharmaceutical formulation comprising diazepam (as well as analogs, or derivatives thereof) results in a formulation that produces a significant increase in transport of such diazepam across a mucosa (e.g., a nasal mucosa). As used herein, excipients include methyl-β-cyclodextrin (Me-β-CD); ethylenediaminetetraacetic acid (EDTA); didecanoylphosphatidyl choline (DDPC); chlorobutanol (CB); sodium benzoate (NaBZ), polysorbate 80, polysorbate 20, sorbitol, cremophor EL, polyethylene glycol (e.g., polyethylene glycol 400), propylene glycol and other such agents as listed in the Handbook of Pharmaceutical Excipients. When used in the manufacture of a pharmaceutical formulation, excipients may be present at concentrations ranging from about 0.1 % to about 2% or from about 2% to about 70% or 85% by weight (w/w or w/v), including 0.15%, about 0.2%, about 0.5%, about 1%, about 1.5%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 45%, about 48%, about 85%, about 78%, and further including about 0.25%, about 0.35%, about 0.5%, about 1.0%, about 2.0%, about 2.5%, about 7.5%, about 13%, about 14%, about 16%, about 17%, about 17.5%, about 18%, about 25%, about 25.5%, about 27%, about 27.5%, about 32%, about 50%, about 53%, about 60%, about 62%, about 70%, about 75%, about 80%, and so on.. As used herein, mucosal delivery-enhancing agents include agents which improve the release (e.g., from a formulation delivery vehicle), solubility, diffusion rate, penetration capacity and timing, uptake, residence time, stability, effective half-life, peak or sustained concentration levels, clearance and other desired mucosal delivery characteristics (e.g., as measured at the site of delivery, or at a selected target site such as the bloodstream or central nervous system) of diazepam or other biologically active compound(s). The ability of drugs to permeate epithelial cell layers of mucosal surfaces, unassisted by delivery-enhancing agents, appears to be related to a number of factors, including molecular size, lipid solubility, and ionization. In general, small molecules, less than about 300-1,000 daltons and dependent upon their particular solubility, are often capable of penetrating mucosal barriers, however, as molecular size increases, permeability decreases rapidly. For these reasons, mucosal drug administration typically requires larger amounts of drug than administration by injection. Thus, in order to deliver large or small molecular weight drugs in therapeutically effective amounts (i.e., an amount capable of favorably modulating, or reducing one or more symptoms of a disease or condition, or otherwise favorably impacting a patient in need of such treatment), one or more mucosal delivery enhancing agent (e.g., a cell permeation enhancing agent) may be employed in order to aid their passage across a mucosal surface and into systemic circulation where they may quickly act on the target tissue.

Additionally, a pharmaceutical formulation for intranasal delivery of diazepam or any other active pharmaceutical ingredient may be preservative free or further comprise one or more preservative in order to improve antimicrobial effectiveness (AE). Improved antimicrobial effectiveness of a formulation disclosed herein may meet USP Antimicrobial Effectiveness Testing (AET) requirements and/or EP Antimicrobial Effectiveness Testing requirements. Suitable preservatives include, but are not limited to, phenol, methylparaben (MP), propylparaben (PP), propylene glycol, benzyl alcohol (BA), phenylethyl alcohol (PA), butylparaben, paraben, m-cresol, ortho-cresol, meta-cresol, par-cresol, thiomersal, chlorobutanol, benzylalkonimum chloride (BZK), benzethonium chloride, sodium benzoate, ethanol, sorbic acid, and the like. Preservatives (including combinations of preservatives) may be used at concentrations deemed effective, for example, benzethonium chloride may be used at concentrations of about 0.1 mg/ml to about 0.2 mg/ml or even 0.5 mg/ml or 1.0 mg/ml. Methylparaben at about 0.33 mg/ml to about 4.2 mg/ml, or 5 mg/ml or higher; propylparaben at about 0.17 mg/ml to about 2.0 mg/ml, or 2.5 mg/ml; phenyethyl alcohol at about 1 mg/ml to about 2.0 mg/ml, 1.5 mg/ml, 2.5 mg/ml or higher concentration; benzyl alcohol at about 1.0 mg/ml, 1.75 mg/ml, 2.5 mg/ml to about 5 mg/ml; ethanol at about 1 mg/ml to about 2.0 mg/ml. A preservative contained in a pharmaceutical formulation for intranasal delivery of diazepam may be present at a concentration from about 0.01% to about 0.5% or to about 1.0% or to about 1.5% by weight. For example, BZK may be used at about 0.01% to about 0.1% including about 0.05%, about 0.02%, about 0.25%, about 0.03% and so on; MP may be used at about 0.8%, about 0.7%, about 0.5%, about 0.45%, and so on; BA may be used at about 0.3%, about 0.4%, about 0.5%, about 0.6% and so on; PA may be used at about 0.5%, about 0.4%, about 0.3%, 0.25%, 0.2%, and so on; PP may be used at about 0.5%, about 0.3%, about 0.25%, about 0.2%, and so on. As disclosed herein, preservatives may be used alone or in any combination.

As disclosed herein, other than or in addition to the inclusion of a preservative, formulations may be sterilized by filtration, gamma irradiation or steam sterilization (e.g., terminal sterilization), or combinations thereof.

In certain aspects disclosed herein, the delivery methods and compositions of the present disclosure provide for therapeutically effective mucosal delivery of diazepam, an analog, or derivative thereof for prevention or treatment of, for example, anxiety, seizures, alcohol withdrawal, and/or muscle spasms. In one aspect of this disclosure, pharmaceutical formulations suitable for intranasal administration comprise a therapeutically effective amount of diazepam, an analog, or derivative thereof, and one or more intranasal mucosal delivery-enhancing agent (e.g., a solubilizing agent, or excipient), may be used for preventing the onset or progression of anxiety, seizures, alcohol withdrawal, and/or muscle spasms in a subject (e.g., patient) in need of such treatment, and provides a benefit to such patient compared to nasal delivery of diazepam in the absence of such agent(s). The formulations and preparative (e.g., manufacturing) and delivery methods of this disclosure provide improved solubility and mucosal delivery of a diazepam, analog, or derivative thereof, to a mammalian subject. These compositions and methods can involve combinatorial formulation or coordinate administration of diazepam with one or more mucosal delivery-enhancing agents including (A) solubilization agents; (B) charge modifying agents; (C) pH control agents; (D) degradative enzyme inhibitors;

(E) mucolytic or mucus clearing agents; (F) ciliostatic agents; (G) membrane penetration- enhancing agents (e.g., (i) a surfactant, (ii) a bile salt, (iii) a phospholipid or fatty acid additive, mixed micelle, liposome, or carrier, (iv) an alcohol, (v) an enamine, (vi) an NO donor compound, (vii) a long-chain amphipathic molecule (viϋ) a small hydrophobic penetration enhancer; (ix) sodium or a salicylic acid derivative; (x) a glycerol ester of acetoacetic acid (xi) a cyclodextrin or beta-cyclodextrin derivative, (xii) a medium-chain fatty acid, (xiii) a chelating agent, (xiv) an amino acid or salt thereof, (xv) an N- acetylamino acid or salt thereof, (xvi) an enzyme capable of degrading a selected membrane component, (xvii) an inhibitor of fatty acid synthesis, (xviii) an inhibitor of cholesterol synthesis; or (xix) any combination of the membrane penetration-enhancing agents of (i)-(xviii)); (H) agents capable of modulating epithelial tight junction physiology, such as nitric oxide (NO) stimulators, chitosan, and chitosan derivatives; (I) vasodilator agents; (J) selective transport-enhancing agents; and (K) stabilizing delivery vehicles, carriers, supports or complex-forming species with which the diazepam, analog, or derivative thereof, is/are effectively combined, associated, contained, encapsulated or bound in a manner which stabilizes the active agent for enhanced mucosal delivery. Thus, in various embodiments of this disclosure, a diazepam, analog, or derivative thereof, is combined with one, two, three, four or more of the mucosal delivery-enhancing agents recited in (A)-(K), above.

Pharmaceutically acceptable formulations, as used herein, may be comprised of a therapeutically effective dose of an active pharmaceutical ingredient (API; e.g., diazepam, an analog, or derivative thereof) and vehicle (comprising one or more pharmaceutically inactive ingredients or agents such as excipients, suspending agents, mucosal deliver enhancing agents and the like).

A mucosally effective dose of diazepam, analog, or derivative thereof within the pharmaceutical formulations disclosed herein may be in a range from about 1.0 mg to about 5 mg, about 10 mg or about 20 mg, as needed to provide a therapeutic benefit to a subject receiving such dose. The pharmaceutical formulations of the present disclosure may be administered one or more times per day, or 3 times per week or once per week for between one week and at least 96 weeks or even for the life of the individual patient or subject. In certain embodiments, the pharmaceutical formulations disclosed herein are administered one or more times daily, two times daily, four times daily, six times daily, or eight times daily. Sequential nasal delivery of a pharmaceutical formulation comprising diazepam may be administered in a dose escalating or de-escalating fashion, as needed to provide sustained therapeutic benefit to a subject in need of such treatment. A pharmaceutical formulation disclosed herein may be capable of providing diazepam solubility of at least about 15 mg/ml to at least about 60 mg/ml, about 70 mg/ml, about 75 mg/ml or about 100 mg/ml, including at least about 25 mg/ml, at least about 50 mg/ml and at least about 60 mg/ml. Diazepam solubilities greater than 100 mg/ml, including about 110 mg/ml, about 115 mg/ml and about 125 mg/ml are also appreciated herein.

Intranasal mucosal delivery-enhancing agents which enhance delivery of diazepam, an analog, or derivative thereof, into or across a nasal mucosal surface may also be employed in the manufacture of a pharmaceutical formulation capable of providing increased diazepam solubility. In certain aspects, the intranasal mucosal delivery-enhancing agent of the present disclosure may be a pH control agent. The pH of a pharmaceutical formulation of the present disclosure may be a factor affecting mucosal absorption of diazepam, an analog, or a derivative thereof, via paracellular and transcellular pathways. The pH of a pharmaceutical formulation may be used to provide improved diazepam stability. In certain embodiments, the pH of a pharmaceutical formulation of the present disclosure may be adjusted to be within a range between about pH 2 to about 10, including a pH of about 3.5, about 4, about 4.5, about 6, about 6.5, about 7, about 8, and about 8.5. In a further embodiment, the pharmaceutical formulation of the present disclosure is pH adjusted to be within a range of from about pH 3.0 to about 6.0. In a further embodiment, the pharmaceutical formulation of the present disclosure is pH adjusted to be within a range of from about pH 4.0 to about 6.0.

A buffer may be used to adjust and/or maintain a desired pH of a pharmaceutical formulation disclosed herein. A "buffer" which as used herein may also be referred to as a "buffering agent" or a "buffering system" is generally used to maintain the pH of a solution at a nearly constant value. A buffer maintains the pH of a solution, even when small amounts of strong acid or strong base are added to the solution, by preventing or neutralizing large changes in concentrations of hydrogen and hydroxide ions. A buffer generally consists of a weak acid and its appropriate salt (or a weak base and its appropriate salt). The appropriate salt for a weak acid contains the same negative ion as present in the weak acid {see Lagowski, Macmillan Encyclopedia of Chemistry, Vol. 1, Simon & Schuster, New York, 1997, p. 273-4). The Henderson-Has selbach Equation, pH = pKa + logio [A^"]/[HA], is used to describe a buffer, and is based on the standard equation for weak acid dissociation, HA ^ H⁺ + A^". Examples of commonly used buffer sources include acetate, tartrate, arginine, phosphate or citrate, and combinations thereof.

The "buffer capacity" means the amount of acid or base that can be added to a buffer solution before a significant pH change will occur. If the pH lies within the range of pK-1 and pK+1 of the weak acid the buffer capacity is appreciable, but outside this range it falls off to such an extent as to be of little value. Therefore, a given system only has a useful buffer action in a range of one pH unit on either side of the pK of the weak acid (or weak base) {see Dawson, Data for Biochemical Research, Third Edition, Oxford Science Publications, 1986, p. 419). Generally, suitable concentrations are chosen so that the pH of the solution is close to the pKa of the weak acid (or weak base) {see Lide, CRC Handbook of Chemistry and Physics, 86^th Edition, Taylor & Francis Group, 2005-2006, p. 2-41).

As used herein peak concentration (C_max) in a blood plasma, area under concentration vs. time curve (AUC) in a blood plasma, and time to maximal plasma concentration (t_maχ) in a blood plasma are pharmacokinetic parameters known to one skilled in the art (e.g., see Laursen et al., Eur. J. Endocrinology, 135:309-315, 1996). The "concentration vs. time curve" measures the concentration of diazepam (an analog, derivative, or functional metabolite thereof) in a blood serum of a subject vs. time after administration of a particular dose by intranasal, intramuscular, subcutaneous or other parenteral route of administration. The term C_max is the maximum concentration of diazepam, an analog, or derivative thereof in a blood serum of a subject following a single administration of a particular dose to a subject. The term t_max is the time to reach maximum concentration of diazepam, analog, or derivative thereof in a blood serum of a subject following administration of a single (or multiple) dose of diazepam, an analog, or derivative thereof to a subject.

As used herein, "area under concentration vs. time curve (AUC) of diazepam, analog, or derivative thereof in a blood plasma" is calculated according to the linear trapezoidal rule and with addition of the residual areas. A decrease of 23% or an increase of 30% between two dosages would be detected with a probability of 90% (type II error β = 10%). The "delivery rate" or "rate of absorption" is estimated by comparison of the time (tmax) to reach the maximum concentration (C_1118x). Both C_max and t_max are analyzed using non-parametric methods. Comparisons of the pharmacokinetics of intramuscular, subcutaneous, intravenous and intranasal diazepam, analog, or derivative thereof administrations were performed by analysis of variance (ANOVA). For pair wise comparisons a Bonferroni-Holmes sequential procedure is used to evaluate significance. The dose-response relationship between the three nasal doses is estimated by regression analysis. P <0.05 is considered significant. Results are given as mean values +/- SEM.

While the mechanism of absorption promotion may vary with different mucosal delivery-enhancing agents, useful reagents in this context will not in a significant manner adversely affect the mucosal tissue and will be selected according to the physicochemical characteristics of the particular diazepam, analog, or derivative thereof or other active or delivery-enhancing agent. In this context, delivery-enhancing agents that increase penetration or permeability of mucosal tissues will often result in some alteration of the protective permeability barrier of the mucosa. For such delivery-enhancing agents to be of value within this disclosure, it is generally desired that any significant changes in permeability of the mucosa be reversible within a time frame appropriate to the desired duration of drug delivery. Furthermore, there should be no substantial, cumulative toxicity, nor any permanent deleterious changes induced in the barrier properties of the mucosa with long-term use.

Within certain aspects herein, penetration-enhancing agents include small hydrophilic molecules including dimethyl sulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and the 2-pyrrolidones. Alternatively, long-chain amphipathic molecules, for example, deacylmethyl sulfoxide, azone, sodium laurylsulfate, oleic acid, and the bile salts, may be employed in order to enhance mucosal penetration of a diazepam, analog, or derivative thereof. In additional aspects, surfactants (e.g., polysorbates) may be employed as an adjunct compound, co-solvent, processing agent, or formulation additive to enhance or otherwise improve intranasal delivery of diazepam, an analog, or derivative thereof.

Additional mucosal delivery-enhancing agents that may be useful within the coordinate administration and manufacturing methods and combinatorial formulations disclosed herein include mixed micelles; enamines; nitric oxide donors (e.g., S-nitroso-N- acetyl-DL-penicillamine, NORl₅ N0R4-- which are preferably co-administered with an NO scavenger such as carboxy-PITO or doclofenac sodium); sodium salicylate; glycerol esters of acetoacetic acid (e.g., glyceryl-l,3-diacetoacetate or 1,2- isopropylideneglycerine-3-acetoacetate); and other release, diffusion, or intra- or trans- epithelial penetration-promoting agents that are physiologically compatible for mucosal delivery. Other absorption-promoting agents are selected from a variety of carriers, bases and excipients that enhance mucosal delivery, solubility, stability, activity or trans- epithelial penetration of diazepam include β-cyclodextrin derivatives (e.g., 2- hydroxypropyl-β-cyclodextrin, methyl-β-cyclodextrin and heptakis(2,6-di-O-methyl-β- cyclodextrin). Such compounds may be optionally conjugated or otherwise complexed with one or more pharmaceutically active ingredient and further optionally formulated in an oleaginous base. Yet additional absorption-enhancing agents adapted for mucosal delivery include medium-chain fatty acids, including mono- and diglycerides (e.g., sodium caprylate, extracts of coconut oil, glyceryl caprylate), and triglycerides (e.g., amylodextrin, Estaram 299, Miglyol 810). The therapeutic and prophylactic pharmaceutical formulations of the present disclosure may be supplemented with any suitable penetration-promoting agent that facilitates absorption, diffusion, or penetration of a diazepam, analog, or derivative thereof across mucosal barriers. Thus, in more detailed aspects of this disclosure pharmaceutical compositions are provided that incorporate one or more penetration- promoting agents selected from sodium salicylate and salicylic acid derivatives (acetyl salicylate, choline salicylate, salicylamide, etc.); amino acids and salts thereof (e.g. monoaminocarboxlic acids such as glycine, alanine, phenylalanine, proline, hydroxyproline, etc. ; hydroxyamino acids such as serine; acidic amino acids such as aspartic acid, glutamic acid, etc; and basic amino acids such as lysine etc — inclusive of their alkali metal or alkaline earth metal salts); and N-acetylamino acids (N-acetylalanine, N-acetylphenylalanine, N-acetylserine, N-acetylglycine, N-acetyllysine, N-acetylglutamic acid, N-acetylproline, N-acetylhydroxyproline, etc.) and their salts (alkali metal salts and alkaline earth metal salts). Also provided as penetration-promoting agents within the methods and compositions of this disclosure are substances which are generally used as emulsifiers {e.g., sodium oleyl phosphate, sodium lauryl phosphate, sodium lauryl sulfate, sodium myristyl sulfate, polyoxyethylene alkyl ethers, polyoxyethylene alkyl esters, etc.), caproic acid, lactic acid, malic acid and citric acid and alkali metal salts thereof, pyrrolidonecarboxylic acids, alkylpyrrolidonecarboxylic acid esters, N-alkylpyrrolidones, proline acyl esters, and the like.

In certain aspects disclosed herein, and in order to improve the taste that may be associated with intranasal administration of a pharmaceutical formulation comprising diazepam, or to improve patient compliance, one or more flavor modifying agents (e.g., sweeteners or masking agents) may be incorporated. Such flavor modifying agent may include acacia syrup, anethole, anise oil, aromatic elixir, benzaldehyde, benzaldehyde elixir, butterscotch, cyclodextrins, compound, caraway, caraway oil, cardamom oil, cardamom seed, cardamom spirit, compound, cardamom tincture, compound, cherry juice, cherry syrup, cinnamon, cinnamon oil, cinnamon water, citric acid, citric acid syrup, clove oil, cocoa, cocoa syrup, coriander oil, dextrose, eriodictyon, eriodictyon fluid extract, eriodictyon syrup, aromatic, ethylacetate, ethyl vanillin, fennel oil, ginger, ginger fluidextract, ginger oleoresin, dextrose, glucose, sugar, maltodextrin, glycerin, glycyrrhiza, glycyrrhiza elixir, glycyrrhiza extract, glycyrrhiza extract pure, glycyrrhiza fluid extract, glycyrrhiza syrup, honey, iso-alcoholic elixir, lavender oil, lemon oil, lemon tincture, mannitol, methyl salicylate, nutmeg oil, orange bitter, elixir, orange bitter, oil, orange flower oil, orange flower water, orange oil, orange peel, bitter, orange peel sweet, tincture, orange spirit, compound, orange syrup, peppermint, peppermint oil, peppermint spirit, peppermint water, phenylethyl alcohol, raspberry juice, raspberry syrup, rosemary oil, rose oil, rose water, rose water, stronger, saccharin, saccharin calcium, saccharin sodium, sarsaparilla syrup, sarsaparilla compound, sorbitol solution, spearmint, spearmint oil, sucrose, sucralose, syrup, thyme oil, tolu balsam, tolu balsam syrup, vanilla, vanilla tincture, vanillin, or wild cherry syrup, or combinations thereof.

The present disclosure provides for the mucosal (e.g., nasal) delivery of a pharmaceutical formulation comprising diazepam, an analog, or a derivative thereof, in combination with one or more mucosal delivery-enhancing agents and an optional sustained release-enhancing agent or agents. Mucosal delivery-enhancing agents of the present disclosure yield an effective increase in delivery, for example, an increase in the maximal plasma concentration (C_max) to enhance the therapeutic activity of mucosally- administered diazepam. Another factor affecting therapeutic activity of diazepam, analog, or derivative thereof, in the blood plasma and/or central nervous system (CNS) is residence time (RT). Sustained release-enhancing agents, in combination with intranasal delivery-enhancing agents, may increase C_max and increase residence time (RT) of diazepam (analog, or derivative thereof). Polymeric delivery vehicles and other agents and methods of the present disclosure that yield sustained release-enhancing formulations, for example, polyethylene glycol (PEG), are disclosed herein.

Within the mucosal delivery formulations and methods of this disclosure, diazepam, an analog, or derivative thereof, may be combined or coordinately administered with a suitable carrier or vehicle for mucosal delivery. As used herein, the term "carrier" means pharmaceutically acceptable solid or liquid (e.g., a filler), diluent or encapsulating material. A water-containing liquid carrier can contain pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffering agents, chelating agents, complexing agents, solubilizing agents, humectants, solvents, suspending and/or viscosity-increasing agents, tonicity agents, wetting agents or other biocompatible materials. A tabulation of ingredients listed by the above categories can be found in the U.S. Pharmacopeia National Formulary, 1857-1859 (1990) and subsequent versions thereof, and Remington: The Science and Practice of Pharmacy (21^st Edition). Some examples of the materials which can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer's solution, ethyl alcohol and phosphate buffer solutions, as well as other non toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in a pharmaceutical composition contemplated herein. Examples of pharmaceutically acceptable antioxidants include water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol and the like; and metal-chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form may vary depending upon the desired effect and/or particular mode of administration.

The pH of a formulation disclosed herein may be from about pH 2.0 to about 10.0, including about 6.5, about 7.0 about 7.25, about 7.5, about 8.0, about 8.5 and about 9.0. The pH of a formulation disclosed herein may be maintained at the desired level by the addition of one or more (a plurality or mixture of) buffer(s), pH control agent, buffering agent or buffering system. Such buffers include phosphate, arginine, acetate, citrate, acetic acid, hydrochloric acid, tartrate, glutamate and sodium hydroxide. A buffer, buffering agent or system may be used at concentrations from about 2.5 mM to about 75mM or about 10OmM including 5mM, 1OmM, 15mM, 2OmM, 25mM, 35mM, 4OmM, 45mM, 50 mM, 6OmM and so on.

As disclosed herein, methodologies used to formulate pharmaceutically acceptable formulations comprising therapeutically effective amounts of diazepam include: Cosolvent and complexing formulations, non-aqueous formulations, solid surface dispersion (SSD) formulations, emulsion formulations, liposomal formulations, micelle formulations, aqueous suspensions, and dry powder formulations.

Examples of cosol vents include ethanol, polyethylene glycol 200, polyethylene glycol 400 (PEG400), tetraethylene glycol, glycofurol, propylene glycol, glycerol, etc. As disclosed herein, a pharmaceutical formulation capable of solubilizing a diazepam may comprise one or more cosolvent. Such cosolvents may be used in a formulation disclosed herein at a concentration of about 0.1 to about 85% by weight (w/w or w/v); including about 0.25%, about 0.35%, about 0.5%, about 1.0%, about 2.0%, about 2.5%, about 5%, about 7.5%, about 10%, about 13%, about 14%, about 15%, about 16%, about 17%, about 17.5%, about 18%, about 20%, about 25%, about 25.5%, about 27%, about 27.5%, about 30%, about 32%, about, about 45%, about 50%, about 53%, about 60%, about 62%, about 70%, about 75%, about 78%, about 80%, and so on.

Examples of complexing agents include Ot-, β-, and γ-cyclodextrins, analogs and derivatives thereof, which may have a circular arrangement of the glucose units of a complexing agent forms a torus- shaped molecule, with a hydrophobic interior cavity and a polar exterior. Examples of β-cyclodextrins include hydroxypropyl-β-cyclodextrins, methyl-β-cyclodextrins, dimethyl-β-cycϊodextrins, sulfobutylether-7-β-cyclodextrins, maltosyl -β-cyclodextrins, and the like. Such complexing agents may be used in a formulation disclosed herein at a concentration of about 0.1 to about 85% by weight (w/w or w/v); including about 0.25%, about 0.35%, about 0.5%, about 1.0%, about 2.0%, about 2.5%, about 5%, about 7.5%, about 10%, about 13%, about 14%, about 16%, about 17%, about 17.5%, about 18%, about 20%, about 25%, about 25.5%, about 27%, about 27.5%, about 30%, about 32%, about 15%, about, about 45%, about 50%, about 53%, about 60%, about 62%, about 70%, about 75%, about 78%, about 80%, and so on.

In respect of pharmaceutically acceptable non-aqueous formulations, the formulation approach employs a composition comprising one or more non-aqueous vehicle. Two distinct cases may be envisioned, one is a non-aqueous solution formulation and another is a non- aqueous suspension formulations. Non-aqueous solution formulations utilize the solubilization capability of one or more non-aqueous solvent to maximize the solubility of diazepam in such formulation vehicle. The non-aqueous solvent can be either water-miscible or non-water-miscible. Examples of non-water- miscible non-aqueous solvents include oils such as almond oil USP, cascara sagrada fluid extract USP, castor oil USP, cod liver oil USP, corn oil USP, cottonseed oil USP, eucalyptus oil USP, lavender oil NF, olive oil NF, peppermint oil NF, safflower oil USP, sesame oil NF, and soybean oil USP.

In other aspects disclosed herein, pharmaceutically-acceptable, water-miscible, non-aqueous solvents include, but are not limited to, N-methyl pyrrolidone (NMP); propylene glycol; ethyl acetate; dimethyl sulfoxide; dimethyl acetamide; benzyl alcohol; 2-pyrrolidone; benzyl benzoate; C_2-6 alkanols; 2-ethoxyethanol; alkyl esters such as 2- ethoxyethyl acetate, methyl acetate, ethyl acetate, triacetin, ethylene glycol diethyl ether, or ethylene glycol dimethyl ether; (s)-(-)-ethyl lactate; acetone; glycerol; alkyl ketones such as methylethyl ketone or dimethyl sulfone; tetrahydrofuran; cyclic alkyl amides such as caprolactam; decylmethylsulfoxide; oleic acid; aromatic amines such as N,N-diethyl- m-toluamide; or l-dodecylazacycloheptan-2-one.

In respect of non-aqueous suspension formulations, the suspended drug (e.g., diazepam) may be in the form of a dried solid suspended within a non-aqueous solvent. The particle size of the solid drug suspended within the non-aqueous solvent can be controlled by mechanical means during the manufacturing process (e.g., by homogenization of the suspension), or can be achieved by providing the dried drag at an appropriate particle size before processing (e.g., pre-milling or pre-sieving the powder, or by creating a spray dried powder). In respect of pharmaceutically acceptable solid surface dispersion (SSD) formulations, the formulation approach requires the manufacture of solid surface dispersions of diazepam in/on, for example, a carboxymethyl cellulose sodium solid surface. The amount of diazepam in solid form recovered after solid surface dispersion may provide a higher solubility. Solid surface dispersion, a technique that provides deposition of the drug on the surface of certain materials, can alter the dissolution characteristics of the drug so deposited. Deposition of drug on the surface of an inert carrier leads to reduction in the particle size of drug, thereby providing a faster rate of dissolution. Various hydrophilic materials with high surface area can be utilized to deposit a drug on their surface. The selection of carrier and method of preparation are critical factors influencing the properties of a drug incorporated in the SSD. Carrier materials for the preparation of solid surface dispersions may include different types of silica, non-porous (Aerosil 200) or porous silica (Sylysia 350) by using spray-drying method. In respect of pharmaceutically acceptable emulsion formulations, an emulsion may be defined as a mixture of two or more immiscible (unblendable) phases (resulting in the formation of dispersed droplets (the dispersed phase) in a continuous phase) with a third component (emulsifier) used in order to stabilize the dispersed droplets. For a non- polar compound, an oil-in-water emulsion may be used to solubilize the non-polar compound in the oil phase with the oil droplets dispersed in the aqueous medium. Thermodynamically stable systems, known as microemulsions are also disclosed herein as a clear dispersion of two immiscible liquids (oil and water) stabilized by an interfacial film of surfactant molecules. The surfactant may be pure, a mixture, or combined with other additives. In the absence of water, mixtures of oil(s) and non-ionic surfactant(s) form clear and transparent isotropic solutions that are known as self-emulsifying drug delivery system (SEDDS) and may used for improving lipophilic drug dissolution and absorption. Examples of the oil component of the emulsion formulations (such as microemulsions and SEDDS) include cottonseed oil, sesame oil, olive oil, corn oil, caster oil, peanut oil, triglyceride oil, polyoxyl 35 caster oil, polyoxyl 40 caster oil, polyoxyl 60 caster oil, mineral oil, soybean oil, vegetable oil, egg lecithin, soybean lecithin, phosphatides, decanol, propylene glycol dicaprate (Captex 200^®, Captex 355^®), capylic/capric triglyeride (Miglyol 812^®, Miglyol 840^®), Myvacet, etc. Examples of the surfactant component of the emulsion, microemulsion or SEDDS formulations include oleic acid, pluronic block copolymer (F68, Fl 28, F108), polyoxyethylene (20) sorbitan monolaurate (polysorbate 20, polysorbate 80), Cremophor EL^®, d-alpha-tocopheryl polyethylene glycol 1000 succinate, polyoxyethylene glycerol trioleate, glycerol monooleate, glycerol monocaprylate, glycerol dioleate, propylene glycol monocaprylate, sorbitan monooleate (Arlacel 80 , Arlacel 186), glyceryl caprylate (Capmul MCM^®), lecithin (Centrophase 31^®), Labrafac CMlO, Labrafil M 1944 CSD, Labrafil M 2125 CS, Labrasol, and the like.

In respect of pharmaceutically acceptable liposomal formulations, the formulation may be comprised of neutral lipids, such as dipalmitoylphosphatidylcholine (DPPC) or distearoyl-phosphatidylcholine (DSPC), and may contain cationic lipids for cell surface and mucoadhesion. The cationic lipids such as N-[l-(2,3-dioleyloxy)propyl]-N, N, N- trimethylamnionium chloride (DOTMA) or 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP) are commonly used in transfection formulations, but in this utilization may provide alternative properties to solubilize diazepam in the liposomes. Various compositions with different T_n, can be made to enable different encapsulation capability, stability properties, and release rates. The length of the lipid tail can be varied from 18 carbons to 14 carbons with different release profiles. Also, different degrees of saturation of the lipids may provide different degrees of solubilization. Liposomes may be prepared from natural, biodegradable, non-toxic, and non-immunogenic lipid molecules, and can efficiently entrap or bind drug molecules, into, or onto, their membranes. A variety of methods are available for preparing liposomes within this disclosure, for example, U.S. Patent Nos. 4,235,871; 4,501,728 and 4,837,028.

In respect of pharmaceutically acceptable micelle formulations, the rationale for this approach to solubilize a non-polar compound is to exclude the non-polar (aromatic) part of the drug molecule from the water, thereby increasing its aqueous solubility. A micelle formulation may contain one or more surfactant(s). Examples of surfactants useful in this regard include pluronic block copolymers (e,g., F68, F128, F108 and others), polysorbates (e.g., polysorbate 20, polysorbate 80, and others), cremophor EL, and bile salts.

In respect of pharmaceutically acceptable aqueous suspensions, diazepam particles may be suspended within an aqueous vehicle. In this case, the homogeneity of the diazepam throughout the suspension can be achieved by maintaining particle buoyancy, for example, in a viscous aqueous solution (e.g., a suspending agent), and/or by agitation of the manufactured product prior to administration. Such suspending agents includefmethylcellulose (MC); hydroxypropylmethylcellulose (HPMC); hydroxypropylmethylcellulose acetate succinate (HPMCAS), carboxymethylcellulose (CMC), HPMC 2910, HPMC 2208, and polyvinyl alcohol (PVA). Suspending agents may be present at a concentration ranging from about 0.25 to about 1, from about 0.25 to about 2, from about 0.25 to about 4, from about 0.25 to about 5 or from about 0.25 to about 10%, by weight. Avoidance of particle agglomeration within the suspension can be achieved by the addition of various agents, for example, surfactants such as polysorbates (e.g., polysorbate 20, polysorbate 80, and the like), and cosolvents (e.g., PEG 400, propylene glycol (PG), and the like), and combinations thereof. Further examples of surfactants that may be useful in formulating a suspension formulation include oleic acid, pluronic block copolymer (F68, F128, F108), cremophor EL, TPGS, tagat TO, glycerol monooleate, glycerol monocaprylate, glycerol dioleate, propylene glycol monocaprylate, Arlacel 80 , Arlacel 186, Capmul MCM, Centrophase 31, Labrafac CMlO, Labrafil M 1944 CSD, Labrafil M 2125 CS, Labrasol, and the like. Examples of suspension nasal products include: beclomethasone (Beconase^®), budesonide (Rhinocort^®), fluticasone, mometasone (Nasonex^®), and triamcinolone.

Dispersing agent may be used to maintain the homogeneity of a suspension formulation disclosed herein by, for example, minimizing or preventing settling of suspension particles. Representative examples of such dispersion agents/excipients are Avicel® microcrystalline cellulose and carboxymethycellulose sodium, poloxamers, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, hypromellose acetate succinate, lecithin, polyethylene alkyl ethers, sorbitan esters, poly(methylvinyl ether/maleic anhydride and the like. In further aspects of this disclosure, the formulations set forth may be prepared or manufactured, using biological polymers such as low molecular weight (-202 kDa) hyaluronic acid, which can have mucoadhesive properties that may enable permeation of the associated drug (e.g., diazepam) across tight junctions. In another aspect of this disclosure, the suspension of drug particles within an anionic polymer may be entrapped within or otherwise associated with a liquid containing one or more viscosity (thickening) or gel forming agent. For example, diazepam may be complexed with CMC, or like compound (e.g., hydroxypropylmethylcellulose acetate succinate (HPMCAS)) , formulated as a suspension within a liquid containing, for example, pectin (or derivative thereof) which may only form a gel following a temperature shift or when in the presence of divalent cations, such as calcium (consistent with the biological environment of a nasal mucosa), resulting in or otherwise allowing for the suspension to be trapped within a gel upon in vivo delivery to the nasal mucosa (or other mucosal surface). In another embodiment, the suspension is formulated within a liquid containing an agent which gels as the temperature is increased (i.e., shifted) from ambient to physiologic. The particles formed between a drug and suitable polymer may be net neutral (ca. -20 to +20 mV zeta potential), negatively charged (ca. < -20 mV zeta potential) or positively charged (ca. > +20 mV zeta potential). The particle distribution within such formulation maybe unimodal, bimodal, or multimodal. The average particle size may range from about 10 nm to about 5000 nm, for example about 10 nm to about 1000 nm. In respect of pharmaceutically acceptable powder formulations, the formulation may be prepared by lyophilizing a dispersion of an aqueous (or non-aqueous) formulation disclosed herein of an appropriate particle size or within an appropriate particle size range. In this regard, it is appreciated that a minimum particle size appropriate for deposition of a diazepam within the lung may be about 0.5 μm mass median equivalent aerodynamic diameter (MMEAD), but is often targeted to be about 1 to about 2 μm MMEAD. Maximum particle size appropriate for deposition within the lung is from about 8 to about 10 μm MMEAD, or about 4 μm MMEAD. In contrast, the minimum particle size appropriate for deposition of a diazepam (an analog, or derivative thereof) within the nose is about 0.5 μm MMEAD, or about 3 μm or about 5 μm MMEAD. Maximum particle size appropriate for effective deposition within the nose is typically about 100 μm MMEAD, but includes particle about 50 μm MMEAD, and about 20 μm MMEAD. In general, the smaller the particle size the more likely deposition will occur in the lungs and larger particle sizes will be deposited within the nasal mucosa. Pharmaceutically acceptable powder formulations comprising a diazepam, analog, or derivative thereof within the preferred size range can be produced by a variety of conventional techniques, such as jet milling, spray drying, solvent precipitation, supercritical fluid condensation, and the like. Because particle size (because larger particle sizes are acceptable) is less important for nasal delivery, crystallization from solution may be sufficient. If it is not sufficient, it may be augmented by jet milling or ball milling.

Such dry powders of appropriate MMEAD can be administered to a patient via a conventional dry powder inhalers (DPI's) which rely on the patient's breath, upon inhalation, to disperse the power into an aerosolized amount. Alternatively, the dry powder may be administered via air assisted devices that use an external power source to disperse the powder into an aerosolized amount, e.g., a piston pump.

Dry powder devices typically require a powder mass in the range from about 1 mg to 20 mg to produce a single aerosolized dose ("puff"). If the required or desired dose of a diazepam is lower than this amount, the diazepam powder may be combined with a pharmaceutical dry bulking powder to provide the required total powder mass. Dry bulking powders include sucrose, lactose, dextrose, mannitol, glycine, trehalose, human serum albumin (HSA), and starch. Other suitable dry bulking powders include cellobiose, dextrans, maltotriose, pectin, sodium citrate, sodium ascorbate, and the like.

When the dry powder is prepared by solvent precipitation, buffers and salts may be used to stabilize a diazepam in solution prior to particle formation. Suitable buffers include, but are not limited to, ascorbate, phosphate, citrate, acetate, and tris-HCl, at concentrations ranging from about 5 mM to about 50 mM. Suitable salts include sodium chloride, sodium carbonate, calcium chloride, and the like.

Effective delivery of biotherapeutic agents (i.e., active pharmaceutically ingredient) via intranasal administration may take into account the decreased drug transport rate across the protective mucus lining of the nasal mucosa, in addition to drug loss due to binding to glycoproteins of the mucus layer. Normal mucus is a viscoelastic, gel-like substance consisting of water, electrolytes, mucins, macromolecules, and sloughed epithelial cells. It serves primarily as a cytoprotective and lubricative covering for the underlying mucosal tissues. Mucus is secreted by randomly distributed secretory cells located in the nasal epithelium and in other mucosal epithelia. The structural unit of mucus is mucin. This glycoprotein is mainly responsible for the viscoelastic nature of mucus, although other macromolecules may also contribute to this property. In airway mucus, such macromolecules include locally produced secretory IgA, IgM, IgE, lysozyme, and bronchotransferrin, which also play an important role in host defense mechanisms.

Accordingly, the coordinate administration methods of the instant disclosure may optionally incorporate effective mucolytic or mucus-clearing agents, which serve to degrade, thin, or clear mucus from intranasal mucosal surfaces to facilitate absorption of intranasally administered biotherapeutic agents. Within these methods, a mucolytic or mucus-clearing agent may be coordinately administered as an adjunct compound to enhance intranasal delivery of diazepam (as well as an analog, or derivative thereof). Alternatively, an effective amount of a mucolytic or mucus-clearing agent is incorporated as a processing agent within a multi-processing method of this disclosure, or as an additive within a combinatorial formulation of this disclosure, to provide an improved formulation that enhances intranasal delivery of biotherapeutic compounds by reducing the barrier effects of intranasal mucus.

A variety of mucolytic or mucus-clearing agents are available for incorporation within the methods and compositions of this disclosure. Based on their mechanisms of action, mucolytic and mucus clearing agents can often be classified into the following groups: proteases (e.g., pronase, papain) that cleave the protein core of mucin glycoproteins; sulfhydryl compounds that split mucoprotein disulfide linkages; and detergents (e.g., Triton X-100, Tween 20) that break non-covalent bonds within the mucus. Additional compounds in this context include, but are not limited to, bile salts and surfactants, for example, sodium deoxycholate, sodium taurodeoxycholate, sodium glycocholate, and lysophosphatidylcholine.

The effectiveness of bile salts in causing structural breakdown of mucus is in the order: deoxycholate > taurocholate > glycocholate. Other effective agents that reduce mucus viscosity or adhesion to enhance intranasal delivery according to the methods of this disclosure include, for example, short-chain fatty acids, and mucolytic agents that work by chelation, such as N-acylcollagen peptides, bile acids, and saponins (the latter function in part by chelating Ca²⁺ and/or Mg²⁺ which play an important role in maintaining mucus layer structure). These and other mucolytic or mucus-clearing agents are contacted with the nasal mucosa, which may be used at a concentration ranging from of about 0.2 to about 20 mM, coordinately with administration of the active pharmaceutically ingredient, to reduce the polar viscosity and/or elasticity of intranasal mucus.

Still other mucolytic or mucus-clearing agents may be selected from a range of glycosidase enzymes, which are able to cleave glycosidic bonds within the mucus glycoprotein; α-amylase and β-amylase are representative of this class of enzymes, although their mucolytic effect may be limited. In contrast, bacterial glycosidases and similar agents which function to assist a microorganism to penetrate mucus layers of their hosts may have a stronger effect. Ciliostatic agents, within the methods and compositions of this disclosure, may increase the residence time of mucosally (e.g., intranasally) administered diazepam. In particular, within the methods and compositions of this disclosure, delivery is significantly enhanced in certain aspects by the coordinate administration or combinatorial formulation of one or more ciliostatic agents that function to reversibly inhibit ciliary activity of mucosal cells, to provide for a temporary, reversible increase in the residence time of the mucosally administered active agent(s). Various bacterial ciliostatic factors isolated and characterized in the literature may be employed within certain embodiments of this disclosure. For example, ciliostatic factors from the bacterium Pseudomonas aeruginosa include a phenazine derivative, a pyo compound (2- alkyl-4-hydroxyquinolines), and a rhamnolipid (also known as a hemolysin). The pyo compound produced ciliostasis at concentrations of 50 μg/ml and without obvious ultrastructural lesions. The phenazine derivative also inhibited ciliary motility but caused some membrane disruption, although at substantially greater concentrations of 400 μg/ml. Limited exposure of tracheal explants to the rhamnolipid resulted in ciliostasis, which is associated with altered ciliary membranes. For use within these aspects of this disclosure, the foregoing ciliostatic factors, either specific or indirect in their activity, are each candidates for successful employment as ciliostatic agents in appropriate amounts (depending on concentration, duration and mode of delivery) such that they yield a transient (i.e., reversible) reduction or cessation of mucociliary clearance at a mucosal site of administration to enhance delivery of a diazepam, analog or derivative thereof, and other biologically active agents disclosed herein, without unacceptable adverse side effects.

Surface active agents (surfactants) are readily incorporated within the mucosal delivery formulations and methods of this disclosure as mucosal delivery- enhancing agents. Examples of surface-active agent are noniom^'c polyoxyethylene ether, bile salts, sodium glycocholate, deoxycholate, derivatives of fusidic acid, sodium taurodihydrofusidate, L-α-phosphatidylcholine didecanoyl (DDPC), poloxamer F68, poloxamer F127, polysorbate 80 (PS80), polysorbate 20, a polyethylene glycol, cetyl alcohol, polyvinylpyrolidone, a polyvinyl alcohol, lanolin alcohol, and sorbitan monooleate. The utility of these surface active agents may include solubilization of a biologically active agent.

In certain aspects of this disclosure, the combinatorial formulations and/or coordinate administration methods herein incorporate an effective amount of a diazepam which may adhere to charged glass thereby reducing the effective concentration in the container. Silanized containers, for example, silanized glass containers, are used to store the finished product to reduce adsorption of a diazepam to a glass container.

In yet additional aspects of this disclosure, a kit for treatment of a mammalian subject comprises a stable pharmaceutical composition of a diazepam (an analog, or derivative thereof) formulated for mucosal delivery to a mammalian subject in need thereof. The kit may further comprise a pharmaceutical reagent bottle containing diazepam. The pharmaceutical reagent bottle is composed of pharmaceutical grade polymer, glass or other suitable material. The pharmaceutical reagent bottle is, for example, a silanized glass bottle. The kit further comprises an aperture for delivery of the composition to a nasal mucosal surface of the subject. The delivery aperture is composed of a pharmaceutical grade polymer, glass or other suitable material.

A silanization technique combines a special cleaning technique for the surfaces to be silanized with a silanization process at low pressure. The silane is in the gas phase and at an enhanced temperature of the surfaces to be silanized. The method provides reproducible surfaces with stable, homogeneous and functional silane layers having characteristics of a monolayer. The silanized surfaces prevent binding to the glass of polypeptides or mucosal delivery enhancing agents of the present disclosure.

The procedure is useful to prepare silanized pharmaceutical reagent bottles to hold formulations comprising diazepam of the present disclosure. For example, glass trays are cleaned by rinsing with double distilled water (ddH₂O) before using. The silane tray is then rinsed with 95% EtOH, and the acetone tray is rinsed with acetone. Pharmaceutical reagent bottles are sonicated in acetone for 10 minutes. After the acetone sonication, reagent bottles are washed in ddH₂O tray at least twice. Reagent bottles are sonicated in 0.1M NaOH for 10 minutes. While the reagent bottles are sonicating in NaOH, the silane solution is made under a hood. (Silane solution: 800 mL of 95% ethanol; 96 L of glacial acetic acid; 25 mL of glycidoxypropyltrimethoxy silane). After the NaOH sonication, reagent bottles are washed in ddH₂O tray at least twice. The reagent bottles are sonicated in silane solution for 3 to 5 minutes. The reagent bottles are washed in 100% EtOH tray. The reagent bottles are dried with prepurified N₂ gas and stored in a 100⁰C oven for at least 2 hours before using.

Compositions according to the present disclosure are often administered in an aqueous solution as a nasal or pulmonary spray and may be dispensed in spray form by a variety of methods known to those skilled in the art. Preferred systems for dispensing liquids as a nasal spray are disclosed in U.S. Patent No. 4,511,069, hereby incorporated by reference. The formulations may be presented in multi-dose containers, for example in the sealed dispensing system disclosed in U.S. Patent No. 4,511,069. Additional aerosol delivery forms may include, for example, compressed air-, jet-, ultrasonic-, and piezoelectric-nebulizers, which deliver the biologically active agent dissolved or suspended in a pharmaceutical solvent, for example, water, ethanol, or a mixture thereof. Examples of nasal delivery devices capable of delivering diazepam to a patient's nasal cavity (including sinuses and olfactory region) include Pfeiffer APF, Valois Equadel™, OptiNose, BD Medical AccuSpray™ and the like. A nasal spray device can be selected according to what is customary in the industry or acceptable by the regulatory health authorities. In certain applications disclosed herein, a metered dose nasal delivery system may be employed with the added aspect of a lock-out system used to regulate a patient's dosing scheme by time. The delivery systems contemplated herein may be single or multi-use devices. A wide range of volumes may be delivered with each actuation of a nasal delivery device, ranging from about 25 μl to about 250 μl. A pharmaceutical formulation comprising a diazepam may be delivered (dosed) at a range from about 5 mg/dose to about 20 mg/dose, or higher. It is contemplated herein that a formulation comprising a concentration of diazepam (analog or derivative thereof) necessary to achieve such dosing may be in a range of from about 20 to about 40 or 45 mg/ml. Even higher concentrations may be achieved including concentrations from about 50 to about 60 mg/ml.

As disclosed herein, a diazepam can be administered intranasally as a nasal spray or aerosol, the particle size of the spray or aerosol may be between 10 - 100 μm (microns) in size, for example 20 - 100 μm in size. The particle size distribution may be monomodal, bimodal or otherwise multimodal. In certain aspects, the particle size of suspension formulations (or micelle formulation, or liposomal formulation, or dry powder formulation) may be adjusted by including stirring, homogenization, glass beads (of different size), or one or more similar methodology into the formulation manufacturing process. Such processes may result in particles of between 1 and 2000 microns or larger in size, including particles with sizes from about 1 micron to about 100 micron. It is also appreciated that such manufacturing processes may produce articles from about 1 nm to about 1000 nm or more in size, including about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 25 nm, about 45 nm, or about 50 nm. In this regard, the following definitions are useful:

Aerosol - A product that is packaged under pressure and contains therapeutically active ingredients that are released upon activation of an appropriate valve system.

Metered aerosol - A pressurized dosage form comprised of metered dose valves, which allows for the delivery of a uniform quantity of spray upon each activation. Powder aerosol - A product that is packaged under pressure and contains therapeutically active ingredients in the form of a powder, which are released upon activation of an appropriate valve system.

Spray aerosol - An aerosol product that utilizes a compressed gas as the propellant to provide the force necessary to expel the product as a wet spray; it is generally applicable to solutions of medicinal agents in pharmaceutically acceptable aqueous solvents.

Spray - A liquid minutely divided as by a jet of air or steam. Nasal spray drug products contain therapeutically active ingredients dissolved or suspended in pharmaceutically acceptable solutions or mixtures of excipients in non-pressurized dispensers.

Metered spray - A non-pressurized dosage form consisting of valves that allow the dispensing of a specified quantity of spray (pharmaceutically acceptable) upon each activation. Suspension spray - A pharmaceutically acceptable liquid preparation containing solid particles dispersed in a liquid vehicle and in the form of course droplets or as finely divided solids.

The fluid dynamic characterization of the pharmaceutically acceptable aerosol spray emitted by metered nasal spray pumps as a drug delivery device ("DDD"). Spray characterization is an integral part of the regulatory submissions necessary for Food and Drug Administration ("FDA") approval of research and development, quality assurance and stability testing procedures for new and existing nasal spray pumps.

Thorough characterization of the spray's geometry has been found to be the best indicator of the overall performance of nasal spray pumps. In particular, measurements of the spray's divergence angle (plume geometry) as it exits the device; the spray's cross- sectional ellipticity, uniformity and particle/droplet distribution (spray pattern); and the time evolution of the developing spray have been found to be the most representative performance quantities in the characterization of a nasal spray pump. During quality assurance and stability testing, plume geometry and spray pattern measurements are key identifiers for verifying consistency and conformity with the approved data criteria for the nasal spray pumps.

In this regard, the following definitions are considered:

Plume Height - the measurement from the actuator tip to the point at which the plume angle becomes non-linear because of the breakdown of linear flow. Based on a visual examination of digital images, and to establish a measurement point for width that is consistent with the farthest measurement point of spray pattern, a height of 30 mm is defined for this study.

Major Axis - the largest chord that can be drawn within the fitted spray pattern that crosses the COMw in base units (mm).

Minor Axis - the smallest chord that can be drawn within the fitted spray pattern that crosses the COMw in base units (mm).

Ellipticity Ratio - the ratio of the major axis to the minor axis.

D₁₀ - the diameter of droplet for which 10% of the total liquid volume of sample consists of droplets of a smaller diameter (μm).

D₅₀ - the diameter of droplet for which 50% of the total liquid volume of sample consists of droplets of a smaller diameter (μm), also known as the mass median diameter. Dgo - the diameter of droplet for which 90% of the total liquid volume of sample consists of droplets of a smaller diameter (μra).

Span - measurement of the width of the distribution, the smaller the value, the narrower the distribution. Span is calculated as — .

D50 % RSD - percent relative standard deviation, the standard deviation divided by the mean of the series and multiplied by 100, also known as % CV.

In further aspects of this disclosure, evaluation of the chemical stability of an API (e.g., diazepam) disclosed herein is important to the preparation of a pharmaceutically acceptable therapeutically effective amount of a formulation comprising diazepam for intranasal spray administration. Chemical stability generally refers to the amount of chemical degradation of a particular material (e.g., diazepam). When administering compounds such as diazepam, a highly stable formulation is desirable. Chemical stability of a pharmaceutically acceptable therapeutically effective preparation depends upon the amount of chemical degradation of the active pharmaceutical ingredient (API) in that preparation. Stability analysis of such a pharmaceutical preparation, such as a liquid nasal spray product, may be performed under accelerated temperature conditions, such as in a 40, 50 or 6O⁰C. The kinetic methods used in the accelerated stability analysis need not involve detailed studies of mechanism of degradation to be able to predict stability, but they are preferably based upon sound scientific principles and compliance with regulatory requirements.

All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, non-patent publications, figures, and websites referred to in this specification are expressly incorporated herein by reference, in their entirety.

EXAMPLES

The above disclosure generally describes the present disclosure, which is further exemplified by the following Examples. These Examples are described solely for purposes of illustration, and are not intended to limit the scope of this disclosure. Although specific terms and values have been employed herein, such terms and values will likewise be understood as exemplary and non-limiting to the scope of this disclosure. EXAMPLE 1 Solution Formulations Comprising Diazepam

The solubility of diazepam was measured in various excipients. A total of 61 samples were evaluated, including co-solvents, surfactants, oils, complexing agents, and multi-component formulations.

Diazepam powder was added directly to each excipient solution. Samples were placed in eppendorf tubes which were then placed on a 360 degree rotator for more than 24 hours. After rotation, the solutions were centrifuged and filtered. The filtrates were then analyzed using RP-HPLC to determine the concentration of diazepam present in each of the samples.

The results are shown in Table 1.

Table 1

Diazepam Solubility Study: Co-solvents, Complexing Agents and Surfactants

Three co-solvents (PEG300, PEG400, and EtOH) provided diazepam solubilities of greater than 40 mg/ml. Ten surfactants tested yielded solubilities of greater than 25 mg/ml. In addition, two multi-component formulations containing a plurality of excipients were identified providing diazepam solubility at greater than 40 mg/ml.

The two multi-component solution formulations containing a plurality of excipients were evaluated to determine the effect of dilution on the solubility of diazepam prepared in various multi-component formulations. Table 2 illustrates the composition of exemplary multi-component formulations with 20% ethanol.

Table 2 Composition of Multi-component Formulations

The solubility results for the multi-component formulations show that all four multi-component solution formulations provided a diazepam solubility of at least about 40 mg/ml. The formulation containing ethanol, PEG400 and PEG400 monolaurate provided a diazepam solubility of about 61 mg/ml.

The solubility of diazepam was further evaluated after dilution in water. Accordingly, multi-component formulations comprising diazepam (at least about 40 mg/ml) initially prepared as presented in Table 1 having the solubilities presented in Table 2 were prepared and diluted with water at diazepam at various ratios. The concentration of diazepam was measured in the diluted samples. The results showed that diazepam solubility decreased non-linearly upon dilution. Various solubilizers were prepared at the maximum concentration currently allowable under FDA guidelines, as listed in the US-FDA CDER inactive ingredients database. The solubility of diazepam was measured in four formulations containing these solubilizers. Every formulation contained 10% polysorbate 80, 20% polyethylene glycol, 2% ethanol, and 20% PEG400, along with other excipients as shown in Table 3. Diazepam solubility was measured as before.

Table 3 Formulation Composition (%w/w)

The results show that the solubility of diazepam was significantly lower than the solubility range obtained from formulations of Table 2. Currently marketed nasally administered products contain 10% PS80, 2% dehydrated alcohol, 20% PEG400, but do not contain caprylocapryol macrogolglycerides, PEG 400 monolaurate, or PEG300. Therefore the inventive formulations of Table 2 provide significantly improved diazepam solubility relative to formulations prepared at current, comparator FDA acceptable levels of non-aqueous components.

EXAMPLE 2 Improving Physical Stability of Solution Formulations

Polysorbate 80 formulations were modified to obtain a physically stable solution formulation. Various excipients were added or modified: ethanol (2-5%), PEG400 (50- 78%), propylene glycol (0-20%), polysorbate 80 (0-10%) and polysorbate 20 (0-2.5%). Formulations and their results are shown in Table 4. Table 4

Stability and Diazepam Solubility in Solution Formulations

These results show, five stable multi-component solution formulation vehicles containing diazepam at concentrations about 32 mg/ml to about 52 mg/ml were identified as suitable for administration to human subjects. Of the five solution formulations so identified, formulation numbers 2, 8 and 19 comprise non-aqueous formulation vehicles, while formulation numbers 6 and 14 comprise aqueous formulation vehicles.

EXAMPLE 3 Suspension Formulations Containing Diazepam

Various suspension formulations of diazepam using suspending agents at various concentrations. The samples tested included suspensions prepared using five suspending agents at three different concentrations. PEG 400 and polyethylene glycol were cosol vents used in this study and polysorbate 80 was the surfactant used (Table 5. Appearance, diazepam content uniformity, viscosity, density, particle size, zeta potential, and dissolution kinetics were measured. A dilution study was also performed at 1: 10 and 1 : 100 with PBS CaCl₂MgCl₂. Samples were evaluated at 0, 10, 20, 30, 60 and 120 minutes. Table 5 Suspension Formulation Content

The results are shown in Table 6.

Table 6 Suspension Formulations Results

Four of the suspension formulations tested in this study were found to be stable after 12 hours of preparation. The appearance of such suspension formulations was initially milky white and then after 24 hours particles settled to the bottom of the glass bottle. Instability of the other formulations was attributed to formation of gels or crystallization of formulation component(s). The suspensions identified contained the suspending agents CMC or HPMC 2910 at 1% or 2% concentrations. Suspension formulations were dispersible after 24 hours. When a drug is nasally administered as a suspension formulation, a dissolution process precedes the absorption process. Reduced particle size increases the surface area; and, therefore may result in faster dissolution rate. It is also believed that nanop articles of 20-1000 nra can be transported across the nasal cavity into the bloodstream without prior dissolution (see Brooking et al. J. Drug Target 9:267-279, 2001). The uptake was size dependent, i.e., the smaller the size the higher the uptake. Additional consideration for the suspension formulations manufactured herein is the particle size distribution. A mono- disperse (e.g., mono-modal) system with small particle size and narrow particle size distribution may be used for intranasal administration of a drug (e.g., diazepam) that requires rapid onset of action. Intranasal administration of a suspension formulation containing diazepam at a concentration from at least about 40 mg/ml to about 100 mg/ml, including about 50 mg/ml, or about 60 mg/ml, or about 75 mg/ml or about 100 mg/ml, may be accomplished using a nasal spray device.

Three manufacturing process approaches (stir bar mixing, homogenization, and glass bead mixing with a stir bar) were evaluated for the ability to impact particle size of the four suspension formulations containing 1 % or 2% of CMC or HPMC. For suspensions manufactured with glass beads, two different sizes of glass beads (0.1 and 0.5 mm) were evaluated. Accordingly, the four suspension formulations with more than 12 hour stability were evaluated for particle size. The data shown in Table 7 is presented as an average of four runs for the formulations manufactured by stirring; Table 8 presents the average data for the formulations manufactured by homogenization; Table 9 presents the average data for the formulations manufactured in the presence of glass beads. The composite data from individual runs presented in Table 10.

The results indicate that particle sizes of 1% CMC, 1% HPMC, and 2% HPMC suspensions were minimally affected by the manufacturing process or glass bead size whereas the particle sizes of the 2% CMC suspension were significantly reduced in the presence of glass beads. For example, the 1% CMC suspension contained mono-disperse particles in the range of 500-600 μm. Both 1 % and 2% HPMC suspensions contained two populations of smaller and larger particles; 1% HMPC suspension was composed of predominantly micron size particles whereas 2% HPMC suspension was composed of predominantly particles in the sub 100 nm range. Specifically, 1% HPMC suspension contained 60-90% volume of 1-2 μm particles and 10-40% volume of 40-50 nm particles. 2% HPMC suspension contained -90% volume of 20-30 nm particles and -10% volume of 1-4 μm particles. For the 2% CMC suspension manufactured with a stir bar or by homogenization, the results showed that it was composed of mono-disperse particles in the 100±50 nm range. For the 2% CMC suspension manufactured with glass beads of 0.5 mm, the particle sizes were reduced to 1-2 nm range.

Taken together, these data indicate that formulations tested in this study containing 1% CMC had a mono-modal (i.e., mono-disperse) particle size distribution, while formulations with 2% CMC were bimodal {i.e., poly-disperse). Formulations containing 1% HPMC also were bimodal in particle size distribution. Formulations containing 2% HPMC were multimodal with particle size peaks at about 23, 945 and 3077 nm. The particle size distribution in the formulation containing 2% HPMC may be of interest in this study as it has about 92% of particles at a relatively small size of about 23 nm, which may allow for a rapid dissolution of diazepam following nasal administration.

In summary, based on the particle size results, the 2% CMC or HPMC suspensions contained particles in the more desirable ranges than the 1% CMC or HPMC suspensions. In terms of physical stability, settling of particles upon storage at room temperature was observed with both 2% CMC or HPMC suspension. Due to the high viscosity of the 2% CMC suspension, it was more difficult to disperse the particles than the 2% HPMC suspension formulation. However, the 2% HPMC suspension vehicle showed phase separation upon storage at room temperature. Further evaluation showed that reduced polysorbate 80 concentration from 10% to 5% improved the physical stability of the suspension vehicle.

All formulations manufactured by stirring were prepared at a diazepam concentration of 50 mg/ml, and were milky white suspension in appearance (density was about 1.1 g/cm³; the viscosity of 1% CMC suspension was about 182 cps and the 2% suspension was about 807 cps, the viscosity of the 1% and 2% HPMC suspensions was 49 and 99 cps, respectively.

Table 7 Particle Size Analysis (averaged) Suspensions Manufactured by Stirring

The suspension formulations manufactured by stirring, indicated that particles settled in each formulation, although the rate of settling decreased with increasing viscosity. Formulation dispersion decreased with an increasing viscosity; formulations containing HPMC were readily dispersible. In a series of subsequent studies with the objective of identifying a manufacturing process that may improve mono-modal particle size distribution of small size formulations containing diazepam, and various co-solvents (PEG 400 at 20%w/w, PG at 20%w/w) and PS80 at 10%w/w (surfactant) were manufactured by homogenization with an automatic homogenizer, or were subjected to glass beads of different sizes. In parallel, a suspension formulation containing 1 % PVA was evaluated. AU suspension formulations manufactured by homogenization were initially milky white in appearance and then settled to the bottom of the glass container after 24 hours. For samples that were homogenized, the data presented in Table 8 indicate that mono-modal suspensions were obtained with formulations containing CMC while smaller particle sizes were obtained with homogenized formulations containing suspensions with HPMC. AU suspensions manufactured by homogenization were milky white in appearance and provided good recovery of diazepam ranging from about 42 to about 75 % of initial.

Table 8

Particle Size Analysis (averaged) of Suspensions Manufactured by Homogenization

The diazepam content uniformity of suspension formulations containing CMC, HPMC or PVA manufactured by homogenization was also determined and shown to be uniformly distributed throughout the formulation.

For formulations prepared (see Table 9) using glass beads of two different sizes and at different ratios relative to drug/vehicle provided the averaged particle size analysis shown in Table 10. These pharmaceutical formulations were manufactured by first preparing the formulation vehicle containing PS 80, PEG 400, PG and water. Diazepam was then added to a concentration of 50 mg/ml, followed by the addition of glass beads. The suspension formulations containing glass beads were stirred overnight, after which time the glass beads (0.1 mm or 0.5mm) were allowed to settle and the supernatant collected for evaluation of particle size, diazepam content (by HPLC), and appearance. Table 9 Formulations of Suspensions Manufactured in Presence of Glass Beads

Table 10

Particle Size Analysis (averaged) for Suspensions Manufactured in Presence of Glass Beads

Table 11 Composite Particle Size Analysis (individual runs)

The results presented in Table 11 indicated that suspension formulation manufactured using glass beads contained smaller particle sizes compared to formulations manufactured by stirring or homogenization. Higher amounts of glass beads seemed to result in particles of smaller size. For formulations containing the suspending agent HPMC, the particle size obtained in the presence of glass beads was the similar to that observed for formulations manufactured by stirring and homogenization. The diazepam content of suspension formulations manufactured using glass beads was determined by HPLC analysis. The results indicated diazepam concentration of about 52.3 mg/ml for formulation number 2 (see Table 10), 114.4 mg/ml for formulation number 3, 66 mg/ml for formulation number 4 and 27.9 mg/ml for formulation number 6.

The appearance of all formulation presented in Table 11 (manufactured in the presence of glass beads) was initially milky white and then showed phase separation after 24 hours at room temperature.

The content uniformity of suspension formulations containing 50 mg/ml diazepam was also determined by measuring diazepam concentration in samples taken from the top and bottom of the formulation. The results indicated that diazepam was uniformly distributed in formulations containing CMC or HPMC as suspending agent, at either 1% or 2% w/w. EXAMPLE 4 Physical Stability Evaluation of Suspension Vehicles

A certain degree of physical instability was observed for suspension formulations comprising 2% HPMC 2910, resulting in a phase separation. Interactions between HPMC and various formulation excipients was evaluated in order to identify a formulation that would significantly reduce or eliminate such phase separation and thus improve the physical stability of pharmaceutical formulations comprising diazepam. Accordingly, in a first experiment, ten suspension formulations containing 2% HPMC 2910 were tested for phase separation in the presence (at varying concentrations) or absence of polysorbate 80 (PC80), PEG 400 and PG (see Table 12).

Table 12 Evaluation of Formulation Physical Instability

The results from this study indicated that the three formulations without 10% PS80 (i.e., formulation numbers 4, 5 and 6) did not show phase separation. Thus, in subsequent studies, formulations containing PS 80 at reduced concentrations were evaluated (see Table 13) along with a separate study in which polysorbate 80 was substituted with polysorbate 20 (PS20) at various concentration (see Table 14).

Table 13

Physical Stability of HPMC Formulation Containing Varying PS 80 Concentrations

The six suspension formulations shown in Table 13 were manufactured with 2% HPMC 2910 as suspending agent, PEG 400 and PG as co-solvents, and PS 80 as surfactant. In this study, formulations containing PS80 at 4% and 5% concentration were shown to be physically unstable, forming a phase separation. The formulations containing PS 80 at 2% and \% concentration were shown to be physically stable and selected for further evaluation in the development of a pharmaceutical formulation for the intranasal delivery of diazepam.

Table 14

Physical Stability of HPMC Formulation Containing Varying PS 20 Concentrations

The six suspension formulations shown in Table 14 were manufactured with 2% HPMC 2910 as suspending agent, PEG 400 and PG as co-solvents, and varying concentrations of PS20 as surfactant. The physical stability of these formulations indicate that all formulations containing PS20 as surfactant were physically stable, as no phase separation was observed. Based on these results, suspension formulation vehicles containing 2.5% and 1% PS20 as surfactant were selected for further evaluation in the development of a pharmaceutical formulation for the intranasal delivery of diazepam {see Table 15).

With the results provided in Tables 13 and 14, a series of pharmaceutical formulations containing 50 mg/ml of diazepam along with PEG 400 and PG as co- solvents and either PS 80 or PS20 as surfactant were manufactured {see Table 15) and examined for physical stability {e.g., phase separation). Table 15

Physical Stability of Pharmaceutical Formulations Containing Diazepam, and PS80 or PS20

The pharmaceutical formulations disclosed in Table 15 were prepared by first mixing co-solvents, surfactant, and SWFI. Diazepam was then weighed and added in an amount necessary to prepare 50 mg/ml suspensions, stirred overnight. The appearance, density and viscosity, particle size, and content represent the study results.

The results from observation of formulation appearance show that after 72 hours, all formulation were physically stable, appearing as milky white suspensions, no phase separation was observed. The density and viscosity measurements for this study concerning suspension formulations prepared using the drug delivery vehicle HPMC are shown in Table 16.

Table 16

Density and Viscosity Measurements of Pharmaceutical Formulations Containing Diazepam, HPMC, and PS80 or PS20

The data presented in Table 16 indicate that there was no change in density in formulations prepared with different surfactants or with differing concentrations of a particular surfactant. For both PS 80 and PS 20, lower concentrations resulted in lower viscosity; for example, the viscosity of the formulation comprising PS20 at 2.5% was about 231.2 whereas when prepared at a concentration of 1% PS20 the viscosity was about 214.8 mPa.s. It was also noted that all formulations prepared in the study as presented in Table 15 and 16 were easily dispersible regardless of any difference in viscosity. In addition, the appearance of all suspension formulations {see Tables 31 and 32) were further evaluated after storage for two weeks at room temperature and shown to be physically stable by visual appearance (e.g., no phase separation observed).

The particle size was also determined for each suspension formulation prepared as shown in Table 15. The results of particle size analysis are shown in Table 17.

Table 17

Particle Size Measurements of Pharmaceutical Formulations Containing Diazepam, HPMC, and PS80 or PS20

The data presented in Table 17 indicate that pharmaceutical formulations containing diazepam at 50 rag/ml in the presence of PS20 at concentrations of either 1% or 2.5% provided a mono-disperse particle size of about 346 nm and about 276 nm, respectively. Formulations comprising PS 80 provided a poly-disperse particle size (particles of about 350 and about 6 nm) at 1% PS80, and a mono-disperse particle size of about 353 nm at a PS80 concentration of about 2%.

The diazepam content of each suspension formulation prepared as shown in Table 15 was also determined (see Table 18) and shown to be within ±10% of the initial concentration set at 50 mg/ml.

Table 18

Diazepam Content Measurements of Pharmaceutical Formulations Comprising HPMC, and PS80 or PS20

A two week stability study was then performed in order to test stability of suspension formulations containing diazepam at two temperature conditions (5⁰C and 25⁰C), and in order to include the analysis of suspension formulations containing diazepam at a concentration of 100 mg/ml, and in order to evaluate stability of formulations manufactured at a batch size of from about 10 to about 25 g. Accordingly, the formulations provided in Table 18 were prepared (by mixing) for analysis. This short term stability study was performed based upon the design shown in Table 19.

Table 19

Pharmaceutical Formulations Comprising Diazepam, HPMC, and PS80 or PS20 Evaluated at 25⁰C and 5⁰C

Table 20

Two Week Stability Study Design Pharmaceutical Formulations Containing Diazepam, HPMC, and PS80 or PS20 Evaluated at 25⁰C and 5⁰C

The six pharmaceutical formulations described in Table 19 were manufactured as homogeneous suspensions that were milky white in appearance (i.e., the initial appearance results). After two weeks at 25⁰C, appearance results indicated that the six suspension formulations disclosed in Table 19 were milky white in appearance. Formulations 2, 5 and 6 were showing a certain degree of settling out while formulations 1, 3 and 4 continued to appear homogeneous. After 2 weeks at 5⁰C, all formulations remained milky white in appearance; formulations 2, 5 and 6 appeared to have a certain degree of settling out (or separated out), while formulations 1, 3 and 4 remained homogeneous in appearance. The average particle size of these HPMC suspension formulations, all of which were mono-disperse, is shown in Table 21.

Table 21

Averaged Particle Size Measurements of Pharmaceutical Formulations Containing Diazepam, HPMC, and PS80 or PS20 (average of 4 measurements)

The particle size data shown in Table 21 indicate that initially all formulations manufactured for this study presented a particle size distribution that was mono-disperse, including the formulation containing diazepam at 100 mg/ml. In this study, it is noted that the formulation manufactured at about 100 mg/ml of diazepam contained a mono- disperse particle size of about 149 nm, which is smaller than the determined particle size of all formulations manufactured at a diazepam concentration of about 50 mg/ml. After 2 weeks of storage at 25⁰C, formulation numbers 1-4 remained mono-disperse while formulations 5 and 6 presented a poly-disperse particle size profile. After two weeks of storage at 5⁰C, all formulation presented a mono-disperse particle size profile.

The data concerning pH and diazepam content of the suspension formulations shown in Table 18 is presented in Table 22.

Table 22 pH and Diazepam Content Measurements of Pharmaceutical Formulations Containing Diazepam, HPMC, and PS 80 or PS20

The data presented in Table 22 indicate that the initial pH of these formulations was in a range from about 7.0 to about 7.6. In this study, increasing the surfactant concentration resulted in the initial formulation having a lower pH. In addition, increasing the diazepam concentration from about 50 mg/ml to about 100 mg/ml resulted in an increased initial pH from about 7.0 to about 7.6. As shown in Table 22, after storage for two weeks at 25⁰C, the pH of these HPMC suspension formulation numbers 1-4 and 6 decreased whereas formulation number 5 remained the same. As shown in Table 22, after storage for two weeks at 5⁰C, suspension formulation numbers 1, 3, 4 and 5 had an increase in pH, whereas formulation numbers 2 and 6 were lower. Analysis of initial diazepam content, samples from the top and bottom of the formulation indicated that all formulations were determined to be within 5% of nominal and were manufactured as homogeneous suspensions. Diazepam content was also determined after two weeks of storage at 5⁰C and 25⁰C (see Table 23). In this aspect of this study, after two weeks of storage, samples were first either shaken briefly or were stirred for about 30 minutes before a sample was removed for determination of diazepam content. As shown in Table 23, diazepam content was in accordance with starting concentration after stirring but not after a brief shaking.

Table 23 Diazepam Content After Two Weeks Storage

An analysis of formulation density and viscosity was also performed on all formulations containing diazepam (50 mg/ml and 100 mg/ml) and either PS20 or PS80. The results are shown in Table 24. Table 24

Density and Viscosity Measurements of Suspension Formulations Containing Diazepam, HPMC, and PS80 or PS20

The results show that all formulations evaluated had the same initial density of about 1.1 g/cm³, which was unchanged after storage for 2 weeks at 5⁰C or at 25⁰C. The results regarding viscosity show that all formulation manufactured at a diazepam concentration of 50 mg/ml had an initial viscosity within a range of from about 207 to about 231 mPa.s. The formulation manufactured at a diazepam concentration of about 100 mg/ml had an initial viscosity determined to be about 371 mPa.s. After 2 weeks of storage at 25⁰C, viscosity of all formulations disclosed in Table 23 increased. All formulations manufactured in the stability study shown in Table 24 were initially homogeneous milky white suspension. After two weeks storage at 25⁰C or 5⁰C all formulations except number 1 showed particle settlement by visual inspection. Settlement at 5⁰C was however less than observed at 25⁰C.

In order to determine if the manufacturing process {i.e., the order of adding various formulation components) has any impact on the particle size distribution of a suspension formulation so prepared, the following manufacturing processes were evaluated. In manufacturing process No. 1 the order of addition was co-solvent, surfactant, concentrated HPMC solution, water, diazepam powder. For manufacturing process No. 2 the order of addition was co-solvent, surfactant, diazepam powder, concentrated HPMC solution, water. For manufacturing process No. 3 the order of addition was cosolvent, surfactant, concentrated HPMC solution, diazepam powder, water. The pharmaceutical suspension formulation prepared by each manufacturing process contained diazepam at 50 mg/ml and 2.5% PS20. Results from particle size analysis are shown in Table 25. Table 25

Manufacturing Order of Addition Particle Size Measurements of Pharmaceutical Suspension Formulations Containing Diazepam, HPMC and PS20

Abbreviations: DZP = diazepam

As shown in Table 25, the particle size of all suspension formulations manufactured by varying the order of excipient addition was mono-disperse and of similar size, from about 235 nm to about 302 nm.

A further study was performed in order to evaluate the impact of including a buffer on diazepam suspension formulations. In this regard, the formulations presented in Table 26 were manufactured.

Table 26 Suspension Formulations Including a Buffer

Upon visual inspection, all formulation manufactured in accordance with Table 26 were initially milky white in appearance. The initial pH, density and viscosity of the formulations shown in Table 26 were also evaluated; the results are shown in Table 27. Table 27 Density, pH and Viscosity of Buffer Adjusted Suspension Formulations

In addition, it is appreciated that any one or more of the five diazepam suspension formulations presented in Table 27 (formulation numbers 2-6) may also be used to prepare a dry powder (i.e., via spray drying) version thereof for intranasal administration. In this case, such dry powder pharmaceutical formulations may be, for example, administered intranasally using a bi-dose dry powder delivery system. Such administration may be passive or active. Passive administration requires that the subject actually initiates delivery by nasal inhalation (e.g., snorting through the nostrils). Active administration may be achieved by actuation of a delivery device (e.g., a pump). Such a dry powder delivery system is available from a number of sources including Pfeiffer (Oschlestr, Germany), and Valois. The manufacturing of such dry powder formulations would include evaluation of feed solution volume (e.g., from about 20 ml to about greater than one liter), inlet and outlet temperatures, aspirator, feed solution flow rate and atomizing pressure, where the percent recovery is calculated based upon volume of feed solution, concentration of solids in the feed solution and weight of the spray dried powder.

In addition, with regard to selection of an intranasal delivery device, suspension formulation containing 100 mg/ml diazepam may be compatible with a single 200 μl dosage form administered via, for example, an AccuSpray™ device (Becton Dickinson, Franklin Lakes, NJ).

EXAMPLE 5 Emulsion Formulations Comprising Diazepam Emulsion formulations containing three oils were evaluated. The solubility of diazepam in these formulations was determined as before. Emulsion formulations were prepared with selected emulsifying agents and the determined diazepam concentrations are shown in Table 28.

Table 28 Study Design and Diazepam Solubility of Oil Emulsion Formulations

As shown in Table 28, the solubility of diazepam in the emulsions containing ethyl laurate, oleic acid or soybean oil ranged from about 7.3 to about 14.8 mg/ml. In this study, a higher diazepam solubility was determined for emulsion formulations containing oleic acid manufactured with PS 80 or PS 20.

Table 29

Study Design and Diazepam Solubility in Emulsion Formulations with Selected Emulsifying Agents

Table 29 shows that the solubility of diazepam emulsion formulations manufactured with selected emulsifying agents ranged from about 7.1 to about 10.7 mg/ml. In this study, the emulsion formulations containing Capmul MCM or Lauroglycol 90 provided the highest diazepam solubility (10.2 and 10.7 mg/ml, respectively).

In a separate study, the solubility of diazepam in emulsion formulations containing either mineral oil or triacetin were evaluated. Accordingly the emulsion formulations shown in Table 30 were manufactured. In this study, solubility was determined by HPLC analysis and stability was determined by visual inspection. The target concentration of diazepam was set at 50 mg/ml. Formulations were mixed by rotation (tumbling) overnight at room temperature, after which formulations were centrifuged at 12,000 rpm for 30 minutes and then a sample of each supernatant analyzed by HPLC.

Table 30 Diazepam Solubility in Emulsion Formulations Containing Triacetin

EXAMPLE 6 Solid Surface Dispersion Formulations Containing Diazepam

Solid surface dispersions of diazepam were prepared in a carboxymethyl cellulose sodium solid medium in order to determine the recovery of diazepam and diazepam solubility. A slurry of diazepam and carboxy methyl cellulose sodium was then prepared using proportions of one to one (1:1) and one to two (1:2). The slurry was dried at 5O⁰C in order to evaporate the chloroform.

Diazepam recovery results are found on Table 31. Table 31 Solid Surface Dispersions: Diazepam Recovery

Table 32 Solid Surface Dispersions: Diazepam Solubility

There was higher recovery with the 1:1 ratio of diazepamxarbyoxy methyl cellulose sodium solid dispersion as compared to the 1:2 ratio of diazepam carboxy methyl cellulose sodium solid dispersion. Diazepam with EtOH and PEG400 in either the surfactant labrasol or PEG400 ML was measured for solubility. Results are found in Table 48.

EXAMPLE 7 Dispersion Agents in Diazepam Suspension Formulations

Water insoluble dispersible colloidal excipients may be used in the manufacture of pharmaceutical suspension or emulsion formulations. Such dispersible colloidal excipients may be used in order to improve the stability of a suspension formulation, providing a structured dispersion vehicle exhibiting a high degree of thixotropy and may prevent drug particles from settling or phase separation in a suspension formulation. Two representative examples of such excipients are Avicel® microcrystalline cellulose and carboxymethycellulose sodium. Other examples of dispersion agents include poloxamers, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, hypromellose acetate succinate, lecithin, polyethylene alkyl ethers, sorbitan esters, poly(methylvinyl ether/maleic anhydride and the like. In this study Avicel was tested in diazepam suspension formulations, alone or in combination with HPMC2910. The composition of the formulations to be tested is listed in Table 33, and will be characterized for appearance, viscosity, density, pH, content, and particle size.

Table 33 Suspension Formulations Containing Dispersion Agents

EXAMPLE 8 Preservative in Formulations Containing Diazepam

A preservative system was developed for diazepam nasal delivery formulations that was suitable to attain USP Antimicrobial Effectiveness Testing requirements and EP Antimicrobial Effectiveness Testing (AET) requirements.

The quality (physical and chemical analysis) of all formulations to be evaluated is monitored for pH, content and appearance at the time of manufacturing. In addition, HPLC analysis was performed at the beginning and end of each study to determine stability of diazepam and of the preservative. The data are used to identify a combination of preservatives that are successful in passing USP AET requirements.

Various preservatives were tested, including benzalkonium chloride, methylparaben, propylparaben, benzyl alcohol, and phenylethyl alcohol. Formulations contained 20% PEG400, 2% hydroxypropyl methylcellulose 2910, 20% polyethylene glycol, and 40 mM phosphate buffer, pH 6.8 (Table 34). Formulations 3 through -10 (including 6a and 9a) containing different levels of surfactant, preservative(s), co-solvent, and/or phosphate buffer. Formulation 11 contains surfactants (polysorbate 20 and polysorbate 80), all preservatives, and co-solvent, in the presence or absence (formulation 6a and 9a) of phosphate buffer, were tested for neutralization, preparatory test and microbial limit. Table 34 AET Testing of Multi-Component Diazepam Suspension Formulations

Abbreviations: PS - Polysorbate; BZK - Benzalkonium chloride; MP - Methylparaben; PP - Propylparaben; BA - Benzyl alcohol; PE - Phenylethy] alcohol;

Results concerning appearance (e.g., physical stability) and pH for this stability study are shown in Table 35 (placebo (i.e., formulation vehicle) means without diazepam). Formulation numbers 1-8 correspond to formulations listed in Table 57. Initial appearance results indicate that all formulations were within product specifications. Initial pH results were within specification (6.8±0.5).

Table 35

Stability Time Points and Testing: Multi-Component Suspension and Solution Diazepam Formulations, Initial Appearance and pH Results

non-aqueous formulation

In a separate AET, a suspension formulation containing 2% ethanol, 78% PEG400, and 20% polyethylene glycol was evaluated. Antϊ microbial testing was also initiated for two solution formulations; one contained 25%EtOH, 55% PEG400, 2.5% PS 20, 17.5% SWFI, and the second contained 20% EtOH , 50% PEG400, 20% PG, 10% SWFI). A composite representation of solution and suspension formulations selected for further development in this study is presented in Table 36.

Table 36 Multi-Component Suspension and Solution Diazepam Formulations

The stability results for diazepam content, purity, density and viscosity of suspension and solution formulations described in Table 36 indicated that each formulation evaluated was mono-modal in its particle size distribution.

The suspension formulation numbers IA through 1OA as provided in Table 36 were evaluated for AET under USP and EP standards. Results for AET for suspension formulation vehicles are summarized in Table 37. AU multi-Component suspension formulations tested in this AET study also contained PG at 20%, PEG 400 at 20%, HPMC at 2%, and were buffered with 40 mM phosphate (pH 6.8).

Based upon data concerning AET results as well as stability data, one or more of suspension formulation numbers 6 A through 1OA identified in Table 37 are suitable for administration to human subjects.

Two additional suspension formulations identified in Table 37 (formulations 12A and 13A), and three solution formulations one containing EtOH (25% w/v), PEG400 55% (w/v), PS 20 (2.5% w/v), SWFI (17.5% w/v), and the second (formulation number 14, see Table 4) containing EtOH (20% w/v), PEG400 50% (w/v), PG (20% w/v), SWFI (10% w/v), and a third containing EtOH (2%), PEG400 (78%) and PG (20%), were also studied under USP and EP AET guidelines. The results of these studies are summarized in Table 38.

Table 38 AET Summary Results: Sus ension and Solution Formulation Vehicles

Taken together, the suspension formulation 3 and 5, aqueous solution formulation numbers 6 or 7, and non-aqueous formulation number 8, passed both USP and EP AET standards and were suited for further development. Suspension formulation 1, 2 and 4 passed USP AET standards only.

The one month stability results, including appearance, content (mean), content (% nominal), impurity, pH_; density and viscosity, and particle size for samples described in Table 38 show that the diazepam content for all formulations is within 5% of their nominal concentrations at the one month time point.

EXAMPLE 9

Evaluation of Delivery Device Performance For Administration of Suspension Formulations Containing Diazepam

In this Example, representative delivery devices for intranasal administration were evaluated for there suitability to deliver suspension formulations containing diazepam at concentration of about 50 to about 100 mg/ml. The suspension formulation evaluated in this representative study also contained HPMC at about 2% by weight, PEG at about 20% by weight, PG at about 20% by weight, and 40 mM phosphate buffer. The nasal pump delivery devices evaluated were a Pfeiffer high viscosity device and a gel pump device. The shot weight from two to three bottles per pump type fitted with the appropriate pump device was determined, averaged from 5 individual sprays from each bottle. Diazepam content in the sprays was determined by reversed-phase HPLC. The average diazepam content in sprays obtained using three Pfeiffer high viscosity pumps (5 sprays each) was about 50 mg/ml; and, the average from two gel pumps was also about 50 mg/ml. Similarly, when filled with the suspension formulation containing 100 mg/ml diazepam, the gel pump device provided the average diazepam content determined (5 sprays each) was about 100 mg/ml.

All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, non-patent publications, figures, tables, and websites referred to in this specification are expressly incorporated herein by reference, in their entirety. Although the foregoing disclosure has been described in detail by way of example for purposes of clarity of understanding, it is apparent to the artisan that certain changes and modifications are comprehended by the disclosure and may be practiced without undue experimentation within the scope of the appended claims, which are presented by way of illustration, not limitation.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical formulation for intranasal delivery of diazepam, comprising an aqueous suspension of diazepam at a concentration of at least about 25 mg/ml, a suspending agent, and a surfactant.

2. The pharmaceutical formulation of claim 1, wherein the suspending agent is one or more molecules selected from the group consisting of methylcellulose; hydroxypropylniethylcellulose; hydroxypropylmethylcellulose acetate succinate, carboxymethylcellulose, HPMC 2910, HPMC 2208, and polyvinyl alcohol.

3. The pharmaceutical formulation of claim 1, wherein the surfactant is selected from nonionic polyoxyethylene ether, bile salts, sodium glycocholate, deoxycholate, derivatives of fusidic acid, sodium taurodihydrofusidate, L-α- phosphatidylcholine didecanoyl, poloxamer F68, poloxamer F127, polysorbate 80, polysorbate 20, a polyethylene glycol, cetyl alcohol, polyvinylpyrolidone, a polyvinyl alcohol, lanolin alcohol, or sorbitan monooleate.

4. The pharmaceutical formulation of claim 1, wherein the surfactant is selected from the group consisting of polysorbate 80, polysorbate 20 and polyethylene glycol 400 monolaurate.

5. The pharmaceutical formulation of any of claims 1-4, further comprising a cosolvent.

6. The pharmaceutical formulation of claim 5, wherein the cosolvent is one or more molecules selected from the group consisting of ethanol, polyethylene glycol, and polyethylene glycol 400 (PEG 400).

7. The pharmaceutical formulation of any of claims claim 1-6, wherein the concentration of diazepam is at least about 40 mg/ml.

8. The pharmaceutical formulation of any of claims claim 1-6, wherein the concentration of diazepam is at least about 50 mg/ml.

9. The pharmaceutical formulation of any of claims 1-8, further comprising a preservative.

10. The pharmaceutical formulation of claim9, wherein the preservative is selected from the group consisting of benzalkonium chloride, chlorobutanol, methylparaben, propylparaben, ethylparpaben, phenol, and m-cresol.

11. The pharmaceutical formulation of any of claims 1-10, wherein the formulation is produced by mixing with glass beads.

12. The pharmaceutical formulation of any of claims 1-10, wherein the formulation is produced by homogenization.

13. The pharmaceutical formulation of any of claims 1-10, comprising particles having diameters from about 1 nanometer to about 1000 nanometers.

14. The pharmaceutical formulation of claim 13, wherein at least about 80 percent of particles are less than about 30 nanometers in size.

15. The pharmaceutical formulation of claim 14, wherein the particle size distribution is mono-disperse.

16. The pharmaceutical formulation of any of claims 1-15, further comprising a dispersion agent.

17. The pharmaceutical formulation of claim 16, wherein the dispersion agent is selected from the group consisting of Avicel® microcrystalline cellulose, carboxymethycellulose sodium, a poloxamer, diethanol amine, ethylene glycol palmitostearate, glycerin monostearate, hypromellose acetate succinate, lecithin, polyethylene alkyl ethers, sorbitan esters, poly(methylvinyl ether/maleic anhydride).

18. A pharmaceutical formulation for intranasal delivery of diazepam, comprising a non-aqueous solution of diazepam at a concentration of at least about 25 mg/ml, one or more suspending agents, one or more mucosal delivery enhancing agents.

19. A pharmaceutical formulation for intranasal delivery of diazepam, comprising an aqueous solution of diazepam at a concentration of at least about 25 mg/ml, a cosolvent and a surfactant.

20. The pharmaceutical formulation of claim 19, wherein the surfactant is selected from the group consisting of polysorbate 80, polysorbate 20, caprylocaproyl macrogolglyceride, and polyethylene glycol 400 monolaurate.

21. The pharmaceutical formulation of claim 19, wherein the cosolvent is one or more molecules selected from the group consisting of ethanol, polyethylene glycol, and polyethylene glycol 400 (PEG 400).

22. A method for delivering diazepam intranasally, comprising preparing a pharmaceutical formulation of any of claims 1-21, and intranasally administering the pharmaceutical formulation to a patient in need thereof.