Diazepam Prodrug; plasma enzymes; As an inactive prodrug, Avizafone itself has no intrinsic activity at benzodiazepine receptors. Its primary target is the enzymatic cleavage site of aminopeptidases (specifically Aminopeptidase B) in the bloodstream. Upon enzymatic conversion, it releases the active metabolite, diazepam. Diazepam then acts as a positive allosteric modulator of the GABAA receptor, enhancing chloride ion channel opening to produce sedative, anxiolytic, and anticonvulsant effects .

Human aminopeptidase B (APB) is a labile enzyme that is being investigated as a biocatalyst for intranasal delivery of prodrug/enzyme combinations. Therefore, the stability of APB is a major concern to ensure a viable drug product. Lyophilization is one technique commonly used to extend shelf life of enzymes. However, the lyophilization process itself can cause conformational changes and aggregation, leading to inactivation of enzymes. In this study, we demonstrate the use of the substrate Avizafone (AVF), a prodrug for diazepam, as a stabilizer to minimize inactivation of APB during lyophilization. Permutations of APB samples combined with AVF, trehalose, and/or mannitol were snap-frozen and lyophilized, and subsequently reconstituted to measure the activity of APB. Of the formulation permutations, an APB + AVF + trehalose combination resulted in minimum degradation with 71% retention of activity. This was followed by APB + AVF and APB + trehalose with 60 and 56% retention of activity, respectively. In comparison, APB + mannitol and APB alone retained only 16 and 6.4% activity, respectively. Lyophilizates of the APB + AVF + trehalose formulation were subjected to a 6 month accelerated stability study, at the end of which negligible reduction in activity was observed. These results suggest that colyophilization of an enzyme with its substrate can impart stability on par with the commonly used lyoprotectant, trehalose, but the combination of substrate and trehalose provides a greater stabilizing effect than either additive alone [1].

Intranasal administration is an attractive route for systemic delivery of small, lipophilic drugs because they are rapidly absorbed through the nasal mucosa into systemic circulation. However, the low solubility of lipophilic drugs often precludes aqueous nasal spray formulations. A unique approach to circumvent solubility issues involves coadministration of a hydrophilic prodrug with an exogenous converting enzyme. This strategy not only addresses poor solubility but also leads to an increase in the chemical activity gradient driving drug absorption. Herein, researchers report plasma and brain concentrations in rats following coadministration of a hydrophilic diazepam prodrug, Avizafone, with the converting enzyme human aminopeptidase B. Single doses of Avizafone equivalent to diazepam at 0.500, 1.00, and 1.50 mg/kg were administered intranasally, resulting in 77.8% ± 6.0%, 112% ± 10%, and 114% ± 7% bioavailability; maximum plasma concentrations 71.5 ± 9.3, 388 ± 31, and 355 ± 187 ng/ml; and times to peak plasma concentration 5, 8, and 5 minutes for each dose level, respectively. Both diazepam and a transient intermediate were absorbed. Enzyme kinetics incorporated into a physiologically based pharmacokinetic model enabled estimation of the first-order absorption rate constants: 0.0689 ± 0.0080 minutes−1 for diazepam and 0.122 ± 0.022 minutes−1 for the intermediate. Our results demonstrate that diazepam, which is practically insoluble, can be delivered intranasally with rapid and complete absorption by coadministering Avizafone with aminopeptidase B. Furthermore, even faster rates of absorption might be attained simply by increasing the enzyme concentration, potentially supplanting intravenous diazepam or lorazepam or intramuscular midazolam in the treatment of seizure emergencies [2].

Enzyme kinetics. [1]
The Michaelis constant (KM) and maximum reaction velocity (Vmax) for the hydrolysis of Avizafone (AVF) by APB in pH 7.4 PBS at 32°C were determined by fitting the Michaelis-Menten equation to the initial substrate depletion rates across a range of AVF concentrations. Reactions were performed in an Eppendorf Thermomixer 5436 at 500 rpm with 62.5–4000 μM Avizafone (AVF) and 15 μg/mL (0.203 μM) APB. After 1 minute, APB was denatured by the addition of methanol. The cyclization of ORI to DZP was allowed to complete before measuring the UV spectra of the quenched reaction mixtures in a Cary 100 Bio-UV/Vis spectrophotometer. The second derivative of the spectra at 338 nm was used to quantify DZP, which was taken as the molar equivalent of AVF consumed.

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Freeze-drying procedure [2]
Aqueous stock solutions of APB, Avizafone (AVF), Tre, Man, pH 7.4 Tris, and pure water were cooled to approximately 0°C in an ice bath. Aliquots of the stock solutions were pipetted in appropriate proportions into chilled 2 mL glass vials to achieve the indicated component concentrations and an equal final volume for each sample. For samples containing AVF, the AVF solution was added last. The cold, pre-frozen solutions were briefly mixed with a pipette before the vials were immersed in liquid nitrogen for 5 minutes to rapidly freeze the contents. The vials were then removed from the liquid nitrogen, transferred to a freeze-drying flask, and dried for 18 hours at room temperature under a vacuum of 0.016 mbar using a FreeZone 6 manifold freeze dryer. The vials containing the lyophilizate were capped and stored in a desiccator until analysis. Unless otherwise stated, storage at room temperature is assumed.

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Assay of active enzyme [2]
The amount of active enzyme remaining after sample processing, freeze-drying, and storage was determined using spectroscopic techniques. Absorbance (Abs) measurements to track substrate hydrolysis were performed using a Cary 100 Bio-UV/Vis double-beam spectrophotometer equipped with a temperature controller. Unless otherwise stated, the temperature controller was set to 32°C, the average temperature of the human nasal cavity. Samples were analyzed in quartz ultra-micro cells with a 1 cm path length and a minimum fill volume of 50 μL. Solutions of APB, Tre, Tris, and Man at the concentrations used in this study were transparent at the wavelengths used for the spectroscopic quantification of substrate and product concentrations. To measure specific enzyme activity, the lyophilizate was reconstituted in PBS heated to 32°C with 1.00 mM LpNA. The resulting solution was immediately transferred to a cuvette already placed in the temperature-controlled block of the spectrophotometer. The change in Abs at 405 nm due to LpNA hydrolysis was monitored. Readings were taken every 100 milliseconds. The slope of the absorbance change (dAbs/dt) during the first 0.25 minutes was used to calculate the reaction rate (d[pNA]/dt) as shown below:
where ε represents the molar absorptivity coefficient. In pH 7.4 PBS at 32°C, εpNA = 9860 M⁻¹ cm⁻¹ and εLpNA = 55.9 M⁻¹ cm⁻¹ at 405 nm (refer to Supporting Information Table S1 for the temperature dependence of ε). The specific enzyme activity was calculated as (d[pNA]/dt)/[APB]. The chromogenic substrate LpNA cannot be used to measure the amount of active APB in samples containing the prodrug substrate Avizafone (AVF). To measure active APB in these samples, the lyophilizate was reconstituted with 100 μL of PBS or a 1.00 mM Avizafone (AVF) solution in PBS that had been heated to 32°C, and the resulting solution was immediately transferred to a cuvette recessed in the spectrophotometer as described above. The change in Abs at 315 nm due to the conversion of AVF to DZP was monitored for 20 minutes. Readings were taken every 0.25 minutes. The concentration of DZP at time t was calculated as follows:
where Abs₀ is the initial absorbance of the sample and Absₜ is the absorbance measured at time t. In pH 7.4 PBS at 32°C, εDZP = 2040 M⁻¹ cm⁻¹ and εAvizafone (AVF) = 753 M⁻¹ cm⁻¹ at a wavelength of 315 nm. The temperature dependence of εDZP and εAvizafone (AVF), as well as the validity of ignoring εORI when calculating DZP concentration from absorbance measurements, has been discussed previously. After obtaining the DZP concentration–time curve, the following set of coupled differential equations was fitted to the data, with [APB] as a separate fitting parameter.

While Avizafone is primarily studied in cell-free systems for enzyme kinetics, researchers focus on its role in overcoming solubility barriers. In epithelial cell models, Avizafone is combined with converting enzymes to create supersaturated drug solutions. This gradient forces high concentrations of the liberated diazepam across cell monolayers, mimicking the enhanced permeation observed in nasal mucosal absorption .

For IN dosing, rats were anesthetized, placed in the supine position, and cannulated. Rats designated for nasal tissue histology were not cannulated. Solutions of Avizafone (AVF) and APB prepared in PBS were admixed to the appropriate concentration for each animal immediately prior to administration. See Table 1 for dose levels. After mixing, the formulation was quickly instilled into the nasal cavity using an Eppendorf pipettor with a gel loading pipette tip inserted to a depth of 14 mm past the nares. A total volume of 30 μl was delivered, 15 μl into the right nostril followed by 15 μl into the left nostril within 0.5 minutes. There were four IN dose groups: Avizafone (AVF)/APB at low, medium, and high doses, and an Avizafone (AVF)-only group at the medium dose. These doses were chosen because DZP near 1 mg/kg is commonly used in rat studies and results in plasma concentrations in rats that are clinically relevant. [1]

Avizafone acts as an efficient delivery vehicle. In human trials, injection of Avizafone results in a higher maximum plasma concentration (Cmax 231 ng/mL) of diazepam compared to equimolar doses of diazepam itself (Cmax 148 ng/mL), achieving peak levels faster (Tmax 0.75h vs 1.5h). In rat intranasal studies, conversion is nearly instantaneous, with absorption half-lives measured in minutes, demonstrating enhanced bioavailability and rapid brain distribution .

Toxicological data is primarily derived from the known safety profile of its active metabolite, diazepam, with the exception of local tolerability. Histological examinations of rat nasal tissues following intranasal administration of Avizafone/APB formulations revealed no acute damage or lesions to the mucosal membrane. The prodrug strategy avoids the use of organic solvents (propylene glycol, benzyl alcohol) required for diazepam injections, potentially reducing local irritation .

[1]. Diazepam Prodrug Stabilizes Human Aminopeptidase B during Lyophilization. Mol Pharm. 2020 Feb 3;17(2):453-460.
[2]. Intranasal Coadministration of a Diazepam Prodrug with a Converting Enzyme Results in Rapid Absorption of Diazepam in Rats. J Pharmacol Exp Ther. 2019 Sep;370(3):796–805.

Avizafen is a peptide prodrug. In summary, the pharmacokinetic results presented in this paper demonstrate that intranasal combination of avizafen (AVF) and apiracetam (APB) is a feasible and rapid method for delivering diazepam (DZP) into the systemic circulation and ultimately to the brain. As this high-concentration formulation contains no organic solvents, it is expected to be better tolerated and absorbed more quickly than intranasal diazepam formulations that use solubilizers. Intranasal avizafen (AVF)/apiraracetam (APB), administered as a non-invasive nasal spray, could be used to rapidly terminate seizures in humans, thereby reducing emergency room visits and improving the quality of life for patients with seizures. Further research progress requires the development of a device that can store the prodrug and enzyme separately and mix them into a spray solution upon administration. [1] Finally, we recognize that although this study demonstrates the effect of the substrate on the stability of the enzyme during lyophilization, its scope is limited to APB. The primary objective of these experiments is to evaluate the feasibility of co-lyophilizing the avizafool (AVF)/APB combination to stabilize the drug formulation for further translational development and to guide the design requirements of dedicated nasal spray devices. Further experiments are currently underway, including scale-up to therapeutically relevant concentrations, optimization of lyophilization process parameters, comprehensive characterization of the lyophilized product, and performance testing in delivery devices.

C22H29CL3N4O3

592.75

429.1819195

60067-15-4

65617-86-9

71968

Typically exists as solid at room temperature

1.253g/cm3

697.6ºC at 760 mmHg

375.7ºC

1.604

4.347

3

5

10

30

583

1

NCCCC[C@@H](C(NCC(C(C1C=CC(Cl)=CC=1C(C1C([2H])=C([2H])C([2H])=C([2H])C=1[2H])=O)C)=O)=O)N

WQRZTXVDPVPVDN-MBABXSBOSA-N

InChI=1S/C23H28ClN3O3/c1-15(21(28)14-27-23(30)20(26)9-5-6-12-25)18-11-10-17(24)13-19(18)22(29)16-7-3-2-4-8-16/h2-4,7-8,10-11,13,15,20H,5-6,9,12,14,25-26H2,1H3,(H,27,30)/t15?,20-/m0/s1

(2S)-2,6-diamino-N-[3-(2-benzoyl-4-chlorophenyl)-2-oxobutyl]hexanamide

Avizafone Dihydrobromide; 60067-15-4; (2S)-2,6-diamino-N-[3-(2-benzoyl-4-chlorophenyl)-2-oxobutyl]hexanamide; N-[3-(2-Benzoyl-4-chlorophenyl)-2-oxobutyl]-L-lysinamide; Prodiazepam Dihydrobromide; (2S)-2,6-Diamino-N-(3-(2-benzoyl-4-chlorophenyl)-2-oxobutyl)hexanamide; L-Lysyl-N-(2-benzoyl-4-chlorophenyl)-N-methyl-glycinamide Dihydrobromide; DTXSID20747061 Avizafone; 65617-86-9; Pro-diazepam; Avizafonum [INN-Latin]; Avizafona [INN-Spanish]; Avizafona; Avizafonum; Ro 03-7355/000;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples