Diazepam Prodrug; lasma enzymes

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 rates of substrate consumption for a series of Avizafone (AVF) concentrations. Reactions with 62.5–4000 μM Avizafone (AVF) and 15 μg/ml (0.203 μM) APB were carried out in an Eppendorf Thermomixer 5436 at 500 rpm. After 1 minute, APB was denatured by addition of methanol. Cyclization of ORI to DZP was allowed to progress to completion before measurement of UV spectra of the quenched reaction mixtures in a Cary 100 Bio UV/Vis spectrophotometer. The second derivative of the spectrum at 338 nm was used to quantify DZP, and this quantity was taken to be the molar equivalent to the amount of Avizafone (AVF) consumed.

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Lyophilization Procedure [2]
Aqueous stock solutions of APB,Avizafone (AVF), Tre, Man, pH 7.4 Tris, and pure water were cooled in an ice bath to ∼0 °C. Aliquots of the stock solutions were pipetted into cold 2 mL glass vials in appropriate ratios to give the indicated component concentrations and an equal final volume for each sample. For samples that contained Avizafone (AVF), the Avizafone (AVF) solution was added last. These cold prelyophilization solutions were mixed briefly with a pipette before placing the vials in liquid nitrogen for 5 min to snap-freeze the contents. The vials were then removed from the liquid nitrogen, transferred to a lyophilization flask, and dried at room temperature under 0.016 mbar vacuum for 18 h using a FreeZone 6 manifold freeze dryer. The vials containing the lyophilizates were capped and stored in desiccators until analysis. Storage was at room temperature unless otherwise stated.

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Measurement of Active Enzyme [2]
The amount of active enzyme remaining after sample processing, lyophilization, and storage was determined using spectroscopic techniques. Absorbance (Abs) measurements to track the hydrolysis of substrates were made using a Cary 100 Bio UV/Vis double-beam spectrophotometer equipped with a temperature controller. Unless stated otherwise, the temperature controller was set to 32 °C, which is the average temperature of the human nasal cavity. Samples were analyzed in a quartz ultramicrocuvette with 1 cm pathlength and 50 μL of minimum fill volume. Solutions of APB, Tre, Tris, and Man at the concentrations used in this study were transparent at the wavelengths used for spectroscopic quantification of the substrate and product concentrations. To measure specific enzyme activity, lyophilizates were reconstituted with 1.00 mM LpNA in PBS that had been warmed to 32 °C. The resulting solutions were immediately transferred to a cuvette already positioned in the temperature-controlled block of the spectrophotometer. The change in Abs due to hydrolysis of LpNA was monitored at 405 nm. Readings were taken every 100 ms. The slope of the absorbance change (dAbs/dt) during the first 0.25 min was used to calculate the reaction rate (d[pNA]/dt) as
where ε denotes molar absorptivity. 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). Specific enzyme activity was calculated by (d[pNA]/dt)/[APB]. The chromogenic substrate, LpNA, could not be used to measure the amount of active APB in samples that contained the prodrug substrate, Avizafone (AVF). To measure active APB in these samples, the lyophilizates were reconstituted with 100 μL of either PBS or 1.00 mM Avizafone (AVF) in PBS that had been warmed to 32 °C, and the resulting solutions were immediately transferred to a cuvette embedded in the spectrophotometer, as described above. The change in Abs due to conversion of Avizafone (AVF) to DZP was monitored for 20 min at 315 nm. Readings were taken every 0.25 min. The concentration of DZP at time t was calculated as
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 315 nm. The temperature dependence of εDZP and εAvizafone (AVF) and the validity of neglecting εORI in the calculation of DZP concentrations from absorbance measurements have been discussed previously. After obtaining DZP concentration–time profiles, the following set of coupled differential equations was fit to the data with [APB] as the lone fitting parameter.

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]

[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.

Avizafone is a peptide prodrug.
In conclusion, the pharmacokinetic results presented here demonstrate that IN coadministration of Avizafone (AVF) with APB is a viable method to rapidly deliver DZP into the systemic circulation and, subsequently, the brain. Since the highly concentrated formulation does not contain organic solvents, it is expected to be better tolerated and absorb faster than IN formulations of DZP that use solubilizing excipients. Administered as a noninvasive nasal spray, IN Avizafone (AVF)/APB could be used to quickly terminate seizure emergencies in humans, resulting in reduced emergency department visits and improved quality of life for patients who experience seizure emergencies. Further progress necessitates development of a device that can store the prodrug and enzyme separately, and then combine them into sprayed solution at the time of administration.[1]

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We conclude by recognizing that this study, while demonstrating the effect of substrate on the stability of an enzyme during lyophilization, was limited in scope to APB. The primary goal of these experiments was to assess the feasibility of colyophilizing the Avizafone (AVF)/APB pair to stabilize the pharmaceutical formulation for further translational development and guide design requirements for a specialized nasal spray device. Additional experiments for scale-up to therapeutically relevant concentrations, optimization of the lyophilization process parameters, full characterization of the lyophilizates, and performance testing in the delivery device are ongoing.

C22H27CLN4O3

430.9

430.177

65617-86-9

60067-15-4

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

CN(C1=C(C=C(C=C1)Cl)C(=O)C2=CC=CC=C2)C(=O)CNC(=O)[C@H](CCCCN)N

LTKOVYBBGBGKTA-SFHVURJKSA-N

InChI=1S/C22H27ClN4O3/c1-27(20(28)14-26-22(30)18(25)9-5-6-12-24)19-11-10-16(23)13-17(19)21(29)15-7-3-2-4-8-15/h2-4,7-8,10-11,13,18H,5-6,9,12,14,24-25H2,1H3,(H,26,30)/t18-/m0/s1

(2S)-2,6-diamino-N-[2-(2-benzoyl-4-chloro-N-methylanilino)-2-oxoethyl]hexanamide

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)