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Avizafone Dihydrobromide is a novel and potent water-soluble peptide prodrug that has to be metabolized by plasma enzymes to produce the active drug diazepam. It is used mainly as an antidote to poisoning with organophosphate nerve agents.
| Targets |
Diazepam Prodrug; lasma enzymes
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|---|---|
| ln Vitro |
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].
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| ln Vivo |
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].
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| Animal Protocol |
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]
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| References |
[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. |
| Additional Infomation |
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] 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. |
| Molecular Formula |
C22H29CL3N4O3
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|---|---|
| Molecular Weight |
592.75
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| CAS # |
60067-15-4
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| Related CAS # |
65617-86-9
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| PubChem CID |
71968
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| Appearance |
Typically exists as solid at room temperature
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| Density |
1.253g/cm3
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| Boiling Point |
697.6ºC at 760 mmHg
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| Flash Point |
375.7ºC
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| Index of Refraction |
1.604
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| LogP |
4.347
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
10
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| Heavy Atom Count |
30
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| Complexity |
583
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| Defined Atom Stereocenter Count |
1
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| SMILES |
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
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| InChi Key |
WQRZTXVDPVPVDN-MBABXSBOSA-N
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| InChi Code |
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
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| Chemical Name |
(2S)-2,6-diamino-N-[3-(2-benzoyl-4-chlorophenyl)-2-oxobutyl]hexanamide
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| Synonyms |
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;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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
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| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6871 mL | 8.4353 mL | 16.8705 mL | |
| 5 mM | 0.3374 mL | 1.6871 mL | 3.3741 mL | |
| 10 mM | 0.1687 mL | 0.8435 mL | 1.6871 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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