Diazepam Prodrug; lasma enzymes

人氨基肽酶B（APB）是一种不稳定的酶，目前正被研究作为前药/酶组合鼻内递送的生物催化剂。因此，APB的稳定性是确保可行药物产品的主要关注点。冻干是一种常用的延长酶保质期的技术。然而，冻干过程本身会引起构象变化和聚集，导致酶失活。在这项研究中，我们展示了使用底物Avizafone（AVF）作为稳定剂，以尽量减少冻干过程中APB的失活，AVF是地西泮的前药。将APB样品与AVF、海藻糖和/或甘露醇混合后进行快速冷冻和冻干，随后进行复溶以测量APB的活性。在制剂排列中，APB+AVF+海藻糖组合的降解率最低，活性保留率为71%。随后是APB+AVF和APB+海藻糖，分别保留了60%和56%的活性。相比之下，APB+甘露醇和单独使用APB分别仅保留了16%和6.4%的活性。对APB+AVF+海藻糖制剂的冻干物进行了为期6个月的加速稳定性研究，研究结束时观察到活性的降低可以忽略不计。这些结果表明，酶与其底物的冻干可以赋予与常用的冷冻保护剂海藻糖相当的稳定性，但底物和海藻糖的组合比单独使用任何一种添加剂都能提供更大的稳定作用[1]。

鼻内给药是一种有吸引力的途径，用于全身输送小型亲脂性药物，因为它们会通过鼻粘膜迅速吸收到全身循环中。然而，亲脂性药物的低溶解度通常排除了水性鼻喷雾剂的可能性。一种规避溶解度问题的独特方法涉及将亲水性前药与外源性转化酶联合给药。这种策略不仅解决了溶解性差的问题，而且导致了驱动药物吸收的化学活性梯度的增加。在此，研究人员报告了亲水性地西泮前药Avizafone与转化酶人氨基肽酶B联合给药后大鼠的血浆和脑浓度。单次剂量相当于0.500、1.00和1.50mg/kg地西泮的Avizafone经鼻给药，生物利用度为77.8%±6.0%、112%±10%和114%±7%；最大血浆浓度分别为71.5±9.3、388±31和355±187 ng/ml；以及每种剂量水平达到血浆浓度峰值的时间分别为5、8和5分钟。地西泮和一种短暂的中间体都被吸收了。将酶动力学纳入基于生理学的药代动力学模型，可以估算一级吸收速率常数：地西泮为0.0689±0.0080分钟-1，中间体为0.122±0.022分钟-1。我们的研究结果表明，实际上不溶的地西泮可以通过与氨基肽酶B联合使用Avizafone进行鼻内给药，快速完全吸收。此外，通过增加酶浓度，可能会取代静脉注射地西泮或劳拉西泮或肌肉注射咪达唑仑治疗癫痫发作紧急情况，从而获得更快的吸收率[2]。

酶动力学。[1]
通过将Michaelis-Menten方程拟合到一系列AVF浓度的初始底物消耗速率，确定了32°C下APB在pH 7.4 PBS中水解Avizafone (AVF) 的米氏常数（KM）和最大反应速度（Vmax）。在Eppendorf Thermomixer 5436中以500 rpm的速度与62.5-4000μM Avizafone (AVF) 和15μg/ml（0.203μM）APB进行反应。1分钟后，通过加入甲醇使APB变性。在Cary 100 Bio-UV/Vis分光光度计中测量淬灭反应混合物的紫外光谱之前，允许ORI向DZP的环化过程完成。338nm光谱的二阶导数用于量化DZP，该量被视为与AVF消耗量的摩尔当量。

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冻干程序[2]
将APB、Avizafone (AVF) 、Tre、Man、pH 7.4 Tris的水储备溶液和纯水在冰浴中冷却至约0°C。将储备溶液的等分试样以适当的比例移入冷的2 mL玻璃瓶中，以获得每个样品的指示成分浓度和相等的最终体积。对于含有AVF的样品，最后加入AVF溶液。在将小瓶放入液氮中5分钟以快速冷冻内容物之前，用移液管将这些冷的预冻溶液短暂混合。然后将小瓶从液氮中取出，转移到冻干烧瓶中，并在室温下在0.016毫巴真空下使用FreeZone 6歧管冷冻干燥器干燥18小时。将装有冻干物的小瓶加盖并储存在干燥器中，直至分析。除非另有说明，否则应在室温下储存。

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活性酶的测定[2]
使用光谱技术测定样品处理、冻干和储存后剩余的活性酶的量。使用配备有温度控制器的 Cary 100 Bio-UV/Vis双光束分光光度计进行用于跟踪底物水解的吸收率（Abs）测量。除非另有说明，否则温度控制器设置为32°C，这是人体鼻腔的平均温度。样品在光程为1cm、最小填充体积为50μL的石英超微毛细管中进行分析。本研究中使用的浓度的APB、Tre、Tris和Man溶液在用于底物和产物浓度光谱定量的波长下是透明的。 为了测量特定的酶活性，将冻干物在加热至32°C的PBS中用1.00 mM LpNA复溶。所得溶液立即转移到已经放置在分光光度计温控块中的试管中。在405nm处监测LpNA水解引起的Abs变化。每100毫秒读取一次读数。前0.25分钟内吸光度变化的斜率（dAbs/dt）用于计算反应速率（d[pNA]/dt），如下所示
其中ε表示摩尔吸光系数。在32°C的pH 7.4 PBS中，405nm处的εpNA=9860 M–1 cm–1和εLpNA=55.9 M–1 cm-1（ε的温度依赖性请参考支持信息表S1）。比酶活性通过（d[pNA]/dt）/[APB]计算。 发色底物LpNA不能用于测量含有前药底物Avizafone (AVF) 的样品中活性APB的量。为了测量这些样品中的活性APB，冻干物用100μL PBS或1.00 mM Avizafone (AVF) 的PBS溶液复溶，PBS溶液已加热至32°C，所得溶液立即转移到分光光度计中嵌入的试管中，如上所述。在315nm下监测AVF转化为DZP引起的Abs变化20分钟。每0.25分钟读取一次读数。时间t时DZP的浓度计算如下：
其中Abs0是样品的初始吸光度，Abst是在时间t测量的吸光度。在32°C的pH 7.4 PBS中，εDZP=2040 M–1 cm–1，εAvizafone (AVF) =753 M–1 cm-1，波长为315 nm。之前已经讨论了εDZP和εAvizafone (AVF) 的温度依赖性，以及在根据吸光度测量计算DZP浓度时忽略εORI的有效性。在获得DZP浓度-时间曲线后，以[APB]作为单独拟合参数，将以下一组耦合微分方程拟合到数据中。

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.

C22H29CL3N4O3

592.75

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

122.01

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