Development of Fc-Fused Cocaine Hydrolase for Cocaine Addiction Treatment: Catalytic and Pharmacokinetic Properties

Abstract

Cocaine abuse is a worldwide public health and social problem without a US Food and Drug Administration (FDA)-approved medication. Accelerating cocaine metabolism that produces biologically inactive metabolites by administration of an efficient cocaine hydrolase (CocH) has been recognized as a promising strategy for cocaine abuse treatment. However, the therapeutic effects of CocH are limited by its short biological half-life (e.g., 8 h or shorter in rats). In this study, we designed and prepared a set of Fc-fusion proteins constructed by fusing Fc(M3) with CocH3 at the N-terminus of CocH3. A linker between the two protein domains was optimized to improve both the biological half-life and catalytic activity against cocaine. It has been concluded that Fc(M3)-GS-CocH3 not only has fully retained the catalytic efficiency of CocH3 against cocaine but also has the longest biological half-life (e.g., ∼ 136 h in rats) among all of the long-acting CocHs identified so far. A single dose (0.2 mg/kg, IV) of Fc(M3)-GS-CocH3 was able to significantly attenuate 15 mg/kg cocaine-induced hyperactivity for at least 11 days (268 h) after the Fc(M3)-GS-CocH3 administration.

The current practice for cocaine-associated chest pain in the Netherlands

“…Currently, potentially more effective cocaine use disorder treatment options are in the pre-clinical and clinical development stage. [ 45 , 46 ] These treatments are based on accelerating cocaine metabolism by administering a cocaine hydrolase (CocH) [ 45 ]. This cocaine-metabolizing enzyme is able to rapidly reverse cocaine toxicity and has already been tested in rats [ 46 ].…”

Development of a long-acting Fc-fused cocaine hydrolase with improved yield of protein expression

“…2), which is consistent with the expected dimeric structure with disulfide bonds formed on the Fc part. CocH3, which was produced predominantly in a tetramer form in our previous study [9] has a k cat of 5700 min −1 ; however, the fusion protein, Fc(M3)-CocH3, which was constructed without any linker in our previous study [32], exhibited only ~30% catalytic activity against cocaine as compared to the corresponding unfused CocH3.…”

“…Q5 ® Site-Directed Mutagenesis Kit was used to introduce each linker between Fc(M3) and CocH3. The pCMV-Fc(M3)-CocH3, constructed in our previous study [32] to encode Nterminal Fc-fused CocH3 without a linker, was used as the template. PCR reactions with Q5 hot start high-fidelity DNA polymerase along with primers listed in Table 1 were utilized to create insertions.…”

“…In this study, directly fusing Fc to the N-terminal of CocH3 increased the protein expression level by ~2-fold. However, as Fc(M3)-CocH3 protein had only ~30% catalytic activity against cocaine as compared to the unfused CocH3 [32]. As Fc(M3) domain sterically interferes with the CocH3 domain activity and lowers its catalytic activity against cocaine, it is also possible that this steric interference affects the efficiency of the protein folding.…”

Cocaine hydrolase blocks cocaine‐induced dopamine transporter trafficking to the plasma membrane

“…The locomotor data are plotted as the mean ± SEM metres travelled in 5-min bin during locomotor activity for 7 h. Particularly for the lethal dose (60 mg/kg), in addition to the hyperactivity, 60-mg/kg cocaine induced convulsion in six out of eight rats, with two rats died after convulsion, 37 Particularly for treatment of cocaine abuse, previous studies 20,23,30,37,41,43 demonstrated that an exogenous CocH (known as a BChE mutant with at least 1000-fold improved catalytic efficiency against cocaine compared to wild-type BChE) can powerfully rescue rats by rapidly detoxifying cocaine and its toxic metabolites 25,27,29,44 at any time point as long as rats are still alive, and also effectively block cocaine-induced hyperactivity. 23,28,30,37,45 However, it was unknown whether an enzyme can also effectively block cocaine-induced DAT trafficking to plasma membrane. According to the animal data with CocH3-Fc(M3) treatment, the enzyme is capable of effectively preventing cocaine from reaching the brain and limiting the brain cocaine concentration to be lower than the threshold (2.0 ± 0.8 μg/g) even with a lethal dose of 60-mg/kg cocaine (ip).…”