Staphopain mediated virulence and antibiotic resistance alteration in co-infection of Staphylococcus aureus and Pseudomonas aeruginosa: an animal model.

Polymicrobial communities lead to worsen the wound infections, due to mixed biofilms, increased antibiotic resistance, and altered virulence production. Promising approaches, including enzymes, may overcome the complicated condition of polymicrobial infections. Therefore, this study aimed to investigate Staphopain A-mediated virulence and resistance alteration in an animal model of Staphylococcus aureus and Pseudomonas aeruginosa co-infection. S. aureus and P. aeruginosa were co-cultured on the L-929 cell line and wound infection in an animal model. Then, recombinant staphopain A was purified and used to treat mono- and co-infections. Following the treatment, changes in virulence factors and resistance were investigated through phenotypic methods and RT-PCR. Staphopain A resulted in a notable reduction in the viability of S. aureus and P. aeruginosa. The biofilm formed in the wound infection in both animal model and cell culture was disrupted remarkably. Moreover, the biofilm-encoding genes, quorum sensing regulating genes, and virulence factors (hemolysin and pyocyanin) controlled by QS were down-regulated in both microorganisms. Furthermore, the resistance to vancomycin and doripenem decreased following treatment with staphopain A. According to this study, staphopain A might promote wound healing and cure co-infection. It seems to be a promising agent to combine with antibiotics to overcome hard-to-cure infections.

A lava-inspired proteolytic enzyme therapy on cancer with a PEG-based hydrogel enhances tumor distribution and penetration of liposomes.

The enhanced permeability and retention (EPR) effect has become the guiding principle for nanomedicine against cancer for a long time. However, several biological barriers severely resist therapeutic agents' penetration and retention into the deep tumor tissues, resulting in poor EPR effect and high tumor mortality. Inspired by lava, we proposed a proteolytic enzyme therapy to improve the tumor distribution and penetration of nanomedicine. A trypsin-crosslinked hydrogel (Trypsin@PSA Gel) was developed to maintain trypsin's activity. The hydrogel postponed trypsin's self-degradation and sustained the release. Trypsin promoted the cellular uptake of nanoformulations in breast cancer cells, enhanced the penetration through endothelial cells, and degraded total and membrane proteins. Proteomic analysis reveals that trypsin affected ECM components and down-regulated multiple pathways associated with cancer progression. Intratumoral injection of Trypsin@PSA Gel significantly increased the distribution of liposomes in tumors and reduced tumor vasculature. Combination treatment with intravenous injection of gambogic acid-loaded liposomes and intratumoral injection of Trypsin@PSA Gel inhibited tumor growth. The current study provides one of the first investigations into the enhanced tumor distribution of liposomes induced by a novel proteolytic enzyme therapy.

Specificity Enhancement of Glutenase Bga1903 toward Celiac Disease-Eliciting Pro-Immunogenic Peptides via Active-Site Modification.

Celiac disease is an autoimmune disease triggered by oral ingestion of gluten, with certain gluten residues resistant to digestive tract enzymes. Within the duodenum, the remaining peptides incite immunogenic responses, including the generation of autoantibodies and inflammation, leading to irreversible damage. Our previous exploration unveiled a glutenase called Bga1903 derived from the Gram-negative bacterium Burkholderia gladioli. The cleavage pattern of Bga1903 indicates its moderate ability to mitigate the toxicity of pro-immunogenic peptides. The crystal structure of Bga1903, along with the identification of subsites within its active site, was determined. To improve its substrate specificity toward prevalent motifs like QPQ within gluten peptides, the active site of Bga1903 underwent site-directed mutagenesis according to structural insights and enzymatic kinetics. Among the double-site mutants, E380Q/S387L exhibits an approximately 34-fold increase in its specificity constant toward the QPQ sequence, favoring glutamines at the P1 and P3 positions compared to the wild type. The increased specificity of E380Q/S387L not only enhances its ability to break down pro-immunogenic peptides but also positions this enzyme variant as a promising candidate for oral therapy for celiac disease.

Engineering of Therapeutic and Detoxifying Enzymes.

Therapeutic enzymes present excellent opportunities for the treatment of human disease, modulation of metabolic pathways and system detoxification. However, current use of enzyme therapy in the clinic is limited as naturally occurring enzymes are seldom optimal for such applications and require substantial improvement by protein engineering. Engineering strategies such as design and directed evolution that have been successfully implemented for industrial biocatalysis can significantly advance the field of therapeutic enzymes, leading to biocatalysts with new-to-nature therapeutic activities, high selectivity, and suitability for medical applications. This minireview highlights case studies of how state-of-the-art and emerging methods in protein engineering are explored for the generation of therapeutic enzymes and discusses gaps and future opportunities in the field of enzyme therapy.

Motor and respiratory decline in patients with late onset Pompe disease after cessation of enzyme replacement therapy during COVID-19 pandemic.

BACKGROUND AND PURPOSE: Data on interruption of enzyme replacement therapy (ERT) are scarce in late onset Pompe disease. Due to the COVID-19 crisis, eight neuromuscular reference centers in France were obligated to stop the treatment for 31 patients. METHODS: We collected the motor and respiratory data from our French registry, before COVID-19 and at treatment restart. RESULTS: In 2.2 months (mean), patients showed a significant deterioration of 37 m (mean) in the 6-min walk test and a loss of 210 ml (mean) of forced vital capacity, without ad integrum restoration after 3 months of ERT restart. CONCLUSIONS: This national study based on data from the French Pompe Registry shows that the interruption of ERT, even as short as a few months, worsens Pompe patients' motor and respiratory function.

Cell type-selective targeted delivery of a recombinant lysosomal enzyme for enzyme therapies.

Lysosomal diseases are a class of genetic disorders predominantly caused by loss of lysosomal hydrolases, leading to lysosomal and cellular dysfunction. Enzyme replacement therapy (ERT), where recombinant enzyme is given intravenously, internalized by cells, and trafficked to the lysosome, has been applied to treat several lysosomal diseases. However, current ERT regimens do not correct disease phenotypes in all affected organs because the biodistribution of enzyme uptake does not match that of the affected cells that require the enzyme. We present here targeted ERT, an approach that utilizes antibody-enzyme fusion proteins to target the enzyme to specific cell types. The antibody moiety recognizes transmembrane proteins involved in lysosomal trafficking and that are also preferentially expressed in those cells most affected in disease. Using Pompe disease (PD) as an example, we show that targeted ERT is superior to ERT in treating the skeletal muscle phenotypes of PD mice both as a protein replacement therapeutic and as a gene therapy.

Gender differences in cocaine-induced hyperactivity and dopamine transporter trafficking to the plasma membrane.

As well known, cocaine induces stimulant effects and dopamine transporter (DAT) trafficking to the plasma membrane of dopaminergic neurons. In the present study, we examined cocaine-induced hyperactivity along with cocaine-induced DAT trafficking and the recovery rate of the dopaminergic system in female rats in comparison with male rats, demonstrating interesting gender differences. Female rats are initially more sensitive to cocaine than male rats in terms of both the DAT trafficking and hyperactivity induced by cocaine. Particularly, intraperitoneal (i.p.) administration of 5 mg/kg cocaine induced significant hyperactivity and DAT trafficking in female rats but not in male rats. After repeated cocaine exposures (i.e., i.p. administration of 20 mg/kg cocaine every other day from Day 0 to Day 32), cocaine-induced hyperactivity in female rats gradually became a clear pattern of two phases, with the first phase of the hyperactivity lasting for only a few minutes and the second phase lasting for over an hour beginning at ~30 min, which is clearly different from that of male rats. It has also been demonstrated that the striatal DAT distribution of female rats may recover faster than that of male rats after multiple cocaine exposures. Nevertheless, despite the remarkable gender differences, our recently developed long-acting cocaine hydrolase, known as CocH5-Fc(M6), can similarly and effectively block cocaine-induced DAT trafficking and hyperactivity in both male and female rats.

Biomimetic microbioreactor-supramolecular nanovesicles improve enzyme therapy of hepatic cancer.

A novel biomimetic nanovesicle-loaded supramolecular enzyme-based therapeutics has been developed. Here, using a biomimetic lipid-D-α-tocopherol polyethylene glycol succinate (TPGS) hybrid semi-permeable membrane, cyclodextrin supramolecular docking, metal-ion-aided coordination complexing, we combined multiple functional motifs into a single biomimetic microbioreactor-supramolecular nanovesicle (MiSuNv) that allowed effective transport of arginine deiminase (ADI) to hepatic tumor cells to enhance arginine depletion. We compared two intercalated enzyme-carrying supermolecular motifs mainly comprising of 2-hydroxypropyl-ß-cyclodextrin and sulfobutyl-ether-ß-cyclodextrin, the only two cyclodextrin derivatives approved for injection by the United States Food and Drug Administration. The ADI-specific antitumor effects were enhanced by TPGS (one constituent of MiSuNv, having synergistic antitumor effects), as ADI was separated from adverse external environment by a semi-permeable membrane and sequestered in a favorable internal microenvironment with an optimal pH and metal-ion combination. ADI@MiSuNv contributed to cell cycle arrest, apoptosis and autophagy through the enhanced efficacy of enzyme treatment against Hep3B xenograft tumors in rats.

Multimodal Enzyme Delivery and Therapy Enabled by Cell Membrane-Coated Metal-Organic Framework Nanoparticles.

Therapeutic enzymes used for genetic disorders or metabolic diseases oftentimes suffer from suboptimal pharmacokinetics and stability. Nanodelivery systems have shown considerable promise for improving the performance of enzyme therapies. Here, we develop a cell membrane-camouflaged metal-organic framework (MOF) system with enhanced biocompatibility and functionality. The MOF core can efficiently encapsulate enzymes while maintaining their bioactivity. After the introduction of natural cell membrane coatings, the resulting nanoformulations can be safely administered in vivo. The surface receptors on the membrane can also provide additional functionalities that synergize with the encapsulated enzyme to target disease pathology from multiple dimensions. Employing uricase as a model enzyme, we demonstrate the utility of this approach in multiple animal disease models. The results support the use of cell membrane-coated MOFs for enzyme delivery, and this strategy could be leveraged to improve the usefulness of enzyme-based therapies for managing a wide range of important human health conditions.

Enzyme Therapy: Current Challenges and Future Perspectives.

In recent years, enzymes have risen as promising therapeutic tools for different pathologies, from metabolic deficiencies, such as fibrosis conditions, ocular pathologies or joint problems, to cancer or cardiovascular diseases. Treatments based on the catalytic activity of enzymes are able to convert a wide range of target molecules to restore the correct physiological metabolism. These treatments present several advantages compared to established therapeutic approaches thanks to their affinity and specificity properties. However, enzymes present some challenges, such as short in vivo half-life, lack of targeted action and, in particular, patient immune system reaction against the enzyme. For this reason, it is important to monitor serum immune response during treatment. This can be achieved by conventional techniques (ELISA) but also by new promising tools such as microarrays. These assays have gained popularity due to their high-throughput analysis capacity, their simplicity, and their potential to monitor the immune response of patients during enzyme therapies. In this growing field, research is still ongoing to solve current health problems such as COVID-19. Currently, promising therapeutic alternatives using the angiotensin-converting enzyme 2 (ACE2) are being studied to treat COVID-19.

Computational Design and Crystal Structure of a Highly Efficient Benzoylecgonine Hydrolase.

Benzoylecgonine (BZE) is the major toxic metabolite of cocaine and is responsible for the long-term cocaine-induced toxicity owing to its long residence time in humans. BZE is also the main contaminant following cocaine consumption. Here, we identified the bacterial cocaine esterase (CocE) as a BZE-metabolizing enzyme (BZEase), which can degrade BZE into biological inactive metabolites (ecgonine and benzoic acid). CocE was redesigned by a reactant-state-based enzyme design theory. An encouraging mutant denoted as BZEase2, presented a >400-fold improved catalytic efficiency against BZE compared with wild-type (WT) CocE. In vivo, a single dose of BZEase2 (1 mg kg-1 , IV) could eliminate nearly all BZE within only two minutes, suggesting the enzyme has the potential for cocaine overdose treatment and BZE elimination in the environment by accelerating BZE clearance. The crystal structure of a designed BZEase was also determined.

Therapeutic Activity of Type 3 Streptococcus pneumoniae Capsule Degrading Enzyme Pn3Pase.

PURPOSE: Streptococcus pneumoniae (Spn) serotype 3 (Spn3) is considered one of the most virulent serotypes with resistance to conventional vaccine and treatment regimens. Pn3Pase is a glycoside hydrolase that we have previously shown to be highly effective in degrading the capsular polysaccharide of type 3 Spn, sensitizing it to host immune clearance. To begin assessing the value and safety of this enzyme for future clinical studies, we investigated the effects of high doses of Pn3Pase on host cells and immune system. METHODS: We assessed the enzyme's catalytic activity following administration in mice, and performed septic infection models to determine if prior administration of the enzyme inhibited repeat treatments of Spn3-challenged mice. We assessed immune populations in mouse tissues following administration of the enzyme, and tested Pn3Pase toxicity on other mammalian cell types in vitro. RESULTS: Repeated administration of the enzyme in vivo does not prevent efficacy of the enzyme in promoting bacterial clearance following bacterial challenge, with insignificant antibody response generated against the enzyme. Immune homeostasis is maintained following high-dose treatment with Pn3Pase, and no cytotoxic effects were observed against mammalian cells. CONCLUSIONS: These data indicate that Pn3Pase has potential as a therapy against Spn3. Further development as a drug product could overcome a great hurdle of pneumococcal infections.

Enzymes for pharmaceutical and therapeutic applications.

Enzymes are highly efficient and selective biocatalysts, present in the living beings. They exist in enormous varieties in terms of the types of reactions catalyzed by them for instance oxidation-reduction, group transfers within the molecules or between the molecules, hydrolysis, isomerization, ligation, bond cleavage, and bond formation. Besides, enzyme based catalyses are performed with much higher fidelity, under mild reaction conditions and are highly efficient in terms of number of steps, giving them an edge over their chemical counter parts. The unique characteristics of enzymes makes them highly applicable fora number of chemical transformation reactions in pharmaceutical industries, such as group protection and deprotection, selective acylation and deacylation, selective hydrolysis, deracemization, kinetic resolution of racemic mixtures, esterification, transesterification, and many others. In this review, an overview of the enzymes, their production and their applications in pharmaceutical syntheses and enzyme therapies are presented with diagrams, reaction schemes and table for easy understanding of the readers.

[Systemic enzyme therapy for treatment of women with chronic recurrent bacterial cystitis].

INTRODUCTION: In most cases, treatment of infectious and inflammatory diseases, including lower urinary tract infections, includes antibacterial drugs. However, their efficiency decreases every year. The absence of new groups of antibiotics makes it necessary to develop alternative treatment schemes and methods for improving efficiency of existing drugs. Systemic enzyme therapy is one of the promising directions in the treatment of lower urinary tract infections, which was shown to be effective for treating a number of diseases. AIM: to evaluate the results of complex treatment of women with chronic recurrent bacterial cystitis using a drug for systemic enzyme therapy. MATERIALS AND METHODS: The examination and treatment of 60 women aged 19 to 45 years with an exacerbation of chronic recurrent bacterial cystitis, who were randomly divided into two groups of 30 patients, was performed. In the group 1, patients received standard antibiotic therapy. In the group 2, women additionally received Phlogenzym. An evaluation of the efficiency was carried out on the 1st, 7th and 14th day and included analysis of lower urinary tract symptoms, according to bladder diaries, assessment of pain intensity and laboratory tests (C-reactive protein [CRP], IL-6, IL-1, TNF. During long-term follow-up (6 months after completion of treatment), the relapse rate was evaluated in both groups. RESULTS: At admission, all women had clinical and laboratory signs of cystitis. By the 14th day, there was normalization of evaluated parameters in both groups. However, in patients receiving systemic enzyme therapy a more rapid relief of the inflammatory process (a decrease in the frequency of urination, urgency, nocturia, severity of inflammatory changes in the blood and urine) by the 7th day of treatment was observed. During a further 6-month follow-up, relapses were detected in 9 (30%) and 4 (13.3%) patients in group 1 and 2, respectively. CONCLUSION: The results of the study demonstrated the high efficiency of systemic enzyme therapy. Its use for complex treatment provides more rapid relief of lower urinary tract symptoms and pain, as well as reduces the number of relapses in women with chronic recurrent bacterial cystitis.

[New opportunities of using gialuronidase in chronic prostatitis].

INTRODUCTION: The use of hyaluronidase contributes to improvement in microcirculation, destruction of biofilms and reducing of congestion in patients with chronic prostatitis. Based on this mechanism, the use Longidaza for the diagnosis of latent bacterial prostatitis was suggested. AIM: To evaluate diagnostic and treatment capabilities of the long-acting enzyme preparation Longidaza (rectal suppositories with an activity of 3000 IU), prescribed to patients with chronic prostatitis in routine clinical practice. MATERIALS AND METHODS: A total of 39 men with chronic prostatitis of categories II and IIIa were included in open, prospective, non-interventional, comparative, single-center study. A microbiological study of expressed prostate secretion (EPS) was performed three times, at baseline, after using two suppositories Longidaza 3000 IU and after 7 weeks of therapy. From the 1st to the 10th suppository, Longidaza was used every 48 hours, then, from the 11th to the 20th suppository, it was prescribed every 72 hours. The isolation of the pathogen was an indication for the antibiotic therapy. The treatment outcome was evaluated by using NIH-CPSI scale and laboratory and microbiological analysis of EPS. RESULTS: Diagnostic stage. Initially, pathogens were isolated in 27 (69.2%) patients. The number of leukocytes in EPS averaged 25.9+/-2.3. The use of two suppositories Longidaza increased the number of patients with isolated pathogens to 33 (82%). In 7 out 12 patients with an initial diagnosis of abacterial prostatitis, the use of two suppositories of Longidaza contributed to isolation of the pathogen in EPS (58.3%). The number of leukocytes in the EPS after enzymatic provocation increased by 50.2%. Treatment stage. The number of patients with isolated pathogens decreased from 33 to 7 (17.9%). The pain intensity decreased on average to 2.2+/-0.4 points, and in 27 (69.2%) men dysuria resolved. The quality of life significantly improved up to 1.3+/-0.2 points. The total score on NIH-CPSI scale decreased on average from 17.3+/-1.9 to 3.9+/-0.3 (p<0.05). An increased number of white blood cells in EPS was seen only in 5 (12.8%) men. CONCLUSION: In many cases, bacterial prostatitis remains undiagnosed. Rectal suppositories Longidaza can be recommended for the diagnosis of the latent bacterial chronic prostatitis, since this drug contributes to detection of pathogen and inflammation.

[Assessment of the efficiency of sialosis treatment in patients with type 2 diabetes mellitus]. / Otsenka effektivnosti lecheniya bol'nykh s sialozom na fone sakharnogo diabeta 2-go tipa.

THE AIM OF THE STUDY: Was to determine changes in the levels of cytokines in the oral fluid in patients with type 2 diabetes and sialosis, depending on the level of glycemia and the duration of type 2 diabetes, as well as in the dynamics of basic and complex treatment. MATERIALS AND METHODS: Forty patients with type 2 diabetes were enrolled in the study and divided into two groups according to disease duration: group 1 comprised 15 patients having diabetes for ≤5 years, group 2 - 25 suffering for more than 5 years. RESULTS: The study revealed changes in pancreatic cancer immunological parameters in patients with sialosis and type 2 diabetes. Long course of diabetes led to an increase in the level of IL-2 and a decrease in IFN-γ in oral fluid. The level of IL-1ß was the highest in the group of patients with a duration of diabetes ≤5 years. According to the data presented, the content of IL-1ß, IL-2 in patients with diabetes is higher, and INF-γ is lower than in healthy individuals. In patients with sialosis on the background of type 2 diabetes before treatment there is a decrease in the level of IFN-γ in the oral fluid and its increase to 7.3±0.2 pg/ml after treatment. At the same time, when enzyme therapy was included in the treatment regimen, the increase in the level of IFN-γ in the main group was more pronounced and averaged 8.3±0.1 pg/ml, which was 1.2 times higher than the initial level (p<0,05). CONCLUSION: The inclusion of enzyme therapy in the complex therapy of patients with sialosis against the background of diabets, unlike traditional methods of therapy, made it possible to provide faster positive clinical dynamics, namely, under the influence of this drug, the pain stopped in a short time, salivation increased and the feeling of dryness disappeared. Along with a faster relief of clinical symptoms, normalization of the levels of IL-1ß, IL-2 and an increase in IFN-γ by the end of treatment were noted.

Development of wheat genotypes expressing a glutamine-specific endoprotease from barley and a prolyl endopeptidase from Flavobacterium meningosepticum or Pyrococcus furiosus as a potential remedy to celiac disease.

Ubiquitous nature of prolamin proteins dubbed gluten from wheat and allied cereals imposes a major challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of hydrolytic glutenases as food supplement is an alternative to deliver the therapeutic agents directly to the small intestine, where sensitization of immune system and downstream reactions take place. The aim of the present research was to evaluate the capacity of wheat grain to express and store hydrolytic enzymes capable of gluten detoxification. For this purpose, wheat scutellar calli were biolistically transformed to generate plants expressing a combination of glutenase genes for prolamin detoxification. Digestion of prolamins with barley endoprotease B2 (EP-HvB2) combined with Flavobacterium meningosepticum prolyl endopeptidase (PE-FmPep) or Pyrococcus furiosus prolyl endopeptidase (PE-PfuPep) significantly reduced (up to 67%) the amount of the indigestible gluten peptides of all prolamin families tested. Seven of the 168 generated lines showed inheritance of transgene to the T2 generation. Reversed phase high-performance liquid chromatography of gluten extracts under simulated gastrointestinal conditions allowed the identification of five T2 lines that contained significantly reduced amounts of immunogenic, celiac disease-provoking gliadin peptides. These findings were complemented by the R5 ELISA test results where up to 72% reduction was observed in the content of immunogenic peptides. The developed wheat genotypes open new horizons for treating celiac disease by an intraluminal enzyme therapy without compromising their agronomical performance.

X-Ray Crystallography in Structure-Function Characterization of Therapeutic Enzymes.

Enzymes are key biological macromolecules that support life by accelerating the conversion of target molecules to desired products in many biochemical reactions. Enzymes are characterized by high affinity, specificity and great catalytic efficiency. Owing to their unique characteristics, enzymes have attracted significant attention for use in therapeutic settings as a distinct class of drugs different from other types of medicines. Enzyme-based therapies are currently in use for the treatment of a wide range of diseases, including leukemia, metabolic disorders, inflammation and cardiovascular disease. However, several challenges, such as immunogenicity and stability, remain. X-ray crystallography has provided key structural insights into the understanding of the molecular basis of diseases and development of enzyme-based therapies. Here, the role of X-ray crystallography in the development of therapeutic enzymes is examined and several examples are provided.

Engineering Strategies for Oral Therapeutic Enzymes to Enhance Their Stability and Activity.

Oral application of therapeutic enzymes is a promising and non-invasive administration that improves patient compliance. However, the gastrointestinal tract poses several challenges to the oral delivery of proteins, including harsh pH conditions and digestive proteases. A promising way to stabilise enzymes during their gastrointestinal route is by modification with polymers that can provide both steric shielding and selective interaction in different digestive compartments. We give an overview of modification technologies for oral enzymes ranging from functionalisation of native proteins, to site-specific mutation and protein-polymer engineering. We specifically focus on enzymes that are active directly in the gastrointestinal lumen and not systemically absorbed. In addition, we discuss examples of microparticle and nanoparticle encapsulated enzymes for improved oral delivery. The modification of orally administered enzymes offers a broad chemical variability and may be a promising tool for enhancing their gastrointestinal stability.

Long-acting cocaine hydrolase for addiction therapy.

Cocaine abuse is a world-wide public health and social problem without a US Food and Drug Administration-approved medication. An ideal anticocaine medication would accelerate cocaine metabolism, producing biologically inactive metabolites by administration of an efficient cocaine-specific exogenous enzyme. Our recent studies have led to the discovery of the desirable, highly efficient cocaine hydrolases (CocHs) that can efficiently detoxify and inactivate cocaine without affecting normal functions of the CNS. Preclinical and clinical data have demonstrated that these CocHs are safe for use in humans and are effective for accelerating cocaine metabolism. However, the actual therapeutic use of a CocH in cocaine addiction treatment is limited by its short biological half-life (e.g., 8 h or shorter in rats). Here we demonstrate a novel CocH form, a catalytic antibody analog, which is a fragment crystallizable (Fc)-fused CocH dimer (CocH-Fc) constructed by using CocH to replace the Fab region of human IgG1. The CocH-Fc not only has a high catalytic efficiency against cocaine but also, like an antibody, has a considerably longer biological half-life (e.g., ∼107 h in rats). A single dose of CocH-Fc was able to accelerate cocaine metabolism in rats even after 20 d and thus block cocaine-induced hyperactivity and toxicity for a long period. Given the general observation that the biological half-life of a protein drug is significantly longer in humans than in rodents, the CocH-Fc reported in this study could allow dosing once every 2-4 wk, or longer, for treatment of cocaine addiction in humans.