What is the Advantage and Disadvantage of Eprinomectin Api Manufacturers

Introduction

Antibiotics are produced by bacteria, molds, or other microorganisms in the course of life, which can interfere with or inhibit the survival of pathogenic microorganisms (Fleming, ; Stachelek et al., ). They can be categorized into seven main kinds, namely, sulfonamides (SAs), quinolones (QNs), tetracyclines (TCs), macrolides (MALs), chloramphenicol (CAPs), β-lactams (BLAs), and aminoglycosides (AGs) (Chen et al., ; Ming et al., ). Besides being widely used to prevent and treat animal and human diseases, antibiotics are also widely used as growth-promoting agents in animal husbandry and aquaculture, playing an important role in improving animal and human health (Ming et al., ). However, with the increasing use and abuse of antibiotics, bacteria are quickly adapted to the antibiotics, and all kinds of 'superbugs' are being born (Stachelek et al., ). Consequently, antibiotic residue has become one of the most important environmental issues in the world. The residues and accumulation through various pathways in animals and the environment not only induce the growth of drug-resistant bacteria with increasing numbers and species (Stachelek et al., ) but also accumulate toxicity through the food chain, causing great harm to the ecological environment and human health (Liu et al., ). It is urgently required to develop effective analytical methods toward antibiotics residues. Chromatography and mass spectrometry (MS) based methods are commonly utilized, especially high-performance liquid-phase chromatography- (HPLC-) and tandem MS (HPLC-MS/MS). Despite high sensitive detection techniques, such as HPLC-MS/MS, it is still quite imperative to use influential sample preparation/pretreatment steps prior to detection, owing to the characteristics of low residual amounts, various interfering factors, and complicated sample matrices (Dugheri et al., ).

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At present, sample pretreatment is widely employed in the analysis of trace analytes in complex matrices to purify and enrich the analytes and thereby will improve the sensitivity and accuracy of analytical methods (Zhang Y. et al., ). Currently, solid-phase extraction (SPE) that uses solid adsorbent to adsorb the target analytes is commonly used in the process of sample pretreatment, since it has many advantages, such as less time-consuming, simple operation, high efficiency, low or without solvent, and good compatibility with different analytical methods (Wu et al., ). SPE has a variety of modes, mainly including conventional SPE, dispersive SPE (DSPE), magnetic SPE (MSPE), matrix solid-phase dispersion (MSPD), solid-phase microextraction (SPME), stir-bar sorptive extraction (SBSE), and pipette-tip SPE (PT-SPE). It is well known that solid adsorbent is the key parameter of SPE efficiency. Nowadays, commercially available solid adsorbents like HLB, C18, and Oasis MCX usually exhibit nonspecific adsorption on the target analytes, which decrease the selectivity and specificity for antibiotics, thus greatly limiting the application of trace analysis of antibiotics in complicated matrices. Molecularly imprinted polymers (MIPs) with structure predictability, recognition specificity and application universality, easy preparation, low cost, and so on have gained great popularity as SPE adsorbents (Li et al., ; Chen et al., ; BelBruno, ; Arabi et al., ).

MIPs are prepared by molecular-imprinting technology based on the principle of antigens and antibodies specifically recognizing and binding, and the technology has often been vividly described as the 'artificial lock' technology for preparing to recognize 'molecular key' (Li et al., ). MIPs have been widely used for sample pretreatment (Płotka-Wasylka et al., ), chromatographic separation (Wang and Cao, ; BelBruno, ), chemical/biosensors (Chen et al., ; Gaudin, ; Arabi et al., ), and other fields (BelBruno, ; Haupt et al., ). Using MIPs as the adsorbents in SPE, molecularly imprinted or molecular-imprinting-based SPE (MISPE) can specifically recognize targets, which can effectively adsorb targets in complex matrices. Therefore, MISPE is extensively used for the highly selective cleanup and enrichment of trace antibiotics in complex samples. Furthermore, MISPE combined with separation and detection technologies such as chromatography has been swiftly developed and applied to achieve rapid, simultaneous, selective enrichment, and sensitive detection of multiple antibiotics contaminants (Li and Row, ; Wang J. et al., ; Wang S. et al., ; Hu et al., ; Zhu et al., b; Lu et al., ).

Therefore, in this work, the application of MISPE coupled with chromatographic analysis of antibiotics is reviewed comprehensively, focusing on the recent advances since . Firstly, the classification of SPE and the commonly used advanced preparation techniques of MIPs are overviewed. Then, the applications of MISPE for the analysis of a variety of antibiotics are emphasized. Finally, some attempts to facilitate the wide application of MISPE in the field of sample pretreatment are proposed.

Various SPE Modes Based on MIPs

SPE is a technology to separate the component to be tested from the interfering component by the difference of the adsorption ability of solid adsorbent on each component in the liquid sample. The separation efficiency is increasing with the improvement of the selectivity of solid adsorbent toward target analyte (Fan, ). Therefore, the use of highly selective MIPs as SPE adsorbent can achieve efficient extraction and enrichment of target analytes, which has been widely used in antibiotics residues detection (Lian and Wang, ; Xie et al., ; Hammam et al., ; Negarian et al., ; Zhu et al., b; Tan et al., ; Yu et al., ). Various SPE modes based on MIPs, i.e., conventional SPE, DSPE, MSPE, MSPD, SPME, SBSE, and PT-SPE, are briefly introduced below.

SPE generally means the conventional SPE, namely, packed SPE, and it is a common method for sample preparation. The column is packed with a solid adsorbent to make the sample be tested flow through the column so that the solid adsorbent can adsorb the target compounds, and then the target compounds can be separated and enriched by chemical reagent elution or heating desorption. The high selectivity of MIPs can greatly improve the extraction efficiency of SPE. The SPE with MIPs as extraction adsorbent has been widely used in the determination of antibiotics (Huang L. et al., ; Ma and Row, ; Qin et al., ).

DSPE does not need the packing and washing steps, the extraction time is short, and the adsorbent can be more fully dispersed into the sample solution to improve the adsorption effect. After the purified sample is shaken and centrifuged, the supernatant can be directly or simply processed into the next step of the analysis. This method proves to be quick, easy, cheap, effective, rugged, and safe and is also known as the QuEChERS method. As an efficient and rapid sample pretreatment technology, DSPE is widely used in the analysis and detection of contaminants and antibiotics residue analysis (Song et al., ; Lu et al., ).

MSPE is a technique in which a magnetic or magnetizable material is used as a solid adsorbent for SPE. Instead of being packed in the extraction column, a magnetic adsorbent is directly added to the sample solution or suspension so as to attain a full dispersion. The target analyte is adsorbed on the surface of the dispersed magnetic adsorbent, and an external magnetic field is utilized to separate the target analyte from the sample matrix (Liang J. et al., ). MSPE requires only low consumption of adsorbents and equilibrium time to realize the enrichment and separation of trace analytes. MSPE can avoid column blockage, which is a very important problem in traditional SPE (Li et al., ). Magnetic MIPs (MMIPs) materials have been widely used in the detection of many types of antibiotics (Liu et al., ; Nazario et al., ; Kunsa-Ngiem et al., ; Li Z. et al., ; Qin et al., ; Dil et al., ; Gao et al., ; Lamaoui et al., ).

MSPD is the basic process as follows: adding solid-phase adsorption materials directly to the sample matrix, mechanical mixing resulting in a semidry mixture, using the obtained mixture as a packed column, cleaning the column with a small amount of reagent to remove impurities, and finally eluting the target analyte by a small amount of eluent. MSPD combines with sample crushing, extraction and purification, which not only avoids sample loss but also save solvent/time. MSPD has the advantages of simple and quick operation, high extraction efficiency, no special equipment, and so on; large quantities of automatic analysis can be carried out through MSPD. Therefore, MSPD is widely used in the analysis of antibiotics residues (Wang S. et al., ; Soares et al., ), contaminants (Liang et al., ), and harmful components (Tang et al., ). As a kind of solid-phase adsorbent with high selectivity, MIP has been used in MSPD, which provides powerful technical support for the analysis of pollutants (Wang S. et al., ) and harmful components (Balsebre et al., ) in a complex matrix.

SPME is a solvent-free pretreatment method developed on the basis of SPE. It is easy to be combined with gas chromatography (GC), HPLC, and capillary electrophoresis (CE), so it has been widely used (Barahona et al., ; Huang S. et al., ; Guo et al., ). SPME has been successfully applied in the analysis of organic and inorganic substances in gas, water, soil, sediment, and other environmental samples (Reyes-Garces et al., ). SPME process is actually the adsorption/desorption process of each component in the sample on the surface of the extracted fiber coating, and its principle depends on the distribution balance between the analyte in the sample matrix and the extraction phase. By selecting different groups of coating heads, SPME can attain ideal extraction efficiency toward targeted components, while other components are restrained. The MIP coating, because of its simple preparation, good repeatability, high mechanical strength, good resistance to high temperature, and solvent resistance, especially the advantage of efficient choice specificity for trace target in complex environment medium analysis, has a broad application prospect and become a research hotspot of selective SPME coatings (Barahona et al., ; Liu et al., ).

SBSE is a variant of SPME, in which the glass tube with an inner magnetic core is coated with an extractive adsorption coating. After the distribution balance between the sample matrix and the coating on the surface of the stirring rod is reached, the sample is analyzed by thermal desorption or solvent desorption technology. Polydimethylsilane (PDMS) is the widely used coating material of SBSE. The coating prepared by the sol-gel method is compact in structure, highly hydrophobic, and stable in chemical properties. It is suitable for the extraction of nonpolar and weak polar compounds in the water phase. The SBSE has been developed to improve the sensitivity by using a larger volume of extraction phase. The difference is that a magnetic core is required for self-stirring to accelerate mass transfer, as thicker extractive coatings are usually prepared. However, since the SBSE format with magnetic agitation is not as convenient for sampling in vivo or in the field as fiber and film shapes, SBSE is commonly used for in-bottle extraction (Rodriguez-Gomez et al., ). However, the MIP coating-based SBSE is rarely used in antibiotic detection (Cui et al., ; Yang et al., ).

PT-SPE fills the pipette head with an adsorbent, making SPE setup smaller and analysis more environmentally friendly. PT-SPE is currently the most concerned SPE modes, mainly because it requires fewer adsorption materials, significantly reduces organic solvents consumption, and saves the cost. This method is simple and does not need extra special instruments. The transfer and distribution mechanism of the analytes to be enriched is roughly the same as that of the traditional SPE. The eluted solution can be directly used for LC or MS analysis without vacuum concentration. The pipetting nozzle can realize sampling, purification, enrichment, and quantification, at the same time, which makes up for the technical defects of traditional pretreatment methods, such as complicated operation, large amount of organic solvent, and easy loss of targets (de Oliveira et al., ; Teixeira et al., ; Zhang Y. et al., ; Hashemi et al., ).

Relative occurrence of the above-mentioned MISPE toward antibiotics residues detection within ' is shown in Figure 1. As seen, conventional SPE is the most frequently used (54%), followed by MSPE and DSPE at 22 and 11%, respectively, and the two modes of SPME and MSPD are not higher than 10%, while PT-SPE and SBSE are both just 1%. The main characteristics, advantages, and disadvantages of the seven types of SPE techniques are listed in Table 1, with a reasonable expansion and modification of Figure 1 of our previous work (Arabi et al., ).

FIGURE 1TABLE 1

Emerging Techniques for MIPs Preparation

Various efficient MIPs toward antibiotics have been prepared by virtue of emerging techniques such as surface imprinting, nanoimprinting, and living/controlled radical polymerization (LCRP) technology and multitemplate, multifunctional monomer, and dummy template imprinting strategy. Figure 2 schematically illustrates the six techniques' basic processes/mechanisms.

FIGURE 2

Surface Imprinting

Surface imprinting technology means preparing imprinted materials by controlling templates to locate at the surface or in the proximity of materials surface to create more effective recognition sites (Chen et al., ). It can overcome the disadvantages of low binding capacity and difficulty in elution of traditional MIPs. Core-shell structured MIPs are the major type of surface imprinting MIPs, owing to the increased surface area and larger binding capacity, and thus, they are widely used for detecting antibiotic residues (Ji et al., ; Liu et al., ; Negarian et al., ; Qin et al., ; Zhu et al., a).

Nanoimprinting

Nanoimprinting technology is the technique of preparing nanosized MIPs. Nanomaterials have a large surface area and more binding sites, which can effectively improve the binding capacity of MIPs. To a certain extent, the problems of fewer target sites and low mass transfer rate of large-size MIPs are solved. Moreover, the nanostructured MIPs can be directly used without grinding, simplifying the experimental operation. The commonly used methods to synthesize nano-MIPs microspheres include precipitation polymerization (Liu et al., ), sol-gel (Li G. et al., ; Diaz-Alvarez et al., ), and core-shell polymerization (Qin et al., ). Nanoimprinting technology can be divided into zero-dimensional, one-dimensional, and two-dimensional types (Li and Wang, ).

LCRP

LCRP overcomes the disadvantage that the growth rate of the traditional radical polymerization chain is not easy to control and causes the particle size distribution of the polymer to be in a narrow range. Among them, atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer (RAFT) polymerization are the most commonly used ones. LCRP technology has been increasingly used for the determination of antibiotic residues (Liang Y. et al., ; Zhao et al., ; Cai et al., ; Li et al., b; Cai et al., ) and other environmental contaminants (Azizi et al., ).

Multitemplate Imprinting

Multitemplate imprinting strategy means that two or more targeted analytes are as templates to prepare MIPs and thereby there are multiple recognition sites of template molecules in one imprinted polymer material (Wang et al., ). Due to the expansion of binding sites and recognition ability of MIPs, simultaneous recognition, enrichment, and separation of multiple targets can be realized, which greatly saves time and improves the utilization efficiency of MIPs. It has great potential in multiresidue and high-throughput analysis of complex samples (Wei et al., ; Xu et al., ; Lu et al., ; Dil et al., ).

Multifunctional Monomer Imprinting

The multifunctional monomer imprinting strategy is to take advantage of two or more functional monomers to interact with template molecules; by giving full play the synergistic effects of multiple functional monomers, MIPs selectivity is significantly enhanced and thereby enrichment ability. The strategy has been increasingly applied in antibiotics determination (Li G. et al., ; Li Z. et al., ; Cai et al., ).

Dummy Template Imprinting

Dummy template imprinting strategy is also called pseudotemplate imprinting strategy, which uses similar compounds to the target compounds as a template in the shape, size, structure, and functionality. It is a high requirement and especially suitable for the target compounds that are costly, flammable, explosive, easy to degrade, and having too low solubility. The strategy can effectively avoid template leakage pollution or inaccurate results. The most common method is the computer-aided design of dummy template toward MIPs (Song et al., ). MISPE prepared by dummy template imprinting strategy is widely used in the detection of antibiotic residues (Song et al., ; Zhang Z. et al., ; Gao et al., ).

Applications of MISPE to Analysis of Antibiotics Residues

The wide applications of MISPE to the analysis of antibiotics residues are summarized, including SAs, QNs, TCs, and other antibiotics. Some typical examples are listed in Table 2.

TABLE 2

SAs

SAs residues are closely related to food and environmental safety levels. The pollution sources of SAs antibiotics mainly include medical sources (patients feces and urine, antibiotics remaining on medical supplies, losses in the production of antibiotics by pharmaceutical enterprises, etc.), animal sources (animal excrement and urine, leakage of sewage from farms, etc.), and aquaculture (overuse of antibiotics in the process of farming, etc.) (Zhang Y. et al., ). The key to detection is to develop fast, efficient, and highly selective pretreatment methods. Common SPE materials for SAs include HLB, C18, and Oasis MCX which are relatively general and commercially available, but they lack selectivity for SAs. MISPE can identify, extract, and enrich the target substances with high selectivity and specificity and has high adsorption capacity and stability. At the same time, various SPE devices have become smaller and are easier to operate. MISPE has been widely used coupled with chromatographic determination toward SAs in complex samples (Wang J. et al., ; Fonte et al., ; Huang et al., ; Kechagia et al., ; Xu et al., ; Zhao et al., ; Zhu et al., a; Zhu et al., c; Gao et al., ; Zhao et al., ).

In order to improve the selective adsorption performance of MIPs in strong polar solvents, Zhu et al. (a) synthesized sulfamethoxazole (SMZ) imprinted polymers in methanol by using a new ionic liquid (IL) functional monomer, namely, 1-butyl-3-vinylbromidazole (BVIM-Br). The MIPs exhibited highly specific recognition properties toward the three commonly found SAs (SMZ, sulfamonomethoxine (SMM), and sulfadiazine (SDZ)) in methanol, while low adsorption ability was displayed for the interferents. Then, a MISPE methodology was developed and successfully applied to effective enrichment of trace SMZ in soil and sediment samples, followed by HPLC analysis. The limits of detection (LODs) were all as low as 5 μg/L. The present research can offer an important reference for influential MIPs preparation in aqueous media. Furthermore, Zhu et al. (c) prepared SMM surface imprinted polymers in the strong polar solvent of methanol with 1-allyl-3-vinylimidazolium chloride (AVIM-Cl) IL as a functional monomer. The developed MISPE coupled with HPLC was established for the selective extraction and sensitive determination of trace SMM in soil and sediment samples. The recoveries were high to 95.0'105.0% and the LOD was low to 1.0 μg/L. Such MIPs materials have a broad application prospect in the pretreatment of various complex samples.

Zhao et al. () synthesized hydrophilic magnetic MIPs (HMMIPs) on the surface of silanated Fe3O4 via surface imprinting technique and using SDZ as a template molecule and employed them as DSPE adsorbents for the enrichment and purification of 10 SAs prior to HPLC-UV detection in chicken, cow milk, and goat milk samples. Under the optimal experimental conditions, good linearity in the range of 5 μg/L to 10 mg/L was exhibited, low LODs ranged from 0.57 to 1.50 μg/L, and high spiked recoveries were between 85.09 and 110.93%. The HMMIPs-DSPE method can provide a potentially applicable way for the sensitive, reliable, simple, and rapid detection of various drug residues. In the previous work (Zhao et al., ), water-compatible MIPs were also prepared by combining RAFT with reflux precipitation polymerization (RPP), and the resulting MISPE coupled with HPLC-MS/MS succeeded in the enrichment and determination of six SAs in animal-derived foods and water samples.

Gao et al. () fabricated magnetic carbon nanotube dummy MIP (MCNTMIP) nanocomposite by surface imprinting technique and used it as MSPE adsorbent to realize the simultaneous separation and enrichment of 13 SAs (SDZ, sulfathiazole (ST), sulfamerazine (SM1), sulfamethazine (SM2), sulfamethizole (SMZO), SMM, sulfachloropyridazine (SCP), sulfadoxine (SO), SMZ, sulfisoxazole (SFZ), and sulfaquinoxaline (SOX), sulfadimethoxine (SDM), and avermectin B1a) in fish and shrimp samples. Figure 3 illustrates the process of MIPs preparation and MSPE application. The MCNTMIP displayed a simple magnetic separation, specific molecular recognition, and high adsorption capacity. Coupled with ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) determination of all the SAs, the LODs were all as low as 0.1 μg/kg, and the recoveries were in a range of 90.2'99.9%. Moreover, the precision values ranged from 0.5 to 9.1%. Consequently, the developed MCNTMIP-MSPE method can be routinely utilized for the trace analysis of SAs to ensure food quality and consumer safety.

FIGURE 3

QNs

QNs are a class of antibacterial drugs with 1,4-dihydro-4-oxyquinoline-3-carboxylic acid structure. They are widely used in clinical diagnosis and treatment, animal disease prevention, and growth promotion because of their advantages such as wide antibacterial spectrum, good efficacy, small toxic and side effects, simple synthesis, and low cost. The residual concentration of QNs is low, mostly ng/L or ng/kg. Excessive or improper use of QNs in animal-derived food has potential carcinogenic, teratogenic, and mutagenic effects, thus threatening human health. Almost all countries have formulated the maximum residual limit standard (MRLS) for quinolone antibiotics. MISPE is widely used in the determination of QNs in complex substrates (Li G. et al., ; Wang J. et al., ; Hu et al., ; Rodriguez-Gomez et al., ; Barahona et al., ; Zhu et al., b; Ma and Row, ; Li et al., b; Tian et al., ; Yu et al., ; Cai et al., ; Soares et al., ).

Our group (Lu et al., ) prepared novel double-template MIPs (dt-MIPs) via a simple and facile precipitation polymerization method with norfloxacin (NOR) and enrofloxacin (ENR) as templates and used them in DSPE combined with HPLC-DAD for the simultaneous selective enrichment and determination of two fluoroquinolones (FQs) in environmental water samples. Figure 4 schematically shows the dt-MIPs preparation and DSPE process. The well-prepared dt-MIPs exhibited good adsorption capacity and selectivity for NOR and ENR, with high enrichment factors of 71 and 61, respectively. Good linearity was obtained in the range of 1'200 µg/L. The LOD and limit of quantification (LOQ) values for NOR were 0.22 and 0.67 µg/L, respectively, and 0.36 and 0.98 µg/L for ENR. Satisfactory recoveries of the two FQs were attained of 80.9'101.0% with relative standard deviations (RSDs) of 0.9'6.9% from spiked lake, sea, and tap water samples. This study not only offered a good method choice for FQs detection but also enriched the research connotation of multitemplate imprinting.

FIGURE 4

Yu et al. () developed a MISPE method for the simultaneous enrichment of 11 FQs [ofloxacin (OFX), gatifloxacin (GAT), NOR, ciprofloxacin (CIP), difloxacin, pefloxacin (PEF), sarafloxacin (SAR), enoxacin (ENX), floxacin, ENR, and lomefloxacin (LOM)] in water by UPLC/MS-MS determination. The attained LODs of FQs were within 6'150 ng/L. The recoveries of all the targeted FQs in sample matrices were higher than 75%, and RSDs were below 15%. The developed MISPE-UPLC/MS-MS proved to be effective for the determination of FQs in wastewater and sludge samples.

Li et al. (b) successfully prepared restricted-access media-imprinted nanomaterials (RAM-MIPs) on the surface of the metal-organic framework (MOF) by RAFT. Figure 5 shows the synthesized route of the RAM-MIPs. Then, they were applied for the DSPE of five FQs (OFX, PEF, NOR, ENR, and GAT) in milk and river water samples prior to HPLC-UV detection. The method attained low LODs, namely, 1.02'3.15 μg/L for milk samples and 0.93'2.87 μg/L for river water samples, respectively, as well as satisfactory recoveries, namely, 80.7'103.5% and 85.1'105.9%, respectively. In comparison with other materials, the RAM-MIP materials are significantly advantageous owing to their simple preparation conditions, uniform and controllable imprinting layer thickness, fast adsorption rate, and so on. The present study demonstrates that RAM-MIP (prepared with MOF as a matrix)-based SPE has broad prospects toward efficient extraction of trace FQs in complex samples.

FIGURE 5

Cai et al. (), using surface-initiated ATRP and sarafloxacin (SAR) as a template, constructed monodisperse RAM-MIPs, and three methods were adopted, as illustrated in Figure 6. The optimum synthesis method was to combine 4-vinylpyridine (VP) and methacrylic acid (MAA) (1:3) as monomers and to select an 8:1:32:8 ratio of a template molecule, cross-linker, and restricted-access functional monomer. The RAM-MIPs showed a high IF (6.05) and the selectivity coefficients were 1.86'2.64 between SAR and other FQs. The RAM-MIP-packed SPE showed higher enrichment ability toward SAR in a complex protein-containing solution than that of traditional MIP-packed one. As a result, the MISPE coupled with the HPLC-UV method achieved a low LOQ for SAR at 4.23 ng/g and the high mean recoveries within 94.0'101.3%. The present study indicated a great application potential of the RAM-MIPs based SPE for trace analysis in complex samples. Furthermore, the proposed functional monomer ratio and rebinding method opened a new way for devising and synthesizing various MIPs.

FIGURE 6

de Oliveira et al. () used ENRO as the template molecule to synthesize MIP1, adopted a multitemplate imprinting strategy (four studied FQs as the template molecules) to synthesize MIP2, and utilized it for the simultaneous PT-SPE of the four FQs (CIP, ENR, marbofloxacin (MARBO), and NOR) in human urine samples. By comparison, MIP1 proved a better adsorbent, and high extraction efficiency was obtained to ENRO (96.0%). Figure 7 schematically shows the apparatus employed for PT-MIP-μ-SPE. It was possible to extract CIPRO ('40%), NOR ('40%), and MARBO ('80%) due to the similarity of the molecular structures. The method attained good linearity from 39 to 1,260 ng/ml for individual FQ, and the LOQ for individual FQ was as low as 39 ng/ml. Finally, the validated PT-MIP-μ-SPE method was proved to be practically applicable, through the preliminary cumulative urinary excretion study after administrating CIPRO to a healthy volunteer.

FIGURE 7

TCs

TCs, as a kind of broad-spectrum antibiotics produced by Streptomyces, have caused serious harm to the ecological environment and human health because of their wide use and hence residues. Huang L. et al. () used TC, chlortetracycline (CTC), and doxycycline (DC) as the templates and magnetic graphene oxide (Fe3O4/GO) as the supporting material to prepare magnetic multi-MIPs. Then chip-based magnetic multi-MIPs monolithic capillary array columns were constructed for simultaneous MSPE and determination of the TCs in eggs samples. High affinity and specificity to TC, CTC, and DC were shown and the IFs reached 86'104-fold. The LOD values ranged from 3.0 to 5.5 μg/kg. Therefore, the MISPE columns afforded hopeful perspectives for the facile extraction of antibiotics from complicated samples.

Aguilar et al. () synthesized the MIPs by precipitation polymerization using TC as template molecule and applied them for dispersive SPME (DSPME) and removal of TC residues in milk samples. The molecular recognition properties and selectivity of MIPs against four TCs (TC, oxytetracycline (OT), CTC and DC) were evaluated, and then high selectivity was demonstrated for the four TCs. The MIP-based DSPME process provided a high removal ratio between 81.83 and 96.44% with RSD<5% in all cases. Compared to classical removal methods, the present method was faster and required lower solvent consumption and minimum sample manipulation. Therefore, a promising prospect can be expected for facilely synthesizing efficient and selective adsorbents and utilizing MISPE for the simultaneous removal of multiple contaminants residues.

Ma et al. () prepared magnetic molecularly imprinted biochar microspheres with specific adsorption of TCs (TC; OT) by Pickering emulsion polymerization. The obtained materials were employed as adsorbents for extraction and purification of TCs in actual samples (fish, chicken, and tap water). This method was simple in preparation process and cost-effective; the synthesized polymer was a regular spherical structure with magnetic response characteristics, which can simplify the extraction and purification of sample pretreatment. It offers a new idea for the application of MIPs based biochar in contaminant detection in food samples.

Other Antibiotics

CAP is a kind of broad-spectrum antibiotics isolated from Streptomyces venezuelae. Because its long-term and high dose use easily caused granulocytosis, aplastic anemia, and other diseases, China has banned its use in feed-animals (especially laying hens and dairy cows) and required CAPs residues to be mandatory test items in all aquatic products, livestock, and poultry products (Zhang et al., ). BLAs are a broad class of antibiotics, and their residues mainly come from agricultural and veterinary drugs, medical drugs, and wastewater treatment discharges from sewage plants. They pose potential threats to the human body and ecology. For example, a few patients will have allergic reactions to BLAs, and the residual antibiotics in the soil will affect the growth of plant roots and seed germination, etc. In addition, the pollution of Antibiotic-Resistant Bacteria (ARB) and Antibiotics Resistance Gene (ARG) caused by the abuse of BLAs and other antibiotics is threatening human health and ecological safety (Li et al., a). MALs are widely used in clinical and veterinary medicine fields, with a broad spectrum of antimicrobial effects, especially for animal husbandry and aquaculture (Liang and Zhang, ). At present, more and more efforts have been devoted to the monitoring of MALs residues.

Using MISPE technology with excellent performance, the trace detection of various antibiotics in diverse complex substrates has been realized (Li Z. et al., ; Lian and Wang, ; Xie et al., ; Negarian et al., ; Qin et al., ; Garza Montelongo et al., ; Tan et al., ). For example, Qin et al. () developed a straightforward method for selective separation of CAP from marine sediment samples. CAP-MMIPs were synthesized via surface imprinting and nanoimprinting technologies. The material has a perfect core-shell structure, excellent thermal stability, and high selectivity toward CAP. The CAP-MMIPs were employed for fast and selective SPE of CAP followed by HPLC-DAD. An excellent linearity was attained from 0.1 to 20 mg/L (R2 = 0.999, n = 3), and the LOD was 0.1 μg/L. The spiked recoveries were between 77.9 and 102.5% with low RSDs (< 6.3%). Good reusability was achieved (at least 5 times) by the regeneration and there was hardly any loss of selectivity and adsorption capability. Such MMIPs-SPE method can provide a vital alternate to traditional extraction ones for preparing environmental samples.

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Teixeira et al. () prepared 1-vinylimidazole-co-trimethylolpropane trimethacrylate- (1-VI-co-TRIM-) based MIPs adsorbent for PT-SPE of abamectin (ABA), eprinomectin (EPR), and moxidectin (MOX), coupled to HPLC-UV determination. The performance criteria for linearity, sensitivity, precision, accuracy, recovery, robustness, and stability were systematically assessed and were within the recommended guidelines. The validated PT-MIP-SPE proved to be economical, simple, easy-to-perform, and potentially applicable for the extraction of MALs in complicated samples.

Nascimento et al. () firstly synthesized a new selective adsorbent based on magnetic molecularly imprinted polypyrrole-conducting polymer (MMIPPy) and applied it to the MSPE of praziquantel (PZQ) enantiomers [(R)-(')-PZQ and (S)-(+)-PZQ] combined with HPLC-DAD determination. Under optimal conditions, excellent linearity was attained in a range of 0.01'10 µg/ml, with correlation coefficients higher than 0.98 and RSDs less than 15%. The LOQ was 0.01 µg/ml for both enantiomers, and RSDs and relative errors were below 20%. The method was applied satisfactorily for the determination of PZQ enantiomers from sheep milk samples with the possibility to other analytes in different complex matrices. The economical, simple, and easy-to-perform MMIPPy-MSPE method suggested great application potential for antibiotics residues determination.

Conclusions and Prospects

To summarize, we review recent advances on both the classification of MISPE and new imprinting techniques for antibiotics coupled with chromatographic analysis, with emphasis on typical examples of MISPE in SAs, QNs, TCs, and other antibiotics. The use of emerging typical imprinting techniques has greatly improved the performance of MISPE and further broadened its application scope. The advancement of new imprinting techniques, especially the combination of multiple techniques, can effectively solve some problems in the practical application of traditional MIPs, such as low binding capacity, template leakage, and difficulties in aqueous phase identification, and provide a better performance, easier separation, and intelligently controlled release of MIPs as solid-phase sorbents. This can provide an effective means to eliminate matrix interference and enrich trace antibiotics with high selectivity. Moreover, the sample pretreatment will definitely develop in the direction of economical, efficient, environmentally friendly, and easy to operate related aspects. In order to further improve the extraction efficiency of MISPE by using ideal MIPs with greater adsorption capacity, higher selectivity, better hydrophilicity, and easier separation, it is necessary to introduce more and advanced preparation techniques, especially to explore the rational synergistic combination of multiple imprinting techniques. Furthermore, the large-scale production and commercialization of the well-prepared MIPs and well-established MISPE should be given more attention, in order to push forward their greater advance and wider applications.

Author Contributions

DS: writing the original draft. ZS: supervision, revising the manuscript, and funding support. YZ: searching for references and writing part of the original draft. YW: reviewing the manuscript. ML: reviewing and revising the manuscript and funding support. HL: reviewing and revising the manuscript. LW: searching for reference and revising the manuscript. JL: supervision, revising the manuscript, and funding support. LC: editing the manuscript and funding support.

Funding

This work was financially supported by the National Natural Science Foundation of China (, , ), the Youth Innovation Promotion Association CAS (), the Science and Technology Innovation Development Plan of Yantai City of China (MSGY112, MSGY077), the Shandong Key Laboratory of Coastal Environmental Processes, YICCAS (SDHADKFJJ17), the National Demonstration Center for Experimental Pharmacy Education (Yantai University), the Natural Science Foundation of Shandong Province of China (ZRQB183, ZRKC032, ZRQC011), and the Taishan Scholar Project Special Funding (ts).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

References

Background

The causative agent of heartworm disease, Dirofilaria immitis, infects many mammals including dogs, cats, and ferrets [1, 2]. This parasitic filarial disease is characterized by the presence of sexually mature dimorphic adults in the pulmonary arteries that produce microfilariae (MF) circulating in the bloodstream [3]. Female mosquitoes ingest circulating MF during a blood meal from animals harboring patent infections, with ingested MF molting to the infective third-stage larvae (L3) within 10'14 days post-blood meal. Thereafter, during subsequent blood meals, the L3s are deposited on the skin of the host and enter the subcutaneous tissues through the bite wound. The L3s molt to the fourth-stage larvae (L4) 3'5 days post-infection in the host, reaching the heart and lungs as early as 70 days post-infection as juvenile adult worms (L5) [1].

The presence of adult worms in the pulmonary arteries causes vascular damage such as thickening of the arteries and hypertrophy of the smooth muscle cells [4, 5]. Vascular changes, including separation of endothelial junctions, loss of endothelial cells, and the adherence of leukocytes due to the presence of adult worms, can be detected as soon as 4 days post-surgical transplantation of adults in heartworm-naïve dogs [6]. This damage appears to be chronic and cumulative, with increasing numbers of worms and duration of infection causing more severe pathological changes. Pathological lesions in the vasculature due to the presence of adult worms is caused by endothelial swelling, precipitating the accumulation of platelet-derived growth factor (PDGF) [7]. This accumulation of PDGF stimulates the migration of smooth muscle cells into the tunica media, causing the rough velvety appearance observed in the pulmonary arteries in heartworm disease [8].

The vascular changes and pathology associated with heartworm disease include right heart enlargement, main and lobar pulmonary artery enlargement and tortuous vasculature, with resulting congestive heart failure, hypertension, and the potential development of caval syndrome over time [4, 9]. While melarsomine dihydrochloride is approved for the treatment of adult heartworms in canine heartworm disease, there are no approved therapeutics for the treatment of adult heartworms in cats. With limited adulticidal treatments available, the severity of damage associated with this disease, and the potential complications associated with its treatment, the prevention of heartworm disease development is crucial [10].

Heartworm disease has been successfully prevented by the proper administration of chemoprophylactic medications in dogs and cats. Current products on the market prevent heartworm disease by targeting the L3 and L4 stages, preventing the development of larval heartworms into adults and subsequent migration of adults to the heart and pulmonary arteries through the use of various macrocyclic lactone (ML) formulations. MLs have been successfully utilized for the prevention of heartworm disease for over 30 years [11'19]. Within this class of molecules are the avermectin and milbemycin subfamilies, of which ivermectin, eprinomectin, abamectin, selamectin, and doramectin are classified under the avermectin subfamily, and milbemycin oxime and moxidectin under the milbemycin subfamily [13]. These molecules are available in various commercial presentations approved by the Food and Drug Administration (FDA) Center for Veterinary Medicine (CVM) as oral, topical, and injectable products.

While MLs have been successfully utilized for the prevention of heartworm disease, and for the treatment and control of numerous other parasitic infections for decades, the exact mechanism of action of this drug class in the prevention of heartworm disease is unclear. MLs have been shown to bind to glutamate-gated chloride ion channels precipitating hyperpolarization, paralysis, and death of heartworms [20'22]. In mammals, MLs bind to gamma-aminobutyric acid type A-gated chloride (GABAA) channels and glycine receptors located in intestinal epithelial cells, central nervous system neurons, brain capillary endothelial cells, renal epithelial cells, placenta, and testes [23]. GABAA channels are protected by ATP-dependent transmembrane efflux pumps, P-glycoproteins (P-gp), which actively pump xenobiotics out of the cell. ABCB1 mutations, such as multidrug resistance-1-mutants (MDR-1) result in improper development of P-gps, featuring a premature stop codon in the ABCB1 gene. Homozygous MDR1-mutant dogs have an increased sensitivity to MLs due to the abnormally high accumulation of drug by the nonfunctional P-gp formation. Additionally, MDR1-mutant dogs are susceptible to increased neurological side effects due to the increased accumulation of drug in the central nervous system [23]. However, all FDA CVM-approved heartworm preventive products are safe to administer to wild-type and MDR1-mutant dogs at the manufacturer's recommended dose.

Despite the high potency of the MLs against D. immitis L3 and L4 stages in vivo, the concentrations of MLs necessary in vitro to kill these stages do not correlate with the in vivo potency, making pharmacokinetic/pharmacodynamic relationships unpredictable. This drug class has been observed to bind at the excretory-secretory (ES) pore of Brugia malayi MF, a filarid closely related to D. immitis, and reduce the concentration of ES products post-treatment [24, 25]. These ES products are theorized to suppress the host immune response and bias the host toward an anti-inflammatory Th2 response in order to evade the host immune system. Therefore, it is hypothesized that the host immune response plays an integral role in the killing of these larvae post-ML treatment [26]. It is possible that these same mechanisms are also involved in the activity of MLs against D. immitis.

Macrocyclic lactone resistance in heartworm disease

Despite the high efficacy of heartworm preventive products, the prevalence of reported heartworm-positive cases increased by 21.7% between and [27], and resistance of D. immitis to MLs has been confirmed against multiple strains of heartworms in the USA [27'33]. Without any new products on the market for heartworm prevention utilizing a novel mechanism of action, the management of ML resistance may currently only be addressed by optimizing the available MLs. This can be achieved by increasing the dose of the ML, the frequency of dosing, and/or improving the bioavailability of the drugs through optimized formulations. Among the MLs, moxidectin has some unique attributes such as high potency, a long half-life and other favorable pharmacokinetic parameters, a wide therapeutic index, and versatility in formulation that may allow the development of new products that could aid in overcoming drug resistance [34].

To date, confirmed cases of ML resistance have been primarily concentrated in the Lower Mississippi River Valley (LMRV) region, but with the continual movement of dogs around the USA and the availability of competent mosquito vectors across the USA, the spread of ML-resistant heartworms is inevitable [35]. Dogs rescued by shelter organizations harboring heartworm infections are regularly transported across state lines, with many of these dogs coming from the LMRV, where ML resistance has been confirmed [36, 37]. This further increases the likelihood of the spread of ML resistance to other areas of the USA. The use of products with proven preventive efficacy against ML-resistant heartworm infections may help slow the spread of these infections and even prevent the establishment of new resistant D. immitis subpopulations.

Moxidectin pharmacology

MLs are 16-member rings fused with both benzofuran and spiroketal functions that target glutamate-gated chloride ion channels in invertebrates, including filarial nematodes. Of all the ML molecules, moxidectin has the most unique characteristics that enable optimal product profiles. Most notably, moxidectin is the most lipophilic and potent of the MLs [34, 38, 39].

With lipophilicity of logPMOX'='6 and logPIVM'='4.8, moxidectin has a higher tissue distribution compared to ivermectin, leading to a lower rate of clearance [38]. As heartworm L3s and L4s migrate through host tissue during their pathway to the pulmonary arteries, higher tissue distribution and longer elimination half-life may play a role in the increased potency of moxidectin as compared to other MLs [2, 34]. Moxidectin was shown to be 100% effective when dosed 60 days post-infection at only 0.5 µg/kg compared to the lowest efficacious doses of 6 µg/kg and 500 µg/kg for ivermectin and milbemycin oxime, respectively, when administered 30 days post-infection against a known susceptible heartworm strain (Table 1) [15, 40'42].

Table 1.

Macrocyclic lactone (FDA-approved dose) Dosage (µg/kg) Treatment time post-inoculation (months)a Preventive efficacy (%) References Moxidectin 0.5 2 100 [15] (3 µg/kg) 0.625 2 100 [15] 1.0 3 47.8 [15] 1.25 1 100 [15] 1.25 2 100 [15] 3.0 1 100 [15] 3.0 3 64.2 [15] Ivermectin 1 1 53.2 [42] (6 µg/kg) 2 1 83.3; 97.2 [41, 42] 2 1.5 63.8 [42] 3.3 1 98.1 [42] 6 1 100 [41] 6 1.5 100 [41] Milbemycin oxime 500 1 100 [40] (500 µg/kg) 500 1.5 100 [40] 500 2 93.9 [40]

In addition to the increased lipophilicity and potency, moxidectin displays a lower affinity to GABAA channels leading to an increased safety profile for this molecule in MDR-1ab('/') P-gp deficient mice [43]. ML toxicity is characterized by the oversaturation of drug binding to P-gps and the accumulation of drug in the brain. P-gps actively transport drugs across membranes, protecting such areas as the blood'brain-barrier. In dogs homozygous for the MDR-1 mutation, moxidectin has demonstrated improved safety compared to ivermectin and doramectin, with only mild neurotoxicity when administered at ''400' µg/kg, while ivermectin and doramectin induce severe neurotoxicity when administered at 200'600 µg/kg [23, 44'47].

Studies characterizing the binding affinity of ivermectin and moxidectin have indicated that while moxidectin and ivermectin bind to glutamate-gated chloride ion channels, moxidectin may interact in a different manner than ivermectin due to structural differences such as the lack of a disaccharide moiety on C-13 of the macrocycle in moxidectin, an olefinic side chain on C-25, and a methoxime moiety on C-23 [34, 38]. These differences between moxidectin and ivermectin in sites of binding to nematode glutamate-gated chloride ion channels may be a contributing factor in the slower development of drug resistance to moxidectin. The mode of action, structural differences, and pharmacodynamics of MLs, including moxidectin, have been reviewed previously [34, 38].

Moxidectin formulations available for heartworm prevention

Due to the versatility of moxidectin, a number of formulations are available for heartworm prevention. These include products for oral, topical, and injectable administration.

Oral moxidectin

Initially, moxidectin was developed as a monthly oral dose product administered at 3 µg/kg labeled for heartworm prevention only. However, this product was marketed in the USA only for a short period of time but remains on the market in Latin America and in some areas of Southeast Asia. More recently, moxidectin has been formulated with other active pharmaceutical ingredients (APIs), sarolaner and pyrantel pamoate, for oral delivery as Simparica Trio® (Zoetis, NJ, USA). This combination of APIs provides additional coverage against multiple ecto- and endoparasites. The Simparica Trio formulation (24 µg/kg moxidectin/1.2 mg/kg sarolaner/5 mg/kg pyrantel) provides a convenient, easy-to-administer option for pet owners, as well as eliminating concerns of potential human and environmental exposure, as is the case with topical products.

Evaluations of the preventive efficacy of a number of MLs, at various oral dosages and treatment times post-experimental inoculation, were performed for the determination of minimum efficacious dosages for heartworm prevention (Table 1). Ivermectin administered 30 days post-inoculation was not 100% efficacious in the prevention of heartworm disease at ''3.3 µg/kg, with decreasing efficacy when administered 45 days post-inoculation compared to the 30-day post-inoculation efficacy [41, 42]. When administered at 30 or 45 days post-inoculation, 6 µg/kg ivermectin provided full protection against heartworm disease [41]. Milbemycin oxime was evaluated at 500 µg/kg when administered 30, 60, 90, and both 60 and 90 days post-inoculation, demonstrating full protection against heartworm disease when administered at 30 days and at both 60 and 90 days post-inoculation [14, 40]. In comparison, moxidectin administered 30 days post-inoculation was 100% efficacious at the lowest dose tested, 1.25 µg/kg [15]. Additionally, moxidectin provided full protection against heartworm disease when administered at 0.5 µg/kg, 2 months post-inoculation, while ivermectin required a ~'12-fold higher dose and milbemycin oxime, a -fold higher dose for full protection. All of these early evaluations were performed against strains of heartworm that were susceptible to MLs.

Studies evaluating the efficacy of MLs have demonstrated that increasing the dose and number of sequential doses administered increases the efficacy against ML-resistant strains of heartworm. Moxidectin and other MLs have been evaluated for efficacy against the genetically confirmed ML-resistant strains JYD-34, ZoeLA, and ZoeMO. Oral moxidectin administered at 3, 6, 12, and 24 µg/kg demonstrated preventive efficacy of 19%, 25.5%, 33.3%, and 53.2%, respectively, when administered as a single dose 30 days post-inoculation with 50 JYD-34 L3. Additionally, in this same study when oral moxidectin was administered every 30 days for three consecutive months at 3 µg/kg, adult heartworm recovery was reduced by 44.4%, demonstrating an increased efficacy at this dose with repeated monthly administration [48]. Against this same strain, JYD-34, 100% efficacy was achieved when oral moxidectin was administered monthly at 40 µg/kg or 60 µg/kg for three consecutive months, and a 24 µg/kg dose administered monthly for three consecutive months was highly effective (98.8%) (Table 2) [48].

Table 2.

Study Straina Treatmentb Dosage (µg/kg) Days of treatment Preventive efficacy (%) 1 JYD-34 Placebo 0 0, 30, 60 ' JYD-34 Moxidectin 3 0 19 JYD-34 Moxidectin 3 0, 30, 60 44.4 JYD-34 Moxidectin 6 0 25.5 JYD-34 Moxidectin 12 0 33.3 JYD-34 Moxidectin 24 0 53.2 2 JYD-34 Placebo 0 0, 28, 56 ' JYD-34 Moxidectin 24 0, 28, 56 98.8 JYD-34 Moxidectin 40 0, 28, 56 100 JYD-34 Moxidectin 60 0, 28, 56 100 ZoeLA Placebo 0 0, 28, 56 ' ZoeLA Moxidectin 3 0 44.4 ZoeLA Moxidectin 24 0, 28, 56 99.5 ZoeLA Moxidectin 40 0, 28, 56 99.5 ZoeLA Moxidectin 60 0 88.2 ZoeLA Moxidectin 60 0, 28, 56 100 3 ZoeMO Placebo 0 0, 28, 56 ' ZoeMO Moxidectin 3 0 82.1 ZoeMO Moxidectin 24 0, 28, 56 99.5 ZoeMO Moxidectin 40 0, 28, 56 100 ZoeMO Moxidectin 60 0, 28, 56 100

Oral moxidectin has also been evaluated against the resistant strains ZoeMO and ZoeLA for efficacy in the prevention of heartworm disease. When administered as three consecutive oral monthly dosages, 60 µg/kg moxidectin was 100% effective in preventing the development of both the ZoeLA and ZoeMO resistant strains. Efficacy was reduced to 88.2% when only a single 60 µg/kg oral moxidectin dose was administered against the ZoeLA strain, demonstrating improved efficacy with consecutive monthly administration (Table 2) [48].

The 24 µg/kg moxidectin dose was selected for further development in Simparica Trio based on a number of scientific and commercial considerations [48]. In two additional laboratory studies, the efficacy of 24 µg/kg moxidectin administered orally for either four or six consecutive months was compared directly to Heartgard® Plus (ivermectin/pyrantel, Boehringer Ingelheim Animal Health, Ingelheim, Germany), Interceptor® Plus (milbemycin oxime/praziquantel, Elanco, Greenfield, IN, USA), with both commercial products administered according to the labels, and an untreated control for a total of six consecutive months [49]. Studies used the ML-resistant strains ZoeLA and JYD-34, with each study animal receiving an inoculation of 50 L3 on day '30. A necropsy was performed approximately 9 months post-inoculation (3 months after the last of the six monthly doses) for all study animals in both studies. In the study with the ZoeLA strain, four or six consecutive monthly treatments of 24 µg/kg moxidectin were ''96.1% efficacious, however, six monthly treatments of Heartgard Plus and Interceptor Plus were only 18.7% and 21.2% efficacious, respectively (Table 3) [49]. In the study with JYD-34, four or six consecutive monthly treatments of 24 µg/kg moxidectin were 95.9% and 99.3% effective, and six monthly treatments of Heartgard Plus and Interceptor Plus were 63.9% and 54.6% effective, respectively (Table 4) [49]. These data indicate that the increased dose of oral moxidectin provides greater efficacy against ML-resistant heartworm strains when compared to commercially available oral heartworm preventives and is a viable strategy for the management of ML resistance in the field.

Table 3.

Study Treatment Dosage (µg/kg) Day of inoculationa Days of treatment Preventive efficacy (%) References 1 Placebo 0 '30 0, 30, 60, 90, 120, 150 ' [50] Simparica Trio (moxidectin) Min. 24 '30 0, 30, 60, 90, 120, 150 97.2 [50] Heartgard Plus (ivermectin) Min. 6 '30 0, 30, 60, 90, 120, 150 8.5 [50] Interceptor Plus (milbemycin oxime) Min. 500 '30 0, 30, 60, 90, 120, 150 35.9 [50] 2 Placebo 0 '30 0, 30, 60, 90, 120, 150 ' [49] Moxidectin 24 '30 0, 30, 60, 90 96.8 [49] Moxidectin 24 '30 0, 30, 60, 90, 120, 150 96.1 [49] Heartgard Plus (ivermectin) Min. 6 '30 0, 30, 60, 90, 120, 150 18.7 [49] Interceptor Plus (milbemycin oxime) Min. 500 '30 0, 30, 60, 90, 120, 150 21.2 [49]

Table 4.

Study Treatment Route of administration Dosage (µg/kg) Day of inoculationa Days of treatment Preventive efficacy (%) References 1 Placebo SC 0 '30 0 ' [54] ProHeart 12 (moxidectin) SC 500 '30 0 100 [54] Heartgard Plus (ivermectin) Oral Min. 6 '30 0, 30, 60, 90, 120, 150 10.5 [54] Interceptor Plus (milbemycin oxime) Oral Min. 500 '30 0, 30, 60, 90, 120, 150 14.6 [54] 2 Placebo SC 0 165 0 ' [54] ProHeart 12 (moxidectin) SC 500 165 0 98.3 [54] Heartgard Plus (ivermectin) Oral Min. 6 165 0, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330 37.7 [54] Interceptor Plus (milbemycin oxime) Oral Min. 500 165 0, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330 34.9 [54] 3 Placebo Oral 0 '30 0, 30, 60, 90, 120, 150 ' [49] Moxidectin Oral 24 '30 0, 30, 60, 90 95.9 [49] Moxidectin Oral 24 '30 0, 30, 60, 90, 120, 150 99.3 [49] Heartgard Plus (ivermectin) Oral Min. 6 '30 0, 30, 60, 90, 120, 150 63.9 [49] Interceptor Plus (milbemycin oxime) Oral Min. 500 '30 0, 30, 60, 90, 120, 150 54.6 [49]

In a recent laboratory study, Simparica Trio, containing 24 µg/kg moxidectin, 1.2 mg/kg sarolaner and 5 mg/kg pyrantel, and Heartgard Plus and Interceptor Plus (n'='6) were administered in six consecutive monthly oral doses at the labeled dose rates after receiving an inoculation of 50 ZoeLA strain D. immitis L3 on day '30 [50]. Dogs were necropsied approximately 9 months post-inoculation and evaluated for the presence of circulating MF and heartworm antigen on days 180, 210, and 236. Preventive efficacy of 97.2% in the Simparica Trio-treated group was demonstrated, with all of the treated dogs having ''3 worms present. No dogs treated with Simparica Trio tested positive for the presence of circulating MF at any time during this study, with only two of the six dogs testing positive for heartworm antigen. All six dogs treated with Heartgard Plus and Interceptor Plus had adult worms present at necropsy, with a geometric mean of 32.5 and 22.8 adults recovered, and preventive efficacy of 8.5% and 35.9%, respectively (Table 3) [50]. On day 236, all Heartgard Plus- and Interceptor Plus-treated dogs were positive for the presence of heartworm antigen, and five of the total six dogs for each group were positive for circulating MF, indicating the possibility of infected dogs being able to transmit ML-resistant heartworm strains despite proper use of these preventive products. With very similar results to those obtained in the previous study [49], using 24 µg/kg moxidectin dose as a standalone, these data confirm the robust efficacy of 24 µg/kg moxidectin against the ML-resistant ZoeLA heartworm strain.

The preventive efficacy of Simparica Trio was also evaluated in a multicenter field study, enrolling a total of 410 dogs (365 included in the efficacy evaluation) from 23 different veterinary clinics across the USA. Dogs were administered 11 consecutive monthly doses of either Heartgard Plus (6'12 µg/kg ivermectin and 5'10 mg/kg pyrantel) or Simparica Trio (24'48 µg/kg moxidectin, 2'4 mg/kg sarolaner, and 5'10 mg/kg pyrantel) [51]. All dogs enrolled in the study tested negative for the presence of circulating MF and adult heartworm antigen on days 1/0 (before treatments), 120, and 240. Compliance in administering Heartgard Plus and Simparica Trio in this study was rigorously documented, ensuring that all dogs in efficacy evaluations received their required doses at the appropriate time. Of the 246 dogs that were eligible for efficacy inclusion that received Simparica Trio with complete compliance, no dogs tested positive for MF or heartworm antigen at any point throughout the study, while two dogs of the total 119 eligible for efficacy evaluation that received Heartgard Plus in complete compliance tested positive for heartworm antigen on day 330, with one of these dogs also testing positive for MF. The two dogs treated with Heartgard Plus that became positive for adult heartworms during the study, even with 11 months of documented compliance were from Livonia, Louisiana (LA) in the LMRV [51]. Based on the data presented above, Simparica Trio, containing a minimum of 24 µg/kg moxidectin, will likely provide robust heartworm prevention against the strains to which most dogs in the USA will likely be exposed, including those that may be resistant to MLs.

Injectable moxidectin

ProHeart® 6 [0.17 mg/kg (170 µg/kg) moxidectin] and ProHeart® 12 [0.5 mg/kg (500 µg/kg) moxidectin] are the only injectable heartworm preventives, and these products offer extended-release moxidectin for the continuous prevention of heartworm disease in dogs for 6 and 12 months, respectively. ProHeart 6 and 12 offer a safe and reliable heartworm preventive option without owners having to comply with monthly administration. Data show that, on average, owners using a monthly product for heartworm prevention administer only half of the prescribed monthly doses necessary to provide complete heartworm prevention [52]. Once ProHeart is administered, compliance is guaranteed to be 100% for the 6- or 12-month dosing interval. In addition to the increased compliance as compared to monthly administered products, data indicate that ProHeart formulations (0.17 mg/kg; ProHeart 6) provide 100% prophylactic activity for one full year against ML-susceptible heartworms, demonstrating the robust, persistent efficacy of the ProHeart formulation against experimentally induced ML-susceptible heartworms [53]. The persistent efficacy observed in ProHeart 6 and 12 is a function of the inherent physicochemical properties of moxidectin, including long half-life and increased lipophilicity and potency, together with the extended-release properties of the formulation [39]. Anaphylactic and anaphylactoid reactions may occur in some dogs following administration of ProHeart 6/12 alone or with vaccines, and they are available through a restricted distribution program to veterinarians that have completed the RiskMAP training and certification module.

The efficacy of ProHeart 12, Heartgard Plus, and Interceptor Plus against the ML-resistant strain JYD-34 was evaluated in two different laboratory studies, in which dogs were either inoculated with 50 D. immitis L3 30 or 165 days after initiating treatment, respectively [54]. In the first study, where dogs were inoculated on day '30, dogs (n'='6) were either treated with a single dose of ProHeart 12 on day 0 or administered six consecutive monthly oral treatments of Heartgard Plus or Interceptor Plus on days 0, 30, 60, 90, 120, and 150. All dogs were necropsied on day 185, approximately 8 months post-inoculation. The preventive efficacy of ProHeart 12 was 100%, with no dogs having any adult worms at necropsy. In comparison, all six dogs in both the Heartgard Plus- and Interceptor Plus-treated groups, had adult worms present at necropsy, demonstrating an overall preventive efficacy of 10.5% and 14.6% respectively (Table 4) [54]. In the second laboratory study, dogs were inoculated on day 165, in order to compare efficacies against an ML-resistant strain in the middle of the treatment regimen. For this study, dogs (n'='6 for each treatment group) were either treated with a single dose of ProHeart 12 on day 0 or administered six consecutive oral monthly doses before and after inoculation with Heartgard Plus or Interceptor Plus, for a total of 12 consecutive monthly treatments [54]. All dogs were necropsied on day 360, approximately 7 months post-inoculation. Adult heartworms were present in all dogs treated with either Heartgard Plus or Interceptor Plus, with preventive efficacies of 37.7% and 34.9% respectively. The preventive efficacy of those dogs treated with ProHeart 12 was 98.3%, with only four of the total six dogs containing a single worm each (Table 4) [54]. These studies indicate that a single injection of ProHeart 12 is highly efficacious (''98%) against a known ML-resistant strain when exposed at both the start and middle of the treatment regimen.

As claims of lack of efficacy for MLs, and confirmed cases of ML resistance continue to increase, field efficacy trials become extremely important in evaluating the efficacy against current natural strains circulating that could be a mixture of susceptible and potentially ML-resistant strains. A field efficacy study evaluated the preventive efficacy of Heartgard Plus and ProHeart 12 with client-owned dogs from 19 veterinary clinics across the USA. Dogs received either 20 consecutive monthly doses of Heartgard Plus or two annual doses of ProHeart 12, with a total of 218 dogs receiving Heartgard Plus and 236 dogs receiving ProHeart 12 [55]. The study was designed to test the efficacy of treatment in dogs over an initial exposure period of 12 months. However, in order to determine if dogs may have been exposed at the end of the 12-month period became infected, all animals needed to be followed for a final antigen test 8 months after the end of the 12-month exposure period. The second ProHeart 12 dose and the additional eight Heartgard Plus doses provided heartworm prevention during this period. Of the 236 dogs that received two annual doses of ProHeart 12, no animals tested positive for the presence of heartworm antigen or circulating MF at any time during this study, indicating complete heartworm prevention for the 12-month evaluation period. Four of the total 218 dogs that received 20 consecutive monthly doses of Heartgard Plus tested positive for the presence of adult heartworm infection during the study. Of these four dogs, three tested positive for heartworm antigen and circulating MF, with the fourth testing positive solely for the presence of heartworm antigen. All four cases of breakthrough infection with Heartgard Plus occurred in the LMRV, with two dogs from Zachary, LA, one dog from Lake Charles, LA, and one dog from Memphis, Tennessee. Compliance for administration of Heartgard Plus in the study was rigorously documented, ensuring that all dogs received their required doses at the appropriate time. Three of the four Heartgard Plus-treated dogs were also positive for MF, even with continued Heartgard Plus treatment, indicating that these treatment failures may have been due to exposure to ML-resistant heartworms. There was a significant difference between the ProHeart 12 and Heartgard Plus groups, indicating that ProHeart 12 performed better than Heartgard Plus in preventing heartworm disease in this field study, confirming the robust efficacy of ProHeart 12 under field conditions [55]. The difference in the performance of these two products in this study is likely related to the inherent properties of moxidectin and the dose of moxidectin in the product, in addition to the continuous presence of the active compound through the dosing period made possible by the ProHeart 12 extended-release formulation.

The transmission of heartworm disease is dependent on the ingestion of MF by female mosquitoes from a patent host. Therefore, the reduction and elimination of circulating MF in infected animals is important in reducing the transmission and prevalence of this disease. Topical moxidectin (Advantage® Multi, Bayer Animal Health, Shawnee Mission, KS, USA) is the only ML FDA-approved for the elimination of circulating MF in canine heartworm disease, with a >'99% reduction in MF observed compared to untreated controls 7 days post-treatment [56]. The 0.5 mg/kg moxidectin dose in ProHeart 12 is also highly microfilaricidal, with >'97% reduction in MF reported 7 days post-treatment [57]. The results for both Advantage Multi and ProHeart 12 are for strains susceptible to MLs. Evaluation of ProHeart 6 and ProHeart 12 in the reduction of circulating MF against an ML-resistant strain, ZoeMO, demonstrated a >'90% reduction in MF by 28 and 42 days post-treatment respectively, and >'96% on day 84, after a single subcutaneous dose [58]. Based on these data, injectable moxidectin may offer the potential for microfilarial reduction, even against ML-resistant strains providing additional opportunity to reduce the transmission of heartworm.

Topical moxidectin

Moxidectin can be administered topically for the prevention of heartworm disease and is available for dogs, cats, and ferrets (Advantage Multi®; Coraxis®, Bayer Animal Health). The increased dose of moxidectin administered in Advantage Multi/Coraxis, 2.5 mg/kg ( µg/kg), provides efficacy for the treatment and control of gastrointestinal nematodes, including hookworm, roundworm, and whipworm species in dogs.

Topical moxidectin is the only FDA-approved treatment for circulating MF in heartworm-positive dogs [56, 59, 60]. In two laboratory studies, naturally infected dogs and dogs experimentally infected by surgical transplantation of adult worms were administered topical Advantage Multi on days 0 and 28, with blood samples collected at various times post-treatment. Fourteen days post-treatment, dogs in both studies had a >'99% reduction in circulating MF observed compared to the untreated control groups. This reduction of >'99% persisted through day 42, the study end, with one dog still testing positive for the presence of circulating MF in the naturally infected dogs and a geometric mean of 7.1 MF/ml in the experimentally infected dogs on day 42 [56, 60].

Due to the high dose of moxidectin administered in Advantage Multi/Coraxis, accumulation occurs with subsequent monthly doses, with a steady state of mean serum concentration reached after four monthly topical applications [61]. This steady state of moxidectin is fully efficacious in the prevention of heartworm disease, as dogs inoculated with 50 susceptible D. immitis L3 28 days after four monthly topical treatments had no adult heartworms recovered at necropsy [61]. The mean terminal phase half-life for the topical administration of moxidectin is 28 days, therefore providing an extended duration of time over the mean efficacious dose (MED) as compared to that of oral ivermectin [61]. Advantage Multi has also demonstrated efficacy against ML-resistant heartworms, with 100% preventive efficacy against the JYD-34 strain with a single dose administered 30 days post-inoculation with D. immitis L3 [30]. There was a report of'<'100% efficacy in a second study using a single dose of Advantage Multi that was not published (McCall, unpublished data).

Despite the benefits of topical moxidectin for the prevention of heartworm disease, there are disadvantages to this topical administration route. Improper owner administration of topical products can lead to inadequate exposure and absorption of the active ingredients, and subsequently sub-efficacious doses to the dog. Additionally, topical products can lead to increased environmental contamination due to exposure of household items and other members of the household to the product. Also important is that accidental oral administration of ''40% of the topical dose (total of 1 mg/kg dose) to a dog with the MDR-1 defect can result in severe adverse effects, resulting in a black box warning on the label.

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