Development of inert coatings to prevent drug retention in 3D-printed diffusion cells.

Diffusion cells play a crucial role in the pharmaceutical and cosmetic fields by assessing the release and permeation of active pharmaceutical ingredients across membranes. However, commercially available glass-based devices, such as Franz diffusion cells, are expensive and fragile. The emergence of three-dimensional (3D) printing technology enables the creation of diffusion cells with cost-effective polymeric materials and resins, offering exceptional precision and custom geometries. Nonetheless, there are challenges associated with interactions between 3D printing materials and drug molecules. This work aimed to develop inert coatings for 3D-printed diffusion models. Diffusion devices were designed and 3D-printed with a stereolithography (SLA) 3D printer, and different coatings were applied. Then, two model drugs were used to evaluate drug retention by coated devices. Among the tested coatings, one of them showed great potential in preventing drug retention and was selected for subsequent experiments with different drugs and conditions. Finally, voriconazole eyedrops were used to confirm the viability of 3D-printed Franz diffusion cells as a drug release diffusion model. The favourable results obtained with the coating promote the use of 3D printing as a cost-effective manufacturing technology, capable of producing diffusion cells tailored to specific study requirements.

Intravitreal implants manufactured by supercritical foaming for treating retinal diseases.

Chronic retinal diseases, such as age-related macular degeneration (AMD), are a major cause of global visual impairment. However, current treatment methods involving repetitive intravitreal injections pose financial and health burdens for patients. The development of controlled drug release systems, particularly for biological drugs, is still an unmet need in prolonging drug release within the vitreous chamber. To address this, green supercritical carbon dioxide (scCO2) foaming technology was employed to manufacture porous poly(lactic-co-glycolic acid) (PLGA)-based intravitreal implants loaded with dexamethasone. The desired implant dimensions were achieved through 3D printing of customised moulds. By varying the depressurisation rates during the foaming process, implants with different porosities and dexamethasone release rates were successfully obtained. These implants demonstrated controlled drug release for up to four months, surpassing the performance of previously developed implants. In view of the positive results obtained, a pilot study was conducted using the monoclonal antibody bevacizumab to explore the feasibility of this technology for preparing intraocular implants loaded with biologic drug molecules. Overall, this study presents a greener and more sustainable alternative to conventional implant manufacturing techniques, particularly suited for drugs that are susceptible to degradation under harsh conditions.

Infliximab microencapsulation: an innovative approach for intra-articular administration of biologics in the management of rheumatoid arthritis-in vitro evaluation.

Microencapsulation of the therapeutical monoclonal antibody infliximab (INF) was investigated as an innovative approach to improve its stability and to achieve formulations with convenient features for intra-articular administration. Ultrasonic atomization (UA), a novel alternative to microencapsulate labile drugs, was compared with the conventional emulsion/evaporation method (Em/Ev) using biodegradable polymers, specifically Polyactive® 1000PEOT70PBT30 [poly(ethylene-oxide-terephthalate)/poly(butylene-terephthalate); PEOT-PBT] and its polymeric blends with poly-(D, L-lactide-co-glycolide) (PLGA) RG502 and RG503 (PEOT-PBT:PLGA; 65:35). Six different formulations of spherical core-shell microcapsules were successfully developed and characterized. The UA method achieved a significantly higher encapsulation efficiency (69.7-80.25%) than Em/Ev (17.3-23.0%). Mean particle size, strongly determined by the microencapsulation method and to a lesser extent by polymeric composition, ranged from 26.6 to 49.9 µm for UA and 1.5-2.1 µm for Em/Ev. All formulations demonstrated sustained INF release in vitro for up to 24 days, with release rates modulated by polymeric composition and microencapsulation technique. Both methods preserved INF biological activity, with microencapsulated INF showing higher efficacy than commercial formulations at comparable doses regarding bioactive tumor necrosis factor-alpha (TNF-α) neutralization according to WEHI-13VAR bioassay. Microparticles' biocompatibility and extensive internalization by THP-1-derived macrophages was demonstrated. Furthermore, high in vitro anti-inflammatory activity was achieved after treatment of THP-1 cells with INF-loaded microcapsules, significatively reducing in vitro production of TNF-α and interleucine-6 (Il-6).

Fighting type 2 diabetes: Formulation strategies for peptide-based therapeutics.

Diabetes mellitus is a major health problem with increasing prevalence at a global level. The discovery of insulin in the early 1900s represented a major breakthrough in diabetes management, with further milestones being subsequently achieved with the identification of glucagon-like peptide-1 (GLP-1) and the introduction of GLP-1 receptor agonists (GLP-1 RAs) in clinical practice. Moreover, the subcutaneous delivery of biotherapeutics is a well-established route of administration generally preferred over the intravenous route due to better patient compliance and prolonged drug absorption. However, current subcutaneous formulations of GLP-1 RAs present pharmacokinetic problems that lead to adverse reactions and treatment discontinuation. In this review, we discuss the current challenges of subcutaneous administration of peptide-based therapeutics and provide an overview of the formulations available for the different routes of administration with improved bioavailability and reduced frequency of administration.

3D printed tacrolimus suppositories for the treatment of ulcerative colitis.

Ulcerative colitis is a global health problem, affecting millions of individuals worldwide. As an inflammatory condition localised in the large intestine, rectal delivery of immunosuppressive therapies such as tacrolimus is a promising strategy to maximise drug concentration at the site of action whilst minimising systemic side effects. Here, for the first time, self-supporting 3D-printed tacrolimus suppositories were prepared without the aid of moulds using a pharmaceutical semi-solid extrusion (SSE) 3D printer. The suppositories were printed vertically in three different sizes using combinations of two lipid pharmaceutical excipients (Gelucire 44/14 or Gelucire 48/16) and coconut oil. Although both suppository formulations had the appropriate viscosity characteristics for printing, the Gel 44 formulation required less energy and force for extrusion compared to the Gel 48 system. The Gel 44 disintegrated more rapidly but released tacrolimus more slowly than the Gel 48 suppositories. Although the tacrolimus release profiles were significantly different, both suppository systems released more than 80% drug within 120 min. DSC and XRD analysis was inconclusive in determining the solid-state properties of the drug in the suppositories. In summary, this article reports on the fabrication of 3D printed self-supporting suppositories to deliver personalised doses of a narrow therapeutic index drug, with potential benefits for patients with ulcerative colitis.

3D Printed Tacrolimus Rectal Formulations Ameliorate Colitis in an Experimental Animal Model of Inflammatory Bowel Disease.

The aim of this study was to fabricate novel self-supporting tacrolimus suppositories using semisolid extrusion 3-dimensional printing (3DP) and to investigate their efficacy in an experimental model of inflammatory bowel disease. Blends of Gelucire 44/14 and coconut oil were employed as lipid excipients to obtain suppository formulations with self-emulsifying properties, which were then tested in a TNBS (2,4,6-trinitrobenzenesulfonic acid) induced rat colitis model. Disease activity was monitored using PET/CT medical imaging; maximum standardized uptake values (SUVmax), a measure of tissue radiotracer accumulation rate, together with body weight changes and histological assessments, were used as inflammatory indices to monitor treatment efficacy. Following tacrolimus treatment, a significant reduction in SUVmax was observed on days 7 and 10 in the rat colon sections compared to non-treated animals. Histological analysis using Nancy index confirmed disease remission. Moreover, statistical analysis showed a positive correlation (R2 = 71.48%) between SUVmax values and weight changes over time. Overall, this study demonstrates the effectiveness of 3D printed tacrolimus suppositories to ameliorate colitis and highlights the utility of non-invasive PET/CT imaging to evaluate new therapies in the preclinical area.

Drug Delivery to the Posterior Segment of the Eye: Biopharmaceutic and Pharmacokinetic Considerations.

The treatment of the posterior-segment ocular diseases, such as age-related eye diseases (AMD) or diabetic retinopathy (DR), present a challenge for ophthalmologists due to the complex anatomy and physiology of the eye. This specialized organ is composed of various static and dynamic barriers that restrict drug delivery into the target site of action. Despite numerous efforts, effective intraocular drug delivery remains unresolved and, therefore, it is highly desirable to improve the current treatments of diseases affecting the posterior cavity. This review article gives an overview of pharmacokinetic and biopharmaceutics aspects for the most commonly-used ocular administration routes (intravitreal, topical, systemic, and periocular), including information of the absorption, distribution, and elimination, as well as the benefits and limitations of each one. This article also encompasses different conventional and novel drug delivery systems designed and developed to improve drug pharmacokinetics intended for the posterior ocular segment treatment.

Enzyme-Loaded Gel Core Nanostructured Lipid Carriers to Improve Treatment of Lysosomal Storage Diseases: Formulation and In Vitro Cellular Studies of Elosulfase Alfa-Loaded Systems.

Mucopolysaccharidosis IVA (Morquio A) is a rare inherited metabolic disease caused by deficiency of the lysosomal enzyme N-acetylgalatosamine-6-sulfate-sulfatase (GALNS). Until now, treatments employed included hematopoietic stem cell transplantation and enzyme replacement therapy (ERT); the latter being the most commonly used to treat mucopolysaccharidoses, but with serious disadvantages due to rapid degradation and clearance. The purpose of this study was to develop and evaluate the potential of nanostructured lipid carriers (NLCs) by encapsulating elosulfase alfa and preserving its enzyme activity, leading to enhancement of its biological effect in chondrocyte cells. A pegylated elosulfase alfa-loaded NLC was characterized in terms of size, ζ potential, structural lipid composition (DSC and XRD), morphology (TEM microscopy), and stability in human plasma. The final formulation was freeze-dried by selecting the appropriate cryoprotective agent. Viability assays confirmed that NLCs were non-cytotoxic to human fibroblasts. Imaging techniques (confocal and TEM) were used to assess the cellular uptake of NLCs loaded with elosulfase alfa. This study provides evidence that the encapsulated drug exhibits enzyme activity inside the cells. Overall, this study provides a new approach regarding NLCs as a promising delivery system for the encapsulation of elosulfase alfa or other enzymes and the preservation of its activity and stability to be used in enzymatic replacement therapy (ERT).

Evaluation of the therapeutic activity of melatonin and resveratrol in Inflammatory Bowel Disease: A longitudinal PET/CT study in an animal model.

Inflammatory Bowel Disease (IBD) is a group of chronic disorders of the gastrointestinal tract, which two main types are Crohn's disease and ulcerative colitis. Although conventional therapeutic strategies have demonstrated to be effective in the IBD treatment, it is necessary to incorporate novel therapeutic agents that target other mechanisms involved in the pathogenesis of the disease, such as oxidative stress. For this reason, the efficacy in vivo of two antioxidant compounds, melatonin and resveratrol, has been investigated in an animal model of TNBS (2, 4, 6-trinitrobenzenesulfonic acid) induced colitis. PET/CT (Positron emission tomography/Computer Tomography) scans were performed to assess disease activity and evaluate treatment response. SUVmax (Standardized Uptake Value) values, body weight changes and histological evaluation were used as inflammatory indices to measure the efficacy of both treatments. SUVmax values increased rapidly after induction of colitis, but after the beginning of the treatment (day 3) a statistically significant decrease was observed on days 7 and 10 in treated animals compared to the non-treated group. This remission of the disease was also confirmed by histological analysis of the colon tissue using the Nancy histological index (p value < 0.05 for differences between non-treated and both groups of treated animals). Moreover, statistical analysis showed a correlation (R2 = 65.52%) between SUVmax values and weight changes throughout the treatment. Overall, this study demonstrates the potential of resveratrol, and melatonin in lower extent, as therapeutic agents in the IBD treatment.

Development, Characterization, and In Vitro Evaluation of Resveratrol-Loaded Poly-(ε-caprolactone) Microcapsules Prepared by Ultrasonic Atomization for Intra-Articular Administration.

: Intra-articular administration of drugs to the joint in the treatment of joint disease has the potential to minimize the systemic bioavailability and the usual side-effects associated with oral drug administration. In this work, a drug delivery system is proposed to achieve an anti-inflammatory local effect using resveratrol (RSV). This study aims to develop microcapsules made of poly-(ε-caprolactone) (PCL) by ultrasonic atomization to preserve the antioxidant activity of RSV, to prevent its degradation and to suppress the inflammatory response in activated RAW 264.7 macrophages. An experimental design was performed to build a mathematical model that could estimate the effect of nozzle power and polymer concentration on particle size and encapsulation efficiency. RSV-loaded microcapsules showed adequate morphology, particle size, and loading efficiency properties. RSV formulations exhibited negligible cytotoxicity and an efficient amelioration of inflammatory responses, in terms of Nitric Oxide (NO), ROS (Reactive Oxygen Species), and lipid peroxidation in macrophages. Thus, RSV-loaded microcapsules merit consideration as a drug delivery system suitable for intra-articular administration in inflammatory disorders affecting the joint.

Longitudinal PET/CT evaluation of TNBS-induced inflammatory bowel disease rat model.

Inflammatory bowel disease (IBD) is a group of chronic disorders of the gastrointestinal tract, which two main types are Crohn's disease and ulcerative colitis. It has multifactorial etiologies, being essential the use of animal models and disease activity measures to develop new therapies. With this aim, the use of animal models in combination with non-invasive molecular imaging can play an important role in the development of new treatments. In this study, IBD was induced in rats using 2,4,6-trinitrobenzenesulfonic acid (TNBS) and longitudinal [18F]FDG PET/CT scans were conducted to assess disease progression post-TNBS administration. Afterwards, [18F]FDG PET/CT scans were carried out after treatment with methylprednisolone to validate the model. In non-treated rats, SUVmax (Standardized Uptake Value) rapidly increased after IBD induction, being particularly significant (p < 0.01) on days 7-13 after induction. There were no significant differences between non-treated and treated IBD rats from days 0-3. Nevertheless, treated IBD rats showed a significant decrease in SUVmax between days 7-13 (p < 0.01). Histological examination showed descending and transverse colon as the most affected regions. There was a moderate (R2 = 0.61) and strong (R2 = 0.82) correlation of SUVmax with Nancy grade (parameter for histological assessment of disease activity) and weight changes, respectively. In this study, we have performed the first longitudinal [18F]FDG PET/CT assessment of TNBS-induced IBD in rats, demonstrating the potential role of preclinical molecular imaging for the evaluation of new therapies in combination with IBD rat models.

Development of particulate drug formulation against C. parvum: Formulation, characterization and in vivo efficacy.

This research aims towards developing an alternative therapy against Cryptosporidium parvum using bioadhesive paromomycin and diloxanide furoate-loaded microspheres. Microspheres were prepared using chitosan and poly(vinyl alcohol) and two types of cyclodextrins (ß-CD and DM-ß-CD) for the potential use of treating cryptosporidiosis. This pathogen is associated with gastrointestinal illness in humans and animals. Microparticle formulations were characterized in terms of size, surface charge, drug release and morphology. In vivo bioadhesion properties of CHI/PVA microspheres were also evaluated in mice. Finally, the in vivo efficacy of CHI/PVA microspheres against C. parvum was tested in neonatal mouse model. In this work, microspheres prepared by spray-drying showed spherical shape, diameters between 6.67±0.11 and 18.78±0.07µm and positively surface charged. The bioadhesion studies demonstrated that MS remained attached at +16h (post-infection) to the intestinal cells as detected by fluorescence. This finding was crucial taking use of the fact that the parasite multiplication occurs between 16 and 20h post-infection. The efficacy of treatment was determined by calculating the number of oocysts recovered from the intestinal tract of mice after 7days of post-infection. Mice receiving orally administered microspheres with and without drug exhibited significantly lower parasite loads compared with the control mice. Ultrastructural observations by TEM bring to light the uptake of smallest particles by enterocytes associated with conspicuous changes in enterocytic cells. Completely recovery of cell morphology was detected after 24h of first inoculation with MS. CHI/PVA microspheres appear to be a safe and simple system to be used in an anticryptosporidial treatment. The distinctive features of neonatal mice requires further work to determine the suppressive effect of this particulate delivery system on C. parvum attachment in other animal models.

Cyclodextrin-based polysaccharidic polymers: an approach for the drug delivery.

The aim of the present review is to give an overview of a new class of polysaccharidic CD-based systems and to address the use of these systems in drug delivery. First, we review miscellaneous methods to obtain and synthesize the structural modification of natural polysaccharides, mainly, with cyclodextrins. In a second part of this review, we provide a summary of recent findings not only in the field of drug delivery, but in also how these systems have displayed different applications in the biomedical and pharmaceutical fields.

Cyclodextrin based rotaxanes, polyrotaxanes and polypseudorotaxanes and their biomedical applications.

For years, great efforts have been made by scientists to construct various novel interlocked supramolecular systems. A wide range of pseudorotaxanes and rotaxanes, full of challenging constructions and potential applications in areas such as nanostructured functional materials, molecular switches, molecular logic gates, molecular wires, memory devices Willner and biomedical applications have been reported recently. Cyclodextrin polyrotaxantes or polypseudorotaxanes there are not only nice and interesting supramolecular architectures but they also have a high interest for biomaterials application that allow to opened up new approaches for tissue regeneration, drug and gene delivery. The use of the supramolecular cyclodextrins complexes has given rise to interesting studies to design and develop new biomaterials with advanced properties. In this review we will update the recent advances in the use of CDs-supramolecular structures for develop new and advanced drug and gene delivery systems and for use in tissue engineering.

NMR techniques in drug delivery: application to zein protein complexes.

Zein is a protein containing a large amount of nonpolar amino acids, which has shown the ability to form aggregates and entrap solutes, such as drugs and amino acids. NMR techniques were used to detect binding interactions and measure affinity between zein and three different drugs: tetracycline, amoxicillin and indomethacin. The release study of zein microparticle formulations containing any of these drugs was confronted with the affinity results, showing a remarkable correlation. The feasible methodology employed, focused in the functionality of the protein-drug interaction, can be very promising for the rational design of appropriate drug vehicles for drug delivery.

In vitro evaluation of the suppressive effect of chitosan/poly(vinyl alcohol) microspheres on attachment of C. parvum to enterocytic cells.

We present a new strategy to suppress the attachment of Cryptosporidium parvum to the enterocytes cell surface by bioadhesive microspheres. An optimized microsphere system based on chitosan/poly(vinyl alcohol) was prepared by experimental design for the delivery of Diloxanide Furoate-cyclodextrin complex. Formulations were characterized in terms of size, surface charge, drug release, IR spectroscopy and morphology. Bioadhesion properties of chitosan/poly(vinyl alcohol) microspheres, evaluated in the human enterocytic HCT-8 model, were concentration and time dependent. In vitro efficacy of chitosan/poly(vinyl alcohol) microspheres against Cryptosporidium was tested in infected cultures and stages of parasite were assessed by immunofluorescence. The degree of adherence to cells and the inhibition of infectivity were directly related with the lowest level of cross-linking. The C. parvum attachment to cells surface was efficiently suppressed by a concentration of 100 µg/ml of microspheres. TEM observations showed no epithelial-cell damage when microspheres were co-incubated in infected cultures. These results were coincident with the lack of toxicity in cytocompatibility studies. Microspheres remained adhered after 72 h to the apical area of enterocytes. The results suggest that chitosan/poly(vinyl alcohol) with adequate size and appropriate surface characteristics suppress by impairment the attachment of sporozoites to enterocytes and may have a great potential in the oral chemotherapy of Cryptosporidium infections.

Effect of zein on biodegradable inserts for the delivery of tetracycline within periodontal pockets.

Treatment with antibiotics within the periodontal pocket against bacterial infections represents a useful and adjunctive tool to conventional therapy for healing and teeth preservation. With this function in view, an implantable, tetracycline delivery device for the treatment of periodontal disease was developed. The aim of this study was to develop biodegradable, tetracycline-loaded microparticles made of two polymers: PLGA and zein which were compressed into monolithic devices. In this polymer delivery system, the encapsulation efficiency, release characteristics, drug-polymer interaction, and antibacterial activity of loaded drug were investigated. The interaction of tetracycline with the corn protein zein was studied by nuclear magnetic resonance (NMR), Fourier transform infrared, and X-ray diffraction. The hydrophobic interaction of tetracycline with zein in the formulations was deduced from the NMR studies, whereas X-ray diffraction studies showed a new crystalline state of the drug in the presence of the protein. Zein was not denatured by preparation of inserts. Sustained release of tetracycline was obtained, and the proportion of zein in the inserts had a great impact on the drug release. Finally, an effective tetracycline release from inserts against Staphylococcus aureus was achieved over 30 days. In conclusion, the PLGA:zein delivery system described in this study was found to be effective in controlled delivery of tetracycline, and hence may be suitable for intra-pocket delivery of antimicrobial agents in the treatment of periodontitis.

Incorporation of PVMMA to PLGA MS enhances lectin grafting and their in vitro activity in macrophages.

Attachment of lectins, especially those that are recognized and bounded by macrophages and other antigen presenting cells onto biodegradable microspheres surface, may serve as support for a wide variety of applications. The aim of this work was: (1) to prepare microspheres (MS) based on two biodegradable copolymers, poly (lactide-co-glicolide) acid (PLGA) and poly[(methyl vinyl ether)-co-(maleic anhydride)] (PVMMA) with their surface functionalized with Concanavalin A (Con A) and, (2) to evaluate their behaviour in macrophage culture. The incorporation of PVMMA was studied in order to augment the lectin coupling efficiency. MS were obtained by spray-drying and bovine serum albumin (BSA) was loaded as protein model. Particles were characterized for size, morphology, surface charge, entrapment efficiency, FTIR, in vitro protein release and conjugation efficiency. Finally, functionalized MS were tested in vitro with raw 264.7 murine macrophages (Mф) in terms of cytotoxicity, phagocytosis, nitric oxide (NO) production, and oxygen consumption. Conjugated Con A microspheres showed increased grafting efficiency up to four times compared to PLGA alone. The retention of Con A after coupling was confirmed by desorption studies. The attachment of Con A to microspheres induced oxygen consumption, increased phagocytosis efficiency and even NO production by macrophages. The results suggest that Con A and possibly, other lectins, grafted onto PLGA-PVMMA microspheres may serve as potential adjuvants by modulating protein delivery and macrophage activation.

Biodegradable microspheres alone do not stimulate murine macrophages in vitro, but prolong antigen presentation by macrophages in vitro and stimulate a solid immune response in mice.

The purpose of this study was to analyze the potential of various types of biodegradable microspheres (MS) (i) to activate in vitro cell line-derived macrophages (RAW 264.7, Mphi), and primary peritoneal and bone marrow-derived mouse Mphi, to prolong the release and presentation of microencapsulated synthetic malaria antigens by Mphi after uptake of antigen-loaded MS, and (ii) to stimulate an immune response in mice against a microencapsulated synthetic malaria antigen. The MS were made of various types of poly(lactide-co-glycolide) (PLGA) or chitosan cross-linked with tripolyphosphate. PLGA, but not chitosan MS, were efficiently ingested by Mphi. Upon exposure to the various MS types, Mphi increased only the production of reactive oxygen intermediates (ROI), while the production of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), and the expression of cyclooxigenase-2 (COX-2), inducible NO synthase (iNOS), the cell surface markers MHC class I and II, and CD 86 remained unaffected. In vitro release of the microencapsulated antigen from PLGA50:50 MS followed a pulsatile pattern and extended over 14 weeks. This prolonged antigen release was also mirrored in the significantly prolonged antigen presentation over more than 7 days by Mphi after uptake of antigen-loaded PLGA MS. Finally, antigen-loaded PLGA MS induced a solid immune response in mice after a single s.c.-injection, which was only slightly inferior to the antibody titers measured with the control formulation with Montanide ISA720. These results suggest that MS are well tolerated by Mphi. The prolonged antigen presentation by Mphi, as measured in vitro, along with the capacity to induce a strong immune response in animals emphasize that biodegradable MS are a very promising delivery system for both preventive and immunotherapeutic vaccines.

Efficacy of alpha-cyclodextrin against experimental cryptosporidiosis in neonatal goats.

The efficacy of orally administered tablets containing alpha-cyclodextrin, an excipient used in the pharmaceutical industry with demonstrated anticryptosporidial activity in vitro and in neonatal mice, was evaluated in neonatal goat kids. The formulation was evaluated for hardness and was subjected to in vitro drug release studies. Twenty goat kids were orally inoculated with 10(6) oocysts of C. parvum within the first 6 days of age. Half of the animals were treated by oral administration of four tablets of alpha-cyclodextrin/day (500 mg/kg of body weight) for six consecutive days, the treatment beginning on the day of inoculation. Infection was monitored by daily examination of faecal samples from the first day to 25 days post-inoculation. The criteria studied in evaluating efficacy were: oocyst shedding, presence of diarrhoea and weight gain at 15 and 25 days post-inoculation. alpha-cyclodextrin was effective when given at the beginning of infection: there was a longer pre-patent period, a reduction in the patent period and a decrease in the intensity of infection, these differences being statistically significant (P < 0.05) compared with untreated neonatal kids. Moreover, except in one animal, the diarrhoea was prevented in infected neonatal kids. Animals from both groups increased the body weight and no significant differences were seen between the two groups.